Application of wheat TaSMAX1 protein and coding gene thereof in regulating and controlling ear characters of wheat

Abstract

The invention discloses a wheat TaSMAX1 protein and an application of a coding gene thereof in regulation and control of wheat ear characters. The invention belongs to the technical field of biology, and particularly relates to application of wheat TaSMAX1 protein and a coding gene thereof in regulation and control of wheat ear characters. The application of the TaSMAX1 protein in regulation and control of wheat ear characters is specifically embodied in that overexpression of the TaSMAX1 protein causes increase of wheat ear length, floret number and grain number per ear, so that gene resources are provided for cultivation of high-yield and high-quality wheat varieties, and the TaSMAX1 protein has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of wheat TaSMAX1 protein and its coding gene in regulating wheat spike traits. Background Art

[0002] Wheat is one of the most important crops in the world. It is estimated that by 2050, wheat production needs to increase by 70% on the current level to meet the demand of the growing world population. China is the largest wheat producer and consumer in the world. Developing wheat production plays an important role in ensuring China's food security and market demand and plays an important role in the sustainable food supply system. The three elements of wheat yield include the number of effective spikes, the number of grains per spike, and the 1000-grain weight. Among them, the number of grains per spike and the 1000-grain weight are both closely related to the spike morphology. Compared with the 1000-grain weight, increasing the number of grains per spike is an effective way to increase wheat yield. The number of grains per spike is determined by the number of spikelets per spike and the number of grains per spikelet, and these are all important wheat spike traits. Under the condition of not changing the spikelet density, increasing the spike length and the number of fertile florets is the key to increasing the number of grains per spike, thereby further increasing the yield. Therefore, discovering genes related to regulating spike traits such as spike length and the number of fertile florets is of great significance for clarifying the molecular basis of wheat yield traits and increasing wheat yield. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to effectively regulate the wheat spike length, the number of florets or the number of grains per spike, so as to increase wheat yield.

[0004] In order to solve the problems existing in the prior art, the present invention provides the application of a protein or a substance for regulating the expression of a gene or a substance for regulating the activity or content of the protein in regulating wheat spike traits.

[0005] The application provided by the present invention of the protein or the substance for regulating the expression of the gene or the substance for regulating the activity or content of the protein is in any one of the following:

[0006] 1) The application of the protein or the substance for regulating the expression of the gene or the substance for regulating the activity or content of the protein in regulating plant spike traits;

[0007] 2) The application of the protein or the substance for regulating the expression of the gene or the substance for regulating the activity or content of the protein in the preparation of regulating plant spike traits;

[0008] 3) The application of the protein or the substance for regulating the expression of the gene or the substance for regulating the activity or content of the protein in cultivating plants with changed spike traits;

[0009] 4) The application of the protein or the substance for regulating the expression of the gene or the substance for regulating the activity or content of the protein in the preparation of products for cultivating plants with changed spike traits;

[0010] 5) Use of a protein or a substance that regulates gene expression or a substance that regulates the activity or content of said protein in plant breeding;

[0011] The protein is any of the following proteins:

[0012] a1) A protein with an amino acid sequence of SEQ ID No. 2;

[0013] a2) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No. 2 and having the same function;

[0014] a3) A protein having more than 80% identity with the amino acid sequence defined in any of a1)-a2) and having the same function;

[0015] a4) A fusion protein obtained by linking a tag to the end of the protein defined in any of a1)-a3).

[0016] The name of the protein described in a1) above is TaSMAX1.

[0017] In order to facilitate the purification or detection of the protein in a1), a tag protein can be linked to the amino terminus or carboxyl terminus of the protein composed of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0018] The above-mentioned protein can be artificially synthesized, or its coding gene can be synthesized first and then expressed biologically.

