WO2021082567A1 - 大豆广谱抗病相关基因的应用 - Google Patents

大豆广谱抗病相关基因的应用 Download PDF

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WO2021082567A1
WO2021082567A1 PCT/CN2020/105801 CN2020105801W WO2021082567A1 WO 2021082567 A1 WO2021082567 A1 WO 2021082567A1 CN 2020105801 W CN2020105801 W CN 2020105801W WO 2021082567 A1 WO2021082567 A1 WO 2021082567A1
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gene
gmlmm1
amino acid
soybean
acid sequence
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PCT/CN2020/105801
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French (fr)
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冯献忠
杨素欣
王东梅
于慧
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中国科学院东北地理与农业生态研究所
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Priority to US17/597,717 priority Critical patent/US20220243218A1/en
Publication of WO2021082567A1 publication Critical patent/WO2021082567A1/zh

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    • 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
    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • 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
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • A01H6/542—Glycine max [soybean]
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination

Definitions

  • the present invention relates to the technical field of plant genetic engineering, in particular to the application of soybean disease resistance related gene GmLMM1 in regulating the PTI immune response of plants and enhancing the ability of plants to resist pathogens.
  • Soybeans are one of the main sources of oil and vegetable protein worldwide. With the rapid growth of the world’s population, the demand for soybeans in food, feed and industrial production has also steadily increased. However, on a global scale, the occurrence of persistent diseases has severely affected its yield and quality. Soybean diseases such as soybean bacterial spot and Phytophthora root rot have caused great losses to global soybean production and quality. Among them, Phytophthora sojae (P.sojae) disease is one of the most harmful diseases in soybean production, which seriously harms soybean production, and the diseases caused by it are widespread in all major soybean planting areas. Phytophthora sojae can infect seeds, roots, stems and leaves.
  • P.sojae Phytophthora sojae
  • the purpose of the present invention is to provide the application of soybean GmLMM1 gene in the regulation of plant PTI immune response and pathogen resistance.
  • the present invention obtains a soybean plant related to broad-spectrum disease resistance by screening an artificial chemically mutagenized soybean mutant library, and named it Gmlmm1 (Glycine max Lesion mimic mutant 1).
  • the soybean plant was confirmed by map-based cloning and sequencing analysis.
  • the enhancement of broad-spectrum disease resistance is caused by artificial mutagenesis in the Glyma.13G054400 gene sequence (the amino acid sequence of the encoded protein is shown in SEQ ID NO. 1, and the CDS sequence is shown in SEQ ID NO. 2).
  • the amino acid sequence of the encoded protein of the gene after artificial mutagenesis is shown in SEQ ID NO.3, and the CDS sequence is shown in SEQ ID NO.4.
  • the present invention finds that GmLMM1 gene negatively regulates plant PTI immune response and pathogen resistance.
  • the present invention provides the application of soybean GmLMM1 gene or its mutant gene or their encoded protein in regulating the resistance of plant pathogenic bacteria.
  • the present invention provides the application of soybean GmLMM1 gene or its mutant gene or their encoded protein in regulating the PTI immune response of plants.
  • the pathogen resistance of the plant can be improved and the PTI immune response of the plant can be enhanced.
  • the present invention provides the application of soybean GmLMM1 gene or its mutant gene or their encoded protein in plant genetic breeding or transgenic plant preparation.
  • the transgenic plant is a disease-resistant transgenic plant. More preferably, it is a transgenic plant resistant to Phytophthora and bacterial spot.
  • the encoded protein of the soybean GmLMM1 gene has any one of the following amino acid sequences:
  • the encoded protein of the mutant gene of the soybean GmLMM1 gene has any one of the following amino acid sequences:
  • the CDS of the soybean GmLMM1 gene has any one of the following nucleotide sequences:
  • nucleotide sequence encoding the same functional protein obtained by replacing, inserting or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 2.
