WO2020173403A1 - 灰斑病抗性相关蛋白ZmWAK-RLK及其编码基因和应用 - Google Patents

灰斑病抗性相关蛋白ZmWAK-RLK及其编码基因和应用 Download PDF

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WO2020173403A1
WO2020173403A1 PCT/CN2020/076320 CN2020076320W WO2020173403A1 WO 2020173403 A1 WO2020173403 A1 WO 2020173403A1 CN 2020076320 W CN2020076320 W CN 2020076320W WO 2020173403 A1 WO2020173403 A1 WO 2020173403A1
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plant
plants
protein
transgenic
sequence
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PCT/CN2020/076320
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French (fr)
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徐明良
钟涛
番兴明
朱芒
张艳
徐凌
刘丽
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中国农业大学
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Priority to BR112021016750A priority Critical patent/BR112021016750A2/pt
Priority to CN202080013982.1A priority patent/CN113490683B/zh
Priority to MX2021010318A priority patent/MX2021010318A/es
Priority to US17/433,159 priority patent/US20220177907A1/en
Priority to EP20762082.4A priority patent/EP3932939A4/en
Publication of WO2020173403A1 publication Critical patent/WO2020173403A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a gray spot disease resistance-related protein
  • ZmWAK-RLK and its coding gene and application.
  • Corn gray leaf spot is a corn leaf disease that affects the yield and quality of corn.
  • gray spot disease was first discovered in Alexandria County, Illinois, USA, and then gradually developed into a serious global leaf disease.
  • Gray spot disease is widely distributed in the main corn producing areas of the United States, Asia, Europe and Africa. In the case of disease, gray spot disease can cause a 20-60% reduction in production, and in a severe case, it can reach 100%, causing serious economic losses to corn production.
  • Corn gray leaf spot is a fungal disease. It is generally believed that the pathogenic bacteria are mainly Cercospora zeae (Czm, Cercospora zeae-maydis) and Cercospora zeae-maydis (Cz, Cercospora zeina). In 2013, Liu et al.
  • the resistance of maize to gray spot disease belongs to quantitative inheritance, controlled by polygenes, with additive effects. Then, if the gray spot disease resistance gene is cloned and introduced into the existing inbred lines using molecular marker-assisted selection technology, the gray spot disease resistance of the promoted varieties will be improved.
  • the present invention provides a gray spot disease resistance-related protein ZmWAK-RLK and its coding gene and application.
  • the protein provided by the present invention obtained from the corn inbred line Y32, named ZmWAK-RLK protein, is as follows (al) or (a2) or (a3) or (a4) or (a5):
  • (a3) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein in (al) or (a2);
  • (A4) A protein related to plant gray spot disease resistance obtained by substituting and/or deleting and/or adding one or several amino acid residues to (al) or (a2);
  • (A5) It is derived from corn and is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to (al) or (a2) and is identical to A protein related to plant gray spot disease resistance.
  • Proteins can be synthesized artificially, or their coding genes can be synthesized first and then biologically expressed.
  • the nucleic acid molecule encoding the ZmWAK-RLK protein also belongs to the protection scope of the present invention.
  • the nucleic acid molecule is as follows (bl) or (b2) or (b3) or (b4) or (b5) or (b6):
  • (B5) It is derived from corn and has 90%, (bl) or (b2) or (b3) or (b4)
  • the stringent conditions are in a solution of 2XSSC, 0.1% SDS, hybridizing and washing the membrane twice at 68 ° C, 5 min each time, in a solution of 0.5XSSC, 0.1% SDS, hybridizing and washing the membrane at 68 ° C Wash the membrane twice, 15min each time.
  • DNA molecules, expression cassettes, recombinant vectors or recombinant microorganisms containing the nucleic acid molecules are all within the protection scope of the present invention.
  • the DNA molecule containing the nucleic acid molecule can be specifically as shown in sequence 4 of the sequence listing.
  • Existing expression vectors can be used to construct recombinant expression vectors containing the nucleic acid molecules.
  • any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, and they can be used alone or in combination with other plants.
  • Promoters are used in combination; in addition, when the nucleic acid molecule is used to construct a recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons However, it must be the same as the reading frame of the coding sequence to ensure correct translation of the entire sequence.
  • the sources of the translation control signals and initiation codons are extensive, and they may be natural or synthetic.
  • the translation initiation region can be derived from a transcription initiation region or a structural gene.
  • the expression vector used can be processed, such as adding genes that express enzymes or luminescent compounds that can produce color changes in plants or microorganisms, resistant antibiotic markers, or Anti-chemical reagent marker genes, etc. Considering the safety of transgene, it is possible to directly screen transformed plants or microorganisms by phenotype without adding any selectable marker genes.
  • the recombinant expression vector can specifically be: Insert the double-stranded DNA molecule shown in sequence 4 of the sequence table into the multiple cloning site of the PCAMBIA3301 vector Site) the resulting recombinant plasmid.
  • the recombinant expression vector can be specifically: inserting the double-stranded DNA molecule shown in nucleotide 87-2084 of sequence 2 in the sequence table into the multiple cloning site of pBCXUN vector
  • the recombinant expression vector may specifically be: Insert the recombinant double-stranded DNA molecule shown in sequence 6 of the sequence table into the multiple cloning site of the pBCXUN vector The resulting recombinant plasmid.
  • the present invention also protects the application of ZmWAK-RLK protein as follows (cl) or (c2) or (c3) or (c4):
  • the present invention also protects the application of the nucleic acid molecule or the DNA molecule containing the nucleic acid molecule as follows (dl) or (d2) or (d3) or (d4):
  • the application of the nucleic acid molecule also includes the realization mode of using the gene through CRISPS/CAS9 technology. For example: genome fragment reset (to introduce disease-resistant alleles into the susceptible genome), allele exchange (to replace susceptible alleles with disease-resistant alleles), and to change susceptible alleles into resistant alleles through gene editing Disease alleles and so on.
  • the application of the nucleic acid molecule also includes other implementation methods aimed at enhancing the expression of the nucleic acid molecule.
  • the expression of the nucleic acid molecule is enhanced by promoter replacement, the expression of the nucleic acid molecule is enhanced by introducing an enhancer, and so on.
  • the present invention also protects a method for preparing a transgenic plant, which includes the following steps: introducing the nucleic acid molecule or the DNA molecule containing the nucleic acid molecule into a starting plant to obtain a transgenic plant with enhanced gray spot disease resistance.
  • the nucleic acid molecule can be specifically introduced into the starting plant through any of the above recombinant expression vectors.
  • the recombinant expression vector carrying the nucleic acid molecule can be transformed into the starting plant by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, and Agrobacterium mediation.
  • Crossing the transgenic plants with existing corn varieties including single crosses and multiple crosses, such as three consecutive crosses
  • the resulting transgenic progeny plants are also transgenic plants with enhanced disease resistance.
  • the existing corn variety may specifically be a corn inbred line Q11.
  • the present invention also protects a plant breeding method, which includes the following steps: increasing the content and/or activity of the ZmWAK-RLK protein in the target plant, thereby improving the disease resistance of the target plant to gray leaf spot.
  • the present invention also protects a method for preparing a transgenic plant, which includes the following steps: introducing the nucleic acid molecule or the DNA molecule containing the nucleic acid molecule into a starting plant to obtain a transgenic plant with enhanced disease resistance; Sex is disease resistance to diseases caused by Cercospora cornae.
  • the nucleic acid molecule can be specifically introduced into the starting plant through any of the above recombinant expression vectors.
  • the recombinant expression vector carrying the nucleic acid molecule can be transformed into the starting plant by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conduction, and Agrobacterium mediation.
  • Crossing the transgenic plants with existing maize varieties including single crosses and multiple crosses, for example, three consecutive crosses
  • the obtained transgenic progeny plants are also transgenic plants with enhanced disease resistance.
  • the existing corn variety may specifically be a corn inbred line Q11.
  • the present invention also protects a plant breeding method, which includes the following steps: increasing the content and/or activity of ZmWAK-RLK protein in the target plant, thereby improving the disease resistance of the target plant; Sex is disease resistance to diseases caused by Cercospora cornae.
  • any of the above-mentioned plants is a dicotyledonous plant or a monocotyledonous plant.
  • the monocot plants may be grasses.
  • the gramineous plant may be a plant of the genus Zea.
  • the Zea may be specifically corn, such as corn inbred line B73, such as corn inbred line B73-329.
  • gray spot diseases may specifically be gray spot disease caused by Cercospora cornae.
  • Figure 1 shows the PCR identification results of some plants in Example 2; the arrow marks the target band (1197bp), the leftmost lane is the molecular weight standard (M), and each of the remaining lanes corresponds to a plant (numbered 1-18); if An amplified product of 1197 bp is obtained, which is identified as positive by PCR, and the plant is a transgenic plant; if no amplified product is obtained, the plant is identified as negative by PCR, and the plant is a non-transgenic plant.
