Dissertation > Industrial Technology > Chemical Industry > Pesticide Industry > Pesticides

The Construction of the Integrated Plasmids and the System of Transformation in Saccharopolyspora Spinosad

Author DuanYueJiao
Tutor LuWenYu
School Tianjin University
Course Biochemical Engineering
Keywords Saccharopolyspora spinosad construct integrated plasmids transtormation system
Type Master's thesis
Year 2012
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Spinosad is a new class of macrolide compounds produced by aerobicfermentation of the Gram-positive bacteria Saccharopolyspora spinosad. Spinosadhas won the the award of the U.S. Presidential Green chemicals Challenge in1999,because of their characteristics of low toxicity, low residue, fast decomposition, theyhave become a new class of highly effective biological insecticide. With the safety ofbio-pesticides and efficiency of chemical pesticides, they have been widely used. Onthe one hand, Its application prospect is perfect, but on the other hand, its productionis very low that is far from being able to achieve the lecel of industrialization due tothe restrictions existing in their own strains. Therefore, with the application of thegenetic engineering technologies, there do have been an important economic andenvironment value to build a stability and high-yield system.As the strict restriction modification system existing in Saccharopolysporaspinosad, which prevents the effective conversion of the exogenous gene. Genetictransformation has become one of the key limiting factors of its genetic manipμlation.It is therefore necessary to establish a stable DNA transfer system, which is aprerequisite for all other works invoving identification and analysis of the spinosadbiosynthetic gene cluster, and other genetic operations. There has been thephenomenon that for different strains of actinomycetes, the genetic manipμlationvaries very much. So this paper aims to construct a stable system that allow thevectors efficiently transform. Furthermore to provide a methodological basis for thegenetically engineering.This study has cloned the genes abaA, afsQ1-Q2, afsR2from Streptomycescoelicolor and gdh from Saccharopolyspora spinosad to establish the transformationsystem. This study has constructed two sets of plasmids, one group from integratedplasmid pIB139are pWY001pWY002, pWY003, pWY013, pWY014, pWY015which are integrated plasmids throμgh the ΦC31insertion sites attP to obtaine thestable transformants with the dapramycin resistance; another group derived from thepOJ260with the erythromycin strong promoter ermEp*are pWY008pWY009,pWY010, pWY012.Because the transformation process is influenced by lots of factors, including stains and plasmids, so we established a transformation process using pWY012.pWY012was integrated on the chromosome of Saccharopolyspora Spinosadsuccessfμlly by conjugation. However the electroporation has not been successfullyused. The established process can be used in Saccharopolyspora spinosad in ourlaboratory.The paper futher did experiments on the effect from plasmids and donor strains,the pIB139series of integration plasmids has no transformants, so did not theET12567as the donor strain.We built the genetic transformation system for Saccharopolyspora spinosad inour laboratory.This system woμld pave the way for reforming these strains.

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