Promoter specifically expressed from upland cotton pollen and application thereof
By developing the GH_D01G2221 promoter specifically expressed on land cotton pollen, the metabolic interference problem caused by constitutive promoters is solved, pollen-specific expression is achieved, pollen breeding and pollination success rate is improved, and it has important application value for breeding and gene function analysis.
Patent Information
- Application Number
- CN202510478448.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-16
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, constitutive promoters are continuously expressed in cotton, leading to the accumulation of heterologous proteins, interfering with plant metabolic balance, affecting plant growth and development, and lacking efficient pollen-specific expression promoters to regulate pollen fertility and create sterile lines.
The GH_D01G2221 promoter specifically expressed by terrestrial cotton pollen was developed and utilized to build a pollen-specific promoter-driven gene expression vector to achieve specific expression in pollen, reduce the adverse effects on plants, and enhance the vitality of pollen in high temperature environments.
Pollen-specific gene expression is achieved, pollen breeding and pollination success rate is improved, and the negative impact on plants is reduced. It has important application potential for breeding and gene function analysis.
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Figure CN120249280A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of plant promoters, and particularly relates to a promoter specifically expressed in pollen of Gossypium hirsutum and its application. Background Art
[0002] A promoter is a specific DNA sequence located upstream of a gene that can be precisely recognized and bound by RNA polymerase. In plant genetic engineering, it is often classified into three categories according to its function and mode of action, namely, constitutive promoters, inducible promoters, and tissue-specific promoters. The expression of structural genes controlled by constitutive promoters is generally constant at a certain level, and there is no obvious difference in the expression level in different tissues and parts, without showing spatio-temporal specificity and not being induced by external factors; the genes initiated by tissue-specific promoters are only expressed in certain specific organs or tissue parts in plants and often show the characteristics of developmental regulation; inducible promoters are a class of promoters that can greatly increase the transcriptional level of genes under certain specific physical or chemical stimuli.
[0003] In most transgenic engineering, constitutive promoters are used to drive the expression of foreign genes. They are not restricted by time and space and are not induced by substances, and have the advantages of high efficiency, universality, stability, etc. However, precisely because they continuously initiate the expression of genes in various tissues and at various times of plants, constitutive promoters will cause plants to accumulate a large amount of heterologous proteins or metabolites in vivo, disturbing the original metabolic balance and even being poisoned by some foreign proteins, and then unable to grow normally or even die. With the development of plant genetic engineering, constitutive promoters can no longer meet the requirements, and researchers have begun to search for tissue-specific promoters with less impact and more specific action and inducible promoters that are easier to control. Tissue-specific promoters can not only increase the regional expression level but also avoid unnecessary waste caused by continuous expression, and have great advantages compared with the commonly used constitutive promoters in the past. Therefore, exploring tissue-specific promoters with strong specificity and precise expression has become a key and difficult point in plant genetic engineering.
[0004] Cotton is one of the important economic crops in the world. Cultivating cotton varieties with high yield, stress resistance, and excellent quality is of great significance to the economic development of our country and the world. During the growth process of cotton, the development of pollen is a key step. The vitality and maturity of pollen will directly affect the success rate and fruit setting rate of cotton self-pollination and cross-pollination, and then affect the yield of cotton fibers. In addition, the driving activity and specificity of pollen promoters also greatly affect the success or failure of regulating pollen fertility, creating plant sterile lines and restorer lines by genetic engineering means.
[0005] At present, the promoters used in genetically modified cotton are mainly constitutive promoters. This type of promoter is not restricted by time, space, or the induction of substances, and has advantages such as high efficiency and strong stability. However, it will also cause the plant to accumulate a large amount of heterologous proteins or metabolites in vivo, disturbing the original metabolic balance. The promoter of the upland cotton gene provided by this patent has the characteristic of pollen-specific expression, which can overcome the disadvantages of constitutive promoters, restrict the expression of genes in specific pollen tissues, not only reduce the energy consumption of plants, mitigate the impact on plant morphology and growth and development, but also increase the expression concentration of foreign genes in specific parts, enhance the effect of genetic transformation, and is of great significance in the functional analysis and identification of genes related to the growth and development of plant anthers, the creation of plant male sterile lines and restorer lines, etc. Summary of the Invention
[0006] The present invention provides a promoter derived from the pollen-specific expression gene GH_D01G2221 of upland cotton and its application, aiming to explore and utilize the pollen-specific promoter in upland cotton to enrich the selectivity of plant tissue-specific promoters.
