m 6 Application of RNA methylation modification genes in the creation of new drought-resistant walnut germplasm

Abstract

The application belongs to the field of molecular breeding, and specifically relates to m 6 A RNA methylation modification gene in creating new drought-resistant walnut germplasm, through the function research of a RNA methyltransferase gene and a demethylase gene, the core regulation role of m Juglans regia A in the drought resistance of walnut is revealed. 6 A methyltransferase gene JrMTA And demethylase gene JrALKBH9B The function research reveals that m 6 A modification plays a core regulation role in the drought resistance of walnut. JrALKBH9B The knockout by using CRISPR / Cas9 technology can significantly improve the m 6 A level of walnut, enhance the ability to remove reactive oxygen species (ROS) and reduce water loss, thereby greatly improving the drought resistance. JrMTA On the contrary, the loss of m 6 A leads to the decrease of the m A level, so that the plant is sensitive to drought. The application provides a key target for the molecular precise improvement of drought-resistant walnut varieties.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding, specifically m 6 Application of RNA methylation modification genes in the creation of new drought-resistant walnut germplasm. Background Technology

[0002] Walnuts are an important member of the Juglandaceae family, renowned as the "top of the four major dried fruits," and are also an important economic tree species native to my country. They are widely loved for their rich oil, nutrients, protein content, and various essential trace elements and minerals. In recent years, drought has become an increasingly prominent problem, a major bottleneck restricting the sustainable development of the walnut industry. In eukaryotic cells, m 6 A-type modifications account for up to 80% of all RNA base modifications, making them the most common type of RNA modification. Studies have shown that m 6 RNA methylation, one of the most common covalent modifications of RNA, is a dynamic and reversible process regulated by methyltransferase complexes (writers such as METTL3, ETTL14, TAP, and MTA) and demethylases (erasers such as FTO and ALKBH5). Studies have shown that posttranscriptional RNA methylation is crucial for plant responses to stress and can improve m... 6 A modification level can enhance the drought resistance of plants. Therefore, it is necessary to explore the relevant m-levels that regulate the drought response of walnut trees. 6 Discovering an RNA methylation modification gene and elucidating its regulatory mechanism is of great significance for breeding new drought-resistant walnut varieties that utilize water resources efficiently.

[0003] However, there is currently no information about walnut m. 6 A homologous gene of RNA methylation modification demethylase. JrALKBH9B Homologous genes of genes and methyltransferases JrMTA Reports on walnut plant responses to drought stress. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.

[0005] The technical solution adopted by this invention to solve its technical problem is: the m described in this invention 6 The application of an RNA methylation modification gene in the creation of new drought-resistant walnut germplasm, the m 6 A RNA methylation modification gene is JrMTA Genes and / or JrALKBH9B Gene; Among them, by suppressing the JrMTA Gene expression or activity is reduced, decreasing the drought resistance of walnuts; by inhibiting the expression or activity of the gene. JrALKBH9BGene expression or activity can enhance the drought resistance of walnuts.

[0006] As a further technical solution of the present invention: the regulation is achieved through CRISPR / Cas9 gene editing technology, RNA interference technology, or homologous recombination technology, affecting the walnut... JrMTA Genes and / or JrALKBH9B This is achieved by knocking out or mutating genes.

[0007] As a further technical solution of the present invention: the CRISPR / Cas9 gene editing technology includes the following steps: S1, targeting JrMTA Gene or JrALKBH9B Gene design and construction of CRISPR / Cas9 knockout vectors; S2. The knockout vector is converted into walnut somatic embryos; S3, after screening and regeneration, obtain JrMTA Genes and / or JrALKBH9B A walnut mutant with its genes edited.

[0008] As a further technical solution of the present invention: In S1, the knockout target designed for the JrMTA gene includes a first target near the N-terminus of its sequence and a second target near the N-terminus of its conserved protein functional domain; the knockout target designed for the JrALKBH9B gene includes a first target near the N-terminus of its sequence and a second target near the N-terminus of its conserved protein functional domain.

[0009] As a further technical solution of the present invention, it also includes inhibiting the expression or activity of the JrALKBH9B gene in walnuts; The inhibition is achieved by knocking out CRISPR / Cas9 technology. JrALKBH9B Genetic achievement, obtaining JrALKBH9B Mutant strains in which both strands of the gene have lost protein activity.

[0010] As a further technical solution of the present invention, it also includes inhibiting the growth of walnuts. JrMTA Gene expression or activity; The inhibition is achieved by knocking out CRISPR / Cas9 technology. JrMTA Genetically derived mutant lines in which at least one strand of the JrMTA gene loses its protein activity.

[0011] As a further technical solution of the present invention: the difference in drought resistance is manifested as follows: JrALKBH9B The mutant strains exhibit stronger drought resistance than the wild type; and / or, JrMTA Mutant strains are less drought resistant than wild-type strains.

