Abstract:
Nucleic acid sequence encoding  Allium fistulosum  leaf agglutinin (AFAL) is disclosed. The invention provides  Allium fistulosum  leaf agglutinin (AFAL) recombinant protein, its encoding nucleotides, primers and the process of preparation thereof, said recombinant protein is useful for insect control and haemagglutination activity. AFAL is found more toxic to sap sucking insect pest  Aphis gossypii  (cotton aphid) and  Bemisia tabaci  (whiteflies) as compared to known  Allium sativum  leaf agglutinin. AFAL can be used in the development of transgenic plants for resistance against sap sucking and chewing pests.

Description:
FIELD OF THE INVENTION 
       [0001]    The invention relates to  Allium fistulosum  leaf agglutinin (AFAL) recombinant protein, its encoding polynucleotide, primers and process of preparation thereof said  Allium fistulosum  leaf agglutinin (AFAL) protein is useful for insecticidal activity and haemagglutination activity. In particular the present invention relates to nucleic acid sequence (afal) encoding for  Allium fistulosum  leaf agglutinin (AFAL) applicable for haemagglutination and insect control. 
       BACKGROUND AND PRIOR ART OF THE INVENTION 
       [0002]    Plant lectins, also known as “agglutinins”, are heterogeneous group of carbohydrate binding proteins, which are able to bind simple sugars and/or complex carbohydrates reversibly (Van Damme et al. 1998;  CRC Crit Rev Plant Sci  17:575-692). They show a marked heterogeneity with respect to their molecular structures, sugar binding specificity and temporal and spatial regulation. Mannose binding lectins are widely found in higher plants and play a significant role in defense due to their ability to recognize high-Mannose-type glycans of microbial pathogens and plant predators (Van Damme et al. 1998, 1998 , CRC Grit Rev Plant Sci  17:575-692; Van Damme et al. 2004  Trends Plant Sci  9:484-9). Mannose binding lectin from garlic leaf [ Allium sativum  leaf agglutinin (ASAL)] is a 25 kDa homodimeric protein, structurally and evolutionarily related to  Galanthus nivalis  agglutinin (GNA) (Smeets et al. 1997a,  Plant Mol Biol  35: 531-535; Van Damme et al. 1992 , Eur J Biochem  206:413-420). Some of the biological properties of ASAL are—(1) it readily agglutinates rabbit erythrocytes but does not affect human erythrocytes (Bandhopadhyay et. al. 2001,  Plant Sci.  161:1025-1033; Smeets et al. 1997a,  Plant Mol Biol  35: 531-535), (2) it has inhibitory effect against retrovirus (HIV1 and HIV2) induced cytopathicity in MT-4 cells (Smeets et. al. 1997b,  Plant Mol Biol  33:223-234) and (3) it is toxic (growth inhibitory) against a spectrum of insects of order Homoptera, Lepidoptera and Coleoptera. Some important pests inhibited by ASAL are aphids [mustard aphid, peach potato aphid, tobacco aphid (Bandhopadhyay et al. 2001,  Plant Mol Biol  33:223-234 Hossain et al. 2006,  Crop Sci  46:2022-2032; Smeets et al. 1997a,  Plant Mol Biol  35: 531-535), red cotton bug (Bandhopadhyay et al. 2001,  Plant. Mol. Biol.  33:223-234), brown plant hopper, green leaf hopper (Saha et al. 2006,  Plant Mol. Biol.  62:735-52) and cotton leaf worm (Sadeghi et al. 2008,  Transgenic Res.  17:9-18). Although exact mechanism of insecticidal action of lectins is still not well understood, three different modes of action have been proposed—(1) binding of the lectins to the peritrophic matrix of the midgut, inhibiting nutrient absorption (Harper et al 1998 , Tissue Cell  30: 166-176), (2) binding to glycoproteins on epithelial cells of the midgut and disrupting tissue integrity (Powell et al. 1998,  J Insect Physiol.  44: 529-539; Sauvion et al. 2004,  J Insect Physiol.  50: 1137-1150) and (3) binding to carbohydrate moieties of the sensory receptors of insect mouth parts, disrupting membrane integrity and interfering in the food detection ability of insects (Murdock et al. 2002,  J Agric Food Chem.  50: 6605-6611). All these mechanisms result in decreased ability of insect to ingest food or absorb nutrients, leading to delayed development and premature death. 
         [0003]    U.S. Pat. No. 5,545,820 (Gatehouse, et al., 1996) discloses the use of lectins having specific mannose-binding ability, derived from family Amaryllidaceae or Alliaceae for the control of insect pests. WO/1992/002139 relates to the use of lectins having specific mannose-binding ability, derived from family Amaryllidaceae or Alliaceae for the control of insect pests and the development of transgenic plants expressing such lectins. U.S. Pat. No. 5,407,454 relates to selected plant lectins having larvicidal activity against a number of common insect pests of agricultural crops. Insect resistance in the transgenic plants is due to insertion of larvicidal lectin gene in all the cells of the plants. 
         [0004]    U.S. Pat. No. 6,127,532 (Raikhel) refers to transgenic plants containing cDNA encoding Gramineae lectin. Such transgenic plants expressed barley lectin and stored in in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal′ properties. 
         [0000]    
       Allium fistulosum  
     
