福建农业学报2025，Vol.40Issue(6)：602-609,8.DOI:10.19303/j.issn.1008-0384.2025.06.008
豆伞滑刃线虫类毒液过敏原蛋白基因Bd-VAP-1的克隆及原位杂交分析
Cloning and Organ-localization by In Situ Hybridization of Bd-VAP-1 in Bursaphelenchus doui
摘要
Abstract
[Objective]A venom allergen-like protein(VAP)gene was cloned from Bursaphelenchus doui with its expressions in organs and function in the nematode-host interaction determined.[Method]A specific cDNA library on B.doui was constructed by solid-phase hybridization using cDNA from the anterior end as the tester and the posterior end of nematode as the driver.It was subsequently scrutinized by the reverse-northern blotting to obtain the sequence of positive clones.The full-length cDNA of Bd-VAP-1 gene was cloned using the rapid amplification of cDNA ends(RACE)technology,nucleotide and amino acid sequences analyzed using bioinformatics methods,and gene expression in organs localized by in situ hybridization.[Result]A specific cDNA library of the anterior end of B.doui was successfully constructed.The full-length cDNA encoding a putative Bd-VAP-1 was identified by the reverse-northern blotting,sequencing,and RACE technologies.The open reading frame of Bd-VAP-1 was 644 bp in length and consisted of two extrons and one intron.The coding region,609 bp in length,encoded a 202-amino acid protein with an average molecular weight of 22.32 kDa and theoretical isoelectric point of 4.86.The Bd-VAP-1 protein was highly similar in sequence to the MIF homologous proteins of B.xylophilus and B.mucronatus.A secretory protein,it contained a typical CAP conservative domain and a signal peptide without a transmembrane structure.The gene expressed predominantly in the esophagus glands of B.doui as shown by the in situ hybridization.[Conclusion]Bd-VAP-1 was successfully cloned and speculated to have its coding protein synthesized in the esophageal gland of B.doui and excreted to affect the host.关键词
豆伞滑刃线虫/特异cDNA文库/类毒液过敏原蛋白/生物信息学/原位杂交Key words
Bursaphelenchus doui/specific cDNA library/venom allergen-like protein/bioinformatics/in situhybridization分类
农业科技引用本文复制引用
林世锋,王仁刚,林英超,王自力,李莉,李国红,吴沙沙..豆伞滑刃线虫类毒液过敏原蛋白基因Bd-VAP-1的克隆及原位杂交分析[J].福建农业学报,2025,40(6):602-609,8.基金项目
国家自然科学基金项目(31360519) （31360519）
贵州师范大学学术新苗基金(黔师新苗[2021]A13号) （黔师新苗[2021]A13号）
上海烟草科技开发项目(2024521300640044) （2024521300640044）
贵州省科技厅农业攻关项目(黔科合NY[2013]3041号) （黔科合NY[2013]3041号）
