松墨天牛肠道细菌多样性和粘质沙雷氏菌木质素降解特性的研究

发布时间：2018-05-09 16:14

本文选题：松墨天牛 + 木质素　；参考：《福建农林大学》2017年硕士论文

【摘要】：松墨天牛(MonnochamusalternatusHope)是松树的重大害虫,除能直接钻蛀危害造成松树衰弱甚至死亡外,还是松材线虫病的主要传播媒介。木质纤维素是木食性昆虫的主要营养来源,包含细菌在内的肠道微生物在帮助昆虫降解吸收木质纤维素中有着重要作用。本研究利用形态学、生理学和分子生物学测定等方法,研究了松墨天牛幼虫、蛹和成虫3个发育阶段肠道细菌的多样性,筛选出具有木质素降解功能的肠道细菌,探讨了粘质沙雷氏菌(Serratiamarcescens)的木质素降解功能,以期为深入理解松墨天牛的营养利用特性及其与寄主的协同关系提供基础。现将主要结果摘要如下:1.利用IlluminaMiSeq技术对松墨天牛肠道细菌16SrDNA-V4变异区序列进行扩增、测序,发现肠道细菌群落共有24个菌属,幼虫、蛹和成虫肠道分别有13、10和17个菌属,分别隶属于拟杆菌门、变形菌门、厚壁菌门、放线菌门、脱铁杆菌门、疣微菌门、蓝藻门和浮霉菌门。松墨天牛肠道优势细菌种类及其相对丰度在不同发育阶段具有差异,幼虫、蛹和成虫的优势菌属(丰度)分别是伊丽莎白菌属(61.12%)、金黄杆菌属(61.38%)和沙雷氏菌属(43.92%)。颤杆菌属、Ruminiclostridium和螺杆菌属为幼虫特有菌属,土地杆菌属、根瘤菌属、贪噬菌属和鞘氨醇盒菌属为蛹特有菌属,土壤杆菌属、肠球菌属、Nubsella、Blautia和短波单胞菌属为成虫特有菌属。Serratia在幼虫(12.6%)、蛹(0.85%)与成虫(43.92%)3个发育阶段均有分布。松墨天牛成虫细菌群落多样性最高,然后依次是幼虫和蛹。2.以滤纸为唯一碳源进行液体培养,经分离、纯化和分子鉴定,从松墨天牛3个不同发育阶段的肠道中筛选出20株具有木质素降解功能的细菌,隶属于变形菌门、厚壁菌门、放线菌门和拟杆菌门的15个菌属,包括沙雷氏菌属、假单胞菌属、芽孢杆菌属、丛毛单胞菌属、克雷伯氏菌属、节杆菌属、金黄杆菌属、根瘤菌属、肠杆菌属、寡养单胞菌属、苍白杆菌属、土壤球菌属、链霉菌属、无色杆菌属和泛菌属。进一步利用羧甲基纤维素钠(CMC)刚果红染色法和滤纸降解实验复筛,发现1株可高效降解木质素优势细菌—粘质沙雷氏菌。3.以粘质沙雷氏菌为对象,通过硫酸盐木质素培养基液体培养并测定该菌株的木质素降解性能,发现在第4天其降解率达到峰值(15.16%),该菌能同时产生木质素过氧化物酶(LiP)、锰过氧化物酶(MnP)、漆酶(Lac)3种胞外木质素降解酶。进一步以其中的优势木质素降解酶—LiP木质素过氧化物酶为研究对象,采用控制变量的单因素试验法,探究体外不同培养条件对源于松墨天牛肠道粘质沙雷氏菌产木质素过氧化物酶活性的影响。结果表明:木质素浓度、pH值、氮源种类、氮源浓度和金属离子及其浓度均对其产酶以及酶活性有显著影响。木质素浓度为39·L-1时酶活性最强,pH值为5时最适于产酶,有机氮源更有利于产酶,且酵母膏为最佳氮源,其最适浓度为5 g·L-1;最佳Mg2+、Ca2+、Fe2+、Mn2+、K+离子浓度分别为 0.20 g·L-1,0.40 g·L-1,0.15 g·L-1,0.04 g·L-1,Og·L-1。
[Abstract]:MonnochamusalternatusHope is a major pest of pine trees. It is the main medium of pine wood nematode disease except that it can lead to the decay and even death of pine trees. Lignocellulose is the main source of nutrition for wood eating insects. Intestinal microbes, including bacteria, help the insects to degrade and absorb lignocellulose This study used morphological, physiological and molecular biological methods to study the diversity of intestinal bacteria in the 3 developmental stages of the larvae, pupae and adult of Pinus Pinus, screening the intestinal bacteria with lignin degradation function, and discussing the lignin degradation function of the Serratiamarcescens (Serratiamarcescens). The main results are summarized as follows: 1. the sequence of 16SrDNA-V4 variation in the intestinal bacteria of Pinus Pinus was amplified and sequenced by IlluminaMiSeq technology. The results showed that there were 24 bacteria, larva, pupae and adult intestinal tract in the intestinal bacterial community. 