TWI379903B - Inducible promoter from lily and use thereof - Google Patents

Description

Invention Patent Application No. 099101394: Specification Revision Replacement Page Supplement, Revision Date: August 22, 2011, Invention Description: ~~~ -- [Technical Field of the Invention] The present invention relates to the selection of a plant promoter, The field of analysis and application of molecular biology, in particular, is related to the reverse promoter of lily and its use. [Prior Art] Plant genetics (4) is a powerful tool for modern breeding and cultivating, which is used to introduce the desired trait into plant body towels. At present, many breeding strategies have been provided to provide plant-derived Resistance (Prins ei α/, toilet; R_ens — Kish〇re, 2000). The selection of appropriate promoters in regulating the production, performance and performance of foreign genes is quite important, and Xu Wei’s attempt to continue the bribery action _ 丨 ep dirty performance exogenous resistance-related SJ study Wei 'sustained height The expression of resistance genes often leads to a decrease in plant yield and stem production (Hammond-Kosack and Parker, 2003; Michelmore, 20〇3; Stuivef and Custers, 2001). e is a non-necessity for reducing resistance-related genes in uninfected cells. S is now 'inducing start-up publication ndueible pro_ef), such as pathogen or stress-induced promoter, as a feasible improvement strategy _Gurrand Rushton, Na). Many important food crops and cash crops, such as rice, wheat, corn, sugar cane, orchids, etc., belong to the scorpion scorpion's current _ sex promoters, such as the maize promoter and the rice promoter have been selected. Colonization, but some reports indicate that the above promoters are not able to perform well in some flowering and non-steroidal monocots (Joung and Kamo, 2006; Kamo fl/., 1995, 2000; Wilmink slaves α/., 1995). In addition, gene silencing in many transgenic plants is attributed to the use of the same promoter to express several genes (Flavell Heart, 1994; Matzke (10)., 1994; Park Heart/., 1996). Because of the 1379903 ____________ invention special-fresh "reviews] Supplementary, revised date: 〗 〖August 22nd, in the molecular breeding, there is an urgent need to isolate novel promoters from different plant species, only a few suitable start Flowering is more important than non-steroidal monocots. The expression of a gene is via transcription, translation, and post-translational modification (PTM), and the promoter is involved in the transcriptional phase 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nstitmive promoter) and an inducible promoter (the former is 35S of broccoli mosaic virus). Promoter (Califlower mosaic virus 35S, CaMV35S promoter), Agrobacterium nos promoter and plant Actl promoter, etc., can stably and continuously perform gene expression; the latter such as light-induced rape LTP promoter, heat-induced heat shock The protein promoter, the wound-inducible potato PIN2 promoter, and the pathogenesis-induced protein promoter are induced by specific factors to enhance the expression activity. In terms of tissue specificity of promoter expression, it can be distinguished as a tissue-specific promoter (tjssue_Speeific pr〇m〇ter) that is only active in some tissues and organs, or can be expressed in various tissues and organs. Non-tissue specific promoter (n〇n tissue-specific promoter). Whether a promoter is persistent or inducible, tissue-specific, etc., 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 vaccination is known. Infected with Lilium oxysporum (and noisy ¢//(7)), treated with salicylic acid (Sa), or probenazole, can make sunflower lily (legs such as Gaze〇 leaves) The number of lesions caused by Botrytis cinerea infection is significantly reduced (Chen βα/., 2003; Chen and Huang, 199 is 4 1379903

补充 Supplement, revision date 8 /2?J

Lu and Chen, 1998, 2005; Lu Xin/·, Cong), showed that lily can be stimulated by extrinsic factors to induce disease resistance, and the induction of resistance can reduce the severity of secondary infection. The identification-related gene is identified and isolated. Lilium 'Star Gazer, glycine-rich protein 1, LsG^/7 is encoded as a 138. Amino acid protein having homology to a number of glycine-rich proteins expressed in an extracellular matrix, having salicylic acid, culling heat, and Botrytis cinerea induction Improve plant resistance to stress and pathogens. Therefore, the inventors of the present invention speculated that the promoter should have an appropriate basic expression of stem and stress, pathogen-inducing, and can be applied to the genetic engineering by screening the sequence of the promoter to enhance the organism for adversity. , or resistance to disease. In view of the importance of developing a specific and inducible promoter in the biotechnology industry, the inventor of this case succeeded in researching and developing the "Lily stress-inducing promoter and its use". Part of the present invention has been disclosed in the Summary of the XIV International Congress on Molecule Plant Microbe Interactions from July 19, 2009 to July 23, 2009. References 1. Abe, H., Urao, T., Ito, Τ., Seki, Μ., Shinozaki, Κ., and Yamaguchi-Shinozaki, K. 2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function Plant transcription 15:63-78. 2. Ashoub, A. and Abdalla, KS 2006. A primer-based approach to genome walking. Plant Mol. Biol. Rep. 24:237-243. 3. Chen, CY, Lu, YY, and Chung, JC 2003. Induced host resistance against Botrytis 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. Natl. Acad. Sci. USA 91:3490-3496. 1379903 Patent Application No. 099101394: Specification Amendment Replacement Page Supplement, Revision Date : August 22, 2011 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. Lu, Y. Y., Liu, Y. H, and Chen, C. Y. 2007. Stomatal closure, callose deposition, and increase of -corresponding transcript in probenazole-induced resistance

Botrytis elliptica in lily. Plant Sci. 172: 913-919. 7. Denekamp, M. and Smeekens, SC 2003. Plant Physiol. Integration of wounding and osmotic stress signals determines the expression of the AtMYB102 transcription factor gene. Plant Physiol. 132 :1415-1423. 8. Du. H., Zhang, L., Liu, L., Tang, XF, Yang, WJ, Wu, YM, Huang, YB, and Tang, YX 2009. Biochemical 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, K., 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, YH and Kamo, K. 20 06. The expression of a polyubiquitin promoter isolated from Gladiolus. Plant Cell Rep. 25:1081-1088. 13. Kapila, J., de Rycke, R., 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., Blowers, A., Smith, F., Van Eck, J., and Lawson, R. 1995. Stable transformation J. Am. Hortic. Sci. 120:347-352. 15. Kamo, K., 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 plants. In Vitro Cell Dev. Biol. Plant. 36:13-20. 16. Kopertekh, L. and Schiemann, J. 2005. Agroinfiltration as Transgenic Res. 14:793-798. 17. Lescot, M., Dehais, P., Moreau, Y., De Moor, B., Rouze, P., and Rombauts , S. 2002. PlantCARE, a database of plant cw-acting regulatory elements and a Nucleic Acids Res. 30:325-327. 18. Lu, YY and Chen, CY 1998. Probenazole-induced resistance of lily leaves against Botrytis elliptica. Plant 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 -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. 244:219-229.

