WO2020177560A1 - 一种培育抗灰斑病植物的方法 - Google Patents

一种培育抗灰斑病植物的方法 Download PDF

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WO2020177560A1
WO2020177560A1 PCT/CN2020/076319 CN2020076319W WO2020177560A1 WO 2020177560 A1 WO2020177560 A1 WO 2020177560A1 CN 2020076319 W CN2020076319 W CN 2020076319W WO 2020177560 A1 WO2020177560 A1 WO 2020177560A1
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plant
sequence
plants
protein
sequence listing
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French (fr)
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徐明良
朱芒
番兴明
钟涛
徐凌
张艳
刘丽
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China Agricultural University
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China Agricultural University
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Priority to US17/434,206 priority Critical patent/US20220170041A1/en
Priority to CN202080018758.1A priority patent/CN113677696A/zh
Priority to EP20767245.2A priority patent/EP3936519A4/en
Priority to MX2021010663A priority patent/MX2021010663A/es
Priority to BR112021017043A priority patent/BR112021017043A2/pt
Publication of WO2020177560A1 publication Critical patent/WO2020177560A1/zh
Priority to MX2025008671A priority patent/MX2025008671A/es
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Priority to US19/072,388 priority patent/US20250250583A1/en
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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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/1255—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4684—Zea mays [maize]
    • 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 method for cultivating gray spot disease resistant plants, and more specifically to a method for genetically improving plants by expressing ZmPK genes to obtain gray spot resistant plants.
  • Corn gray leaf spot is a worldwide fungal disease of corn leaves. It was first discovered in Alexandria County, Illinois, USA in 1925, which seriously affected corn yield. Maize gray spot disease first occurred in Dandong City, Liaoning City, my country in 1991, and has been reported in Jilin, Hebei and Yunnan since then. In recent years, gray spot disease has become one of the main leaf diseases in maize production in my country, especially in the southwest maize producing areas. The occurrence of maize gray spot disease generally causes a 10-30% reduction in maize production, and in severe cases, it can reach 60-80% or even no harvest, which severely restricts the production of maize in my country.
  • the protein provided by the present invention obtained from corn and named ZmPK protein, is as follows (a1) or (a2) or (a3) or (a4) or (a5) or (a6) or (a7) or (a8):
  • (a6) A protein composed of the following three segments in sequence from N-terminal to C-terminal: the protein, connecting peptide, and EGFP protein in any one of (a1) to (a4);
  • (a7) 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 the protein in any one of (a1) to (a6);
  • (a8) It is derived from corn and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the protein described in (a1) to (a4).
  • Protein can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression.
  • the nucleic acid molecule encoding the ZmPK protein also belongs to the protection scope of the present invention.
  • the nucleic acid molecule is any one of the following (b1) to (b15):
  • the coding region is a DNA molecule represented by nucleotides 56-1624 in sequence 10 of the sequence listing;
  • (b14) is derived from corn and has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with any of (b1) to (b13) DNA molecules with the above identity and encoding the protein;
  • (b15) A DNA molecule that hybridizes to any one of (b1) to (b13) under stringent conditions and encodes the protein.
  • the stringent conditions are in a solution of 2 ⁇ SSC, 0.1% SDS, hybridizing and washing the membrane twice at 68°C, 5 min each time, and hybridizing in a solution of 0.5 ⁇ SSC, 0.1% SDS at 68°C And wash the membrane 2 times, 15min each time.
  • the expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule all belong to the protection scope of the present invention.
  • the recombinant expression vector may specifically be: a recombinant plasmid obtained by inserting the double-stranded DNA molecule shown in sequence 12 of the sequence table into the multiple cloning site (for example, BamHI site) of the pCAMBIA3301 vector.
  • the recombinant expression vector may specifically be a recombinant plasmid obtained by inserting the nucleic acid molecule at the multiple cloning site (for example, the XcmI restriction site) of the pBCXUN vector.
  • the present invention also protects the application of ZmPK protein, which is as follows (c1) or (c2): (c1) regulating the gray spot disease resistance of plants; (c2) reducing the gray spot disease resistance of plants.
  • the present invention also protects the application of the nucleic acid molecule as follows (d1) or (d2): (d1) cultivating transgenic plants with altered gray spot disease resistance; (d2) cultivating transgenic plants with reduced gray spot disease resistance .
  • the application of the nucleic acid molecule further includes using the nucleic acid molecule as a target to reduce the expression amount of the nucleic acid molecule.
  • Implementation methods include but are not limited to: RNAi interference, gene knockout, etc.
  • Implementation methods also include: insertion, deletion or editing of the promoter region, and promoter interchange. Ways to achieve the target include, but are not limited to: using edited or mutant alleles with lower expression or weaker activity.
  • the present invention also protects the application of substances for inhibiting the transcription of the nucleic acid molecule and/or for inhibiting the expression of the nucleic acid molecule and/or for the gene editing of the nucleic acid molecule, in order to enhance the disease resistance of plants against gray spot Sex.
  • the "substance for gene editing the nucleic acid molecule” may specifically be any interference vector described below or any gene editing vector described below.
  • the present invention also protects a method for preparing a transgenic plant, which includes the steps of: introducing the nucleic acid molecule into a starting plant to obtain a transgenic plant with reduced 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, for example, three consecutive crosses), the obtained transgenic progeny plants are also transgenic plants with reduced gray spot 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 ZmPK protein in the target plant, thereby reducing the gray spot disease resistance of the target plant.
  • the present invention also protects a method for preparing a transgenic plant, which includes the following steps: inhibiting the expression of the nucleic acid molecule in the starting plant to obtain a transgenic plant with increased gray spot disease resistance. Inhibiting the expression of the nucleic acid molecule in the starting plant can be specifically achieved by introducing an interference vector.
  • the interference vector may specifically be the following recombinant plasmid: a recombinant plasmid having a forward fragment, a spacer fragment, and a reverse fragment; the spacer fragment is used to space the forward fragment and the reverse fragment; the forward fragment and the reverse fragment are reverse The relationship is complementary; the forward fragment is shown in sequence 11 of the sequence listing.
  • the interference vector may specifically be the following recombinant plasmid: using the pGreen-HY104 vector as the starting vector, a recombinant plasmid obtained by inserting forward fragments and reverse fragments into different multiple cloning sites; the forward fragments and reverse fragments are reverse complementary The relationship; the forward fragment is shown in sequence 11 of the sequence listing.
  • the interference vector may specifically be the following recombinant plasmid: using the pGreen-HY104 vector as the starting vector, insert a forward fragment between the BamHI and XbaI restriction sites, and insert a reverse fragment between the HindIII and EcoRI restriction sites, Obtain the RNAi interference vector; the forward fragment and the reverse fragment are in a reverse complementary relationship.
  • the forward fragment is shown in sequence 11 of the sequence listing.
  • the interference vector can be transformed into the starting plant by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electric conduction, Agrobacterium mediation and the like.
  • Crossing the transgenic plants with existing corn varieties including single crosses and multiple crosses, such as three consecutive crosses, the obtained transgenic progeny plants are also transgenic plants with increased gray spot 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: gene-editing a specific gene in the genome of the starting plant (causing a frameshift mutation in the specific gene), so as to increase the gray spot disease resistance of the target plant;
  • the specific gene encodes the ZmPK protein.