         [0005]      Allium fistulosum  L. (Welsh onion, Japanese bunching onion) is a perennial onion. Other names that may apply to this plant include green onion, spring onion, escallion, and salad onion. These names are ambiguous, as they may also be used to refer to any young green onion stalk, whether grown from Welsh onions, common bulb onions, or other similar members of the genus  Allium . The species is very similar in taste and odor to the related bulb onion,  Allium cepa , and hybrids between the two (tree onions). The Welsh onion, however, does not develop bulbs, and possesses hollow leaves (“ fistulosum ” means “hollow”) and scapes. Large varieties of the Welsh onion resemble the leek, such as the Japanese ‘negi’, whilst smaller varieties resemble chives. Many Welsh onions can be multiplied by forming perennial evergreen clumps. Next to culinary use, it is also grown as an ornamental plant. Historically, the Welsh onion was known as the cibol. 
         [0006]    The name “Welsh onion” has become a misnomer in modern English, as  Allium fistulosum  is not indigenous to Wales. “Welsh” preserves the original meaning of the Old English word “welisc”, or Old German “welsche”, meaning “foreign” (compare wal- in “walnut”, of the same etymological origin). The species originated in Asia, possibly Siberia or China. 
       Culinary Use 
       [0007]    In the West, the Welsh onion is primarily used as a scallion or salad onion, but is widely used in other parts of the world, particularly East Asia. 
         [0000]    Russia: Welsh onion is used in Russia in the spring for adding green leaves to salads.
 
Asia: The Welsh onion is an ingredient in Asian cuisine, especially in East and Southeast Asia. It is particularly important in China, Japan, and Korea, hence the other English name for this plant, ‘Japanese bunching onion’. Bulb onions were introduced to East Asia in the 19th century, but  A. fistulosum  remains more popular and widespread. In Japan, it is used in miso soup, negimaki (beef and scallion rolls), among others, and it is widely sliced up and used as a garnish on teriyaki or takoyaki.
 
Jamaica: Known as escallion, the Welsh onion is an ingredient in Jamaican cuisine, in combination with thyme, scotch bonnet pepper, garlic and allspice (called pimenta). Recipes with escallion sometimes suggest leek as a substitute in salads. Jamaican dried spice mixtures using escallion are available commercially. The Jamaican name is probably a variant of scallion, the term used loosely for the spring onion and various other plants in the genus  Allium.  
 
       Lacking in the Prior Art 
       [0008]      Allium  lectin disclosed in present invention shows high insecticidal activity and therefore novel. Homology of the nucleotide sequence with available nucleotide sequences in database shows more than 90% homology. 
       OBJECTIVES OF THE INVENTION 
       [0000]    
       
         
           
             a) The main objective of the invention is to provide nucleic acid sequence which encodes for  Allium fistulosum  leaf agglutinin 
             b) Another objective of the present invention is to provide gene specific premiers GSP1 (5′-ATGGACAGTACTCCATCTCCTAAAC-3′) and GSP2 (5′-TTAGCCCCTTGGCCTCCTGCA-3′), useful for amplification of the gene. 
             c) Another objective of the present invention is to provide agglutinin recombinant protein having high insecticidal activity 
             d) Another objective of the present invention is the application of  Allium fistulosum  leaf agglutinin protein for insect control. 
           