[0007] To achieve the above object, the technical solution of the present invention is as follows: In the first aspect, the present invention provides a promoter of the pollen-specific expression GH_D01G2221 gene derived from upland cotton, and its nucleotide sequence is shown in SEQ ID NO.1. Primers were designed according to the sequence of the upland cotton GH_D01G2221 gene, and a 1208bp sequence (SEQ ID NO.1) upstream of the GH_D01G2221 gene was cloned and named promoter fragment P1. A plant recombinant expression vector pBI121-P1 was constructed based on the promoter fragment P1. The above vector was transferred into Arabidopsis thaliana Columbia type Col-0 and Nicotiana benthamiana by the method of Agrobacterium infection to create stably transformed materials and verify the expression specificity of the promoter fragment.
[0008] In the second aspect, based on the above pollen-specific promoter, the present invention provides a pollen-specific gene expression cassette, including a pollen-specific promoter, a target gene initiated by this promoter, and a termination codon. In actual production, the target gene can be selected according to actual needs. For example, if the target gene has the characteristic of high temperature resistance, connecting this gene into the above expression cassette can enhance the high temperature resistance of pollen tissues during plant growth and development, thereby achieving the effects of enhancing the vitality and fertility of pollen in high temperature environments and increasing the pollination success rate.
[0009] In the third aspect, the present invention also provides a plant recombinant expression vector containing the above pollen-specific promoter, as well as recombinant Escherichia coli and Agrobacterium containing this recombinant expression vector. In the examples, the expression vector that can be used to insert the pollen-specific promoter is pBI121, and the bacteria that can be used to contain this pollen-specific promoter are Agrobacterium or Escherichia coli.
[0010] Fourthly, the present invention also provides the above-mentioned pollen-specific promoter, or pollen-specific gene expression cassette, or plant recombinant expression vector, or the application of the recombinant bacterium in initiating the expression of a target gene in pollen in plants, which may specifically include the following steps: 1. Clone the pollen-specific promoter and construct a recombinant expression vector containing the pollen-specific promoter and the target gene; 2. Transfer the recombinant expression vector into Agrobacterium tumefaciens and then infect it into plants. For Arabidopsis thaliana, the floral dipping method is adopted, which includes dipping flowers, harvesting seeds, screening and identification to obtain transgenic plant lines.
[0011] In the above solution, the method for cloning the pollen-specific promoter is: using the genomic DNA of the upland cotton standard line TM-1 as a template and amplifying it with the primer pair shown in SEQ ID NO.2-3.
[0012] In the above solution, the object of the floral dipping method is the fully blooming flowers at the top of Arabidopsis thaliana.
[0013] In the above solution, the target gene recombined into the expression cassette is specifically expressed in pollen.
[0014] Pollen usually contains plant genetic information and is crucial for plant reproduction. The viability and maturity of pollen will directly affect the success rate of plant self-pollination, cross-pollination and fruit setting rate. Selecting a suitable pollen-specific promoter in plants can effectively regulate the expression of functional genes in pollen, reduce adverse effects on plants, maintain the normal growth of plants, and thus improve plant traits.
[0015] The beneficial effects of the present invention include: The present invention discovers a promoter for pollen-specific expression in upland cotton, which is used for the specific expression of foreign genes in cotton pollen, and further develops and utilizes it, providing technical support for the application of the upland cotton pollen-specific expression promoter in production. In addition, in-depth research helps to improve the application of pollen-specific promoters in basic research such as plant genetic breeding and improvement of agronomic traits. Description of the Drawings
[0016] Figure 1 It is a comparison diagram of the expression levels of GH_D01G2221 in different tissues of upland cotton TM-1;
[0017] Figure 2 It is a detection diagram of colony PCR of the PBI121 expression vector containing the GH_D01G2221 promoter transformed into Escherichia coli;
[0018] Figure 3 It is a detection diagram of colony PCR of the PBI121 expression vector containing the GH_D01G2221 promoter transformed into Agrobacterium tumefaciens GV3101;
[0019] Figure 4This is a photo taken by a stereomicroscope for the GUS activity detection of transgenic Arabidopsis thaliana positive seedlings. Among them, (A) shows photos of different parts of transgenic Arabidopsis thaliana positive seedlings, blank controls, and positive controls, and (B) shows photos of anthers of transgenic Arabidopsis thaliana positive seedlings at different stages. The GUS gene is significantly expressed in anthers at different stages. Specific Embodiments
[0020] Example 1: Expression Analysis of the GH_D01G2221 Gene in Different Tissues of Gossypium hirsutum
[0021] Previously, through the analysis of transcriptome data of different tissues of Gossypium hirsutum, it was found that the GH_D01G2221 gene has tissue-specific expression. Therefore, a comprehensive analysis of the spatio-temporal expression of GH_D01G2221 was decided.
