Application of fluorescent protein to garden plant
View Patent ↗The present invention provides a process for generation of a transformed plant capable of emitting fluorescence by introducing a gene encoding a non-plant-derived fluorescent protein into a plant such that the fluorescent protein is recombinantly expressed in the active form of its mature protein in the leaf or petal of the plant, and also provides a transformed garden plant capable of emitting fluorescence that is generated by using the process. For example, cDNA encoding the full-length amino acid sequence of a Chiridius poppei -derived fluorescent protein CpYGFP or its H52F modified protein CpYGFP H52F is inserted into a T-DNA-based expression vector system, which is in turn introduced into the chromosomal DNA of a plant. As a result, the transformed plant thus generated can exhibit fluorescence attributed to these fluorescent proteins and exhibit no substantial difference in the other phenotypes from wild-type one of the plant.
1. A method for using a Chiridius poppei -derived fluorescent protein for generation of a transformed plant capable of emitting fluorescence, characterized in that
said Chiridius poppei -derived fluorescent protein used therefor is a Chiridius poppei -derived wild-type yellowish-green fluorescent protein (CpYGFP) comprising the following amino acid sequence (SEQ ID NO: 1):
MTTFKIESRI HGNLNGEKFE LVGGGVGEEG RLEIEMKTKD KPLAFSPFLL SHCMGYGFYH
60
FASFPKGTKN IYLHAATNGG YTNTRKEIYE DGGILEVNFR YTYEFNKIIG DVECIGHGFP
120
SQSPIFKDTI VKSCPTVDLM LPMSGNIIAS SYARAFQLKD GSFYTAEVKN NIDFKNPIHE
180
SFSKSGPMFT HRRVEETHTK ENLAMVEYQQ VFNSAPRDM
219
as its full-length amino acid sequence,
a wild-type plant used in the process for generation of the aimed transformed plant capable of emitting fluorescence is a plant which is capable of undergoing Agrobacterium infection and producing a harvestable T1 seed through the self-pollination of each plant individual, and
the process for generation of the aimed transformed plant capable of emitting fluorescence comprises steps of:
inserting a DNA fragment encoding the full-length amino acid sequence of the Chiridius poppei -derived fluorescent protein into the cloning site of a T-DNA-based binary vector;
introducing the obtained T-DNA-based binary vector into a host bacterium of the genus Agrobacterium such that the bacterium of the genus Agrobacterium is transformed therewith;
infecting the wild-type plant with the obtained transformant of the bacterium of the genus Agrobacterium such that a T-DNA region contained in the T-DNA-based binary vector is recombinantly introduced in the chromosomal DNA of the wild-type plant to obtain a transformed plant;
subjecting each individual of the obtained transformed plant to self-pollination, and then harvesting a T1 seed from the plant individual; and
sowing the obtained T1 seed and screening each grown plant to select a transformed plant individual to which a fluorescent phenotype attributed to the recombinant expression of the fluorescent protein in the leaf surface of the plant has been imparted.
2. The method of using the Chiridius poppei -derived fluorescent protein according to claim 1 , wherein
the DNA fragment encoding the full-length amino acid sequence of the Chiridius poppei -derived fluorescent protein comprises the following nucleotide sequence (SEQ ID NO: 2):
ATG ACA ACC TTC AAA ATC GAG TCC CGG ATC CAT GGC AAC CTC AAC GGG
48
GAG AAG TTC GAG TTG GTT GGA GGT GGA GTA GGT GAG GAG GGT CGC CTC
96
GAG ATT GAG ATG AAG ACT AAA GAT AAA CCA CTG GCA TTC TCT CCC TTC
144
CTG CTG TCC CAC TGC ATG GGT TAC GGG TTC TAC CAC TTC GCC AGC TTC
192
CCA AAG GGG ACT AAG AAC ATC TAT CTT CAT GCT GCA ACA AAC GGA GGT
240
TAC ACC AAC ACC AGG AAG GAG ATC TAT GAA GAC GGC GGC ATC TTG GAG
288
GTC AAC TTC CGT TAC ACT TAC GAG TTC AAC AAG ATC ATC GGT GAC GTC
336
GAG TGC ATT GGA CAT GGA TTC CCA AGT CAG AGT CCG ATC TTC AAG GAC
384
ACG ATC GTG AAG TCG TGT CCC ACG GTG GAC CTG ATG TTG CCG ATG TCC
432
GGG AAC ATC ATC GCC AGC TCC TAC GCT AGA GCC TTC CAA CTG AAG GAC
480
GGC TCT TTC TAC ACG GCA GAA GTC AAG AAC AAC ATA GAC TTC AAG AAT
528
CCA ATC CAC GAG TCC TTC TCG AAG TCG GGG CCC ATG TTC ACC CAC AGA
576
CGT GTC GAG GAG ACT CAC ACC AAG GAG AAC CTT GCC ATG GTG GAG TAC
624
CAG CAG GTT TTC AAC AGC GCC CCA AGA GAG ATG TAG
660
as the nucleotide sequence of an open-reading frame encoding the full-length amino acid sequence.
