小麦磷脂酶Dδ基因的克隆及表达分析  

Cloning and Expression Analysis of Phospholipase Dδ Gene from Wheat

作　　者：王俊斌[1,2] 丁博[1] 李明[1] 陈帅君[1] 李欣[1] 张卫国[3] 彭四八 谢晓东[1] 

机构地区：[1]天津农学院天津一布里斯托环境变化对农作物影响研究中心,天津300384 [2]天津农学院基础科学学院,天津300384 [3]内蒙古民族大学农学院,内蒙古通辽028000

出　　处：《麦类作物学报》2015年第7期888-895,共8页Journal of Triticeae Crops

基　　金：天津市应用基础与前沿技术研究计划项目(14JCYBJC30600);天津市高等学校科技发展基金计划项目(20130606);天津市高等学校创新团队培养计划项目(TD12-5017)

摘　　要：磷脂酶D(Phospholipase D,PLD)是植物体内重要的信号转导酶。为了深入了解小麦PLD基因功能,利用同源克隆的方法从普通小麦扬麦158中克隆出TaPLDδ基因后进行序列分析,同时对TaPLDδ基因在不同组织及不同胁迫条件下的表达模式进行分析。结果表明,TaPLDδ基因编码区长为2 589bp,编码862个氨基酸,等电点为6.93,分子量约为97kDa。氨基酸序列分析显示,TaPLDδ基因编码的氨基酸序列含有N端C2结构域及两个保守的HKD活性中心。预测分析表明,TaPLDδ蛋白属于亲水性稳定蛋白,在细胞质中合成后,不进行蛋白转运;其二级结构包含27.49%的α-螺旋、19.14%的延伸链、6.73%的β-转角和46.64%的不规则卷曲。不同物种间TaPLDδ蛋白的同源性分析比较表明,TaPLDδ与谷子、玉米和拟南芥的PLDδ氨基酸序列之间具有高度的保守性,且与乌拉尔图小麦、二穗短柄草和水稻PLDδ亲缘关系密切。此外,qRT-PCR分析表明,TaPLDδ在叶、根、茎、花和种子中均有不同程度表达,并受ABA、NaCl和PEG诱导表达增强,推测该基因参与小麦渗透胁迫应答。以上这些研究结果为进一步研究TaPLDδ的生物学功能奠定了基础。Phospholipase D is the major signaling transduction enzyme.In order to deeply understand the function of a novel PLD gene,named TaPLDδ,it was isolated based on homologous cloning and reverse transcription PCR（RT-PCR）from common wheat（Triticum aestivum L.cv.Yangmai 158）,and its coding sequence was predicted with the bioinformatics analysis.Furthermore,qRT-PCR assay were performed to investigate the tissue-specific expression of TaPLDδ,and its expressing patterns under various abiotic stresses and signaling molecules treatments.The results showed that the open reading frame of TaPLDδconsisted of 2 589 bp and encodes a polypeptide of 862 amino acid residues.The estimated molecular weight and isoelectric point of the putative protein was 97 kDa and 6.93,respectively.Sequence analysis showed that the amino acid sequence encoded by TaPLDδcontained N-terminal C2 domain and two HKD motifs.TaPLDδprotein was a stable hydrophilic protein and located in the cytoplasm.The potential secondary structure of the protein included about 27.49% alpha helixes,19.14% extended strand,6.73% beta turn and 46.64% random coil as predicted with software.There was no signal peptide and transmembrane helices in TaPLDδ.Compared with that from Setaria italica,Zea mays and Arabidopsis,the amino acid sequence encoded by TaPLDδwas highly conserved.The phylogenic tree indicated that TaPLDδshared high similarity to PLDδproteins from Triticum urartu,Brachypodium distachyon and Oryza sativa.TaPLDδexpressed differentially in various organs such as leaves,roots,stems,flowers and seeds of wheat.After treatment with ABA,NaCl and PEG,TaPLDδshowed higher expression levels than the control.The fact implies that it might be involved in osmotic stress signaling pathways,which may provide a basis for further functional studies of TaPLDδ.

关 键 词：小麦 磷脂酶Dδ基因 克隆 基因表达 生物信息学 

分 类 号：S512.1[农业科学—作物学] S330