Rice mesophyll cell specific promoter POs0495C02 and application thereof

Abstract

The invention discloses a specific promoter POs0495C02 of a rice mesophyll cell and application of the specific promoter POs0495C02, and belongs to plant genetic engineering. The specific promoter provided by the invention is located at the 5'end and upstream of the rice Os0495C02 gene, the nucleotide sequence is as shown in SEQ ID NO.1, and the length is 1888bp. Experimental results show that the specific promoter provided by the invention has necessary transcription initiation sites and starting points, and can effectively promote specific expression of a target gene only in rice mesophyll cells; the specific promoter can overcome waste caused by non-specific, continuous and efficient expression of an exogenous gene in a receptor plant, reduces the influence of a target gene on other tissues, enhances the expression effect of the target gene in mesophyll cells, provides a new tool for regulating and controlling accurate spatial specific expression of a functional gene in a plant body, and has a wide application prospect. And the method has an extremely wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and particularly to a rice mesophyll cell-specific promoter POs0495C02 and its application. Background Art

[0002] The entire growth and development stage of a plant is the result of the specific expression of genes at specific times and in specific spaces. The expression of genes is affected and regulated in multiple aspects and at multiple levels. Among them, gene transcription is the most important regulatory process, and the promoter is the "gene switch" of gene transcription. The promoter can regulate the expression of genes by recognizing RNA polymerase and regulating transcription factors. Plant genetic engineering aims to introduce genes with excellent traits into target plants and express them during their genetic development process to achieve the expected genetic characteristics of the recipient plants. Generally, promoters are divided into three categories according to different regulatory gene expression patterns: constitutive promoters, inducible promoters, and tissue-specific promoters.

[0003] Tissue-specific promoters refer to promoters that can initiate the specific expression of foreign genes only in the required parts of the recipient. In addition to the general elements that control tissue-specific expression, such promoters also possess the general characteristics of enhancers and silencers. The expression specificity is jointly determined by the number, type, and relative position of cis-acting elements inside the promoter. Compared with constitutive promoters, specific promoters avoid the accumulation of metabolites and the waste of plant energy, and maintain the metabolic balance of plants. Therefore, the basic research and practical application of tissue-specific expression promoters have been increasingly emphasized. So far, some promoters with excellent functions have been obtained from rice, but there are few reports on tissue-specific expression promoters, which do not meet the needs of regulating the tissue-specific expression of foreign genes in transgenic breeding practice.

[0004] As the world's most typical cereal food crop, the yield and quality of rice directly affect food security and the living quality of people. The main biological functions of rice mesophyll cells are photosynthesis and gas exchange. They synthesize organic substances through photosynthesis to provide energy and nutrients for plants; through gas exchange, they regulate the water balance and the absorption of carbon dioxide, and are the main part of plant photosynthesis. In addition, mesophyll cells supply the growth and metabolism of other tissues and organs by accumulating nutrients; maintain the normal functions of cells through water regulation; and achieve internal communication and coordination of plants through signal conduction. It can be seen that to make a foreign gene specifically expressed in rice mesophyll tissue, a promoter that drives the specific expression of this gene in mesophyll cells is needed. Therefore, isolating and identifying natural or artificially optimized and synthesized mesophyll cell tissue-specific expression promoters is of great significance for rice transgenic breeding. Summary of the Invention

[0005] The object of the present invention is to provide a rice mesophyll cell-specific promoter POs0495C02 and its application, so as to solve the problems existing in the above-mentioned prior art. The specific promoter POs0495C02 provided by the present invention only drives the target gene to be highly expressed in mesophyll cells, overcomes the waste caused by the expression of foreign genes by constitutive promoters, and promotes the exertion of the biological functions of the target gene.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a rice mesophyll cell-specific promoter POs0495C02, and the nucleotide sequence of the rice mesophyll cell-specific promoter POs0495C02 is as shown in SEQ ID NO.1.

