CA2502460A1 - Use of the green fluorescent protein as a screenable marker for plant transformation - Google Patents
Use of the green fluorescent protein as a screenable marker for plant transformation Download PDFInfo
- Publication number
- CA2502460A1 CA2502460A1 CA002502460A CA2502460A CA2502460A1 CA 2502460 A1 CA2502460 A1 CA 2502460A1 CA 002502460 A CA002502460 A CA 002502460A CA 2502460 A CA2502460 A CA 2502460A CA 2502460 A1 CA2502460 A1 CA 2502460A1
- Authority
- CA
- Canada
- Prior art keywords
- plant cell
- expression vector
- promoter
- dna molecule
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A method for the production of transgenic plants is provided in which a vector carrying a gene encoding the green fluorescent protein is introduced into cells, the cells are screened for the protein and transformed cells are selected and regenerated. The cellular toxicity of the green fluorescent protein is circumvented by regulating expression of the gene encoding the protein or directing the protein to a subcellular compartment where it is not toxic to the cell. DNA constructs are provided for cell transformation in which the expression of a gene encoding the green fluorescent protein is placed under the control of an inducible promoter. In addition, DNA constructs are provided in which a nucleotide sequence encoding the green fluorescent protein is operably linked to a signal sequence which directs the expressed protein to a subcellular compartment where the protein is not toxic to the cell. Oxidative stress to plant cells transformed with GFP also can be ameliorated by transforming cells with an expression vector comprising genes encoding GFP and an oxygen scavenger enzyme such as superoxide dismutase. The toxicity of GFP in transformed plants can be eliminated by excising the screenable marker gene following detection of transformed cells or sectors. The FLP/FRT system is used in conjunction with GFP as a visible marker for transformation and FRT excision. A nucleotide sequence optimized for expression of the green fluorescent protein in plants is also provided.
Claims (36)
1. An isolated DNA molecule comprising a nucleotide sequence selected from the group consisting of:
(a) SEQ ID NO: 1, wherein said sequence encodes a modified green fluorescent protein (GFPm);
(b) SEQ ID NO: 1, wherein the nucleotides TCC at positions 193-195 are changed to ACG, and wherein said sequence encodes a red shifted green fluorescent protein (GFPr);
(c) SEQ ID NO: 1, wherein nucleotides TAC at positions 196-198 are changed to CAC, and wherein said sequence encodes a blue fluorescent protein (BFP);
and (d) SEQ ID NO: 1, wherein nucleotides TTC at positions 295-297 are changed to AGC, nucleotides ATG at positions 457-459 are changed to ACC, and nucleotides GTG at positions 487-489 are changed to GCC, and wherein said sequence encodes a soluble green fluorescent protein (GFPs).
(a) SEQ ID NO: 1, wherein said sequence encodes a modified green fluorescent protein (GFPm);
(b) SEQ ID NO: 1, wherein the nucleotides TCC at positions 193-195 are changed to ACG, and wherein said sequence encodes a red shifted green fluorescent protein (GFPr);
(c) SEQ ID NO: 1, wherein nucleotides TAC at positions 196-198 are changed to CAC, and wherein said sequence encodes a blue fluorescent protein (BFP);
and (d) SEQ ID NO: 1, wherein nucleotides TTC at positions 295-297 are changed to AGC, nucleotides ATG at positions 457-459 are changed to ACC, and nucleotides GTG at positions 487-489 are changed to GCC, and wherein said sequence encodes a soluble green fluorescent protein (GFPs).
2. The isolated DNA molecule according to claim 1, wherein said isolated DNA
molecule is operably linked to a nucleotide sequence encoding a signal sequence for subcellular localization which directs the protein encoded by said DNA
molecule to a subcellular compartment.
molecule is operably linked to a nucleotide sequence encoding a signal sequence for subcellular localization which directs the protein encoded by said DNA
molecule to a subcellular compartment.
3. The isolated DNA molecule according to claim 1 operably linked to a nucleotide sequence encoding a sequence for mitochondrial localization which directs the expression of said green fluorescent protein to the mitochondria.
4. A method for producing a transgenic plant, wherein said transgenic plant is produced by positive selection, by screening for a transformed cell without selection of a cell carrying a gene that confers resistance to a toxic substance, comprising the steps:
(a) providing at least one expression vector comprising (i) a first promoter, operably linked to the isolated DNA molecule of claim 1, and (ii) a second promoter operably linked to a foreign gene;
(b) introducing said expression vector into regenerable plant cells;
(c) screening for a transformed plant cell containing said green fluorescent protein, wherein said screening does not depend on negative selection of cells carrying a gene that confers resistance to a toxic substance; and (d) regenerating a transformed plant from the transformed plant cell identified by said screening.
(a) providing at least one expression vector comprising (i) a first promoter, operably linked to the isolated DNA molecule of claim 1, and (ii) a second promoter operably linked to a foreign gene;
(b) introducing said expression vector into regenerable plant cells;
(c) screening for a transformed plant cell containing said green fluorescent protein, wherein said screening does not depend on negative selection of cells carrying a gene that confers resistance to a toxic substance; and (d) regenerating a transformed plant from the transformed plant cell identified by said screening.