Michelmore, RW 2003. The impact zone: genomics and breeding for durable disease 6 22. 1379903 Patent application No. 099101394: Specification revision replacement page supplement, revision date: August 22, 2011 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, YD, Papp, I., Moscone, EA, Iglesias, VA, Vaucheret, H., Matzke, AJ, 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, CM, and Kishore, G M. 2000. Exploiting the full potential of disease-resis Cur. Opin. Biotechnol. 11:120-125. 27. Stuiver, Μ. H. and Custers, J. Η. Η. V. 2001. Engineering disease resistance 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, JL, 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., BCE, and Dons, JJM 1995. Activity of constitutive promoters In various species from the Liliaceae. Plant Mol. Biol. 28:949-955. 31. Yang, Y., Li, R., and Qi, M. 2000. In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco [Abstract] The object of the present invention is to provide a tissue-specific high expression intensity promoter with high surface strength and specific tissue in plants. Medium (leaves, bulbs, flowers) drive a large number of target genes linked to them. A second object of the present invention is to provide a variety of organisms, particularly

丨: I A promoter with high expression intensity in various plant types (eg, ferns, gymnosperms, monocotyledons, and dicotyledonous angiosperms). 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, 7 1379903 ___ invention patent application No. 099101394: manual amendment replacement page supplement, revision date: August 22, 2011 Various types of application methods or application strategies applicable to the present invention, as well as various types of products, compositions or organisms comprising the promoters of the present invention. A lily stress-inducible promoter (human 5 (^7 promoter), which is derived from the genomic DNA of the lily hybrid cv. Star Gazer). Since lily is rich in glycine protein i (Lilium 'star Gazer' glycine-rich protein l, LsGi? P7) with salicylic acid, culling heat and inducibility of Botrytis cinerea, the inventors of this case speculated that the promoter should be It has the property of being induced by stress. 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 was ligated to the reporter gene β-glucuronidase (p_glucur〇nidase, GUS) gene sequence, column 5 & as the reporter gene a promoter, constructed in a transformation vector to form a recombinant transplastosome; and then, by agroin injection, the recombinant protoplast containing the promoter is transferred to tobacco and lily, by Analysis (31; 3 activity to assess the promoter activity of the test promoter; the results show that the lily promoter can drive the target gene linked to it to a large extent in the specific tissues of the plant (leaves, flowers and bulbs); The promoter of the invention has a high expression intensity and is tissue specific. α wherein the nucleotide sequence of the promoter is selected from: (8) a nucleotide sequence as shown in SEQ ID NO: 1 (868 bp) And (b) having the nucleotide sequence (714 bp) as shown in SEQ ID NO: 2, (c) having the nucleotide sequence (586 bp) as shown in SEQ ID NO: 3, (d) having The nucleotide sequence (362 bp), (e) shown in SEQ ID NO: 4 has SEQ ID NO: ID NO: The nucleotide sequence (125 bp) shown in Figure 5, at least one of the group consisting of. In addition, the present invention 8 1379903 ___ invention patent application No. 101394: the specification correction replacement page supplement, revision date The nucleotide sequence of the LsGM/promoter described in Aug. 22, 101 can also be modified by appropriate nucleotide substitution, mutation, etc., and the nucleotide sequence of the promoter is also included. It has at least 80% identity with the nucleotide sequences (a) to (e) above, and still has the same performance characteristics as the promoter of the present invention.

I • In addition, different treatments are used to assess the inducibility of the promoter. The treatment includes: chemical treatment, stress treatment, and pathogen treatment. The chemical substance includes auxin, gibberellin, cytokinin and abseil acid, methyl jasmonate, salicylic acid. The adversity includes: salt 'high temperature, low temperature, heavy metal environment; the pathogen contains · · gray 'mold mold (price noisy, lily gray mold bacteria (price noisy Xianchong (four), Solanaceae bacterial spot plaque 'JCanthomonas Campestris pv. vesicatoria), soft rot disease (Erwinia c/wya « ewz·). According to the invention, the promoter has chemical substances, stress, and pathogen-inducing properties. "Chemical substance inducing" means that after being induced by chemical recklessness (such as 'plant hormone, environmental hormone, compound, etc.), it can effectively enhance the promoter activity of the promoter, and the activation activity is higher than that of the non-chemical substance. (eg: plant hormones - hormones, compounds, etc.) inducers. f • The towel "hearing" for plants, too high a degree of turbidity will lead to an imbalance of osmotic pressure, which seriously affects the plant body, so ,#Phyto body violence In high-concentration salt environments (such as nano-ion (4) and gas-ion (cr)), it is an environmental stress for plants, and each plant species has different tolerances for salt concentration. The "salt treatment" or "salt-inducing" according to the present invention includes but is not limited to: (1) the plant 9 invention patent application No. 099101394: the specification correction replacement page supplement, revision date: August 22, 2011 After the salt concentration treatment of the body weight, it will cause the osmotic pressure imbalance to cause the growth of the plant body to be affected, thereby inducing the promoter activation activity, or (7) after the salt induction, the promoter can be effectively promoted. The high-temperature treatment is generally performed at a temperature higher than 30 〇c. One embodiment is a treatment at 37 ° C, and the low temperature treatment is generally performed under the temperature of (4) 〇c卩. It is treated by 4C. Another 'high temperature or low temperature inducibility" according to the present invention can also be defined as: after a temperature induction, it can effectively increase the promoter activity of the mover, which is higher than that which is not induced by temperature. Detailed : If it is "high temperature material", the temperature of the material is higher than that of the uninduced one, and the / phosgene can effectively induce the promoter activation activity, and the activation activity Γ7 is not, and the worker induces. "low temperature inducibility" means that the induced spill is lower than that of the uninduced, and the temperature can promote the promoter's initiation, and the activation activity is higher than that of the uninduced one. However, it is secret: the sulfur-(CuS〇4>K solution is used to evaluate the stress inducibility of the LsG brother P/promoter to copper or other heavy metal ions. The "heavy metal inducibility" and "copper" described in the present invention. Inducibility can also be defined as: after induction by heavy metals or heavy metal ions (such as copper, copper ions), can effectively enhance the promoter activation activity, and the activation activity is higher than the heavy metal or heavy metal ions (such as copper, Copper ion) inducer. The "disease-conductivity" as used in the present invention means that after the infection or induction by the pathogen, the promoter is effectively enhanced, and the grade activity is higher than that of the uninfected county or inducer. Further, the present invention also introduces a recombinant transplastosome containing a LsG/W promoter into different plant species by Agrobacterium injection to evaluate the applicable range of the promoter. The plant species comprises: ferns, gymnosperms, monocots, and dicotyledonous angiosperms. The 1379903 invention patent application No. 099101394: Specification revision replacement page _ Supplementary, revision date: August 22, 2011 The fern contains BirdVnest fem; the gymnosperm contains: bamboo cypress (9) 咱p〇d〇carp) With Ginkgo (Gmgo); the monocotyledonous angiosperm includes: Maize, Butterfly orchid, Oncidium orchid, Chinese taro, Snake plant, Ginger lotus ( Siam Tulip), 'Star Gazer' lily and Taiwanese lily (F〇rmosa%), the monocotyledonous angiosperm contains: Maize, Butterfly orchid, Oncidium orchid, aunt Chinesetaro, Snakeplant and Siam Tulip; the dicotyledonous angiosperm contains Lettuce, spinach

