GRF-GIF fusion gene and application thereof in promoting leaf regeneration and fruit expansion of pear

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a GRF-GIF gene in promoting pear leaf regeneration and fruit expansion. According to the invention, GRF and GIF genes are cloned from leaves of pyrus ussuriensis, and the two genes are connected together through overlap PCR (polymerase chain reaction). The GRF-GIF gene is stably overexpressed in the pyrus ussuriensis by a leaf disc method, and the result shows that the overexpressed GRF-GIF gene can promote the regeneration of the callus and bud of the leaf of the pyrus ussuriensis; the GRF-GIF gene is stably overexpressed in pear fruit calluses, and the result shows that the fusion gene can effectively promote expansion of the calluses.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the GRF-GIF fusion gene and its application in promoting pear leaf regeneration and fruit enlargement. The invention cloned the GRF and GIF genes from leaves of 'Autumn Pear Seedling Progeny' and ligated the two genes together using overlap PCR. The GRF-GIF gene was stably overexpressed in Autumn Pear Seedling Progeny using the leaf disc method. Results showed that overexpression of the GRF-GIF gene promoted the regeneration of pear leaf callus and buds. Stable overexpression of the GRF-GIF gene in pear fruit callus tissue showed that the gene effectively promoted callus enlargement. Background Art

[0002] Genetic transformation is a method of introducing foreign genes into recipient cells, thereby endowing the recipient cells with new genetic characteristics. Genetic transformation has a wide range of applications, including genetic engineering, drug development, and agricultural production. In agricultural production, genetic transformation can be used to improve crop varieties, enhance crop resistance to pests and diseases and increase yield, and breed new varieties.

[0003] The earliest reports of pear gene transformation were made by foreign researchers who used Agrobacterium-mediated transformation to obtain transgenic plants. The establishment of transgenic systems is crucial and fundamental for the generation of new germplasm through gene modification. Although the number of transgenic plants among pear varieties is gradually increasing, most are European pear varieties. Establishing a stable transformation system depends on many factors, and the required factors differ between different plants or even between different varieties of the same plant.

[0004] GRF (Growth Regulatory Factor) is a family of transcription factors unique to plants. It can form functional complexes with the transcriptional coactivator GIF (Growth Regulatory Factor), playing a crucial regulatory role in plant growth and development. In recent years, research on its role in promoting regeneration and transformation has deepened. This invention clones and constructs the GRF-GIF complex and applies it to pear genetic transformation research, providing new insights for pear genetic transformation studies and also benefiting gene function research and agricultural breeding. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the GRF-GIF gene or biomaterials related to the GRF-GIF gene in promoting pear leaf regeneration and fruit enlargement.

[0006] The applicant screened GRF and GIF genes from 'pear seedling progeny'. GRF, a plant-specific transcription factor family, is involved in regulating various biological processes such as plant growth, development, and responses to environmental stress. GIF, a transcriptional coactivator, can form a functional complex with GRF, playing a crucial role in plant growth and development. Applying the GRF-GIF fusion gene to pear leaf genetic transformation yielded transgenic lines with enhanced leaf regeneration capacity. Furthermore, overexpression of this fusion gene promoted callus enlargement.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention seeks protection for the use of the GRF-GIF fusion gene or biological materials associated with the GRF-GIF fusion gene in at least one of the following:

[0009] (a1) Application in promoting pear leaf regeneration;

[0010] (a2) Application in the preparation of products for promoting the regeneration of pear leaves;

[0011] (a3) Application in promoting pear fruit enlargement;

[0012] (a4) Application in the preparation of products for promoting pear fruit enlargement;

[0013] (a5) Application in improving pear conversion efficiency;

[0014] (a6) Application in the preparation of products for improving pear conversion efficiency;

[0015] The GRF-GIF fusion gene is a fusion gene constructed by linking the growth regulator GRF (nucleotide sequence as shown in SEQ ID NO.1) and the transcription coactivator GIF (nucleotide sequence as shown in SEQ ID NO.2).

[0016] Furthermore, in the above applications, the growth regulator GRF and the transcriptional coactivator GIF are linked using GCGGCCGCTGCC as the linker. Even further, the nucleotide sequence of the GRF-GIF fusion gene is shown in SEQ ID NO. 3.

[0017] Furthermore, the biological material associated with the GRF-GIF fusion gene is at least one of the following (b1)-(b7):

[0018] (b1) The protein encoded by the GRF-GIF fusion gene;

[0019] (b2) An expression cassette containing the GRF-GIF fusion gene;

[0020] (b3) A recombinant vector containing the GRF-GIF fusion gene, or a recombinant vector containing the expression cassette described in (b2);

[0021] (b4) A recombinant microorganism containing the GRF-GIF fusion gene, or a recombinant microorganism containing the expression cassette of (b2), or a recombinant microorganism containing the recombinant vector of (b3);

[0022] (b5) A transgenic plant cell line containing the GRF-GIF fusion gene, or a transgenic plant cell line containing the expression cassette described in (b2), or a transgenic plant cell line containing the recombinant vector described in (b3);

[0023] (b6) Transgenic plant tissue containing the GRF-GIF fusion gene, or transgenic plant tissue containing the expression cassette of (b2), or transgenic plant tissue containing the recombinant vector of (b3);

[0024] (b7) A transgenic plant organ containing the GRF-GIF fusion gene, or a transgenic plant organ containing the expression cassette of (b2), or a transgenic plant organ containing the recombinant vector of (b3);

[0025] (b8) A transgenic plant containing the GRF-GIF fusion gene, or a transgenic plant containing the expression cassette of (b2), or a transgenic plant containing the recombinant vector of (b3);

[0026] (b9) Tissue cultures produced from regenerative cells of the transgenic plant described in (b8);

[0027] (b10) Protoplasts produced from the tissue culture described in (b9);

[0028] (b11) Transformation reagent containing the GRF-GIF fusion gene.

