菠萝AcMYB44基因的克隆及植物表达载体构建

Cloning and Expression Vector Construction of AcMYB44 Gene from Ananas comosus

为研究R2R3-MYB转录因子家族成员AcMYB44基因在菠萝非生物胁迫过程作用机制,利用PCR技术从菠萝品种‘手撕菠萝’叶片中克隆AcMYB44并利用生物信息学对其结构及功能进行分析。结果表明,克隆获得的AcMYB44基因cDNA序列全长1048 bp,开放阅读框(ORF)为771 bp,编码256个氨基酸。AcMYB44编码一个无信号肽、无跨膜结构且定位于细胞核的不稳定亲水蛋白,存在两个SANT保守结构域,属于R2R3-MYB家族。系统进化分析表明,AcMYB44与AtMYB44、AtMYB70、AtMYB73、AtMYB77、GmMYB73、MsMYB44、CeMYB44、DcMYB44亲缘关系较近且聚类为S22亚族。模式植物中研究表明S22亚族成员与抗逆相关。利用同源重组的方法构建了绿色荧光蛋白融合表达载体pGREEN-AcMYB44-ORF-GFP。以上结果为深入研究AcMYB44基因在菠萝中的功能奠定基础。

In order to study the mechanism of R2R3-MYB transcription factor gene family member in abiotic stress in Ananas comosus,the AcMYB44 gene was cloned from the leaves of Ananas comosus’Shousi pineapple’cultivar by PCR technique and analyzed its structure and function by bioinformatics method.The results showed that the cloned cDNA sequence of AcMYB44 gene was 1048 bp in length,and the open reading frame(ORF)was 771 bp,encoding 256 amino acids.AcMYB44 encoded an unstable hydrophilic protein with no signal peptide,no transmembrane structure and localization in the nucleus.There were two SANT conserved domains,belonging to the R2R3-MYB family.Phylogenetic analysis showed that,AcMYB44 and AtMYB44,AtMYB70,AtMYB73,AtMYB77,GmMYB73,MsMYB44,CeMYB44,Dc MYB44 were clustered into S22 subfamily.Studies in model plants have shown that members of the S22 subgroup are associated with stress tolerance.The green fluorescent protein fusion vector pGREEN-AcMYB44-ORF-GFP was constructed by homologous recombination.These results laid a foundation for further study on the function of AcMYB44 gene in Ananas comosus.