JP2000516812A - Peptides with inhibitory activity against phytopathogenic fungi - Google Patents

Abstract

  (57) [Summary] Lactoferricin was found to have antifungal activity against plant pathogens. A genetic construct containing a synthetic bovine lactoferricin gene is prepared and used to provide a transgenic disease resistant plant. Also described is a method of transforming a plant to express lactoferrici, which is expressed in the plant and confers a disease regime on the plant.

Description

DETAILED DESCRIPTION OF THE INVENTION              Peptides with inhibitory activity against phytopathogenic fungi   The present invention provides by genetically modifying a plant to produce a peptide. For controlling plant pathogens with an isolated peptide, and genetics useful in the method Regarding offspring and plants. In particular, the present invention relates to the control of lactoferricin and plant pathogens. About its use in yours.   Many fungi and bacteria are important pathogens of crops of economic importance. plant Many methods of controlling this disease have been used with varying degrees of success. One person The law is the application of antimicrobial agents to crops. This method has many, recognized in the art. Have problems. Alternatively, more recent approaches have been Relates to the use of biological control organisms ("biological control") that are suppressors. But However, it is difficult to apply biocontrol to large areas, and these live It is more difficult to leave in the period processing area. More recently, recombinant DNA technology has Opportunity to insert cloned genes expressing antimicrobial compounds into plant cells providing. However, this technology has evolved into known naturally occurring antimicrobial agents. Raise concerns about the ultimate resistance of microorganisms. Therefore, one gene The need to identify naturally occurring antimicrobial compounds that can form plant cells directly in translation Still exists.   Because peptides with antipathogen and antimicrobial activity are already known in the art Of particular interest is the identification of novel peptides and DNA encoding such peptides deep. For example, mammalian defensins, cecropin, thionine, melittin, insect Many so-called cells from animal, plant, insect and microbial sources, including fensin etc. The vesicle lytic peptide is present.   Other classes of peptides with antipathogenic activity have been shown to inhibit fungal growth. Hydrolyzing enzymes such as chitinase and β-1,3-glucanase Table (Schlumbaum, et al., 1986; Mauch, et al., 1988).   Enzymatic hydrolysis of the milk protein lactoferrin also has antimicrobial properties. Are shown. Lactoferrin is used in mammals, including milk, tears, and mucosal secretions. It is an iron-binding glycoprotein present in biological fluids (Brock, J., Arch. Dis. Child. 55: 417 (1980); Bullen, J .; Rev. Infect. Dis. 3: 1127 (1981)). Lactoferrin Promotes cell growth, regulates hematopoiesis and protects against microbial infection in vitro And immunomodulatory properties. However, lactoferrin in vivo The function is almost unknown. Extracellular lactoferrin is an antimicrobial and anti- It has been reported to have lupus activity. In experiments with bovine lactoferrin, The antimicrobial activity of the enzymatic hydrolyzate produced by digestion with tapesin was It has been found to be stronger against Escherichia coli lysate than protein (Tom ita, etal., J. DairySci. 74: 4137-4142 (1991)).   The antifungal activity of lactoferrin has also been demonstrated in humans. Lactoferricin B Sensitivity of Candida albicans to inhibition and inactivation of Bellamy et a l., Med. Microbiol. Immunolog. 182: 97-105 (1993). The work Use is lethal and results in a rapid loss of colony-forming ability, lactoferrin May contribute to host defense against C. albicans Show.   Human lactoferrin cDNA has recently shown the antibacterial activity of lactoferrin in plants. To be tested, it was expressed on tobacco floating cells. Transgenic callus is complete Produces a single 48 kD peptide that is significantly smaller than the full length lactoferrin protein did. Total protein extract produced by transgenic tobacco callus is As demonstrated by a decrease in colony forming units when tested with four pathogens Showed higher antibacterial activity than commercially available purified lactoferrin. Complete Full-length lactoferrin is not detected in transgenic calli, Indicates that the phosphogene product has undergone post-translational processing. Human lactoferrin The gene is expressed in Aspergillus nidulans and produces large amounts of full-length lactoferri To produce lactoferrin, full-length plant-produced lactoferrin is properly folded. It was explained that the unfolded part was decomposed. Cal Cleavage proteins that exhibit antimicrobial activity in proteins are based on the amino- or It was identified as the sea end.   The present invention relates to lactoferricin that can be safely expressed in plants and confer disease resistance Or an enzymatic hydrolyzate of Gene structure including lactoferricin synthetic gene A building is prepared and used to provide a transgenic disease resistant plant. Expressing lactoferrin expressed in plants to confer disease resistance on plants Also described is a method of transforming plants for this purpose.   Figure 1: Plasmid pCIB7703, ubiquitin promoter (15-1995) and no s Terminator (2079-2339) under control of the lactoferricin B gene (1995-2079 )).   Figure 2: Plasmid pCIB7704 with ubi-SynPat-nos selection cassette (positions 15-2845) And the same as pCIB7703.   Figure 3: Plasmid 7705, ubi-SynPat-nos-ubi-lac in plasmid containing NptII. Includes tofericin B-nos fragment.   FIG. 4: Plasmid 7706, ubi-lactoferricin B-n in NptII containing plasmid Includes os fragment.   The present invention relates to a lactoferrin that has antipathogenic activity and corresponds to the N-terminal region of lactoferrin. Lactoferricin, an enzymatic hydrolyzate of ctoferrin, is utilized. Lactov Wellin is a protein produced in mammalian milk that has structural and Although having functional homology, there are slight differences in the exact amino acid sequence between species. The present invention For use in lactoferricin, lactoferricin B (bovine lactoferricin B) is preferred. Toferricin) or lactoferricin H (human lactoferricin). Easy Tofericin, according to the present invention, inhibits the growth of many agronomically important pathogens, It was found to be expressed in plants, thereby conferring disease resistance.   Lactoferricin B (or bovine lactoferricin) is a 25 amino acid long peptide. Do: And has complete homology to the N-terminus of the entire bovine lactoferrin sequence. Lacto Ferricin H (or human lactoferricin) is the amino acid 1 of human lactoferrin It corresponds to -33 and is therefore 33 amino acids of the following sequence: When expressed, these peptides have the N-terminal methionine corresponding to the start codon. May contain residues.   Lactoferricin gene is expressed in plants based on protein sequence To be designed. The codon usage of this synthetic gene is the highest among maize expressed genes. By using each frequently used codon, amino acids in monocots Optimized for expression of Maize codon usage tables are available from Murray, Lotzer and Eberle , Nucl. Acids Res. 17, 477-498, 1989. Alternatively, synthetic genetics Codon usage of offspring is determined according to different concepts, such as those described in EP-A-359472. Can be optimized.   The sequences designed for the plant-optimized lactoferricin B gene are as follows: Including the array If a stop codon (TAA) is added to the 3 'end, the sequence is as follows: The plant optimized sequence for lactoferricin H is as follows: Preferably, a methionine start codon and a stop codon (TAG) are added. In addition Adds the ACC sequence derived from Kozak consensus sequence to enable efficient translation To improve. This results in the Met-lactoferricin H gene sequence shown below, Start and stop codons are underlined:   Structural coding sequence encoding lactoferricin can function in plant cells It is expressed in plant cells under the control of a promoter, Produce a 3 'untranslated region that results in liadenylation. In plants or plant cells Promoters that can function in plants, ie, related to lactoferricin in plant cells Examples of those that can result in the expression of a structural gene Virus (CaMV) 19S or 35S promoter and CaMV double Motor; nopaline synthase promoter; pathogen-related (PR) protein pro Motor; small subunit of ribulose bisphosphate carboxylase promoter Including units (ssuRUBISCO). Preferred is the rice actin promoter (McElroy).  et al., Mol. Gen. Genet. 231: 150 (1991)), Maze ubiquitin promoter (EP0342926; Taylor et al., Plant Cell Rep. 12: 491 (1993)) Promoter and Pr-1 promoter from tobacco, Arabidopsis or maize Motor. Also preferred are the 35S promoter and Kay et al., Sc. ience 236: 1299-1302 (1987). Double 35, deposited at ATCC 40587, cloned into DNA and pCGN2113 The S promoter. The promoter itself may be ligated according to methods known in the art. Engineered to modify promoter power to increase ctofericin expression obtain.   A signal or transit peptide can be added to a chimeric DNA construct of the invention to Lactoferricin peptide at the desired site of action. Transport directly. Examples of signal peptides are naturally associated with plant pathogen-related proteins. Include, for example, PR-1 and PR-2. For example, Payne et al., Plant Mol. B iol. 11: 89-94 (1988). Examples of transit peptides are described in Von Heijne et al., Plant. M ol. Biol. Rep. 9: 104-126 (1991); Mazur et al., Plant Physiol. 85: 1110 (1987 ); A chloroplast transit peptide and Bo as described in Vorst et al., Gene 65:59 (1988). utry et al., Nature 328: 340-342 (1987). Including peptides. The relevant descriptions of these publications are incorporated herein by reference in their entirety. To be included. A methionine start codon is added for cytoplasmic expression.   The chimeric DNA constructs of the present invention may contain multiple copies of the promoter or It may contain multiple copies of the ricin structural gene. In addition, the construct is Coding sequences of other peptides such as signal sequences and signal or transit peptides. And a suitable reading frame, each with other functional elements within the DNA molecule It is. The manufacture of such a construct is within the skill of the art.   Useful markers include, for example, hygromycin, kanamycin, G418, gen Tamycin, lincomycin, methotrexate, glyphosate, phosphinos Peptides that confer herbicide, antibiotic or drug resistance, such as resistance to ricin Including These markers were transformed with the chimeric DNA constructs of the present invention. It can be used to select cells from non-transformed cells. Other useful markers are A peptide enzyme, such as luciferase, which can be detected in a visible reaction, for example, a chromogenic reaction, β-glucuronidase or β-galactosidase.   Transgenic plants (a) inserting the recombinant DNA molecule of the present invention into the genome of a plant cell; (b) obtaining a transformed plant cell; (c) then expressing an amount of lactoferricin effective to reduce injury from the disease Regenerating transgenic plants Gain by doing.   Recombinant DNA molecules can be inserted into plant cells in a number of ways known in the art. One of skill in the art will appreciate that the choice of method depends on the type of plant targeted for transformation, i.e., monocotyledonous or bicotyledonous. Recognize that it depends on cotyledons. A suitable method for transforming plant cells is micro Injection (Crossway et al., BioTechniques 4: 320-334 (1986)), Elec Troporation (Riggs et al., Proc. Natl. Acad. Sci. USA 83: 5602-5606 (1 986)), Agrobacterium-mediated transformation (Hinchee et al., Biotechnology 6:91). 5-921 (1988)), direct gene transfer (Paszkowski et al., EMBO J. 3: 2717-2722 (1984). )) And Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmingto n, Delaware (eg, Sanford et al., US Patent No. 4,945,050; and Mc Using equipment available from Cabe et al., Biotechnology 6: 923-923 (1988)) Ballistic particle acceleration. Also, Weissinger et al., Annual Rev. Genet. 22: 4 21-477 (1988); Sanford et al., Particulate Science and Technology 5: 27-37 ( 1987) (Onion); Christou et al., Plant Physiol. 87: 671-674 (1988) (soybean ); McCabe et al., Bio / Technology 6: 923-923. (1988) (soy); Datta et al., Bio / Technology 8: 736-740 (1990) (US); Klein e t al., Proc. Natl. Acad. Sci. USA, 85: 4305-4309 (1988) (Maize); Klein et. al., Bio / Technology 6: 559-563 (1988) (maize); Klein et al., Plant Physiol. . 91: 440-444 (1988) (Maize); Fromm et al., Bio / Technology 8: 833-839 (1990). And Gordon-Kamm et al., Plant Cell. See also 2: 603-618 (1990) (Maize).   Selection of the promoter used for the expression cassette depends on the transgenic plant. Determine the spatial and temporal expression patterns of the transgene. Select promoter (leaves Specific cell types or tissues (such as epidermal cells, mesophyll cells, Expresses the transgene in an organ (e.g., root, leaf or flower) and this selection To reflect the desired location of expression. Alternatively, the selectable promoter is the gene expression To induce expression under a light-induced or other transiently regulated promoter. A further alternative is , Chemical regulation of the selected promoter. This leads to the induction of transgene expression, Offers the possibility of limiting it only when it is desired and allows for treatment with chemical inducers Brought from.   