CN110669119A - EjAGL17 protein for regulating loquat flowering time and coding gene and application thereof - Google Patents
EjAGL17 protein for regulating loquat flowering time and coding gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of plant molecular biology, and particularly relates to an EjAGL17 gene related to loquat flowering time regulation and application thereof. The full length of the coding region sequence of the cDNA of EjAGL17 gene is shown as SEQ ID No.1, and the amino acid sequence of the coding protein is shown as SEQ ID No. 2. The EjAGL17 gene is transiently expressed in tobacco lamina cells and is positioned in the nucleus, and the protein coded by the gene has typical transcription factor characteristics. Meanwhile, the expression of the gene in different periods of loquat flower development has significant difference. The EjAGL17 gene overexpression vector is transferred into wild type Arabidopsis thaliana by an agrobacterium-mediated inflorescence dip-dyeing method. The results show that the wild Arabidopsis thaliana over-expression of EjAGL17 gene can promote the flowering time of Arabidopsis thaliana to be about 14 days earlier. The transgenic plant material obtained by utilizing the EjAGL17 gene overexpression vector can obviously advance the flowering phase of the plant, further promote the fruiting time to be advanced, can be used for the directional breeding of early-flowering and early-maturing varieties of the plant, and has good application prospect.
Description
Technical Field
The invention belongs to the field of plant molecular biology, and particularly relates to loquat EjAGL17 protein, and a coding gene and application thereof.
Background
Loquat (Eriobotrya japonica) is a plant of the genus Eriobotrya of the family rosaceae and is an important evergreen fruit tree in the south of China; the fruits of the fresh-keeping tea are ripe in the late spring and early summer, the pulp is fresh, tender and juicy, and the fresh-keeping tea has the effects of moistening lung, reducing phlegm and relieving cough and is popular with consumers. Unlike most other rosaceous plants, loquat flower buds begin to differentiate in summer and usually flower in late autumn and early winter. The flower development of the plant is carried out through a flower bud morphological differentiation period, an inflorescence main shaft and fulcrum differentiation period and a lateral growth fulcrum rapid elongation period to form a conical inflorescence; further, the differentiation stage and flowering stage of florets occur on the fulcrum. Therefore, the research on the molecular regulation mechanism of the loquat flowering phase is beneficial to comprehensively understanding the flowering process of the rosaceous plants.
Flowering is a key conversion process of angiosperms from vegetative growth to reproductive growth, relates to a network approach of multi-gene molecule regulation, and is influenced by the external environment and expression regulation of various endogenous flowering related genes. The MADS-box gene family is ubiquitous in angiosperms and is closely associated with the regulation of floral development, such as: inducing flowering, formation and development of floral organs, etc. MADS-box gene is a kind of gene with specific structure domain and function, and has important effect on signal transduction and plant growth and development. Wherein, AGAMOUS-Like 17 and homologous genes are key MADS-box genes involved in the regulation of angiosperm flower development. In recent years, only the research of model plants, namely arabidopsis thaliana and rice, shows that AGL17 and homologous genes are involved in the regulation and control of the flower development process. However, no report has been found on the research on the control of the loquat AGAMOUS-Like 17(EjAGL17) gene on the flower development process and the flowering time.
Disclosure of Invention
The invention aims to provide loquat EjAGL17 protein, and a coding gene and application thereof.
First, the present invention provides loquat EjAGL17 protein which is:
1) a protein consisting of the amino acids shown in SEQ ID No. 2; or
2) Protein which is derived from the protein 1) and has the same activity by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.2, wherein the protein derived from the protein 1) comprises a MADS structural domain, an I structural domain, a K structural domain and a C terminal structural domain.
The invention also provides a gene for coding the loquat EjAGL17 protein.
The sequence of the gene is shown as SEQ ID No. 1.
The invention also provides an overexpression vector containing the gene, a host cell and an engineering bacterium.
The invention also provides the application of the gene in regulating and controlling the flowering time of angiosperm.
