Ginkgo biloba gbgrf2 gene and gbgrf2-gbgif2 fusion gene in plant tissue culture

Abstract

The present application relates to the technical field of plant genetic engineering, and particularly relates to a ginkgo biloba L. GbGRF2 gene and application of a ginkgo biloba L. GbGRF2-GbGIF2 fusion gene in plant tissue culture; a full-length coding region of a ginkgo biloba L. GRF gene family GbGRF2 gene is successfully cloned from ginkgo biloba L., the gene presents a specific expression mode in a ginkgo biloba L. callus regeneration process; overexpression of the GbGRF2 and GbGRF2-GbGIF2 fusion gene can significantly promote regeneration efficiency of adventitious buds in an in-vitro culture process of a plant; the gene provided by the present application has important theoretical significance and application potential in a plant tissue regeneration regulation network, and plays a key role in improving in-vitro regeneration efficiency of gymnosperms.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a ginkgo biloba. GbGRF2 Genes and their GbGRF2- GbGIF2 Application of fusion genes in plant tissue culture. Background Technology

[0002] ginkgo( Ginkgo biloba As a gymnosperm unique to my country ( Gymnospermae Ginkgo biloba, or ginkgo seedlings, has significant economic and scientific research value. Under natural conditions, its propagation primarily relies on traditional methods such as grafting, cuttings, or seedling cultivation. However, these methods have limitations, including long propagation cycles, high costs, and susceptibility to environmental factors. Large-scale propagation of ginkgo seedlings through in vitro tissue culture can effectively compensate for the shortcomings of traditional methods. However, the prevalent microbial contamination and browning phenomena during tissue culture severely affect the induction and differentiation efficiency of callus tissue, resulting in the lack of a successful establishment of a complete in vitro regeneration system for ginkgo. Therefore, establishing an efficient in vitro regeneration and genetic transformation system has become an urgent priority for ginkgo molecular breeding research, providing crucial technical support for solving key problems such as low regeneration efficiency and excessively long propagation cycles.

[0003] GRF Developmental regulators possess two highly conserved domains in their N-terminal region: the QLQ domain and the WRC domain. The QLQ domain is the functional domain mediating protein-protein interactions. GRF Interacting factors GIF Interactions form transcription activators. WRC, as a plant-specific domain, has a more conserved amino acid site than QLQ, and the zinc finger structure in WRC can bind DNA. The GIF gene family is small but highly conserved, typically containing no more than five members. Its proteins contain two key domains, SNH and QG. The SNH domain specifically binds to the QLQ domain of the GRF protein, forming the GRF-GIF complex. This complex participates in key biological processes such as plant growth and development, cell proliferation, and signal transduction by regulating the expression of downstream target genes, and exhibits high conservation in evolution. Studies have shown that... GRF Genes play a crucial role in regulating cell proliferation and differentiation, promoting plant tissue regeneration, and responding to abiotic stress. Furthermore, studies have shown that individual growth regulators... GRF or GRF With interaction factors GIF The fusion protein can significantly promote the regeneration of tissue cultures from multiple species without causing defects in transgenic plants.

[0004] Currently, it has been reported that transplantation can be used in multiple species.GRF Genes promote tissue differentiation. Ginkgo, this ancient seed plant, provides us with valuable material for studying the family system and functional evolution of plant transcription factors. However, currently, there is still limited information available about… GRF Research on growth regulators in Ginkgo biloba is still insufficient, lacking a comprehensive and systematic exploration. Given... GRF Growth regulators play a crucial role in biological processes such as regulating plant growth and development, responding to abiotic stresses, and participating in the biosynthesis of secondary metabolites, particularly in Ginkgo biloba. GRF Systematic bioinformatics analysis of gene families and in-depth exploration GRF Genes and GbGRF2-GbGIF2 The molecular regulatory mechanism of fusion genes in Ginkgo tissue culture regeneration system is of great theoretical significance and application value for establishing an efficient Ginkgo tissue culture system and genetic transformation technology platform. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention aims to provide a ginkgo biloba... GbGRF2 Genes and their GbGRF2- GbGIF2 The application of fusion genes in plant tissue culture aims to promote the regeneration of plant tissues.

[0006] To solve the above problems, the present invention adopts the following technical solution: Firstly, the present invention provides a ginkgo GbGRF2 The gene, whose nucleotide sequence is shown in SEQ ID No. 1.

