黄瓜T-DNA插入突变体库的构建  

Construction of cucumber(Cucumis sativus L. )T-DNA insertion mutant library

作　　者：李蕾[1] 李季[1] 孟永娇[1] 张璐[1] 娄群峰[1] 钱春桃[1] 陈劲枫[1] 

机构地区：[1]南京农业大学作物遗传与种质创新国家重点实验室/园艺学院,江苏南京210095

出　　处：《南京农业大学学报》2016年第1期40-47,共8页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金重点项目(31430075);国家重点基础研究发展计划项目(2012CB113904);国家863计划项目(2012AA100202);国家公益性行业(农业)科研专项(201403032);国家自然基金青年基金项目(31301781)

摘　　要：[目的]为了进一步研究黄瓜基因的功能,本文构建了黄瓜T-DNA插入突变体库。[方法]以黄瓜栽培品种‘长春密刺’子叶节为转化外植体,通过农杆菌介导的遗传转化方法,将T-DNA插入突变载体pROK2后转化黄瓜;通过筛选压力梯度试验,确定遗传转化体系最适合的卡那霉素(Kan)筛选浓度;使用PCR检测和斑点杂交方法鉴定T0和T1代转化植株;以‘长春密刺’植株为对照,统计T1代植株表型。[结果]确定100 mg·L-1为最适合的Kan抗性芽筛选浓度。T0代植株PCR检测结果初步证明,pROK2成功整合到14S1和14S2两个T0代株系基因组中。14S1自交后获得55个T1代单株,对其进行PCR检测,发现其中12个单株均扩增出了35S和NPT-Ⅱ片段。以Dig标记的35S和NPT-Ⅱ为探针,对这12个单株及随机从剩余T1代中选取的11个单株进行斑点杂交检测,得到了2个含有35S和NPT-Ⅱ杂交信号的单株:14S1-27和14S1-34。性状调查统计显示:T1代植株在生长各阶段相对于野生型无明显突变表型。[结论]pROK2重组质粒成功整合到了14S1株系的基因组中。对14S1自交后代的PCR检测和斑点杂交检测结果进一步证明了14S1为转化植株,确定14S1为插入突变体。结合T-DNA插入位点鉴定技术可进一步开展相关基因分离及功能研究工作。[Objectives]For the purpose of further study on the cucumber gene function,this article carried out the research that is the construction of cucumber T-DNA insertion mutant library. [Methods]With the cotyledon nodes of cucumber cultivars‘Changchunmici＇as the explants and the Agrobacterium-mediated transformation method,T-DNA was inserted into vector pROK2 and cucumber was transferred. The most suitable selection pressure concentration of kanamycin was confirmed by setting selection pressure gradient.PCR and dot blotting were used to identify the T0 and T1transgenic plants. The phenotypes of T1 plants were statistically investigated with ‘Changchunmici＇used as control. [Results]The selection pressure concentration of kanamycin was 100 mg·L- 1. PCR detection results of T0 plants indicated that pROK2 was integrated into 14S1 line and 14S2 line successfully. 55 T1 plants were gained from 14S1 self-pollinated and 12 of them could amplify 35 S promoter and NPT-Ⅱ gene sequence. The results of dot blotting detection of the 12 plants and 11 plants randomly selected from the other T1 plants by dig labeled 35 S probe and NPT-Ⅱ probe were that 14S1-27 and 14S1-34 had 35 S and NPT-Ⅱ hybridization signals. Traits investigation showed the phenotype of T1 generation from 14S1 selfpollinated had no difference with‘Changchunmici＇in every period of growth. [Conclusions]pROK2 recombinant plasmid was integrated into 14S1 successfully. Results of PCR and dot blotting detection of 14S1 selfed progeny further proved that 14S1 were transgenic plants that are T-DNA insertion mutant. Then gene isolation and function analysis will be studied with T-DNA insertion site identification methods.

关 键 词：黄瓜 T-DNA 插入突变 转基因 pROK2载体 

分 类 号：S642.2[农业科学—蔬菜学]