TW200416286A - A novel nucleic acid encoding β-1,3-glucanase from lily - Google Patents

Abstract

The present invention relates to a novel nucleic acid encoding β-1,3-glucanase polypeptide of lily, and an expression vector, host cell and transgenic plant comprising the nucleic acid of the invention. The expression of the nucleic acid of the invention in the plant will enhance resistance against a wide variety of stresses, in particular fungal attack.

Description

200416286 发明 Description of the invention (The description of the invention should state: the technical field to which the invention belongs, the prior art, the content, the embodiments, and the drawings) [Technical field to which the invention belongs] The present invention relates to a coded lily -Novel nucleic acids of glucanase (/ 3-1, 3_glucanase), the polypeptides encoded by them and their applications. [Previous Technology] Plants have many mechanisms to fight pathogen attack, such as hypersensitive response (HR), systemic acquired resistance (SAR; induced systemic resistance, ISR), etc. Plant defense response is often accompanied by the expression of pathogenesis-related (PR) gene. Some of the pathogenesis-related proteins produced have bacteriostatic activity, and some of them have enzyme functions such as glucanase and glucanase. Chitinase. Tobacco infected with Tobacco mosaic virus (TMV) has accumulated a number of disease-related proteins in leaf tissue, of which four proteins have W, 3_ glucanase activity (Kauffmann β α /. 1987, EMBO J. 6: 3209-3212). β-1,3-glucanase is widely present in many monocotyledonous and dicotyledonous plants, and some are known to have the ability to inhibit true growth (Sela_Buurlage β α / ·, 1993, Plant Physiol. 101: 857-863 ). A glycanase purified from soybean breaks down fungal cell walls and releases carbohydrates that induce soybean to synthesize plant antibiotics (Keen & Yoshikawa, 1983, Plant

Physiol. 71: 460-465). Using genetic engineering technology to make plants continuously express β-υ-glucan transfection can increase the plant's resistance to pathogenic fungal infections (Kusso & Clip, 1996; Physiol. Mol. Plant Pathol. 49: 267-283; Masoud et al., 1996, Transgenic Res. 5: 313-323; Nakamura et al., 1999, Plant Cell Reports 18: 527-532). In addition to this, EP 440304 200416286 improves the resistance of plants to pathogenic fungi. These plants have at least one gene transduced to produce chitinase or glucanase intracellularly or extracellularly. β_1,3-glucanase and its genes have been extensively studied. si_〇ns, c · R · details the plant υ-β-D-glucanase and U; i + pa glucanase Physiology and Molecular Biology (si_ons, cr_, 1994, Critical Rev. Plant Sci. 13: 325-387). U.S. Patent No. 6,066,491 states that when plant infections cause gangrene lesions, the amount of these enzymes in the entire plant is higher than before infection, including uninfected parts, and the increase in the synthesis of these enzymes can also be Induced by microorganism-derived factors are generally true ii cell wall components. The most specific glucanase gene expression is known to be tissue-specific and pathogen-inducible (Castresana accounts for α /, 1990, Plant Cell 2: 1131-1143) cThimmapuram, J. et al. (2001) and Maher, E.A. et al. Human ((1993) studied the characteristics and performance of peach trees and alfalfa β-1> glucanase and found that their gene expression was pathogenic (Mol · Gen · Genet · 265: 469-479; Physiol. Mol · Plant Pathol. 43: 329-342).