(Spinach), Bottle Gourd, Angled Luffa, Chinese Kale, Snap bean, Coffee, Basil, Hot Pepper, Petunia Petunia), Pepper, Orange, Madagascar Periwinkle, Arabidopsis, and Tobacco, TV·Benthamiana N. tabaccum. In addition to providing a lily stress-inducible promoter promoter, the present invention also provides a gene expression composition. The gene expression composition comprises: (1) a promoter of the present invention, and (2) a segment having a polynucleotide of an open reading frame (ORF), that is, a target gene; the polynucleotide is linked to the 3' end of the promoter of the present invention, and the promoter system can contain the The organism of the gene expression composition 'initiates the transcription of the polynucleotide; in one embodiment, the target gene is the reporter gene β-glucuronidase (GUS). Furthermore, the lily stress-inducible promoter promoter of the present invention is constructed into a general commercial carrier, including but not limited to: ρΒΙΙΟΙ, pBI121, pBIN 19 (ClonTech), pCAMBIA1301, pCAM IA1305, pGREEN (GenBank u 1379903 ___ Invention patent application No. 099101394: Supplementary replacement page supplement, date of revision: August 22, 101

Accession No: AJ007829) > pGREEN II (GenBank Accession No: EF590266), pGreen0029 (John Innes Centre), can form a gene expression vector, or recombinant transfer vector, and insert the target gene into the gene expression vector. , 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 ligated to the 3' thereof by transfection. 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 performance of the target gene in the target transgenic organism and its progeny. The present invention also provides a method for allowing target gene specificity to be expressed on leaves, flowers and bulbs of plants, the method comprising: linking the 3, end of the aforementioned promoter to the 5' end of the target gene, to prepare at least one The promoter sequence and the gene expression vector of 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 leaf of the plant , flowers and bulbs. The methods for transfection include, but are not limited to, Agrobacterium mediation, Agrobacterium injection, recombinant virus infection, jumper vector transfer, gene grab transfer, electroporation, microinjection, pollen tube Method, liposome mediation method, ultrasonic mediation method, carbonization fiber medium; silicon carbide fiber-mediated tmsformation, electrophoresis, laser micr〇beam , polyethylene glycol (PEG), calcium phosphate transfer method, DEA dextran transfer method. Because of the promoter provided by the present invention, the target gene can be expressed in a large amount, with tissue specificity, stress inducibility, and a wide range of characteristics of the organism, and therefore, in addition to the invention patent application No. 099101394 : Specification Amendment Replacement page Supplement, Revision Date: August 22, 2011 e eH ZriGitP/promoter to initiate the defense-related gene: the amount of glycine-rich glycine protein 1 is so Transgenic plants can also be used to produce a variety of desired proteins in large quantities, or to regulate transcriptional activity to achieve various applications in which the present invention is applicable. 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 readily available commercially, and the following are merely examples of available pipelines. [Embodiment] Abstract: Firstly, the full-length sequence and partial sequence fragments of the promoter were selected from sunflower lily, and the selected LsGRPi promoter was analyzed by Agrobacterium injection method to understand the tissue performance characteristics of the anti-p7 promoter. Responses to various adversities, pathogens, and plant hormones, and applicability to different plant species. Example 1 Selection of Lily Promoter 1.1 Plant Material A commercially available sunflower lily (U/wm oriental hybrid cv. Star Gazer, Fukuoka Seedling Co., Ltd.) was planted with a ball of P/. After surface disinfection of sodium hypogasate, it is planted in a plastic pot containing cultivation medium (peat soil [BVB]: No. 2 pearl stone = 3: 1) at 20-25. (: cultivation for about one month to be used. 1.2 Lily genomic DNA purification 13 1379903 Patent application No. 099101394: Specification revision replacement page supplement, revision date: August 22, 2011, modified CTAB Method (Taylor and Powell, 1982) Purification of the nucleic acid of P. sylvestris L. After 2 g of sunflower lily leaves were ground and pulverized with liquid nitrogen, add 8 mi of CTAB extraction solution preheated to 65 «>c (6% cetyitrimethyammonium bromide, 100 mM Tris-HCl, 1·4 M NaCl, 200 mM EDTA, pH 8. Add protease κ (proteinase K) and 2-mercaptoethanol (2-mercapt〇ethanoal) to a final concentration of 300 pg/mi before use. 30/〇 (v/v)) Mix well and rotate at 5 μm for 2 hours at 65 ° C. Add an equal volume of Cl (chloroform: octanol = 24:1) and mix gently, then centrifuge at 40C, 6000 g. After 1 minute, the supernatant was collected, and 1/10 volume of CTAB/NaCl solution (1〇〇/0 CTAB, 0.7 MNaCl) preheated to 65T was added, gently mixed and extracted with CI. Extraction of CTAB/NaCl solution and CI After several times of repeated steps, the nucleus in the supernatant of the chamber was dissolved in 0.7 volumes of isopropanol (iSOprOpan0i). 'The resulting nucleic acid precipitate is rinsed with 80% alcohol and air dried, then dissolved in an appropriate amount of sterile deionized water. See the method of Ashoub and Abadalla (2006) for selection. Use restriction enzymes ρηΐ, ΛίΙ SacI and (Roche) separately fermented the whole-genome nucleic acid of K. chinensis and recovered the DNA fragment with 3, end-pointing, and then separately linked it to the linker with the complementary sequence of each restriction enzyme (adapter_primer) OPH-Kpnl, OPItPstl, OPH-SacI and OPH-SacII for ligation [recovering their products as a polymerase chain reaction ^p〇iymerase_chain reacti〇n, PCR) template, with the conserved sequence of each linker The primer Ap and the specific reverse primer p329 are increased. 'Recycling the product as a PCR template, using each linker 14 · · · 1379903 Patent Application No. 099101394: Specification Revision Replacement Page Supplement, Revision Date: August 2011 On the 22nd, the conserved sequence primer NP and another specific reverse primer p37i were subjected to nested PCR to recover the PCR product and colonize it to E. coli. In the u-Biue or τ〇ρ1〇F' strains, the selected strains were sequenced for subsequent sequence and functional analysis, and the primers were designed according to the results of the sequence. pCR was generated from the ginseng nucleus nucleic acid Add "Nakata" and select the 868 bp promoter (mouth (five) muscle) sequence full length, with seq id

