Strong promoter CP17 specifically expressed in anther of plants at various periods and application thereof

Abstract

The application discloses a strong promoter CP17 specifically expressed in anthers of plants at various periods, a gene expression cassette containing the CP17, a recombinant expression vector, a recombinant bacterium and a transgenic plant cell line, and the strong promoter CP17, the gene expression cassette, the recombinant expression vector, the recombinant bacterium and the transgenic plant cell line specifically expressed in the anthers of the plants at various periods can efficiently drive specific expression of an exogenous gene in the anthers of the plants at various periods, the expression level is accurate, adverse effects caused by continuous expression of a target gene in other tissues of the plants can be avoided, the strong promoter CP17 can be used for creating a plant male sterile line, and has a good application prospect in plant genetic engineering and utilization of hybrid vigor. In addition, the nucleotide sequence of the strong promoter CP17 specifically expressed in the anthers of the plants at various periods is short, with a full length of only 737 bp, is easy to clone, can effectively reduce cloning difficulty and cost of the promoter, is helpful to improve construction and transformation efficiency of the recombinant expression vector, and is helpful to quickly obtain the transgenic plants.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular biology, specifically to a strong promoter CP17 that is specifically expressed in anthers at various stages of plant growth and its applications. Background Technology

[0002] Plant gene expression regulation is primarily at the transcriptional level, coordinated by various cis-acting elements and trans-acting factors. Promoters are crucial cis-acting elements, playing a key role in transcriptional regulation. Tissue-specific promoters drive gene expression only in certain specific tissues or organs, enabling precise, time- and quantitative regulation of exogenous gene expression within plants. This effectively reduces the adverse effects on plant growth and development caused by the continuous expression of constitutive promoters in different sites or the excessive accumulation of exogenous proteins, thus possessing broad application value in plant genetic engineering. Although many plant promoters have been disclosed, the development of tissue-specific promoters remains relatively slow and cannot yet meet the ever-expanding demands of the transgenic industry.

[0003] Anthers are crucial organs in the sexual reproduction stage of plants. Anther-specific promoters are commonly used for creating male-sterile germplasm resources, restoring male fertility, and studying certain metabolic processes or gene regulatory pathways during anther development. On one hand, anther-specific promoters can be chimeric with cytotoxin genes to construct expression vectors, which can then be transformed to block pollen development and create male-sterile plants. Fertility can also be restored by driving the specific expression of exogenous genes in the anther. On the other hand, when studying certain metabolic processes or gene regulatory pathways in the anther, anther-specific promoters can be used to drive gene overexpression or gene silencing, allowing for targeted research on the role of genes in anther development. Sometimes, to improve transgenic efficiency and research speed, different anther-specific promoters with low homology are chimeric with different genes in the same expression vector, effectively preventing gene silencing that might be caused by highly homologous promoter sequences. However, currently, very few anther-specific promoters with strong driving activity and good specificity have been publicly disclosed, making it difficult to meet the above needs.

[0004] Furthermore, the publicly disclosed anther-specific promoter nucleotide sequences are generally quite long, mostly ranging from 1000 to 3000 bp. For example, CN108486112B discloses a promoter with anther tissue specificity of 2147 bp; CN108753777B discloses a promoter with anther tissue specificity and its application of 2384 bp; and CN106480026B discloses a promoter with anther early development specificity and its application of 3007 bp. Although these promoters exhibit good anther specificity, their long promoter nucleotide sequences increase the difficulty and cost of promoter cloning, reduce the efficiency of expression vector construction and transformation, and are not conducive to rapid cloning and obtaining transgenic plants.

[0005] Therefore, developing promoters with short nucleotide sequences that are specifically expressed in anthers at different stages of plant growth can not only help in the functional analysis and identification of genes related to anther development, but also contribute to the creation of germplasm resources for male sterile lines, and has great application prospects in plant genetic engineering and the utilization of heterosis. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a promoter CP17 with a short nucleotide sequence that is specifically expressed in anthers at different stages of plant growth and its application.

[0007] To achieve the above objectives, the present invention provides a strong promoter CP17 specifically expressed in anthers at various stages of plant growth, having a nucleotide sequence as shown in SEQ ID No. 1.

[0008] The present invention also provides a gene expression cassette containing the strong promoter CP17 specifically expressed in anthers at various plant stages. The gene expression cassette is composed of the strong promoter CP17 sequence specifically expressed in anthers at various plant stages, an exogenous target gene, and a transcription termination sequence connected in a specific direction. The exogenous target gene includes, but is not limited to, structural genes, regulatory genes, antisense genes of structural genes, antisense genes of regulatory genes, or small RNA genes, long non-coding RNA (lncRNA) genes, and circular RNA (circRNA) genes that can interfere with the expression of endogenous genes. In one embodiment of the present invention, the exogenous target gene is specifically the β-glucuronidase gene (GUS).

