阅读说明：本技术 一种通过抑制OsSDM基因表达提高水稻耐盐性的方法 (Method for improving salt tolerance of rice by inhibiting OsSDM gene expression ) 是由 种康 高莹 唐永严 徐云远 于 2019-02-01 设计创作，主要内容包括：本发明公开了一种通过抑制OsSDM基因表达提高水稻耐盐性的方法。本发明提供了一种培育抗非生物胁迫的植物的方法,包括如下步骤：抑制出发植物中甲基转移酶基因的表达,从而使植物对非生物胁迫的耐逆性增强。本发明还保护一种制备转基因植物的方法,包括如下步骤：在出发植物中导入特异质粒,得到对非生物胁迫的耐逆性增强的转基因植物；所述特异质粒表达Cas9蛋白基因和特异sgRNA基因；所述特异sgRNA的靶序列如序列表的序列6所示。本发明对于以培育耐逆植物为目的的植物育种,特别是以培育耐盐水稻为目的的水稻育种,具有重大的应用推广价值。(The invention discloses a method for improving salt tolerance of rice by inhibiting OsSDM gene expression. The invention provides a method for cultivating a plant resisting abiotic stress, which comprises the following steps: inhibiting the expression of a methyltransferase gene in a starting plant, thereby enhancing the stress tolerance of the plant to abiotic stress. The invention also provides a method for preparing a transgenic plant, which comprises the following steps: introducing specific plasmids into an original plant to obtain a transgenic plant with enhanced stress tolerance to abiotic stress; the specific plasmid expresses a Cas9 protein gene and a specific sgRNA gene; the target sequence of the specific sgRNA is shown as a sequence 6 in a sequence table. The invention has important application and popularization values for plant breeding aiming at cultivating stress-tolerant plants, in particular for rice breeding aiming at cultivating salt-tolerant rice.)

1. A method of growing plants resistant to abiotic stress comprising the steps of: inhibiting the expression of a methyltransferase gene in a starting plant, thereby enhancing the stress tolerance of the plant to abiotic stress.

2. The method of claim 1, wherein: the methyltransferase is (a1) or (a2) or (a3) as follows:

(a1) protein shown in a sequence 1 in a sequence table;

(a2) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) and having the same function;

(a3) a protein derived from rice, which has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to (a1) and has the same function.

3. The method of claim 2, wherein: the methyltransferase gene is (b1) or (b2) or (b3) or (b4) as follows:

(b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(b2) a DNA molecule shown in a sequence 3 of a sequence table;

(b3) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (b1) or (b2) and which encodes a methyltransferase;

(b4) a DNA molecule derived from rice, having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to (b1) or (b2), and encoding methyltransferase.

4. A method as claimed in claim 1, 2 or 3, characterized by: "inhibiting the expression of the methyltransferase gene in the starting plant" is achieved by the CRISPR/Cas9 system.

5. A method of making a transgenic plant comprising the steps of: introducing a DNA molecule for expressing a Cas9 protein gene and a DNA molecule for expressing a specific sgRNA gene into a starting plant to obtain a transgenic plant with enhanced stress tolerance to abiotic stress; the target sequence of the specific sgRNA is shown as a sequence 6 in a sequence table.

6. A method of making a transgenic plant comprising the steps of: introducing specific plasmids into an original plant to obtain a transgenic plant with enhanced stress tolerance to abiotic stress; the specific plasmid expresses a Cas9 protein gene and a specific sgRNA gene; the target sequence of the specific sgRNA is shown as a sequence 6 in a sequence table.

7. Use of a substance for inhibiting the expression of a methyltransferase gene for the cultivation of plants having increased stress tolerance to abiotic stress.

8. A method of breeding plants with increased stress tolerance to abiotic stress comprising the steps of: reducing the level of and/or inhibiting the activity of methyltransferases in plants, thereby increasing the stress tolerance of plants to abiotic stress.

9. The target sequence of the sgRNA is shown as a sequence 6 in a sequence table.

10. Use of the sgRNA of claim 9 in the preparation of a transgenic plant with increased stress tolerance to abiotic stress.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for improving salt tolerance of rice by inhibiting OsSDM gene expression.

Background

During the growth and development of rice, various abiotic stresses, such as high salt, high temperature, drought, cold damage and the like, are applied to the rice. The rice originally originates from a fresh water swamp environment, is a crop which is moderately sensitive to salt stress, and naturally salinization also becomes one of the main adverse factors influencing the rice yield.

