Screening Of The Related-genes For Antibiotic Biosynthesis In Micromonospora Carbonacea JXNU-1

Micromonospora carbonacea JXNU-1 is a strain of actinomyces with broad-spectrum antibacterial activity, and isolated from soil samples. The technology for purification of antibiotics from the fermentation broth of M. carbonacea JXNU-1 has been established in former studies, and the antibiotics was initially featured nucleosides by scientific test equipments, however it is not clear about the mechanism for antibiotic biosynthesis. The related-genes for antibiotic biosynthesis in M. carbonacea JXNU-1 were screened by genomic methods in this study. The specific contents and results are as follows:1.The genomic DNA of M. carbonacea JXNU-1 was extracted by optimized SDS methods, and sequenced by high-throughput DNA sequencing techniques. The genome was assembled with SOAPdenovo software. Multiplex PCR was used to close the gaps, and the genome sequence information was analyzed by bioinformatics methods. Finally a fine genome map of M. carbonacea JXNU-1 was completed. This Whole Genome Shotgun project has been deposited at Gen Bank under the accession number JXSX00000000.2.The total RNA of M. carbonacea JXNU-1 after cultivation for 36 h(before antibiotics secretion) and 108 h(after antibiotics secretion) were extracted by RNAiso reagent respectively. Two different c DNA libraries of M. carbonacea JXNU-1 MC_CT(cultivation for 36 h) and MC_ST(cultivation for 108 h) were constructed, and sequenced using Illumina Hiseq 2500 sequencer respectively, the obtained sequences have been deposited in the NCBI SRA database under the accession number SRP066689. Based on the sequences above, the differentially expressed genes were analyzed. Compared with cultivation for 36 h, 1072 genes in M. carbonacea JXNU-1 of cultivation for 108 h were differentially expressed. A total of 573 genes were up-regulated and 499 genes were down-regulated. Compared with the whole genome sequence of M. carbonacea JXNU-1, thirty most significant differentially expressed genes can be located into 13 biosynthetic gene clusters. Finally the gene cluster 12 related to antibiotic biosynthesis in M. carbonacea JXNU-1 was screened.3. The total RNA of M. carbonacea JXNU-1 from the mycelium of M. carbonacea JXNU-1 at the cultivation time of 108 h(antibiotics secretion) as tester and 36 h(non-secretion) as driver were extracted, and then double strand c DNA were synthesized by reverse transcriptions respectively. The subtractive c DNA library between antibiotics secretion and non-antibiotics secretion in M. carbonacea JXNU-1 was constructed sucessfully. Parts of positive clones in the library were sequenced and analyzed by BLAST software in GenBank. A total of 26 differential fragments were acquired, and then 5 genes were screened and identified by quantitative real-time PCR(q RT-PCR). According to the whole genome sequence of M. carbonacea JXNU-1, the 5 genes above can be located into 4 biosynthetic gene clusters. Eventually, the gene cluster 2 was screened as the most likely related to the biosynthesis of antibiotics in M. carbonacea JXNU-1.4.The gene cluster 12 from comparative transcriptome analysis was same as the gene cluster 2 from SSH analysis by BLSAT, therefore, it can be deduced tha the cluster is related to the biosynthesis of antibiotics in M. carbonacea JXNU-1, and the gene sequences have been deposited at Gen Bank under the accession number KU355271.The results may provide the experimental basis for further exploration of metabolic pathways and regulatory mechanism for bio-synthesis of antibiotic in M. carbonacea JXNU-1.