[0008] The present invention also provides a primer pair for amplifying the above-mentioned rice mesophyll cell-specific promoter POs0495C02, which is characterized by including an upstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a downstream primer as shown in SEQ ID NO.3.

[0009] The present invention also provides a recombinant vector containing the above-mentioned rice mesophyll cell-specific promoter POs0495C02.

[0010] The present invention also provides an engineered bacterium containing the above-mentioned recombinant vector.

[0011] The present invention also provides an application of the above-mentioned rice mesophyll cell-specific promoter POs0495C02, the above-mentioned recombinant vector or the above-mentioned engineered bacterium in initiating the specific expression of a plant target gene.

[0012] Preferably, the specific expression is specifically expressed in the leaves of the plant; the plant is rice.

[0013] The present invention also provides a method for initiating the specific expression of a plant target gene, including the following steps:

[0014] Synthesize the rice mesophyll cell-specific promoter POs0495C02, construct a recombinant vector containing the rice mesophyll cell-specific promoter POs0495C02 and the target gene, and transform the recombinant vector into the plant;

[0015] Among them, the nucleotide sequence of the rice mesophyll cell-specific promoter POs0495C02 is as shown in SEQ ID NO.1.

[0016] The present invention also provides an application of the above-mentioned rice mesophyll cell-specific promoter POs0495C02, the above-mentioned recombinant vector or the above-mentioned engineered bacterium in constructing a transgenic plant with specific expression of a target gene.

[0017] Preferably, the specific expression is specific expression in the leaves of the plant; the plant is rice.

[0018] The present invention also provides a method for constructing a transgenic plant with specific expression of a target gene, comprising the following steps:

[0019] Synthesize the rice mesophyll cell-specific promoter POs0495C02, construct a recombinant vector containing the rice mesophyll cell-specific promoter POs0495C02 and the target gene, and transform the recombinant vector into the plant;

[0020] Wherein, the nucleotide sequence of the rice mesophyll cell-specific promoter POs0495C02 is as shown in SEQ ID NO.1.

[0021] The present invention discloses the following technical effects:

[0022] The rice mesophyll cell tissue-specific expression promoter POs0495C02 provided by the present invention is derived from the japonica rice variety Nipponbare. The rice mesophyll cell-specific expression promoter POs0495C02 has the following characteristics: 1. It is located at the 5' end and its upstream of the gene Os0495C02; 2. The base length is 1888bp; 3. It has the necessary sites for initiating transcription and the transcription start point; 4. It initiates the specific expression of the downstream target gene in the rice mesophyll cell tissue, overcomes the waste caused by the constitutive promoter initiating the non-specific, continuous and efficient expression of the foreign gene in the recipient plant, reduces the influence of the target gene on the normal growth and development of other tissues, and enhances the expression effect of the target gene in the mesophyll cells. It can not only serve for the genetic breeding of transgenic rice, but also reserve resources for the subsequent modification and design of promoters.

[0023] The present invention also discloses a recombinant expression vector containing the rice mesophyll cell tissue-specific expression promoter POs0495C02, which enables the specific expression of the downstream target gene in the mesophyll cells under the regulation of the mesophyll cell tissue-specific expression promoter POs0495C02 of the recombinant expression vector, provides a practical tool for the research in the field of plant genetic engineering to accurately regulate the spatial-specific expression of functional genes in plants, and has great application prospects. Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It is the electrophoresis result diagram of the PCR amplification product; among them, A is the result of the amplification product of the target DNA fragment, M is DL3000 Marker, and 1 is the PCR amplification product of the target DNA fragment; B is the result of the double digestion product of the plasmid vector pCAMBIA1391Z, M is DL5000 Marker, and 2 is the double digestion product of the plasmid vector pCAMBIA1391Z.

