Patent Application
20150073126
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.
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.
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.
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.
Allium fistulosum
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.
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.
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.
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.
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.
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.
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—
In another embodiment of the present invention the primers GSP1 and GSP2 comprising
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.
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.
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.
In yet another embodiment of the present invention the polypeptide has the minimum haemagglutination value of in the range of 6-10 ng/ml.
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.
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.
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,
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,
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. The 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 the Seq. ID No. 1, to obtain the mature protein encoding gene sequence, similar to the 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 (
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
Allium sativum agglutinin (standard)
Allium fistulosum leaf agglutinin
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 LC50 using probit analysis.
Allium sativum
Galanthus nivalis
Allium fistulosum
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′).
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. 1 Nucleic acid sequences encoding full-length AFAL
Seq. ID no. 2 Nucleic acid sequences encoding mature AFAL.
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
The gene encoding mature AFAL was cloned in E. coli expression vector in fusion with SUMO peptide under T7 promoter. SUMO had (His6) 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 (
Purification of SUMO-AFAL from E. coli
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 (
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 LC50 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 3850/DEL/2011 | Dec 2011 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IN2012/000822 | 12/17/2012 | WO | 00 |
