Gingko GbGRF2 gene and application of GbGRF2-GbGIF2 fusion gene thereof in plant tissue culture

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a ginkgo GbGRF2 gene and application of a GbGRF2-GbGIF2 fusion gene of the ginkgo GbGRF2 gene in plant tissue culture. According to the invention, the GbGRF2 gene coding region full length of the GRF gene family of gingko is successfully cloned from gingko, and the gene presents a specific expression mode in the regeneration process of gingko callus; the overexpression of the fusion gene of GbGRF2 and GbGRF2-GbGIF2 can significantly promote the regeneration efficiency of adventitious buds in the in-vitro culture process of plants; the gene provided by the invention 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 gymnosperm.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly relates to the application of a Ginkgo biloba GbGRF2 gene and its GbGRF2-GbGIF2 fusion gene in plant tissue culture. Background Art

[0002] Ginkgo biloba, as a unique gymnosperm in China, has important economic value and scientific research significance. Under natural conditions, the reproduction of Ginkgo biloba mainly relies on traditional methods such as grafting, cutting or seedling. However, these methods have limitations such as long reproduction cycle, high cost and being easily restricted by environmental factors. Large-scale propagation of Ginkgo biloba seedlings through in vitro tissue culture technology can effectively make up for the deficiencies of traditional reproduction methods. However, during the tissue culture process, the common problems of microbial contamination and browning seriously affect the induction and differentiation efficiency of callus, resulting in the failure to successfully establish a complete in vitro regeneration system for Ginkgo biloba. Therefore, establishing an efficient in vitro regeneration and genetic transformation system has become an urgent task in the molecular breeding research of Ginkgo biloba, which will provide important technical support for solving key problems such as low regeneration efficiency and long reproduction cycle of Ginkgo biloba.

[0003] GRF developmental regulators have two highly conserved domains in the N-terminal region: the QLQ domain and the WRC domain. Among them, QLQ is a functional domain that mediates protein-protein interaction and interacts with the GRF interacting factor GIF to form a transcriptional activator. And WRC, as a unique domain of the object, has more conserved amino acid sites than QLQ. The zinc finger structure in WRC can bind to DNA. The GIF gene family is small in size but highly conserved, usually containing no more than five members. Its protein contains two key domains, SNH and QG. Among them, the SNH domain can specifically bind to the QLQ domain of the GRF protein to form a 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 is highly conserved in evolution. Research shows that GRF genes play an important role in regulating cell proliferation and differentiation, promoting plant tissue regeneration and responding to stress. In addition, some studies have shown that the single growth regulator GRF or the fusion protein of GRF and its interacting factor GIF can significantly promote the regeneration of tissue cultures of multiple species and will not cause defects in transgenic plants.

[0004] At present, it has been reported in multiple species that the transfer of GRF genes can promote tissue differentiation. Ginkgo biloba, an ancient seed plant, provides us with valuable materials for studying the system and functional evolution of plant transcription factor families. However, current research on GRF growth regulators in Ginkgo biloba is still insufficient, lacking a comprehensive and systematic discussion. Given the key role of GRF growth regulators in biological processes such as regulating plant growth and development, responding to abiotic stresses, and participating in the biosynthesis of secondary metabolites, a systematic bioinformatics analysis of the Ginkgo biloba GRF gene family and an in-depth exploration of the molecular regulatory mechanisms of GRF genes and GbGRF2-GbGIF2 fusion genes in the Ginkgo biloba tissue culture regeneration system have important theoretical significance and application value for establishing an efficient Ginkgo biloba tissue culture system and genetic transformation technology platform. Summary of the Invention

[0005] Aiming at the deficiencies of the above-mentioned prior art, the present invention aims to provide an application of Ginkgo biloba GbGRF2 gene and its GbGRF2-GbGIF2 fusion gene in plant tissue culture to promote plant tissue culture regeneration.

[0006] To solve the above problems, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a Ginkgo biloba GbGRF2 gene, whose nucleotide sequence is shown in SEQ ID No.1.

[0008] As an implementable embodiment, the Ginkgo biloba GbGRF2 gene is highly expressed in Ginkgo biloba female reproductive organs (female cones, embryos, ovules, mature seeds), stems, and roots, and is lowly expressed or not expressed in other tissues.

