| Abscisic acid (ABA), an important signaling molecule, can regulate a variety of physiological and molecular responses for plants against stresses, including drought, chilling and salt. Previous studies showed that one model of ABA action was related to plant oxidative stress signaling. The two-component system (TCS), which works on the principle of histidine kinase (HK) phosphorelay signaling, is known to play an important role in diverse physiological processes in lower organisms and has recently emerged as an important signaling system in plants. But the relationship between ABA-induced oxidative stress signaling pathways and HKs gene is reported little. Our lab previous studies found out OsHK3which responsed significantly in the expressions induced by ABA, was an ABA-regulated OsHK of the OsHK gene family(OsHK1-6).In view of the above-mentioned facts, in this study, to analyse the function of OsHK3, the ORF of OsHK3gene was amplified by RT-PCR from rice leaves which were treated with ABA. The ORF of OsHK3was3072bp, which deduced994aa. Sequence analysis showed that OsHK3consisted typical HK conserved domains:a CHASE domain, a transmitter domain and a receive domain. According to the forecast analysis, OsHK3had two transmembrane domains. Amino acid sequence analysis among OsHK3and Arabidopsis thaliana hybrid histidine kinases by ClustalW2showed that OsHK3contained key conserved sites:conserved His residual base in HisKA domain and DDK sequences in REC domain, which suggested OsHK3might have complete histidine kinase activities.In order to study the function of OsHK3in ABA-induced antioxidant defense, we have established a rice seedling protoplasts over-expression system. We inserted OsHK3’s ORF into the sites of pXZP008vector. The construct (35S-OsHK3-YFP) was then transformed into rice protoplasts by PEG method. We found that, the gene expressions of SODCc2, APX, CATB significantly increased after eight hours. Sixteen hours later, the total activities of SOD, APX and CAT largely increased. The total activities of SOD, APX increased2-3times, while CAT’s dramatically increased. These results suggested that OsHK3might be involved in ABA-induced antioxidant defense reaction.To investigate further, we also have established a rice seedling protoplasts system designed for the rapid characterization of large numbers of genes by transient double-stranded RNA interference (RNAi) which used for the analysis of the relationship between OsHK3and ABA-induced antioxidant defense. The protoplasts were transfected with an in vitro-synthesized dsRNA against OsHK3, and treated with ABA after24hours’ culture. The results showed that, ABA could significantly increase the expressions of SODCc2, APXA, CATB, and the the total activities of SOD, CAT and APX. When OsHK3was silenced, the expression levels of SODCc2, APXA, CATB and the total activities of SOD, CAT, APX dramatically reduced. The total activities of SOD and APX reduced about50%and40%, and CAT’s reduced significantly too, which restored little by ABA treatment. These results suggested OsHK3might be involved in ABA-induced antioxidant defense reaction on the other hind.To investigate subcellular location of OsHK3, we inserted the ORF of OsHK3into pXZP008. On one hand we transformed the construct (35S-OsHK3-YFP) into rice protoplasts, on the other hand we incorporated pXZ-OsHK3into onion (Allium cepa L.)epidermis by particle bombardment. Images of YFP-OsHK3fusion were then captured with the confocal laser scanning microscope.We initially identified that YFP-OsHK3was located to cytoplasm and cytomembrane.As mentioned above, we clarified that during the ABA-induced signal transduction, OsHK3played important roles. This study provided new evidence to the relationship between ABA and OsHK3, so as to affluent the ABA-induced signal transduction in plant cell antioxidant defense system. |
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