Application of negative regulation of ZmSWEET4b gene or its coded protein in improving resistance of corn to sheath blight
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENYANG AGRI UNIV
- Filing Date
- 2026-03-10
- Publication Date
- 2026-05-29
Smart Images
Figure CN122104780A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of plant molecular biology and genetic engineering technology, specifically involving negative regulation. ZmSWEET4b Application of genes or their encoded proteins in improving maize resistance to sheath blight. Background Technology
[0002] Corn sheath blight is caused by Rhizoctonia solani (… Rhizoctonia solani ) caused by occurring in corn ( Zea mays L. Sheath blight is a disease that primarily affects the base of the corn stem and roots, but can also damage the leaves and ears. Initially, the infection site of *Rhizoctonia solani* appears as water-soaked lesions, gradually developing into brown spots with tissue rotting. Later, brown to black sclerotia are produced, irregularly scattered. Upon maturity, the epidermis of the diseased area ruptures, releasing sclerotia or mycelium. To effectively control corn sheath blight, agricultural workers typically take the following measures: strengthening field sanitation and eliminating diseased plant debris to reduce the source of pathogens; and timely spraying of fungicides at the early stages of disease. However, currently, chemical fungicides are mainly used to control corn sheath blight, but long-term use may lead to fungal resistance, reducing the effectiveness of the fungicides. Furthermore, the use of chemical fungicides may have negative impacts on the environment and human health. To reduce the use of chemical agents, planting disease-resistant varieties is the most economical, safe, and effective measure for controlling sheath blight. Therefore, identifying corn disease-resistant genes to promote the breeding of corn sheath blight-resistant varieties is particularly important in this field. Summary of the Invention
[0003] The purpose of this invention is to discover disease-resistant genes and improve resistance to corn sheath blight.
[0004] This invention provides negative regulation ZmSWEET4b The application of genes or their encoded proteins in improving resistance to maize sheath blight, the ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44.
[0005] Preferably, the ZmSWEET4b The nucleotide sequence of the gene is shown in SEQ ID NO:45.
[0006] This invention provides suppression ZmSWEET4b The application of transcription factors, including genes or their encoded proteins, in enhancing resistance to maize sheath blight, wherein the transcription factors include ZmMYB20 The amino acid sequence is shown in SEQ ID NO:4.
[0007] Preferably, the transcription factor ZmMYB20 The nucleotide sequence of the encoding gene is shown in SEQ ID NO:2.
[0008] Preferably, the transcription factor also includes ZmbHLH164 The amino acid sequence is shown in SEQ ID NO:5.
[0009] Preferably, the transcription factor ZmbHLH164 The nucleotide sequence of the encoding gene is shown in SEQ ID NO:3.
[0010] The present invention provides a method for improving resistance to corn sheath blight, comprising the following steps (1) and / or (2); (1) Knockout or knockdown of maize receptors ZmSWEET4b Genes; the stated ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44; (2) Overexpression of transcription factors in maize receptors; said transcription factors include transcription factors ZmMYB20 The transcription factor ZmMYB20 The amino acid sequence is shown in SEQ ID NO:4.
[0011] Preferably, the transcription factor further includes transcription factors. ZmbHLH164 The transcription factor ZmbHLH164 The amino acid sequence is shown in SEQ ID NO:5.
[0012] This invention provides a maize material resistant to sheath blight, which, compared to wild-type maize, exhibits [advantages / benefits]. ZmSWEET4b Gene expression levels were significantly reduced, the aforementioned ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44.
[0013] Preferably, the corn material includes any one or more of the following (1) to (3); (1) Compared to wild-type maize, there are maizes as shown in SEQ ID NO:40 and SEQ ID NO:41. ZmSWEET4b Mutant sequence; (2) Compared to wild-type maize, transcription factors ZmMYB20 Expression levels increased significantly; (3) Compared to wild-type maize, transcription factors ZmMYB20 and transcription factors ZmbHLH164 Expression levels increased significantly; The ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44; The transcription factor ZmMYB20 The amino acid sequence is shown in SEQ ID NO:4; The transcription factor ZmbHLH164 The amino acid sequence is shown in SEQ ID NO:5.
[0014] Beneficial effects: This invention provides negative regulation ZmSWEET4b The application of genes or their encoded proteins in improving resistance to maize sheath blight, the ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44. This invention discovered that under the induction of *Rhizoctonia solani* in maize... ZmSWEET4b Gene expression was significantly upregulated, as verified by both EMS-induced mutants and CRISPR / Cas9 gene-editing mutants. ZmSWEET4b Negative regulation of maize resistance to sheath blight. Furthermore, this invention also discovered that individual transcription factors... ZmMYB20 inhibition ZmSWEET4b Gene promoter activity, individual transcription factors ZmbHLH164 Promote ZmSWEET4b Gene promoter activity, but yeast two-hybrid assays showed that when both transcription factors were present... ZmSWEET4b Promoter region activity was significantly lower than in the wild type. Overexpression of transcription factors ZmMYB20 Or co-expression of transcription factors ZmMYB20 and ZmbHLH164 Able to inhibit ZmSWEET4b Gene promoter activity, thereby inhibiting ZmSWEET4b Expression of the gene or its encoded protein can enhance resistance to corn sheath blight. Attached Figure Description
[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0016] Figure 1 Changes in gene expression levels in maize inoculated with Rhizoctonia solani at 0, 24, 48, and 72 h. Figure 2 for pZmSWEET4b and ZmMYB20, ZmbHLH164 Results of yeast one-hybrid assay; Figure 3 for pZmSWEET4b and ZmMYB20 , ZmbHLH164 Results of dual-luciferase assay; Figure 4 for pZmSWEET4b and ZmMYB20 , ZmbHLH164 LUC / REN values of dual luciferase assay; Figure 5 for ZmMYB20 and ZmbHLH164 Results of yeast two-hybrid assay; Figure 6 for ZmMYB20 andZmbHLH164 Results of fluorescence complementarity assay; Figure 7 For EMS- ZmSWEET4a1 Sequencing-aligned mutation sites and peak plots; Figure 8 For EMS- ZmSWEET4a2 Sequencing-aligned mutation sites and peak plots; Figure 9 For EMS- ZmSWEET4b1 Sequencing-aligned mutation sites and peak plots; Figure 10 For EMS- ZmSWEET4b2 Sequencing-aligned mutation sites and peak plots; Figure 11 For EMS- ZmSWEET4c1 Sequencing-aligned mutation sites and peak plots; Figure 12 For EMS- ZmSWEET4c2 Sequencing-aligned mutation sites and peak plots; Figure 13 Phenotypic diagrams of wild-type maize and EMS-induced variants after inoculation with Rhizoctonia solani; Figure 14 Analysis of lesion length after inoculation of wild-type maize and EMS-induced variants with Rhizoctonia solani; Figure 15 Phenotypic diagrams of wild-type maize and CRISPR mutants after inoculation with Rhizoctonia solani; Figure 16 Analysis of lesion length after inoculation of wild-type maize and CRISPR mutants with Rhizoctonia solani; ns indicates no significant difference. express P <0.01, express P <0.0001. Detailed Implementation
[0017] This invention provides negative regulation ZmSWEET4b The application of genes or their encoded proteins in improving resistance to maize sheath blight, the ZmSWEET4bThe amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44, specifically: MVSSDTIRTAIGVIGNGTALVLFLSPVPTFIRIWKKGSVEQYSPIPYVATLLNCMMWVLYGLPLVHPHSMLVITINGTGMLIQLTYVALFLVYSAGAARRKVSLLLAAEVAFVGAVAALVLALAHTHERRSMVVGILCVLFGTGMYAAPLSVMKMVIQTKSVEYMPLFLSLASLVNGICWTAYALIRFDLYITIPNGLGVLFALAQLLLYAIYYKNTQKIVEARKRKAGQVAMTEVVVDGSRASNNNNNGGSGTY .
