| Studies show that Monascus spp. can Proudce different kinds of bio-active subsrtates, such as a group of edible pigments, Monacolins, γ-Aminobutyric Acid, sterols, monounsaturated fatty acids, protease and isoflavones, which has been widely used in food industries and medicin, but few studies were related to the study on molecular biology of Monascus.In this research, the M. purpureus strain Mp-41which was isolated from red rice was used for transformation experiment, and constructed a transformation library of Mp-41mediated by Agrobacterium tumefaciens. According to observing the biological characteristics, Some transformations were selected and the main metabolites-Monascus pigment was analyzed. After then, two DNA fragments from the transcriptional start region and the stop codon region of pksCT gene were gained from M. Purpureus transformation by genomic PCR.As follows were the main results of this research:1. Optimizing condition of A. tumefaciens-mediated transformation, some critical factors which could obviously affect transformation rate, were evaluated.the factors containe:medium, culture time, and spores of strain Mp-41; concentration of A. tumefaciens; co-cultivation membrane, temperature and time of spores of strain Mp-41and A. tumefaciens; concentration of inducer acetosyringone, and so on. The result indicated that the effective transformation contains were as follows:at30℃, cultivating original strain M. purpureus Mp-41on the DE medium for20d,100μmol/L AS, optical density value at600nm of A. tumefaciens was0.7, concentration of strain Mp-41spores was104/mL co-cultured with A. Tumefaciens for3d at 25℃on NC membrane. More than1000transformants were amplified under this condition in the transformation library.2. Genetic stability and PCR identification of transformants.60transformants were choosed randomly from transformation library, cultured six generations on DE medium without hygromycin B, after then, transferred all the transformants to DE medium, which contains200μg/mL hygromycin B. As control, original strain Mp-41with the same. Meanwhile, detection of gene hph in transformants was performed through PCR analysis. The results showed that almost all the tested transformants keeped the resistance stability, and what’s more, the PCR identification confirmed that the T-DNA has successfully integrated into transformants genome.3. Biological characters research of transformants. the expression of certain genes would be differently affected after integration of T-DNA into M. purpureus genome. In order to studying the colony morphology and mycelial morphology of transformants, culturing the transformants for7days at30℃on DE、ME A and CYA medium with original strain M. purpureus Mp-41as control, and then, observing the biological characteristics of the transformants. The results showed that compared with original strain Mp-41, most transformants were the same with it, howere, there’re a few transformants’ mycelial morphology and colony morphology had significantly changed.4. Analysis of Monascus pigment and citrinin produced by transformants.33randomly selected transformants whose colonial and mycelial morphology diversified obviously from original strain M. purpureus Mp-41were fermental cultivated for7days at30℃under liquid fermentation and solid-stated fermentation.Using UV-VIS, TLC, HPLC and other methods to measure transformants’ability of producing pigment and citrinin. The results told that, compared with the original strain M. purpureus Mp-41, the32transformants’ capacity to produce pigment and citrinin was differently changed. Among32transformants,17transformants’colour value were obvioustly higher than the original strain Mp-41at505nm,especially, the colour value of Mp-41-155was up to115.98U/mL under liquid fermentation,which was almost1.75times than that of Mp-41, and about1.58times than that of Mp-41under solid-stated fermentation; and the other15transformants were obvioustly lower than Mp-41, especially, Mp-41-62’s color value of was2.71U/mL, which was the lowest, having been decreased by0.04fold. Meanwhile, transformants changed not only in color value, the result of UV-VIS scan map in300-600nm showed that they also changed in absorption wavelength and peak. For instance, in the original strain Mp-41, there were two absorption peaks, but there were three absorption peaks in Mp-41-81、Mp-41-83、Mp-41-84、 Mp-41-85、Mp-41-88、Mp-41-89、Mp-41-90、Mp-41-91、Mp-41-92、Mp-41-150、Mp-41-151、 Mp-41-152、Mp-41-153、Mp-41-155、Mp-41-156、Mp-41-157、Mp-41-160、Mp-41-161、 Mp-41-162、Mp-41-163、Mp-41-164、Mp-41-165、Mp-41-169, and only one absorption peak in Mp-41-62and Mp-41-86. The ability to produce Citrinin33were significantly different from the original strain M. purpureus Mp-41,7transformants were lower than Mp-41, further more, Mp-41-62and Mp-41-86’s Citrinin was not detected, which might because their ability to produce Citrinin were lower than0.2μg/g.5. Analyzing conservativeness for citrinin biosynthesis-related gene pksCT from transformants. Recent studies shows that the synthesis of Monascus pigment shares the same biosynthetic pathway with citrinin biosynthesis in M. Purpureus. pksCT gene was a related-gene responsible for citrinin biosynthesis in M. Purpureus. In this research,32strains selected from transformants library were used. Two DNA fragments from the transcriptional start region and the stop codon region of pksCT gene were obtained from M. Purpureus transformation by genomic PCR,which were659bp and591bp respectively. Results of being compared with genome databases at GenBank of the NCBI shows that PCR products exhibited high identity with each other, and the similarity of the sequences was up to99%with the partial sequences of pksCT gene in M. Purpureus which was announced in the GenBank. So, pksCT gene was a general gene responsible for citrinin biosynthesis in Monascus spp.,the results can be used as research subject of Monascus pigment-related genes in the future. |
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