CN112608924A - Inducible promoter PCHI and application thereof - Google Patents
Inducible promoter PCHI and application thereof Download PDFInfo
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Abstract
The invention discloses an inducible promoter PCHI, which is derived from Lilium regale and has a nucleotide sequence shown as SEQ ID NO. 1, and the molecular biology and genetic engineering related technology researches prove that the Lilium regale promoter PCHI responds to methyl jasmonate and biological stress; the expression frame constructed by the Lilium regale promoter PCHI and the beta-glucuronidase gene is transferred into tobacco for expression, and the glucuronidase activity of the transgenic tobacco is quantitatively detected by a fluorescence method, so that the result shows that the glucuronidase activity of the transgenic tobacco is obviously enhanced after the treatment of methyl jasmonate, fusarium oxysporum, fusarium solani and nigrospora oryzae; the Lilium regale promoter PCHI is induced by methyl jasmonate and biological stress factors and can be used in plant disease-resistant genetic engineering.
Description
Technical Field
The invention relates to the field of molecular biology and genetic engineering related research, in particular to an inducible promoter PCHI and application thereof.
Background
A promoter is a DNA sequence located upstream of a gene and specifically recognized by RNA polymerase. It acts like a "switch" controlling the initial time and extent of gene expression. The promoter composition includes a core promoter and upstream promoter elements. The core promoter consists of a transcription initiation site, a TATA box and 5' UTR sequences. Upstream promoter elements include the CAAT box, the GC box and some constitutive and specific elements that bind to the corresponding protein factor to increase transcription efficiency. Promoters can be classified according to function and mode of action into constitutive promoters (the expression of a gene is not controlled by temporal and spatial limitations and exogenous factors), inducible promoters (the expression of a gene is induced by exogenous physical and chemical factors, no inducer exists, the expression level of the gene is low or even no), and tissue-specific promoters (the expression of a gene is distributed in a certain tissue or organ of a plant). However, under certain conditions, a promoter may have the characteristics of both types of promoters, e.g., most fruit-specific promoters have ethylene response elements present in combination, and thus may also be considered ethylene-inducible.
Plant genetic engineering is a method of introducing foreign genes into recipient cells, integrating them with recipient chromosomes, and altering the genetic characteristics of the recipient plants. It can not only overcome the reproductive isolation between species, but also greatly accelerate the plant breeding process. In genetic engineering, the expression of a foreign gene must be driven by a promoter. Most of the traditional genetic engineering uses constitutive promoters, which are expressed with high intensity throughout the life cycle of the plant, resulting in excessive accumulation of gene products and consequent metabolic disorders and even plant death. The inducible promoter is formed in the process of adapting to the environment and evolving for a long time of plants, and can respond to special biological, physical and chemical signals so as to improve the transcription level of a specific gene to adapt to the environment change in a certain range. Under the condition of no inducing factor, the coding gene controlled by the gene is not expressed or is expressed in a background mode. Once the inducing factor appears in the environment, the expression of the coding gene is rapidly increased, and the regulation of the gene is stopped after the inducing factor is removed. In genetic engineering, if the foreign gene transferred into a plant is not controlled, the foreign gene can be expressed in the plant in a large amount, so that a large amount of protein is accumulated and energy is wasted. The addition of the inducible promoter on the vector can regulate the expression of the target gene when being stimulated by the outside, well solves the problem of unlimited expression of the exogenous gene, and the inducible promoter drives the expression of the exogenous gene to be controlled by specific physical or chemical signals, so that the expression of the exogenous gene can be more finely controlled. Therefore, research on inducible promoters has been one of the hot spots in plant molecular biology and genetic engineering research. (Yangzui, Baijiarong, Lirui, Changli. research on inducible promoter in plant genetic engineering progress [ J ] Shanxi agricultural science, 2018, 46(02): 292-.
