| Objective (1) The aim of this research is to explore the effect of L-DOPA on Yeast phase Penicillium marneffei(PM), from the observation of the culture liquid color, colony morphology and color, morphology by optical microscope and Tyrosinase gene expression by fluorescence quantitative PCR,for study the role of L-DOPA in different culture medium on Yeast phase Penicillium marneffei. (2) A method for simultaneous determination of Aflatoxin, gliotoxin and josamycin was established by Ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)and whether Penicillium marneffei can produce them.Methods (1) The yeast phase of PM standard strains of FRR and clinical isolated strains of GXMUDXR were grown on Sabouraud medium, brain heart infusion agar(BHI)and DMEM medium which containing 10% fetal bovine serum supplemented with and without 1.0mM L-Asparagine(L-DOPA), after cultured protected from light at 37℃ for 3 days, the color of colony and culture medium, morphological changes was observed by optical microscope, the expression of Tyrosinase gene was determined by fluorescence quantitative PCR. (2) The UPLC separation was performed on a Zorbax RRHD Eclipse Plus C18 column(2.1 mm×50 mm,1.8μm)by using methanol and water which contained 0.1% formic acid as mobile phase with the gradient elution at a flow rate of 0.3 mL/min. The analytes were detected by tandem mass spectrometry under the positive ion mode with the ESI source and carried out in the multiple reaction monitoring(MRM)mode. The yeast phase of PM standard strains of FRR and clinical isolated strains of GXMUDXR were yeast phase cultured on DMEM liquid culture with 10% fetal bovine serum for 7 days,after pre-processed and then detected of active ingredients such as aflatoxin, gliotoxin, josamycin.Results (1)Two tested strains on Salmonella solid medium grown at 37℃ protected from light for 3 days,there are no difference in the color of the solid medium with and without L-DOPA.(2)The tested strains without L-DOPA colony on brain heart infusion agar grown at 37℃ protected from light for 3 days,the color was lighter than which with L-DOPA, the difference was significantly.(3)The tested strains without L-DOPA colony on DMEM solid medium which containing 10% fetal bovine serum grown at 37℃ protected from light for 3 days, the color was lighter than which with L-DOPA group and the DMEM with 10% FBS culture which only with L-DOPA also became dark.(4)Optical microscope observation:Two PM yeast cells on all medium of with and without L-DOPA at 37℃ after 3 days, there were no significantly difference in the shape, size and color. (5)The expression of TYR gene by fluorescence quantitative PCR:a.BHI culture medium group:with and without L-DOPA of the PM strains of Bamboo rat and clinical isolate,TYR gene expression were higher than those of Sabouraud medium group, the difference was statistically significant(P< 0.05); and BHI with L-DOPA group was higher than that without L-DOPA group, the difference was statistically significant (P< 0.05).(b) The DMEM group with 10% fetal bovine serum,neither with or without L-DOPA of the PM strains of Bamboo rat and clinical isolate,there were no difference in TYR gene expression compared with Salmonella group(P> 0.05). There were also no difference between DMEM group with and without L-DOPA(P>0.05).(5)Under the optimum conditions, compare to standard,the peak curve of similar to aflatoxin and gliotoxin were not detected, but each sample even blank control,a peak curve similar to josamycin was detected, the intensity is far less than the standard.Conclusion (1)PM strains produce more pigment on BHI culture when supplemented with L-DOPA.(2) The supplemented with exogenous L-DOPA increase PM expression of Tyrosinase gene on BHI culture.(3)The ingredients such as aflatoxin, gliotoxin, josamycin unable detected in Penicillium marneffei in this research. |
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