Application of RNA binding protein / Y-motif regulation model in plant growth-resistance tradeoff

Abstract

The invention discloses application of an RNA binding protein / Y-motif regulation and control model in plant growth-resistance tradeoff, and relates to the technical field of plant molecular biology and agricultural biology. The RNA binding protein (RBP) is a protein with a typical RNA recognition motif (RRM), and the Y-motif is an RNA element widely existing in a plant growth gene. And regulating and controlling the mRNA stability of the growth gene through an RBP / Y-motif model so as to realize the effect of plant growth-resistance balancing. The method provided by the invention is not only applied to regulation and control of plant growth-resistance tradeoff and deepening understanding of the plant growth-resistance tradeoff, but also provides a brand new target and theoretical basis for genetic improvement of crops, lays a key technical foundation for cultivation of disease-resistant high-yield new varieties, and has a wide application prospect. And reference value can be provided for research of other adversity stress.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biology and agricultural biotechnology, and in particular to the application of RNA-binding protein / Y-motif regulatory models in regulating the stability of plant growth gene mRNA and balancing resistance and yield. Background Technology

[0002] In the field of genetic improvement for crop disease resistance, maintaining growth while effectively resisting disease is a core biological problem in plant adaptation to environmental stress. Activation of the plant immune system typically leads to a shift in resources towards defense responses, resulting in a significant "growth penalty"—suppressed growth and reduced yield. Therefore, achieving precise temporal and dimensional control of immune output and the expression of growth-related genes, and establishing a post-transcriptional molecular framework for regulating the growth-resistance tradeoff, are crucial issues that urgently need to be addressed in this field.

[0003] Currently, regulatory strategies in this field mainly focus on the transcriptional level, specifically regulating gene transcriptional activity through transcription factors. In contrast, posttranscriptional regulation, particularly the regulation of mRNA stability, translation, and localization mediated by RNA-binding proteins (RBPs), can more rapidly and dynamically control the timing and dosage of protein output, providing a more crucial link in achieving the aforementioned balance. Existing technologies have revealed the fundamental principle by which RBPs regulate resistance gene expression by recognizing specific cis-acting elements (such as purine-rich elements) on target mRNAs. For example, in pattern-triggered immunity (PTI), specific PAB proteins can bind to AG-rich elements to promote selective translation of resistance genes.

[0004] However, current research generally focuses on how RBP regulates the expression of resistance genes. There is still a lack of in-depth understanding and effective intervention methods regarding how the post-transcriptional fate of growth gene mRNA is regulated in the context of immune export, so as to establish a fine post-transcriptional molecular mechanism for regulating the growth-resistance tradeoff. Summary of the Invention

[0005] This invention provides an application of an RNA-binding protein / Y-motif regulatory model in the plant growth-resistance tradeoff. This invention first discovered a specific RNA sequence element called Y-motif in plants. This element is widely present in the mRNA of various key growth genes (especially members of the growth regulator gene family that regulate cell proliferation and organ size). Such elements may serve as an important post-transcriptional regulatory node. Based on this, this invention proposes a novel solution: by specifically intervening in the binding of RBPs (e.g., PTBP3) that can recognize Y-motifs, the stability of the mRNA of core growth genes such as GRFs can be directly regulated. During immune activation, appropriate regulation of the expression of the mRNA of such growth genes allows plants to maintain effective resistance while maintaining a certain level of growth, ultimately achieving a synergistic balance between plant disease resistance and yield potential. This invention is specifically achieved through the following techniques.

[0006] In a first aspect, the present invention provides an application of an RNA-binding protein / Y-motif regulatory model in balancing plant resistance and yield. The RNA-binding protein / Y-motif regulatory model comprises an RNA-binding protein and Y-motif elements, and its structure is as follows:

[0007] ;

[0008] The RNA-binding protein specifically recognizes and binds to the Y-motif element on the mRNA of plant growth genes, thereby regulating the expression of the plant growth genes.

[0009] In a second aspect, the present invention provides a method for balancing plant resistance and yield by transferring a gene expressing an RNA-binding protein into a plant to overexpress the RNA-binding protein; upon immune activation, the RNA-binding protein specifically recognizes and binds to the Y-motif element on the mRNA of a plant growth gene, thereby regulating the expression of the plant growth gene.

[0010] Furthermore, the RNA-binding protein is the Arabidopsis thaliana PTBP3 protein, and the amino acid sequence of the Arabidopsis thaliana PTBP3 protein is shown in SEQ ID NO.43.

[0011] Furthermore, the nucleotide sequence encoding the PTBP3 protein of the Arabidopsis thaliana is shown in SEQ ID NO.37.

[0012] Optionally, the plant growth gene is the GRF3 gene.

[0013] A third aspect of the present invention provides a method for regulating the expression of plant growth genes, wherein a gene fragment expressing an RNA-binding protein is transferred into a plant to overexpress the RNA-binding protein; upon immune activation, the RNA-binding protein specifically recognizes and binds to the Y-motif element on the mRNA of the plant growth gene, thereby regulating the expression of the plant growth gene.

[0014] A fourth aspect of the present invention provides an RNA-binding protein / Y-motif regulatory model, wherein the RNA-binding protein / Y-motif regulatory model is composed of an RNA-binding protein and a Y-motif element, and its structure is as follows:

[0015] ;

[0016] The Y-motif element is a widely present and conserved RNA element in plants and animals; the RNA-binding protein is the PTBP3 protein of Arabidopsis thaliana, which specifically recognizes and binds to growth genes containing the Y-motif element.

[0017] This invention screened a PTBP3 protein and a class of Y-motifs that are widely present and conserved in plants and animals in Arabidopsis thaliana. Furthermore, through co-expression experiments in tobacco, in vitro RNA pull-down experiments, and transgenic complementation experiments, it was demonstrated that the RNA-binding protein / Y-motif regulatory model is effective in regulating the stability of plant growth gene mRNA and balancing resistance and yield.

[0018] A fifth aspect of the invention provides a nucleic acid molecule comprising a gene encoding an RNA-binding protein.

[0019] In a sixth aspect, the present invention provides a recombinant expression vector, characterized in that it comprises the above-mentioned nucleic acid molecule.

[0020] In a seventh aspect, the present invention provides a biological material comprising the above-described nucleic acid molecule or comprising the above-described recombinant expression vector.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] 1. This invention provides an application of an RNA-binding protein / Y-motif regulatory model in regulating the stability of growth gene mRNA and achieving a balance between plant resistance and yield.

[0023] This strategy fills a technical gap in balancing crop traits by manipulating the stability of growth gene mRNA, elucidates the mechanism of action of this RNA-binding protein / Y-motif regulatory model, and deepens our understanding of the intrinsic homeostatic regulatory network in plants.

[0024] 2. The method provided by this invention can regulate the stability of plant growth gene mRNA. This invention is of great significance for exploring the molecular mechanism of PTBP3 / Y-motif, which can not only deepen the understanding of plant resistance and yield balance, but more importantly, provide new targets and theoretical basis for crop genetic improvement.

[0025] 3. This invention can also achieve predictable synergistic improvement of crop disease resistance and yield by intervening in the specific interaction of RBP-RNA elements, providing a new molecular target and theoretical tool that directly acts on the post-transcriptional level for crop genetic improvement, and laying a key technical foundation for breeding disease-resistant and high-yielding new varieties. Attached Figure Description

[0026] Figure 1 Phenotypic analysis of the PTBP3 gene and ptbp3 mutants.

[0027] Figure (a) shows the 3D protein structure of PTBP3 predicted using the Alphafold2 database; the start and end points (amino acids 187 to 237) of its low complexity domain (LCD) are indicated by arrows. Figure (b) shows PTBP3-YFP and PTBP3... ΔLCD (c) Schematic diagram of YFP vector construction. (d) Figure shows the ease with which salicylic acid (SA) induces PTBP3-YFP to form phase transition aggregates in tobacco. Scale bar 10 µm. (e) Figure shows the growth status of wild-type plants (Col-0) and ptbp3 mutants two days after watering and SA application. White arrows indicate leaf survival status. (f) Figure shows the quantification of wilting symptoms in Col-0 and ptbp3 leaves two days after SA treatment. Type 1: no obvious wilting; Type 2: partial wilting; Type 3: complete wilting. (f) Figure shows the growth of Psm ES4326 in Col-0 and ptbp3 plants after exogenous watering and SA application (n=6). npr1-1 is a salicylic acid signaling defective mutant used as a control. Significance was determined using two-way ANOVA.

[0028] Figure 2The PTBP3 gene regulates the expression of the growth gene GRF3. (a) Figure shows the gene ontology analysis of the biological function of the upregulated gene in the SA-treated ptbp3 mutant. The color scale represents the logarithm of the RPKM value to base 2. (b) Figure shows that in SA-treated ptbp3, the 5′-leader regions of a large number of GRF genes are rich in Y-motif. The gray box represents the 5′-leader sequence, and the orange box represents the Y-motif. (c)-(d) Figures show the relative expression levels of GRF3 mRNA in ptbp3 and Col-0 after SA treatment (as shown in (c)) or infection with Psm ES4326 pathogen (as shown in (d)) (n=3). HPT was used as an incorporation internal control, and a two-tailed Student's t-test was used to determine statistical significance. (e) Figure shows the mRNA degradation experiment of ptbp3 and Col-0 after SA treatment. In the experiment, 1 mM cordycepin was used to inhibit the transcription of newly generated mRNA.

