【作者】 黄钦耿；

【导师】 黄建忠； 田宝玉；

【作者基本信息】 福建师范大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 经济适用的木质纤维素水解工艺是实现纤维素乙醇商品化的关键环节。研究土壤中多样性的木质纤维素分解微生物及其酶基因资源,将为开发新型木质纤维素水解酶系奠定基础。本文通过对一系列不同环境不同来源的森林土壤的理化及生物酶活性分析,筛选出一组具有着良好木质纤维素分解能力的酸性森林土壤样品SD。提取样品SD总基因组DNA为模板,用特异的16S rDNA及18S rDNA引物进行PCR扩增,构建环境微生物的16S rDNA及18S rDNA基因文库,并对文库进行RFLP分析(restriction fragment length polymorphism)、测序、序列分析和构建系统进化树。对该土壤细菌和真菌的菌群结构及生态功能进行分析。结果表明,从挑取的112个克隆中一共鉴定出66个不同细菌类群,主要类群包括Acidobacteria(包含Gp1、Gp2、Gp3、Gp10以及Gp13等5个簇),Proteobacteria(包含alpha,beta,delta和gamma四亚门),Verrucomicrobia和Unclassified Bacteria。其中,Acidobacteria是最大的类群,含有80个克隆,占总克隆数的71.5%;Proteobacteria次之,含有27个克隆,占总克隆数的24.1%。相对细菌来说,真菌菌群具有更为丰富的多样性则,鉴定出的40个不同的真菌带型包括6大类,即Ascomycota(36.2%),Basidiomycota(42.8%),Zygomycota(13.8%),Chytridiomycota(2.9%)和Fungi incertae sedis(4.3%)。其中Basidiomycota和Ascomycota是18S rDNA文库克隆中的主要菌型,占总菌群的79%。分析表明SD土壤样品中包含有丰富的木质纤维素分解微生物,细菌有Sphingomonas和Burkholderia以及一些固氮细菌;真菌中包含有更多的参与木质纤维素分解的微生物种类,主要包括担子菌门的Tricholomataceae,Strophariaceae和Agaricaceae;子囊菌门的Orbilia,Aspergillus,Phialocephala,Epicoccum,Phoma和Zygomycota的Mucorales等。为了开发这些土壤微生物所蕴含的丰富的木质纤维素分解酶基因资源,我们构建了森林土壤的宏基因组文库。通过对土壤样品总基因组DNA大量抽提、剪切、浓缩,首先获得适合文库构建的DNA。纯化的基因组片段插入Cosmid粘粒,包装,转染大肠杆菌宿主,构建成功森林土壤宏基因组文库。文库包含约230000个克隆,随机酶切和测序分析显示,插入片段平均大小为35kb,总库容为8.03 Gb。根据NCBI数据库公布的木聚糖酶基因和漆酶基因的保守序列分别设计特异性引物,扩增获得包含有漆酶基因保守区域长121bp的DNA片段,利用该片段对宏基因组文库进行原位杂交筛选。同时通过高通量功能筛选方法对文库进行了木聚糖酶、纤维素酶、淀粉酶以及蛋白酶等工业用酶的初步筛选,得到了35个具有明显蛋白酶水解圈的阳性克隆,进一步的分析正在进行中。我们的研究为以后对文库的进一步筛选以及将来从土壤中鉴定更多的木质纤维素水解酶奠定了坚实的基础。更多还原

【Abstract】 The emerging technology about conversion of lignocellulosic biomass to biofuels has not yet been demonstrated at commercial scale.The central question is the general absence of commercial technology for biodegrading lignocellulosic biomass to fermentable sugars.Fundamental understanding of microbial lignocellulose utilization will drive biotechnology innovations in developing commercial technologies for converting lignocellulosic biomass to biobased fuels and products.SD-1,a soil sample, with excellent wood-decaying performance,screened from the acidic decomposed-hollowed stump ecosystem of native forest in Fujian in China during the surveys of lignocelluse-decomposing abilities of the forest soils.Microbial communities of SD-1 were characterized by constructing and analyzing rRNA gene clone libraries.66 phylotypes were identified from 112 bacterial clones,including Acidobacteria(71.5%), Proteobacteria(24.1%) and Verrucomicrobia(0.9%).Acidobacteria were the predominant group,suggested their ecologically importance in this ecosystem. Proteobacteria included alpha,beta,delta and gamma subdivisions.They were belonged to members of photosynthetic,nitrogen-fixing,and bacteriolytic bacteria.None of there bacteria has been reported to be able to degrade lignocellulose in nature and/or in the laboratory.Total 40 phylotypes were obtained from 138 fungal clones,including Ascomycota(36.2%),Basidiomycota(42.8%),Zygomycota(13.8%),Chytridiomycota (2.9%) and Fungi incertae sedis(4.3%).RFLP and phylogenetic analysis showed a variety of clones related to the strains reported to have lignocellulose-degrading capability. They included some important bacterial decomposers,such as Sphingomonas and Burkholderia,and a number of wood-decaying fungi,such as Tricholomataceae, Strophariaceae and Agaricaceae of Basidiomycota;Orbilia,Aspergillus,Phialocephala, Epicoccum and Phoma of Ascomycota;and Mucorales of Zygomycota.To explore the abundant lignocellulolytic enzyme genes from the diverse soil microorganisms,a soil metagenomic library has been constructed.Total soil genomic DNA was extracted from the acid forest soil sample SD-1,sheared and concentrated.The target total genomic DNA was ligated with Cosmid,packaged,and transformed into the E. coil to lead to a forest soil metagenome library.The metagenome library contained a total of 230000 clones,the average length of foreign insert DNA fragments about 35kb,the capacity of foreign DNA of the library was 8.03Gb(8.03×10~6 kb.The digestion by restriction enzyme and sequencing for the inserted fragments from the positive clones showed that the lengths of inserted DNA fragments were big and random.The xylanase genes and laccase genes were screened from the library by PCR sequence-based method.A 21-base pairs laccase gene fragment contained two conservative region was first amplified by designed specific primer pairs.However,the xylanase gene PCR products cannot find similar sequence through blast analysised.The cloned laccase gene fragment was used in the next noethern blot to identify the positive clones contained laccase genes.And also,based on functional screening method,we screened for novel enzymes which used in industrial enzyme,included xytanase,cellulose, amylase and proteases.Meanwhile we obtained 35 positive clones with protease activity. Our works provided a solid basis for the future library screening and evaluation,also follow-up of other environmental samples metagenome library construct to identify the potential industrial enzyme sources.更多还原