Preliminary Study On The Promoter Of PsbD Gene Of Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is a traditional Chinese medicinal herb with high medicinal value.At present,S.miltiorrhiza is mainly cultivated artificially to meet the market demand,but often suffer from seasonal drought,resulting in dead seedlings affecting yield.Past studies have found that the chloroplasts of S miltiorrhiza are very sensitive to drought stress at moderate level,and the photosynthetic function is usually reduced.Photosystem ? has the ability to absorb light energy,initiate charge separation,transfer electrons,and catalyze water decomposition.D2 protein is one of the core components of photosystem ? complex.It is important to study the encoded psbD gene to reveal the mechanism of photosynthesis in S.miltiorrhiza.This study selected Shaanxi Shangluo Tasly provided excellent Salvia SH-B,cloning of psbD gene upstream 1355 BP,and the full-length promoter as a template to design the 5 '-end deletion promoter fragments,respectively with GUS gene fusion constructs multiple expression vector,using the GV3101 of Agrobacterium infection of tobacco leaf's analysis of the promoter fragment of transient expression differences,in order to verify psbD gene promoter function,to lay a foundation for further study on the mechanism of photosynthesis and male sterility in S.miltiorrhiza.The main conclusions are as follows:(1)Cloning and analysis of psbD gene promoter of S.miltiorrhiza.The upstream promoter region of Salvia miltiorrhiza psbD gene was cloned with the chloroplast DNA of S.miltiorrhiza(1355 bp).On the PLACE site,the full-length promoter was found to contain multiple cis acting elements related to the external environment,indicating that the promoter was an inducible promoter.(2)Construction of 5' deletion vector of psbD gene promoter of S.miltiorrhiza.Using Primer 5 to design specific primers,we cloned 1147 bp,891 bp,746 bp,582 bp,451 bp,323 bp,230 bp,142 bp and 8 5' deletion fragments by using full-length promoter as template.The full-length promoter and eight 5'-terminal promoter deletion sequences were ligated with pCAMBIA1304,which removed the CaMV 35 S promoter,and the expression vectors were successfully constructed.The sequences were named SalP-1355,SalP-1147,SalP-891,SalP-746,SalP-582,SalP-451,SalP-323,SalP-230,SalP-142.(3)S.miltiorrhiza psbD gene promoter expression vector converted tobaccoThe constructed expression vector was transferred into Agrobacterium tumefaciens,and the tobacco leaves were transiently transfected.GUS staining was performed on the leaves.The results showed that the full-length promoter and the 5 'deletion promoter could drive GUS gene expression and promoter activity were significantly different.(4)Functional analysis of psbD gene promoter of S.miltiorrhizaThe tobacco leaves were transfected with Agrobacterium tumefaciens GV3101 containing various expression vectors.The total RNA was extracted and transcribed into cDNA and analyzed by RT-PCR.The results showed that SalP-1355 and SalP-451 had the highest expression level and lower SalP-230.In combination with bioinformatics analysis,it was concluded that it has enhancers between 1355 bp and 891 bp,and silence in the range of 891 bp to 746 bp.There were enhancers in the domains of 1355 bp-891 bp?746 bp-582 bp?451 bp-230 bp,and silencers in the domains of 891 bp-746 bp?582 bp-451 bp?230 bp-142 bp.