The Targeted-deletion Technology in the Monascus ruber Mediated via Agrobacterium tumefaciens / 微生物学通报

Monascus spp.,a kind of filamentous fungi,produce abundant of important metabolites which were widely used in the fields of food and medicine.Until now,there are few reports on the important functional genes of the Monascus spp.due to little genetic information.In this paper,the feasibility of gene deletion mediated via Agrobacterium tumefaciens on the basis of homologous recombination was analyzed by studying on the deletion of the RGS domain of putative G-protein signaling regulator gene mrfA in Monascus ruber.The length of homologous arms of deletion vector pC805S were 958 bp and 824 bp,respectively.There were 26 transformants in which homologous recombination occurred in 138 transformants and the recombination rate was 18.8%.The result showed it was feasible to identify the function of unknown gene in M.ruber with the targeted-deletion technology mediated via A.tumefaciens.

Detection of Salmonella spp.,Escherichia coli and Staphylococcus aureus by Multiplex PCR / 微生物学通报

According to DNA sequences of the invA gene of Salmonella spp.,the phoA gene of Escherichia coli and the nuc gene of Staphylococcus aureus,three pairs of oligonucleotide primers were designed and synthesized to amplify the special DNA sequences by multiplex PCR. Moreover,the reaction conditions of multiplex PCR were optimized. The results showed the multiplex PCR using the three pairs of primers produced specific amplicons of expected sizes,284bp for Salmonella spp.,622bp for Escherichia coli,484bp for Staphylococcus aureus. The optimized reaction conditions followed as the concentration of primer 40nmol/L for Salmonella spp.,40nmol/L for Escherichia coli,80nmol/L for Staphylococcus aureus,2.4mmol/L Mg 2+ ,200?mol/L dNTP,1.5U Taq DNA polymerase,anneal temperature from 55.0℃ to 57.4℃. Under the condition,the detection limits for DNA template were 10.2pg,10.2pg and 102.0pg for Salmonella spp.,Escherichia coli and Staphylococcus aureus,respectively. The whole process could be completed within 4h. The multiplex PCR assay was a specific,sensitive,rapid and reliable method for detecting Salmonella spp.,Escherichia coli and Staphylococcus aureus,which establish important foundation for simultaneous detection for these three bacteria in food.

Screening Citrinin Mutants from the Transformants Library of Monascus ruber M-7 by Agrobacterium-mediated DNA transfer / 微生物学通报

200 citrinin mutants were screened with the inhibition zone method from the transformants library of Monascus ruber M-7 by Agrobacterium-mediate DNA transfer, which contains more than 5,000 transformants.Then 53 mutants, whose citrinin contents ranged from 0.04?g/g to 154.57?g/g in the red fermented rice (RFR), were achieved by high performance liquid chromatography (HPLC).Color values of RFR prepared by these mutants were also detected.The results showed that there was a positive correlation between the citrinin content and the color value among the mutants.These results provide materials and research bases for ferrther studying the relationship between the production of citrinin and pigment of Monascus ruber at molecular level.