13,10 and 17 genera belong to the bacteriobacteria, deformable bacteria, actinomycetes, actinomycetes, deformiobacterium, verruca microbacteria, cyanobacteria, and floating mold. The species and relative abundance of the intestinal dominant bacteria in the intestinal tract of the Pinus Pinus are different at different developmental stages, and the dominant bacteria (abundance) of larvae, pupae and adult are Elizabeth bacteria, respectively. Genus (61.12%), chrysobacterium (61.38%) and salebacter (43.92%). Ruminiclostridium and Helicobacter are larval endemic genera, land bacilli, rhizobia, phagocybacilli and sphingosine as endemic genera, soil bacilli, enterococci, Nubsella, Blautia, and short wave monocytic genus.S Erratia was distributed in the larvae (12.6%), the pupa (0.85%) and the adult (43.92%) 3 developmental stages. The diversity of the bacteria community was the highest. Then the larvae and pupae.2. were used as the only carbon source for the liquid culture. After separation, purification and molecular identification, 20 strains were screened from the intestine of the 3 different developmental stages of the Pinus Pinus. The 15 bacteria belonging to the lignin degradation function, belonging to the deformable bacteria gate, the thick wall bacteria gate, the actinomycetes and the bacteriobacteria, including the genus seracella, Pseudomonas, bacillus, hairy monomonas, Klebsiella, bacillus, rhizobia, Enterobacteriaceae, oligomonas, pallid, soil The genus, Streptomyces, colorless and pan fungi. Further using CMC Congo red staining and filter paper degradation experiment, we found that 1 strains of lignin dominant bacteria - salebium mucilagus.3., which can efficiently degrade lignin, were used as the salebium mucilagus, and the sulfate lignin culture medium was used to culture and determine the bacteria The degradation of lignin was found to reach a peak (15.16%) in fourth days. The bacteria could produce lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac), 3 kinds of extracellular lignin degrading enzymes. The effects of different culture conditions on the activity of lignin peroxidase from the intestinal mucilage of the intestinal mucilage were investigated in vitro. The results showed that the concentration of lignin, pH, the type of nitrogen source, the concentration of nitrogen source and the concentration of metal ions and their concentration had significant effects on their enzyme production and enzyme activity. The concentration of lignin was 39. L. At the time of -1, the enzyme activity was the best. When the pH value was 5, it was the most suitable for producing enzymes. The organic nitrogen source was more beneficial to the enzyme production, and the yeast ointment was the best nitrogen source. The optimum concentration was 5 g. L-1. The optimum Mg2+, Ca2+, Fe2+, Mn2+, and K+ ions were 0.20 G. L-1,0.40 G.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S763.38

【参考文献】