Comparison Of The Electrophysiologigal Properties And Functional Study Of KAT1 Channels Among Cucumis Melon, Cucumis Sativus L. And Citrullus Lanatus

Two Shaker genes,named CsKAT1 and ClKAT1 in this research,were cloned from two kinds of vegetable crop,Cucumis sativus and Citrullus lanatus.Analyses of their amino acids and nucleotides,the deduced proteins encoding by both genes have three typical Shaker putative conserved domains.According to the phylogenetic tree,CsKAT1and ClKAT1 were belonged to KAT1 subgroup and showed the highest homology to that of potassium channel,MIRK,which is from the same cucurbitaceae family,muskmelon.These two genes were speculated to encode the inward rectifier potassium channels belonging to Shaker-like family.To verify the speculation above,CsKAT1 and ClKAT1 were constructed with the oocyte expression vector pGEMXho firstly.After linearization and in vitro transcription,each cRNA was invidually injected to Xenopus oocytes,then,two electrodes voltage-clamped system was used to characterize the electrical properties.The experiment revealed that both proteins functionally present slow-activating inwardly rectifying currents,one of the typical inward Shaker features.Besides,their open probabilities were independent of the external potassium concentration.The analyses of the affinity of both channels to K⁺and their cationic selectivity indicated that both genes encoded the K⁺-selective channel with low affinity to K⁺.Moreover,the increase of external proton concentration resulted in the increase of the macroscopic inward currents of both channels.In addition,the sensitivity of both channels to the external Na⁺was tested via the same technique.Both channels shared the same principle with that of MIRK,of which the activity was prohibited by the external Na⁺,meanwhile,the inhibition lev_Ie_Vl induced with the external sodium concentration without voltage dependency.The inhibition level of these three channels gave as sequence:ClKAT1?MIRK>CsKAT1,which was independent on the external potassium concentration.With aims of investigating the molecular bases that modulate this external Na⁺-induced current inhibition,S5-P-S6 amino acid domain of MIRK was replaced by the corresponding region of KAT2 from Arabidopsis,resulting in a new recombination DNA,named?KAT2/MIRK.The electrophysiological study proven that the activity of the novel channel encoding by?KAT2/MIRK kept the same kinetic as that of MIRK in terms of K⁺sensitivity.However,the inward?KAT2/MIRK currents was not any more affected by the external Na⁺,implying that the molecular region that leads to the external Na⁺-induced current inhibition phenomenon was located at the position of S5-P-S6.By transformation,MIRK and?KAT2/MIRK have been integrated into the genome of Arabidopsis thaliana?kat1?.The phenotype of transgenic plants?T3-generation?and kat1-mutant with salt treatments?0,50 and 100mmol/L?were investigated.Under 50 mM NaCl treatment,there wasn't noticeable difference regarding the appearance of these three lines of plants.Under 100 mM NaCl,the growth of kat1-mutant was prohibited while compared with the other two types of transgenic plants.These results indicated that MIRK or?KAT2/MIRK transformation compensates part of physiological capacity of mutant line,thereby,further improve salt tolerance of plant.Two melon cultivars differing in salt tolerance were used to evaluate MIRK?melon inward rectifying K⁺channel?transcription,K⁺and Na⁺distribution,as well as stomatal aperture under various salt treatments?0,100,and 200mM NaCl?for exploring the mechanism of salinity tolerance in melon.The salt-tolerant cultivar,cv.BXC?Bingxuecui?showed a smaller reduction in biomass due to salt treatments than the salt-sensitive cultivar,cv.YL?Yulu?.Although the level of total Na⁺in the whole plant was the same in both melon cultivars after salinity treatments,a lower Na⁺level with higher K⁺/Na⁺ratio was observed in the leaves of the BXC cultivar,which is likely to promote salt tolerance in melon.Salinity also resulted in down-regulation of MIRK transcription and stomatal closure in the leaves of both varieties,however,compared to the YL cultivar,a lesser extent of MIRK transcription and stomatal aperture reduction were observed in the BXC cultivar,both of which remained steadier at most sampling times.The high correlation observed between stomatal aperture closure and MIRK transcription level suggests that the regulation of the MIRK either in mRNA level or channel acitivity under salt stress in melon may be resulted in the less K⁺uptake into guard cell,and subsequently involved in modulation of the stomatal apertures,ultimately contributed to balancing the leaf transpiration and CO₂ exchange.