Patent Application
20150139976
The following specification particularly describes the invention and the manner in which it is to be performed:
The invention relates to a novel insecticidal chitinase protein from fern Tectaria sp. process for preparation of the insecticidal protein and nucleic acid sequence encoding for said insecticidal protein and its application for insect control purposes.
Loss of crop yields due to the attack of various insects is a worldwide problem. Insect pests are of mainly of two different types; biting and chewing type (e.g. Lepidopteran insects) & piercing and sucking type (Hemipteran insects). Transgenic insect resistant cultivars expressing Bacillus thuringiensis (Bt) protein have been developed for the control of insect pests of order Lepidoptera and Coleoptera group. However, the plants expressing Bt protein are not toxic to sap sucking pests. Hence, an alternative approach is required to control sap sucking pests. One of the approaches would be screening of plant biodiversity for insect toxic proteins.
Ferns are the most ancient vascular plants. Fossil records of the ferns extend back to Devonian periods. Ferns are vascular plants, differing from the more primitive lycophytes in having true leaves, and they differ from seed plants (gymnosperms and angiosperms) in their mode of reproduction, absence of flowers and seeds. Ferns show great degree of diversity than any other plant phyla except angiosperms. Success of ferns is often attributed to their less susceptibility to insect attack. Although 9300 species of insects are reported to use ferns as a food source (Crooper-Driver 1978; Entomol. Exp. Appl. 24: 110-116), ferns have not been reported to suffer severe insect attacks, which is mainly due to the high concentration of secondary metabolites and possible presence of insect resistant macromolecules. Ferns are known to contain insect resistant secondary metabolites such as ferulic acid, hydrolysable tannins, terpenes, and alkaloids (Schaufelberger and Hostettmann, 1983; Planta Med. 48:105-107; Asakawa 1990; Biologically active substances from bryophytes. Pages 259-287. In: R chopra, B Satish (eds). Bryophyte development: Physiology and Biochemisrty. CRC, Boston.) and ecdysones mimics like insect hormone (Jones and Firn 1978; J Chem. Ecol. 4: 117-138; Lafont and Horn 1989. Phytoecdysteroids: structure and occurrence. Pages 39-64. In: J. Koolman (ed). Ecdysone: from chemistry to mode pf action. Thieme, Stuttgart.). Nevertheless ferns and mosses serve as the important source of insecticidal proteins, the crude protein extracts of several ferns and mosses caused 70-100% mortality of Spodoptera frugiperda and Helicoverpa zea and also resulted in significant growth reduction of both the insect species (Markham et al., 2006; Int. J. Plant Sci. 167: 111-117). Many insecticidal lectins have been isolated from ferns. Enzyme thiaminase derived form ferns and moss has been demonstrated for IR (Insect Resistance) activity. Thiaminase deterred feeding by southern armyworm Nephrolepis exaltata (Hendrix, 1977; Am. Nat. 115-171-196.).
Plants have evolved sophisticated defense mechanisms including a wide array of defensive compounds that confer resistance against phytophagous predators and infection by viruses, bacteria, fungi, nematodes, etc. The best known plant proteins supposedly involved in defense mechanisms are lectins, ribosome-inactivating proteins (RIPs) of types 1 and 2, inhibitors of proteolytic enzymes and glycohydrolases (Ryan, 1990; Annu. Rev. Phytopathol. 28, 425-449; Bowles, 1990; Ann. Rev. Biochem. 59, 873-907; Chrispeels and Raikhel, 1991; Plant Cell 3, 1-9; Barbieri et al., 1993; Biochem. J. 185, 203-210; Peumans and Van Damme, 1995; Plant Physiol. 109, 347-352.). Other plant proteins involved in the complex mechanisms of defense are the arcelins (Osborn et al., 1988; Science 240, 207-210.), chitinases (Herget et al., 1990; Mol. Gen. Genet. 224, 469-476), canatoxin (Carlini et al., 1997; J. Econ. Entomol. 90, 340-348.) and modified forms of storage proteins (Macedo et al., 1993; Comp. Biochem. Physiol. 105C, 89-94).
