Application of TaSMAX1 protein and coding gene thereof in improving gelatinization characteristic of wheat starch

Abstract

The invention discloses application of a TaSMAX1 protein and a coding gene thereof in improving the gelatinization characteristic of wheat starch, and belongs to the technical field of biology. The protein is shown as SEQ ID NO: 2, and the coding gene of the protein is 58th to 3123th sites of SEQ ID NO: 1. Experiments prove that compared with receptor wheat Fielder, the stability index breakdown value and low ebb viscosity of TaSMAX1 protein overexpressed strain starch are obviously improved, the gelatinization temperature is reduced, the food processing performance is improved, and it is proved that overexpressed TaSMAX1 protein can effectively improve the gelatinization characteristic of wheat starch. The invention provides an excellent gene resource and a technical path for cultivating high-yield and high-quality wheat varieties, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of TaSMAX1 protein and its encoding gene in improving the gelatinization properties of wheat starch. Background Technology

[0002] Wheat is the most widely cultivated food crop, providing energy and protein for about half of the world's population. With societal development, people's demands for wheat flour quality have significantly increased. Starch is the main component of wheat grains, accounting for approximately 75% of the total weight. The gelatinization characteristics of starch are one of the important indicators reflecting flour quality. Numerous studies have shown that the gelatinization characteristics of wheat starch have a significant impact on the quality of flour products, especially on the quality of processed noodles. Therefore, using modern molecular breeding techniques to precisely, rapidly, and efficiently improve the gelatinization characteristics of wheat starch is of great significance for improving people's living standards. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to improve the gelatinization properties of wheat starch. To this end, this invention provides the application of a protein, or a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the activity or content of the protein, wherein the protein is TaSMAX1 protein, and is any one of the following:

[0004] a1) A protein with the amino acid sequence SEQ ID NO: 2. a2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 2, but with one or more amino acid residues substituted and / or deleted and / or added. Proteins that share more than 80% identity with the amino acid sequence defined by a3) and a1) or a2) and have the same function. a4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in a1)-a3); The application is any one of the following: b1) Regulating the starch gelatinization characteristics of plant seeds b2) Prepare products that regulate the starch gelatinization properties of plant seeds. b3) Cultivate plants with altered starch gelatinization characteristics in their grains. b4) Prepare products from plants whose grain starch gelatinization properties have been altered. b5) Plant breeding, b6) Prepare products for plant breeding.

[0005] The indicator for plant breeding can be the gelatinization characteristics of wheat starch. The purpose of plant breeding can be to cultivate plants with altered grain starch gelatinization characteristics.

[0006] To facilitate the purification or detection of the protein in a1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

[0007] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0008] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) 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 (monomer red fluorescent protein), or AviTag tag protein.

[0009] Those skilled in the art can readily mutate the nucleotide sequence of the protein TaSMAX1 encoded by this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that have 75% or more of the nucleotide sequence identity with the protein TaSMAX1 isolated by this invention, as long as they encode and function as protein TaSMAX1, are derived from and equivalent to the nucleotide sequence of this invention.

[0010] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0011] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0012] In this document, the 80% or more of identity 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.

[0013] In this document, the above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0014] In the above applications, the protein is derived from wheat ( Triticum aestivum L.).

[0015] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein TaSMAX1.

[0016] In the above text, the substance that regulates gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the coding gene, 2) regulation after transcription of the coding gene, 3) regulation of RNA transport of the coding gene, 4) regulation of translation of the coding gene, 5) regulation of mRNA degradation of the coding gene, and 6) regulation after translation of the gene.

[0017] In this invention, the regulation can be up-adjusted, enhanced, or increased.

[0018] In the above applications, the substance regulating the expression of the protein-coding gene or the substance regulating the activity or content of the protein can be a biological material related to the protein described above, and the biological material can be any of the following: C1) The nucleic acid molecule that encodes the protein described above. C2) expresses the gene encoding the nucleic acid molecule described in C1). C3) contains the expression cassette of the gene described in C2). C4) A recombinant vector containing the gene described in C2), or a recombinant vector containing the expression cassette described in C3). C5) Recombinant microorganisms containing the gene described in C2), or recombinant microorganisms containing the expression cassette described in C3), or recombinant microorganisms containing the recombinant vector described in C4). C6) A transgenic plant cell line containing the gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4). C7) Transgenic plant tissue containing the gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4). C8) A transgenic plant organ containing the gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4); In the above applications, the nucleic acid molecule described in C1) can be any of the following: D1) The coding region sequence is the DNA molecule shown in positions 58-3123 of SEQ ID NO: 1 in the sequence listing. D2) has 90% or more identity with the nucleotide sequence defined by D1), and is derived from wheat DNA molecule encoding the protein described above. D3) hybridizes under strict conditions with a nucleotide sequence defined by D1) or D2) and encodes a DNA molecule that encodes the protein described above.

