Study of dimorphism and the DNA fragment homologous to S1 region of FLO11 gene on yeast Pseudozyma sp. SE4-R

• Main Authors: Shang-jung Lin, 林尚蓉
• Other Authors: Pi-han Wang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/46600723559375985893

碩士 === 東吳大學 === 微生物學系 === 93 === The environmental stress causes some changes of cell adhesion or cell physiology and even cell morphology﹙such as dimorphism﹚of fungal cells. Nitrogen depletion activates two signalling transduction pathway, cAMP-dependent PKA pathway and MAPK pathway in S. cerevisiae, positively regulating the transcription of downstream target gene FLO11 result in filamentous growth of yeast cell. A dimorphic yeast strain, SE4-R was obtained from screening laboratory-stored yeast strains collected from the environment. Growing on rich YM medium, SE4-R has ovoid, round, lemon-shaped or spindle-shape yeast form cells, they are polar or bipolar budding. SE4-R was identified as Pseudozyma sp. based on cell morphology, physiological and biochemical characteristics, also on LSU 26S rDNA and ITS sequence information. Growing on poor medium MBS, SE4-R changes it’s morphology from yeast form to hypha cell (R1) and hyphae morphology remain stable; when growing on TMBS medium, a MBS medium with ten fold calcium concentration (6.8mM), appeared pseudo-/hyphae filament (R2) in the edge of cell colony and yeast form cells remain as budding cell in the middle of the R2 colony. Designing STA1/STA2 primer pair according to the sequence of conserved S1 region in FLO11 gene in S. cerevisiae, STA1/STA2 primers can amplify a 476 bp fragment (C1-5) from S. cerevisiae genomic DNA. After sequencing, fragment C1-5 is surely part of the S1 region sequence. Using DIG-labeled C1-5 fragment doing dot-blot hybridization to SE4-R genomic DNA, a slight hybridization signal can be detected which indicate that the homology sequence may exist in SE4-R genome but in quite small amount. Indeed, STA1/STA2 primer can amplify a 549 bp fragment (SEpp) from SE4-R genomic DNA by booster PCR.The sequence of SEpp has 94% similarity to the one of C1-5. In order to verify if FLO11 gene expressed differently in yeast form cell and filament cell, SEpp fragment was DIG-labeled and hybridized to the RNA of YF, R1, R2 cells. But FLO11 gene expression can’t be detected in any one of three morphology type cell. In addition, Affymetrix Yeast S98 microarray is used to analyze filamentation associated genes in wider aspect. It has high dendity probes arrayed on the Affymetrix chip. After analysis of the microarray, there is only one gene in R1 with 2-fold chang higher than YF cells. PHD1 is a transcription activator with main function in regulating the expression of FLO11 gene and enhancing pseudohyphae growth in S. cerevisiae. It is homologous to STUA gene of Aspergillus nidulans and EFG1 gene lf Candida albicans. There is no any result in microarray analysis shows any gene similar to FLO11 which can correspond to the result in the previous study. There’re still some genes found related to filamentous growth induced in R1 and R2 cells, indicated that although there may be some difficulties to identify more genes with 2-fold change expression, some useful information still can be obtained.