阅读说明：本技术 SiMYB61蛋白及其相关生物材料在调控植物抗逆性中的应用 (Application of SiMYB61 protein and related biological material thereof in regulation and control of plant stress resistance ) 是由 陈明 孙黛珍 马有志 黎毛毛 张玥玮 唐文思 周永斌 徐兆师 陈隽 于 2020-05-13 设计创作，主要内容包括：本发明公开了SiMYB61蛋白及其相关生物材料在调控植物抗逆性中的应用。本发明为水稻逆境基因工程提供了新的基因,同时通过遗传转化导入粳稻优良品种,培育抗逆转基因新品种,可以有效地提高水稻的抗逆性,减少逆境胁迫对水稻生产的影响,将有重要的经济效益和社会效益。(The invention discloses an application of SiMYB61 protein and related biological materials thereof in regulation and control of plant stress resistance. The invention provides a new gene for rice stress genetic engineering, and simultaneously introduces japonica rice fine varieties through genetic transformation to culture new stress-resistant transgenic varieties, so that the stress resistance of rice can be effectively improved, the influence of stress on rice production is reduced, and important economic and social benefits are achieved.)

Use of a SiMYB61 protein or related biomaterial thereof in (a1) and/or (a2) as follows:

(a1) regulating and controlling plant yield-related traits;

(a2) regulating and controlling the stress resistance of the plants;

the related biological material is a nucleic acid molecule capable of expressing the SiMYB61 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the SiMYB61 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 4;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.4 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

2. The use of claim 1, wherein:

said plant yield-related traits comprise ear number, ear length, grain per ear number and/or biomass;

the plant stress resistance is the resistance of a plant to low nitrogen stress.

Use of a SiMYB61 protein or a related biomaterial thereof in plant breeding;

the related biological material is a nucleic acid molecule capable of expressing the SiMYB61 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the SiMYB61 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 4;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.4 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

4. Use according to claim 3, characterized in that: the breeding aims to breed plants with high yield and/or high stress resistance.

5. A method for increasing plant yield and/or increasing plant stress tolerance, comprising the step of increasing the expression level and/or activity of a SiMYB61 protein in a recipient plant.

6. A method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing a SiMYB61 protein into a recipient plant to obtain a transgenic plant with increased SiMYB61 protein expression; the transgenic plants have increased yield and/or stress resistance as compared to the recipient plant.

7. The method of claim 6, wherein: the "introducing into a recipient plant a nucleic acid molecule capable of expressing a SiMYB61 protein" is effected by introducing into the recipient plant an expression cassette comprising a gene encoding the SiMYB61 protein.

8. The method of claim 7, wherein:

the encoding gene of the SiMYB61 protein is a DNA molecule described in any one of the following items:

(B1) DNA molecule shown in SEQ ID No. 1;

(B2) DNA molecule shown in SEQ ID No. 2;

(B3) a DNA molecule shown as SEQ ID No. 3;

(B4) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) or (B2) or (B3) and encodes the SiMYB61 protein;

(B3) a DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the DNA sequence defined in (B1) or (B2) or (B3) and encoding the SiMYB61 protein.

9. The method of any of claims 5-8, wherein: the stress resistance is the resistance of a plant to low nitrogen stress.

10. Use or method according to any of claims 1-9, wherein: the plant is a dicotyledonous plant or a monocotyledonous plant;

further, the monocotyledon is a gramineous plant;

further, the gramineous plant is rice or millet.

Technical Field

The invention relates to an application of SiMYB61 protein and related biological materials thereof in regulation and control of plant stress resistance.

Background

China is one of countries with the lowest grain input and output of unit fertilizers in the world, the utilization rate of nitrogen fertilizers is only 30-35% (45% in developed countries), and the utilization rate of phosphate fertilizers is only 10-20%. According to the current consumption, for example, 10 percent of nitrogen fertilizer and 20 percent of phosphate fertilizer are saved, 241 million yuan of capital can be saved every year. The low-efficiency utilization of nitrogen and phosphorus fertilizers enables agricultural non-point source pollution to become the most important factor for water system eutrophication, soil acidification and heavy metal pollution, and threatens ecological safety and sustainable development. Therefore, our country urgently needs to cultivate new crop varieties with high nutrient utilization efficiency, thereby greatly improving the utilization efficiency of nitrogen and phosphorus fertilizers in our country.

