Application of PaBBX28 / 29-2 protein and coding gene thereof in promoting plant dormancy breaking

Abstract

The invention relates to application of a PaBBX28 / 29-2 protein and a coding gene thereof in promoting plant dormancy breaking, and belongs to the technical field of plant genetic engineering. The amino acid sequence of the protein PaBBX28 / 29-2 is as shown in SEQ ID No. 2. Experiments prove that the PaBBX28 / 29-2 gene can promote plant dormancy release by up-regulating expression of a bud dormancy key gene PdeuFT1 for the first time, and the action mechanism of the PaBBX28 / 29-2 gene as a positive regulation factor is disclosed. The invention also provides an amino acid sequence of the PaBBX28 / 29-2 protein, a nucleic acid sequence of a gene for coding the PaBBX28 / 29-2 protein, a functional primer, an over-expression vector and a genetically engineered bacterium. The PaBBX28 / 29-2 protein can be widely applied to growth rhythm regulation and molecular improvement of garden plants, forest trees and other perennial plants.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of a PaBBX28 / 29-2 protein and its encoding gene in promoting dormancy release in plants. Background Technology

[0002] Terminal bud dormancy is a typical adaptive growth strategy and an important physiological mechanism developed by perennial plants in response to environmental stress. Terminal bud dormancy is mainly regulated by external environmental signals such as photoperiod and temperature, as well as internal factors such as hormone levels. Under unfavorable growth conditions such as low temperature and short day length, the terminal bud actively enters a dormant state to reduce metabolic levels, resist adverse environments, and ensure survival and continuation. However, when environmental conditions return to a suitable state, the speed and extent of dormancy release directly determine whether the terminal bud can germinate in a timely manner, thus affecting the plant's annual growth rhythm and overall growth performance.

[0003] Seasonal dormancy is widespread in perennial plants, especially deciduous trees. This process is not only closely related to the plant's survival during the cold season, but also largely determines the plant's economic value and ornamental effect in applications such as landscaping, forestry production, and fruit tree cultivation. For example, delayed dormancy termination at the terminal bud can lead to uneven budding and branch growth, thus reducing the plant's ornamental value and economic benefits; while premature or unstable dormancy termination can expose young tissues to environmental stresses such as late frosts.

[0004] Current research on plant dormancy regulation mainly focuses on photoperiod signals, low-temperature accumulation, and plant hormones (such as ABA (abscisic acid) and GA (gibberellin)). Some transcription factors have also been shown to play a role in dormancy induction and release. However, the molecular mechanisms of dormancy release remain unclear, limiting the development of molecular breeding and horticultural regulation techniques. Therefore, it is urgent to identify and analyze key regulatory factors to provide a theoretical basis and technical approach for the molecular improvement and application of plant dormancy. Summary of the Invention

[0005] Based on the above description, this invention provides an application of the PaBBX28 / 29-2 protein and its encoding gene in promoting dormancy release in plants, aiming to solve the problem of how to regulate the release of dormancy in plant buds.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The first objective of this invention is to provide an application of the PaBBX28 / 29-2 protein in promoting dormancy release in plants, the amino acid sequence of which is shown in SEQ ID No. 2.

[0007] A second objective of this invention is to provide the application of a gene encoding the PaBBX28 / 29-2 protein in promoting dormancy release in plants, the amino acid sequence of which is shown in SEQ ID No. 2.

[0008] In a preferred embodiment, the nucleotide sequence of the gene encoding the PaBBX28 / 29-2 protein is shown in SEQ ID No. 1.

[0009] A third objective of this invention is to provide an application of an overexpression vector in promoting dormancy release in plants, the overexpression vector containing a gene encoding the protein shown in SEQ ID No. 2.

[0010] A fourth object of the present invention is to provide the application of a genetically engineered bacterium in promoting dormancy release in plants, said genetically engineered bacterium comprising an overexpression vector of a gene encoding the protein shown in SEQ ID No. 2; or, The genome of the genetically engineered bacteria integrates a gene encoding the protein shown in SEQ ID No. 2.

[0011] The fifth objective of this invention is to provide a method for promoting dormancy breaking in plants, comprising the following steps: S10. Construct an overexpression vector containing a gene encoding the protein shown in SEQ ID No. 2; S20. The overexpression vector was introduced into plants using Agrobacterium-mediated transformation, and positive transgenic lines were screened using kanamycin.

