Cloning And Expression Analysis Of The Dehydrin Gene PlDHNs In Peony (Paeonia Lactiflora)

Dehydrins（DHNs）belong to group 2 of the late embryogenesis-abundant（LEA）proteins,also known as LEA-II proteins,which typically accumulate in plant during dehydrative stresses caused by low/freezing temperature,drought,salinity,and abscisic acid（ABA）.DHNs are a group of intrinsically disordered proteins due to high proportions of hydrophilic amino acids.It is assumed that intrinsically disordered character of DHNs allows them to change their conformation according to the changes in their ambient microenvironment,resulting in multiple functions.In this study,stress-responsive dehydrin gene Pl DHN1 and Pl DHN2 were cloned from the buds of the Paeonia lactiflora‘Dafugui’.Using bioinformatics methods for identification and analysis of its comprehensive utilization;Using laser scanning confocal microscope to observe its subcellular localization;Their expression patterns in different tissues were analyzed by semi quantitative PCR method;The expression patterns of them under different abiotic stress were analyzed by real-time fluorescence quantitative PCR;The main results of this study are as follows:1.Isolated the 2 new DHN genes of peony:PlDHN1,GeneBank accession number is KX524516,a total length of 783 bp,encoding a total of 260 amino acids;PlDHN2,GeneBank accession number is KY272747,a total length of 399 bp,encoding 132 amino acids.Sequence alignment showed that the PlDHN1 sequence possessed one S segment and three K segments,belonging to the typical SK₃-type de-hydrin;the PlDHN1 sequence possessed three Y segments,one S segment and three K segments,belonging to the typical Y₃SK₂-type de-hydrin.2.Using DNA as template to clone the 2 genes,and analyse their gene structure.PlDHN1 and Pl DHN2 contained one 334-bp and 84-bp intron respectively,which were inserted in the nucleotide sequence encoding the S-motif and characterised by a GT-AG border.3.Bioinformatics analysis showed that the predicted molecular weight of PlDHN1 and PlDHN2 is 29.476 kDa and 14.703 kDa respectively,an isoelectric point（pI）is 5.29 and 8.63 respectively,grand average of hydropathicity（GRAVY）index is-1.344 and-1.325 respectively.A phylogenetic tree analysis with the related DHNs indicated that the same type of DHNs have close phylogenetic relationship.4.Constructed an expression vector of pROKII-PlDHNs-GFP and expressed in the leaves of Nicotiana benthamiana transiently.Subcellular localization showed that the PlDHNs protein was localized in the nucleus and cytomembrane.The results indicate that PlDHNs is a nuclear localization protein,which may play an important biological role in the nucleus and cytomembrane.5.The expression of DHN in different tissues was analyzed by RT-PCR.The results showed that the 2 genes were expressed in different tissues.DHN gene is involved in all aspects of plant growth and development.6.qRT-PCR analysis indicated that the PlDHN1 and PlDHN2 genes in buds accumulated during the winter and peaked in harsh December with gradual decrease prior to spring.7.The expression of 2 PlDHNs genes in Paeonia lactiflora Pall at low temperature,high temperature,waterlogging and ABA was analyzed by fluorescence quantitative PCR,The results showed that PlDHNs transcript accumulation were all induced observably.The relative sensitivity of PlDHN1 expression to various stresses was waterlogging>heat>ABA>cold,and The relative sensitivity of Pl DHN2expression to various stresses was ABA>waterlogging>heat>cold.