  • Figure 2 shows the photos of representative leaves of each grade; the disease-resistant parent Y32 has fewer disease spots, and the susceptible parent Q11 has more disease spots.
  • Figure 3 shows the results of the disease resistance identification of the offspring isolated from the backcross in Example 2; in the BCA generation and the BCA generation, the disease index of the non-transgenic plants and the transgenic plants in the offspring of the two transgenic events WAK1-15 and WAK1-17 were counted respectively ;
  • the gray bar graphs are non-transgenic plants, the black bar graphs are transgenic plants, and the numbers in the bar graph indicate the number of plants; *: corpse ⁇ 0.05; **: corpse ⁇ 0.01.
  • Figure 4 shows the disease resistance identification results of the homozygous transgenic lines in Example 2; the disease index of the homozygous transgenic positive plants and the transgenic recipient material (B73-329), the disease index of the homozygous transgenic positive plants was significantly lower than that of the transgene Recipient material:
  • the gray bar graph is the transgenic recipient material B73-329, the black bar graph is the pure line of transgenic plants, and the numbers in the bar graph indicate the number of plants.
  • Figure 5 shows the PCR identification results of some plants in Example 3; the arrow marks the target band (530bp), the leftmost lane is the molecular weight standard (M), and each of the remaining lanes corresponds to a plant (numbered 1-19); if An amplified product of 530 bp is obtained, and the plant is identified as positive by PCR, and the plant is a transgenic plant; if no amplified product is obtained, the plant is identified as negative by PCR, and the plant is a non-transgenic plant.
  • the target band 530bp
  • M molecular weight standard
  • Figure 6 shows the results of the disease resistance identification of the offspring separated from the backcross in Example 3.
  • the non-transgenic plants and transgenic plants in the offspring of the three transgenic events of WAK2-6, WAK2-7 and WAK2-8 were counted respectively
  • the disease index of the plant the gray bar graphs are non-transgenic plants
  • the black bar graphs are transgenic plants
  • the numbers in the bar graphs indicate plants . 001;
  • Fig. 7 is the disease resistance identification result (sickness index) of the B73 background complementary transgene pure line in Example 4; the gray bar graph represents the transgenic receptor material B73, and the black bar graph represents the transgenic pure line C#1, C#2 And C#3; the number in the bar graph indicates the number of plants; *: K0.05; **: K0.01.
  • Figure 8 is the resistance identification result (sickness index) of the B73 background overexpression transgenic pure line in Example 5; the gray bar graph represents the recipient material B73, and the black bar graph represents the transgenic pure line 0#1, 0#2 0#3 and 0#4; The numbers in the bar graph indicate the number of plants; *: K0.05.
  • Figure 9 shows the PCR identification results of some plants in Example 6; the arrow marks the target band (357bp), the leftmost lane is the molecular weight standard (M), and each of the remaining lanes corresponds to a plant (numbered 1-14); if An amplified product of 357 bp is obtained, and the plant is identified as positive by PCR, and the plant is a transgenic plant; if no amplified product is obtained, the plant is identified as negative by PCR, and the plant is a non-transgenic plant.
  • M molecular weight standard
  • Figure 10 shows the resistance identification results (sickness index) of the Q11 background overexpressed chimeric gene in Example 6:
  • the progeny of the three transgenic events R1, R2 and R3 were respectively counted for non-transgenic plants and transgenic plants Disease index; gray bar graphs are non-transgenic plants; black bar graphs are transgenic plants; the numbers in the bar graph indicate the number of plants; *: K0.05; **: K0.01.
  • the following embodiments facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • the maize inbred line Y32 is a maize inbred line with high resistance to gray leaf spot of maize.
  • Maize inbred line Y32 (line Y32), recorded in the following literature: Theoretical and Applied Genetics, 2012, 25 (8): 1797-1808. Zhang, Y., et al. ⁇ QTL mapping of resistance to gray leaf spot in maize . ⁇
  • the corn inbred line Qll is a corn inbred line highly susceptible to gray spot disease.
  • Maize inbred line Qll (line Q11), recorded in the following literature: Theoretical and Applied Genetics, 2012, 25 (8): 1797-1808. Zhang, Y., et al. ⁇ QTL mapping of resistance to gray leaf spot in maize . ⁇
  • Maize inbred line B73-329 (B73-329 inbred lines), recorded in the following literature: New Phytologist, 2018, 217 (3): 1161-1176. Zhang, M,, et al. "A retrotransposon in an HKT 1 family sodium transporter causes Variation of leaf Na+ exclusion and salt tolerance in maize. Maize inbred line B73 (B73 inbred lines), recorded in the following documents:
  • Cercospora zeina recorded in the following documents: Plant Disease, 2013, 97 (12): 1656-1656. Liu, KJ, et al.''First Report of Gray Leaf Spot of Maize Caused by Cercospora zeina in China.”.
  • pCAMBIA3301 vector bivalent expression vector pCAMBIA3301 vector (bivalent expression vector pCAMBIA3301), described in the following literature: Theoretical and Applied Genetics 131.10 (2016): 2145-2156. Zhu, X, et al. "Pyramiding of nine transgenes in maize generates high-level resistance against necrotrophic maize pathogens . ⁇ .
  • pBCXUN vector (pBCXUN vector), described in the following documents: Journal of integrative plant biology 61.6 (2019): 691-705. Qin, YJ, et al. "ZmHAK5 and ZmHAKl function in K+ uptake and distribution in maize under low K+ conditions. ⁇ .
  • the high-resistance to gray spot disease maize inbred line Y32 (as the donor parent) and the highly susceptible gray spot disease maize inbred line QU (as the recurrent parent) were used to construct the initial positioning population and the fine positioning population.
  • Bird and qRglsl are located between IDP2 and M2 of maize chromosome 8, and the physical location is about 120kb.
  • the Y32 BAC library of disease-resistant parents was screened by PCR. Perform BAC clone fingerprint analysis to construct BAC contigs covering the entire gene segment. Select the clone that can cover the least gene region for sequencing. Through sequence alignment and expression analysis, a new gene was discovered, which encodes the protein shown in sequence 1 of the sequence list.
  • the protein shown in sequence 1 of the sequence listing is named ZmWAK-RLK protein.
  • the gene encoding the ZmWAK-RLK protein is named ZmWAK-RLK protein.
  • the gene encoding the ZmWAK-RLK protein is shown in sequence 2 of the sequence table (wherein the 87th-2084th nucleotides are open Put in reading frame) (in sequence 2, nucleotides 87-1058 are used to construct chimera genes).
  • the AWPU gene in the genomic DNA of the maize inbred line Y32 is shown in sequence 3 of the sequence table.
  • Maize inbred line The open reading frame sequence of is shown in sequence 5 in the sequence listing (in sequence 5, nucleotides 1102-2115 are used to construct chimera genes).
  • the chimeric gene is shown in Sequence 6 in the Sequence Listing, and expresses the chimeric protein shown in Sequence 7 in the Sequence Listing.
  • a fragment of about 7.2 kb from the maize inbred line Y32 (the fragment is shown in sequence 4 of the sequence table; in sequence 4, nucleotides 1-2103 are promoters, and nucleotides 2104-4316 are The acid is the same as sequence 3 in the sequence list) Insert the BamH I restriction site of pCAMBIA3301 vector to obtain a recombinant plasmid.
  • step 2 Introduce the recombinant plasmid obtained in step 1 into Agrobacterium EHA105 to obtain recombinant Agrobacterium.
  • step 3 Take the recombinant Agrobacterium obtained in step 2, and use the Agrobacterium-mediated method to genetically transform the immature embryos of the maize inbred line B73-329 to obtain T0 generation plants.
  • the T0 generation plants are selfed, the seeds are harvested, and the seeds are cultivated into plants, which are the T1 generation plants.
  • the T1 generation plants were identified by PCR, and transgenic plants were screened.
  • the transgenic plants selected from the T1 generation plants are the T1 transgenic plants.
  • Several transgenic plants were selected from the T1 generation plants, two of which were named WAK1-15 plants and WAK1-17 plants.
  • PCR identification method Take plant leaves, extract genomic DNA, and use a primer pair composed of F1 and R1 for PCR amplification. If a 1 197bp amplified product is obtained, the PCR identification is positive, the plant is a transgenic plant; if no amplification is obtained The product is identified as negative by PCR, and the plant is a non-transgenic plant.
  • FI CGAGGAGGTTTCCCGATATTAC; R1: CACGTCAATCCGAAGGCTATTA.
  • the PCR identification results of some plants are shown in Figure 1.
  • the arrow marks the target band, the leftmost lane (M) is the molecular weight standard, and each of the remaining lanes (numbered 1-18) corresponds to a plant.
  • the T1 transgenic plants are selfed and seeds are harvested, and the seeds are cultivated into plants, which are T2 generation plants.
