【作者】 龙雁华；

【导师】 樊美珍； 黄勇平；

【作者基本信息】 安徽农业大学 ， 微生物学， 2009， 博士

【摘要】 培菌白蚁属于高等白蚁白蚁科（Termitidae）、大白蚁亚科（Macrotermitinae）,因与Termitomyces属真菌共生而得名。由于在热带干旱地区可消耗90%以上的干木、在多雨的热带草原地区能矿化20%的初级产物而引起研究学者们的广泛研究兴趣。在初步了解高等白蚁与其培养真菌的共生关系后,本研究利用分子生物学技术调查了共生系统中白蚁肠道及菌圃共生生物的多样性,通过cDNA文库的构建从共生真菌中筛选与木质纤维素降解相关的基因。主要研究内容如下:在安徽省、湖南省、江苏省、云南省等地广泛采集白蚁标本,建立了白蚁的形态鉴定和分子鉴定平台,利用ISSR分子标记调查了在上述地区广泛分布的高等、低等白蚁种群的遗传多样性及其与系统发育的关系。得到如下结果:①候选的24个ISSR引物中筛选出10个能得到清晰条带、重复性好、多态性高的引物,在14个白蚁群体中共扩增出145条带,平均每个引物扩增出14.5条带,多态性条带有144条,占总条带的99.31%,可见平均等位基因数（n_a）为1.9931±0.0830,平均有效等位基因数（n_e）为1.5661±0.3043,Nei’s基因多样性指数（h）和Shannon’s信息指数（Ⅰ）在分别为0.3355±0.1363和0.5065±0.1645,表明所有供试种群具有较高的遗传多样性;而三个不同种类种群多样性受地理条件影响较大,地理上的分隔很可能阻碍了种群内的基因交流;②一个种群内不同品级问的ISSR-PCR扩增格局是相同的,并且在一个品级内不同个体间的扩增也是稳定的,从而确定在缺失兵蚁的情况下也可以利用工蚁进行分子鉴定和种群多样性的调查;③利用PAUP软件分别对所有种群的COⅡ序列及ISSR-PCR电泳条带生成的0/1矩阵构建系统发育树,结果表明,两个系统发育树的拓扑结构相似,且都较好地与形态分类结果相吻合,显示了物种遗传多样性与其进化是保持一致的,同时也显示了ISSR分子标记是一个进行系统发育分析的有力工具。对广泛采集得到的白蚁种群进行高通量酶活测定,通过比较不同地区采集的17个种群、5种高等白蚁的4种纤维素酶酶活后,选择在西双版纳地区广泛分布的云南土白蚁（Odontotermes yunnanensis）及其共生Termitomyces属真菌作为研究对象。为探讨共生系统原核生物所发挥的作用,分别构建白蚁肠道细菌16s rDNA全长序列克隆文库和菌圃细菌16s rDNA全长序列克隆文库进行微生物区系的调查。其中,白蚁肠道细菌16s rDNA文库分析显示,623条可用序列被分成187个系统发育型,其中丰度最高的是厚壁菌门（Firmicutes）（占38%）,94.4%的系统发育型比对上梭菌纲（Clostridia）、梭菌目（Clostridiales）的纯培养或未培微生物;其次分别是拟杆菌门（Bacteroidetes）（占26.7%）、变形菌门（Proteobacteria）（占11.8%）、螺旋体门（Spirochaetes）（占8%）,拟杆菌目（Bacteroidales）、δ-变形菌纲（Deltaproteobacteria）、密螺旋体属（Treponema）分别是它们相应的优势群体;而浮霉菌门（Planctomycetes）、放线菌门（Actinobacteria）、TM7门、脱铁杆菌门（Deferribacteres）、绿菌门（Chlorobi）及TG1门的丰度则较低。而菌圃细菌文库分析表明,菌圃上细菌菌群的多样性明显低于肠道菌群,但厚壁菌门、变形菌门及拟杆菌门仍为主要组成。在成功分离得到菌圃上的共生真菌Termitomyces sp.菌株后,对其展开了一系列基础研究。得到以下结论:①生长于菌圃上的野生菌大部分情况下成小白球状,经显微镜观察可见由无数产孢结构和长椭圆形分生孢子构成,并判断分生孢子是由庞大的球状胞原生质体浓缩而分隔开形成的;②纯化菌株的ITS区序列（GenBANK号:FJ769410）与野生菌株的ITS区序列（GenBANK号FJ769409）基本一致,表明两者为同一菌株,同时在分类地位上都与Termitomyces sp.Group 8（AB073529）较近,但由于缺乏野外形成的子实体形状等分类信息,只能将该菌株鉴定为Termitomyces sp.;③菌圃及白蚁肠道真菌多样性的分析表明,在野外良好的共生状态下,菌圃上只生长一种Termitomyces属真菌,未见其他真菌生存,且白蚁肠道真菌与其为同一种,表明该共生真菌为白蚁食物组成的一部分。考虑到在共生状态下,共生真菌极可能在木质纤维素降解过程中发挥着重要作用,本研究首次利用直接在野外菌圃上采集的野生菌为材料,通过SMART^TM cDNA文库构建试剂盒构建了一个全长cDNA文库。所构建的文库经检测,文库滴度为7×10⁶Pfu/μl,库容量为3.5×10⁶Pfu,文库重组率达97%,平均插入片段长度约为1.33 Kb,文库质量合格。在此基础上,建立了引物PCR筛选目的基因的技术平台,经过两轮PCR筛选成功获得一个含木聚糖酶基因的阳性克隆,记为Xyl_C5。该目的基因序列在3’端存在明显的polyA结构,可编码226个氨基酸,预测分子量大小约为24 KDa,在43位氨基酸至223位氨基酸处为GHF11的功能域,并且129氨基酸和220氨基酸两个位点为活性位点,均为谷氨酸,NCBI比对后多数与曲霉属Aspergillus真菌木聚糖酶基因的序列一致性在58%-60%,可判定这是首个来自Termitomyces属真菌的木聚糖酶基因。最后,构建了一个pET22b_XylC5重组质粒、转化感受态大肠杆菌BL21进行诱导表达,经SDS-PAGE分析表明,目的蛋白表达出两条大小分别约为17 KDa、15 KDa的条带,且为不可溶性蛋白。该结果与预测的蛋白大小存在差距,可能是由于原核表达体系不适合表达真核来源基因或表达过程的各种折叠、修饰等步骤的原因导致的。更多还原

