A transcription factor OsERF44 regulating rice quality and its application

Abstract

This invention discloses a transcription factor OsERF44 that regulates rice quality and its applications, relating to the fields of biotechnology and plant genetic engineering. This invention clones a transcription factor OsERF44 that regulates rice quality using reverse genetics, its nucleotide sequence being shown in SEQ ID NO.1. This transcription factor can serve as a rice quality-related gene, used to regulate rice phenotypic indicators, including important nutritional indicators of rice: total starch content and total protein content. This invention demonstrates that knockout of this transcription factor leads to a decrease in starch content and an increase in protein content in rice. The OsERF44 transcription factor of this invention provides an important gene resource and theoretical basis for the genetic improvement of rice quality, while also expanding the biological functions of rice AP2 / ERF family transcription factors in regulating rice quality.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and plant genetic engineering, and in particular to a transcription factor OsERF44 that regulates rice quality and its applications. Background Technology

[0002] With global population growth and rising living standards, the market demand for nutritious and healthy foods is constantly increasing. Rice, as one of the world's most important food crops, has a significant impact on human health due to its nutritional value and functional components. Traditional rice varieties are mainly high in starch, which, while meeting energy needs, is insufficient in protein and other nutrients. In recent years, the market demand for rice varieties with low starch and high protein content has been growing.

[0003] Modern research indicates that excessive intake of high-starch foods may lead to metabolic diseases such as obesity and diabetes. Therefore, developing low-starch rice varieties can help improve people's dietary structure and reduce the risk of these diseases. Furthermore, high-protein rice can provide people with more high-quality protein sources, enhancing physical fitness and promoting health.

[0004] From an agricultural and economic perspective, planting low-starch, high-protein rice varieties can not only improve land utilization but also increase farmers' income. For example, these rice varieties can be used to produce specialty rice products (such as infant rice cereal and health foods for the elderly) or animal feed, thereby opening up new market areas and increasing product added value.

[0005] Currently, many studies are dedicated to improving the nutritional composition of rice using advanced technologies such as gene editing and molecular breeding. Despite some progress, existing varieties still struggle to simultaneously meet the requirements of low starch and high protein content. Therefore, there is an urgent need to develop a new rice variety that combines low starch and high protein content to fill the market gap and meet consumer demand.

[0006] Transcription factors, also known as trans-acting factors, can specifically bind to cis-acting elements associated with gene promoter regions, activating gene expression. Currently, various plant-related transcription factors have been reported, such as MYB, bHLH, AP2 / ERF, WRKY, and NAC, among which AP2 / ERF (APETALA2 / ethylene responsive factor) is a plant-specific class of transcription factors. Based on the number of AP2 / ERF domains and the presence of other domains, they are divided 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 stresses, and biosynthesis. Transcription factors play a decisive role in the regulation of starch biosynthesis, but currently only a few transcription factors have been reported to participate in the regulation of starch synthesis in rice endosperm. AP2 / ERF family transcription factors have complex functions, participating in multiple pathways and regulating numerous genes. However, there are few reports on the role of AP2 / ERF family transcription factors in regulating rice yield and quality traits. Therefore, in-depth exploration of rice starch synthesis-related genes and expansion of the biological functions of rice AP2 / ERF family transcription factors are of great significance for rice breeding and improvement. Summary of the Invention

[0007] The purpose of this invention is to provide a transcription factor, OsERF44, that regulates rice quality and its applications, in order to solve the problems existing in the prior art. Transcription factor OsERF44 provides an important gene resource and theoretical basis for the genetic improvement of rice quality, while also expanding the biological functions of rice AP2 / ERF family transcription factors in regulating rice quality.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] This invention provides the application of biomaterials with the OsERF44 gene knocked out in the construction of rice varieties with low starch content and high protein content, wherein the nucleotide sequence of the OsERF44 gene is shown in SEQ ID NO.1.

[0010] Furthermore, the biomaterial is the substance described in (1) or (2) below:

[0011] (1) Gene knockout vector for knocking out the OsERF44 gene;

[0012] (2) Recombinant microbial strains containing the gene knockout vector.

[0013] Furthermore, the base strain of the recombinant microbial strain is Agrobacterium.

[0014] Furthermore, the gene knockout vector is a CRISPR / Cas9 gene knockout vector, and its target site sequence is shown in SEQ ID NO.5.

