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
- Publication number
- CN116042612B CN116042612B CN202210882447.0A CN202210882447A CN116042612B CN 116042612 B CN116042612 B CN 116042612B CN 202210882447 A CN202210882447 A CN 202210882447A CN 116042612 B CN116042612 B CN 116042612B
- Authority
- CN
- China
- Prior art keywords
- plant
- mint
- promoter
- vector
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000000762 glandular Effects 0.000 title claims abstract description 31
- 108010078791 Carrier Proteins Proteins 0.000 title claims abstract description 27
- 210000004209 hair Anatomy 0.000 title claims abstract description 27
- 150000002632 lipids Chemical class 0.000 title claims abstract description 27
- 235000016257 Mentha pulegium Nutrition 0.000 title description 12
- 235000004357 Mentha x piperita Nutrition 0.000 title description 12
- 235000001050 hortel pimenta Nutrition 0.000 title description 12
- 241001479543 Mentha x piperita Species 0.000 title 1
- 241000196324 Embryophyta Species 0.000 claims abstract description 44
- 235000006679 Mentha X verticillata Nutrition 0.000 claims abstract description 27
- 235000002899 Mentha suaveolens Nutrition 0.000 claims abstract description 27
- 235000001636 Mentha x rotundifolia Nutrition 0.000 claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 23
- 239000013604 expression vector Substances 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 239000013598 vector Substances 0.000 claims description 16
- 230000014509 gene expression Effects 0.000 claims description 13
- 230000009261 transgenic effect Effects 0.000 claims description 7
- 241000589158 Agrobacterium Species 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 5
- 210000000056 organ Anatomy 0.000 claims description 5
- 108700008625 Reporter Genes Proteins 0.000 claims description 4
- 241000207923 Lamiaceae Species 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 241000208292 Solanaceae Species 0.000 claims description 2
- 241000208125 Nicotiana Species 0.000 abstract description 21
- 235000002637 Nicotiana tabacum Nutrition 0.000 abstract description 21
- 238000011160 research Methods 0.000 abstract description 8
- 210000002268 wool Anatomy 0.000 abstract description 8
- 238000011161 development Methods 0.000 abstract description 6
- 230000002503 metabolic effect Effects 0.000 abstract description 6
- 238000012269 metabolic engineering Methods 0.000 abstract description 3
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 108700019146 Transgenes Proteins 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 244000246386 Mentha pulegium Species 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 210000004907 gland Anatomy 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- 239000000341 volatile oil Substances 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000003248 secreting effect Effects 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000002955 secretory cell Anatomy 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 241001619326 Cephalosporium Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 235000014435 Mentha Nutrition 0.000 description 1
- 241001072983 Mentha Species 0.000 description 1
- 235000016278 Mentha canadensis Nutrition 0.000 description 1
- 244000245214 Mentha canadensis Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 101100268446 Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950 / KT2440) proR gene Proteins 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- -1 acyl saccharides Chemical class 0.000 description 1
- 238000007846 asymmetric PCR Methods 0.000 description 1
- 210000000270 basal cell Anatomy 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000012881 co-culture medium Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 230000001254 nonsecretory effect Effects 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 229930000223 plant secondary metabolite Natural products 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
- C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
- C12N15/8212—Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/34—Vector systems having a special element relevant for transcription being a transcription initiation element
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210882447.0A CN116042612B (en) | 2022-07-25 | 2022-07-25 | Peppermint lipid transport protein gene promoter specifically expressed by glandular hairs and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210882447.0A CN116042612B (en) | 2022-07-25 | 2022-07-25 | Peppermint lipid transport protein gene promoter specifically expressed by glandular hairs and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116042612A CN116042612A (en) | 2023-05-02 |
CN116042612B true CN116042612B (en) | 2024-04-16 |
Family
ID=86112035
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
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 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116042612B (en) |
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 |
-
2022
- 2022-07-25 CN CN202210882447.0A patent/CN116042612B/en active Active
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 * |
Also Published As
Publication number | Publication date |
---|---|
CN116042612A (en) | 2023-05-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108374012B (en) | Rubber powdery mildew endogenous promoter WY51 and application thereof | |
MXPA02003456A (en) | Method for regulating transcription of foreign genes in the presence of nitrogen. | |
CN107974454B (en) | Rubber powdery mildew endogenous promoter WY193 and application thereof | |
CN112852820B (en) | Lilium regale inducible promoter PD1 and application thereof | |
CN110029113B (en) | Encoding gene related to rice grain type growth and development and application thereof | |
CN112708625A (en) | Lilium regale inducible promoter PG1 and application thereof | |
Dolgov et al. | Agrobacterial transformation of chrysanthemum | |
CN113234726B (en) | Tobacco glandular hair specific promoter pNtTCP9a and application thereof | |
CN110938617A (en) | Lilium regale LrPAL-1 gene and application thereof | |
CN111621504B (en) | Stress-resistant gene BjuIBS of tumorous stem mustard and application thereof | |
CN107058317B (en) | Pollen specific promoter and application thereof | |
CN116042612B (en) | Peppermint lipid transport protein gene promoter specifically expressed by glandular hairs and application thereof | |
CN116103290A (en) | Tomato root specific expression promoter pSlROOT2 and application thereof | |
CN107058324B (en) | Rice root specific expression promoter POsRO4 and corresponding rice cultivation method | |
CN106434659B (en) | Soybean low-temperature inducible promoter, recombinant expression vector containing promoter and application | |
CN114480447A (en) | Application of kenaf thioredoxin analog protein gene HcTrx and recombinant vector thereof in VIGS silencing system | |
CN115354042A (en) | Promoter and preparation method and application thereof | |
KR100862599B1 (en) | Promoter for the high level expression in plant-cell culture and vector using the same | |
CN117721111B (en) | Mangrove plant avicennia marina endogenous promoter AMGT P5 and application thereof | |
CN116064509B (en) | Plant constitutive promoter CEPro432 and application thereof | |
CN114561387B (en) | Peanut promoter and application thereof | |
CN117924451B (en) | Transcription factor CIB3 and application of coding gene thereof | |
CN112322621B (en) | Eucommia DIR1 gene MeJA response promoter and application thereof | |
CN109825507B (en) | Plant drought-induced synthetic promoter SP18 and application thereof | |
KR101825960B1 (en) | Root―specific promoter derived from Oryza sativa and use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |