大豆孢囊线虫果胶酸裂解酶基因Hg-pel-5的克隆与分析

doi:10.3864/j.issn.0578-1752.2012.05.005

摘要/Abstract

摘要： 【目的】克隆和分析与大豆孢囊线虫寄生密切相关的果胶酸裂解酶新基因，为研究大豆孢囊线虫寄生和致病的分子机理提供依据，并为探讨大豆孢囊线虫的防控新途径提供理论基础。【方法】通过EST分析结合RACE-PCR扩增方法，从大豆孢囊线虫中克隆出1个果胶酸裂解酶新基因；通过原位杂交和半定量PCR的方法确定基因的表达部位和分析不同发育阶段的基因表达；采用Southern杂交方法分析基因的拷贝数。【结果】从大豆孢囊线虫中克隆出1个全长为957个碱基编码227个氨基酸残基的新果胶酸裂解酶基因Hg-pel-5（GenBank 登录号HQ123259）。Hg-pel-5 基因组由2个外显子和1个内含子组成。预测蛋白含有20个氨基酸残基的信号肽和4段细菌结构的果胶酸裂解酶第三家族的保守位点。原位杂交确定Hg-pel-5在大豆孢囊线虫亚腹食道腺中表达。半定量RT-PCR结果表明Hg-pel-5在寄生前和寄生过程早期的2龄幼虫大量表达。Southern杂交结果显示Hg-pel-5存在于大豆孢囊线虫基因组中并以多拷贝形式存在。【结论】对大豆孢囊线虫中1个新的果胶酸裂解酶基因Hg-pel-5进行克隆和分析，表明该基因在大豆孢囊线虫早期寄生过程中发挥重要作用。

关键词: 大豆孢囊线虫, 果胶酸裂解酶, 基因克隆, 原位杂交, Southern 杂交

Abstract: Abstract:【Objective】The objective of this study is to clone and analyze the pectate lyase genes from soybean cyst nematode (Heterodera glycines), understand the parasitism and pathogenicity of plant parasitic nematodes, and to develop new control strategies for soybean cyst nematode. 【Method】A noval pectate lyase gene Hg-pel-5 was cloned from H. glycines by EST analysis and RACE. The tissue localization and developmental expression of pectate lyase were analysed at different life stages of H. glycines by in situ mRNA hybridization and RT-PCR amplification, and the copy number of the gene was identified by Southern blot. 【Result】 A novel parasitism gene encoding pectate lyase, named Hg-pel-5, was cloned from H. glycines. The full length cDNA of Hg-pel-5 consists of 957 bp, with a 681 bp ORF encoding a 227 amino acid residues. The structure of Hg-pel-5 genomic DNA includes 2 extons and 1 intron. The predicted protein includes a signal peptide of 20 amino acids on the N-terminal and 4 conserved regions characteristic of class Ⅲ pectate lyases of microbes. In situ mRNA hybridization analyses showed that the transcripts of Hg-pel-5 was expressed in subventral oesophageal gland cells of H. glycines. RT-PCR analysis confirmed that Hg-pel-5 transcriptions were mainly expressed at the pre-parasitic and early parasitic second-stage juveniles. Southern blotting confirmed that Hg-pel-5 was of nematode origin and a member of a small multigene family. 【Conclusion】 A novel pectate lyase gene was cloned and analyzed from H. glycines, and these results imply that the Hg-pel-5 might play an important role during the early stages of plant parasitism.

Key words: Heterodera glycines, pectate lyase, gene cloning, in situ hybridization, Southern blot

彭焕, 彭德良, 黄文坤, 贺文婷, 胡先奇. 大豆孢囊线虫果胶酸裂解酶基因Hg-pel-5的克隆与分析[J]. 中国农业科学, 2012, 45(5): 854-866.

PENG Huan, PENG De-Liang, HUANG Wen-Kun, HE Wen-Ting, HU Xian-Qi. Molecular Cloning and Analysis of a Novel Pectate Lyase Gene Hg-pel-5 from Soybean Cyst Nematode[J]. Scientia Agricultura Sinica, 2012, 45(5): 854-866.

