【作者】 尹立伟；

【导师】 池玉杰；

【作者基本信息】 东北林业大学 ， 森林保护学， 2013， 博士

【摘要】 本研究对猴头菌Hericium erinaceum(Bull.:Fr.)Pers.菌株CB1进行了系统发育分析和分类鉴定,对其主要木质素降解酶系统进行了的检测,用其酶液对4种不同类型的染料茜素红、中性红、刚果红、结晶紫进行了脱色处理；以菌株CB1为基因供体,克隆得到锰过氧化物酶(MnP)同工酶基因Hemnpl和Hemnp2的全长cDNA基因及基因组全长DNA基因,通过荧光定量PCR检测了Hemnp1和Hemnp2基因的差异表达,并构建了重组质粒,通过原生质体转化方法将2个MnP同工酶基因转化到了构巢曲霉中,最终获得2个有效表达的MnP同工酶基因的构巢曲霉工程菌株。主要研究结果如下：1.首先对猴头菌菌株CB1进行了培养特性的观察,结果表明,菌株的宏观培养特性与猴头菌子实体的颜色和形态非常相似。对包括猴头菌菌株CB1在内的5种猴头菌属17个菌株进行了基于ITS序列的系统发育分析,结果表明菌株CB1与猴头菌同种其他菌株遗传距离较近并聚类在一起,该菌属于猴头菌科、猴头菌属的猴头菌。2.明确了该菌株的分类地位后,采用低氮天冬酰胺-琥珀酸(LNAS)培养基,在填加不同浓度的Mn2+和木屑为底物的情况下,对其进行了木质素降解酶系统的检测。结果表明,猴头菌可同时产生MnP和漆酶,但不产生LiP。不同锰离子为底物的检测中,以Mn2+的浓度600μmol·L-1为底物,MnP酶活性最高达259.55U-Lq,表明Mn2+对MnP的酶活性起到诱导作用,是猴头菌产生MnP的必要因子。通过酶活性的检测,了解了菌株CB1木质素降解酶系统的主要酶系及其与培养基的成分和酶作用的底物等条件的关系。当胞外酶的酶活达到最佳时,将4种不同结构的染料加入培养体系进行共同培养,从猴头菌固／液体培养基对染料的脱色结果来看,在4个染料浓度5mg/L、20mg/L、50mg/L、100mg/L中,对茜素红和中性红的脱色效果最好,其次是刚果红,对结晶紫脱色效果表现出较难被降解。3.采用简并PCR、RT-PCR、RACE、Genome Walking-PCR等方法,克隆得到2个猴头菌MnP同工酶基因,命名为He-mnp1和He-mnp2。He-mnp1的cDNA基因含有1080bp,编码359aa;He-mnp2的cDNA基因含有1086bp,编码361aa.He-mnp1与He-mnp2两个同工酶DNA和cDNA基因之间同源性分别为67%和81%,He-mnp1和He-mnp2的基因组全长都含有11个外显子和10个内含子。使用BioEdit软件、ClustalW2.1软件、3D软件等预测并分析了猴头菌He-mnp1和He-mnp2的蛋白质序列。4.采用实时荧光定量PCR技术,检测了猴头菌2个MnP同工酶基因之间的差异表达水平。结果猴头菌He-mnp1基因在第10d Mn2+浓度为200μm/1的表达水平最高,He-mnp2基因在第7d Mn2+浓度为600μm/1下转录水平达到最高He-mnp2基因最高表达量比He-mnp1基因最高表达量高出8倍,可见He-mnp2基因受Mn2+的影响较大。猴头菌MnP同工酶基因之间存在不同的转录调控机制,随时间的变化He-mnp1和He-mnp2同工酶基因表达水平达到最高后,无论提高锰离子浓度或者增加诱导时间表达量都受到抑制,He-mnp1基因和He-mnp2基因都受锰离子浓度的不同程度的诱导调控。5.利用RT-PCR方法克隆得到猴头菌He-mnp1和He-mnp2完整MnP同工酶CDS基因转化于构巢曲霉中进行异源表达,构建了重组质粒pLB01/He-mnp1和pLB01/He-mnp2。采用PEG/CaCl2介导的原生质体转化方法,将两个重组质粒转入到了构巢曲霉缺陷体菌株TN02A7的原生质体中,获得了转化子菌株TN02A7-He-mnpl和TN02A7-He-mnp2。将2个转化子菌株、缺陷体菌株TN02A7、WJA01、猴头菌菌株CB1在相同的木质素环境下进行了MnP活性的检测,结果TN02A7-He-mnpl在有血红素存在的情况下,诱导96h后酶活最高为38.31U.L-1,比不加血红素的酶活力高出8.64倍；而TN02A7-He-mnp2在有血红素存在的情况下,诱导96h后酶活最高为64.06U-L-1,比不加血红素的酶活力高出15.88倍,结果表明血红素是重组MnP基因异源表达的限制性因素之一。但在144h时转化子菌株都比猴头菌菌株CB1的酶活低,而TN02A7与WJA01始终无MnP酶活性,证明基因He-mnpl和He-mnp2已经成功地被转化到构巢曲霉中,并在木质素环境下已得到表达。更多还原

