CN116769052A - Method for promoting soybean flowering time to advance under long-day condition - Google Patents
Method for promoting soybean flowering time to advance under long-day condition Download PDFInfo
- Publication number
- CN116769052A CN116769052A CN202310562581.7A CN202310562581A CN116769052A CN 116769052 A CN116769052 A CN 116769052A CN 202310562581 A CN202310562581 A CN 202310562581A CN 116769052 A CN116769052 A CN 116769052A
- Authority
- CN
- China
- Prior art keywords
- soybean
- gmcdf1
- gfp
- protein
- fusion protein
- 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.)
- Pending
Links
- 244000068988 Glycine max Species 0.000 title claims abstract description 123
- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 121
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims abstract description 27
- 230000001737 promoting effect Effects 0.000 title abstract description 9
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 42
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 42
- 230000009261 transgenic effect Effects 0.000 claims abstract description 23
- 108090000623 proteins and genes Proteins 0.000 claims description 60
- 102000004169 proteins and genes Human genes 0.000 claims description 28
- 125000003729 nucleotide group Chemical group 0.000 claims description 27
- 239000002773 nucleotide Substances 0.000 claims description 26
- 108020004414 DNA Proteins 0.000 claims description 24
- 102000053602 DNA Human genes 0.000 claims description 17
- 230000014509 gene expression Effects 0.000 claims description 17
- 150000007523 nucleic acids Chemical class 0.000 claims description 17
- 102000039446 nucleic acids Human genes 0.000 claims description 16
- 108020004707 nucleic acids Proteins 0.000 claims description 16
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 15
- 230000001105 regulatory effect Effects 0.000 claims description 9
- 108010075254 C-Peptide Proteins 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 8
- 125000000539 amino acid group Chemical group 0.000 claims description 7
- 238000009395 breeding Methods 0.000 claims description 5
- 210000004899 c-terminal region Anatomy 0.000 claims description 4
- 108091026890 Coding region Proteins 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims 3
- 239000013612 plasmid Substances 0.000 abstract description 22
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000012408 PCR amplification Methods 0.000 description 25
- 238000006243 chemical reaction Methods 0.000 description 16
- 239000013598 vector Substances 0.000 description 15
- 230000002018 overexpression Effects 0.000 description 9
- 239000002299 complementary DNA Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 description 6
- 238000010276 construction Methods 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 241000219194 Arabidopsis Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 241000589158 Agrobacterium Species 0.000 description 1
- 108700024611 Arabidopsis GI Proteins 0.000 description 1
- 101100020619 Arabidopsis thaliana LATE gene Proteins 0.000 description 1
- 101150038243 CLOCK gene Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 1
- 101150066002 GFP gene Proteins 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 101710185720 Putative ethidium bromide resistance protein Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002567 autonomic effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000000422 nocturnal effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000036561 sun exposure Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Physiology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The application discloses a method for promoting the flowering time of soybeans to advance under the condition of long sunlight. Experiments prove that the recombinant plasmid pTF101-GFP-C17 is introduced into soybean variety Jack, and then the T is obtained through selfing 1 Transgenic GFP-GmCDF1-C17 soybean. Under the condition of long sunlight, compared with soybean variety Jack, T 1 Flowering time of the transgenic GFP-GmCDF1-C17 soybean strain is remarkably advanced. From this, the fusion protein GFP-GmCDF1-C17 can regulate the flowering time of soybean. The application has important application value.
Description
Technical Field
The application belongs to the field of soybean molecular genetic breeding, and particularly relates to a method for promoting the flowering time of soybeans to be advanced under a long-day condition.
Background
Flowering marks the transition of plants from vegetative to reproductive growth, a process regulated by complex genetic networks and environmental conditions. There have been studies showing 6 floral transition pathways in the model plant Arabidopsis, photoperiod, autonomic regulation, vernalization, seedling-age dependent, gibberellin and environmental temperature-affecting pathways, respectively. Soybeans are typical short-day crops, and photoperiod is an important factor affecting soybean flowering and maturation and ecological fitness. In recent years, a lot of breakthrough progress is made in the field of soybean photoperiod regulation and flowering, and a genetic network for regulating soybean flowering in photoperiod is gradually established.
