Application of OsABCG6 gene in increasing rice yield
By cloning and overexpressing the rice OsABCG6 gene, and using the OsABCG6 gene overexpression vector and genetic transformation technology, the problem of insufficient regulation of rice tillering development was solved, rice yield was significantly increased, and genetic resources and breeding methods for new high-yield rice varieties were provided.
Patent Information
- Application Number
- CN202510760421.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-09
- Publication Date
- 2025-09-09
AI Technical Summary
Existing technologies lack effective means to regulate rice tillering development, resulting in the inability to increase rice yields to meet global food security needs.
By cloning and overexpressing the rice OsABCG6 gene, using the OsABCG6 gene overexpression vector and Agrobacterium-mediated genetic transformation technology, the activity or expression level of the OsABCG6 protein is increased, the rice tiller development is promoted, and the tiller number and yield are increased.
The OsABCG6 gene overexpression lines had more tillers, significantly improved yield, and no significant change in plant height, providing genetic resources and breeding methods for new high-yield rice varieties.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to application of rice OsABCG6 gene in improving rice yield. Background Art
[0002] Against the backdrop of continued global population growth and intensified food security challenges, increasing the yield of rice (Oryza sativa L.), a staple food crop, has become a strategic need to ensure national food security. From the perspective of yield components, rice yield is mainly regulated by the coordinated regulation of three key agronomic traits: the number of effective tillers, the number of grains per panicle, and grain weight. Among them, the number of effective tillers is an important factor determining yield stability due to its significant environmental plasticity and large genetic variation. Tillering development is a complex biological process involving signal transduction of multiple plant hormones (such as strigolactones, auxins, and cytokinins), the dynamic distribution of carbon and nitrogen nutrients, and the precise regulation of multi-level gene expression networks. To increase rice yield to meet global food security needs.
[0003] The ABCG transporter family (ATP-binding cassette subfamily G) is widely found in bacteria, fungi, plants, and animals, playing key roles in physiological processes such as transmembrane transport, drug efflux, lipid metabolism, and antioxidant defense. Prior research has reported that AtABCG16 mediates the efflux of jasmonic acid, AtABCG25 transports abscisic acid (ABA) and abscisic acid-glucose ester (ABA-GE) from roots to aerial tissues and regulates stomatal movement. In Arabidopsis, AtABCG14 controls the apical transport of cytokinins. ABCG36 and ABCG40, among others, play important roles in heavy metal resistance and cellular detoxification in Arabidopsis. However, their impact on rice development is currently unknown. This study used molecular biology techniques to investigate the effects of these transporters on rice yield, identify key functional genes regulating rice tillering, and, combined with molecular design breeding techniques, develop new rice varieties with high yield potential and superior agronomic traits, providing a theoretical basis and genetic resources for rice genetic improvement and variety innovation. Summary of the Invention
[0004] One of the objectives of the present invention is to provide an OsABCG6 gene for increasing rice yield.
[0005] A second object of the present invention is to provide the application of the above gene OsABCG6 in increasing rice yield.
[0006] In order to achieve the above object, the technical solution of the present invention is as follows:
[0007] The present invention has found a rice OsABCG6 gene, the gene sequence number (Sequence ID) of which is LOC_Os03g17370 in the Rice Gene Index, the CDS sequence of which is shown in SEQ ID NO.1, the coding sequence of the OsABCG6 gene is 2298 bp in length, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.
[0008] Through cloning and preliminary research on the rice OsABCG6 gene, the inventors discovered that OsABCG6 has a certain impact on rice yield. The rice OsABCG6 gene is positively correlated with rice yield; that is, overexpressing the OsABCG6 gene increases rice yield. This provides a genetic resource foundation for the cultivation of new high-yield rice varieties.
[0009] The present invention also constructs a series of plant expression vectors, and the functions of the expression vectors containing the above genes, transgenic plant lines and host cells containing the vectors in increasing rice yield also fall within the protection scope of the present invention.
[0010] Furthermore, those skilled in the art can readily mutate the OsABCG6 gene described herein using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the nucleotide sequence encoding the transporter protein OsABCG6 are derived from and are equivalent to the nucleotide sequence described herein, as long as they encode the same protein and have the same function. Primer pairs for amplifying the full-length coding sequence or fragments thereof encoding the OsABCG6 protein also fall within the scope of the present invention.
