Study On Drought-resistance And Salt-tolerance Of ThASR3 Gene From Tamarix.hispida

Abscisic acid-,stress-,and ripening-induced proteins(ASRs)are a family of proteins involved in osmoregulation in a wide range of organisms,and have basic features of transcription factors and LEA proteins,which play important roles in regulating the stress resistance of plants.In this experiment,a stress-responsive gene was identified from the Tamarix hispida transcriptome data,which has a typical ABA/WDS domain and belongs to the plant abscisic acid-mature-stress response gene,named as ThASR3.The gene is 309 bp in length and encodes 103 amino acids with a molecular weight of 11.45 kDa and the isoelectric point of 9.1.The qRT-PCR analysis showed that the expression of the ThASR3 gene was up-regulated by high salt and drought,suggesting that it may play an important role in drought and salt tolerance of Tamarix hispida.Overexpression vector of ThASR3 gene was constructed and transformed into T3 generation transgenic lines of Arabidopsis thaliana performed by floral dip method.Under NaCl and Manntiol treatment,the transgenic lines of ThASR3 gene had higher root length,fresh weight and growth than WT plants,which demonstrated that ThASR3 Overexpression can significantly improve tolerance to salt and drought stress.Under the stress of NaCl and Manntiol,the analyses of DAB,NBT and Evans blue stainings of ThASR3 overexpression and WT showed that O2· and H2O2 were the lowest in the transgenic plants and ThASR3 overexpression lines had the best cell membrane integrity,which indicated that the ThASR3 genecan significantly enhance the stress tolerance in transgenic plants.A suppression vector of pFGC5941-ThASR3 was constructed.Three kinds of transgenic T.hispida plants were generated,including plants overexpressing ThASR3(transformed with pROKII-ThASR3),RNAi-silencing ThASR3 plants(transformed with pFGC5941-ThASR3),and control plants(transformed with empty pROKII)by the transient transformation method.DAB,NBT,and Evans Blue stainings showed that overexpressing ThASR3 has lowest ROS content and lowest cell membrane damage compared with control plants and RNAi-silenced ThASR3 plants.These results suggested that overexpression of ThASR3 significantly improved abiotic stress tolerance of plants.The results of the physiological indexes of the ThASR3 overexpressing,RNAi-silencing and control plants showed that the H2O2,ROS and MDA contents were the lowest,While POD and SOD activities,betaine,soluble sugar and PRO contents were the highest in the overexpressing plants(OE),however,the H2O2,ROS and MDA contents were the highest,While POD and SOD activities,betaine,soluble sugar and PRO contents were the lowest in the RNAi plants(IE).These proved that overexpression of ThASR3 increased the reactive oxygen species(ROS)scavenging capability,decreased cell membrane damage,and improved tolerance to stress in transgenic T.hispida plants.The plant expression vector pBI121-GFP-ThASR3 was constructed,and was transiently transformed into the onion epidermis using particle bombardment to study subcellular localization.The results showed that ThASR3 protein was expressed both in the nucleus and cytoplasm.Yeast two-hybrid technique was used to study ThASR3 transcriptional activation activity.The results showed that ThASR3 had no transcriptional activation activity in yeast and could not form homodimer,suggesting that it may not function as a transcription factor.In the future study,yeast two-hybrid library will be constructed under salt and drought conditions to screen ThASR3 interaction proteins,and further reveal the regulating mechanism of ThASR3 in response to stress in T.hispida.