[0007] As one possible implementation method, the ginkgo GbGRF2 The gene is highly expressed in the female reproductive organs of Ginkgo (female cones, embryos, ovules, and mature seeds) and in the stems and roots, but lowly expressed or not expressed in other tissues.

[0008] As one possible implementation method, the ginkgo GbGRF2 The nucleotide sequences of the full-length primers for gene cloning are shown in SEQ ID No. 5-SEQ ID No. 6; the Ginkgo biloba... GbGRF2 The nucleotide sequences of the primers for qRT-PCR amplification of the gene are shown in SEQ ID No. 9-SEQ ID No. 10.

[0009] Secondly, the present invention provides the aforementioned ginkgo GbGRF2 Gene fusion gene GbGRF2-GbGIF2 ,in GbGIF2 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0010] As one possible implementation method, the GbGIF2The sequence of the amino acids encoded by the gene is shown in SEQ ID NO.4.

[0011] Thirdly, the present invention provides the aforementioned ginkgo GbGRF2 The amino acids encoded by the gene, the ginkgo GbGRF2 The sequence of the amino acids encoded by the gene is shown in SEQ ID NO.3.

[0012] Fourthly, the present invention provides the aforementioned fusion gene. GbGRF2-GbGIF2 The encoded amino acids, the fusion gene GbGRF2-GbGIF2 The sequence of the encoded amino acid is shown in SEQ ID NO.4.

[0013] Fifthly, the present invention provides a recombinant plasmid comprising the aforementioned Ginkgo biloba. GbGRF2 Genetic pCAMBIA1300-GbGRF2 Recombinant plasmids or plasmids containing the aforementioned fusion gene GbGRF2-GbGIF2 of pCAMBIA1300- GbGRF2-GbGIF2 Recombinant plasmid.

[0014] Sixthly, the present invention provides a method for promoting adventitious shoot regeneration during plant tissue culture regeneration, comprising transforming the recombinant plasmid into GV3101 Agrobacterium competent cells to prepare positive clones, infecting plant leaves with the clones, and screening... GbGRF2 and GbGRF2-GbGIF2 Plant callus tissue overexpressed was cultured.

[0015] Seventhly, the present invention provides the aforementioned ginkgo GbGRF2 Gene or the aforementioned fusion gene GbGRF2-GbGIF2 Or the application of the recombinant plasmids in plant tissue culture.

[0016] As one possible implementation method, the plant is large-leaved tobacco (Tobacco Monotamogeton pv. pubescens). Nicotiana tabacum (or ginkgo)

[0017] As one possible implementation method, overexpression of Ginkgo biloba GbGRF2 Gene or GbGRF2-GIF2 Fusion genes and their vectors promote the regeneration of adventitious buds during plant tissue culture regeneration.

[0018] The beneficial effects of this invention are: this invention successfully clones ginkgo from ginkgo. GRF Gene family GbGRF2 The gene coding region is full-length, and this gene exhibits a specific expression pattern during Ginkgo callus regeneration. Transgenic functional verification experiments confirmed that overexpression... GbGRF2 and GbGRF2-GbGIF2 Fusion genes can significantly improve the regeneration efficiency of adventitious shoots during plant in vitro culture. Therefore, GbGRF2 Genes and GbGRF2-GbGIF2Fusion genes have important theoretical significance and application potential in the plant tissue regeneration regulatory network, and play a key role in improving the in vitro regeneration efficiency of gymnosperms. Attached Figure Description

[0019] Figure 1 Ginkgo GbGRF Genes and GbGIF A map showing the location of chromosomes.

[0020] Figure 2 for pCAMBIA1300-GbGRF2 and pCAMBIA1300-GbGRF2-GbGIF2 Electrophoresis diagram for identification of overexpression vector.

[0021] Figure 3 for pCAMBIA1300-GbGRF2 and pCAMBIA1300-GbGRF2-GbGIF2 Overexpression vector map.

[0022] Figure 4 In the culture medium for adding hormones GbGRF2 and GbGRF2-GbGIF2 The in vitro regeneration of converted tobacco; scale bar: 1 cm.

[0023] Figure 5 In hormone-free culture medium GbGRF2 and GbGRF2-GbGIF2 The in vitro regeneration of converted tobacco; scale bar: 1 cm.

