【摘要】：萊氏野村菌(Nomuraea rileyi)作為一種能夠侵染多種鱗翅目夜蛾科害蟲的昆蟲病原真菌,能在田間引發(fā)夜蛾科害蟲的流行病故而受到人們的高度重視。但是萊氏野村菌作為殺蟲的主要有效成分--分生孢子發(fā)酵生產(chǎn)所需的條件較為苛刻,從而限制了其規(guī)�；a(chǎn)及大規(guī)模應(yīng)用。重慶大學(xué)王中康的科研團隊借鑒植物病原真菌微菌核的誘導(dǎo)方法,成功誘導(dǎo)培養(yǎng)出能代替能傳統(tǒng)分生孢子的侵染體結(jié)構(gòu),即易于規(guī)�；a(chǎn)、耐高溫、耐儲存、抗逆性強等優(yōu)點的萊氏野村菌微菌核,并采用比較轉(zhuǎn)錄組的方法從分子生物學(xué)方面探索了萊氏野村菌微菌核的形成機理。研究發(fā)現(xiàn),在優(yōu)化誘導(dǎo)培養(yǎng)基(AM)時發(fā)現(xiàn)添加不同濃度Ca2+能顯著影響微菌核的產(chǎn)量。因此,本論文從萊氏野村菌比較轉(zhuǎn)錄組中選取與鈣調(diào)控相關(guān)的鈉鈣交換體(NCX)與鈣離子通道抑制子(CCI)基因研究鈣與萊氏野村菌產(chǎn)生微菌核的關(guān)系。主要研究結(jié)果如下:①通過在萊氏野村菌微菌核AM培養(yǎng)基中不添加Ca2+發(fā)現(xiàn):不添加Ca2+的AM培養(yǎng)基中菌絲團大小不均一,菌絲團數(shù)量減少了34.4%,且菌絲纏繞疏松,邊緣松散并有菌絲飄散,紫褐色色素積累明顯降低;但統(tǒng)計生物量發(fā)現(xiàn)Ca2+添加與否對發(fā)酵生物量沒有明顯影響;②根據(jù)比較轉(zhuǎn)錄組NCX和CCI的EST序列,利用普通PCR方法獲得NCX和CCI的c DNA全長序列和基因組序列,其開放閱讀框(ORF)分別為3006bp和405bp,各自編碼1001和134個氨基酸。利用生物信息學(xué)軟件和網(wǎng)站分析NCX和CCI發(fā)現(xiàn):NCX編碼蛋白的分子量為109.563k Da,主要由α螺旋和隨機卷曲組成,理論等電點為5.98,無信號肽,但有跨膜結(jié)構(gòu)域;CCI編碼蛋白的分子量為13.63k Da,主要由α螺旋、β折疊和隨機卷曲組成,理論等電點為7.5,無跨膜結(jié)構(gòu)域,但有信號肽,其信號肽為1-16個氨基酸。利用Clustal X和MEGA4.0同源比對和系統(tǒng)進化分析發(fā)現(xiàn)萊氏野村菌NCX和CCI蛋白與綠僵菌的親緣關(guān)系最近。③實時熒光定量PCR(q PCR)分析NCX和CCI的表達模式發(fā)現(xiàn):NCX和CCI在微菌核形成的各個時期均有表達,其中NCX的表達量在微菌核形成初期顯著性的增高(48-72h),在72h時達最大,然后在微菌核成熟過程(72-120h)中其表達量又逐漸降低;CCI的表達量在微菌核發(fā)育期(48-120h)表達量逐漸增高,在120h時達到最大。這說明NCX與CCI均參與了微菌核的形成,但二者參與的調(diào)控有差異,NCX主要在微菌核形成初期起作用,參與微菌核形成的啟動及發(fā)育過程,而CCI主要在微菌核成熟末期高表達,可能參與微菌核成熟過程。④利用RNA干擾技術(shù)(RNAi)研究了NCX和CCI功能:通過篩選發(fā)現(xiàn)NCX和CCI的最適干擾濃度均為400n M,干擾效率分別為55.5%、93.6%。對基因的表達檢測發(fā)現(xiàn),干擾NCX基因后,CCI的表達量上升了2.1倍;干擾CCI基因后NCX的表達量上升了5.3倍;在固體培養(yǎng)基上,NCX和CCI干擾菌株相對于野生型(WT)和陰性對照(EGFP)的菌落形態(tài)變小,產(chǎn)孢時間延遲,且產(chǎn)孢量分別減少為60.6%和88.2%。在微菌核液體誘導(dǎo)培養(yǎng)基AM中,NCX和CCI干擾菌株微菌核的形態(tài)和大小不均勻,AM培養(yǎng)液的黏稠度明顯降低,無紫褐色色素積累。統(tǒng)計分析微菌核的產(chǎn)量發(fā)現(xiàn)干擾菌株所形成微菌核數(shù)量急劇減少,與對照菌株相比,分別減少了81.8和90.4%。同時統(tǒng)計培養(yǎng)液中的生物量,發(fā)現(xiàn)NCX和CCI干擾菌株的生物量分別降低25.8%和30.5%。⑤采用點滴接種法接種發(fā)酵產(chǎn)生的微菌核到三齡斜紋夜蛾幼蟲表皮上,測定NCX和CCI對各菌株的毒力的影響:結(jié)果顯示,干擾菌株對斜紋夜蛾幼蟲的致死率比對照菌株分別下降4.45%、6.1%,LT50都延長了2d以上。結(jié)論:萊氏野村菌NCX和CCI基因都參與了微菌核形成,其中NCX主要參與微菌核形成的啟動及微菌核發(fā)育過程,CCI基因主要參與微菌核成熟過程;NCX和CCI基因不僅嚴(yán)重影響微菌核的形成,而且對萊氏野村菌分生孢子產(chǎn)生及菌株毒力也有明顯影響,但其影響機理還需要進一步研究。
[Abstract]:Nomuraya rileyi, a kind of insect-borne fungus capable of infecting various lepidopteran insect pests, is highly valued by people in the field. But the conditions required for the production of the conidia are more severe as the main active components of the insect-killing main active components of the Lehlet, thereby limiting the large-scale production and the large-scale application. the scientific research team of the wang zhongkang of the university of chongqing, using the induction method of the microsclerotium of the plant pathogenic fungi, successfully induced the culture of the bacterial micro-sclerotium of the lehlet, which can replace the infection body structure which can replace the traditional conidia, A comparative transcriptome was used to study the mechanism of the formation of the micro-sclerotium in the field of Lehlet's field from the aspect of molecular biology. It was found that when the induction medium (AM) was optimized, it was found that the addition of different concentration of Ca2 + could significantly affect the yield of the microsclerotia. In this paper, the relationship between calcium and calcium channel suppressor (CCI) gene was selected to study the relationship between calcium and calcium channel suppressor (CCI) gene. The main results of the study were as follows: The mycelium mass in the AM medium without Ca2 + was not increased, the number of hyphae was reduced by 34.4%, and the hyphae were loose, the edges were loose and the hyphae were dispersed. the accumulation of the purple brown pigment is obviously reduced; but the statistical biomass is found that the addition of Ca2 + has no obvious effect on the fermentation biomass, and the full-length sequence and the genome sequence of the c DNA of the ncx and the cci are obtained by using the common PCR method according to the EST sequence of the comparative transcriptome NCX and the CCI, The open reading frame (ORF) is 