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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 clone the full-length sequence of MYB gene in Breviscapus and lay a foundation for the analysis of the function of Breviscapus MYB gene. Methods According to the related information of transcriptome of Erigeron breviscapus, a MYB gene possibly involved in the synthesis of scutellarin was cloned from Erigeron breviscapus by RACE method. The cDNA sequence, nucleotide sequence similarity, physicochemical properties, hydrophobicity , Transmembrane structure, secondary structure and tertiary structure analysis and prediction based on the multiple sequence alignment and the construction of the system tree. At the same time, a fusion expression vector of this gene and green fluorescent protein was also constructed and a preliminary transformation study was carried out. Results The Breviscapus MYB gene was cloned and named ebMYB06. The open reading frame of the MYB gene was 783 bp, encoding a 260 amino acid residue with a relative molecular mass of 63 800 and a theoretical isoelectric point (pi) of 5.18. Its protein secondary structure is mainly composed of random coil, α-helix and β-sheet. According to the results of phylogenetic tree analysis between Erigeron breviscapus MYB and Arabidopsis thaliana MYB (AtMYB), it was found that the ebMYB gene was associated with the gene homology of AtMYB4,7,32,6,8 and AtMYB11,12,111 2 subgroups in Arabidopsis Species, suggesting that the cloned gene in the structure or function, may be common to both groups. Experiments also show that the constructed expression can be used for the efficient transformation of Breviscapus. Conclusions For the first time, the MYB gene, which may be involved in the regulation of phenylpropanoid metabolism or environmental response, was cloned from the Erigeron breviscapus.