摘要
为了提高农杆菌介导的水稻茎尖遗传转化效率,笔者以贵州特有的地方水稻品种‘平塘黑糯’茎尖为材料,以含有Ubiqutin启动子驱动苦瓜几丁质酶基因McCHIT1表达的植物表达载体p Cambia-UbiMcCHIT1为转化载体,采用正交试验设计,系统研究了菌液OD600值、真空处理时间和真空渗透压强对水稻茎尖遗传转化效率的影响。结果表明:在3个因素中,真空处理时间是影响水稻转化效率和死亡率的最关键因素。农杆菌介导‘平塘黑糯’茎尖遗传转化的最优条件为农杆菌菌液浓度OD600为1.0、真空处理时间为7 min、真空渗透压强为15 k Pa。GUS组织化学染色和PCR分子鉴定表明,通过本研究优化的农杆菌介导水稻茎尖遗传转化体系,已将苦瓜的几丁质酶基因McCHIT1已整合到水稻基因组中,获得转McCHIT1基因‘平塘黑糯’新种质。
To improve the efficiency of genetic transformation of rice shoot tips via Agrobacterium-mediatedmethod, a glutinous rice variety＂Pingtang Heinuo＂was used as the research material, the vector p CambiaUbi-McCHIT1 with Ubi promotor driving bitter melon chitinase gene McCHIT1 was used as the transformationvector, we investigated systematically the influence of Agrobacterium concentration, vacuum treatment timeand vacuum osmotic pressure on the efficiency of Agrobacterium-mediated genetic transformation of rice shoottips by orthogonal experimental design method. The results showed that, among the three factors, the vacuumtreatment time was the key factor which influenced the transformation efficiency and death rate. The optimumAgrobacterium concentration was OD600=1.0, the optimum vacuum treatment time was 7 min and the optimumvacuum osmotic pressure was 15 k Pa. The histochemical staining of GUS activity and the identifying of PCR amplification confirmed that McCHIT1 gene was integrated into the rice genome. It demonstrated that we hadcreated a novel transgenic rice germplasm with McCHIT1, and developed a high efficiency genetic transformation system for rice shoot tips by Agrobacterium-mediated method.
出处
《中国农学通报》
2016年第27期114-120,共7页
Chinese Agricultural Science Bulletin
基金
国家转基因新品种培育重大专项"安全转基因技术研究"(2014ZX08010-003)
贵州省重大科技专项"贵州特有水稻种质资源优异基因克隆及育种技术研究应用"(黔科合重大专项字[2012]6005号)
