TaIIP1 gene for regulating wheat grain starch synthesis and application of TaIIP1 gene

Abstract

The invention discloses a TaIIP1 gene for regulating and controlling wheat grain starch synthesis and application of the TaIIP1 gene. Belongs to the technical field of gene function regulation. According to the invention, the important effect of the wheat isoamylase TaISA1 gene on the grain starch synthesis quality is determined, the wheat sucrose non-fermentation related protein kinase TaIIP1 gene which has a positive regulation effect on grain starch synthesis and grain phenotype is found through a yeast double-impurity method and the like, and the function of the wheat sucrose non-fermentation related protein kinase TaIIP1 gene is verified through overexpression; the excellent variation of the TaIIP1 gene is excavated through haplotype analysis, and a theoretical basis and an excellent gene resource are provided for creating or improving a high-quality wheat material by using the TaIIP1 gene.

Description

Technical Field

[0001] The present invention relates to the technical field of gene function regulation, and more specifically to a TaIIP1 gene for regulating starch synthesis in wheat grains and an application thereof. Background Art

[0002] Wheat is an important food crop in the world. Wheat grains are mainly composed of protein, starch, lipids, crude fiber and ash. Among them, starch accounts for about 65% to 75% of the dry weight of wheat grains, and also affects the processing quality and edible quality of wheat. Starch exists in the form of granules, and is divided into two types: amylose and amylopectin, but the proportions of the two are different. Amylose accounts for about 25% of starch granules, and amylopectin accounts for about 74%. Amylose is a polysaccharide chain formed by D-glucose groups connected by α-1,4 glycosidic bonds, which is approximately linear. It can be dissolved in hot water, turns blue when exposed to iodine, can be completely decomposed by β-amylase, and the solution is easy to precipitate and form a semisolid gel. Amylopectin is a highly branched glucose polymer formed by short α-1,4 glycosidic bond-linked glucose chains connected by α-1,6 glycosidic bonds. The aqueous solution is extremely viscous, turns purple or red when exposed to iodine, and can only be partially decomposed by β-amylase.

[0003] Different enzymes are needed to play a role in each stage of starch synthesis. The synthesis of amylose requires the action of granule-bound starch synthase (GBSS). GBSS can specifically bind to starch granules, allowing amylose to maintain its original unbranched state. The synthesis of amylopectin requires the joint action of starch branching enzyme (SBE), soluble starch synthase (SSS), and starch debranching enzyme (DBE). Among them, SSS catalyzes the extension of the linear chain of amylopectin; SBE introduces α-1,6 glycosidic branch bonds to form branch points on the linear glucose chain; and the function of DBE is to hydrolyze α-1,6 branch bonds, accurately "trim" the amylopectin and package it into a higher-level ordered structure-starch granule crystals. The starch debranching enzyme in plants is called R enzyme, which is divided into pullulanase (PUL) and isoamylase (ISA).

[0004] There are three isoenzymes of ISA, which can remove branch chains in amylopectin and plant and animal glycogen. Potato StISA1 is a product of the same gene family (ISA1) as maize Sul and rice Sug-1. Chimeric RNAi of three potato isoamylase genes showed tissue-specific damage in starch metabolism in tubers, resulting in a significant decrease in starch content and changes in starch granule size in tubers, but did not affect starch in leaves. In addition, it was found that ISA1 directly interacts with FLO6 (a starch-binding CBM domain protein) in rice, affecting starch synthesis during rice seed development, and ISA1 and FLO6 simultaneously interact with OsLESV, a key regulator of rice starch biosynthesis, to form a functional module that synergistically regulates the synthesis of storage starch in rice endosperm and endosperm development.

[0005] In durum wheat, RNAi interference was used to find that downregulation of ISA1 led to a significant decrease in starch content, while soluble α-glucan and phytoglycogen content increased significantly, while fructose and galactose decreased significantly. The glucan content in the endosperm of ISA1 knockout offspring was significantly higher than that of the wild type, which may reflect the cross-talk between starch and glucan synthesis pathways. In addition, ISA1 in durum wheat contributes to starch synthesis, which is very important for ensuring the correct packaging of amylopectin in starch granules.

[0006] Previous studies on the role of isoamylase genes in the starch synthesis pathway of rice and corn have yielded certain research results, proving that the isoamylase ISA1 gene plays an important role in the initial stage of starch granule formation. However, the specific function of the TaISA1 gene in starch synthesis and its mechanism for regulating starch synthesis in the complex background of common wheat have not been explored. Therefore, the present invention creates TaISA1 gene editing materials in the background of common wheat to further study the function of the TaISA1 gene. In vivo and in vitro interaction experiments found the TaIIP1 protein that interacts with TaISA1, and sequence alignment revealed that TaIIP1 is a SnRK1b protein, which belongs to the SnRK protein kinase family and is a key kinase for signal transduction and protein modification. By further studying the interaction pattern of TaISA1 and TaIIP1 proteins, the basic model for their involvement in regulating grain starch synthesis is explored.

