格特隐球菌HOG1基因的敲除及重建

中国真菌学杂志　 2013, Vol. 8 　Issue (3): 137-141.

格特隐球菌HOG1基因的敲除及重建

皇幼明, 娄永华, 朱红梅, 温海

上海长征医院皮肤病与真菌病研究所全军真菌病重点实验室第二军医大学附属长征医院皮肤科, 上海 200003

收稿日期:2013-05-02 出版日期:2013-06-28 发布日期:2013-06-28
通讯作者: 朱红梅,E-mail:hmzhu_cn@163.com.cn;温海,E-mail:wenhai98@sohu.com E-mail:hmzhu_cn@163.com.cn;wenhai98@sohu.com
作者简介:皇幼明,男 (汉族),硕士研究生在读.E-mail:fhguangming@hotmail.com
基金资助:
国家自然基金(81071336,81271800,31270181)

Deletion and reconstruction of HOG1 gene in Cryptococcus gattii

HUANG You-ming, LOU Yong-hua, ZHU Hong-mei, WEN mei

Institute of dermatology and fungal disease, Key Laboratory of the army fungal disease, Department of dermatology, Changzheng Hospital, Shanghai 200003, China

Received:2013-05-02 Online:2013-06-28 Published:2013-06-28

摘要/Abstract

摘要： 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。

关键词: 格特隐球菌, HOG1基因, 缺陷株, 重建株

Abstract: Objective To construct the HOG 1 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 pGAPzα-A respectively. Finally, pGAPzα-dHOG 1 was transferred into Crypcococcus gattii original 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 1 reconstituted strain would be useful in further research of virulence and pathogenesis of Cryptococcus gattii.

Key words: Cryptococcus gattii, HOG 1 gene, mutant, reconstituted strain

中图分类号:

R379.5

皇幼明, 娄永华, 朱红梅, 温海. 格特隐球菌HOG1基因的敲除及重建[J]. 中国真菌学杂志, 2013, 8(3): 137-141.

HUANG You-ming, LOU Yong-hua, ZHU Hong-mei, WEN mei. Deletion and reconstruction of HOG1 gene in Cryptococcus gattii[J]. Chinese Journal of Mycology, 2013, 8(3): 137-141.

参考文献

[1] Fyfe M, MacDougall L, Romney M, et al. Cryptococcus gattii infections on Vancouver Island, British Columbia, Canada: emergence of a tropical fungus in a temperate environment[J]. Can Commun Dis Rep,2008, 34(6): 1-12.
[2] Hohmann S, Krantz M, Nordlander B. Yeast osmoregulation[J]. Methods Enzymol,2007, 428: 29-45.
[3] Reed RH, Chudek JA, Foster R, et al. Osmotic significance of glycerol accumulation in exponentially growing yeasts[J]. Appl Environ Microbiol,1987, 53(9): 2119-2123.
[4] Escote X, Miranda M, Rodriguez-Porrata B, et al. The stress-activated protein kinase HOG1 develops a critical role after resting state[J]. Mol Microbiol,2011, 80(2): 423-435.
[5] Hull CM,Heitman J. Genetics of Cryptococcus neoformans[J]. Annu Rev Genet,2002, 36: 557-615.
[6] Chaturvedi S, Ren P, Narasipura SD, et al. Selection of optimal host strain for molecular pathogenesis studies on Cryptococcus gattii[J]. Mycopathologia,2005, 160(3): 207-215.
[7] Chen S, Sorrell T, Nimmo G, et al. Epidemiology and host-and variety-dependent characteristics of infection due to Cryptococcus neoformans in Australia and New Zealand. Australasian Cryptococcal Study Group[J]. Clin Infect Dis,2000, 31(2): 499-508.
[8] Sorrell TC. Cryptococcus neoformans variety gattii[J]. Med Mycol,2001, 39(2): 155-168.
[9] Speed BDunt D.Clinical and host differences between infections with the two varieties of Cryptococcus neoformans[J]. Clin Infect Dis,1995, 21(1): 28-34; discussion 35-36.
[10] Chowdhary A, Randhawa HS, Sundar G, et al. In vitro antifungal susceptibility profiles and genotypes of 308 clinical and environmental isolates of Cryptococcus neoformans var. grubii and Cryptococcus gattii serotype B from north-western India[J]. J Med Microbiol,2011, 60(Pt 7): 961-967.
[11] Tseng HK, Liu CP, Ho MW, et al. Microbiological, epidemiological, and clinical characteristics and outcomes of patients with cryptococcosis in taiwan, 1997-2010[J]. PLoS One,2013, 8(4): e61921.
[12] Bahn YS, Geunes-Boyer S, Heitman J. Ssk2 mitogen-activated protein kinase kinase kinase governs divergent patterns of the stress-activated HOG1 signaling pathway in Cryptococcus neoformans[J]. Eukaryot Cell,2007, 6(12): 2278-2289.
[13] Bahn YS, Kojima K, Cox GM, et al. Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans[J]. Mol Biol Cell,2005, 16(5): 2285-2300.
[14] Perfect JR. Cryptococcus neoformans: a sugar-coated killer with designer genes[J]. FEMS Immunol Med Microbiol,2005, 45(3): 395-404.
[15] Frealle E, Noel C, Viscogliosi E, et al. Manganese superoxide dismutase in pathogenic fungi: an issue with pathophysiological and phylogenetic involvements[J]. FEMS Immunol Med Microbiol,2005, 45(3): 411-422.
[16] Latouche GN, Sorrell TC, Meyer W. Isolation and characterisation of the phospholipase B gene of Cryptococcus neoformans var. gattii[J]. FEMS Yeast Res,2002, 2(4): 551-561.
[17] Ren P, Springer DJ, Behr MJ, et al. Transcription factor STE12alpha has distinct roles in morphogenesis, virulence, and ecological fitness of the primary pathogenic yeast Cryptococcus gattii[J]. Eukaryot Cell,2006, 5(7): 1065-1080.
[18] Narasipura SD, Ault JG, Behr MJ, et al. Characterization of Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant of Cryptococcus neoformans var. gattii: role in biology and virulence[J]. Mol Microbiol,2003, 47(6): 1681-1694.
[19] Ngamskulrungroj P, Himmelreich U, Breger JA, et al. The trehalose synthesis pathway is an integral part of the virulence composite for Cryptococcus gattii[J]. Infect Immun,2009, 77(10): 4584-4596.
[20] Petzold EW, Himmelreich U, Mylonakis E, et al. Characterization and regulation of the trehalose synthesis pathway and its importance in the pathogenicity of Cryptococcus neoformans[J]. Infect Immun,2006, 74(10): 5877-5887.
[21] D'Souza CA, Alspaugh JA, Yue C, et al. Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans[J]. Mol Cell Biol,2001, 21(9): 3179-3191.
[22] Hicks JK, Heitman J. Divergence of protein kinase A catalytic subunits in Cryptococcus neoformans and Cryptococcus gattii illustrates evolutionary reconfiguration of a signaling cascade[J]. Eukaryot Cell,2007, 6(3): 413-420.
[23] Kidd SE, Hagen F, Tscharke RL, et al. A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada)[J]. Proc Natl Acad Sci U S A,2004, 101(49): 17258-17263.