The invention separates 1 EjAGL17 gene closely related to loquat flower development regulation from loquat flower buds. The EjAGL17 gene was found to be located in the nucleus by transient expression in tobacco leaf cells, indicating that the gene encodes a typical transcription factor. The real-time fluorescent quantitative PCR proves that the EjAGL17 gene expresses obviously different in diploid and triploid loquat early and late flower varieties, and the expression mode of the EjAGL17 gene is closely related to the flowering phase of loquat. A plant over-expression vector of EjAGL17 gene is constructed by means of genetic engineering, and is transferred into wild arabidopsis thaliana for over-expression, so that the arabidopsis thaliana can be promoted to bloom in advance, and the early fruiting of the arabidopsis thaliana is promoted. The invention provides good application prospect for the transformation of the florescence and the fruit period of the angiosperm.
Drawings
FIG. 1 is an electrophoretogram showing the 3'RACE and 5' RACE of loquat Ejagl17 gene and the sequence verification of the coding region of the gene. Wherein, A is an electrophoresis picture of 3' RACE, M is DL2000DNA marker, 3R1 is PCR product of step 1 of 3' RACE, and 3R2 is PCR product of step 2 of 3' RACE; b is an electrophoresis photograph of 5'RACE, M is DL2000DNA marker, and 5R2 is PCR product of step 2 of 5' RACE; c is a PCR electrophoresis photograph for verifying EjAGL17 gene ORF, M is DL2000DNA marker, and 1 is a PCR product of EjAGL17 gene ORF; the black arrows indicate the bands of the target gene amplified by PCR.
FIG. 2 is a cDNA nucleotide sequence diagram of the coding region of EjaGL17 gene related to loquat flowering phase control.
FIG. 3 is an amino acid sequence and domain partition diagram of loquat EjaGL17 protein.
FIG. 4 is a comparison of the amino acid sequence of the protein encoded by loquat Ejagl17 with the sequences of pear, coffee tree, Arabidopsis thaliana and Brassica napus, and the protein sequence has obvious sequence difference compared with the sequences of its kindred species and other angiosperms, especially the amino acid sequence of C structural domain has the largest difference, indicating the specificity of the protein sequence. The 1 st horizontal line is the M domain; the 2 nd horizontal line is the K domain; between the M and K domains is the I domain; the K domain is followed by the C domain.
FIG. 5 is the subcellular localization of transient expression of the loquat EjAGL17 gene in tobacco leaves, showing that the expression product of the gene is localized to the nucleus. GFP: green fluorescent protein; DAPI: 4, 6-diamidine-2-phenylindole; BF: bright field imaging; merged: combined images of GFP, DAPI and BF.
FIG. 6 shows that the expression of the loquat EjaGL17 gene shows significant difference in different periods of loquat flower development.
FIG. 7 shows the restriction electrophoresis of EjAGL17 gene overexpression vector. Wherein a is a pBI121 carrier enzyme cutting electrophoresis picture, M is DL15000 DNA marker, and 1 is a pBI121 carrier after double enzyme cutting; b is a double-enzyme digestion verification electrophoretogram of an EjAGL17 overexpression vector pBI121-EjAGL17, M is DL15000 DNA marker, and 1 is a pBI121-EjAGL17 vector after double enzyme digestion.
FIG. 8 is a photograph of flowering-time of Arabidopsis thaliana before and after 6 transgenes. Wherein, compared with the non-transgenic wild arabidopsis, the overexpression of the EjAGL17 gene can promote the flowering time of the transgenic arabidopsis to be about 14 days earlier.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory manual,2001), or the conditions suggested by the manufacturer's instructions.