The chitin-binding plant proteins are defined as a group of protein comprising of chitinases, chitin-binding lectins and hevein (Raikel and Broekaert, 1991, in Control of plant gene expression, Verma DP (ed), Telford Press). All these proteins contain a conserved cysteine/glycine rich domain. This common region may confer the chitin binding activity. The domain is 40-43 amino acids in length and is either repeated twice, four-fold or fused to an unrelated domain. The chitin-binding plant proteins known to affect the growth of fungi or insects that contain chitin. However, the chitin binding proteins isolated from different sources differ in the specificity. The wheat/barley/rice-type lectins are toxic to insects, but are inactive to fungi in vitro (Murdock et al, 1990, Phytochem, 29: 85-89). The chitinases are inhibitory to the growth of certain pathogenic fungi. A chitinase with antifungal property has been isolated from the fern Pteris ryukyuensis (Onaga and Taira, 2008, Glycobiology, 18; 414-423).
Chitinases so far sequenced are classified into two different families, family 18 and 19, in the classification system of Glycoside hydrolases, based on amino acid sequence similarity of their catalytic module (Henrissat and Bairoch, 1993; Biochem. J. 293: 781-788: Davies and Henrissat, 1995, Structure, 3: 853-859). Family 18 contains chitinases from bacteria, fungi, viruses and some plant chitinases (class III and V). Family 19 contains plant chitinases (class I, II and IV), chitinases from purple bacteria, actinobacteria, certain nematodes, arthropods and protists (Udaya Prakash et al. 2010, J. Mol Evol. DOI 10.1007/s00239-010-9345-z). Plants synthesize various chitinases (Collinge et al. 1993, Plant J, 3: 31-40) and they are divided into five classes on the basis of their primary structures, independent of glycoside hydrolase classification (Kezuka et al. 2006, J. Mol. Biol, 358: 472-484).
Chitin-binding plant proteins are being used for the protection of plants against fungal disease and transgenic plants expressing chitin biding protein has been developed to confer resistance for fungal pathogen. U.S. Pat. No. 5,514,779 describes an antimicrobial protein which can be isolated from seeds of Amaranthus, seeds of Capsicum and seeds of Briza, has an amino acid sequence containing the common cysteine/glycine domain of Chitin-binding plant proteins and posses substantially better activity against plant pathogenic fungi than that of the Chitin-binding plant proteins. U.S. Pat. No. 6,710,228 discloses chimeric genes encoding lectins exhibiting insecticidal and/or fungicidal activity or which can be used to transform cotton to yield cotton cells, plants, and seeds in which the chimeric genes are expressed. The cotton embryogenic callus transformed, suppresses the growth of Heliothis larvae, and killed some larvae, when 25 mg of lyophilized transformed callus mixed into the artificial diet. U.S. Pat. No. 4,940,840 describe fungus (Alternaria longipes) resistant tobacco plants, expressing a chitinase gene from the bacterium Serratia marcescens. European Patent Application Number 418695 describes the use of regulatory DNA sequences from tobacco chitinase gene to drive expression of introduced genes producing transgenic plants with improved resistance to pathogens. Patent Application Number WO9007001 describes chitinase gene over-express transgenic plants for improved resistance to fungal pathogens.
The chitin binding proteins usually posses a catalytic module (CM) and one or two chitin binding module (CtBM). Class II chitinases of family 19 are known to lack CtBM and posses enzymatic activity (Udaya Prakash et al. 2010, J. Mol Evol. DOI 10.1007/s00239-010-9345-z), likewise chitinase (Chi NCTU2) from Bacillus cereus, belongs to family 18 also posses only CM (Yin-Cheng Hsieh et al., JBC. in Press. Aug. 4, 2010 as Manuscript M110.149310).
