CN117126885B - Litchi F-Box gene and application of F-Box protein - Google Patents
Litchi F-Box gene and application of F-Box protein Download PDFInfo
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- CN117126885B CN117126885B CN202311273804.4A CN202311273804A CN117126885B CN 117126885 B CN117126885 B CN 117126885B CN 202311273804 A CN202311273804 A CN 202311273804A CN 117126885 B CN117126885 B CN 117126885B
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- 108010066805 F-Box Proteins Proteins 0.000 title claims abstract description 68
- 102000018700 F-Box Proteins Human genes 0.000 title claims abstract description 21
- 241001629511 Litchi Species 0.000 claims abstract description 88
- 241000196324 Embryophyta Species 0.000 claims abstract description 42
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 claims abstract description 37
- 230000001737 promoting effect Effects 0.000 claims abstract description 15
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 8
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
- 241000219195 Arabidopsis thaliana Species 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 12
- 239000013604 expression vector Substances 0.000 claims description 11
- 238000003259 recombinant expression Methods 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 6
- 241000589158 Agrobacterium Species 0.000 claims description 5
- 230000004883 flower formation Effects 0.000 abstract description 10
- 241000219194 Arabidopsis Species 0.000 description 18
- 230000009261 transgenic effect Effects 0.000 description 10
- 239000002299 complementary DNA Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
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- 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]
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
The invention discloses a litchi F-Box gene and application of F-Box protein, wherein the litchi F-Box gene is applied to promoting plant flowering, the nucleotide sequence of the litchi F-Box gene is shown as SEQ ID NO.1, or the litchi F-Box gene codes protein with the amino acid sequence shown as SEQ ID NO. 2. The invention confirms that the litchi F-Box gene is an important regulation gene for litchi flower formation, and the litchi F-Box gene can promote litchi flower formation and stabilize flower formation, thereby realizing litchi stable yield and having important application value.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to application of a litchi F-Box gene and a coded protein thereof in regulating and controlling flowering time of plants.
Background
The litchi belongs to tropical subtropical evergreen arbor which is widely cultivated in the south of Guangdong, guangxi, hainan and Fujian of China, and is also one of the most characteristic fruits with international market competitiveness in China.
The flower forming is the precondition of the litchi fruit, and the litchi flower forming is influenced by external factors besides the self gene regulation. In the global scope, because the flower formation of litchi has extremely strict requirements on low-temperature conditions, the economic cultivation area of litchi in the world is mainly distributed in the latitude zone of 17-32 degrees in the north and south.
The genetic basis of litchi is relatively complex, the environment is changeable, the phenomenon of 'size and year' is easily caused in litchi production, and low yield and unstable yield caused by unstable flower formation are main reasons of the 'size and year' result of litchi.
Therefore, the method for determining the regulation and control mechanism of the litchi flower formation, and the method for excavating and utilizing the regulation and control gene of the litchi flower formation are important contents of litchi research.
Disclosure of Invention
Based on the above, the invention aims to provide an application of litchi F-Box gene and F-Box protein.
The technical scheme for realizing the aim of the invention comprises the following steps.
In a first aspect of the invention, an application of a litchi F-Box gene in promoting plant flowering is provided, wherein the nucleotide sequence of the litchi F-Box gene is shown as SEQ ID NO.1, or the litchi F-Box gene codes a protein with the amino acid sequence shown as SEQ ID NO. 2.
In a second aspect of the invention, the application of litchi F-Box protein in promoting flowering of plants is provided, and the amino acid sequence of the litchi F-Box protein is shown as SEQ ID NO. 2.
In a third aspect, the invention provides an application of a recombinant expression vector for over-expressing litchi F-Box genes in promoting flowering of plants.
In a fourth aspect, the invention provides the use of a recombinant bacterium transformed with a recombinant expression vector as described above for promoting flowering in plants.
In a fifth aspect of the present invention, there is provided a biological agent for promoting flowering in plants, the active ingredient of the biological agent comprising the recombinant expression vector described above.
In a sixth aspect of the present invention, there is provided a method for promoting flowering in a plant, the method comprising increasing expression of a litchi F-Box gene or litchi F-Box protein in the plant.
The invention confirms that the litchi F-Box gene is an important regulation gene for litchi flower formation, and the litchi F-Box gene can promote litchi flower formation and stabilize flower formation, thereby realizing litchi stable yield and having important application value.
Drawings
FIG. 1 shows the results of detection of Arabidopsis positive plants overexpressing the litchi F-Box gene in example 2 of the present invention.