Inducible promoters are often referred to by an inducing signal, such as light-inducible promoters, heat-inducible promoters, wound-inducible promoters, hormone-inducible promoters and fungal-inducible promoters. Using chromosome walking technique from lily (A), (B), (C)Lilium regale) Is/are as followsPR10-51489bp promoter is amplified from the end of gene and connected with GUS reporter gene to be transferred into tobacco: (Nicotiana tabacum) In the middle, the results show that lily is usedPR10-5The promoter is a multiple stress-inducible promoter. Gibberellin, abscisic acid and ethylene pairsPR10-5The promoters have positive regulation and control functions, wherein gibberellin has the strongest induction effect on the promoters; after treatment of abiotic stresses such as salt stress and injury stress, the GUS activity of the transgenic tobacco is obviously enhanced, which shows that the salt stress and the injury stress are also positively regulated and controlledPR10-5A promoter; fusarium oxysporum (F.), (Fusarium oxysporum) Sclerotinia sclerotiorum (A) and (B)Sclerotinia sclerotiorum) And Botrytis cinerea (Botrytis cinerea) Processing pairPR10-5The induction of GUS activity of the promoter is also very significant (Chen)R, He H, Yang Y, et al, Functional characterization of a pathogenesis-related protein family 10 gene, LrPR10-5, from Lilium regale Wilson, Australia Plant Path, 2017, 46(3): 1-9). From Erythrochloe arundinacea (Erianthus arundinaceus) The PR10 promoter obtained by cloning is transferred into tobacco and rice after being connected with reporter gene GUSOryza sativa) And sugar caneSaccharum officinarum). GUS enzyme activity determination results show that the treatment of wound stress, methyl jasmonate and abscisic acid induces the festuca arundinaceaPR10Expression of the promoter (Chakravarthi M, Syamaladevi DP, Harunipriya P, Augustine SM, Subramonian N. A novel PR10 promoter fromErianthus arundinaceus directs high constitutive transgene expression and is enhanced upon wounding in heterologous plant systems. Mol Biol Rep, 2016, 43(1):17-30）。
Disclosure of Invention
The invention aims to provide an inducible promoter PCHI which is derived from Lilium regale and the nucleotide sequence of which is shown as SEQ ID NO. 1.
The other purpose of the invention is to apply the promoter in gene engineering, namely to induce the expression promoter to regulate the specific high-efficiency expression of the exogenous gene in the transgenic receptor plant under the biotic stress or the action of plant hormone.
The invention relates to a separation inducible promoter fragment and identification of the expression activity thereof, the invention obtains the inducible promoter from Lilium regale by cloning, and the length of the promoter is 491 bp; bioinformatic analysis indicated that the inducible promoter contains a series of different cis-acting elements, such as light regulatory element (SORLIP 1 AT), gibberellin response element (GT 1 CONSENSUS), abscisic acid response Element (EBOXBNNAPA), high salt and dark inducible element (ABRELATED 1), and the like. The WRKY transcription factor plays an important regulation role in the disease-resistant defense response of plants, and the inducible promoter sequence has 1W-box (C/TTGACC/T) cis-acting element of WRKY.
The inducible promoter fragment separated and cloned by the invention is used for replacing 35s promoter of CaMV on a pBI121 vector, and the inducible promoter drives a reporter geneGUSExpression cassette of (1), by root cancerAgrobacterium (A) and (B)Agrobacterium tumefaciens) The promoter is transferred into model plant tobacco for expression, and the expression characteristic of the inducible promoter is revealed through further experiments, thereby laying a foundation for regulating the high-efficiency specific expression of the exogenous gene in the transgenic plant by utilizing the promoter at the later stage. The inventors named this promoter PCHI.
Transferring the expression frame of GUS driven by the PCHI promoter into tobacco, treating the transgenic tobacco plant by adopting phytohormone and biological stress, and carrying out fluorescence quantitative analysis on GUS activity, wherein the detection result shows that the PCHI promoter responds to the treatment of the phytohormone and the biological stress, and the methyl jasmonate and the fusarium oxysporum (F.) (B.) are subjected to the treatment of the phytohormone and the biological stressFusarium oxysporum) Fusarium solani (F.solani) (II)Fusarium solani) "Black rice spore (Nigrospora oryzae) Can obviously induce the activity of promoter PCHI.
The promoter PCHI can be applied to the induced expression of exogenous genes in genetic engineering, and the specific operation is as follows:
(1) extracting genome DNA from young tissues of Lilium regale by using a specific primer for amplifying the PCHI, amplifying the PCHI by Polymerase Chain Reaction (PCR), connecting the PCHI to a pGEM-T vector, and sequencing to obtain a clone with a correct sequence;
(2) the pGEM-T-PCHI vector is cut by restriction enzyme, and a promoter fragment is recovered; meanwhile, a constitutive expression promoter on the plant expression vector is removed by adopting proper restriction enzyme digestion, and a large vector segment is obtained by glue recovery; then the obtained PCHI fragment and pBI121-GUSConnecting the vector segments to construct a plant induction expression vector; then the constructed plant induction expression vector is transferred into a receptor plant through the mediation of agrobacterium tumefaciens. When a transgenic plant is infected by fusarium oxysporum, fusarium solani and nigrospora oryzae, a target gene driven by the promoter PCHI can induce and up-regulate the expression level, and in addition, methyl jasmonate in vivo and in vitro can also induce the high-level expression of the target gene.