The prior art lacks a chitinase with CtBM only (and lacking CM). No prior art is available on isolation of insecticidal Chitin-binding proteins from ferns. No experimental data is available to show the toxicity of any plant derived Chitin-binding proteins against sap sucking pest like whiteflies (Bemisia labaci).
The Novelty of the Disclosed Protein has been Established on the Basis of Below Mentioned Points
The object of the present invention is to provide insecticidal Chitinase proteins having a chitin binding module and lacking Catalytic Module, from fern Tectaria sp.
Another object of present invention is to prepare the said insecticidal chitinase protein.
Another object of the present invention is to isolate pure insecticidal chitinase protein from fern Tectaria sp. and to prepare nucleic acid sequence encoding the said insecticidal protein.
Still another object of the present invention is to prepare recombinant chitinase protein having CtBM only and lacking CM for its application for insect control purposes.
Accordingly, the present invention is directed to novel insecticidal Chitinase proteins from fern Tectaria sp., having a chitin binding module and lacking Catalytic Module a process for preparation of the insecticidal protein and to prepare nucleic acid sequence encoding the said insecticidal protein to produce recombinant chitinase proteins which substantially obviate one or more problems due to limitations of the related art.
These and other features, aspects and advantages of the present invention will be better understood with reference to the following description, drawings and claims.
The present invention provides purification and isolation of insecticidal chitinase protein from the fern Tectaria sp. process for preparation of an insecticidal protein isolated from the fern Tectaria sp. and DNA sequence encoding the said protein. The insecticidal protein was purified from leaves of Tectaria sp. The method of protein purification involves, extraction of total soluble protein; fractionation of crude extract using differential ammonium sulfate precipitation and different steps of chromatography. Each stage of purification was guided by insecticidal activity. The protein defined as insecticidal protein is toxic to at least one of the insects-whitefly (Bemisia tabaci), cotton boll worm (Helicoverpa armigera), aphid (Aphis gossypii) and Spodoptera litura. Insecticidal activity includes a range of antagonistic effects such as mortality (death), growth reduction and feeding deterrence. Gene encoding the purified insecticidal protein was cloned using N-terminal sequencing data of the purified protein by designing degenerate primers. The pI of the protein was in range of 5-6. The protein is of 216 amino acids (Sequence I.D. No. 4) and the mature peptide is of 192 amino acids (Sequence I.D. No. 5) with respective molecular weight of 23.684 kDa and 21.270 kDa. The cloned cDNA consisted of 828 nucleotides (Sequence I.D. No. 1), of which the protein encoding ORF sequence was of 651 nucleotides (Sequence ID No. 2) and the mature peptide encoding ORF is of 579 nucleotides (Sequence ID No. 3). The gene encoding the insecticidal protein was cloned in E. coli and plant expression vector. The insecticidal protein was expressed in E. coli and purified. Like native protein, the recombinant protein also showed the insecticidal activity. The purified native protein as well as the recombinantly expressed protein showed the chitinase activity. The amino acid sequence of the protein was compared with the available data base of chitinases by BlustlW analysis, to establish its novelty. The bio-safety of the protein was evaluated using online allergic domain search and pepsin digestibility test. The protein has no allergic domains and hence does not cause any allergic response and is quickly digested by enzyme pepsin. This indicated bio-safety of the protein.
Accordingly present invention provides an isolated novel insecticidal protein characterized in that it contain chitin binding module without having catalytic module, from fern Tectaria sp., process for preparation of the insecticidal chitinase protein comprising the step of:
In the embodiment of the invention, the nucleotide sequence encoding an insecticidal protein as shown in sequence SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3.
In the other embodiment of the invention, the amino acid residues of the insecticidal polypeptide is shown in sequences SEQ ID No. 4 or SEQ ID No.5.
In yet another embodiment of the invention, the insecticidal protein can be produced by the expression of recombinant DNA.