EXAMPLE 1 cloning of cDNA sequence of EjAGL17 Gene of loquat
Extraction of loquat flower bud total RNA
Collecting fresh flower buds of loquat in differentiation period of about 1.0cm, quickly placing into a freezing tube, quickly freezing in liquid nitrogen for 2h, and placing into an ultra-low temperature refrigerator at-80 deg.C for use. Extracting the total RNA of the loquat flower buds by adopting an RNA extraction kit: taking out the collected flower bud material from an ultralow temperature refrigerator at minus 80 ℃, putting the flower bud material into a mortar which is frozen in advance and is added with 1mL of RLT lysate and 100 mu of LPLANTaid, and fully grinding the flower bud material at room temperature; transferring the grinding fluid into a 1.5mL eppendorf centrifuge tube, centrifuging at 13000rpm for 10min, sucking 500 mu L of supernatant fluid, and transferring the supernatant fluid into a new 1.5mL centrifuge tube; adding 250 μ L of anhydrous alcohol into the supernatant, sucking, mixing, adding into adsorption column, and placing into collection tube; adding 500 μ L deproteinized solution into adsorption column, centrifuging at 13000rpm for 2 min; adding 500 μ L of rinsing solution, centrifuging at 13000rpm for 2min, pouring off waste liquid in the collecting pipe, adding rinsing solution again and centrifuging at 13000rpm for 2 min; putting the adsorption column back into the empty collection pipe, centrifuging at 13000rpm for 3min, removing residual rinsing liquid, and placing the adsorption column in a super clean bench for 2min to volatilize the residual rinsing liquid; placing the adsorption column back into empty RNase-free centrifuge tube, adding 50 μ L RNase free water, standing at room temperature for 2min, and centrifuging at 13000rpm for 2 min; and adding the first eluent into the adsorption column again, and centrifuging again to improve the extraction concentration of the RNA. 2. mu.L of the diluted RNA sample was aspirated, and the RNA concentration was detected by a trace nucleic acid concentration detector.
3' RACE experiment of EjAGL17 gene of loquat
The extracted loquat flower bud total RNA is used as a 3' RACE experiment template, and 3' RACE adapter is used as a joint primer to carry out reverse transcription reaction, so as to synthesize the first chain cDNA of the 3' RACE experiment. The specific operation is as follows: suction of Total RNA 1. mu.L, 3' RACEAdaptor 1. mu.L, DEPC-ddH2O4.5 mu L, uniformly mixing, denaturing at 70 ℃ for 10min, and carrying out ice bath for 2 min; after the RNA denaturation reaction is finished, 0.25 mu L of RNase inhibitor, 1 mu L of 10mM dNTP, 2 mu L of 5 XM-MLV buffer and 0.25 mu L of M-MLV are sequentially added, mixed uniformly and placed at 42 ℃ for reaction for 60 min; then, reacting for 10min at 70 ℃; ice-cooling for 2min, and storing at-20 deg.C.
According to conserved regions of homologous gene sequences of loquat kindred pear (KP164022) published by NCBI website and model plant Arabidopsis (AB763910) AGAMOUS-Like 17, upstream specific primers 3REjAGL17F1 and 3REjAGL17F2, 3REAGL17F1 of 3' RACE experiment are directly designed: 5'-GAAGGCTAAGGGGCTATCAATCC-3' and 3REjAGL17F 2: 5'-GCGGAGCATCAACTGCCGAATCCG-3' are provided. 3'RACE reverse transcription product was used as template, using high fidelity EX-taq enzyme, upstream Outer specific Primer 3REjAGL17F1 and 3' RACE Outer Primer: 5'-TACCGTCGTTCCACTAGTGATTT-3', performing first chain PCR reaction at 95 deg.C for 4 min; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 40 s; 10min at 72 ℃. Next, using the first strand PCR reaction product as a template, the high fidelity EX-taq enzyme, the upstream outer specific Primer 3REjAGL17F2 and the 3' RACE Inner Primer: 5'-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3', performing a second chain nest type PCR reaction at 95 ℃ for 4 min; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 40 s; 10min at 72 ℃. After the second strand PCR reaction was completed, the band was cut by 1% agarose gel electrophoresis (FIG. 1A), and the PCR product was recovered with an agarose gel DNA recovery kit according to the instructions. And connecting the recovered PCR product to a pMD18-T vector, transferring the PCR product into an escherichia coli competent cell, picking a monoclonal and sequencing.
