Functional Analysis Of Stilbene Synthase Fruit-specific Promoter From Chinese Wild Vitis Quinquangularis Accession Danfeng-2

Grape is an important worldwide economic crop used for table fruit, juice and wine. As a kind of phytoalexin in plant, resveratrol has a significant function in plant disease resistance. Particularly, it has a variety of biological health-care activities, including anti-cancer and anti-cardiovascular disease. The most widely cultivated cultivars are Eurasian species(Vitis vinifera), which have high quality, but poor stress resistance.The content of resveratrol in Vitis vinifera is limited. Based on the previous studies, Vitis quinquangularis accession Danfeng-2 not only has a good resistance, but also a high content of resveratrol during the fruit ripening. Stilbenes are formed via the phenylalanine/polymalonate-route, the last step of which is catalyzed by the enzyme stilbene synthase. One of the most important stilbenes is resveratrol. In this research, Chinese wild Vitis quinquangularis accession Danfeng-2 was used as experimental material to investigate the clonal analysis and regulating expression of fruit-specific promoter(PvqSTS6) and bidirectional stilbene synthase promoters(PvqDSTS12/PvqDSTS33), the results are as follows:1. In previous study, our team analyzed the expression levels of Chinese wild Vitis quinquangularis accession Danfeng-2 stilbene synthase gene family(STSs) 41 members using real-time quantitative PCR method in young leaves, young fruit, ripe fruit, pulp, peel and seeds. Among them, VqSTS6 has the highest expression level in ripe fruit and pulp. VqSTS6 promoter(PvqSTS6) was amplified using nested-PCR, whose length is 1259 bp. Through predictive analysis, the promoter contains four Skn-1_motifs and a GCN4_motif, these cis-acting elements associated with endosperm development.2. GUS gene was fused to the 3’ end sequences of promoter PvqSTS6, recombinant plasmid was transferred to tomato(Solanaceae lycopersicum L. Micro-Tom). GUS fluorescent quantitation analysis and RT-PCR quantitative analysis was accomplished to test GUS expression profiles in root, leaf, stem and fruit. It turned out that PvqSTS6 is a fruit-specific promoter.3. Deletion promoter PvqSTS6 with GUS genetransferred into Micro-Tom was detected. Among them, PvqSTS6-2 has the highest starting activity by histochemical staining of GUS, GUS fluorescent quantitation analysis and RT-PCR quantitative analysis.-518 bp ~-411 bp of the promoter sequence may be the core region, which contained two closed Skn-1_motifs.4. Bidirectional stilbene synthase promoters cloned from Chinese wild Vitis quinquangularis accession Danfeng-2 by genome walking were named as PvqDSTS12(reverse) and PvqDSTS33(forward), respectively. The corresponding cis-elements of bidirectional promoters were analyzed and GUS gene was fused to the 3’ end sequences of bidirectional promoter. The expression patterns of GUS gene in grape leaves under different stress treatments, including powdery mildew, 40℃, 4℃, methyl jasmonate(MeJA) and salicylic acid(SA), were analyzed using GUS quantification and RT-PCR. The results showed that PvqDSTS12 has a higher level activity than PvqDSTS33. PvqDSTS12 was positively regulated by powdery mildew incubation, MeJA, cold and heat stress. While PvqDSTS33 was positively regulated by MeJA and negatively by heat and cold stress but not affected with powdery mildew incubation. Both promoters had no significant changes in response to SA.