In the soybean photoperiod flowering pathway, soybean E3 (GmPhyA 3) and E4 (GmPhyA 2) are homologous genes of the Arabidopsis photopigment phytochromen A, which act to inhibit flowering. Under long sunlight, the e3 mutant blooms earlier than the control; under far-red light conditions, the e4 mutant hypocotyl was longer than the control. E1 is a site controlling the maturity of soybean, and the E1 mutant has an early flowering phenotype under natural conditions; the E1 protein is a nuclear protein containing a B3 domain, and inhibits soybean flowering by inhibiting expression of FLOWERINGLOCUS T homologous genes (GmFT 2a and GmFT5 a). E2 is a homologous gene of Arabidopsis GIGANTEA, and the expression level of GmFT2a in the E2 mutant is increased to cause early flowering. Overexpression of the PseudoResponseRegulator homologous genes (GmPRR 3a and GmPRR3 b) promoted expression of E1 by LATE ELONGATED HYPOCOTYL (LHY) led to soybean late flowers. Homologous genes GmCOL1a and GmCOL1b of CONSTANS inhibit soybean flowering under long-day irradiation. Overexpression of GmFT1a and GmFT4 inhibited soybean flowering, and under short-day exposure, the plants overexpressing GmFT1a were flowering 4-7 days later than the wild type, while under long-day exposure, the plants overexpressing GmFT1a were flowering 3-5 days later than the wild type. Over-expression of GmRAV inhibited soybean flowering both on long and short days. Overexpression of GmSPA3c inhibits soybean flowering, and mutants with GmSPA3c loss of function have early flowering in long sunlight, but have no significant difference in flowering time in short sunlight.
In addition to the several flowering-inhibiting genes described above, some flowering-promoting genes are also found in the soybean photoperiod pathway. Overexpression of soybean GmFT2a resulted in early flowers, whereas the GmFT2a mutant showed late flowers. Overexpression of soybean GmFT5a promotes flowering under long sun exposure. Soybean long childhood gene J (earlyflowerin 3 homologous gene) is a night composite body weight essential component of biological clock, and is used for promoting flowering and J mutation by inhibiting E1 expressionAnd the plant is flowering late, and is suitable for low-latitude planting. Soybean GmGBP1 is a flowering promoting factor, and transgenic lines with the expression of GmGBP1 down-regulated are opened later than wild type under the conditions of short day, long day and nature. LUXARRYTHMO is another important component of the biological clock nocturnal complex, which interacts with J, inhibiting E1 expression; the soybean lux1lux2 double mutant showed extremely late flowers. Time offlowing 16 (Tof 16)/LHY is a site for controlling the maturity of soybean, also a biological clock gene, at low latitude, tof16 CR The soybean mutant blooms early. Soybean GmFUL (FRUITFUL) promotes flowering by promoting expression of GmFT2a and GmFT5 a. Soy SOC1a/b promoted flowering, and the single mutant of SOC1a, the single mutant of SOC1b and the double mutant of SOC1asoc1b had late flowering phenotype on both long and short days. Overexpression of soybean GmMDE06 promotes flowering by promoting expression of GmFT2a and GmFT5 a. Recent studies indicate that soybean QNE1 (QTLnear E1) is a transcription factor of the TCP type; overexpression of soybean QNE1 causes soybean to bloom early.
It follows that the function of some important flowering genes of the soybean photoperiod pathway has been initially determined, but the functions of many genes are still unknown, and specific regulatory mechanisms thereof are still to be further explored.
Disclosure of Invention
The application aims to promote the flowering time of soybeans to be advanced under long-day conditions.
Firstly, protecting a fusion protein GFP-GmCDF1-C17, wherein the fusion protein comprises GFP protein, connecting peptide protein and GmCDF1-C17 from the N end to the C end in sequence;
the connecting peptide protein consists of 9-13 amino acid residues;
the GmCDF1-C17 protein is b 1) or b 2) or b 3) or b 4) as follows:
b1 Amino acid sequence is the protein shown in 250 to 266 positions from the 5' end of SEQ ID NO. 1;
b2 A fusion protein obtained by ligating a tag to the N-terminal or/and C-terminal of the protein shown in positions 250 to 266 from the 5' -terminal of SEQ ID NO. 1;
b3 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in b 1) or b 2), is derived from soybean and is related to the flowering time of the soybean;
b4 A protein which has 80% or more homology with the amino acid sequence defined from 250 to 266 from the 5' -end of SEQ ID NO. 1, is derived from soybean and is related to the flowering time of soybean.
In order to facilitate purification of the protein of b 1), the amino-or carboxyl-terminal end of the protein shown in SEQ ID NO. 1 from position 250 to 266 from the 5' -end may be linked to a tag as shown in Table 1.