3006 bp and 405 bp, respectively, and each encodes 1001 and 134 amino acids. NCX and CCI found that the molecular weight of NCX-encoded protein was 109.563k Da, which was mainly composed of microspiral and random crimp, with a theoretical isoelectric point of 5.98, no signal peptide, but a transmembrane domain; the molecular weight of the CCI-encoded protein was 13.63 k Da, which was mainly composed of a spiral-type spiral, The theoretical isoelectric point was 7.5, without a transmembrane domain, but with a signal peptide with a signal peptide of 1 to 16 amino acids. The relationship between NCX and CCI protein and Metarhizium anisopliae was found by using the homologous comparison and phylogenetic analysis of Clustal X and MEGA4.0. The expression patterns of NCX and CCI were analyzed by real-time fluorescence quantitative PCR (q-PCR). The expression of NCX and CCI in the microsclerotia was significantly increased (48-72 h) at the initial stage of the formation of microsclerotia, and reached the maximum at 72 h. The expression of CCI was gradually decreased in the process of microsclerotium maturation (72-120 h), and the expression of CCI was gradually increased at the development stage of microsclerotia (48-120 h) and reached the maximum at 120 h. This indicates that both the NCX and CCI are involved in the formation of the microsclerotium, but the control of the two is different, and the NCX plays an important role in the initiation and development of the formation of the microsclerotia, and the CCI is mainly expressed at the late stage of the microsclerotia, and may be involved in the process of microsclerotium maturation. The functions of NCX and CCI were studied by RNA interference (RNAi): the optimal interference concentration of NCX and CCI was 400n M, and the interference efficiency was 55.5% and 93.6%, respectively. The expression of the gene found that the expression of CCI increased by 2.1 times after interfering with the NCX gene, and the expression of the NCX after interfering with the CCI gene increased by 5.3 times; on the solid medium, the colony morphology of the NCX and CCI-interfering strains with respect to the wild-type (WT) and the negative control (EGFP) was reduced, The time delay of sporulation was 60.6% and 88.2%, respectively. In the micro-sclerotium liquid-induced culture medium AM, the morphology and the size of the micro-sclerotium of the NCX and CCI-interfering strains were not uniform, and the viscosity of the AM culture medium was significantly reduced, and no purple-brown pigment was accumulated. The results of the statistical analysis of the microsclerotia showed that the number of microsclerotia formed by the interfering strain decreased dramatically, and the number of microsclerotia was 81.8 and 90.4%, respectively, as compared to the control strain. At the same time, the biomass in the culture solution was counted, and the biomass of the NCX and CCI interference strains was found to be 25.8% and 30.5%, respectively. The effects of NCX and CCI on the virulence of each strain were determined by the inoculation of the microsclerotium produced by the inoculation with the drip-inoculation method to the skin of the three-instar-old Spodoptera litura. The results showed that the strain of the interfering strain on the larvae of the Spodoptera litura was reduced by 4.45% and 6.1%, respectively. LT50 has been extended by more than 2d. Conclusion: The NCX and CCI genes of the NCX and CCI genes are involved in the formation of microsclerotia. The NCX is mainly involved in the initiation of the formation of microsclerotia and the process of microsclerotium development. The CCI gene is mainly involved in the process of microsclerotia. The NCX and CCI genes not only affect the formation of the microsclerotia, It also has a significant effect on the production of the conidia and the virulence of the strains of the strains of the strains of the field, but the mechanism of its influence also needs to be further studied.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S476.12;Q78

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