Method for producing bioluminescent plants
The present invention concerns bioluminescent plants and methods for producing them. Among said methods, the invention relates, in particular, to a method of introducing luciferase genes and luciferin biosynthesis genes into the chloroplasts of a plant. In addition, the invention concerns a method of producing light comprising adding chemical compounds to a culture medium of the plant according to the invention.
1 . An autoluminescent Nicotiana benthamiana plant in which all cells comprising at least one chloroplast are such that the at least one chloroplast comprises 5 constructs each comprising a promoter operably linked to a nucleic acid encoding a protein, wherein the proteins are
a Neonothopanus nambi hispidin-3-hydroxylase (H3H),
a N. nambi luciferase (Luz),
a N. nambi caffeoylpyruvate hydrolase (CPH),
a N. nambi hispidin synthase (HispS), and
an Aspergillus nidulans phosphopantetheinyl transferase (NpgA).
2 . The autoluminescent plant according to claim 1 , wherein the nucleic acids comprise codons adapted for expression in the at least one chloroplast.
3 . The autoluminescent plant according to claim 1 , wherein the promoters are selected from the group consisting of: SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:12.
4 . The autoluminescent plant according to claim 1 , wherein the constructs are integrated into the genome of the at least one chloroplast at trnI and trnA sites, represented by SEQ ID NO: 6 and SEQ ID NO: 7, respectively.
5 . The autoluminescent plant according to claim 1 , wherein the constructs have been integrated into the genome of the at least one chloroplast by homologous recombination.
6 . A method of producing the autoluminescent plant according to claim 1 , wherein the method comprises
transforming chloroplasts of a Nicotiana benthamiana plant cell the 5 constructs and a 6 th construct comprising a resistance gene selected from the group consisting of: nptII, badh, hph, aadA, cat, aphA6, aacCl, pat, gox, epsp, bxn and als,
wherein the 6 constructs are inserted into the genome of the chloroplasts of plant cells by homologous recombination,
producing callus from the transformed cell grown on a selective medium comprising:
an antibiotic selected from the group consisting of: neomycin, kanamycin, betain, hygromycin B, spectinomycin, streptomycin, chloramphenicol, amikacin, and gentamycin; or
an herbicide selected from the group consisting of: bialophos, phosphinotricin, glufosinate, glyphosate, bromoxynil, sulfonylureas, imidazolines, triazolopyrimidines and pyrimidylbenzoates; and
regenerating a plant from the callus.
7 . The method according to claim 6 , wherein transformation is performed by bombarding plant leaves using a particle gun.
8 . The method according to claim 6 , wherein transformation is performed by destabilizing plasma membranes with polyethylene glycol (PEG).
9 . The method according to claim 6 , wherein the resistance gene is aadA and the selective medium comprises spectinomycin and/or streptomycin.
10 . A method of producing light, wherein the method comprises adding hispidin to a culture medium on which the autoluminescent plant according to claim 1 is growing.