Study On Molecular Cloning And Function Of Mrr In Monascus Ruber

Monascus spp., used for red rice fermentation, is a kind of fungi with high commercial values due to its production of many kinds of physiological active substances, such as monascus pigment, Monacolin K,γ-aminobutyric acid and ergosterol. At present, the investigation about Monascus spp. was focused on its classification, breeding, fermentation conditions and citrinin detection, but the study on molecular biology of Monascus spp. is at the beginning stage. The investigation of functional genes about Monascus spp. was focused on the genes related to the metabolic regulation of pigment, citrinin and Monacolins K.The two component system which exists in bacteria, plants and fungi comprises two protein components, a histidine protein kinase and a response regulator protein. Study showed that the roles of the two component system were involved in oxidative stress response, osmotic response and spore formation in filamentous fungi. Until now, there was no report about the genes involves in the two component system in Monascus spp..The T-DNA inserted transformant library of Monascus ruber M-7 containing more than 10000 transformants was constructed in preliminary studies of our laboratory. In this study, 8 T-DNA inserted mutans were selected, and the colony morphologies, microscopic morphologies were observed. After that, the T-DNA flanking sequences of four mutants were isolated by TAIL-PCR, and one of the four isolated sequence was extended by SON-PCR. 5 genes were cloned and the coding protein of mrr, one of the five, has high homology to the response regulator protein of Aspergillus spp.. Finally, the function of mrr was analyzed by gene knock-out. The main research contents are as follows:1 Isolation of the the T-DNA flanking sequences of Monascus ruber mutantAccording to the comparison between 143 T-DNA inserted mutans and M-7, 8 mutants with different colony morphologies were selected. The microscopic morphologies were observed, and the DNA sequences flanked T-DNA border of 8 mutants were amplified by TAIL-PCR and the T-DNA flanked sequences of 3440^#, 2341^# and 3725^# were successfully isolated. 726bp isolated from 3192^# has a 101bp region of overlap with Aspergillus nidulans stress response regulator. A sequence with length of 13649bp was cloned by extending the flanked sequences of the 726bp region by SON-PCR, and in the sequence, 5 genes were found to have more than 80% homology with Cog6, Nif3, BolA, AhpC/TSA family and response regulator protein, respectively. 2 gene knock-out of mrrThe mrr(Monascus ruber response regulator) encoding the response regulator protein contains 2356bp with 6 exons and 5 introns, and the dedued amino acids sequence of mrr contained 634 amino acids with a receiver domain and a DNA binding domain which both were the basic components of response regulator protein of the two component system. To analyze the funtion of mrr in Monascus spp., the gene knock-out vector pCMRR was constructed and transformated into Monascus ruber M-7 by the method of Agrobacterium tumefaciens mediated transformation. Finally, 3â–³mrr mutants was obtained.3 Primarily study on the function of mrrThe characters ofâ–³mrr mutant were studied and the results show that the growing rate ofâ–³mrr mutant was fast than M-7, while the sexual development ofâ–³mrr mutant was slower. Mrr was not involved in osmostress resistance but was required for H₂O₂ resistance, the maximum tolerated concentration ofâ–³mrr mutant was less than 0.3mmol/L, while the M-7 is higher the 0.75mmol/L, mrr was presumed to be related to oxidative stress signal transduction and sexual reproduction of Monascus spp..