PsASH2R gene for promoting dormancy breaking of peony buds and application of PsASH2R gene

Abstract

The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to a PsASH2R gene for promoting dormancy breaking of peony buds and application of the PsASH2R gene. The nucleotide sequence of the PsASH2R gene is as shown in SEQ ID NO. 1 (sequence identifier number 1). In-depth study proves that the dormant state of buds can be effectively relieved by overexpressing the PsASH2R gene in the peony, and germination and growth of the buds are promoted; and when the gene is silenced, the release of bud dormancy can be inhibited. The discovery clearly reveals that the PsASH2R gene plays an important role in the peony bud dormancy regulation and control process through H3K4me3 apparent modification. The invention lays a solid technical foundation for improving the dormancy habit of the peony buds and cultivating novel peony varieties through a genetic engineering means.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and genetic engineering, specifically relating to a method for promoting the breaking of dormancy in peony buds. PsASH2R Genes and their applications. Background Technology

[0002] Nucleosomes are the basic structural units of eukaryotic chromatin, consisting of an octamer composed of two molecules each of histones H2A, H2B, H3, and H4, and a DNA double helix. Histone modifications mainly occur at their N-terminus, including methylation, acetylation, and phosphorylation. These modifications alter chromatin structure, thereby regulating gene expression. Histone methylation primarily occurs at lysine and arginine residues, a process catalyzed by histone methyltransferases. H3K4me3 modification, in particular, is typically positively correlated with gene transcription levels. Histone methyltransferases usually require cofactors to catalyze H3K4 methylation in the form of a complex. In yeast, this complex is called the COMPASS complex.

[0003] In Arabidopsis thaliana, there exists an H3K4 methylation complex centered around the histone methyltransferases ATX1 / 2, supplemented by cofactors such as WDR5a, RBL, and ASH2R. Distinguished from the yeast COMPASS complex, this is termed the COMPASS-like complex. The COMPASS-like complex in Arabidopsis promotes flowering by reducing H3K4me3 methylation levels at the FLC locus. Silencing the ASH2R gene leads to a decrease in genome-wide H3K4me3 levels, thereby inducing an early flowering phenotype. In rice, ASH2-like1 and ASH2-like2 synergistically affect flowering time and plant height with VIL4. In sorghum, SbASH2B / C exhibits endosperm-specific expression, suggesting their potential involvement in the formation of an endosperm-specific COMPASS-like complex. However, to date, no studies have reported on the involvement of ASH2R in bud dormancy release. Summary of the Invention

[0004] To address the problems existing in breaking dormancy in peony buds, this invention provides a method for promoting the breaking of dormancy in peony buds. PsASH2R Genes and their applications.

[0005] The present invention specifically adopts the following technical solution: In a first aspect, the present invention provides a method for promoting the breaking of dormancy in peony buds. PsASH2R Genes, the ones mentioned PsASH2R The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] This invention represents a major breakthrough in the field of peony research, marking the first successful excavation of... PsASH2R The study investigated the genes and explored their key role in the dormancy process of peony buds. The results showed that... PsASH2R Genes play a crucial role in breaking dormancy in peony buds: overexpression PsASH2R The gene can effectively promote the breaking of dormancy in peony buds, while silencing the gene will inhibit the breaking of bud dormancy.

[0007] Secondly, the present invention provides a PsASH2R protein that promotes the breaking of dormancy in peony buds, wherein the PsASH2R protein is composed of... PsASH2R Gene encoding.

[0008] Furthermore, the amino acid sequence of the PsASH2R protein is shown in SEQ ID NO.2.

[0009] Thirdly, the present invention provides a carrier comprising the aforementioned... PsASH2R Gene.

[0010] Fourthly, the present invention provides a recombinant bacterium comprising the aforementioned vector.

[0011] Fifthly, the present invention provides the aforementioned PsASH2R The application of the gene, the PsASH2R protein, the vector, or the recombinant bacteria in promoting the breaking of dormancy in peony buds.