  • the CDS of the mutant gene of the soybean GmLMM1 gene has any of the following nucleotide sequences:
  • nucleotide sequence encoding the same functional protein obtained by replacing, inserting or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.4.
  • SEQ ID NO. 1 is the amino acid sequence of the protein encoded by the soybean GmLMM1 gene.
  • Those skilled in the art can use the amino acid sequence disclosed in the present invention and the conservative substitution of amino acids and other conventional technical means in the field. Under the premise of affecting its activity, one or several amino acids are substituted, deleted and/or added to obtain a mutant of the GmLMM1 gene encoding protein with the same activity as the GmLMM1 gene encoding protein disclosed in the present invention.
  • SEQ ID NO. 2 The aforementioned nucleotide sequence shown in SEQ ID NO. 2 is the CDS sequence of GmLMM1 gene in soybean. Taking into account the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for expression in specific species as needed.
  • amino acid sequence shown in SEQ ID NO. 3 is the amino acid sequence of the protein encoded by the mutant gene of soybean GmLMM1 gene.
  • amino acid sequence disclosed in the present invention can be used and conservative substitution of amino acids and other conventional technical means in the field Under the premise of not affecting its activity, one or several amino acids can be substituted, deleted and/or added to obtain a GmLMM1 gene coding protein mutant having the same activity as the GmLMM1 gene coding protein disclosed in the present invention.
  • the aforementioned nucleotide sequence shown in SEQ ID NO. 4 is the CDS sequence of the mutant gene of GmLMM1 gene in soybean. Taking into account the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for expression in specific species as needed.
  • the GmLMM1 gene knockout transgenic plant is obtained by CRISPR/Cas9 technology, and the obtained transgenic plant is a disease-resistant transgenic plant.
  • the present invention provides a gRNA for targeting a specific position of the GmLMM1 gene, the gRNA comprising the nucleotide sequence shown in SEQ ID NO.5.
  • the gRNA can cooperate with gene editing tools such as Cas9 to realize the knockout of soybean GmLMM1 gene.
  • GmLMM1 gene or its mutant gene or their encoded protein can be applied in the form of GmLMM1 gene or its mutant gene or their encoded protein itself, or in the form of an expression cassette containing the encoded protein of the GmLMM1 gene or the encoded protein of the mutant gene.
  • Vector, and host cell containing the expression cassette or the vector.
  • the present invention provides a method for regulating the resistance of plant pathogens, including: regulating the expression of GmLMM1 gene in soybeans to obtain mutants with increased pathogen resistance;
  • the encoded protein of the soybean GmLMM1 gene has any one of the following amino acid sequences:
  • the encoded protein of the mutant gene of the soybean GmLMM1 gene has any one of the following amino acid sequences:
  • the above method includes: reducing the expression level of the GmLMM1 gene in the plant to increase the pathogen resistance of the plant.
  • the aforementioned reduction in the expression of the GmLMM1 gene in plants can be achieved by conventional technical means in the art, for example: using CRISPR/Cas9 technology to knock out the GmLMM1 gene in plants.
  • the present invention utilizes CRISPR/Cas9 technology to knock out the GmLMM1 gene in plants by using the nucleotide sequence shown in SEQ ID NO. 5 as gRNA, which can knock out the GmLMM1 gene in plants efficiently.
  • the plant is a monocotyledonous plant or a dicotyledonous plant.
  • the plants include, but are not limited to, soybean, wheat, rice, corn, cotton, rape, peanut, legume crops, vegetable crops and the like.
  • the present invention clones the GmLMM1 gene involved in plant immune response and pathogen resistance regulation in soybean for the first time.
  • GmLMM1 gene can negatively regulate plant PTI immune response and pathogen resistance: reducing GmLMM1 gene expression can effectively enhance plant PTI immune response and increase plant pathogen resistance (reducing pathogen susceptibility and disease).
