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目的考察以His1-Leu2-Arg4基因敲除策略敲除白假丝酵母菌基因后,异源性筛选标记C.dubliniensis HIS1(C.d.HIS1)、C.maltosa LEU2(C.m.LEU2)和C.dubliniensis ARG4(C.d.ARG4)对菌株耐受各种应激的影响。方法以SN152为亲本菌,融合PCR法构建重组片段,醋酸锂法转染白假丝酵母菌SN152,分别构建上述3个标记的回复菌。采用套式PCR法鉴定回复菌的构建,spot assay实验考察菌株对各种应激的敏感性。结果 PCR验证7种菌株构建成功,spot assay结果显示各回复菌对pH应激、氧化应激、各种临床抗菌药应激、金属离子应激、细胞壁损伤相关药物应激、渗透应激、DNA损伤化合物应激的敏感性与亲本菌SN152一致。而给予0.02%SDS时各菌株的敏感性不一致,缺乏C.d.HIS1标记的菌株在0.02%SDS的应激条件下不能生长。结论C.d.HIS1、C.m.LEU2和C.d.ARG4 3个异源性筛选标记不影响白假丝酵母菌对大部分应激的敏感性,不同标记基因敲除菌间可以相互比较,且可以统一用SN152作为对照菌株。在考察SDS应激时,所用筛选标记不同,对结果有影响,不能用SN152代替作为统一对照,若要相互比较,要保证C.d.HIS1标记必须一致。
Objective To investigate the heterologous selection markers C.dubliniensis HIS1 (CdHIS1), C.maltosa LEU2 (CmLEU2) and C.dubliniensis ARG4 (Candida albicans) after the Candida albicans gene was knocked out by His1-Leu2- CdARG4) on strains resistant to various stress. Methods SN152 was used as the parent bacterium, and the fusion fragment was constructed by fusion PCR method. The Candida albicans SN152 was transfected by lithium acetate method, and the three replicative strains were constructed respectively. The nested PCR method was used to identify the pathogenic bacteria. The spot assay assayed the susceptibility of the strains to various stresses. Results The results of PCR showed that seven strains were successfully constructed. The results of spot assay showed that each strain of bacteria had significant effects on pH stress, oxidative stress, stress of various antibacterial drugs, stress of metal ions, drug stress related to cell wall damage, osmotic stress, The sensitivity of the injury compound to stress is consistent with the parental strain SN152. When 0.02% SDS was given, the sensitivity of each strain was inconsistent. The strain lacking C.d.HIS1 could not grow under the stress of 0.02% SDS. Conclusion The three heterologous markers CdHIS1, CmLEU2 and CdARG4 do not affect the susceptibility of Candida albicans to most of the stress. Mutants of different marker genes can be compared with each other, and SN152 can be used as a control Strain. Screening SDS stress, the screening of different markers, the results have an impact, can not be replaced by SN152 as a unified control, to be compared with each other, to ensure that C. d.HIS1 markers must be consistent.