5169
Baiome Abd El-Maguid Ali
CLONING AND CHARACTERIZATION OF INSECTICIDAL PROTEIN ENCODING GENES FROM THE SYMBIOTIC BACTERIA WITH ENTOMOPATHOGENIC NEMATODE
Photorhabdus , Xenorhabdus, RAPD, ISSR, SRAP,Txp40
Three symbiotic bacterial strains isolated, from Heterorhabditis and two from Steinernema were identified as Photorhabdusluminescens HRM1, P. luminescens HS1, P. luminescens HP88, Xenorhabdusindica and X. nematophila based on 16S rDNA sequence analysis. The genetic diversity among these bacterial strains was resolved using three molecular markers (RAPD, ISSR and SRAP). RAPD analysis showed 73.8 and 54.5 polymorphism percentages for the PhotorhabdusandXenorhabdusstrains respectively. ISSR analysis resulted in 70.1 and 75.2 polymorphism percentages among the Photorhabdus and Xenorhabdus strains, respectively. Twenty five SRAP combinations of five forward and five reverse primers were used to characterize these bacterial strain. The Photorhabdusstrains showed 75.6 polymorphism percentages among them while Xenorhabdus strains showed 61.2% polymorphism. Txp40 toxin gene from P. luminescens HP88 strain was identified. The data indicated that, the the sequence obtained was highly similar to the complete sequence of the txp40 genes of P. luminescens strains with the score of (99%) with P. luminescens TTO1 strain, (98%) with P. luminescens V16 strain and (98%) with P.luminescens HI strain. Total recombinant toxin protein was shown to be active against G. mellonella by direct injection into the larvae. Syringed proteins extracted from transformed E.coli DH5α strain killed a high percentage of G. mellonella larvae. Whereas 10mM phosphate buffer at pH 7.2used as a control did not. The recombinant protein at the concentration of 19.4 mg/ml, 9.7 mg/ml and 4.85 mg/ml injected per larvae showed 100%, 93% and 20%, respectively. While the other total syringed proteins extracted from wild type E. coli bacteria strain DH5α did not have the same impact on the larvae. These results indicated that the expression of Txp40 gene in E. coli strain DH5α caused significant mortality to G. mellonella larvae and this results indicated that this recombinant protein can be used in future IPM programs to control the economic pests and can be used as a replacement to insecticides in agriculture application.
2015
M.Sc
Cairo
Agriculture