Application of PaBBX30 protein and coding gene thereof in regulating dormancy delay of plant terminal bud

Abstract

The invention provides an application of a PaBBX30 protein and a coding gene thereof in regulating dormancy delay of a plant terminal bud, and belongs to the technical field of plant genetic engineering. The amino acid sequence of the protein PaBBX30 is as shown in SEQ ID No. 2. The method comprises the following steps: connecting a gene for coding the PaBBX30 protein to a p2301s vector by a homologous recombination method to construct an over-expression vector; the overexpression vector is introduced into a plant by adopting an agrobacterium-mediated transformation method, a positive transgenic line is screened out through kanamycin, and whether the PaBBX30 gene in the transgenic line is expressed or not is detected; the overexpression strain is subjected to short-day treatment, the dormancy performance of the overexpression strain is observed, and the terminal bud dormancy of the overexpression PaBBX30 gene strain is obviously delayed. The method is suitable for being applied to growth rhythm regulation and breeding improvement technologies of landscaping plants, fruit trees or other perennial plants.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the application of PaBBX30 protein and its encoding gene in regulating the delay of apical bud dormancy in plants. Background Technology

[0002] Plant dormancy refers to the temporary cessation of plant growth and is an important adaptive strategy for perennial plants to cope with adverse environments (such as low temperature and short day). Timely initiation of dormancy helps plants avoid winter stress, while the regulation of delayed dormancy, under certain conditions, can help extend the growing season, improve photosynthetic efficiency, enhance resource accumulation and reproductive capacity, and has potential agricultural and horticultural applications. Therefore, elucidating the molecular regulatory mechanisms of apical bud dormancy initiation and delay has potential significance for agriculture, horticulture, and greenhouse cultivation.

[0003] B-box domain transcription factors (BBXs) are a class of zinc finger-containing regulatory proteins that are widely involved in plant biological processes such as light signal transduction, temperature response, flowering regulation, and stress resistance. In recent years, studies have shown that BBX family members play core functions in various plant physiological regulations. However, current research on the role of BBX transcription factors in plant dormancy regulation is limited, especially regarding the mechanisms of dormancy initiation and delay in woody plants, where systematic research and functional validation are lacking. Existing studies have largely focused on model tree species such as poplar, primarily concentrating on the functions of genes such as MADS-box, CBF, and DAM, while the role of the BBX family in dormancy is not clearly reported.

[0004] In non-model landscaping tree species such as the sycamore, the dormancy rhythm of the terminal bud directly affects its overwintering ability, growth cycle, and horticultural value. However, current research has not clearly indicated whether and how BBX family genes participate in the regulation of sycamore terminal bud dormancy, particularly regarding the molecular basis of delayed dormancy initiation and regulation of annual growth rhythm. This deficiency limits a deeper understanding of the dormancy regulation mechanism of sycamore and restricts its potential application in molecular breeding and precision cultivation. Therefore, in-depth research on the functional roles of BBX family members in sycamore terminal bud dormancy, clarifying their regulatory pathways and biological significance, can provide a theoretical basis and technical foundation for subsequent molecular manipulation of plant terminal bud dormancy rhythm, optimization of plant growth cycles, and stress resistance. This is of great significance for promoting germplasm improvement, cultivation regulation, and the breeding of highly adaptable varieties of sycamore and other landscaping tree species. Summary of the Invention

[0005] This invention focuses on the PaBBX30 gene, a member of the BBX family of Platanus orientalis, constructs an overexpression vector for it, and introduces it into the model plant Populus to analyze its function in regulating the dormancy process of apical buds. It was found that heterologous overexpression of the PaBBX30 gene can significantly delay the time when the plant's apical buds enter dormancy.

[0006] The first objective of this invention is to provide the application of a protein with an amino acid sequence as shown in SEQ ID No. 2 in regulating the delay of dormancy in plant apical buds.

[0007] A second objective of this invention is to provide the application of a gene encoding the protein shown in SEQ ID No. 2 in regulating the delay of dormancy in plant apical buds.

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

[0009] A third objective of this invention is to provide an application of an overexpression vector in regulating delayed dormancy of plant apical buds, wherein the overexpression vector contains a gene encoding the protein shown in SEQ ID No. 2.