[0029] Furthermore, the protein encoded by the GRF-GIF fusion gene is as follows (c1) or (c2) or (c3):

[0030] (c1) A protein with the amino acid sequence shown in SEQ ID NO. 6;

[0031] (c2) The amino acid residue sequence shown in SEQ ID NO.6 is modified by substitution and / or deletion and / or addition of one or more amino acid residues and is associated with a protein derived from SEQ ID NO.6 that promotes pear leaf regeneration and fruit enlargement;

[0032] (c3) A fusion protein that links a protein tag to the N-terminus and / or C-terminus of (c1) or (c2).

[0033] Furthermore, the above applications include: stably overexpressing the GRF-GIF fusion gene in pear leaves to improve the regeneration capacity of pear leaves, or overexpressing it in pear fruit callus tissue to promote the enlargement of fruit callus tissue.

[0034] Secondly, the present invention claims protection for a method to improve the regeneration ability of pear leaves, wherein the above-mentioned GRF-GIF fusion gene is overexpressed in pear plants, which can improve the regeneration ability of pear leaves.

[0035] Thirdly, the present invention claims protection for a method for promoting pear fruit enlargement, wherein the above-mentioned GRF-GIF fusion gene is overexpressed in pear plants and / or fruits, which can promote pear fruit enlargement.

[0036] Fourthly, the present invention claims protection for a GRF-GIF fusion gene, which is a fusion gene constructed by linking the growth regulator GRF with the nucleotide sequence shown in SEQ ID NO.1 and the transcription coactivator GIF with the nucleotide sequence shown in SEQ ID NO.2.

[0037] Fifthly, the present invention seeks protection for biological materials related to the above-mentioned GRF-GIF fusion gene, wherein the biological material is at least one of the following (b1)-(b7):

[0038] (b1) The protein encoded by the GRF-GIF fusion gene;

[0039] (b2) An expression cassette containing the GRF-GIF fusion gene;

[0040] (b3) A recombinant vector containing the GRF-GIF fusion gene, or a recombinant vector containing the expression cassette described in (b2);

[0041] (b4) A recombinant microorganism containing the GRF-GIF fusion gene, or a recombinant microorganism containing the expression cassette of (b2), or a recombinant microorganism containing the recombinant vector of (b3);

[0042] The protein encoded by the GRF-GIF fusion gene is as described above.

[0043] Sixthly, the present invention claims protection for a method for constructing the above-mentioned GRF-GIF fusion gene, the method comprising the following steps:

[0044] (1) Using cDNA from leaves of 'Pyrus pyrifolia seedlings' as templates, the GRF and GIF genes were amplified by PCR using GRF-F / R and GIF-F / R primers, respectively; the primer sequences are as follows:

[0045] GRF-F:TCCAAAGAATTCAAAAAGCTTATGAGCGGGTCGTCGGT(SEQ ID NO.7)

[0046] GRF-R:ATCTGCATCAGGTGCTGCTGCATGGCAGCGGCCGCAGTATAGGCATCAC TGGGTGACT(SEQID NO.8)

[0047] GIF-F:AGTCACCCAGTGATGCCTATACTGCGGCCGCTGCCATGCAGCAGCACCTG ATGCAGAT(SEQID NO.9)

[0048] GIF-R:TCTAGAGTCCTGCTTTAATGAATTCCCATCATCGGTCGATTT(SEQ ID NO.10);

[0049] (2) Using the amplified GRF and GIF genes as templates, the GRF-GIF fusion gene containing the GCGGCCGCTGCC linker was amplified using GRF-F and GIF-R primers;

[0050] (3) The linearized PSAK277 vector was double-digested and ligated with the GRF-GIF fusion gene fragment using Exnase II;

[0051] (4) The ligation product was transformed into DH5α, positive clones were selected for sequencing verification, and the GRF-GIF recombinant plasmid of the correctly sequenced clone was extracted using a plasmid extraction kit and transformed into Agrobacterium LBA4404 competent cells for culture.

[0052] (5) After verifying positive clones by colony PCR, pick the colonies, shake them, add glycerol, and store them at -80℃.

[0053] The applicant screened a fusion protein, GRF-GIF, to improve regeneration rate from 'pear seedlings' and cloned the GRF and GIF genes from 'pear seedlings' using gene cloning technology. The nucleotide sequence of the GRF gene is shown in SEQ ID NO.1, containing a 987 bp open reading window encoding 328 amino acids, and its encoded amino acid sequence is shown in SEQ ID NO.4. The nucleotide sequence of the GIF gene is shown in SEQ ID NO.2, containing a 651 bp open reading window encoding 216 amino acids, and its encoded amino acid sequence is shown in SEQ ID NO.5. This invention uses GCGGCCGCTGCC as a linker to connect the GRF and GIF genes to obtain the GRF-GIF fusion gene, the nucleotide sequence of which is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.6.