[0011] The main purpose of the present invention is to protect the above-mentioned OsABCG6 gene, the transport protein OsABCG6 encoded by the OsABCG6 gene, and the use of biological materials containing the above-mentioned coding sequence in improving rice yield.
[0012] The improvement in rice yield is specifically manifested in that, compared with the wild type, the OsABCG6 gene overexpression strain has better growth, more tillers, increased yield, and no significant change in plant height.
[0013] In order to increase crop yield, the present invention also protects a novel plant breeding method, which is selected from method (1) or method (2):
[0014] Method (1) is to increase the activity of OsABCG6 protein in the target plant to obtain a plant with a higher yield than the target plant;
[0015] Method (2) is to obtain plants with higher yield than the target plant by promoting the expression of the OsABCG6 gene in the target plant;
[0016] Method (2) is achieved by promoting the expression of the OsABCG6 gene in the target plant, which is selected from the following methods:
[0017] Method (1) is to introduce the OsABCG6 gene into the target plant;
[0018] Method (2) is to introduce a strong promoter and / or enhancer.
[0019] Method (3) includes other common methods in the art, including small RNA regulation, methylation / demethylation, phosphorylation / dephosphorylation, promoter binding site regulation, etc.
[0020] Among them, preferably, the target plant of the present invention is rice, more specifically the conventional japonica rice variety Zhonghua 11 (Oryza sativa ssp. japonica).
[0021] More specifically, the present invention discloses a method for increasing rice yield, comprising the following steps:
[0022] (1) Construction of rice OsABCG6 gene overexpression vector pC1300s-OsABCG6;
[0023] (2) Genetic transformation of rice: The plasmid containing the overexpression vector pC1300s-OsABCG6 was transformed into rice using Agrobacterium-mediated callus genetic transformation;
[0024] (3) Screening of positive plants: After PCR amplification verification, positive plants are screened to obtain rice plants with increased yield.
[0025] The method for constructing the overexpression vector pC1300s-OsABCG6 in step (1) is specifically as follows:
[0026] First, primers with restriction endonuclease Kpn I and BamHI sites were designed;
[0027] Second, PCR amplification is performed using the cDNA sample as a template, and the amplified product is purified and recovered;
[0028] Third, the pC1300s vector was double-digested with Kpn I and Bam HI, and the digestion product was purified;
[0029] Fourth, the PCR amplification product and the enzyme-digested vector were ligated by homologous recombination to construct the pC1300s-OsABCG6 overexpression vector;
[0030] Fifth, the ligation product was transformed into Escherichia coli DH5α using the heat shock transformation method and Spec + Resistance screening: select positive colonies for PCR detection, amplify the colonies identified correctly by PCR, send them for sequencing, and extract plasmids from the bacteria solution that are sequenced correctly for future use;
[0031] Sixth, the extracted plasmid was transformed into Agrobacterium competent cells GV3101.
[0032] Among them, the primer sequences with restriction endonuclease Kpn I and BamHI restriction enzyme sites are as follows:
[0033] OsABCG6-F:
[0034] 5′-GAGCTTTCGCGAGCTCGGTACCATGTCGAGTAGCTTTCGCAG-3′;
[0035] OsABCG6-R:
[0036] 5′-CTGCAGGTCGACTCTAGAGGATCCTCACCTCCTCCTGTTCCTG-3′.
[0037] In the above method, positive plants are screened by obtaining homozygous OsABCG6 gene-overexpressing plants from the progeny of OsABCG6 gene-overexpressing plants; homozygous OsABCG6 gene-overexpressing plants are plants with higher yields. The selfed progeny can be the progeny of the first selfing generation, the progeny of the second selfing generation, the progeny of the third selfing generation, and so on, until homozygous OsABCG6 gene-overexpressing plants are obtained. The lines of the selfed progeny of the homozygous OsABCG6 gene-overexpressing plants are high-yield rice lines.
