Development Of A Screening System For The Diacyihydrazine Compounds

Ecdysteroids cause a molt by combining with the ecdysone receptor（EcR）and theultraspiracle protein（USP） heteredimer to initiate a molting cascfide including expression oftranscription factors, which in turn regulate tissue-specific genes involved in molting andmetanaorphosis. Bishydrazide insect growth regulator acting in a similar fashion of ecdysone,interaction with the protein of EcR but difficult to separate, it can also effects kinds of genesexpression,and enzymes’ activity. So, those insecticides can made the pests continue moltingand control them.The availability of cDNA clones for EcR and USP from several insectspecies has opened up possibilities for the development of high throughput EcR/USP basedscreening assays. EcR/USP proteins can be produced by in vitro transcription and transition ofcDNAs in bacteria, yeast and insect cells.Either crude or purified proteins can be used indisplacement assays．In the study, we cloned EcR and USP genes from Spodoptera litura, and the full-lengthcDNA of EcR from Laodelphgax striatellus. Use prokaryotic expression biotechnology, to getthe purified protein and Structuring simple system of screening the diacyihydrazinecompounds. The results as follow:With RT-PCR technique, we cloned target gene segments with length of1149bp and1062bp, Sequence alignment analysis proof that target gene segments have high homologouswith the insect of Lepidoptera. arget gene segments contains DNA-binding domain, hingeregion, ligand-bind domain. construct prokaryotic expression vector pEHISEGFPTEV-EcR|_cdeand pEHISEGFPTEV-USPcdesuccessfully.After induction expression,we obtain the proteinwith the molecular weight of67kD and64kD.1. The full-length EcR gene from Laodelphgax striatellus were cloned by Racetechniques. The full-length EcR cDNA was3403bp, An open reading frame （ORF） cotains1521bp, encoding506amino acids.it’s the first full-length cDNA from the homoptera insect.We also construct prokaryotic expression vector pEHISEGFPTEV-EcR|_cde-H. The reseachselectd Seven kinds of insects from different species, after equence alignment analysis wefound some differences sites of amino acids.3. Use the Radioligand receptor binding assays and the purified protein from the vectorpEHISEGFPTEV-EcR|_cdeand pEHISEGFPTEV-USPcde, the system of screening theDiacyihydrazine compounds were established.