CN116042612B - Peppermint lipid transport protein gene promoter specifically expressed by glandular hairs and application thereof - Google Patents
Peppermint lipid transport protein gene promoter specifically expressed by glandular hairs and application thereof Download PDFInfo
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Abstract
The invention discloses a mint lipid transport protein gene promoter specifically expressed by glandular hairs and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the mint lipid transport protein gene promoter is shown as SEQ ID NO.1, and the recombinant plant expression vector using the promoter is used for plant transgene, so that the downstream gene can be specifically expressed in tobacco glandular wool, and the mint lipid transport protein gene promoter has important application value in the functional research of glandular wool development and metabolic synthesis related genes and the regulation and control research of plant metabolic engineering.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a mint lipid transport protein gene promoter specifically expressed by glandular hairs and application thereof.
Background
Mint MENTHA CANADENSIS L is a perennial herb of Mentha of Labiatae, and its stem and leaf contains abundant essential oil, and is one of the spice crops with largest cultivation area. The peppermint essential oil is widely applied to the fields of food, chemical industry, medicine and the like, and has important economic value. Peppermint is also a traditional Chinese medicinal material in China, and has the effects of dispelling wind and heat, clearing head and eyes and the like. The peppermint essential oil is mainly synthesized, secreted and stored in glandular hairs on the surfaces of stems and leaves of the peppermint essential oil. The glandular Mao Shuyu specialized plant epidermal hair can be divided into secretory and non-secretory glandular hair according to its functions, the secretory glandular hair includes head glandular hair and shield glandular hair, and is mainly composed of basal cell, stem cell and one or more secretory cells at the top end, and their forms have a certain correlation with their synthesized metabolic products. For example, the hair of the head gland secretes non-volatile substances like acyl saccharides, resins and the like secreted by tobacco and tomato hair of the head gland through the surface of the top secretion cells, and plays a role in the plant defense process; whereas the scutellum gland hair mainly produces volatile substances, such as a sub-epidermal space covered by a horny layer outside the secretory cells of the scutellum gland hair, for storing the produced essential oil.
The promoter is a nucleic acid sequence with transcription initiation activity at the upstream of the 5' end of the functional gene, and is an important cis-acting element for regulating gene expression. In general plant research, a large number of constitutive promoters, such as 35S promoter and Ubiqutin promoter, are used for over-expression of genes and gene function verification. The disadvantage is that ectopic expression of genes may affect plant growth and development and metabolism, and the research result is not ideal. The tissue-specific promoter can regulate and control the expression of the target gene only in specific organs or tissue parts of the plant, thereby being beneficial to the targeted scientific research.
Glandular wool is taken as an important tissue part for generating and secreting plant secondary metabolites, and in functional research on genes related to glandular wool development and metabolic synthesis, a target functional gene is transferred into a plant, particularly expressed in the glandular wool system, through genetic transformation, corresponding functional verification is carried out, and regulation and control of a plant secondary metabolic network are deeply known. Therefore, the development of the promoter for the specific expression of the glandular wool, the research of gene functions in the glandular wool system, and the development of the promoter has important significance and wide application prospect in the research of plant metabolic engineering by utilizing the expression of the glandular wool system and the production of metabolic products of plants.
Disclosure of Invention
Aiming at the problems existing in the prior art, the technical problem to be solved by the invention is to provide a mint lipid transporter gene promoter. The invention also solves the other technical problem of providing application of the mint lipid transporter gene promoter.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
Aiming at the problems existing in the prior art, the technical problem to be solved by the invention is to provide a mint lipid transporter gene promoter. The invention also solves the other technical problem of providing application of the mint lipid transporter gene promoter.
A mint lipid transporter gene promoter has a nucleotide sequence shown in SEQ ID NO. 1.
Expression cassettes, vectors, transgenic cell lines or host bacteria containing said mint lipid transporter gene promoters.
Further, the host bacteria are agrobacterium.
Further, the vector is a recombinant plant expression vector.
Further, the vector assembles the gene of interest at the 3' end of the promoter of the peppermint lipid transporter.
Further, the target gene is a GUS reporter gene.
The application of the mint lipid transport protein promoter in regulating and controlling the specific expression of a target gene in plant glandular hair tissue.
Further, the application specific steps are as follows: and connecting a target gene to the downstream of the mint lipid transporter gene promoter, constructing a recombinant plant expression vector, introducing the recombinant plant expression vector into plant cells, tissues or organs, and culturing the transformed plant cells, tissues or organs into plants to obtain the transgenic plants.
Further, the plant in the application is a plant with glandular hairs.
Further, the plant is a Labiatae plant or a Solanaceae plant.
