Cloning Of GABA And Ctirinin Meatbolism-Related Genes

Monascus spp. is a important genus of brewing microbiology in China, and has been used for centuries. In recent years, multiple efficacities of Monascus spp. successively were confirmed. It was widely used in medical care, food processing and so on due to its production of many kinds of metabolites, such as y-aminobutyric acid (GABA), monacolins, Monascus pigment. However, in 1995 professor Blanc found that there was a mycotoxin citrinin in red fermented rice, which led to the certain limitation to Monascus products both at home and abroad. Recently, production, regulation and application of some metabolites of Monascus, such as GABA, citrinin have become a research hotspot in China and abroad. Although progress was relatively slow, namely, the majority of research findings temporarily cann’t be applied to manufacture and technological means was in establishment stage, study on metabolic genes regulating GABA and citrinin will be a essential foundation for product-regulating of Monascus metabolites.The Monascus ruber strain Mr-5(CGMCC NO. M208043) was used as the original strain for transformation, and 1478 transformants was generated by Agrobacterium tumefaciens-mediated transformation technology(ATMT). TLC and HPLC of GABA in Monascus spp. were established.50 GABA mutants were selected from 1478 mutants, which were markedly different from the original strain Mr-5 in colony morphologies. Then, the flanked T-DNA sequences of some GABA mutants were isolated by thermal asymmetric interlaced PCR and high-efficient thermal asymmetric interlaced PCR. Finally, software was used to analyze the function of the cDNA sequence. The main research contents are as follows:1. Agrobacterium-mediated Monascus transformationIn this study, some critical factors which influenced transfer efficiency, such as concentration of Agrobacterium tumefaciens, induction time, concentration of inducer acetosyringone, incubation time of Monascus, concentration of Monascus spores, co-cultivation time, tempertature and so on, were evaluated. The optimal transformationconditons were as follows:The original strain of Monascus was cultivated on the PDA medium for 21 d at 28 ℃ then the Monascus spores were collected and diluted to 105/mL with Agrobacterium tumefaciens AGL-1 suspension, whose optical density at 600 nm was 0.5. The mixture was co-cultured at 25℃ for 2.5 d with 50-200 μmol/L inducer acetosyringone (AS).2. Genetic stability, colony and mycelial morphology of transformants50 transformants randomly chose from 1478 transformants library were respectively cultured six generations on PDA medium, and then transferred to PDA medium containing 150 μg/mL hgyromycin B with original strain Mr-5 as control. PCR analysis for detection of gene hph in transformants was performed. The results showed that 96% transformants was stability, and the T-DNA inserted successfully. After observing the colony and mycelial morphology, the results displayed that most transformants were the same as the original strain, and only a few changed greatly in those areas including colony color, colony character, colony growth rate and microscopic features.3. Selection of GABA-producing mutantsPaper chromatography and TLC was established for determination of GABA and citrinin in fermentation broth of 250 mL erlenmeyer flask, respectively. After finding some transformants changed comparatively in GABA and citrinin, the broth belonging to these strains was analyzed with HPLC. Finally,50 GABA and citrinin mutants, which had great different in GABA contents, were selected from 1478 mutants by paper chromatography, TLC and HPLC.4. Amplification of transformants T-DNA flanking sequence and bioinformatics analysisInsertional T-DNA flanking sequence of M. ruber transformants was amplified with TAIL-PCR and hiTAIL-PCR. Three nested primers, seven degenerate primer and five long degenerate primer were designed by right-flanking sequence of T-DNA. After amplification, sequencing and analysis by Blast, The results shown that the comparability of T-DNA flanking sequence of transformant strain 82 and anthranliate isomerase(trpC) tryptophan synthase beta subunit were 66-99%, comparability of T-DNA flanking sequence of transformant strain 1358 and putative cytochrome C peroxidase were 85%, comparability of T-DNA flanking sequence of transformant strain 1338 and probable 6-phospho-beta-glucosidase were 99%, T-DNA flanking sequence of transformant strain 660 had highly similar with glutamate decarboxylase(GAD) of various bacteria and fungus, especially, had 79% similarity with Aspergillus niger, had 78% similarity with Aspergillus fumigatus, had 76% similarity with Aspergillus clavatus.5. The amplification, validation and bioinformatics analysis of GADComparing amplification sequence of TAIL-PCR with cDNA of Mr-5, design primers and genome as a template, G2-1were amplified by PCR. Upstream and downstream sequences has already validated, intron and exome was confirmed, proved G2-1 was integrated DNA section of Mr-5 genome. Analysis of the gene sequence and deduced amino acid sequence by Orf Find、Conserved Domains、Blastp、ClustalX and others.