| Red fermented rice is a traditional fermentation food of rice by Monascus spp., which has been used for more than one thousand years in China. It is widely used in vintage, food colorant, function food, food fermentation starter and medicine. Besidesγ-Aminobutyric Acid (GABA), Monascus spp. can also product hongqu pigment, monacolins and ergosterol. At present, the investigation about Monascus spp. was focused on its mutation breeding, metabolites isolation and fermentation conditions to improve useful metablites, but the study on molecular biology of Monascus spp. is still at the beginning stage. The research on GABA of Monascus spp. is also about the fermentation conditions.In this paper, TLC and HPLC-FL of GABA in Monascus spp. were established. 10 GABA mutants were selected from 53 mutants, which were markedly different from the original strain M-7 in colony morphologies. These 53 mutants were selected from the transformants library of Monascus ruber M-7 before. Then, the left flanked T-DNA sequences of 3 GABA mutants were isolated by thermal asymmetric interlaced PCR. Finally, RNAi was used to identify the function of the cDNA sequence from 805#. The main research contents are as followes:1. TLC was established for determination of GABA in red fermented rice. Monascus ruber. The optimum conditions of TLC were ascertained by comparing the extraction method and the type of developing solvent, which was the water as the extraction reagent, V95% ethanol:V25% ammonia=4:1 as developing solvent and 0.2% ninhydrin-ethanol as chromogenic reagent. Under this condition, the detection limit was 0.2μg.2. HPLC-FL was established for determination of GABA in red fermented rice. The HPLC system with fluorescence detection (FL) was used to examine GABA formed by a reaction with o-phthalaldehyde (OPA). The optimal chromatographical conditions were as follows: column (chromasil C18, 5μm, 250×4.6mm); Mobile phase (K2HPO4(0.1 mol/L, pH6.0): CH3OH:CH3CN=6:3:1); flow rate (1.0 mL/min); column temperature (35℃); injection volume (20μL); fluorescence detector (λex =340 nm,λem =512nm). It can detect the GABA concentration ranged from 0.1μmol/L～100μmol/L, the relative standard deviation (RSD) and average recovery were 0.48% and 95.12%, respectively.3. 10 GABA mutans, including 189#, 192#, 533#, 635#, 733#, 805#, 1164#, 1263#, 3257#, and 4096#, which had great different in GABA contents, were selected from 53 mutants by TLC and HPLC-FL. The highest was 1263#, which was 6.184 mg/g and was 14.2 times of that of M-7. The amount of 635# was the lowest to 0.156 mg/g, and was just 0.33 times of that of M-7. The citrinin contents of those GABA mutants were also analysed. The amount of citrinin were between 0.18μg/g to 149.51μg/g.4. PCR with primers designed according to the Gus gene of T-DNA was performed to identify weather the 10 mutants had been inserted by T-DNA. The results showed all the mutants were inserted by T-DNA.5. The DNA sequences flanked T-DNA left border of were amplified by TAIL-PCR and the DNA sequences of 189#, 805# and 3257# were successfully isolated and the length of isolated DNA fragments was ranged from 500 bp to 1 300 bp. After Blast analysis, we found that the amplified DNA from 805# carried the RGS functional domain of regulator of G-protein signaling, the similarity of which was up to 84% with that of Aspergillus fumigatus. There was no significant homology in other 2 strains.6. The function of the total eDNA sequence of 805#, which was obtained by Dr. Shao Yanchun, was identified by RNAi. The results showed that the mutants with RGS domain deletion produced little hongqu pigment and the content of GABA was higher than M-7. The isolated flb A may regulate the GABA and hongqu pigment.
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