5' RACE experiment of EjAGL17 gene of loquat
Specific primers 5REjAGL17R1 and 5REjAGL17R2 for 5'RACE experiments were designed based on the sequence of the 3' RACE experiments, wherein 5REAGL17R 1: 5'-CTTCTCATTCTCTTTACTTACGAGGT-3', 5REjAGL17R 2: 5'-TATTTCATCATTAAATATCTGGTTC-3' are provided. According to the 5' RACE experimental procedure: first Strand Synthesis Buffer Mix was prepared by adding 1.0. mu.L dNTP Mix (10mM), 2.0. mu.L 5 Xfirst-strand Buffer, and 1.0. mu.L DTT (20mM) in this order, mixing well, and standing at room temperature.
mu.L of total RNA, 1.0. mu.L of 5' -CDS primer A, and 1.75. mu.L of H were added to 200. mu.L of eppendorf tubes2O, mixing uniformly, after instantaneous centrifugation, cooling to 42 ℃ for 2min at 72 ℃ for 3min, after cooling, centrifuging for 10s at 14000g, adding 1 μ L of SMARTER IIA oligo, 1.0 μ L of SMARTscrube Reverse transcriptase (100U), 4.0 μ L of Buffer Mix, 0.25 μ L of RNase inhibitor (400U/. mu.L), the total volume is 10 μ L, mixing uniformly, after instantaneous centrifugation, reacting at 42 ℃ for 90min, and denaturing at 70 ℃ for 10min to obtain control 5' -E-Ready cDNA.
5' RACE amplification System: 34.5 μ L of PCR-Grade water, 5.0 μ L of 10 × Advantage 2PCRbuffer, 1.0 μ L of 50 × Advantage 2polymerase Mix, 1.0 μ L of dNTP Mix, 2.5 μ L of control 5' -RACE-Ready cDNA, 1.0 μ L of 5REjAGL17R1 primer, 5.0 μ L of UPM (10 ×). The procedure for touchdown PCR was: 30s at 95 ℃, 3min at 72 ℃ and 5 cycles; 30s at 95 ℃ and 30s at 70 ℃ for 5 cycles; 3min at 72 ℃, 30s at 95 ℃ and 30s at 68 ℃ for 30 cycles; 5min at 72 ℃. After the PCR reaction is finished, performing second-strand PCR reaction by using a PCR reaction product of first-strand 5' RACE as a template, high-fidelity LA-taq enzyme, an upstream outer side specific Primer 5REjAGL17R2 and a UPM Primer, wherein the reaction program is 95 ℃ for 5 min; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s and 72 ℃ for 30 s; 10min at 72 ℃. After the PCR reaction was completed, the second strand of the PCR reaction was detected by electrophoresis on a 1% agarose gel (FIG. 1B). The band of interest was excised and the PCR product was recovered using an agarose gel DNA recovery kit. After being connected to pMD18-T vector, the vector is transferred into Escherichia coli competent cells, and a single clone is picked up for sequencing analysis.
The PCR sequencing results of 3'RACE and 5' RACE experiments were spliced and analyzed by using DNAMAN software to obtain the sequence of the coding region of cDNA of loquat EjaGL17 gene (SEQ ID No.1), and the sequence picture thereof is shown in FIG. 2.
Primers FLEjAGL17F:5'-ATGGGGAGAGGAAAGATTGTGATTAGA-3' and FLEjAGL 17: 17R:5'-ATTGTTCGAAGGCCCAAGCCCATG-3' are designed in the full length of the loquat EjAGL17 gene sequence, and the reaction condition is 95 ℃ for 4 min; 30 cycles of 95 ℃ for 40s, 56 ℃ for 40s and 72 ℃ for 40 s; 10min at 72 ℃. After completion of the PCR reaction, the band of interest was cut out by 1% agarose gel electrophoresis (FIG. 1C), and the PCR product was recovered by using an agarose gel DNA recovery kit. After being connected to a pMD18-T vector, the vector is transferred into an escherichia coli competent cell, and a single clone is selected and sequenced to verify the sequence of the coding region of the EjAGL17 gene.
