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CLC number: R379

On-line Access: 2024-08-27

Received: 2023-10-17

Revision Accepted: 2024-05-08

Crosschecked: 2015-11-18

Cited: 1

Clicked: 4599

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Qing-bi Hu

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

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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.

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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

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