Characteristics And Expression Of AP2/ERF Transcription Factor Family In Hemerocallis ’Here Lies Butch’ Under Waterlogging Stress

Water is one of the most important factors for the growth of Hemerocallis fulva,which are perennial hosta flowers.It is of great significance for Hemerocallis breeding to study the response mechanism of Hemerocallis to waterlogging stress and excavate the waterlogging tolerance related genes for molecular breeding of Hemerocallis.AP2/ERF(APETALA2/Ethylene Responsive Factor)transcription factors play an important role in the response of plants to waterlogging stress.In this study,we took flood-tolerant Hemerocallis variety ’ Here Lies Butch ’ as the research material,RNA was extracted from root tissues at 0h,3h,6h,12 h and 24 h of waterlogging treatment,respectively,and sequenced the transcriptome of 0h and 24 h samples to screen out AP2/ERF transcription factor family members,with’Stella de Oro’ three generations of the transcriptome sequencing data correction.Finally,27 complete gene sequences were obtained.The AP2/ERF transcription factor family members of Hemerocallis were analyzed by bioinformatics.The key gene Hf RAV2 was screened and studied.The following are the main conclusions:1.Hemerocallis AP2/ERF family members are mainly divided into three subfamilies,ERF,AP2 and RAV.The number of amino acid residues of the proteins encoded by the member genes ranges from 153 to 551,including 9 basic proteins and 21 acidic proteins,and all are unstable,non-transmembrane,non-secretory hydrophilic proteins.Except for Hf AP2/ERF-15、27,all of them contain conserved structural domains motif1 and motif2.The core domains(YRG and RAYD elements)are highly conserved and contain highly conserved WLG motifs in AP2.According to phylogenetic tree analysis,the members that may participate in the response of Hemerocallis to waterlogging stress mainly belong to ERF subfamily.Hf AP2/ERF-5(Hf RAV2)belongs to the RAV subfamily.It has not been reported whether members of the RAV subfamily are involved in waterlogging stress.The gene differential expression data showed that Hf RAV2 expression level was significantly up-regulated when Hemerocallis was subjected to waterlogging stress,so it was speculated that Hf RAV2 played a certain regulatory role in this process.2.Bioinformatics analysis of the protein encoded by Hf RAV2 revealed that it is a hydrophilic protein localised in the cytoplasm that does not span membranes,is a non-signalling peptide and has a predominantly irregularly convoluted structure.Functional predictions suggest that this gene may have functions to enhance waterlogging tolerance,drought tolerance,salt tolerance and cold tolerance in plants.The result of function prediction indicated that the gene might enhance the tolerance of waterlogging,drought,salt and cold.The results of real-time fluorescence quantitative PCR showed that the expression level of Hemerocallis was significantly up-regulated after waterlogging stress,which was consistent with transcriptome data,suggesting that Hemerocallis participates in the waterlogging stress process.3.In order to explore the function of Hf RAV2 in Hemerocallis in response to waterlogging stress,the full-length c DNA of Hf RAV2(The total length was 1037 bp and encoded amino acid quantity was 345aa)was cloned,and the overexpression vector was constructed,which was transformed into Arabidopsis thaliana by agrobacterium immersion method.It provides a material basis for further research on the function of Hf RAV2.In this study,the AP2/ERF transcription factor family of Hemerocallis was analysed and key member genes were screened for relevant experiments to explore the role of AP2/ERF transcription factor family members in the response to waterlogging stress in Hemerocallis,which will help to screen and study the key genes regulating waterlogging tolerance in Hemerocallis and provide a basis for guiding the molecular breeding of Hemerocallis for waterlogging tolerance.