高粱SbCYP79A1基因表达载体的构建及转化玉米研究  

Construction of Expression Vector of SbCYP79A1 Gene in Sorghum and Study on Transformation of Maize

作　　者：李金红[1,2] 霍岩 付莉[3] 翁倩 陶承光 史振声[2] Li Jinhong;Huo Yan;Fu Li;Weng Qian;Tao Chengguang;Shi Zhensheng(Innovation Center, Liaoning Academy of Agricultrual Science, Shenyang, 110161;Special Corn Institute of Shenyang Agriculture University, Shenyang, 110866;Institute of Food, Jinzhou Medical University, Jinzhou, 121000;Technology Academy of Fine Art, Liaoning Economic Voca- tional Technolgical Institute, Shenyang, 110400)

机构地区：[1]辽宁省农业科学院创新中心,沈阳110161 [2]沈阳农业大学特种玉米研究所,沈阳110866 [3]锦州医科大学食品学院,锦州121000 [4]辽宁经济职业技术学院工艺美术学院,沈阳110400

出　　处：《分子植物育种》2018年第9期2859-2866,共8页Molecular Plant Breeding

基　　金：辽宁省博士后企业项目(137301);国家现代农业产业技术体系(CARS-06-01-06);主要粮食作物分子设计育种项目玉米分子设计育种课题(2016YFD0101803)共同资助

摘　　要：为研究SbCYP79A1基因功能及其转化玉米抗虫能力的研究,本试验根据NCBI公布的SbCYP79A1基因序列设计PCR引物,利用高粱品种BTX623克隆到同源性达到100%的SbCYP79A1基因全长cDNA序列,该序列开放阅读框包含1 887 bp,由629个氨基酸组成。构建SbCYP79A1基因植物超表达载体,PCR及测序结果均显示植物表达载体pMDC141-CYP79A1构建成功。将该表达载体导入农杆菌EHA105中,热激法转化到玉米A188幼胚,共培养7 d后,在幼胚检测到GUS基因瞬时表达,说明该农杆菌介导的玉米转化体系的转化效率具有可靠性;经选择培养获得54株转化植株,经PCR鉴定获得38株阳性植株,同时利用GUS基因对阳性植株的叶片和根部进行组织定位分析,均观测到较强的GUS基因表达,进一步验证了PCR检测结果。以上结果均证明高粱的SbCYP79A1基因已经整合到玉米基因组中,该研究结果为进一步获得玉米抗虫的新材料提供依据。In order to st udy the function of SbCYP79 A1 gene and its ability for the insect resistance of maize, PCR primers were designed according to the SbCYP79 A1 sequence recorded in NCBI. Full-length cDNA sequence of CYP79 A1 was cloned with the homology of 100% from Sorghum bicolor cultivar BTX623,and the open reading frame contained 1 887 bp sequence and encoded 629 amino acids. Plant over-expression vector of SbCYP79 A1 gene was constructed, and PCR and sequencing results both showed that plant expression vector pMDC141-CYP79 A1 was built successfully. This expression vector was transformed into Agrobacterium tumefacience EHA105 first and then into immature-embryo A188 by heat-stock. The transient expression of GUS gene was detected in immature-embryo after co-culture for 7 days, indicating that the conversion efficiency of maize transformation system mediated by Agrobacterium tumefaciens was reliable.54 transformed plants were obtained by selective culture, in which 38 plants were identified as positive plants by PCR. At the same time, the GUS gene was used to locate the leaves and roots of the positive plants, and strong GUS gene expression was observed. The results of PCR detection were further verified. The above results all demonstrated that SbCYP79 A1 gene had been integrated into the maize genome, which could provide reference for the obtain of the new materialsfor insect-resistant maize.

关 键 词：高粱 基因 表达载体 玉米 遗传转化 

分 类 号：S513[农业科学—作物学]