Genetic Transformation Of Hsp24 Gene From Trichoderma Asperellum ACCC30536 To Populus Davidiana×P. Alba Var. Pyramidalis To Cultivate Resistant-pathogen Transgenic Poplar Plant

Poplar species, which have the capacity for efficient asexual reproduction, rapid growth and a short production cycle, are cultivated throughout the world. However, there are many biotic stresses (Cytospora chrysosperma, Alternaria alternate and Melampsora laricipopulina) and abiotic stresses (salt, drought and heat shock) that cause damage to poplars and reduce their production. Biocontrol Trichoderma spp. contain lots of defense response genes and pathogen-resistant genes, such as heat protein genes(Hsps) which play an important role in Biological control mechanism of Trichoderma.The open reading frame (ORF) of Hsp24 genes was 657bp, encoding 218 aa, its molecular weight is 24.3kD. Due to without intron, the GenBank Accession Number of DNA and cDNA is all KP337939. Hsp24 protein belong to Hsp20 protein family (pfam00011), its pI is 5.50. The expression of Hsp24 gene from T. asperellum ACCC30536 under ten different condition was detected by RT-PCR, the result showed that Hsp24 was up-regulated under the stress of alkali, salt, drought, high temperature and the fermentation broth of C. chrysosperma and A. alternate, especially under the high temperature and A. alternate stress, reaching to 51.6 and 31.1 folds.The cDNA of heat shock protein 24 (Hsp24) gene was cloned from T. asperellum ACCC30536, and transformed into the genome of Populus davidiana × P. alba var. pyramidalis (Pdpap) by the Agrobacterium tumefacie ns-mediated transformation system, and RT-qPCR analysis proved that Hsp24 gene transcripted successfully in poplar transformants. Pathogen-related genes (PRs) in four Pdpap-Hsp24s were up-regulated compared with those in the control Pdpap (Pdpap-Con).After co-culture of Pdpap-Hsp24s with the weak parasite C. chrysosperma, the transcription of genes related to hormone signal pathway (JA and SA) were up-regulated in Pdpap-Hsp24s, and ethidium bromide (EtBr) and Nitro-blue tetrazolium (NBT) staining assays indicated that the cell membrane permeability and the active oxygen content of Pdpap-Hsp24s leaves were lower than that of the control Pdpap-Con. And when the Pdpap-Hsp24s were under the A. alternate stress, the activities of superoxide dismutase (SOD) and peroxidase (POD) got higher in Pdpap-Hsp24s than that in Pdpap-Con, and the disease spots in Pdpap-Con leaves were obviously larger than those in Pdpap-Hsp24s leaves.In summary, Hsp24 of T. asperellum ACCC30536 is important defense respond gene, and its heterologous expression improved the resistance of transformant Pdpap-Hsp24s to C. chrysosperma and A. Alternate.The findings of this study provided a further understanding to the role of Hsp24 in biocontrol and a practical reference for the application of transgenic technology to enhance the resistance of woody plants against biotic and abiotic stresses.