[0019] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0020] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the PC186 vector.

[0021] Existing wheat expression vectors can be used to construct structures containing... TaSMAX1 Recombinant gene expression vectors. The wheat expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for wheat microbombardment. The wheat expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall tumor-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0022] use TaSMAX1When constructing a recombinant wheat expression vector, any enhancing or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CaMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing a wheat expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0023] To facilitate the identification and screening of transgenic wheat cells or wheat, the wheat expression vectors used can be processed, such as by adding genes that can be expressed in wheat, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective for transgenic plants, no selective marker genes may be added, and transformed plants can be directly selected by stress screening.

[0024] Using any vector capable of guiding the expression of exogenous genes in wheat, the present invention can be used to express the gene. TaSMAX1 Transgenic cells and transgenic plants with improved starch gelatinization properties can be obtained by introducing genes or gene fragments into wheat cells or recipient wheat. TaSMAX1 Gene expression vectors can be used to transform wheat cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed wheat tissues can be cultured into plants.

[0025] The present invention also provides a method for cultivating plants with improved starch gelatinization properties, the method comprising step P, wherein step P is to enhance, increase or upregulate the activity and / or content of the aforementioned proteins in the target plant, or / and enhance, increase or upregulate the expression level of the encoding genes of the aforementioned proteins, to cultivate plants with improved starch gelatinization properties; the plant is a seed plant.

[0026] The present invention also provides a method for improving the starch gelatinization properties of plant seeds, the method comprising step P, wherein step P is to enhance, increase or upregulate the activity and / or content of the aforementioned proteins in the target plant, or / and enhance, increase or upregulate the expression level of the encoding genes of the aforementioned proteins, thereby improving the starch gelatinization properties of plant seeds; wherein the plant is a seed plant.

[0027] In the above method, increasing the expression level and / or activity of the gene encoding the protein TaSMAX1 in the target plant can be achieved by overexpressing the gene encoding the protein TaSMAX1 in the genome of the target plant to enhance its activity.

[0028] In one embodiment of the present invention, the method may include the following steps: f1) Construct recombinant expression vectors for the coding genes of the proteins described above to enhance, improve, or upregulate their expression. f2) The recombinant expression vector constructed in step f1) is transferred into the recipient (wheat) to obtain plants with starch gelatinization properties superior to those of the recipient.

[0029] In this invention, the starch gelatinization characteristics of the plant seeds can be starch gluten viscosity and / or thinning value and / or gelatinization temperature.

[0030] In this invention, regulating the gelatinization characteristics of plant seed starch can be achieved by increasing the trough viscosity of plant seed starch and / or decreasing the starch dilution value and / or decreasing the starch gelatinization temperature.

[0031] In this invention, the plant may be any of the following: N1) monocotyledonous or dicotyledonous plants; N2) grasses; N3) grasses; N4) wheat; N5) wheat.

[0032] The present invention also provides a product, which may be the protein or biological material described above.

[0033] This invention provides a gene for improving the gelatinization properties of wheat starch. TaSMAX1 The cloning method was developed, and it was revealed that TaSMAX1 is an important protein regulating the gelatinization properties of wheat starch, improving... TaSMAX1 The expression level of this substance can significantly improve the starch gelatinization properties of wheat. This study investigated the effect of overexpression of this substance. TaSMAX1 These findings can improve the starch gelatinization properties of wheat, providing genetic resources for breeding high-yield and high-quality wheat varieties. Attached Figure Description

[0034] Figure 1 for TaSMAX1 Cloning of the full-length cDNA of the target gene; lane M is the D2000 plus DNA Ladder; lanes 1 and 2 are... TaSMAX1 The full-length cDNA is 3139 bp in size.

