CN115976045A - Cotton GaTFL1 gene and application and identification method thereof - Google Patents
Cotton GaTFL1 gene and application and identification method thereof Download PDFInfo
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- CN115976045A CN115976045A CN202211253429.2A CN202211253429A CN115976045A CN 115976045 A CN115976045 A CN 115976045A CN 202211253429 A CN202211253429 A CN 202211253429A CN 115976045 A CN115976045 A CN 115976045A
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Abstract
The invention provides a cotton GaTFL1 gene and an application and identification method thereof, belonging to the technical field of gene engineering.A base at 201 th site of No. 7 chromosome of the Asian cotton GaTFL1 gene is mutated from G to A, so that a cotton apical meristem is differentiated into flower buds to form a stop flower, a main stem is changed from infinite growth to limited growth, the plant height is reduced, lateral branch axillary buds grow into flowers, and the limited growth is shown.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a cotton GaTFL1 gene and an application and identification method thereof.
Background
The regulation and control of the corresponding gene/protein function through single base change is the basic requirement and target of gene editing technology, and the key base and potential mutation type of the target gene are the basis for precise gene editing. The site-directed controllable gene editing is carried out in crops, which is beneficial to quickly and efficiently obtaining plants with excellent characters. In the morphogenesis of higher plants, inflorescence development is of vital importance. The artificial control of inflorescence development process is an important step for creating plant model suitable for human production requirement. The TFL1 gene belongs to one of members of the PEBP (phosphatic ethylene diamine binding protein) family, and its main function is to inhibit the initiation of flowering. The TFL1 gene mutation leads to the formation of a stop flower at the end of a plant, changes an unlimited inflorescence of the plant into a limited inflorescence, and lays a foundation for realizing molecular regulation and design breeding of plant inflorescence development.
In 2018, chen et al identified 5 mutation sites in the TFL1 gene of Gossypium hirsutum and Gossypium barbadense. The mutation is exon 2G 217A (Ghnb-1), exon 1 194 single base T deletion (Ghnb-2), exon 2 254-255 two base deletion (Ghnb-3), exon 4 491 single base T deletion (Ghnb-4) and exon 4C 337T mutation (Gbnb-1). The mutation can cause the limited growth of cotton lateral branches, the cotton lateral branches are in short fruit branch type or zero type, and main stems still keep unlimited growth. However, other mutation sites of the GaTFL1 gene of the Asian cotton have not been reported in the prior art.
Disclosure of Invention
In view of the above, the invention aims to provide a cotton GaTFL1 gene and an application and identification method thereof, wherein the 201 th base of the No. 7 chromosome of the Asian cotton GaTFL1 gene is mutated from G to A, so that the apical meristem of cotton is differentiated into flower buds to form stop flowers, the main stem is changed from infinite growth to limited growth, the plant height is reduced, and lateral branch axillary buds grow into flowers and show limited growth.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a cotton GaTFL1 gene, which is characterized in that the 201 st base of No. 7 chromosome of Asian cotton GaTFL1 gene is mutated from G to A.
Preferably, the nucleotide sequence of the cotton GaTFL1 gene is shown in SEQ ID No. 1.
Preferably, the CDS sequence of the cotton GaTFL1 gene is shown in SEQ ID No. 2.
Preferably, the amino acid sequence of the cotton GaTFL1 protein is shown in SEQ ID No. 3.
Preferably, the nucleotide sequence of the mutant Asian cotton GaTFL1 gene is shown in SEQ ID No. 4.
Preferably, the CDS sequence of the mutant Asian cotton GaTFL1 gene is shown as SEQ ID No. 5.
Preferably, the amino acid sequence of the mutant Asian cotton GaTFL1 protein is shown in SEQ ID No. 6.
The invention also provides application of the cotton GaTFL1 gene in cotton plant type improvement.
The invention also provides application of the cotton GaTFL1 gene in the technical scheme in changing the main stem of cotton from infinite growth to limited growth.
