绵羊卷曲受体1基因克隆、表达谱与多态性分析

doi:10.16431/j.cnki.1671-7236.2023.12.003

Abstract

Abstract: 【Objective】 The aim of this study was to obtain the CDS sequence of frizzled 1 (FZD1) gene in sheep,determine its biological function,explore its polymorphism and clarify its expression differences in different breeds and tissues.【Method】 The heart,liver,spleen,lung,kidney,muscle and skin tissues of Small-tail Han sheep and Xinji Fine Wool sheep were collected and total RNA was extracted and reverse transcribed to synthesize cDNA.According to the predicted sequence of sheep FZD1 gene in NCBI database(accession No.:XM_027968435.2),the target gene sequence was obtained by TA cloning using the Small-tail Han sheep skin tissue cDNA as the template,and bioinformatics analysis was performed on the sequence to predict its possible encoded protein structure and function.Real-time quantitative PCR was used to detect the difference of FZD1 gene expression in different tissues of Small-tail Han sheep and Xinji Fine Wool sheep.Sequencing and high resolution melting analysis (HRM) were used for polymorphism detection to determine the possible single nucleotide polymorphisms(SNP) sites.【Result】 The CDS region of sheep FZD1 gene was successfully cloned,and the total length of the sequence was 1 941 bp,encoding 646 amino acids.The amino acid sequence of FZD1 of sheep had the highest similarity with Capra hircus (99.5%),and the lowest similarity with Homo sapiens (96.7%).The phylogenetic tree showed that FZD1 of sheep had the closest genetic relationship with Capra hircus and the farthest genetic relationship with Homo sapiens.The FZD1 protein contained 7 transmembrane domains,42 phosphorylation sites,and 2 N-glycosylation sites,mainly distributed on the cell membrane.The prediction results of the secondary structure of FZD1 protein showed that random coil,alpha helix,extended chain,and beta turn accounted for 48.30%,35.15%,15.02% and 1.55%,respectively,which was consistent with the predicted tertiary structure.The Real-time quantitative PCR results showed that the expression of FZD1 gene in the muscle and skin tissues of Small-tail Han sheep were significantly higher than that of Xinji Fine Wool sheep,while the expression in the heart and lung were significantly lower than that of Xinji Fine Wool sheep (P＜0.05).The expression of FZD1 gene were the highest in the lungs and skin of both breeds of sheep,and were significantly higher than other tissues (P＜0.05).The sequencing results and HRM validation showed that there were two SNPs (g.9443458 or g.9443458) at the 1 273 and 1 276 bp loci of the FZD1 gene.These two SNPs were in a fully linked imbalanced state,with only two haplotypes present,and both SNPs were moderately polymorphic.【Conclusion】 The total length of FZD1 gene CDS was 1 941 bp,encoding 646 amino acids.As seven transmembrane receptor proteins located on the cell membrane,FZD1 protein had 42 possible phosphorylation sites.It was speculated that the signaling of FZD1 was conducted through phosphorylation and dephosphorylation.The expression of FZD1 gene was the highest in the skin tissue of Small-tailed Han sheep,suggesting that FZD1 gene might be involved in the regulation of hair follicle development.

Key words: Small-tail Han sheep; FZD1 gene; bioinformatics; tissue expression; SNP

CLC Number:

S823.83

ZHANG Shuangshuang, JIA Qi, WANG Dan, WANG Zhuo, LIU Jianqiang, ZHANG Lichun, JI Shuang. Cloning,Expression Profile and Polymorphism Analysis of FZD1 Gene in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(12): 4805-4815.

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Cloning,Expression Profile and Polymorphism Analysis of FZD1 Gene in Sheep

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