农杆菌介导棉花遗传转化及纯合株系的筛选论文论文.docVIP

摘 要 提高棉花自身利用土壤潜在磷的能力意义重大。本研究以 4 个优良的陆地棉品种 为受体，利用农杆菌介导的方法，将含有根特异表达启动子的 PhyA 基因转入胚性愈 伤组织和下胚轴，以期获得转植酸酶基因棉花材料，同时对课题组前期转化获得的转 PhyA 基因 T3 代 3 230 株材料进行 PCR 检测，筛选纯合株系，并进一步测定根系植酸 酶活性。获得主要结果如下： 1 2 3 用农杆菌侵染棉花胚性愈伤后，卡那霉素浓度、胚性愈伤组织生长状态、受体材 料的基因型等因素对目的基因的转化效率影响较大。本研究表明：卡那霉素的筛 选最佳浓度为 75 mg/L，且黄色疏松，处于活跃分裂状态的棉花胚性愈伤，有助 于提高转化效率，筛选出晋棉 14、陕 724 是两个优良的转基因受体材料。 利用优化的条件，以陆地棉品种晋棉 14、陕 724 的胚性愈伤组织为受体，用农杆 菌介导的方法，从农杆菌侵染后经过 6 个月获得了经 PCR 鉴定含 PhyA 基因的转 基因植株。以中棉所 18 的下胚轴为受体，用农杆菌介导的方法获得含 PhyA 基因 的抗性愈伤组织。 对前期转化获得的转 PhyA 基因 T3 代 3 230 株进行 PCR 检测，筛选得到 1 个转基 因纯合株系，进一步测定其植酸酶活性，结果表明，转基因 T3 单株根系分泌植酸 酶活性明显高于未转化的对照单株，是对照的 1.18-5.13 倍。研究还发现，外源基 因大都发生了分离，各株系阳性植株比例明显低于 63：1，不符合孟德尔遗传规 律。 关键词：棉花；农杆菌介导；植酸酶基因；遗传转化；纯合株系 The Genetic Transformation of Cotton via Agrobacterium-mediated and Screening of Homozygous Lines Author: Guo Caiju Major: Crop Genetics and Breeding Supervisor: Prof. Ma Zhiying Prof. Duan Huijun Abstract It is great significant that Improving inorganic phosphorus use efficiency in cotton. This study used the upland cotton Jin mian 14, Shan 724, Zhong 6331 and Zhong mian suo 18 as target materials and transformed the expression vector PhyA gene into the embryogenic callus and hypocotyls of these varieties via Agrobactetium-mediated. The objective of this study is to increase new content and new germplasm for cotton. The aim of the thesis is to find out some homozygous lines by PCR methods. The activity analysis of roots excreted phytase was also be detected. The results were as follows: 1. Effect of kanamycin concentration, the growth status of embryogenic callus and the genotypes of the target material on the transformation efficicency had been assayed. Examines the research to indicate that the card that mildew element’s screening best density should be 75 mg/L. We used embryogenic callus coloured in yellow as target materials. It helps to improve the transformation efficiency. We selected cotton Jin mian 14 and Shan 724 are two excellent receptor transgenic materials. 2. Embryogenic callus o