Taiip1 gene for regulating wheat grain starch synthesis and use thereof

Abstract

A TaIIP1 gene for regulating wheat grain starch synthesis and use thereof. The present invention belongs to the technical field of gene function regulation. Firstly, the important effect of the wheat isoamylase TaISA1 gene on the quality of grain starch synthesis is clarified. Moreover, the wheat sucrose non-fermentation-related protein kinase TaIIP1 gene, which has a positive regulatory effect on the grain starch synthesis and the phenotype of grains, is found by means of the yeast two-hybrid method and other methods, the function thereof is verified by means of overexpression, and excellent variants thereof are found by means of haplotype analysis. The present invention provides a theoretical basis and excellent gene resources for the use of the TaIIP1 gene to create or improve high-quality wheat materials.

Description

The TaIIP1 gene regulating wheat grain starch synthesis and its application Technical Field

[0001] This invention relates to the field of gene function regulation technology, and more specifically to the TaIIP1 gene that regulates starch synthesis in wheat grains and its applications. Background Technology

[0002] Wheat is an important food crop worldwide. Wheat grains are mainly composed of protein, starch, lipids, crude fiber, and ash. Starch accounts for approximately 65%–75% of the dry weight of wheat grains and significantly influences both processing and consumption quality. Starch exists in granular form and is classified into two types: amylose and amylopectin, but in different proportions. Amylose accounts for about 25% of starch granules, while amylopectin accounts for about 74%. Amylose is a polysaccharide chain composed of D-glucose units linked by α-1,4 glycosidic bonds, and is approximately linear. It is soluble in hot water, turns blue upon contact with iodine, and can be completely decomposed by β-amylase. The solution easily aggregates, forming a semi-solid gel. Amylopectin is a highly branched glucose polymer composed of short α-1,4 glycosidic bonds linked by α-1,6 glycosidic bonds. Its aqueous solution is extremely viscous, turns purple or red upon contact with iodine, and can only be partially decomposed by β-amylase.

[0003] Different enzymes are required at each stage of starch synthesis. Amylose synthesis requires granule-bound starch synthase (GBSS). GBSS specifically binds to starch granules, maintaining the unbranched state of amylose. Amylopectin synthesis requires the combined action of starch branching enzyme (SBE), soluble starch synthase (SSS), and starch debranching enzyme (DBE). SSS catalyzes the elongation of the linear chain of amylopectin; SBE introduces α-1,6 glycosidic branching bonds, forming branch points on the linear glucose chain; and DBE hydrolyzes α-1,6 branching bonds, precisely "pruning" the amylopectin and packaging it into a higher-order, ordered structure—starch granule crystals. Starch debranching enzymes in plants are called R enzymes, and are divided into pullulanase (PUL) and isoamylase (ISA).

[0004] ISA has three isoenzymes capable of removing branched chains from amylopectin and glycogen in plants and animals. Potato StISA1, along with proteins such as maize Sul and rice Sug-1, belongs to the same gene family (ISA1). Chimeric RNAi of the three potato isoamylase genes exhibits tissue-specific damage in tuber starch metabolism, leading to a significant decrease in starch content and altered starch granule size in tubers, but without affecting starch in leaves. Furthermore, it was found that ISA1 directly interacts with FLO6 (a starch-binding CBM domain protein) in rice, affecting starch synthesis during rice seed development. ISA1 and FLO6 also interact with OsLESV, a key regulator of rice starch biosynthesis, forming a functional module that synergistically regulates the synthesis of stored starch in the rice endosperm and endosperm development.

[0005] In durum wheat, RNAi interference revealed that downregulation of ISA1 significantly decreased starch content, while soluble α-glucan and glycogen content significantly increased, and fructose and galactose content relatively decreased. The endosperm of ISA1 knockout progeny showed significantly higher glucan content than the wild type, possibly reflecting a cross-interaction between starch and glucan synthesis pathways. Furthermore, ISA1 in durum wheat contributes to starch synthesis and is crucial for ensuring proper packaging of amylopectin within starch granules.

[0006] Previous studies have yielded some results on the role of isoamylase genes in the starch synthesis pathway of rice and maize, demonstrating that the isoamylase ISA1 gene plays an important role in the initiation stage of starch granule formation. However, the specific function of the TaISA1 gene in starch synthesis and its regulatory mechanism in the complex context of common wheat have not yet been explored. Therefore, this invention creates TaISA1 gene-edited materials in the context of common wheat to further study the function of the TaISA1 gene. In vivo and in vitro interaction experiments identified the TaIIP1 protein that interacts with TaISA1. Sequence alignment revealed that TaIIP1 is a SnRK1b protein, belonging to the SnRK protein kinase family, and is a key kinase in signal transduction and protein modification. By further investigating the interaction pattern between TaISA1 and TaIIP1 proteins, we can explore the basic model by which it participates in regulating grain starch synthesis.

[0007] Sucrose nonfermenting 1-related kinase (SnRK) is highly conserved and ubiquitous in plants. Based on its protein structure, it can be divided into three subfamilies: SnRK1, SnRK2, and SnRK3. SnRK1 is a homologous protein of mammalian AMP-activated protein kinase (AMPK) and yeast sucrose nonfermenting 1 (SNF1) in plants. The structure of SNF1 / AMPK / SnRK1 is highly conserved, comprising three subunits: α, β, and γ, which form a heterotrimeric complex. The α subunit primarily functions as the catalytic unit of the kinase complex; the β subunit acts as a bridge between the α and γ subunits, playing a regulatory role; and the γ subunit primarily functions as the activator. As a key regulator of energy metabolism, SnRK1 is widely involved in plant growth and development, sugar metabolism, starch synthesis, plant stress responses, and plant-pathogen interactions.