Renault, A. S. et al. (2000) also observed β · glucanase expression in grape leaves infected with fungi (Am · J_Enol. Vitic · 51: 81-87). In addition, Didierjean, L. et al. (1996) reported that β-1,3-glucanase also increased when corn leaves were subjected to abiotic stress (planta 199: 1-8)> W0 92/16632 revelation-soybean Recombinant DNA sequence, which encodes a novel protein with glucanase activity; for the needs of various applications, it is still necessary to continue to breed novel nucleic acids that can express m glucanase. [Summary of the Invention] Summary of the Invention 200416286 The invention provides an isolated nucleic acid molecule, which encodes a polypeptide having glucanase activity, wherein the polypeptide is selected from the group consisting of: Wei of the amino acid sequence shown in the sequence identification number ·· 丨; and -A polypeptide encoded by a nucleic acid that hybridizes under high stringency to a nucleotide sequence of sequence identification number · 2. The present invention provides-Lai, which comprises the isolated molecule of the present invention. The present invention also provides- The co-cell contains privately-marked probabilistic molecules. The present invention also provides a transgenic cultivar, which is _ 本 发. 分 _ 岁 子 狮. The present invention also provides an isolated polypeptide, which is selected from the following group: Identification number: 1 peptide showing amino acid sequence ; And ⑼ a polypeptide encoded by a nucleic acid that hybridizes with a nucleotide sequence of sequence identification number · 2 under highly stringent conditions. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to-encoding lily lily_1,3_ Novel nucleic acids of glucanase, the polypeptides encoded by them and their applications. Nucleic acids and polypeptides encoded by: ^ The aspect of the present invention provides-isolated nucleic acid sequences, which encode- Polypeptide, wherein the polypeptide is selected from the group consisting of: a polypeptide having an amino syllabic sequence represented by i; and (b) a polypeptide encoded by a nucleic acid, which is under high stringency Hybridizes with the nucleotide sequence of sequence identification number: 2. Here, "nucleic acid" refers to a polymer formed by DNA or ribonucleic acid in single or double stranded form, and includes a polymer that can hybridize with single stranded nucleic acid. Similar nucleic acid sequences (such as polypeptide nucleic acids)., 8 200416286 Selective nucleic acid, refers to a nucleic acid that does not include accompanying or interacting parts under natural conditions; or the nucleic acid has been * mutated, "on the cell locus (E.g. genome or Organelles), not a nuclear "polynuclear acid" under natural conditions, refers to a deoxyribose-only version of ribose ribozyme or its analogues that have natural nucleic acid characteristics and can be hybridized in a highly severe environment, and The natural nucleic acid sequence is the same or its sequence can translate into the same amino acid sequence as the natural nucleic acid. 杂交 Hybridization under stringency '' refers to a double-stranded nucleic acid formed by hybridizing two single-stranded nucleic acids. A double-stranded region may include- Or the full length of two single-stranded nucleic acids, or-the full length of one single-stranded nucleic acid and another part of single-stranded nucleic acid 'or two parts of 胄 -stranded nucleic acid. High stringent conditions such as 卩 10 (000,000 nuclear The ordinary acid molecule is a probe and hybridized at 68C. The hybrid solution contains 5xSSpE, 1% sodium lauryl sulfate (SDS), 5xDenhardt's reagent and 100 pg / mi denatured salmon sperm DNA. 1χ sspE and 0.1% sodium dodecyl sulfate, rinsed at 68 ° C; or hybridized at 50% formamide and 42C. High severity rinsing can be performed in 0xSSC_〇2xSSC, 1% sodium lauryl sulfate, C for 15-20 minutes. An example of a severe hybrid rinsing in the South was rinsed at 0 2xSSC at 65 ° C for 15 minutes. Cold Spring Harbor Laboratory, Cold

