Use of protein derived from wheat and biomaterial related thereto in increasing yield per plant and grain protein content of crop

Abstract

Provided is the use of a protein derived from wheat and a biomaterial related thereto in increasing the yield per plant and grain protein content of a crop. The protein is designated as SWEET11, and may be a protein having an amino acid sequence as shown in sequence 9, sequence 6 or sequence 3 in the sequence listing. The wheat SWEET11 protein (TaSWEET11) is over-expressed in the Zhengmai 7698 wheat variety (nationally approved), and it is found that the over-expression of TaSWEET11 can significantly improve the thousand-grain weight of wheat grains and increase the yield. In addition, the over-expression of TaSWEET11 also increases the protein content in the wheat grain, significantly improving the grain quality while increasing the yield. The TaSWEET11 protein can be used for breeding and quality improvement of crops.

Description

Application of wheat-derived proteins and related biomaterials in increasing crop yield per plant and grain protein content Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the application of wheat-derived protein and related biological materials in increasing crop yield per plant and grain protein content. Background Art

[0002] Wheat is one of the world's most important food crops. With the continuous growth of the global population, the continuous reduction of arable land, and the frequent occurrence of extreme weather events, biotic and abiotic stresses on wheat are intensifying throughout its growth cycle, placing tremendous pressure on increasing wheat yield. With the continuous advancement of technology, effective improvement of crop varieties has achieved significant increases in yield. However, in recent years, yield increases have reached a certain bottleneck, necessitating the exploration of new strategies and approaches to increase wheat yield. Wheat yield is composed of three factors: the number of ears per unit area, the number of kernels per ear, and 1000-kernel weight. Therefore, increasing 1000-kernel weight is crucial for increasing yield. Grain filling is a key process in determining wheat grain yield. During grain filling, carbon source produced by the photosynthetic organs is transported long distances through the phloem to the grain, where it is synthesized into storage substances such as starch in the endosperm. Currently, the mechanisms that control grain filling in wheat are not fully understood. Understanding the regulatory mechanisms of grain development is crucial for achieving high-yield wheat breeding.

[0003] SUMMARY OF THE INVENTION

[0004] The technical problem to be solved by the present invention is how to increase the spike length, number of spikelets and / or number of grains per spike of the plant and / or how to increase the yield of wheat and enhance the protein content of the grains and / or how to increase the grain length, grain width and / or thousand-grain weight of the plant grains and / or how to increase the single-plant yield of the plant.

[0005] In order to solve the above technical problems, the present invention first provides any of the following applications of a protein or a substance for regulating protein expression or a substance for regulating protein activity:

[0006] P1. Application in regulating the grain length, grain width and / or thousand-grain weight of plant seeds.

[0007] P2. Application in increasing the grain length, grain width and / or thousand-grain weight of plant seeds.

[0008] P3. Application in regulating plant spike length, spikelet number and / or grain number per spike,

[0009] P4. Application in increasing the spike length, spikelet number and / or grain number of a plant.

[0010] P5. Application in regulating the yield of individual plants.

[0011] P6. Application in increasing the yield of individual plants.

[0012] P7. Application in regulating protein content in plant seeds,

[0013] P8. Application in increasing protein content of plant seeds,

[0014] P9. Application in plant high-yield breeding and / or high grain protein content breeding,

[0015] P10. Application in improving plant quality with high yield and / or high grain protein content.

[0016] The protein may be the following protein:

[0017] A1) The amino acid sequence is the protein of sequence 9 in the sequence listing;

[0018] A2) The amino acid sequence is the protein of sequence 3 in the sequence listing;

[0019] A3) The amino acid sequence is the protein of sequence 6 in the sequence listing;

[0020] A4) a protein derived from the amino acid sequence of A1), A2) or A3) by substitution and / or deletion and / or addition of amino acid residues, or a protein having 80% or more identity with the protein of A1), A2) or A3) and having the same function;

[0021] A5) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1), A2) or A3).

[0022] In the above application, the protein may be derived from wheat.

[0023] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0024] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0025] In the above-mentioned proteins, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, blastp can be used as the program, with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0026] In the above proteins, the above 80% or greater identity may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0027] In the above application, the plant may be any of the following:

[0028] D1) Dicotyledons;

[0029] D2) Monocotyledons,

[0030] D3) Gramineae,

[0031] D4) Grasses,

[0032] D5) Triticum plants,

[0033] D6) Wheat.

[0034] In order to solve the above technical problems, the present invention further provides any of the following applications of the biomaterial related to the protein described above:

[0035] Q1. Application in regulating the grain length, grain width and / or thousand-grain weight of plant seeds.

[0036] Q2. Application in increasing the grain length, grain width and / or thousand-grain weight of plant seeds.

[0037] Q3. Application in regulating plant spike length, spikelet number and / or grain number per spike,

[0038] Q4. Application in increasing the spike length, spikelet number and / or grain number of a plant.

[0039] Q5. Application in regulating the yield of individual plants.

[0040] Q6. Application in increasing the yield of single plant,

[0041] Q7. Application in regulating protein content in plant seeds.

