Rice constitutive promoter OSCAX1a and application thereof
By developing the rice constitutive promoter OsCAX1a, the problem of lack of constitutive promoter in rice is solved, the stable expression of exogenous genes in various rice tissues is achieved, and the effect of transgenic breeding is improved.
Patent Information
- Application Number
- CN202510665626.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-22
- Publication Date
- 2025-08-15
AI Technical Summary
The lack of effective constitutive promoters in rice leads to problems with transgene silencing and overexpression, affecting the effectiveness of transgene breeding.
A rice constitutive promoter OsCAX1a and its application were developed. The promoter sequence was isolated from the rice japonica variety ZH11 by designing primers and ligating it with the target gene to construct a recombinant vector. The constitutive expression was achieved in rice plants using Agrobacterium mediated method.
The stable and constitutive expression of exogenous genes in various rice tissues has been achieved, the disease resistance, stress resistance and yield have been improved, and the application potential of promoters in rice transgenic breeding has been verified.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a rice constitutive promoter, a recombinant adeno-associated virus and applications thereof. Background Art
[0002] During the long process of growth and development, organisms have evolved the ability to selectively and programmatically regulate the appropriate expression of related genes to adapt to environmental changes. Gene expression regulatory mechanisms enable organisms to flexibly respond to complex environments. From single-celled organisms such as viruses and bacteria to multicellular organisms and even higher mammals, gene expression exhibits strict temporal and spatial specificity. The higher the species, the more complex and sophisticated the expression patterns. This precise regulatory process is achieved through the interaction between the promoter sequence or enhancer of a specific gene and regulatory proteins.
[0003] Promoters, located in the noncoding region upstream of the DNA coding region, are DNA sequences that recognize and bind to the RNA transcription initiation complex, thereby determining the start site of transcription. They are crucial components of gene expression, regulating gene activity in various ways. They can provide temporal and spatial regulation of transgenic expression in plants, and various types of promoters have been identified over the years for use in plant transgenesis in diverse organisms. Gene expression depends largely on the use and effective selection of promoters, and even the same promoter can drive the expression of multiple genes in a single plant. In modern biotechnology, promoters are primarily divided into four categories: constitutive promoters, tissue-specific promoters, inducible promoters, and synthetic promoters. Among them, constitutive promoters are the earliest and most widely used promoters in plant genetic engineering. They can enable efficient, stable and long-lasting expression of foreign genes in plants. Typical representatives include the 35S promoter of cauliflower mosaic virus (CaMV) (Zheng, X., Deng, W., Luo, K. et al. The cauliflower mosaic virus (CaMV)35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Rep 26,), the maize ubiquitin promoter (Anonymous, et al. (2017). Maize ubiquitin promoters. Official Gazette of the United States Patent and Trademark Office), and the rice actin promoter (Anonymous, et al. (2016). Chimeric promoters comprising a rice Actin 1 promoter and 35Senhancers for use in plants. Official Gazette of the United
[0004] Constitutive promoters are mainly used in plant transformation to promote the expression of transgenic genes with different purposes. Their characteristic is that they maintain gene control during most of the plant development process, and the expression level depends on the cell type in which they work (Villao-Uzho L, Chávez-Navarrete T, Pacheco-Coello R, Sánchez-Timm E,Santos-Ordóñez E. Plant Promoters: Their Identification, Characterization,and Role in Gene Regulation. Genes. 2023; 14(6):1226.). The use of constitutive promoters has driven a significant increase in the yield and resistance of transgenic crops, resulting in many crop varieties with important economic value, and promoting research in many areas of plant biology, such as gene function, hormone signaling, disease resistance mechanisms, metabolic pathways, developmental regulation and epigenetics. However, the fact that genes driven by constitutive promoters are expressed in the same way in different tissues also brings some problems, such as transgene silencing, excessive accumulation of certain proteins leading to crop poisoning, and crop deformity or even death due to overexpression (Zhang, CX., et al. (2004). "Review on plant gene promoters." Acta GeneticaSinica 31(12):).
