Construction Of The Avermectins-producing Streptomyces Avermitilis With AveD Gene Knock-out

This dissertation take Streptomyces avermitilis 2266 as the object of the research. In genome DNA, aveD from avermectins biosynthetic gene clusters was knocked out. Construct the recombinated plasmid pHD3 with aveD knock-out, and then pHD3 was used to transformed protoplasts of Streptomyces avermitilis. With exchange and recombination between aveD’and aveD in genome DNA, the aveD in genome DNA was knock-out, the recombination colonies were screened. So the components of fermentent product were changed.First, primers of aveD were designed and used to amplify the aveD from the genome DNA of Streptomyces avermitilis. The sequencing result of aveD was conformed. Then we cloned the aveD into pMD18-T(pHD1). Acoording to sequencing result of aveD PCR product, two restriction sites of BssHII were confirmed.BssHII was used to digest the aveD, aveD was knocked out(aveD’). The T4 DNA Ligase was used to recombine larger restriction fragment by itself to form a new recombination plasmid pHD2. The sequencing result of aveD’ conforms that pHD2 is correct.Shuttle plasmid pKC1139 was used as a carrier. Plasmid pKC1139 and pHD2 were both digested by HinIII and EcoRI, the digestion fragments of pKC1139 and aveD’were ligated by T4 DNA Ligase to construct the recombinated plasmid pHD3. According to sequencing result of PCR product of aveD’(incompleted aveD), pHD3 is correct.Plasmid pHD3 was used to transform E.coli ET12567. Plasmid pHD3 was get from E.coli ET12567 according to the color of colony.The protoplasts of Streptomyces avermitilis were made with strain 2266 by lysozyme. pHD3 was then transformed to protoplasts of Streptomyces avermitilis. The recombination colonies were got with the resistantce of Apramycin. Then the spore of the clones was used to culture on the YMS plates with apramycin and 40℃for 9 days to select out recombination colonies.And then the recombination colonies passaged by tow or three generations on the plates of YMS. The aim is to select the double cross-over colonies with aveD in genome DNA was knocked out and have no resistantce of Apr. The aveD of the double cross-over colonies was amplified by PCR.According to sequencing results of aveD from genome DNA of strain 489, it’s clear that aveD from genome DNA was double cross-over with aveD’.So strain 489 was the double cross-over recombination colonies and it’s successful we construct the recombination colony with aveD knock-out.