阅读说明：本技术 SiTGAL6蛋白质的新用途 (Novel application of SiTGAL6 protein ) 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 2020-05-14 设计创作，主要内容包括：本发明公开了SiTGAL6蛋白质的新用途。本发明公开了SiTGAL6蛋白质在调控植物耐逆性和产量相关性状中的应用。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SiTGAL6和终止子的DNA片段侵染水稻愈伤组织(转化片段中没有任何载体骨架序列,最大程度减少了由于载体骨架序列可能带来的安全风险),得到转SiTGAL6水稻。实验证明：在低氮胁迫下,转SiTGAL6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SiTGAL6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 a new application of SiTGAL6 protein. The invention discloses application of SiTGAL6 protein in regulation and control of plant stress tolerance and yield-related traits. By adopting a minimum expression frame transformation method, the rice callus is infected by the DNA fragment only containing the promoter, the target gene SiTGAL6 and the terminator (the transformation fragment does not have any vector skeleton sequence, so that the safety risk possibly brought by the vector skeleton sequence is reduced to the maximum extent), and the SiTGAL6 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 the SiTGAL 6-transformed rice are higher than those of wild rice. The SiTGAL6 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 ear number and/or ear length and/or kernel per ear and/or total kernel per ear and/or thousand kernel 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) -Y10):

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

y2) the transgenic plant has more ears than the recipient plant;

y3) the spike length of the transgenic plant is longer than that of the recipient plant;

y4) the transgenic plant has more kernels per ear than the recipient plant;

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

y6) the transgenic plant has a thousand kernel weight higher than that of the recipient plant;

y7) the transgenic plant has a higher straw weight than the recipient plant;

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

y9) the transgenic plant has a higher content of nitrogen in rice than the recipient plant;

y10) 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 a new application of SiTGAL6 protein.

Background

With the rapid growth of the world population, food supply is becoming an increasingly serious global problem. In recent years in China, with the rapid growth of industrial land and residential land, the agricultural land area is rapidly reduced. In addition, with the improvement of life of people, the occupation ratio of non-food production agricultural land in the total agricultural land is increased day by day, which further influences the self-sufficiency of domestic food and aggravates the domestic food safety problem.

Rice is one of the most important crops in the world and is also one of the main grains in China. The rice yield of the rice is about 50 percent of the total amount of the commercial grains in China, so the key influence of the rice yield on the grain yield in China is caused. Effectively improving the rice yield has important significance for improving the total grain yield in China and ensuring the national grain safety.

Disclosure of Invention

The invention aims to provide application of SiTGAL6 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 sital 6 protein.

The invention provides application of SiTGAL6 protein in regulation and control of plant stress tolerance and/or plant yield related traits;

the SiTGAL6 protein is derived from millet (Setaria italica) and is a protein represented by A1) or A2) or A3) or A4) 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.

Wherein, the sequence 2 in the sequence table is composed of 401 amino acid residues.

The labels are specifically shown in table 1.

TABLE 1 sequences of tags

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 the sital 6 protein.

The invention provides application of biological materials related to SiTGAL6 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 sital 6 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 by C1) or C2) and encoding a sital 6 protein;

C4) a DNA molecule which hybridizes with the DNA molecule defined by C1) or C2) or C3) under stringent conditions and encodes the SiTGAL6 protein.

Wherein, the sequence 1 in the sequence table is composed of 1206 nucleotides.

The nucleotide sequence encoding the SiTGAL6 protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence encoding the sital 6 protein are derived from and identical to the nucleotide sequence of the present invention as long as they encode the sital 6 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 application, the stringent conditions are hybridization and membrane washing 2 times at 68 ℃ for 5min in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing 2 times at 68 ℃ for 15min in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In the application, the expression cassette sequentially comprises a promoter, the encoding gene of the SiTGAL6 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 sital 6 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 obtained by inserting the nucleic acid molecule into an expression vector to express the protein. When the nucleic acid molecule is used for constructing a recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added in front of the transcription initiation nucleotide, and can be used alone or combined with other plant promoters; in addition, when recombinant expression vectors are constructed using the nucleic acid molecules, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or adjacent regions initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene. In a specific embodiment of the invention, the recombinant vector is obtained by cloning the SiTGAL6 gene fragment shown in sequence 1 into the SpeI and BamH I enzyme cutting sites of the vector LP 0471118-Bar-ubi-EDLL.