[0012] As a further technical solution of the present invention: constructing a walnut JrALKBH9B Gene mutants; The obtained mutants were evaluated for drought resistance. The evaluation included measuring at least one of the following indicators: plant height, internode length, degree of wilting of detached leaves, dry and fresh weight, stomatal aperture, chlorophyll content, proline content, malondialdehyde content, superoxide anion free radical content, or hydrogen peroxide content. Select mutant strains with higher drought resistance than wild-type walnuts; these are the drought-resistant walnut plants.

[0013] The beneficial effects of this invention are as follows: This invention, by constructing a genetic transformation system for walnuts, elucidated the genetic characteristics of walnut m... 6 A mechanism by which methylation regulates drought resistance in walnuts was demonstrated by knocking out key genes for methyltransferases. JrALKBH9B It can effectively improve the drought resistance of plants and knock out key genes for methyltransferase. JrMTA It can effectively reduce the drought resistance of plants, and has important theoretical value and practical significance for breeding drought-resistant walnut varieties. Attached Figure Description

[0014] The invention will now be further described with reference to the accompanying drawings.

[0015] Figure 1 This is a schematic diagram of the knockout vector construction process; among which, Figure 1 A-1 in the middle: JrMTA- Schematic diagram of CRISPR / Cas9 knockout vector construction; Figure 1 A-2 in the middle: JrMTA Agarose gel electrophoresis image of the amplified products of the four primers; Figure 1 A-3 in the middle: JrMTA Agarose gel electrophoresis image for bacterial testing; Figure 1 B-1 in the middle: JrALKBH9B - Schematic diagram of CRISPR / Cas9 knockout vector construction; Figure 1 B-2 in the middle: JrALKBH9B Agarose gel electrophoresis image of the amplified products of the four primers; Figure 1 B-3 in the middle: JrALKBH9B Agarose gel electrophoresis image for bacterial testing; Note: M is a 2000 bp marker, lanes 1-5 are PCR electrophoresis bands of the correct size.

[0016] Figure 2 It's a walnut. JrMTA and JrALKBH9B Diagram showing the base changes at the target site in the knockout mutant; among which... Figure 2 A in the text: walnut JrMTA Knockout mutant target site base changes; Figure 2 B in the text: walnut JrALKBH9B Knockout mutant target base changes.

[0017] Figure 3 It is walnut WT and JrMTA, JrALKBH9B A schematic diagram of somatic embryonic development and plant regeneration in mutants; Figure 4 It's a walnut. JrMTA and JrALKBH9B Schematic diagram illustrating the effects of gene mutations on plant height and internode length; Figure 4 A in the figure: Phenotypic diagrams of test-tube seedlings at different time points under normal culture conditions; Figure 4 B in the text refers to the plant height after 2 weeks of normal culture. Figure 4 C in the figure represents the intersegmental length at 2 weeks under normal culture conditions; Note: abbreviation in the legend. MTA -1、 MTA -2 indicates a mutant. JrMTA-1, JrMTA-2 , ALKBH9B-1, ALKBH9B-2, ALKBH9B -3 indicates a mutant. JrALKBH9B -1 、JrALKBH9B -2 、JrALKBH9B -3; the letters indicate the significance of one-way ANOVA, and different lowercase letters indicate significant differences (P<0.05).

[0018] Figure 5 It is a 5% PEG-8000 simulating drought stress in walnuts JrMTA and JrALKBH9B Schematic diagram of the effects of gene mutation on detached leaves and test-tube seedlings; Figure 5 A in the figure: Phenotypic diagram of detached leaves under simulated drought stress with 5% PEG-8000; Figure 5 B in the figure: Phenotypic images of test-tube seedlings under simulated drought stress at different times with 5% PEG-8000; Figure 5 C-1:5% PEG-8000 in the formula simulates the fresh weight of test-tube seedlings at different times under drought stress; Figure 5 C-2:5% PEG-8000 in the formula simulates the dry weight of test-tube seedlings at different times under drought stress; Figure 6 It's a walnut. JrMTA and JrALKBH9B Schematic diagram of the effect of gene mutation on the natural water loss rate of plant leaves; Figure 7 It is a 5% PEG-8000 simulating drought stress in walnut JrMTA and JrALKBH9B Schematic diagram illustrating the effect of gene mutation on stomatal morphology; Figure 7 A in the figure: Stomatal phenotypes at different time points under simulated drought stress using 5% PEG-8000; Figure 7 B in the formula represents 5% PEG-8000, which simulates the stomatal aperture ratio at different time points under drought stress. Figure 8 This is a schematic diagram simulating leaf color under drought stress using 5% PEG-8000; Figure 8 A: Leaf color phenotype diagram under simulated drought stress using 5% PEG-8000; Figure 8 B-1: Chlorophyll content in the control group; Figure 8 B-2:5% PEG-8000 simulates chlorophyll content under drought stress; Figure 9 It's a walnut. JrMTA and JrALKBH9B Schematic diagram showing the effect of gene mutation on proline and MDA content; Figure 9 A-1 in the control group: proline content; Figure 9 The A-2 component contains 5% PEG-8000, which simulates proline content under drought stress. Figure 9 B-1: Malondialdehyde content in the control group; Figure 9 B-2:5% PEG-8000 simulates malondialdehyde content under drought stress; Figure 10 It is a 5% PEG-8000 simulating drought stress in walnut JrMTA and JrALKBH9B Schematic diagram of the effect of gene mutation on leaf chemical staining; Figure 10 A-1 in the figure: DAB staining phenotypes at different time points; Figure 10 A-2: Gray value of DAB staining phenotype; Figure 10 B-1 in the text: NBT staining phenotypes at different time points; Figure 10 B-2: Gray value of NBT staining phenotype; Figure 11 It is a 5% PEG-8000 simulating drought stress in walnut JrMTA and JrALKBH9B Schematic diagram illustrating the effect of gene mutations on ROS levels; Figure 11 A-1 in the control group: H2O2 content; Figure 11 A-2 in the formula simulates the H2O2 content under drought stress (5% PEG-8000). Figure 11 B-1 in the control group: O2• － content; Figure 11 B-2:5% PEG-8000 in the formula simulates O2• under drought stress. － content. Detailed Implementation