[0015] Furthermore, the nucleotide sequences of the upstream and downstream primers of the gRNA oligonucleotide chain of the CRISPR / Cas9 gene knockout vector are shown in SEQ ID NO.6-7.

[0016] The present invention also provides 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 a transgenic rice; wherein the transgenic rice is the rice variety with low starch content and high protein content.

[0017] The nucleotide sequence of the OsERF44 gene is shown in SEQ ID NO.1.

[0018] Furthermore, the gene knockout is performed using a CRISPR / Cas9 gene knockout vector.

[0019] Furthermore, the OsERF44 gene was knocked out using Agrobacterium genetic transformation.

[0020] Furthermore, the target site sequence of the CRISPR / Cas9 gene knockout vector is shown in SEQ ID NO.5.

[0021] Furthermore, the nucleotide sequences of the upstream and downstream primers of the gRNA oligonucleotide chain of the CRISPR / Cas9 gene knockout vector are shown in SEQ ID NO.6-7.

[0022] The present invention discloses the following technical effects:

[0023] This invention cloned a transcription factor, OsERF44, that regulates rice quality using reverse genetics. This transcription factor, OsERF44, can serve as a rice quality-related gene, used to regulate rice phenotypic indicators, including important nutritional indicators such as total starch content and total protein content. This invention demonstrates that knocking out this transcription factor leads to a decrease in starch content and an increase in protein content in rice. The OsERF44 transcription factor of this invention provides an important gene resource and theoretical basis for the genetic improvement of rice quality, while also expanding the biological functions of rice AP2 / ERF family transcription factors in regulating rice quality. Attached Figure Description

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a plot showing the predicted tissue expression analysis of the OsERF44 gene on a website.

[0026] Figure 2 The structure prediction diagram of the OsERF44 protein;

[0027] Figure 3 Subcellular localization map of OsERF44 protein; ad represents GFP green fluorescence signal, nuclear marker (dye DAPI) blue fluorescence signal, bright field, and fluorescence superposition signal, respectively;

[0028] Figure 4 A map of CRISPR / Cas9 knockout vectors containing the OsERF44 gene target site sequence;

[0029] Figure 5 This is a schematic diagram of gene editing of OsERF44 under the Nipponbare (NIP) background; where a is a schematic diagram of the gene structure and the sequences of the wild-type target site and the gene mutation target site; b is a sequencing peak diagram of the sequences of the wild-type target site and the gene mutation target site.

[0030] Figure 6 The graphs show the phenotypic characteristics of mature seeds of wild-type NIP and mutant oserf44; where a is the graph showing the length of rice seeds and b is the graph showing the diameter of rice seeds.

[0031] Figure 7 The graphs show the physicochemical properties of wild-type NIP and mutant oserf44 rice; where a is the total starch content graph; b is the total protein content graph; and c is the starch viscosity curve. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] 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.

[0038] SEQ ID NO.1:

[0039] ATGATTCTGATACATCGATACAATCAAGCTAGCATGGAGATGGACATCGGCGAGGGCGAGAGCTGCTGCGGCAGGCGAAAGCAGCAGCAGCAGCAGAATATTAGCAGCAGCAAGTCACGCAAGTGCTGCCCGCTGCGGCGGTCGCGGAAGGGGTGCATGAAGGGGAAGGGCGGGCCGGAGAACCAGCGTTGCCCCTTCCGCGGCGTCCGGCAGCGCACCTGGGGCAAGTGGGTGGCCGAGATCCGCGAGCCCAACCGCGGCGCCCGCCTCTGGCTCGGCACCTTCAACACCGCCCTCGACGCCGCCCGCGCCTACGACTCCGCCGCCAGGGCCCTCTACGGCGACTGCGCCCGCCTCAACCTACTCCTCGCCGCCGCCACCGCCGGTGCTCCTCCTGCTGCTGCTACCCCTTCCGTGGCCACGCCCTGCAGCACCAACGACGACTCCAACAACTCGTCTTCCACGACGCATCAGCAGCAGCTGACGACGATGCTGCAGCTGGACGACGACAACTACACGCTGCAGCCGTCGTCGTCAGATCAAGAGGACTTCGAGACGTACGTCACGCGGCTACCCAAGGCGGAGGACTTTGGGCTGGAGGGCTTCCAGGAGGTTCCACTCGACGTCCTCGACGAAGCCGGCGGTGGCATCAGCATCTGGGACCTCTCCATCTGCCCCGCCGATTTCATGGCCACCGCCGCCACCACCACCGCCAAATCATCTTAA。