  • the gene editing is specifically realized by Cas9 technology.
  • the gene editing is specifically realized by introducing a gene editing vector.
  • the gene editing vector can specifically be the following recombinant plasmid: insert the double-stranded DNA molecule shown in sequence 16 of the sequence table into the BsaI restriction site of the pBUE411 vector.
  • Figure 1 is a schematic diagram of the structure of pBCXUN vector.
  • Figure 2 is the result of identification of gene expression level in Example 2.
  • Figure 3 shows the results of the identification of disease resistance in Example 2.
  • FIG. 5 shows the results of the gene expression level identification in Example 3.
  • Figure 6 shows the results of the identification of disease resistance in Example 3.
  • Figure 10 shows the results of the identification of disease resistance in Example 5.
  • Figure 11 is a schematic diagram of the structure of recombinant plasmid E and recombinant plasmid F.
  • Figure 12 shows the results of the gene expression level identification in Example 6.
  • Figure 13 shows the results of the identification of disease resistance in Example 6.
  • Figure 14 shows the sequencing results of two gene-edited plants.
  • Figure 15 shows the results of the identification of disease resistance in Example 7.
  • the following examples 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 corn inbred line Y32 is a corn inbred line with high resistance to gray leaf spot of corn.
  • the corn inbred line Y32 (line Y32) is recorded in the following documents: QTL mapping of resistance to gray leaf spot in maize, Yan Zhang ect., Theor Appl Genet DOI 10.1007/s00122-012-1954-z.
  • the maize inbred line Q11 is a maize inbred line highly susceptible to gray leaf spot.
  • the corn inbred line Q11 (line Q11) is recorded in the following documents: QTL mapping of resistance to gray leaf spot in maize, Yan Zhang ect., Theor Appl Genet DOI 10.1007/s00122-012-1954-z.
  • Maize inbred line B73 (B73inbred lines), recorded in the following literature: The B73 maize geneome: complexity, diversity, and dynamics, Patrick S. Schnable ect., Science(2009) 326:1112-1115.DOI:10.1126/science.1178534 ;The tin1 gene retains the function of promoting tilling in maize, Zhang ect., Nature communities(2019) 5608. DOI:10.1038/s41467-019-134225-6..
  • Corn inbred line B73-329 (B73-329inbred lines), recorded in the following documents: A retrotransposon in an HKT1 family sodium transporter causes variation of leaf Na + exclusion and salt tolerance in maize, Ming Zhang ect., New Phytologist (2018) 217:1161–1176doi:10.1111/nph.14882.
  • Cercospora zeina recorded in the following documents: First Report of Gray Leaf Spot of Maize Caused by Cercospora zeina in China, Plant Disease/Vol.97 No. 12.
  • the pGreen-HY104 vector (vector pGreen-HY104) is recorded in the following document: A maize wall-associated kind confers quantitative resistance to head smut, Nature Genetics (2015) 47:151-157.
  • the pCAMBIA3301 vector (bivalent expression vector pCAMBIA3301) is recorded in the following document: Pyramiding of nine transgenes in maize generate high-level resistance against necrotrophic maize pathogens.
  • the maize inbred line Y32 (as the donor parent) and the maize inbred line Q11 (as the recurrent parent) were used to construct the initial positioning population and the fine positioning population.
  • the Y32BAC library of disease-resistant parents was screened by PCR.
  • the ZmPK gene in the genomic DNA of the maize inbred line Y32 is shown in sequence 1 of the sequence table. There are two transcripts of the ZmPK gene, the first transcript encodes the protein shown in sequence 2 of the sequence table, and the second transcript encodes the protein shown in sequence 4 of the sequence table.
  • the open reading frame corresponding to the first transcript in the cDNA of the maize inbred line Y32 is shown in nucleotides 56-1618 in the sequence 3 of the sequence listing.
  • the open reading frame corresponding to the second transcript in the cDNA of the maize inbred line Y32 is shown in nucleotides 56-1624 in the sequence 5 of the sequence listing.
  • the ZmPK gene in the genomic DNA of the three maize inbred lines (the three maize inbred lines refer to the maize inbred line B73, the maize inbred line B73-329 and the maize inbred line Q11, the same below) are as shown in the sequence table Sequence 6 is shown.
  • the first transcript encodes the protein shown in sequence 7 in the sequence table
  • the second transcript encodes the protein shown in sequence 9 in the sequence table.
  • the open reading frame corresponding to the first transcript in the cDNA of the three maize inbred lines is shown in nucleotides 56-1618 in sequence 8 of the sequence listing.
  • the open reading frame corresponding to the second transcript in the cDNAs of the three maize inbred lines is shown at nucleotides 56-1624 in sequence 10 of the sequence listing.
  • a primer pair composed of ZmPK-OE-F and ZmPK-OE-R is used for PCR amplification to obtain PCR amplification products.
  • ZmPK-OE-F 5’-ATGGGCGCTTGCTTCTCCTC-3’;
  • ZmPK-OE-R 5'-TCACAGAGCCTGAGGGTTTGG-3'.
  • the recombinant plasmid A and the recombinant plasmid B are constructed in the same way and produced at the same time, and they need to be identified by sequencing.
  • two exogenous fragments can also be directly synthesized and inserted into the XcmI restriction site of the pBCXUN vector to obtain recombinant plasmid A and recombinant plasmid B.
  • a primer pair composed of ZmPK-OE-F and ZmPK-OE-R is used for PCR amplification to obtain PCR amplification products.
  • step 7 Connect the PCR amplification product obtained in step 6 and the vector backbone obtained in step 3 to obtain recombinant plasmid C.
  • the structure of the recombinant plasmid C is described as follows: the DNA molecule shown in nucleotides 56-1618 in sequence 8 of the sequence table is inserted into the XcmI restriction site of the pBCXUN vector.
  • step 2 Take the recombinant Agrobacterium obtained in step 1, and use Agrobacterium-mediated method to genetically transform the immature embryos of the corn inbred line B73-329 to obtain T0 generation plants.
  • PCR identification method Take plant leaves, extract genomic DNA, and use a primer pair consisting of bar-F and bar-R for PCR amplification. If an amplified product of about 262 bp is obtained, the PCR is identified as positive, and the plant is a transgenic plant. No amplified product was obtained, PCR was identified as negative, and the plant was a non-transgenic plant.
  • bar-F GAAGGCACGCAACGCCTACGA
  • bar-R CCAGAAACCCACGTCATGCCA.
