栽培草莓果实中特异表达的bHLH78基因的克隆及过量表达载体构建  

Cloning of bHLH78 Gene Expressed Specifically in Fruits of Cultivated Strawberry and Construction of Overexpression Vector

作　　者：豆玉娟[1] 曹飞[1] 马跃[1] 李贺[1] 刘月学[1] 张志宏[1] 

机构地区：[1]沈阳农业大学园艺学院,沈阳110866

出　　处：《分子植物育种》2014年第3期456-465,共10页Molecular Plant Breeding

基　　金：国家科技支撑计划课题(2013BAD02B04)资助

摘　　要：根据二倍体野生森林草莓(Fragaria vesca)预测的bHLH78-like基因序列设计特异性引物,利用RT-PCR技术从八倍体栽培凤梨草莓(Fragaria×ananassa)品种‘幸香’果实中克隆出FabHLH78基因全长CDS。测序和分析结果表明,FabHLH78基因的CDS全长1 653 bp,编码550个氨基酸残基,在线软件Compute pI/Mw分析编码的蛋白质理论分子量约为59.6 kD、等电点(pI)6.17,经软件DNAMAN分析,核酸序列和氨基酸序列与二倍体草莓FvbHLH78-like基因的一致性分别为98.31%和96.55%。利用半定量PCR检测FabHLH78基因在草莓根、匍匐茎、叶、果实中的表达情况,发现其只在果实中特异表达;实时定量RT-PCR结果显示随着果实发育FabHLH78基因表达量逐渐增加。FabHLH78全长CDS被整合到植物表达载体pRI101-AN上,构建出FabHLH78基因的过量表达载体pRI101-bHLH78,为进一步验证FabHLH78基因的功能奠定了基础。The full-length coding sequence （CDS） of FabHLH78 gene was cloned from octoploid cultivated strawberry （Fragariaxananassa） cultivar ‘Sachinoka＇ by RT-PCR with the specific primers designed according to the sequence of bHLH78-1ike gene sequence from diploid wild strawberry （Fragaria vesca）. The full length of FabHLH78 CDS was 1 653 bp and the deduced amino acid sequence contained 550 amino acid residues. After analysis DNAMAN, it shared 98.31% and 96.55% identity with the nucleotide sequence and amino acid sequence of bHLH78-like gene from F. vesca, respectively. The molecular weight of FabHLH78 was 59.6 kD and its isoelectric point （pI） was 6.17 predicted with online software Compute pI/Mw. The expressions of FabHLH78 were analyzed in root, runner, leave and fruit of strawberry by semi-quantitative PCR, and the result showed that FabHLH78 was expressed specifically in the fruit. The data of real time PCR showed that the expression level of FabHLH78 increased during fruit ripening. The overexpression vector of FabHLH78, pRI101-bHLH78, was constructed by integrating the CDS into a plant expression vector pRI101-AN. It would be used in functional analysis in the future research.

关 键 词：bHLH78 草莓 基因克隆 实时定量PCR 过量表达载体 

分 类 号：S668.4[农业科学—果树学]