简并PCR结合RACE技术克隆申克孢子丝菌未知过氧化氢酶基因

目的 克隆孢子丝菌未知过氧化氢酶基因,命名为Sscat基因.方法 根据生物信息库中7种已知真菌过氧化氢酶氨基酸序列的高度保守区域设计简并引物,PCR扩增获得部分Sscat基因cDNA片段,随后应用RACE技术分别扩增其3’端和5’端未知序列.结果 Sscat基因cDNA序列全长1746 bp,其中包括5’端121 b...

Cloning a novd catalase gene of Sporothrix schenckii with degenerate PCR and RACE

Objective To isolate a novel catalase homologous gene from yeast-form Sporothrix schenckii and to make a designation.Methods Oligonucleotide primers were designed according to the conserved areas of the other 7 fungal catalase genes.Partial Sscat cDNA was amplified by PCR,and special primers were designed by RACE method to amplify the 3′cDNA and 5′cDNA.Results The full-length Sscat cDNA sequence was 1 746 bp with an open reading frame of 1 500 bp encoding 499 amino acids.The predicted molecular mass of Sscat was 56.07 kDa.The deduced amino acid sequence of Sscat showed 66.3% and 56.6% identity with those of Aspergillus oryzae and A.clavatus.An intron was identified within the 933-1 063 bp Sscat genomic DNA sequence of S.schenckii.Conclusions Degenerate PCR combined with RACE is effective in searching and isolating novel genes of S.schenckii.