Full Text:   <750>

Summary:  <405>

CLC number: R379

On-line Access: 2015-12-04

Received: 2015-06-21

Revision Accepted: 2015-11-09

Crosschecked: 2015-11-18

Cited: 1

Clicked: 1729

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Qing-bi Hu

http://orcid.org/0000-0002-0404-4778

-   Go to

Article info.
Open peer comments

Journal of Zhejiang University SCIENCE B 2015 Vol.16 No.12 P.991-997

http://doi.org/10.1631/jzus.B1500151


Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes


Author(s):  Qing-bi Hu, Yu He, Xun Zhou

Affiliation(s):  Department of Dermatology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; more

Corresponding email(s):   zhouxun123@sina.com

Key Words:  Sporothrix globosa, Dimorphism, Suppression subtractive hybridization (SSH), Real-time PCR


Qing-bi Hu, Yu He, Xun Zhou. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes[J]. Journal of Zhejiang University Science B, 2015, 16(12): 991-997.

@article{title="Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes",
author="Qing-bi Hu, Yu He, Xun Zhou",
journal="Journal of Zhejiang University Science B",
volume="16",
number="12",
pages="991-997",
year="2015",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1500151"
}

%0 Journal Article
%T Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes
%A Qing-bi Hu
%A Yu He
%A Xun Zhou
%J Journal of Zhejiang University SCIENCE B
%V 16
%N 12
%P 991-997
%@ 1673-1581
%D 2015
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1500151

TY - JOUR
T1 - Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes
A1 - Qing-bi Hu
A1 - Yu He
A1 - Xun Zhou
J0 - Journal of Zhejiang University Science B
VL - 16
IS - 12
SP - 991
EP - 997
%@ 1673-1581
Y1 - 2015
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1500151


Abstract: 
Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

中国球形孢子丝菌双相cDNA消减文库构建及其差异基因的筛选和验证

目的:为进一步研究中国球形孢子丝菌的双向转换机制,明确目的差异基因对我国球形孢子丝菌形态和双相转换的影响,探讨可能参与的信号传导通路,以期较全面地探讨孢子丝菌双相转换相关的分子机制,也为筛选抗真菌药物研究提供新的靶位和切入点。
创新点:以中国球形孢子丝菌双向转换为立足点,应用抑制性消减文库成功构建的高特异性的中国球形孢子丝菌酵母相(Y)和菌丝相(M)的正反cDNA基础上,对中国球形孢子丝菌双相转换相关基因进行生物信息学分析并对部分有意义的差异表达基因进行验证。
方法:本实验通过抑制性消减杂交技术构建中国球形孢子丝菌酵母相和菌丝相的正反cDNA消减文库,并对差异基因进行生物信息学分析,获得部分有意义的差异表达基因,再通过实时荧光聚合酶链反应(PCR)技术验证目的基因在双相中的差异表达。
结论:通过抑制性消减杂交技术对中国球形孢子丝菌的正向和反向两个文库进行筛选,获得可能参与球形孢子丝菌细胞壁合成、信号转到、新陈代谢类等相关的片段重叠群(contigs),结果表明中国球形孢子丝菌在双相型转换中存在明显表达差异基因,同时利用相对定量的实时荧光定量技术对其中两个基因(细胞表面分子类基因细胞周期检测点激酶CHK和结构基因类核糖体RNA基因)进行验证,认为它们可能与双相转换有关。

关键词:球形孢子丝菌;双相;抑制性消减杂交技术(SSH);实时荧光PCR技术

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

[1]Aquino-Piñero, E.E., Rodriguez, D.V.N., 1997. Different protein kinase C isoforms are present in the yeast and mycelium forms of Sporothrix schenckii. Mycopathologia, 138(3):109-115.

[2]Arrillaga-Moncrieff, I., Capilla, J., Mayayo, E., et al., 2009. Different virulence levels of the species of Sporothrix in a murine model. Clin. Microbiol. Infect., 15(7):651-655.

[3]Chen, Y., Sanchez, Y., 2004. Chk1 in the DNA damage response: conserved roles from yeasts to mammals. DNA Repair (Amst), 3(8-9):1025-1032.

[4]de Beer, Z.W., Harrington, T.C., Vismer, H.F., et al., 2003. Phylogeny of the Ophiostoma stenocerasSporothrix schenckii complex. Mycologia, 95(3):434-441.

[5]de Jesus-Berrios, M., Rodriguez-del, V.N., 2002. Expression of a Pho85 cyclin-dependent kinase is repressed during the dimorphic transition in Sporothrix schenckii. Fungal Genet. Biol., 37(1):39-48.

[6]de Meyer, E.M., de Beer, Z.W., Summerbell, R.C., et al., 2008. Taxonomy and phylogeny of new wood- and soil-inhabiting Sporothrix species in the Ophiostoma stenocerasSporothrix schenckii complex. Mycologia, 100(4):647-661.

[7]Diatchenko, L., Lau, Y.F., Campbell, A.P., et al., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. PNAS, 93(12):6025-6030.

[8]Fernandes, G.F., Dos, S.P., Rodrigues, A.M., et al., 2013. Characterization of virulence profile, protein secretion and immunogenicity of different Sporothrix schenckii sensu stricto isolates compared with S. globosa and S. brasiliensis species. Virulence, 4(3):241-249.

[9]Fleischmann, J., Liu, H., Wu, C.P., 2004. Polyadenylation of ribosomal RNA by Candida albicans also involves the small subunit. BMC Mol. Biol., 5:17.

