Application of OsSAP3 gene in regulating high temperature resistance in rice seedlings

Abstract

The present invention belongs to the technical field of plant genetic engineering and specifically relates to OsSAP3 Application of genes in regulating high temperature resistance of rice seedlings, the OsSAP3 The CDS sequence of the gene is shown in SEQ ID NO.1. OsSAP3 The full-length sequence of the gene is shown in SEQ ID NO. 2. OsSAP3 Gene, found OsSAP3 The survival rate of gene knockout mutants was significantly increased after high temperature treatment at the rice seedling stage, confirming its use in regulating high temperature resistance of rice seedlings and providing an important genetic resource for breeding high temperature resistant rice varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering and specifically relates to OsSAP3 Application of genes in regulating high temperature resistance in rice seedlings. Background Art

[0002] In recent years, high temperatures have occurred frequently around the world, which has had a significant impact on rice ( Oryza Sativa L.) poses a serious threat to rice production safety. Rice is sensitive to high temperatures throughout its entire growth period. High temperatures during the germination period will reduce seed vitality, preventing seeds from germinating normally, resulting in a lower seedling rate; high temperatures during the seedling stage will cause poor seedling growth or even dryness and death; high temperatures during the flowering period will reduce crop pollen vitality, resulting in a lower fruit set rate, seriously affecting yields; and high temperatures during the filling period will accelerate seed dry matter accumulation and shorten the filling time, thereby affecting the thousand-grain weight and grain quality, ultimately leading to reduced yields and a decline in rice quality. Therefore, exploring high-yield and high-resistance rice varieties is crucial for rice breeding.

[0003] Under high temperature stress, plants produce a large amount of reactive oxygen species (ROS), and chloroplasts and peroxisomes are the main ROS producers. Excessive ROS can cause oxidative damage to plants, resulting in excessive accumulation of harmful substances, which in turn affects cell homeostasis and inhibits plant growth. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) play a vital role in maintaining the balance between ROS production and scavenging in plants and protecting plants from oxidative damage. It is particularly noteworthy that APX is a class I heme-containing peroxidase in higher plants, which has ROS scavenging activity and the strongest affinity for ROS. APX uses ascorbic acid (AsA) as an electron donor to catalyze the conversion of H2O2 to H2O and O2. High temperature stress significantly induced the expression of APXs in almost all developmental stages of Arabidopsis, and the H2O2 content was significantly increased in the corresponding deletion mutants.

[0004] It is reported that plant stress-associated proteins (SAPs) are a class of zinc finger proteins with A20 / AN1 zinc finger domains, also known as ZFPs (Zinc-finger proteins) family proteins, which play an important role in plant growth and development and stress response. There are 18 SAP proteins in rice, and OsSAP1 is the first reported zinc finger protein with A20 / AN1 domain in plants. It is overexpressed in tobacco. OsSAP1 , the plants are more resistant to cold, salt and drought. OsSAP8 Overexpression of tobacco and rice seeds enhanced tolerance to salt, drought, and cold stress. OsSAP9 ( ZFP177 ) respond to both cold and high temperature stress. OsSAP9Transgenic plants are more sensitive to salt and drought stress. OsSAP4 ( ZFP185 ) can affect the biosynthesis of gibberellins and abscisic acid and increase the sensitivity of rice to abiotic stress. OsSAP7 ( ZFP173 ) overexpression in rice plants showed reduced drought tolerance. Both OsSAP1 and OsSAP11 can interact with the receptor-like protein kinase OsRLCK253 to enhance the plant's tolerance response to abiotic stress. OsSAP16 The plant's stomatal conductance decreased, photosynthesis increased, and resistance to drought stress increased. OsSAP16 The germination rate of the mutant decreased at low temperature, and the overexpression OsSAP16 The low temperature germination of rice seeds was enhanced. OsSAP5 was reported to be involved in the high temperature response of rice during the grain filling period and positively regulates the high temperature tolerance of rice. However, OsSAP3, as a member of the rice SAP family, has not been reported in the regulation of rice high temperature stress response. Summary of the invention

[0005] In view of the problem that the prior art is unclear about the ability of OsSAP3 to regulate the response of rice to high temperature stress, the present invention provides OsSAP3 The application of genes in regulating high temperature resistance of rice seedlings, the specific technical scheme is as follows:

[0006] In a first aspect, the present invention provides OsSAP3 Application of genes in regulating high temperature resistance of rice seedlings, the OsSAP3 The CDS sequence of the gene is shown in SEQ ID NO.1. OsSAP3 The full-length sequence of the gene is shown in SEQ ID NO.2.