[0007] Sucrose nonfermenting 1-Related Kinase (SnRK) is highly conserved and ubiquitous in plants. According to its protein structure, it can be divided into three subfamilies: SnRK1, SnRK2 and SnRK3. SnRKI is the 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, including three subunits, α, β and γ, which form a heterotrimeric complex. Among them, the α subunit mainly performs the catalytic activity of the kinase complex; the β subunit acts as a bridge that can connect the α and γ subunits and has a regulatory effect; the γ subunit mainly plays an activating role. As a key regulator of energy metabolism, SnRK1 is widely involved in plant growth and development, sugar metabolism, starch synthesis, plant stress response, and the interaction between plants and pathogens.

[0008] In rice, the three genes encoding SnRK1 are divided into two subfamilies, namely SnRK1a (OSK1) and SnRK1b (OSK24 and OSK35). SnRK1 participates in regulating starch and sucrose metabolism in plants through sugar signal transduction and post-translational modification. Under sugar starvation conditions in rice seedlings, SnRK1a and SnRK1b phosphorylate the transcription factor MYBS1 upstream, prompting it to transcriptionally activate the downstream α-AMY3 SRC region, alleviating sugar inhibition. Overexpression of the SnRK1 gene in potato increased the expression of two key enzyme genes in the starch synthesis pathway, sucrose synthase and ADP-glucose pyrophosphorylase, resulting in a significant increase in the starch content in potato tubers, indicating that the SnRK1 gene controls starch synthesis by regulating the expression of the sucrose synthase gene at the transcriptional level and affecting the activity of AGPase.

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

[0010] The process of starch synthesis is extremely complex and requires the synergistic action of multiple enzymes and starch synthesis-related proteins. Although multiple genes directly involved in encoding starch synthesis-related enzymes have been cloned in a variety of cereals, and mutants have been created through EMS mutagenesis or single target genes have been knocked out through CRISPR-Cas9 and other technologies to study their functions, due to the complexity of starch synthesis, other genes may interact with the amylase-encoding genes and participate in the starch synthesis process together. Therefore, the complex functions and overall synergistic regulatory mechanisms of starch-related genes in wheat still need to be further studied, so as to clarify the starch synthesis mechanism and the regulation mode of starch synthesis-related enzymes, and provide strong theoretical support for wheat quality improvement and yield increase. Summary of the invention

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

[0012] The problem to be solved by the present invention is to clarify the biological function of 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 solve the above problems, the present invention first creates an isoamylase TaISA1 gene editing material for controlling the quality of starch synthesis in wheat, and clarifies the important role of the TaISA1 gene on grain phenotype, internal starch synthesis and flour quality; the nucleotide sequence of the isoamylase TaISA1 gene is shown in SEQ ID No.1.

[0014] Furthermore, a protein kinase TaIIP1 protein related to sucrose non-fermentation that interacts with the isoamylase ISA1 protein and is related to the starch synthesis process was found. The application is (1) overexpressing the TaIIP1 gene encoding the TaIIP1 protein can increase the grain weight of wheat grains and affect the starch synthesis of wheat grains; (2) screening TaIIP1 excellent haploid materials that can increase grain weight from natural populations and apply them in actual breeding. The nucleotide sequence of the sucrose non-fermentation protein kinase TaIIP1 gene is shown in SEQ ID No.2.

[0015] In order to achieve the above object, the present invention adopts the following technical solution:

[0016] The nucleotide sequence of the TaIIP1 gene that regulates starch synthesis in wheat grains is shown in SEQ ID No.2.

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

[0018] Further, it is used in the following aspects:

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

[0020] (2) Increase the thousand-grain weight of 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] The application of overexpressing the above-mentioned TaIIP1 gene in constructing a wheat model with increased starch content and grain weight.

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

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

[0026] (1) The present invention first clarifies the important role of the wheat isoamylase TaISA1 gene in the quality of grain starch synthesis. At the same time, through yeast two-hybrid methods, the wheat sucrose non-fermentation-related protein kinase TaIIP1 gene that has a positive regulatory effect on grain starch synthesis and grain phenotype is found, providing a theoretical basis for using the TaIIP1 gene to create or improve high-quality wheat materials.

[0027] (2) The present invention provides a specific application of the TaIIP1 gene in breeding work. The application is to construct an overexpression recombinant vector containing the TaIIP1 gene, transform wheat embryos, and obtain transgenic plants overexpressing the TaIIP1 gene. It has been verified that overexpressing the TaIIP1 gene can significantly increase starch content and grain weight. At the same time, molecular markers are used to screen for excellent haplotypes of the TaIIP1 gene that can increase grain width and grain weight in natural populations, and the haplotypes can be used as important germplasm resources in breeding work.

