Identification Of Interaction Between Transcription Factor CsERF1 In Citrus Sinensis And CTV Protein P20 And Construction Of RNAi Vector

CTV is a worldwide citrus virus disease caused by Citrus tristeza virus.It is widely distributed all over the world and seriously threatens the development of the global citrus industry.p20,as one of the silencing suppressors of CTV forms inclusion bodies in amorphous form in host cells.p20 affects the expression of related genes in the host salicylic acid signal pathway.AP2/ERF transcription factors are involved in a variety of signal transduction pathways such as salicylic acid,jasmonic acid,ethylene and abscisic acid,and regulate many kinds of biological processes simultaneously including plant growth,seed development,biotic and abiotic stresses.Virus infection can lead to changes in the balance of hormones such as salicylic acid and jasmonic acid,and other physiological activities in the host plant,which in turn will affect the adaptability of vector insects to the host and their interaction.In the previous research of our group,it was shown that the population growth of toxoptera citricida was obviously slow on sweet oranges infected with CTV and treated with exogenous SA,and it was related to the expression of p20 in different isolates.The content of endogenous SA in susceptible plants increased,and the expression of genes related to SA signaling pathway increased significantly.p20 is involved in the host SA signaling pathway.In the early stage,our research group used p20 as bait protein to screen the library of C.sinensis（L.）Osbeck.cv.Symons,and screened a gene fragment of transcription factor AP2/ERF1a interacting with it.It is necessary to clarify which subfamily transcription factor AP2/ERF1a belongs to,and verify the interaction between p20 and transcription factor AP2/ERF1a and lay a foundation for studying the mechanism in CTV regulating host SA,JA and other signaling pathways to inhibit the population growth of toxoptera citricida,which is expected to provide new ideas for developing green prevention and control of new aphid-borne viruses.On the basis of the previous research of our group,the CTV silencing suppressor p20 and AP2/ERF1a protein were used as the research object to further clone the CDS sequences of AP2/ERF1a,verify the interaction between p20 and CsERF1 by Yeast Two-hybrid point-to-point interaction,Bimolecular Fluorescence Complementation and Luciferase Complementation Assay.The subcellular localization of p20 and CsERF1 was observed by laser confocal microscope,and Sequence of CsERF1 bioinformatics analysis.Transgenic citrus plants based on RNAi CsERF1 were constructed.The results lay a good foundation for deep studying the molecular mechanism of CsERF1 mediated virus-host plant-vector insect interaction.The main researchresults are as follows:1.Through cloning and bioinformatics analysis of AP2/ERF1a,the results showed that the open reading frame of AP2/ERF1a gene is 816 bp,it encodes 271amino acids and has a typical AP2 domain.The 14^thand 19^thamino acids in the domain are alanine and aspartic acid,respectively,belonging to the ERF subfamily,The transcription factor AP2/ERF1a was formally named CsERF1.The amino acid sequence alignment showed that the whole sequence of CsERF1 was48.34%∽100%identical with that of Citrus genus,and the identity was also extremely high between theamino acid sequence in the conserved domain of CsERF1 and that of Citrus genus,which suggested that its physiological function was speculated to be close to them.The phylogenetic tree showed that the highest homology of CsERF1 gene with apple was 59.66%,and the lowest homology with Arabidopsis thaliana was 24.55%.CsERF1 gene had the closest genetic relationship with apple,which suggested that CsERF1 protein might have similar functions with apple ERF1A-like protein.2.The interaction between p20 and CsERF1 was actually existed by Yeast two-hybrid point-to-point verification,Bi FC and LCA.3.We use RFP gene as an autophagosome marker to visualize the subcellular localization of p20 and CsERF1.The subcellular localization was observed with laser confocal microscope.The results showed that CsERF1 was located in nucleus and CTV p20 was located in nucleus,cytoplasm and membrane.4.We constructed RNAi vector of sweet orange gene CsERF1 firstly.Then,we obtained one RNAi interference CsERF1-transgenic citrus by ordinary PCR and real-time fluorescence RT-PCR.