FIG. 2 shows flowering phenotype (A) and flowering days statistics (B) of Arabidopsis positive plants and wild type Arabidopsis plants over-expressing litchi F-Box gene in example 3 of the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Green and Sambrook et al, molecular cloning, an experimental guideline (Molecular Cloning: A Laboratory Manual, 2013), or by the manufacturer's recommendations. The various chemicals commonly used in the examples are commercially available.
In the invention, total RNA of litchi tender leaves is firstly reversely transcribed into cDNA, the cDNA is used as a template, a primer pair shown as SEQ ID NO.3 and SEQ ID NO.4 is used for carrying out PCR amplification to obtain litchi F-Box genes, then the litchi F-Box genes are cloned to a pCAMBIA1390 vector to obtain the pCAMBIA1390-F-Box vector which over-expresses the litchi F-Box genes, arabidopsis thaliana is transformed by agrobacterium to obtain Arabidopsis thaliana transgenic plants, and the discovery is carried out in the process of counting the flowering time of the Arabidopsis thaliana transgenic plants: compared with wild arabidopsis, the transgenic arabidopsis has obviously advanced flowering time, which indicates that the litchi F-Box gene is very likely to promote the flowering of litchi.
In some embodiments of the invention, an application of a litchi F-Box gene in promoting plant flowering is disclosed, wherein the nucleotide sequence of the litchi F-Box gene is shown as SEQ ID NO.1, or the litchi F-Box gene encodes a protein with the amino acid sequence shown as SEQ ID NO. 2.
In some embodiments, the plant is litchi or arabidopsis thaliana.
In other embodiments of the invention, application of litchi F-Box protein in promoting flowering of plants is disclosed, wherein the amino acid sequence of the litchi F-Box protein is shown as SEQ ID NO. 2.
In some embodiments, the plant is litchi or arabidopsis thaliana.
SEQ ID NO.1
ATGTTAACCTCAGTACTGGTGAGCTGTGCTCAATTGACAAAGTTGCTTGTA
CTGCCCTTTGCTCTGCTGCCTCCACCTGATATCGTGAGAGAAAATTATCACT
ATTTTCTTGGACTTTTGGGAGGGTGTCTTTGCTTTTCAAAGGGAAGGAATA
GATATTGTGGCGAATTTTGGGTTATGAAGGAATATGGTGCGAAAAAATCGT
GGACTAGAATTATGATTATACCTAATGTGTTCTATATATTGAAGCCATTGTGT
TATTTAAATACTAGTGATACAGTGTTGCTGTTTATGAATGGACACATTGGTG
TTTCACAATTGAAGAGATGGGAATTTGAAGAATATAGAGATGATCAGGTTG
ATGGCATTAATGATGCTGAGTGGCTTGGCATCAATATTAGAGGGCTAATGCA
GAACGCGAAGTGCTCTAATAGAGTGAAGGTAGAAGGATAG
It is understood that modifications of the base sequence of the above cDNA reading frame without changing the amino acid sequence, taking into account the degeneracy of the codons, are also within the scope of the present invention.
SEQ ID NO.2
MLTSVLVSCAQLTKLLVLPFALLPPPDIVRENYHYFLGLLGGCLCFSKGRNRY
CGEFWVMKEYGAKKSWTRIMIIPNVFYILKPLCYLNTSDTVLLFMNGHIGVS
QLKRWEFEEYRDDQVDGINDAEWLGINIRGLMQNAKCSNRVKVEG
It is understood that the amino acid sequence of the litchi F-Box protein is subjected to various substitutions, deletions or additions of one or more amino acids or terminal modification without affecting the structure and activity of the litchi F-Box protein, and the litchi F-Box protein also belongs to the protection scope of the invention.
In other embodiments of the present invention, the use of a recombinant expression vector over-expressing the litchi F-Box gene to promote flowering in plants is disclosed.
In some of these embodiments, the recombinant expression vector is pCAMBIA1390-F-box.
In other embodiments of the present invention, the use of a recombinant bacterium transformed with the recombinant vector described above to promote flowering in plants is disclosed.
In some embodiments, the recombinant bacterium is a recombinant agrobacterium.
In other embodiments of the invention, application of litchi F-Box genes, litchi F-Box proteins, recombinant expression vectors for over-expressing litchi F-Box genes and recombinant bacteria transformed with the recombinant expression vectors in genetic breeding for improving flowering time of plants is disclosed.
