741杨双Bt基因的遗传转化及转基因株系的抗虫性  

Transformation of 741 Poplar with Double Bt Genes and the Insect-Resistance of the Transgenic Plant

作　　者：王桂英[1,2] 杨敏生[1] 霍雪梅[1] 王艳平[1] 李珊珊[1] 

机构地区：[1]河北农业大学林学院林业生物实验室保定071001 [2]廊坊市农林科学院廊坊065000

出　　处：《林业科学》2012年第9期42-49,共8页Scientia Silvae Sinicae

基　　金：林业公益性行业科研专项经费重大项目(201004004);国家高技术研究发展计划“863”计划(2011AA100201)

摘　　要：以含有Cry3Aa基因的重组质粒pBCC3为基础,利用PCR和DNA重组技术,从pBCC3中克隆出抗虫基因Cry3Aa,将其正向插入载体pCAMBIA1305的CaMV35S启动子和NOS终止子之间,构建成pCAMBIA1305-Cry3Aa植物表达载体,并导入根癌农杆菌EHA105。采用农杆菌介导的遗传转化法,将Cry3Aa基因转入已转Cry1Ac+API基因的741毛白杨无性系pB29中,获得转双Bt基因的741杨。在含潮霉素的培养基中进行多次继代筛选,获得抗性稳定的无性系9个,编号为pCCA1—pCCA9。采用特异引物分别对转基因植株进行PCR检测,结果显示Cry1Ac基因稳定存在于pB29中,Cry3Aa基因已整合到各无性系的基因组DNA中。ELISA毒蛋白检测,转基因株系都有Cry1Ac和Cry3Aa杀虫蛋白表达。用转基因植株叶片进行柳蓝叶甲(鞘翅目)和美国白蛾(鳞翅目)室内饲虫试验结果表明,转基因植株具有双抗性。根据对测试昆虫的致死率划分高中低3个抗性水平,其中pCCA2,pCCA5,pCCA6,pCCA9具有双高抗;pCCA3,pCCA4和pCCA7对柳蓝叶甲表现出中、低抗性,对美国白蛾则高抗;而pCCA1表现对美国白蛾的极低抗性,对柳蓝叶甲则高抗。In this study, plasmid pBCC3 with Cry3Aa gene was used. A Cry3Aa gene was cloned and inserted into the middle of CaMV35S promoter and NOS terminator of pCAMBIA1305 by using PCR and DNA recombination technology, and inserted into the pCAMBIA1305-Cry3Aa plant expression vector. This vector was introduced into Agrobacterium tumefaciens EHA105. Through Agrobacterium-mediated transformation, the target Cry3Aa gene was transferred into the genome of transgenic poplar pB29 （hybrid 741 line expressing CryIAc＋API gene）. Nine regenerated lines with hygromycin resistance were obtained and named pCCA1-pCCA9. The presence and expression of Cry1Ac gene and Cry3Aa gene have been verified using PCR and ELISA analysis. Toxicity evaluation on Plagiodera versicolora （Coleoptera） and Hyphantria cunea （Lepidoptera） by feeding fresh detached leaves showed double resistance. The deleterious ability was classified into three groups with high, medium and low level according to mortality. Toxicity of pCCA2, pCCA5, pCCA6 and pCCA9 showed high level to both P. versicolora and H. cunea; pCCA3, pCCA4 and pCCA7 showed medium or low toxicity to P. versicolora,but high to H. cunea; while pCCA1 showed extremely low toxicity to H. cunea, but high to P. versicolora.

关 键 词：载体构建 CRY1AC基因 Cry3Aa基因 双Bt741杨 抗虫性 

分 类 号：S718.46[农业科学—林学] S722.36