[0009] As an implementable embodiment, the nucleotide sequence of the full-length cloning primer of the Ginkgo biloba GbGRF2 gene is shown in SEQ ID No.5 - SEQ ID No.6; the nucleotide sequence of the qRT-PCR amplification primer of the Ginkgo biloba GbGRF2 gene is shown in SEQ ID No.9 - SEQ ID No.10.

[0010] In the second aspect, the present invention provides a fusion gene GbGRF2-GbGIF2 of the Ginkgo biloba GbGRF2 gene, wherein the nucleotide sequence of the GbGIF2 gene is shown in SEQ ID No.2.

[0011] As an implementable embodiment, the amino acid sequence encoded by the GbGIF2 gene is shown in SEQ ID NO.4; the nucleotide sequence of the full-length cloning primer of the GbGIF2 gene is shown in SEQ ID No.7 - SEQ ID No.8.

[0012] Thirdly, the present invention provides the amino acids encoded by the Ginkgo biloba GbGRF2 gene, and the sequence of the amino acids encoded by the Ginkgo biloba GbGRF2 gene is shown in SEQ ID NO.3.

[0013] Fourthly, the present invention provides the amino acids encoded by the fusion gene GbGRF2-GbGIF2, and the sequence of the amino acids encoded by the fusion gene GbGRF2-GbGIF2 is shown in SEQ ID NO.4.

[0014] Fifthly, the present invention provides a recombinant plasmid, which is the pCAMBIA1300-GbGRF2 recombinant plasmid containing the Ginkgo biloba GbGRF2 gene or the pCAMBIA1300-GbGRF2-GbGIF2 recombinant plasmid containing the fusion gene GbGRF2-GbGIF2.

[0015] Sixthly, the present invention provides a method for promoting adventitious bud regeneration during plant tissue culture regeneration. The recombinant plasmid is transformed into competent GV3101 Agrobacterium to prepare positive clones, and plant leaves are infected and cultured, and the plant callus overexpressing GbGRF2 and GbGRF2-GbGIF2 is screened and cultured.

[0016] Seventhly, the present invention provides the application of the Ginkgo biloba GbGRF2 gene or the fusion gene GbGRF2-GbGIF2 or the recombinant plasmid in plant tissue culture.

[0017] As an implementable mode, the plant is Nicotiana tabacum or Ginkgo biloba.

[0018] As an implementable mode, overexpressing the Ginkgo biloba GbGRF2 gene or the GbGRF2-GIF2 fusion gene and its vector promotes the regeneration of adventitious buds during plant tissue culture regeneration.

[0019] The beneficial effects of the present invention are as follows: The full-length coding region of the GbGRF2 gene of the Ginkgo biloba GRF gene family was successfully cloned from Ginkgo biloba, and this gene showed a specific expression pattern during the regeneration of Ginkgo biloba callus. Through transgenic functional verification experiments, it was confirmed that overexpressing the GbGRF2 and GbGRF2-GbGIF2 fusion genes can significantly promote the regeneration efficiency of adventitious buds during in vitro culture of plants. Therefore, the GbGRF2 gene and the GbGRF2-GbGIF2 fusion gene have important theoretical significance and application potential in the plant tissue regeneration regulation network and play a key role in improving the in vitro regeneration efficiency of gymnosperms. Description of the Drawings

[0020] Figure 1It is a position map of the Ginkgo biloba GbGRF gene and GbGIF on the chromosome.

[0021] Figure 2 It is an identification electrophoresis map of the overexpression vectors of pCAMBIA1300-GbGRF2 and pCAMBIA1300-GbGRF2-GbGIF2.

[0022] Figure 3 It is a map of the overexpression vectors of pCAMBIA1300-GbGRF2 and pCAMBIA1300-GbGRF2-GbGIF2.

[0023] Figure 4 It is the in vitro regeneration of tobacco transformed with GbGRF2 and GbGRF2-GbGIF2 in hormone-added medium; the scale bar is 1 cm.

[0024] Figure 5 It is the in vitro regeneration of tobacco transformed with GbGRF2 and GbGRF2-GbGIF2 in hormone-free medium; the scale bar is 1 cm.