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 SiTGAL6 protein or the biological material in cultivating transgenic plants with improved stress tolerance and/or yield.

The invention also provides application of the SiTGAL6 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 the SiTGAL6 protein in a plant is reduced, the low nitrogen tolerance of the plant is reduced; when the content and/or activity of the SiTGAL6 protein in the plant is increased, the low nitrogen tolerance of the plant is increased.

Said plant yield-related traits comprise dry grain weight and/or ear number and/or ear length and/or kernel number per ear and/or total kernel number per ear and/or thousand kernel 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 SiTGAL6 protein in a plant is reduced, the dry grain 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 sital 6 protein in a plant is increased, the dry grain 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 increasing the content and/or activity of SiTGAL6 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 for increasing the content and/or activity of the sital 6 protein in the recipient plant is to overexpress the sital 6 protein in the recipient plant. The overexpression method is to introduce a gene encoding the SiTGAL6 protein into a recipient plant.

Further, the nucleotide sequence of the encoding gene of the SiTGAL6 protein is any one of the C1) -C4). In a specific embodiment of the invention, the gene encoding the sital 6 protein is introduced into the recipient plant via the expression cassette 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) -Y10):

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

y2) the transgenic plant has more ears than the recipient plant;

y3) the spike length of the transgenic plant is longer than that of the recipient plant;

y4) the transgenic plant has more kernels per ear than the recipient plant;

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

y6) the transgenic plant has a thousand kernel weight higher than that of the recipient plant;

y7) the transgenic plant has a higher straw weight than the recipient plant;

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

y9) the transgenic plant has a higher content of nitrogen in rice than the recipient plant;

y10) 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 SiTGAL6 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 SiTGAL6 and a terminator (no vector skeleton sequence exists in a transformation fragment, so that safety risk possibly brought by the vector skeleton sequence is reduced to the maximum extent) by adopting a transformation method of a minimum expression frame to obtain the SiTGAL6 transformed rice. 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 the SiTGAL 6-transformed rice are higher than those of wild rice. The SiTGAL6 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 molecular characterization of rice transgenic for SiTGAL 6. Note: marker: DL1000 Marker; negative control: kitaake; positive control: the SiTGAL6 plasmid.

Fig. 2 is 2018 field data arrangement. Wherein CK is wild rice Kitaake; OE2, OE7 and OE17 are trans-sital 6 rice.

FIG. 3 shows the identification result of low nitrogen stress of SiTGAL6 rice transferred from Jiangxi in 2019. Wherein, the receptor (Kitaake) is wild rice Kitaake; m23193/17 is SiTGAL6 transformed 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; OE7 and OE17 are trans-sital 6 rice.

FIG. 5 shows the results of 2019 measurements of biological yields. Wherein, WT is wild rice Kitaake; OE7 and OE17 are trans-sital 6 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.

The LP 0471118-Bar-ubi-EDLL expression vector in the following examples is described in the literature: ning bud, Wang Shuang, ju Peng Gao, Bai Xin Xuan, Ge Lin Hao, Qixin, Jiangqin, Sun Su Jun, Chenming, Sun Dazhen, over-expressed millet SiANT1 has influence on rice salt tolerance [ J ] in Chinese agricultural science, 2018,51(10): 1830) 1841, the public can obtain from the institute of crop science of Chinese academy of agricultural sciences, and the biological material is only used for repeating the related experiments of the invention, and can not be used for other purposes.

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.