Method for creating high amylose corn germplasm using gene editing technology

Abstract

This invention relates to the field of plant genetic engineering technology, and provides a method for creating high-amylose maize germplasm using gene editing technology. This invention designs sgRNA for creating high-amylose maize germplasm, comprising: a specific target maize sequence as shown in SEQ ID NO:1 or 2. Ae1 The sgRNA1 or sgRNA2 of the gene; and / or, sequences specifically targeting maize as shown in SEQ ID NO:3 or 4. SBEI The sgRNA3 or sgRNA4 of the gene. The sgRNA and CRISPR / Cas9 gene editing vector provided by this invention can significantly improve maize... Ae1 and SBEI Gene editing efficiency. The method of this invention creates high-amylose maize germplasm, which can significantly increase the amylose content in maize kernels, facilitating its application in food, healthcare, materials, textiles, papermaking, packaging, environmental protection, and other fields.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to a method for creating high amylose maize germplasm using gene editing technology. Background Technology

[0002] Corn is an important food and feed crop. The main component of corn endosperm is starch, which is divided into amylose and amylopectin. In ordinary corn, amylose accounts for 22-25% of the total starch content, while amylopectin accounts for 75-78%. Due to its unique molecular structure and physicochemical properties, amylose has very high application value and is widely used in the manufacture of more than 5,000 industrial products in over 30 fields, including food, healthcare, materials, textiles, papermaking, packaging, and environmental protection. Currently, China's domestic consumption of amylose is approximately 2 million tons per year. A small amount is extracted from ordinary corn varieties, with an extraction efficiency of only 30%-50%, resulting in high processing costs and failing to meet the requirements of industries such as pharmaceuticals. Extracting amylose from corn with high amylose content can significantly reduce costs and improve efficiency. Currently, the United States has corn varieties with an amylose content as high as 100%. In contrast, my country is highly dependent on imports of corn amylose, with prices reaching 18,000-20,000 yuan per ton. Furthermore, there are currently no high-quality, high-yield commercially available high-amylose corn varieties, which is one of the prominent problems restricting the development of my country's corn seed industry. Therefore, breeding maize varieties with high amylose content is beneficial to the industrial demand for amylose. Developing high amylose maize breeding and production and processing has significant economic and social benefits.

[0003] Adenosine diphosphate glucose (ADPG) is a precursor in starch synthesis. It is synthesized into amylose and amylopectin through the combined action of granule-bound starch synthase (GBSS), soluble starch synthase (SSs), starch branching enzyme (SBE), and starch debranching enzyme (DBE). The starch branching enzyme SBEIIb is encoded in maize endosperm. Ae1 The gene is currently the most significant determinant of amylose content; it is located on the long arm of chromosome 5 in maize and is a recessive gene. In maize with different backgrounds... Ae1In homozygous mutants of the gene, the amylose content can be increased to 55%-65%. SBEI is a key enzyme in the starch synthesis pathway, and inhibiting SBEI activity is an effective way to increase amylose content. Studies have found that inactivation or inhibition of SBEI protein expression reduces amylopectin synthesis or branching to free up more space for amylose synthesis.

[0004] Gene editing technology, as a core means of targeted modification of biological genetic material, is characterized by precision, efficiency, and customizability, providing a revolutionary approach for crop genetic improvement and targeted trait creation. CRISPR / Cas9, as the core technology of gene editing, is a precise genome editing tool derived from the adaptive immune systems of bacteria and archaea, enabling targeted modification of the genomes of eukaryotic and prokaryotic organisms. The core of this system consists of the Cas9 nuclease and single-guide RNA (sgRNA): sgRNA recognizes the target DNA sequence through complementary base pairing; Cas9, with the assistance of the PAM motif, binds to and cleaves the target, generating a DNA double-strand break (DSB). The cell then repairs the break through non-homologous end joining (NHEJ) or homologous targeted repair (HDR) pathways, thereby achieving gene knockout, site-specific insertion, base substitution, or expression regulation. Compared to traditional editing tools such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR / Cas9 not only boasts significant advantages such as simple design, flexible targeting, high editing efficiency, low cost, and simultaneous multi-target editing, but also possesses the core characteristic of not requiring the introduction of exogenous genes. During the editing process, the target site and the insertion site of the gene editing element are independent, and they can be separated through hybridization and marker screening. This allows for precise screening of individual plants in the offspring that exhibit the target trait mutation but do not contain transgenic elements. After several generations of backcrossing, the original genetic background can be restored, effectively avoiding linkage burden during the transgenic process and obtaining the desired homozygous mutant in a relatively short time. This technology has been widely applied to gene function research, disease model construction, biomedical development, plant and animal breeding, and pathogen control, becoming the most mainstream gene manipulation platform in the current life sciences and biotechnology fields. The gene editing technology described in this invention, with CRISPR / Cas9 as its core framework, can significantly improve editing accuracy and safety, meeting the needs of basic research and industrial applications. Furthermore, this technology allows for the targeted knockout of negative regulatory genes. Ae1 and SBEI This is an important way to obtain new materials with high amylose content. Summary of the Invention

[0005] The purpose of this invention is to provide a method for creating high amylose maize germplasm using gene editing technology.