[0028] (3) The present invention is of great significance in revealing the important role of genes related to non-starch synthesis pathways in regulating starch synthesis and increasing grain weight. The acquisition of transgenic wheat overexpressing the TaIIP1 gene and excellent haploid germplasm resources in natural populations provides guidance for the breeding of high-yield and high-quality wheat varieties from a molecular biology perspective. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0030] Figure 1 This is a flow chart of a verification method for the application of the wheat TaIIP1 gene in regulating grain starch synthesis and grain weight of the present invention;

[0031] Figure 2 It is a grain phenotype and starch deficiency phenotype diagram produced after wheat TaISA1 gene editing in Example 2 of the present invention, wherein A is a result diagram of single plant yield of different editing types of TaISA1; B is a result diagram of thousand-grain weight of different editing types of TaISA1; C is a result diagram of grain length; D is a result diagram of grain width; E is a phenotype diagram of grain length and grain width of different editing types of TaISA1; F is the result of total starch content; G is the result of soluble sugar content; H is the result of amylose content; I is a scanning electron microscopic comparison diagram of starch granules of wild-type and isa1-abd triple-mutant material grains at different stages after flowering; WT represents the wild-type control Fielder; isa1-a represents gene-edited ISA1-A single-deficient material; isa1-b represents gene-edited ISA1-B single-deficient material; isa1-d represents gene-edited ISA1-D single-deficient material; isa1-ab represents gene-edited ISA1-AB double-deficient material; isa1-ad represents gene-edited ISA1-AD double-deficient material; isa1-abd represents gene-edited ISA1-ABD triple-deficient material;

[0032] Figure 3 The results of the interaction between TaIIP1 protein and TaISA1 protein proved by the three methods in Example 3 of the present invention, wherein A is the result of yeast two-hybrid experiment; B is the result of luciferase complementation experiment; C is the result of in vitro MBP pull-down experiment;

[0033] Figure 4 Schematic diagram of the recombinant vector for overexpressing the TaIIP1 gene in Example 4 of the present invention;

[0034] Figure 5 The phenotype diagram of wheat plants and grains overexpressing the TaIIP1 gene in Example 4 of the present invention, wherein A is a plant morphology diagram, and the scale in the figure is 10 cm; B is a grain morphology diagram, and the scale in the figure is 0.5 cm; C is a grain morphology diagram, and the scale in the figure is 1 cm; D is the result of 1000-grain weight; E is the result of grain length; F is the result of grain width; G is the result of grain thickness; OE-1, OE-2, and OE-3 represent three strains overexpressing the TaIIP1 gene, respectively, the same below;

[0035] Figure 6 The figure is a graph showing the starch granule morphology and starch content changes of wheat grains with overexpression of the TaIIP1 gene in Example 4 of the present invention, wherein 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 This is the natural population association analysis based on the TaIIP1 locus molecular marker in Example 5 of the present invention, wherein A is the PCR test result of 4 different varieties of wheat by molecular markers, and "CS" represents the Chinese Spring variety; B is a schematic diagram of the existence of SNP sites identified in natural populations using three molecular markers;

[0037] Figure 8 This is a difference significance analysis of the natural population haplotype analysis in Example 5 of the present invention, wherein 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 single plant yield result. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0040] SPSS22 statistical software was used to process the data, and GraphPad Prism 10.1.2 was used to plot the data. The experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 indicated a significant difference compared with the control.

[0041] Figure 1 A flow chart of the verification method for the application of the wheat TaIIP1 gene of the present invention in regulating grain starch synthesis and increasing grain weight and cultivating and improving wheat varieties is given. The embodiment of the present invention provides a verification method for the function of the wheat sucrose non-fermentation-related protein kinase TaIIP1 gene, including: S101, using gene editing materials to study the function of the TaISA1 gene; S102, obtaining the TaIIP1 protein that interacts with TaISA1 and verifying it through experiments; S103, creating TaIIP1 transgenic wheat, and performing molecular detection on TaIIP1 transgenic wheat; S104, investigating the grain phenotype and starch-related phenotype of TaIIP1 overexpression materials, and determining the important effect of overexpression of TaIIP1 on starch synthesis; S105, performing association analysis on natural populations based on TaIIP1 site molecular markers to find excellent haplotypes that can significantly increase grain weight.

[0042] Example 1

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

[0044] In this example, the protein sequence of the IIP1 gene and its coding gene sequence and promoter sequence were obtained by BLAST from the EnsemblPlants website (https: / / plants.ensembl.org / Triticum_aestivum / Info / Index). Primers were designed to amplify the cDNA sequence of the TaIIP1 gene, and the cDNA of the Fielder plant was used as a template to amplify the TaIIP1 gene and sequence it. 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 used to amplify the cDNA sequence of the TaIIP1 gene 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 of different editing types with stable inheritance (hexaploid common wheat contains three genomes, A, B, and D, and the three genomes of the wild-type material, A, B, and D, all contain complete TaISA1 genes. ISA1-A means that the ISA1 gene in the A genome is knocked out by gene editing technology, so that the ISA1 gene in the A genome is deleted, and the TaISA1 gene in the B and D genomes still exists, so it is called ISA1-A single-deficient material;

[0047] ISA1-B is a knockout of the TaISA1 gene in the B genome alone, while the TaISA1 genes in the A and D genomes exist; ISA1-AB is a knockout of the TaISA1 genes in the A and B genomes at the same time, while the ISA1 gene in the D genome exists, so it is called the ISA1-AB double-deficient material; ISA1-ABD is a knockout of the TaISA1 genes in the A, B, and D genomes at the same time, so that there is no TaISA1 gene in the entire wheat genome, so it is called the ISA1-ABD triple-deficient material).

