黔北麻羊RARRES1基因的克隆表达及功能初步研究

doi:10.11843/j.issn.0366-6964.2020.07.008

摘要/Abstract

摘要： 旨在对黔北麻羊视黄酸受体应答蛋白1（retinoic acid receptor responder 1，RARRES1）基因的功能进行初步探究。本研究首先利用PCR扩增克隆得到黔北麻羊RARRES1基因编码的完整序列，应用生物信息学方法分析黔北麻羊RARRES1蛋白理化性质、高级结构、疏水性、氨基酸同源性及亚细胞定位，并构建系统进化树，同时应用qRT-PCR法检测RARRES1基因在单、多羔黔北麻羊不同组织中的表达水平，随后构建目的基因pEGFP-N3-RARRES1真核表达载体与pGPH1/GFP/Neo-RARRES1 RNA干扰载体，并转染至黔北麻羊卵泡颗粒细胞，利用qRT-PCR法从细胞水平检测并分析目的基因真核表达载体与RNA干扰载体对目的基因RARRES1及多胎性状候选基因BMPR-IB mRNA表达的影响。qRT-PCR结果显示，RARRES1 mRNA在卵巢中的表达量最高，单羔组黔北麻羊卵巢组织中的表达量极显著高于多羔组（P<0.01）。生物信息学分析表明，黔北麻羊RARRES1基因共编码289个氨基酸，RARRES1是一种定位在细胞质中的亲水性蛋白，蛋白质二级结构分析显示，其主要是由α-螺旋与无规则卷曲构成，三级结构预测与二级结构分析一致；同源性以及系统进化树结果显示，黔北麻羊RARRES1基因序列与绵羊、牛的同源性高、遗传距离近，与鸡的同源性最低、遗传距离最远。经双酶切、测序验证后，表明目的基因pEGFP-N3-RARRES1真核表达载体与pGPH1/GFP/Neo-RARRES1干扰载体构建成功。将各载体转染至黔北麻羊卵泡颗粒细胞后，与空白对照相比，重组质粒pEGFP-N3-RARRES1能极显著提高RARRES1与BMPR-IB的表达量（P<0.01）。在各干扰载体中，重组质粒pGPH1/GFP/Neo-RARRES1-4的抑制效率最好，能够显著与极显著下调RARRES1与BMPR-IB mRNA在卵泡颗粒细胞中的表达量（P<0.05，P<0.01）。本研究克隆了黔北麻羊RARRES1基因编码的完整序列并进行了生物信息学分析。将重组质粒pEGFP-N3-RARRES1、pGPH1/GFP/Neo-RARRES1成功转染至黔北麻羊卵泡颗粒细胞并验证其表达变化。结果表明，RARRES1可能对山羊多胎性状具有促进作用，为进一步探究RARRES1基因对山羊多胎性状的调控机理提供依据。

关键词: 黔北麻羊, RARRES1基因, 超表达载体, 干扰载体, 卵泡颗粒细胞

Abstract: The aim of this study was to explore the function of the retinoic acid receptor responder 1 gene in Qianbei Ma goat. In this study, the complete sequence encoded by RARRES1 gene of Qianbei Ma goat was obtained by PCR amplification. The bioinformatics method was used to analyze the physical and chemical properties, higher protein structure, hydrophobicity, amino acid homology, subcellular localization of RARRES1 protein, and the phylogenetic tree was constructed. At the same time, qRT-PCR was used to detect the expression level of RARRES1 gene in different tissues of monotocous and polytocous of Qianbei Ma goat, and then the eukaryotic expression vector pEGFP-N3-RARRES1 and pGPH1/GFP/Neo-RARRES1 RNA interference vector were constructed and transfected into the follicular granulosa cells of Qianbei Ma goat. The effect of the eukaryotic expression vector pEGFP-N3-RARRES1 and pGPH1/GFP/Neo-RARRES1 RNA interference vector on the expression of RARRES1 and candidate genes for fecundity BMPR-IB mRNA were detected by qRT-PCR at cell level. qRT-PCR results showed that the expression of RARRES1 mRNA was the highest in the ovary, the expression level in ovarian tissue of Qianbei Ma goat in the monotocous group was extremely significantly higher than that in the polytocous group(P<0.01). Bioinformatics analysis showed that the RARRES1 gene encoded 289 amino acids, RARRES1 was a hydrophilic protein localized in the cytoplasm. The secondary structure analysis of the protein showed that it was mainly composed of α-helix and random coil. Its tertiary structure predicted was consistent with the secondary structure. The results of homology and phylogenetic tree showed that the RARRES1 gene sequence of Qianbei Ma goat had high homology with sheep and cattle, and their genetic distance was closer, the homology with chicken was the lowest and the genetic distance was the farthest. After double enzyme digestion and sequencing verification, the eukaryotic expression vector pEGFP-N3-RARRES1 and pGPH1/GFP/Neo-RARRES1 interference vector were successfully constructed. After the vectors were transfected into follicular granulosa cells of Qianbei Ma goat, compared with the blank control, recombinant plasmid pEGFP-N3-RARRES1 extremely significantly increased the expression of RARRES1 and BMPR-IB (P<0.01). Among the interfering vectors, the recombinant plasmid pGPH1/GFP/Neo-RARRES1-4 had the best interference efficiency, and it could significantly and extremely significantly down-regulate the expression of RARRES1 and BMPR-IB mRNA in follicular granulosa cells (P<0.05, P<0.01). In this study, the complete sequence of RARRES1 gene of Qianbei Ma goat was cloned and analyzed by PCR amplification and bioinformatics analysis, respectively. The recombinant plasmids pEGFP-N3-RARRES1, pGPH1/GFP/Neo-RARRES1 were successfully transfected into follicular granulosa cells of Qianbei Ma goat, and their expression changes were verified. The results showed that RARRES1 might have a promoting effect on fecundity in goats, which provided a basis for further exploring the regulation mechanism of RARRES1 gene on fecundity in goats.

Key words: Qianbei Ma goat, RARRES1 gene, overexpression vector, interference vector, follicular granulosa cells

中图分类号:

S827.2

周志楠, 陈祥, 敖叶, 洪磊, 唐文, 陈浩瀚. 黔北麻羊RARRES1基因的克隆表达及功能初步研究[J]. 畜牧兽医学报, 2020, 51(7): 1548-1562.

ZHOU Zhinan, CHEN Xiang, AO Ye, HONG Lei, TANG Wen, CHEN Haohan. Cloning and Expression of RARRES1 Gene in Qianbei Ma Goat and Preliminary Study on Its Function[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1548-1562.

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