[0035] Figure 2 for TaSMAX1 Identification results of the T0 generation of transgenic strains.

[0036] Figure 3 forTaSMAX1 Overexpression wheat OETaSMAX1 Gene expression levels were detected in the control group Fielder.

[0037] Figure 4 for TaSMAX1 Overexpression wheat OETaSMAX1 The detection of various indicators of grain starch gelatinization characteristics compared with the control Fielder. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0041] The wheat material “Fielder” used in the following examples was donated by Professor 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 wheatgrain quality improvement. Plant Biotechnology Journal, 19(9):1684–1686. This biological material is available to the public from the applicant and is used only for repeating the experiments of this invention and shall not be used for any other purpose.

[0042] The Escherichia coli strain DH5α in the following examples was purchased from Beijing TransGen Biotech Co., Ltd., China.

[0043] In the examples described below, Agrobacterium strain EHA105 was purchased from Beijing Bomed Gene Technology Co., Ltd., China.

[0044] The vector pBM27 used in the following examples was purchased from Beijing Biomed Biotechnology Co., Ltd., China.

[0045] The carrier PC186 used in the following embodiments was donated by Professor Fu Daolin of the College of Agriculture, Shandong Agricultural University. It is described on page 11, fourth line from the bottom right column of the following document. This biological material is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only for repeating the experiments of this invention and should not be used for other purposes: 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 basalresistance to Fusarium graminearum in barley. Molecular Plant Pathology. 19(8):1995–2010.

[0046] The following examples used SPSS 19.0 statistical software to process the data. The experimental results are expressed as mean ± standard deviation, using Student's... t The test results and These represent significant differences at the 0.05 and 0.01 levels, respectively.

[0047] Example 1 TaSMAX1 Cloning of genes 1. Total RNA extraction from wheat leaves RNA was extracted using the Trizol method, and the specific procedures are as follows: (1) Take 150 mg-200 mg of Fielder wheat seedling leaves and put them into an RNase-free mortar that has been pre-cooled in liquid nitrogen. Grind the material into a uniform powder in liquid nitrogen and then add it into a 2.0 mL RNase-free centrifuge tube. (2) Add 1 mL of Trizol to the centrifuge tube, vortex, and let stand at room temperature for 5 min; (3) Add 200 μL of chloroform, shake vigorously for 30 s, and let stand at room temperature for 5 min; (4) Centrifuge at 12,000 rpm and 4℃ for 15 min, and transfer the supernatant (550-650 μL) to a new 1.5 mL RNase-free centrifuge tube; (5) Add an equal volume of isopropanol to the supernatant, mix thoroughly by inverting, let stand at room temperature for 5-10 min, centrifuge at 12,000 rpm and 4℃ for 15 min, pour off the supernatant and keep the precipitate. (6) Add 1 mL of 75% alcohol to wash the precipitate, centrifuge at 12,000 rpm and 4℃ for 5 min, and discard the supernatant; (7) Repeat step (6), use the nozzle to absorb the alcohol and then place it in the clean bench to dry; (8) Add 50 μL of RNase-free ddH2O to dissolve RNA. This can be done at 50℃ for 5-10 min to aid dissolution and remove polysaccharides and polyphenols. (9) Centrifuge at 12,000 rpm and 4℃ for 5-10 min, and aspirate the supernatant into a new 1.5 mL RNase-free centrifuge tube. Measure the concentration or store at -80℃ for later use.

[0048] 2. cDNA synthesis Reverse transcription was performed using the Novizan HiScript® III RT SuperMix for qPCR (+gDNA wiper) kit, with the entire process conducted on ice. The reaction mixture consisted of 1.5 μg RNA template, 4 μL of 4×gDNA wiperMix, and RNase-free ddH2O to a final volume of 16 μL. The mixture was then pipetted and incubated at 42°C for 2 min.

[0049] The reverse transcription system consisted of 16 μL of the product from the previous reaction and 4 μL of 5×HiScript III qRT SuperMix. After mixing with a pipette, the following reverse transcription reaction program was performed in a PCR instrument: 50℃ for 15 min, 85℃ for 5 sec. The reaction was immediately placed on ice after completion. The solution was diluted with 180 μL of ddH2O and stored at -20℃.