         
       
     
       SUMMARY OF THE INVENTION 
       [0013]    Accordingly, the present invention relates to  Allium fistulosum  leaf agglutinin (AFAL) recombinant protein, its encoding nucleotides, primers and the process of preparation thereof, said protein is useful for insect control and haemagglutination activity. 
         [0014]    In an embodiment of present invention, the process of preparation of  Allium fistulosum  leaf agglutinin (afal) recombinant protein, useful for insecticidal activity and haemagglutination activity, comprising of following steps—
       a) Extracting of total RNA from the  Allium fistulosum  leaves   b) Synthesizing of cDNA from total RNA extracted from leaves of  Allium fistulosum      c) Designing of primers GSP1 and GSP2 from 5′- and 3′-RACE fragment of c DNA to clone full-length protein encoding DNA, designing of primers GSP3 and GSP4 for cloning of the DNA encoding mature polypeptide of AFAL protein,   d) Expressing the DNA encoding the mature AFAL in  E. coli  SUMO expression vector where SUMO peptide is fused with AFAL at the N-terminus and expressed in  E. coli  under T7 promoter to get desired AFAL recombinant protein.       
 
         [0019]    In another embodiment of the present invention the primers GSP1 and GSP2 comprising
       1. GSP1 represented by sequence I.D:5   2. GSP2 represented by sequence I.D:6       
 
         [0022]      Allium fistulosum  leaf agglutinin (AFAL) recombinant protein is 8-10 fold more toxic to cotton aphid ( Aphis gossypii ) and 6-8 fold to whiteflies ( Bemisia tabaci ) as compared to well-known  Allium sativum  leaf agglutinin. 
         [0023]    According to the invention a DNA fragment of 651 bp was cloned from total cDNA of  A. fistulosum  leaves, consisted of, 585 bp long open reading frame encoding AFAL precursor protein of 194 amino acid residues with 28 amino acid long N-terminal signal peptide and 56 amino acid long C-terminal peptide. The amino acid sequences of AFAL are different from the other reported  Allium  lectin sequences. The cloned genomic DNA sequence of afal showed absence of intron. 
         [0024]    In another embodiment of the present invention the nucleic acid sequence having sequence ID. 2, encodes a polypeptide as represented in Seq ID No 4, which is a 110 amino acid residue long mature peptide of  Allium fistulosum  leaf agglutinin (AFAL), having a molecular weight of ˜12 kDa. 
         [0025]    In yet another embodiment of the present invention the polypeptide has the minimum haemagglutination value of in the range of 6-10 ng/ml. 
         [0026]    In yet another embodiment of the present invention the polypeptide exhibits insecticidal activity selected from the group comprising haemagglutination, insecticidal, antifungal and anti-prolific activity. 
         [0027]    In still yet another embodiment of the present invention the polypeptide exhibits haemagglutination activity selected from the group comprising haemagglutination, insecticidal, antifungal and anti-prolific activity. 
     
    
     
