Rice osatg4b gene and application thereof
By using gene editing technology on the OsATG4b gene to regulate the chalky trait in rice, transgenic rice with high or low chalky phenotypes was created, solving the problem of insufficient resources of rice chalky regulation genes and achieving significant improvement in rice quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YANGZHOU UNIV
- Filing Date
- 2025-04-14
- Publication Date
- 2026-04-28
AI Technical Summary
In the current technology, there is a lack of gene resources to regulate the chalkiness trait in rice, which makes it difficult to improve rice quality. In particular, excessive chalkiness and chalky grain rate affect the appearance and processing quality of rice, thus hindering the progress of rice breeding.
By mining and utilizing the rice OsATG4b gene, and using the CRISPR/Cas9 system to target and knock out or overexpress the OsATG4b gene, the chalkiness trait in rice was regulated, and transgenic rice lines with high or low chalkiness phenotypes were created, revealing its role in the regulation of rice chalkiness.
It has achieved significant regulation of rice chalkiness and chalky grain rate, created new, efficient and stable rice quality improvement gene resources, and enhanced the market competitiveness of rice.
Smart Images
Figure CN120272495B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of rice molecular breeding and gene editing technology, specifically, to a type of rice. OsATG4b Genes and their applications. Background Technology
[0002] In recent years, my country has made remarkable achievements in rice breeding to increase yields. Advances in technologies ranging from high-yield breeding to green super rice breeding have made significant contributions to ensuring national food security. However, rice quality, especially the optimization of chalkiness, remains a key bottleneck in current rice breeding. Chalkiness is the opaque portion of the rice endosperm, which severely affects the appearance, processing, and eating quality of rice, thereby reducing its market competitiveness (Fitzgerald et al., 2009).
[0003] Existing research indicates that the chalkiness level of rice varieties promoted in my country remains high, with average chalky grain rates of 23.2% and 16.2% for indica and japonica rice, and average chalkiness of 4.6% and 2.9%, respectively (Chen et al., 2024). According to the national standard for high-quality rice (GB / T 17891-2017), the chalkiness of Grade 1 indica and japonica rice must be below 2.0%, and the Grade 2 standard only allows for chalkiness ≤5.0% for indica rice and ≤4.0% for japonica rice. Currently, the chalkiness of most promoted varieties still fails to meet the Grade 2 standard for high-quality rice, severely restricting the improvement of rice quality and international competitiveness in my country.
[0004] Although research on genes regulating chalkiness has been reported, efficient and stable techniques for effectively improving chalkiness traits still lack. This is because the number of discovered chalkiness-regulating genes in rice is insufficient, and their molecular regulatory mechanisms are poorly understood. Therefore, identifying new gene resources capable of precisely regulating chalkiness formation and utilizing new breeding methods or molecular techniques based on these genes is of great significance for promoting rice quality breeding in my country.
[0005] Autophagy is a highly conserved intracellular degradation mechanism in eukaryotes. Through the encapsulation of cytoplasmic material by autophagosomes, a double-membrane structure, the cytoplasm eventually fuses with lysosomes (in animals) or vacuoles (in plants), achieving the degradation and recycling of its contents. In the 1990s, Japanese scientist Yoshinori Ohsumi identified the first autophagy-related gene (ATG1) using a yeast model and systematically elucidated the key steps in autophagy formation. More than 40 ATG proteins in yeast work synergistically in processes such as autophagy induction, membrane nucleation, phagosome expansion, autophagosome closure, and degradation, and these core mechanisms are highly conserved in plants.
[0006] Although the functions of autophagy in yeast, animals, and model plants have been extensively studied, its role in rice quality regulation (such as chalkiness, starch metabolism, and protein accumulation) remains unclear. Therefore, exploring the regulatory mechanisms of the autophagy pathway on rice quality and developing molecular breeding technologies based on key autophagy genes hold promise for providing new strategies for cultivating high-quality rice varieties. Summary of the Invention
[0007] To address the incomplete understanding of the genetic basis and molecular mechanisms of chalkiness, which hinders breakthroughs in yield and quality improvement, this invention provides a rice... OsATG4b The study focuses on genes and their applications, exploring and utilizing autophagy-related genes to investigate their potential role in regulating rice chalkiness, and targeting and regulating rice quality-related metabolic pathways to provide new gene resources for solving the current industrial problems of high chalkiness rate and substandard quality in rice.
