阅读说明：本技术 SiMYB30蛋白质及其相关生物材料在调控植物耐逆性和产量中的应用 (Application of SiMYB30 protein and related biological material thereof in regulation and control of stress tolerance and yield of plants ) 是由 陈明 马有志 黎毛毛 张玥玮 唐文思 周永斌 徐兆师 陈隽 于 2020-05-12 设计创作，主要内容包括：本发明公开了SiMYB30蛋白质及其相关生物材料在调控植物耐逆性和产量中的应用。本发明通过采用最小表达框的转化方法,将仅含有启动子、目的基因SiMYB30和终止子的DNA片段侵染水稻愈伤组织,得到转SiMYB30水稻。实验证明：在低氮胁迫下,转SiMYB30水稻的产量相关性状(穗数、穗长、粒数、千粒重、稻草重、稻谷重等)及含氮量均高于野生型水稻。说明SiMYB30蛋白质具有调控植物耐逆性和产量相关性状的功能,尤其是提高植物的低氮耐性和产量,为培育耐逆和高产植物品种奠定基础。(The invention discloses application of SiMYB30 protein and related biological materials thereof in regulation and control of stress tolerance and yield of plants. According to the invention, by adopting a minimum expression frame transformation method, the rice callus is infected by the DNA fragment only containing the promoter, the target gene SiMYB30 and the terminator, so that the SiMYB 30-transgenic rice is obtained. Experiments prove that: under the low nitrogen stress, the yield-related traits (spike number, spike length, grain number, thousand grain weight, straw weight, rice grain weight and the like) and nitrogen content of SiMYB 30-transgenic rice are higher than those of wild rice. The SiMYB30 protein has the function of regulating and controlling the stress tolerance and yield-related traits of plants, particularly improves the low-nitrogen tolerance and yield of the plants, and lays a foundation for cultivating stress-tolerant and high-yield plant varieties.)

1. The application of the protein shown in A1) or A2) or A3) or A4) in regulating and controlling plant stress tolerance and/or plant yield related traits is as follows:

A1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

A2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

A3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table;

A4) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in any one of A1) -A3) and having the same function.

2. Use of a biological material related to a protein as defined in claim 1 for modulating stress tolerance and/or plant yield-related traits in a plant;

the biological material is any one of the following B1) -B10):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic cell line comprising the nucleic acid molecule of B1);

B10) a transgenic cell line comprising the expression cassette of B2).

3. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is any one of the following C1) -C4):

C1) the coding sequence is a DNA molecule shown in a sequence 1 in a sequence table;

C2) DNA molecule shown in sequence 3 in the sequence table;

C3) a DNA molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA molecule sequence defined in C1) or C2) and encoding the protein of claim 1;

C4) a DNA molecule which hybridizes under stringent conditions with a DNA molecule defined in C1) or C2) or C3) and which encodes a protein as claimed in claim 1.

4. Use according to any one of claims 1 to 3, characterized in that: the modulation is an increase.

5. Use of a protein according to claim 1 or a biological material according to claim 2 or 3 for the cultivation of transgenic plants with increased stress tolerance and/or yield;

or, use of the protein of claim 1 or the biological material of claim 2 or 3 in plant breeding.

6. Use according to any one of claims 1 to 5, characterized in that: the stress tolerance is low nitrogen tolerance;

or, said plant yield-related traits comprise dry grain weight and/or dry straw weight and/or ear number and/or ear length and/or number of grains per ear and/or total grains per ear and/or thousand grain weight and/or rice weight and/or straw weight;

alternatively, the breeding is aimed at breeding plants with high stress tolerance and/or high yield.

7. A method for producing a transgenic plant with improved stress tolerance and/or yield, comprising the steps of increasing the content and/or activity of the protein of claim 1 in a recipient plant to obtain a transgenic plant; the transgenic plant has higher stress tolerance and/or higher yield than the recipient plant.

8. The method of claim 7, wherein: the method for increasing the content and/or activity of the protein of claim 1 in a recipient plant comprises overexpressing the protein of claim 1 in the recipient plant;

alternatively, the overexpression method is to introduce a gene encoding the protein of claim 1 into a recipient plant.

