Ramesh S, Nagadhara D, Pasalu I C, Kumari A Padma, Sarma N P, Reddy V D, Rao K V
Centre for Plant Molecular Biology, Osmania University, Hyderabad 500 007, India.
J Biotechnol. 2004 Jul 15;111(2):131-41. doi: 10.1016/j.jbiotec.2004.04.004.
Stem borer resistant transgenic parental lines, involved in hybrid rice, were produced by Agrobacterium-mediated gene transfer method. Two pSB111 super-binary vectors containing modified cry1Ab/cry1Ac genes driven by maize ubiquitin promoter, and herbicide resistance gene bar driven by cauliflower mosaic virus 35S promoter were, used in this study. Embryogenic calli after co-cultivation with Agrobacterium were selected on the medium containing phosphinothricin. Southern blot analyses of primary transformants revealed the stable integration of bar, cry1Ab and cry1Ac coding sequences into the genomes of three parental lines with a predominant single copy integration and without any rearrangement of T-DNA. T1 progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses. Furthermore, the co-segregation of bar and cry genes in T1 progenies suggested that the transgenes are integrated at a single site in the rice genome. In different primary transformants with alien inbuilt resistance, the levels of cry proteins varied between 0.03 and 0.13% of total soluble proteins. These transgenic lines expressing insecticidal proteins afforded substantial resistance against stem borers. This is the first report of its kind dealing with the introduction of Bacillus thuringiensis (Bt) cry genes into the elite parental lines involved in the development of hybrid rice.
通过农杆菌介导的基因转移方法培育出了参与杂交水稻的抗螟虫转基因亲本系。本研究使用了两个pSB111超双元载体，其中包含由玉米泛素启动子驱动的修饰cry1Ab/cry1Ac基因，以及由花椰菜花叶病毒35S启动子驱动的抗除草剂基因bar。与农杆菌共培养后的胚性愈伤组织在含有草丁膦的培养基上进行筛选。对初级转化体的Southern杂交分析表明，bar、cry1Ab和cry1Ac编码序列稳定整合到三个亲本系的基因组中，主要为单拷贝整合，且T-DNA没有任何重排。分子分析证实，T1代子代植株呈现出转基因分离的单基因模式（3:1）。此外，T1代子代中bar和cry基因的共分离表明转基因整合在水稻基因组的单个位点。在具有外源内在抗性的不同初级转化体中，cry蛋白水平在总可溶性蛋白的0.03%至0.13%之间变化。这些表达杀虫蛋白的转基因系对螟虫具有显著抗性。这是首次将苏云金芽孢杆菌（Bt）cry基因导入参与杂交水稻培育的优良亲本系的此类报道。