[0018] As one implementation method, the present invention described ZmSWEET4bThe nucleotide sequence of the gene is shown in SEQ ID NO:45, specifically:
[0019] This invention provides suppression ZmSWEET4b The application of transcription factors, including genes or their encoded proteins, in enhancing resistance to maize sheath blight, wherein the transcription factors include ZmMYB20 The amino acid sequence is shown in SEQ ID NO:4.
[0020] As one implementation method, the transcription factor of the present invention ZmMYB20The nucleotide sequence of the encoding gene is shown in SEQ ID NO:2. As one embodiment, the transcription factor of the present invention also includes... ZmbHLH164 The amino acid sequence is shown in SEQ ID NO:5. As one embodiment, the transcription factor of the present invention... ZmbHLH164 The nucleotide sequence of the encoding gene is shown in SEQ ID NO:3.
[0021] SEQ ID NO:3:ATGGCCTCCCCCGAGGGCACAACGTGGGTCTTCGACTGTCCCCTTATGGACGACCTCGCGGTCGCCGCCGACTTCGCGGCAGCCCCCGCGGGAGGATTTTTCTGGGCAGCGCCGCCGTCGCTGCAGCCGCAGGCGCCAGTGCAGTCTGTCGTTGCCGCGTCGGCTCCCAACCCATGTATGGAAATCAGTAGCTCTGTGGACTGTGGTCAGGAAAAAGAACAGCCAACAAATAAACGTCCAAGGTCAGAAAGTACTACAGAATCAAGCACAAAAGCATCCAGGGAGAAAATTAGAAGGGACAAGCTGAACGAGAGATTCTTGGAATTGGGTGCCATTTTGGAGCCAGGGAAAACTCCTAAAATGGACAAAACAGCTATATTGAGTGATGCTATTCGTGTAGTAGGTGAATTGCGTAGTGAAGCAAAAAAGCTCAAGGATTCAAATGAGAATCTCCAAGAGAAGATTAAAGAGCTGAAGGCCGAGAAGAATGAGCTGCGAGACGAGAAGCAAAGGCTGAAGGCCGAGAAGGAGAGCCTGGAGCAGCAGATCAAGTTCCTGAATGCCCGGCCAAGCCTCGTACCACACCACCCAGTGATCCCAGCCTCTGCGTTCCCTGCTCCCCAGGGGCCAGCAGCCGCCGCCAGGCACAAGCTGATGATGCCTGTGATTGGCTACCCTGGATTCCCGATGTGGCAGTTCATGCCGCCTTCAGATGTTGACACCTCTGATGACCCTAGGTCTTGTCCTCCTGTGGCGTAG; SEQ ID NO:4:MARFQARSNQRPAADHVGHRDFMDHLKNTLSSGDMDLPGGARAPKARKPYTISKQREKWTEDEHKLFLEALQQHGRAWRRIQEHIGSKTAVQIRSHAQKFFSKVIR ESSGDNSNNSVGAPPQLQIPPPRPKRRPTHPYPRKLGNSVGKDASAAIKQLRKPQLQAQSLSEQENCSPKSVLTTAQMCSEALPAEGSGSPASSVHMEDKCLTPNTSVGES SVQVALSTDSNDAACEIPEAEGPVLRLFGKRVVVSNLDQQPSSNTGSLQHAADMELDDASAETPTSGTGKLSSHAAEEAETWCPWLVGSTQQFLYYLPRGEVFSMHPGCQF LNYGNGSISYTALDAQTVTSNKQRSRPWTESIETSSSVPGTAQNSDPAESTKVNRGEDKVAVPVPGSRKCAGAIPACRRGFVPYKKCTARSKVLQPVAPGEEADRELTRLCL ; SEQ ID NO:5:MASPEGTTWVFDCPLMDDLAVAADFAAAPAGGFFWAAPPSLQPQAPVQSVVAASAPNPCMEISSSVDCGQEKEQPTNKRPRSESTTESSTKASREKIRRDKLNERFLELGAILEPGKTPKMDK TAILSDAIRVVGELRSEAKKLKDSNENLQEKIKELKAEKNELRDEKQRLKAEKESLEQQIKFLNARPSLVPHHPVIPASAFPAPQGPAAAARHKLMMPVIGYPGFPMWQFMPPSDVDTSDDPRSCPPVA .
[0022] In one embodiment, the maize described in this invention is the standard maize inbred line B73. The standard maize inbred line B73 is the type line of maize, and its whole genome sequence has been fully resolved, making it an important platform for functional genomics research. This material can be obtained through public platforms such as the USDA Germplasm Bank (GRIN-Global) (accession number: PI 550473).
[0023] This invention provides a method for improving maize resistance to sheath blight, comprising steps (1) and / or (2); (1) knocking out or knocking down the receptor in maize ZmSWEET4bGenes; the stated ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44; (2) overexpression of transcription factors in maize receptors; the transcription factors include transcription factors ZmMYB20 The transcription factor ZmMYB20 The amino acid sequence is shown in SEQ ID NO:4. As one embodiment, the transcription factor of the present invention further includes transcription factors. ZmbHLH164 The transcription factor ZmbHLH164 The amino acid sequence is shown in SEQ ID NO:5.
[0024] This invention provides a maize material resistant to sheath blight, which, compared to wild-type maize, exhibits [advantages / benefits]. ZmSWEET4b Gene expression levels were significantly reduced, the aforementioned ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44.
