Construction Of T-DNA Insertional Library Of Monascus Ruber And Cloning Of γ-Amino-Butyric Acid Metabolism-related Genes

Monascus ruber is an important microbial resource in China, and has been widely used for centuries. Recent studies shows that Monascus contained many kinds of useful compounds, such as y-aminobutyric acid (GABA), Monacolins, sterols, isoflavones, monounsaturated fatty acids, protease, and a group of edible pigments, which could promote the isolation of novel strains for the use in food industries and medicine. As one of the metabolites of M. ruber, GABA has several functions such as fighting anxiety, decreasing blood pressure, improving brain function, promoting growth hormone secretion, activating hepatorenal function and so on, and has a very broad prospect in food industry and pharmaceutical industry. At present, the investigation about M. ruber was focused on its mutation breeding, etabolites isolation and fermentation conditions to improve useful metablites, and the study on molecular biology of M. ruber had gradually started, but many studies were still in the stage of exploration and establishment of methods. Currently, the genes involved in biosynthesis of citrinin and other secondary metabolites of M. ruber have triggered scientific interests. But, there has been little information available regarding the genetic information involved in metabolites synthesis in Monascus species, the genetic background of M. ruber was little known, and the biosynthesis genes of GABA in Monascus was still not clear.In this study, the M. ruber strain Mr-5isolated from red yeast rice was used as the original strain for transformation, and a T-DNA insertional library of M. ruber with more than1300transformants was generated by Agrobacterium tumefaciens-mediated transformation technology (ATMT). Some of them with significant changes were selected through the colony and micro-morphological observation, and the main metabolites of them were analyzed. On this basis, the genomic DNA fragment right-flanking the insertional T-DNA of M. ruber transformants was amplified with TAIL-PCR. The research contents were as follows: 1. Agrobacterium-mediated transformationIn this study, some critical factors which influenced transfer efficiency, such as concentration of Monascus spores, concentration of inducer acetosyringone, co-cultivation time and temperature and so on, were evaluated, and the effective transformation was acquired when original strain of Monascus was cultivated on the PDA medium for21d at30℃, concentration of Monascus spores was106/mL, optical density value at600nm of Agrobacterium was0.5, concentration of inducer AS was100μmol/L, Agrobacterium and Monascus spores were co-cultured on NC membrane for3d at25℃. On this condition, the number of transformants in the library was amplified to more than1300.2. Genetic stability, PCR identification, colony morphology and mycelial morphology of transformants50transformants randomly choosed from transformation library were respectively cultured six generations on PDA medium without hygromycin B, and then transferred to PDA medium containing200μg/mL hygromycin B with original strain Mr-5as control. At the same time, PCR analysis for detection of gene hph in transformants was performed. The results showed that the resistance stability of transformants was up to98%, and the PCR identification confirmed the successful integration of T-DNA into M. ruber genome.When T-DNA was inserted into the genome of M. ruber, it would affect the expression of certain genes, causing phenotypic variation, and these variations would be reflected in all aspects of M. ruber. So, it was important to study the colony morphology and mycelial morphology of transformants. The transformants were respectively cultured on PDA medium for7days at30℃, and were observed the colony morphology and mycelial morphology. The results showed that most transformants were the same as the original strain, and only a few changed in those areas including colony color, colony character, colony growth rate, form of aerial hyphae, conidia and ascospores.3. Analysis of main metabolites of transformantsPaper chromatography, UV-VIS, TLC and other methods were used on the mensuration of GAB A, pigment and citrinin produced by14transformants. The results showed that, the capacity of transformants to produce metabolites was changed in different degrees compared with the original strain. Among14transformants, the ability to produce GABA of13transformants were stronger than the original strain Mr-5, and GABA output of Mr-5-50was up to3.161g/L, which was almost4.37times than that of Mr-5; The color value of4transformants at505nm were significantly higher than the original strain Mr-5, and2transformants were significantly lower than Mr-5. Among them, the color value of Mr-5-155was the highest, which was almost3.2times than that of Mr-5, and Mr-5-33was the lowest, which was less than fourth of Mr-5. At the same time, the results of UV-VIS scan map in300-600nm indicated that transformants changed not only in color value, but also in absorption wavelength and the absorption peak. For example, there were three absorption peaks in Mr-5-7、Mr-5-35、Mr-5-38、Mr-5-54、Mr-5-59、Mr-5-97、Mr-5-155, and only one absorption peak in Mr-5-46and Mr-5-50, almost no absorption peak in Mr-5-158, but in the original strain Mr-5, there were two absorption peaks. Citrinin production of5transformants were lower than the original strain Mr-5, and citrinin production of Mr-5-50was not detected under the experimental condition, which might lower than0.2μg/g.4. Identification of T-DNA flanking sequences and Bioinformatical analysisThe genomic DNA fragment right-flanking the insertional T-DNA of M. ruber GABA transformants was amplified with Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). A405bp right-flanking sequence of transformant Mr-5-72named gabaX-72was obtained and compared with genome databases at GenBank of the NCBI. The similarity of its protein sequence was up to98%with the hypothetical protein AN7090.2of Aspergillus nidulans FGSC A4. According to the previous study, transformant Mr-5-72exhibited fluffy aerial hyphae, and its conidia forming capability, radial growth rate, pigment and citrinin production was almost the same as the original strain Mr-5, but the content of GABA in liquid fermentation by transformant Mr-5-72was four times as that in the original strain Mr-5. As a result, T-DNA intergration most likely affected GABA synthesis pathway in transformant Mr-5-72. It was necessary to study its potential information deeply.