5. An expression vector comprising said DNA molecule of claim 1.
6. An expression vector comprising the DNA molecule of claim 2 or 3.
7. A method of using said expression of vector of claim 5 to produce a transformed plant, comprising the steps of introducing said expression vector into regenerable plant cells, screening for cells containing said GFPm, GFPr, BFP or GFPs, and regenerating a transformed plant from cells identified by said screening as containing GFPm, GFPr, BFP or GFPs.
8. The method of claim 7, wherein said regenerable plant cells are selected from the group consisting of Zea, Brassica and Helianthus cells.
9. The expression vector according to claim 5, wherein said DNA molecule is operably linked to a first promoter and the expression vector optionally comprises a second promoter linked to a foreign gene.
10. The expression vector of claim 9, wherein the first promoter is an inducible, constitutive or tissue-specific promoter.
11. The expression vector of claim 10, wherein the first promoter is an estrogen-inducible promoter, an estradiol-inducible promoter, the ACE1 promoter, the IN2 promoter or the tetracycline repressor promoter.
12. The expression vector of claim 10, wherein the first promoter is a CaMV, actin, ubiquitin, pEMU, MAS or histone promoter.
13. The expression vector of any one of claims 5 or 9, further comprising a nucleotide sequence for subcellular localization that directs the protein encoded by said DNA molecule to a subcellular compartment.
14. The expression vector of claim 13, wherein the subcellular localization sequence directs expression to the mitochondria, chloroplast, cytoplasm, peroxisome, endoplasmic reticulum, cell wall, apoplast or nucleus.
15. The expression vector of claim 14, wherein the DNA molecule is operably linked to a mitochondrial promoter and said subcellular localization sequence directs expression of said protein to the mitochondria.
16. The expression vector of claim 13, wherein the subcellular localization sequence directs expression to the chloroplast.
17. The expression vector of any one of claims 5 or 9, further comprising a recombinase-specific target sequence wherein the target sequence flanks the 5-prime and 3-prime ends of the first promoter, the DNA molecule or both.
18. The expression vector of claim 17, wherein the recombinase-specific target sequence comprises FLP/FRT, Ac/DS or cre/lox.
19. A transgenic plant cell containing said DNA molecule of claim 1.
20. A transgenic plant cell containing said DNA molecule of any one of claims 1, 2, or 3, wherein said plant cell is from a plant selected from the group consisting of the genera Zea, Brassica, and Helianthus.