Spring Harbor Press, NY). Another example of high stringency rinsing is sodium lauryl sulfate 70/0, ο.% ΜL scale buffer, pH 7.0-7.2, 0.25 M NaCl at 65-68 ° C or 50% amine acid Formamidine is hybridized at 42 ° C. Examples of moderately severe hybrids are 35% formamide and 42. (: Heterozygous, rinsed at 55 ° C. This colony nucleic acid encodes a polypeptide with β-1,3-glucanase activity, including the sequence of the gene encoding the glucanase and its regulation | M, 3 -Transcription and translation of a glucanase coding sequence, and includes a cDNA sequence encoding 9 200416286 polypeptide with β-1,3-glucanase activity. Preferably, the nucleic acid of the invention comprises a polynucleotide, which encodes For example, the amino acid sequence of the sequence identification number: 丨. Preferably, the nucleic acid of the present invention comprises a polynucleic acid, which encodes a polypeptide. Nucleus: encoded by a nucleic acid sequence of sequence hybridization. More preferably, the nucleic acid of the present invention comprises a polynucleotide having a sequence as shown in the sequence identification number: 2. According to the present invention, a preferred polynucleoside of sequence identification number: 2 The acid includes 011 nucleotides. Another aspect of the present invention is to provide an isolated polypeptide, which is selected from the following group ya) a polypeptide having an amino acid sequence as shown in the sequence identification number: 1; and A polypeptide encoded by a nucleic acid that is edited with sequence recognition under highly stringent conditions No .: 2 nucleotide sequence hybridization. "Polypeptide" and "peptide" proteins, which can be used interchangeably, refer to amino acid polymers. Amino acid polymers include one or more amino acids that are chemical analogs or are natural amino acid polymers. Proteins containing amino acid analogs can specifically react with antibodies prepared from proteins containing natural amino acids. According to the present invention, the sequence of the sequence identification number: 2 encodes 337 amino acids, and its predicted molecule ϊ is 35.328 A specific embodiment of the polypeptide of the present invention is derived from lily, which is a pathogen-induced glucanase. The N-terminal amino acid of this glucanase protein is MDGDNLPQPADWNLY. Therefore, this new polymerized polypeptide is named LPGlul (lily The pathogen-induced glucanase !, Lily Pathogen-induced glucanase 1). This LPGlul is an acidic β-1,3-glucanase with a pi value of about 4.0. It is known that acid glucanase The enzyme has the function of releasing β-glucan oligomer, which can induce the defense ability of plants (Ebel & Mithofer, 1998, Planta 206: 335-348). Acid glucanase infects plants with pathogens Can be induced systematically and is water Acid is one of the hallmarks of plant defense response (Henning 1993, Plant J. 4: 481-493). Therefore, this LPGlul can be used to strengthen the plant's resistance to adversity 10 200416286, especially resistance to fungal infections. Table j See Zhao Huiman's Master System. Another aspect of the present invention is to provide a vector comprising the isolated nucleic acid molecule of the present invention. "Vector" means a nucleic acid that can be used for transplantation or transfer into a host cell and the nucleic acid can be Inserted into a nucleic acid. The carrier is often a unit of replication. Expression vectors allow transcription and translation of an inserted nucleic acid. The present invention further provides a host cell comprising the isolated nucleic acid molecule of the present invention. "Host cells" refer to Eubacteria & Archea (e.g. Pentagram / • and cyanobacteria), eukaryotic microbes (e.g. fungi) and plant cells. These host cells can be used as recipients of introduction vectors. The invention nucleic acid, the selected nucleic acid encoding the polypeptide of the invention, can be inserted into an appropriate expression