目的 克隆丹参碱性螺旋-环-螺旋(bHLH)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cDNA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qPCR,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bHLH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bHLH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 and characterize a basic helix-loop-helix (bHLH) transcription factor SmbHLH93 from Salvia miltiorrhiza, and to predict its probable function. Methods SmbHLH93 was cloned by PCR and RT-PCR from genomic DNA and cDNA, while its 5' promoter region was cloned by BD Walking. Analysis on physico-chemical property, structure characteristic, and phylogenetic relationships of SmbHLH93 protein was carried out by bioinformatic method. Gene expression in different organs and inducing conditions was detected by qPCR. Results Gene sequences of SmbHLH93 (954 bp) were obtained, including three introns and four exons, and the open reading frame was 657 bp, encoding 218 amino acids. Its promoter region had 1 583 bp nucleotides. The putative SmbHLH93 protein contains bHLH and ACT_UUR-ACR-like domains, without transmembrane helices, and located in the nucleus. The gene expression was highest in roots and lowest in stems. With the development of flowers, its expression decreased gradually. Light and low temperature could induce high expression of SmbHLH93, while salicylicacid (SA) inhibited its expression. Conclusion A new member of bHLH transcription factor, SmbHLH93, is cloned from S. miltiorrhiza, and it could be involved in the development of flower and regulation of secondary metabolic pathways in S. miltiorrhiza.

国家自然科学基金资助项目(31270338);陕西省科技厅科技计划项目(2012K19-02-04);陕西学前师范学院基金(2014QNKJ078,2014QNKJ079)