       BRIEF DESCRIPTION OF FIGURES 
         [0028]      FIG. 1A . Lane 1, Molecular weight markers; Lane 2, Total soluble protein from leaves of  A. fistulosum ; Lane 3, Unbound total soluble protein; Lane 4, Column wash (before elution); Lanes 5-8, eluted fractions.  FIG. 1B . Lane 1, Molecular weight markers; Lane 2, Purified AFAL concentrated on 10 kDa cut-off filtration device. 
           [0029]      FIG. 2 . Peptide mass fingerprinting of AFAL 
           [0030]      FIG. 3 . V-bottom plate showing haemagglutination of rabbit erythrocytes by AFAL 
           [0031]      FIG. 4  Clustalw analysis of AFAL with other  Allium lectins . Amino acid identity of AFAL with other mannose binding lectins vary from 67% to 75%. Dark area represents mannose binding domain. 
           [0032]      FIG. 5 . Lane 1, Molecular weight markers; lane 2. uninduced bacterial lysate; lane 3-5, sample after 1 h, 2 h and 3 h induction; lane 6, supernatant of 3 hr induced culture; lane 7, protein in inclusion. 
           [0033]      FIG. 6 . Lane 1, molecular weight markers; lane 2, total  E. coli  protein containing SUMO-AFAL; lanes 3-4 represent purified fusion protein. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0034]    The leaves of  Allium fistulosum  were collected from the National Bureau of Plant Genetic Resources, Bhowali, Nainital, Uttarakhand, India, and used for the purification of  Allium fistulosum  leaf agglutinin (AFAL). It was purified on mannose-agarose affinity column, followed by cut-off filtration device (Example 1,  FIGS. 1 &amp; 2 ). The purified protein was used for brief characterization. 
         [0035]    AFAL shows several times better insecticidal activity against sap sucking pest  Aphis gossypii  (cotton aphids),  Bemisia tabaci  (whitefly) as compared to ASAL (Example 3). Large amount of purified protein is required for further characterization. The purification of AFAL from plant leaves in bulk amount is difficult due to unavailability of plant material and very low accumulation of the lectin in leaves. Expression of protein in a re combinant system is an alternative approach to produce the desired protein in large amount, which required the cloning of AFAL encoding gene. The gene (afal) was cloned from cDNA prepared from total RNA of leaves of  A. fistulosum . The cloning was done by RACE (Rapid Amplification of cDNA Ends) using degenerate primers designed from the conserved mannose binding domain. 
         [0036]    The cloned DNA fragment gene was of 651 bp, consisted of 585 bp open reading frame, 66 bp 5′ untranslated leader sequence. The full-length gene encoding AFAL precursor protein of 194 amino acid residues had 28 amino acid long N-terminal signal and 56 amino acid long C-terminal peptide. It contains three mannose binding domains as reported in the case of other mannose binding lectins (Example 5). The cloned genomic DNA sequence of afal had no intron. The amino acid sequences of AFAL are different from the other reported  Allium  lectin sequences (Example 5,  FIG. 4 ). 
         [0037]    The gene encoding insecticidal protein was cloned in  E. coli  expression vector in fusion with SUMO peptide and the recombinant insecticidal protein was expressed. The protein was purified on Ni-NTA column. The recombinant protein showed the insecticidal activity against cotton aphids and whiteflies. The gene encoding the mature peptide was cloned in plant expression vector pBI121 under CaMVE35S promoter (Example 6-8). 
         [0038]    In the embodiment of the invention, the cloned full-length gene sequence of  Allium fistulosum  leaf agglutinin (afal) similar to Seq. ID No. 1. The Seq. ID no. 1 contains DNA sequence which encodes N-terminal signal peptide, mature peptide and C-terminal peptide. 
         [0039]    In another embodiment of the invention, nucleotides encoding N-terminal signal peptide and C-terminal peptide are removed from the Seq. ID No. 1, to obtain the mature protein encoding gene sequence, similar to the Seq. ID No. 2. 
         [0040]    In another embodiment of the invention, the gene sequence of Seq. ID No. 1 was translated to obtain the full-length amino acid sequences of AFAL similar to Seq. ID No. 3. 
         [0041]    In yet another embodiment of the invention, the gene sequence of Seq. ID No. 2 was translated to obtain mature AFAL with amino acid sequence similar to Seq. ID No. 4. 
         [0042]    In yet another embodiment of the invention, the gene encoding the mature AFAL is cloned in  E. coli  SUMO expression vector where SUMO peptide is fused with AFAL at the N-terminus and expressed in  E. coli  under T7 promoter. 
         [0043]    In yet another embodiment of the invention, the AFAL agglutinated rabbit erythrocytes. 
         [0044]    In yet another embodiment of the invention, the AFAL was tested against insect pests like cotton aphid ( Aphis gossypii ) and whiteflies ( Bemisia tabaci ). 
         [0045]    In yet another embodiment of the invention, the gene encoding the mature AFAL was cloned in plant expression vector under constitutive promoter CaMV35S and phloem specific promoters CoYMoV, RSS1, RolC etc. and expressed in transgenic plants for insect control. 
       (5) Examples 
     Example 1 
     Isolation and Purification of the Protein 
       [0046]      Allium fistulosum  leaves were collected from National Bureau of Plant Genetic Resources, Regional Centre, Bhawali, Uttaranchal, India. The protein was purified according to the protocol described (Smeets et al., 1997b). The purified protein was further purified on 50 kDa cut-off filtration device. The purified protein was concentrated on 10 kDa cut-off filter and stabilized in PBS for experiments. 
       Example 2 
     Peptide Mass Finger Printing 
       [0047]    The purified protein was resolved on SDS-PAGE. The protein band was excised and digested with trypsin and used for peptide mass finger printing. The data was analyzed on MASCOT search. The peptides were found matching to the mannose binding lectins ( FIG. 2 ). 
       Example 3 
     Haemagglutination Assay 
       [0048]    Haemagglutination assays with rabbit RBCs were carried out in V-bottomed microtitre plates. Total volume of assay was 100 μl, 50 μl aliquot of two-fold serially diluted lectin in PBS was mixed with 50 μl 1 of 2% trypsinized rabbit erythrocytes suspension. Microtitre plate was incubated for 1 hour at room temperature. Agglutination was assessed visually. Reciprocal of the highest dilution of lectin showing detectable agglutination was taken as titer of the haemagglutination 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Haemagglutination assay of AFAL and its comparision with ASAL 
               