[0006] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides sgRNA for creating maize germplasm with high amylose content, selected from any one, any two, or a combination thereof from sgRNA1 to sgRNA4. The specific target maize sequence is shown in SEQ ID NO:1. Ae1 sgRNA1 (5'-GATACACATTACTTTCACAGTGG-3') of gene (Zm00001d016684); The specific target maize sequence is shown in SEQ ID NO:2. Ae1 sgRNA2 (5'-CTCGAGGAATATAAGTTTGATGG-3') of gene (Zm00001d016684); The specific target maize sequence is shown in SEQ ID NO:3. SBEI sgRNA3 (5'-CTACAACACAGTTCAGTTGATGG-3') of gene (Zm00001d014844).

[0007] The specific target maize sequence is shown in SEQ ID NO:4. SBEI sgRNA4 (5'-TCGATGTCCACACTTATTGACGG-3') of gene (Zm00001d014844).

[0008] Secondly, the present invention provides a CRISPR / Cas9 gene editing vector containing the sgRNA.

[0009] Preferably, the CRISPR / Cas9 gene editing vector is a pBUE411 vector containing the sgRNA.

[0010] Thirdly, the present invention provides a kit for creating high amylose maize germplasm, comprising any one of the following ① to ③: ①The sgRNA used to create maize germplasm with high amylose content; ②The DNA molecule encoding the sgRNA; ③ A CRISPR / Cas9 gene editing vector containing the sgRNA.

[0011] Fourthly, the present invention provides the sgRNA, the CRISPR / Cas9 gene editing vector, or the kit for editing maize. Ae1 Genes and / or SBEI Applications in genes.

[0012] Fifthly, the present invention provides the application of the sgRNA, the CRISPR / Cas9 gene editing vector, or the kit in the creation of high amylose maize germplasm.

[0013] Sixthly, this invention provides a method for creating high amylose maize germplasm using gene editing technology, utilizing genetic engineering techniques to modify maize... Ae1 Genes and / or SBEI Genes are modified to lose their function, thereby increasing the amylose content of corn.

[0014] For example, gene editing can be performed using any of the following gene editing technologies: zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), CRISPR / Cas technology, transposon technology, etc.

[0015] Further, the method includes: introducing the CRISPR / Cas9 gene editing vector into Agrobacterium competent cells to obtain recombinant Agrobacterium; infecting maize callus tissue with the recombinant Agrobacterium; and then inducing and culturing the obtained positive callus tissue to obtain regenerated plants, which are transgenic maize plants.

[0016] Preferably, the specific PCR primers used for detecting transgenic maize plants are as follows: Targeted corn Ae1 The PCR primers for transgenic maize plants containing sgRNA1 and sgRNA2 of the gene are: 5'-AGCTCTTTGGTTTCATACCT-3' (SEQ ID NO:5) and 5'-CCAGTCAGACTTAGCAAGTG-3' (SEQ ID NO:6). Targeted corn SBEI The transgenic maize plants containing sgRNA3 of the gene were obtained using PCR primers: 5'-TACACATTTAAGCATCCTCG-3' (SEQ ID NO:7) and 5'-GCATGGCTATGGACAACATC-3' (SEQ ID NO:8). Targeted corn SBEI The transgenic maize plants containing the gene sgRNA4 were obtained using PCR primers: 5'-AGTCCAAGTATAGGATGAGC-3' (SEQ ID NO:9) and 5'-CGCTGCAAAGCCATGTGATC-3' (SEQ ID NO:10).

[0017] In a seventh aspect, the present invention provides the application of transgenic maize plants obtained according to the method in plant breeding.

[0018] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0019] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention utilizes the CRISPR / Cas9 gene editing system for maize Ae1 Genes and SBEI Gene editing. Through the specific design of sgRNA, highly efficient editing of maize genes was achieved. Ae1 Genes and SBEI The addition of sgRNA and CRISPR / Cas9 vectors to the gene significantly improved the targeting efficiency of CRISPR / Cas9, thereby significantly improving maize... Ae1 Genes and SBEI Gene editing efficiency.

[0020] The high amylose corn germplasm created according to the method of this invention can significantly increase the amylose content in corn kernels, which is beneficial for its promotion and application in food, medical care, materials, textiles, papermaking, packaging, environmental protection and other fields. Attached Figure Description

[0021] Figure 1 In a preferred embodiment of the present invention Ae1 Genes and SBEI A schematic diagram of sgRNA target design sites. Black and white boxes represent exons and UTR regions, respectively, and dashed lines represent introns.

[0022] Figure 2 This is a map of the gene-editing pBUE411 vector in a preferred embodiment of the present invention.

[0023] Figure 3 Gene editing is a preferred embodiment of the present invention. Ae1 and SBEI Mutations occurring in the regions where genes sgRNA1 (A), sgRNA2 (B), sgRNA3 (C), and sgRNA4 (D) bind.

[0024] Figure 4 In a preferred embodiment of the present invention Ae1 Genes and SBEI Amylose content in gene-edited corn kernels. express P <0.01, express P <0.0001.

[0025] Figure 5In a preferred embodiment of the present invention Ae1 Genes and SBEI Electron microscopic observation of starch granules in corn kernels after gene editing.

[0026] Figure 6 Genes in a preferred embodiment of the present invention Ae1 The amylose content (A) and 100-grain weight of the four target sites were analyzed (B).