[10]Furuya, K., Niki, H., 2010. The DNA damage checkpoint regulates a transition between yeast and hyphal growth in Schizosaccharomyces japonicus. Mol. Cell. Biol., 30(12):2909-2917.

[11]Han-Yaku, H., Naka, W., Tajima, S., et al., 1996. Differential expression of the 45 kDa protein (actin) during the dimorphic transition of Sporothrix schenckii. J. Med. Vet. Mycol., 34(3):175-180.

[12]Huang, G.S., Zhang, J.M., Xi, L.Y., et al., 2007. The differential expressions of HSP70 gene from yeast phaseand mycelia phase of Penicillium marneffei. Chin. J. Zoonoses, 23(12):1191-1193 (in Chinese).

[13]Li, F.Q., Yang, X., Bao, B.L., 2007. The research and prospect of Sporothrix schenckii. Jilin Med. J., 28(5):581-583 (in Chinese).

[14]Liu, T.T., Zhang, K., Zhou, X., 2014. Molecular identification of Sporothrix clinical isolates in China. J. Zhejiang Univ. -Sci. B (Biomed. & Biotechnol.), 15(1):100-108.

[15]Ma, J.B., Zhou, Q.H., 2005. The application of suppression subtractive hybridization in tumor. Chin. J. Lung Cancer, 8(6):563-566 (in Chinese).

[16]Madrid, H., Gene, J., Cano, J., et al., 2010. Sporothrix brunneoviolacea and Sporothrix dimorphospora, two new members of the Ophiostoma stenocerasSporothrix schenckii complex. Mycologia, 102(5):1193-1203.

[17]Marimon, R., Gene, J., Cano, J., et al., 2006. Molecular phylogeny of Sporothrix schenckii. J. Clin. Microbiol., 44(9):3251-3256.

[18]Marimon, R., Cano, J., Gene, J., et al., 2007. Sporothrix brasiliensis, S. globosa, and S. mexicana, three new Sporothrix species of clinical interest. J. Clin. Microbiol., 45(10):3198-3206.

[19]Marques, E.R., Ferreira, M.E.S., Drummond, R.D., et al., 2004. Identification of genes preferentially expressed in the pathogenic yeast phase of Paracoccidioides brasiliensis, using suppression subtraction hybridization and differential macroarray analysis. Mol. Genet. Genomics, 271(6):667-677.

[20]Martinho, R.G., Lindsay, H.D., Flaggs, G., et al., 1998. Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses. EMBO J., 17(24):7239-7249.

[21]Mesa-Arango, A.C., del Rocío, R.M., Pérez-Mejía, A., et al., 2002. Phenotyping and genotyping of Sporothrix schenckii isolates according to geographic origin and clinical form of Sporotrichosis. J. Clin. Microbiol., 40(8):3004-3011.

[22]Pappas, P.G., Tellez, I., Deep, A.E., et al., 2000. Sporotrichosis in Peru: description of an area of hyperendemicity. Clin. Infect. Dis., 30(1):65-70.

[23]Ran, Y.P., Liu, D.C., Li, Z.Y., et al., 1999. The analysis of 10982 cases of dermatomycosis analysis from 1981 to 1997. Chin. J. Dermatol., 32(5):336 (in Chinese).

[24]Schubach, A., Barros, M.B., Wanke, B., 2008. Epidemic sporotrichosis. Curr. Opin. Infect. Dis., 21(2):129-133.

[25]Sipiczki, M., Takeo, K., Grallert, A., 1998. Growth polarity transitions in a dimorphic fission yeast. Microbiology, 144(Pt 12):3475-3485.

[26]Su, H.C., Cheng, B., 2013. Effects of histidine kinase gene CHK1 on some biological characteristics of Candida albicans. Chin. Med. J., 93(14):1109-1113 (in Chinese).

[27]Tan, J.W., Liu, W., Wan, Z., et al., 2013. Reclassification of 33 clinical strains of Sporothrix from northern China based on phenotypic and molecular characters. Mycosystema, 32(2):161-167 (in Chinese).

[28]Valentin-Berrios, S., Gonzalez-Velazquez, W., Perez-Sanchez, L., et al., 2009. Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii. BMC Microbiol., 9:100.

[29]Valle-Aviles, L., Valentin-Berrios, S., Gonzalez-Mendez, R.R., et al., 2007. Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii. BMC Microbiol., 7:107.

[30]Wang, J.F., Zhu, M.J., Jiang, L.X., et al., 1998. Epidemiological investigation of Sporotrichosis in Tongyu County of Jilin Province. Chin. J. Public Health, 17(5):317-318 (in Chinese).

[31]Yap, F.B., 2011. Disseminated cutaneous Sporotrichosis in an immunocompetent individual. Int. J. Infect. Dis., 15(10):e727-e729.

[32]Yu, X., Wan, Z., Zhang, Z., et al., 2013. Phenotypic and molecular identification of Sporothrix isolates of clinical origin in Northeast China. Mycopathologia, 176(1-2):67-74.

[33]Zeng, F., Zhang, X., Zhu, L., et al., 2006. Isolation and characterization of genes associated to cotton somatic Embryogenesis by suppression subtractive hybridization and macroarray. Plant Mol. Biol., 60(2):167-183.

[34]Zhou, X., Rodrigues, A.M., Feng, P., et al., 2013. Global ITS diversity in the Sporothrix schenckii complex. Fungal Divers., 66(1):153-165.

Open peer comments: Debate/Discuss/Question/Opinion

<1>

Please provide your name, email address and a comment





Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - Journal of Zhejiang University-SCIENCE