NGF基因在黔北麻羊卵巢颗粒细胞及组织的表达分析  

Analysis of NGF gene expression in ovary granulosa cells and tissues of Qianbei Ma goats

作　　者：陈祥[1] 敖叶 李诚兰 赵佳福[1] 周志楠 韦仕南 CHEN Xiang;AO Ye;LI Chenglan;ZHAO Jiafu;ZHOU Zhinan;WEI Shinan(Key Laboratoryof Animal Genetics,Breeding and Reproduction,Ministry of Education/Guizhou Key Laboratory of Animal Genetics,Breeding and Reproduction,College of Animal Science,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州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,贵州贵阳550025

出　　处：《中国兽医学报》2023年第7期1558-1566,共9页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31760652);贵州省优秀青年科技人才资助项目([2017]5624)。

摘　　要：旨在验证NGF基因在转录组测序中单羔组对多羔组上调的结果,并探究NGF基因对黔北麻羊产羔性状的影响。选取健康、体质量45 kg、4岁左右的单、多羔黔北麻羊母羊为研究对象,采用qPCR法检测NGF基因在单、多羔组黔北麻羊母羊不同组织的表达水平,构建pEGFP-N3-NGF重组质粒,利用脂质体转染法转染pEGFP-N3-NGF重组质粒进入卵巢颗粒细胞,分为超表达NGF试验组和pEGFP-N3空载体对照组,通过检测培养基中雌二醇、孕酮的浓度,及对卵巢颗粒细胞增殖、凋亡、NGF、GnRHR、FSHR、BMP4、TIMP3基因mRNA表达的影响,来探究NGF基因对卵巢颗粒细胞的影响。结果表明,NGF基因在黔北麻羊各组织中均有表达,组间比较可知,卵巢和输卵管在多羔组表达极显著高于单羔组(P<0.01)。成功将pEGFP-N3-NGF重组质粒转染进入卵巢颗粒细胞,在12 h对E2的分泌具有显著促进作用(P<0.05),而对P4的分泌在12,24 h具有显著抑制作用(P<0.05),对卵巢颗粒细胞增殖在24,48 h具有极显著抑制作用(P<0.01),对卵巢颗粒细胞的凋亡具有促进作用。而对繁殖相关基因GnRHR、FSHR、BMP4、TIMP3 mRNA的表达有极显著促进作用(P<0.01)。综上表明,NGF基因可提高E2的分泌,抑制P4的分泌,有助于黔北麻羊发情及妊娠的维持,抑制细胞增殖,促进细胞凋亡,也极显著促进繁殖相关基因mRNA的表达,而这些繁殖相关基因都能够调控卵泡数、卵泡的发育与形成以及排卵,因此,NGF基因能够促进这些基因表达,进而促进卵泡的发育、形成及排卵,提高山羊的产羔力,这为产羔性状相关基因的研究提供了理论基础,初步认为NGF基因可作为探究影响山羊产羔性状的候选基因。To verify the results of up-regulation of NGF gene in transcriptome sequencing by single-lamb group to multi-lamb group,and to explore the effect of NGF gene on lamb traits in northern Qianbei Ma goats.In this experiment,single and multi-lamb Qianbei Ma female goats with a healthy weight of 45kg and about 4 years of age is selected as the research subjects.The qPCR method was used to detect the expression level of NGF gene in different tissues of single-lamb and multi-lamb be Qianbei Ma female goats.Constructed pEGFP-N3-NGF recombinant plasmid,and use liposome transfection method to transfect pEGFP-N3-NGF recombinant plasmid into ovarian granulosa cells,divided into overexpression NGF test group and pEGFP-N3 empty vector control group,pass the test the concentrations of estradiol and progesterone in the medium and their effects on ovarian granulosa cell proliferation,apoptosis,NGF,GnRHR,FSHR,BMP4,and TIMP3 gene mRNA expression was investigated to explore the effect of NGF gene on ovarian granulosa cells.The results showed that the NGF gene was expressed in all tissues of Qianbei Ma goats,and comparison between groups showed that ovaries and fallopian tubes were highly expressed in the single lamb group(P<0.01).The pEGFP-N3-NGF recombinant plasmid was successfully transfected into ovarian granulosa cells,which significantly promoted the secretion of E2 at 12 h(P<0.05),while the secretion of P4 was significantly inhibited at 12 and 24 h(P<0.05),which has a very significant inhibitory effect on the proliferation of ovarian granulosa cells at 24 h and 48 h(P<0.01),and promotes the apoptosis of ovarian granulosa cells.The expression of GnRHR,FSHR,BMP4,and TIMP3 mRNA in the genes related to reproduction was extremely significant(P<0.01).In summary,the NGF gene can increase the secretion of E2,inhibit the secretion of P4,help the maintenance of estrus and pregnancy in Qianbei Ma goats,inhibit cell proliferation,promote apoptosis,and also significantly promote the expression of reproduction-related gene mRNA.These reprod

关 键 词：黔北麻羊 NGF 卵巢颗粒细胞 差异表达 

分 类 号：S826[农业科学—畜牧学]