Patent Application: US-201214368443-A

Abstract:
nucleic acid sequence encoding allium fistulosum leaf agglutinin is disclosed . the invention provides allium fistulosum leaf agglutinin 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 and bemisia tabaci 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:
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 , fig1 & amp ; 2 ). the purified protein was used for brief characterization . 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 . 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 , fig4 ). 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 ). in the embodiment of the invention , the cloned full - length gene sequence of allium fistulosum leaf agglutinin ( afal ) similar to seq id no : 1 . seq id no : 1 contains dna sequence which encodes n - terminal signal peptide , mature peptide and c - terminal peptide . in another embodiment of the invention , nucleotides encoding n - terminal signal peptide and c - terminal peptide are removed from seq id no : 1 , to obtain the mature protein encoding gene sequence , similar to seq id no : 2 . 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 . 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 . 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 . in yet another embodiment of the invention , the afal agglutinated rabbit erythrocytes . in yet another embodiment of the invention , the afal was tested against insect pests like cotton aphid ( aphis gossypii ) and whiteflies ( bemisia tabaci ). 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 . 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 . 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 ( fig2 ). 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 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 . 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 ′; seq id no : 11 }( primer sequence was derived from the mannose - binding site , qxdxnxvxy ( seq id no : 10 ), 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 ′; seq id no : 5 ) gsp2 ( 5 ′- gccccttggcctcctgca - 3 ′; seq id no : 9 ). the full - length gene was amplified and cloned . the mature afal encoding dna was amplified with primers 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 . seq id no : 3 amino acid sequence of the allium fistulosum leaf agglutinin seq id no : 4 amino acid sequence of the mature allium fistulosum leaf agglutinin expression of afal with n - terminal fusion of sumo in e . coli and purification of sumo - afal 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 ( fig5 ). approximately half of the recombinant protein was in the soluble form and rest as inclusion in the pellet . 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 ( fig6 ). 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 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 . bandyopadhyay s , roy a , das s ( 2001 ) binding of garlic ( allium sativum ) leaf lectin to the gut receptors of homopteran pests is correlated to its insecticidal activity . plant sci 161 : 1025 - 1033 harper s m , hopkins t l , czapala t h ( 1998 ) effect of wheat germ agglutinin on formation and structure of the peritrophic membrane in european corn borer ( ostrina nubilalis ) larvae . tissue cell 30 : 166 - 176 hossain m a , maiti m k , basu a , sen s , ghosh a k , sen s k ( 2006 ) transgenic expression of onion leaf lectin gene in indian mustard offers protection against aphid colonization . crop sci 46 : 2022 - 2032 murdock l l , shade r e ( 2002 ) lectins and protease inhibitors as plant defenses against insects . j agric food chem 50 : 6605 - 6611 powell k s , spence j , bharathi m , gatehouse j a , gatehouse a m r ( 1998 ) immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper nilaparvata lugens ( stal ). j insect physiol 44 : 529 - 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