[0024] Figure 6 for GbGRF2 and GbGRF2-GbGIF2 Observation of GFP fluorescence in transgenic tobacco; tobacco observed under natural light (top), tobacco explants emitting GFP fluorescence (bottom), scale bar 1cm.

[0025] Figure 7 for GbGRF2 and GbGRF2-GbGIF2 DNA identification diagram of genetically modified tobacco; Marker is DL5000bp.

[0026] Figure 8 for GbGRF2 and GbGRF2-GbGIF2 A graph showing the gene expression levels in genetically modified tobacco.

[0027] Figure 9 for GbGRF2 Figures showing the callus induction of transformed Ginkgo biloba leaves; (A) Ginkgo biloba leaves after two days of co-culture; (B) Ginkgo biloba leaves and callus on day 14 after co-culture, transferred to callus induction medium; (C) Ginkgo biloba leaves and callus on day 14 after transfer to screening medium; Scale bar is 1 cm.

[0028] Figure 10 for GbGRF2 andGbGRF2-GbGIF2 DNA identification image of transgenic Ginkgo biloba; Marker is DL5000bp.

[0029] Figure 11 for GbGRF2 and GbGRF2-GbGIF2 A graph showing the expression level of transgenic Ginkgo biloba genes. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments.

[0031] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0032] 1. Materials and Reagents Unless otherwise specified in this embodiment, all experimental methods can be performed according to conventional methods, such as those described in J. Sambrook et al.'s "Molecular Cloning: A Laboratory Manual" and F. Osber et al.'s "A Concise Laboratory Manual of Molecular Biology," or according to the manufacturer's instructions for use of the products used.

[0033] The ginkgo leaves used in the experiment were tender leaves of two-month-old ginkgo seedlings, which were obtained by germinating seeds collected from a 20-year-old female ginkgo tree on the campus of Nanjing Forestry University.

[0034] RNA extraction kit and plasmid extraction kit were purchased from OMEGA (Catalog numbers R6827 and D6943, respectively); reverse transcription reagent was purchased from Monad (Catalog number MR05101M); high-fidelity enzymes for cloning PCR were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. (TaKaRa China) (Catalog number D2215); gel extraction kit was purchased from Novizan Biotechnology Co., Ltd. (Catalog number DC301); 2×Rapid Taq Master Mix for conventional PCR was purchased from Novizan Biotechnology Co., Ltd. (Catalog number P222); restriction endonucleases BamH I and Xba I were purchased from TransGen Biotech Co., Ltd. (Catalog numbers JB101 and JX101, respectively); homologous recombinase was purchased from Novizan Biotechnology Co., Ltd. (Catalog number C112); plant expression vector was pCAMBIA1300-GFP; E. coli competent cells DH5α, Agrobacterium competent cells GV3101 and Y2H were used. Gold competent cells were purchased from Beijing Qingke Biotechnology Co., Ltd., with catalog numbers DLC301 and DLC402 respectively; MS and LB media are commonly used media in this field, and their formulations are based on J. Sambrook et al.'s "Molecular Cloning: A Laboratory Manual".

[0035] Unless otherwise specified, all materials and reagents used in this embodiment are commercially available.

[0036] 2. GbGRF2 and GbGIF2 Chromosomal localization Chromosomal localization shows that GbGRF Genes are distributed relatively dispersedly across chromosomes, unevenly distributed across the seven chromosomes, with one gene each for chr2, chr3, chr5, chr6, chr7, and chr11. GbGRF The chr4 gene contains two GbGRF genes. GbGIF The gene family members are located on chromosomes chr3, chr4, and chr10. Ginkgo biloba. GbGRF2 and GbGIF2 All genes are located on chromosome chr3 ( Figure 1 ).

