CN113322257A

CN113322257A - Panax notoginseng inducible promoter PPO1 and application thereof

Info

Publication number: CN113322257A
Application number: CN202110599949.8A
Authority: CN
Inventors: 刘迪秋; 郑锂蕾; 梁婷婷; 苏琳琳; 邓婕; 葛锋; 崔秀明
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2021-08-31
Anticipated expiration: 2041-05-31
Also published as: CN113322257B

Abstract

The invention discloses a pseudo-ginseng inducible promoter PPO1 and application thereof, wherein the nucleotide sequence of PPO1 is shown as SEQ ID NO:1, the research of related technologies of molecular biology and genetic engineering proves that the pseudo-ginseng promoter PPO1 responds to several plant hormones, biological stresses and abiotic stresses. The expression frame constructed by the pseudo-ginseng inducible promoter PPO1 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, and the result shows that the glucuronidase activity of the transgenic tobacco is obviously enhanced after the treatment of gibberellin, indoleacetic acid, abscisic acid, injury, fusarium solani and alternaria. Therefore, the panax notoginseng promoter PPO1 is induced by several hormones, biotic and abiotic stress factors and can be used for plant stress resistance gene engineering.

Description

Panax notoginseng inducible promoter PPO1 and application thereof

Technical Field

The invention relates to the field of related researches of molecular biology and genetic engineering, in particular to a pseudo-ginseng inducible promoter PPO1 and application thereof.

Background

The plant promoter is a DNA sequence which can be specifically combined with RNA polymerase and transcription factors thereof and determines the initiation of gene transcription. Promoters are the regulatory centers for gene transcription, and act like "switches" that control the initiation time and extent of gene expression. The promoter composition not only comprises basic action elements such as CAAT-box, TATA-box and the like, but also comprises certain cis-action elements responding to adversity stress so as to ensure that the transcription of genes can be regulated and controlled under different conditions under the influence of factors such as environment, exogenous treatment and the like. Plant promoters can be classified according to their transcription pattern as: constitutive promoters (the expression of the gene is not limited by time and space and controlled by exogenous factors), inducible promoters (the expression of the 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 the gene is distributed in a certain tissue or organ of the plant).

The constitutive promoter can continuously and efficiently drive gene transcription in different types of cells and different stages of cell development, has relatively constant transcription activity, is typically cauliflower mosaic virus (CaMV) 35S promoter, and is widely applied to dicotyledonous plant transgenic engineering. However, if the target gene is expressed in high intensity in the whole life cycle of the plant, the gene product is excessively accumulated to cause unnecessary waste of resources, and meanwhile, the accumulation of a large amount of heterologous protein can break the original metabolic balance of the plant, prevent the normal growth of the plant and cause the subsequent metabolic disturbance and even death of the plant. To improve this problem, the development and utilization of tissue-specific promoters and inducible promoters have become a hot spot in genetic engineering research.

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. The addition of the inducible promoter on the vector can regulate the expression of the target gene under external stimulation, and the problem of unlimited expression of the foreign gene is well solved. And the expression of the exogenous gene driven by the inducible promoter is controlled by specific physical or chemical signals, and the characteristic can ensure that the expression of the exogenous gene can be more finely controlled (Yangyuan, Baijiarong, Lirui, everlasting).

