Application of osbtb05 gene or osbtb05 protein in regulating rice seed storage tolerance and related breeding method

Abstract

The application belongs to the field of plant seed biology and rice breeding, and discloses OsBTB05 application of a gene or OsBTB05 protein in regulating rice seed storage tolerance and a related breeding method. Specifically disclosed is a kind of OsBTB05 application of a gene or OsBTB05 protein in regulating rice seed storage tolerance, OsBTB05 the nucleotide sequence of the gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2, OsBTB05 the amino acid sequence of the protein is shown in SEQ ID NO: 3. A molecular marker assisted selection method for breeding rice varieties with high storage tolerance and a method for reducing rice seed storage tolerance are disclosed. The application proves that OsBTB05 the gene is a key factor for regulating rice seed storage tolerance, and the expression level thereof is significantly positively correlated with seed storage tolerance. This provides a clear molecular target and a brand-new gene resource for improving seed storage tolerance from a genetic source.

Description

Technical Field

[0001] This invention belongs to the field of plant seed biology and rice breeding, and particularly relates to a... OsBTB05 Gene or OsBTB05 Application of proteins in regulating the storage tolerance of rice seeds and related breeding methods. Background Technology

[0002] Seeds play a crucial role in the plant life cycle, serving as both the starting point of agricultural sowing and the end point of harvest, and thus possess significant economic importance. With the advancement of rice breeding technology in my country, rice yields have increased year by year. Ensuring the safe storage of rice seeds has become critical to guaranteeing seed security and food security. Rice seed storage tolerance (i.e., seed lifespan or anti-aging ability) is a key agronomic trait for ensuring germplasm resource security and a stable food supply. Currently, extending seed storage time mainly relies on controlling external conditions such as temperature and humidity, but this method is costly and cannot fundamentally solve the problem. Therefore, improving seed storage tolerance from a genetic perspective has become a more fundamental and economical breeding direction. This trait is jointly regulated by multiple genes, among which the enhancement of the function of positive regulatory genes can directly improve the seed's ability to maintain viability, resist oxidative damage, and maintain physiological stability during storage, making it the most efficient and direct genetic target for improving storage tolerance in molecular breeding.

[0003] However, the genetic regulatory mechanism of this trait is not yet fully understood, and related breeding progress is limited. Although several quantitative trait loci have been located, a considerable number of positively regulatory genes have not yet been effectively cloned and functionally verified, resulting in a lack of key genetic tools that can be directly used in molecular design breeding. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a... OsBTB05 Gene or OsBTB05 Application of proteins in regulating the storage tolerance of rice seeds and related breeding methods.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0006] A sort of OsBTB05 Gene or OsBTB05 The application of proteins in regulating the storage tolerance of rice seeds, the aforementioned OsBTB05 The nucleotide sequence of the gene is shown in SEQ ID NO:1 or SEQ ID NO:2. OsBTB05 The amino acid sequence of the protein is shown in SEQ ID NO:3.

[0007] SEQ ID NO:1:

[0008]

[0009] SEQ ID NO: 2:

[0010]

[0011] SEQ ID NO:3:

