CN112708625A - Lilium regale inducible promoter PG1 and application thereof - Google Patents
Lilium regale inducible promoter PG1 and application thereof Download PDFInfo
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Abstract
The invention discloses a Lilium regale inducible promoter PG1, the nucleotide sequence of the Lilium regale inducible promoter PG1 is shown as SEQ ID NO 1, the molecular biology and genetic engineering related technology research proves that the Lilium regale promoter PG1 responds to several plant hormones, biological and abiotic stresses; the fusion expression frame constructed by the Lilium regale promoter PG1 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 abscisic acid, salicylic acid, fusarium oxysporum, nigrospora oryzae, alternaria compacta and injury stress treatment, and the Lilium regale promoter PG1 is induced by several phytohormones, biological and abiotic stress factors, so that the Lilium regale promoter PG1 has wide application prospect in genetic engineering of biological or abiotic stress resistance.
Description
Technical Field
The invention relates to the field of molecular biology and genetic engineering related research, in particular to a Lilium regale inducible promoter PG1 and application thereof.
Background
The promoter is a DNA sequence which is positioned in the upstream region of the gene and used for regulating gene transcription, is an important cis-regulatory factor and determines the space-time accuracy and the transcription efficiency of the gene transcription, thereby regulating and controlling the expression of downstream genes. 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 of plant genes can be classified into constitutive promoters, inducible promoters and tissue-specific promoters according to expression patterns. Often, gene expression driven by tissue-specific promoters occurs only in certain specific organs or tissue sites, exhibiting developmentally regulated properties. Inducible promoters generally do not initiate gene transcription or have very low transcriptional activity, which is significantly increased under stimulation by certain specific signals. The specific expression of tissue-specific promoters is not absolute, and most fruit-specific promoters have ethylene response elements, so that the promoters can be regarded as ethylene-inducible promoters.
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 that may even produce severe dysplasia. The application of the inducible promoter reduces the accumulation of metabolites such as heterologous proteins and the like generated during the expression of exogenous genes and the waste of plant energy, thereby stabilizing the metabolic balance of plants. The inducible promoter can not only avoid the excessive consumption of plant energy caused by the continuous expression of a target gene, but also eliminate the damage to the plant itself caused by the accumulation of gene products, and becomes a research hotspot of plant genetic engineering in recent years (Sunxiang, Songhuangyan. research on plant inducible promoter. southern gardening, 2014, 25(02): 51-56).
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. The inducible promoter drives the expression of the exogenous gene to be controlled by a specific physical or chemical signal, and the characteristic enables the expression of the exogenous gene to be more finely controlled, so the research aiming at the inducible promoter is one of the hot spots of the research of plant molecular biology and genetic engineering. Using chromosome walking technique from lily (A), (B), (C)Lilium regale) Is/are as followsPR10-51489 bp 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 activity of the promoter has the positive regulation function, wherein the gibberellin has the strongest induction effect on the promoter; after treatment of abiotic stresses such as salt stress, injury stress and the like, 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 controlledPR10-5The activity of the promoter; fusarium oxysporum (F.), (Fusarium oxysporum) Sclerotinia sclerotiorum (A) and (B)Sclerotinia sclerotiorum) And Botrytis cinerea (Botrytis cinerea) Infection pairPR10-5The induction of GUS activity in the promoter was also significant (Chen R, He H, Yang Y, et al, Functional characterization of a pathogenic-related protein family 10 gene,LrPR10-5, from Lilium regalewilson, Australias Plant Path, 2017, 46(3): 1-9). From powdery mildew germs (Oidium heveae) Separating a WY7 promoter from the genome, and transferring the WY7 promoter connected with a reporter gene GUS into tobacco to be expressed in a transient mannerExpression of the GUS gene driven by WY7 was achieved and given appropriate stress treatment, low temperature and salt stress, indicating that the WY7 promoter was induced by low temperature and salt stress factors (Wang Y, Wang C, Rajaopera N, et al. WY7 is a new identified promoter from the rubber powder promoter and expression of gene expression in cells and diodes. PloS one, 2020, 15(6): e 0233911).
