Study On Transformation Of BcWRKY Ⅱ Gene To Maize And The Improvement Of Maize Stress-Resistance

Because of its importance as a food, economic and feed crop maize (Zea Mays.L.) is planted all over the world. Maize plays an important role on national economy and people’s lives. In2012, the maize yield of China was208.12million tons that was3.83million tons more than rice and became China’s first big grain crop. Adversity environment, such as drought, salinity and low temperature stress factor are the main limiting factors of maize growth and impact on maize yield seriously. It has become a hotspot that transforming exogenous resistant genes to maize by genetic engineering methods and breeding varieties for stress resistance in further.In this study, some maize inbred lines which are used widely in Heilongjiang province were used for the optimization of maize immature embryo and shoot tip regeneration system. A transcription factors gene,BcWRKY II was transformed into maize inbred lines by infecting embryonic callus with agrobacterium, infecting of maize shoot tip in vitro, infecting of maize shoot tip in Planta and the pollen tube pathway method respectively.The main research results are as follows:1. The optimization of maize immature embryo and shoot tip regeneration system.The optimal regeneration system of maize immature embryo is as follows:the genotype zheng58, inducing medium:salt N6+120mg/L inositol+500mg/L L-pro+500mg/L CH+2mg/L2,4-D+10mg/L AgNO3+30g/L sucrose+6.5g/L agar, pH5.8; Subculture medium medium N6+2mg/L2,4-D+15g/L mannitol. Differentiation medium N6+120mg/L inositol+690mg/L L-pro+30g/sucrose L+6.5g/L Agar+2.0mg/L6-BA, pH5.8.The optimal regeneration system shoot tip is as follows:the genotype k10, inducing medium MS+1.0mg/L2,4-D+2.0mg/L6-BA; multiple shoot clumps differentiation media:MS+0.5mg/L6-BA+1.0mg/L IBA.2. The sensitivity of callus and shoot tip meristem to selective agents were measured:The sensitivity order of callus to selective antibiotics is as follows:Geneticin G418> paromomycin (Par)> kanamycin (Kan)> Neomycin (Neo), and the appropriate selection concentration of G418was15-30mg/L, the concentration of Par was30～50mg/L, the suitable concentration of PPT for selection was3～5mg/L.The sensitivity order of shoot tip meristem was the same as that of callus. The appropriate selection concentration of G418was15～30mg/L, the concentration of Par was30～50mg/L, the suitable concentration of PPT for selection was1～3mg/L. 3. Different genetic transformation methods were optimizedThe optimized factors of agrobacterium-mediated transformation of embryonic callus is as follows:genotype zheng58, agrobacterium concentration OD600=0.8, Acetosyringone concentration200μmol/L, vacuum processing time was5min, an average rate of resistant callus was12.55%;The optimized factors of agrobacterium-mediated transformation of shoot tip in vitro is as follows:genotype K.10,agrobacterium concentration QD600=0.8, Acetosyringone concentration150μmol/L,vacuum processing time was25minutes, the average resistant seedling rate was34.00%;The optimized factors of agrobacterium mediated transformation of shoot tip in planta was as follows:the genotype zheng58, vacuum processing time was20min, OD600=0.9, Acetosyringone concentration was150μmol/L, Silwet-77concentration was0.05%, the average resistant seedling rate was9.84%;the optimized factors of pollen tubepathway method is as follows:genotype Longkang11, transforming time was16h after pollination, and the concentration of DNA was200ng/ul, average resistant seedling rate was4.81%.4^The homozygous transgenic lines with BcWRKYⅡ gene were gainedThe exogenous BcWRKYⅡ gene has been certificated to be integrated into the maize genome by PCR detection, Southern hybridization and RT-PCR detection,Four homozygous lines were gained.5.The transformation of exogenous BcWRKYⅡ gene increased maize drought resistance and salt resistance.It showed that drought resistance of four transformed lines with BcWRKYⅡ gene increased from11%to41%after transformed. The increase of grain number might be the main phenomenon for the increase of drought resistance.It also showed that the transformation of BcWRKYⅡ gene could increase the content of chlorophyll and decrease the damage degree of leaves and the content of MDA,and eventually promoted the biomass of transgenic plants under NaCl stress.