[0010] A fourth objective of this invention is to provide an application of a genetically engineered bacterium in regulating delayed dormancy of plant apical buds, wherein the genetically engineered bacterium comprises an overexpression vector, the overexpression vector comprising a gene encoding the protein shown in SEQ ID No. 2; or the genome of the genetically engineered bacterium 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 regulating the delayed dormancy of plant apical buds, comprising the following steps: constructing an overexpression vector, wherein the overexpression vector contains a gene encoding the protein shown in SEQ ID No. 2; introducing the overexpression vector into plants using Agrobacterium-mediated transformation; and screening for positive transgenic lines using kanamycin.

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

[0013] In a preferred embodiment, the method for constructing the overexpression vector includes the following steps: ligating the gene encoding the protein shown in SEQ ID No. 2 into the p2301s vector using homologous recombination; the primer sequences with specific homologous arms are as follows: forward primer P5: 5'-tctagaggatccccgggtaccatgtgcagaggaagggatgaag-3', reverse primer P6: 5'-agctttcgcgagctcggtaccaagaaacagaaaaggccacttc-3'.

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

[0015] In a preferred embodiment, the plant is a poplar.

[0016] The technical solution of the present invention has the following beneficial effects: (1) This invention, through stable transformation of model plants and combined with phenotypic and molecular detection, clarified that the PaBBX30 gene can significantly delay the dormancy initiation time of plant apical buds; it revealed the role of the PaBBX30 gene as a potential negative regulator in the dormancy initiation process of plant apical buds, and provided new theoretical support for understanding the function of the BBX family in the seasonal growth regulation of woody plants.

[0017] (2) This invention provides a complete breeding tool, including the nucleotide sequence of the PaBBX30 gene and its encoded protein, functional primers, and expression vectors. It has good operability and reproducibility and is suitable for application in the phenotypic regulation and breeding improvement technology of garden plants, fruit trees or other perennial plants. Attached Figure Description

[0018] Figure 1 Phenotypic diagrams of wild-type (WT) poplar plants and poplar plants with PaBBX30 gene overexpression; Figure 2 This is a statistical graph showing the dormancy time of apical buds in wild-type (WT) poplar plants and poplar plants overexpressing the PaBBX30 gene after short-day (SD) treatment. In the graph, WT represents wild-type poplar plants, #1 represents line 1 of the 35S::BBX30 transgenic poplar, #3 represents line 2 of the 35S::BBX30 transgenic poplar, and #4 represents line 3 of the 35S::BBX30 transgenic poplar. Figure 3 The figure shows the results of qRT-PCR detection of the expression levels of genes related to dormancy pathways in wild-type (WT) poplar plants and poplar plants overexpressing the PaBBX30 gene (PaBBX30-OE). Figure 4 This is a spectrum of the p2301s vector used in this invention. Detailed Implementation

[0019] 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.

[0020] Example 1 1. Isolation and cloning of the PaBBX30 gene 1.1 Extraction of total RNA: Mixed buds of Platanus orientalis were collected and total RNA was extracted using the modified CTAB (hexadecyltrimethylammonium bromide) method. The components of the CTAB extraction solution were as follows: 1.4 mol / L NaCl, 20 mmol / L EDTA, 100 mmol / L Tris-HCl, 2% (w / v) polyvinylpyrrolidone (PVP-K30), and 2% (v / v) β-mercaptoethanol. The specific extraction steps were as follows: (1) After grinding the mixed bud tissue of Platanus orientalis with liquid nitrogen, 4 mL of preheated (65℃) CTAB extraction solution was immediately added and the mixture was placed in a water bath at 65℃ for 5 minutes. (2) An equal volume of a mixture of chloroform and isoamyl alcohol (the volume ratio of chloroform to isoamyl alcohol was 24:1) was added and mixed well. After standing for 5 minutes, the mixture was centrifuged at 10,000 rpm for 10 minutes at 4℃. (3) The supernatant was collected and step (2) was repeated once. (4) Take the supernatant again, add 8 mol / L LiCl solution accounting for 1 / 3 of the total volume of the supernatant, precipitate at -20℃ for 12 hours, centrifuge at 10000 rpm at 4℃, discard the supernatant, wash twice with 75% (v / v) ethanol, dissolve the extracted total RNA in RNase-free water for later use.

