Evolution And Expression Of The Ethylene-Induced Esterase Gene Family Identified From Citrus Sinensis

Ethylene plays important roles in the responses of plants to biotic and abiotic stresses and in plant growth and development. The Ethylene Induced Esterase(CsEIE) gene can be highly induced by ethylene and wounding, suggesting an important role in stress tolerance of citrus. Similar to other plant species, CsEIE has experienced repetitive gene duplication events during evolution, forming gradually a gene family. The release of the on-line databases such as sweet orange genome draft, TAIR and Phytozome makes it possible to analyze the history and subfunctionalization of the gene family members.In this study, we analyzed of CsEIE gene family members, and obtained the following results:1. In this study, through multiple alignments of EIE genes of different plant species, a total of18CsEIE gene family members were identified in Citrus sinensis by means of bioinformatics analysis. These CsEIE genes were distributed on4different chromosomes, mostly as tandem repeats. The number of exons of the members varied from1to5. Most of the members possessed3conserved motifs. Phylogenetic trees showed that the CsEIE gene family was clustered into three clads which were also prsenent in all analysed angiosperms, suggesting that the gene family had at least3ancient copies before the branching of angiosperms. Eight genes, CsEIE1～CsEIE8, were clustered into an independent clade, indicating they were derived from introspecific gene duplication events after citrus speciation. Expression analysis showed that the gene members have differed from each others in transcriptional activities and expression patterns.2. An analysis for cis-acting elements was carried out on the promoter sequences of the eitht members, CsEIE1-CsEIE8, respectively. The length of each promoter is around3.0kb. Results showed that the cis-elements were different in both types and quantites, but they contained mostly the followings:those responsive to hormones including abscisic acid, auxin, methyl-jasmonate, gibberellic acid, salicylic acid, and ethylene, those responsive to stresses such as high temperature,low temperature, hypoxia, drought, wounding and pathogen attacks, those controlling tissue-specific expression in endosperm, seed, mesophyll, meristem and other plant tissues. These results suggested that these genes could be involved in the responses of plants to hormones and to growth signals.3. The expression patterns of the gene family members under the treatments of salicylic acid, abscisic acid, ethylene and mechanical wounding were analyzed using qRT-PCR in combination with transcriptomoc analysis. Results showed that CsEIE(1,3), CsEIE2, and CsEIE4were induced by all treatments with ET and wounding as the two most potent inducers, and CsEIE2was the most highly induced gene. CsEIE5was induced only by SA, while CsEIE(6,8) were induced only by both SA and ABA. CsEIE1was nearly undetectable under all treatments, indicating it might be a pseudogene.4. The promoters of CsEIE1, CsEIE3and CsEIE4were cloned. A series of5’deletion expression vectors were construsted by ligating the deleted promoters to GUS gene, and were used to transform Arabidopsis by dipping the flowers in vector-containing agrobacterium liquid culture.These results should pave the way for further analysis of the subfunctionaliztion of the CsEIE gene family members.