The invention also provides a method for identifying the single base G201A mutation of cotton GaTFL1 gene chromosome 7, which comprises the following steps:
1) Performing PCR amplification by taking the cDNA of the cotton root as a template to obtain a PCR product;
2) And (2) carrying out agarose gel electrophoresis on the PCR product obtained in the step 1), and carrying out single base G201A mutation on the chromosome 7 of the cotton GaTFL1 gene when the amplified band is 432bp.
In the invention, the single-base G201A mutation of the cotton GaTFL1 gene is located in the No.1 exon of GaTFL1, the 5' end RNA shearing site is changed, and a shorter exon is generated, so that 31 amino acids of GaTFL1 protein are deleted. Compared with wild WT, the mutant cotton plant has its apical meristem differentiated into flower bud to form terminal flower, the main stem is changed from infinite growth to limited growth, the plant height is lowered obviously, and lateral branch axillary bud is also grown directly into flower with limited growth.
The beneficial effects of the invention are as follows:
the invention has the advantages that a novel single base mutation site G201A is found in the TFL1 gene of cotton, so that the RNA shearing site at the 5' end is changed, the main stem and the lateral branches of the cotton are limited to grow, and a new thought is provided for directionally improving the plant type of the cotton. The prior TFL1 gene mutation site can only regulate the inflorescence development of cotton side branches, so that the side branches are short or zero fruit branches are formed, and the unlimited growth habit of main stems cannot be effectively changed.
Drawings
FIG. 1 is a genomic sequence alignment of GaTFL1 in mutant dt1 and wild-type WT;
FIG. 2 is a CDS sequence alignment of the GaTFL1 gene in mutant dt1 and wild type WT;
FIG. 3 shows that a single base G201A mutation results in an altered RNA cleavage site at the 5' end of the GaTFL1 gene;
FIG. 4 shows that a single base G201A mutation results in shortening of the GaTFL1 protein;
FIG. 5 shows that the single base G201A mutation results in limited growth of the dt1 mutant. Compared with wild WT, the strain height of the dt1 mutant is reduced by 47% because the 201 st base of chromosome 7 of the GaTFL1 gene is mutated from G to A (A in FIG. 5); further observing the top end and the lateral branches of the main stem of the cotton, the G201A mutation causes the direct differentiation of the meristem at the top end of the main stem of the dt1 mutant to form a flower bud, and the unlimited growth is stopped (B in figure 5 and C in figure 5); in addition, the dt1 mutant cotton lateral shoot axillary buds also developed directly into flower buds, as shown by limited growth (D in fig. 5, E in 5) compared to wild-type WT;
FIG. 6 is a graph showing the identification of single base G201A mutation, and as shown in FIG. 6, the single base G201A mutant material dt1 has a cDNA sequence deletion of 93bp due to the change of the 5' end RNA cleavage site of the GaTFL1 gene, so that the electrophoresis band is slightly smaller than that of wild type WT.
Detailed Description
The invention provides a cotton GaTFL1 gene, wherein the 201 st base of the No. 7 chromosome of the Asian cotton GaTFL1 gene is mutated from G to A.
