牛卵泡AGTR2序列结构及表达特性分析

doi:10.3864/j.issn.0578-1752.2020.07.016

摘要/Abstract

摘要： 【目的】研究牛卵泡发育过程中关键调控蛋白CART的候选受体AGTR2的分子特征和立体结构,并结合AGTR2在不同生理状态牛卵泡的表达特性分析其功能。【方法】 同期发情处理后,经B超声波连续监测（每12 h 一次）采集牛双侧卵巢,分离优势卵泡（dominant follicles, DF）和从属卵泡（subordinate follicles, SF）;其中3头牛DF和SF分别分离颗粒细胞（granulosa cells, GCs）,抽提总RNA后,经反转录、引物特异性扩增、胶回收及测序,获得AGTR2基因全CDS区;生物信息学方法对其序列结构、亲缘关系及立体结构进行分析;设计AGTR2和内参基因RPLP0 qRT-PCR引物,分析AGTR2在牛DF和SF的差异表达情况;另1头牛DF和SF经4%多聚甲醛固定后,设定阳性、阴性对照组和试验组,应用免疫组织化学技术对AGTR2进行表达和定位分析。【结果】 AGTR2 CDS区全长1 089 bp,编码362个氨基酸;牛卵泡AGTR2氨基酸序列与NCBI数据库获得的其他24种动物对应的氨基酸序列BLAST分析表明,该序列与印度水牛序列相似性最高（99.4%）,与其他动物序列相似性为92.0%—98.9%;立体结构和功能域分析表明,AGTR2立体结构中包含有7个横跨细胞膜的平行的α螺旋结构,符合G蛋白偶联受体（G-protein-coupled receptors, GPCRs）的典型特征;qRT-PCR分析结果表明AGTR2 mRNA在DF的表达量极显著高于SF（P<0.01）,且差异表达倍数达7.47倍;免疫组织化学分析结果表明AGTR2在牛DF和SF颗粒层、膜层细胞均有表达,特异性显色强度表明AGTR2在SF膜层细胞表达量高于DF。【结论】 AGTR2属于G蛋白偶联受体,符合神经肽CART受体的基本特征;本试验为进一步研究AGTR2在牛卵泡发育过程中调控信号通路和激素分泌的机理奠定基础,同时,对后期鉴定CART的受体、深入阐明CART调控牛卵泡发育的作用机理具有重要意义。

关键词: 牛, 卵泡发育, CART, G蛋白偶联受体, AGTR2

Abstract: 【Objective】 The aim of study was to identify molecular characteristics and three-dimensional structure of AGTR2 in the bovine follicular development, and the function was analyzed by combining expression characteristics of AGTR2 in different physiological follicles.【Method】Ovaries in the bovine follicular were observed by B-type ultrasonography (twice a day), removed from cows when DF and SF appeared, and then separated DF and SF; GCs were isolated, total RNA was extracted and detected by RT-PCR, specific primers were amplified and sequenced, and CDS region sequence structure was obtained; the bioinformatics method was used to analyze its sequence structure, relationship and three-dimensional structure; the qRT-PCR primers of AGTR2 and reference gene RPLP0 were designed to analyze differential expression level of AGTR2 in DF and SF; DF and SF of another cow were fixed with 4% paraformaldehyde, positive, negative control groups and experimental groups were set, expression level and localization of AGTR2 were analyzed by immunohistochemistry. 【Result】The results showed that total length of AGTR2 CDS region was 1 089 bp, encoding 362 amino acids; BLAST analysis of amino acid sequence of AGTR2 and the corresponding amino acid sequence of other 24 animals obtained by NCBI database indicated that the sequence had the highest similarity with buffalo (99.4%) and 92.0%-98.9% with other animals; The three-dimensional structure and functional domain analysis showed that AGTR2 possessed 7 parallel alpha helical structures across the cell membrane, which was a typical G protein-coupled receptor; the results of qRT-PCR analysis showed that expression level of AGTR2 mRNA in DF was significantly higher than SF (P<0.01), and the differential expression multiple was up to 7.47 times. The immunohistochemical analysis showed that AGTR2 was expressed in GCs and membrane cells layer of DF and SF, and the specific color intensity showed that expression of AGTR2 in SF membrane cells was higher than DF. 【Conclusion】AGTR2 belonged to G protein-coupled receptor and conforms to basic characteristics of CART receptor, and the study laid a foundation for further study on mechanism of AGTR2 regulating signal pathway and hormone secretion during bovine follicular development, meanwhile, it was of great significance for identification of CART receptor and in-depth explanation of mechanism of CART regulating bovine follicular development.

Key words: bovine, follicular development, CART, G protein-coupled receptor, AGTR2

朱芷葳,侯淑宁,郝庆玲,景炅婕,吕丽华,李鹏飞. 牛卵泡AGTR2序列结构及表达特性分析[J]. 中国农业科学, 2020, 53(7): 1482-1490.

ZhiWei ZHU,ShuNing HOU,QingLing HAO,JiongJie JING,LiHua LÜ,PengFei LI. Sequence Structure and Expression Characteristics Analysis of AGTR2 in Bovine Follicle[J]. Scientia Agricultura Sinica, 2020, 53(7): 1482-1490.

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https://www.chinaagrisci.com/CN/Y2020/V53/I7/1482

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引物名称 Primer name	引物序列 Sequence (5′ to 3′)	退火温度 Tm (℃)	大小 Size (bp)
RT-PCR
AGTR2-F	ATGAAGACCAACTTCTCCCTTGCCAC	58.6	1089
AGTR2-R	TTAAGACACAAAGGTCTCCATTTCTCTAAGGG	62.9	1089
qRT-PCR
AGTR2-R-F	TTCCCTTCCATGTTCTGACC	59.9	96
AGTR2-R-R	AAGGAAGTGCCAGGTCAATG	60.1	96
RPLP0-F	CAACCCTGAAGTGCTTGACAT	59.3	115
RPLP0-R	AGGCAGATGGATCAGCCA	57.1	115