Functional Analysis Of The Class ? Hydrophobin Gene HFBII-4 From The Biocontrol Agent Trichoderma Asperellum ACCC30536

Plant diseases cause significant economic losses to agriculture and forestry production.Biological control has great potential in plant disease control because of its low toxicity,low residue and environmental friendliness.To study the function of hydrophobin gene HFBII-4from Trichoderma asperellum ACCC30536.Firstly,the HFBII-4 gene was obtained from T.asperellum ACCC30536.Its cDNA sequence is 294 bp in length,encoding 97 aa with a predicted protein molecular weight of 9.74kDa and pI of 4.41.The GenBank Accession Number of its cDNA is KY914555.The HFBII-4protein belongs to Hydrophobin-2 superfamily and can secrete into the extracellular.Multiple sequence alignment of 29 hydrophobins from different fungal species was conducted using the ClustalX program,revealing that the eight cysteine residues that form four disulfide bridges were highly conserved in all 29 hydrophobins.Secondly,to study the functions of HFBII-4 protein,the recombinant eukaryotic expression vector p PIC9K-HFBII-4 was successful structured and the recombinant strain GS115-HFBII-4 was obtained.The recombinant eukaryotic protein r HFBII-4 was obtained under the methanol inducing,a clearly visible band with molecular weight of approximately9.74 kDa on the Tricine-SDS-PAGE gel.The result indicated that the HFBII-4 gene had been successfully expressed in Pichia Pastoris.Lastly,the genes related to the auxin,jasmonic acid and salicylic acid signal transduction pathways were up-regulated after the interaction with renatured rHFBII-4.The results showed that the expression level of AUX1,LAX2,TIR1,IAA8 and GH3 genes were higher than that of the control and showed a peak expression of 2^-1.43,2^2.00,2^1.66,2^1.12 and 2^1.52 folds at 24 h,respectively.The expression level of PR1 and NPR1 genes were higher than that of the control and showed a peak expression of 2^6.26 and 2^1.72 folds at 24 h,respectively.The expression level of JAR1,COI,COA,JAZ and MYC2 genes were higher than that of the control and showed a peak expression of 2^1.53,2^6.18,2^9.55and 2^3.85folds at 24 h,respectively.The expression level of ORCA3 gene increased at 2 h and reached the maximum of 2^1.10folds.Compared with control,the enzymatic activity of Pdpap poplar was strongly changed.The activity of PPO and PAL were higher than that of the control under the inducing of recombinant protein r HFBII-4.The chlorophyll content and the soluble protein content were higher than that of the control,and reached a peak of 1.98 and 16.61 mg^-1.g at 5 and 3 d,respectively.At the same time,the Evans BLue and Nitro-blue tetrazolium?NBT?staining assays indicated that the cells membrane permeability and the active oxygen content of leaves were lower than that of the control.The disease spots in the Pdpap poplar leaves were obviously smaller than that of control under the inducing of recombinant protein r HFBII-4.To sum up,our results show that the rHFBII-4 can promote Pdpap poplar growth and enhance the resistance of Pdpap poplar against pathogen.These results will provide a theoretical basis and practical reference for the development of biological small hydrophobins and establish a good foundation for production and application of new pesticides.