s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Transcriptomic Analysis Reveals Key Candidate Genes Related to Seed Abortion in Chinese Jujube (Ziziphus jujuba Mill)

Background Seed abortion is a common phenomenon in Chinese jujube that seriously hinders the process of cross-breeding. However, the molecular mechanisms of seed abortion remain unclear in jujube. Methods Here, we performed transcriptome sequencing using eight flower and fruit tissues at different developmental stages in Ziziphus jujuba Mill. ‘Zhongqiusucui’ to identify key genes related to seed abortion. Histological analysis revealed a critical developmental process of embryo abortion after fertilization. Results Comparisons of gene expression revealed a total of 14,012 differentially expressed genes. Functional enrichment analyses of differentially expressed genes between various sample types uncovered several important biological processes, such as embryo development, cellular metabolism, and stress response, that were potentially involved in the regulation of seed abortion. Furthermore, gene co-expression network analysis revealed a suite of potential key genes related to ovule and seed development. We focused on three types of candidate genes, agamous subfamily genes, plant ATP-binding cassette subfamily G transporters, and metacaspase enzymes, and showed that the expression profiles of some members were associated with embryo abortion. Conclusion This work generates a comprehensive gene expression data source for unraveling the molecular mechanisms of seed abortion and aids future cross-breeding efforts in jujube.

INTRODUCTION
Chinese jujube (Ziziphus jujuba Mill.), a member of the Rhamnaceae family, is thought to have been cultivated for more than 7,000 years and is one of the most economically important fruit trees in China [1]. It is widely distributed and rich in germplasm resources [2]; it also has high nutritional and medicinal value, as it contains several biologically active components required by the human body, including vitamin C, phenolics, flavonoids, triterpenic acids, polysaccharides, and microelements [3,4]. Jujube fruits can be consumed fresh, dried, or processed in various forms [5]. In addition to its high economic value, the Chinese jujube also provides ecological benefits. Because of its ability to grow in diverse environments, the jujube tree has played a critical role in restructuring the agricultural industry and in the economic development of poverty-stricken regions in China. Some of the disadvantages associated with the production of Chinese jujube fruit include the severe abscission *Address correspondence to this author at the College of Landscape Architecture, Central South University of Forestry and Technology, Changsha 410004, China; Tel: +86-137-8612-3158; E-mail: csuftwangsen@163.com of flowers and fruits and the ease with which the fruit can crack. Although improvements in jujube varieties are essential for enhancing the quality, yield, and efficiency of jujube cultivation [6], embryo abortion is a common phenomenon in jujube, which prevents the formation of viable seeds, restricts cross-breeding efficiency, and makes obtaining hybrid progeny a major challenge. There is thus an urgent need to understand the causes of seed abortion at the molecular level, as such information is critically important for breeding new cultivars specifically and improving the jujube industry more generally.
The factors affecting plant seed abortion have been studied extensively. In model plants, genetic studies have greatly improved our understanding of embryo development, and mutations of genes related to embryo development have been shown to cause seed abortion. In Arabidopsis thaliana, a great number of genes required for seed development have been identified through large-scale mutagenesis approaches [7,8], which have provided valuable resources for functional analyses. Biotic and abiotic stresses, the partitioning of carbohydrates, and phytohormones (e.g., auxin, gibberellin) can affect the development of seed and fruit, and even lead to seed abortion [9]. Studies on invertases, enzymes hydrolyzing sucrose into glucose and fructose, have revealed that the elevated expression of invertase and high glucose concentrations enhance embryo development and reduce seed abortion in maize and tomato [10,11]. In fruit crops, studies on seedlessness have identified many genes that may be selected during domestication. For example, in grapevine, VvAGL11 (a homologous gene of Arabidopsis Seedstick) has been found to play a central role during the selection of seedless grapevine cultivars [12]; sequence variations in the promoter and coding regions of VvAGL11 have been shown to be correlated with seed abortion [13].
In recent years, the advent of high-throughput 'omics' studies has greatly facilitated our understanding of the molecular regulation of seed abortion at the transcriptional, biochemical, and metabolic levels. Based on large-scale transcriptomic and proteomic analyses, 140 differentially expressed genes (DEGs) and 41 differentially expressed proteins were identified between normal and aborted embryos in chrysanthemum [14]. Comparative proteomic analysis in longan (Dimocarpus longan Lour.) has yielded more than 1000 differentially expressed proteins [15]. In peanut, transcriptomics studies have also been used to study embryo abortion and have identified thousands of DEGs, including genes involved in cell division, the stress response, embryonic development, and signal transduction [16,17]. These results have highlighted the complexity of the regulation of embryo development, which involves the coordination of multiple pathways; more generally, these findings have emphasized the need for a deeper understanding of the molecular functions of critical genes.
Previous studies have primarily focused on the physiology, biochemistry, and embryology aspects of the mechanisms of seed abortion in jujube [18][19][20]. By comparison, little is known of the molecular mechanisms. Z. jujuba Mill. 'Zhongqiusucui' is a newly bred cultivar with large, highly sweet fruits [21]. We have previously observed defects in the abnormal embryo sac, despite the fact that the development of microsporogenesis appeared normal, which led to incomplete embryo development [22]. Here, we used RNAseq technology to sequence and analyze the transcriptome of jujube flowers and fruits at different developmental stages. A great number of DEGs were identified for further functional analysis, which revealed a suite of genes potentially involved in seed abortion. Generally, this work provides a foundation for understanding the regulation of seed development in jujube.

Plant Materials
The experimental jujube cultivar was Z. jujuba Mill. 'Zhongqiusucui', which was selected from a bud mutation of Z. jujuba Mill. 'Tangzao' in Qidong County, Hunan Province, China [21]. Its flowers are hermaphroditic and are composed of the flower stalk, calyx, petal, flower disk, sta-men, and pistil. It has a high seed abortion rate (approximately 95%) under natural pollination according to our previous study (unpublished data).
The experimental site was the jujube tree experimental base of Central South University of Forestry and Technology (Qidong County,Hunan Province;112.12°E,26.78°N). There were about 300 five-year-old jujube trees that showed good growth and developmental conditions, relatively uniform degrees of tree vigor, and no signs of diseases or pests. Flower samples were collected at five developmental stages: floral bud oblate stage (BO), floral bud-yellowing stage (BY), sepal-flattening stage (SF), stamen-wilting stage (SW), and ovary enlargement stage (OE) [23,24]. We randomly collected whole flowers at the same developmental stage from flower-bearing branches in the middle of jujube trees and placed them into 1.5 ml centrifuge tubes; the tubes were placed into liquid nitrogen when they were full. A total of nine tubes of flowers were collected for each stage. Each stage had three biological replicates, and the mixed samples of every three tubes were considered to represent one biological replicate. Fruit samples were harvested at three developmental stages. At the FrI stage, there were fruits with a normal embryo. In the FrII stage, the jujube fruit embryo began to abort. The samples for the FrII stage were jujube fruits with light embryo abortion. The FrIII stage is the period when the embryo begins mass abortion. The jujube fruit samples for the FrIII stage exhibited a higher embryo abortion degree. We randomly collected whole fruits at the same stage from fruit-bearing branches in the middle of the jujube trees. Each fruit was cut with a razor blade to determine whether it was bearing seeds or not. The ovaries were cut and placed into a 1.5 ml centrifuge tube; tubes were placed into liquid nitrogen when they were full. A total of nine tubes of flowers were collected for each stage. Each stage had three biological replicates, and the mixed samples of every three tubes were considered to represent one replicate. All plant tissues were transferred to and stored in a -80°C ultra-low temperature freezer until analyses were performed.

Microscopic Observations
The routine paraffin section method [25] was used to generate 8-μm sections. Sections were stained using the modified Ehrlich's haematoxylin staining method [26] and mounted with neutral gum. A Leica DMi8 inverted microscope was used to observe and take photographs.