[0025] Figure 6 It is a GFP fluorescence observation map of GbGRF2 and GbGRF2-GbGIF2 transgenic tobacco; tobacco observed under natural light (upper), tobacco explants emitting GFP fluorescence (lower), the scale bar is 1 cm.

[0026] Figure 7 It is a DNA identification map of GbGRF2 and GbGRF2-GbGIF2 transgenic tobacco; the Marker is DL5000bp.

[0027] Figure 8 It is a detection map of the gene expression levels of GbGRF2 and GbGRF2-GbGIF2 transgenic tobacco.

[0028] Figure 9 It is a map of the callus induction of Ginkgo biloba leaves transformed with GbGRF2; (A) the state of Ginkgo biloba leaves two days after infection and co-culture; (B) the state of Ginkgo biloba leaves and callus on the 14th day after transfer to callus induction medium after the end of co-culture; (C) the state of Ginkgo biloba leaves and callus on the 14th day after transfer to screening medium; the scale bar is 1 cm.

[0029] Figure 10 It is a DNA identification map of GbGRF2 and GbGRF2-GbGIF2 transgenic Ginkgo biloba; the Marker is DL5000bp.

[0030] Figure 11 It is a detection map of the gene expression levels of GbGRF2 and GbGRF2-GbGIF2 transgenic Ginkgo biloba. Specific implementation methods

[0031] The present invention will be further described in detail below in conjunction with specific embodiments.

[0032] It should be noted that these embodiments are only used to illustrate the present invention, rather than limiting the present invention. Under the premise of the concept of the present invention, simple improvements to this method all fall within the scope of protection required by the present invention.

[0033] 1. Materials and Reagents

[0034] In this embodiment, if no specific experimental method is specified, it can be carried out according to conventional methods. Such as the conditions described in "Molecular Cloning: A Laboratory Manual" by J. Sambrook et al. and "Current Protocols in Molecular Biology" by F. Ausubel et al., or according to the instructions of the product manufacturers.

[0035] The ginkgo leaves used in the experiment were the young leaves of two-month-old ginkgo seedlings, which were obtained by germinating and planting the seeds collected from a 20-year-old female ginkgo tree on the campus of Nanjing Forestry University.

[0036] The RNA extraction kit and plasmid extraction kit were purchased from OMEGA Company, and their product numbers were R6827 and D6943 respectively; the reverse transcription reagent was purchased from Monad Company, and its product number was MR05101M; the high-fidelity enzyme used for cloning PCR was purchased from Takara Biotechnology (Beijing) Co., Ltd. (TaKaRa China), and its product number was D2215; the gel extraction kit was purchased from Nanjing Novoprotein Scientific Co., Ltd., and its product number was DC301; the 2×Rapid Taq Master Mix for ordinary PCR reaction was purchased from Nanjing Novoprotein Scientific Co., Ltd., and its product number was P222; the restriction endonucleases BamHⅠ and XbaⅠ were purchased from TransGen Biotech Co., Ltd., and their product numbers were JB101 and JX101 respectively; the homologous recombination enzyme was purchased from Nanjing Novoprotein Scientific Co., Ltd., and its product number was C112; the plant expression vector was pCAMBIA1300-GFP; the Escherichia coli competent cell DH5α, the Agrobacterium competent cells GV3101 and Y2H Gold competent cells were all purchased from Beijing Tsingke Biotechnology Co., Ltd., and their product numbers were DLC301 and DLC402 respectively; MS and LB media are common media in the art, and their formulations refer to "Molecular Cloning: A Laboratory Manual" by J. Sambrook et al.

[0037] The materials, reagents, etc. used in this embodiment can all be obtained through commercial channels without special instructions.

[0038] 2. Chromosomal Localization of GbGRF2 and GbGIF2

[0039] Chromosomal localization showed that the GbGRF genes were relatively dispersed on the chromosomes. They were unevenly distributed on 7 chromosomes, with 1 GbGRF gene on each of chr2, chr3, chr5, chr6, chr7, and chr11, and 2 GbGRF genes on chr4. The members of the GbGIF gene family were located on three chromosomes, namely chr3, chr4, and chr10. The GbGRF2 and GbGIF2 genes of Ginkgo biloba were both located on chromosome chr3( Figure 1 ).

