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

摘要/Abstract

摘要： 【目的】获得绵羊卷曲受体1(frizzled 1,FZD1)基因CDS序列,确定其生物学功能,探索其多态性并明确其在不同品种不同组织中的表达差异。【方法】采集小尾寒羊和新吉细毛羊心脏、肝脏、脾脏、肺脏、肾脏、肌肉和皮肤组织,提取总RNA并反转录成cDNA。依据NCBI数据库中绵羊FZD1基因预测序列(登录号:XM_027968435.2)设计引物,以小尾寒羊皮肤组织cDNA为模板,采用TA克隆获得目的基因序列,并对序列进行生物信息学分析,预测其可能编码的蛋白质结构和功能;利用实时荧光定量PCR检测FZD1基因在小尾寒羊和新吉细毛羊不同组织中的表达差异;采用测序和高分辨熔解曲线(high resolution melting analysis,HRM)进行多态性检测,确定可能存在的单核苷酸多态性(single-nucleotide polymorphisms,SNP)位点。【结果】成功克隆出绵羊FZD1基因CDS区,序列全长为1 941 bp,编码646个氨基酸。绵羊FZD1氨基酸序列与山羊相似性最高,为99.5%,与人的相似性最低,为96.7%;系统进化树结果显示,绵羊FZD1与山羊亲缘关系最近,与人亲缘关系最远。FZD1蛋白含有7个跨膜结构域,42个磷酸化位点和2个N-糖基化位点,主要分布在细胞膜上。FZD1蛋白二级结构预测结果显示,无规则卷曲、α-螺旋、延伸链和β-转角分别占48.30%、35.15%、15.02%和1.55%,与预测的三级结构一致。实时荧光定量PCR结果显示,小尾寒羊FZD1基因在肌肉和皮肤组织中的表达量显著高于新吉细毛羊,在心脏和肺脏中的表达量显著低于新吉细毛羊(P＜0.05)。FZD1基因在2个品种羊的肺脏和皮肤中的表达量均最高,且显著高于其他组织(P＜0.05)。测序结果及HRM验证显示,FZD1基因第1 273和1 276 bp位点处存在2个SNPs(g.9443455和g.9443458),这2个SNPs为完全连锁不平衡状态,仅存在2种单倍型,2个SNPs位点均为中度多态。【结论】FZD1基因CDS全长1 941 bp,编码646个氨基酸。FZD1蛋白作为位于细胞膜上的七跨膜受体蛋白,存在42个可能的磷酸化位点,推测FZD1的信号传导是通过磷酸化和去磷酸化进行的;FZD1基因在小尾寒羊的皮肤组织中表达量最高,提示FZD1基因可能参与毛囊发育的调控。

关键词: 小尾寒羊; FZD1基因; 生物信息学; 组织表达; SNP

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

中图分类号:

S823.83

张双双, 贾琪, 王丹, 王卓, 柳俭强, 张立春, 戢爽. 绵羊卷曲受体1基因克隆、表达谱与多态性分析[J]. 中国畜牧兽医, 2023, 50(12): 4805-4815.

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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