TW201125978A - Inducible promoter from lily and use thereof - Google Patents

Abstract

The present invention relates to a novel non-coding nucleic acid from the upstream region of LsGRP1 from lily, which encoding a glycine-rich protein. The sequence of this nucleic acid has promoter activity, capable of driving target gene expression in host cells. The hosts include ferns, gymnosperms, monocot and dicot angiosperms. This sequence is involved in stress-induced gene expression, including abiotic and biotic stresses, and capable of driving high expression upon induction of plant hormones, resistance-related signal molecules, and pathogens.

Description

201125978 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to the field of colonization, analysis, and molecular biology of planting detents, and in particular to the stress-inducible promoter of lily and its use. [Prior Art] The Plant Gene Guardian is a modern f-reader that is used to introduce the desired trait into plant body towels; there are many anti-disease strategies for gene transfer technology. Plants have novel resistance (Prim β a/., 2008; Rommens and Kishore, 2000). Choosing (4) When Qi Xuan is very important in the export of silk, since the performance, the performance and the timing of the performance, many of the attempts to use the persistent promoter (_titutive pr_ter) to express the exogenous resistance-related research, Wei, sustained silk resistance Sex genes often result in reduced plant yields and yields (Hammond-Kosack and Parker, 2001 Miehelmo% 2003; Stuivet and Custers, 2001). In order to reduce the non-essential performance of resistance-related genes in uninfected cells, an inducible pr〇m〇ter, such as a pathogen or a reverse-inducible promoter, was chosen as a viable improvement strategy (Gurr andRushton, 2〇〇5). Many important food crops and cash crops, such as rice, wheat, corn, sugar cane, orchids, etc., belong to monocotyledonous plants. Currently, there are several monocotyledonous promoters, such as maize t/6z7 promoter and rice a. ^The promoter has been cloned, but some reports indicate that the above promoters are not able to perform well in some flowering and non-cereal monocots (J〇ung and Kamo, 2006; Kamo shackles; /, 1995, 2000; Wilmink Ei α/·, 1995). In addition, gene silencing in many transgenic plants is attributed to the use of the same promoter to express several genes (Flavelleu/, 1994; Matzkeea/_, 1994; Parkeia/, 19%). Because of 201125978, there is an urgent need to isolate novel promoters from different plant species in molecular breeding, and it is more important in flowering and non-cereal monocots with only a few suitable promoters. The genes are expressed through transcription, translation, and post-translational modification (PTM), and the promoter is involved in transcription to regulate gene expression. The functional classification of the promoter can be changed according to whether it is stimulated by a specific factor, and is divided into a persistent promoter (c〇nstitutive promoter) and an inducible promoter (inducibie pr〇m〇ter), the former such as broccoli mosaic The 35S promoter (Califlower mosaic virus 35S, CaMV35S promoter), the Agrobacterium nos promoter and the plant AcU promoter can stably and continuously perform gene expression; the latter such as photoinducible rape LTP promoter, heat induction A sexual heat shock protein promoter, a wound-inducible potato PIN2 promoter, a pathogenesis-induced protein promoter, etc. are induced by specific factors to enhance expression activity. In terms of tissue specificity of promoter expression, it can be distinguished as a tissue-specific promoter (tissue_speciflc piOm〇ter) that is only active in some tissues and organs, or non-tissue specific in various tissues and organs. Promoter (n〇n tissue-specific pfomoter). Whether a promoter is persistent or inducible, whether it is tissue specific#, is a very important basic data in the application of promoters (Gurr and Rushton, 2005) ° In the case of monocot resistance related research, vaccination or Infected with Lilium oxysporum (5 〇 沿 e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e 葵 葵 葵 百合 百合 百合 百合 百合 百合 百合 百合 百合 百合 百合 百合 百合 百合 百合 百合 百合The number of lesions caused by Botrytis cinerea infection is significantly reduced (Cheneia/., 2003; Chen and Huang, 1997; 201125978

Lu and Chen' 1998' 2005; Lu α/., 2007)' shows that the lily can be stimulated by extrinsic factors to induce a disease-resistant response, and the induction of resistance can reduce the severity of secondary infection. An inducible lily-related gene was identified and isolated: Lily 'Lilium 'Star Gazer, glycine-rich protein 1, encodes a protein of 138 amino acids The protein is homologous to a number of glycine-rich proteins expressed in the extracellular matrix, and has salicylic acid, chlorpyrifos, and Botrytis cinerea induction to enhance plant tolerance, φ Pathogen resistance. Therefore, the inventors of the present invention speculated that the promoter should have appropriate basic expression and adversity, pathogen-inducible, and can be applied to the genetic engineering by screening the sequence of the promoter. Enhance the resistance of organisms to adversity or disease. In view of the importance of developing a specialized, stimulating and inducible promoter in the biotechnology industry, the inventor of the case has successfully developed and completed this piece. Lily stress-inducible promoter and its use." Part of the invention is in the tenth country of the bio-molecular interaction of the plant ship • International Congress 〇n Molecular Plant Microbe