The invention provides a new promoter for inducing expression in plant genetic engineering application. The 35S promoter from cauliflower mosaic virus is commonly used as a plant over-expression vector in genetic engineering, the promoter is a constitutive expression promoter, the expression of a target gene is approximately constant at a certain level, and the expression levels of different tissues and parts are not obviously different, so that the expression of an exogenous gene transferred into a plant is not controlled, and a large amount of protein is accumulated and energy is wasted. The inducible promoter can improve the expression quantity of genes when a plant is influenced by external stress or chemical factors, and can regulate the expression of target genes after stress removal or chemical treatment, so that the effects of protecting the plant and resisting external stimulation can be ensured when the plant is stressed by adversity, and otherwise, the energy of the plant is not wasted in a proper environment. In addition, in the genetic engineering application, the inducible promoter not only can avoid the excessive consumption of plant energy caused by the continuous expression of the target gene, but also can eliminate the damage to the plant itself caused by the accumulation of gene products. The expression activity of the PCHI promoter is obviously induced by methyl jasmonate and biological stress (fusarium oxysporum, fusarium solani and black sporotrichum oryza), so the invention has wide application prospect in genetic engineering of biological stress resistance.
Drawings
FIG. 1 shows the results of gel recovery detection of the promoter PCHI (Panel A) and pBI121 vector (Panel B) in the present invention;
FIG. 2 is a diagram showing pBI121-PCHI-GUSConverting a positive clone detection result of the escherichia coli, wherein the positive control is PCR reaction taking pGEM-T-PCHI plasmid as a template, and the blank control is PCR reaction taking sterile water as a template;
FIG. 3 is a diagram showing a part pBI121-PCHI-GUSPCR screening result of transgenic tobacco, wherein the positive control is plasmid pBI121-PCHI-GUSPCR reaction as template; WT: PCR with total DNA of non-transgenic tobacco (wild type) as template; the blank control is PCR reaction with sterile water as a template;
FIG. 4 is a standard curve for the assay of GUS enzyme activity in the present invention;
FIG. 5 is a diagram showing pBI121-PCHI-GUSGUS Activity of transgenic tobacco after methyl jasmonate treatment, wherein the control is normally grown pBI121-PCHI-GUSGUS activity of transgenic tobacco;
FIG. 6 shows pBI121-PCHI-GUSGUS activity of transgenic tobacco after inoculation of Fusarium oxysporum, Fusarium solani and Neurospora oryzae, wherein the control is pBI121-PCHI-GUSGUS activity of transgenic tobacco.
Detailed Description
The present invention is further illustrated by the following figures and examples, without limiting the scope of the invention thereto, wherein the process is carried out in a conventional manner unless otherwise specified, and wherein reagents are used, such as reagents used or formulated in a conventional manner, unless otherwise specified.
Example 1: cloning and sequence analysis of Lilium regale inducible promoter PCHI
The extracted genomic DNA of Lilium regale root is used as a template, and a specific primer (upstream primer: 5' ATCATACGTGTGTCCCCTATCATG3 ' ' and downstream primer: 5' GGAATGAAGGGCGAGGGTGG3 ') for amplifying the promoter PCHI is used for cloning the sequence of the promoter PCHI by PCR. The reaction system (20. mu.L) was Lilium regale genomic DNA 0.5. mu.g, 2. mu.L of 10 Xadavitage 2 PCR Buffer, 1.8. mu.L of dNTP Mix (10mM each), 0.2. mu.L of forward primer (10. mu.M), 0.2. mu.L of reverse primer (10. mu.M), 0.2. mu.L of Advantage 2 PCR Polymerase Mix, and 14.6. mu.L of PCR-Grade water. And (3) PCR reaction conditions: 5min at 94 ℃; 30s at 94 ℃, 30s at 63 ℃, 30s at 72 ℃ and 32 cycles; 5min at 72 ℃. After the PCR was completed, 8. mu.L of the mixture was subjected to agarose gel electrophoresis to examine the specificity and size of the amplified product.
