Heterologous Expression Hydrophobins Of Trichoderma Harzianum TH33 And Induction Of Tobacco Defense Response

Hydrophobins are a kind of small molecular proteins in higher filamentous fungi.They can automatically form amphiphilic monolayers at the two-phase interface to reduce surface tension,and therefore help fungi to form aerogenic structure,and facilitate spore differentiation and diffusion.In recent years,it has been found that hydrophobins are closely related to the surface interaction of fungi-pathogens and fungi-plants,such as participating in the pathogenicity of pathogenic fungi and inducing plant disease resistance by beneficial fungi.In order to further develop and utilize the beneficial gene resources of Trichoderma,researches on hydrophobin of T.harzianum TH33 were carried out in this study.Six genes annotated as hydrophobin coding genes were cloned from TH33.The functions of some of those genes in TH33 were studied by gene knockout,heterologous expression,non target metabonome sequencing and transcriptome sequencing.The results are as follows:1.According to the whole genome sequence information of T.harzianum TH33,there were six hydrophobins coding genes in T.harzianum TH33.The sequence numbers were Tha₀0773?thhyd1?,Tha₀2696?thhyd2?,Tha₀3110?thhyd3?,Tha₀4012?thhyd4?,Tha₀4345?thhyd5?and Tha₀9745?thhyd6?.The cDNA fragments of these six genes were amplified with TH33 genomic cDNA as template,and were verified by sequenceing.2.The thhyd4 knockout mutant ?thhyd4 and thhyd6 knockout ?thhyd6 were obtained through double-crossover homologous recombination strategy.Comparing the biological characteristics of ?thhyd4 and ?thhyd6 to wild-type TH33,there were no significant differences in morphology growth rate,sporulation,hydrophobicity and antagonistic ability to pathogens among wild strain and the mutants.3.The E.coli expression vectors of thhyd2,thhyd3,thhyd4,thhyd5 and thhyd6 were constructed by using pET-28 a vector,and were transfered into E.coli BL21?DE3?,The fusion proteins THhyd4 and THhyd6 were obtained by inducing expression and purification in E.coli BL21?DE3?transformants.The optimal induction conditions were as follows: IPTG concentration was 0.5 mM,28 ? and 200 rpm for 4 hours.The eukaryotic expression vectors of thhyd2?thhyd3 and thhyd5 were constructed by using plasmid pPICZ?A,and the fusion protein of THhyd3 was induced and purified by using the expression system of Pichia pastoris KM71 H.4.Tobacco was treated with purified fusion proteins THhyd3,THhyd4 and THhyd6,the results showed that THhyd4 and THhyd6 could cause HR reaction,active oxygen generation,callose and phenolics accumulation in tobacco.It means that the hydrophobins THhyd4 and THhyd6 might be protein elicitors and could induce the immune defense reaction of tobacco.5.The non target metabolome and transcriptome of tobacco leaves treated with THhyd6 were sequenced.The analysis results showed that THhyd6 induced the expression of genes related to ?-linolenic acid metabolism,linoleic acid metabolism,glutathione metabolism,amino acid metabolism and the accumulation of metabolites in tobacco.In addition,THhyd6 induced the accumulation of JA signaling substances,up-regulated expression of key genes in JA signal transduction pathway,and differential expression of various defense related genes in tobacco.It is speculated that THhyd6 can induce systemic disease resistance?ISR?of tobacco by activating JA signal pathway as an elicitor.