拟南芥WUS基因的克隆及植物表达载体的构建与鉴定  

Cloning of Arabidopsis WUS Gene, Construction and Identification of Plant Expression Vectors

作　　者：毕政鸿[1,2] 李哲[2] 

机构地区：[1]海南大学农学院,海南儋州571737 [2]中国热带农业科学院橡胶研究所/农业部橡胶树生物学重点开放实验室,海南儋州571737

出　　处：《中国农学通报》2013年第18期119-126,共8页Chinese Agricultural Science Bulletin

基　　金：科技部国际科技合作项目"橡胶树易碎愈伤组织长期培养植株再生和超低温保存研究"(2008DFA32020);农业部"948"项目"橡胶树易碎胚性愈伤组织长期继代培养;过表达Lec1基因促进胚状体发生和植株再生"(2010-S7);国家自然科学基金项目"橡胶树胚性细胞悬浮培养及其植株再生的研究"(30760128)

摘　　要：为了研究AtWUS基因在橡胶树胚性易碎愈伤组织中的表达对体细胞胚发生的生物学作用,用构建的根癌农杆菌EHA105(携带pCAMBIA2301-35S-AtWUS)对橡胶树胚性易碎愈伤组织进行转化。根据已报道的AtWUS序列设计特异引物,进行RT-PCR和序列分析,同时用重叠延伸PCR技术(gene splicing by overlap extension PCR,简称)构建AtWUS-EGFP融合基因。结果表明:与GenBank上EGFP基因和拟南芥AtWUS序列同源性为100%,分别构建了pCAMBIA2301-35S-AtWUS(含有GUS和NPTII基因)和pER8-AtWUS-EGFP(含有HPTII基因)载体,采用电击转化法转进根癌农杆菌EHA105中。最后,通过GUS检测法鉴定了pCAMBIA2301-35S-AtWUS已经整合到橡胶树中。本研究分别成功构建了组成型植物表达载体pCAMBIA2301-35S-AtWUS和诱导型植物表达载体pER8-AtWUS-EGFP,为橡胶树遗传转化的研究奠定了基础。In order to study the effect of expression of AtWUS in embryonic friable calli on embryogenesis, calli were co-cultivated with the A. turaefaciens strain EHA105 harboring a binary vector pCAMBIA2301-35S-AtWUS. According to the reported AtlVUS gene sequence, a pair of special primers was designed, being carried out RT- PCR and sequence analysis. At the same time, SOE PCR （gene splicing by overlap extension PCR） was used to form fusion gene AtWUS-EGFP. The results suggested that two fragments cloned had a homology of 100% with AtWUS and EGFP gene respectively logged on GenBank. The pCAMBIA2301-35S-AtWUS with uidA and nptH genes and pER8-AtWUS-EGFP containing hptⅡ gene were constructed. At last, GUS detection indicated that pCAMBIA2301-35S-AtWUS had been inserted into genome of rubber tree. In a word, successful construction of constitutive plant expression vector pCAMBIA2301-35S-AtWUS and induced plant expression vector pER8-AtWUS-EGFP laid foundations for Agrobacterium tumefaciens mediated transformation of rubber tree.

关 键 词：AtWUS基因 EGFP基因 融合基因 重叠延伸PCR 橡胶树 

分 类 号：S59[农业科学—作物学]