  • the cloning and functional analysis of the GmLMM1 gene is a breakthrough in the exploration of soybean disease resistance mechanisms. It provides important genetic basis and theoretical support for the research on plant disease resistance related mechanisms, and promotes the research and application of plant defense systems and the cultivation of high New disease-resistant soybean varieties provide valuable genetic resources.
  • the GmLMM1 gene and its encoded protein have great application value in soybean disease-resistant genetic engineering breeding.
  • Figure 1 shows the observation of pathogen infection under ultraviolet light 48 hours after wild-type Williams 82 leaves and Gmlmm1 mutant leaves were inoculated with Phytophthora sojae P7076 hyphae in Example 1 of the present invention. Ruler, 1 cm. Inside the dashed circle are the lesions infected by pathogenic bacteria.
  • Figure 2 shows the location of GmLMM1 gene on soybean chromosome 13 in Example 1 of the present invention.
  • Fig. 3 shows the observation of infection and the statistics of the diseased spot area of the GmLMM1 gene knockout transgenic plant leaves inoculated with Phytophthora sojae P.sojae P7076 hyphae block 60 hours after the GmLMM1 gene knock-out transgenic plant in Example 2 of the present invention.
  • Fig. 4 shows the statistics of bacterial colony counts of the GmLMM1 gene knockout transgenic plants in Example 2 of the present invention after 4 days of inoculation of soybean bacterial spot pathogen Pseudomonas syringae Psg (Pseudomonas syringae pv.glycinea). ** represents a significant difference at the p ⁇ 0.01 level.
  • Fig. 5 is the statistics of bacterial colony number of GmLMM1 gene knockout transgenic plant leaves inoculated with the pathogen Pseudomonas Syringae pv.phaseolicola (Psp) 4 days after the leaves of the GmLMM1 gene knockout transgenic plant in Example 2 of the present invention. *** represents a significant difference at the p ⁇ 0.001 level.
  • FIG. 6 shows that GmLMM1 inhibits flg22-induced reactive oxygen species burst in the PTI immune response in Example 3 of the present invention.
  • GFP represents the control group
  • GmLMM1-HA represents the experimental group
  • H 2 O represents the blank control group treated with H 2 O
  • flg22 represents Treated with flg22 small peptide
  • Western blot was used to detect the expression of GmLMM1, and Rubisco was the internal control.
  • the mutant Gmlmm1 was obtained by screening the mutant library obtained by EMS mutagenesis of Williams 82. Phytophthora sojae P7076 hyphae were used to inoculate wild-type "Williams 82" leaves and Gmlmm1 mutant leaves. After 48 hours of infection, observe the pathogen infection on the leaves under ultraviolet light ( Figure 1). The results showed that the mutants The pathogen infection area of Gmlmm1 was significantly smaller than that of the wild-type "Williams 82", indicating that compared with the wild-type Williams 82, the mutant Gmlmm1 significantly improved the resistance to Phytophthora sojae. The above experimental results indicate that the Gmlmm1 mutant has resistance to Phytophthora sojae.
  • GmLMM1 may be involved in the regulation of plant immune response.
  • Gmlmm1 and "Hedou 12" hybrid F2 segregated population was used for gene mapping.
  • the GmLMM1 gene was located in the range of 131kb between 15.08Mb-15.21Mb of chromosome 13 ( Figure 2), combined with the whole genome resequencing result of Gmlmm1, finally confirmed
  • the GmLMM1 gene is Glyma.13G054400 (the amino acid sequence of the encoded protein is shown in SEQ ID NO. 1, and the CDS sequence is shown in SEQ ID NO.
  • Gmlmm1 is in the second exon of Glyma.13G054400 (GmLMM1) gene
  • GmLMM1 Glyma.13G054400
  • GFP control group
  • GmLMM1 experimental group
  • flg22 a small peptide of 22 amino acids that is conserved at the N-terminus of flagellin commonly used in plant immunity research
  • the present invention provides the application of soybean broad-spectrum disease resistance related gene GmLMM1.