[0048] 2. Dry the harvested seeds and weigh each plant to obtain the single plant yield of materials of different editing types.

[0049] 3. Use the Wanshen seed measuring instrument to examine the thousand-grain weight, grain length, grain width and other indicators of the grains; and display the grain morphology by arranging and photographing the grains.

[0050] 4. Determination of total starch content: Use the Total Starch Assay Kit from Megazyme for detection.

[0051] 5. Determination of amylose content: Use K-AMYL kit from Megazyme Company for detection.

[0052] 6. Determination of soluble sugar content:

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

[0054] The specific experimental operation process is strictly carried out according to the instructions, as follows:

[0055] (1) Sample treatment: Weigh 0.1-0.2 g of sample, add 1 mL of distilled water and grind into a homogenate, pour into a covered centrifuge tube, boil in boiling water for 10 min (cover tightly to prevent water loss), cool, centrifuge at 8000g for 10 min at room temperature, take the supernatant into a 10 mL test tube, dilute to 10 mL 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 620 nm, and zero the spectrophotometer with distilled water;

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

[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 addition table (reaction in EP tube):

[0060] Table 1 Sample addition table

[0061] Reagent (μL) Blank tube Determination tube Standard tube sample - 40 - Standards - - 40 Distilled water 80 40 40 Working fluid 20 20 20 Concentrated sulfuric acid 200 200 200

[0062] Mix well, place in a 95°C water bath for 10 min (cover tightly to prevent water loss), cool to room temperature, transfer 200 μL to a micro-cuvette or 96-well plate, and measure the absorbance at 620 nm, record them as A blank tube, A assay tube, and A standard tube, and calculate ΔA = A assay tube - A blank tube, ΔA standard = A standard tube - A blank tube.

[0063] 7. Scanning electron microscopy observation of starch granules:

[0064] The morphology of starch granules was observed using an environmental scanning electron microscope (Q45 SEM, FEI, The Czech Republic).

[0065] Figure 2 This is the grain phenotype and starch deficiency phenotype produced after wheat TaISA1 gene editing. Figure 2 A. Figure 2 B shows that the yield per plant and 1000-grain weight decreased significantly after TaISA1 gene editing, indicating that TaISA1 deletion can affect grain weight; Figure 2 C. Figure 2 D is the data of grain length and grain width, the grain length increases significantly and the grain width decreases significantly; Figure 2 As shown in E, the grain morphology changed significantly and was severely wrinkled; further investigation revealed that the change in grain morphology was mainly caused by changes in starch content. Figure 2 F. Figure 2 G. Figure 2 It can be seen from H that the loss of TaISA1 led to a significant decrease in the total starch content and amylose content, and an increase in the soluble sugar content; Figure 2 I shows that starch granules were extracted from the seeds of the wild type and the isa1-abd complete deletion material at 10, 15, and 25 days after flowering, and their morphology was observed under a scanning electron microscope, showing that the synthesis of starch granules was blocked, the starch granules were wrinkled and incomplete, and the size of type A starch granules changed significantly. This indicates that TaISA1 gene editing will affect the starch synthesis process of the seeds, resulting in the synthesis of excess soluble sugars and a decrease in grain weight.

[0066] The TaIIP1 protein that interacts with TaISA1 was found through yeast two-hybrid screening library method:

[0067] 1. Amplify the coding region of ISA1 gene and insert it into the yeast vector pGBKT7 digested with BamHI and EcoRI as bait plasmid;

[0068] 2. The constructed bait plasmid and library plasmid were co-transformed into the yeast strain Y2H Gold. The preparation of competent cells and the detailed transformation process were performed according to the method provided by the yeast two-hybrid transformation kit of Coolaber;

[0069] 3. Spread the transformed bacterial solution on SD / -Leu / -Trp, SD / -His / -Leu / -Trp, and SD / -Ade / -His / -Leu / -Trp plates respectively. After placing them in a 30°C incubator for 2 to 3 days, send the grown colonies for testing to obtain the TaIIP1 protein that interacts with the bait protein TaISA1.

[0070] Example 3

[0071] This example provides a method for verifying the interaction between TaIIP1 protein and TaISA1 protein.

[0072] 1. Yeast two-hybrid:

[0073] The coding regions of TaIIP1 and ISA1 genes were amplified and inserted into the yeast vectors pGADT7 and pGBKT7 digested with BamHI and EcoRI, respectively. After successful sequencing, the plasmids were extracted for use. The pGADT7 yeast expression vector was constructed with forward primer TaIIP1-AD-F (nucleotide sequence as shown in SEQ ID No.8) and reverse primer TaIIP1-AD-R (nucleotide sequence as shown in SEQ ID No.9); the pGBKT7 yeast expression vector was constructed with forward primer ISA1-BD-F (nucleotide sequence as shown in SEQ ID No.10) and reverse primer TaISA1-BD-R (nucleotide sequence as shown in SEQ ID No.11).

