大豆胚尖再生体系的优化及EPSPS基因遗传转化的研究-生物工程专业论文.docx

中文摘要 大豆是中国的五大作物之一，是油料作物和植物性蛋白的重要来源。杂草防 除是大豆栽培中一项费时费力的工作，抗除草剂大豆的种植成功地解决了这一问 题。由于大豆再生体系的再生率低、重复性差、基因型依赖性强，且大豆的遗传 转化方法和受体系统的匹配性较差，因此，大豆转化是植物基因工程领域的难点 之一，限制了利用基因工程手段对大豆进行遗传改良。 本论文以大豆胚尖为外植体，对不同组织培养阶段培养基中添加的激素浓度 进行优化，并对农杆菌介导 EPSPS 基因的转化体系进行优化，优化的结果为： 胚尖不定芽诱导培养基中最适的 6-BA 浓度为 2 mg/L，不定芽伸长培养基中添加 1 mg/L 6-BA 和 0.2 mg/L IBA，不定芽在添加 1 mg/L IBA 的培养基中培养 7 d 后 转入不加任何激素的培养基中生根快且移栽成活率高。黑农 37、吉育 91 和北京 小粒豆是大豆胚尖再生体系比较理想的基因型，且不同大豆基因型对草甘膦的耐 受能力不同，黑农 37 的草甘膦筛选浓度为 12.5 mg/L，吉育 91 的草甘膦筛选浓 度为 15 mg/L。用农杆菌 EHA101 将 EPSPS 基因转入大豆中，在侵染前先将胚尖 在不定芽诱导培养基中预培养 24 h，然后在 O D600 为 0.6～0.8 的农杆菌侵染液中 侵染 1～4 h，在共培养培养基中添加 200 μ mol/L AS 和 6 mg/L 6-BA，25 ℃暗培 养 3 天，该转化体系的抗性苗再生率较高。再生的抗性植株用除草剂涂抹进行检 验，经除草剂涂抹检验为阳性的植株用 PCR 进一步检测证明，有 11 株大豆植株 中整合有 EPSPS 基因。 关键词：大豆；胚尖；再生体系；农杆菌介导；草甘膦 ABSTRACT Soybean is one of the five important crops in China, and also important source of oil crop and vegetable protein. Removing weeds is a laborious and time-consuming work. Planting herbicide-resistant soybean can successfully solve this problem. Soybean transformation is one of the difficulties in the field of plant genetic engineering, because soybean regeneration systems are systems of low rate, poor reproducibility, genotype dependent, and the genetic transformation methods are not well matched with soybean regeneration system. In this paper, soybean embryonic tips were used as explants to investigate the regeneration system. The herbicide resistant gene (EPSPS) was transferred into soybean embryonic tip using the Agrobacterium-mediated transformation, and the transformation system was optimized. The results show that the medium with 2 mg/L 6-BA can well induce adventitious shoots. The results also indicated that 1 mg/L 6-BA and 0.2 mg/L IBA which could improve regeneration rate of the adventitious shoots were the optimal concentrations for adventitious shoots elongated. The shoots rooted quickly when cultured in medium with 1 mg/L IBA for 7 days and then transferred to the medium without any hormones. Heinong37 and Jiyu91 were su