小麦 Tappc3A 基因克隆及功能预测

Cloning and Functional Prediction of Tappc3A Gene in Wheat

PEPC可催化磷酸烯醇式丙酮酸(PEP)生成草酰乙酸(OAA)进入三羧酸循环,对植物的生长发育和逆境适应发挥重要作用。但目前尚无关于PEPC参与植物器官发育的报道。发掘并研究小麦Tappc3A在花发育中的生物学功能,为探究小麦雄蕊同源转化为雌蕊性状的分子机制提供新的线索。通过PCR技术从CM28TP和HTS-1中克隆Tappc3A基因,使用生物信息学工具分析基因序列及系统进化关系,利用qRT-PCR技术分析Tappc3A在小麦幼穗不同发育阶段和不同生殖器官中的表达水平,通过原核表达分析Tappc3A基因编码的蛋白功能是否正常,利用小麦RNA-Seq数据库分析Tappc3A与其他调控花器官发育基因之间的共表达情况。Tappc3A基因的ORF长2901 bp,编码966个氨基酸残基,具有典型的磷酸烯醇式丙酮酸羧化酶(PEPase)保守结构域,N端具有保守的丝氨酸(Ser,S)可逆磷酸化位点(SIDAQLR),C端具有植物型PEPC蛋白特征序列(QNTG),第774位氨基酸为C3植物PEPC典型的丙氨酸。聚类分析也表明,Tappc3A属于C3型PEPC家族。qRT-PCR分析表明,在小麦幼穗发育的3个阶段,二棱期至小花分化期和药隔时期,HTS-1中的Tappc3A基因的表达量高于CM28TP,且Tappc3A在雌蕊(P)和雌蕊化雄蕊(PS)中的表达量显著高于雄蕊(S)。原核表达结果显示,Tappc3A基因编码的蛋白能催化PEP生成OAA,并且在IPTG诱导后活性明显增强。基因共表达分析显示,Tappc3A可能参与了小麦花器官的形态发生。小麦Tappc3A可能参与小麦雌蕊发育,其在雄蕊中的过量表达可能与雄蕊同源转化为雌蕊性状形成有一定的关联。

PEPC catalyzes phosphoenolpyruvate(PEP)to generate oxaloacetate(OAA)to participate the tricarboxylic acid(TCA),which plays an important role in development and stress adaptation of plant.However,there have been no reports of PEPC involvement in plant organ development.Exploration and study on biological function of Tappc3A gene in wheat flower development,and provides new clues to explore the molecular mechanism of homologous conversion of stamens into pistillody in wheat.The Tappc3A gene was cloned from CM28TP and HTS-1 by PCR,and the sequence and phylogenetic tree were analyzed by bioinformatics tools,and the expression level of Tappc3A gene in different developmental stages and different reproductive organs of wheat young spikes was analyzed by using qRT-PCR,and whether the protein function encoded by Tappc3A gene was analyzed by prokaryotic expression.The wheat RNA-Seq database was used to analyze the co-expression of Tappc3A gene and other genes that regulate flower organ development.The ORF of Tappc3A gene was 2901 bp in length,encoding 966 amino acid residues,with a typical phosphoenolpyruvate carboxylase(PEPase)conserved domain,a conserved serine(Ser,S)reversible phosphorylation site(SIDAQLR)at the N-terminal,a plant-type PEPC protein signature sequence(QNTG)at the C-terminal,and the 774th amino acid was the typical alanine of C3 plant PEPC.Cluster analysis also showed that Tappc3A belonged to the C3 type PEPC family.qRT-PCR analysis,in three stages of wheat young spike development,showed that the expression of Tappc3A gene in HTS-1 was higher than that in CM28TP at the dichotomous stage to the florescence differentiation stage and the pharmacophore period,and the expression of Tappc3A gene was significantly higher in pistils(P)and pistillody stamens(PS)than stamens(S).The prokaryotic expression results showed that the protein encoded by Tappc3A gene,which catalyzes the production of OAA from PEP,and its activity was significantly enhanced after IPTG induction.The gene co-expression analysis showed that Tappc3A gene might be involved in the morphogenesis of wheat floral organs.Tappc3A gene might be involved in wheat pistil development,and its overexpression in stamens might be associated with the homologous transformation of stamens into pistillody trait formation.