[0042] Q8. Application in increasing protein content of plant seeds,

[0043] Q9. Application in plant high-yield breeding and / or high grain protein content breeding,

[0044] Q10. Application in improving plant quality with high yield and / or high grain protein content,

[0045] The biological material may be any of the following:

[0046] B1) a nucleic acid molecule encoding the protein according to claim 1;

[0047] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0048] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0049] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0050] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2);

[0051] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

[0052] B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2);

[0053] B8) a nucleic acid molecule that promotes or increases the gene expression of the protein according to claim 1;

[0054] B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

[0055] In the above application, the nucleic acid molecule in B1) may be the gene encoding the protein shown in b1), b2) or b3) below:

[0056] b1) The coding sequence of the coding strand is a cDNA molecule or DNA molecule of Sequence 8, Sequence 2, and Sequence 5 in the sequence listing;

[0057] b2) The nucleotides are DNA molecules of sequence 7, sequence 1 and sequence 4 in the sequence list,

[0058] b3) A cDNA molecule or DNA molecule that hybridizes with the cDNA or DNA molecule defined in b2) and encodes a protein having the same function.

[0059] In the above application, the plant may be any of the following:

[0060] D1) Dicotyledons;

[0061] D2) Monocotyledons,

[0062] D3) Gramineae,

[0063] D4) Grasses,

[0064] D5) Triticum plants,

[0065] D6) Wheat.

[0066] In the aforementioned biological material, the expression cassette containing the nucleic acid molecule described in B2) refers to DNA capable of expressing the protein described in the aforementioned application in a host cell. This DNA may include not only a promoter for initiating transcription of the protein-encoding gene, but also a terminator for terminating transcription of the protein-encoding gene. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0067] Available existing plant expression vector construction contains the recombinant expression vector of described protein encoding gene expression cassette.Described plant expression vector comprises binary agrobacterium vector and the carrier that can be used for plant microprojectile bombardment etc.Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc.Described plant expression vector can also comprise the 3 ' end non-translated region of foreign gene, promptly comprise polyadenylic acid signal and any other DNA fragment that participates in mRNA processing or gene expression.Described polyadenylic acid signal can guide polyadenylic acid to join the 3 ' end of mRNA precursor, and the non-translated region of transcribed as agrobacterium crown gall induction (Ti) plasmid gene (such as nopaline synthase gene Nos) and plant gene (such as soybean storage protein gene) 3 ' end all has similar function. When using the gene of the present invention to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. These enhancer regions can be the ATG start codon or the start codon of the adjacent region, etc., but must be consistent with the reading frame of the coding sequence to ensure the correct translation of the entire sequence.

[0068] In the above-mentioned biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

[0069] In order to solve the above technical problems, the present invention also provides a method for increasing the grain length, grain width, thousand-grain weight, number of grains per ear, yield per plant and grain protein content of plants, comprising enhancing or increasing the activity of the protein mentioned above or / and the expression level of the gene encoding the protein mentioned above in the target plant, thereby increasing the grain length, grain width, thousand-grain weight, number of grains per ear, yield per plant and grain protein content of the target plant.

[0070] In the above method, the enhancement or increase of the activity of the above-mentioned protein or / and the expression level of the gene encoding the above-mentioned protein in the target plant is achieved by introducing the gene encoding the above-mentioned protein into the target plant.

[0071] In the above method, the gene encoding the protein can be modified as follows before being introduced into the target plant to achieve better expression effect:

[0072] 1) Connected to various plant-expressed promoters to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the temporal and spatial requirements of expression and also depends on the target species; for example, a tissue- or organ-specific expression promoter, depending on the stage of development at which the receptor is required; although many promoters derived from dicots have been shown to function in monocots and vice versa, ideally, dicot promoters are selected for expression in dicots and monocot promoters are selected for expression in monocots;

[0073] 2) Linking to a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml from CaMV, E9 from rbcS; any available terminator known to function in plants can be linked to the gene of the present invention;

[0074] 3) Introducing enhancer sequences, such as intron sequences (e.g., from Adhl and bronze) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0075] The present invention also provides a method for producing transgenic plants, which includes enhancing or increasing the activity of the protein or / and the expression level of the gene encoding the protein in the target plant to obtain the transgenic plant. Compared with the target plant, the transgenic plant has increased grain length, grain width, thousand-grain weight, number of grains per ear, single plant yield and / or grain protein content.

[0076] In the above method, the comparison is performed under comparable conditions.

[0077] The comparable conditions refer to the same or similar environmental conditions. Environmental conditions include, for example, light, temperature, water, humidity, soil, and nutrients (e.g., nitrogen and phosphorus). The transgenic plant contains a cDNA gene encoding the protein. The cDNA gene is a cDNA molecule that includes the coding sequence (CDS) of the protein. The coding sequence can be Sequence 8, Sequence 2, and / or Sequence 5 in the sequence listing.