No. 1 shows the sequence in which the primer sequence used is as shown in Table 1. Another design 4 primers were used to select the sputum to the milk partial sequence fragment (sequence deletion group: 714

(SEQ ID 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 deleted the reverse of the group (milk The primers are all p400; the forward primers are: dpGRP-714, dpGRP-586, dpGRP-362 'dpGRP-125, and the primer sequences used are shown in Table 1. Table 1 Primer name primer sequence SEQ ID No ΟΡΗ-ΚρηΙ 5'-gaattcgagctcgcccgggatcctctagagtac -3' 6 OPH-PstI 5r-gaattcgagctcgcccgggatcctctagatgca-3* 7 OPH-SacI 5'-gaattcgagctcgccgggatcctctagaagct-3' 8 OPH-SacII 5'-gaattcgagctcgccgggatcctctagagc -3ς 9 AP 5'-gaattcgagctcgcccgggatcc-3' 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 dpGRP-714 5-tcttctcctagggctctcaagtgtatttag-31 16 dpGRP-586 5'-tacatgtaatagttttggatcgag-3' 17 dpGRP-362 5f-tcagtatcctctattgcccccttaacg -3* 18 dpGRP-125 5 '-cgccaactgcatgtctgtcgcc-3' 19 Promoter sequence analysis

The selected LsGRP1 promoter sequence of the genus Lilium sinensis L. is an online software T; §sp analysis possible 15 1379903 _ Patent application No. 099101394: Specification revision replacement page supplement, correction period: August 22, 2011 RNApolymerase II The binding region, TATA box and transcription initiation site (Shahmuradov β α/., 2003), and the online plant cis-acting element database PLAce (Higo βα/., 1999) for comparative analysis, the prediction may have a smooth Sequence of action elements. After the alignment prediction, it was found that the multiple cis-acting elements of the LsGRP1 promoter (p^m) are related to pathogenic and abiotic stresses, plant hormones, signaling, and physiological regulation. Multiples are associated with light-induced performance and mesophyll tissue specificity, • indicating that it should be a valid promoter sequence. ·-' Example 2 LG/m promoter (functional analysis of Pis (JW) 2.1 Binary expression vector) See Figure 1 for the 'binary vector' pBI121 to limit enzymes/ //«dill and such as wffl (Roche) to remove the CaMV 35S promoter, recycling and removal

The 14 kb pBI121 backbone of the CaMV 35S promoter, with Klenow enzyme (New

England Biolab) cuts the restriction enzymes at both ends to a Tj DNALigase with 868 bp and 4 groups of PjLiG^^ sequence deletions (125 bp, 362 bp, 586 bp, and 714 bp) amplified by polymerase chain reaction. (Promega) performs the binding reaction, and then transforms and transforms the E. coli ToplOF' strain for screening and sequencing, and determines that the construction is correct, and five sets of recombinant double-coupled vectors having different promoter sequences can be prepared. Five sets of recombinant bicone vectors were introduced into Agrobacterium tumefaciens (CfeWww ί(10)^/(10)) C58C1 strain for Agroinfiltration to analyze the transcriptional functions of the promoter sequences. In the function analysis of the promoter, the maize promoter is used and the (:^358 promoter double 16 1379903 _ invention patent application No. 099101394: the specification correction replacement page supplement, revision date: August 22, 2011, even carrier As a control group. 2·2 Agroinfiltration, the five sets of recombinant double-coupled vector and pBI121 as a control group were respectively transferred into Agrobacterium by electroporation (jgraZwcim.ww C5) The 8C1 strain was inoculated separately into 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 250 C, 200 rpm φ for 16 hours. , then move the cells to the induction medium (0.3% K2HP〇4, 0.1%

NaHP045 0.1% NH4CI, 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), incubated at 25 〇C, 200 rpm for 6 hours. The cells were recovered and suspended at a concentration of 0.8 () ()

