农杆菌介导热激转录因子8基因转化大豆  

Agrobacterium-Mediated Transformation of hsf8 into Soybean

作　　者：唐晓飞[1] 刘丽君[1] 高明杰[1] 杨喆[1] 蒲国峰[1] 张雷[1] 魏崃[2] 

机构地区：[1]黑龙江省农业科学院大豆研究所,哈尔滨150086 [2]东北农业大学,啥尔滨150030

出　　处：《分子植物育种》2009年第3期444-450,共7页Molecular Plant Breeding

基　　金：国家863计划资助项目(2006AA1021F9);国家转基因重大专项(2008ZX08004-002)共同资助

摘　　要：环境胁迫严重影响作物的生长和发育,热激转录因子(heat shock factor 8,HSF)是一类在热应激反应中起重要作用的蛋白质,主要功能是在热休克基因的表达过程中与相应热激元件结合,启动基因的转录过程,最终促进热休克蛋白(HSP)基因的表达。本文将热激转录因子8(hsf8)基因构建在植物表达载体pCAMBIA3300中,以大豆子叶节作为受体,通过农杆菌介导法将hsf8基因导入品质性状优良的两个高产大豆新品系哈交5337和哈交5489中,最后获得转基因植株。在开展大豆遗传转化过程中,探讨了农杆菌介导大豆子叶节转化体系的影响因素,优化了转化条件。在共培养后,采用延迟筛选的方法来提高选择效率。并确定草胺磷筛选浓度为3.5mg/L。获得转化质粒pCAMBIA3300-HSF8的转基因植株,其中T1代PCR阳性植株17株。采用Real-timePCR的方法对T1代抗性植株进行hsf8基因的转录水平的相对定量分析,确定9株较非转基因植株哈交5337表达量明显提高。有1株明显低于哈交5489的hsf8基因表达量。Abiotic stresses, such as drought, salinity, extreme temperatures, chemical toxicity and oxidative stress are serious threats to agriculture and the natural status of the environment. Heat-shock transcription factors （HSFs） are important in regulating heat stress response, which mediate expression of heat shock protein （HSP）. This research used genetic engineering techniques to insert hsf8 gene to dicotyledon expression vector pCAMBIA3300 that contains the selection marker gene bar. And pCAMBIA3300-HSF8 has been transfered into new soybean lines Hajiao5337 and Hajiao5489 by Agrobacterium tumefaciens. In the process of soybean genetic transformation, we researched the impact of factors on the cotyledons of soybean Agrobocterium-mediated transformation, optimized the conversion conditions. We discussed the PGR concentrations, After the co-culture cultivated, delayed selection was used to improve the efficiency of selection. The optimal concentration of glufosinate-ammonium was 3.5 mg/L in selection culture medium. Obtained the T1 transgenic plant of Hajiao5337 and Hajiao5489 with pCAMBIA3300-HSF8 by Agrobacterium-mediated technology, in which 17 plants were confirmed as transgenic plants by PCR. Real-time PCR was employed to investigate the transcription level of hsf8 gene in T1 transgenic plants with glufosinate-ammonium resistance, and 9 of them indicated a much higher expression level than that of the control Hajiao5337. Only one transgenic soybean, which expression was lower than Hajiao5489.

关 键 词：大豆 农杆菌转化 热激转录因子8基因 耐热性 

分 类 号：S565.1[农业科学—作物学]