The cultivation of new stress-resistant rice varieties improves the stress resistance of the rice varieties, and is an important measure for improving the yield level of the rice under the conditions of reduced application amount of chemical fertilizers and drought and water shortage.

Disclosure of Invention

The invention aims to provide SiMYB61 protein related to plant stress resistance, a related biological material and application thereof.

In a first aspect, the present invention first protects the use of a SiMYB61 protein or related biomaterial as follows (a1) and/or (a 2):

(a1) regulating and controlling plant yield-related traits;

(a2) regulating and controlling the stress resistance of the plants;

the related biological material is a nucleic acid molecule capable of expressing the SiMYB61 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the SiMYB61 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 4;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.4 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, etc.

The nucleic acid molecule may specifically be a gene encoding a SiMYB61 protein. The encoding gene of the SiMYB61 protein is a DNA molecule described in any one of the following items:

(B1) DNA molecule shown in SEQ ID No. 1;

(B2) DNA molecule shown in SEQ ID No. 2;

(B3) a DNA molecule shown as SEQ ID No. 3;

(B4) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) or (B2) or (B3) and encodes the SiMYB61 protein;

(B3) a DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the DNA sequence defined in (B1) or (B2) or (B3) and encoding the SiMYB61 protein.

The stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA in a mixed solution at 50 ℃ 2 × SSC, 0.1%Rinsing in SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridizing with 1mM EDTA, rinsing in 1 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 0.5 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 0.1 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridization with a mixed solution of 1mM EDTA, rinsing in 0.1 × SSC, 0.1% SDS at 65 ℃ or 6 × SSC, 0.5% SDS at 65 ℃ followed by washing once each with 2 × SSC, 0.1% SDS and 1 × SSC, 0.1% SDS.

The expression cassette can be specifically an expression cassette consisting of a ubiquitin constitutive promoter, the coding gene of the SiMYB61 protein and a terminator nos 3'. The expression cassette can be obtained by double enzyme digestion of a recombinant vector by Hind III and EcoRI. The recombinant vector can be specifically a recombinant vector obtained by cloning SEQ ID NO.3 into BamHI and SpeI sites of the vector LP 0471118-Bar-ubi-EDLL.

The recombinant strain can be obtained by introducing the expression cassette or the recombinant vector into an agrobacterium strain. The agrobacterium strain may specifically be agrobacterium strain EHA 105.

In said use, said plant yield-related traits comprise ear number, ear length, grain per ear and/or biomass; the biomass comprises the weight of rice straw and/or the weight of rice.

The plant stress resistance is the resistance of a plant to low nitrogen stress.

The modulation is a forward modulation.

In a second aspect, the invention features the use of a SiMYB61 protein or a related biomaterial thereof in plant breeding;

the related biological material is a nucleic acid molecule capable of expressing the SiMYB61 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the SiMYB61 protein is as shown above.

In the application, the breeding aims to breed plants with high yield and/or high stress resistance. The high yield can be embodied in particular as a high number of ears and/or a high ear length and/or a high number of grains per ear and/or a high biomass. The biomass comprises the weight of rice straw and/or the weight of rice. The stress tolerance is in particular resistance to low nitrogen stress. The high resistance to low nitrogen stress is reflected by high yield and/or plant height under low nitrogen stress. The high resistance to low nitrogen stress is reflected by a high nitrogen content.

In a third aspect, the present invention provides a method for increasing plant yield and/or increasing plant stress tolerance, comprising the step of increasing the expression level and/or activity of a SiMYB61 protein in a recipient plant.

The yield is specifically the ear number, ear length, grain number per ear, grain weight and/or biomass.

The biomass comprises the weight of rice straw and/or the weight of rice.

The stress tolerance is in particular resistance to low nitrogen stress.

The improved stress tolerance can be embodied as an increased yield and/or an increased plant height under low nitrogen stress.

The high resistance to low nitrogen stress is reflected by a high nitrogen content.

The SiMYB61 protein is as shown above.

