| 黔北麻羊SRD5A2基因干扰载体构建及其对产羔相关基因表达的影响 |
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| 引用本文： | 洪磊,陈祥,唐文,周志楠,段志强,赵佳福.黔北麻羊SRD5A2基因干扰载体构建及其对产羔相关基因表达的影响[J].畜牧兽医学报,2020,51(12):2980-2990. |
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| 作者姓名： | 洪磊 陈祥 唐文 周志楠 段志强 赵佳福 |
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| 作者单位： | 1. 贵州大学 高原山地动物遗传育种与繁殖教育部重点试验室, 贵州省动物遗传育种与 繁殖重点试验室, 贵阳 550025;2. 贵州大学动物科学学院, 贵阳 550025 |
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| 基金项目： | 国家重点研发计划项目（2018YFD0502005）；国家自然科学基金（31760652）；贵州省优秀青年科技人才项目（〔2017〕5624） |
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| 摘 要： | 旨在探究SRD5A2基因对山羊产羔性状相关基因表达的影响。本研究选择健康的（2~3岁）黔北麻羊经产母羊3只，体重约32 kg，采集卵巢组织并成功培养卵巢颗粒细胞。通过在线软件设计SRD5A2基因的4对siRNA干扰序列和1对阴性对照序列，连接pGPH1/GFP/Neo载体后，将构建成功的干扰载体转染至黔北麻羊卵巢颗粒细胞。利用qRT-PCR方法筛选出干扰效率最佳的载体，检测其对SRD5A2基因表达的影响。运用qRT-PCR检测沉默SRD5A2基因对山羊产羔性状相关基因骨形态发生蛋白15（BMP15）、生长分化因子9（GDF9）、骨形态发生蛋白1B（BMPR-1B）和卵泡刺激素β亚基（FSHβ）表达的影响。结果表明，本试验在培养黔北麻羊卵巢颗粒细胞的基础上，成功构建了黔北麻羊SRD5A2基因的干扰载体，并筛选出shRNA-SRD5A2-1干扰效果最佳（P<0.01）；抑制SRD5A2基因表达后，qRT-PCR检测产羔性状相关基因BMP15、BMPR-1B、GDF9和FSHβ的表达量均显著降低，BMP15的表达量极显著低于shRNA-NC对照组（P<0.01），BMPR-1B、GDF9和FSHβ的表达量显著低于shRNA-NC对照组（P<0.05）。本研究成功构建了SRD5A2基因干扰载体并转染至卵巢颗粒细胞，发现体外沉默SRD5A2基因可抑制产羔性状相关基因的表达，提示SRD5A2基因与黔北麻羊产羔性状密切相关，本试验结果为进一步研究SRD5A2基因对黔北麻羊产羔性状的调控机制提供了基础。
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| 关 键 词： | 黔北麻羊 SRD5A2基因 RNA干扰 卵巢颗粒细胞 产羔性状 |
| 收稿时间： | 2020-06-01 |
Construction of SRD5A2 Gene Interference Vector and Its Effect on the Expression of Lambing Related Genes in Qianbei Ma Goats |
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| HONG Lei,CHEN Xiang,TANG Wen,ZHOU Zhinan,DUAN Zhiqiang,ZHAO Jiafu.Construction of SRD5A2 Gene Interference Vector and Its Effect on the Expression of Lambing Related Genes in Qianbei Ma Goats[J].Acta Veterinaria et Zootechnica Sinica,2020,51(12):2980-2990. |
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| Authors: | HONG Lei CHEN Xiang TANG Wen ZHOU Zhinan DUAN Zhiqiang ZHAO Jiafu |
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| Institution: | 1. Guizhou Key Laboratory of Plateau Mountain Animal Genetics, Breeding and Reproduction, Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Education, Guizhou University, Guiyang 550025, China;2. College of Animal Science, Guizhou University, Guiyang 550025, China |
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| Abstract: | The purpose of this study was to investigate the effect of SRD5A2 gene on the expression of genes related to goat lambing traits. In this study, 3 healthy (2-3 years old) and female Qianbei Ma goat with a weight of approximate 32 kg were selected. Ovary tissue was collected and granulosa cells were successfully cultured. Four pairs of siRNA interference sequence of SRD5A2 gene sequence and a pair of negative control sequence were designed by online software. After connecting pGPH1/GFP/Neo vector, the constructed interference vectors were transfected into ovarian granulosa cells of Qianbei Ma goats. The interference vector with optimal interference efficiency was screened by qRT-PCR, and its effect on the expression of SRD5A2 gene was detected. The effects of silencing SRD5A2 gene on the expression of bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), bone morphogenetic protein-1B (BMPR-1B) and follicle-stimulating hormone lactin (FSHβ) were investigated. In this experiment, the interference vector of SRD5A2 gene was successfully constructed on the basis of cultivating ovarian granulosa cells of Qianbei Ma goats. shRNA-SRD5A2-1 interference vector with the optimal interference efficiency was screened out (P<0.01). After inhibiting SRD5A2 gene expression, the expression levels of BMP15, BMPR-1B, GDF9 and FSHβ were significantly reduced. The expression level of BMP15 was extremely significantly lower than that in shRNA-NC control group (P<0.01), and the expression levels of BMPR-1B, GDF9 and FSHβ were significantly lower than those in shRNA-NC control group (P<0.05). In this study, the constructed SRD5A2 gene interference vector was successfully transfected into ovarian granulosa cells. The silencing of SRD5A2 gene can inhibit the expression of genes related to lambing traits, which indicate that SRD5A2 gene is closely related to lambing traits of Qianbei Ma goats, the result will provide a basis for further studies on the regulatory mechanism of SRD5A2 gene on lambing traits in Qianbei Ma goats. |
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| Keywords: | Qianbei Ma goat SRD5A2 gene RNA interference ovarian granulosa cells lambing trait |
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