籽鹅卵泡颗粒细胞α-烯醇化酶基因RNA干扰表达质粒的构建及鉴定

Construction and identification of α-enolase gene RNAi expression plasmid in Zi goose granulosa cells

目的构建籽鹅卵泡颗粒细胞α-烯醇化酶(α-enolase,ENO1)基因RNA干扰表达质粒,并进行鉴定。方法利用RNAi Designer等网络在线RNA干扰设计软件设计3条可能会干扰籽鹅卵泡颗粒细胞ENO1基因表达的插入DNA序列:shRNA-ENO1-350、shRNA-ENO1-892、shRNA-ENO1-591,将体外合成的3条干扰序列分别与线性化的pGPU6/GFP/Neo载体连接,构建ENO1 RNA干扰表达质粒,在Lipofectamine 2000的介导下转染原代培养的籽鹅卵泡颗粒细胞,转染48 h后,荧光倒置显微镜下观察GFP的表达;实时荧光定量PCR和Western blot法检测转染细胞中ENO1基因mRNA的水平及蛋白的表达水平。结果 pGPU6/GFP/Neo-shDNA重组质粒经单酶切及测序证实构建正确;转染后48 h,转染重组质粒的颗粒细胞在荧光倒置显微镜下可见较强的绿色荧光,转染效率可达60%;与培养液组、转染试剂组和无关序列干扰组(shNC)相比,shRNA-ENO1-350组颗粒细胞中ENO1基因mRNA水平和蛋白表达水平均显著降低(P<0.01),其他各组之间差异无统计学意义(P>0.05)。结论成功构建了籽鹅卵泡颗粒细胞ENO1基因RNA干扰表达质粒,在原代培养的籽鹅卵泡颗粒细胞中,其表达量显著下降,为后续有关ENO1功能的研究奠定了基础。

Objective To construct and identify α-enolase （ENO1） gene RNAi expression vector in Zi goose granulosa cells. Methods Three cDNA sequences named as shRNA-ENO1-350, shRNA-ENO1-892 and shRNA-ENO1-591, which might interfere in the expression of ENO1 gene in Zi goose granulose cells, were designed by online software such as RNAi Designer, then synthesized in vitro and inserted into linearized vector pGPU6/GFP/Neo respectively. The constructed ENO1 RNA interference expression plasmids were transfected into primary Zi goose granulosa cells in mediation of Lipofectamine 2000, and observed for expression of green fluorescent protein （GFP） under inverted fluorescent microscope 48 h later. The expressions of ENO1 at mRNA and protein levels were determined by real-time fluorescent quantitative PCR and Western blot respectively. Results Restriction analysis and sequencing proved that recombinant plasmid pGPU6/GFP/Neo-shDNA was constructed correctly. Strong green fluorescence was observed in the cells 48 h after transfection with the recombinant plasmid, with a transfection efficiency of 60%. The expression levels of ENO1 mRNA and protein in were significantly lower in shRNA-ENO1-350 group than in culture medium, transfection reagent and shNC groups （P 〈 0.01）, while showed no significant difference in the latter three groups （P 〉 0.05）. Conclusion The RNA interference expression vector for ENO1 gene in Zi goose granulosa cells was constructed suceessfuly, of which the expression level decreased significantly in primary Zi goose guanulosa cells. It laid a foundation of further study on the function of ENO1.