[0008] To achieve the above objectives, the present invention provides, in one aspect, a rice... OsATG4b The gene, whose coding region has a nucleotide sequence as shown in SEQ ID No. 1 and an encoded amino acid sequence as shown in SEQ ID No. 2.
[0009] The second aspect of the present invention provides the above-mentioned rice. OsATG4b Any of the following applications of genes:
[0010] (1) Regulating the chalky trait in rice; specifically involving the targeted knockout of autophagy-related genes in rice using the CRISPR / Cas9 system. OsATG4b Create rice mutants with a high chalky phenotype; simultaneously overexpress... OsATG4b Creating transgenic rice lines with low chalky white phenotype reveals OsATG4b The regulatory effect on chalkiness in rice.
[0011] (2) Used for rice variety improvement;
[0012] (3) Used to prepare genetically modified rice.
[0013] Preferably, the chalkiness trait of rice includes chalkiness degree and chalky grain rate.
[0014] A third aspect of this invention provides a method for improving the chalkiness and chalky grain rate of rice, comprising: using genetic engineering techniques to knock out [a specific gene] in rice. OsATG4b Gene.
[0015] Specifically, with OsATG4b Using genes as targets, CRISPR / Cas9-based sgRNA primer sequences were designed. DNA fragments encoding these sgRNA primer sequences were ligated into vectors carrying CRISPR / Cas, and rice was transformed to obtain... OsATG4bGenetically modified rice with missing gene function.
[0016] Specifically, OsATG4b The target site is located in exon 1, and the sequence is shown in SEQ ID No. 3.
[0017] Specifically, the sgRNA primers include: OsATG4b The primer sequence for the gRT1 gene is shown in SEQ ID NO.4. OsATG4b The OsU3T1 primer sequence for the gene is shown in SEQ ID NO.5.
[0018] A fourth aspect of this invention provides a method for reducing the chalkiness and chalky grain rate of rice, comprising: using genetic engineering techniques to enhance the... OsATG4b Gene.
[0019] Preferably, the enhancement method is selected from one or an optional combination of the following (1)-(5):
[0020] (1) Enhancement by introducing a plasmid containing the gene;
[0021] (2) Enhanced by increasing the copy number of the aforementioned genes on the rice chromosome;
[0022] (3) Enhancement is achieved by altering the promoter sequence of the aforementioned genes on the rice chromosome;
[0023] (4) Enhancement is achieved by operatively linking a strong promoter to the gene;
[0024] (5) Enhancement by introducing enhancers.
[0025] The fifth aspect of this invention provides the application of the transgenic rice obtained by the above-described method in plant breeding.
[0026] Through the above technical solution, the present invention achieves the following beneficial effects:
[0027] This invention is the first to discover core genes in the autophagy pathway. OsATG4b Influencing the formation of chalkiness in rice, created through gene editing technology. OsATG4b Gene knockout mutants are obtained through transgenic technology. OsATG4b Gene overexpression lines were verified. OsATG4b The gene is a new gene that regulates chalkiness in rice, providing new genetic resources for improving rice quality. Attached Figure Description
[0028] Figure 1 yes OsATG4b Genotyping of knockout mutants, where A is... osatg4b-1 and osatg4b-2 The target mutation mode of the mutant, B is osatg4b-1 and osatg4b-2 The corresponding mutant protein coding sequence of the mutant;
[0029] Figure 2 yes osatg4b-1 and osatg4b-2 The main agronomic traits of the mutants were compared, with AH being, in descending order, heading date, number of tillers, seed setting rate, number of grains per panicle, thousand-grain weight, plant height, number of primary branches, and number of secondary branches;
[0030] Figure 3 yes osatg4b-1 and osatg4b-2 The chalky phenotype of the mutant, where A represents the wild-type WT. osatg4b-1 and osatg4b-2 Photograph of chalky rice from mutant strains, B represents wild-type WT. osatg4b-1 and osatg4b-2 Statistical analysis of the chalkiness of polished rice in mutants, C represents wild-type WT, osatg4b-1 and osatg4b-2 Statistics on the chalky grain rate of milled rice in mutants;
[0031] Figure 4 It is a wild-type WT. OsATG4b-OE1 and OsATG4b-OE2 Gene expression analysis of overexpression plants;
[0032] Figure 5 It is a wild-type WT. OsATG4b-OE1 and OsATG4b-OE2 The main agronomic traits of the plant were overexpressed, with AF being the number of tillers, seed setting rate, grain length, grain width, thousand-grain weight, and plant height, in descending order.
