中国农业科学2017，Vol.50Issue(19)：3723-3732,10.DOI:10.3864/j.issn.0578-1752.2017.19.009
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
Identification and Expression Analysis of a New Pectate Lyase GeneHa-pel-1 fromHeterodera avenae
摘要
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 fromH. avenae.[Method]A novel pectate lyase gene Ha-pel-1 was cloned fromH. 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, Signal P 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-1were analyzed byin situhybridization 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 1717 bp in length which contained a 1563 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 7199 bp. Gene structure analysis showed that theHa-pel-1genome 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 ofHa-pel-1were mainly expressed in the two subventral gland cells ofH. 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 geneHa-pel-1 from H. avenae, closely related to the infection and parasitic process of cereal cyst nematode, was found and analyzed.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Key words
Heterodera avenae/pectate lyase/gene cloning/in situhybridization/developmental expression analysis引用本文复制引用
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):3723-3732,10.基金项目
国家"973"计划(2013CB127502)、国家公益性行业(农业)科研专项(201503114) （2013CB127502）