【Abstract】 So called "fungus-growing" termites belong to Termitidae,Macrotermitinae.The special name is got from the symbiosis between termite and fungi of genus Termitomyces. For its ability of consuming more than 90%of dry wood in some arid tropical areas and mineralizing up to 20%of the net primary products in wetter savannas,they aroused extensive interests of many researchers.Under the preliminary study of this symbiosis,we surveyed the diversity of symbiotic organisms using molecular biology techniques and constructed one cDNA library in order to screening for genes related to the degradation of lignocelluloses.The main results were listed as below.Firstly,termites samples were widely collected in South China（e.g.Anhui Province, Hunan Province,Jiangsu Province and Yunnan Province）,which were well identified through traditional morphological classification and molecular method.The genetic diversity of five major peregrinuses was analyzed by inter-simple sequence repeat（ISSR） markers.10 primers,which generated bright,polymorphic and reproducible amplification products were selected.145 discernible DNA fragments were produced,of which 144 （99.31%） were polymorphic loci.The observed number of alleles（n_a） was 1.9931±0.0830, the effective number of alleles（n_e） was 1.5661±0.3043,the Nei’s gene diversity（h） was 0.3355±0.1363 and the Shannon’s information index（I） was 0.5065±0.1645.These data suggested high genetic diversity in the overall populations.Some population genetic diversities were severally affected by geographic separation,which blocked intercourses of genes.The ISSR-PCR pattern was uniform between different castes of one colony and the amplification bands of different individuals in one caste were also stable.So,when soldiers were absent,it is feasible to use workers as objects in molecular identification and survey of diversity.Phylogenetic trees were constructed on sequence of cytochrome oxidase subunitⅡ（COⅡ） and 0/1 matrix generated by bands of ISSR-PCR electrophoresis.The results showed that topology of two trees partially coincided with each other and they were also congruous with the result of morphologic identification.Next,enzyme activities of 4 kinds of cellulases were compared in 17 colonies,5 species of higher termite.As a result,Odontotermes yunnanensis,which was widely distributed in Xishuangbanna,was chosen as our specimen for the highest enzymes activity of the symbiotic system.In order to explore the possible role of prokaryote in the symbiosis,two bacterial 16S rDNA gene clone libraries of microbial community from termite’s gut and fungus combs were constructed respectively.Analysis of library from the gut showed 623 usable sequences were divided into 187 phylotypes.Among these representative phylotypes,most were affiliated to Firmicutes（38%） and 94.4%of this phylum were regarded as the pure cultured or uncultured microorganisms from Clostridiales.Most of the rest phylotypes were mainly consisted of Bacteroidetes（26.7%）, Proteobacteria（11.8%）,Spirochaetes（8%） and Bacteroidales,Deltaproteobacteria, Treponema