3. A method for using a Chiridius poppei -derived fluorescent protein for generation of a transformed plant capable of emitting fluorescence, characterized in that
said Chiridius poppei -derived fluorescent protein used therefor is a Chiridius poppei -derived modified yellowish-green fluorescent protein (CpYGFP H52F) comprising the following amino acid sequence (SEQ ID NO: 3):
MTTFKIESRI HGNLNGEKFE LVGGGVGEEG RLEIEMKTKD KPLAFSPFLL SFCMGYGFYH
60
FASFPKGTKN IYLHAATNGG YTNTRKEIYE DGGILEVNFR YTYEFNKIIG DVECIGHGFP
120
SQSPIFKDTI VKSCPTVDLM LPMSGNIIAS SYARAFQLKD GSFYTAEVKN NIDFKNPIHE
180
SFSKSGPMFT HRRVEETHTK ENLAMVEYQQ VFNSAPRDM
219
as its full-length amino acid sequence,
a wild-type plant used in the process for generation of the aimed transformed plant capable of emitting fluorescence is a plant which is capable of undergoing Agrobacterium infection and producing a harvestable T1 seed through the self-pollination of each plant individual, and
the process for generation of the aimed transformed plant capable of emitting fluorescence comprises steps of:
inserting a DNA fragment encoding the full-length amino acid sequence of the Chiridius poppei -derived fluorescent protein into the cloning site of a T-DNA-based binary vector;
introducing the obtained T-DNA-based binary vector into a host bacterium of the genus Agrobacterium such that the bacterium of the genus Agrobacterium is transformed therewith;
infecting the wild-type plant with the obtained transformant of the bacterium of the genus Agrobacterium such that a T-DNA region contained in the T-DNA-based binary vector is recombinantly introduced in the chromosomal DNA of the wild-type plant to obtain a transformed plant;
subjecting each individual of the obtained transformed plant to self-pollination, and then harvesting a T1 seed from the plant individual; and
sowing the obtained T1 seed and screening each grown plant to select a transformed plant individual to which a fluorescent phenotype attributed to the recombinant expression of the fluorescent protein in the leaf surface of the plant has been imparted.
4. The method for using the Chiridius poppei -derived fluorescent protein according to claim 3 , wherein
the DNA fragment encoding the full-length amino acid sequence of the Chiridius poppei -derived fluorescent protein comprises the following nucleotide sequence (SEQ ID NO: 4):
ATG ACA ACC TTC AAA ATC GAG TCC CGG ATC CAT GGC AAC CTC AAC GGG
48
GAG AAG TTC GAG TTG GTT GGA GGT GGA GTA GGT GAG GAG GGT CGC CTC
96
GAG ATT GAG ATG AAG ACT AAA GAT AAA CCA CTG GCA TTC TCT CCC TTC
144
CTG CTG TCC TTC TGC ATG GGT TAC GGG TTC TAC CAC TTC GCC AGC TTC
192
CCA AAG GGG ACT AAG AAC ATC TAT CTT CAT GCT GCA ACA AAC GGA GGT
240
TAC ACC AAC ACC AGG AAG GAG ATC TAT GAA GAC GGC GGC ATC TTG GAG
288
GTC AAC TTC CGT TAC ACT TAC GAG TTC AAC AAG ATC ATC GGT GAC GTC
336
GAG TGC ATT GGA CAT GGA TTC CCA AGT CAG AGT CCG ATC TTC AAG GAC
384
ACG ATC GTG AAG TCG TGT CCC ACG GTG GAC CTG ATG TTG CCG ATG TCC
432
GGG AAC ATC ATC GCC AGC TCC TAC GCT AGA GCC TTC CAA CTG AAG GAC
480
GGC TCT TTC TAC ACG GCA GAA GTC AAG AAC AAC ATA GAC TTC AAG AAT
628
CCA ATC CAC GAG TCC TTC TCG AAG TCG GGG CCC ATG TTC ACC CAC AGA
576
CGT GTC GAG GAG ACT CAC ACC AAG GAG AAC CTT GCC ATG GTG GAG TAC
624
CAG CAG GTT TTC AAC AGC GCC CCA AGA GAC ATG TAG
660
as the nucleotide sequence of an open-reading frame encoding the full-length amino acid sequence.
