| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 05.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 30.05.00 |
| PATENT TITLE |
Fungus and insect control with chitinolytic enzymes |
| PATENT ABSTRACT |
The present invention relates to chitinolytic enzymes which have chitinolytic activity under alkaline conditions as well as DNA molecules encoding these enzymes and expression systems, host cells, and transgenic plants and plant seeds transformed with such DNA molecules. A chitinolytic enzyme can be applied to a plant or plant seed under conditions effective to control insects and/or fungi on the plant or plants produced from the plant seed. Alternatively, transgenic plants or transgenic plant seeds transformed with a DNA molecule encoding a chitinolytic enzyme can be provided and the transgenic plants or plants resulting from the transgenic plant seeds are grown under conditions effective to control insects and/or fungi. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 18.02.98 |
| PATENT REFERENCES CITED |
Lorito, M. Chitinolytic Enzymes and Their Genes, In Trichoderma and Gliocladium, vol. II, Haren and Kubicek, Eds., pp. 73-75, 1998. Lorito et al, MPMI, vol. 9, pp. 206-213, 1996. Neuhaus et al, Plant Mol. Biol., vol. 16, pp. 141-151, 1991. Kramer et al, In Transgenic Plants for the Control of Insect Pests, N. Carrozzi & M. Koziel, eds., Washington, D.C., pp. 185-193, 1996. Wang et al, Insect Biochem. Molec. Biol., vol. 26., pp. 1055-1064, 1996. Zhu et al, J. Biochem., vol. 112, pp.163-167, 1992. Monreal et al., "The Chitinase of Serratia marcescens," Canadian Journal of Microbiology, 15:689-696 (1969). Regev et al., "Synergistic Activity of a Bacillus thuringiensis .delta.-Endotoxin and a Bacterial Endochitinase Against Spodoptera littoralis Larvae," Applied and Environmental Microbiology, 62(10):3581-3586. Park et al., "Production and Some Properties of Chitinolytic Enzymes by Antagonistic Bacteria," Korean J. Plant Pathol. 11(3):258-264 (1995). Tsujibo et al., "Cloning and Sequence Analysis of the Gene Encoding a Thermostable Chitinase from Streptomyces thermoviolaceus OPC-520," Gene 134:113-117 (1993). Pleban et al., "Chitinolytic Activity of an Endophytic Strain of Bacillus cereus, " Letters in Applied Microbiology 25(4):284-288 (1997). Bolar et al., "Endochitinase--Transgenic McIntosh Apple Lines Have Increased Resistance to Scab," Phytopathology 87(6):S10 (1997) (abstract). Broadway et al., "Novel Chitinolytic Enzymes with Biological Activity Against Herbivorous Insects," J. Chem. Ecol. 24(6):985-998 (1998). Wong et al., "Chitinase-Transgenic Lines of `Royal Gala` Apple Showing Enhanced Resistance to Apple Scab," ACTA Hortic, 484:595-599 (1996). Xue, B., et al., "Development of Transgenic Tomato Expressing a High Level of Resistance to Cucumber Mosaic Virus Strains of Subgroups I and II," Plant Disease 78(11):1038-41 (1994). Pang, S., et al., "Different Mechanisms Protect Transgenic Tobacco Against Tomato Spotted Wilt and Impatiens Necrotic Spot Tospoviruses, " Bio/Technology 11:819-24 (1993). Broadway, R.M., et al., "Partial Characterization of Chitinolytic Enzymes from Streptomyces albidoflavus," Applied Microbiology 20:271-76 (1995). |
| PATENT GOVERNMENT INTERESTS | The present invention was developed with support under USDA/NRI Grant No. 95-37302-1904. The U.S. Government may have certain rights. |
| PATENT CLAIMS |
What is claimed: 1. An isolated DNA molecule encoding a Streptomyces albidoflavus chitinolytic enzyme having chitinolytic activity under alkaline conditions. 2. An isolated DNA molecule according to claim 1, wherein the chitinolytic enzyme is selected from the group consisting of chitobiosidase and endochitinase. 3. An isolated DNA molecule according to claim 2, wherein the chitinolytic enzyme is chitobiosidase. 4. An isolated DNA molecule according to claim 3, wherein the chitobiosidase has a molecular mass of 34 kD and an isoelectric point of less than 3.0. 5. An isolated DNA molecule according to claim 3, wherein the chitobiosidase has an amino acid sequence comprising SEQ. ID. No. 1. 6. An isolated DNA molecule according to claim 2, wherein the chitinolytic enzyme is an endochitinase. 7. An isolated DNA molecule according to claim 6, wherein the endochitinase has a molecular mass of 45 kD and an isoelectric point of about 6.5. 8. An isolated DNA molecule according to claim 6, wherein the endochitinase has an amino acid sequence comprising SEQ. ID. No. 3. 9. An expression system transformed with the DNA molecule according to claim 1. 10. An expression system according to claim 9, wherein the chitinolytic enzyme is selected from the group consisting of chitobiosidase, endochitinase, and combinations thereof. 11. An expression system according to claim 9, wherein the DNA molecule is in proper sense orientation and correct reading frame. 12. A host cell transformed with the DNA molecule according to claim 1. 13. A host cell according to claim 12, wherein the chitinolytic enzyme is selected from the group consisting of chitobiosidase, endochitinase, and combinations thereof. 14. A host cell according to claim 12, wherein the host cell is a bacterial cell. 15. A host cell according to claim 12, wherein the host cell is a plant cell. 16. A host cell according to claim 12, wherein the host cell contains an expression system transformed with the DNA molecule. 17. A transgenic plant transformed with the DNA molecule according to claim 1. 18. A transgenic plant according to claim 17, wherein the chitinolytic enzyme is selected from the group consisting of chitobiosidase, endochitinase, and combinations thereof. 19. A transgenic plant according to claim 18, wherein the chitinolytic enzyme is chitobiosidase. 20. A transgenic plant according to claim 18, wherein the chitinolytic enzyme is an endochitinase. 21. A transgenic plant according to claim 17, wherein the plant is selected from the group consisting of Gramineae, Liliaceae, Iridaceae, Orchidaceae, Salicaceae, Ranunculaceae, Magnoliaceae, Cruciferae, Rosaceae, Leguminosae. Malvaceae, Umbelliferae, Labitatae, Solanaceae, Cucurbitaceae, Compositae, and Rubiaceae. 22. A transgenic plant seed transformed with the DNA molecule according to claim 1. 23. A transgenic plant seed according to claim 22, wherein the chitinolytic enzyme is selected from the group consisting of chitobiosidase, endochitinase, and combinations thereof. 