Im_i〇nS), published in the abstract from July 19th to July 2nd, 2nd year. SUMMARY OF THE INVENTION The object of the present invention is to provide a high expression intensity promoter with tissue specificity. The promoter has high expression intensity and can be driven by plants (leaves, bulbs, flowers). The target genes linked are expressed in large numbers. The second objective of the present invention is to provide a high expression intensity in various organisms, in particular, in each of the complements (eg, Lai, Naked Mi, Mono-F and Dicotyledonous angiosperms) 201125978. Promoter. Another object of the present invention is to provide a promoter which can be induced by stress to initiate strength. A further object of the present invention is to provide various uses of the stress-inducing promoter. The use comprises various application methods or application strategies applicable to the present invention, and various products, compositions or organisms comprising the promoter of the present invention. body. A lily stress-inducible promoter (LsG brother P/promoter) capable of achieving the above object, the source of which is obtained from the genomic nucleic acid of _ 〇 / w w oriental hybrid cv. Star Gazer DNA). Since lily is rich in glycine protein 1 (Lilium 'Star Gazer' glycine-rich protein has salicylic acid, culling heat and Liriomyza sativa inducing', the inventors of the present invention speculated that the promoter should be induced by stress. Characteristic, therefore, the promoter is selected by a conventional method such as cloning. In order to analyze the ability of the promoter to initiate a downstream target gene, the promoter sequence is ligated to the reporter gene β-glucosic acid. The 5th gene sequence of the enzyme (P_glucuronidase, GUS) is used as a promoter of the reporter gene, and is constructed in a transformation vector to form a recombinant transplastosome; and then, the promoter is contained by Agrobacterium injection method. The recombinant transgenic plastids were transferred into tobacco and lily, and the activation activity of the LsGitP/promoter was evaluated by analyzing Gus activity; the results showed that the lily promoter can drive the target gene linked to it to be expressed in a large amount in plants. Specific tissues (leaves, flowers and bulbs); therefore, the sibling 7 promoter of the present invention has a high expression intensity of initiation ability and is tissue specific. The LsG is like 7 The nucleotide sequence of the promoter is selected from the group consisting of: (a) having a nucleotide sequence as shown in SEq ID 2011 201125978 1 (868 bp), (b) having a nuclear bitterness as shown in SEQ ID NO: 2. The acid sequence (714 bp), (c) has the nucleotide sequence (586 bp) as shown in SEQ ID NO: 3, and (d) has the nucleotide sequence (362 bp) as shown in SEQ ID NO: 4. (4) having a nucleotide sequence (?25 bp) as shown in SEQ ID NO: 5, at least one of the group consisting of. Further, the nucleotide sequence of the promoter of the present invention may also be appropriately Nucleotide substitution, mutation, etc. are designed to change part of the sequence, and therefore, the nucleotide sequence of the promoter also includes an identity having at least 80% identity with the nucleotide sequences (a) to (e) above, and It still has the same performance characteristics as the promoter of the present invention. In addition, it is used to evaluate the inducibility of the promoter by different treatment methods, including: chemical substance treatment, stress treatment, and pathogen treatment. Substance contains: auxin, gibberellin, cyt〇kinin and abscisic add, methyl Methyl jasm〇nate, salkylic acid; the stress includes: salt, high temperature, low temperature, heavy metal environment; the pathogen includes: gray mold (5 〇 noisy cmemz), lily gray mold (such as The noisy sentence along the ca), Solanaceae bacterial spot disease (Xanthomonas campestris pv. vesicat〇ria), soft rot pathogen 汍em〇. According to the invention, the promoter has chemical substances, stresses, and pathogen-inducing properties. The "chemically inducible" and "chemically inducible" as used in the present invention means that the promoter is activated by a chemical substance (e.g., plant hormone, environmental hormone, a compound, etc.), and the promoter activity is effectively enhanced, and the promoter activity is promoted. Higher than those induced by chemical substances (eg, plant hormones, environmental hormones, compounds, etc.). Among them, "salt treatment" for plant body, too high salt concentration will lead to osmotic pressure 201125978 imbalance, and harsh plant body 'thus, when the plant is exposed to high concentration of salt environment (such as: sodium Ions (Na+) and gas ions (cr) are an environmental stress for plants. Each of the plant species has different tolerances for the concentration of the salt. Therefore, the "salt treatment" or "salt induced" according to the present invention includes but is not limited to: (1) the concentration of the plant body-concentration After treatment, it will cause the osmotic pressure to be unbalanced, which will lead to the growth of the plant body, and then the initiator will be able to effectively promote the start-up activity of the start-up, or (2) after the guide, the activity of the start-up activity can be effectively enhanced. The priming activity is higher than that of the priming inducer The temperature is processed on the temperature of the general system #3〇τα, and the first embodiment is the 370C treatment. The low temperature treatment generally refers to a temperature of 1 〇{>c or less, and an embodiment is 4 °C treatment. . Further, the "high temperature or low temperature inducibility" of the present invention can also be defined as: after a temperature induction, the promoter activation activity can be effectively enhanced, which is higher than that which is not induced by temperature. Description of the transfer: If "high temperature inducibility" means that the temperature of induction is higher than that of the uninduced, and the temperature is effective to induce the promoter activation activity, and the activation activity is souther than the uninduced one. Conversely, '^ is low temperature inducible' means that the temperature of induction is lower than that of the non-inducer, and the temperature is effective to induce the promoter activation activity, and the promoter activity is higher than that of the non-inducer. Wherein the heavy metal treatment comprises, but is not limited to, evaluating the stress inducibility of the hG brother Pi promoter for copper or other heavy metal ions by an aqueous solution of copper sulfate (CuS?4). In addition, the "heavy metal inducibility" and "copper inducibility" described in the present invention may also be defined as: after being induced by heavy metals or heavy metal ions (eg, copper or copper ions), the promoter activity of the promoter can be effectively enhanced, and The starting activity is higher than that induced by heavy metals or heavy metal ions (eg copper, copper ions). The "pathogenic inducibility" as used in the present invention means that after the infection or induction of the pathogen, the promoter of the promoter can be effectively enhanced by 201125978, and the activation is higher than that of the uninfected county. In addition, the present invention also contains Ls (recombinant transgenic plastids of 7i?P/promoter by Agrobacterium; a major plant species introduced by the main method) to evaluate the applicable range of the promoter. Species include: fine, naked, _, and gemini. The fern contains BirdVnest fern; the gymnosperm contains: cypress (9) 咱p〇d〇carp) and ginkgo (Ging.); Miscellaneous 働 contains n (Maize), __utterfiy (10) hid), wenxinlan (0ncidium orchid), auntie (Chinese crown), tigertail orchid (such as Pi when Siam Tulip, sunflower lily ('Star Gazer, %) and Taiwan Lily (F_〇sa lily) '泫 Monocotyledonous angiosperm contains: corn _ze), Phalaenopsis _), Oncidium orchid, Apo 芋 (Chi tar〇), Tigertail (Snakeplant) and Siam Tulip; the dicotyledonous angiosperm contains: Lettuce, Spinach, Bottle Gourd, Angled Mfa, Chinese Kale, Green Bean (Snap bean), coffee (c〇ffee), nine-story tower (Basi), chili (H〇t pepper) petunia, sweet pepper (Pepper), Liu (Orange), Hunchun

(Madagascar Periwinkle), Arabidopsis and Tobacco (W benthamiana gas N. tabaccum) 〇 In addition to providing a lily stress-inducible promoter promoter, the present invention also provides a gene expression composition (expressi〇n cassette) The gene expression composition comprises: (1) the LsG brother P7 promoter of the present invention, and a polynucleotide having an open reading frame (ORF), that is, a target gene; a nucleotide line is ligated to the 3' end of the promoter of the present invention, which can initiate transcription of the polynucleotide (transcripti〇n) in an organism containing the gene expression composition; In an embodiment 201125978, the gene is the reporter gene β-glucosidase (gus). Further, the lily stress-inducible promoter promoter of the present invention is constructed into a general commercial carrier, including but not limited to: pBI1〇1, pBI12l, pBIN19 (ClonTech), pCAMBIA1301, pCAMBIA1305, pGREEN (GenBank)

