| Cucumber, as one of the most important horticultural crops is widely cultivated in the world andaccount for a pivotal position of vegetable production and consumption in China. However, the yieldand quality of cucumber are seriously affected by the environment stresses in the greenhouse such aslow-temperature.Transcription factors play important roles in environmental stresses. WRKYtranscription factors family is widely involved in biotic and abiotic stresses response in the whole life ofplant. Therefore, with the completing of the cucumber genome sequencing, it is more convenience forus to make clear the function of WRKY transcription factors in cold signal transduction and regulation.In this study, we investigated the WRKY family factors in cucumber and the expression patterns of thegenes involved in cold stress. Also we focus on the two cucumber CsWRKY46and CsWRKY21transcription factors, whose gene function in cold stress and the roles of the cold signal passway wereresearched. The results are as follows:(1)2WRKY genes were cloned from cucumber, and the CsWRKY46and CsWRKY21wereanalyzed. The length of CsWRKY46open reading frame is786bp, containing two introns and threeexons; encoding262amino acids, protein molecular weight is28.6kDa, belongs to the Class II familyof WRKY transcription factor; phylogenetic analysis shows that CsWRKY46and AtWRKY71arehomologous genes. The length of CsWRKY21open reading frame is948bp, containing four intronsand five exons; encoding316amino acids, protein molecular weight is34.5kDa, belongs to the Class IIfamily of WRKY transcription factor; phylogenetic analysis shows CsWRKY21and AtWRKY40arehomologous genes.(2) Expression characteristics of CsWRKY46and CsWRKY21were analyzed. CsWRKY46andCsWRKY21are response to low temperature stress and exogenous ABA, the up-regulate expression aredetected by real time PCR. Semi-RT-PCR results showed that CsWRKY46and CsWRKY21genes areexpressed in10tissues of cucumber, while CsWRKY21has high expression in flowers.(3) CsWRKY46and CsWRKY21localized in nucleus. The CsWRKY46and CsWRKY21withGFP-fusion protein expression vector were successfully constructed separately, CsWRKY46andCsWRKY21localized in the nucleus by transient expressing of GFP-fusion protein in Arabidopsisprotoplasts.(4) CsWRKY46and CsWRKY21over-expression vectors and the RNAi vectors were constructedsuccessfully, and over-expression transgenic Arabidopsis were obtained. CsWRKY46positivelyregulates cold transduction pathway, cold resistance of CsWRKY21over-expression plants(WRKY21-OE) is also enhanced. The transgenic plants were subjected to freezing treatment, andshowed the survival rates of the CsWRKY46over-expressing plants (WRKY46-OE) were higher thanthat of wild-type plants by30.67%. Following cold treatment electrolyte leakage (EC) of WRKY46-OEwas significantly lower than that of WT; the proline of WRKY46-OE accumulated significant higher levels compared with that of WT. Meanwhile, MDA were increased after chilling stress in both WT andWRKY46-OE plants; however, there was less MDA accumulation in WRKY46-OE plants than in WTplants after chilling stress. These results indicated that the WRKY46-OE was more tolerant to coldstress. Expression of ABA/stress-responsive genes such as COR47,RD29A,KIN1and ABI5wassignificantly up-regulated in WRKY46-OE plants than that of WT. The survival rate of WRKY21-OEwas higher than that of wild-type plants in frozen treatment, but no significant difference. The EC,proline and MDA of WRKY21-OE were changed significantly compared with the WT plants, theseresults indicated that the WRKY21-OE was more tolerant to cold stress. Expression of coldtress-responsive genes such as COR47,RD29A,KIN,1CBF1,CBF2and CBF3were significantlyup-regulated in WRKY21-OE plants than that of WT, suggesting that CsWRKY21may response to CBFdependent cold signal pathway.(5) WRKY46-OE were more hypersensitive to ABA. Compared with WT plants, WRKY46-OEenhanced the plants sensitivity to ABA by effect on the germination but not seedling growth, thisdemonstrated that CsWRKY46positively regulated the ABA signal pathway.(6) The constructed plasmid pET-CsWRKY46and pET-CsWRKY21were transformed into E. coliBL21(DE3), and the expression of recombinant protein His-CsWRKY46and His-CsWRKY21arehighly induced.In summary, this study identified the gene function of two cucumber WRKY transcription factorCsWRKY46and CsWRKY21in cold stress resistance. CsWRKY46as a positive regulator involved inthe response to cold stress by ABA-dependent pathway. The CsWRKY21is also a positive regulator incold stress. This research will give further insight for understanding the resistance regulation ofmolecular mechanisms in horticultural crops and provide a theoretical base for improving the coldresistance by applying the transcription factors in the genetic engineering in the future. |
