Cloning Of Chitinase Gene And Development Of Wettable Powders From Xenorhabdus Nematophila HB310Strain

Xenorhabdus nematophila HB310(Xn HB310) is insect pathogen bacteriasymbiotically associated with entomopathogenic nematode Steinernema carpocapsae,which is motile gram-negative bacteria belonging to the family of Enterobacteriaceae. XnHB310had high and broad spectrum insecticidal activity and antifungal activity. In orderto clarify the antifungal ingredient, the chitinases from Xn HB310was analyzed. Inaddition, the wettable powder formulation for Xn HB310was developed. The resultswere as the followings.1. The bioassay results to antifungal activities of Xn HB310showed that Culturebroth and Intracellular proteins had obvious antifungal activities on mycelia growth ofpathogenic fungus of apple valsa canker. A chitinase was indentified from Xn HB310intracellular protein by mass spectrometry. These data illustrate that the antifungalactivities of Xn HB310could be related with the chitinase.2. According to the chitinase genes of X. nematophila ATCC19061strain, two pairsof primers were designed. Two chitinase genes, chi60and chi70, were cloned by PCRfrom Xn HB310genomic DNA. Sequencing results indicated that chi60and chi70had1647bp open reading frame (ORF) and1947bp ORF with deduced535amino acidsequence and648amino acids, respectively. The sequences of chi60and chi70had beendeposited in GenBank with accession no. KC701470and KC701470. In order to get therecombinant CHI60protein and CHI70protein, the expression vector pET-21b-chi60andpET-21b-chi70were constructed and transformed into Escherichia coli BL21(DE3).After IPTG induction, neither chi60nor chi70genes were successfully expressed inE.coli.3. A bioinformatic analysis suggested that CHI60protein was a stable water-solubleprotein with a molecular weight of59.3kDa isoelectric point of4.51, but no signalpeptide. The CHI70was also a stable water-soluble protein with a molecular weight of72.4kDa, isoelectric point of4.89and no signal peptide. The secondary structurepredictions of CHI60and CHI70had been performed by four different methods. At thesame time, the accurate three-dimensional structures of the two proteins were predictedby homology modeling. It was found that chitinases have certain conserved regions by multiple sequence alignment. At last the phylogenetic trees of the two proteins werebuild, which indicated that the chitinases have potential application in bacterial taxonomyand phylogeny.4. The formulation of50%Xn HB310wettable powder was obtained through thescreening carriers, wetting agents and dispersers. The optimal formulation was confirmedas follows:50.0%Xn HB310bacterium powder,5.0%sodium lignin sulfonate as disperser,5.0%nekal BX as wetting agent,0.2%skimmed milk powder as ultraviolet protectant,39.8%white carbon black as carrier. The quality detecting results showed that all indicatorsof this product accorded with the national standards of wettable powder formulation. Theexperiment in field showed that the diluted500times of50%Xn HB310WP still had82.9%lethallty rate to2ndinstar larvae of Plutella xylostella after spraying72hours. Thiswork illustrated that Xn HB310wettable powder had commercial potential.