9. The method according to claim 7 or 8, characterized in that: the stress tolerance is low nitrogen tolerance;

or, the transgenic plant has higher stress tolerance and/or higher yield than the recipient plant in any one of the following Y1) -Y11):

y1) the transgenic plant has a higher dry grain weight than the recipient plant;

y2) the transgenic plant has a higher straw dry weight than the recipient plant;

y3) the transgenic plant has more ears than the recipient plant;

y4) the spike length of the transgenic plant is longer than that of the recipient plant;

y5) the transgenic plant has more kernels per ear than the recipient plant;

y6) the transgenic plant has more total grains per ear than the recipient plant;

y7) the transgenic plant has a thousand kernel weight higher than that of the recipient plant;

y8) the transgenic plant has a higher straw weight than the recipient plant;

y9) the transgenic plant has a higher rice weight than the recipient plant;

y10) the transgenic plant has a higher content of nitrogen in rice than the recipient plant;

y11) the transgenic plant has a higher nitrogen content in the straw than in the recipient plant.

10. The use according to any one of claims 1 to 6 or the method according to any one of claims 7 to 9, wherein: the plant is a monocotyledon or a dicotyledon.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an application of SiMYB30 protein and related biomaterials in regulation and control of stress tolerance and yield of plants.

Background

Adversity stress is a barrier factor affecting plant growth and development. Under the stress of adversity, a series of response reactions are generated in plants, and a plurality of physiological, biochemical and developmental changes are accompanied. The reaction mechanism of the plant to the stress is determined, and scientific data is provided for the research and application of the stress-resistant gene engineering. At present, the research on plant stress resistance has been advanced to the cellular and molecular level, and combined with the research on genetics and genetic engineering, the research on improving the growth characteristics of plants by biotechnology is aimed at improving the adaptability of plants to stress.

Nitrogen is a mineral nutrient element with the largest demand in plant growth and development, and the nitrogen content in plant dry matter and plant total nitrogen is 1.5% -2% and 16%, respectively. Elemental nitrogen plays a very important role in crop growth, being a constituent of amino acids in the plant body, being a constituent of proteins, and also being a constituent of chlorophyll, which is decisive for photosynthesis in plants.

In the second half of the 20 th century, in order to improve the productivity of crops, the requirements of main high-yield crops on nitrogen fertilizer and other nutrients are greatly improved, and the global nitrogen fertilizer consumption is improved by ten times. However, the absorption and utilization of nitrogen fertilizer by plants are far less than half of the application amount of the plants, and most of the nitrogen fertilizer is N₂And NH₃The volatilization of gases, nitrification, denitrification, leaching and the like are wasted. Moreover, nitrogen fertilizer is now the most important cost for crop production, and to a greater extentAnd influence the income of farmers. Therefore, how to make the plants use the nitrogen fertilizer more efficiently becomes a problem to be solved urgently.

Disclosure of Invention

The invention aims to provide application of SiMYB30 protein and related biological materials thereof in regulation and control of plant stress tolerance and/or plant yield related traits.

In order to achieve the above object, the present invention firstly provides a novel use of the SimYB30 protein.

The invention provides an application of SiMYB30 protein in regulation and control of plant stress tolerance and/or plant yield related traits;

the SiMYB30 protein is derived from millet (Setaria italica) and is any one of the following proteins A1) or A2) or A3) or A4):

A1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

A2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

A3) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table;

A4) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in any one of A1) -A3) and having the same function.

Wherein, the sequence 2 in the sequence table is composed of 318 amino acid residues.

The labels are specifically shown in table 1.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL
HA	9	YPYDVPDYA

The protein represented by any one of A1) -A4) above may be artificially synthesized, or may be obtained by synthesizing the encoding gene and then performing biological expression.

In order to achieve the above object, the present invention also provides a novel use of a biomaterial related to a SiMYB30 protein.

The invention provides application of biological materials related to SiMYB30 protein in regulation and control of plant stress tolerance and/or plant yield related traits;

the biological material is any one of the following B1) -B10):

B1) a nucleic acid molecule encoding a SiMYB30 protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic cell line comprising the nucleic acid molecule of B1);

B10) a transgenic cell line comprising the expression cassette of B2).

In the above application, the nucleic acid molecule of B1) is any one of the following C1) -C4):

C1) DNA molecule shown in sequence 1 in the sequence table;

C2) DNA molecule shown in sequence 3 in the sequence table;

C3) a DNA molecule having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA molecule sequence defined in C1) or C2) and encoding a SiMYB30 protein;

C4) a DNA molecule which hybridizes under stringent conditions with a DNA molecule defined in C1) or C2) or C3) and which encodes a SiMYB30 protein.

