Cloning And Function Analysis Of PeuAZF2 Encoding A C2H2-type Zinc Finger Protein From Populus Euphratica Oliv

C2H2-type zinc finger proteins(ZFPs)family is one of the largest families of transcription factors in plants.It plays an important role in regulating growth and development of plants,as well as in various biotic and abiotic stress responses.Populus euphratica is an important model tree species with high tolerance to salt and drought stresses.In this study,we identified 78 full-length C2H2-ZFPs in the genome of P.euphratica underlined sequences alignment and gene structure analysis.Of these Peu ZFPs,one ZFP gene named as PeuAZF2 was cloned and identified,and its function analysis was also performed via transgenetic in Populus species.The results are showed as below:(1)Phylogenetic and evolutionary analysis: a total of 78 putative genes coding C2H2-ZFPs were identified using blast methods from the genome of P.euphratica,and confirmed by Pfam database based on gene feature and structure.All C2H2-ZFP genes were verified the presence of the C2H2-ZF domain and a plant-specific conserved motif QALGGH.78 C2H2 zinc finger genes in P.euphratica have 1 to 5 zinc finger domains.NJ tree analysis showed that all C2H2-ZFPs were classed into four groups(I,II,III and IV)containing 3,15,33,and 27 members,respectively.The number of exon was conservative among different groups,but the number of ZF domain and conservative motif was different significantly.Phylogenetic tree built using ML method showed that Peu AZF2 and PalAZF2 are both in one branch in ML tree with AtAZF2.(2)Structure analysis and function prediction of AZF2 genes in Populus species.Transcriptome analysis showed that expression of PeuAZF2 was induced by salt treatment.As a member of C2H2-ZFPs familly,Peu AZF2 was classified into motif-rich group IV,and its expression was induced by salt treatment.Gene cloning found that t he length of Peu AZF2 is 753 bp,while PalAZF2(from Populus alba)is 756 bp.The proteins sequence similarity of the two genes is very high(93.2%)with 17 amino acid sites difference.PeuAZF2 and PalAZF2 both have two conservative zinc finger domains,similar with AZF2 in A.thaliana,and they are classified into C1 subfamily of C2H2-ZFP family and also identified as Q-type C2H2 ZFPs for conservative QALGGH motif in their zinc finger domain.The expression pattern analysis of AZF2 were performed in P.alba and P.euphratica seedlings treated with salt solution respectively.After salt treatment,the expression level of Peu AZF2 was up-regulated in roots,while that of PalAZF2 were up-regulated in leaves significantly,suggesting that the two orthologous genes expression were regulated in response to salt stress but displayed diverse in expression patterns under salt stress.(3)To explore the biological functions of AZF2 in Populus species,two transgenic lines: generated by transgenic P.alba plants,35S::Peu AZF2 that overexpress Peu AZF2 in P.alba plants and multi-target knockout mutant plants of PalAZF2 gene,palazf2 were used to further function analysis.Compared to WT(wild type),35S::Peu AZF2 plants exhibited more rapidly in roots growth and lateral roots formation,than that of palazf2 and WT.These results indicate that PeuAZF2 gene may involves in the process of roots growth and formation of lateral roots.(4)Under salt treatment,roots growth of 35S::Peu AF2 was faster than that of palazf2 and WT,and the number of lateral roots of 35S::Peu AF2 was more than that of palazf2 and WT.Some physiological parameters were measured,proline content and soluble sugar content in leaf of 35S::Peu AZF2 obviously increased and were higher than that of WT and palazf2.The leaf growth of 35S::Peu AZF2 was better than that of WT and palazf2.The maximum photochemical efficiency of PSII(Fv/Fm)was also higher in leaf of 35S::PeuAZF2 than that of WT and palazf2 under NaCl treatment with same concentration.These results indicate that 35S::Peu AZF2 showes better salt tolerance than in that of WT and palazf2.In conclusion,as a C2H2 zinc finger transcription factor,the expression of Peu AZF2 was induced under salt treatment.Overexpression of PeuAZF2 in P.alba enhances the tolerance of transgenic plants to salt stress.Peu AZF2 plays an important role in salt stress response,and it probably functions as a positive regulator in salt stress response.