[0033] Figure 6 It is a wild-type WT. OsATG4b-OE1 and OsATG4b-OE2 Overexpression of the chalky phenotype of plants, where A represents wild-type WT, OsATG4b-OE1 and OsATG4b-OE2 Chalky white rice from overexpressing plants, B represents wild-type WT. OsATG4b- OE1 and OsATG4b-OE2 Statistical analysis of chalkiness in polished rice from overexpressing plants, where C represents wild-type WT. OsATG4b-OE1 and OsATG4b- OE2 Statistics on the chalky grain rate of polished rice in overexpressing plants. Detailed Implementation
[0034] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.
[0035] Example 1: Target gene analysis and sgRNA design
[0036] 1.1 Gene Sequence Acquisition:
[0037] OsATG4b The gene is located on chromosome 4 of the rice genome, with the gene number Os04g0682000 (RAP-DB naming rule) or LOC_Os04g58560 (MSU naming rule). OsATG4b The nucleotide sequence length of the gene coding region is 1437 bp, and the sequence is shown in SEQ ID No. 1. OsATG4b The gene encodes an amino acid sequence of 479 amino acids, as shown in SEQ ID No. 2. The protein size is approximately 52.5 kDa.
[0038] SEQ ID NO.1:
[0039]
[0040] SEQ ID NO.2:
[0041] MTSLPDRGVSSSSSSDPLCEGNIAPCSSSSEQKEDCSLKQSKTSILSCVFNSPFNIFEAHQDSSANKSPKSSSGSYDWSRVLRRIVCSGSMWRFLGTSKVLTSSDVWFLGKCYKLSSEESS SDSDSESGHATFLEDFSSRIWITYRRGFDAISDSKYTSDVNWGCMVRSSQMLVAQALIFHHLGRSWRRPLEKPYNPEYIGILHMFGDSEACAFSIHNLLQAGNSYGLAAGSWVGPYAMCR AWQTLVRTNREQHEVVDGNESFPMALYVVSGDEDGERGGAPVVCIDVAAQLCCDFNKGQSTWSPILLLVPLVLGLDKINPRYIPLLKETFTFPQSLGILGGKPGTSTYIAGVQDDRALYL DPHEVQMAVDIAADNIEADTSSYHCSTVRDLALDLIDPSLAIGFYCRDKDDDFDDFCSRATELVDKANGAPLFTVVQSVQPSKQMYNQDDVLGISGDGNINVEDLDASGETGEEEWQIL*.
[0042] Note: * represents the stop codon.
[0043] 1.2 Target Selection:
[0044] Targets were screened using the CRISPR target prediction website (http: / / crispr.dbcls.jp / ) to ensure they were located in conserved functional domains and had a low off-target risk. Based on the website's prediction results, suitable target sequences were selected. OsATG4b The target site 1 is located in the first exon, and the sequence SEQ ID No. 3 is as follows: GGAGCCGAGCTGGTCCACGG.
[0045] Example 2 OsATG4b Knockout vector construction and rice transformation
[0046] 2.1 Primer Design:
[0047] sgRNA primers were synthesized using the CRISPR primer design platform (http: / / skl.scau.edu.cn / primerdesign / vector / ). OsATG4bThe gRT1 primer sequence SEQ ID No. 4 is as follows: GAGCCGAGCTGGTCCACGGgttttagagctagaaat. OsATG4b The OsU3T1 primer sequence SEQ ID No. 5 is as follows: CCGTGGACCAGCTCGGCTCCggcagccaagccagca.
[0048] 2.2 Vector Construction:
[0049] First, a first round of PCR amplification was performed. Using the U3 vector as a template, PCR amplification was performed using OsATG4b-gRT1 and B1' primers to obtain DNA fragment a, and PCR amplification was performed using OsATG4b-OsU3T1 and B2 primers to obtain DNA fragment b. The PCR amplification reaction system included the following: template DNA, 0.2 μM primers, 0.2 mM dNTPs, Taq DNA polymerase, and PCR buffer. The amplification program was as follows: (1) Initial denaturation: 95°C, 5 minutes; (2) Cyclic amplification (35 cycles): Denaturation: 95°C, 30 seconds. Annealing: 55°C, 30 seconds. Extension: 72°C, 30 seconds.
[0050] The DNA fragments from the PCR products were recovered by agarose gel electrophoresis. The B1' primer sequence (SEQ ID NO. 6) was: TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG, and the B2 primer sequence (SEQ ID NO. 7) was: AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC.
