【作者】 刘玉冰； 郑维发；

【Author】 LIU YuBing ZHENG WeiFa (Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province,Xuzhou Normal University,Xuzhou 221116)

【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(Zheng et al.2009b)。该真菌分布在寒冷地区,自然生境下生长十分缓慢,这表明野生桦褐孔菌已无法成为这类化合物的可靠来源。人工培养往往导致桦褐孔菌酚类化合物的积累量大幅降低。为了提高酚类化合物积累,Zheng et al.(2009a)向桦褐孔菌培养液中添加40μg/L的真菌激发子,导致得菌丝体产生大量的NO,同时苯丙氨酸解氨酶(phenylalanine ammonia lyase,PAL)的活性随之提高,酚类积累也大幅度提高。进一步提高激发子浓度,酚类化合物的积累又大幅下降,其原因尚未明了。NO除了对真菌的生长发育起调节作用外,过高水平的NO与超氧阴离子形成ONOO~-,对细胞产生亚硝基化胁迫,导致细胞凋亡(Zheng et al.2010)。本研究在桦褐孔菌培养液中加入不同浓度的激发子以观测菌丝体、多酚的积累、NO和S-亚硝基硫醇(S-nitrosothiols,SNO)水平以及S-亚硝基谷胱甘肽还原酶(S-nitrosoglutathione reductase,GSNOR)活性的变化情况,从而阐明激发子诱导的桦褐孔菌酚类化合物合成的调控机制。结果表明,加入60μg/L的激发子使NO的水平达到690 nM,PAL最高活性达205.13U/mgprot,菌丝体总酚最高水平为25.38 mg/g。加入40μg/L激发子使NO的最高水平降为489.67nM,PAL最高活性达到272.96 U/mgprot,总酚积累的最高水平达到39.8725.38 mg/g。为了进一步阐明NO的作用机理,本研究在桦褐孔菌培养液中分别加入氨基胍(AG,NOS抑制剂)、硝普钠(SNP,NO供体),结果在加有激发子的培养液中加入AG使PAL活性及总酚的水平均显著下降。加入SNP则使PAL和总酚提高。这进一步证明NO参与桦褐孔菌酚类化合物合成的调控。随着AG和SNP的加入,SNO积累和GSNOR活性也分别出现下降和提高。这说明NO在调控桦褐孔菌酚类合成时需要GSNOR的参与。当NO水平过高时形成高水平的SNO蛋白,导致多种与亚硝基结合的酶(如PAL)活性下降。另外,过高水平的NO还能与超氧阴离子形成毒性极大的过亚硝基化物,可导致菌丝体死亡。因此,激发子诱导的桦褐孔菌酚类合成是由NO介导的。NO使PAL和GSNOR表达得以提高,而后者能使可能形成的SNO-PAL转换为PAL,从而提高酚类化合物积累。更多还原

【Abstract】 Inonotus obliquus is a medicinal fungus able to treat various diseases,and phenolic compounds produced by the fungus are believed to be the active components to treat or protect oxidative stress-induced diseases including diabetes,hypertension,cancer and neurodegenerative diseases(Zheng et al.2009b).In nature,I.obliquus is restricted to the cold districts and grows very slowly,indicating that this fungus is not a reliable source for producing these phenolic compounds.Previous attempts to grow this fungus all resulted in a reduced production of phenolic compounds.In order to up-regulate production of phenolic compounds,Zheng et al (2009a) added 40μg/L fungal elicitor into the culture,which led to a drastic increase in nitric oxide(NO) generation,PAL activity and subseqent accumulation of phenolic compounds. Further increase in elicitor concentration,however,resulted in substantial decrease in phenolic compounds with reasons unknown.It has been evidenced that NO is a signal molecule capable of modulating fungal growth and development.But high NO level tends to combine with superoxide anion to produce peroxynitrite leading to nitrosative stress and subsequent aptotosis (Zheng et al.2010).In this study,different levels of elicitor were added in the culture of I. obliquus to measure the accumulation of mycelial biomass and phenolic compounds,generation of NO and SNO,and the changes in activity of phenylalanine ammonia lyase(PAL) and S-nitrosoglutathine reductase(GSNOR),and finally to clarify mechanisms of elicitor-induced biosynthesis of phenolic compounds.In our results,an addition of elicitor at a concentration of 60μg/L was responded by the maximum level of NO at 690 nM/mgprot,PAL at 205.13U/mgprot and phenolic compounds at 25.38 mg/g.In contrast,addition of elicitor at a concentration of 40μg/L coincided with the attainment of maximum level of NO at 489.67 nM,PAL at 272.96 U/mgprot and phenolic compounds at 39.87 mg/g.To further elucidate the mechanisms of biosynthesis of phenolic compounds,aminoguanidine(AG,NOS inhibitor) and sodium nitroprusside(SNP,NO donor) were respectively added into the culture to observe the changes in PAL activity and production of phenolic compounds.The results showed that addition of AG at a concntration of 40 mM in the presence of elicitor(40μg/L) decreased PAL activity and accumulation of phenolic compounds;while addition of SNP only at a concentration of 0.2 mM brought about significant increase in PAL activity and accumulation of phenolic compounds. This implicates that NO is involved in governing biosynthesis of phenolic compounds by I. obliquus.The presence of elicitor and SNP also triggered the increase in SNO accumulation and GSNOR activity.Whereas the supplement of AG reduced SNO production and GSNOR activity. GSNOR catalyses the release of NO from NO-binded proteins and thereby recovers their activity (Wang et al.2008).Thus,NO-enhanced biosynthesis of phenolic compounds can be realized only in the presence of antinitrosative mechanisms.Excessive production of NO will lead to an enhanced accumulation of SNO and subsequent reduction in protein functions(i.e.PAL activity) and production of phenolic compounds.In addition,high level of NO will also combine with superoxide anion to produce highly toxic substance leading to cell damage.Thus elicitor-induced biosynthesis of phenolic compounds in I.obliquus is mediated by the production of NO able to enhance expression of PAL and GSNOR,the latter can transform the posibly formed SNO-PAL into PAL to maintain its activity for biosynthesis of phenolic compounds.更多还原