Cloning And Functional Analysis Of Citrus CitERF34 Transcription Factor In Response To Abiotic Stresses

Citrus belongs to the Rutaceae family.It is cultivated in more than 140 countries in the world,as one of the most important economic fruit crops.As original center,our country has abundant citrus resources and excellent varieties,and its cultivation area and output also rank first in the world.During citrus growth and development such as sprouting,flowering and fruit ripening,the external environment including low temperature,drought,waterlogging,salt stress etc.would result in economic loss.Therefore,it is particularly important to study the molecular mechanism of citrus in response to adverse conditions with aim to improve its tolerance.Under biotic or abiotic stresses,a large number of defence-related genes are been induced or repressed.Among them,ERF genes(Ethylene-responsive factor)as a type of plant-specific transcription factors participate in plant growth and development and adaptation to stresses.Compared with model plants,few data about ERF gene involving in stresses is available in citrus.’Ziyang’ orange(Citrus junoscv.’Ziyang’)is widely used as a rootstock in the citrus industry,due to high disease resistance,strong alkali tolerance and good compatibility with scion.Because this,we used ’Ziyang’ orange as experimental material to clone and analyze CitERF34 gene.Using bioinformatics,the physical and chemical properties,gene structure,and phylogenetic relationship of CitERF34 had been analyzed.With gene expression,genetic transformation,subcellular localization,and physiological and biochemical analysis,the expression pattern of CitERF34 gene under different stresses and in different tissues was detected,and finally its stresses-related biological functions were also tested.The main results were as follows:(1)CitERF34(ID: Ciclev10032575m)was cloned from ’Ziyang’ orange and analyzed by bioinformatics.The results showed that the open reading frame(ORF)of this gene was 735 bp in length,containing 1 intron and 2 exons,and could encode atranscription factor with 244 amino acids.The predicted relative molecular weight of the protein is 92939.50 D,the theoretical isoelectric point is 5.04,the instability coefficient is 46.77,and the fat coefficient is 28.12.CitERF34 contains a AP2 conserved domain in which has WLG,YRG and LAYD motifs.The 14 th and 19 th positions are alanine and aspartic acid respectively.Phylogenetic tree showed that CitERF34 was closely related to cassava Me ERF113,apple Md ERF113 and grape Vq ERF114.Previous studies have reported that these ERF genes were all involved in biotic stresses,indicating that CitERF34 has a potential role in response to stresses.(2)Tissue-specific expression analysis showed that CitERF34 gene was expressed in roots,stems,leaves and pericarp,with the highest expression in pericarp,followed by roots,and almost no expression in stems,indicating that this gene plays an important role in the growth and development of citrus and its fruits.(3)Through the treatments of different hormones(ABA,ACC)and abiotic stress(drought,low temperature and NaCl),it was found that the expression level of CitERF34 was upregulated by ABA in roots and in leaves was increased to maximum value at 3 h,and then decreased to the level lower than the control at 6 h.After ACC treatment,the expression level of CitERF34 in roots showed a up-regulated pattern and in leaves was significantly inhibited.Under abiotic stresses,the expression levels of CitERF34 were all up-regulated in leaves and inhibited in roots.The above results indicate that CitERF34 gene could respond to a variety of hormones and abiotic stresses,showing important roles in stress tolerance of citrus.(4)Subcellular localization analysis revealed that CitERF34 protein is located in the nucleus.Compared to WT plants,the tobacco transgenic plants overexpressing CitERF34 showed higher levels of soluble sugar and proline under abiotic stresses including drought,salt stress,and low temperature,and the transcription level of Nt SOD,astress-related gene,was higher in CitERF34-overexpressing tobacco plants than that of WT plants.Taken together,all there results suggested that CitERF34 play an important role in citrus stress tolerance.