荧光定量PCR检测小鼠卵母细胞中Mad2mRNA的表达王建英1,杭苏琴2,李云龙13(11山东师范大学生命科学学院动物抗性重点实验室,山东济南250014;21南京农业大学动物科技学院,江苏南京210095)摘要:目的:应用SYBRGreenI染料法建立检测Mad2mRNA表达的实时荧光定量PCR的方法,并探讨小鼠卵母细胞减数分裂成熟中Mad2mRNA的表达。方法:采用实时荧光定量PCR的方法,以SYBRGreenI为荧光染料,梯度稀释的重组质粒为模板制作标准曲线,并以此方法分析了小鼠卵母细胞减数分裂成熟过程中Mad2mRNA表达的动态变化。结果:建立了实时荧光定量检测方法分析小鼠卵母细胞中Mad2mRNA的表达,该方法灵敏、特异,扩增效率接近100%。进行了标准曲线的制作,以及对目的基因Mad2转录水平的绝对定量。结论:实时荧光定量PCR是检测基因表达水平的理想选择,在小鼠卵母细胞减数分裂成熟过程中Mad2mRNA表达存在动态变化。关键词:SYBRGreenI;实时荧光定量PCR;小鼠;卵母细胞;Mad2中图分类号:S865113文献标识码:A文章编号:052925130(2006)1020011204Mad2mRNAlevelinmouseoocytemeiosismaturationbySYBRGreenIreal2timeRT2PCRWANGJian2ying1,HANGSu2qin2,LIYun2long13(1.KeyLaboratoryofAnimalResistanceResearch,CollegeofLifeScience,ShandongNormalUniversity,Jinan250014,China;2.CollegeofAnimalScienceandTechnology,NanjingAgriculturalUniversity,Nanjing210095,China)Abstract:Objectives:TostudyMad2mRNAlevelinmousemeiosismaturationinvitro.Methods:Real2timePCRmethodwasusedfor收稿日期:2006205218作者简介:王建英(19712),女,畜牧师,在读博士研究生。3通讯作者。激活始于2细胞期,此后其转录活动逐渐增强。但热应激对卵裂早期胚胎的基因组具有较强的抑制作用[5],使得热应激后的2细胞胚胎虽然可以转录并合成HSP70[8],却不具备在转录水平上调控HSP70诱导合成的能力,只有到4细胞阶段才可能获得此能力。然而,也有人发现[9],体外发育的附植前早期鼠胚的短暂热应激可诱导8细胞以上胚胎合成HSP70,这与本试验结果(4细胞)有所不同。因此,对于体内发育的附植前早期鼠胚而言,在母体遭受应激后,胚胎HSP70的诱导表达本质上决定于胚胎细胞内相关基因(HSP基因)的合成和转录活性,同时与热应激强度和胚胎发育环境等因素相关。参考文献:[1]李玉保,付旭彬,鲍恩东.热休克蛋白研究进展[J].畜牧与兽医,2004,36(11):38240.[2]王小军,鲍恩东.热应激蛋白(HSPs)的表达[J].畜牧与兽医,2004,36(1):41243.[3]EdwardsJL,KingWA,KawarskySJ,etal.Responsivenessofearlyembryostoenvironmentalinsults:potentialprotectiverolesofHSP70andglutathione[J].Theriogenology,2001,55(1):2092223.[4]HansenPJ,DrostM,RiveraRM,etal.Adverseimpactofheatstressonembryoproduction:causesandstrategiesformitigation[J].Theriogenology,2001,55(1):912103.[5]Al2KatananiYM,HansenPJ.Inducedthermotoleranceinbovinetwo2cellembryosandtheroleofheatshockprotein70inembryonicdevel2opment[J].MolecularReproductionandDevelopment,2002,62:1742180.[6]RiveraRM,HansenPJ.Developmentofculturedbovineembryosaf2terexposuretohightemperaturesinthephysiologicalrange[J].Re2production,2001,121(1):1072115.[7]FederME,HofmannGE.Heatshockproteins,molecularchaper2ones,andthestressresponse:evolutionaryandecologicalphysiology[J].AnnuRevPhysiol,1999,61:2432282.[8]ChandoliaRK,PeltierMR,TianWR,etal.Transcriptionalcontrolofdevelopment,proteinsynthesisandheat2inducedheatshockpro2tein70synthesisin22cellbovineembryos[J].BiolReprod,1999,61:164421648.[9]MullerWU,LiGC,GoldsteinLS.Heatdoesnotinducesynthesisofheatshockproteinsorthermotoleranceintheearlieststageofmouseembryodevelopment[J].IntJHyperthermia,1985,1(1):972102.·11·畜牧与兽医2006年第38卷第10期quantitativeanalysisofMad2mRNAlevelinmouseoocyteusingSYBRGreenIasreporterdyeandrecombinedplasmidasstandardcurvetemplate.Results:Aspecific,sensitivequantitativ...