Inducible promoters have been used in various plants, for example, in rice: (A), (B), (C) and (C)Oryza sativa) InOsAAA1The gene is a novel gene having induced resistance to rice blast: (Magnaporthe oryzae) And bacterial blight diseaseXanthomonas oryzae pv. oryzae) All have strong resistance. In order to research the function and disease resistance mechanism, Wang et al construct an inducible promoter vector PPR1b-ADH-OsAAA1, the inducible promoter can obviously reduce the adverse effect on the agronomic traits of plants caused by the expression of resistance genes, when the inducible expression of rice blast bacteria is carried out on positive plants, and qPCR detection is carried out to detect the adverse effectOsAAA1The result of the change of the gene expression indicates that the inducible promoter fusion vector PPR1b-ADH-OsAAA1 is successfully induced by rice blast bacteria,OsAAA1the expression level of (Wang Z, Chen J, Wang C, Liu X. Genetic transformation and expression of promoter structure of rice resistance gene OsAAA1. Molecular Plant Breeding, 2018, 16(21): 7021) 7026) was significantly increased. From Erythrochloe arundinacea (Erianthus arundinaceus) The PR10 promoter obtained by cloning is transferred into tobacco, rice and sugarcane after being connected with reporter gene GUSSaccharum officinarum). The GUS enzyme activity test result shows that the hypochondrium is damagedImperata arundinacea induced by treatment with methyl jasmonate and abscisic acidPR10Expression 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 PPO1 which is derived from pseudo-ginseng and has a nucleotide sequence 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 the condition of biotic stress.

The invention relates to a method for separating an inducible promoter fragment and identifying the expression activity of the inducible promoter fragment, wherein the inducible promoter is obtained by cloning from pseudo-ginseng and has the length of 965 bp. 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 geneGUSThe expression cassette of (1) by Agrobacterium tumefaciens (A)Agrobacterium tumefaciens) Mediating the transfer into the model plant Nicotiana tabacum: (Nicotiana tabacum) The expression is carried out, the expression characteristic of an inducible promoter is revealed through further experiments, the foundation is laid for regulating the high-efficiency specific expression of an exogenous gene in a transgenic plant by utilizing the promoter in the later period, and the promoter is named as PPO1 by the inventor.

The PPO1 promoter is driven by the inventionGUSThe expression cassette is transferred into tobacco, a transgenic tobacco plant is treated by adopting plant hormone, biotic stress and abiotic stress, and the fluorescence quantitative analysis of GUS activity is carried out, and the detection result shows that the PPO1 promoter responds to the treatment of several plant hormones, biotic stress and abiotic stress, abscisic acid, indoleacetic acid, gibberellin, injury stress and fusarium solani (Fusarium solani)Fusarium solani) Alternaria alternata (Alternaria compacta) Can obviously induce the activity of promoter PPO 1.

The promoter PPO1 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 tender tissue of panax notoginseng, adopting a specific primer for amplifying PPO1 to amplify a PPO1 sequence through Polymerase Chain Reaction (PCR), then connecting the PPO1 sequence to a pGEM-T carrier, and obtaining clone with correct sequence through sequencing;

(2) the pGEM-T-PPO1 vector is cut by restriction enzyme, and the 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 PPO1 fragment and pBI121-GUSConnecting the vector segments to construct a plant induction expression vector; then transferring the constructed plant induction expression vector into a receptor plant through the mediation of agrobacterium tumefaciens; when the transgenic plant is infected by injury stress or fusarium solani and alternaria compacta, a target gene driven by a promoter PPO1 can be induced and the expression level can be up-regulated, and in addition, abscisic acid, indoleacetic acid and gibberellin in vivo and in vitro can also induce the high-level expression of the target gene.

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 the target gene when the plant is influenced by external stress or chemical factors, and can regulate the expression of the target gene after stress removal or chemical treatment, thereby ensuring that the plant can be protected and the external stimulation can be resisted when the plant is stressed by adverse circumstances, 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, indoleacetic acid and gibberellin), abiotic stress (injury) and biotic stress (fusarium solani and alternaria) can obviously induce the expression activity of the PPO1 promoter, so the invention has wide application prospect in genetic engineering of biotic stress resistance.