[0009] The present invention also provides a recombinant expression vector containing the above-mentioned gene expression cassette. The recombinant expression vector is any vector known in the prior art that can be expressed in plants, including but not limited to pCAMBIA3301, pCAMBIA1300, pBI101, pBI121, pRI201, pBin19, pCAMBIA2301, and pCAMBIA1301 plant expression vectors. The recombinant expression vector may also contain a selection marker gene, which typically includes genes that provide antibiotic resistance or herbicide resistance, such as hygromycin resistance genes, glufosinate-ammonia-phosphorus or glufosinate-ammonia-phosphorus resistance genes, etc. In one embodiment of the present invention, the recombinant expression vector is specifically pBI101.

[0010] The present invention also provides recombinant bacteria and transgenic plant cell lines containing the above-mentioned gene expression cassettes.

[0011] This invention also provides the application of the above-mentioned strong promoter CP17 specifically expressed in anthers at various stages of plant growth, the above-mentioned gene expression cassette containing the strong promoter CP17 specifically expressed in anthers at various stages of plant growth, recombinant expression vector, transgenic plant cell line and recombinant bacteria in driving the specific expression of exogenous target genes in plant anthers.

[0012] The present invention also provides the application of the above-mentioned strong promoter CP17 specifically expressed in anthers at various stages of plant growth, the above-mentioned gene expression cassette containing the strong promoter CP17 specifically expressed in anthers at various stages of plant growth, recombinant expression vector, transgenic plant cell line and recombinant bacteria in the preparation of transgenic plants.

[0013] The transgenic plants described in this invention can be obtained by any known transgenic technology capable of introducing exogenous genes into plant cells or plant tissues; in one embodiment of this invention, the transgenic technology is Agrobacterium-mediated transformation.

[0014] The transgenic plant described in this invention is a transgenic plant in which the exogenous target gene is specifically expressed in the anther, preferably a transgenic plant with enhanced or weakened pollination or fertilization ability, and more preferably a male-sterile transgenic plant.

[0015] The plants mentioned in this invention include, but are not limited to, Arabidopsis thaliana, Chinese cabbage, kale, rapeseed, citrus, chili pepper, beet, kiwifruit, cucumber, carrot, sunflower, tobacco, tomato, potato, and soybean; preferably, Chinese cabbage, kale, rapeseed, and Arabidopsis thaliana; in one embodiment of this invention, the plant is Arabidopsis thaliana.

[0016] The present invention also provides primer pairs for PCR amplification of the strong promoter CP17 specifically expressed in anthers at various stages of plant growth, the nucleotide sequences of which are shown in SEQ ID No. 2 and SEQ ID No. 3.

[0017] The present invention also provides a method for isolating or identifying the strong promoter CP17 specifically expressed in anthers at various stages of plant growth, comprising using primer pairs SEQ ID No. 2 and SEQ ID No. 3 to amplify the nucleotide sequence of the strong promoter CP17 specifically expressed in anthers at various stages of plant growth by PCR amplification from genomic DNA of Arabidopsis thaliana of Columbia type.

[0018] DNA molecules complementary to the strong promoter CP17, which is specifically expressed in anthers at various stages of plant growth, are also part of this invention. Using such DNA molecules, the same purpose as the strong promoter CP17, which is specifically expressed in anthers at various stages of plant growth, can also be achieved.

[0019] The beneficial effects of this invention are as follows:

[0020] 1) This invention discloses for the first time a strong promoter CP17 that is specifically expressed in anthers at various stages of plant growth, and provides a new method for efficiently driving the specific expression of exogenous genes in plant anthers.

[0021] 2) The strong promoter CP17 provided by this invention is specifically expressed in anthers at various stages of plant growth. It has strong driving activity and tissue specificity. CP17 can efficiently drive the specific expression of exogenous genes in plant anthers with precise expression levels. It can avoid the adverse effects of continuous expression of target genes in other plant tissues and has broad application value in the field of plant genetic engineering.

[0022] 3) The nucleotide sequence of the strong promoter CP17, which is specifically expressed in anthers at various stages of plants, provided by this invention is relatively short, with a full length of only 737 bp. It is easy to clone, which can effectively reduce the difficulty and cost of promoter cloning, help improve the construction and transformation efficiency of recombinant expression vectors, and help to quickly obtain transgenic plants.

[0023] 4) The strong promoter CP17 specifically expressed in anthers at different stages of plant growth provided by this invention can be used for functional analysis and identification of anther development-related genes, which helps to understand the molecular mechanism of anther development regulation.