Total RNA Extraction, cDNA Library Construction, and RNA Sequencing
Three biological replicates of each sample at each stage were used for the RNA-Seq experiments. Total RNA was extracted using the CTAB method. The quality and quantity of RNA were assessed by electrophoresis on 1% agarose gels and a Nanodrop 2500 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to ensure that the samples were suitable for transcriptome sequencing. RNA integrity was assessed by an Agilent2100 Bioanalyzer (Agilent, Palo Alto, CA, USA) in conjunction with an Agilent RNA 6000 Nano Kit. RNA samples were sent to Wuhan Frasergen Co., Ltd. (Donghu New and High-tech Development Zone, Wuhan, China), where the libraries were produced and sequenced. Oligo (dT) magnetic beads were used to enrich the poly(A) tails of mRNA, and the enriched mRNA was randomly interrupted by divalent cations in the NEB fragmentation buffer. First-strand cDNA was synthesized in an M-MuLV reverse transcriptase system with the fragmented mR-NA as the template and random oligonucleotides as the primers. Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. The purified double-stranded cDNA was repaired, A-tailed, and connected to the sequencing connector. To select cDNA fragments ~240 bp in length, the library fragments were purified with AMPure XP beads (Beckman Coulter, Beverly, MA, USA). The cDNA fragments were amplified by PCR and purified by AMPure XP beads to obtain the library. Next, a Qubit 2.0 Fluorometer was used for preliminary quantification, and the library was diluted to 1.5 ng/ul. An Agilent 2100 Bioanalyzer was used to detect the insert size of the library. After the insert size met expectations, qRT-PCR was used to accurately quantify the effective concentration of the library to ensure its quality (the effective concentration of the library needed to be higher than 2 nm). Sequencing was performed on an Illumina Hiseq 2500 as per the manufacturer's protocol.

RNA-Seq Analysis
The software Trimmomatic (v0.4.5) [27] was used to filter the raw reads in the FASTQ format by removing lowquality reads, which consisted of reads in which more than 20% of the bases had a quality lower than 10, reads containing adaptors, or reads with more than 5% of unknown bases. Next, the quality of clean reads was verified using FastQC, including the Q20, Q30, and GC content of the clean data. All downstream analyses were based on clean data of high quality. Clean data were aligned to the Z. jujuba genome (https://www.ncbi.nlm.nih.gov/genome/?term=Zizyphus+jujuba) using Tophat2 [28] and bowtie2 [29], with the mapping parameters "-library-type fr-unstranded -p 6 -G". The gene expression levels of all samples were estimated by RSEM [30] for each sample, and fragments per kilobase of transcript per million mapped reads (FPKM) were used to estimate gene expression levels.

Differential Gene Expression Analysis
Comparisons were made among seven groups: BO-vs-BY, BY-vs-SF, SF-vs-SW, SW-vs-OE, OE-vs-FrI, FrI-vs-FrII, and FrII-vs-FrIII. The genes of three biological replicates were pooled in each group. DEGs between two groups were identified using the R package DEGseq2 [31] with the following threshold values: |log 2 (fold change) | > 1 and FDR < 0.05. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed for the DEGs. Hypergeometric tests of GO analysis and pathway functional enrichments of the DEGs were performed with the Stats R package (version 3.7.0). GO functional enrichment analysis and KEGG pathway functional enrichment analysis were performed via phyper, a function in R. The false discovery rate (FDR) for each p-value was calculated; in general, terms with an FDR < 0.05 were defined as significantly enriched.

WGCNA
WGCNA (weighted gene co-expression network analysis) is a systems biology method for describing the correlations among genes across multiple samples. This method finds clusters (modules) of highly correlated genes and relates modules to external sample traits. Co-expression networks were constructed using the WGCNA (v1.47) package in R [32]. After filtering genes not expressed in more than half of the samples, gene expression values were imported into WGCNA to construct co-expression modules using the automatic network construction function 'blockwiseModules' with default settings, except that the power was 15; TOM Type was unsigned, 'mergeCutHeight' was 0.25, and 'minModuleSize' was 50. Genes were clustered into 12 correlated modules. To identify biologically significant modules, module eigengenes were used to calculate the correlation coefficients with samples. Intramodular connectivity (K.in) and the module correlation degree (MM) of each gene were calculated by the WGCNA R package, and genes with high connectivity tended to be hub genes that might have important functions. For genes in each module, GO and KEGG pathway enrichment analyses were conducted to analyze the biological functions of modules.

Quantitative Real-time PCR (qRT-PCR) Analysis
To validate the accuracy of the RNA-seq results, 15 DEGs were selected for quantitative real-time PCR (qRT-PCR) analysis, and ZjEF1α (GeneID: 107422504) was used as the housekeeping gene to normalize the expression levels. Gene-specific primers were designed by Primer Premier 6.0 software (http://www.PremierBiosoft.com) (Supplementary file 1). The cDNA was synthesized from 500 ng of the total RNA in a 10-μL reaction system by PrimeScript™ RT Master Mix Kit (Takara, Beijing, China). The qRT-PCR reactions were carried out using a TB Green™ Premix Ex Taq ™ II (Tli RNaseH Plus) Kit (TaKaRa, Beijing, China). The 20-μL reaction volume contained 5 μL of TB Green Premix Ex Taq II (Tli RNaseH Plus) (2×), 0.4 μL of ROX reference Dye II (50 ×), 0.8 μL of each primer (10 μM), 2 μL of cD-NA, and 6 μL of sterile distilled water. The PCR procedure was 95°C for 30 s, 40 cycles of 95°C for 5 s, and 60°C for 30 s, followed by a dissociation stage. The qRT-PCR reaction was performed on an ABI 7500 Real-time PCR System. The relative expression level of each gene was calculated using the 2 -ΔΔCt method. Each reaction was performed with three biological replicates.

Morphological Characteristics of Jujube Seeds at Different Developmental Stages
Studies of early embryology have identified ovule abortion to be one of the main causes of seed abortion in Z. jujuba Mill. 'Zhongqiusucui' [22]. The characteristic signs of embryo abortion in jujube were the cessation of development and the gradual shrinkage of kernels or seeds, leaving only seed traces (Fig. 1A). Normally well-developed jujube seeds grew as the fruit developed and filled each ventricle of the ovary (Fig. 1A). In contrast, abortive seeds ceased growing in the middle, despite the continued development of the fruit. Abortive seeds then gradually withered and shriveled as the jujube fruit matured. We hypothesized that seed abortion in Z. jujuba Mill. 'Zhongqiusucui' was caused by ovule abortion; this leads to the prediction that crucial genes mediating the process of abortion should be misexpressed. Therefore, we sequenced the transcriptomes of jujube flowers (BO, BY, SF, SW, and OE; Fig. 1B) and fruits (FrI, FrII, and FrIII; Fig. 1C) at different developmental stages to explore the relationship between the expression of genes during ovule development and embryo or seed abortion.

RNA-seq, Reads Mapping, and DEG Analyses
To reveal the molecular mechanisms underlying seed abortion in jujube, a total of 24 samples (including three biological replicates) were sequenced using the Illumina Hiseq 2500 platform. After quality control, 81.12 Gb of high-quality clean reads were obtained. The number of clean reads was between 21,441,794 and 30,551,956. Q20 ranged from 97.4% to 97.6%, Q30 from 93.5% to 93.9%, and GC content from 44.1% to 44.9%. Statistical results are shown in Supplementary file 2. Clean data were aligned to the Z. jujuba genome (https://www.ncbi.nlm.nih.gov/genome/?term=Zizyphus). The mapping ratio of all clean reads in the sample to the reference genome was greater than 75%, indicating that the 24 samples had a high degree of matching with the Z. jujuba genome (https://www.ncbi.nlm.nih.gov/genome/?ter-m=Zizyphus) (Supplementary file 3). The high mapping ratio also demonstrated the reliability of the sequencing results. Thus, transcriptome sequencing data in this study were accurate, and the sequencing quality was high.
We calculated the FPKM of each sample to explore differences in gene expression among different jujube tissues. To obtain the DEGs, seven comparison groups were analyzed, including BO-vs-BY, BY-vs-SF, SF-vs-SW, SW-vs-OE, OE-vs-FrI, FrI-vs-FrII, and FrII-vs-FrIII. We used 'FDR < 0.05 & |log 2 FC| > 1' as the criterion for assessing signifi-cant differences in gene expression. When log 2 FC > 1, the DEG was considered up-regulated. In contrast, when log 2 FC < -1, the DEG was considered down-regulated. We conducted up-and down-regulated analyses of DEGs for the seven comparison groups, and the results are shown in Fig.  (2A-2H). Overall,5,696,4,722,7,651,1,972,1,254,5,422,and 762 DEGs were detected for BO-vs-BY, BY-vs-SF, SF-vs-SW, SW-vs-OE, OE-vs-FrI, FrI-vs-FrII, and FrII-vs-FrIII, respectively. We found that SF-vs-SW had the largest number of DEGs, indicating that this is a critical developmental event during floral development. There were overlapped DEGs among the seven comparisons. After removing duplicate overlapping genes, a total of 14,012 DEGs were obtained.

