双型真菌莱氏野村菌Nrcdc24与Nrbem1基因的克隆和功能研究

发布时间：2018-07-09 20:53

  本文选题：莱氏野村菌 + 微菌核　； 参考：《重庆大学》2015年硕士论文

【摘要】：莱氏野村菌是一种在自然界广泛存在的能有效控制昆虫种群的重要病原真菌。对鳞翅目夜蛾科害虫有较强的感染力和致病力,莱氏野村菌杀虫制剂的有效成分为分生孢子。在莱氏野村菌发酵生产中,它对碳源的要求较高、生长周期长、产孢量不高,且在培养过程中需要持续光照,限制了对该菌的推广和应用。本实验室通过液体发酵成功诱导出了抗逆性强、毒力与传统的分生孢子没有明显差异且耐储存的微菌核,成为了一个新的具有高效杀虫活性的杀虫制剂的有效成分。显微观察发现,诱导培养基AM诱导产生的微菌核是由菌丝体特化形成的休眠体构造;而用比较转录组测序分析的方法证实,微菌核的形成是一种氧胁迫的过程,伴随色素的累积、活性氧(ROS)的产生及大量功能基因的表达变化。对这一调控机制的深入研究可以让我们更好的了解微菌核形成的分子特性,也可为后期对微菌核发酵生产及制剂的开发等提供理论参考依据。本论文根据莱氏野村菌比较转录组库中的EST序列克隆得到了在微菌核形成期高表达的Nrcdc24与Nrbem1基因的全长序列,经生物信息学分析、基因在微菌核形成过程中的表达测定以及外源RNA干扰的方法证明了两个基因的功能。主要研究结果如下:①采用SMART RACE RT-PCR技术获得Nrcdc24与Nrbem1的c DNA和基因组全长序列,其ORF分别为3021 bp和1689 bp,各自编码1006和562个氨基酸;②对目的基因编码蛋白的生物信息学分析显示,Nrcdc24编码的蛋白含有与Rho家族结合的功能位点Rho GTP;Nrbem1含有两个SH3(Src Homology 3)结合位点以及一个磷酸肌醇结合位点(phox homology,PX)。此外,Nrcdc24与Nrbem1蛋白都有PB1(phox and Bem1)结合位点;通过对近缘物种的系统进化树分析发现莱氏野村菌的Nrcdc24与Nrbem1蛋白与绿僵菌的亲缘关系最近;③显微观察莱氏野村菌液体培养形成微菌核过程发现,在培养至84 h左右时,培养基中极性生长的菌丝体开始局部缩聚,微菌核开始大量形成;采用实时荧光定量PCR的方法对Nrcdc24与Nrbem1基因在微菌核不同发育时期表达模式进行分析发现,Nrcdc24与Nrbem1均在微菌核大量形成初期(84 h左右)表达量高;④为了进一步研究Nrcdc24与Nrbem1基因在微菌核形成过程中所起到的作用,设计同源干扰片段,以e GFP干扰片段的干扰菌株作为阴性对照,采用外源RNAi的方法进行研究发现:在SMAY固体培养基上,Nrcdc24与Nrbem1干扰菌株(命名为cdc24RNAi和bem1RNAi)的酵母态阶段生长菌落比野生型(WT)和阴性对照(e GFP)的菌落生长相对缓慢,菌体由酵母态向菌丝态转化(两型转变)延迟0.5-1d,产孢时间也随之延迟;经显微计数发现,cdc24RNAi和bem1RNAi菌株的产孢量较对照显著降低,产孢量分别降低95%和56%。而双干扰菌株酵母态时期的菌落更小,产孢量较出发菌株降低了99.04%。在AM液体培养基中,相对于对照组,各干扰菌株的色素积累明显减少,同步培养5-7d的发酵液粘稠度也明显降低。显微观察发现,各干扰菌株的菌丝体均变粗且分枝增多,bem1RNAi菌株及双干扰菌株的菌体上产生许多不脱落的芽质状孢子,而cdc24RNAi菌株的菌体形态同对照相比则无明显差异。此外,在同步培养条件下,对照组菌株在4d左右时便开始有微菌核产生,而干扰菌株中无微菌核产生;继续培养观察发现,各干扰菌株微菌核形成的时间会延迟1-2d。干重法测微菌核的生物量显示,cdc24RNAi和bem1RNAi菌株的生物量降低20-32%,双干扰菌株的生物量降低约为40%。另外,各处理菌株培养至7d左右(常规诱导培养微菌核收获)时,相对于对照组,cdc24RNAi和bem1RNAi及双干扰菌株的微菌核形成的数量分别下降了93.2%、90.7%和97.3%;⑤采用q PCR的方法检测在cdc24RNAi与bem1RNAi菌株中与活性氧的nox R与nox A基因的表达量发现,同干扰rac A与cdc42基因一样nox R与nox A表达量均明显下降;此外,在沉默Nrcdc24基因以后,rac A与cdc42基因的表达量也降低,同时,在干扰的cdc42与rac A菌株中Nrcdc24基因的表达量显著升高,暗示Nrcdc24基因可能为rac A与cdc42基因的上游基因,调控着二者的表达,进而影响微菌核的形成;⑥采用点滴接种法接种斜纹夜蛾3龄幼虫,发现与对照相比,cdc24RNAi与bem1RNAi菌株对斜纹夜蛾幼虫的致死率下降,LT50延长。通过对斜纹夜蛾幼虫的存活曲线分析,野生型,e GFP,Nrcdc24,Nrbem1和双干扰菌株LT50的平均值分别为9.32,9.53,11.74,11.06和13.60天。结论:成功克隆得了Nrcdc24与Nrbem1基因的全长序列,证明了两基因均对微菌核的发育形成、菌丝的生长、产孢量及菌株毒力等有一定的影响;Nrcdc24与Nrbem1基因与NADPH复合体有互作关系。而在微菌核过程中Nrcdc24可调控rac A与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.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.12

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