The transcription factor OsERF44 that regulates rice quality and its use, the use of biomaterials that knock out the OsERF44 gene in the construction of rice varieties with low starch content and high protein content, and methods for constructing rice varieties with low starch content and high protein content.

Abstract

Providing the transcription factor OsERF44, which regulates rice quality, and its use. [Solution] The present invention provides the use of a biomaterial to knock out the OsERF44 gene in the construction of a rice variety with low starch content and high protein content, characterized in that the nucleotide sequence of the OsERF44 gene is as shown in a specific sequence.

Description

Technical Field

[0001] The present invention relates to the technical fields of biotechnology and plant genetic engineering, and particularly to a transcription factor OsERF44 for regulating rice quality and its use.

Background Art

[0002] With the increase in the world population and the improvement of living standards, the market demand for nutritious and healthy foods is constantly increasing. As one of the most important cereal crops in the world, rice's nutritional value and functional components have an important impact on people's health. Conventional rice varieties are mainly characterized by high starch content and can meet energy needs, but they are lacking in terms of protein and other nutritional components. In recent years, the market demand for rice varieties with low starch content and high protein content has been increasing day by day.

[0003] According to modern research, excessive intake of high-starch foods may cause metabolic diseases such as obesity and diabetes. Therefore, developing rice varieties with low starch content can help improve people's diet and reduce the risk of these diseases. Furthermore, rice with high protein content can provide people with more high-quality protein sources, strengthen the constitution, and promote health.

[0004] From the perspectives of agriculture and economy, cultivating low-starch and high-protein rice varieties can not only improve land utilization rate but also increase farmers' income. For example, this type of rice variety can be used in the production of special rice products (such as rice flour for infants, health foods for the elderly) or animal feed, thereby opening up new market fields and increasing the added value of products.

[0005] Currently, much research is being conducted to improve the nutritional content of rice using advanced technologies such as gene editing and molecular breeding. Although some progress has been made, existing varieties still struggle to simultaneously meet the requirements for both low starch and high protein. Therefore, there is an urgent need to develop new rice varieties that combine low starch and high protein content to fill the market gap and meet consumer demand.

[0006] Transcription factors are so-called transcription activators that can specifically bind to cis-acting elements associated with the promoter region of a gene, thereby activating gene expression. To date, numerous plant-related transcription factors have been reported, including MPB, bHLH, AP2 / ERF, WRKY, and NAC. Among these, AP2 / ERF (APETALA2 / ethylene responsive factor) is a transcription factor unique to plants. Based on the number of AP2 / ERF domains and the presence or absence of other domains, it is classified into five subfamilies: AP2 (APETALA2), ethylene responsive factor (ERF), dehydration response element-binding protein (DREB), RAV, and Soloist. AP2 / ERF family proteins play important roles in plant growth and development, biotic and abiotic stress responses, and biosynthesis. While transcription factors play a crucial role in regulating starch biosynthesis, only a small number of transcription factors involved in regulating starch synthesis in rice endosperm have been reported to date. AP2 / ERF family transcription factors have complex functions, are involved in multiple pathways, and can regulate multiple genes. However, there are few reports indicating that AP2 / ERF family transcription factors in rice are involved in regulating rice yield and quality traits. Therefore, delving deeper into rice starch synthesis-related genes and expanding the biological functions of rice AP2 / ERF family transcription factors is crucial for rice breeding and improvement. [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide the transcription factor OsERF44 and its use in regulating rice quality in order to solve the problems of the prior art described above. The transcription factor OsERF44 provides an important genetic resource and theoretical basis for improving the genetics of rice quality, and expands the biological function of rice AP2 / ERF family transcription factors in regulating rice quality. [Means for solving the problem]

[0008] To achieve the above objective, the present invention provides the following scheme.

[0009] The present invention provides the use of a biomaterial that knocks out the OsERF44 gene in the construction of rice varieties with low starch content and high protein content. The nucleotide sequence of the OsERF44 gene is as shown in SEQ ID NO.1.

[0010] Furthermore, the biological material is the substance described in (1) or (2) below: (1) A gene knockout vector for knocking out the OsERF44 gene, (2) Recombinant microbial strain containing the gene knockout vector.