Functional Characterization of DEGs by GO and KEGG Enrichment Analyses
To identify the molecular functions of DEGs at different developmental stages in jujube flowers and fruits, the DEGs in seven comparison groups were analyzed by GO enrichment. The top 50 GO terms enriched in each comparison are listed in Fig. (3), Supplementary file 4 and 5. We analyzed the GO terms enriched in BO-vs-BY and FrI-vs-FRII in detail. According to the classification of GO terms in biological process, most DEGs were enriched in 'metabolic process (GO:0008152)', 'cellular process (GO:0009987)', and 'single-organism process (GO:0044699)' (Figs. 3A and 3B). These biological processes were likely related to flower and seed development. According to the classification of GO terms in molecular functions, most DEGs were enriched in 'catalytic activity (GO:0003824)', 'binding (GO:0005488)', 'transporter activity (GO:0005215)', and 'nucleic acid binding transcription factor activity (GO:0001071)'. Among cellular component GO terms, DEGs were primarily enriched in 'membrane (GO:0016020)', 'plasma membrane part (GO:0044459)', and 'membrane part (GO:0044425)' (Figs. 3A and 3B). All of the above GO terms were closely related to the regulation of biological processes and the synthesis and metabolism of functional substances. The numbers of DEGs enriched in these GO terms in each comparison were large, and both up-regulated and down-regulated DEGs were contained in these GO terms (Supplementary file 5).

Gene Co-expression Network Analysis
Gene co-expression networks were constructed based on pairwise correlations between genes using WGCNA [33]. Before WGCNA, we filtered the selected genes to remove low-quality genes or samples that had an unstable impact on the results to improve the accuracy of network construction. A total of 13,155 genes were filtered, and there were 20,037 effective genes after filtering. Modules were defined as clusters of highly interconnected genes; genes within the same cluster had high correlation coefficients. A total of 12 distinct modules (labeled by different colors) were identified and illustrated by a dendrogram (Fig. 5A). The size of these modules depended on the number of genes that they contained (Fig. 5B). Among them, the number of genes clustered in the dark green module was the highest (9,394); the coral 2 module contained the lowest number of genes (130). The grey module contained a set of genes that could not be classified into any other modules (Fig. 5B).
The module eigengene is the first principal component of a given module and can be considered to represent the module's gene expression profile. A heat map of the expression patterns of samples was performed based on the 12 module eigengenes for the 12 distinct modules (Fig. 6). We also performed a gene expression trend analysis for each module separately (Supplementary file 8). GO and KEGG pathway enrichment analyses on the genes of these modules were performed (Supplementary files 9 and 10). These 12 modules were screened based on the expression patterns and the results of GO and KEGG pathway enrichment analyses of each module. Dark green and yellow-green modules were composed of genes that were highly expressed in the FrII and FrIII seed abortion stages. The genes clustered in dark orange 2, medium orchid, and saddle brown modules were highly expressed during the key period of seed abortion (FrI). Indiana red 4, plum, and light pink 4 modules contained genes that were highly expressed during the critical stages of the embryo sac (BY and SF) but were expressed at low levels during seed abortion stages (FrII and FrIII). The expression patterns of these seven modules had periodic correlations, with high or low expression levels during the critical period of ovule or seed development (Supplementary file 8). The dark green module contained the largest number of DEGs and was significantly enriched for a larger number of biological process GO terms (Supplementary file 9). Many biological processes related to development (14 terms), flower development (7 terms), embryo development (7 terms file 9). GO terms enriched in the yellow-green module were primarily related to the synthesis, metabolism, and transportation process of cell biosynthesis (10 terms), proteins (28 terms), saccharides (10 terms), and vitamins (6 terms), as well as transport (10 terms) (Supplementary file 9). In addition, GO terms related to cell wall formation were significantly enriched, such as 'cell wall polysaccharide metabolic process (GO:0010383)' and 'cell wall organization or biogenesis (GO:0071554)' (Supplementary file 9). The above analysis indicated that key modules were involved in multiple biological processes, including flower development, embryo and seed development, and metabolism, that might be essential for the regulation of seed abortion.

Expression Profiling of Key Genes Related to Ovule and Seed Development
The seed abortion of jujube occurs during the process of ovule and seed development. The regulatory pathway of ovule and seed development has been clarified in the model plant A. thaliana. A series of important genes affecting either ovule or seed development have been identified in A. thaliana [34], providing a valuable reference for similar studies on other plant species. Based on previously collected data [34][35][36][37], our study used the ovule and seed development in A. thaliana as a reference, including related genes as precursors, and identified homologous genes in jujube based on sequence similarity searches (BioEdit version 7.0.9.0) (Supplementary file 11). A total of 38 homology genes in jujube were selected (Supplementary file 11). There were 7 modules (dark green, plum, indian red 4, medium orchid, light green, dark orange 2, and saddle brown modules) for all of the homologous genes; most of them were in the module, and the pink module had the second highest number of homologous genes Supplementary file 11). We then used transcriptome data to analyze the expression patterns of these genes during ovule and seed development in jujube. The ex-pression profiles of genes related to jujube ovule and seed development in different functional categories were evaluated, including ovule development in the early stage, seed coat development, and endosperm development (Fig. 7).
Many ovule identity genes have been identified in A. thaliana, such as SEEDSTICK(STK)/AG11, SHATTER-PROOF1 (SHP1), SHP2, AGAMOUS (AG), BEL1, and SE-PALLATA(SEP) [38][39][40], which regulate ovule development primarily through formatting complexes [34,41]. The expression levels of ovule identity genes (ZjAG, ZjSEP1/2, ZjSEP3, ZjBEL1, and ZjSHP1/2) were higher during the jujube flower stages (BO, BY, SF, SW, and OE) than during the seed development stages (FrI, FrII, and FrIII) (Fig. 7). However, the lowest transcript levels of the STK/AGL11 homolog were detected in the BO stage, and the highest levels were observed in the FrI stage (the early developmental stage of ovules); the expression of the STK/AGL11 homolog was low in the FrII and FrIII stages (Fig. 7). These results suggest that STK/AGL11 plays an important role in the regulation of ovule development in jujube.

Potential Genes Related to Ovule and Seed Abortion in Jujube
The seed abortion process has been extensively studied in various plant species, and the number of potential related genes is vast. The MADS-box gene is one of the most important transcription regulators in plants and plays an important role in regulating growth, development, and signal transduction [42,43]. As previously discussed, some ovule identity genes, such as STK/AGL11, SHP1/AGL1, and SHP2/AGL5 identified in A. thaliana, belong to the MADS-box family of genes [39]. The AG family consists of class-D MADS-box genes that play important roles in ovule development [44]. The ABCG subfamily represents the largest group of ABC transporters, which are widely distributed in various plants and play an important role in growth and development [45]. Studies of A. thaliana, Oryza sativa L., V. vinifera, and other plants have shown that ABCG subfamily members are primarily involved in the formation of the vascular system, lipid transport, pollen wall development, and epidermal cuticle formation [46][47][48][49], which may be related to ovule or seed development. Plant metacaspases (MCs) are a family of cysteine proteases that are structurally related to caspase enzymes but differ in the specific sites of their substrates [50]. MC family genes have been shown to play a critical role in the process of programmed cell death (PCD) related to embryo development [51][52][53]. The abortion of jujube ovules or seeds may also be caused by a similar PCD process in normal embryo development. Thus, we investigated these three types of genes in jujube, including the AG subfamily of MADS-box genes, ABCG transporters, and MC enzymes. Eight AG subfamily genes that may affect ovule or seed development were identified. We found that two AGL61-like, one AGL29-like, one AGL18-like, and one AGL6-like transcripts displayed similar expression patterns with peak expression in the BO sample (Fig. 8A); the expression of the AGL29-like transcript peaked in the OE sample (Fig. 8A). The expression of the AGL23-like gene was down-regulated   during the developmental stage of the embryo sac (from SF to SW) and then was significantly up-regulated during the seed abortion stages (FrII and FrIII) (Fig. 8A). Based on sequence similarity analysis, we uncovered 22 ABCG-like transcripts, and five groups with distinctive expression profiles were identified (Fig. 8B). Four genes, including GeneI-D:107412473, GeneID:107411471, GeneID:107414661, and GeneID:107412484, were highly expressed during the embryo development stages (FrII and FrIII), suggesting that they play critical regulatory roles (Fig. 8B). We identified seven MC genes in jujube (Fig. 8C), and expression analysis revealed that five genes were up-regulated during the late stage of embryo development (Fig. 8C).
To confirm the accuracy of our RNA-seq data, 15 genes were randomly selected, and their expression patterns were validated by qRT-PCR. The results showed that the expression patterns of these genes were essentially consistent with the expression patterns observed in the RNA-seq data, indicating that our RNA-seq data were highly reliable and reproducible ( Fig. 9, Supplementary file 1). Further mining and functional characterization of potential causal genes are needed to elucidate the mechanism underlying seed abortion in jujube.

