周明帅, 温晓艳, 陆情梅, 刘彬, 杨永鲜, 赵佳福. 2023: 贵州黑山羊COL1A1基因真核表达载体构建及其对颗粒细胞中胶原蛋白家族基因和产羔基因的影响. 南方农业学报, 54(1): 261-272. DOI: 10.3969/j.issn.2095-1191.2023.01.026
引用本文: 周明帅, 温晓艳, 陆情梅, 刘彬, 杨永鲜, 赵佳福. 2023: 贵州黑山羊COL1A1基因真核表达载体构建及其对颗粒细胞中胶原蛋白家族基因和产羔基因的影响. 南方农业学报, 54(1): 261-272. DOI: 10.3969/j.issn.2095-1191.2023.01.026
ZHOUMing-shuai, WENXiao-yan, LUQing-mei, LIUBin, YANGYong-xian, ZHAOJia-fu. 2023: 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. Journal of Southern Agriculture, 54(1): 261-272. DOI: 10.3969/j.issn.2095-1191.2023.01.026
Citation: ZHOUMing-shuai, WENXiao-yan, LUQing-mei, LIUBin, YANGYong-xian, ZHAOJia-fu. 2023: 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. Journal of Southern Agriculture, 54(1): 261-272. DOI: 10.3969/j.issn.2095-1191.2023.01.026

贵州黑山羊COL1A1基因真核表达载体构建及其对颗粒细胞中胶原蛋白家族基因和产羔基因的影响

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

  • 摘要: 【目的】构建I型胶原蛋白α1链基因(COL1A1)过表达载体,并分析其对颗粒细胞中胶原蛋白家族基因和产羔相关基因的影响,为后续深入研究COL1A1基因影响山羊产羔性状的分子机制打下基础。【方法】通过重叠延伸PCR克隆COL1A1基因A和B片段,然后分别重组连接至pUC57和pcDNA3.1(+)线性化载体上,再通过酶切连接获得COL1A1基因全长,构建pcDNA3.1(+)-COL1A1过表达载体;转染山羊卵巢颗粒细胞后,通过Western blotting检测COL1A1蛋白表达效率,采用免疫荧光检测其在颗粒细胞中的定位情况,并以实时荧光定量PCR检测COL1A1基因过表达对胶原蛋白家族基因COL1A1COL2A1COL3A1及产羔相关基因[骨形态发生蛋白15(BMP15)、骨形态发生蛋白受体1B(BMPR1B)、生长分化因子9(GDF9)和促卵泡激素β亚基(FSHB)]表达的影响。【结果】COL1A1基因全长环化质粒检测大小为1847 bp,连接至pcDNA3.1(+)线性化载体上成功构建获得COL1A1基因过表达载体。Westernblotting检测结果表明,pcDNA3.1(+)-COL1A1转染颗粒细胞48 h后,COL1A1蛋白表达量极显著高于pcDNA3.1(+)空载体转染组(P<0.01,下同);免疫荧光检测结果表明,COL1A1蛋白在颗粒细胞细胞核及细胞质中均有表达,但在细胞核中的表达水平更高;实时荧光定量PCR检测结果表明,pcDNA3.1(+)-COL1A1转染组颗粒细胞中COL1A1COL2A1COL3A1胶原蛋白家族基因及GDF9FSHBBMP15产羔相关基因的相对表达量极显著高于pcDNA3.1(+)空载体转染组,但对BMPR1B基因的表达无显著影响(P>0.05)。【结论】COL1A1基因在卵巢颗粒细胞中广泛表达,其过表达能极显著促进胶原蛋白家族基因COL2A1COL3A1及产羔相关基因GDF9FSHBBMPR1B在颗粒细胞中的表达,即COL1A1基因可协同胶原蛋白家族基因的表达促进ECM合成来影响卵巢组织结构、胚胎发育及促进性腺激素表达,进而影响山羊产羔性状,可作为影响贵州黑山羊多羔候选基因进行深入研究。

     

    Abstract: 【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 COL1A1COL2A1 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 COL1A1COL2A1 and COL3A1 collagen family genes and GDF9FSHB 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 GDF9FSHB 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.

     

/

返回文章
返回