[0027] Figure 7 Genes in a preferred embodiment of the present invention SBEI The amylose content (A) and 100-grain weight of the four target sites were analyzed (B). Detailed Implementation

[0028] This invention utilizes gene editing technology to provide a gene editing vector and method for creating high amylose maize germplasm, capable of editing maize knockout... Ae1 (Zm00001d016684) gene and SBEI (Zm00001d014844), rapidly creating high amylose maize germplasm.

[0029] The present invention adopts the following technical solution: First, this invention provides sgRNA for creating high amylose maize germplasm, comprising: Specific Targeted Corn Ae1 The nucleotide sequence of the sgRNA1 gene's target site is: 5'-GATACACATTACTTTCACAGTGG-3' (SEQ ID NO:1); or, Specific Targeted Corn Ae1 The nucleotide sequence of the sgRNA2 gene at its site of action is: 5'-CTCGAGGAATATAAGTTTGATGG-3' (SEQ ID NO:2); And / or, Specific Targeted Corn SBEI The nucleotide sequence of the gene's sgRNA3, at its site of action, is: 5'-CTACAACACAGTTCAGTTGATGG-3' (SEQ ID NO:3). And / or, Specific Targeted Corn SBEI The nucleotide sequence of the gene's sgRNA4 at its site of action is: 5'-TCGATGTCCACACTTATTGACGG-3' (SEQ ID NO:4).

[0030] The present invention further provides a CRISPR / Cas9 gene editing vector containing the sgRNA.

[0031] Preferably, the CRISPR / Cas9 gene editing vector is a pBUE411 vector containing the sgRNA described above.

[0032] The present invention further provides a kit for creating maize germplasm with high amylose content, comprising any one of the following: 1) The sgRNA mentioned above; 2) The DNA molecule encoding the sgRNA; 3) The CRISPR / Cas9 gene editing vector described above.

[0033] This invention further provides the sgRNA, the CRISPR / Cas9 gene editing vector, or the kit described herein for editing knockout... Ae1 Genes and SBEI Applications in genes.

[0034] The present invention further provides the application of the sgRNA, the CRISPR / Cas9 gene editing vector, or the kit in the creation of high amylose maize germplasm.

[0035] This invention also provides a method for creating high amylose maize germplasm, comprising: introducing the CRISPR / Cas9 gene editing vector into BMEHA105 Agrobacterium competent cells to obtain recombinant Agrobacterium; infecting maize callus tissue with the recombinant Agrobacterium; and then inducing and culturing the obtained positive callus tissue to obtain regenerated plants.

[0036] The present invention further provides primer combinations for detecting the mutation effects of genes associated with high amylose maize germplasm, including primers with sequences as shown in SEQ ID NO:5-10.

[0037] Among them, the primers shown in SEQ ID NO:5 and 6 are for detection. Ae1 Primers for detecting gene target site sequence mutations; the primers shown in SEQ ID NO:7-10 are for detection. SBEI Primers for gene target site sequence mutations.

[0038] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0039] Example 1: Method for Creating High Amylose Maize Germplasm Using Gene Editing Technology 1. Materials and Methods 1.1 Construction of CRISPR / Cas9 gene editing vector (1) Target sequence design and sgRNA synthesis: Reference genome sequence B73_RefGen_v4 Ae1 (Zm00001d016684) SBEI The (Zm00001d014844) gene was used to design sgRNAs using the CRISPR-P 2.0 online tool, including a specific target for maize as shown in SEQ ID NO:1. Ae1 The sgRNA1 of the gene; its sequence is shown in SEQ ID NO:2, specifically targeting maize. Ae1 The sgRNA2 of the gene; the sequence of which is shown in SEQ ID NO:3 specifically targets maize. SBEI The sgRNA3 of the gene; its sequence is shown in SEQ ID NO:4, specifically targeting maize. SBEI The sgRNA4 of the gene. The sgRNA sequence was verified by Sanger sequencing in the B73-329 (LH244) receptor. Ae1 Genes and SBEI Gene sgRNA target design sites such as Figure 1 As shown. The structure of the CRISPR / Cas9 gene editing vector pBUE411 is as follows. Figure 2 As shown, sgRNA single-stranded DNA was prepared by chemical synthesis, and then annealed to form a double-stranded sgRNA intermediate.

[0040] (2) Carrier linearization processing The expression vector pBUE411 (addgene, catalog number 62200) was digested with the restriction endonuclease BssHII (NEB, catalog number R0199V). The reaction mixture consisted of 5 μL of 10×CutSmart Buffer, 20 μL of pBUE411 plasmid (50 ng / μL), 2 μL of BssHII restriction endonuclease (10 U / μL), and 50 μL of ultrapure water. The mixture was digested at 37°C for 2 h, followed by heat inactivation at 65°C for 10 min. The digestion products were separated by 1% agarose gel electrophoresis. The linearized vector backbone was recovered by gel extraction and purified using a gel extraction kit to obtain a high-purity linearized vector for later use.

