白羽番鸭LIF基因克隆及其组织表达差异分析

doi:10.16431/j.cnki.1671-7236.2023.04.002

摘要/Abstract

摘要： 【目的】探究白血病抑制因子(leukemia inhibitory factor,LIF)基因在白羽番鸭中的生物学功能,阐明其在下丘脑、垂体、卵巢、输卵管膨大部、子宫组织中的表达模式。【方法】以白羽番鸭卵巢组织为研究对象,采用cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术克隆白羽番鸭LIF基因序列,并进行相似性比对和系统进化树构建;通过生物信息学方法分析LIF蛋白理化性质及结构。利用实时荧光定量PCR技术检测LIF基因在白羽番鸭下丘脑、垂体、卵巢、输卵管膨大部、子宫5个组织中的相对表达量。【结果】克隆得到白羽番鸭LIF基因序列长1820 bp,其中CDS区长636 bp,编码211个氨基酸。核苷酸序列相似性比对结果表明,白羽番鸭LIF基因序列与凤头潜鸭、绿头鸭、棕硬尾鸭、黑天鹅、金丝雀、鹌鹑、鸡、山雀的相似性分别为99.36%、99.21%、99.21%、98.11%、92.03%、90.68%、90.58%和87.60%。系统进化树分析表明,白羽番鸭与凤头潜鸭的遗传距离最近,与鸡的遗传距离最远。白羽番鸭LIF蛋白是碱性亲水的稳定蛋白,有16个磷酸化位点、7个N-糖基化位点及1个信号肽,主要定位于细胞外和溶酶体;LIF蛋白质二级结构主要由α-螺旋和无规则卷曲构成,蛋白质三级结构预测结果与二级结构一致。白羽番鸭LIF蛋白可能与IL6ST、CNTFR、IL6、OSMR、LIFR、IL6R、STAT3、CNTF、IFNGR1、IFNGR2蛋白具有相互作用。组织表达图谱显示,LIF基因在白羽番鸭5种组织中均有表达,其中就巢期白羽番鸭LIF基因表达量在输卵管膨大部、卵巢组织显著高于产蛋期(P＜0.05);两个时期LIF基因在输卵管膨大部的表达量均最高,且显著高于其他组织(P＜0.05)。【结论】试验成功克隆了白羽番鸭LIF基因,其在输卵管膨大部的表达量显著高于其他组织,且就巢期表达量高于产蛋期。结果为探究白羽番鸭卵巢发育的调控机制提供理论依据。

关键词: 白羽番鸭; LIF基因; 生物信息学; 组织表达

Abstract: 【Objective】 This study was aimed to explore the biological function of LIF gene in White muscovy duck,and clarify its expression pattern in hypothalamus,pituitary,ovary,oviduct enlargement and uterus.【Method】 In this study,the ovarian tissue of White muscovy duck was used as the research object,and the LIF gene sequence of White muscovy duck was cloned by rapid amplification of cDNA ends technology (RACE),and the similarity comparison and phylogenetic tree were constructed.The physicochemical properties and structure of LIF protein were analyzed by bioinformatics methods.The relative expression of LIF gene in the hypothalamus,pituitary,ovary,oviduct enlargement and uterus of White muscovy duck was detected by Real-time quantitative PCR.【Result】 The cloned LIF gene sequence of White muscovy duck was 1 820 bp,and the CDS region was 636 bp,encoding 211 amino acids.The results of nucleotide sequence similarity comparison showed that the sequence similarity of LIF gene between White muscovy duck and Aythya fuligula,Anas platyrhynchos,Oxyura jamaicensis,Cygnus atratus,Serinus canaria, Coturnix japonica,Gallus gallus,Nothoprocta perdicaria were 99.36%,99.21%,99.21%,98.11%,92.03%,90.68%,90.58%,87.60%,respectively.The analysis of phylogenetic tree showed that the genetic distance between White muscovy duck and Aythya fuligula was the closest,and the genetic distance between White muscovy duck and Gallus gallus was the farthest.The LIF protein in White muscovy duck was an alkaline hydrophilic stable protein with 16 phosphorylation sites,7 N-glycosylation sites and 1 signal peptide,which was mainly located in the extracellular and lysosome.The secondary structure of LIF protein was mainly composed of alpha helix and random coli,and the prediction result of protein tertiary structure was consistent with that of secondary structure.LIF protein in White muscovy duck might interact with IL6ST,CNTFR,IL6,OSMR,LIFR,IL6R,STAT3,CNTF,IFNGR1 and IFNGR2 proteins.The tissue expression showed that LIF gene was expressed in five tissues of White muscovy duck. LIF gene of White muscovy duck in the oviduct enlargement and ovarian tissue at nest stage was significantly higher than that at laying stage (P＜0.05).The expression of LIF gene in the oviduct enlargement was significantly higher than other tissues (P＜0.05) in two periods.【Conclusion】 The LIF gene in White muscovy duck was successfully cloned in the experiment,and its expression in the oviduct enlargement was significantly higher than that in other tissues,and the nesting stage was higher than that at laying stage.The results provided a theoretical basis for exploring the regulatory mechanism of ovarian development in White muscovy duck.

Key words: White muscovy duck; LIF gene; bioinformatics; tissue expression

中图分类号:

S834

陶庆华, 黄彩云, 陈超, 刘威, 孙宁宁, 李昂. 白羽番鸭LIF基因克隆及其组织表达差异分析[J]. 中国畜牧兽医, 2023, 50(4): 1296-1306.

TAO Qinghua, HUANG Caiyun, CHEN Chao, LIU Wei, SUN Ningning, LI Ang. Cloning and Tissue Expression Difference Analysis of LIF Gene in White Muscovy Duck[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1296-1306.

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