Genes Related to Ovule and Seed Development are Essential Regulators of Seed Abortion
To explore the potential mechanisms underlying ovule and seed abortion in jujube, we exploited the potential regulatory genes based on three aspects: ovule development during the early stages, seed coat development, and endosperm development (Fig. 7). The ovule identity genes (STK/AGL11, SHP1, SHP2, and SEP) primarily regulate ovule development through the formation of a complex in A. thaliana [34,41,54]. BEL1 interacts with the AG-SEP complex to form the BEL1-AG-SEP complex, which plays an important regulatory role [38]. Similarly, the ovule has been shown to develop abnormally and change into carpellate structures in stk, shp1, shp2, and bel1 mutants [38,39]. In this study, we found that during the abortion of ovules (FrIvs-FrII), homologs of STK/AGL11, INO, and BEL1 showed significantly down-regulated expression patterns in jujube (Fig. 7). These results suggested that the functions of the ovule identity genes were conserved, and suppression of their expression levels might be a molecular signature of embryo abortion during the early developmental stages.
The down-regulation of VvAGL11 expression in Vitis vinifera L. fruits can promote the formation of seedless fruit [55,56]. Moreover, in Punica granatum L., the expression level of PgAGL11 initially increased and then decreased during the development of fertile flowers but showed the opposite pattern in abortive flowers [57]. Transcript levels of ZjSTK/AGL11 were low during the seed abortion stages (FrII and FrIII) and fluctuated during the flower developmental stages (Fig. 7). Therefore, ZjAGL11 likely played a role in the regulation of ovule and seed development and might be involved in jujube seed abortion. The up-regulated or down-regulated expressions of these ovule identity genes during the critical periods of ovule and seed abortion suggested that these genes might be related to ovule and seed abortion in jujube.

ABCG Transporters are involved in Seed Abortion
Plant ATP-binding cassette subfamily G (ABCG) members are membrane-localized transporters that play an important role in transporting various molecules [58]. Members of ABCG are primarily involved in the formation of the vascular system, lipid transport, pollen wall development, and epidermal cuticle formation [46][47][48][49]. In A. thaliana, AtABCG11 and AtABCG12 have been shown to be involved in the formation of the epidermal cuticle [49,[59][60][61][62], specifically the transportation of epidermal lipid precursors from epidermal cells to the surface of plant organs [49,[59][60][61][62]. Although mutations of ABCGs often lead to male fertility and pollen defects [63], ABCG genes have been found to be involved in seed abortion. VvABCG20 has been shown to be specifically expressed in ovules of V. vinifera L. and participate in the ovule abortion process in seedless grapes; its expression level was significantly higher in seeded grapes than in the seedless variety [64]. When the LeABCG20 gene was silenced in Lycopersicum esculentum, the number of seeds decreased significantly [64]. We previously have shown that microsporogenesis can proceed normally and that embryo abortion occurred during the ovule development stages [22]. In this study, the putative ZjABCG20 (GeneID:107435601) showed up-regulated expression during 'FrI-vs-FrII' and had relatively higher transcript levels during the seed abortion stages (FrII and FrIII) (Fig. 8). A putative ZjABCG26 (GeneI-D:107416341) had an expression pattern similar to the putative ZjABCG20 (GeneID:107435601) (Fig. 8). These results indicated that these members could be important regulators for seed development. However, additional studies are needed to establish the biochemical functions of ZjABCG26 and ZjABCG20, including their roles in ovule and seed abortion in jujube.

Expression Profiles of MCs are a Molecular Signature of PCD and Seed Abortion
PCD is a major mechanism of seed abortion in various plants, which is regulated by various internal and external factors [65]. For example, in A. thaliana, salt stress can induce PCD at multiple stages of gametophyte development and cause seed abortion [66]. The MC family genes play an important role in the process of PCD, and MC-dependent PCD is one of the most important processes in plant embryo development [51][52][53]. The abundance and enzymatic activity of MCs are one of the most characteristic molecular signs of PCD [51]. In a previous study in grape, Zhang et al. [67] found that the MC family genes VvMC1, VvMC3, and VvMC4 were differentially expressed during ovule development, and their expression patterns coincided with the endosperm abortion period of seedless grape. In this study, we identified seven MC-like transcripts that were highly expressed through transcriptomic analysis (Fig. 8). We found that five MC-like genes displayed similar expression patterns: expression was low during early stages of ovule development (SW, OE, and FrI) and significantly up-regulated during 'FrI-vs-FrII'; expression levels of these genes were highest during the seed abortion stages (FrII and FrIII) (Fig.  8). There were multiple annotated copies of MC genes corresponding to the Arabidopsis reference (Fig. 8C), which indicated that the MC gene family might be expanded in jujube. The differential expression patterns of copies suggested that the functions of MCs were diversified during flower and seed development. Overall, we speculate that some MC genes regulate seed abortion by mediating the PCD pathway.

CONCLUSION
Here, we conducted an integrative transcriptomic analysis of ovule and seed development in jujube to explore the mechanisms underlying ovule and seed abortion. RNA-seq data were used to identify DEGs as well as GO terms and pathways during different stages of ovule and seed development. Our study revealed a suite of potential key genes related to ovule and seed development, as well as their regulatory patterns, and mapped the networks of potential genes related to jujube ovule and seed development in three aspects: ovule development during the early stages, seed coat development, and endosperm development. Our data will aid future efforts to explore the mechanisms underlying ovule and seed abortion. We also screened related genes from the AG subfamily as well as ABCG and MC family genes. Seed development is a complex process with many layers of regulation. This RNA-Seq dataset promises to provide insight into the molecular mechanisms affecting ovule and seed abortion, contribute to future genetic studies, and enhance the quality of breeding in jujube.

ETHICS APPROVAL AND CONSENT TO PARTICI-PATE
Not applicable.

HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.

CONSENT FOR PUBLICATION
Not applicable.

RESEARCH INVOLVING PLANTS
The Non-wood Forestry Teaching and Research Department is a professional institute engaged in the teaching and research on non-wood forestry, which is subordinate to the Central South University of Forestry and Technology. Its business mainly includes the introduction and breeding of improved varieties, research on high-yield cultivation techniques, breeding and promotion of improved varieties and strong seedlings, and technical guidance services of nonwood forestry. It undertook the formal identification of the plant material used in this study. There were no voucher specimens of this material deposited in a publicly available herbarium.

AVAILABILITY OF DATA AND MATERIALS
Not applicable.

FUNDING
None.

CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.


provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

text:
Component Analysis and Anti-Colorectal Cancer Mechanism via AKT/mTOR Signalling Pathway of Sanghuangporus vaninii Extracts

Sanghuangporus vaninii (Ljub.) L.W. Zhou & Y.C. Dai (SV) is a major cultivar of Sanghuang, which is well known as an excellent anti-tumour drug and reaches the mainstream market in China. Water, 60% ethanol and 95% ethanol were used to extract the drug, and three kinds of polar extracts were obtained separately. Compared with water extracts and 95% ethanol extracts, the 60% ethanol extract had the highest flavonoid content, and its polysaccharide content was greater than that in the 95% ethanol extract and lower than that in the water extract. Its essential components were phenolics whose majority were phenolic acids, flavonoids and phenylpropanoids. This extract has better inhibition effects on the proliferation of SW480 human colon cancer cells, inducing cell apoptosis and blocking G2/M period cells. It can significantly inhibit gene expression and reduce the activation of the AKT/mTOR signalling pathway. The anti-cancer activity of the 60% ethanol extract is satisfactory and may be a result of the combined effects of polysaccharides and flavonoids. The data suggest that the 60% ethanol extract can be used as an adjuvant for chemotherapy and as a potential anti-cancer agent with broad development prospects.