Using primer 5 software, the sequence of the coding region of cDNA of loquat EjaGL17 gene was translated into a protein sequence (SEQ ID No.2), and the loquat EjaGL17 protein sequence was subjected to division of MADS domain, I domain, K domain and C-terminal domain according to the characteristics of MADS-box protein (FIG. 3). Furthermore, comparing the amino acid sequence of the protein encoded by loquat Ejagl17 with the sequences of pear, coffee tree, Arabidopsis thaliana and Brassica napus, the sequence of the protein has obvious sequence difference compared with the sequences of the related species and other angiosperms, especially the amino acid sequence of the C structural domain has the largest difference, which indicates the specificity of the protein sequence (FIG. 4).
Example 2 subcellular localization analysis of loquat EjAGL17 Gene
The ORF sequence of EjAGL17 gene is subjected to enzyme cutting site analysis by using software Oligo7, enzyme cutting site primers at two ends are designed, EjAGL 17-SacI: 5'-cgagctcATGGGGAGAGGAAAGATTGTGAT-3', respectively; EjAGL 17-BamHI: 5'-cgggatccCTAAGTTTGAATCTGTGGCTGGC-3' are provided. And (3) amplifying by using a pMD18-EjAGL17 plasmid with correct sequencing as a template to obtain an EjAGL17 gene ORF sequence containing SacI and BamHI enzyme cutting sites. Respectively extracting target gene and modified vector pCAMBIA1300 plasmid, respectively carrying out double enzyme digestion reaction by using restriction enzymes SacI and BamHI, and recovering after agarose gel electrophoresis. By T4DNA ligase double-digested target gene EjAGL17 andthe modified pCAMBIA1300 vector is connected, the recombinant vector is transferred into an escherichia coli competent cell, and then bacterial liquid PCR and double enzyme digestion verification are carried out, and sequencing is carried out to ensure that a target gene sequence is successfully connected to the vector. The extracted and constructed vector plasmid is transferred into agrobacterium GV1301 competent cells by a freeze-thaw method.
A single colony of Agrobacterium was picked from the solid LB medium plate, inoculated into 10mL of liquid medium (containing Rif + kan), cultured at 28 ℃ and 250rpm until OD600 ═ 0.5. 5mL of culture solution is taken and centrifuged for 10min to collect thalli, then 2mL of penetrating fluid is added to resuspend the thalli, and then centrifugation is carried out for 10min to add 2mL of penetrating fluid to suspend the thalli. Finally, the tobacco leaves were diluted to an OD600 of 0.03 to 0.1, and the transformed tobacco leaves were transformed, and after culturing for 16 hours in a low light, normal growth was resumed, and after 3 to 4 days, GFP fluorescence was observed (fig. 5).
The results show that: the expression product of the loquat EjaGL17 gene is positioned in a nucleus, and the fact that the protein coded by the gene has typical transcription factor characteristics is shown.
Example 3 real-time fluorescent quantitative PCR expression analysis of EjAGL17 Gene of Eriobotrya japonica
The flowering process of loquat comprises: (A) the method comprises the following steps of (1) a physiological differentiation period of flower buds, (B) a morphological differentiation period of flower buds, (C) a major axis differentiation period of inflorescences, (D) a branch axis differentiation period of inflorescences, (E) a lateral branch axis rapid elongation period of inflorescences, (F) a floret differentiation period, (G) a flower bud blooming period, (H) a full-bloom period and (I) a late flowering period. Total RNA of loquat in the 9 developmental stages is respectively extracted, and after trace DNA in the total RNA is removed, cDNA is obtained through reverse transcription. Real-time fluorescent quantitative PCR primers qRTEjAGL17F:5'-TTGACGAGCAGGCAAGTGAC-3' and qRTEjAGL17R:5'-TAGCATAATCGTAAAGCCTG-3' are designed by utilizing oligo 6.0 software according to loquat cDNA as a template. Taking loquat actin gene as reference gene, the primer is qRTEjactinF: 5'-AATGGAACTGGAATGGTCAAGGC-3' and qRTEjactinR: 5'-TGCCAGATCTTCTCCATGTCATCCCA-3', the specificity is detected by PCR, and the real-time fluorescent quantitative PCR experiment can be carried out on the premise of ensuring the PCR specific amplification, and each reaction is provided with 3 biological repeats. The PCR reaction program is pre-denaturation at 94 ℃ for 5 min; 94 ℃ 20s, 55 ℃ 20s, 72 ℃ 20s, 41 cycles, then, a dissolution curve was taken: adjusting the temperature to 60 ℃ for 90s, and pre-dissolving; then the temperature is increased at the speed of 1.0 ℃/s, and the temperature is kept at 1 ℃ per liter for 5s until the temperature reaches 95 ℃.