Wherein, the sequence 1 in the sequence table is composed of 957 nucleotides.

The nucleotide sequence encoding the SiMYB30 protein of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides that have been artificially modified to have 75% or greater identity to the nucleotide sequence encoding the SiMYB30 protein are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode the SiMYB30 protein and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 of the present invention. 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 the identity between related sequences.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the stringent conditions are hybridization and washing of the membrane at 68 ℃ for 2 times, 5min each, in a solution of 2 × SSC, 0.1% SDS, and hybridization and washing of the membrane at 68 ℃ for 2 times, 15min each, in a solution of 0.5 × SSC, 0.1% SDS, or at 65 ℃ in a solution of 0.1 × SSPE (or 0.1 × SSC), 0.1% SDS.

In the application, the expression cassette sequentially consists of a promoter, the coding gene of the SiMYB30 protein and a terminator. In a specific embodiment of the invention, the expression cassette consists of a constitutive promoter of ubiquitin, the gene encoding the above-mentioned SiMYB30 protein, and the terminator nos 3' in that order.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

The recombinant vector is constructed by inserting the nucleic acid molecule into an expression vector to obtain the recombinant vector for expressing the protein, any enhanced, constitutive, tissue-specific or inducible promoter can be added before the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters, furthermore, when the nucleic acid molecule is used for constructing the recombinant expression vector, an enhancer including a translation enhancer or a transcription enhancer can be used, and the enhancer region can be an ATG initiation codon or initiation codon of a contiguous region, but is required to be identical to the reading frame of the coding sequence to ensure the correct translation of the whole sequence, the translation control signal and the initiation codon can be widely derived, either naturally or synthetically.

In the above application, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium. The recombinant microorganism is a microorganism containing the expression cassette or the recombinant vector. In a specific embodiment of the present invention, the recombinant microorganism is agrobacterium EHA105 containing the above expression cassette.

The invention also provides application of the SiMYB30 protein or the biological material in cultivating transgenic plants with improved stress tolerance and/or yield.

The invention also provides application of the SiMYB30 protein or the biological material in plant breeding. The breeding aims to breed plants with high stress tolerance and/or high yield.

Further, the modulation is an increase.

Further, the stress tolerance is low nitrogen tolerance.

The regulation and control of the plant stress tolerance are specifically embodied in that: when the content and/or activity of a SiMYB30 protein in a plant is reduced, the plant's low nitrogen tolerance is reduced; when the content and/or activity of a SiMYB30 protein is increased in a plant, the plant has increased low nitrogen tolerance.

Said plant yield-related traits comprise dry grain weight and/or dry straw weight and/or number of ears and/or ear length and/or number of grains per ear and/or total number of grains per ear and/or thousand grain weight and/or rice weight and/or straw weight.

The plant yield-related traits are specifically embodied as follows: when the content and/or activity of the SimYB30 protein in a plant is reduced, the dry grain weight and/or the dry straw weight and/or the number of ears and/or the ear length and/or the number of grains per ear and/or the total number of grains per ear and/or the thousand-grain weight and/or the rice weight and/or the straw weight of the plant is reduced; when the content and/or activity of the SiMYB30 protein in a plant is increased, the dry grain weight and/or the dry straw weight and/or the number of ears and/or the ear length and/or the number of grains per ear and/or the total number of grains per ear and/or the thousand-grain weight and/or the rice weight and/or the straw weight of the plant is increased.

To achieve the above object, the present invention finally provides a method for breeding transgenic plants with improved stress tolerance and/or yield.

The method for cultivating the transgenic plant with improved stress tolerance and/or yield comprises the steps of improving the content and/or activity of SiMYB30 protein in a receptor plant to obtain a transgenic plant; the transgenic plant has higher stress tolerance and/or higher yield than the recipient plant.

Further, the method of increasing the content and/or activity of a SiMYB30 protein in a recipient plant is by overexpressing a SiMYB30 protein in the recipient plant. The overexpression method is to introduce a gene coding for a SimYB30 protein into a recipient plant.

Further, the nucleotide sequence of the coding gene of the SiMYB30 protein is any one of C1) -C4). In a particular embodiment of the invention, the gene encoding the SiMYB30 protein is introduced into the recipient plant via an expression cassette as described above.