[0007] In a second aspect, the present invention provides an application of OsSAP3 protein in regulating high temperature resistance of rice at seedling stage, wherein the amino acid sequence of the OsSAP3 protein is shown in SEQ ID NO.3.

[0008] In a third aspect, the present invention provides an application of a recombinant vector in regulating high temperature resistance of rice seedlings, wherein the recombinant vector comprises OsSAP3 Gene, the OsSAP3 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0009] In a fourth aspect, the present invention provides an application of a genetically engineered bacterium in regulating high temperature resistance of rice seedlings, wherein the genetically engineered bacterium comprises OsSAP3 Gene, the OsSAP3 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0010] Furthermore, the application method is:

[0011] By silencing, knocking down or knocking out OsSAP3 gene, thereby improving the resistance of rice to high temperature stress at the seedling stage.

[0012] Furthermore, the knockout OsSAP3 The genetic method is:

[0013] (1) Based on OsSAP3 Genome sequence, design target sequence sgRNA, and construct using CRISPR / Cas9 technology OsSAP3 Knockout mutant vector;

[0014] (2) The CRISPR / Cas9 vector is transferred into Agrobacterium competent cells to construct a knockout vector OsSAP3 Agrobacterium strains with gene CRISPR / Cas9;

[0015] (3) Transforming the rice callus with the Agrobacterium strain to obtain a knockout OsSAP3 Gene homozygous strains.

[0016] Furthermore, the nucleotide sequence of the sgRNA is shown as SEQ ID NO.6 or SEQ ID NO.8.

[0017] In a fifth aspect, the present invention provides OsSAP3 The application of genes in screening and creating high temperature resistant rice germplasm resources, OsSAP3 The CDS sequence of the gene is shown in SEQ ID NO.1. OsSAP3 The full-length sequence of the gene is shown in SEQ ID NO.2.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention knocks out the OsSAP3 Gene, found OsSAP3 The survival rate of gene knockout mutants was significantly increased after high temperature treatment at the rice seedling stage, confirming its use in regulating high temperature resistance of rice seedlings and providing an important genetic resource for breeding high temperature resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 for OsSAP3 The genotype identification results of mutant plants; OsSAP3 represents the wild-type genotype, Ossap3-2 , Ossap3-3 , Ossap3-5 Indicates different OsSAP3 Knockout mutants.

[0021] Figure 2 forOsSAP3 Schematic diagram of the results of physiological indicators of mutant plants after strong high temperature treatment at 45℃; control is the control group, 45℃-48h represents the experimental group treated at 45℃ for 48h, 45℃-54h represents the experimental group treated at 45℃ for 54h, A represents the wild-type plants and Ossap3-2, Ossap3-3, Ossap3-5 Phenotypes of the three mutants after high temperature treatment, B is the wild-type plant and Ossap3-2, Ossap3-3, Ossap3-5 Survival statistics of three mutants after high temperature treatment, C is the wild type plant and Ossap3-2, Ossap3-3, Ossap3-5 Schematic diagram of DAB and NBT staining of three mutants after high temperature treatment, scale bar = 5 cm, values ​​are mean ± SD (n = 3), statistical analysis was performed using one-way ANOVA, *P< 0.05；**P<0.01 . DETAILED DESCRIPTION

[0022] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0023] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0024] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0025] SEQ ID NO.1:

[0026]

[0027] SEQ ID NO.2:

[0028]

[0029] SEQ ID NO.3:

[0030] MGQQVQHESRINVGEATHVSKAEMGANTMFATSRLNSNNNKVGPELAYLSGVASSASDSSTAAPSPCYLCHKPAALHVFGLAGRYVFGSVKREAYLSQEGPRSGRTPNRIAESLPVRVVNDFGLRLRVVTNQGPIKPRPPRPIDAIVFASIETRNRLRGFDRSFCCSAPPETYVFLPRA RETIVLRANIIKMSSEQQASAGQPVLCASGCGFYGNPATLDMCSVCYRQHCLLNGATMATGPSSSVAAASAATVATGAVTSDSCSVPSAEVNGAAFSSKNNPEPATVVEKKAPANRCASCKKKVGLLGFACRCGATYCGTHRYPEKHACGFDFKGASRDAIARANPLIKGEKLTNKI*.

[0031] Example 1 Rice OsSAP3 Gene cloning, construction and transformation of gene knockout vectors and gene overexpression vectors

[0032] 1. OsSAP3 CRISPR / Cas9 knockout vector construction and Agrobacterium transformation

[0033] Based on the genome (SEQ ID NO.2) sequence, we searched for specific target sequences online (http: / / skl.scau.edu.cn / ), and selected appropriate knockout target sequences based on the target sites with high score values, low off-target rates, and reasonable GC%. OsSAP3 Two pre-mutation target sites, Target1 and Target2, were designed in the open reading frame, and sgRNA (SEQ ID NO.6 and SEQ ID NO.8) was designed based on the pre-mutation target sites.