Agrobacterium injection buffer (100 mM acetosyringone, 10 mM MgS04, 10 mM MES, pH 5.6) was injected from the plant leaf back or other tissue with a 1 ml syringe. • 2.3 β-glucuronidase (GUS) active tissue staining analysis Take appropriate amount of tissue samples to be stained, add GUS active group staining solution (1% X-Gluc (5-bromo-4) that can submerge sample volume -chloro-3-indolyl-pD-glucuronic acid, Biosynth), 100 mM phosphate buffer, pH 8.0, 10 mM EDTA, 0.5 mM ferrocyanide, 0.5 mM ferricyanide, 10% Triton X-100, 10% methanol) ' at 37 After 16 hours of staining at °C, 'chlorophyll discoloration was performed with 70% alcohol until the sample tissue was completely whitened. 17 1379903 Patent Application No. 099101394: Specification Revision Replacement Page Supplement, Revision Date: August 22, 2001 2.4 GUS Activity Fluorescence Quantitative Analysis Take 5 〇mg of plant tissue sample and add 2〇0 μΐ 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, centrifuged at 40C, 1200 g to remove plant debris, 50 μM The supernatant was added to a 200 ml GUS extraction solution preheated to 37 ° C and 250 ml of GUS analytical solution (GUS extraction solution containing 2 mM 4-methylumbelliferyl-bD-glucuronide), and reacted at 37 ° C for 30 minutes to 1 hour. The ΙΟΟμΙ reaction product was added to 900 μl 0.2 M sodium carbonate to terminate the reaction, and Plate CHAMELEON V (Hidex) was used for fluorescence detection and quantification of 〇D365. 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 ZsGiiW promoter 3.1 Plg/w enables the reporter gene to be transiently expressed in the blade of grass. The recombinant haplotype vector containing the 868 bp promoter (P^Gm::GUS-pBI121) The rod and the control group (p35S::GUS-pBI121) were sent to the grass by Agrobacterium injection (5 days after iV/coi/awa expression, the GUS protein was stained by GUS active tissue staining to detect r* r· product (as shown in Figure 2A and Figure 2B). The results show that the promoter (卩^奶) can make the reporter gene GUS highly expressed in tobacco (Fig. 2A), and its cumulative performance is almost universal. Sub-(Fig. 2B) 3.2 Organization-specificity 18 1379903 Patent application No. 099101394 No. Revision of the manual Replacement date, date of revision: August 22, 2011 To understand the LsG/ePi promoter (Ρζ^ΛΡ/ In the performance characteristics of different plant tissues, various tissues or organs (eg, bulbs, leaves, flowers) of sunflower plants are selected, and the aforementioned recombinant gemini vector containing the promoter (SEQ ID Ν ο: 1) is introduced by Agrobacterium injection method. (PLsG^^/^GUS-pBId) and perform 're-growth analysis by GUS activity to understand LsGMi The tissue characteristics of the promoters. The results showed that the analysis of the bulbs, leaves and flowers of the sunflower lily showed the basic performance of the bulbs, leaves and flowers of the sunflower lily, and the highest expression in the leaves was about bulbs. With the flower, it is stronger (as shown in Figure 3). The predictive analysis of the cis-acting factor (^-actingelement) shows that piiGm should have mesophyll cell specificity and light-inducing; this prediction is inferred with PlsG/w The results of in vivo performance analysis of different tissues are consistent, which proves that it is tissue-specific and can initiate the target gene expression in leaves, bulbs, flowers, etc. ' ^•3 ^LsGRPI is a chemical substance, stress and pathogen-inducible promoter In order to compare the inducibility of the promoter under different stimuli: First, as shown in Figure 4a, the recombinant gemini vector containing the promoter (SEQ ID No: 1) prepared above was injected with Agrobacterium. The method was transferred to the final grass (eight) W38), and the same ship was used to evaluate the inducibility and possible inducing factors of the promoter. The different stimuli are: (4) the control group is the leaf watering and the normal washing of the grass plant; (b) the disease material: the foliar injection is floating in (1) the humiliation (7) ^mmmrwirna chrysanthemi^n%^ (OD6〇〇 =0.2) ; Quality induction: foliar spray with 1 mM, pH 6.5 salicylic acid; (4) salt induction: (iv) 〇mi i M NaC1 aqueous solution for root zone; (4) thermal induction: plant at 37〇c Growth box. 19 1379903 "_____ Invention patent application No. 099101394: Specification revision replacement page _ Supplementary, revision date: August 22, 2011, each group of treatments carried out the number of repeats of 3 plants, after 48 hours of treatment, collected in grass blades for GUS Quantitative analysis of active fluorescence. The results were significantly higher than those in the control group (9) to (7). The activity of the promoter in the heat-induced group was the highest, and the salt-induced group. Chemical substance (salicylic acid)-inducing group, pathogen (soft rot) induction group. Confirmation of the promoter of the present invention (SEQ Ι£) N〇: ^ 868 sounding chemical substance 'adversity, pathogen inducibility>> In addition, the present invention This promoter (SEQ ID N: 1} was also distinguished into fragments of different sizes to evaluate the possibility of each fragment sequence as an inducible promoter, and to analyze possible regulatory units on the promoter (SEQ ID No: 1). First, the above five recombinant haplotype vectors (125 (A), 362 bp (B), 586 bp (C), 714 bp (D), 868 bp (E) were prepared by Agrobacterium injection method. a, B, C, D are partial fragments of the promoter, and E is the full length of the promoter. Column) was introduced into tobacco W38). Plant hormones were used: auxin (Αυχ), gibberellm (GA), cytokinin (CK) and detached acid (abscisic add, ΑΒΑ), plants Resistance-related signaling molecules: methyl jasmonic acid (me%1 MeJA), salt (Salt) treatment, high and low temperature (37〇C and 4. 〇, copper (Cu) treatment, phytopathogenic fungi: Botrytis cinerea Noisy fishing rod buckle, Be), bacteria: Xamhomofias cwnpestHs pv. vesicatoria, Xcv, etc., and observe and analyze the performance of the GUS gene to understand the inducing performance characteristics of the promoter. When the recombinant symposium vector containing the cockroach is introduced into the lily, the induced phytopathogenic fungus is preferably Botrytis cinerea (price ¢ ¢ / / (4) Ζ · Λ 7). First, the Agrobacterium injection method for 6 weeks Dazhi Tobacco (#_W38) carries the full length of 20 1379903 ----- Invention patent application No. 099101394: Specification revision replacement page - Supplementary, revision date: August 22, 2011 • (868 bp) and sequence deletion group Analysis of the performance of the recombinant double-coupled vector, respectively, after 24 hours The following 9 groups were treated: (4) foliar spray with 50 mM methyl jasmonic acid (MeJA); (b) treatment with lyophilic acid (ΑΒΑ), foliar spray with 1 μμL of separation acid; (c) 1〇〇ppm, indole-3-acet丨e acid auxin (AUX) for foliar spraying; (d) foliar spraying with 1〇〇ppm.gibberellic acid-3 gibberellin (GA); e) foliar spraying with 1 〇〇 ppm 6-benzylaminopurine cytokinin (CK); (f) high temperature treatment (heat treatment), placing the plants in a growth chamber at 37 ° C; (g) low temperature treatment (cold treatment), Plants were placed in a 4 〇c φ growth chamber; (h) copper (Cu) treatment (heavy metal treatment), foliar spray using 1 mM aqueous solution of copper sulphate (CuS04); (1) salt treatment 'with 150 ml 200 mM NaCl aqueous solution Root ring watering; (〇 pathogen treatment, foliar injection of 10 niM MgS04 of Xanthomonas campestris pv. vesicatoria, Xcv) fine sputum suspension (〇〇60〇-〇·2), (k ) Pathogen treatment 'foliate spray 1 χ 1 〇 5 Sp〇res / ml Botrytis cinerea (price noisy, Be). The control group was leaf sprayed with water and normally watered tobacco plants. The number of replicates of 3 plants was treated in each group, and 48 hours after treatment, the leaves were collected for quantitative analysis of GUS activity fluorescence. Referring to Figure 4B, the results indicate that compared to the control group, after various treatments - especially along with the promoter (whether it is a fragment of partial sequence deletion (A, :·Β, C, D)' or The full-length sequence-column can effectively induce the driving function of the promoter, in which the pathogen (especially caw/7 such as rw pv_ and 5. and high and low temperature stress can highly induce the driving ability of the promoter (P·,), thioglycolic acid Plant hormone, copper sulfate and high-salt treatment are the second. Another 'compared from the full-length and sequence deletion performance analysis results of the sand/promoter, 125 21 1379903 invention patent application No. 099101394: the manual revised replacement page supplement, correction Date: August 22, 曰 bp has a high performance characteristic, 586 bp and 868 bp second, 362 bp and 714 bp are worse. Example 4 hG / W promoter (PisG milk) application range 4.1 can be applied Many plant species are tested for the degree of performance of promoters in different plant species, selecting a variety of bio-research-related patterns or generic species, and crops with high economic value and agricultural importance. Agrobacterium injection will contain promoters. (S The recombinant haplotype vector of EQ ID No: l) was introduced into the plant (leaf) to analyze the performance characteristics of the promoter in different species, and was used in the promoters of molecular breeding, molecular farm and biotechnology research: broccoli mosaic The performance characteristics of the 35S promoter and the maize Actl promoter were compared to understand the characteristics of the promoter. Each promoter was subjected to 3 replicates per plant, and the mean and standard deviation of the intensity of the performance were calculated. The average performance intensity of the 35S promoter in each plant species was set to 1, and the relative intensity of the promoter and Ubil promoter in each plant species was listed in Table 2. The numbers in parentheses represent the relative standard deviation values. 'Expression is not detected. Gas refers to Table 2 lion, silk hG/W promoter in the native plant species 'performance can be known' in 2 kinds of lily: sunflower lily ('Star Gazer, my) and Taiwan lily (F_〇sa%) , Bird's Nest fern; 2 species of gymnosperms: P. sylvestris) and Gingo, 6 monocotyledonous angiosperms: Maize, Butterucker (Butter£|y orchid) , Wen Xinlan (〇ncidium orchid), aunt (Chinese Rugao), Huweilan (9) such as plant) and ginger lotus (Siam Tulip); 16 kinds of dicotyledonous angiosperms: lettuce 1 coffee (four), spine 22 1379903 invention patent application No. 099101394: manual correction replacement page supplement, correction Date: August 22, 2011 Spinach, Bottle Gourd, Angled Luffa, Chinese Kale, Snap bean, Coffee, Basil, Hot pepper, Petunia, Pepper, Orange, Madagascar Periwinkle, Arabidopsis and Tobacco, bent. benthamiana 氙N. tabaccum ) f, LsGRP1 promoters have excellent performance, even more widely used in genetic engineering and molecular breeding CaMV35S promoter (P35S) and corn Ubil Kai · mover is high, showing promoter (P〇Gm) can be applied In addition to molecular breeding, there are many possibilities for plant species to drive the performance of target genes. In addition to molecular breeding, there are also possibilities for their application on molecular farms, which can be used to produce a wide variety of desired proteins in different plant species. Table 2 Analysis of PlsGRPJ and other general-purpose promoters in different plant species