In other embodiments of the present invention, a biological agent for promoting flowering in plants is disclosed, the active ingredient of which comprises a recombinant expression vector that overexpresses the litchi F-Box gene.
In other embodiments of the present invention, a method of promoting flowering in a plant is disclosed, the method comprising increasing expression of a litchi F-Box gene or litchi F-Box protein in the plant.
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1 preparation of litchi F-Box Gene and its encoded protein
The method comprises the following steps:
1. RNA extraction and cloning of cDNA
The young leaf of Feizixiao litchi is selected to be about 1g. After adding liquid nitrogen, tissues and cells were lysed with a mortar and rapidly transferred into a 2.0mL centrifuge tube, and litchi leaf RNA was extracted using a plant total RNA extraction kit (RNAprep pure Tissue Kit, TIANGEN). The concentration of RNA was detected by spectrophotometry, while the integrity of RNA was detected by agarose gel electrophoresis.
Next, using a reverse transcription kit (Prime Script Reverse Transcriptase kit, takara) from Dalianbao biological company, the total volume of the reverse transcription primer, dNTPs, and template RNA was mixed to 20. Mu.L, and 5 minutes at 65℃was added with 5X reverse transcriptase, 30℃for 10 minutes, 42℃for 25 minutes, 95℃for 5 minutes, and reverse transcribed to cDNA, which was stored in a refrigerator at-20℃for use.
2. Amplification of litchi F-Box gene
The total cDNA after reverse transcription is used as a template, and F-Box-F: ATGTTAACCTCAGTACTG (SEQ ID NO. 3); F-Box-R: CTATCCTTCTACCTTCACTCT (SEQ ID NO. 4) is a forward primer and a reverse primer, and the litchi F-Box gene fragment is obtained by cloning.
The PCR reaction system is as follows: the forward and reverse primers were 1. Mu.L each, the cDNA template was 2. Mu.L, superMIX was 25. Mu.L, and dd H was supplemented 2 O to 50. Mu.L.
The PCR reaction procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94℃for 30 seconds, renaturation at 58℃for 2 minutes, extension at 72℃for 45 seconds, and 34 cycles followed by 8 minutes at 72 ℃.
And sequencing the final PCR product to obtain the sequence of the F-Box, wherein the nucleotide sequence of the F-Box is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.
Example 2 construction of vector overexpressing litchi F-Box Gene and detection of Positive plants
The method comprises the following steps:
1. the PCR amplified product of purified example 1 was recovered, the F-Box gene was ligated to the vector pCAMBIA1390 plasmid (stored In the applicant's laboratory and commercially available) by In-fusion technique, and positive clones were selected for sequencing after transformation.
2. The correct plasmid was transformed into Agrobacterium GV3101 by liquid nitrogen freeze thawing, 50mg/L Rif and 50mg/L Kan were used as resistance selection, and the obtained monoclonal was identified as positive by PCR.
3. Adding agrobacterium into 50mL LB culture medium containing 50mg/L Rif and 50mg/L Kan resistance, placing into a shaking table for overnight culture at 28 ℃, measuring OD, and stopping shaking when the OD value of the bacterial liquid reaches 0.8-1.0. Centrifugation was performed at 6000rpm at 28℃for 10min.
4. 50ml of bacterial liquid is mixed with 50ml of invasion liquid (containing 0.11g of MS, 2.5g of sucrose and 10uL of 400 mu L/L Silwet-77), and the arabidopsis thaliana is infected by an agrobacteria-mediated flower dipping method for 20-30s and is shaded for 24h.
5. The transformed Arabidopsis thaliana was re-planted in an incubator, and T0 generation Arabidopsis thaliana seeds were collected.
6. Planting T0 generation Arabidopsis seeds in a sterilized matrix, and culturing in an incubator until the plants are mature under the condition that the light intensity is 120-150 umol/(m) 2 S), photoperiod 16h light, 8h dark. Obtaining T1 generation plants.
7. Collecting T1 generation seeds, continuously planting the T1 generation seeds, detecting T1 generation arabidopsis positive plants through detection primers, and detecting the primer F: CGGCGACGAGCCAGGGATA (SEQ ID NO. 5); detection primer R: GCACCATCGTCAACCACTACAT (SEQ ID NO. 6).
The results are shown in FIG. 1, and 31T 1-generation Arabidopsis thaliana detected was positive.