  • the T0 generation plants are selfed, the grains are harvested, and the seeds are cultivated into plants, that is, the T1 generation plants; the T1 generation plants are screened for transgenic plants, and the PCR identification method is the same as step 2 3; the T1 generation transgenic plants are used as the female parent, and the maize
  • the cross line Q11 is used as the male parent, the seeds are harvested, and the seeds are cultivated into plants, that is, the F1 generation plants; the F1 generation plants are screened for the transgenic plants, and the PCR identification method is the same as that of step 2 3; the F1 generation transgenic plants are used as the mother
  • the maize inbred line Q11 was used as the male parent, crossed, harvested the kernels, and cultivated the kernels into plants, that is, BC 1 F 1 generation plants; BC 1 F 1 generation plants were screened for transgenic plants and non-transgenic plants, and PCR identification The method is the same as step 2 of 3; the BC 1 F 1 generation transgenic plant is used as the female parent, and the
  • the T0 generation plants are: YT1-1 plant, YT1-2 plant, YT1-3 plant, YT2-1 plant, YT2-2 plant, YT2-3 plant, QT1-1 plant, QT1-2 plant, QT1-3 plant .
  • the tested plants were: BC 3 F 1 generation transgenic plants and non-transgenic plants of YT1-1 plants, BC 3 F 1 generation transgenic plants and non-transgenic plants of YT1-2 plants, and BC 3 F 1 generation transgenic plants of YT1-3 plants Plants and non-transgenic plants, BC 1 F 1 generation of transgenic plants and non-transgenic plants of YT2-1 plants, BC 1 F 1 generation of transgenic plants and non-transgenic plants of YT2-2 plants, BC 1 F 1 generation of YT2-3 plants Transgenic plants and non-transgenic plants, BC 2 F 1 generation transgenic plants and non-transgenic plants of QT1-1 plants, BC 1 F 1 generation transgenic plants and non-transgenic plants of QT1-2 plants, BC 3 F 1 of QT1-3 plants Generation of transgenic plants and non-transgenic plants.
  • RNA from the leaves of the tested plants was extracted and reverse transcribed to obtain cDNA.
  • GAPDH gene as an internal reference gene, the relative expression level of ZmPK gene was detected by qPCR. The results are shown in Figure 2.
  • test plants were cultured normally, and pathogenic bacteria were inoculated at the bell mouth stage, and then the normal culture was continued. Phenotypic investigation was carried out two weeks after pollination, and the disease index (DSI) was calculated by graded investigation.
  • the specific method of inoculating pathogenic bacteria (bacterial fluid filling method): Suspend the spores of gray spot disease pathogenic bacteria in sterile water to obtain a spore suspension with a spore concentration of 2-4 ⁇ 10 5 /mL. The spore suspension was poured into the corn bell mouth, and 5ml was poured per corn plant.
  • RNAi#806 plants Three randomly selected transgenic plants were named as RNAi#806 plants, RNAi#1065 plants, and RNAi#581 plants.
  • step 2 Take the recombinant Agrobacterium obtained in step 1, and use the Agrobacterium-mediated method to genetically transform the immature embryos of the maize inbred line B73 to obtain T0 generation plants.
  • the PCR identification method is the same as step 2 of Example 2 in 3.
  • the method is the same as Step 4 of Example 2.
  • brackets indicate the number of plants, the number before the dividing line is the number of non-transgenic plants, and the number after the dividing line is the number of transgenic plants.
  • step 2 Take the recombinant Agrobacterium obtained in step 1, and use the Agrobacterium-mediated method to genetically transform the immature embryos of the maize inbred line B73 to obtain T0 generation plants.
  • the PCR identification method is the same as step 2 of Example 2 in 3.
  • Recombinant plasmid E was used for step two, and four randomly selected transgenic plants were named: Y1#349 plant, Y1#350 plant, Y1#351 plant, Y1#354 plant.
  • Recombinant plasmid F was used to perform step two, and three randomly selected transgenic plants were named, namely Y2#731 plant, Y2#732 plant, Y2#735 plant.
  • the following homozygous transgenic lines were obtained: Y1#349 line, Y1#350 line, Y1#351 line, Y1#354 line, Y2#731 line, Y2#732 line, Y2#735 line .
  • the tested plants are: T3 generation plants of Y1#349 line, T3 generation plants of Y1#350 line, T3 generation plants of Y1#351 line, T3 generation plants of Y1#354 line, Y2#731 line
  • the gene editing vector is constructed, that is, the following recombinant plasmid: insert the double-stranded DNA molecule shown in sequence 16 of the sequence list at the BsaI restriction site of the pBUE411 vector.
  • the recombinant plasmid has been verified by sequencing.
  • the gene editing vector encodes two sgRNAs, the target sequence binding region of one sgRNA is shown in sequence 17 of the sequence listing, and the target sequence binding region of the other sgRNA is shown in sequence 18 of the sequence listing.
  • the gene editing vector contains the Cas9 gene and expresses the Cas9 protein.
  • PCR identification method Take plant leaves, extract genomic DNA, and use primer pair consisting of bar-F and bar-R for PCR amplification. If no amplified product is obtained, PCR identification is negative.
  • ZmPK-KO#1 plant and ZmPK-KO#2 plant Two gene-edited plants were obtained, named ZmPK-KO#1 plant and ZmPK-KO#2 plant respectively.
  • step (2) of the two gene editing plants are shown in Figure 14.
  • two nucleotides were missing in ZmPK-KO#1 plant, and one nucleotide was inserted in ZmPK-KO#2 plant, both of which caused frameshift mutations.
  • ZmPK-KO#1 strain Two gene editing strains were obtained, named ZmPK-KO#1 strain and ZmPK-KO#2 strain respectively.
  • the tested plants were: T2 generation plants of ZmPK-KO#1 line, T2 generation plants of ZmPK-KO#2 line, and corn inbred line B73 plants.

