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

Abstract

The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to a PsRBL gene for promoting dormancy breaking of peony buds and application of the PsRBL gene. The nucleotide sequence of the PsRBL gene is as shown in SEQ ID NO. 1. In-depth study proves that overexpression of the PsRBL gene in peony can effectively promote release of a bud dormancy state and promote germination and growth of buds; and when the gene is silenced, the release of bud dormancy can be inhibited. The discovery clearly reveals that the PsRBL 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] The application belongs to the field of molecular biology and genetic engineering technology, and particularly relates to a gene for promoting bud dormancy release of Paeonia suffruticosa PsRBL and application thereof. BACKGROUND

[0002] In eukaryotes, the basic unit of chromatin is nucleosome, which is assembled by DNA double helix and histone octamer, including two molecules of H2A, H2B, H3 and H4, which are accompanied by various epigenetic modifications, including histone modification, DNA methylation, non-coding RNA and chromatin remodeling, etc., which regulate gene expression and play an important role in various physiological processes. Among them, histone modification often occurs at the N-terminus, including methylation, acetylation, phosphorylation, etc., which regulates gene expression by changing chromatin structure. Histone methylation modification mainly occurs on lysine residues and arginine residues, which is catalyzed by histone methyltransferase, and H3K4me3 is usually positively correlated with gene transcription level. Histone methyltransferase usually binds to cofactors in the form of a complex to catalyze H3K4 methylation, which is called COMPASS complex, the full name of English is Complex Proteins Associated with Set1, and the reference is:

[0003] Takahashi Y, Westfield GH, Oleskie AN, et al. Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human. Proceedings of the National Academy of Sciences, USA, 2011, 108:20526-20531. There is a H3K4 methylation complex in Arabidopsis thaliana, which takes histone methyltransferase ATX1 / 2 as the core, WDR5a, RBL, ASH2R as the cofactor, which is different from the COMPASS complex in yeast, and is called COMPASS-like complex. As one of the core components, RBL has fewer reports on directly regulating gene expression and related physiological processes. Jiang et al. found that RBL directly regulates the expression of genes related to the response to abscisic acid and the response to drought stress in Arabidopsis thaliana RbBP5Gene silencing leads to the decrease of H3K4me3 level at genome level, which in turn causes early flowering phenotype, reference: Jiang D, Kong NC, Gu X, et al. Arabidopsis COMPASS-like complexes mediate histone H3 lysine-4 trimethylation to control floral transition and plant development. PLoS Genetics, 2011, 7: e1001330. However, whether the components of the Paeonia suffruticosa COMPASS-like complex are involved in the release of bud dormancy has not been verified. PsRBL Whether the components of the Paeonia suffruticosa COMPASS-like complex are involved in the release of bud dormancy has not been verified. SUMMARY

[0004] In order to solve the problems existing in the release of Paeonia suffruticosa bud dormancy, the application provides a Paeonia suffruticosa bud dormancy release promoting PsRBL gene and application thereof.

[0005] The application specifically adopts the following technical scheme: In a first aspect, the application provides a Paeonia suffruticosa bud dormancy release promoting PsRBL gene, wherein the PsRBL nucleotide sequence of the gene is shown as SEQ ID NO. 1.

[0006] In a second aspect, the application provides a Paeonia suffruticosa bud dormancy release promoting PsRBL protein, wherein the PsRBL protein is encoded by the PsRBL gene.

[0007] Further, the amino acid sequence of the PsRBL protein is shown as SEQ ID NO. 2.

[0008] In a third aspect, the application provides a vector comprising the PsRBL gene.

[0009] In a fourth aspect, the application provides a recombinant bacterium comprising the vector.

[0010] In a fifth aspect, the application provides the PsRBL gene, the PsRBL protein, the vector or the recombinant bacterium for use in promoting the release of Paeonia suffruticosa bud dormancy.

[0011] In a sixth aspect, the application provides a method for promoting the release of Paeonia suffruticosa bud dormancy, comprising: In the Paeonia suffruticosa dormant bud, the Agrobacterium-mediated vacuum infiltration method is used to transform the PsRBL gene to overexpress, so as to promote the release of Paeonia suffruticosa bud dormancy.

