多浪羊LIN28A基因启动子区克隆、序列分析及蛋白表达研究

doi:10.16431/j.cnki.1671-7236.2025.07.003

摘要/Abstract

摘要： 【目的】探究多浪羊LIN28A基因的启动子区序列特征及蛋白表达规律，为进一步研究其在初情期的转录调控机理奠定基础。【方法】根据NCBI中绵羊LIN28A基因序列(登录号：NC_056055.1)设计引物进行PCR扩增、克隆测序及序列比对，获取多浪羊LIN28A基因启动子区序列，并利用在线分析软件对其进行生物信息学分析。采集幼年期、初情期前、初情期及初情期后多浪羊的下丘脑、垂体和卵巢组织，利用Western blotting检测LIN28A蛋白在下丘脑-垂体-卵巢(HPO)轴中的表达情况。【结果】多浪羊LIN28A基因启动子区序列长2 089 bp，GC含量为56.28%。LIN28A基因启动子序列包含MEIS1、STAT3、COMP1等65个转录因子结合位点，3个TATA-box，1个CpG岛，以及9个潜在转录起始位点。LIN28A蛋白在多浪羊3个组织中均有表达，以卵巢中表达水平最高，下丘脑次之，垂体中表达量最低。在初情期前多浪羊下丘脑中LIN28A蛋白表达量显著高于幼年期(P＜0.05)，在幼年期和初情期前多浪羊垂体中LIN28A蛋白表达量显著高于初情期及初情期后(P＜0.05)，卵巢中LIN28A蛋白表达量在初情期启动前后呈现稳定趋势(P＞0.05)；在同一时期，LIN28A蛋白在多浪羊下丘脑、垂体和卵巢组织中的表达量均无显著差异(P＞0.05)。【结论】多浪羊LIN28A基因启动子区有多个与初情期相关的转录因子结合位点，为后续转录因子验证和启动子活性检测提供了试验材料。LIN28A蛋白在多浪羊HPO轴表达差异显著，为进一步研究LIN28A基因功能及调控机制提供了科学依据。

关键词: 多浪羊; LIN28A基因; 启动子; 初情期; 下丘脑-垂体-卵巢轴

Abstract: 【Objective】 The aim of this study was to explore the promoter sequence characteristics and protein expression of LIN28A gene in Duolang sheep,so as to lay a foundation for further study of its transcriptional regulation mechanism at puberty stage. 【Method】 Primers were designed for PCR amplification,cloning sequencing,and sequence alignment based on the LIN28A gene sequence of sheep in NCBI (accession No.:NC_056055.1),and the promoter sequence of LIN28A gene in Duolang sheep was obtained,which was analyzed by online analysis software.The hypothalamus,pituitary,and ovarian tissues of Duolang sheep during juvenile,prepuberty,puberty,and post-puberty periods were collected,and the expression of LIN28A protein in hypothalamic-pituitary-ovarian (HPO) axis was detected by Western blotting. 【Result】 The length of promoter sequence of LIN28A gene in Duolang sheep was 2 089 bp,and the GC content was 56.28%.The promoter sequence of LIN28A gene covered a total of 65 transcription factor binding sites,such as MEIS1,STAT3,and COMP1,3 TATA-boxes,1 CpG island,and 9 potential transcription start sites.LIN28A protein was expressed in all three tissues of Duolang sheep,with the highest expression in ovarian,followed by hypothalamus,and the lowest expression in pituitary.The expression of LIN28A protein in hypothalamus of Duolang sheep in prepuberty was significantly higher than that in juvenile and puberty (P＜0.05).The expression of LIN28A protein in pituitary of Duolang sheep in juvenile and prepuberty was significantly higher than that in puberty and post-puberty (P＜0.05).The expression of LIN28A protein in ovarian of Duolang sheep showed a stable trend before and after of puberty (P＞0.05).During the same period,there was no significant difference in the expression of LIN28A protein in hypothalamus,pituitary and ovarian of Duolang sheep (P＞0.05). 【Conclusion】 The promoter sequence of LIN28A gene in Duolang sheep had multiple transcription factor binding sites associated with puberty,providing experimental materials for transcription factor validation and promoter activity detection in the future.The expression of LIN28A protein in HPO axis of Duolang sheep was significantly different,providing a scientific basis for further study of the function and regulatory mechanism of LIN28A gene.

Key words: Duolang sheep; LIN28A gene; promoter; puberty; HPO axis

中图分类号:

S826.2

李伟, 黄巧艳, 王鑫昆, 谷若怀, 孙慧萍, 朱乐萧, 邢凤. 多浪羊LIN28A基因启动子区克隆、序列分析及蛋白表达研究[J]. 中国畜牧兽医, 2025, 52(7): 2992-3003.

LI Wei, HUANG Qiaoyan, WANG Xinkun, GU Ruohuai, SUN Huiping, ZHU Lexiao, XING Feng. Cloning, Sequence Analysis and Protein Expression of LIN28A Gene Promoter in Duolang Sheep[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 2992-3003.

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