【作者】 郑维发； 刘玉冰； 赵艳霞； 张梅梅；

【Author】 ZHENG Wei-Fa LIU Yu-Bing ZHAO Yan-Xia ZHANG Mei-Mei (Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province,Xuzhou Normal University,Xuzhou 221116,China)

【机构】 徐州师范大学江苏省药用植物生物技术重点实验室；

【摘要】 桦褐孔菌是一种能够治疗多种疾病的药用真菌,其中酚类化合物被认为是治疗和预防氧化胁迫诱导的糖尿病、高血压、癌症及老年痴呆症的有效成分。该真菌分布在寒冷地区,自然生境下生长十分缓慢,野生桦褐孔菌无法成为这类化合物的可靠来源。而人工培养的桦褐孔菌多酚的积累量大幅降低(Zheng et al.2010)。最近,Zheng et al.(2009a,2009b,2011b)先后使用不同光质、真菌激发子以及与其他真菌混合培养等方式对桦褐孔菌多酚生物合成进行调控,结果使酚类特别是硬毛素类多酚的含量明显提高,但与野生桦褐孔菌相比仍有很大差距。人工培养条件下制约桦褐孔菌多酚生物合成的关键因素尚不清楚。本研究主要观察桦褐孔菌多酚合成关键酶苯丙氨酸解氨酶(phenylalanine ammonia lyase,PAL)的表达以及活性与NO水平间的关系、PAL的亚硝基化与多酚积累的关系,以阐明桦褐孔菌多酚生物得到调控体系及其作用机制。结果表明,NO供体硝普钠(SNP)浓度低于0.05μM对桦褐孔菌PAL的活性有促进作用,高于0.5μM时,PAL虽然表达量增加但活性下降。原核表达的桦褐孔菌PAL与NO的反应表明,NO能对PAL进行亚硝基化修饰。修饰后的PAL活性显著下降。因此,NO对桦褐孔菌多酚生物合成有双向调控作用,PAL的去亚硝基化是进一步提高多酚生物的关键。但正常生理条件下,去亚硝基化酶亚硝基谷氨酰胺还原酶(S-nitrosoglutathione reductase,GSNOR),硫氧还蛋白还原酶(thioredoxin reductase,TrxR)活性处于动态平衡是不表现去亚硝基化作用。当GSNOR活性被抑制,亚硝基化蛋白(SNO)含量明显减少,PAL的活性明显增加,多酚积累则进一步提高(Zheng et al.2011a)。因此,NO介导的桦褐孔菌多酚生物合成调控体系由NO、PAL,SNO、GSNOR以及去亚硝基化酶复合体硫氧还蛋白/硫氧还蛋白还原酶/NADPH组成,正常生理条件下,GSNOR与TrxR活性处于动态平衡,多高的NO对PAL进行亚硝基化修饰,以防止多酚的过量合成。当GSNOR活性被抑制则平衡被打破,去亚硝基化酶开始去亚硝基化,多酚的积累增加。更多还原

【Abstract】 The medicinal fungus Inonotus obliquus is traditionally used to treat various diseases,polyphenols and produced by the fungus are evidenced to be the active components to treat or protect oxidative stress-induced diseases including diabetes,hypertension,cancer and neurodegenerative diseases.In nature,I.obliquus is restricted to the cold districts and grows very slowly,indicating that this fungus is not a reliable source for these polyphenols.In cultured conditions,however,it grows fast but accumulates little polyphenols(Zheng et al.2010). Recently,Zheng et al.(2009a,2009b,2011b) regulated the biosynthesis of polyphenols by the fungus using different light quality,fungal elicitor and co-culture with other fungus,and did increase the production of these polyphenols especially hispidin analogs under submerged culture conditions.The content of polyphenols found in the culture,nevertheless,is still far less than those found in field-grown I.obliquus.Yet,the factors inhibiting biosynthesis of polyphenols by the fungus remain unknown.In this study,we observed the relationship between expression of phenylalanine ammonia lyase(PAL) and NO level,S-nitrosylation of PAL and accumulation of polyphenols to elucidate mechanisms controlling biosynthesis of polyphenols by the fungus.The results showed that sodium nitroprusside(SNP) enhanced the expression and activity of PAL at concentrations below 0.05μM, and enhanced PAL expression but decreased PAL activity at concentrations above 0.5μM.The interaction of prokaryotic expressed PAL of I.obliquus with NO suggested that the cysteine residue(s) in PAL was(were) combined with NO and thus S-nitrosylated,resulting in PAL inactivation.Therefore,NO has dual regulation on biosynthesis of polyphenols by I.obliquus,and denitrosylation of S-nitrosylated PAL is the key for further enhancing the production of polyphenols.Under normal physiological conditions,denitrosylating enzymes S-nitrosoglutathione reductase(GSNOR) and thioredoxin reductase(TrxR) do not show any denitrosylation when their activity is in homeostasis.Inhibiting GSNOR led to a reduction of S-nitrosylated proteins but an enhanced activity of PAL and an increased production of polyphenols(Zheng et al.2011a).Thus,regulation on biosynthesis of polyphenols by I.obliquus involves production of NO and SNO,S-nitrosylation and denitrosylation of PAL, and the changes in GSNOR and TrxR activity.Under normal physiological conditions,GSNOR and TrxR are homeostatic in activity,and over-produced NO S-nitrosylates PAL to prevent excessive biosynthesis of polyphenols.Inhibiting GSNOR triggered the start of denitrosylation,leading to an enhanced accumulation of polyphenols by I.obliquus.更多还原