Transcription factor OsERF44 for regulating and controlling rice quality and application of transcription factor OsERF44

Abstract

The invention discloses a transcription factor OsERF44 for regulating and controlling rice quality and application of the transcription factor OsERF44, and relates to the technical field of biotechnology and plant genetic engineering. The transcription factor OsERF44 for regulating and controlling the rice quality is cloned through reverse genetics, and the nucleotide sequence of the transcription factor OsERF44 is shown as SEQ ID NO. 1. The transcription factor can be used as a rice quality related gene for regulating and controlling rice character indexes, including important rice nutrition indexes: total starch content and total protein content. The invention proves that the knockout of the transcription factor can cause the reduction of the starch content and the increase of the protein content in the rice. The transcription factor OsERF44 provides important gene resources and theoretical basis for rice quality genetic improvement, and expands the biological function of the rice AP2 / ERF family transcription factor in rice quality regulation and control.

Description

Technical Field

[0001] The invention relates to the technical fields of biotechnology and plant genetic engineering, and in particular to a transcription factor OsERF44 for regulating rice quality and an application thereof. Background Art

[0002] With the growth of the global population and the improvement of living standards, the market demand for nutritious and healthy food continues to increase. As one of the most important food crops in the world, the nutritional value and functional ingredients of rice have an important impact on people's health. Traditional rice varieties are mainly high in starch content. Although they can meet energy needs, they are insufficient in protein and other nutrients. In recent years, the market demand for rice varieties with low starch content and high protein content has been growing.

[0003] Modern research shows that excessive intake of high-starch foods may lead to metabolic diseases such as obesity and diabetes. Therefore, the development of rice varieties with low starch content can help improve people's dietary structure and reduce the risk of these diseases. In addition, high-protein rice can provide people with more high-quality protein sources, enhance physical fitness and promote 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, such rice varieties can be used to produce specialty rice products (such as baby rice cereal, elderly health food) or animal feed, thereby opening up new market areas and increasing product added value.

[0005] Currently, many studies have been devoted to improving the nutritional content of rice through advanced technologies such as gene editing and molecular breeding. Despite some progress, existing varieties still find it difficult to meet the requirements of low starch and high protein at the same time. Therefore, there is an urgent need to develop a new rice variety with both low starch content 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 related to gene promoter regions to activate gene expression. At present, a variety of 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 type of transcription factor unique to plants. According to the number of AP2 / ERF domains and whether it contains other domains, it is divided into five subfamilies: AP2 (APETALA2), ethylene response factor (ERF), dehydration response element binding protein (DREB), RAV and Soloist. AP2 / ERF family proteins play an important role 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 be involved in the regulation of rice endosperm starch synthesis. The functions of AP2 / ERF family transcription factors are complex, and they can participate in multiple pathways and regulate multiple genes. However, there are few reports on the involvement of AP2 / ERF family transcription factors in rice in regulating rice yield and quality traits. Therefore, it is of great significance to deeply explore the genes related to rice starch synthesis and expand the biological functions of rice AP2 / ERF family transcription factors for rice breeding and improvement. Summary of the invention

[0007] The purpose of the present invention is to provide a transcription factor OsERF44 for regulating rice quality and its application to solve the problems existing in the above-mentioned prior art. The transcription factor OsERF44 provides an important gene resource and theoretical basis for genetic improvement of rice quality, and at the same time expands the biological function of rice AP2 / ERF family transcription factors in rice quality regulation.

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

[0009] The present invention provides an application of a biological material with an OsERF44 gene knocked out in constructing a rice variety with low starch content and high protein content. The nucleotide sequence of the OsERF44 gene is shown in SEQ ID NO.1.

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

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

[0012] (2) A recombinant microbial strain containing the gene knockout vector.

[0013] Furthermore, the basic 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 steps of knocking out the OsERF44 gene of rice to construct transgenic rice; 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 uses a CRISPR / Cas9 gene knockout vector.

