Selection Of Appropriate Reference Genes For Quantitative Real-time PCR And Functional Study Of Squalene Epoxidase Gene In Panax Vietnamensis Var.fuscidiscus

Objective Screening and verifying suitable reference genes in different tissue parts of Panax vietnamensis var.fuscidiscus,so as to provide a reference for the gene expression related research of P.vietnamensis var.fuscidiscus in the later studies,at the same time,functional studies on squalene epoxidase of P.vietnamensis var.fuscidiscus.Methods Based on the transcriptomic data of P.vietnamensis var.fuscidiscus,the candidate genes were screened and specific primers were designed.The expression level of these candidate genes in different tissues(root,rhizome,stem and leaf)of P.vietnamensis var.fuscidiscus was analyzed by q RT-PCR.The ge Norm,Norm-Finder and Best Keeper softwares were used for comprehensive analysis of the expression stability of the candidate genes.Based on previous research,we redesigned the specific primers of Pvf SE2 and Pvf SE2 d,constructed the prokaryotic expression vector with p Mal-c2 X,and constructed the prokaryotic expression vector p ET-28a-Sm CPR,p ET-28a-Sm CPRd of Salvia miltiorrhiza P450 reductase(Sm CPR)with His-Sm CPR as the template.The recombinant plasmid was induced to express in E.coli BL21(DE3),SDS-PAGE was used to detect the expression of the protein,and q RT-PCR was used to detect the expression level of squaleneepoxidase gene in different tissues of P.vietnamensis var.fuscidiscus.Result1.Based on the transcriptome data of P.vietnamensis var.fuscidiscus,13 candidate reference genes(ACT1,ACT2,b TUB1,b TUB2,a TUB,GADPH,UBQ,el F,EF-1a,F-box,CYP,QCR and TCTP)were selected.The expression trends of these candidate genes in P.vietnamensis var.fuscidiscus were analyzed by q RT-PCR.The results of software analysis show that both ACT1 and a TUB were the suitable internal reference genes for different tissues of P.vietnamensis var.fuscidiscus.2.In the previous research,we cloned four SEs genes,named Pvf SE1,Pvf SE2,Pvf SE3,Pvf SE4.The truncated sequences of Pvf SE1,Pvf SE3 and Pvf SE4 were successfully expressed and the corresponding fusion proteins were obtained.In this experiment,we successfully constructed the prokaryotic expression vector p Mal-c2X-Pvf SE2 d,p ET-28a-Sm CPR,p ET-28a-Sm CPRd were successfully constructed.The recombinant plasmid was induced to express in E.coli BL21(DE3).SDS-PAGE electrophoresis results showed that Pvf SE2 d,Sm CPR,and Sm CPRd were successfully expressed in supernatant,and the corresponding fusion protein was obtained.Using q RT-PCR technology,ACT1 was used as the reference gene to detect the expression level of Pvf SEs gene in different tissue parts.The results showed that the expression level of Pvf SE1 was the highest in the root and the lowest in the stem;Pvf SE2 and Pvf SE3 had the same expression level trend in different tissue parts,the expression level in the rhizome was significantly higher than that in other tissue parts,and the lowest expression in roots,the second in stems and leaves;Pvf SE4 expression in roots is slightly higher than other tissue parts,and its expression levels in the four tissue parts of roots,rhizomes,stems and leaves are significantly lower than that of the other three SEs genes.In general,Pvf SEs are mainly expressed in roots and rhizomes,and lower in stems and leaves.Conclusion This study screened and verified the appropriate reference genes for different tissue parts of P.vietnamensis var.fuscidiscus were ACT1 and a TUB at the first time,which laysa foundation for further research on the functional gene expression level of P.vietnamensis var.fuscidiscus.Successfully constructed p Mal-c2X-Pvf SE2 d,p ET-28a-Sm CPR,p ET-28a-Sm CPRd prokaryotic expression vectors,and successfully expressed them in E.coli to obtain soluble protein.It will lay a foundation for further study on the function of SEs and analysis of triterpenoids biosynthesis pathway.