Establishment Penicilium Marneffei Gene Transformation Technology & Deletion HOG1 Gene In Penicillium Marneffei And Study Its Functions

Objective Through established DNA-mediated gene transformation technology in Penicillium marneffei (P.marneffei), deleted H0Gl gene and observed the dimorphism switched and other function changes in P.marneffei hogl deletion strain compared to its parents strain. This research work will help us understand the function of HOG1 gene in P.marneffei and shed light on the molecular mechanism of pathogenesis in P.marneffei infection.Methods P.marneffei strain SPM4 (pryG, niaD) was used in the study, which is unable to grow on nitrate as a sole nitrogen source and is a uracil auxotroph. We extracted DNA and RNA, total RNA reversely transcribed into cDNA and cloned HOGl gene in P.marneffei. Constructed HOGl gene deletion cassette and transformation vector by fusing PCR. SPM4 protoplast was prepared by chemical method, on this base we established gene transformation technology and deleted HOGl gene of P.marneffei. Positive clones of hogl deletion strains which grown on the selection medium were certification by PCR. In the meantime we observed the dimorphism switched and other function changes in the hogl deletion strains compared to its parents strain. Results P. marneffei HOGl gene full-length is 2446bp, comparative analysis of the nucleotide sequence of genomic DNA and cDNA confirmed the presence of contents 8 exons and 7 introns, mRNA length is 2046bp and open reading frame extended to 1035 bp, encoding putative protein of 344 amino acid protein. The deduced amino acid sequence was homologous to HOGl from others fungi and had the highest similarity with Aspergillus fumigatus, was 96%(Figure 10). We succeed in constructed HOGl gene deletion cassette and vector by fusion PCR and transform the vector into P. marneffei protoplasts prepared by chemical method used uracil (pryG) as the selection marker, cultured in selection growth medium without uracil to screen positive transformants and further verified using PCR. five hogl deletion strains was successfully obtained. However, preliminary functional studies showed HOGl gene in P. marneffei had no effect on dimorphic switch.Conclusion We succeed in establishment the DNA-mediated gene transformation and gene knockout technology of P. marneffei. Cloned the HOGl gene of P. marneffei and found it was homology with other species of fungi. Preliminary functional studies showed hogl deletion strains had no effect on the dimorphic switch of P. marneffei.