[0074] 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 the yeast strain Y2H Gold, and a strict experimental control group was set up. The preparation of competent cells and the detailed transformation process were performed according to the method provided by the yeast two-hybrid transformation kit of Coolaber.

[0075] The plasmid combinations are as follows:

[0076] pGADT7+pGBKT7: empty control combination;

[0077] pGADT7+pGBKT7-ISA1: self-activation detection combination;

[0078] pGADT7-IIP1+pGBKT7-ISA1: experimental combination.

[0079] 2. Luciferase complementation assay (LUC):

[0080] The coding regions of TaISA1 and TaIIP1 genes were constructed into pCAMBIA1300-nLUC vector and pCAMBIA1300-cLUC vector respectively, and Agrobacterium was transformed to express in tobacco, and the target gene was introduced into tobacco. Tobacco with good growth and thick green leaves was selected for transient transformation, and samples were taken after 2 to 3 days. The reverse side of the tobacco leaf was evenly coated with LUC luminescent substrate, and the leaves were left in the dark for 5 minutes, and the fluorescence signal was observed with a live imaging device. The pCAMBIA1300-nLUC vector was constructed with the forward primer TaISA1-nLUC-F (nucleotide sequence as shown in SEQ ID No.12) and the reverse primer TaISA1-nLUC-R (nucleotide sequence as shown in SEQ ID No.13); the pCAMBIA1300-cLUC vector was constructed with the forward primer TaIIP1-cLUC-F (nucleotide sequence as shown in SEQ ID No.14) and the reverse primer TaIIP1-cLUC-R (nucleotide sequence as shown in SEQ ID No.15).

[0081] 3. In vitro MBP pull-down assay:

[0082] (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, 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 on a shaker at 4°C for 0.5 to 1 h.

[0083] (2) The beads were adsorbed on a magnetic rack and washed three times with Binding Buffer;

[0084] (3) Add equal amounts of His-TaIIP1 protein (about 2 μg) to each centrifuge tube and incubate on a shaker at 4°C for 2 to 3 h;

[0085] (4) The beads were adsorbed on a magnetic rack and washed 8 times with Binding Buffer;

[0086] (5) Resuspend the beads with 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 pMal-MBP vector forward primer TaISA1-MBP-F (nucleotide sequence as shown in SEQ ID No.16) and reverse primer TaISA1-MBP-R (nucleotide sequence as shown in SEQ ID No.17); construct the pCold-His vector forward primer TaIIP1-His-F (nucleotide sequence as shown in SEQ ID No.18) and reverse primer TaIIP1-His-R (nucleotide sequence as shown in SEQ ID No.19).

[0087] Figure 3 The results of the interaction between TaIIP1 protein and TaISA1 protein were proved by three methods. Figure 3 As shown in A, yeast two-hybrid experiments proved that pGADT7-TaIIP1 and pGBKT7-TaISA1 could grow normally on four-deficient plates, while the control empty vector combination could not grow; Figure 3 B shows that the expression of TaISA1-nLUC and TaIIP1-cLUC was detected in the in vivo imaging instrument after co-injection of tobacco by luciferase complementation experiment (LUC); Figure 3 C shows that the in vitro MBP pull-down experiment further proves that after adding TaISA1-MBP and TaIIP1-His at the same time, MBP beads are used to enrich TaISA1-MBP protein, and TaIIP1-His can be detected by His (MBL, D291-7) antibody. The above experimental results all indicate that TaISA1 protein and TaIIP1 protein interact.

[0088] Example 4

[0089] This example provides an application of a method for overexpressing wheat TaIIP1 gene in regulating grain starch synthesis and grain weight.

[0090] 1. Construct an overexpression recombinant vector containing the TaIIP1 gene, transfer it to wheat embryos using Agrobacterium-mediated genetic transformation technology, obtain transgenic wheat overexpressing the TaIIP1 gene, and perform PCR detection and electrophoresis verification to screen out positive plants.

[0091] The method for constructing the overexpression recombinant vector containing the TaIIP1 gene is as follows:

[0092] (1) Using the forward primer TaIIP1-NP-F and the reverse primer TaIIP1-NP-R with Fielder genomic DNA as a template, the promoter sequence of the TaIIP1 gene was amplified; using the forward primer TaIIP1-GFP-F and the reverse primer TaIIP1-GFP-R with the synthesized TaIIP1-GFP gene plasmid as a template, the TaIIP1 gene and the GFP gene were amplified;

[0093] (2) The two fragments and the overexpression vector were homologously recombined using a seamless cloning kit (Vazyme, C115-01). The system is as follows:

[0094] Table 2 Homologous recombination system

[0095] Components Volume (μL) Clip 1 1.32 Clip 2 2.44 Overexpression vector 1 2×ClonExpress Mix 5 <![CDATA[ddH2O]]> 0.24

[0096] After successful sequencing, transfer to Agrobacterium for future use.

[0097] Primers for constructing overexpression recombinant vector:

[0098] TaIIP1-NP-F (nucleotide sequence is shown in SEQ ID No. 20);

[0099] TaIIP1-NP-R (the nucleotide sequence is shown in SEQ ID No. 21).