[0021] 1.2. cDNA Synthesis: Using the total RNA extracted in step 1.1 as a template, first-strand cDNA was synthesized by reverse transcription using the PrimeScript Reverse Transcriptase Kit (R423-01) from Nanjing Novizan Biotechnology Co., Ltd. The experimental procedures were performed according to the kit's instructions. The reaction conditions were: 42℃ for 2 min, 37℃ for 15 min, and 85℃ for 5 s.

[0022] 1.3 Gene Amplification: Specific primers P1 and P2 were designed based on transcriptome sequencing information to amplify the PaBBX30 gene fragment by PCR. Forward primer P1: 5'-ccacatcttctgccccgttc-3'; Reverse primer P2: 5'-cggctgtagggtctttgctc-3'. The PCR amplification reaction system was 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. The PCR amplification program was as follows: 94℃ pre-denaturation for 4 min; 35 cycles of 94℃ for 30 s, 56℃ for 30 s, and 72℃ for 30 s; extension at 72℃ for 10 min.

[0023] 1.4 Purification and Sequence Analysis: After PCR amplification in step 1.3, the products were detected by 1% agarose gel electrophoresis and then purified using the MolPure® Fast Gel Extraction Kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 19101ES70). The purified products were then ligated into pMD... ® The 18-T vector (purchased from Takara) was used to recombinantly transform *E. coli* DH5α. The recombinant vector PaBBX30-T was transformed into *E. coli* DH5α using a heat shock method. The bacterial culture was spread evenly on LB agar (main components: 10 g / L tryptone + 5 g / L yeast extract + 10 g / L sodium chloride + 15 g / L agar) containing 100 mg / L ampicillin and incubated at 37°C. After colony growth, positive single colonies were selected for sequencing. Positive clones were sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the nucleotide sequence of the PaBBX30 gene is shown in SEQ ID No. 1. The amino acid sequence encoded by this gene is shown in SEQ ID No. 2.

[0024] Nucleotide sequence SEQ ID of PaBBX30 gene No.1: atgtgcagaggaagggatgaagagaataaacaaagcggacttagccagaatgaggtcccaatggaggtgggtttgggccaccatgaaaccatg gccttggtgttgtgcgaactctgcagctcacaagcttcattgtattgtcaagctgatgatgcctatctatgtagaagatgtgacaaacaggtccatgg agcaaatttcttggcactcaggcacattagatgcttgctatgtgatacatgccaaaatctgacagaaagatacttgattggaacttcacttgaattggtgattccgaccatcacgagttggacagagaggtgtaatcccaaggtgactgtgaagtgctcaagaatgcagaagaagtggccttttctgtttctttaa.

[0025] The amino acid sequence encoded by the PaBBX30 gene is SEQ ID No. 2: MCRGRDEENKQSGLSQNEVPMEVGLGHHETMALVLCELCSSQASLYCQADDAYLCRRCDKQVHGANFLALRHIRCLLCDTCQNLTERYLIGTSLELVIPTITSWTERCNPKVTVKCSRMQKKWPFLFL.

[0026] 2. Construction and genetic transformation of PaBBX30 gene overexpression vector 2.1 Construction of overexpression vectors Using the PaBBX30-T positive clone obtained in step 1 as a template, based on the nucleotide sequence of the PaBBX30 gene and the p2301s vector (vector map as shown in Figure 1), the following steps were performed. Figure 4 As shown, the vector contains the nucleotide sequence of the 35S promoter and the kanamycin resistance gene. Homologous recombination primer pairs P5 and P6 containing the Kpn I restriction site were designed (the underlined part of the primer sequence is the sequence on the p2301s vector). The PaBBX30 gene fragment was amplified by PCR.

[0027] Homologous recombination forward primer P5: 5'- tctagaggatccccgggtacc atgtgcagaggaagggatgaag-3'; Homologous recombination reverse primer P6: 5'- agctttcgcgagctcggtacc aagaaacagaaaaggccacttc-3'.

[0028] The PCR reaction system is as follows: 1 μL PaBBX30-T positive clone, 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.

[0029] The PCR amplification program is as follows: 94℃ pre-denaturation for 4 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s, for 35 cycles; 72℃ extension for 10 min.

[0030] The recombinant plasmid p2301s-PaBBX30 was constructed by ligating the PCR amplification product after Kpn I digestion into the p2301s vector linearized by Kpn I using the ClonExpress II One Step Cloning Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: C112-01).