[0012] In a preferred embodiment, step S10 includes amplifying the gene encoding the protein shown in SEQ ID No. 2, wherein the primers for gene amplification are: forward primer P1: 5'-gagagaatcaggtggttccgtgg-3'; reverse primer P2: 5'-cgtgttgttgtttgggtgagaag-3'.

[0013] In the preferred embodiment, step S10 includes the following steps: The gene encoding the protein shown in SEQ ID No. 2 was ligated into the p2301s vector using homologous recombination. The primer sequences with specific homologous arms are as follows: forward primer P3: 5'-tctagaggatccccgggtaccatgaaggaatgcgagctctgc-3', reverse primer P4: 5'-agctttcgcgagctcggtacccctcagatcggagggtttagg-3'.

[0014] In a preferred embodiment, the Agrobacterium is GV3101.

[0015] Compared with the prior art, the technical solution of this application has the following beneficial technical effects: (1) This application is the first to identify the function of the PaBBX28 / 29-2 gene of Platanus diversicolor in regulating the release of dormancy in plant apical buds, and the experiment proves that it has a clear phenotypic regulatory role in transgenic plants; (2) This application confirms that the PaBBX28 / 29-2 gene promotes dormancy release by upregulating the expression of PeduFT1, a key gene for apical bud growth, revealing its mechanism of action as a positive regulator. (3) This application provides the nucleic acid sequence of the PaBBX28 / 29-2 gene, as well as the amino acid sequence of the protein encoded by the gene, functional primers and overexpression vectors, which can be widely used for phenotypic regulation and molecular improvement of garden plants, forest trees and other perennial plants. Attached Figure Description

[0016] Figure 1 This is a comparison diagram of the dormancy release of poplar trees transformed with the PaBBX28 / 29-2 gene (#1, #2, and #3 represent three independent transgenic poplar trees) and the control group poplar trees (WT) in Example 1 of this invention; Figure 2 This is a statistical comparison of the time required for dormancy to break in poplar plants transformed with the PaBBX28 / 29-2 gene in Example 1 (#1, #2, and #3 represent three independent transgenic poplar plants, respectively) and the control group poplars (WT). Figure 3 This is a comparison of the expression levels of related dormancy-breaking genes in poplar plants transformed with the PaBBX28 / 29-2 gene in Example 1 (PaBBX28 / 29-2#1, PaBBX28 / 29-2#2, and PaBBX28 / 29-2#3 represent three independent transgenic poplar plants) and poplar plants in the control group (WT1 and WT2 represent two independent groups of wild-type poplar plants). Figure 4 This is a spectrum of the carrier p2301s used in this invention. Detailed Implementation

[0017] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.

[0018] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0019] The main components of the culture medium used in this application are as follows: LB liquid medium: 10 g / L peptone + 5 g / L yeast extract + 10 g / L NaCl + 100 mg / L kanamycin; WPM liquid culture medium: 1.9 g / L commercially available WPM powder + 20 g / L sucrose; WPM solid culture medium: 1.9 g / L commercially available WPM powder + 20 g / L sucrose + 5 g / L agar powder; CIM medium: WPM solid medium + 0.3 mg / L furfurylaminopurine + 1 mg / L 2,4-D + 50 mg / L kanamycin + 300 mg / L cephalosporin + 300 mg / L termethin.

[0020] SIM medium: WPM solid medium + 0.02 mg / L TDZ + 50 mg / L kanamycin + 300 mg / L cephalosporin + 300 mg / L termethin.

[0021] EM medium: WPM solid medium + 50 mg / L kanamycin + 300 mg / L cephalosporin.

[0022] RM medium: WPM solid medium + 50 mg / L kanamycin + 250 mg / L cephalosporin.

[0023] Example 1 Isolation and cloning of the PaBBX28 / 29-2 gene This embodiment aims to illustrate how to isolate and clone the PaBBX28 / 29-2 gene from Platanus orientalis and verify its nucleotide sequence.

[0024] The specific operation steps of this embodiment are as follows: 1. Select the lower bud tissue of *Platanus orientalis* and extract total RNA using the CTAB method. The CTAB buffer formulation is as follows: NaCl 1.4 mol / L, EDTA (ethylenediaminetetraacetic acid) 20 mmol / L, Tris-HCl 100 mmol / L, 2% (w / v) polyvinylpyrrolidone K30 (pvp) and 2% (v / v) β-mercaptoethanol.

[0025] The extraction steps are as follows: 1) CTAB extract was placed in a 65°C water bath.

[0026] 2) After grinding the sample with liquid nitrogen, add 4 mL of preheated CTAB.

