绿僵菌Mid1基因功能研究

发布时间：2019-05-27 18:23

【摘要】：昆虫病原真菌是一类重要的生防微生物,与其他农药相比,具有选择性强、环境污染小、易于改造等优点。然而绿僵菌的致病力弱、产品储存期短以及较强的环境依赖性等缺点严重限制了微生物农药的发展,因此,虫生真菌致病机制的相关研究成为热点,有关致病基因的研究也为提高绿僵菌毒力和菌株改造奠定了基础。许多病原真菌的致病性或耐受性都是受到Ca~(2+)信号调控。Mid1,作为一种延伸激活的钙离子通道蛋白,是外界钙离子在一定条件下进入细胞内的必经之路,是引起钙离子变化从而影响真菌生理机能的重要因子。目前在昆虫病原真菌中研究较少。本文主要以蝗绿僵菌为材料,利用已知的Mid1基因序列构建敲除与回复载体分析该基因的功能。主要研究结果如下:Mid1基因的克隆与功能分析(1)Mid1基因信息学分析根据绿僵菌基因组序列设计引物,克隆得到Mid1基因,登录号为XP007808776.1,开放阅读框(ORF)全长1818bp,在线预测此蛋白编码606个氨基酸,等电点为6.27,蛋白质分子量为87.43KDa。生物信息学分析表明Mid1蛋白一级结构包含一个跨膜结构域和一个保守的典型的半胱氨酸富集区C1和C2,C端含有一个信号肽。系统发育树分析显示绿僵菌Mid1与金龟子绿僵菌Mid1亲缘关系较近(95%相似性)。(2)Mid1敲除菌株、回复菌株的获得为了研究绿僵菌Mid1基因的功能,利用同源重组的方法构建了敲除载体,并在此基础上构建了回复载体。利用农杆菌介导转化绿僵菌,经抗性筛选PCR验证筛及Southen杂交验证,得到正确的敲除与回复菌株。(3)Mid1基因表达模式为了分析Mid1在绿僵菌不同生长时期的表达量,分别提取了48h菌丝,3、6、9、12天孢子时期,附着胞以及虫菌体时期的RNA,RT-qPCR分析结果表明Mid1在附着胞时期表达量最高,也进一步分析Mid1对毒力的影响提供依据。(4)Mid1的缺失导致绿僵菌毒力降低以东亚飞蝗五龄虫为实验材料,对其进行体表点滴和体内注射实验。结果表明:体表点滴实验中敲除菌株与野生型和回复菌株相比毒力下降,LT50推迟1天左右,而注射实验中敲除与野生型和回复相比毒力无显著差异,我们推测Mid1的缺失影响了绿僵菌对寄主的体壁穿透过程。(5)敲除Mid1影响绿僵菌在蝗虫血淋巴中的生长为了研究Mid1基因对绿僵菌在蝗虫血淋巴中生长的影响,利用体表点滴和体内注射方法分析不同时间点血腔中虫菌体的生长情况。显微镜下观察发现点滴接种时,血淋巴中ΔMid1出现虫菌体时间晚于野生型与回复菌株,数量也显著低于野生型与回复。而体内注射实验中,ΔMid1缺失菌株和野生型之间无显著差异。(6)Mid1基因影响绿僵菌附着胞形成率对绿僵菌孢子在后翅上的萌发率和附着胞的形成率进行了分析。结果表明ΔMid1菌株的孢子萌发率与野生型和回复菌株相比,无显著差异;而ΔMid1菌株的附着胞形成率显著低于野生型和回复菌株。说明Mid1的缺失影响绿僵菌在蝗虫后翅上附着胞的形成,但不影响后翅上孢子的萌发。(7)Mid1的缺失影响穿透体壁相关酶基因的表达生测实验结果表明Mid1可能影响蝗绿僵菌穿透昆虫体壁的过程,因此通过qRT-PCR对穿透体壁过程中的相关基因枯草蛋白酶Pr1,Pr2以及几丁质酶CHI,CHII,酯酶等转录水平进行检测。分析发现,Pr1,CHI与酯酶的表达量均下调,结果表明Mid1通过调控相关穿透基因的表达量从而影响毒力。(8)ΔMid1对细胞破坏剂敏感但与绿僵菌抗逆性无关在加入了细胞壁破坏剂刚果红(CR)与荧光增白剂(CFW)的1/4SDAY固体培养基上,敲除菌株ΔMid1菌落明显小于WT与CP,说明Mid1影响菌株对CR和CFW的抗性。然而,紫外照射和湿热处理分析显示,Mid1的缺失不影响绿僵菌对紫外照射和湿热的抗逆能力。(9)ΔMid1菌株金属离子敏感性增强在加入了不同金属离子如Ca~(2+)、Fe~(2+)、Mg~(2+)、Mn~(2+)以及钙离子螯合剂EGTA等的1/4SDAY固体培养基上,ΔMid1长势均弱于WT和CP。说明ΔMid1菌株在1/4SDAY固体培养基上对不同金属离子具有敏感性。(10)Mid1调控绿僵菌胞内钙离子运输利用特异性的钙离子荧光染料Fluo-3AM对ΔMid1,WT和CP菌株进行染色。通过显微镜观察发现ΔMid1缺失菌株的荧光强度远远弱于WT和CP菌株,说明Mid1基因在蝗绿僵菌中调控胞内钙离子的运输。通过RT-qPCR对不同金属离子对Mid1基因转录水平的影响进行分析,发现在Ca~(2+)诱导下,Mid1上调表达,而在Fe~(2+)、Na~+、K~+、EGTA等金属离子的诱导下Mid1下调表达。表明在营养丰富条件下,Ca~(2+)对Mid1基因起上调表达的作用,也说明Mid1基因能够调控钙离子运输。综上所述,Mid1基因与绿僵菌侵染致病密切相关,参与金属离子,特别是钙离子流通。因此,明确该基因的功能为进一步深入理解病原真菌致病机制具有重要意义。
[Abstract]:The insect pathogenic fungi are a kind of important biological and anti-microbial. Compared with other pesticides, it has the advantages of high selectivity, little environmental pollution, easy to be modified and so on. However, the pathogenic force of the Metarhizium anisopliae, the short storage period of the product and the strong environmental dependence of the product severely restrict the development of the microbial pesticide, and therefore, the related research of the pathogenic mechanism of the entomogenous fungi has become the hot spot, The research of the disease-related gene has laid the foundation for improving the virulence of the Metarhizium anisopliae and the transformation of the strain. The pathogenicity or tolerance of many pathogenic fungi is regulated by Ca ~ (2 +) signal. Mid1, as an extended activated calcium ion channel protein, is a necessary pathway for external calcium ions to enter the cell under certain conditions, which is an important factor that causes the change of calcium ions to affect the physiological function of the fungus. There are currently less studies in insect pathogenic fungi. In this paper, the function of the gene was analyzed