[0050] 3. TaSMAX1 Gene cloning Using the wheat leaf cDNA obtained in the previous step as a template, primers were designed based on the UTR region of the gene for amplification. TaSMAX1 The gene and primer sequences are as follows: TaSMAX1-F: 5'-AGAATTCGTGCCCTTTGATC-3', TaSMAX1-R: 5'-CTTTCTCAAATGCCCTCTACA-3'.

[0051] The PCR amplification system was 50 μL and included the following components: 2×PCR Buffer 25 μL, dNTPs (2 mM) 10 μL, cDNA 1 μL, KOD Fx 1 μL, Primer-F (10 μM) 1.5 μL, Primer-R (10 μM) 1.5 μL, and ddH2O 10 μL.

[0052] PCR reaction program: 94℃ pre-denaturation for 5 min; 98℃ denaturation for 10 sec, 56℃ annealing for 30 sec, 68℃ extension for 3 min 30 sec, 35 cycles; 68℃ extension for 10 min, reaction terminated at 12℃. Amplification products were obtained, including... TaSMAX1 The coding region sequence was separated by 1% agarose gel electrophoresis, and a band of approximately 3100 bp was observed. Figure 1 ), where lane M is the D2000 plus DNA Ladder; lanes 1 and 2 are TaSMAX1 The full-length cDNA is 3149 bp in size, and its nucleotide sequence is SEQ ID NO: 1, as follows:

[0053] The target fragment was rapidly cut into strips using a UV gel cutter and chopped into small pieces. The strips were then placed into clean centrifuge tubes for gel recovery to obtain the gel recovery product.

[0054] TaSMAX1 The coding sequence (CDS) of the gene in the wheat material Fielder is positions 58-3123 of SEQ ID NO: 1, and its encoded amino acid sequence is SEQ ID NO: 2 (1021aa). TaSMAX1 Proteins, specifically as follows:

[0055] 4. TaSMAX1 Connecting pBM27 vector Using the gel recovery product obtained in the previous step as a template, amplification was performed using primers pBM27-TaSMAX1-F: 5'-CACCATGAGGGCGGATCTCAGCA-3' and pBM27-TaSMAX1-R: 5'-CATTCCATCGATGGCAATCG-3'. In pBM27-TaSMAX1-F, "CACC" represents the pBM27 vector adapter sequence. The amplification system was the same as in the previous step, with 50 ng of template added, and water was added to bring the volume to 50 μL. The PCR reaction procedure was the same as in the previous step. After the reaction, the bands were separated by 1% agarose gel electrophoresis, and the target bands were excised and recovered from the gel.

[0056] The gel-recovered product was ligated into the pBM27 vector using a pBM27 cloning kit. The reaction mixture was as follows: 100 ng gel-recovered product, 1 μL pBM27 Vector, 1 μL 10×Toposmart, and ddH2O to a final volume of 10 μL. After addition, the mixture was gently swirl-mixed and centrifuged. The reaction was carried out at 25°C for 30 min using a PCR instrument. After the reaction, the cells were transformed into competent E. coli DH5α cells. Positive clones were identified and cultured, and plasmids were extracted and sent to the company for sequencing. The correctly sequenced plasmid was named pBM27- TaSMAX1 Store at -20℃.

[0057] Recombinant vector pBM27- TaSMAX1 The structure 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 positions 58-3123 of SEQ ID NO: 1, while keeping the other nucleotides of the pBM27 vector unchanged. Recombinant vector pBM27- TaSMAX1 Contains attL1- TaSMAX1 -attL2.

[0058] Example 2 TaSMAX1 Construction of overexpression vectors vector pBM27- TaSMAX1 via LR reaction TaSMAX1 Connected to the PC186 support. The reaction system is pBM27- TaSMAX1 150 ng of PC186, 150 ng of LR enzyme, and ddH2O to a final volume of 5 μL were added. The mixture was incubated overnight at 25°C and then transformed into *E. coli* DH5α competent cells. Single clones were picked for identification, followed by culture and plasmid extraction. The plasmid was sent to the company for sequencing, and the correctly sequenced plasmid was named PC186-. TaSMAX1 .