vector, and the compilation contains the necessary elements for translation. The method of conforming the silkworm body includes in vitro DNA recombination technology, DNA fusion technology and Bio-recombinant recombination technology. A good selection and manipulation vector is a plastid, which can be propagated in an appropriate microbial host (such as E. mirabilis). A good vector can be used for neutron gland oil and lion cells. A good carrier for the transformation of agricultural rods and media is Ti plastids. Any of the above vectors can be used to introduce direct DNA into the protoplasts. In any operation, a suitable vector is used as the starting material. Both can be used to contain and express the cells that encode the glucanase polypeptide of the present invention. Various techniques can be used to present this nucleic acid to the host cell towel. However, the transformation method has practical effects on the hairpin of the hairpin. The use is not important. The host cell is preferably a bacterial cell or a plant cell, especially an Agrobacterium cell. Transgenic plants The present invention also includes transgenic plants that transform the nucleic acid molecules of the present invention. 200416286 * A 1 ', Λ > The genome of a cell, cell line, tissue, plant part or plant body is changed by the introduction of a foreign coding region. The foreign coding region is sent to the mother cell or plant cell by genetic engineering technology, It can be transmitted to the next generation by sexual or asexual reproduction. Many methods can transform plants or plant tissues (that is, the stable transfer of foreign DNA into plants), including transformation by Agrobacterium and direct gene transfer The construction of the inventive nucleic acid transgenic plant containing the glucanase polypeptide is derived from the introduction of the selected nucleic acid into plant cells. Generally, the inventive nucleic acid is present on the mentioned vector. In plant cells, This vector can be replicated independently. It can maintain a large number of vectors outside the chromosome and has a high peptide capacity, or can be inserted into human # chromosomal DNA. The best vector can Into the chromosomal DNA of plant cells. The transgenic plant of the present invention can resist the infection of various pathogenic bacteria by expressing the new gene. The transgenic plant has the ability to resist stress, especially the damage caused by fungi, such as plant withering and Gray mold. The nucleic acid of the present invention, which encodes the β-1,3-glucanase polypeptide finely, can make plants resistant to disease. Glucanase is-pathogen-inducible, 7-square purple-related protein. "M 3_glucanase can decompose the pathogenic bacteria, and release the inducing substance that activates the plant's defense against green. It can decompose plants and react with other anti-purple reactions such as lignin and disease-related proteins. The performance of the nucleic acid of the present invention will Increasing the resistance of plants to adversity, mainly against the resistance of true ugly crickets, the present performance can especially inhibit the infection of Ling Qing pathogen. The selection and reduction molecule of the present invention can be used as a domain defense machine for selecting disease-resistant varieties. Disease-resistant varieties can be screened by detecting the performance of the nucleic acid of the invention. _ The performance of the nucleic acid of the present invention can also be measured by the enzyme activity of glucanase. This issue 12 200416286 shows that the polypeptide encoded by the nucleic acid can catalyze the hydrolysis of dextran and has the ability to break down the fungal cell wall, so it can be used as an antifungal substance. [Embodiment] Example 1 Isolate the β-1,3-glucanase plant material encoded by the present invention _ Lily bulbs (Oriental hybrid ‘Star GaZer,’) were planted in cultivated soil _ Β_η, appeal