[0008] In rice, the three genes encoding SnRK1 are divided into two subfamilies: SnRK1a (OSK1) and SnRK1b (OSK24 and OSK35). SnRK1 participates in regulating starch and sucrose metabolism in plants through sugar signaling and post-translational modifications. Under sugar starvation conditions in rice seedlings, SnRK1a and SnRK1b phosphorylate the transcription factor MYBS1 upstream, promoting its transcriptional activation of the downstream α-AMY3 SRC region, thus alleviating sugar repression. In potatoes, overexpression of the SnRK1 gene increases the expression of two key enzyme genes in the starch synthesis pathway—sucrose synthase and ADP-glucose pyrophosphorylase—leading to a significant increase in starch content in potato tubers. This indicates that the SnRK1 gene controls starch synthesis at the transcriptional level by regulating the expression of sucrose synthase genes and influencing AGPase activity.

[0009] In current crop production, wheat yield and quality are closely related to national food security, the improvement of people's living standards, and national economic development. During wheat grain growth and development, starch accounts for a large proportion of the grain, and the majority of grain weight comes from starch content. Therefore, how to increase the proportion of starch and thus increase grain weight is an important issue that needs to be considered in current production.

[0010] Starch synthesis is an extremely complex process requiring the synergistic action of multiple enzymes and starch synthesis-related proteins. Although several genes directly encoding starch synthesis-related enzymes have been cloned from various cereals, and their functions can be studied by creating mutants through EMS mutagenesis or knocking out individual target genes using technologies such as CRISPR-Cas9, the complexity of starch synthesis means that other genes may interact with amylase-encoding genes to jointly participate in the process. Therefore, the complex functions and overall synergistic regulatory mechanisms of starch-related genes in wheat require further investigation to clarify the starch synthesis mechanism and the regulatory patterns of starch synthesis-related enzymes, providing strong theoretical support for improving wheat quality and yield. Summary of the Invention

[0011] In view of this, the present invention provides the TaIIP1 gene that regulates starch synthesis in wheat grains and its application.

[0012] The problem to be solved by this invention is to clarify the biological function of the wheat TaIIP1 gene and its important role in regulating grain starch synthesis and increasing grain weight, so as to provide theoretical support and gene resources for the creation or improvement of high-yield and high-quality wheat.

[0013] To address the aforementioned issues, this invention first creates an edited material of the TaISA1 gene, which controls the quality of starch synthesis in wheat, clarifying the important role of the TaISA1 gene in grain phenotype, internal starch synthesis, and flour quality; the nucleotide sequence of the TaISA1 gene is shown in SEQ ID No. 1.

[0014] Furthermore, the TaIIP1 protein, a sucrose non-fermentation-related protein kinase, was identified as interacting with the isoamylase ISA1 protein and involved in starch synthesis. The applications are: (1) overexpression of the TaIIP1 gene encoding the TaIIP1 protein increases wheat grain weight and affects wheat grain starch synthesis; (2) screening for superior haplotypes of TaIIP1 that can increase grain weight from natural populations and applying them in actual breeding. The nucleotide sequence of the sucrose non-fermentation-related protein kinase TaIIP1 gene is shown in SEQ ID No. 2.

[0015] To achieve the above objectives, the present invention adopts the following technical solution:

[0016] The TaIIP1 gene, which regulates starch synthesis in wheat grains, has the nucleotide sequence shown in SEQ ID No. 2.

[0017] The above-mentioned application of the TaIIP1 gene in regulating wheat grain starch synthesis.

[0018] Furthermore, it is used for the following purposes:

[0019] (1) Increase the length, width and thickness of the grains;

[0020] (2) Increase the thousand-grain weight of the grains;

[0021] (3) Increase the total starch content, amylose content and resistant starch content of the grains.

[0022] Furthermore, the TaIIP1 interacts with the TaISA1 protein.

[0023] Application of overexpression of the TaIIP1 gene in constructing a wheat model that increases starch content and grain weight.

[0024] The above-mentioned application of the TaIIP1 gene in wheat breeding.

[0025] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0026] (1) This invention first clarified the important role of the wheat isoamylase TaISA1 gene in the quality of grain starch synthesis. At the same time, through yeast double hybridization and other methods, the wheat sucrose non-fermentation related protein kinase TaIIP1 gene, which has a positive regulatory effect on grain starch synthesis and grain phenotype, was found, providing a theoretical basis for creating or improving high-quality wheat materials using the TaIIP1 gene.

[0027] (2) This invention provides a specific application of the TaIIP1 gene in breeding work. This application is achieved by constructing an overexpression recombinant vector containing the TaIIP1 gene, transforming wheat embryos, and obtaining transgenic plants that overexpress the TaIIP1 gene. It has been verified that overexpression of the TaIIP1 gene can significantly increase starch content and grain weight. At the same time, in natural populations, excellent haplotypes that can improve grain width and grain weight by screening for the TaIIP1 gene through molecular markers can be used as an important germplasm resource in breeding work.