[0031] 2.2 Recombinant plasmid amplification and extraction The recombinant plasmid p2301s-PaBBX30 obtained in step 2.1 was transformed into DH5α competent Escherichia coli. The transformation procedure was the same as in step 1.4. Positive clones were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0032] Recombinant plasmids were extracted from correctly sequenced clones using a high-purity plasmid DNA mini-extraction kit (purchased from Beijing Qingke Biotechnology Co., Ltd., catalog number: TSP502-200).

[0033] 2.3 Constructing genetically engineered bacteria The recombinant plasmid extracted in step 2.2 was introduced into Agrobacterium GV3101 via electroporation. Single clones were identified by PCR, using the same primers, reaction system, and amplification procedure as in step 2.2. The amplified products were detected by 1% agarose gel electrophoresis; colonies showing a target band near the 400 bp marker were considered positive clones.

[0034] 2.4 Genetic Transformation 2.4.1 Take 10 mL of the colonies identified as positive by PCR in step 2.3 and add them to LB liquid medium (the main components of the medium are: 10 g / L tryptone + 5 g / L yeast extract + 10 g / L sodium chloride), and incubate at 28°C until OD500. 600nm =0.5~0.8. Dilute the bacterial culture 10 times with WPM liquid medium containing 100 μmol / L acetylsyleugenol (main components: 1.9 g / L WPM powder + 20 g / L sucrose), and resuspend and culture for 1~2 h.

[0035] 2.4.2 After making incisions on the midrib of the 3rd to 5th leaves of the sterile poplar seedlings with a sterile scalpel, infect them with the bacterial solution obtained in step 2.4.1 for 15 min. Then, incubate them in the dark for 2 days in WPM solid medium (main components: 1.9 g / L WPM powder + 20 g / L sucrose + 5 g / L agar) containing 100 μm / L acetylsyringone.

[0036] 2.4.3 Transfer to CIM callus induction medium containing screening agents (main components: WPM + 0.3 mg / L furfurylaminopurine + 1 mg / L 2,4-dichlorophenoxyacetic acid + 50 mg / L kanamycin + 300 mg / L cephalosporin + 300 mg / L termethin) and culture in the dark; change the medium every 2 weeks.

[0037] 2.4.4 After small callus formation, transfer to SIM bud induction medium (main components: WPM + 0.02 mg / L phenylthiazolidinyl urea + 50 mg / L kanamycin + 300 mg / L cephalosporin + 300 mg / L termethin).

[0038] 2.4.5 After the shoots emerge, they are transferred to hormone-free EM medium (main components: WPM + 50 mg / L kanamycin + 300 mg / L cephalosporin) for elongation culture.

[0039] 2.4.6. After the shoots grow to more than 1 cm, transfer them to RM rooting medium (main components: WPM + 50 mg / L kanamycin + 250 mg / L cephalosporin) and culture until roots develop.

[0040] 2.4.7 After acclimatization treatment, the rooted seedlings were transplanted into the soil to obtain 35S::PaBBX30 overexpressing plants for subsequent phenotypic and molecular detection.

[0041] 3. Phenotypic observation of poplar trees overexpressing the PaBBX30 gene To verify the function of the PaBBX30 gene in regulating the dormancy process of poplar, transgenic poplar plants grown in soil for 2.5 months were treated under short-day conditions (24℃, photoperiod of 8h light / 16h darkness). The time from the start of short-day treatment until the apical meristem stopped growing, formed dormant buds, and no longer produced new leaves was recorded to assess the dormancy period.

[0042] like Figure 1 and Figure 2As shown, the observation results indicate that the dormancy initiation time of PaBBX30 gene-overexpressing poplar plants was significantly later than that of the control wild-type (WT) plants. In the fourth week of short-day treatment, WT plants began to form dormant buds, and all buds were formed by the fifth week. In contrast, 35S::PaBBX30-overexpressing plants did not begin to form dormant buds until the sixth week, and all buds were formed by the eighth week. These results demonstrate that PaBBX30 gene overexpression can significantly delay the dormancy initiation time in poplars.

