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目的克隆丹参碱性螺旋-环-螺旋(b HLH)类转录因子SmbHLH93,并初步探究该基因的功能。方法采用PCR和RT-PCR技术分别从DNA和c DNA水平克隆得到了SmbHLH93基因,进一步通过BD Walking获得了该基因的5’启动子区。生物信息学分析SmbHLH93蛋白的理化性质、结构特点和系统进化关系。结合启动子区分析软件和q PCR,研究SmbHLH93在丹参不同部位和环境诱导下的表达模式。结果获得SmbHLH93基因序列954 bp和5’启动子区1 583 bp,其中开放阅读框(ORF)657 bp,有3个内含子和4个外显子,编码218个氨基酸,含有b HLH和ACT_UUR-ACR-like结构域,无跨膜区域,可能定位于细胞核。表达模式分析结果显示该基因的表达量在根中最高,茎中最低,且随着花的形成逐渐降低。光和低温诱导SmbHLH93的高表达,而水杨酸(SA)抑制其表达。结论克隆得到丹参b HLH类转录因子新成员SmbHLH93,初步预测其参与了丹参花的发育过程和丹参次生代谢途径的调控。
Objective To clone SmbHLH93, a basic class of b HLH transcription factor, and to explore the function of this gene. Methods The SmbHLH93 gene was cloned from the DNA and c DNA by PCR and RT-PCR, respectively. The 5 ’promoter region of the gene was further obtained by BD Walking. Bioinformatics analysis of SmbHLH93 protein physical and chemical properties, structural characteristics and phylogenetic relationships. Combined with promoter region analysis software and q PCR, SmbHLH93 expression patterns in different parts of Salvia miltiorrhiza induced by the environment. Results The SmbHLH93 gene sequence was 954 bp in length and 1 583 bp in the 5 ’promoter region. The open reading frame (ORF) was 657 bp with 3 introns and 4 exons encoding 218 amino acids, including b HLH and ACT_UUR -ACR-like domain, no transmembrane region, may be located in the nucleus. Expression pattern analysis showed that the expression level of the gene was the highest in the root, the lowest in the stem, and decreased gradually as the flower formed. Light and hypothermia induced high expression of SmbHLH93, whereas salicylic acid (SA) suppressed its expression. Conclusion SmbHLH93, a new member of b HLH transcription factor of Salvia miltiorrhiza, was cloned. It is preliminarily predicted that it is involved in the development of Salvia miltiorrhiza and the regulation of the secondary metabolic pathway of salvia miltiorrhiza.