[0012] MKVEEKKRSITVAPFECAWDEEFRFRETGRGCITFEASAHNDVTLVFREQPGSQHYHYKMDNSRHYIVILGSHRNKRLKIEVDGKTVVDVAGIGLCCSSSFQSYWISIYDGLISIGQGRHPNNNILFQWLDPDPNRNVQYVGLSSWDKHVGYRNISLMPSAPQNSILWSQIECAYVEPDGAGGHTRKQESKDGLDQRALANFLENWDFSDSIFVVGSERKVVPAHKVVLGSCGDFPFNLMMSRPAIELPSVSYPVLHSLLEYIYTGSTQISEWQLVSLLELSSQFKVKPLVMYCEEIIGCLKMSDAVSESSKKIQLSSGGSQAHQFYYFPFKAPLNTQKIEQFLVNGEHSDVNIYVNGHGLVTHAHKLILSLWSMTFDKMFTNGMKESSASNVFFEDVPVEAFFLLIQFMYSGELKVDIEEITPVLVELLLLSDQFGITALQFECCKRIMEFLSKDTVCSVLRAVSSIPSCKLLEEMCKRNFAEHFDYCTTACTDFVLLDEATFKDILQHGHMTVTSEERVLDAILTWCMEACDCFNWTSVHELLSTSRPEKLFGGRLTAINTLLPFVRFPLVQPSVLHLMEKSNLAKNIEAFRQLVAEAIEFSNAGLRMATNTCERFHHRRSSYKELQYISDGDNNGVIYYAGTSFGKHQWINPVLAKNITVTASSPNSRYTDPKALVSKNYQATCFAGPRLEDGKMCSWWMVDIGPDHQLMCNYYTVRQDGSATFMRSWVLQGSMDGRSWTSLHVHEDDQTICQPGQFASWPITGQTALLPFRFFRVMLTAPATGVSNTWNLCICFLELYGYFR。

[0013] Among them, SEQ ID NO: 1 is OsBTB05 the full-length gene sequence, SEQ ID NO: 2 is OsBTB05The cDNA sequence of the gene, which is located on chromosome 5 of rice, is LOC_Os05g27880 (Os05g0345500) in the rice gene database.

[0014] The above-mentioned applications, preferably, are any of the following uses:

[0015] (a) Rice varieties used for screening seed storage tolerance;

[0016] (b) As molecular markers, used in marker-assisted selection breeding for rice seed storage tolerance.

[0017] In the above applications, preferably, in use (a), the selection criteria are as follows: OsBTB05 The presence of mutations in the gene that cause loss or reduction of its function, or OsBTB05 Protein activity is reduced.

[0018] In the above applications, preferably, in use (b), the molecular marker is... OsBTB05 The Hap3 haplotype of the gene; the Hap3 haplotype is defined by the following combination of single nucleotide polymorphisms (SNPs): in the cDNA sequence shown in SEQ ID NO: 2, the base at position 82 (relative to the start codon ATG) is C and the base at position 2075 is T.

[0019] As a general inventive concept, this invention also provides a molecular marker-assisted selection method for breeding rice varieties with high storage tolerance, in order to OsBTB05 The haplotype of the gene is used as a molecular marker to select rice plants carrying the Hap3 haplotype; the Hap3 haplotype is defined by the following combination of single nucleotide polymorphisms: in the cDNA sequence shown in SEQ ID NO: 2, the 82nd base is C and the 2075th base is T.

[0020] As a general inventive concept, this invention also provides a method for reducing the storage tolerance of rice seeds, thereby reducing the storability of rice seeds in the rice plant. OsBTB05 Reduced or absent gene expression levels, or OsBTB05 The activity of the protein is reduced or lost; OsBTB05 The gene has a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, wherein OsBTB05 The amino acid sequence of the protein is shown in SEQ ID NO:3.

[0021] The preferred method for reducing the storage tolerance of rice seeds described above involves using gene editing technology to knock out or knock down the aforementioned gene. OsBTB05 Genes cause rice plants to have OsBTB05 The expression level of the gene is absent or reduced.

[0022] Preferably, the above-mentioned method for reducing the storage tolerance of rice seeds includes:

[0023] (1) Construction OsBTB05 Gene knockout vectors;

[0024] (2) The above OsBTB05 Gene knockout vectors were transformed into rice;

[0025] (3) Screening OsBTB05 Rice plants with reduced or absent gene expression.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) This invention is the first to identify and confirm the encoding of E3 ubiquitin ligase. OsBTB05 Genes are key positive factors regulating the storage tolerance of rice seeds, and the expression level of these genes is significantly positively correlated with seed storage tolerance. This discovery provides a clear molecular target and novel gene resource for improving seed storage tolerance from a genetic perspective, and has significant theoretical value.

