Cosmid Vector for Transforming Plant and Use Thereof

Abstract

The present invention provides novel cosmid vectors for plant transformation. The cosmid vectors have a full length of 15 kb or less and contain: 1) an origin of replication of an IncP plasmid, but not any origin of replication of other plasmid groups; 2) the trfA1 gene of an IncP plasmid; 3) an oriT of an IncP plasmid; 4) the incC1 gene of an IncP plasmid; 5) a cos site of lambda phage, which is located outside the T-DNA; 6) a drug resistance gene expressed in E. coli and a bacterium of Agrobacterium ; 7) a T-DNA right border sequence of a bacterium of Agrobacterium ; 8) a T-DNA left border sequence of a bacterium of Agrobacterium ; 9) a selectable marker gene for plant transformation located between 7) and 8) and expressed in a plant; and 10) restriction endonuclease recognition site(s) located between 7) and 8) for cloning a foreign gene.

1 . A cosmid vector having a full length of 15 kb or less characterized in that:
 1) it contains an origin of replication (oriV) of an IncP plasmid, but does not contain any origin of replication of other plasmid groups;   2) it contains the trfA1 gene of an IncP plasmid;   3) it contains an origin of conjugative transfer (oriT) of an IncP plasmid;   4) it contains the incC1 gene of an IncP plasmid;   5) it contains a cos site of lambda phage and the cos site is located outside the T-DNA;   6) it contains a drug resistance gene expressed in  E. coli  and a bacterium of the genus  Agrobacterium;      7) it contains a T-DNA right border sequence of a bacterium of the genus  Agrobacterium;      8) it contains a T-DNA left border sequence of a bacterium of the genus  Agrobacterium;      9) it contains a selectable marker gene for plant transformation located between 7) and 8) and expressed in a plant; and   10) it contains restriction endonuclease recognition site(s) located between 7) and 8) for cloning a foreign gene, wherein the cosmid vector is selected from the group consisting of:   the cosmid vector pLC40 consisting of the nucleotide sequence of SEQ ID NO: 2 or an equivalent thereof consisting of a nucleotide sequence having at least 95% identity to the nucleotide sequence;   the cosmid vector pLC40GWH consisting of the nucleotide sequence of SEQ ID NO: 3 or an equivalent thereof consisting of a nucleotide sequence having at least 95% identity to the nucleotide sequence;   the cosmid vector pLC40 bar consisting of the nucleotide sequence of SEQ ID NO: 4 or an equivalent thereof consisting of a nucleotide sequence having at least 95% identity to the nucleotide sequence;   the cosmid vector pLC40GWB consisting of the nucleotide sequence of SEQ ID NO: 5 or an equivalent thereof consisting of a nucleotide sequence having at least 95% identity to the nucleotide sequence;   the cosmid vector pLC40GWHKorB consisting of the nucleotide sequence of SEQ ID NO: 65 or an equivalent thereof consisting of a nucleotide sequence having at least 95% identity to the nucleotide sequence;   the cosmid vector pLCleo consisting of the nucleotide sequence of SEQ ID NO: 66 or an equivalent thereof consisting of a nucleotide sequence having at least 95% identity to the nucleotide sequence; and   the cosmid vector pLC40GWHvG1 consisting of the nucleotide sequence of SEQ ID NO: 7 or an equivalent thereof consisting of a nucleotide sequence having at least 95% identity to the nucleotide sequence;   
       wherein the cosmid vector is stably maintained in  E. coli  and  Agrobacterium  cells. 
     
     
         2 . A method for transforming a plant, comprising transforming the plant with a bacterium of the genus  Agrobacterium  harboring an expression vector containing a nucleic acid fragment of a plant inserted into the cosmid vector of  claim 1 . 
     
     
         3 . A method for transforming a plant, comprising transforming the plant with a bacterium of the genus  Agrobacterium  harboring the cosmid vector according to  claim 1  and a plasmid vector characterized in that:
 1) it contains an element necessary for the replication of an IncW plasmid, but does not contain any origin of replication of other plasmid groups; 
 2) it contains the repA gene necessary for the replication of an IncW plasmid; 
 3) it contains a drug resistance gene expressed in  E. coli  and a bacterium of the genus  Agrobacterium ; and 
 4) the virG gene of a bacterium of the genus  Agrobacterium.    
 
     
     
         4 . The method for transforming a plant according to  claim 2  or  3 , wherein the selectable marker gene for plant transformation is selected from the group consisting of a hygromycin resistance gene, a phosphinotricin resistance gene and a kanamycin resistance gene.