TABLE 1 sequence of tags
| Label (Label) | Residues | Sequence(s) |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| FLAG | 8 | DYKDDDDK |
| Strep-tagII | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
The protein of b 3) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.
The protein in b 3) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.
The coding gene of the protein in b 3) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in positions 748 to 798 of SEQ ID NO. 2 from the 5' end and/or by performing one or several base pair missense mutation and/or by ligating the coding sequences of the tags shown in Table 1 at the 5' and/or 3' end thereof.
The protein of b 4) may be a fusion protein as shown in Table 2
The amino acid sequence of GFP-GmCDF 1-C17-1-GFP-GmCDF 1-C17-14 is shown as SEQ ID NO. 3-SEQ ID NO. 16 in sequence. The nucleotide sequence of the fusion protein GFP-GmCDF 1-C17-1-GFP-GmCDF 1-C17-14 shown in the encoding table 2 is shown as SEQ ID NO. 17-SEQ ID NO. 30 in sequence.
TABLE 2
In the fusion protein GFP-GmCDF1-C17, the amino acid sequence of the GFP protein is shown in positions 1 to 238 from the 5' end of SEQ ID NO. 1.
In the fusion protein GFP-GmCDF1-C17, the amino acid sequence of the connecting peptide protein is shown in positions 239-249 from the 5' end of SEQ ID NO. 1.
The amino acid sequence of the fusion protein GFP-GmCDF1-C17 is shown as SEQ ID NO. 1.
Nucleic acid molecules encoding any of the fusion proteins GFP-GmCDF1-C17 mentioned above are also within the scope of the application.
The nucleic acid molecule may be a DNA molecule as shown in c 1) or c 2) or c 3) or c 4):
c1A DNA molecule with a coding region shown as SEQ ID NO. 2;
c2 A DNA molecule with a nucleotide sequence shown as SEQ ID NO. 2;
c3 A DNA molecule which has 75% or more homology with the nucleotide sequence defined in C1) or C2), is derived from soybean and encodes any one of the fusion proteins GFP-GmCDF 1-C17;
c4 Under stringent conditions with the nucleotide sequence defined under C1) or C2), a DNA molecule derived from soybean and encoding any of the fusion proteins GFP-GmCDF1-C17 described above.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
Wherein, SEQ ID NO. 2 is composed of 801 nucleotides, and the nucleotide of SEQ ID NO. 2 codes for the amino acid sequence shown as SEQ ID NO. 1.
The nucleotide sequence of the fusion protein GFP-GmCDF1-C17 according to the application encoding any of the above can be mutated easily by the person skilled in the art using known methods, for example directed evolution and point mutation. Those artificially modified nucleotides having 80% or more identity to the nucleotide sequence of any one of the above-mentioned fusion proteins GFP-GmCDF1-C17 isolated by the present application are all nucleotide sequences derived from the present application and are equivalent to the sequences of the present application, as long as they encode any one of the above-mentioned fusion proteins GFP-GmCDF1-C17.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences which have 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the fusion protein GFP-GmCDF1-C17 of the application, which encodes the amino acid sequence shown in SEQ ID NO. 1. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The application also protects the application of any fusion protein GFP-GmCDF1-C17 in regulating and controlling the flowering time of soybeans or cultivating transgenic soybeans with early flowering time.
The application also protects the application of any one of the nucleic acid molecules in regulating and controlling the flowering time of soybeans or culturing transgenic soybeans with early flowering time.
In any of the above applications, the regulating soybean flowering time may be a soybean flowering time advance.
In any of the above applications, the soybean culture conditions may be long-day (e.g., 16h light/8 h dark).
The application also provides a method for cultivating transgenic soybean, which can comprise the following steps: increasing the expression level and/or activity of any one of the fusion proteins GFP-GmCDF1-C17 in the starting soybean to obtain transgenic soybean; the flowering time of the transgenic soybeans is advanced compared to the starting soybeans.
In the above method, the effect of increasing the expression level and/or activity of the above fusion protein GFP-GmCDF1-C17 in the starting soybean can be achieved by methods known in the art such as transgenesis, multicopy, promoter and regulatory factor modification.
In the above method, the improvement of the expression level and/or activity of the above fusion protein GFP-GmCDF1-C17 in the starting soybean is achieved by introducing a nucleic acid molecule encoding the above fusion protein GFP-GmCDF1-C17 into the starting soybean.