[0054] This invention constructs a GRF-GIF fusion gene and uses Agrobacterium tumefaciens-mediated stable genetic transformation to verify that GRF-GIF has the function of promoting callus enlargement and improving leaf regeneration rate.

[0055] Compared with the prior art, the present invention has advantages and effects:

[0056] (1) By referring to the transgenic system of Qiuzi pear, the formulation of the infection solution was optimized, and the genetic transformation of Qiuzi pear seedlings was realized, which effectively improved the leaf regeneration ability.

[0057] (2) The discovery of the GRF-GIF fusion gene provides a new approach to the realization of pear genetic transformation, which is conducive to gene function research and variety breeding. Attached Figure Description

[0058] Figure 1 To identify the DNA level of the transgenic line.

[0059] In this context, A represents tissue culture seedlings, and B represents callus tissue.

[0060] Figure 2 To identify the RNA level of the transgenic line.

[0061] In this context, A represents tissue culture seedlings, and B represents callus tissue.

[0062] Figure 3 This is a comparison of the regeneration capacity of transgenic leaves and wild-type leaves.

[0063] Figure 4 This represents the callus phenotype.

[0064] Figure 5 The genetic transformation process of leaves from seedlings of *Pyrus pyrifolia*. Detailed Implementation Plan

[0065] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and these embodiments, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the invention.

[0066] Example 1: Cloning of the GRF-GIF gene and construction of its overexpression vector

[0067] In this invention, the plasmid vector is the PSAK277 vector (a commercially available vector from New Zealand, with spectinomycin as the bacterial resistance marker and kanamycin as the plant resistance marker). Double digestion was performed using EcoRI and HindIII (purchased from NEB). The optimal digestion reaction system and conditions were: 1000 ng PSAK277 empty plasmid, 10 μL 10×Cutsmart Buffer, 2 μL EcoRI restriction enzyme, 2 μL HindIII restriction enzyme, and ddH2O to bring the volume to 100 μL. The reaction was carried out at 37°C for 2 h. The GRF and GIF genes were cloned using conventional methods. To fuse the two genes together, GCGGCCGCTGCC was used as the linker, and the specific primer design is as follows.

[0068] The primer pairs for PCR amplification of the gene are:

[0069] GRF-F:TCCAAAGAATTCAAAAAGCTTATGAGCGGGTCGTCGGT(SEQ ID NO.7)

[0070] GRF-R:ATCTGCATCAGGTGCTGCTGCATGGCAGCGGCCGCAGTATAGGCATCAC TGGGTGACT(SEQID NO.8)

[0071] GIF-F:AGTCACCCAGTGATGCCTATACTGCGGCCGCTGCCATGCAGCAGCACCTG ATGCAGAT(SEQID NO.9)

[0072] GIF-R:TCTAGAGTCCTGCTTTAATGAATTCCCATCATCGGTCGATTT(SEQ ID NO.10)

[0073] PCR amplification system: 2 μL of leaf cDNA from 'Qiuzi pear seedlings' (Qiuzi pear seedlings obtained from Qingdao Agricultural University), 2 μL each of forward and reverse primers, 25 μL of 2×... Max Master Mix (Dye) (purchased from Nanjing Jujiang Biotechnology Co., Ltd.), add up to 50 μL with ddH2O.

[0074] The amplification program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, 35 thermal cycles, 72℃ extension for 10 min, and storage at 4℃.

[0075] After amplification, the PCR product with a single target band was detected by 1.5% agarose gel electrophoresis, and the specific band was recovered according to the instructions of the gel recovery kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.).

[0076] GRF was cloned using the GRF-F and GRF-R primer pairs according to the cloning system described above. GIF was cloned using the GIF-F and GIF-R primer pairs. Then, using the cloned GRF and GIF as templates, the amplification system consisted of: 1 μL each of template GRF and GIF, 2 μL each of GRF-F and GIF-R primers, and 25 μL of 2× [amount missing]. Max Master Mix (Dye) was added to a final volume of 50 μL with ddH2O. The amplification program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 10 s, 55°C annealing for 15 s, 72°C extension for 50 s, 35 thermal cycles, 72°C extension for 10 min, and storage at 4°C. After amplification, the PCR product was detected as a single target band by 1.5% agarose gel electrophoresis, and the specific band was recovered according to the gel extraction kit instructions.