  • the present invention clones the GmLMM1 gene involved in the regulation of PTI immune response, Phytophthora resistance, soybean bacterial spot, and kidney bean halo disease in soybeans.
  • GmLMM1 gene can negatively regulate plant PTI immune response and pathogen resistance. By reducing the expression of GmLMM1 gene, it can effectively enhance plant PTI immune response and increase plant pathogen resistance.
  • the cloning and functional discovery of the GmLMM1 gene provide important genetic basis and theoretical support for the research of soybean disease resistance-related mechanisms, and provide valuable genetic resources for the advancement of the research and application of plant defense systems and the cultivation of new soybean varieties with high disease resistance. Soybean disease resistance genetic engineering has important application value and prospects.

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Abstract

植物基因工程技术领域，具体涉及大豆广谱抗病相关基因GmLMM1的应用。在大豆中克隆了参与PTI免疫反应和疫霉病抗性、大豆细菌性斑点病以及菜豆光晕病等调控的GmLMM1基因。GmLMM1基因能够负调控植物PTI免疫反应和病原菌抗性，通过降低GmLMM1基因的表达量，能够有效增强植物PTI免疫反应，提高植物的病原菌抗性。GmLMM1基因的克隆和功能发现为大豆抗病相关机制的研究提供了重要的基因基础及理论支持，为推进植物防御系统的研究和应用以及培育高抗病的大豆新品种提供宝贵的基因资源，在大豆抗病基因工程育种中具有重要的应用价值。

Description

大豆广谱抗病相关基因的应用
交叉引用
本申请要求2019年10月30日提交的专利名称为“大豆抗疫霉病相关基因GmLMM1的应用”的第201911044883.5号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及植物基因工程技术领域，具体涉及大豆抗病相关基因GmLMM1在调控植物PTI免疫反应和提升植物抵抗病原菌能力的应用。
背景技术
大豆是世界范围内油脂和植物蛋白的主要来源之一。随着世界人口的迅速增长，食品、饲料和工业生产中的大豆需求量也稳步增加。但在全球范围内，持续病害的发生对其产量和品质造成了严重影响。大豆疾病如大豆细菌斑点病和疫霉根腐病等对全球大豆产量和品质造成了极大的损失。其中大豆疫霉Phytophthora sojae(P.sojae)病是大豆生产上最具危害性的病害之一，严重危害大豆的生产，其引发的病害广泛存在于所有主要大豆种植区。大豆疫霉能侵染种子、根、茎和叶，对于大豆疫霉高度敏感的品种，该病会造成大豆绝产。传统的病害防治主要依靠化学方法和育种方法，然而，病原体的进化速度超越了这些防治方法的改良优化速度，使这些传统的防治方法不能发挥有效的防治病害的作用。因此，利用抗病基因工程育种是解决该难题的最有效途径。
