發(fā)布時間：2018-07-09 20:53

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

【摘要】：萊氏野村菌是一種在自然界廣泛存在的能有效控制昆蟲種群的重要病原真菌。對鱗翅目夜蛾科害蟲有較強的感染力和致病力,萊氏野村菌殺蟲制劑的有效成分為分生孢子。在萊氏野村菌發(fā)酵生產(chǎn)中,它對碳源的要求較高、生長周期長、產(chǎn)孢量不高,且在培養(yǎng)過程中需要持續(xù)光照,限制了對該菌的推廣和應用。本實驗室通過液體發(fā)酵成功誘導出了抗逆性強、毒力與傳統(tǒng)的分生孢子沒有明顯差異且耐儲存的微菌核,成為了一個新的具有高效殺蟲活性的殺蟲制劑的有效成分。顯微觀察發(fā)現(xiàn),誘導培養(yǎng)基AM誘導產(chǎn)生的微菌核是由菌絲體特化形成的休眠體構造;而用比較轉錄組測序分析的方法證實,微菌核的形成是一種氧脅迫的過程,伴隨色素的累積、活性氧(ROS)的產(chǎn)生及大量功能基因的表達變化。對這一調(diào)控機制的深入研究可以讓我們更好的了解微菌核形成的分子特性,也可為后期對微菌核發(fā)酵生產(chǎn)及制劑的開發(fā)等提供理論參考依據(jù)。本論文根據(jù)萊氏野村菌比較轉錄組庫中的EST序列克隆得到了在微菌核形成期高表達的Nrcdc24與Nrbem1基因的全長序列,經(jīng)生物信息學分析、基因在微菌核形成過程中的表達測定以及外源RNA干擾的方法證明了兩個基因的功能。主要研究結果如下:①采用SMART RACE RT-PCR技術獲得Nrcdc24與Nrbem1的c DNA和基因組全長序列,其ORF分別為3021 bp和1689 bp,各自編碼1006和562個氨基酸;②對目的基因編碼蛋白的生物信息學分析顯示,Nrcdc24編碼的蛋白含有與Rho家族結合的功能位點Rho GTP;Nrbem1含有兩個SH3(Src Homology 3)結合位點以及一個磷酸肌醇結合位點(phox homology,PX)。此外,Nrcdc24與Nrbem1蛋白都有PB1(phox and Bem1)結合位點;通過對近緣物種的系統(tǒng)進化樹分析發(fā)現(xiàn)萊氏野村菌的Nrcdc24與Nrbem1蛋白與綠僵菌的親緣關系最近;③顯微觀察萊氏野村菌液體培養(yǎng)形成微菌核過程發(fā)現(xiàn),在培養(yǎng)至84 h左右時,培養(yǎng)基中極性生長的菌絲體開始局部縮聚,微菌核開始大量形成;采用實時熒光定量PCR的方法對Nrcdc24與Nrbem1基因在微菌核不同發(fā)育時期表達模式進行分析發(fā)現(xiàn),Nrcdc24與Nrbem1均在微菌核大量形成初期(84 h左右)表達量高;④為了進一步研究Nrcdc24與Nrbem1基因在微菌核形成過程中所起到的作用,設計同源干擾片段,以e GFP干擾片段的干擾菌株作為陰性對照,采用外源RNAi的方法進行研究發(fā)現(xiàn):在SMAY固體培養(yǎng)基上,Nrcdc24與Nrbem1干擾菌株(命名為cdc24RNAi和bem1RNAi)的酵母態(tài)階段生長菌落比野生型(WT)和陰性對照(e GFP)的菌落生長相對緩慢,菌體由酵母態(tài)向菌絲態(tài)轉化(兩型轉變)延遲0.5-1d,產(chǎn)孢時間也隨之延遲;經(jīng)顯微計數(shù)發(fā)現(xiàn),cdc24RNAi和bem1RNAi菌株的產(chǎn)孢量較對照顯著降低,產(chǎn)孢量分別降低95%和56%。而雙干擾菌株酵母態(tài)時期的菌落更小,產(chǎn)孢量較出發(fā)菌株降低了99.04%。在AM液體培養(yǎng)基中,相對于對照組,各干擾菌株的色素積累明顯減少,同步培養(yǎng)5-7d的發(fā)酵液粘稠度也明顯降低。顯微觀察發(fā)現(xiàn),各干擾菌株的菌絲體均變粗且分枝增多,bem1RNAi菌株及雙干擾菌株的菌體上產(chǎn)生許多不脫落的芽質(zhì)狀孢子,而cdc24RNAi菌株的菌體形態(tài)同對照相比則無明顯差異。此外,在同步培養(yǎng)條件下,對照組菌株在4d左右時便開始有微菌核產(chǎn)生,而干擾菌株中無微菌核產(chǎn)生;繼續(xù)培養(yǎng)觀察發(fā)現(xiàn),各干擾菌株微菌核形成的時間會延遲1-2d。干重法測微菌核的生物量顯示,cdc24RNAi和bem1RNAi菌株的生物量降低20-32%,雙干擾菌株的生物量降低約為40%。另外,各處理菌株培養(yǎng)至7d左右(常規(guī)誘導培養(yǎng)微菌核收獲)時,相對于對照組,cdc24RNAi和bem1RNAi及雙干擾菌株的微菌核形成的數(shù)量分別下降了93.2%、90.7%和97.3%;⑤采用q PCR的方法檢測在cdc24RNAi與bem1RNAi菌株中與活性氧的nox R與nox A基因的表達量發(fā)現(xiàn),同干擾rac A與cdc42基因一樣nox R與nox A表達量均明顯下降;此外,在沉默Nrcdc24基因以后,rac A與cdc42基因的表達量也降低,同時,在干擾的cdc42與rac A菌株中Nrcdc24基因的表達量顯著升高,暗示Nrcdc24基因可能為rac A與cdc42基因的上游基因,調(diào)控著二者的表達,進而影響微菌核的形成;⑥采用點滴接種法接種斜紋夜蛾3齡幼蟲,發(fā)現(xiàn)與對照相比,cdc24RNAi與bem1RNAi菌株對斜紋夜蛾幼蟲的致死率下降,LT50延長。通過對斜紋夜蛾幼蟲的存活曲線分析,野生型,e GFP,Nrcdc24,Nrbem1和雙干擾菌株LT50的平均值分別為9.32,9.53,11.74,11.06和13.60天。結論:成功克隆得了Nrcdc24與Nrbem1基因的全長序列,證明了兩基因均對微菌核的發(fā)育形成、菌絲的生長、產(chǎn)孢量及菌株毒力等有一定的影響;Nrcdc24與Nrbem1基因與NADPH復合體有互作關系。而在微菌核過程中Nrcdc24可調(diào)控rac A與cdc42基因的作用。
[Abstract]:Brunella is an important pathogenic fungus which can effectively control the insect population in nature. It has strong infection and pathogenicity to the Lepidoptera nocturnal insect pests. The effective component of the insecticidal preparation of the Nomura lauriti is the conidium. In the fermentation production, it has high requirements for carbon source and long growth cycle. The production of sporulation is not high, and it needs continuous light in the