Various transcription terminators can be used in the expression cassette. These are It is responsible for terminating transcription in addition to the transgene and its correct polyadenylation. Appropriate transfer Terminators and those known to function in plants are CaMV3 5S terminator, tml terminator, nopaline synthase terminator , Including the pea rbcS E9 terminator. These are monocots and dicots Can be used for both things.   Many sequences have been found to promote gene expression within the transcription unit, and these sequences The row, together with the gene of the invention, increases its expression in transgenic plants Can be used for   Various intron sequences have been shown to promote expression, especially in monocot cells Have been. For example, the intron of the maize Adh1 gene Is found to significantly enhance the expression of wild-type genes under the same promoter are doing. Intron 1 is particularly effective, and chloramphenicol acetyl tra It was found that the fusion construct with the luciferase gene promoted expression (Callis et al., Genes Develop. 1: 1183-1200 (1987)). In the same experimental system, Maize bronze1 remains Gene-derived introns had a similar effect in promoting expression (Callis et al., Supra). . Intron sequences are routinely added to plant transformation vectors, typically with an untranslated leader. Inserted in the   Many untranslated leader sequences from the virus also drive expression, It is also known to be effective in dicotyledonous plants. In particular, tobacco mosaic wheels (TMV, "W sequence"), maize vitiligo virus (MCMV) and alfalfa -Make sure that the leader sequence derived from mosaic virus (AMV) is effective in promoting expression. (Eg, Gallie et al., Nucl. Acids Res. 15: 8693-8711 (19 87); Skuzeski et al., Plant Molec. Biol. 15: 65-79 (1990)).   Various mechanisms of gene product targeting are known to exist in plants, The sequences that control the functioning of these mechanisms have been characterized to some extent. For example, Targeting of gene products to chloroplasts is found at the amino terminus of various proteins. Chloroplasts transport mature proteins that are produced, controlled by signal sequences (See, for example, Comai et al., J. Biol. Chem. 263: 15104-15109). 1988)). These signal sequences can be fused to a heterologous gene product, resulting in a heterologous chloroplast. Acts on the transport of seed products (van den Broeck et al., Nature 313: 358-363 (1985)) ). DNA encoding a suitable signal sequence is RUBISCO protein, CAB protein. Localized to protein, EPSP synthase enzyme, GS2 protein and chloroplast Isolated from the 5 'end of the cDNA of many other proteins known to be Wear.   Other gene products are found in other cells such as mitochondria and peroxisomes. Localized to organelles (see, eg, Unger et al., Plant Molec. Biol. 13: 411-418 (1 989)). The cDNAs encoding these products are also It can be manipulated to target these organelles. Of an array like this Examples are nuclear-encoded ATPase and specific aspartate amino from mitochondria Transferase. Targeting cellular protein bodies is Rogers et al., Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).   In addition, the sequence may provide for targeting of the gene product to other cellular compartments. And is characterized by: The amino terminal sequence is ER, the target to apoplast. It is responsible for targeting and extracellular secretion from aleurone cells (Koehler & Ho, Pla nt Cell 2: 769-783 (1990)). In addition, the amino terminal sequence is shared with the carboxy terminal sequence. Is responsible for vacuolar targeting of gene products (Shinshi et al., Plant Molec. Bi ol. 14: 357-368 (1990)).   By fusing the appropriate targeting sequence described above with the transgene of interest, The gene product can be directed to any organelle or cell compartment. For chloroplast targeting, for example, RUBISCO gene, CAB gene, EP Frame chloroplast signal sequence derived from SP synthase gene or GS2 gene In the amino terminal ATG of the transgene. The selected signal sequence is The fusion constructed contains a known cleavage site and contains the amino acid after the cleavage site required for cleavage. Must be taken into account. In some cases, the requirement is that the cleavage site and transgene A By adding a small number of amino acids between TGs, or May be satisfied by substitution of certain amino acids within. Melts constructed for chloroplast transport The compound is an in vitro translation of the in vitro transcription construct, followed by in vitro chloroplast uptake. The efficiency of chloroplast uptake by humans (Bartlett et al., In: Edelmann et al. (Eds.) M ethods in Chloroplast Molecular Biology, Elsevier, 1081-1091 (1982); Wasma nn et al., Mol. Gen. Genet. 205: 446-453 (1986)). Wear. These construction methods are known in the art and include mitochondria and peroxisomers. The same can be applied to the system. Selection of targeting that may be required for transgene expression The choice depends on the cellular localization of the precursor required as a starting point for certain pathways. This is Permanent cytosol or chloroplast, but sometimes mitochondria or peroxy It can be a sisome. The product of transgene expression is usually ER, apoplast or Does not require targeting to vacuoles.   The above method of cellular targeting is not only with its cognate promoter. And can be used with heterologous promoters to target specific cell targeting purposes. Has a different expression pattern from the promoter, although the origin of the signaling signal This is performed under the transcriptional control of a promoter.   Methods for transforming dicotyledonous plants are known in the art and are based on Agrobacterium-based And methods that do not require Agrobacterium. Non-Agrobacterium Method involves the direct uptake of exogenous genetic material by protoplasts or cells. No. This includes PEG or electroporation mediated uptake, bombardment mediated transfer This can be achieved by feeding or microinjection. Examples of these methods are Pa szkowski et al., EMBO J .; 3: 2717-2722 (1984), Potrykus et al., Mol. Gen. G enet. 199: 169-177 (1985), Reich et al., Biotechnology 4: 1001-1004 (1986). And Klein et al., Nature 327: 70-73 (1987). Any place Once again, the transformed cells are regenerated into whole plants using methods known in the art.   Agrobacterium-mediated transformation is characterized by its high transformation efficiency and many different It is a preferred method for transforming dicotyledonous plants due to its wide availability to certain species. Idiomatic Many crop species that can be selectively transformed with Agrobacterium are tobacco, tomato, Contains mawari, cotton, rape, potato, soybean, alfalfa and poplar (EP0317511 (cotton); EP0249432 (tomato, Calgene); WO 87/072 99 (Brassica, Calgene); US 4,795,855 (Poplar). Agrobacterium Transformation is typically performed using a binary vector carrying the heterologous DNA of interest (eg, (E.g. pCIB200 or pCIB2001), depending on the host Agrobacterium strain A suitable that may depend on the complementarity of the vir gene carried on the rasmid or chromosomally Includes transfer to relevant Agrobacterium strains (e.g., pCIB200 and pCIB2001 Strain CIB542 (Uknes et al., Plant Cell 5: 159-169 (1993)). Recombinant binary vector Transfer of the vector into Agrobacterium ・ Recombinant binary vectors carrying plasmids such as E. coli and pRK2013 Interaction Using a Helper E. coli Strain That Can Be Transferred to a Typical Agrobacterium Strain Achieved by distribution. Alternatively, the recombinant binary vector is Res. 16: 9877 (1988)).   Transformation of target plant species with recombinant Agrobacterium Including co-culture of lium with explants of plant origin and following methods known in the art. Trait The transformed tissue is treated with the antibiotic or antibiotic present between the binary plasmid T-DNA boundaries. Regenerate on a selective medium containing a herbicide resistance marker.   Transformation of most monocotyledonous plants is also now routine. Preferred one Methods include cytoplasmic and microscopic using PEG or electroporation methods. Includes direct transfer to callus tissue by particle gun. Transformation can be a single DNA species or It can be performed on multiple DNA species (ie, co-transformation) and both of these methods Suitable for use in. Co-transformation avoids complex vector construction, Transgenes having loci that do not belong to the same linkage group with respect to offspring and selectable markers This allows for the production of transgenic plants and the removal of selectable markers at passage. Can have advantages if deemed desirable. However, using co-transformation The disadvantage is that less than 100% of other DNA species are integrated into the genome. (Schocher et al., Biotechnology 4: 1093-1096 (1986)).   Patent applications EP 0 292 435, EP 0 392 225 and WO 93/0727 8 shows a method for producing callus and protoplasm from maize selected inbred lines, PEG and Is Protoplasm Transformation Using Electroporation and Transformation Protoplasm? The regeneration of these maize plants is described. Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) and Fromm et al., Biotechnology 8: 833-839 (1990) 1 shows the published method of transformation of the A118-derived maize strain used. Further, the application WO 93/07278 and Koziel et al., Biotechnology 11: 194-200 (1993). A method for transformation of a selected inbred strain of Isuzu with a microprojectile gun is described. This method is 1.5-2.5 mm long immature maize embryos collected from maize ears 14-15 days after milling And use a PDS-1000He Biolistics instrument for irradiation.   Transformation of rice can also be performed by direct transfer using protoplasm or biolistics. Can be. Protoplasm-mediated transformation is based on japonica type and indica (Zhang et al., Plant Cell Rep. 7: 379-384 (1988); Shima moto et al., Nature 338: 274-277 (1989); Datta et al., Biotechnology 8: 736-. 740 (1990)). Both types can also be transformed conventionally using a biolistic gun (C hristou et al., Biotechnology 9: 957-962 (1991)).   Patent application EP 0 323 581 discloses the passage, transformation and regeneration of P. ideae protoplasm. Write raw. These methods are used in the form of Puideae, such as dactylis and wheat. Enables quality change. In addition, using a particle gun for type C long-term reproducible calli Wheat transformation has been described by Vasil et al., Biotechnology 10: 667-674 (1992)) and Vascular et al. Using immature embryos and immature embryo-derived callus l., Biotechnology 11: 1553-1558 (1993) and Weeks et al., Plant Physiol. 1 02: 1077-1084 (1993). The preferred method for wheat transformation is However, including immature embryo microprojectile bombardment of wheat, Includes sucrose or high maltose stages. Prior to irradiation, any number of embryos (0.75- 1 mm long) with 3% sucrose and 3 mg / l 2,4-D for transfer of somatic embryos Medium (Murashige & Skoog, Physiologia Plantarum 15: 473-497 (1 962)) and processed in the dark. On the day of irradiation, the embryos are recovered from the transfer medium and Place on a pressure regulating substance (ie sucrose or at the desired concentration, typically 15%) Transfer medium supplemented with maltose). Embryos are separated for 2-3 hours and then irradiated I do. Twenty embryos per target plate are typical but not critical. Suitable inheritance The child-bearing plasmid (such as pCIB3064 or pSG35) is transferred to the microphone using standard methods. Agglomerates into metric gold particles. Place each embryo plate on a standard 80 mesh screen. Use a burst pressure of ~ 1000 psi with a lean to DuPont Biolistics. Shoot with a lium device. After irradiation, the embryos are returned to the dark and allowed to recover for approximately 24 hours (still penetrating On pressure regulating substances). After 24 hours, the embryos are recovered from the osmolyte and returned to the transfer medium. , Put it for about a month before playing. About one month later, embryo explants of embryogenic callus that developed Are further combined with a suitable selective agent (10 mg / l busta for pCIB3064 and 2 mg for pSOG35) / L Methotrexate) (MS + 1 mg / l NAA, 5 mg / l) (GA). Approximately one month later, the grown shoots were replaced with half strength MS, 2% Large sterilization known as "GA7s" containing sucrose and selective medium of the same concentration Transfer to container.   