[0029] Figure 3 This study analyzed the Y-motif binding of PTBP3 and GRF3. Figures (a)-(d) show the effect of PTBP3 on the relative levels of target gene mRNAs containing Y-motifs in the 5′ leader region. Figures (a) and (c) are schematic diagrams of the vectors used in the co-expression experiment. Figure (b) shows the relative levels of FLUC mRNAs (n=7) with or without Y-motifs in the 5′ leader. Figure (d) shows the relative levels of Arabidopsis GRF3 mRNAs with or without Y-motifs in the 5′ leader. 5′ leader: 5′ leader region, 5′ leader ΔY : Removal of the 5′-leader of the Y-motif (n=3). Figure (e) shows the RNA pull-down assay demonstrating the interaction between PTBP3 and the Y-motif of the Arabidopsis GRF3 gene. Biotin: Biotin modification; MBP-PTBP3-3xHA represents the PTBP3 fusion protein expressed in prokaryotes, and MBP-3xHA represents the control. The bars in the figure show the mean ± standard deviation of mRNA levels normalized to the first bar (Figures (b) and (d)). Statistical significance was determined using a two-tailed Student's t-test (Figure (b)) and two-way ANOVA (Figure (d)). AtUBQ5 mRNA was used as an incorporation internal control.

[0030] Figure 4The growth of Psm ES4326 pathogen in complementary materials of the grf3 mutant is shown. (a) The figure shows a schematic diagram of the miR396a binding site in the CDS region of the GRFs gene. rGRF3 is an artificially introduced mutation to disrupt miR396a binding ability while maintaining the encoded amino acid sequence. (b) The figure shows a comparison of the growth phenotypes of Col-0, ptbp3 mutants, and 35S::rGRF3 transgenic lines under control and SA treatment. Two independent GRF3 overexpression lines are shown in the figure. (c) The figure shows the quantification of wilting symptoms in the leaves of Col-0 and 35S::rGRF3 transgenic lines two days after SA treatment. Type 1: no obvious wilting; Type 2: partial wilting; Type 3: complete wilting. (d) The figure shows the growth of Psm ES4326 in Col-0 and 35S::rGRF3 transgenic lines (n=6). npr1-1 is a salicylic acid signaling defect mutant used as a control. Two-tailed Student's t-tests were used to determine statistical significance. Figure (e) shows a schematic diagram of vector construction for GRF3 gene expression with (or without) Y-motif driven by the GRF3 autopromoter. Figure (f) shows the susceptibility phenotype of the grf3 mutant with 5′-leader (Y-motif or Y-motif deletion) GRF3 gene complementation driven by the GRF3 autopromoter (n=6). Statistical significance was determined using one-way ANOVA. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0033] This invention provides an application of an RNA-binding protein / Y-motif regulatory model in regulating target gene expression. The overall approach is as follows:

[0034] We explored the application of RNA-binding protein / Y-motif regulatory models in plants, and here we propose a method for regulating growth gene expression using RNA-binding protein / Y-motif regulators. We identified an RNA-binding protein (PTBP3) in the model plant Arabidopsis thaliana, and further demonstrated in tobacco that PTBP3 can bind to the Y-motif of the GRF3 5′-leader, inhibiting GRF3 expression; in vitro experiments also confirmed that PTBP3 can bind to the Y-motif of the GRF3 5′-leader.

[0035] The RNA-binding protein / RNA regulation model, which regulates the expression level of target genes, is a method of gene regulation at the transcriptional level and has important potential application value for crop disease resistance breeding.

[0036] As one embodiment, the present invention provides an RNA-binding protein PTBP3, which can bind to genes containing Y-motif elements to regulate gene expression levels.

[0037] Y-motif elements are RNA elements rich in the base "CU" found in plant growth-related genes, typically around 5 bp in length. Specifically, the sequence of a Y-motif element can be one of "CUUCU", "UUUCU", "CCUCU", etc.

[0038] The PTBP3 protein is an RNA-binding protein with Y-motif binding function. Our research has shown that the PTBP3 / Y-motif model can regulate the expression of plant growth genes containing Y-motif elements. Table 1 below lists the primer pair numbers required for PCR amplification.

[0039] Table 1

[0040] In Table 1, (1) primer pair P1 was used to amplify the PTBP3 gene from Arabidopsis leaf cDNA and ligated to a 35S::X–YFP vector using the LIC method. The full-length PTBP3 gene fragment encoding the Arabidopsis PTBP3 protein is 1296 bp, which is the nucleic acid molecule encoding the Arabidopsis PTBP3 protein. The nucleotide sequence is shown in SEQ ID NO.37, and the amino acid sequence of the PTBP3 protein is shown in SEQ ID NO.43.

[0041] (2) Primer pair P2 was used to amplify the GRF3 gene from Arabidopsis leaf cDNA and ligated it into a vector containing YFP using a recombination method. The full-length GRF3 gene fragment encoding the GRF3 protein is 1194 bp, and its nucleotide sequence is shown in SEQ ID NO.38.

[0042] (3) First, primers SEQ ID NO.28, 29 and SEQ ID NO.30, 31 were used to amplify two small rGRF3 DNA fragments (GRF3-BsaI and BsaI-GRF3) (DNA sequences that mutate the miR396 binding site but do not change the amino acid sequence). Then, primer pair P2 was used to amplify the full-length rGRF3 fragment, which was ligated into a vector containing YFP using the LIC method. The full-length rGRF3 gene fragment encoding Arabidopsis rGRF3 protein is 1194 bp, and its nucleotide sequence is shown in SEQ ID NO.39.

[0043] (4) Primer pair P3 was used to amplify the GRF3 gene promoter from Arabidopsis leaf DNA and ligated to a vector containing YFP using T4 DNase. The full-length DNA fragment of the Arabidopsis GRF3 gene promoter is 1243 bp, and its nucleotide sequence is shown in SEQ ID NO. 40.

[0044] (5) Primer pair P4 was used to amplify the 5′-leader of the GRF3 gene from Arabidopsis leaf DNA. GRF3 The sequence was ligated into a vector containing FLUC using T4 DNase. The 5′-leader of the Arabidopsis GRF3 gene. GRF3 The full-length sequence is 217 bp, and the nucleotide sequence is shown in SEQ ID NO.41.

[0045] (6) Primer pair P5, synthesized from Beijing Qingke Biotechnology Co., Ltd., GRF3 gene 5′-leader ΔY GRF3 Amplification of 5′-leader in the sequence ΔY GRF3 The fragment was ligated into a vector containing FLUC using T4 DNase. Arabidopsis GRF3 gene 5′-leader ΔY GRF3 The fragment is 155 bp in length, and its nucleotide sequence is shown in SEQ ID NO.42.

[0046] The following will provide a detailed explanation of the application of the RNA-binding protein / Y-motif regulatory model provided in this application in regulating the stability of plant growth gene mRNA and balancing resistance and yield, in conjunction with examples and experimental data.

[0047] Example 1: Functional study of the PTBP3 gene

[0048] This embodiment screened an RNA-binding protein, PTBP3, from Arabidopsis thaliana. This protein has a typical RNA recognition domain and a low-complexity domain (LCD), such as... Figure 1 As shown in a.

[0049] I. Carrier Construction

[0050] 1. Primer design and synthesis

[0051] The PTBP3 gene sequence was downloaded from the Arabidopsis thaliana website (tair), and specific amplification primers were designed. We used wild-type Arabidopsis thaliana (Col-0) cDNA as a template and performed PCR amplification using the following primers. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The sequences of each primer are as follows.

[0052] (1) Primer pair P1, used to amplify the PTBP3 gene.

[0053] Upstream primer PTBP3-F:

[0054] 5′-CGACGACAAGACCGTACCATGGCGGAATCTTCCAAAGTC-3′, as shown in SEQ ID NO.1.

[0055] Downstream primer PTBP3-R:

[0056] 5′-GAGGAGAAGAGCCGTGCAATCGTCTGTAGCTGGGAG-3′, as shown in SEQ ID NO.2.

[0057] (2) Use the following 4 primers to amplify the PTBP3 gene with LCD removed.

[0058] Upstream primer PTBP3-F: as shown in SEQ ID NO.1.

[0059] Downstream primer PTBP3 ∆LCD -R:

[0060] 5′-GGTCTCCTTATATTGTTGTAATTTACTTGCAGCTCC-3′, as shown in SEQ ID NO.3.

[0061] upstream primer PTBP3 ∆LCD -F:

[0062] 5′-GGTCTCCATAACCGGCACAAATGATAG-3′, as shown in SEQ ID NO.4.

[0063] Downstream primer PTBP3-R: as shown in SEQ ID NO.2.

[0064] 2. PCR amplification

[0065] (1) Amplify the PTBP3 fragment using primer pair P1. The amplification reaction system and specific reaction conditions are shown in Table 2 below.