[0040] SEQ ID NO.2:

[0041] MILIHRYNQASMEMDIGGESCCGRRKQQQQQNISSSKSRKCCPLRRSRKGCMKGKGGPENQRCPFRGVRQRTWGKWVAEIREPNRGARLWLGTFNTALDAARAYDSAARALYGDCARLNL LLAAATAGAPPAAATPSVATPCSTNDDSNNSSSTTHQQQLTTMLQLDDDNYTLQPSSSDQEDFETYVTRLPKAEDFGLEGFQEVPLDVLDEAGGGISIWDLSICPADFMATAATTTAKSS*.

[0042] Example 1: Tissue expression pattern analysis of predicted OsERF44 gene in rice

[0043] Tissue expression pattern analysis was performed using the RAP-DB website (https: / / rapdb.dna.affrc.go.jp) by inputting the RAP_Locus number of OsERF44. The prediction results showed that OsERF44 was highly expressed in rice embryos and endosperm. Figure 1 ).

[0044] Example 2: Prediction of the structure of rice transcription factor OsERF44 protein

[0045] Using the SMART website (http: / / smart.embl-heidelberg.de / ), the protein sequence corresponding to OsERF44 was input for domain prediction analysis. The prediction results showed that the OsERF44 protein contains an AP2 domain between amino acids 65 and 128. Such domains are generally involved in binding DNA to regulate the expression of downstream target genes. Figure 2 ).

[0046] Example 3 Subcellular localization of rice transcription factor OsERF44

[0047] Recombinant primers were designed based on the nucleotide sequence of the OsERF44 gene. PCR amplification was performed using cDNA from Nipponbare rice (NIP) as a template. After agarose gel electrophoresis detection, the PCR product was purified by gel extraction. The green fluorescent tag vector pAN580 was double-digested with Spe I and Xba I to obtain a purified linearized vector for later use. The PCR product was then ligated to the linearized pAN580 vector using infusion enzyme and transformed into *E. coli*. The correctly sequenced plasmid was extracted and transformed into rice protoplasts. Fluorescent expression sites were observed and photographed using laser confocal microscopy (LSM710, Zeiss, Germany). The specific operational steps are as follows:

[0048] Rice seedling culture: Prepare a 1 / 2 MS medium and sterilize the culture flasks simultaneously. After sterilization and cooling, add kanamycin and pour the medium into the culture flasks to a thickness of approximately 3 cm. Select clean rice grains (93-11 mm) with intact embryos and free of melanin. Disinfect with 75% ethanol for 1 minute, then wash three times with sterile water. Disinfect with 50% pasteurized solution for 30 minutes, shaking on a shaker. After pasteurization, wash with sterile water approximately four times until the air bubbles disappear. Place the sterilized seeds on a laminar flow hood and air dry. Transfer to 1 / 2 MS medium and incubate at 30°C. After 2 weeks, the seedlings are ready for protoplast extraction.

[0049] Protoplast extraction: Take 15 seedlings aged 10-13 days and cut them into fragments smaller than 0.5 mm on clean A4 paper. Immediately transfer them to 0.6 M mannitol solution. After all seedlings have been cut, place them in a vacuum chamber for 30 minutes to remove air from the leaves. Remove the 0.6 M mannitol by passing the fragments through a 40 μm filter. Add room temperature enzymatic hydrolysate and incubate at 28°C and 40 rpm for 5 hours on a shaker. After enzymatic hydrolysis, remove the hydrolysate through a 40 μm filter. Add 10 mL of W5 (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES (pH 5.7), and bring the volume to 100 mL with ddH2O). Incubate at 28°C and 40 rpm for 10 minutes on a shaker. Filter into a 50 mL centrifuge tube (labeled tube 1). Add another 10 mL of W5 and incubate at 28°C and 40 rpm for 10 minutes on a shaker. Filter into another 50 mL centrifuge tube. Add the filtrate to a centrifuge tube (labeled tube 2); centrifuge at 70 g room temperature for 5 minutes with the centrifuge tube horizontally and the acceleration / deceleration setting set to 1, then discard the supernatant; add 1 mL of W5 solution to suspend the protoplasts, and gently agitate the centrifuge tube to allow the protoplasts to be fully released into the W5 solution. Pipette 10 μL of the protoplast suspension and count the protoplasts, adjusting the protoplast concentration to 0.5–1 × 10⁻⁶. 7 / mL; centrifuge at 70 g at room temperature for 5 minutes, discard the supernatant, resuspend the protoplasm in MMG medium, and adjust the concentration to 0.5-1×10⁻⁶ mL. 7Prepare 5 μg (or 10 μg) of plasmid to be transformed, dilute it to 10 μL, add 200 μL of protoplast suspension to each round-bottom centrifuge tube, gently tap to mix, then add 210 μL of PEG-CaCl2 solution and gently tap to mix. Incubate at room temperature for 15 minutes; then add 840 μL of W5 solution to stop the transformation; transfer the centrifuge tube to a centrifuge (horizontal rotor, 100 g) and centrifuge for 5 minutes, add 0.5 mL of WI solution to resuspend the protoplasts, and transfer them to a culture plate pre-filled with 300 μL of WI solution; incubate for 16 hours. Centrifuge the cultured protoplasts for 5 minutes (horizontal rotor, 100 g), remove the supernatant, and use the remaining portion for fluorescence signal observation.