In the above method, said introducing a nucleic acid molecule encoding any one of the fusion proteins GFP-GmCDF1-C17 into the starting soybean may be effected by introducing a recombinant vector into the starting soybean; the recombinant vector may be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding any one of the fusion proteins GFP-GmCDF1-C17 into an expression vector. The recombinant vector may be specifically the recombinant plasmid pTF101-GFP-C17 mentioned in the examples.
In the above method, the nucleic acid molecule encoding the fusion protein GFP-GmCDF1-C17 may be a DNA molecule as shown in C1) or C2) or C3) or C4):
c1A DNA molecule with a coding region shown as SEQ ID NO. 2;
c2 A DNA molecule with a nucleotide sequence shown as SEQ ID NO. 2;
c3 A DNA molecule which has 75% or more homology with the nucleotide sequence defined in C1) or C2), is derived from soybean and encodes any one of the fusion proteins GFP-GmCDF 1-C17;
c4 Under stringent conditions with the nucleotide sequence defined under C1) or C2), a DNA molecule derived from soybean and encoding any of the fusion proteins GFP-GmCDF1-C17 described above.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
Wherein, SEQ ID NO. 2 is composed of 801 nucleotides, and the nucleotide of SEQ ID NO. 2 codes for the amino acid sequence shown as SEQ ID NO. 1.
The application also provides a soybean breeding method, which comprises the following steps: increasing the content and/or activity of the fusion protein GFP-GmCDF1-C17 in soybeans, thereby advancing the flowering time of the soybeans.
In any of the above methods, the soybean culture conditions may be long-day (e.g., 16h light/8 h dark).
Experiments prove that the recombinant plasmid pTF101-GFP-C17 is introduced into soybean variety Jack, and then the T is obtained through selfing 1 Transgenic GFP-GmCDF1-C17 soybean. Under the condition of long sunlight, compared with soybean variety Jack, 3T 1 Flowering times of the transgenic GFP-GmCDF1-C17 soybean lines (GFP-C17-OE 1, GFP-C17-OE2 and GFP-C17-OE 3) were all significantly advanced by about 20 days. From this, the fusion protein GFP-GmCDF1-C17 can regulate the flowering time of soybean. The application has important application value.
Drawings
FIG. 1 shows the result of agarose gel electrophoresis of the 3PCR amplification product in step one of example 1.
FIG. 2 shows the result of agarose gel electrophoresis of the 2PCR amplified product in step two of example 1.
FIG. 3 shows the result of agarose gel electrophoresis of the 2PCR amplification product in step four of example 1.
FIG. 4 shows a real-time fluorescent quantitative determination of T 1 Expression level of fusion gene GFP-GmCDF1-C17 in soybean with transgenic GFP-GmCDF1-C17 gene.
FIG. 5 is T 1 Flowering time statistics of transgenic GFP-GmCDF1-C17 soybean.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 construction of recombinant plasmid pTF101-GFP-C17
1. Construction of recombinant plasmid pLB-GmCDF1
1. Extracting total RNA of the leaves of the soybean variety Williams 82 to obtain the total RNA of the Williams 82. Reverse transcription was performed using the total RNA of Williams 82 as a template to obtain cDNA of Williams 82.
2. The cDNA of Williams 82 is used as a template, and a primer GmCDF1-F is adopted: 5'-ATGCAGGAGAATAAGGATCC-3' and primer GmCDF1-R: and 5'-TCAAGAGTTCTCATGAAAGT-3', performing PCR amplification to obtain a PCR amplification product.
The reaction system was 20. Mu.l composed of 1. Mu.l of Williams 82 cDNA, 1. Mu.l of an aqueous solution of the primer GmCDF1-F (concentration: 10. Mu.M), 1. Mu.l of an aqueous solution of the primer GmCDF1-R (concentration: 10. Mu.M), 10. Mu.l of KOD one (TOYOBO, KMM-101S) and 7. Mu.l of ddH 2 O composition.
The reaction procedure is: 95 ℃ for 2min; 15s at 95 ℃,30 s at 60 ℃,20 s at 68 ℃ and 30 cycles; and at 68℃for 5min.
3. And (3) performing agarose gel electrophoresis on the PCR amplification product obtained in the step (2). The result showed that the size of the PCR amplification product obtained in step 2 was 1377bp. Recovering the PCR amplification product.
The result of electrophoresis is shown in FIG. 1 (1 is PCR amplification product, marker is DNAmarker).