[0034] Target1: CACGAGAGTCGCATCAATGT AGG (SEQ ID NO.4);

[0035] Target2: GTCAGGCACCCGAGGGAGTC CGG (SEQ ID NO.5);

[0036] OsSAP3-gRT1: CACGAGAGTCGCATCAATGTgttttagagctagaaat (SEQ ID NO. 6);

[0037] OsSAP3- OsU3T1: ACATTGATGCGACTCTCGTGTgccacggatcatctgc (SEQ ID NO.7);

[0038] OsSAP3- gRT2: TCAGGCACCCGAGGGAGTCgttttagagctagaaat (SEQ ID NO. 8);

[0039] OsSAP3- OsU6aT2: GACTCCCTCGGGTGCCTGACggcagccaagccagca (SEQ ID NO. 9).

[0040] OsU3 and OsU6a promoters are the promoters of the sgRNA expression cassette. The pYLgRNA-OsU3 and pYLgRNA-OsU6a plasmids are used as templates to amplify the sgRNA expression cassette. After PCR amplification, gel electrophoresis is performed to detect bands. The first half of the promoter and the second half of the scaffold gel of OsU3 with expected band sizes are placed in the same centrifuge tube (OsU3+sgRNA), and the first half of the promoter and the second half of the scaffold gel of OsU6a are placed in the same centrifuge tube (OsU6a+sgRNA). The gels are crushed and 100 µL ddH2O is added. They are stored at 4°C as template materials for the next step of sgRNA complete expression cassette amplification. sgRNA complete expression cassette amplification The above gel products (OsU3+sgRNA and OsU6a+sgRNA) are used as templates for PCR amplification. The above two-step PCR reaction can connect the promoter and sgRNA to form a complete expression cassette. After the PCR program is completed, a gel electrophoresis test is performed to observe whether the amplified bands are in line with expectations. The target band of U3-gRNA is 564 bp, and the target band of U6a-gRNA is 629 bp. If the product is correct, gel recovery is performed and stored at -20°C.

[0041] The CRISPR / Cas9 vector (pYLCRISPR / Cas9 Pubi-H, pCAMBIA-1300) was digested with restriction endonuclease BsaI (#R0535V) from NEB Biotechnology, and the sgRNA expression cassette gel recovery product and the digested vector were recombined with T4 ligase (#M0202V) from NEB. The recombinant vector was transformed into Escherichia coli DH5α, and positive clones were picked for colony PCR and gel electrophoresis tests. Positive bands were selected for sequencing. After successful sequencing, Agrobacterium was transformed. The transformation of Agrobacterium was performed by freeze-thaw method according to the instructions of EHA105 competent cells from Weidi Company. Single colonies were picked for PCR identification, and Agrobacterium with correct bands was used for genetic transformation.

[0042] Example 2 Rice OsSAP3 Obtaining homozygous gene knockout plants

[0043] 1. Agrobacterium-mediated genetic transformation of rice

[0044] Use containing OsSAP3 Agrobacterium carrying the CRISPR / Cas9 knockout vector infected rice callus and after screening with differentiation medium and rooting medium, the corresponding genetically transformed plants were obtained.

[0045] 2. Obtaining homozygous gene-edited plants

[0046] The young leaves of T0 transgenic rice were selected when they grew to 3-4 leaves, and genomic DNA was extracted by CTAB method. OsSAP3 Mutant plants were subjected to T-DNA identification test. Primers were designed based on the 610 bp sequence in the CRISPR / Cas9 vector (forward primer: 5'-GCTTCATGGCCCACTACGAAATGCTT-3'; reverse primer: 5'- CGCATCTATGGGCCTGGACGGAATAG -3'). PCR reaction was performed using the genomic DNA of the T0 generation OsSAP3 mutant as a template.

[0047] The T-DNA identification results in the previous step are consistent with expectations. OsSAP3 The mutant plants were sequenced for mutation analysis. Primers were designed 100 bp before the first target site and 100 bp after the second target site (forward primer: 5'-TCCTCGCATCGTTGTGCCAG-3'; reverse primer: 5'- CGAAGCCTCGAAGGCGATTG-3') to amplify the transgenic rice OsSAP3 Gene, through sequence comparison, the results are as follows Figure 1 As shown, it can be seen that OsSAP3 The gene has been deleted, i.e. OsSAP3Transgenic lines with loss of gene function are named ossap3-2 , ossap3-3 and ossap3-5 .

