| RATIONALES:Onjisaponin is an important secondary metabolites of Polygala tenuifolia, which as potential active substance has been shown to have calm, expectorant, fall blood sugar, and other pharmacological activities. Currently, the studies on onjisaponin more concentrated in the chemical structure identification, pharmacological activity of compounds isolated areas, and less in molecular biology. The molecular biology research of P. tenuifolia mainly have the following two questions:(1). There are only five mRNA genes of P. tenuifolia in NCBI database (data to 2014.02). (2) It is clearly that the triterpenoid saponins from acetyl coenzyme A to squalene. But the triterpenoid saponins synthesis pathway from squalene cyclooxygenase (SQE) formed triterpenoid saponins nucleus has its own laws to each species. The triterpenoid saponins synthesis pathway of P. tenuifolia is not yet clear. Therefore, this paper was to explore these issues that aimed to improving polygala gene database, and to better understand onjisaponin synthesis pathway. The goal of this study was to provide data to support its regulatory and genetic transformation in P. tenuifolia.OBJECTIVE:1. To rich genetic resources of P. tenuifolia. based on high-throughput sequencing technology to perform transcriptome and 2. To screening suitable reference genes based on transcription database, which would lay the foundation for qRT-PCR technique in gene expression studies of of P. tenuifolia.3. To find differentially expressed function genes in different tissues of P. tenuifolia through the correlation of polygala triterpenoid saponins peak intensity and triterpenoid saponins biosynthesis functional genea expression. It.would be helpful for future regulation onjisaponin content through the bioengineering.METHOD:1. Performing the transcriptome sequencing via Illumina high-throughput sequencing technology, and get a lot genetic information of P. tenuifolia. by bioinformatics research.2. Using UPLC technology to analyze different parts, different lives and different origin of P. tenuifolia. and screening the next subjects by content analysis.3 Based UPLC/Q-TOF MS metabolomics technology to identify and analyze the content of saponins compounds in P. tenuifolia. different tissues.4. Using qRT-PCR technique to screen P. tenuifolia. reference gene based on previous transcriptome database. Using 2-ΔΔCT method to analyze which involved in saponins biosynthetic pathway of different tissues of P. tenuifolia. Then combined saponin biosynthesis genes expression with saponin peak intensity based on the correlation UPLC/Q-TOF MS to elucidate the molecular mechanisms resulting in econdary metabolism differences in different tissues of the P. tenuifolia,RESULTS:1 In this study, transcriptome sequencing data of P. tenuifolia. were assembled and statisticed. The statistical results obtained 115,477 Unigenes, total length of 149 Mb. The unigenes were performed functional alignment and annotation via comparison on the NCBI, respectively Nt, Nr, Swissprot protein database, COG database and KEGG protein database.2. When UPLC technology-based screening study were performed, the findings show that the similarity in different tissues of UPLC fingerprint polygala in 318nm is significantly smaller than the different origin and different lives. It was found that the difference about saponins content was biggest in different tissues of P. tenuifolia.3. Through the identified compounds based on UPLC/Q-TOF MS,25 compounds were identified, including four polygala 6 sucrose esters,8 oligosaccharide multiesters and 7 triterpenoid saponins substances. There were 22 biomarkers via metabolomics analysis in different tissues of P. tenuifolia. Which except Lancerin and 7-O-Methylmangiferin, other compounds were highest concentrations in the root of P. tenuifolia. The statistical analysis showed that all 7 triterpenoid saponins in root were significant higher than in stems, leaves and seeds. (P<0.05).4. The study screened reference gene from the 18s RNA, UBC 2, ACT 11, GAPDH, TUA, ACT1 and EFla seven commonly used reference genes based on transcriptome database P. tenuifolia. The results showed that GAPDH gene was the most stable gene in different tissues of P. tenuifolia. which alculated by NormFinder software and GeNorm software. Using qRT-PCR technique to screen P. tenuifolia. reference gene based on previous transcriptome database. Using 2-ΔΔCT method to analyze which involved in saponins biosynthetic pathway of different tissues of P. tenuifolia. Then combined saponin biosynthesis genes expression with saponin peak intensity based on the correlation UPLC/Q-TOF MS to elucidate the molecular mechanisms resulting in econdary metabolism differences in different tissues of the P. tenuifolia. The results showed that the correlation between the saponin peak intensity and the expression of squalene synthas (SQS), squalene epoxidase (SQE) and β-amyrin synthase (β-AS) was highest in different tissues of P. tenuifolia. But the correlation between the saponin peak intensity and the expression of 6 CYP450 and UGT enzymes was not significant positive relationship.Conclusion:In this study, the transcriptome sequencing data were assembled and bioinformatics were analyzed of P. tenuifolia. GAPDH gene was screened as reference gene in different tissues of P. tenuifolia. We found that the correlation between the saponin peak intensity and the expression of SQS, SQE and P-AS was highest combined saponin biosynthesis genes expression with saponin peak intensity based on the correlation UPLC/Q-TOF MS to elucidate the molecular mechanisms resulting in econdary metabolism differences in different tissues of P. tenuifolia. The results may provide useful information for deeper understanding of the biosynthesis pathway of triterpenoid saponins in P. tenuifolia. The metabolomic analysis by UPLC/Q-TOF MS combined with the gene expression analysis by qRT-PCR technique provide useful information in studying gene discovery and an effective approach in understanding the mechanism of onjisaponin biosynthesis. |
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