[0019] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0020] Example 1 This invention provides a method for constructing walnuts JrMTA and JrALKBH9BPlant materials, experimental reagents, and plasmids for the CRISPR / Cas9 genetic transformation system; Among them, the plant material: walnut somatic embryos came from the State Key Laboratory for Subtropical Forest Cultivation jointly established by the province and the ministry, and were derived from California black walnuts (Kiwifruit). J.hindsi × J.regia Secondary somatic embryos induced from immature zygote embryos were used as wild-type walnut somatic embryos, and well-grown walnut somatic embryos were selected as gene editing materials. Walnut somatic embryos were cultured in a dark chamber at (25±2)℃.

[0021] Experimental reagents: Plasmid DNA mini-extraction kit purchased from Tiangen Biotech Co., Ltd.; Column DNA gel extraction kit purchased from Sangon Biotech Co., Ltd.; DKW medium (solid and liquid); Auxin (IAA); Indole-3-butyric acid (IBA); PrimeSTAR® Max DNA Polymerase (Takara, R045Q); Agarose gel; 1× TAE buffer; 4× TAE buffer; YeaRed nucleic acid dye (Yeasen); 2000 DAN Marker; 10× DAN / RAN Loading Buffer; T4 DNA Ligase (Takara). Bsa I (NEW ENGLOAD Biolabs, R0535).

[0022] Plasmid vectors: pCBC-DT1DT2 (amplification vector), pHSN401 vector (knockout vector). pEASY -Blunt vector (identification vector).

[0023] Strains: GV3101 Agrobacterium competent cells, BL21 Escherichia coli competent cells, T1 Escherichia coli competent cells.

[0024] This invention provides walnuts JrMTA and JrALKBH9B The method for constructing a CRISPR / Cas9 genetic transformation system for genes includes the following steps: S1. Target Selection Criteria: The sgRNA consists of a 19 bp target knockout site and NGG (N can be any ATCG, with priority order G>C>A>T). Of the two designed target sites, the first knockout target is located near the N-terminus of the entire sequence, aiming to inactivate the entire protein after knockout. The second knockout target is located near the N-terminus of the protein's conserved functional domain, aiming to inactivate the conserved functional domain after knockout. Finally, the knockout target sequence is unique in the entire walnut genome and contains no more than three off-target mismatches to minimize off-target effects in subsequent experiments and ensure successful knockout. S2. Target Design Methodology: Target sites are designed using the online tool Cas-Designer website and submitted. JrMTA as well as JrALKBH9B After obtaining the CDS sequence and the walnut genome sequence file, information on multiple target sites containing the PAM sequence on this gene was obtained. Primer sequences for the pCBC-DT1DT2 amplification vector were designed and are shown in Table 1. Table 1 CRISPR / Cas9 target cloning primer sequences

[0025] S3. Target amplification and ligation: Four-primer PCR amplification was performed using pCBC-DT1T2 diluted 100 times as a template. JrMTA / JrALKBH9B -BsF and JrMTA / JrALKBH9B -BsR is used at the normal primer concentration. JrMTA / JrALKBH9B -F0 and JrMTA / JrALKBH9B - R0 was diluted 20-fold and added to the system; the amplified PCR product was mixed with 6× DNA / RNA Loading Buffer and electrophoresed simultaneously with DL 2000 Marker in a 1.35% agarose gel. The electrophoresis voltage was set to 135 V. After electrophoresis for 20 min, the target gel band was cut and recovered under UV light. The target band size was about 750 bp.