[0011] Furthermore, the base strain of the recombinant microbial strain is Agrobacterium.

[0012] Furthermore, the gene knockout vector is a CRISPR / Cas9 gene knockout vector, and its target site sequence is as shown in SEQ ID NO. 5.

[0013] Furthermore, the nucleotide sequences of the upstream and downstream primers of the gRNA oligonucleotide chain of the CRISPR / Cas9 gene knockout vector are as shown in SEQ ID NO. 6-7.

[0014] The present invention further provides a method for constructing a rice variety with low starch content and high protein content, comprising the steps of knocking out the OsERF44 gene of rice to construct genetically modified rice, wherein the genetically modified rice is the low starch content and high protein content rice variety. The nucleotide sequence of the OsERF44 gene is as shown in SEQ ID NO.1.

[0015] Furthermore, knocking out the aforementioned gene involves employing a CRISPR / Cas9 gene knockout vector.

[0016] Furthermore, the OsERF44 gene is knocked out using Agrobacterium gene transformation.

[0017] Furthermore, the target site sequence of the CRISPR / Cas9 gene knockout vector is as shown in SEQ ID NO. 5.

[0018] Furthermore, the nucleotide sequences of the upstream and downstream primers of the gRNA oligonucleotide chain of the CRISPR / Cas9 gene knockout vector are as shown in SEQ ID NO. 6-7. [Effects of the Invention]

[0019] This invention discloses the following technical effects.

[0020] This invention cloned the transcription factor OsERF44, which regulates rice quality, using reverse genetics. As a rice quality-related gene, OsERF44 can be used to regulate rice trait indicators, including total starch content and total protein content, which are important nutritional indicators of rice. This invention demonstrates that knocking out this transcription factor reduces starch content and increases protein content in rice. The transcription factor OsERF44 of this invention provides an important genetic resource and theoretical basis for improving rice quality genetically, and expands the biological function of rice AP2 / ERF family transcription factors in regulating rice quality.

Brief Description of Drawings

[0021] To more clearly explain the embodiments of the present invention or the technical solutions of the prior art, the drawings necessary for the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative labor. [Figure 1] It is a tissue expression analysis diagram by website prediction of the OsERF44 gene. [Figure 2] It is a structure prediction diagram of the OsERF44 protein. [Figure 3] It is a subcellular localization diagram of the OsERF44 protein. a - d respectively show the GFP fluorescence signal, the fluorescence signal of the nuclear marker (dye DAPI), the bright field of view, and the fluorescence overlay signal. [Figure 4] It is a map of the CRISPR / Cas9 knockout vector containing the target site sequence of the OsERF44 gene. [Figure 5] It is a schematic diagram of gene editing of OsERF44 in the Nipponbare (NIP) background. Here, a shows the schematic diagram of the gene structure, the wild - type target site, and the gene - mutated target site sequence, and b is the sequencing chromatogram of the wild - type target site and the gene - mutated target site sequence. [Figure 6] It is a statistical chart of the traits of mature seeds of wild - type NIP and mutant oserf44. Here, a is the statistical chart of the grain length of rice seeds, and b is the statistical chart of the grain diameter of rice seeds. [Figure 7] It is a measurement and analysis diagram of the physicochemical indexes of rice of wild - type NIP and mutant oserf44. Here, a is the statistical chart of the total starch content, b is the statistical chart of the total protein content, and c is the starch viscosity curve diagram.

Modes for Carrying Out the Invention

[0022] Various exemplary embodiments of the present invention will be described in detail, but this detailed description should not be construed as limiting the invention, but rather as a more detailed description of specific aspects, features, and embodiments of the invention.

[0023] It should be understood that the terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the invention. Furthermore, with respect to numerical ranges in the invention, it should be understood that all intermediate values ​​between the upper and lower limits of that range are also specifically disclosed. Any numerical value or intermediate value within the described range, and each narrower range between any other numerical value or intermediate value within the said range, are also included in the invention. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range.

