A New Recombineering System For Agrobacterium Tumefaciens

Agrobacterium tumefaciens is a gram-negative soil bacterium that can infect wounds in plants causing crown gall tumours in most dicotyledons.It can infect the injured part of plants and induce crown gall.A.tumefaciens C58 is a wild strain of A.tumefaciens,and its genome sequencing has been completed.The genome is 5.67M,including a circular chromosome,a linear chromosome,and two plasmids(pAtC58 and pTiC58).The Ti plasmid encodes virulence genes and transfer DNA(T-DNA).The gene in the T-DNA can be replaced by any DNA sequence,making A.tumefaciens an ideal vehicle for gene transfer and an essential tool for plant research and transgenic crop production.In order to improve the gene transfer function of A.tumefaciens,more profound genome research is required,followed with genetic modification.In 2014,ShengBiao Hu et.al applied the ?Red recombination system in A.tumefaciens.The Ti plasmid and linear chromosome of A.tumefaciens were successfully modified by this system.However the efficiency was still suboptimum.The aim of this study was to establish and optimize the new recombination systems in the A.tumefaciens C58 and EHA105 based on the recombinase from its native phage,so as to carry out simple and efficient genetic modification for A.tumefaciens.Detecting optimal inducible promoters for A.tumefaciens.In this study,we constructed a series of expression plasmids containing reporter genes green fluorescent protein(GFP)and luciferase((firefly),to detect the optimal induction promoter in A tumefaciens C58 and EHA105.GFP is simply qualitative.Firefly is used for accurate quantification.The experimental results showed that the Ptet was the strictest promoter of A.tumefaciens C58 and EHA105.The Ptet promoter can turn on the recombination activity only in a small window when it is required.The stability of the recombination system is ensured.This inducible promoter reporter system is useful for development of recombineering systems for the other gram negative bacteria.Based on the principle Red/ET recombineering,a series of homologous recombination systems are constructed.In the Agrobacterium,Rhizobium and its phage genome,we searched for homologous proteins of the Red?/Red? from the Escherichia coli lambda bacteriophage or the RecE/RecT from Escherichia coli Rac prophage.The results of NCBI Blast showed that there are 4 operons containing both 5'-3' exonuclease and single strand annealing protein.Then a series of recombinase expression plasmids were constructed based on these 4 operons.The efficiency of different homologous recombination systems was compared and the best recombination system was selected to modify the genome of A.tumefaciens.The recombinase expression plasmids were transformed into A.tumefaciens C58 and EHA105.By electroporation of PCR products which had 80bp homology arms and a antibiotic selectable marker,the expression plasmids were modified and the efficiencies were compared.The results showed that the expression of red? or pluy in the A tumefaciens could significantly improve the accuracy of the 4 homologous recombination systems.Using the newly developed recombineering systems the genome of A.tumefaciens C58 and EHA105 was modified successfully.Because 3 recombinases were from the rhizobia phage,this provide a great potential to develop efficient recombineering systems for rhizobia after systematic comparison and optimization.In this study,a series of new recombination systems were established in A.tumefaciens.After comparison and optimization,a simple and efficient genome engineering methodology is now available.The application of the new recombineering will open up a new prospect for functional genomics of A.tumefaciens and optimize the utilization of A.tumefaciens as a vehicle for plant transgenesis.