Cloning And Analysis Of The Genes Flanking The Avermectin Biosynthetic Gene Cluster

Polyketides are produced by various organisms such as bacteria, fungi, or plants, a subgroup of natural products. Polyketides are among the most important microbial metabolites in human medicine, since they exhibit the largest diversity in chemical structures and biological activities. They are in clinical use as antibiotics, anticancer drugs, cholesterol-lowering agents, antiparasitics, antifungals, insecticides and immunosuppressants. As one of the antiparasitics, avermectin is produced by Streptomyces avermitilis, a 16-membered macrolide antibiotic which is composed by a series of eight related homologous compound. Avermectin exhibits extraordinarily potent anthelmintic activity, is widely used for the treatment of diseases caused by nematodes and arthropods in veterinary and agricultural fields. So avermectin owns a very high value in research, development and the commercial field. However, with the increasing of resistant strains and avermectin has no antibacterial activity to the bacterium and fungi, it is high time that we pay much attention to the promotion of the avermectin or the production of the new compounds which hold the similarity of avermectin.The experiment is the preliminary work to the whole topic. According to the feature that Streptomyces avermitilis genome is rich of GC base pairs, the system and procedure of PCR were established and optimized. According to the genome sequence (GI: 57833846) of Streptomyces avermitilis which is provided by GenBank database, two pairs a total of four primers, averU-A, averU-B, averL-A and averL-B were designed by using Oligo 6.69 software. Using Streptomyces avermitilis chromosome as template, the target gene, designated"averU", between SAV934 and aveR of the avermectin biosynthetic gene cluster upstream of which the length 1011bp and another target gene, designated"averL", between aveG and yerD1 of the avermectin biosynthetic gene cluster downstream of which the length 651bp were amplified. Two plasmids, pAVERU and pAVERL, were constructed by using averU and averL with vector, respectively. Similarly, using plasmid pAVERU as template, two target genes, designated"averU1"and"averU2", were amplified by usage of averU1-A, averU1-B, averU2-A and averU2-B as primers. The length of averU1 and averU2 is 504bp and 507bp, respectively. Two plasmids, pAVERU1 and pAVERU2, were constructed by using averU1 and averU2 with vector, respectively. Based on the experiment above, the plasmid pAVERU1 and pUC119_KanR were doubledigested by endonuclease of EcoRâ… and Kpnâ… . And the recombinant plasmid pkAVERU1 was constructed by using T4 DNA ligase after reclaiming target genes, averU1 and pUC119_KanR (cut). The recombinant plasmid pkAVERU2 was constructed by usage of the similar methods as well as the recombinant plasmid pkAVERU1.In summary, this study has successful completed the expected task, providing a nice surrounding for the followe-up experiment, and laid a good foundation for the experimental work of the whole issue.