Introduction
Colon cancer is the third most common cancer in the world and ranks fourth in cancer mortality [1,2]. The mammalian target of rapamycin (mTOR) is a 289 kDa serine/threonine kinase which belongs to the Akt phosphorylated downstream effector family. It can control cell growth and regulate cancer cell survival and proliferation [3,4]. The Akt/mTOR signalling pathway plays an important role in the survival and growth of cancer cells, such as colon cancer cells [5]. Phytomedicine has become the focus of colon cancer research due to its mild medicinal effect and few side effects [6].
Sanghuang is a large perennial medicinal fungus that parasitizes the woody substrate of Morus plants. Sanghuang has strong inhibitory effects on tumours, and it is widely used in Japan and South Korea [7]. It was first recorded in Shen Nong's Materia Medica and was considered to be a high-grade herb. Sanghuang is stated to have an "elimination of toxicant" ("pai du qi" in Chinese) function in the New Compendium of Materia Medica (Xin Xiu Ben Cao) and Chinese Compendium of Materia Medica (Ben Cao Gang Mu) [8,9]. Pharmacological studies have revealed that Sanghuang possesses a variety of biological activities, including anti-cancer, immunoregulation, anti-diabetes, antioxidation and antiinflammation activities. Many kinds of compounds, such as polysaccharides, flavones, triterpenes, aromatic acids and amino acids, have been reported in Sanghuang, and the largest proportion of compounds in the drug are polysaccharides [10]. According to the research of the Japanese scholar Tetsuro Ikekawa, the wild fruit body extract of Sanghuang has a 96.7% inhibitory rate on mouse sarcoma S180, with little toxicity towards normal cells [11]. Many studies have also shown that Sanghuang polysaccharides have significant anti-cancer activity [12,13]. Ge Li et al. reported that polysaccharides have an inhibitory effect on the proliferation and colony formation of SW480 human colon cancer cells, and the effect is associated with decreased Bcl-2 expression, increased release of cytochrome c and reduced cyclin B1 expression [14]. The study also showed that Sanghuang polyphenols have a significant anti-cancer effect, as the polyphenols had cytotoxic activities against various cancer cells [15]. In the treatment of medullary thyroid cancer, researchers found that citric acid did improve symptoms caused by cancer [16]. Takuji Tanaka et al. [17] reported that PA can protect against the development of epithelial malignancy in different tissues and cardiovascular diseases. The mechanism of action mostly involves antioxidant activity, such as inhibiting free radical generation and scavenging and upregulating antioxidant enzymes. By performing a caffeic acid-mediated facile synthesis of silver nanoparticles, the study demonstrated that PA could enhance anti-cancer activity [18].
Sanghuangporus vaninii is responsible for the largest amount of Sanghuang in markets. However, many studies have mainly focused on the extraction, isolation and component analysis of S. vaninii, and there are few studies regarding its bioactivity [7]. Qiong Guo et al. found that S. vaninii exhibits a strong capacity for free radical scavenging, can effectively alleviate cellular oxidative stress and provides therapeutic effects on gout [19]. Xilin Wan et al. reported that polysaccharide from S. vaninii have anti-tumour functions through the activation of the p53 signalling pathway in breast cancer MCF-7 cells. The anti-colon mechanism of S. vaninii has been reported scarcely. In this article, three kinds of extracts were extracted by different polar solvents (water, 60% ethanol and 95% ethanol). The effects on the proliferation, cell cycle and apoptosis of SW480 colon cancer cells in vitro were used as indicators. qRT-PCR and Western blotting were used to explore the effects of the extracts on the AKT/mTOR pathway and preliminarily explain their mechanism of inhibiting human colon cancer cells.

Total Polysaccharide and Flavonoids Contents
The total polysaccharide and flavonoid contents of SVW (water extract of S. vaninii), SVE60 (60% ethanolic extract of S. vaninii) and SVE95 (95% ethanolic extract of S. vaninii) were 0.626, 0.342 and 0.337 mg/mg and 0.0481, 0.5574 and 0.4843 mg/mg, respectively. The polysaccharide content of SVW was the highest, and that of flavonoids was the lowest. The polysaccharide content of SVE60 was slightly higher than that of SVE95, and the total flavonoid content was the highest in SVE60, which was 11.58 times as high as that in SVW and 1.15 times as that in SVE95.

The Result of CCK8 Cell Proliferation
Cancer cells are unmanageable, chronic and have obvious characteristics, including a high proliferation ability and an ability to rapidly migrate [40,41]. To demonstrate whether the extracts of S. vaninii have an inhibitory effect on SW480 human colon cell proliferation in a dose-dependent manner, the experiments involved three groups. The SVW group (the control group, group 1), SVE60 (group 2) and SVE95 (group 3) were each treated with the same dosages (0, 1.2, 3.7, 11.1, 33.3, 100 and 300 μg/mL) and effective times (72 h).
Compared with that of the control group, the OD values of the three extracts gradually decreased with increasing dose levels after administration for 72 h. There were significant differences in the SVW group (300, 100, 33.3 μg/mL dose groups) (Figure 3a), SVE60 group (300, 100, 33.3, 11.1 and 3.7 μg/mL dose groups) ( Figure 3b) and SVE95 group (300, 100 and 33.3 μg/mL dose groups) (Figure 3c). The three extracts could significantly

The Result of CCK8 Cell Proliferation
Cancer cells are unmanageable, chronic and have obvious characteristics, including a high proliferation ability and an ability to rapidly migrate [40,41]. To demonstrate whether the extracts of S. vaninii have an inhibitory effect on SW480 human colon cell proliferation in a dose-dependent manner, the experiments involved three groups. The SVW group (the control group, group 1), SVE60 (group 2) and SVE95 (group 3) were each treated with the same dosages (0, 1.2, 3.7, 11.1, 33.3, 100 and 300 μg/mL) and effective times (72 h).
Compared with that of the control group, the OD values of the three extracts gradually decreased with increasing dose levels after administration for 72 h. There were significant differences in the SVW group (300, 100, 33.3 μg/mL dose groups) (Figure 3a

The Result of CCK8 Cell Proliferation
Cancer cells are unmanageable, chronic and have obvious characteristics, including a high proliferation ability and an ability to rapidly migrate [40,41]. To demonstrate whether the extracts of S. vaninii have an inhibitory effect on SW480 human colon cell proliferation in a dose-dependent manner, the experiments involved three groups. The SVW group (the control group, group 1), SVE60 (group 2) and SVE95 (group 3) were each treated with the same dosages (0, 1.2, 3.7, 11.1, 33.3, 100 and 300 µg/mL) and effective times (72 h).
Compared with that of the control group, the OD values of the three extracts gradually decreased with increasing dose levels after administration for 72 h. There were significant differences in the SVW group (300, 100, 33.3 µg/mL dose groups) (Figure 3a), SVE60 group (300, 100, 33.3, 11.1 and 3.7 µg/mL dose groups) ( Figure 3b) and SVE95 group (300, 100 and 33.3 µg/mL dose groups) (Figure 3c). The three extracts could significantly reduce the cell survival rate with increased dosage, especially the 300, 100 and 33.3 µg/mL dose groups (p < 0.01).
proliferation of colon cancer cells SW480 at a lower concentration (3.7 μg/mL), with a significant difference (p < 0.05). The IC50 values of SVW, SVE60 and SVE95 were 209.5, 7.91 and 27.14 μg/mL. The effect of two ethanol extracts was more obvious than that of the water extract, particularly the 60% ethanol extract of S. vaninii, whose IC50 was only one third of 95% ethanol extract and one twenty-sixth of water extract, approximately.

Induction of Apoptosis by the Ethanol Extracts of S. vaninii
Apoptosis is the key process in inhibiting the proliferation of cancer cells [42]. Annexin-V can specially bind with the early stage of apoptosis and provide FITC labels [43]. Propidine iodide (PI) is a nucleic acid dye that can penetrate the cell membrane in the middle and advanced stages of apoptosis to incarnadine nuclei. Therefore, Annexin-V and PI are used simultaneously in experiments to identify and differentiate early, middle and advanced apoptosis and necrotic cells [44]. This procedure was adopted in our research, and flow cytometry was applied to detect the apoptosis rates.
There were two groups in the experiment, the SVE60 (Figure 4a-d) and SVE95 group (Figure 4e-h), which were divided into four dose groups (the control group, 3, 30 and 300 μg/mL dose groups). Then, the cells were stained with Annexin V and PI.
Compared with the control group, the numbers of late apoptotic cells and early apoptotic cells were greater with multiplied dosages (10 times) of SVE60 and SVE95 ( Figure 3). The results showed that the ethanol extracts of S. vaninii induced the SW480 apoptosis in a certain dose-dependent manner, and the effect of SVE60 was slightly superior to that of SVE95 (Table 2). Compared with the SVW and SVE95, SVE60 showed an inhibitory effect on the proliferation of colon cancer cells SW480 at a lower concentration (3.7 µg/mL), with a significant difference (p < 0.05). The IC 50 values of SVW, SVE60 and SVE95 were 209.5, 7.91 and 27.14 µg/mL. The effect of two ethanol extracts was more obvious than that of the water extract, particularly the 60% ethanol extract of S. vaninii, whose IC 50 was only one third of 95% ethanol extract and one twenty-sixth of water extract, approximately.

