BMH2调控新型隐球菌致病感染能力的功能研究  

Functional study of BMH2 in regulating the pathogenic infection ability of Cryptococcus neoformans

作　　者：李洁[1] 何智春 郑芳林 徐新平 LI Jie;HE Zhichun;ZHENG Fanglin;XU Xinping(Jiangxi Institute of Respiratory Disease,the First Affiliated Hospital of Nanchang University,Jiangxi Province,Nanchang 330006,China;Department of Respiratory,Yichun People’s Hospital,Jiangxi Province,Yichun 336000,China)

机构地区：[1]南昌大学第一附属医学院江西省呼吸疾病研究所,江西南昌330006 [2]江西省宜春市人民医院呼吸科,江西宜春336000

出　　处：《中国医药导报》2023年第21期9-13,18,共6页China Medical Herald

基　　金：国家自然科学基金资助项目(8177216431760038);江西省自然科学基金资助项目(20202ACBL206024)。

摘　　要：目的研究BMH2基因在新型隐球菌中的功能作用。方法采取CRISPR-Cas9联合电穿孔技术,将BMH2基因的原始启动子替换为诱导性启动子CTR4构建条件敲除型突变株。回补BMH2基因原始启动子构建回补株。将菌株在基础YPD/富铜YPD固体培养基中培养,观察菌株的生长情况;在富铜YPD液体培养基中培养,绘制生长曲线;在基础YPD/富铜YPD液体培养基中培养,观察细胞形态;在基础V8/富铜V8固体培养基中培养,观察菌丝生长情况;在25℃、30℃和37℃的基础YPD/富铜YPD固体培养基中培养,观察不同温度下生长情况;在富铜YPD和富铜YPD+应激压力固体培养基中培养,观察生长情况。结果在基础YPD固体培养基中,所有菌株生长无差异;在富铜YPD固体培养基中,突变株生长减弱而野生型与回补株的生长无差异;与野生型比较,突变株生存曲线降低,回补株与其走势一致。在基础YPD液体培养基中,所有菌株的细胞形态无差异;在富铜YPD液体培养基中,野生型和回补株的细胞形态无差异,而突变株的细胞变大且形态不规整。在富铜YPD液体培养基中,突变株细胞直径大于野生型和回补株(P<0.05),回补株和野生型细胞直径比较,差异无统计学意义(P>0.05)。在基础V8固体培养基中,与野生型比较,突变株有微量菌丝产生。在富铜V8固体培养基中,与野生型比较,突变株完全不产生菌丝。在基础YPD固体培养基中,所有菌株在不同温度下生长无差异;在富铜YPD固体培养基中,回补株和野生型生长无差异,但突变株生长呈现出高温敏感。在不同应激压力条件下,回补株和野生型的生长无差异。相对于野生型及无应激压力正常生长的突变株而言,突变株在0.005%SDS、0.250%CR、1 mol/L KCL、1 mol/L NaCl及1 mol/L Sorbitol应激下表现出生长抑制;相对于野生型而言,突变株在10、15、20 mmol/L DTT的应激压力下表现为生长抑制;而相对于无应激压力正常培养Objective To study the functional role of BMH2 gene in Cryptococcus neoformans.Methods Using CRISPR-Cas9 combined electroporation,the original promoter of BMH2 gene was replaced with the inducible promoter CTR4 construction conditional knockout mutant strain.The original promoter of BMH2 gene was supplemented to construct a complement strain.The strains were cultured in basic YPD/copper-rich YPD solid medium to observe the growth of the strains;the growth curve was plotted in copper-rich YPD liquid medium;the cell morphology was observed in basic YPD/copper-rich YPD liquid medium;the hypha growth was observed in basic V8/copper-rich V8 solid medium;Basic YPD/copper-rich YPD solid medium was cultured at 25℃,30℃,and 37℃,and the growth was observed at different temperatures;the growth was observed in copper-rich YPD and copper-rich YPD+stress solid medium.Results There was no difference in the growth of all strains in basic YPD solid medium;in copper-rich YPD solid medium,the growth of mutant train was weakened,but there was no difference between wild type and complement strain.Compared with the wild type,the survival curve of mutant train decreased,and the trend of complement strain was consistent with that of wild type.There was no difference in cell morphology of all strains in basic YPD liquid medium;in copium-rich YPD liquid medium,there was no difference in cell morphology between wild-type and complement strain,but the cells of mutant strain became larger and irregular.In copper-rich YPD liquid medium,the cell diameter of mutant strain was larger than that of wild type and complement strain(P<0.05),but there was no significant difference in cell diameter between wild type and complement strain(P>0.05).In base V8 solid medium,the mutant strain produced a small amount of hypha compared with the wild type.In copper-rich V8 solid medium,the mutant strains did not produce hypha at all compared with the wild type.In basic YPD solid medium,all strains showed no difference in growth at different temperatures;in

关 键 词：新型隐球菌 生长 菌丝生长 温度 应激 

分 类 号：R3[医药卫生—基础医学]