In the further embodiment of the invention, the plant expression cassette containing the nucleotide encoding the insecticidal protein is useful for transformation of cotton and other crop plants for the development of transgenic plants resistant to whiteflies.
Ferns are vascular plants differing from the more primitive lycophytes in having true leaves, and seed plants (gymnosperms and angiosperms) in their mode of reproduction, absence of flowers and seeds. Ferns show great degree of diversity than any other plant phyla except angiosperms. Success of ferns is often attributed to their less susceptibility to insect attack. These have not been reported to suffer from severe insect attacks, which is mainly due to the high concentration of secondary metabolites and possible presence of insect resistance macromolecules. Ferns are known to contain insect resistant secondary metabolites such as ferulic acid, hydrolysable tannins, terpenes, alkaloids and ecdysones that mimic insect hormones. The crude protein extracts of several ferns and mosses caused mortality and also significant growth reduction of insects. Many insecticidal lectin proteins have been isolated from ferns.
In this present invention, we purified a new insecticidal protein from the leaves of fern Tectaria. The method of insecticidal activity guided purification of protein involved extraction of total soluble protein from leaves; fractionation of total soluble protein with differential ammonium sulfate precipitation and further purification involving different chromatography as explained in detail (Example 1). At each stage of purification, every fraction was dialyzed, evaluated for insecticidal activity and the fractions which were found effective were taken to the next step of purification. The purified protein was evaluated for toxicity against whiteflies (Bemisia tabaci) by incorporating the protein in the artificial diet (Example 4). The protein caused mortality of whiteflies (Table 2). The purity and pI of the purified insecticidal protein was further determined by 2-D PAGE (
The gene encoding the insecticidal protein was cloned in E. coli expression vector in fusion with SUMO peptide and the recombinant protein was expressed and purified (Example 5). The recombinant protein also showed the insecticidal activity against whiteflies.
In the embodiment of the invention, the nucleotide sequence encoding an insecticidal protein as shown in sequence SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3.
In the other embodiment of the invention, the amino acid residues of the insecticidal protein is shown in sequences SEQ ID No. 4 or SEQ ID No. 5.
In another embodiment of the invention, an isolated protein as claimed in claim 1 is toxic to whitefly (Bemisia tabaci).
In yet another embodiment of the invention, the protein can be used for the control of other insect pests.
The preferred use of the protein according to the invention is to insert the genes encoding these proteins into the plants using various methods available for the introduction and expression of the foreign genes in transgenic plants. The method of gene insertion and expression may include methods such as Agrobacterium mediated gene transfer, microinjection of DNA into cells or protoplasts, DNA transfer via growing pollen tubes, DNA uptake by imbibing zygotic embryos, silicon carbide fiber mediated delivery, microprojectile bombardment and direct DNA uptake employing polyethylene glycol, liposomes or electroporation. Once a line of transgenic plants is established, the character may be transferred to other cultivars by conventional plant breeding.
Plants which can be protected, preferably by transformation, according to the methods of this invention include, but are not limited to rice, wheat, maize, cotton, potato, sugarcane, tobacco, soybean, cabbage, cauliflower, beans, apple, tomato, mustard, rape seed and sunflower etc.
The protein useful in insect control and the corresponding genes can be obtained from, all the above ground and below ground plant parts of any fern not necessarily limited to Tectaria sp.
In yet another embodiment of the invention, an insecticidal protein can be produced by the expression of recombinant DNA.
In further embodiment of the invention, the gene encoding the insecticidal protein was cloned in E. coli expression vector in fusion with SUMO peptide. The recombinant insecticidal protein was expressed in E. coli and purified by affinity chromatography. The recombinant protein was digested with SUMO-Protease I to liberate the desired protein from SUMO peptide. The recombinant protein also showed the insecticidal activity against whiteflies.
In the further embodiment of the invention, the plant expression cassette was transformed in cotton for the development of transgenic plants resistant to whiteflies.
