含PxIxIT模序烟曲霉KpsF基因作用初探

发布时间：2018-05-15 13:15

本文选题：烟曲霉 + KpsF基因　；参考：《山西医科大学》2017年硕士论文

【摘要】：背景:烟曲霉是一种常见的腐生真菌,侵袭性曲霉病(Invasive aspergillosis,IA)约90%由烟曲霉引起,其病死率呈上升趋势,其治疗成为当前临床常见的疑难问题。钙调磷酸酶(Calcineurin,Ca N)在病原真菌的形态发生和毒性方面等的重要作用使其成为一个有吸引力的抗真菌靶点。在烟曲霉研究中,Ca N定位于菌丝的尖端和分隔处,对烟曲霉致病性有显著的影响。研究表明Ca N发挥作用同Px Ix IT模序密切相关,钙调磷酸酶底物或靶蛋白中如Crz1、SIm1、SIm2、Hph1、Cbp A中都含有类似Px Ix IT模序,而在烟曲霉Kps F基因序列中也有类似模序存在。虽然钙调磷酸酶抑制剂FK506和环孢素A在现代移植医学应用非常成功;但是与钙调磷酸酶相关的系统毒副作用使其临床抗真菌应用受到极大限制。因此,研究烟曲霉Kps F基因功能,以期发现新的内源性钙调磷酸酶抑制剂,为侵袭性曲霉病的治疗提供新的思路。目的:1.构建烟曲霉Kps F基因缺陷株和Kps F基因过表达株,初步了解Kps F基因对烟曲霉径向生长和形态学的影响。2.通过体外抗真菌药物实验初步明确Kps F基因对常用抗真菌药物敏感性影响。3.初步明确Kps F基因与钙调磷酸酶和钙离子体内平衡的相互关系。4.通过构建绿色荧光定位株初步探究Kps F基因在菌丝生长中的定位情况。方法:1.使用原生质体法构建烟曲霉Kps F基因缺陷株,并用Southern blot验证。2.使用p UCGH质粒作为载体,以潮霉素作为筛选标记,并通过原生质体法构建Kps F基因过表达株。3.将对照菌株Ku80、△Kps F和Kps F过表达菌株这三种菌株配成不同浓度菌悬液,接种一定浓度于固体培养基上,分别于24 h,48 h,72 h,96 h,120 h记录菌落直径,观察菌落生长状况,并作统计分析;使用小培养的方法对菌株进行形态学研究,培养48 h,滴加乳酸酚棉兰染液进行观察。4.使用斑点法探究对照菌株Ku80、△Kps F和Kps F过表达菌株这三种菌株抗真菌药物的敏感性,在加有抗真菌药物的基础培养基上点种菌悬液,分别于24 h,48 h,72 h,96 h,120 h记录菌落直径,观察菌落生长状况,并统计分析。5.RT-PCR检测不同浓度Ca2+液体培养基条件下三种不同菌株的Cna A和Vcx A基因m RNA表达水平,并作统计分析。6.使用激光共聚焦显微镜观察定位株Kps F-EGFP在菌丝生长中的定位情况。结果:1.成功构建烟曲霉?Kps F,Kps F基因过表达菌株,Kps F基因绿色荧光定位株(实验室已经构建并保存)。2.烟曲霉?Kps F和Ku80径向生长结果未见明显差异(P0.05),过表达株径向生长结果相对于对照菌株Ku80下降约45%。3.烟曲霉?Kps F和对照菌株Ku80形态差别不大,Kps F过表达株菌丝分枝较多,排列不整齐,其隔膜相对较少;分生孢子头相对短小,顶囊发育不良,顶囊上的单层小梗不明显。4.当卡泊芬净药物浓度为1.0~5.0 ug/ml时,?Kps F出现较小的矛盾生长变化,Ku80和Kps F过表达菌株未见明显变化。?Kps F,Ku80和Kps F过表达菌株对伊曲康唑、伏立康唑、两性霉素B和FK506四种抗真菌药体外药物敏感性,经过多次重复测量方差分析统计未见明显差异(P0.05)。5.在10 m M Ca Cl2浓度下,?Kps F的Cna A基因表达量显著高于对照菌株Ku80和Kps F过表达株,?Kps F的Cna A表达量是Ku80的29.76倍(P0.01),Kps F过表达株是对照菌株Ku80的1.56倍(P0.05)。而在100 m M浓度下,三种菌株Cna A表达量差别不明显。普通GMM培养基,?Kps F的Cna A表达量是Ku80的2.81倍,Kps F过表达株和Ku80无差别。6.在10 m M Ca Cl2浓度下,Kps F过表达株Vcx A基因表达量明显高于Ku80和?Kps F,Kps F过表达株是Ku80的2.65倍,差异有统计学意义(P0.01)。?Kps F和Ku80相比无明显变化。而在普通GMM培养基和100 m M条件下三种菌株Vcx A基因表达差异无统计学意义。7.烟曲霉Kps F-EGFP的绿色荧光均匀分布在菌丝细胞膜处。结论:1.Kps F基因缺陷对于烟曲霉径向生长没有影响,Kps F基因过表达使烟曲霉生长受限。2.Kps F基因缺陷对烟曲霉生长形态没有明显影响,但是Kps F基因过表达影响菌丝排列,分枝的角度,其分生孢子头相对短小,顶囊发育不良,顶囊上的单层小梗不明显。3.Kps F基因缺陷与Kps F基因过表达均对体外抗真菌药物敏感性无明显影响。4.在低浓度Ca2+条件下,Kps F基因可能参与钙调磷酸酶的负反馈调节,并且在10 m M Ca2+条件下负反馈调节作用明显,在高浓度Ca2+条件下该负反馈调节作用却受到抑制;Kps F基因对于钙离子体内平衡有一定的影响。5.烟曲霉Kps F基因可能与菌丝的细胞膜有关。
[Abstract]:Background: Aspergillus fumigatus is a common saprophytic fungus. Invasive aspergillosis (IA) is caused by Aspergillus fumigatus, which is caused by Aspergillus fumigatus. The mortality of Aspergillus fumigatus is on the rise, and its treatment has become a common difficult problem. The important role of calcineurin (Calcineurin, Ca N) in the morphogenesis and toxicity of pathogenic fungi makes it become an important factor. In the study of Aspergillus fumigatus, Ca N is located at the tip and division of mycelium, which has a significant effect on the pathogenicity of Aspergillus fumigatus. The study shows that Ca N plays a close relationship with the Px Ix IT motif, and the calcineurin substrate or target protein, such as Crz1, SIm1, SIm2, Hph1, contains similar patterns in Cbp. Similar motif exists in the Kps F gene sequence of Aspergillus fumigatus. Although calcineurin inhibitor FK506 and cyclosporine A are very successful in modern transplant medicine, the systemic toxic side effects associated with calcineurin can greatly restrict the clinical antifungal application of Aspergillus fumigatus. Therefore, the study of the function of Aspergillus fumigatus Kps F gene is expected to be found. New endogenous calcineurin inhibitors provide new ideas for the treatment of invasive aspergillosis. Objective: 1. construction of Aspergillus fumigatus Kps F gene defect strain and Kps F gene overexpressed strain, preliminary understanding of the effect of Kps F gene on the radial growth and morphology of Aspergillus fumigatus,.2. through an in vitro antifungal drug test, preliminarily clarified the common resistance of Kps F gene to the common antifungal agent. The influence of fungal drug susceptibility on the interaction between Kps F gene and calcineurin and calcium ions in the body.4. by constructing a green fluorescent location plant to explore the location of Kps F gene in the growth of mycelium. Method: 1. using protoplast method to construct Aspergillus fumigatus Kps F gene defective strain, and use Southern blot to verify.2.. 