Cloning And Biological Activity Study Of Tc Toxin Complexs From Photorhabdus Luminescens

Photorhabdus luminescens are facultative anaerobic,gram-negative Entomopathogenic bacteria which can produce biological fluorescence.It is symbiotic with Heterorhabditis and colonize the gut of nematode.Tc toxin complexes are the most main insecticidal protein with broad-spectrum,high efficiency insecticidal activity.When cultured alone,it will not produce such toxins or less toxic.Because of the clustering,coordination and complexity of Tc toxin gene cluster,there is no precedent for the full sequence heterologous expression.In my thesis we find six secret Tc gene sequences in TT01 named TcaZCB 6804 bp),TccAB3(7419 bp),TccAB1(10954 bp),Tcd(51406 bp),TccC4(2891 bp),TccC7(2871 bp)according to the NCBI data analysis.Using direct cloning technology,we cloned 4 Tc genes TcaZCB(plu0514-05150),TccAB3(plu0805-0806),TccAB1(plu4165-4169),Tcd(plu0960-0971)from Photorhabdus luminescens TT01 genome and constructed the recombinant vector p 15A-cm-TcaZCB,p15A-cm-TccAB3,p15A-cm-TccAB1,p15A-cm-Tcd.Later,the p15A-cm-TcaZCB recombinant vector was modified by LCHR to form the plasmid p15A-OriT-TnpA-Genta-TcaZCB.The recombinant vector p 15A-OriT-TnpA-Genta-TcaZCB was introduced into a widely distributed,fast-growing,multi-nutrient,simple and competitive plant growth promoting rhizobacteria-Pseudomonas fluorescens Pf5 by the conjugation.The recombinant strain Pf5-tcaZCB was successfully obtained by resistance screening and colony PCR and make growth curve.The results showed that the introduction of TcaZCB complex genes had no effect on Pf5 growth.In this study,we can provide more evidence for the heterologous expression host of Tc toxin complexs,and lay a foundation for the further study of genetic modification and insecticide mechanism of different Tc toxins.