本文選題：萊氏野村菌 + 微菌核��； 參考：《重慶大學(xué)》2015年碩士論文

【摘要】：蟲生真菌因在防治農(nóng)林害蟲時(shí)具有對(duì)環(huán)境無污染、無殘留和使害蟲不易產(chǎn)生抗藥性等優(yōu)點(diǎn),是一種環(huán)境友好型真菌。隨著人們對(duì)環(huán)保意識(shí)的增強(qiáng),利用蟲生真菌防治農(nóng)林害蟲受到人們的廣泛關(guān)注及重視。萊氏野村菌在田間能夠自然侵染多種夜蛾科害蟲并引起域內(nèi)流行病,是一種重要的昆蟲病原真菌。萊氏野村菌作為殺蟲劑的有效成分是分生孢子,但是由于其產(chǎn)孢條件比較苛刻如需要苛刻的碳源(麥芽糖)和持續(xù)光照,其工業(yè)化生產(chǎn)方面一直受到極大限制,所以尋找該菌的其他繁殖體來代替分生孢子行使殺蟲作用具有十分重要的意義。液體發(fā)酵產(chǎn)生的芽生孢子雖然有一定侵染力,但因其細(xì)胞壁比較薄、不耐儲(chǔ)存以及毒力低等特點(diǎn)也不能用于生產(chǎn)實(shí)踐。微菌核是真菌菌絲聚集而形成特異休眠體結(jié)構(gòu),它具有耐儲(chǔ)存、抗逆性強(qiáng)、在適宜的環(huán)境下能萌發(fā)產(chǎn)生菌絲和孢子。重慶大學(xué)生命科學(xué)學(xué)院微生物實(shí)驗(yàn)室成功的誘導(dǎo)出萊氏野村菌的微菌核,并對(duì)其的抗逆性和毒力等進(jìn)行了測試,實(shí)驗(yàn)結(jié)果證實(shí)該微菌核可以作為有效的活性成分來代替分生孢子用于害蟲的生物防治制劑。其后,采用比較轉(zhuǎn)錄組技術(shù)從分子層面來探討微菌核形成機(jī)理,發(fā)現(xiàn)在微菌核形成與氧脅迫密切相關(guān),并且在微菌核形成的過程中大量的還原性酶或合成還原性物質(zhì)的基因上調(diào)表達(dá)。依據(jù)此,本課題從比較轉(zhuǎn)錄組上調(diào)表達(dá)的c DNA文庫中挑取Nraox基因的EST序列,將其克隆并采用RNA干擾技術(shù)來研究該基因在微菌核發(fā)育過程中的作用。主要研究結(jié)果如下:①萊氏野村菌Nraox基因的克隆與序列分析根據(jù)萊氏野村菌轉(zhuǎn)錄組庫中Nraox基因的EST序列,克隆得到該基因的c DNA全長序列(Gen Bank登錄號(hào):No.KM978957)。序列分析顯示其開放閱讀框?yàn)?068bp,編碼355個(gè)氨基酸;生物信息學(xué)分析表明該蛋白的理論分子量為40.517KD,理論等電點(diǎn)為9.53;該蛋白具有親水性,無信號(hào)肽,亞細(xì)胞定位分析該蛋白定位于線粒體內(nèi)。Nraox的基因組全長為1512 bp,該基因含有3外顯子,2內(nèi)含子。利用DNAMAN和MEGA軟件進(jìn)行同源比對(duì)和系統(tǒng)進(jìn)化分析發(fā)現(xiàn),萊氏野村菌的AOX蛋白與綠僵菌的同源性最高,并與麥角菌科的真菌聚為一組,同源性都達(dá)到88.17%。②萊氏野村菌Nraox的表達(dá)分析收集萊氏野村菌微菌核發(fā)育不同時(shí)期的樣品,采用熒光定量q PCR的方法對(duì)Nraox基因的表達(dá)模式進(jìn)行分析,結(jié)果表明該基因在微菌核發(fā)育的不同時(shí)期均有表達(dá),并在微菌核形成初期表達(dá)量最高,然后降低;采用q PCR技術(shù)檢測Nraox在氧脅迫誘導(dǎo)劑處理后的基因表達(dá),研究結(jié)果表明,氧化劑H2O2和甲萘醌都能引起該基因的高度上調(diào)表達(dá),H2O2處理后Nraox表達(dá)量是對(duì)照組的7倍,甲萘醌處理后是對(duì)照組的20倍。③抑制劑處理對(duì)交替氧化酶功能的驗(yàn)證采用交替氧化酶的特殊抑制劑SHAM處理后,檢測細(xì)胞內(nèi)過氧化氫含量變化,結(jié)果顯示,與對(duì)照組相比,抑制劑SHAM處理后前10min中內(nèi)細(xì)胞內(nèi)H2O2較高,處理15min后沒有明顯變化;但是,顯微觀察菌絲形態(tài)發(fā)現(xiàn)與對(duì)照組相比,處理組菌絲表現(xiàn)為彎曲和很多芽狀結(jié)構(gòu)的不正常生長狀態(tài);而誘導(dǎo)培養(yǎng)基中的微菌核表現(xiàn)為微菌核的形成延遲、微菌核顆粒直徑較大以及微菌核結(jié)構(gòu)松散、表面比較蓬松。由此可以確定,抑制劑處理后減緩了細(xì)胞內(nèi)過氧化氫的清除速度,并對(duì)細(xì)胞產(chǎn)生了一定的氧毒害。④RNA干擾技術(shù)驗(yàn)證Nraox基因功能利用RT-q PCR技術(shù)對(duì)不同濃度si RNA的干擾效率進(jìn)行檢測,結(jié)果顯示800 n M si RNA能夠有效下調(diào)Nraox基因的表達(dá),并且該基因表達(dá)量下調(diào)了約76.1%。與對(duì)照組相比,干擾菌株在含有脅迫誘導(dǎo)劑的固體SMAY培養(yǎng)基上生長時(shí),菌落生長緩慢,菌落變小,產(chǎn)孢量降低。而在微菌核誘導(dǎo)培養(yǎng)基AM中生長時(shí),Nraox基因干擾后的微菌核表型發(fā)生了明顯變化,主要表現(xiàn)為:與野生菌相比,微菌核的形成推遲,微菌核顆粒的直徑變大,質(zhì)地較為疏松;微菌核的生物量和產(chǎn)量顯著性降低;顯微觀察發(fā)現(xiàn)相對(duì)于對(duì)照組,干擾后的萊氏野村菌菌絲顯示畸形生長,表現(xiàn)為菌絲上生出很多芽狀結(jié)構(gòu);這些結(jié)果表明,Nraox參與了萊氏野村菌微菌核的形成調(diào)控。⑤干擾菌株對(duì)靶標(biāo)害蟲的致病性鑒定采用點(diǎn)滴法測定干擾菌株微菌核對(duì)三齡斜紋夜蛾幼蟲的侵染和致病能力,結(jié)果發(fā)現(xiàn),干擾菌株相對(duì)于野生型菌株對(duì)斜紋夜蛾幼蟲的致死率下降,半致死時(shí)間LT50顯著增長,較野生菌株LT50增加26.2%。結(jié)論:首次克隆得到萊氏野村菌的交替氧化酶基因,并命名為Nraox。研究了該基因在萊氏野村菌菌絲生長、微菌核的形成、產(chǎn)孢、毒力和氧脅迫等方面的作用,結(jié)果表明Nraox通過調(diào)節(jié)細(xì)胞內(nèi)的氧化還原平衡和菌絲生長來影響萊氏野村菌微菌核的形成、影響到萊氏野村菌產(chǎn)孢,并且在微菌核的毒力方面Nraox也起一定的作用。