[0028] (3) This invention is of great significance in revealing the important role of genes related to non-starch synthesis pathways in starch synthesis regulation and grain weight improvement. It has obtained transgenic wheat overexpressing the TaIIP1 gene and excellent haplotype germplasm resources from natural populations, providing guidance for the breeding of high-yield and high-quality wheat varieties from a molecular biology perspective. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 is a flowchart of the verification method for the application of the wheat TaIIP1 gene in regulating grain starch synthesis and grain weight in this invention.

[0031] Figure 2 shows the grain phenotypes and starch deficiency phenotypes of wheat after TaISA1 gene editing in Example 2 of this invention. A represents the yield per plant for different TaISA1 editing types; B represents the thousand-grain weight for different TaISA1 editing types; C represents the grain length; D represents the grain width; E represents the grain length and width phenotypes for different TaISA1 editing types; F represents the total starch content; G represents the soluble sugar content; H represents the amylose content; I represents a comparison of scanning electron microscopy images of starch granules at different post-flowering stages between wild-type and isa1-abd triple-mutant materials; WT represents the wild-type control Fielder; isa1-a represents the single-deficient ISA1-A gene-edited material; isa1-b represents the single-deficient ISA1-B gene-edited material; isa1-d represents the single-deficient ISA1-D gene-edited material; isa1-ab represents the double-deficient ISA1-AB gene-edited material; isa1-ad represents the double-deficient ISA1-AD gene-edited material; and isa1-abd represents the triple-deficient ISA1-ABD gene-edited material.

[0032] Figure 3 shows the results of three methods in Example 3 of this invention to demonstrate the interaction between TaIIP1 protein and TaISA1 protein. Among them, A is the result of the yeast two-hybrid experiment; B is the result of the luciferase complementation experiment; and C is the result of the in vitro MBP pull-down experiment.

[0033] Figure 4 is a schematic diagram of the recombinant vector overexpressing the TaIIP1 gene in Example 4 of the present invention;

[0034] Figure 5 shows the phenotypes of wheat plants and grains overexpressing the TaIIP1 gene in Example 4 of this invention. In the figure, A represents plant morphology (scale bar: 10cm); B represents grain morphology (scale bar: 0.5cm); C represents grain morphology (scale bar: 1cm); D represents thousand-grain weight; E represents grain length; F represents grain width; G represents grain thickness; OE-1, OE-2, and OE-3 represent three lines overexpressing the TaIIP1 gene, and the same applies below.

[0035] Figure 6 shows the changes in starch granule morphology and starch content in wheat grains overexpressing the TaIIP1 gene in Example 4 of this invention. In the figure, A is a scanning electron microscope image; B is the total starch content result; C is the amylose content result; D is the amylopectin content result; and E is the resistant starch content result.

[0036] Figure 7 shows the natural population association analysis based on the TaIIP1 site molecular marker in Example 5 of the present invention. In this figure, A shows the PCR detection results of four different wheat varieties using molecular markers, where "CS" represents the Chinese spring variety; B is a schematic diagram of the SNP sites identified in the natural population using three molecular markers.

[0037] Figure 8 shows the significance analysis of haplotype analysis in the natural population in Example 5 of the present invention, where A is the plant height result; B is the thousand-grain weight result; C is the grain length result; D is the grain width result; and E is the yield per plant result. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[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] SPSS 22 statistical software was used to process the data, and GraphPad Prism 10.1.2 was used to plot the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 was considered to be statistically significant compared with the control.

[0041] Figure 1 shows a flowchart of the verification method for the application of the wheat TaIIP1 gene in regulating grain starch synthesis and increasing grain weight, as well as in the breeding and improvement of wheat varieties. The embodiments of this invention provide a method for verifying the function of the wheat sucrose non-fermentation-related protein kinase TaIIP1 gene, including: S101, studying the function of the TaISA1 gene using gene-edited materials; S102, obtaining the TaIIP1 protein that interacts with TaISA1 and verifying it experimentally; S103, creating TaIIP1 transgenic wheat and performing molecular detection on the TaIIP1 transgenic wheat; S104, examining the grain phenotype and starch-related phenotype of TaIIP1 overexpression materials to determine the significant impact of TaIIP1 overexpression on starch synthesis; S105, performing association analysis on natural populations based on TaIIP1 site molecular markers to identify superior haplotypes that can significantly increase grain weight.

[0042] Example 1

[0043] This embodiment provides the amino acid sequence of wheat sucrose non-fermentation-related protein kinase IIP1 and its encoding gene.

[0044] In this embodiment, the protein sequence, coding gene sequence, and promoter sequence of the TaIIP1 gene were obtained using BLAST from the Ensembl Plants website (https: / / plants.ensembl.org / Triticum_aestivum / Info / Index). Primers for amplifying the TaIIP1 gene cDNA sequence were designed, and the TaIIP1 gene was amplified and sequenced using cDNA from Fielder plants as a template. The protein-coding CDS sequence of the TaIIP1 gene is shown in SEQ ID No. 3, the promoter sequence is shown in SEQ ID No. 4, and its amino acid sequence is shown in SEQ ID No. 5. The forward primer TaIIP1-cDNA-F for amplifying the TaIIP1 gene cDNA sequence is shown in SEQ ID No. 6, and the reverse primer TaIIP1-cDNA-R is shown in SEQ ID No. 7.