[0005] To avoid the potential for gene silencing or co-suppression caused by using the same promoter to drive two or more exogenous genes simultaneously, different promoters are generally required to drive different exogenous genes, screening genes, and reporter genes. Although some constitutive promoters have been isolated and identified in previous studies, reports on constitutive promoters in rice are relatively rare, far from matching the increasing number of genes. Therefore, this invention is of great significance to transgenic rice breeding in my country.
[0006] Therefore, it is necessary to develop a promoter that can drive the constitutive expression of exogenous genes in rice tissues, which is of great significance to my country's rice transgenic breeding. Summary of the Invention
[0007] The purpose of the present invention is to provide a rice constitutive promoter and its application. The promoter can guide the constitutive expression of exogenous genes in various rice tissues, laying a foundation for the practical application of the promoter.
[0008] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions: In a first aspect of the present invention, a rice constitutive promoter is provided, the sequence of which is shown in SEQ ID NO.1 or any one of the following: a) a nucleotide sequence having at least 80% homology to the nucleotide sequence of SEQ ID NO:1 and having the same constitutive promoter function; b) a mutant, allele or derivative generated by adding, substituting, inserting or deleting one or more nucleotides to the nucleotide sequence of SEQ ID NO:1, and having the same constitutive promoter function as SEQ ID NO:1.
[0009] Furthermore, the promoter can drive the constitutive expression of exogenous genes in rice tissues.
[0010] In a second aspect of the present invention, a recombinant vector is provided, comprising the rice constitutive expression promoter OsCAX1a, wherein the promoter is operably linked to the upstream of a target gene.
[0011] Furthermore, the target gene is a reporter gene, a disease resistance gene, a stress resistance gene or a metabolic regulation gene.
[0012] In a third aspect of the present invention, a set of primer pairs for amplifying the rice constitutive expression promoter OsCAX1a is provided, and the nucleotide sequences of the primer pairs are shown in SEQ ID NO: 2 to SEQ ID NO: 3.
[0013] In a fourth aspect of the present invention, a rice constitutive promoter function verification kit is provided, comprising the primer pair.
[0014] In the fifth aspect of the present invention, a method for regulating the expression of exogenous genes using the rice constitutive expression promoter OsCAX1a is provided, the method comprising: connecting the promoter with the target gene to construct a recombinant vector, and transforming it into rice plants through Agrobacterium-mediated method to drive the constitutive expression of the target gene in various rice tissues.
[0015] In a sixth aspect of the present invention, there is provided the use of the rice constitutive expression promoter in regulating or initiating constitutive expression of a target heterologous gene in rice tissue.
[0016] In a seventh aspect of the present invention, there is provided the use of the rice constitutive expression promoter OsCAX1a in breeding transgenic rice varieties, wherein the use includes improving disease resistance, stress resistance, yield or metabolite accumulation.
[0017] Furthermore, the target gene is a β-glucuronidase gene, which is used to verify the constitutive expression function of the promoter.
[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: The constitutively expressed rice promoter of the present invention was isolated from the japonica rice variety ZH11 using PCR using designed primers and a 1614-bp sequence upstream of the OsCAX1a gene as a template. Expression-specific experiments in rice demonstrated that the promoter directed constitutive expression of the β-actin glucuronidase (GUS) reporter gene in rice tissues. This demonstrates that the promoter of the present invention can drive constitutive expression of exogenous genes in rice tissues. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 :DX2181 plasmid map.
[0019] Figure 2 : Staining results of the GUS gene driven by the OsCAX1a promoter in various tissues of transformed plants. A: old leaves, B: new leaves, C: stems, D: old roots, E: new roots, F: panicles, G: anthers, H: leaf sheaths. DETAILED DESCRIPTION
[0020] The following will be combined with specific implementation methods and examples to specifically describe embodiments of the present invention, and the advantages and various effects of the embodiments of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementation methods and examples are used to illustrate embodiments of the present invention, rather than to limit the embodiments of the present invention.
