目的 利用生信技术分析秦艽WRKY家族，为进一步研究GmWRKY转录因子对秦艽次生代谢产物生物合成的分子调控机制提供信息。方法 基于秦艽转录组注释结果挖掘WRKY家族成员，通过NCBI-tBlastx和SMART保守结构域预测进一步筛选得到41条WRKY序列并进行生信分析。基于getorf预测开放阅读框（open reading frame，ORF）区，对41条序列的ORF片段进行MEME保守基序分析和ClustalW比对，采用MEGAX构建邻接法（neighbor-joining，NJ）系统发育树。利用Omicshare GO对41个WRKY成员进行富集分析，利用String进行WRKY成员蛋白互作网络预测分析。通过转录组FPKM值对GmWRKY成员的基因表达情况进行热图分析，并对蛋白互作核心成员进行基因相对表达分析。结果 41个秦艽WRKY成员中大部分成员的保守基序为“WRKYGQK”，仅GmWRKY27保守基序发生氨基酸突变，为“WRKYGKK”。依据WRKY特征结构域将41条序列的51个结构域分为3大类和8个亚类型，第I大类为氮端（N）和碳端（C），第II大类下设5个亚类型，以及第III大类；此外，系统进化树可将51个WRKY结构域分成4大组，GroupI[I(N)]、GroupII（IIa＋IIb）、GroupIII[I(C)＋IIc]、GroupIV（IId＋IIe和III），该结果与经典的WRKY家族分类特点基本一致。GO分析发现秦艽WRKY家族成员广泛参与代谢调控过程。当茉莉酸甲酯（jasmonic acid methyl ester，MeJA）诱导秦艽后，蛋白网络互作的核心成员GmWRKY1和GmWRKY17表达显著提高，推测这些WRKY成员与秦艽次生代谢产物的调控关系密切。结论 为进一步开展秦艽WRKY转录因子功能研究奠定基础。

Objective To analyze the WRKY family of Gentiana macrophylla (GmWRKY) using biotransformation techniques in order to provide information for further research on the molecular regulation mechanism of GmWRKY transcription factor on the biosynthesis of the secondary metabolites of G. macrophylla.Methods Based on the annotation results of WRKY family members, 41 WRKY sequences were further screened by NCBI-tBLastx and SMART domains for bioinformatics analysis. Based on getorf prediction of open reading frame (ORF) region, MEME conservative motif analysis and ClustalW alignment were performed on ORF fragments of 41 sequences, and the neighbor-joining (NJ) phylogenetic tree was constructed by MEGAX. The enrichment analysis of 41 WRKY members was performed using Omicshare GO. And prediction analysis of WRKY member protein interaction network was performed by String. The expression of WRKY members was analyzed by heat map based on the FPKM value of the transcriptome, and the relative gene expression of the core members of GmWRKY protein interaction was analyzed.Results Most of the 41 GmWRKY members had a conserved motif of "WRKYGQK", and only THE GmWRKY27 conserved motif had an amino acid mutation of "WRKYGKK". According to the WRKY characteristic domain, 51 domains of 41 sequences were divided into three categories and eight subtypes. The first category consisted of nitrogen terminal (N) and carbon terminal (C), the second category consisted of 5 subtypes and the third category. Phylogenetic tree can divide 51 WRKY domains into four groups: GroupI [I(N)], GroupII (IIa+IIb), GroupIII [I(C)+IIc], GroupIV (IId+IIe and III). These results were consistent with the classic WRKY family classification. GO analysis revealed that members of the GmWRKY family were extensively involved in metabolic regulation. The expression of GmWRKY1 and GmWRKY17, the core members of protein network interaction, were significantly increased when G. macrophylla was induced by MeJA, suggesting that these WRKY members were closely related to the regulation of secondary metabolites of G. macrophylla.Conclusion This study can lay a foundation for further research on the function of GmWRKY transcription factors.

R286.12

国家自然科学基金项目(81903753);陕西中医药大学科学基金项目(2017PY06)