In the invention, the nucleotide sequence of the cotton GaTFL1 gene is shown as SEQ ID No.1, and specifically comprises the following steps:
5’-ATGGCAAAACTGTCAGATCCTCTTGTGGTGGGGAGAGTGATTGGGGATGTTATTGATGCCCTCTCCCCATCTGTGAAAATGTCAGTCACTTTCAACACCAACAAGCAGGTATATAATGGCCATGAATTTTTTCCATCTGCAGTTACTAACAAGCCTAAGGTTGAGGTTCATGGAGGTGATATGAGATCCTTTTTCACCCTGgtaactatacatgtcactaatactctcttttcttttcttttttttttttctggattttcaagagaaaatgtctatcagcaatcatatttttatattgcagGTGATGACAGACCCAGATGTTCCTGGTCCTAGTGACCCTTACCTGAGGGAGCACTTACACTGgtacttctcttaatcaaactacatgtataaaaataaaaatatatattaaccattaataccaatatataacttccatagggaagctgacaatgatctatctatctatatctatatgaaatgactgttagggttcataatttccttggcttgataaagctaaaaaaaaaaggaaataagatttcatcaaaacaagaagagcacaaatttaagcaatctactgaatatttgtacttgaattgacaatctcacagGATAGTGACAGATATCCCCGGCACAACAGATGCCACATTTGgtaagtatcctcttcattcttcatagagagagagagagagagagagagagagagcattatatgatagaaaatggactaactttgatggttaaaaatataattataatatatatatatatatatatatatatacacacacagGAAGGGAAATGGTGAACTACGAAATGCCAAGGCCAAACATAGGGATCCATAGGTTTGTGTTCCTCCTCTTCAAGCAGAAAGGCAGGCAAACAGTGAGAAGCATACCATCATCAAGGGATCGTTTCGATACCAGGAAGTTTGCAGAAGAAAACGAACTAGGGGTTCCTGTTGCAGCTGTCTATTTCAATGCTCAAAGGGAAACAGCTGCTAGAAGACGCTAA-3’。
in the invention, the CDS sequence of the cotton GaTFL1 gene is shown as SEQ ID No.2, and specifically comprises the following steps:
5’-ATGGCAAAACTGTCAGATCCTCTTGTGGTGGGGAGAGTGATTGGGGATGTTATTGATGCCCTCTCCCCATCTGTGAAAATGTCAGTCACTTTCAACACCAACAAGCAGGTATATAATGGCCATGAATTTTTTCCATCTGCAGTTACTAACAAGCCTAAGGTTGAGGTTCATGGAGGTGATATGAGATCCTTTTTCACCCTGGTGATGACAGACCCAGATGTTCCTGGTCCTAGTGACCCTTACCTGAGGGAGCACTTACACTGGATAGTGACAGATATCCCCGGCACAACAGATGCCACATTTGGAAGGGAAATGGTGAACTACGAAATGCCAAGGCCAAACATAGGGATCCATAGGTTTGTGTTCCTCCTCTTCAAGCAGAAAGGCAGGCAAACAGTGAGAAGCATACCATCATCAAGGGATCGTTTCGATACCAGGAAGTTTGCAGAAGAAAACGAACTAGGGGTTCCTGTTGCAGCTGTCTATTTCAATGCTCAAAGGGAAACAGCTGCTAGAAGACGCTAA-3’。
in the invention, the amino acid sequence of the cotton GaTFL1 protein is shown as SEQ ID No.3, and specifically comprises the following steps:
MAKLSDPLVVGRVIGDVIDALSPSVKMSVTFNTNKQVYNGHEFFPSAVTNKPKVEVHGGDMRSFFTLVMTDPDVPGPSDPYLREHLHWIVTDIPGTTDATFGREMVNYEMPRPNIGIHRFVFLLFKQKGRQTVRSIPSSRDRFDTRKFAEENELGVPVAAVYFNAQRETAARRR。
in the invention, the nucleotide sequence of the mutated Asian cotton GaTFL1 gene is shown in SEQ ID No.4, and specifically comprises the following steps:
