Identification Of Drought-induced Di19 Transcription Factors In Moso Bamboo And Function Analysis Of PeDi19-4

Moso bamboo(Phyllostachys edulis),which is an important bamboo species that has variety values,such as ecological,economic and cultural.The planting area of moso bamboo is the largest among bamboos.And the requirements of climate and soil for moso bamboo growth is very high.However,with the global climate change increasingly severe,the adverse environmental quality limits the normal growth and development of plants.Di19 protein is one of the drought-induced protein and mainly adjust the abiotic stress of plants.Nowadays,a large number of Di19 proteins have been experimentally identified from several plant species.However,a drought-induced 19 protein(Di19)has not been isolated from moso bamboo.In this paper,ten Di19 proteins were identified from moso bamboo by the genome analysis.First,the structure characteristics and evolution of these ten genes were observed and the their expression levels under abiotic stress treatments were detected.So identified a candidate genes-PeDi19-4.Through the analysis of subcellular localization,transcriptional activity and DNA binding sites,it was found that PeDi19-4 had typical transcription factor characteristics.Subsequently,the functions of PeDi19-4 gene in abiotic stress response was studied by overexpressing this gene in plants and DEGs were identified and analyzed by transcriptome sequencing analysis.Furthermore,the target genes of PeDi19-4 were identified by Ch IP-PCR experiment.The primary results are obtained.1.A total of 10 non-redundant Di19 genes were identified from moso bamboo genome.The sequence characteristics and gene structure showed that there are two highly conserved and unusual Cys2/His2(C2H2)zinc-finger-like motifs in the N-terminal and a conserved Di19?C domain in the C-terminal.The comprehensive phylogenetic tree showed that all Di19 proteins were divided into five groups(group1-5).Groups 1 and 2 only contained the dicotyledonous(Arabidopsis,Glycine max and Gossypium spp).Groups 3,4,and 5 were formed by all the monophyletic proteins.2.qRT-PCR experiments showed that under drought,salt and ABA threatments,the expression level of all the ten PeDi19 genes were increased,and PeDi19-4 was dramatically unregulated.3.The PeDi19-4 gene was cloned from moso bamboo genome and the sequencing result was consistent with the published sequence in moso bamboo genome database.4.The subcellular localization results suggest that PeDi19-4 is a nuclear-localized protein.But PeDi19-4 has no transcriptional activity in the yeast.The EMSA assay verified that PeDi19-4 protein could bind to the conserved element TACA(A/G)T.5.PeDi19-4 could interact with a calcium-dependent protein kinases(Pe CDPK22)by yeast two hybrid assays,and Pe CDPK22 was strongly induced by drought,salt and ABA stresses.6.The transgenic rice(Arabidopsis)of PeDi19-4 have no significant differences in the morphology compared with WT.After drought and salt treatments,the transgenic rice(Arabidopsis)showed significantly increased the drought and salt tolerance.Under stress conditions,the soluble sugar and proline content in the transgenic plants were higher than that in WT plants.However,the REL and MDA in the transgenic plants were lower than that in WT plants.7.At both germination and postgermination stages,the overexpression rice(Arabidopsis)of PeDi19-4 were showed sensitivity to ABA.Stomata closure of PeDi19-4-overexpressing plants is also regulated by ABA.8.The RNA-seq analysis revealed 736 up-and 214 down-regulated genes in the PeDi19-4 transgenic plants relative to WT after drought stress,and 598 up-and 126down-regulated genes compared to the control after salt stress(Fold?2,P < 0.01).The overlap of 214 up-regulated and 76 down-regulated genes between drought and salt stress.The biological processes of these 290 DEGs were predicted by GO analysis,they mainly responded to stimuli.More importantly,many of them have been annotated or evidenced for their functions in stress-related or ABA signaling.The expression levels of the 12 chosen genes in PeDi19-4-overexpressing rice were higher than that in the WT plants,which was similar to the expression patterns derived from the RNA-seq data.These results confirmed that PeDi19-4 enhanced drought and salt tolerance in transgenic plants by ABA-dependent signaling pathway.