24. A transgenic plant seed according to claim 22, wherein the plant seed is selected from the group consisting of Gramineae, Liliaceae, Iridaceae, Orchidaceae, Salicaceae, Ranunculaceae, Magnoliaceae, Cruciferae, Rosaceae, Leguminosae, Malvaceae, Umbelliferae, Labitatae, Solanaceae, Cucurbitaceae, Compositae, and Rubiaceae. 25. A method of insect control for plants comprising: providing a transgenic plant or plant seeds transformed with a DNA molecule according to claim 1 and growing the transgenic plant or transgenic plants produced from the transgenic plant seeds under conditions effective to control insects. 26. A method according to claim 25, wherein a transgenic plant is provided. 27. A method according to claim 25, wherein a transgenic plant seed is provided. 28. An isolated DNA molecule encoding a chitinolytic enzyme having chitinolytic activity under alkaline conditions, wherein said DNA molecule has a nucleotide sequence of SEQ. ID. Nos. 2 or 4 or a nucleotide sequence which hybridizes to the nucleotide sequence of SEQ. ID. Nos. 2 or 4 under stringent conditions. 29. An isolated DNA molecule according to claim 28, wherein the DNA molecule has a nucleotide sequence which hybridizes to the nucleotide sequence of SEQ. ID. Nos. 2 or 4 under stringent conditions. 30. An isolated DNA molecule according to claim 29, wherein the stringent conditions are 65.degree. C. for 20 hours in a medium containing 1M NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodium dodecyl sulfate, 0.2% ficoll 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, 50 .mu.m g/ml E. coli DNA. 31. An expression system transformed with the DNA molecule according to claim 28. 32. A host cell transformed with the DNA molecule according to claim 28. 33. A host cell according to claim 32, wherein the host cell is a bacterial cell. 34. A host cell according to claim 32, wherein the host cell is a plant cell. 35. A host cell according to claim 32, wherein the host cell contains an expression system transformed with the DNA molecule. 36. A transgenic plant transformed with the DNA molecule according to claim 28. 37. A transgenic plant according to claim 35, wherein the plant is selected from the group consisting of Gramineae, Liliaceae, Iridaceae, Orchidaceae, Salicaceae, Ranunculaceae, Magnoliaceae, Cruciferae, Rosaceae, Leguminosae, Malvaceae, Umbelliferae, Labitatae, Solanaceae, Cucurbitaceae, Compositae, and Rubiaceae. 38. A transgenic plant seed transformed with the DNA molecule according to claim 28. 39. A transgenic plant seed according to claim 38, wherein the plant seed is selected from the group consisting of Gramineae, Liliaceae, Iridaceae, Orchidaceae, Salicaceae, Ranunculaceae, Magnoliaceae, Cruciferae, Rosaceae, Leguminosae, Malvaceae. Umbelliferae, Labitatae, Solanaceae, Cucurbitaceae, Compositae, and Rubiaceae. 40. A method of insect control for plants comprising: providing a transgenic plant or plant seeds transformed with a DNA molecule according to claim 28 and growing the transgenic plant or transgenic plants produced from the transgenic plant seeds under conditions effective to control insects. 41. A method according to claim 40, wherein a transgenic plant is provided. 42. A method according to claim 40, wherein a transgenic plant seed is provided. 43. A DNA molecule according to claim 1, wherein the chitinoltic enzyme has chitinolytic activity only under alkaline conditions. 44. A DNA molecule according to claim 28, wherein the chitinolytic enzyme has chitinolytic activity only under alkaline conditions. 45. An isolated DNA molecule according to claim 29, wherein the DNA molecule has a nucleotide sequence which hybridizes to the nucleotide sequence of SEQ. ID. No. 2 under stringent conditions. 46. An isolated DNA molecule according to claim 29, wherein the DNA molecule has a nucleotide sequence which hybridizes to the nucleotide sequence of SEQ. ID. No. 4 under stringent conditions. 47. An isolated DNA molecule according to claim 28, wherein the DNA molecule has a nucleotide sequence of SEQ. ID. No. 2. 48. An isolated DNA molecule according to claim 28, wherein the DNA molecule has a nucleotide sequence of SEQ. ID. No. 4. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates to chitinolytic enzymes which are active against insects under alkaline conditions. BACKGROUND OF THE INVENTION The introduction of synthetic organic pesticides following World War II brought inestimable benefits to humanity and agricultural economic profitability. Application of broad-spectrum pesticides is the primary method used for controlling fungal and insect pests. For example, the widescale deployment of DDT resulted in the complete riddance, from entire countries, of serious public pests such as malaria mosquitoes. However, there were warnings about the hazard of unilateral approaches to pest control. The development of new pesticides and the increasing amounts of pesticides used for pest control are closely correlated with the development of pest resistance to chemicals. The number of pesticide resistant species has greatly increased since the adoption of DDT in 1948. As a result, by the 1980s, the number of reports of pesticide resistance for arthropod pests was listed as 281, for plant pathogens 67, and for weeds 17. These numbers have steadily increased to the present day. Thus, the need for biological control agents, especially those with broadbase activity is especially important. One approach that is gaining significant attention is the use of agricultural cultivars that are resistant to pests. These cultivars can be developed by the transgenic introduction of target specific natural resistance factors. However, to enhance host-plant resistance, it is necessary first to identify and to characterize target-specific factors that will significantly reduce the population(s) of herbivorous insect(s). Only a limited number of natural products have been characterized and identified as effective defensive agents against herbivorous insects, few of these are proteins (e.g., proteinase inhibitors, arcelin, alpha-amylase inhibitors, lectins, endotoxin from Bacillus thuringiensis, and lipoxygenases), and even fewer are target specific (Duffey, et al., "Plant Enzymes in Resistance to Insects," In J. R. Whitaker and P. E. Sonnet (eds.), Biocatalysis in Agricultural Biotechnology, American Chemical Society, Washington, D.C. (1989); Gill, et al., "The Mode of Action of Bacillus thuringiensis