Construction Of CDNA Subtractive Library Between Yeast And Mycelium Phase Of Sporothrix Schenckii And Bioinformatics Analysis

Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis, with an increasing worldwide incidence. It enters through small cuts and abrasions in the skin to cause the infection. Symptoms of sporotrichosis include localized nodular leisions, bumps and chronic ulcers at the point of entry and also along lymph nodes and vessels. However, it can disseminate in immunocompromised patients .S.schenckii is a pathogenic fungus that undergoes a dimorphic transition from yeast to mycelium in response to environment conditions such as temperature. It appears as mycelium form at 25°C and yeast form at 37°C. Sporotrichosis is caused by the multiplication of yeast form colonies.By some approaches, several genes have been identified that are expressed differently in transition from yeast to mycelium. Signal transduction modulated by some elements such as mitogen-activated protein kinases, cAMP and pH-responsive modules, appear to regulate this transition. However, the mechanism of dimorphic transition is still unclear. Therefore, study on the mechanism of dimorphic transition is important to reveal the pathogenesis of S.schenckii.To screen the other differentially expressed genes about dimorphic transition of S.Schenckii, cDNA subtractive library between yeast and mycelium phase was constructed by suppression subtractive hybridization(SSH)and bioinformatics analysis was performed to profile the relationship between those differently expressed genes and dimorphic transition.In this study, CMCC(F)D1a, a S. schenckii standard strain, was induced to form mycelium(M)and yeas(Y)cells. Total RNA isolated from M and Y cells was reverse transciptased and amplified respectively, the average length of cDNA after purification and RsaI enzyme digestion is 500bp. cDNA of M and Y cells after RsaI enzyme restriction was regarded as tester and driver respectively, and then were divided into two fragments to ligate adaptor1 and adaptor2. The PCR products were ligated with pGEM-T plasmid vectors, transformed into E.coli TOP10. The positive recombinant clones were picked up through the blue-white screening system, and confirmed by PCR method with P7 primer. Finally, the differentially expressed cDNA fragments were sequenced and compared with EST database in Genbank.Results: 751 expressed sequence tags(ESTs)were obtained in M+Y library(that means ESTs were over expressed in M-phase, low expressed or no expressed in Y-phase), the average length is 690.98bp. Meanwhile, 875 ESTs were obtained in Y+M library, the average length is575.9bp. After splicing of ESTs, 101 unigenes were obtained in M+Y library and 249 unigenes in Y+M library. However, several repeated sequences were found by Blastn analysis in both two subtractive libraries. Finally, 45 unigenes in M+Y library and 91 unigenes in Y+M library were obtained after the removal of repetitive sequences.During the construction of cDNA subtractive libraries, the ?distribution of differently expressed genes varied with dimorphic transition. The over expressed genes with diversity and complexity were divided into four types.(1)Structural genes, such as 18S, 25sRNA gene_AJ496242.1,_FJ882050.1,_AF343676.1, some expressed only in yeast or mycelium phase, were the consequence of phase transition of Sporothrix schenckii.(2)Metabolic enzymes,such as gb|CP001656.1, _ref|NM₀01006571.1, _DQ298384.1, which participated in carbohydrate, amino acid, lipid and coenzyme transport and metabolism(.3)Molecule on cell surface, such as gi|30721613|,_gi|45383668,_gb|GQ379464.1|, which participated in signaling transduction of cell, could modulate the expression of genes or morphogenesis of cells(.4)Molecule with indistinct function, such as gb|DQ510714.1| _gb|EU923089.1| _gb|DQ298391.1|.Construction of cDNA subtractive libraries between yeast and mycelium phase of S.schenckii and bioinformatics analysis could play a role in the understanding of the phase transition mechanisms.