[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as that generally understood by an ordinary person skilled in the art to which this invention pertains. While this invention describes only preferred methods and materials, any similar or equivalent methods and materials may be used in the implementation or testing of this invention. All references made herein are incorporated by reference to disclose and explain methods and / or materials related to those references. In the event of any conflict between incorporated references and the content of this specification, the content of this specification shall prevail.

[0025] Numerous modifications and variations can be made to the specific embodiments of the specification of the present invention without departing from the scope or spirit of the present invention, which will be apparent to those skilled in the art. Other embodiments derived from the specification of the present invention will also be apparent to those skilled in the art. The specification and examples of the present invention are merely illustrative.

[0026] As used herein, "includes," "contains," "possesses," and "contains" are all non-restrictive expressions, meaning "includes but not limited to these."

[0027] The nucleotide sequence of the OsERF44 gene is shown in SEQ ID NO.1, and the amino acid sequence of the OsERF44 protein is shown in SEQ ID NO.2.

[0028] SEQ ID NO.1: ATGATTCTGATACATCGATACAATCAAGCTAGCATGGAGATGGACATCGGCGAGGGCGAGAGCTGCTGCGGCAGGCGAAAGCAGCAGCAGCAGCAGAATATTAGCAGCAGCAAGTCACGCAAGTGCTGCCCGCTGCGGCGGTCGCGGAAGGGGTGCATGAAGGGGAAGGGCGGGCCGGAGAACCAGCGTTGCCCCTTCCGCGGCGTCCGGCAGCGCACCTGGGGCAAGTGGGTGGCCGAGATCCGCGAGCCCAACCGCGGCGCCCGCCTCTGGCTCGGCACCTTCAACACCGCCCTCGACGCCGCCCGCGCCTACGACTCCGCCGCCAGGGCCCTCTACGGCGACTGCGCCCGCCTCAACCTACTCCTCGCCGCCGCCACCGCCGGTGCTCCTCCTGCTGCTGCTACCCCTTCCGTGGCCACGCCCTGCAGCACCAACGACGACTCCAACAACTCGTCTTCCACGACGCATCAGCAGCAGCTGACGACGATGCTGCAGCTGGACGACGACAACTACACGCTGCAGCCGTCGTCGTCAGATCAAGAGGACTTCGAGACGTACGTCACGCGGCTACCCAAGGCGGAGGACTTTGGGCTGGAGGGCTTCCAGGAGGTTCCACTCGACGTCCTCGACGAAGCCGGCGGTGGCATCAGCATCTGGGACCTCTCCATCTGCCCCGCCGATTTCATGGCCACCGCCGCCACCACCACCGCCAAATCATCTTAA

[0029] SEQ ID NO.2: MILIHRYNQASMEMDIGEGESCCGRRKQQQQQNISSSKSRKCCPLRRSRKGCMKGKGGPENQRCPFRGVRQRTWGKWVAEIREPNRGARLWLGTFNTALDARRAYDSAARALYGDCARLN LLLAATAGAPPAAATPSVATPCSTNDDSNNSSSTTHQQQLTTMLQLDDDNTTLQPSSSDQEDFETYVTRLPKAEDFGLEGFQEVPLDVLDEAGGGISIWDLSICPADPMATAATTTAKSS*

[0030] Example 1: Analysis of predicted tissue expression patterns of the rice OsERF44 gene

[0031] Using the RAP-DB website (https: / / rapdb.dna.affrc.go.jp), we entered the RAP_Locus number for OsERF44 and performed tissue expression pattern analysis. The prediction results showed that OsERF44 is highly expressed in rice embryos and endosperm (Figure 1).

[0032] Example 2: Structural prediction of the rice transcription factor OsERF44 protein

[0033] Using the SMART website (http: / / smart.embl-heidelberg.de / ), we input the protein sequence corresponding to OsERF44 and performed a domain prediction analysis. The prediction results indicated that one AP2 domain exists between amino acids 65 and 128 of the OsERF44 protein, and that this type of domain generally binds to DNA and regulates the expression of downstream target genes (Figure 2).