             
          
           
               
                 S.N 
                 Agglutinin 
                 Haemagglutination 
               
               
                   
               
               
                 1 
                   Allium sativum  agglutinin (standard) 
                 200 ng/ml 
               
               
                 2 
                   Allium fistulosum  leaf agglutinin 
                  8 ng/ml 
               
               
                   
               
             
          
         
       
     
       Example 4 
     Insect Bioassay 
       [0049]    Insect bioassay was carried out against sap sucking pest, cotton aphid ( Aphis gossypii ) and whiteflies ( Bemisia tabaci ). The known amount of purified protein was mixed in synthetic diet and insect mortality data was recorded at different time interval. The data was used for the calculation of LC 50  using probit analysis. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Insecticidal activity of AFAL purified from leaves of  A. fisrulosum.   
               
             
          
           
               
                   
                   
                 Aphids 
                 Whiteflies 
               
               
                   
                   
                 ( Aphis gossypii ), 
                 ( Bemisia tabaci ), 
               
               
                 S.N 
                 Agglutinin 
                 LC 50   
                 LC 50   
               
               
                   
               
               
                 1 
                 
                   Allium sativum 
                 
                   68 μg/ml 
                   76 μg/ml 
               
               
                   
                 agglutinin (Standard) 
               
               
                 2 
                 
                   Galanthus nivalis 
                 
                 51.39 μg/ml 
                 53.39 μg/ml 
               
               
                   
                 agglutinin 
               
               
                 3 
                 
                   Allium fistulosum 
                 
                  7.1 μg/ml 
                  8.5 μg/ml 
               
               
                   
                 agglutinin 
               
               
                   
               
             
          
         
       
     
