利用抑制消减杂交初步构建犬小孢子菌在不同诱导底物表达的差异基因文库

目的比较犬小孢子菌在儿童与成人头皮组织、儿童头皮组织与儿童光滑皮肤组织为诱导底物表达的差异基因,探讨犬小孢子菌蛋白酶在头癣中的发病机制。方法将犬小孢子菌菌株分别接种于含儿童光滑皮肤组织(driver1)、儿童头皮组织(tester)和成人头皮组织(driver2)三种底物诱导培养基中培养,经抑制消减杂交技术(SSH)分别构建在tester与driver1,tester与driver2培养基中培养的犬小孢子菌菌株正反向差异基因文库,菌液PCR筛选鉴定,对阳性克隆进行测序分析。结果在tester与driver1的正向差异基因文库中初步筛选出1个差异表达基因与编码红色毛癣菌线粒体细胞色素b和NADH1-5具有高度同源性,另15个差异表达基因在真菌界均未知。结论应用SSH技术成功构建了不同底物诱导培养基犬小孢子菌表达的差异基因文库;这个与编码红色毛癣菌线粒体细胞色素b和NADH1-5具有高度同源性的差异表达基因,可能在犬小孢子菌所致头癣中起一定作用,进一步研究该基因的功能对研究头癣的发病机制具有重要意义。

Construction and Analysis of Differentially Expressed Genes Libraries of Microsporum Canis Incubated in Different Induced Medium by Using SSH

Objective To compare differential expressed cDNA genes of M.canis incubated on mineral medium with child's glabrous skin,scalp tissue or adult's scalp tissue by using SSH technique and to study the pathogenic mechanism of tinea capitis caused by microsporum canis.Methods Microsporum canis was cultivated on mineral medium with child's glabrous skin,scalp tissue and adult's scalp tissue as sole substrates.The forward and reverse cDNA library from microsporum canis incubated on mineral medium with tester and driver1,tester and driver2 were constructed by using SSH.The four subtractive library were screened firstly by PCR.The positive clones were sequenced and analysed.Results One of the twenty clones was high homologous with Trichophyton rubrum mitochondrial cytb gene and NADH1 to NADH5 genes.Fifteen of the twenty clones were unknown in fungus.Conclusion The cDNA libraries were constructed successfully from microsporum canis incubated in different proteases-induced medium successfully by using suppression subtractive hybridization(SSH).The screened clone that was high homologous with Trichophyton rubrum mitochondrial cytb gene and NADH1 to NADH5 genes may be on the role of tinea capitis.It is very meaningful to further study on this gene's function.