东方山羊豆GoMIPS双元表达载体的构建及鉴定  

Construction and Indentification of Binary Expression Vector of Galega Orientalis GoMIPS Gene

作　　者：李春艳[1] 刘建宁[1] 高洪文[2] 

机构地区：[1]山西省农业科学院畜牧兽医研究所,山西太原030032 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《家畜生态学报》2013年第1期25-28,共4页Journal of Domestic Animal Ecology

基　　金：山西省农业科学院畜牧兽医研究所基金项目(YGG0853)

摘　　要：从东方山羊豆(Galega.orientalis L.)中克隆出的GoMIPS(肌醇-1-磷酸合成酶)基因序列设计合成1对带有NcoI和SpeI酶切位点的引物,用RT-PCR方法从东方山羊豆幼嫩叶片总RNA中扩增出GoMIPS基因的cDNA序列,经纯化后,克隆至pMD19-T载体上,构建pMD-MIPS的中间载体,测序鉴定。然后用NcoI和SpeI限制性酶对含有目的基因的pMD-MIPS和pCAMBIA1302空载体进行双酶切,通过酶切鉴定和测序分析表明,已成功构建了CaMV35S启动子驱动GFP报告基因的双元植物表达载体pCAMBIA1302-MIPS。A pair of primers containing the restriction sites for NcoI/SpeI were designed according to the cloned GoMIPS （inositol-1 - phosphate synthase） gene sequence of Galega orientalis. The GoMIPS cD- NA was amplified by RT-PCR from the total RNA of the young leaves of the Oriental Galega. After purifi- cation, RT-PCR products were cloned to the pMD19-T vector were dealt to construct the pMD-MIPS in the middle of the carrier DNA sequencing, pMD-MIPS containing the target gene and pCAMBIA1302 emp- ty vector with NcoI / SpeI restriction enzyme digestion,the restriction endonuclease and sequence analysis shows that a dual CaMV35S promoter-d~,iven GFP reporter geneplant expression vector pCAMBIA1302- MIPS has been successfully constructed.

关 键 词：东方山羊豆(Galega.orientalis L.) GoMIPS pMD19-T pCAMBIA1302 

分 类 号：S811.6[农业科学—畜牧学]