Disclosure of Invention
The invention aims to provide an inducible promoter PG1 which is derived from Lilium regale and the nucleotide sequence of which is shown as SEQ ID NO. 1.
The invention also aims to apply the promoter in gene engineering, namely, the promoter is used as an induced expression promoter to regulate the specific high-efficiency expression of an exogenous gene in a transgenic receptor plant under biotic and abiotic stress.
The invention relates to a method for separating inducible promoter fragments and identifying the expression activity of the inducible promoter fragments, wherein the inducible promoter is obtained by cloning from Lilium regale, and the length of the promoter is 904 bp. The inducible promoter fragment separated and cloned by the invention is used for replacing the CaMV 35s promoter on the pBI121 vector, and the inducible promoter drives the reporter geneGUSThe expression cassette of (1) by Agrobacterium tumefaciens (A)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 PG 1.
The PG1 promoter is drivenGUSThe expression frame is transferred into tobacco, a transgenic tobacco plant is treated by adopting several plant hormones, biotic and abiotic stresses, the fluorescence quantitative analysis of GUS activity is carried out, and the detection result shows that the PG1 promoter response adopts several plant hormones, biotic and abiotic stresses for treatment, abscisic acid, salicylic acid and fusarium oxysporum (F. (B.), (B.))Fusarium oxysporum) Alternaria densa (C. densa)Alternaria compact) "Black rice spore (Nigrospora oryzae) And the injury stress can obviously induce the activity of the promoter PG 1.
The promoter PG1 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 tissue of Lilium regale by using specific primers for amplifying PG1, amplifying PG1 by Polymerase Chain Reaction (PCR), connecting the PG1 to a pGEM-T carrier, and sequencing to obtain clone with correct sequence;
(2) the pGEM-T-PG1 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; the obtained PG1 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 the transgenic plant is subjected to fusarium oxysporum, alternaria densa, nigrospora oryzae and injury stress, a target gene driven by a promoter PG1 can induce and up-regulate the expression level, and in addition, abscisic acid and salicylic acid 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. Several hormones (abscisic acid and salicylic acid), abiotic stress (injury) and biotic stress (fusarium oxysporum, alternaria densa and nigrospora oryzae) obviously induce the expression activity of the PG1 promoter, so the invention has wide application prospect in genetic engineering of biological or abiotic stress resistance.
Drawings
FIG. 1 shows the results of gel recovery detection of promoter PG1 (Panel A) and pBI121 vector (Panel B) in the present invention;
FIG. 2 shows pBI121-PG1-GUSConverting a positive clone detection result of the escherichia coli, wherein the positive control is PCR reaction taking pGEM-T-PG1 plasmid as a template, and the negative control is PCR reaction taking sterile water as a template;
FIG. 3 shows a part pBI121-PG1-GUSPCR screening result of transgenic tobacco, wherein the positive control is plasmid pBI121-PG1-GUSPCR reaction as template; WT: PCR reaction with total DNA of non-transgenic tobacco (wild type) as template.
FIG. 4 is a standard curve for the assay of GUS enzyme activity in the present invention;
FIG. 5 shows pBI121-PG1-GUSGUS Activity of transgenic tobacco after abscisic acid and salicylic acid treatments, wherein the control is normally grown pBI121-PG1-GUSGUS activity of transgenic tobacco;
FIG. 6 shows pBI121-pBI121-PG1-GUSGUS Activity of transgenic tobacco after injury treatment, wherein the control was pBI121-PG1-GUSGUS activity of transgenic tobacco;
FIG. 7 shows pBI121-PG1-GUSGUS activity of transgenic tobacco after inoculation of fusarium oxysporum, alternaria densa and nigrospora oryzae, wherein the control is normally grown pBI121-PG1-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 PG1
Taking the extracted Lilium regale root genomic DNA as a template, cloning the sequence of a promoter PG1 by PCR (20 muL) with a specific primer (an upstream primer is 5 'GCCCCATAGACCCTATCCAAGTA 3' 'and a downstream primer is 5' CAGGGGCAGAGGGTTGAC3 '') of an amplification promoter PG1, wherein the specific primer is 0.5 mug, 2 muL 10 × Advantage 2 PCR Buffer, 1.8 muL dNTP Mix (10mM each), 0.2 muL upstream primer (10 muM), 0.2 muL downstream primer (10 muM), 0.2 muL Advantage 2 PCR Polymerase Mix and 14.6 muL PCR-Grade water, and after PCR reaction conditions are 94 ℃ for 5min, 94 ℃ for 30s, 63 ℃ for 30s and 72 ℃ for 50s and 32 cycles, 8 muL is taken for gel electrophoresis after PCR is finished at 72 ℃ for detecting the specificity and the size of an amplification product.