[0034] Example 3: Subcellular localization of the rice transcription factor OsERF44

[0035] Recombinant primers were designed based on the nucleotide sequence of the OsERF44 gene. PCR amplification was performed using cDNA from rice variety Nipponbare (NIP) as a template. After detection by agarose gel electrophoresis, the product was excised from the gel, recovered, and purified to obtain the PCR-purified product. Using the green fluorescent tag vector pAN580, dual enzymatic digestion with Spe I and Xba I was performed to obtain a purified linearized vector, which was set aside as a reserve. Furthermore, the PCR product and the linearized pAN580 vector were ligated using an infusion enzyme, and E. coli was transformed to prepare a large plasmid with correct sequencing, which was then transformed into rice protoplasts. Fluorescence expression sites were observed and imaged using a laser confocal microscope (LSM710, Zeiss, Germany). The specific operating procedure is as follows.

[0036] Rice seedling culture: 1 / 2 MS medium was prepared and sterilized simultaneously with the culture bottles. After sterile cooling, kanamycin was added, and the medium was poured into the culture bottles to a thickness of approximately 3 cm. Complete 93-11 rice grains containing embryos were selected, clean and free of black pigment, disinfected with 75% ethanol for 1 minute, washed three times with sterile water, disinfected with 50% pasteurized disinfectant solution for 30 minutes, and shaken up and down in a shaker. After pasteurization, the seeds were washed approximately four times with sterile water until no more bubbles remained, and the disinfected seeds were air-dried on a clean bench. They were transferred to 1 / 2 MS medium and cultured in an incubator at 30°C, and seedlings were used for protoplast extraction after 2 weeks.

[0037] Protoplast Extraction: Fifteen seedlings aged 10-13 days were taken out, cut into pieces less than 0.5 mm on clean A4 paper, and immediately transferred to a 0.6 M mannitol solution. After cutting all the seedlings, they were left in a vacuum chamber for 30 minutes to remove air from the leaves. The 0.6 M mannitol was removed using a 40 μm filter, and the enzymatic hydrolysate at room temperature was added. Enzymatic digestion was carried out in a 28°C shaker at 40 rpm for 5 hours. After the enzymatic digestion was complete, the enzymatic hydrolysate was removed using a 40 μm filter, and 10 mL of W5 (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES (pH 5.7) diluted to 100 mL with ddH2O) was added. The mixture was shaken in a 28°C shaker at 40 rpm for 10 minutes and filtered into a 50 mL centrifuge tube (labeled as tube 1). Add another 10 mL of W5 solution and shake in a shaker at 28°C and 40 rpm for 10 minutes. Filter into another 50 mL centrifuge tube (labeled as tube 2). Centrifuge the filtrate at room temperature with 70 g for 5 minutes using a horizontal rotor with acceleration / deceleration set to 1, and discard the supernatant by aspirate. Add 1 mL of W5 solution to suspend the protoplasts and carefully shake the centrifuge tube to ensure that the protoplasts are well dispersed in the W5 solution. Take a 10 μL protoplast suspension and count the protoplasts to determine a protoplast concentration of 0.5–1 × 10⁶. 7 The solution was adjusted to 0.5 / mL. 70g was centrifuged at room temperature for 5 minutes, and the supernatant was discarded. The protoplasts were resuspended in MMC medium, and the concentration was adjusted to 0.5–1 × 10⁶. 7 The solution was adjusted to 1 / mL. 5 μg (or 10 μg) of plasmid required for transformation was prepared and diluted to 10 μL. 200 μL of protoplast suspension was added to each round-bottom centrifuge tube, and after gently tapping with the thumb, 210 μL of PEG-CaCl2 solution was added to the centrifuge tube and mixed by gently flicking. The tubes were left at room temperature for 15 minutes. Then, 840 μL of W5 solution was added to the centrifuge tubes to stop the transformation. The centrifuge tubes were transferred to a centrifuge (horizontal rotor, 100 g) and centrifuged for 5 minutes. 0.5 mL of WI solution was added to resuspend the protoplasts, and they were transferred to a culture plate to which 300 μL of WI solution had been added beforehand. They were cultured for 16 hours. The cultured protoplasts were centrifuged (horizontal rotor, 100 g) for 5 minutes, the supernatant was removed, and the remaining portion was used for fluorescence signal observation.