Endotoxins," Ann. Rev. Entomol., 37:615-36 (1992); Hedin, P. A., "Plant Resistance to Insects," American Chemical Society, Washington, D.C., p. 375 (1983); Rosenthal, et al., "Herbivores--Their Interaction with Secondary Plant Metabolites," Academic Press, New York, p. 718 (1979). Identification and characterization of proteins as resistance factor(s) enables the isolation of gene(s) that encode(s) these proteins. These genes can be transgenically inserted into agricultural crops, which may enhance the resistance of these crops against herbivorous insects without altering desirable characteristics of the cultivar(s) (Fraley, et al., "Genetic Improvements of Agriculturally Important Crops," Cold Spring Harbor Laboratory, p. 120 (1988); Hilder, et al., "A Novel Mechanism of Insect Resistance Engineered into Tobacco," Nature, 330:160-63 (1987); Ryan, C. A., "Proteinase Inhibitor Gene Families: Strategies for Transformation to Improve Plant Defenses Against Herbivores," BioEssays, 10:20-24 (1989); Vaeck, et al., "Transgenic Plants Protected from Insect Attack," Nature, 328:33-27 (1987). One target that has been selected is a structural polymer, chitin, which is present in insects and some fungi that attack plants, but is absent in higher plants and vertebrates. U.S. Pat. No. 4,751,081 follows this approach and is directed to novel chitinase producing bacteria strains for use in inhibiting chitinase sensitive plant pathogens (i.e. fungi and nematodes). However, the approach of U.S. Pat. No. 4,751,081 lacks flexibility. The present invention is directed to controlling fungi and insects that attack plants. SUMMARY OF THE INVENTION The present invention relates to isolated DNA molecules encoding chitinolytic enzymes, which have chitinolytic activity under alkaline conditions, as well as to vectors, host cells, and transgenic plants and plant seeds transformed with these DNA molecules. Another aspect of the present invention relates to a method of insect and/or fungus control for plants. This method involves applying the chitinolytic enzymes to plants or plant seeds under conditions effective to control insects and/or fungi on the plants or plants grown from the plant seeds. As an alternative to applying the chitinolytic enzymes to plants or plant seeds in order to control insects and/or fungi on plants or plants grown from the seeds, transgenic plants or plant seeds can be utilized. When utilizing transgenic plants, this involves providing a transgenic plant transformed with a DNA molecule encoding a chitinolytic enzyme and growing the plant under conditions effective to control insects and/or fungi. Alternatively, a transgenic plant seed transformed with a DNA molecule encoding the chitinolytic enzyme can be provided and planted in soil. A plant is then propagated from the planted seed under conditions effective to control insects and/or fungi. The present invention is directed to effecting any form of insect and/or fungus control for plants. For example, insect control or fungus control according to the present invention encompasses preventing insects or fungi from contacting plants to which the chitinolytic enzymes have been applied, preventing direct damage to plants by feeding injury, causing insects or fungi to depart from such plants, killing insects or fungi proximate to such plants, interfering with feeding on plants by insects or fungi, preventing insects or fungi from colonizing host plants, preventing colonizing insects or fungi from releasing phytotoxins, etc. The present invention also prevents subsequent disease damage to plants resulting from insect or fungal invasion. As a result, the present invention provides significant economic benefit to growers. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A to C show a time course of chitinolytic enzyme activity in culture filtrate from Streptomyces albidoflavus NRRL B-16746 and NRRL 21918. The liquid medium contained 0.5% chitin, while the glucose component was 0% (solid square), 0.01% (open triangle), 0.1% (open circle), 0.25% (solid triangle), and 0.5% (open square). FIG. 1A indicates endochitinase activity (units/ml), FIG. 1B indicates chitobiosidase activity (% nkatals), and FIG. 1C indicates glucosaminidase activity (% nkatals) at pH 9. These are representative data; the experiment was replicated twice with similar results. FIGS. 2A-B show the polyacrylamide gel electrophoresis of dialyzed culture filtrate from Streptomyces albidoflavus NRRL B-16746. Lanes 1-8 contain samples collected after 0, 24, 48, 72, 96, 120, 144, 168, or 192 hrs. of culture, respectively. FIG. 2A shows native gel with Coomassie stain, while FIG. 2B shows native gel with overlay of fluorogenic substrate for endochitinases and chitobiosidases. C indicates the chitobiosidase band, G indicates the glucosaminidase band, and E indicates the endochitinase bands. These are representative data; the experiment was replicated 2 times with similar results. FIG. 3 shows the influence of pH on chitinolytic activity in the culture medium from Streptomyces albidoflavus NRRL B-16746. Glucosaminidase activity is indicated by the open circles, chitobiosidase activity is indicated by open triangles, and endochitinase activity is indicated by the solid squares. FIG. 4 shows the low pressure anion exchange chromatography of chitinolytic enzymes from Streptomyces albidoflavus NRRL B-16746. The solid line indicates total protein (optical density, 280 nm), the dashed line indicates chitobiosidase activity (% nkatals), and the dotted line indicates endochitinase activity (units/ml). These are representative data; the analysis was replicated more than 10 times. FIGS. 5A-B show the polyacrylamide gel electrophoresis of chitinolytic enzymes from anion exchange chromatography. Lanes 1 and 2 contain endochitinases (peaks III and IV, respectively, from FIG. 8), and lane 3 contains chitobiosidases (peak II from FIG. 4). The gels were stained with Coomassie (FIG. 5A), or a chitinase-specific fluorogenic substrate (FIG. 5B). These are representative data; the determination of the number of chitinolytic enzymes was replicated more than 10 times. FIGS. 6A-B show the isoelectric focusing gel of chitinolytic enzymes from anion exchange chromatography. The gels were stained with Coomassie (FIG. 6A), or a chitinase-specific fluorogenic substrate (FIG. 6B). For FIG. 6A, lane 1 contains endochitinases (peaks III from FIG. 8), lane 2 contains