[Abstract]:Entomophyte fungi are a kind of environmentally friendly fungi for the advantages of no pollution to the environment, no residue and resistance to insecticide resistance in the control of agroforestry. With the increasing awareness of environmental protection, the use of entomophyll fungi in the control of agroforestry is widely concerned and paid attention to. A variety of nocturum pests and causing the epidemic in the domain is an important entomopathogenic fungus. As an effective component of the insecticide, nocturum is a conidium, but its industrial production has been greatly restricted because of its severe sporulation conditions such as the demanding carbon source (maltose) and continuous light. The other propagating bodies of the bacteria are of great significance to replace the conidium. Although the spore produced by liquid fermentation has certain infectivity, it can not be used in production practice because of its thin cell wall, poor storage and low toxicity. The micro sclerotium is a specific dormant structure formed by the accumulation of fungal mycelium. It is resistant to storage, strong resistance, and can germinate mycelium and spores in a suitable environment. The microbiological laboratory of the Chongqing University Institute of life science has successfully induced the micromycelium of Nomura Brunei, and tested its resistance and virulence. The results of the experiment confirmed that the microsclerotium could be used as an effective active ingredient to replace the micromycelium. The conidium is used as a biological control agent for the pest. Subsequently, the mechanism of the sclerotium formation is discussed from the molecular level by the comparative transcriptional technique. It is found that the formation of the sclerotium is closely related to the oxygen stress, and a large number of reductive or reductive substances are up-regulated in the process of the formation of the micro sclerotium. The EST sequence of the Nraox gene was selected from the C DNA library, which was up-regulated in the comparative transcriptional group. The gene was cloned and used to study the role of the gene in the development of the sclerotia. The main results were as follows: (1) the cloning and sequence analysis of the Nraox gene of Nomura Brunelli was based on the EST of the Nraox gene in the village of Nomura's transcriptional group. The sequence analysis showed that the C DNA full-length sequence of the gene (Gen Bank login number: No.KM978957). The sequence analysis showed that the open reading frame was 1068bp and encoded 355 amino acids; bioinformatics analysis showed that the molecular weight of the protein was 40.517KD and the theoretical isoelectric point was 9.53; the protein was hydrophilic, no signal peptide and subcellular localization analysis. The total length of the protein located in the mitochondrial.Nraox is 1512 BP, which contains 3 exons and 2 introns. Using DNAMAN and MEGA software for homologous comparison and phylogenetic analysis, it is found that the AOX protein of M. lazeri is the most homologous to the Bacillus anisopliae, and it is together with the fungi of the family ergodaceae, and the homology is 88.17%. 2. The expression analysis of Nraox was collected and analyzed at different stages of the growth of the sclerotia of Nomura brunellus. The expression pattern of Nraox gene was analyzed by the method of fluorescence quantitative Q PCR. The results showed that the gene was expressed at different stages of the development of the sclerotia, and the expression was highest in the early stage of the sclerotium formation and then decreased, and Q PCR was used. The technique was used to detect the gene expression of Nraox after the oxygen stress inducer treatment. The results showed that both the oxidant H2O2 and the naphthoquinone could cause the high expression of the gene. The expression of Nraox was 7 times as high as that of the control group after H2O2 treatment, and 20 times when the naphthoquinone was treated as the control group. The changes in the intracellular hydrogen peroxide content were detected after the treatment of SHAM, a special inhibitor of oxidase. The results showed that compared with the control group, the intracellular H2O2 was higher in the anterior 10min after SHAM treatment than in the control group, and there was no significant change in the treatment of 15min. However, the microscopic observation of mycelium morphology found that the mycelium in the treatment group was curved and much more than that of the control group. The micro sclerotium in the inducible medium was delayed by the micro sclerotium, the diameter of the sclerotia was larger and the micro sclerotium was loose and the surface was loosely loose. Thus, it could be determined that the removal rate of hydrogen peroxide in the cells was slowed down and certain oxygen toxicity was produced to the cells. RNA interference technique verified that Nraox gene function was used to detect the interference efficiency of Si RNA with different concentrations by RT-q PCR technique. The results showed that 800 n M Si RNA could reduce the expression of Nraox gene effectively, and the expression of this gene down regulated 76.1%. and the control group in the solid medium containing the stress inducer. At the time of growth, the growth of the colonies was slow, the colony became smaller and the sporulation decreased. The micro sclerotium phenotype of the micro sclerotium induced by the micro sclerotium induced AM was obviously changed. The main manifestations were: the formation of the micro sclerotium was delayed, the diameter of the sclerotia was larger, the texture was loose, and the biomass of the micro sclerotium, compared with the wild bacteria. The microscopical observation showed that the mycelium of the mycelium of brunellite showed abnormal growth compared with the control group. The results showed that Nraox was involved in the formation and regulation of the micromycelium of the mycelium of the mycelium. 5 The interference strain microbacteria checked the infection and pathogenicity of the larvae of the three instar Spodoptera Spodoptera. The results showed that the lethal rate of the interfered strain was lower than that of the wild type. The semi lethal time LT50 increased significantly and the wild strain LT50 increased the 26.2%. conclusion: the first cloning of the alternation oxidase gene of the nomacella laeriae was first cloned, and the name was named. The effects of the gene on the growth of the mycelium, formation, sporulation, virulence and oxygen stress of the mycelium of the mycelium of the brunellite were studied for Nraox.. The results showed that Nraox could affect the formation of the sclerotia of the brunellite by regulating the redox balance and the growth of the mycelium in the cells, affecting the sporulation of the brunellite and the virulence of the sclerotia. Surface Nraox also plays a role.

【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S476.12

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