Promoters of different plant species Relative activity Promoter Sunflower Lily Taiwan Lily Wuchao Bamboo Cypress Ginkgo P35S 1.00 ±0.38 1.00 ±0.16 1.00 ±0.20 1.00 ±0.08 1.00 ±0.20 ^LsCRPl 3.03 ± 0.58 2.27 ± 0.66 2.98 ± 1.18 2.58 ± 0.20 1.92 ±0.66 Ubil — — — — — Promoter 虎尾兰 Auntie corn phalaenopsis cymbidium P35S l.oo ±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 ± 0.47 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 Promoter Ginger Lotus Lettuce Spinach Flat Pain Loofah 'P35S i.〇〇±0.ll 1'00 ±0.02 1.00 ±0.03 1.00 ±0.10 1.00 ±0.26~ ^LsGRPl 4.19 ±0.28 2.89 ± 0.07 1.95 ±0.25 2.40 ± 0.06 3.10 ±0.34 Ubil 1.69 ±〇.〇8 0.30 ±0.02 0.09 ±0.04 0.22 ± 0.01 0.17 ±0.06 Promoter Cabbage Green Bean Coffee Nine Layer Pepper Pepper P35S 1.00 ± 0.35 1.00 ±0.08 1.00 ±0.20 1.00 ±0.18 1.00 Soil 0.13~ ^LsGRPI 2.86 ±0.59 2.32 ±0.11 1.90 ±0.22 2·40±0.12 2.23 ± 0.42 Ubil 0.2 4 ± 0.02 0.12 ± 0.02 — — — 23 1379903 __-__ Invention Patent Application No. 099101394: Manual Amendment Replacement Page Supplement 'Revised Period: August 22, 2010 曰 Promoter Petunia Sweet Pepper Orange Orange Spring Arabidopsis P35S 1.00 ±0.51 1.00 ±0.64 1.00 ±〇.〇8 1.00 ±0.09 1.00±5ϋ~~ ^LsGRPI 1.14 ±0.14 3.95 ±0.34 11.62 ±0.17 2.890 ±0.17 1.38 ±0.27 Ubil O il ±0.01 0.52 ± 0.05 0.45 ± 0.04 0,07 ± 0.02 — N. benthamiana, tabaccuni, P35S 1.00 ± 0.22 1.00 ± 0.20 ^LsGRPI 1.14 ± 0.14 1.90 ± 0.22 Ubil 0.11 ± 0.01 0.06 ± 0.00 Lily stress induction provided by the present invention The sex promoter and its use, when compared with other conventional promoters, have the following advantages: 1. The promoter of the present invention can activate the target gene behind the 3, end, and display it in a specific tissue of the plant body. Medium (leaves, bulbs, flowers), tissue-specific. 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 raise the yield of the target gene, and its intensity is even higher than that of the general CaMV35S promoter (P35S) and the corn ubil. The child is higher. 3. The promoter of the present invention can be transferred in the form of a carrier. In all kinds of plants, regardless of the strict type, gymnosperm, single red and gemini plants, the ability to start the money is large. 4. The stress-inducible promoter provided by the present invention can be driven by abiotic and biological stress factors, and the user can control the promoter to drive the target gene expression degree connected thereto according to the needs thereof. The detailed description above is a detailed description of one of the possible embodiments of the present invention, which is intended to be limited to the scope of the invention. The patent scope of this case. In summary, this _ in the gene sequence is indeed a unique performance uniqueness of riding 1, should be fully in line with the novelty and progressiveness of the statutory invention patent requirements, love according to law 24 1379903 invention patent application No. 099101394: specification Amendment of the replacement page, date of amendment: On August 22, 101, the application was submitted, and you are requested to approve the application for the invention patent to encourage the invention. [Simplified illustration] The picture shows the promoter gene expression group: gene eXpressi〇rt. cassette construction schematic diagram 'PBH2l (i3kb) CaMV35S promoter replaced with LsGRPl 旬 + 〇 Figure 2A is containing LsGiiP7 The promoter (868 bp) recombinant gemini vector (pLsGRP1::GUS-PBI121) was stained with GUS activity in the leaves of the grass; Figure 2B is the bis-couple vector of the control group (p35S::GUS-PBI121) in tobacco ( Maxima leaves (JUS activity staining results. Figure 2 shows the LsGTiP7 promoter (PAiGm) in the different tissues of the sunflower lily (leaves, flowers, bulbs) analysis, the average performance intensity and standard deviation of each treatment after 3 replicates Gus activity unit: nmole MU min-1 mg "protein. Figure 4A is the proton activity of the full-length promoter (Pwm) under different treatments. The induction treatment method includes: soft rot fungus treatment, salicylic acid treatment , salt treatment, heat treatment. Figure 4B shows the promoter activity analysis of the promoter (Ρ^_) under different treatments, wherein the 尨GM/promoter (Ρ&c/w) is divided into: 125 bp (A), 362 bp (9), 586 bp (C), 714 bp (D), 868 bp (E), the A The B, C, and D groups are promoter partial sequence fragments (sequence deletion group), and the e group is the full length sequence of the promoter; the induction treatment method includes: methyl jasmonic acid (MeJA) treatment, separation acid (ΑΒΑ) treatment, Auxin (AUX) treatment, Gibbs (GA) treatment, cytokinin (CK) treatment, heat treatment, 25 1379903 Patent application No. 099101394: Specification revision replacement page supplement, revision date: August 22, 2011 '♦ After the complex, the good, the salt, the Xanthomonas campestrispv. vesicatoria (Kcv) treatment, 50 noisy (Be) treatment" GUS activity unit: nmole MU min · 1 mg-1 protein ° [main symbol Description] *