[0040] 3. Full-length cloning of GbGRF2 and GbGIF2 and construction of overexpression vectors

[0041] RNA of Ginkgo biloba was extracted 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 detected by NanoDrop 2000, and it was stored at -80 °C for later use. Reverse transcription was performed using the reverse transcription reagent from Monad Company to obtain the cDNA of Ginkgo biloba. The CDS sequence information of GbGRF2 and GbGIF2 was searched in the Ginkgo biloba whole-genome CDS file. According to the sequence information, the forward primers for cloning the full length designed by Oligo software were: 5′-ATGGATTTCTCACATAGTTC AGTG-3′ (SEQ ID No.5) and 5′-ATGGGGCTTCTCACTATCTTCA-3′ (SEQ ID No.7); and the reverse primers were: 5′-CACGAGGAGAGCTTGAATTT-3′ (SEQ ID No.6) and 5′-CTAGCTTCCTTCTTCTTCAGAGC-3′ (SEQ ID No.8). The operation steps are as follows:

[0042] (1) Using cDNA as a template, high-fidelity enzyme was used for amplification. The PCR reaction program was: 98 °C for 3 min, followed by 35 cycles, namely 98 °C for 10 s, 58 °C for 5 s, 72 °C for 1 min, and finally 72 °C for 5 min. After PCR, the products were detected by electrophoresis( Figure 2 ), and the products were recovered using a gel recovery kit. The specific operation is shown in the instruction manual.

[0043] (2) The recovered fragments were subjected to secondary PCR with homologous arm primers using the same program and then recovered. The expression vector pCAMBIA1300-GFP was double-digested with restriction enzymes BamHⅠ and XbaⅠ. The double-digestion reaction program was: incubate at 37 °C for 15 min and then terminate the reaction by heating at 80 °C for 20 min.

[0044] (3) The linearized pCAMBIA1300-GFP vector and the product after the second PCR were recombinantly ligated at 37 °C for 30 min under the action of a homologous recombinase, and after the reaction ended, it was cooled on ice.

[0045] (4) The ligation product was transferred into competent Escherichia coli DH5α cells. The transformation procedure is described in the instruction manual. Finally, the resuscitation solution was evenly spread on an LB solid medium with Kana resistance and cultured inverted at 37 °C for 16 h.

[0046] (5) After the culture ended, monoclonal colonies on the medium were picked for identification. First, a monoclonal colony was picked with a sterile toothpick and mixed evenly in 10 μL of sterile water. Then, 2 μL of the bacterial solution was taken for bacterial solution PCR identification, and the remaining 8 μL was stored at 4 °C. Bacterial solution PCR identification used 2×Rapid Taq Master Mix, and the PCR reaction program was: 95 °C for 3 min, and then 33 cycles were carried out, namely 95 °C for 15 s, 58 °C for 15 s, 72 °C for 30 s, and finally 72 °C for 5 min.

[0047] (6) The remaining 8 μL of the bacterial solution with successful PCR identification was sent to Qingke Company for sequencing to identify whether the target fragment was completely cloned successfully. Sequence alignment was performed using DNAMAN software. The bacterial solution with successful alignment was cultured at 37 °C and 200 rpm for amplification, and then plasmid extraction was carried out. The plasmid extraction procedure is described in the instruction manual. The obtained pCAMBIA1300-Gb GRF2 and pCAMBIA1300-GbGRF2-GbGIF2 plasmids ( Figure 3 ) were stored at -20 °C. The nucleotide sequences of the amplified target fragments GbGRF2 and GbGIF2 are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, with lengths of 1744 bp and 594 bp, encoding 593 and 195 amino acids respectively, and the amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively. A nucleotide linker was added between the two genes in the GbGRF2-GbGIF2 fusion gene, encoding four amino acids. The nucleotide sequence and the encoded amino acid sequence of the GbGRF2-GbGIF2 fusion gene are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0048] The nucleotide sequence of the amplified target fragment GbGRF2 is as follows (SEQ ID NO.1):

[0049]

[0050] The amino acid sequence of the amplified target fragment GbGRF2 is as follows (SEQ ID NO.3):