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参考文献

[1]刘维志. 植物病原线虫学. 北京: 中国农业出版社, 2000: 281-293.

Liu W Z. Plant Nematology. Beijing: China Agriculture Press, 2000: 281-293. (in Chinese)

[2]卢为国, 盖钧镒, 李卫东. 黄淮地区大豆胞囊线虫生理小种的抽样调查与研究. 中国农业科学, 2006, 39(2): 306-312.

Lu W G, Gai J Y, Li W D. Sampling survey and identification of races of soybean cyst nematode (Heterodera glycines lchinohe) in Huang-Huai Valleys. Scientia Agriculture Sinica, 2006, 39(2): 306-312. (in Chinese)

[3]张俊立, 彭德良, 曹克强. 河北省大豆胞囊线虫分子鉴定及其分布. 植物保护, 2005, 31(1): 40-43.

Zhang J L, Peng D L, Cao K Q. Molecular identification and distribution of Heterodera glycines in Hebei. Plant Protection, 2005, 31(1): 40-43. (in Chinese)

[4]Davis E L, Hussey R S, Baum T J, Bakker J, Schots A, Rosso M N, Abad P. Nematode parasitism genes. Annual Review of Phytopathology, 2000, 38: 365-396.

[5]Davis E L, Hussey R S, Baum T J. Getting to the roots of parasitism by nematodes. Trends in Parasitology, 2004, 20(3): 134-141.

[6]彭德良, 郑经武, 廖金铃, 万方浩. 重要植物线虫致病相关基因研究进展//彭友良. 中国植物病理学会: 2006年学术年会论文集. 北京: 中国农业科技出版社, 2006: 221-229.

Peng D L, Zheng J W, Liao J L, Wan F H. Advanced on putative patasitim genes of plant-parasitic nematodes and prospective//Peng Y L. Proceedings of the Annual Meeting of Chinese Society for Plant Pathology. Beijing: China Agricultural Scientech Press, 2006: 221-229. (in Chinese)

[7]Popeijus H, Overmars H, Jones J, Blok V, Goverse A, Helder J, Schots A, Bakker J, Smant G. Degradation of plant cell walls by a nematode. Nature, 2000, 406: 36-37.

[8]Doyle E A, Lambert K N. Cloning and characterization of an esophageal-gland-specific pectate lyase from the root-knot nematode Meloidogyne javanica. Molecular Plant-Microbe Interactions, 2002, 15(6): 549-556.

[9]De Boer J M, McDermott J P, Davis E L, Hussey R S, Popeijus H, Smant G, Baum T J. Cloning of a putative pectate lyase gene expressed in the subventral esophageal glands of Heterodera glycines. Journal of Nematology, 2002, 34(1): 9-11.

[10]Bakhetia M, Urwin P E, Atkinson H J. qPCR analysis and RNAi define pharyngeal gland cell-expressed genes of Heterodera glycines required for initial interactions with the host. Molecular Plant-Microbe Interactions, 2007, 20(3): 306-312.

[11]Sukno S A, McCuiston J, Wong M Y, Wang X H, Thon M R, Hussey R, Baum T, Davis E. Quantitative detection of double-stranded RNA-mediated gene silencing of parasitism genes in Heterodera glycines. Journal of Nematology, 2007, 39(2): 145-152.

[12]Huang G, Dong R, Allen R, Davis E L, Baum T J, Hussey R S, Developmental expression and molecular analysis of two Meloidogyne incognita pectate lyase genes. International Journal for Parasitology, 2005, 35: 685-692.

[13]Vieira P, Danchin E G J, Neveu C, Crozat C, Jaubert S, Hussey R S, Engler G, Abad P, de Almeida-Engler J, Castagnone-Sereno P, Rosso M N. The plant apoplasm is an important recipient compartment for nematode secreted proteins. Journal of Experimental Botany, 2011, 62(3):1241-1253.