[0019] The tag proteins include but are not limited to: GST (glutathione S-transferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0020] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein TaSMAX1 of the present invention by using known methods, such as directed evolution or site-directed mutagenesis. Those nucleotides that have been artificially modified and have 75% or more identity with the nucleotide sequence of the protein TaSMAX1 isolated from the present invention, as long as they encode the protein TaSMAX1 and have the function of the protein TaSMAX1, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0021] The above-mentioned identity of 75% or more can be an identity of 80%, 85%, 90% or more than 95%.

[0022] In this article, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively and performing a search, the identity value (%) of a pair of amino acid sequences can be calculated.

[0023] In this article, the identity of more than 80% can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0024] In this article, the identity of more than 90% can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0025] In the above application, the protein is derived from wheat (Triticum aestivum L.).

[0026] In this article, the substance that regulates the activity and / or content of the protein can be a substance that regulates gene expression, and the gene encodes the protein TaSMAX1.

[0027] In the above text, the substance that regulates gene expression can be a substance that performs at least one of the following six regulations: 1) regulation at the transcriptional level of the gene; 2) regulation after gene transcription (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the gene's mRNA from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated by the gene).

[0028] In the present invention, the regulation can be up-regulation, enhancement or increase.

[0029] In the above application, the substance for regulating gene expression or regulating the activity or content of the protein may be a biological material related to the protein described above, and the biological material may be any one of the following:

[0030] c1) A nucleic acid molecule encoding the protein described above;

[0031] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0032] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0033] c4) A recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3);

[0034] c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2);

[0035] c6) A transgenic plant tissue containing the nucleic acid molecule described in c1), or a transgenic plant tissue containing the expression cassette described in c2);

[0036] c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2);

[0037] In the above application, the nucleic acid molecule described in c1) may be a DNA molecule shown as any of the following:

[0038] d1) A DNA molecule whose nucleotide sequence is the DNA molecule shown in SEQ ID No. 3;

[0039] d2) A DNA molecule whose coding region sequence is the DNA molecule shown in positions 58-3123 of SEQ ID No. 1 in the sequence listing;

[0040] d3) A DNA molecule having 90% or more identity with the nucleotide sequence defined in d1) or d2), derived from wheat and encoding the protein described above;

[0041] d4) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described above.

[0042] The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0043] The vectors described in this article are well-known to those skilled in the art, including but not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), Ti plasmids or viral vectors. Specifically, it can be vector PC186.

[0044] Existing plant expression vectors can be used to construct recombinant expression vectors containing the TaSMAX1 gene. The plant expression vectors include but are not limited to binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. The plant expression vector may also contain the 3'-untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, it includes but is not limited to the Agrobacterium tumefaciens (Ti) plasmid gene (such as the nopaline synthase Nos gene), and the 3'-untranslated regions transcribed from plant genes (such as soybean storage protein genes) all have similar functions.

[0045] When using the TaSMAX1 gene to construct a recombinant plant expression vector, any one of the enhanced promoters or constitutive promoters can be added before its transcription start nucleotide, including but not limited to the cauliflower mosaic virus (CaMV) 35S promoter, the ubiquitin promoter of maize (ubiquitin), and they can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and start codons are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0046] In order to facilitate the identification and screening of transgenic plant cells or plants, the used plant expression vectors can be processed, such as adding genes encoding enzymes or luminescent compounds that can produce color changes and can be expressed in plants (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or anti-chemical reagent marker genes (such as herbicide-resistant genes), etc. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0047] Using any vector capable of guiding the expression of foreign genes in plants, introducing the TaSMAX1 gene or gene fragment provided by the present invention into plant cells or recipient plants can obtain transgenic cell lines and transgenic plants with altered spike traits. The expression vector carrying the TaSMAX1 gene can transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroconductivity, and Agrobacterium-mediated transformation, and cultivate the transformed plant tissues into plants.

[0048] The present invention also provides a method for regulating wheat spike traits, the method includes step P, and the step P is to positively regulate wheat spike traits by enhancing, increasing or upregulating the activity and / or content of the aforementioned protein in the target plant, and / or, enhancing, increasing or upregulating the expression level of the coding gene of the aforementioned protein.

