格特隐球菌HOG1基因的敲除及重建

Deletion and reconstruction of HOG1 gene in Cryptococcus gattii

目的构建格特隐球菌HOG1基因缺陷株和HOG1基因重建株。方法从格特隐球菌基因组扩增HOG1基因,通过部分基因缺失方法,获得缺陷基因dHOG1。将获得的HOG1基因及其缺陷基因dHOG1分别亚克隆到真核表达载体pGAPzα-A,构建pGAPzα-HOG1及pGAPzα-dHOG1质粒。将pGAPzα-dHOG1质粒转染隐球菌原始株,通过筛选获得HOG1基因缺陷菌株;同样方法将pGAPzα-HOG1质粒转染格特隐球菌HOG1基因敲除菌株,获得HOG1基因重建株。结果 RT-PCR结果示:格特隐球菌HOG1基因缺陷株不转录表达完整的HOG1基因,而HOG1基因重建株可以转录表达完整的HOG1基因片段。结论成功获得格特隐球菌HOG1缺陷株和HOG1基因重建株,为后续格特隐球菌毒力和致病机制的研究奠定了基础。

Objective To construct the HOG l mutant strain and the HOG 1 reconstituted strain. Methods Cloning HOG 1 gene according to the gene sequences, we got dHOG 1 gene by deleting part of HOG 1 gene. Then the HOG 1 gene and dHOG 1 gene were subcloned into eukaryotic vectors pGAPzcc-A respectively. Finally, pGAPzα- dHOG 1 was transferred into Crypcococcus gattii origi- nal strain to get HOG 1 mutant strain. By the same method, pGAPzα-HOG 1 vector was transferred into HOG 1 mutant strain to get HOG 1 reconstituted strain. Results Results of RT-PCR showed that the HOG 1 mutant strain did not express whole HOG 1 gene fragment, while HOG 1 reconstituted strain expressed intact HOG 1 gene. Conclusion Successful construction of HOG 1 mutant strain and HOG I reconstituted strain would be useful in further research of virulence and pathogenesis of Cryptococcus gattii.