本文選題：松墨天牛 + 木質(zhì)素��； 參考：《福建農(nóng)林大學(xué)》2017年碩士論文

【摘要】：松墨天牛(MonnochamusalternatusHope)是松樹(shù)的重大害蟲(chóng),除能直接鉆蛀危害造成松樹(shù)衰弱甚至死亡外,還是松材線蟲(chóng)病的主要傳播媒介。木質(zhì)纖維素是木食性昆蟲(chóng)的主要營(yíng)養(yǎng)來(lái)源,包含細(xì)菌在內(nèi)的腸道微生物在幫助昆蟲(chóng)降解吸收木質(zhì)纖維素中有著重要作用。本研究利用形態(tài)學(xué)、生理學(xué)和分子生物學(xué)測(cè)定等方法,研究了松墨天牛幼蟲(chóng)、蛹和成蟲(chóng)3個(gè)發(fā)育階段腸道細(xì)菌的多樣性,篩選出具有木質(zhì)素降解功能的腸道細(xì)菌,探討了粘質(zhì)沙雷氏菌(Serratiamarcescens)的木質(zhì)素降解功能,以期為深入理解松墨天牛的營(yíng)養(yǎng)利用特性及其與寄主的協(xié)同關(guān)系提供基礎(chǔ)。現(xiàn)將主要結(jié)果摘要如下:1.利用IlluminaMiSeq技術(shù)對(duì)松墨天牛腸道細(xì)菌16SrDNA-V4變異區(qū)序列進(jìn)行擴(kuò)增、測(cè)序,發(fā)現(xiàn)腸道細(xì)菌群落共有24個(gè)菌屬,幼蟲(chóng)、蛹和成蟲(chóng)腸道分別有13、10和17個(gè)菌屬,分別隸屬于擬桿菌門(mén)、變形菌門(mén)、厚壁菌門(mén)、放線菌門(mén)、脫鐵桿菌門(mén)、疣微菌門(mén)、藍(lán)藻門(mén)和浮霉菌門(mén)。松墨天牛腸道優(yōu)勢(shì)細(xì)菌種類(lèi)及其相對(duì)豐度在不同發(fā)育階段具有差異,幼蟲(chóng)、蛹和成蟲(chóng)的優(yōu)勢(shì)菌屬(豐度)分別是伊麗莎白菌屬(61.12%)、金黃桿菌屬(61.38%)和沙雷氏菌屬(43.92%)。顫桿菌屬、Ruminiclostridium和螺桿菌屬為幼蟲(chóng)特有菌屬,土地桿菌屬、根瘤菌屬、貪噬菌屬和鞘氨醇盒菌屬為蛹特有菌屬,土壤桿菌屬、腸球菌屬、Nubsella、Blautia和短波單胞菌屬為成蟲(chóng)特有菌屬。Serratia在幼蟲(chóng)(12.6%)、蛹(0.85%)與成蟲(chóng)(43.92%)3個(gè)發(fā)育階段均有分布。松墨天牛成蟲(chóng)細(xì)菌群落多樣性最高,然后依次是幼蟲(chóng)和蛹。2.以濾紙為唯一碳源進(jìn)行液體培養(yǎng),經(jīng)分離、純化和分子鑒定,從松墨天牛3個(gè)不同發(fā)育階段的腸道中篩選出20株具有木質(zhì)素降解功能的細(xì)菌,隸屬于變形菌門(mén)、厚壁菌門(mén)、放線菌門(mén)和擬桿菌門(mén)的15個(gè)菌屬,包括沙雷氏菌屬、假單胞菌屬、芽孢桿菌屬、叢毛單胞菌屬、克雷伯氏菌屬、節(jié)桿菌屬、金黃桿菌屬、根瘤菌屬、腸桿菌屬、寡養(yǎng)單胞菌屬、蒼白桿菌屬、土壤球菌屬、鏈霉菌屬、無(wú)色桿菌屬和泛菌屬。進(jìn)一步利用羧甲基纖維素鈉(CMC)剛果紅染色法和濾紙降解實(shí)驗(yàn)復(fù)篩,發(fā)現(xiàn)1株可高效降解木質(zhì)素優(yōu)勢(shì)細(xì)菌—粘質(zhì)沙雷氏菌。3.以粘質(zhì)沙雷氏菌為對(duì)象,通過(guò)硫酸鹽木質(zhì)素培養(yǎng)基液體培養(yǎng)并測(cè)定該菌株的木質(zhì)素降解性能,發(fā)現(xiàn)在第4天其降解率達(dá)到峰值(15.16%),該菌能同時(shí)產(chǎn)生木質(zhì)素過(guò)氧化物酶(LiP)、錳過(guò)氧化物酶(MnP)、漆酶(Lac)3種胞外木質(zhì)素降解酶。進(jìn)一步以其中的優(yōu)勢(shì)木質(zhì)素降解酶—LiP木質(zhì)素過(guò)氧化物酶為研究對(duì)象,采用控制變量的單因素試驗(yàn)法,探究體外不同培養(yǎng)條件對(duì)源于松墨天牛腸道粘質(zhì)沙雷氏菌產(chǎn)木質(zhì)素過(guò)氧化物酶活性的影響。結(jié)果表明:木質(zhì)素濃度、pH值、氮源種類(lèi)、氮源濃度和金屬離子及其濃度均對(duì)其產(chǎn)酶以及酶活性有顯著影響。木質(zhì)素濃度為39·L-1時(shí)酶活性最強(qiáng),pH值為5時(shí)最適于產(chǎn)酶,有機(jī)氮源更有利于產(chǎn)酶,且酵母膏為最佳氮源,其最適濃度為5 g·L-1;最佳Mg2+、Ca2+、Fe2+、Mn2+、K+離子濃度分別為 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.

【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S763.38

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