[14]Kikuchi T, Shibuya H, Aikawa T, Jones J T. Cloning and characterization of pectate lyases expressed in the esophageal gland of the pine wood nematode Bursaphelenchus xylophilus. Molecular Plant-Microbe Interactions, 2006, 19(3): 280-287.

[15]Vanholme B, Van Thuyne W, Vanhouteghem K, De Meutter J, Cannoot B, Gheysen G. Molecular characterization and functional importance of pectate lyase secreted by the cyst nematode Heterodera schachtii. Molecular Plant Pathology, 2007, 8(3): 267-278.

[16]Kudla U, Milac A L, Qin L, Overmars H, Roze E, Holterman M, Petrescu A J, Goverse A, Bakker J, Helder J, Smant G. Structural and functional characterization of a novel, host penetration-related pectate lyase from the potato cyst nematode Globodera rostochiensis. Molecular Plant Pathology, 2007, 8(3): 293-305.

[17]Stare B G, Fouville D, Sirca S, Gallot A, Urek G, Grenier E. Molecular variability and evolution of the pectate lyase (pel-2) parasitism gene in cyst nematodes parasitizing different Solanaceous plants. Journal of Molecular Evolution, 2011, 72(2): 169-181.

[18]Karim N, Jones J T, Okada H, Kikuchi T. Analysis of expressed sequence tags and identification of genes encoding cell-wall- degrading enzymes from the fungivorous nematode Aphelenchus avenae. BMC Genomics, 2009, 10: 525.

[19]Abad P, Gouzy J, Aury J M, Castagnone-Sereno P, Danchin E G J, Deleury E, Perfus-Barbeoch L, Anthouard V, Artiguenave F, Blok V C, Caillaud M C, Coutinho P M, Dasilva C, De Luca F, Deau F, Esquibet M, Flutre T, Goldstone J V, Hamamouch N, Hewezi T, Jaillon O, Jubin C, Leonetti P, Magliano M, Maier T R, Markov G V, McVeigh P, Pesole G, Poulain J, Robinson-Rechavi M, Sallet E, Ségurens B, Steinbach D, Tytgat T, Ugarte E, Van Ghelder C, Veronico P, Baum T J, Blaxter M, Bleve-Zacheo T, Davis E L, Ewbank J J, Favery B, Grenier E, Henrissat B, Jones J T, Laudet V, Maule A G, Quesneville H, Rosso M N, Schiex T, Smant G, Weissenbach J, Wincker P. Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita. Nature Biotechnology, 2008, 26(8): 909-915.

[20]Opperman C H, Bird D M, Williamsond V M, Rokhsar D S, Burke M, Cohn J, Cromera J, Diener S, Gajan J, Graham S, Houfek T D , Liu Q L, Mitros T , Schaff J, Schaffer R, Scholl E, Sosinski B R, Thomas V P, Windham E. Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(39): 14802-14807.

[21]Kikuchi T, Cotton J A, Dalzell J J, Hasegawa K, Kanzaki N, McVeigh P, Takanashi T, Tsai I J, Assefa S A, Cock P J A, Otto T D, Hunt M, Reid A J, Sanchez-Flores A, Tsuchihara K, Yokoi T, Larsson M C, Miwa J, Maule A G, Sahashi N, Jones J T, Berriman M. Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus. PLoS Pathogens, 2011, 7(9): e1002219.

[22]Haegeman A, Joseph S, Gheysen G. Analysis of the transcriptome of the root lesion nematode Pratylenchus coffeae generated by 454 sequencing technology. Molecular and Biochemical Parasitology, 2011, 178(1/2): 7-14.

[23]Jaubert S, Laffaire J B, Abad P, Rosso M N. A polygalacturonase of animal origin isolated from the root-knot nematode Meloidogyne incognita. FEBS Letters, 2002, 522: 109-112.