[0025] As one embodiment, the corn material of the present invention includes any one or more of the following (1) to (3); (1) compared with wild-type corn, it has the characteristics shown in SEQ ID NO:40 and SEQ ID NO:41. ZmSWEET4b Mutated sequences; (2) Compared to wild-type maize, transcription factors ZmMYB20 Expression levels were significantly increased; (3) compared to wild-type maize, transcription factors... ZmMYB20 and transcription factors ZmbHLH164 The expression level increased significantly; ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44; the transcription factor ZmMYB20 The amino acid sequence is shown in SEQ ID NO:4; the transcription factor ZmbHLH164 The amino acid sequence is shown in SEQ ID NO:5. In this invention, the maize material is, compared to wild-type maize, ZmSWEET4b Non-stable genetic materials with significantly reduced gene expression levels are different from maize varieties and do not possess uniformity and stability, meaning that there is phenotypic segregation in other phenotypic traits.
[0026] To further illustrate the present invention, the negative regulation provided by the present invention will be described below with reference to the accompanying drawings and embodiments. ZmSWEET4b The application of genes or their encoded proteins in improving maize sheath blight resistance is described in detail, but should not be construed as limiting the scope of protection of this invention.
[0027] The culture medium formulation, PCR primer information, and main instruments used in this invention are as follows: LB medium: Weigh 2 g sodium chloride, 1 g yeast extract, and 2 g tryptone, bring the volume to 200 mL, and sterilize at 121℃ for 20 min (for solid medium, add 4 g agar powder).
[0028] YPD medium: 1 g yeast extract, 2 g peptone, bring to a final volume of 90 mL, then add 2 g agar powder. Weigh 2 g anhydrous glucose and bring to a final volume of 10 mL. Sterilize separately at 121℃ for 20 min. After the solution cools to about 50℃, pour the anhydrous glucose solution into a 90 mL tube in a clean bench.
[0029] SD / -Leu, SD / -Ura, SD / -His media: 14 g / 500 mL, 115℃, 20 min. Add nutrient-deficient media powder in equal proportions according to the required amount of media; add 2 g / 100 mL of agar when preparing solid media.
[0030] Two-stage plate culture medium: Do Supplement Leu- / Trp 1.19 g / L, minimal SD Base 26.7 g / L. First, add the powder and distilled water to adjust the pH to 5.8, and then add 2 g / 100 mL of agar.
[0031] Four-cell plate culture medium: Do Supplement Ade / His Leu- / Trp 1.15 g / L, minimal SD Base 26.7 g / L. First add the powder and distilled water to adjust the pH to 5.8, then add 2 g / 100 mL of agar.
[0032] Table 1. PCR primer sequences used in this invention
[0033] GI54DS Automatic Pressure Steam Sterilizer, manufactured by Zealway.
[0034] Centrifuge 5418R refrigerated centrifuge, manufactured by Eppendorf.
[0035] S1000™ PCR instrument, manufactured by BIO-RAD.
[0036] The QuantStudio real-time quantitative PCR instrument is manufactured by Applied Biosystems.
[0037] Electronic analytical balance, manufactured by Sartorius.
[0038] TGL ice maker, manufactured by Shanghai Medical Analytical Instrument Factory.
[0039] Vortex oscillator, manufactured by Thermo Fisher Scientific.
[0040] DYY electrophoresis apparatus, manufactured by Beijing Liuyi Company.
[0041] Alliance 4.7 Chroma UV gel imaging system, manufactured by Uvitec.
[0042] H2O3-100C metal bath, manufactured by Kayodi Biotechnology Co., Ltd.
[0043] Spectrophotometer, manufactured by Shanghai Youke Instrument Co., Ltd.
[0044] 30℃ shaking incubator, manufactured by Dalong Xingchuang Experimental Instruments (Beijing) Co., Ltd.
[0045] FV3000 laser scanning confocal microscope (OLYMPUS), manufactured by Olympus Corporation.
[0046] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and are of laboratory purity. Unless otherwise specified, the experimental methods and conditions used in the following examples are conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, public literature, or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0047] Example 1 1. Extraction of maize RNA, synthesis of cDNA, and extraction of gDNA. (1) Extraction of maize RNA: Total RNA was extracted from B73 maize using the Trizol method. The specific steps are as follows: 0.1 g of maize leaf sample was placed in a 2 mL centrifuge tube (with 2 sterilized steel balls) and placed in liquid nitrogen. After 5 min, the frozen sample was ground in a grinder (45 Hz) for 90 s until the sample was crushed and placed on ice. 1 mL of RNAisoPlus (Takara) was quickly added to the centrifuge tube and mixed by inverting until the sample was completely dissolved in RNAisoPlus. 200 µL of chloroform (trichloromethane) was added, shaken for 15 s, and centrifuged at 12000 rpm for 5 min. The supernatant (approximately 530 µL) was transferred to a new 1.5 mL centrifuge tube. An equal volume of isopropanol was added, and the mixture was mixed by inverting. The mixture was allowed to stand at 4℃ for 20 min, and centrifuged at 12000 rpm for 20 min. The supernatant was discarded, and the white precipitate was retained. 350 µL of 75% ethanol was added, and the mixture was mixed by pipetting and centrifuged at 12000 rpm for 5 min. After 5 min, remove the ethanol; open the lid and place it in a clean bench (level 3 fan) for 5 min; add 20 µL of sterile water and mix well to obtain the RNA solution, and store it in a -80℃ freezer.
[0048] (2) cDNA synthesis: The total maize RNA obtained was reverse transcribed using the Novizan RNA Reverse Transcription Kit (catalog number R333-C1) according to the kit instructions to synthesize the first strand of cDNA, which was used as a template for gene cloning. The obtained maize cDNA was stored at -20℃.
[0049] (3) Extraction of maize gDNA: The CTAB method was used to extract maize gDNA. The specific steps are as follows: 0.1 g of maize leaf sample was placed in a 2 mL centrifuge tube (with 2 sterilized steel balls) and placed in liquid nitrogen. After 5 min, the frozen sample was ground in a grinder (45 Hz) for 90 s until the sample was crushed and placed on ice. 600 µL of 2×CTAB was added and the mixture was placed in a 60℃ metal bath for 30 min (mixing once every 10 min). 120 µL of chloroform (trichloromethane) was added and vortexed. The mixture was centrifuged at 13000 rpm for 5 min. Approximately 500 µL of the supernatant was transferred to a new 1.5 mL centrifuge tube. An equal volume of isopropanol was added and the mixture was inverted and mixed. The mixture was allowed to stand for 10 min and centrifuged at 13000 rpm for 15 min. The supernatant was then discarded. 350 µL of 75% ethanol (freshly prepared) was added for extraction and washing. The mixture was centrifuged at 13000 rpm for 5 min. After 1 minute, discard the supernatant, centrifuge again using a small centrifuge, and discard the supernatant once more. Place the centrifuge tubes in a clean bench at level 3 for 5-30 minutes until the ethanol has completely evaporated. Add 30 µL of sterile water and store at -20°C for later use.
