大豆孢囊线虫扩展蛋白新基因（Hg-exp-1、Hg-exp-2）的鉴定及功能分析

doi:10.3864/j.issn.0578-1752.2018.17.006

摘要/Abstract

摘要： 【目的】大豆孢囊线虫(Heterodera glycines)是大豆上最重要的土传病原物之一，由孢囊线虫口针分泌的扩展蛋白（expansin）在线虫侵染过程中发挥了重要作用。本研究旨在鉴定大豆孢囊线虫扩展蛋白基因，并对其结构、组织定位和发育表达特性进行研究，为进一步明确大豆孢囊线虫的寄生、致病机制打下基础。【方法】利用同源基因克隆技术，根据已报道的线虫expansin基因的保守序列设计上下游简并引物，从大豆孢囊线虫2龄幼虫cNDA中克隆expansin基因的EST片段，根据EST片段序列设计RACE特异性引物，通过RACE技术扩增测序后采用DNAstar 7.1和DNAman软件对序列进行比对和拼接，得到大豆孢囊线虫expansin基因cDNA全长；采用CLC sequence viewer 6进行开放阅读框查找、蛋白质翻译和序列比对；使用EBI在线软件SignalP 3.0 Server和TMHMM软件进行预测蛋白质前体信号肽和跨膜结构域预测，GSDS在线软件进行基因组结构分析；使用PHYML软件和MEGA5.0软件最大似然法对获得的基因与其他线虫expansin基因进行比对与系统进化树构建；采用southern杂交技术分析Hg-exp-1。在大豆孢囊线虫基因组中的拷贝数；通过原位杂交技术确定2个expansin基因的表达部位；提取卵、侵染前2龄幼虫、侵染后2龄幼虫、3龄幼虫、4龄幼虫与白雌虫的cDNA作为模板，通过半定量PCR技术分析expansin基因在线虫不同龄期的发育表达特性；根据Hg-exp-1基因序列设计引物扩增并合成dsRNA，对大豆孢囊线虫2龄幼虫浸泡处理24 h后接种大豆植株，分析Hg-exp-1 RNA干扰后对线虫侵染的影响。【结果】从大豆孢囊线虫2龄幼虫中成功克隆出2个扩展蛋白基因全序列，命名为Hg-exp-1和Hg-exp-2，长度分别为1 047和1 037 bp，分别编码长度为288和295个氨基酸的多肽，2个预测蛋白N端均含有信号肽，无跨膜结构域，表明其为分泌型蛋白。序列比对发现大豆孢囊线虫HG-EXP-1序列与马铃薯金线虫（Globodera rostochiensis）GR-EXPB1（CAC83611）和GR-EXPB2（CAC84564）以及非洲茎线虫（Ditylenchus africanus）DA-EXPB1（ADJ57307）等具有高度一致性。Southern杂交分析表明，expansin基因在大豆孢囊线虫中可能以多拷贝方式或多基因家族存在。原位杂交显示它们特异地在大豆孢囊线虫亚腹食道腺表达。Hg-exp-1体外RNA干扰后，2龄幼虫浸泡在dsRNA 24 h，靶基因的转录水平下调，接种后大豆根内线虫2龄幼虫数和雌虫数分别下降了38.3%和43.4%。【结论】从大豆孢囊线虫中成功分离鉴定出2个expansin基因，同时明确了其在大豆孢囊线虫寄生早期过程中起重要作用。