[0041] (3) Recombinant vector linkage and transformation The double-stranded sgRNA intermediate prepared in step (1) was mixed with the linearized vector obtained in step (2) at a molar ratio of 3:1. T4 DNA ligase (Thermo Scientific, catalog number EL0011) and 10×T4 DNA ligation buffer were added, and ultrapure water was added to a final volume of 20 μL. The ligation reaction was carried out overnight at 16°C. The ligation product was transformed into E. coli DH5α competent cells (preserved in our laboratory), plated on LB solid medium containing 50 mg / L kanamycin, and incubated upside down at 37°C for 12–16 h. Single colonies were picked and inoculated into LB liquid medium, and cultured at 37°C with shaking at 200 r / min for 8 h. Plasmid DNA was then extracted.

[0042] (4) Identification of recombinant vectors Positive clones were identified using a combination of colony PCR and enzyme digestion verification: Extracted plasmid DNA was used as a template for PCR amplification with sgRNA-specific primers (primer sequences shown in SEQ ID NO: 5-10). The PCR reaction system consisted of: 0.5 μL plasmid template, 1 μL upstream primer (10 μmol / L), 1 μL downstream primer (10 μmol / L), 5 μL 5×PCR Mix (Kangwei Century, catalog number cw0690m), and 2.5 μL ultrapure water. The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 51℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 36 cycles; and a final extension at 72℃ for 5 min. The PCR products were detected by 3% agarose gel electrophoresis; colonies showing the expected band size were considered positive clones. Simultaneously, plasmids from positive clones were selected for BssHII digestion verification, and the correctness of the vector construction was further confirmed by the size of the electrophoretic bands. The recombinant plasmids that have been validated by sequencing were used as CRISPR / Cas9 gene editing recombinant expression vectors for maize genetic transformation.

[0043] (5) Extraction and concentration determination of recombinant vector The correctly sequenced positive E. coli clones were inoculated into 50 mL of LB liquid medium containing 50 mg / L kanamycin and cultured at 37°C with shaking at 200 rpm for 12 h. The recombinant plasmid was extracted using a plasmid kit (Invitrogen, catalog number K210002), and the plasmid concentration and purity were determined by Nanodrop 2000. The concentration was adjusted to 100 ng / μL and stored at -20°C for later use in Agrobacterium transformation experiments.

[0044] 1.2 Maize genetic transformation (1) Construction and activation of recombinant bacteria The constructed gene-editing vector plasmid was introduced into BMEHA105 Agrobacterium competent cells (preserved in our laboratory) using the heat shock method. The cells were then plated on LB solid medium containing the appropriate antibiotics and incubated upside down at 28°C. Positive recombinant Agrobacterium single colonies were selected. Single colonies were picked and inoculated into LB liquid medium, and cultured at 28°C with shaking at 200 rpm until the bacterial culture reached OD. 600 The nm value reached 0.8. The cells were then collected by centrifugation at 5000 rpm for 10 min, resuspended in pretreated infection buffer, and the cell concentration was adjusted to OD. 600 The nm value is approximately 0.5. It is placed in a constant temperature shaking environment at 28℃ and 150 r / min for 0.5 h to induce activation, and then set aside for later use.

[0045] The aforementioned infection buffer consists of the following components per 1 L: 4 g of N6 salt containing N6 vitamin, 1.5 mg of 2,4-D, 0.7 g of proline, 68.4 g of sucrose, 36 g of glucose, 0.1 g of inositol, 10 mg of AgNO3, 100 μmol of acetylsuccinone (As), with the balance being ultrapure water, and the pH adjusted to 5.2 with hydrochloric acid or sodium hydroxide solution.

[0046] (2) Explant infection and co-culture Callus tissue from the maize inbred line B73-329 (LH244) in good growth condition was selected as the transformation recipient. First, the callus tissue was pre-soaked in the aforementioned infection buffer for 1 hour to ensure thorough saturation. Then, the callus tissue was transferred to the activated infection solution and gently soaked at room temperature for 15 minutes, with appropriate agitation to ensure uniform infection. After infection, the callus tissue was removed, excess bacterial solution was blotted off with sterile filter paper, and then air-dried. The dried callus tissue was then transferred to a co-culture medium and co-cultured at 20°C in the dark for 3 days.

[0047] The co-culture medium consists of the following components per 1L: 4g N6 salt containing N6 vitamin, 1.5 mg 2,4-D, 0.7 g proline, 30 g sucrose, 8 g agar, 0.1 g inositol, 0.3 g L-cysteine, 10 mg AgNO3, 100 μmol acetylsuccinone (As), with the remainder being ultrapure water, and the pH adjusted to 5.8 with hydrochloric acid or sodium hydroxide solution.

[0048] (3) Recovery culture and resistance screening After co-culture, the callus tissue was rinsed 3-5 times with sterile water to remove surface Agrobacterium, and then blotted dry with sterile filter paper. It was then transferred to recovery medium and cultured in the dark at 28°C for 10 days to restore callus viability and inhibit the growth of residual Agrobacterium. After recovery culture, the callus tissue was transferred to selection medium containing 1.5 mg / L glufosinate and cultured in the dark at 28°C for another 7 days. Well-grown positive callus tissue was obtained through resistance selection.

[0049] The recovery medium (screening medium) consists of the following components per 1 L: 4 g N6 salt, 1 mL N6 vitamin (1000×), 1.5 mg 2,4-D, 0.7 g proline, 30 g sucrose, 0.85 mg AgNO3, 0.5 g MES, 100 mg cefotaxime, 100 mg vancomycin, 8 g agar, and the balance being ultrapure water. The pH is adjusted to 5.8 with hydrochloric acid or sodium hydroxide solution.