Induction of Apoptosis by the Ethanol Extracts of S. vaninii
Apoptosis is the key process in inhibiting the proliferation of cancer cells [42]. Annexin-V can specially bind with the early stage of apoptosis and provide FITC labels [43]. Propidine iodide (PI) is a nucleic acid dye that can penetrate the cell membrane in the middle and advanced stages of apoptosis to incarnadine nuclei. Therefore, Annexin-V and PI are used simultaneously in experiments to identify and differentiate early, middle and advanced apoptosis and necrotic cells [44]. This procedure was adopted in our research, and flow cytometry was applied to detect the apoptosis rates.
There were two groups in the experiment, the SVE60 (Figure 4a-d) and SVE95 group (Figure 4e-h), which were divided into four dose groups (the control group, 3, 30 and 300 µg/mL dose groups). Then, the cells were stained with Annexin V and PI.
Compared with the control group, the numbers of late apoptotic cells and early apoptotic cells were greater with multiplied dosages (10 times) of SVE60 and SVE95 (Figure 3). The results showed that the ethanol extracts of S. vaninii induced the SW480 apoptosis in a certain dose-dependent manner, and the effect of SVE60 was slightly superior to that of SVE95 (

Effect of SVE60 on Cell Cycle Progression
The cell cycle is an important factor in cell proliferation, and its process begins with one cell division and ends when the next cell division is finished [45]. The cell cycle process was analysed via flow cytometry, and the study used SVE60 as the drug. The control group, and 3, 30 and 300 μg/mL dose groups were used. Compared with that of the control group, the proportion of cells in the G0/G1 phase was decreased by 6.08%, 1.82% and 9.53% at 3, 30 and 300 μg/mL, and those in G2/M phase increased by 7.75%, 0.61% and 9.36%, respectively. The proportion of cells in S phase was increased by 1.21% and 0.16% at 30 and 300 μg/mL, respectively, but decreased by 1.68% at 3 μg/mL.
The proportion of cells in the S or G2/M phase was influenced by different drug concentrations. The 3 μg/mL dose decreased the proportion of cells in the S phase and increased the proportion of cells in the G2/M phase. The 30 μg/mL dose group induced apoptosis of cells derived from the G0/G1 phase. The results ( Figure 5) showed that SVE60 was able to inhibit cancer cell proliferation by causing cell arrest in different phases.

Effect of SVE60 on Cell Cycle Progression
The cell cycle is an important factor in cell proliferation, and its process begins with one cell division and ends when the next cell division is finished [45]. The cell cycle process was analysed via flow cytometry, and the study used SVE60 as the drug. The control group, and 3, 30 and 300 µg/mL dose groups were used. Compared with that of the control group, the proportion of cells in the G0/G1 phase was decreased by 6.08%, 1.82% and 9.53% at 3, 30 and 300 µg/mL, and those in G2/M phase increased by 7.75%, 0.61% and 9.36%, respectively. The proportion of cells in S phase was increased by 1.21% and 0.16% at 30 and 300 µg/mL, respectively, but decreased by 1.68% at 3 µg/mL.
The proportion of cells in the S or G2/M phase was influenced by different drug concentrations. The 3 µg/mL dose decreased the proportion of cells in the S phase and increased the proportion of cells in the G2/M phase. The 30 µg/mL dose group induced apoptosis of cells derived from the G0/G1 phase. The results ( Figure 5) showed that SVE60 was able to inhibit cancer cell proliferation by causing cell arrest in different phases.

The Result of qRT-PCR
The study observed the influence of SVE on the mTOR and AKT gene expression in the mTOR signalling pathway ( Figure 6). There were significant differences between the SVE60 0.01 μg/mL dose group, which had an obvious inhibitory effect on mTOR gene expression, and the control group. Three dose groups showed a decreasing trend in AKT gene expression. The data demonstrated that SVE60 had a significant inhibitory effect on the expression of key genes in the mTOR signalling pathway.

The Result of SVE60 on the Protein Expression via mTOR Signalling Pathway
Tubulin was used as an internal reference protein to observe the influence of SVE60 on the mTOR, AKT and p-AKT proteins in the mTOR signalling pathway (Figure 7). The results showed that the three dose groups significantly decreased the expression of mTOR, AKT and p-AKT proteins. SVE60 exhibited an obvious dose-dependent relationship with the expression of AKT and p-AKT proteins. This result indicated that the inhibitory effect of SVE60 on the proliferation of SW480 cells was related to the activation of mTOR signalling.

The Result of qRT-PCR
The study observed the influence of SVE on the mTOR and AKT gene expression in the mTOR signalling pathway ( Figure 6). There were significant differences between the SVE60 0.01 µg/mL dose group, which had an obvious inhibitory effect on mTOR gene expression, and the control group. Three dose groups showed a decreasing trend in AKT gene expression. The data demonstrated that SVE60 had a significant inhibitory effect on the expression of key genes in the mTOR signalling pathway.

The Result of qRT-PCR
The study observed the influence of SVE on the mTOR and AKT gene expression in the mTOR signalling pathway ( Figure 6). There were significant differences between the SVE60 0.01 μg/mL dose group, which had an obvious inhibitory effect on mTOR gene expression, and the control group. Three dose groups showed a decreasing trend in AKT gene expression. The data demonstrated that SVE60 had a significant inhibitory effect on the expression of key genes in the mTOR signalling pathway.

The Result of SVE60 on the Protein Expression via mTOR Signalling Pathway
Tubulin was used as an internal reference protein to observe the influence of SVE60 on the mTOR, AKT and p-AKT proteins in the mTOR signalling pathway (Figure 7). The results showed that the three dose groups significantly decreased the expression of mTOR, AKT and p-AKT proteins. SVE60 exhibited an obvious dose-dependent relationship with the expression of AKT and p-AKT proteins. This result indicated that the inhibitory effect of SVE60 on the proliferation of SW480 cells was related to the activation of mTOR signalling.

The Result of SVE60 on the Protein Expression via mTOR Signalling Pathway
Tubulin was used as an internal reference protein to observe the influence of SVE60 on the mTOR, AKT and p-AKT proteins in the mTOR signalling pathway (Figure 7). The results showed that the three dose groups significantly decreased the expression of mTOR, AKT and p-AKT proteins. SVE60 exhibited an obvious dose-dependent relationship with the expression of AKT and p-AKT proteins. This result indicated that the inhibitory effect of SVE60 on the proliferation of SW480 cells was related to the activation of mTOR signalling.

Materials
The

Preparation of the Phellinus Linteus Extracts
The powder (20 g, approximately) of S. vaninii was extracted with 400 mL of pure water for 1 h in a water bath. The extract was centrifuged at 5000 r/min for 5 min. The supernatant was concentrated under vacuum and then lyophilized using a vacuum freeze dryer. The water extract of S. vaninii (SVW) was stored for further use. The 60% and 95% ethanolic extracts (SVE60 and SVE95) were prepared with the same method as SVW, and the supernatants were evaporated in vacuum until dry.

Total Polysaccharide Content
The polysaccharide content was analysed by the glucose calibration, which was determined using the anthrone-sulfuric acid method [46]. SVW, SVE60 and SVE95 were diluted with distilled water (w/v, 1:5), and 1.0 mL of aqueous solution was used for

Preparation of the Phellinus Linteus Extracts
The powder (20 g, approximately) of S. vaninii was extracted with 400 mL of pure water for 1 h in a water bath. The extract was centrifuged at 5000 r/min for 5 min. The supernatant was concentrated under vacuum and then lyophilized using a vacuum freeze dryer. The water extract of S. vaninii (SVW) was stored for further use. The 60% and 95% ethanolic extracts (SVE60 and SVE95) were prepared with the same method as SVW, and the supernatants were evaporated in vacuum until dry.

Total Polysaccharide Content
The polysaccharide content was analysed by the glucose calibration, which was determined using the anthrone-sulfuric acid method [46]. SVW, SVE60 and SVE95 were diluted with distilled water (w/v, 1:5), and 1.0 mL of aqueous solution was used for measurement. The solution was added to 2.0 mL with distilled water, and 6.0 mL of 0.2% anthrone-sulfuric acid was added slowly and heated at 100 • C for 15 min, and absorbance measurements were recorded at 625 nm.

Total Flavonoids Content
The total flavonoid content was measured by sodium nitrite-aluminium nitrate colorimetry through UV spectroscopy using rutin as a reference substance at a concentration of 0.20 mg/mL [47]. The control solutions 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mL were added, and distilled water was added to 6.0 mL. Then, 1.0 mL of 5% sodium nitrite was added to the solutions and incubated for 6 min separately. Next, 1.0 mL of 10% aluminium nitrate was added and reacted for 6 min. Finally, 10.0 mL of NaOH was added, distilled water was added to increase the liquid level, and the solution was shaken and developed for 15 min. The test solution and water were the blank controls, and a 500 nm wavelength was used. To formulate the standard curve, the concentration of rutin (C) was used as the X-axis and the Y-axis was the absorbance (A). The extracts were dissolved in methanol (w/v, 1:1), and the filtrates were measured in the same way.

UPLC-Q-TOF-MS Analytical Conditions
The

Cell Culture
The human colon cancer cell line SW480 was cultured in 1640 culture fluid with 10% foetal bovine serum. The cells displayed monolayer growth in a 37 • C atmosphere of 95% relative humidity and 5% CO 2 . Cells at logarithmic growth phase were used in the experiment.

