【作者】 李聪； 张启军； 宗寿余； 孙永华； 赖东； 刘少奎； 吕川根；

【Author】 LI Cong~(1,2),ZHANG Qi-jun~2,ZONG Shou-yu~2,SUN Yong-hua~2,LAI Dong~2,LIU Shao-kui~2,Lü Chuan-gen~2 (1 Agricultural college of Nanjing Agricultural University,Nanjing,210095;2 Institute of Food Crops,Jiangsu Academy of Agricultural Sciences,Jiangsu High Quality Rice R&D Center,Nanjing,210014)

【机构】 南京农业大学农学院； 江苏省农业科学院粮食作物研究所江苏省优质水稻工程技术研究中心；

【摘要】 【目的】将双价抗虫基因(修饰后的Lgc1A(c),sbk和改良的CpTI,sck)导入栽培稻南粳45中,并对其进行分子和抗虫鉴定,明确其抗虫性能,为应用打下基础。【方法】将整合有抗虫基因sbk和scA双T-DNA质粒pCDMARUBA-Hyg转化农杆菌EHA105后,感染南粳45的愈伤组织,经再生获得植株后,通过PCR、RT-PCR、Southern杂交、Bt抗虫基因检测试纸条和人工接虫等方法进行转基因后代植株的筛选。【结果】通过对56个再生植株PCR分析,筛选出去选择标记的且具有双价抗虫基因的阳性单株42株,然后通过Southern杂交分析,发现其中35株为单拷贝插入,6株为2个拷贝,1株为3个拷贝;对单拷贝的单株进行RT-PCR、Bt抗虫基因检测试纸条表明外源基因能正常表达,人工接虫结果也表明转基因植株的抗虫能力大大提高,对螟虫的毒杀效果达97%~100%。【结论】通过转入抗虫基因,大大提高了南粳45的抗虫能力。更多还原

【Abstract】 [Objective]Two insect resistant genes（sbk,modified Cry1A（c） and sck,modified CpTI） were transferred into Oryza sativa L.（Nanjing45）.Molecular and insect resistant identification were carried out to put it into practical use in the future.[Method]Plasmid of pCDMARUBA-Hyg which contained two insect resistant genes（sbk and sck） was transformed into Agrobacterium EHA105,then to infect the callus of Nanjing45.After gained the regeneration plants,a series of methods,such as PCR,RT-PCR,Southern-blotting,Bt insect resistant gene test paper,and feeding rice stem borer by manual,were used to identify and filter excellent individuals. [Result]Through PCR analysis with the primers of sbk and sck gene,42 marker-free positive individuals which contained the target genes were selected from 56 regeneration plants.Results of Southern-blotting indicated that 35 plants had single copy,6 plants had two copies and only one was three copies of exogenous gene.Analysis with RT-PCR and Bt insect resistant gene test paper of single copy insert plants indicated that the two insect resistant genes could normally express in T₄ generation plants.Results of feeding rice stem borer by manual also indicated that the ability of transgenic plants were greatly increased,and the insecticidal effect was 97%to 100% on the rice stem borer.[Conclusion]It greatly increased the insect resistant ability of Nanjing45 after being transferred into two insect resistant genes.更多还原