process of culture, which restricts the popularization and application of the bacteria. The laboratory has successfully induced the strong resistance, the virulence and the traditional conidium, which have no obvious difference between the conidium and the stored micro sclerotium, have become a new effective insecticidal agent. Microscopical observation found that the micromycelium induced by the inducible medium AM was a dormant structure formed by the mycelium, and it was confirmed by the method of comparative transcriptional sequence analysis that the formation of the microsclerotium was a process of oxygen stress, with the accumulation of pigment, the production of active oxygen (ROS) and the change of the expression of a large number of functional genes. The in-depth study of the control mechanism can make us better understand the molecular characteristics of the microsclerotium formation, and provide theoretical reference for the fermentation production and preparation of the microsclerotium in the later period. In this paper, the high expression of Nrcdc24 and Nrbem1 in the formation period of the microsclerotium is obtained on the basis of the EST sequence clones in the comparative transcriptional group library. The full length sequence of the gene, through bioinformatics analysis, the expression of gene in the formation of sclerotia and the method of exogenous RNA interference, proved the function of two genes. The main results are as follows: (1) SMART RACE RT-PCR technique is used to obtain C DNA and the whole length sequence of Nrcdc24 and Nrbem1, and the ORF is 3021 BP and 1689 B, respectively. P, each encoded 1006 and 562 amino acids; 2. The bioinformatics analysis of the encoded protein of the target gene showed that the Nrcdc24 encoded protein contained the functional locus Rho GTP associated with the Rho family; Nrbem1 contained two SH3 (Src Homology 3) binding sites and a phosphoric inositol junction site (phox homology, PX). White have PB1 (phox and Bem1) binding site; by phylogenetic tree analysis of the proximal species, the relationship between the Nrcdc24 and Nrbem1 protein of Nomura brunellae and the relationship between Nrbem1 and Bacillus anisopliae was found. At the beginning of local polycondensation, microsclerotium began to form in large quantities; the expression patterns of Nrcdc24 and Nrbem1 genes in different developmental stages of microsclerotia were analyzed by real-time fluorescence quantitative PCR, and Nrcdc24 and Nrbem1 were both high in the early stage of microsclerotium formation (about 84 h). (4) to further study the microbacteria of Nrcdc24 and Nrbem1 gene. In the process of nuclear formation, homologous interference fragments were designed, and the interfering strains of E GFP interference fragments were used as negative controls. The method of exogenous RNAi was used to study that the yeast state growth of Nrcdc24 and Nrbem1 interfering strains (named cdc24RNAi and bem1RNAi) in the SMAY solid medium was more than that of the wild type (WT) and the Yin. The colony growth of E GFP was relatively slow, the