[0012] Further, the Agrobacterium-mediated vacuum infiltration method is used to transform the PsRBL The gene overexpression is specifically as follows: The branch of the peony containing the dormant bud is immersed into the infection solution containing the gene overexpression vector, and the vacuum infiltration method is used to make the PsRBL gene overexpressed. PsRBL

[0013] Compared with the prior art, the beneficial effects of the present application at least include: The present application has made a major breakthrough in the peony research field, and successfully excavates the PsRBL gene for the first time, and deeply explores the key role of the gene in the bud dormancy process of the peony. PsRBL The research results show that the PsRBL gene plays a crucial role in the bud dormancy mechanism of the peony: overexpression of the PsRBL gene can effectively promote the release of the bud dormancy state of the peony, and silencing the gene can inhibit the release of the bud dormancy. Further, the present application deeply analyzes the regulation mechanism of the PsRBL gene in the peony, and proves by experiments that the gene regulates the release process of the peony bud dormancy in a positive way by promoting H3K4me3 modification. This discovery not only reveals the molecular mechanism of the peony bud dormancy regulation, but also provides a solid theoretical basis and strong technical support for the improvement of the peony bud dormancy release habit and the creation of new germplasm, and has important scientific significance and broad application prospect.

[0014] Figure 1 is the phylogenetic tree of PsRBL.

[0015] Figure 2 is the homology alignment of PsRBL.

[0016] Figure 3 is the expression pattern of the PsRBL gene in the release process of the peony bud dormancy under low temperature and GA3 treatment, A: different days of low temperature treatment, B: different times of GA3 treatment.

[0017] Figure 4 is the PsRBL overexpression promoting the release of the peony bud dormancy, A: PsRBL morphological changes of the bud after overexpression of the PsRBL gene; Bar=1.0cm; B: PsRBL relative growth amount of the bud after overexpression of the PsRBL ​Relative expression analysis of dormancy release marker genes after gene overexpression; B, C, D significant analysis using two-tailed Student's-t test, asterisks indicate significant differences, *P<0.05; **P<0.01; ***P<0.001.

[0018] Figure 5 For PsRBL Inhibition of dormancy release in Paeonia suffruticosa buds after silencing; A: morphological changes of buds after silencing using VIGS PsRBL Buds after gene silencing; Bar = 1.0 cm; B: PsRBL Relative growth of buds after gene silencing; C: PsRBL Relative expression analysis of genes in Paeonia suffruticosa buds after their silencing; D: PsRBL Relative expression analysis of dormancy release marker genes after gene silencing; B, C, D significant analysis using two-tailed Student's-t test, asterisks indicate significant differences, *P<0.05; **P<0.01; ***P<0.001.

[0019] Figure 6 For overexpression and VIGS silencing PsRBL Changes in H3K4me3 levels in Paeonia suffruticosa buds after silencing, anti-H3K4me3 antibody was used for detection, anti-H3 antibody was used for control; Standardized relative amount as shown by the number under the band. DETAILED DESCRIPTION

[0020] The present application will be described in detail below in conjunction with the accompanying drawings and specific examples, but should not be understood as limiting the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0021] The materials, catalog numbers, and compositions involved in the following examples are as follows: SteadyPure Plant RNA Extraction Kit: AG21019, Accurate Biotechnology, Hunan.

[0022] Evo M-MLV Reverse Transcription Premix Kit: AG11728, Accurate Biotechnology, Hunan.

[0023] SYBR Green Pro Taq HS Premix qPCR Kit II: AG11702, Accurate Biotechnology, Hunan.

[0024] The infection buffer consisted of 10 mM MgCl2, 10 mM MES, and 200 μM Acetosyringone.

[0025] Plant Histone Extraction Kit: Solarbio, R0050.

[0026] 10% SDS-PAGE: Vazyme, Nanjing.

[0027] monmethyl-histone H3-K4 antibody: A2355, ABclonal, Wuhan.

[0028] Anti-dimethyl-histone H3-K4 antibody: A2356, ABclonal, Wuhan.

[0029] Anti-trimethyl-histone H3-K4 antibody: A2357, ABclonal, Wuhan.

[0030] Anti-histone H3 antibody: A2348, ABclonal, Wuhan.

[0031] ECL Super Reagent Kit: ED0015-B, Sparkjade, Shandong.

[0032] The co-culture medium consisted of 1 / 2 MS + 200 μM acetylsyringone + 0.1 g / L activated carbon.

[0033] The buds were transferred to the growth medium consisting of MS + 200 mg / mL termethin.

[0034] Example 1: PsRBL Clonal analysis of genes.