[0066] Table 2

[0067]

[0068] After briefly centrifuging and mixing the above components, the following reaction was performed on a PCR thermal cycler: denaturation at 95℃ for 5 min, followed by 35 cycles of 95℃ for 30 sec, 56℃ for 30 sec, and 72℃ for 1 min (1 kb / 30 sec); and 72℃ for 5 min.

[0069] (2) Using primers PTBP3-F and PTBP3 ∆LCD -R, and PTBP3 ∆LCD -F and PTBP3-R amplify PTBP3 respectively. ∆LCD The two fragments, the amplification reaction system and specific reaction conditions are the same as those in step (1) above.

[0070] (3) The two fragments were digested with BsaI and then ligated with T4 DNA; then, using this as a template, PTBP3 was amplified with primer pair P1. ∆LCD Excerpt.

[0071] (4) The amplified PCR products were detected by 1% agarose gel electrophoresis and the target fragment was purified and recovered. The recovered fragment was ligated using the LIC method.

[0072] ① First, the target fragment is subjected to LIC treatment. The specific reaction system and steps are shown in Table 3 below.

[0073] Table 3

[0074]

[0075] After briefly centrifuging to mix the above components, perform the following reaction on a PCR thermal cycler: 12℃ for 30 min, 75℃ for 20 min.

[0076] ② The 35S::X-YFP vectors stored in the laboratory were subjected to LIC treatment. The specific reaction system and steps are shown in Table 4 (Step 1) and Table 5 (Step 2).

[0077] Table 4

[0078]

[0079]

[0080] Mix well, incubate at 37℃ for 1 h, then at 65℃ for 20 min.

[0081] Table 5

[0082]

[0083] After briefly centrifuging to mix the above components, perform the following reaction on a PCR thermal cycler: 12℃ for 40 min, 75℃ for 20 min.

[0084] ③ Take 1.5 μl of each LIC vector and fragment, mix well, and place in a PCR thermal cycler for reaction: 75℃ for 5 min, 22℃ for 10 min. After the reaction, place on ice or store at -20℃.

[0085] 3. Transformation of the constructed carrier

[0086] The LIC ligation product (3 μl) was transferred to 50 μl of E. coli supercompetent DH5α cells, and the cells were gently tapped with a finger to mix. The cells were then incubated on ice for 20-30 min.

[0087] After standing, it was placed in a 42°C heat shock for 45 s, and immediately transferred to ice and left to stand for 2 min. 200 μl of antibiotic-free LB was added, and it was resuscitated for 1 h on a shaker at 37°C and 220 rpm.

[0088] E. coli was precipitated at 6000 rpm for 2 min and then evenly spread on a culture dish with Kana resistance. The dish was then incubated overnight at 37°C.

[0089] The following day, four colonies were streaked onto new plates with the corresponding antibiotics, and colony PCR was performed in the afternoon. One positive colony was selected for sequencing. After confirming the sequencing results were correct, the plasmid was returned from the company for Agrobacterium transformation.

[0090] The plasmids were transformed into Agrobacterium competent cells GV3101. The specific Agrobacterium transformation steps are as follows: 3 μl of the plasmid was transferred to 30 μl of highly competent Agrobacterium GV3101, and the mixture was gently tapped with a finger to mix. The mixture was then incubated on ice for 15 min. It was then flash-frozen in liquid nitrogen for 2 min, followed by heat shock at 42°C for 1 min, and immediately transferred to ice and incubated for 2 min. 200 μl of antibiotic-free LB medium was added, and the mixture was incubated on a shaker at 220 rpm and 28°C for 1 h. Agrobacterium was precipitated at 6000 rpm for 2 min and evenly spread onto solid LB agar plates containing Kana, Gent (gentamicin), and Rif (rifampin) antibiotics. The plates were incubated at 28°C for two days. Single colonies of Agrobacterium were picked, shaken, and mixed with 80% glycerol at a 3:1 ratio, and stored at -80°C.

[0091] II. Observation of PTBP3 and PTBP3 in tobacco∆LCD Subcellular localization

[0092] 1. Tobacco Injection

[0093] Nicotiana benthamiana was grown in a greenhouse under the following conditions: 12 hours of light / 12 hours of darkness, temperature 25°C, and relative humidity 70%. The tobacco could be used for injection after about 4 weeks of cultivation.

[0094] The *Agrobacterium* bacteria from the above steps were streaked overnight on solid LB agar plates containing Kana, Gent, and Rif antibiotics, respectively. The next day, a portion of the *Agrobacterium* culture was picked up with a pipette tip and transferred to 2 ml of LB agar containing Kana, Gent, and Rif antibiotics, and incubated overnight at 200 rpm in a shaker at 28°C (OD200). 600 Approximately 1.0-2.0).

[0095] Pilosulate 100 μl of bacterial culture into 2 ml centrifuge tubes and centrifuge at 6000 rpm for 5 min at room temperature to collect the bacterial cells. Resuspend the bacterial cells in 2 mL of infiltration buffer (containing 10 mM MgCl2, 10 mM MES, 150 μM acetylsylgenone (AS), pH 5.6). Then allow to stand at room temperature for 2–3 h (at least 0.5 h, and no more than 3 h).

[0096] Draw up the bacterial solution with a syringe and co-inject different combinations of Agrobacterium into tobacco leaves. Mark the injection sites with a marker.

[0097] 2. Subcellular localization observation

[0098] Two days after tobacco injection culture, 5 mM SA (salicylic acid) was injected, and water was used as a control; 3 hours later, the subcellular localization of PTBP3 was observed under a confocal microscope.

[0099] The results showed that PTBP3 and PTBP3 ∆LCD Under normal circumstances, the protein is located in the cytoplasm and nucleus. Upon SA treatment, the PTBP3 protein undergoes a phase transition, forming dot-like structures of varying sizes in the cytoplasm and nucleus. ∆LCD The subcellular localization of the protein remained unchanged, indicating that the phase transition of the PTBP3 protein is dependent on its LCD structure, as shown in the figure. Figure 1 b、 Figure 1 As shown in c.

[0100] III. The ptbp3 mutant is insensitive to SA.

[0101] 1. Arabidopsis thaliana cultivation

[0102] The PTBP3 gene T-DNA insertion mutant “Salk_207185C” was purchased from the Arabidopsis thaliana biological resource center. The Col-0 and ptbp3 mutants were grown in a greenhouse under the following conditions: 12 h light / 12 h dark, temperature 22℃, relative humidity 70%; the plants were ready for use after approximately 22-24 days of cultivation.

[0103] 2. Observe the effect of exogenous SA spraying on ptbp3 mutant.

[0104] A 5 mM SA solution was prepared and sprayed onto the leaves of wild-type Arabidopsis thaliana (Col-0) and the ptbp3 mutant, respectively; water was sprayed simultaneously as a control. Two days after treatment, the wilting of the leaves was quantified. The severity of wilting was divided into three types: Type 1, no obvious wilting; Type 2, partial wilting; and Type 3, complete wilting.

[0105] The results showed that the wilting of the ptbp3 mutant leaves was significantly less than that of the Col-0 mutant, with most leaves surviving. This indicates that the ptbp3 mutant leaves are not sensitive to SA and exhibit good growth. Figure 1 d、 Figure 1 As shown in e.

[0106] IV. The ptbp3 mutant exhibits good disease resistance.

[0107] (1) Planting and treatment

[0108] Wild-type Col-0, ptbp3 mutants and npr1-1 (a salicylic acid signaling defect mutant used as a control) were grown in a greenhouse with 12 h light / 12 h darkness, temperature 22℃ and relative humidity 70%.

[0109] Arabidopsis thaliana plants aged 22-24 days were selected for injection of the Psm ES4326 pathogen. One day prior to injection, 1 mM SA was sprayed, and water was sprayed as a control.

[0110] (2) Activation and injection of Psm ES4326 pathogen

[0111] The pathogen Psm ES4326 (i.e., Pseudomonas syringae pathovar (pv.) maculicola ES4326) was cultured on KB medium containing streptomycin (Str) antibiotic two days prior to injection. An appropriate amount of bacterial cells was scraped from the culture using a pipette tip into a 2 mL centrifuge tube, resuspended in 10 mM MgCl2, and the bacterial concentration was measured. The culture was then further diluted to OD. 600 nm =0.001. The bacterial solution was injected into the Col-0 and ptbp3 leaves using a needle-free injector, and any remaining liquid on the leaf surface was blotted dry with absorbent paper towels.

[0112] (3) Leaf collection, stenciling and statistical analysis of Psm ES4326 injected

[0113] Three days after the pathogen injection, the leaves injected with Psm ES4326 were removed with tweezers, sprayed with 75% alcohol for surface disinfection, and then wiped dry with absorbent paper towels.

[0114] Small discs were obtained by punching holes using a 0.6 cm diameter punch. Three discs were randomly selected as one biological replicate, and each treatment included six biological replicates.

[0115] Add 200 µL of 10 mM MgCl2 and a sterile steel ball to each sample using a tube gun, tighten the cap, and then homogenize the sample using a plant tissue homogenizer (45 Hz for 4 minutes).

[0116] Use 10 mM MgCl2 for gradient dilution, that is, add 180 µL of 10 mM MgCl2 to each well of the 96-well plate in advance using a pipette, and then take 20 µL of the blade slurry and mix it thoroughly by suction and blow.