植物抗病虫害的能力与自身的免疫系统密切相关，因此，研究大豆的免疫系统和探索大豆如何保护自己抵御微生物病原体的侵害非常重要，同时，研究大豆抗病机理对于培育抗病大豆新品种具有重要意义。
发明内容
为解决现有技术中存在的技术问题，本发明的目的在于提供大豆GmLMM1基因在调控植物PTI免疫反应和病原菌抗性中的应用。
为实现上述目的，本发明的技术方案如下：
本发明通过筛选人工化学诱变大豆突变体库，获得了一个与广谱性抗病相关的大豆植株，将其命名为Gmlmm1(Glycine max lesion mimic mutant 1)，经图位克隆及测序分析证实大豆广谱性抗病性的增强是由于Glyma.13G054400基因序列(其编码蛋白的氨基酸序列如SEQ ID NO.1所示，CDS序列如SEQ ID NO.2所示)的人工诱变改变造成的，人工诱变后的基因的编码蛋白的氨基酸序列如SEQ ID NO.3所示，CDS序列如SEQ ID NO.4所示。本发明发现GmLMM1基因负调控植物PTI免疫反应以及病原菌抗性。
第一方面，本发明提供大豆GmLMM1基因或其突变基因或它们的编码蛋白在调控植物病原菌抗性中的应用。
第二方面，本发明提供大豆GmLMM1基因或其突变基因或它们的编码蛋白在调控植物PTI免疫反应中的应用。
上述应用中，通过降低植物中所述GmLMM1基因的表达量，可提高植物的病原菌抗性、增强植物PTI免疫反应。
第三方面，本发明提供大豆GmLMM1基因或其突变基因或它们的编码蛋白在植物遗传育种或转基因植物制备中的应用。
优选地，所述转基因植物为抗病转基因植物。更优选为抗疫霉病和细菌性斑点病转基因植物。
本发明中，所述大豆GmLMM1基因的编码蛋白具有如下任一种氨基酸序列：
(1)如SEQ ID NO.1所示的氨基酸序列；
(2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列；
(3)与如SEQ ID NO.1所示的氨基酸序列具有至少80％同源性的氨基酸序列；优选地，所述同源性为至少90％；更优选为95％。
本发明中，所述大豆GmLMM1基因的突变基因的编码蛋白具有如下 任一种氨基酸序列：
(1)如SEQ ID NO.3所示的氨基酸序列；
(2)如SEQ ID NO.3所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列；
(3)与如SEQ ID NO.3所示的氨基酸序列具有至少80％同源性的氨基酸序列；优选地，所述同源性为至少90％；更优选为95％。
本发明中，所述大豆GmLMM1基因的CDS具有如下任一种核苷酸序列：
(1)如SEQ ID NO.2所示的核苷酸序列；
(2)如SEQ ID NO.2所示的核苷酸序列经一个或多个核苷酸的替换、插入或缺失得到的编码相同功能蛋白的核苷酸序列。
本发明中，所述大豆GmLMM1基因的突变基因的CDS具有如下任一种核苷酸序列：
(1)如SEQ ID NO.4所示的核苷酸序列；
(2)如SEQ ID NO.4所示的核苷酸序列经一个或多个核苷酸的替换、插入或缺失得到的编码相同功能蛋白的核苷酸序列。
上述如SEQ ID NO.1所示的氨基酸序列为大豆GmLMM1基因的编码蛋白的氨基酸序列，本领域技术人员可根据本发明公开的氨基酸序列以及氨基酸的保守性替换等本领域常规技术手段，在不影响其活性的前提下，取代、缺失和/或增加一个或几个氨基酸，得到与本发明公开的GmLMM1基因的编码蛋白具有相同活性的GmLMM1基因编码蛋白的突变体。
上述如SEQ ID NO.2所示的核苷酸序列为大豆中GmLMM1基因的CDS序列。考虑到密码子的简并性以及不同物种密码子的偏爱性，本领域技术人员可以根据需要使用适合特定物种表达的密码子。
上述如SEQ ID NO.3所示的氨基酸序列为大豆GmLMM1基因的突变基因的编码蛋白的氨基酸序列，本领域技术人员可根据本发明公开的氨基酸序列以及氨基酸的保守性替换等本领域常规技术手段，在不影响其活性 的前提下，取代、缺失和/或增加一个或几个氨基酸，得到与本发明公开的GmLMM1基因的突变基因的编码蛋白具有相同活性的GmLMM1基因编码蛋白的突变体。