[0028] (2) This invention utilizes a reverse genetics strategy, employing technologies such as CRISPR / Cas9 to knock out or knock down [certain genes]. OsBTB05 Genes were studied to investigate their regulatory function on seed storage tolerance; after treatment with artificially accelerated aging (45℃, 90% relative humidity) to simulate harsh storage conditions, OsBTB05 The germination rate of seeds from gene knockout mutants was significantly reduced, indicating that... OsBTB05 The deletion of this gene leads to a decrease in the storage tolerance of rice seeds. Further experiments showed that under standard germination conditions without aging treatment, OsBTB05 There was no significant difference in germination rate between gene knockout mutants and wild-type seeds. This indicates that... OsBTB05 Gene knockout did not affect the seed's basal vigor under normal conditions, but its function is crucial for maintaining seed survival and growth under aging stress. This finding clarifies that... OsBTB05 Genes are positive regulators of seed resistance to aging stress, providing new potential targets for molecular breeding to improve storage tolerance.

[0029] (3) This invention identifies, through haplotype analysis of large-scale natural germplasm populations, a key characteristic significantly associated with high storage tolerance. OsBTB05 The superior haplotype Hap3 was identified, and its key molecular markers (SNP combinations) were determined. This led to the development of a complete marker-assisted selection (MAS) breeding program, which can directly utilize these markers for rapid screening of germplasm resources. This allows for efficient and targeted improvement of storage tolerance traits without involving complex transgenic processes, significantly reducing breeding costs and time, and broadening application scenarios. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 for OsBTB05 Schematic diagram of gene knockout mutant identification results: A, OsBTB05 Gene knockout target location and target mutation site; B, wild type (ZH11) and osbtb5-1 Sequencing chromatogram alignment of the mutant in the target 1 region shows that the arrow indicates the mutation location is at position 16269316 on chromosome 5, which is the 1900th base position of SEQ ID NO:1. osbtb5-1 Insert a "T" (red box) here; C: Wild type (ZH11) and osbtb5-2 Sequencing chromatogram alignment of the mutant in the target 2 region shows that the arrow indicates the mutation location is at position 16270647 on chromosome 5, corresponding to bases 3231 and 3232 of SEQ ID NO:1. osbtb5-2 The two bases "GA" are missing here (red box); D: osbtb5-1 Prediction of protein sequence alterations caused by mutations; E: osbtb5-2 Prediction of protein sequence changes caused by mutations.

[0032] Figure 2 Background of the 11-flower pattern OsBTB05 Schematic diagram of standard germination test and storage tolerance phenotype identification results for gene knockout mutant seeds, A: Wild type and OsBTB05 Phenotypic results of knockout mutant seeds on day 7 after standard germination test (CK) and artificial aging for 7 days (AA7), scale bar = 2 cm; B: wild type and OsBTB05 Line graph of germination rate of knockout mutant seeds from 1 to 7 days (n=3); C: wild type and OsBTB05 Germination rate bar chart of standard germination test of knockout mutant seeds (n=6); D: wild type and OsBTB05 After artificial aging (AA7) of knockout mutant seeds, a standard germination test was conducted, and a line graph of germination rate from 1 to 7 days was plotted (n=3); E: wild type and... OsBTB05 Germination rate bar graph (n=6) of standard germination test after artificial aging (AA7) of knockout mutant seeds. In the graph, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001, and ns indicates no significant difference.

[0033] Figure 3 for OsBTB05 A schematic diagram of the results of haplotype storage tolerance analysis. Detailed Implementation

[0034] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0035] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0036] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0037] Example 1: OsBTB05 Creation and identification of gene knockout mutants

[0038] This embodiment aims to create rice using CRISPR-Cas9 gene editing technology. OsBTB05 Knockout mutants of genes provide materials for subsequent functional studies.