Compared with the prior art, the invention has the beneficial effects that:
The invention provides a mint lipid transport protein gene promoter sequence for the first time, and constructs a recombinant expression vector of the promoter to carry out plant transgenosis, so that the downstream gene can be specifically expressed in tobacco leaf secretory gland hair and hardly expressed in other tissues or development stages; the promoter sequence is derived from peppermint, and the specific expression of the promoter sequence in the leaf glandular hair of tobacco suggests that the promoter sequence can specifically regulate and control the target gene to reach the glandular hair of peppermint or the glandular Mao Zhongbiao of other plants; the promoter is used for researching gene functions in a plant glandular hair system, and has important value for researching plant metabolic engineering by utilizing the expression of the plant glandular hair system and the production of metabolic products.
Drawings
FIG. 1 is an electrophoretogram of a lipid transporter gene promoter obtained by PCR amplification from the mint genome, M is a standard molecular weight marker (DNA MARKER), and the arrow indicates a promoter band;
FIG. 2 is a diagram of constructing a promoter and pGC-GUS recombinant vector;
FIG. 3 PCR identification map of transgenic tobacco; 1. 4-9 strains are positive plants;
FIG. 4 is a GUS staining chart of transgenic homozygous cationic plants obtained after transformation of tobacco, A: tobacco leaves; b: the tobacco leaves are provided with glandular hairs; the GUS reporter gene is expressed in the leaf glandular hairs of tobacco.
Detailed Description
The invention is further described below in connection with specific embodiments. The experimental methods in which specific conditions are not specified in the examples are generally carried out according to conventional methods, for example, the conditions described in the guidelines for molecular cloning experiments (third edition, J. Sam Brookfield et al), or according to the methods recommended by the manufacturers of kits.
Example 1: obtaining of mint lipid transport protein gene promoter
(1) The genomic DNA of peppermint was extracted by CTAB (peppermint leaves were collected from the plant institute of China academy of sciences of Jiangsu province in five months of two zeros and one year).
(2) The following specific primers were designed and synthesized based on the mint lipid transporter sequence:
SP1：5′-CACTTCACCTTAGAGCAATCGG-3′：
SP2：5′-GAATATCCGCTGGCCAAAGATT-3′；
SP3：5′-TGACTTCATTACTCCGGCCAT-3′。
The mint genomic DNA was used as a template, subjected to a thermal asymmetric PCR reaction with the degenerate primers in a chromosome Walking kit (Genome Walking, takara, dalian), flanking sequences were obtained by three times of nested PCR, and the PCR products were recovered and DNA sequenced. Specific procedures and PCR amplification conditions were performed according to the instructions in the kit.
(3) Sequencing results showed that a 1704bp promoter region (SEQ ID NO. 1) upstream of the lipid transporter gene ATG was obtained in the mint genome. Designing the promoter sequence amplification primer ProF:5'-TGTCAATAGACACCCCTTAAA-3': proR:5'-TGTTGAAAGCGAGAACGTTT-3' PCR amplification verification is performed using mint genomic DNA as a template, and the verification result is shown in FIG. 1.
Example 2: constructing a plant recombinant expression vector containing the promoter sequence.
According to the mint lipid transporter gene promoter sequence and pGC-GUS (G5) vector map obtained in example 1, kpnI single enzyme cleavage site is selected for carrying out homologous recombination method vector construction, mint genomic DNA is taken as a template, homologous recombination primers are designed, and PCR amplification and vector connection are carried out. The ligation product was transferred into E.coli competent cells, cultured overnight at 37℃on plates of 50mg/L kana antibiotic, and positive single colony plasmid was picked for sequencing verification. The obtained recombinant vector drives the expression of beta-glucuronidase Gene (GUS) by a peppermint lipid transporter gene promoter (SEQ ID NO. 1), and the schematic diagram of the recombinant vector is shown in figure 2.
Example 3: expression of exogenous proteins in tobacco using the plant recombinant expression vectors described above
The recombinant vector constructed in example 2 was transferred into Agrobacterium GV3101 for Agrobacterium-mediated genetic transformation and identification of tobacco. The method comprises the following specific steps:
(1) Preparing a conversion bacterial liquid: selecting positive monoclonal agrobacterium into YEB culture medium containing 50 mug/mL rifampicin and 50 mug/mL kanamycin, and shaking at 28 ℃ and 200rpm until turbidity; sucking 1mL of bacterial liquid into 25mL of new YEB culture medium, and performing amplification culture until OD600 is 0.6-0.8; centrifuging, discarding the supernatant, and suspending and precipitating with 20mLMS salt solution to obtain the infection liquid for later use.