21. A transgenic Zea mays plant cell containing said DNA molecule of claim 1.
22. A transgenic plant cell comprising the expression vector of any one of claims 5 or 9.
23. A transgenic plant cell from a plant regenerated from the plant cell of claim 22.
24. The plant cell of claim 23, wherein the plant cell is a monocot or a dicot plant cell.
25. The plant cell of claim 24, wherein the monocot plant cell is of the genera Zea.
26. The plant cell of claim 24, wherein the dicot plant cell is of the genera Brassica or Helianthus.
27. A transgenic cell from a transgenic seed of a plant regenerated from the plant cell of claim 19.
28. A transgenic plant cell comprising the expression vector of any one of claims 10, 13 or 17.
29. A transgenic plant cell from a plant regenerated from the plant cell of claim 28.
30. The plant cell of claim 28, wherein the plant cell is a monocot or a dicot plant cell.
31. The plant cell of claim 30, wherein the monocot plant cell is of the genera Zea.
32. The plant cell of claim 30, wherein the dicot plant cell is of the genera Brassica or Helianthus.
33. A transgenic cell from a transgenic seed of a plant regenerated from the plant cell of claim 28.
34. The transgenic plant cell of any of claims 19-33 wherein the cell is isolated.
35. Use of the DNA molecule of claim 1 to transform a plant cell.
36. Use of the expression cassette of any one of claims 5, 9, 10, 13 or 17 to transform a plant cell.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US1634596P | 1996-05-01 | 1996-05-01 | |
| US60/016,345 | 1996-05-01 | ||
| CA002252412A CA2252412C (en) | 1996-05-01 | 1997-05-01 | Use of the green fluorescent protein as a screenable marker for plant transformation |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002252412A Division CA2252412C (en) | 1996-05-01 | 1997-05-01 | Use of the green fluorescent protein as a screenable marker for plant transformation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2502460A1 true CA2502460A1 (en) | 1997-11-06 |
| CA2502460C CA2502460C (en) | 2011-06-28 |
Family
ID=34620935
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2502657A Expired - Fee Related CA2502657C (en) | 1996-05-01 | 1997-05-01 | Use of the green fluorescent protein as a screenable marker for plant transformation |
| CA2502460A Expired - Fee Related CA2502460C (en) | 1996-05-01 | 1997-05-01 | Use of the green fluorescent protein as a screenable marker for plant transformation |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2502657A Expired - Fee Related CA2502657C (en) | 1996-05-01 | 1997-05-01 | Use of the green fluorescent protein as a screenable marker for plant transformation |
Country Status (1)
| Country | Link |
|---|---|
| CA (2) | CA2502657C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110343716A (en) * | 2018-04-04 | 2019-10-18 | 南京农业大学 | A kind of genetic transformation method for soybean using green fluorescent protein |
| CN110582200A (en) * | 2017-04-03 | 2019-12-17 | 孟山都技术公司 | Compositions and methods for transferring cytoplasmic or nuclear traits or components |
| CN110656125A (en) * | 2019-09-23 | 2020-01-07 | 四川育良生物科技有限公司 | Genetic transformation method of drought-resistant corn |
| CN114088669A (en) * | 2021-10-28 | 2022-02-25 | 大连理工大学 | Preparation of lectin fluorescent fusion protein and application of lectin fluorescent fusion protein in glycosylation detection |
-
1997
- 1997-05-01 CA CA2502657A patent/CA2502657C/en not_active Expired - Fee Related
- 1997-05-01 CA CA2502460A patent/CA2502460C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110582200A (en) * | 2017-04-03 | 2019-12-17 | 孟山都技术公司 | Compositions and methods for transferring cytoplasmic or nuclear traits or components |
| CN110343716A (en) * | 2018-04-04 | 2019-10-18 | 南京农业大学 | A kind of genetic transformation method for soybean using green fluorescent protein |
| CN110656125A (en) * | 2019-09-23 | 2020-01-07 | 四川育良生物科技有限公司 | Genetic transformation method of drought-resistant corn |
| CN114088669A (en) * | 2021-10-28 | 2022-02-25 | 大连理工大学 | Preparation of lectin fluorescent fusion protein and application of lectin fluorescent fusion protein in glycosylation detection |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2502460C (en) | 2011-06-28 |
| CA2502657A1 (en) | 1997-11-06 |
| CA2502657C (en) | 2010-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Empty pericarp5 encodes a pentatricopeptide repeat protein that is required for mitochondrial RNA editing and seed development in maize | |
| CA2252412A1 (en) | Use of the green fluorescent protein as a screenable marker for plant transformation | |
| Hiratsu et al. | The SUPERMAN protein is an active repressor whose carboxy-terminal repression domain is required for the development of normal flowers | |
| Yang et al. | Arabidopsis MYB26/MALE STERILE35 regulates secondary thickening in the endothecium and is essential for anther dehiscence | |
| Dafny-Yelin et al. | Delivery of multiple transgenes to plant cells | |
| Yang et al. | TETRASPORE encodes a kinesin required for male meiotic cytokinesis in Arabidopsis | |
| O’Malley et al. | Linking genotype to phenotype using the Arabidopsis unimutant collection | |
| Ding et al. | Arabidopsis GLUTAMINE-RICH PROTEIN23 is essential for early embryogenesis and encodes a novel nuclear PPR motif protein that interacts with RNA polymerase II subunit III | |
| Esch et al. | A contradictory GLABRA3 allele helps define gene interactions controlling trichome development in Arabidopsis | |
| Yoshida et al. | EMBRYONIC FLOWER2, a novel polycomb group protein homolog, mediates shoot development and flowering in Arabidopsis | |
| Boussardon et al. | Two interacting proteins are necessary for the editing of the NdhD-1 site in Arabidopsis plastids | |
| Cai et al. | LPA66 is required for editing psbF chloroplast transcripts in Arabidopsis | |
| Huang et al. | An E4 ligase facilitates polyubiquitination of plant immune receptor resistance proteins in Arabidopsis | |
| Kim et al. | A defective signal peptide in a 19-kD α-zein protein causes the unfolded protein response and an opaque endosperm phenotype in the maize De*-B30 mutant | |
| Zhang et al. | FERTILIZATION-INDEPENDENT SEED-Polycomb Repressive Complex 2 plays a dual role in regulating type I MADS-box genes in early endosperm development | |
| Hancock et al. | The rice miniature inverted repeat transposable element mPing is an effective insertional mutagen in soybean | |
| CN106749574A (en) | One plant male fertility-associated MS6021 and its encoding gene and application | |
| Zhang et al. | Albino Leaf1 that encodes the sole octotricopeptide repeat protein is responsible for chloroplast development | |
| Kitahara et al. | Ectopic expression of the rose AGAMOUS-like MADS-box genes ‘MASAKO C1 and D1’causes similar homeotic transformation of sepal and petal in Arabidopsis and sepal in Torenia | |
| Wu et al. | Ascorbic acid-mediated reactive oxygen species homeostasis modulates the switch from tapetal cell division to cell differentiation in Arabidopsis | |
| CA2502460A1 (en) | Use of the green fluorescent protein as a screenable marker for plant transformation | |
| Li et al. | Mutation of the ALBOSTRIANS ohnologous gene HvCMF3 impairs chloroplast development and thylakoid architecture in barley | |
| Calderon-Villalobos et al. | LucTrap vectors are tools to generate luciferase fusions for the quantification of transcript and protein abundance in vivo | |
| Zhao et al. | Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA | |
| Dafny-Yelin et al. | Blocking single-stranded transferred DNA conversion to double-stranded intermediates by overexpression of yeast DNA REPLICATION FACTOR A |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |
Effective date: 20160502 |