  • the T2 generation plants are selfed and the seeds are harvested, and the seeds are cultivated into plants, that is, the T3 generation plants.
  • the T3 generation plants are identified by PCR (the PCR identification method is the same as that of step 5).
  • the T2 generation plants are homozygous transgenic plants.
  • the offspring obtained by selfing of homozygous transgenic plants are homozygous transgenic lines.
  • the PCR identification method is the same as that of Step 1.
  • WAK1-15 plant or WAK1-17 plant
  • the maize inbred line Q11 as the female parent
  • cross-breed harvest the kernels, and cultivate the kernels into plants, namely BCh plants, and screen transgenic plants through PCR identification And non-transgenic plants.
  • WAK1-15 plant or WAK1-17 plant
  • Cross-breed harvest the kernels, and cultivate the kernels into plants, namely BCh plants, and screen transgenic plants through PCR identification ;
  • the transgenic plants in the BC ⁇ plant were used as the male parent, and the maize inbred line Q11 was used as the female parent, crossed, harvested the grains, and cultivated the grains into plants, namely BCK plants.
  • the transgenic plants were screened by PCR identification; BC 2 Fi
  • BC 2 Fi The transgenic plant in the plant is used as the male parent, and the maize inbred line Q11 is used as the female parent.
  • Crossing is performed, the grains are harvested, and the grains are cultivated into plants, which are BC ⁇ plants, and the transgenic plants and non-transgenic plants are screened by PCR.
  • the pathogenic bacteria of gray spot disease Cercospora zeina).
  • DSI disease index
  • the specific method of inoculating pathogenic bacteria (bacterial fluid filling method): Suspend the spores of the gray spot disease-causing bacteria in sterile water to obtain a spore suspension with a spore concentration of l X 10 5 cfu/mL, and use a syringe to suspend the spores The solution was poured into the corn leaf heart, and 5 mL per corn plant was poured.
  • the grading standard of disease grade (X represents the percentage of diseased spot area in leaf area):
  • Level 1 (assigned as 0): X 5%;
  • Level 3 (assigned as 0.25): 5% ⁇ X ⁇ 10%;
  • Level 5 (assignment value is 0.5): 10% ⁇ X ⁇ 30%;
  • Level 7 (assignment value of 0.75): 30% ⁇ X ⁇ 70%; Level 9 (assigned as 1): 70% ⁇ X ⁇ 100%.
  • the first set of test materials BC ⁇ plants obtained from the WAK1-15 plant as the male parent in step 3, transgenic and non-transgenic plants in the BC ⁇ plant obtained from the WAK1-17 plant as the male parent in step 3 Transgenic plants and non-transgenic plants in.
  • the second group of test materials BC a F plants obtained from the WAK1-15 plant as the male parent in step 3, transgenic plants and non-transgenic plants, and BC ⁇ obtained from the WAK1-17 plant in step 3 as the male parent. Transgenic plants and non-transgenic plants in plants.
  • the first group of test materials were identified for disease resistance according to the method in step 1.
  • the disease index of transgenic plants was significantly lower than that of non-transgenic plants, and DSI decreased by 10. 5-11. 6%.
  • the second group of test materials were identified for disease resistance according to the method in step 1.
  • the disease index of transgenic plants was significantly lower than that of non-transgenic plants, and DSI decreased by 9. 5-10. 6%.
  • Test materials T3 generation plants of the homozygous transgenic line obtained in step 2, and maize inbred line B73-329 plants.
  • the test materials were identified for disease resistance according to the method in step 1.
  • the results are shown in Figure 4 (the gray bar graph is the transgenic recipient material (B73-329); the black bar graph is pure transgenic plants; the numbers in the bar graph indicate the number of plants; *: K0. 05).
  • the disease index of transgenic plants was significantly lower than that of maize inbred line B73-329 plants, and the disease index of 051 was reduced by 9.5%.
  • AWPU gene is a functional gene of IL-qRlsl.
  • the introduction of AWPU gene into maize can significantly reduce its gray spot disease index by about 10%.
  • Embodiment 3. Verify the function of the open reading frame
  • step 2 Introduce the recombinant plasmid obtained in step 1 into Agrobacterium EHA105 to obtain recombinant Agrobacterium.
  • step 3 Take the recombinant Agrobacterium obtained in step 2, and use the Agrobacterium-mediated method to genetically transform the immature embryos of the maize inbred line B73-329 to obtain T0 generation plants.
  • the T0 generation plants are selfed, the seeds are harvested, and the seeds are cultivated into plants, which are the T1 generation plants.
  • the T1 generation plants were identified by PCR, and transgenic plants were screened.
  • the transgenic plants selected from the T1 generation plants are the T1 transgenic plants.
  • Several genetically modified plants were selected from the T1 generation plants, three of which were named WAK2-6 plants, WAK2-7 plants and WAK2-8 plants.
  • PCR identification method Take plant leaves, extract genomic DNA, and use primer pair composed of F2 and R2 to perform PCR amplification. If a 530bp amplified product is obtained, the PCR identification is positive, the plant is a transgenic plant; if no amplified product is obtained , PCR identification is negative, the plant is non-transgenic plant.
  • F2 TTTTAGCCCTGCCTTCATACGC
  • R2 CGACATCGAATTCGGATAAAGGA.
  • the PCR identification results of some plants are shown in Figure 5.
  • the arrow marks the target band, the leftmost lane is the molecular weight standard (M), and each of the remaining lanes corresponds to a plant (numbered 1-19).
  • the PCR identification method is the same as step 5 of step 1.
  • WAK2-6 plants or WAK2-7 plants or WAK2-8 plants
  • WAK2-7 plants or WAK2-8 plants as the male parent
  • WAK2-8 plants the maize inbred line Q11 as the female parent.
  • Cross-breed harvest the kernels, and cultivate the kernels into plants, that is, BCA plants. PCR identification and screening of transgenic plants and non-transgenic plants.
  • WAK2-6 plants or WAK2-7 plants or WAK2-8 plants
  • WAK2-7 plants or WAK2-8 plants as the male parent
  • the maize inbred line Q11 as the female parent
  • crossbreed harvest the kernels, and cultivate the kernels into plants, that is, BCA plants.
  • transgenic plants in BC ⁇ plants are used as the male parent
  • maize inbred line Q11 is used as the female parent
  • crosses are performed, the grains are harvested, and the seeds are cultivated into plants, namely BC ⁇ plants, and the transgenic plants and non-transgenic plants are screened by PCR Plant.
  • the first group of test materials Transgenic plants and non-transgenic plants in the BC ⁇ plants obtained from the WAK2-6 plant in step 1 as the male parent, and BC ⁇ plants in the BC ⁇ plant obtained from the WAK2-7 plant as the male parent in step 2 Transgenic plants and non-transgenic plants in Step 2, transgenic plants and non-transgenic plants in BCA plants obtained by using WAK2-8 plants as the male parent in Step 2 1.
  • the second group of test materials the transgenic plants and non-transgenic plants in the BC ⁇ plants obtained from the WAK2-6 plant in step 2 as the male parent, and the BC 3 F obtained from the WAK2-7 plant in step 2 as the male parent.
  • the first group of test materials were identified for disease resistance according to the method in step 1.
  • the disease index of transgenic plants was significantly lower than that of non-transgenic plants, and DSI decreased by 8.3-10. 5%.
  • the second group of test materials were identified for disease resistance according to the method in step 1.
  • the disease index of transgenic plants was significantly lower than that of non-transgenic plants, and DSI was reduced by 10. 8-11. 9%.
  • the AWPU gene is a functional gene in the main ⁇ L qRglsl segment for resistance to gray spot disease.
  • Backcrossing it into the Q11 genetic background can significantly improve the resistance of maize to gray spot disease.
  • Example 4. Verify the function of the 7.2 kb fragment on the B73 genetic background
  • step 3 Take the recombinant Agrobacterium obtained in step 2, and use the Agrobacterium-mediated method to genetically transform the immature embryos of the maize inbred line B73 to obtain T0 generation plants.
  • the T0 generation plants are selfed, the seeds are harvested, and the seeds are cultivated into plants, which are the T1 generation plants.
  • T1 generation plants were identified by PCR, and transgenic plants were screened.
  • the PCR identification method is the same as step 5 of Example 2.
  • the transgenic plants selected from the T1 generation plants are the T1 transgenic plants.
  • Test materials T3 generation plants of C#1 line, T3 generation plants of C#2 line, T3 generation plants of C#3 line, and corn inbred line B73 plants.
  • test materials shall be identified for disease resistance according to the method in step 1.
  • the disease index of the C#1 line was significantly reduced (a decrease of 21.3%), and the disease index of the C#2 line was significantly reduced (a decrease of 28.6). %), the plant disease index of the C#3 strain was significantly reduced (the reduction was 10.5%).
  • Figure 7 the gray bar graph is the transgenic recipient material; the black bar graph is pure transgenic plants; the numbers in the bar graph indicate the number of plants; *: K0. 05; **: K0. 01).