[0050] (4) Plant regeneration and hardening off The obtained positive callus tissue was transferred to embryoid induction medium and cultured at 28°C in the dark for 2 weeks to induce callus differentiation into embryoids. The embryoids were then transferred to differentiation medium and cultured at 25°C under 16h light / 8h dark conditions until green seedlings of approximately 2-3cm in height emerged. The differentiated seedlings were cut and transferred to rooting medium, cultured at 25°C under 16h light / 8h dark conditions to induce root formation. Once the regenerated plants were robust and had well-developed root systems, they were removed from the culture bottles, the root culture medium was washed off, and they were transplanted into sterilized substrate and placed in a greenhouse for 3 days to harden off, completing the transplanting process.

[0051] The embryoid induction medium consists of the following components per 1 L: 4.43 g MS salt containing MS vitamins (including inositol), 1.5 mg 2,4-D, 0.7 g proline, 30 g sucrose, 0.1 g inositol, 0.85 mg AgNO3, 3 g plant gel, and the balance being ultrapure water, with the pH adjusted to 5.8 using hydrochloric acid or sodium hydroxide solution.

[0052] The differentiation medium consists of the following components per 1 L: 4.43 g MS salt containing MS vitamins (containing inositol), 0.1 g inositol, 30 g sucrose, 3 g plant gel, 0.5 mg 6-BA, and the remainder is ultrapure water, with the pH adjusted to 5.8 using hydrochloric acid or sodium hydroxide solution.

[0053] The rooting medium consists of the following components per 1 L: 2.22 g MS salt, 30 g sucrose, 51.55 mg MS vitamins, 3 g plant gel, and the remainder is ultrapure water, with the pH adjusted to 5.8 using hydrochloric acid or sodium hydroxide solution.

[0054] 1.3 Identification of positive plants DNA was extracted using a plant genomic DNA extraction kit. zCas9 The PCR amplification primers were 5'-CAACCGGAAGGTCACGGTTA-3' (SEQ ID NO:11) and 5'-CACAACCTTCACGGTCTGCA-3' (SEQ ID NO:12). The PCR program was: 95℃ for 5 min; 95℃ for 30 s, 53℃ for 30 s, 72℃ for 30 s, for 36 cycles; and a final extension at 72℃ for 5 min.

[0055] 1.4 Detection of gene editing sites Primers designed targeting sgRNA1-4 (SEQ ID NO: 5-10) were used to detect positive plant leaves by PCR. The PCR reaction system contained: 3 μL DNA, 4 μL upstream primer (10 μmol / L), 4 μL downstream primer (10 μmol / L), 25 μL 5×PCR Mix, and 14 μL ultrapure water. The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 51℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 36 cycles; and a final extension at 72℃ for 5 min. The PCR products were detected by 3% agarose gel electrophoresis.

[0056] 1.5 Determination of amylose content Amylose content was determined using an amylose / amylopectin assay kit (Megazyme, catalog number: K-AMYL). The principle is that the starch sample is completely dissolved in dimethyl sulfoxide (DMSO) upon heating. Ethanol is used to precipitate the starch to remove lipids, and the precipitated starch is recovered. The precipitated sample is dissolved in acetic acid / salt solution, and concanavalin A (ConA) is added to specifically precipitate amylopectin. The precipitate is then removed by centrifugation. Amylose in a unit volume of the supernatant is enzymatically hydrolyzed to D-glucose, and then measured using a glucose oxidase / peroxidase reagent. Similarly, total starch in a unit volume of acetic acid / salt solution is enzymatically hydrolyzed to D-glucose, then glucose oxidase / peroxidase is added, and the result is determined colorimetrically. The amylose content in total starch is determined based on the ratio of the absorbance of GOPOD at 510 nm in the supernatant of the ConA precipitate sample to that in the total starch sample.

[0057] 1.5.1 Sample Pretreatment (1) Grind the corn kernels finely and weigh 20-25 mg (accurate to 0.1) of the sample powder into a 10 mL centrifuge tube.

[0058] (2) While gently stirring the sample tube at low speed with a vortex mixer, add 1 mL of DMSO. Tighten the cap and heat the test tube in a boiling water bath until the sample is completely dispersed, about 1 minute. Make sure there are no lumps of starch.

[0059] (3) Use a vortex mixer to mix the sample in the test tube at high speed and vigorously, and make sure the sample tube is sealed. Place the sample tube in a boiling water bath and heat for 15 minutes, during which time use a vortex mixer to mix it at high speed and vigorously intermittently.

[0060] (4) Let stand at room temperature for 5 minutes. While continuously vortexing, add 2 mL of 95% (v / v) ethanol, then add 4 mL of ethanol, tighten the cap, and mix by inverting to form starch precipitate. Place the sample tube upright for 15 minutes (or overnight).

[0061] (5) Centrifuge at 2000g for 5 min, discard the supernatant, and invert the test tube on a paper towel for 10 min to remove any residual supernatant. Ensure that all ethanol has been removed. The flocculent clump at the bottom of the sample tube will be used in the subsequent determination of amylose and starch.

[0062] (6) While gently stirring at low speed with a vortex mixer, add 2 mL of DMSO to the starch flocculent. Heat the sample tube in a boiling water bath for 15 minutes, mixing intermittently during the process. Ensure that no gel-like lumps form.