Drawings

FIG. 1 shows the results of gel recovery product detection of promoter PPO1 (Panel A) and pBI121 vector (Panel B) in the present invention;

FIG. 2 shows pBI121-PPO1-GUSThe positive clone detection result of the transformed Escherichia coli, wherein the positive control is the plasmid pBI121-PPO1-GUSPCR reaction as template; the blank control is PCR reaction with sterile water as a template;

FIG. 3 shows the section pBI121-PPO1-GUSThe PCR screening result of the transgenic tobacco, wherein the positive control is the plasmid pBI121-PPO1-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 shows pBI121-PPO1-GUSGUS activity of transgenic tobacco treated with abscisic acid, indoleacetic acid and gibberellin, wherein the control is normally grown pBI121-PPO1-GUSGUS activity of transgenic tobacco;

FIG. 6 shows pBI121-PPO1-GUS transgenic tobacco is treated by fusarium solani, alternaria and injured GUS activity respectively, wherein the contrast is pBI121-PPO1-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 panax notoginseng inducible promoter PPO1

The extracted genome DNA of the pseudo-ginseng root is used as a template, and a specific primer (an upstream primer is 5 ' ACATCCTACCGTGAAGA3 ' ' and a downstream primer is 5 ' GAGGAGTGAAAGGGCAT3 ') of a promoter PPO1 is amplified to clone a sequence of the promoter PPO1 through PCR. The reaction system (20. mu.L) was 0.5. mu.g of Panax notoginseng genomic DNA, 2. mu.L of 10 × Advantage 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: 94 ℃ for 5 min; 94 ℃, 1min, 56 ℃, 30s, 72 ℃, 50s, 32 cycles; after PCR was completed at 72 ℃ for 5min, 8. mu.L of the mixture was subjected to agarose gel electrophoresis to detect the specificity and size of the amplified 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 PPO1 were tested using specific primers that amplified PPO 1. Sequencing the obtained positive clones to finally obtain the promoter PPO1 which is 965bp in length.

Example 2: PPO1-GUSExpression vector construction

pBI121 has multiple cloning sitesScaI andXbai cleavage site, so that specific primers for amplification promoter are added separatelyScaI andXbai recognition site. A small extraction kit (Shanghai worker) of the column type plasmid DNA is adopted to extract the plasmid pGEM-T-PPO1 of the Escherichia coli inserted with the PPO1 and the plasmid pBI121 of the plant expression vector, and 1 mu L of the plasmid is used for agarose gel electrophoresis to detect the integrity and the concentration of the extracted plasmid. Using restriction endonucleasesScaI andXbathe plasmid pGEM-T-PPO1 and pBI121 are subjected to double enzyme digestion (50 mu.L system) respectively by the following reaction system and operation process: 20 μ L of pGEM-T-PPO1 and pBI121 plasmid were taken, and 3 μ L of 10 XH buffer and 2 μ L of plasmid were added in sequenceScaI、5μL ddH₂O, mixing, centrifuging for a short time, and standing at 37 deg.CAfter 2h of reaction, a further 2. mu.L of XbaI、5μL 10×M buffer、13μL ddH₂And O, mixing uniformly, centrifuging for a short time, and reacting at 37 ℃ for 2 h. 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 PPO1 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 PPO1 by using a bacterial liquid as a template, selecting a clone of successfully connected PPO1 and pBI121, adding glycerol into a positive clone shown in figure 2, and storing at-80 ℃ for later use.

Extracting and purifying pBI121-PPO1 from Escherichia coli DH5 alpha-GUSPlasmid, and then freezing and thawing the plant expression vector pBI121 constructed above by using liquid nitrogen-PPO1-GUSTransferred into the prepared agrobacterium tumefaciens LBA4404 competent cells. The operation steps are as follows: 0.2. mu.g of pBI121-PPO1 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 was spread on LB solid medium containing 50mg/L kanamycin and cultured in an inverted state at 28 ℃. Selecting single colony shake bacteria, carrying out PCR reaction by using specific primers for amplifying PPO1, and detecting pBI121-PPO1-GUSWhether it is transferred into agrobacterium. Adding glycerol into the obtained positive strain, and storing at-80 deg.C for 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.

pBI121-PPO 1-containing was 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 the amplification promoter PPO 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 results of part of transgenic tobacco plants are shown in fig. 3, and 45 positive transgenic plants are screened from the transgenic tobacco with the panax notoginseng inducible promoter PPO 1.