  • step 3 Take the recombinant Agrobacterium obtained in step 2, and use the Agrobacterium-mediated method to genetically transform the immature embryos of the maize inbred line B73 to obtain T0 generation plants.
  • the T0 generation plants are selfed, the seeds are harvested, and the seeds are cultivated into plants, which are the T1 generation plants.
  • T1 generation plants were identified by PCR, and transgenic plants were screened.
  • the PCR identification method is the same as step 5 of Example 3.
  • the transgenic plants selected from the T1 generation plants are the T1 transgenic plants.
  • Test materials T3 generation plants of 0#1 line, T3 generation plants of 0#2 line, T3 generation plants of 0#3 line, T3 generation plants of 0#4 line, B73 plants of maize inbred line .
  • test materials shall be identified for disease resistance according to the method in step 1.
  • the disease index of the 0#1 line was significantly reduced (a reduction of 22.5%), and the disease index of the 0#2 line was significantly reduced (the reduction was 16. 3 %), the plant disease index of the 0#3 line was significantly reduced (28.3% reduction), and the plant disease index of the 0#4 line was significantly reduced (22.2% reduction).
  • Figure 8 the gray bar graph is the transgenic recipient material; the black bar graph is pure transgenic plants; the number in the bar graph indicates the number of plants; *: K0. 05).
  • step 3 Take the recombinant Agrobacterium obtained in step 2, and use the Agrobacterium-mediated method to genetically transform the immature embryos of the maize inbred line B73 to obtain T0 generation plants.
  • the T0 generation plants are selfed, the seeds are harvested, and the seeds are cultivated into plants, which are the T1 generation plants.
  • the T1 generation plants were identified by PCR, and transgenic plants were screened.
  • the transgenic plants selected from the T1 generation plants are the T1 transgenic plants.
  • Several transgenic plants were selected from the T1 generation plants, three of which were named R1 plants, R2 plants and R3 plants.
  • PCR identification method Take plant leaves, extract genomic DNA, and use a primer pair composed of F3 and R3 for PCR amplification. If a 357bp amplified product is obtained, the PCR identification is positive, the plant is a transgenic plant; if no amplified product is obtained , PCR identification is negative, the plant is non-transgenic plant.
  • R3 TCGGTGACGGGCAGGACCGG.
  • the PCR identification method is the same as step 5 of step 1.
  • R1 plant or R2 plant or R3 plant
  • the maize inbred line Q11 as the female parent
  • the transgenic plants in the BCA plants were used as the male parent
  • the maize inbred line Q11 was used as the female parent.
  • Crossing was performed, the grains were harvested, and the grains were cultivated into plants, namely BCK plants, and the transgenic plants and non-transgenic plants were screened by PCR identification.
  • Test materials transgenic plants and non-transgenic plants in the BC 2 Fi plant obtained from the R1 plant as the male parent in Step 2, transgenic plants and non-transgenic plants in the BC 2 Fi plant obtained from the R2 plant as the male parent in Step 2, Second, the transgenic plants and non-transgenic plants in the BC 2 Fi plants obtained from the R3 plant as the male parent.
  • test materials shall be identified for disease resistance according to the method in step 1.
  • the disease index of transgenic plants was significantly lower than that of non-transgenic plants, and DSI was reduced by 8.4-14. 1%.
  • the inventors of the present invention provided the ZmWAK-RLK protein and its coding gene, through the overexpression transgene experiment Significantly reduce the disease index of corn gray spot disease.
  • the invention has great application value for the breeding of maize against gray spot disease.

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Abstract