[0050] The results showed that fluorescent signals were distributed in both the cytoplasm and nucleus of rice protoplasts transformed with the empty vector plasmid. The green fluorescence of OsERF44::GFP coincided with the blue fluorescence of the nuclear dye DAPI, indicating that OsERF44 is located in the nucleus as a typical transcription factor (such as...) Figure 3 (As shown).

[0051] The primers for constructing the OsERF44 subcellular localization vector are as follows:

[0052] The upstream primer is OsERF44-GFP-F (SEQ ID NO.3):

[0053] 5′-GCCCAGATCAACTAGTATGATTCTGATACATCGATACAA-3′;

[0054] The downstream primer is OsERF44-GFP-R (SEQ ID NO.4):

[0055] 5′-TCGAGACGTCTCTAGAAGATTGATTTGGCGGTGGTG-3′.

[0056] Example 4: Construction of OsERF44 gene knockout transgenic rice

[0057] Selection of gRNA target sequence: Based on CRISPR / Cas9 related experimental methods, the 5′-GTCGCGGAAGGGGTGCATGAAGG-3′ sequence (SEQ ID NO.5) containing NGG as the recognition site was selected as the knockout target site on the exon of the OsERF44 gene, and the PAM sequence is AGG;

[0058] Design of upstream and downstream primers for gRNA oligonucleotide chains:

[0059] The upstream primer is OsERF44_gRNA_F (SEQ ID NO.6):

[0060] 5′-TGTGTGGTCGCGGAAGGGTGCATGA-3′;

[0061] The downstream primer is OsERF44_gRNA_F (SEQ ID NO.7):

[0062] 5′-ACTCATGCACCCCTTCCCGACCAAA-3′.

[0063] CRISPR / Cas9 vector construction: This example uses a plant Cas9 / gRNA plasmid construction kit (Catalog. No. BGK03, e.g.) Figure 4 As shown in the figure, the target sequence is loaded to form a recombinant vector containing the OsERF44 gene target site. The specific operation method is as follows:

[0064] (1) Preparation of oligo dimers: Take 1 μL of each of the upstream and downstream primers of 10 μM target site and add 18 μL of Buffer Aneal. After mixing, treat at 95℃ for 3 minutes, and then slowly reduce to 20℃ at about 0.2℃ / second to obtain the double-stranded sequence containing the knockout target site, i.e., oligo dimers.

[0065] (2) Constructing oligo dimers into CRISPR / Cas9: Take 2 μL of Cas9 / gRNA vector, 1 μL of oligo dimer from step (1), 1 μL of Enzyme Mix, add 6 μL of ddH2O, mix well and react in a metal bath at 20℃ for 1 hour.

[0066] (3) E. coli transformation: Take 10 μL of the final product from step (2) and add it to 50 μL of freshly thawed DH5α competent cells. Gently mix, incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, and let stand on ice for 2 minutes. Then add 200 μL of antibiotic-free LB and place in a 37°C constant temperature shaker at 200 rpm. After one hour of recovery, apply kanamycin resistance (Kana) assay. + (a flat plate.)

[0067] (4) PCR detection of bacterial culture: The next day, single clones were picked and cultured in kanamycin-resistant liquid medium at 37°C until the bacterial culture became turbid. Sequencing was performed using the BGK03 vector-specific sequencing primers provided in the kit. The sequencing results were analyzed using Snapgene software, and plasmids of positive clone bacterial cultures were extracted for later use.

