莱氏野村菌钠钙交换体和钙离子通道抑制子基因的克隆与功能分析

发布时间：2019-07-04 17:00

【摘要】：莱氏野村菌(Nomuraea rileyi)作为一种能够侵染多种鳞翅目夜蛾科害虫的昆虫病原真菌,能在田间引发夜蛾科害虫的流行病故而受到人们的高度重视。但是莱氏野村菌作为杀虫的主要有效成分--分生孢子发酵生产所需的条件较为苛刻,从而限制了其规模化生产及大规模应用。重庆大学王中康的科研团队借鉴植物病原真菌微菌核的诱导方法,成功诱导培养出能代替能传统分生孢子的侵染体结构,即易于规模化生产、耐高温、耐储存、抗逆性强等优点的莱氏野村菌微菌核,并采用比较转录组的方法从分子生物学方面探索了莱氏野村菌微菌核的形成机理。研究发现,在优化诱导培养基(AM)时发现添加不同浓度Ca2+能显著影响微菌核的产量。因此,本论文从莱氏野村菌比较转录组中选取与钙调控相关的钠钙交换体(NCX)与钙离子通道抑制子(CCI)基因研究钙与莱氏野村菌产生微菌核的关系。主要研究结果如下:①通过在莱氏野村菌微菌核AM培养基中不添加Ca2+发现:不添加Ca2+的AM培养基中菌丝团大小不均一,菌丝团数量减少了34.4%,且菌丝缠绕疏松,边缘松散并有菌丝飘散,紫褐色色素积累明显降低;但统计生物量发现Ca2+添加与否对发酵生物量没有明显影响;②根据比较转录组NCX和CCI的EST序列,利用普通PCR方法获得NCX和CCI的c DNA全长序列和基因组序列,其开放阅读框(ORF)分别为3006bp和405bp,各自编码1001和134个氨基酸。利用生物信息学软件和网站分析NCX和CCI发现:NCX编码蛋白的分子量为109.563k Da,主要由α螺旋和随机卷曲组成,理论等电点为5.98,无信号肽,但有跨膜结构域;CCI编码蛋白的分子量为13.63k Da,主要由α螺旋、β折叠和随机卷曲组成,理论等电点为7.5,无跨膜结构域,但有信号肽,其信号肽为1-16个氨基酸。利用Clustal X和MEGA4.0同源比对和系统进化分析发现莱氏野村菌NCX和CCI蛋白与绿僵菌的亲缘关系最近。③实时荧光定量PCR(q PCR)分析NCX和CCI的表达模式发现:NCX和CCI在微菌核形成的各个时期均有表达,其中NCX的表达量在微菌核形成初期显著性的增高(48-72h),在72h时达最大,然后在微菌核成熟过程(72-120h)中其表达量又逐渐降低;CCI的表达量在微菌核发育期(48-120h)表达量逐渐增高,在120h时达到最大。这说明NCX与CCI均参与了微菌核的形成,但二者参与的调控有差异,NCX主要在微菌核形成初期起作用,参与微菌核形成的启动及发育过程,而CCI主要在微菌核成熟末期高表达,可能参与微菌核成熟过程。④利用RNA干扰技术(RNAi)研究了NCX和CCI功能:通过筛选发现NCX和CCI的最适干扰浓度均为400n M,干扰效率分别为55.5%、93.6%。对基因的表达检测发现,干扰NCX基因后,CCI的表达量上升了2.1倍;干扰CCI基因后NCX的表达量上升了5.3倍;在固体培养基上,NCX和CCI干扰菌株相对于野生型(WT)和阴性对照(EGFP)的菌落形态变小,产孢时间延迟,且产孢量分别减少为60.6%和88.2%。在微菌核液体诱导培养基AM中,NCX和CCI干扰菌株微菌核的形态和大小不均匀,AM培养液的黏稠度明显降低,无紫褐色色素积累。统计分析微菌核的产量发现干扰菌株所形成微菌核数量急剧减少,与对照菌株相比,分别减少了81.8和90.4%。同时统计培养液中的生物量,发现NCX和CCI干扰菌株的生物量分别降低25.8%和30.5%。⑤采用点滴接种法接种发酵产生的微菌核到三龄斜纹夜蛾幼虫表皮上,测定NCX和CCI对各菌株的毒力的影响:结果显示,干扰菌株对斜纹夜蛾幼虫的致死率比对照菌株分别下降4.45%、6.1%,LT50都延长了2d以上。结论:莱氏野村菌NCX和CCI基因都参与了微菌核形成,其中NCX主要参与微菌核形成的启动及微菌核发育过程,CCI基因主要参与微菌核成熟过程;NCX和CCI基因不仅严重影响微菌核的形成,而且对莱氏野村菌分生孢子产生及菌株毒力也有明显影响,但其影响机理还需要进一步研究。
[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.
【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;Q78

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