[0063] (7) Immediately add 4 mL of ConA solution and mix well. Quantitatively transfer all the sample from the sample tube (which can be washed repeatedly with ConA solvent) to a 25 mL volumetric flask. Make up to volume with ConA solvent (this is solution A). (The entire process must be completed within 2 hours).

[0064] 1.5.2 Con A precipitation of amylopectin and determination of amylose (1) Transfer 1.0 mL of solution A to a 2.0 mL centrifuge tube, add 0.50 mL of Con A solution, tighten the cap, and gently invert to mix. Avoid foaming of the sample.

[0065] (2) Let stand at room temperature for 1 h. Centrifuge at 14000 g at room temperature for 10 min.

[0066] (3) Take 1 mL of supernatant into a 15 mL centrifuge tube. Add 3 mL of 100 mM sodium acetate buffer (pH 4.5). Mix well, gently cover the tube, and heat it in a boiling water bath for 5 min to denature Con A.

[0067] (4) Place the centrifuge tube in a 40°C water bath for 5 min. Add 0.1 mL of amylase / α-amylase mixture and incubate at 40°C for 30 min. Centrifuge at 2000 g for 5 min.

[0068] (5) Transfer 1.0 mL of the supernatant to a new centrifuge tube and add 4 mL of GOPOD reagent. Incubate at 40℃ for 20 min. Simultaneously incubate the blank reagent and D-glucose control.

[0069] (6) Read the absorbance value of each sample, including the control, at 510 nm relative to the blank reagent.

[0070] 1.5.3 Determination of total starch (1) Mix 0.5 mL of solution A with 4 mL of 100 mM, pH 4.5 sodium acetate buffer.

[0071] (2) Add 0.1 mL of amylase / α-amylase solution and incubate at 40°C for 10 min.

[0072] (3) Take 1 mL of supernatant, add 4 mL of GOPOD reagent, and incubate at 40 °C for 20 min. This incubation should be carried out simultaneously with the Con A precipitation of amylopectin and the determination of the amylose fraction of the sample and standard.

[0073] 1.5.4 Result Calculation Amylose content (%) = Absorption value of Con A supernatant / Total starch absorption value × 66.8.

[0074] 1.6 Scanning electron microscopy observation of starch granules (1) Starch extraction and crude purification: Take mature corn kernels, remove the seed coat and germ, add an appropriate amount of ultrapure water and grind thoroughly into a homogenate, filter through a 100 μm nylon filter and collect the filtrate. Centrifuge at 2000 r / min for 15 min, discard the supernatant and collect the starch precipitate at the bottom.

[0075] (2) Defatting and impurity removal: Add 20 mL of 0.5% NaOH solution to the starch precipitate, let it stand and soak for 4 h, centrifuge at 4000 r / min for 10 min, and discard the supernatant; repeat the NaOH washing until the supernatant is colorless to remove protein and soluble impurities. Wash the precipitate three times each with 70% ethanol, 95% ethanol and anhydrous ethanol, vortexing and centrifuging after each washing, and discard the supernatant.

[0076] (3) Drying and refining: The washed starch precipitate was transferred to a vacuum freeze dryer and freeze-dried for 24 h to obtain purified corn starch powder. It was sealed and stored in the dark for subsequent scanning electron microscopy observation.

[0077] (4) Scanning electron microscopy sample preparation: Take a small amount of purified corn starch powder and sprinkle it evenly and lightly on the sample stage surface with conductive double-sided tape. Use a bulb syringe to gently blow away any excess powder that is not adhered, so that the starch particles are spread out in a single layer without stacking or agglomeration. Place the sample stage in an ion sputtering instrument and perform gold sputtering under vacuum conditions to form a uniform conductive film on the particle surface and eliminate charge interference.

[0078] (5) Electron microscopy observation and image acquisition: The prepared sample is loaded into the scanning electron microscope sample chamber, vacuumed to the working state, and the accelerating voltage is set to 10 kV. The morphology, surface structure, particle size uniformity and damage of starch granules are observed at different magnifications such as 500×, 1000× and 2000×. Typical fields of view are selected to take pictures and save images for phenotypic comparison analysis.

[0079] 2 Results 2.1 Construction and genetic transformation of CRISPR / Cas9 gene editing vectors This invention successfully constructed a vector that simultaneously targets maize using pBUE411 as the base vector. Ae1 Genes and SBEI CRISPR / Cas9 dual-target gene editing vector ( Figure 2 Using an Agrobacterium-mediated genetic transformation system for maize callus, recombinant editing vectors were introduced into the maize inbred line B73-329 (LH244) recipient material. After resistance selection and plant regeneration, multiple independent positive transformation events were successfully obtained. Target site PCR amplification and Sanger sequencing verification were performed on the T1 generation regenerated plants. The results showed that the sgRNA1, sgRNA2, sgRNA3, and sgRNA4 designed in this invention can effectively guide Cas9 nuclease to precisely cleave the target region and generate the expected gene mutations during cell repair, demonstrating high editing efficiency and strong target specificity. Figure 3 Sequencing and alignment revealed that the editing type was predominantly frameshift mutations, all of which were base deletion mutations. Specific mutation types are as follows: Ae1 Gene (sgRNA1 target): 3 bases deleted (TCA); Ae1 Gene (sgRNA2 target): 4 bases deleted (TTTG); SBEI Gene (sgRNA3 target): 2 bases (AG) deleted; SBEI Gene (sgRNA4 target): 2 bases (CA) are missing.