Accession No: AJ007829) ^ pGREEN II (GenBank Accession No: EF590266), PGreen〇029 (John Innes centre), can form a gene expression vector, or recombinant transfer vector, and insert the target gene into the gene expression vector In the above, the target gene is ligated to the 3' end of the promoter of the present invention to form the above expression cassette; and the promoter of the present invention can be linked to it by translocation. The target gene behind the end is transferred to the target organism, thereby changing the genomic composition of the transgenic organism, so that the promoter and the target gene of the present invention can effectively initiate the expression of the target gene in the target transgenic organism and its progeny. The present invention also provides a method for allowing a target gene to be exclusively expressed on a f, a flower and a bulb of a plant, the method comprising: ligating the 3' end of the aforementioned promoter to the end of the target gene 5, to prepare - a gene expression vector containing at least the promoter sequence and the target gene; the gene expression vector is further transferred into the plant body, or a part of the organ, tissue or cell of the plant body, so that the target gene can be specifically expressed on the plant Leaves, flowers and bulbs. The methods for transfection include, but are not limited to, Agrobacterium mediation, Agrobacterium injection, recombinant virus infection, porcine vector transfer, base gambling, electroporation, microinjection, fresh Method, lipid (four) age transcription, ultrasonic media transfer method, broken sputum fiber age transition (4) (7) n e_de f fine side tmsf_ti ridge electrophoresis 201125978 method (electrophoresis), laser microbeam (laser microbeam), polyethylene glycol ( Polyethylene glycol; peg), calcium phosphate transfer method, DEAE-dextran transfer method. Due to the promoter provided by the present invention, the target gene can be characterized by a large amount of expression, tissue specificity, stress induction, and a wide range of applicable organisms, and therefore, the lily can be activated by inducing a mysterious LsGRPi promoter. The purple-related gene (defense_reiated gene>: ι 〇畐3 glycine from the protein 1 (LsGJRP·/), in order to increase the resistance of the transgenic plants to adversity, can also be applied to mass production of various types of The present invention is exemplified by the following examples, but the present invention is not limited by the following examples. The materials used in the present invention are all exemplified by the following examples. Commercially available, the following are only examples of the available channels. [Embodiment] The first summary of the DG/ep/promoter full-length sequence and partial sequence fragments from the sunflower lily was selected by Agrobacterium injection method. Analysis was performed with the _7 promoter to understand the tissue performance characteristics of the promoter, the response to various stresses, pathogens, and plant hormone treatments' and applicability to different plant species. Example 1 Selection of Lily Promoter 1.1 Plant Material The commercially available sunflower lily (four) is called oriental hybrid cv. Star Gazer, the seed of the plant is sterilized by 1/〇_human sodium, and then planted. It is cultivated at 20-25 ° C for about one month in a plastic pot containing cultivation medium (Peat 201125978 \ Soil [BVB]: No. 2 Pearl Stone = 3: 1). 1.2 Ligenomic genomic DNA purification The modified lily phytase nucleic acid was purified by modified CTAB method (Taylor and Powell, 1982). After the 2 g Po Li leaves were ground and pulverized with liquid nitrogen, 8 ml was added to preheat to 65. CT CTAB extraction solution (6% cetyltrimethyammonium bromide, 100 mM Tris-HCl l.4 M NaCl, 2 〇 0 mM EDTA, pH 8. Add protease κ (proteinase jg and 2-mercaptoethanoal) before use. The final concentration was 300 and 3% (ν/ν) were mixed uniformly, and after rotating at 5 rpm for 2 hours at 65 ° C, an equal volume of Cl (chl〇r〇form: octanol=24:l was added. ) Mix gently, then take the supernatant after centrifugation for 10 minutes at (:, 6000 g, add 1/10 volume of CTAB/NaCl solution (1〇% CTAB, OJMNaCl) preheated to 65〇C, gently mix and Extraction with α. After the extraction step of CTAB/NaC1 solution and α is repeated several times, the nucleic acid in the supernatant is dissolved in 0.7 times volume of isopropanol (is〇pr〇pan〇1), and the obtained nucleic acid is precipitated with 80% alcohol. After rinsing and air drying, dissolve in an appropriate amount of sterile deionized water. · 1.3 Selection of LsGRPi promoter (PLsGRp!) Refer to the method of Ashoub and Abadalla (2006) for selection. Select endogenous restriction enzymes (4) riction coffee yme ) such as [, Can, such as 1 and fcII (R〇che) separately fermented the whole body of Cai Lili and recovered with 3' end After the DNA fragment is extracted, it is linked to the linker (4) with the relative complementary sequence of each _restricted enzyme, and the reaction is carried out (2011): 2011-ΚρηΙ, OPH-PstI, OPH-SacI and OPH-SacII (Hgati〇) n) 'Recover the product as a polymerase-chain reaction (PCR) template, increase the amplitude of the conserved sequence primer AP and the specific reverse primer p329 of each linker, and recover the product as a PCR template. Nested PCR was performed on the conserved sequence primer NP on each linker and another specific reverse primer p371. The PCR product was recovered and cloned into E. coli XL1-Blue or ToplO F' strain, and the result was obtained. The selected strains were sequenced for subsequent sequence and functional analysis, and the primers were designed according to the results of the sequence to p99 and p400 from the sunflower nucleic acid nucleic acid with pCr amplification and colonization of the 868 bp LsG/iPi promoter (PiiGm) sequence. Full length, having the sequence shown in SEQ ID No: 1, wherein the primer sequence used is shown in Table 1. Another primer was designed to select PisG/?/>/partial sequence fragment (sequence deletion group: 714) Bp (S EQ Π) No : 2), 586 bp (SEQ ID No: 3), 362 bp (SEQ ID No: 4) and 125 bp (SEQ ID No: 5) fragment; wherein the sequence deletes the reverse of the group (reverse) The primers are all p400; the forward primers are: dpGRP-714, dpGRP-586, dpGRP-362, and dpGRP-125, respectively, and the primer sequences used are shown in Table 1. Table 1 Introduction of primers for introduction of primers SEQ ID No ΟΡΗ-ΚρηΙ 5 '-gaattcgagctcgcccgggatcctctagagtac -3' 6 OPH-PstI 5*-gaattcgagctcgcccgggatcctctagatgca-3* 7 OPH-SacI 5'-gaattcgagctcgcccgggatcctctagaagct-3' 8 OPH- SacII 5 '-gaattcgagctcgcccgggatcctctagagc -3s 9 AP S-gaattcgagctcgcccgggatcc-S1 10 NP 5'-gctcgcccgggatcctctaga-3' 11 p329 5 '-cctcagccagctcccgaccagcgtcggagg-3' 12 p371 5 '-cttaccctatttatacacagagatgcgc-3' 13 p391 5-ggaagcttg atttacgga attatagtctcattgg -3 ' 14 p400 5'-cgtcgacagaggccaggactcaggacc-3' 15 13 201125978 dpGRP-714 5 '-tcttctcctagggctctcaagtgtatttag-3' 16 dpGRP-586 5 -tacatgtaatagttttggatc gag-3' 17 dpGRP-362 S'-tcagtatcctctattgcccccttaacg -3* 18 dpGRP-125 5 '-cgccaactgcatfitctgtcgcc-3f 19 1.4 Promoter Sequence Analysis The LsGRP1 promoter sequence of the selected Lilium sinensis L. was analyzed by the online software TSSP for possible RNA polymerasell binding regions, TATAbox and transcription initiation sites (Shahmuradove, 2003), and on-line plants. Cis-acting element database PLAce (Higo β α/· 1999) for comparison and analysis' prediction of possible cis-acting element sequence of. After comparison prediction, it was found that the multiple cis-acting elements of the LsGRP1 promoter (4) _acting elements are related to pathogen and abiotic stress, plant hormone action, signal transmission and physiological regulation. There are also multiple light-induced expressions and mesophyll tissue specificity. Relevant to sex, showing that it should be a valid promoter sequence. Example 2 Functional analysis of the promoter (PisG/w) 2·1 Constructing a binary expression vector) Please refer to Figure 1 for the binary vector pBI121 to limit the enzymes //zmllll and 5amHI (Roche Glycolysis to remove the CaMV 35S promoter, recover and remove the 14 kb pBI121 backbone of the CaMV 35S promoter, and fill the restriction enzymes at both ends with Klenow enzyme (New England Biolab), respectively, with 868 bp and 4 groups of polymerization The sequence deletion group of the enzyme chain reaction (125 bp, 362 bp, 586 匕卩, and 71 Park 0) was bound by Ding 4〇^1^336 〇11^3), and then transferred to the large intestine. Screening and sequencing of Bacillus ToplOF' strains, confirming the construction is correct, 14 201125978 can produce five sets of recombinant gemini vectors with different promoter sequences, and the five sets of recombinant gemini vectors are sent into Agrobacterium C58C1 strain, Agroinfiltration is performed to analyze the transcriptional functions of the promoter sequences. In the promoter function analysis, the maize Ubil promoter and the CaMV35S promoter double vector were used as the control group. 2.2 Agrobacterium injection method (Agr〇infihration) The five sets of recombinant bicone vectors and the double-coupled vector pBI121 as a control group were respectively transferred into Agrobacterium tumefaciens iwwe/acz.ew by the electrotransformation method (C58C1 strain) They were inoculated separately in 3 ml YEP liquid medium (10 g/1 bactopeptone, 10 g/1 yeast extract, 5 g/1 NaCl) with 50 ppm kanamycin, and cultured at 25 ° C, 200 rpm for 16 hours, then the bacteria Body transfer to induction medium (〇3% κ2Ηρ〇4 〇1%