4. Sequencing the PCR amplification product recovered in the step 3. Sequencing results show that the nucleotide sequence of the PCR amplification product is shown as SEQ ID NO. 31. The PCR amplification product is the CDS sequence of GmCDF1 gene. The GmCDF1 gene encodes GmCDF1 protein.
5. The PCR amplification product recovered in step 3 was ligated with pLB-Simple Vector (pLB components of the rapid cloning kit with zero background) to give recombinant plasmid pLB-GmCDF1.pLB zero background rapid cloning kit is a product of Tiangen corporation, and the product catalog number is VT206-01.
The reaction system was 10. Mu.l, consisting of 1. Mu.l of pLB-Simple Vector, 5. Mu.l of 2X ReactionSolution, 1. Mu. l T 4 DNA Ligase, 50ng of PCR amplification product recovered in step 3 and ddH 2 O composition.
The reaction procedure is: the reaction was carried out at 22℃for 15min.
2. Construction of recombinant plasmid pDONR207-GmCDF1-C17
1. The recombinant plasmid pLB-GmCDF1 constructed in the step one is taken as a template, and a primer GmCDF1-C17-attB1-F is adopted: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGGA-3' and primer GmCDF1-C17-attB2-R:
and 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAAGAGTTCTCATGAAAGT-3', performing PCR amplification to obtain a PCR amplification product.
2. And (3) performing agarose gel electrophoresis on the PCR amplification product obtained in the step (1). The result showed that the size of the PCR amplification product obtained in step 1 was 105bp. Recovering the PCR amplification product. The PCR amplification product consisted of a linker of 61bp in size and a GmCDF1-C17 of 54bp in size.
The result of electrophoresis is shown in FIG. 2 (1 is PCR amplification product, marker is DNAmarker).
3. The PCR amplified product recovered in step 2 and the vector pDONR207 (nucleotide sequence shown as SEQ ID NO:32, circular) were subjected to BP reaction (with the objective of cloning GmCDF1-C17 into the vector pDONR 207) to obtain an entry clone pDONR207-GmCDF1-C17.
The reaction system was 2.5. Mu.l, which consisted of 100ng of the vector pDONR207, 100ng of the PCR amplification product recovered in step 2 and 0.5. Mu.l of BP enzyme (Invitrogen Co., product, catalog number 11789013).
The reaction procedure is: the reaction was carried out at 25℃for 1h.
3. Construction of recombinant plasmid pGWB6-GmCDF1-C17
1. The full length of the entry clone pDONR207-GmCDF1-C17 and pGWB6 vector (circular double-stranded DNA) is 17956bp, the nucleotide sequence of1 st to 8320 th positions of one strand is shown as SEQ ID NO. 33, the nucleotide sequence of 8321 st to 17956 th positions is shown as SEQ ID NO. 34, and the pGWB6 vector contains GFP tag) are subjected to LR reaction (aiming at cloning GmCDF1-C17 to pGWB6 vector) to obtain recombinant plasmid pGWB6-GmCDF1-C17.
The reaction system was 2.5. Mu.l, consisting of 100ng of the entry clone pDONR207-GmCDF1-C17, 100ng of pGWB6 vector, 0.5. Mu.l of LR enzyme (Invitrogen, cat. No. 11791043).
The reaction procedure is: the reaction was carried out at 25℃for 1h.
2. The recombinant plasmid pGWB6-GmCDF1-C17 was sequenced. Sequencing results showed that the recombinant plasmid
pGWB6-GmCDF1-C17 contains the fusion gene GFP-GmCDF1-C17 with the nucleotide sequence shown in SEQ ID NO. 2. In SEQ ID NO. 2, the GFP gene (nucleotide sequence encoding GFP protein) is located from the 5' -end at positions 1 to 714, the nucleotide sequence encoding the connecting peptide is located at positions 715 to 747, and the nucleotide sequence encoding 17 amino acids of the C-terminal of the GmCDF1 protein is located at positions 748 to 798.
The recombinant plasmid pGWB6-GmCDF1-C17 expresses fusion protein GFP-GmCDF1-C17 shown in SEQ ID NO. 1. In SEQ ID NO. 1, the amino acid sequence of the GFP protein is from the 5' end at positions 1 to 238, the amino acid sequence of the connecting peptide is from 239 to 249, and the 17 amino acids at the C-terminal end of the GmCDF1 protein are from 250 to 266.
