转新型抗草甘膦基因棉花的转化效率及相关分子特征分析  

Transgenic Efficiency and the Molecular Characteristics of Genetically Modified Cottons with a New Glyphosate-Resistant Gene

作　　者：马燕斌[1] 孙璇[1] 吴霞[1] 林朝阳[2] 刘程毅[2] 王霞[3] 张树伟[1] 王新胜[1] 张林水[1] 沈志成[2] 李燕娥[1] 

机构地区：[1]山西省农业科学院棉花研究所,山西运城044000 [2]浙江大学农业与生物技术学院,浙江杭州310029 [3]运城学院生命科学系,山西运城044000

出　　处：《棉花学报》2016年第4期418-424,共7页Cotton Science

基　　金：国家科技重大专项--转基因生物新品种培育(2014ZX08010-003);国家自然科学基金项目(31201253);山西省自然科学青年基金(2013021024-3);山西省农业科学院博士基金(YBSJJ1204)

摘　　要：培育抗草甘膦除草剂棉花品种对棉花生产具有重要意义。利用农杆菌介导法获得了大量抗除草剂草甘膦棉花的再生植株,对其进行阳性检测。通过确定PCR检测目标条带大小、蛋白杂交条带大小分子特征,对65个独立转化事件在DNA水平、蛋白水平进行分析和筛选。结果表明,经PCR检测再生植株DNA得到的阳性率平均值为81.5%,经过蛋白检测后阳性率确定为55.4%,该类阳性植株即为外源基因正常表达的转基因植株。在此基础上,结合田间抗性检测,对其中9个株系进行了不同世代的选择,经过综合鉴定筛选,发现2个抗性优良的转基因株系具有极高的利用价值,其余还需进一步筛选鉴定。It is important to cultivate transgenic cotton varieties with glyphosate-resistance to enhance cotton production in China.This research aimed to obtain transgenic cotton with a new glyphosate-resistant gene that was initially obtained by Zhejiang University. All of the 65 independent transgenic events of the regeneration cotton plants were obtained through the tissue culture technology using a glyphosate-resistant screen. For these transgenic regeneration plants, improving the transgenic efficiency of the regenerated plant is very important in transgenic research. First, a 1.7-kb fragment of exogenous gene of all 65 regeneration plants was detected respectively through a special primer of G10evo-EPSPS with PCR amplification. The result showed that the rate of positive transgenic plants could reach the high level of 66.7% ― 100%, with the average value of 81.5%. We then used these fragment characters to verify the positive segregating progenies of transgenic cotton with PCR amplification. In addition,protein antibodies were used in the western-blot experiment for selecting the resistant plants, in which the 46-k Da hybridization bands were tested. The statistical result of the test showed that the positive transgenic plants rate reached 55.4%. Based on the positive lines verifiedby PCR and western-blot, the generations of transgenic cotton were selected by spraying glyphosate herbicides onto the test field. On this basis, two transgenic lines with excellent resistance were confirmed, both of which have a high utilization value for the yield and resistance performance of cotton breeding materials in the future.

关 键 词：棉花 抗草甘膦基因 转化效率 阳性率 抗性表现 

分 类 号：S562[农业科学—作物学]