[0045] Example 2

[0046] 1. Field planting and propagation to obtain TaISA1 gene-edited materials with different editing types and stable genetic inheritance (hexaploid common wheat contains three genomes: A, B, and D; wild-type materials contain the complete TaISA1 gene in all three genomes: A, B, and D. ISA1-A means that the ISA1 gene in genome A is knocked out using gene editing technology, resulting in the absence of the ISA1 gene in genome A, while the TaISA1 gene in genomes B and D remains. Therefore, it is called ISA1-A single-deficient material;

[0047] ISA1-B is a material in which the TaISA1 gene is knocked out in the B genome alone, while the TaISA1 gene is present in the A and D genomes; ISA1-AB is a material in which the TaISA1 gene is knocked out in both the A and B genomes simultaneously, while the ISA1 gene is present in the D genome, hence it is called ISA1-AB double-deficient material; ISA1-ABD is a material in which the TaISA1 gene is knocked out in the A, B, and D genomes simultaneously, resulting in the complete absence of the TaISA1 gene in the entire wheat genome, hence it is called ISA1-ABD triple-deficient material.

[0048] 2. The harvested seeds are dried and each plant is weighed to obtain the yield per plant for different types of materials.

[0049] 3. The 1000-grain weight, grain length, and grain width of the seeds were examined using a deep seed testing instrument; and the morphology of the seeds was displayed by taking pictures of the seed arrangement.

[0050] 4. Determination of total starch content: The Total Starch Assay Kit from Megazyme was used for the determination.

[0051] 5. Amylose content determination: The K-AMYL kit from Megazyme was used for detection.

[0052] 6. Determination of soluble sugar content:

[0053] Sample preparation: The seeds were ground into whole wheat flour using a sample grinder and dried at 65°C until the moisture content was uniform.

[0054] The specific experimental procedures must be strictly followed in accordance with the instruction manual, as detailed below:

[0055] (1) Sample processing: Weigh 0.1-0.2g of sample, add 1mL of distilled water and grind into a homogenate, pour into a covered centrifuge tube, boil in a water bath for 10min (tightly cover to prevent water loss), cool, centrifuge at 8000g at room temperature for 10min, take the supernatant into a 10mL test tube, dilute to 10mL with distilled water, shake well and set aside.

[0056] (2) Preheat the spectrophotometer or microplate reader for more than 30 minutes, adjust the wavelength to 620nm, and zero the spectrophotometer with distilled water.

[0057] (3) Adjust the water bath to 95℃;

[0058] (4) Preparation of standards: Dilute the standards with distilled water to 0.3, 0.2, 0.1, 0.05, 0.025, and 0.0125 mg / mL;

[0059] (5) Sample loading table (reaction in EP tube):

[0060] Table 1 Sample Addition Table

[0061] Mix well, place in a 95℃ water bath for 10 min (tightly cover to prevent moisture loss), cool to room temperature, take 200 μL and transfer to a micro-volume cuvette or 96-well plate, measure the absorbance at 620 nm, and record them as A blank tube, A test tube, and A standard tube, respectively, and calculate ΔA = A test tube - A blank tube and ΔA standard = A standard tube - A blank tube.

[0062] 7. Scanning electron microscopy observation of starch granules:

[0063] The morphology of starch granules was observed using an environmental scanning electron microscope (Q45 SEM, FEI, The Czech Republic).

[0064] Figure 2 shows the grain phenotypes and starch-deficient phenotypes resulting from TaISA1 gene editing in wheat. Figures 2A and 2B show that TaISA1 gene editing significantly reduced yield per plant and thousand-grain weight, indicating that TaISA1 deletion affects grain weight. Figures 2C and 2D show grain length and width data, with a significant increase in grain length and a significant decrease in grain width. Figure 2E shows a marked change in grain morphology with severe wrinkling. Further investigation revealed that the changes in grain morphology were mainly caused by changes in starch content. Figures 2F, 2G, and 2H show that TaISA1 deletion led to a significant decrease in total starch and amylose content, and an increase in soluble sugar content. Figure 2I shows starch granules extracted from wild-type and isa1-abd completely deleted materials at 10, 15, and 25 days after flowering. Scanning electron microscopy revealed that starch granule synthesis was inhibited, the starch granules were wrinkled and incomplete, and the size of type A starch granules was significantly altered. This indicates that editing the TaISA1 gene affects the starch synthesis process in the grain, resulting in the synthesis of excess soluble sugars and a decrease in grain weight.

[0065] The TaIIP1 protein interacting with TaISA1 was identified using a yeast double-hybrid screening library method.

[0066] 1. The coding region of the ISA1 gene was amplified and inserted into the yeast vector pGBKT7, which was double-digested with BamHI and EcoRI, as a bait plasmid;

[0067] 2. The constructed bait plasmid and library plasmid were co-transformed into yeast strain Y2H Gold. The preparation of competent cells and the detailed transformation process were performed according to the method provided by Coolaber's yeast two-hybrid transformation kit.

[0068] 3. The transformed bacterial culture was plated on SD / -Leu / -Trp, SD / -His / -Leu / -Trp and SD / -Ade / -His / -Leu / -Trp plates respectively. After incubating at 30℃ for 2-3 days, the colonies were sent for testing to obtain the TaIIP1 protein that interacts with the bait protein TaISA1.

[0069] Example 3

[0070] This embodiment provides a method for verifying the interaction between TaIIP1 protein and TaISA1 protein.