[0027] 3) Add an equal volume of chloroform:isoamyl alcohol (volume ratio 24:1) to the sample, mix well, let stand, centrifuge at 10000 rpm / min for 10 min at 4℃, and repeat the step once.

[0028] 4) Take the supernatant, add 0.25 mL of 8mol / L LiCl, let stand at -20℃ for 12 hours, centrifuge at 10000 rpm / min at 4℃ for 10 min, discard the supernatant, wash the precipitate twice with 75% ethanol, and dissolve it in an appropriate amount of RNase-free water for later use.

[0029] 2. Using the extracted RNA as a template, the first strand of cDNA was synthesized using a reverse transcription kit. The reaction conditions were: 42℃ for 2 min, 37℃ for 15 min, and 85℃ for 5 s.

[0030] 3. Based on transcriptome data, the ORF region of the PaBBX28 / 29-2 gene was obtained, and the PaBBX28 / 29-1 gene was amplified from Platanus orientalis using specific primers P1 / P2. Forward primer P1: 5'-gagagaatcaggtggttccgtgg-3'; Reverse primer P2: 5'-cgtgttgttgtttgggtgagaag-3'.

[0031] The PCR amplification reaction system is as follows: 1 μL cDNA template, 0.5 μL 10 mmol / L forward primer, 0.5 μL 10 mmol / L reverse primer, 5 μL 2×Taq enzyme Mix, and 3 μL ddH2O.

[0032] The PCR amplification program was as follows: 94℃ pre-denaturation for 4 min, 94℃ for 30 s, 57℃ for 30 s, 72℃ for 30 s, 35 cycles, and 72℃ extension for 10 min.

[0033] The amplified product was purified by agarose gel electrophoresis, ligated into the pMD®18-T vector, and transformed into E. coli DH5α for positive detection. Positive clones were selected for sequencing verification, and the target sequence was obtained and named PaBBX28 / 29-2-T. Its complete sequence is shown in SEQ ID No. 1, and its encoded amino acid sequence is shown in SEQ ID No. 2.

[0034] The nucleotide sequence of the PaBBX28 / 29-2 gene is SEQ ID No. 1: atgaaggaatgcgagctctgcaattttccggcgaggatgtattgtgaatcggatcaagcgagcttgtgttgggactgtgatgcgaaggttcacaccgcgaatttccttgttgctcggcattccaggtgtctgctttgccatgtttgtcaatcgccgacgccatggaaagcttcgggatcgaagcttgggcctacggtctcggtttgcgagaggtgtgttggtagctgtgacgggagggaggagagaggtggagtggatgcggaaagtgaaggaggaagtgacgacgacgagattgatggaggcgatgatgaagatgatgaggaggaggaggaggatagcgactgtgatgatgaagaagatggagagaatcaggtggttccgtggtcttctacgccccctccgccggtcgcgagttcttcgagtggtgatgaatcttcttgtagtggtgggcgaggtgttgctaattcagcgatgtcgttttacttgaaacggatgcgtgagaatgcagatctcggttctcaagatgaaatcggctactcatcttctcacccaaacaacaacacggcgttagcagcggcatcgacctctggggcctcagccaacgacgatgccaagtattatggcttgttcaggccttccaaggtccaaaagagaaacataacgttccggtcaactcaagccgaatccacaccggcggcgctcgtctgctcgctcaagagattccagcaagacaaactctccagcgaagacgcgtccgcaacgatactcggtatctgcaaactaagcaaagaccctagagccgttgatctcttttccaccaactctcctcctaaaccctccgatctgaggtag。

[0035] Amino acid sequence SEQ ID No.2 encoded by the PaBBX28 / 29-2 gene: MKECELCNFPARMYCESDQASLCWDCDAKVHTANFLVARHSRCLLCHVCQSPTPWKASGSKLGPTVSVCERCVGSCDGREERGGVDAESEGGSDDDEIDGGDDEDDEEEEEDSDCDDEEDGENQVVPWSSTPPPPVASSSSG DESSCSGGRGVANSAMSFYLKRMRENADLGSQDEIGYSSSHPNNNTALAAASTSGASANDDAKYYGLFRPSKVQKRNITFRSTQAESTPAALVCSLKRFQQDKLSSEDASATILGICKLSKDPRAVDLFSTNSPPKPSDLR.