[0019] Furthermore, the OsERF44 gene is knocked out by 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] The present invention cloned a transcription factor OsERF44 that regulates rice quality through reverse genetics. The transcription factor OsERF44 can be used as a rice quality-related gene to regulate rice trait indicators, including important rice nutritional indicators: total starch content and total protein content. The present invention proves that knocking out the transcription factor will lead to a decrease in starch content and an increase in protein content in rice. The transcription factor OsERF44 of the present invention provides an important gene resource and theoretical basis for the genetic improvement of rice quality, and at the same time expands the biological function of rice AP2 / ERF family transcription factors in rice quality regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0025] Figure 1 This is a diagram of tissue expression analysis for site prediction of the OsERF44 gene;

[0026] Figure 2 This is the predicted structure of OsERF44 protein;

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

[0028] Figure 4 This is a map of the CRISPR / Cas9 knockout vector containing the OsERF44 gene target site sequence;

[0029] Figure 5 Schematic diagram of gene editing of OsERF44 in the Nipponbare (NIP) background; 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 wild-type target site and the gene mutation target site sequence;

[0030] Figure 6 The trait statistical diagrams of mature seeds of wild-type NIP and mutant oserf44; a is the statistical diagram of rice seed length; b is the statistical diagram of rice seed diameter;

[0031] Figure 7 The figure shows the analysis of the physical and chemical indicators of wild-type NIP and mutant oserf44 rice; a is the statistical graph of total starch content; b is the statistical graph of total protein content; and c is the starch viscosity curve. DETAILED DESCRIPTION

[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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are 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] ATGATTCTGATACATCGATACAATCAAGCTAGCATGGAGATGGACATCGGCGAGGGCGAGAGCTGCTGCGGCAGGCGAAAGCAGCAGCAGCAGAATATTAGCAGCAGCAAGTCACGCAAGTGCTGCCCGCTGCGGCGGTCGCGGAAGGGGTGCATGAAGGGGAAGGGCGGGCCGGAGAACCAGCGTTGCCCCTTCCGCGGCGTCCGGCAGCGCACCTGGGGCAAGTGGGTGGCCGAGATCCGCGAGCCCAACCGCGGCGCCCGCCTCTGGCTCGGCACCTTCAACACCGCCCTCGACGCCGCCCGCGCCTACGACTCCGCCGCCAGGGCCCTCTACGGCGACTGCGCCCGCCTCAACCTACTCCTCGCCGCCGCCACCGCCGGTGCTCCTCCTGCTGCTGCTACCCCTTCCGTGGCCACGCCCTGCAGCACCAACGACGACTCCAACAACTCGTCTTCCACGACGCATCAGCAGCAGCTGACGACGATGCTGCAGCTGGACGACGACAACTACACGCTGCAGCCGTCGTCGTCAGATCAAGAGGACTTCGAGACGTACGTCACGCGGCTACCCAAGGCGGAGGACTTTGGGCTGGAGGGCTTCCAGGAGGTTCCACTCGACGTCCTCGACGAAGCCGGCGGTGGCATCAGCATCTGGGACCTCTCCATCTGCCCCGCCGATTTCATGGCCACCGCCGCCACCACCACCGCCAAATCATCTTAA。

[0040] SEQ ID NO.2:

[0041] MILIHRYNQASMEMDIGGESCCGRRKQQQQQNISSSKSRKCCPLRRSRKGCMKGKGGPENQRCPFRGVRQRTWGKWVAEIREPNRGARLWLGTFNTALDAARAYDSAARALYGDCARLNL LLAAATAGAPPAAATPSVATPCSTNDDSNNSSSTTHQQQLTTMLQLDDDNYTLQPSSSDQEDFETYVTRLPKAEDFGLEGFQEVPLDVLDEAGGGISIWDLSICPADFMATAATTTAKSS*.

[0042] Example 1 Analysis of the predicted tissue expression pattern of rice OsERF44 gene

[0043] The RAP-DB website (https: / / rapdb.dna.affrc.go.jp) was used to input the RAP_Locus number of OsERF44 and analyze the tissue expression pattern. The prediction results showed that OsERF44 was highly expressed in rice embryo and endosperm ( Figure 1 ).