[0026] Figure 2 It is the structural schematic diagram of the recombinant plasmid vector pCAMBIA1391Z-POs0495C02 and the promoter POs0495C02; among them, the upper figure is the structural schematic diagram of the recombinant plasmid vector pCAMBIA1391Z-POs0495C02, LB and RB respectively represent the left and right boundaries of T-DNA, Hyg represents the hygromycin resistance gene, POs0495C02 represents the promoter, and NOS represents the terminator; the lower figure is the promoter POs0495C02 loaded into the multiple cloning site (MCS) of the plasmid vector pCAMBIA1391Z.

[0027] Figure 3 It is the electrophoresis result diagram of the PCR amplification product of the transgenic rice plant; among them, M is DNA Marker, and 1-10 are the PCR amplification products of the exogenous target gene of the transgenic plants.

[0028] Figure 4 It is the histochemical staining result diagram of the expression of the GUS gene driven by the promoter POs0495C02; among them, A is the result of the root, the upper figure is the root segment, and the lower figure is the cross-section of the root; B is the result of the stem, the upper figure is the stem segment, and the lower figure is the cross-section of the stem; C is the cross-section of the leaf; D is the result of the seed, the upper figure is the seed including the hull, and the lower figure is the seed after removing the hull. Detailed implementation manners

[0029] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms used in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0034] Tissue-specific expression promoters can precisely initiate the expression of exogenous target genes in specific tissue parts of transgenic plants, making the gene expression more controllable, safer, and more efficient, and having great potential in genetic engineering breeding. For example, the tobacco root-specific promoter pNtREL1, the Pharbitis green tissue-specific promoter PNZIP, the peanut seed-specific promoter LEC1A, the rice pollen-specific promoter OsLSP, etc. are all tissue-specific expression promoters commonly used by scientific researchers in the practice of genetic engineering breeding.

[0035] In the quantitative tests in the embodiments of the present invention, three repeated experiments were set up, and the results were averaged.

[0036] The GUS staining solution in the embodiments of the present invention: the solvent is 200 mmol / L PBS buffer, and the solutes and their concentrations in the staining solution are respectively: 100 mmol / L potassium ferrocyanide, 100 mmol / L potassium cyanide, 0.5 mmol / L EDTA (pH = 8.0), 10 mg / mL X-Gluc, 0.1% (volume fraction) Triton X-100; adjust the pH to 7.0.

[0037] All primers used in the embodiments of the present invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; DNA sequencing was completed by Beijing Qingke Biotechnology Co., Ltd.; Taq enzyme, Trans5α competent cells and related experimental kits were all purchased from Beijing TransGen Biotech Co., Ltd.; restriction enzymes Hind III and EcoR I were purchased from Thermo Fisher Scientific; In-Fusion ligase was purchased from Beijing Baori Biotechnology Co., Ltd.; antibiotics were purchased from Sigma, USA; pCAMBIA1391Z vector and Agrobacterium tumefaciens AGL1 were both purchased from Beijing Boai Yonghua Biotechnology Co., Ltd.; the rest of the reagents were all domestic analytical pure.

[0038] Example 1 Cloning of the tissue-specific expression promoter POs0495C02 of rice mesophyll cells

[0039] 1. Primer design

[0040] Based on the published whole genome sequence of Oryza sativa L cv. Nipponbare on the NCBI website, PCR amplification primers were designed according to the 5'-terminal promoter region sequence of the rice gene Os0495C02. The upstream primer was added with the AAGCTT restriction enzyme site of HindIII, and the downstream primer was added with the GAATTC restriction enzyme site of EcoR I. The specific primer sequences are as follows:

[0041] Upstream primer: 5'-GATTACGCC AAGCTT AGGAGCAAATGGCTGCTTAT-3' (SEQ ID NO.2);

[0042] Downstream primer: 5'-ACGGCCAGT GAATTC AGCCCATGTGAACAGCTATG-3' (SEQ ID NO.3).

[0043] 2. PCR amplification

[0044] Using the genomic DNA of Oryza sativa L cv. Nipponbare extracted by the plant genomic kit of TransGen Biotech Co., Ltd. as a template, PCR amplification was carried out with the upstream and downstream primers shown in SEQ ID NO.2-3 to obtain a PCR product.