strain from yeast state to hyphae (type two transformation) delayed 0.5-1d, and the sporulation time was delayed. The microcount showed that the sporulation of cdc24RNAi and bem1RNAi strains decreased significantly compared with the control, and the sporulation decreased by 95% and 56%. respectively. Compared with the control group, the accumulation of 99.04%. in the liquid culture medium was lower than that of the starting strain. Compared with the control group, the accumulation of pigments in all the interfered strains decreased obviously, and the consistency of the fermentation broth of 5-7d was obviously reduced. The microscopic observation showed that the mycelia of all the interfered strains were all thicker and more branched, and the strains of bem1RNAi and double interfering strains were produced on the bacteria. There were many non shedding sprout spore, but there was no obvious difference in the morphology of cdc24RNAi strain. In addition, under the synchronous culture condition, the control group began to have micro sclerotium when the strain was around 4D, and the interfering strains had no sclerotia. The biomass of the delayed 1-2d. dry weight micrometer Sclerotinia showed that the biomass of cdc24RNAi and bem1RNAi strains decreased by 20-32%, and the biomass of the double interfering strains decreased by about 40%.. The microsclerotium formation of cdc24RNAi, bem1RNAi and double interfering strains was compared to the control group when the treated strains were cultured to 7d (the conventional cultivation microsclerotium harvest). The number of Q PCR was 93.2%, 90.7% and 97.3% respectively. 5. The expression of NOx R and NOx A gene in cdc24RNAi and bem1RNAi strains was detected by using the method of cdc24RNAi and bem1RNAi. At the same time, the expression of Nrcdc24 gene in the Cdc42 and Rac A strains was significantly increased, suggesting that the Nrcdc24 gene might be the upstream gene of Rac A and Cdc42 gene, regulating the expression of the two, and then affecting the formation of the micro sclerotium; 6. Inoculation of the 3 instar larvae of the Spodoptera Spodoptera by drip inoculation, and found that cdc24RNAi and bem1 were compared with the control. The death rate of the RNAi strain to the larva of the Spodoptera litura decreased and LT50 prolonged. The average value of the wild type, e GFP, Nrcdc24, Nrbem1 and double interfering strain LT50 was 9.32,9.53,11.74,11.06 and 13.60 days respectively by analyzing the survival curve of the larva of the Spodoptera litura. Conclusion: the full-length sequence of the Nrcdc24 and Nrbem1 genes was cloned successfully, and the two gene was proved. Both the growth of mycelium, the growth of mycelium, the amount of sporulation and the virulence of the strain have a certain influence. Nrcdc24 and the Nrbem1 gene have mutual relationship with the NADPH complex. While in the process of micro sclerotium, Nrcdc24 can regulate the role of Rac A and the Cdc42 gene.
【學位授予單位】：重慶大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S476.12

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