The stress tolerance is low nitrogen tolerance.

The transgenic plant has higher stress tolerance and/or yield than the acceptor plant and is embodied in any one of the following Y1) -Y11):

y1) the transgenic plant has a higher dry grain weight than the recipient plant;

y2) the transgenic plant has a higher straw dry weight than the recipient plant;

y3) the transgenic plant has more ears than the recipient plant;

y4) the spike length of the transgenic plant is longer than that of the recipient plant;

y5) the transgenic plant has more kernels per ear than the recipient plant;

y6) the transgenic plant has more total grains per ear than the recipient plant;

y7) the transgenic plant has a thousand kernel weight higher than that of the recipient plant;

y8) the transgenic plant has a higher straw weight than the recipient plant;

y9) the transgenic plant has a higher rice weight than the recipient plant;

y10) the transgenic plant has a higher content of nitrogen in rice than the recipient plant;

y11) the transgenic plant has a higher nitrogen content in the straw than in the recipient plant.

In any of the above uses or methods, the plant may be a monocot or a dicot. Further, the monocotyledon may be a gramineae. Further, the gramineous plant is specifically rice (e.g., rice variety Kitaake) or millet (e.g., millet variety yu-gu No.).

The invention provides SiMYB30 protein related to plant stress tolerance and yield traits, and rice callus is infected by a DNA fragment only containing a promoter, a target gene SiMYB30 and a terminator by adopting a transformation method of a minimum expression frame (no vector skeleton sequence exists in the transformation fragment, so that the safety risk possibly brought by the vector skeleton sequence is reduced to the maximum extent), and then the transformed SiMYB30 rice is obtained. Experiments prove that: under the low nitrogen stress, the yield-related traits (spike number, spike length, grain number, thousand grain weight, straw weight, rice grain weight and the like) and nitrogen content of SiMYB 30-transgenic rice are higher than those of wild rice. The SiMYB30 protein has the function of regulating and controlling the stress tolerance and yield-related traits of plants, particularly improves the low-nitrogen tolerance and yield of the plants, and lays a foundation for cultivating stress-tolerant and high-yield plant varieties.

Drawings

FIG. 1 shows the molecular identification of SiMYB 30-transgenic rice, wherein the Marker is D L1000 Marker, the negative control is Kitaake, and the positive control is SiMYB30 plasmid, wherein the P is less than 0.05, indicating that the difference is obvious.

Fig. 2 is 2018 field data arrangement. Wherein CK is wild rice Kitaake; OE24 was SiMYB30 transgenic rice.

FIG. 3 shows the low nitrogen stress identification result of SiMYB30 rice transferred in Jiangxi in 2019. Figure 3A is a field phenotype for the low nitrogen treated group (no nitrogen fertilizer applied group). FIG. 3B is a phenotypic graph of 3 strains of each material taken under low nitrogen treatment (no nitrogen fertilizer application group) and normal treatment (nitrogen fertilizer application group). Wherein, the receptor (Kitaake) is wild rice Kitaake; m29893/24 is SiMYB30 transgenic rice.

FIG. 4 shows the results of the test data of 2019. FIG. 4A shows the results of the test data of the normal treatment group (nitrogen fertilizer application group). FIG. 4B shows the results of the test data of the low nitrogen treatment group (nitrogen fertilizer non-application group). Wherein, WT is wild rice Kitaake; OE24 was SiMYB30 transgenic rice.

FIG. 5 shows the results of 2019 measurements of biological yields. Wherein, WT is wild rice Kitaake; OE24 was SiMYB30 transgenic rice.

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.

L P0471118-Bar-ubi-ED LL expression vector in the following examples is described in the literature, Ning bud, Wan Shuang Shi, Ju Peng lifting, Bai Xin Xuan, Ge Lin Hao, Qi Xin, Jiangqi, Sun Xijun, Cheng Ming, Sun Dai Zhen, over-expressed millet SiANT1 has influence on the salt tolerance of rice [ J ] China agricultural science, 2018,51(10): 1830-.

The marker gene bar expression vector pSBAR in the following examples is described in the literature: threepo, drought-resistant transgenic wheat [ D ] obtained by using an improved minimum expression cassette technology, university of inner mongolia agriculture, 2012, the public can be obtained from the institute of crop science of the Chinese academy of agricultural sciences, and the biomaterial is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.