| 郭凯阳,林世锋,王仁刚,等.豆伞滑刃线虫扩展蛋白基因Bd-exp-1的克隆与分析[J].广东农业科学,2025,(3-4):- |
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豆伞滑刃线虫扩展蛋白基因Bd-exp-1的克隆与分析 |
| Cloning and Analysis of the Expansin Gene Bd-exp-1 from Migration Plant-parasitic Nematode (Bursaphelenchus doui) |
| 投稿时间：2024-12-13 修订日期：2025-03-15 |
| DOI： |
| 中文关键词: 豆伞滑刃线虫 扩展蛋白 序列分析 Southern杂交 原位杂交 |
| 英文关键词: Bursaphelenchus doui Expansin Sequence analysis Southern blot In situ hybridization |
| 基金项目:中国烟草总公司贵州省公司科技项目(2023XM04),贵州省科技厅农业攻关项目(黔师新苗[2021]A13号),贵州省林业科研项目(黔林科合[2014]02号) |
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| 中文摘要: |
| 【目的】克隆豆伞滑刃线虫类毒液过敏原蛋白(EXP)基因Bd-exp-1,并进行表达特性分析,为深入研究Bd-exp-1基因在豆伞滑刃线虫与寄主互作过程中的作用提供参考。【方法】以包括食道腺、分泌孔和头感器在内的豆伞滑刃线虫体前端为测验子(tester),以包括肠道在内的豆伞滑刃线虫体后端为驱赶子(driver),利用固相消减杂交方法构建豆伞滑刃线虫体前端特异cDNA文库。利用反向Northern杂交和测序技术筛选文库,结合RACE技术获得目的基因全长序列,并利用生物信息学方法进行序列分析,Southern杂交确定基因拷贝数,原位杂交进行基因表达定位。【结果】成功构建1个豆伞滑刃线虫体前端特异cDNA文库。通过反向Northern杂交、测序与RACE技术相结合的方法克隆获得一个豆伞滑刃线虫类毒液过敏原蛋白全长基因Bd-exp-1,GenBank登录号为PQ276072。Bd-exp-1基因开放阅读框全长为499 bp,包括2个外显子和1个内含子,为多拷贝基因；编码区全长为450 bp,编码149个氨基酸。预测蛋白质的相对分子量为16.16 kD,理论等电点为7.47,与松材线虫的一致性最高,含有1个典型且保守的DPBB_SPI-like结构域,存在信号肽但无跨膜结构域,属于分泌型蛋白。原位杂交结果表明,Bd-exp-1基因在豆伞滑刃线虫体前端的食道腺特异表达。【结论】成功克隆了豆伞滑刃线虫Bd-exp-1基因,初步推测其编码蛋白在线虫食道腺合成,并在线虫与宿主互作过程中分泌到体外发挥作用。 |
| 英文摘要: |
| 【Objective】A venom allergen-like protein gene Bd-exp-1 was cloned from Bursaphelenchus doui, and its expression characteristics were investigated in order to provide reference for the in-depth study of the function of Bd-exp-1 gene in the interactions between B. doui and its hosts.【Method】A specific cDNA library of the anterior end of B. doui was constructed by solid-phase hybridization using cDNA from the anterior end of nematode as tester and those from the posterior end of nematode as driver. The cDNA library was screened by reverse-northern blotting and the positive clones were sequenced. The full-length cDNA sequence of EXP homologue was cloned by using RACE technology. Further, the nucleotide sequence and amino acid sequence were analyzed using bioinformatics methods, the gene copy number was identified by Southern blot and the gene expression was localized by in situ hybridization.【Result】A specific cDNA library of the anterior end of B. doui was constructed successfully. The full-length cDNA encoding a putative EXP protein, named Bd-exp-1, was identified by using reverse-northern blotting, sequencing and RACE technologies. Bd-exp-1 was a multi-copy gene, its open reading frame sequence was 499 bp with one intron and two exons. The coding region, 450 bp in length, encoded a 149-aminoacid protein with an average molecular mass of 16.16 kD and theoretical isoelectric point of 7.47. The Bd-EXP-1 protein had the highest sequence similarity with the homologous protein of B. xylophilus, and contained a typical DPBB_SPI-like conservative domain and a signal peptide but no transmembrane structure, indicating that it was a secretory protein. In situ hybridization analysis showed that the transcripts of Bd-exp-1 accumulated exclusively within the esophageal gland of B. doui.【Conclusion】The Bd-exp-1 gene was successfully cloned, and it is speculated that its coding protein was synthesized in the esophageal gland of B. doui and excreted outside of the body to played a role in interactions with the host. |
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