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

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

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

[0047] According to the nucleotide sequence of the OsERF44 gene, recombinant primers were designed, and PCR amplification was performed using the cDNA of rice Nipponbare (NIP) as a template. After agarose electrophoresis detection, the gel was cut and purified to obtain the PCR purified product. The green fluorescent label vector pAN580 was used, and SpeI and Xba I were selected to perform double enzyme digestion to obtain a purified linearized vector for standby use. The PCR product was further connected to the linearized pAN580 vector using an infusion enzyme and transformed into Escherichia coli. The correctly sequenced large plasmid was extracted and transformed into rice protoplasts, and the fluorescent expression site was observed and photographed using laser confocal microscopy (LSM710, Zeiss, Germany). The specific operation steps are as follows:

[0048] Rice seedling culture: Prepare 1 / 2MS, sterilize the culture bottle at the same time, add kanamycin after sterilization and cooling, and pour the culture medium into the culture bottle about 3cm thick; select 93-11 rice grains with complete embryos, clean and without melanin, disinfect with 75% ethanol for 1 minute, wash three times with sterile water, change to 50% pasteurized solution for 30 minutes, and shake up and down on a shaker. After pasteurization, wash with sterile water about 4 times until the bubbles disappear, and place the sterilized seeds on the clean bench to dry. Transfer to 1 / 2MS culture medium, and then place it in a 30℃ incubator for culture. After 2 weeks, the seedlings can be used for protoplast extraction.

[0049] Protoplast extraction: Take out 15 10-13 day old seedlings, cut them into pieces smaller than 0.5 mm on clean A4 paper, and transfer them to 0.6 M mannitol solution in time. After all the seedlings are cut, place them in a vacuum box for 30 minutes, remove the air in the leaves, remove the 0.6 M mannitol through a 40 μm filter, add room temperature enzymolysis solution, shake at 28 ° C, 40 rpm enzymolysis for 5 hours; after the enzymolysis is completed, remove the enzymolysis solution with a 40 μm filter, add 10 mL W5 (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES (pH 5.7) was diluted to 100 mL with ddH2O.) After that, it was released on a shaker at 28°C and 40 rpm for 10 minutes, filtered into a 50 mL centrifuge tube (marked as tube 1), and then 10 mL W5 was added. After that, it was released on a shaker at 28°C and 40 rpm for 10 minutes, filtered into another 50 mL centrifuge tube (marked as tube 2); the filtrate was centrifuged at room temperature for 5 minutes at 70 g, horizontally rotated, the acceleration and deceleration were set to 1, and the supernatant was discarded; 1 mL W5 solution was added to suspend the protoplasts, and the centrifuge tube was carefully shaken to allow the protoplasts to be fully released in the W5 solution. 10 μL of the protoplast suspension was aspirated and counted, and the protoplast concentration was adjusted to 0.5-1×10 7 / mL; centrifuge at 70g for 5 minutes at room temperature, discard the supernatant, resuspend the protoplasts in MMG medium, and adjust the concentration to 0.5-1×10 7 / mL, prepare 5μg (or 10μg) of the plasmid to be transformed, dilute it to 10μL, add 200μL of protoplast suspension to each round-bottom centrifuge tube, flick with your thumb to mix, then add 210μL of PEG-CaCl2 solution to the centrifuge tube and flick to mix. Place at room temperature for 15 minutes; then add 840μL of W5 solution to the centrifuge tube to stop the transformation; transfer the centrifuge tube to a centrifuge (horizontal rotor, 100g) and centrifuge for 5 minutes, add 0.5mL WI solution, resuspend the protoplasts, and transfer them to a culture plate, in which 300μL WI solution is pre-added; culture for 16 hours. Centrifuge the cultured protoplasts for 5 minutes (horizontal rotor, 100g), remove the supernatant, and the remaining part is used to observe the fluorescence signal.