[0012] Sixthly, the present invention provides a method for promoting the breaking of dormancy in peony buds, comprising: In dormant peony buds, the Agrobacterium-mediated vacuum infiltration method was used to transform the target bacteria. PsASH2R Gene overexpression promotes the breaking of dormancy in Danshen buds.

[0013] Furthermore, the Agrobacterium-mediated vacuum impregnation method was used to transform the target... PsASH2R Gene overexpression specifically refers to: Immerse peony branches containing dormant buds in water containing... PsASH2R In the infection solution of the gene overexpression vector, vacuum impregnation method is used to... PsASH2R Gene overexpression.

[0014] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention provides an in-depth analysis PsASH2R The regulatory mechanism of genes in peonies has been experimentally confirmed. PsASH2RThe gene positively regulates the process of breaking peony bud dormancy by promoting H3K4me3 modification. This discovery not only reveals the molecular mechanism of peony bud dormancy regulation, but also provides a solid theoretical foundation and strong technical support for improving the dormancy-breaking behavior of peony buds and creating new germplasm, which has important scientific significance and broad application prospects. Attached Figure Description

[0015] Figure 1 This is the phylogenetic tree of PsASH2R.

[0016] Figure 2 This is a homology comparison for PsASH2R.

[0017] Figure 3 During the process of breaking dormancy in peony buds treated with low temperature and GA3 PsASH2R Gene expression patterns: A: Low temperature treatment for different numbers of days; B: GA3 treatment for different durations.

[0018] Figure 4 for PsASH2R Overexpression promotes the breaking of dormancy in peony buds. A: PsASH2R Morphological changes in shoots after gene overexpression; Bar = 1.0 cm; B: PsASH2R Relative bud growth after gene overexpression; C: PsASH2R Gene expression level analysis in overexpressed peony buds; D: PsASH2R Expression levels of marker genes released from dormancy after gene overexpression were analyzed; the significance analysis of B, C, and D was performed using the two-tailed Student's t test, with asterisks indicating significant differences, *P<0.05; **P<0.01; ***P<0.001.

[0019] Figure 5 for PsASH2R Silencing inhibits dormancy in peony buds; A: Silencing using VIGS. PsASH2R Morphological changes in the bud after gene generation; Bar = 1.0 cm; B: PsASH2R The relative growth of gene-silenced buds; C: PsASH2R Analysis of gene expression levels in its silenced peony buds; D: PsASH2R Expression levels of marker genes after gene silencing and release of dormancy were analyzed. The significance of B, C, and D was analyzed using the two-tailed Student's t test. Asterisks indicate significant differences. *P<0.05; **P<0.01; ***P<0.001.

[0020] Figure 6 For overexpression and VIGS silence PsASH2RThe changes in H3K4me3 levels in peony buds were investigated. Anti-H3K4me3 antibody was used for detection, and anti-H3 antibody was used as a control. The standardized relative amounts are shown in the numbers below the bands. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0022] The following examples involve the source, part number, and composition of materials: SteadyPure Plant RNA Extraction Kit: AG21019, Accurate Biotechnology, Hunan.

[0023] Evo M-MLV Reverse Transcription Premixed Kit: AG11728, Accurate Biotechnology, Hunan.

[0024] SYBR Green Pro Taq HS Premixed qPCR Kit II: AG11702, Accurate Biotechnology, Hunan.

[0025] The infection buffer consisted of 10 mM MgCl2, 10 mM MES, and 200 μM Acetosyringone.

[0026] Plant Histone Extraction Kit: Solarbio, R0050.

[0027] 10% SDS-PAGE: Vazyme, Nanjing.

[0028] monmethyl-histone H3-K4 antibody: A2355, ABclonal, Wuhan.

[0029] Anti-dimethyl-histone H3-K4 antibody: A2356, ABclonal, Wuhan.