isoelectric focusing markers, lane 3 contains endochitinases (peaks IV from FIG. 8), and lane 4 contains chitobiosidases (peak II from FIG. 4). For FIG. 6B, lane 1 contains endochitinases (peaks IV from FIG. 8), lane 2 contains endochitinases (peaks III from FIG. 8), and lane 3 contains chitobiosidases (peak II from FIG. 4). The isoelectric points of the chitobiosidases were <3.0, while the isoelectric points of the endochitinases were 6.4, 5.8-5.9, 5.7, 5.3, 5.1, and 5.0. These are representative data; the isoelectric point determinations were replicated more than 10 times. FIG. 7 shows the SDS-polyacrylamide gel electrophoresis of chitinolytic enzymes from anion exchange chromatography. Lane 1 contains endochitinases (peak III from FIG. 8), lane 2 contains molecular weight markers, lane 3 contains endochitinases (peak IV from FIG. 8), lane 4 contains chitobiosidases (peak II, FIG. 4). The gel was stained with Coomassie. The proteins with chitobiosidase activity have molecular mass of 59, 45, 38.5, 27, and 25.5 kD. These are representative data; the molecular weight determinations were replicated more than 10 times. FIG. 8 shows the perfusion anion exchange chromatography of endochitinases (FIG. 4, peak I) from Streptomyces albidoflavus NRRL B-16746. The solid line indicates total protein (optical density, 280 nm), the dashed line indicates sodium chloride gradient. Endochitinase activity occurred in peaks III and IV. These are representative data; the chromatographic separation was replicated 6 times. FIG. 9 shows the strong anion exchange perfusion chromatography of semipurified chitinolytic enzymes. Total chitinolytic activity, as measured by hydrolysis of p-nitrophenyl .beta.-D-N,N'-diacetylchitobiose, was 0.14 nkat/peak I, 1.52 nkat/peak II, 0.26 nkat/peak III, <0.01 nkat/peak IV, 2.45 nkat/peak V, and 0.09 nkat/peak VI. FIGS. 10A-B show polyacrylamide gel electrophoresis of the peaks collected from anion exchange perfusion chromatography (FIG. 9). FIG. 10A shows Coomassie stain to detect all proteins. FIG. 10B shows fluorogenic overlay to locate enzymes with chitinolytic activity. Based on previous experiments (Broadway, et al., "Partial Characterization of Chitinolytic Enzymes from Streptomyces albidoflavus," Lett. Appl. Microbiol., 20:271-76 (1995), which is hereby incorporated by reference), the upper (most acidic) two bands were chitobiosidases, while the lower (most alkaline) five bands were endochitinases. FIG. 11 shows the development of Trichoplusia ni larvae on artificial diet supplemented with semipurified chitinolytic enzymes. Solid bars indicate larval weight as a percent of the mean weight of untreated controls. Stippled bars indicate the percent larvae that pupated. Hatched bars indicate the percent pupae that molted to adults. Vertical lines indicate .+-.1 SEM. Columns associated with an insect growth stage having similar letters are not significantly different (LSD test). FIG. 12 shows the effect of dietary chitinolytic enzymes on survival of Myzus persicae, Bemisia argentifolii, and Hypothenemus hampei. For B. argentifolii, solid bars indicate % mortality after 18 h exposure, stippled bars indicate % mortality after 42 h exposure. For M. persicae, solid bars indicate % mortality after 24 h exposure, while stippled bars indicate % mortality after 48 h exposure. For H. hampei, solid bars indicate % mortality after 30d exposure. Vertical lines indicate .+-.1 SEM. Columns associated with a single time of observation and having similar letters are not significantly different (LSD test). FIG. 13 shows the effect of dietary endochitinases or chitobiosidases on survival of Bemisia argentifolii. Endochitinase treatments I and II contained 10% sucrose and 0.5% peak I or II, respectively, from the anion exchange perfusion chromatography (FIG. 9), while chitobiosidase treatments IV and V contained 10% sucrose and 0.5% peak IV or V, respectively, from the anion exchange column (FIG. 9). Two controls were included: treatment VI contained 10% sucrose and 0.5% peak VI (no chitinolytic activity), and treatment C, which contained 10% sucrose. Vertical lines indicate .+-.1 SEM. Columns with similar letters are not significantly different (LSD test). FIG. 14 shows the biological activity of chitobiosidases against Botrytis cinerea and Fusarium oxysporum. Square indicates % inhibition of spore germination, triangle indicates % inhibition of germ tube elongation, dashed line indicates Botrytis cinerea, and solid line indicates Fusarium oxysporum. DETAILED DESCRIPTION OF THE INVENTION Chitin, an insoluble linear .beta.-1,4-linked polymer of N-acetyl-.beta.-D-glucosamine, is a structural polysaccharide that is present in all arthropods, yeast, most fungi, and some stages of nematodes. Chitinolytic enzymes are proteins that catalyze the hydrolysis of chitin by cleaving the bond between the C1 and C4 of two consecutive N-acetylglucosamines. There are three types of chitinolytic enzyme activity: (1) N-acetyl-.beta.-glucosaminidase (i.e., EC 3.2.1.30, abbreviated glucosaminidase), which cleaves monomeric units from the terminal end of chitin, (2) 1,4-.beta.-chitobiosidase (i.e., abbreviated chitobiosidase), which cleaves dimeric units from the terminal end of chitin, and (3) endochitinase (EC 3.2.1.14), which randomly cleaves the chitin molecule internally (Sahai, et al., "Chitinases of Fungi and Plants: Their Involvement in Morphogenesis and Host-Parasite Interaction," FEMS Microbiol. Rev., 11:317-38 (1993), which is hereby incorporated by reference). Two or three types of enzymes are often synthesized by a single organism (Harman, et al., "Chitinolytic Enzymes of Trichoderma harzianum: Purification of Chitobiase and Endochitinase," Phytopathology, 83:313-18 (1993), Neugebauer, et al., "Chitinolytic Properties of Streptomyces lividans," Arch. Microbiol, 156:192-97 (1991), Romaguera, et al., "Protoplast Formation by a Mycolase from Streptomyces olivaceoviridis and Purification of Chitinases," Enzyme Microb. Technol., 15:412-17 (1993), which are hereby incorporated by reference), which may enhance the speed and/or efficiency of degradation of chitin. The chitinolytic enzymes of the present invention are particularly effective in controlling insects, because they are active under alkaline conditions. As a result, these enzymes can be ingested by insects and then attack the insects by degrading their chitin-containing, alkaline digestive