[0039] 1. Gene knockout target design and primer synthesis

[0040] OsBTB05 The gene is located in rice ( Oryza sativa L. On chromosome 5 (gene ID: LOC_Os05g27880), a design was created within its coding region (CDS) using the online CRISPR design website (http: / / crispr.hzau.edu.cn / CRISPR2). OsBTB05 Knockout targets were selected based on the following criteria: targets not containing more than four consecutive T bases, GC content between 45% and 70%, location within gene exons, and high targeting efficiency. Target specificity was then assessed using a BLAST platform, and two targets were selected (e.g.,...). Figure 1 (A) The knockout primers are: front primer: GCCG+ target sequence; back primer: reverse complementary sequence of AAAC+ target sequence, as shown in Table 1 and SEQ ID NO:4 to SEQ ID NO:7.

[0041] Table 1 Target Primers

[0042]

[0043] The primer sequences MF / gR-R and Pps-GGL / Pgs-GGR are used to construct universal primers for Crispr-Cas9, and the sequences are shown in Table 2 and SEQ ID NO:8 to SEQ ID NO:11.

[0044] Table 2 Construction of CRISPR-Cas9 universal primers

[0045]

[0046] The designed primers with known sequences were sent to the Hunan branch of Beijing Qingke Biotechnology Co., Ltd. for primer synthesis.

[0047] 2. Knockout vector construction and transformation

[0048] The vector used in this embodiment is the pYLCRISPR / Cas9 binary vector system, with the sgRNA intermediate vector pYLgRNA-OsM6a.

[0049] sgRNA expression cassettes were constructed using the pYLCRISPR / Cas9 binary vector system and target-adaptor ligation amplification method. The specific steps are as follows:

[0050] (1) Annealing of the adapter primers: Dilute the synthesized target primers to 10 μM, take 1 µL of each primer, add 8 µL of 0.5×TEBuffer, and mix them in a 10 µL system. After mixing, heat in a metal bath at 90℃ for 30 seconds, and then cool to room temperature to complete the annealing process.

[0051] (2) gRNA expression cassette ligation and amplification: The sgRNA expression cassette was digested and ligated using the Golden Gate assembly method. A 10 μL reaction system was prepared on ice, containing: 1 μL of 10× T4 DNA ligase buffer, 1 μL of 10× CutSmart buffer, 1 μL of linearized pYLgRNA-OsU6a empty vector plasmid (20 ng / μL), 0.5 μL of annealed target-specific adapter primers, 0.1 μL of restriction endonuclease BsaI, 0.1 μL of T4 DNA ligase, and sterile ddH2O to a total volume of 10 μL. The reaction system was placed in a PCR instrument and the following program was run: incubation at 37°C for 5 minutes, followed by incubation at 20°C for 5 minutes, repeated 5 times, to efficiently complete the digestion of the vector backbone and the directional ligation of the target fragment.

[0052] To amplify and obtain high-purity sgRNA expression cassette fragments, two rounds of PCR were performed using the prepared ligation reaction product as a template.

[0053] (1) First round of PCR amplification

[0054] Using the ligation product as a template, initial amplification was performed using target-specific adapter primers. A 15 μL reaction mixture was prepared, containing: 10× Kod Plus Buffer, dNTPs, ligation product, adapter primers (MF / gR-R), Kod Plus DNA polymerase, and sterile ddH2O. The reaction program was set as follows: 95℃ pre-denaturation for 1 min; followed by 30 cycles (95℃ denaturation for 10 sec, 60℃ annealing for 15 sec, 68℃ extension for 30 sec); and a final extension at 68℃ for 5 min. After the reaction, 5 μL of the product was analyzed by agarose gel electrophoresis.