were corresponding dominant colonies.Only a few phylotypes belonged to Planctomycetes,Actinobacteria,TM7 phylum,Deferribacteres,Chlorobi and TG 1 phylum. Results showed lower bacterial diversity appeared in fungus comb than that in termite flora,and the combs were also consisted mostly of phylotypes belonging to Firmicutes.A series of fundamental research were carried out after pure cultured strain was isolated from fungus comb.Microexamination of one nodule collected in the wild showed many typical infllated conidiogenous structures and abundant conidia with the shape of long oval.It suggested that conidia were comparted from sphere cells as protoplasm concentrating.The ITS sequences of fungus nodule（GenBANK No.:FJ769409） and cultured strain（FJ769410） were the same and on the status of classification,they were also near to Termitomyces sp.Group 8（AB073529）.But it was still hard to give the accurate species name due to the absence of fruiting bodies formed in the field.Analysis of fungal diversity revealed that only Termitomyces sp.fungi were cultivated on fungus comb by termites and any other non-Termitomyces fungi cannot be detected.Uniform fungus appeared in intestinal tract and that combs also indicated that Termitomyces sp.was partial constituent of termites’ food.Considering the significant role of symbiotic fungi in the degradation of lignocelluloses,a cDNA library of Termitomyces sp.collected directly from fungus combs was constructed via SMART^TM cDNA library construction kit,which provided a valuable library for screening of cellulase genes and laid a foundation for basic biology research of this fungi.The library has a high titer of 7×10⁶ Pfu/μl and the capacity was 3.5×10⁶ Pfu. The recombination rate was 97%.The size of average insert cDNAs was 1.33 Kb.These experiment data showed the good quality of this library.A positive clone containing xylanase gene,named as Xyl_C5,was obtained using PCR screening method from library successfully.This gene coded 226 amino acids and had an obvious polyA structure existing at the 3’ end.The molecular weight of protein coded by this gene was about 24 KDa and from 43 to 223 amino acids was functional domain of glycoside hydrolase family 11.The 129 and 220 amino acids were two active sites,made up of glutamate.The amino acids sequence was 58%-60%identical to some other xylanase proteins from Aspergillus,indicating the first report of xylanase from Termitonyces.Last,a recombinant plasmid,pET22b-XylC5 was constructed and induced.SDS-PAGE showed two insoluble target proteins expressed with sizes of 17 KDa and 15 KDa,respectively.The difference between this result and the prediction perhaps was due to the unsuitable prokaryotic expression system or the folding and modification of the proteins.更多还原