Enhancement of nematicidal potential through cloning and expression of chitinase gene from Bacillus subtilis subsp. Subtilis BTN7A strain

Abstract

A gene encoding chitinase from B. subtilis has been isolated after optimization of PCR conditions. It was cloned with two different prometers, T7 promoter of the pJET1.2/blunt cloning vector and the SP6 promoter of pGEM®-T Easy vector. After transforming E. coli DH5α, two transformants were selected, CHI-NRC-4 from the first vector and T-CHI-NRC-6 from the second vector, and used for further studies. The complete CDS sequence of chitinase gene was determined and submitted to GenBank with the accession number KX268692.1. Culture supernatants of E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) were investigated for their inhibitory effect on M. javanica egg hatch under laboratory conditions. Result showed up to 96% inhibition in egg hatching due to both E. coli transformants as compared to control which reflect the same expression efficiency of both used prometers. A greenhouse experiment was carried out to evaluate the nematicidal effect of culture supernatants of the two transformts E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) against M. javanica infected eggplant. Obtained results showed a significant reduction in nematode population in soil and roots and enhancement in eggplant growth parameters as compared to control.

Keywords: Chitinase; Eggplant; Gene cloning; Meloidogyne javanica; Nematode.

Fig. 1

Agarose gel electrophoreses of chitinase gene amplification from recombinant plasmids. Lane 1: T-NRC-CHI-6, lane 2: T-NRC-CHI-101 and lane 3: CHI-NRC-4.

Fig. 2

Plasmid CHI-NRC-4.

Fig. 3

The recombinant plasmid T-NRC-CHI-6.

Fig. 4

Inhibition effect of supernatant of transformed E. coli with chitinase gene on M. javanica eggs.