[0050] 2. Cloning of the target gene (1) The inventors discovered a gene in maize that was upregulated under the induction of Rhizoctonia solani. ZmSWEET4b ( Figure 1 ), and found in the literature ZmMYB20 and ZmbHLH164 Expression patterns of two genes under Rhizoctonia solani infection and ZmSWEET4b Both were induced to upregulate expression. ZmSWEET4bZmMYB20 and ZmbHLH164 The nucleotide sequences of the open reading frames (ORFs) of the genes are shown in SEQ ID NO:2 and SEQ ID NO:3, and the amino acid sequences of the proteins they encode are shown in SEQ ID NO:4 and SEQ ID NO:5.
[0051] (2) ZmSWEET4b The gene was cloned using the extracted gDNA as a template. ZmMYB20 Genes and ZmbHLH164 Gene cloning was performed using maize cDNA synthesized via reverse transcription as a template. PCR amplification was carried out using the corresponding primers, which were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR amplification reaction system is shown in Table 2. The high-fidelity enzyme PhantaMax Super-Friendly DNA Polymerase was purchased from Vazyme Biotech Co., Ltd. (Nanjing Novizan Biotechnology Co., Ltd.). The PCR amplification reaction program was: 95℃ for 3 min; 95℃ for 15 s, Tm for 15 s, 72℃ for 1 kb / 45 s, 72℃ for 5 min, 33 cycles; stored at 4℃ (the reaction program was set according to the primer annealing temperature Tm and fragment size).
[0052] Table 2 PCR reaction system
[0053] (3) The PCR amplification products were detected by 2.0% agarose gel electrophoresis (160V, 15 min). The target band was recovered using a DNA purification and recovery kit (Vamyze, catalog number DC301-01) according to the kit instructions. The obtained target fragment was stored at -20℃.
[0054] 3. Construction of the carrier Using the maize cDNA synthesized via reverse transcription as a template, the target fragment was amplified using the corresponding primers (as shown in Table 1). Following the system described in Table 3, the process was carried out in advance using… EcoRThe intermediate vector pBlueScriptSK(+) was linearized using a V rapid digestion enzyme and recovered by agarose gel electrophoresis. The target fragment, stored at -20℃, was ligated to the linearized intermediate vector using T4 DNA Ligase. After transformation with *E. coli*, X-gal and IPTG were plated together on the antibiotic resistance medium during the transformation process. The next day, blue-white screening was performed, and single white colonies were streaked onto fresh antibiotic resistance medium. Colony PCR was performed using universal primers M13. Colonies with correct bands were cultured overnight at 37℃ with shaking. The next day, plasmids were extracted using a plasmid mini-extraction kit I (Omega Bio-Tek, USA). Double digestion was performed according to the system in Table 4. Recombinant plasmids with correct bands were sent for sequencing. Recombinant plasmids with correct sequences were double digested according to the system in Table 5 and recovered by gel digestion. The double digestion test was based on the primers in Table 1, with the appropriate rapid digestion enzyme selected for different experiments corresponding to different primers. For example, MYB20 and bHLH164 on the single-hybrid PGADT7 vector were double-digested using Nde1 and BamH1 fast digestion enzymes listed in Table 1. The T4 ligase reaction system was prepared according to Table 6, and the vector was ligated to the final vector. The ligation product was transformed with DH5α, and single colonies were picked and streaked. Colony PCR was performed using the designed universal primers for the final vector for verification. Correct bands on the gel indicate successful ligation of the target gene to the final vector. Depending on the specific experimental requirements for the vector, the final vector was linearized beforehand using restriction enzymes corresponding to the restriction sites added to the primers for the target fragment, and then purified and recovered.
[0055] Table 3 pBlueScriptSK(+) linearization system
[0056] Table 4 Double enzyme digestion verification system
[0057] Table 5. Double enzyme digestion and recovery system
[0058] Note: Select the optimal digestion temperature based on the characteristics of the restriction endonuclease. If the two enzymes have different optimal temperatures, use the enzyme with the lower temperature first, and then use the enzyme with the higher temperature for digestion. Digest for 1 hour each, for a total digestion time of 2 hours.
[0059] Table 6. T4 ligase reaction system
[0060] 4. (Y1H) Yeast Transformation (1) Activation of yeast strain: Y1H Gold yeast strain frozen at -80℃ was streaked on YPD plate and cultured upside down at 28~30℃ until single colonies were produced. Y1H Gold single colonies were picked and cultured overnight at 30℃ and 220 rpm in YPD liquid medium until the bacterial solution became turbid.
[0061] (2) Yeast transformation: In a clean bench, add the turbid yeast culture to a 2 mL centrifuge tube, centrifuge at 10,000 rpm for 1 min, and discard the culture medium; add 1 mL ddH2O to resuspend, centrifuge at 10,000 rpm for 1 min; repeat once, using 300 μL (variable) of resuspended culture as yeast. Add solutions according to the system in Table 7. Vortex the prepared system, incubate at 28℃ for 30 min; incubate at 42℃ for 13 min; incubate at 28℃ for 1~2 min. Centrifuge for 2 min using a mini centrifuge, discard the supernatant, add 50 μL ddH2O to resuspend, spread on the corresponding nutrient-deficient plate, and incubate upside down at 30℃ for 36~48 h. The appearance of colonies proves that the plasmid has been transformed into yeast. Note: For yeast transformation, first transform into the linearized PABAi: promoter, and then use the PABAi: promoter transformed into yeast as yeast. When introducing the promoter, spread on SD / -Ura solid medium and shake culture on SD / -Ura liquid medium; when introducing the PGADT7 transcription factor based on the PABAi promoter, spread on SD / -Leu solid medium and shake culture on SD / -Leu liquid medium.
[0062] Table 7 (Y1H) Yeast Transformation System
[0063] 5. (Y2H) Yeast Transformation (1) Yeast activation: AH109 yeast stored at -80℃ was spread on YDP solid medium and incubated upside down at 30℃ for 24~36 h.
