禾谷孢囊线虫果胶酸裂解酶新基因Ha-pel-1的鉴定与表达特征分析

doi:10.3864/j.issn.0578-1752.2017.19.009

摘要/Abstract

摘要： 【目的】禾谷孢囊线虫（Heterodera avenae。）是一种严重危害麦类作物的重要植物病原线虫，对农业生产造成巨大的经济损失，然而其致病机制和有效防控方法还有待进一步研究。通过克隆禾谷孢囊线虫的果胶酸裂解酶新基因Ha-pel-1，并对其表达特性进行分析，为后续探究Ha-pel-1的基因功能及其与寄主的互作提供理论依据，并为探讨禾谷孢囊线虫防控途径提供新思路。【方法】采用同源克隆结合RACE技术从禾谷孢囊线虫中克隆出一个新的果胶酸裂解酶基因；采用DNAMAN、Clustal、SignalP 4.0 Server和GSDS等相关生物信息学软件和在线工具分析该基因的核苷酸和氨基酸序列，并使用MEGA 5.0构建系统进化树；采用原位杂交和半定量PCR方法确定该基因的在禾谷孢囊线虫中的表达部位及其在线虫不同龄期中的表达情况。【结果】从禾谷孢囊线虫中成功克隆出一个果胶酸裂解酶基因Ha-pel-1（GenBank登录号GQ998895)，该基因cDNA全长1 717 bp，包含一个长度为1 563 bp的开放阅读框，编码一个长度为521个氨基酸残基的蛋白，其理论分子量为57.5 kD，理论等电点为8.52。从线虫基因组DNA中扩增获得长度为7 199 bp的Ha-pel-1基因组全长，基因结构显示分析发现，Ha-pel-1基因组包含14个外显子和13个内含子，除第3个内含子剪接位点是GC-AG外，其余12个内含子都符合真核生物基因剪接位点GT-AG规则。同源比对结果表明，预测蛋白Ha-PEL-1的C端与大豆孢囊线虫果胶酸裂解酶HG-PEL-1、甜菜孢囊线虫果胶酸裂解酶HS-PEL-1均有67%的一致性和83%的相似性；此外，其N端信号肽后比报道的其他植物寄生线虫果胶酸裂解酶多出一段长度为254个氨基酸残基的序列，这段序列中，靠近N端的184个氨基酸残基与数据库中的蛋白均无相似性，而靠近C端有70个氨基酸残基（Lys205—Glu274）与韦塞尔斯布朗病毒NS5蛋白（注册号3ELD）的甲基转移酶区域有32%的一致性和47%的相似性。氨基酸序列分析发现，预测蛋白Ha-PEL-1包含一个长度为20个氨基酸残基的信号肽和4个果胶酸裂解酶第3家族（PL3）的高度保守区域以及多个保守的半胱氨酸残基；系统进化分析发现，Ha-pel-1及其他已报道的线虫果胶酸裂解酶基因与细菌和真菌来源的PEL聚在一个大的分支中；原位杂交结果显示Ha-pel-1主要在禾谷孢囊线虫亚腹食道腺中表达；半定量RT-PCR确定Ha-pel-1在寄生前和寄生后的2龄幼虫中大量表达。【结论】通过对禾谷孢囊线虫中一个新果胶酸裂解酶基因Ha-pel-1的克隆和表达特征分析，揭示该基因与禾谷孢囊线虫的侵染和寄生过程密切相关。

关键词: 禾谷孢囊线虫, 果胶酸裂解酶, 基因克隆, 原位杂交, 发育表达分析

Abstract: 【Objective】The cereal cyst nematode (Heterodera avenae) is one of the important plant parasitic nematodes which seriously threatened cereal crops and caused huge economic losses to agricultural production in China. However, its pathogenic mechanism and effective prevention and control methods still need to be further studied. The objective of this study is to provide a theoretical basis for further study on the gene function of Ha-pel-1 and its interaction with host plants, and to give new ideas for the control strategies of cereal cyst nematode based on the cloning and expression analysis of a new pectate lyase gene Ha-pel-1 from H. avenae.【Method】A novel pectate lyase gene Ha-pel-1 was cloned from H. avenae using homology cloning combined with RACE technology, and its nucleotide sequence and amino acid sequence were analyzed by related bioinformatics softwares and online tools, such as DNAMAN, Clustal, SignalP 4.0 Server and GSDS. a phylogenetic tree was also constructed using MEGA 5.0. the tissue localization and developmental expression characteristics of Ha-pel-1 were analyzed by in situ hybridization and semi-quantitative PCR method. 【Result】 A novel pectate lyase gene (Ha-pel-1, GenBank accession number GQ998895) was cloned successfully from H. avenae. Ha-pel-1 was 1 717 bp in length which contained a 1 563 bp open reading frame (ORF) encoding a protein of 521 amino acid residues. The molecular weight of Ha-pel-1 encoding protein was 57.5 kD and isoelectric point was 8.52. The full length of genomic sequence of Ha-pel-1 was amplified from the nematode genome DNA which contains 7 199 bp. Gene structure analysis showed that the Ha-pel-1 genome contains 14 exons and 13 introns, except for the 3rd intron splice sites are GC-AG, the other 12 introns are in line with the rules of the eukaryotic gene splicing site GT-AG. The results of homologous comparison showed that the C-terminal sequence of the putative Ha-PEL-1 had a 67% identity and a similarity of 83% with that of soybean cyst nematode HG-PEL-1 and beet cyst nematode HS-PEL-1. In addition, after the end of N-terminal signal peptide, the putative Ha-PEL-1 had a sequence of 254 amino acid residues more than other reported plant parasitic nematodes pectate lyases. In this sequence, 184 amino acid residues closing to the N-terminal had no similarity with protein database, while 70 amino acid residues (Lys205-Glu274) closing to the C-terminal had an identity of 32% and a similarity of 47% with the methyltransferase domain of Wesselsbron virus NS5 (Registration No. 3ELD). The amino acid sequence analysis revealed that the predicted protein contained a signal peptide of 20 amino acid residues, as well as 4 highly conserved regions and several conserved cysteine residues characteristic of class Ⅲ pectate lyases (PL3). A phylogenetic analysis revealed that Ha-pel-1 and other nematodes pectate lyase genes are gathered in a large branch with bacterial and fungal sources PEL. In situ hybridization analyses showed that the transcripts of Ha-pel-1 were mainly expressed in the two subventral gland cells of H. avenae. a semi-quantitative RT-PCR analysis confirmed that its transcriptions were highly expressed at the pre-parasitic and parasitic 2nd stage juveniles.【Conclusion】A new pectate lyase gene Ha-pel-1 from H. avenae, closely related to the infection and parasitic process of cereal cyst nematode, was found and analyzed.

Key words: Heterodera avenae, pectate lyase, gene cloning, in situ hybridization, developmental expression analysis

李新，顾晓川，龙海波，彭焕，黄文坤，彭德良. 禾谷孢囊线虫果胶酸裂解酶新基因Ha-pel-1的鉴定与表达特征分析[J]. 中国农业科学, 2017, 50(19): 3723-3732.

LI Xin, GU XiaoChuan, LONG HaiBo, PENG Huan, HUANG WenKun, PENG DeLiang. Identification and Expression Analysis of a New Pectate Lyase Gene Ha-pel-1 from Heterodera avenae[J]. Scientia Agricultura Sinica, 2017, 50(19): 3723-3732.

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