26 1379903 _ Invention Patent Application No. 099101394: Sequence Table Revision Replacement Page' Supplementary, Correction Period: August 22, 曰 Sequence Listing &lt;110&gt; National Taiwanese Taixue&lt;120&gt; Lily Inducible Promoter and Use thereof &lt;160> 19 &lt;210> 1 &lt;211> 868 &lt;212> DNA <213> wilted lily (hh(10) oriental hybrid cv·Star Gazer) &lt;400> 1 60 120 180 240 300 360 420 480 540 600 660 720 780 840 868 ggaagcttga tgaagaatta tgttataatt ttagtgtaga tcttcatgct atcgagaatc acaaaatctt ccaagagctt ggatatttcc cagtagaaaa tggtaatggg gttgccacgc cttgaagaaa ttaatactaa tacaatgcat tttacggaat gcttgttgtc gaactcaaga gacaaccgct ttgtcctctt tatatgacca ttcctataaa cactcgtctt tatttccatg agcactgatg gtccatgaat tattaattca tgaggtaccc cagaagttca tcctcatccc tatagtctca aaatttagaa ccttttataa gatggagtag tagaccatag taaatatttt tctgttccgg cagagaattt gaaacctcag acgtggcatc gcaaattgat cttattgtct atccgccaac ctgaacatgc aaacagcc ttggcaatta Atatgataac attttcttct aggatatatc gttacccaca gaacatatat tttaattaac cacaaatggg tatcctctat aatcggaaga c accaattcc gtgttcaatc tgcatgtctg gcatctctgt ttaaaatatc aattggtaca cctagggctc attataagac ggtacatgta ttcatcttgt caaattccaa aggatatttc tgccccctta caatacgtcc aaacctatct tctccacgac tcgcctaccc gtataaatag gttcacattt ttgttttata tcaagtgtat gatttcatca atagttttgg gtgcctacag caactgctgg ctatttccat acgtgatatc gagctgataa tggctcttct tgttgataag cacgtaatct ggtaagaggg &lt; 210> 2 &lt; 211> 714 <212> DNA &lt; 213 &gt; Isa lily oriental hybrid cv Star Gazer) &lt; 400> 2 tcttctccta gggctctcaa tatatcatta taagacgatt cccacaggta catgtaatag atatatttca tcttgtgtgc attaaccaaa ttccaacaac aatgggagga tatttcctat gtgtatttag tgtagagaca tcatcatctt catgctttgt ttttggatcg agaatctata ctacagacaa aatcttttcc tgctggccaa gagcttcact ttccatggat atttcctatt accgctgatg gagtagagga cctctttaga ccataggtta tgaccataaa tattttgaac tataaatctg ttccggttta cgtcttcaga gaatttcaca tccatggaaa cctcagtatc 60 120 180 240 300 360 1 1379903 -- Invention Patent Application No. 099101394: Sequence Listing Revision Replacement Page Supplement, Amendment Date: August 22, 曰ctctatt gcc cccttaacgt gatatccagt agaaaaagca ctgatgacgt ggcatcaatc 420 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 &gt; 586

&lt;212> DNA &lt;213> Oriental hybrid cv. Star Gazer) <400> 3

tacatgtaat agttttggat cgagaatcta tatgaccata aatattttga acatatattt 60 catcttgtgt gcctacagac aaaatctttt cctataaatc tgttccggtt taattaacca 120 aattccaaca actgctggcc aagagcttca ctcgtcttca gagaatttca caaatgggag 180 gatatttcct atttccatgg atatttccta tttccatgga aacctcagta tcctctattg 240 cccccttaac gtgatatcca gtagaaaaag cactgatgac gtggcatcaa tcggaagaca 300 atacgtccga gctgataatg gtaatggggt ccatgaatgc aaattgatca ccaattccaa 360 acctatcttg gctcttctgt tgccacgcta ttaattcact tattgtctgt gttcaatctc 420 tccacgactg Ttgataagct tgaagaaatg aggtacccat ccgccaactg catgtctgtc 480 gcctacccca cgtaatcttt aatactaaca gaagttcact gaacatgcgc atctctgtgt 540 ataaataggg taagagggta caatgcattc ctcatcccaa acagcc 586 &lt;210> 4 &lt;211> 362

&Lt; 212> DNA &lt; 213> sunflower lily oriental hybrid cv · Star Gazer) <400> 4 tcagtatcct catcaatcgg tgatcaccaa gtctgtgttc caactgcatg atgcgcatct cc ctattgcccc aagacaatac ttccaaacct aatctctcca tctgtcgcct ctgtgtataa cttaacgtga gtccgagctg atcttggctc cgactgttga accccacgta atagggtaag tatccagtag ataatggtaa ttctgttgcc taagcttgaa atctttaata agggtacaat aaaaagcact tggggtccat acgctattaa Gaaatgaggt ctaacagaag gcattcctca gatgacgtgg gaatgcaaat ttcacttatt acccatccgc ttcactgaac tcccaaacag 60 120 180 240 300 360 362 &lt;210> 5 &lt;211> 125

