Cloning and expression of chitinase A, B, and C (chiA, chiB, chiC) genes from Serratia marcescens originating from Helicoverpa armigera and determining their activities

Authors: MEHTAP DANIŞMAZOĞLU, İSMAİL DEMİR, KAZIM SEZEN, HACER MURATOĞLU, REMZİYE NALÇACIOĞLU

Abstract: Three genes encoding chitinase A (chiA), B (chiB), and C (chiC) were amplified from a bacterium that was isolated from a naturally dead Helicoverpa armigera larva and identified as Serratia marcescens based on 16S rRNA gene sequence analysis (KF823633 accession number). The open reading frames (ORFs) were identified as 1692, 1500, and 1443 base pairs for chiA, chiB, and chiC genes, respectively. These sequences were submitted to the GenBank with accession numbers KF823630 (chiA), KF823631 (chiB), and KF823632 (chiC). Comparison of the deduced amino acid sequences with those of other bacterial chitinases revealed that the 3 chitinases contain the catalytic domain. Furthermore, all 3 chitinases showed 99% similarity to the S. marcescens WW4 strain at the amino acid level. The chitinases were overexpressed in Escherichia coli. Expressed proteins were purified and their activities were tested using colloidal chitin as substrate. Reasonable pH and temperature ranges were also determined as 7-11 and 33-37 °C, respectively. Insecticidal activities of these proteins were tested on the larvae of Malacosoma neustria and H. armigera. Test results showed that 1000 U/mL ChiA, ChiB, and ChiC have 47%, 50%, and 66% insecticidal activities on M. neustria, and 80%, 45%, and 50% insecticidal activities on H. armigera larvae within 10 days, respectively.

Keywords: Serratia marcescens, chiA, chiB, chiC, insecticidal activity

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