[0078] The target plant may contain the protein and the cDNA gene of the protein. The target plant may contain an expression cassette. An expression cassette refers to a DNA capable of expressing the protein in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary for expressing the nucleic acid molecule of the protein. The regulatory sequence can guide the coding sequence to express the protein in a suitable host cell under compatible conditions. The regulatory sequence includes, but is not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequence must include a promoter and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites into the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulatory sequence with a linker may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence can be operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of an mRNA that is important for translation in the host cell. The leader sequence can be operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of the protein that directs the encoded protein into the cell's secretory pathway. Any signal peptide coding region that directs the expressed protein into the secretory pathway of the selected host cell can be used in the present invention. It may also be desirable to add regulatory sequences that can regulate protein expression based on the growth conditions of the host cell. Examples of regulatory sequences are those that can turn gene expression on or off in response to chemical or physical stimuli (including in the presence of regulatory compounds). Other examples of regulatory sequences are those that enable gene amplification. In these examples, the nucleic acid sequence encoding the protein should be operably linked to the regulatory sequence. In one embodiment of the present invention, the promoter can be the strong constitutive promoter Ubi from corn.

[0079] In the above method, the plant sensitive to adverse stress may be a transgenic plant or a plant obtained by conventional breeding techniques such as hybridization.

[0080] In the above methods, the transgenic plants are understood to include not only first- and second-generation transgenic plants, but also their progeny. Transgenic plants can be propagated within their species or transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. Transgenic plants include seeds, callus, whole plants, and cells.

[0081] In the above method, the plant and / or target plant may be any of the following:

[0082] D1) Dicotyledons;

[0083] D2) Monocotyledons,

[0084] D3) Gramineae,

[0085] D4) Grasses,

[0086] D5) Triticum plants,

[0087] D6) Wheat.

[0088] The above-mentioned proteins and / or the above-mentioned biological materials also fall within the scope of protection of the present invention.

[0089] With the global population increasing, increasing wheat yield remains a pressing goal in wheat breeding. Over the past few decades, 1000-grain weight, number of grains per spike, and number of tillers have been of great concern to breeders. Studies on increasing wheat yield by genetically modifying the expression characteristics of grain weight genes have been widely reported. Sugar transport is a prerequisite for starch synthesis in the developing endosperm of grains, and efficient sucrose transport is the material basis for grain filling. Loss of sugar transporter function can lead to defective grain filling and the inability to form a functional endosperm, significantly reducing grain yield.

[0090] The present invention significantly reduces plant yield by knocking out the gene for the wheat SWEET11 protein through gene editing. Further overexpression of the gene has been found to increase the 1000-grain weight, protein content, and yield per plant of the overexpressed material. This invention, through the use of genetic engineering techniques and methods, has important theoretical and practical significance for increasing wheat yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 shows a phenotypic comparison of the wild-type Fielder (WT) and its TaSWEET11 gene-edited (KO) materials, including plant height, ear length, single plant yield, and grain morphology. A shows the comparison of plant height between the wild-type and TaSWEET11 gene-edited materials, with plant height as the ordinate. B shows the comparison of spikelet length between the wild-type and TaSWEET11 gene-edited materials, with spikelet length as the ordinate. C shows the comparison of spikelet number between the wild-type and TaSWEET11 gene-edited materials, with spikelet number per plant as the ordinate. D shows the comparison of grain number per spikelet between the wild-type and TaSWEET11 gene-edited materials, with grain number per spikelet as the ordinate. E shows the comparison of 1000-grain weight between the wild-type and TaSWEET11 gene-edited materials, with 1000-grain weight as the ordinate. F shows the comparison of grain length between the wild-type and TaSWEET11 gene-edited materials, with grain length as the ordinate. G shows the comparison of grain width between the wild-type and TaSWEET11 gene-edited materials, with grain width as the ordinate. H shows the comparison of yield per plant between the wild-type and TaSWEET11 gene-edited materials, with yield per plant as the ordinate. I shows a photograph comparing grain length, grain width, and grain morphology between the wild-type and TaSWEET11 gene-edited materials.

[0092] Figure 2 shows a phenotypic comparison of plant height, ear length, and yield per plant for ZM7698 and its TaSWEET11 overexpressing OE materials. A shows a comparison of plant height for ZM7698 and its TaSWEET11 overexpressing OE materials, with the vertical axis representing plant height; B shows a comparison of ear length for ZM7698 and its TaSWEET11 overexpressing OE materials, with the vertical axis representing ear length; C shows a comparison of spikelet number for ZM7698 and its TaSWEET11 overexpressing OE materials, with the vertical axis representing the number of spikelets per plant; D shows a comparison of grain number per spike for ZM7698 and its TaSWEET11 overexpressing OE materials, with the vertical axis representing the number of grains per spike. E shows a comparison of grain length between ZM7698 and its TaSWEET11-overexpressing OE ancestry, with grain length plotted on the ordinate. F shows a comparison of grain width between ZM7698 and its TaSWEET11-overexpressing OE ancestry, with grain width plotted on the ordinate. G shows a comparison of 1000-grain weight between ZM7698 and its TaSWEET11-overexpressing OE ancestry, with 1000-grain weight plotted on the ordinate. H shows a comparison of yield per plant between ZM7698 and its TaSWEET11-overexpressing OE ancestry, with yield per plant plotted on the ordinate. I shows a photograph comparing grain length and width between ZM7698 and its TaSWEET11-overexpressing OE ancestry.