[0059] Recombinant vector PC186- TaSMAX1 The structure is described as follows: After the LR reaction, the recombinant vector PC186- TaSMAX1 This involves replacing the fragment between the attR1 and attR2 sites in the PC186 vector with attB1- TaSMAX1 -attB2, a recombinant expression vector obtained by keeping other nucleotides of the PC186 vector unchanged. PC186- TaSMAX1 Contains bits 58-3123 of SEQ ID NO: 1 in the sequence list. TaSMAX1 The gene's CDS sequence, Ubi promoter, and NOS terminator enable the expression of the TaSMAX1 protein, whose expression is driven by the Ubi promoter.

[0060] Example 3 TaSMAX1 Overexpression of the gene in wheat significantly enhances the starch gelatinization properties of wheat. 1. TaSMAX1 Obtaining transgenic plants by overexpression Will build the correct PC186- TaSMAX1 The vector was transformed into Agrobacterium competent cells EHA105. Single clones were selected for positive clone identification. The correctly identified positive clones were transferred into wheat Fielder callus using Agrobacterium-mediated genetic transformation. Positive plants were selected using the Bar selection marker provided by the vector. They were then transferred to flower pots and cultured in a greenhouse under normal conditions (16 h light, 8 h dark) to obtain T0 generation positive plants.

[0061] 2. TaSMAX1 Identification of overexpression positive plants 1) PCR identification DNA was extracted from wheat leaves using the CTAB method. 1 cm × 3 cm wheat leaves were cut and placed in a 2.0 mL centrifuge tube with steel balls. The tube was immediately frozen in liquid nitrogen for 2 min, then pulverized into powder using a high-throughput grinder. Immediately after removal, 600 μL of preheated CTAB (at 65°C) was added, and the mixture was thoroughly mixed. The mixture was then incubated at 65°C for 45 min, inverting the tube every 15 min. After removing the centrifuge tube, 200 μL of chloroform was added, and the mixture was repeatedly inverted until thoroughly mixed. The tube was centrifuged at 12000 rpm at room temperature for 10 min. 600 μL of the supernatant was carefully transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added. The mixture was thoroughly mixed and centrifuged at 12000 rpm at room temperature for 10 min. The supernatant was discarded, and 1 mL of 70% ethanol was added to wash the precipitate. The tube was centrifuged at 12000 rpm for 10 min, and this washing process was repeated once. Residual ethanol was removed using a pipette tip, and the tube was opened and inverted in a clean bench to air dry. 60 μL of CTAB was added to the centrifuge tube. DNA was dissolved in ddH2O, and its concentration and quality were measured using a Nano Drop 2000 micro-ultraviolet spectrophotometer. The DNA was then diluted with ddH2O to 50 ng / μL for later use.

[0062] The transgenic positive plants were identified by PCR. DNA was amplified using two pairs of primers (Primer pair 1: Primer-F1: 5'-ACTCGGTGGGGATGTTATCA -3', Primer-R1: 5'-GCCAAATGTTTGAACGATCGG-3'; Primer pair 2: Primer-F2: 5'-ACCAGGAAGCCCAGTGAAA -3', Primer-R2: 5'-GCCAAATGTTTGAACGATCGG-3'). The PCR system was as follows: 7.5 μL of 2×M5 HiPer plus Taq HiFi PCR Mix, 1 μL of DNA, 0.5 μL of Primer-F (10 μM), 0.5 μL of Primer-R (10 μM), and 5.5 μL of ddH2O.

[0063] PCR reaction procedure: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 72℃ extension for 1 min (2 kb / min), 35 cycles; 72℃ extension for 10 min, reaction terminated at 12℃. PCR products were separated by 1.5% agarose gel electrophoresis, and the results are as follows: Figure 2 As shown, lane M is the D2000 plus DNA Ladder; lanes 1 and 2 are negative control Fielders; and lanes 3 and 4 are T0 generation positive plants. OETaSMAX1The top and bottom images represent two different pairs of primers for identification (primer pair 1 is used in the top image; primer pair 2 is used in the bottom image). The product size in the top image is 2028 bp, and the product size in the bottom image is 1536 bp. Positive plants were selected and self-crossed to obtain the T2 generation for subsequent phenotypic analysis.