【Abstract】 The study on Hericium erinaceum (Bull.:Fr.) Pers. CB1strains were classified identification and phylogenetic analyses, its major ligninolytic enzymes were detected. Four different types dyes of Alizarin red, Neutral red, Congo red, Crystal violet were decolored treatment by enzyme liquid. Strain CB1for gene donor, cloning manganese peroxidase(MnP) isozyme genes Hemnpl and Hemnp2full length of cDNA genes and DNA genes, and through the fluorescence quantitative PCR Hemnpl and Hemnp2were detected differential expression,and then to construction recombinant plasmid,through the protoplast transformation methods two MnP isozyme gene transformation into the constitutive A. nidulans, They were obtained effectivee expression of two MnP isozyme Aspergillus nidulans engineering strain.The research conclusions of the present study as follows:1. Firstly, Cultural characteristic of H. erinaceum strain CB1was described. The results indicated that the appearance and microscopic characteristics of strain CB1and the color and forms of H. erinaceum fruiting body are very similar. The phylogenetic analysis of Hericium five species17different regional strains using the internal transcribed spacer (ITS) region, results showed that strain CB1and other strains from H. erinaceum were nearer in genetic distance and clustered together with them. The strain belongs to Hericiaceae, Hericium, Hericium erinaceum.2. The classification status of H. erinaceum strain CB1was fixed, Then,add the with different Mn2+concentrations and sawdust as the substrate by LNAS (Low Nitrogen Asparagine Succinic acid) culture solution in such situation,and its the major ligninolytic enzymes were detected.The results indicated that H. erinaceum could produce MnP and laccase simultaneously, but no LiP. Detection on different manganese ion as the substrate,The highest MnP activity were259.55U-L"1when sawdust was added to LNAS culture solution with600umol-L"1Mn2+substrates. Results indicated that Mn2+to MnP enzyme activity play induction.Mn2+was proved to be the essential factor for H. erinaceum to produce MnP,the lignin-degrading enzymes of the strain CB1and the relationships between enzyme activities and medium composition and substrates were gained. When the extracellular enzymes achieve optimum, the4different structure dyes add to the culture system for common culture, from H. erinaceum solid and liquid culture medium for dyes degradation decoloring, and results show that4dyes concentration5mg/L,20mg/L,50mg/L and100mg/L, Both alizarin red and neutral red are best decolorization effect, Congo red took second place, Crystal violet decolorization effect show the difficulty of degradation.3. Using degenerate PCR, RT-PCR, RACE, and Genome walking-PCR methods,etc. Molecular cloning of gene encoding two MnP isozyme genes from H. erinaceum. the full length cDNA were obtained then named He-mnpl and He-mnp2, respectively, He-mnpl and He-mnp2cDNA genes contains1080bp and1086bp,encoding for precursor polypeptide of359and361amino acids.Both two isozyme DNA and cDNA gene homology67%and81%,respectively. They genomes contain the11exon and10introns.Forecast and analysis amino acids of He-mnpl and He-mnp2by BioEdit、ClustalW2.1and3D software.4. The inequality expression levels of H. erinaceum two MnP isozyme genes were detected by quantitative Real-time PCR method. The results show that He-mnp1genes is the highest expression level for200μm/1Mn2+concentrations in10d, He-mnp2genes transcription level to reach the highest for600μm/1Mn2+concentrations in7d,He-mnp2gene expression than the highest amount He-mnpl gene expression highest amount was more than8times, Thus it can be seen that He-mnp2gene is strongly influenced by Mn2+.H. erinaceum MnP isozyme genes exist between different transcription regulation mechanism, He-mnp1and He-mnp2isozyme gene expression after reaching the highest level with time changes, regardless of the manganese ion concentration and increased induction time expression is restrained,He-mnp1and He-mnp2by Mn ion concentration of different process induction control.5. The coding complete MnP isozyme CDS gene, He-mnp1and He-mnp2were cloned by RT-PCR from H. erinaceum, and heterologous expression in A nidulans. the recombinant plasmid pLB01/He-mnp1and He-mnp2were constructed. And, using PEG/CaCl2protoplast transformation method, He-mnp1and He-mnp2were successfully transferred to auxotrophic stain TN02A7of A.nidulans. The two new tranformant stains, Auxotrophic stain TN02A7, WJA01and stain CB1were detected the MnP activity in the same shaking medium containing lignin,result showed that TN02A7-He-mnpl could produce MnP activity in the absence and presence of heme, but the MnP activity was up to38.31U-L-1on96h with heme which was8.64times higher than that without heme;TN02A7-He-mnp2could produce MnP activity in the absence and presence of heme, but the MnP activity was up to64.06U-L-1on96h with heme which was15.88times higher than that without heme,but tranformant stains produce MnP activity less than that of H. erinaceum CB1on144h, Because of TN02A7and WJA01could not produce MnP activity at any time, indicating that the gene He-mnpl and He-mnp2had been successfully transformed into A.nidulans expressed in lignin environment. The results of heme was one of restrictive factor for rescombinant mnp gene to express in A.nidulans.更多还原