[0048] Example 3 OsSAP3 High temperature treatment and determination of physiological and biochemical indices of gene knockout rice

[0049] 1. High temperature treatment of rice plants and statistics of phenotype and survival rate after treatment

[0050] The different rice background materials obtained in Example 2 OsSAP3 Knockout clones ossap3-2 , ossap3-3 and ossap3-5 , and the control ordinary rice material was treated with high temperature: the seeds were disinfected with 2% NaClO for 20 min and then rinsed with clean water, soaked at 37℃ for 3 days until they turned white, and the seeds with the same whiteness were sown in a 96-well hydroponic box, and hydroponically cultured using the Yoshida rice nutrient solution formula (Table 1), and the hydroponic solution was changed every 4 days. The culture conditions are as follows: constant temperature of 25℃, 12 h light / 12 h dark, environmental relative humidity of 75%, and light intensity of 22000 lux. When the rice seedlings grew to the four-leaf stage, they were treated with high temperature in a light incubator: constant temperature of 45℃, 12 h light / 12 h dark, relative humidity of 75%, the temperature of the control group was constant at 25℃, and the light and humidity conditions were consistent with the treatment group. After 48 h and 54 h of high temperature treatment, the growth was restored for 7 days, and the survival rate was counted, with 3 biological replicates.

[0051] Table 1 Yoshida rice nutrient solution formula

[0052]

[0053] Note: Add 5 mL of each of the above I-VI stock solutions to every 4000 mL of culture medium and adjust the pH to 5.5-6.0

[0054] The results are as follows Figure 2 As shown in A, when the four-leaf rice was treated with high temperature at 45℃ for 48 h, Ossap3 The leaves of the plants partially curled and dried up, while the leaves of the wild-type plants completely curled and dried up. The treated plants were transferred to normal temperature 25℃ to resume growth for 7 days. ossap3-2 , ossap3-3 and ossap3-5 The survival rates were 54.9%, 68.4% and 83.1%, respectively, which were significantly higher than the 26% of the wild type. ossap3-2, ossap3-3 and ossap3-5 The survival rate was still significantly higher than that of the wild type.

[0055] 2. DAB and NBT staining

[0056] DAB staining: Wash the leaves from the same part after high temperature treatment with distilled water and place them in a cell culture plate or 35 mm culture dish, add 2 mL of 1 mg / mL DAB reaction solution for H2O2 staining. Place at 28℃ away from light for 4-6 hours. When obvious brown spots are observed in the tissue, discard the staining solution, add 80% ethanol for decolorization, and incubate in a 60℃ oven overnight. During this period, replace 80% ethanol until the tissue completely loses its green color and then take pictures.

[0057] NBT staining: Cut the rice leaves after high temperature treatment into 1 cm lengths, place them in a small culture dish, add 2 mL of 6 mM NBT dye solution, vacuum impregnate the samples for 10 min at a pressure of 60 kPa, and incubate them under light at room temperature for 2 h. If there is no vacuum infiltration device, immerse and incubate the samples under light at room temperature for 8 h. Add the leaves to anhydrous ethanol and dechlorophyllize them to white in a 100 ° C water bath for about 2 h. After cooling, soak them in 20% glycerol. Place the leaves on a slide and take pictures using a microscope.

[0058] The results are as follows Figure 2 As shown in C, it can be seen that OsSAP3 The degree of leaf staining in the knockout mutant plants was significantly lower than that in the wild type, indicating that OsSAP3 The knockout mutant of the gene is more resistant to high temperature stress.

Claims

1. Knockout OsSAP3 The application of the gene in improving the high temperature resistance of rice at the seedling stage is characterized in that: Said OsSAP3 The CDS sequence of the gene is shown in SEQ ID NO.

1. OsSAP3 The full-length sequence of the gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The knockout OsSAP3 The genetic method is: (1) Based on OsSAP3 Genome sequence, design target sequence sgRNA, and construct using CRISPR / Cas9 technology OsSAP3 Knockout mutant vector; (2) Transform the CRISPR / Cas9 vector into Agrobacterium competent cells to construct knockout OsSAP3 Agrobacterium strains with gene CRISPR / Cas9; (3) The Agrobacterium strain is used to mediate the transformation of rice callus tissue to obtain knockout OsSAP3 Gene homozygous strains.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the sgRNA is shown as SEQ ID NO.6, or as shown by SEQ ID NO.6 and SEQ ID NO.

8.

4. Knockout OsSAP3 The application of the gene in screening and creating high temperature resistant rice germplasm resources is characterized in that: Said OsSAP3 The CDS sequence of the gene is shown in SEQ ID NO.

1. OsSAP3 The full-length sequence of the gene is shown in SEQ ID NO.2.

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