Molecular Identification Of Steinernema Sp. YL001 And Cloning Gene Of Insecticidal Toxin

Symbiotic bacteria of entomopathogenic nematodes are symbiose in intestines of entomopathogenic nematodes. Symbiotic bacteria play an important role in the pathogenicity of insect. The relationship of symbiotic bacteria and nematode can be summed up as: nematodes carry symbiotic bacteria into insect hosts and release to haemocoel of insect; Symbiotic bacteria reproduces and produces insecticidal toxin protein with high activity which leads to die from septicemia within 48h. The insecticidal toxin protein has the advantage of highly active, broad spectrum, high speed and so on. Cloned the gene of insecticidal toxin from symbiotic bacteria is used to develop the new biological insecticidal resources in order to prevent the resistant. Symbiotic bacteria have become the biological resources which remainto be developd.So accuracy of classification and identification is the basis to carry out the relevant studies.Molecular identification: Steinernema sp. YL001 was separated by research and development center of biorational pesticide Northwest Agriculture and Forestry University which is high-drug strain. Based on the sequence of 16S rDNA reported previously a pair of primers P1P2 was designed. A specific band(568bp in length)was amplified from DNA of Steinernema sp. YL001. The accession number in Genbank is EF206655 which had 99.5% similarity on nucleotides level to XN16RNA0. Construct the phylogenetic trees showed the relationship with Xenorhabdus nematophilus was the nearest. Compared the 16S rDNA from Steinernema sp. YL001 with other symbiotic bacteria showed that Steinernema sp.YL001 had 99.5% similarity on nucleotides level to Xenorhabdus nematophilus and its divergence was 0.5. So the Steinernema sp. YL001 was identified as Xenorhabdus nematophilus.Clone the gene of insecticidal toxin: Based on the sequence of Pilin Subunit reported previously a pair of primers P3P4 was designed. A specific band was amplified from DNA of Xenorhabdus nematophilus YL001.The segment was cloned into T-easy vector. Analyzed the results of sequencing showed that the length of PSYL001 was 537bp, 179 amino acids were encoded. The predicted MW and pI are 18.9ku and 6.94.The accession number in Genbank is DQ537944.Comparison showed that this gene has 99% similarity on nucleotides level to other Pilin Subunit gene.