Study Of Function And Regulatory Mechanism Of EgrZFP7 Under Abiotic Stress In Eucylptus Grandis

Eucalyptus is one of the three fast-growing trees in the world,as well as important economic tree species.Abiotic stresses,such as low tempreture,drought and salt can destroy the growth and development of plants.To elucidate the function and regulatory mechanism of key genes which related with abiotic stresses will be helpful for molecular assisted breeding in Eucalyptus.EgrZFP7 is a classic C2H2 type zinc finger transcription factor which is involved in abiotic stresses response in Eucalyptus grandis.There are 2 zinc finger domains containing QALGGH motif which is specific to plants in the protein sequence of it.An ERF(Ethylene responsive element binding factor)associated amphiphilic repression(EAR)motif and another L-box motif related with protein interaction were also found in it.Furthermore,Several cis-elements which are involved stresses response were found in the promoter sequence of EgrZFP7,such as ABRE、 DREB and NACF.Different time course treatment at 4℃(0,2,6,12,24,48h)can promote the expression of EgrZFP7.832 genes that co-expressed with EgrZFP7 were screened under this treatment with WGCNA software based on the DGE(digital gene expression).117 co-expression genes were distributed in different KEGG pathways,71 genes were involved in metabolic pathways and biosynthesis of secondary metabolites pathway.To the biosynthesis and metabolism pathway of amino acids and sugar,there are 22 and 19 genes in them respectively.Analysis of the 20 genes of top correlation value showed most of them are involved in the stress responses,including 5 transcription factors,such as MYB,WRKY and zinc finger protein,and 4 glycosyltransferase.To determine whether over-expression of EgrZFP7 gene could enhance resistance to abiotic stress,we construct 35S∷EgrZFP7over-expression vector and transformed it into Arabidopsis thaliana.Finally we obtain 2 homozygous transgenic plant lines EgrZFP7-OX1 and EgrZFP7-OX2.Both of them showed longer lateral roots and the number of lateral roots were also increased compared to the wide type under normal condition.The sensitivity to the low temperature of them were also enhanced.The sensitivity to salt was not significantly changed under salt treatments,but the tolerance to PEG was found in the two over-expression lines.To study the regulatory mechanism of EgrZFP7,the protein expression vector in prokaryotic was constructed based on PMAL-C2X(with MBP fusion tag).For the better results,it should be transformed into E.coli Transetta(DE3)and the fusion protein expression was induced for 14 hours under 20℃ by 0.4mmol?L-1 IPTG.Anti-MBP Magnetic Beads was used a to purify the fusion protein.Finally,CASTing method was used to select the core sequence that EgrZFP7 binds.For the sequences isolated,there are a high level consecutive consistency fragments containing two thymine(T),the possible core sequence of [TG][TG][CA]AA[AG][TC][TG] was thought to be the cis-element that EgrZFP7 bind,but it should be further tested with EMSA.