[0064] (2) Yeast co-transformation: Pick a single colony of AH109 and culture it in 2 mL of YPD liquid medium at 30℃ and 220 rpm for about 16 h. Transfer the bacterial solution to a 2 mL tube in a clean bench and centrifuge at 6000 rpm for 1 min, discard the supernatant; add 1 mL of ddH2O to resuspend, centrifuge at 6000 rpm for 1 min, discard the supernatant, and repeat the resuspension operation; add 100 μL of 0.1mol LiAc to resuspend, incubate in a metal bath at 30℃ for 5 min, centrifuge at 6000 rpm for 1 min, discard the supernatant; add the system solution in Table 8 in sequence, vortex until the precipitate is evenly dispersed in the system, incubate at 42℃ for 20 min, incubate at 12000 rpm for 30 s, discard the supernatant with a pipette, resuspend with 100~200 μL of ddH2O, spread on SD / -Leu / -Trp plates, and incubate upside down at 30℃ for 36~48 h.
[0065] Table 8 (Y2H) Yeast Conversion Solution System
[0066] 6. Transformation of Agrobacterium (GV3101) competent cells Remove GV3101 competent cells from the -80℃ freezer, thaw on ice, add 10 µL of plasmid, gently aspirate and mix, and incubate on ice for 30 min; flash freeze in liquid nitrogen for 5 min, thaw at 37℃ for 5 min, and incubate on ice for 5 min; add 400 µL of LB liquid medium in a clean bench, and incubate at 28℃ and 220 rpm for 2 h; centrifuge at 5000 rpm for 2 min, discard most of the supernatant, retain about 50 µL of liquid to resuspend the cells, and spread them on LB solid medium containing the corresponding antibiotics (PGD3GGm and pGreenⅡ0800-LUC vectors both have kanamycin resistance), seal, and incubate upside down at 28~30° for 48 h.
[0067] 7. pZmSWEET4b and ZmMYB20 , ZmbHLH164 Yeast one-hybrid test (1) Carrier construction: pZmSWEET4b The target fragment was amplified using primers P4b-XhoⅠ-F and P4b-KpnⅠ-R from Table 1 and ligated into the PABAi vector. The two transcription factors were amplified using primers bHLH164-PGADT7-Nde1-F and bHLH164-PGADT7-BamH1-R, and ZmMYB20-ADT7-Nde1-F and ZmMYB20-ADT7-BamH1-R from Table 1 and ligated into the PGADT7 vector. Note that the transcription factor amplification should not include stop codons. The target fragment must first be ligated into the intermediate vector, and only the correctly sequenced target fragment should be ligated into the final vector.
[0068] Cis-acting elements cloned onto the bait vector pABAi may bind to endogenous yeast transcription factors, causing autoactivation and resulting in false positives. The Y1H Gold-pABAi yeast one-hybrid system uses aureobasidin A (AbA) to inhibit autoactivation, but the selection concentration should not exceed 800 ng / mL; concentrations above 1000 ng / mL are not suitable for this system. AbA is diluted with anhydrous ethanol. Different concentrations of AbA are prepared as follows: Single-absence medium SD / -Ura or SD / -Leu solid medium, autoclaved (115℃, 20 min, 103.4 kPa), cooled to approximately 55℃, then AbA is added, 10 mL per plate.
[0069] (2) Yeast Transformation: The pABAi:pZmSWEET4b recombinant plasmid was linearized according to the system in Table 9. The linearized pABAi:pZmSWEET4b recombinant plasmid was then integrated into Y1H Gold yeast using the yeast transformation method described above. The plasmid was plated on SD / -Ura medium and incubated at 30℃ for 36-48 h. The emergence of colonies indicated that the pABAi:pZmSWEET4b recombinant plasmid had been integrated into the yeast. Single colonies of the recombinant plasmid integrated into the yeast were picked, resuspended in ddH2O, and the OD was adjusted. 600 To a concentration of 0.02. Spread 100 µL onto SD / -Ura plates with different AbA concentrations and incubate upside down at 30°C for 36–48 h. The concentration with the fewest yeast cells or the fewest single colonies is the optimal AbA concentration selected. Pick single colonies of the validated self-activated pABAi:pZmSWEET4b and incubate them in SD / -Ura liquid medium at 30°C and 220 rpm for 12–16 h. Then, follow the yeast transformation procedure to transform PGADT7 and PGADT7: ZmMYB20 PGADT7: ZmbHLH164 Transfer to the integrated pABAi respectively: pZmSWEET4b In yeast, the samples were finally plated on SD / -Leu medium and incubated upside down at 30°C for 36-48 h. The growth of colonies proved that the transcription factors and promoters had been integrated into the yeast.
[0070] Table 9 ABAi: Promoter linearization system
[0071] (3) Plate test: After yeast cells grow on SD / -Leu medium, pick pABAi: pZmSWEET4b pABAi: pZmSWEET4b +PGADT7: ZmMYB20 pABAi: pZmSWEET4b +PGADT7:ZmbHLH164 Single colony OD diluted with ddH2O 600 Dilute to 0.2 with ddH2O sequentially by 10. -1 10 -2 10 -3 Take 5 μL of each diluted bacterial solution and add them at 0.2 and 0.2 × 10⁻⁶ ppm respectively. -1 0.2×10 -2 0.2×10 -3 The components were added sequentially onto SD / -Leu plates with the optimal AbA concentration to verify binding. Figure 2 As shown, ZmMYB20 and ZmbHLH164 All can and ZmSWEET4b Initiate interaction between sub-regions.
[0072] 8. pZmSWEET4b and ZmMYB20 , ZmbHLH164 Dual-luciferase assay (1) Carrier construction: pZmSWEET4b The target fragment was amplified using primers P4b-pGreen2-Kpn1-F and P4b-pGreen2-Hind3-R and ligated into the pGreenⅡ0800-LUC vector. Two transcription factors were amplified using primers MYB20-PGD3GGm-XhoI-F and MYB20-PGD3GGm-HindⅢ-R, bHLH164-pGD3GGm-XhoⅠ-F and bHLH164-pGD3GGm-SalⅠ-R, and finally ligated into the pGD3GGm vector. Note that the transcription factor amplification should not include stop codons. The target fragment must first be ligated to the intermediate vector, and only the correctly sequenced target fragment is then ligated to the final vector.
[0073] (2) Agrobacterium transformation: The vector pGreenⅡ0800-LUC was transformed into Agrobacterium: pZmSWEET4b pGD3GGm: ZmMYB20 pGD3GGm: ZmbHLH164 pGD3GGm was transformed into GV3101 using the Agrobacterium transformation method described above.
