Generation and identification of DNA sequence flanking T-DNA integration site of Trichoderma atroviride mutants with high dichlorvos-degrading capacity.
A protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of biocontrol fungus Trichoderma atroviride strain T23 was developed to construct mutants with improved dichlorvos-degradation ability. A transformation frequency of 5x10(-6) was achieved. Among 110 genetically stable T-DNA transformants of T. atroviride T23, two transformants, AMT-12 and AMT-28, confirmed by Southern blot analysis to have single-copy inserts of T-DNA, showed an increase in dichlorvos-degradation ability of more than 10% compared to that of the wild type, exhibited similar tolerance to the pesticide, but lower spore formation ability. Five transformants exhibited a reduction in degradation of more than 70%, exhibited wild-type spore formation, and tolerated up to 800 microg/mL of dichlorvos. The left-flanking sequence of the insertion site in AMT-12 was cloned as a 1845-bp fragment and shown to have 89% identity to the DNA from T. atroviride IMI 206040; however, the involvement of this DNA in dichlorvos degradation remains still to be determined. This study can promote both a more efficient isolation of DNA sequence flanking T-DNA integration site in T. atroviride mutants and a more rational utilization of these transformants in dichlorvos degradation.
Sun
Wenliang
W
Department of Resources and Environmental Sciences, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, China.
Liu
Lixing
L
Hu
Xiaolu
X
Tang
Jun
J
Liu
Peng
P
Chen
Jie
J
Chen
Yunpeng
Y
D001673:Biodegradation, Environmental; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004006:Dichlorvos / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D008938:Mitosis; D009154:Mutation; D010758:Phosphorus / Q000633:toxicity; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods; D013696:Temperature; D013997:Time Factors; D014242:Trichoderma / Q000235:genetics*; D014873:Water Pollutants / Q000737:chemistry
D004269:DNA, Bacterial; C036483:T-DNA; D014873:Water Pollutants; D010758:Phosphorus; D004006:Dichlorvos