[0030] Anti-trimethyl-histone H3-K4 antibody: A2357, ABclonal, Wuhan.

[0031] Anti-histone H3 antibody: A2348, ABclonal, Wuhan.

[0032] ECL Super Reagent Kit: ED0015-B, Sparkjade, Shandong.

[0033] The co-culture medium consisted of 1 / 2 MS + 200 μM acetylsyringone + 0.1 g / L activated carbon.

[0034] The buds were transferred to the growth medium consisting of MS + 200 mg / mL termethin.

[0035] Example 1: PsASH2R Clonal analysis of genes.

[0036] To identify the core components of the peony COMPASS-like complex, this embodiment used the Arabidopsis thaliana ASH2R protein sequence as the seed sequence and searched for homologous sequences in the peony genome database built by Qingdao Agricultural University. One homologous gene for ASH2R was obtained, as shown below. Figure 1 As shown, it is named PsASH2R, Genbank accession no. PX021628. PsASH2R The coding region contains 1296 bp, as shown in SEQ ID NO.1, encoding 432 amino acids, as shown in SEQ ID NO.2. The molecular weight is 47.537 kDa, and the theoretical isoelectric point is 5.28. Homology alignment analysis shows that PsASH2R contains one SPRY domain from the ASH2 family, as shown in SEQ ID NO.2. Figure 2 As shown in the figure. These results indicate that the core component ASH2R of the peony COMPASS-like complex is highly homologous and structurally conserved, suggesting that its function may be similar to that of homologous proteins in other species.

[0037]

[0038] SEQ ID NO.2: MDTLRATYRDEEEEDGLALTTTTTTTDDSPNGSPPPIATTPNTEIHTPNGFEPSNSNNLSEDETCGEDPKKSPKFSSISDEEEDEDDRPPKKQKQLSLLTAQGPASEPSELLQATPESAIIAHATPKAITSTATTTIVKKSTKKKNINVWKSTSRKGKKKGKANNHSATTEDTVLITPISRFPDKSDDAPDMKICLSKGYKAEKVELSDDRM CAGSTKGYRMVRATRGVTEGAWYFEIKVLKLGETGHTRLGWSTDKGDLQAPVGYDSHSFGYRDIDGSKIHKALREKYGEEGYKEGDVIGFYINLPEGGLYVPKPPNLVW YKGQKYVYAADAKDDPPKIVPGSELSFFKNGACQGVAFKDLFGGRYYPAASMYTLPNQPNCTVKFNFGPDFEFFPEDLNERPVPRPMVEVPYHGFDIQIENGVSNEKKT.

[0039] Example 2: PsASH2R Gene expression analysis during the dormancy-breaking process in peonies.

[0040] 1. Method.

[0041] Peonies cultivated at Qingdao Agricultural University for 4 years Paeonia suffruticosa 'Luhehong', following the method of Gao et al. (reference: Gao LQ, Niu DM, Chi TY, et al. PsRGL1 negatively regulates schilling-and gibberellin-induced dormancy release by PsF-box1-mediated targeting for proteolytic degradation in tree peony. Horticulture Research, 2023, 10: uhad044), was treated at 0–4℃ for 0, 7, 14, 21, and 28 days. After 7 days of low-temperature treatment, the plants were moved to a greenhouse at 18–22℃ in darkness for 8 hours, followed by treatment with 200 mM GA3. Terminal buds were collected at 0, 12, 24, 48, and 72 hours and stored at -80℃.

[0042] To assess dormancy status, following the method of Zhang et al. (reference: Zhang YX, Niu DM, Yuan YC, et al. PsSOC1 is involved in the gibberellin pathway to trigger cell proliferation and budburst during endodormancy release in tree peony. New Phytologist, 2024, 243:1017-1033), plants under the same treatment were cultured and observed in a greenhouse at 18-22℃ in the dark for 8 hours, and analyzed by real-time quantitative PCR. PsASH2R Expression patterns during dormancy release. Actin gene was used as an internal control. Real-time quantitative primers were designed online using NCBI (see Table 1) and synthesized by Shanghai Sangon Biotech.