[0074] (3) Agrobacterium-mediated transient expression system in tobacco: Agrobacterium colonies that have been correctly transformed into the corresponding vector and detected by PCR were picked and inoculated into 4 mL of liquid LB medium with the corresponding resistance. The culture was carried out at 30℃ with shaking at 220 rpm for about 16 h. After centrifugation at 4000 rpm for 10 min, the supernatant was discarded and the precipitate was retained. The bacterial cells were gently resuspended in 2 mL of Agrobacterium suspension buffer (preparation method as shown in Table 10) for each sample and the OD was adjusted. 600 To 0.8, pGreenⅡ0800-LUC:pZmSWEET4b Separately with pGD3GGm: ZmMYB20 pGD3GGm: ZmbHLH164 The pGD3GGm bacterial suspension was mixed at a 1:1 volume ratio and allowed to stand in the dark at room temperature for at least 3 hours. One-month-old Nicotiana Bunsenata plants were selected, and an equal volume of the settled bacterial suspension was drawn using a 1 mL syringe (needle removed) and injected into different parts of the underside of the same tobacco leaf. Each treatment was repeated at least three times. The plants were then incubated at 25°C under low light for 24 hours, followed by 24 hours of light incubation. Fluorescence intensity was detected using a bioluminescence imaging system, and luciferase activity was measured. The LUC and REN values were recorded, and their ratio was calculated.
[0075] Table 10. Preparation system of Agrobacterium suspension buffer
[0076] The results are as follows Figure 3 and Figure 4 As shown, ZmMYB20 inhibition ZmSWEET4b Promoter region activity, while ZmbHLH164 Promote ZmSWEET4b Promoter region activity, when both transcription factors are present ZmSWEET4b Promoter region activity is higher than that of only ZmMYB20 When present, below only ZmbHLH164 When it exists.
[0077] 9. ZmMYB20 and ZmbHLH164 Yeast two-hybrid test (1) Carrier construction: ZmMYB20 The target fragment was amplified using primers ZmMYB20-ADT7-Nde1-F and ZmMYB20-ADT7-BamH1-R, and then ligated into the PGADT7 vector. ZmbHLH164 The target fragment was amplified using primers bHLH164-PGBKT7-Nde1-F and bHLH164-PGBKT7-BamH1-R and ligated into the PGBKT7 vector. Note that the transcription factor amplification process must not include stop codons. The target fragment must first be ligated to the intermediate vector, and only after the sequenced fragment is correctly identified should it be ligated to the final vector.
[0078] (2) Self-activation verification: Before introducing the prey protein, it was confirmed that the constructed pGBKT7-transcription factor bait plasmid itself would not activate the reporter gene. In this study, 3-AT, a competitive inhibitor of the histidine metabolic pathway, was used to suppress potential background growth (weak self-activation). PGBKT7: ZmbHLH164 +PGADT7 and PGADT7: ZmMYB20When +PGBKT7 is applied to SD / -Trp / -Leu / -His plates (with 3-AT added, up to 100 mM), the optimal concentration for self-activation inhibition is reached when neither of the two colonies grows.
[0079] (3) Yeast co-transfer: PGADT7: ZmMYB20 +PGBKT7: ZmbHLH164 (Interaction group), PGADT7-T+PGBKT7-Lam (positive control), PGADT7-T+PGBKT7-53 (negative control), PGBKT7: ZmbHLH164 +PGADT7 (Auto-activation group), PGADT7: ZmMYB20 +PGBKT7 (self-activating group) was transferred into AH109 using the yeast co-transformation method described above. The growth of colonies indicates that the corresponding vector has been transferred into AH109 yeast.
[0080] (4) Plate validation: After yeast cells grow on SD / -Leu / -Trp medium, pick single colonies from the above five groups and dilute OD with ddH2O. 600 Dilute to 0.2 with ddH2O sequentially by 10. -1 10 -2 10 -3 Take 5 μL of each diluted bacterial solution and add them at 0.2 and 0.2 × 10⁻⁶ ppm respectively. -1 0.2×10 -2 0.2×10 -3 The transcription factor binding was verified by sequentially adding the sample onto SD / -Leu / -Trp / -His / -Ade plates containing the optimal 3-AT concentration. The results are as follows: Figure 5 As shown, bacteria grew in the positive control group, while no bacteria grew in the negative control and self-activation group. PGADT7: ZmMYB20 +PGBKT7: ZmbHLH164 Bacterial growth, ZmbHLH164 Can be harmonious ZmMYB20 Combined.
[0081] 10. ZmMYB20 and ZmbHLH164 Complementary fluorescence (BiFC) assay (1) Carrier construction: ZmMYB20 The target fragment was amplified using primers MYB20-PDoner-R and MYB20+PDoner-F. ZmbHLH164 Both target fragments were ligated to pDONER using primers bHLH164-pDONR221-F and bHLH164-pDONR221-R. TM221 The BP reaction was performed on the vector (as shown in Table 11), and then verified using the M13 universal primer. ZmMYB20Connect to PXC-cCFP, ZmbHLH164 Link to PXN-nYFP for LR reaction (as shown in Table 12). Note that the transcription factor amplification process must not include a stop codon.
[0082] Table 11 BP Reaction System
[0083] Table 12 LP Reaction System
[0084] (2) Agrobacterium transformation: The vectors ZmbHLH164-nYFP, ZmMYB20-cCFP, H2B-mRFP1, and p19 were transformed into GV3101 according to the Agrobacterium transformation method described above.
[0085] (3) Agrobacterium-mediated transient expression system in tobacco: Agrobacterium was injected into the leaves of Tobacco Bunsen according to the dual-luciferase method in step 8. It should be noted that the OD value adjustment is different, as shown in Table 13.
[0086] Table 13 BiFC Tests
[0087] The results are as follows Figure 6 As shown, ZmbHLH164 and ZmMYB20 They interact and are located in the cell nucleus.
[0088] Example 2 Obtaining EMS-induced variants and validating resistance to sheath blight. 1. Maize mutant seeds obtained through EMS mutagenesis were planted under conventional greenhouse conditions. When the plants reached the 3-4 true leaf stage, fresh leaf tissue was collected for subsequent genomic DNA extraction. Genomic DNA (gDNA) was extracted from the leaf tissue using the CTAB method described in Example 1, and the integrity and concentration of the DNA were detected by agarose gel electrophoresis and spectrophotometry. Based on the target gene EMS- ZmSWEET4a EMS ZmSWEET4b and EMS- ZmSWEET4c Based on the gene sequence information, specific primers as described in Table 14 were designed for their respective mutation regions to amplify gene fragments containing mutated bases. Each mutated gene contains 2 EMS-induced mutation sites, for a total of 6 mutation detection sites.
