The Characteristics And Functions Of Chitinases And Chitin-Binding Protein From Xenorhabdus Nematophila

Xenorhabdus nematophila is gram-negative bacteria symbiotic with Steinernema carpocapsae,belonging to the Enterobacteriaceae.It has a wide spectrum of insecticidal activity and antifungal activity.The insecticidal toxins and antifungal substances from X.nematophila include chitinases and chitin-binding protein.In order to clarify the characteristics and function of chitinases and chitin-binding protein from X.nematophila,a bioinformatics analysis of all chitinase genes and chitin-binding protein genes in X.nematophila was performed.Then,the recombinant chitinases and chitin-binding protein were obtained by prokaryotic expression system of Escherichia coli,and the physiological and biochemical characteristics and biological activities of the recombinant chitinases and chitin-binding protein were measured.Finally,the relationship between chitinases(Xn-Chi60 and Xn-Chi70)and Xn-Tc toxin was understood by knocking out Xn-chi60 and Xn-chi70 genes.The main results are as follows:1.The genome of X.nematophila contain four chitinase genes and one chitin-binding protein gene.The chitinases encoded by these genes belong to glycoside hydrolases(GH)family 18 and GH family 20,respectively.Genome analysis results showed that X.nematophila in GenBank contained two family 18 chitinase genes Xn-chi60 and Xn-chi70(accession numbers:KC701470 and KC701471),two family 20 chitinase genes Xn-chi101 and Xn-chi25(accession numbers:MT199128 and MT219905)and one chitin-binding protein gene Xn-cbp(accession number:KY817118).The results of bioinformatics analysis showed that Xn-chi60 had an open reading frame(ORF)of 1608bp,coding 535 amino acid.The deduced protein Xn-Chi60 has a 59.3kDa molecular weight,4.51 isoelectric point and no signal peptide.Xn-chi70 had a 1947bp ORF,coding 648 amino acid.The deduced protein Xn-Chi70 has a 72.4kDa molecular weight,4.89 isoelectric point and no signal peptide.Xn-chi101 had a 2700bp ORF,coding 899 amino acid.The deduced protein Xn-Chi 101 has a 101.43kDa molecular weight,6.94 isoelectric point and one signal peptide.Xn-chi25 had a 3678bp ORF,coding 225 amino acid.The deduced protein Xn-Chi25 has a 25.31kDa molecular weight,10.21 isoelectric point and one signal peptide.Xn-cbp had a 600bp ORF,coding 199 amino acid.The deduced protein Xn-CBP has a 22.08kDa molecular weight,8.00 isoelectric point and one signal peptide.These five proteins were stable water-soluble protein and no transmembrane domain.The secondary structure predictions showed that Xn-Chi60 had 19 ?-helix and 14 ?-pleated sheets,Xn-Chi70 had 24 ?-helix and 20?-pleated sheets,Xn-Chi101 had 25 ?-helix and 36 ?-pleated sheets,Xn-Chi25 had 8?-helix and 6 ?-pleated sheets,while Xn-CBP had 4 ?-helix and 7 ?-pleated.The three-dimensional structures predictions showed that Xn-Chi60 and Xn-Chi70 have typical(?/?)8 double barrel structures and belong to GH family 18,while Xn-Chil01 and Xn-Chi25 have ?/? barrel folds and belong to GH family 20.The phylogenetic tree showed that Xn-Chi60 and Xn-Chi70 were from the same branch,Xn-Chi101 and Xn-Chi25 were from the same branch,Xn-CBP and the chitin-binding proteins of bacteria from Xenorhabdus and Photorhabdus were formed a branch.2.Expression of chitinase genes and chitin-binding protein gene from X.nematophila and the purification of recombinant proteins.The prokaryotic expression vectors of pET-28a-chi101 and pET-28a-cbp were constructed and transferred into E.coli Transetta(DE3)and Trans BL21(DE3),respectively.The recombinant proteins Xn-Chi101 and Xn-CBP were successfully expressed as inclusion bodies.At the condition of 28? and final concentration of 0.5mmmol/L IPTG,the expression of inclusion bodies was the highest.After denaturation-renaturation of inclusion bodies,a single band of soluble protein was obtained.Substrates binding assays showed that Xn-CBP had the strongest binding ability to colloidal chitin.3.Clarified the enzymatic properties of the chitinases from X.nematophila.The enzymatic properties of the recombinant chitinases Xn-Chi60,Xn-Chi70 and Xn-Chi101 were measured using DNS method.The results showed that the optimal pH and the optimal temperature of Xn-Chi60 were 6.0 and 50?,respectively,Ca2+,Cu2+,Fe3+,DTT,SDS and EDTA inhibited the enzyme,while Zn2+ and Mn2+stimulated the enzyme.The optimal pH and temperature of Xn-Chi70 were 6.0 and 50?,respectively,Ca2+,Cu2+,Fe3+,Fe2+,DTT,SDS and EDTA inhibited the enzyme,while Zn2+ and Mn2+stimulated the enzyme.The optimal pH and the optimal temperature of Xn-Chi101 were 7.0 and 40?,respectively.Ca2+,Cu2+,Fe3+,DTT and SDS inhibited the enzyme,but Mg2+stimulated the enzyme.The results of substrate specificity and enzyme kinetics showed that chitinases Xn-Chi60,Xn-Chi70 and Xn-Chi101 could specifically bind to colloidal chitin,and the binding ability of Xn-Chi70 was higher than Xn-Chi60 and Xn-Chi101.4.The insecticidal activities and antifungal activities of recombinant chitinases and chitin-binding protein.The results of insecticidal activities showed that the inhibition rates of chitinases and chitin-binding protein on the growth of Helicoverpa armigera larvae were all more than 80%at the concentration of 1mg/mL.Similar to Bt-Chi73,Xn-Chi70 had the strongest inhibitory effect on the growth of H.armigera,and the growth inhibition rate was over 90%.The results of synergistic effects showed that different chitinases had different synergistic effects on Bt Cry1Ac toxin and Xn-Tc toxin,Xn-Chi70 have the highest synergistic effect on Bt Cry1Ac toxin and Xn-Tc toxin that was similar to Bt-Chi73.Xn-Chi101 had no obvious synergistic effect,while Xn-Chi60 and Xn-CBP showed no synergistic effect.However,Xn-CBP could increase the synergistic effect of all chitinases on two toxins.The results of antifungal activities showed that chitinases and chitin-binding protein could inhibit the mycelial growth and spore germination of Coniothyrium diplodiella,Fusarium oxysporum,Alternaria solani and Rhizoctonia solani.Overall,the antifungal activities of several chitinases from X.nematophila were significantly lower than Bt-Chi73 from B.thuringiensis.5.Clarified the relationship between two chitinases(Xn-Chi60 and Xn-Chi70)of X.nematophila and Xn-Tc toxin.The single Xn-chi60 gene mutant strain,single Xn-chi70 gene mutant strain,double Xn-chi60 and Xn-chi70 gene mutant strain were successfully screened by pJQ200SK suicide plasmid knockout system.Then,the insecticidal activity of Xn-Tc toxin from wild-type strain and mutant strain were determined.The results showed that the toxicity of Xn-Tc toxin from single Xn-chi60 gene mutant strain,single Xn-chi70 gene mutant strain,double Xn-chi60 and Xn-chi70 gene mutant strain were 3.861 times,4.517 times and 102.240 times lower than the wild-type strain,respectively.These results indicate that the presence of these two chitinases is indispensable for the toxicity of Xn-Tc toxin.