KTI基因转化花椰的分析.docx

摘要摘要 摘要 摘要 花椰菜(Brassica oleracea vat．botryis)是我国常见的蔬菜之一，然而虫害的发 生造成花椰菜品质下降和大幅度减产，已成为制约花椰菜生产的重要因素之 一。利用基因工程技术培育抗虫农作物品种，已成为当今植物虫害防治的重要 手段。一些重要抗虫基因已经成功分离并应用于基因工程，为培育植物抗虫品 种开辟了一条安全有效的新途径，具有重要的应用价值和巨大的市场潜力。 本研究将杨树来源的抗虫基因Kunitz型丝氨酸胰蛋白酶抑制剂(．Im)基因 连入具有人工合成的GlO．90启动子的无抗生素抗性筛选标记的表达载体pX6 中，构建了双元表达载体pX6．KTI，具有在植物中表达的新霉素磷酸转移酶基因 NPTII以及cre基因，可在甾二醇诱导下去除朋叮Ⅱ抗性标记，实现marker free。 以花椰菜云山品种为供试材料，在原有报道的基础上，建立和优化了花椰菜再 生、遗传转化体系，通过根癌农杆菌介导法将目的基因KTI成功转入受试品种， 以获得高抗虫性的转基因植株。获得子叶柄分化培养基MS+6．BA 1 mg／L+NAA 0．025 mg／L+Agar 0．7％，下胚轴分化培养基MS+6．BA 1 mg／L+NAA 0．1 m朗H Agar 0．7％，生根培养基1／2 MS+Agar 0．5％；最优的转化条件为预培养2d，菌液 稀释后浓度为OD600值0．2一O．3，浸染30s，共培养2d。在脱菌和筛选阶段添加 Cef200 mg／L，分化阶段的Kan筛选浓度为25 mg／1．,，生根阶段的筛选浓度为15 mg／L。通过Kan初步筛选得到23株抗性苗，其中有lO株移栽成活。4株经PCR 检测呈阳性，2株经Southern blotting检测证实KTI基因成功整合到受体基因组 中；其中1株经RT-PCR检测证实目的基因已成功表达。 关键词：花椰菜；抗虫；KTI基因；遗传转化 AbstractAbstract Abstract Abstract Cauliflower(Brassica oleracea vaL Botryis)is one ofthe ordinary vegetables in China． However in recent years pest outbreak area has increased year by year,which results in a significant decline in quality and production．Using genetic engineering tO cultivate new crops resistant tO pest more effective than traditional methods which make an important mean of pest control for plants．Some important inst-resistant genes have been isolated and succcssfu!ly applied to genetic enginc燃ing and breeding,which opened a new,safe and effective new way to enrich germplasm resources and has important great market potential．In this study,a weak inst-resistant auliflower strain Yunshan was used as the test material to devdop a inst-resistant cultivar．Using Agrobactcdum-mcdiated method,a highly-effective and stable genetic transformation system was established．The ftmctional gene KT／is cloned from Populus trichocarpa． was insert into the vector px6，and a hi．gh efficient expression vector pX6·KTI is obtained．The acccptor system cauliflower regeneration system and related gene transformation appear in some literature．The research optimizes the leaf regenerat