[0037] 3. GbGRF2 and GbGIF2 Construction of full-length clones and overexpression vectors RNA was extracted from Ginkgo biloba using an RNA extraction kit. The extracted RNA was subjected to agarose gel electrophoresis to check its integrity. Then, the concentration and purity of the RNA were determined using a NanoDrop 2000, and the RNA was stored at -80℃ for later use. Ginkgo biloba cDNA was obtained by reverse transcription using Monad's reverse transcription reagent. The cDNA was then searched in the Ginkgo biloba whole genome CDS file. GbGRF2 and GbGIF2 The CDS sequence information is used to design the design using Oligo software. GbGRF2 The forward primers for cloning the full length were 5′-ATGGATTTCTCACATAGTTCAGTG-3′ (SEQ ID No. 5), and the reverse primers were 5′-CACGAGGAGAGCTTGAATTT-3′ (SEQ ID No. 6). The procedure is as follows: (1) Using cDNA as a template, amplification was performed using a high-fidelity enzyme. The PCR reaction program was: 98℃ for 3 min, followed by 35 cycles: 98℃ for 10 s, 58℃ for 5 s, 72℃ for 1 min, and finally 72℃ for 5 min. After PCR, the products were detected by electrophoresis. Figure 2 The product was recovered using a gel recovery kit; see the instruction manual for specific instructions.

[0038] (2) The recovered fragments were subjected to a second PCR using homologous arm primers according to the same procedure and then recovered. The expression vector pCAMBIA1300-GFP was double-digested with restriction endonucleases BamH Ⅰ and XbaⅠ. The double digestion reaction procedure was as follows: incubation at 37℃ for 15 min followed by heating at 80℃ for 20 min to terminate the reaction.

[0039] (3) The linearized pCAMBIA1300-GFP vector and the product after the second PCR were recombined and ligated at 37°C for 30 min under the action of homologous recombinase. After the reaction was completed, the mixture was placed on ice to cool.

[0040] (4) Transfer the ligation product to Escherichia coli DH5α competent cells. See the instruction manual for the transformation steps. Finally, spread the revival solution evenly on LB solid medium with Kana resistance and incubate at 37°C upside down for 16 hours.

[0041] (5) After the culture is completed, single clones on the culture medium are picked for identification. First, a single clone is picked with a sterile toothpick and mixed in 10 μL of sterile water. Then, 2 μL of bacterial solution is taken for bacterial PCR identification, and the remaining 8 μL is stored at 4℃. Bacterial PCR identification uses 2×Rapid Taq Master Mix. The PCR reaction program is: 95℃ for 3 min, then 33 cycles, i.e., 95℃ for 15 s, 58℃ for 15 s, 72℃ for 30 s, and finally 72℃ for 5 min.

[0042] (6) The remaining 8 μL of bacterial culture that was successfully identified by PCR was sent to Qingke Company for sequencing to determine whether the target fragment was completely cloned. Sequence alignment was performed using DNAMAN software. After successful alignment, the bacterial culture was expanded at 37℃ and 200 rpm, followed by plasmid extraction. The plasmid extraction procedure is detailed in the instruction manual. pCAMBIA1300-GbGRF2 and pCAMBIA1300-GbGRF2-GbGIF2 plasmid ( Figure 3 Store at -20℃. Amplify the target fragment. GbGRF2 and GbGIF2 The nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, with lengths of 1744bp and 594bp, encoding 593 and 195 amino acids, respectively. The amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. GbGRF2-GbGIF2 A nucleotide linker is added between the two genes in a fusion gene, encoding four amino acids. GbGRF2-GbGIF2 The nucleotide sequence and encoded amino acid sequence of the fusion gene are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0043] The target fragment to be amplified GbGRF2 The nucleotide sequence is as follows (SEQ ID NO.1): ATGGATTTCTCACATAGTTCAGTGTCGGTGTCTGGAAGTTTGTCTGGTTTTAGCTCAGAGCTGGGAGATCACCGACACAGAATGCCTGGGCTGTCATCTCCTGGAGGAATTGGAATACTGACTGTGGGAGATCCTTACAAGCAATGTCGTTCAGCTTCAGAAATGGACTTGGATGAGAGGACCAGAGGACCCATCAAGATTGCACGCACTGATTCGTTCCCATGTACCAACAGCAGACAGCAGAATACAAGTAGTAATAATAGTAATAATAATGCTACTAATAACAATAATAATGCCATTGGATCTCTGTTGAGGAGTAATTCTATAATGTCTGATGGGCGTTTGGCTAATTGTTCTCCTACCAATTCCAGTGCTAATTCAGTGCTTTGATGAGTA ATGAAGGGTGTGTTAGTGTGTCTGAGCATAGGCTTATGAGGAGTGACTCGGTTGTGTCTGCATATGGTGGTGGTGCAAGGACTTTGAGCTTTCATCAACC