[0117] For each new dilution, the leaf homogenate from the previous stage is extracted using the same procedure. Depending on the treatment, the leaf homogenate is eventually diluted to 10. -2 -10 -5 class.

[0118] Using a pipette, 10 µL of the diluted plant homogenate was evenly spread onto KB solid medium containing the corresponding antibiotic. After the surface of the medium dried, it was incubated upside down in a 28°C incubator for 1-2 days. The number of colonies on the medium was counted, and the results were analyzed and plotted using GraphPad Prism software.

[0119] The results showed that the ptbp3 mutant exhibited strong resistance to Psm ES4326, such as Figure 1 As shown in f.

[0120] Example 2: PTBP3 binds to γ-motif mRNA and regulates target gene stability

[0121] I. RNA-seq data analysis of ptbp3 mutant under SA treatment conditions

[0122] 1. Culture of ptbp3 mutant and wild-type Col-0 plants and collection of RNA-seq samples

[0123] Both Col-0 and ptbp3 mutant plants were grown in a greenhouse under the following conditions: 12 h light / 12 h dark, temperature 22℃, and relative humidity 70%. After about 22 days of cultivation, plants with uniform growth were selected and treated with 2 mM SA (water spraying was used as a control). After 4 h of treatment, leaves were collected for RNAseq samples (two replicates were collected for each mutant).

[0124] 2. A large number of growth-related genes contain Y-motif.

[0125] RNAseq data analysis revealed that the ptbp3 mutant showed upregulated expression of numerous growth-related genes after SA treatment, such as... Figure 2 As shown in a.

[0126] Our careful analysis revealed that a large number of growth-related genes contain Y-motifs in their 5′-leader sequences. Among these upregulated growth-related genes, growth regulatory factor (GRF) genes showed particularly prominent expression. In Arabidopsis, the GRF family comprises nine members, named GRF1 to GRF9, and numerous Y-motifs are present in the 5′-leader sequences of GRF genes, such as... Figure 2 As shown in b.

[0127] II. Detection of GRF3 gene expression level in ptbp3 mutant using real-time quantitative PCR (qPCR)

[0128] The expression level of the GRF3 gene in the ptbp3 mutant was detected using the following method:

[0129] (1) Collection of leaf RNA samples after SA treatment

[0130] Col-0 and ptbp3 mutants were cultured in a greenhouse for about 22 days. They were then sprayed with 2 mM SA (water spraying was used as a control). After 4 h of treatment, 3 leaves were collected and placed in 2 ml centrifuge tubes without RNase containing 1 steel ball, and then frozen in liquid nitrogen.

[0131] (2) Collection of leaf RNA samples after injection of Psm ES4326

[0132] Col-0 and ptbp3 mutants, cultured in a greenhouse for approximately 22 days, were directly injected with Psm ES4326 (OD). 600 nm = 0.001) to study the changes in plant RNA during pathogen invasion. 24 h after injection, 3 leaves were collected and placed in a 2 ml centrifuge tube containing 1 steel ball and free of RNase, and then frozen in liquid nitrogen.

[0133] (3) RNA extraction

[0134] Total RNA was extracted from Arabidopsis thaliana using a total RNA extraction reagent. The specific steps are as follows:

[0135] ① Place the centrifuge tube containing the blades in liquid nitrogen for rapid freezing, and then grind the sample using a sample grinder. Next, add 1 mL of total RNA extraction reagent and allow to stand. After the sample has completely thawed, continue to pipette until the lysate is clear. Centrifuge at 11,200 rpm (12,000 × g) at 4℃ for 5 min, and collect the supernatant.

[0136] ② In a fume hood, add 200 μL (1 / 5 volume) of chloroform to the above lysis buffer. Shake vigorously for 15 seconds to form an emulsion, and let stand at 4 ℃ for 5 min.

[0137] ③ Centrifuge at 4℃, 11,200 rpm (12,000 × g) for 10 min. The mixture separates into three layers, including the bottom phenol-chloroform organic phase, the intermediate phase, and the upper aqueous phase. Carefully transfer the upper aqueous phase into a new EP tube.

[0138] ④ Add an equal volume of pre-cooled isopropanol, mix by inverting the container, and let stand at 4°C for 10 min.

[0139] ⑤ Centrifuge at 4℃, 11,200 rpm (12,000 × g) for 10 min. Discard the supernatant and retain the white precipitate.

[0140] ⑥ Carefully discard the supernatant and add 1 ml of 75% ethanol (prepared with RNase-free ddH2O). Gently tap the bottom of the tube to suspend the precipitate, and invert it several times. Let it stand at room temperature for 3-5 minutes.

[0141] Centrifuge at 4℃, 11,200 rpm (12,000 × g) for 5 min, and discard the supernatant.

[0142] ⑧ Dry the precipitate in a clean environment at room temperature for 2-5 minutes. Be careful not to over-dry it, otherwise the RNA will be difficult to dissolve. Add an appropriate amount of RNase-free ddH2O to dissolve the precipitate. After complete dissolution, take a small amount for testing, and store the rest of the sample at -80 ℃.

[0143] (4) cDNA synthesis

[0144] The concentration and integrity of the extracted RNA were detected using Nanodrop and agarose gel electrophoresis, and cDNA was synthesized using the Novizan reverse transcription kit. The specific procedures are as follows:

[0145] ① RNA template denaturation. Prepare the following mixture in an RNase-free EP tube and gently pipette to mix. Heat at 65°C for 5 min and immediately place on ice for 2 min. The specific reaction system is shown in Table 6 below.

[0146] Table 6

[0147]

[0148] ② Removal of genomic DNA. Prepare the following mixture and gently pipette to mix. Incubate at 42°C for 2 min. The specific reaction system is shown in Table 7 below.

[0149] Table 7

[0150]

[0151]

[0152] ③ Prepare the first-strand cDNA synthesis reaction solution and gently mix by pipetting. The specific reaction system is shown in Table 8 below.

[0153] Table 8

[0154]

[0155] ④ Synthesize the first-strand cDNA according to the following reaction program: 25℃, 5 min; 37℃, 45 min; 85℃, 5 sec.

[0156] (5) qPCR detection of GRF3 expression levels in ptbp3 mutant and Col-0

[0157] ① First, synthesize qPCR detection primers.

[0158] Upstream primer GRF3-F:

[0159] 5′-GTCTTCGCTGGCCACAAGTATT-3′, as shown in SEQ ID NO.5.

[0160] Downstream primer GRF3-R:

[0161] 5′-TGTTGCTGTTGTAGTGGTGGCT-3′, as shown in SEQ ID NO.6.

[0162] Upstream primer HPT-F:

[0163] 5′-TGGTTGGCTTGTATGGAGCAGCAG-3′, as shown in SEQ ID NO.7.

[0164] Downstream primer HPT-R:

[0165] 5′-TGGTCAAGACCAATGCGGAGCATA-3′, as shown in SEQ ID NO.8.

[0166] ②qPCR reaction

[0167] Expression levels were detected using the Novizan SYBR qPCR Mix kit. The reaction mixtures shown in Table 9 below were prepared.

[0168] Table 9

[0169]

[0170] The PCR reaction program is as follows: Pre-denaturation: 95℃, 30 sec. Cycling reaction: 95℃, 10 sec; 60℃, 10 sec; 40 cycles. Melting curve: 95℃, 15 sec; 60℃, 1 min; 95℃, 15 sec.

[0171] ③ Data Analysis. Using the exogenously added HPT gene as an internal reference, a 2... -ΔΔCt The Ct values ​​for each data set were analyzed using a GraphPad Prism software. The results showed that under normal conditions, the stability of GRF3 gene mRNA was not different between the Col-0 and ptbp3 mutants; however, under exogenous SA spraying or pathogen invasion, the expression level of the GRF3 gene in the ptbp3 mutant was significantly higher than that in the Col-0 mutant. Figure 2 c and Figure 2 d.

[0172] III. During SA treatment, PTBP3 regulates the stability of GRF3 mRNA.

[0173] The stability of GRF3 mRNA was studied using mRNA degradation experiments.

[0174] Col-0 and ptbp3 seedlings cultured on 1 / 2 MS medium for 5 days were treated with 0.5 mM SA (water was used as a control).

[0175] After 5 h of treatment, about 70 seedlings were taken from each sample and placed in a 24-well cell culture plate. They were immersed in 2 mL of incubation buffer (containing 15 mM sucrose, 1 mM KCl, 1 mM PIPES [pH 6.25], and 1 mM sodium citrate) and pre-incubated at 80 rpm for 15 minutes.

[0176] Then, the buffer solution was replaced with 2 mL of fresh buffer solution containing 1 mM cordycepin (Solepro, #SC8470), and vacuum permeation was performed immediately for 1 minute.

[0177] After vacuuming, the collected samples were immediately flash-frozen in liquid nitrogen, and this time point was recorded as T0. The remaining samples were then subjected to two more 1-minute vacuum permeation cycles (with a 1-minute decompression interval between each cycle). Samples were collected at 15, 30, 60, and 120 min and flash-frozen in liquid nitrogen. Total RNA was extracted and analyzed by RT-qPCR.

