玉米ZmCKX基因的克隆及分析  

Cloning and Preliminary Analysis of ZmCKX in Maize

作　　者：刘建怡 张彦彦[1] 宋玉峰[2] 潘金豹[1] 韩俊[1] 南张杰[1] 王维香[1] 史利玉[1] 王晔[1] 孙清鹏[1] 卢敏[1] 

机构地区：[1]北京农学院植物科学技术学院,北京102206 [2]山东省产品质量监督检验研究院,济南250102

出　　处：《中国农学通报》2015年第11期130-136,共7页Chinese Agricultural Science Bulletin

基　　金：北京市教委"zmNAC在玉米响应干旱胁迫反应中的作用"(CIT&TCD201304096)

摘　　要：细胞分裂素氧化酶降解细胞分裂素,从而调控植物的生长发育,克隆和分析玉米细胞分裂素氧化酶基因(Zm CKX),为研究Zm CKX在玉米抗逆反应中的作用奠定基础。运用RT-PCR和RACE技术克隆了Zm CKX基因的c DNA全长,并对其进行了初步的生物信息学分析。琼脂糖凝胶电泳鉴定结果表明,PCR扩增出一条约1035 bp的片段,与目的片段大小相似。将目的片段与p MD-19t构建的p MDZm CKX重组质粒并转化为DH5α,经检测片段与原始片段大小相同。Blastn分析结果表明,其与Zm CKX1的相似性为99%。对该基因序列分析表明,Zm CKX基因编码的蛋白的开放阅读框为1035 bp,起始位点为23 bp,终止位点为1057 bp。Zm CKX基因长1035 bp,编码344个氨基酸。它与Zm CKX1基因核苷酸同源性99%。Zm CKX基因编码的蛋白是存在于细胞质中的亲水蛋白。Cytokinin oxidase could degrade cytokinins, and thus influence various aspects of plant growth anddevelopment. Zm CKX gene function could be understood from Zm CKX gene cloning, which can provide thebasis for maize＇s resistance reaction. Prokaryotic expression vector of Zm CKX gene was constructed and wasexpressed. Zm CKX full length c DNA was cloned by using RT-PCR and RACE technique, and preliminaryanalysis of the bioinformatics was carried out on it. A pair of primers was designed according to digestion sitesin plasmid p MD- 19 t and the Zm CKX gene sequence by Gen Bank. The DNA fragment of 1035 bp wasamplified by PCR from the p MD-Zm CKX recombinant plasmid with Zm CKX gene, then cloned into p MD-19 tand transformed into the host E.coli strain DH5α. The fragment was conformed to the original sequence. Itindicated that fusion expression vector p MD- Zm CKX was constructed. The p MD- Zm CKX plasmid wastransformed into DH5α for expression. The expression product of Zm CKX gene was identified by AGE（agarosegel electrophoresis）. The open reading frame of Zm CKX was 1035 bp in length and encoded a predicted proteinof 344 amino acids. The analysis of c DNA sequence shows that Zm CKX shares 99% identity with Zm CKX1.Zm CKX genes encoding protein exists in the cytoplasm, and Zm CKX is hydrophilic protein.

关 键 词：玉米 细胞分裂素氧化酶 克隆 原核表达载体 

分 类 号：Q752[生物学—分子生物学]