黑曲霉mRNA的分离及其cDNA的合成和克隆

Isolation of Aspergillus niger mRNA and Synthesis and Cloning of its cDNA

采用酚一蛋白酶K抽提和氯化锂沉淀法,从10g黑曲霉细胞中获得8mg总RNA,经二次寡聚(dT)-纤维素亲和层析纯化得60μg poly(A)^+mRNA。经紫外分光,6M尿素—琼脂糖凝胶电泳分析及体外翻译试验结果证明:纯化的poly(A)^+mRNA的纯度及完整性好,无降解并具翻译活性。以纯化的poly(A)^+mRWA为模板,以寡聚(dT)_(12-18)及随机的寡聚核苷酸为引物,用AMV反向转录酶合成了第一链cDNA。用RNaseH及DNA聚合酶Ⅰ水解模板mRNA并合成双链cDNA。第一链及第二链cDNA合成的产率分别为25.8％及96％。合成的cDNA的长度在200～5000bp之间与纯化的poly(A)^+mRNA的长度相等。合成的双链cDNA,经酚-氯仿抽提及2M醋酸钠-乙醇沉淀,除去游离的核苷酸后与EcoRI接头连接,再经EcoRI消化后与λgt10DNA连接,然后进行体外包装,包装物感染大肠杆菌BNN93及BNN102。λgt10及重组的λgt10都能在大肠杆菌BNN93上长出噬菌斑,而只有重组的噬菌体才能在BNN102上长出噬菌斑,按此法筛选,每μgcDNA可获得8×10~4个重组噬菌体。

Total RNA of Aspergillus niger was isolated by phenol-proteinase K extract- ion and LiCl precipitation.Poly(A)^+ mRNA was purified by two cycles of oligo dT)-cellulose affinity chromatography.The poly(A)^+ mRNA thus prepared was electrophoresed on denaturing agarose-6M urea gel to estimate the length and in vitro translated in a rabbit reticulocyte lysate supplemented with L-(^(35)S)methio- nine,which showed enough translation activity.The first and second strand cDNA were synthesized by AMV reverse transcriptase and RNase H-DNA polymerase I respectively using the total poly(A)^+ mRNA as template and oligo(dT)12-18 as primer.The yield rates of first and second strand cDNA cynthesis are 25.8% and 96% respectively.The length of ds cDNA is about 200-5000bp which is similar to that of poly(A)^+ mRNA.The ds cDNA was first ligated with EcoRI linker,after digested with EcoRI,it was mixed with EcoRI-digested λgt10 and ligated with T4- DNA ligase.The resultant DNA molecules were packaged in vitro and then infe- cted E.coli BNN93 and BNN102.8x10^4 recombinant phages were obtained per ug cDNA.Rapid isolation and restriction analysis of two recombinant phages show the existence of 4.3Kb and 3.5Kb inserts respectively.