关键词: 大豆孢囊线虫, 扩展蛋白, 发育表达, RNA干扰

Abstract: 【Objective】Soybean cyst nematode (Heterodera glycines) is a devastating disease all over the world. The expanded protein (expansin) secreted by stylet plays an important role in the parasitism of H. glycines. The objective of this study is to identify the expansin gene from H. glycines, understand its structure, tissue localization and the expression characteristics at different developmental stages, so as to lay a foundation for further clarifying the parasitic and pathogenic mechanism of H. glycines. 【Method】According to the conserved sequence of the reported expansin gene, upstream and downstream degenerate primers were designed and the EST fragment of expansin gene from the 2nd stage juveniles of H. glycines was cloned. According to the sequence of EST fragment, RACE specific primers were designed. After amplified and sequenced by RACE technique, the sequence was compared and spliced by DNAstar 7.1 and DNAman software. The full length of expansin gene cDNA of H. glycines was obtained. CLC sequence viewer 6 was used for open reading frame search, protein translation and sequence alignment. On-line software SignalP 3.0 Server and TMHMM were used to predict protein precursor signal peptide and transmembrane domain, and GSDS was used to analyze the genome structure. Using PHYML and MEGA 5.0 software maximum likelihood method, the obtained genes were compared with other nematode expansin genes to construct phylogenetic tree. Southern hybridization was used to analyze the number of the Hg-exp-1 copies in the genome. The expression sites of two genes were confirmed by in situ hybridization. The cDNAs of eggs, pre-parasitic 2nd stage juvenile, parasitic 2nd stage juvenile, parasitic 3rd stage juvenile, parasitic 4th stage juvenile and females were extracted as templates, the developmental expression characteristics were analyzed by semi quantitative PCR. According to the sequence of Hg-exp-1, primers were designed to amplify and synthesize dsRNA. Soybean plantlets were inoculated with 2nd stage juvenile after immersion for 24 h. RNA interference in vitro method was used to identify the function of Hg-exp-1. 【Result】The full-length cDNAs of two expansin genes named Hg-exp-1 and Hg-exp-2 were successfully cloned from the 2nd stage juveniles of H. glycines, with a length of 1 047 and 1 037 bp, and the peptides with length of 288 and 295 amino acids were encoded. Both of the two predicted proteins contained a signal peptide in N-terminal and had no transmembrane domain, indicating that they were secretory proteins. Sequence alignment showed that the HG-EXP-1 sequence of H. glycines was highly consistent with GR-EXPB1 (CAC83611) and GR-EXPB2 (CAC84564) in Globodera rostochiensis and DA-EXPB1 (ADJ57307) in Ditylenchus africanus. Southern blot analysis showed the expansin genes might exist in H. glycines in multi-copy mode or members of a small multi-gene family. In situ hybridization analyses showed that the transcripts of them accumulated exclusively in the subventral oesophageal gland cells of H. glycines. The results of Hg-exp-1 interference in vitro showed that the transcriptional level of the target gene was down-regulated in nematode treated with dsRNA for 24 h. After Hg-exp-1 was silenced, the number of 2nd stage juveniles infected in soybean root and females decreased by 38.3% and 43.4% than the control, respectively.【Conclusion】 two expansin genes were successfully isolated and identified from H. glycines, and their important role in the early parasitic process of H. glycines was also clarified.

Key words: Heterodera glycines, expansin, developmental expression, RNA interference

张瀛东，孔详超，黄文坤，孔令安，李红梅，彭焕，彭德良. 大豆孢囊线虫扩展蛋白新基因（Hg-exp-1、Hg-exp-2）的鉴定及功能分析[J]. 中国农业科学, 2018, 51(17): 3302-3314.

ZHANG YingDong, KONG XiangChao, HUANG WenKun, KONG LingAn, LI HongMei, PENG Huan, PENG DeLiang. Identification and Functional Analysis of Two Expansin Genes Hg-exp-1 and Hg-exp-2 from the Soybean Cyst Nematode (Heterodera glycines)[J]. Scientia Agricultura Sinica, 2018, 51(17): 3302-3314.

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参考文献

[1] 彭德良, 朱振东. 大豆病害防治//王连铮. 大豆研究50年. 北京: 中国农业科学技术出版社, 2010: 319-334.

Peng D L, Zhu Z D. Control of soybean disease//Wang L Z. 50 years of Soybean Research. Beijing: China Agricultural Science and Technology Press, 2010: 319-334. (in Chinese)

[2] 张俊立, 彭德良, 曹克强. 河北省大豆孢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)

[3] Davis E L, Hussey R S, Baum T J. Parasitism genes: What they reveal about parasitism//Plant Cell Monographs. Springer, 2008: 15-44.

[4] Gheysen G, Mitchum M G. How nematodes manipulate plant development pathways for infection. Current Opinion in Plant Biology, 2011, 14(4): 415-421.

[5] Qin L, Kudla U, Roze E H, Goverse A, Popeijus H, Nieuwland J, Overmars H, Jones J T, Schots A, Smant G, Bakker J, Helder J. Plant degradation: A nematode expansin acting on plants. Nature, 2004, 427: 30.

[6] Kikuchi T, Li H M, Karim N, Kennedy M W, Moens M, Jones J T.Identification of putative expansin-like genes from the pine wood nematode, Bursaphelenchus xylophilus, and evolution of the expansin gene family within the nematoda. Nematology, 2009, 11(3): 355-364.

[7] Haegeman A, Kyndt T, Gheysen G. The role of pseudo- endoglucanases in the evolution of nematode cell wall-modifying proteins. Journal of Molecular Evolution, 2010, 70: 441-452.

