摘要
【目的】galectin-1是凝集素的一种,广泛存在于各种生物体内,在生长发育、免疫调节方面起重要作用。本研究克隆和表达了松材线虫的galectin-1蛋白,并分析了各个龄期的表达量。【方法】设计引物,扩增松材线虫的galectin-1基因,使用双酶切的方法连接pET-28a载体和目的基因,转化到大肠杆菌DH5α感受态细胞,筛选阳性克隆;在不同温度下,用不同浓度的异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,Western blot检验蛋白表达情况;采用RT-PCR技术检验松材线虫各个龄期galectin-1基因的表达情况。【结果】由SMART和PredictProtein软件分析可知,该蛋白有2个结构域,并且主要由无规卷曲和β折叠构成;生物学信息分析显示,松材线虫的galectin-1与小卷蛾斯氏线虫的相似性更高;与日本血吸虫相比,线虫能很好地聚集在一个分支上。Western blot检测纯化蛋白大小与查询所得蛋白分子质量一致。RT-qPCR结果显示,以繁殖型2龄松材线虫(L 2)为对照,galectin-1基因在繁殖型3龄(L 3)、繁殖型4龄(L 4)、扩散型3龄(LⅢ)和扩散型4龄(LⅣ)松材线虫中的表达量高,尤其是在LⅢ中的表达量最高;雌雄成虫没有显著性差异。【结论】松材线虫的galectin-1基因在pET-28a原核表达系统中呈可溶性表达,在不同龄期的表达量有差异。本研究为进一步研究松材线虫的galectin-1基因奠定了基础,为松材线虫的防治提供了新的方向。
【Aim】Galectins(Gal),a family of lectins,are widely distributed in various organisms and play pivotal roles in many biological processes,including growth,development and immunity.In the present study,a galectin-coding gene was cloned and identified from Bursaphelenchus xylophilus(Bx),and we analyzed its expression patterns in different developmental stages.【Method】Primers were designed to amplify the galectin-1 gene of in B.xylophilus,while the pET-28a vector and the target gene were ligated by double enzyme digestion,then transformed into Escherichia coli DH5αcompetent cells,and positive clones were screened.The expressioned galectin-1 protein was induced with isopropyl-β-D-thiogalactoside(IPTG)at various temperatures and Western blot was used to detect the expression of galectin-1 protein.Quantitative real time-polymerase chain reaction(RT-qPCR)was used to analyse the expression of this gene in different developmental stages of B.xylophilus.【Result】The cDNA of BxGal was cloned(Gen Bank Gene ID:GU130138.1).SMART and PredictProtein softwares indicated that the BxGal had two CRDs and was mainly composed of random coils andβ-sheets.Bioinformatics analysis showed that BxGal was more similar to that in the insect parasitic nematode Steinernema carpocapsae.The phylogenetic tree indicated that nematodes clustered well,compared to Schistosoma japonicum.Developmental expression profiles revealed that the expression level of BxGal was the highest in the dispersingal third-larval instar(LⅢlarvae),and lower in propagative larval stages,dispersing fourth-larval stage(LⅣ)and in the reproductive adult.There was no difference between activity in males vs.females.【Conclusion】The galectin-1 gene of B.xylophilus was soluble expressioned in the pET-28a prokaryotic expression system,and its expression level varied in the different developmental stages.This study provides the basis cornerstones for further research of the BxGal and provides a new direction for the control of B.xylophilus.
作者
任万兰
张驰
周娇
侯有明
赵莉蔺
REN Wanlan;ZHANG Chi;ZHOU Jiao;HOU Youming;ZHAO Lilin(State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops,College of Plant Protection,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;Fujian Province Key Laboratory of Insect Ecology,College of Plant Protection,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;State Key Laboratory of Integrated Management of Pest Insects and Rodents,Institute of Zoology,Chinese Academy of Sciences,Beijing 100101,China;CAS Center for Excellence in Biotic Interactions,Beijing 100049,China)
出处
《生物安全学报》
CSCD
2020年第2期99-105,122,共8页
Journal of biosafety
基金
中国科学院前沿科学重点研究项目(QYZDB-SSW-SMC014)
国家自然科学基金面上项目(31572272)。