4. Construction of recombinant plasmid pTF101-GFP-C17
1. The recombinant plasmid pGWB6-GmCDF1-C17 constructed in the step three is used as a template, and a primer is adopted
GmCDF1-C17-XbaI-F：5’-AGAACACGGGGGACTCTAGAATGGTGAGCAAGGGCGAGGA-3' (underlined as a point of recognition for the restriction enzyme XbaI) and primer GmCDF1-C17-SacI-R:5' -GATCGGGGAAATTCGAGC TCTCAAGAGTTCTCATGAAAGT-3' (underlined as a dot for recognition of restriction enzyme SacI) to obtain a PCR amplified product.
The reaction system was 20. Mu.l, and the reaction system consisted of 10. Mu.g of recombinant plasmid pGWB6-GmCDF1-C17, 1. Mu.l of primer
GmCDF1-C17-XbaI-F aqueous solution (concentration: 10. Mu.M), 1. Mu.l of primer GmCDF1-C17-SacI-R aqueous solution (concentration: 10. Mu.M), 10. Mu.l of KOD one (TOYOBO, KMM-101S) and 7. Mu.l of ddH 2 O composition.
The reaction procedure is: 95 ℃ for 2min; 15s at 95 ℃,30 s at 60 ℃,20 s at 68 ℃ and 30 cycles; and at 68℃for 5min.
2. And (3) performing agarose gel electrophoresis on the PCR amplification product obtained in the step (1). The result shows that the size of the PCR amplified product obtained in the step 1 is 801bp. Recovering the PCR amplification product.
The result of electrophoresis is shown in FIG. 3 (1 is PCR amplification product, marker is DNAmarker).
3. pTF101 vector (nucleotide sequence shown in SEQ ID NO:35, circular) was digested with restriction enzymes HindIII and SacI, and the vector backbone of about 9kb was recovered.
4. And (3) connecting the PCR amplification product recovered in the step (2) with the carrier skeleton recovered in the step (3) by adopting a seamless connection kit (product of the product catalog number TSV-S2 of the Optimaceae company to obtain the recombinant plasmid pTF101-GFP-C17.
The recombinant plasmid pTF101-GFP-C17 was sequenced. Sequencing results show that the recombinant plasmid pTF101-GFP-C17 is obtained by replacing a small DNA fragment between restriction enzymes HindIII and SacI of the pTF101 vector with the DNA molecule shown in SEQ ID NO. 2 (i.e. fusion gene GFP-GmCDF 1-C17).
The recombinant plasmid pTF101-GFP-C17 expresses the fusion protein GFP-GmCDF1-C17 shown in SEQ ID NO. 1.
Example 2 application of fusion protein GFP-GmCDF1-C17 in promoting early flowering of soybeans under long sunlight
1、T 1 Production of transgenic GFP-GmCDF1-C17 Gene soybean
Recombinant plasmid pTF101-GFP-C17 was transformed into soybean variety Jack by Agrobacterium-mediated method (Chen et al 2018), and then by selfing, T was obtained 1 Transgenic GFP-GmCDF1-C17 soybean. The method for screening positive seedlings comprises the following steps: extracting genome DNA of soybean seedling to be detected and using it as template, adopting
Primer pairs composed of 5'-GCTACCCCGACCACATGAAG-3' and 5'-AGGGATCTAGACAAAGCCGC-3' are subjected to PCR amplification to obtain PCR amplification products; then, the following judgment is made: if a PCR amplified product contains a DNA fragment of about 559bp, the soybean seedling corresponding to the PCR amplified product is a positive seedling.
2. Real-time fluorescent quantitative detection T 1 Fusion gene in soybean with GFP-GmCDF1-C17 gene transfer
Expression level of GFP-GmCDF1-C17
(1) Respectively T each 1 Seedlings grown to 10 days from the GFP-GmCDF1-C17 gene transgenic soybean are put into liquid nitrogen for preservation, and corresponding samples to be tested are obtained. Taking soybean seeds Jack, alternately culturing at 28 ℃ for 10 days to obtain seedlings; and (5) placing the seedlings into liquid nitrogen for preservation, and obtaining corresponding samples to be tested.
(2) Extracting total RNA of a sample to be detected by adopting a Trizo1 method, then carrying out reverse transcription to obtain first-strand cDNA, diluting the cDNA by 5 times with sterile water as a template, and detecting the relative expression quantity of GFP-GmCDF1-C17 gene (GmACTIN gene is an internal reference gene) by real-time quantitative PCR.
