Identification Of WRKY Family Genes And Function Characterization Of Low Temperature Responsive WRKYs In Watermelon

Watermelon?Citrullus lanatus?is an important horticultural crop cultivated all over the world.Low temperature stress is frequently encountered in early spring and off-season protected cultivation in watermelon production.Studying gene expression patterns of watermelon suffering low temperature stress and identifiying the function of differentially expressed genes will be beneficial to understand the regulation mechanism of watermelon in response to low temperature stress and provide gene resources for watermelon breeding aiming at improving low temperature tolerance.WRKY transcription factor family has a large number of members with multi functions,which are not only involved in the growth and development of plants,but also regulate the response of plants suffering biological or abiotic stresses.Transcriptome of watermelon under low temperature stress showed that expression patterns of several watermelon WRKY family genes significantly changed,indicating that WRKY transcription factors participated in the response of watermelon to low temperature stress.However,whether these genes have functions in regulating the response of watermelon to low temperature stress and their underlying mechanism are still unknown.To address this problem,genome wide identification of WRKY family genes was preformed firstly,and then the functions of two low temperature responsive WRKY genes Cla018059 and Cla021170 were characterized in this study.The main results are as follows:1.The identification and characterization of watermelon WRKY transcription factors ware conducted.A total of 57 WRKY transcription factors were identified on watermelon genome using the Hidden Markov Mode.These genes were named as ClWRKY1-ClWRKY57 according to their position on the chromosomes.Protein sequence alignment and phylogenetic analysis showed that 55 ClWRKY transcription factors had conserved seven-peptide WRKYGQK DNA binding domains,and the other 2 had WRKYGKK and KSRKRQV respectively;Zinc-finger motifs of all the ClWRKYs were C₂H₂ or C₂HC type.According to the number of WRKY domains and the type of zinc-finger motif,watermelon ClWRKY transcription factors were divided into three groups,namely I,II,and III with 11,39,and 7 members respectively.Gene structure analysis showed that the length of watermelon ClWRKY genes ranged from 0.7 kb to 7 kb,mainly in 1 kb³ kb.The exon numbers of ClWRKY family genes ranged from 2 to 6.Collinearity analysis showed that there were 18 duplication events in watermelon ClWRKY family genes,3 of which were transposable duplication and 15 were segmental duplication,suggesting that segmental duplication plays a major role in amplification of watermelon ClWRKY family genes.All of the Ka/Ks values of the duplicated ClWRKY genes were less than 1,indicating that the duplicated ClWRKY family genes experienced purfying selection during the evolution process.2.Expression profiles of ClWRKY transcription factors were analysed.Transcriptome analysis on watermelon fruit development showed that 56 ClWRKYs expressed in fruit except for ClWRKY1,50,52,and 54 ClWRKY genes expressed in flesh,rind,and seeds,respectively.Transcriptome analysis on watermelon suffering low temperature stress showed that 19 ClWRKYs were significantly induced and differentially expressed,including 7 genes which were down regulated and 11 genes that were up regulated.Among the significantly unregulated genes,Cla018059?ClWRKY55?and Cla021170?ClWRKY20?exhibited the highest log₂FC values,implying that they were sensitive to low temperature stress.These two genes were selected and further study.3.The functions of low temperature related ClWRKYs were analysed.The subcellular localization expression vectors pH7LIC5.0-N-eGFP-ClWRKY20 and pH7LIC5.0-N-eGFP-ClWRKY55 were constructed and injected into epidemal cells of tobacco leaves,respectively.Both ClWRKY20 and ClWRKY55 were observed in the nucleus under the laser confocal microscope.The over-expression vectors of ClWRKY20and ClWRKY55 were constructed and transformed into tobacco,generating the transgenetic plants of OE20 and OE55,respectively.The T₁ plants were subjected to 4?treatment.The phynotypic and physiological traits such as cold injury index,relative electric conductivity,and the content of malondialdehyde were measured.The results showed that OE20 had higher low temperature tolerance and OE55 had lower low temperature tolerance compared with the wild type.The activities of reactive oxygen scavenging enzyme were determined.The results showed that the OE20 plants suffering low temperature stress had higher activities of SOD,POD,and CAT compared with the wild type plants,indicating that ClWRKY20 improves the low temperature tolerance putatively by increasing the antioxidant ability of the plants.While the antioxidant enzyme activities of OE55 plants were lower than that of the wild type plants,which suggested that ClWRKY55 decreased the low temperature tolerance of plants putatively by reducing their antioxidant ability.