The Cloning,Subcellular Localization And Expression Patten Analysis Of Heat Shock Transcription Factor HaHsfA3,HaHsfB3 In Haloxylonammodendron

Temperature is one of the three essential conditions for plant growth,and it is consider a main environmental factor affecting plant growth and development.High temperature stress caused damage to plant growth and development,thus breeding heat resistant varieties can facilitate plants to better respond to high temperature hazards.Heat shock transcription family is a class of family that showed sensitive response towards temperature.HSF possessed anti inverse function against heat and drought.Haloxylonammodendron is a perennial shrub or small tree,belonging to the Chenopodiaceae of ammodendron Bunge.Geographically it's mainly distributed in the tropical and subtropical areas of Asia and Africa,grows in desert,saline soil and gravel gobi.It is also resistant to wind erosion and have tolerance for drought.In our study,H.ammodendron was taken as a test material.Two heat shock transcription factors were cloned and their sequences were analyzed and compared.The subcellular localization was also analyzed.In addition,the function of HSF in adversity,H.ammodendron seedlings were treated under high and low temperature and for drought stress treatment.Furthermore,its expression pattern was analyzed afterwards.In conclusion,the expression and regulation mechanisms of assimilating branch and root of H.ammodendron under different stress were studied at gene level.The main results are as follows:1.According to the data of transcriptome sequencing results of H.ammodendron,bioinformatics method was used to identify HaHSFs genes.Cloned heat shock transcription factor HaHsfA3,HaHsfB3 from H.ammodendron,the Two ORF of 1083 bp and 768 bp encoded for 361 and 256 amino acids,respectively.The molecular weight was 29.3 kDa and 39.76 kDa whereas theoretical isoelectric point was 5.38 and 8.21.HaHsfA3 and HaHsfB3 genes have specific DNA binding domain which encoded hydrophilic proteins.The homologous evolution tree showed that evolution relationship of HaHsfA3 was closest to HsfA3 in solanaceae,while HaHsfB3 was found closest to HsfB3 in cucurbitaceae.2.Softberry and PSORT WOLF websites were searched to predict the subcellular localization of HaHsfA3 and HaHsfB3 genes.Their results revealed that they are mostly likely located in the nucleus and have highest score.Based on above forecasting results,here the GFP-PA7 vector was constructed,and the gene gun bombard method of tobacco leaves was used for subcellular localization.Their results confirmed that the protein localization of HaHsfA3,HaHsfB3 were in the nucleus.3.For one-month the seedlings of H.ammodendron were treated under high and low temperature effect and also for drought stress at different-time sampling.After that qRT-PCR was performed to study the expression level of HaHsfA3 and HaHsfB3 genes.The results showed that under different stress,there were significant differences in the expression patterns of HaHsfA3 and HaHsfB3 genes.Under 45? treatment,the expression patterns of HaHsfA3 and HaHsfB3 genes in H.ammodendron leaf both increased at first and then decreased,but the expression level of HaHsfA3 was significantly higher than of HasfB3.Under 4? treatment,the expression patterns of HaHsfA3 and HaHsfB3 genes were increased at first and then decreased;the two genes have reached the maximum in 1 h.The gene expression level of HaHsfB3 was higher than of HaHsfA3 totally.Under drought treatment,expression of HaHsfA3 and HaHsfB3 in leaf reached maximum in 1 h,significantly higher than of control and slightly higher than the control after 1h,but in root,the expression of HaHsfA3 and HaHsfB3 significantly raised.HaHsfA3 raised and firstly reached the maximum in 8 h,there was second rising after falling until 48 h,the expression of 48 h slightly less than 8 h.While in general,the expression of HaHsfB3 raised until reached maximum in 48 h.These results clarify that HaHsfA3 and HaHsfB3 in leaf response to high temperature stresses significantly,but the two genes in root significantly response to drought stress.4.SOD,POD,CAT,are important components for antioxidant enzyme system.They have important roles for drought tolerance in plants.In this study,one-month H.ammodendron seedlings were treated under 20%PEG treatment which simulated drought stress for different time of sampling.Physiological indexes,Such as SOD,POD,CAT and MDA were detected to analyze the changes of these enzymes activity and the content of MDA in drought conditions,found that SOD,POD and CAT enzyme activity tended to raise and decrease afterwards under drought treatment,the content of MDA raised slowly within 4 h and raised rapidly after 4 h.The result indicated that,at the early stage of drought stress,the drought damage to plants was not serious,because the antioxidant enzyme system of the plants were enhanced,enzymes activities of SOD,POD and CAT were also enhanced but the ROS injury to cell membrane was reduced.ROS damaged cell membrane lightly,so MDA,as the membrane lipid per oxidation products raised slowly and maintained at a low level.With the drought stress continuing,the damage to plant increased and might destroyed the normal physiological process,ROS removal ability was weaken,activity of antioxidant enzyme reduced,and due to serious damage to cell membrane,MDA content raised sharply.These results preliminary analyzed the physiological and biochemical mechanisms against drought stress of H.ammodendron and provided preliminary theoretical platform for breeding drought resistant varieties of H.ammodendron.In summary,HSFs of H.ammodendroncan respond rapidly under high temperature,low temperature,and for drought treatments.It begins to activate the expression of downstream protein HSP.In this study,the function of HSFs was preliminary analyzed which will provide the foundation for future comprehensive understanding of the molecular regulation mechanism against heat resistance of H.ammodendron.