[0089] Table 14 Primers used for EMS-induced mutant mutation site detection
[0090] 2. Using the extracted gDNA as a template, PCR amplification was performed using the primers described above. The reaction system and procedure were set up according to standard molecular biology methods. The amplified products were detected by 1.0%–1.5% agarose gel electrophoresis to confirm that the band size was consistent with the expectation. The PCR amplified products were then sent for bidirectional Sanger sequencing to obtain the nucleotide sequence peak map and base sequence map of the corresponding mutation region. The sequencing results were compared with the wild-type sequence. If only a single base mutation peak appeared at the target mutation site without overlapping peak signals, the mutant was determined to be a homozygous mutant; if double peak signals of wild-type and mutant bases appeared simultaneously at the mutation site, it was determined to be a heterozygous mutant. The results are as follows: Figures 7 - 12 As shown, in order to investigate ZmSWEET4b Closest to homology ZmSWEET4a and ZmSWEET4c To investigate the possibility of functional redundancy, EMS mutants of these three genes were obtained from Qilu Normal University. They were named EMS- ZmSWEET4a EMS ZmSWEET4b and EMS- ZmSWEET4c .in, t-4a1-F / R amplification ZmSWEET4a1 The mutated sequence is shown in SEQ ID NO:38, specifically: ATGCATGGCATGTATGAATGCATATATAACACTCACATATATATACATGTGACGTACAGGCCTACGTTCATCCGCATCTGGAAGAAAAGGTCGGTGGAGCAGTACTCGCCGATCCCGTACGTGGCGACGCTGCTGAACTGCATGATGTGGGTGCTGTACGGGCTGCCGGCGGTGCACCCGCACAGCATGCTGGTGATCACCATCAACGGCACCGGCATGGCGATCCAGCTGACCTACGT, where the bolded part is the mutation site, where G is mutated to A; t-4a2-F / R amplification ZmSWEET4a2 The mutated sequence is shown in SEQ ID NO:39, specifically: TGGTCGTCGACGGCGGCAAGACCAACAACCAAGCCGGCGCGGGCTAATACTGACCGACGATCAAGTGACGATCGACACCAATATGCTCAAGCAAGAAGCCAGCGGCAAATCAATTATGTCGCCGCTGGGTTTTATGTATTTTGTGATTCTATATGCACGCTGCTTGTCCGTCCCGGCCAATGGAAAAAAAAGGGGGAAGA, where the bolded part is the mutation site, which is mutated from C to T; t-4b1-F / R amplification ZmSWEET4b1 The mutated sequence is shown in SEQ ID NO:40, specifically: GGGTGTCTCGCAGGCAATGGGACCGCCCTGGTGCTCTTCCTCTCCCCGGTGTAAGAACAGCCAATTAATACAGATACTCATACAACATGTGTGTGTGTTTGAAGGAGGATGTACGTACTAACCGAAGGACGTATTATACAACGTACGTACGTACGTACGTACGAACATGCAGGCCTACGTTCATCCGCATCTGAAAGAAGGGGTCGGTGGAGCAGTACTCGCCGATCCCGTACGTGGCGACGCTGCTGAACTGCATGATGTGGGTGTTGTACGGGCTGCCGCTGGTGCACCCGCACAGCATGCTGGTGATCACCATCAACGGCACCGGCATGCTCATCCAGCTGACCTACGTGGCGCTCTTCCTCGT, where the bolded part is the mutation site, where G is mutated to A; t-4b2-F / R amplification ZmSWEET4b2 The mutated sequence is shown in SEQ ID NO:41, specifically: GCTCTCCGTCATGGTAAGTACTGTAGTATATGTATGCCTTTGTTCCTACATGTACGCCGCGCCTAATGCCCTTCTTTTAATTTGTTACCCTCGTATGATGCGTGCGTGCGTGGATCAGAAAATGGTGATCCAGACGAAGAGCGTGGAGTACATGCCGCTGTTCCTGTCACTGGCTTCCCTGGTGAACGGCATCTGCTGAACCGCCTACGCGCTCATCCGCTTCGACCTCTACATCACCGTAAGCAACTACAACCCTGCTGGACCTGGACATGGCACGCGCGCCTGTCCGCCGCACATGTACAGTACGATTCCTATCCTGTGCAGTTAGATCATATCACCCGGTTCAAATGCCCATCTATCCAGCGGGCGGCGACCATGCCGCACAGGCACGCATGCAGCAGCAGCAGTA, where the bolded part is the mutation site, where G is mutated to A, resulting in premature termination of translation; t-4c1-F / R amplification ZmSWEET4c1The mutant sequence is shown in SEQ ID NO: 42, specifically: ACTGTGCTCTGCTTATGGATACATACATGCATACTAGTAGCTCATATATGCGTACAAATGCGCCCCGCAGGCCCACCTTCGTGGGCATCTGGAAGAAGCGTGCGGTGGAGCAGTACTCGCCGATCCCGTACGTGGCGACGCTGCTGAACTGCATGATGTGGGTGCTGTACGGGCTGCCGCTGGTGCACCCGCACAGCATGCTGGTGGTGACCATCAACGGCACCGGCATGCTCATCCAGCTGACCTACGTGGCGCTCTTCATCCTCTGCTCCGCGGGGGCGGTGCACCGCAGGGTCGTGCTCCTGTTCGCCGCCGAGGTCGCCTTCGTCGTCGCCTTGGCCGCGCTGGTGCTCACCCTGGCGCACACGCACGAGCGCAGGTCCATGCTCGTCGGCATCGTCTCCGTCTTCTTCGGCACCGGCATGTACGCTGCGCCGCTCTCGGTCATGGTACGTACGTTGTTTTCACATACATGTCGTTGATTCTTTCTCTCTATGATATGACACGAAGGTAATGTTTGTTTGTTTTCTTACTTACTTACTTACTTGGTGTCTCTGATCTGCTG. Among them, the bold part is the mutation site, which mutates from G to A; amplified by t-4c2-F / R ZmSWEET4c2The mutated sequence is shown in SEQ ID NO:43, specifically: GGTGTCTCTGATCTGCTGTGTGTGTCTCTTTTTTTGTATATGGTTGATCAGAAATTGGTGATCCAGACAAAGAGCGTGGAATACATGCCCCTGTTCCTGTCCCTGGCCTCCCTCGCCAACAGCATCTGCTGGACCGCCTACGCGCTCATCCGCTTCGACCTCTACATCACCATAAGCGACTATTACTAGTATAACGTGTGTGTTGGATAAATTTTACATTCATTTTTGTTTAAACACCTAGTATGCATTCGAACTACCCCGAGCCGCTGTAGCTACTACTACATCATCAGTGGGAATCTGGCAGTAACAAGTCAGGGCATGCATGCTTCAGTTAGCAG, where the bolded part is the mutation site, where G is mutated to A.