No. 2) in a medium mixed with perlite (3: 1 ratio). Plants were planted in semi-open net cages covered with black nets and planted for 25-45 days depending on the weather. Sporulation of Botrytis cinerea The costly beta / coupling and c⑻mz strain spore suspension was inoculated on the leaves and petals of lily (6 drops per leaf or petal, 20 μΐ per drop). The median leaf of the lily plant was picked (eighth to tenth #leaf from the top) and the leaf was placed on a glass dish towel with the back of the leaf facing up, and the veins of the leaf were moistened with wet cotton. Lily petals are taken from flowers that bloom for 1-2 days and placed in a petri dish.的 Recovery of inoculum and inoculum of e // button ζ · αζ and 5. d / zerea strains were recovered from the inoculated leaves and petals, and then stored in 0.2 pg / ml protease inhibitor APMSF and placed in _2. . 〇.

SDS-PAGF recovered from lily leaves and e // zhongru ^ inoculum was reddish-brown, this reddish-brown material could be Ce_c0n YM-K-3 (removing molecules smaller than 3-discussion) (Amic〇n, Bedf (Ord, Zu, usa) column removal. The recovered inoculum (approximately 200 μ1) was concentrated five times by freeze-drying and subjected to denatured polypropylene 13 200416286 amidine colloidal electrophoresis analysis (SDS-PAGE). Under denaturing conditions, denatured polyacrylamide colloid electrophoresis was performed according to the method reported by Laemmli, U.K. (Laemmli, 1970, Nature 227: 680-685). 4% (w / v) stacking gel and 12% running gel were used to run the gel at 120V. The protein in the film was stained according to the Bradford method (Bradford, M.M. 1976, Anal. Biochem. 72: 248-254), protein stain (Coomassie Brilliant Blue, CBR-250), and pre-stained proteins were used as molecular markers. (GIBCOBRL) 'The results show that there are more than four protein bands in the inoculated recovery solution (Figure 1), and their molecular weights are estimated to be 49, 33, 27, and 16 kDa. During inoculation and e // z ice · 72 These proteins were clearly observed in the recovered solution (Figure 1A, line 2) after 96 hours (Figure 1A, line 5). There were no protein bands in the recovery solution from 72 hours (Figure 1) and stigma hours (Figure 1A, line 4). The 72 hours of recovery (Figure 1A, line 3) and The 96-hour recovered inoculum (Figure 1B, row 6) was used as a control, and the inoculation on the flap was 12 hours (Figure 1B, row 2), 24 hours (Figure 1B, row 5), and 48 hours (Figure B). Row 7) There is no visible protein band in the recovery solution. The same is true for the inoculation recovery solution on the petals (Figure 1B 'lines 4 and 8). Place on the petals for 12 hours (Figure 1B, line 3), 24 hours (Figure IB, line 6) and 48 hours (Figure IB, line 9) were used as controls. After N-terminal amine farm brewing, the gel # 33 containing 33 kDa egg self-quality was used in Yu Li ml buffer solution was rubbed with '〇.i ° / 〇 (w / v) sodium lauryl sulfate, adjusted to pH 8 with 1M NaOH to infiltrate for one hour, and then the protein of this towel was transferred to a buffer solution (Trisamino group dirty, Tris_boric 50 mM each) was transferred to a nylon transfer film pVDF) overnight at a voltage of 35 v. After the transfer, the nylon transfer film was rinsed in distilled water, and then placed in 〇1% (w / v) 14 200416286 naphthol blue black, 45〇 / 〇 (v / v) f? 0 / 〇 (y / v) ^ ^ 5 ^ 〇 ^ This 33 kDa protein band was cut out, de-stained in steamed water, air-dried, and stored in the passage for N-terminal amino acid digestion sequence. The sequencing results show that the n-terminus of this 33 protein f has 16 consecutive amino acids MDGDNLPQPADVVNLY and six discontinuous amino acid sequences. This continuum of 16 amino acid sequences has 86_93% _ similarity to β_1,3, polyfluorene of cotton and Arabidopsis thaliana. @This, named this 33 kDa protein as LPGlul (lily pathogen-induced glucanase 1). 〇1,3-glucanase 塍 M meat activity uses two β-1 , 3-glucanase in-vivo staining method, a method modified from the method described by Shimoni, M. (Shimoni, 1994, Anal. Biochem. 220: 36-38). The other is a method performed in a denatured polypropylene-amine colloidal electrophoresis analysis system (Trudd et al, 1998, Electrophosis 19.1788-1792). 2,3,5-triphenyltetrazolium chloride and 2% kumbudo St (lanunarm) were used as the coloring agent and reaction matrix. The film was analyzed by denatured polyacrylamide colloid electrophoresis and placed in a reducing solution (10 mM Triton X-100 in 10 mM acid buffer solution, pH 6.0) to increase protein reduction efficiency. The glucanase activity was demonstrated by the second method / 3-1,3 · glucanase activity (Fig. 2A), and the inoculation of the leaf inoculation recovery solution in the film showed β-U- The glucanase activity (row 丨); and the leaf inoculation and recovery solution (row 2) and control inoculum (row 3) of Yumeno did not show β · 1,3-glucanase activity. And r-tomycey glutamate%, 1,3-glucanase was used as a positive control (line 4). The CBR protein staining results are shown in Figure 2b, and the low molecular weight proteins (97, 66, 45, 30, 21, and MkDa) are used as marks. 15 200416286 Analysis by isoelectrofocusing electrophoresis (IEF) and inoculation of e / you α leaves. About 200 pg of protein was analyzed by two-dimensional electrophoresis according to the instruction manual provided by the manufacturer (Bio_Rad). The results showed that LPGlul (indicated by a circle) is an acid β_1,3-glucanase with an isoelectric point of about 4.0 (Figure three). Example 2 Selection of Complementary DNA (cDNA) Encoding Lily β-1,3-Glucanase To select a full-length complementary DNA encoding Lily β-1,3-glucanase, reverse transcription polymerase chain reaction was performed. (Reverse transcription-polymerase chain reaction, RT-PCR) and RACE (rapid amplification of cDNA ends). Reverse Transcriptase Polymerase Chain 4 »and 廄 Extraction Inoculation and Total RNA of Lily Leaves with e // Kelly (Ausubel et al. 1995, Current