[0077] The double-digested vector was ligated with purified DNA. Exnase II ligase was purchased from Nanjing Novizan Biotechnology Co., Ltd. The reaction mixture consisted of 20 μL: 150 ng PSAK277 linear vector, 50 ng gene fragment, 4 μL 5×CE II Buffer, 2 μL Exnase II, and the remainder was added with ddH2O. The mixture was incubated at 37℃ for 30 min. The ligation product was then transferred to *E. coli* DH5α (purchased from Nanjing Novizan Biotechnology Co., Ltd.), incubated on ice for 30 min, heat-shocked for 45 s, and incubated on ice for 2 min. 600 μL of LB liquid medium was added, and the mixture was activated at 37℃ and 220 rpm for 50 min. After centrifugation at 6000 rpm for 5 min, approximately 100 μL of supernatant was retained. The cells were gently resuspended by pipetting and evenly spread onto LB agar plates containing 100 μg / ml spectinomycin. After complete absorption of the liquid, the plates were inverted and incubated at 37℃ for 16 hours. Five positive clones were then picked and sequenced (performed by Shanghai Sangon Biotech Co., Ltd.). The recombinant plasmid that had been successfully sequenced was extracted using a plasmid extraction kit (purchased from Shanghai Sangon Biotech Co., Ltd.). The expression vector plasmid was transformed into Agrobacterium LBA4404. After the Agrobacterium was thawed, 1 μg of plasmid DNA was added to every 100 μL of competent cells and gently mixed. The cells were incubated in an ice-water bath for 5 minutes, then flash-frozen in liquid nitrogen for 5 minutes, then incubated in a 37°C water bath for 5 minutes, and finally incubated in an ice-water bath for 5 minutes. 800 μl of LB liquid medium was added, and the cells were incubated at 28-30°C with shaking for 2-3 hours. The cells were collected by centrifugation at 5000 rpm for 5 minutes, and about 100 μL of supernatant was retained. The cells were gently resuspended by pipetting and evenly spread onto LB solid medium plates containing 100 μg / ml spectinomycin + 100 μg / ml rifampin. After the liquid in the plates was completely absorbed, the plates were inverted and incubated at 28-30°C for 48-72 hours. Colony PCR was performed to verify the growth of colonies. Positive colonies were labeled, and a small number were picked, shaken, and stored at -80°C with glycerol for later use.

[0078] Example 2: Genetic transformation of leaves from seedlings of *Pyrus pyrifolia*

[0079] The tissue culture seedlings of 'Qiuzi pear seedlings' were preserved at the Pear Center of Nanjing Agricultural University. The growing conditions were 25℃, 16h light / 8h darkness, and subcultured approximately every 30 days.

[0080] Subculture medium formula (1L): MS powder + 30g sucrose + 1.0mg / L 6-BA + 0.2mg / L LIBA + 7.5g agar powder, pH 5.8.

[0081] Conversion process as follows Figure 5 As shown.

[0082] Detailed operation process:

[0083] (1) Take tender leaves in a clean bench, make three even cuts perpendicular to the main vein, and then place the leaves face up on NN69 regeneration medium and pre-culture for 5 days in the dark at 25°C.

[0084] (2) Agrobacterium was activated by streaking on LB solid medium (containing 100 mg / L Kan + 50 mg / L LRif) plates and then incubated in the dark at 28°C for 48 hours.

[0085] (2) Pick colonies in LB broth (containing 100 mg / L kanamycin and 50 mg / L rifampin), and incubate at 28°C and 200 r / min with shaking until OD600 reaches 0.6-0.8.

[0086] (4) Centrifuge at 6000 rpm to recover the bacterial cells, discard the supernatant, resuspend the precipitate in MS liquid medium, adjust the OD600 of the resuspended solution to 0.4-0.6, and activate in a shaker at 28℃ for 1 h.

[0087] (5) Place the pre-cultured leaves in the resuspension solution and incubate for 15-20 minutes. Remove the leaves and place them on sterile, dry filter paper to absorb excess liquid. Then, place them face up on NN69 regeneration medium and incubate in the dark at 25°C for 2 days. After two days, place the leaves on selection medium and continue incubating in the dark at 25°C for 20-30 days. Once new shoots have regenerated, place them under 16h light / 8h darkness conditions for growth.

[0088] Culture medium formulations:

[0089] NN69 regeneration medium formula (1L): NN69 + 30g sucrose + 3.0mg / L TDZ + 0.2mg / L IAA + 7.5g agar powder, pH 5.8.

[0090] Resuspension formulation (1L): NN69 + 20g sucrose, pH 6.0

[0091] Screening medium formula (1L): NN69 + 30g sucrose + 3.0mg / L TDZ + 0.2mg / L IAA + 7.5g agar powder + 15mg / L kanamycin + 200mg / L cephalosporin, pH 5.8.

[0092] Example 3: Identification of overexpression positive lines

[0093] After the tissue culture seedlings grew, leaf DNA was extracted for DNA level identification experiments of transgenic plants (purchased from Nanjing Novizan Biotechnology Co., Ltd., and the operation was performed according to the instructions provided with the kit). Using the extracted genomic DNA from the screened tissue culture seedlings as a template, PCR identification was performed using psak-277-F (CATCGAAAGGACAGTAGAAAAGG, SEQ ID NO.11) and GRF-R as primers. Figure 1 Electrophoresis showed that the tissue culture seedlings could amplify the target band of the same size as the positive control, which confirmed that they were transgenic positive.

[0094] RNA was extracted from leaves of tissue culture seedlings and reverse transcribed to obtain first-strand cDNA for qRT-PCR experiments. RNA extraction was performed using a plant total RNA extraction kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., and the procedure was followed according to the kit's instructions). Reverse transcription was performed using TransScript One-Step RT-PCR SuperMix (purchased from Beijing TransGen Biotech Co., Ltd., and the procedure was followed according to the kit's instructions). Specific quantitative primers for the GRF gene were designed.