[0026] The total volume of the PCR reaction system was 50 µL, including 25 µL of PrimeSTAR Max Premix (2×) at a final concentration of 1×; upstream outer primers. JrMTA / JrALKBH9B -DT1-BsF and downstream outer primer JrMTA / JrALKBH9B The final concentration of -DT2-BsR was 0.2 µM, and the upstream inner primer... JrMTA / JrALKBH9B -DT1-F0 and downstream inner primer JrMTA / JrALKBH9B The final concentration of DT2-R0 was 0.02 µM; the amount of template pCBC-DT1T2 added did not exceed 200 ng, and the remaining volume was made up with double-distilled water. The PCR amplification program was as follows: First, pre-denaturation was performed at 95℃ for 5 minutes; then, the amplification cycle began, with each cycle consisting of denaturation at 98℃ for 10 seconds, annealing at 55℃ for 5 seconds, and extension at 72℃ for 5 seconds, for a total of 30 cycles; after each cycle, a final extension at 72℃ for 10 minutes was performed. The amplified products were separated by 1.35% agarose gel electrophoresis, and the target band of approximately 750 bp was excised under UV light, purified, and used for subsequent vector construction.

[0027] S4. Obtaining the knockout plasmid: The recovered PCR product and the pHSN401 vector plasmid were simultaneously linearized by single-enzyme digestion with restriction endonuclease Bsa I, and incubated at 37°C for 1 h. The digestion products were recovered by DNA gel electrophoresis, following the same procedure as above. The recovered products were mixed with T4 ligase in a certain ratio for sticky end ligation, and incubated at 16°C for 3 h.

[0028] The total volume of the enzyme digestion system is 50 µL, which includes 20 µL (approximately 2 µg) of PCR product or pHSN401 plasmid, and restriction endonuclease. Bsa I 1 µL, 10× NEBuffer 5 µL, and the remaining volume is made up with double-distilled water.

[0029] The total volume of the ligation reaction system was 20 µL, which included 1 µL of T4 DNA ligase, 2 µL of 10× T4 DNA ligase buffer, 12 µL of target PCR product after enzyme digestion, and 5 µL of pHSN401 vector fragment after enzyme digestion.

[0030] The ligation product was introduced into T1 E. coli competent cells via heat shock transformation. Single colonies with good growth and appropriate size were selected and incubated in 700 mL of LB+Kan liquid medium at 37°C for 3 h in a shaker. Sequencing primers U626-IDF are shown in Table 3.1. The sequencing results were compared with the constructed vector sequence. The two target sequences and the CAS9 protein sequence were completely identical, confirming the successful construction of the binary knockout vector pHSN401. The recombinant plasmid pHSN401-DT1DT2 and the E. coli strain were obtained. A portion of the engineered bacteria was cultured overnight in 10 mL of LB+Kan liquid medium, and the plasmid was then extracted.

[0031] S5. Agrobacterium-mediated transformation of wild-type walnut embryos: The recombinant plasmid pHSN401-DT1DT2 was transformed into wild-type walnut embryos using the Agrobacterium-mediated transformation method. Specifically: Preparation of Agrobacterium: 100 μL of competent Agrobacterium cells were removed from a -80℃ cryogenic freezer and thawed on ice. The extracted pHSN401-DT1DT2 recombinant plasmid was mixed with GV3101 and 50 μL of competent Agrobacterium cells in a 1.5 mL sterile EP tube and incubated on ice for 30 min. The mixed competent cells were then quickly transferred to liquid nitrogen and incubated for 5 min. The liquid nitrogen-frozen competent cells were then quickly transferred to a preheated 37℃ water bath and incubated for 5 min. The incubated product was then quickly transferred to ice and incubated for 2 min.

[0032] Add 500 mL of antibiotic-free LB medium, pre-seasoned to room temperature, to a 1.5 mL EP tube. Incubate competent cells in a 28°C shaker for 2 h. Centrifuge at 8000 rpm for 1 min at room temperature to allow bacterial aggregates to settle on the sidewalls of the EP tube, discarding most of the culture medium waste. Resuspend Agrobacterium using the remaining LB medium liquid, then evenly spread it onto LB solid medium containing rifampicin (RIF) and kanamycin (KAN) resistance. Invert the spread LB+Kan+RIF solid medium and incubate overnight at 28°C.

[0033] Select healthy, appropriately sized, milky-white single colonies from a clean bench and incubate them in 500 mL of LB+Kan+RIF liquid medium at 37°C for 5 h on a shaker. Then, take 1 mL of the medium, add 400 μL of 50% glycerol, and store at -80°C for later use.

[0034] Preparation of infecting bacterial culture: Take 100 μL of prepared Agrobacterium, stored at -80℃, into a 10 mL sterile tube, add 5 mL of LB+Kan+RIF liquid medium, and incubate overnight at 28℃ on a shaker for 12 h. Pour the obviously turbid bacterial culture after overnight incubation into a 50 mL sterile tube, add another 20 mL of LB+Kan+RIF liquid medium, and continue to propagate Agrobacterium at 28℃ on a shaker. Measure the OD of the bacterial culture every 1 h. 600 Value, until the bacterial culture OD 600 The value is the optimal value. 50 mL of the correct OD... 600 Centrifuge the bacterial suspension at room temperature at 6000 rpm for 10 min, discard the supernatant culture medium, and allow the bacterial clump to settle at the bottom of the tube. Add 30 mL of AS liquid culture medium to the settled bacterial clump, while gently shaking the test tube to accelerate the dissolution of the bacterial clump, until no obvious bacterial clump is visible and the Agrobacterium suspension is milky white.