&lt;212> DNA 2 1379903 __ Invention Patent Application No. 099101394: Sequence Listing Revision Replacement Page' Supplementary, Revision Date: August 22, 2011 • &lt;213> Cai Lili (&quot;&quot;(10) oriental hybrid cv. Star Gazer) &lt;400&gt; 5 60 120 125 33 cgccaactgc atgtctgtcg cctaccccac gtaatcttta atactaacag aagttcactg aacatgcgca tctctgtgta taaatagggt aagagggtac giatgcattcc tcatcccaaa cagcc &lt;210&gt; 6 &lt;211> 33 &lt;212&gt; DNA &lt;213> Artificial Sequence &lt;220&gt; <223> Forward introduction &lt;400&gt; 6

Gaattcgagc tcgcccggga tcctctagag tac <210> 7 <211> 33

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

&lt;210〉 8 &lt;211&gt; 33

&lt;212> DNA &lt;213> Artificial sequence &lt;220> &lt;223> Forward introduction &lt;400> 8 gaattcgagc tcgcccggga tcctctagaa get 33

&lt;210> 9 &lt;211> 31 &lt;212> DNA 1379903 Patent Application No. 099101394: Sequence Listing Correction Replacement Page Supplement, Revision Date: August 22, 2011 &lt;213> Artificial Sequence &lt;220> &lt;223&gt; Forward introduction &lt;400&gt; 9 31 gaattcgagc tcgcccggga tcctctagag c &lt;210&gt; 10 &lt;211> 23 &lt;212> DNA &lt;213> Artificial sequence <220> &lt;223> Forward introduction &lt; 400> 10 gaattcgagc tcgcccggga tcc 23 &lt;210> 11 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; forward index &lt;400&gt; 11 gctcgcccgg gatcctctag a 21 &lt; 210> 12 &lt;211> 30 &lt;212&gt; DNA &lt;213> Artificial sequence &lt;220&gt;&lt;223&gt; Reverse introduction &lt;400> 12 cctcagccag ctcccgacca gcgtcggagg

&lt;210&gt; 13 &lt;211&gt; 28 &lt;212&gt; DNA 30 1379903 Patent Application No. 099101394: Sequence Listing Correction Replacement Page Supplement, Revision Period: August 22, 2011 &lt;213> Artificial Sequence &lt; 220> &lt;223> Reverse introduction &lt;400> 13 cttaccctat ttatacacag agatgcgc 28

&lt;210> 14 &lt;211&gt; 34 &lt;212> DNA &lt;213&gt; Artificial sequence &lt;220> &lt;223> Forward introduction &lt;400> 14 ggaagcttga tttacggaat tatagtctca ttgg 34

&lt;210&gt; 15 &lt;211> 27 &lt;212> DNA &lt;213&gt;&lt;220&gt; Artificial sequence &lt;223&gt; Reverse introduction &lt;400&gt; 15 cgtcgacaga ggccaggact caggacc &lt;210> 16 &lt;211&gt; 30 &lt;212> DNA &lt;213&gt;&lt;220> Artificial sequence &lt;223> Forward introduction &lt;400&gt; 16 tcttctccta gggctctcaa gtgtatttag 27 &lt;210&gt; 17 &lt;211> 24 &lt;212> DNA &lt;213&gt; Artificial sequence 30 1379903 - Invention patent application No. 099101394: Sequence table correction replacement page supplement, revision date: August 22, 2011 &lt;220&gt;&lt;223> Forward introduction &lt;400&gt; 17 tacatgtaat agttttggat cgag 24 &lt ;210&gt; 18 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Forward introduction &lt;400> 18 tcagtatcct ctattgcccc cttaacg 27 &lt;210> 19 &lt;211&gt;&lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Forward introduction &lt;400> 19 22 cgccaactgc atgtctgtcg cc

Claims (1)

Invention patent towel request No. 0^9101394: Replace the towel with Li Fanling. I ____ Supplement, date of revision: August 2011 _22曰__ VII. Patent application scope: 1. Induction from lily a promoter having a nucleotide sequence selected from the group consisting of: a nucleotide sequence as shown in SEQ ID NO: 1, and (b) a nucleotide having SEQ ID NO: 2 The sequence, (c) has the nucleotide sequence shown as SEQ ID NO: 3, (d) has the nucleotide sequence shown as SEQ ID NO: 4, and (e) is as true as SEq ID N〇: 5辧One of the group consisting of the nucleotide sequence shown. 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, auxin, and gibberellin. ), cytokinin, abscisic acid, methyl jasmonate; the stress includes salt, high temperature, low temperature, heavy metal treatment; the pathogen contains gray ash disease c/werea), lily ash pathogen (and along e//(4)/ca), bacterial spot disease (Abwi/zomcwos cawpej/riiy pv. vesi'cfltonVz), soft rot (Erwinia chrysanthemi) ° 3. The promoter described in claim 1 can drive the target gene linked thereto to be expressed in the leaves, flowers and bulbs of the plant body. 4. A gene expression cassette comprising: a promoter as described in claim 1; and - a polynucleotide having an open reading frame; The polynucleotide is ligated to the 3' end of the promoter which is capable of 'initiating the transcription of the polynucleotide' in an organism containing the gene expression composition. 5. A gene expression vector comprising the promoter sequence 1379903 as described in claim 1 of the patent application. Patent application No. 09910 Brain number: Replacement of the patent _______ :1〇August 22nd 6. A method for allowing a target gene to be exclusively expressed in the leaf flower and bulb of a plant, comprising: a promoter as described in claim 1 of the scope of claim π, 3 And the target is ligated to the target gene to prepare a gene expression vector containing at least the promoter sequence and the target gene; and the gene expression vector is further transferred into the plant body, or a part of the organ, tissue or cell of the plant body, In order to make the target gene uniquely expressed in the leaves, flowers and bulbs of the secret. - 7. The method described in claim 6, wherein the method of agglutination includes Agrobacterium mediation, Agrobacterium injection, Gene recombinant virus infection, Jumper vector transfer, Gene cloning Method, electroporation, microinjection, pollen tube method, liposome mediation, supersonic _ wave media transfer method, carbonized sputum fiber media transfer method (smc〇n coffee sulfur mediated _f〇_i〇n ), electrophoresis (electr〇ph〇resis), laser microbeam (1·μ—·), polyvinyl alcohol (polyethylene glyC〇i;. peg) ' scalloped mother transfer method, DEAE-dextran transfer method . 2 1379903 Invention patent application No. 099101394: Manual revision and replacement page Supplementary, revised period: August 22, 101. IV. Designated representative map: (1) The designated representative figure of this case is · (B). (2) A brief description of the symbol of the representative figure: None Chemical formula: 5. If there is a chemical formula in the case of t, please reveal the best indication of the invention.