[0035] To identify the core components of the peony COMPASS-like complex, this embodiment used the Arabidopsis thaliana RBL protein sequence as the seed sequence and searched for homologous sequences in the peony genome database built by Qingdao Agricultural University. Only one homologous gene of RBL was obtained. Figure 1 As shown, it is named PsRBL, Genbank accession no. PX021627. PsRBL The coding region contains 1650 bp, as shown in SEQ ID NO.1, encoding 550 amino acids, as shown in SEQ ID NO.2. The molecular weight is 60.491 kDa, and the theoretical isoelectric point is 4.62. Homology alignment analysis shows that PsRBL contains five WD40 repeating domains of the RBBP5 family, such as... Figure 2The results showed that the core component of the COMPASS-like complex, PsRBL, has high homology and structural conservation, suggesting that its function might be similar to that of the homologous proteins in other species.

[0036]

[0037] SEQ ID NO. 2: MNAPIIDPLQGDFPEVIEEYLEHGVMKCIAFNRRGTLLAAGCSDGSCIVWDFETRGIAKELRDKDTVAAITSVCWSKYGHRILVSAADKSLTLWDVVTGTKISRTTLQQTPLQARLHPGSSTPSLCLVCPLSSAPMIVDLNTGTTTAIPVTVSDTNTGLAPPLRNGSPSITPTAACFNKYGDLVYVANSKGEILVIDHKNMKVRAVVSSQGGAVIKNIIFSRNGQHLLTNSNDRTIRIYENLLPLKDSLKAINELNETMDDLDRVEKLKSVGLKCLTLFREFQDSITKIHWKAPCFSGDGEWVIGGSASKGEHKIYIWDRAGHLVKILEGPKEALIDLAWHPVRPIVVSVSLTGLAYIWAKDYTENWSAFAPDFKELEENEEYIEREDEFDLIPETEKVKESDINEDDELDIETVEKDSDFSDSDMSQEELCFLPAVPCPDVNEQQDKFGGSSSKLVDSESAFSEEGGLQNGQVNHASSPAEEDTEETPLKRRRKPSEKALELQAEKGRRPASLKMKSSGKSSKVKSKSSVDQDSSICVFADDVIDEFN.

[0038] Example 2: PsRBL Expression analysis of genes during dormancy release of Paeonia suffruticosa.

[0039] 1. Method.

[0040] Four-year-old Paeonia suffruticosa were cultivated at Qingdao Agricultural University Paeonia suffruticosa'Luhehong', according to the method of Gao et al. Reference: Gao LQ, Niu DM, Chi TY, et al. PsRGL1 negatively regulates chilling- and gibberellin-induced dormancy release by PsF-box1-mediated targeting for proteolytic degradation in tree peony. Horticulture Research, 2023, 10: uhad044, were treated at 0-4 °C for 0, 7, 14, 21 and 28 days, respectively. After 7 days of low temperature treatment, the plants were moved to a greenhouse (18-22 °C, 8 hours of darkness), and then treated with 200 mM GA3. After 0, 12, 24, 48, 72 hours, the terminal buds were collected and stored at -80 °C.

[0041] The dormancy state was evaluated according to 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. Specifically, the same treated plants were cultured in a greenhouse (18-22 °C, 8 hours of darkness) to observe the morphology, and the expression of PsRGL1 was analyzed by real-time quantitative PCR PsRBL Expression pattern during dormancy release induced by low temperature and exogenous GA3. The actin gene actin was used as an internal reference. Real-time quantitative primers were designed online using NCBI and synthesized by Shanghai Biosciences.

[0042] RNA extraction and real-time quantitative PCR: Total RNA was extracted using the SteadyPure Plant RNA Extraction Kit. First-strand cDNA synthesis used the Evo M-MLV Reverse Transcription Premix Kit. Real-time quantitative PCR analysis was performed using the SYBR Green Pro Taq HS Premix qPCR Kit II. The 2 -ΔΔCt Method for calculating relative gene expression level.

[0043] Table 1: Real-time quantitative PCR primers.

[0044] 2. Results.

[0045] The results show PsRBL 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, PsRBL The transcriptional level of [the substance] increased rapidly 24 hours after GA3 treatment and then maintained a high expression level, such as [the following]. Figure 3 As shown in B.

[0046] Example 3: PsRBL Its function in breaking the dormancy of peony buds.

[0047] 1. Method.

[0048] (1) Overexpression.

[0049] Will PsRBL The complete coding region is inserted into the pBI121 vector. Xba Ⅰ / Sma The primers between site I are shown in Table 2. The recombinant vector was transformed into Agrobacterium. A. tumefaciens 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 achieved. Subsequently, bacterial cells were collected using a high-speed centrifuge and resuspended in infection buffer until an OD600 of 0.8 was achieved. Peony buds, after 7 days of cryogenic treatment, were immersed in 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 incubated 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 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.