[0051] MDFSHSSVSVSGSLSGLGSELGDHRHRMPGLSSPGGIGILTVGDPYKQCRSASEMDLDERTRGPIKIARTDSFPCTNSRQQNTSSNNSNNNATNNNNNAIGSLLRSNSIMSDGRLANCSPTNSSANSDGGLMSNEGCVSVSEHRLMRSDSVVSAYGGGARTLSFHQPYHYKSAGLPLTMIRESATHMGGMHAIVAGSRPPFTQSQWQELEHQALIFKYMMAGVSVPSDLIIPIRKSVAALSVALSAGSYHPNMAWGSFHLGFANNTDPEPGRCRRTDGKKWRCSRDVVPDQKYCERHMHRGRHRSRKPVEGQTGASSQSHLGGPTTTTTTTANLSSNGPSSVSLAAAARNSSSNLRPSISMNNQQQHNHSGSNNSALGMNSSLLLQIASGSASPLESNKEYRYMNGGMKGGADNVDEQVFFSEVSGSSRGLGQDAMLSSVNNNGWRSSMPSKVSQVKATDQQNGSLLSYNSPQLRTLLAQDFGLMSETNQMNLPSHHQHSFRNTGFGVVESVNVGRESEGQGQHLRHFFDDWPRSRDASALSWSDVEEDRSNRSSSTTQLSISIPAMTSSDFSATNSSSPRX。

[0052] The nucleotide sequence of the amplified target fragment GbGIF2 is as follows (SEQ ID NO.2):

[0053] ATGTATCTGGCTGCCATTGCTGATTCTCAACCACAACCACCAACTGCACATACTCAGATTCCTCCAAATGCAGTGATGCAGTCTGGTGCACATTACATGCAACACCAGCAGGCACAACAACAAGTGACGCCTCAGTCCCTCATGTCATCCAGGGCTCCCATGCTGTATGCTCAGCAGCCAATCGCTGCTTTGCATCAGGCCCAGCAGCAACAGCAGCAGCAGCAACAACACCAGTCTCTTCACAGCCAACTGGGCATGAATTCTGGAGGAAGCAATGGCCTACACATGTTGCACGGTGATACAAACATGGGAGGTAATGGGCCTCTCTCATCTGGGGGCTTCCCTGACTTTGGGCGTGGTAATTCTGGCAGCTCTGGGGATGGCATGCATGCAAACAGGGGCTTGGGTGCAGATCGTGGGGCAAATAAACAGGATGGAGGAATAGGATCAGAGAATGCACATCCAGGTTCTGGTGATTGTAGGGGGAGCTCAGCTGGAGGGCCGAATGCTGACGAGTCCGAACCATCATACCTGAAAGCCTCCGAAGAAGAGGGGAACTAG。

[0054] The amino acid sequence of the amplified target fragment GbGIF2 is as follows (SEQ ID NO.4):

[0055] MYLAAIADSQPQPPTAHTQIPPNAVMQSGAHYMQHQQAQQQVTPQSLMSSRAPMLYAQQPIAALHQAQQQQQQQQQHQSLHSQLGMNSGGSNGLHMLHGDTNMGGNGPLSSGGFPDFGRGNSGSSGDGMHANRGLGADRGANKQDGGIGSENAHPGSGDCRGSSAGGPNADESEPSYLKASEEEGN。

[0056] The nucleotide sequence of the fusion gene GbGRF2 - GbGIF2 is as follows (SEQ ID NO.11):

[0057]

[0058] The amino acid sequence of the fusion gene GbGRF2-GbGIF2 is as follows (SEQ ID NO.12):