[0080] The aforementioned deletions and mutations all lead to frameshifts in the target gene, causing premature termination of the encoded protein or disruption of its functional domains, ultimately resulting in... Ae1 Genes and SBEI The knockout of gene function laid a reliable material foundation for the subsequent creation of maize germplasm with high amylose content.

[0081] 2.2 Amylose content in corn kernels The amylose content of seeds from gene-edited positive plants was determined, and the results showed that ( Figure 4 Compared with control B73-329 (LH244), Ae1 Single mutants and Ae1 and SBEI The amylose content in the seeds of both mutants was significantly increased. The average amylose content in the control seeds was 21.18%. Ae1 After loss-of-function mutations in the genes (sgRNA1 and sgRNA2), the amylose content in the grains increased significantly to 55.33% and 53.67%, respectively. SBEI After loss-of-function mutations in the genes (sgRNA3 and sgRNA4), the amylose content in the grains increased to 29.57% and 31.82%, respectively. Ae1 (sgRNA2) and SBEI After double mutation of (sgRNA3, sgRNA4) genes, the amylose content of the grains reached 49.61%. This was significantly different from the control. P <0.001), indicating that the knockout was achieved through CRISPR / Cas9 technology. Ae1 and SBEI The gene can significantly increase the amylose content of maize kernels, and a new maize germplasm with high amylose content has been successfully created.

[0082] 2.3 Observation of starch content in corn kernels The microscopic morphology of starch granules in control and gene-edited mutant seeds was observed and compared using scanning electron microscopy. Figure 5 The results showed that wild-type starch granules were mostly spherical or ellipsoidal, with smooth and rounded surfaces, uniform granule size, and a relatively regular overall structure. In contrast, Ae1 Single mutant and SBEIThe morphology of starch granules in the mutant strain is significantly altered, with more irregular outlines, increased surface roughness, and some granules exhibiting wrinkled, sunken, or angular features. The differences in size and shape between granules are also more pronounced. These changes in the microstructure of starch granules are correlated with an increased proportion of amylose, directly reflecting… Ae1 and SBEI The significant impact of gene loss of function on starch synthesis, branching structure, and starch grain development further confirms that gene editing can effectively regulate starch structure and properties.

[0083] Example 2: sgRNA Sequence Design and Optimization Ae1 The gene was designed with four targets in total: Ae1 Target 1 (i.e., sgRNA1) Ae1 Target 2 (i.e., sgRNA2) Ae1 Target 3 (5'-AATACTGGATTATGCCTGG-3', SEQ ID NO:13) and Ae1 Target 4 (5'-ACTTCAATGAGTATTTTGG-3', SEQ ID NO:14). CRISPR / Cas9 gene editing vectors were constructed, and through genetic transformation, screening, and validation, different results were obtained. Ae1 Mutant plants. The content of amylose in the grains was determined. Ae1 The mutant plants targeting target 1 (i.e., sgRNA1) had the highest amylose content in their seeds (55.33%), followed by [other types of amylose]. Ae1 The mutant plants targeting 2 (i.e., sgRNA2) had a seed amylose content of 53.67%. Ae1 The mutant plants targeting point 3 had a grain amylose content of 56.67%. Ae1 The amylose content in the seeds of mutant plants targeting point 4 was 36.98%. Figure 6 (A). Meanwhile, by comparing agronomic traits, Ae1 Target 1 (i.e., sgRNA1) and Ae1 The mutant targeting 2 (i.e., sgRNA2) had the highest seed weight per 100 seeds, and there was no significant difference compared with the control. Ae1 Although target 3 has the highest amylose content, its 100-grain weight is significantly reduced. Figure 6 (Middle B) Ae1 Target 4 has an amylose content of less than 40%, therefore, through comprehensive comparison, Ae1 Target 1 (i.e., sgRNA1) and Ae1 The mutant of target 2 (i.e. sgRNA2) has the advantage of both high amylose content and yield.

[0084] SBEI The gene was designed with four targets in total: SBEI Target 1 (5'-AGCCAGCAGTAAGCACATATAGG-3', SEQ ID NO:15) SBEI Target 2 (5'-TCAGATGCTTGGCCTAAGAGTGG-3', SEQ ID NO:16) SBEI Target 3 (i.e., sgRNA3) and SBEI Target 4 (i.e., sgRNA4). CRISPR / Cas9 gene editing vectors were constructed for each of the four targets. Through genetic transformation, screening, and validation, different results were obtained. SBEI Mutant plants. The content of amylose in the grains was determined by... Figure 7 (A) SBEI The mutant plants of target 1 and target 2 had relatively low amylose content in their grains, at 21.83% and 23.58%, respectively. SBEI The mutant plants targeting target 3 (i.e., sgRNA3) and target 4 (i.e., sgRNA4) had relatively higher amylose content in their seeds, at 29.57% and 31.82%, respectively. Meanwhile, agronomical traits showed no significant difference in the 100-seed weight between the four mutants and the control. Figure 7 (Middle B). Therefore, through comprehensive comparison, SBEI Target 3 (i.e., sgRNA3) and SBEI Target 4 (i.e., sgRNA4) has significant application value.

