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

Abstract

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

CLC Number:

S827.2

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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Cloning and Expression of RARRES1 Gene in Qianbei Ma Goat and Preliminary Study on Its Function

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