NaHP04> 0.1% NH4C1, 0.03% MgS04.7H20, 0.015% g/1 KC1, 0.001% CaCl2, 0.0025% FeS〇4.7H2〇, 2% glucose, 10 mM 2-(N-morpholino) ethanesulfonic acid (MES), 100 mM acetosyringone, pH 5.6), cultured for 6 hours at 25 〇C, 200 rpm. The cells were recovered and suspended in Agrobacterium injection buffer (100 mM acetosyringone, 10 mM MgS04, 10 mM MES, pH 5·6) at a concentration of 0.8 ,d6GG, with 1 ml syringe from the leaf back of the plant or other tissues. Inject. 2.3 β-glucuronidase (GUS) active tissue staining analysis

Take appropriate amount of tissue sample to be stained, add GUS active group staining solution (1% X-Gluc (5-bromo-4-chloro-3-indolyl-PD-glucuronic acid, Biosynth), 100 mM phosphate buffer , pH 8.0, 10 mM EDTA, 0.5 mM 15 201125978 • ferrocyanide, 0.5 mM ferricyanide, 10°/. Triton X-100, 10°/methanol), after 16 hours of staining at 37 ° C, 70% alcohol The chlorophyll is decolored until the sample tissue is completely whitened. 2.4 GUS activity fluorescence quantitative analysis Take 50 mg plant tissue samples and add 200 μL of GUS extraction solution (50 mM phosphate buffer, pH 8.0, 10 mM 2-mercaptoethanol, 10 mM EDTA, 0.1% sodium lauryl sacosine, 1.0% Triton) X-100) Uniformly ground, remove the plant residue by centrifugation at 4 ° C, 1200 g, take 50 μl of the supernatant and add 200 ml of GUS extraction solution preheated to 37 °C with 250 ml of GUS analysis solution (containing 2 mM 4 -methylumbelliferyl-bD-glucuronide GUS extraction solution), after reacting at 37 °C for 30 minutes to 1 hour, taking 100μ1 reaction product and adding 900μ1 0.2 M sodium carbonate to terminate the reaction. 'Plate CHAMELEON V (Hidex) for OD365 fluorescence detection With quantification. The quantification method was performed by interpolating the sample readings with the standard curve after making a standard curve using 4-methyl umbelliferone. Example 3 Analysis of the performance of the hG brother W promoter 3_1 PisG/w allows the reporter gene to be transiently expressed in the grass leaf. A recombinant symplet vector containing the 868 bp promoter (PLGmzGUS-pBmi), and The control group (p35S::GUS-pBI121) was fed into tobacco by Agrobacterium injection (10) a) After 5 days of expression, the accumulation of GUS protein was measured by GUS active tissue staining (as shown in Figure 2A and Figure 2B). ). The results show that the promoter (P^Gm) can be used to report the high-level performance of GUS in tobacco (Fig. 2A), and its cumulative performance is almost universally used in the 35S promoter (Fig. 2B). 3 · 2 with tissue specificity In order to understand the performance characteristics of ZjG brother ρ / promoter in different plant tissues, select various tissues or organs (such as: bulbs, leaves, flowers) of sunflower lily plants, and introduce the above-mentioned contents by Agrobacterium injection method. Promoter (SEQ ID No: 1) recombinant double-coupled vector (PiiG/w::GUS-PBI121) and expressed, and then analyzed by GUS activity fluorescence analysis

The tissue characteristics of the L7G brother P7 promoter. The results showed that the performance of the bulbs, leaves and flowers of the sunflower lily ' PhG/w has a basic performance on the bulbs, leaves and flowers of the sunflower lily, and the highest expression in the leaves is about two bulbs and flowers. The results of the predictive analysis of the cis-acting element (汾-actingelement) shown in Figure 3 indicate that 匕(4) should have mesophyll cell specificity and light-inducedness; this predictive inference and PisG/w expression in different tissues The results of the analysis were consistent, demonstrating that PiiG/w is indeed tissue-specific and can initiate the expression of the target gene in leaves, bulbs, flowers, etc. 3.3 ^isGRPl is a chemical, stress and pathogen-inducible promoter for comparison hG/ Induction of cadaver/promoter under different stimuli: First, as shown in Figure 4A, the recombinant gemini vector containing the promoter (SEQ ro No. 1) prepared above was injected into Agrobacterium. The method was transferred to tobacco (ΛΓ W38) to re-administer different stimuli to evaluate the inducibility and possible inducing factors of the promoter. The different stimuli were: (a) the control group was sprayed with water. With 17 • foliar 201125978 10mMMgS〇4 shot suspended in water and in the normal fishing grass plants; (b) inducing pathogen:; (c) chemicals