[0050] The results showed that the fluorescent signals were distributed in the cytoplasm and nucleus of rice protoplasts transformed with the empty vector plasmid, and the green fluorescence of OsERF44::GFP overlapped with the blue fluorescence of the nuclear dye DAPI, indicating that OsERF44 is located in the nucleus of typical transcription factors (such as Figure 3 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′-TCGAGACGTCTCTAGAAGATGATTTGGCGGTGGTG-3′.

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

[0057] Selection of gRNA target sequence: According to 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 was AGG;

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

[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, Figure 4 As shown in the figure), the target sequence is loaded into the 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 10 μM target site upstream and downstream primers and add 18 μL of Buffer Aneal. Mix well and treat at 95°C for 3 minutes. Then slowly reduce the temperature to 20°C at a rate of about 0.2°C / s to obtain a double-stranded sequence containing the knockout target site, i.e., oligo dimer.

[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 20°C metal bath for 1 hour.

[0066] (3) E. coli transformation: Take 10 μL of the final product of step (2) and add it to 50 μL of DH5a competent cells that have just been thawed, flick to mix, place on ice for 30 minutes, heat shock at 42°C for 45 seconds, let stand on ice for 2 minutes, then add 200 μL of antibiotic-free LB, place in a constant temperature shaker at 37°C, 200 rpm, and recover for one hour before applying kanamycin resistance (Kana + ) tablet.

[0067] (4) PCR detection of bacterial liquid: The next day, a single clone was picked and placed in a kanamycin-resistant liquid culture medium. The culture was cultured in a shaker at 37°C until the bacterial liquid 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 the plasmid of the positive clone bacterial liquid was extracted and set aside.

[0068] (5) Agrobacterium transformation and rice genetic transformation: The successfully constructed plasmid was transformed into Agrobacterium (EHA105): 1 μL of the plasmid was injected into the frozen and thawed Agrobacterium competent cells on ice, and then placed on ice for 5 minutes, liquid nitrogen for 5 minutes, and 37°C for 5 minutes. 300 μL of antibiotic-free LB was added and revived at 28°C for 4 hours, and then evenly applied to the (kanamycin + rifampicin) K + / Rif-resistant plates were plated and cultured at 28°C for 2 days before picking single clones. Positive clones were detected with hygromycin primers and expanded with 3 mL of liquid K + / Rif medium, further positive K + / Rif bacterial liquid was sent to the company for genetic transformation of rice under the Nipponbare background.

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

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

[0071] Knockout transgenic seedling detection: 20 T0 generation transgenic seedlings were obtained, placed in a normal temperature light incubator for about a week, and then DNA of the 20 seedlings was taken. After PCR amplification with OsERF44cas9TF and OsERF44cas9TR, the DNA was sent to the company for sequencing. The sequencing results were analyzed to obtain transgenic plants in which the translation of two proteins was terminated prematurely. The nucleotide sequences of the two homozygous mutants of the knockout OsERF44 gene obtained by the present invention are shown in SEQ ID NO.8 and SEQ ID NO.9, and the encoded proteins are shown in SEQ ID NO.10 and SEQ ID NO.11.

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

[0073] ATGATTCTGATACATCGATACAATCAAGCTAGCATGGAGATGGACATCGGCGAGGGCGAGAGCTGCTGCGGCAGGCGAAAGCAGCAGCAGCAGCAGAATATTAGCAGCAGCAAGTCACGCAAGTGCTGCCCGCTGCGGCGGTCGCGGAAGGGGTAGGGGAAGGGCGGGCCGGAGAACCAGCGTTGCCCCTTCCGCGGCGTCCGGCAGCGCACCTGGGGCAAGTGGGTGGCCGAGATCCGCGAGCCCAACCGCGGCGCCCGCCTCTGGCTCGGCACCTTCAACACCGCCCTCGACGCCGCCCGCGCCTACGACTCCGCCGCCAGGGCCCTCTACGGCGACTGCGCCCGCCTCAACCTACTCCTCGCCGCCGCCACCGCCGGTGCTCCTCCTGCTGCTGCTACCCCTTCCGTGGCCACGCCCTGCAGCACCAACGACGACTCCAACAACTCGTCTTCCACGACGCATCAGCAGCAGCTGACGACGATGCTGCAGCTGGACGACGACAACTACACGCTGCAGCCGTCGTCGTCAGATCAAGAGGACTTCGAGACGTACGTCACGCGGCTACCCAAGGCGGAGGACTTTGGGCTGGAGGGCTTCCAGGAGGTTCCACTCGACGTCCTCGACGAAGCCGGCGGTGGCATCAGCATCTGGGACCTCTCCATCTGCCCCGCCGATTTCATGGCCACCGCCGCCACCACCACCGCCAAATCATCTTAA。

