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Molecular Cloning of the Cellobiohydrolase(CBHI)cDNA Gene Derived from Tricholderma viride

u Tong; Zhang Manfu(Beijing Agricultural University,Beijing 100094)  
Total RNA was isolated from Trichoderma viride 3-3711,with Guanidinium ThiOcvanate fol-lowed by centrifugation in cesium chloride solutions,and poly(A)+ RNA was purified through oligo(dT)cel-lulose affinity chromatography. Using oligo(dT)12-18 as primer,poly(A)+ RNA as template,the first strandof cDNA was synthesized by AMV reverse transcriptase,the second-strand of cDNA was synthesized byRNaseH and E.coli DN A polymerase I.The longer ds-cDNA selected through sepharose 4B column chro-Tnatography was inserted into Smal site of pUC19 by blunt-end ligation. After transformation of E. coliJM107 by the recombinant plasmid,positive clones were screened out by colony,dot and southern hybridiza-tions using 32P labelled two synthetic probes. it was evidenced by restriction endonuclease analysis that fulllength cellobiohydrolase cDNA had been cloned from Trichoderma viride.
【Fund】: 北京农业大学生物技术基金
【CateGory Index】: Q78
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