When expressed in plants, lactoferricin can cause viral, bacterial and fungal plant diseases. Controls or inhibits a broad spectrum of plant pathogens, including the drug substance. Ease in the plant Plant diseases that are important as products suitable for control by expression of tofericin include the following: Including:Corn pathogen Fungus: Gibberella Zeae             Aspergillus Flavas             Aspergillus Paralytics             Fusarium Moniliforme             Giprodia Maidis             Coretotricum Graminola             Cephalosporium acremonium             Macrofomina Phaseolina             Sercospora Zeae-Maidis             Exelofolium Torusikum             Bipolaris Maidis             Cavatierella Zeae             PUSSYNA Sorghi             Poussinia Polysora             Sphaceloseca Leliana             Ustylago Maidiz             Coretotricum Graminola             Helmin Sosporium Carbonam             Pernos Clerospora Solgi             Skulerovsola Laixiae             Pernos Clerospora Sacchari             Pernos clerospora filipinensis             Pernos Clerospora Maidis             Pernos Clerospora Spontanea             Pernosclerospora heteropogoni             Sclerospora Graminola Bacteria: Erwinia Stewarty             Corynebacterium Nebrasquence Virus: Maze Dwarf Mosaic Virus             Maze Luff Dwarf (Maze Rio Quart) Virus             Maize Streak Virus             Maize Chlorotic Dwarf Virus             Maize Chlorotic Motor Virus             Burleigh Yellow Dwarf Virus             Corn lethal necrosis virus             High plane virus             Maize Stripe VirusSweet corn pathogen: A. Bacterial disease             Erwinia Stewarty             Pseudomonas avenae             Erwinia Krysansemi B. Fungal disease             Bipolaris Maidis             Exeloholium Tulsicum             Coretotricum Graminola             Philostikta Maidis             Cavatierella Zeae             Sercospora Zeae-Maidice             Skullofsola SPP             Pernos clerospora spp             Ustylago Maidiz             Sphaceloseca Leliana             PUSSYNA Sorghi             Poussinia Polysora             Fissopera Zeae             Fusarium Moniliforme             Picium spp             Penicillium oxalicum             Fusarium spp             Giprodia Maidis             Gibberella Zeae C. Viral disease             Sugar Cane Mosaic             Maize Dwarf Mosaic             Mays Chlorotic Dwarf             High plane virus             Maize Chlorotic Motor             Wheat streak mosaic             Burleigh Yellow DwarfWheat pathogen Fungal pathogens:             Poussinian Graminis FSP Tritici             Poussian Recondita FSP Tritici             Poussinian Striformis             Chiletia Trichi             Chiletia Contrasa             Chiletia Indica             Ustylago Tritici             Urocistis Tritici             Gaeumannomyces Graminis             Picium spp             Fusarium Kurumoram             Fusarium Graminaelm             Fusarium Avenaceum             Dreschere Tritici-Repentis             Rhizoctonia spp             Coretotricum Graminola             Helminsosporium spp             Microdosium Nivale             Pseudocell cosporella herpotrichoides             Elishife Graminis FSP Tritici             Skulerovsola Macrospora             Septoria Tritici             Septoria Nodrum (Stago Nospora Nodrum)             Bipolaris Solokiniana             Fusarium Roseum             Cephalosporium and Gramineum             Kochirio bolus sativas             Krabicepts Purpurea Bacterial pathogens:             Pseudomonas syringae pibui atrofaciens             Pseudomonas syringae             Xanthomonas campestris pveii translusens Viral pathogen:             Burleigh Yellow Dwarf Virus             Soilborn Wheat Mosaic Virus             Wheat spindle streak mosaic virus             Wheat yellow mosaic virusSoybean pathogen 1. fungus             Phytosora megasperma variant soiae             Koretotricum Trancatum             Septoria Grycines             Sercospora Kikuchi             Sercospora Sodina             Peronospora Manshlika             Microsfaera diphtha             Rhizoctonia Solani             Facopsora Oasiridi             Korinespora Kashikora             Caronectria crotalariae             Fusarium Solani             Fusarium Oxysporum             Macrofomina Phaseolina             Diaporse Phaseolum Variant Kauribola & Melide             Ionaris             Diapolse Phaseolum Variant Soiae             Fiarophora Gregata             Picium spp             Sclerothinia sclerothiolam             Sucrerotiun Rolfushi             Sierrabiopsis Vasicola 2. Bacteria             Pseudomonas             Xanthomonas campestris pavei phaseoli 3. Virus             Soy Bean Mosaic             Bean Pod Motor             Bad Bright             Peanut motor             Cowpy Chlorotic Motor             Yellow mosaicPea pathogen I. Seed and seedling diseases             Pisium Ultimum             Rhizoctonia Solani II. Bacterial diseases             Pseudomonas syringae pibui pishi             Pseudomonas syringae III. Fungal leaf disease             Ascocita Pisi             Mikosfaerela Pinodes             Ascocita Pinodella             Sclerothinia sclerothiolam             Peronospora Pissi             Erichife Pisi             Koretotricum Pishi             Alternaria Alternata             Botulitis Cinerea IV. Fungal root disease             Fusarium Oxysporum FSP             Aphanomyces Utjeches FSP Pisi             Pisium Ultimum             Fusarium Solani FSP Pisi             Sierrabiopsis Vasicola V. Diseases caused by viruses             P Enathion Mosaic             Bean (Pee) Leaf Roll             P. seedbone mosaic             P Streak P Mosaic             Kukumbar Mosaic             Peary Browning Virus             Red Clover Bain MosaicKidney beans pathogen I. Root disease             Afano Myces Utecheses FSP Fateoli             Pisium Ultimum             Rhizoctonia Solani             Fusarium Solani FSP Faseoli             Sierrabiopsis Vasicola             Sclelotium rolfushi II. Fungal diseases in parts of the atmosphere             Koretotricum Indemsianum             Uromiyas appendicultas             Sclerothinia sclerothiolam             Fusarium Oxysporum FSP Faseoli             Foma Exigua Variant Exigua             Sercospora Canesence             Kaetceptria Wellmani             Phytosora nicotianae             Erichife Polygoni             Pisium Ultimam & Devariam             Artemaria alternata, various species             Phaeoisariopsis griseola             Macrofomina Phaseolina             Botulitis Cinerea             Rhizoctonia Solani III. Bacterial diseases             Pseudomonas Syringae Phoebe Faseolicora             Pseudomonas syringae             Xanthomonas phaseoli IV. Diseases caused by nematodes             Meloidzine Incognita             Platinex species V. Diseases caused by viruses             Bean Common Mosaic             Bean Goiden Mosaic             Cooley Top             Bean Cooley Dwarf Mosaic             Bean Pod Motor             Clover Yellow Bain             Red node             Kukumbar Mosaic             Peanut stuntSugar beet pathogen 1. fungus:             Sercospora beticola spot disease             Aphanomyces Cothioides             Rhizoctonia solani blight, root rot             Erichife Betta powdery mildew             Rhizoctonia violacea (Helicobacterium purpurium)             Ramulalia Beticola             Picium spp             Foma Betae             Uromyses Betae             Peronospora Farinosa             Alternaria Tenuis             Fusarium oxysporium             Berthicilium dariae             Sclelotium rolfushi             Erichife Polygoni Powdery Mildew             Polymixer Beta 2. Bacteria:             Pseudomonas syringae             Erwinia cartobola Erwinia root rot 3. Virus:             Rhizomania Beat Necrotic Yellow Bain Will             Su (BNYVV)             Beat Mild Yellowing Virus (BMYV)             Beat Yellows Virus (BYV)             Beat Cooly Top Virus             Beat mosaic virus   The present invention provides plants that express lactoferricin when germinated and cultivated, and Provide seeds. Preferably, lactoferricin is stably integrated into the genome Expressed from recombinant DNA. Transgenic plants or seeds can be monocotyledonous or Or dicotyledonous plants, preferably maize, rice, wheat, barley, sorghum Mu, rye, oats, millet, cotton, soybeans, peas, kidney beans, sunflowers , Grass and rape. Transgenic seeds are preferably planted. Bags labeled for use in controlling pathogens may be packaged.   The genetic traits engineered into the transgenic seeds and plants described above Alternatively, they can be passaged in asexual growth and thus be maintained and inherited in progeny plants. General In particular, such maintenance and breeding can be carried out in specific ways, such as cultivation, sowing or harvesting The use of known agronomic methods that have been developed to suit different purposes. Hydroponic Or the greenhouse method can also be used. The growing crop is attacked and wounded by insects or infectious agents Because of its vulnerability to harm and competition from weeds, the method is controlled weeds, plant diseases, insects, It is performed under the control of nematodes and other conditions which are disadvantageous for improving the yield. These are the soil Methods such as cultivation or removal of weeds and infected plants and herbicides, pesticides Includes the application of pesticides such as fungicides, nematicides, growth regulators, ripening agents and pesticides.   By using the advantageous genetic properties of the transgenic plants and seeds of the invention , Resistance to pathogens, herbicides or loads, improved nutritional value, increased yield or Development of plants with improved structure resulting in reduced losses due to lodging or brittleness To The intended plant breeding can be further performed. The various breeding stages depend on the mating line. Well-defined humans, such as selection, direct pollination of parental lines, or selection of suitable progeny plants Characterized by the inclusion of Different breeding methods are performed, depending on the desired purpose. versus Corresponding methods are known in the art and include hybridization, inbreeding, backcrossing. Including crossbreeding, multibreeding, various crosses, interspecies hybridization, aneuploidy, etc. Are not limited to these. The hybridization method involves sterility of the plant species, Produce male or female sterile plants by mechanical, chemical or biochemical means. Male Cross-pollination of pollen different from that of the sterile plant is due to the male sterile but the female fertile plant genotype. Ensure that both parents have the homogeneity of the properties of both parents. Therefore, the present invention Transgenic seeds and plants can, for example, have their modified genetic properties. Increase the effect of conventional methods such as herbicide or pesticide treatment, or It can be used for improved plant breeding which eliminates such methods. Or improved New crops that are stress tolerant are equally disadvantaged due to their optimized gene “equipment”. Better yields than products that are not resistant under mild growing conditions .   In seed production, seed germination quality and homogeneity are essential product characteristics. However, the quality and homogeneity of germination of seeds harvested and sold by farmers is important. There is no. Separating crops from other crops and weed seeds, controlling seed-borne diseases, and Fairly large and well-defined seeds due to the difficulty of producing seeds with good germination Seed production whose manufacturing method is expert in the field of growing, conditioning and selling pure seeds Developed by developers. Thus, for the farmer, the seeds harvested from his own crop It is common to buy certified seeds that meet specific quality standards instead of using offspring It is a target. Propagation materials used as seed are commonly used as herbicides, insecticides, fungicides Protective coatings containing germicides, nematicides, molluscicides or mixtures thereof Have been treated. Conventionally used protective coatings include captan, Le Controls to provide protection against disorders caused by fungal or animal pathogens Additional carriers, surfactants or application-enhancing adjuvants conventionally used in the field of Dispensing with the drug. The protective coating can be used to soak the propagation material in a liquid formulation or It can be applied by coating with a complex wet or dry formulation. Bud or fruit Other applications are possible, such as direct treatment in the fruit.   A transgenic plant of the invention for providing control against plant disease, Use and characteristics of transgenic plant material or transgenic seed The provision of novel agricultural methods, such as the methods exemplified above, is a further aspect of the present invention. You.   To breed progeny from plants transformed according to the method of the present invention, Such a method may be used: plants produced as described in the Examples below are known in the art. It grows in pots or soil in a greenhouse as above, and blooms. Get pollen and the same plant Used to pollinate, sister plants or any desired plants. Similarly, transformed plants Can be pollinated with pollen obtained from the same plant, sister plant or any desired maize plant You. The transformed progeny obtained by this method can be used for transgene and / or associated DNA (Transgenic), or the conferred phenotype can distinguish it from non-transformed progeny . Transformed progeny are also self-determined, as is usually done with plants having the desired properties. May be crossed with pollination or other plants. Similarly, other transformants produced by this method Plants are self-pollinated as is known in the art to produce progeny of the desired characteristics. Or can be crossed.   