农杆菌敏感小麦基因型的筛选及其转化(英文)  

Screening of Wheat Genotypes Sensitive to Agrobacterium tumefaciens Infection and Transformation

作　　者：叶兴国[1] 王艳丽[1] 康乐[1] 杜丽璞[1] 徐惠君[1] 

机构地区：[1]中国农业科学院作物科学研究所

出　　处：《作物学报》2005年第12期1552-1556,T0001,共6页Acta Agronomica Sinica

基　　金：SupportedbytheNationalSpecialProgramforResearchandIndustrializationofTransgenicPlants(JY03B22)andNationalBasicResearchProgram(2002CB1130).

摘　　要：利用C58c1农杆菌菌株(其携带的pPTN290质粒载体上含有nptⅡ基因和GUS基因)为供体材料和小麦幼胚为受体材料,对我国35个春小麦基因型进行了农杆菌敏感性研究和转化研究。组织化学染色结果表明,PM97034、P187、巴140105、扬麦158、新春9号和克丰6号等基因型农杆菌感染后GUS,基因瞬时表达率达到了15.0%～30.0%,显著高于模式基因型Bobwhite。其余幼胚分别在含10～25mg/LG418培养基上诱导愈伤组织和分化植株,抗性再生植株经ELISA检测、PCR检测、叶片离体退绿检测、XGluc染色检测和Southernblot检测,最终从新春9号、PM97034和Bobwhite获得了转基因植株,转化效率分别为3.3%、2.0%和1.1%,新春9号和PM97034的农杆菌转化效率高于Bobwhite。Southernblot检测结果同时表明,外源基因在多数转基因植株中表现为单拷贝整合。T1代植株ELISA检测结果和PCR检测结果表明,外源基因在个别株系中的分离符合孟德尔遗传规律,而在多数株系中的分离偏离了孟德尔遗传规律。阳性植株与阴性植株分离比经χ2c适合性检测,表明外源基因在多数株系中的遗传符合3∶1分离规律。本文首次对不同小麦基因型进行了农杆菌敏感性和转化效率研究,筛选到新春9号和PM97034两个对农杆菌比较敏感且转化效率比较高的基因型,可望广泛用于小麦转基因研究。Thirty-five Chinese spring wheat genotypes were tested for their sensitivity to C58cl Agrobacterium strain＇ s （harboring a binary vector pPTN290 with npt Ⅱ gene and GUS gene） infection and final transformation. Six cultivars including PM97034, P187, Ba1401051, Yangmai 158, Xinchun 9 and Kefeng 6 were proved to be more sensitive than Bobwhite to the Agro-infection according to X-Gluc staining, with GUS gene transient expression frequency of 15.0% to 30.0%. Regeneration plants obtained from selection medium were identified by ELISA, PCR, leaf bleach and X-Gluc, and stable transformation events were obtained from Xinchun 9, PM97034 and Bobwhite by 3.3 %, 2.0% and 1.1%, respectively. Southern blot analysis showed the alien genes integrated in most transgenic plants just by single copy. The alien genes in most T1 transgenic lines segregated by Mendelian regulation, but the segregation ratio in a few T1 transgenic lines was a little bit away from the expecting value, according to Xc^2 test. Xinchun 9 and PM97034 are suggested to be used in wheat Agrobacterium mediated transformation to replace Bobwhite for better result.

关 键 词：小麦 农杆菌转化 基因型筛选 瞬时表达 分子检测 

分 类 号：S512.1[农业科学—作物学]