Preliminary Study On The Application Of Novel Fusion Fluorescent Protein IEGFP As A Fluorescence Indicator Molecule

Fluorescent proteins are important indicator molecules,which have been widely used in the biological research.In order to adapt to different needs,researchers have made efforts to improve the fluorescent protein in many ways.Previously,the N-terminal secretory signal peptide(Iasp)of Cry1 Ia protein of Bacillus thuringiensis(Bt),was used to guide the expression of EGFP.The resulted fusion fluorescent protein was designated as IEGFP.The p H stability of IEGFP was consistent with that of EGFP.Its expression in Escherichia coli and Bt was significantly higher than that of EGFP and consequently,provided stronger fluorescence signal for the host.More importantly,most of the expressed products retained intact size which would be attribute to the low transmembrane transport efficiency of IEGFP.These results indicated that IEGFP can be used as a noval fluorescent indicator.In this study,we first compared the effect of Iasp to other two well-known signal peptides on translocation of EGFP.Then,IEGFP was used to compare the activity differences of three typical Bt insecticidal gene promoters P_v(vip3A),P_ac(cry1Ac)and P_i(cry1Ia),and to monitor the P_v's regulation patterns in E.coli and Bt.Finally,in view of the similarity between Vip3 A protein and Cry1 Ia protein,we test whether the signal peptide of Vip3 A protein can improve the expression of IEGFP.The results are as follows:First,the encoding sequences of Cry1 Ia,pectate lyase B(pel B)and trimethylamine nitrogen oxide reductase(tor A)were fused to the egfp gene,respepectively.These fusion genes were placed at the 3' end of the promoter P_ac and expressed in E.coli.Using spectrophotometer and laser confocal microscopy,had the strongest fluorescence signal of the host cells expressing IEGFP were observed and mainly located in the cytoplasm.Tor A-EGFP could be translocated into the periplasmic space of E.coli,but fluorescence signal was so weak.Pel B-EGFP protein could also transport efficiently,but it could not fluores normally,and the expression of pel B-EGFP protein could inhibit the growth of host cells.Therefore,we believed that Iasp was different from the two typical signal peptides,which could improve the expression of EGFP protein in cytoplasm of the prokaryotic host,and IEGFP had the potential as a fluorescent indicator molecule.Furthermore,IEGFP was used to compare the activity differences of the three representative Bt insecticidal gene promoters P_v,P_ac and P_i.These promoters were connected to the 5?end of iegfp gene respectively to guide its expression in E.coli TG1 strain.The result showed that 12 hours after inoculation,the fluorescence signal intensity regulated by the three promoters decreased in turn(P_ac > P_i > P_v).We also noted that even the weakest P_v is about two times more active than the control(promoter of lac I gene of lactose operon repressor protein in E coli,P_lacI).The P_ac activity with the highest activity was about 75% of that of P_lacIq.IEGFP was also used to monitor the expression pattern of P_v.It was found that the promoter was a constitutive promoter in E.coli.In Bt cells,the fluorescence intensity of IEGFP peaked 24 hours after inoculation,which was consistent with the previous report.In order to further understand the sensitivity of IEGFP to the change of weak promoter activity,we modified the sequences between SD of P_v.and initial codon AUG.The result showed that IEGFP could indicate the effect of different connection modes on P_v activity.Finally,this paper explored whether the Vip3 A signal peptide(Vsp)had similar effect with Iasp.The result showed that under the control of P_v,only the fluorescence signal of IEGFP was detected in E.coli.The vegfp gene was also placed under the strong promoter T₇ regulation and expressed in BL21-Star(DE3).The result showed that VEGFP could express normally.But about half of the expressed products were degraded to EGFP molecular size.The results suggested that the transport efficiency of VEGFP was higher than that of IEGFP.Although the secretory pathway of Vip3 A protein was not clear,it was speculated from the fluorescence signal monitoring results of VEGFP protein that it may be transported to the periplasmic space of E.coli.These results indicated that Vsp did not have the function of increasing EGFP expression by Iasp.Interestingly,as a secretory insecticidal protein,the N-terminal signal peptide of Vip3 A was not removed during or after transport.However,this study found that the signal peptide of VEGFP could be removed.Of course,the result still needed to be further confirmed.In conclusion,IEGFP has lower transmembrane transport efficiency and higher expression than EGFP,so it can be used as a novel fluorescent indicator molecule.