[0071] 1. Yeast two-hybrid:

[0072] The coding regions of the TaIIP1 and ISA1 genes were amplified and inserted into the yeast vectors pGADT7 and pGBKT7, which were double-digested with BamHI and EcoRI, respectively. After successful sequencing, plasmids were extracted for later use. The forward primer TaIIP1-AD-F (nucleotide sequence shown in SEQ ID No. 8) and the reverse primer TaIIP1-AD-R (nucleotide sequence shown in SEQ ID No. 9) were constructed for the pGADT7 yeast expression vector; the forward primer ISA1-BD-F (nucleotide sequence shown in SEQ ID No. 10) and the reverse primer TaISA1-BD-R (nucleotide sequence shown in SEQ ID No. 11) were constructed for the pGBKT7 yeast expression vector.

[0073] All defective culture media and other reagents used in the yeast two-hybrid experiment were purchased from Coolaber. Different combinations of plasmids were co-transformed into yeast strain Y2H Gold, and strict experimental control groups were set up. The preparation of competent cells and the detailed transformation process were performed according to the methods provided in Coolaber's yeast two-hybrid transformation kit.

[0074] The plasmid combination is as follows:

[0075] pGADT7+pGBKT7: Empty control combination;

[0076] pGADT7+pGBKT7-ISA1: Self-activation detection combination;

[0077] pGADT7-IIP1+pGBKT7-ISA1: Experimental combination.

[0078] 2. Luciferase Complementation Assay (LUC):

[0079] The coding regions of the TaISA1 and TaIIP1 genes were constructed into the pCAMBIA1300-nLUC and pCAMBIA1300-cLUC vectors, respectively, and transformed into Agrobacterium to express the target genes in tobacco. The target genes were exogenously introduced into tobacco. Tobacco plants with excellent growth and thick, dark green leaves were selected for transient transformation, and samples were collected 2–3 days later. The underside of the tobacco leaves was evenly coated with a LUC luminescent substrate, incubated in the dark for 5 minutes, and the fluorescence signal was observed using a live-cell imaging system. Construct the forward primer TaISA1-nLUC-F (nucleotide sequence shown in SEQ ID No. 12) and the reverse primer TaISA1-nLUC-R (nucleotide sequence shown in SEQ ID No. 13) for the pCAMBIA1300-nLUC vector; and the forward primer TaIIP1-cLUC-F (nucleotide sequence shown in SEQ ID No. 14) and the reverse primer TaIIP1-cLUC-R (nucleotide sequence shown in SEQ ID No. 15) for the pCAMBIA1300-cLUC vector.

[0080] 3. In vitro MBP pull-down experiment:

[0081] (1) Add equal amounts of MBP-TaISA1 protein and empty MBP protein (about 2 μg) to a 2.0 mL centrifuge tube, add 20 μL of MBP beads, and make up to 1 mL with Binding Buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% Triton X-100 and protease inhibitor Cocktail), and incubate at 4 °C on a rotating shaker for 0.5–1 h;

[0082] (2) Adsorb the beads with a magnetic rack and wash the beads three times with Binding Buffer;

[0083] (3) Add equal amounts of His-TaIIP1 protein (about 2 μg) to centrifuge tubes and incubate at 4°C on a shaker for 2-3 hours.

[0084] (4) Adsorb the beads with a magnetic rack and wash the beads 8 times with Binding Buffer;

[0085] (5) Resuspend the beads in an equal volume of 1×SDS-PAGE Loading Buffer and denature at 100℃ for 10 min; perform SDS-PAGE analysis on the samples using antibodies MBP (MBL, M091-3) and His (MBL, D291-7), respectively. Construct the forward primer TaISA1-MBP-F (nucleotide sequence as shown in SEQ ID No. 16) and the reverse primer TaISA1-MBP-R (nucleotide sequence as shown in SEQ ID No. 17) for the pMal-MBP vector; construct the forward primer TaIIP1-His-F (nucleotide sequence as shown in SEQ ID No. 18) and the reverse primer TaIIP1-His-R (nucleotide sequence as shown in SEQ ID No. 19) for the pCold-His vector.

[0086] Figure 3 shows the results of three methods demonstrating the interaction between TaIIP1 and TaISA1 proteins. Figure 3A shows that the yeast double-hybrid assay demonstrated that pGADT7-TaIIP1 and pGBKT7-TaISA1 could grow normally on a four-cell plate, while the control empty vector combination could not grow. Figure 3B shows that the luciferase complementation (LUC) assay revealed that TaISA1-nLUC and TaIIP1-cLUC were co-injected into tobacco and expressed in vivo using an in vivo imaging system. Figure 3C shows that the in vitro MBP pull-down assay further confirmed that after simultaneously adding TaISA1-MBP and TaIIP1-His, TaISA1-MBP protein was enriched using MBP beads, and TaIIP1-His was simultaneously enriched using the His(MBL,D291-7) antibody. All these experimental results demonstrate the interaction between TaISA1 and TaIIP1 proteins.

[0087] Example 4

[0088] This embodiment provides the application of overexpression of the wheat TaIIP1 gene in regulating grain starch synthesis and grain weight.

[0089] 1. Construct a recombinant vector containing the TaIIP1 gene overexpression, and transfer it to wheat embryos using Agrobacterium-mediated genetic transformation technology to obtain transgenic wheat overexpressing the TaIIP1 gene. Positive plants were screened out by PCR detection and electrophoresis verification.

[0090] The construction method of the recombinant vector containing the TaIIP1 gene overexpression is as follows:

[0091] (1) Using Fielder genomic DNA as a template, the promoter sequence of the TaIIP1 gene was amplified using the forward primer TaIIP1-NP-F and the reverse primer TaIIP1-NP-R; the TaIIP1 gene and the GFP gene were amplified using the forward primer TaIIP1-GFP-F and the reverse primer TaIIP1-GFP-R as a template.

