期刊文献+

KpsF基因对烟曲霉生长的影响以及抗真菌药物作用下的生长变化 被引量:1

Effect of KpsF gene on the growth of Aspergillus fumigatus and its growth change under antifungal drug stress
下载PDF
导出
摘要 目的KpsF基因在烟曲霉生长中的作用目前还未明确。文中旨在构建烟曲霉KpsF基因荧光定位菌株和过表达菌株,明确该基因对烟曲霉生长和在抗真菌药物作用下的生长变化情况。方法构建烟曲霉KpsF基因敲除株(ΔKpsF)、KpsF过表达菌株。光学显微镜观察菌落特征,利用激光共聚焦显微镜观察绿色荧光在烟曲霉生长中的定位变化;采用斑点法观察菌株在抗真菌药物作用下生长变化情况。结果同Ku80和△KpsF相比,烟曲霉KpsF基因过表达株菌落直径显著缩小(P<0.05)。低倍显微镜下观察可见:△KpsF和Ku80菌丝细长,排列整齐,舒展,菌丝分支角度多为45°;而KpsF过表达菌株菌丝生长较致密,分枝繁多,且菌丝短小,分支角度相对较大。高倍显微镜下观察可见:△KpsF和Ku80的分生孢子头规整,呈短柱状,梗壁光滑;KpsF过表达菌株分生孢子头明显发育不良,孢子头欠饱满,顶囊单层小梗不清晰。激光共聚焦显微镜观察示烟曲霉孢子状态下绿色荧光弥漫分布于整个细胞中,而在菌丝状态下可见荧光在隔膜处有明显聚集。KpsF过表达株菌菌落直径分别和卡泊芬净、伊曲康唑、两性霉素B浓度呈负相关(r=-0.568、-0.743、-0.813,P<0.05)。卡泊芬净浓度>1.0μg/mL时,△KpsF、Ku80菌株菌落直径分别和卡泊芬净浓度呈正相关(r=0.927、0.915,P<0.05),卡泊芬净浓度<1.0μg/mL时,△KpsF、Ku80菌株菌落直径分别和卡泊芬净浓度呈负相关(r=-0.929、-0.923,P<0.05)。△KpsF、Ku80菌株菌落直径同伊曲康唑、两性霉素B浓度均呈负相关(P<0.05)。结论烟曲霉KpsF基因过表达会影响烟曲霉的生长,且该基因可能参与烟曲霉生长形成分隔过程。KpsF基因缺陷在含卡泊芬净培养基上存在矛盾生长现象,过表达菌株无此现象。 Objective Through the construction of fluorescent localization strains and overexpression strains of KpsF gene in A.fumigatus,the role of KpsF gene in the growth and development of A.fumigatus and reaction to the pressure of antifungal drugs were preliminarily identified.Methods The sequence of KpsF gene was found by molecular biological method.Enhanced green fluorescent protein(eGFP)was used as the reporter gene.A pUCGH plasmid,containing hygromycin resistant gene,was used to construct the vector.The localization strain and overexpress strain of KpsF gene were obtained by protoplast transformation and verified by PCR.Morphological changes of different strains were observed by microscope.The distribution of eGFP in spore,spore germination and mycelium of A.fumigatus was observed by confocal laser microscopy.The growth of each strain under the pressure of different antifungal drugs was observed by spot assay.Results The KpsF gene of A.fumigatus was amplified and cloned into pUCGH.Transformants of localization strain and overexpression strains were obtained by protoplast transformation of A.fumigatus,which were confirmed by PCR.The result of colony observation showed that the diameter of overexpression strain was smaller than that of Ku80 andΔKpsF.And the colony of overexpression strain had deep folds.Under the low magnification of microscope,the mycelium of the overexpression strain was disorderly distributed than Ku80 andΔKpsF.The common 45 o branch of A.fumigatus was lost.The relative angle became larger,and the mycelial branches were numerous and short.Under high magnification of microscope,the overexpression strain was relatively short of conidial head,dysplasia of apical sac and no obvious single-layer peduncle.The green fluorescence was diffusely distributed in the whole cell in the spore state,while the fluorescence was obviously concentrated at the septum in the mycelial state of KpsF gene location strain by laser confocal microscopy.When the concentration of caspofungin was 1.0-10.0μg/mL,there was a contradictory growth betweenΔKpsF and Ku80,but not in the overexpression strain.The colony growth diameter ofΔKpsF,Ku80 and overexpression strains on the medium containing itraconazole and amphotericin B retarded the increase of drug concentration,showing a significant negative correlation.Conclusion Overexpression of KpsF gene in A.fumigatus can significantly affect the growth of A.fumigatus.The KpsF gene may play a role in the process of formation of septum in the growth of A.fumigatus.The growth ofΔKpsF and Ku80 on the GMM containing caspofungin also show paradoxical effect.
作者 马彦 冀英 杨静 岑雯 曹雪 李佳娟 冯文莉 MA Yan;JI Ying;YANG Jing;CEN Wen;CAO Xue;LI Jia-juan;FENG Wen-li(Department of Dermatology,The Second Hospital of Shanxi Medical University/Key Research Lab of Airway neuroimmunology,Taiyuan 030001,Shanxi,China;The Second Clinical Medical College,Shanxi Medical University,Taiyuan 030001,Shanxi,China)
出处 《医学研究生学报》 北大核心 2021年第9期906-913,共8页 Journal of Medical Postgraduates
基金 山西省重点研发计划(201803D31115) 山西省高等学校科学研究优秀成果培育项目(2019KJ025)。
关键词 KpsF基因 烟曲霉 过表达 生长变化 KpsF aspergillus fumigatus overexpression growth change
  • 相关文献

参考文献4

二级参考文献6

共引文献4

同被引文献8

引证文献1

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部