灰皮支黑豆GmPUB24基因的生物信息学与胞囊线虫诱导表达分析

Bioinformatics of Huipizhi black soybean GmPUB24 gene and its expression under Heterodera glycines infection

为明确大豆U-box型E3泛素连接酶介导植物抗病性的机理,以抗胞囊线虫品种灰皮支黑豆为材料,利用RT-PCR技术克隆GmPUB24基因的蛋白质编码区序列(coding sequence,CDS),对该基因进行生物信息学分析,并接种大豆胞囊线虫,进行诱导表达分析。结果表明,GmPUB24基因CDS总长1254 bp,编码417个氨基酸,分子量为46.78 ku。蛋白质二级结构分析显示,GmPUB24编码的蛋白质含有α螺旋、无规则卷曲、延伸链、β折叠,α螺旋占比最高为58.9%,为亲水蛋白质且无跨膜结构域,无信号肽;蛋白质系统进化树表明,其与野生大豆亲缘性最高,亚细胞定位显示其定位于细胞质中。GmPUB24基因上游1500 bp启动子区含有CGTCA-motif、TGACG-motif等响应抗病通路的作用元件,以及ABRE、WUN-motif、TATA-box等响应非生物胁迫的作用元件。实时荧光定量PCR(qRT-RCR)结果显示,GmPUB24在接种大豆胞囊线虫1~3 d持续上调表达,接种3 d时根部表达量为未接种线虫样本的6.14倍,表明GmPUB24基因可被大豆胞囊线虫诱导表达,可能参与大豆抵御胞囊线虫的过程。研究结果为阐明大豆U-box家族基因在抗大豆胞囊线虫病中的调控机制奠定了基础。

In order to clarify the mechanism of soybean U-box E3 ubiquitin ligase mediating plant disease resistance,coding sequence(CDS)of GmPUB24 gene from resistant cultivar Huipizhi black bean was cloned by RT-PCR.Bioinformatics and expression analysis of this gene after inoculation with soybean cyst nematode were carried out.The results showed that the length of CDS was 1254 bp,encoded a total of 417 amino acids with a molecular weight of 46.78 ku.Protein secondary structure analysis showed that the protein encoded by GmPUB24 contained alpha helix,random coil,extended chain and beta turn,and the alpha helix accounted for the highest proportion of 58.9%.Protein encoded by GmPUB24 gene was a hydrophilic protein without transmembrane domain and signal peptide.Protein phylogenetic tree displayed that it had the highest affinity with wild soybean.Subcellular location predicted that it localized in the cytoplasm.Promoter analysis of 1500 bp upstream of GmPUB24 gene showed that it contained CGTCA-motif,TGACG-motif and other elements that respond to disease resistance pathways,as well as ABRE,WUN-motif,TATA-box and other corresponding abiotic stress elements.Quantitative real-time PCR(qRT-RCR)results showed that the expression of GmPUB24 was induced by soybean cyst nematode,and the highest expression level in roots was 6.14 times compared with the uninoculated sample on the third day,indicating that GmPUB24 gene was induced by soybean cyst nematode and might be involved in the process of soybean defense against cyst nematode.These results provided a basis for clarifying the regulatory mechanism of soybean U-box family gene in resistance to soybean cyst nematode disease.