GUS activity fluorescence spectroscopy analysis. The results showed that compared with the control group, the promoters of each of the induction groups (b) to (e) were higher than the dragon group, and the towel was the highest in the pyrolysis group, and the salt was induced in the order. Substance (salicylic acid) induction group, soft rot _ conduction group. It has been confirmed that the sputum of the present invention (SEQ ID Nq: 秘, 秘 p) has chemical substances, stress, and pathogen-inducing properties. Furthermore, the present invention also applies this. The NSID promoter (SEQ ID NO: U is distinguished by different sizes). The abundance is used to evaluate the possibility of each fragment sequence as an inducible promoter, and can be used to analyze possible regulatory units on the LsC^/7 promoter (SEQ ID No: 1). First, the Agrobacterium injection method will be used. The above five recombinant haplotype vectors (125 bp (4), 362 bp (B), 586 bp (C), 714 bp (D), 868 bp (E), wherein A, b, C, and D are promoters. Partial sequence fragment, e is the full length sequence of the promoter) was introduced into grass (TV·(10)W38). Plant hormones were used: auxin, AlD〇, gibberellin (GA), cytokinin (cytokinin, CK) ) Signaling molecules related to abscisic acid (植物), plant resistance: methyi jasm〇nate (MeJA), salt (Salt) treatment, high and low temperature (37 ° C and 4. 〇, Copper (Cu) treatment, phytopathogenic fungi: Soiryfe Be, bacteria: Solanaceae bacterial spot disease bacteria (meaning <3« recognitionowow As cowpesirk pv_ vesi'eaton.a, Xcv), etc., and observe the analysis of the performance of 201125978 GUS-based SI, the induction of brittle and milk promoter. If the above-mentioned promoter-containing recombinant bis-vector is introduced into lily The induced phytopathogenic fungus is preferably Botrytis cinerea (and > noisy. First, the full length (868 bp) and sequence deletion of 6 weeks old grass (tv; __ W38) by Agrobacterium injection method Analysis of the performance of the recombinant double-coupled vector, the following 9 groups were treated after 24 hours: (4) foliar spraying with 5 mM methyl jasmonic acid (MeJA); (b) treatment with broad acid (ΑΒΑ), 1〇〇μΜseed acid for foliar spray; (4) foliar spray with exo-indole-3-acetic add auxin (AUX); (4) gibberdlic acid-3 (gi) with 1〇〇ppm Foliar spraying; (8) foliar sprinkler irrigation with 1〇〇ppm ό-benzylaminopurine cytokinin (CK); (7) high temperature treatment (heat treatment), placing the plants in a growth chamber at 37 ° C; (g) low temperature treatment (cold treatment) ), placing the plants in a 4 〇c growth chamber; (h) copper (Cu) treatment (heavy metal treatment), using 丨 mM sulphuric acid (CuS〇4) aqueous solution for foliar spraying; (丨) salt treatment, using 150 ml 200 mM NaCl aqueous solution for root ring watering; (j) pathogen treatment, foliar injection suspension of 1 mM MgS〇4 Solanaceae bacteria Xanthomonas campestris (vesicatoria, Xcv) fine mirror % sputum (〇D6〇q-〇.2) '(k) pathogen treatment 'foliar spray ΐχίο5 Sp〇res/mi Botrytis cinerea ( Wan (10) Qing Be). The control group was leaf sprayed with water and normally watered tobacco plants. For each group of treatments, the number of replicates of 3 plants was carried out. After 48 hours of treatment, tobacco leaves were collected for quantitative analysis of GUS activity fluorescence. Referring to Figure 4B, the results indicate that the promoters after various treatments (whether partial deletions (A, b, c, D) or full-length sequences (E)) are compared to the control group. It can effectively induce the driving function of &G brother P/promoter (PiiG/w), in which the pathogen 201125978 (h-ipv. Wei (10) coffee and fish 6 and high and low temperature stress can be highly induced promoter (P_) The driving ability, methyl jasmonic acid, plant hormone, copper sulfate and high salt treatment is second. In addition, the results of the analysis of the full length and sequence deletion performance of (5) are shown, 125 bp has a high performance characteristic, 586 bp and The fine bp is second, 362 bp and 714 bp PiiG/y5/ is worse. Example 4 IsGIWV promoter (ρ_ρ/) application range 4.1 PisGRPf can be applied to many plant species | To detect the LyGm promoter in different plant species The degree of performance is selected from a variety of bio-research-related models or generic species, and crops with high economic value and agricultural importance are introduced into the re-planted double-couple vector containing the deletion promoter 1n〇:i) by Agrobacterium injection method. In the plant (leaf), the analysis starts The performance in different species! · Biosynthesis and ubiquitous use in molecular breeding, molecular farm and bio-development of the promoter: the flowering vegetables embedded virus 35S promoter, corn killer and other performance characteristics to compare The turn of the 7 7 7 promoter. Each promoter was subjected to three weights for each sputum, and the mean and standard deviation values of the intensity of the performance were calculated. The 35S promoter in each plant species is shown as the intensity of the scorpion promoter (p plus milk) in each plant species. The relative intensity of the mover is shown in Table 2, and the hourly number represents the relative standard deviation. Value, indicating no detection. Please refer to Table 2 for analysis of the promoter (p_) in a variety of plant species. The table can be found in two species of lily: Cai Lily (‘Star Gazer,%) and Taiwan Lily (F〇rmosa 20 201125978

Uly), Birds-nest fern, 2 gymnosperms: Nagi podocarp and Gingo, 6 early cotyledon angiosperms: Maize, Butterfly orchid, Oncidium (Oncidium orchid), Chinese taro, Snake plant and Siam Tulip; 16 dicotyledonous angiosperms: Lettuce, Spinach, Bottle Gourd, Angled luffa, Chinese Kale, Snap bean, Coffee, Basil, Hot Pepper, Petunia, Pepper, Willow 〇range, ada春 φ (Madagascar Periwinkle), Arabidopsis and tobacco (T〇bacc〇, # benthamiana 氙N. tabaccum) f ' LsGRPi promoter (PLsG brush) have excellent performance 'Even even more widely used in genetic engineering and molecular breeding, the CaMV35S promoter (P35S) and the maize Ubil promoter are high 'display promoters can be applied to a variety of plant species to drive the potential of target gene expression, in addition to molecular breeding, in the molecule There are also possibilities for application on the farm' Different plant species for the mass production of various types of desired protein.

