摘要： 为防治水稻干尖线虫病提供理论支持,研究了水稻干尖线虫氨基肽酶基因特征,并对其编码蛋白结构与功能进行分析。基于水稻干尖线虫转录组测序,并通过RACE技术,从水稻干尖线虫中克隆得到了一氨基肽酶基因 Ab-Ampep。生物信息学进行基因分析,原位杂交实验进行表达定位分析。该基因CDS序列全长为2 787 bp,含有2个开放阅读框(ORF)。最长开放阅读框(ORF)大小2 661 bp,可编码886个氨基酸,蛋白分子量为101.74 kDa,理论等电点(PI)为5.78。编码蛋白氨基酸序列中具有2个Zn²⁺结合位点和1个PepN保守结构域,有特征信号序列TIS HE LA H FW,属于谷氨酸锌蛋白(GluZincin)超家族。Ab-Ampep氨基酸序列同挑选的其他9种动物的氨基肽酶氨基酸序列进行多重序列分析,发现功能区域的氨基酸序列较为保守,Ab-Ampep与猪蛔虫(Ascaris suum)的氨基肽酶P(ERG83308.1)蛋白相似度最高为50%。系统进化树分析表明,Ab-Ampep与猪蛔虫(A.suum)的氨基肽酶P(ERG83308.1)蛋白在同一分支上,亲缘关系最近。蛋白质结构分析显示,α-螺旋为Ab-Ampep主要结构元件,能够形成金属离子Zn²⁺结合位点。原位杂交实验显示,经反义链探针处理的线虫在其生殖部位有颜色加深的杂交信号,该基因的表达位置主要在水稻干尖线虫的生殖部位。通过对水稻干尖线虫氨基肽酶基因研究,利用分子技术改造氨基肽酶或寻找氨基肽酶抑制剂,抑制氨基肽酶的表达,影响线虫生长发育,为防治水稻干尖线虫病提供新思路。

关键词: 水稻干尖线虫, 氨基肽酶基因, 基因克隆, 生物信息学分析, 原位杂交

Abstract: To study the characteristics of the aminopeptidase gene in white-tip nematode,and to provide theory support for the prevention.According to the transcriptome resequencing,an aminopeptidase gene of A.besseyi was isolated by RACE method,and designed as Ab-Ampep.Bioinformatic and in-situ hybridization were used for the gene analysis.The coding sequence was 2 787 bp including a 2 661 bp open reading frame (ORF),which encoded an 886 amino acid protein.The putative protein molecular weight was 101.74 kDa and its theoretical isoelectric point was 5.78.The deduced amino acid sequence contains two conserved zinc-binding sites and a PepN Multi-domain.It belongs to GluZincin Superfamily,which contained the characteristic conserved sequence of HE xx H.The multiple sequence alignment showed that the functional region was conserved,and the highest level of similarity was 50% to a aminopeptidase(ERG83308.1)from Ascaris suum in GenBank.The phylogenetic tree constructed on the basis of amino acid sequences clearly showed that the relationship of Ab-Ampep was most intimate with aminopeptidase(ERG83308.1)from A.suum. Protein structure analysis indicated that the main structural element in Ab-Ampep protein was α-Helix.In situ hybridization indicated that enhanced hybridization signals were observed in genital area,and the expression of Ab-Ampep was similarly in genital area of A.besseyi.Though the functional analysis of aminopeptidase gene,molecular technology or enzyme inhibitor was used to inhibit the expression of aminopeptidase.It may affect the nematode development and provide a new way for prevention.

Key words: Aphelenchoides besseyi, Aminopeptidase gene, Gene cloning, Bioinformatics analysis, In situ hybridization

中图分类号: 

• S435.111.48

• Q786