In still another embodiment of the invention, the protein is biologically safe to use because it can be completely digested by pepsin in less than 30 seconds under the experimental conditions i.e.; at pH 1.2 and pH 2.0 SGF buffer. The online search using allergen data revealed that the protein has no allergic domains and does not cause any allergic responses.
Plant material was collected from the fern house of National Botanical Research Institute, Lucknow, India. Total soluble protein was prepared by following the procedures of Markham et al., (2006). Leaves were crushed into fine powder under liquid nitrogen. Powdered leaf was suspended in ice cold protein extraction buffer (20 mM HEPES, 0.5 mM DTT, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonylfluoride and 1 mm benzamidine, pH 8.0) in 1:4 (w/v) ratio. The suspension was homogenized at 4° C. and incubated for 1 h and then filtered through cheesecloth. The homogenate was centrifuged (3000×g, 4° C., 30 min). The total soluble protein was fractionated with differential ammonium sulfate precipitation at the interval of 20% saturation. Each fraction was dialyzed and evaluated for insecticidal activity. The effective fraction was further dialyzed in 20 mM TrisCl (pH 8.0) and loaded on Q sepharose (FF) column, pre-equilibrated with 20 mM TrisCl (pH 8.0). The column was washed with the same buffer until OD280 reached to less than 0.02. The column bound proteins were eluted with a linear gradient of 0-1 M NaCl in 20 mM TrisCl (pH 8.0). The eluted fractions were dialyzed against 20 mM TrisCl (pH 8.0) and used for insect bioassay. Fractions causing mortality to the insect were pooled and dialyzed against 20 mM Tris (pH 8.0) containing 200 mM NaCl. The pooled protein sample was resolved on Superdex 200 equilibrated with protein sample buffer. Eluted fractions were again dialyzed to remove salt and insect bioassay was performed. Purified insecticidal protein was further analysed by 2 dimensional gel electrophoresis for purity and pI determination. The pI of the protein was between 5-6.
Peptide Mass Finger Printing:
The purified protein was electrophorased on SDS-PAGE. The protein band was cut and digested with trypsin and used for peptide mass finger printing. The data was analyzed on MASCOT search. No match with the peptide/protein was found in the database.
N-Terminal Sequencing:
For N-terminal sequencing, the purified protein was run on SDS-PAGE and transferred onto the PVDF membrane and used for N-terminal sequencing.
Total RNA was isolated from the plant leaves. The cDNA synthesis was performed for 5′ and 3′ rapid amplification of cDNA ends. For 3′ RACE, RNA was reversely transcribed with the 3′ RACE CDS Primer A. The primary PCR was performed with degenerate primer (designed on the basis of N-terminal sequencing data) and Universal primer A mix. For 5′ RACE, RNA was reversely transcribed with the 5′ RACE CDS Primer and SMART II A Oligonucleotide. Based on the sequence of the 3′ RACE product, the gene specific primers (GSP1 and GSP2) were designed and synthesized. The first round of PCR was performed with GSP1 and Universal Primer A Mix (UMP, provided in the kit). The PCR product was diluted 50-fold for a second round of amplification of the gene with GSP2 and Nested Universal Primer A (NUP).
Bioassay was carried out with >1 day old adult whiteflies (Bemisia tabaci). Whiteflies were reared on cotton plants grown in pots in the laboratory at 26±2° C. and 80% relative humidity. Cotton plants having large number of nymphs and pupae were selected, adult whiteflies were removed and plants were kept in isolation for the emergence of fresh adults. Bioassays were carried out as per Upadhyay et al., 2011 (J. Biosciences. 36: 153-161). The whiteflies were directly collected into specimen tubes. The leaf containing freshly emerged adults was kept close to the open end of the tube. Insects were stimulated to move inside the tube by gentle tapping (
The gene encoding the insecticidal protein was cloned in E. coli expression vector in fusion with SUMO peptide under T7 promoter. The recombinant insecticidal protein was expressed after induction with IPTG and expression profile was observed for every hour after induction for 3 h. After 3 h of induction, the cells were harvested by centrifugation and lysed by lysozyme and broken by sonication. The inclusion bodies containing the desired protein were washed with 20 mM TrisCl (pH 8).