The obtained PCR product has only one DNA band, so TA cloning is directly carried out on the PCR product, the kit used is pGEM-T vector system (Promega), and the reaction system and the operation process are as follows: mu.L of the PCR product was taken, and 1. mu.L of pGEM-T vector (50 ng/. mu.L) and 2.5. mu.L of 2 × Ligation solution I were sequentially added thereto, mixed well and then left to react at 16 ℃ overnight. The ligation product was transformed into E.coli DH 5. alpha. competence by heat shock transformation. Positive clones were selected on LB solid medium containing ampicillin. Selecting several single colonies, shaking, and detecting the clone with the multiple cloning site inserted with PCHI by using specific primers for amplifying PCHI. Sequencing the obtained positive clones to finally obtain 491bp promoter PCHI.
Example 2: PCHI-GUSExpression vector construction
pBI121 has multiple cloning sitesHindIII andBamHI cleavage site, so that specific primers for amplification promoter are added separatelyHinddiii andBamrecognition sites for HI. The plasmid pGEM-T-PCHI of the Escherichia coli inserted with the PCHI and the plasmid pBI121 of the plant expression vector are extracted by a SanPrep column type plasmid DNA small extraction kit (Shanghai worker), and 1 mu L of the extracted plasmid is used for agarose gel electrophoresis to detect the integrity and the concentration of the extracted plasmid. Using restriction endonucleasesBamHI andHinddiII to plasmid pGEM-T-PCHI and pBI121 respectively carry out double enzyme digestion (100 muL system), and the reaction system and the operation process are as follows: 20 μ L of pGEM-T-PCHI and pBI121 plasmid were taken, and 10 μ L of 10 XH buffer and 5 μ L ofBamHⅠ、5μL HindⅢ、60μL ddH2And O, mixing uniformly, centrifuging for a short time, and reacting at 37 ℃ overnight. All the enzyme digestion products were subjected to agarose gel electrophoresis, then the promoter fragment and the pBI121 vector large fragment were subjected to gel recovery using a SanPrep column type DNA gel recovery kit (Shanghai Prov.), and 1. mu.L of the recovered product was subjected to agarose gel electrophoresis to detect the size and concentration of the recovered fragment, with the results shown in FIG. 1.
The recovered promoter DNA fragment and pBI121 vector fragment were ligated by using T4 DNA Ligase (TaKaRa) in a reaction system (20. mu.L) by the procedure: mu.L of the PCHI DNA fragment was taken and added to 2. mu.L of the pBI121 vector DNA, 2. mu.L of 10 XT 4 DNA Ligase Buffer, 1. mu. L T4 DNA Ligase, 5. mu.L of ddH in this order2And O, mixing uniformly, centrifuging for a short time, and then carrying out water bath at 16 ℃ for overnight reaction. The ligation product was then transformed into E.coli DH 5. alpha. by heat shock transformation, and positive clones were selected on solid medium containing 50mg/L kanamycin. Selecting single colony shake bacteria, carrying out PCR by using a specific primer of an amplification promoter PCHI by using a bacteria liquid as a template, selecting a clone of the PCHI and pBI121 which are successfully connected, adding glycerol into the obtained positive strain, and storing at-80 ℃ for later use.
Extracting and purifying pBI121-PCHI in the Escherichia coli DH5 alpha by using a SanPrep column type plasmid extraction kit-GUSA plasmid. Subsequent freezing and thawing with liquid nitrogenThe plant expression vector pBI121 constructed above is used-PCHI-GUSTransferred into the prepared agrobacterium tumefaciens LBA4404 competent cells. The operation steps are as follows: 0.2. mu.g of pBI121-PCHI was taken-GUSThe plasmid is added into a centrifuge tube containing 200 mu L of competent cells, the mixture is gently mixed and then is subjected to ice bath for 5min, then the mixture is transferred into liquid nitrogen to be frozen for 1 min, then the mixture is rapidly placed in a water bath at 37 ℃ for 5min and then is subjected to ice bath for 2min, and then 500 mu L of LB liquid culture medium is added to be subjected to shaking culture at 28 ℃ for 4 h. The activated Agrobacterium was spread on LB solid medium containing 50mg/L kanamycin and cultured in an inverted state at 28 ℃. Selecting single colony shake bacteria, performing PCR reaction with specific primer for amplifying PCHI, and detecting pBI121-PCHI-GUSWhether it is transferred into agrobacterium. For the positive clones shown in FIG. 2, glycerol was added and stored at-80 ℃ until use.
