Cloning And Expression Analysis Of E3Ubiquitin Ligases In Cucumis Sativus L.

Cucumber is one of the most important vegetables in the world. The largestplanting area all over the world is in China. Many diseases and insects damagecucumber production, among which, downy mildew, caused by Pseudoperonosporacubensis（Berk. and Curk.）Rostov., is a devastating foliar disease of cucumber（Cucumis sativus L.）prevalent in humid regions of the world. Our early researchsuggested that the hypersensitive response（HR）is a defense mechanism of cucumberagainst downy mildew. Moreover, a resistant-relative gene（GD254261）was earlysuppressed in the resistant inbred line during the HR, suggesting that this gene may beinvolved in defense mechanism. In order to testify its function in the HR, the genewill be cloned and its function will be identified. The results in this study will provideinsights into understanding the molecular mechanism underlying the HR. The mainresults are as follows:1. The biquitin gene was isolated and identified using gDNA and cDNA astemplates, respectively, according to EST（GenBank accession number: GD254261）,designated as CsFBXL（GenBank accession number: KC493560）.The full length ofCsFBXL is of1288bp with an open reading frame（ORF）of957bp encoding318amino acid residues. CsFBXL locates on the chromosome3with an intron of232bp.The deduced amino acid sequence contains an F-box domain and seven Leucine RichRepeat motifs belonging to a member of the FBXL of F-box gene family. Thepredicated protein of CsFBXL had a theoretical isoeletric point（pI）of4.45, and amolecular mass of36.74kDa. The putative amino acid sequence of CsFBXL hassubstantial homology with other proteins including Vitis vinifera, Arabidopsisthaliana and so on.2. The expression pattern of CsFBXL gene in resistant and susceptible inbredlines between cucumber and P. cubensis was monitored by qRT-PCR. In the resistantinbred line, the expression level of CsFBXL was significantly suppressed at48hpiand72hpi, whereas its up-regulated significantly at24h in the susceptible inbred line, suggesting that CsFBXL may be involved in the HR of cucumber to the attack of P.cubensis.CsFBXL was expressed constitutively in mature leaves, whereas it expressed atsignificantly low level in stems, fruits, roots and flowers than leaves, respectively,exhibiting a tissue-specific expression pattern.The expression of CsFBXL was induced significantly as early as24h aftertreated with SA, JA, and ABA, indicated that expression of CsFBXL may be regulatedunder SA, JA and ABA mediated pathways.The expression was suppressed extremely significantly at4h after the coldtreatments and the heat shock. Whereas the transcripts were also significantly inducedat2.0%and5.0%NaCl treatments, suggesting that this gene may be involved in thepathways responding to environmental stresses.3. The result of SDS-PAGE electrophoresis indicated that the molecular mass ofrecombinant protein was about36.74kDa and CsFBXL gene had expressed in E.coli.4. CsFBXL gene is localized in cytoplasm of cucumber from confocalmicroscopy.