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

[0075] ATGATTCTGATACATCGATACAATCAAGCTAGCATGGAGATGGACATCGGCGAGGGCGAGAGCTGCTGCGGCAGGCGAAAGCAGCAGCAGCAGCAGAATATTAGCAGCAGCAAGTCACGCAAGTGCTGCCCGCTGCGGCGGTCGCGGAAGGGGTGCTGAAGGGGAAGGGCGGGCCGGAGAACCAGCGTTGCCCCTTCCGCGGCGTCCGGCAGCGCACCTGGGGCAAGTGGGTGGCCGAGATCCGCGAGCCCAACCGCGGCGCCCGCCTCTGGCTCGGCACCTTCAACACCGCCCTCGACGCCGCCCGCGCCTACGACTCCGCCGCCAGGGCCCTCTACGGCGACTGCGCCCGCCTCAACCTACTCCTCGCCGCCGCCACCGCCGGTGCTCCTCCTGCTGCTGCTACCCCTTCCGTGGCCACGCCCTGCAGCACCAACGACGACTCCAACAACTCGTCTTCCACGACGCATCAGCAGCAGCTGACGACGATGCTGCAGCTGGACGACGACAACTACACGCTGCAGCCGTCGTCGTCAGATCAAGAGGACTTCGAGACGTACGTCACGCGGCTACCCAAGGCGGAGGACTTTGGGCTGGAGGGCTTCCAGGAGGTTCCACTCGACGTCCTCGACGAAGCCGGCGGTGGCATCAGCATCTGGGACCTCTCCATCTGCCCCGCCGATTTCATGGCCACCGCCGCCACCACCACCGCCAAATCATCTTAA。

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

[0077] MILIHRYNQASMEMDIGEGESCCGRRKQQQQQNISSSKSRKCCPLRRSRKG*。

[0078] Protein sequence encoded by the homozygous mutant 2 with deletion of the 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 inherited T2 knockout strain, further sequence the target site to obtain a homozygous mutant (such as Figure 5 The mature seeds were placed in a 65°C oven and dried to a constant weight. The mature seeds were analyzed for grain type. The results are shown in Figure 6 The results showed that there was no significant difference in rice grain length and size between the wild type and mutant.

[0085] Example 6 Rice Quality Identification of OsERF44 Gene Knockout Strains

[0086] The wild-type NIP and the T2 homozygous mutant lines with knockout OsERF44 gene were cultivated in the field with conventional sunlight, water and fertilizer management, and the mature seeds were harvested and used in the following experiments.

[0087] Determination of total starch content: The mature seeds of wild-type NIP and knockout homozygous mutants were shelled and ground into rice flour, and the rice flour was carefully sieved into a clean ziplock bag using a 100-mesh sieve for later use. 50 mg of each sample was weighed, three replicates were set, and placed in a 50 mL centrifuge tube. 5 mL of 80% anhydrous ethanol was gently added to the centrifuge tube along the tube wall, and it was placed in an 85°C water bath for 5 minutes. 5 mL of 80% anhydrous ethanol was added again, and then centrifuged at a speed of 4000g for 10 minutes. The supernatant was carefully sucked off with a gun, and 10 mL of 80% anhydrous ethanol was added to the precipitate, and gently mixed. It was further centrifuged at room temperature at 4000g for 10 minutes, and then the centrifuged 50 mL centrifuge tube was quickly turned upside down on the filter paper to naturally air dry the residual ethanol inside.