[0049] In the above method, the increasing of the expression level and / or activity of the coding gene of the protein TaSMAX1 in the target plant can be achieved by overexpression to enhance the activity of the coding gene of the protein TaSMAX1 in the genome of the target plant.

[0050] The present invention provides a method for cultivating plants with increased spike length and grain number per spike, including overexpressing the expression of the coding gene of the above protein and / or the content and / or activity of the above protein in the target plant, to obtain plants with increased spike length, increased number of fertile florets, and ultimately increased yield.

[0051] In one embodiment of the present invention, the breeding method for cultivating plants with improved spike traits includes the following steps:

[0052] (1) Construct a recombinant expression vector that enhances, increases or upregulates the coding gene of the aforementioned protein;

[0053] (2) Transfer the recombinant expression vector constructed in step (1) into a recipient plant (such as a crop or wheat) to obtain a plant with better spike traits than the recipient plant.

[0054] In the present invention, the purpose of plant breeding may include cultivating plants with improved spike traits.

[0055] In the present invention, the improvement of spike traits may be to increase spike length, floret number and grain number per spike.

[0056] In the above application or method, the plant may be any of the following plants:

[0057] N1) Monocotyledonous plants or dicotyledonous plants;

[0058] N2) Plants of Poales;

[0059] N3) Gramineous plants;

[0060] N4) Triticum plants;

[0061] N5) Wheat.

[0062] In this study, overexpression of TaSMAX1 can increase the spike length, floret number and grain number per spike of wheat, indicating that TaSMAX1 can improve the spike traits of wheat. These findings provide gene resources for breeding high-yield and high-quality wheat varieties. Description of the Drawings

[0063] Figure 1 For the cloning of the full-length cDNA of the TaSMAX1 target gene; where the M lane is D2000 plus DNA Ladder; lanes 1 and 2 are the full-length TaSMAX1 cDNA, and its size is 3139 bp.

[0064] Figure 2 For the identification results of the T0 generation of TaSMAX1 transgenic lines; where the M lane is D2000 plus DNA Ladder; lanes 1 and 2 are the negative control Fielder, lanes 3 and 4 are TaSMAX1-OE1, lanes 5 and 6 are TaSMAX1-OE2, and the CK+ lane represents the positive plasmid. The upper and lower figures respectively represent two pairs of different identification primers (the primers used in the upper figure are primer 1: F1: 5’-ACTCGGTGGGGATGTTATCA-3’, R: 5’-GCCAAATGTTTGAACGATCGG-3’; the lower figure is primer 2: F2: 5’-ACCAGGAAGCCCAGTGAAA-3’, R: 5’-GCCAAATGTTTGAACGATCGG-3’). The size of the product in the upper figure is 2028 bp, and the size of the product in the lower figure is 1536 bp.

[0065] Figure 3 For the detection of the gene expression level of TaSMAX1 overexpression materials.

[0066] Figure 4 For the spike phenotypes of TaSMAX1 overexpression materials at the flowering stage; where A is the overall plant phenotype of WT and TaSMAX1 overexpression material TaSMAX1-OE1 at the flowering stage; B is the spike length phenotype of WT and TaSMAX1 overexpression material TaSMAX1-OE1 at the flowering stage; C and D are the floret phenotypes of WT and TaSMAX1 overexpression material TaSMAX1-OE1; the scale bar is 5 cm.

[0067] Figure 5 For the spike phenotypes of TaSMAX1 overexpression materials TaSMAX1-OE1 and TaSMAX1-OE2 at the maturity stage; the spike length, spikelet number and grain number per spike of WT and TaSMAX1 overexpression materials were respectively counted, and the scale bar is 5 cm. Detailed implementation manners

[0068] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0069] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0070] In the following embodiments, the quantitative experiments are all set with three repeated experiments and the results are averaged unless otherwise specified.