[0178] Experimental results showed that GRF3 mRNA exhibited better stability in SA-treated ptbp3 seedlings; however, GRF3 mRNA degraded faster in the other samples, showing a relatively consistent trend. Figure 2 As shown in e, this confirms that PTBP3 regulates the stability of GRF3 mRNA during treatment with the immune hormone SA.

[0179] Example 3: PTBP3 protein binding to 5′-leader GRF3 Y-motif, inhibiting GRF3 expression

[0180] I. Construction of Expression Carrier

[0181] 1. Primer design and synthesis

[0182] The PTBP3 and GRF3 genome sequences were downloaded from the Arabidopsis thaliana website (tair). Specific amplification primers were designed, using Arabidopsis thaliana Col-0 cDNA as a template for gene fragment amplification and Arabidopsis thaliana Col-0 DNA as a template for 5′-leader fragment amplification. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the sequences and numbers of each primer are as follows.

[0183] (1) Primer pair P1 for amplifying the PTBP3 gene fragment, the sequence of which is the same as in Example 1.

[0184] (2) Amplification of 5′-leader GRF3 The primer pair for the fragment is P4.

[0185] upstream primer 5′-leader GRF3 -F:

[0186] 5′-GGTCTCCAGGACCTTTCTCTCTCTGCAGAAGAAG-3′, as shown in SEQ ID NO.9.

[0187] Downstream primer 5′-leader GRF3 -R:

[0188] 5′-GGTCTCCCCATTGAAGAAAGAGAGAGAGAAGTGTT-3′, as shown in SEQ ID NO.10.

[0189] (3) Amplify 5′-leaderΔY GRF Primer pair P5 of the fragment

[0190] upstream primer 5′-leader ΔY GRF3 -F:

[0191] 5′-GGTCTCCAGGAGCAGAAGAAGCTCAGATACAGAAAC-3′, as shown in SEQ ID NO.11.

[0192] Downstream primer 5′-leader ΔY GRF3 -R:

[0193] 5′-GGTCTCCCCATTTGTTGGTTACTTTAAGGATAATGG-3′, as shown in SEQ ID NO. 12.

[0194] (4) Amplify 5′-leader GRF3- The GRF3 fragment contains a partial homologous arm sequence at the 5′ end of the primer, which is used for recombination reactions.

[0195] First, amplify the 5′-SacI-leader. GRF3 -PstI-3' fragment.

[0196] upstream primer SacI-leader GRF3 -F:

[0197] 5′-TCATTTGGAGAGAACACGGGGGACGAGCTCCCTTTCTCTCTCTGCAGAGAAG-3′, as shown in SEQ ID NO. 13.

[0198] Downstream primer 5'-leader GRF3 -PstI-R:

[0199] 5′-CCATTGTTTCAGTTGCAAATCCATCTGCAGTGAAGAAAGAGAGAGAGAAGTGTT-3′, as shown in SEQ ID NO. 14.

[0200] Secondly, the PstI-GRF3-PstI fragment was amplified.

[0201] upstream primer PstI-leader GRF3 -F:

[0202] 5′-ACACTTCTCTCTCTCTTCTTCACTGCAGATGGATTTGCAACTGAAACAAT-3′, as shown in SEQ ID NO. 15.

[0203] Downstream primer PstI-leader GRF3 -R:

[0204] 5′-CCGATGATACGAACGAAAGCTCTGCAGTCAATGAAAGGCTTGTGTCGAGAC-3′, as shown in SEQ ID NO. 16.

[0205] (5) Amplify 5′-leader ΔY GRF3 -GRF3 fragment (the 5' end of the primer contains a partial homologous arm sequence for recombination reaction).

[0206] First, amplify the 5′-SacI-leader. ΔY GRF3 -PstI-3' fragment.

[0207] upstream primer SacI-leader GRF3 -F:

[0208] 5′-TCATTTGGAGAGAACACGGGGGACGAGCTCGCAGAAGAAGCTCAGATACAGAAAC-3′, as shown in SEQ ID NO.17.

[0209] Downstream primer 5'-leader GRF3 -PstI-R:

[0210] 5′-AAACCGATGATACGAACGAAAGCTCTGCAGTTGTTGGTTACTTTAAGGATAATGG-3′, as shown in SEQ ID NO.18.

[0211] Secondly, the PstI-GRF-PstI fragment was amplified.

[0212] upstream primer PstI-leader GRF3 -F:

[0213] 5′-CCATTATCCTTAAAGTAACCAACAACTGCAGATGGATTTGCAACTGAAACAA-3′, as shown in SEQ ID NO. 19.

[0214] Downstream primer PstI-leader GRF3-R: As shown in SEQ ID NO.16.

[0215] (6) Amplify the NdeI-PTBP3-3x-HA-PstI fragment (the 5' end of the primer contains a partial homologous arm sequence for recombination).

[0216] Upstream primer PTBP3-3x-HA-F:

[0217] 5′-GGGATCGAGGGAAGGATTTCACATATGCGGTCTAGACTCGACGACAA-3′, as shown in SEQ ID NO.20.

[0218] Downstream primer PTBP3-3x-HA-R:

[0219] 5′-TTGAAGCTTCTGAATAGCATGCctgcagGGTGGTGGTGCTCGAGCTAG-3′, as shown in SEQ ID NO.21.

[0220] 2. PCR amplification

[0221] (1) PCR amplification using Arabidopsis cDNA as a template

[0222] For details of the PCR amplification and product recovery procedure using Arabidopsis cDNA as a template, please refer to Example 1.

[0223] (2) PCR amplification using Arabidopsis DNA or synthetic DNA fragments as templates

[0224] The PCR amplification reaction system is shown in Table 10 below.

[0225] Table 10

[0226]

[0227] After briefly centrifuging and mixing the above components, the following reaction was performed on a PCR thermal cycler: denaturation at 95℃ for 5 min; followed by 35 cycles of 95℃ for 30 sec, 56℃ for 30 sec, and 72℃ for 1 min (1 kb / 30 sec); and finally 72℃ for 5 min.

[0228] The amplified PCR products were detected using 1% agarose gel electrophoresis, and the target fragment was purified and recovered.

[0229] (3) PCR amplification using plasmid as template

[0230] The NdeI-PTBP3-3x-HA-PstI fragment was amplified on a plasmid containing the PTBP3-3x-HA fragment preserved in our laboratory. The amplification reaction system is shown in Table 11 below.

[0231] Table 11

[0232]

[0233] After briefly centrifuging and mixing the above components, the following reaction was performed on a PCR thermal cycler: denaturation at 95℃ for 5 min; followed by 35 cycles of 95℃ for 30 sec, 56℃ for 30 sec, and 72℃ for 1 min (1 kb / 30 sec); and finally 72℃ for 5 min.

[0234] The amplified PCR products were detected using 1% agarose gel electrophoresis, and the target fragment was purified and recovered.

[0235] 3. Connection between the target fragment and the vector

[0236] (1) Ligation of the PTBP3 gene fragment and the 35S::X-YFP vector.

[0237] The 35S::PTBP3-YFP plasmid was obtained in Example 1.

[0238] (2) Ligation of the target fragment with the 35S::FLUC vector

[0239] ① Digest the target fragment and the 35S::FLUC vector with BsaI restriction endonuclease.

[0240] The 5′-SacI-leader recovered from the PCR amplification in step 2 above GRF3 -PstI-3' fragment, 5'-leader ΔY GRF The fragments and the laboratory-preserved 35S::FLUC vector were digested with BsaI, and the specific reaction system is shown in Table 12 below.

[0241] Table 12

[0242]

[0243] Briefly centrifuge to mix the above components, then digest with enzymes at 37°C for 60 min. Detect the digested PCR product and vector using a 1% agarose gel electrophoresis to recover the target fragment and vector fragment.

[0244] ②Connection

[0245] The recovered PCR products and vectors were ligated using T4 DNA ligase (NEB, M0202), and the ligation system is shown in Table 13 below.

[0246] Table 13

[0247]

[0248] Briefly centrifuge to mix the above components, and then connect at room temperature for 3 h.

[0249] (3) Construction of a vector to drive the GRF3 gene by 35S

[0250] ①35S::5′-leader GRF3 -GRF3 vector construction

[0251] The vector containing the 35S promoter, preserved in our laboratory, was digested with SacI and PstI restriction endonucleases. The specific reaction system is shown in Table 14 below.

[0252] Table 14

[0253]

[0254]

[0255] After briefly centrifuging to mix the above components, digest them at 37°C for 60 min, excise the gel, and recover the vector fragment.

[0256] The 5′-leader recovered in step 2 GRF3 The fragment, the PstI-GRF3-PstI fragment, and the recovered vector fragment were ligated using recombinase. The ligation system is shown in Table 15 below.

[0257] Table 15

[0258]

[0259] After briefly centrifuging to mix the above components, incubate at 55°C for 30 min in a PCR instrument, then quickly place on ice to cool or temporarily store at -20°C.

[0260] ②35S::5′-leader ΔY GRF3 -GRF3 vector construction

[0261] The vector containing the 35S promoter, preserved in our laboratory, was digested with SacI and PstI restriction endonucleases. The specific reaction system is shown in Table 16 below.

[0262] Table 16

[0263]

[0264] Briefly centrifuge to mix the above components, digest at 37°C for 60 min, and recover the vector fragment.

