Biochemical Characterization and Molecular cloning of Lectin Protein Isolated from Garlic (Allium Sativum)

Abstract

One of the most important direct defense responses in plants against the attack by phytophagous insects is the production of insecticidal peptides or proteins known as Iectins. Plant Iectins have been shown to be toxic to some insects, including sucking pests, aphids, in particular Iectins from garlic. Structural studies have demonstrated that the mannose-binding specificity of Iectins is mediated by distinct structural scaffolds. Thus, in the present study attempt was made to biochemically characterize Allium satium agglutinin and to clone LECASAI gene in an expression vector pCAMBIA 1302. The lectin protein from garlic (Allium sativwn) was successfully extracted using PBS buffer and partially purified by dialysis. The presence of Allium sativwn Agglutinin was verified using a blood agglutination test with trypsin treated rabbit erythrocytes having a titre value 6 in dilution process. ASA possesses carbohydrate binding site for mannose sugar was determined by haemagglutination inhibition test. Using SDS-PAGE analysis, the molecular weight was determined to be approximately 11 .5 kDa. The protein content of ASAI was determined by Lowry assay method and was 323 J.lg/mg whereas, the carbohydrate content was 3.59%. ASA protein was most stable between 20-60°C, having maximum activity at pH 7. Further, the metal ions have no effect on haemagglutination property of agglutinin however, CaClz and CuS04 salts inhibited the agglutination property completely and slightly respectively. A 1033bp DNA fragment amplified using modified primers construct Lee 1 F & construct Lee 1 R was eluted from recombinant pTZ57R!f vector. The pCAMBIA plasmid was prepared by the restriction digestion by removing gus region. The restricted gene was then ligated in prepared plasmid and then subjected to clone into E.coli DH5a. The band of 1033bp was showing positive result in colony PCR. It can be concluded that genetic engineering of insecticidallectins will be useful tools that can contribute to the development of integrated pest management strategies with minimal effects on non-target organisms. So there is needed to make transgenic crops containing lectin to fulfill the requirement of increasing world's population.