[0045] PCR reaction system (40 μL): 20 μL of 2×Phanta Flash Master Mix (Dye Plus), 2 μL of forward primer, 2 μL of reverse primer, 2 μL of DNA template, 14 μL of ddH2O.

[0046] PCR reaction program: Pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 60°C for 5 s, extension at 72°C for 20 s, for a total of 32 cycles; extension at 72°C for 1 min.

[0047] 3. Detection of PCR products

[0048] After the PCR reaction, the PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that a DNA fragment of 1888 bp was amplified by PCR ( Figure 1 in A). After recovering and purifying the DNA fragment with the DNA purification and recovery kit from TransGen Biotech Co., Ltd., it was sent to Beijing Tsingke Biotechnology Co., Ltd. for sequence determination. Its nucleotide sequence is shown in SEQ ID NO.1, with a size of 1888 bases. This DNA fragment was named POs0495C02.

[0049] SEQ ID NO.1:

[0050]

[0051] Example 2: Functional Verification of the POs0495C02 Promoter

[0052] 1. Obtaining Transgenic Lines

[0053] 1.1 Construction of the Expression Vector

[0054] The plasmid vector pCAMBIA1391Z was digested with the restriction enzymes Hind III and EcoR I to obtain a linear pCAMBIA1391Z vector backbone fragment ( Figure 2 ); the POs0495C02 fragment was ligated with the pCAMBIA1391Z vector backbone fragment to obtain the recombinant plasmid pCAMBIA1391Z-POs0495C02.

[0055] The above recombinant plasmid pCAMBIA1391Z-POs0495C02 was transformed into Escherichia coli DH5α competent cells by heat shock method. After verification by colony PCR ( Figure 1 in B), the bacterial liquid corresponding to the single colony of the positive clone was picked and sent to the company for sequencing.

[0056] The correctly sequenced recombinant plasmid pCAMBIA1391Z-POs0495C02 is a vector obtained by inserting the POs0495C02 promoter with the nucleotide sequence shown in SEQ ID NO.1 between the HindIII and EcoRI restriction sites of the pCAMBIA1391Z vector, and keeping the other sequences of the pCAMBIA1391Z vector unchanged; the POs0495C02 promoter is used to initiate the expression of the GUS gene in the pCAMBIA1391Z vector.

[0057] 1.2 Agrobacterium-Mediated Genetic Transformation of Rice

[0058] After the recombinant plasmid pCAMBIA1391Z-POs0495C02 was introduced into Agrobacterium tumefaciens AGL1, the Agrobacterium was resuspended in a liquid co-culture medium (AAM liquid medium + 50 mg / L acetosyringone, pH = 5.2) and cultured. After adjustment, an OD 600nm = 0.15 bacterial liquid was obtained.

[0059] Calli of the rice variety Nipponbare (induced from Nipponbare seeds on MS medium supplemented with 2 mg / L 2,4-D for about 28 days) were taken and soaked in the above OD 600nm = 0.15 bacterial liquid for 30 min, and then through co-culture, screening, rooting and seedling strengthening, T0 generation transgenic plants were obtained.

[0060] Extract the DNA of the T0 generation transgenic plants, and perform PCR amplification using the upstream primer and downstream primer in Example 1. The PCR reaction system and procedure are the same as in Example 1. The DNA fragment with a size of 1888 bp obtained by PCR amplification is the positive transgenic plant with the POs0495C02 promoter gene. Finally, the PCR detection results are as Figure 3 shown, and all 10 T0 generation test-tube seedlings obtained are transgenic positive plants.