The invention relates in a first aspect to 1. As a functional array: (a) promoters that function in plant cells and result in the production of RNA sequences, for example, The above promoters, such as the maize ubiquitin promoter; (b) lactoferricin, such as lactoferricin B or lactoferricin H, For example, a peptide of SEQ ID NO: 1 or 2 optionally including an N-terminal methionine A structural coding sequence that encodes the production of 5, or 6 plant-optimized coding sequences; and (c) a polyadenylation nucleoside that functions in plant cells and is attached to the 3 ′ end of the RNA sequence 3 'untranslated region that results in the addition of a transcript, such as the transcription terminator described above, eg, For example, the nopaline synthase transcription factor from Agrobacterium tumefaciens -Minator; And optionally one or more enhancers, as further described herein. Sir or targeting sequence And a recombinant DNA molecule comprising:   In a second aspect, the present invention provides 2. (a) in the genome of a plant cell, for example (i) in a plant cell, to produce an RNA sequence (Ii) a structural promoter encoding lactoferricin production. And (iii) polyadenylation at the 3 'end of the RNA sequence, which functions in plant cells. The above recombinant DNA fragment containing a 3 'untranslated region resulting in the addition of Insert a child; (b) obtaining transformed cells; and (c) transforming plant cells in an amount sufficient to reduce damage from pathogen infection; Regenerate a genetically transformed plant expressing lactoferricin: Transgenic disease resistant plants capable of expressing lactoferricin, including the steps A process for the production of a plant, for example a maize or wheat plant as described above.   In a third aspect, the present invention provides 3. Plants containing cells expressing lactoferricin (eg, maize, wheat or sausage) (Radish plant), for example, as a functional sequence (a) a promoter that is fused in a plant cell and results in the production of an RNA sequence; (b) a structural coding sequence encoding the production of lactoferricin; and (c) a polyadenylation nucleoside that functions in plant cells and is attached to the 3 ′ end of the RNA sequence A 3 'untranslated region that results in the addition of a code; To provide a plant in which the recombinant DNA containing is stably integrated into its genome. Here, for example, the plants are developmentally and morphologically normal).   In a fourth aspect, the present invention provides 4. Plants containing cells expressing lactoferricin when germinated and cultivated, eg For example, a seed for growing the above plant is provided (where, for example, the seed is More (for example, with polymers, fungicides, insecticides, pigments or other seed coatings) Coated and / or in, for example, a bag or other container for sale or transport Packed).   In a fifth aspect, the present invention provides 5. For example, as described above, a plant pathogen, such as a plant bacterium, fungus or virus Use of lactoferricin to control pathogens; And exposing the pathogen to a plant that expresses lactoferricin, e.g., A method for controlling a plant pathogen as described above is provided.   The following description further illustrates the methods and compositions of the present invention. However, this business It is reasonable that other methods known to be equivalent by the Understood.                                  Example Example 1: Antimicrobial activity of lactoferricin B in phytopathogenic fungi and other microorganisms Physical activity a.Inhibition of spore germination   Lactoferricin B was administered to Diprodia Maidis and Colletotrichum Laminocola spores were used and tested in a spore germination inhibition assay. Loral fungal strain Obtained from Castor, CIBA-GEIGY, Illinois. Lactoferricin B peptide and LA Kutoferin was obtained from Morinaga Milk Products Co., Ltd., Tokyo, Japan.   Fungal spores grown on potato dextrose agar (PDA, Difco) plates Recovered from the sporulated culture. Keep Dee Maidis spores fresh Was used in 25% glycerol stored at -80 ° C. Sea Gramino Cola Spore Was used fresh.   For the assay, 10 ml lysis medium (1.5% agar, containing 8% carrot extract) Was cooled to about 50 ° C. 10 microliters of streptomycin (250 mg / Ml) followed by either Dee Maidis or Sea Gramino Cola spores About 10⁶/ Ml (Dee Maidis) and 10⁷/ Ml (sea gramino cola) concentration Was added. Mix the solution by gentle swirling and pour into sterile Petri dishes It is. After solidifying the medium, place a sterile 1/4 inch diameter filter disc on top of the medium. The test solution was pipetted onto these disks. The test solution was Lactof (10 micrograms), lactoferricin B (10 micrograms), Lothionine (positive control, 0, 5, 10 and 20 micrograms; Calbiochem? And buffer (20 mM Tris pH 7.5).   After 2-5 days incubation at room temperature, prothionine and lactoferricin Fungal growth is clearly visible on the plate except around the filter disc containing B I was able to. The size of the inhibition zone is shown in FIG. In the Dee Maidis experiment The filter disk of 10 micrograms of lactoferricin B is 4.95 m m, while 20 micrograms of prothionine show a 4.5 mm inhibition zone. did. Lactoferricin B inhibits 1.7 mm in Sea-Graminocora experiment , While prothionine produced a zone of inhibition of 2.1 mm. Lactofe Phosphorus did not inhibit fungal germination and growth.   Erwinia Stewarti was also tested in this system and lactoferricin B was also shown to inhibit the growth of this bacterium. Table 1: Inhibition of spore germination by lactoferricin B b.Inhibition of mycelium growth   The second method used to evaluate antimicrobial activity involves measuring inhibition of mycelial growth. Book In the assay, around a 1.5% agarose plate containing 4% carrot extract Drilled the hole with the wide end of the Pasteur pipette. Bottom of hole with drop of molten agar solution Enclosed. Individual fungal plugs for Bipolaris Maidis, Fusarium monorif Olme, Gibberella Zeae, Pichium aphanidermatium or Rhizoctoni Take one of the A. Solani master plates and place it in the center of each test plate. Was. The test solution in this well (40 microliter) was lactoferricin B (10 microliters). 0 micrograms), lactoferrin (100 micrograms) or buffer (20 micrograms). mM Tris pH 7.5). At room temperature the fungus grows from the inoculation plug into the hole After incubation for the required time, inhibition of fungal growth was scored. All fungi Showed growth inhibition near the hole containing lactoferricin B, but lactoferrin Or not shown around the buffer control well. Table 2 shows the results. Table 2: Inhibition of fungal growth by lactoferricin Bc.Minimum inhibitory concentration in liquid culture   Lactoferricin B and two derivatives of Fusarium curmolam and sep Activity in Tria nodrum was tested in a liquid inhibition assay. Peptide is easy Tofericin B (as described above), Met-lactoferricin B (additional N-terminal methionine) N-lactoferricin B) and Gln-lactoferricin B (added N-terminal Lactoferricin B) with terminal glutamine. All peptides are described in M . Purchased from Keck Biotechnology Resource Center, New Haven, CT. Pepti Was dissolved in double distilled sterile water at 1.5 mg / ml.   The fungal spores are plated on potato dextrose agar (PDA, Difco) as described above. Recovered from sporulated cultures grown at a rate of 15,000 in 1/2 strength PDA. Diluted at 0 / ml. To 50 microliter spores, serially dilute 25 microliters. Tol peptides were added in all 96 well plates. After mixing the wells The OD at 590 nm was measured (measurement of fungal growth). Plates are then incubated at 28 ° C. Cuvated and OD at 590 nm was measured at regular intervals over 2 days. Completely raw The minimum concentration of lactoferricin B peptide that inhibits growth is defined as the minimum inhibitory concentration (MIC). Shown. Table 3 shows these values.   Pichia pastoris on YPD plate, Pseudomonas syringae BL882 And E. coli DH5α were grown in L-broth. Small amounts of P. pastoris cells Is resuspended in YPD, the cell concentration is counted, and adjusted to 30,000 cells / ml with YPD. Saved. The OD at 600 nm was measured in cultures of DH5α and BL882. Cell concentration Adjusted to 30,000 cells / ml in loss, OD = 1 at 600 nm for DH5α 109 cells / ml, 5 × 10 for BL882⁸It was assumed to be cells / ml.   Serial dilutions of lactoferricin B peptide to 80 microliter cells Microliters were added in all 96 well plates. 590nm of plate The OD was read and the plate was then incubated at 28 ° C. OD at 590 I read it regularly over two days. Lactoferricin B pepti that completely inhibits growth The minimum inhibitor concentration was shown as the minimum inhibitory concentration (MIC). Table 3 shows these values. Table 3: Minimum inhibitory concentration of lactoferricin B Example 2: Stability of Gln-lactoferricin B peptide in extracellular fluid of plant   The lactoferricin B peptide was placed in several different subcellular locations (see below for details). (Described in Examples 3-6) using a regulatory sequence to direct the peptide I let you. For extracellular expression, the lactoferricin B coding sequence is It was fused with the leader sequence of the maize PR-1 gene, which leads to secretion into the extracellular region. This The leader sequence used for the purpose of Lactoferricin B containing an N-terminal glutamine residue This results in secretion of the peptide (Gln-lactoferricin B). Synthetic Gln-lactoferri Syn-B peptide is stable in extracellular washings of wheat, corn and tobacco Was tested.   Extracellular wash (ECF) is obtained by sterilizing leaf tissue and immersing it in double distilled water. This was collected by centrifugation of the immersed tissue. For wheat, leave the leaves of the UC703 plant For sorghum, 6N615 plants were used. Cut these leaves into small pieces, Placed in the barrel of a 20 ml syringe containing wadding at the bottom. Wash sections with water Extracellular exudate was removed from the surface. Next, add water and remove the plunger from the syringe I put it in the barrel. Place the syringe upside down in the lyophilization container, then freeze-dryer Connected to. After a gentle vacuum of 30-40 seconds, the vacuum was released. Repeat this once did. The leaf pieces were then placed in the barrel of a 10 ml syringe and placed in a centrifuge tube. this Spin at 1000-2000 rpm in a clinical centrifuge, collect extracellular wash, It was then frozen.   For tobacco (cultivar Xanthi N.C.), using a 10 ml syringe, Water was injected into the leaves. The leaves are then harvested from the plant, blotted and dried, rolled, short And cut into a 10 ml syringe barrel. Put syringe into centrifuge tube Spun at 1000 rpm in a clinical centrifuge. Collect the washing solution collected from the tube and freeze I let it.   Run an aliquot of ECF on a 10% Tricine gel according to manufacturer's instructions (Novex). Was. Estimates of relative protein concentration were made for each plant ECF. Ink that follows this Required to recover a liquid with approximately equal amounts of total ECF protein. It was used to estimate the amount of ECF required. That is, each 18.5 microliters ECF from wheat, 31 microliters of corn ECF and tobacco E 60 ml of CF is mixed with 10 mM Tris pH 7 in a total volume of 75 microliter. 2.5, 50 mM NaCl, 2.5 mM, MgCl_TwoAnd 2.5 mM CaCl_Two Each containing 11.25 micrograms of Gln-lactoferricin B peptide And incubated. The reaction was incubated at 37 ° C and 10 microliters The torr amounts were taken at 0, 15, 30, 60, 90, 120 and 180 minutes. these Samples were immediately frozen.   After heat inactivation and alkylation, the sample was gel- The presence of tofericin B was analyzed. For incubation at 100 ° C for 10 minutes Subsequently, the sample was cooled and spun rapidly in an Eppendorf centrifuge. Each sun The alkylation of the pull is accomplished by first sampling the sample with 2 microliters of 1 mM DTT. , N_TwoThe reduction was performed in an atmosphere at 100 ° C. for 10 minutes. Then yaw Doacetamide (2 microliters of 10 mM) and 1.4 microliters 1 M Tris (pH 8.5) was added to make the solution more basic. Test tube N_TwoWith After washing, the samples were incubated in the dark at 50 ° C. for 50 minutes. Control Peptide sample (Gln-lactoferrin B peptide without buffer or ECF) Was alkylated in the same manner. Until the sample is analyzed by gel electrophoresis Stored frozen.   To each sample, add an equal volume of 2x Tricine / SDS sample buffer (Novex) And then incubated at 100 ° C. for 5 minutes. The sample is then 10-20% Run on ricin / SDS gel (Novex) according to manufacturer's instructions. Gel, pepti Stain was removed by staining with Coomassie (Pharmacia) to visualize the band.   Gln-lactoferricin B peptide was quite stable in wheat FCF. 3 o'clock After incubation between the two, a significant amount of peptide is still present and the amount of peptide It shows that the solution is relatively slow at this ECF. For corn and tobacco ECF Gln-lactoferricin B peptide after 3 hours incubation , Even in reduced quantities, still existed.   