Protocols in Molecular Biology, John Wiley and Sons, Inc., USA), and mRNA was purified according to the procedures described in the Oligotex ™ kit Handbook. Based on the alignment of the recovered amino acid sequence of the 33 kDa protein with similar amino acid sequences, degenerate primers were designed to amplify the corresponding complementary DNA from the purified mRNA by reverse transcription polymerase chain reaction. Polymerase chain reaction temperature cycle is 95 ° C, 3 minutes, 1 cycle; 95 ° C 1 minute, 55 ° C 1 minute, 72 ° C 1.5 minutes, 30

Cycles; 72 ° C for 10 minutes, 1 cycle (Robocycler temperature cycler, Stratagene). RACE uses MarathonTM cDNA Amplification Kit (Clontech) for RACEcRACE PCR program at 94 ° C, 1 minute, 1 cycle; 94 ° C 30 seconds, 72 ° C 3 minutes, 5 cycles; 94 ° C 30 seconds, 70 ° C 30 seconds, 72 ° C 3 minutes, 5 cycles; 94 ° C 30 seconds, 68 ° C 30 seconds, 72 ° C 2 minutes, 20 cycles. 200416286 In reverse transcription polymerase chain reaction, 3, RACE and 5, RACE have one major amplified PCR product (700, 750, and 300 bp). After molecular selection, the DNA is sequenced and combined into a full-length complementary DNA sequence. Sequence analysis revealed that the full-length complementary DNA encoding LPGlui (Figure 4) has a 1,010 bp open reading frame that encodes 337 amino acids and has a predicted molecular weight of 35.328 (Figure 5). In the complementary DNA sequence, the predicted translation start code ATG and termination code TGA are indicated by boxes. The predicted polyadenylation (polia (ienylation) message sequence is underlined. The predicted position of the cleavage of the message enzyme ⑼㈣ peptidase) is between serine_isoleucine (equivalent to 28th). Between -29 amino acids. Example 3 Observation of the infection of Botrytis cinerea on lily leaves and petals by an inverted fluorescence microscope. The inoculated lily leaves and petals were placed in 30-50 ml of 1 M KOH, and subjected to high pressure and high temperature treatment at 121 ° C for 15 minutes. Rinse three times with steamed water. A few drops of dye were added to the sample and observed with an inverted light microscope (Leica DM IL microscope, Wetzlar, Germany). A dye solution (0.05% aniline blue formulated in a 0.067 M dipotassium phosphate solution, pH 90) was prepared more than two hours before use (Hood & Shew, 1996, Phytopathology 86: 704-708). The light source of the inverted fluorescent microscope was a mercury lamp, and the filter accessory was a G340-380 nm exciter: LP (10ng pass) 425 nm fluorescent cross-pass. The picture was taken with a digital camera (Nikon Co. PIX990, Tokyo, Japan). Under microscope observation, it can be seen that the infection of the leaves of Lily and the minor ill lesions caused by the disease appear in half of the observed invasion sites, and often appear around the 5 · e // 切 々 ca hypha growing in the tissue of the lily . However, tiny gangrene spots did not appear in the lily dagger that was infected with e // cutting ^. On the other hand, tiny gangrene spots appeared only on a few leaf guard cells adjacent to and czVzerea's germinated spores, but not on dner from infected lily petals. Lily leaf cell death 17 200416286 The growth may be limited to the infection of the dance face. These dead cells may prevent the death of the six A_ cells of the fungal hypha and do not appear on the "_ " infected lily petals (Figure, D) .12 hours after inoculation, observe in bright field (Figure 6B and D) or glory field (Figure, and C) (Bar = 24.5pm). [Schematic description] Figure 1 Figure 2 is from the lily leaves The sum of the flower petal recovery and 7 is along the spp. The inoculation recovery solution is the β-1,3-glucanase activity of the lily leaf inoculation recovery solution. Protein map.