[0265] The 5′-leader recovered in step 2 ΔY GRF3The fragments were ligated to the recovered vector fragments using recombinase. The ligation system is shown in Table 17 below.

[0266] Table 17

[0267]

[0268] After briefly centrifuging to mix the above components, incubate at 55°C for 30 min in a PCR instrument, then quickly place on ice to cool.

[0269] Take 5 μl of the ligation product and transform it with supercompetent E. coli DH5α (detailed Example 1). Obtain 5S::5′-leader. ΔY GRF3 The vector was then digested with a single PstI restriction endonuclease. The specific reaction system and steps are shown in Table 18 below.

[0270] Table 18

[0271]

[0272] Briefly centrifuge to mix the above components, then digest at 37°C for 60 min to recover the 35S::5′-leader. ΔY GRF3 Carrier fragment.

[0273] The PstI-GRF3-PstI fragment recovered in step 2 was compared with the recovered 35S::5′-leader ΔY GRF3 The vector fragments were ligated using recombinase. The ligation system is shown in Table 19 below.

[0274] Table 19

[0275]

[0276] After briefly centrifuging to mix the above components, incubate at 55°C for 30 min in a PCR instrument, then quickly place on ice to cool or temporarily store at -20°C.

[0277] (4) Ligation of NdeI-PTBP3-3x-HA-PstI fragment with pMAL-C5X vector

[0278] The NdeI-PTBP3-3x-HA-PstI fragment was amplified (the 5' end of the primer contains a partial homologous arm sequence for recombination), and then ligated into the pMAL-C5X vector using recombination methods.

[0279] The pMAL-C5X vector stored in our laboratory was digested with NdeI and PstI. The reaction system is shown in Table 20 below.

[0280] Table 20

[0281]

[0282] After briefly centrifuging to mix the above components, digest them at 37°C for 60 min, and then detect and recover the vector fragments using 1% agarose gel.

[0283] The NdeI-PTBP3-3x-HA-PstI fragment recovered in step 2 was ligated with the recovered pMAL-C5X vector fragment using recombinase. The ligation system is shown in Table 21 below.

[0284] Table 21

[0285]

[0286] After briefly centrifuging to mix the above components, incubate at 55°C for 30 min in a PCR instrument, then quickly place on ice to cool or temporarily store at -20°C.

[0287] 4. Transformation of the constructed carrier

[0288] For details on the transformation methods of Escherichia coli and Agrobacterium, please refer to Example 1.

[0289] II. Tobacco co-expression experiments demonstrate the effective inhibition of PTBP3 on the expression levels of target genes.

[0290] 1. Tobacco Injection

[0291] In the above steps, 35S::PTBP3-YFP and 35S::5′-leader GRF3 -FLUC、35S::5′-leader ΔY GRF3 -FLUC、35S::5′-leader GRF3 -GRF3 and 35S::5′-leader ΔY GRF - GRF3 Agrobacterium was activated and shaken, as detailed in Example 1.

[0292] Take 100 μl of bacterial culture from each of the following combinations and place them into 2 ml centrifuge tubes.

[0293] Group 1: 35S::PTBP3-YFP and 35S::5′-leader GRF3 -FLUC.

[0294] Group 2: 35S::PTBP3-YFP and 35S::5′-leader ΔY GRF3 -FLUC.

[0295] Group 3: 35S::PTBP3-YFP and 35S::5′-leader GRF3 -GRF3.

[0296] Group 4: 35S::PTBP3-YFP and 35S::5′-leader ΔY GRF3 -GRF3.

[0297] Group 5: 35S::YFP and 35S::5′-leader GRF3 -GRF3 (control group).

[0298] Group 6: 35S::YFP and 35S::5′-leader ΔY GRF3 -GRF3 (control group).

[0299] For details on the methods of Agrobacterium tumefaciens bacterial suspension collection and tobacco injection, please refer to Example 1.

[0300] 2. qPCR detection of co-expressed genes

[0301] After culturing the injected plants for 48 hours, samples from each combination were collected for RNA extraction. The methods for RNA extraction, reverse transcription, and qPCR were as described in Example 2. The qPCR primers are as follows:

[0302] ① FLUC gene expression level detection.

[0303] The upstream primer FLUC-F: 5′-GGATTACAAGATTCAAAGTGCG-3′, as shown in SEQ ID NO.22.

[0304] Downstream primer FLUC-R: 5′-TGATACCTGGCAGATGGAAC-3′, as shown in SEQ ID NO.23.

[0305] ② Detection of GRF3 gene expression level.

[0306] Upstream primer GRF3-F: as shown in SEQ ID NO.5.

[0307] Downstream primer GRF3-R: as shown in SEQ ID NO.6.

[0308] ③The HPT gene was used as an internal reference.

[0309] Upstream primer HPT-F: as shown in SEQ ID NO.7.

[0310] Downstream primer HPT-R: as shown in SEQ ID NO.8.

[0311] qPCR analysis revealed that the PTBP3 gene significantly inhibited the expression of FLUC and GRF3 genes, such as Figure 3 As shown in a-3d.

[0312] III. RNA pull-down experiments demonstrate that PTBP3 protein binds to the Y-motif of the GRF3 gene.

[0313] 1. Prokaryotic expression and purification of proteins

[0314] (1) Transform the above pMAL-C5X-PTBP3-3x-HA plasmid and pMAL-C5X-3x-HA (preserved in our laboratory and used as a control) into Escherichia coli BL21 competent cells. Pick the colonies that were correctly detected by PCR and put them into 5 ml LB medium containing 50 mg / L Carb, and incubate overnight at 37°C and 220 rpm / min.

[0315] (2) The next day, the bacterial solution was diluted at a ratio of 1:100. 5 ml of the bacterial solution was added to 500 mL of CarbLB containing 50 mg / L. The solution was incubated at 37°C and 220 rpm / min for 2 h until the OD600nm reached 0.6.

[0316] (3) The protein was induced to express overnight using 0.3 mM IPTG at 16℃ and 180 rpm / min.

[0317] (4) The next day, the bacterial cells were collected by centrifugation and resuspended in 50 mL of lysis buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 200 mM NaCl, 1 mM DTT, 10% glycerol, 1 mM PMSF). The bacteria were then lysed using a high-pressure homogenizer.

[0318] (5) Centrifuge the lysed protein at 11,000 rpm at 4℃ for 20 min. Collect the supernatant and store it for later use.

[0319] 2. Synthesis of biotin-labeled nucleotides (Biotin-RNA)

[0320] The following Biotin-RNA was synthesized (by Beijing Qingke Biotechnology Co., Ltd.) and adjusted to a concentration of 0.1 mM.

[0321] RNA1: 5′-GAAAAGAAAAGAAAAGAAAA-3′, as shown in SEQ ID NO.24.

[0322] RNA2: 5-GAAAACUUCUGAAAAGAAAA-3′, as shown in SEQ ID NO.25.

[0323] RNA3: 5′-GAAAACUUCUCUUCUGAAAA-3′, as shown in SEQ ID NO.26.

[0324] RNA4: 5′-GAAAACUUCUGAAAACUUCU-3′, as shown in SEQ ID NO.27.

[0325] 3. Streptavidin magnetic beads bind to biotin-RNA

[0326] (1) Place the magnetic beads on a mixer and vortex for 20 seconds to fully suspend them. Take 250 μL of the magnetic beads into a new 1.5 mL centrifuge tube, place it on a magnetic rack, and magnetically separate it. Discard the supernatant.

[0327] (2) Add 1 mL of Buffer I (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 M NaCl, 0.01% Tween-20) to the centrifuge tube, cap the tube, and vortex the magnetic beads for 15 seconds to thoroughly wash them. Magnetic separation is achieved, and the supernatant is discarded. Repeat the above steps 3 times.

[0328] (3) Add 250 μL of Buffer I to suspend the magnetic beads and divide them into 5 tubes. Add 200 μL of Buffer I to each tube to make the magnetic bead concentration 2 mg / mL.

[0329] (4) Take 5 μL of Biotin-RNA (RNA1-RNA4) and add it to a 1.5 mL centrifuge tube containing magnetic beads. Add 5 μL of RNase-free water to another tube as a control.

[0330] (5) Shake and suspend thoroughly, then incubate at 4°C for 2 h on a rotary mixer.

[0331] (6) Magnetic separation, remove supernatant and resuspend in 1 mL Buffer I.

[0332] (7) Vortex the magnetic bead for 15 seconds to separate the magnetism, and discard the supernatant. Repeat the above steps twice. Store the magnetic bead for later use.

[0333] 4. PTBP3 protein binds to magnetic beads-Biotin-RNA

[0334] (1) Take 1 mL of the supernatant of the above centrifuged and lysed MBP-PTBP3-3×HA or MBP-3×HA protein and add it to the bound Biotin-RNA magnetic beads, then add 2 μL (20U / μL) RNase inhibitor and incubate at 4℃ for 2 hours.

[0335] (2) Wash the magnetic beads three times with washing buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 200 mM NaCl, 1 mM MTT, 0.01% Tween-20).

[0336] (3) Add 50 μL of 2×SDS loading buffer and incubate at 95℃ for 10 min to denature the RNA-bound protein. Separate the protein using a 10% SDS-PAGE gel and detect it by immunoblotting.