[0043] RNA extraction and real-time quantitative PCR: Total RNA was extracted using the SteadyPure Plant RNA Extraction Kit. First-strand cDNA synthesis was performed using the Evo M-MLV Reverse Transcription Premix Kit. Real-time quantitative PCR analysis was performed using the SYBR Green Pro Taq HS Premixed qPCR Kit II. -ΔΔCt The method calculates relative gene expression levels.

[0044] Table 1: Primers for real-time quantitative PCR.

[0045] 2. Results.

[0046] The results show PsASH2R The temperature was increased during the first 0-14 days of low-temperature treatment, reaching its maximum value after 14 days. Figure 3 As shown in A. During the gibberellin-induced cessation of dormancy, compared with the control group, PsASH2R The transcriptional level of [the substance] increased rapidly 12 hours after GA3 treatment and then maintained a high expression level, such as [the following]. Figure 3 As shown in B.

[0047] Example 3: PsASH2R Its function in breaking the dormancy of peony buds.

[0048] 1. Method.

[0049] (1) Overexpression.

[0050] Will PsASH2R The complete coding region is inserted into the pBI121 carrier. Xba Ⅰ / SmaThe primers between site I are shown in Table 2. The recombinant vector was transformed into Agrobacterium. A.tummefaciens EHA105 cells were used, with the empty vector pBI121 as a control. Positive colonies were cultured in LB broth at 28°C on a shaker until an OD600 of 0.6–0.8 was reached. Subsequently, bacterial cells were collected using a high-speed centrifuge and resuspended in infection buffer with an OD600 of 0.8. After 3 hours of culture, peony buds that had undergone 7 days of cryogenic treatment were immersed in the infection buffer and then transformed using a vacuum infiltration method at -0.07 MPa. After infection, the buds were washed three times with distilled water, inserted into co-culture medium, and treated in the dark at 8°C for 3 days. The buds were then transferred to growth medium for further culture. The growth of the buds, including height and width, was recorded daily. After 10 days of normal culture, buds with distinct phenotypes were selected and cryopreserved in liquid nitrogen at -80°C. At least 10 peony buds were used per combination, with 3 replicates per group. Seven days after transformation, the peony buds were immediately frozen in liquid nitrogen and stored at -80°C for later use.

[0051] Table 2: PsASH2R Primers for amplifying the complete coding region.

[0052] (2) Virus-mediated gene silencing Will PsASH2R A specific fragment of approximately 300 bp from ORF was cloned into the pTRV2 vector. Xba Ⅰ / Sma Between I and II, the primers are shown in Table 3, and then the conversion is performed. A.tummefaciens In EHA105 strain, pTRV2- PsASH2R The pTRV1 combination was used to infect peony buds that had been subjected to low-temperature treatment for 10 days, with the pTRV1+pTRV2 combination serving as a control.

[0053] Table 3: PsASH2R Primers for specific fragment amplification.

[0054] Histone extraction and Western blotting: Histones were extracted from peony buds after gene overexpression and silencing using the Plant Histone Extraction Kit. Histone analysis was performed using Western blotting. Histones were separated using 12% SDS-PAGE, and then detected by Western blotting using anti-monmethyl-histone H3-K4, anti-dimethyl-histone H3-K4, anti-trimethyl-histone H3-K4, and anti-histone H3 antibodies. Goat anti-rabbit antibody was used as a secondary antibody, and the final detection was performed using the ECL Super Kit. Relative protein levels were calculated using ImageJ (https: / / imagej.net / ij / ).

[0055] RNA extraction and real-time quantitative PCR: Same as in Example 2.

[0056] 2. Results.

