| The retinoid X receptor（RXR）is a member of nuclear receptors superfamily that function as transcription factors with roles in metabolism,development,cell differentiation and death.More and more studies indicated that the imposex in gastropodas induced by organotin was highly related to RXR.Baseing the full cDNA sequence of RXR from small abalone（Haliotis diversicolor,HdRXR）,the present study established a transcriptional activation system of HdRXR.The natural and enviromental ligands of HdRXR also were screened and the binding activities were compared with that of zebrafish RXR（Danio rerio,DrRXR）.The results are as follows:1)Retinoid X receptor ORF was cloned and recombined into an expressed plasmid of pcDNA3.1（+）and the DNA fragment containing retionid X response element（RXRE）was synthesised and recombined into a luciferease reporter gene plasmid of pGL3-promoter,making the reporter gene regulated by RXR.The reconstructed reporter plasmid and the corresponding expression plasmid of RXR were co-transfected into 293FT cell line to create a transcriptional activation system.2)The natural and enviromental ligands of HdRXR were secreened using the above transcriptional activation system.The results showed that 9cRA,TBT and TPT significantly induced the expression of luciferease respectively in the concentration of 1×10-7 M,1×10-8 M and 1×10-7M,respectivly.The maximum stimulatory concentrations were 1×10-6M,1×10-7M and 1×10-6 M,with the inducing fold of 2.25,2.67 and 3.23 times of the control（DMSO）respectively.The significant induced concentration of DHA is 1×10-8 M and the induced fold were 1.51 times of the control.3)The transcriptional activation system of DrRXR also were established in order to check the species specificity of RXR ligands.The effective concentrations of 9cRA to HdRXR were 10folds lower than that of DrRXR and The effective concentrations of TBT to HdRXR were 100folds lower than that of DrRXR,while in terms of TPT,these two RXR share the same minimum stimulatory concentrations but the induced fold of DrRXR was lower than that of HdRXR.DHA could not induce the transcriptional activitiesof DrRXR.These results confirmed that the binding charicteristics of RXR have species specificity.4)Different host cells were compared during the experiment.Cos-1 cells showed better adherence than 293FT and easy to handle,the expression stability of target gene and reporter gene in cos-1 cell also were good.However,293FT cells exhibited higher transfection rate,shorter expression time and higher level of expresssion than those of cos-1,the data from 293FT cells were used in present study.In addition,the expression of an reference reporter gene of renilla luciferase changed with the TBT concentrtion added into the HdRXR transcriptional activation system.Therefore,the renilla luciferase reporter gene is not suitable for all transcriptional activation system although it’s the most widely used double reporter gene assay at present. |
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