Molecular Cloning And Genetic Transformation Analysis Of WRKY54 Gene In Poplar

Having advantages, such as rapid growth,early foresting,yield rich and the wide application of lumber, poplar is regarded as reforestation tree species of short-term rotational felling. In addition, Poplar plantation also occupies an important position in the ecological environment governance. However, poplar is perennial species. In the whole growth cycle, poplar will be threatened by different disease. Various disease seriously impediment the growth and development of poplar, result in serious economic loss. WRKY gene family members broadly involve in a series of plant physiological activities, for example, responses to environmental stresses, plant growth and development, metabolic regulation and plant defense responses to distinct pathogens.In this research,we conducted phylogenetic analysis, sequence analysis, expression profile analysis, subcellular localization research, transcription activity analysis. Besides, we cloned PtrWRKY54 gene from Populus deltoids cv. Nanlin 95, constructed over expression vector of PtrWRKY54 and transformed to Populus deltoids cv. Nanlin 95 by leaf-mediated method. And transgenic plants were filtered through antibiotics screening, PCR identification and GUS staining identification. The main results are as follows:1. We cloned PtrWRKY54 gene from Populus deltoids cv. Nanlin 95. Possessing coding region of of 969 bp, this gene encodes 322 amino acids. Besides the predictive coding protein contains one WRKY domain and one C2 HC zinc finger motif.2. Phylogenetic analysis showed that 16 WRKY homologous genes clustered into monocotyledon and dicotyledone two different large branches; PtrWRKY54 was closed to two disease resistance related WRKY genes PtrWRKY89 and At WRKY70. Sequence analysis indicated that PtrWRKY54 was highly similar to At WRKY70 in gene structure and conserved motif; cis-element analysis showed that PtrWRKY54 obtained fungal elicitor responsive element and cis-acting element involved in defense and stress responsiveness.3. Tissue expression pattern analysis found PtrWRKY54 had high expression quantity in differentiating xylem. PtrWRKY54 was obviously induced by SA with highest gene expression, followed by wounding, But PtrWRKY54 gene expression of Me JA treatment is not obviously elevated; gene expression with two mixed rust pathogens treatment is higher than the it of individual rust pathogen treatment.4. We constructed PtrWRKY54-GFP fusion expression vector and injected it into the leaves of Nicotiana benthamianai, subcellular localization experiment showed that PtrWRKY54 located in the nucleus.5. To study transcription activity of the PtrWRKY54 we constructed pGBKT7-PtrWRKY54 vector, yeast hybrid assays exhibited that PtrWRKY54 owned transcription activity.6. We constructed over-expression vector pRI201-AN-GUS-PtrWRKY54 and transformed it into “Nanlin 95”. Eventually we obtained 2 transformed plants after screening.