农杆菌介导bar基因转化水稻胚性愈伤组织的研究  

Transformation of the bar gene into embryogenic calli of rice via Agrobacterium tumefaciens

作　　者：何少海[1] 姚盛存 丰宇凯 汪轲[1] 李飞飞[1] 

机构地区：[1]浙江农林大学农业与食品科学学院浙江省农产品品质改良技术研究重点实验室,浙江临安311300

出　　处：《浙江农林大学学报》2017年第1期129-136,共8页Journal of Zhejiang A&F University

基　　金：国家自然科学基金青年基金资助项目(31401461);浙江省大学生科技创新活动计划(新苗人才计划)项目(2015R412002);2015年浙江农林大学农学院大学生创新训练计划项目

摘　　要：为建立以草丁膦为选择标记的农杆菌Agrobacterium tumefaciens介导的水稻Oryza sativa胚性愈伤组织转化体系,通过农杆菌介导法将bar基因导入了‘日本晴’Oryza sativa‘Nipponbare’‘秀水134’‘Xiushui 134’和‘中花11’‘Zhonghua 11’等3个水稻品种的胚性愈伤组织。在分别以选择压15.0,10.0和10.0 mg·L^(-1)的草丁膦质量浓度进行了3次选择后,聚合酶链式反应(polymerase chain reaction,PCR)发现‘日本晴’‘秀水134’和‘中花11’的愈伤组织转化效率分别是77.9%,67.5%和92.2%,获得的抗性愈伤组织可以进一步分化成苗。它们的分化率依次是76.1%,44.4%和86.7%;成苗率分别为65.6%,66.7%和30.7%。在以草丁膦作为筛选标记时,首选‘日本晴’作为转化受体进行遗传转化。该体系为以抗除草剂基因作为筛选标记的水稻遗传转化和基因功能验证提供了技术基础,获得的抗草丁膦转化植株为水稻育种提供了材料。To establish the embryogenic calli （EC） transformation system of rice with Basta as a selectable a-gent, the glufosinate-resistant gene （bar gene） was transformed into EC of rice cultivars, ‘ Nipponbare’ , ‘ Xiushui 134’ and ‘ Zhonghua 11’ by Agrobacterium-mediated transformation. After 3 cycles of glufosi-nate-resistant screening with 15, 10 and 10 mg.L^-1 glufosinate, transgenic EC was detected by PCR. The trans-formation efficiency of ‘ Nipponbare’ , ‘ Xiushui 134’ and ‘ Zhonghua 11’ was 77.9%, 67.5% and 92.2% respectively. The results showed that the selectable concentrations were effective for resistant EC screening and the resistant EC subsequently differentiated into seedlings. The frequency of EC differentiation of ‘ Zhonghua 11 ’ was 86.7% and the seedling rate was 30.7%; the frequency of EC differentiation of ‘ Xiushui 134’ was 44.4%, and the seedling rate was 66.7%; the frequency of EC differentiation of ‘Nipponbare ’ was 76.1%; and the seedling rate was 65.6%. Therefore, of the 3 cultivars, the EC of ‘ Nipponbare’ for genetic transformation with glufosinate as a selectable agent was most efficient. The glufosinate selection transformation system not only provides a technical basis for rice genetic transformation and functional validation, but also provides transgenic materials with herbicide resistance for rice breeding. [Ch, 5 fig. 3 tab. 32 ref.]

关 键 词：分子生物学 草丁膦 BAR基因 胚性愈伤组织 粳稻 农杆菌介导法转化 

分 类 号：S511[农业科学—作物学] Q785[生物学—分子生物学]