[0038] The above-mentioned method for increasing rice yield is also applicable to other recipient plants that share homologous genes with rice. There are no particular limitations on the recipient plants suitable for the present invention, including not only rice but also other plants with high homology, as long as they are suitable for gene transformation, such as various crops, flowers, or forestry plants. Examples of such plants include (but are not limited to): dicots, monocots, woody plants, plants of the Rosales order, plants of the Rosaceae family, Prunus, peaches, cruciferous plants, Arabidopsis, and Arabidopsis thaliana.
[0039] As used herein, "plant" includes the entire plant, its parent and progeny plants, and various parts of the plant, including seeds, fruits, stems, buds, leaves, roots, flowers, tissues, and organs, all of which contain the gene or nucleic acid of interest. "Plant" as used herein also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, and pollen, each of which may contain the gene / nucleic acid of interest.
[0040] The present invention encompasses any plant cell, or any plant obtained or obtainable by any of the methods herein, and all plant parts and propagules thereof. The present invention also encompasses transfected cells, tissues, organs, or whole plants obtained by any of the aforementioned methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics, and that the progeny obtained using the methods of the present invention have the same characteristics.
[0041] The present invention also extends to the harvestable parts of the plants described above, but is not limited to seeds, leaves, flowers, stems and roots. It further relates to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starch or protein. The present invention also relates to foods or food additives obtained from the plants. Advantages of the present invention:
[0042] During the research process, the present invention discovered a new OsABCG6 gene that regulates plant yield in rice. Experiments have shown that compared with the wild type, the OsABCG6 gene overexpression strain has better growth, more tillers, higher yield, and no significant change in plant height. In other words, the development of the OsABCG6 gene overexpression strain is significantly better than that of the wild type material, indicating that the OsABCG6 gene plays an important regulatory function in the process of improving rice yield, which has important guiding significance for the genetic improvement of rice. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure 1 Schematic diagram of the OsABCG6 overexpression vector construction. LB: left border, RB: right border.
[0044] Figure 2 This is the detection result of the transcription level of OsABCG6 in the OsABCG6 overexpression line.
[0045] Figure 3 Comparison of the growth phenotypes of OsABCG6-overexpressing strains and wild-type plants. Panel A shows the growth phenotype of wild-type plants. Panel B shows the growth phenotype of OsABCG6-overexpressing strains. Scale bar = 20 cm.
[0046] Figure 4 is the single plant yield of OsABCG6 overexpressing lines and wild-type plants.
[0047] Figure 5 is the tiller number of OsABCG6 overexpression lines and wild-type plants.
[0048] Figure 6 is the plant height of OsABCG6 overexpression lines and wild-type plants. DETAILED DESCRIPTION
[0049] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. However, the specific experimental methods involved in the following examples, unless otherwise specified, are all conventional methods or are performed under the conditions recommended by the manufacturer's instructions.
[0050] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be purchased from the market.
[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.
[0052] As used herein, the terms "nucleic acid," "nucleic acid sequence," "nucleotide," "nucleic acid molecule," or "polynucleotide" are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences in non-coding regions. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in a genomic sequence, and / or include coding sequences in a cDNA, and / or include cDNA and its regulatory sequences. In specific embodiments, such as with respect to isolated nucleic acid sequences, it is preferably assumed to be cDNA.
[0053] Unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.
[0054] During their research, the inventors discovered a new rice yield-related gene in rice, named OsABCG6. The gene is listed in the Rice Gene Index I database as OsABCG6 and its sequence ID is LOC_Os03g17370. The CDS sequence of the OsABCG6 gene in rice is shown in SEQ ID NO. 1 in the sequence listing. The protein encoded by OsABCG6 is named OsABCG6 protein, and the amino acid sequence of the OsABCG6 protein in rice is shown in SEQ ID NO. 2 in the sequence listing.
[0055] Example 1 Cloning of OsABCG6 gene
[0056] RNA was extracted from leaves of wild-type rice Zhonghua 11 and reverse transcribed into cDNA. The gene sequence obtained from the Rice Gene Index I database was analyzed by Oligo Analyzer. TM Tool-PrimerAnalysis was used to design specific primers. Using cDNA as a template, the coding sequence of the OsABCG6 gene (shown as SEQ ID NO. 1) was cloned through PCR reaction using the high-fidelity enzyme Primerstar.