[24]Davis E L, Haegeman A, Kikuchi T. Degradation of the Plant Cell Wall by Nematodes//Jones J, Gheysen G, Fenoll G. Genomics and Molecular Genetics of Plant-Nematode Interactions. London: Springer, 2011, Part 3: 255-272.

[25]彭焕, 彭德良, 黄文坤. 甘薯茎线虫 (Ditylenchus destructor) β-1,4内切葡聚糖酶基因 (Dd-eng-1b) cDNA全长的克隆与序列分析. 农业生物技术学报, 2009, 17(6): 1035-1041.

Peng H, Peng D L, Huang W K. Molecular cloning and sequence analysis of a new β-1, 4-endoglucanase gene (Dd-eng-1b) from plant-parasitic nematode Ditylenchus destructor on sweetpotato in China. Chinese Journal of Agricultural Biotechnology, 2009, 17(6): 1035-1041. (in Chinese)

[26]Bendtsen J D, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. Journal of Molecular Biology, 2004, 340(4): 783-795.

[27]Anisimova M, Gascuel O. Approximate likelihood-ratio test for branchs: A fast, accurate and powerful alternative. Systematic Biology, 2006, 55(4): 539-552.

[28]Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.

[29]Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics, 2006, 22(2): 195-201.

[30]De Boer J M, Yan Y, Smant G, Davis E L, Baum T J. In situ hybridization to messenger RNA of Heterodera glycines. Journal of Nematology, 1998, 30(3): 309-312.

[31]Elling A A, Mitreva M, Recknor J, Gai X, Martin J, Maier T R, McDermott J P, Hewezi T, Bird D M, Davis E L, Hussey R S, Nettleton D, McCarter J P, Baum T J. Divergent evolution of arrested development in the dauer stage of Caenorhabditis elegans and the infective stage of Heterodera glycines. Genome Biology, 2007, 8: R211.

[32]Shevchik V, Robert-Baudouy J, Hugouviux-Cotte-Pattat N. Pectate lyase PelI of Ervinia chrysanthemi 3937 belongs to a new family. The Journal of Bacteriology, 1997, 179(23): 7321-7330.

[33]Akita M, Suzuki A, Kobayashi T, Ito S, Yamane T. The first structure of pectate lyase belonging to polysaccharide lyase family 3. Acta Crystallographica Section D, 2001, 57: 1786-1792.

[34]Herron S R, Benen J A E, Scavetta R D, VisserJ, Jurnak F. Structure and function of pectic enzymes: virulence factors of plant pathogens. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(16): 8762-8769.

[35]Vanholme B, De Meutter J, Tytgat T, Van Montagu M, Coomans A, Gheysen G. Secretions of plant-parasitic nematodes: a molecular update. Gene, 2004, 332: 13-27.

[36]Garron M L, Cygler M. Structural and mechanistic classification of uronic acid-containing polysaccharide lyases. Glycobiology, 2010, 20(12): 1547-1573.

[37]Danchin E G J , Rosso M N, Vieira P, de Almeida-Engler J, Coutinho P M, Henrissat B, Abad P. Multiple lateral gene transfers and duplications have promoted plant parasitism ability in nematodes. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(41): 17651-17656.

[38]Haegeman A, Jones J T, Danchin E G J. Horizontal gene transfer in nematodes: a catalyst for plant parasitism? Molecular Plant-Microbe Interactions, 2011, 24(8): 879-887.

[39]Jones J T, Furlanetto C, Kikuchi T. Horizontal gene transfer from bacteria and fungi as a driving force in the evolution of plant parasitism in nematodes. Nematology, 2005, 7(5): 641-646.

[40]Smant G, Stokkermans J P W G, Yan Y T, de Boer J M, Baum T J, Wang X H, Hussey R S, Gommers F J, Henrissat B, Davis E L, Helder J, Schots A, Bakker J. Endogenous cellulases in animals: Isolation of β-1,4-endoglucanase genes from two species of plant-parasitic cyst nematodes. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(9): 4906-4911.