[0044] Amplified target fragment GbGRF2 has the amino acid sequence shown below (SEQ ID NO. 3): MDFSHSSVSVSGSLSGLGSELGDHRHRMPGLSSPGGIGILTVGDPYKQCRSASEMDLDERTRGPIKIARTDSFPCTNSRQQNTSSNNSNNNATNNNNNAIGSLLRSNSIMSDGRLANCSPTNSSANSDGGLMSNEGCVSVSEHRLMRSDSVVSAYGGGARTLSFHQPYHYKSAGLPLTMIRESATHMGGMHAIVAGSRPPFTQSQWQELEHQALIFKYMMAGVSVPSDLIIPIRKSVAALSVALSAGSYHPNMAWGSFHLGFANNTDPEPGRCRRTDGKKWRCSRDVVPDQKYCERHMHRGRHRSRKPVEGQTGASSQSHLGGPTTTTTTTANLSSNGPSSVSLAAAARNSSSNLRPSISMNNQQQHNHSGSNNSALGMNSSLLLQIASGSASPLESNKEYRYMNGGMKGGADNVDEQVFFSEVSGSSRGLGQDAMLSSVNNNGWRSSMPSKVSQVKATDQQNGSLLSYNSPQLRTLLAQDFGLMSETNQMNLPSHHQHSFRNTGFGVVESVNVGRESEGQGQHLRHFFDDWPRSRDASALSWSDVEEDRSNRSSSTTQLSISIPAMTSSDFSATNSSSPRX。

[0045] Amplified target fragment GbGIF2 has the nucleotide sequence shown below (SEQ ID NO. 2): ATGTATCTGGCTGCCATTGCTGATTCTCAACCACAACCACCAACTGCACATACTCAGATTCCTCCAAATGCAGTGATGCAGTCTGGTGCACATTACATGC AACACCAGCAGGCACAACAACAAGTGACGCCTCAGTCCCTCATGTCATCCAGGGCTCCCATGCTGTATGCTCAGCAGCCAATCGCTGCTTTGCATCAGGC CCAGCAGCAACAGCAGCAGCAGCAACAACACCAGTCTCTTCACAGCCAACTGGGCATGAATTCTGGAGGAAGCAATGGCCTACACATGTTGCACGGTGAT ACAAACATGGGAGGTAATGGGCCTCTCTCATCTGGGGGCTTCCCTGACTTTGGGCGTGGTAATTCTGGCAGCTCTGGGGATGGCATGCATGCAAACAGGG GCTTGGGTGCAGATCGTGGGGCAAATAAACAGGATGGAGGAATAGGATCAGAGAATGCACATCCAGGTTCTGGTGATTGTAGGGGGAGCTCAGCTGGAGG GCCGAATGCTGACGAGTCCGAACCATCATACCTGAAAGCCTCCGAAGAAGAGGGGAACTAG.

[0046] The target fragment to be amplified GbGIF2 The amino acid sequence is as follows (SEQ ID NO.4): MYLAAIADSQPQPPTAHTQIPPNAVMQSGAHYMQHQQAQQQVTPQSLMSSRAPMLYAQQPIAALHQAQQQQQQQQHQSLHSQLGMNSGGSNGLHMLHGDTNMGGNGPLSSGGFPDFGRGNSGSSGDGMHANRGLGADRGANKQDGGIGSENAHPGSGDCRGSSSAGGPNADESEPSYLKASEEEGN.

[0047] The nucleotide sequence of the fusion gene GbGRF2-GbGIF2 is as follows (SEQ ID NO.11): ATGGATTTCTCACATAGTTCAGTGTCGGTGTCTGGAAGTTTGTCTGGTTTTAGCTCAGAGCTGGGAGATCACCGACACAGAATGCCTGGGCTGTCATCTCCTGGAGGAATTGGAATACTGACTGTGGGAGATCCTTACAAGCAATGTCGTTCAGCTTCAGAAATGGACTTGGATGAGAGGACCAGAGGACCCATCAAGATTGCACGCACTGATTCGTTCCCATGTACCAACAGCAGACAGCAGAATACAAGTAGTAATAATAGTAATAATAATGCTACTAATAACAATAATAATGCCATTGGATCTCTGTTGAGGAGTAATTCTATAATGTCTGATGGGCGTTTGGCTAATTGTTCTCCTACCAATTCCAGTGCTAATTCAGTGCTTTGATGAGTA

[0048] The amino acid sequence of the fusion gene GbGRF2-GbGIF2 is as follows (SEQ ID NO.12): .

