Obtaining vacuolar protein sorting 34 gene in Sporothrix globosa by RACE method and analysis of its expression during the mycelium-to-yeast transition

Abstract

Objective To obtain the full-length sequence of the vacuolar protein sorting 34 coding gene (vps34) of Sporothrix globosa (S.globosa) and to investigate the role of vps34 gene during the phase transition from mycelium to yeast in S. globosa. Methods The 3′ end and 5′ end of the vps34 gene of S. globosa were amplified by rapid amplification of cDNA ends (RACE). The obtained sequences were spliced and analyzed by bioinformatics software. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze the expression of vps34 gene in mycelial and yeast phases. Results The vps34 gene of S. globosa was 3 228 bp in length. The coding sequence was 3 000 bp and encoded 999 amino acids with a molecular mass of 111.49×103 and an isoelectric point of 6.38. It contained three domains including C2_PI3K_class_Ⅲ, PI3Ka_Ⅲ and PI3Kc_Ⅲ. The results of qRT-PCR showed that the expression of vps34 gene in yeast-phase S. globosa was higher than that in mycelial phase at 24 h (P<0.05), and the greatest difference between them was observed at 48 h (P<0.01). Conclusions Vps34p participates in the process of dimorphic transformation of S. globosa. The obtainment of the full-length vps34 gene of S. globosa lays the foundations for further study on the function of Vps34p. Key words: Sporothrix globosa; Sporotrichosis; Vacuolar protein sorting 34; Rapid amplification of cDNA ends; Quantitative reverse transcription PCR; Dimorphic transformation