Table 2 and other general-purpose promoters in different plant species. Analysis of promoters in different plant species Relative activity Promoter Sunflower Lily Taiwan Lily Bird Nest Fern Bamboo Cypress Ginkgo P35S ^LsGRPl Ubil 1.00 ±0.38 3.03 ± 0.58 1.00 ±0.16 2.27 ± 0.66 1.00 ±0.20 2.98 ± 1.18 1.00 ±0.08 2.58 ±0.20 1.00 ±0.20 1.92 ±0.66 Promoter Huwei Langupo Maize Butterfly Phalaenopsis P35S 1.00±0.11 1.00 ±0.06 1.00 ±0.28 1.00 ±0.09 1.00 ±0.20 ^LsGRPI 1.03 ± 0.12 3.00 ±〇.47 3.35 ± 0.45 3.48 ±0.50 6.09 ± 0.54 Ubil — 1.72 ±0.37 0.24 ±0.12 0.55 ±0.05 Start on 畺 lotus lettuce spinach ί 看 Look edged loofah P35S 1.00±0.11 1.00 ±0.02 1.00 ±0.03 1.00 ±0.10 1.00 ±026 21 201125978 ^LsGRPi Ubil 4.19 ±0.28 1.69 ±0.08 2.89 ± 0.07 0.30 ± 0.02 1.95 ±0.25 0.09 ± 0.04 2.40 ±〇.〇6 3.10 ±0.34 .0.22 ±〇.〇i 0.17 ±0.06 Promoter Green Bean Coffee P35S P rLsGRPI Ubil 1·00±0·35 2.86 ± 0.59 0.24 ± 0.02 1.00 ±0.08 2.32 ±0.11 0.12 ±0.02 1.00 ±0.20 1.90 ±0.22 1.00±0.1 8 1.00 ±0.13 2-40 ±0.12 2.23 ±0.42 — Promoter petunia sweet pepper orange ^ Hunchun Arabis P35S p 1 LsGRPI Ubil 1.00 ±0.51 U4±〇.14 0.11 ±0.01 1.00 ±0.64 3.95 ± 0.34 0.52 ±0.05 1.00 ±0.08 11·62±0·17 0.45 ± 0.04 1.00 ±0.09 1.00 ±0.18 2.890 ±〇.i7 1.38 ±0.27 0.07 ± 0.02 - Promoter P35S P 1 LsGRPI Ubil in grass (N, beyithcmiicmci) 1.00 ± 0.22 1.14±0.14 0.11 ±0.01 Yu Cui (N. [abbaccum] 1.00 ± 0.20 1-90 ± 0.22 0.06 ± 0.00 • The lily stress-inducible promoter provided by the present invention and its use are compared with other conventional promoters The time 'has the following advantages: 1. The promoter of the present invention can activate the target gene behind the 3' end to be expressed in a specific tissue (leaf, bulb, flower) of the plant body, and has tissue specificity. 2. In addition to the high expression intensity, the promoter of the present invention can make the target gene abundantly expressed in the target & plant to increase the yield of the target gene, and its intensity is even higher than the general use of the 35 § promoter (P35S) and The corn ubil promoter is higher. · 3. The promoter of the present invention can carry the _ form to the difficult things (four), regardless of the genus, naked, monocotyledonous and dicotyledonous fresh plants, both of which have the ability to start. 4. The stress-inducible promoter provided by the present invention can be driven by abiotic and biological stress factors to allow the user to control the degree of expression of the target gene to which the promoter is driven, as needed. The detailed description above is a detailed description of one of the possible embodiments of the present invention, but the embodiment of the present invention is not intended to limit the scope of the patents of the present invention, and is not equivalent to the spirit of the present invention. Silk changes are included in the patent scope of this case. Not the above-mentioned case is not only innovative in the genetic sequence, but also has a unique performance and uniqueness. It should be filled with money and money, and it is shameful to make an application according to law. You have approved this special invention for this invention, in order to invent the invention, to the sensation [simple description of the schema] Figure 1 is the 启动 启动 promoter _) gene expression cassette (gene expression cassette) schematic diagram, the system will be simple 21〇 The promoter of 3 just replaced with a promoter. Figure 1A shows the results of (10) activity staining of the recombinant symplet vector (pLsGm::GUS-PBI121) containing the promoter (868 bp) in the leaves of the tobacco (from the surface of the face); Figure 2B shows the control group. The double-coupled vector (p35S::GUS_pBI121) was obtained from the GUS activity of the leaves of Nicotiana benthamicmcO. Figure 2 shows the expression of the promoter (piiG/w) in different tissues (leaves, flowers, bulbs) of sunflower lily. The average table height and standard deviation value were calculated after 3 replicates. The unit of GUS activity: (10) the mu mirf丨mg_1 protein. Figure 4A shows the promoter of the full-length promoter (PiiG/?/v) under different treatments. Activity analysis, wherein the induction treatment method includes: soft rot fungus (five ~ such as .flc as only), salicylic acid treatment, salt treatment, heat treatment. Figure 4B is the promoter (ρΖίσ /? / ν) in different treatments Promoter activity analysis, wherein the promoter (Pw^/} is divided into: 125 23 201125978 bp (A), 362 bp (B), 586 bp (C), 714 bp (D), 868 bp (E) The A, B, C, and D groups are promoter partial sequence fragments (sequence deletion group), and the E group is a full-length sequence of the promoter; Methods include: mercapto jasmonic acid (MeJA) treatment, separation acid (ABA) treatment, auxin (AUX) treatment, gibberellin (GA) treatment, cytokinin (CK) treatment, heat treatment, cold good, steel Good reason, salt nitrogen, Xanth〇m〇nas campestfis

Processing, fine-grained (Bc) processing. Unit of Gus activity: facial 匕 round ―·丨mgJ protein 〇 [Main component symbol description]

twenty four

201125978 Sequence Listing <11〇> National Taiwan University <120> Lily-inducible promoter and its use <160> 19 <210> 1 <211> 868

&Lt; 212 &gt; DNA <213> sunflower lily is the ⑽ oriental hybrid cv Star Gazer) &lt;. 400> 1 ggaagcttga tttacggaat tatagtctca ttggcaatta ttaaaatatc gttcacattt tgaagaatta gcttgttgtc aaatttagaa atatgataac aattggtaca ttgttttata tgttataatt gaactcaaga ccttttataa attttcttct cctagggctc tcaagtgtat ttagtgtaga gacaaccgct gatggagtag aggatatatc attataagac gatttcatca tcttcatgct ttgtcctctt tagaccatag gttacccaca ggtacatgta atagttttgg atcgagaatc tatatgacca taaatatttt gaacatatat ttcatcttgt gtgcctacag acaaaatctt ttcctataaa tctgttccgg tttaattaac caaattccaa caactgctgg ccaagagctt cactcgtctt cagagaattt cacaaatggg aggatatttc ctatttccat ggatatttcc tatttccatg gaaacctcag tatcctctat tgccccctta acgtgatatc cagtagaaaa agcactgatg acgtggcatc aatcggaaga caatacgtcc gagctgataa tggtaatggg gtccatgaat gcaaattgat caccaattcc aaacctatct tggctcttct gttgccacgc tattaattca cttattgtct gtgttcaatc tctccacgac tgttgataag cttgaagaaa tgaggtaccc atccgccaac tgcatgtctg Tcgcctaccc cacgtaatct ttaatactaa cagaagttca ctgaacatgc gcatctctgt gtataaatag Ggtaagaggg tacaatgcat tcctcatccc aaacagcc &lt;210〉 2 &lt;211&gt; 714

&lt;212> DNA &lt;213&gt; Lily oriental hybrid cv. Star Gazer) 60 120 180 240 300 360 420 480 540 600 660 720 780 840 868 &lt;400> 2 tcttctccta tatatcatta cccacaggta atatatttca attaaccaaa aatgggagga ctctattgcc gggctctcaa taagacgatt catgtaatag tcttgtgtgc ttccaacaac tatttcctat cccttaacgt gtgtatttag tcatcatctt ttttggatcg ctacagacaa tgctggccaa ttccatggat gatatccagt tgtagagaca catgctttgt agaatctata aatcttttcc gagcttcact atttcctatt agaaaaagca accgctgatg cctctttaga tgaccataaa tataaatctg cgtcttcaga tccatggaaa ctgatgacgt gagtagagga ccataggtta tattttgaac ttccggttta gaatttcaca cctcagtatc ggcatcaatc 60 120 180 240 300 360 420 25 201125978 ggaagacaat acgtccgagc tgataatggt aatggggtcc atgaatgcaa attgatcacc 480 aattccaaac ctatcttggc tcttctgttg ccacgctatt Aattcactta ttgtctgtgt 540 tcaatctctc cacgactgtt gataagcttg aagaaatgag gtacccatcc gccaactgca 600 tgtctgtcgc ctaccccacg taatctttaa tactaacaga agttcactga acatgcgcat 660 ctctgtgtat aaatagggta agagggtaca atgcattcct catcccaaac agcc 714 &lt;210> 3 &Lt;211> 586

