向叶绿体中导入双价抗虫蛋白获得无选择标记的抗虫小桐子

Pests such as Lepidoptera,Homoptera,ticks and mites may cause severe damage of the biodiesel plant Jatropha curcas production. Introducing pest resistance to the jatropha by transgene technology is efficient and ecnomic. (1) Insect will get resistance to a pesticide protein after a long-term exposure. People try to transfer two or more types of insect-resistant genes simultaneously into the plant. It will help to delay the pest insect getting resistance. Another advantage is the complementary roles of different pest-resistant genes will increase the pest-resistant ability of a transgenic plant. Development and utilization of weed origined new pesticide genes is another important way to win the bat with the pest. In this project, we plan to transfer SaPIN2a, which was isolated from the weed Solanum americanum, together with CryIA(c) from Bacillus thuringiensis to jatropha. (2) Usually, pest resistance of the transgenic plant is determined by the expression and accumulation level of the pesticide proteins. High expression and accumulation level will lead high pest resistance. However, over-expression of transgene often cause abnormal growth and agronomic traits lost of transgenic plant. In this project, we plan to fuse chloroplast transit peptide to the pesticide protein CryIA(c) and SaPIN2a. Thus the over expressed fusion protein will be transferred from cytoplasm to chloroplast. By this way, higher accumulation of pesticide proteins in transgenic jatropha plant with less affection of agronomic traits can be obtained. (3) In this project, CRE/loxP site specific recombination system induced by beta-estradiol will be used to excise marker genes. A marker free transgenic plant can provide more convenience to further transgene as well as enhance the biosafety of genomic modification.

鳞翅目、同翅目、节肢类等害虫威胁着能源植物小桐子的生产。创建转基因小桐子抗虫新品种意义重大。1、虫长期接触同一种抗虫蛋白易产生抗性。同时导入不同抗虫基因，既可延缓害虫对其产生抗性，又可通过不同基因间的互补作用提高植物抗虫范围和能力。抗虫策略还包括从野生植物中寻找新抗虫基因。本项目采用来自野生龙葵的龙葵蛋白酶抑制剂SaPIN2a与苏云金杆菌杀虫晶体蛋白CryIA(c)一起转化小桐子。2、转基因植物抗虫性高低取决于抗虫基因表达水平及累积量，但外源基因超量表达往往导致植物表型异常。本项目采用叶绿体转运肽与抗虫蛋白融合，从而将超量表达的CryIA(c)和SaPIN2a定位于叶绿体内，利于在增强转化体抗虫性同时保持其优良农艺性状。3、本项目还将采用β-雌二醇诱导的CRE/loxP定位重组系统切除选择标记基因，在提高生物安全性的同时为今后向该转基因小桐子中引入更多优良性状提供方便。

小桐子（Jatropha curcas）是重要生产生物柴油的能源植物，提高其抗虫性对规模化种植生产意义重大。针对严重危害小桐子额度两种田间害虫斜纹夜蛾（Prodenia litura）和粉蚧（Pseudococcidae），本课题组开展了抗虫基因工程育种研究，利用农杆菌介导转基因技术在小桐子体内表达苏云金杆菌杀虫晶体蛋白CryIA(c)和雪花莲凝集素蛋白GNA，并通过对转基因个体人工自交繁育，获得抗虫性显著增强的纯合株系。此外，课题组利用原有天然药物研究平台和研究过程中田间产生的大量小桐子叶片、枝条、果壳、种皮等废料，进行了开发药用价值和制备灵芝环保培养基的有益探索，取得积极进展。通过本项目研究，发表核心期刊论文3篇，申报国内专利1项，培养研究生2名，参加国内学术会议2次。