Cloning And Functional Analysis Of Stress Responsive Genes,PbeNAC1,PbeMTB2,PubHLH1 And PuALDH2,in Pyrus

Pear is one of important fruit trees and widely distributed throughout of China,which play a pivotal role in promoting regional economy.In recent decades,tremendous progress has been achieved in the pear industry,the developments of quality and yield are impeded by various abiotic stresses,such as cold,drought and salt,which making how to enhance abiotic stress tolerance of pear become an urgent problem to be solved.With the development of genetic engineering,there is a new insight for pear resistance of breeding.In the present study,NAC and MYB,bHLH and ALDH were respectively cloned from pear cultivars of Pyrus betulaefolia Bunge and Pyrus ussuriensis Maxim for functional analysis in transgenic tobacco.The main results as follows:1.PbeNACl was cloned from Pyrus betulaefolia Bunge,containing an open reading frame(ORF)of 927bp and encoding 308 amino acids with predicted protein molecular weight(Mw)35.6KDa and isoelectric point(pI)6.46.Phylogenetic tree analysis revealed that PbeNACl has higher homology with the members of ATAF subfamily in Arabidopsis and rice.Multiple alignments between PbeNAC1 and other NAC proteins of different plants displayed that there were high sequence identities in their N-terminal regions and possessed a conserved nuclear localization signal sequence.PbeNAC1 is a nuclear protein and has transactivation activity.PbeNAC1 was induced by cold,dehydration and salt stresses.Overexpression of PbeNACl in tobacco,the transgenic lines showed enhanced cold and drought tolerance.Exposure to chilling or drought stresses,the transgenic lines accumulated lower ROS and had higher antioxidant enzyme activities compared with the WT.Meanwhile,15 of ROS-scavenging or stress-related genes were observed higher transcription levels in transgenic tobacco before and after chilling or drought stress.Further studies showed that PbeNAC1 physically interacts with PbeDREBl and PbeDREB2A,respectively,involved in the DREB-mediated signaling pathway,which might be related to the improved tolerance in transgenic lines.These results showed that overexpression of PbeNACl in transgenic lines enhanced cold and drought tolerance.2.PbeMYB2 was obtained from Pyrus betulaefolia Bunge,containing an open reading frame(ORF)of 873bp and encoding 290 amino acids with predicted protein molecular weight(Mw)32.8KDa and isoelectric point(pI)5.1.Phylogenetic tree analysis revealed that PbeMYB2 is most closely related to AtMYB2.PbeMYB2 is a nuclear protein and has transactivation activity.PbeMYB2 could be induced by cold,dehydration and salt stresses.Overexpression of PbeMYB2 in tobacco enhanced the transgenic lines salt tolerance.Under salt stress,the transgenic lines scavenged hydrogen peroxide(H2O2)more effectively compared with the WT,and 5 of ROS-scavenging or stress-related genes were observed higher expression in transgenic tobacco before and after salt stress.All the results demonstrated that overexpression of PbeMYB2 conferred salt tolerance in transgenic plants.3.PubHLH1 was obtained from Pyrus ussuriensis Maxim,containing an open reading frame(ORF)of 1638bp and encoding 545 amino acids with predicted protein molecular weight(Mw)59KDa and isoelectric point(pI)6.05.Phylogenetic tree analysis revealed that PubHLHl is most closely related to AtbHLH33.Motif scanning demonstrated that PubHLH1 included a typical bHLH domain.PubHLH1 localizes to the nucleus and has transactivation activity.PubHLH1 responses to cold,dehydration and salt stresses.Overexpression of PubHLH1 in tobacco,the transgenic lines showed enhanced cold tolerance.Subjected to chilling stresses,the transgenic lines accumulated lower reactive oxygen species(ROS)and had higher antioxidant enzyme activities in comparison with the WT.In addition,the expression of ROS-scavenging or stress-related genes were higher in transgenic tobacco before and after chilling stress.The results showed that PubHLH1 improved cold tolerance.4.PuALDH2 was isolated from Pyrus ussuriensis Maxim,containing an open reading frame(ORF)of 1506bp and encoding 501 amino acids with predicted protein molecular weight(Mw)54.7KDa and isoelectric point(pI)6.13.Phylogenetic tree analysis revealed that PuALDH2 has a higher homology with AtALDH2C4 and PtrALDH2C4.PuALDH2 was induced by cold,dehydration and salt stresses.Overexpression of PuALDH2 in tobacco,the transgenic lines showed enhanced cold tolerance.Subjected to chilling stresses,the transgenic lines exhibited higher antioxidant enzyme activities in comparison with the WT.In addition,the expression of ROS-scavenging or stress-related genes were higher in transgenic tobacco before and after chilling stress.The results showed that overexpression of PuALDH2 improved cold tolerance in transgenic plants.