tracts. The present invention relates to chitinolytic enzymes which are active under alkaline conditions (i.e. at a pH greater than 7) alone but may also be active under neutral and/or acid conditions. The present invention also encompasses the DNA molecules encoding these enzymes. Examples of such chitinolytic enzymes are the following enzymes isolated from Streptomyces albidoflavus which have either chitobiosidase or endochitinase activity. The chitobiosidase isolated from Streptomyces albidoflavus has an amino acid sequence of SEQ. ID. No. 1 as follows: Ala Pro Ala Ala Val Pro Ala His Ala Val Thr Gly Tyr Trp Gln Asn 1 5 10 15 - Phe Asn Asn Gly Ala Thr Val Gln Thr Leu Ala Asp Val Pro Asp Ala 20 25 30 - Tyr Asp Ile Ile Ala Val Ser Phe Ala Asp Ala Thr Ala Asn Ala Gly 35 40 45 - Glu Ile Thr Phe Thr Leu Asp Ser Val Gly Leu Gly Gly Tyr Thr Asp 50 55 60 - Glu Gln Phe Arg Ala Asp Leu Ala Ala Lys Gln Ala Asp Gly Lys Ser 65 70 75 80 - Val Ile Ile Ser Val Gly Gly Glu Lys Gly Ala Val Ala Val Asn Asp 85 90 95 - Ser Ala Ser Ala Gln Arg Phe Ala Asp Ser Thr Tyr Ala Leu Met Glu 100 105 110 - Glu Tyr Gly Phe Asp Gly Val Asp Ile Asp Leu Glu Asn Gly Leu Asn 115 120 125 - Ser Thr Tyr Met Thr Glu Ala Leu Thr Lys Leu His Glu Lys Ala Gly 130 135 140 - Asp Gly Leu Val Leu Thr Met Ala Pro Gln Thr Ile Asp Met Gln Ser 145 150 155 160 - Pro Glu Asn Glu Tyr Phe Lys Thr Ala Leu Val Thr Lys Asp Phe Leu 165 170 175 - Thr Ala Val Asn Met Gln Tyr Tyr Asn Ser Gly Ser Met Leu Gly Cys 180 185 190 - Asp Gly Gln Val Tyr Ala Gln Gly Thr Val Asp Phe Leu Thr Ala Leu 195 200 205 - Ala Cys Ile Gln Leu Glu Asn Gly Leu Asp Ala Ser Gln Val Gly Ile 210 215 220 - Gly Val Pro Ala Ser Pro Lys Ala Ala Gly Gly Gly Tyr Val Glu Pro 225 230 235 240 - Ser Val Val Asn Asp Ala Leu Asp Cys Leu Thr Arg Gly Thr Gly Cys 245 250 255 - Gly Ser Phe Lys Pro Glu Lys Thr Tyr Pro Ala Leu Arg Gly Ala Met 260 265 270 - Thr Trp Ser Thr Asn Trp Asp Ala Asp Thr Gly Asn Ala Trp Ser Asn 275 280 285 - Val Val Gly Pro His Val Asp Asp Leu Pro 290 295 The chitobiosidase has a molecular mass of 34 kD and an isoelectric point of less than 3.0. The chitobiosidase isolated from Streptomyces albidoflavus having an amino acid sequence of SEQ. ID. No. 1 is encoded by a DNA molecule having a nucleotide sequence of SEQ. ID. No. 2 as follows: GCGGCCGCTC CGGGCGGACG ACCGTACGGA CTCCTCGGCC GACCCCTGCG GGAACCCTTG 60 - ACAACCCCAT TGGTCTGGAC CAGTTTGGTG CCCATCGCGG TGGCCACCG T GCGCCAACTC 120 - CCCGCCCCCT CCCGGGTGGC GGGCCCCGTC GGCGCGTCCC CCCACGTCCG TGACTCCCCC 180 - CACCGGAGGC AGCAGTGGTA CGCACCTACC CCCTTCCGCA CCCCGGCCGG CGCCCCTCCA 240 - CGCCCGGCCT CCACCGCAGG GGCCGGCTGA CCGCCGCCCT CACCGCGGCC GTCCTCGGCG 300 - CCTCCGGGCT CGCCCTCACC GGCCCCGCGA CCGCCGGCGA GGGGGCCCCC GCCGCCCAGG 360 - CCGCCCCGGC CGCCGTACCG GCCCACGCGG TGACCGGTTA CTGGCAGAAC TTCAACAACG 420 - GCGCGACCGT GCAGACCCTC GCCGACGTGC CGGACGCCTA CGACATCATC GCCGTCTCCT 480 - TCGCCGACGC CACGGCCAAC GCGGGCGAGA TCACCTTCAC CCTCGACTCG GTCGGGCTCG 540 - GCGGCTACAC CGACGAGCAG TTCCGCGCCG ACCTCGCCGC CAAGCAGGCC GACGGCAAGT 600 - CGGTGATCAT CTCGGTCGGC GGCGAGAAGG GCGCGGTCGC CGTCAACGAC AGCGCCTCCG 660 - CCCAGCGCTT CGCCGACAGC ACCTACGCGC TGATGGAGGA GTACGGCTTC GACGGCGTCG 720 - ACATCGACCT GGAGAACGGC CTCAACTCCA CCTACATGAC CGAGGCCCTC ACCAAGCTCC 780 - ACGAGAAGGC CGGGGACGGC CTGGTCCTCA CCATGGCGCC GCAGACCATC GACATGCAGT 840 - CGCCCGAGAA CGAGTACTTC AAGACGGCGC TGGTCACGAA AGACTTCCTG ACCGCCGTCA 900 - ACATGCAGTA CTACAACAGC GGCTCGATGC TCGGCTGCGA CGGCCAGGTC TACGCGCAGG 960 - GCACCGTCGA CTTCCTCACC GCGCTCGCCT GCATCCAGCT GGAGAACGGT CTCGACGCCT1020 - CCCAGGTCGG CATCGGTGTC CCCGCCTCCC CGAAGGCGGC CGGCGGCGGC TACGTCGAGC1080 - CCTCCGTGGT CAACGACGCG CTGGACTGCC TGACCCGGGG CACCGGTTGT GGCTCGTTCA1140 - AGCCGGAGAA GACCTACCCG GCGCTGCGTG GCGCCATGAC CTGGTCGACC AACTGGGACG1200 - CCGACACCGG CAACGCCTGG TCGAACGTGG TCGGCCCGCA CGTCGACGAC CTGCCGTAAC1260 - CCCGGAGCCG GGCACCCGTC CGCTTCCCCC GCAC1294 The endochitinase isolated from Streptomyces albidoflavus has an amino acid sequence of SEQ. ID. No. 3 as follows: Gly Pro Gly Pro Gly Pro Arg Glu Lys Ile Asn Leu Gly Tyr Phe Thr 1 5 10 15 - Glu Trp Gly Val Tyr Gly Arg Asn Tyr His Val Lys Asn Leu Val Thr 20 25 30 - Ser Gly Ser Ala Glu Lys Ile Thr His Ile Asn Tyr Ser Phe Gly Asn 35 40 45 - Val Gln Gly Gly Lys Cys Thr Ile Gly Asp Ser Phe Ala Ala Tyr Asp 50 55 60 - Lys Ala Tyr Thr Ala Ala Glu Ser Val Asp Gly Val Ala Asp Thr Trp 65 70 75 80 - Asp Gln Pro Leu Arg Gly Asn Phe Asn Gln Leu Arg Lys Leu Lys Ala 85 90 95 - Lys Tyr Pro His Ile Lys Val Leu Trp Ser Phe Gly Gly Trp Thr Trp 100 105 110 - Ser Gly Gly Phe Thr Asp Ala Val Lys Asn Pro Ala Ala Phe Ala Lys 115 120 125 - Ser Cys His Asp Leu Val Glu Asp Pro Arg Trp Ala Asp Val Phe Asp 130 135 140 - Gly Ile Asp Leu Asp Trp Glu Tyr Pro Asn Ala Cys Gly Leu Ser Cys 145 150 155 160 - Asp Ser Ser Gly Pro Ala Ala Leu Lys Asn Met Val Gln Ala Met Arg 165 170 175 - Ala Gln Phe Gly Thr Asp Leu Val Thr Ala Ala Ile Thr Ala Asp Ala 180 185 190 - Ser Ser Gly Gly Lys Leu Asp Ala Ala Asp Tyr Ala Gly Ala Ala Gln 195 200 205 - Tyr Phe Asp Trp Tyr Asn Val Met Thr Tyr Asp Phe Phe Gly Ala Trp 210 215 220 - Asp Lys Thr Gly Pro Thr Ala Pro His Ser Ala Leu Asn Ser Tyr Ser 225 230 235 240 - Gly Ile Pro Lys Ala Asp Phe His Ser Ala Ala Ala Ile Ala Lys Leu 245 250 255 - Lys Ala Lys Gly Val Pro Ala Ser Lys Leu Leu Leu Gly Ile Gly Phe 260 265 270 - Tyr Gly Arg Gly Trp Thr Gly Val Thr Gln Asp Ala Pro Gly Gly Thr 275 280 285 - Ala Thr Gly Pro Ala Thr Gly Thr Tyr Glu Ala Gly Ile Glu Asp Tyr 290 295 300 - Lys Val Leu Lys Asn Thr Cys Pro Ala Thr Gly Thr Val Gly Gly Thr 305 310 315 320 - Ala Tyr Ala Lys Cys Gly Ser Asn Trp Trp Ser Tyr Asp Thr Pro Ala 325 330 335 - Thr Ile Lys Thr Lys Met Thr Trp Ala Lys Asp Gln Gly Leu Gly Gly 340 345 350 - Ala Phe Phe Trp Glu Phe Ser Gly Asp Thr Ala Gly Gly Glu Leu Val 355 360 365 - Ser Ala Met Asp Ser Gly Leu Arg 370 375 The endochitinase has a molecular mass of 45 kD and an isoelectric point of about 6.5. The endochitinase isolated from Streptomyces albidoflavus having an amino acid sequence of SEQ. ID. No. 3 is encoded by a DNA molecule having a nucleotide sequence of SEQ. ID. No. 4 as follows: GTCGACTGGT ACAACGTGAT GACCTACGAC TACTTCGGCA CCTGGGCCGC CCAGGGCCCG 60 - ACGGCGCCCC ACTCGCCGCT CACCGCCTAC CCGGGCATCC AGGGCGAGC A CAACACCTCC 120 - TCGGCCACCA TCGCCAAGCT GCGGGGCAAG GGCATCCCGG CGAAGAAGCT GCTGCTGGGC 180 - ATCGGCGCCT ACGGCCGCGG CTGGACCGGC GTCACCCAGG ACGCCCCCGG CGGCACCGCC 240 - ACCGGCCCGG CCGCCGGCAC CTACGAGGCG GGCAACGAGG AGTACCGGGT GCTGGCCGAG 300 - AAGTGCCCGG