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Abstract

本发明公开了一种培育抗灰斑病植物的方法。本发明提供的蛋白质，获自玉米，命名为ZmPK蛋白，为序列表中序列2、序列4、序列7或序列9所示的蛋白质。编码ZmPK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，得到灰斑病抗病性降低的转基因植物。本发明还保护一种制备转基因植物的方法，包括如下步骤：敲除或抑制出发植物中所述核酸分子的表达，得到灰斑病抗病性增高的转基因植物。本发明对于玉米的抗灰斑病育种具有重大的应用价值。

Description

一种培育抗灰斑病植物的方法 技术领域
本发明属于生物技术领域，具体涉及一种培育抗灰斑病植物的方法，更具体涉及一种通过表达ZmPK基因对植物进行遗传改良以得到抗灰斑病植物的方法。
背景技术
玉米灰斑病是一种世界性的玉米叶部真菌性病害，最早于1925年在美国伊利诺伊州亚历山大县发现，严重影响玉米的产量。玉米灰斑病于1991年首次在我国辽宁省丹东市发生，之后在吉林、河北以及云南等地均有报道。近年来，灰斑病已经成为我国玉米生产上主要的叶部病害之一，其中西南玉米产区尤为严重。玉米灰斑病的发生一般会导致玉米减产10-30％，严重时可达60-80％甚至绝收，严重制约着我国玉米的生产。
目前研究认为造成玉米灰斑病的致病菌主要有玉蜀黍尾胞菌(Cercospora zeae-maydis,Czm)和玉米尾胞菌(Cercospora zeina,Cz)两种。长期以来，国内研究者认为我国玉米灰斑病的致病菌是Czm。Liu等在云南地区取样并进行病菌形态学、致病性、ITS序列和组蛋白H3的基因序列分析后发现，我国云南地区玉米灰斑病的致病菌是Cz。病斑首先出现在下部叶片，且叶片上的症状最为明显。发病初期，病斑呈水渍状褪绿斑，之后扩展成灰褐色，近似矩形且与叶脉平行，发病严重时，病斑会扩展成片，导致叶片枯萎。使用杀真菌剂等化学防治手段效果不明显，培育抗灰斑病品种是控制此病最经济有效的途径。
发明公开
本发明提供了一种培育抗灰斑病植物的方法。。
本发明提供的蛋白质，获自玉米，命名为ZmPK蛋白，为如下(a1)或(a2)或(a3)或(a4)或(a5)或(a6)或(a7)或(a8)：
(a1)序列表中序列2所示的蛋白质；
(a2)序列表中序列4所示的蛋白质；
(a3)序列表中序列7所示的蛋白质；
(a4)序列表中序列9所示的蛋白质；
(a5)在(a1)至(a4)中任一所述蛋白质的N端或/和C端连接标签得到的融合蛋白；
(a6)自N末端至C末端依次由如下三个区段组成的蛋白质：(a1)至(a4)中任一所述蛋白质、连接肽、EGFP蛋白；
(a7)将(a1)至(a6)中任一所述蛋白质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；
(a8)来源于玉米且与(a1)至(a4)中任一所述蛋白质具有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
标签 残基 序列
Poly-Arg 5-6(通常为5个) RRRRR
Poly-His 2-10(通常为6个) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
HA 9 YPYDVPDYA
EGFP 239 序列15
EGFP蛋白具体如序列表的序列15所示。连接肽具体可如序列表的序列19所示。
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ZmPK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(b1)至(b15)中的任意一种：
(b1)编码区如序列表的序列3中第56-1618位核苷酸所示的DNA分子；
(b2)序列表中序列3所示的DNA分子；
(b3)编码区如序列表的序列5中第56-1624位核苷酸所示的DNA分子；
(b4)序列表中序列5所示的DNA分子；
(b5)序列表中序列1所示的DNA分子；
(b6)编码区如序列表的序列8中第56-1618位核苷酸所示的DNA分子；
(b7)序列表中序列8所示的DNA分子；
(b8)编码区如序列表的序列10中第56-1624位核苷酸所示的DNA分子；
(b9)序列表中序列10所示的DNA分子；
(b10)序列表中序列6所示的DNA分子；
(b11)序列表中序列12所示的DNA分子；
(b12)序列表中序列13所示的DNA分子；
(b13)序列表中序列14所示的DNA分子；
(b14)来源于玉米且与(b1)至(b13)中的任意一种具有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；
(b15)在严格条件下与(b1)至(b13)中的任意一种杂交且编码所述蛋白质的DNA分子。
所述严格条件是在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min。
含有所述核酸分子的表达盒、重组载体或重组微生物均属于本发明的保护范围。
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。
所述重组表达载体具体可为：将序列表的序列12所示双链DNA分子插入pCAMBIA3301载体的多克隆位点(例如BamHI位点)得到的重组质粒。
所述重组表达载体具体可为在pBCXUN载体的多克隆位点(例如XcmI酶切位点)插入所述核酸分子得到的重组质粒。
本发明还保护ZmPK蛋白的应用，为如下(c1)或(c2)：(c1)调控植物的灰斑病抗病性；(c2)降低植物的灰斑病抗病性。
本发明还保护所述核酸分子的应用，为如下(d1)或(d2)：(d1)培育灰斑病抗病性改变的转基因植物；(d2)培育灰斑病抗病性降低的转基因植物。
所述核酸分子的应用，还包括以所述核酸分子为靶标降低所述核酸分子的表达量。实现方式包括但不限于：RNAi干扰、基因敲除等。实现方式还包括：启动子区域的插入、缺失或编辑，启动子互换等。等方式对靶标实现方式包括但不限于：使用表达较低或活性较弱的编辑或突变等位基因等。
本发明还保护用于抑制植物中ZmPK蛋白的活性和/或用于降低植物中ZmPK蛋白的丰度的物质的应用，为增强植物的灰斑病抗病性。
本发明还保护用于抑制所述核酸分子转录和/或用于抑制所述核酸分子表达和/或用于对所述核酸分子进行基因编辑的物质的应用，为增强植物的灰斑病抗病性。所述“对所述核酸分子进行基因编辑的物质”具体可为后面任一所述的干扰载体或者后面任一所述的基因编辑载体。
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，得到灰斑病抗病性降低的转基因植物。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灰斑病抗病性降低的转基因植物。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ZmPK蛋白的含量和/或活性，从而降低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：抑制出发植物中所述核酸分子的表达，得到灰斑病抗病性增高的转基因植物。抑制出发植物中所述核酸分子的表达具体可通过导入干扰载体实现。所述干扰载体具体可为如下重组质粒：具有正向片段、间隔片段和反向片段的重组质粒；所述间隔片段用于间隔正向片段和反向片段；正向片段和反向片段为反向互补的关系；正向片段如序列表的序列11所示。所述干扰载体具体可为如下重组质粒：以pGreen-HY104载体为出发载体，不同多克隆位点分别插入正向片段和反向片段得到的重组质粒；正向片段和反向片段为反向互补的关系；正向片段如序列表的序列11所示。所述干扰载体具体可为如下重组质粒：以pGreen-HY104载体为出发载体，在BamHI和XbaI酶切位点之间插入正向片段，在HindIII和EcoRI酶切位点之间插入反向片段，得到RNAi干扰载体；正向片段和反向片段为反向互补的关系。正向片段如序列表的序列11所示。所述干扰载体可通过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灰斑病抗病性增高的转基因植物。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：降低目的植物中ZmPK蛋白的含量和/或活性，从而增高目的植物的灰斑病抗病性。
本发明还保护一种植物育种方法，包括如下步骤：对出发植物基因组中的 特定基因进行基因编辑(引起特定基因内发生移码突变)，从而增高目的植物的灰斑病抗病性；所述特定基因编码ZmPK蛋白。