Primers for detecting GFP-GmCDF1-C17 gene are 5'-CAAGATCCGCCACAACATCG-3' and 5'-GACTGGGTGCTCAGGTAGTG-3'.
The detection results are shown in FIG. 4. The results show that each T is compared with soybean variety Jack 1 The relative expression amount of GFP-GmCDF1-C17 genes in the soybean with the transgenic GFP-GmCDF1-C17 genes is increased to different degrees, wherein 3T are 1 The soybean strain with GFP-GmCDF1-C17 gene transferred by substitution has the highest relative expression level of GFP-GmCDF1-C17 gene, and is named as GFP-C17-OE1 and GFP-C17-OE2 and GFP-C17-OE3.
3、T 1 Flowering time statistics of transgenic GFP-GmCDF1-C17 soybean
The soybean seed to be tested is GFP-C17-OE 1T 1 T of seed of the generation GFP-C17-OE2 1 Seed generation, GFP-C17-OE 3T 1 A seed of a soybean variety Jack.
The experiment was repeated three times to average the values, and the procedure for each repetition was as follows: 20 soybean seeds to be tested are planted in a greenhouse and are cultivated under the condition of long sunlight (16 h illumination/8 h darkness) until flowering. 3 soybean plants to be tested are randomly selected, flowering time is counted and an average value is obtained. Wherein flowering time is calculated from the start of the emergence of cotyledons until the first flower appears at any node on the soybean plant, and the number of days elapsed in this process is the flowering time (unit: day).
The phenotype of a portion of soybean plants grown under long-day conditions for 28 days is shown in fig. 5a (LD indicates long-day, WT indicates soybean variety Jack, GFP-C17-OE is GFP-C17-OE1, scale = 10 cm, white arrow indicates white petals of soybean).
The statistical results of flowering time of a part of the soybean to be tested are shown in FIG. 5B (WT indicates soybean variety Jack, GFP-C17-OE is GFP-C17-OE 1).
The results show that compared with soybean variety Jack, 3T 1 Flowering times of the transgenic GFP-GmCDF1-C17 soybean lines (GFP-C17-OE 1, GFP-C17-OE2 and GFP-C17-OE 3) were all significantly advanced by about 20 days.
The results show that the over-expression of GFP-GmCDF1-C17 gene in soybean variety Jack can significantly advance the flowering time of soybean by about 20 days.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. The fusion protein GFP-GmCDF1-C17 sequentially comprises GFP protein, connecting peptide protein and GmCDF1-C17 protein from the N end to the C end;
the connecting peptide protein consists of 9-13 amino acid residues;
the GmCDF1-C17 protein is b 1) or b 2) or b 3) or b 4) as follows:
b1 Amino acid sequence is the protein shown in 250 to 266 positions from the 5' end of SEQ ID NO. 1;
b2 A fusion protein obtained by ligating a tag to the N-terminal or/and C-terminal of the protein shown in positions 250 to 266 from the 5' -terminal of SEQ ID NO. 1;
b3 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in b 1) or b 2), is derived from soybean and is related to the flowering time of the soybean;
b4 A protein which has 80% or more homology with the amino acid sequence defined from 250 to 266 from the 5' -end of SEQ ID NO. 1, is derived from soybean and is related to the flowering time of soybean.
2. The fusion protein GFP-GmCDF1-C17 of claim 1, wherein:
the amino acid sequence of the GFP protein is shown in positions 1 to 238 from the 5' end of SEQ ID NO. 1;
the amino acid sequence of the connecting peptide protein is shown in the 239 th to 249 th positions from the 5' end of SEQ ID NO. 1.
3. A nucleic acid molecule encoding the fusion protein GFP-GmCDF1-C17 of claim 1 or 2.
4. A nucleic acid molecule according to claim 3, wherein: the nucleic acid molecule is a DNA molecule shown in c 1) or c 2) or c 3) or c 4):
c1A DNA molecule with a coding region shown as SEQ ID NO. 2;
c2 A DNA molecule with a nucleotide sequence shown as SEQ ID NO. 2;
c3 A DNA molecule derived from soybean and encoding the fusion protein GFP-GmCDF1-C17 of claim 1 or 2, having 75% or more homology to the nucleotide sequence defined in C1) or C2);
c4 A DNA molecule derived from soybean and encoding the fusion protein GFP-GmCDF1-C17 of claim 1 or 2) hybridizing under stringent conditions to the nucleotide sequence defined in C1) or C2).
