Study On Agrobactirium Tumefaciens-mediated Sbe Ⅱb Gene RNAi Segments Transformation Of Maize Inbred-lines

Maize is one of the most important crops,staple food and industry materials.Highly efficient regeneration system is always the focus of scientist researches.Starch,as the main component of maize seed,is widely applied in energy sources and chemistry industry. Improving maize starch quality is the main object of maize breeding.RNA interference (RNAi),as an important gene silencing methods,has a significant application to the research of gene function and genetic melioration.In order to select and establish the regeneration and transformation system of maize elite inbred lines,to investigate effects of RNAi in maize starch improvement and to found a new molecular breeding method in maize,we systematically explored the characteristics and parameters of seven maize elite inbred lines which induced by mature and immature embryos as explants,researched inheritable transformation system by Agrobacterium,and constructed one RNAi expression vector of she fib and Agrobactirium tumefaciens-mediated transformation.The integration of targeted gene in transgenic plants was proved by molecular analysis.The main results are summarized as follow:1.A total of seven widely cultivated maize elite inbred lines were researched in regeneration abilities.Primary callus were easily induced in seven maize cultivars,but the frequencies of embryogenesis callus were significantly different.The results showed maize regeneration abilities were highly genotype-dependent.The embryogenesis callus were induced in four maize elite inbred lines,which were 178,R18h,Zong 3 and Gaoyou 116 respectively,and the highest frequencies was 78%in 178.2.By studying on inducing culture such as LS,N6B5,MB,MS,the optimal inducing medium is LS+2,4-D 2.0 mg/L+L-pro 700 mg/L+CH 500 mg/L+3%sucrose.Through a serious of research,the optimal culture and program of inducing,subculture,regeneration and rooting using immature embryos were established,and optimal culture condition with 96%frequencies in regeneration was established in maize elite inbred line 178.3.Using orthogonal design and the protocol of GUS infection,the four factors (fermenting liquor of OD,infection time,inclusion of acetosyringone and co-cultivation time) in Agrobactirium tumefaciens-mediated transformation of immature embryos were optimized,and the factors in optimized transformation edifice were 0.6 of fermenting liquor of OD,30 minutes of infection time,200μmol/L of inclusion of acetosyringone and 2 days of co-cultivation time.The program of screening culture was optimized by three times which the concentration of hygromycin were 3 mg/L,5 mg/L,5 mg/L.Positive transgenic plants of a 1.8%transformation ratio were confirmed by PCR analysis using hyg-specific primers.The Agrobacterium tumefaciens-mediated inheritable transformation system of in maize elite was primarily constructed.4.Double fragment expression vector pCAMBIA 1301+Glu+2F was successfully constructed through the bridge vectors as pCAMBIA 1301+Glu and pCAMBIA 1300+HMW+2F.The vector contains endosperm specific promoter(Glu promoter from wheat glutenin),double fragment expression of the 16 and 20 exon in sheⅡb gene,and hygromycin resistance gene.The constructed interference vector was directly transformed to Agrobacterium tumefaciens EHA105 by protocol of frozen and thawing transformation. The Agrobacterium transformant can be used in corn transformation.5.RNA interference expression vector pCAMBIA 1301+Glu+2F of starch branch enzyme gene was transformed into maize inbred lines using optimal transformation system. Five transgenic plants were survival,and the results of PCR primarily proved integration of the targeted segment into the 178 genome successfully.In sum,integrated studies demonstrated the immature embryos of 178 quality maize inbred -lines highly effective regeneration is been developed by selecting in seven maize inbred -lines,and Agrobactirium tumefaciens-mediated transformation was optimized. RNAi expression vector has been constructed to improve starch quality of maize.The five transgenic plants which were PCR positive were obtained by transformation..These researches provide important means to transformation of maize and improving starch quality by gene engineering.