[0088] Add 1 mL of ddH2O to the prepared sample and dissolve it in a boiling water bath for 30 minutes; cool to room temperature, add 4 mL of 2M KOH, and oscillate at room temperature for 30 minutes (to prevent caking); add 16 mL of sodium acetate (1.2 M, pH = 3.8) and 200 μL of amyloglucosidase (3000 U / mL), and bathe in a 60°C water bath for 45 minutes, oscillating 2 to 3 times during the period, and finally adjust the volume to 100 mL and mix gently.

[0089] Glucose content was determined by GOD-PAP method. 1 mL of the above treated sample was placed in a 1.5 mL centrifuge tube, centrifuged at 4,000 rpm for 10 minutes, 100 μL of supernatant was placed in a 10 mL test tube or 10 mL centrifuge tube, 3 mL of GOD-PAP (taken out in advance, wrapped in tin foil, and placed on ice to melt) was added, and the sample was placed in a 37°C constant temperature water bath for 20 minutes, inverted, and fully mixed; the absorbance was measured at a wavelength of 510 nm using an ELISA reader (Infinite 200PRO, TECAN, Switzerland), and blank zeroing was performed using 100 μL of 0.1 M (pH 4.75) sodium acetate and 3 mL of GOD-PAP reagent treated under the same conditions. All samples must be determined within 60 minutes.

[0090] The gradient glucose solution standard sample reaction solution is shown in Table 1.

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

[0092]

[0093] According to the absorbance value of the standard sample measured in the microplate reader and the known concentration of the standard sample, a standard curve and a regression equation were established. Then, the absorbance value of each sample was substituted into the regression equation to obtain the corresponding glucose content, and finally converted into the total starch content (total starch content equals 0.9 times the glucose content, and the sample water content is calculated as 12%). Each sample was repeated three times, and the average 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 (such as Figure 7 (shown in a).

[0094] Determination of total protein content: Turn on the digestion furnace, set the temperature to 290℃, weigh 0.2g of sieved rice flour into a 100mL digestion tube (try not to let the rice flour touch the tube wall), add 5mL H2SO4, and when the temperature reaches 290℃, place the sample in the digestion furnace and boil for 20 minutes. Then take out the digestion tube and shake it gently, put it back into the digestion furnace, treat it at 290℃ for 1 hour, take out the digestion tube every 15 minutes and shake it well. After 1 hour, take out the digestion tube and cool it to room temperature. Add 1mL H2O2 to mix it thoroughly, then put the digestion tube back into the digestion furnace, and check whether the sample becomes clear after 10 minutes (if the sample still does not become clear after 10 minutes, add 0.5mL H2O2 and put it back into the digestion furnace until it becomes clear), take it out and cool it to room temperature. Finally, adjust the volume to 100mL and measure it on the FOSS 250 Kjeldahl nitrogen analyzer. The results showed that compared with the 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 spectrum analysis: 3g of wild-type and knockout homozygous rice flour were accurately weighed, and 25mL of distilled water was added to each. The viscosity characteristics of the rice flour were measured using the TechMaster RVA rapid viscosity analyzer from Perten, Sweden. The results showed that the viscosity curve of the knockout mutant starch had a similar trend to that of the wild-type, but there were significant differences between them (e.g. Figure 7 As shown in c). Starch has a maximum viscosity peak as the temperature rises, which is basically consistent with the wild type, but the maximum 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 the wild type.

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

[0097] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a biomaterial with knocked-out OsERF44 gene in constructing a rice variety 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 use according to claim 1, characterized in that: The biological material is the substance described in (1) or (2) below: (1) a gene knockout vector for knocking out the OsERF44 gene; (2) A recombinant microbial strain containing the gene knockout vector.

3. The use according to claim 2, characterized in that: The basic strain of the recombinant microorganism strain is Agrobacterium.

4. The use 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 use 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 a rice variety with low starch content and high protein content, characterized in that: The method comprises the steps of knocking out the OsERF44 gene of 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 uses a CRISPR / Cas9 gene knockout vector.

8. The method according to claim 7, characterized in that The OsERF44 gene was knocked out by 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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