[0095] GRF-qPCR-F:5'-GGCCGCAATCGTTCAAGAAA-3'(SEQ ID NO.12)

[0096] GRF-qPCR-R:5'-CTGGCTCCCCTGTTAGACAC-3'(SEQ ID NO.13)

[0097] Using the pear UBQ gene as an internal reference, the specific quantitative primers are as follows:

[0098] PbUBQ-F:5'-CCCTTCACTTGGTTCTCCGT-3'(SEQ ID NO.14)

[0099] PbUBQ-R:5'-TAATCAGCAAGCGTGCGACC-3'(SEQ ID NO.15)

[0100] qRT-PCR experiments were performed using the LC480 SYBR Green Mix kit (Roche), following the kit instructions. A 20 μL qRT-PCR reaction mixture consisted of: 10 μL 2×SYBR Green Mix, 0.4 μM forward and reverse primers, 20 ng cDNA, and the remainder was made up with sterile water. A 96-well qRT-PCR plate was used, and PCR was performed using a qRT-PCR instrument (LightCycler 480, Roche). The qRT-PCR program was: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 10 s; 60℃ annealing for 30 s; 72℃ extension for 20 s; 45 cycles. Three biological replicates and three technical replicates were set up for each cDNA sample. The average Ct value of each cDNA sample was calculated, and the results were analyzed by 2... -ΔΔCt The relative expression levels of genes were determined.

[0101] like Figure 2 As shown in Figure A, the expression level of GRF in the overexpression line was significantly higher than that in the wild type. Statistical analysis of leaf regeneration rate revealed that the regeneration rate of transgenic leaves was 19.8% higher than that of the wild type (e.g., ...). Figure 3 (As shown).

[0102] Example 4: Application of GRF-GIF gene in promoting callus enlargement in pear fruit

[0103] Pear fruit callus was preserved from the Pear Center of Nanjing Agricultural University. The growth conditions were dark culture at 25℃, with subculture every 20 days. The culture medium formula (1L) was: MS powder + 30g sucrose + 1.5mg / L 2,4-D + 0.5mg / L 6-BA + 7.5g agar powder, pH 5.8.

[0104] The specific process of stable transformation of pear callus tissue:

[0105] (1) Agrobacterium was activated by streaking on LB solid medium (containing 50 mg / L Kan + 50 mg / L Rif) plates and then incubated in the dark at 28°C for 48 hours.

[0106] (2) Pick colonies in LB broth (containing 50 mg / L kanamycin and 50 mg / L rifampin), and culture at 28°C and 200 r / min with shaking until the bacterial solution turns orange-yellow.

[0107] (3) Collect bacterial cells by centrifugation at 6000 rpm, then resuspend in infection medium (MS liquid medium containing 100 uM / LAS), and adjust the OD of the resuspended solution. 600 Up to 0.4-0.6.

[0108] (4) After inducing the resuspension at 28℃ and low speed (120 rpm) for 2 hours, add the pear callus tissue and continue shaking for 20 minutes. Then place the pear callus tissue on sterile and dry filter paper to absorb excess liquid.

[0109] (5) Incubate the callus tissue on MS co-existing medium (MS subculture medium + 100 uMAS) at 25°C in the dark for 2 days. After two days, spread the callus tissue evenly on MS selection medium (MS subculture medium + 150 mg / L kanamycin + 200 mg / L cephalosporin) and wait for the callus tissue to grow.

[0110] Example 5: Identification of positive callus overexpression lines

[0111] The identification method is the same as in Example 3, such as... Figure 2 As shown in Figure B, the expression level of the GRF gene in the overexpression lines was significantly higher than that in the wild-type lines. Phenotypic experiments were conducted on wild-type and transgenic lines, as shown in Figure B. Figure 4 As shown, the results indicate that the callus of the overexpression line was significantly larger than that of the wild type, suggesting that the fusion gene can promote the expansion of callus in pear fruit.