by using the known Mid1 gene sequence and using the known Mid1 gene sequence as the material. The main results are as follows: the cloning and functional analysis of the Mid1 gene (1) Mid1 gene informatics analysis is based on the genomic sequence of Metarhizium bassiana to design the primer and clone to obtain the Mid1 gene. The accession number is XP007808776.1, the full length of the open reading frame (ORF) is 1818bp, and the on-line prediction of the protein encodes 606 amino acids. The isoelectric point was 6.27 and the molecular weight of the protein was 87.43 KDa. Bioinformatics analysis shows that the first-order structure of the Mid1 protein comprises a transmembrane domain and a conserved, typical cysteine-rich region C1 and C2, and the C-terminal contains a signal peptide. The phylogenetic tree analysis showed that the relationship between the mid1 of Metarhizium anisopliae and the mid1 of Metarhizium anisopliae was closer (95% similarity). (2) Mid1 knockout strain and the recovery strain were obtained in order to study the function of the Mid1 gene of Metarhizium bassiana, and the knockout vector was constructed by homologous recombination. Using Agrobacterium-mediated transformation of Metarhizium anisopliae, the screening and Southern hybridization were verified by the resistance screening PCR, and the correct knock-out and response strains were obtained. (3) The expression pattern of Mid1 gene was used to analyze the expression of Mid1 in different growth stages of Metarhizium anisopliae. The results of RT-qPCR showed that the expression of Mid1 was the highest in the period of attachment. The effect of Mid1 on virulence was also analyzed. (4) The deletion of Mid1 resulted in the decrease of the virulence of Metarhizium anisopliae, which was used as the experimental material for the five-instar larvae of the migratory locust in East Asia, and the body surface and in vivo injection experiments were carried out. The results showed that the virulence of the knockout strain in the body surface drop experiment was lower than that of the wild type and the restoring strain, and the LT50 was delayed for about 1 day, and the virulence of the knockout in the injection experiment was not significantly different than that of the wild type and the response, and the deletion of the Mid1 in the injection experiment affected the body wall penetration of the host by the Metarhizium anisopliae. (5) In order to study the effect of Mid1 gene on the growth of Metarhizium anisopliae in the blood lymph of the locust, the growth of the cell in the blood cavity of different time points was analyzed by the method of body surface drip and in-vivo injection. Under the microscope, in the case of drip inoculation, the time of the occurrence of the mid1 in the blood lymph node was later than that of the wild type and the recovery strain, and the number was also significantly lower than that of the wild type and the recovery strain. In that in vivo injection experiment, there was no significant difference between the deletion and the wild-type of the yeast mid1. (6) The effect of Mid1 gene on the rate of germination of Metarhizium anisopliae and the rate of formation of the adherent cells of Metarhizium anisopliae were analyzed. The results showed that there was