[0059] MDFSHSSVSVSGSLSGLGSELGDHRHRMPGLSSPGGIGILTVGDPYKQCRSASEMDLDERTRGPIKIARTDSFPCTNSRQQNTSSNNSNNNATNNNNNAIGSLLRSNSIMSDGRLANCSPTNSSANSDGGLMSNEGCVSVSEHRLMRSDSVVSAYGGGARTLSFHQPYHYKSAGLPLTMIRESATHMGGMHAIVAGSRPPFTQSQWQELEHQALIFKYMMAGVSVPSDLIIPIRKSVAALSVALSAGSYHPNMAWGSFHLGFANNTDPEPGRCRRTDGKKWRCSRDVVPDQKYCERHMHRGRHRSRKPVEGQTGASSQSHLGGPTTTTTTTANLSSNGPSSVSLAAAARNSSSNLRPSISMNNQQQHNHSGSNNSALGMNSSLLLQIASGSASPLESNKEYRYMNGGMKGGADNVDEQVFFSEVSGSSRGLGQDAMLSSVNNNGWRSSMPSKVSQVKATDQQNGSLLSYNSPQLRTLLAQDFGLMSETNQMNLPSHHQHSFRNTGFGVVESVNVGRESEGQGQHLRHFFDDWPRSRDASALSWSDVEEDRSNRSSSTTQLSISIPAMTSSDFSATNSSSPRXAAAAMYLAAIADSQPQPPTAHTQIPPNAVMQSGAHYMQHQQAQQQVTPQSLMSSRAPMLYAQQPIAALHQAQQQQQQQQQHQSLHSQLGMNSGGSNGLHMLHGDTNMGGNGPLSSGGFPDFGRGNSGSSGDGMHANRGLGADRGANKQDGGIGSENAHPGSGDCRGSSAGGPNADESEPSYLKASEEEGN。

[0060] 4. Effects of overexpressing GbGRF2 and GbGRF2-GbGIF2 genes on in vitro regeneration of Nicotiana benthamiana

[0061] 4.1. Transformation of the recombinant plasmids pCAMBIA1300-GbGRF2 and pCAMBIA1300-GbGRF2-GbGIF2 into Agrobacterium tumefaciens GV3101

[0062] Referring to the instruction manual of GV3101 Chemically Competent Cell of Tsingke Biotechnology Co., Ltd., the two overexpression vectors were transformed into Agrobacterium tumefaciens GV3101 competent cells by the freeze-thaw method. The specific steps are as follows:

[0063] (1) Place the Agrobacterium tumefaciens competent cells stored at -80°C on ice to melt into an ice-water mixture, add 1 μL of the recombinant plasmid, gently mix, and then let it stand on ice for 5 min, in liquid nitrogen for 5 min, in a water bath at 37°C for 5 min, and in an ice bath for 5 min in sequence.

[0064] (2) Add 700 μL of LB liquid medium without antibiotics to the centrifuge tube, mix well, and recover at 28°C and 200 rpm for 3 h.

[0065] (3) Pipette the recovered liquid and spread it evenly on the 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 colonies for identification.

[0066] Colonies that can amplify the target band are positive clones. Culture them in LB liquid medium containing 50 mg / L Kan until the OD600 is about 1, add 50% sterile glycerol, freeze them quickly in liquid nitrogen for 2 min, and store them at -80°C for later use.

[0067] 4.2. Agrobacterium-mediated genetic transformation of Nicotiana tabacum leaves

[0068] (1) Select healthy tobacco sterile seedlings as the propagation material, perform propagation operations under sterile conditions, cut the sterile seedlings into stem segments of appropriate size, inoculate them onto the new medium MS + 30 g / L sucrose + 7 g / L agar, and place the inoculated tobacco seedlings under the conditions of 2000 lux light, 24°C, and 50% humidity for cultivation.

[0069] (2) Use the leaves of sterile seedlings grown for about 26 days after propagation as the materials for genetic transformation of tobacco leaves. Cut the sterile seedling leaves into squares about 0.5 - 1 cm in size, remove the leaf margins and main veins. Immerse the leaves in the resuspension solution with OD600 = 0.6 and infect for about 10 minutes. After the timing ends, blot the liquid on the surface of the leaves with sterile filter paper, lay them flat with the front side down on the co-cultivation medium MS + 0.1 mg / L NAA + 1 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar + 100 μmol / L AS, and incubate them in the dark at 25°C for 2 days.

[0070] (3) After the co - cultivation, transfer them to the 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 culture them under light at 25℃ for 30 days.

[0071] (4) Cut the emerged buds and transfer them to a new screening medium, and continue to culture them under light at 25℃ for 30 days. Transfer the well - growing buds to the rooting medium MS + 30 g / L sucrose + 7 g / L agar + 20 mg / L Tim + 10 mg / L Hygr, and culture them under light at 25℃.

[0072] (5) After the co - cultivation, continuously observe and take pictures, record the growth status of tobacco leaves in different media and different transgenic treatments, and count the budding time and the number of buds.

