丹参转录因子SmWRKY14基因的克隆及表达分析

Cloning and expression analysis of WRKY family member SmWRKY14 in Salvia miltiorrhiza

目的克隆丹参Salvia miltiorrhiza转录因子基因SmWRKY14,分析其在不同组织及应答环境因子和植物激素过程中的表达特征。方法以丹参转录组数据中Unigene(c50007_g1)序列为参考,利用PCR扩增获得SmWRKY14基因序列。通过生物信息软件及在线工具预测基因编码蛋白的理化性质、蛋白质结构等分子特征,采用DNAMAN和MEGA 6.0分别进行氨基酸多序列比对和进化树构建,运用实时荧光定量PCR检测该基因的表达模式。结果克隆得到SmWRKY14基因cDNA全长1103 bp,编码244个氨基酸,相对分子质量27600,等电点8.19。该蛋白含有WRKY特征基序,不含信号肽及跨膜结构域,其高级结构主要由无规卷曲构成。进化树分析表明SmWRKY14蛋白与柿WRKY14蛋白的亲缘关系最近。SmWRKY14基因在丹参中各器官中为组成型表达,且该基因与丹参酮合成关键酶基因DXS、GGPPS、CPS的表达模式相似,且受茉莉酸甲酯、脱落酸、赤霉素等植物激素及机械损伤的强烈诱导。结论获得丹参SmWRKY14基因序列、生物信息及表达特征,为研究WRKY家族成员调控丹参酮类化合物的合成、应答植物激素及参与防御反应的分子机制提供理论依据。

Objective To clone a WRKY protein gene SmWRKY14 with full length cDNA from Salvia miltiorrhiza and carry out bioinformatics and expression analysis in different tissues and response to environmental factors and phytohomone.Methods The PCR was preformed based on the sequence of Unigene(c50007_g1)searched from our transcriptome database,and characteristics of physiochemical properties,conserved domains and structure prediction of the protein were determined using a series of bioinformatics tools.The analyses of multiple alignment and phylogenetic tree were performed using DNAMAN and MEGA 6.0,respectively.Real-time quantitative PCR was used for gene expression analysis.Results In this study,the full length cDNA of SmWRKY14 was 1103 bp in size,encoding a 244-aa protein with a molecular weight of 27.6 KDa and an isoelectric point of 8.19.SmWRKY14 was an unstable hydrophilic protein containing characteristic and conserved WRKY domain without signal peptide or transmembrane domain.The main secondary structure of the amino acid sequences was random coil.Moreover,multiple sequence alignments and phylogenetic trees showed that SmWRKY14 protein had high homology with WRKY14 of Diospyros kaki.Quantitative real-time PCR indicated that SmWRKY14 constitutively expressed in the roots,stems,leaves and flowers of S.miltiorrhiza and was strongly induced by methyl jasmonate,abscisic acid,gibberellins,and mechanical wound,which indicated SmWRKY14 could participate in regulation of biosynthesis of tanshinones and defense process.Conclusion The gene sequences of SmWRKY14 was successfully cloned and the bioinformatics and expression pattern analysis was carried out,which will provide a foundation for further research on the molecular mechanism of regulation of tanshinones synthesis and response to defense process in S.miltiorrhiza.