黔北麻羊<em>HSD3B1</em>基因真核表达载体构建及其对卵巢颗粒细胞的影响

doi:10.3969/j.issn.1674-7968.2020.09.009

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摘要 3β-羟基类固醇脱氢酶-1 (3β-hydroxysteroid dehydrogenase 1, HSD3B1)是类固醇激素家族中的关键成员之一,与动物的繁殖性状密切相关。为了克隆黔北麻羊(Capra hircus) HSD3B1基因,构建真核表达载体,探究其对产羔性状相关基因在卵巢颗粒细胞表达的影响,本研究采集黔北麻羊卵巢组织进行卵巢颗粒细胞培养并提取组织中的总RNA,反转录为cDNA,克隆黔北麻羊HSD3B1基因(GenBank No. NM_001135932.1) CDS区全长序列,并进行生物信息学分析;同时将构建的HSD3B1基因真核表达载体,转染至卵巢颗粒细胞,qRT-PCR检测HSD3B1、山羊产羔性状相关的骨形成蛋白15基因(bone morphogenetic protein 15, BMP15)、生长分化因子9基因(growth differentiation factor 9, GDF9)、骨形态发生蛋白受体1B基因(bone morphogenetic protein receptor type 1B, BMPR-1B)的mRNA表达水平。结果发现：T-克隆的黔北麻羊HSD3B1基因CDS区为1 122 bp,编码373个氨基酸,与NCBI上登录的绵羊(Ovis aries)的mRNA序列相同;蛋白预测结果显示：黔北麻羊HSD3B1蛋白的分子式为C₁₉₄₀H₃₀₀₂N₅₀₆O₅₄₃S₁₅,相对分子质量为42.583 14 kD;等电点为7.96;遗传进化树分析得出,黔北麻羊HSD3B1基因与牛(Bos taurus)的遗传距离最近;qRT-PCR结果显示,转染实验组HSD3B1表达量极显著高于空白组(P<0.01),产羔性状相关的GDF9基因表达量极显著高于空白对照组(P<0.01),BMP15基因的表达量显著高于空白对照组(P<0.05),表明HSD3B1基因的超表达可以促进产羔相关的GDF9基因和BMP15基因在卵巢颗粒细胞的表达。本研究成功构建pEGFP-N3-HSD3B1真核表达载体,检测HSD3B1基因对黔北麻羊产羔性状相关基因的表达影响,为进一步研究HSD3B1基因对黔北麻羊产羔性状的影响提供基础资料。

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	洪磊
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	韦仕南

关键词 ：黔北麻羊, 3β, -羟基类固醇脱氢酶-1 (HSD3B1), T-克隆, 生物信息学分析, 真核载体构建

Abstract：3β-hydroxysteroid dehydrogenase 1 (HSD3B1) is a key member of the steroid hormone family and is closely related to animal reproductive traits. In order to clone the HSD3B1 gene of the Qianbei Ma goat (Capra hircus), construct a eukaryotic expression vector, and explore its effect on the expression of genes, in this study, Qianbei Ma goats ewes were selected to collect ovarian tissue for ovarian granulosa cell culture and extract the total RNA in the tissue for reverse transcription into cDNA. The full-length sequence of HSD3B1 (GenBank No. NM_001135932.1) gene CDS region of Qianbei Ma Goats was cloned and analyzed by bioinformatics. Meanwhile, eukaryotic expression vector of HSD3B1 gene was constructed and transfected into ovarian granulosa cells. mRNA expression levels of HSD3B1, bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), and bone morphogenetic protein receptor type 1B (BMPR-1B) genes were detected by qRT-PCR. The results of protein prediction analysis showed that the molecular formula of HSD3B1 was C₁₉₄₀H₃₀₀₂N₅₀₆O₅₄₃S₁₅, and the relative molecular weight was 42.583 14 kD, isoelectric point 7.96; The genetic tree analysis showed that the genetic distance between the HSD3B1 gene and the cattle (Bos taurus) was the closest. The results of qRT-PCR showed that the expression of HSD3B1 and GDF9 gene in the transfection experimental group were significantly higher than those in the blank group (P<0.01), and the mRNA expression level of BMP15 gene was significantly higher than that of the control group (P<0.05). The results showed that the overexpression of HSD3B1 gene could promote the expression of GDF9 and BMP15. In this study, the pEGFP-N3-HSD3B1 eukaryotic expression vector was successfully constructed to detect the effect of HSD3B1 gene on the expression of genes related to lambing traits of Qianbei Ma goats, and provide basic data for further research on the effect of HSD3B1 gene on lambing traits of Qianbei Ma Goats..

Key words： Qianbei Ma goat 3β-hydroxysteroid dehydrogenase 1 (HSD3B1); T-clone Bioinformatics analysis Eukaryotic vector construction

收稿日期: 2020-01-19

ZTFLH:

S813.3

通讯作者: *,xchen2@gzu.edu.cn

引用本文:

洪磊, 陈祥, 敖叶, 周志楠, 唐文, 韦仕南. 黔北麻羊HSD3B1基因真核表达载体构建及其对卵巢颗粒细胞的影响[J]. 农业生物技术学报, 2020, 28(9): 1604-1613.
HONG Lei, CHEN Xiang, AO Ye, ZHOU Zhi-Nan, TANG Wen, WEI Shi-Nan. Construction of Eukaryotic Expression Vector of HSD3B1 Gene and Its Effect on Ovarian Granulosa Cells of Qianbei Ma Goat (Capra hircus). 农业生物技术学报, 2020, 28(9): 1604-1613.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2020.09.009 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2020/V28/I9/1604

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