[0073] 4.3 Effects of overexpression of GbGRF2 and GbGRF2 - GbGIF2 on the tissue - culture regeneration process of Nicotiana benthamiana

[0074] Agrobacterium containing GbGRF2 and GbGRF2 - GbGIF2 was used to infect Nicotiana benthamiana, and the infected leaf explants were placed in media with hormones (0.1 mg / l NAA + 1 mg / l 6 - BA) and media without hormones respectively, and the tissue - culture process was continuously observed. At the same time, tobacco infected with the resuspension without Agrobacterium was set as a control (mock) and an empty - vector control, and the number of leaf discs in each group was 40 - 60.

[0075] Observation found that in the medium with hormones, adventitious buds began to grow on the transgenic tobacco leaves on the 11th day, while adventitious buds began to appear in the empty - vector control group on the 18th day, and the number of adventitious buds grown on the transgenic tobacco leaves was much more than that of the control group ( Figure 4 ); in the medium without hormones, adventitious buds first appeared on the GbGRF2 - transformed tobacco on the 14th day, while the control did not grow adventitious buds throughout the process ( Figure 5 ). The tobacco transformation results show that the GbGRF2 and GbGRF2 - GbGIF2 genes can shorten the regeneration time of tobacco excised leaves, improve the regeneration efficiency, and can also promote the regeneration of tobacco excised leaves without exogenous hormone stimulation. It should be noted that compared with the single GbGRF gene, the promotion effect of the GbGRF - GbGIF fusion gene on the tobacco regeneration efficiency has not been significantly improved.

[0076] 4.4 Identification and expression level detection of tobacco transgenic plants

[0077] (1) The pCAMBIA1300 vector has a GFP green fluorescent protein, which can emit green fluorescence under blue light excitation. Under blue light irradiation, when observed with yellow glasses, green fluorescence can be observed at the edges and wounds of transgenic tobacco leaves, indicating transgenic positivity, and no fluorescence was observed in the control. Figure 6 )

[0078] (2) Twelve adventitious buds were selected from the gene-treated ones in the hormone-free medium, and DNA was extracted for PCR identification. The results showed that 4 and 9 samples of the plants with GRF2 and GbGRF2-GbGIF2 genes respectively had positive bands. Figure 7 )

[0079] (3) Expression level detection was performed on the transgenic samples. The qRT-PCR results showed that extremely high expression levels of the corresponding genes were detected in the GbG RF2 and GbGRF2-GbGIF2 transgenic tobacco leaves compared to the control. Figure 8 )

[0080] 5. Effects of Overexpressing GbGRF2 and GbGRF2-GbGIF2 Genes on the In Vitro Regeneration of Ginkgo biloba

[0081] 5.1 Preliminary Experiments on Ginkgo biloba Genetic Transformation

[0082] Two groups of controls were set up in the experiment: the mock control group (Ginkgo biloba leaves without any treatment) and the empty vector control group (Ginkgo biloba leaves infected with Agrobacterium tumefaciens carrying only the empty vector). The operation steps are as follows:

[0083] (1) The Ginkgo biloba leaf discs were pre-cultured for 4 days before infection, and the medium was MS + 1 mg / L NAA + 1 mg / L KT + 30 g / L sucrose + 7 g / L agar.

[0084] (2) Co-culture and infection were carried out for two days, and the medium was MS + 1 mg / L NAA + 1 mg / L KT + 30 g / L sucrose + 7 g / L agar + 100 μmol / L AS.

[0085] (3) They were transferred to the 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 cultured for 14 days. After 14 days, they were transferred to the selection 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.

[0086] (4) Seven days and 14 days after the end of co-culture, the growth of callus was observed, and the callus induction rate (the number of leaf discs with callus / the total number of leaf discs) was counted (Table 1).

[0087] The results showed that the callus induction rates of GbGRF2- and GbGRF2-GbGIF2-transformed Ginkgo biloba leaves were higher than those of the empty vector control group at 7 days and 14 days after infection, indicating that GbGRF2 or GbGRF2-GbGIF2 could promote callus induction during the tissue culture of Ginkgo biloba. However, during the infection process, Agrobacterium might cause certain damage to the young Ginkgo biloba leaves, resulting in a lower callus induction rate of the transgenic materials than that of the mock control ( Figure 9 , Table 1). Among all treatments, the GbGRF2-GbGIF2-transformed Ginkgo biloba leaves had the highest callus induction rate. At 14 days after infection, the callus state of the leaves was significantly better than that of other transgenic materials, and also better than that of the mock and empty vector controls.

