| Seedless breeding is an efficient way to obtain excellent traits in Siraitia grosvenorii. Our team previously obtained a new triploid variety by hybridization between diploid and tetraploid, its female parent was normal diploid and male parent was male sterile tetraploid. However, the knowledge of these processes is comparatively deficient. Here, we use Illumina sequencing to gain insights into the transcriptome of triploid and diploid fruits of S. gorsvenorii. In total,26.4 million transcript reads with an average length of 90 bp were generated from a cDNA library that combined the two fruits. Following assembly,64,732 unigenes with gene expression strictly controlled by fruit and seed features were obtained. We identified differentially expressed genes, with 329 genes specific to the diploid fruit, and 330 specific to the triploid. In all,1,748 genes were up-regulated, and 2,037 were down-regulated in the triploid fruit. Functional analysis revealed that 41,678 unigenes were annotated with putative functions based on the protein databases using BLASTx; the other 22,694 unigenes were considered as putative novel genes. KEGG analysis mapped 17,593 unigenes to 119 pathways, and genes with altered expression were involved in 118 pathways. Notably, the TCA cycle, fatty biosynthesis, and brassinolide biosynthesis were up-regulated, while gibberellic acid biosynthesis, glycolysis, fatty acid metabolism and nitrogen metabolism were down-regulated. Interestingly, a gene encoding T-cytoplasm male sterility factor 2, two genes annotated as ATP synthase, and two genes involved in male meiosis were down-regulated, these genes may be associated with male sterile. We also found five classes of candidate genes related with embryo abortion. GA and zeatin content were coherent with the expressions of genes involved in their biosynthesis respectively. There were numerous differences between diploid and triploid fruit including size, seedy or seedless, phytohormone content and differentially expressed genes. These results further our understanding of triploid fruit and seed formation, and are expected to be helpful for seedless breeding of S. grosvenorii and of other plants in the future.Siraitia grosvenorii, an importantly traditional Chinese medicine and sweeterner crop, has great potential for development. The major bioactive component and sweet gradient is mogroside V, and it is extraordinarily high and has good effect for cough and expectorant. Due to high sweetness, low calorie, non-toxic, good taste and other advantages, mogroside V has broad prospect. However the low content and high cost of production restrict its development. Cucurbitadienol is precursor of mogroside V and the enzymes invovoled in its biosynthesis can regulate its content. HMGR upstream in MVA pathway restricts MVA content, which is the first rate-limiting enzyme; SQS is a key enzyme for squalene biosynthesis; CS can generate cucurbitadienol directly, which directly control its content; CAS has the common substrate and catalyzes it to cycloartenol, which brings a different branch and influence mogorside content indirectly. Study of gene functions involved in mogroside biosynthesis will help us understand its biosynthesis mechanism, lay the foundation for regulation of mogroside biosynthesis, provide scientific basis and target genes for tranSgenic breeding. Base on the unigene sequences of these enzymes in S. grosvenorii, we attempted to study functions of these genes including gene full-length cloning, bioinformatics analysis, spatial and temporal expression by qRT-PCR, heterologous expression, subcellular localization, etc. The main findings are as follows:1) Full-length of SgCS gene with 2280 bp was obtained, and the protein encoded had no transmembrane region, no signal peptide, and had high identity with cucumis sativus and cucumis pelo. SgCS gene expression in different stages showed that there were two peak, 10 d and 20 d, respectively; and there was low abundance from 30 d to 50 d. SgCS protein could be expressed in prokaryotic system, but there was no activity because of inclusion body form. Further study of SgCS in yeast demonstrated that it can catalyze 2,3-oxidosqualen into cucurbitadienol, which has the function of cucurbitadienol synthase. We overexpressed SgCS in Arabidopsis thaliana and obtained positive tranSgenic plant, and found upregulation of SgCS after transformation. We observed the subcellular location in tobacco and found SgCS was mainly in nucleus and cytoplasm, which was consistant with the results predicted by bioinformatics softwares.2) Full-length of SgCAS gene was obtained after RACE-PCR with 2298 bp, it has no transmembrane region and no signal peptide, was probably lacated in chloroplast and nucleus. SgCS can be expressed in prokaryotic system in the form of inclusion body, and can catalyze 2,3-oxidosqualene into cycloartenol in yeast expression system pYES2/IVF, which demonstrated that SgCAS has oxidosqualen cyclase function. SgCAS gene has highest level in 15 d and lowest level in 5 d, and can be detected in stem, root, leave and fruit. For further understanding of SgCAS, we obtained positive tabacco after RNA interference of SgCAS, and found its down-regulation in tranSgenic tobacco. Finally, we found SgCAS was mainly located in nucleus and cytoplasm.3) We obtained 1254 bp SgSQS-ORF, and it was probably located in cytoskeleton and cytoplasm predicted by software. Similar with SgCS gene expression pattern in fruits, SgSQS also had two peak in 10 d and 20 d, and it had minimum level in 50 d. After IPTG induction, SDS-PAGE and western blot, we detected expression of SgSQS in prokaryotic system with no activity. We transformed subcellular location vector SgSQS-pN-YFP into tabacoo epidermal cells and SgSQS protein was lacated in cytoplasm, nucleus and cytoplasm membrane, which is more consistent with results predicted by software. However, lack of cytoskeleton markers, we could not determine whether SgSQS was in cytoskeleton and which type of skeleton it was in. Besides, one overexpression vector and two RNAi vectors were also constructed.4) We obtained 5’RACE and 3’RACE of SgHMGR by means of RACE-PCR, and got SgHMGR-ORF after sequence assembly and PCR amplification. The protein encoded by SgHMGR had no signal peptide, but had two transmembrane reigons in N-terminal, therefore we constructed SgHMGR 1-pET32a avoiding transmembrane regions, and detected target protein in supernatant after IPTG induction. We determined SgHMGR was expressed by Western blot for further testing. In order to understand SgHMGR gene expression in different tissues, we detected its expression level in tissue cultures of S. grosvenorii by qRT-PCR and found there was highest level in stem and lowest in root. The result of subcellular location in tabacco showed that it was located in cytoplasm membrane which was basically the same with predicted results. We also successfully constructed one overexpression vector and two RNAi vectors of SgHMGR. |
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