大豆查尔酮还原酶1基因的克隆及转化  

Cloning and transformation of chalcone reductase 1 gene of soybean

作　　者：李丹[1] 吴楠[1] 郑成忠[1] 张琳[1] 马建[1] 曲静[1] 张卓[1] 付永平[1] 王丕武[1] 

机构地区：[1]吉林农业大学农学院,吉林长春130118

出　　处：《西北农林科技大学学报（自然科学版）》2015年第8期71-78,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金面上项目"大豆查尔酮还原酶(CHR)在大豆苷元合成中的作用机理研究"(31771568)

摘　　要：【目的】克隆大豆查尔酮还原酶1(CHR1)基因,以构建的过表达载体pCAMBIA3301-CHR1转化大豆,获得含有CHR1基因的阳性植株,为研究该基因的功能奠定基础。【方法】以大豆基因组DNA为模板,通过PCR扩增克隆CHR1基因,构建植物过表达载体pCAMBIA3301-CHR1,对其进行PCR及双酶切鉴定。采用农杆菌介导法将该载体转入大豆"吉农28"中,对转基因植株进行PCR、Southern杂交检测,对CHR1基因mRNA相对表达量进行荧光定量PCR检测。【结果】克隆得到的CHR1基因大小为1 100bp。成功构建了植物过表达载体pCAMBIA3301-CHR1,将其转化大豆后,通过PCR检测获得T1代转基因大豆植株12株;Southern杂交检测结果显示,CHR1基因以单拷贝形式整合入大豆基因组中;荧光定量PCR检测结果显示,转基因植株的CHR1mRNA相对表达量高于非转基因大豆植株。【结论】成功构建了过表达载体pCAMBIA3301-CHR1,并获得了CHR1基因表达量高的转基因大豆。[Objective] The chalcone reductasel （CHR1） gene was cloned from soybean,and the over- expression vector pCAMBIA3301-CHR1 was constructed and transformed into soybean to obtain transgenic plants. [Method] The CHR1 gene was cloned by PCR using soybean genomic DNA as template. Then the plant over-expression vector of pCAMBIA3301-CHR1 was constructed and identified by PCR and double digestion before being transformed into soybean variety ＂Jinong 28＂ by Agrobacterium mediation. The transgenic plants were detected by PCR and Southern blotting,and mRNA expression of CHR1 gene was detected by real-time fluorescence quantitative PCR. [Result] The cloned CHR1 gene was 1 100 bp. The plant over-expression vector pCAMBIA3301-CHR1 was constructed successfully and transformed into soybean. A total of 12 T1 generation transgenic plants were obtained by PCR detection. Southern blotting showed that the CHR1 gene was integrated into soybean genome by single copy and real-time fluorescence quantitative PCR showed that mRNA expression of transgenic plants was higher than in non-transgenic plants. [Conclusion] The plant over-expression vector pCAMBIA3301-CHR1 was successfully constructed,and transgenic soybeans with high expression level of CHR1 gene were obtained.

关 键 词：大豆查尔酮还原酶1基因 克隆 表达载体构建 农杆菌介导 

分 类 号：Q78[生物学—分子生物学] S565.103.3[农业科学—作物学]