Construction Of Plant Expression Vector Of Blueberry LAR,ANS,ANR And Study On Its Transgene

In this study,the leucoanthocyanidin reductase gene and anthocyanin synthase gene were cloned from leaves of ‘Brightwell'(V.ashei‘Brightwell')which containedmore procyanidins and ‘Herbert'(V.corymbosum ‘Herbert')which contained less procyanidins.And the obtained genes were named VaLAR1?VaLAR2?VaANS ? VcLAR1?VcLAR2?VcANS based on their sources,respectively.The sequence analysisresulted that only a few base sites were different betweenthe homologous genes from two cultivars,and the similarity of the homologous genes were all over99%.The phylogenetic treesof the genes cloned constructed in this study indicating that the closestgenetic distance to the above geneswas tea tree.The virus-induced gene silencing(VIGS)vectorscontaining VcLAR1,VcLAR2,VcANS and VcANR(cloned by Wang Lijin)of 'Herbert'were constructed.The vectors were transfered into the explants from 'Herbert'tissue culture seedlings mediating by Agrobacterium tumefaciens,and the virus-induced gene of 'Herbert' tissue cultureseedlings silencing system was exploredinitially.The results showed that the optimal system of VIGS-mediated: the OD600 value of Agrobacterium tumefaciens was 2.0,the infection time was 4 min,and the darkness culture time was 3 d before transferred to normal culture and samples were collected after 4 weeks.In this study,the expression of VcLAR1 gene in 'Herbert' tissue culture seedlings was successfully silenced,and the content of procyanidins in leaves was decreased.The recombinant plasmids of plant bivariate vector pCAMBIA 1302 containing VaLAR1,VaLAR2,VaANS and VaANR genes of ‘Brightwell'were constructed,and the explants from 'Herbert' tissue culture seedlings were transformed by Agrobacterium tumefaciens containing the recombainant vectors,respectively.And the genetic transformation system of 'Herbert' was explored initially.The Hyg resistance of 'Herbert' explants was determined by screening.The optimum infection method of 'Herbert' was determined by orthogonal experiment: the OD600 value representing the concentration of Agrobacterium tumefacienswas 0.7,the infection time was 10 min,and the darkness culturetimeof co-cultured was 6 d.The 'Herbert resistance callus of VaLAR1,VaLAR2,VaANS,VaANR gene was obtained,respectively.