Position: Home > Articles > Optimizing conditions for Agrobacterium tumefaciens mediated transformation of ‘Wink’ grapevine
Journal of Fruit Science
2012
(3)
343-349+524
根癌农杆菌介导的‘魏可’葡萄遗传转化体系的优化
作 者：
余智莹;张平;徐志胜;孙兴民;章镇;陶建敏
单 位：
南京农业大学园艺学院
关键词：
‘魏可’葡萄;农杆菌介导法;nptⅡ基因;转化体系;分子检测;卡那霉素抗性鉴定
摘 要：
以‘魏可’葡萄离体叶片为外植体,利用植物表达载体上含有卡那霉素抗性基因(nptⅡ)的根癌农杆菌LBA4404(pCAMBIA2301)对影响‘魏可’葡萄遗传转化效率的因素进行系统研究。结果表明,最佳农杆菌介导‘魏可’葡萄遗传转化体系为农杆菌侵染4 min,共培养3 d,卡那霉素质量浓度5 mg·L-1,羧苄青霉素质量浓度200 mg·L-1。采用此体系对‘魏可’葡萄进行转化,共获得9株抗性苗。利用GUS组织化学染色、PCR扩增的方法检测,结果表明,有4株通过了PCR检测,1株通过了RT-PCR检测,初步证明nptⅡ基因已经整合进入‘魏可’葡萄基因组中。将PCR产物回收,经测序验证nptⅡ基因完全整合进入‘魏可’葡萄基因组中。对CK及经PCR检测呈阳性的株系进行卡那霉素抗性鉴定,发现PCR呈阳性的株系均比CK抗Kan的能力明显增强。
译 名：
Optimizing conditions for Agrobacterium tumefaciens mediated transformation of ‘Wink’ grapevine
作 者：
YU Zhi-ying,ZHANG Ping,XU Zhi-sheng,SUN Xing-min,ZHANG Zhen,TAO Jian-min*(College of Horticulture,Nanjing Agricultural University,Nanjing,Jiangsu 210095 China)
关键词：
'Wink' grape;Agrobacterium-mediated transformation;nptⅡ gene;Transformation systems;Molecular detection;Kanamycin-resistant experiment
摘 要：
Several factors affecting the efficiency of Agrobacterium-mediated transformation of 'Wink'(Vitis vinifera L.) were studied using leaves of in vitro plants and Agrobacterium strain LBA4404 containing a vector pCAMBIA2301 with the kanamycin Kan resistance gene(nptⅡ).The results showed that the optimal conditions were: infected 4 min by Agrobacterium,cocultured 3 d,Km 5 mg·L-1 and Cb 200 mg·L-1.Nine Kan resistant plants of 'Wink' grape were obtained by optimized Agrobacterium-mediated transformation.GUS assay on these resistant shoots showed that there were four PCR positive plants and one RT-PCR positive plant.It proved that nptⅡ gene were integrated into 'Wink' grape.PCR products were harvested,purified and sequenced,and the results also confirmed that nptⅡ gene were integrated into the host genome of 'Wink' grape.The result of kanamycin-resistant experiment showed that the ability of Kan-resistance for the PCR positive plants was much better than that of CK.
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