Application of OsCATA gene in delaying rice heading date

Abstract

The application discloses application of an OsCATA gene in controlling rice heading stage and belongs to the technical field of genetic engineering. The application finds for the first time that the OsCATA gene (cDNA sequence as shown in SEQ ID NO:1) derived from rice Nipponbare is involved in the regulation of the rice heading stage, and knocking out the gene can significantly delay the heading of rice under short-day and long-day conditions; under long-day, the OsCATA gene regulates the heading stage in a manner dependent on OsELF3-1, down-regulates Ghd7 to inhibit the expression of Ehd1, Hd3a and RFT1, and finally delays the heading. The application also provides a knocking-out vector and method of the OsCATA gene and obtained mutant plants, the oscata single mutant delays the heading by 12.4 days under the long-day in Hangzhou and by 7.2 days under the short-day in Hainan, and the oself3-1 oscata double mutant has a more significant delay effect. The application provides a new gene target for the regulation of the rice heading stage, perfects the photoperiodic flowering regulation network of rice, and the provided method and mutant can be used for the directional breeding of new rice varieties suitable for different photoperiod regions, and has important breeding application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically the application of the OsCATA gene in delaying the heading stage of rice. Background Technology

[0002] Rice is a vital global food crop, providing staple food for more than half the population. As a facultative short-day crop, the heading date is a core agronomical trait determining the wide applicability and environmental adaptability of rice, directly impacting yield, quality, and the choice of cultivation season. Analyzing the molecular regulatory network of rice heading date and using genetic engineering to directionally improve this trait is of great significance for breeding new rice varieties adapted to different photoperiods and ensuring food security.

[0003] Existing research indicates that the molecular network regulating flowering in rice through photoperiod mainly comprises two core signaling pathways: First, the OsGI-Hd1-Hd3a pathway, conserved with the Arabidopsis GI-CO-FT pathway, where Hd1 has a dual regulatory function: under short-day conditions, it promotes the expression of the florigen gene Hd3a to accelerate flowering, while under long-day conditions, it interacts with Ghd7 and other genes to inhibit heading. Second, the Ehd1-centric regulatory pathway, which integrates multiple upstream signals to directly promote the expression of downstream Hd3a and RFT1, driving flowering in rice under both long and short-day conditions. Ghd7, a key flowering inhibitor under long-day conditions, can bind to the Ehd1 promoter region to suppress its expression. Furthermore, Ghd7's expression is regulated by genes such as OsELF3-1 and Ehd3; under long-day conditions, OsELF3-1 can form the EC complex to promote Ghd7 expression, thereby delaying flowering. Ultimately, the flowering proteins Hd3a and RFT1 are transported to the shoot apical meristem to form the FAC complex, which drives the rice to transition from vegetative growth to reproductive growth.

[0004] Gene editing technology provides an efficient means for targeted improvement of the heading stage trait in rice. By targeting and knocking out or editing genes related to heading stage, superior alleles can be created, enabling precise regulation of rice growth stages. Existing technologies have demonstrated the feasibility of gene editing in improving rice heading stage by knocking out genes such as Hd2, Hd4, Hd5, Phyb, and Ehd1 using CRISPR / Cas9 technology. However, research on the role of catalase-encoded genes in regulating rice heading stage has not yet been reported. Discovering new genes regulating heading stage and clarifying their mechanisms of action can provide new targets and strategies for molecular design breeding of rice varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a new application of the OsCATA gene in controlling the heading period of rice, to clarify that knocking out this gene can delay rice heading under both long and short day conditions, and to provide corresponding gene editing methods, recombinant vectors and transgenic rice plants, providing new gene targets and technical solutions for the targeted improvement of rice heading period.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: the application of the OsCATA gene in delaying the heading period of rice, characterized in that knocking out the OsCATA gene can inhibit rice heading under both short-day and long-day conditions, thereby delaying the heading period of rice; the OsCATA gene is derived from the rice variety Nipponbare, and its cDNA sequence is shown in SEQ ID NO:1.

