Bioinformatics And Preliminary Expression Analysis Of Eucalyptusgrandis WRKY Genes Family

WRKY gene,as an important transcription factor,is mainly involved in response to plant biological and abiotic stresses.Eucalyptus as the firstly major afforestation in southern China,which is stressed by the dry blight,bacterial wilt,cold damage and other hazards.The WRKY protein in Eucalyptus may be involved in the response of eucalyptus to stress.Therefore,this study the system of Eucalyptus urophylla WRKY family genes were analyzed in this study,based on the sequencing of the eucalyptus genome,combined with bioinformatics analysis,.The expression of EgrWRKY gene in different tissues was analyzed by RT-PCR.Fluorescence quantitative PCR was used to analyze the genetic differences in response to different hormone treatments(SA,MeJA,BR)and stress treatments(cold stress,salt stress)Expression analysis.Seventy-nine putative WRKY genes identified in E.grandisi were systematicall analyzed.Chromosomal localization revealed that EgrWRKY genes were enriched on chromosomes Chl3,Chl7 and Chi11,and approximately 35 percent of the WRKY genes participated in gene duplication.All EgrWRKY transcription factors were assigned into groups ?,?,and ?,and a second group was classified into sub-groups ?a through ?e).Promoter analysis of EgrWRKY genes identified various cis-acting elements associated with stress and defense responses.Eight EgrWRKY genes showed significantly higher expression in flowers,mature leaves and roots.Moreover,expression profiles in leaves showed various expression patterns in response to phytohormones and abiotic stress.All EgrWRKY genes showed varying dEgrees of responsiveness to at least one abiotic stress treatment,with the exception that three EgrWRKY genes were undetected.Most EgrWRKY genes were upregulated in response to phytohormone treatment.Moreover,numerous EgrWRKY gene were upregulated following 168 hours of salt stress.Further,EgrWRKY genes were mainly induced following two and six hours of cold stress.Notably,several EgrWRKY gene expression profiles showed co-responsiveness to different treatments,suggesting multiple stress resistance-related functions.The findings helped identify functional EgrWRKY genes and strengthened understanding of the complex regulatory mechanisms of WRKYs following stress exposure in Eucalyptus.Moreover,EgrWRKY70 was cloned and analyzed by using reverse transcription polymerase chain reaction(RT-PCR)and real time quantitative PCR(qRT-PCR).The results showed that EgrWRKY70 encodes a predict protein of 321 amino acids.The theoretical molecular weight of EgrWRKY70 is 36.18 kDa with pI of 5.28,unstability index of 63.67.EgrWRKY70 contained a conserved WRKY domain and belonged to group ?a.The results of multiple alignments and phylogenetic analysis revealed that EgrWRKY70 is closed to JcWRKY70 with respect to evolutionary relationship,with 58.2%similarity.The semi-quantitative reverse transcription and polymerase chain reaction indicated that Theexpression of EgrWRKY70 was detected in in flowers,leaves and roots,whereas the EgrWRKY70 did not nearly expressed in stem including xylem and phloem.Real-time analysis of RT=PCR concluded that expression of EgrWRKY70 increased firstly and then decreased with the duration of the treatment of salt and cold.While the EgrWRKY70 expression maintained at a high level under salt stress.The levels of EgrWRKY70 expression responsed rapidly with the treatments of salicylic acid and brassinolide,whereas there is no distinct change in EgrWRKY70 expression with the treatment of methyl jasmonate.These concluded that EgrWRKY70 may take part in biotic and abiotic stress responses 'by various ways.