[0022] Sampling: Samples such as pollen, petals, receptacles, bracts, sepals, stems, leaves, roots, ovules and fibers at different stages of Gossypium hirsutum standard line TM-1 were selected. Total RNA of the above samples was extracted respectively. After detecting the integrity of RNA by 1% agarose gel electrophoresis, the first strand of cDNA was reverse transcribed. Using this cDNA as a template, according to the exon sequence of the GH_D01G2221 gene (SEQ ID NO.10) aligned from the cotton genome database, with the Y8991 gene as an internal reference, primers were designed for RT-PCR analysis. The results are as Figure 1 shown that the GH_D01G2221 gene is almost only expressed in Gossypium hirsutum pollen, and the expression level in other tissue parts of Gossypium hirsutum is extremely low or not expressed.
[0023] Table 1 RT-PCR Primer Sequence Table
[0024]
[0025] Example 2: Cloning of Gene Promoter and Obtaining of Recombinant Bacteria
[0026] Using the genomic DNA of Gossypium hirsutum standard line TM-1 as a template, recombinant primers (restriction enzyme sites Hind III, BamH I) were designed, and a 1208bp fragment was amplified from the template by PCR and ligated to the pBI121 vector. The PCR recombinant primers and reaction system are shown in Table 2 and Table 3; DH5α competent cells were transformed. After 12 hours, single colonies were picked for shaking culture and sent for sequencing, and the sequences returned by sequencing were correctly aligned.
[0027] Table 2 PCR Recombinant Primer Sequence Table
[0028]
[0029] Table 3 PCR Reaction System Table
[0030] 20 μL system <![CDATA[ddH2O]]> 7 μL 2×KOD Buffer 10 μL cDNA 1 μL Primer F 1 μL Primer R 1 μL
[0031] The PCR conditions were pre-denaturation at 95 °C for 5 min, denaturation at 98 °C for 10 s, annealing at 57 °C for 10 s, extension at 68 °C for 10 s, for 34 cycles, and finally extension at 68 °C for 5 min.
[0032] After the obtained amplified DNA fragment was confirmed to have the correct sequence by sequencing, it was ligated into the PBI121 vector that had been digested with Hind III and BamH I in advance, and Escherichia coli DH5α was transformed. After the positive clones obtained by transformation were identified to have the correct sequence by PCR and sequencing ( Figure 2 ), they were named pBI121-P1, and plasmid DNA was extracted and transformed into competent Agrobacterium tumefaciens GV3101 cells by the liquid nitrogen freeze-thaw method. Screening was carried out on LB medium containing 50 mg / L Kana and 50 mg / L Rif. Positive clones were obtained by colony PCR identification, and PCR detection was performed as Figure 3 shown and could be used for subsequent experiments.
[0033] Example 3: Stable expression transformation of Arabidopsis thaliana by promoter-driven GUS reporter gene analysis
[0034] In this example, the recombinant expression vector constructed in Example 2 was transformed into wild-type Arabidopsis thaliana Col-0 by Agrobacterium to initiate the specific expression of the target gene GUS in pollen. The specific process is as follows:
[0035] The Arabidopsis thaliana material was Columbia (Col-0). The plant growth chamber conditions were 22 degrees Celsius, and the photoperiod conditions were 16 hours of light / 8 hours of darkness. Escherichia coli DH5α was used in the vector construction stage, and the Agrobacterium tumefaciens strain GV3101 containing the plant recombinant expression vector.
[0036] Take an appropriate amount of Arabidopsis thaliana seeds and place them in a 2 mL centrifuge tube. Inject 75% alcohol (v / v) into the tube and surface sterilize for 5 min. Aspirate the alcohol, wash with sterile water 6 - 7 times, aspirate the sterile water, add 1000 μL of agar at an appropriate temperature to the tube, mix well by pipetting, then pour the seeds and agar in the tube onto a pre-cooled 1 / 2 MS solid medium plate. Gently push the plate so that the liquid agar drives the seeds to spread evenly on the surface of the plate. After the agar solidifies, seal it with a sealing film. After vernalization in the dark at 4 °C for 3 d, take out the plate and place it in an incubator at a constant temperature of 22 °C under long-day conditions (light period duration 16 h, dark period duration 8 h) for cultivation. After 7 d, transplant the Arabidopsis thaliana seedlings into the culture soil (nutrient soil: vermiculite = 1:1, volume ratio), cover with a plastic wrap to keep moisture and slow down the seedlings. After 7 d, remove the plastic wrap and continue cultivation. Four to six weeks after transplantation, Arabidopsis thaliana bolts and flowers, and it is ready for infection. Water the Arabidopsis thaliana one day before infection, which is beneficial for subsequent infection work.
