Construction of eukaryotic expression vector of COL1A1 gene in Guizhou black goat and its effect on collagen family genes and kidding genes in granulosa cells

【Objective】To construct the overexpression vector of type I collagen α1 chain gene（COL1A1）and analyze its effect on collagen family genes and lambing genes in granulosa cells，so as to lay a foundation for further study on the molecular mechanism of COL1A1 gene affecting goat kidding traits. 【Method】The A and B fragments of COL1A1 gene were cloned by overlap extension PCR，and then recombined and ligated into pUC57 and pcDNA3.1（+）linearized vectors respectively. The full length of COL1A1 gene was obtained by double enzyme digestion and ligation，and the pcDNA3.1（+）-COL1A1 overexpression vector was constructed. After transfection of goat ovarian granulosa cells，the expression efficiency of COL1A1 protein was detected by Western blotting，and its localization in granulosa cells was detected by immunofluorescence. The effects of COL1A1 gene overexpression on the expression of collagen family genes COL1A1， COL2A1 and COL3A1 and kidding related genes［bone morphogenetic protein 15（BMP15），bone morphogenetic protein receptor 1B（BMPR1B），growth differentiation factor 9（GDF9）and follicle stimulating hormone β subunit（FSHB）］were detected by real-time fluorescence quantitative PCR. 【Result】The full-length circular plasmid of COL1A1 gene was 1847 bp，the overexpression vector of COL1A1 gene was successfully constructed after it was ligated into pcDNA3.1（+）linearized vectors. Western blotting results showed that the expression of COL1A1 protein in pcDNA3.1（+）-COL1A1 transfection group after 48 h was extremely significantly higher than that in pcDNA3.1（+）empty vector transfection group（P< 0.01，the same below）. Immunofluorescence results showed that COL1A1 protein was expressed in the nucleus and cytoplasm of granulosa cells，but the expression level in nucleus was higher. The results of real-time fluorescence quantitative PCR showed that the expression levels of COL1A1，COL2A1 and COL3A1 collagen family genes and GDF9，FSHB and BMP15 kidding-related genes in granulosa cells of pcDNA3.1（+）-COL1A1 transfection group were extremely significantly higher than those of pcDNA3.1（+）empty vector transfection group（P<0.01）. However，there was no significant effect on the expression of BMPR1B gene（P>0.05）. 【Conclusion】COL1A1 gene is widely expressed in ovarian granulosa cells， and its overexpression can extremely significantly promote the expression of collagen family genes COL2A1 and COL3A1 and lambingrelated genes GDF9，FSHB and BMPR1B in granulosa cells. That is，COL1A1 gene can cooperate with the expression of collagen family genes to promote ECM synthesis to affect ovarian tissue structure，embryonic development and promote gonadal hormone expression，thereby affecting the kidding traits of goats，which can be used as a candidate gene affecting the prolificacy of Guizhou black goats.