Cloning, Expression And Insecticidal Spectrum Analysis Of The PirAB Gene From Photorhabdus Luminescens TT01

Bacteria of the genera Xenorhabdus and Photorhabdus have a mutual symbioticrelationship with entomopathogenic nematodes (EN) of the genus Steinernema andHeterorhabditis, respectively。In nature, this type of bacteria lives in the intestine of ENinfective larvae. The nematodes carry the symbiotic bacteria into the insect haemocoel, wherethe bacteria grow and reproduce and then secret insecticidal protein toxins to kill the insects.Cloning of symbiotic insecticidal toxin genes and understanding their insecticidal mechanismhelp to understand the pathogenesis of EN and the development of transgenic insect-resistantcrops.There are two loci，each institutes two ORF, in the genome of Photorhabdus luminescensTT01: ORF plu4093-plu4092(referred to as pirA1B1) and ORF plu4437-plu4436(referred toas pirA2B2). The predicted amino acid sequence of ORF plu4437and plu4436has aconsistency of50%and45%with ORF plu4093and plu4092, respectively. PirA1B1has beenconfirmed with oral insecticidal activity against Plutella xylostella, Aedes aegypti, Culex pipiens and Gangya anopheles, but the insecticidal activity disappeared if pirB1wasknockouted. Follow-up studies demonstrated that pirA1B1is a binary insecticidal toxin geneand it existed in other bacteria. Is pirA2B2a binary insecticidal toxin gene? If so, why theexistence of two binary insecticidal toxin genes in the same genome? Are there any difference(such as insecticidal spectrum) between them?In order to answer the above questions, pirA2, pirB2, pirA2B2and pirA1B1genes werePCR amplified and cloned. The recombinant expression vector pQE-pirA2, pQE-pirB2,pQE-pirA2B2and pQE-pirA1B1were constructed and transferred into E. coli M15,individually. The soluble PirA2, PirB2, PirA2B2and PirA1B1proteins were detected fromthe supernatant of the recombinant M15induced with IPTG by both SDS-PAGE andWestern-blot. The proteins expressed in the four recombinant E. coli M15strains werepurified by affinity chromatography combined with desalination technology and thenquantified individually. The heamocoal and oral insecticidal activities of the expressedproteins were analyzed against the larvae of Galleria mellonella、Spodoptera litura、Culexquinquefasciatus and Aedes albopictus. The results showed:1. PirA2B2has heamocoalinsecticidal activity against the fifth instar larvae of both G. mellonella and S. litura, with anLD50of4.0and2.8μg/larvae, respectively,2. neither PirA2nor PirB2alone has heamocoalinsecticidal activity against the insects tested, while the mixture of PirA2and PirB2reconstituted full activity;3. PirA2B2show oral insecticidal activity to C. quinquefasciatusand A. albopictus, with a LC50of37.3and26.5μg/ml, respectively. The results abovedemonstrate that the pirA2B2is another binary insecticidal toxin gene presented in the TT01genome.Comparison on the insecticidal virulence of PirA1B1and PirA2B2showed that theformer displayed stronger haemocoel insecticidal activity to G. mellonella, S. litura(Lepidoptera), T. molitor (Coleoptera) and the Blattella. germanica (Blattellidae) thanPirA2B2, and the same on oral insecticidal activity to C. quinquefasciatus and A. albopictus.For all insects tested, none was more susceptive to PirA2B2than Pir A1B1, which isinconsistent with the hypothesis that the presence of two binary toxin genes in TT01genomeis to kill different insect hosts. But this maybe due to the number of insect species tested is not enough.Bioinformatics analysis showed that, pirAB genes distributed widely in Photorhabdusbacteria, Xenorhabdus bacteria and Yersinia bacteria. further analysis results show that pirABgenes being transfered by mean of horizontal transfer. BT toxin_N superfamily domain andJacalin-like lectin domain were detected in PirB amino acid sequence, indicating that thePirAB may kill the insects by pore-forming and/or heamaglutiningThe present study has successfully1. cloned and expressed pirA2，pirB2，pirA2B2andpirA1B1genes and purified the resulted protein products by prokaryotic gene expression andprotein purification technologies;2. confirmed that pirA2B2gene is a binary insecticidal toxin;and3. compared the insecticidal activities of PirA2B2to pirA1B1. This study laid afoundation for further study on the function, insecticidal mechanism and expressionregulation of the binary toxins.