[0049] 4. Overexpression GbGRF2 and GbGRF2-GbGIF2 Effects of genes on in vitro regeneration of Tobacco monotypic leaves 4.1 Recombinant plasmids pCAMBIA1300-GbGRF2 and pCAMBIA1300-GbGRF2-GbGIF2 Transformed into Agrobacterium GV3101 Referring to the instructions for GV3101 Chemically Competent Cell from Qingke Company, the two overexpression vectors were transformed into GV3101 Agrobacterium competent cells using the freeze-thaw method. The specific steps are as follows: (1) Place the competent Agrobacterium cells stored at -80℃ on ice to melt them into an ice-water mixture, add 1µL of recombinant plasmid, mix gently, and then place on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and in an ice bath for 5 min.

[0050] (2) Add 700 μL of antibiotic-free LB liquid medium to the centrifuge tube, mix well, and revive at 28°C and 200 rpm for 3 h.

[0051] (3) Take the resuscitation solution and spread it evenly on LB solid medium containing Kana and Rif resistance. Invert the plate and incubate it in the dark at 28°C for 3 days. Then pick single clones for identification.

[0052] Colonies that can amplify the target band are considered positive clones. They are cultured on 50 mg / L Kan LB liquid medium until the OD600 is about 1, then 50% sterile glycerol is added, and the medium is flash-frozen in liquid nitrogen for 2 min and stored at -80℃ for later use.

[0053] 4.2 Agrobacterium-mediated genetic transformation of large-leaf tobacco leaves (1) Select vigorous aseptic tobacco seedlings as propagation material and carry out propagation under aseptic conditions. Cut the aseptic seedlings into stem segments of appropriate size and inoculate them onto a new medium MS+30g / L sucrose+7g / L agar. Place the inoculated tobacco seedlings under 2000 lux light, 24℃ and 50% humidity for cultivation.

[0054] (2) Leaves of sterile seedlings that have grown for about 26 days after propagation were used as materials for genetic transformation of tobacco leaves. The sterile seedling leaves were cut into squares of about 0.5-1cm, and the leaf margins and main veins were removed. The leaves were soaked in a resuspension with OD600=0.6 for about 10 minutes. After the time was up, the liquid on the surface of the leaves was wiped off with sterile filter paper, and the leaves were laid flat with the upper side down on the co-culture medium MS + 0.1mg / L NAA + 1mg / L 6-BA + 30g / L sucrose + 7g / L agar + 100µmol / L AS, and incubated in the dark at 25℃ for 2 days.

[0055] (3) After co-culture, the culture medium was transferred to screening medium MS + 0.1 mg / L NAA + 1 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar + 20 mg / L Tim + 10 mg / L Hygr and cultured at 25°C for 30 days.

[0056] (4) Cut the sprouts and transfer them to a new selection medium and continue to grow them under light at 25°C for 30 days. Transfer the sprouts with good growth to rooting medium MS + 30 g / L sucrose + 7 g / L agar + 20 mg / L Tim + 10 mg / L Hygr and grow them under light at 25°C.

[0057] (5) After co-culture, continue to observe and take pictures to record the growth status of tobacco leaves under different culture media and different transgenic treatments, and count the budding time and number of buds.

[0058] 4.3 Overexpression GbGRF2 and GbGRF2-GbGIF2 Effects on the tissue culture regeneration process of Tobacco denudata Will contain GbGRF2 and GbGRF2-GbGIF2 Agrobacterium was used to infect Tobacco grandiflora, and the infected leaf explants were placed in culture media supplemented with hormones (0.1 mg / L NAA + 1 mg / L 6-BA) and without hormones, respectively, and the tissue culture process was continuously observed. Tobacco leaves infected with Agrobacterium-free suspension served as a mock control and an empty control. The number of leaf discs in each group was 40-60.

