高羊茅黔草1号SAMDC基因的克隆及植物表达载体的构建  

Cloning of SAMDC gene from Festuca arundinacea Qiancao No.1 and construction of its plant expression vector

作　　者：曾庆飞 杨春燕 李小冬 吴佳海 王小利 

机构地区：[1]贵州省草业研究所,贵州贵阳550006 [2]贵州阳光草业科技有限责任公司,贵州贵阳550006

出　　处：《草业科学》2015年第8期1237-1242,共6页Pratacultural Science

基　　金：贵州省农业科技攻关项目(黔科合NY[2013]3060号);贵州省农业科学院博士科研启动基金(2013-06);贵州省重大科技专项基金[(2014)6017号]

摘　　要：采用高保真平末端法从高羊茅"黔草1号"(Festuca arundinacea cv.Qiancao No.1)叶片c DNA中扩增S-腺苷甲硫氨酸脱羧酶基因(SAMDC)序列。基因测序分析表明,扩增片段含有1 835个核苷酸,包含有9 bp的微型上游阅读框、147 bp的小型上游阅读框和1 185 bp的主阅读框。微型上游阅读框和小型上游阅读框存在一个碱基重叠,主阅读框编码395个氨基酸。整个克隆片段与Gen Bank上注册的高羊茅Fa SAMDC基因的核苷酸同源性为95.05%,编码氨基酸的同源性为98.22%。利用Kpn I和Pst I酶切位点将该片段插入到经贵州省草业研究所分子生物学实验室改造过的p CAMBIA1300植物表达载体的多克隆位点内,经琼脂糖凝胶电泳检测、酶切鉴定和序列测定,获得具有SAMDC基因和潮霉素抗性基因的适合于单子叶植物遗传转化的表达载体p CAM-SAMDC。通过冻融法将构建的重组质粒导入根癌农杆菌GV3101和EHA105感受态细胞中,并通过PCR反应对所获得的工程菌株进行鉴定,为进一步通过农杆菌介导法培养抗逆禾草新材料奠定了基础。SAMDC gene was amplified from c DNA of Festuca arundinacea Qiancao No. 1 leaves and DNA sequencing analysis indicated that the amplified fragment contained 1 835 nucleotides,which involved a tiny upstream open reading frame（ ORF） of 9 bp,a small upstream ORF of 147 bp and a main ORF of 1 185 bp encoding 395 amino acids. There was one overlap base between the tiny upstream ORF and the small upstream ORF. The homology between the cloned fragment and the Fa SAMDC gene of Festuca arundinacea registered in Gen Bank was 95. 05%and the homology of their encoded amino acid was 98. 22%. This cloned fragment was inserted into modified plant expression vector p CAMBIA1300 with multiple cloning sites using the Kpn I and PstI enzyme. The plant expression vector p CAM-SAMDC with genes of SAMDC and hygromycin resistance was obtained by agarose gel detection,restriction enzyme digestion and sequencing which was suitable for monocotyledons genetic transformation. The recombinant plasmid was transmitted into Agrobacterium tumefaciens component cells GV3101 and EHA105 and the positive clone was selected by PCR. These works laid a foundation for further stress tolerance Poaceae grass via Agrobacterium-mediated method.

关 键 词：SAMDC 基因克隆 过表达载体 农杆菌导入 

分 类 号：Q943.2[生物学—植物学]