[0057] Example 2 Analysis of the expression pattern of OsABCG6
[0058] In order to analyze the expression pattern of OsABCG6, the inventors constructed an OsABCG6 overexpression vector pC1300s-OsABCG6 ( Figure 1 The specific process is briefly described as follows.
[0059] First, primers with restriction endonuclease Kpn I and BamHI cleavage sites were designed. The sequences are as follows:
[0060] OsABCG6-F:
[0061] 5′-GAGCTTTCGCGAGCTCGGTACCATGTCGAGTAGCTTTCGCAG-3′;
[0062] OsABCG6-R:
[0063] 5′-CTGCAGGTCGACTCTAGAGGATCCTCACCTCCTCCTGTTCCTG-3′;
[0064] Second, PCR amplification was performed using the cDNA sample prepared in Example 1 as a template, and the amplified product was purified and recovered;
[0065] Third, the pC1300s vector was double-digested with Kpn I and Bam HI, and the digestion product was purified;
[0066] Fourth, the PCR amplification product and the enzyme-digested vector were ligated by homologous recombination to construct the pC1300s-OsABCG6 overexpression vector;
[0067] Fifth, the ligation product was transformed into Escherichia coli DH5α using the heat shock transformation method and Spec + (Spectinomycin, 75 μg / mL) resistance screening, select positive colonies for PCR detection, amplify the colonies identified correctly by PCR, send them for sequencing, and extract plasmids from the bacteria solution with correct sequencing for later use;
[0068] Sixth, the extracted plasmid was transformed into Agrobacterium competent cells GV3101.
[0069] Example 3 Overexpression of rice to verify the function of OsABCG6 gene in rice
[0070] To obtain homozygous transgenic rice materials, the wild-type rice Zhonghua 11 (ZH11, WT) was transformed using Agrobacterium-mediated callus genetic transformation. Based on the above Example 2, after the Agrobacterium containing the recombinant vector was cultured for 2-3 days, a single colony was picked and transferred to YEP culture medium and cultured at 28°C and 220 rpm for about 14 hours. After positive identification, the bacterial solution was inoculated into 200 mL of YEP culture medium for propagation. The bacterial solution was incubated until the OD 600 = 1.2, collect Agrobacterium by centrifugation at 3800 rpm at room temperature, and resuspend the bacteria in a resuspension solution to OD 600 =0.8. Agrobacterium containing the recombinant plasmid was used to infect mature embryonic callus of rice Zhonghua 11 and cultured in the dark at 28°C for 2-3 days. The callus was washed with cephalosporin and transferred to a screening medium containing kanamycin (50 mg / L) to obtain resistant calli. Subsequently, differentiation (6-BA+NAA) and rooting (IBA) were induced by hormones to regenerate complete plants (T0 generation). The integration and expression levels of the exogenous gene were verified by PCR and qRT-PCR. Finally, stable genetic strains were selected in the T1 generation, and their phenotypic differences with the wild type (such as tiller number, plant height, yield, etc.) were analyzed.
[0071] The expression levels of OsABCG6 in different plants overexpressing OsABCG6 created by the pC1300s-OsABCG6 system were detected by qRT-PCR. The results showed that the expression levels of the four transgenic rice lines tested were all more than 15 times higher than those of the control group ( Figure 2 ), these results indicate that transgenic rice plants overexpressing OsABCG6 have been successfully generated.
[0072] The obtained overexpression lines were planted in the field, and water and fertilizer management was unified. The indicators of tiller number and plant height were investigated during the tillering and heading stages. Plant height was measured from the natural height of the stem base to the highest panicle top, and the number of tillers counted the main stem and effective tillers with more than 3 leaves. The yield trait survey was carried out at maturity, mainly measuring the thousand-grain weight, and selecting full grains for weighing. The results showed that rice plants overexpressing OsABCG6 grew better, the number of tillers was significantly higher than that of ZH11 (WT), and the yield was significantly increased, but the plant height did not change significantly. This result shows that OsABCG6 is a key gene that regulates rice yield. It can positively regulate tiller development, thereby increasing rice yield. At the same time, its overexpression does not change the plant height, which gives it a unique advantage in high-yield breeding ( Figure 3-6 ).