上述如SEQ ID NO.4所示的核苷酸序列为大豆中GmLMM1基因的突变基因的CDS序列。考虑到密码子的简并性以及不同物种密码子的偏爱性，本领域技术人员可以根据需要使用适合特定物种表达的密码子。
优选地，通过CRISPR/Cas9技术获得GmLMM1基因敲除转基因植株，所述获得的转基因植物为抗病转基因植物。
作为示例，本发明提供一种用于靶向GmLMM1基因特定位置的gRNA，所述gRNA包含如SEQ ID NO.5所示的核苷酸序列。该gRNA可与Cas9等基因编辑工具配合作用，实现大豆GmLMM1基因的敲除。
上述GmLMM1基因或其突变基因或它们的编码蛋白的应用可以以GmLMM1基因或其突变基因或它们的编码蛋白本身的形式应用，或者以含有GmLMM1基因的编码蛋白或其突变基因的编码蛋白的表达盒、载体、含有所述表达盒或所述载体的宿主细胞的形式应用。
第四方面，本发明提供一种调控植物病原菌抗性的方法，包括：调控大豆中GmLMM1基因的表达量以获得病原菌抗性提高的突变体；
所述大豆GmLMM1基因的编码蛋白具有如下任一种氨基酸序列：
(1)如SEQ ID NO.1所示的氨基酸序列；
(2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列；
(3)与如SEQ ID NO.1所示的氨基酸序列具有至少80％同源性的氨基酸序列；优选地，所述同源性为至少90％；更优选为95％。
所述大豆GmLMM1基因的突变基因的编码蛋白具有如下任一种氨基酸序列：
(1)如SEQ ID NO.3所示的氨基酸序列；
(2)如SEQ ID NO.3所示的氨基酸序列经一个或多个氨基酸的替换、 插入或缺失得到的具有相同功能蛋白的氨基酸序列；
(3)与如SEQ ID NO.3所示的氨基酸序列具有至少80％同源性的氨基酸序列；优选地，所述同源性为至少90％；更优选为95％。
优选地，上述方法包括：通过降低所述植物中GmLMM1基因的表达量，提高所述植物的病原菌抗性。
上述降低植物中GmLMM1基因的表达量可通过本领域常规技术手段实现，例如：利用CRISPR/Cas9技术敲除植物中GmLMM1基因。
优选地，本发明利用CRISPR/Cas9技术，以如SEQ ID NO.5所示的核苷酸序列为gRNA敲除植物中GmLMM1基因，可高效敲除植物中GmLMM1基因。
本发明中，所述植物为单子叶植物或双子叶植物。所述植物包括但不限于大豆、小麦、水稻、玉米、棉花、油菜、花生、豆类作物、蔬菜作物等。
本发明的有益效果在于：
本发明首次在大豆中克隆了参与植物免疫反应和病原菌抗性调控的GmLMM1基因。GmLMM1基因能够负调控植物PTI免疫反应和病原菌抗性：降低GmLMM1基因表达量，能够有效增强植物PTI免疫反应，提高植物的病原菌抗性(降低病原菌的易感性和发病情况)。GmLMM1基因的克隆及其功能解析是大豆抗病机理探究里程中的突破性进展，为植物抗病相关机制研究提供了重要的基因基础及理论支持，为推进植物防御系统的研究和应用以及培育高抗病的大豆新品种提供宝贵的基因资源，GmLMM1基因及其编码蛋白在大豆抗病基因工程育种中具有重大的应用价值。
附图说明
图1为本发明实施例1中野生型Williams 82叶片与Gmlmm1突变体叶片接种大豆疫霉菌P.sojae P7076菌丝块48小时后，紫外灯下病菌侵染观察。标尺，1厘米。虚线圈内为病原菌侵染的病斑。
图2为本发明实施例1中GmLMM1基因在大豆13号染色体上的定位。
图3为本发明实施例2中GmLMM1基因敲除转基因植株叶片接种大豆疫霉菌P.sojae P7076菌丝块60小时后的侵染观察及病斑面积统计。标尺，1厘米。***代表在p<0.001水平上差异显著。
图4为本发明实施例2中GmLMM1基因敲除转基因植株叶片接种大豆细菌性斑点病病原菌丁香假单胞菌大豆致病变种Psg(Pseudomonas syringae pv.glycinea)4天后的细菌菌落数统计。**代表在p<0.01水平上差异显著。
图5为本发明实施例2中GmLMM1基因敲除转基因植株叶片接种菜豆光晕病的病原菌Pseudomonas syringae pv.phaseolicola(Psp)4天后的细菌菌落数统计。***代表在p<0.001水平上差异显著。