本发明公开了一种灰斑病抗性相关蛋白ZmWAK-RLK及其编码基因和应用。本发明提供的蛋白质，获自玉米自交系Y32，命名为ZmWAK-RLK蛋白，为序列表中序列1所示的蛋白质。编码ZmWAK-RLK蛋白的核酸分子也属于本发明的保护范围。本发明还保护一种制备转基因植物的方法，包括如下步骤：在出发植物中导入所述核酸分子，得到灰斑病抗病性增强的转基因植物。本发明还保护一种植物育种方法，包括如下步骤：增加目的植物中ZmWAK-RLK蛋白的含量和/或活性，从而提高目的植物的灰斑病抗病性。本发明对于玉米的抗灰斑病育种具有重大的应用价值。

Description

灰斑病抗性相关蛋白 ZmWAK-RLK及其编码基因和应用 技术领域
本发明属于生物技术领域， 具体涉及一种灰斑病抗性相关蛋白
ZmWAK-RLK及其编码基因和应用。
背景技术
玉米灰斑病 (GLS) 是一种玉米叶部病害， 影响玉米的产量和品质。 20世纪 20年代， 灰斑病首次在美国伊利诺伊州亚历山大县发现， 随后逐 渐发展成为一种严重的全球性叶部病害。 灰斑病广泛分布在美国、 亚洲、 欧洲以及非洲的玉米主产区。在发病情况下， 灰斑病能够导致 20-60%的减 产， 在发病严重的情况下可达 100%， 对玉米生产造成严重的经济损失。
玉米灰斑病是一种真菌性病害， 普遍认为其致病菌主要有玉蜀黍尾孢 菌 ( Czm, Cercospora zeae-maydis)和玉米尾抱菌 ( Cz, Cercospora zeina) 两种。 2013年 Liu等人广泛采集了我国玉米灰斑病发生区的病样， 利用单 孢分离方法获得大量菌株， 采用病菌形态学、 培养特征生长状态和分子生 物学的手段， 准确的鉴定了我国不同地区的玉米灰斑病致病种， 引起我国 北方地区玉米灰斑病的是玉蜀黍尾孢菌， 而引起西南地区玉米灰斑病的是 玉米尾孢菌。
据报道， 玉米对灰斑病的抗性属于数量遗传， 由多基因控制， 以加性 效应为主。 那么如果克隆到灰斑病抗病基因， 利用分子标记辅助选育技术 导入现有的自交系， 将能够提高推广品种的灰斑病抗性。
发明公开
本发明提供了一种灰斑病抗性相关蛋白 ZmWAK-RLK及其编码基因和应 用。
本发明提供的蛋白质， 获自玉米自交系 Y32 , 命名为 ZmWAK-RLK蛋白， 为如下 (al) 或 (a2 ) 或 (a3 ) 或 (a4 ) 或 (a5 ) :
(al) 序列表中序列 1所不的蛋白质；
(a2 ) 序列表中序列 7所不的蛋白质；
(a3 ) 在 (al) 或 (a2 ) 所述蛋白质的 N端或 /和 C端连接标签得到的 融合蛋白； （a4） 将 （al） 或 （a2） 经过一个或几个氨基酸残基的取代和 /或缺失 和 /或添加得到的与植物灰斑病抗病性相关的蛋白质；
（a5） 来源于玉米且与 （al） 或 （a2） 具有 90%、 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%或 99%以上同一性且与植物灰斑病抗病性 相关的蛋白质。
标签具体如表 1所示。
表 1 标签的序列
Figure imgf000004_0001
蛋白质可人工合成， 也可先合成其编码基因， 再进行生物表达得到。 编码 ZmWAK-RLK蛋白的核酸分子也属于本发明的保护范围。
所述核酸分子为如下（bl）或（b2）或（b3）或（b4）或（b5）或（b6） :
（bl）编码区如序列表中序列 2第 87-2084位核苷酸所示的 DNA分子; （b2） 序列表中序列 2所示的 DNA分子；
（b3） 序列表中序列 3所示的 DNA分子；
（b4） 编码区如序列表中序列 6所示的 DNA分子；
（b5） 来源于玉米且与 （bl） 或 （b2） 或 （b3） 或 （b4） 具有 90%、
91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%或 99%以上同一性且 编码所述蛋白质的 DNA分子；
（b6） 在严格条件下与 （bl） 或 （b2） 或 （b3） 或 （b4） 限定的核苷 酸序列杂交且编码所述蛋白质的 DNA分子。
所述严格条件是在 2XSSC， 0.1% SDS 的溶液中， 在 68°C下杂交并洗 膜 2次， 每次 5min， 又于 0.5XSSC， 0.1% SDS 的溶液中， 在 68°C下杂交 并洗膜 2 次， 每次 15min。
含有所述核酸分子的 DNA分子、 表达盒、 重组载体或重组微生物均属 于本发明的保护范围。
含有所述核酸分子的 DNA分子具体可如序列表的序列 4所示。 可用现有的表达载体构建含有所述核酸分子的重组表达载体。 使用所 述核酸分子构建重组表达载体时， 可在其转录起始核苷酸前加上任何一种 增强型、 组成型、 组织特异型或诱导型启动子， 它们可单独使用或与其它 的植物启动子结合使用； 此外， 使用所述核酸分子构建重组表达载体时， 还可使用增强子， 包括翻译增强子或转录增强子， 这些增强子区域可以是 ATG起始密码子或邻接区域起始密码子等， 但必需与编码序列的阅读框相 同， 以保证整个序列的正确翻译。 所述翻译控制信号和起始密码子的来源 是广泛的， 可以是天然的， 也可以是合成的。 翻译起始区域可以来自转录 起始区域或结构基因。 为了便于对转基因植物或转基因微生物进行鉴定及 筛选， 可对所用表达载体进行加工， 如加入在植物或微生物中表达可产生 颜色变化的酶或发光化合物的基因、 具有抗性的抗生素标记物或是抗化学 试剂标记基因等。 从转基因安全性考虑， 可不加任何选择性标记基因， 直 接以表型筛选转化植物或微生物。
重组表达载体具体可为： 将序列表的序列 4 所示双链 DNA 分子插入 PCAMBIA3301载体的多克隆位点
Figure imgf000005_0001
位点) 得到的重组质粒。
重组表达载体具体可为： 将序列表的序列 2第 87-2084位核苷酸所示 的双链 DNA分子插入 pBCXUN载体的多克隆位点
Figure imgf000005_0002
得到的重组质粒。
重组表达载体具体可为： 将序列表的序列 6所示重组双链 DNA分子插 入 pBCXUN载体的多克隆位点
Figure imgf000005_0003
得到的重组质粒。
本发明还保护 ZmWAK-RLK蛋白的应用， 为如下 (cl)或 (c2)或 (c3) 或 (c4) :
(cl) 调控植物的灰斑病抗病性；
(c2) 提高植物的灰斑病抗病性；
(c3) 调控植物对玉米尾孢菌引发的病害的抗病性；
(c4) 提高植物对玉米尾孢菌引发的病害的抗病性。
本发明还保护所述核酸分子或所述含有所述核酸分子的 DNA分子的应 用， 为如下 (dl) 或 (d2) 或 (d3) 或 (d4) :
(dl) 培育灰斑病抗病性改变的转基因植物；
(d2) 培育灰斑病抗病性增强的转基因植物； （d3） 培育对玉米尾孢菌引发的病害的抗病性改变的转基因植物； （d4） 培育对玉米尾孢菌引发的病害的抗病性增强的转基因植物。 所述核酸分子的应用， 还包括通过 CRISPS/CAS9技术使用该基因的实 现方式。 例如： 基因组片段重置 （将抗病等位基因导入感病基因组） ， 等 位基因交换 （用抗病等位基因替换感病等位基因） ， 通过基因编辑将感病 等位基因改成抗病等位基因等等。
所述核酸分子的应用， 还包括以增强所述核酸分子的表达为目的的其 它实现方式。 例如， 通过启动子置换增强所述核酸分子的表达， 通过引入 增强子增强所述核酸分子的表达， 等等。
本发明还保护一种制备转基因植物的方法， 包括如下步骤： 在出发植 物中导入所述核酸分子或所述含有所述核酸分子的 DNA分子， 得到灰斑病 抗病性增强的转基因植物。 所述核酸分子具体可通过以上任一所述重组表 达载体导入所述出发植物。携带有所述核酸分子的重组表达载体可通过 Ti 质粒、 Ri质粒、 植物病毒载体、 直接 DNA转化、 显微注射、 电导、 农杆菌 介导等常规生物学方法转化到出发植物中。 将所述转基因植物与现有玉米 品种进行杂交 （包括单次杂交和多次杂交， 例如连续杂交三次） ， 得到的 转基因后代植株同样为抗病性增强的转基因植物。 所述现有玉米品种具体 可为玉米自交系 Q11。
本发明还保护一种植物育种方法， 包括如下步骤： 增加目的植物中 ZmWAK-RLK蛋白的含量和 /或活性， 从而提高目的植物的灰斑病抗病性。
本发明还保护一种制备转基因植物的方法， 包括如下步骤： 在出发植 物中导入所述核酸分子或所述含有所述核酸分子的 DNA分子， 得到抗病性 增强的转基因植物； 所述抗病性为对玉米尾孢菌引发的病害的抗病性。 所 述核酸分子具体可通过以上任一所述重组表达载体导入所述出发植物。 携 带有所述核酸分子的重组表达载体可通过 Ti质粒、 Ri质粒、 植物病毒载 体、 直接 DNA转化、 显微注射、 电导、 农杆菌介导等常规生物学方法转化 到出发植物中。 将所述转基因植物与现有玉米品种进行杂交 （包括单次杂 交和多次杂交， 例如连续杂交三次） ， 得到的转基因后代植株同样为抗病 性增强的转基因植物。 所述现有玉米品种具体可为玉米自交系 Q11。
本发明还保护一种植物育种方法， 包括如下步骤： 增加目的植物中 ZmWAK-RLK蛋白的含量和 /或活性， 从而提高目的植物的抗病性； 所述抗病 性为对玉米尾孢菌引发的病害的抗病性。
以上任一所述植物为双子叶植物或单子叶植物。 所述单子叶植物可为 禾本科植物。 所述禾本科植物可为玉蜀黍属植物。 所述玉蜀黍属植物具体 可为玉米， 例如玉米自交系 B73， 例如玉米自交系 B73-329。
以上任一所述灰斑病具体可为玉米尾孢菌引起的灰斑病。
附图说明及图注
图 1 为实施例 2 中部分植株的 PCR 鉴定结果； 箭头标注目标带 （1197bp） ， 最左侧的泳道为分子量标准 （M） ， 其余每个泳道对应一株 植株 （编号 1-18） ; 如果得到 1197bp的扩增产物、 PCR鉴定为阳性、 该 植株为转基因植株； 如果没有得到扩增产物、 PCR鉴定为阴性、 该植株为 非转基因植株。
