【作者】 圣平；

【导师】 张宏宇；

【作者基本信息】 华中农业大学 ， 农业昆虫与害虫防治， 2014， 博士

【摘要】 暗黑鳃金龟(Holotrichia parallela)幼虫是我国重要的地下害虫,主要以植物根、茎等高纤维素含量的食物为食,其后肠发酵腔中富含丰富多样的微生物,其可能在宿主纤维性食物的消化、降解过程中发挥着重要的作用。本文研究了暗黑鳃金龟不同发育阶段肠道中微生物的群落组成及多样性,同时,利用简并引物同源克隆的方法对其肠道微生物源纤维素酶、半纤维素酶基因家族的多样性进行了研究,分离、筛选了可培养的、高活性的纤维素和半纤维素分解菌株并克隆了其纤维素酶、半纤维素酶基因,以期为纤维素和半纤维素的生物降解提供菌株及基因资源。1.利用16S rRNA基因宏基因组测序分析技术研究了暗黑鳃金龟成虫肠道(AL)、卵巢(LC)、卵(OL)、一龄(FL)、二龄(SL)、三龄幼虫(TL)肠道以及室内饲养土壤(TR)中微生物的群落组成及多样性。比较不同样本中细菌群落组成及多样性,结果表明：(1)幼虫不同发育阶段,二龄、三龄幼虫肠道微生物多样性显著高于一龄幼虫肠道微生物；(2)成虫肠道、卵巢、卵、一龄、二龄和三龄幼虫肠道细菌群落组成中,拟杆菌门细菌(Bacteroidetes)为主要的类群,厚壁菌门(Firmicutes)和变形菌门细菌(Protebacteria)所占比例次之。而土壤(TR)样本细菌群落组成中,变形菌门(Protebacteria)、拟杆菌门(Bacteroidetes)以及Candidate division TM7细菌占有绝对的优势；(3)暗黑鳃金龟幼虫肠道部分微生物来自垂直传递,其中梭菌纲OTU220、OTU461;p-变形菌纲OTU105、OTU856、OTU1130;拟杆菌纲,Dysgonomonas属OTU114、OTU774、OTU1157、OTU931所代表的细菌遗传型为卵巢与幼虫肠道或卵与幼虫肠道所共有,为蛴螬肠道的共生微生物。2.通过扫描电镜观察了暗黑鳃金龟幼虫野生种群和所有不同处理实验种群后肠发酵腔超微结构并比较了实验种群间三龄幼虫的体重,从实验结果可以看出不同处理组实验种群与野生种群幼虫后肠均附着着大量的圆形瘤突,野生种群幼虫后肠瘤表面形成菌-膜复合结构,而所有的实验种群处理组幼虫后肠瘤表面无明显的菌膜结构形成。对实验种群三龄幼虫体重进行多重比较分析,结果表明,幼虫生活的土壤不灭菌以及卵表面不消毒处理组,其三龄幼虫体重最高,土壤灭菌以及卵表面不消毒处理组和土壤不灭菌以及卵表面消毒处理组幼虫体重次之,而土壤灭菌,卵表面酒精消毒处理组,幼虫体重最低,且四个处理组幼虫体重之间存在显著的差异(P<0.05)。上述实验结果说明卵表面以及外界环境微生物显著影响暗黑鳃金龟幼虫的生长发育。3.采用简并引物同源克隆的方法研究了暗黑鳃金龟幼虫肠道微生物源纤维素酶、半纤维素酶基因的组成及多样性,成功克隆了5个糖苷水解酶家族酶基因序列,共包含101个OTUs,其中GH2(p-半乳糖苷酶)、GH36(a-半乳糖苷酶)、GH8(p-1,4-内切葡聚糖苷酶)、GH10以及GH11(内切木聚糖酶)家族分别包含24、27、19、14和17个OTUs。序列比对分析表明,GH2、GH8、GH10、GH11及GH36家族中分别有93.94%、10%、52.48%、28.95%以及19.32%的基因片段与已知序列相似性小于80%。其中,GH2家族中10.61%、GH8家族中3.33%以及GH11家族中18.42%的基因序列与已知序列的相似性小于60%,这些结果说明在暗黑鳃金龟幼虫后肠中富含丰富多样的纤维素酶,半纤维素酶基因,且部分基因序列有着很高的新颖性。进一步系统发育分析表明纤维素酶家族中GH8家族酶基因序列大多来源于变形菌门(Protebacteria)细菌,半纤维素酶家族中GH2、GH10、GH11以及GH36家族酶基因序列大多来源于拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)的细菌。4.从暗黑鳃金龟幼虫肠道分离、筛选出93株纤维素分解菌,经过复筛获得—株高产纤维素酶(HP207)的菌株,鉴定该菌株属于假单胞菌属Pseudomonas sp.。通过研究发现,该菌株发酵产酶最适培养基组成(1L)为CMC-Na15g,麸皮40g,酵母粉15g, NaC110g, KH2PO40.94g, K2HPO41.9g。最适培养条件为pH7.0,温度为30℃,100mL装液量,3%的接种量,培养时间24h。在最佳培养基以及培养条件下,菌株HP207β-1,4-内切葡聚糖苷酶(CMC酶)酶活可达到1.432U/mL。相关酶学性质分析表明,该酶有着较强的温度耐受性,其在70℃处理1h后,剩余酶活力仍保持在55%以上。5.从暗黑鳃金龟幼虫肠道分离、筛选得到103株木聚糖分解菌,通过复筛得到一株高产木聚糖酶(HP455)的菌株,鉴定该菌株属于鞘脂杆菌属Sphingobacteriumsp.。通过简并引物同源克隆并结合染色体步移的方法,从该菌株中成功克隆了1个内切木聚糖酶和1个p-木糖苷酶编码基因,分别命名为xyn455和xyl455。其中xyn455编码的内切木聚糖酶属于糖苷水解酶第10家族,xyl455编码的β-木糖苷酶属于第43家族。对xyn455和xyl455酶基因进行原核表达,并进行相关酶学性质分析发现,Xyn455酶不仅可以降解木聚糖类底物,对阿拉伯聚糖类底物也有着一定的降解作用,且Xy1455酶对阿拉伯聚糖也存在着较高的降解活性,表明Xyn455和Xy1455酶均具有阿拉伯糖苷酶活性,属于多功能酶。两者共同作用于木聚糖底物时,其降解效率显著增高,可完全降解木聚糖生成62.8%的木糖终产物,表明两者之间存在着强的协同降解作用。此外,研究还发现Xy1455酶对于终产物木糖有着较强的耐受力,抑制常数ki为247mM,其在pH3.0-9.0范围内酶活力也非常稳定。Xyn455以及Xy1455酶的性质特点使其在饲料添加剂、造纸等行业具有一定的应用潜力。更多还原

