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

Identification and Functional Analysis of Two Expansin Genes Hg-exp-1 and Hg-exp-2 from the Soybean Cyst Nematode(Heterodera glycines)

【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c DNA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Signal P 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c DNA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。

【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 2 nd 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 c DNA of H. glycines was obtained. CLC sequence viewer 6 was used for open reading frame search, protein translation and sequence alignment. On-line software Signal P 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 c DNAs of eggs, pre-parasitic 2 nd stage juvenile, parasitic 2 nd stage juvenile, parasitic 3 rd stage juvenile, parasitic 4 th 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 2 nd 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 c DNAs of two expansin genes named Hg-exp-1 and Hg-exp-2 were successfully cloned from the 2 nd 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 2 nd 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.