图 2为各个分级的代表性叶片的照片； 抗病亲本 Y32叶片病斑较少， 感病亲本 Q1 1病斑较多。
图 3为实施例 2中回交分离后代的抗病性鉴定结果;在 BCA代和 BCA 代， 分别统计了 WAK1-15和 WAK1-17两个转基因事件后代中非转基因植株 和转基因植株的病情指数； 灰色柱形图为非转基因植株， 黑色柱形图为转 基因植株， 柱形图内数字表示植株数量； * : 尸＜0.05 ; **: 尸＜0.01。
图 4为实施例 2中纯合的转基因株系的抗病性鉴定结果； 纯合转基因 阳性植株及转基因受体材料 （B73-329） 病情指数， 纯合转基因阳性植株 的病情指数显著低于转基因受体材料； 灰色柱形图为转基因受体材料 B73-329 , 黑色柱形图为纯系转基因植株， 柱形图内数字表示植株数量。
图 5为实施例 3中部分植株的 PCR鉴定结果;箭头标注目标带（530bp）， 最左侧的泳道为分子量标准（M），其余每个泳道对应一株植株（编号 1-19）； 如果得到 530bp的扩增产物、 PCR鉴定为阳性、 该植株为转基因植株； 如 果没有得到扩增产物、 PCR鉴定为阴性、 该植株为非转基因植株。
图 6为实施例 3中回交分离后代的抗病性鉴定结果;在 BCA代和 BCA 代， 分别统计了 WAK2-6、 WAK2-7和 WAK2-8三个转基因事件后代中非转基 因植株和转基因植株的病情指数； 灰色柱形图为非转基因植株， 黑色柱形 图为转基因植株， 柱形图内数字表示植株
Figure imgf000007_0001
. 001；
NS： 无显著差异。 图 7为实施例 4中 B73背景互补转基因纯系的抗病性鉴定结果 (病情 指数) ； 灰色柱形图表示转基因受体材料 B73， 黑色柱形图表示转基因纯 系 C#1、 C#2和 C#3； 柱形图内数字表示植株数量； *: K0.05; **: K0.01。
图 8为实施例 5中 B73背景过表达转基因纯系的抗性鉴定结果 (病情 指数) ； 灰色柱形图表示受体材料 B73, 黑色柱形图表示转基因纯系 0#1、 0#2、 0#3和 0#4; 柱形图内数字表示植株数量； *: K0.05。
图 9为实施例 6中部分植株的 PCR鉴定结果;箭头标注目标带 (357bp)， 最左侧的泳道为分子量标准 (M)，其余每个泳道对应一株植株 (编号 1-14)； 如果得到 357bp的扩增产物、 PCR鉴定为阳性、 该植株为转基因植株； 如 果没有得到扩增产物、 PCR鉴定为阴性、 该植株为非转基因植株。
图 10为实施例 6中 Q11背景过表达嵌合基因的抗性鉴定结果 (病情 指数) ： 在 BC^代， 分别统计了 Rl、 R2和 R3三个转基因事件后代中非转 基因植株和转基因植株的病情指数； 灰色柱形图为非转基因植株； 黑色柱 形图为转基因植株； 柱形图内数字表示植株数量； *: K0.05; **: K0.01。
实施发明的最佳方式
以下的实施例便于更好地理解本发明， 但并不限定本发明。 下述实施 例中的实验方法， 如无特殊说明， 均为常规方法。 下述实施例中所用的试 验材料， 如无特殊说明， 均为自常规生化试剂商店购买得到的。 以下实施 例中的定量试验， 均设置三次重复实验， 结果取平均值。
玉米自交系 Y32， 为高抗玉米灰斑病的玉米自交系。 玉米自交系 Y32 ( line Y32)，记载于如下文献： Theoretical and Applied Genetics, 2012, 25 (8) : 1797-1808. Zhang, Y. , et al. 〃QTL mapping of resistance to gray leaf spot in maize.〃
玉米自交系 Qll， 为高感灰斑病的玉米自交系。 玉米自交系 Qll (line Q11 ) , 记载于如下文献： Theoretical and Applied Genetics, 2012, 25 (8) : 1797-1808. Zhang, Y. , et al. 〃QTL mapping of resistance to gray leaf spot in maize.〃
玉米自交系 B73-329 (B73-329 inbred lines) ， 记载于如下文献： New Phytologist, 2018, 217 (3) : 1161-1176. Zhang, M, , e t al. " A r e t r o t r a n s p o s o n in an HKT 1 family sodium transporter causes variation of leaf Na+ exclusion and salt tolerance in maize. 玉米自交系 B73 (B73 inbred lines) ， 记载于如下文献：
Plant Science, 1985， 41 (2) : 140. Everett, N. P. , et al. "Biochemical markers of embryogenesis in tissue cultures of the maize inbred B73. " 。
玉米尾孢菌 ( Cercospora zeina) ,记载于如下文献： Plant Disease, 2013， 97 (12) : 1656-1656. Liu, K. J.， et al. ''First Report of Gray Leaf Spot of Maize Caused by Cercospora zeina in China."。
pCAMBIA3301载体 (bivalent expression vector pCAMBIA3301) , 记载于如下文献： Theoretical and Applied Genetics 131.10 (2018) : 2145-2156. Zhu， X， et al. "Pyramiding of nine transgenes in maize generates high-level resistance against necrotrophic maize pathogens.〃 。
pBCXUN载体 (pBCXUN vector) ， 记载于如下文献： Journal of integrative plant biology 61.6 (2019) : 691-705. Qin， Y. J. , et al. "ZmHAK5 and ZmHAKl function in K+ uptake and distribution in maize under low K+ conditions.〃 。 实施例 1、 ZmWAK-RLK蛋白及其编码基因的发现
用高抗灰斑病玉米自交系 Y32 （作为供体亲本） 和高感灰斑病玉米自 交系 QU （作为轮回亲本） ， 构建初定位群体和精细定位群体。 鳥、 qRglsl 定位到玉米 8号染色体 IDP2和 M2之间， 物理位置约为 120kb。
利用位于精细定位的 m^qRglsl区域的分子标记， 通过 PCR的方法 筛选抗病亲本 Y32 BAC文库。 进行 BAC克隆指纹图谱分析， 构建覆盖整个 基因区段的 BAC重叠群。 选择能够覆盖基因区域最少的克隆， 用于测序。 通过序列比对和表达分析发现了一个新基因， 编码序列表的序列 1所示的 蛋白质。
将序列表的序列 1 所示的蛋白质命名为 ZmWAK-RLK 蛋白。 将编码 ZmWAK-RLK蛋白的基因命
Figure imgf000009_0001
。玉米自交系 Y32的 cDNA中 的 AWPU基因如序列表的序列 2所示 （其中第 87-2084位核苷酸为开 放阅读框） （序列 2中， 第 87-1058位核苷酸用于构建嵌合体基因） 。 玉 米自交系 Y32的基因组 DNA中的 AWPU基因如序列表的序列 3所示。 玉米自交系
Figure imgf000010_0001
的开放阅读框序列如序列表 的序列 5所示（序列 5中， 第 1102-2115位核苷酸用于构建嵌合体基因）。 嵌合体基因如序列表中的序列 6所示， 表达序列表的序列 7所示的嵌合蛋 白。 实施例 2、 验证 7. 2kb的片段的功能
转基因植株的获得
1、 将玉米自交系 Y32 中约 7. 2kb 的片段 （该片段如序列表的序列 4 所示； 序列 4中， 第 1-2103位核苷酸为启动子， 第 2104-4316位核苷酸 与序列表的序列 3相同） 插入 pCAMBIA3301载体的 BamH I酶切位点， 得 到重组质粒。
2、 将步骤 1得到的重组质粒导入农杆菌 EHA105， 得到重组农杆菌。
3、 取步骤 2 得到的重组农杆菌， 采用农杆菌介导法对玉米自交系 B73-329的幼胚进行遗传转化， 得到 T0代植株。
4、 T0代植株自交， 收获籽粒， 将籽粒培育为植株， 即为 T1代植株。
5、 将 T1代植株进行 PCR鉴定， 筛选转基因植株。 从 T1代植株中筛 选到的转基因植株， 即为 T1转基因植株。 从 T1代植株中筛选到若干转基 因植株， 其中两株命名为 WAK1-15植株和 WAK1-17植株。
PCR鉴定方法： 取植株叶片， 提取基因组 DNA， 采用 F1和 R1组成的 引物对进行 PCR扩增， 如果得到 1 197bp的扩增产物、 PCR鉴定为阳性、 该植株为转基因植株； 如果没有得到扩增产物、 PCR鉴定为阴性、 该植株 为非转基因植株。