[0051] Next, a second round of PCR amplification was performed. Using the amplification products from the first round as a template, DNA fragments a and b were mixed and used as a template. PCR amplification was then performed using primers B1' and B2 to obtain DNA fragment A. The PCR product A was then recovered by agarose gel electrophoresis.
[0052] The Golden Gate cloning method was used to insert DNA fragment A into the pYLCRISPR / Cas9-MH vector. First, the circular pYLCRISPR / Cas9-MH plasmid vector was digested with BstB1, and the linearized pYLCRISPR / Cas9-MH vector was recovered by agarose gel electrophoresis. Then, DNA fragment A was ligated into the linearized vector using T4 DNA ligase. The reaction products were transformed into competent *E. coli* cells, and positive colonies were selected by kanamycin resistance screening. Single colonies were then selected for culture by shaking and Sanger sequencing verification. The sequencing process was as follows: the plasmid to be tested was extracted and sent to a biotechnology company for sequencing. The sequencing results were compared with the expected sequence using DNAMAN tool; a 100% match indicated successful vector construction. The sequencing primer MH-F sequence (SEQ ID NO. 8) was as follows: CGGTGTCATCTATGTTACTAG; the sequencing primer MH-R sequence (SEQ ID NO. 9) was as follows: CCGACATAGATGCAATAACTTC. Finally, it was confirmed that DNA fragment A was correctly constructed into the pYLCRISPR / Cas9-MH vector.
[0053] 2.3 Rice Conversion:
[0054] Using the japonica rice variety Nipponbare as the recipient, the constructed pYLCRISPR / Cas9-MH vector was transformed into rice callus tissue via Agrobacterium-mediated transformation (strain EHA105). Positive transgenic plants were screened using hygromycin (50 mg / L), ultimately yielding 20 T0 generation transgenic plants. Rice transformation was performed by Wuhan Boyuan Company. The pYLCRISPR / Cas9-MH vector was initially detected by PCR. The experimental procedure was as follows: DNA was extracted from leaves using the CTAB method, and the specific sequence on the vector was amplified by PCR. The PCR reaction system and procedure were the same as above. The PCR product was detected by agarose gel electrophoresis. The target band was consistent with the expected size, indicating that the plant contained the pYLCRISPR / Cas9-MH vector, and was preliminarily identified as a transgenic positive plant. The detection primers were: Cas9-F sequence SEQ ID NO.10, e.g., GTCGCCTACCACGAGAAGTA; Cas9-R sequence SEQ ID NO.11, e.g., GTGAGGTCCTGGTGGTGCTC. The vector has been successfully transferred into the transgenic plants.
[0055] Example 3 OsATG4b Mutant identification and phenotypic analysis
[0056] 3.1 Gene Editing Detection:
[0057] Genomic DNA was extracted from T0 generation transgenic plants, and PCR amplification and sequencing were performed using primers near the target site. OsATG4b The sequencing primers OsATG4b-F sequence (SEQ ID NO. 12) are as follows: AGAGACCCAATTGCGGATG; the OsATG4b-R sequence (SEQ ID NO. 13) is as follows: AAGTGCGACAAGCACCTGAGC. PCR sequencing results showed... osatg4b-1 The mutant contains a 1 bp base insertion at the target site ( Figure 1 A), causing the 76th bit to terminate prematurely ( Figure 1 B). osatg4b-2 The mutant contains a 2 bp deletion at the target site ( Figure 1 A), causing the 75th bit to terminate prematurely ( Figure 1 B). The T0 generation transgenic mutant was self-crossed to obtain the T1 generation, which was then further validated by sequencing. OsATG4b Gene knockout results in homozygous mutant lines.
[0058] 3.2 OsATG4b Phenotypic determination of gene knockout mutants:
[0059] Wild-type Nipponbare was cultivated under summer field conditions in Yangzhou. OsATG4b Knockout mutant, normal water and fertilizer management. Observation of agronomic traits revealed that, compared with the wild type, OsATG4b The knockout mutants showed no significant differences in thousand-grain weight, grain type, plant height, and tiller number. Figure 2 ).
[0060] Further studies on wild-type and OsATG4b Seeds of the knockout mutant were milled into polished rice, and observation of the chalky phenotype revealed that, compared to the wild-type Nipponbare, it exhibited a chalky white phenotype. OsATG4b The knockout mutant showed a highly significant increase in chalkiness and chalky grain rate, with an increase of up to 50%. Figure 3 ).
