4种重金属对灵芝漆酶活性及转录表达的影响

Enzymatic activity and transcription of Ganoderma lucidum laccases following treatment with four heavy metals

漆酶是一种分布广泛的多酚氧化酶,在食品、能源和环保等领域具有重要的应用价值.灵芝作为重要的白腐真菌,可以合成分泌包括漆酶在内的多种木质纤维素降解酶,这些酶的活性会随着菌株、发酵条件等不同而不同.以灵芝荣保1号为材料,利用平板实验分析4种重金属(Pb^(2+)、Cd^(2+)、Cu^(2+)和Fe^(2+))在不同浓度下对灵芝菌丝体生长及氧化带形成的影响,用分光光度法测定重金属发酵液中漆酶的活性,并通过实时定量PCR检测重金属离子胁迫下灵芝漆酶同工酶表达的差异性和特异性.结果表明:低浓度Pb^(2+)和Cu^(2+)促进菌丝体生长和氧化带形成,而Cd^(2+)和Fe^(2+)抑制菌丝生长及氧化带形成;Pb^(2+)、Cd^(2+)、Cu^(2+)和Fe^(2+)分别以终浓度为100、20、100、100 mg/kg时对菌丝生长及氧化带的增强作用较大,培养至第11天、第9天、第7天、第7天时,漆酶活性达到最大值,分别为20.83±0.32、15.89±0.21、30.56±0.42、9.44±0.25 U/L,Cu^(2+)促进了发酵液中漆酶酶活,而Pb^(2+)、Cd^(2+)和Fe^(2+)则抑制漆酶酶活.荧光定量PCR结果显示,4种重金属离子在液体培养条件下对15个灵芝漆酶同工酶的转录表达影响存在差异性,其中5个漆酶基因(Glac4、Glac6、Glac10、Glac11和Glac12)转录表达发生上调.综上,灵芝漆酶对重金属离子作用的不同响应表明其具有较复杂的生理功能及调控机制,结果可为进一步阐明漆酶作用机制提供基础.

Laccases are widely distributed polyphenol oxidases that have important applications in the fields of food, energy, and environmental protection. Ganoderma lucidum is an important white rot fungus that can synthesize and secrete a variety of lignin cellulose-degrading enzymes, including laccases. Activities of these enzymes can be affected by strain and fermentation conditions. The transcriptional and isoenzymes activities of G. lucidum （No. 1 Rongbao） were tested in different concentrations of Cu2＋, Pb2＋ , Cd2＋, or Fe2＋. Mycelial growth and oxidation zones were assayed by plate experiments, laccase activity was tested by spectrophotometry, and the specific or differential expression profiles of laccases were determined by real-time quantitative PCR （RT-PCR）.The results showed that low concentrations of Pb2＋ and Cu2＋ promoted mycelial growth and oxidation, whereas Cd2＋ and Fe2＋ inhibited mycelial growth and oxidation. The highest laccase activities-20.83 ± 0.32, 15.89 ± 0.21, 30.56 ± 0.42, and 9.44±0.25 U/L-were obtained on the 11th, 9th, 7th, and 7th day of cultivation by adding 100 mg/kg Pb2＋, 20 mg/kg Cd2＋, 100 mg/kg Cu2＋, and 100 mg/kg Fe2＋ to the medium, respectively. The results of RT-PCR showed that the four metal ions affected the expression of 15 G. lucidum laccase genes in liquid culture conditions, and that five laccase genes （Glac4, Glac6, Glac10, Glac11, and Glac12） were upregulated in response to heavy metal treatment. In summary, the varied responses of G. lucidum laccase to different heavy metals show its complicated physiological functions and regulation mechanisms.