| Pinosylvin synthase (PS), a key enzyme in the biosynthesis of pinosylvin-type phytoalexins, catalyzes the formation of pinosylvin from p-coumaroyl-CoA and malonyl-CoA.In the study, a full-length PS cDNA (PSR) fragment was cloned from total RNA of Pinus massoniana by RT-PCR method. Sequence analysis indicated that the gene encoded 393aa and the identity of the derived protein with several PS genes registered in Genbank is between 98% and 82%.The plant expression vector, driven by CaMV35S promoter and terminated by NOS,was constructed and transformed into tobacco discs via the mediation of Agrobacterium tumefaclens to study the situation of PS gene expression in plant.Two full-length PS gDNA fragments were cloned from genomic DNA of Pinus massoniana by PCR method. The sequence is 1667 bp and 1349 bp in length respectively. Analysis of the nucleotide sequence indicated that both of the sequences contained two extrons with the same length (184 bp and 998 bp), but one intron with different length (481 bp or 163 bp).The upstream sequence of PS gene was cloned by genomic walking based on ligation-mediated PCR method. This sequence was 1768 bp in length, containing one TATA-box, one CAAT-box, two GATA-boxes, one GAGA-box, one G-box, and three activators P of flavonoid biosynthetic gene and several TGAC-like sequences. These cis-acting elements, played very important role in the gene regulation of transcription, were respectively identified regions of RNA polymerase II, bZIP protein and AT enrich sequences binding protein.A series of vectors were construted by PCR meathod, in which 0 bp, 350 bp, 775 bp, 1051 bp were deleted from 5' end of the upstream sequence of PS by PCR method, respectively. Then they were ligated to GUS reporter gene to form four plant expression vectors, which will be transferred into tobacco. All these research will play the key role in the analysis of regulation structure and function of PS gene.
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