Gynostemma Transcriptome Sequencing, Excavation Of Triterpene Synthesis Genes And Construction Of Expression Vectors With Mutations In The Positive Selection Site Of Squalene Synthase

PART 1 INVESTIGATION OF GENE AND ITS PATHWAY RELATED TO TRITERPENOID SYNTHESIS OF GYNOSTEMMA PENTAPHYLLUM BY TRANSCRIPTOME SEQUENCINGObject:We performed transcriptome sequencing on fibous root,stem and leaf of Gynostemma pentaphyllum(G.pentaphyllum)to annotate genes and pathways related to triterpenoid synthesis.q PCR technology was used to test the post-transcription expression pattern of enzyme FPS,SS and SE,which showed different expression in transcriptome sequencing.The content of Gypenoids was tested simultaneously to futher identify the expression of enzyme FPS,SS and SE.We concluded that the tendency of the differential expression of FPS,SS and SE are similar to the pattern of differential distribution of Gypenoids among fibous root,stem and leaf.Our investigation contributes to the research of regulation mechanism of genes expression on Gypenoids biostynthesis.Method:We isolated the RNA from the fibrous root,stem and leaf of G.pentaphyllum to perform transcriptome sequencing.Based on the KEGG pathway database,we worked to find out the RPKM value of FPS,SS and SE,which were the key enzymes in the triterpenoid synthesis.We identified the different expression of FPS,SS and SE based on the standard value of |Fold Change| >2 and concluded the differential pattern of these three genes.The full-length ORF sequences of FPS,SS and SE were cloned as the standard substance of the qPCR test.To take c DNA of G.pentaphyllum as the reaction template,we performed qPCR test on FPS,SS and SE to verify their expression in fibrous root,stem and leaf.Gypenoside was isolated and purified from G.pentaphyllum and regarded as sample group while panoxadiol was treated as standard group.UV-Vis spectrophotometry on color-test was used to detect the content of Gypenoside among sample groups and standard groups.Based on the standard curve of panoxadiol constructed,we calculated the content of Gypenoside among fibrous root,stem and leaf.Finally,we got the insight into the different distribution characteristics of Gypenoside in G.pentaphyllum.Result:Transcriptome sequencing and Gypenoside content-test results showed a same expressional tendency: FPS,SS and SE exhibited a same order on expression level among leaf,stem and fibrous root(leaf had the highest RPKM value among three tissues,leaf,stem or fibrous root,while qPCR result confirmed this pattern).Finally,the test results with UV-Vis spectrophotometry showed that the content of Gypenoside was the highest in leaf while it was the lowest in fibrous root.The results of transcriptome sequencing,q PCR and UV-Vis spectrophotometry test were highly consistent.Conclusion:qPCR test exhibited the tendency of FPS,SS and SE expression among fibrous root,stem and leaf,which was consistent to the transcriptome sequencing result.UV-Vis spectrophotometry testing also conformed this tendency.Combinated the differential expression of FPS,SS and SE with the differential distribution of Gypenoside,this research will contribute to the futher investigation of regulation mechanism of genes expression related to Gypenoside biostynthesis.This research provides theory support on improving the Gypenoside content of G.pentaphyllum.PART ? CONSTRUCTION OF EXPRESSION VECTOR OF SQUALENE SYNTHASE OF G.PENTAPHYLLUM UNDER POSITIVE-SELECTION SITE MUTATIONObject: Combined positive-selection sites with PCR mutation technology,we constructed wild type and positive-selection sites mutation type vector for prokaryotic or eukaryotic expression of squalene synthase of G.pentaphyllum.This research help us get insight into the association between the mutation on positive-selection-site and the function of squalene synthase.This research also help us deeply understand about the association between the structure and the function of squalene synthase.Method: Based on the sequences analyse from transcriptome sequencing result,we designed primers to clone the full length of SS ORF by PCR.After ligation with T-Vector,SS ORF was sequenced for obtaining wild SS sequence.We redesigned the primers based on the wild sequence of SS.Ligation was performed on tagert gene and eukaryotic expression vector after RE digestion.Then we gained the reconstructed eukaryotic expression vector pc DNA3.1(+)-SS.In addition,SS ORF was connected with prokaryotic expression vector directly to build the prokaryotic expression vector p EASY-E1-SS.We made mutation to the expression vector on the positive selection sites(S109,S196,T390 and S407)by mutation PCR technology.We changed serine and threonine into proline to obtain the mutational type expression vector.Result: We successfully constructed the expression vector p EASY-E1-SS and pc DNA3.1(+)-SS.In addition,we performed the mutation successfully and gained the wild type and mutational type expression vectors of G.pentaphyllum squalene synthase.Conclusion: The construction of expression vector of G.pentaphyllum squalene synthase with mutation of positive-selection-sites is the foundation to understand to relationship between positive-selection-sites of SS and its enzyme activity.Our work contributes to the expolation between the primary structure of G.pentaphyllum squalene synthase and its biofunction.