Insecticidal chitinase protein its encoding nucleotide and application thereof

Abstract

A novel insecticidal chitinase protein from fern Tectaria sp., a process for preparation of the insecticidal protein and nucleic acid sequence encoding for said insecticidal protein and its application for insect control purposes.

Claims

1. A cDNA molecule encoding an insecticidal chitinase protein containing chitin binding module but lacking catalytic module wherein the nucleotide sequence encoding the insecticidal protein is comprised by any one of the SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 3 and amino acid sequence of the insecticidal protein is comprised by any one of the sequences SEQ ID NO. 4 or SEQ ID NO. 5.

2. The cDNA molecule of claim 1, wherein said insecticidal chitinase protein is of 216 amino acid residues long pro-protein and 192 amino acid residues long mature protein with respective molecular weight 23.684 and 21.270 kDa.

3. The cDNA molecule of claim 1, wherein said insecticidal chitinase protein comprises of chitin binding module (CtBM), and shows exo- and endo-chitinase activity.

4. The cDNA molecule of claim 1, wherein the said insecticidal chitinase protein consists of chitin binding module (CtBM), and shows exo- and endo-chitinase activity.

5. The cDNA molecule of claim 1, wherein the said insecticidal chitinase protein is useful for the control of insects from order homoptera, heteroptera, diptera, coleoptera and lepidoptera, particularly toxic to white fly (Bemisia tabaci).

6. A process for preparation of the insecticidal chitinase protein encoded by the cDNA molecule of claim 1, comprising the steps of: (i) isolating chitinase protein from fern Tectaria sp. in a manner such as herein described, (ii) cloning the cDNA of claim 1 from purified protein, using N-terminal sequencing data of the purified protein by designing degenerate primers, (iii) identifying ORF sequence encoding a mature polypeptide of insecticidal chitinase from cloned cDNA sequence, (iv) cloning the cDNA encoding the insecticidal protein in an E. coli expression vector in fusion with SUMO peptide to get expression of recombinant protein followed by purification of recombinant protein by conventional manner.

7. The process as claimed in claim 6, wherein the insecticidal protein is being produced by expressing its encoding nucleotide in homologous or heterologous system using recombinant DNA technology.

8. A transgenic crop comprising the cDNA molecule encoding an insecticidal chitinase protein according to claim 1, wherein the transgenic crop expresses the insecticidal chitinase protein causing toxicity to insect and exhibiting protection against insect pest.

9. An expression vector comprising the cDNA molecule encoding an insecticidal chitinase protein according to claim 1.

10. A method of controlling insects using the cDNA molecule encoding an insecticidal chitinase protein according to claim 1.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1: chromatogram of the protein fraction separated on Q-sepharose (fast flow) column. The arrow indicates the fractions showing high insecticidal activity.

(2) FIG. 2: SDS-PAGE of protein fraction separated on Q-sepharose (fast flow) column M: marker, BL: before loading, UB and W: Unbound proteins, 12-42 different fractions eluted from column.

(3) FIG. 3: Purified protein separated on 2-D PAGE.

(4) FIG. 4: MALDI-TOF-TOF analysis of the isolated protein

(5) FIG. 5: Expression and purification of the insecticidal protein of Tectaria in E. coli. M: protein molecular weight marker; lane 1, uninduced sample; lane 2, 1 h post induction; lane 3, 2 h post induction; lane 4, 3 h post induction; lane 5, Ni-NTA purified protein; lane 6, fusion protein digested with SUMO-Protease I; lane 7, Negative purification of insecticidal protein on Ni-NTA. Arrowhead in lanes 2-5 indicates the desired fusion protein and in lanes 6 and 7 indicates desired protein after digestion with SUMO protease and after purification, respectively.

(6) FIG. 6: pepsin digestibility and thermal stability of the insecticidal protein.

DETAILED DESCRIPTION OF THE INVENTION

(7) 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.

(8) 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: (a) isolating chitinase protein from leaf of fern Tectaria sp. in a manner such as herein described, (b) cloning c-DNA from purified protein, using N-terminal sequencing data of the purified protein by designing degenerate primers, (c) identifying ORF sequence encoding mature polypeptide of insecticidal chitinase from cloned cDNA sequence, (d) cloning the DNA encoding the insecticidal protein in E. coli expression vector in fusion with SUMO peptide to get expression of recombinant protein followed by purification of recombinant protein by conventional manner.

(9) 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.

(10) 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.

(11) In yet another embodiment of the invention, the insecticidal protein can be produced by the expression of recombinant DNA.

(12) 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.

(13) 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.