[0088] Table 1 Callus induction of Ginkgo biloba leaves at 7 days and 14 days after co-culture

[0089]

[0090] 5.2 Identification and relative expression level detection of Ginkgo biloba transgenic plants

[0091] Ginkgo biloba calli cultured on the selection medium for 20 days were selected, with 24 and 27 samples of materials infected with GbGRF2 and GbGRF2-GbGIF2 respectively, 18 samples of materials infected with the empty vector, and 2 samples of the mock control group. DNA was extracted and PCR identification was carried out, obtaining 18 and 9 positive bands respectively ( Figure 10 ).

[0092] The gene expression levels of the samples with positive bands identified were detected. The steps of RNA extraction and reverse transcription were the same as in 3.1. According to the instructions of MonAmp TM Green qPCR Mix (None / Low / High ROX) (product number MQ10201), qRT-PCR was performed using ABI7500 Real time PCR Systems (Applied Biosystems). Three biological replicates and three technical replicates were set up in the experiment. The Ginkgo biloba GADPH gene was used as the internal reference gene. The upstream primer for the internal reference qRT-PCR was: 5′-ATCCACGGGAGTCTTCAC-3′, and the downstream primer was: 5′-CTCATTCACGCCAACAAC-3′; the upstream primer for the qRT-PCR of GbGRF2 designed with PrimerPremier6 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:

[0093] Table 2. qRT-PCR reaction system

[0094]

[0095] The qRT-PCR program was set as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, annealing at 58°C for 10 s, and extension at 72°C for 30 s. Among them, denaturation, annealing, and extension were set for 40 cycles. The relative quantitative expression level of GbGRF2 was determined by calculating the fluorescence quantitative results using the 2-ΔΔCT method. The qRT-PCR results showed that, compared with the empty vector control, the GbGRF2 and GbGRF2-GbGIF2 in the transgenic Ginkgo biloba leaf callus were increased ( Figure 11 ).

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described by referring to the preferred embodiments of the present invention, those of ordinary skill in the art should understand that various changes can be made in form and details without departing from the spirit and scope of the present invention defined by the appended claims.

Claims

1. A Ginkgo biloba GbGRF2 gene, characterized in that, Its nucleotide sequence is as shown in SEQ ID No.

1.

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

10.

3. The fusion gene GbGRF2-GbGIF2 of the ginkgo GbGRF2 gene according to claim 1, characterized in that, Among them, the nucleotide sequence of the GbGIF2 gene is as shown in SEQ ID No.

2.

4. The fusion gene GbGRF2-GbGIF2 according to claim 3, characterized in that, The amino acid sequence encoded by the GbGIF2 gene is as shown in SEQ ID NO.4; the nucleotide sequences of the full-length cloning primers of the GbGIF2 gene are as shown in SEQID No.7-SEQ ID No.

8.

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

3.

6. The amino acid encoded by the fusion gene GbGRF2-GbGIF2 according to claim 3.

7. A recombinant plasmid, characterized in that, It is the pCAMBIA1300-GbGRF2 recombinant plasmid containing the Ginkgo biloba GbGRF2 gene according to claim 1 or the pCAMBIA1300-GbGRF2-GbGIF2 recombinant plasmid containing the fusion gene GbGRF2-GbGIF2 according to claim 3.

8. A method for promoting adventitious bud regeneration during plant tissue culture regeneration, characterized in that, The recombinant plasmid according to claim 7 is transformed into GV3101 Agrobacterium competent cells to prepare positive clones, and the plant leaves are infected and cultured, and the plant callus overexpressing GbGRF2 or GbGRF2-GbGIF2 is screened and cultured.

9. The application of the Ginkgo biloba GbGRF2 gene according to claim 1 or the fusion gene GbGRF2-GbGIF2 according to claim 3 or the recombinant plasmid according to claim 7 in plant tissue culture.

10. The application according to claim 9, wherein, The plant is Nicotiana benthamiana or Ginkgo biloba.

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