异源性筛选标记对白假丝酵母菌耐受各种应激的影响

Effect of heterologous auxotrophy markers on adaptation to various stresses in Candida albicans

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 effect of heterologous auxotrophy markers from non-Candida albicans （C. albicans） yeasts （C. dubliniensis HIS1 [C. d. HIS1], C. maltosa LEU2 [C. m. LEU2] and C. dubliniensis ARG4 [C. d. ARG4]） on adaptation to various stresses of C. albicans knockout mutants （by Hisl-Leu2-Arg4 strategy）. Methods Fusion PCR strategy was used to construct recombination fragment, which was then used to transform SN152 by lithium acetate method. Genomic PCR was used to confirm the integration of the three selection markers. Spot assays were employed to assess the sensitivities of different reintegrated strains to different stresses. Results The results of PCR showed that seven reintegrated strains containing C. rn. LEU2, C.d. HIS1 and C.d. ARG4 were successfully constructed. The results of spot assay showed that there were no differences between the seven reintegration strains and SN152 in responses to the tested stresses, including pH stress, oxidative stress, antifungal agent stress, metal ion stress, cell wall stress, osmotic stress and DNA damage agent stress. We also found that only strains which integrated C.d. HIS1 marker could survive from the 0. 02% SDS stress. Conclusion The three heterologous selection marker C. m. LEU2, C.d. HIS1 and C. d. ARG4 do not influence the sensitivity of C. albicans to most stresses; comparison can be made between C. albicans knockout mutants with different markers, and SN152 can be used as parental control. While assessing the sensitivity to SDS, the results vary with different selection markers, and SN152 can not be used as a unified control; therefore comparison should be made with C.d. HIS1 kept consistent.