5-氮杂胞苷对桦褐孔菌多酚积累的影响

Effects of 5-azacytidine on accumulation of polyphenols in Inonotus obliquus

本文旨在研究DNA甲基转移酶抑制剂5-氮杂胞苷对桦褐孔菌多酚合成的调控。以液体摇瓶法培养桦褐孔菌,并在培养液中添加5-氮杂胞苷。采用荧光定量PCR测定与多酚合成相关的编码苯丙氨酸解氨酶(pal)、4-香豆酸辅酶A连接酶(4cl)和硬毛素合成酶(sps)基因表达水平,染色质免疫沉淀技术对编码硬毛素合成酶基因启动子区的组蛋白甲基化修饰进行检测,Folin-Ciocalteu法测定桦褐孔菌细胞内和发酵液中多酚的含量。结果显示,5-氮杂胞苷的添加提高了桦褐孔菌体内pal、4cl和sps基因的表达水平,改变了sps启动子区的组蛋白甲基化修饰,即降低了H3K9三甲基化修饰水平,提高了H3K4和H3K36三甲基化修饰水平,显著提高了桦褐孔菌细胞内和发酵液中多酚的积累量。5-氮杂胞苷诱导下桦褐孔菌细胞内多酚积累量达(46.6±2.8)mg/g,明显高于对照组多酚积累量(28.7±1.0)mg/g,并且胞外多酚的含量由对照组的(66.9±1.3)mg/L提高至(92.3±2.3)mg/L。此外,经5-氮杂胞苷处理后胞内多酚清除DPPH自由基、超氧阴离子和羟自由基的能力显著提高。可见,5-氮杂胞苷可以作为调节因子激发桦褐孔菌液体培养条件下多酚的合成,可以成为进一步提高桦褐孔菌液体发酵产物中多酚产量的技术手段之一。

The purpose of this study was to investigate the regulation of the DNA methyltransferase inhibitor 5-azacytidine(5-AC)on the biosynthesis of phenolic compounds in Inonotus obliquus.I.obliquus was cultured by liquid shake flask in the presence of 5-AC.For determining the effects of 5-AC on biosynthesis of polyphenols by I.obliquus,the mRNA expression level of genes encoding phenylalanine ammonium lyase(pal),4-coumarate CoA ligase(4 cl)and styrylpyrone synthase(sps)were assayed by real-time PCR.In addition,the histone methylation modification of the promoter region of sps was measured by chromatin immunoprecipitation technique.For assaying the phenolic content of I.obliquus under submerged culture conditions,Folin-Ciocalteu method wasutilized to determine the accumulation of total polyphenols either in mycelia or in culture broth.The results showed that the addition of 5-AC enhanced the expression levels of pal,4 cl and sps in I.obliquus.The addition of 5-AC reduced the histone modification of H3 K9 Me3,but increased H3 K4 Me3 and H3 K36 Me3 modification of the sps promoter region.Consistent with the increase of gene expression of pal,4 cl and sps and the change of histone modification at promoter region of sps,the accumulation of total polyphenols reached(46.6±2.8)mg/g for mycelial polyphenols and(92.3±2.3)mg/L for extracellular polyphenols,being obviously higher than the yield of mycelial polyphenols(28.7±1.0)mg/g and extracellular polyphenols(66.9±1.3)mg/L found in the experimental control.5-AC treatment enhanced the capacity of mycelial polyphenols to scavenge DPPH,superoxide anion and hydroxyl radicals.In summary,5-AC can be used as a regulator to stimulate the synthetic metabolism of I.obliquus under submerged culture conditions,and the application of 5-AC might be one of the measures to further increase the accumulation of polyphenols in artificial culture of I.obliquus.