       Example 5 
     Gene Cloning and Characterization 
       [0050]    cDNA was synthesized following standard protocol. The 3′ RACE was performed with degenerate primer {5′-ATGCA(A/G)(C/G)A(G/T)GACTGCAACC-3′} (primer sequence was derived from the mannose-binding site, QXDXNXVXY, conserved among most of the monocot mannose-binding lectins) and universal primer. For 5′ RACE, RNA was reversely transcribed with the 5′-RACE CDS Primer. Based on the 3′ and 5′ RACE results, primers were designed for amplification of full length gene, GSP1 (5′-ATGGACAGTACTCCATCTCCTAAAC-3′) GSP2 (5′-GCCCCTTGGCCTCCTGCA-3′). The full-length gene was amplified and cloned. The mature AFAL encoding DNA was amplified with primers GSP3 (5′-AGAAACGTATTGGTGAACAACG-3′) and GSP4 (5′-TTATCTTCTGTA GGTACCAGTAGAC-3′). 
         [0051]    The amino acid sequence of AFAL was deduced with Expasy translate tool. The analysis and comparison of the deduced amino acid sequences and nucleotide sequences obtained in RACE was performed with blast p (Standard Protein-Protein BLAST), blastn (Standard Nucleotide-Nucleotide BLAST) on NCBI (www.ncbi.nlm.nih.gov) and clustal W. 
         [0052]    Seq. ID no. 1 Nucleic acid sequences encoding full-length AFAL 
         [0053]    Seq. ID no. 2 Nucleic acid sequences encoding mature AFAL. 
         [0054]    Seq. ID no. 3 Amino acid sequence of the  Allium fistulosum  leaf agglutinin 
         [0055]    Seq. ID no. 4 Amino acid sequence of the mature  Allium fistulosum  leaf agglutinin 
         [0000]    
       
         
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
           
               
                 Nucleic acid sequences encoding full length AFAL 
               
               
                 Seq. ID no. 1 
               
             
          
           
               
                 ATGGACAGTA CTCCATCTCC TAAACTAATG AGCATGACCA 
                 60 
               
               
                 CTGTAGCCAC CATCCTAACC 
                   
               
               
                 ATTTTGGCAT CTACATGCAT GGCCAGAAAC GTATTGGTGA 
                 120 
               
               
                 ACAACGAAGG ACTGTACGCA 
                   
               
               
                 GGCCAATCCC TAGTCGTAGA ACAGTACACT TTTACAATGC 
                 180 
               
               
                 AGGATGACTG CAACCTTGTA 
                   
               
               
                 CTCTACGAAT ACTGCGCCCC AATCTGGGCC TCAAACACGG 
                 240 
               
               
                 GCGTCACCGG CAAAAATGGG 
                   
               
               
                 TGCAGGGCCG TGATGCAGGC TGATGGCAAC TTTGTGGTCT 
                 300 
               
               
                 ACGATGTTAA CGGGCGTGCC 
                   
               
               
                 GTCTGGGCCA GTAACAGCAG AAGAGGGAAC GGAAACTATA 
                 360 
               
               
                 TCCTGGTGCT TCAGGAGGAC 
                   
               
               
                 AGGAACGTTG TTATTTACGG ATCTGATATT TGGTCTACTG 
                 420 
               
               
                 GTACGTACAG AAGAGGGCCC 
                   
               
               
                 GGTCCTGGTC CTGGTGCCGC CTGCAAGTGC GATGACGATG 
                 480 
               
               
                 GTCCTGACAT TCGCAGTGCT 
                   
               
               
                 ACTTTGACAG GCACTGTCGA TTTGGGAAGC TGCAACGAGG 
                 540 
               
               
                 GATGGGAGAA GTGCGCATCT 
                   
               
               
                 TTCTACACCA TCCTCGCGGA TTGCTGCAGG AGGCCAAGGG 
                 585 
               
               
                 GCTAA 
                   
               
               
                   
               
             
          
           
               
                 Nucleic acid sequences encoding mature AFAL. 
               
               
                 Seq. ID no. 2 
               
             
          
           
               
                 AGAAACGTAT TGGTGAACAA CGAAGGACTG TACGCAGGCC 
                 60 
               
               
                 AATCCCTAGT CGTAGAACAG 
                   
               
               
                 TACACTTTTA CAATGCAGGA TGACTGCAAC CTTGTACTCT 
                 120 
               
               
                 ACGAATACTG CGCCCCAATC 
                   
               
               
                 TGGGCCTCAA ACACGGGCGT CACCGGCAAA AATGGGTGCA 
                 180 
               
               
                 GGGCCGTGAT GCAGGCTGAT 
                   
               
               