Figure 2 is a two-dimensional electrophoretic analysis of lily leaf inoculation and recovery solution. Figure 4 shows the nucleic acid sequence encoding the full-length complementary DNA of LPGlul. Figure 5 shows the predicted amino acid sequence of LPGlul. Figure 6 is a comparison of the infection of lily leaves and petals.

18 200416286 P03001-seq list.ST25 Sequence Listing < 110> Chen Zhaoying < 120> A novel nucleic acid derived from lily code / 3 -1,3-glucanase < 130> p03001 < 160> 2 < 170 > Patentln version 3.2 < 210〉 1 < 211〉 337

< 212〉 PRT < 213〉 Lily < 400〉 1

Met Ala Ala Gin His lie lie Ser Met Ala Ala Met Ala Ser Leu Leu 15 10 15

Val Val Leu Ser Ala lie Pro Arg Gly Val Glu Ser lie Gly Val Cys 20 25 30 \ sn Gly Met Asp Gly Asp Asn Leu Pro Gin Pro Ala Asp Val Val Asn 35 40 45

Leu Tyr Lys Ser Asn Asn lie Ala Gly Met Arg Leu Tyr Ser Pro Asp 50 55 60

Gin Ala Thr Leu Gin Ala Leu Gin Gly Ser Asn lie Tyr Leu lie Leu 65 70 75 80

Asp Val Pro Asn Ser Asp Leu Gin Asn lie Ala Ser Asp Gin Ser Ala 85 90 95

Ala Thr Asn Trp Val Gin Thr Asn Val Gin Ala Tyr Pro Asn Val Ala 100 105 110

Phe Arg Tyr lie Ala Val Gly Asn Glu Val lie Pro Gly Gly Gin Ala 115 120 125 200416286 P03001-seq list.ST25