[0068] (5) Agrobacterium-mediated transformation and rice genetic transformation: The successfully constructed plasmid was transformed into Agrobacterium (EHA105): 1 μL of plasmid was injected into the frozen and thawed Agrobacterium competent cells, then placed on ice for 5 minutes, in liquid nitrogen for 5 minutes, at 37°C for 5 minutes, and then 300 μL of antibiotic-free LB was added and the cells were thawed at 28°C for 4 hours. The mixture was then evenly spread on (kanamycin + rifampin) K + On Rif-resistant plates, after incubation at 28°C for 2 days, single colonies were picked, and positive clones were obtained by detection with hygromycin primers. The colonies were then expanded by incubation in 3 mL of liquid K. + In Rif medium, further positive K+ will be added. + / Rif bacterial solution was sent to the company for genetic transformation of rice under the Nipponbare background.

[0069] Example 5 Phenotypic Analysis of Rice OsERF44 Gene Knockout Lines

[0070] To identify the knockout transgenic lines obtained in Example 4, the transgenic seedlings were cultured in a room temperature and light incubator for about a week, and then the positive seedlings were identified. The specific steps are as follows:

[0071] Detection of knockout transgenic seedlings: Twenty T0 generation transgenic seedlings were obtained and cultured in a room temperature light incubator for about one week. DNA from these 20 seedlings was then collected and amplified by PCR using OsERF44cas9TF and OsERF44cas9TR. The DNA was then sent to a company for sequencing. Analysis of the sequencing results revealed transgenic plants with premature termination of translation for two proteins. The nucleotide sequences of the two homozygous mutants with the OsERF44 gene knockout obtained in this invention are shown in SEQ ID NO.8 and SEQ ID NO.9, and their encoded proteins are shown in SEQ ID NO.10 and SEQ ID NO.11.

[0072] The nucleotide sequence of OsERF44 gene deletion homozygous mutant 1 (SEQ ID NO.8)

[0073] ATGATTCTGATACATCGATACAATCAAGCTAGCATGGAGATGGACATCGGCGAGGGCGAGAGCTGCTGCGGCAGGCGAAAGCAGCAGCAGCAGCAGAATATTAGCAGCAGCAAGTCACGCAAGTGCTGCCCGCTGCGGCGGTCGCGGAAGGGGTAGGGGAAGGGCGGGCCGGAGAACCAGCGTTGCCCCTTCCGCGGCGTCCGGCAGCGCACCTGGGGCAAGTGGGTGGCCGAGATCCGCGAGCCCAACCGCGGCGCCCGCCTCTGGCTCGGCACCTTCAACACCGCCCTCGACGCCGCCCGCGCCTACGACTCCGCCGCCAGGGCCCTCTACGGCGACTGCGCCCGCCTCAACCTACTCCTCGCCGCCGCCACCGCCGGTGCTCCTCCTGCTGCTGCTACCCCTTCCGTGGCCACGCCCTGCAGCACCAACGACGACTCCAACAACTCGTCTTCCACGACGCATCAGCAGCAGCTGACGACGATGCTGCAGCTGGACGACGACAACTACACGCTGCAGCCGTCGTCGTCAGATCAAGAGGACTTCGAGACGTACGTCACGCGGCTACCCAAGGCGGAGGACTTTGGGCTGGAGGGCTTCCAGGAGGTTCCACTCGACGTCCTCGACGAAGCCGGCGGTGGCATCAGCATCTGGGACCTCTCCATCTGCCCCGCCGATTTCATGGCCACCGCCGCCACCACCACCGCCAAATCATCTTAA。

[0074] Nucleotide sequence of homozygous mutant 2 with deletion of OsERF44 gene (SEQ ID NO.9)