CCACCGGCAC CGCCGGCGGC ACCGCGTACG CCAAGTGCGG CGACGACTGG 360 - TGGAGTTACG ACACCCCTGA GACGGTGACG GGCAAGATGG CCTGGGCGAA GAAGCAGAAG 420 - CTCGGCGGTG CCTTCCTCTG GGAGTTCGCC GGCGACGGCG CCAAGGGCGA TCTGTTCAGG 480 - GCGATGCACG AGGGGCTGCG CTGACCGGCC GGGCACTCAC CCGGAACTGA CCCTTCCCGC 540 - ACGGCCGTCC GCCGTGGCAC CGGAGCTCCG GTCGCCGCGG CGGGCGGCCG TGTCCGCATG 600 - TCGCCACCCC CGCGCACCAG GCGCGATCCG GCCGAACTTT CCTTTGGTCC AGACCTCTTG 660 - ACCTCTGGTC CAGACCTTTT CTACTCTCGC CCCACTGCGG TGGGCACATC GGTCGTCGGT 720 - GCTCACGGGC GTCGCAGGGT TCCGCCCCCA TACGTCCGGA CCTCTTGAGG AGTACGCCTT 780 - GAGTACGGTT TCCCCCAGCA CCGACGGCGC CCGCAGCCGT CCCAGACCCC TCAGCCGCTT 840 - CCGCCGGCGC GCGCTGGCCG CGCTCGTCGG CCTCGCGGTC CCCTTCGCCG GGATGGTCGG 900 - CCTCGCCGCC CCCACCCAGG CCGCCGAGGC CGCGGCCGAC CCCAGCGCCT CCTACACCAG 960 - GACGCAGGAC TGGGGCAGCG GCTTCGAGGG CAAGTGGACG GTGAAGAACA CCGGCACCGC1020 - CCCCCTCAGC GGCTGGACCC TGGAGTGGGA CTTCCCCGCC GGAACCAAGG TGACCTCGGC1080 - CTGGGACGCC GACGTCACCA ACAACGGCGA CCACTGGACC GCCAAGAACA AGAGCTGGGC1140 - GGGGAGCCTC GCCCCCGGCG CCTCGGTCAG CTTCGGCTTC AACGGCACCG GCCCCGGCAC1200 - CCCCTCGGGC TGCAAGCTCA ACGGCGCCTC CTGCGACGGC GGCAGCGTCC CCGGCGACAC1260 - CCCGCCCACC GCCCCCGGCA CCCCCACCGC CAGTGACCTC ACCAAGAACT CGGTGAAGCT1320 - CTCCTGGAAG GCGGCCACCG ACGACAAGGG CGTCAAGAAC TACGACGTCC TGCGCGACGG1380 - CGCCAAGGTC GCCACCGTCA CCGCCACCAC CTTCACCGAC CAGAACCTCG CCCCCGGCAC1440 - CGACTACTCC TACTCGGTCC AGGCCCGCGA CACCGCCGAC CAGACCGGCC CGGTCAGCGC1500 - CCCCGTCAAG GTCACCACCC CCGGCGACGG CACGGGCCCC GGCCCCGGCC CCCGCGAGAA1560 - GATCAACCTC GGCTACTTCA CCGAGTGGGG CGTCTACGGC CGCAACTACC ACGTCAAAAA1620 - CCTGGTGACC TCCGGCTCCG CCGAGAAGAT CACCCACATC AACTACTCCT TCGGCAACGT1680 - CCAGGGCGGC AAGTGCACCA TCGGTGACAG CTTCGCCGCC TACGACAAGG CGTACACCGC1740 - CGCCGAGTCG GTCGACGGCG TCGCCGACAC CTGGGACCAG CCGCTGCGCG GCAACTTCAA1800 - CCAGCTCCGC AAGCTCAAGG CCAAGTACCC GCACATCAAG GTCCTCTGGT CCTTCGGCGG1860 - CTGGACCTGG TCCGGCGGCT TCACCGACGC CGTGAAGAAC CCGGCCGCCT TCGCCAAGTC1920 - CTGCCACGAC CTGGTCGAGG ACCCGCGCTG GGCCGACGTC TTCGACGGCA TCGACCTCGA1980 - CTGGGAGTAC CCGAACGCCT GCGGCCTCAG CTGCGACAGC TCCGGTCCGG CCGCGCTGAA2040 - GAACATGGTC CAGGCGATGC GCGCCCAGTT CGGCACCGAC CTGGTCACCG CCGCCATCAC2100 - CGCCGACGCC AGCTCCGGCG GCAAGCTCGA CGCCGCCGAC TACGCGGGCG CCGCCCAGTA2160 - CTTCGACTGG TACAACGTGA TGACGTACGA CTTCTTCGGC GCCTGGGACA AGACCGGCCC2220 - GACCGCGCCC CACTCGGCCC TGAACTCCTA CAGCGGCATC CCCAAGGCCG ACTTCCACTC2280 - GGCCGCCGCC ATCGCCAAGC TCAAGGCGAA GGGCGTCCCG GCGAGCAAGC TCCTGCTCGG2340 - CATCGGCTTC TACGGCCGCG GCTGGACCGG CGTCACCCAG GACGCCCCGG GCGGCACCGC2400 - CACCGGCCCG GCCACCGGCA CCTACGAGGC GGGCATCGAG GACTACAAGG TCCTCAAGAA2460 - CACCTGCCCC GCCACCGGCA CCGTCGGCGG CACCGCGTAC GCCAAGTGCG GCAGCAACTG2520 - GTGGAGCTAC GACACCCCGG CCACCATCAA GACCAAGATG ACCTGGGCCA AGGACCAGGG2580 - CCTCGGCGGC GCCTTCTTCT GGGAGTTCAG CGGTGACACC GCGGGCGGCG AACTGGTCTC2640 - CGCGATGGAC TCCGGCCTCC GCTAGCCCCG GACCGGCACC CCGCCCGAAC CACTAGCACG2700 - ACCTCCCCCG GA2712 Fragments of the above chitinolytic enzymes are encompassed by the present invention. Suitable fragments can be produced by several means. In the first, subclones of the gene encoding the chitinolytic enzymes of the present invention are produced by conventional molecular genetic manipulation by subcloning gene fragments. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or peptide that can be tested for chitinolytic activity according to the procedure described below. As an alternative, fragments of a chitinolytic enzyme can be produced by digestion of a full-length chitinolytic enzyme with proteolytic enzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin. Different proteolytic enzymes are likely to cleave chitinolytic enzymes at different sites based on the amino acid sequence of the chitinolytic enzyme. Some of the fragments that result from proteolysis may be active chitinolytic enzymes. In another approach, based on knowledge of the primary structure of the protein, fragments of a chitinolytic enzyme encoding gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These then would be cloned into an appropriate vector for increased expression of a truncated peptide or protein. Chemical synthesis can also be used to make suitable fragments. Such a synthesis is carried out using known amino acid sequences for a chitinolytic enzyme being produced. Alternatively, subjecting a full length chitinolytic enzyme to high temperatures and pressures will produce fragments. These fragments can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE). Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the properties, secondary structure and hydropathic nature of an enzyme. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide. Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of SEQ. ID. Nos. 2 or 4 under stringent conditions. An example of suitable stringency conditions is when hybridization is carried out at 65.degree. C. for 20 hours in a medium containing 1M NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodium dodecyl sulfate, 0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, 50 .mu.m g/ml E. coli DNA. A chitinolytic enzyme of the present invention is preferably produced in purified form (preferably at least about 80%, more preferably 90%, pure) by conventional techniques. Typically, a chitinolytic enzyme of the present invention is secreted into the growth medium of recombinant host cells. Alternatively, a chitinolytic enzyme of the present invention is produced but not secreted into growth medium. In such cases, to isolate a chitinolytic enzyme, the host cell (e.g., E. coli) carrying a recombinant plasmid is propagated, lysed by sonication, heat, differential pressure, or chemical treatment, and the homogenate is centrifuged to remove bacterial debris. The supernatant is then subjected to sequential ammonium sulfate precipitation. The fraction containing a chitinolytic enzyme of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC. The DNA molecule encoding a chitinolytic enzyme can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture. Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus. Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK.+-. or KS.+-. (see "Stratagene Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see F. W. Studier et. al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes," Gene Expression Technology vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1989), which is hereby incorporated by reference. A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used. Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation). Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promoters differ from those of procaryotic promoters. Furthermore, eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells. Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno ("SD") sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3'-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference. Promotors vary in their "strength" (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the P.sub.R and P.sub.L promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene. Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced. In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls. Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promotor, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires an SD sequence about 7-9 bases 5' to the initiation codon ("ATG") to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used. Once an isolated DNA molecule encoding a chitinolytic enzyme has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like. The present invention further relates to a method of effecting insect and/or fungus control for plants. This method involves applying a chitinolytic enzyme to all or part of a plant or a plant seed under conditions effective to control insects and/or fungi. Alternatively, the chitinolytic enzyme can be applied to plants such that seeds recovered from such plants themselves are able to effect insect and/or fungus control. As an alternative to applying a chitinolytic enzyme to plants or plant seeds in order to control insects and/or fungi on the plants or plants grown from the seeds, transgenic plants or plant seeds can be utilized. When utilizing transgenic plants, this involves providing a transgenic plant transformed with a DNA molecule encoding a chitinolytic enzyme and growing the plant under conditions effective to permit that DNA molecule to control insects and/or fungi. Alternatively, a transgenic plant seed transformed with a DNA molecule encoding a chitinolytic enzyme can be provided and planted in soil. A plant is then propagated from the planted seed under conditions effective to control insects and/or fungi. The embodiment of the present invention where the chitinolytic enzyme is applied to the plant or plant seed can be carried out in a number of ways, including: 1) application of an isolated chitinolytic enzyme and 2) application of bacteria which do not cause disease and are transformed with genes encoding a chitinolytic enzyme. In one embodiment of the present invention, a chitinolytic enzyme of the present invention can be isolated (i.e. separated from the bacteria which naturally produce it) as described in the Examples infra. Preferably, however, an isolated chitinolytic enzyme of the present invention is produced recombinantly and purified (i.e. made substantially free of contaminants) as described supra. In another embodiment of the present invention, a chitinolytic enzyme of the present invention can be applied to plants or plant seeds by applying bacteria containing genes encoding a chitinolytic enzyme. Such bacteria must be capable of secreting or exporting the enzyme so that the enzyme can effect fungus and/or insect control. In these embodiments, the chitinolytic enzyme is produced by the bacteria in planta or on seeds or just prior to introduction of the bacteria to the plants or plant seeds. In the bacterial application mode of the present invention, the bacteria do not cause the disease and have been transformed (e.g., recombinantly) with a gene encoding a chitinolytic enzyme. For example, E. coli can be transformed with a gene encoding a chitinolytic enzyme and then applied to plants. Bacterial species other than E. coli can also be used in this embodiment of the present invention. The method of the present invention can be utilized to treat a wide variety of plants or their seeds to control fungi and/or insects. Suitable plants include dicots and monocots. Monocots treatable in accordance with the present invention include Gramineae (e.g., grass, corn, grains, bamboo, sugar cane), Liliaceae (e.g., onion, garlic, asparagus, tulips, hyacinths, day lily, and aloes), Iridaceae (e.g., iris, gladioli, freesia, crocus, and watsonia), and Orchidacea (e.g., orchid). Examples of dicots which can be treated pursuant to the present invention include Salicaceae (e.g., willow, and poplar), Ranunculaceae (e.g., Delphinium, Paeonia, Ranunculus, Anemone, Clematis, columbine, and marsh marigold), Magnoliaceae (e.g., tulip tree and Magnolia), Cruciferae (e.g., mustards, cabbage, cauliflower, broccoli, brussel sprouts, kale, kohlrabi, turnip, and radish), Rosaceae (e.g., strawberry, blackberry, peach, apple, pear, quince, cherry, almond, plum, apricot, and rose), Leguminosae (e.g., pea, bean, peanut, alfalfa, clover, vetch, redbud, broom, wisteria, lupine, black locust, and acacia), Malvaceae (e.g., cotton, okra, and mallow), Umbelliferae (e.g., carrot, parsley, parsnips, and hemlock), Labiatae (e.g., mint, peppermints, spearmint, thyme, sage, and lavender), Solanaceae (e.g., potato, tomato, pepper, eggplant, and Petunia), Cucurbitaceae (e.g., melon, squash, pumpkin, and cucumber), Compositae (e.g., sunflower, endive, artichoke, lettuce, safflower, aster, marigold, dandelions, sage brush, Dalia, Chrysanthemum, and Zinna), and Rubiaceae (e.g., coffee). The present invention is effective against a wide variety of insect pests including the orders of Lepidoptera, Coleoptera, Diptera, Homoptera, Hemiptera, Thysanoptera, and Orthoptera. Examples of Lepidoptera include butterflies and moths. Coleoptera include beetles. Examples of Diptera are flies. Examples of Homoptera are aphids, whiteflies, scales, psyllids, leafhoppers, plant hoppers, cicadas, and treehoppers. The Hemiptera which are treatable in accordance with the present invention include true bugs. Thysanoptera which can be treated in accordance with the present invention include thrips. Examples of Orthoptera which can be treated in accordance with the present invention are grasshoppers, crickets, and katydids. Collectively, these orders of insect pests represent the most economically important group of pests for vegetable production worldwide. The chitin-containing fungi inhibited by the purified chitinases of the present invention include, for example, species from the genera including