燕子花WRKY11基因克隆、结构分析及功能验证

Cloning,Structure Analysis and Functional Verification of IlWRKY11 in Iris laevigata

为了开发燕子花WRKY家族的功能基因资源,验证WRKY11转录因子与花期调控相关性,更好地服务于燕子花定向分子育种。以东北林业大学苗圃的燕子花(Iris laevigata)为材料,燕子花花被片为cDNA模板,克隆燕子花IlWRKY11基因,对IlWRKY11蛋白质结构进行生物信息学分析、亚细胞定位,并通过该基因转化模式植物拟南芥,开展WRKY11转录因子对花期调控方面的功能解析。结果表明:以燕子花总RNA反转录的cDNA为模板,克隆获得IlWRKY11基因的长度为888 bp,编码295个氨基酸;IlWRKY11蛋白的相对分子质量为32291,分子式为C_(1398)H_(2237)N_(419)O_(421)S_(20),属于不稳定亲水性蛋白,pCAMBIA1300-IlWRKY11-GFP植物过表达载体确定IlWRKY11蛋白定位在细胞核,IlWRKY11蛋白与单子叶植物深圳拟兰(Apostasia shenzhenica)、石刁柏(Asparagus officinalis)的关系较近。过表达IlWRKY11基因的拟南芥抽薹时间和开花时间比对照提前2~3 d。通过对转基因拟南芥和转空载对照的成花期相关基因表达量进行qRT-PCR检测,推测IlWRKY11基因通过调控赤霉素途径相关基因GA20OX和春化途径基因VRN1以及年龄途径基因SPL3参与花期调控,说明IlWRKY11基因(ON399551)对燕子花花期调控具有重要作用。

In order to develop functional gene resources of WRKY family of Iris laevigata,verify the relevance of WRKY11 transcription factor to flowering regulation and better serve I.laevigata targeted molecular breeding.The I.laevigata from the Northeast Forestry University was used as the material,and the I.laevigata tepals were used as cDNA templates to clone the IlWRKY11 gene,perform bioinformatics analysis and subcellular localization of IlWRKY11 protein structure,and the functional elucidation of WRKY11 transcription factor on flowering regulation by transforming the model plant Arabidopsis thaliana was conducted with this gene.The results showed that the cDNA reverse-transcribed from total RNA of I.laevigata was used as a template to clone the IlWRKY11 gene with a length of 888 bp and encoding 295 amino acids.The relative molecular mass of IlWRKY11 protein is 32291 kDa and the molecular formula is C_(1398)H_(2237)N_(419)O_(421)S_(20),belonging to unstable hydrophilic protein.The pCAMBIA1300-IlWRKY11-GFP plant overexpression vector determined that IlWRKY11 protein is localized in the nucleus,and IlWRKY11 protein is more closely related to the monocotyledonous plants such as Apostasia shenz-henica and Asparagus officinalis.Bolting time and flowering time are 2-3 d earlier in IlWRKY11 gene overexpressed arabidopsis compared in the control.The IlWRKY11 gene is found to be involved in flowering regulation through regulation of the gibberellin pathway-related gene GA20OX and the vernalization pathway-related gene VRN1 as well as the age pathway-related gene SPL3 by qRT-PCR assay of flowering-phase related gene expression in transgenic arabidopsis and trans-null control.It is suggested that the IlWRKY11 gene(ON399551)plays an important role in regulating blooming time for I.laevigata.