根癌农杆菌介导的‘魏可’葡萄遗传转化体系的优化

Optimizing conditions for Agrobacterium tumefaciens mediated transformation of ‘Wink’ grapevine

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。

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 （npt Ⅱ ）. The results showed that the optimal conditions were： infected 4 min by Agrobacterium, eocultured 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 npt Ⅱ gene were integrated into ＇Wink＇ grape. PCR products were harvested, purified and sequenced, and the results also confirmed that npt Ⅱ 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.