[0021] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the embodiments of the present invention belong. In the event of any conflict, the present specification shall take precedence.
[0022] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be purchased on the market or obtained through existing methods.
[0023] The water-soluble self-assembled chitosan nanocomposite and its preparation method of the present application will be described in detail below with reference to examples and experimental data.
[0024] Example 1: Cloning of the promoter OsCAX1a and construction of a GUS fusion vector 1. Experimental Methods 1. Primer design and PCR amplification: Using ZH11 genomic DNA as a template, the OsCAX1a promoter was amplified using the forward primer: 5'-GAGAAGCAGTACTCGATGCCTTCAT-3' (SEQ ID NO. 2) and the reverse primer: 5'-CACCACTAGATCAAATTCGATCCCC-3' (SEQ ID NO. 3). The amplified fragment was a 1614 bp sequence upstream of OsCAX1a. BamH The OsCAX1a promoter sequence was cloned into the promoter-fusion GUS vector DX2181 by seamless ligation (homologous recombination) and then introduced into the normal rice variety ZH11.
[0025] In this study, when using genomic DNA or cDNA as template amplification, high-fidelity DNA polymerase Phanta Max Super-Fidelity DNA Polymerase (Norwegian) was used to ensure high fidelity and reduce mutations during PCR amplification. The PCR reaction system used (10 μL) was: Table 1. PCR reaction system
[0026] The PCR amplification program was as follows: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, and 72°C for 45 s, for 36 cycles; and 72°C for 5 min.
[0027] Sequencing of the PCR amplified product showed that its sequence was as shown in SEQ ID NO.1.
[0028] 2. Vector construction and verification: The PCR product was digested with Bam-HI and then linked to the GUS reporter vector DX2181-G1 (purchased from Wuhan Tianwen Biotechnology Co., Ltd.) by homologous recombination. Figure 1 ), construct the recombinant plasmid pOsCAX1a::GUS.
[0029] The recombinant plasmid was transformed into Escherichia coli DH5α, and a single colony was picked for sequencing verification.
[0030] 3. Experimental results Agarose gel electrophoresis showed that the size of the amplified fragment was 1614 bp, which was consistent with the length of the target promoter.
[0031] Sequencing comparison confirmed that the cloned sequence was completely consistent with SEQ ID NO: 1, with no base mutation.
[0032] Enzyme digestion verification showed that the recombinant plasmid was inserted in the correct direction and the pOsCAX1a::GUS vector was successfully constructed.
[0033] Example 2: Obtaining transgenic rice plants and hygromycin screening 1. Experimental Methods 1. Agrobacterium transformation and plant regeneration: The pOsCAX1a::GUS plasmid was transformed into Agrobacterium tumefaciens EHA105 by electroporation and plated on YEP plates containing kanamycin (50 mg / L) and rifampicin (25 mg / L) for selection.
[0034] Positive Agrobacterium was used to infect rice ZH11 callus, and after co-cultivation, the callus was transferred to a selection medium containing hygromycin (50 mg / L) and cultured for 4 weeks to obtain resistant calli.
[0035] Induce regeneration in differentiation medium to obtain pOsCAX1a::GUS , T0 generation transgenic plants.
[0036] 2. Nutrient solution culture and screening: T0 generation seeds were soaked for 3-4 days until they turned white, and sown in a 96-well plate with a clean bottom after germination for 1 day. At the 3-leaf and 1-heart stage, they were transplanted into a rice nutrient solution containing hygromycin (50 mg / L) and cultured for 3 days to eliminate negative plants.
[0037] Nutrient solution formula (pH 5.4): 1.44 mmol / L NH4NO3, 0.3 mmol / L NaH2PO4, 0.5 mmol / L K2SO4, 1.0 mmol / L CaCl2, 1.6 mmol / L MgSO4, 0.17 mmol / L Na2SiO3, 50 μmol / L Fe(II)-EDTA, 0.06 μmol / L (NH4)6Mo7O 24 , 15 μmol / L H3BO3, 8 μmol / L MnCl2, 0.12 μmol / L CuSO4 / ZnSO4, 29 μmol / L FeCl3, 40.5 μmol / L citric acid.
[0038] 2. Experimental Results After hygromycin selection, approximately 75% of the seedlings survived, indicating that the positive plants successfully integrated the hygromycin resistance gene.
[0039] Thirty independent T0 transgenic plants were obtained by greenhouse soil culture, and PCR detection confirmed that the pOsCAX1a::GUS vector was stably integrated into the rice genome.
[0040] Example 3: Verification of constitutive expression of the OsCAX1a promoter (GUS staining) 1. Experimental Methods 1. GUS histochemical staining: Roots, stems, leaves (old and new leaves), glumes, anthers, and leaf sheaths of T0 transgenic plants were taken and immersed in GUS staining solution (containing 1 mM X-Gluc, 0.1% Triton X-100, and 50 mM phosphate buffer, pH 7.0), and incubated at 37°C in the dark for 24 h.
[0041] 2. Decolorization: Wash with 70% ethanol until there is no background staining in the negative control (wild-type ZH11).
[0042] 3. Microscopic observation: Observe the distribution of blue precipitate under a stereomicroscope to evaluate the expression level of GUS gene in various tissues.
[0043] 2. Experimental Results The GUS staining results of pOsCAX1a::GUS transgenic plants are shown in Figure 2. Figure 2 As shown, pOsCAX1a::GUS has obvious GUS signals in old leaves, new leaves, old roots, new roots, stems, lemmas, anthers, leaf sheaths and other parts. These results indicate that the pOsCAX1a promoter can drive the constitutive expression of related genes in rice tissues, which is of great significance to transgenic rice breeding in my country.
[0044] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0045] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.
[0046] Obviously, those skilled in the art may make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Thus, if such changes and modifications fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are intended to include such changes and modifications.
Claims
1. A rice constitutive promoter OsCAX1a, characterized in that The sequence of the promoter is as shown in SEQ ID NO.1 or any one of the following: a) a nucleotide sequence that has at least 80% homology to the nucleotide sequence of SEQ ID NO:1 and has the same constitutive promoter function; b) a mutant, allele or derivative generated by adding, substituting, inserting or deleting one or more nucleotides to the nucleotide sequence of SEQ ID NO:1, and has the same constitutive promoter function as SEQ ID NO:
1.
2. A rice constitutive promoter according to claim 1, characterized in that The promoter can drive the constitutive expression of foreign genes in rice tissues.
3. A recombinant vector, characterized in that The method comprises the rice constitutive expression promoter OsCAX1a according to claim 1 or 2, and the promoter is operably linked to the upstream of the target gene.
4. The recombinant vector according to claim 3, characterized in that The target gene is a reporter gene, a disease resistance gene, a stress resistance gene or a metabolic regulation gene.
5. A set of primer pairs for amplifying the constitutively expressed rice promoter OsCAX1a according to claim 1, characterized in that: The nucleotide sequences of the primer pairs are shown in SEQ ID NO: 2-SEQ ID NO:
3.
6. A rice constitutive promoter function verification kit, characterized in that: Comprising the primer pair according to claim 5.
7. A method for regulating the expression of exogenous genes using the rice constitutive expression promoter OsCAX1a according to claim 1 or 2, characterized in that: The method comprises: connecting the promoter and the target gene to construct a recombinant vector, and transforming the vector into rice plants through Agrobacterium-mediated method to drive the target gene to be constitutively expressed in various rice tissues.
8. Use of the rice constitutive expression promoter according to claim 1 or 2 in regulating or initiating constitutive expression of a target heterologous gene in rice tissue.
9. Use of the rice constitutive expression promoter OsCAX1a according to claim 1 or 2 in breeding transgenic rice varieties.
10. The use according to claim 9, characterized in that The applications include improving disease resistance, stress resistance, yield or metabolite accumulation.