[Cloning, expression and insecticidal activity of the pirA2B2 gene from Photorhabdus luminescens TT01]

The predicted amino acid sequence of locus plu4437-plu4436 in the genome of Photorhabdus luminescens TT01 (referred to as pirA2B2) has a consistency of 50% and 45% with locus plu4093-plu4092 (referred to as pirA1B1) , respectively. PirA1B1 has been confirmed with oral insecticidal activity against Plutella xylostella and mosquito. The purpose of the present study was to examine whether pirA2B2 possesses insecticidal activity.

pirA2, pirB2 and pirA2B2 genes were PCR amplified and cloned. The recombinant expression vector pQE-pirA2, pQE-pirB2 and pQE-pirA2B2 were constructed and transferred into E. coli M15, individually. The soluble PirA2, PirB2 and PirA2B2 proteins were detected from the supernatant of the recombinant M15 induced with IPTG by both SDS-PAGE and Western-blot. The proteins expressed in the three recombinant E. coli M15 strains were purified by affinity chromatography combined with desalination technology and then quantified individually. The heamocoal and oral insecticidal activities of the expressed proteins were analyzed against the larvae of Galleria mellonella and Spodoptera litura.

The results show: 1. PirA2B2 had heamocoal insecticidal activity against the fifth instar larvae of both G. mellonella and S. litura, with an LD50 of 4.0 and 2.8 microg/larvae, respectively, 2. neither PirA2 nor PirB2 alone had heamocoal insecticidal activity against the insects tested, while the mixture of PirA2 and PirB2 reconstituted full activity, and 3. PirA2B2 showed no oral insecticidal activity to the first instar larvae of either G. mellonella or S. litura.

pirA2B2 in the genome of P. luminescens TT01 is a binary insecticidal toxin gene.

The successful expression and purifcation of PirA2B2 laid a foundation for further study on the function, insecticidal mechanism and expression regulation of the binary toxins.