Vector Construction Of The Organ-specific Expressing Promoter With Genes Of SGAs Biosynthesis Metabolismic Pathway And Transformation In Solanum Tuberosum L.

The steroidal glycoalkaloids (SGAs) is one of the toxicant secondary metabolites inSolanaceae and some Liliaceae, that has a strong physiological and drug activity andenhance plant resistance to pathogens and abictic stresses. If SGAs content exceeds a certainvalue in potato tuber, it will produce toxicity to humans and animals. It is important andsignificant to increase SGAs content in the aerial parts by regulation SGAs synthesis andspatial distribution for improving disease resistant in potato. In this study, therefore, plantexpression vectors of SGTs were constructed and driven by organ-specific andlight-inducible promoter rbcS. Then, leading them into tobacco and potato throughtAgrobacterium-mediated method to aim at regulating SGAs spatial distribution in potato andspecific improving SGAs content on potato aerial parts. The main results of the research areas follows:1. Subcloned genes sgt1, sgt2and sgt3from plasmid pMD-T/sgt1, pMD-T/sgt2,pMD-T/sgt3comtaining SGTs, and shared a high level of similarity with thereported sgt1, sgt2and sgt3in GenBank are99.3%、99.5%、99.5%respectively.The length of open reading frame (ORF) was1470bp、1470bp、1500bp.2. The organ-specific and light-inducible promoter rbcS was cloned from potatogenomic DNA by polymerase chain reaction (PCR) and shared a high level ofsimilarity with the reported rbcS in GenBank is99.8%.3. Introduced rbcS promoter, sgt1、sgt2and sgt3genes in the multicloning sitesequentially based on the plant expression vector pCEPSPS. To construct plantexpression vectors pCENr-sgt1、 pCENr-sgt2and pCENr-sgt3drived byorgan-specific and light-inducible promoter rbcS, and lead-in pCENr-sgt3into theagrobacterium tumefaciens LBA4404to gain engineering bacteria.4. Tobacco line T12was transformated by agrobacterium strain with pCENr-sgt3,56regenerated plants were obtained and11positive plants were screened by PCR. Thetransformation frequence was19.6%. Herbicide resistance screening resultsshowed that transgnic tobacco has resistance to glyphosate, indicating that targetgene has been integrated into tobacco plants and can express. And the SGT3geneexpression levels of the transgenic tobacco were measured by RT-PCR. The results showed that sgt3can express in leaves specifically with rbcS promoter, and theexpression was not obvious in roots.5. The pCENr-sgt3was introduced into two outstanding varieties, Shepody and L-3,via agrobacterium-mediated genetic transformation using microtuber as receptormaterial. Atotal of9regenerated plants were obtained. But the expression level ofsgt3will be further determinated out in transgenic plants.