(2) Genetic transformation and identification of tobacco leaf disc method: taking out the aseptic tobacco leaves obtained by tissue culture on an ultra-clean workbench, cutting the aseptic tobacco leaves into small pieces with the length of 0.5cm multiplied by 0.5cm, putting the cut tobacco leaves into agrobacterium tumefaciens infection liquid, and slightly shaking for 10min; the redundant bacterial liquid is sucked by filter paper, the leaves are put into a co-culture medium (MS+2 mg/L6-BA+0.1 mg/L NAA+3% sucrose+0.8% agar+50 mg/L acetosyringone) and grown for two days under the dark condition at 25 ℃; after two days, the culture medium was transferred to a screening medium (MS+2 mg/L6-BA+0.1 mg/L NAA+3% sucrose+0.8% agar+200 mg/L Cephalosporium+50 mg/L kanamycin), and the culture was carried out at 25℃under light for 16 hours/dark for 8 hours. The medium was changed every 15-20 days. Cutting off the resistant buds growing to more than 1cm along the basal part, transferring to a MS culture medium containing 50mg/L kanamycin, and hardening seedlings and transplanting after rooting. And taking leaves for PCR detection of transgenic plants. The carrier detection primer is F:5'-ACGGCTAAAACACTGGCACT-3' r:5'-GTGTGTGTTTTGGCATGGAG-3'. The PCR detection result shows that the target DNA fragment can be amplified by using the designed detection primer, and the target fragment is not amplified when wild type tobacco or non-transformed tobacco genome DNA is used as a template, as shown in figure 3.
(3) The detected positive plants were observed by beta-Glucosidase (GUS) staining. The results are shown in fig. 4, and the tobacco leaves are stained; b tobacco glandular hairs; GUS reporter gene is expressed in transgenic tobacco leaf gland Mao Zhongbiao, and the blue color of the leaf non-glandular hair tissue is hardly seen, which indicates that the promoter can drive the specific expression of target gene in tobacco glandular hair.
Claims (8)
1. A mint lipid transporter gene promoter has a nucleotide sequence shown in SEQ ID NO. 1.
2. An expression cassette, vector or host bacterium comprising the mint lipid transporter gene promoter of claim 1.
3. The host bacterium of claim 2, wherein the host bacterium is agrobacterium.
4. The vector of claim 2, wherein the vector is a recombinant plant expression vector.
5. The vector of claim 2, wherein the vector assembles the gene of interest at the 3' end of the mint lipid transporter promoter.
6. The vector of claim 5, wherein the gene of interest is a GUS reporter gene.
7. The use of the mint lipid transporter gene promoter according to claim 1 for regulating the specific expression of a target gene in glandular hair tissue of a plant, wherein the plant is a Labiatae plant or a Solanaceae plant.
8. The use according to claim 7, wherein a desired gene is linked downstream of the mint lipid transporter gene promoter to construct a recombinant plant expression vector, and the recombinant plant expression vector is introduced into a plant cell, tissue or organ, and the transformed plant cell, tissue or organ is cultivated into a plant to obtain a transgenic plant.
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| CN116042612B true CN116042612B (en) | 2024-04-16 |
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| CN202210882447.0A Active CN116042612B (en) | 2022-07-25 | 2022-07-25 | Peppermint lipid transport protein gene promoter specifically expressed by glandular hairs and application thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009097823A1 (en) * | 2008-02-04 | 2009-08-13 | Shanghai Institutes For Biological Sciences, Cas | Plant epidermal hair specific expression promoter fif1 and its use |
| CN103966216A (en) * | 2014-04-18 | 2014-08-06 | 浙江大学 | Specific promoter ZFP6 of tobacco glandular hair and application of specific promoter |
| CN104059915A (en) * | 2014-06-10 | 2014-09-24 | 浙江大学 | Tobacco glandular trichome specific promoter GIS2 and application thereof |
| CN108070594A (en) * | 2017-12-22 | 2018-05-25 | 河南农业大学 | Tobacco glandular hairs TTR1 promoters, its expression vector and its application |
| CN116334094A (en) * | 2022-07-25 | 2023-06-27 | 江苏省中国科学院植物研究所 | Mint nonspecific lipid transporter gene and expression protein and application thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009097823A1 (en) * | 2008-02-04 | 2009-08-13 | Shanghai Institutes For Biological Sciences, Cas | Plant epidermal hair specific expression promoter fif1 and its use |
| CN103966216A (en) * | 2014-04-18 | 2014-08-06 | 浙江大学 | Specific promoter ZFP6 of tobacco glandular hair and application of specific promoter |
| CN104059915A (en) * | 2014-06-10 | 2014-09-24 | 浙江大学 | Tobacco glandular trichome specific promoter GIS2 and application thereof |
| CN108070594A (en) * | 2017-12-22 | 2018-05-25 | 河南农业大学 | Tobacco glandular hairs TTR1 promoters, its expression vector and its application |
| CN116334094A (en) * | 2022-07-25 | 2023-06-27 | 江苏省中国科学院植物研究所 | Mint nonspecific lipid transporter gene and expression protein and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 薄荷McHD-Zip3基因的克隆及其调控腺毛发育的功能分析;陈泽群 等;植物资源与环境学报;20201231;第29卷(第3期);第1-10页 * |
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