[0100] TaIIP1-GFP-F (nucleotide sequence is shown in SEQ ID No. 22);

[0101] TaIIP1-GFP-R (the nucleotide sequence is shown in SEQ ID No. 23).

[0102] Figure 4 Schematic diagram of the recombinant vector for overexpressing the TaIIP1 gene. Figure 4 In the figure, “Native promoter” indicates the promoter of TaIIP1 gene, “TaIIP1” indicates the inserted TaIIP1 gene sequence, “GFP” indicates green fluorescent protein, “T-NOS” indicates NOS promoter, and “Bar” indicates phosphinothricin acetyl-CoA transferase gene, which is tolerant to the herbicide phosphinothricin.

[0103] 2. Plant wheat plants that stably overexpress the TaIIP1 gene in the field and observe their plant height after they are fully mature. After harvest, observe and count the phenotypes of grain length, grain width, grain thickness, and thousand-grain weight. Grind the harvested grains, and further measure and count the physiological and biochemical indicators of the grains, such as starch content, starch particle size distribution, and starch granule morphology.

[0104] Figure 5The field phenotype and grain trait statistics of wheat plants overexpressing the TaIIP1 gene are shown in Figure 2. Figure 5 As shown in A, the plant morphology and plant height of the overexpressing plants were not significantly different from those of the wild-type plants; Figure 5 B. Figure 5 C shows that the grain morphology of plants overexpressing the TaIIP1 gene changed significantly, with larger grains, longer grains, and increased grain width and thickness; Figure 5 D~ Figure 5 G is the result of grain testing, from which we can know that overexpression of TaIIP1 gene can increase the thousand-grain weight of grains, significantly increase the grain length and width compared with the wild-type material, and significantly increase the grain thickness compared with the wild-type material, indicating that overexpression of TaIIP1 gene can make grains larger and increase grain weight.

[0105] 3. Determination of total starch content:

[0106] The Total Starch Assay Kit from Megazyme was used for detection.

[0107] Sample preparation: The seeds were ground into whole wheat flour using a grinding machine and dried at 65°C until the moisture content was consistent.

[0108] The specific experimental operation process is strictly carried out according to the instructions, as follows:

[0109] (1) Take a 10 mL glass test tube, rinse it with deionized water and dry it, accurately weigh 0.1000 g of sample using a 1 / 10,000 balance, and carefully place it at the bottom of the 10 mL glass test tube;

[0110] (2) Add 200 μL of 80% alcohol to thoroughly soak the sample in the test tube and gently mix using a vortex machine;

[0111] (3) Dilute the reagent in bottle 1 of the kit 30 times with 100 mM, pH 5.0 sodium acetate buffer to prepare solution A. Add 3 mL of solution A to the glass tube and place in a boiling water bath for 6 to 10 minutes, shaking every 2 minutes.

[0112] (4) Place the glass tube in a 50℃ water bath, add 100μL of the reagent in bottle 2, mix thoroughly, and place in a 50℃ water bath for 30 min, shaking gently every 5 min.

[0113] (5) Rinse the sample in the test tube in step (4) thoroughly with a wash bottle, transfer it to another clean 100 mL volumetric flask, dilute to volume with deionized water, take 1 mL of the solution into a 1.5 mL Eppendorf centrifuge tube, centrifuge at 12,000 rpm for 10 min, and then aspirate 100 μL of the supernatant into another clean glass test tube;

[0114] (6) Prepare two clean glass test tubes and add 100 μL of deionized water and the reagent in bottle 3 (D-glucose) as a control. Add 3 mL of GOPOD solution to each glass test tube and warm it in a 50°C water bath for 30 min.

[0115] (7) Use an ELISA reader to measure the absorbance at 510 nm and substitute the result into the formula provided in the instructions to calculate the starch percentage.

[0116] 4. Determination of amylose and amylopectin content:

[0117] The K-AMYL kit of Megazyme was used for the test. The sample preparation was the same as the total starch content determination method.

[0118] The specific experimental operation process is strictly carried out according to the instructions, as follows:

[0119] A. Sample pretreatment

[0120] (1) Accurately weigh the sample into a 10 mL sample tube and record the sample weight to the nearest 0.1 mg.

[0121] (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 (approximately 1 minute) to ensure that the starch does not clump.

[0122] (3) Seal the test tube, mix at high speed, heat in a boiling water bath for 15 min, and mix again at high speed intermittently.

[0123] (4) Leave at room temperature for 5 minutes, add 2 mL of 95% ethanol, mix well, then add 4 mL of ethanol, cover with lid, and mix well by inversion. Starch precipitate is formed, and the test tube is left to stand for 15 minutes (if conditions permit, leave it to stand overnight).

[0124] (5) Centrifuge at 2000 rpm for 5 min, discard the supernatant, and invert for 10 min to ensure that all ethanol evaporates. Use this precipitate in the subsequent determination of amylose and total starch.

[0125] (6) Add 2 mL of DMSO to the starch precipitate and place the test tube in a boiling water bath for 15 min (mixing occasionally) to ensure that there are no lumps.

[0126] (7) Remove the test tube from the boiling water bath and immediately add 4 mL of ConA solution. Transfer the drug in the test tube to a 25 mL volumetric flask and adjust the volume with ConA solution. This solution is Solution A. (The entire process must be completed within 2 hours.)

[0127] B. Determination of amylopectin-ConA precipitate and amylose

[0128] (1) Measure 1 mL of solution A into a 2 mL test tube, add 0.54 mL of Con A solution, cover the tube, and invert repeatedly to mix. Avoid bubbling of the sample.

[0129] (2) Let stand at room temperature for 1 hour. Centrifuge at 14000g for 10 minutes.

[0130] (3) Transfer 1 mL of the supernatant to a 15 mL centrifuge tube, add 3 mL of 100 mM, pH 4.5 sodium acetate buffer, mix the drugs, gently plug the tube, and boil in a boiling water bath for 5 min to denature Con A.

[0131] (4) The test tube was placed in a water bath at 40°C for 5 min, 1 mL of a mixture of amylotransferase and α-amylase was added, the reaction was continued at 40°C for 30 min, and centrifuged at 2000 rpm for 5 min.

[0132] (5) Accurately measure 1 mL of supernatant and add 4 mL of GOPOD reagent. React at 40°C for 20 min to allow the blank reagent and D-glucose standard solution to react simultaneously.

[0133] (6) Measure the absorbance of each sample and D-glucose standard solution at 510 nm.

[0134] C. Determination of total starch

[0135] (1) Mix 0.5 mL of solution A and 4 mL of 100 mM, pH 4.5 sodium acetate buffer.

[0136] (2) Add 1 mL of a mixture of amylotransferase and α-amylase and react at 40°C for 10 min.

[0137] (3) Accurately measure 1 mL of 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 standard solution as in Part B.

[0138] (4) Measure the absorbance of each sample and D-glucose standard solution at 510 nm, and substitute the result into the formula provided in the instruction manual to calculate the starch percentage.

[0139] 5. Starch granule extraction:

[0140] The starch granule extraction method was improved based on the previous experimental method (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:

[0141] (1) Grind the seeds in a mortar, add the crude starch extract and continue grinding until the liquid is turbid and has a moderate concentration (the solution is milky white when poured into a sieve and can be sieved smoothly without obvious precipitation).

[0142] (2) Pass the liquid in the mortar through a 300-mesh sieve and a 500-mesh sieve in sequence to obtain a sieved mixed liquid.

[0143] (3) Add about 2 mL of the sieved mixture to a 2 mL centrifuge tube and centrifuge at 5000 g for 3 min at 4°C. Discard the supernatant.

[0144] (4) Add two phases: first add 600 μL of 90% ethanol, shake thoroughly, then add 1200 μL of 2.7 M sodium dihydrogen phosphate solution, shake thoroughly, centrifuge at 5000 g for 3 minutes at 4°C, and discard the supernatant.

[0145] (5) Add an appropriate amount of water to the centrifuge tube, shake it, and centrifuge it at 5000g for about 3 minutes.

[0146] (6) Pour off the supernatant and air-dry at room temperature. The white precipitate at the bottom is the extracted starch. After air-drying, store at -20°C for future use.

[0147] Table 3 Starch crude extract formula (200mL)

[0148]

[0149] 6. Scanning electron microscopy observation of starch granules:

[0150] The morphology of starch granules was observed using an environmental scanning electron microscope (Q45 SEM, FEI, The Czech Republic).

[0151] 7. Resistant starch content detection:

[0152] The K-AMYL kit of Megazyme was used for the test. The sample preparation was the same as the total starch content determination method.

[0153] The specific experimental operation process is strictly carried out according to the instructions, as follows:

[0154] A. Hydrolysis and solubilization of non-resistant starch:

[0155] (1) Accurately weigh 100 mg of sample and pour directly into a 2 mL tube;

[0156] (2) Add 400 μL of α-pancreatic amylase (10 mg / mL) containing AMG (3 U / mL) (Solution 2) to each test tube;

[0157] (3) Close the lid tightly, mix on a vortex oscillator, and place in a 20°C shaker horizontally, parallel to the direction of movement, and shake accurately for 16 h;

[0158] (4) After the test tube is removed from the shaker, add 400 μL of anhydrous ethanol and vortex to mix to terminate the reaction;

[0159] (5) Centrifuge at 1500 g for 10 min and collect the supernatant; add 200 μL of 50% ethanol, resuspend and then add 600 μL of 50% IMS (90% anhydrous ethanol + 10% methanol), vortex mix and centrifuge at 1500 g for 10 min, collect the supernatant;

[0160] (6) Repeat the previous step to fully dissolve the non-resistant starch and lipids.

