甘肃红豆草BAN基因克隆与双标记选择表达载体构建  

Cloning of BAN Gene and Construction of Double Marking Expression Vector in O. viciaefolia Scop. cv. Gansu

作　　者：陈春艳[1,2] 马晖玲[1] 董文科[1] 杨江伟[3] 李玉珠[1] 姜寒玉[3] 冯玉兰[4] 

机构地区：[1]甘肃农业大学草业学院/草业生态系统教育部重点实验室/中-美草地畜牧业可持续发展研究中心,甘肃兰州730070 [2]河南科技大学农学院,河南洛阳471003 [3]甘肃农业大学生命科学院,甘肃兰州730070 [4]西北民族大学生命科学与工程学院,甘肃兰州730030

出　　处：《核农学报》2016年第10期1880-1888,共9页Journal of Nuclear Agricultural Sciences

基　　金：甘肃省科技厅科技支撑计划项目(1104NKCA087);国家自然科学基金项目(31502012)

摘　　要：为探讨编码花青素还原酶BAN基因的功能及其与缩合单宁合成的关系,根据BAN基因序列设计特异引物,采用RT-PCR技术获得甘肃红豆草BAN的相似基因;利用生物信息学相关软件分析,预测其c DNA序列编码的蛋白质结构和功能;采用Real-time PCR法检测该基因在甘肃红豆草各组织中的表达;构建含有BAN基因的双标记选择载体。结果表明,克隆的BAN基因与报道的BAN基因序列相似性达到99.41%,其ORF长为1 020 bp,可编码339个氨基酸残基,具有花青素还原酶保守结构域和重要功能位点;三级结构预测显示,预测模型与花青素还原酶单体结构(C2RH8A)相似,属于短链脱氢/还原酶超家族成员,表明其可能具有花青素还原酶的功能。BAN基因在各组织中均表达,其表达量依次为荚果、叶、蕾、花、茎。以此为靶序列,构建携带抗除草剂bar和绿色荧光蛋白GFP基因的双标记选择植物表达载体,将为缩合单宁合成、代谢的进一步研究及抗除草剂和抗臌胀病牧草新品种的培育奠定基础。To investigate the function of BAN gene coding anthocyanin reductase and the relationship between BAN gene and the biosynthesis of condensed tannins. Similar c DNA sequence to the BAN was obtained from the total RNA of Sainfoin（ O. viciaefolia Scop. cv. Gansu） by reverse transcription polymerase chain reaction（ RT-PCR） using specific primers designed according to the conserved sequence of BAN gene. The protein function and structure encoded by the similar BAN gene c DNA sequence was predicted and analyzed by related software of bioinformatics technology. BAN gene expression was determined in the tested tissues in O. viciaefolia Scop. cv. Gansu by Real-time PCR. The doublemarker expression vector of BAN gene was constructed. The results showed that,the gene sequence shared a high level of similarity with the BAN gene in Gen Bank（ accession No： HM152980） and its homology was 99. 41%. The open reading frame（ ORF） was 1 020 bp in length,which encoded 339 amino acids containing anthocyanin reductase conserved domain and many important functional sites. The 3D structure of predicted protein showed that it was highly similar to the structure of the monomer of anthocyanin reductase. It was inferred that the protein encoded by BAN should be a member of Short-Chain Dehydrogenase / Reductase family and possibly have function of anthocyanin reductase.Real-time PCR indicated that BAN gene expressed in legume,leaf,bud,flower and stem with the expression level decreased in turn. Based on the c DNA sequence,the double-marker expressing vector of BAN gene with herbicide resistance bar gene and green fluoresce protein gene（ GFP） was constructed. The research will provide the fundamental information and method for studying the synthesis and metabolism of condensed tannins and further cultivation of transgenic alfalfa varieties with resistance to herbicide and bloat disease.

关 键 词：甘肃红豆草 基因克隆 表达分析 载体构建 

分 类 号：S541.4[农业科学—作物学]