Molecular cloning and expression of lectin gene (srl) from sclerotium rolfsii sacc.
| dc.contributor.advisor | Ramesh Bhat | |
| dc.contributor.author | T.M.Chandrashekar | |
| dc.date.accessioned | 2016-10-18T09:11:30Z | |
| dc.date.available | 2016-10-18T09:11:30Z | |
| dc.date.issued | 2007 | |
| dc.description.abstract | In the present study, an effort was made to clone srl gene encoding Sclerotium rolfsii lectin, and express it in Escherichia coli and Saccharomyces cerevisiae. The presence of lectin in sclerotial bodies was confirmed by haemagglutination assay. Hapten inhibition assay indicated that it had sugar specificity for Mucin and Asialofetuin. A PCR product of ~450bp amplified from S. rolfsii DNA using degenerate primers was cloned into pTZ57R/T. The sequence showed an open reading frame (ORF) of 426bp, encoding 142 amino acids. BLASTp with deduced protein of srl (DP-srl) showed high homology with SRL, ABL (Agaricus bisporus lectin) and XCL (Xerocomus chrysenteron) confirming that it is a fungal lectin. DP-srl showed a maximum of 75% identity and 89% similarity with the SRL (Acc. No. 2OFC_A). Structural comparison between deduced protein of srl (DP-srl) and SRL at primary (Tyr27, Ala28, Ser47, Gly48, His70, Asn71, Tyr72, Arg105) and secondary (Asp77, Ile78, Thr80, Arg101, Tyr112, Val114) carbohydrate-binding sites showed no difference for primary structure. Of the 35 mismatches between DP-srl and SRL, 7 were conservative and 6 were ambiguous substitutions. Amino acid sequence alignment of related fungal lectins identified two conserved regions (Ser47 to Gly51 and Gly68 to Lys73). DP-srl had higher similarity (73%) with XCL than SRL (72%). Coding sequence of srl gene was cloned into pET-32b(+), the resulting vector (pCR29) was transferred to BL21(DE3)pLysS. Similarly, pCR14 yeast expression vector containing the SRL coding region in pYES2/CT and transferred to S. cerevisiae strain, INVSc1. Quantity of heterologous protein produced in E. coli and yeast was 7.50 and 0.78μg/μl respectively. SDS-PAGE analysis of the purified heterologous proteins from pCR29 and pCR14 showed protein bands of corresponding sizes. Hemagglutination assay and hepten inhibition assay confirmed that the expressed protein is Sclerotium rolfsii lectin | en_US |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/80771 | |
| dc.publisher | UAS, Dharwad | en_US |
| dc.research.problem | Molecular cloning and expression of lectin gene (srl) from sclerotium rolfsii sacc. | en_US |
| dc.sub | Plant Biotechnology | en_US |
| dc.theme | Molecular cloning and expression of lectin gene (srl) from sclerotium rolfsii sacc. | en_US |
| dc.these.type | M.Sc | |
| dc.title | Molecular cloning and expression of lectin gene (srl) from sclerotium rolfsii sacc. | en_US |
| dc.type | Thesis | en_US |