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0113] sequence list

[0114] SEQ ID NO.1

[0115] ATGAGCGGGTCGTCGGTGGGCACTGGAGGTCACGCGCCGCTTCACAGTGGCGCAA

[0116] TGGGCTGAGATGGAGCATCAAGCTTTGATATTCAAGTATCTCAAGGCAGGAGTCCCTG

[0117] TTCCTTCTGAGCTCCTCGCCCCTATTCGCACCAGTTTTCAACCTCATTTCCCCCAATTTC

[0118] TTACCTCACCACCCCACCTGGGGTTATGCTTCCTATTGCGGGAAGAAGATAGACCCAG

[0119] AGCCGGGGCGGTGCCGGAGGACGGATGGCAAGAAATGGAGGTGCTCCAAAGATGCA

[0120] TATCAGGACTCCAAGTATTGCGAGCGGCACATGAACCGCGGCCGCAATCGTTCAAGA

[0121] AAGCTTGTGGAATCACAAACTGCCTCTCAATCTTTGTCAACTGTGACGTCAGACAGT

[0122] GTGATCGGGAGCTCCAGCCACGGTGGGAGCTTCCAGACCATGCCCTTACACTCAATT

[0123] GGAAATAGTGGAGGCCTATGCTTTGGAGGCAATGGATCTGAGTTGAAGATGGAAGCT

[0124] ATACCCTATGGGGTGTCTAACAGGGGAGCCAGGGGGTGCAATAGATCTGGATCAGAA

[0125] GAACACAATTTCATGTCAGAAGCTAAGAGGTGTGTTCAGGCGCACAGTACTGTAGAC

[0126] AGTCCGTGGCGTCTAATGGAGCCTCAAGTTCCCTTAAAATCAGAATTGAGAAATAGTT

[0127] CTCTTTGGCTAAACAACAGCTCTCAGCTACAGACATTACAAACTATGGAGTCATTGAG

[0128] CGGTGCTGCTGCAATCACCAAACAGGAGTATGTTGGTGGAGAATTTGGCTTACCAGG

[0129] ATCTCAAAAACACGAACAAAATTCTCTTCAGCCTTTATTTGATGAGTGGCCTAAGTCG

[0130] AGGGATCTGGGGTTCCATCTGAATGATCATAGATCAAACACGACTGAGCTGTCAATGT

[0131] CTAATGCAGGGGCCACCCCTAAATATTGCGCAAGGAGTGCTGAGTCACCCAGTGATG

[0132] CCTATACTTGA

[0133] SEQ ID NO.2

[0134] ATGCAGCAGCACCTGATGCAGATGCAGCCTATGATGGCAGCCTATTATCCCAACAACG

[0135] TCACTACTGATCACATTCAGCAGTACTTGGACGAGAACAAGTCGTTGATTCTGAAGAT

[0136] TGTTGAGAGCCAGAATACCGGGAAACTGAGTGAATGTGCAGAGAACCAAGCAAAGC

[0137] TACAGCGGAATCTGATGTACCTGGCTGCCATTGCTGATTCACAACCCCAGCCTCCTAC

[0138] CATGCATCCTCAGTACTCTTCCACCGGCATGATGCAGCCAGGAGCACATTACATGCAG

[0139] CAACAAGCAGCTCAGCAGATGACACCACAATCGCTAATGGCTGCACGCTCTTCCATG

[0140] ATGTACTCCCAGCATCCGTTTTCAGCTCTGCAGCAACAAGCCCTGCATAGCCAACTTG

[0141] CTATGACTGCTGGAGGAAGCGCGGGACTGCACATGCTCGGGAATGAGGGAAATAATG

[0142] CTGGAGGCAGCGGGCAACTTGGGGCTGGAGGGTTTGGTGATTACGGACGCAGCTCTC

[0143] CTGGAGAAGGCATGCATAGGAGGATGGCCGGTGGGAGTAAGCAAGATATGGATGGGC

[0144] GAGGCGGGAGCTCTGGAAGCCATGGTGGAGATGGGGGTGAGACTCTTTACCTGAAAT

[0145] CGACCGATGATGGGAATTAA

[0146] SEQ ID NO.3

[0147] ATGAGCGGGTCGTCGGTGGGCACTGGAGGTCACGCGCCGCTCTTCACAGTGGCGCAA

[0148] TGGGCTGAGATGGAGCATCAAGCTTTGATATTCAAGTATCTCAAGGCAGGAGTCCCTG

[0149] TTCCTTCTGAGCTCCTCGCCCCTATTCGCACCAGTTTCAACCTCATTTCCCCCAATTTC

[0150] TTACCTCACCACCCCACCTGGGGTTATGCTTCCTATTGCGGGAAGAAGATAGACCCAG

[0151] AGCCGGGGCGGTGCCGGAGGACGGATGGCAAGAAATGGAGGTGCTCCAAAGATGCA

[0152] TATCAGGACTCCAAGTATTGCGAGCGGCACATGAACCGCGGCCGCAATCGTTCAAGA

[0153] AAGCTTGTGGAATCACAAACTGCCTCTCAATCTTTGTCAACTGTGACGTCAGACAGT

[0154] GTGATCGGGAGCTCCAGCCACGGTGGGAGCTTCCAGACCATGCCCTTACACTCAATT

[0155] GGAAATAGTGGAGGCCTATGCTTTGGAGGCAATGGATCTGAGTTGAAGATGGAAGCT

[0156] ATACCCTATGGGGTGTCTAACAGGGGAGCCAGGGGGTGCAATAGATCTGGATCAGAA

[0157] GAACACAATTTCATGTCAGAAGCTAAGAGGTGTGTTCAGGCGCACAGTACTGTAGAC

[0158] AGTCCGTGGCGTCTAATGGAGCCTCAAGTTCCCTTAAAATCAGAATTGAGAAATAGTT

[0159] CTCTTTGGCTAAACAACAGCTCTCAGCTACAGACATTACAAACTATGGAGTCATTGAG

[0160] CGGTGCTGCTGCAATCACCAAACAGGAGTATGTTGGTGGAGAATTTGGCTTACCAGG

[0161] ATCTCAAAAACACGAACAAAATTCTCTTCAGCCTTTATTTGATGAGTGGCCTAAGTCG

[0162] AGGGATCTGGGGTTCCATCTGAATGATCATAGATCAAACACGACTGAGCTGTCAATGT

