大豆子叶节再生体系的优化及转EPSPS基因的研究-遗传学专业论文.docx

摘 要 利用基因工程技术将抗草甘膦基因导入大豆中具有重要的经济意义，草甘磷 的广泛使用使克隆草甘磷抗性基因、培育抗草甘磷转基因作物成为研究热点。大 豆是公认的难转化的作物之一，其遗传转化体系还不完善，较低的转化效率限制 了大豆功能基因组的研究和转基因大豆新品种研发，所以探索建立一套高效的转 化途径对于大豆遗传转化有着重要的意义。 本研究在优化大豆子叶节再生体系研究中，筛选出了再生能力强的大豆基因 型黑农 37、黑农 35、合丰 35、合丰 25；5 d 苗龄的大豆子叶节再生能力较其他 苗龄好；向发芽培养基中添加 1～2 mg/L 的 6-BA，不定芽诱导效果好；不定芽 诱导培养时加入 1.6 mg/L 6-BA 最有利于不定芽的诱导；生根培养中 IBA 的浓度 为 0.5～1.0 mg/L 时，不定根诱导效果好。通过对合丰 35 和黑农 37 的草甘膦除 草剂抗性实验，确定了合丰 35 和黑农 37 筛选剂草甘膦浓度分别为 5～7 mg/L 和 7～9 mg/L；抑菌剂头孢霉素的有效抑菌浓度为 500 mg/L。 在优化农杆菌介导的转化体系中，确定了光下共培养 3 d 有利于抗性不定芽 的形成；共培养培养基中添加抗氧化剂为 400 mg/L L-cys 和 154.2 mg/L DTT；伸 长培养过程中，合丰 35 和黑农 37 伸长培养基中 ZT 浓度分别为 0.5 mg/L 和 1.0 mg/L。通过草甘膦除草剂的喷洒和涂抹实验，确定了黑农 37 和合丰 35 再生植 株的抗性鉴定浓度。 最后，建立了一个稳定可行的农杆菌介导子叶节遗传转化体系，并通过此遗 传转化体系将 EPSPS 基因导入大豆中，得到经过草甘膦抗性和 PCR 扩增鉴定的 阳性再生植株。目前，已获得 11 株 PCR 阳性及草甘膦抗性再生植株，合丰 35 为 6 株、黑农 37 为 5 株，均已结实。 关键词： 大豆 子叶节 再生 EPSPS 基因 遗传转化 农杆菌介导 ABSTRACT EPSPS gene was transformed into soybean for boosting up herbicide resistant, having important economic significance. Glyphosate has been extensively used for broad-spectrum control of weeds in the word, and much attention has been paid to the search for glyphosate-tolerant enzymes for transgenic crops. Soybean is one of the crops whose transformation is difficult，the low transformation efficiency in soybean is limited to soybean gene function and gene transformation research. So, it is meaningful to establish a high efficiency transformation method. In this study, the EPSPS gene was transformed into soybean by Agrobactrium- mediated genetic transformation system with cotyledonary node as explants in soybean. Some major factors, which affect the regeneration efficiency and transformation efficiency of soybean were assessed. The main conclusion of this experiment was as following: Four varieties of soybean were chosen and the cotyledonary nodes of Hefeng35 and Heinong37 were used to be the target explants. The best seed germination condition is to place soybean seeds on germination medium for five days. In addition, we found it could improv