The inclusion bodies were again suspended in 20 mM Tris (pH 8) containing 8M Urea and kept at room temperature for 2 h for solubilization. The suspension was centrifuged (13000×g, 15 min, room temperature) and supernatant was collected. The recombinant protein was purified by Ni-affinity chromatography in denatured condition. The purified recombinant protein was refolded. The protein was dialyzed in PBS and digested with SUMO-Protease I to liberate the desired protein from SUMO peptide. The purified insecticidal recombinant protein was tested in insect bioassay.
The biosafety of protein was evaluated using online allergic domain search and pepsin digestibility test.
Allergen Search
The online search using allergen data based revealed that the protein has no allergic domains and therefore expected not to cause allergic responses.
Pepsin Digestibility
Purified porcine pepsin has been used to evaluate the stability of a number of food allergens and non-allergenic proteins in a multi-laboratory study that demonstrated the rigor and reproducibility in nine laboratories (Thomas et al 2004., Regulatory Toxicology and Pharmacology,. 37:87-98). Porcine pepsin is an aspartic endopeptidase with broad substrate specificity. Pepsin is optimally active between pH 1.2 and 2.0, but inactive at pH 3.5 and irreversibly denatured at pH 7.0. The assay is performed under standard conditions of 10 units of pepsin activity per microgram of test protein. The original assay described by Astwood et al. (Nature Biotechnology,. 14:1269-1273, 1996) recommends performing the digestion at pH 1.2. However, the FAO/WHO (2001) recommends using two pH conditions (pH 1.2 and pH 2.0). The assay is performed at 37° C. and samples are removed at specific times (0, 0.5, 1, 2, 5, 10, 20, 30, 60 minutes) and the activity of pepsin is quenched by neutralization with carbonate buffer and sodium dodecyl sulfate (SDS-) polyacrylamide gel electrophoresis (PAGE) loading buffer and heating at >70° C. for 3-5 minutes. The timed digestion samples are electrophorased on SDS-PAGE and stained with Coomassie Brilliant Blue to evaluate the extent of digestion. Assessment of the digestibility assays developed by Bannon et al. (2002, Comments Toxicol. 8:271-285.) and by Thomas et al. (2004) indicate that the most of the non-allergenic food proteins are digested in approximately 30 seconds, while the major food allergens are stable, or produce pepsin-stable fragments that are detectable for 8-60 minutes. The protein was completely digested by pepsin in less than 30 seconds under both the experimental conditions (at pH 1.2 and pH 2.0 SGF buffer).
Thermal Stability
A 1 mg/ml solution of the protein was prepared in 20 mM TrisCl (pH 8.0). The protein was incubated at the 30° C., 40° C., 50° C., 60° C., 70° C., 80° C., 90° C. and 100° C. 2.5 μl aliquot that contained 2.5 μg of protein was analyzed on a 12% SDS-PAGE gel. The experiment was performed in triplicate. 2.5 μg of treated protein was used for the enzymes assay also. The protein was found unstable at the temperatures beyond 90° C. (
Protein coding ORF sequences was predicted by ORF finder software of NCBI (http://www.ncbi.nlm.nlh.gov/proiects/gorf/orfig.cgi).
ORF sequence was translated to the amino acid sequences by Expasy translate tools http://www.expasy.ch/tools/dna.html.
Amino acid sequences was further analyzed by signal iP software http://www.cbs.dtu.dk/services/SignalP/ for signal peptide. Signal peptide was 24 amino acid long.
| Number | Date | Country | Kind |
|---|---|---|---|
| 3851/DEL/2011 | Dec 2011 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2012/000860 | 12/28/2012 | WO | 00 |
