Cloning And Expression Analysis Of CqWRKY1Gene Induced By Fusarium Acid In Chieh-qua

Chieh-qua (Benincasa hispida Cogn.var. Chieh-qua How.) is one of specialty vegetables inChina and is also the main vegetable crops in south China. Fusarium wilt of Chieh-qua, causingby fungal pathogen Fusarium oxysporum, is one of the most economically damaging diseasesworldwide. It can decrease the yields of Chieh-qua severely. Fusarium wilt is a soil-bornedisease which is difficult to control using chemical prevention or crop rotation.Breeding varieties with resistance against fusarium wilt disease is the fundamental way to solvethis problem. Because of narrow genetic background, few variant types and the lack of resistantresources against fusarium wilt, research on resistance mechanism of Chieh-qua against fusariumwilt and discovering the resistance related gene is of particular importance for promotingthe breeding for disease resistance.WRKY transcription factor is a supergene family, which plays an importantrole in plant stress resistance. In our previous study, by suppression subtractive hybridizationtechnique, a suppression subtractive hybridization cDNA library was constructed with resistantChieh-qua cultivar induced by Fusarium oxysporum. Our result showed that some defenserelated EST sequences in cDNA library were significantly similar to WRKY transcription factorsof other species. In order to explain the role of WRKY in resistance to Fusarium wilt, thefull-length cDNA sequence of a WRKY transcription factor was amplified and itsexpression pattern was studied. In addition, in order to accurately analyze the expression oftarget gene, we selected appropriate reference genes of Chieh-qua based on differentexperimental conditions through real-time quantitative PCR technique. The main results are asfollows:1. Full length of one WRKY gene was obtained from Chieh-qua using RACE techniquebased on a putative EST sequence, and was designated as CqWRKY1. The length of CqWRKY1cDNA was2088bp, containing an open reading frame of1746bp encoding a protein of581amino acids. CqWRKY1protein belongs to group I, of the WRKY family, including two typicalconserved WRKY domains and C2H2(C-X4-C-X22-23-H-X1-H) zinc finger structure.2. The bioinformatic analysis showed that the tertiary structure of CqWRKY1proteinmainly contains four antiparallel β sheets, three random coils and a zinc-binding pocket at the end of β sheet. CqWRKY1transcription factor localizes in the nucleus and contains two nuclearlocalization signals PTKKKVE and PEAKRWR. It may have transcription, transcriptionregulation and other functions. Sequence alignment and phylogenetic tree analysis indicated thatCqWRKY1had the closest evolutionary relationship with WRKYP2protein of cucumber andtheir similarity was about92%.3. Four commonly used housekeeping genes GAPDH, Actin, UBQ, CYP and two novelreference genes CACS, F-box were chosen for identifying the expression stability in Chieh-quasamples from various plant tissues (roots, stems and leaves) or under different treatments. Thestability of the expression of the candidate genes was determined and ranked using RefFindersoftware. The results revealed a remarkably stable expression pattern of Actin when sampleswere under fusaric acid stress. High stability was obtained for CACS and UBQ during salicylicacid treatment, while CACS was also found to be most suitable in methyl jasmonate treatment.F-box protein gene showed the most stable expression level in different tissues.Additionally, expression of tradition housekeeping genes GAPDH and CYP showed greatervariations and therefore considered unsuitable as reference genes.4. The expression patterns of CqWRKY1in different tissues (root, stem, leaf) andtreatments were analyzed by real-time quantitative PCR. The results showed that the expressionof CqWRKY1gene was strongly induced under fusarium acid stress. The expressionof CqWRKY1reached the peak at12h after being induced, about80.26times of controls.Besides, the expression of CqWRKY1was also induced by signal molecules SA or MeJA.Therefore, CqWRKY1transcription factor may involved in the defense response of fusariumwilt disease, and its regulation may depend on the signal pathway mediated by SA or JA.