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

Abstract

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

CLC Number:

S839

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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Cloning and Bioinformatics Analysis of TLR4 Gene in Cygnus olor

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