[0007] Furthermore, the coding region sequence of the OsCATA gene is shown in SEQ ID NO:2, which encodes a catalase containing 493 amino acids, participates in the scavenging of reactive oxygen species in rice, and its expression is rhythmic.

[0008] Furthermore, under long-day conditions, OsCATA relies on OsELF3-1 to regulate the heading date of rice. OsCATA relieves the inhibition of Ehd1 by downregulating the expression of Ghd7, thereby promoting the expression of Hd3a and RFT1 mediated by Ehd1. After knocking out the OsCATA gene, the expression of Ghd7 is upregulated, inhibiting the expression of Ehd1, Hd3a and RFT1, ultimately delaying the heading of rice.

[0009] Further, the target sequence of the OsCATA gene was constructed into the pC1300-Cas9 knockout vector to obtain the recombinant vector; the target sequence of the OsCATA gene is shown in SEQ ID NO:3.

[0010] A method for obtaining delayed-heading rice plants includes the following steps: S1. Transform Agrobacterium EHA105 with the recombinant vector described in claim 4 to obtain an engineered strain of Agrobacterium. S2. Using Agrobacterium-mediated genetic transformation, the Agrobacterium engineered strain was transformed into rice callus tissue. After co-culture, screening, differentiation, rooting, hardening, and transplanting, positive transgenic rice plants with OsCATA gene knockout were obtained. The rice variety in question is Nipponbare.

[0011] Furthermore, in step S2, hygromycin was used to screen resistant callus tissues, and positive transgenic rice plants were identified by PCR sequencing.

[0012] Furthermore, the OsCATA gene knockout positive transgenic rice plants obtained headed 12.4 days later than the wild-type Nipponbare under natural long-day conditions in Hangzhou and 7.2 days later under natural short-day conditions in Hainan.

[0013] A method for constructing a rice double mutant involves knocking out the OsCATA gene and the OsELF3-1 gene to obtain the oself3-1oscata double mutant; the target sequence of the OsCATA gene is shown in SEQ ID NO:4, and the target sequence of the OsELF3-1 gene is shown in SEQ ID NO:5.

[0014] Furthermore, a fragment containing the OsCATA and OsELF3-1 dual gRNA expression cassettes was constructed into the pC1300-Cas9 knockout vector to obtain a double knockout recombinant vector. This vector was then transformed into Nipponbare rice using Agrobacterium EHA105-mediated transformation, and double mutants were obtained through screening. Under natural long-day conditions in Hangzhou, the double mutants headed 24.0 days later than the wild-type Nipponbare. Beneficial effects

[0015] This invention is the first to discover and confirm that the OsCATA gene is involved in the regulation of rice heading stage, clarifying the role and molecular mechanism of this gene in the rice photoperiodic flowering regulatory network, providing a new gene target for the regulation of rice heading stage, and filling the gap in the research on the involvement of catalase-encoded genes in the regulation of rice heading stage.

[0016] The method for delaying rice heading by knocking out the OsCATA gene provided by this invention utilizes CRISPR-Cas9 gene editing technology to achieve precise regulation of the rice heading period. This method is simple to operate and highly efficient. The knockout mutants can stably exhibit the delayed heading phenotype under both long and short day conditions. Furthermore, double knockout of OsCATA and OsELF3-1 can further delay the heading period. This method can create rice germplasm resources with different heading periods according to the photoperiod requirements of different planting areas.

