含loxP农杆菌介导Bt基因转化水稻的研究-生物化学与分子生物学专业论文.docxVIP

摘 摘 要 I I 摘 要 水稻是最重要的粮食作物之一。培育抗虫品种是水稻品种选育的重要目标。 近年来，植物转基因技术的发展为培育高效抗虫水稻品种开辟了新的途径。Bt 基因是目前应用最为广泛的高效抗虫基因。 目前多数转化方法都是通过标记基因筛选转化细胞和非转化细胞。标记基因 在提高转化子筛选效率的同时，可能会带来一些潜在的安全性问题。因此建立无 选择标记的转基因体系是转基因研究的重要方向。来自噬菌体 P1 的 Cre/loxP 重 组系统是一种有效的标记删除方法。在这个系统中，Cre 酶可以精确的识别和切 割位于两个 loxP 位点之间的筛选标记基因。 本研究我们使用了能定向切除潮霉素抗性标记基因 loxP 位点识别系统，利 用农杆菌介导法成功地将 bt 抗虫基因导入安徽主栽水稻品种“晚粳 97”中，主 要结果概括如下： 1. 本文研究了水稻组织培养的起始材料和培养条件：幼胚的出愈率明显高于成 熟胚；愈伤组织诱导的 2,4-D 最佳浓度为 2.0 mg·L-1，在此浓度下诱导的愈伤 组织生长能力强；愈伤组织分化前适度的干燥处理有助于提高转化效率。 2. 在十批水稻转化实验中，经 50mg/l 潮霉素浓度筛选，共获得 58 个抗性愈伤 组织，平均转化率为 0.74%。通过继代分化，最终得到了 24 棵转 loxp-hpt-loxp-bt 抗性植株。对潮霉素抗性植株进行 PCR 检测和 Southern 杂交分析，结果显示外 源基因已经整合到水稻基因组中。 关键词：水稻；农杆菌；loxP；转基因；标记剔除；Bt 含 含 loxP 农杆菌介导 Bt 基因转化水稻的研究 I II Abstract Rice is one of the most important crops. Plant transgenic technology is an useful method to breed insect-resistant rice. In recent years transgenic plants with insect- resistant gene have been widely popularized and applied. Bt is the most effective insect-resistant gene. Marker genes are used to screen transgenic cells from non-transformed cells. Though it could improve the efficiency of selection, meanwhile, it may result in potential bio-safety issues. Therefore it is important to establish a marker-free system for transformation. Cre/loxP site-specific DNA excision system from the bacteriophage P1 is an effective method of removing marker gene. In the system, Cre recombinase can specifically recognizes and perform precise recombination and DNA excision between two loxP recognition site flanking the selectable marker gene. In this study, we used hygromycin-resistance gene as selection marker flanked with loxp recognition site (loxp-hpt-loxp-bt). Bt gene as aim gene was introduced into the rice cells of Wanjing 97 mediated by Agrobacterium tumefaciens. Main results as follows: 1.In this paper, different initiative explants and cultural conditions were compared. Callus induction from young embryo was better than that from mature embryo. The proper concentrati