[8] Peng H, Gao B L, Kong L A, Yu Q, Huang W K, He X F, Long H B, Peng D L. Exploring the host parasitism of the migratory plant-parasitic nematode Ditylenchus destuctor by expressed sequence tags analysis. PloS One, 2013, 8(7): e69579.

[9] Liu J, Peng H, Cui J, Huang W K, Kong L A, Clarke J L, Jian H, Wang G L, Peng D L. Molecular characterization of a novel effector expansin-like protein from Heterodera avenae that induces cell death in Nicotiana benthamiana. Scientific Reports, 2016, 6: 35677.

[10] Ali S, Magne M, Chen S, Côté O, Stare B G, Obradovic N, Jamshaid L, Wang X, Bélair G, Moffett P. Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses. PloS One, 2015, 10(1): e0115042.

[11] Eves-van den Akker S, Laetsch D R, Thorpe P, Lilley C J, Danchin E G, Da Rocha M, Rancurel C, Holroyd N E, Cotton J A, Szitenberg A, Grenier E, Montarry J, Mimee B, Duceppe M O, Boyes I, Marvin J M, Jones L M, Yusup H B, Lafond-Lapalme J, Esquibet M, Sabeh M, Rott M, Overmars H, Finkers-Tomczak A, Smant G, Koutsovoulos G, Blok V, Mantelin S, Cock P J, Phillips W, Henrissat B, Urwin P E, Blaxter M, Jones J T. The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence. Genome Biology, 2016, 17: 124.

[12] Opperman C H, Bird D M, Williamson V M, Rokhsar D S, Burke M, Cohn J, Cromer J, Diener S, Gajan J, Graham S, Houfek T D, Liu Q, 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.

[13] Abad P, Gouzy J, Aury J M, Castagnone-Sereno P, Danchin E G, Deleury E, Perfus-Barbeoch L, Anthouard V, Artiguenave F, Blok VC, 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 TJ, 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.

[14] 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, 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.

[15] Bird D M, Williamson V M, Abad P, McCarter J, Danchin E G, Castagnone-Sereno P, Opperman C H. The genomes of root-knot nematodes. Annual Review of Phytopathology, 2009, 47: 333-351.

[16] 彭焕, 彭德良, 黄文坤. 甘薯茎线虫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. Journal of Agricultural Biotechnology, 2009, 17(6): 1035-1041. (in Chinese)

[17] 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.

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

[19] 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.

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

[21] Elling A A, Mitreva M, Recknor J, Gai X, Martin J, Maier T R, McDermott J P, Hewezi T, McK Bird D, 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.

[22] Urwin P E, Lilley C J, Atkinson H J. Ingestion of double- stranded RNA by preparasitic juvenile cyst nematodes leads to RNA interference. Molecular Plant-Microbe Interactions, 2002, 15(8): 747-752.

[23] Cosgrove D J. Loosening of plant cell walls by expansins. Nature, 2000, 407: 321-326.

[24] Long H B, Peng H, Huang W K, Wang G F, Gao B L, Moens M, Peng D L. Identification and molecular characterization of a new β-l,4-endoglucanase gene (Ha-eng-la) in the cereal cyst nematode Heterodera avenae. European Journal of Plant Pathology, 2012, 134(2): 391-400.

[25] Huang W, Long H, Liu Y, Peng D L, Peng H. Identification of a putative expansin gene expressed in the subventral glands of the cereal cyst nematode Heterodera avenae. Nematology, 2012, 14(5): 571-577.

[26] Peng H, Cui J, Long H, Huang W K, Kong L A, Liu S, He W, Hu X, Peng D L. Novel pectate lyase genes of Heterodera glycines play key roles in the early stage of parasitism. PloS One, 2016, 11(3): e0149959.

[27] Long H, Peng D, Huang W, Peng H, Wang G. Molecular characterization and functional analysis of two new β-1,4- endoglucanase genes (Ha-eng-2, Ha-eng-3) from the cereal cyst nematode Heterodera avenae. Plant Pathology, 2013, 62(4): 953-960.

[28] Kudla U, Qin L, Milac A, Kielak A, Maissen C, Overmars H, Popeijus H, Roze E, Petrescu A, Smant G, Bakker J, Helder J. Origin, distribution and 3D-modeling of Gr-EXPB1, an expansin from the potato cyst nematode Globodera rostochiensis. FEBS Letters, 2005, 579: 2451-2457.

[29] 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.

[30] 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.