【Abstract】 Holotrichia parallela larvae are the severe agricultural pests, they feed on plant roots and organic matter of high cellulose content. Its hindgut typically contains a wide diversity of microorganisms, which may play an important role in food digestion of the host. Here, the microbial abundance and diversity of different developmental stages of the H. parallela were investigated, and we used a culture-independent method to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the larval hindgut. Furthermore, we isolated a large number of cellulolyitc and xylanolytic bacteria from the hindgut of H. parallela larvae, which were expected to be applied to the biodegradation of cellulose and hemicellulose.1. In our study, the microbial abundance and diversity of different life stages of the scarab larvae, H. parallela, were investigated using bacteria culture-independent method based on the sequence of bacteria16S rRNA V1-V3region gene. After comparing microbial community richness and composition among these different samples, the results were as follows:(1) Among the different life stages of the H. parallela larvae, the hindgut microbial diversities of the second instar, third instar larvae were significantly higher than the microbial diversity of the first instar larval hindgut.(2) Adult, ovary, egg, first-instar, second-instar and third-instar larvae libraries included the similar phyla composition, where Bacteroidetes were the most important groups, followed by Firmicutes and Proteobacteria. However, the soil library was dominated by Proteobacteria, Bacteroidetes and Candidate division TM7.(3) Many bacteria residented in hindgut of H. parallela larvae were from parent-to-offspring transmission, OTU220, OTU461from Clostridia, OTU105, OTU856, OTU1130from Betaproteobacteria,OTU114, OTU774, OTU1157, OTU931from Bacteroidetes were common in LC, OL, FL, SL, and TL samples, respectively, they are strong candidates for being members of H. parallela larvae core microbiome.2. The morphology and ultrastructure of the hindgut fermentation chamber of H. parallela, were examined using scanning electron microscopy, and the weight of third instar larvae was determined. The results showed that a special lobe-like structure was formed in the wild-caught and lab-reared third-instar larvae. The lobe-like structure of wild-caught population constituted by bacteria and the bacteria-covered cuticular intima, however, we could not observed the obviously cuticular intima structure in the lab-reared population. Furthermore, we found that there is a significant difference between the lab-reared populations (p<0.05). The weight of the third instar larvae was the highest under the unsterile soil and eggs treatment group, followed by sterile soil and unsterile eggs, unsterile soil and sterile eggs treatment groups. If the soil and the surface of eggs were sterilized, the larval weight became the lowest. Our results showed that the eggshell surface and environmental microbial populations significantly affect the growth and development of H. parallela larvae.3. We used the homologous cloning strategy with degenerate primers to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of H. parallela larvae. PCR with degenerate primers using metagenomic DNA from H. parallela larvae hindgut content yielded amplicons for