利用Cre/lox重组系统获得无选择标记的SKTI抗虫转基因烟草  

Construction of Selectable Marker-free SKTI Transgenic Tobacco Plants Conferring Resistance Against to Insect by Cre/lox System

作　　者：宋洪元[1] 任雪松[1] 司军[1] 李成琼[1] 宋明[1] 雷建军[2] 

机构地区：[1]重庆市蔬菜学重点实验室,西南大学园艺园林学院,重庆400715 [2]华南农业大学园艺学院,广州510642

出　　处：《西北植物学报》2010年第1期21-29,共9页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(30200185);重庆市教委应用基础项目(030208)

摘　　要：将置于两个同向lox位点之间的Bar基因表达盒与大豆胰蛋白酶抑制剂SKTI基因表达盒融合后获得相应植物表达载体,转化烟草Wisconsin 38后获得对棉铃虫具有明显抗性的SKTI转基因植株。SKTI转基因植株通过叶盘二次转化法导入Cre基因,对再生植株叶盘进行Basta的抗性检测,检测Bar基因的删除情况。结果表明：绝大多数再生植株对应叶盘在含8 mg/L PPT的筛选培养基上无法再生,Bar基因被删除的效率在38%-100%之间。对Bar基因删除区域进行PCR及克隆测序后发现Bar基因表达盒被精确删除。对Bar基因删除植株开花自交获得的分离后代进行NPTⅡ抗性检测,5株NPTⅡ敏感植株分子检测显示均只含有SKTI基因而无Cre基因存在,为无选择标记基因的SKTI转基因植株。A binary expression vector was constructed containing the insecticidal gene,soybean kunitz trypsin inhibitor（SKTI）,and a selectable Bar marker gene cassette,flanked by lox sites.The expression vector was transformed into Wisconsin 38 tobacco plants mediated by Agrobacterium tumefaciens.The transgenic tobacco plants showed well resistance to cotton bollworm.The Cre gene was subsequently introduced into SKTI transgenic tobacco plants by re-transformation.The Bar gene deletion status was detected by test the resistance to Basta of leaf discs from regeneration plants on the medium with PPT 8 mg·L^-1 in it.The results showed few discs were able to regenerate normally on the medium.The excision efficiency of Bar marker gene cassette was 38% to 100%,depending on individual re-transformation events.Farther PCR analyses and sequencing to the recombination region indicated the excision of the Bar gene in highly precised manner.The Bar gene excised plants were self-fertilized to make the SKTI gene segregated from Cre-NPTⅡ locus,5 Kan senstive plants were performed PCR analysis for both the SKTI gene and Cre gene,the results indicated all the 5 Kan senstive plants were marker free SKTI transgenic tobacco plants,by reason of SKTI gene presented but Cre gene absented in them.

关 键 词：Cre/lox重组系统 SKTI基因 无选择标记 抗虫转基因烟草 

分 类 号：Q789[生物学—分子生物学]