5. The method for using the Chiridius poppei -derived fluorescent protein according to claim 1 , wherein
any plant belonging to the family Brassicaceae, Poaceae, Solanaceae, or Leguminosae is selected as said wild-type plant used in the process for generation of the aimed transformed plant capable of emitting fluorescence.
6. The method for using the Chiridius poppei -derived fluorescent protein according to claim 1 , wherein
Any of a rose, Dianthus caryophyllus , a chrysanthemum, Gerbera cvs., Eustoma grandiflorum, Petunia×hybrida, Torenia fournieri, Nierembergia hippomanica , garden verbena, Calibrachoa hybrid Cultivar, Cyclamen persicum, Cactaceae , and an orchid is selected as said wild-type plant used in the process for generation of the aimed transformed plant capable of emitting fluorescence.
7. The method for using the Chiridius poppei -derived fluorescent protein according to claim 1 , wherein
Agrobacterium tumefaciens is selected as the host bacterium of the genus Agrobacterium used in the step of introducing the obtained T-DNA-based binary vector into the host bacterium of the genus Agrobacterium such that the bacterium of the genus Agrobacterium is transformed therewith.
8. The method for using the Chiridius poppei -derived fluorescent protein according to claim 7 , wherein
An electro-competent cell of an Agrobacterium GV3101 strain is selected as said host bacterium of the genus Agrobacterium.
9. A process for generation of a transformed plant capable of emitting fluorescence using a Chiridius poppei -derived fluorescent protein which is recombinantly expressed by genetic recombination and intracellularly produced, characterized in that
the Chiridius poppei -derived fluorescent protein used therefor is a Chiridius poppei -derived wild-type yellowish-green fluorescent protein (CpYGFP) comprising the following amino acid sequence (SEQ ID NO: 1):
MTTFKIESRI HGNLNGEKFE LVGGGVGEEG RLEIEMKTKD KPLAFSPFLL SHCMGYGFYH
60
FASFPKGTKN IYLHAATNGG YTNTRKEIYE DGGILEVNFR YTYEFNKIIG DVECIGHGFP
120
SQSPIFKDTI VKSCPTVDLM LPMSGNIIAS SYARAFQLKD GSFYTAEVKN NIDFKNPIHE
180
SFSKSGPMFT HRRVEETHTK ENLAMVEYQQ VFNSAPRDM
219
as its full-length amino acid sequence,
a wild-type plant used in the process for generation of the aimed transformed plant capable of emitting fluorescence is a plant which is capable of undergoing Agrobacterium infection and producing a harvestable T1 seed through the self-pollination of each plant individual, and
the process for generation of the aimed transformed plant capable of emitting fluorescence comprises the steps of:
inserting a DNA fragment encoding the full-length amino acid sequence of the Chiridius poppei -derived fluorescent protein into the cloning site of a T-DNA-based binary vector;
introducing the obtained T-DNA-based binary vector into a host bacterium of the genus Agrobacterium such that the bacterium of the genus Agrobacterium is transformed therewith;
infecting the wild-type plant with the obtained transformant of the bacterium of the genus Agrobacterium such that a T-DNA region contained in the T-DNA-based binary vector is recombinantly introduced in the chromosomal DNA of the wild-type plant to obtain a transformed plant;
subjecting each individual of the obtained transformed plant to self-pollination, and then harvesting a T1 seed from the plant individual; and
sowing the obtained T1 seed and screening each grown plant to select a transformed plant individual to which a fluorescent phenotype attributed to the recombinant expression of the fluorescent protein in the leaf surface of the plant has been imparted.