This result indicates that the Gln-lactoferricin B peptide is the ECF of these three plant species. Indicates that it is only slowly decomposed. The amount of peptide in ECF The solution takes more than 3 hours under these conditions.   Example 3: Construction of a gene for cytoplasmic expression of lactoferricin B in plants   Coding and non-coding DNA for cytoplasmic expression of synthetic lactoferricin B The sequence was obtained by the synthesis of two partially complementary oligonucleotides. (code (5 'to 3' of the strand) the BamHI restriction enzyme terminus, which encodes the Kozak consensus site The lignonucleotide is a methionine initiation codon, a lactoferricin B peptide. Includes 75 base pairs encoding amino acid sequence, stop codon and NotI restriction enzyme end .   The oligo nucleotide sequence is: Met.   Purchase these synthetic oligonucleotides and add 10% urea / polyacrylamide After electrophoresis on a gel, a band containing the full-length oligonucleotide was excised. Oligo Nucleotide eluted, annealed, PC using oligos 1-1 and 2-1 Used for R amplification.   Ligate the PCR product to pCRII, identify clones containing the insert, insert The fragments were sequenced to confirm deletion of the introduced mutation. BamHI of clone of correct sequence -The NotI insert was cloned into BamHI and NotI predigested Bluescript. You 2 kb Hi containing biquitin promoter (Toki, et al., Plant Physiol. 100: 1503) The ndIII-BamHI fragment was cloned into the HindIII-BamHI site of this clone. Was. The nos terminator was ligated to the NotI-SacI site and ubiquitin-lac PCIB7703, a plasmid having a tofericin B-nos gene cassette (see FIG. 1) (Shown) was produced. The DNA fragment containing ubi-SynPAT-nos was converted to Hin It was cloned into the dIII site. The resulting plasmid, pCIB7704 (shown in FIG. 2), Ubi-SynPAT-nos and ubi-lacto-nos gene cassettes. This plasmid is Includes SynPAT gene for selection in plants and uses maize ubiquitin promoter It is useful for transformation of plants with lactoferricin B under control.   Plasmid pCIB7704 was cleaved with KpnI and SacI and ubi-SynPAT-nos--ubi-lacto- The fragment containing the nos gene fusion was released. Use this fragment for KpnI, Sac It was cloned into I digested pCIB6848 to form pCIB7705 (shown in FIG. 3). Plasmi De pCIB7705 is the NPTII (kanamycin resistance) gene and plant for selection in bacteria Containing the SynPAT gene for selection in the maize ubiquitin promoter Useful for transforming plants with lactoferricin B under control.   Plus the KpnI-SacI fragment from pCIB7703 containing the ubi-lacto-nos construct It was cloned into mid pCIB6848 to produce pCIB7706 (shown in FIG. 4). This allows The ubi-lacto-nos gene construct is placed in a plasmid containing the namycin resistance gene. Example 4: Construction of a synthetic gene for extracellular expression of lactoferricin B in plants   For extracellular expression of lactoferricin B, a leader sequence was added to lactoferricin. It was located at the 5 'end of the B gene. Leader sequence is derived from maize PR1 cDNA (PR-1mz) Yes, this is from a maize cDNA library (using a barley PR-1 gene probe). Cloned and sequenced (Patent Publication WO 95/19443). Synthetic Ligonucleotide and PCR amplification in two steps for the production of the gene construct used. Oligonucleotide 3 is a PR-1 leader and lactoferricin B Sequence, but with one silent nucleotide change (possible To prevent some secondary structure problems). Oligo 4 is lactoferricin B code Sequence (this design is described in the detailed description) and the 3 'end of the PR-1 leader sequence Complementary to the edge. Oligo 3-1 was identical to the 5 'end of the PR-1 coding sequence .   The nucleotide sequence of the synthetic oligonucleotide is: Met.   First, a maize PR1 leader sequence was constructed using oligos 3-1 and 4, PCR amplification was performed using the PR-1 cDNA clone as a template. This is a complete BamHI restriction PCR containing position, PR-1 leader, lactoferricin B and part of a NotI restriction site The fragment was produced. The lactoferricin B template was used for the PCR reaction, Oligo 2-1 (described in Example 3 above) and Oligo 3 were used as primers. The two PCR fragments are then mixed, denatured at 94 ° C, and the Taq DNA Extension was performed at 72 ° C. for 5 minutes with limase. Next, this reaction product was treated with oligo 2-1 and oligo 2-1. And 3-1 for 10 cycles of PCR amplification.   The PCR fragment was cloned into PCRII. Clone containing insert The insert was sequenced to confirm the deletion of the introduced mutation. Leader arrangement Plasmid # 10 containing the insert with one mutation in the row was isolated (No. 12 GCC is changed to GAC by the amino acid of (). Repeat the above experiment to Plasmid # 11 having one mutation in the syn B gene sequence was isolated (15th Amino acid changes from GCC to ACC). These two, each containing a different mutation The correct sequence was produced using the plasmid. Insert the BamHI-NotI insert into pBluescri At pt, it was cloned as a BamHI-NotI insert. BstXI fragment containing mutation Was removed from the Bluescript plasmid containing insert # 11 and insert # 10 was removed. Was replaced with the corresponding BstXI fragment from the Bluescript plasmid containing You 2 kb containing the biquitin promoter (Toki, et al., Plant Physiol. 100: 1503)   The HindIII-BamHI fragment was cloned into the HindIII-BamHI site of this clone. And the nos terminator was ligated to the NotI-SacI site. The obtained plastic The sumid was named p # 10/11.   This accurate plasmid is then used as a template in a PCR reaction and used as a primer. Oligo 5 and Oligo 2-1 were used. Oligo 5 is the last of lactoferricin B One amino acid glutamine is added before the first amino acid. This is PR1 A reliable leader cleavage site is generated at the junction between the leader and lactoferricin B. I went there. Removal of the PR1 leader in plant cells, therefore, results in an additional N-terminal glue. We anticipate the production of a lactoferricin B peptide with thamin.   The resulting PCR fragment is cloned into pCRII and the clone is sequenced did. The BstXI-NotI insert of the correct sequence (including additional glutamine) was isolated and BstXI was cloned into NotI digested p # 10/11 to produce pCIB7707. This It converts the maize PR1 leader-lactoferricin B coding sequence to a ubiquitin-pro (Ubi-PR1-lactoferri) under control of motor and nos terminator B-nos). The DNA fragment containing ubi-SynPAT-nos was converted to HindIII of this plasmid. Site and cloned the SynPat and PR1-lactoferricin B gene cassettes. The resulting plasmid pCIB7708 was obtained.   This plasmid is cut with KpnI and SacI, and ubi-SynPat-nos--ubi-PR1-lacto-no The insert containing the s gene fusion was released. This fragment is digested with KpnI and SacI pCI Cloned into B6848, producing pCIB7709. This plasmid is suitable for bacterial selection. NPTII (kanamycin resistance) gene for synthesizing, and SynPAT gene for selection in plants. Lactoferricin B inheritance under the control of the maize ubiquitin promoter Useful for transforming plants in offspring.   Purify the KpnI-SacI fragment from pCIB7707 containing the ubi-lacto-nos cassette. It was cloned into the rasmid pCIB6848 to produce pCIB7710. This is ubi-lacto-no The s gene construct is placed in a plasmid containing the kanamycin resistance gene. Example 5: Construction of a gene for lactoferricin B vacuole expression in plants   For vacuolar expression of lactoferricin B, a sequence for vacuolar targeting was used. Example 4 (of a synthetic gene for extracellular expression of lactoferricin B in plants) Construction) added to the 3 'end of the PR1 leader lactoferricin B gene cassette described in Was. This additional 18 base pair sequence is a C-terminal Val-Phe-Ala-Glu-Ala-Ile vacuole target. Coding sequence (Dombrowski et al., Plant Cell 5, 587-596, 1993), Following the stop codon. In addition, restriction ends were inserted at the 5 'and 3' ends of the synthetic gene. And facilitated cloning.   Two oligonucleotides were used in the construction of this synthetic gene. Oligo 3-1 Is described in Example 4 above. The sequence of oligo 6 is lactoferricin Complement at the 3 'end of the B coding sequence, codon for vacuole targeting sequence, stop Includes stop codon and NotI restriction enzyme site. This sequence is shown below.   Oligo 3-1 and oligo 6 were replaced with plasmid pCIB7707 described in Example 4 above. The PR1 leader lactoferricin B gene construct was used for PCR amplification. PCR The fragment was cloned into pCRII and the clone containing the insert was selected. Insertion The incoming fragment was sequenced and the clone with the correct sequence was replaced with the BamHI-NotI fragment. Was used for extraction. This fragment was cloned into Bluescript and ubiquitin 2 kb HindIII-B containing promoter (Toki, et al., Plant Physiol. 100: 1503) The amHI fragment was cloned into the HindIII-BamHI site of this clone and nos The terminator was ligated to the NotI-SacI site. This results in plasmid pCIB 7712 was produced. This is the maize PR1 leader-lactoferricin B-vacuole target The sequence is placed under the control of a ubiquitin promoter and a nos terminator (u bi-PR1-lactoB-vac-nos). ubi-SynPAT-nos DNA fragment The plasmid was ligated to the HindIII site of plasmid to obtain plasmid pCIB7713.   Cleavage of pCIB7713 with KpnI and SacI, ubi-SynPAT-nos--ubi-PR1-lacto-vac-no The insert containing the s gene fusion was released. Fragment KpnI, SacI digested pCI Clone into B6848 to form pCIB7714. This plasmid is suitable for bacterial selection. NPTII (kanamycin resistance) gene for synthesizing, and SynPAT gene for selection in plants. Lactoferricin B inheritance under the control of the maize ubiquitin promoter Useful for transforming plants in offspring.   Purify the KpnI-SacI fragment from pCIB7712 containing the ubi-lacto-nos cassette. Cloned into the rasmid pCIB6848 to produce pCIB771015. This is ubi-lacto- The nos gene construct is placed in a plasmid containing the kanamycin resistance gene. Example 6: Construction of a gene for lactoferricin B plastid expression in plants   For expression of lactoferricin B plastid, ribulose-1,5-bifos was used. Clones the chloroplast target sequence of the fate carboxylase small subunit (ssu) gene. And placed 5 'of the lactoferricin B gene sequence. This transport peptide arrangement The rows direct small subunit proteins to the chloroplast, where they are cleaved to mature proteins. Releases the substance. The designed transit peptide-lactoferricin B gene construct is (5 'to 3') BamHI restriction site, Kozak consensus sequence (methionine start codon ), A chloroplast target sequence fused to the lactoferricin B coding sequence, a stop codon And a NotI restriction site. The lactoferricin B gene construct is Cis (ast1A; Wong et al., Plant Mol. Biol. 20, 81-93, 1992) and maize (Ma tsuoka et al. Biochem. 102, 673-676, 1987) chloroplast target of ssu gene Manufactured from the target array. The coding sequences of the transit peptides of these ssu genes (Wong et al., Plant Mol. Biol., 20, 81-93, 1992) by PCR amplification of genomic DNA, Clone from cDNA or cDNA library. A series of three transport boxes Peptide sequences (TP1, TP2 and TP3) were prepared according to Wong et al. (Plant Mol. Biol. 2). 0, 81-93, 1992), both Arabidopsis and maize ssu genes It was built from. Therefore, TP1 binds the coding sequence of only the transit peptide (up to the cleavage site). TP2 and 3 contain the complete coding sequence of the transit peptide and the mature ssu protein A portion of the coding sequence of the protein is included, with a double cleavage site. TP2 is the double cleavage site Has additional two amino acid codons. TP3 ends exactly at the double cleavage site Wrong. a.Arabidopsis chloroplast target sequence-lactoferricin B gene construct   The first transit peptide sequence contains the oligonucleotides ATP-5 'and And ATP1-3 ′. The arrangement of these oligonucleotides The columns are: Met.   ATP-5 'oligonucleotides are BamHI restricted to facilitate cloning. Site, Kozak consensus sequence 5 ′ of start codon and coding sequence of ats1A gene Included the first 18 nucleotides in the row. Genomic DNA was used as a template. Increase The width product was cloned into pCR-Script (SK +), the clone was sequenced, and the ats1A Insertion of the sending peptide coding sequence containing 165 bp extending from +1 to +165 bp The piece was confirmed. The clone with the correct sequence was named pATP1.   Fusion of the transit peptide sequence to lactoferricin B involves four primers: , ATP1 amplification production From the 5 'end of the product;ATP-LF3 ' : Same as oligo 2-1 described in Example 3 above; Then, the ats1A / lactoferricin B fusion was crosslinked and 18 nucleosides at the 3 ′ end of ATP1. Lactoferricin B coding sequence at the 18 nucleotides at the 5 'end. The following two crosslinking primers. The sequence of the 5 'bridge primer is: The sequence of the 3 'bridge primer (complement of the 5' bridge primer) is: Was achieved by PCR using   Two rounds of amplification were performed for each transport fusion construct. The first round Two reactions were involved. Reaction 1 is specific for short lactoferricin B sequence extension ATP1 PCR fragment was generated. This reaction was performed using primers ATP-LF5 'and A TP1-LFB3 'and plasmid pATP1 DNA were included. Reaction 2 is a short 5 'ATP1 sequence A lactoferricin B PCR fragment with extension was generated. This reaction , Containing either primers ATP-LF3 'and ATP-1-LFB5' Ferricin B was used as a template. The second round of amplification involves the production of reactions 1 and 2. The product is denatured at 94 ° C. for 1 minute, then annealed product For 5 minutes at 72 ° C. in the presence of Taq DNA polymerase and nucleotides. A "full length" mold was formed. This is for primers ATP-LF5 'and ATP-LF3'. Addition and PCR amplification were followed. Thus, the ATP1-lactoferricin B gene fusion Produced. The PCR product was cloned into the PCRII vector.   