[0043] 4. Transcriptome data analysis of poplar trees overexpressing the PaBBX30 gene To further elucidate the molecular mechanism by which the PaBBX30 gene regulates dormancy release in poplar, leaves from 35S::PaBBX30 transgenic plants and WT plants treated for 2 weeks under short-day conditions (24℃, 8h light / 16h dark) were collected. RNA was extracted (using the same method as in step 1.1) and reverse transcribed to obtain cDNA. Real-time quantitative PCR (qRT-PCR) was used to detect the expression levels of genes involved in key dormancy-related pathways, focusing on changes in genes related to the gibberellin (GA) signaling pathway, PedFT2, and PedSVL genes. qRT-PCR was performed using a highly sensitive dye-based quantitative PCR kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number Q331). The amplification reaction system and procedure followed the kit instructions. The PdeUBQL gene was used as an internal control gene for poplar. 2 −ΔΔCT The expression levels of relevant genes in qRT-PCR were calculated using a method that was repeated three times for each dataset.

[0044] The primers for qRT-PCR amplification of the PdeSVL gene are as follows: Forward primer PdeSVL-F: tgagagactcaaacagcaagtgg; Reverse primer PdeSVL-R: actgcccttcctcgtaaccaac.

[0045] The primers for qRT-PCR amplification of the PdeFT2 gene are as follows: Forward primer PdeFT2-F: aactttgggcaagaggttgtg; Reverse primer PdeFT2-R: tcatggtctccttccaccggagccac.

[0046] The primers for qRT-PCR amplification of the PdeGA20OX2 gene are as follows: Forward primer PdeGA20OX2-F: tcgtccagatgccctagttgt; Reverse primer PdeGA20OX2-R: ttctctcgctgtccctgttca.

[0047] The primers for qRT-PCR amplification of the PdeGA20X1B gene are as follows: Forward primer PdeGA20X1B-F: ccacagagttcatttggccca; Reverse primer PdeGA20X1B-R: cagcctaacaagctcagccg.

[0048] The primers for qRT-PCR amplification of the PdeGA2OX8 gene are as follows: Forward primer PdeGA2OX8-F: gacccacctttcctagagacct; Reverse primer PdeGA2OX8-R: gaaagaacccccactcttgtga.

[0049] The primers for qRT-PCR amplification of the PdeUBQL gene are as follows: Forward primer PdeUBQL-F: gcaagatggatgccactacc; Reverse primer PdeUBQL-R: agtgggagacgaaggggct.

[0050] qRT-PCR test results are as follows Figure 3 As shown, Figure 3 The "**" indicates a statistically significant difference (P < 0.01). The results showed that under short-day conditions, the expression of GA synthesis-related genes PdeGA20X1B and PdeGA20OX2, GA degradation-related genes PdeGA2OX8 and PdeFT2, and other genes in transgenic plants did not change significantly, while the expression of the dormancy-promoting factor PdeSVL was significantly reduced.

[0051] In conclusion, overexpression of the PaBBX30 gene in plants may inhibit the accumulation of the dormancy-promoting factor PdeSVL in transgenic plants, thereby delaying dormancy.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Various modifications and variations can be made to the present invention by any person skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection of this invention and the content of the specification should be included within the scope of protection of the present invention.

Claims

1. An application of PaBBX30 protein in regulating delayed dormancy of plant apical buds, characterized in that, The amino acid sequence of the PaBBX30 protein is shown in SEQ ID No.

2.

2. Application of the gene encoding the protein shown in SEQ ID No. 2 in regulating delayed dormancy of plant apical buds.

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

1.

4. The application of an overexpression vector in regulating delayed dormancy of plant apical buds, 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 regulating the delay of dormancy in plant apical buds, 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 regulating the delay of dormancy in plant apical buds, characterized in that, The process includes the following steps: constructing the overexpression vector as described in claim 4; introducing the overexpression vector into plants using Agrobacterium-mediated transformation; and screening for positive transgenic lines using kanamycin.

7. The method according to claim 6, characterized in that, The construction of the overexpression vector includes the step of amplifying the gene encoding the protein shown in SEQ ID No.

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

8. The method according to claim 6, characterized in that, The method for constructing the overexpression vector includes the following steps: ligating the gene encoding the protein shown in SEQ ID No. 2 into the p2301s vector using homologous recombination; the primer sequences with specific homologous arms are as follows: forward primer P5: 5'-tctagaggatccccgggtaccatgtgcagaggaagggatgaag-3', reverse primer P6: 5'-agctttcgcgagctcggtaccaagaaacagaaaaggccacttc-3'.

9. The method according to claim 6, characterized in that, The Agrobacterium species in question is GV3101.

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