5’-ATGGCAAAACTGTCAGATCCTCTTGTGGTGGGGAGAGTGATTGGGGATGTTATTGATGCCCTCTCCCCATCTGTGAAAATGTCAGTCACTTTCAACACCAACAAGCAGgtatataatggccatgaattttttccatctgcagttactaacaagcctaaggttgaggttcatggaggtgatatgagatcctttttcaccctagtaactatacatgtcactaatactctcttttcttttcttttttttttttctggattttcaagagaaaatgtctatcagcaatcatatttttatattgcagGTGATGACAGACCCAGATGTTCCTGGTCCTAGTGACCCTTACCTGAGGGAGCACTTACACTGgtacttctcttaatcaaactacatgtataaaaataaaaatatatattaaccattaataccaatatataacttccatagggaagctgacaatgatctatctatctatatctatatgaaatgactgttagggttcataatttccttggcttgataaagctaaaaaaaaaaggaaataagatttcatcaaaacaagaagagcacaaatttaagcaatctactgaatatttgtacttgaattgacaatctcacagGATAGTGACAGATATCCCCGGCACAACAGATGCCACATTTGgtaagtatcctcttcattcttcatagagagagagagagagagagagagagagagcattatatgatagaaaatggactaactttgatggttaaaaatataattataatatatatatatatatatatatatatacacacacagGAAGGGAAATGGTGAACTACGAAATGCCAAGGCCAAACATAGGGATCCATAGGTTTGTGTTCCTCCTCTTCAAGCAGAAAGGCAGGCAAACAGTGAGAAGCATACCATCATCAAGGGATCGTTTCGATACCAGGAAGTTTGCAGAAGAAAACGAACTAGGGGTTCCTGTTGCAGCTGTCTATTTCAATGCTCAAAGGGAAACAGCTGCTAGAAGACGCTAA-3’。
in the invention, the CDS sequence of the mutated Asian cotton GaTFL1 gene is shown as SEQ ID No.5, and specifically comprises the following steps:
5’-ATGGCAAAACTGTCAGATCCTCTTGTGGTGGGGAGAGTGATTGGGGATGTTATTGATGCCCTCTCCCCATCTGTGAAAATGTCAGTCACTTTCAACACCAACAAGCAGGTGATGACAGACCCAGATGTTCCTGGTCCTAGTGACCCTTACCTGAGGGAGCACTTACACTGGATAGTGACAGATATCCCCGGCACAACAGATGCCACATTTGGAAGGGAAATGGTGAACTACGAAATGCCAAGGCCAAACATAGGGATCCATAGGTTTGTGTTCCTCCTCTTCAAGCAGAAAGGCAGGCAAACAGTGAGAAGCATACCATCATCAAGGGATCGTTTCGATACCAGGAAGTTTGCAGAAGAAAACGAACTAGGGGTTCCTGTTGCAGCTGTCTATTTCAATGCTCAAAGGGAAACAGCTGCTAGAAGACGCTAA-3’。
in the invention, the amino acid sequence of the mutant Asian cotton GaTFL1 protein is shown as SEQ ID No.6, and specifically comprises the following steps:
MAKLSDPLVVGRVIGDVIDALSPSVKMSVTFNTNKQVMTDPDVPGPSDPYLREHLHWIVTDIPGTTDATFGREMVNYEMPRPNIGIHRFVFLLFKQKGRQTVRSIPSSRDRFDTRKFAEENELGVPVAAVYFNAQRETAARRR。
the invention also provides application of the cotton GaTFL1 gene in cotton plant type improvement. The invention preferably specifically changes the base at the 201 st position of the GaTFL1 gene from G to A by means of gene editing and the like, and can change the function of the GaTFL1 gene, thereby reducing the plant height of cotton, shortening fruit branches and properly changing the plant type. When the operation is carried out on a specific variety, the plant type can be directionally changed without influencing other characters.
The invention also provides application of the cotton GaTFL1 gene in the technical scheme in changing the main stem of cotton from infinite growth to limited growth.
The invention also provides a method for identifying the single base G201A mutation of cotton GaTFL1 gene chromosome 7, which comprises the following steps:
1) Performing PCR amplification by taking the cDNA of the cotton root as a template to obtain a PCR product;
2) And (2) carrying out agarose gel electrophoresis on the PCR product obtained in the step 1), and carrying out single base G201A mutation on the chromosome 7 of the cotton GaTFL1 gene when the amplified band is 432bp.