[0059] Observations revealed that in the hormone-added culture medium, the transgenic tobacco leaves began to sprout adventitious buds on day 11, while the untreated control group began to show adventitious buds on day 18. Furthermore, the transgenic tobacco leaves produced significantly more adventitious buds than the control group. Figure 4 In a culture medium without added hormones, GbGRF2 Adventitious buds first appeared in the converted tobacco on day 14, while the control group did not produce any adventitious buds at all. Figure 5 The results of tobacco conversion indicate that... GbGRF2 and GbGRF2-GbGIF2 Genes can shorten the regeneration time of detached tobacco leaves, improve regeneration efficiency, and promote the regeneration of detached tobacco leaves without exogenous hormone stimulation. It is worth noting that... GbGRF Compared to single genes, GbGRF-GbGIF The fusion gene did not significantly improve the efficiency of tobacco regeneration.

[0060] 4.4 Identification and Expression Level Detection of Tobacco Genetically Modified Substances (1) The pCAMBIA1300 vector contains GFP green fluorescent protein, which emits green fluorescence under blue light excitation. Under blue light irradiation and observed with yellow glasses, green fluorescence can be observed on the edges and wounds of transgenic tobacco leaves, indicating transgenic positivity. No fluorescence was observed in the control group. Figure 6 ).

[0061] (2) Twelve adventitious shoots were selected from the hormone-free culture medium after gene treatment, and DNA was extracted for PCR identification. The results showed that... GRF2 and GbGRF2-GbGIF2 Four and nine samples from the genetically modified plants, respectively, showed positive bands. Figure 7 ).

[0062] (3) The expression levels of the transgenic samples were detected. The qRT-PCR results showed that, compared with the control, GbGRF2 and GbGRF2-GbGIF2 Extremely high expression levels of the corresponding gene were detected in transgenic tobacco leaves. Figure 8 ).

[0063] 5. Overexpression GbGRF2 and GbGRF2-GbGIF2 The Influence of Genes on the In Vitro Regeneration of Ginkgo 5.1 Preliminary Experiment on Genetic Transformation of Ginkgo The experiment included two control groups: a mock control group (untreated ginkgo leaves) and an empty vector control group (ginkgo leaves infected only with Agrobacterium carrying the empty vector). The procedures were as follows: (1) Before infection, Ginkgo leaf discs were pre-cultured for 4 days. The culture medium was MS + 1 mg / L NAA + 1 mg / L KT + 30 g / L sucrose + 7 g / L agar.

[0064] (2) Co-culture infection for two days, the culture medium is MS + 1mg / L NAA + 1mg / L KT + 30g / L sucrose + 7g / L agar + 100µmol / L AS.

[0065] (3) Transfer to callus induction medium MS + 1 mg / L NAA + 1 mg / L KT + 30 g / L sucrose + 7 g / L agar + 400 mg / L CEF and culture for 14 days. After 14 days, transfer to screening medium MS + 1 mg / L NAA + 1 mg / L KT + 30 g / L sucrose + 7 g / L agar + 400 mg / L CEF + 8 mg / L Hygr.

[0066] (4) Seven and fourteen days after the end of co-culture, the growth of callus tissue was observed and the callus induction rate (number of leaf discs that emerged from callus / total number of leaf discs) was calculated (Table 1).

[0067] The results showed that GbGRF2 and GbGRF2-GbGIF2 The cure rate of transgenic ginkgo leaves was higher than that of the empty control group at both 7 and 14 days after infection, indicating that... GbGRF2 or GbGRF2-GbGIF2It can promote callus induction during Ginkgo tissue culture, but Agrobacterium may cause some damage to young Ginkgo leaves during infection, resulting in a lower callus rate in transgenic materials compared to the mock control. Figure 9 (See Table 1). In each treatment... GbGRF2-GbGIF2 The transformed Ginkgo leaves had the highest callus formation rate. 14 days after infection, the callus formation on the leaves was significantly better than that of other transgenic materials, as well as the mock and empty control.

[0068] Table 1. Callus induction of Ginkgo biloba leaves 7 and 14 days after co-culturing.

[0069] 5.2. Identification of Ginkgo Transgenic Genes and Detection of Relative Expression Levels Ginkgo callus cultured on a screening medium for 20 days was selected. GbGRF2 , GbGRF2-GbGIF2 24 and 27 samples of materials were infected, and 18 samples of materials were infected with empty vector, along with 2 samples of mock control materials. DNA was extracted and PCR identification was performed, yielding 18 and 9 positive bands respectively. Figure 10 ).

