灵芝染料脱色过氧化物酶基因GlDyP1和GlDyP2的克隆及表达分析

Cloning and Expression analysis of Dye-Decolorization Peroxidase Gene of Ganoderma Lucidum

摘要:

本研究以灵芝(Ganoderma lucidum)ZJ-1菌株为研究对象,通过生物信息学分析和表达分析,探究灵芝染料脱色过氧化物酶(Dye-decolorizing peroxidase,DyP)基因潜在的作用。本研究克隆得到了灵芝染料脱色过氧化物酶基因GlDyP1和GlDyP2,它们均含有6个内含子,7个外显子,所有内含子的剪切位点均符合GT-AG规律。GlDyP1编码框长度为1488 bp,编码495个氨基酸；GlDyP2编码框长度为1461 bp,编码486个氨基酸。通过qRT-PCR分析,GlDyP1和GlDyP2在菌丝阶段的表达量均显著高于其它时期,表明这两个基因有可能在菌丝阶段参与木质纤维素的降解。在不同基质培养和不同染料诱导的条件下,对灵芝GlDyP酶活进行了测定,同时对GlDyP1和GlDyP2进行了表达模式分析。发现基质和染料均能诱导GlDyP的产生,GlDyP1基因在花生壳中的表达量最高,GlDyP2基因在木屑中的表达量最高；GlDyP1在活性黑5中的表达量最高,而GlDyP2基因在台盼蓝中的表达量最高。以上结果表明GlDyP可参与灵芝对木质纤维素和染料的降解。本研究为探究灵芝GlDyP在降解木质素和染料方面的生理功能和开发GlDyP的应用奠定基础。

Abstract:

In this study, Ganoderma lucidum ZJ-1 strain was used as the research object. Bioinformatics analysis and expression analysis were used to explore the potential role of dye-decolorizing peroxidase (DyP) gene in G. lucidum. The dye decolorizing peroxidase genes GlDyP1 and GlDyP2 were cloned in this study. Both GlDyP1 and GlDyP2 contain 6 introns and 7 exons, and the splicing sites of all introns conform to the GT-AG rule. The length of GlDyP1 coding frame was 1488 bp, encoding 495 amino acids; the length of the GlDyP2 coding frame was 1461 bp and encodes 486 amino acids. By qRT-PCR analysis, the expression levels of GlDyP1 and GlDyP2 in mycelium stage were significantly higher than other stages, indicating that these two genes may be involved in the degradation of lignocellulose in mycelium stage. The GlDyP activity in G. lucidum was determined under different substrates cultures and different dye induction conditions, and the expression patterns of GlDyP1 and GlDyP2 were analyzed. Both substrates and dyes were found to induce the production of GlDyP. The expression of GlDyP1 gene was the highest in peanut shell and GlDyP2 gene was the highest in sawdust. The expression level of GlDyP1 in reactive black 5 was the highest, while the expression level of GlDyP2 in trypan blue was the highest. These results indicated that GlDyP could participate in the degradation of lignocellulose and dyes by G. lucidum. This study lays a foundation for exploring the physiological function of GlDyP in degrading lignin and dyes and developing the application of GlDyP.