目的 预测分析并克隆丹参E3泛素连接酶SmCOP1（constitutively photomorphogenic1），并对其靶基因进行初筛，为丹参次生代谢关键调控因子MYB以及bHLH等转录因子泛素化研究做准备。方法 本研究利用拟南芥AtCOP1蛋白序列，用HMMER3程序和在线程序SMART，在丹参全基因组数据库筛选到丹参中的E3泛素连接酶SmCOP1序列，经RT-PCR技术获得丹参cDNA，以其为模板设计引物扩增，并利用生物信息学方法对该基因及其编码蛋白进行相关分析。通过酵母双杂实验对其调控丹参酮和酚酸物质机制进行初步研究。结果 结果表明SmCOP1的2个蛋白均为不稳定亲水性蛋白，其N-端为环形锌指结构域（RING–finger），C-端为WD40重复结构域。通过建立进化树表明SmCOP1b和AtCOP1是直系同源的关系，而SmCOP1a和SmCOP1b是旁系同源的关系。Y2H实验显示SmCOP1a与SmPAP1互作。结论 成功克隆并预测分析丹参E3泛素连接酶SmCOP1，并初步发现其可能通过靶向MYB转录因子SmPAP1对其泛素化修饰参与调控酚酸类物质的代谢。

Objective To predict and clone SmCOP1 (constitutively photomorphogenic1) of E3 ubiquitin ligase in Salvia miltiorrhiza, and preliminarily screen its target genes, so as to prepare for the study of ubiquitin of MYB, a key regulatory factor of secondary metabolism and bHLH.Methods In this study, AtCOP1 protein sequence of Arabidopsis thaliana was used to screen SmCOP1 sequence of E3 ubiquitin-ligase from Salvia miltiorrhiza genome database by HMMER3 program and SMART online program. CDNA of Salvia miltiorrhiza was obtained by RT-PCR, and primers were designed and amplified using it as template. The gene and its encoded protein were analyzed by bioinformatics method. The regulation mechanism of tanshinone and phenolic acid was studied by yeast dihybrid experiment.Results The results showed that the two proteins of SmCOP1 were unstable hydrophilic proteins, whose N-terminus were Ring-finger and C-terminus were WD40 repeating domains. Phylogenetic tree analysis showed that SmCOP1b was a direct homolog of AtCOP1 and SmCOP1a was a side homolog of SmCOP1b. Y2H showed that SmCOP1a interacted with SmPAP1.Conclusion The E3 ubiquitin ligase SmCOP1 was successfully cloned and predicted and analyzed, and it was preliminarily found that SmPAP1 may be involved in regulating the metabolism of phenolic acids through targeting the MYB transcription factor SmPAP1.

S567.53

国家自然科学基金委员会面上项目（31670295）：JAZ蛋白与转录因子互作调控丹参酮类物质合成的研究，负责人：麻鹏达。