普通小麦TaADF8基因的克隆及表达分析

肌动蛋白解聚合因子(actin-depolymerizing factor,ADF)普遍存在于真核细胞中,为低分子量的肌动蛋白结合蛋白,在调控细胞内肌动蛋白纤维的聚合和解聚中起关键作用。为给深入研究TaADF8基因在小麦中的功能机理奠定基础,并为进一步丰富小麦ADF基因研究内容提供理论参考,本研究利用电子克隆策略从小麦品种CP53中克隆出TaADF8基因(GenBank登录号为KJ864962)后对其进行序列分析,并进一步采用荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术对其在小麦不同组织间的表达差异及不同非生物胁迫下的表达模式进行分析。核酸序列分析表明,该基因全长695bp,拥有完整的ORF,编码142个氨基酸。氨基酸序列分析表明,该蛋白含有保守的ADF同源区和PIP2结合结构域,且在氨基端有核定位信号。进化和聚类分析表明,小麦TaADF8基因与大麦HvADF2基因、HvADF3基因和水稻OsADF3基因亲缘关系较近,蛋白相似度分别为75.35%、93.66%和67.86%。qRT-PCR表达特性分析显示,该基因为组成型表达,在根、茎、叶、颖壳和雄蕊中均表达,且在根、叶和雄蕊中表达量较高;该基因表达受低温的强烈诱导,同时也受水分、高盐和外源脱落酸胁迫诱导。

Cloning and Expression Analysis of a Novel Actin-depolymerizing Factor Gene ( TaADF8) from Wheat (Triticum aestivum L.)

Actin-depolymerizing factors are low molecular weight actin binding proteins that exist widely in eukaryocyte. They play an important regulation role in polymerization and depolymerization of actin filaments in cells. In order to study the function and mechanism of TaADF8 (GenBank Accession: KJ864962) deeply in common wheat, and to provide theoretical reference to enrich the knowledge of ADF genes in wheat, the strategy of in silico cloning was used to clone this gene from wheat line CP53 genome. The methods of bioinformatics and quantitative real-time PCR(qRT-PCR) were used to analyze the sequences and expression patterns in different wheat tissues and abiotic stresses. The analysis of nucietide sequence indicated that TaADF8 contains a 695 bp complete open reading frame (ORF) which encodes a peptide of 142 amino acids. The amino acids analysis indicated that the predicted protein sequence contained a typical ADF gene family domain (ADF homology region) and a PIP2 binding domain. Evolution and clustering analysis revealed that TaADF8 protein shared 75.35%, 93.66% and 67.86% sequence similarities with HvADF2 (Hordeum vulgare), HvADF3 (Hordeum vulgare) and OsADF3 (rice) protein.The expression pattern analysis carried out by qRT-PCR indicated that TaADF8 was constitutively expressed in various tissues of wheat seedlings, but with higher in root, leaf and stamen. And meanwhile, it was induced by cold stress, water stress, high salinity and exogenous abscisic(ABA), but under cold stress it was strongly up-regulated.