Gene Cloning And Function Study Of Mcf Insecticidal Gene From Photorhabdus Luminescens

Xenorhabdus spp. and Photorhabdus spp. are harbored as symbionts in the intestine of the infective juvenile stage of nematodes belongs to the families steinernematidae and Heterorhabditide, respectively. The insecticidal protein toxins produced by these two bactria have oral insecticidal activity and Hemocoel insecticidal activity. The photorhabdus luminescens W14 toxin (Mcf) which makes caterpillars floppy (mcf) was discovered due to its ability to kill caterpillars when was expressed in Escherichia coli. Mcf causes shedding of the insect midgut epithelium and destructive blebbing of haemocytes suggesting it may trigger apoptosis. In this study the insecticidal toxin Mcf was heteroexpressed by recombinant technique for insect control.Through searching for amino acids homology sequences in the protein Databases. BH3 domain was found in the N-terminal of Mcf toxin. It was speculated that the C-terminal truncated Mcf toxin with BH3 domain in the N-terminal might have the insecticidal activity because of the function of apoptosis of BH3 domain. The 5’-end of mcf insecticidal toxin gene from Photorhabdus luminescens subsp. Laumondii, which was 3 960 bp in size, was cloned into pET28a vector and transformed into BL21(DE3). After induced by IPTG, a 145 kDa protein in size was detected on SDS-PAGE gel. The oral insecticidal activity of truncated Mcf toxin was tested against the 1th-instar of Spodoptera exigua. Hemocoel insecticidal activity of truncated Mcf toxin was determined against 4th-instar of Spodoptera exigua. The present study demonstrated that the C-terminal truncated Mcf toxin had the insecticidal activity when was expressed in E. coli.The expression of mcf gene was driven by the crylAc promoters in Cry-B. Constructing the expression vector pHT1AcPTMcf through Red/ET homologous recombination techniques. Transformant the plasmid pHT1AcPTmcf into Cry-B, expression of Mcf was indentified by the technique of SDS-PAGE and Western-blotting. The result showed that the engineering strain espressed the truncated insecticidal Mcf toxin successfully. The oral insecticidal activity of truncated Mcf toxin was tested against the 1st-instar of Spodoptera exigua. The present study demonstrated that the C-terminal truncated Mcf toxin had the insecticidal activity when expressed in Cry-B.This work the C-terminal truncated Mcf toxin was hetero-expressed successfully. It is important foundation for constructing the engineering strains for insect contol. Applied this technique could contribute to the delay of resistance development among insects and to improve efficiency toxicity of biological pesticide.