[0092] (2) Homologous recombination of the two fragments and the overexpression vector was performed using a seamless cloning kit (Vazyme, C115-01). The system is as follows:

[0093] Table 2 Homologous Recombination System

[0094] After successful sequencing, transfer to Agrobacterium for later use.

[0095] Primers for constructing overexpression recombinant vectors:

[0096] TaIIP1-NP-F (nucleotide sequence as shown in SEQ ID No. 20);

[0097] TaIIP1-NP-R (nucleotide sequence as shown in SEQ ID No. 21).

[0098] TaIIP1-GFP-F (nucleotide sequence shown in SEQ ID No. 22);

[0099] TaIIP1-GFP-R (nucleotide sequence shown in SEQ ID No. 23).

[0100] Figure 4 is a schematic diagram of the recombinant vector overexpressing the TaIIP1 gene. In Figure 4, "Native promoter" represents the promoter of the TaIIP1 gene, "TaIIP1" represents the inserted TaIIP1 gene sequence, "GFP" represents green fluorescent protein, "T-NOS" represents the NOS promoter, and "Bar" represents the glufosinate acetyl-CoA transferase gene, which is resistant to the herbicide glufosinate.

[0101] 2. Wheat plants that stably overexpress the TaIIP1 gene were planted in the field, and their plant height was observed after full maturity. After harvest, the phenotypic characteristics of the grains, such as grain length, grain width, grain thickness, and thousand-grain weight, were examined and statistically analyzed. The harvested grains were milled, and the physiological and biochemical indicators, such as starch content, starch granule size distribution, and starch granule morphology, were further measured and statistically analyzed.

[0102] Figure 5 shows the field phenotype and grain trait statistics of wheat plants stably overexpressing the TaIIP1 gene. As shown in Figure 5A, the plant morphology and height of the overexpressing plants were not significantly different from those of the wild-type plants. Figures 5B and 5C show that the grain morphology of the overexpressing TaIIP1 gene plants changed significantly, with larger grains, increased grain length, and increased grain width and thickness. Figures 5D to 5G show the grain testing results, indicating that overexpression of the TaIIP1 gene increases the thousand-grain weight, and the grain length and width are significantly increased compared to the wild-type material, while the grain thickness is extremely significantly increased compared to the wild-type material. This demonstrates that overexpression of the TaIIP1 gene can increase grain size and grain weight.

[0103] 3. Determination of total starch content:

[0104] The test was performed using the Total Starch Assay Kit from Megazyme.

[0105] Sample preparation: The seeds were ground into whole wheat flour using a sample grinder and dried at 65°C until the moisture content was uniform.

[0106] The specific experimental procedures must be strictly followed in accordance with the instruction manual, as detailed below:

[0107] (1) Take a 10mL glass test tube, rinse it with deionized water and dry it. Accurately weigh 0.1000g of sample using a balance of 0.01g and carefully place it at the bottom of the 10mL glass test tube.

[0108] (2) Add 200 μL of 80% alcohol to thoroughly wet the sample in the test tube and mix gently with a vortex mixer;

[0109] (3) Dilute the reagent in bottle 1 of the kit 30 times with 100mM, pH 5.0 sodium acetate buffer to prepare solution A. Add 3mL of solution A to the glass tube, boil in water for 6-10 minutes, and shake once every 2 minutes.

[0110] (4) Place the glass tube in a 50°C water bath, add 100 μL of the reagent from bottle 2, mix thoroughly, and incubate at 50°C for 30 min, gently shaking once every 5 min.

[0111] (5) Rinse the sample in the test tube in step (4) thoroughly with a wash bottle, transfer it to another clean 100mL volumetric flask, dilute to volume with deionized water, take 1mL of the solution into a 1.5mL Eppendorf centrifuge tube, centrifuge at 12,000rpm for 10min, and then aspirate 100μL of the supernatant into another clean glass test tube.

[0112] (6) Prepare two clean glass test tubes, and add 100 μL of deionized water and the reagent (D-glucose) from Bottle 3 to each test tube as controls. Add 3 mL of GOPOD solution to each glass test tube and incubate in a 50°C water bath for 30 min;

[0113] (7) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 510 nm, and substitute the result into the formula provided in the instruction manual to calculate the starch percentage content.

[0114] 4. Determination of amylose and amylopectin content:

[0115] The K-AMYL kit from Megazyme was used for detection. Sample preparation was the same as that used for the determination of total starch content.

[0116] The specific experimental procedures must be strictly followed in accordance with the instruction manual, as detailed below:

[0117] A. Sample pretreatment

[0118] (1) Accurately weigh the sample into a 10 mL sample tube and record the sample weight to an accuracy of 0.1 mg.

[0119] (2) Add 1 mL of DMSO to the test tube, mix slowly in a vortex mixer, and heat in a boiling water bath until dispersed (about 1 minute) to ensure that the starch does not clump together.

[0120] (3) Seal the test tube, mix at high speed, heat in a boiling water bath for 15 minutes, and then mix at high speed intermittently.

[0121] (4) Let stand at room temperature for 5 minutes, add 2 mL of 95% ethanol, mix well, then add 4 mL of ethanol, cover and invert to mix well. Starch precipitate will form. Let the test tube stand for 15 minutes (or overnight if conditions permit).

[0122] (5) Centrifuge at 2000 rpm for 5 min, discard the supernatant, invert for 10 min to ensure complete evaporation of ethanol. Use this precipitate in the subsequent determination of amylose and total starch.

