水牛p21基因在卵母细胞体外成熟过程中的表达及其真核表达载体构建
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摘要
旨在检测p21基因在水牛卵母细胞体外成熟过程中的表达变化,并克隆摩拉水牛p21基因CDs序列,构建真核表达载体。收集体外成熟培养不同时期(0、6、12、24h)和不同形态[有第1极体(first polar body,PB1)PB1和无PB1]的水牛卵母细胞,通过RT-qPCR检测其p21mRNA相对表达量。同时利用RTPCR技术从摩拉水牛卵巢颗粒细胞中扩增出p21基因编码序列,插入pEGFP-N1真核表达载体中,构建出重组质粒。将重组质粒pEGFP-N1-p21转染水牛颗粒细胞,并于培养24h和48h后观察转染细胞的荧光表达情况。收集转染48h后的颗粒细胞,通过RT-qPCR检测p21mRNA相对表达量。结果显示,成熟培养6h的卵母细胞p21mRNA表达量显著高于成熟培养0、12、24h的表达量(P<0.05),有PB1的卵母细胞中p21mRNA的表达量显著高于无PB1的卵母细胞(P<0.05);重组质粒pEGFP-N1-p21转染颗粒细胞后,有绿色荧光蛋白表达,且与转染pEGFP-N1和空白组相比,pEGFP-N1-p21转染组的p21mRNA表达量显著升高。结果表明,在体外成熟培养过程中均能检测到水牛卵母细胞p21mRNA表达量,成功构建了摩拉水牛p21基因真核表达载体,为下一步研究p21基因在水牛卵母细胞成熟和胚胎发育过程中的功能和调控奠定了基础。
The objective of this study was to determine the expression of p21 gene of buffalo oocytes during IVM and to clone the CDs of p21 gene to construct the eukaryotic expression vector of Murrah buffalo.The buffalo oocytes of different periods(0,6,12,24 h)and different morphologies(with PB1 and no PB1)were collected during in vitro oocyte maturation(IVM),and the expression of p21 gene was detected by qRTPCR.The coding sequence(CDs)of p21 gene was amplified from the ovarian granulosa cells of Murrah buffalo using RT-PCR technique.The p21 sequence was inserted into the pEGFP-N1 to construct the pEGFP-N1-p21 eukaryotic expression vector.The plasmids were transfected into ovarian granulosa cells and investigated by the green fluorescence under microscope after 24 hand 48 h.The relative transcription levels of p21 gene mRNA were determined by qRT-PCR 48 hafter transfecting ovarian granulosa cells.The results showed that the expression of p21 gene in 6 hduring IVM was significantly increased compared with other groups(P<0.05),and the expression of p21 gene in oocytes that emitted the first polar body was significantly higher than those oocytes that did not(P<0.05);The pEGFP-N1-p21 vector was constructed successfully and the green fluorescence was observed in the transfected cells.Compared with pEGFP-N1,NC,the relative level of p21 gene mRNA increased significantly in ovarian granulosa cells which transfected with pEGFP-N1-p21.In conclusion,these data demonstrated that expression of p21 gene was detected in different phases of buffalo ooctyes,and the pEGFP-N1-p21 was constructed successfully.This greatly facilitated the further study of function and regulation of p21 gene,especially its role on oocytes maturation and early embryo development in buffalo.
The objective of this study was to determine the expression of p21 gene of buffalo oocytes during IVM and to clone the CDs of p21 gene to construct the eukaryotic expression vector of Murrah buffalo.The buffalo oocytes of different periods(0,6,12,24 h)and different morphologies(with PB1 and no PB1)were collected during in vitro oocyte maturation(IVM),and the expression of p21 gene was detected by qRTPCR.The coding sequence(CDs)of p21 gene was amplified from the ovarian granulosa cells of Murrah buffalo using RT-PCR technique.The p21 sequence was inserted into the pEGFP-N1 to construct the pEGFP-N1-p21 eukaryotic expression vector.The plasmids were transfected into ovarian granulosa cells and investigated by the green fluorescence under microscope after 24 hand 48 h.The relative transcription levels of p21 gene mRNA were determined by qRT-PCR 48 hafter transfecting ovarian granulosa cells.The results showed that the expression of p21 gene in 6 hduring IVM was significantly increased compared with other groups(P<0.05),and the expression of p21 gene in oocytes that emitted the first polar body was significantly higher than those oocytes that did not(P<0.05);The pEGFP-N1-p21 vector was constructed successfully and the green fluorescence was observed in the transfected cells.Compared with pEGFP-N1,NC,the relative level of p21 gene mRNA increased significantly in ovarian granulosa cells which transfected with pEGFP-N1-p21.In conclusion,these data demonstrated that expression of p21 gene was detected in different phases of buffalo ooctyes,and the pEGFP-N1-p21 was constructed successfully.This greatly facilitated the further study of function and regulation of p21 gene,especially its role on oocytes maturation and early embryo development in buffalo.