[0036] Example 2 Construction and transformation of PaBBX28 / 29-2 gene overexpression vector into poplar To clarify the biological function of the PaBBX28 / 29-2 gene in plants, this embodiment constructs a plant overexpression vector for this gene and introduces it into poplar trees to observe phenotypic changes in transgenic plants. The specific steps are as follows: 1. Using the PaBBX28 / 29-2-T plasmid obtained in Example 1 as a template, and p2301s (plasmid map see...) Figure 4 Using a CaMV 35S promoter as the backbone, the target gene expression was driven by the CaMV 35S promoter. Homologous recombination primers containing a kanamycin resistance selection marker were designed and synthesized, and Kpn I restriction sites were used for PCR amplification of the target gene fragment. The DNA sequences of the primer pairs are shown below. The forward homologous recombination primer P3: 5'- tctagaggatccccgggtacc atgaaggaatgcgagctctgc-3'; Homologous recombination reverse primer P4: 5'- agctttcgcgagctcggtacc cctcagatcggagggtttagg-3', where the underlined part is the sequence on the p2301s vector.

[0037] The PCR amplification system is as follows: 1 μL PaBBX28 / 29-2-T plasmid, 0.5 μL 10 mmol / L homologous recombination forward primer P3, 0.5 μL 10 mmol / L homologous recombination reverse primer P4, 5 μL 2×Taq enzyme Mix, and 3 μL H2O.

[0038] The PCR amplification program was as follows: 94℃ pre-denaturation for 4 min, 94℃ for 30 s, 57℃ for 30 s, 72℃ for 30 s, 35 cycles, and 72℃ extension for 10 min.

[0039] Simultaneously, the plant expression vector p2301s was linearized by Kpn I single-enzyme digestion. Subsequently, the digested amplification product and the linearized vector were ligated using a homologous cloning enzyme and transformed into Escherichia coli.

[0040] After antibiotic resistance screening and sequencing to identify positive clones, the successfully constructed expression vector was 35S::PaBBX28 / 29-2. This vector was then introduced into Agrobacterium GV3101 strain via electroporation for subsequent poplar transformation.

[0041] 2. This invention uses the leaf disc transformation method to genetically transform poplar trees. The specific steps are as follows: 1) Inoculate a single colony of Agrobacterium containing the target fragment onto 5 mL of LB liquid medium and incubate at 28°C and 200 r / min until OD. 600nm The value is 0.5-0.8.

[0042] 2) Take 1 mL of the above bacterial culture and transfer it to 30 mL of LB liquid medium. Incubate at 28℃ and 200 r / min until OD reaches 100%. 600nm The value was 0.5-0.8. The bacterial culture was diluted 10-fold with WPM liquid medium containing 100 μM / L acetylsuccinone and resuspended at 28℃ and 200 r / min for 1-2 h.

[0043] 3) Transformation: Take the 3rd to 5th leaves from the top of the sterile seedlings, make several cuts on the midrib with a sharp sterile blade, and then immerse them in the above resuspension for 15 min. Use sterile filter paper to remove excess bacterial solution and place them in WPM solid medium containing 100 μM / L acetylsyringone for dark culture for 2 days (with the underside of the leaves facing up).

[0044] 4) Transfer the infected leaves to selective CIM medium and incubate in the dark for at least 2 weeks. Change the medium every 2 weeks.

[0045] 5) When the callus reaches the size of a grain of rice, cut it off and transfer it to the SIM medium for selection.

[0046] 6) After the callus sprouts, place the callus in hormone-free EM medium to allow it to elongate.

[0047] 7) When the bud elongates to 1 cm, cut off the bud and place it in RM medium to cultivate roots.

[0048] 8) Take rooted positive seedlings, cut off the leaves, divide the stem into stem segments of about 2 cm, and inoculate them into WPM medium.

[0049] 9) Select about 10 healthy seedlings with good root growth from each strain for hardening-off treatment. Unscrew the cap of the tissue culture bottle and leave it for 1 day, then leave it open for 1 day, add water to the culture medium and leave it for 1 day, finally wash off the culture medium, place the seedlings directly in water for 2 days, mark them, and then plant them in the growth chamber.

[0050] Example 3 Phenotypic observation of PaBBX28 / 29-2 transgenic poplar To assess the effect of the PaBBX28 / 29-2 gene on the process of breaking apical dormancy, 35S::PaBBX28 / 29-2 transgenic poplar plants and wild-type (WT) plants that had been grown in the ground for 2.5 months were selected and subjected to short-day (SD) treatment for 14 weeks to induce dormancy. Subsequently, the plants were transferred to a low-temperature (LT) environment with 0 light / 24 h darkness and a constant temperature of 24°C for 3 weeks, and then restored to a long-day (LD) environment with 24°C, 16 h light / 8 h darkness.

