芍药分生组织决定基因APETALA2(AP2)的克隆及生物信息学分析

摘要

Flower-meristem-identity gene APETALA2(AP2) belonging to ABCDE model plays a role in regulating the development of plant floral organ.In order to understand the biological function of P.lactiflora AP2 gene,the plAP2 gene sequence of Paeonia lactiflora was cloned using RACE and sequencing technique technology,meanwhile the sequence signature,protein structure and function,subcellular localization were analyzed by bioinformatics methods.Molecular phylogenetic tree of AP2 from different plants were constructed by MEGA 5.0 software.Finally,we detected the differential expression between inner-petal and outer-petal by qPCR.Results showed,the full-length cDNA sequence of plAP2 gene was 1 935 bp and consisted of a 1 578 bp open reading frame (ORF) encoding 525 amino acid proteins.Analysis of protein structure and function showed,plAP2 was a hydrophilic and unstable protein,meanwhile plAP2 was also non-secretory protein without signal peptide and transmembrane structure.Nuclear localization signal located between 139th and 147th amino acid (KKSRRGPRS).The secondary structure of plAP2 protein contained 24% α-helices,19% β-sheet,28% β-turn and 28% random coil.plAP2 protein had eight glycosylation sites and sixty-four phosphorylation sites.plAP2 protein had two same conservative domain:AP2/ Ethylene-Responsive factors (ERF), which was located in the region between 151 and 213,between 243 and 306,respectively.Prediction of subcellular localization showed that plAP2 located mostly in cytoplasm (45.0%),rarely in microbody, mitochondrial matrix and lysosome.Analysis of evolutionary tree showed that the AP2 of Paeonia lactiflora were highly homologous and close to Paeonia suffruticosa.qPCR showed the expression of AP2 in outer-petal was extremely significant than that in inner-petal (P<0.01).In this study,we cloned the complete cDNA sequence of AP2 gene in Paeonia lactiflora and systematically revealed the basic protein structure,functional sites,domains,cellular localization and expression level,which will provide basic materials and theoretical references for further studying plAP2 gene function.%花分生组织决定基因APETALA2(AP2)属于植物ABCDE模型基因,在花器官发育过程中起着重要的调控作用.为进一步了解芍药AP2基因的生物学功能,利用RACE扩增和测序技术克隆plAP2基因序列,利用生物信息学在线程序对其序列特征、蛋白结构及功能、亚细胞定位进行预测,并利用MEGA 5.0构建不同植物AP2分子进化树,最后利用qPCR检测其在内外花瓣中的差异表达情况.结果显示,克隆获得芍药AP2基因(plAP2)cDNA序列全长1 935 bp,其ORF全长为1 578 bp,编码525个氨基酸.蛋白结构与功能分析表明,plAP2蛋白为亲水性不稳定蛋白,无跨膜结构和信号肽,表明为非分泌蛋白;核定位信号位于氨基酸序列139-147(KKSRRGPRS);二级结构包括α-螺旋(24%)、β-折叠(19%)、β-转角(28%)和无规则卷曲(28%);plAP2蛋白存在8个糖基化位点和64个磷酸化位点,plAP2蛋白包含2个相同的保守结构域:AP2/(Ethylene-Responsive factors,ERF)(151-213aa和243-306aa).亚细胞定位主要在细胞质(45.0%)中,少量分布于微体、线粒体基质间隙和溶酶体;进化树分析表明,芍药AP2基因与牡丹高度同源且亲缘关系最近;qPCR检测显示外瓣AP2表达量均极显著高于内瓣(P<0.01).克隆出芍药AP2全长cDNA序列,系统地揭示了plAP2蛋白基本结构、功能位点区域、细胞定位以及组织表达情况,为今后深入研究plAP2基因功能提供基础素材和理论参考.