Expression Profile Analysis Of Ammopiptanthus Mongloicus Under Drought Stress And Functional Characterization Of Three Genes For AmERF Transcription Factors

ERFs(ethylene responsive factors),subfamily in the AP2/EREBP superfamily,participate in multiple plant biological processes and signal transduction pathways.Ammopiptanthus mongolicus has very strong stress tolerance that the research on its resistance mechanisms and related genes has been the focus of attention.In this paper,Illumina technology was used to analyze the transcriptome profile of drought treated A.mongolicus seedlings,and multiple drought-induced AmERF transcription factor genes were identified.Then three genes for the AmERFs were cloned and their functions in stress tolerance were analyzed.The work in this paperincludes three parts:(1)transcriptome profile analysis of the drought-treated A.mongolicus seedlings and screening of the differentially expressed genes(DEGs);(2)the expression analysis of six AmERF genes under different stress conditions and in different organs;(3)cloning of the AmERF4,AmERF9 and AmERF1 genes and functional characterization of AmERF4 and AmERF9 under stress conditions.The main results are as follows:(1)The expression profiles of A.mongloicus after 2 h,8 h and 24 h of drought stress were obtained,and 2028 common DEGs across all three time points were identified,including 779 DEGs withup-regulation,1185 DEGs with down-regulation,and 64 DEGs with complex regulation.Besides,600 to 1178 DEGs with up-regulation and 591 to 1038 DEGs with down-regulation were screened at a single time point.(2)The transcription levels of AmERFs in A.mongloicus seedlings under drought or cold treatment were analyzed by semi-quantitative RT-PCR.The analyses showed that AmERF1,AmERF4,AmERF5,AmERF9 and AmERF61 were up-regulated by drought treatment.And AmERF1,AmERF4,AmERF5,AmERF9 and AmERF1 06 were up-regulated by cold stress.(3)The transcription levels of AmERFs in different organs of A.mongolicus shrubs naturally growing in the wild were analyzed by semi-quantitative RT-PCR.The results showed that AmERF1 was most abundantly expressed in the flowers,young leaves,twigs,lateral roots and immature pods;AmERF4 was abundantly expressed in the young leaves,twigs,lateral roots and immature pods but was not obviously expressed in the flowers;AmERF61 was most abundantly expressed in the flowers,lateral roots and immature pods.(4)The transcription changes of AmERFs in young leaves of A.mongolicus shrubs naturally growing in the wild in different months were analyzed by RT-PCR.The analysis showed that the transcription levels of AmERF1,AmERF4,AmERF9 and AmERF61 changed obviously with the seasons,while that of AmERF5 did not.(5)The complete coding regions of AmERF4,AmERF9 and AmERF1 consist of 708 bp,633 bp and 669 bp,respectively,and none of them contain intron.(6)The results of subcellular localization showed that all of the AmERF4,AmERF9 and AmERF1 proteins are located in the nucleus.(7)The germination of AmERF4 transgenic lines' seeds was slower than that of the control wild type seeds under salt stress treatment.Under low osmotic stress treatment,the germination of the AmERF4 transgenic lines' seeds was better than that of the wild type seeds.However,with the increase of the stress intensity,osmotic stress tolerance of the transgenic lines disappeared.In addition,the AmERF4 transgenic lines showed tolerance to both high concentrations of sucrose and Ultraviolet stress treatment.(8)Under salt treatment,the germination of AmERF9 transgenic lines' seeds was slower than that of the control wild type seeds.