Clones were isolated and sequenced. BamHI and N It was inserted with otI and the insert was cloned into BamHI, NotI pre-digested Bluescript. You HindIII-BamHI fragment containing biquitin promoter (Toki, et al., Plant  Physiol. 100: 1503) into the HindIII-BamHI site of this clone. The os terminator was ligated to the NotI-SacI site. The resulting plasmid is Contains the ATP1 chloroplast target sequence upstream of the lactoferricin B coding sequence. This gene The construct is located between the ubiquitin promoter and the nos terminator. The DNA fragment containing the ubi-SynPAT-nos gene construct was Clone into HindIII site to produce pCIB7721. The resulting plasmid is called KpnI Ubi-SynPat-nos--ubi-ATP1 / 2 / 3-lacto-nos gene fusion The containing insert is released. These fragments were digested with KpnI and SacI digested pCIB6848. Cloned to form pCIB7722. This plasmid is NPTI for bacterial selection. The I (kanamycin resistance) gene contains the SynPAT gene for selection in plants, Planting with the lactoferricin B gene under the control of the isubiquitin promoter Useful for transformation of products. pCIB7720 containing ubi-ATP1-lacto-nos gene fusion The KpnI-SacI fragment from was cloned into plasmid pCIB6848, and pCIB7723 To produce This is because the ubi-ATP1-lacto-nos gene fusion Place in the containing plasmid.   ATP2, the second transit peptide coding sequence, is cloned in the same manner as ATP1. Genomic DNA or oligonucleotides using oligonucleotides ATP-5 'and ATP2-3'. Rigo dT-primed, reverse transcribed RNA was used as a template.   The PCR product was cloned and the clone with the insert was changed from +1 to +261. Confirm that it contains a 261 bp ats1A transit peptide coding sequence that extends to The clone containing the correct sequence is named pATP2.   ATP3, the third transit peptide coding sequence, is cloned in a similar manner to ATP2. And use oligonucleotides ATP-5 'and ATP3-3'.   The PCR product was cloned and the clone with the insert was changed from +1 to +255. Confirm that it contains a 255 bp ast1A transit peptide coding sequence that extends to 255 bp.   These transit peptide sequences were converted to lactoferricin B residues as described above for ATP1. Fused with gene sequence. The sequence of the set of 5 'bridge primers is: It is.   The sequence of the set of 3 'bridge primers (complementary to the 5' bridge primer) is: It is.   The resulting PCR fusion product is cloned. The correct sequence clone is ATP2 -With lactoferricin B and ATP3-lactoferricin B gene fusion. This These fragments were cloned into Bluescript and ubiquitin promoter and And nos terminator are added as described above to generate pCIB7730 and pCIB7740. Produce. The ATP1 / 2 / 3-lactoferricin B gene construct is a ubiquitin promoter. Located between the motor and the nos terminator. Ubiquitin-SynPat-nos A new construct with the fragment was produced as described above for pCIB7720, plus The mides pCIB7731 and pCIB774l are obtained. b.Maize chloroplast target sequence-lactoferricin B gene construct   Additional transit peptide-lactoferricin B constructs were transferred to Arabidopsis ssu transport In the same manner as described above for the peptide-lactoferricin B gene construct, Transport vector for the maize ssu gene (Matsuoka et al., J. Biochem. 102, 673-676, 1987) Constructed using peptide sequences. The only difference is the casting for the production of the transit peptide. Source of type DNA and oligonucleotide sequence. Maize ssu transport peptide The template DNA for PCR amplification of the sequence may be maize genomic DNA, cDNA or c It is a DNA library. The oligonucleotide sequence (5 'to 3') is as follows Is:   The resulting plasmid was transfected with maize fused to the lactoferricin B coding sequence. Contains protein sequences (MTP1, 2 and 3). Example 7: Expression of lactoferricin B gene in maize protoplasm   Contains lactoferricin B, a protoplasm known to be capable of transient expression Used for transient expression of plasmid. The protoplasm was determined by Shillitto et al. (U.S. Pat. , 350, 689: Zea mays plants and transgenic Zea mays plants regen erated from protoplasts or protoplast deribed cells. 1989) Isolated from suspension culture cells, 20 × 10⁶/ M at a concentration of 0.5M mannitol / 15 mM MgCl_TwoWas resuspended. Plasmid DNA used for transformation Is: pUbi-bar, ubiquitin promoter + first exon and intron, Contains bar gene and nos terminator; pUbi-GUS, ubiquitin promoter -First exon and intron, GUS gene and nos terminator And pCIB7703.   PEG precipitation of plasmid DNA (Kramer et al., Planta 190, 454-458, 1993) Was introduced into the protoplasm as follows: The protoplasm was introduced in a 45 ° C. Heat shock was applied with gentle stirring. 500 milliliters containing 10 million protoplasm The amount of the bottle was transferred to a sterile test tube, and the following additions were made to each test tube: 25-50 μg Of each plasmid and 450 ml 40% PEG. PEG to 8.0 pH adjusted 0.4M mannitol and 0.1M Ca (NO_Three) 2.4H_TwoO From PEG 8000 (Sigma Chemical Co., St. Louis, MO) dissolved in Made. The test tube was left for 20 minutes with occasional gentle agitation, then at 5 minute intervals, 1 ml, 2 ml and 5 ml of W6 solution (5 mM KCl, 61 mM CaCl_Two・ H_TwoO 154 mM NaCl, 51 mM glucose, 0.1% 2- (N-morpholino) (Ethanesulfonic acid, pH 6.0). After the last dilution, the protoplasm was Centrifuge at 60g-100g to remove most of the suspension and reconstitute about 0.5ml of liquid. Left on the trait. The protoplasm was resuspended in the remaining solution by gentle shaking. After insertion of the protoplasm into DNA for analysis of RNA and protein expression, 20 The cells were resuspended at a density of 10,000 / ml in FV medium and cultured at room temperature in the dark for 18 hours.   For subsequent GUS activity measurements, protein extraction was performed according to the manufacturer's description (Tropix). Effluent was prepared from an aliquot of all transformants. The result is the combination of pCIB7703 and ubi-GUS Transformed cells were combined with ubi-GUS (10 × 10⁶Count / min / 1x10⁶ High GUS activity (10 × 1), equivalent to GUS activity in cells transformed with 0⁶Count / min / 1 × 10⁶Protoplasm). This is Lactov Expression of the hericin B gene construct increases GUS gene expression in transiently expressing cells It appeared to have no effect, ie no cytotoxicity. ubi-bar and pCIB770 Transformation at 3 showed only background levels of GUS activity (5000 Count / min / 1 × 10⁶Protoplasm).   RNA samples were prepared for reverse transcription-PCR (RT-PCR) analysis and The presence of syn B mRNA was assessed. For this purpose, all and poly (A) RN A was isolated from transfected plasma. RT-PCR product kit (Stratagene) Was performed according to the manufacturer's instructions. ubi-lactoferricin B-nos cDN For the detection of A, just the nos terminator polyadenylation signal PCR complementary to the 5 'sequence and the first exon of the ubiquitin promoter Primers were used. In the pCIB7703 construct, this exon and lactoferri Due to the intron between the syn B coding sequence / nos terminator, PCR bands should be derived from RNA based on their size. It was recognized. For RT-PCR reactions, 1 μl of total RNA was prepared in a 50 μl reaction volume. Reverse transcription was performed according to the manufacturer's instructions. 1 μl of this was added to Primer-176 (SEQ ID NO: 39: 5'-TTCCCCAACCTCGTGTTGTTC-3 ') and 179 (SEQ ID NO: 40: 5'-CCAAATG TTTGAACGATCGCG-3 ') or primer 177 (SEQ ID NO: 41: 5'-CACAACCAGATC TCCCCCAAA-3 ') and 180 (SEQ ID NO: 42: 5'-AAATTCGCGGCCGCTTAGAAG-3') Used for the PCR amplification used. The reaction conditions were: 94 ° C for 45 seconds, 60 ° C for 45 seconds and And 43 cycles at 72 ° C. for 2 minutes. Size fractionate sample on agarose gel did. Primers 176 and 179 were used when the template DNA was formed in the pCIB7703 plasmid. As expected for cDNA from transformed cells, an approximately 220 bp P A CR fragment was produced. The plasmid-derived PCR band is 1.3 kb in size. Was predicted.   For Northern analysis, RNA samples were size-fractionated on an agarose gel and Blotted on iron membrane. This blot was then used to construct the lactoferricin B gene. Hybridized with a radioactive probe containing the sequence.   Protein extracts for immunoblotting are prepared by converting the protoplast suspension into a suitable buffer. Prepared by manufacturing. The suspension is microcentrifuged for 5 minutes, and the supernatant and pellet are removed. Suspend in Laemli sample buffer and run on Laemli SDS gel electrophoresis. Lacto Disulfide bonds of ferricin B to proteins can be problematic, so DTT or Or 2-ME, followed by iodoacetamido in the protein-SH group of the extract. Blocking may be required before electrophoresis. Run the electrophoresis gel with anti-lactoferricin B The antisera was used to electroblot for Western analysis.   Vigorously agitate protein suspension for protoplast suspension in mass buffer for mass spectrometry And centrifuge twice for 10 minutes, then collect the supernatant at each hour You. Appropriate dilutions can be made using MALDI-MS (Voy ager, Perceptive Biosystems). The sample needs to be further purified In some cases, the extract can be fractionated on a C18 column. Then fract the fraction Analyze by MALDI-MS for the presence of ferricin B peptide.   Protein extracts are also prepared for evaluation of antimicrobial activity. In this case, the protoplasm With 1.5% PVPP, 5 mM DTT and 10 μM AEBSF (4- (2- Aminoethyl) -benzenesulfonyl fluoride, hydrochloride) containing 20m Homogenize in M Tris buffer, pH 7.5. The extract was prepared as described in Example 1 above. As such, antimicrobial activity is assessed based on an in vitro fungal inhibition assay.   Transient expression analysis of other lactoferricin B gene-containing constructs is described in this example. Perform using the method described above. Example 8: Plant transformation, selection and regeneration a.Description of plasmid and selectable marker   The lactoferricin B gene contains a ubiquitin promoter and a nos terminator. Under the control of the Noether. Selectable markers for corn transformation Cloned into these plasmids, the synthetic phosphoinothricin acetyl tra The transferase gene (designated SynPAT) is used for the maize ubiquitin promoter and And under the control of the nos terminator (ubi-SynPAT-nos). SynPAT Strep Bar gene derived from a strain of Tomyces (Thompson et al., EMBO 6: 2519-2326, 1987) And optimized for expression in plants (US Pat. No. 5,276, No. 268; Phosphoinotricin-resistance gene and its use). ubi-SynPAT-nos Plasmid containing the cassette (pUBIAc) was transferred to Hoechst Aktiengesellschaft, Frankfurt, Obtained from Germany. ubi-lactoferricin B-nos and ubi-SynPAT-nos cassette Used for maize transformation. The presence of the SynPAT gene cassette indicates that the herbicide BA Enables selection of transformed plants using STA.   For wheat transformation, a plasmid containing the ubi-lacto-nos gene cassette was Selectable bar under control of ubiquitin promoter and nos terminator Co-bombard with a plasmid (pUBA or pUBA / kan) containing a functional marker gene (T oki et al., 1992 Plant Physiol. 100: 1503-1506). Existence of bar gene cassette Now allows the selection of transformed plants with the herbicide BASTA. pUBA plasmid Includes ampicillin resistance gene for selection in bi-bar-nos and E. coli. Plasmid pUBA / kan contains the ampicillin resistance gene for selection in E. coli. Instead of the kanamycin resistance gene.   The hygromycin gene from E. coli (Gritz and Davies, Gene 25, 179-18) 8, 1983), ligated a BglII linker to this end, and Was then cloned into the BamHI site of vector pCIB710, producing pCIB712. two Oligonucleotides (5'-AGTAGGATCCATGAAAAAGCCTGAACTC-3 '(SEQ ID NO: 43) And 5′-TACTGGATCCCTATTCCTTTGCCCTC-3 ′ (SEQ ID NO: 44)) derived from pCIB712 Used for PCR amplification of the gene. The PCR fragment was digested with BamHI and pPEH3 It was cloned into the BamHI site. Clones containing the insert are sequenced and The one with the sequence and the correct orientation of the gene was named pCIB7613. This plasmid is The hygromycin resistance gene is converted to a ubiquitin promoter and a nos terminator. Included under control. Following bombardment of plants with these plasmids, transformation Plants can be selected using hygromycin as a selection agent.   