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 for catalyzing 4-MUG to generate 1pmol 4-MU in one minute is taken as an activity unit, and the GUS enzyme activity is calculated by the enzyme activity per MU g total protein, namely, the GUS enzyme activity is expressed as 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 panax notoginseng promoter PPO1 to plant hormones, biotic stress and abiotic stress, leaves of transgenic tobacco are respectively treated by using a plurality of plant hormones, biotic stress and abiotic 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 figure 5, after being respectively treated by abscisic acid, indoleacetic acid and gibberellin, the GUS activity of the transgenic tobacco leaf with the pseudo-ginseng promoter PPO1 is obviously up-regulated. Wherein the induction degree of the indoleacetic acid and the gibberellin is obviously greater than that of the abscisic acid; the leaf blades of transgenic tobacco inoculated with fusarium solani and alternaria substantially up-regulated the activity of the promoter PPO1 (fig. 6). When the transgenic tobacco leaves are subjected to wound stress, the expression level of GUS genes can also be obviously induced. The experimental results show that the pseudo-ginseng promoter PPO1 responds to plant hormone, biotic and abiotic stresses, and obviously, the pseudo-ginseng promoter PPO1 is a plant hormone, biotic stress and abiotic stress factor inducible promoter and can be applied to plant stress resistance genetic engineering.

Sequence listing

<110> university of Kunming science

<120> pseudo-ginseng inducible promoter PPO1 and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 965

<212> DNA

<213> Panax notoginseng (Panax notogeng)

<400> 1

acatcctacc gtgaagaaat atatttttct tttgaccgac tatggtgagg gatgaaaaag 60

caaattaaaa ctttaaaaaa taaaattaat agttgttttt tttttttggt aaaataaaag 120

atcgtcttaa ggcggaaggt gcattgttta ttgcttaatg gttcatattt gagattaaaa 180

tggtccataa cttttttgta ttcaatttgt gaccagcatt ttgtgatatg tggattcatt 240

ggacacatgg gaagctggga gttgagtcca aattgtttgg atatccataa cagaagaaaa 300

ccgacacagg caggctccct tatccatatt ggcgctacat ttgcacgata aaaaattaca 360

ttttgaataa agtaggagta ttatacaaat aattctaact ttttaatata tatatatata 420

tcatttcaat aaaattgttc gaatttgaca atgacacaag ggcccaagat aaaaacagaa 480

aacagctaaa agcaaactaa aggctacgtg aatttcgatc agattttaaa agaatgacat 540

gctttggata ttcggcttta tgcttttagg ttacattttt cctttgctag tagtagtatt 600

tatcttttac tttactttta acatatacta gtataatata gcttacaata acttataact 660

tggcatgcat taggttaggt tctagctggc tagaattcgg gtaagattaa cgatataaac 720

gtaaaaatat tattttcacg taatcctttt ttacataaag aaaattcaca ttctattaca 780

acaggctaat aaacaaaaga attatttttt gaaaaatatt ctattatatt ttttctgttt 840

ttacataata ccaaacttta aacaatctca aatgaacaaa atatttcctc aacaaataaa 900

aaaggacact aaatatcagt ttttcacaaa aaatagccta agatcaacat gccctttcac 960

tcctc 965

<210> 2

<211> 17

<212> DNA

<213> Artificial sequence (Artificial)

<400> 2

acatcctacc gtgaaga 17

<210> 3

<211> 17

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

gaggagtgaa agggcat 17

Claims

1. An inducible promoter PPO1 is derived from pseudo-ginseng, and the nucleotide sequence of the inducible promoter PPO1 is shown as SEQ ID NO. 1.

2. The use of the inducible promoter PPO1 as claimed in claim 1 in plant stress resistance gene engineering.

3. 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 adverse stress.