Salt stress is a harmful effect on crops caused by high concentration of salt ions in soil or solution, and includes direct ion poisoning effect and indirect osmotic stress, ion imbalance, nutrient deficiency and the like. Salt stress affects almost all the growth metabolic processes of rice. Under the salt stress, rice can generate various physiological and biochemical changes, and according to the research of predecessors, the damage mechanism of the salt stress to the rice mainly comprises the following aspects: (1) excessive salt ion destroys the integrity of rice cytoplasmic membrane, leading to decrease of selective permeability until disappearance of the membrane, thereby leading to Na in cells⁺、Cl^-Plasma mass accumulation, K⁺、Ca²⁺The nutrient elements are largely exosmosed to cause the dynamic balance of the ion concentration in the cells to be disordered, the structures of cell membranes and organelles are damaged, the stomatal conductance, the chlorophyll content, the nucleic acid content and the like of leaves are reduced, a series of metabolic disorders are caused, the functions of the cells are reduced, and the aging or death is accelerated; (2) the high salt reduces the water potential of the soil solution, forms water stress, makes the rice root difficult to absorb water, and increases the salt concentration in the cell, thereby causing physiological metabolism disorder and further influencing the seedling emergence and growth development of the rice; (3) salinity stress encourages reactive oxygen species O₂ ^-、H₂O₂OH, etc., and the activity or content of intracellular antioxidant systems such as superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POX), Glutathione (GSH), ascorbic Acid (ASC), etc., is damaged or weakened, thereby affecting the balance of the active oxygen metabolic system in the body; the increase of the active oxygen content can intensify membrane peroxidation, destroy the integrity of the membrane, lose selective permeability, cause a great amount of electrolyte and some small molecular organic matters to permeate, destroy the material exchange balance and damage rice; (4) salt stress reduces photosynthetic rate, supply of assimilate and energy is reduced, and protein is inhibitedIn addition, in order to adapt to a high-salt environment and maintain the growth of the rice, more energy is consumed to carry out active absorption and transportation of ions, regionalization and matching and synthesis of osmoregulation substances, so that the growth and development of the rice are limited, and the production capacity and the quality of the rice are greatly influenced.

The rice has different varieties, different periods and different organs with different degrees of damage caused by salt stress and different salt tolerance. In the germination stage of rice seeds, the water absorption is inhibited by high salt, the seeds germinate unevenly, the germination rate is reduced, and even the seeds can not germinate at all and deteriorate and rot in soil. After the seeds germinate, the seeds are extremely sensitive to salt stress, and the tips of the buds are withered and yellow and bent, and cannot be greened in time until seedlings die after emergence. After emergence, the rice transits from the heterotrophic stage to the autotrophic stage, and when subjected to salt damage, leaf rolling and withering occur, and leaf elongation and formation of new leaves are inhibited. When the rice is subjected to salt stress in the tillering stage, the young ear forming stage and the heading flowering stage, tillering and elongation of the rice are inhibited, ineffective tillering is increased, heading is delayed, ears are short and few, seeds are not full, and finally yield is reduced.

The response adaptation and salt tolerance of rice to salt stress is a key point and difficulty of research in recent years. Salt tolerance is the ability of rice to grow on a high salt substrate and tolerate or resist salt stress and complete the entire life cycle. The salt tolerance of the rice is the expression of the comprehensive effect of a plurality of physiological processes, the research on the adaptation of the rice to the salt stress and the salt tolerance is deeply carried out, and the salt tolerance rice has important significance for cultivating salt-tolerant rice varieties, further enlarging the plantable area of the rice and ensuring the safe production and quality of grains.

Disclosure of Invention

The invention aims to provide a method for improving salt tolerance of rice by inhibiting OsSDM gene expression.

The invention provides a method for cultivating a plant resisting abiotic stress, which comprises the following steps: inhibiting the expression of a methyltransferase gene in a starting plant, thereby enhancing the stress tolerance of the plant to abiotic stress.

"inhibiting the expression of a methyltransferase gene in a starting plant" is achieved by gene editing.

"inhibiting the expression of the methyltransferase gene in the starting plant" is achieved by the CRISPR/Cas9 system. The target sequence of sgRNA in the CRISPR/Cas9 system is shown as sequence 6 in the sequence table. The sgRNA in the CRISPR/Cas9 system is shown as a sequence 5 in a sequence table.

"inhibiting the expression of a methyltransferase gene in a starting plant" is achieved by introducing a recombinant plasmid; the recombinant plasmid expresses a Cas9 protein gene and a specific sgRNA gene; the target sequence of the specific sgRNA is shown as a sequence 6 in a sequence table. The specific sgRNA is shown as a sequence 5 in a sequence table.

The invention also provides a method for preparing a transgenic plant, which comprises the following steps: and introducing a DNA molecule for expressing a Cas9 protein gene and a DNA molecule for expressing a specific sgRNA gene into the starting plant to obtain a transgenic plant with enhanced stress tolerance to abiotic stress. The DNA molecule expressing the Cas9 protein gene and the DNA molecule expressing the specific sgRNA gene may be present in the same plasmid or may be present in different plasmids. The target sequence of the specific sgRNA is shown as a sequence 6 in a sequence table. The specific sgRNA is shown as a sequence 5 in a sequence table.

The invention also provides a method for preparing a transgenic plant, which comprises the following steps: introducing specific plasmids into an original plant to obtain a transgenic plant with enhanced stress tolerance to abiotic stress; the specific plasmid expresses a Cas9 protein gene and a specific sgRNA gene; the target sequence of the specific sgRNA is shown as a sequence 6 in a sequence table. The specific sgRNA is shown as a sequence 5 in a sequence table.