In a fourth aspect, the present invention provides a method of growing a transgenic plant, comprising the steps of: introducing a nucleic acid molecule capable of expressing a SiMYB61 protein into a recipient plant to obtain a transgenic plant with increased SiMYB61 protein expression; the transgenic plants have increased yield and/or stress resistance as compared to the recipient plant.

The yield is specifically the ear number, ear length, grain number per ear, grain weight and/or biomass.

The biomass comprises the weight of rice straw and/or the weight of rice.

The stress tolerance is in particular resistance to low nitrogen stress.

The improved stress tolerance can be embodied as an increased yield and/or an increased plant height under low nitrogen stress.

The high resistance to low nitrogen stress is reflected by a high nitrogen content.

The "introducing into a recipient plant a nucleic acid molecule capable of expressing a SiMYB61 protein" is effected by introducing into the recipient plant an expression cassette comprising a gene encoding the SiMYB61 protein.

The gene encoding the SiMYB61 protein is as indicated above.

The expression cassette can be specifically an expression cassette consisting of a ubiquitin constitutive promoter, the coding gene of the SiMYB61 protein and a terminator nos 3'. The expression cassette can be obtained by double enzyme digestion of a recombinant vector by Hind III and EcoRI. The recombinant vector can be specifically a recombinant vector obtained by cloning SEQ ID NO.3 into BamHI and SpeI sites of the vector LP 0471118-Bar-ubi-EDLL.

The number of grains per spike is the number of solid grains per spike and/or the total grains per spike.

Any one of the plants is a dicotyledonous plant or a monocotyledonous plant;

further, the monocotyledon is a gramineous plant;

further, the gramineous plant is rice or millet.

The rice can be rice variety Kitaake.

The millet can be Yu Gu I.

The invention provides a new gene for rice stress genetic engineering, and simultaneously introduces japonica rice fine varieties through genetic transformation to culture new stress-resistant transgenic varieties, so that the stress resistance of rice can be effectively improved, the influence of stress on rice production is reduced, and important economic and social benefits are achieved.

Drawings

FIG. 1 shows SSR detection results of stress-resistant rice transformed with SiMYB61 gene. Marker: DL1000 Marker; negative control: kitaake; positive control: the plant expression vector psSiMYB 61.

Fig. 2 shows the statistical result of 18-year field test data.

FIG. 3 is a comparison of transgenic plants with wild type phenotype.

FIG. 4 shows the statistical results of the test data under the condition of normal field treatment for 19 years.

FIG. 5 is a statistical result of detection data under 19-year field low nitrogen stress conditions.

FIG. 6 shows the statistical results of 19 years field straw and rice weight measurement data.

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.

LP0471118-Bar-ubi-EDLL vector: are described in the literature: ning bud, Wang Shuang, ju Peng Gao, Bai Xin Xuan, Ge Lin Hao, Qixin, Jiangqin, Sun Xijun, Chenming, Sun Dazhen, over-expression millet SiANT1 has influence on rice salt tolerance [ J ]. Chinese agricultural science, 2018,51(10): 1830) 1841.

Yugu I: are described in the literature: ning bud, Wang Shuang, ju Peng lifting, Bai Xin Xuan, Ge Lin Hao, Qixin, Jiangqin, Sun Shi Jun, Chenming, Sun Dazhen, overexpression of millet SiANT1 on rice salt tolerance [ J ]. Chinese agricultural science, 2018,51(10):1830 one 1841.; the public is available from the institute of crop science, academy of agricultural sciences, china.

bar gene expression vector pSBAR: are described in the literature: obtaining drought resistant transgenic wheat [ D ] using an improved minimum expression box technique, university of inner mongolia agriculture, 2012; the public is available from the institute of crop science, academy of agricultural sciences, china.

Agrobacterium strain EHA 105: beijing Ongke New Biotechnology Co.

Rice variety Kitaake: are described in the literature: ning bud, Wang Shuang, ju Peng lifting, Bai Xin Xuan, Ge Lin Hao, Qixin, Jiangqin, Sun Shi Jun, Chenming, Sun Dazhen, overexpression of millet SiANT1 on rice salt tolerance [ J ]. Chinese agricultural science, 2018,51(10):1830 one 1841.; the public is available from the institute of crop science, academy of agricultural sciences, china.