Example 3: agrobacterium-mediated genetic transformation of plants and transgenic plant screens
The transgenic receptor of the experiment was tobacco, tobacco seeds were soaked in 75% alcohol for 30s, washed with sterile water and then washed with 0.1% HgCl2Soaking for 8min, washing with sterile water for several times, sowing on 1/2 MS culture medium, dark culturing at 28 deg.C for 5-8d, germinating, transferring to light incubator (25 deg.C, 16h/d light), and subculturing with MS culture medium once a month.
pBI 121-PCHI-containing food stored in refrigerator at-80 deg.C-GUSThe plasmid Agrobacterium LBA4404 bacterial liquid was taken out, 10. mu.L of bacterial liquid was inoculated into 1mL LB liquid medium containing 20mg/L rifampicin and 50mg/L kanamycin, and the liquid was cultured with shaking at 200rpm at 28 ℃ until it became turbid. Sucking 500. mu.L of bacterial liquid, uniformly spreading on LB solid culture medium containing 20mg/L rifampicin and 50mg/L kanamycin, and carrying out inverted culture at 28 ℃ until lawn grows out. Scraping 3-5 ring thallus Porphyrae with inoculating loop, inoculating into 40mL MGL culture medium containing 25mg/mL acetosyringone, and shake culturing at 28 deg.C and 220rpm until OD600About 0.6. Cutting the leaves of the sterilized tobacco tissue culture seedling to about 1cm2And soaking the leaf disks with the sizes in an MGL culture medium containing suspended agrobacterium tumefaciens, and performing shake culture at 25 ℃ for 15 min. After the bacterial liquid on the surface of the leaf disc is sucked dry by sterile filter paper, the leaf disc is transferred into a tobacco co-culture medium and cultured in dark at 22 ℃ for 2 days. Transferring the co-cultured leaf disc to a tobacco screening culture medium, and culturingCulturing in light incubator (25 deg.C, 16h/d light). After culturing for about 3 weeks, the differentiated tobacco seedlings were cut out and subcultured on a rooting medium containing 50mg/L kanamycin and 300mg/L cephamycin for rooting culture.
Extracting genome DNA of transgenic tobacco plant leaves by a CTAB method, and carrying out agarose gel electrophoresis on 1 mu L of the genome DNA to detect the integrity and the concentration of the genome DNA. And carrying out PCR reaction by using the genome DNA of the transgenic plant as a template and using a specific primer of the amplification promoter PCHI. After the PCR was completed, 8. mu.L of the product was subjected to agarose gel electrophoresis to detect positive transgenic plants. The amplification result of part of transgenic tobacco plants is shown in figure 3, and 29 positive transgenic plants are screened from the Lilium regale inducible promoter PCHI transgenic tobacco.
Example 4: GUS fluorescent quantitative detection of transgenic tobacco
For The quantitative fluorescent analysis of GUS activity in transgenic tobacco leaves, reference is made to The method of Jefferson et al (Jefferson R. assay genetic genes in plants: The GUS gene fusion system. Plant Mol Biol Rep. 1987,5(4): 387-405), The reaction mechanism is: GUS can react with a substrate 4-MUG to catalyze and generate 4-MU, the 4-MU generates fluorescence under the conditions that the excitation wavelength is 365nm and the emission wavelength is 455nm, and the generated fluorescence value can be quantitatively measured by a fluorescence spectrophotometer.
The pre-treated tobacco leaves were ground into powder in a mortar containing liquid nitrogen, 400. mu.L of GUS extraction buffer was added, and the homogenate was transferred to a 1.5mL centrifuge tube and centrifuged at 12000g for 10min at 4 ℃. After centrifugation, the supernatant was collected in a new centrifuge tube. 1mL of 4-MUG solution (1 mmol/L) was pre-heated in a 2.0mL centrifuge tube at 37 ℃ for 10 min. 50 μ L of the supernatant was added to pre-warmed GUS reaction buffer, shaken rapidly and 200 μ L of the reaction mixture was immediately added to 1.8mL of stop buffer (run time less than 30s) and spotted as enzymatic reaction 0 (blank at fluorescence measurement) and the remaining liquid was allowed to continue the reaction at 37 ℃ and time was started. At the time of reaction for 15min, 30min and 45min, 200. mu.L of the reaction mixture was added to 1.8mL of the stop buffer for fluorometry. The fluorescence value of each sample was measured using a fluorescence spectrophotometer under the conditions that the excitation wavelength was 365nm and the emission wavelength was 455 nm. Making a 4-MU standard curve: 1mM 4-MU was diluted with the reaction-terminated solution to 5nM, 10nM, 20nM, 40nM, 60nM, 80nM and 100nM each of the different gradients, and the fluorescence of each gradient was measured at 365nM excitation wavelength and 455nM emission wavelength, and a standard curve was plotted using the measured fluorescence and 4-MU concentration as a blank (see FIG. 4). 10 μ L of the supernatant was taken and the protein content of the sample was determined by a modified Coomassie Brilliant blue method. The enzyme amount for catalyzing 4-MUG to generate 1pmol 4-MU in one minute is taken as an activity unit, the GUS enzyme activity is calculated by the enzyme activity per MU g total protein, namely 4-MU pmol/min/MU g (protein), and the GUS activity of the transgenic tobacco is calculated through a standard curve.