[0061] 2. Staining of the plants with the POs0495C02 promoter

[0062] Perform GUS histochemical staining on different parts of the positive plants with the POs0495C02 promoter gene. The specific operation steps are as follows: Take different tissues (roots, stems, leaves, seeds) of the positive transgenic plants with the POs0495C02 promoter, and put each tissue block into a test tube containing an appropriate amount of GUS staining solution, so that the GUS staining solution submerges the tissue block, and incubate at 37 °C for 4 - 10 h; after staining, first place the stained tissue in 75% ethanol for rinsing and decolorization, and then soak it in 50% and 20% ethanol for more than 20 min respectively until the tissue material becomes white; finally, observe the stained tissue block or the section of the tissue block under a microscope, and the tissue part stained blue indicates that the GUS gene is expressed in this tissue part.

[0063] The staining results are as Figure 4 shown, where A is the result of the root, the upper part is the root segment, and the lower part is the cross-section of the root; B is the result of the stem, the upper part is the stem segment, and the lower part is the cross-section of the stem; C is the cross-section of the leaf; D is the result of the seed, the upper part is the seed including the hull, and the lower part is the seed without the hull. It can be seen from Figure 4 that only Figure 4 the cross-section of the leaf in C shows blue, indicating that the GUS gene is only expressed in the mesophyll cells and mesophyll tissues of the plants with the POs0495C02 promoter. This shows that the promoter POs0495C02 of the present invention is specifically expressed only in the mesophyll cell tissues of rice, and can be used to initiate the specific expression of the target gene in the mesophyll cell mesophyll tissues in transgenic breeding research.

[0064] The promoter POs0495C02 obtained in the present invention, as an endogenous mesophyll cell mesophyll tissue-specific promoter of rice, is more conducive to the recognition of exogenous target genes by rice recipient cell regulatory factors, promotes the integration of exogenous genes, and enhances the function of the target gene in specific tissues in future rice transgenic breeding research and practice.

[0065] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A rice mesophyll cell-specific promoter POs0495C02, characterized in that, The nucleotide sequence of the rice mesophyll cell-specific promoter POs0495C02 is shown in SEQ ID NO.

1.

2. A primer pair for amplifying the rice mesophyll cell-specific promoter POs0495C02 recited in claim 1, characterized in that, It includes an upstream primer with a nucleotide sequence shown in SEQ ID NO.2 and a downstream primer with a nucleotide sequence shown in SEQ ID NO.

3.

3. A recombinant vector containing the rice mesophyll cell-specific promoter POs0495C02 described in claim 1.

4. An engineered bacterium containing the recombinant vector described in claim 3.

5. Use of the rice mesophyll cell-specific promoter POs0495C02 described in claim 1, the recombinant vector described in claim 3, or the engineered bacterium described in claim 4 in initiating specific expression of a plant target gene.

6. The application according to claim 5, characterized in that, The specific expression is specific expression in the leaves of the plant; The plant is rice.

7. A method for initiating the specific expression of a target gene in a plant, characterized in that, It includes the following steps: Synthesize the rice mesophyll cell-specific promoter POs0495C02, construct a recombinant vector containing the rice mesophyll cell-specific promoter POs0495C02 and the target gene, and transform the recombinant vector into the plant; Among them, the nucleotide sequence of the rice mesophyll cell-specific promoter POs0495C02 is shown in SEQ ID NO.

1.

8. Use of the rice mesophyll cell-specific promoter POs0495C02 described in claim 1, the recombinant vector described in claim 3, or the engineered bacterium described in claim 4 in constructing a transgenic plant with specific expression of a target gene.

9. The application according to claim 8, wherein The specific expression is specific expression in the leaves of the plant; The plant is rice.

10. A method for constructing a transgenic plant with specific expression of a target gene, characterized in that, It includes the following steps: Synthesize the rice mesophyll cell-specific promoter POs0495C02, construct a recombinant vector containing the rice mesophyll cell-specific promoter POs0495C02 and the target gene, and transform the recombinant vector into the plant; Among them, the nucleotide sequence of the rice mesophyll cell-specific promoter POs0495C02 is shown in SEQ ID NO.1.

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