Study On The Expression Analysis And Preliminary Function Of The PgRFK And PgEXO70B1 In Ginseng

As a traditional Chinese medicine,Panax ginseng C.A.Meyer,and one of the main medicinal herbs in JiLin province,has been utilized for long history.it has been demonstrated that ginsenoside Rb₁,which is one of the main active ingredients of Panax ginseng,plays an important role in dealing with the problem of nervous system,cardiovascular system,relieving aging,anti-tumor and diabetes by Modern research.Ginseng has a long growth cycle and low Saponin production.With the increasing market need of ginseng-related products and the rapid development of modern biotechnology,it is of great theoretical and practical significance to further explore the key genetic resources of ginsenoside Rb₁biosynthesis,at the molecular level.In this study,the ginsenoside Rb₁ biosynthesis pathway gene cluster was obtained by analyzing the ginseng transcriptome database,and was named PgRb₁?A2?and Analyzed the expression patterns of them.The expression pattern of ginsenoside Rb₁ synthesis-related gene PgRb₁?A2?was investigated,and there was a close relationship between PgRb₁?A2?and 16 key enzyme genes.And through correlation analysis with ginsenoside Rb₁ content,the important candidate gene PgRb₁S05 and PgRb₁S08 were further obtained in PgRb₁?A2?gene group.The qRT-PCR technique was used to analyze the relationship between the expression of PgRb₁S05 and PgRb1S08 genes and the content of ginsenosides induced by methyl jasmonate.Then,the biological function of PgRb₁S08 gene was isolated and verified by gene cloning and DNA recombinant technology,and the PgRb₁S05enzyme protein was successfully expressed by a prokaryotic expression system.The results are as follows:1.The expression analysis of the PgRb₁?A2?gene group related to the synthesis of ginsenoside Rb₁,Results showed that there was a significantly correlation between Rb₁ content with the expression of each gene in PgRb₁?A2?gene group,and they belong to different protein families,and having their own expression characteristics.2.By analyzing the gene expression interaction network between PgRb₁?A2?and16 key enzyme genes,it was found that there was a close relationship between Pg Rb₁?A2?and 16 key enzyme genes,with the increased of Rb₁,the link between genes and genes and genes become more and more closely,and more and more genes were involved.And the important candidate genes related to Rb₁ high content group PgRb₁S05 and PgRb₁S08 were obtained.3.The results showed that the content of total ginsenosides and the Rb₁ of ginseng hair root were significantly increased in the induction of methyl jasmonate.Gene expression of PgRb₁S05 and PgRb₁S08 in methyl jasmonic acid induced ginseng root hair by qRT-PCR detection,the results show that the expression patterns of genes in PgRb₁S05 and PgRb₁S08 in different content of saponins were consistent with the analysis results based on transcriptome database.4.PgRb₁S08 were separated by gene cloning,the length is 1836 bp,encoding 515amino acids,through prediction analysis of related bioinformatics,the protein of PgRb₁S08 had a typical structure of exocyst.it is the highest homologous with Arabidopsis EXO70B1 protein subunit by sequence alignment,the result of subcellular localization in Arabidopsis protoplast showed that the protein of PgRb₁S08 was located in the cytoplasm,and was consistent with the predicted result,and transgenic Arabidopsis T₂ plants was obtained by Agrobacterium transformationand,and the characteristic of promoting stomatal opening was found.5.PgRb₁S05 were separated by 3'RACE and PCR technology,the length is 1200bp,encoding 399 amino acids,the protein as riboflavin kinase in plants through sequence alignment,protein and owned multiple function,it is successfully construct the prokaryotic expression vector,it is identified the protein of expressed in E.coli,by Western blotting,this progect lay a foundation for the further research of the protein function.