Allium fistulosum leaf agglutinin rcombinant protein, its encoding polynucleotide, primer and process for preparation thereof

一种重组蛋白、凝集素的技术，应用在葱叶凝集素的核酸序列编码领域，能够解决破坏膜完整性、昆虫摄取食物或吸收营养物质的能力降低、破坏组织完整性等问题

Inactive Publication Date: 2015-01-21
COUNCIL OF SCI & IND RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the exact mechanism of the insecticidal action of the lectin is not fully understood, three different modes of action have been proposed - (1) the lectin binds to the peritrophic matrix of the midgut, inhibiting the absorption of nutrients ( Harper et al. 1998, Tissue Cell 30:166–176), (2) binds to glycoproteins on epithelial cells of the midgut and disrupts tissue integrity (Powell et al. 1998, J Insect Physiol.44:529–539; Sauvion et al. 2004, J Insect Physiol.50:1137–1150), and (3) bind to carbohydrate moieties of sensory receptors in the insect's mouth, disrupting membrane integrity and interfering with the insect's ability to detect food (Murdock et al. 2002 , J Agric Food Chem. 50:6605–6611)
All of these mechanisms result in a reduced ability of the insect to ingest food or absorb nutrients, resulting in stunted growth and premature death

Method used

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  • Allium fistulosum leaf agglutinin rcombinant protein, its encoding polynucleotide, primer and process for preparation thereof
  • Allium fistulosum leaf agglutinin rcombinant protein, its encoding polynucleotide, primer and process for preparation thereof
  • Allium fistulosum leaf agglutinin rcombinant protein, its encoding polynucleotide, primer and process for preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Isolation and purification of proteins

[0055] Onion leaves were collected from the National Institute of Plant Genetic Resources at Regional Centre, Bhawali, Uttaranchal, India. Its protein was purified according to a published protocol (Smeets et al., 1997b). The purified protein was further purified using a 50 kDa cut-off filter unit. Purified proteins were pooled on 10 kDa cut-off filters and stabilized in phosphate buffered saline (PBS) for experiments.

Embodiment 2

[0056] Example 2. Peptide Mass Fingerprinting Method

[0057] The purified protein was resolved by SDS-PAGE. Protein bands were excised and trypsinized prior to peptide mass fingerprinting. Bioinformatics analysis (MASCOT search) was performed on the data. Its peptide was found to match the mannose-binding lectin ( figure 2 ).

Embodiment 3

[0058] Example 3. Hemagglutination test

[0059] Hemagglutination tests were performed using rabbit erythrocytes in V-bottom microtitre plates. The total volume tested was 100 μl, and 50 μl aliquots of 2-fold serial dilutions of the lectin in PBS were mixed with 50 μl of a 2% trypsinized suspension of rabbit erythrocytes. The microplate was incubated for 1 hour at room temperature. Agglutination was assessed visually. The titer of cell agglutination was taken as the reciprocal of the maximum dilution of the lectin with measurable agglutination.

[0060] Table 1. Hemagglutination tests for AFAL and their comparison with ASAL

[0061] S.N.

[0062] 2

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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

technical field [0001] The present invention relates to Allium fistulosum leaf agglutinin (AFAL) recombinant protein, its encoding polynucleotide, primers, and a preparation method thereof. The allium fistulosum leaf agglutinin (AFAL) protein has insecticidal activity and hemagglutination activity . In particular, the present invention relates to the nucleic acid sequence (afal) coding for allium agglutinin (AFAL) which can be used in hemagglutination and insect control. Background technique [0002] Plant lectins, also called "lectins", are a heterogeneous group of sugar-binding proteins capable of reversibly binding monosaccharides and / or complex carbohydrates (Van Damme et al. 1998; CRC Crit Rev Plant Sci 17:575–692). They exhibit marked heterogeneity in molecular structure, sugar-binding specificity, and spatiotemporal regulation. Mannose-binding lectins are ubiquitous in higher plants and exert remarkable defense against microbial pathogens and plant predators due to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/11C12N15/70C07K14/42A01P7/04A01N63/50
CPCA01N63/02C07K14/42A01N37/46C12Q1/6895A01N63/50A01N47/44
Inventor 普拉丹优纳·库马尔·辛格普里提·拉伊拉赫尔·辛格桑托什·库马尔·乌帕德亚沙拉德·绍拉巴哈尔帕·辛格普拉韦恩·钱德拉·维尔马钱德拉谢卡尔·克里希纳帕拉凯什·图利
Owner COUNCIL OF SCI & IND RES
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