[0075] ATGATTCTGATACATCGATACAATCAAGCTAGCATGGAGATGGACATCGGCGAGGGCGAGAGCTGCTGCGGCAGGCGAAAGCAGCAGCAGCAGCAGAATATTAGCAGCAGCAAGTCACGCAAGTGCTGCCCGCTGCGGCGGTCGCGGAAGGGGTGCTGAAGGGGAAGGGCGGGCCGGAGAACCAGCGTTGCCCCTTCCGCGGCGTCCGGCAGCGCACCTGGGGCAAGTGGGTGGCCGAGATCCGCGAGCCCAACCGCGGCGCCCGCCTCTGGCTCGGCACCTTCAACACCGCCCTCGACGCCGCCCGCGCCTACGACTCCGCCGCCAGGGCCCTCTACGGCGACTGCGCCCGCCTCAACCTACTCCTCGCCGCCGCCACCGCCGGTGCTCCTCCTGCTGCTGCTACCCCTTCCGTGGCCACGCCCTGCAGCACCAACGACGACTCCAACAACTCGTCTTCCACGACGCATCAGCAGCAGCTGACGACGATGCTGCAGCTGGACGACGACAACTACACGCTGCAGCCGTCGTCGTCAGATCAAGAGGACTTCGAGACGTACGTCACGCGGCTACCCAAGGCGGAGGACTTTGGGCTGGAGGGCTTCCAGGAGGTTCCACTCGACGTCCTCGACGAAGCCGGCGGTGGCATCAGCATCTGGGACCTCTCCATCTGCCCCGCCGATTTCATGGCCACCGCCGCCACCACCACCGCCAAATCATCTTAA。

[0076] Protein sequence encoded by homozygous mutant 1 with deletion of OsERF44 gene (SEQ ID NO.10):

[0077] MILIHRYNQASMEMDIGEGESCCGRRKQQQQQNISSSKSRKCCPLRRSRKG*。

[0078] Protein sequence encoded by homozygous mutant 2 with deletion of OsERF44 gene (SEQ ID NO.11):

[0079] MILIHRYNQASMEMDIGGESCCGRRKQQQQQNISSSKSRKCCPLRRSRKGC*.

[0080] Upstream primer OsERF44cas9TF (SEQ ID NO.12):

[0081] 5′-TGTGTGGTCGCGGAAGGGTGCATGA-3′;

[0082] Downstream primer OsERF44cas9TR (SEQ ID NO.13):

[0083] 5′-AAACTCATGCACCCCTCCGCGACCA-3′.

[0084] Phenotypic identification: After obtaining a stable T2 generation knockout line, further sequencing of the target site was performed to obtain homozygous mutants (e.g., Figure 5 As shown in the figure, mature rice seeds were harvested. The mature seeds were dried in a 65℃ oven to constant weight. Grain shape analysis of the mature seeds was performed, and the results are shown in the figure. Figure 6 The results showed no significant differences in grain length and diameter between wild-type and mutant rice.

[0085] Example 6: Rice quality identification of OsERF44 gene knockout rice lines

[0086] Wild-type NIP and OsERF44 knockout T2 generation homozygous mutant lines were cultivated in the field, and mature seeds were harvested under conventional daylight and water and fertilizer management for the following experiments.

[0087] Total starch content determination: Mature seeds of wild-type NIP and knockout homozygous mutants were dehulled and ground into rice flour. The rice flour was carefully sieved through a 100-mesh sieve into a clean resealable bag for later use. 50 mg of each sample was weighed, with three replicates, and placed in a 50 mL centrifuge tube. 5 mL of 80% anhydrous ethanol was gently added along the tube wall. The tube was placed 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 with a pipette. 10 mL of 80% anhydrous ethanol was added to the precipitate and gently mixed. The tube was then centrifuged again at 4000 g for 10 minutes at room temperature. The centrifuged 50 mL centrifuge tubes were then quickly inverted onto filter paper and allowed to air dry to remove any remaining ethanol.

[0088] Dissolve the prepared sample in 1 mL of ddH2O and boil in a water bath for 30 minutes. Cool to room temperature, add 4 mL of 2M KOH, and shake at room temperature for 30 minutes (to prevent clumping). Add 16 mL of sodium acetate (1.2 M, pH=3.8) and 200 μL of amylase (3000 U / mL), and incubate in a water bath at 60℃ for 45 minutes, shaking 2-3 times during the process. Finally, bring the volume to 100 mL and mix gently.

[0089] To determine glucose content using the GOD-PAP method, transfer 1 mL of the prepared sample to a 1.5 mL centrifuge tube and centrifuge at 4,000 rpm for 10 minutes. Transfer 100 μL of the supernatant to a 10 mL test tube or centrifuge tube, add 3 mL of GOD-PAP (pre-thawed by wrapping in aluminum foil on ice), and incubate at 37°C for 20 minutes, inverting the tube to mix thoroughly. Measure the absorbance at 510 nm using an ELISA reader (Infinite 200 PRO, TECAN, Switzerland). Zero the sample using a blank prepared with 100 μL of 0.1 M (pH 4.75) sodium acetate and 3 mL of GOD-PAP reagent under the same conditions. All samples must be measured within 60 minutes.