[0038] Results: In rice protoplast cells introduced with an empty vector plasmid, fluorescence signals were distributed in both the cytoplasm and the nucleus. The green fluorescence of OsERF44::GFP overlapped with the blue fluorescence of the nuclear dye DAPI, indicating that OsERF44 is a typical transcription factor localized in the nucleus (shown in Figure 3).

[0039] The primers for constructing the OsERF44 subcell localization vector are as follows: Upstream primer: OsERF44-GFP-F (SEQ ID NO.3) 5′-GCCCAGATCAACTAGTATGATTCTGATACATCGATACAA-3′ Downstream primer: OsERF44-GFP-R (SEQ ID NO.4) 5′-TCGAGACGTCTCTAGA AGATGATTTGGCGGTGGTG-3′.

[0040] Example 4: Construction of genetically modified rice with OsERF44 gene knockout.

[0041] Selection of gRNA target sequence: Based on CRISPR / Cas9-related experimental methods, the 5′-GTCGCGGAAGGGGTGCATGAAGG-3′ sequence (SEQ ID NO. 5), which contains NGC as the recognition site, was selected as the knockout target site on the exon of the OsERF44 gene, and the PAM sequence was set to AGG.

[0042] Design of upstream and downstream primers for gRNA oligonucleotide chains: Upstream primer: OsERF44_gRNA_F (SEQ ID NO.6) 5′-TGTGTGGTCGCGGAAGGGTGCATGA-3′ Downstream primer: OsERF44_gRNA_R (SEQ ID NO.7) 5′-ACTCATGCACCCCTTCCGCGACCAAA-3′.

[0043] CRISPR / Cas9 Vector Construction: In this example, a plant Cas9 / gRNA plasmid construction kit (Catalog. No. BGK03, shown in Figure 4) was used. After loading the target sequence, a recombinant vector containing the target site of the OsERF44 gene was formed. The specific procedure is as follows.

[0044] (1) Preparation of oligodimers: 1 μL each of 10 μM upstream and downstream primers of the target site were taken, 18 μL of Buffer Aneal was added, and after mixing, the mixture was treated at 95°C for 3 minutes, and then slowly cooled to 20°C at approximately 0.2°C / second to obtain a double-stranded sequence containing the knockout target site, i.e., an oligodimer.

[0045] (2) Construction of the oligodimer into the CRISPR / Cas9 vector: Take 2 μL of Cas9 / gRNA vector, 1 μL of the oligodimer from step (1), and 1 μL of Enzyme Mix, add 6 μL of ddH2O, mix, and react on a metal bath at 20°C for 1 hour.

[0046] (3) Transformation of E. coli: Add 10 μL of the final product from step (2) to 50 μL of DH5α-competent cells immediately after thawing, mix by gently flicking, and leave in an ice bath for 30 minutes. Heat shock was applied at 42°C for 45 seconds, and then left to stand on ice for 2 minutes. Then, 200 μL of antibiotic-free LB medium was added, and the mixture was placed in a constant temperature shaker at 37°C and cultured at 200 rpm for 1 hour to induce kanamycin resistance (Kana + It was applied to a flat plate.

[0047] (4) Bacterial suspension PCR detection: The following day, single colonies were picked and cultured in kanamycin-resistant liquid medium in a shaker at 37°C until the suspension became cloudy. Sequencing was performed using the BGK03 vector-specific sequencing primers provided in the kit. The sequencing results were analyzed using Snapgene software, and plasmids were extracted from the suspension of positive clones and set aside as a reserve.

[0048] (5) Agrobacterium transformation and rice gene transformation: The successfully constructed plasmid was introduced into Agrobacterium (EHA105): 1 μL of plasmid was injected into Agrobacterium competent cells that had been frozen and thawed on ice, and left on ice for 5 minutes. Treatment was carried out in liquid nitrogen for 5 minutes and at 37°C for 5 minutes. 300 μL of antibiotic-free LB medium was added, and recovery culture was carried out at 28°C for 4 hours. (Kanamycin + Rifampicin) K + The sample was uniformly spread onto Rif-resistant plates and incubated at 28°C for 2 days, after which a single colony was picked. Testing was performed using hygromycin primers to obtain a positive clone, which was then subjected to 3 mL of liquid potassium. + Amplified culture was performed in / Rif medium. Furthermore, positive K + I sent the / Rif bacterial solution to the company and requested rice gene transformation under a Nipponbare background.

[0049] Example 5: Phenotypic analysis of rice OsERF44 gene knockout lines

[0050] To identify the knockout transgenic lines obtained in Example 4, the transformed seedlings were cultured in a room-temperature, light-activated incubator for approximately one week, and then positive seedlings were identified. The specific procedure was as follows: Testing of knockout transformed seedlings: Twenty T0 generation transformed seedlings were obtained and cultured in a room-temperature, light-up incubator for approximately one week. DNA was then collected from the 20 seedlings, amplified by PCR using OsERF44cas9TF and OsERF44cas9TR, and sent to the company for sequencing. The obtained sequencing results were analyzed to obtain two types of transformed plants in which protein translation terminated prematurely. The nucleotide sequences of the two homozygous mutants lacking the knockout OsERF44 gene obtained by the knockout according to the present invention are shown in SEQ ID NO.8 and SEQ ID NO.9, and the proteins they encode are shown in SEQ ID NO.10 and SEQ ID NO.11.

[0051] Nucleotide sequence of OsERF44 gene deletion homozygous mutant 1 (SEQ ID NO. 8): ATGATTCTGATACATCGATACAATCAAGCTAGCATGGAGATGGACATCGGCGAGGGCGAGAGCTGCTGCGGCAGGCGAAAGCAGCAGCAGCAGCAGAATATTAGCAGCAGCAAGTCACGCAAGTGCTGCCCGCTGCGGCGGTCGCGGAAGGGGTAGGGGAAGGGCGGGCCGGAGAACCAGCGTTGCCCCTTCCGCGGCGTCCGGCAGCGCACCTGGGGCAAGTGGGTGGCCGAGATCCGCGAGCCCAACCGCGGCGCCCGCCTCTGGCTCGGCACCTTCAACACCGCCCTCGACGCCGCCCGCGCCTACGACTCCGCCGCCAGGGCCCTCTACGGCGACTGCGCCCGCCTCAACCTACTCCTCGCCGCCGCCACCGCCGGTGCTCCTCCTGCTGCTGCTACCCCTTCCGTGGCCACGCCCTGCAGCACCAACGACGACTCCAACAACTCGTCTTCCACGACGCATCAGCAGCAGCTGACGACGATGCTGCAGCTGGACGACGACAACTACACGCTGCAGCCGTCGTCGTCAGATCAAGAGGACTTCGAGACGTACGTCACGCGGCTACCCAAGGCGGAGGACTTTGGGCTGGAGGGCTTCCAGGAGGTTCCACTCGACGTCCTCGACGAAGCCGGCGGTGGCATCAGCATCTGGGACCTCTCCATCTGCCCCGCCGATTTCATGGCCACCGCCGCCACCACCACCGCCAAATCATCTTAA。

[0052] Nucleotide sequence of OsERF44 gene deletion homozygous mutant 2 (SEQ ID NO.9): .

[0053] Protein sequence encoded by OsERF44 gene deletion homozygous mutant 1 (SEQ ID NO. 10): MILIHRYNQASMEMDIGEGESCCGRRKQQQQQNISSSKSRKCCPLRRSRKG*.

[0054] Protein sequence encoded by OsERF44 gene deletion homozygous mutant 2 (SEQ ID NO. 11): MILIHRYNQASMEMDIGEGESCCGRRKQQQQQNISSSKSRKCCPLRRSRKGC.

[0055] Upstream primer OsERF44cas9TF (SEQ ID NO.12): 5′-TGTGTGGTCGCGGAAGGGTGCATGA-3′ Downstream primer OsERF44cas9TR (SEQ ID NO.13): 5′-AAACTCATGCACCCCTTCCGCGACCA-3′.