[0085] Example 3: Application of this method Based on the parent inbred lines PH4CV and PH6WC of Xianyu 335, a high-yield and multi-resistant maize hybrid widely planted in my country, and using... Ae1 Using sgRNA1 mutant plants as donors, F1 generations were obtained through hybridization. Then, two generations of backcrossing were performed using the two parent lines of Xianyu 335 as the male parent to obtain at least 200 BC2F1 ears. The developed [technology / method / technology] was then utilized. Ae1 Molecular marker screening for target 1 (i.e., sgRNA1) was conducted, and individuals with high amylose content were selected through amylose detection. At least 800 DH pure lines were obtained through haploid induction and doubling. Pure lines with an amylose content greater than 50% in the grains were selected from these lines, and combining ability was tested simultaneously. Finally, the high amylose inbred line HAF001, which has excellent overall traits, is stable and consistent, and has high combining ability, was obtained.

[0086] Furthermore, based on the parent-cross lines Chang 7-2 and Zheng 58 of Zhengdan 958, a high-yield and multi-resistant maize hybrid widely planted in my country, and using... Ae1Using sgRNA1 mutant plants as donors, F1 generations were obtained through hybridization. Then, two generations of backcrossing were performed using the Zhengdan 958 double parent inbred line as the male parent to obtain at least 200 BC2F1 ears. The developed [technology / method / technology] was then used to [achieve this goal]. Ae1 Target 1 (i.e., sgRNA1) molecular marker screening was conducted using amylose detection to select individuals with high amylose content. Then, at least 800 DH pure lines were obtained through haploid induction and doubling. Among them, pure lines with an amylose content of more than 50% in the grain were selected, and combining ability was tested simultaneously. Finally, the high amylose inbred line HAF002 with excellent comprehensive traits, stable and consistent, and high combining ability was obtained.

[0087] The experimental results above show that the inbred lines with Xianyu 335 and Zhengdan 958 as the genetic background have a grain amylose content of more than 50%, and can be directly used as core parents for high amylose maize breeding and hybridization.

[0088] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An sgRNA for creating high amylose maize germplasm, characterized in that, Selected from any one, any two, or a combination of sgRNA1 to sgRNA4; The specific target maize sequence is shown in SEQ ID NO:

1. Ae1 sgRNA1 of the gene; The specific target maize sequence is shown in SEQ ID NO:

2. Ae1 sgRNA2 of the gene; The specific target maize sequence is shown in SEQ ID NO:

3. SBEI sgRNA3 of the gene; The specific target maize sequence is shown in SEQ ID NO:

4. SBEI sgRNA4 of the gene.

2. A CRISPR / Cas9 gene editing vector containing the sgRNA of claim 1.

3. A reagent kit for creating high-amylose maize germplasm, characterized in that, Including any one of the following ①~③: ①The sgRNA as described in claim 1; ②The DNA molecule encoding the sgRNA; ③The CRISPR / Cas9 gene editing vector as described in claim 2.

4. The sgRNA of claim 1, the CRISPR / Cas9 gene editing vector of claim 2, or the kit of claim 3 for editing maize Ae1 Genes and / or SBEI Applications in genes.

5. The application of the sgRNA of claim 1, the CRISPR / Cas9 gene editing vector of claim 2, or the kit of claim 3 in the creation of high amylose maize germplasm.

6. A method for creating high amylose maize germplasm using gene editing technology, characterized in that, Using genetic engineering techniques to modify corn Ae1 Genes and / or SBEI Genes are modified to lose their function, thereby increasing the amylose content of corn.

7. The method according to claim 6, characterized in that, Gene modification can be performed using any of the following gene editing technologies: zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR / Cas technology, or transposon technology.

8. The method according to claim 7, characterized in that, The method includes: introducing the CRISPR / Cas9 gene editing vector of claim 2 into Agrobacterium competent cells to obtain recombinant Agrobacterium; infecting maize callus tissue with the recombinant Agrobacterium; and then inducing and culturing the obtained positive callus tissue to obtain regenerated plants, which are transgenic maize plants.

9. The method according to claim 8, characterized in that, The specific PCR primers used for detecting transgenic maize plants are as follows: Targeted corn Ae1 The transgenic maize plants containing sgRNA1 and sgRNA2 of the gene were obtained using PCR primers: 5'-AGCTCTTTGGTTTCATACCT-3' (SEQ ID NO:5) and 5'-CCAGTCAGACTTAGCAAGTG-3' (SEQ ID NO:6). Targeted corn SBEI The transgenic maize plants containing sgRNA3 of the gene were obtained using PCR primers: 5'-TACACATTTAAGCATCCTCG-3' (SEQ ID NO:7) and 5'-GCATGGCTATGGACAACATC-3' (SEQ ID NO:8). Targeted corn SBEI The transgenic maize plants containing the gene sgRNA4 were obtained using PCR primers: 5'-AGTCCAAGTATAGGATGAGC-3' (SEQ ID NO:9) and 5'-CGCTGCAAAGCCATGTGATC-3' (SEQ ID NO:10).

10. The use of transgenic maize plants obtained by the method according to any one of claims 6-9 in plant breeding; Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

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