[0090] The reaction solutions for the gradient glucose solution standard samples are shown in Table 1.

[0091] Table 1 Gradient glucose solution standard sample reaction solution

[0092]

[0093] Based on the absorbance values ​​of standard samples measured by an ELISA reader and their known concentrations, a standard curve and regression equation were established. Then, the absorbance value of each sample was substituted into the regression equation to obtain the corresponding glucose content, which was then converted into the total starch content (total starch content equals 0.9 times the glucose content, and sample moisture content is calculated as 12%). Each sample was tested three times, and the average value was taken 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 (e.g., ...). Figure 7 (as shown in a).

[0094] Total protein content determination: Turn on the digestion furnace and set the temperature to 290℃. Weigh 0.2 g of sieved rice flour into a 100 mL digestion tube (avoid touching the tube wall with the rice flour as much as possible). Add 5 mL of H2SO4. When the temperature reaches 290℃, place the sample in the digestion furnace and boil for 20 minutes. Then remove the digestion tube, gently shake it, and put it back into the digestion furnace. Treat at 290℃ for 1 hour, shaking the digestion tube every 15 minutes. After 1 hour, remove the digestion tube and cool it to room temperature. Add 1 mL of H2O2 and mix thoroughly. Then put the digestion tube back into the digestion furnace and treat for 10 minutes. Check if the sample has become clear (if the sample has not become clear after 10 minutes, add 0.5 mL of H2O2 and put it back into the digestion furnace until it becomes clear). Remove and cool to room temperature. Finally, bring the volume to 100 mL and determine the content using a FOSS 250 Kjeldahl nitrogen analyzer. The results showed that, compared with wild-type NIP, the total protein content of the knockout homozygous mutant was significantly higher than that of the wild-type (e.g., Figure 7 (As shown in b).

[0095] RVA spectral analysis: 3 g of wild-type and knockout homozygous rice flour were accurately weighed and added to 25 mL of distilled water respectively. The viscosity characteristics of the rice flour were measured using a TechMaster RVA rapid viscosity analyzer from Perten, Sweden. The results showed that the viscosity curves of the knockout mutant starch exhibited similar trends to those of the wild type, but significant differences existed between them (e.g., ...). Figure 7 (As shown in c). Starch reaches its maximum viscosity peak as temperature increases, which is basically consistent with the wild type. However, the highest viscosity of the knockout mutant is lower than that of the wild type. When the temperature decreases, the viscosity values ​​of the three mutants begin to decrease like the wild type, but are still lower than those of the wild type.

[0096] Based on the above experimental results, it can be concluded that the loss of function of the OsERF44 transcription factor leads to a significant decrease in starch content and a significant increase in total protein content in rice seeds.

[0097] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. The application of a biomaterial with the OsERF44 gene knocked out in the construction of rice varieties with low starch content and high protein content, characterized in that, The nucleotide sequence of the OsERF44 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The biomaterial is the substance described in (1) or (2) below: (1) Gene knockout vector for knocking out the OsERF44 gene; (2) Recombinant microbial strains containing the gene knockout vector.

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

4. The application according to claim 2, characterized in that, The gene knockout vector is a CRISPR / Cas9 gene knockout vector, and its target site sequence is shown in SEQ ID NO.

5.

5. The application according to claim 4, 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 shown in SEQ ID NO. 6-7.

6. A method for constructing rice varieties with low starch content and high protein content, characterized in that, The process includes the step of knocking out the OsERF44 gene in rice to construct transgenic rice; the transgenic rice is the rice variety with low starch content and high protein content. The nucleotide sequence of the OsERF44 gene is shown in SEQ ID NO.

1.

7. The method according to claim 6, characterized in that, The gene knockout was performed using a CRISPR / Cas9 gene knockout vector.

8. The method according to claim 7, characterized in that, The OsERF44 gene was knocked out using Agrobacterium genetic transformation.

9. The method according to claim 7, characterized in that, The target site sequence of the CRISPR / Cas9 gene knockout vector is shown in SEQ ID NO.

5.

10. The method according to claim 9, 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 shown in SEQ ID NO. 6-7.

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