&Lt; 212> DNA &lt;. 213> sunflower lily oriental hybrid cv Star Gazer) &lt; 400> 3 tacatgtaat catcttgtgt aattccaaca gatatttcct cccccttaac atacgtccga acctatcttg tccacgactg gcctacccca ataaataggg agttttggat gcctacagac actgctggcc atttccatgg gtgatatcca gctgataatg gctcttctgt ttgataagct cgtaatcttt taagagggta cgagaatcta tatgaccata aatattttga acatatattt aaaatctttt cctataaatc tgttccggtt taattaacca aagagcttca ctcgtcttca gagaatttca caaa / tgggag atatttccta tttccatgga aacctcagta tcctctattg gtagaaaaag cactgatgac gtggcatcaa tcggaagaca gtaatggggt ccatgaatgc aaattgatca ccaattccaa tgccacgcta ttaattcact tattgtctgt gttcaatctc tgaagaaatg aggtacccat ccgccaactg catgtctgtc aatactaaca gaagttcact gaacatgcgc atctctgtgt caatgcattc ctcatcccaa acagcc 60 120 180 240 300 360 420 480 540 586

&lt;210〉 4 &lt;211〉 362

&lt;212&gt; DNA &lt;213> Kale Lily (10) oriental hybrid cv. Star Gazer)

& Lt 4 60 120 180 240 300 360 362 tcagtatcct catcaatcgg tgatcaccaa gtctgtgttc caactgcatg atgcgcatct cc ctattgcccc cttaacgtga tatccagtag aaaaagcact gatgacgtgg aagacaatac gtccgagctg ataatggtaa tggggtccat gaatgcaaat ttccaaacct atcttggctc ttctgttgcc acgctattaa ttcacttatt aatctctcca cgactgttga taagcttgaa gaaatgaggt acccatccgc tctgtcgcct accccacgta atctttaata ctaacagaag ttcactgaac ctgtgtataa atagggtaag agggtacaat gcattcctca tcccaaacag; &lt; 400 &gt;210> 5 &lt;211> 125

&lt;212> DNA &lt;213> Oriental hybrid cv· Star Gazer) 26 201125978 &lt;400> 5 cgccaactgc atgtctgtcg cctaccccac gtaatcttta atactaacag aagttcactg 60 aacatgcgca tctctgtgta taaatagggt aagagggtac aatgcattcc tcatcccaaa 120 cagcc 125 &lt;210> 6 &lt;211〉 33

&lt;212&gt; DNA &lt;213> Artificial sequence &lt;220> &lt;223> Forward introduction

&lt;400&gt; 6 gaattcgagc tcgcccggga tcctctagag tac 33 &lt;210〉 7 〈211〉 33

<212> DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223> Forward introduction &lt;400&gt; 7 gaattcgagc tcgcccggga tcctctagat gca 33

&lt;210〉 8 &lt;211〉 33

&lt;212> DNA &lt;213> Artificial sequence &lt;220&gt;&lt;223> Forward introduction &lt;400&gt; 8 gaattcgagc tcgcccggga tcctctagaa get &lt;210> 9 &lt;211&gt; 31 &lt;212> DNA &lt;213&gt; Artificial sequence 27 33 201125978 &lt;220&gt;&lt;223> Forward introduction <400> 9 gaattcgagc tcgcccggga tcctctagag c 31 &lt;210&gt; 10 &lt;211> 23

&lt;212&gt; DNA <213> Artificial sequence &lt;220&gt; <223> Forward introduction &lt;400&gt; 10 gaattcgagc tcgcccggga tcc 23 <210> 11 &lt;211> 21

&lt;212> DNA &lt;213> Artificial sequence &lt;220&gt; <223> Forward introduction &lt;400> 11 gctcgcccgg gatcctctag a 21 &lt;210&gt; 12 &lt;211&gt; 30

&lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Reverse introduction &lt;400> 12 cctcagccag ctcccgacca gcgtcggagg 30 &lt;210> 13 &lt;211> 28

&lt;212> DNA &lt;213> Artificial sequence 28 201125978 &lt;220> &lt;223> Reverse primer &lt;400> 13 cttaccctat ttatacacag agatgcgc 28 &lt;210> 14 &lt;211> 34 &lt;212> DNA &lt; 213> &lt;220> artificial sequence &lt;223> forward introduction &lt;400> 14 ggaagcttga tttacggaat tatagtctca ttgg 34 &lt;210&gt; 15 &lt;211> 27 &lt;212&gt; DNA &lt;213&gt;&lt;220> artificial sequence &lt;223> Reverse introduction &lt;400> 15 cgtcgacaga ggccaggact caggacc 27 &lt;210&gt; 16 • &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt;&lt;220&gt; Artificial sequence &lt;223> Forward introduction &lt;223&gt;;400&gt; 16 tcttctccta gggctctcaa gtgtatttag 30 &lt;210&gt; 17 &lt;211> 24 &lt;212&gt; DNA &lt;213&gt;&lt;220&gt; Artificial sequence 29 201125978 &lt;223&gt; Forward introduction &lt;400&gt; 17 tacatgtaat agttttggat cgag &lt;210> 18 &lt;211&gt; 27 &lt;212> DNA &lt;213&gt;&lt;220> Artificial sequence &lt;223> Forward introduction <400> 18 tcagtatcct ctattgcccc cttaacg &lt;210> 19 &lt;211&gt; 22 &lt ;212> DNA &lt;213〉 &l t;220&gt; Artificial sequence &lt;223&gt; Forward introduction &lt;400&gt; 19 cgccaactgc atgtctgtcg cc 201125978 [Reference] 1. Abe, H·, Urao, T” Ito, T·, Seki, M·, Shinozaki, K ·, and Yamaguchi-Shinozaki, Κ· 2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15:63-78. 2. Ashoub, A. and Abdalla, KS 2006. Plant Mol. Biol. Rep. 24:237-243. 3. Chen, C· Y” Lu, YY, and Chung, J_ C. 2003. Induced host resistance against less &quot;5 Leaf blight. In 'Advances in Plant Disease Management' (eds) HC Huang and SN Acharya. pp. 259-267. 4. Flavell, RB 1994. Inactivation of gene expression in plants as a consequence of specific sequence duplication. Proc. Acad. Sci. USA 91:3490-3496. 5. Lu, YY and Chen, CY 2005. Molecular analysis of lily leaves in response to salicylic acid effective towards protection against Botrytis elliptica. Plant Sci. 169 :1-9. 6. Lu, Y. Y.5 Liu, Y. H? and Chen, C. Y. 2007. Stomatal closure, callose deposition, and