Example 3 flowering phenotype detection of transgenic plants overexpressing the litchi F-Box Gene
Transgenic arabidopsis T2 generation plant seeds and control wild arabidopsis seeds are sown into a sterilized matrix (peat soil: vermiculite=1:1), and when the seeds are flowering and heading, the whole growth days of the arabidopsis plants are counted during flowering, 6 plants are investigated for each transgenic strain, and 3 times of repetition are performed in parallel.
The results are shown in FIG. 2. A in fig. 2 is the flowering phenotype of arabidopsis plants at 25d growth, as evident from the figure: transgenic Arabidopsis thaliana OE1 and OE2 overexpressing the litchi F-Box gene had been flowering and heading at 25d, while wild Arabidopsis thaliana WT had not yet flowering and heading at this time. B in FIG. 2 is the statistical result of the growing days of the Arabidopsis plants at the time of flowering, and the result shows that: the flowering time of transgenic arabidopsis OE1 and OE2 of the over-expressed litchi F-Box gene is 25d on average, and the flowering time of wild type arabidopsis plant WT is 28d, and the growth days of the transgenic arabidopsis plant WT and the wild type arabidopsis plant WT are obviously different in flowering time, so that the flowering time of the transgenic arabidopsis is earlier than that of the wild type arabidopsis, and the litchi F-Box gene is very likely to participate in regulating and controlling the flowering time of litchi.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (7)
1. The application of the litchi F-Box gene in promoting plant flowering is characterized in that the nucleotide sequence of the litchi F-Box gene is shown as SEQ ID NO.1 or the litchi F-Box gene codes protein with the amino acid sequence shown as SEQ ID NO. 2; the plant is litchi or Arabidopsis thaliana.
2. The application of the litchi F-Box protein in promoting plant flowering is characterized in that the amino acid sequence of the litchi F-Box protein is shown as SEQ ID NO. 2; the plant is litchi or Arabidopsis thaliana.
3. The application of the recombinant expression vector over-expressing the litchi F-Box gene in promoting the flowering of plants is characterized in that the nucleotide sequence of the litchi F-Box gene is shown as SEQ ID NO.1 or the litchi F-Box gene encodes a protein with the amino acid sequence shown as SEQ ID NO. 2; the plant is litchi or Arabidopsis thaliana.
4. The use according to claim 3, wherein the recombinant expression vector is pCAMBIA1390-F-box.
5. Use of a recombinant bacterium transformed with the recombinant expression vector of claim 3 or 4 to promote flowering in plants; the plant is litchi or Arabidopsis thaliana.
6. The use according to claim 5, wherein the recombinant bacterium is a recombinant agrobacterium.
7. A method for promoting flowering of plants is characterized by comprising the steps of improving expression of litchi F-Box genes or litchi F-Box proteins in plants, wherein the nucleotide sequence of the litchi F-Box genes is shown as SEQ ID NO. 1; the amino acid sequence of the litchi F-Box protein is shown as SEQ ID NO. 2; the plant is litchi or Arabidopsis thaliana.
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Citations (2)
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| CN109053871A (en) * | 2018-08-09 | 2018-12-21 | 中国农业科学院作物科学研究所 | Application of the AtBIX gene in the regulation flowering of plant time |
| CN113832164A (en) * | 2021-10-29 | 2021-12-24 | 海南大学 | Broad bean salt-resistant gene F-box and application thereof |
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| KR100350213B1 (en) * | 2000-12-20 | 2002-08-28 | (주)제노마인 | Novel gene encoding an F-box protein which regulates leaf longevity in Arabidopsis thaliana and mutant genes thereof |
| US7655835B2 (en) * | 2004-12-10 | 2010-02-02 | The Salk Institute For Biological Studies | Regulating the ethylene response of a plant by modulation of F-box proteins |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109053871A (en) * | 2018-08-09 | 2018-12-21 | 中国农业科学院作物科学研究所 | Application of the AtBIX gene in the regulation flowering of plant time |
| CN113832164A (en) * | 2021-10-29 | 2021-12-24 | 海南大学 | Broad bean salt-resistant gene F-box and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| De novo transcriptome assembly for rudimentary leaves in Litchi chinesis Sonn. and identification of differentially expressed genes in response to reactive oxygen species;Xingyu Lu等;《BMC Genomics》;第15卷;805:1-14 * |
| 植物F-box 基因家族的研究进展;许克恒等;《生物技术通报》;第34卷(第1期);26-32 * |
| 荔枝miRNA和phasiRNA以及碳水化合物胁迫诱导幼果脱落相关miRNA的调控途径鉴定;马武强;《中国博士学位论文全文数据库电子期刊》(第08期);D048-29 * |
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