no significant difference between the spore germination rate and the wild type and the restoring strain of the first strain, and the formation rate of the strain of the first strain was significantly lower than that of the wild type and the restoring strain. Note that the deletion of Mid1 affects the formation of the attachment of Metarhizium anisopliae on the hind wings of the grasshoppers, but does not affect the germination of the spores on the hind wings. (7) The deletion of Mid1 affects the expression of the relevant enzyme gene in the body wall. The results show that Mid1 may affect the process of penetrating the body wall of the siana bassiana, so that the related gene subtilisin Pr1, Pr2 and the chitinase CHI and CHII in the body wall process are caused by the qRT-PCR. And the transcription level of the esterase and the like is detected. The results showed that the expression of Pr1, CHI and esterase was down-regulated, and the results showed that the expression of mid1, CHI, and esterase was affected by the regulation of the expression of the related penetrating gene. (8) In the 1/4 SDAY solid culture medium of the cell wall destruction agent (CR) and the fluorescent whitening agent (CFW), the colony of the knockout strain was significantly smaller than that of WT and CP, and the resistance of the strain to CR and CFW was described in the Mid1 strain. However, the analysis of ultraviolet irradiation and moist heat treatment shows that the deletion of Mid1 does not affect the resistance of Metarhizium anisopliae to ultraviolet irradiation and moist heat. (9) The sensitivity of the metal ion of the strain was enhanced in 1/4 SDAY solid culture medium with different metal ions such as Ca ~ (2 +), Fe ~ (2 +), Mg ~ (2 +), Mn ~ (2 +), and Ca ~ (2 +), and Ca ~ (2 +), Ca ~ (2 +), and Ca ~ (2 +). It is demonstrated that the BMid1 strain is sensitive to different metal ions on a 1/4 SDAY solid culture medium. (10) Mid1 was used to control the intracellular calcium ion transport of green and stiff cells, and the specific calcium ion fluorescent dye Fluo-3AM was used to dye the strains of the first, the WT and the CP strains. It was found that the fluorescence intensity of the missing strain was much weaker than that of WT and CP, and the expression of the mid1 gene in the control of the intracellular calcium ion in the Metarhizium anisopliae was described. The effect of different metal ions on the transcription level of the Mid1 gene was analyzed by RT-qPCR, and the expression of Mid1 was up-regulated under the induction of Ca ~ (2 +), and the expression of Mid1 was down-regulated under the induction of Fe ~ (2 +), Na ~ +, K ~ +, EGTA and other metal ions. The effect of Ca ~ (2 +) on the up-regulation of the Mid1 gene under the condition of rich nutrition also indicates that the Mid1 gene can regulate the transport of calcium ions. In conclusion, the Mid1 gene is closely related to the infection of Metarhizium anisopliae, and is involved in the circulation of metal ions, especially calcium ions. Therefore, it is of great significance to clarify the function of the gene to further understand the pathogenic mechanism of the pathogenic fungi.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S476.12

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