[0017] This invention clarifies that OsCATA regulates rice heading time through the molecular pathway of OsELF3-1-Ghd7-Ehd1-Hd3a / RFT1, improves the molecular network of photoperiod regulation of flowering in rice, provides new theoretical basis for in-depth analysis of the regulatory mechanism of rice heading time, and provides a new technical approach for molecular design breeding of rice varieties. The selected delayed-heading rice varieties can adapt to the planting needs of high-latitude long-day regions, expand the planting area of ​​rice, and improve the yield potential and environmental adaptability of rice. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 The images show the phenotypes of OsCATA and OsELF3-1 gene knockout positive plants and the ofself3-1oscata double mutant of this invention. A, B, and C represent the phenotypes of the oscata single mutant, ofself3-1 single mutant, and ofself3-1oscata double mutant plants under natural long-day conditions. D, E, and F represent the statistical results of the heading date of the corresponding mutants. G represents the mutant sequence of the homozygous oscata family (red dashed lines indicate the deletion of 4 bases). H shows the OsELF3-1 knockout site in the ofself3-1oscata double mutant and the comparison with sequencing results. I shows the OsCATA knockout site in the ofself3-1oscata double mutant and the comparison with sequencing results (red dashed lines indicate the deletion of 4 bases).

[0020] Figure 2 This is a subcellular localization map of the OsCATA protein of the present invention; wherein, A is the subcellular localization of OsCATA in rice protoplasts (mCherry as a marker); B is the subcellular localization of OsCATA in tobacco (mCherry or mCherry-NLS as a nuclear marker), confirming that OsCATA is localized in the nucleus and cytoplasm.

[0021] Figure 3 This diagram illustrates the rhythmic expression patterns of OsCATA and OsELF3-1 in this invention. A and C represent the rhythmic expression patterns of OsCATA under short-day and long-day conditions in a clear Japanese background; B and D represent the rhythmic expression patterns of OsELF3-1 under short-day and long-day conditions in a clear Japanese background; E and G represent the expression patterns of OsCATA under short-day and long-day conditions in a clear Japanese background and the oself3-1 mutant; F and H represent the expression patterns of OsELF3-1 under short-day and long-day conditions in a clear Japanese background and the oself3-1 mutant, confirming that both expressions are rhythmic and that OsCATA expression is positively regulated by OsELF3-1.

[0022] Figure 4 This diagram shows the protein-protein interaction verification of OsELF3-1 and OsCATA in this invention; where A is the yeast double hybridization experiment to verify the interaction between the two; B, C, and D are the LAC, BiFC, and Co-IP experiments, respectively, to further verify the protein-protein interaction relationship between the two.

[0023] Figure 5 This is a graph showing the expression levels of genes related to the heading stage in different mutants according to the present invention. Among them, AD represents the expression levels of Hd3a, RFT1, Ehd1, and Ghd7 in wild-type Nip and oscata mutants; EH represents the expression levels of the above genes in wild-type Nip and oself3-1 mutants; IL represents the expression levels of the above genes in wild-type Nip and oself3-1oscata double mutants (samples were taken from the field in Fuyang, Hangzhou). This confirms that knocking out OsCATA upregulates Ghd7 and inhibits the expression of Ehd1 and florigen genes.

[0024] Figure 6 This diagram shows the expression levels of OsCATA and OsELF3-1 in the ghd7 mutant. A and B represent the expression levels of OsCATA in wild-type Nip and ghd7 mutants under short-day and long-day conditions, respectively. C and D represent the expression levels of OsELF3-1 in wild-type Nip and ghd7 mutants under short-day and long-day conditions, confirming that OsCATA and OsELF3-1 are located upstream of Ghd7.

[0025] Figure labeling: Nip-Nipponbare wild-type rice; oscata-OsCATA gene knockout single mutant; oself3-1-OsELF3-1 gene knockout single mutant; oself3-1oscata-OsCATA and OsELF3-1 double knockout mutant. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0027] Unless otherwise specified, all experimental materials, reagents and instruments used in this invention are conventional commercially available products; and all experimental methods used are conventional methods in the field of molecular biology.

[0028] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0029] Unless otherwise specified, all experimental materials, reagents and instruments used in this invention are conventional commercially available products; and all experimental methods used are conventional methods in the field of molecular biology.

