【摘要】：昆蟲病原真菌是一類重要的生防微生物,與其他農(nóng)藥相比,具有選擇性強、環(huán)境污染小、易于改造等優(yōu)點。然而綠僵菌的致病力弱、產(chǎn)品儲存期短以及較強的環(huán)境依賴性等缺點嚴重限制了微生物農(nóng)藥的發(fā)展,因此,蟲生真菌致病機制的相關研究成為熱點,有關致病基因的研究也為提高綠僵菌毒力和菌株改造奠定了基礎。許多病原真菌的致病性或耐受性都是受到Ca~(2+)信號調控。Mid1,作為一種延伸激活的鈣離子通道蛋白,是外界鈣離子在一定條件下進入細胞內的必經(jīng)之路,是引起鈣離子變化從而影響真菌生理機能的重要因子。目前在昆蟲病原真菌中研究較少。本文主要以蝗綠僵菌為材料,利用已知的Mid1基因序列構建敲除與回復載體分析該基因的功能。主要研究結果如下:Mid1基因的克隆與功能分析(1)Mid1基因信息學分析根據(jù)綠僵菌基因組序列設計引物,克隆得到Mid1基因,登錄號為XP007808776.1,開放閱讀框(ORF)全長1818bp,在線預測此蛋白編碼606個氨基酸,等電點為6.27,蛋白質分子量為87.43KDa。生物信息學分析表明Mid1蛋白一級結構包含一個跨膜結構域和一個保守的典型的半胱氨酸富集區(qū)C1和C2,C端含有一個信號肽。系統(tǒng)發(fā)育樹分析顯示綠僵菌Mid1與金龜子綠僵菌Mid1親緣關系較近(95%相似性)。(2)Mid1敲除菌株、回復菌株的獲得為了研究綠僵菌Mid1基因的功能,利用同源重組的方法構建了敲除載體,并在此基礎上構建了回復載體。利用農(nóng)桿菌介導轉化綠僵菌,經(jīng)抗性篩選PCR驗證篩及Southen雜交驗證,得到正確的敲除與回復菌株。(3)Mid1基因表達模式為了分析Mid1在綠僵菌不同生長時期的表達量,分別提取了48h菌絲,3、6、9、12天孢子時期,附著胞以及蟲菌體時期的RNA,RT-qPCR分析結果表明Mid1在附著胞時期表達量最高,也進一步分析Mid1對毒力的影響提供依據(jù)。(4)Mid1的缺失導致綠僵菌毒力降低以東亞飛蝗五齡蟲為實驗材料,對其進行體表點滴和體內注射實驗。結果表明:體表點滴實驗中敲除菌株與野生型和回復菌株相比毒力下降,LT50推遲1天左右,而注射實驗中敲除與野生型和回復相比毒力無顯著差異,我們推測Mid1的缺失影響了綠僵菌對寄主的體壁穿透過程。(5)敲除Mid1影響綠僵菌在蝗蟲血淋巴中的生長為了研究Mid1基因對綠僵菌在蝗蟲血淋巴中生長的影響,利用體表點滴和體內注射方法分析不同時間點血腔中蟲菌體的生長情況。顯微鏡下觀察發(fā)現(xiàn)點滴接種時,血淋巴中ΔMid1出現(xiàn)蟲菌體時間晚于野生型與回復菌株,數(shù)量也顯著低于野生型與回復。而體內注射實驗中,ΔMid1缺失菌株和野生型之間無顯著差異。(6)Mid1基因影響綠僵菌附著胞形成率對綠僵菌孢子在后翅上的萌發(fā)率和附著胞的形成率進行了分析。結果表明ΔMid1菌株的孢子萌發(fā)率與野生型和回復菌株相比,無顯著差異;而ΔMid1菌株的附著胞形成率顯著低于野生型和回復菌株。說明Mid1的缺失影響綠僵菌在蝗蟲后翅上附著胞的形成,但不影響后翅上孢子的萌發(fā)。(7)Mid1的缺失影響穿透體壁相關酶基因的表達生測實驗結果表明Mid1可能影響蝗綠僵菌穿透昆蟲體壁的過程,因此通過qRT-PCR對穿透體壁過程中的相關基因枯草蛋白酶Pr1,Pr2以及幾丁質酶CHI,CHII,酯酶等轉錄水平進行檢測。分析發(fā)現(xiàn),Pr1,CHI與酯酶的表達量均下調,結果表明Mid1通過調控相關穿透基因的表達量從而影響毒力。(8)ΔMid1對細胞破壞劑敏感但與綠僵菌抗逆性無關在加入了細胞壁破壞劑剛果紅(CR)與熒光增白劑(CFW)的1/4SDAY固體培養(yǎng)基上,敲除菌株ΔMid1菌落明顯小于WT與CP,說明Mid1影響菌株對CR和CFW的抗性。然而,紫外照射和濕熱處理分析顯示,Mid1的缺失不影響綠僵菌對紫外照射和濕熱的抗逆能力。(9)ΔMid1菌株金屬離子敏感性增強在加入了不同金屬離子如Ca~(2+)、Fe~(2+)、Mg~(2+)、Mn~(2+)以及鈣離子螯合劑EGTA等的1/4SDAY固體培養(yǎng)基上,ΔMid1長勢均弱于WT和CP。說明ΔMid1菌株在1/4SDAY固體培養(yǎng)基上對不同金屬離子具有敏感性。(10)Mid1調控綠僵菌胞內鈣離子運輸利用特異性的鈣離子熒光染料Fluo-3AM對ΔMid1,WT和CP菌株進行染色。通過顯微鏡觀察發(fā)現(xiàn)ΔMid1缺失菌株的熒光強度遠遠弱于WT和CP菌株,說明Mid1基因在蝗綠僵菌中調控胞內鈣離子的運輸。通過RT-qPCR對不同金屬離子對Mid1基因轉錄水平的影響進行分析,發(fā)現(xiàn)在Ca~(2+)誘導下,Mid1上調表達,而在Fe~(2+)、Na~+、K~+、EGTA等金屬離子的誘導下Mid1下調表達。表明在營養(yǎng)豐富條件下,Ca~(2+)對Mid1基因起上調表達的作用,也說明Mid1基因能夠調控鈣離子運輸。綜上所述,Mid1基因與綠僵菌侵染致病密切相關,參與金屬離子,特別是鈣離子流通。因此,明確該基因的功能為進一步深入理解病原真菌致病機制具有重要意義。
[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.
【學位授予單位】：重慶大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S476.12

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