[0071] The wheat material "Fielder" in the following embodiments was a gift from Teacher Li Genying of the Crop Research Institute of Shandong Academy of Agricultural Sciences, and has been recorded in: Zhang, S., Zhang, R., Gao, J., Song, G., Li, J., Li, W., Qi, Y., Li, Y., & Li, G. (2021). CRISPR / Cas9-mediated genome editing for wheat grain quality improvement. Plant Biotechnology Journal, 19(9): 1684–1686. The public can obtain this biological material from the applicant. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0072] The Escherichia coli strain DH5α in the following embodiments was purchased from TransGen Biotech Co., Ltd., Beijing, China.

[0073] The Agrobacterium strain EHA105 in the following embodiments was purchased from Beijing Bomed Gene Technology Co., Ltd., China.

[0074] The vector pBM27 in the following embodiments was purchased from Beijing Bomed Biotechnology Co., Ltd., China.

[0075] The vector PC186 in the following examples was a gift from Teacher Fudao Lin of the College of Agronomy, Shandong Agricultural University. It has been recorded in: Hao Q, Wang W, Han X, Wu J, Lyu B, Chen F, Caplan A, Li C, Wu J, Wang W, Xu Q, Fu D. (2018) Isochorismate-based salicylic acid biosynthesis confers basal resistance to Fusarium graminearum in barley. Molecular Plant Pathology. 19(8): 1995–2010. The public can obtain this biological material from the applicant. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0076] In the following examples, SPSS 19.0 statistical software was used to process the data. The experimental results were expressed as mean ± standard deviation. Student's t-test was used. *, **, and *** in the results represent significant differences at the 0.05, 0.01, and 0.001 levels, respectively.

[0077] Example 1. Cloning of TaSMAX1 gene

[0078] 1. Extraction of total RNA from wheat leaves

[0079] Trizol method was used to extract RNA

[0080] (1) Take 150 mg - 200 mg of the seedling leaves of Fielder material and put them into a mortar pre-cooled with liquid nitrogen and free of RNase. Quickly grind the material into a uniform powder in liquid nitrogen and then add it to a 2.0 mL centrifuge tube free of RNase.

[0081] (2) Add 1 mL of Trizol to the centrifuge tube, vortex and then let it stand at room temperature for 5 min.

[0082] (3) Add 200 μL of chloroform, shake vigorously for 30 s and then let it stand at room temperature for 5 min.

[0083] (4) Centrifuge at 12,000 rpm and 4 °C for 15 min, transfer the supernatant (550 - 650 μL) to a new 1.5 mL centrifuge tube free of RNase.

[0084] (5) Add isopropanol with the same volume as the supernatant, mix well by inverting thoroughly, then let it stand at room temperature for 5 - 10 min, centrifuge at 12,000 rpm and 4 °C for 15 min, pour out the supernatant and retain the precipitate.

[0085] (6) Add 1 mL of 75% alcohol to wash the precipitate, centrifuge at 12,000 rpm at 4 °C for 5 min, and discard the supernatant.

[0086] (7) Repeat step (6), pipette the alcohol dry with a pipette tip and place it in the laminar flow hood to dry.

[0087] (8) Add 50 μL of RNase-free ddH 2 O to dissolve the RNA, and it can be assisted in dissolving at 50 °C for 5 - 10 min, while removing polysaccharides and polyphenols.

[0088] (9) Centrifuge at 12,000 rpm at 4 °C for 5 - 10 min, pipette the supernatant into a new 1.5 mL RNase-free centrifuge tube, measure the concentration or store it at -80 °C for later use.

[0089] 2. cDNA synthesis

[0090] Use Novoprotein III RT SuperMix for qPCR(+gDNA wiper) kit for reverse transcription, operate on ice throughout the process. The reaction system is as follows: The gDNA removal reaction system includes 1.5 μg of RNA template, 4 μL of 4×gDNA wiperMix, and RNase-free ddH 2 O is made up to 16 μL. After pipetting and mixing well with a pipette, control the temperature with a PCR instrument at 42 °C for 2 min.

[0091] The reverse transcription system includes 16 μL of the product of the previous reaction system and 4 μL of 5×HiScript III qRT SuperMix. After pipetting and mixing well with a pipette, perform the following reverse transcription reaction program in a PCR instrument: 50 °C for 15 min, 85 °C for 5 sec. Immediately place it on ice after the reaction. Add 180 μL of ddH 2 O to dilute and store it for later use at -20 °C.

[0092] 3. Cloning of TaSMAX1 gene

[0093] Use the wheat leaf cDNA obtained in the previous step as a template, design primers according to the UTR region of this gene to amplify the TaSMAX1 gene. The primer sequences are as follows: TaSMAX1-F: 5’-AGAATTCGTGCCCTTTGATC-3’, TaSMAX1-R: 5’-CTTTCTCAAATGCCCTCTACA-3’.

[0094] The PCR amplification system was 50 μL and included the following components: 25 μL of 2×PCR Buffer, 10 μL of dNTPs (2 mM), 1 μL of cDNA, 1 μL of KOD Fx, 1.5 μL of Primer-F (10 μM), 1.5 μL of Primer-R (10 μM), ddH 2 O 10 μL.

[0095] PCR reaction program: pre-denaturation at 94 °C for 5 min; denaturation at 98 °C for 10 sec, annealing at 56 °C for 30 sec, extension at 68 °C for 3 min 30 sec, 35 cycles; extension at 68 °C for 10 min, and the reaction ended at 12 °C. The amplification product was obtained. The amplification product included the coding region sequence of TaSMAX1. It was separated by 1% agarose gel electrophoresis, and a band of about 3100 bp could be seen ( Figure 1 ), and the specific nucleotide sequence was SEQ ID No.1. The band of the target fragment was quickly cut and chopped under an ultraviolet gel cutting instrument and placed into a clean centrifuge tube for gel recovery.

[0096] The coding sequence (CDS) of the TaSMAX1 gene in the wheat material Fielder was from the 58th to 3123rd positions of SEQ ID No.1, and the encoded amino acid sequence was the TaSMAX1 protein of SEQ ID No.2. In the genomic DNA of wheat, the genomic sequence encoding the TaSMAX1 protein was as shown in SEQ ID No.3 of the sequence listing. The 1st to 1211th positions of SEQ ID No.3 were the first exon, the 2764th to 3028th positions were the second exon, and the 3117th to 4706th positions were the third exon.

[0097] 4. Ligation of TaSMAX1 to the pBM27 vector

[0098] Using the above gel recovery product as a template, the following primers were used for amplification (pBM27-TaSMAX1-F: 5’-CACCATGAGGGCGGATCTCAGCA-3’, pBM27-TaSMAX1-R: 5’-CATTCCATCGATGGCAATCG-3’, and these primers contained the pBM27 vector adapter sequence). The amplification system was the same as the previous step, 50 ng of template was added, and finally water was added to make up to a 50 μL system. The PCR reaction program was the same as the previous step. After the reaction ended, it was separated by 1% agarose gel electrophoresis, and the target band was cut and recovered.

[0099] The above gel recovery product was ligated to the pBM27 vector using the pBM27 cloning kit. The reaction system was as follows: 100 ng of gel recovery product, 1 μL of pBM27 Vector, 1 μL of 10×Toposmart, ddH 2Make up to 10 μL with O. After adding, gently flick to mix and centrifuge. Control the temperature with a PCR instrument, react at 25°C for 30 min. After the reaction, transform Escherichia coli competent DH5α. After identifying the positive clones, shake the bacteria, extract the plasmid and send it to the company for sequencing. Name the plasmid with correct sequencing as pBM27-TaSMAX1 and store it at -20°C.

[0100] The structure of the recombinant vector pBM27-TaSMAX1 is described as follows: It is a recombinant vector obtained by replacing the fragment between 5'-CCCTT-3' and 5'-AAGGG-3' of the pBM27 vector with the 58th to 3123rd positions of SEQ ID No.1, while keeping the other nucleotides of the pBM27 vector unchanged.

[0101] Example 2: Construction of TaSMAX1 overexpression vector

[0102] The TaSMAX1 in the vector pBM27-TaSMAX1 was ligated to the PC186 vector through an LR reaction. The reaction system was 150 ng of pBM27-TaSMAX1, 150 ng of PC186, 1 μL of LR enzyme, and ddH 2 Make up to 5 μL with O. After reacting overnight at 25°C, transform Escherichia coli DH5α competent cells. Pick monoclonal colonies for identification, then shake the bacteria, extract the plasmid and send it to the company for sequencing. Name the plasmid with correct sequencing as PC186-TaSMAX1.

[0103] The structure of the recombinant vector PC186-TaSMAX1 is described as follows: The recombinant vector PC186-TaSMAX1 is a recombinant expression vector obtained by replacing the fragment between the attR1 site and the attR2 site of the PC186 vector with attL1-TaSMAX1-attL2, while keeping the other nucleotides of the PC186 vector unchanged. PC186-TaSMAX1 contains the TaSMAX1 gene CDS sequence shown in the 58th to 3123rd positions of SEQ ID No.1 in the sequence listing, the Ubi promoter, and the NOS terminator, and can express the TaSMAX1 protein, and the expression of this protein is driven by the Ubi promoter.

[0104] Example 3: Application of TaSMAX1 protein in regulating wheat spike traits

[0105] 1. Obtaining of TaSMAX1 overexpression transgenic plants

[0106] The correctly constructed PC186-TaSMAX1 vector was transformed into Agrobacterium competent EHA105. Single colonies were picked for positive clone identification. The correctly identified positive clones were transferred into the callus of wheat material Fielder by the Agrobacterium-mediated genetic transformation method. Positive plants were screened through the Bar selection marker carried by the vector. Subsequently, they were transplanted into flower pots and normally cultured in the greenhouse (16 h light, 8 h dark), which were the T0 generation plants.

[0107] 2. Identification of TaSMAX1 overexpression positive plants

[0108] 1) PCR identification

[0109] The CTAB method was used to extract wheat leaf DNA. Cut a 1 cm × 3 cm wheat leaf and place it in a 2.0 mL centrifuge tube with steel beads. Immediately freeze it in liquid nitrogen for 2 min, then use a high-throughput grinder to crush the leaf into powder. After taking it out, immediately add 600 μL of CTAB preheated at 65 °C in advance, mix well, and incubate in a water bath at 65 °C for 45 min, during which invert and mix every 15 min; after taking out the centrifuge tube, add 200 μL of chloroform, invert repeatedly until fully mixed, and centrifuge at 12000 rpm at room temperature for 10 min; carefully transfer 600 μL of the supernatant to a new 1.5 mL centrifuge tube, add isopropanol equal in volume to the supernatant, mix well, and centrifuge at 12000 rpm at room temperature for 10 min; pour out the supernatant, add 1 mL of 70% ethanol to wash the precipitate, centrifuge at 12000 rpm for 10 min, and repeat the washing once; use a pipette tip to suck out the residual ethanol, open the lid and invert it to dry in the ultra-clean bench; add 60 μL of ddH 2 O to dissolve the DNA, and use a micro ultraviolet spectrophotometer Nano Drop 2000 to detect the DNA concentration and quality, and dilute the DNA with ddH 2 O to 50 ng / μL for standby.

[0110] The identification of transgenic positive plants was carried out by PCR method. Two pairs of primers (Primer 1: Primer-F1: 5’-ACTCGGTGGGGATGTTATCA-3’, Primer-R1: 5’-GCCAAATGTTTGAACGATCGG-3’; Primer 2: Primer-F2: 5’-ACCAGGAAGCCCAGTGAAA-3’, Primer-R2: 5’-GCCAAATGTTTGAACGATCGG-3’) were used to amplify the DNA. The PCR system was as follows: 2×M5 HiPer plus Taq HiFi PCR Mix 7.5 μL, DNA 1 μL, Primer-F (10 μM) 0.5 μL, Primer-R (10 μM) 0.5 μL, ddH 2 O 5.5 μL.

[0111] PCR reaction procedure: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 1 min (2 kb / min), 35 cycles; extension at 72°C for 10 min, and the reaction ended at 12°C. The PCR products were separated by 1.5% agarose gel electrophoresis( Figure 2 ). Positive plants were selected and self-crossed to obtain the T2 generation for subsequent phenotypic analysis.

[0112] 2) Identification at the RNA level

[0113] The leaf RNA of the T1 generation of TaSMAX1 transgenic overexpression materials was extracted by the Trizol method, reverse transcribed into cDNA, and the expression level of the target gene was analyzed by RT-qRCR. The primers used were TaSMAX1-qF1: 5’-GCGTCGTCCTCTGAGGGT-3’, TaSMAX1-qR1: 5’-AGATCTTACACCTGAGGTAT-3’. The internal reference gene was TaActin, and its primers were TaActin-F: 5’-ACCTTCAGTTGCCCAGCAAT-3’, TaActin-R: 5’-CAGAGTCGAGCACAATACCAGTTG-3’.

[0114] The results were as Figure 3 shown. Compared with the transgenic receptor material Fielder, the expression levels of the TaSMAX1 gene in the two TaSMAX1 overexpression lines were up-regulated by about 14-fold and 8-fold respectively, indicating that the overexpression transgenic plants were successfully constructed. The two obtained overexpression lines were named TaSMAX1-OE1 and TaSMAX1-OE2.

[0115] 3. Phenotypic study of TaSMAX1 overexpression plants

[0116] The T1 generation seeds of TaSMAX1 overexpression materials TaSMAX1-OE1 and TaSMAX1-OE2 obtained in the above steps and the transgenic receptor material Fielder (abbreviated as WT) were sown in the field during the normal growth period of wheat. 20 plants of each material were sown, and phenotypic observations were carried out when they grew normally to the flowering stage of wheat. It was found that the spike phenotypes of TaSMAX1-OE1 and TaSMAX1-OE2 were consistent, and TaSMAX1-OE1 was selected for photography.

[0117] The results were as Figure 4 shown. At the flowering stage of wheat, compared with Fielder, the spike length of the TaSMAX1 overexpression line (OE1) became longer( Figure 4 in A and B), and the number of florets increased significantly( Figure 4 in C and D).

[0118] At the same time, the panicle traits of the overexpression materials TaSMAX1-OE1 and TaSMAX1-OE2 that had grown normally to the mature stage and the receptor material Fielder were investigated and phenotypic photographs were taken. Table 1 shows the statistical results of the panicle phenotypes of TaSMAX1-OE at the mature stage in the field; more than 6 individual plants of different lines were measured respectively, and the values represent the mean ± SD, ***P<0.001, *P<0.05 (Student's t-test). When investigating the panicle traits at the wheat mature stage, it was found that ( Figure 5 , Table 1), compared with the control, the panicle length of the TaSMAX1-OE lines was significantly increased, while the number of spikelets was less than that of the control Fielder (abbreviated as WT). However, due to the significant increase in the number of florets, the total number of grains per panicle of TaSMAX1-OE was significantly higher than that of the control.

[0119] Table 1. Statistical results of the panicle phenotypes of TaSMAX1-OE at the mature stage in the field

[0120] Panicle traits Fielder TaSMAX1-OE1 TaSMAX1-OE2 Panicle length (cm) 12.23±0.61 14.05±0.33** 14.49±0.69** Spikelet number 20.1±1.04 17.7±0.78*** 17.8±0.87*** Grain number per panicle 73.2±3.44 81.8±7.22* 81.7±6.65*

[0121] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made by using conventional techniques known in the art.

Claims

1. Use of a protein, a substance for regulating the expression of a gene, or a substance for regulating the activity or content of the protein in any of the following: 1) Use of a protein, a substance for regulating the expression of a gene, or a substance for regulating the activity or content of the protein in regulating the ear traits of plants; 2) Use of a protein, a substance for regulating the expression of a gene, or a substance for regulating the activity or content of the protein in the preparation of a product for regulating the ear traits of plants; 3) Use of a protein, a substance for regulating the expression of a gene, or a substance for regulating the activity or content of the protein in cultivating plants with altered ear traits; 4) Use of a protein, a substance for regulating the expression of a gene, or a substance for regulating the activity or content of the protein in the preparation of a product for cultivating plants with altered ear traits; 5) Use of a protein, a substance for regulating the expression of a gene, or a substance for regulating the activity or content of the protein in plant breeding; The protein is any of the following proteins: a1) A protein with an amino acid sequence of SEQ ID No. 2; a2) A protein with the amino acid sequence shown in SEQ ID No. 2 having one or several amino acid residue substitutions and / or deletions and / or additions and having the same function; a3) A protein having more than 80% identity with the amino acid sequence defined in a1) or a2) and having the same function; a4) A fusion protein obtained by linking a tag to the end of the protein defined in any of a1)-a3); The gene encodes the protein.

2. The use according to claim 1, wherein: The protein is derived from wheat.

3. The use according to claim 1 or 2, wherein: The substance for regulating the expression of a gene or the substance for regulating the activity or content of the protein is a biological material related to the protein described in claim 1 or 2, and the biological material is any of the following: c1) A nucleic acid molecule encoding the protein described in claim 1; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) A recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) A transgenic plant tissue containing the nucleic acid molecule described in c1), or a transgenic plant tissue containing the expression cassette described in c2).

4. The use according to claim 3, wherein: c1) The nucleic acid molecule is any of the following DNA molecules, d1) A DNA molecule with a nucleotide sequence shown in SEQ ID No. 3; d2) A DNA molecule with a coding region sequence shown in positions 58-3123 of SEQ ID No. 1 in the sequence listing.

5. A method for increasing the ear length of plants, wherein: The method includes step P, where step P is to enhance, increase or up-regulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, and / or, enhance, increase or up-regulate the expression level of the coding gene of the protein described in claim 1 or 2, so as to increase the spike length of the plant.

6. A method for increasing the number of grains per spike of a plant, characterized in that: the method includes step P, where step P is to enhance, increase or up-regulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, and / or, enhance, increase or up-regulate the expression level of the coding gene of the protein described in claim 1 or 2, so as to increase the number of grains per spike of the plant.

7. A breeding method for cultivating a plant with improved spike traits, characterized in that: it includes enhancing, increasing or up-regulating the expression level of the coding gene of the protein described in claim 1 or 2 in the target plant, and / or, the activity and / or content of the protein, to obtain a plant with improved spike traits.

8. The method according to claim 7, characterized in that: it includes the following steps: (1) Construct a recombinant expression vector that enhances, increases or up-regulates the coding gene of the protein described in claim 1 or 2; (2) Transfer the recombinant expression vector constructed in step (1) into a recipient plant (such as a crop or wheat) to obtain a plant with better spike traits than the recipient plant.

9. The biological material described in claim 3.

10. The application according to any one of claims 1-4, and / or, the method according to any one of claims 5-8, characterized in that: the plant is any one of the following plants: N1) Monocotyledonous plants or dicotyledonous plants; N2) Plants of the order Poales; N3) Gramineous plants; N4) Plants of the genus Triticum; N5) Wheat.

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