CCK-8 Assay
The human colon cancer cell line SW480 was inoculated in 96-well culture plates at a density of 1 × 10 5 per mL per well. The clear supernatant of SW480 cells that would be used for detection was poured out, and the cells were washed twice with PBS. A total of 100 µL of fresh L-15 complete medium and 10 µL of CCK-8 reagent was used per well. Then, the plates were placed in an incubator for 3 h, and the optical density (OD) was detected at 450 nm using a microplate reader.

Flow Cytometry Cell Apoptosis Analysis
The cells were pre-treated before the formal experiment. The colon cell SW480 was digested with trypsin. The adherent cells were washed with PBS three times and collected into centrifuge tubes. The supernatant was discarded after centrifugation at 2600 r/min for 5 min. The binding buffer was diluted 10 times with deionized water. The single-cell suspension was prepared by mixing the primed cells with 250 µL of diluted binding buffer. Then, the 50 µL single-cell suspension was removed from the 1.5 mL centrifuge tube, and the cells were stained with 2.5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) for 15 min at ambient temperature. Apoptosis was detected through flow cytometric analysis.

Cell Cycle Analysis
The colon cell line SW480 was prepared as a single-cell suspension through the same method as described in 2.8. The pre-treated cells were put into a tube, strained with 1 mL of DNA staining solution and 10 µL of permeabilization solution, and were mixed by vortex oscillation for 5-10 s. Then, the cells were incubated for 30 min at room temperature and were subjected to flow cytometry.

Quantitative RT-PCR
Total RNA was extracted from the cells with TRIzon reagent. The effect on the pathway mediated by mTOR and AKT was determined using the qRT-PCR method using a variety of extracts doses, such as blank, 0.01 µg/mL, 0.1 µg/mL and 1 µg/mL. A quantitative PCR instrument (Bio-Rad CFX96, Hercules, CA, USA) was used for the PCR, and the following primer sequences were used for the qRT-PCR (Table 3).

Western Blot
The expression of mTOR, p-AKT and AKT protein was detected after the extracts reacted with SW480 cells for 48 h and the antibodies, including mTOR, p-AKT and AKT, were diluted at a ratio of 1:1000. SW480 cells were washed with PBS three times, RIPA protein lysis buffer was added, the mixture rested for 30 min, and the cells were centrifuged at 12,000 rpm at 4 • C.
The supernatant liquid was obtained and the concentration of protein in the samples was detected using BCA test kits. Total protein was obtained and rinsed with TBST after sample filling, electrophoresis, transmembrane and closure was performed. Extracts with different concentrations, such as 0, 0.01, 0.1 and 1 µg/mL, were added, and then the mixture was stored overnight at 4 • C. Then, the mixture was rinsed with TBST again. After exposure and imaging, tubulin was used as a reference protein (at 1:1000 dilution) and Image Quant TL software was used to analyse the grey value of every belts.

Statistical Analysis
All quantitative data are presented as the means ± standard deviation (SD). The data were analysed by t-tests using GraphPad Prism 9.0 software. Differences were considered statistically significant when p < 0.05.

Conclusions and Discussion
Sanghuang has long been known to possess anti-cancer abilities. S. vaninii is its most widespread cultivar and provides rich resources for the application of this plant. To explore the medical usage of this drug, the relationship between the active compounds and anti-colon cancer was studied in this experiment.
Phenolic compounds and polysaccharides are the two main bioactive chemical groups with medicinal properties of Sanghuang [48]. In the study of Yang, the proximate compositions and microelements of Sanghuang were determined, including the contents of total phenolics, flavonoids and polysaccharides, SOD-like and nutrient compositions of water extracts. The content of total polysaccharides was obviously higher than flavonoids [49]. SVW, SVE60 and SVE95 were three extracts of S. vaninii which were separately extracted by water, 60% ethanol and 95% ethanol in this experiment. The SVW had the richest content in polysaccharides and lowest content in flavonoid compounds in those extracts. SVW60 had the highest flavonoid content, and its polysaccharide content was greater than that in SVE95. Therefore, the composition of SVE60 was analysed via UPLC-MS. The results suggested that the essential components were phenolics, and the majority were phenolic acids, flavonoids and phenylpropanoids. Many phenolic compounds have been shown to inhibit proliferation and angiogenesis of tumour cells in vitro [50].
The 95% ethanolic extract from the fruiting body of Sanghuang inhibited the proliferation of both cell lines in a dose-dependent manner, and the IC 50 values at 48 h were 72 and 103 g/mL for SK-Hep-1 cells and RHE cells [51]. According to the CCK-8 cell proliferation results, the three extracts significantly reduced the cell survival rate with a good dose-effect relationship, and the effect of the two ethanol extracts was better than the water extract, especially SVE60, which had a much higher IC 50 than SVW and SVW95. It is well known that apoptosis is an evolutionarily suicide program regulated by many genes that activates the cell death process, resulting in the removal of damaged tissue [52]. Furthermore, the two ethanol extracts induced SW480 apoptosis, particularly SVE60. SVE60 also had the ability to inhibit cancer cell proliferation by causing cell arrest in different phases.
Actually, while obvious inhibitory trends for cancer cells were observed in all dosages, there was very little apoptosis at high concentrations in this experiment, survival rates were low in all concentrations, and similar results were also found in cell cycle. They were probably caused by apoptosis or the death of cancer cells, due to the short detection time in these tests. SVE60 could significantly decrease the expression of mTOR, AKT and p-AKT proteins, which was related to the proliferation of SW480 cells and the activation of mTOR signalling. This study analysed the extract components of S. vaninii and explored its anti-colon cancer mechanism. These results showed that SVE60 had good potential for usage as an adjuvant chemotherapeutic and anti-colon cancer drug, in postoperative nutrition and in the prevention of recurrence.
The study demonstrated SVE60 had an obvious effect on anti-colon cancer. In a further study, we plan to prolong the determining time in apoptosis and cell cycle experiments so a clearer trend can be observed. Furthermore, we need to isolate and purify components from the 60% ethanol extract and observe the inhibition effect of the extract on human colon cancer in vivo via an orthotopic model of colon cancer in nude mice.  Data Availability Statement: The study did not report any data.


text:
Effects of Black Shade Net on Yield of Brassica rapa and Brassica oleracea Cabbages in Kilifi County

Cabbage is an important vegetable to humans since it provides crucial nutrients such as fiber, minerals, and vitamins. They also help manage cancer, cardiovascular diseases, prevent type 2 diabetes, and inflammation of the digestive systems. However, its production in the tropics is constrained by climatic and edaphic factors that include soil moisture stress, high soil and air temperatures, high evapotranspiration, and unbalanced nutrition. Due to the above challenges, sustainable cabbage production requires adoption of technologies that can modify the growth environment with minimal environmental effects. A field study was conducted at Pwani University Crop Science farm, Kilifi County, to evaluate the effects of black shade net on yield of Brassica rapa and Brassica oleracea. A randomized complete block design experiment with three replications was set. The treatments included: 50%, 70% black shade net intensity and open field as control. Data collected included, leaf chlorophyll content, fresh head weight, and dry matter weight. The data obtained was subjected to analysis of variance using General Linear Model (GLM) SAS Computer package version 9.1. The results indicated that, shading resulted in decreased chlorophyll content, and that use of 50% and 70% black net shading intensity significantly resulted in 55% and 47.5%, more number of cabbage heads for Brassica rapa than open field respectively, while in Brassica oleracea, 70% shading and 50% resulted in 60% and 52% more cabbage heads than open field respectively.

Background Information
Cabbage is one of the crucial vegetables widely consumed in the world and which is in high demand, especially in the Coastal region of Kenya where it is second in importance after Amaranthus [1]. It is consumed in most households for its nutritional benefits such as the supply of fiber, minerals ions such as calcium, iron, sodium, zinc, magnesium, phosphorus, potassium, and a good source of vitamins A, C, E, and K. It also contains glucosinolates and phenolics that are anti-carcinogenic and prevent type 2 diabetes, cardiovascular diseases, and inflammation of digestive systems [2,3,4].
Despite the high demand and important role played by this vegetable, its local production is limited by fluctuating ecological factors such as temperature, relative humidity, soil moisture, as well as unreliable and erratic rainfall that are modulated by global warming and climate change [5]. Since cabbage is sourced from other distant up-country regions of Kenya into the coastal region, increased demand in these other counties has resulted in scarcity and high prices, making it unaffordable to the majority of the rural poor. It is in this regard that this study was conceived to evaluate technologies such as the use of black shade net that have been used elsewhere to modify the plant growing environment for successful cabbages production [6,7].