[0030] Experimental materials: Rice variety Nipponbare (Oryzasativa L. ssp. Japonicacv. Nipponbare); Agrobacterium strain EHA105 (purchased from CAMBIA); CRISPR-Cas9 knockout vector pC1300-Cas9, intermediate vector SK-gRNA; subcellular localization vectors pYBA1132 and pYBA1152; yeast double hybrid vectors pGBKT7 (BD) and pGADT7 (AD); LAC experimental vectors Nluc and cLuc; BiFC experimental vectors HYNE and HYCE; Co-IP experimental vectors GFP and MYC.

[0031] Experimental reagents: Restriction endonucleases such as AarI, KpnI, BglII, BamHI, EcoRI, and SalI; T4 DNA ligase and homologous recombinase; PCR amplification kits and RT-qPCR kits; hygromycin; GFP antibody and MYC antibody; Agrobacterium infection solution, rice callus induction medium, differentiation medium, and rooting medium.

[0032] Experimental apparatus: PCR instrument, real-time PCR instrument, laser confocal microscope, gel imaging system, constant temperature incubator, artificial climate chamber, shaker, etc. Example 1:

[0033] Construction of the OsCATA gene knockout vector: Target sequence design and primer synthesis: Based on the CRISPR / Cas9 target sequence design database (http: / / cbi.hzau.edu.cn / CRISPR2 / ), a specific target sequence for the OsCATA gene was designed, as shown in SEQ ID NO:3. GGCA and AAAC were added before the forward and reverse complementary sequences of the target sequence, respectively, to form primers aF and aR. Simultaneously, amplification primers adF and adR were designed on both sides of the target site for subsequent genotype detection of transgenic positive plants. The primer sequences are shown in the sequence listing.

[0034] Construction of the gRNA expression cassette: Primers aF and aR were annealed at 99°C for 5 min in a PCR instrument to form double strands; the intermediate vector SK-gRNA was digested with AarI enzyme at 37°C, and the digestion product was purified by agarose gel electrophoresis; the annealed double-stranded DNA and the purified SK-gRNA digestion product were recombinantly ligated at room temperature, the ligation product was transformed into E. coli DH5α, single clones were picked, and PCR identification was performed using T7 primers. Sequencing verified that the gRNA expression cassette was constructed correctly.

[0035] Construction of the recombinant knockout vector: The correctly sequenced SK-gRNA-OsCATA recombinant plasmid was digested with restriction endonucleases KpnI and BglII, and the fragment containing the gRNA expression cassette was recovered; the pC1300-Cas9 vector was digested with KpnI and BamHI, linearized, and then recovered; the two recovered fragments were ligated, the ligation product was transformed into E. coli DH5α, single clones were picked, and PCR identification was performed using pC1300-F primers. Sequencing verification yielded the CRISPR-Cas9 knockout recombinant vector pC1300-Cas9-OsCATA for the OsCATA gene. Example 2:

[0036] Agrobacterium-mediated genetic transformation of rice and screening of positive plants: Agrobacterium transformation: The recombinant vector pC1300-Cas9-OsCATA constructed in Example 1 was transformed into Agrobacterium EHA105 competent cells by freeze-thaw method. Single clones were picked, and positive Agrobacterium engineered strains were identified by PCR. The cells were cultured in a shaker until OD600 = 0.6~0.8 to prepare Agrobacterium infection solution.

[0037] Rice callus transformation: Mature rice seeds of Nipponbare were dehulled, surface-sterilized, and inoculated into callus induction medium. They were cultured in the dark at 28°C for 2 weeks. Pale yellow, loose callus tissue was selected as transformation recipients. The callus tissue was immersed in Agrobacterium infection solution for 30 min. After the bacterial solution was dried, it was inoculated into co-culture medium and cultured in the dark at 25°C for 3 days. The co-cultured callus tissue was transferred to selection medium containing hygromycin and cultured in the dark at 28°C for 3 rounds (2 weeks per round) to screen for resistant callus tissue.

[0038] Differentiation and transplanting: The resistant callus was inoculated into the differentiation medium and cultured at 28°C under light (16h light / 8h dark). After the green seedlings differentiated, they were transferred to the rooting medium and cultured for 2 weeks. Transgenic seedlings with well-developed root systems were selected and transplanted to the field after 3 days of hardening off to obtain T0 generation transgenic rice plants.

[0039] Identification of positive plants: Genomic DNA was extracted from T0 generation transgenic plants and amplified by PCR using primers adF and adR. The amplified products were verified by sequencing, and positive transgenic plants with OsCATA gene knockout (oscata single mutant) were obtained by screening. Example 3:

[0040] Phenotypic identification of heading stage in OsCATA gene knockout plants: OsCATA gene knockout positive plants (oscata single mutant) and wild-type Nipponbare were planted in natural long-day conditions in Hangzhou (around 30°N latitude, summer daylight hours >14h) and natural short-day conditions in Hainan (around 18°N latitude, winter daylight hours <12h), respectively. At the same time, artificial long-day (14h light / 10h darkness) and artificial short-day (10h light / 14h darkness) conditions were set in artificial climate chambers for planting. Each condition was set up with 3 replicates, and 20 plants were planted in each replicate.

[0041] When the plants began to flower, the flowering period of each plant was recorded, and the results showed: Under natural long-day conditions in Hangzhou, the oscata single mutant headed 12.4 days later than the wild type; Under natural short-day conditions in Hainan, the oscata single mutant headed 7.2 days later than the wild type; Under artificial long-day conditions, the oscata single mutant was delayed in heading by 5.5 days compared to the wild type; Under artificial short-day conditions, the oscata single mutant was delayed in heading by 4.7 days compared to the wild type.

[0042] It was confirmed that knocking out the OsCATA gene significantly delayed the heading period of rice under both long and short day conditions.

[0043] Example 4: Construction of OsCATA and OsELF3-1 double knockout vectors and acquisition of double mutants Dual-target sequence design and primer synthesis: Target sequences (SEQ ID NO:4) of the OsCATA gene and (SEQ ID NO:5) of the OsELF3-1 gene were designed. GGCA and AAAC were added before the forward and reverse complementary sequences of the two target sequences, respectively, to form primers a-1-F / a-1-R and 3-1-F / 3-1-R. Simultaneously, amplification primers a-1-dF / a-1-dR and 3-1-dF / 3-1-dR were designed on both sides of the two target sites for genotype detection of the double mutants. Primer sequences are shown in the sequence listing.

[0044] Construction of dual gRNA expression cassettes: Following the method in Example 1, SK-gRNA recombinant plasmids containing OsCATA and OsELF3-1 gRNA expression cassettes were constructed respectively; SK-gRNA-OsCATA was digested with KpnI and BglII, the gRNA expression cassette fragments were recovered, and ligated with SK-gRNA-OsELF3-1 digested with KpnI and BamHI to obtain the recombinant plasmid SK-gRNA-OsCATA-OsELF3-1 containing dual gRNA expression cassettes.

[0045] Construction of the double knockout recombinant vector: SK-gRNA-OsCATA-OsELF3-1 was double-digested with KpnI and BglII, the double gRNA expression cassette fragment was recovered, and ligated with the pC1300-Cas9 vector digested with KpnI and BamHI. Sequencing verified the acquisition of the double gene knockout recombinant vector pC1300-Cas9-OsCATA-OsELF3-1.

[0046] Obtaining and identifying the double mutant: Following the Agrobacterium-mediated genetic transformation method in Example 2, the double knockout recombinant vector was transformed into rice Nipponbare callus tissue. After screening, differentiation, and transplanting, genomic DNA of the transgenic plants was extracted, and PCR sequencing was performed using the corresponding amplification primers to identify the oself3-1oscata double mutant.

[0047] Heading date phenotype of double mutants: The double mutants were planted under natural long-day conditions in Hangzhou, and the heading date was statistically analyzed. The results showed that the double mutants headed 24.0 days later than the wild-type Nipponbare, and the degree of delay was significantly higher than that of the oscata single mutant and the oself3-1 single mutant. Moreover, the heading date phenotype of the double mutants was similar to that of the oself3-1 single mutant, confirming that OsCATA depends on OsELF3-1 to regulate the heading date of rice. Example 5:

[0048] Subcellular localization of OsCATA protein: Subcellular localization vector construction: Using the full-length cDNA of the OsCATA gene (SEQ ID NO: 1) as a template, PCR amplification was performed using primers OsCATA-GFP-F / OsCATA-GFP-R and GFP-OsCATA-F / GFP-OsCATA-R to obtain the complete coding region fragment of the OsCATA gene. The amplified fragments were then ligated into EcoRI-digested pYBA1132 and pYBA1152 vectors, respectively, through homologous recombination to construct OsCATA-GFP (N-terminal fusion) and GFP-OsCATA (C-terminal fusion) recombinant expression vectors. Sequencing verified the correct vector construction.

[0049] Transient expression in rice protoplasts: The constructed recombinant expression vector was transformed into rice protoplasts via PEG-mediated transformation and cultured in the dark at 28°C for 20-24 h. The GFP fluorescence signal was observed using a laser confocal microscope, with mCherry as the cell marker.

[0050] Transient expression in tobacco: The recombinant expression vector was transformed into Agrobacterium GV3101, mixed with Agrobacterium containing mCherry (cytoplasmic marker) or mCherry-NLS (nuclear marker), injected into tobacco leaves, and cultured at 25°C under light for 2 days. The GFP fluorescence signal was observed using a laser confocal microscope.

[0051] The results showed that the fluorescence signals of the OsCATA-GFP and GFP-OsCATA fusion proteins appeared simultaneously in the cell nucleus and cytoplasm, confirming that the OsCATA protein is located in the cell nucleus and cytoplasm of rice cells. Example 6:

[0052] Rhythmic expression analysis of the OsCATA gene: Material cultivation and sampling: The wild-type Nipponbare rice and the oself3-1 mutant were planted in artificial climate chambers and cultured for 50 days under long-day conditions (14h light / 10h darkness) and 40 days under short-day conditions (10h light / 14h darkness). Sampling was carried out every 4 hours from the start of light exposure for 2 consecutive days. Rice leaves were collected each time, flash-frozen in liquid nitrogen, and stored at -80℃.

[0053] RNA extraction and RT-qPCR detection: Total RNA was extracted from samples at each time point and reverse transcribed into cDNA; using the UBQ gene as an internal control, quantitative real-time PCR was performed using RT-OsCATA-F / RT-OsCATA-R and RT-OsELF3.1-F / RT-OsELF3.1-R primers to detect the expression levels of OsCATA and OsELF3-1 genes. Primer sequences are shown in the sequence listing.

[0054] Rhythmic expression analysis: Gene expression rhythm curves were plotted based on RT-qPCR results. The results showed that: In wild-type Japanese Harmony, the expression of the OsCATA gene exhibits a distinct rhythmicity, peaking at the light-dark transition time under short-day conditions and after dawn under long-day conditions. The rhythmic expression patterns of OsCATA and OsELF3-1 are highly consistent; Regardless of whether the day length was long or short, the expression level of OsCATA in the oself3-1 mutant was significantly lower than that in the wild type, confirming that the expression of OsCATA is regulated by the biological clock, and that OsELF3-1 is located upstream of OsCATA and positively regulates its expression. Example 7:

[0055] Verification of the protein interaction between OsCATA and OsELF3-1: Yeast hybridization experiment: Construction of BD-OsCATA recombinant vector: The OsCATA gene coding region fragment was amplified using BD-OsCATA-F / BD-OsCATA-R primers and ligated with the pGBKT7 vector digested with EcoRI to construct the BD-OsCATA fusion vector; Constructing a series of AD-OsELF3-1 vectors: The full-length OsELF3-1 and different truncated fragments were amplified using AD-OsELF3.1-F / R primers, and then ligated with pGADT7 vector digested with EcoRI to construct a series of AD-OsELF3-1 fusion vectors. Yeast transformation and screening: BD-OsCATA was co-transformed into yeast AH109 with AD-OsELF3-1 series vectors, respectively. The transformation products were plated on two-deficient medium (-Trp / -Leu) and four-deficient medium (-Trp / -Leu / -His / -Ade) and incubated at 30℃ for 5-7 days. Yeast growth was observed. The results showed that yeast co-transformed with BD-OsCATA and AD-OsELF3-1 could grow normally on four-deficient medium, confirming the protein interaction between OsCATA and OsELF3-1.

[0056] LAC (luciferase complementation) experiment: Constructing nLuc-OsCATA and cLuc-OsELF3-1 recombinant vectors: The target gene was amplified using nLuc-OsCATA-F / R and cLuc-OsELF3.1-F / R primers, respectively, and ligated with Nluc vector digested with SalI and cLuc vector digested with KpnI / SalI. Tobacco co-transformation and detection: The two recombinant vectors were transformed into Agrobacterium GV3101, and after being mixed in equal amounts, they were injected into tobacco leaves. Two days later, the activity of luciferase was detected using an in vivo fluorescence imaging system. The results showed obvious fluorescence signals, confirming the existence of protein-protein interaction between the two vectors.

[0057] BiFC (Bimolecular Fluorescence Complementary) Experiment: Construct YN-OsCATA and YC-OsELF3-1 recombinant vectors: Amplify the target gene using YN-OsCATA-F / R and YC-OsELF3.1-F / R primers, respectively, and ligate it with HYNE and HYCE vectors digested with BamHI / SalI enzymes. Tobacco co-transformation and detection: Two recombinant vectors were co-transformed into tobacco leaves. Two days later, the fluorescence signal was observed using a laser confocal microscope. Green fluorescence was detected, confirming that the two vectors interact with each other in plant cells.

[0058] Co-IP (Co-precipitation Immunoprecipitation) Assay: Constructing OsCATA-GFP and OsELF3-1-MYC recombinant vectors: The target gene was amplified using OsCATA-GFP-F / R and OsELF3-1-MYC-F / R primers and ligated into GFP and MYC tag vectors, respectively; Rice protoplast co-transformation and protein detection: Rice protoplasts were co-transformed with two recombinant vectors. Total protein was extracted after 24 hours and immunoprecipitated with MYC magnetic beads. The GFP tag was detected by Western blot. The results showed that OsCATA-GFP protein was detected in the precipitate, confirming the in vivo protein interaction between the two. Example 8:

[0059] Validation of the molecular mechanism by which OsCATA regulates the heading stage of rice: Expression detection of genes related to heading stage: Wild-type rice Nip, oscata single mutant, oself3-1 single mutant, and oself3-1oscata double mutant were planted in a field in Fuyang, Hangzhou (long-day conditions). Leaf samples were taken before heading, and samples were taken every 4 hours for 48 consecutive hours. Total RNA was extracted and reverse transcribed into cDNA. Using UBQ as an internal control, the expression levels of the florigen genes Hd3a and RFT1, as well as the upstream regulatory genes Ehd1 and Ghd7, were detected by RT-qPCR. Primer sequences are shown in the sequence listing.

[0060] The results show: Compared with the wild type, the expression levels of Hd3a, RFT1, and Ehd1 were significantly downregulated and the expression level of Ghd7 was significantly upregulated in the oscata single mutant. In the oself3-1 single mutant and the oself3-1oscata double mutant, the expression levels of Hd3a, RFT1, and Ehd1 were lower than those in the oscata single mutant, while the expression level of Ghd7 was higher. The expression patterns of the above genes in the double mutant are similar to those in the oself3-1 single mutant.

[0061] Verification of the upstream and downstream relationship between OsCATA and Ghd7: Wild-type rice Nip and ghd7 mutants were planted under long-day and short-day conditions in an artificial climate chamber, respectively. After sampling, the expression levels of OsCATA and OsELF3-1 were detected by RT-qPCR. The results showed that, regardless of long-day or short-day conditions, the expression levels of OsCATA and OsELF3-1 did not differ significantly between the wild-type and ghd7 mutants, confirming that OsCATA and OsELF3-1 are upstream of Ghd7 and influence downstream pathways by regulating Ghd7 expression.

[0062] In summary, the molecular mechanism by which OsCATA regulates the heading stage of rice is as follows: under long-day conditions, OsELF3-1 positively regulates the expression of OsCATA, and the two proteins interact; OsCATA downregulates the expression of Ghd7, thereby relieving the inhibitory effect of Ghd7 on Ehd1, and thus promoting the expression of Hd3a and RFT1 mediated by Ehd1, which in turn promotes rice flowering; after knocking out OsCATA, the expression of Ghd7 is upregulated, inhibiting the expression of Ehd1, Hd3a, and RFT1, ultimately leading to delayed heading of rice.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of the OsCATA gene in delaying the heading stage of rice, characterized by, Knocking out the OsCATA gene can inhibit rice heading under both short-day and long-day conditions, thus delaying the heading period of rice. The OsCATA gene is derived from the rice variety Nipponbare, and its cDNA sequence is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The coding sequence of the OsCATA gene is shown in SEQ ID NO:

2. It encodes a catalase containing 493 amino acids, which participates in the scavenging of reactive oxygen species in rice, and its expression is rhythmic.

3. The application according to claim 1, characterized in that, Under long-day conditions, OsCATA relies on OsELF3-1 to regulate the heading date of rice. OsCATA relieves the inhibition of Ehd1 by downregulating the expression of Ghd7, thereby promoting the expression of Hd3a and RFT1 mediated by Ehd1. After knocking out the OsCATA gene, the expression of Ghd7 is upregulated, inhibiting the expression of Ehd1, Hd3a and RFT1, and ultimately delaying the heading of rice.

4. A recombinant vector for knocking out the OsCATA gene of claim 1, characterized in that, The recombinant vector was obtained by constructing the target sequence of the OsCATA gene into the pC1300-Cas9 knockout vector; the target sequence of the OsCATA gene is shown in SEQ ID NO:

3.

5. A method for obtaining delayed-heading rice plants, characterized in that, Includes the following steps: S1. Transform Agrobacterium EHA105 with the recombinant vector described in claim 4 to obtain an engineered strain of Agrobacterium. S2. Using Agrobacterium-mediated genetic transformation, the Agrobacterium engineered strain was transformed into rice callus tissue. After co-culture, screening, differentiation, rooting, hardening, and transplanting, positive transgenic rice plants with OsCATA gene knockout were obtained. The rice variety in question is Nipponbare.

6. The method according to claim 5, characterized in that, In step S2, hygromycin was used to screen resistant callus tissues, and positive transgenic rice plants were identified by PCR sequencing.

7. The method according to claim 5, characterized in that, The OsCATA gene knockout positive transgenic rice plants obtained headed 12.4 days later than the wild-type Nipponbare under natural long-day conditions in Hangzhou and 7.2 days later under natural short-day conditions in Hainan.

8. A method for constructing a rice double mutant, characterized in that, The OsCATA and OsELF3-1 genes were knocked out to obtain the oself3-1oscata double mutant; the target sequence of the OsCATA gene is shown in SEQ ID NO:4, and the target sequence of the OsELF3-1 gene is shown in SEQ ID NO:

5.

9. The construction method according to claim 8, characterized in that, Fragments containing OsCATA and OsELF3-1 dual gRNA expression cassettes were constructed into the pC1300-Cas9 knockout vector to obtain a double knockout recombinant vector. The vector was then transformed into Nipponbare rice using Agrobacterium EHA105-mediated transformation, and double mutants were obtained through screening. The double mutants headed 24.0 days later than wild-type Nipponbare under natural long-day conditions in Hangzhou.

10. The application of the OsCATA gene knockout positive transgenic rice plant obtained by the method of claim 5, or the oself3-1oscata double mutant obtained by the method of claim 8, in rice breeding, characterized in that... Used for breeding delayed-heading rice varieties adapted to long-day conditions.

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