[0055] (2) Second round of PCR amplification

[0056] Using the first-round PCR product as a template, PCR amplification was performed using universal primers Pps-GGL and Pgs-GGR, located outside the multiple cloning site of the vector. A 50 μL reaction mixture was prepared, containing: 25 μL of 2× Kod FX Buffer, 1 μL of Kod FX DNA polymerase, 10 μL of dNTPs, 1.5 μL each of primers Pps-GGL and Pgs-GGR, 2 μL of the first-round PCR product, and brought to a final volume of 50 μL with sterile ddH2O. The reaction program was set as follows: 94℃ pre-denaturation for 2 minutes; followed by 25 cycles (94℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds, 68℃ extension for 50 seconds); and a final extension at 68℃ for 5 minutes. After amplification, the product was subjected to agarose gel electrophoresis, and the correctly sized target band was excised and purified. The purified fragment is the sgRNA expression cassette, used for subsequent assembly with the Cas9 vector.

[0057] (3) Assembly and cloning of sgRNA expression cassette and Cas9 vector

[0058] To construct a complete CRISPR-Cas9 knockout vector, the purified sgRNA expression cassette fragment was subjected to an in vitro enzyme digestion-ligation reaction with the pYLCRISPR / Cas9 vector backbone pretreated with the same restriction endonuclease.

[0059] (a) Golden Gate assembly reaction

[0060] Prepare a 20 μL reaction mixture on ice, comprising: 3 μL 10× T4 DNA ligase buffer, 1 μL 10× CutSmart buffer, 0.2 μL purified sgRNA expression cassette fragment, 1 μL linearized Cas9 backbone plasmid (pYLCRISPR / Cas9), 0.5 μL 10× restriction endonuclease BsaI, 0.3 μL T4 DNA ligase, and bring the volume to 20 μL with sterile ddH2O. Place the reaction mixture in a PCR instrument and run the following cycle: incubation at 37°C for 15 minutes; followed by 13 cycles, each consisting of incubation at 37°C for 5 minutes, 10°C for 5 minutes, and 20°C for 5 minutes; finally, store at 16°C.

[0061] (b) E. coli transformation and plasmid amplification

[0062] Take 5 μL of the ligation product and gently mix it with 50 μL of DH5α competent cells on ice. After incubating on ice for 30 minutes, heat shock it in a 42°C metal bath for 45 seconds, then quickly transfer it to ice and let it stand for 2 minutes. Add 500 μL of preheated (37°C) LB liquid medium and incubate at 220 rpm for 20 minutes in a shaker at 37°C. Spread an appropriate amount of bacterial culture onto LB agar plates containing kanamycin (50 mg / L) and incubate overnight at 37°C. Pick single colonies for expansion culture, extract plasmids, and perform PCR and enzyme digestion identification to verify the correct recombinant plasmid.

[0063] (c) Preparation of Agrobacterium-mediated bacterial culture

[0064] The validated recombinant plasmid was transformed into Agrobacterium tumefaciens EHA105 competent cells for plant transformation. Approximately 3 μL of plasmid (about 100 ng) was mixed with 50 μL of Agrobacterium competent cells on ice and incubated for 30 minutes. After a heat shock at 42°C for 90 seconds, the cells were incubated on ice for 2 minutes. 500 μL of sterile LB broth was added, and the cells were incubated at 28°C and 200 rpm for 3 hours using a shaker. The bacterial culture was then spread onto LB agar plates containing both kanamycin (50 mg / L) and rifampin (50 mg / L) and incubated upside down at 28°C for 3 days. Single colonies were picked and inoculated into LB broth containing the corresponding antibiotics and cultured with shaking at 28°C and 200 rpm until the logarithmic growth phase. The culture was then ready for subsequent rice genetic transformation or stored in glycerol at -80°C.

[0065] 3. Agrobacterium-mediated genetic transformation of rice

[0066] The Agrobacterium-mediated transformation of the prepared culture containing the recombinant knockout vector was carried out using the rice variety Zhonghua 11 as the recipient. The specific process included: inducing embryogenic callus tissue as the recipient using mature rice embryos; infecting the recipient material with Agrobacterium carrying the target vector; after co-culturing, screening with corresponding antibiotics to obtain resistant callus tissue; and then differentiating and regenerating it to obtain T0 generation transgenic positive plants.

