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纸质出版:2013
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申克孢子丝菌SPDS基因的生物信息学分析及分子克隆[J]. 中山大学学报(医学科学版), 2013,34(6).
Bioinformatics Analysis and Molecular Cloning of Spermidine Synthase Gene fromSporothrix schenckii[J]. Journal of Sun Yat-sen University (Medical Sciences), 2013, 34(6).
【目的】 分析预测申克孢子丝菌cDNA中编号为Locus_168_Contig_1序列编码的基因及蛋白质结构、功能特征,并进行分子克隆。 【方法】 利用NCBI及Expasy网站提供的BLASTx、SignalIP、InterPro Scan等生物信息学软件分析其序列,判断其是否为全长编码序列、同源序列名称,并分析其结构、定位及功能等;将其中的全长编码区利用PCR方法进行扩增后克隆到pET-30a(+)载体,测序验证。 【结果】 该序列是亚精胺合成酶(SPDS)的同源基因,全长序列为1062 bp,包含完整的编码区190 ~ 1 062 bp,编码291个氨基酸,在GenBank中,其与粗糙脉孢菌的SPDS基因同源性最高,达88%。理化性质预测其编码蛋白疏水性高;无质体、线粒体等定位序列,不存在跨膜螺旋结构,为非分泌型蛋白。进化树分析结果显示与粗糙脉孢菌同源性最高;PCR扩增及测序结果证实目的基因成功克隆进入pET-30a(+)质粒。【结论】 获得申克孢子丝菌SPDS的基因及蛋白结构、功能特点,构建了其重组表达质粒,将为进一步明确其在孢子丝菌病中的致病作用奠定理论基础。
【Objective】 To identify the characteristics of the sequence Locus_168_Contig_1 from full cDNA sequence of Sporothrix schenckii by bioinformatics analysis and obtain its recombinant plasmid. 【Methods】The characterizations of gene structure and its deduced amino acid sequence encoding by the insert sequence of EST clone (Locus_168_Contig_1) from the S.schenckii full-length cDNA sequence was analyzed by using bioinformatics software packages such as BLASTx, SignalIP, InterProScan provided by websites of NCBI or Expasy. 【Results】 A complete open reading frame of 873bp was included in the full cDNA sequence encoding SPDS. The sequence had high homology with the amino acids sequence from other species in GenBank. It has an 88% homology with SPDS from Neurospora crassa. The SPDS homolog from Sporothrix schenckii was predicted to be an aliphatic, highly hydrophobic protein without plastid or mitochondrial localization sequence. It was predicted as a non-secreted protein. Phylogenetic tree showed that it has the highest homology with Neurospora crassa. The recombinant plasmid pET-30a(+)-SPDS was confirmed by PCR amplification and sequencing. 【Conclusion】 The characterizations of structure and function of SPDS from S. schenckii were obtained and the recombinant plasmid pET-30a (+)-SPDS was constructed successfully. Our study established a cornerstone for the research on the role of SPDS in the pathogenicity of S. schenckii.
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