5. Use of the fusion protein GFP-GmCDF1-C17 of claim 1 or 2 for regulating the flowering time of soybean or for breeding transgenic soybeans with advanced flowering time.
6. Use of the nucleic acid molecule of claim 3 or 4 for regulating flowering time of soybean or for breeding transgenic soybean with advanced flowering time.
7. A method of breeding transgenic soybean comprising the steps of: increasing the expression level and/or activity of the fusion protein GFP-GmCDF1-C17 of claim 1 or 2 in the starting soybean to obtain transgenic soybean; the flowering time of the transgenic soybeans is advanced compared to the starting soybeans.
8. The method according to claim 7, wherein: the increase in the expression level and/or activity of the fusion protein GFP-GmCDF1-C17 according to claim 1 or 2 in the starting soybean is achieved by introducing into the starting soybean a nucleic acid molecule encoding the fusion protein GFP-GmCDF1-C17.
9. A soybean breeding method comprising the steps of: increasing the content and/or activity of the fusion protein GFP-GmCDF1-C17 of claim 1 or 2 in soybean, thereby advancing the flowering time of soybean.
10. The use according to claim 5 or 6 or the method according to any one of claims 7 to 9, characterized in that: the culture condition of the soybean is long sunlight.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310562581.7A CN116769052A (en) | 2023-05-18 | 2023-05-18 | Method for promoting soybean flowering time to advance under long-day condition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310562581.7A CN116769052A (en) | 2023-05-18 | 2023-05-18 | Method for promoting soybean flowering time to advance under long-day condition |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116769052A true CN116769052A (en) | 2023-09-19 |
Family
ID=88009000
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310562581.7A Pending CN116769052A (en) | 2023-05-18 | 2023-05-18 | Method for promoting soybean flowering time to advance under long-day condition |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116769052A (en) |
-
2023
- 2023-05-18 CN CN202310562581.7A patent/CN116769052A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113430212B (en) | Apple rootstock salt stress resistance related gene MdLysMe3 and encoding protein and application thereof | |
| CN114774439B (en) | Tea tree CsFAAH6 gene and application thereof | |
| CN110804090B (en) | Protein CkWRKY33 and coding gene and application thereof | |
| CN109486830A (en) | Rice SNB gene and application, the method for regulating and controlling seed size | |
| CN114369147B (en) | Application of BFNE gene in tomato plant type improvement and biological yield improvement | |
| CN113403325B (en) | Tea tree orphan gene CsOG3 and application thereof in improving cold resistance of tea trees | |
| CN112724213B (en) | Sweet potato anthocyanin synthesis and stress resistance related protein IbMYB4, and coding gene and application thereof | |
| CN105585623B (en) | The disease-resistant breeding method for turning TaMYB-KW DNA triticum and relevant biological material and application | |
| CN113088526B (en) | Heat shock related gene ZmHsf11 and application thereof in regulation and control of plant heat resistance | |
| CN110698550A (en) | Molecular detection method for rapidly identifying real plum/apricot plum strain | |
| CN111662366A (en) | Preparation method of early-flowering high-yield tomato material | |
| CN114703199B (en) | Plant drought resistance related gene TaCML46 and application thereof | |
| CN109207485B (en) | Application of OsAPS1 gene in improving disease resistance of rice | |
| CN111171127A (en) | Astragalus sinicus LHY gene and application thereof | |
| CN114292855B (en) | PagARR9 gene for regulating and controlling growth of xylem of poplar and application thereof | |
| CN106978499B (en) | Transgenic soybean event GC1-1 external source Insert Fragment flanking sequence and its application | |
| CN112795580B (en) | Pitaya gene HuAAE3 and application thereof in regulation and control of high temperature stress resistance of plants | |
| CN113563439B (en) | Fruit shape development related protein, and coding gene and application thereof | |
| CN116769052A (en) | Method for promoting soybean flowering time to advance under long-day condition | |
| CN112409465B (en) | Application of protein M57 in regulation and control of ammonium resistance of rice | |
| CN109371036B (en) | An alfalfa salt tolerance gene MsPIP 2; 2 and uses thereof | |
| CN109722441B (en) | Cucumber small heat shock protein Cu-sHSP gene and application thereof | |
| CN108103075B (en) | Switchgrass gene PvC3H29 for delaying plant senescence and application thereof | |
| CN114672493B (en) | Method for cultivating drought-resistant plant by ZmPHT1, 7 protein or encoding gene thereof | |
| CN115094072B (en) | Populus tomentosa PtYABBY7 gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |