Cloning, expression and prediction of insecticidal toxin Txp40 from Photorhabdus luminescens NLK-1

Abstract:

[Background] Photorhabdus sp. is preserving in the intestine of Heterorhabditis sp., and it can produce variety efficient and broad spectrum insecticidal proteins and toxins. In recent years, it’s the novel research object on new insecticidal protein and gene after Bacillus thuringiensis. [Objective] To clone Txp40 toxin gene of Photorhabdus luminescens (NLK-1) and analyze the differences on gene sequence, protein composition, physicochemical properties and conformation similarity to other symbiotic bacteria. Txp40 toxin gene was cloned in prokaryotic expression vector of Escherichia coli and induced its to express, then utilized Galleria mellonella larvae to preliminary detect its insecticidal activity. [Methods] The primary symbiotic bacteria were isolated from the infected larvae by Heterorhabditis bacteriophora, and the primers were designed according to the reported sequences. The target gene was amplified and sequenced by the cloning plasmid pMD19-T. The Expasy Online ProtParam Tool to predict its basic physical and chemical properties parameters, NPS@-Network Protein Sequence Analysis online tools for secondary structure prediction. Txp40 toxin gene was detected by cloning, digestion and ligation on pET28a and transformed into Escherichia coli BL21 then induced by IPTG, and identified by blue-white blotting. Utilizing Photorhabdus luminescens (NLK-1) Txp40 protein crude extract to test the insecticidal activity to Galleria mellonella larvae by fed and hemocole injection. [Results] Txp40 toxin gene open reading frame is 1 008 bp (335 amino acid), and the similarity to the known gene was 94%. The similarity with the known 40 kD related protein was 99%, molecular weight was 37.9 kD, pI 8.37, and it was mainly composed of Alpha helix 35.71%, Random coil 54.46%, Extended strand 9.52% by secondary structure predicting. The expression of pET28a-Txp40 was detected by SDS-PAGE, which showed that there were specific band at 38 kD, and the molecular weight of protein was similar with prediction, and the expression was relatively single and the level was higher. The insecticidal activity tests showed that Photorhabdus luminescens (NLK-1) Txp40 protein had high virulence to Galleria mellonella larvae, and the lethality reached 100% at 48 h under 5 μL Txp40 protein crude extract per larvae, but oral toxicity was not detected. [Conclusion] Photorhabdus luminescens (NLK-1) Txp40 toxin gene was successfully obtained, and the physicochemical properties of the protein were analyzed by comparison to the known gene, and secondary structure were predicted and analyzed to other known protein. At the same time, the prokaryotic expression vector was constructed, the preliminary insecticidal test showed Photorhabdus luminescens (NLK-1) Txp40 protein had high hemocole toxicity to Galleria mellonella larvae. The research laid the foundation for the further study and discovery of insecticidal genes and toxic protein from Photorhabdus luminescens (NLK-1).