Cloning Of Resveratrol Synthase CDNAs From Arachis Hypogaea L.and Transformation Of Resveratrol Synthase Gene Into Daucus Carota L.

Resveratrol is a member of stilbenoids with the capcity of disease-resistantance for plants and health benefit for human. Carrot is one of the most important vegetables rich in β-carotene. To further promote carrot's heath care value by expression of foreign resveratrol, we conducted the genetic transformation of RS to carrot. To achieve this goal, the cDNA of peanut pods which is rich in resveratrol was constructed and 2 postive cDNA clones were screened by a PCR based 96-well method. With a cDNA of RS, the transformation vecter was constructed by inserting the cDNA into a transformation vector pCB. On the other hand, a efficiency Agrobacterium mediaed transformation system was established and Agrobacterium mediaed carrot transformation was conducted to acquire the RS transgenic carrot plants for further use. The main results were as followings:1) The total RNA was extracted from the pods of peanut cultivar 'JinHua1012' by combining the method of Trizol and the column based RNA extract method from Huashun Company. The acquired total RNA, whose ratio of A260/A280 was 1.89, and ratio of A260/A230 was 2.03, was good enough for the cDNA library construction. With the total RNA, a cDNA library was constructed with SMART? cDNA library kit, titer of the cDNA library was more than 1×106 pfu/ml, and the recombinating rate of the cDNA library was about 99%.2) By the PCR Based 96 well method, the cDNA library was screened with a pair of primers designed according to the high homology sequences of RS genes in plants,and two positive cDNA clones, CAG\ and CAG2, with 94% of the homologies in their nucloetide sequences and more than 90% homology with other RS gene cDNAs from grapes and peanut, were acquired. CAG1 and CAG2 were infered as two cDNAs of multiple resveratrol synthase gene family in the genome of peanut.3) The transformation vecter was constructed by inserting a cDNA of grape RS to the position of BamHI/SacI in the transformation vector pCB, and the cDNA of RS was put under the control of CaMV 35S-promoter, a promoter suitable for genetic transformation in the dicotyledon.4) A efficiency Agrobacterium mediaed transformation system for Minghui 86 was established, and Agrobacterium mediaed carrot transformation was conducted by this system to acquire the RS transgenic carrot plants for further use. 200 transgenic plants and more than 100 other resistant callus were acquired, and the plants and the resistant calli were positive by GUS assay and PCR amplification with the specific primers based on the sequence of RS gene.