草莓NCED基因正反义表达载体和RNAi载体的构建  

Sense,Antisense and RNAi Expression Vectors Construction of NCED Gene of Strawberry

作　　者：朱海生[1] 陈敏氡[1] 李严曼[1] 温庆放[1] 

机构地区：[1]福建省农业科学院作物研究所 福建省农业科学院蔬菜研究中心 福建省蔬菜工程技术研究中心,福建福州350003

出　　处：《热带作物学报》2012年第3期459-466,共8页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.31101534);福建省自然科学基金项目(No.2010J0111);福建省科技重大专项(No.2008NZ0002);福建省属公益类科研院所基本科研专项(No.2010R1028-5);福建省农业科学院科技创新团队重点科研项目(No.CXTD2011-20)

摘　　要：根据草莓(Fragaria ananassa Duch)NCED基因序列(GenBank:HQ399498),克隆NCED基因开放阅读框,将该片段插入植物表达载体pBI 121的CaMV 35S启动子和NOS终止子之间,构建了正义表达载体pBI 121NCED。克隆NCED基因正义、反义片段和作为内含子的gusA基因片段,以植物表达载体pBI 121为基础,以pCAMBIA2301作为中间载体,通过多次酶切和连接,成功构建了草莓NCED基因RNAi表达载体pBI121NCEDRNAi和反义表达载体pBI 121NCEDF。经PCR、限制性内切酶酶切和测序鉴定后,成功将pBI121NCED、pBI 121NCEDF和pBI 121NCEDRNA 3个重组表达质粒导入农杆菌EHA105中。研究结果为进一步研究草莓NCED基因的功能奠定了基础。By using the specific primers designed on the basis of NCED gene sequence of strawberry（GenBank accession number： HQ399498） , open reading frame（ORF）was amplified by RT-PCR. The ORF was inserted between the CaMV 35S promoter and NOS terminator into the expression vector pBI121, and plant sense vectors called pBI121NCED was obtained. The three gene fragments of RNAi structure including the sense and antisense fragments of NCED gene and the fragment of gusA gene which used as intron were cloned by PCR method, Then the RNAi expression vector pBI121NCEDRNAi containing a hairpin structure was constructed based on the vector of pBI121with pCAMBIA2301 as the bridging vector by many times of enzyme digestion and connection. In addition, the antisense expression vector pBI121NCEDF was also constructed. Vectors of pBI121NCED, pBI121NCEDF and pBI121NCEDRNAi were checked by PCR,restriction enzymes analysis and sequencing, and transformed into Agrobactrium tumefaciens EHA 105. The results provide a foundation for further studying the function of NCED gene.

关 键 词：草莓 NCED 表达载体 RNA干扰 

分 类 号：S668.4[农业科学—果树学]