[0067] 4. Molecular identification of mutant plants

[0068] The T0 generation transgenic positive plants obtained above were cultivated normally to maturity at the Hunan Agricultural University transgenic base in Huanghua Town, Changsha County. Seeds of the T0 generation plants were harvested through self-pollination, and these seeds are the T1 generation seeds. The T1 generation seeds were sown, and the seedlings were used for subsequent molecular identification when they reached the appropriate growth stage.

[0069] Genomic DNA was extracted from T1 generation transgenic plants and... OsBTB05 Specific identification primers were designed on both sides of the gene target region, as shown in Table 3 and SEQ ID NO:12 to SEQ ID NO:15.

[0070] Table 3. Design of specific primers to identify mutation types in mutant plants.

[0071]

[0072] The target region was amplified by PCR (PCR reaction system: 45 µL Gold Mix, 2 µL each of pre- and post-prime extracts, 1 µL DNA. PCR program: 98℃ 3 min, 98℃ 15 s, 60℃ 15 s, 72℃ 30 s, 72℃ 5 min, 16℃ 10 min, 35 cycles) and Sanger sequencing was performed. The sequencing results were compared with the wild-type Zhonghua 11 sequence. Figure 1 As shown.

[0073] Select plants in the T1 generation that were found to be homozygous (or biallelic). OsBTB05 Gene editing mutations were named respectively. osbtb5-1 and osbtb5-2, Used for subsequent identification of seed storage tolerance phenotypes. osbtb5-1 The transgenic line is located at the 1900th base position of the sequence shown in SEQ ID NO:1 (corresponding to target site 1 region). osbtb5-1 Insert a "T" here ( Figure 1 (See B in SEQ ID NO:3). This insertion mutation causes a frameshift and premature translation termination at amino acid position 35 of the amino acid sequence shown in SEQ ID NO:3, ultimately producing a truncated, non-functional protein (see B in SEQ ID NO:3). Figure 1 (D in the middle).osbtb5-2 The transgenic line is located at base positions 3231 and 3232 of the sequence shown in SEQ ID NO:1 (corresponding to target site 2 region). osbtb5-2 The two bases "GA" are missing here (see Figure 1 The deletion mutation causes a frameshift and premature translation termination at amino acid position 414 of the amino acid sequence shown in SEQ ID NO:3, ultimately producing a truncated, non-functional protein (see C). Figure 1 (E in the text).

[0074] Example 2: OsBTB05 Effects of gene knockout on the storage tolerance of rice seeds

[0075] This embodiment systematically evaluates the results through standard germination tests and artificial accelerated aging tests. [[ID=·69]]OsBTB05 The effect of gene knockout on the storage tolerance of rice seeds.

[0076] 1. Standard germination test

[0077] To assess the basal viability of the mutants, a standard germination test was first performed. Wild-type Zhonghua 11 (ZH11) harvested at the same time was compared with two mutants (…). osbtb5-1 and osbtb5-2 Mature seeds were dried in a 40℃ oven for 7 days to break dormancy. One hundred plump seeds were selected from each line, with three biological replicates. After soaking the seeds in distilled water for 24 hours (changing the water every 12 hours), they were placed on two layers of moist germination paper, spaced approximately 2 cm apart, and cultured in a light incubator (30℃, 16h light / 8h darkness). Germination rate was recorded daily for 7 consecutive days, and the final germination rate was calculated.

[0078] Result: As Figure 2 As shown, under standard germination conditions, there was no statistically significant difference in germination rates between wild-type and the two mutant seeds. This indicates that knockout... OsBTB05 Genes do not affect the germination of rice seeds under normal conditions.

[0079] 2. Artificial accelerated aging treatment and storage tolerance assessment

[0080] To simulate the stresses of long-term storage, an artificial accelerated aging treatment was conducted. Wild-type Zhonghua 11 (WT) grapes harvested at the same time were compared with two mutant varieties (…). osbtb5-1 and osbtb5-2 Mature seeds were placed in an aging chamber and treated for 7 days at 45℃ and 90% relative humidity. After aging, the seeds were removed and allowed to equilibrate at room temperature for 1 day. Germination was then tested according to the standard germination test procedure described above, and all indicators were recorded. This experiment was performed in triplicate.

