根据白腐菌多功能过氧化物酶（Versatile peroxidase，VPs）基因序列的保守区域设计引物，利用简并PCR、cDNA末端的快速扩增（RACE）和融合引物巢式结合PCR（FPNI-PCR）等方法，扩增得到虎奶菇多功能过氧化物酶基因（GenBank登录号MN701764），命名为Pt-vp1。该基因DNA全长1833 bp，含有15个外显子和14个内含子，cDNA和完整开放阅读框1077 bp，编码358个氨基酸。多种白腐菌VPs氨基酸序列的系统发育结果表明，Pt-vp1蛋白与平菇VP（GenBank登录号ASU87523.1）亲缘关系最近。在发酵培养基中静止培养4 d后虎奶菇VP达到最大酶活，为43.83 U/L。在发酵培养基中加入不同浓度Mn2+，当其浓度为200 μmol/L时，虎奶菇VP酶活达到最大值，为91.98 U/L，表明Mn2+不是虎奶菇产生VP的必要因子，但可以显著提高产酶量。利用实时荧光定量qRT-PCR技术检测不同浓度Mn2+对Pt-vp1基因转录水平的影响，结果显示Mn2+对Pt-vp1蛋白的表达具有促进作用，当Mn2+浓度为300 μmol/L时，其转录水平增加了5.79倍。本研究结果为研究虎奶菇VP基因的表达机制及功能研究提供了重要信息。

Primers were designed according to the conserved domain of versatile peroxidase gene cDNA sequences of white-rot fungi, and the gene of versatile peroxidase from Pleurotus tuber-regium (termed Pt-vp1; GenBank accession number: MN701764) was obtained via amplification using degenerate PCR, rapid amplification of cDNA end (RACE), and fusion primer and nested integrated PCR (FPNI-PCR). The full-length DNA of Pt-vp1 was 1833 bp, with 15 exons and 14 introns. The length of cDNA and open reading frame were both 1077 bp and encoded 358 amino acids. Phylogenetic analysis of the amino acid sequences of VPs from various white-rot fungi showed that Pt-vp1 protein was the closest to Pleurotus ostreatus VP (GenBank accession number: ASU87523.1). The VP activity of P. tuber-regium reached the highest to 43.83 U/L after 4 days of stationary culture in the fermentation medium. Mn2+ was added at different concentrations. When its concentration was 200 μmol/L, the VP activity was the highest 91.98 U/L. The results indicated that Mn2+ was not the necessary factor for producing VP in Pleurotus tuber-regium, but could increase significantly the enzyme production. The effect of Mn2+ at different concentrations on the transcription level of Pt-vp1 was examined using quantitative real-time PCR (qRT-PCR), and the results showed that Mn2+ promoted the expression of Pt-vp1. When the concentration of Mn2+ was 300 μmol/L, the transcription level of Pt-vp1 increased 5.79 times. This study provides important information for investigating the expression mechanism and function of the VP gene from P. tuber-regium.

国家自然科学基金项目（31772375）