Application of OsABCG21 transporter in regulation and control of tillering and yield of rice

Abstract

The invention discloses an application of an OsABCG21 transporter in regulation and control of tillering and yield of rice, an amino acid sequence of the OsABCG21 transporter is shown as SEQ ID NO.1, a gene for coding the OsABCG21 transporter is named as OsABCG21, and a CDS sequence of the OsABCG21 gene is shown as SEQ ID NO.2. The invention further discloses an application of the OsABCG21 transporter in regulation and control of tillering and yield of rice. According to the application, an OsABCG21 gene overexpression plant is obtained by constructing an OsABCG21 overexpression vector. Based on observation of phenotype of the overexpressed strain, the inventor preliminarily determines that the OsABCG21 gene is positively correlated with growth and development of rice and regulates tillering. Compared with a wild type, the rice plant overexpressed with the OsABCG21 gene has the advantages that the growth vigor is better, the tillering is increased, and the yield is increased. It is shown that the OsABCG21 gene plays an important role in positive regulation and control of rice tillering and yield. The invention lays a good gene genetic resource foundation for the cultivation of a new high-yield rice variety.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of OsABCG21 transporter in regulating rice tillering and yield. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, and increasing its yield is crucial for ensuring food security. Rice yield is primarily determined by three key agronomic traits: effective tiller number, grain number per panicle, and 1000-grain weight. Tiller number, due to its remarkable phenotypic plasticity and high heritability, is an important target for yield regulation. Tiller development involves a complex regulatory network, encompassing processes such as plant hormone signaling, nutrient distribution, and the coordinated regulation of multiple genes.

[0003] The ABCG transporter family (ATP-binding cassette subfamily G), the largest membrane transporter subfamily in plants, participates in regulating plant growth and development by mediating the transmembrane transport of a variety of bioactive molecules, such as phytohormones and secondary metabolites. However, the functional role of ABCG family members in rice tillering development and yield formation is still lacking. This study integrates molecular biology and bioengineering techniques to identify key OsABCG family genes that regulate rice tillering. This study provides a theoretical basis and genetic resources for molecular design breeding of high-yield and high-quality rice varieties, and aims to develop new rice varieties with superior tillering characteristics and high-yield potential through genetic engineering. Summary of the Invention

[0004] One of the objectives of the present invention is to provide a transporter protein OsABCG21 that regulates rice tillering development and thus affects yield.

[0005] A second object of the present invention is to provide the use of the above-mentioned transport protein OsABCG21 in increasing the tiller number and yield of rice.

[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 OsABCG21 gene, which has a gene sequence number (Sequence ID) of LOC_Os09g23640 in the Rice Gene Index, a CDS sequence as shown in SEQ ID NO.2, a CDS sequence length of 2625 bp, and an amino acid sequence as shown in SEQ ID NO.1, comprising 874 amino acids.

[0008] Through cloning and preliminary research on the rice gene OsABCG21, the inventors discovered that OsABCG21 has a certain impact on rice yield. The rice gene is positively correlated with rice yield; that is, overexpression of the gene increases rice yield. This provides a genetic resource foundation for the development 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] The main purpose of the present invention is to protect the above-mentioned transport protein OsABCG21, the OsABCG21 gene encoding the transport protein OsABCG21, and the use of biological materials containing the above-mentioned coding sequence in increasing the tiller number and yield of rice.

[0011] The improvement in rice tiller number and yield is specifically manifested in that, compared with the wild type, the OsABCG21 gene overexpression strain has better growth, more tillers, increased yield, and no significant change in plant height.

[0012] In order to increase crop yield, the present invention also protects a new breeding method for increasing the number of rice tillers and yield, wherein the method is to obtain plants with a higher number of crop tillers and yield than the target plant by promoting the expression of the OsABCG21 gene in the target plant.

[0013] The method of promoting the expression of the OsABCG21 gene in the target plant is to overexpress or superexpress the OsABCG21 gene.

