Cloning Of The CaM And Ets Gene From Hyriopsis Cumingii And Their Expression Under Lanthanum Exposure

Calmodulin?CaM?is a Ca²⁺ receptor protein exists widely in eukaryotic cells,which participating in regulation of a variety of enzyme activity,and mediating various biological processes including inflammation,metabolization,apoptosis,muscle contraction,intracellular movement,and the immune response.It also has a clinical application in hypermnesia,antipsychotic,anti-tumor and so on,which shows a good prospect in medicine.Accordingly,it becomes more widely and deeply in current research on the regulation mechanism of calmodulin,but little is known about it in mollusks.In this paper,for the study of Ca²⁺ signal transduction mechanism mediated by CaM in Hyriopsis cumingii,we clone and identify the genes of CaM?designated hcCaM?and Ets transcription factor?designated hcEts?,and we investigate the effect of Lanthanum ion(La³⁺,its spatial structure similar to that of Ca²⁺)on expression of hcCaM and hcEts,and the acitivity of AKP?Alkaline phosphatase,a downstream protein of CaM?.The results are as shown below.1?The partial length of hcCaM was 562 bp,contained an open reading fame of 450 bp which encoded a hypothetical protein of 149 amino acids?aa?with 4 characteristic structure EF-hand domain of CaM.The comparison of hcCaM with those of other CaM sequences revealed a high similarity with published CaM sequences from invertebrates and vertebrates?95.3% to 96.6%?.Besides,the molecular phylogenetic tree also demonstrated that CaM is conserved from invertebrates to vertebrates.The total length of hcEts was 777 bp,contained a complete reading fame,encoded a hypothetical protein of 258 amino acids?aa?with a characteristic structure of Ets family protein—ETS domain.hcEts was a Ets transcription factor gene firstly cloned and identified in invertebrates,freshwater pearl mussel?Hyriopsis cumingii?.It showed a different identity from others animals?27.91%-87.1%?.The lowest identity was 27.91%?Xenopus laevis and Gallus gallus?;the most identity was 87.1%?Azumapecten farreri?.In addition,the molecular phylogenetic tree based on sequences of Ets also demonstrated that H.cumingii has a simiar the evolutionary position with A.farreri.2?The result of the effect on expression of hcCaM under La³⁺ exposure: the expression of hcCaM was significantly up-regulated at 24,72,and 168 h?P<0.01?.In the LaCl₃ plus Trifluoperazine?TFP,a CaM special inhibitor?and TFP treated group,it was not significantly affected.All the results revealed that hcCaM transcription was induced by La³⁺ exposure in freshwater pearl mussels,which was blocked by TFP exposure.3?The result of the effect on expression of hcEts under La³⁺ exposure: in the LaCl₃ treated group,expression of hcEts was significantly decreased after LaCl₃ exposure at 6 h compared with that in the control group?0h??P<0.01?,but was significantly up-regulated after 24 h exposure?P<0.01?,but was not significantly affected after 3,12 and 48 h exposure,which was similar to the result of hcCaM.It demonstrated that La³⁺increased expression of hcEts by Ca²⁺/CaM signal transduction.While in the TFP treated group,expression of hcEts was significantly up-regulated after 3,6 and 12 h exposure compared with that in the control group?0h??P<0.01?,indicating expression of hcEts was significantly decreased when Ca²⁺/CaM signal transduction was blocked by TFP.No significant difference with that in the control group after 24 and 48 h exposure,which revealing the decrease in expression of hcEts active the other signal transduction,and the results of TFP plus LaCl₃ treated group further suggested there may be other signaling pathways regulating hcEts expression mediated by Ca²⁺.4?The resulty of the effect on activity of AKP under La³⁺exposure: in LaCl₃ plus TFP treated group,the activity of AKP was significantly increased by LaCl₃ exposure 12h after the addition of TFP?P<0.05?,but no significant effect was observed at 24,72,and 168 h,indicating that CaM regulates the activation of AKP by competitively binding to La³⁺to regulate calcium metabolism.