Expression And Analysis On The Gene Of Stilbene Synthase And Its Promoter From Chinese Wild V. Quinquangularis Rehd. ‘Danfeng-2’

Due to its great economic and ecological benefits, grapevine(Vitis L.) has become one of the most important fruit trees cultured worldwide. However, the growth and yield of many grapevine varieties, especially the European grape which is a main grape cultivar, suffer seriously from grapevine powdery mildew caused by Uncinula necator(Schw.)Burr. In China, there are diverse wild grape germplasm resources, including some highly resistant species to powdery mildew, such as wild V.quinquangularis Rehd.accession‘Danfeng-2’. Resveratrol is a phytoalexin, and can be quickly induced by fungi,strong ultraviolet or in pathological condition to accumulate in response tobiotic and abioticstresses. Stilbene synthase(STS) is one restrict enzymein thebiosynthesis of resveratrol, and its expression is regulated by its promoter upstream.In this study, V.quinquangularis Rehd.accession‘Danfeng-2’was used as experimental material. The research focus is to investigate the expression of three stilbene synthase genes Vq STS21, Vq STS30 and Vq STS32 and their corresponding promoters Pro-Vq STS21, Pro-Vq STS31, Pro-Vq STS32 and also three other STS promoters Pro-Vq STS3, Pro-Vq STS6 and Pro-Vq STS33. The results are as follows:1. Three STS genes were cloned from V.quinquangularis Rehd.accession‘Danfeng-2’ and designed as Vq STS21, Vq STS30 and Vq STS32. The Gene Bank numbers were JQ868677, JQ868668 and JQ868666.Sequence analysis showed that their c DNA had equal length of 1179 bp, encoding a polypeptide of 392 amino acid residues. RT-PCR indicated that Vq STS21,Vq STS30 and Vq STS32 constitutively expressed in roots, stems, tendrils, young and old leaves of grape species V.quinquangularis Rehd.accession‘Danfeng-2’ and Cabernet Sauvignon. However, the expression conditions of each of the three genes were different in the same tissue; it also varied in different grape species.2. Real-time PCR was employed to real the expression patterns of Vq STS21, Vq STS30 and Vq STS32 from V.quinquangularis Rehd.accession‘Danfeng-2’ and Cabernet Sauvignon, after treated by ABA, SA, Me JA and abiotic stress of high-temperature, low-temperature and high salinity.The results indicated that all three genes Vq STS21, Vq STS30 and Vq STS32 could be induced in the two grape species but had different expression patterns.3. Real-time PCR was employed to real the expression patterns of Vq STS21, Vq STS30 and Vq STS32 from V.quinquangularis Rehd.accession‘Danfeng-2’ and Cabernet Sauvignon, after treated by powdery mildew,.The results indicated that all three genes could be induced in the two grape species but had different expression patterns.Except Vq STS21, Vq STS30 and Vq STS32 showed higher expression level in V.quinquangularis Rehd.accession‘Danfeng-2’ than in Cabernet Sauvignon.4. Plant expression vectors of constructed by ligating the cloned c DNA of Vq STS21, Vq STS30 and Vq STS32 to vector p BI121.Using leaf disc method, the constructed vectors were transformed into tobacco NC89 mediated by Agrobacterium tumefaciens.The successfully transformed plants were verified by PCR. Then the contents of stilbenes in transgenic seedlings were determined by HPLC. The results were that, trans-piceid was only measured in seedlings expressing Vq STS30 and Vq STS32, and its accumulations in the two transgenic seedlings were similar, with 26.087μg/g FW and 26.371μg/g FW respectively.5.Cluster analysis was done for 32 STS genes of V.quinquangularis Rehd.accession ‘Danfeng-2’obtained from Gene Bank. These genes were classified into 6 groups. The promoter of one typical STS gene from each group was cloned and connected onto the vector p CAMBIA0390 with a GUS gene in its downstream. Through transient transformation mediated by agrobacterium tumefaciens, these recombined plant expression vectors were transformed into the leaves and somatic embryo of Thompson Seedless. Histochemical stain and fluorescence quantitative methods displayed that the expression and activity of GUS in leaves transformed by 6 Pro-Vq STS were significantly different, while they were not intransformed somaticembryo.