[0163] CTAATGCAGGGGCCACCCCTAAATATTGCGCAAGGAGTGCTGAGTCACCCAGTGATG

[0164] CCTATACT GCGGCCGCTGCC ATGCAGCAGCACCTGATGCAGATGCAGCCTATGATGG

[0165] CAGCCTATTATCCCAACAACGTCACTACTGATCACATTCAGCAGTACTTGGACGAGAA

[0166] CAAGTCGTTGATTCTGAAGATTGTTGAGAGCCAGAATACCGGGAAACTGAGTGAATG

[0167] TGCAGAGAACCAAGCAAAGCTACAGCGGAATCTGATGTACCTGGCTGCCATTGCTGA

[0168] TTCACAACCCCAGCCTCCTACCATGCATCCTCAGTACTCTTCCACCGGCATGATGCAG

[0169] CCAGGAGCACATTACATGCAGCAACAAGCAGCTCAGCAGATGACACCACAATCGCTA

[0170] ATGGCTGCACGCTCTTTCCATGATGTACTCCCAGCATCCGTTTTCAGCTCTGCAGCAAC

[0171] AAGCCCTGCATAGCCAACTTGCTATGACTGCTGGAGGAAGCGCGGGACTGCACATGC

[0172] TCGGGAATGAGGGAAATAATGCTGGAGGCAGCGGGCAACTTGGGGCTGGAGGGTTT

[0173] GGTGATTACGGACGCAGCTCTCCTGGAGAAGGCATGCATAGGAGGATGGCCGGTGGG

[0174] AGTAAGCAAGATATGGATGGGCGAGGCGGAGCTCTGGAAGCCATGGTGGAGATG

[0175] GGGTGAGACTCTTTACCTGAAATCGACCGATGATGGGAATTAASEQ ID NO.4

[0176] MSGSSVGTGGHAPLFTVAQWAEMEHQALIFKYLKAGVPPVPSELLAPIRTSFNLISPNFLP

[0177] HHPTWGYASYCGKKIDPEPGRCRRTDGKKWRCSKDAYQDSKYCERHMNRGRNRSRKL

[0178] VESQTASQSLSTVTSDSVIGSSSHGGSFQTMPLHSIGNSGGLCFGGGNGSELKMEAIPYGVS

[0179] NRGARGCNRSGSEEHNFMSEAKRCVQAHSTVDSPWRLMEPQVPLKSELRNSSLWLNNS

[0180] SQLQTLQTMESLSGAAAITKQEYVGGEFGLPGSQKHEQNSLQPLFDEWPKSRDLGFHLNDHRSNTTELSMSNAGATPKYCARSAESPSDAYT*

[0181] SEQ ID NO.5

[0182] MQQHLMQMQPMMAAYYPNNVTTDHIQQYLDENKSLILKIVESQNTGKLSECAENQAK

[0183] LQRNLMYLAAIADSQPQPPTMHPQYSSTGMMQPGAHYMQQQAAQQMTPQSLMAARS

[0184] SMMYSQHPFSALQQQALHSQLAMTAGGSAGLHMLGNEGNNAGGSGQLGAGGFGDYGRSSPGEGMHRRMAGGSKQDMDGRGGSSGSHGGDGGETLYLKSTDDGN*

[0185] SEQ ID NO.6

[0186] MSGSSVGTGGHAPLFTVAQWAEMEHQALIFKYLKAGVPVPSELLAPIRTSFNLISPNFLP

[0187] HHPTWGYASYCGKKIDPEPGRCRRTDGKKWRCSKDAYQDSKYCERHMNRGRNRSRKL

[0188] VESQTASQSLSTVTSDSVIGSSSHGGSFQTMPLHSIGNSGGLCFGGNGSELKMEAIPYGVS

[0189] NRGARGCNRSGSEEHNFMSEAKRCVQAHSTVDSPWRLMEPQVPLKSELRNSSLWLNNS

[0190] SQLQTLQTMESLSGAAAITKQEYVGGEFGLPGSQKHEQNSLQPLFDEWPKSRDLGFHLN

[0191] DHRSNTTELSMSNAGATPKYCARSAESPSDAYT AAAA MQQHLMQMQPMMAAYYPNN

[0192] VTTDHIQQYLDENKSLILKIVESQNTGKLSECAENQAKLQRNLMYLAAIADSQPQPPTM

[0193] HPQYSSTGMMQPGAHYMQQQAAQQMTPQSLMAARSSMMYSQHPFSALQQQALHSQL

[0194] AMTAGGSAGLHMLGNEGNNAGGSGQLGAGGFGDYGRSSPGEGMHRRMAGGSKQDMDGRGGSSGSHGGDGGETLYLKSTDGN*.

Claims

1. The GRF-GIF fusion gene or biomaterials associated with the GRF-GIF fusion gene are used in at least one of the following: (a1) Application in promoting pear leaf regeneration; (a2) Application in the preparation of products for promoting the regeneration of pear leaves; (a3) Application in promoting pear fruit enlargement; (a4) Application in the preparation of products for promoting pear fruit enlargement; The GRF-GIF fusion gene is a fusion gene constructed by linking the growth regulator GRF (nucleotide sequence as shown in SEQ ID NO.1) and the transcription coactivator GIF (nucleotide sequence as shown in SEQ ID NO.2).

2. The application according to claim 1, characterized in that, The growth regulator GRF and the transcription coactivator GIF are linked using GCGGCCGCTGCC as the linker, and the nucleotide sequence of the GRF-GIF fusion gene is shown in SEQ ID NO.

3.