所述基因编辑具体通过Cas9技术实现。
所述基因编辑具体通过两个sgRNA和Cas9蛋白实现。其中一个sgRNA的靶序列结合区如序列表的序列17所示，另一个sgRNA的靶序列结合区如序列表的序列18所示。
所述基因编辑具体通过导入基因编辑载体实现。基因编辑载体具体可为如下重组质粒：在pBUE411载体的BsaI酶切位点插入序列表的序列16所示的双链DNA分子。
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或玉米自交系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
图1为pBCXUN载体的结构示意图。
图2为实施例2中基因表达水平鉴定的结果。
图3为实施例2中抗病性鉴定的结果。
图4为RNAi干扰载体的结构示意图。
图5为实施例3中基因表达水平鉴定的结果。
图6为实施例3中抗病性鉴定的结果。
图7为ZmPK基因的表达量与灰斑病病情等级之间的相关性的结果。
图8为pCAMBIA3301载体的结构示意图。
图9为实施例5中基因表达水平鉴定的结果。
图10为实施例5中抗病性鉴定的结果。
图11为重组质粒E和重组质粒F的结构示意图。
图12为实施例6中基因表达水平鉴定的结果。
图13为实施例6中抗病性鉴定的结果。
图14为两株基因编辑植株的测序结果。
图15为实施例7中抗病性鉴定的结果。
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：QTL mapping of resistance to gray leaf spot in maize，Yan Zhang ect.,Theor Appl Genet DOI 10.1007/s00122-012-1954-z.。
玉米自交系Q11，为高感灰斑病的玉米自交系。玉米自交系Q11(line Q11)，记载于如下文献：QTL mapping of resistance to gray leaf spot in maize，Yan Zhang ect.,Theor Appl Genet DOI 10.1007/s00122-012-1954-z.。
玉米自交系B73(B73inbred lines)，记载于如下文献：The B73maize genome:complexity,diversity,and dynamics,Patrick S.Schnable ect.,Science(2009)326:1112-1115.DOI:10.1126/science.1178534；The tin1 gene retains the function of promoting tillering in maize,Zhang ect.,Nature communictions(2019)5608.DOI:10.1038/s41467-019-134225-6.。
玉米自交系B73-329(B73-329inbred lines)，记载于如下文献：A retrotransposon in an HKT1 family sodium transporter causes variation of leaf Na +exclusion and salt tolerance in maize，Ming Zhang ect.,New Phytologist(2018)217:1161–1176doi:10.1111/nph.14882。
玉米尾孢菌(Cercospora zeina)，记载于如下文献：First Report of Gray Leaf Spot of Maize Caused by Cercospora zeina in China，Plant Disease/Vol.97 No.12。
pBCXUN载体(pBCXUN vector)，记载于如下文献：ZmHAK5 and ZmHAK1 function in K+uptake and distribution in maize under low K+conditions.Journal of Intergrative Plant Biology(2018)doi:10.1111/jipb.12756。pBCXUN载体的结构示意图见图1。
pGreen-HY104载体(vector pGreen-HY104)，记载于如下文献：A maize wall-associated kinase confers quantitative resistance to head smut,Nature Genetics(2015)47:151-157。
pCAMBIA3301载体(bivalent expression vector pCAMBIA3301)，记载于如下文献：Pyramiding of nine transgenes in maize generates high-level resistance against necrotrophic maize pathogens。
pBUE411载体(pBUE411 vector),记载于如下文献：ZmCCT9 enhances maize adaptation to higher latitudes.Huang ect.,PNAS(2018)115(2):E334-E341 DOI:10.1073/pnas.1718058115。
实施例1、ZmPK蛋白及其编码基因的发现
用玉米自交系Y32(作为供体亲本)和玉米自交系Q11(作为轮回亲本)，构建初定位群体和精细定位群体。利用位于精细定位的区域的分子标记，通过PCR的方法筛选抗病亲本Y32BAC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。
玉米自交系Y32的基因组DNA中的ZmPK基因如序列表的序列1所示。ZmPK基因存在两个转录本，第一个转录本编码序列表的序列2所示的蛋白质，第二个转录本编码序列表的序列4所示的蛋白质。玉米自交系Y32的cDNA中第一个转录本对应的开放阅读框如序列表的序列3中第56-1618位核苷酸所示。玉米自交系Y32的cDNA中第二个转录本对应的开放阅读框如序列表的序列5中第56-1624位核苷酸所示。
三个玉米自交系(三个玉米自交系指的是玉米自交系B73和玉米自交系B73-329和玉米自交系Q11，下同)的基因组DNA中的ZmPK基因如序列表的序列6所示。ZmPK基因存在两个转录本，第一个转录本编码序列表的序列7所示的蛋白质，第二个转录本编码序列表的序列9所示的蛋白质。三个玉米自交系的cDNA中第一个转录本对应的开放阅读框如序列表的序列8中第56-1618位核苷酸所示。三个玉米自交系的cDNA中第二个转录本对应的开放阅读框如序列表的序列10中第56-1624位核苷酸所示。
实施例2、过表达植物的获得和鉴定
一、重组表达载体的构建
1、取玉米自交系Y32的新鲜叶片，提取总RNA，反转录得到cDNA。
2、以步骤1得到的cDNA为模板，采用ZmPK-OE-F和ZmPK-OE-R组成的引物对进行PCR扩增，得到PCR扩增产物。
ZmPK-OE-F：5’-ATGGGCGCTTGCTTCTCCTC-3’；
ZmPK-OE-R：5’-TCACAGAGCCTGAGGGTTTGG-3’。
3、取pBCXUN载体，采用限制性内切酶XcmI进行酶切，回收载体骨架。
4、将步骤2得到的PCR扩增产物和步骤3得到的载体骨架连接，得到重组质粒甲和重组质粒乙。根据测序结果，对重组质粒甲进行结构描述如下：在pBCXUN载体的XcmI酶切位点插入了序列表的序列3中第56-1618位核苷酸所示的DNA分子。根据测序结果，对重组质粒乙进行结构描述如下：在pBCXUN载体的XcmI酶切位点插入了序列表的序列5中第56-1624位核苷酸所示的DNA分子。由于模板中同时存在两种序列且差异仅为6个核苷酸，重组质粒甲和重组质粒乙的构建方式相同并且同时产生，需要通过测序鉴别。实际应用中，也可以分 别直接合成两个外源片段然后插入pBCXUN载体的XcmI酶切位点，从而得到重组质粒甲和重组质粒乙。
5、取玉米自交系Q11的新鲜叶片，提取总RNA，反转录得到cDNA。
6、以步骤5得到的cDNA为模板，采用ZmPK-OE-F和ZmPK-OE-R组成的引物对进行PCR扩增，得到PCR扩增产物。
7、将步骤6得到的PCR扩增产物和步骤3得到的载体骨架连接，得到重组质粒丙。根据测序结果，对重组质粒丙进行结构描述如下：在pBCXUN载体的XcmI酶切位点插入了序列表的序列8中第56-1618位核苷酸所示的DNA分子。
二、过表达植物的获得
1、将重组质粒导入农杆菌EHA105，得到重组农杆菌。