The obtained PCR product has only one DNA band, TA cloning is directly carried out on the PCR product, the used kit 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 screened on LB solid medium containing ampicillin (Ampicillin, Amp). Several single colonies were picked and shaken and clones with multiple cloning sites inserted into PG1 were tested using specific primers for amplification of PG 1. Sequencing the obtained positive clones to finally obtain the promoter PG1 with the length of 904 bp. PLANTCARE is adopted to predict cis-acting elements of the promoter, and cis-acting elements related to adversity stress in the promoter sequence are predicted.
Example 2: PG1-GUSExpression vector construction
pBI121 has multiple cloning sitesHinddiii andBamHI cleavage site, so that specific primers for amplification promoter are added separatelyHinddiii andBamrecognition sites for HI. Extracting Escherichia coli plasmid pGEM-T-PG1 inserted with PG1 and plant expression vector pBI121 plasmid with small amount of extraction kit (Shanghai worker) for SanPrep column type plasmid DNA, and collecting 1 μ LThe method is used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. Using restriction endonucleasesHinddiii andBamh I carries out double enzyme digestion (5 mu L system) on plasmids pGEM-T-PG1 and pBI121 respectively, and the reaction system and the operation process are as follows: 20 μ L of pGEM-T-PG1 and pBI121 plasmid were taken, and 10 μ L of 10 XH buffer and 5 μ L of 5 μ L were added in sequenceBamHI、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 PG1 DNA fragment was added to 2. mu.L of pBI121 vector DNA, 2. mu.L of 10 XT 4 DNA Ligase Buffer, 1. mu. L T4 DNA Ligase, and 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 PG1 by using a bacterial liquid as a template, selecting a clone of successfully connected PG1 and pBI121, adding glycerol into the obtained positive bacterial strain, and storing at-80 ℃ for later use.
Extracting and purifying pBI121-PG1 in Escherichia coli DH5 alpha by using SanPrep column type plasmid extraction kit-GUSA plasmid. Then the plant expression vector pBI121 constructed above is frozen and thawed by liquid nitrogen-PG1-GUSTransferred into the prepared agrobacterium tumefaciens LBA4404 competent cells. The operation steps are as follows: 0.2. mu.g of pBI121-PG1 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 1min, 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 is coated on the surface of the substrate containing 50mg/L kanamycinThe cultured cells were inverted at 28 ℃ on LB solid medium. Selecting single colony shake bacteria, performing PCR reaction with specific primer for amplifying PG1, and detecting pBI121-PG1-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.
The extract containing pBI121-PG1 stored in a 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 mixture was cultured at 28 ℃ with shaking at 200rpm 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, performing shake culture 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. The leaf discs after co-culture are transferred to a tobacco screening culture medium and cultured in a light incubator (25 ℃, 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 an amplification promoter PG 1. 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 PG1 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 12000 g 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, the fluorescence of each gradient was measured at 365nM excitation wavelength and 455nM emission wavelength, and the fluorescence and 4-MU concentration were plotted against the reaction-terminated solution 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 catalyzing 4-MUG to generate 1pmol 4-MU in one minute is taken as an activity unit, and GUS enzyme activity is calculated by enzyme activity per MU g total protein, namely, 4-MU pmol/min/MU g (protein). And calculating the GUS activity of the transgenic tobacco through a standard curve.
