SMAD1基因对黔北麻羊卵巢颗粒细胞的影响及组织表达分析

Effect of SMAD1 Gene on Ovarian Granulosa Cells and Its Tissue Expression Analysis in Qianbei Ma Goats

旨在验证SMAD1基因在转录组测序中单羔组对多羔组上调的结果,并探究SMAD1基因对黔北麻羊产羔性状的影响。本试验选取健康、体重45 kg、4岁左右的单、多羔黔北麻羊母羊为研究对象,采用qRT-PCR法检测SMAD1基因在单、多羔组黔北麻羊母羊子宫、输卵管、垂体、下丘脑及卵巢组织的表达水平,PCR扩增黔北麻羊SMAD1基因CDS区,构建pEGFP-N3-SAMD1重组质粒,利用脂质体转染法转染pEGFP-N3-SAMD1重组质粒进入卵巢颗粒细胞,分为超表达SMAD1试验组和pEGFP-N3空载体对照组,通过检测培养基中雌二醇和孕酮的浓度,利用CCK-8法和Annexin V-FITC法检测超表达SMAD1基因对卵巢颗粒细胞增殖、凋亡的影响,qRT-PCR检测超表达SMAD1基因对SAMD1、GnRHR、FSHR、BMP4、TIMP3基因mRNA表达的影响,来探究SMAD1基因对卵巢颗粒细胞的影响。结果表明,SMAD1基因在黔北麻羊各组织中均有表达。组间比较可知,卵巢和下丘脑组织单羔组极显著高表达于多羔组(P<0.01),输卵管组织中单羔组显著高表达于多羔组(P<0.05),其他组织未达到差异显著性(P>0.05)。本研究成功克隆了SMAD1基因CDS序列,未发现突变位点,并将pEGFP-N3-SAMD1重组质粒转染进入卵巢颗粒细胞,转染试验组培养基中E2含量在12、48 h显著高于对照组(P<0.05),P4含量在12 h显著高于对照组(P<0.05);超表达SMAD1基因对细胞增殖在24 h达到显著促进作用(P<0.05),48、72 h达到极显著促进作用(P<0.01),并且发现超表达SMAD1基因对卵巢颗粒细胞的凋亡具有抑制作用,与SMAD1基因对卵巢颗粒细胞增殖具有促进的结果是相符的。SMAD1基因超表达后繁殖相关基因GnRHR、FSHR、BMP4、TIMP3 mRNA的表达极显著降低(P<0.01)。综上表明,SMAD1基因超表达可提高E2、P4的生成,有助于黔北麻羊发情及妊娠的维持,促进细胞增殖,抑制细胞凋亡,也极显著抑制繁殖相关基因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,SMAD1基因能够抑制这些基因的表达,致使黔北麻羊单羔组受胎率低,进而降低产羔数,这为产羔性状相关基因的研究提供了理论基础,初步认为SMAD1基因可作为探究影响山羊产羔性状的候选基因。

The purpose of this study was to verify the results of SMAD1 gene up-regulation comparing single lamb group to multiple lambs group in transcriptome sequencing,and to explore the effect of SMAD1 gene on lambing traits of Qianbei Ma goats.In this experiment,healthy,4-year-old single,multi-lamb Qianbei Ma goats ewes with 45 kg were selected as the research objects.qRT-PCR method was used to detect the expression of SMAD1 gene in uterus,fallopian tubes,pituitary,hypothalamus and ovarian tissues of Qianbei Ma goats ewes in the single and multi-lamb groups.CDS region of SMAD1 gene of Qianbei Ma goats was amplified,recombinant plasmid pEGFP-N3-SAMD1 was constructed and was transfected into ovarian granulosa cells by liposome transfection method.The SMAD1 overexpression test group and pEGFP-N3 empty vector control group were set up.By detecting the concentration of estradiol and progesterone in the culture medium,the effect of overexpressing SMAD1 gene on the proliferation and apoptosis of ovarian granulosa cells was detected by CCK-8 and Annexin V-FITC methods.qRT-PCR was used to detect the effect of overexpressing SMAD1 gene on the mRNA expressions of SAMD1,GnRHR,FSHR,BMP4 and TIMP3 genes to explore the influence of SMAD1 gene on ovarian granulosa cells.The results showed that SMAD1 gene was expressed in various tissues of Qianbei Ma goats.Comparison between the groups showed that the expression of SMAD1 gene in ovary and hypothalamus tissues were extremely significantly higher in the single lamb group than those in the multiple lambs group(P<0.01),in the fallopian tube tissues,it was significantly higher in the single lamb group than that in the multiple lambs group(P<0.05),and there were no significant difference in other tissues between the groups(P>0.05).The CDS sequence of SMAD1 gene was successfully cloned,no mutation site was found.The recombinant plasmid pEGFP-N3-SAMD1 was transfected into ovarian granulosa cells.The E2 content in the culture medium in transfection test group was significantly higher than that in the control group at 12 and 48 h(P<0.05),the content of P4 in transfection test group was significantly higher than that in the control group at 12 h(P<0.05);Overexpressing SMAD1 gene significantly promoted cell proli-feration at 24 h(P<0.05),and extremely significantly promoted cell proliferation at 48 and 72 h(P<0.01).It was found that overexpression of SMAD1 gene inhibited the apoptosis of ovarian granulosa cells,which was consistent with the result of SMAD1 gene promoting the proliferation of ovarian granulosa cells.However,the mRNA expressions of GnRHR,FSHR,BMP4,and TIMP3 were extremely significantly decreased after overexpressing SMAD1 gene(P<0.01).In summary,the overexpression of SMAD1 gene can increase the production of E2 and P4,help to maintain the estrus and pregnancy of Qianbei Ma goats,promote cell proliferation,inhibit cell apoptosis,and also significantly inhibit the expression of reproduction-related genes mRNA,and these reproduction-related genes can regulate the number,development and formation of follicles and ovulation.Therefore,the SMAD1 gene can suppress the expressions of reproduction-related genes,which results in a low conception rate in the single lamb group of Qianbei Ma goats,and further reduces the number of lambs.The result of this study provide a theoretical basis for studying the lambing traits related genes,and SMAD1 gene can be used as a candidate gene to explore goat lambing traits.