3. The application according to claim 1 or 2, characterized in that, The biological material associated with the GRF-GIF fusion gene is at least one of the following (b1)-(b7): (b1) The protein encoded by the GRF-GIF fusion gene; (b2) An expression cassette containing the GRF-GIF fusion gene; (b3) A recombinant vector containing the GRF-GIF fusion gene, or a recombinant vector containing the expression cassette described in (b2); (b4) A recombinant microorganism containing the GRF-GIF fusion gene, or a recombinant microorganism containing the expression cassette of (b2), or a recombinant microorganism containing the recombinant vector of (b3); (b5) A transgenic plant cell line containing the GRF-GIF fusion gene, or a transgenic plant cell line containing the expression cassette described in (b2), or a transgenic plant cell line containing the recombinant vector described in (b3); (b6) Transgenic plant tissue containing the GRF-GIF fusion gene, or transgenic plant tissue containing the expression cassette of (b2), or transgenic plant tissue containing the recombinant vector of (b3); (b7) A transgenic plant organ containing the GRF-GIF fusion gene, or a transgenic plant organ containing the expression cassette of (b2), or a transgenic plant organ containing the recombinant vector of (b3); (b8) A transgenic plant containing the GRF-GIF fusion gene, or a transgenic plant containing the expression cassette of (b2), or a transgenic plant containing the recombinant vector of (b3); (b9) Tissue cultures produced from regenerative cells of the transgenic plant described in (b8); (b10) Protoplasts produced from the tissue culture described in (b9); (b11) Transformation reagent containing the GRF-GIF fusion gene.

4. The application according to claim 3, characterized in that, The protein encoded by the GRF-GIF fusion gene is as follows (c1) or (c2) or (c3): (c1) A protein with the amino acid sequence shown in SEQ ID NO. 6; (c2) The amino acid residue sequence shown in SEQ ID NO.6 is modified by substitution and / or deletion and / or addition of one or more amino acid residues and is associated with a protein derived from SEQ ID NO.6 that promotes pear leaf regeneration and fruit enlargement; (c3) A fusion protein that links a protein tag to the N-terminus and / or C-terminus of (c1) or (c2).

5. The application according to claim 1, characterized in that, Stable overexpression of the GRF-GIF fusion gene in pear leaves enhances the regeneration capacity of pear leaves, or overexpression in pear fruit callus tissue promotes the enlargement of fruit callus tissue.

6. A method for improving the regeneration ability of pear leaves, characterized in that, Overexpression of the GRF-GIF fusion gene as described in claim 1 or 2 in pear plants can improve the regeneration capacity of pear leaves.

7. A method for promoting pear fruit enlargement, characterized in that, Overexpression of the GRF-GIF fusion gene as described in claim 1 or 2 in pear plants and / or fruits can promote pear fruit enlargement.

8. A GRF-GIF fusion gene, wherein the GRF-GIF fusion gene is a fusion gene constructed by linking the growth regulator GRF with the nucleotide sequence shown in SEQ ID NO.1 and the transcription coactivator GIF with the nucleotide sequence shown in SEQ ID NO.

2.

9. Biomaterials related to the GRF-GIF fusion gene of claim 8, characterized in that... The biomaterial is at least one of the following (b1)-(b7): (b1) The protein encoded by the GRF-GIF fusion gene; (b2) An expression cassette containing the GRF-GIF fusion gene; (b3) A recombinant vector containing the GRF-GIF fusion gene, or a recombinant vector containing the expression cassette described in (b2); (b4) A recombinant microorganism containing the GRF-GIF fusion gene, or a recombinant microorganism containing the expression cassette of (b2), or a recombinant microorganism containing the recombinant vector of (b3); The protein encoded by the GRF-GIF fusion gene is as follows (c1) or (c2) or (c3): (c1) A protein with the amino acid sequence shown in SEQ ID NO. 6; (c2) The amino acid residue sequence shown in SEQ ID NO.6 is modified by substitution and / or deletion and / or addition of one or more amino acid residues and is associated with a protein derived from SEQ ID NO.6 that promotes pear leaf regeneration and fruit enlargement; (c3) A fusion protein that links a protein tag to the N-terminus and / or C-terminus of (c1) or (c2).

10. The method for constructing the GRF-GIF fusion gene according to claim 1, characterized in that, The method includes the following steps: (1) Using leaf cDNA of 'Autumn Pear Seedling Progeny' as a template, the GRF and GIF genes were amplified by PCR using GRF-F / R and GIF-F / R primers, respectively; The primer sequences are as follows: GRF-F:TCCAAAGAATTCAAAAAGCTTATGAGCGGGTCGTCGGT GRF-R:ATCTGCATCAGGTGCTGCTGCATGGCAGCGGCCGCAGTATAGGCATCACTGGGTGACT GIF-F:AGTCACCCAGTGATGCCTATACTGCGGCCGCTGCCATGCAGCAGCACCTGATGCAGAT GIF-R:TCTAGAGTCCTGCTTTAATGAATTCCCATCATCGGTCGATTT; (2) Using the amplified GRF and GIF genes as templates, the GRF-GIF fusion gene containing GCGGCCGCTGCClinker was amplified using GRF-F and GIF-R primers; (3) The linearized PSAK277 vector was double-digested and ligated with the GRF-GIF fusion gene fragment using Exnase II; (4) The ligation product was transformed into DH5α, positive clones were selected for sequencing verification, and the GRF-GIF recombinant plasmid of the correctly sequenced clone was extracted using a plasmid extraction kit and transformed into Agrobacterium LBA4404 competent cells for culture. (5) After verifying positive clones by colony PCR, pick the colonies, shake them, add glycerol, and store them at -80℃.

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