[0061] Example 4 OsATG4b Construction of overexpression vectors and rice transformation
[0062] according to OsATG4b Based on the coding region sequence, primers for the overexpression vector were designed. OE-F primer sequences (SEQ ID NO. 14) were used, such as: ACGGGGGACGAGCTCGGTACCATGACGAGCTTGCCTGATAGG. OE-R primer sequences (SEQ ID NO. 15) were used, such as: ATGGTCTTTGTAGTCTTCGAACTAAAGAATCTGCCATTCCT. PCR amplification was then performed. OsATG4b The coding region sequence was obtained by glue recovery. OsATG4bThe DNA fragment was obtained. The pCAMBIA1300 vector was digested with Kpn1 and BamH1 enzymes, and the linearized pCAMBIA1300 vector was obtained by gel purification. Homologous recombinase was used to... OsATG4b The gene fragment was homologously recombinated into the overexpression vector pCAMBIA1300. The reaction product was transformed into competent *E. coli* cells, and positive colonies were selected by kanamycin resistance screening. Single colonies were then selected for culture by shaking and Sanger sequencing for verification, ultimately confirming the positive results. OsATG4b It was correctly constructed onto the pCAMBIA1300 vector.
[0063] Using the japonica rice variety Nipponbare as the recipient, the pCAMBIA1300 vector constructed above was transformed into rice callus tissue via Agrobacterium-mediated transformation. Positive transgenic plants were screened using hygromycin, and 20 T0 generation transgenic plants were finally obtained. Rice transformation was completed by Wuhan Boyuan Company.
[0064] Example 5 OsATG4b Identification and phenotypic analysis of overexpressing plants
[0065] 5.1 OsATG4b Gene expression level detection:
[0066] RNA was extracted from leaves of wild-type and transgenic plants and reverse transcribed into cDNA. The primers for quantitative real-time PCR (qRT-PCR) were: qRT-PCR-F sequence (SEQ ID NO. 16, e.g., ATGACGAGCTTGCCTGATAG) and qRT-PCR-R sequence (SEQ ID NO. 17, e.g., GGCTACAATCCTCCTTCTGT). qRT-PCR experimental results showed... OsATG4b-OE1 and OsATG4b-OE2 Overexpression of plant OsATG4b Expression levels were significantly higher compared to wild type ( Figure 4 ).
[0067] 5.2 OsATG4b Phenotypic determination of plants with gene overexpression:
[0068] Wild-type Nipponbare was cultivated under summer field conditions in Yangzhou. OsATG4b Overexpressing plants were managed with normal water and fertilizer. Observation of agronomic traits revealed that, compared with the wild type, OsATG4b There were no significant differences in thousand-grain weight, grain type, plant height, and tiller number among the gene-overexpressing plants. Figure 5 ).
[0069] Further analysis of the chalky phenotype by grinding the seeds of wild-type and overexpressing plants into polished rice revealed that, compared with the wild-type Nipponbare, the chalkiness and chalky grain rate of the overexpressing plants were significantly reduced. Figure 6 The reduction reached 30%.
[0070] The above research shows OsATG4b It is a novel gene that regulates chalkiness. Overexpression of this gene can significantly reduce the occurrence of chalkiness in rice, which plays an important role in improving rice quality.
[0071] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
[0072] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.
[0073] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.
Claims
1. A type of rice OsATG4b The application of genes in regulating the chalky trait in rice is characterized by, Through overexpression OsATG4b To create transgenic rice lines with a low chalky phenotype, OsATG4b The nucleotide sequence of the coding region of the gene is shown in SEQ ID No. 1, and the encoded amino acid sequence is shown in SEQ ID No.
2.
2. The application according to claim 1, characterized in that, The chalkiness trait of rice includes chalkiness degree and chalky grain rate.
3. A method for reducing the chalkiness and chalky grain rate of rice, characterized in that, include: Using genetic engineering techniques to enhance the properties of rice OsATG4b Gene expression levels, OsATG4b The nucleotide sequence of the coding region of the gene is shown in SEQ ID No. 1, and the encoded amino acid sequence is shown in SEQ ID No.
2.
4. The method according to claim 3, characterized in that, The enhancement method is selected from one or an optional combination of the following (1)-(3): (1) Enhancement by introducing a plasmid containing the gene; (2) Enhanced by increasing the copy number of the aforementioned genes on the rice chromosome; (3) Enhancement is achieved by altering the promoter sequence of the genes described on the rice chromosome.