2、取步骤1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。
3、将T0代植株进行PCR鉴定。
PCR鉴定方法：取植株叶片，提取基因组DNA，采用bar-F和bar-R组成的引物对进行PCR扩增，如果得到约262bp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。
bar-F：GAAGGCACGCAACGCCTACGA；bar-R：CCAGAAACCCACGTCATGCCA。
采用重组质粒甲进行步骤二，对随机取的三个转基因植株进行命名，分别为YT1-1植株、YT1-2植株、YT1-3植株。采用重组质粒乙进行步骤二，对随机取的三个转基因植株进行命名，分别为YT2-1植株、YT2-2植株、YT2-3植株。采用重组质粒丙进行步骤二，对随机取的三个转基因植株进行命名，分别为QT1-1植株、QT1-2植株、QT1-3植株。
三、回交分离后代的获得
T0代植株自交，收获籽粒，将籽粒培育为植株，即为T1代植株；从T1代植株中筛选转基因植株，PCR鉴定方法同步骤二的3；将T1代转基因植株作为母本，玉米自交系Q11作为父本，进行杂交，收获籽粒，将籽粒培育为植株，即为F1代植株；从F1代植株中筛选转基因植株，PCR鉴定方法同步骤二的3；将F1代转基因植株作为母本，玉米自交系Q11作为父本，进行杂交，收获籽粒，将籽粒培育为植株，即为BC 1F 1代植株；从BC 1F 1代植株中筛选转基因植株和非转基因植株，PCR鉴定方法同步骤二的3；将BC 1F 1代转基因植株作为母本，玉米自交系Q11作为父本，进行杂交，收获籽粒，将籽粒培育为植株，即为BC 2F 1代植株；从BC 2F 1代植株中筛选转基因植株和非转基因植株，PCR鉴定方法同步骤二 的3；将BC 2F 1代转基因植株作为母本，玉米自交系Q11作为父本，进行杂交，收获籽粒，将籽粒培育为植株，即为BC 3F 1代植株；从BC 3F 1代植株中筛选转基因植株和非转基因植株，PCR鉴定方法同步骤二的3。
T0代植株分别为：YT1-1植株、YT1-2植株、YT1-3植株、YT2-1植株、YT2-2植株、YT2-3植株、QT1-1植株、QT1-2植株、QT1-3植株。
四、植株的抗病性鉴定
供试植株为：YT1-1植株的BC 3F 1代转基因植株和非转基因植株、YT1-2植株的BC 3F 1代转基因植株和非转基因植株、YT1-3植株的BC 3F 1代转基因植株和非转基因植株、YT2-1植株的BC 1F 1代转基因植株和非转基因植株、YT2-2植株的BC 1F 1代转基因植株和非转基因植株、YT2-3植株的BC 1F 1代转基因植株和非转基因植株、QT1-1植株的BC 2F 1代转基因植株和非转基因植株、QT1-2植株的BC 1F 1代转基因植株和非转基因植株、QT1-3植株的BC 3F 1代转基因植株和非转基因植株。
1、基因表达水平鉴定
提取供试植株叶片的总RNA并反转录得到cDNA,以GAPDH基因为内参基因，通过qPCR检测ZmPK基因的相对表达水平。结果见图2。
用于检测ZmPK基因的引物对：
ZmPK-F：GCGTTGCCGTCAAGCGCAT；ZmPK-R：GCTCCATCACAATGTACACGT。
用于检测GAPDH基因的引物对：
GAPDH-F：ATCAACGGCTTCGGAAGGAT；GAPDH-R：CCGTGGACGGTGTCGTACTT。
2、抗病性鉴定
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(Cercospora zeina)。
正常培养供试植株，喇叭口期接种致病菌，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2-4×10 5个/mL的孢子悬液，利用注射器将孢子悬液灌注于玉米喇叭口中，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％。
Figure PCTCN2020076319-appb-000001
结果见图3。图3中，括号标注的是植株数量，分隔线前的数字为非转基因植株的数量，分隔线后的数字为转基因植株的数量。
结果表明，与非转基因植株相比，转基因植株中ZmPK基因的表达量显著增高，相应的转基因植株的病情指数显著增高。
实施例3、抑制表达植物的获得和鉴定
一、抑制表达植物的获得
1、以pGreen-HY104载体为出发载体，在BamHI和XbaI酶切位点之间插入正向片段，在HindIII和EcoRI酶切位点之间插入反向片段，得到RNAi干扰载体。RNAi干扰载体已进行测序验证。正向片段和反向片段为反向互补的关系。正向片段如序列表的序列11所示。RNAi干扰载体的结构示意图见图4。
2、将步骤1得到的RNAi干扰载体导入农杆菌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T0代植株进行PCR鉴定。
PCR鉴定方法同实施例2的步骤二的3。
对随机取的三个转基因植株进行命名，分别为RNAi#806植株、RNAi#1065植株、RNAi#581植株。
二、回交分离群体的获得
PCR鉴定方法均同实施例2的步骤二的3。
T0代植株自交，收获籽粒，将籽粒培育为植株，即为T1代植株；从T1代植株中筛选转基因植株(PCR鉴定)；将T1代转基因植株作为母本，玉米自交系Q11作为父本，进行杂交，收获籽粒，将籽粒培育为植株，即为F1代植株；从F1代植株中筛选转基因植株(PCR鉴定)；将F1代转基因植株作为母本，玉米自交系Q11作为父本，进行杂交，收获籽粒，将籽粒培育为植株，即为BC 1F 1代植株；从BC 1F 1代植株中筛选转基因植株和非转基因植株(PCR鉴定)；将BC 1F 1代转基因植株作为母本，玉米自交系Q11作为父本，进行杂交，收获籽粒，将籽粒培育为植株，即为BC 2F 1代植株；从BC 2F 1代植株中筛选转基因植株和非转基因植株(PCR鉴定)。
T0代植株分别为：RNAi#806植株、RNAi#1065植株、RNAi#581植株。
三、植株的抗病性鉴定
供试植株为：RNAi#806植株的BC 2F 1代转基因植株和非转基因植株、RNAi#1065植株的BC 2F 1代转基因植株和非转基因植株、RNAi#581植株的BC 2F 1代转基因植株和非转基因植株。
1、基因表达水平鉴定
方法同实施例2的步骤四的1。
结果见图5。
2、抗病性鉴定
方法同实施例2的步骤四的2。
结果见图6。图6中，括号标注的是植株数量，分隔线前的数字为非转基因植株的数量，分隔线后的数字为转基因植株的数量。
结果表明，与非转基因植株相比，转基因植株中ZmPK基因的表达量显著降低，相应的转基因植株的病情指数显著降低。
实施例4、基因表达量与抗性相关性分析
定位群体的原始亲本是玉米自交系Y32和玉米自交系Q11。通过不断杂交和回交构建高世代的回交分离群体。
随机选取了定位群体中病情等级为1、3、5、7、9的植株各3株，进行基因表达水平鉴定(方法同实施例2的步骤四的1)和抗病性鉴定(方法同实施例2的步骤四的2)，计算ZmPK基因的表达量与灰斑病病情等级之间的相关性。结果见图7。ZmPK基因的表达量与灰斑病病情等级之间显著正相关，再次证明了ZmPK基因的表达量与植物灰斑病抗病性之间的负相关性。
实施例5、互补转基因植物的获得和鉴定
一、重组表达载体的构建
制备重组质粒。根据测序结果，对重组质粒进行结构描述如下：在pCAMBIA3301载体的BamHI酶切位点插入了序列表的序列12所示的DNA分子。pCAMBIA3301载体的结构示意图见图8。
二、互补转基因植物的获得
1、将步骤一制备的重组质粒导入农杆菌EHA105，得到重组农杆菌。
2、取步骤1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。
3、将T0代植株进行PCR鉴定。
PCR鉴定方法同实施例2的步骤二的3。
对随机取的一个转基因植株进行命名，为C#596植株。
三、回交分离后代的获得
T0代植株自交，收获籽粒，将籽粒培育为植株，即为T1代植株；从T1代植株中筛选转基因植株，PCR鉴定方法同步骤二的3；将T1代转基因植株作为母本，玉米自交系Q11作为父本，进行杂交，收获籽粒，将籽粒培育为植株，即为F1代植株；从F1代植株中筛选转基因植株，PCR鉴定方法同步骤二的3；将F1代转基因植株作为母本，玉米自交系Q11作为父本，进行杂交，收获籽粒，将籽粒培育为植株，即为BC 1F 1代植株。
四、植株的抗病性鉴定
供试植株为：C#595植株的BC 1F 1代转基因植株和非转基因植株，C#596植株的BC 1F 1代转基因植株和非转基因植株。
1、基因表达水平鉴定
方法同实施例2的步骤四的1。
