Insecticidal Activity And Function Of Chitinases And Secretory Proteins Posp2 And Posp9 From Penicillium Ochrochloron

As an important entomopathogenic and wood-degrading fungus,Penicillium ochrochloron plays an important role in maintaining the balance of natural ecosystem and has great biocontrol potential.P.ochrochloron Q-3-1 strain isolated from the larvae of the Hepialidae in Kangding,Sichuan Province.Papilio xuthus,Dialeurodes citri,Panonychus citri,Galleria mellonella and Diaphorina citri insecticidal activity was tested.DGEs data the strains infect citrus psyllid.Ananlysis of chitinase and small molecules secreted proteins Posp2,Posp9 function was carried.In order to lay a theoretical foundation for the molecular mechanism,genetic improvement and application research of insecticidal and stress resistance of P.ochrochloron.The results are as follows:1.Insecticidal activity of P.ochrochloronImpregnation method is used to determine the virulence of P.ochrochloron Q-3-1strain of Diaphorina citri adults,nymphs and Dialeurodes citri nymphs.The results showed that the LT₅₀ of Diaphorina citri adults was 16 h(1×10⁸ spores m L^-1).The LC₅₀values for adults were 1.2×10⁴ spores m L^-1,when the spore suspension of Q-3-1 strain was treated for 24 h,and the LT₅₀ of Diaphorina citri adults was 16 h when the spore suspension was treated with 1×10⁸ spores m L^-1.The LC₅₀ of Q-3-1 strain to Dialeurodes citri nymphs was 1.5×10⁷ spores m L^-1,and the LT₅₀ was 96 h.The toxicity of Q-3-1 strain to the female adults and eggs of P.citri was determined by spray method.After treatment for 12 h,the LC₅₀ of female adults of P.citri was 1×10⁵ spores m L^-1,and the LT₅₀ was 16.1 h.The LC₅₀ for eggs was 2.25×10⁷ spores m L^-1.The insecticidal activity of Q-3-1 against the 4th instar larvae of P.xuthus was determined by impregnation method.The LC₅₀ was 5.57×10⁵ spores m L^-1,and the LT₅₀ was 48 h.The insecticidal activity of Q-3-1 strain against the 3rd instar larvae of G.mellonella was determined by injection method.The LC₅₀ of the Q-3-1 strain against the 3rd instar larvae of G.mellonella was 1.29×10⁷ spores m L^-1,and the LT₅₀ of the spore suspension of G.mellonella were 15.3 h.P.ochrochloron has a widely insecticidal spectrum,strongly insecticidal activity and good quickly efficacy,and has the potential to be a biocontrol agent.2.Analysis of DGEs of Diaphorina citri infected by P.ochrochloronIn this study,high-throughput sequencing technology was used to establish the expression profile of P.ochrochloron Q-3-1 strain 24 hours after infection with D.citri adults,and a large number of reliable data were obtained（141.55 G in the experimental group）.Six hundred differentially expressed genes were obtained after 24h infection with Q-3-1 strain,among which 451 genes were up-regulated and 139 genes were down-regulated.The results showed that the secretory proteins was related to the fungal defense response,bacterial defense response,carbohydrate metabolism in response to stress biological process,and even had chitinin-binding molecular hydrolase activity.In addition,the secretory protein genes and chitinase genes of P.ochrochloron were induced to express.Chitinase genes in P.ochrochloron were mainly concentrated in carbohydrate metabolism,epithelial cell development,and catalytic hydrolase activity on chitin.Analysis the RT-q PCR eleven secretory protein genes and eleven chitinase genes of expression in the haemolymph of G.mellonella larvae,the results showed that Posp2,Posp9,Poch4,Poch5,Poch8,Poch10 and Poch11 genes with higher expression.They may be closely related to insecticidal activity,so mainly on these two five secreted protein gene and chitinase gene were studied.3.Functional study of the chitinase from P.ochrochloronEleven chitinases gene were found from the genome data of P.ochrochloron.Eleven chitinases belonging to the GH18 family,clustering analysis of the protein sequences showed that Poch1,Poch2 and Poch3 were closely related to each other and clustered into a branch.Poch4,Poch6,Poch10 and Poch11 were closely related and grouped into a branch.There are four chitinase gene clustered with plant pathogenic fungi together,while three chitinase gene clustered with entomopathogenic fungi together.There other genes clustered into the unknown.RT-q PCR was used to analyze the spatiotemporal expression of 11 chitinase genes in the epidermis,hemolymphoid,adipose tissue and midgut of during Q-3-1 strain infection of G.mellonella larvae.The relative expression levels of six genes,such as Poch1,Poch2,Poch4,Poch8,Poch9 and Poch11,were relatively high in the tissues of G.mellonella larvae.The relative expression levels of Poch3,Poch5,Poch6,Poch7 and Poch10 in infected larvae were very low.The Poch1 gene mainly acted on the epidermis and midgut of the larva.Poch2mainly acted on the hemolymph of G.mellonella larvae.As time goes on,the expression of Poch4 in adipose tissue increased gradually,and reached the highest expression level in adipose tissue at 40 h.The expression of Poch8 gene in hemolymph and midgut reached the highest level after infection 32 h,and then decreased rapidly in hemolymph.While Poch9 gene mainly acts on epidermis,Poch11 gene mainly acts on epidermis and fat tissue.We selected Poch1,Poch8 and Poch11 genes for prokaryotic expression,and obtained Pc1,Pc8 and Pc11 recombinant proteins.All three recombinant chitinases could degrade chitin,citrus pectin,apple pectin,dextran and cellobiose.The utilization rate of insect chitin by pc11 was higher than that of plant pectin,and different p NP substrates could be degraded randomly.Pc1 can effectively degrade insect chitin and plant pectin,has high activity to p NP-β-D-glucopyranoside and p NP-β-xylopyranside.Pc8 has a very high enzyme activity for cellobiose,and its activity for different p NP substrates is similar.It is speculated that the main mode of action of Pc8 is to specifically recognize and catalyze disaccharide.4.Functional study of the Posp2 and Posp9 from P.ochrochloronThe full length of Posp2 was 615bp with signal peptide,protein was nighty-nigh percent of similar to the hypothetical protein of Beauveria bassiana.Posp2 protein has three cysteines and the protein was mainly composed of chain proteins formed byβ-folded sheets,which formed the tertiary structure.RT-q PCR analysis showed that the expression level of Posp2 gene was higher in midgut and adipose body at the late stage of infection.Westenblot analysis showed that Posp2 protein could not be detected in wild plants,epidermis and adipose tissues.In the midgut,a large number of proteins were detected in 12 h,24 h,32 h and 40 h tissue samples,suggesting that Posp2 protein mainly acts in the midgut of insects.The total length of Posp9 was 519bp,and protein was nighty-nigh percent of similar to the hypothesized protein of B.bassiana.There are five cysteines and signal peptides.The tertiary structure is mainly formed by the chain proteins formed byβ-folding andβ-turning.The conformation after folding shows pseudo-double symmetry.RT-q PCR analysis showed that the expression level of Posp9 gene was high in the epidermis and blood at the early stage of infection.Westenblot analysis showed that the target protein could not be detected in the epidermis in the antibody-binding test of Posp9,but could be detected in the wild strains,midgut and adipose tissues.There were obvious bands in the midgut tissues at 12 h,24 h,32 h and 40 h,and the bands were obvious in the adipose body at 12 h.These results indicate that Posp9 plays an important role in host immune defense in the early stage of infection.