[0073] In summary, OsABCG6-overexpressing lines showed improved growth, increased tillering, and higher yield compared to wild-type plants, with no significant change in plant height. This suggests that OsABCG6 positively regulates tillering development in rice, thereby increasing yield. This suggests that OsABCG6 plays an important role in regulating rice yield and could provide a valuable genetic resource foundation for developing new high-yield rice varieties using molecular genetic breeding techniques.
[0074] The embodiments described above are merely preferred embodiments of the present invention and are intended only to explain the present invention, not to limit the scope of implementation of the present invention. Those skilled in the art can, of course, easily make other implementations by replacing or changing the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of application of the present invention.
Claims
1. Application of OsABCG6 gene in increasing rice yield and rice production, characterized in that: The CDS sequence of the OsABCG6 gene is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that The increase in rice yield is specifically manifested as follows: compared with the wild type, the OsABCG6 overexpression strain has better growth, more tillers, increased yield, and no significant change in plant height.
3. Use of an overexpression vector containing the OsABCG6 gene according to claim 1 in increasing rice yield.
4. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the OsABCG6 gene is shown in SEQ ID NO.
2.
5. A plant breeding method, characterized in that: The method is selected from method (1) or method (2): Method (1) is to increase the activity of OsABCG6 protein in the target plant to obtain a plant with a higher yield than the target plant; Method (2) is to obtain plants with higher yield than the target plant by promoting the expression of the OsABCG6 gene in the target plant; The CDS sequence of the OsABCG6 gene is shown in SEQ ID NO.1, and the amino acid sequence of the OsABCG6 protein is shown in SEQ ID NO.2; and the target plant is rice.
6. The breeding method according to claim 5, characterized in that Method (2) is achieved by promoting the expression of the OsABCG6 gene in the target plant, which is selected from the following method (1) or method (2): Method (1) is to introduce the OsABCG6 gene into the target plant; Method (2) is to introduce a strong promoter and / or enhancer.
7. A method for increasing rice yield, characterized in that: The method comprises the following steps: (1) Construction of rice OsABCG6 gene overexpression vector pC1300s-OsABCG6; (2) Genetic transformation of rice: The plasmid containing the overexpression vector pC1300s-OsABCG6 was transformed into rice using Agrobacterium-mediated callus genetic transformation; (3) Screening of positive plants: After PCR amplification verification, positive plants are screened to obtain rice plants with increased yield.
8. The method for increasing rice yield according to claim 7, characterized in that: The method for constructing the overexpression vector pC1300s-OsABCG6 in step (1) is specifically as follows: First, primers with restriction endonuclease Kpn I and BamHI sites were designed; Second, PCR amplification is performed using the cDNA sample as a template, and the amplified product is purified and recovered; Third, the pC1300s vector was double-digested with Kpn I and Bam HI, and the digestion product was purified; Fourth, the PCR amplification product and the enzyme-digested vector were ligated by homologous recombination to construct the pC1300s-OsABCG6 overexpression vector; Fifth, the ligation product was transformed into Escherichia coli DH5α using the heat shock transformation method and Spec + Resistance screening: select positive colonies for PCR detection, amplify the colonies identified correctly by PCR, send them for sequencing, and extract plasmids from the bacteria solution that are sequenced correctly for future use; Sixth, the extracted plasmid was transformed into Agrobacterium competent cells GV3101.
9. The method for increasing rice yield according to claim 8, characterized in that: The primer sequences with restriction endonuclease Kpn I and BamHI restriction sites are as follows: OsABCG6-F: 5′-GAGCTTTCGCGAGCTCGGTACCATGTCGAGTAGCTTTCGCAG-3′: OsABCG6-R: 5′-CTGCAGGTCGACTCTAGAGGATCCTCACCTCCTCCTGTTCCTG-3′.
10. The method for increasing rice yield according to claim 8, characterized in that: The method for screening positive plants is: obtaining homozygous OsABCG6 gene overexpression plants from the self-pollinated progeny of OsABCG6 gene overexpression plants; homozygous OsABCG6 gene overexpression plants are plants with higher yield.