Site Description
The study was done at Pwani University Crop Science farm for two trials, during the period July 2020 to September 2020 and from December 2020 to February 2021. The farm lies about 30m above sea level (ASL) and is located at 39.85°E and 3.62°S in Kilifi County. The area experiences a bimodal type of rainfall, amounting to 900 to 1100mm per annum, with long rains occurring from April to June and short rains occurring from September to December. The temperature ranges between 29 o C to 32 o C [8]. The soils are predominantly sandy loam, specifically ferralic and dystric cambisols characterized by low organic matter, poor nutrients, and low water holding capacity [9].

Materials
The black shade nets used were of 50% and 70% light reduction and were sourced from Graduate Farmers Ltd-Eldoret, Kenya. The cabbage seeds used were Brassica oleracea (white cabbage, cv. Rossy F1) and Brassica rapa, (Chinese cabbage, cv. Nice F1) and were sourced from Continental seed company, Nairobi. These cabbage varieties were chosen because of their fast growth and early maturity ranging from 60-70 days after transplanting.

Nursery Establishment
The nursery beds of the Brassica oleracea and Brassica rapa cabbages were established using standard nursery preparation methods as described by KALRO [10]. The land was cleared, ploughed, and harrowed to a fine tilth, using a panga, Jembes, and rakes respectively. The nursery beds dimensions were 1 m by 2 m. 200 g Diammonium phosphate (DAP) fertilizer (grade: 18% N: 46% P 2 O 5 : 0% K) was broadcasted on both nursery beds, thoroughly mixed with the soil, and drills were done using a stick, at intervals of 10cm then seeds were evenly placed into the drills and covered with some soil. Dry grass mulch was applied on the surface of the seedbed, and an overhead shade was erected. Watering to field capacity was done and maintained in the morning and evening each day until germination and establishment. All other nursery management practices were carried out as recommended including, weeding, pest control, and hardening.

Transplanting
On attaining a height of 10 to 12 cm, the seedlings were transplanted during the evening when temperatures were low. Each nursery was watered with 5 litres of water 30 minutes before transplanting to minimize root injury and shock. During transplanting, a spacing of 40 cm by 40 cm was adopted. From the margins, 30 cm was left as guard rows, resulting in five (5) rows and ten (10) plants per row amounting to 50 plants per cabbage type in every block as recommended by Burt et al. [11]. Ten (10) grams or a rate of 250 kg/ha of DAP fertilizer was applied to every hole before the seedlings were transplanted after which watering was done [12]. Grass mulch 15 cm thick was applied one month after transplanting as recommended by Kelley et al. [13]. Topdressing was done using calcium ammonium nitrate (CAN) when bolting started, at a rate of 215 kg ha -1 or two teaspoonfuls per plant as recommended by Muleke et al. [5] (2014). Weeds, pests, and diseases were monitored and controlled regularly, as they appeared.

Experimental Design and Treatments
A completely randomized block design experiment replicated three times was set. The treatments included: i) three levels of shading intensity at 70%, 50% (using black shade nets), and open field; and ii) two cabbage types, namely, Brassica oleracea (white cabbage) and Brassica rapa (Napa cabbage) ( Table 1 and Figs. 1 and 2).
Each experimental block measured 4m by 4m and was then divided at the centre, where each half was planted with Brassica oleracea and the other with Brassica rapa (Figures 1 and 2). At the edges of the two treatments designated for 50% and 70% shading, metallic frames were erected, and cross ties were fixed on top from one metal pole to another. The black shade nets were then placed on top and sideways of each metallic frame. A small entrance was made on one side. The plots under treatment were cultivated manually using a Jembe. After one week, farmyard manure was applied to every treatment plot and thoroughly mixed with soil at a rate of 2.5 t ha -1 as recommended by Saha & Muli [14] before transplanting was done.

Sampling and Data Collection
After transplanting, five plants per plot were randomly selected from which data was collected every week, until harvesting when final cabbage fresh and dry weights were determined. Other parameters measured included chlorophyll content (using chlorophyll meter, model SPAD 502 PLUS, Decagon devices, as described by Rodriguez & Miller [15]), and the number of fullyformed cabbage heads per plot. At the harvest stage, final fresh and dry matter head weights for each treatment were determined using an electronic weighing balance (Model PM 200, Mettler Instrument Limited, Switzerland). The dry matter was obtained by cutting the cabbage into smaller pieces and drying at 105 0 C, until constant dry weight.

Data Analysis
The collected data was then subjected to analysis of variance (ANOVA) using the General Linear Model (GLM) procedure of SAS, 13th Edition. The means so obtained were compared using Tukey's honest test for significant difference (THSD) at a 5% level of significance.

RESULTS
It is important to point out that, during the experimentation period, the first trial which ran from July to September, was fairly wet and cooler than the second trial from December to February, which was hot and fairly dry. Also during the second trial, the transplanted seedlings in the open field all dried during the third week due to high ambient temperatures, despite adequate watering. However, those planted under shade nets survived to harvest.

Effect of Black Shade Net on Leaf Chlorophyll Content of Brassica rapa and Brassica oleracea
Black shade net had significant effects on leaf chlorophyll content of Brassica rapa and Brassica oleracea during the first and second trial. Open field Brassica rapa cabbages maintained higher chlorophyll content of about 6.4% throughout the growing period compared to cabbages under 50% shading. On the other hand, 50% shading had about 6.0% higher chlorophyll content than 70% shading throughout the growing period in the first trial ( th, and 7 th week of the first trial (Table 3). During the second trial, Brassica oleracea under 50% shading, maintained significantly more chlorophyll content averagely 9.7% than 70% shading throughout the growing period.

Effects of Black Shade Net on the Number of Cabbage Heads Formed
For Brassica rapa, the use of 70% black shade net resulted in 73 % more cabbage heads than the open field during the first trial, while the use of 50% black shade net resulted in 26 %more cabbage heads than the open field. For Brassica oleracea, the use of 70% and 50% black shade net during the first trial resulted in 92% and 88% more cabbage heads, respectively, than in the open field (Fig. 3).   Despite the high air temperature experienced during the second trial, the performance of the cabbages was remarkable (Fig. 4).

Effect of Black Shade Net on Fresh Cabbage Head Weight
The use of black shade nets had significant effects on fresh cabbage head weight in both trials ( . A comparison of 50% and 70% shading of Brassica rapa during the first trial indicated that 50% shading resulted in significantly 27.4% (or 9.6 tonnes) higher yields than 70% shading, while during the drier second trial, 70% shading gave 82% more fresh weight yields equivalent to 6.7 tonnes, than 50% shading (Table 4) . During the second trial, 70% shading resulted in 75% more fresh cabbage weight yield compared to 50% shading that yielded 1.5 tonnes ha -1 (Table 4).   ) under 50% and 70% shading, respectively (Table 5). Thus, 50% shading resulted in more dry matter than 70% shading when compared with open field, for Brassica rapa in the first trial. However, in both trials, 50% and 70% shading resulted in comparable amounts of dry matter yields. For Brassica oleracea, open field and 50% shading resulted in comparable amounts of dry matter yields, while 70% shading resulted in significantly 93.4% higher dry matter yields than open field during the first trial. However, during the second trial, 50% and 70% shading resulted in comparable dry matter yields.

Effect of Black Shade Net on the Number of Cabbage Heads
In both trials, more cabbage heads were harvested from 70% shading, followed by 50%

Effect of Black Shade Net on Cabbage Dry Matter
From the study, cabbages grown under shade nets had more dry matter yield than those grown in the open field. However, 50% shading during the first trial resulted in higher Brassica rapa dry matter yields than 70% shading and open field. This suggests that 50% shading intensity was the most ideal for maximum dry matter formation for Brassica rapa. For Brassica oleracea, 70% shading resulted in the highest dry matter yields than 50% shading and open field during the first trial. This implies that for Brassica oleracea, 70% shading provided the most ideal shading intensity and micro-climate for maximum production of dry matter yields. During the second trial, for both types of cabbages, 70% shading resulted in the highest dry matter yields. This suggests that 70% shading provided the most ideal shading intensity and micro-climate for maximum dry matter production. These findings are in agreement with those reported by Muleke et al. [5] and Ngelenzi et al. [22], who found that cabbages grown under agro net covers accumulated more dry matter than those grown in the open field. The conducive microclimate created by the black shade nets possibly led to increased growth vigour, leading to more accumulation of total fibre and therefore dry matter yields.

CONCLUSION AND RECOMMENDA-TION
The study has shown that it is possible to grow cabbages in Kilifi County and the Coastal region of Kenya as a whole or even in most Arid and semi-arid areas by using a black shade net, that moderates temperature and light intensity creating a micro-climate conducive to growth, dry matter accumulation and formation of cabbage heads, so long as soil moisture is not limiting and other factors are not limiting.

DISCLAIMER
The products used for this research are commonly and predominantly used products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.