                 GGCAACTTTG TGGTCTACGA TGTTAACGGG CGTGCCGTCT 
                 240 
               
               
                 GGGCCAGTAA CAGCAGAAGA 
                   
               
               
                 GGGAACGCAA ACTATATCCT GGTGCTTCAG GAGGACAGGA 
                 300 
               
               
                 ACGTTGTTAT TTACGGATCT 
                   
               
               
                 GATATTTGGT CTACTGGTAC GTACAGAAGA 
                 330 
               
               
                   
               
             
          
           
               
                 Amino acid sequence of AFAL 
               
               
                 Seq. ID no. 3 
               
             
          
           
               
                 MET ASP SER THR PRO SER PRO LYS LEU MET SER 
                 20 
               
               
                 MET THR THR VAL ALA THR ILE LEU THR 
                   
               
               
                 ILE LEU ALA SER THR CYS MET ALA ARG ASN VAL 
                 40 
               
               
                 LEU VAL ASN ASN GLU GLY LEU TYR ALA 
                   
               
               
                 GLY GLN SER LEU VAL VAL GLU GLN TYR THR PHE 
                 60 
               
               
                 THR MET GLN ASP ASP CYS ASN LEU VAL 
                   
               
               
                 LEU TYR GLU TYR CYS ALA PRO ILE TRP ALA SER 
                 80 
               
               
                 ASN THR GLY VAL THR GLY LYS ASN GLY 
                   
               
               
                 CYS ARG ALA VAL MET GLN ALA ASP GLY ASN PHE 
                 100 
               
               
                 VAL VAL TYR ASP VAL ASN GLY ARG ALA 
                   
               
               
                 VAL TRP ALA SER ASN SER ARG ARG GLY ASN GLY 
                 120 
               
               
                 ASN TYR ILE LEU VAL LEU GLN GLU ASP 
                   
               
               
                 ARG ASN VAL VAL ILE TYR GLY SER ASP ILE TRP 
                 140 
               
               
                 SER THR GLY THE TYR ARG ARG GLY PRO 
                   
               
               
                 GLY PRO GLY PRO GLY ALA ALA CYS LYS CYS ASP 
                 160 
               
               
                 ASP ASP GLY PRO ASP ILE ARG SER ALA 
                   
               
               
                 THR LEU THR GLY THE VAL ASP LEU GLY SER CYS 
                 180 
               
               
                 ASN GLU GLY TRP GLU LYS CYS ALA SER 
                   
               
               
                 PHE TYR THR ILE LEU ALA ASP CYS CYS ARG ARG 
                 194 
               
               
                 PRO ARG GLY 
                   
               
               
                   
               
             
          
           
               
                 Amino acid sequence of the mature AFAL 
               
               
                 Seq. ID no. 4 
               
             
          
           
               
                 ARG ASN VAL LEU VAL ASN ASN GLU GLY LEU TYR 
                 20 
               
               
                 ALA GLY GLN SER LEU VAL VAL GLU GLN 
                   
               
               
                 TYR THR PHE THR MET GLN ASP ASP CYS ASN LEU 
                 40 
               
               
                 VAL LEO TYR GLU TYR CYS ALA PRO ILE 
                   
               
               
                 TRP ALA SER ASN THR GLY VAL THR GLY LYS ASN 
                 60 
               
               
                 GLY CYS ARG ALA VAL MET GLN ALA ASP 
                   
               
               
                 GLY ASN PHE VAL VAL TYR ASP VAL ASN GLY ARG 
                 80 
               
               
                 ALA VAL TRP ALA SER ASN SER ARG ARG 
                   
               
               
                 GLY ASN GLY ASN TYR ILE LEU VAL LEU GLN GLU 
                 100 
               
               
                 ASP ARG ASN VAL VAL ILE TYR GLY SER 
                   
               
               
                 ASP ILE TRP SER THR GLY THR TYR ARG ARG 
                 110 
               
               
                   
               
             
          
           
               
                 Primer sequence GSP1: 
               
               
                 Seq. ID no. 5 
               
               
                 (5′-ATGGACAGTACTCCATCTCCTAAAC-3′). 
               