The results show that: in the loquat flower development process, EjAGL17 gene is expressed in 9 different development stages of flower development, wherein the expression level is the highest in the morphological differentiation stage of flower buds, and the expression levels are obviously different in the different development stages (figure 6); the EjAGL17 gene is closely related to the loquat flower development process.
Example 4 construction of plant transgenic vector pBI121-EjAGL17 for loquat EjAGL17 Gene
And introducing enzyme cutting sites at two ends of a CDS region of the loquat EjaGL17 gene by adopting PCR amplification. Taking cDNA reverse transcribed by total RNA of loquat flower buds as a template, and taking TEjAGL 17-F: 5' -TCTAGAATGGGGAGAGGAAAGATTGTGATTAG-3' (introduction of Xba I cleavage site) and TEjAGL 17-R: 5' -CCCGGGCTAAGTTTGAATCTGT GGCTGGC-3' (incorporating SmaI cleavage sites) as primers, PCR amplification was carried out using Ex-taq enzyme. PCR reaction procedure: 4min at 95 ℃; 30 cycles of 95 ℃ for 40s, 56 ℃ for 40s and 72 ℃ for 40 s; 10min at 72 ℃. After the PCR reaction was completed, the PCR product was subjected to 1% agarose gel electrophoresis, and the PCR product was recovered using an agarose gel DNA recovery kit. And connecting the recovered PCR product with a pMD18-T vector, transferring into an escherichia coli competent cell, picking a monoclonal, and sequencing. And (5) extracting the plasmid according to the analysis of the sequencing result. The pMD18-EjAGL17 recombinant plasmid and pBI121 vector were double-digested with XbaI and SmaI restriction enzymes, respectively, detected by 1% agarose gel electrophoresis, and recovered using an agarose gel DNA recovery kit. Using T4The EjAGL17 gene after double enzyme digestion is connected with pBI121 by DNA ligase, and then transferred into escherichia coli competent cells to obtain a plant transgenic expression vector pBI121-EjAGL 17. The pBI121 (control) and the pBI121-EjAGL17 expression vector were digested with XbaI and SmaI, respectively, to obtain pBI121 with only 1 band (FIG. 7A), and pBI121-EjAGL17 with only 2 bands (FIG. 7B) of pBI121 vector and EjAGL17 gene, respectively, which confirmed successful ligation of the EjAGL17 gene to pBI121 vector.
Example 5 transfer of the transgenic expression vector pBI121-EjAGL17 into Arabidopsis thaliana
Adding 100 mu L of agrobacterium tumefaciens competent cells into 1 mu g of pBI121-EjAGL17 plasmid, and mixing uniformly; performing ice bath for 10min, transferring into liquid nitrogen, rapidly freezing for 2min, rapidly placing at 37 deg.C, and performing water bath for 10 min; adding 800 μ L LB liquid culture medium, oscillating at 28 deg.C and 250rpm for 5 h; the bacterial liquid is transferred to LB (50mL LB + 50. mu.g/mL Kan + 50. mu.g/mL Rif) solid selection medium, evenly coated and inversely cultured for 48h at the temperature of 28 ℃.
Agrobacterium containing pBI121-EjAGL17 positive clones were streaked on 25mL solid plate medium (containing 25. mu.g/mLKan + 25. mu.g/mL Rif), and cultured in an inverted state at 28 ℃ for 48 h; selecting a single clone, and inoculating the single clone into 10mL of liquid LB culture medium (containing 10 mu g/mL Kan +10 mu g/mL Rif); the cells were cultured overnight at 28 ℃ and 250rpm with shaking until OD was 0.7-0.8. Uniformly coating 1mL of culture solution on a 25mL solid LB medium plate (containing 25 mu g/mL Kan +25 mu g/mLRif), and carrying out inverted culture at 28 ℃ for 48 h; agrobacterium on solid medium was scraped off using a sterilized glass triangle rod, and the pellet was resuspended in 1/2MS liquid medium containing 5% sucrose and 3% Silwet L-77 to an OD of 0.2 for arabidopsis transgenesis.