The acetohydroxyacid synthase (AHAS) gene was replaced with a sulfonylurea (Beaco n) From the λ-Zap genomic library derived from resistant maize tissue, Using the Dopsis gene (K. Sathasivan et al., Nucl. Acid Res. 18, 2188) Cloned. Oligonucleotides with appropriate single base changes by PCR amplification Using pide, a G to A change was inserted at position 1862 of the gene. This nucleotide change resulted in a Ser to Asn change. This gene as a vector Cloned to produce plasmid pCIB4247. Maize AHAS remains in pCIB4247 Two oligonucleotides specific for the start and end of the gene (5'-TATCTCTCTCTATA AGGATCCATGGTCACC-3 '(SEQ ID NO: 45) and 5'-TACTGGATCCTCAGTACACAGTCCTGCC -3 ′ (SEQ ID NO: 46)) to generate fragments by PCR amplification. I cloned. Inserts are sequenced to confirm deletion of mutation and BamHI flag with AHAS sequence using a plasmid with a unique insert Was isolated. This fragment was cloned into the BamHI site of pPEH3, CIB7612 was produced. This plasmid contains the maize ubiquitin promoter and Contains the mutated AHAS gene under the control of the nos terminator. This gene When transformed, imidazolinone (imazaquin / Scepter) is provided.   Protoporphyrinogen oxidase from Arabidopsis and maize (Protox) cDNA and protox gene gene derived from Arabidopsis The motor was isolated. Protox cDNA from Arabidopsis and Maize Was cloned by complementation of a Protox deficient E. coli strain. Mutated arabid Select psis and maize protox genes, which are protox inhibitory herbicides Provides resistance to the agent. Two herbicide-resistant Arabidopsis protox cDNAs The plasmid is cloned between the heavy 35S promoter and the tml terminator. You.   Using the Arabidopsis protox promoter as a probe Genomic license using fragments from the 5 'end of Isolated from Bally. Plasmid containing promoter with NcoI and BamHI Digestion leaves the promoter attached to the vector, but removes downstream sequences. Weeding NcoI-BamHI flag containing drug-resistant protox cDNA and tml terminator Was cloned into it. This results in an Arabidop of approximately 600 nucleotides. The cis-protox promoter and the herbicide-resistant Arab protox gene Plasmids containing the nucleotide sequence and the tml terminator were produced. This plus Plants transformed with the mid can be selected on media containing herbicides. b.Transformation of lactoferricin B gene construct into maize   The method used for maize transformation into cells of immature embryos using microprojectile bombardment is described in Ko ziel et al. (Biotechnology 11, 194-200, 1993). May Immature embryos of the inbred CGA00526 were isolated by aspiration about 10 days after pollination and In a “D” medium (Duncan et al., Planta 165, 322-332, 1985) with the scutellum up Plated. Chloramben (5 mg / L) was added to the medium instead of dicamba. After about two weeks, the callus was isolated from the embryo and 2,4-dichloro Plating on a medium supplemented with phenoxyacetic acid, followed by secondary culture at 10-14 intervals Nourished. 4 to 6 hours before transformation, callus of 1-4 months old was transformed into 12% sucrose medium Was transferred to a fresh medium containing as an osmotic regulator.   Plasmid DNA or isolated fragment DNA from DuPont Biolistic Manual Precipitated on the gold particles as described in Table 1. Duplicate each tissue plate twice with DuPont Bioli Shot with stics helium equipment. Burst pressure 600-1100 psi and standard 80-10 A 0 μm mesh screen was used. After bombardment, leave tissue in the dark for 12-24 hours Placed. After this time, the tissue was added to the selective agent BASTA at a minimum of 20 mg / L. Return to modified Duncan "D" medium and grow in the dark. Modified Duncan “D” medium , Modified Koa amino acid addition (KW Kao and MR Michaelyluk, Planta 126, 105-110) Glutamine and asparagine. Was completely removed. The tissue is administered at intervals of more than 14 days for 60-80 days, a minimum of 20 mg / Transferred to fresh medium containing BASTA at L. During this subculture period, non-embryogenic calli Removed.   After this dark selection period, the tissue germinates following hormones and embryonic somatic embryo development MS media (Murashige and Skoog, Physiol. Plant 15 , 473-439, 1962): ancimiddle (0.25 mg / L), kinetin (0.5 mg / L) L), naphthaleneacetic acid (1 mg / L) and a minimum concentration of 5 mg / L BASTA. Tissues in this medium Cultivated for 10-14 days under light control for 16 hours, and then BASTA (without plant hormones) Secondary culture was carried out in an MS basic medium supplemented with 3 mg / L) to grow seedlings. Once the seedlings have developed Transferred to 3/4 strength MS medium to develop roots. Transfer the rooting plant to the soil and in the greenhouse Grew. c.Transformation of lactoferricin B gene construct into wheat   Vasil transforms immature embryos and calli from immature embryos using microprojectile bombardment et al. (Biotechnology 11: 1553-1558, 1993) and Weeks et al. (Plant Physi ology 102: 1077-1084, 1933). Immature embryo of spring or winter wheat Approximately two weeks after pollination, they are isolated by aspiration, and 2,4-D (dichloromethane) for callus induction Rofenoxyacetic acid) glutamine and asparagine supplemented MS basic medium (Murashige And Skoog, 1962 Physiol. Plant 15: 473-439), 6-10 days with scutellum up The plate was cultured. Four to six hours before transformation, lungs were selected for embryogenic reactions and Was transferred to a fresh medium containing 15% maltose as osmotic agent. The construct used for transformation contained the ubi-bar-nos cassette (pUBA or pUBA / kan). To allow selection of transformed tissues with BASTA. Alternatively, other herbicides (e.g., , Sulfonylureas, imidazolinones) or antibiotics (e.g., hygromycin ) Can be used. This construct was implemented as described above. PCIB7706, pCIB7710, pCIB7715, pCIB7723, pCIB7733, and pCIB7706, described in Example 2-6. Can be co-transformed with one of the lactoferricin B-containing gene constructs containing pCIB7743 . In this regard, the plasmid DNA was prepared as described in the Dupont Biolistic manual. To fine gold particles. Each plate of embryos was subjected to a 1100 psi burst pressure and standard Shoot with a DuPont PDS1000 helium device using a quasi-80 mesh screen. 24 After an hour, the embryos are recovered from the osmotic agent and returned to the transfer medium. About a month later, it grew The embryo explants having embryogenic calli were regenerated in a regeneration medium containing 1 mg / L BASTA (MS + 1 mg / L-naphthalene acetic acid and 5 mg / L gibberellic acid) for 2 weeks, then 3 mg / L Transfer to BASTA-containing hormone-free regeneration medium for 2-6 weeks until shoots emerge. Sprouting embryo To half strength MS supplemented with 0.5 mg / L NAA and 3 mg / L BASTA. Transfer until roots develop, at which point the seedlings are transferred to soil and matured. Plant tissue Harvested for analysis (as described in Example 10) and plants were self-retained to produce seeds. To powder. d.Transformation of lactoferricin B gene construct into sugar beet   Guard cells are described in Hall et al., Nature Biotechnology, vol. 14,1133-1138,1 Isolated from leaf sections without central vein as described in 996. Leaf section, Ficoll Medium (100 g / l Ficoll, 735 mg / l calcium chloride.2H_TwoO and 1 g / l PVP40) on ice and in a blender (23,000 rpm, 60 seconds). You. After filtration through a nylon mesh (300 micrometers), the homogenate was filtered for 5 hours. Wash with 00 ml of cold water, 3.8% (w / v) calcium chloride.2H_TwoO-containing CPW9M1 Transfer to 0 ml. Next, the epidermis was removed by centrifugation, and the cell wall degrading enzyme (0.5% cellulase) was removed. And 3% macerozyme, Yakult Honsha, Japan) for 16 hours. The vesicles were released. Following filtration through a 55 micrometer filter, the suspension Mix with an amount of Percoll containing 15% sucrose. 1 ml of CPW15S in test tube Followed by 0.5 ml of 9% mannitol / 1 mM potassium chloride in the protoplast suspension Layer on top. Guard cells were recovered from the upper layer after spinning at 55 g for 10 min and again Reisolate in the same way.   0.75 ml of DNA (50 micrograms / million cells) 9% mannitol / 1 mM calcium chloride (mannitol solution) added to the resuspended guard cells, Subsequently, 0.75 ml of 40% PEG6000 is added. After 30 minutes at room temperature, 2 ml of F medium The amount is added a total of four times every 5 minutes. Wash protoplasm with mannitol solution, then Layer on Ca alginate. They are cultured in a modified K8P medium and grown in Bialaphos. One week later, start with 200 micrograms / liter. Eleven days later, Layer a small amount of alginate on agarose and add 250 micrograms in PGIB medium. Incubate with mbiaarafos / liter. Bialofos added to new callus Transfer to a fresh medium and culture for 2 weeks. After this they are kept at 25 ° C under light / dark control (16 (8 hours) without any selection. Subculture is performed every 2 weeks, and seedlings are cultured for 4 weeks. Play for a while. They root and plant in the soil. Example 9: Analysis of transgenic plants expressing lactoferricin B   Tissue derived from the transformed plant surviving the selection method (Example 8) was Gene construct (PCR), RNA from this transgene (Northern blot Hybridization, RT-PCR) and proteins from this transgene (Wester Analyze the presence of a.PCR analysis   Transfer the DNA to the IsoQuick Nucleic Acid Extraction Kit (ORCA Re extract Inc.) from the transformed plant tissue. Standardized PCR analysis Follow the protocol. Plasmid used for amplification of lactoferricin B gene construct Imers are designed from the known sequence of the transgene construct. b.Northern analysis   Transformed plants were analyzed for RNA by Northern blot hybridization. Analyzed for presence. RNA from leaf tissue for Northern blot analysis Extracted. Following trituration of the frozen tissue, the extraction buffer (0.1 M LiCl, 100 mM M Tris, pH 8, 10 mM EDTA, 1% SDS), followed by an equal volume of water Saturated phenol and chloroform were added. RNA to sodium acetate / eta And precipitated on a formaldehyde-containing agarose gel. Get Of the lactoferricin B gene Hybridized with respect to the derived probe. Continue with overnight hybridization Blots were washed at high stringency (65 ° C) and exposed to X-ray film. Was. c.RT-PCR analysis   The RNA sample was also subjected to lactoferricin B RNA as described in Example 7. Are analyzed by RT-PCR for the presence of d.Western analysis   The transformed plant is transformed with lactoferricin B or lactoferricin derived from the transgene. Tested for the presence of B-derived protein. For Western analysis, Leaves were snap frozen in liquid nitrogen and ground to a fine powder. Then the sample is placed in acetone Treated with cold 10% trichloroacetic acid at -20 ° C for 1 hour, spin down, and Washed with tons and air dried. The dried sample was then added to 0.05M sulfuric acid (3 mg / g Fresh weight) for 1 hour on ice. Extracts are extracted with 0.01N sodium hydroxide And dried. Samples were run on a 10% -20% Tricine SDS gel (No. vex) according to the manufacturer's instructions. Protein is wetted on nitrocellulose Transferred in cell at 200 mA constant current for 3 hours, then stained with Ponceau red Then, the membrane-bound proteins were visualized. Transfer the protein transferred to nitrocellulose Immunoaffinity purified goat anti-lactoferricin B antibody followed by secondary and alkaline phos The probe was probed with a phosphatase-conjugated tertiary antibody. Anti-lactoferricin B reactive band To a nitro blue triazolium / bromo-chloro-indolyl phosphate substrate Detected after addition of the solution. e.Mass spectroscopy   Protein extracts prepared from the transformed plants were also prepared as described in Example 7 above. Using MALDI-MS to determine the presence of lactoferricin B peptide And analyze. f.Antimicrobial activity in lactoferricin B transgenic plant extracts Evaluation   The protein extract was harvested with 1.5% PVPP, 5 mM DTT and 10 μl tissue. M AEBSF (4- (2-aminoethyl) -benzenesulfonyl fluoride, hydroxy (Chloride) containing 20 mM Tris buffer, pH 7.5 by homogenization. To make. The protein extract was subjected to ammonium sulfate precipitation, HPLC chromatography. Established such as immunopurification using anti-lactide and anti-lactoferricin B antisera Further purify by protocol. Measure protein concentration in each extract, and Used for fungal assays.   Using the three assays described in Example 1 above, crude and partially purified Assess the presence of antimicrobial activity in the dentin extract. Testing for the presence of antifungal activity The organisms used for these are those described in Example 1 and other important corn and Wheat pathogens, such as E. coli and Staphylococcus aureus Lactoferricin B-sensitive bacteria laboratory strain (W. Bellamyetal., J. Appl. Bacteri ol. 73, 472-479, 1992). Example 10: Increased disease resistance of lactoferricin B transgenic plants test a.Testing of transgenic maize plants 1. Southern corn black leaf blight (SCLB) assay   SCLB is Bipolaris Maidis (formerly Cochliobolus Heterostrof) Asus and Helmin Sosporium Madeis) It is. Bee Maidis cultures were grown on PDA (potato dextrose agar). Grow under continuous near-ultraviolet light to induce pup formation. 