               
                   
               
               
                 Primer sequence GSP2: 
               
               
                 Seq. ID no. 6 
               
               
                 (5′-GCCCCTTGGCCTCCTGCA-3′). 
               
               
                   
               
               
                 Primer sequence GSP3: 
               
               
                 Seq. ID no. 7 
               
               
                 (5′-AGAAACGTATTGGTGAACAACG-3′). 
               
               
                   
               
               
                 Primer sequence GSP4: 
               
               
                 Seq. ID no. 8 
               
               
                 (5′-TTATCTTCTGTAGGTACCAGTAGAC-3′).. 
               
             
          
         
       
     
       Example 6 
     Expression of AFAL with N-Terminal Fusion of SUMO in  E. coli  and Purification of SUMO-AFAL 
       [0056]    The gene encoding mature AFAL was cloned in  E. coli  expression vector in fusion with SUMO peptide under T7 promoter. SUMO had (His 6 ) tag attached which helped in the purification of recombinantly expressed protein on Ni-NTA resin. SUMO-AFAL was expressed after induction with IPTG. The expression of the recombinant protein was observed every hour for 3 hours. After 3 hours of induction, cells were harvested by centrifugation; suspended in 20 mM TrisCl (pH 8). Bacterial cells were lysed by lysozymen and disrupted by sonication. The lysed bacterial cells were spun and supernatant and pellet were collected and electrophorased on denaturing PAGE ( FIG. 5 ). Approximately half of the recombinant protein was in the soluble form and rest as inclusion in the pellet. 
         [0000]    Purification of SUMO-AFAL from  E. coli    
         [0057]    Recombinantly expressed SUMO-AFAL was purified on metal-affinity column. Total bacterial protein was loaded on Ni-column, pre-equilibrated with the buffer (20 mM Tris pH 8, 300 mM NaCl and 10 mM Imidazole). NaCl and Imidazole were used to prevent the binding of non-specific proteins to the column. The column was washed with same buffer having 20 mM imidazole to remove low affinity bound proteins. Finally, the protein was eluted with 200 mM Imidazole ( FIG. 6 ). 
       Example 7 
     Insect Bioassay with Recombinant Fusion Protein 
       [0058]    Insect bioassay was carried out against sap sucking pest, cotton aphid ( Aphis gossypii ) and whiteflies ( Bemisia tabaci ). The known amount of SUMO-AFAL was mixed in synthetic diet and insect mortality data was recorded at different time interval. The data was used for the calculation of LC 50  using probit analysis. SUMO-ASAL served as positive control. The results of insect bioassay is shown in the table 3 
         [0000]    
       
         
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
             
             
               
                   
                   
               
               
                   
                 LC 50   
               
             
          
           
               
                   
                   
                   
                 Whiteflies 
               
               
                 S.N 
                 Agglutinin 
                 Aphids ( Aphis gossypii ) 
                 ( Bemisia tabaci ) 
               
               
                   
               
               
                 1 
                 Recombinant ASAL 
                 55.05 μg/ml 
                 51.2 μg/ml 
               
               
                   
                 (SUMO-ASAL) 
               
               
                 2 
                 Recombinant AFAL 
                  4.97 μg/ml 
                 8.80 μg/ml 
               
               
                   
                 (SUMO-AFAL) 
               
               
                   
               
             
          
         
       
     
       Advantages of the Invention 
       [0059]    The lectin protein being disclosed in the present invention (AFAL) is 6-10 folds more toxic to insects like aphids and whiteflies as compared to the standard  Allium sativum  leaf lectin (ASAL). AFAL also showed 25 folds higher haemagglutination activity as compared to ASAL. AFAL binds to lesser number of carbohydrate residues on glycans array as compared to ASAL. This assures fewer non-specific binding of AFAL to carbohydrates and therefore expected to be safe as compared to ASAL. 
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                 WO/1992/002139 
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                 Gatehouse et al 
               
               
                   
                 5,407,454 
                 Apr. 18, 1995 
                 Anthony et al 
               
               
                   
                   
               
             
          
         
       
     
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