Placing Arabidopsis seeds on wet filter paper, placing the filter paper at 4 ℃ for 48h, then sowing the seeds into nutrient soil (perlite: vermiculite: nutrient soil: 1:4:5), and culturing the seeds under the conditions of temperature of 22 ℃, humidity of 70% and 14h light/10 h dark; before transgenosis, arabidopsis thaliana (purchased from arabidopsis thaliana mutant library) plants are watered thoroughly; cutting off existing siliques on an arabidopsis plant to be used during dip dyeing, and soaking flower buds into PBI121-EjAGL17 agrobacterium tumefaciens dip dyeing solution for about 90 s; covering a black sealing film, maintaining a high-temperature and high-humidity environment in the film, and uncovering the film after dark culture for 2 d; the method is used for infecting 4 times with the interval time of 7 d.
Example 6 transgenic Arabidopsis thaliana screening and phenotypic characterization of the loquat EjAGL17 Gene
And (4) collecting EjAGL17 transgenic arabidopsis mature seeds, and cleaning the seeds. Performing vernalization treatment in a refrigerator at 4 deg.C for 14 d; placing Arabidopsis seeds into a collecting pipe, adding 800 μ L of absolute ethyl alcohol into the seeds, and shaking for 6 min; centrifuging at 5000rpm for 2 min; pouring off alcohol in the collecting pipe, adding 800 μ L70% ethanol into the collecting pipe, and shaking for 5 min; centrifuging at 5000rpm for 2 min; airing the seeds; the suspension was spread evenly on 1/2MS medium (pH 5.8, 50. mu.g/mL Kan, 3% sucrose and 0.8% agar) plates. Putting the inoculated flat plate into a refrigerator at 4 ℃ for vernalization for 2 d; and (4) placing the vernalized seeds in an artificial climate box for normal culture. After 6 true leaves grow, the leaves are moved into nutrient soil, and after hardening and strengthening seedlings, the seedlings are managed according to conventional water and fertilizer until the flowers bloom.
Extracting EjAGL17 transgenic Arabidopsis DNA, placing 1 piece of Arabidopsis leaf in 1.5mL eppendorf tube, placing in liquid nitrogen for quick freezing, and grinding; adding 600 μ L of extraction buffer solution, vortex shaking, and placing on ice; after all samples are treated, placing the samples in a water bath at 65 ℃ for 25 min; taking out the sample from the water bath, placing the sample to room temperature, adding 340 mu L of potassium acetate solution after cooling to the room temperature, carrying out vortex oscillation and carrying out ice bath for 20 min; 13000rpm, high speed centrifugation for 5min, transfer the supernatant to a new eppendorf tube; adding equal volume of isopropanol, centrifuging at 4 deg.C and 13000rpm for 10min, removing clear liquid, and rinsing with ice anhydrous ethanol (anhydrous ethanol is placed in a refrigerator at-20 deg.C 2h in advance); rinsing the precipitate with 70% and 100% ethanol in sequence; after the precipitate was blown dry, it was dissolved in 50. mu.L of sterile water.
The DNA of the non-transgenic wild type Arabidopsis thaliana is used as a control, primers TEjAGL17-F and TEjAGL17-R constructed by a carrier are used for carrying out PCR screening on the DNA of a positive plant of the transgenic Arabidopsis thaliana. PCR amplification procedure: 4min at 95 ℃; 30 cycles of 95 ℃ for 40s, 56 ℃ for 40s and 72 ℃ for 40 s; 10min at 72 ℃. 31 positive EjAGL17 transgenic wild type Arabidopsis plants were obtained altogether.
