[Construction of a yeast model for screening Aedes albopictus ecdysone agonist pesticides].
OBJECTIVE
To reconstitute a transactivation system in yeast (yeast model) for screening the pesticides acting on ecdysone metabolism route and eventually influencing the process of ecdysis.


METHODS
The fragment of 5 times repeated EcRE from Drosophila melanogaster was synthesized and the HSP27 promoter from D. melanogaster genome was amplified with PCR. The two sequences were connected and followed by a reporting gene--green fluorescence protein (GFP) gene. The EcRE-HSP27 promoter-GFP fragment was inserted into the expression plasmid pPIC3.5 and integrated into the yeast chromosome to construct yeast A. EcR and USP coding sequences of Aedes albopictus were synthesized, and these two fragments were inserted into Pichia pastoris expression plasmid pGAPZ as two respective reading frames. The two reading frames were integrated into Pichia pastoris chromosome in another recombinant site (pGAPZ and pPIC3.5k share different recombinant sites while being integrated into Pichia pastoris yeast chromosome). EcR and USP were constituted and expressed in the yeast. This recombinant yeast was called yeast B. The model yeast was thus constructed. A known ecdysone agonist-tebufenozide was used to test the yeast model. The effect of tebufenozide on the model yeast was observed under fluorescent microscope. Semi-quantitative RT-PCR was used to test the transcription level of GFP in the tebufenozide affected yeast and the control.


RESULTS
In the model yeast, the intracellular expressed EcR and USP constituted EcR/USP heterodimer interacting with EcRE, the expression of GFP was activated, and green fluorescence was observed in model yeast under fluorescent microscope. Tebufenozide affected model yeast showed less fluorescence in comparison to the control model yeast, indicating that the transcription of GFP was suppressed by tebufenozide. Yeast housekeeping gene Actin-1 was used as inner control, semi-quantitative RT-PCR was operated and the result was scanned. The ratio of the brightness of GFP to Actin-1 was calculated automatically, and that of tebufenozide added yeast and the control yeast was 0.614 and 1.134 respectively. This result showed a low transcription level of GFP in tebufenozide affected model yeast, comparing to that of the control.


CONCLUSIONS
The ecdysone-related transacting system in yeast has been constructed, and the model yeast can be used to screen the ecdysone agonists which can act on the ecdysone metabolic route.
Gu
Jin-bao
JB
Department of Pathogen Biology, Southern Medical University, Guangzhou 510515, China.
Sun
Yan-tao
YT
Peng
Hong-juan
HJ
D000330:Aedes / Q000378:metabolism*; D000818:Animals; D015195:Drug Design; D004440:Ecdysone / Q000819:agonists* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006834:Hydrazines / Q000494:pharmacology; D007306:Insecticides / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015003:Yeasts / Q000235:genetics*
D006834:Hydrazines; D007306:Insecticides; D049452:Green Fluorescent Proteins; D004440:Ecdysone; C082026:tebufenozide