结果见图9。
2、抗病性鉴定
方法同实施例2的步骤四的2。
结果见图10。图10中，括号标注的是植株数量，分隔线前的数字为非转基因植株的数量，分隔线后的数字为转基因植株的数量。
结果表明，与非转基因植株相比，转基因植株中ZmPK基因的表达量显著增高，相应的转基因植株的病情指数显著增高。
实施例6、融合过表达植株纯系的获得和鉴定
一、重组表达载体的构建
分别制备重组质粒E和重组质粒F。根据测序结果，对重组质粒E进行结构描述如下：在pBCXUN载体的XcmI酶切位点插入了序列表的序列13所示的DNA分子。根据测序结果，对重组质粒F进行结构描述如下：在pBCXUN载体的XcmI酶切位点插入了序列表的序列14所示的DNA分子。重组质粒E和重组质粒F的结构示意图见图11。
二、融合过表达植株纯系的获得
1、将重组质粒导入农杆菌EHA105中，得到重组农杆菌。
2、取步骤1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。
3、将T0代植株进行PCR鉴定。
PCR鉴定方法同实施例2的步骤二的3。
采用重组质粒E进行步骤二，对随机选取的四个转基因植株进行命名，分别为：Y1#349植株、Y1#350植株、Y1#351植株、Y1#354植株。采用重组质粒F 进行步骤二，对随机选取的三个转基因植株进行命名，分别为Y2#731植株、Y2#732植株、Y2#735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。从T1代植株中筛选转基因植株(PCR鉴定)进行自交，收获籽粒，将籽粒培育为植株即为T2代植株。从T2代植株中筛选转基因植株(PCR鉴定)进行自交，收获籽粒，将籽粒培育为植株即为T3代植株。PCR鉴定方法同实施例2的步骤二的3。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。
得到如下纯合的转基因株系：Y1#349株系、Y1#350株系、Y1#351株系、Y1#354株系、Y2#731株系、Y2#732株系、Y2#735株系。
三、植株的抗病性鉴定
供试植株为：Y1#349株系的T3代植株、Y1#350株系的T3代植株、Y1#351株系的T3代植株、Y1#354株系的T3代植株、Y2#731株系的T3代植株、Y2#732株系的T3代植株、Y2#735株系的T3代植株，以及玉米自交系B73植株。
1、基因表达水平鉴定
方法同实施例2的步骤四的1。
结果见图12。
2、抗病性鉴定
方法同实施例2的步骤四的2。
结果见图13。图13中，括号标注的是植株数量。
结果表明，与玉米自交系B73植株(对照非转基因植株)相比，转基因植株中ZmPK基因的表达量显著增高，相应的转基因植株的病情指数显著增高。
实施例7、基因编辑植株的获得和鉴定
一、基因编辑载体的构建
构建基因编辑载体，即如下重组质粒：在pBUE411载体的BsaI酶切位点插入序列表的序列16所示的双链DNA分子。重组质粒已进行测序验证。基因编辑载体编码两个sgRNA,其中一个sgRNA的靶序列结合区如序列表的序列17所示，另一个sgRNA的靶序列结合区如序列表的序列18所示。基因编辑载体中具有Cas9基因，并表达得到Cas9蛋白。
二、基因编辑植株的获得
1、将基因编辑载体导入农杆菌EHA105，得到重组农杆菌。
2、取步骤1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。
3、从T0代植株中筛选目标序列发生变异的植株。
具体方法：取植株叶片，提取基因组DNA，采用F和R组成的引物对进行PCR扩增，回收PCR扩增产物并进行测序，将测序结果与野生型序列进行比较(野生型序列如序列表的序列12中第9599-10985所示)，筛选得到具有差异序列的植株。
F：5’-TTGAGGTCATTGTCTCAGCC-3’；
R：5’-AGCAGCTTGGCAGCGTATG-3’。
4、取步骤3筛选的T0代植株，自交并收获籽粒，将籽粒培育为植株，即为T1代植株。
5、从T1代植株中筛选基因编辑植株。
(1)将植株进行以基因编辑载体中的bar基因为靶标的PCR鉴定。PCR鉴定方法：取植株叶片，提取基因组DNA，采用bar-F和bar-R组成的引物对进行PCR扩增，如果没有得到扩增产物、PCR鉴定为阴性。
(2)将植株进行以基因编辑目标区域为靶标的鉴定。鉴定方法同步骤3。
如果某一植株步骤(1)PCR鉴定为阴性，且步骤(2)的鉴定结果显示与野生型序列具有差异且为纯合型(且两条染色体一致)，该植株为基因编辑植株。
获得了两株基因编辑植株，分别命名为ZmPK-KO#1植株、ZmPK-KO#2植株。
两株基因编辑植株步骤(2)的测序结果见图14。与B73相比，ZmPK-KO#1植株中缺失了两个核苷酸，ZmPK-KO#2植株中插入了一个核苷酸，均造成移码突变。
6、取基因编辑植株，自交，获得后代植株，即为基因编辑株系。
获得了两个基因编辑株系，分别命名为ZmPK-KO#1株系、ZmPK-KO#2株系。
三、基因编辑植株的抗病性鉴定
供试植株为：ZmPK-KO#1株系的T2代植株、ZmPK-KO#2株系的T2代植株，玉米自交系B73植株。
方法同实施例2的步骤四的2。
结果见图15。图15中，括号标注的是植株数量。
相对于玉米自交系B73植株，基因编辑植株的病情指数显著降低。
工业应用
本发明提供了一个抗玉米灰斑病的主效基因ZmPK，进一步通过转基因互补、过表达、CRISPR敲除和RNAi干扰实验证明了该基因的抗灰斑病功能-负调控灰斑病抗性。本发明对于玉米的抗灰斑病育种具有重大的应用价值。

Claims (22)

  1. 一种蛋白质，为如下(a1)或(a2)或(a3)或(a4)或(a5)或(a6)或(a7)或(a8)：
    (a1)序列表中序列2所示的蛋白质；
    (a2)序列表中序列4所示的蛋白质；
    (a3)序列表中序列7所示的蛋白质；
    (a4)序列表中序列9所示的蛋白质；
    (a5)在(a1)至(a4)中任一所述蛋白质的N端或/和C端连接标签得到的融合蛋白；
    (a6)自N末端至C末端依次由如下三个区段组成的蛋白质：(a1)至(a4)中任一所述蛋白质、连接肽、EGFP蛋白；
    (a7)将(a1)至(a6)中任一所述蛋白质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；
    (a8)来源于玉米且与(a1)至(a4)中任一所述蛋白质具有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(b1)至(b15)中的任意一种：
    (b1)编码区如序列表的序列3中第56-1618位核苷酸所示的DNA分子；
    (b2)序列表中序列3所示的DNA分子；
    (b3)编码区如序列表的序列5中第56-1624位核苷酸所示的DNA分子；
    (b4)序列表中序列5所示的DNA分子；
    (b5)序列表中序列1所示的DNA分子；
    (b6)编码区如序列表的序列8中第56-1618位核苷酸所示的DNA分子；
    (b7)序列表中序列8所示的DNA分子；
    (b8)编码区如序列表的序列10中第56-1624位核苷酸所示的DNA分子；
    (b9)序列表中序列10所示的DNA分子；
    (b10)序列表中序列6所示的DNA分子；
    (b11)序列表中序列12所示的DNA分子；
    (b12)序列表中序列13所示的DNA分子；
    (b13)序列表中序列14所示的DNA分子；
    (b14)来源于玉米且与(b1)至(b13)中的任意一种具有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；
    (b15)在严格条件下与(b1)至(b13)中的任意一种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(c1)或(c2)：
    (c1)调控植物的灰斑病抗病性；
    (c2)降低植物的灰斑病抗病性。
  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(d1)或(d2)：
    (d1)培育灰斑病抗病性改变的转基因植物；
    (d2)培育灰斑病抗病性降低的转基因植物。
  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. 用于抑制植物中权利要求1所述蛋白质的活性和/或用于降低植物中权利要求1所述蛋白质的丰度的物质的应用，为增强植物的灰斑病抗病性。
  10. 如权利要求9所述的应用，其特征在于：所述植物为玉蜀黍属植物。