Fusarium, Gliocadium, Rhizoctonia, Trichoderma, Uncinula, Ustilago, Erysiphe, Botrytis, Saccharomyces, Sclerotium, and Alternaria. The method of the present invention involving application of a chitinolytic enzyme can be carried out through a variety of procedures when all or part of the plant is treated, including leaves, stems, roots, etc. This may (but need not) involve infiltration of the chitinolytic enzyme into the plant. Suitable application methods include topical application (e.g., high or low pressure spraying), injection, and leaf abrasion proximate to when enzyme application takes place. When treating plant seeds, in accordance with the application embodiment of the present invention, a chitinolytic enzyme can be applied by low or high pressure spraying, coating, immersion, or injection. Other suitable application procedures can be envisioned by those skilled in the art provided they are able to effect contact of the chitinolytic enzyme with the plant or plant seed. Once treated with a chitinolytic enzyme of the present invention, the seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants. After plants have been propagated from seeds treated in accordance with the present invention, the plants may be treated with one or more applications of a chitinolytic enzyme to control insects and/or fungi on the plants. The chitobiosidase or endochitinase can be applied to plants or plant seeds in accordance with the present invention individually, in combination with one another, or in a mixture with other materials. Alternatively, a chitinolytic enzyme can be applied separately to plants with other materials being applied at different times. A composition suitable for treating plants or plant seeds in accordance with the application embodiment of the present invention contains a chitinolytic enzyme in a carrier. Suitable carriers include water, aqueous solutions, slurries, or dry powders. In this embodiment, the composition contains greater than 500 nM chitinolytic enzyme. Although not required, this composition may contain additional additives including fertilizer, insecticide, fungicide, nematacide, and mixtures thereof. Suitable fertilizers include (NH.sub.4).sub.2 NO.sub.3. An example of a suitable insecticide is Malathion. Useful fungicides include Captan. Other suitable additives include buffering agents, wetting agents, coating agents, and abrading agents. These materials can be used to facilitate the process of the present invention. In addition, a chitinolytic enzyme can be applied to plant seeds with other conventional seed formulation and treatment materials, including clays and polysaccharides. In the alternative embodiment of the present invention involving the use of transgenic plants and transgenic seeds, a chitinolytic enzyme need not be applied topically to the plants or seeds. Instead, transgenic plants transformed with a DNA molecule encoding a chitinolytic enzyme are produced according to procedures well known in the art. In producing transgenic plants, the DNA construct in a vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics, 202:179-85 (1985), which is hereby incorporated by reference. The genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incorporated by reference. Another approach to transforming plant cells with the DNA construct is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, a vector containing the DNA construct can be introduced into the cell by coating the particles with the vector containing that heterologous DNA construct. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA construct) can also be propelled into plant cells. Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies. Fraley, et al., Proc. Natl. Acad. Sci. USA, 79:1859-63 (1982), which is hereby incorporated by reference. The DNA molecule may also be introduced into the plant cells by electroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824 (1985), which is hereby incorporated by reference. In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate. Another method of introducing the DNA molecule into plant cells is to infect a plant cell with Agrobacterium tumefaciens or A. rhizogenes previously transformed with the gene. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots or roots, and develop further into plants. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28.degree. C. Agrobacterium is a representative genus of the gram-negative family Rhizobiaceae. Its species are responsible for crown gall (A. tumefaciens) and hairy root disease (A. rhizogenes). The plant cells in crown gall tumors and hairy roots are induced to produce amino acid derivatives known as opines, which are catabolized only by the bacteria. The bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes. In addition, assaying for the presence of opines can be used to identify transformed tissue. Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-83 (1987), which is hereby incorporated by reference. After transformation, the transformed plant cells must be regenerated. Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III (1986), which are hereby incorporated by reference. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes. Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable. After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the DNA construct is present in the resulting plants. Alternatively, transgenic seeds are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants. When transgenic plants and plant seeds are used in accordance with the present invention, they additionally can be treated with the same materials as are used to treat the plants and seeds to which a chitinolytic enzyme is applied. These other materials, including a chitinolytic enzyme, can be applied to the transgenic plants and plant seeds by the above-noted procedures, including high or low pressure spraying, injection, coating, and immersion. Similarly, after plants have been propagated from the transgenic plant seeds, the plants may be treated with one or more applications of a chitinolytic enzyme to control fungi and/or insects. Such plants may also be treated with conventional plant treatment agents (e.g., insecticides, fertilizers, etc.). The transgenic plants of the present invention are useful in producing seeds or propagules (e.g., cuttings) from which fungus and/or insect resistant plants grow. |
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