1. P UCGH plasmid was used as a carrier, hygromycin was used as a screening marker, and the Kps F overexpression strain.3. was constructed by Protoplast.3., and the control strain Ku80, Delta Kps F and Kps F overexpressed strain were mixed with different concentration strains, and inoculated on solid medium, respectively, in 24 h, 48 h, 72, 96 and 120. Diameter, observe the colony growth condition, and make statistical analysis; use the small culture method to carry on the morphological study to the strain, culture 48 h, add the lactate cotton blue dye to observe.4. using dot method to explore the control strain Ku80, Delta Kps F and Kps F overexpressed strain the sensitivity of antifungal drugs, in addition to antifungal drugs 24 h, 48 h, 72 h, 96 h, and 120 h were recorded on the basic culture medium, and the colony diameter was recorded, and the growth status of the colony was observed. The expression level of Cna A and Vcx A gene of three different strains of Ca2+ liquid culture medium with different concentration Ca2+ was statistically analyzed, and a statistical analysis was made by laser confocal microscopy. Location of Kps F-EGFP in the growth of mycelium. Results: 1. the successful construction of Aspergillus fumigatus, Kps F, Kps F gene overexpressed strain, Kps F gene green fluorescent location strain (laboratory established and preserved).2. Aspergillus fumigatus, Kps F and Ku80 radial growth results, the results of overexpressed plant radial growth relative to the control bacteria The strain Ku80 decreased about 45%.3. Aspergillus fumigatus? Kps F and the control strain Ku80 had little difference in morphology, Kps F overexpressed mycelium branches, irregular arrangement and less septum; the spore head was relatively short, the top capsule was poorly developed, the single peduncle on the top capsule was not obviously.4. when the concentration of cappofinn was 1.0~5.0 ug/ml, and Kps F appeared smaller. The overexpressed strains of Ku80 and Kps F did not change significantly. The sensitivity of the four antifungal agents of Kps F, Ku80 and Kps F to itraconazole, voriconazole, amphotericin B and FK506 in vitro was no significant difference (P0.05) under the concentration of 10 The expression of a A gene was significantly higher than that of the control strain Ku80 and Kps F overexpressed strain, and the Cna A expression of Kps F was 29.76 times of Ku80 (P0.01), and the Kps expressed strain was 1.56 times as high as that of the control strain. The expression of F overexpressed strain and Ku80.6. at the concentration of 10 m M Ca Cl2, the Vcx A gene expression of Kps F overexpression strain was significantly higher than that of Ku80 and 2.65 times. The green fluorescence of Kps F-EGFP of Aspergillus fumigatus.7. was distributed uniformly at the membrane of mycelium cell. Conclusion: 1.Kps F gene defect has no effect on the radial growth of Aspergillus fumigatus. Kps F gene overexpression makes the F gene defect of Aspergillus fumigatus.2.Kps F gene has no obvious influence on the growth morphology of Aspergillus fumigatus, but the Kps F gene overexpression affects the growth of Aspergillus fumigatus. Mycelium arrangement, branching angle, its conidium head is relatively short, the top capsule is dysplastic, the single layer on the top capsule is not obvious.3.Kps F gene defect and Kps F gene overexpression have no obvious effect on anti fungal drug sensitivity in vitro.4. under the low concentration Ca2+ condition, Kps F basis may participate in the negative feedback regulation of calcineurin. Under the condition of 10 m M Ca2+, the negative feedback regulation is obvious, and the negative feedback regulation is inhibited under the high concentration of Ca2+, and the Kps F gene has a certain effect on the balance of calcium ions in the body of calcium ions, and the F gene of.5. Aspergillus fumigatus Kps may be related to the cell membrane of the mycelium.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R519

【参考文献】