Increase of -corresponding transcript in probenazole-induced resistance against

Botrytis elliptica in lily. Plant Sci. 172: 913-919. 7. Denekamp, M. and Smeekens, S. C, 2003. Plant Physiol. Integration of wounding and osmotic stress signals determines the expression of the AtMYB102 transcription factor gene. Physiol. 132:1415-1423. 8. Du·H·, Zhang, L·, Liu, L” Tang, X· F., Yang, W. J” Wu, Y. M·, Huang, YB, and Tang Biochemistry and molecular characterization of plant MYB transcription factor family. Biochemistry 74:1-11. 9. Gurr, SJ and Rushton, PJ 2005. Engineering plants with increased disease resistance: how are we going to express it? Trends Biotechnol 23:283-90. 10. Hammond-Kosack, KE and Parker, JE 2003. Deciphering plant pathogen communication: fresh perspectives for molecular resistance breeding. Curr. Opin. Biotechnol. 14:177-193. 11. Higo, Ks Ugawa , Y., Iwamoto, M., and Korenaga T. 1999. Plant cw-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res. 27:297-300. 12. Joung s Y H. and Kamo, K. 2006. The expression of a polyubiquitin promoter isolated from Gladiolus. Plant Cell Rep. 25:1081-1088. 13. Kapila, J.5 de Rycke, R.5 van Montagu, M., and Angenon, G. 1997. An Agrobacterium mediated transient gene expression system for intact leaves. Plant Sci. 122:101-108. 14. Kamo, K.5 Blowers, A., Smith, F., Van Eck, J., and Lawson, R. 1995. Stable transformation of Gladiolus using suspension cells and callus. J. Am. Hortic. Sci. 120:347-352. 15. Kamo, K.3 Blowers, A., and McElroy, D. 2000. Effect Of the cauliflower mosaic virus 35S, actin, and ubiquitin promoters on uidA expression from a bar-uidA fusion gene in transgenic Gladiolus pleints. In Vitro Cell Dev. Biol. Plant. 36:13-20. 16. Kopertekh, L. and Schiemann , J. 2005. Agroinfiltration as a tool for transient expression of ere recombinase in vivo. Transgenic Res. 14:793-798. 17. Lescot, M.? Dehais, P., Moreau, Y.5 De Moor, B., Ronze, P., and Rombauts, S. 2002. PlantCARE, a database of plant c/5-acting regulato Ry elements and a portal to tools for in 201125978 . silico analysis of promoter sequences. Nucleic Acids Res. 30:325-327. 18. Lu, YY and Chen, CY 1998. Probenazole-induced resistance of lily leaves against Botrytis elliptica. Pathol. Bull. 7: 134-140. 19. Lu, YY and Chen? CY 2005. Molecular analysis of lily leaves in response to salicylic acid effective towards protection against Botrytis elliptica. Plant Sci. 169:1-9. 20. Lu ,YY,Liu,YH,and Chen,CY 2007. Stomatal closure,callose deposition,and increase of Z^G^PZ-corresponding transcript in probenazole-induced resistance against Botrytis elliptica in lily. Plant Sci. 172:913-919 21 Matzke, AJ, Neuhuber, F., Park, YD, Ambros, PF, and Matzke, MA 1994. Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes. Mol. Gen. Genet. :219-229. 22. Michelmore, RW 2003. The impact zone: genomics and breeding for Curative. resistance. Curr. Opin. Plant Biol. 6:397-404. 23. Nagaoka, S., and Takano, T. 2003. Salt tolerance-related protein STO binds to a Myb transcription factor homologue and confers salt tolerance in Arabidopsis J. Exp. Bot. 54:2231-2237. 24_ Park, Y. D., Papp, I., Moscone, EA, Iglesias, VA, Vaucheret, H., Matzke, A. J·, and Matzke, MA 1996. Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity. Plant J. 9: 183-194. 25. Prins, M., Laimer, M., Noris, E ., Schubert, J., Wassenegger, M., and Tepfer, M. 2008. Strategies for antiviral resistance in transgenic plants. Mol. Plant Pathol. 9:73-83. 26. Rommens, CM5 and Kishore, GM 2000. Cur. Opin. Biotechnol. 11:120-125. 27. Stuiver, Μ. H. and Custers, J. Η. Η. V. 2001. Engineering disease resistanc e in plants. Nature 411:865-868. 28. Taylor, B and Powell, A. 1982 Isolation of plant DNA and RNA. Focus 4: 4-6. 29. Vailleau, F., Daniel, X., Tronchet, M., Montillet, J. L., Triantaphylides, C., and Roby, D. 2002. A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack. Proc Natl. Acad. Sci. USA 99:10179-10184. 30. Wilmink, A., van de Ven·, B. CE, and Dons, J. J. M. 1995. Activity of constitutive promoters in various species from the Liliaceae. Plant Mol. Biol. 28:949-955. 31. Yang, Y.5 Li, R., and Qi, M. 2000. In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J. 22:543-551.

Claims (1)

201125978 VII. Patent application scope: 1. An inducible promoter isolated from lily, the nucleotide sequence of the promoter is selected from: (a) having the nucleotide sequence as shown in SEQ ID NO. a nucleotide sequence as shown in SEQ ID NO: 2, (c) having a nucleotide sequence as shown in SEQ ID Ν α 3, (d) having a nucleotide sequence as shown in SEQ ID NO: 4, (e) And at least one of the group consisting of nucleotide sequences as shown in SEqidn〇: 5. 2. The promoter of claim 1, wherein the promoter has a chemical substance, a stress, and a pathogen-inducing property, wherein the chemical substance comprises salicylic acid, growth, glbberellin, cells Cytokinin and abscisic acid, methyl jasmonate; the stress includes salt, high temperature, low temperature, heavy metal treatment; the pathogen contains gray mold pathogen (5〇 no tomm, lily ash Mold fungus (β(10) Shaozhi (4) (4) (3)), Solanomonas campestris ipv. vesicatoria 'Erwinia chrysanthemi. 3. 3. The promoter described in claim 1 can be driven to connect with it. The target gene is expressed in the leaves, flowers and bulbs of the plant body. 4. A gene expression composition comprising: a promoter as described in claim 1; and an open reading frame (open reading frame) of a polynucleotide; wherein the polynucleotide is linked to the 3' end of the promoter, and the promoter is capable of producing a composition containing the gene In vivo, the transcription of the polynucleotide is initiated (transcripti〇n). 5. A gene expression vector comprising the promoter sequence as described in claim 1. 31 201125978 6. -_ The base contains the (4) patent range body or part of the organ, tissue or cell of the organism. The promoter of the organism 7. The species can be used to turn the target - the sex leaf, flower and sputum method, Including the promoter as described in the first item of the application, the 3, the end is ligated to the target gene to prepare a gene expression vector containing at least the promoter sequence and the target gene; It is planted into a plant, or part of an organ, tissue or cell of the plant, so that the target gene can be specialized in the leaves, flowers and bulbs of the lion. 8. For the method described in item 7 of the patent (4) of the application, the method for the transformation of the towel includes the Agrobacterium vector method, the Agrobacterium tumefaciens primary method, the genetic recombinant virus infection method, the jumper vector transfer method, and the gene cloning. Method, electroporation, microinjection, pollen tube method, liposome media transfer method, ultrasonic medium &quot;transfer method "silic〇n carbide fiber-mediated tmsformation" , electrophoresis (electr〇ph〇resis), laser microbeam (iaser micr〇beam), polyethylene glycol (PEG), calcium phosphate transfer method, DEAE-dextran transfer method. 32