[0024] 5) The strong promoter CP17, which is specifically expressed in the anthers of plants at various stages, provided by this invention can be used to create germplasm resources of male sterile lines in plants, and has great application prospects in plant genetic engineering and the utilization of heterosis. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the construction of the CP17 recombinant expression vector pCP17-GUS;

[0026] Figure 2These are images of pCP17-GUS transgenic seedlings and GUS staining of various tissues. A: Seedling; B: Stem and mature leaves; C: Siliques and seeds; D: Inflorescence; E: Flower. Detailed Implementation

[0027] The present invention will now be clearly and completely described through specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Modifications or substitutions made by those skilled in the art to the methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention are all within the protection scope of the present invention.

[0028] Unless otherwise specified, the experimental methods in the following examples are all conventional methods; the materials, reagents and instruments in the following examples can be purchased commercially; unless otherwise specified, the first position of each nucleotide sequence in the following examples is the 5′ terminal nucleotide of the corresponding DNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA.

[0029] Example 1: Obtaining the strong promoter CP17 specifically expressed in anthers at different plant stages

[0030] 1) Preparation of genomic DNA from Arabidopsis thaliana of Columbia type

[0031] Genomic DNA was extracted from leaves of Arabidopsis thaliana using FN-C cell lysis buffer (CN114426966A, a cell lysis buffer, kit, and method for rapid and non-toxic extraction of genomic DNA from plants, animals, or microorganisms) and stored at -20°C for later use.

[0032] 2) Cloning of the CP17 promoter

[0033] The Arabidopsis thaliana reference genomic DNA sequence was obtained from the http: / / www.gramene.org / microsat database. Specific PCR amplification primers for CP17 were designed using SnapGene software. The forward and reverse primer sequences are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively. PCR amplification was performed using the prepared Arabidopsis thaliana genomic DNA as a template. The amplification product was cloned into the pMD19-T vector, and the CP17 promoter sequence was determined as shown in SEQ ID No. 1. The correctly sequenced plasmid was named pMD19-CP17 and used as a PCR amplification template for constructing various recombinant expression vectors of the CP17 promoter. The specific PCR reaction system and procedure used are as follows:

[0034] a) PCR reaction system (50 μL):

[0035]

[0036] b) PCR reaction procedure:

[0037]

[0038] Example 2: Construction of the recombinant expression vector pCP17-GUS with promoter CP17 (construction diagram shown in Figure 1) Figure 1 )

[0039] 1) Using the same primer design method as in Example 1, PCR amplification primer pairs for promoter CP17 were obtained. HindIII and BamHI restriction sites were added to the 5′ ends of the forward and reverse primers, respectively. Then, 15bp sequences overlapping with the corresponding ligation sites of the pBI101 vector were added to the 5′ ends of the forward and reverse primers, respectively, to obtain PCR amplification primer pairs for ligating promoter CP17 to the pBI101 vector. The forward and reverse primer sequences are shown in SEQ ID No. 4 and SEQ ID No. 5, respectively.

[0040] 2) Using the CP17 promoter amplification primer pair obtained above (sequences shown in SEQ ID No. 4 and SEQ ID No. 5), PCR amplification was performed with pMD19-CP17 plasmid as template. The PCR product was recovered by gel extraction to obtain the target fragment. The PCR reaction system and procedure were the same as in Example 1.

[0041] 3) The pBI101 vector was digested with HindIII and BamHI, and the linear vector was obtained by gel recovery.

[0042] 4) The target fragment described in 2) and the linear vector described in 3) were efficiently ligated using a seamless ligation recombinase, transformed into competent E. coli cells, and single clones were screened and sequenced for verification. The sequencing primer sequences are shown in SEQ ID No. 6 and SEQ ID No. 7. The vector with correct sequencing was named pCP17-GUS.

[0043] Example 3: Transformation of Arabidopsis thaliana with recombinant expression vector pCP17-GUS

[0044] 1) Transform the recombinant expression vector pCP17-GUS into Agrobacterium.

[0045] Take 1-2 μg of the recombinant expression vector pCP17-GUS plasmid obtained in Example 2, transform it into Agrobacterium GV3101 competent cells using the freeze-thaw method, and culture and screen single clones on sterile YEB solid plates containing rifampicin (25 μg / mL) and kanamycin (50 μg / mL). PCR verification of the pCP17-GUS recombinant bacteria was performed using specific primers SEQ ID No. 6 and SEQ ID No. 7 to identify positive clones. The PCR reaction system and procedure were the same as in Example 1.

[0046] 2) Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

[0047] Agrobacterium tumefaciens broth was activated at 28℃, and wild-type Arabidopsis thaliana was transformed using the inflorescence immersion method. Transgenic seeds were cultured and harvested under normal culture conditions and placed in 1.5mL centrifuge tubes. 3-8 desiccant particles of color-changing silica gel were added, and the tubes were sealed with sealing film and stored at 4℃ for long-term storage.

