Serine recombinases mediating stable integration into plant genomes
Described is a serine recombinase that integrates large sections of foreign DNA into plant chromosomes through non-homologous recombination through attP or attB sites on the foreign DNA but without the engineering of corresponding attB or attP site on the plant chromosome. The serine recombinase-based method has several advantages over other systems, such as Agrobacterium . Also described are related methods, composition, and plants containing exogenous DNA.
1. A method for incorporating an exogenous DNA into a plant genome, comprising:
co-transfecting a plant cell with:
(a) an exogenous DNA comprising an attP or attB site and
(b) a polynucleotide encoding an SPβc2 serine recombinase comprising a nucleotide sequence of SEQ ID NO:36, or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:36, operably linked to a promoter that is active in the plant;
selecting for integration of the exogenous DNA into the plant cell genome at a plant att pseudosite comprising a core sequence of SEQ ID NO:99 or SEQ ID NO:101 or a consensus sequence of SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO: 130, or SEQ ID NO: 131; and
growing the plant cell.
2. The method of claim 1 , wherein the exogenous DNA is up to 25 kb, up to 50 kb, up to 75 kb up to 100 kb, up to 150 kb, up to 200 kb, up to 250 kb or up to 300 kb.
3. The method of claim 1 , wherein the target att pseudosite is a pseudo attB or pseudo attP site.
4. The method of claim 1 , wherein the exogenous DNA comprises an attP site.
5. The method of claim 4 , wherein the attP site has the sequence of SEQ ID NO:7.
6. The method of claim 1 , wherein said plant cell comprises the exogenous DNA and the polynucleotide, further comprising introducing into the plant cell a polynucleotide encoding a second SPβc2 serine recombinase and a cognate Recombinase Directionality Factor (RDF) wherein the second SPβc2 serine recombinase and RDF are under the control of an inducible promoter or gene switch, wherein when activated, the promoter drives expression of the second SPβc2 serine recombinase and the RDF to excise the exogenous DNA from the plant genome.
7. The method of claim 6 wherein the RDF comprises the amino acid sequence of SEQ ID NO:31.
8. A transgenic plant cell obtained by the method of claim 1 .
9. A transgenic plant obtained by culturing the transgenic plant cell of claim 8 .
10. A method for obtaining site-specific recombination in a lettuce plant cell comprising:
contacting a lettuce plant cell with:
(a) an exogenous DNA comprising an attP or attB site; and
(b) a serine recombinase polypeptide that is active in a lettuce plant, wherein the serine recombinase is SPβc2;
selecting for integration of the exogenous DNA into the lettuce plant cell genome at a lettuce plant att pseudosite; and
growing the lettuce plant cell.
11. The method of claim 10 , further comprising selecting for the integration of the exogenous DNA into one or more target att pseudosites on the lettuce chromosome.
12. The method of claim 10 , wherein the exogenous DNA is up to 25 kb, up to 50 kb, up to 75 kb, up to 100 kb, up to 125 kb, up to 150 kb, or more than 150 kb, up to 150 kb, up to 200 kb, up to 250 kb or up to 300 kb.
13. The method of claim 10 , wherein the target att pseudosite is a pseudo attB or pseudo attP site.
14. The method of claim 10 , wherein the exogenous DNA comprises an attP site.
15. The method of claim 14 , wherein the attP site has the sequence of SEQ ID NO:7.
16. The method of claim 10 , wherein the SPβc2 serine recombinase has an amino acid sequence at least 65%, 70%, 75%, 80%, 85% or 90% identical to SEQ ID NO: 1.
17. The method of claim 10 , wherein the SPβc2 serine recombinase is encoded by a plasmid that is co-transfected into the lettuce plant cell with the exogenous DNA.
18. A transgenic lettuce plant cell obtained by the method of claim 10 , wherein said lettuce plant cell comprises the exogenous DNA and the polypeptide.
19. A transgenic lettuce plant obtained by culturing the transgenic lettuce plant cell of claim 18 .
20. The method of claim 1 , wherein said plant att pseudosite comprises the nucleic acid sequence of any one of SEQ ID NOs: 56, 66-96, 107-116, 132-139, or 140-146.
21. The method of claim 10 , wherein the lettuce plant att pseudosite comprises the nucleic acid sequence of any one of SEQ ID NOs: 66-96, 130, 131, or 140-146.