摘要
漆酶是一种含铜的多酚氧化酶，普遍存在于白腐菌中，具有降解木质素和使苯氧基类除草剂等有毒酚类物质无毒化的作用，还可去除石油工业废水的毒性。在造纸、饮料加工、和环保等方面具有广泛的应用前景。大多数白腐菌在水中的生长速度较慢，结球结块，仅能生活在水体表面，这些都限制了其在处理废水等方面的直接应用。克隆白腐菌漆酶的结构基因，对今后培育白腐菌漆酶基因大量表达的工程菌株和用工程菌处理酚类污染的废水等具有十分重要的意义。本研究以25种白腐真菌为材料，测定了不同菌株漆酶的活性，并对6种白腐菌漆酶基因进行了克隆和序列分析，结果如下：
(1) 对25种白腐真菌进行了漆酶活性测定，结果有11个菌种具漆酶活性，它们分别是裂蹄木层孔菌(Phellinus linteus)、朱红密孔菌(Pycnoporus cinnabarinus)、环带干酪菌(Tyromyces zonatulus)、栎迷孔菌(Daedalea quercina)、三色革裥菌(Lenzites tricdor)、北方梭囊孔菌(Climacocystis borealis)、桦革裥菌(Lenzites betulina)、血红密孔菌(Pycnoporus sanguineus)、白耙齿菌(Irpex lactea)、单色下皮黑(Cerrenaunicolor)、梭孔菌(Favolus alveolaris)。
(2) 建立了快速提取高纯度完整丝状真菌RNA、DNA的方法。
(3) 通过RT-PCR实验证明了扁芝菌的mRNA不具有Poly(A)序列。
(4) 设计并合成了用于克隆漆酶基因的一对简并引物，在6个菌种中扩增和克隆了与漆酶基因及其类似基因有同源性的特异片段，它们分别是朱红密孔菌(1227 bp)、血红密孔菌(1227 bp，AY331189)、单色下皮黑(1207 bp，AY454305)、环带干酪菌(1223 bp，AY454306)、北方梭囊孔菌(1201 bp，AY333124)、枥迷孔菌(1232 bp，AY333125)。片段的获得为这些菌种全长漆酶基因的克隆奠定了基础。
(5) 获得了血红密孔菌漆酶基因全长cDNA序列。根据已克隆得到的血红密孔菌漆酶基因片段(AY331189)，通过RACE技术从血红密孔菌中扩增得到漆酶全长cDNA序列(AY458017)，共1902 bp，包括完整的开放读码框(1554 bp)，编码518个氨基酸。其5′非编码区为51 bp，3′非编码区为297 bp。其编码的氨基酸序列与朱红密孔菌漆酶(1cc3-2)的同源性为96％，与其它担子菌漆酶的同源性也很高。蛋白质结构域预测结果表明在该基因的氨基酸序列中具有4个的铜离子结合区域，7个潜在的糖基化位点，其相对分子量为56313.2，等电点为5.59。
(6) 获得了血红密孔菌漆酶基因完整DNA序列。根据血红密孔菌漆酶cDNA序列，设计一对特异性引物，经PCR扩增得到了该酶全长的DNA序列(AY510604)，长2154 bp，该序列除包含全部血红密孔菌漆酶cDNA编码区外，还包括10个内含子序列，长度在52～
70 bp之间，是典型的真核生物内含子，包括明显的5‘端的供体剪切位点、.3‘端的
受体剪切位点和分支点序列。
Laccases is a kind of Cu-contained polyphenol oxidase produced by many white-rot fungi. It can degrade lignin and phenoxy containing herbicide and waste water in petroleum industry and affect toxic phenolic materials, so laccases can be widely applied in paper making industry drinking processing and environment protection. Most of white-rot fungi propagate slowly in water and agglomerate quickly, so they can only live on the surface of water, these characteristic directly restrict their applied for waste water treatment. Research on the cloning laccase structure gene of white-rot fungi has an important significance for cultivate engineering bacilli, which express high laccase activity and treat waste water containing phenolic materials by those engineering bacilli. In this paper, the activity of laccase in selected 25 strains were mensurated, the sequence of six white-rot fungi laccase gene have been cloned and analyzed. Main results as followed:
(1) The activity of laccase from 25 selected white-rot fungi was mensurated. The results indicated that 11 strains have the activity of laccase, including Phellinus linteus Pycnoporus cinnabarinus Tyromyces zonatulus Daedalea quercina Lenzites tricdor Climacocystis borealis Lenzites betulina Pycnoporus sanguineus Irpex lactea Cerrena unicolor Favolus alveolaris.
(2) The rapid methods of RNA and DNA extraction from mycelial fungi were established. Entire and high purity DNA and RNA can be isolated by these methods.
(3) The results of RT-PCR experiment showed that mRNA of Eljvngia applanata has no Poly (A) tail sequence.
(4) Six specific fragments were obtained from 6 fungi that have laccase activity using a pair of degenerate primer. These fragments share high identity with laccase gene from other organism, which were Pycnoporus cinnabarinus ( 1227 bp ) Pycnoporus sanguineus ( 1227 bp, AY331189) Cerrena unicolor (1207 bp, AY45.4305) Tyromyces zonatulus (1223 bp, AY454306) Climacocystis borealis (1201 bp, AY333124) Daedalea quercina (1232 bp, AY333125) .
(5) The full-length cDNA of Pycnoporus sanguineus laccase gene was cloned. According to the laccase gene fragment of Pycnoporus sanguineus (AY331189), The full-length cDNA of Pycnoporus sanguineus laccase gene was isolated by using RACE (rapid amplification of cDNA ends) technique. The full length of sequence was 1902 bp, containing an open reading frame(1554 bp), encoding a peptide of 518 amino acids.5' untranslated region was 51 bp, 3'
untranslated region was 297 bp. BLAST analysis revealed that the laccase cDNA and amino acids sequence from Pycnoporus sanguineus shared high identity with laccase gene from other organisms in nucleotides and amino acids respectively. The laccase protein has four catalytic cupric ions domain and seven glycosylation sites, the relative molecular weight was 56313.2, and theoretical pI was 5.59.
(6) The complete DNA sequence of laccase from Pycnoporus sanguineus was obtained. Based on the Pycnoporus sanguineus laccase cDNA sequence, a pair of specific primer had been designed; the complete DNA sequence of 2154 bp (AY510604) was isolated by PCR. Besides the coding area of cDNA, the sequence containing 10 introns regions, with length between 52~70 bp, which showed typical eukaryotes intron characteristic, including 5' donor splicing site3' receptor splicing site and bifurcate sequence.
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