In order to detect the response of the Lilium regale promoter PCHI to phytohormone and biological stress, the leaves of transgenic tobacco are respectively treated by methyl jasmonate and biological stress factors, the GUS activity before and after treatment is determined by the method, and the GUS activity of the leaves of the transgenic tobacco which are not treated in normal growth is used as a control. As shown in FIG. 5, after methyl jasmonate treatment, GUS activity of transgenic tobacco leaves of the Lilium regale promoter PCHI is obviously up-regulated. Three pathogenic fungi, namely fusarium oxysporum, fusarium solani and fusarium oryzae are used for inoculating leaves of transgenic tobacco, so that the activity of the promoter PCHI is obviously up-regulated. From the point of view of induction, fusarium solani > fusarium oxysporum > nigrospora oryzae (fig. 6). The GUS activity after infection of fusarium solani is about 3 times that before infection, the GUS activity after infection of fusarium oxysporum is about 2.4 times that before infection, and the GUS activity after infection of rice black cell mold is about 2.4 times that before infection. The experimental result shows that the Lilium regale promoter PCHI responds to the treatment of plant hormone and biological stress, and the methyl jasmonate, the fusarium oxysporum, the fusarium solani and the nigrospora oryzae can obviously up-regulate GUS activity driven by the PCHI. Obviously, the Lilium regale promoter PCHI is a plant hormone and biological stress factor inducible promoter, and can be applied to plant stress resistance genetic engineering.
Sequence listing
<110> university of Kunming science
<120> inducible promoter PCHI and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 491
<212> DNA
<213> Lilium regale (Lilium regale)
<400> 1
atcatacgtg tgtcccctat catgtgtcgt tatcttcaac atccttcctc ctagtatata 60
tgcagaacat ccatacattt ttcattacaa ctgttcaaaa ttgttagtga gacactcatt 120
tttaaagaac tgtcttaaaa taccgtctca taaaaatttg ccacaataaa ttcacaaagg 180
tttgccaaat catacctatg tacaagctga gcaccagtgt acgtctggaa caggttgaat 240
atacgagggt ggtaccgagg ccacacacgg aggttggcac agaagattat ttctcctgtt 300
aatttatatt tctggcttat ctttaagcct gccgattcaa catttagggt agttcaatcg 360
ctgctcatta cgtacacagt tgtctcactg ctcgttacca cgttttacaa cacacggcaa 420
acttttaggc caagagattg actagaaccc tcacaatctc tatataagcc cccaccctcg 480
cccttcattc c 491
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<213> Artificial sequence (Artificial)
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atcatacgtg tgtcccctat catg 24
<210> 3
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<212> DNA
<213> Artificial sequence (Artificial)
<400> 3
Claims (4)
1. An inducible promoter PCHI is derived from Lilium regale, and the nucleotide sequence of the inducible promoter PCHI is shown as SEQ ID NO. 1.
2. The use of the inducible promoter PCHI of claim 1 in plant disease resistance genetic engineering.
3. Use according to claim 2, characterized in that: fusarium oxysporum (F.), (Fusarium oxysporum) Fusarium solani (F.solani) (II)Fusarium solani) "Black rice spore (Nigrospora oryzae) The specific high-efficiency expression of the stress down-regulation exogenous gene in the transgenic receptor plant.
4. Use according to claim 2, characterized in that: the specific high-efficiency expression of the exogenous gene in the transgenic receptor plant is controlled under the action of plant hormone.
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| CN112708625B (en) * | 2021-03-16 | 2023-06-16 | 昆明理工大学 | Lilium regale inducible promoter PG1 and application thereof |
| CN112852820B (en) * | 2021-03-16 | 2023-06-20 | 昆明理工大学 | Lilium regale inducible promoter PD1 and application thereof |
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