灯盏花MYB基因克隆及其荧光表达载体的构建

Cloning of MYB gene and construction of greenfluorescent protein expression vector in Erigeron breviscapus

目的克隆灯盏花MYB基因的全长序列,为解析灯盏花MYB基因的功能奠定基础。方法根据灯盏花转录组的相关信息,利用RACE方法从灯盏花中克隆到一个可能参与灯盏花乙素合成的MYB基因,在对其cDNA序列、核苷酸序列的相似性、理化性质、疏水性、跨膜结构、二级结构及三级结构进行分析预测的基础上,对其进行多序列比对并构建系统树。同时,还构建了该基因与绿色荧光蛋白的融合表达载体,并进行了初步转化研究。结果克隆获得灯盏花MYB基因,命名为ebMYB06,其开放阅读框为783 bp,编码260个氨基酸残基,相对分子质量为63 800,理论等电点(p I)为5.18,属稳定蛋白。其蛋白二级结构主要由无规卷曲、α-螺旋和β-折叠构成。根据灯盏花MYB与拟南芥MYB(AtMYB)的系统树比对分析结果,发现ebMYB基因与拟南芥中的AtMYB4、7、32、6、8和AtMYB11、12、111 2亚群的基因聚类,推测所克隆基因在结构或功能上,可能与这两组具有共同性。实验还表明所构建的表达可用于灯盏花的高效转化。结论首次从灯盏花中克隆到可能参与其苯丙烷代谢或环境响应调控的MYB基因。

Objective To study the role of MYB transcription factor in phenylpropane biosynthesis pathway and the response to biotic or abiotic stress, a full length sequence of putative MYB gene was cloned in Erigeron breviscapus. Methods Based on a partial sequence of putative MYB gene from transcriptome we have previous reported, the full length c DNA was cloned by RACE method in E. breviscapus. According to the sequence of cloning c DNA, the nucleotide sequence similarity, physicochemical properties, hydrophobicity, transmembrane structure, secondary structure and tertiary structure were predicted and analyzed by various softwares. Meanwhile, multiple sequence alignment of cloned MYB gene was performed, and the phylogenetic tree was constructed. In addition, the fusion expression vector of this gene and green fluorescent protein was constructed. Results The MYB gene was cloned and named e BMYB06. The open reading frame was 783 bp, encoding 260 amino acid residues with a relative molecular mass of 63.80 k Da and a theoretical pI of 5.18 which proved to be a stable protein. The secondary structure of the protein is mainly composed of irregular coil, alpha helix and beta fold. According to the result of phylogenetic tree alignment of E.breviscapus with R2R3 MYBs from Arabidopsis thaliana, the cloned MYB gene was clustered to two subsets of R2R3-MYB genes from A. thaliana, which suggested that the cloned MYB gene would be similar with the two groups in structure and function, which would be involved in response to biotic and abiotic stress or phenylpropane biosynthesis pathway, respectively. Further experimental results show that the constructed expression can be used for the efficient transformation in E. breviscapus. Conclusion For the first time, a MYB gene was cloned may be involved in the phenylpropanoid metabolism or gene metabolism or gene regulation in response to the environment in E. breviscapus.