(14) 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 (FIG. 3). The purified protein was subjected to Mass Spectrometric analysis and N-terminal sequencing (Example 2). Mass spectrometric analysis on MALDI-TOF TOF platform (FIG. 4) established novelty of the molecule. Its insecticidal activity has not been reported earlier. The degenerate primers were designed using the N-terminal sequencing data (Table 1) and the gene encoding protein was cloned from the cDNA, synthesized from the total RNA, isolated from the plant leaves (Example 3). The protein was of 216 amino acid residues (Sequence ID No. 4) and the mature peptide of 192 amino acid residues (Sequence ID No. 5) with respective molecular weight of 23.684 kDa and 21.27 kDa. The cloned cDNA consisted of 828 nucleotides (Sequence ID No. 1), of which protein encoding ORF sequence is of 651 nucleotides (Sequence ID No. 2) and mature peptide encoding ORF is of 579 nucleotides (Sequence ID No. 3).

(15) 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.

(16) 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.

(17) In another embodiment of the invention, an isolated protein as claimed in claim 1 is toxic to whitefly (Bemisia tabaci).

(18) In yet another embodiment of the invention, the protein can be used for the control of other insect pests.

(19) 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.

(20) 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.

(21) 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.

(22) In yet another embodiment of the invention, an insecticidal protein can be produced by the expression of recombinant DNA.

(23) 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.

(24) 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.

(25) In the further embodiment of the invention, the plant expression cassette was transformed in cotton for the development of transgenic plants resistant to whiteflies.

(26) 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.

Example 1

Extraction of Total Soluble Protein and Insecticidal Activity Guided Purification

(27) 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 (3000g, 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 OD.sub.280 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.

Example 2

Peptide Mass Finger Printing and N-Terminal Sequencing

(28) Peptide Mass Finger Printing:

(29) 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.

(30) N-Terminal Sequencing:

(31) For N-terminal sequencing, the purified protein was run on SDS-PAGE and transferred onto the PVDF membrane and used for N-terminal sequencing.

(32) TABLE-US-00001 TABLE 1 N-terminal sequencing data of the insecticidal protein Position 1st choice 2nd choice, X - no clear signal 1. H 2. G 3. S 4. M 5. E 6. D 7. P 8. I 9 S 10. X R 11. X V 12. X Y 13. X Y 14. X Y, R 15. X 16. X 17. X L 18. X E

Example 3

Cloning of the Toxin Encoding Gene

(33) 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).

Example 4

Insect Bioassay Against Whiteflies (Bemisia tabaci)

(34) 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 262 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 (FIG. 1). After the collection of whiteflies, tubes were capped and kept in inverted position. Artificial diet (with/without insecticidal protein) was filter sterilized through syringe filter (0.22 m) and sandwiched (100 l) between the two layers of sterilized stretched parafilm on inner surface of the sterile specimen tube caps aseptically. The caps of the bioassay tubes containing insects were replaced with the diet containing caps. The tubes were kept in upright position so that the caps faced toward light. The old caps were replaced with caps containing fresh diet of respective test sample on alternate days to minimize the chances caused by degradation of test sample and contamination in diet. Perforations were made on the bioassay vial for air exchange.

(35) TABLE-US-00002 TABLE 2 Toxicity of purified protein against whitefly (Bemisia tabaci) Protein conc. % Mortality g/ml 2.sup.nd day 3.sup.rd day 4.sup.th day 5.sup.th day 6.sup.th day 7.sup.th day 100 87.5 96.87 100 50 56.75 78.37 93.7 100 25 16.07 56.75 78.37 94.64 100 12 15.62 21.87 28.12 53.12 62.5 76.34 5 13.15 15.78 21.05 28.94 31.57 36.84 2 13.63 13.63 22.72 27.27 27.27 34.09 Control 0 6.25 9.37 12.5 18.75 18.75

Example 5

Expression and Purification of Insecticidal Protein in E. coli

(36) 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).

(37) 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 (13000g, 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.

Example 6

Biosafety Evaluation of the Insect Toxic Protein

(38) The biosafety of protein was evaluated using online allergic domain search and pepsin digestibility test.

(39) Allergen Search

(40) The online search using allergen data based revealed that the protein has no allergic domains and therefore expected not to cause allergic responses.

(41) Pepsin Digestibility

(42) 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).

(43) Thermal Stability

(44) 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 l aliquot that contained 2.5 g of protein was analyzed on a 12% SDS-PAGE gel. The experiment was performed in triplicate. 2.5 g of treated protein was used for the enzymes assay also. The protein was found unstable at the temperatures beyond 90 C. (FIG. 6)