beta-galactosidase (GH2), beta-1,4-endoglucanase (GH8), endo-xylanase (GH10,11), alpha-galactosidase (GH36) enzymes. All of these genes were grouped into101OTUs. Among them,24,27,19,14and17OTUs were identified in the GH2,8,10,11, and36families, respectively. Of these reads,93.94%,10%,52.48%,28.95%, and19.32%of GH2, GH8, GH10, GH11, and GH36sequences were<80%identical with those of known GH enzymes, respectively. Moreover,10.61%,3.33%, and18.42%of GH2, GH8, and GH11sequences had<60%identities with the available sequences in the NCBI database. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela, and many of them originate from unknown micro-organisms. Based on phylogenetic analysis, fragments from cellulase family (GH8) were most associated with the phylum of Proteo bacteria, and sequences from hemicellulase families (GH2, GH10, GH11and GH36) were related to enzymes from Bacteroidetes and Firmicutes.4. From the hindgut contents of H. parallela,93cellulolytic bacterial strains were isolated after enrichment in CMC medium. Among these isolates, a novel bacterium, designated HP207, with high endoglucanase productivity was selected for further study. This bacterium was identified as Pseudomonas sp. based on the results of the16S rRNA gene analysis, morphological characteristics and biochemical properties. The composition of the optimal fermentation medium was as follows (in g/L):CMC-Na (15), wheat bran (40), yeast extract (15), NaCl (10), KH2PO4(0.94) and K2HPO4(1.9). The optimal initial medium pH, temperature, medium volume, inoculum size and incubation period were7.0,30℃,100mL/250mL (v/v),3%(v/v) and24h, respectively. The maximum endoglucanase yield of1.432U/mL (expressed as enzyme activity) was obtained at the shake flask level. Moreover, this enzyme was also highly thermostable; approximately55%of the original activity was maintained after pretreatment at70℃for1h.5. From the hindgut contents of H. parallela,103xylanolytic bacterial strains were isolated. Among these isolates, a highly xylanolytic bacterium, designated HP455, was selected for further study. This bacterium was identified as Sphingobacterium sp. based on the results of the16S rRNA gene analysis. The endo-xylanase (xyn455) gene of the glycoside hydro lase (GH) family10and β-xylosidase (xyl455) gene of the GH43family were cloned and expressed in vitro from this highly xylanolytic bacterium. Both the Xyn455and Xy1455enzymes could degrade the arabinan, they are bifunctional enzymes with both endo-xylanase/β-xylosidase and a-L-arabinofuranosidase activities. And we also found that the in vitro degradation of xylan by Xyn455and Xy1455showed a strong synergistic hydrolysis action between these two enzymes. Furthermore, Xy1455enzyme also exhibits high xylose tolerance and a broad pH stability. These results suggest that the recombinant Xyn455and Xy1455enzymes are potential candidates to be used in feed additives, paper and other industries.更多还原