疣鼻天鹅TLR4基因克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2023.09.003

摘要/Abstract

摘要： 【目的】克隆疣鼻天鹅Toll样受体4(Toll-like receptor 4,TLR4)基因,并对其编码蛋白进行生物信息学分析,为进一步探索疣鼻天鹅TLR4蛋白抗病原微生物免疫机制提供重要参考依据。【方法】利用PCR扩增并克隆疣鼻天鹅TLR4基因CDS序列,应用NCBI-BLAST、Mega-X软件进行序列比对并构建系统进化树;利用ProtParam、SWISS-MODEL等软件预测TLR4基因编码蛋白的结构、功能及与宿主蛋白的相互作用关系,并进行GO功能和KEGG通路富集分析。【结果】试验成功克隆疣鼻天鹅TLR4基因CDS区序列,全长2 550 bp,已提交至NCBI,登录号为:OP908241。疣鼻天鹅TLR4基因CDS区序列中存在79个稀有密码子,其中含6段连续稀有密码子,编码849个氨基酸,与灰雁和鸿雁相似度较高,亲缘关系最近。TLR4蛋白的跨膜结构域位于第647―667位氨基酸处,N-端包含由36个氨基酸组成的信号肽,信号肽切割位点在第36―37位氨基酸之间,置信度为67.85%,含有10个N-糖基化位点和85个磷酸化位点,是亲水性蛋白,主要存在于细胞质中,与人TLR4蛋白有相似的三级结构,相似率为45.48%。TLR4蛋白共存在10个主要互作蛋白,富集得到96个GO功能条目和7条KEGG通路,其中GO功能条目包括物种间相互作用、免疫反应、应激反应、信号转导等;KEGG通路包括甲型流感、单纯疱疹病毒Ⅰ型感染及沙门氏菌感染等。【结论】疣鼻天鹅TLR4基因与灰雁和鸿雁亲缘关系最近,有明显的密码子种属选择性,含有6段连续稀有密码子,其蛋白存在跨膜结构域和信号肽,并与天然免疫应答、信号转导和应激反应密切相关。

关键词: 疣鼻天鹅; TLR4基因; 克隆; 蛋白相互作用网络; GO功能; KEGG通路

Abstract: 【Objective】 This study was aimed to clone the Toll-like receptor 4 (TLR4) gene in Cygnus olor and analyze its encoded protein by bioinformatics,so as to provide an important reference basis for further exploring the immune mechanism of TLR4 protein against pathogenic microorganisms in Cygnus olor.【Method】 The sequence of TLR4 gene CDS in Cygnus olor was amplified by PCR and cloned.NCBI-BLAST and Mega-X were used for sequence comparison and phylogenetic tree construction.The structure and function of TLR4 protein and its interaction with host protein were predicted using ProtParam,SWISS-MODEL,etc.,GO functional and KEGG pathway enrichment analysis were conducted.【Result】 The sequence of TLR4 gene CDS in Cygnus olor was successfully cloned,the complete length of the gene was 2 550 bp,which was submitted to NCBI with the accession No.:OP908241.There were 79 rare codons of the TLR4 gene CDS sequence in Cygnus olor,including 6 consecutive rare codons,encoding 849 amino acids.It had high similarity with Anser anser and Anser cygnoides,and had the closest genetic relationship.The transmembrane domain of TLR4 protein was located at amino acids 647-667,and the N-terminal contained a signal peptide composed of 36 amino acids.The signal peptide cleavage site was between amino acids 36 and 37,with a confidence of 67.85%.TLR4 protein contained 10 N-glycosylation sites and 85 phosphorylation sites,which had similar tertiary structures to TLR4 protein in human,and the similarity was 45.48%.TLR4 protein had a total of 10 major interaction proteins,and 96 GO functional items and 7 KEGG pathways were enriched.GO functional entries included species interaction,innate immune response,stress response,signal transduction,etc.The KEGG pathway included influenza A,Herpes simplex virus type Ⅰ infection and Salmonella infection,etc.【Conclusion】 TLR4 gene in Cygnus olor was closely related to Anser anser and Anser cygnoides,with obvious codon species selectivity,and contained 6 consecutive rare codons.TLR4 protein had transmembrane domains and signal peptides,and was closely related to innate immune response,signal transduction and stress response.

Key words: Cygnus olor; TLR4 gene; cloning; protein interaction network; GO function; KEGG pathway

中图分类号:

S839

于鑫, 胡玉苗, 李芳兵, 杨亚东, 赵金, 冯亚莉, 张莹. 疣鼻天鹅TLR4基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(9): 3469-3479.

YU Xin, HU Yumiao, LI Fangbing, YANG Yadong, ZHAO Jin, FENG Yali, ZHANG Ying. Cloning and Bioinformatics Analysis of TLR4 Gene in Cygnus olor[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3469-3479.

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