FI： CGAGGAGGTTTCCCGATATTAC； Rl： CACGTCAATCCGAAGGCTATTA。 部分植株的 PCR鉴定结果见图 1，箭头标注目标带，最左侧的泳道（M） 为分子量标准， 其余每个泳道 （编号 1-18） 对应一株植株。
二、 B73-329遗传背景转基因纯系的获得
T1转基因植株自交并收获种子， 将种子培育为植株， 即为 T2代植株。 T2代植株自交并收获种子， 将种子培育为植株， 即为 T3代植株。 T3代植株进行 PCR鉴定 （PCR鉴定方法同步骤一的 5） 。
对于某一 T2代植株， 如果其自交得到的 T3代植株均为转基因植株， 该 T2 代植株为纯合的转基因植株。 纯合的转基因植株自交得到的后代为 纯合的转基因株系。
三、 回交分离后代的获得
PCR鉴定方法同步骤一的 5。
1、 将 WAK1-15植株 （或 WAK1-17植株） 作为父本， 玉米自交系 Q11 作为母本， 进行杂交， 收获籽粒， 将籽粒培育为植株， 即为 BCh植株， 通 过 PCR鉴定筛选转基因植株和非转基因植株。
2、 将 WAK1-15植株 （或 WAK1-17植株） 作为父本， 玉米自交系 Q11 作为母本， 进行杂交， 收获籽粒， 将籽粒培育为植株， 即为 BCh植株， 通 过 PCR鉴定筛选转基因植株； 将 BC^植株中的转基因植株作为父本， 玉米 自交系 Q11作为母本，进行杂交，收获籽粒，将籽粒培育为植株，即为 BCK 植株，通过 PCR鉴定筛选转基因植株；将 BC2Fi植株中的转基因植株作为父 本， 玉米自交系 Q11作为母本， 进行杂交， 收获籽粒， 将籽粒培育为植株， 即为 BC^植株， 通过 PCR鉴定筛选转基因植株和非转基因植株。
四、 植株的抗病性鉴定
1、 抗病性鉴定的方法
抗病性鉴定在中国农业大学上庄实验基地进行。
灰斑病致病菌： 玉米尾孢菌 i Cercospora zeina） 。
正常培养供试材料， 10叶期接种致病菌， 然后继续正常培养， 授粉两 周后进行表型调查， 采用分级调查计算病情指数 （DSI） 。 接种致病菌的 具体方法 （菌液灌心法） ： 用无菌水悬浮灰斑病致病菌的孢子， 得到孢子 浓度为 l X 105cfu/mL 的孢子悬液， 利用注射器将孢子悬液灌注于玉米叶 心， 每株玉米灌注 5mL。
病情等级的分级标准 （X代表病斑面积占叶片面积的百分比） ：
1级 （赋值为 0） : X 5%;
3级 （赋值为 0. 25） : 5%<X^ 10%；
5级 （赋值为 0. 5） : 10%<X^ 30%；
7级 （赋值为 0. 75） : 30%<X^ 70%； 9级 （赋值为 1） : 70%<X< 100%。
各个分级的代表性叶片的照片见图 2。
Figure imgf000012_0001
（病情等级財应的赋值 X该等级的植株数） K 100 病情指敷 ( )
Figure imgf000012_0002
X总株敷
2、 回交分离后代 （BCh植株和 BCA植株） 的抗病性鉴定
第一组供试材料： 步骤三的 1 中 WAK1-15植株作为父本得到的 BC^ 植株中的转基因植株和非转基因植株、 步骤三的 1中 WAK1-17植株作为父 本得到的 BC^植株中的转基因植株和非转基因植株。
第二组供试材料： 步骤三的 2 中 WAK1-15植株作为父本得到的 BCaF 植株中的转基因植株和非转基因植株、 步骤三的 2中 WAK1-17植株作为父 本得到的 BC^植株中的转基因植株和非转基因植株。
第一组供试材料按照步骤 1的方法进行抗病性鉴定。 转基因植株的病 情指数显著低于非转基因植株， DSI降低 10. 5-11. 6%。
第二组供试材料按照步骤 1的方法进行抗病性鉴定。 转基因植株的病 情指数显著低于非转基因植株， DSI降低 9. 5-10. 6%。
结果见图 3（灰色柱形图为非转基因植株； 黑色柱形图为转基因植株; 柱形图内数字表示植株数量； * : K0. 05 ; **: K0. 01） 。
3、 纯合的转基因株系的抗病性鉴定
供试材料： 步骤二得到的纯合的转基因株系的 T3 代植株、 玉米自交 系 B73-329植株。
供试材料按照步骤 1 的方法进行抗病性鉴定。 结果见图 4 （灰色柱形 图为转基因受体材料 （B73-329） ; 黑色柱形图为转基因纯系植株； 柱形 图内数字表示植株数量； * : K0. 05） 。 转基因植株的病情指数显著低于 玉米自交系 B73-329植株， 051降低9. 5%。
以上结果表明， AWPU基因为 ^IL-qRlsl 的功能基因。 将 AWPU基因导入玉米， 可以显著的降低其灰斑病病情指数 10%左右。 实施例 3、 验证开放阅读框的功能
转基因植株的获得
1、 将序列表的序列 2第 87-2084位核苷酸所示的双链 DNA分子插入 pBCXUN载体的
Figure imgf000013_0001
得到重组质粒。
2、 将步骤 1得到的重组质粒导入农杆菌 EHA105， 得到重组农杆菌。
3、 取步骤 2 得到的重组农杆菌， 采用农杆菌介导法对玉米自交系 B73-329的幼胚进行遗传转化， 得到 T0代植株。
4、 T0代植株自交， 收获籽粒， 将籽粒培育为植株， 即为 T1代植株。
5、 将 T1代植株进行 PCR鉴定， 筛选转基因植株。 从 T1代植株中筛 选到的转基因植株， 即为 T1转基因植株。 从 T1代植株中筛选到若干转基 因植株， 其中三株命名为 WAK2-6植株、 WAK2-7植株和 WAK2-8植株。
PCR鉴定方法： 取植株叶片， 提取基因组 DNA， 采用 F2和 R2组成的 引物对进行 PCR扩增， 如果得到 530bp的扩增产物、 PCR鉴定为阳性、 该 植株为转基因植株； 如果没有得到扩增产物、 PCR鉴定为阴性、 该植株为 非转基因植株。
F2： TTTTAGCCCTGCCTTCATACGC；
R2： CGACATCGAATTCGGATAAAGGA。
部分植株的 PCR鉴定结果见图 5 , 箭头标注目标带， 最左侧的泳道为 分子量标准 （M） ， 其余每个泳道对应一株植株（编号 1-19）。
二、 回交分离后代的获得
PCR鉴定方法同步骤一的 5。
1、 将 WAK2-6植株 （或 WAK2-7植株或 WAK2-8植株） 作为父本， 玉米 自交系 Q11作为母本，进行杂交，收获籽粒，将籽粒培育为植株，即为 BCA 植株， 通过 PCR鉴定筛选转基因植株和非转基因植株。
2、 将 WAK2-6植株 （或 WAK2-7植株或 WAK2-8植株） 作为父本， 玉米 自交系 Q11作为母本，进行杂交，收获籽粒，将籽粒培育为植株，即为 BCA 植株，通过 PCR鉴定筛选转基因植株；将 BC^植株中的转基因植株作为父 本， 玉米自交系 Q11作为母本， 进行杂交， 收获籽粒， 将籽粒培育为植株， 即为 BC^植株， 通过 PCR鉴定筛选转基因植株； 将 BC^植株中的转基因 植株作为父本， 玉米自交系 Q11作为母本， 进行杂交， 收获籽粒， 将籽粒 培育为植株， 即为 BC^植株，通过 PCR鉴定筛选转基因植株和非转基因植 株。
三、 植株的抗病性鉴定 1、 抗病性鉴定的方法
同实施例 2的步骤四的 1。
2、 回交分离后代 （BCA植株和 BCA植株） 的抗病性鉴定
第一组供试材料： 步骤二的 1中 WAK2-6植株作为父本得到的 BC^植 株中的转基因植株和非转基因植株、 步骤二的 1 中 WAK2-7植株作为父本 得到的 BC^植株中的转基因植株和非转基因植株、 步骤二的 1 中 WAK2-8 植株作为父本得到的 BCA植株中的转基因植株和非转基因植株。
第二组供试材料： 步骤二的 2中 WAK2-6植株作为父本得到的 BC^植 株中的转基因植株和非转基因植株、 步骤二的 2 中 WAK2-7植株作为父本 得到的 BC3F植株中的转基因植株和非转基因植株、 步骤二的 2 中 WAK2-8 植株作为父本得到的 BCK植株中的转基因植株和非转基因植株。
第一组供试材料按照步骤 1的方法进行抗病性鉴定。 转基因植株的病 情指数显著低于非转基因植株， DSI降低 8. 3-10. 5%。
第二组供试材料按照步骤 1的方法进行抗病性鉴定。 转基因植株的病 情指数显著低于非转基因植株， DSI降低 10. 8-11. 9%。
结果见图 6（灰色柱形图为非转基因植株； 黑色柱形图为转基因植株; 柱形图内数字表示植株数量； * : K0. 05； *** : K0. 001； NS： 无显著差 异） °
以上结果表明， ? AWPU基因为抗灰斑病主效 ^L qRglsl区段内的 功能基因，将其回交导入 Q11遗传背景可以显著提高玉米对灰斑病的抗性。 实施例 4、 在 B73遗传背景验证 7. 2kb的片段的功能
转基因植株的获得
1、 同实施例 2的步骤一的 1。
2、 同实施例 2的步骤一的 2。
3、取步骤 2得到的重组农杆菌， 采用农杆菌介导法对玉米自交系 B73 的幼胚进行遗传转化， 得到 T0代植株。
4、 T0代植株自交， 收获籽粒， 将籽粒培育为植株， 即为 T1代植株。
5、 将 T1代植株进行 PCR鉴定， 筛选转基因植株。
PCR鉴定方法同实施例 2的步骤一的 5。 从 T1代植株中筛选到的转基因植株， 即为 T1转基因植株。
二、 B73遗传背景转基因纯系的获得
同实施例 2的步骤二。
分别获得三个的纯系转基因材料， C#1株系、 C#2株系和 C#3株系。 三、 植株的抗病性鉴定
1、 抗病性鉴定的方法
同实施例 2的步骤四的 1。
2、 B73遗传背景纯合的转基因株系的抗病性鉴定
供试材料： C#1株系的 T3代植株、 C#2株系的 T3代植株、 C#3株系的 T3代植株、 玉米自交系 B73植株。
供试材料按照步骤 1的方法进行抗病性鉴定。