(45) TABLE-US-00003 NucleotidesequenceofthecompletecDNAofthebio-active proteinencodinggene SequenceI.D.NO.1 acgcggggatcggtcatagtgtgagccttgaggatggggaggtcatggggagttgtggct 60 gttatggtgttgtgcgccagtggcctgctgggcatagtgcgcggccatggcagcatggag 120 gaccccatcagtcgcgtctacagatgccgtctagagaatccggagcgtcccacgtcgcca 180 gcttgccaagcggcggtggcgctcagtggcactcaagccttctatgattggaatgaggcg 240 aacattcctaacgccgctggccggcaccgcgagctcattccggatggccaactgtgcagc 300 gccgggcggttcaagtttcggggcctcgacttggcacgctccgactggatagccaccccc 360 tcgccctccggcgccagcagcttcccattccgctacatagccaccgccgcgcacttgggc 420 ttcttcgagttctacgtcaccagggaaggttaccagcccactgtaccgcttaaatgggca 480 gacttggaggagttgccgttcatcaacgtcaccaaccccccgcttgtcagcggctcctac 540 caaatcaccggcaccacgccttcctgcaagtccggcagccacgtcatgtacgtcatatgg 600 cagcgcaccgacagccccgaagccttccactcctgctccgacgtctacttcactgatgcc 660 ctctctctccactctaccacctaggaggagggcgctctgttgggccacttctctctctct 720 ctctctctctctctctcggggcagtgctctcgtgctcggaatgctcctgtaattacaata 780 agaaatgaacatgtttctttcgcctctctaaaaaaaaaaaaaaaaaaa. 828

(46) Protein coding ORF sequences was were predicted by ORF finder software of NCBI

(47) TABLE-US-00004 Nucleotidesequenceofthefull-lengthbioactiveproteinencoding ORF SequenceIDNo.2 atggggaggtcatggggagttgtggctgttatggtgttgtgcgccagtggcctgctgggc 60 atagtgcgcggccatggcagcatggaggaccccatcagtcgcgtctacagatgccgtcta 120 gagaatccggagcgtcccacgtcgccagcttgccaagcggcggtggcgctcagtggcact 180 caagccttctatgattggaatgaggcgaacattcctaacgccgctggccggcaccgcgag 240 ctcattccggatggccaactgtgcagcgccgggcggttcaagtttcggggcctcgacttg 300 gcacgctccgactggatagccaccccctcgccctccggcgccagcagcttcccattccgc 360 tacatagccaccgccgcgcacttgggcttcttcgagttctacgtcaccagggaaggttac 420 cagcccactgtaccgcttaaatgggcagacttggaggagttgccgttcatcaacgtcacc 480 aaccccccgcttgtcagcggctcctaccaaatcaccggcaccacgccttcctgcaagtcc 540 ggcagccacgtcatgtacgtcatatggcagcgcaccgacagccccgaagccttccactcc 600 tgctccgacgtctacttcactgatgccctctctctccactctaccacctag. 651 Nucleotidesequenceencodingmaturebio-activeprotein SequenceIDNo.3 catggcagcatggaggaccccatcagtcgcgtctacagatgccgtctagagaatccggag 60 cgtcccacgtcgccagcttgccaagcggcggtggcgctcagtggcactcaagccttctat 120 gattggaatgaggcgaacattcctaacgccgctggccggcaccgcgagctcattccggat 180 ggccaactgtgcagcgccgggcggttcaagtttcggggcctcgacttggcacgctccgac 240 tggatagccaccccctcgccctccggcgccagcagcttcccattccgctacatagccacc 300 gccgcgcacttgggcttcttcgagttctacgtcaccagggaaggttaccagcccactgta 360 ccgcttaaatgggcagacttggaggagttgccgttcatcaacgtcaccaaccccccgctt 420 gtcagcggctcctaccaaatcaccggcaccacgccttcctgcaagtccggcagccacgtc 480 atgtacgtcatatggcagcgcaccgacagccccgaagccttccactcctgctccgacgtc 540 tacttcactgatgccctctctctccactctaccacctag. 579

(48) ORF sequence was translated to the amino acid sequences by Expasy translate tools http://www.expasy.ch/tools/dna.html.

(49) TABLE-US-00005 Aminoacidsequenceofthefull-lengthbio-activeprotein SequenceIDNo.4 MGRSWGVVAVMVLCASGLLGIVRGHGSMEDPISRVYRCRLENPERPTSPACQAAVALSGT 60 QAFYDWNEANIPNAAGRHRELIPDGQLCSAGRFKFRGLDLARSDWIATPSPSGASSFPFR 120 YIATAAHLGFFEFYVTREGYQPTVPLKWADLEELPFINVTNPPLVSGSYQITGTTPSCKS 180 GSHVMYVIWQRTDSPEAFHSCSDVYFTDALSLHSTT. 216

(50) 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.

(51) TABLE-US-00006 Aminoacidsequenceofthematurebio-activeprotein SequenceIDNo.5 HGSMEDPISRVYRCRLENPERPTSPACQAAVALSGTQAFYDWNEANIPNAAGRHRELIPD 60 GQLCSAGRFKFRGLDLARSDWIATPSPSGASSFPFRYIATAAHLGFFEFYVTREGYQPTV 120 PLKWADLEELPFINVTNPPLVSGSYQITGTTPSCKSGSHVMYVIWQRTDSPEAFHSCSDV 180 YFTDALSLHSTT. 192