[0050] Table 2: PsRBL Primers for amplifying the complete coding region.

[0051] (2) Virus-mediated gene silencing.

[0052] Will PsRBL 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. tumefaciens In EHA105 strain, pTRV2- PsRBLpTRV1, combined infection 10 days low temperature treatment of peony bud, pTRV1 + pTRV2 as a control.

[0053] Table 3: PsRBL Specific fragment amplification primer.

[0054] Histone extraction and Western blotting: The peony bud after gene overexpression and silencing, using Plant Histone Extraction Kit to extract histone. Western blotting analysis of histone. Using 12% SDS-PAGE separation of histone, and then using anti-monomethyl-histone H3-K4, anti-dimethyl-histone H3-K4, anti-trimethyl-histone H3-K4 and anti-histone H3 antibody for immunoblotting detection. Goat anti-rabbit antibody as a secondary antibody, finally using ECL super kit for detection. Using ImageJ to calculate the relative amount of protein level, ImageJ website: https: / / imagej.net / ij / .

[0055] RNA extraction and real-time quantitative PCR: same as example 2.

[0056] 2. Results.

[0057] PsRBL From 0 days to 14 days of low temperature treatment, indicating that they may play a role in low temperature induced dormancy release. In order to study their function, we constructed PsRBL Overexpression vector, and overexpressed in 7 days low temperature treatment of peony bud, pBI121 empty vector as a control. After 3 days of 4℃ dark culture, transfer to normal temperature conditions, 22℃, 16 hours light / 8 hours dark culture. After 7 days of transformation, using RT-qPCR technology to identify the infection efficiency, the results show that PsRBL The expression level is significantly higher than that of the control group, as shown in Figure 4 C. PsRBL The morphological changes of the bud after gene overexpression are shown in Figure 4 A, the relative growth of positive buds was measured, which was significantly higher than that of the control group at 14 days and 28 days after transformation, as shown in Figure 4 B. In addition, the transcription level of dormancy release related genes PsEBB3, PsCYCD3.1, PsCYCD3.3 and PsBG6 in positive buds were significantly up-regulated, as shown in Figure 4 D. Meanwhile, overexpression of PsRBLThe levels of H3K4me3 in buds showed that the levels in positive buds were significantly higher than those in the control group. Figure 6 As shown.

[0058] clone PsRBL 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 subjected to 10 days of low-temperature treatment. RT-qPCR was used to detect the gene. PsRBL The efficiency of silence, such as Figure 5 As shown in C. At 14 and 28 days post-infection, the relative growth of silent buds was significantly lower than that of the control group, consistent with phenotypic changes, such as... Figure 5 A and Figure 5 As shown in B. Dormancy release-related genes. PsEBB3 , PsCYCD3.1 , PsCYCD3.3 and PsBG6 Transcriptional levels were significantly downregulated in these silent buds, such as Figure 5 As shown in D. Ten days after transformation, PsRBL The H3K4me3 level in silent buds was significantly lower than that in the control group, such as Figure 6 As shown. In summary, PsRBL 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 PsRBL Genes, characterized by, The PsRBL The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A PsRBL protein that promotes the breaking of dormancy in peony buds, characterized in that, The PsRBL protein is as described in claim 1. PsRBL Gene encoding.

3. The PsRBL protein for promoting dormancy breaking in peony buds according to claim 2, characterized in that, The amino acid sequence of the PsRBL protein is shown in SEQ ID NO.

2.

4. A carrier, characterized in that, Includes the claims 1 PsRBL Gene.

5. A recombinant bacterium, characterized in that, It includes the carrier as described in claim 4.

6. The claim 1 PsRBL The application of the gene or the PsRBL protein of claim 2, the vector of claim 4, or the recombinant bacteria of claim 5 in promoting the breaking of dormancy in peony buds.

7. A method for promoting the breaking of dormancy in peony buds, characterized in that, include: In dormant peony buds, the Agrobacterium-mediated vacuum infiltration method was used to transform the target bacteria. PsRBL Gene overexpression promotes the breaking of dormancy in Danshen buds.

8. The method for promoting dormancy breaking in peony buds according to claim 7, characterized in that, Transformation of the target bacteria using Agrobacterium-mediated vacuum impregnation method PsRBL Gene overexpression specifically refers to: Immerse peony branches containing dormant buds in water containing... PsRBL In the infection buffer of the gene overexpression vector, vacuum impregnation was used to... PsRBL Gene overexpression.

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