Cloning And Functional Characterization Of AnYAP1 Gene From Formaldehyde-resistant Fungi

Decoration brought about by indoor formaldehyde pollution is affecting people’s health"invisible killer",its purification is a top priority.Many creatures have the ability to efficiently absorb and metabolize formaldehyde,in which fungi not only metabolize formaldehyde,but also tolerate a higher degree of formaldehyde stress.But its genetic resources have not been effectively developed and used.YAP1 is a class of b-ZIP-type activators,and it has been reported that PpYAP1 is able to metabolize formaldehyde and remove ROS by activating glutathione redox reactions during the methanol metabolism of Pichia pastoris.In the previous work of this laboratory,the anti-formaldehyde fungi strain（Aspergillus nomius SGFA1）was isolated.In this study,the AnYAP1 gene was cloned and its plant expression vector was constructed and the tobacco was genetically transformed.To study the function of transcription factor YAP1 in the metabolism of formaldehyde.The main findings are as follows:1.Cloning and sequence analysis of AnYAP1 geneThe AnYAP1 gene was cloned from the fungus by RT-PCR.Sequence analysis showed that the AnYAP1 gene was 1752bp in length and encoded 584 amino acids,which contained the BRLZ and PAP1 domains and belonged to the bZIP family.Blast comparison showed that Aspergillus nomius SGFA1 AnYAP1 amino acid sequence had high similarity with Aspergillus bombycis AbYAP1 amino acid sequence.There were 16amino acid difference sites between AnYAP1 and AbYAP1,and there were eight differences in the PAP1 domain,one in the BRLZ domain and seven in the other positions.The results of molecular phylogenetic analysis showed that AnYAP1 had the closest genetic relationship with AbYAP1.2.Genetic transformation of tobacco by An YAP1 gene and molecular identification of transgenic plantsThe pCBM-AnYAP1 plant expression vector was constructed and the plant tobacco was genetically transformed by Agrobacterium tumefaciens.A total of 48 resistant tobacco plants were obtained.The tobacco plants were identified by PCR and qRT-PCR.The results showed that 13 transgenic lines were identified by PCR.Among which the expression of the target gene was high in transgenic lines 1.3.Analysis of the function of transgenic tobacco with AnYAP1 geneThe three transgenic lines with higher expression were analyzed for formaldehyde resistance.Transgenic tobacco can grow normally on solid medium containing 4 mM formaldehyde,while wild type all albino death.After 12 days of formaldehyde treatment,the leaf damage of transgenic tobacco was significantly lower than that of wild type.Transgenic tobacco seeds can grow normally on medium containing 1500 mg L^-1 formaldehyde,while wild type tobacco is only partially germinated and can not survive.A series of physiological indexes of tobacco under formaldehyde stress were measured.The results showed that transgenic tobacco showed better formaldehyde resistance.The total chlorophyll content,soluble protein and soluble sugar content of the transgenic tobacco and wild type tobacco leaves were significantly higher than those of the latter under the same formaldehyde stress.Under the same formaldehyde concentration,the oxidative damage indexes of transgenic tobacco,including malondialdehyde,hydrogen peroxide and superoxide anion,were significantly lower than those of wild type.Indicating that the AnYAP1 gene reduced the degree of oxidative damage.Under the stress of formaldehyde,the activities of antioxidant enzymes（SOD,POD,CAT）in transgenic tobacco were significantly higher than those in control wild.In the experiment of liquid formaldehyde absorption capacity analysis,the ability of transgenic tobacco to metabolize formaldehyde was 2.77-2.92 times that of wild type.As a result of the analysis of gaseous formaldehyde absorption capacity,the ability of transgenic tobacco to metabolize gas formaldehyde was 2.287-4.417 times that of wild type.