图6为本发明实施例3中GmLMM1抑制flg22诱发PTI免疫反应中的活性氧爆发。经1μM flg22或水处理瞬时表达GFP或GmLMM1基因的烟草叶片后的活性氧爆发情况，GFP代表对照组，GmLMM1-HA代表实验组，H 2O代表采用H 2O处理的空白对照组，flg22代表采用flg22小肽处理；Western blot检测GmLMM1的表达情况，其中，Rubisco为内参。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用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材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1 Gmlmm1突变体抗疫霉病表型鉴定
通过筛选经EMS诱变Williams 82获得的突变体库，获得突变体 Gmlmm1。采用大豆疫霉菌P.sojae P7076菌丝块接种野生型“Williams 82”叶片与Gmlmm1突变体叶片，侵染48小时后，在紫外光下观察病原菌侵染叶片情况(图1)，结果显示突变体Gmlmm1的病原菌侵染面积要显著小于野生型“Williams 82”的病原菌侵染面积，说明与野生型Williams 82相比，突变体Gmlmm1显著提高了对大豆疫霉的抗性。以上实验结果说明Gmlmm1突变体具有抗大豆疫霉病的特性。因此，推测GmLMM1基因可能参与了植物免疫反应调控。利用Gmlmm1与“菏豆12”杂交的F2分离群体进行基因定位将GmLMM1基因定位在13号染色体的15.08Mb-15.21Mb之间131kb区间内(图2)，结合Gmlmm1的全基因组重测序结果最终确定GmLMM1基因即为Glyma.13G054400(编码蛋白的氨基酸序列如SEQ ID NO.1所示，CDS序列如SEQ ID NO.2所示)，Gmlmm1在Glyma.13G054400(GmLMM1)基因的第二个外显子区域内存在单碱基C到T的突变(突变基因的编码蛋白的氨基酸序列如SEQ ID NO.3所示，突变基因的CDS序列如SEQ ID NO.4所示)(图2)。
实施例2 GmLMM1基因敲除转基因植株显著增强病原菌的抗性
构建GmU6启动子驱动gRNA(序列如SEQ ID NO.5所示)和GmUbi3启动子驱动Cas9蛋白的CRISPR/cas9重组质粒，通过农杆菌介导的遗传转化系统将重组质粒导入野生型大豆品种东农50(DN50)大豆中。经遗传转化，最终获得T1代转基因植株。用大豆疫霉菌P.sojae P7076菌丝块接种野生型DN50叶片与GmLMM1基因敲除转基因植株CRISPR的叶片，侵染60小时后，采用台盼蓝染色法观察侵染差异并统计病斑形成面积(图3)，结果显示转基因植株CRISPR叶片的病斑面积要显著小于野生型DN50的病斑面积，说明GmLMM1基因敲除后显著提高了对大豆疫霉病的抗性。采用注射法对野生型DN50叶片与GmLMM1基因敲除转基因植株CRISPR的叶片接种大豆细菌性斑点病病原菌Psg(图4)和菜豆光晕病的病原菌Psp(图5)，4天后统计病原菌菌落形成数目，经统计结果显示转基因植株CRISPR叶片中的病原菌菌落数目显著少于野生型 DN50叶片中的菌落数目，说明GmLMM1基因敲除后显著提高了对大豆细菌性斑点病和菜豆光晕病的抗性。遗传转化实验结果进一步说明GmLMM1基因在免疫反应中的重要作用。GmLMM1基因的新功能的发现为植物遗传育种提供了宝贵的基因资源。
实施例3 GmLMM1基因抑制植物PTI免疫反应
