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

发布时间：2018-04-07 17:18

本文选题：禾谷孢囊线虫　切入点：果胶酸裂解酶　出处：《中国农业科学》2017年19期

【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、Signal P 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(Gen Bank登录号GQ998895),该基因c DNA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 k D,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。
[Abstract]:[objective] Heterodera avenaeae is an important plant nematode that seriously endangers wheat crops, which causes huge economic losses to agricultural production. However, its pathogenic mechanism and effective control methods need to be further studied.A new Pectinate lyase gene Ha-pel-1 was cloned and its expression characteristics were analyzed in order to provide theoretical basis for further study on gene function of Ha-pel-1 and its interaction with host.Methods A new gene of Pectinic acid lyase was cloned from C. graminearum by homologous cloning combined with RACE technique.The nucleotide and amino acid sequences of the gene were analyzed by using the bioinformatics software such as DNAMAN Clustalal signal P 4.0 Server and GSDS, and the phylogenetic tree was constructed by MEGA 5.0.In situ hybridization and semi-quantitative PCR were used to determine the expression site of the gene in cereal cyst nematode and its expression in different ages of nematode. [results] A pectinic acid was cloned successfully from cereal cyst nematode.Ha-pel-1(Gen Bank accession number GQ998895GQ998895A, c DNA is 1 717 BP long and contains an open reading frame of 1 563 BP.A protein encoding 521 amino acid residues with a theoretical molecular weight of 57.5 KD and a theoretical isoelectric point of 8.52.The length of Ha-pel-1 genome was 7 199 BP, which was amplified from the genomic DNA of nematode. The gene structure analysis showed that the Ha-pel-1 genome contained 14 exons and 13 introns, with the exception of the third intron splicing site (GC-AG).The other 12 introns all accord with the GT-AG rule of splicing site of eukaryotic gene.The results of homology comparison showed that the C terminal of the predicted protein Ha-PEL-1 was 67% consistent with that of soybean cyst nematode Pectinic acid lyase HG-PEL-1 and beet cyst nematode Pectinic acid lyase HS-PEL-1, and the similarity was 83% between the predicted protein and soybean cyst nematode Pectinic acid lyase HG-PEL-1.The N-terminal signal peptide produced a length of 254 amino acid residues from the Pectinic lyase of other plant parasitic nematodes. In this sequence, the 184 amino acid residues near the N-terminal were not similar to the proteins in the database.There was 32% agreement and 47% similarity between the 70 amino acid residues Lys 205-Glu274 and the methyltransferase region of Wessels Brown virus NS5 protein (registration number 3ELDD).Amino acid sequence analysis showed that the predicted protein Ha-PEL-1 contained a signal peptide with a length of 20 amino acid residues and a highly conserved region of the third family of Pectinic acid lyase, as well as a number of conserved cysteine residues.Phylogenetic analysis showed that Ha-pel-1 and other reported PEL genes of nematode Pectinase were clustered in a large branch of PEL from bacteria and fungi, and in situ hybridization showed that Ha-pel-1 was mainly expressed in the subventral esophagus of C. graminearum.Semi-quantitative RT-PCR confirmed the high expression of Ha-pel-1 in pre-parasitic and post-parasitic second instar larvae. [conclusion] the cloning and expression characteristics of a new Pectinic acid lyase gene Ha-pel-1 in cereal cyst nematode were analyzed.It was revealed that the gene was closely related to the infection and parasitism of cereal cyst nematode.
【作者单位】：中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室;中国热带农业科学院橡胶研究所;中国热带农业科学院环境与植物保护研究所农业部热带作物有害生物综合治理重点实验室;
【基金】：国家“973”计划(2013CB127502) 国家公益性行业(农业)科研专项(201503114)
【分类号】：S432.45

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