1% spores Using a glass rod, collect the plate by gently scraping the plate. . The solution is recovered, the spore concentration is double distilled, and the spores are adjusted to 500-1000 spores / ml with sterile water. Adjust child concentration. Warm transgenic and wild-type control plants Grow in the room to 3-5 weeks of age. The spore suspension on the three fully extended leaves on top, Spray by applying a fine mist of spore suspension using an aerosol sprayer I do. The plants are then placed in a closed chamber and overnight (16-20 hours) in a fog chamber. And incubate under high humidity conditions. Plant growing chamber from fog chamber Move to Approximately one week after inoculation, the affected lesions were counted and 10-15 representative lesions were identified. Sa The size is measured and compared between control and transgenic plants. Plants also Visually assess disease progression at 3-4 day intervals over 14 days after inoculation.   Plants of the T1 type, B10b, which express Met-lactoferricin B mRNA, Less disease symptoms than transgenic and wild-type controls Thus, they show increased disease resistance. 2. Anthrax blight blight   Colletotrichum glaminicora is the causative agent of anthracnose blight. It feels Causes necrosis of dyed leaves. For this disease assay, Spores were collected from sporulated agar plates as described. 3-5 week old plant leaves Was sprayed with a spore suspension at a concentration of about 10E6 spores / ml. Inoculated plants, Be Meide Incubate overnight in a fog chamber as described for , And then transferred to a growth chamber. Necrotic at 3-4 day intervals over 2 weeks Measure the percentage of leaf foci, transgenic and non-transgenic Compared between controls. 3. Carbonam spot disease assay   Helminsosporium carbonum is the causative agent of maize carbonam spot disease is there. For this disease assay, cells were prepared as described for B. Offspring were recovered from sporulation cultures. 2.5 to the leaves of 3-5 week old corn plants Sprayed with a spore suspension at a concentration of 10 × 10E4 / ml. Overnight. Next, the plants are grown in a growth chamber for about 14 days. I let it. The disease signal was followed as described for anthrax blight.   T1 line B10b plants expressing Met-lactoferricin B RNA increased. Showed disease resistance. In expressing plants, local inoculation of carbonam leaves followed by tiger Reduced disease compared to transgenic and wild-type controls Disease symptoms were observed. 4. Fusarium root assay   Fusarium moniliforme is the source of the fungal fungus of corn ear It is a causal agent. F. Moniliforme is grown on a PDA plate. The spores are recovered from the plate as described for this. Fold the perforated filter paper Fold, place in sachets and pour surface sterilized corn seeds through Place it in the fold so that it extends to the bottom of the bag. 12ml spores of F Moniliforme Place the suspension in the bottom of the sachet and incubate the sachet in a growth chamber at 25-30 ° C. To Inhibition of root growth and root necrosis by fungal infection were assayed after 7-14 days. Compare between transgenic and non-transgenic controls. 5. Pysium root assay   Pysium afinadermatum is a cause of maize root and root rot One of the living things. For disease assays, as described above for Fusarium , Using a perforated format. Inoculum is prepared from spermatozoa or mycelium. Sperm trait Contact the mycelium with a tall Festuka leaf section for continuous fluorescence Incubate below and collect the developed spermatozoa. Or potato de A mycelium plug from a Pythium PDA plate was inoculated into a dextrose medium, and 2-3 Grow for a week. Remove the mycelium from the culture and place the blender in sterile double distilled water. Use and disperse.   The corn is placed on the folds of the filter paper as described and the inoculum (at the appropriate concentration Spores or dispersed mycelium) with holes under the sachet. About F Moniliforme 1-2 weeks after inoculation, root length and necrosis are assayed and Compare sgenic and non-transgenic controls. 6. Stewart bacterial wilt   Erwinia Stewarty is a corn plant stewart blight The causative organism. For disease assays, bacteria are grown at high density in nutrient media and 10⁷Dilute to bacteria / ml. Corn by stinging leaves with a needle soaked in a bacterial suspension Infects the leaves of sorghum plants with bacteria. Lesions are measured at 1, 2 and 3 weeks after inoculation You. 7. Leaf disk assay   The whole plant assay described above can also be performed on leaf discs placed in a wet petri dish. it can. b. Testing of transgenic wheat plants 1. Pseudomonas disease assay   Pseudomonas Silingae Phi Vui Silingae Fun Hall is a small It is the causative agent of bacterial bacterial blight of wheat. Disease assays for scoring visible disease symptoms For growth of bacteria (strain BL882) on L-agar plates overnight. A small band of cells Sharp off the plate with a toothpick, 10 mM MgCl_TwoResuspend in the above solution. 6 Take the absorbance of the suspension at 00 nm and determine the bacterial concentration in the suspension, which is 0.1 1 × 10 per OD600⁸Calculated as cfu / ml. Culture 1 × 10⁷Bacteria / m Dilute to 1 l and aspirate a small amount into a 1 ml plastic Pasteur pipette. Approximately 50 μl of bacteria are injected into the leaves of 3-week old wheat plants. Change the size of the inoculation area to black Lightly mark with a felt-tip pen. Using a common garden sprayer to plant the internal environment Put into a plastic box in a humid environment by spraying a large amount of water. box And placed in a 18-20 ° C percival chamber for 16 hours per day, Shine light. Five days later, the severity of the disease is surrounded by a thin layer of whitening that develops in the inoculation zone It is scored by calculating the percentage of the lesion area apparent by the black necrotic spots.   For a more precise quantification of the extent of disease onset, 1 × 10^FiveFor bacteria at cfu / ml concentration By soaking, BL882 containing the kanamycin resistance gene plasmid was uniformly applied to the leaves. Inoculate. On the day of immersion and the following days, the bacterial titer of the injection area is measured. this is Align the leaf holes in the immersion area, which collectively covers an area of 1 cm, M MgCl_TwoLB plate with 50 microgram / ml kanamycin This is achieved by spreading the extract on a plate (this BL882 isolate is (Transformed with a syn-resistance gene). After three days of growth, a leaf-derived bacterium Forms a knee, which is easy to count. In this method, the transformed plant Growth rate can be quantified and compared with control and non-transformed plants. 2. Septoria Nodrum Assay   S. nodrum is a pathogen of wheat leaf stains and wheat ear glue spots. Disease Spores are harvested from sporulation plates or liquid cultures for assay and 2 week old plants Spray the leaves at the appropriate concentration. Put the inoculated plants in a high humidity fog chamber for 2-3 days, Subsequently, transfer to a growth chamber. The percentage of diseased leaf area was measured over 3 weeks, Record the presence or absence of the offspring. 3. Septoria Tritici Assay   S. trictis is the causative agent of wheat leaf spots. Vesicles for disease assays The spores are recovered from the sporulation plate or liquid culture and the 1-2 week old wheat plants Spray leaves at appropriate concentration. Put the inoculated plants in a high humidity fog chamber for 3 days, then Transfer to growth chamber. The percentage of diseased leaf area was measured 2-3 weeks after inoculation, Record the presence or absence of the vessel. 4. Leaf disk assay   The whole plant assay described above can also be performed on leaf discs placed in a wet petri dish. it can. 5. Fusarium sachet assay   This is due to F Moniliforme and P. Similar to the lumat assay. Spores from F. Kurmoram Collect from the plate as described for Fold the perforated filter paper and remove Put in a bag, surface-sterilized wheat seeds, rooting roots grow through holes, at the bottom of the sachet Crease as shown. Hewitt's medium (for germinating and growing wheat seeds) 12 ml of a spore suspension of F. cromolam in a nutrient medium for The bags are incubated in a growth chamber at 10-15 ° C for about one week, Continue the incubation at 15-18 ° C. After 4 days, the root length and root browning are scored. 6. Powdery mildew leaf assay   Elishefe Graminis FSP Tritici The causative organism. This is an obligate biotrophy, thus maintaining the inoculum with wheat leaves You. Plants for disease testing were grown at 12-14 years of age and 2.5 cm pieces were cut from leaves. , 50 mg / ml benzimidazole on agar plates. Recovered from infected leaves The spores are used at appropriate concentrations for inoculation of leaf sections on agar plates. Next, play The plate is incubated at 17 ° C. under low light conditions. About 1 inoculation of the disease symptoms of leaf sections Evaluate after 0 days. Example 11: Human lactoferricin (Lacto H)   Human lactofin, also called antibacterial and antifungal peptide, also referred to as lactoferricin H Isolated from Elysin. This peptide corresponds to amino acids 1-33 of the mature protein. Respond. The synthetic gene has codons optimized for expression in maize (SEQ ID NO: Produced to encode lactoferricin H peptide (SEQ ID NO: 2) containing 5) Do:   To allow transcription and translation of this gene sequence, a methionine start codon and And a stop codon. In addition, the ACC sequence derived from the Kozak consensus sequence Added at the beginning of the sequence to improve the possibility of efficient translation. This is as shown below Produce a unique Met-lactoferricin H gene sequence, with start and stop sequences underlined. K:   This gene sequence is similar to that described in the previous example for lactoferricin B Is expressed in plant cells under the control of a suitable promoter.

[Procedure of Amendment] Article 184-8, Paragraph 1 of the Patent Act [Submission Date] July 21, 1998 (7.27.21) [Correction contents]   11. The seed of claim 10, which is packaged or coated.   12. 12. The method according to claim 7, wherein the lactoferricin is lactoferricin B. A plant or seed according to any of the preceding claims.   13. Use of lactoferricin to control fungal plant pathogens.   14． A control of a plant pathogen comprising exposing the pathogen to a plant according to claim 7. Law.   15. A transgenic plant according to claim 7, or a progeny thereof, comprising a plant pathogen. Agricultural method used to control.   16. A commercial package comprising the seed of claim 10.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AM, AT , AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, F I, GB, GE, GH, HU, IL, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, M X, NO, NZ, PL, PT, RO, RU, SD, SE , SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Chandler, Daniel Blost             United States 27607 North Carolina             Raleigh, Wake County Diki             Sea Trail 1213 (72) Inventor Kramer, Catherine May             United States 27278 North Carolina             Hillsborough, Orange County, Da             Na Court No. 608

Claims (1)

[Claims]   1. As a functional array: (a) a promoter that functions in a plant cell to effect the production of an RNA sequence; (b) a structural coding sequence encoding the production of lactoferricin; and (c) attachment of polyadenylation nucleotides to the 3 'end of the RNA sequence in plant cells A 3 'untranslated region that functions to effect addition; A recombinant DNA molecule comprising:   2. The promoter is a ubiquitin promoter and the 3 'untranslated region is Nopaline synthase transcription terminator from Lactobacillus tumefaciens 2. The DNA molecule of claim 1, which is a promoter.   3. 3. The method according to claim 1, wherein the lactoferricin is lactoferricin B. DNA molecule.   4. (a) inserting the recombinant DNA molecule of claim 1 into the genome of a plant cell; (b) obtaining transformed cells; and (c) transforming plant cells in an amount sufficient to reduce damage from pathogen infection; Regenerate a genetically transformed plant expressing lactoferricin: A method for producing a transgenic, disease-resistant plant, comprising the steps of:   5. The plant is selected from the group consisting of maize, wheat and sugar beet 5. The method of claim 4, wherein   6. The lactoferricin is lactoferricin B, according to claim 4 or 5. the method of.   7. A plant containing cells expressing lactoferricin.   8. Having the recombinant DNA of claim 1 stably integrated in its genome. The plant according to claim 7.   9. Selected from the group consisting of maize, wheat and sugar beet The plant according to claim 7 or 8.   10. 10. The plant according to claim 7, 8 or 9 when germinated and cultivated. Seeds that produce things.   11. The seed of claim 10, which is packaged or coated.   12. 12. The method according to claim 7, wherein the lactoferricin is lactoferricin B. A plant or seed according to any of the preceding claims.   13. Use of lactoferricin to control plant pathogens.   14． A control of a plant pathogen comprising exposing the pathogen to a plant according to claim 7. Law.   15. A transgenic plant according to claim 7, or a progeny thereof, comprising a plant pathogen. Agricultural method used to control.   16. A commercial package comprising the seed of claim 10.