[0014] The target plant of the present invention is a conventional japonica rice variety Zhonghua 11 (Oryza sativa ssp. japonica).

[0015] The target gene, also known as the target gene, is used in genetic engineering design and manipulation to achieve gene recombination, alter the characteristics of recipient cells, and obtain the desired expression product. It can be native to the organism or from a different organism.

[0016] Furthermore, those skilled in the art can readily mutate the OsABCG21 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 OsABCG21 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 OsABCG21 protein also fall within the scope of the present invention.

[0017] In the above-mentioned method for increasing rice tiller number and yield, the OsABCG21 gene is overexpressed in the plant genome to obtain OsABCG21 gene-overexpressing plants, and homozygous OsABCG21 gene-overexpressing plants are obtained from the progeny of the OsABCG21 gene-overexpressing plants; the homozygous OsABCG21 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 a homozygous OsABCG21 gene-overexpressing plant is obtained. The line of selfed progeny of the homozygous OsABCG21 gene-overexpressing plants is a rice line with increased tiller number and high yield.

[0018] The above-mentioned method for increasing rice tiller number and 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, and the plants include 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.

[0019] 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.

[0020] The present invention encompasses any plant cell, or any plant obtained or obtainable by any of the methods herein, as well as all plant parts and propagules thereof. Transfected cells, tissues, organs, or whole plants obtained by any of the aforementioned methods are also encompassed by this patent. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics, and that the progeny obtained using the methods described herein have the same characteristics.

[0021] 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:

[0022] The present invention discovered a new OsABCG21 transporter protein in rice that regulates crop tillering and yield during the research process, and experimentally proved that compared with the wild type, the OsABCG21 gene overexpression strain had better growth, more tillers, higher yield, and no significant change in plant height. In other words, the development of the OsABCG21 gene overexpression strain was significantly better than that of the wild type material, indicating that the OsABCG21 gene plays an important regulatory function in the tillering development and formation process of rice, and has important guiding significance for the genetic improvement of high-yield rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the detection result of the transcription level of OsABCG21 in the OsABCG21 overexpression line.

[0024] Figure 2 Comparison of the growth phenotypes of OsABCG21-overexpressing and wild-type plants. Panel A shows the growth phenotype of the wild-type plant; Panel B shows the growth phenotype of the OsABCG21-overexpressing plant. Scale bar = 20 cm.

[0025] Figure 3 is the single plant yield of OsABCG21 overexpressing lines and wild-type plants.

[0026] Figure 4 is the tiller number of OsABCG21 overexpression lines and wild-type plants.

[0027] Figure 5 is the plant height of OsABCG21 overexpression lines and wild-type plants. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] During their research, the inventors discovered a new transporter protein in rice that regulates tillering development in crops. The protein was named OsABCG21, and its amino acid sequence is shown in SEQ ID NO. 1 in the sequence listing. The gene encoding the OsABCG21 transporter protein was named OsABCG21, and its sequence ID in the Rice Gene Index I database is LOC_Os09g23640. The CDS of the OsABCG21 gene in rice is shown in SEQ ID NO. 2 in the sequence listing.

[0034] Example 1 Cloning of OsABCG21 gene

[0035] RNA was extracted from wild-type rice leaves and reverse transcribed into cDNA. The gene sequences obtained from the Rice Gene Index I database were analyzed by Oligo Analyzer. TM Tool-PrimerAnalysis was used to design specific primers. Using cDNA as a template, the high-fidelity enzyme Primerstar was used to perform PCR reaction to clone the CDS sequence of the OsABCG21 gene (as shown in SEQ ID NO. 2).

[0036] Example 2 Analysis of the expression pattern of OsABCG21

[0037] To analyze the expression pattern of OsABCG21, the inventors constructed an OsABCG21 overexpression vector pC1300s-OsABCG21. The specific process is briefly described below.

