Studies On Construction Of Plant Expression Vector Carrying Stilbene Synthase Gene Of Wild Grape Species In China And Its Genetic Transformation In Tobacco

Resveratrol, a major stress metabolite (phytoalexin) in grapevine, is a kind of secondary metabolites accumulated in cells in response to pathogen stress or other various elicitors. The accumulation of resveratrol in plants can greatly improve plant disease resistance. In this paper, with STS gen cloned, the recombinant plasmid pWR-STS carring STS gene in wild grape species in China was constructed, and introduced into Agrobacterium tumefaciens strain GV3101. Subsequently STS gene was transformed to tobacco by Agrobacterium-mediated method. We initially discussed the transformation and expression of STS gene through studying tobacco genetic transformation, which will provide some beneficial foundation in grape in the future. The results were as following: 1. The cloning,sequencing and analysis of STS gene came from wild grape species in China According to our obtained sequence of STS gene full-length cDNA from Vitis pesudoreticulata Baihe35-1, two specific primers were designed, then were made some modification, adding restriction enzyme sites to match plant middle expression vector. The PCR products were called back and cloned into pGEM-TEasy vector, then recombinant plasmid pGEM-T-STS was constructed. By enzyme digest analysis, the result showed the plasmid containing STS gene was transformed into E.coli. After DNA sequencing, it showed target fragment was 1179 bp in length, which was correspond to our obtained sequence. 2. Construction and identification of plant intermediate expression vector With the application of DNA recombinant technology, the nucleotide sequence of plasmid pSB166 containing ED35s-O-MCS-UTT-TNOS was specifically cloned into plasmid pCAMBIA1303, then plant intermediate expression vector pWR306 was successfully constructed by enzyme digestion and electrophoresis. 3. Construction of plant expression vector carrying STS gene from wild grape species in China After plasmid pGEM-T-STS and plant expression vector pWR306 were double-digested by HindIII and EcoRI, the target fragments were collected and purified respectively, after being ligated, recombinant plasmid pWR-STS was constructed. 4. Introduction of plasmid pWR-STS into Agrobacterium tumefaciens The recombinant plasmid pWR-STS was intruoduced into Agrobacterium tumefaciens strain GV3101 by means of improved freezing-thawing. By screening in the medium containing kanamycin and Gentamycin, and clonoy PCR identification, it was confirmed the result was successful. 5. Study on tobacco genetic transformation by Agrobacterium-mediated system and detection of transformed explants Factors affecting the transformation frequency were studied, and some parameters were determinated as following description. Tobacco leaf explants had been pre-cultured for 2 days; inoculated with active Agrobacterium suspension (absorbance at 600nm reached 0.5) for 5 minutes; co-cultured for2 days on culture medium appended with 100μM AS. The sensitivity of tobacco's leaf explant to HygB, was used to 25 mg/L,which could effectively inhibit the growth of untransformed tobacco explants. With green fluorescent protein (GFP) detection system, transformed and untransformed explants were separated and identificated in advance, then PCR assay showed that the STS gene had been integrated into tobacco genome.