相对受体材料 （玉米自交系 B73植株） ， C#1株系植株病情指数显著 降低 （降低幅度为 21. 3%） ， C#2 株系植株病情指数显著降低 （降低幅度 为 28. 6%） ， C#3株系植株病情指数显著降低 （降低幅度为 10. 5%） 。 结果 见图 7 （灰色柱形图为转基因受体材料； 黑色柱形图为转基因纯系植株； 柱形图内数字表示植株数量； * : K0. 05 ; **: K0. 01） 。
整体而言， 转入抗
Figure imgf000015_0001
可以显著的降低玉米的灰斑病病 情指数， 提高玉米对灰斑病的抗性。 实施例 5、 在 B73遗传背景验证开放读码框的功能
转基因植株的获得
1、 同实施例 3的步骤一的 1。
2、 同实施例 3的步骤一的 2。
3、取步骤 2得到的重组农杆菌， 采用农杆菌介导法对玉米自交系 B73 的幼胚进行遗传转化， 得到 T0代植株。
4、 T0代植株自交， 收获籽粒， 将籽粒培育为植株， 即为 T1代植株。
5、 将 T1代植株进行 PCR鉴定， 筛选转基因植株。
PCR鉴定方法同实施例 3的步骤一的 5。
从 T1代植株中筛选到的转基因植株， 即为 T1转基因植株。
二、 B73遗传背景转基因纯系的获得 同实施例 2的步骤二。
分别获得四个的纯系转基因材料， 0#1 株系、 0#2 株系、 0#3 株系和 0#4株系。
三、 植株的抗病性鉴定
1、 抗病性鉴定的方法
同实施例 2的步骤四的 1。
2、 B73遗传背景纯合的转基因株系的抗病性鉴定
供试材料： 0#1株系的 T3代植株、 0#2株系的 T3代植株、 0#3株系的 T3代植株、 0#4株系的 T3代植株、 玉米自交系 B73植株。
供试材料按照步骤 1的方法进行抗病性鉴定。
相对受体材料 （玉米自交系 B73植株） ， 0#1株系植株病情指数显著 降低 （降低幅度为 22. 5%） ， 0#2 株系植株病情指数显著降低 （降低幅度 为 16. 3%） , 0#3株系植株病情指数显著降低 （降低幅度为 28. 3%） ， 0#4 株系植株病情指数显著降低 （降低幅度为 22. 2%） 。 结果见图 8 （灰色柱 形图为转基因受体材料； 黑色柱形图为转基因纯系植株； 柱形图内数字表 示植株数量； *: K0. 05） 。
结果表明， AWPU基因为抗灰斑病主效 L-qRglsl区段内的功能 基因， 能够显著提高玉米对灰斑病的抗性。 实施例 6、 Q11背景验证 Y32中 ZmWAK-RLK发挥功能的区段
转基因植株的获得
1、 将序列表的序列 6所示的双链 DNA分子插入 pBCXUN载体的
Figure imgf000016_0001
I 酶切位点， 得到重组质粒。
2、 同实施例 3的步骤一的 2。
3、取步骤 2得到的重组农杆菌， 采用农杆菌介导法对玉米自交系 B73 的幼胚进行遗传转化， 得到 T0代植株。
4、 T0代植株自交， 收获籽粒， 将籽粒培育为植株， 即为 T1代植株。
5、 将 T1代植株进行 PCR鉴定， 筛选转基因植株。 从 T1代植株中筛 选到的转基因植株， 即为 T1转基因植株。 从 T1代植株中筛选到若干转基 因植株， 其中三株命名为 R1植株、 R2植株和 R3植株。 PCR鉴定方法： 取植株叶片， 提取基因组 DNA， 采用 F3和 R3组成的 引物对进行 PCR扩增， 如果得到 357bp的扩增产物、 PCR鉴定为阳性、 该 植株为转基因植株； 如果没有得到扩增产物、 PCR鉴定为阴性、 该植株为 非转基因植株。
F3： GGTGGACGGCGAGGTCGCCG；
R3： TCGGTGACGGGCAGGACCGG。
部分植株的 PCR鉴定结果见图 9 , 箭头标注目标带， 最左侧的泳道为 分子量标准 （M） ， 其余每个泳道对应一株植株（编号 1-14）。
二、 回交分离后代的获得
PCR鉴定方法同步骤一的 5。
将 R1植株 （或 R2植株或 R3植株） 作为父本， 玉米自交系 Q1 1作为 母本， 进行杂交， 收获籽粒， 将籽粒培育为植株， 即为 BC^植株， 通过 PCR鉴定筛选转基因植株； 将 BCA植株中的转基因植株作为父本， 玉米自 交系 Q11作为母本， 进行杂交， 收获籽粒， 将籽粒培育为植株， 即为 BCK 植株， 通过 PCR鉴定筛选转基因植株和非转基因植株。
三、 植株的抗病性鉴定
1、 抗病性鉴定的方法
同实施例 2的步骤四的 1。
2、 回交分离后代 （BCK植株） 的抗病性鉴定
供试材料： 步骤二中 R1植株作为父本得到的 BC2Fi植株中的转基因植 株和非转基因植株、 步骤二中 R2植株作为父本得到的 BC^植株中的转基 因植株和非转基因植株、 步骤二中 R3植株作为父本得到的 BC2Fi植株中的 转基因植株和非转基因植株。
供试材料按照步骤 1的方法进行抗病性鉴定。 转基因植株的病情指数 显著低于非转基因植株， DSI降低 8. 4-14. 1%。
结果见图 10（灰色柱形图为非转基因植株；黑色柱形图为转基因植株; 柱形图内数字表示植株数量； * : K0. 05 ; **: K0. 01） 。
以上结果表明， 将 AWPU基因回交导入 Q11遗传背景可以显著提 高玉米对灰斑病的抗性， 同时也表明 ZmWAK-RLK蛋白的 N端区段 （序列表 的序列 1的第 1-324位氨基酸） 在玉米灰斑病抗性中发挥重要功能。 工业应用
本发明的发明人提供了 ZmWAK-RLK蛋白及其编码基因， 通过过表达转 基因实验
Figure imgf000018_0001
显著的降 低玉米灰斑病的病情指数。 本发明对于玉米的抗灰斑病育种具有重大的应 用价值。

Claims

权利要求
1、 一种蛋白质， 为如下 (al) 或 (a2) 或 (a3) 或 (a4) 或 (a5) : (al) 序列表中序列 1所不的蛋白质；
(a2) 序列表中序列 7所不的蛋白质；
(a3) 在 (al) 或 (a2) 所述蛋白质的 N端或 /和 C端连接标签得到的 融合蛋白；
(a4) 将 (al) 或 (a2) 经过一个或几个氨基酸残基的取代和 /或缺失 和 /或添加得到的与植物灰斑病抗病性相关的蛋白质；
(a5) 来源于玉米且与 (al) 或 (a2) 具有 90%、 91%、 92%、 93%、
94%、 95%、 96%、 97%、 98%或 99%以上同一性且与植物灰斑病抗病性 相关的蛋白质。
2、 编码权利要求 1所述蛋白质的核酸分子。
3、 如权利要求 2 所述的核酸分子， 其特征在于： 所述核酸分子为如 下 (bl) 或 (b2) 或 (b3) 或 (b4) 或 (b5) 或 (b6) :
(bl)编码区如序列表中序列 2第 87-2084位核苷酸所示的 DNA分子; (b2) 序列表中序列 2所示的 DNA分子；
(b3) 序列表中序列 3所示的 DNA分子；
(b4) 编码区如序列表中序列 6所示的 DNA分子；
(b5) 来源于玉米且与 (bl) 或 (b2) 或 (b3) 或 (b4) 具有 90%、
91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%或 99%以上同一性且 编码所述蛋白质的 DNA分子；
(b6) 在严格条件下与 (bl) 或 (b2) 或 (b3) 或 (b4) 限定的核苷 酸序列杂交且编码所述蛋白质的 DNA分子。
4、 含有权利要求 2或 3所述核酸分子的 DNA分子、 表达盒、 重组载 体或重组微生物。
5、 权利要求 1 所述蛋白质的应用， 为如下 (cl) 或 (c2) 或 (c3) 或 (c4) :
(cl) 调控植物的灰斑病抗病性；
(c2) 提高植物的灰斑病抗病性； (c3) 调控植物对玉米尾孢菌弓 I发的病害的抗病性；
(c4) 提高植物对玉米尾孢菌引发的病害的抗病性。
6、 如权利要求 5 所述的应用， 其特征在于： 所述植物为玉蜀黍属植 物。
7、 权利要求 2或 3所述核酸分子的应用， 为如下 (dl) 或 (d2) 或 (d3) 或 (d4) :
(dl) 培育灰斑病抗病性改变的转基因植物；
(d2) 培育灰斑病抗病性增强的转基因植物；
(d3) 培育对玉米尾孢菌引发的病害的抗病性改变的转基因植物； (d4) 培育对玉米尾孢菌引发的病害的抗病性增强的转基因植物。
8、 如权利要求 7 所述的应用， 其特征在于： 所述植物为玉蜀黍属植 物。
9、 一种制备转基因植物的方法， 包括如下步骤： 在出发植物中导入 权利要求 2或 3所述核酸分子， 得到灰斑病抗病性增强的转基因植物。
10、 如权利要求 9所述的方法， 其特征在于： 所述出发植物为玉蜀黍 属植物。
11、 一种植物育种方法， 包括如下步骤： 增加目的植物中权利要求 1 所述蛋白质的含量和 /或活性， 从而提高目的植物的灰斑病抗病性。
12、 如权利要求 11 所述的方法， 其特征在于： 所述目的植物为玉蜀 黍属植物。
13、 一种制备转基因植物的方法， 包括如下步骤： 在出发植物中导入 权利要求 2或 3所述核酸分子， 得到抗病性增强的转基因植物； 所述抗病 性为对玉米尾孢菌引发的病害的抗病性。
14、 如权利要求 13 所述的方法， 其特征在于： 所述出发植物为玉蜀 黍属植物。
15、 一种植物育种方法， 包括如下步骤： 增加目的植物中权利要求 1 所述蛋白质的含量和 /或活性， 从而提高目的植物的抗病性； 所述抗病性 为对玉米尾孢菌引发的病害的抗病性。
16、 如权利要求 15 所述的方法， 其特征在于： 所述目的植物为玉蜀 黍属植物。
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