[0161] B. Determination of resistant starch:

[0162] (1) Place the test tube in an ice bath and add 200 μL of 2 M KOH while stirring vigorously for 20 min to dissolve the resistant starch;

[0163] (2) Add 800 μL of 1.2 M sodium acetate buffer and stir with a magnetic stirrer, then immediately add 10 μL AMG (bottle 1);

[0164] (3) 50°C water bath for 30 min, vortexing intermittently;

[0165] (4) After centrifugation at 1500 g for 10 min, the supernatant was aspirated in 10 μL units, and 300 μL of GOPOD was added in duplicate. The mixture was reacted at 50°C for 20 min.

[0166] (5) Measure the absorbance of each sample and D-glucose standard solution at 510 nm, and substitute the result into the formula provided in the instruction manual to calculate the percentage of resistant starch.

[0167] Figure 6 The following are scanning electron microscope images of starch granules and statistical graphs of starch content in grains. Figure 6 A shows that the morphology of starch granules in materials overexpressing TaIIP1 changes significantly. Type A starch granules become significantly larger, and the diameter of starch granules increases significantly compared with the wild type; the number of type B starch granules increases, and the volume surface area increases significantly. Figure 6 B~ Figure 6 E shows that the starch content of overexpressed grains has changed significantly compared with the wild-type material, and the total starch content, amylose content and resistant starch content of the overexpressed material have increased significantly. This proves that the TaIIP1 gene may be involved in regulating the synthesis of grain starch and affect the final starch content of the grain. It further shows that overexpressing the TaIIP1 gene can significantly increase the starch content of the grain, thereby increasing the grain weight.

[0168] Example 5

[0169] This embodiment provides an association analysis of natural populations based on TaIIP1 locus molecular markers to find an excellent haplotype that can significantly increase grain weight and use it in improving high-quality wheat varieties to further increase grain weight.

[0170] By comparing the reference genome sequences of the gene region and promoter region of TaIIP1 in different varieties (Chinese Spring (CS), Fielder, Avian Anti-58 (AK58), Jimai 22), it was found that there were single nucleotide differences between different varieties, which were divided into two types: Hap1 and Hap2. The single nucleotide differences of the two types are as follows Figure 7 As shown in B. Therefore, 3 pairs of molecular markers were designed for detection based on these differences.

[0171] The molecular marker sequences are as shown in SEQ ID No.24 to SEQ ID No.29.

[0172] First, the three molecular markers were tested using genomic DNA from four varieties. The test results are as follows: Figure 7 As shown in A, agarose gel electrophoresis revealed that the three molecular markers could all detect the target bands and could be sequenced.

[0173] The next step is to identify and sequence the wheat varieties in natural populations based on these three molecular markers. The identification method is: after sequencing the PCR products, the wheat varieties to be identified that contain nucleotide sequences consistent with the Chinese Spring reference genome are Hap1 types, and the wheat varieties with single nucleotide differences are Hap2 types, and the varieties are associated with the plant height, 1000-grain weight, grain length, grain width, yield and other indicators of the variety.

[0174] By using the molecular markers of the TaIIP1 gene to perform haplotype detection on 120 AM3 natural populations, it was found that 32 materials were of Hap1 type and 88 materials were of Hap2 type. The haplotype identification results and trait performance of the natural population are shown in the following table.

[0175] Table 4 Hap1 haplotype results identified by TaIIP1 molecular marker in AM3 population

[0176]

[0177] Table 5 Hap2 haplotype results identified by TaIIP1 molecular marker in AM3 population

[0178]

[0179]

[0180]

[0181] In natural populations, wheat materials with the Hap1 haplotype all have nucleotide sequences consistent with the Chinese Spring reference genome; the nucleotide sequences of wheat materials with the Hap2 haplotype all have single nucleotide differences from the reference genome. Figure 8 shown. Figure 8 A~ Figure 8 E shows that the plant height of Hap2 type has a downward trend, the 1000-grain weight and single plant yield are significantly increased, the grain morphology is changed, and the grain length and width are significantly increased compared with Hap1 type. This shows that the presence of different haplotypes of IIP1 gene in natural populations can significantly increase grain weight and can be further applied in breeding.

[0182] In summary, the application of the wheat TaIIP1 gene of the present invention in regulating grain starch synthesis and increasing grain weight is mainly from the following two aspects:

[0183] (1) The grain phenotype of the overexpression material was significantly changed, with grain length, width and thickness all significantly increased; the total starch content, amylose content and resistant starch content were significantly increased; the size of type A starch granules and the number, volume and surface area of ​​type B starch granules were affected, thereby affecting the distribution of important short chain lengths of starch and increasing grain weight. This material provides an important theoretical basis and germplasm resource for breeding high-yield and high-quality wheat varieties.

[0184] (2) The excellent haploid varieties found in natural varieties can significantly improve the thousand-grain weight and grain width, providing important breeding materials for the further cultivation and improvement of new varieties.

[0185] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0186] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

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

2.

2. Use of the TaIIP1 gene according to claim 1 in regulating starch synthesis in wheat grains.

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

4. The use according to claim 2, characterized in that The TaIIP interacts with the TaISA1 protein.

5. Use of overexpressing the TaIIP1 gene according to claim 1 in constructing a wheat model with increased starch content and grain weight.

6. Use of the TaIIP1 gene according to claim 1 in wheat breeding.

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