[0123] (6) Add 2 mL of DMSO to the starch precipitate and heat the test tube in a boiling water bath for 15 min (mix occasionally) to ensure there are no lumps.

[0124] (7) Remove the test tube from the boiling water bath and immediately add 4 mL of ConA solution. Transfer the reagent in the test tube to a 25 mL volumetric flask and dilute to volume with ConA solution. This solution is solution A. (The entire process must be completed within 2 hours.)

[0125] B. Determination of amylopectin-ConA precipitate and amylose

[0126] (1) Measure 1 mL of solution A into a 2 mL test tube, add 0.54 mL of ConA solution, cover the tube, and invert it repeatedly to mix well. Avoid foaming of the sample.

[0127] (2) Let stand at room temperature for 1 hour. Centrifuge at 14000g for 10 minutes.

[0128] (3) Transfer 1 mL of supernatant to a 15 mL centrifuge tube, add 3 mL of 100 mM, pH 4.5 sodium acetate buffer, mix the reagents, gently plug the tube opening, and boil in a water bath for 5 min to denature ConA.

[0129] (4) Place the test tube in a water bath at 40°C for 5 min to equilibrate. Add 1 mL of a mixture of starch-to-glucosidase and α-amylase. React at 40°C for 30 min and centrifuge at 2000 rpm for 5 min.

[0130] (5) Accurately measure 1 mL of supernatant, add 4 mL of GOPOD reagent, and react at 40℃ for 20 min to allow the blank reagent and D-glucose standard solution to react simultaneously.

[0131] (6) Measure the absorbance of each sample and the D-glucose standard solution at 510 nm.

[0132] C. Determination of total starch

[0133] (1) Mix 0.5 mL of solution A with 4 mL of 100 mM, pH 4.5 sodium acetate buffer.

[0134] (2) Add 1 mL of a mixture of starch-transferase and α-amylase, and react at 40°C for 10 min.

[0135] (3) Accurately measure 1 mL of the supernatant, add 4 mL of GOPOD reagent, and react at 40 °C for 20 min. This reaction must be carried out simultaneously with the sample and the standard solution as described in Part B.

[0136] (4) Measure the absorbance of each sample and the D-glucose standard solution at 510 nm, and use the results to calculate the starch percentage content using the formula provided in the instruction manual.

[0137] 5. Starch granule extraction:

[0138] The starch granule extraction method was improved based on previous experimental methods (Niu L, Ding H, Hao R, et al. A rapid and universal method for isolating starch granules in plant tissues. Plant Cell Environ. 2019 Dec; 42(12):3355-3371.). The specific steps are as follows:

[0139] (1) Grind the seeds in a mortar and grind them in a coarse starch extract until the liquid is turbid and the concentration is appropriate (the solution is milky white when poured into a sieve and can pass through the sieve smoothly without obvious sediment).

[0140] (2) Pass the liquid in the mortar through a 300-mesh sieve and a 500-mesh sieve in sequence to obtain a sieved mixture.

[0141] (3) Add about 2 mL of the sieved mixture to a 2 mL centrifuge tube, centrifuge at 5000 g for 3 minutes at 4℃, and discard the supernatant.

[0142] (4) Add two phases: First add 600 μL of 90% ethanol, shake well, then add 1200 μL of 2.7M sodium dihydrogen phosphate solution, shake well and mix thoroughly, centrifuge at 5000g for 3 minutes at 4℃, and discard the supernatant.

[0143] (5) Add an appropriate amount of water to the centrifuge tube, shake, and centrifuge at 5000g for about 3 minutes.

[0144] (6) Discard the supernatant, air dry at room temperature, and the white precipitate at the bottom is the extracted starch. After air drying, store at -20℃ for later use.

[0145] Table 3. Formula for crude starch extract (200 mL)

[0146] 6. Scanning electron microscopy observation of starch granules:

[0147] The morphology of starch granules was observed using an environmental scanning electron microscope (Q45 SEM, FEI, The Czech Republic).

[0148] 7. Detection of resistant starch content:

[0149] The K-AMYL kit from Megazyme was used for detection. Sample preparation was the same as that used for the determination of total starch content.

[0150] The specific experimental procedures must be strictly followed in accordance with the instruction manual, as detailed below:

[0151] A. Hydrolyzed and soluble non-resistant starch:

[0152] (1) Accurately weigh 100 mg of sample and pour it directly into a 2 mL tube;

[0153] (2) Add 400 μL of α-pancreatic amylase (10 mg / mL) to each test tube, which contains AMG (3 U / mL) (solution 2);

[0154] (3) Tighten the lid, mix well with the vortex shaker, place horizontally in a 20℃ shaker, parallel to the direction of motion, and shake accurately for 16 hours.

[0155] (4) After removing the test tube from the shaker, add 400 μL of anhydrous ethanol, vortex to mix, and terminate the reaction.

[0156] (5) After centrifuging at 1500g for 10 min, collect the supernatant; add 200μL of 50% ethanol, resuspend, add 600μL of 50% IMS (90% anhydrous ethanol + 10% methanol), vortex to mix, centrifuge at 1500g for 10 min, and collect the supernatant.

[0157] (6) Repeat the previous step to fully dissolve the non-resistant starch and lipids.