引文
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[15]赵晓鹏.山羊卵母细胞体外成熟与老化的研究[D].陕西杨凌:西北农林科技大学,2012.
[16]Kumar S,Kumar M,Dholpuria S,et al.Transient arrest of germinal vesicle breakdown improved in vitro development potential of buffalo(Bubalus bubalis)oocytes[J].J Cell Biochem,2017,119(1):1-12.
[17]Shang J H,Zheng H Y,Yang C Y,et al.Cloning and bioinformatics analysis of the coding sequence of water buffalo P21gene[J].Transylvanian Rev,2016,5(special):382-388.
[2]魏如雪,郝海生,赵学明,等.抑制体细胞衰老促进诱导性多潜能干细胞(iPSC)生成的研究进展[J].中国农业科学,2015,48(16):3258-3265.
[3]Karimian A,Ahmadi Y,Yousefi B.Multiple functions of p21in cell cycle,apoptosis and transcriptional regulation after DNA damage[J].DNA Repair(Amst),2016,42:63-71.
[4]闫海龙,龚勇珍.氧化应激及p16和p53/p21与细胞衰老关系的研究进展[J].医学综述,2011,11(5):682-685.
[5]牛育鸿,董靖.肿瘤抑制基因与衰老的研究进展[J].基因组学与应用生物学,2015,34(7):1549-1554.
[6]Guo Q,Wang D,Liu Z,et al.Effects of p21gene down-regulation through RNAi on antler stem cells in vitro[J].PloS One,2015,10(8):e134268.
[7]Moore G D,Ayabe T,Kopf G S,et al.Temporal patterns of gene expression of G1-S cyclins and cdks during the first and second mitotic cell cycles in mouse embryos[J].Mol Reprod Develop,1996,45(3):264-275.
[8]武迪迪,具英花,张杰,等.PKC、PKB通过p21~(CIP1/WAF1)蛋白对小鼠受精卵G2期向M期转换的影响[J].生殖与避孕,2006,26(7):387-391.
[9]李鹂,黄衡宇,谢永芳.p21基因在小鼠胚胎发育过程中的表达[J].吉首大学学报:自然科学版,2006,27(2):80-83.
[10]陈五妍,王正虹,陈树林,等.p21^WAF1/CIP1蛋白在小鼠卵泡与黄体中的表达[J].西北农林科技大学学报:自然科学版,2009(12):29-33.
[11]赵贵民,杨文琳,吴苏君,等.p21基因在牛卵成熟中的表达检测及其真核表达载体构建[J].畜牧兽医学报,2011,42(8):1071-1080.
[12]赵贵民.P21蛋白在牛卵成熟过程中作用的初步研究[D].陕西杨凌:西北农林科技大学,2011.
[13]Frank-vaillant M,Haccard O,Ozon R,et al.Interplay between Cdc2Kinase and the c-Mos/MAPK pathway between metaphaseⅠand metaphaseⅡin xenopus oocytes[J].Develop Biol,2001,231(1):279-288.
[14]许晓磊.牛卵母细胞中的DNA双链损伤和P21基因的相关性研究[D].陕西杨凌:西北农林科技大学,2015.
[15]赵晓鹏.山羊卵母细胞体外成熟与老化的研究[D].陕西杨凌:西北农林科技大学,2012.
[16]Kumar S,Kumar M,Dholpuria S,et al.Transient arrest of germinal vesicle breakdown improved in vitro development potential of buffalo(Bubalus bubalis)oocytes[J].J Cell Biochem,2017,119(1):1-12.
[17]Shang J H,Zheng H Y,Yang C Y,et al.Cloning and bioinformatics analysis of the coding sequence of water buffalo P21gene[J].Transylvanian Rev,2016,5(special):382-388.