[0035] Infection: Select 20 wild-type walnut embryos that are translucent, healthy, and have many small growth points, and culture them in DKW medium in a dark incubator for 3 days to ensure that their growth status is roughly the same before use.

[0036] In a clean bench, walnut embryos with uniform growth status are immersed in the milky white Agrobacterium bacterial solution prepared in step 2. After 15 minutes, they are removed with tweezers and placed on pre-sterilized and dried filter paper. The air speed of the clean bench is adjusted and the embryos are dried for 30 minutes until the excess bacterial solution is dried and no obvious bacterial solution is visible on the walnut embryos.

[0037] Dried walnut embryos were inoculated into AS solid medium, and labeled with numbers on the bottom of the petri dishes. They were then co-cultured in a dark incubator at 28°C. After co-culture, the embryos should be promptly destered to prevent affecting subsequent embryo growth. During co-culture, it is important to monitor for contamination and the amount of embryo bacteria in the medium.

[0038] Sterilization: Prepare 6 sterilized empty bottles, 1 bottle of 3 L sterile water, 1 bottle of 1 L antibiotic solution, several sterile filter papers, and 3 pairs of tweezers. Sterilize the laminar flow hood for at least 40 minutes beforehand. In the laminar flow hood, remove the co-cultured walnut embryos and place them in a sterilized 500 mL Erlenmeyer flask. Pour in 300 mL of sterile water, continuously shaking the flask to accelerate the elution process. Each time, transfer the embryos to a new sterilized empty Erlenmeyer flask. Then, transfer the embryos to a new Erlenmeyer flask, add the antibiotic solution, and continue shaking until the eluted antibiotic solution is clear. Each time, replace the flask with a new clean, sterilized empty Erlenmeyer flask. Place the thoroughly eluted and cleaned infected walnut embryos on sterilized filter paper and blot dry in the laminar flow hood. Inoculate the fully dried walnut embryos into antibiotic culture medium, subculturing daily. After one week, when little new bacterial growth has occurred, transfer them to normal DKW medium for subsequent positive identification.

[0039] Walnut JrMTA and JrALKBH9B Mutant somatic embryonic site identification: Twenty labeled primary infective embryos were designated as generation E0. Newly formed embryos from the E0 generation were labeled as generation E1 and cultured separately. After multiple subcultures, the E3 generation embryos were labeled to differentiate the strains. Tissue samples from each individually cultured E3 generation walnut mutant embryo were taken, and embryonic DNA was extracted using the Trizol method. respectively JrMTA / JrALKBH9B -DT1-F / R, JrMTA / JrALKBH9B Using DT2-F / R as primers, and DNA extracted from each strain as templates, PCR amplification was performed on the DNA sequences flanking the knockout target. The PCR product was recovered via DNA gel extraction, following the same recovery steps as above. 3 μL of the recovered product was mixed with 0.2 μL of pre-linearized... pEASY- The Blunt vector was mixed and ligated at room temperature for 15 min. T1 competent cells were then transformed using heat shock transformation, following the same procedure as above. The bacterial culture was then evenly spread onto LB+Kan plates and incubated upside down overnight. After overnight incubation, uniformly sized, milky-white single colonies were picked and cultured in 700 mL LB+Kan liquid medium for 5 h. The culture was then sent to Qingke Biotechnology Co., Ltd. for sequencing, using M13-F / R primers. The successfully sequenced sequence corresponds to the walnut genome. JrMTA , JrALKBH9B DNA sequence alignment was performed to determine whether the knockout target sequence was deleted or inserted. Based on the three-codon translation principle, the edited sequence was converted into a protein sequence and compared with the original walnut JrMTA and JrALKBH9B protein sequences to determine the knockout status of the target sequence and the knockout status of conserved protein domains. The CRISPR / Cas9 plasmid identification primers are shown in Table 2 below. Table 2 Primers for CRISPR / Cas9 plasmid identification

[0040] Somatic embryos of walnut mutants and wild-type walnut (WT) strains that have been identified were selected and cultured at a constant temperature of 25°C in a dark room. They were transferred to DKW solid medium at one-week intervals and cultured for approximately one month. The growth and development of the embryos were observed using a stereomicroscope, and photographs were taken to record the developmental stages. Once the mutant and WT wild-type embryos had matured, they were transferred to dry, sterile culture dishes and dehydrated for 4-7 days. The dehydration status of the embryos was observed, and they were then transferred to test tubes containing DKW+IAA+IBA solid medium to germinate into walnut plants. The light-dark culture interval was 12 hours, and photographs were taken to record the results.

