Structural Analysis And Functional Study Of The Promoter Region Of Dirigent1 Gene In Eucommia Ulmoides Oliv.

Eucommia ulmoides Oliv.is a tertiary relic plant unique to my country.It has extremely high medicinal and economic value.The main medicinal part is the bark,which is usually adopted by peeling the bark.In this study,the BDGP website was used to predict the transcription start site(Transcription initiation site,TIS)of EuDIR1 gene promoter online,and the cis-acting element of the promoter was analyzed by Plant CARE website.PCR-specific primers were designed by oligo7.0 software and cloned into the EuDIR1 gene promoter.The reporter gene is GUS,and a total of 4 plant expression vectors are constructed.Agrobacterium tumefaciens-mediated genetic transformation of tobacco,GUS histochemical staining analysis and PCR identification of transgenic plants;using hormones 100 ?mol/L methyl jasmonate(MeJA)and 100 ?mol/L abscisic acid(ABA)and 10 g/L high-concentration salt(Na Cl)and 300 mmol/L mannitol(D-mannitol)simulated drought treatment of E.ulmoides seedlings and transgenic tobacco.The regulatory characteristics of the promoter were preliminarily analyzed by q RT-PCR technology.The following results are obtained:(1)Cloning and bioinformatics analysis of EuDIR1 gene promoterIt was cloned into 4 segments of the EuDIR1 gene promoter deleted at the 5? end,and the 1495 bp promoter sequence containing the transcription start site was named EuDIR1p-1,and after 3 times of 5? deletion,804 bp was named EuDIR1p-2;549 bp named EuDIR1p-3 and 212 bp named EuDIR1p-4.Bioinformatics analysis found that there is a transcription initiation site TIS(+1)at-50 bp upstream of the ATG of the EuDIR1 gene.TATA-box and corresponding CAAT-box exist at-25?-35 bp upstream of TIS.Other components include MYB,MYC,Myb,CGTCA-motif(MeJA),etc.(2)GUS Histochemical Staining Analysis of EuDIR1 Gene Promoter in TobaccoThe GUS staining solution was used to analyze the transgenic tobacco seedlings,roots,stems and leaves.The results showed that the promoter activity was different with different degrees of deletion at the 5'end,and it was clear that the core region of the promoter was within 212 bp containing TIS.(3)Expression Analysis of GUS Gene in Transgenic TobaccoTransgenic tobacco with CGTCA-motif promoter was used to spray transgenic tobacco with MeJA,3 h was 5.065 times that of 0 h,6 h was 4.234 times that of 0 h,and 12 h was 1.449 times that of 0 h,At 24 h,it was 2.0599 times that of treatment 0 h,and the expression of GUS gene was significantly up-regulated compared with before treatment;using genetically modified tobacco containing Myb element as the material,the GUS gene after ABA spray treatment was before treatment at 3 h 2.33 times,2.52 times before treatment at 6 h,and significantly increased at 3 h and 6 h compared with treatment at 0 h.Using transgenic tobacco containing MYB cis-acting elements as the material,mannitol-stressed transgenic tobacco was 2.071 times before treatment at 6 h,which was significantly lowered after 12 h compared to before treatment and reached a very significant level at 48 h.Using transgenic tobacco containing STRE elements as material,under high salt stress,the GUS gene was 0.104 times before treatment at 12 h and 0.0997 times before treatment at 48 h,and its expression was significantly inhibited compared with treatment at 0 h.(4)EuDIR1 Gene Expression AnalysisUsing E.ulmoides seedlings as materials,MeJA and ABA were used to spray E.ulmoides seedlings,and the expression of EuDIR1 gene was detected by real-time quantitative PCR.MeJA treatment was 0.276 times before treatment at 3h,and was increased by 3.335 times at 24 h compared to before treatment.Compared with the expression of EuDIR1 gene in E.ulmoides,it was significantly lowered at 3?12 h,and significantly increased at 24 h;At 12 h,it was 0.092 times before treatment,and there was a trend of recovery at 24 h,which was significantly lower than before treatment,and reached a very significant after 12 h.Mannitol simulated drought stress E.ulmoides seedlings gene expression was first up-regulated and then down-regulated,and significantly increased at 8 h,and significantly decreased to 0.12 times before treatment at 24 h;high concentration of salt stress,the expression of EuDIR1 gene was down-regulated and significantly suppressed,12 h was 0.35 times before treatment,and showed a trend of recovery after 24 h.The above results preliminarily revealed the characteristics of the EuDIR1 gene promoter and clarified the core region of the promoter.By exploring the regulatory characteristics of the cis-acting element of the promoter,it provides a theoretical basis for the efficient expression of the EuDIR1 gene promoter in E.ulmoides,in order to provide a theoretical basis for revealing the expression regulation mechanism of the EuDIR1 gene.