[0093] Figure 3 shows a comparison of grain protein content in wild-type wheat ZM7698 and TaSWEET11 overexpressing OE materials. The vertical axis represents grain protein content (%).

[0094] Figure 4 shows the PCR amplification and identification of the SWEET11 gene in wild-type wheat ZM7698 and TaSWEET11 overexpressing OE materials.

[0095] Figure 5 shows the expression level detection of gene TaSWEET11 in wild-type wheat ZM7698 and TaSWEET11 overexpressing OE materials. Modes for Carrying Out the Invention

[0096] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0097] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0098] Example 1. CRISPR target search and vector construction

[0099] 1. Target and Primer Design

[0100] Wheat SWEET11 protein includes three homologous proteins TaSWEET11-7A, TaSWEET11-7B and TaSWEET11-7D, which are distributed in the three subchromosome groups of wheat A, B and D, respectively. The genomic nucleotide sequence of the wheat TaSWEET11-7A gene is shown in sequence 1 in the sequence listing, the CDS sequence of the TaSWEET11-7A gene is shown in sequence 2 in the sequence listing, and the amino acid sequence of the TaSWEET11-7A protein is shown in sequence 3 in the sequence listing; the genomic nucleotide sequence of the wheat TaSWEET11-7B gene is shown in sequence 4 in the sequence listing, the CDS sequence of the TaSWEET11-7B gene is shown in sequence 5 in the sequence listing, and the amino acid sequence of the TaSWEET11-7B protein is shown in sequence 6 in the sequence listing; the genomic nucleotide sequence of the wheat TaSWEET11-7D gene is shown in sequence 7 in the sequence listing, the CDS sequence of the TaSWEET11-7D gene is shown in sequence 8 in the sequence listing, and the amino acid sequence of the TaSWEET11-7D protein is shown in sequence 9 in the sequence listing.

[0101] The E-CRISP Design (http: / / www.e-crisp.org / E-CRISP / designcrispr.html) website was used to search for two gRNA target sites (gRNA1 sequence (sequence 10): 5'-gACGACGGGCGGGTTCAGCT-3', gRNA2 sequence (sequence 11): 5'-TCCCGATCCTCGGCGTGCAC-3') that were conserved in the A, B, and D subchromosomes of wheat in the genomic sequence of wheat TaSWEET11. The specificity of the target sites was compared using the Ensembl Plants (http: / / plants.ensembl.org / index.html) website. Primers (Primer-F (sequence 12): 5′-aataatggtctcAAGCgACGACGGGCGGGTTCAGCT-3′; Primer-F0 (sequence 13): 5′-gACGACGGGCGGGTTCAGCTgttttagagctagaaatagc-3′; Primer-R (sequence 14): 5′-ATTATTGGTCTCTAAACTCCCGATCCTCGGCGTGCAC-3′; Primer-R0 (sequence 15): 5′-TCCCGATCCTCGGCGTGCACCGCTTCTTGGTGCC-3′) were designed to amplify the plasmid pCBC-MT1T2 (kept in our laboratory, related literature: Du D, Zhang D, Yuan J, Feng M, Li Z, Wang Z, Zhang Z, Li X, Ke W, Li R, Chen Z, Chai L, Hu Z, Guo W, Xing J, Su Z, Peng H, Xin M, Yao Y, Sun Q, Liu J,Ni Z.FRIZZY PANICLE defines a regulatory hub for simultaneously controlling spikelet formation and awn elongation in bread wheat.New Phytol.2021Jul;231(2):814-833. Publicly available from applicant for the sole purpose of reproducing the invention).

[0102] 2. PCR amplification and product purification and recovery

[0103] PCR amplification used Tks-DNA polymerase (Takara, R060A), and the reaction system was as follows:

[0104] The PCR reaction program was set as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 10 s, annealing at 58°C for 15 s, and extension at 68°C for 30 s, for a total of 35 cycles; extension at 68°C for 5 min, and storage at 4°C.

[0105] Agarose gel recovery: Add Loading Buffer to the PCR reaction product and perform agarose gel electrophoresis at 180V for 10 minutes. After electrophoresis, the target band is excised and recovered under UV light. The steps are as follows:

[0106] 1) Place the cut gel block into a 2.0 mL optimized centrifuge tube, add 300 μL of the sol solution, incubate in a 55°C water bath for 10-15 minutes, and mix by inverting 1-2 times until the gel block is completely dissolved.

[0107] 2) After cooling to room temperature, place the adsorption column in a collection tube, draw the sol mixture onto the adsorption column, let it stand for 3 minutes, and centrifuge at 12,000 rpm for 1 minute;

[0108] 3) Add 500 μL of rinse solution W1 to the adsorption column and centrifuge at 12,000 rpm for 1 min;

[0109] 4) Add 700 μL of rinse solution W2, let stand for 1 min, centrifuge at 12,000 rpm for 1 min, and repeat once;

[0110] 5) Incubate at room temperature for 5-10 minutes and place the adsorption column in a new 1.5 mL centrifuge tube.

[0111] 6) Add 20-30 μL of ddH2O, incubate at room temperature for 2 minutes, and centrifuge at 12,000 × rpm for 1 minute. Measure the concentration and store at -20°C.

[0112] 3. CRISPR vector construction

[0113] The recovered gum product obtained in step 2 was reacted with the vector pBUE411 (preserved in this laboratory, related literature: Du D, Zhang D, Yuan J, Feng M, Li Z, Wang Z, Zhang Z, Li X, Ke W, Li R, Chen Z, Chai L, Hu Z, Guo W, Xing J, Su Z, Peng H, Xin M, Yao Y, Sun Q, Liu J, Ni Z. FRIZZY PANICLE defines a regulatory hub for simultaneously controlling spikelet formation and awn elongation in bread wheat. New Phytol. 2021 Jul; 231(2): 814-833. Available to the public from the applicant for use only in repeating the present invention) by the following system:

[0114] Reaction procedure: 37°C, 5h; 50°C, 5min; 80°C, 10min.

[0115] After the reaction product was transformed into competent E. coli, PCR amplification was performed on the transformed bacteria using primers 411-F (sequence 16: 5'-TTTCCCAGTCACGACGTTGT-3') and 411-R (sequence 17: 5'-GGATTCATGAGCAGCAAGCA-3'). Transformants containing the target sequences of gRNA1 and gRNA2 were considered positive monoclonal bacteria. The positive monoclonal bacteria were inoculated into LB liquid medium, and the plasmid was extracted from the culture medium to obtain the CRISPR-Cas9 recombinant plasmid pBUE411-TaSWEET11.

[0116] Example 2: Gene amplification and overexpression vector construction of TaSWEET11

[0117] 1. Target gene amplification

[0118] Using the cDNA of wheat variety XX329 as a template (stored in this laboratory, related literature: Zhang H, Zhu B, Qi B, Gou X, Dong Y, Xu C, Zhang B, Huang W, Liu C, Wang X, Yang C, Zhou H, Kashkush K, Feldman M, Wendel JF, Liu B. Evolution of the BBAA component of bread wheat during its history at the allohexaploid level. Plant Cell. 2014 Jul; 26(7): 2761-76. Publicly available from the applicant for the sole purpose of reproducing the present invention), PCR amplification was performed using Tks-DNA polymerase to obtain the CDS sequence of the TaSWEET11-7D gene. The primer sequences are as follows:

[0119] Primer-F1 (SEQ ID NO: 18): 5′-ACACTAGTTCTTGGCTAA-3′;

[0120] Primer-R1 (SEQ ID NO: 19): 5′-GTCGTCGTCGATGTTGGTG-3′.

[0121] The reaction system is as follows:

[0122] The PCR reaction program was set as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 10 s, annealing at 58°C for 15 s, and extension at 68°C for 30 s, for a total of 35 cycles; extension at 68°C for 5 min, and storage at 4°C.

[0123] 2. PCR Product Purification and Recovery

[0124] The PCR amplification product obtained in step 1 is purified and recovered to obtain a purified and recovered product. The purification and recovery steps of the PCR product are the same as those in step 2 of Example 1.

[0125] 3. Construction of TaSWEET11 gene overexpression vector:

[0126] 3.1 Expression vector linearization

[0127] According to the multiple cloning site information of vector pWMB110 (preserved in this laboratory, related literature: Liu J, Chen Z, Wang Z, Zhang Z, Xie X, Wang Z, Chai L, Song L, Cheng X, Feng M, Wang X, Liu Y, Hu Z, Xing J, Su Z, Peng H, Xin M, Yao Y, Guo W, Sun Q, Liu J, Ni Z. Ectopic expression of VRT-A2 underlies the origin of Triticum polonicum and Triticum petropavlovskyi with long outer glumes and grains. Mol Plant. 2021 Sep 6; 14(9): 1472-1488. The public can obtain it from the applicant and is only used to repeat the present invention), the restriction endonuclease BamHI was used to cut the vector pWMB110 to obtain a linearized vector plasmid.

[0128] 3.2 PCR amplification

[0129] Using the recovered product of step 2 as a template, PCR amplification was performed using primers ubi-SWT-F (sequence 20): 5'-AGGTCGACTCTAGAGGATCCATGGCTGGGGGCCTCTTC-3' and ubi-SWT-R (sequence 21): 5'-AGCTCGGTACCCGGGGATCCTCACACGGCGGGGGCGGGGA-3'. The amplification system and procedure were the same as the gene amplification in step 1. The vector homologous sequence was connected to both ends of the TaSWEET11-7D sequence to obtain a PCR product. The PCR product was recovered using the same method as in step 2 to obtain the PCR purified recovered product, which was the TaSWEET11-7D target sequence containing the homologous sequences at both ends of the vector.

[0130] 4. Seamless cloning and ligation reaction

[0131] The ligation system was as follows: 2× Seamless Cloning Mix (Biomed, CL117-01), 2.5 μL; pWMB110 linearized vector obtained in step 3.1, 1.5 μL; 1 μL of the PCR-purified product obtained in step 3.2. The reaction was incubated at 50°C for 30 min to obtain the recombinant plasmid pWMB110-Ubi-TaSWEET11.

[0132] 5. Obtaining positive recombinant bacteria

[0133] The recombinant plasmid pWMB110-Ubi-TaSWEET11 obtained in step 4 was transformed into competent Escherichia coli to obtain recombinant Escherichia coli.

[0134] PCR was used to detect recombinant Escherichia coli. The detection primers were: Ubi-F (sequence 22): 5'-TCGATGCTCACCCTGTTGTTTG-3'; OS-R (sequence 23): 5'-GGACGATCTCGATGACCT-3'. The bacterial solution containing the target fragment of approximately 700 bp (containing the CDS sequence of the TaSWEET11-7D gene shown in sequence 8 in the sequence table) in the PCR product was sent for testing. Based on the sequencing results, the positive bacterial solution was retained to obtain the positive recombinant Escherichia coli.

[0135] The experimental steps for transforming the ligation product into competent E. coli are as follows:

[0136] 1) Place the competent E. coli cells on ice. When the cells are frozen and thawed, add the recombinant plasmid pWMB110-Ubi-TaSWEET11, mix gently, and place on ice for 30 minutes.

[0137] 2) After completion, incubate in a 42°C water bath for 1 min; then in an ice bath for 2 min, add 500 μL of LB liquid medium, and culture at 37°C with shaking for 1 h.

[0138] 3) Centrifuge and retain 200 μL of bacterial solution to be spread on solid medium containing LB resistance, and incubate inverted at 37°C for 1 day.

[0139] 4) Pick a single colony and place it in 1 mL of LB liquid medium. Culture at 37°C with shaking. Screen the positive bacteria by PCR. Mix with 50% glycerol (1:1 volume ratio) and store at -80°C until use.

[0140] After the positive recombinant Escherichia coli was propagated by shaking, the recombinant plasmid pWMB110-Ubi-TaSWEET11 was extracted using a plasmid extraction kit (Zhuangmeng, ZP101-1).

[0141] Example 3: Obtaining TaSWEET11 recombinant Agrobacterium

[0142] The CRISPR-Cas9 recombinant plasmid pBUE411-TaSWEET11 obtained in Example 1 and the recombinant overexpression plasmid pWMB110-Ubi-TaSWEET11 obtained in Example 2 were transformed into Agrobacterium, respectively. The experimental steps are as follows:

[0143] 1) When the competent Agrobacterium cells EHA105 (Ubi, AC1010) are in a freeze-thaw state, add a certain volume of the target plasmid (about 1 μg, pBUE411-TaSWEET11 or pWMB110-Ubi-TaSWEET11), mix gently, and place on ice for 5 minutes.

[0144] 2) Place the competent cells in liquid nitrogen for 5 minutes and then in a 37°C water bath for 5 minutes.

[0145] 3) Place on ice for 5 minutes, add 750 μL LB liquid medium, and culture with shaking at 28°C for 2-3 hours.

[0146] 4) Pipette 200 μL of bacterial solution onto LB solid medium containing Rif and Kan, and incubate at 28°C for 1-2 days.

[0147] Pick a single clone plaque, add 1 mL of LB liquid medium, and culture with shaking at 28°C for 1 day to obtain positive recombinant Agrobacterium EHA105 / pBUE411-TaSWEET11 bacterial liquid and positive recombinant Agrobacterium EHA105 / pWMB110-Ubi-TaSWEET11 bacterial liquid, mix them with 50% glycerol (volume ratio of 1:1), and store at -80°C for use.

[0148] The positive recombinant Agrobacterium EHA105 / pWMB110-Ubi-TaSWEET11 contains the CDS sequence of the TaSWEET11-7D gene shown in Sequence 8 in the sequence listing, and can express TaSWEET11-7D shown in Sequence 9 in the sequence listing.

[0149] Example 4: Positive identification of transgenic wheat materials and determination of agronomic traits

[0150] 1. Acquisition of transgenic wheat materials

[0151] The recombinant Agrobacterium EHA105 / pBUE411-TaSWEET11 and the recombinant Agrobacterium EHA105 / pWMB110-Ubi-TaSWEET11 obtained in Example 3 were transformed into wheat callus tissues using the Agrobacterium-mediated method. The recombinant Agrobacterium EHA105 / PBUE411-TaSWEET11 containing the CRISPR vector was transformed into wheat Fielder to obtain the T0 generation TaSWEET11 gene-edited material KO strain, which was genetically transformed by the transgenic platform of the Wheat Research Center of China Agricultural University; the recombinant Agrobacterium EHA105 / pWMB110-Ubi-TaSWEET11 was transformed into the nationally approved Zhengmai 7698 (related literature: Luo J, Li S, Xu J, Yan L, Ma Y, Xia L. Pyramiding favorable alleles in an elite wheat variety in one generation by CRISPR-Cas9-mediated multiplex gene editing. Mol Plant. 2021 Jun 7; 14(6): 847-850. The public can obtain it from the applicant for the purpose of repeating the present invention only) to obtain T0 generation TaSWEET11 overexpression OE materials OE1, OE2 and OE3. Wheat callus transformation was entrusted to the Gene Editing and New Materials Creation Research Group of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences for genetic transformation (experimental steps reference: Luo J, Li S, Xu J, Yan L, Ma Y, Xia L. Pyramiding favorable alleles in an elite wheat variety in one generation by CRISPR-Cas9-mediated multiplex gene editing. Mol Plant. 2021 Jun 7; 14(6): 847-850).

[0152] 2. Identification of gene-edited plants:

[0153] The DNA of the T0 generation gene-edited material was extracted, and the gene-edited material of the T0 generation was amplified by PCR using three sets of CRISPR detection primers. The amplified products were sequenced, and plants with mutations in different chromosome subgroups of ABD were screened. After cross-generation culture, homozygous triple mutant plants were obtained and planted to obtain three T2 generation plants, KO1, KO2 and KO3, and their agronomic traits were investigated.

[0154] The sequences of the three sets of CRISPR detection primers are as follows:

[0155] SWTCSA-F (SEQ ID NO: 24): 5'-AGTGGGTGGATCTCTGACTCC-3',

[0156] SWTCSA-R (SEQ ID NO: 25): 5′-TCGTGTCGTCGTCGATGTTT-3′;

[0157] SWTCSB-F (SEQ ID NO: 26): 5'-CTGTCGCCTGGTTCTGCT-3',

[0158] SWTCSB-R (SEQ ID NO: 27): 5′-ACACCAACCTGCGGACTAATA-3′;

[0159] SWTCSD-F (sequence 28): 5'-TCCATCATCGTACGTCGTTAGC-3',

[0160] SWTCSD-R (SEQ ID NO: 29): 5'-TATTGTCACCGCACCAACG-3'.

[0161] 3. Identification of positive overexpression plants:

[0162] The DNA of the T0 generation of OE materials overexpressing the TaSWEET11 gene was amplified by PCR using the positive identification primers for OE materials (Ubi-F: 5'-TCGATGCTCACCCTGTTGTTTG-3'; OS-R: 5'-GGACGATCTCGATGACCT-3'). The positive lines were identified (Figure 4) and screened for two generations for planting to obtain the T2 generation pure line plants OE6, OE8 and OE10. The expression level of the TaSWEET11-7D gene was also measured. The results are shown in Figure 5. Compared with the wild-type recipient wheat Zhengmai 7698 (ZM7698 in Figure 5), the three positive OE lines showed significant overexpression of the TaSWEET11-7D gene, which was subsequently used for phenotypic studies.

[0163] 4. Determination of agronomic traits:

[0164] During the wheat maturity stage, the agronomic traits such as plant height, spike length, number of spikelets, number of grains per spike, single plant yield, grain length, grain width, and 1000-grain weight of the T2 generation TaSWEET11 gene-edited plant KO line, T2 generation overexpression material OE line, and wild-type materials were investigated; the crude protein content in the seeds was determined by the Kjeldahl method.

[0165] The results of agronomic characterization are as follows:

[0166] The agronomic traits of the KO materials (KO1, KO2 and KO3) were significantly reduced compared with the wild-type recipient material (WT in Figure 1) in terms of plant height (A in Figure 1), spike length (B in Figure 1), number of spikelets (C in Figure 1) and number of grains per spike (D in Figure 1). The grain length (F in Figure 1), grain width (G in Figure 1) and 1000-grain weight (E in Figure 1) were extremely significantly reduced, resulting in a serious reduction in yield per plant (H in Figure 1) (Figure 1).

[0167] Using the Ubi promoter to drive heterologous overexpression of the TaSWEET11 gene in the nationally approved winter wheat variety Zhengmai 7698, we found that the OE lines (OE6, OE8, and OE10) of Zhengmai 7698 showed reduced plant height (Figure 2A), increased spike length (Figure 2B), spikelet number (Figure 2C), and kernel number per spike (Figure 2D) compared to Zhengmai 7698 (ZM7698 in Figure 2). Grain length (Figure 2E) and width (Figure 2F) of the OE lines were significantly increased, leading to an increase in 1000-grain weight (Figure 2G) of approximately 1.50-1.82 g. Yield per plant was also significantly increased, with increases ranging from 20% to 49% (Figure 2H). Analysis of grain protein content revealed that the OE lines had significantly higher protein content than Zhengmai 7698 (ZM7698 in Figure 3) (Figure 3).

[0168] Therefore, overexpression of TaSWEET11 can significantly increase the thousand-grain weight of wheat grains and improve yield; at the same time, overexpression of TaSWEET11 also increases the protein content of wheat grains, significantly improving the quality of grains while increasing yield. Wheat TaSWEET11 protein can be used in crop breeding and quality improvement.

[0169] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art. Industrial Applicability

[0170] The present invention significantly reduces plant yield by knocking out the gene for the wheat SWEET11 protein through gene editing. Furthermore, overexpressing the wheat SWEET11 protein in the nationally approved wheat variety Zhengmai 7698 revealed an increase in 1,000-grain weight, protein content, and yield per plant, significantly improving grain quality while boosting yield. Therefore, the TaSWEET11 protein of the present invention has important theoretical and practical implications for increasing wheat yield, and the TaSWEET11 protein and its related biomaterials can be applied to crop breeding and quality improvement.

[0171] CROSS-REFERENCE TO RELATED APPLICATIONS

[0172] This application claims priority to the Chinese patent application (application number 202410086663.3) filed on January 22, 2024, the entire contents of which are incorporated herein by reference.

Claims

1. Use of any one of the following substances that regulate protein expression or regulate protein activity: P1. Application in regulating the grain length, grain width and / or 1000-grain weight of plant seeds; P2. Application in increasing the grain length, grain width and / or 1000-grain weight of plant seeds; P3. Application in regulating the spike length, number of spikelets / or number of grains per spike of plant plants; P4. Application in increasing the spike length, number of spikelets / or number of grains per spike of plant plants; P5. Application in regulating the yield per plant of plant plants; P6. Application in increasing the yield per plant of plant plants; P7. Application in regulating the protein content of plant seeds; P8. Application in increasing the protein content of plant seeds; P9. Application in high-yield breeding of plants and / or breeding of seeds with high protein content; P10. Application in improving the quality of high-yield and / or high-protein content of plant seeds; The protein is as follows: A1) A protein with an amino acid sequence that is Sequence 9 in the Sequence Listing; A2) A protein with an amino acid sequence that is Sequence 3 in the Sequence Listing; A3) A protein with an amino acid sequence that is Sequence 6 in the Sequence Listing; A4) A protein obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in A1), A2) or A3), and having the same function, a derivative of A1), A2) or A3), or a protein having more than 80% identity with the protein shown in A1), A2) or A3) and having the same function; A5) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1), A2) or A3).

2. The application according to claim 1, characterized in that: The protein is derived from wheat.

3. The application according to claim 1, wherein: The plant is any one of the following: D1) Dicotyledonous plants; D2) Monocotyledonous plants, D3) Plants of the order Poales, D4) Gramineous plants, D5) Plants of the genus Triticum, D6) Wheat.

4. Use of any one of the following biological materials related to the protein described in any one of Claims 1-3: Q1. Application in regulating the grain length, grain width and / or 1000-grain weight of plant seeds; Q2. Application in increasing the grain length, grain width and / or 1000-grain weight of plant seeds; Q3. Application in regulating the spike length, number of spikelets / or number of grains per spike of plant plants; Q4. Application in increasing the spike length, number of spikelets / or number of grains per spike of plant plants; Q5. Application in regulating the yield per plant of plant plants; Q6. Application in increasing the yield per plant of plant plants; Q7. Application in regulating the protein content of plant seeds; Q8. Application in increasing the protein content of plant seeds; Q9. Application in high-yield breeding of plants and / or breeding of seeds with high protein content; Q10. Application in improving the quality of high-yield and / or high-protein content of plant seeds; The biological material is any one of the following: B1) A nucleic acid molecule encoding the protein described in Claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) A transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2); B8) A nucleic acid molecule that promotes or enhances the gene expression of the protein described in claim 1; B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

5. The application according to claim 4, wherein: The nucleic acid molecule described in B1) is the coding gene of the protein shown in the following b1), b2) or b3): b1) A cDNA molecule or DNA molecule whose coding sequence of the coding strand is the nucleotide of sequence 8, sequence 2 and / or sequence 5 in the sequence listing; b2) A DNA molecule whose nucleotide is sequence 7, sequence 1 and / or sequence 4 in the sequence listing, b3) A cDNA molecule or DNA molecule that hybridizes with the cDNA or DNA molecule defined in b2) and encodes a protein with the same function.

6. The application according to claim 5, wherein: The plant is any one of the following: D1) Dicotyledonous plants; D2) Monocotyledonous plants, D3) Plants of the order Poales, D4) Gramineous plants, D5) Plants of the genus Triticum, D6) Wheat.

7. The application according to claim 4, characterized in that: The plant is any one of the following: D1) Dicotyledonous plants; D2) Monocotyledonous plants, D3) Plants of the order Poales, D4) Gramineous plants, D5) Plants of the genus Triticum, D6) Wheat.

8. A method for increasing the grain length, grain width, 1000-grain weight, number of grains per spike, yield per plant and / or grain protein content of plants, comprising enhancing or increasing the activity of the protein described in claim 1 and / or the expression level of the coding gene of the protein described in claim 1 in the target plant, thereby increasing the grain length, grain width, 1000-grain weight, number of grains per spike, yield per plant and / or grain protein content of the target plant.

9. The method according to claim 8, characterized in that: The enhancement or increase of the activity of the protein described in claim 1 and / or the expression level of the coding gene of the protein described in claim 1 in the target plant is achieved by introducing the coding gene of the protein described in claim 1 into the target plant.

10. The method according to claim 9, characterized in that: The plant and / or the target plant is any one of the following: D1) Dicotyledonous plants; D2) Monocotyledonous plants, D3) Plants of the order Poales, D4) Gramineous plants, D5) Plants of the genus Triticum, D6) Wheat.

11. The method according to claim 8, wherein: The plant and / or the target plant is any one of the following: D1) Dicotyledonous plants; D2) Monocotyledonous plants, D3) Plants of the order Poales, D4) Gramineous plants, D5) Plants of the genus Triticum, D6) Wheat.

12. The protein described in claim 1.

13. The biological material described in claim 4 or 5.

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