[0041] Results and Analysis Walnut JrMTA and JrALKBH9B Construction of the knockout vector: JrMTA and JrALKBH9B The construction of the knockout vector mainly includes two knockout sites, a 35S promoter, the Cas9 protein, and two sgRNAs. The 35S promoter initiates the expression of the Cas9 protein sequence in the plasmid. DT1-sgRNA and DT2-sgRNA are two 19bp target sites selected through screening that bind to the sgRNA. At the same time, there are two U626 and U629 promoters before and after the sgRNA, which enhance sgRNA expression. At this point, the sgRNA binds and guides the Cas9 protein to bind to and cleave the gene at the target site. The products of four-primer PCR amplification using pCBC-DT1T2 as a template were detected by agarose gel electrophoresis. The results showed... JrMTA and JrALKBH9B The band sizes are all around 750bp, which is as expected, and they can be recovered for further experiments (see...). Figure 1A-2 in Figure 1 (B-2 in the middle).

[0042] The obtained target fragment and the pHSN401 vector plasmid were simultaneously passed through restriction endonucleases. Bsa Linearization by I single enzyme digestion was performed, and the obtained product was transformed into E. coli for bacterial testing. The results showed... JrMTA and JrALKBH9B The colony PCR results were all 750 bp in size, as expected. The bacterial cultures with the corresponding bands were then sent for testing. Plasmids with the correct sequences were transformed into Agrobacterium and used for infection (see...). Figure 1 A-3 in Figure 1 (B-3 in the middle).

[0043] The construction of the walnut somatic embryo genetic transformation system includes: Walnut JrMTA and JrALKBH9B Somatic embryo site identification: for identifying walnuts JrMTA Gene editing details of the mutants, based on the walnut genome. JrMTA Identification primers were designed based on the DNA sequences from both sides. After sequencing, the sequencing results were compared with the WT gene using DNAMAN 6.0 software, and two strains were found to be positive. JrMTA -1、 JrMTA -2) Successfully edited. Based on protein function retention from high to low, [the following were listed]: JrMTA Mutants are named JrMTA -1、 JrMTA -2, where JrMTA Both chains of -1 can retain their protein activity. JrMTA -2 One of the chains loses part of its protein activity. JrALKBH9B Mutants are named JrALKBH9B -1, 2, 3, among which JrALKBH9B Both chains of -1 can retain their protein activity. JrALKBH9B -2 One of the chains loses its protein activity. JrALKBH9B -3 double strands both lose their protein activity (see...) Figure 2 ).

[0044] Walnut JrMTA and JrALKBH9B Plant regeneration from positive somatic embryos: Walnut JrMTA and JrALKBH9B After multiple subcultures, the mutant E3 generation embryos showed stable growth and development. Walnut embryos were observed and recorded using a stereomicroscope. JrMTA and JrALKBH9B The growth and development stages of the mutant somatic embryo and the wild-type walnut somatic embryo were compared. The mutant embryo sequentially progressed through four developmental stages: globular embryo, heart-shaped embryo, torpedo-shaped embryo, and cotyledonous embryo. This process is consistent with the growth and development process of the wild-type walnut somatic embryo, indicating that the mutant walnut... JrMTA and JrALKBH9B Gene editing did not affect its somatic embryonic growth and development stage.

[0045] Walnuts JrMTA and JrALKBH9B After being dehydrated and dried for 2-6 days, the mutant positive somatic embryos were transferred to DKW solid medium for further culture. Plant regeneration was then observed and recorded. It was found that after one week, the cotyledons of the mature somatic embryos gradually turned green, and true leaves formed. Following this, after 2-3 weeks, the mature somatic embryos gradually grew radicles downwards, and young buds and stems began to form upwards, gradually developing into complete plants. This process is similar to the regeneration process of wild-type walnut somatic embryos. This indicates that gene editing did not affect the somatic embryo to plant regeneration stage (see...). Figure 3 ).

[0046] Example 2 This invention provides walnuts JrMTA and JrALKBH9B The effect of gene mutation on drought resistance of plants was studied using plant materials: walnut seedlings were all from the National Key Laboratory for Subtropical Forest Cultivation jointly established by the Ministry of Agriculture and Rural Affairs. J.hindsii × J.regia ), including wild-type walnut plants (WT), and identified JrALKBH9B Three mutant lines ( JrALKBH9B -1, 2, 3), JrMTA Two mutant lines ( JrMTA -1, 2). Walnut seedlings were cultivated in a culture room at a constant temperature of 25 (±2)℃, humidity of 80-85%, light intensity of 15000 lx-20001 lx, and a photoperiod of 8 / 16 h. Afterwards, robust and uniformly shaped walnut seedlings were selected for simulated drought stress and other experimental treatments.

[0047] And walnuts JrMTA and JrALKBH9B The effect of gene mutations on plant drought resistance was analyzed as follows: Under drought stress JrMTA and JrALKBH9B The process of analyzing the phenotype of mutant lines is as follows: Morphological analysis of in vitro tissue culture seedlings under drought stress: Walnut WT seedlings with good growth and uniform vigor were selected. JrALKBH9B , JrMTA Tissue culture seedlings were cut and placed in a culture medium simulating a drought environment for 3 weeks, retaining 2-3 pairs of leaflets from the apical bud downwards. The control group was prepared using DKW solid medium without 5% PEG-8000. During this period, the plant condition was observed and photographed every week, and the number of internodes was recorded. At the same time, the plant height and internode length were measured and recorded with a ruler.

[0048] Dry and fresh weight determination under drought stress: Two weeks after drought treatment, WT (weight and dry weight) of walnuts with good growth and uniform growth were selected. JrALKBH9B , JrMTA The fresh weight of the tissue culture seedlings was measured on a balance with a weight of 0.01%. The plants were then placed in disposable petri dishes and dried in an oven for 6 hours. During this period, the plants were weighed every hour until the weight no longer changed, and this was recorded as the dry weight of the plants. Each group was set up with 3 biological replicates.

[0049] Phenotypic analysis of detached leaves under drought stress: Walnut WT and walnut leaves of the same growth cycle were selected respectively. ALKBH9B , MTA Leaves of uniform size from the apical bud to the 3rd-5th leaves of the mutant lines were placed in an aqueous solution containing 10 mL of 5% PEG-8000 for in vitro leaf stress treatment until wilting of the WT leaves was observed, at which point the stress treatment ended. Sterile water without PEG-8000 was set up as a control group.

[0050] Analysis of stomatal aperture of regenerated plants under drought stress: Using the nail polish imprinting method, after treatment with 0 W and 2 W in simulated drought environments, WT and W samples were taken from the same part of the walnut plant. JrALKBH9B , JrMTA Apply a layer of nail polish evenly to the lower surface of the first leaf below the apical bud of the gene knockout lineage in a clean disposable culture dish. Wait 5-10 minutes at room temperature until the nail polish has completely dried into a film. Carefully peel off this film with tweezers, place it on a glass slide, add a drop of water, cover with a coverslip, and observe and photograph it under a biological microscope.

[0051] Under drought stress JrMTA and JrALKBH9B The analytical process for histochemical staining of mutant lines is as follows: NBT staining: NBT staining solution 0.5 mg / mL, freshly prepared and used immediately. Cut samples from plants subjected to drought stress for 0 W, 1 W, and 2 W, respectively. JrALKBH9B , JrMTA Leaves from mutant lines and WT walnut tissue culture seedlings were transferred to large test tubes, and 20 mL of prepared NBT staining solution was added. Staining was carried out at room temperature in the dark for 1 h, with observation every 10 min until a significant color change was observed. Then, 30 mL of anhydrous ethanol was added, and the tubes were placed in a boiling water bath until the leaves showed no green color. After staining, the leaves were fixed in a petri dish containing filter paper with 5% glycerol, the glycerol was discarded, and the tubes were allowed to air dry before photographing.

[0052] DAB staining: Prepare DAB staining solution at 1 mg / mL, use immediately after preparation, and follow the same steps as NBT staining.

[0053] Under drought stress JrMTA and JrALKBH9B Physiological parameters of mutant plants were measured, including leaf color, chlorophyll content, natural water loss rate, proline content, MDA content, and superoxide anion radical (O2•) content. - Content determination of hydrogen peroxide (H2O2) content.

[0054] Results and Analysis like Figure 4 As shown, walnuts JrMTA and JrALKBH9B The effects of gene mutation on plant height and internode length: The results showed that... JrALKBH9B Compared to WT, the plants after gene knockout were taller and had longer internodes; JrMTA The plant height and internode length after gene knockout were not significantly different from those after WT.

[0055] like Figure 5 As shown, phenotypic analysis of detached leaves and test-tube seedlings under drought stress: Detached leaves of plants were placed in PEG solution for 12 h, and walnut WT and test-tube seedlings were subjected to drought stress. JrMTA and JrALKBH9B The mutant lines were cultured in 5% PEG-8000 DKW solid medium for 3 weeks, and their dry and fresh weights were measured, providing preliminary results. JrMTA The mutant strains were less drought-tolerant than the WT strains. JrALKBH9B The mutant strains are more drought resistant than the WT strains.

[0056] like Figure 6 As shown, walnuts JrMTA and JrALKBH9B The effect of gene mutation on natural water loss rate: Studies have shown that plants with strong stress resistance can enhance their drought tolerance by reducing the rate of water loss from leaves. As can be seen from the figure, the natural water loss rate of each strain increases continuously over time, especially in walnut plants. JrMTA , JrALKBH9B The natural water loss rate of each mutant line and WT was significantly increased.

[0057] like Figure 7 As shown, walnuts JrMTA and JrALKBH9B Effects of gene mutations on stomatal morphology: Results show that knockout JrALKBH9B Genes can reduce stomatal aperture in plants, thereby increasing the drought tolerance of walnut trees. Knocking out… JrMTA Genes can increase the pore size, thus reducing the plant's drought resistance.