[0337] 5. Western blot (protein immunoblotting)

[0338] Using anti-HA as the primary antibody, Western blotting was used to analyze the expression of MBP-PTBP3-3×HA or MBP-3×HA proteins. Specific steps included:

[0339] (1) Separation of protein components by SDS-PAGE electrophoresis

[0340] Electrophoresis was performed using a 5% stacking gel and a 10% separating gel. 10 μL of protein sample was loaded into each lane for easy comparison. The sample was run at 80V for 20 min, then the voltage was adjusted to 120V and run for 2 h.

[0341] (2) Transfer membrane and antibody reaction

[0342] After electrophoresis, remove the gel and wash it with water. Simultaneously, soak the PVDF membrane in alcohol for 1 min and filter paper in transfer buffer for 5 min. Lay the gel and PVDF membrane in the following order: positive electrode - filter paper - PVDF membrane - gel - filter paper - negative electrode. Place the membrane in a transfer apparatus and transfer at 25V, 1.3A for 12 min. Remove the PVDF membrane and wash it with 1×TBST (TBS containing 0.1% Tween-20) for 1 min. Add TBST blocking buffer containing 5% skim milk and incubate at room temperature for 1 h. Add mouse anti-HA (1:5000, diluted with TBST containing 5% skim milk) and incubate at room temperature for 1 h. Wash the membrane thoroughly three times with TBST for 10 min each time. Add HRP-labeled goat anti-mouse IgG antibody (TBST 1:10000 dilution) and incubate at room temperature for 1 h. Wash the membrane thoroughly three times with TBST for 10 min each time. Develop with Thermo Fisher Scientific developing solution for 2-5 min and save the experimental results.

[0343] We found that PTBP3 can bind slightly to the Y-motif of RNA2, and binds more strongly to the Y-motifs of RNA3 and RNA4, but does not bind to RNA1, which contains only the R-motif. Meanwhile, the control proteins MBP-3×HA do not bind to any of the four RNAs. This further indicates that PTBP3 can bind to Y-motifs to regulate the expression of target genes, such as… Figure 3 As shown in e.

[0344] Example 4: 5′-leader of the GRF3 gene GRF3 Study on immune function of Y-motif in Arabidopsis thaliana

[0345] I. Carrier Construction

[0346] 1. Primer design and synthesis

[0347] The promoter and coding region sequences of the GRF3 gene were downloaded from the Arabidopsis thaliana website (tair). Specific amplification primers were designed, and the coding region fragment of the GRF3 gene was amplified using Arabidopsis thaliana Col-0 cDNA as a template. The promoter and 5′-leader fragments of the GRF3 gene were amplified using Arabidopsis thaliana Col-0 DNA as a template. Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the sequences and numbers of each primer are as follows:

[0348] (1) Amplification of the rGRF3 fragment. Because the GRF3 gene is regulated by miR396a, in order to eliminate the interference of miR396a, we mutated the miR396a regulatory site of the rGRF3 gene, but did not change the amino acid sequence, such as... Figure 4 As shown in a.

[0349] ① First, amplify the GRF3-BsaI fragment.

[0350] Upstream primer GRF3-BsaI-F:

[0351] 5′-CGACGACAAGACCGTACCATGGATTTGCAACTGAAACAAT-3′, as shown in SEQ ID NO.28.

[0352] Downstream primer GRF3-BsaI-R:

[0353] 5′-ggtctccCTCTACCGGTTTCCTGCTCCTGTTGCGGCCACGGTGCAT-3′, as shown in SEQ ID NO.29.

[0354] ②Then, the BsaI-GRF3 fragment was amplified.

[0355] Upstream primer BsaI-GRF3-F:

[0356] 5′-GGTCTCCAGAGACTCCAACCACCGTCAATG-3′, as shown in SEQ ID NO.30.

[0357] Downstream primer BsaI-GRF3-R:

[0358] 5′-GAGGAGAAGAGCCGTGCATGAAAGGCTTGTGTCGAGAC-3′, as shown in SEQ ID NO.31.

[0359] ③ Finally, the full-length rGRF3 fragment was amplified using primer pair P2.

[0360] Upstream primer GRF3-BsaI-F: as shown in SEQ ID NO.28.

[0361] Downstream primer BsaI-GRF3-R: as shown in SEQ ID NO.31.

[0362] (2) Amplification of Pro GRF3 -5′-leader GRF -GRF3 fragment.

[0363] ① First. Amplify EcoRI-Pro GRF3 -5′-leader GRF3 -SacI fragment.

[0364] Upstream primer EcoRI-Pro GRF3 -5′-leader GRF3 -F:

[0365] 5′-CCTTAATTAACGTGAGCAAGGGCGAGGAGC-3′, as shown in SEQ ID NO.32;

[0366] Downstream primer Pro GRF3 -5′-leader GRF3 -SacI-R:

[0367] 5′-CCTTAATTAACTTGTACAGCTCGTCCATGCC-3′, as shown in SEQ ID NO.33;

[0368] ② Next, the GRF-LIC fragment was amplified using primer pair P2.

[0369] Upstream primer GRF-LIC-F: as shown in SEQ ID NO.28.

[0370] Downstream primer GRF-LIC-R: as shown in SEQ ID NO.31.

[0371] (3) Amplification of Pro GRF3 -5′-leader ΔY GRF3 -GRF3 fragment.

[0372] ① First, primer pair P3 amplifies EcoRI-Pro GRF Excerpt.

[0373] Upstream primer EcoRI-Pro GRF -F: As shown in SEQ ID NO.32;

[0374] Downstream primer EcoRI-Pro GRF -R:

[0375] 5′-CTGTATCTGAGCTTCTTCTGCACAGAGTAGAGGGAAAGAGGTTT-3′, as shown in SEQ ID NO.34;

[0376] ②Then, amplify the 5′-leader ΔY GRF3 Excerpt.

[0377] upstream primer 5′-leader ΔY GRF3 -F:

[0378] 5′-AACCTCTTTCCCTCTACTCTGTGCAGAAGAAGCTCAGATACAGAAAC-3′, as shown in SEQ ID NO.35;

[0379] Downstream primer 5′-leader ΔY GRF3 -R:

[0380] 5′-GGTCTTGTCGTCGAAGGTACCGAGCTCTTGTTGGTTACTTTAAGGATAATGG-3′, as shown in SEQ IDNO.36;

[0381] ③ Finally, the GRF-LIC fragment was amplified.

[0382] Upstream primer GRF-LIC-F: as shown in SEQ ID NO.28.

[0383] Downstream primer GRF-LIC-R: as shown in SEQ ID NO.31.

[0384] 2. PCR amplification

[0385] (1) PCR amplification using Arabidopsis cDNA as a template

[0386] Using the primers synthesized in step 1, the GRF3-BsaI, BsaI-GRF3, and GRF-LIC fragments were amplified by PCR. The amplification reaction system and specific reaction conditions are detailed in Example 1. The amplified PCR products were detected using a 1% agarose gel electrophoresis, and the target fragments were purified and recovered.

[0387] To obtain the full-length rGRF3 fragment, the GRF3-BsaI and BsaI-GRF3 fragments were digested and ligated. The reaction system is shown in Table 22 below.

[0388] Table 22

[0389]

[0390] The reaction procedure was: 37℃ for 2 h, 50℃ for 5 min, and 80℃ for 10 min.

[0391] Then, using the above ligation product as a template, PCR amplification was performed with primers GRF3-BsaI-F and BsaI-GRF3-R, and the full-length rGRF3 fragment was obtained after recovery.

[0392] (2) PCR amplification using plasmid as template.

[0393] The 5′-leader has already been constructed in this laboratory GRF3 With 5'-leader ΔY GRF3 The plasmid fragment was amplified by PCR using the primers synthesized in step 1. GRF3 With 5'-leader ΔY GRF3 The fragments, amplification reaction system, and PCR program are shown in Table 11 and the corresponding PCR program in Example 3.

[0394] (3) PCR amplification using Arabidopsis DNA as a template. Using the primers synthesized in step 1, PCR amplification of Pro... GRF3 The fragments, amplification reaction system, and PCR program are shown in Table 10 and the corresponding PCR program in Example 3.

[0395] 3. Connection between the target fragment and the vector

[0396] The amplified PCR products were detected using 1% agarose gel electrophoresis, and the target fragment was purified and recovered.

[0397] (1) Ligation of rGRF3 fragment and 35S::X-YFP vector

[0398] The rGRF fragment recovered in step 2 and the laboratory-preserved 35S::X-YFP vector were ligated using the LIC method. For details of the specific reaction system and steps, please refer to Example 1.

[0399] Take 1.5 μl each of the LIC-prepared 35S::X-YFP vector and rGRF3 fragment, mix well, and place in a PCR thermal cycler for reaction: 75℃ for 5 min, 22℃ for 10 min. After the reaction, store on ice or at -20℃.

[0400] (2) Pro GRF3 -5′-leader GRF3 - GRF3 fragment conjugation with 35S::X-YFP vector

[0401] ①Pro was digested with EcoRI and SacI restriction endonucleases. GRF3 -5′-leader GRF3 The fragment and the 35S::X-YFP support. The specific reaction system is shown in Table 23 below.