[0037] Agrobacterium liquid containing the GV3101 bacterial liquid of the plant recombinant plasmid pBI121-P1 in Example 2 was inoculated into a liquid LB medium containing 50 μg / mL kanamycin and 50 μg / mL rifampicin at a volume ratio of 1:100, and cultured overnight at 28 °C with shaking at 200 rpm. When the OD 600 of the bacterial liquid was about 1.2, shaking was stopped. The bacteria were collected by centrifugation at 4000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended with the resuspension solution to adjust the OD 600 to 0.6 - 0.8 (the resuspension solution requires weighing 0.443 g of MS salts, 1% sucrose to 200 mL of distilled water, and adding 100 μL of silwet L-77). The resuspended bacterial liquid was poured into a 15 cm culture dish, and the inflorescences of Arabidopsis thaliana were immersed in the resuspension solution containing Agrobacterium for about 90 s, and the excess resuspension solution on the surface of the plant was gently wiped with a clean paper towel. After the infection, the Arabidopsis thaliana plants were cultured in the dark for 18 - 22 h and then grown under normal light conditions.
[0038] Subsequently, Arabidopsis thaliana was continuously cultured at 22 °C under a 16 h light / 8 h dark condition. The harvested mature seeds were fully dried and vernalized in a 4 °C refrigerator for 3 d. After alcohol sterilization, they were evenly sown on a 1 / 2 MS plate containing 50 μg / mL hygromycin to screen for transgenic positive plants. The positive seedlings were transplanted into the culture soil (nutrient soil: vermiculite = 1:1, volume ratio) for culture, and the seeds were collected individually and continued to be screened until a homozygous high-generation line was obtained.
[0039] The T3-generation seeds received were sown on a 1 / 2 MS medium containing hygromycin, and the lines that could grow roots were screened for positive identification. The mixed samples of roots, stems, and leaves of the lines were used to extract DNA by the CTAB method, and using this DNA as a template, PCR amplification was performed with the combination of pBI121-F (5'-CACTGACGTAAGGGATGACGCAC-3', SEQ ID NO.4) and pBI121-R (5'-CACCAACGCTGATCAATTCCACA-3', SEQ ID NO.5). Successful DNA-level verification was considered when the correct band position (at about 1200 bp) was detected by agarose gel electrophoresis.
[0040] Example 4: GUS staining of Arabidopsis thaliana transgenic materials
[0041] Using the GUS reporter gene in the pBI121 vector, the position where the promoter expresses the GUS gene can be observed through GUS staining, thereby further determining whether it has the characteristics of pollen-specific expression. In the positive lines identified at the DNA level, different parts were taken for GUS histochemical staining. The plant materials were soaked in a solution containing the substrate X-Gluc, and the positions with GUS activity in the plants were stained blue spots or showed blue. The specific steps of GUS staining are as follows: Fix with 90% (v / v) acetone at 4°C for 20 min, wash twice with GUS washing solution, cover the leaves with GUS staining solution, incubate overnight in a 37°C incubator, and after the color is developed, decolorize with 95% (v / v) alcohol. Observe with the naked eye or under a microscope, and the stained blue parts are the GUS expression sites.
[0042] From Figure 4 It can be seen from (A)-(B) that blue appears in different tissues of the positive control, and colorless appears in different tissues of the blank control. Only blue pollen was observed in pBI121-P1, indicating that the 1-1208 bp of the promoter of the GH_D01G2221 gene screened in the present invention has pollen-specific promoter activity.
[0043] In summary, the pollen-specific gene expression cassette of the 1-1208 bp fragment of the GH_D01G2221 promoter derived from Gossypium hirsutum developed in the present invention can be specifically expressed in pollen, so it has great application potential in the fields of plant new variety breeding, functional analysis and identification of genes related to plant pollen growth and development, creation of plant male sterile lines and restorer lines, etc.
[0044] The above is the optimized implementation mode of the present invention, and the scope of the rights of the present invention cannot be limited by this. For those of ordinary skill in the art, any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A pollen-specific promoter derived from Gossypium hirsutum, characterized in that, The nucleotide sequence of the pollen-specific promoter is shown in SEQ ID NO.
1.
2. A pollen-specific gene expression cassette, characterized in that, It includes the pollen-specific promoter described in claim 1, a gene transcribed by this promoter, and a stop codon.
3. A plant recombinant expression vector, characterized in that, It includes the pollen-specific promoter described in claim 1.
4. The application of the pollen-specific promoter described in claim 1, or the expression cassette described in claim 2, or the recombinant expression vector described in claim 3 in initiating the expression of a target gene in pollen in a plant.
5. The application according to claim 4, characterized in that Clone the pollen-specific promoter described in claim 1, construct a plant recombinant expression vector containing the pollen-specific promoter; transfer the plant recombinant expression vector into Agrobacterium, and then infect it into the plant to screen for stable transgenic plant lines.
6. The application according to claim 4, characterized in that The plant includes: Arabidopsis thaliana.
7. The application according to claim 5, characterized in that, When constructing the Arabidopsis thaliana transgenic line, the floral dip method is used.