根癌农杆菌介导马铃薯试管薯转化体系的优化及AtNHX1基因的导入  

Optimization of Transformation Conditions of Potato by Agrobacterium tumefaciens and Introduction of AtNHX1 Gene

作　　者：王丽[1] 杨宏羽[1] 张俊莲[1] 王蒂[1] 

机构地区：[1]甘肃省作物遗传改良与种质创新重点实验室

出　　处：《西北植物学报》2008年第6期1088-1094,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家科技支撑计划(2006BAD21B05);“973”计划(2006CB708203);国家“863”计划;甘肃省农业生物技术(GNSW-2006-01)

摘　　要：以马铃薯栽培品种甘农薯2号的试管薯薄片为转化受体材料,通过根癌农杆菌LBA4404介导拟南芥液泡膜Na+/H+逆向转运蛋白基因(AtNHX1)进行转化,获得了抗卡那霉素的再生植株,并对转化植株进行了PCR-Southern检测.结果表明,薯片分化和根再生的卡那霉素选择压为50mg/L;乙酰丁香酮对薯片转化率无影响;优化的试管薯片转化方法是:不经预培养的薯片用OD600值为0.5的菌液侵染8～10min,然后经过2d共培养,在抗性芽分化培养基上生长40d,可获得30%卡那抗性绿苗.对抗性植株的PCR和PCR-Southern检测证明,外源At-NHX1基因已整合到马铃薯基因组中.By mediation of Agrobacterium tumefaciens LBA4404,AtNHX1 gene was transferred into the microtubers of ‘Gannongshu 2＇ potato, The key factors affecting the transformation were assessed and transgenic plants were generated and further tested using PCR-Southern blot hybridization. The results show that optimal kanamycin concentration for microtuber＇s differentiation and root＇s regeneration was 50 mg/L and acetosyringone had no impact on transformation of the microtubers. The optimized transformation conditions and procedures were as follows：microtubers were firstly immersed into liquid bacterium at OD600 of 0.5 for 8-10 rain prior to pre-culture,followed by a two-day long co-culture before being transferred to and cultured in a differentiation medium for 40 days. A proportion 30% of the transgenic plants was kanamycin resistant. The PCR and PCR-Southern blot hybridization eventually detected the AtNHX1 gene inserted into the potato genome that could be used as raw materials for the development of salt resist- ant potato varieties.

关 键 词：马铃薯 根癌农杆菌 转化 ATNHX1基因 转基因植株 

分 类 号：Q789[生物学—分子生物学]