[0081] Seeds that underwent artificial aging treatment were dynamically observed and photographed for 7 consecutive days, and the results were recorded as follows: Figure 2 As shown, after artificially accelerated aging treatment, the two OsBTB05 Knockout mutants exhibit significantly reduced storage tolerance; mutants osbtb5-1 and osbtb5-2 Germination rate on day 7 was significantly lower than that of wild type (see Figure 2 D and Figure 2 (E in the middle)

[0082] This shows OsBTB05 Gene knockout can significantly reduce the survival and germination ability of rice seeds under artificial accelerated aging stress, that is, significantly reduce the storage tolerance of seeds, and this effect does not affect their basic vigor.

[0083] Example 3: OsBTB05 Gene haplotype analysis and identification of superior haplotypes

[0084] This embodiment aims to explore OsBTB05 Genetic variation in natural populations and identification of superior haplotypes associated with storage tolerance provide targets for molecular marker-assisted breeding.

[0085] Data was obtained from RiceVarMap2 (ncpgr.cn), a database website published by Huazhong Agricultural University. OsBTB05 All single nucleotide polymorphisms (SNPs) within the genomic sequence region of the gene (LOC_Os05g27880) were analyzed. SNPs with a minor allele frequency <5% and a deletion rate >8% were removed. Finally, the germplasm genotypes corresponding to the seed storage tolerance phenotype were sorted, and classified into different haplotypes based on the base pairs of the SNP sites. R software was used to plot and compare the seed storage tolerance among different haplotypes. Analysis revealed that this gene mainly exists in three haplotypes: Hap1, Hap2, and Hap3. Figure 3 ). After artificially accelerated aging treatment (7 days at 45℃ and 90% relative humidity) on 127 natural germplasm materials, their seed germination rates were measured. Phenotypic association analysis showed ( Figure 3The average germination rate of seeds carrying the Hap3 haplotype after aging treatment was significantly higher than that of seeds carrying the Hap1 and Hap2 haplotypes, indicating that it is a superior haplotype. Furthermore, observations after 18 months of natural aging also showed a consistent trend. This suggests that the Hap3 haplotype is a superior haplotype associated with stable storage tolerance. The key sequence characteristic of the Hap3 haplotype is that the nucleotide sequence shown in SEQ ID NO: 2 contains specific alleles at the following single nucleotide polymorphism (SNP) sites: C at site 82 (relative to the start codon ATG) and T at site 2075. When these two specific alleles are detected at the aforementioned SNP sites, the gene is identified as the Hap3 haplotype.

[0086] This indicates that, OsBTB05 Genes undergo natural variation, among which OsBTB05 The Hap3 gene is a superior haplotype significantly associated with high storage tolerance. This haplotype can be used as a molecular marker for screening and assisted breeding of rice varieties with high storage tolerance.

[0087] The above embodiments fully demonstrate that the invention has discovered... OsBTB05 The gene is a key gene that positively regulates the storage tolerance of rice seeds. Knocking out this gene using gene editing technology, or using its superior haplotype Hap3 for marker-assisted selection, can effectively create or screen new rice materials with significantly enhanced storage tolerance, which has important breeding application value.

Claims

1. A molecular marker-assisted selection method for breeding rice varieties with high storage tolerance, characterized in that, by OsBTB05 The haplotype of the gene was used as a molecular marker to select rice plants carrying the Hap3 haplotype; OsBTB05 The nucleotide sequence of the gene is shown in SEQ ID NO:

2. The Hap3 haplotype is defined by the following combination of single nucleotide polymorphisms: in the cDNA sequence shown in SEQ ID NO:2, the 82nd base is C and the 2075th base is T.

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