And (3) carrying out phenotype identification on the flowering time of the transgenic wild arabidopsis thaliana, and carrying out observation statistics and photographing on the flowering time phenotype of the arabidopsis thaliana. The phenotypic results show that: compared with wild arabidopsis thaliana, the transgenic wild arabidopsis thaliana over-expressing EjAGL17 gene can promote the flowering time of arabidopsis thaliana to be about 14d earlier (figure 8). The result shows that the EjAGL17 gene has the function of promoting the flowering time of arabidopsis thaliana in advance, and the transgenic arabidopsis thaliana material of the EjAGL17 gene can be used for modifying the flowering time of plants, so that the plants can bear fruits in advance, and the fruit ripening time of the plants can be effectively advanced.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> university of southwest
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agcatgaaat cagttaccga tcgtttcaac aaactgaaag cggagcatca actgccgaat 240
ccggcttcag aagtcaagtt ttggcaaagg gaaacagcaa gcttgaggca acaattgtgg 300
tacttggaag aatgtcacag gcagttaatg ggggaagaac tttctggttt gagtgccaac 360
gatctacaga atttggaaag ccagcttgag atgagtttga aaggtgtccg aacgaaaaag 420
aaccagatat ttaatgatga aataaaagaa ctgaaccaga agggatcttt catacatcaa 480
gagaatatag aacttcataa gaagctggac ctcgtaagta aagagaatga gaagctgaaa 540
aagaaggctt ataaagcaag ggatgtgaat caaggaaaca aaagttccca gcctccatat 600
agcatcagca atgaagatga tttgcatgca cccatccatt tgcagctaag ccagccacag 660
attcaaactt agcatgggct tgggccttcg aacaatgaag taccagcaaa agttatatat 720
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Gln Gln Leu Trp Tyr Leu Glu Glu Cys His Arg Gln Leu Met Gly Glu
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Leu Glu Met Ser Leu Lys Gly Val Arg Thr Lys Lys Asn Gln Ile Phe
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Asn Asp Glu Ile Lys Glu Leu Asn Gln Lys Gly Ser Phe Ile His Gln
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Claims (10)
1. Loquat EjAGL17 protein, which is:
1) a protein consisting of the amino acids shown in SEQ ID No. 2; or
2) Protein which is derived from the protein 1) and has the same activity by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No.2, wherein the protein derived from the protein 1) comprises a MADS structural domain, an I structural domain, a K structural domain and a C terminal structural domain.
2. A gene encoding the loquat EjaGL17 protein of claim 1.
3. The gene of claim 2, having the sequence shown in SEQ ID No. 1.
4. A vector containing the gene according to claim 2 or 3.
5. A host cell comprising the vector of claim 4.
6. An engineered bacterium comprising the gene of claim 2 or 3.
7. Use of the gene of claim 2 or 3 for regulating flowering-time of angiosperms.
8. Use according to claim 7, wherein the EjAGL17 gene is transferred into the genome of an angiosperm plant and overexpressed in the transgenic plant to promote early flowering and fruiting in the plant.
9. A construction method of transgenic plant, adopting Agrobacterium mediated method, transferring the over-expression vector containing the gene of claim 2 or 3 into plant genome, and screening to obtain transgenic plant.
10. The method of claim 9, wherein the transgenic plant has an earlier timing of differentiation and development of inflorescences and floral organs than the wild type.
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| CN111440233A (en) * | 2020-05-25 | 2020-07-24 | 浙江省农业科学院 | Transcription factor EjCA L participating in loquat flower bud differentiation regulation and control and application thereof |
| CN112626081A (en) * | 2020-12-23 | 2021-04-09 | 重庆文理学院 | Longan flowering regulation gene DlWRKY25, and regulation protein and application thereof |
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| CN114671932A (en) * | 2022-04-24 | 2022-06-28 | 西南大学 | EjAGL6 gene for advancing loquat flowering time and encoding protein and application thereof |
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| CN115058435A (en) * | 2022-06-24 | 2022-09-16 | 中国林业科学研究院华北林业实验中心 | Kernel apricot Pasdehydrin-3 gene and application thereof in cold resistance, flowering promotion or seed setting |
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