[0038] First, primers with restriction endonuclease KKpn I and BamHI restriction sites were designed. The sequences are as follows:

[0039] OsABCG21-F:

[0040] 5′-GAGCTTTCGCGAGCTCGGTACCATGCAGCAGCAGCAGC-3′;

[0041] OsABCG21-R:

[0042] 5′-CTGCAGGTCGACTCTAGAGGATCCTCACTTCCTCTCATTCTTGGAG-3′;

[0043] Second, PCR amplification was performed using the cDNA sample prepared in Example 1 as a template, and the amplified product was purified and recovered;

[0044] Third, the pC1300s vector was double-digested with Kpn I and Bam HI, and the digestion product was purified;

[0045] Fourth, the PCR amplification product and the enzyme-digested vector were ligated by homologous recombination to construct the pC1300s-OsABCG21 overexpression vector;

[0046] 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;

[0047] Sixth, the extracted plasmid was transformed into Agrobacterium competent cells GV3101.

[0048] Example 3 Overexpression of rice to verify the function of OsABCG21 gene in rice

[0049] To obtain homozygous transgenic rice materials, the wild-type rice Zhonghua 11 (ZH1 1, WT) was transformed using Agrobacterium-mediated callus genetic transformation. Based on the above Example 2, after culturing the Agrobacterium containing the recombinant vector 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 OD 600 = 1.2, collect Agrobacterium by centrifugation at 3800 rpm at room temperature, and resuspend the bacteria in 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.

[0050] The expression levels of OsABCG21 in different plants overexpressing OsABCG21 created by the pC1300s-OsABCG21 system were detected by qRT-PCR. The results showed that the expression levels of the two transgenic Arabidopsis lines tested were more than 8 times ( Figure 1 ), these results indicate that transgenic rice plants overexpressing OsABCG21 have been successfully generated.

[0051] 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 OsABCG21 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 indicates that OsABCG21 is a key transport protein that regulates rice tillering, positively regulating 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 2-5 ).

[0052] In summary, OsABCG21-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 OsABCG21 positively regulates tillering development in rice, thereby promoting yield. This suggests that the OsABCG21 transporter plays an important role in increasing rice yield and provides a valuable genetic resource foundation for developing new high-yield rice varieties using molecular genetic breeding techniques.

[0053] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. Application of OsABCG21 transporter in regulating rice tillering and yield, characterized in that: The amino acid sequence of the OsABCG21 transporter is shown in SEQ ID NO.

1. The OsABCG21 transporter can increase the number of rice tillers and improve rice yield.

2. The use according to claim 1, characterized in that The increase in rice tiller number and rice yield is specifically manifested in that, compared with the wild type, the OsABCG21 gene overexpression strain has better growth, more tillers, increased yield, and no significant change in plant height.

3. Use of a gene expression cassette, a recombinant vector, a recombinant microorganism, or a transgenic plant cell line containing the OsABCG21 gene for increasing the number of rice tillers and improving rice yield, characterized in that: The CDS sequence of the OsABCG21 gene is shown in SEQ ID NO.

2.

4. A method for increasing the number of rice tillers and improving rice yield, characterized in that: Transgenic rice plants with increased tiller number and yield are obtained by promoting the expression of the OsABCG21 gene in rice. The CDS sequence of the OsABCG21 gene is shown in SEQ ID NO.

2.

5. The method according to claim 4, characterized in that The method of promoting the expression of OsABCG21 gene in rice is overexpression or overexpression.

6. The method according to claim 5, characterized in that The OsABCG21 gene in the plant genome is overexpressed to obtain OsABCG21 gene-overexpressing plants, and homozygous OsABCG21 gene-overexpressing plants are obtained from the self-pollinated progeny of the OsABCG21 gene-overexpressing plants; the homozygous OsABCG21 gene-overexpressing plants are rice plants with more tillers and higher yield.

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