Gin Tyr Val Leu Pro Ala Met Asn Asn lie Gin Ser Ala Leu Ser Ser 130 135 140

Ala Gly Leu Gin Asn lie Lys Val Ser Thr Ser Val Ser Phe Gly Val 145 150 155 160

Val Gly Thr Ser Tyr Pro Pro Ser Ala Gly Ser Phe Ser Ser Asp Ala 165 170 175

Ser Ser Thr Leu Gly Pro lie lie Gin Phe Leu Ala Ser Asn Gly Ser 180 185 190

Pro Leu Leu Ala Asn lie Tyr Pro Tyr Leu Ser Tyr Ala Gly Asn Ser 195 200 205

Gly Ser lie Asp Leu Ser Tyr Ala Leu Phe Thr Ala Ser Gly Thr Val 210 215 220

Val Gin Asp Gly Ser Tyr Ala Tyr Asn Asn Leu Phe Asp Ala Met Val 225 230 235 240

Asp Ala Leu Tyr Ser Ala Leu Glu Ser Ala Gly Gly Pro Asn Val Pro 245 250 255

Val Val Val Ser Glu Ser Gly Trp Pro Ser Ala Gly Gly Thr Ala Ala 260 265 270

Thr Val Ser Asn Ala Gin Thr Tyr Asn Ser Asn Leu lie Asn His Val 275 280 285

Gly Gin Gly Thr Pro Lys Arg Pro Gly Ala lie Glu Thr Tyr lie Phe 290 295 300

Ala Met Phe Asn Glu Asp Gin Lys Gin Pro Gin Gly lie Glu Asn Asn 305 310 315 320

Phe Gly Leu Phe Tyr Pro Asn Glu Gin Pro Val Tyr Ser lie Ser Phe 325 330 335

Thr Page 2 200416286 P03001-seq list.ST25 < 210〉 2 < 211 > 1011 < 212 > DNA < 213〉 Lily &400; 2 atggcagctc agcacatcat ctccatggct gccatgggg ccctccttgt agtactctcg 60 gcaatcggga tga gagg ccccagcccg ccgacgtcgt caacctctac aagtccaaca acatagctgg catgcgactc 180

tacagccccg accaagccac tctccaggcc ctccagggct ctaacatcta cctcatcctc 240 gacgtcccca actccgacct ccaaaacatt gcctccgacc aatccgccgc caccaactgg 300 gtccaaacca acgtccaagc ctacccaaac gttgccttcc gatacatcgc cgtcggaaac 360 gaagtcatcc ccggcggcca agctcagtac gtcctcccag ccatgaacaa catacagtcc 420 gccctctcct ctgccggcct tcagaacatc aaggtctcca catcagtctc cttcggcgtc 480 gtcggtacct catatccccc ctcagctggc tccttctctt ccgatgcatc gtcgacattg 540 ggtccaatca tacagtttct agccagcaat ggctccccat tacttgccaa catctacccc 600 tacttgagct atgctggcaa ctccggatcc atcgacctct catacgccct ctttactgca 660 tctggtacag tcgtacagga cgggtcctac gcttacaaca acctcttcga tgccatggtc 720 gacgcattgt actcggccct ggagagcgcc ggagggccc gtgtctct

gagagtggct ggccgtcagc gggcgggaca gcggcgacgg tgtctaatgc gcagacttac 840 aattccaatt tgatcaacca tgtgggtcag gggacgccga agaggccagggg ggcgattgaggtgagt tgagat cagacat

Claims (1)

200416286 Patent application scope, 1 1 An isolated nucleic acid molecule that encodes a polypeptide with right β-1 3 q, /, 'glycosidase activity, wherein the polysaccharide is selected from the following group: such as sequence identification number: 1 A polypeptide of the amino acid sequence shown; and () a polypeptide encoded by a nucleic acid, the core hybridizing with a nucleotide sequence of sequence identification number · 2 under high stringency. 2. If you apply for _ 1 slave points _ break, for example, carefully identify the sequence number 2. 3. A vector comprising an isolated nucleic acid molecule as set forth in the patent application. "Rushen paste outline 3 slave separation_molecule has a sequence such as sequence identification number ... 2. 5_-A host cell comprising a vector according to item 3 of the patent application. Shishen's “Wounded Primary Cells in Item 5 of the Monthly Patent Scope”, which includes the carrier of item 4 in the scope of the patent application, as stated in the patent application. The host cells are bacterial cells or plant cells. 8. The host cell according to item 7 of the application, wherein the bacterial cell is an Agrobacterium cell. 9. The host cell of item 7 of the patent application, wherein the host cell is a plant cell. 10. A transgenic plant transformed with an isolated nucleic acid molecule as described in item i of the patent application. 11. The genetically modified plant as claimed in item 10 of the patent application is transformed with the isolated nucleic acid molecule as described in item 2 of the patent application. 12. An isolated polypeptide, which is selected from the group consisting of: a sequence identification number: a polypeptide of the amino acid sequence shown by i and (b) a polypeptide encoded by a nucleic acid, which is highly stringent Condition 19 200416286 Hybridized with a nucleotide sequence of sequence identification number: 2. 13. The isolated polypeptide according to item 12 of the scope of patent application, which has the sequence of sequence identification number: 1 20