Construction of knockout strains of The COP9 signalosome subunit 6 (CSN6) in Cryptococcus neoformans

Abstract

Abstract: Objective To construct the homologous recombination cassette of COP9/signalosome component gene CSN 6 and generate its deletion mutant via biolistic transformation.Methods We first got the DNA sequence of COP9/signalsome components by bioinformatics analysis. On the basis, we constructed the homologous recombination cassette of CSN 6 via overlap PCR and transformed them into Cryptococcal competent cells via Particle Delivery System.The positive transformants were screened via YPD argar including geneticin (G418), which were then identified via diagnostic PCR and DNA sequencing.Results CSN 6 deletion strains were successfully constructed.Conclusions This study laid the groundwork for following functional studies of COP9/signalosome in Cryptococcus neoformans.

Key words: Cryptococcus neoformans, COP9/signalosome, CSN6, pathogenesis

CLC Number:

R379.5

YANG Ya-li, ZHANG Chao, MENG Yun-fang, FA Zhen-zong, ZHOU Zhao-jing, ZHAO Jing-yu, FANG Wei, LIAO Wan-qing. Construction of knockout strains of The COP9 signalosome subunit 6 (CSN6) in Cryptococcus neoformans[J]. Chinese Journal of Mycology, 2014, 9(6): 321-324,330.

References

[1] Fang W, Fa Z, Liao W. Epidemiology of Cryptococcus and cryptococcosis in China[J].Fungal Genet Biol, 2014.
[2] Yuchong C, Fubin C, Jianghan C, et al. Cryptococcosis in China (1985-2010): review of cases from Chinese database[J]. Mycopathologia, 2012,173(5-6):329-335.
[3] Braus GH, Irniger S, Bayram O. Fungal development and the COP9 signalosome[J]. Curr Opin Microbiol, 2010,13(6):672-676.
[4] Wei N, Serino G, Deng XW. The COP9 signalosome: more than a protease[J]. Trends Biochem Sci, 2008,33(12):592-600.
[5] Ma H, Hagen F, Stekel DJ, et al. The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation[J]. Proc Natl Acad Sci U S A, 2009,106(31):12980-12985.
[6] Fang W, Price MS, Toffaletti DL, et al. Pleiotropic Effects of Deubiquitinating Enzyme Ubp5 on Growth and Pathogenesis of Cryptococcus neoformans[J]. PLoS One, 2012,7(6):e38326.
[7] Chamovitz DA. Revisiting the COP9 signalosome as a transcriptional regulator[J]. EMBO Rep, 2009,10(4):352-358.
[8] Liu OW, Chun CD, Chow ED, et al. Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans[J]. Cell, 2008,135(1):174-188.
[9] Lee A, Toffaletti DL, Tenor J, et al. Survival defects of Cryptococcus neoformans mutants exposed to human cerebrospinal fluid result in attenuated virulence in an experimental model of meningitis[J]. Infect Immun, 2010,78(10):4213-4225.
[10] Tseng HK, Liu CP, Price MS, et al. Identification of genes from the fungal pathogen Cryptococcus neoformans related to transmigration into the central nervous system [J]. PLoS One, 2012,7(9):e45083.
[11] Wei N, Deng XW. The COP9 signalosome. Annu Rev Cell Dev Biol, 2003,19:261-286.
[12] Birol M, Enchev RI, Padilla A, et al. Structural and biochemical characterization of the Cop9 signalosome CSN5/CSN6 heterodimer[J]. PLoS One, 2014,9(8):e105688.

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