[0048] Example 4: Screening and identification of pCP17-GUS transgenic Arabidopsis thaliana

[0049] The transgenic seeds obtained in Example 3, which were dried and preserved at low temperature, were sterilized and then sown in a sterile 1 / 2 CS solid plant tissue medium (CN110754366B, a widely adaptable plant tissue medium and 1 / 2 medium) containing kanamycin (50 μg / mL). The medium was cultured for 10-15 days under the conditions of temperature 22-24℃, humidity 50%-70%, light intensity 1500-2000 Lux, and alternating light and dark (16h light time, 8h dark time). At this time, dark green resistant seedlings were visible, while yellow or white seedlings were non-resistant wild-type seedlings. The resistant seedlings were then transplanted into a nutrient substrate (vermiculite: nutrient soil = 1:2, V / V) for further culture. Tender tissues of the selected T1 generation positive transgenic plants were taken, and genomic DNA was extracted for PCR detection (primer sequences are shown in SEQ ID No. 6 and SEQ ID No. 7). The results showed that the target band was present in all positive transgenic plants. After repeated screening, T2 generation plants and seeds were obtained. The T2 generation seeds were further screened, and those whose offspring were all dark green resistant seedlings were the pCP17-GUS transgenic homozygous lines.

[0050] Example 5: Histochemical detection of GUS gene expression in various tissues and organs of transgenic Arabidopsis plants

[0051] Seedlings of the pCP17-GUS transgenic homozygous line and different tissues and organs were immersed in X-Gluc staining solution, vacuumed for 5 minutes, and then incubated overnight at 37°C. The staining solution was removed, and the samples were destained 2-3 times with 70% ethanol. The results were observed and photographed under a stereomicroscope and microscope. Figure 2 As shown, no blue color was detected in the seedlings (including roots, hypocotyls, and young leaves), stems, mature leaves, siliques and seeds, sepals, petals, filaments, and pistils of pCP17-GUS transgenic Arabidopsis thaliana. Dark blue was only detected in the anthers at various stages, indicating that the promoter CP17 can efficiently drive the specific expression of the GUS gene in the anthers of Arabidopsis thaliana at various stages.

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Of course, derivative nucleotide sequences that have one or more nucleotides substituted, deleted, or added to the nucleotide sequence shown in SEQ ID No. 1 and have the same pollen-specific promoter function are also within the scope of protection of this application, especially derivative nucleotide sequences that have more than 75% homology with the nucleotide sequence shown in SEQ ID No. 1 and have the same pollen-specific promoter function.

Claims

1. A strong promoter CP17 specifically expressed in anthers of Arabidopsis thaliana at various stages, the nucleotide sequence of which is shown in SEQ ID No.

1.

2. A gene expression cassette containing the strong promoter CP17, which is specifically expressed in the anthers of Arabidopsis thaliana at various stages as described in claim 1.

3. A recombinant expression vector containing the gene expression cassette of claim 2.

4. Recombinant bacteria containing the gene expression cassette of claim 2.

5. A transgenic plant cell line containing the gene expression cassette of claim 2, wherein, The plant in question is Arabidopsis thaliana.

6. The application of the strong promoter CP17 specifically expressed in anthers of Arabidopsis thaliana at various stages as described in claim 1, or the gene expression cassette as described in claim 2, or the recombinant expression vector as described in claim 3, or the recombinant bacteria as described in claim 4 in driving the specific expression of exogenous target genes in anthers of plants at various stages, wherein, The plant in question is Arabidopsis thaliana.

7. The application of the strong promoter CP17 specifically expressed in the anthers of Arabidopsis thaliana at various stages as described in claim 1, or the gene expression cassette as described in claim 2, or the recombinant expression vector as described in claim 3, or the recombinant bacteria as described in claim 4, in the preparation of transgenic plants, wherein, The plant in question is Arabidopsis thaliana.

8. PCR amplification of the primer pair for the strong promoter CP17 specifically expressed in the anthers of Arabidopsis thaliana at various stages, as described in claim 1, wherein the nucleotide sequences of the primer pair are shown in SEQ ID No. 2 and SEQ ID No.

3.

9. A method for isolating or identifying the strong promoter CP17 specifically expressed in the anthers of Arabidopsis thaliana at various stages, as described in claim 1, characterized in that, The method includes using primer pairs SEQ ID No. 2 and SEQ ID No. 3 to amplify the nucleotide sequence of the strong promoter CP17, which is specifically expressed in the anthers of Arabidopsis thaliana at various stages as described in claim 1, from genomic DNA of Arabidopsis thaliana of Columbia type by PCR.

10. A DNA molecule that is complementary to the nucleotide sequence of the promoter CP17 of claim 1 and has the function of an anther-specific promoter of Arabidopsis thaliana.

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