根癌农杆菌介导Barnase基因转化‘美人指’葡萄的研究  

Studies on Agrobacterium-mediated transformation of 'Manicure Finger' grape with Barnase gene

作　　者：杨丽娜[1] 刘捷[1] 庄智敏[1] 章镇[1] 周蓓蓓[1] 陶建敏[1] 

机构地区：[1]南京农业大学园艺学院,江苏南京210095

出　　处：《南京农业大学学报》2009年第1期31-35,共5页Journal of Nanjing Agricultural University

基　　金：江苏省农业资源开发局项目(2007kj-22);江苏省科技厅科技推广项目(BG2007309);江苏省农林厅三项工程项目

摘　　要：以‘美人指’葡萄离体叶片及叶柄为农杆菌介导转化Barnase基因的受体,对农杆菌侵染时间、共培养时间、预培养时间、卡那霉素选择压以及头孢霉素浓度等因素进行了研究。结果表明:最佳的农杆菌介导‘美人指’葡萄遗传转化体系为农杆菌侵染4 min,共培养3 d,预培养3 d,卡那霉素选择压3.0 mg.L-1,头孢霉素质量浓度250 mg.L-1。采用此体系对‘美人指’葡萄进行转化,共获得了12株抗性苗,农杆菌感染后的不定芽再生率为1.78%。利用GUS组织化学染色、PCR扩增的方法进行检测,结果表明,其中只有2株GUS染色呈阳性,阳性率为16.67%;有3株通过了PCR检测,初步证实Barnase基因已经整合进入‘美人指’葡萄基因组中。Transgenic plants were generated from leaves and petiole of ‘ Manicure Finger＇ grape by Agrobacterium-mediated transformation with Barnase gene, and the following factors were investigated ： Agrobacterium dipping time, pre-cuhure time, co-culture time, concentration of Kanamycin （ Km）, and concentration of Cefotaxine （Cef）. The results showed that the optimal conditions were ： infected 4 rain by Agrobacteriam, pre-cuhured 3 d, co-cultured 3 d, Km 3.0 mg·L^-1 and Cef 250 mg·L^-1. Twelve Km-resistant plants of ‘ Manicure Finger＇ grape were obtained by optimized Agrobacterium-mediated transformation and the rate of resistant shoots was 1.78%. GUS assay on these resistant shoots showed that there were two GUS positive plants and the GUS positive rate was 16. 67%. Three plantlets confirmed the conclusion that Barnase gene were integrated into the host genome of ‘ Manicure Finger＇ grape by PCR analysis.

关 键 词：'美人指’葡萄 Barnase基因 GUS组织化学染色 PCR检测 

分 类 号：S663.1[农业科学—果树学]