[0058] like Figure 8 As shown, walnuts JrMTA and JrALKBH9B The effect of gene mutation on chlorophyll content: Experimental results show that drought inhibits chlorophyll synthesis in plants, and knockout... JrALKBH9BThe gene leads to increased chlorophyll content, affecting walnut photosynthesis and thus increasing the walnut's drought resistance. Knockout... JrMTA Genes can cause a decrease in chlorophyll content, reducing the drought resistance of walnuts.

[0059] like Figure 9 As shown, walnuts JrMTA and JrALKBH9B Effects of gene mutations on proline and MDA content: Experimental results show that knockout JrALKBH9B Genes can enhance the stability of plant cells, thereby improving the drought resistance of walnut trees. Knockout... JrMTA Genes can lead to a decrease in proline content in cells, reducing plant cell stability and decreasing the drought resistance of walnut trees.

[0060] like Figure 10 As shown, walnuts JrMTA and JrALKBH9B The effect of gene mutations on ROS levels: knockout JrALKBH9B The gene reduced the level of reactive oxygen species in walnut trees under drought stress, thereby improving the plant's drought resistance. Knockout... JrMTA Genes can cause walnuts to have increased reactive oxygen species levels under drought stress, thus reducing their drought resistance.

[0061] like Figure 11 As shown, walnuts JrMTA and JrALKBH9B Gene mutations affect H2O2 and O2• - Effects on content: knockout JrALKBH9B Genes can reduce O2• in walnut leaves - The accumulation of H2O2 enhances the plant's tolerance to drought stress. Knocking out... JrMTA Genes can increase O2• in walnut leaves under drought stress - Increased accumulation of H2O2 reduces tolerance to drought stress.

[0062] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. m 6 The application of an RNA methylation modification gene in the creation of new drought-resistant walnut germplasm is characterized by... The m 6 A RNA methylation modification gene is JrMTA Genes and / or JrALKBH9B Gene; Among them, by suppressing the JrMTA The expression or activity of the gene reduces the drought resistance of walnuts; the drought resistance of walnuts is improved by inhibiting the expression or activity of the JrALKBH9B gene.

2. The m according to claim 1 6 The application of an RNA methylation modification gene in the creation of new drought-resistant walnut germplasm is characterized by: The regulation is achieved through CRISPR / Cas9 gene editing technology, RNA interference technology, or homologous recombination technology, affecting the walnut... JrMTA Genes and / or JrALKBH9B This is achieved by knocking out or mutating genes.

3. The m according to claim 2 6 The application of an RNA methylation modification gene in the creation of new drought-resistant walnut germplasm is characterized by: The CRISPR / Cas9 gene editing technology includes the following steps: S1, targeting JrMTA Gene or JrALKBH9B Gene design and construction of CRISPR / Cas9 knockout vectors; S2. The knockout vector is converted into walnut somatic embryos; S3, after screening and regeneration, obtain JrMTA Genes and / or JrALKBH9B A walnut mutant with edited genes.

4. The m according to claim 3 6 The application of an RNA methylation modification gene in the creation of new drought-resistant walnut germplasm is characterized by: In S1, targeting JrMTA Knockout targets designed for gene editing include a first target near the N-terminus of the gene and a second target near the N-terminus of the conserved functional domain of the gene; targeting JrALKBH9B Knockout targets in gene design include a first target near the N-terminus of the gene and a second target near the N-terminus of the conserved functional domain of the gene.

5. The m according to claim 4 6 The application of an RNA methylation modification gene in the creation of new drought-resistant walnut germplasm is characterized by: It also includes inhibiting the growth of walnuts. JrALKBH9B Gene expression or activity; The inhibition is achieved by knocking out CRISPR / Cas9 technology. JrALKBH9B Genetic control was used to obtain mutant strains in which both strands of the JrALKBH9B gene lost protein activity.

6. The m according to claim 5 6 The application of an RNA methylation modification gene in the creation of new drought-resistant walnut germplasm is characterized by: It also includes inhibiting the growth of walnuts. JrMTA Gene expression or activity; The inhibition is achieved by knocking out CRISPR / Cas9 technology. JrMTA Genetic achievement, obtaining JrMTA A mutant strain in which at least one strand of a gene has lost its protein activity.

7. The m according to claim 6 6 The application of an RNA methylation modification gene in the creation of new drought-resistant walnut germplasm is characterized by: Differences in drought resistance are manifested as follows: JrALKBH9B The mutant strains exhibit stronger drought resistance than the wild type; and / or, JrMTA Mutant strains are less drought resistant than wild-type strains.

8. The m according to claim 7 6 The application of an RNA methylation modification gene in the creation of new drought-resistant walnut germplasm is characterized by: Building Walnuts JrALKBH9B Gene mutants; The obtained mutants were evaluated for drought resistance. The evaluation included measuring at least one of the following indicators: plant height, internode length, degree of wilting of detached leaves, dry and fresh weight, stomatal aperture, chlorophyll content, proline content, malondialdehyde content, superoxide anion free radical content, or hydrogen peroxide content. Select mutant strains with higher drought resistance than wild-type walnuts; these are the drought-resistant walnut plants.

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