[0070] Gene expression levels were detected in samples that showed positive bands. RNA extraction and reverse transcription were performed as described in section 3.1. qRT-PCR was conducted using ABI 7500 Real-time PCR Systems (Applied Biosystems) according to the MonAmp™ SYBR® Green qPCR Mix (None / Low / High ROX) (catalog number MQ10201) instructions. Three biological replicates and three technical replicates were set up. Ginkgo biloba was used as the starting material. GADPH The gene was used as an internal reference gene. The upstream primer for the internal reference qRT-PCR was 5′-ATCCACGGGAGTCTTCAC-3′, and the downstream primer was 5′-CTCATTCACGCCAACAAC-3′. Primer Premier6 was used to design the qRT-PCR. GbGRF2 The upstream primer for qRT-PCR was 5′-GCGTTGTTCCAGAGACGTTG-3′ (SEQ ID No. 9), and the downstream primer was 5′-TTGCGGTGGTAGTGGTAGTG-3′ (SEQ ID No. 10). The qRT-PCR reaction system is shown in Table 2. Table 2. qRT-PCR reaction system

[0071] The qRT-PCR program was set as follows: 95℃ pre-deformation for 30 s, 95℃ denaturation for 10 s, 58℃ annealing for 10 s, and 72℃ extension for 30 s, with 40 cycles for each of the denaturation, annealing, and extension steps. Quantitative PCR results were calculated using the 2-ΔΔCT method. GbGRF2 The relative quantitative expression level of Ginkgo biloba leaf callus was determined. qRT-PCR results showed that, compared to the empty vector control, the expression level of Ginkgo biloba leaf callus was significantly higher. GbGRF2 and GbGRF2-GbGIF2 There is an improvement ( Figure 11 ).

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A type of ginkgo GbGRF2 Genes, characterized by, Its nucleotide sequence is shown in SEQ ID No.

1.

2. The Ginkgo biloba according to claim 1 GbGRF2 Genes, characterized by, The Ginkgo GbGRF2 The nucleotide sequences of the full-length primers for gene cloning are shown in SEQ ID No. 5-SEQ ID No. 6; the Ginkgo biloba... GbGRF2 The nucleotide sequences of the primers for qRT-PCR amplification of the gene are shown in SEQ ID No. 9-SEQ ID No.

10.

3. The Ginkgo biloba according to claim 1 GbGRF2 Gene fusion gene GbGRF2-GbGIF2 Its characteristics are, in GbGIF2 The nucleotide sequence of the gene is shown in SEQ ID No. 2, and the fusion gene is described. GbGRF2-GbGIF2 The nucleotide sequence is shown in SEQ ID No.

11.

4. The fusion gene according to claim 3 GbGRF2-GbGIF2 Its characteristics are, The GbGIF2 The sequence of the amino acids encoded by the gene is shown in SEQ ID NO.

4.

5. The Ginkgo biloba according to claim 1 GbGRF2 Gene-encoded amino acids, characterized in that, The Ginkgo GbGRF2 The sequence of the amino acids encoded by the gene is shown in SEQ ID NO.

3.

6. The fusion gene according to claim 3 GbGRF2-GbGIF2 The encoded amino acids.

7. A recombinant plasmid, characterized in that, Ginkgo biloba as described in claim 1 GbGRF2 Genetic pCAMBIA1300-GbGRF2 Recombinant plasmids or plasmids containing the fusion gene as described in claim 3 GbGRF2-GbGIF2 of pCAMBIA1300-GbGRF2-GbGIF2 Recombinant plasmid.

8. A method for promoting adventitious bud regeneration during plant tissue culture regeneration, characterized in that, Positive clones were prepared by transforming the recombinant plasmid described in claim 7 into GV3101 Agrobacterium competent cells, and then subjected to infection culture of plant leaves for screening. GbGRF2 or GbGRF2-GbGIF2 The overexpressed plant callus tissue was cultured, and the plant was either Tobacco macrocarpa or Ginkgo biloba.

9. The Ginkgo biloba according to claim 1 GbGRF2 Gene or the fusion gene as described in claim 3 GbGRF2-GbGIF2 Or the application of the recombinant plasmid according to claim 7 in plant tissue culture, wherein the plant is large-leaved tobacco or ginkgo.

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