[0057] PsASH2R The duration of cryogenic treatment was increased from 0 days to 14 days, suggesting that these treatments may play a role in cryogenic-induced dormancy release. To investigate their function, we constructed... PsASH2R The overexpression vector was used and overexpressed in peony shoots treated with 7 days of low temperature, with the empty pBI121 vector as a control. After 3 days of dark culture at 4℃, the cells were transferred to room temperature conditions and cultured at 22℃ with 16 hours of light / 8 hours of dark. Seven days after transformation, the infection efficiency was identified using RT-qPCR, and the results showed... PsASH2R The expression level was significantly higher than that of the control group, such as Figure 4 As shown in C. PsASH2R Morphological changes in shoots after gene overexpression, such as Figure 4 As shown in A, the relative growth of positive buds was measured. Fourteen days after transformation, both the height and width of the buds were higher than the control. Figure 4 As shown in B. Furthermore, the dormancy-release marker gene... PsEBB3, PsCYCD3.1, PsCYCD3.3 and PsBG6 Transcriptional levels were significantly upregulated, such as Figure 4 As shown in D. Simultaneously, in overexpression... PsASH2R The level of H3K4me3 was detected in the buds, and the results showed that the level of H3K4me3 in the positive buds was significantly higher than that in the control group. Figure As shown.

[0058] clone ​ A silencing vector was constructed using a specific 300bp fragment of the gene, and this vector was used to infect peony buds that had been treated with low temperature for 10 days. RT-qPCR was used to detect the gene. ​ The efficiency of silence, such as ​As shown in C. At 28 days post-infection, the relative growth of both height and width of silent buds was lower than that of the control group, consistent with phenotypic changes, such as... ​ A and ​ As shown in B. Dormancy release-related genes. ​ , ​ , ​ and ​ Transcriptional levels were significantly downregulated in these silent buds, such as ​ As shown in D. Ten days after transformation, ​ The H3K4me3 level in silent buds was significantly lower than that in the control group, such as ​ As shown. In summary, ​ Promoting H3K4me3 modification positively regulates the release of dormancy in peony buds.

[0059] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A method to promote the breaking of dormancy in peony buds PsASH2R Genes, characterized by, The PsASH2R The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. A PsASH2R protein for promoting the dormancy release of peony buds, characterized in that, The PsASH2R protein is encoded by the gene of claim 1 PsASH2R gene. 3.The PsASH2R protein for promoting the bud dormancy of peony according to claim 2, wherein, The amino acid sequence of the PsASH2R protein is shown as SEQ ID NO.

2.

4. A vector, characterized in that, comprising the polypeptide of claim 1 PsASH2R gene.

5. A recombinant bacterium, characterized in that, The vector of claim 4.

6. The use of the gene of claim 1, the PsASH2R protein of claim 2, the vector of claim 4 or the recombinant bacteria of claim 5 for promoting the dormancy release of peony buds. PsASH2R The gene of claim 1, the PsASH2R protein of claim 2, the vector of claim 4 or the recombinant bacteria of claim 5 for promoting the dormancy release of peony buds.

7. A method for promoting the release of bud dormancy of Paeonia suffruticosa, characterized by, including: In the dormant bud of Paeonia suffruticosa, the Agrobacterium-mediated vacuum infiltration method was used to transform the gene into the bud to promote the dormancy release of the bud. PsASH2R Gene overexpression.

8. The method for promoting the dormancy release of peony buds according to claim 7, characterized in that, The genes were overexpressed by using Agrobacterium-mediated vacuum infiltration method PsASH2R The overexpression of the genes is specifically: The Paeonia stems with dormant buds are immersed in the infection solution containing the gene overexpression vector, and the vacuum infiltration method is used to make the gene overexpression. PsASH2R The Paeonia stems with dormant buds are immersed in the infection solution containing the gene overexpression vector, and the vacuum infiltration method is used to make the gene overexpression. PsASH2R The Paeonia stems with dormant buds are immersed in the infection solution containing the gene overexpression vector, and the vacuum infiltration method is used to make the gene

Images