粗毛栓菌cDNA文库的构建和漆酶基因的表达分析

Cloning of Laccase Gene from a Constructed cDNA Library of Trametes gallica

粗毛栓菌(Trametesgallica)能够分泌多种胞外氧化酶并且快速降解木质纤维素.为了快速高效分离鉴定粗毛栓菌木质纤维素降解酶相关基因,用Trizol试剂提取不同培养条件下粗毛栓菌总RNA,用CreatorTM SMARTTM cDNA Library Construction Kit和Advantage 2PCR Kit成功构建了该菌全长cDNA文库.原始文库滴度为1.5×105cfu,重组率达99%,插入片段在0.7～2.0kb之间,平均大小约1kb.随机取16个重组子进行测序,全长cDNA序列完整性率为85.7%;并筛选到1个漆酶基因,编码区长1551bp,预测的蛋白质由517个氨基酸残基组成,分子量为55.41kD,等电点为4.76.用半定量RT-PCR法分析了该漆酶基因在不同培养条件下的表达水平.结果显示,高浓度的碳源,氮源,Cu2+均能诱导此基因的表达,该结果为漆酶基因的表达调控机制的深入研究奠定了基础.

Tramets gallica secrete a series of extracellular oxidases to rapidly degrade lignocellulose. To study the relevant lignocellulolytic enzymes under different cultural conditions, a cDNA library was constructed by using the SMART （Switching Mechanism At 5′end of RNA Transcript, SMART^TM ） Kit and the Advantage 2 PCR Kit using the total RNA was extracted from T. gallica. Over 99% of 1.5 × 10^5 independent clones in the cDNA library contained insertions of 0.7 - 2.0 kb. 16 clones were selected randomly for sequencing and 14 of which contained full-length cDNA sequences, including a laccase that encodes a protein of 517 amino acid residues with molecular weight 55.41 kD and pI 4.76. The expression levels of the laccase in different culture conditions was analyzed by semi-quantitative RT-PCR. The result demonstrated that the gene expression was induced by high concentrations of carbon, nitrogen source and Cu^2＋ . This laccase-expressing construct appeared to be useful for further studies of the enzyme expression regulations.