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 in Ganoderma lucidum

作　　者：刁文彤 刘冬梅 亓希武[2] 房海灵[2] 于盱[2] 李莉 柏杨 刘群 陈泽群 梁呈元 DIAO Wentong;LIUDongmei;QI Xiwu;FANG Hailing;YUXu;LI Li;BAI Yang;LIUQun;CHEN Zequn;LIANG Chengyuan(Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Key Laboratory for the Research and Utilization of Plant Resources,Institute of Botany,Jiangsu Province and Chinese Academy of Sciences,Nanjing 210014,China)

机构地区：[1]南京中医药大学,江苏南京210023 [2]江苏省中国科学院植物研究所,江苏省植物资源研究与利用重点实验室,江苏南京210014

出　　处：《现代食品科技》2024年第8期140-149,共10页Modern Food Science and Technology

基　　金：国家自然科学基金青年基金项目(32102457)。

摘　　要：该研究以灵芝(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。该研究克隆得到了灵芝DyP基因GlDyP1和GlDyP2,编码框长度分别为1488 bp和1461 bp,分别编码495和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。结果显示:在不同基质中培养,木屑中酶活最高,达到7330 U/L;经不同染料诱导,在甲基橙染料诱导下酶活最高,达到1466 U/L;GlDyP1在花生壳中的表达量最高,是木屑的6.5倍;GlDyP2在木屑中的表达量最高,是其余基质的2倍左右;GlDyP1和GlDyP2分别在活性黑5和台盼蓝中的表达水平最高,与对照相比,分别提高了4.8倍和3.7倍。以上结果表明GlDyP可参与灵芝对木质纤维素和染料的降解,为探究灵芝GlDyP的生理功能和开发GlDyP的应用奠定基础。To elucidate the potential role of dye-decolorizing peroxidase(DyP)gene in Ganoderma lucidum,G.lucidum ZJ-1 was evaluated using bioinformatics and expression analysis.To this end,the dye-decolorizing peroxidase genes GlDyP1 and GlDyP2 were cloned. The coding frame lengths were 1 488 bp and 1 461 bp, encoding 495 and 486 amino acids, respectively. The results of qRT-PCR analysis indicated that the expression levels of the two genes in the mycelium stage were significantly higher than in other stages, suggesting that GlDyP1 and GlDyP2 may be involved in the degradation of lignocellulose in the mycelium stage. The activity of GlDyP in G. lucidum was determined under different substrate cultures and dye induction conditions, and then the expression patterns of GlDyP1 and GlDyP2 were analyzed. The results attributed the highest enzyme activity of 7 330 U/L and 1,466 U/L to wood chips cultured in different substrates and methyl orange dye when induced by different dyes, respectively. GlDyP1 expression was highest in peanut shells, 6.5 times higher than that in sawdust. By contrast, GlDyP2 expression was highest in sawdust, 2 times higher than that in the other substrates. Compared with the control, the GlDyP1 and GlDyP2 genes showed the highest expression levels in reactive black 5 and trypan blue, with a 4.8-fold and 3.7-fold increase, respectively. These results indicate that GlDyP may participate in the degradation of lignocellulose and dyes by G. lucidum. These findings provide a foundation for exploring the physiological function of GlDyP in degrading lignin and dyes, and development of applications for GlDyP.

关 键 词：灵芝 染料脱色过氧化物酶 基因克隆 生物信息学 表达分析 

分 类 号：S567.31[农业科学—中草药栽培]