Gene targeting in plants using a chimeric multi domain recombination protein

Abstract

An isolated polynucleotide sequence that encodes for a multi domain recombination protein, comprising a donor DNA-Binding domain (BDB); and a chromosomal binding domain (CBD). A plant cell can be transformed by introducing into the cell a first nucleic acid sequence, which comprises a plant nuclear promoter operably linked to a first nucleic acid sequence comprising the stated polynucleotide sequence, and a second nucleic acid sequence, which comprises a donor nucleic acid sequence and at least two flanking sequences capable of binding to a donor binding domain of the protein, the sequences being located adjacent to each end of the donor polynucleotide sequence forming left and right borders thereto.

1 - 37 . (canceled) 
     
     
         38 . An isolated polynucleotide sequence that encodes for a multi domain recombination protein that comprises
 i) a donor DNA-binding domain; and   ii) a chromosomal binding domain.   
     
     
         39 . An isolated polynucleotide sequence according to  claim 38 , further comprising at least one domain selected from the group of a third domain that is a chromosomal target binding domain and a fourth domain that is a recombination inducing domain. 
     
     
         40 . A method of transforming a plant cell that comprises:
 1) introducing into the said plant cell a nucleic acid sequence that comprises a plant nuclear promoter operably linked to a first nucleic acid sequence that comprises a polynucleotide sequence according to  claim 38 ; and   2) introducing into the said plant cell a second nucleic acid sequence that comprises   i) a donor nucleic acid sequence;   ii) at least two flanking sequences that are capable of binding to a donor binding domain of the said multi domain recombination protein that are located adjacent to each end of the donor polynucleotide sequence forming left and right borders thereto.   
     
     
         41 . A method according to  claim 40 , wherein the second nucleic acid sequence further comprises a viral origin of replication located 5′ and 3′ to each of the said left and right borders. 
     
     
         42 . A method according to  claim 41 , further comprising introducing a nucleic acid sequence that comprises one of a plant nuclear promoter and a viral native promoter operably linked to a third nucleic acid sequence that encodes for a viral replicase protein. 
     
     
         43 . A method according to  claim 41 , wherein the said second nucleic acid sequence is operably linked to a plant nuclear promoter and further comprises a primer binding site that is fused to the 3′ end of a right flanking border sequences. 
     
     
         44 . A method according to  claim 43 , further comprising introducing a nucleic acid sequence that comprises a plant nuclear promoter operably linked to a third nucleic acid sequence that encodes for a reverse transcriptase protein. 
     
     
         45 . A plant cell comprising a multi domain recombination protein that comprises:
 i) a donor DNA-binding domain; and   ii) a chromosomal binding domain.   
     
     
         46 . A plant cell according to  claim 45 , further comprising at least one domain selected from the group of a third domain that is a chromosomal target binding domain and a fourth domain that is a recombination inducing domain. 
     
     
         47 . A plant cell according to  claim 46 , wherein the plant cell is selected from tobacco ( Nicotiana tabacum ) and other  Nicotiana  species,  arabidopsis , potato, corn(maize), canola (rape), rice, wheat, barley,  brassica  sp. selected from the group of cauliflower, broccoli (e.g. green and purple sprouting), cabbage (e.g. red, green and white cabbages), curly kale and Brussels sprouts, cotton, algae (e.g. blue green species), lemnaspora, or moss (e.g.  Physcomitrella patens ), tomato,  capsicum , squashes, sunflower, soyabean, carrot, melons, grape vines, lettuce, strawberry, sugar beet, peas and sorghum. 
     
     
         48 . A method of producing at least a heterologous or exogenous RNA species in a plant that comprises:
 1) introducing into a regenerable plant cell a nucleic acid sequence that comprises a plant nuclear promoter operably linked to a first nucleic acid sequence that comprises a polynucleotide sequence according to  claim 38 ;   2) introducing into the said regenerable plant cell a second nucleic acid sequence that comprises   i) a donor nucleic acid sequence; and   ii) at least two flanking sequences that are located adjacent to each end of the donor polynucleotide sequence forming left and right borders thereto; and   iii) a viral origin of replication located 5′ and 3′ to each of the said left and right borders, wherein said second nucleic acid sequence is operably linked to a plant nuclear promoter; and   3) introducing into the said regenerable plant cell a third nucleic acid sequence that encodes for a viral replicase protein, wherein the third nucleic acid sequence is operably linked to a plant nuclear or native viral promoter;   4) growing said regenerable plant cell of steps 1) to 3);   5) selecting a plant cell of 4);   6) regenerating a plant from the plant cell of 5); and   7) growing the plant of 6).   
     
     
         49 . A method of producing at least a heterologous or exogenous RNA species in a plant that comprises:
 1) introducing into a regenerable plant cell a nucleic acid sequence that comprises a plant nuclear promoter operably linked to a first nucleic acid sequence that comprises a polynucleotide sequence according to  claim 38 ;   2) introducing into the said regenerable plant cell a second nucleic acid sequence that comprises   i) a donor nucleic acid sequence;   ii) at least two flanking sequences that are located adjacent to each end of the donor polynucleotide sequence forming left and right borders thereto, wherein said second nucleic acid sequence is operably linked to a plant nuclear promoter; and   iii) a primer binding site that is fused to the 3′ end of the said right flanking border sequence;   3) introducing into the said regenerable plant cell a third nucleic acid sequence that encodes for a reverse transcriptase protein, wherein the third nucleic acid sequence is operably linked to a plant nuclear promoter;   4) growing said regenerable plant cell of steps 1) to 3);   5) selecting a plant cell of 4);   6) regenerating a plant from the plant cell of 5); and   7) growing the plant of 6).   
     
     
         50 . An isolated polynucleotide sequence that comprises a donor nucleic acid sequence, at least two flanking sequences located adjacent to each end of the said donor polynucleotide sequences forming left and right borders thereto, and a viral origin of replication located 5′ and 3′ to each of the said left and right borders, respectively, for use in a method according to  claim 40 . 
     
     
         51 . An isolated polynucleotide sequence according to  claim 50 , further comprising a third nucleic acid sequence that comprises one of a plant nuclear and a viral native promoter operably linked to a first nucleic acid sequence that encodes for a viral replicase protein. 
     
     
         52 . A nucleic acid vector suitable for transformation of a plant or bacterial cell and including an isolated polynucleotide sequence that encodes for a multi domain recombination protein that comprises domains selected from the group:
 i) a donor DNA-binding domain;   ii) a chromosomal binding domain;   iii) a chromosomal target binding domain; and   iv) a recombination inducing domain;   said selected domains comprising at least domain i) and domain ii).   
     
     
         53 . A plant cell containing a heterologous polynucleotide according to  claim 38 . 
     
     
         54 . A host cell containing a nucleic acid vector according to  claim 52 . 
     
     
         55 . A method of producing a plant cell according to  claim 53 , the method including incorporating said polynucleotide into the cell by means of transformation. 
     
     
         56 . A method of producing a host cell according to  claim 54 , the method including incorporating said nucleic acid vector into the cell by means of transformation. 
     
     
         57 . A plant comprising a plant cell according to  claim 53  that is selected from the group consisting of tobacco ( Nicotiana tabacum ),  Nicotiana benthamiana , carrot, vegetable and oilseed  Brassica 's, melons, Capsicums, grape vines, lettuce, strawberry, sugar beet, wheat, barley, (corn)maize, rice, soybean, peas, sorghum, sunflower, tomato, cotton and potato.