In the present invention, the cotton preferably comprises Asian gossypium subnumber 1.
In the present invention, the primers used in the PCR amplification are as follows:
GaTFL1-F(SEQ ID No.7):ATGGCAAAACTGTCAGATCC；
GaTFL1-R(SEQ ID No.8):TTAGCGTCTTCTAGCAGCTG。
in the present invention, the PCR amplification reaction system (50. Mu.L) is preferably: cDNAstrate X. Mu.L (750 ng), primerF (10 mM) 1. Mu.L, primerR (10 mM) 1. Mu.L, KOD One TM PCRMasterMix 25μL，ddH 2 O make up to 50. Mu.L. In the present invention, the PCR amplification reaction procedure is preferably: high-temperature denaturation at 98 ℃ for 10sec; annealing at 55 ℃ for 5sec; the temperature extension is 68 ℃ and 5sec (40 cycles); 4 ℃ for 2min. In the present invention, the agarose gel electrophoresis conditions preferably include: 120V,2% agarose gel, electrophoresis 15min. The invention preferably uses an ultraviolet gel imager to observe DNA bands, judges whether the tested material has G201A single base mutation or not according to the size of the bands, and amplifies normal strainsThe band is 525bp, and the amplified band in the mutant strain is 432bp.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1) Acquisition of GaTFL1 single-base G201A mutant dt1
Ethyl Methane Sulfonate (EMS) mutagenesis test selects Asian cotton-series sub-1 seeds as test materials, 23000 seeds are presoaked in phosphate buffer (100 mmol/L, pH 7.0) at 28 ℃ for 12h, then EMS solution with volume fraction of 0.6% is prepared by the phosphate buffer, and the seeds are treated in dark at 28 ℃ for 8h, and are overturned and lightly shaken in the process. After treatment, the cells were rinsed 3 times with distilled water, each for 30min, to remove the residual EMS solution. The treated seeds were then sown in a row-by-row arrangement in the field according to the space ratio method, with 1 row of controls being placed every 12 rows (control group is phosphate buffer treated stone-type sub-seed No. 1). And investigating the variation condition of the plant field characters in the cotyledon stage, the seedling stage, the bud stage, the flowering stage, the boll stage and the boll opening stage respectively. Harvesting M according to single plant after boll opening 1 And (5) seed generation. From M 1 Selecting morphological variation material from generation seeds, sowing, and then selecting M 2 Selecting morphological variation materials from the generation strains, dibbling according to the strains, investigating and recording the offspring M in the whole growth period 3 Variant phenotype and variant strain number and proportion, 36 mutant materials are finally obtained, including the limited growth mutant dt1 involved in the invention.
2) GaTFL1 single-base G201A mutation sequencing verification
As shown in FIG. 1, the genomic sequence of Ga07G1189 (GaTFL 1) was amplified in EMS mutagenesis material dt1 and wild type material WT, respectively, and the PCR products obtained were recovered by cutting gel, ligated and transformed into TOPO blunt-ended cloning vector, and subjected to sequencing analysis. The results show that the 201bp position of the GaTFL1 gene does generate G-to-A mutation in the EMS mutagenesis material dt1. Similarly, the CDS sequence of Ga07G1189 (GaTFL 1) was further amplified in the above material, and the sequencing result is shown in FIG. 2, wherein the CDS sequence of Ga07G1189 (GaTFL 1) was subjected to 93bp base deletion after G201A single base mutation.
3) GaTFL1 single-base G201A mutation site analysis
The single base G201A mutation is located in exon 1 of GaTFL1, and the 5' end RNA shearing site is changed to generate a shorter exon (figure 3) through combining sequencing results, so that 31 amino acids of GaTFL1 protein are deleted (figure 4).