[0064] 2) RNA level identification Extraction using the Trizol method TaSMAX1 Transgenic wheat T2 generation leaf RNA was overexpressed, and cDNA was obtained by reverse transcription. The expression level of the target gene was analyzed by RT-qRCR. The primers used were TaSMAX1-qF1: 5'-GCGTCGTCCTCTGAGGGT-3' and TaSMAX1-qR1: 5'-AGATCTTACACCTGAGGTAT-3'. The internal reference gene was... TaActin The primers are TaActin-F: 5'-ACCTTCAGTTGCCCAGCAAT-3' and TaActin-R: 5'-CAGAGTCGAGCACAATACCAGTTG-3'.

[0065] The results are as follows Figure 3 As shown, compared to the transgenic recipient wheat Fielder, TaSMAX1 In overexpression lines TaSMAX1 The gene expression level was upregulated by approximately 14-fold, indicating that the overexpression transgenic plant was successfully constructed. The obtained overexpression line was named... OETaSMAX1 .

[0066] 3. TaSMAX1 Overexpression enhances wheat starch gelatinization properties Get the T2 generation TaSMAX1 Genetically modified overexpression wheat OETaSMAX1 The seeds of the control variety Fielder were dried to constant weight and then ground into flour.

[0067] The moisture content of the finished flour was determined using the constant weight method at 105℃, in accordance with the method in GB / T5497-1985. The starch gelatinization characteristics were determined using a rapid viscosity analyzer (Perten, RVA4500) according to the following steps: (1) Start the rapid viscosity analyzer and the connected computer, open the software TCW3, and select the standard method standard1 as the measurement mode; (2) Weigh 3.50 g (14% wet basis, accurate to 0.01 g) each of the control and genetically modified wheat flour samples, and add 25.0 g of water (accurate to 0.1 g) to the sample container. Then pour the flour samples into their respective sample containers, stir evenly with a plastic stirrer, and place them in the rapid viscosity analyzer; (3) Start stirring and mixing. The speed is 960 rpm for the first 10 seconds, and then 160 rpm until the end; (4) The initial temperature of the rapid viscosity analyzer is 50℃, and it is maintained for 1 minute. Then the temperature is increased to 95℃ at a speed of 12℃ / minute, maintained at 95℃ for 2.5 minutes, and then decreased to 50℃ at a speed of 12℃ / minute. The temperature is maintained for 13 minutes to complete the test and obtain the peak viscosity ( Figure 4 Medium A), Low trough viscosity ( Figure 4 (B) Sparse value ( Figure 4 C), final viscosity ( Figure 4 D), Resurrection Value ( Figure 4 (Middle E), peak time ( Figure 4 (F), gelatinization temperature ( Figure 4 The test results of G).

[0068] Peak viscosity is the viscosity of the paste as it gradually increases with temperature, reaching its highest point, representing the thermal stability of starch. Trough viscosity is the value at which the viscosity reaches its lowest point during starch gelatinization as temperature increases; it is typically used to assess the rheological properties and processing performance of starch, reflecting the stability of the starch paste at high temperatures and representing the shear resistance of the hot paste, playing an important role in improving the cooking resistance of noodles. The thinning value reflects the stability of the starch paste under high temperature and shear force; the higher the value, the easier the hot paste decomposes and the worse the stability, playing an important role in maintaining the elasticity of noodles. Final viscosity is the starch viscosity at which it reaches stability, related to the starch's ability to form a viscous paste or gel after cooking and cooling. Retrogradation value is the difference between the final viscosity and the viscosity at the cooling starting point, representing the starch's aging trend. Peak time is the time required for the paste viscosity to gradually increase with temperature and reach its highest point. Gelatinization temperature is the temperature at which starch begins to gelatinize, reflecting the thermal stability of starch granules; the higher the value, the more difficult the starch is to gelatinize, requiring higher temperatures for processing.

[0069] The results showed that TaSMAX1 Overexpression plants ( OETaSMAX1 The starch obtained from milled wheat grains has a low gluten viscosity. Figure 4The mean positivity rate (CPR) for the intermediate B sample was 1834 ± 46.7 cP, significantly higher than that for the control Fielder sample (1543 ± 59.4 cP); the dilution value (CPR) was... Figure 4 The C content (C) was 823±19.8 cP, significantly lower than the control Fielder's 1170±42.4 cP; the gelatinization temperature (C) was 823±19.8 cP. Figure 4 The mean temperature for medium (G) was 64.8 ± 0.6℃, significantly lower than the control Fielder's 69.8 ± 0.4℃, therefore... TaSMAX1 Overexpression can significantly increase the trough viscosity of wheat starch, reduce the dilution value of wheat starch, and lower the gelatinization temperature of wheat starch.