[0091] 3. Live inoculation during corn planting and the large trumpet stage. (1) Planting of EMS mutant maize: Mix the substrate soil and normal soil in a 1:1 volume ratio, add water until it can be formed into a ball by hand and crumble when released, fill it into small square flower pots (press down the bottom 1 / 3 slightly, and fill the pot normally with the rest), and sow one EMS mutant maize seed (obtained from Qilu Normal University) in each flower pot. ZmSWEET4a EMS ZmSWEET4b and EMS- ZmSWEET4c Place them in a greenhouse for cultivation, observe the soil condition and water them (the surface soil should not crumble when pinched). Water them about once every 3-4 days during the seedling stage, and once every other day after the five leaves are formed. Water in the morning or after dusk to prevent burning the seedlings.
[0092] (2) Preparation before inoculation: Cut the thin veneer into small pieces of equal size (0.8 cm × 1 cm × 0.22 mm), sterilize under high temperature and high pressure, and then dry in an oven at 65℃; 2-3 days in advance, activate Rhizoctonia solani on PDA solid medium ( R. solani For strain AG-1IA, use a sterilized 1 mL pipette tip to press uniformly sized circles onto the outermost layer of an unactivated plate. Use the tip of a disposable syringe to pick up the circular bacteria and place them in the center of a new PDA medium (mycelial spreading side down). Place small wooden chips evenly on the outermost side of the new PDA medium and incubate at 28°C for 36-48 hours until the mycelium has fully colonized the wooden chips. These wooden chips can be used for live inoculation of corn.
[0093] (3) Inoculation: When the corn reaches the large trumpet stage, a small wooden piece covered with mycelium is gently inserted into the corn (the fourth leaf sheath from the bottom) for live inoculation. An appropriate amount of sterile water is sprayed to ensure humidity and prevent the wooden piece from falling off. The lesion of the corn is photographed 72 hours after inoculation. Wild type B73 is the control group, and the mutant is the treatment group.
[0094] The results are as follows Figure 13 and Figure 14 As shown, ZmSWEET4b After EMS-induced variants were inoculated with Rhizoctonia solani, compared with the standard maize inbred line B73 (WT), ZmSWEET4a EMS-induced variants and ZmSWEET4c EMS-induced variants showed a significant reduction in lesion length. ZmSWEET4b Negative regulation of maize resistance to sheath blight.
[0095] Example 3 Obtaining CRISPR / Cas9 gene-edited mutants and validating resistance to sheath blight. 1. ZmSWEET4b Target design guidelines for CRISPR / Cas9 mutants: Target design utilized the high-throughput CRISPR-Cas9 target design program developed by Weimi Biotechnology (Jiangsu) Co., Ltd. The program's target design principles are as follows: 1) Knockout sites should be located in the coding sequence (CDS) region and preferably at the protein's front end or in an important functional domain; 2) Coverage should be maximized across a higher proportion of transcripts; 3) No off-target effects or off-target effects should be located in intergenic regions; 4) Targets with high editing efficiency should be prioritized; 5) Sequences should have a relatively balanced GC content and be less prone to secondary structure formation. The successful application of this program in whole-genome target design in rice has proven its feasibility. For non-coding proteins, the Huazhong Agricultural University CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR2 / ) was used for design, selecting targets with high target scores, low off-target rates, and suitable locations. In this embodiment, the target design for the relevant genes or non-coding proteins is a single-gene dual-target transgenic maize B73: 1) TCTTCCTCGTCTACTCCGCGGGG (SEQ ID NO:46); 2) CCAGCAGGAGGGACACCTTGCGG (SEQ ID NO:47). CRISPR / Cas mutant maize was obtained from Weimi Biotechnology Co., Ltd. 2. CRISPR / Cas mutant maize was planted and inoculated according to the steps in Example 2 to verify resistance to sheath blight.
[0096] The results are as follows Figure 15 and Figure 16 As shown, ZmSWEET4b CRISPR mutants inoculated with Rhizoctonia solani showed a significant reduction in lesion length compared to the standard maize inbred line B73 (WT).ZmSWEET4b Negative regulation of maize resistance to sheath blight.
[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. Negative regulation ZmSWEET4b The application of genes or their encoded proteins in improving resistance to maize sheath blight, the ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:
44.
2. The application according to claim 1, characterized in that, The ZmSWEET4b The nucleotide sequence of the gene is shown in SEQ ID NO:
45.
3. Inhibition ZmSWEET4b The application of transcription factors, including genes or their encoded proteins, in enhancing resistance to maize sheath blight, wherein the transcription factors include ZmMYB20 The amino acid sequence is shown in SEQ ID NO:
4.
4. The application according to claim 3, characterized in that, The transcription factor ZmMYB20 The nucleotide sequence of the encoding gene is shown in SEQ ID NO:
2.
5. The application according to claim 3, characterized in that, Transcription factors also include ZmbHLH164 The amino acid sequence is shown in SEQ ID NO:
5.
6. The application according to claim 5, characterized in that, The transcription factor ZmbHLH164 The nucleotide sequence of the encoding gene is shown in SEQ ID NO:
3.
7. A method for improving resistance to corn sheath blight, characterized in that, Includes the following steps (1) and / or (2); (1) Knockout or knockdown of maize receptors ZmSWEET4b Genes; the stated ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44; (2) Overexpression of transcription factors in maize receptors; said transcription factors include transcription factors ZmMYB20 The transcription factor ZmMYB20 The amino acid sequence is shown in SEQ ID NO:
4.
8. The method according to claim 7, characterized in that, The transcription factors also include transcription factors. ZmbHLH164 The transcription factor ZmbHLH164 The amino acid sequence is shown in SEQ ID NO:
5.
9. A corn material resistant to sheath blight, characterized in that, Compared to wild-type maize, the maize material described above... ZmSWEET4b Gene expression levels were significantly reduced, the aforementioned ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:
44.
10. The corn material according to claim 9, characterized in that, The corn material includes any one or more of the following (1) to (3); (1) Compared to wild-type maize, there are maizes as shown in SEQ ID NO:40 and SEQ ID NO:
41. ZmSWEET4b Mutant sequence; (2) Compared to wild-type maize, transcription factors ZmMYB20 Expression levels increased significantly; (3) Compared to wild-type maize, transcription factors ZmMYB20 and transcription factors ZmbHLH164 Expression levels increased significantly; The ZmSWEET4b The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:44; The transcription factor ZmMYB20 The amino acid sequence is shown in SEQ ID NO:4; The transcription factor ZmbHLH164 The amino acid sequence is shown in SEQ ID NO:5.