[0056] Phenotypic identification: After obtaining a stable T2 generation knockout line, target site sequencing was performed to obtain homozygous mutants (shown in Figure 5). Mature rice seeds were harvested. The mature seeds were dried in an oven at 65°C until a constant weight was reached, and grain type analysis was performed on the mature seeds. The results are shown in Figure 6. The results showed no significant difference in grain length and particle size between wild-type and mutant rice.

[0057] Example 6: Evaluation of rice quality of rice OsERF44 gene knockout lines

[0058] T2-generation homozygous mutant lines with wild-type NIP and OsERF44 genes knocked out were cultivated in the field, under conventional sunlight, water, and fertilizer management, and mature seeds were harvested and used for the following experiments.

[0059] Measurement of total starch content: After threshing, mature seeds of wild-type NIP and knockout homozygous mutants were ground into rice flour. The rice flour was carefully sieved using a 100-mesh sieve and transferred to a clean resealable bag for reserve. 50 mg of each sample was weighed, and three replicates were set up and placed in a 50 mL centrifuge tube. 5 mL of 80% anhydrous ethanol was gently added along the wall of the centrifuge tube and left in an 85°C water bath for 5 minutes. Another 5 mL of 80% anhydrous ethanol was added and the tube was centrifuged at 4000 g for 10 minutes. The supernatant was carefully aspirated and removed with a pipette, and 10 mL of 80% anhydrous ethanol was added to the precipitate and gently mixed. Further centrifugation was performed at 4000 g for 10 minutes at room temperature. The centrifuged 50 mL centrifuge tube was then quickly inverted onto filter paper and allowed to air dry to remove any remaining ethanol.

[0060] The sample prepared above was dissolved in 1 mL of ddH2O and heated in a boiling water bath for 30 minutes. After cooling to room temperature, 4 mL of 2 M KOH was added and shaken at room temperature for 30 minutes (to prevent clumping). 16 mL of sodium acetate (1.2 M, pH=3.8) and 200 μL of amyloglucosidase (3000 U / mL) were added and the mixture was heated in a 60°C water bath for 45 minutes, shaking 2-3 times during that time. Finally, the volume was reduced to 100 mL and gently mixed.

[0061] Glucose content was measured using the GOD-PAP method. 1 mL of the treated sample was transferred to a 1.5 mL centrifuge tube and centrifuged at 4,000 rpm for 10 minutes. 100 μL of the supernatant was transferred to a 10 mL test tube or 10 mL centrifuge tube, 3 mL of GOD-PAP (pre-dissolved, wrapped in aluminum foil, and dissolved on ice) was added, and the mixture was heated in a 37°C water bath for 20 minutes, then inverted and thoroughly mixed. Absorbance was measured at a wavelength of 510 nm using a microplate reader (Infinite 200 PRO, TECAN, Switzerland), and blank zero preparation was performed using a reaction mixture treated under the same conditions with 100 μL of 0.1 M (pH 4.75) sodium acetate and 3 mL of GOD-PAP reagent. All samples must be measured within 60 minutes.

[0062] Table 1 shows the standard sample reaction solutions of gradient glucose solution.

[0063] [Table 1]

[0064] Standard curves and regression equations were established based on the absorbance values ​​of standard samples measured with a microplate reader and the known concentrations of the standard samples. Next, the absorbance values ​​of each sample were substituted into the regression equation to obtain the corresponding glucose content, and finally converted to total starch content (total starch content was equal to 0.9 times the glucose content, and the moisture content of the sample was calculated as 12%). Each sample was repeated three times, and the mean value was finally used as the total starch content. The results showed that the total starch content of the knockout homozygous mutant was significantly lower than that of the wild type (shown in Figure 7a).

[0065] Measurement of total protein content: The digester was opened and the temperature was set to 290°C. 0.2g of sieved rice flour was weighed into a 100mL digester tube (taking care to prevent the rice flour from adhering to the tube wall), and 5mL of H2SO4 was added. After the temperature reached 290°C, the sample was placed in the digester and boiled for 20 minutes. Then, the digester tube was removed, gently shaken to homogenize, and placed back into the digester at 290°C for 1 hour. During this time, the digester tube was removed and shaken to homogenize at 15-minute intervals. After 1 hour, the digester tube was removed and cooled to room temperature. 1mL of H2O2 was added and thoroughly mixed. The digester tube was returned to the digester and processed for 10 minutes, after which it was checked whether the sample was clear (if the sample was not clear after 10 minutes, 0.5mL of H2O2 was added and it was returned to the digester until it was clear). The digester tube was removed and cooled to room temperature. Finally, the sample was diluted to 100 mL and measured using a FOSS Model 250 Kjeldahl nitrogen analyzer. The results showed that the total protein content of the knockout homozygous mutant was significantly higher than that of the wild-type NIP (shown in Figure 7b).

[0066] RVA spectral analysis: 3g each of rice flour from wild-type and knockout homozygous mutants was accurately weighed, 25mL of distilled water was added to each, and the viscosity properties of the rice flour were measured using a TechMaster RVA viscosity analyzer from Perten, Sweden. The results showed that the viscosity curve of the knockout mutant starch showed a similar trend to that of the wild-type, but there were significant differences between them (shown in Figure 7c). The starch showed a maximum viscosity peak with increasing temperature, which was basically consistent with the wild-type, but the maximum viscosity of the knockout mutant was lower than that of the wild-type. After the temperature decreased, the viscosity values ​​of the three mutants began to decrease in a similar manner to the wild-type, but were still lower than that of the wild-type.

[0067] In summary, the experimental results above indicate that functional loss of the OsERF44 transcription factor significantly reduces the starch content and significantly increases the total protein content in rice seeds.

[0068] The embodiments described above merely illustrate preferred embodiments of the present invention and do not limit the scope of the invention. Any modifications or improvements made by those skilled in the art to the technical solutions of the present invention, provided that they do not depart from the spirit of the invention, are all included within the scope of protection defined in the claims of the present invention.

Claims

1. The use of a biological material to knock out the OsERF44 gene in the construction of a low-starch, high-protein rice variety, characterized in that the nucleotide sequence of the OsERF44 gene is as shown in SEQ ID NO. 1, wherein the following (1) or (2): (1) A gene knockout vector for knocking out the OsERF44 gene, (2) Use of a recombinant microbial strain containing the gene knockout vector.

2. The use according to claim 1, characterized in that the base strain of the recombinant microbial strain is Agrobacterium.

3. The use according to claim 1, characterized in that the gene knockout vector is a CRISPR / Cas9 gene knockout vector, and its target site sequence is as shown in SEQ ID NO.

5.

4. The use according to claim 3, characterized in that the nucleotide sequences of the upstream and downstream primers of the gRNA oligonucleotide chain of the CRISPR / Cas9 gene knockout vector are as shown in SEQ ID NO. 6-7.

5. A method for constructing a rice variety with low starch content and high protein content, comprising the step of knocking out the OsERF44 gene of rice to construct genetically modified rice, wherein the genetically modified rice is the rice variety with low starch content and high protein content. The nucleotide sequence of the OsERF44 gene is as shown in SEQ ID NO. 1, and is characterized by the following: Knockout of the OsERF44 gene in the aforementioned rice can be performed using either (1) or (2) below: (1) A gene knockout vector for knocking out the OsERF44 gene, (2) A method for constructing a rice variety with low starch content and high protein content, using a recombinant microbial strain containing the gene knockout vector.

6. The method according to claim 5, characterized in that knocking out the aforementioned gene involves employing a CRISPR / Cas9 gene knockout vector.

7. The OsERF44 gene was knocked out using Agrobacterium genetic transformation. The method according to claim 6, characterized by doing the following.

8. The target site sequence of the aforementioned CRISPR / Cas9 gene knockout vector is SEQ I The method according to claim 6, characterized in that it is as shown in D No.

5.

9. The gRNA oligonucleotide of the aforementioned CRISPR / Cas9 gene knockout vector The nucleotide sequences of the upstream and downstream primers of the dove chain are given in SEQ ID NO. 6-7. The method according to claim 8, characterized in that it is as shown.

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