10. The process for generation of the transformed plant capable of emitting fluorescence according to claim 9 , wherein
a garden plant which is capable of undergoing Agrobacterium infection and producing a harvestable T1 seed through the self-pollination of each plant individual is selected as the wild-type plant used in the process for generation of the aimed transformed plant capable of emitting fluorescence.
11. The method for using the Chiridius poppei -derived fluorescent protein according to claim 3 , wherein
any plant belonging to the family Brassicaceae, Poaceae, Solanaceae, or Leguminosae is selected as said wild-type plant used in the process for generation of the aimed transformed plant capable of emitting fluorescence.
12. The method for using the Chiridius poppei -derived fluorescent protein according to claim 3 , wherein
any of a rose, Dianthus caryophyllus , a chrysanthemum, Gerbera cvs., Eustoma grandiflorum, Petunia×hybrida, Torenia fournieri, Nierembergia hippomanica , garden verbena, Calibrachoa hybrid Cultivar, Cyclamen persicum , Cactaceae, and an orchid is selected as said wild-type plant used in the process for generation of the aimed transformed plant capable of emitting fluorescence.
13. The method for using the Chiridius poppei -derived fluorescent protein according to claim 3 , wherein
Agrobacterium tumefaciens is selected as the host bacterium of the genus Agrobacterium used in the step of introducing the obtained T-DNA-based binary vector into the host bacterium of the genus Agrobacterium such that the bacterium of the genus Agrobacterium is transformed therewith.
14. The method for using the Chiridius poppei -derived fluorescent protein according to claim 13 , wherein
an electro-competent cell of an Agrobacterium GV3101 strain is selected as said host bacterium of the genus Agrobacterium.
15. A process for generation of a transformed plant capable of emitting fluorescence using a Chiridius poppei -derived fluorescent protein which is recombinantly expressed by genetic recombination and intracellularly produced, characterized in that
the Chiridius poppei -derived fluorescent protein used therefor is
a Chiridius poppei -derived modified yellowish-green fluorescent protein (CpYGFP H52F) comprising the following amino acid sequence (SEQ ID NO: 3):
MTTFKIESRI HGNLNGEKFE LVGGGVGEEG RLEIEMKTKD KPLAFSPFLL SFCMGYGFYH
60
FASFPKGTKN IYLHAATNGG YTNTRKEIYE DGGILEVNFR YTYEFNKIIG DVECIGHGFP
120
SQSPIFKDTI VKSCPTVDLM LPMSGNIIAS SYARAFQLKD GSFYTAEVKN NIDFKNPIHE
180
SFSKSGPMFT HRRVEETHTK ENLAMVEYQQ VFNSAPRDM
219
as its full-length amino acid sequence,
a wild-type plant used in the process for generation of the aimed transformed plant capable of emitting fluorescence is a plant which is capable of undergoing Agrobacterium infection and producing a harvestable T1 seed through the self-pollination of each plant individual, and
the process for generation of the aimed transformed plant capable of emitting fluorescence comprises the steps of:
inserting a DNA fragment encoding the full-length amino acid sequence of the Chiridius poppei -derived fluorescent protein into the cloning site of a T-DNA-based binary vector;
introducing the obtained T-DNA-based binary vector into a host bacterium of the genus Agrobacterium such that the bacterium of the genus Agrobacterium is transformed therewith;
infecting the wild-type plant with the obtained transformant of the bacterium of the genus Agrobacterium such that a T-DNA region contained in the T-DNA-based binary vector is recombinantly introduced in the chromosomal DNA of the wild-type plant to obtain a transformed plant;
subjecting each individual of the obtained transformed plant to self-pollination, and then harvesting a T1 seed from the plant individual; and
sowing the obtained T1 seed and screening each grown plant to select a transformed plant individual to which a fluorescent phenotype attributed to the recombinant expression of the fluorescent protein in the leaf surface of the plant has been imparted.
16. The process for generation of the transformed plant capable of emitting fluorescence according to claim 15 , wherein
a garden plant which is capable of undergoing Agrobacterium infection and producing a harvestable T1 seed through the self-pollination of each plant individual is selected as the wild-type plant used in the process for generation of the aimed transformed plant capable of emitting fluorescence.