  11. 用于抑制权利要求2或3所述核酸分子转录和/或用于抑制权利要求2或3所述核酸分子表达和/或用于对权利要求2或3所述核酸分子进行基因编辑的物质的应用，为增强植物的灰斑病抗病性。
  12. 如权利要求11所述的应用，其特征在于：所述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，得到灰斑病抗病性降低的转基因植物。
  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，从而降低目的植物的灰斑病抗病性。
  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。
  17. 一种制备转基因植物的方法，包括如下步骤：抑制出发植物中权利要求2或3所述核酸分子的表达，得到灰斑病抗病性增高的转基因植物。
  18. 如权利要求17所述的方法，其特征在于：所述出发植物为玉蜀黍属植物。
  19. 一种植物育种方法，包括如下步骤：降低目的植物中权利要求1所述蛋白质的含量和/或活性，从而增高目的植物的灰斑病抗病性。
  20. 如权利要求19所述的方法，其特征在于：所述目的植物为玉蜀黍属植物。
  21. 一种植物育种方法，包括如下步骤：对出发植物基因组中的特定基因进行基因编辑，从而增高目的植物的灰斑病抗病性；所述特定基因编码权利要求1所述蛋白质。
  22. 如权利要求21所述的方法，其特征在于：所述出发植物为玉蜀黍属植物。
PCT/CN2020/076319 2019-03-04 2020-02-24 一种培育抗灰斑病植物的方法 Ceased WO2020177560A1 (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113444160A (zh) * 2021-04-25 2021-09-28 东北农业大学 ZmNuC基因及其编码蛋白在抗玉米粗缩病中的应用
CN114410651A (zh) * 2022-01-06 2022-04-29 中国农业大学 玉米灰斑病抗性相关蛋白及其编码基因与应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109705202B (zh) * 2019-03-04 2022-05-10 中国农业大学 一种培育抗灰斑病植物的方法
CN114262369B (zh) * 2021-12-15 2023-05-16 中国农业大学 ZmDi19基因及其靶标基因ZmPR10在培育抗灰斑病植物中的应用
CN117209575B (zh) * 2023-05-29 2024-12-24 中国农业大学 蛋白质及其编码基因在调控玉米大斑病和小斑病中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009088756A1 (en) * 2007-12-31 2009-07-16 E. I. Du Pont De Nemours And Company Gray leaf spot tolerant maize and methods of production
CN106701972A (zh) * 2017-01-23 2017-05-24 华中农业大学 玉米抗灰斑病主效qtl的连锁分子标记和应用
WO2018013323A1 (en) * 2016-07-11 2018-01-18 Pioneer Hi-Bred International, Inc. Methods of making gray leaf spot resistant maize
CN109705202A (zh) * 2019-03-04 2019-05-03 中国农业大学 一种培育抗灰斑病植物的方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001084911A1 (en) * 2000-05-05 2001-11-15 The General Hospital Corporation Calcium dependent protein kinase polypeptides as regulators of plant disease resistance
CN103525780B (zh) * 2013-10-08 2015-06-10 北京大学 水稻钙依赖性蛋白激酶基因及其应用
CN105218651B (zh) * 2015-09-01 2019-05-28 中国农业大学 玉米抗禾谷镰刀菌茎腐病基因ZmAuxRP1的克隆及功能分析
CN105821016A (zh) * 2016-04-21 2016-08-03 南京农业大学 一种大豆cdpk类激酶及其编码基因与应用
CN105859860A (zh) * 2016-05-18 2016-08-17 中国科学院遗传与发育生物学研究所 抗病相关蛋白在调控植物抗病性中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009088756A1 (en) * 2007-12-31 2009-07-16 E. I. Du Pont De Nemours And Company Gray leaf spot tolerant maize and methods of production
CN101970669A (zh) * 2007-12-31 2011-02-09 纳幕尔杜邦公司 耐灰叶斑病玉米及其生产方法
WO2018013323A1 (en) * 2016-07-11 2018-01-18 Pioneer Hi-Bred International, Inc. Methods of making gray leaf spot resistant maize
CN106701972A (zh) * 2017-01-23 2017-05-24 华中农业大学 玉米抗灰斑病主效qtl的连锁分子标记和应用
CN109705202A (zh) * 2019-03-04 2019-05-03 中国农业大学 一种培育抗灰斑病植物的方法

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank 5 May 2018 (2018-05-05), Database accession no. NM_001359579 *
HUANG, PNAS, vol. 115, no. 2, 2018, pages E334 - E341
JOURNAL OF INTERGRATIVE PLANT BIOLOGY, 2018
MING ZHANG, NEW PHYTOLOGIST, vol. 217, 2018, pages 1161 - 1176
NATURE GENETICS, vol. 47, 2015, pages 151 - 157
PATRICK S. SCHNABLE, SCIENCE, vol. 326, 2009, pages 1112 - 1115
See also references of EP3936519A4
XU LING: "Fine-mapping a Major QTL Resistance to Gray Leaf Spot in Maize, Gene Prediction and Transcriptome Analysis", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 1 May 2014 (2014-05-01), pages 1 - 93, XP055851589 *
YAN ZHANG, HEOR APPL GENET
YAN ZHANG, THEOR APPL GENET
ZHANG, NATURE COMMUNICTIONS, vol. 5608, 2019

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113444160A (zh) * 2021-04-25 2021-09-28 东北农业大学 ZmNuC基因及其编码蛋白在抗玉米粗缩病中的应用
CN114410651A (zh) * 2022-01-06 2022-04-29 中国农业大学 玉米灰斑病抗性相关蛋白及其编码基因与应用
CN114410651B (zh) * 2022-01-06 2024-02-20 中国农业大学 玉米灰斑病抗性相关蛋白及其编码基因与应用

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