Cloning And Functional Analysis Of Citrus CitERF108 Transcription Factor In Response To Drought Stress

Citrus as an important fruit crop is widly planted in China due to its high economic value.Because mainly planted in hilly and mountainous areas,it's often threaten by drought stress,which negative affect fruit yield and quality,.In order to respond drought,Plants have evolved a large number of drought-related genes,among which are belonged to transcription factors,such as:NAC,ERF,CAMTA,MYB,MYC,bZIP,etc.The ERF transcription factors are known to be unique in plant,which is widely involved in stress tolerance during plant growth and development.In recent years,a large body of studies on ERFs have been reported in model plants such as,Arabidopsis,rice and tomato,but rarely reported in citrus.In this study,CitERF108 and its promoter?Q108?were cloned from citrus junos cv.“Ziyang”.The genetic structure,genetic relationship,and protein characteristics were analysized and the cis-acting elements on Q108 were also predicted by bioinformatics.The biological function of CitERF108 in drought resistance were studied using gene expression,subcellular localization and genetic transformation.The main results are as follows:?1?CitERF108 was cloned from citrus junos cv.“Ziyang”,which contained 1intron and could encode 448 amino acids.The predicted CitERF108 protein possessed of the AP2 conserved domain where14th and 19th positions are alanine and aspartate,which belong to hydrophilic protein.Becides AP2 domain,CitERF108 also contains other conserved elements including WLG,YRG,RAYD.Cluster analysis showed that CitERF108 is closely related to Arabidopsis AtERF111,indicating CitERF108 may be involved in ABA-dependent stress response.Subcellular localization indicates that CitERF108 is located in the nucleus.?2?Expression analysis showed that CitERF108 could be expressed in roots,leaves,cotyledons,pericarps and seeds,with the highest expression level in seeds and almost undetectable in stems.In roots,CitERF108 was induced by hormones including SA,ETH,6-BA,IAA,ABA,JA and GA₃ as well as abiotic stresses including drought and salt damage.In leaves,CitERF108 was induced by ETH,JA.After SA treatment,the expression level was increased firstly,then decreased and increased again,the highest expression level observed at 3h,sharp decrease at 6h and 12h,and rising again at 24h.By 6-BA,ABA and GA₃ treatments,the expression levels were also up-regulated and down-regulated at 24h.The highest expression levels were observed at 6h after either 6-BA or ABA treatment,and at 3h after the GA₃ treatment.In IAA treatment,the expression level was decreased at 3h,and then increased at 6h.In salt treatment,the upward trend was not obvious at 3h,6h and 12h,but reached the highest point at 24h.Under drought treatment,the expression decreased slightly at 3h,increased at 6h and reached the peak at 12h.Taken together,these results indicated that CitERF108 plays an important role in citrus growth and stress response.?3?Overexpressing CitERF108 in tobaccos made transgenic plants show stronger drought tolerance than wild type.After drought treatment,the proline contents of 3 transgenic lines were 117.3?g,221.5?g and 112.3?g,respectively,much higher that of wild tobacco,109.5?g.The expression levels of drought-related NtSOD and NtCAT genes were significantly higher in transgenic plants than that in wild.Trypan blue staining revealed that the transgenic lines overexpressing CitERF108 maintain better cell membrane integrity than wild lines,indicating the transgenic plants were more drought-resistant than the wild type.?4?Q108 sequence mainly contained the cis-acting elements in response to salicylic acid,auxin,jasmonic acid,abscisic acid,ethylene and other hormones as well as cis-acting elements in response to temperature and light.The Q108 expression vector carrying the GUS gene was constructed and transformed into tobacco.The GUS histochemical staining showed that at 24 hours after 6mmol/L ACC treatment,the veins were stained blue,but the deep blue part was on the edge of the leaf;after 24hours of treatment with 1mmol/L ABA solution,the vein was dyed blue,and the staining was more uniform;After 4 hours of chilling treatment at 4°C,the veins were stained blue;After 24 hours of treatment with 20%PEG6000,the staining sites were all on the tip of the tobacco gland,None of the treatment and control treatments were not stained blue.