两型性莱氏野村菌交替氧化酶（AOX）基因的克隆及功能研究

发布时间：2018-04-30 12:54

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】：虫生真菌因在防治农林害虫时具有对环境无污染、无残留和使害虫不易产生抗药性等优点,是一种环境友好型真菌。随着人们对环保意识的增强,利用虫生真菌防治农林害虫受到人们的广泛关注及重视。莱氏野村菌在田间能够自然侵染多种夜蛾科害虫并引起域内流行病,是一种重要的昆虫病原真菌。莱氏野村菌作为杀虫剂的有效成分是分生孢子,但是由于其产孢条件比较苛刻如需要苛刻的碳源(麦芽糖)和持续光照,其工业化生产方面一直受到极大限制,所以寻找该菌的其他繁殖体来代替分生孢子行使杀虫作用具有十分重要的意义。液体发酵产生的芽生孢子虽然有一定侵染力,但因其细胞壁比较薄、不耐储存以及毒力低等特点也不能用于生产实践。微菌核是真菌菌丝聚集而形成特异休眠体结构,它具有耐储存、抗逆性强、在适宜的环境下能萌发产生菌丝和孢子。重庆大学生命科学学院微生物实验室成功的诱导出莱氏野村菌的微菌核,并对其的抗逆性和毒力等进行了测试,实验结果证实该微菌核可以作为有效的活性成分来代替分生孢子用于害虫的生物防治制剂。其后,采用比较转录组技术从分子层面来探讨微菌核形成机理,发现在微菌核形成与氧胁迫密切相关,并且在微菌核形成的过程中大量的还原性酶或合成还原性物质的基因上调表达。依据此,本课题从比较转录组上调表达的c DNA文库中挑取Nraox基因的EST序列,将其克隆并采用RNA干扰技术来研究该基因在微菌核发育过程中的作用。主要研究结果如下:①莱氏野村菌Nraox基因的克隆与序列分析根据莱氏野村菌转录组库中Nraox基因的EST序列,克隆得到该基因的c DNA全长序列(Gen Bank登录号:No.KM978957)。序列分析显示其开放阅读框为1068bp,编码355个氨基酸;生物信息学分析表明该蛋白的理论分子量为40.517KD,理论等电点为9.53;该蛋白具有亲水性,无信号肽,亚细胞定位分析该蛋白定位于线粒体内。Nraox的基因组全长为1512 bp,该基因含有3外显子,2内含子。利用DNAMAN和MEGA软件进行同源比对和系统进化分析发现,莱氏野村菌的AOX蛋白与绿僵菌的同源性最高,并与麦角菌科的真菌聚为一组,同源性都达到88.17%。②莱氏野村菌Nraox的表达分析收集莱氏野村菌微菌核发育不同时期的样品,采用荧光定量q PCR的方法对Nraox基因的表达模式进行分析,结果表明该基因在微菌核发育的不同时期均有表达,并在微菌核形成初期表达量最高,然后降低;采用q PCR技术检测Nraox在氧胁迫诱导剂处理后的基因表达,研究结果表明,氧化剂H2O2和甲萘醌都能引起该基因的高度上调表达,H2O2处理后Nraox表达量是对照组的7倍,甲萘醌处理后是对照组的20倍。③抑制剂处理对交替氧化酶功能的验证采用交替氧化酶的特殊抑制剂SHAM处理后,检测细胞内过氧化氢含量变化,结果显示,与对照组相比,抑制剂SHAM处理后前10min中内细胞内H2O2较高,处理15min后没有明显变化;但是,显微观察菌丝形态发现与对照组相比,处理组菌丝表现为弯曲和很多芽状结构的不正常生长状态;而诱导培养基中的微菌核表现为微菌核的形成延迟、微菌核颗粒直径较大以及微菌核结构松散、表面比较蓬松。由此可以确定,抑制剂处理后减缓了细胞内过氧化氢的清除速度,并对细胞产生了一定的氧毒害。④RNA干扰技术验证Nraox基因功能利用RT-q PCR技术对不同浓度si RNA的干扰效率进行检测,结果显示800 n M si RNA能够有效下调Nraox基因的表达,并且该基因表达量下调了约76.1%。与对照组相比,干扰菌株在含有胁迫诱导剂的固体SMAY培养基上生长时,菌落生长缓慢,菌落变小,产孢量降低。而在微菌核诱导培养基AM中生长时,Nraox基因干扰后的微菌核表型发生了明显变化,主要表现为:与野生菌相比,微菌核的形成推迟,微菌核颗粒的直径变大,质地较为疏松;微菌核的生物量和产量显著性降低;显微观察发现相对于对照组,干扰后的莱氏野村菌菌丝显示畸形生长,表现为菌丝上生出很多芽状结构;这些结果表明,Nraox参与了莱氏野村菌微菌核的形成调控。⑤干扰菌株对靶标害虫的致病性鉴定采用点滴法测定干扰菌株微菌核对三龄斜纹夜蛾幼虫的侵染和致病能力,结果发现,干扰菌株相对于野生型菌株对斜纹夜蛾幼虫的致死率下降,半致死时间LT50显著增长,较野生菌株LT50增加26.2%。结论:首次克隆得到莱氏野村菌的交替氧化酶基因,并命名为Nraox。研究了该基因在莱氏野村菌菌丝生长、微菌核的形成、产孢、毒力和氧胁迫等方面的作用,结果表明Nraox通过调节细胞内的氧化还原平衡和菌丝生长来影响莱氏野村菌微菌核的形成、影响到莱氏野村菌产孢,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.

【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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