利用农杆菌注射烟草瞬时表达GFP(对照组)和GmLMM1(实验组)，24小时后用水和flg22(常用于植物免疫研究中的鞭毛蛋白N端保守的一个含22个氨基酸的小肽)分别处理，检测处理后的活性氧爆发情况，发现瞬时过量表达GmLMM1可抑制flg22诱导的活性氧爆发(图6)。以上实验说明GmLMM1基因能够抑制PTI免疫反应中活性氧的爆发，负调控植物的PTI免疫反应。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对其做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供大豆广谱抗病相关基因GmLMM1的应用。本发明在大豆中克隆了参与PTI免疫反应和疫霉病抗性、大豆细菌性斑点病以及菜豆光晕病等调控的GmLMM1基因。GmLMM1基因能够负调控植物PTI免疫反应和病原菌抗性，通过降低GmLMM1基因的表达量，能够有效增强植物PTI免疫反应，提高植物的病原菌抗性。GmLMM1基因的克隆和功能发现为大豆抗病相关机制的研究提供了重要的基因基础及理论支持，为推进植物防御系统的研究和应用以及培育高抗病的大豆新品种提供宝贵的基因资源，在大豆抗病基因工程育种中具有重要的应用价值和前景。

Claims (9)

  1. 大豆GmLMM1基因或其突变基因或它们的编码蛋白在调控植物病原菌抗性中的应用。
  2. 大豆GmLMM1基因或其突变基因或它们的编码蛋白在调控植物PTI免疫反应中的应用。
  3. 根据权利要求1～2任一项所述的应用，其特征在于，通过降低GmLMM1基因或其编码蛋白的表达量，提高植物病原菌抗性、增强植物PTI免疫反应。
  4. 大豆GmLMM1基因或其突变基因或它们的编码蛋白在植物遗传育种或转基因植物制备中的应用。
  5. 根据权利要求1～4任一项所述的应用，其特征在于，所述大豆GmLMM1基因的编码蛋白具有如下任一种氨基酸序列：
    (1)如SEQ ID NO.1所示的氨基酸序列；
    (2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列；
    (3)与如SEQ ID NO.1所示的氨基酸序列具有至少80％同源性的氨基酸序列；优选地，所述同源性为至少90％；更优选为95％。
    所述大豆GmLMM1基因的突变基因的编码蛋白具有如下任一种氨基酸序列：
    (1)如SEQ ID NO.3所示的氨基酸序列；
    (2)如SEQ ID NO.3所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列；
    (3)与如SEQ ID NO.3所示的氨基酸序列具有至少80％同源性的氨基酸序列；优选地，所述同源性为至少90％；更优选为95％。
  6. 根据权利要求1～5任一项所述的应用，其特征在于，所述大豆GmLMM1基因经CRISPR/cas9技术利用gRNA靶向敲除后，降低GmLMM1基因的表达量，可提高大豆病原菌的抗性。
  7. 根据权利要求6所述的应用，其特征在于，所述gRNA包含如SEQ ID NO.5所示的核苷酸序列。
  8. 一种调控植物病原菌抗性的方法，其特征在于，包括：调控植物中GmLMM1基因或其突变基因或它们的编码蛋白的表达量；
    所述大豆GmLMM1基因的编码蛋白具有如下任一种氨基酸序列：
    (1)如SEQ ID NO.1所示的氨基酸序列；
    (2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列；
    (3)与如SEQ ID NO.1所示的氨基酸序列具有至少80％同源性的氨基酸序列；优选地，所述同源性为至少90％；更优选为95％。
    所述大豆GmLMM1基因的突变基因的编码蛋白具有如下任一种氨基酸序列：
    (1)如SEQ ID NO.3所示的氨基酸序列；
    (2)如SEQ ID NO.3所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列；
    (3)与如SEQ ID NO.3所示的氨基酸序列具有至少80％同源性的氨基酸序列；优选地，所述同源性为至少90％；更优选为95％。
  9. 根据权利要求8所述的方法，其特征在于，通过降低所述植物中GmLMM1基因或其编码蛋白的表达量，提高所述植物的病原菌抗性。
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