| Grape(Vitis L.)is one of the four largest fruits in the world,which enjoys a high economic status.At present,the main grape variety Vitis vinifera in the world is of good quality but poor disease resistance,especially susceptible to powdery mildew.Studies have shown that resveratrol not only has a certain disease resistance function in plants,but also has anti-cancer,anti-oxidation and other health care functions for the human body.The stilbene synthase is a key enzyme in the resveratrol metabolic pathway.Therefore,the study of stilbene synthase(STS)gene and its expression product resveratrol is of great significance for grape disease resistance breeding.The previous research of our research group found that the fruit of Chinese Wild Vitis.quinquangularis ‘Danfeng-2' not only has a high resveratrol content,but also has a strong resistance to powdery mildew.In this study,‘Danfeng-2' was used as a material to clone the stilbene synthase gene Vq STS9,which is highly expressed in mature fruits,and the stilbene synthase gene,Vq STS21,which is highly expressed in young leaves.The mediated method was transferred to Thompson Seedless,and the gene expression characteristics were studied.The main research and results are as follows:1.After artificial inoculation of powdery mildew on leaves,the expressions of Vq STS9 and Vq STS21 at different time periods of 0,12,24,48,72,96,120,and 144 h were analyzed by real-time fluorescence quantitative PCR(q RT-PCR).Using homologous sequence cloning,the Chinese wild grape stilbene synthase genes Vq STS9(JQ868689)and Vq STS21(JQ868677)were obtained.The c DNA sequence size was 1179 bp,encoding 392 amino acids,located on chromosome 16,which were similar to Vitis vinifera Vv STS6,Vv STS2 highest,respectively.2.With the single-bud stem segments of Thompson Seedless,Red Globe,and Cabernet Sauvignon as explants,callus was gradually induced on IM0-IM3 medium.The overexpression vectors with the target genes Vq STS9 and Vq STS21 were transformed into Thompson Seedless by Agrobacterium-mediated method,and a total of 419 Thompson Seedless resistant plants were obtained,including Vq STS9 and Vq STS21 gene resistant seedlings,respectively 223 and 196 strains.After PCR detection and Western blot identification,4 strains of Vq STS9 transgenic Thompson Seedless lines and 3 strains of Vq STS21 transgenic Thompson Seedless lines were identified.3.The expression of transcription factors(MYB14,MYB15)and related genes(STS,PAL,RSGT,CHS)related to resveratrol synthesis in transgenic plants were analyzed by q RT-PCR.The results showed that the stilbene synthase gene(STS),phenylalanine ammonia lyase gene(PAL),resveratrol glycosyltransferase gene(RSGT),transcription factor(MYB14,MYB15)expression increased in varying degrees,while the expression of chalcone synthase(CHS)gene that competed with the grape STS substrate decreased.4.After artificial inoculation of powdery mildew on Thompson Seedless transgenic plants and wild-type plants,through observation and comparison,it was found that compared with wild-type,the mycelium grows slowly on the leaves of transgenic plants;q RT-PCR results showed that under the induction of powdery mildew,The STS expression of transgenic plants was up-regulated.With the development of the inoculation time,the transgenic plant astragalus species increased viniferin on the basis of two types of products of trans-piceid and trans-resveratrol before inoculation,and the accumulation amount increased significantly.Through the expression and accumulation of the product,the stilbene synthase gene improve the resistance to powdery mildew in Thompson Seedless.In summary,Vq STS9 and Vq STS21 gene from Chinese Wild Vitis.quinquangularis‘Danfeng-2' s were induced by biological and abiotic stress,and the expression was up-regulated in response to induction of powdery mildew.So the Chinese Wild Vitis.quinquangularis ‘Danfeng-2' could be used as a germplasm resource to improve the disease resistance of Vitis vinifera for the research and application of disease resistance breeding. |
PDF Full Text Request