[0070] also, TaSMAX1 Overexpression had no significant effect on the peak viscosity, final viscosity, peak time, and setback value of wheat starch. The above experiments were performed in triplicate, and the values ​​are expressed as mean ± SD. P <0.05, (Student's t test).

[0071] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a protein or a substance regulating the expression of the protein-coding gene or a substance regulating the activity or content of the protein, characterized in that, The protein is TaSMAX1 protein, and is any one of the following: a1) A protein with the amino acid sequence SEQ ID NO:

2. a2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 2, but with one or more amino acid residues substituted and / or deleted and / or added. Proteins that share more than 80% identity with the amino acid sequence defined by a3) and a1) or a2) and have the same function. a4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in a1)-a3); The application is any one of the following: b1) Regulating the starch gelatinization characteristics of plant seeds b2) Prepare products that regulate the starch gelatinization properties of plant seeds. b3) Cultivate plants with altered starch gelatinization characteristics in their grains. b4) Prepare products from plants whose grain starch gelatinization properties have been altered. b5) Plant breeding, b6) Prepare products for plant breeding.

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

3. The application according to claim 1 or 2, characterized in that, The substance that regulates the expression of the protein-coding gene or the substance that regulates the activity or content of the protein is a biological material related to the protein of claim 1 or 2, wherein the biological material is any one of the following: C1) A nucleic acid molecule encoding the protein described in claim 1 or 2, C2) expresses the gene encoding the nucleic acid molecule described in C1). C3) contains the expression cassette of the gene described in C2). C4) A recombinant vector containing the gene described in C2), or a recombinant vector containing the expression cassette described in C3). C5) Recombinant microorganisms containing the gene described in C2), or recombinant microorganisms containing the expression cassette described in C3), or recombinant microorganisms containing the recombinant vector described in C4). C6) A transgenic plant cell line containing the gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4). C7) Transgenic plant tissue containing the gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4). C8) A transgenic plant organ containing the gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

4. The application according to claim 3, characterized in that, C1) The nucleic acid molecule is any one of the following: D1) The coding region sequence is the DNA molecule shown in positions 58-3123 of SEQ ID NO: 1 in the sequence listing. D2) has 90% or more identity with the nucleotide sequence defined by D1), and is derived from wheat DNA molecule encoding the protein described above. D3) hybridizes under stringent conditions with the nucleotide sequence defined by D1) or D2) and encodes a DNA molecule that encodes the protein of claim 1 or 2.

5. A method for cultivating plants with improved starch gelatinization properties, characterized in that, The method includes step P, which is to enhance, increase or upregulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein described in claim 1 or 2, so as to cultivate plants with improved starch gelatinization properties; the plant is a seed plant.

6. A method for improving the starch gelatinization properties of plant seeds, characterized in that, The method includes step P, which is to enhance, increase or upregulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein described in claim 1 or 2, so as to improve the starch gelatinization characteristics of plant grains; the plant is a seed plant.

7. The method according to claim 5 or 6, characterized in that, The regulation is at least one of the following six types of regulation: e1) Regulation at the transcriptional level of the encoded gene e2) Regulation that occurs post-transcriptionally in the encoded gene e3) Regulation of RNA transport in the encoded gene. e4) Regulation of the translation of the encoded gene e5) Regulation of mRNA degradation of the encoded gene e6) Post-translational regulation of the gene.

8. The application according to any one of claims 1-4, and / or the method according to any one of claims 5-7, characterized in that, The starch gelatinization characteristics of the plant seeds are starch trough viscosity and / or thinning value and / or gelatinization temperature.

9. The application according to any one of claims 1-4, and / or the method according to any one of claims 5-7, characterized in that, The plant is any of the following: N1) Monocotyledonous or dicotyledonous plants; N2) Plants of the order Poales; N3) Gramineae plants; N4) Plants of the Triticum genus; N5) Wheat.

10. The product, characterized in that, The product is the protein described in claim 1 or 2 and / or the biomaterial described in claim 3.

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