[0402] Table 23

[0403]

[0404] Briefly centrifuge to mix the above components, then digest with enzymes at 37°C for 60 min. Detect the digested PCR products and vector using a 1% agarose gel electrophoresis to recover the target fragment and vector fragment.

[0405] ②Connection

[0406] The recovered PCR products and vectors were ligated using T4 DNA ligase (NEB), and the ligation system is shown in Table 24 below.

[0407] Table 24

[0408]

[0409] Briefly centrifuge to mix the above components, and ligate at room temperature for 3 h. Take 5 μl of the ligation product and transform it with supercompetent E. coli DH5α to obtain Pro. GRF3 -5′-leader GRF3 -YFP vector. Then, for Pro... GRF3 -5′-leader GRF3 The YFP vector and the GRF-LIC fragment are joined using the LIC method, and the specific steps are detailed in Example 1.

[0410] (3) Pro GRF3 -5′-leader ΔY GRF3- GRF3 fragment conjugation with 35S::X-YFP vector

[0411] ①Use EcoRI-Pro GRF Fragments and 5′-leader ΔY GRF3 The fragment is recombined with 35S::X-YFP.

[0412] The 35S::X-YFP vector was digested with EcoRI and SacI restriction endonucleases. The reaction system and process are shown in Table 23 above.

[0413] ②Connection

[0414] Recycled EcoRI-Pro GRF Fragments and 5′-leader ΔY GRF3 The fragment was ligated to the recovered 35S::X-YFP vector fragment using a recombinase. The ligation system is shown in Table 25 below.

[0415] Table 25

[0416]

[0417] After briefly centrifuging to mix the above components, incubate at 55°C for 30 min in a PCR instrument, then quickly place on ice to cool.

[0418] Take 5 μl of the ligation product and transform it with supercompetent E. coli DH5α (see step 4 for detailed steps) to obtain Pro. GRF3 -5′-leader ΔY GRF3-YFP The vector is then linked to the GRF3-LIC fragment using the LIC method. Specific steps are detailed in section 3, ultimately yielding the Pro. GRF3 -5′-leader ΔY GRF3 -GRF3-YFP plasmid vector.

[0419] 4. Transformation of the constructed vector. The transformation methods for *E. coli* and *Agrobacterium* are detailed in Example 1.

[0420] II. Agrobacterium-mediated genetic transformation of Arabidopsis thaliana and screening of positive plants

[0421] 1. Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

[0422] (1) Wild-type Arabidopsis plants Col-0 or grf3 (SALK_026786) mutant materials were grown in a greenhouse under the following conditions: 12 h light / 12 h dark, temperature 22℃, and relative humidity 70%. Plants in full bloom were used for genetic transformation.

[0423] (2) The 35S::rGRF3-YFP and ProGRF3-5′-leader in the above steps GRF3 -GRF3-YFP and Pro GRF3 -5′-leader ΔY Agrobacterium GRF3-GRF3-YFP was cultured overnight in liquid LB medium containing Kana, Gent, and Rif antibiotics (OD600 approximately 1.0-2.0).

[0424] (3) The next day, centrifuge at 4,000 rpm / min for 10 min at room temperature and collect the bacterial cells. Resuspend Agrobacterium in 50 ml of osmotic culture medium (5% sucrose solution, 0.02% Silwet L-77) to make the OD600 around 0.8.

[0425] (4) Before infection, cut off the siliques and open flowers. Immerse the plant inflorescence in Agrobacterium suspension for 20-30 seconds. 35S::rGRF3-YFP bacterial solution is used for transformation of Col-0, Pro GRF3 -5′-leader GRF3 -GRF3-YFP and Pro GRF3 -5′-leader ΔY GRF3 -GRF3-YFP bacterial culture was used to transform the grf3 mutant.

[0426] (5) After infection, the plants should be cultured in the dark for 24 hours with a certain level of humidity to facilitate successful transformation.

[0427] 2. Plant selection

[0428] After the infected plant seeds mature, harvest the seeds and dry them in a 37℃ incubator for 3-4 days. Soak the seeds in sterile water and let them stand at 4℃ for 3 days. Sow the seeds in greenhouse potting soil and screen them using a 1 / 3000 Bastar solution after germination. Transplant the positive plants for subsequent experiments.

[0429] III. Observation of Disease Resistance Phenotypes in Transgenic Materials

[0430] 1. Observe the effects of exogenous SA spraying on 35S::rGRF3 plants.

[0431] Col-0, ptbp3 mutant, and 35S::rGRF3 plants were cultured in a greenhouse for approximately 21 days (Col-0 served as a control). A 5 mM SA solution was prepared and sprayed onto the leaves of the Col-0, ptbp3 mutant, and 35S::rGRF3 Arabidopsis plants, respectively; water was sprayed simultaneously as a control. Two days after treatment, the wilting of the leaves was quantified. The severity of wilting was categorized into three types: Type 1, no obvious wilting; Type 2, partial wilting; and Type 3, complete wilting. It was found that the wilting of the leaves of the ptbp3 mutant and 35S::rGRF3 plants was significantly less than that of the Col-0 plants, with most leaves surviving. This indicates that the leaves of the ptbp3 mutant and 35S::rGRF3 plants are not sensitive to SA and exhibit good growth. Figure 4 b and Figure 4 As shown in c.

[0432] 2. The 35S::rGRF3 plant showed resistance to the pathogen Psm ES4326.

[0433] (1) Plant culture

[0434] Col-0, grf3 mutant, npr1-1 (used as a control), 35S::rGRF3-1 and 35S::rGRF3-2 materials were cultured in a greenhouse for 22-24 days under the following conditions: 12 h light / 12 h dark, temperature 22℃ and relative humidity 70%.

[0435] (2) Activation and injection of Psm ES4326 pathogen

[0436] The activation and injection method for the Psm ES4326 pathogen is detailed in Example 1. Colony counts on the culture medium were recorded and analyzed using GraphPad Prism software. Experimental results showed that the 35S::rGRF3 plant exhibited resistance to the Psm ES4326 pathogen, a phenotype consistent with that of the ptbp3 mutant. Figure 4 As shown in d.

[0437] 3. The grf3 complementary material exhibited resistance to Psm ES4326 pathogen.

[0438] Col-0, grf3 mutants, and grf3 complementary materials were cultured in a greenhouse for 22-24 days under the following conditions: 12 h light / 12 h dark, temperature 22°C, and relative humidity 70%. The methods for activation, injection, leaf collection, plating, and statistical analysis of the Psm ES4326 pathogen were the same as in Example 1. Experimental results were analyzed and plotted using GraphPad Prism software. The results showed that the pathogen contained a complete 5′-leader... GRF3The sequence material was able to reconstruct the susceptibility phenotype of the grf3 mutant, while the 5′-leader ΔY GRF3 The material not only restored the susceptibility phenotype of the grf3 mutant, but also showed resistance to the Psm ES4326 pathogen, such as Figure 4 As shown in e and 4f.

[0439] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. The application of the RNA-binding protein / γ-motif regulatory model in balancing plant resistance and yield, characterized by: The RNA-binding protein / Y-motif regulatory model consists of RNA-binding proteins and Y-motif elements, and its structure is as follows: ； The RNA-binding protein specifically recognizes and binds to the Y-motif element on the mRNA of plant growth genes, thereby regulating the expression of the plant growth genes.

2. A method for balancing plant resistance and yield, characterized in that, The gene expressing the RNA-binding protein is transferred into a plant, causing the RNA-binding protein to be overexpressed. Upon immune activation, the RNA-binding protein specifically recognizes and binds to the Y-motif element on the plant growth gene mRNA, thereby regulating the expression of the plant growth gene.

3. The method for balancing plant resistance and yield according to claim 2, characterized in that, The RNA-binding protein is the Arabidopsis thaliana PTBP3 protein, and the amino acid sequence of the Arabidopsis thaliana PTBP3 protein is shown in SEQ ID NO.

43.

4. The method for balancing plant resistance and yield according to claim 3, characterized in that, The nucleotide sequence encoding the PTBP3 protein of the Arabidopsis thaliana is shown in SEQ ID NO.

37.

5. The method for balancing plant resistance and yield according to claim 3, characterized in that, The plant growth gene is the GRF3 gene.

6. A method for regulating the expression of plant growth genes, characterized in that, A gene fragment expressing an RNA-binding protein is transferred into a plant to overexpress the RNA-binding protein; the RNA-binding protein specifically recognizes and binds to the Y-motif element on the mRNA of a plant growth gene, thereby regulating the expression of the plant growth gene.

7. An RNA-binding protein / γ-motif regulatory model, characterized in that, The RNA-binding protein / Y-motif regulatory model consists of RNA-binding proteins and Y-motif elements, and its structure is as follows: ； The Y-motif element is a widely present and conserved RNA element in plants and animals; the RNA-binding protein is the PTBP3 protein of Arabidopsis thaliana, which specifically recognizes and binds to growth genes containing the Y-motif element.

8. A nucleic acid molecule, characterized in that, It contains genes that encode RNA-binding proteins.

9. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 8.

10. A biomaterial, characterized in that, It comprises the nucleic acid molecule of claim 8, or the recombinant expression vector of claim 9.

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