[0051] The bud resuming growth and sprouting new leaves is considered bud splitting; the result is as follows Figure 1 and Figure 2 As shown, transgenic poplar plants exhibited a significant dormancy-breaking advantage, with all terminal buds sprouting within one week of restoring LD conditions. In contrast, control WT plants under the same treatment conditions did not show any bud recovery until week 7. These phenotypic results clearly demonstrate that PaBBX28 / 29-2 overexpression can significantly accelerate the dormancy-breaking process of plant terminal buds.

[0052] Example 4 Quantitative expression data analysis of PaBBX28 / 29-2 transgenic poplar To further explore the molecular mechanism of the PaBBX28 / 29-2 gene, leaves from 35S::PaBBX28 / 29-2 transgenic poplar plants treated with LT for 2 weeks and WT plants were selected for RNA extraction and real-time quantitative PCR. The expression differences of dormancy-breaking genes PdeuFT1, PdeuCEN1, and PdeuSVL were analyzed. The results are as follows: Figure 3 As shown, the expression of the dormancy-breaking promoter PdeuFT1 in transgenic plants was significantly higher than that in WT plants; while the expression of negative regulators such as PdeuCEN1 and PdeuSVL showed no significant change. These data indicate that PaBBX28 / 29-2 can activate the dormancy-breaking mechanism of apical buds by regulating the expression of the dormancy-breaking promoter PeduFT1 gene.

[0053] In summary, the technical solution of this application has the following beneficial technical effects: (1) This application is the first to identify the function of the PaBBX28 / 29-2 gene of Platanus diversicolor in regulating the release of dormancy in plant apical buds, and the experiment proves that it has a clear phenotypic regulatory role in transgenic plants; (2) This application confirms that the PaBBX28 / 29-2 gene promotes dormancy release by upregulating the expression of PdeuFT1, a key gene for apical bud growth, revealing its mechanism of action as a positive regulator. (3) This application provides the nucleic acid sequence of the PaBBX28 / 29-2 gene, as well as the amino acid sequence of the protein encoded by the gene, functional primers and overexpression vectors, which can be widely used for phenotypic regulation and molecular improvement of garden plants, forest trees and other perennial plants.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a PaBBX28 / 29-2 protein in promoting dormancy breaking in plants, characterized in that, The amino acid sequence of the PaBBX28 / 29-2 protein is shown in SEQ ID No.

2.

2. The application of a gene encoding the PaBBX28 / 29-2 protein in promoting dormancy breaking in plants, characterized in that, The amino acid sequence of the PaBBX28 / 29-2 protein is shown in SEQ ID No.

2.

3. The application according to claim 2, characterized in that, The nucleotide sequence of the gene encoding the PaBBX28 / 29-2 protein is shown in SEQ ID No.

1.

4. The application of an overexpression vector in promoting dormancy release in plants, characterized in that, The overexpression vector contains a gene encoding the protein shown in SEQ ID No.

2.

5. The application of a genetically engineered bacterium in promoting the breaking of plant dormancy, characterized in that, The genetically engineered bacteria comprises the overexpression vector as described in claim 4; or... The genome of the genetically engineered bacteria integrates a gene encoding the protein shown in SEQ ID No.

2.

6. A method for promoting dormancy breaking in plants, characterized in that, Includes the following steps: S10. Construct the overexpression vector as described in claim 4; S20. The overexpression vector was introduced into plants using Agrobacterium-mediated transformation, and positive transgenic lines were screened using kanamycin.

7. The method for promoting plant dormancy breaking according to claim 6, characterized in that, Step S10 includes amplifying the gene encoding the protein shown in SEQ ID No.

2. The primers for gene amplification are: forward primer P1: 5'-gagagaatcaggtggttccgtgg-3'; reverse primer P2: 5'-cgtgttgttgtttgggtgagaag-3'.

8. The method for promoting plant dormancy breaking according to claim 6, characterized in that, Step S10 includes the following steps: The gene encoding the protein shown in SEQ ID No. 2 was ligated into the p2301s vector using homologous recombination. The primer sequences with specific homologous arms are as follows: forward primer P3: 5'-tctagaggatccccgggtaccatgaaggaatgcgagctctgc-3', reverse primer P4: 5'-agctttcgcgagctcggtacccctcagatcggagggtttagg-3'.

9. The method for promoting plant dormancy breaking according to claim 6, characterized in that, The Agrobacterium species in question is GV3101.

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