Plant plastid transformation method

Abstract

Method for heterologous protein production in plant cell plastids comprising introducing into plant cells nucleic acid components that encode heterologous proteins under the control of promoters operative in plastids, vectors, host cells, plants and uses thereof.

1 - 41 . (canceled) 
     
     
         42 . A method of transforming a plant cell, the method comprising:
 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 plastid transgene cassette, a plastid translocation sequence (PTS), and a primer binding domain (PBD);   2) introducing into the said plant cell a second nucleic acid sequence that encodes for a plastid translocation sequence binding protein fused to a first plastid transit peptide (PTSBP-TP), wherein said second nucleic acid sequence is operably linked to a plant nuclear promoter; and   3) introducing into the said plant cell a third nucleic acid sequence that encodes for a reverse transcriptase protein fused to a second plastid transit peptide, wherein the third nucleic acid sequence is operably linked to a plant nuclear promoter that drives expression in a plant cell nucleus.   
     
     
         43 . A method according to  claim 42 , wherein the plant plastid transgene cassette comprises:
 i) A left flanking sequence (LFS) and a right flanking sequence (RFS); and   ii) at least one isolated nucleic acid of interest.   
     
     
         44 . A method according to  claim 42 , wherein the plant plastid transgene cassette comprises at least one plastid specific promoter and at least one plastid specific terminator sequence. 
     
     
         45 . A method according to  claim 42 , wherein the said isolated nucleic acid sequence is a recombinant DNA sequence (e.g. cDNA) or an introduced native, isolated genomic DNA sequence selected from isolated mammalian or plant nucleic acid sequences. 
     
     
         46 . A method according to  claim 42 , wherein the primer binding domain is selected from that of a retrotransposon or of a retrovirus, such as from the Ty1 retrotransposon from yeast. 
     
     
         47 . A method according to  claim 42 , wherein the PTS sequence is selected from naked RNA viroids, viruses, viral coat protein binding domains, group I and group II intron RNA, retrotransposon primer binding sites, or RNA harbouring a domain that is recognised by RNA binding proteins, such as the group II intron-derived PTS from the  Lactococcus lactis  LltrB intron. 
     
     
         48 . A method according to  claim 42 , wherein the plastid reverse transcriptase nucleic acid sequence is selected from retrotransposon RNA or retroviral RNA such as from the yeast retrotransposon Ty1, for example, reverse transcriptase-RNase H. 
     
     
         49 . A method according to  claim 42 , wherein the chloroplast transit peptide of the second and third nucleic acid sequence is independently selected from the tobacco rsbc-cTP and the  Arabidopsis  HSP70-cTP protein. 
     
     
         50 . A transformed plant chloroplast comprising:
 i) an exogenous or heterologous left flanking sequence (LFS) and an exogenous or heterologous right flanking sequence (RFS);   ii) at least one exogenous or heterologous chloroplast specific promoter and at least one exogenous or heterologous chloroplast specific terminator sequence; and   iii) at least one exogenous or heterologous isolated nucleic acid of interest.   
     
     
         51 . A population of transformed plant chloroplasts according to  claim 50  comprised in a plant cell such as a plant cell selected from tobacco ( Nicotiana tabacum ) and other  Nicotiana  species,  arabidopsis , potato, corn (maize), canola (rape), rice, wheat, barley,  brassica  sp. such as cauliflower, broccoli (e.g. green and purple sprouting), cabbage (e.g. red, green and white cabbages), curly kale, Brussels sprouts, cotton, algae (e.g. blue green species), lemnospora, or moss (e.g.  physcomitrella patens ), tomato, capsicum, squashes, sunflower, soybean, carrot, melons, grape vines, lettuce, strawberry, sugar beet, peas, and sorghum. 
     
     
         52 . A method of producing at least a heterologous or exogenous RNA species in a plant, the method comprising:
 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 plant plastid transgene cassette, a plant plastid translocation sequence, and a primer binding domain;   2) introducing into the said regenerable plant cell a second nucleic acid sequence that encodes for a translocation sequence binding protein fused to a plant plastid transit peptide, wherein said second nucleic acid sequence is operably linked to a plant nuclear promoter;   3) introducing into the said regenerable plant cell a third nucleic acid sequence that encodes for a reverse transcriptase protein fused to a plant plastid transit peptide, 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), wherein the transgene comprised within the plant plastid transgene cassette is integrated into the plastid genome;   6) regenerating a plant from the plant cell of (5); and   7) growing the plant of (6), wherein the heterologous or exogenous RNA species encoded by the transgene that is integrated into the plastid is expressed as a heterologous or exogenous protein.   
     
     
         53 . An isolated polynucleotide sequence that comprises a plant nuclear promoter operably linked to a first nucleic acid sequence that comprises a plant plastid transgene cassette, a plant plastid translocation sequence, and a primer binding domain for use in a method according to  claim 42 . 
     
     
         54 . An isolated polynucleotide sequence that comprises a plant nuclear promoter operably linked to a first nucleic acid sequence that comprises a plant plastid transgene cassette, a plant plastid translocation sequence, and a primer binding domain for use in a method according to  claim 52 . 
     
     
         55 . An isolated polynucleotide sequence that encodes for a plastid translocation sequence-binding protein fused to a plant plastid transit peptide, wherein the polynucleotide sequence is operably linked to a plant nuclear promoter for use in a method according to  claim 42 . 
     
     
         56 . An isolated polynucleotide sequence that encodes for a plastid translocation sequence-binding protein fused to a plant plastid transit peptide, wherein the polynucleotide sequence is operably linked to a plant nuclear promoter for use in a method according to  claim 52 . 
     
     
         57 . An isolated polynucleotide sequence that encodes for a reverse transcriptase protein fused to a plant plastid transit peptide wherein the polynucleotide sequence is operably linked to a plant nuclear promoter for use in a method according to  claim 42 . 
     
     
         58 . An isolated polynucleotide sequence that encodes for a reverse transcriptase protein fused to a plant plastid transit peptide wherein the polynucleotide sequence is operably linked to a plant nuclear promoter for use in a method according to  claim 52 . 
     
     
         59 . An isolated polynucleotide sequence according to  claim 53 , comprising genomic DNA or cDNA. 
     
     
         60 . A host cell containing an heterologous polynucleotide according to  claim 53  comprised in a plant, a plant part or a plant propagule, or an extract or derivative of a plant or in a plant cell culture. 
     
     
         61 . A plant comprising a plant cell according to  claim 60  that is selected from the group consisting of tobacco ( Nicotiana tabacum ) and other  Nicotiana  species, such as  Nicotiana benthamiana , carrot, vegetable and oilseed  Brassicas , melons, Capsicums, grape vines, lettuce, strawberry, sugar beet, wheat, barley, (corn) maize, rice, soybean, peas, sorghum, sunflower, tomato, cotton and potato. 
     
     
         62 . A method of producing a plant, the method including incorporating a polynucleotide according to  claim 53  into a plant cell and regenerating a plant from said cell.