The invention also protects the application of the substance for inhibiting the expression of the methyltransferase gene in cultivating plants with enhanced stress tolerance to abiotic stress. The "substance inhibiting the expression of a methyltransferase gene" may be a substance inhibiting the expression of a methyltransferase gene by gene editing. The "substance inhibiting the expression of a methyltransferase gene" may be a substance inhibiting the expression of a methyltransferase gene by the CRISPR/Cas9 system. The target sequence of sgRNA in the CRISPR/Cas9 system is shown as sequence 6 in the sequence table. The sgRNA in the CRISPR/Cas9 system is shown as a sequence 5 in a sequence table. The "substance inhibiting the expression of the methyltransferase gene" may be a DNA molecule expressing a Cas9 protein gene and a DNA molecule expressing a specific sgRNA gene. The "substance inhibiting the expression of the methyltransferase gene" may be a specific plasmid; the specific plasmid expresses a Cas9 protein gene and a specific sgRNA gene. The target sequence of the specific sgRNA is shown as a sequence 6 in a sequence table. The specific sgRNA is shown as a sequence 5 in a sequence table.

The present invention also provides a method for cultivating a plant having enhanced stress tolerance to abiotic stress, comprising the steps of: reducing the level of and/or inhibiting the activity of methyltransferases in plants, thereby increasing the stress tolerance of plants to abiotic stress.

The invention also protects a sgRNA, and the target sequence of the sgRNA is shown as the sequence 6 in the sequence table. The sgRNA is shown as a sequence 5 in a sequence table.

The invention also protects the application of the sgRNA in preparing transgenic plants with enhanced stress tolerance to abiotic stress. The invention also protects the application of the sgRNA coding gene and the Cas9 protein coding gene in preparing transgenic plants with enhanced stress tolerance to abiotic stress.

Any of the methyltransferases described above is an SDM protein.

Any one of the above methyltransferases is an OsSDM protein.

The OsSDM protein is (a1) or (a2) or (a3) as follows:

(a1) protein shown in a sequence 1 in a sequence table;

(a2) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) and having the same function;

(a3) a protein derived from rice, which has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to (a1) and has the same function.

The methyltransferase gene is (b1) or (b2) or (b3) or (b4) as follows:

(b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(b2) a DNA molecule shown in a sequence 3 of a sequence table;

(b3) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (b1) or (b2) and encodes the methyltransferase;

(b4) a DNA molecule derived from rice and having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to (b1) or (b2) and encoding the methyltransferase.

The term "identity" refers to sequence similarity to a native nucleotide or amino acid sequence. "identity" can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess identity between related sequences.

Any of the above plants is a gramineae plant.

Any of the above plants is rice.

Any one of the above plants is japonica rice,

any one of the plants is Nipponbare.

Any of the above abiotic stresses is salt stress.

The invention has important application and popularization values for plant breeding aiming at cultivating stress-tolerant plants, in particular for rice breeding aiming at cultivating salt-tolerant rice.

Drawings

FIG. 1 is a schematic diagram of the elements of recombinant plasmid pCRISPR-OsSDM.

FIG. 2 shows the target sequences and their surrounding sequences in the ossdm-1 and ossdm-2 individuals.

FIG. 3 is a photograph in the determination of salt tolerance.

FIG. 4 shows the survival results in salt tolerance determinations.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. The Nipponbare of rice variety is abbreviated as Nipponbare and is expressed by NIP.

CRISPR/Cas vector: hangzhou Baige Biotechnology Inc., catalog number BGK 03.

N6D2 medium: solid MS medium containing 300mg/L hydrolyzed casein, 500mg/L proline, 500mg/L glutamine, 30g/L sucrose and 2 mg/L2, 4-D.

N6D2S1 medium: N6D2 medium containing 25mg/L hygromycin and 600mg/L cephamycin.

N6D2S2 medium: N6D2 medium containing 50mg/L hygromycin and 300mg/L cephamycin.

Differentiation medium a: N6D2 culture medium containing 300mg/L hydrolyzed casein, 50mg/L hygromycin, 1 mg/L6-BA, 0.5mg/L KT, 0.2mg/L ZT, 0.25mg/L NAA, 30g/L sucrose and 30g/L sorbitol.

Differentiation medium B: N6D2 culture medium containing 300mg/L hydrolyzed casein, 50mg/L hygromycin, 1 mg/L6-BA, 0.5mg/L KT, 0.2mg/L ZT, 0.5mg/L NAA, 30/L sucrose and 20g/L sorbitol.

Rooting and seedling strengthening culture medium: solid 1/2MS medium containing 1mg/L paclobutrazol and 0.5mg/L NAA.

The formulation of Mucuna B culture solution is shown in Table 1, pH is 5.8, and the solvent is water.

TABLE 1

The OsSDM protein in the rice Nipponbare is shown as a sequence 1 in a sequence table. The open reading frame of the OsSDM protein coded in the cDNA of the rice Nipponbare is shown as a sequence 2 in a sequence table. The gene of OsSDM protein coded in the genome DNA of the Nipponbare is shown as a sequence 3 in a sequence table.