Constructing Of Plant Expression Vectors Of Chalcone Synthase Gene Form Scutellaria Viscidula Bunge And Its Function Verification In Tobacco

The dried root of Scutellaria viscidula Bunge is one of commonly used Chinese medicinal materials, baicalin that its main active ingredient remains with flavonoids. Currently, the demand of baicalin exceeds its supply, it urgently need to increase its output. Chalcone synthase(CHS) is the first key enzyme of flavonoids biosynthesis pathway, play an important role in the flavonoids synthesis metabolism. In recent years, the application of plant gene engineering develops vigorously, so it has great practical significance and broad application prospect by the means that CHS genetic engineering improves the contents of baicalin of Scutellaria viscidula Bunge.This study was to construct the sense and antisense CHS gene of Scutellaria viscidula Bunge into plant expression vector pCAMBIA1304+, got plant expression vectors of sense and antisense CHS gene.And then, verified the effect of CHS gene in flavonoids biosynthesis by genetic transformation of tobacco. It lay a foundation for obtain the Scutellaria viscidula Bunge with high yield baicalin in the future. The content in this research were as below:1. Construction of plant expression vectors of sense and antisense CHS geneAttained sense and antisense CHS gene with restriction sites of BlgII and BstEII by PCR, linking objective fragments with pMD18-T, got sense and antisense cloned vectors:pMD18-schs and pMD18-achs. They and pCAMBIA1304+ were digested with BlgII and BstEII, constructed pCAMBIA1304+-schs and pCAMBIA1304+-achs through linking sense and antisense CHS gene and big fragments of pCAMBIA1304+. Then, pCAMBIA1304+-schs and pCAMBIA1304+-achs were transferred into LBA4404.2. CHS genes were transformed into tobacco mediated by Agrobacterium LBA4404 and molecule indentificationYoung leaves chosen from sterile seedlings of tobacco, cut into 0.5 cm×0.5 cm, soaked into fit Agrobacterium LBA4404 for 5-8 min. Sucked dry on the aseptic paper, placed to medium(MS+1.0 mg/L 6-BA+0.1 mg/L NAA) and co-cultivation for 48 h in darkness. Then transferred into medium (MS+1.0 mg/L 6-BA+0.1 mg/L NAA+50 mg/L Hyg+300 mg/L Cef) for germination and selection culture, resistant bud were obtained after light-culture about 20 days, when grown to 2～3 cm, cut and rooted on medium(MS+0.1 mg/L NAA+25 mg/L Hyg+300 mg/L Cef), roots began to generate after 7 days. After 2～3 weeks again, tobacco seedlings that had developed roots were attained. The results showed that obtained transformed tobacco 41 lines, including transformed pCAMBIA1304+-schs(sense) tobacco 20 lines, transformed pCAMBIA1304+-achs(antisense) tobacco 13 lines, transformed pCAMBIA1304+(vector) tobacco 8 lines.Take the leaf of transgene tobacco that has 3-4 pieces of leaves to GUS staining, positive plants were transplanted to the greenhouse. Got positive transgene tobacco(sense has 5 lines;antisense has 6 lines; vector has 3 lines) by PCR from GUS positive plants. Then RT-PCR of transgenic tobacco which were PCR positive. The RT-PCR results revealed that CS3,CS4 and CS6 had ochs(1170 bp), but CA2,CA4 and CA6 hadn't ochs, compared with the control(WT, P3). It suggested that schs had been transcribed into mRNA in sense transgenic tobacco, caused excess expression of CHS gene, whereas achs that was integrated into genome of antisense transgenic tobacco inhibited expression of CHS gene.3. The determination of flavonoids and rutin in transgenic tobaccoThe content of flavonoids and rutin of positive transgenic tobacco by molecular tested positive and untransformed tobacco(control) were determined by ultraviolet spectrophotometry and HPLC. The results showed that, the content of total flavonoids and rutin of transformed vector tobacco and control were close; them of sense transgenic tobacco were all higher than control, and CS4's was the highest, enhanced about 4 mg/g DW compared with the control; them of antisense transgenic tobacco were all lower than control, and CA2's was the lowest, reduced about 6 mg/g DW compared with the control. It indicated that CHS gene whose expression affected the synthesis of flavonoids was an important target of regulation of flavonoids biosynthesis metabolism.