利用Cre/lox重组系统获得烟草TA29-Barnase基因工程雄性不育恢复系  

Construct the Restoring Line of Tobacco Engineered Male Sterile Line Induced by TA29-Barnase GeneUsing Cre/lox Site-specific Recombination 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：《农业生物技术学报》2010年第3期468-475,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(30200185);重庆市科委应用基础项目(渝科计字200217)共同资助

摘　　要：结合F1杂种的生产原理,利用Cre/lox重组系统,建立了一种完全有别于Barnase/Barstar恢复途径的基因工程雄性不育恢复系。将TA29-Barnase雄性不育基因表达盒置于两个同向lox位点之间并与Bar基因融合后获得植物表达载体pBinBarloxTABn,通过农杆菌介导的叶盘转化法将其转入Wisconsin38烟草获得了雄性不育转基因烟草植株。雄性不育转基因植株表现花药瘦小,不饱满,花粉量明显偏少,花粉畸形、皱缩、塌陷、极少萌发,自交无法正常座果结子。TA29-Barnase雄性不育转基因植株用来自pBinCre转基因植株的花粉授粉后则能正常结果结子。分别从pBinCre转基因植株C1与转基因雄性不育植株BN1、BN6杂交后代中筛选到23株、22株Kan和PPT双抗性植株,分子检测结果显示两个杂交组合后代中凡同时含Cre基因和Bar基因的F1植株,其TA29-Barnase雄性不育基因均被删除,删除效率达到100%。F1植株中的TA29-Barnase基因被删除后,育性被恢复,能正常开花结果。According to the principle of F1 hybrid production, a new approach to restore the fertility of engineered male sterility induced by Barnase gene was constructed using the Cre/lox site-specific recombination system, which was distinct from the Barnase/Barstar restoring approach. In this study, the TA29-Barnase male sterile gene expression cassette that flanked by two directly oriented lox sites and the Bar gene expression cassette were assembled into pBinBarloxTABn plant expression vector. The vector was transformed into Wisconsin 38 tobacco genome to produce male sterile plants by Agrobacterium tumefaciens-mediated leave disc transformation method. The male sterile plants appeared anther thinness, unfull and poor pollen produced. The pollen of male sterile plants was unnormal, crimpy, and lost the germination ability, and no normally expanded fruits and seeds formation were observed after self-pollinated. However, there were the normally fruits and seeds appeared on the TA29-Barnasemale sterile plants after pollinated using pollens from pBinCre transgenic plant. Total 23 F1 progenies from the cross combination between pBinCre transgenic plant C1 and male sterile transgenic plant BN1, 22 F1 progenies from cross combination C1 and BN6 with resistance to kanamycin and PPT were screened out respectively, the PCR analysis results indicated the TA29-Barnase gene expression cassette in the genome of those progenies con tained both Cre and Bar genes had been excised efficiently, the deletion rate reached 100% for both the BN1×C1 and BN6×C1 cross combinations. Those progenies that TA29-Barnase gene deleted could flower and fruit normally, indicated the male sterility had been restored.

关 键 词：TA29-Barnase雄性不育基因 Cre/lox重组系统 雄性不育 育性恢复 烟草 

分 类 号：S635.036[农业科学—蔬菜学]