[0158] B. Determination of resistant starch:

[0159] (1) Place the test tube in an ice bath, add 200 μL of 2M KOH while stirring vigorously, and stir for 20 min to dissolve the resistant starch;

[0160] (2) Add 800 μL of 1.2 M sodium acetate buffer and stir with a magnetic stirrer, then immediately add 10 μL of AMG (bottle 1);

[0161] (3) Mix in a 50℃ water bath for 30 minutes, then vortex intermittently.

[0162] (4) After centrifuging at 1500g for 10 min, aspirate the supernatant in 10 μL increments, make two portions, add 300 μL of GOPOD, and react at 50℃ for 20 min.

[0163] (5) Measure the absorbance of each sample and the D-glucose standard solution at 510 nm, and use the results to calculate the percentage of resistant starch content using the formula provided in the instruction manual.

[0164] Figure 6 shows scanning electron microscope images of starch granules and a statistical chart of starch content detection in grains. As shown in Figure 6A, the morphology of starch granules in the TaIIP1 overexpression material changed significantly. Type A starch granules were significantly larger, with a diameter significantly increased compared to the wild type; Type B starch granules were more numerous and had a significantly increased volume and surface area. Figures 6B-6E show that the starch content of the overexpression grains was significantly different from that of the wild type, with a significant increase in total starch content, amylose content, and resistant starch content. This demonstrates that the TaIIP1 gene may be involved in regulating the starch synthesis process in grains, affecting the final starch content. This further illustrates that overexpression of the TaIIP1 gene can significantly increase grain starch content, thereby increasing grain weight.

[0165] Example 5

[0166] This embodiment provides an association analysis of natural populations based on the TaIIP1 site molecular marker to identify superior haplotypes that can significantly increase grain weight, and their application in further improving the grain weight of high-quality wheat varieties.

[0167] By comparing the reference genome sequences of the TaIIP1 gene region and promoter region of different varieties (Chinese Spring (CS), Fielder, AK58, and Jimai 22), single nucleotide differences were found among the varieties, which were divided into two types: Hap1 and Hap2, as shown in Figure 7B. Therefore, three pairs of molecular markers were designed based on these differences for detection.

[0168] Molecular marker sequences are shown in SEQ ID No. 24 to SEQ ID No. 29.

[0169] First, the genomic DNA of four varieties was used to detect three molecular markers. The detection results are shown in Figure 7A. Agarose gel electrophoresis showed that the target bands of all three molecular markers could be detected, and sequencing could be performed.

[0170] The next step is to identify and classify these three molecular markers in natural populations using sequencing. The identification method is as follows: after sequencing the PCR products, wheat varieties containing nucleotide sequences consistent with the Chinese spring reference genome are classified as Hap1 type, while wheat varieties with single nucleotide differences are classified as Hap2 type. Correlation analysis is then performed with indicators such as plant height, thousand-grain weight, grain length, grain width, and yield of the variety.

[0171] Haplotype analysis of 120 AM3 natural populations using the TaIIP1 gene as a molecular marker revealed that 32 accessions were of the Hap1 type and 88 accessions were of the Hap2 type. The haplotype identification results and phenotypic characteristics of the natural populations are shown in the table below.

[0172] Table 4. Hap1 haplotype results identified by TaIIP1 molecular marker in the AM3 population.

[0173] Table 5. Hap2 haplotype results identified by TaIIP1 molecular marker in the AM3 population.

[0174] In natural populations, wheat materials with the Hap1 haplotype all contained nucleotide sequences consistent with the Chinese spring reference genome; while wheat materials with the Hap2 haplotype all exhibited single-nucleotide differences from the reference genome. The results of the significance analysis are shown in Figure 8. Figures 8A-8E show that the plant height of the Hap2 type tends to decrease, while the thousand-grain weight and yield per plant are significantly increased. Grain morphology also changes, with grain length and width significantly increasing relative to the Hap1 type. This indicates that the presence of different haplotypes of the IIP1 gene in natural populations can significantly increase grain weight and can be further applied in breeding.

[0175] In summary, the application of the wheat TaIIP1 gene in regulating grain starch synthesis and increasing grain weight in this invention mainly focuses on the following two aspects:

[0176] (1) The overexpression material showed significant changes in grain phenotype, with a significant increase in grain length, width, and thickness; a significant increase in total starch content, amylose content, and resistant starch content; and affected the size of type A starch grains and the number, volume, and surface area of ​​type B starch grains, thereby influencing the distribution of important short chain lengths of starch and increasing grain weight. This material provides important theoretical basis and germplasm resources for breeding high-yielding and high-quality wheat varieties.

[0177] (2) Superior haplotype varieties found in natural varieties can significantly increase the thousand-grain weight and grain width, providing important breeding materials for the further breeding and improvement of new varieties.

[0178] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0179] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The TaIIP1 gene, which regulates starch synthesis in wheat grains, is characterized by, Its nucleotide sequence is shown in SEQ ID No.

2.

2. The application of the TaIIP1 gene as described in claim 1 in regulating starch synthesis in wheat grains.

3. The application as described in claim 2, characterized in that, Used for the following purposes: (1) Increase the length, width and thickness of the grains; (2) Increase the thousand-grain weight of the grains; (3) Increase the total starch content, amylose content and resistant starch content of the grains.

4. The application as described in claim 2, characterized in that, The TaIIP interacts with the TaISA1 protein.

5. The application of overexpression of the TaIIP1 gene as described in claim 1 in constructing a wheat model that increases starch content and grain weight.

6. The application of the TaIIP1 gene as described in claim 1 in wheat breeding.

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