In order to detect the response of the Lilium regale promoter PG1 to phytohormone, biotic stress and abiotic stress, leaves of transgenic tobacco were treated with several phytohormones, biotic stress and abiotic stress factors, respectively, and GUS activity before and after treatment was determined by the above method, and GUS activity of leaves of transgenic tobacco which had not been treated with normal growth was used as a control. As shown in figure 5, after the abscisic acid and the salicylic acid are treated, the GUS activity of the transgenic tobacco leaf with the Lilium regale promoter PG1 is obviously up-regulated, and the abscisic acid is greater than the salicylic acid in the aspect of the induction degree of the promoter activity. GUS activity of the transgenic tobacco after the damage treatment is shown in figure 6, and the damage to the abiotic stress factor remarkably up-regulates the activity of a promoter PG 1. The activity of a promoter PG1 (figure 7) is obviously up-regulated by inoculating the leaves of transgenic tobacco with three pathogenic fungi of fusarium oxysporum, alternaria densiflora and fusarium graminearum, and from the aspect of induction, the fusarium oxysporum densiflora and the fusarium nigrosporium are obtained. The experimental results show that the Lilium regale promoter PG1 can respond to treatment of several plant hormones, abiotic stress and biotic stress, and abscisic acid, salicylic acid, fusarium oxysporum, alternaria compact, nigrospora oryzae and traumatic stress can obviously up-regulate GUS activity driven by PG 1. Obviously, the Lilium regale promoter PG1 is a plant hormone, biological stress and abiotic stress factor inducible promoter, and can be applied to plant stress resistance gene engineering.
Sequence listing
<110> university of Kunming science
<120> Lilium regale inducible promoter PG1 and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 904
<212> DNA
<213> Lilium regale Wilson
<400> 1
tttttacccc gttggggcct ttttaataag gggattctat ttttctggat aaggccccat 60
agaccctatc caagtattgg aattggggat aaccactaag gtgggtaccg gttattatcc 120
ctaataggcc atcgggtgga taagggcccg gttggtaatt ggataaatta agggcttggt 180
ggtggttccc ccaatacggc taacttcttc gggtatgaaa acgtattcgg aattcatcgg 240
ggatacccag tttttagata agggatttcc cttgggctat attaagggga ctaagggatt 300
caacccagag ggcatacgac cagattccaa gaagttggct gaaggaagat tccttcccgg 360
ttccccgttc cgccggggaa gttcaccttc gccggaggtt cgcattgttt tcgggccgcc 420
ataggaggag gccccttatt cgcctccagc caggattccg gttcgcaagg attattcgga 480
gccgcaaggg gtggttagat tcttggggtg ttttgtaagg gtaaggtttt ttggaagggt 540
aaggtttttt atcctccccc caaaggtttt tattaagtta aaggattctt accaaaatta 600
gggaaggacc atttgggttt ttccaaccag gattggtaat acaaggccat tttaattaat 660
tggggggaaa tccattcacc tattttgggg aaattttaaa aaataaatta aagaaattcc 720
aattttaaat tttttttttt taaaatatga tgaatcatct atatcacgca ttatgtaagg 780
gtacttctag aaaattcaaa aaagaaacaa agaaggagaa cccaaacatg aaaactatgg 840
caagccagcc tacccaccct tgtcctcaag tcaaccctct gcccctgaaa aaaagaaccc 900
aatg 904
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (Artificial)
<400> 2
gccccataga ccctatccaa gta 23
<210> 3
<211> 18
<212> DNA
<213> Artificial sequence (Artificial)
<400> 3
caggggcaga gggttgac 18
Claims (2)
1. An inducible promoter PG1 is derived from Lilium regale, and the nucleotide sequence of the inducible promoter PG1 is shown as SEQ ID NO. 1.
2. The inducible promoter PG1 of claim 1, which controls the specific high-efficiency expression of exogenous gene in transgenic receptor plant under the adverse stress.
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