Gene Cloning And Primary Functional Analysis Of CsERF36 And CsERF43 In Citrus Sinensis Osbeck

Citrus is an important fruit crop in China,which has high economic value.However,in its whole life,it encounters some inescapable abiotic stresses such as low temperature,dehydration and salinity,which seriously affect the growth and development of plants.To resist the adverse effects of these factors,plants often evolve into a set of complex and precise regulatory mechanisms of environmental response,involving the network of transcription factors.AP2/EREBP family is a large family of transcription factors that widely respond to abiotic stresses such as salinity,low temperature and dehydration in plants.‘Longhuihong’navel orange（Citrus sineses Osbeck cv.’Longhuihong’）is a bud mutation variety of‘Newhall’navel orange（Citrus sineses Osbeck cv.’Newhall’）which is selected from freeze injury.Relative to wild type,‘Longhuihong’has little variation in genetic background,but has stronger stress resistance.In this study,the analysis of transcriptome showed the gene expression levels of these CsERF36 and CsERF43 were significantly up-regulated in’Longhuihong’compared with wild type,which is respected to regulate abiotic-mediated response.Based on the analysis of the phylogenetic relationship of AP2/EREBP family in sweet orange,the clone and subcellular localization of CsERF36 and CsERF43 genes were analyzed,to clarify their structural characteristics.The biological functions of CsERF36 and CsERF43 were preliminarily performed by heterologous over-expression of CsERF36 and CsERF43in Arabidopsis and the RNAi interference of CsERF36 and CsERF43 in’Jincheng’orange.This study provides the key material and partial theoretical basis for the follow-up study of the role of CsERF36 and CsERF43 in citrus under abiotic stress.The main results are as follows:1.The results of whole-genome sequencing showed that the genetic similarity of’Longhuihong’navel orange and’Newhall’navel orange was 99.84%,indicating that’Longhuihong’was a bud variety of’Newhall’navel orange.The analysis of transcriptome showed that the expression levels of99 genes were more than 2-fold change in the fruits of’Longhuihong’compared with’Newhall’,in which 53 genes were up-regulated and 46 genes were down-regulated in’Longhuihong’.In particular,the expression levels of transcription factors CsERF36 and CsERF43 were significantly up-regulated by 2.92 and 2.63 times in’Longhuihong’fruit relative to in’Newhall’,respectively.2.A total of 127 AP2/EREBP transcription factors were identified from the whole genome of sweet orange,and the DNA similarity with Arabidopsis AP2/EREBP transcription factors ranged from 27.75%to 88.89%.According to their distribution on chromosomes,they were named by CsERF1～CsERF127,and were mainly distributed on chromosome 1 and 5.The results of promoter prediction showed that they mainly were divided into 10 types of cis-acting elements,which were related to hormone and stress response.By analyzing the protein sequence,it was found that AP2/EREBP transcription factors of sweet orange encode 68-804 amino acids with 1-29 exons,and62.20%of its members contain 5’UTR and 3’UTR.In addition,a total of 20 motifs were identified,and Motif 1/2/3/4 is the main motif of AP2 conserved domain.To further predict the function of each member,127 AP2/EREBP transcription factors of sweet orange and Arabidopsis were used to construct NJ phylogenetic tree,and they were divided into 27 subfamilies.CsERF36 and CsERF43are located in 11 subfamilies,which are related to plant metabolic synthesis and abiotic stress response.The results of conservative domain analysis showed that there were 41 highly conserved amino acid residues in the conserved domain of AP2/EREBP transcription factors.The 41 amino acid residues were mainly distributed over three regions,which contained threeβ-folds and oneα-helix.3.Based on the sweet orange genome database,the CDS sequences of CsERF36 and CsERF43were cloned from’Newhall’and’Longhuihong’respectively.The sequencing results showed that there was no difference between the two genes in’Newhall’and’Longhuihong’;The CDS lengths of CsERF36 and CsERF43 were 741 bp and 600 bp,respectively,encoding 247 and 200 amino acids,respectively.The 14th amino acid of CsERF36 and CsERF43 conservative domain is valine,which indicates that CsERF36 and CsERF43 are CBF/DREB transcription factors and they can specifically bind to DRE cis-acting elements.The results of subcellular localization showed that CsERF43 was a nuclear AP2/EREBP transcription factor,while CsERF36 was expressed on both membrane and nucleus,which might be a membrane-bound transcription factor.4.CsERF36 was overexpressed in Arabidopsis thaliana,and 6 overexpression strains OE2、OE4、OE5、OE10、OE12 and OE19 were identified through resistance screening and PCR identification.Compared with the wild type,the expression of CsERF36 in the overexpressed plants was up-regulated by 2 to 284 times,and OE10 has the highest up-regulation multiple.The analysis of routine physiological parameters showed that the germination rate of OE10 was the highest and significantly higher than that of wild type.The root length of OE10 and OE12 lines was significantly higher than that of wild type.The content of chlorophyll b of OE5 was significantly lower than that of wild-type plants,but the contents of chlorophyll a and chlorophyll a+b of overexpression plants were not significantly different from that of wild-type plants.The response ability of CsERF36overexpressed plants to Na Cl and ABA treatment was further analyzed.The seedlings that germinated for 5 days were transferred to MS solid medium and treated with 200 m M Na Cl for 10days.Some wild-type plants showed albinism,while OE2,OE5,OE10 and OE12 overexpression plants that grew on the same plate showed stronger vitality.After treatment with 3μmol·L^-1 ABA for10 days,the roots of the Arabidopsis seedlings grew well,but the newly grown leaves showed yellowing symptoms.However,there was no significant difference in phenotype between CsERF36overexpression plants and wild-type plants.Overexpression of CsERF36 can not change the sensitivity of Arabidopsis seedlings to ABA.After low temperature treatment（-1℃）,the relative conductivity and MDA content of overexpression plants and wild-type plants increased significantly on the 0 d,2 d and 4 d,but there was no significant difference.It has not been observed that overexpression of CsERF36 improves the freezing tolerance of Arabidopsis at-1°C.The three overexpressed lines（OE2、OE4、OE10）and wild-type plants were subjected to drought treatment and rewatering treatment,and the phenotypic differences among the plants were not observed.Overexpression of CsERF36 in Arabidopsis thaliana could not improve the drought tolerance of the plants.In addition,T2 generation seeds of CsERF43 have been obtained,which provides important materials for further research.5.The pGN-RNAi-CsERF36 and p GN-RNAi-CsERF43 interference vectors were constructed to transform’Jincheng’epicotyls.The resistant buds were obtained by co culture and screening culture.After GUS staining and identification,the positive buds were cultured in bud reproduction medium and used for rooting culture after growing to 3 cm high.