4) Phenotypic identification of GaTFL1 single-base G201A mutation
The GaTFL1 gene in the dt1 mutant generates single base G201A mutation, compared with wild WT, the dt1 mutant apical meristem is differentiated into flower buds to finally form a stop flower, the main stem is changed from infinite growth to limited growth, and the plant height is obviously reduced (A in figure 5, B in 5, and C in 5); at the same time, lateral axillary buds also developed directly into flowers, showing limited growth (D in fig. 5, E in 5).
5) Application of GaTFL1 single-base G201A mutation site in plant type improvement
The function of the GaTFL1 gene can be changed by specifically changing the base at the 201 th site of the GaTFL1 gene from G to A through means of gene editing and the like, so that the plant height of cotton is reduced, fruit branches are shortened, and the plant type is properly changed. In view of the importance and function conservation of the TFL1 gene in plants, the plant type (plant height is shortened and fruit branches are shortened) can be directionally changed by performing the operation on different varieties including cotton without influencing other characters.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A cotton GaTFL1 gene is characterized in that the 201 th base of No. 7 chromosome of the Asian cotton GaTFL1 gene is mutated from G to A.
2. The cotton GaTFL1 gene according to claim 1, wherein the nucleotide sequence of the cotton GaTFL1 gene is shown in SEQ ID No. 1.
3. The cotton GaTFL1 gene according to claim 1 or 2, wherein the CDS sequence of the cotton GaTFL1 gene is shown in SEQ ID No. 2.
4. The cotton GaTFL1 gene according to claim 1 or 2, wherein the amino acid sequence of the cotton GaTFL1 protein is shown in SEQ ID No. 3.
5. The cotton GaTFL1 gene according to claim 1, wherein the nucleotide sequence of the mutated Asian cotton GaTFL1 gene is shown in SEQ ID No. 4.
6. The cotton GaTFL1 gene according to claim 1 or 5, wherein the CDS sequence of the mutated Asian cotton GaTFL1 gene is shown in SEQ ID No. 5.
7. The cotton GaTFL1 gene according to claim 1 or 5, wherein the amino acid sequence of the mutant Asian cotton GaTFL1 protein is shown in SEQ ID No. 6.
8. The use of the cotton GaTFL1 gene of any one of claims 1 to 7 in the plant type improvement of cotton.
9. Use of the cotton GaTFL1 gene of any of claims 1 to 7 in the alteration of the main stem of cotton from unlimited growth to limited growth.
10. A method for identifying single-base G201A mutation of cotton GaTFL1 gene chromosome 7, which is characterized by comprising the following steps:
1) Performing PCR amplification by taking the cDNA of the cotton root as a template to obtain a PCR product;
2) And (2) carrying out agarose gel electrophoresis on the PCR product obtained in the step 1), and carrying out single base G201A mutation on the chromosome 7 of the cotton GaTFL1 gene when the amplified band is 432bp.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6939596A (en) * | 1995-09-13 | 1997-04-01 | Pioneer Hi-Bred International, Inc. | Flowering genes |
| CN114729357A (en) * | 2019-08-02 | 2022-07-08 | 联邦科学技术研究组织 | RNA molecules for modulating flowering in plants |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6939596A (en) * | 1995-09-13 | 1997-04-01 | Pioneer Hi-Bred International, Inc. | Flowering genes |
| CN114729357A (en) * | 2019-08-02 | 2022-07-08 | 联邦科学技术研究组织 | RNA molecules for modulating flowering in plants |
Non-Patent Citations (3)
| Title |
|---|
| ANAGNOSTIS ARGIRIOU等: "Characterization and expression analysis of TERMINAL FLOWER1 homologs from cultivated alloteraploid cotton (Gossypium hirsutum) and its diploid progenitors", 《J PLANT PHYSIOL》 * |
| GENBANK: "PREDICTED: Gossypium arboreum CEN-like protein 2 (LOC108486309), transcript variant X1, mRNA", 《GENBANK》 * |
| 王云梦等: "FT和TFL1基因调控植物开花的分子机理", 《植物生理学报》 * |
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