TGFβ2和PPP3CA基因在黔北麻羊性腺轴组织表达及对卵巢颗粒细胞的调控研究

doi:10.11843/j.issn.0366-6964.2020.11.009

Abstract

Abstract: The aim of this study was to verify the up-regulation and down-regulation of TGFβ2 and PPP3CA genes in transcriptome sequencing in monotocous group compared to polytocous group, and to explore the regulatory mechanism of TGFβ2 and PPP3CA genes on ovarian granulosa cells of Qianbei Ma goat. The monotocous and polytocous Qianbei Ma goat ewes were used as the research object. The expression levels of TGFβ2 and PPP3CA genes in the gonadal axis of monotocous and polytocous Qianbei Ma goat were detected by RT-qPCR technology; The eukaryotic expression vector of target genes were constructed. Qianbei Ma goat ovarian granular cells were cultured, the pEGFP-N3-TGFβ2 and pEGFP-N3-PPP3CA recombinant plasmids were transfected into ovarian granular cells by liposome transfection method, and the concentrations of estradiol(E2) and progesterone(P4) in the medium were detected. The effects of TGFβ2 and PPP3CA genes on the proliferation and apoptosis of ovarian granulosa cells were detected using CCK-8 and Annexin V-FITC methods. Then RT-qPCR was used to detect the effect of overexpressing TGFβ2, PPP3CA genes on TGFβ2, PPP3CA, GnRHR, FSHR, BMP4, TIMP3 genes expression at the cell level. RT-qPCR results showed that TGFβ2 and PPP3CA genes were expressed in the tested tissues, and the expression levels in the monotocous group were generally higher than those in the polytocous group; Immunofluorescence result confirmed that the cultured cells were Qianbei Ma goat ovarian granulosa cells; After the eukaryotic expression plasmids of TGFβ2 and PPP3CA genes were transfected into ovarian granulosa cells, the expressions of TGFβ2 and PPP3CA genes significantly increased while the expressions of FSHR, GnRHR, BMP4, TIMP3 genes were significantly inhibited in ovarian granulosa cells (P<0.01). The results of cell proliferation and apoptosis showed that TGFβ2 and PPP3CA genes could promote the apoptosis of granulosa cells and inhibit their proliferation. The result of enzyme-linked immunosorbent assay showed that TGFβ2 and PPP3CA genes could significantly promote the secretion of E2, but had no significant effect on the secretion of P4. The eukaryotic expression vector of TGFβ2 and PPP3CA genes were successfully constructed in this study. Fluorescence quantitative results showed that overexpression of TGFβ2 and PPP3CA genes inhibited the expression of reproduction-related genes, suggesting that TGFβ2 and PPP3CA genes had a certain effect on goat reproduction-related genes expression. These results will provide basic data for further research on the influence of TGFβ2 and PPP3CA genes on the fecundity of Qianbei Ma goat.

Key words: Qianbei Ma goats, TGFβ2 gene, PPP3CA gene, ovarian granulosa cells, differential expression

CLC Number:

AO Ye, CHEN Xiang, ZHOU Zhinan, LUO Jinhong, WEI Shinan, ZHANG Yan, HUI Maomao, WU Yu, YANG Peifang. Expression of TGFβ2 and PPP3CA Genes in Gonadal Axis of Qianbei Ma Goat and Their Regulation on Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(11): 2699-2709.

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Expression of TGFβ2 and PPP3CA Genes in Gonadal Axis of Qianbei Ma Goat and Their Regulation on Ovarian Granulosa Cells

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