Expression And Identification Of Odrant Receptor2from Codling Moth, Cydia Pomonella L.

Codling moth cydia pomonella L. is a serious fruit-boring pest, widely distributed inorchards on six continents, and has been identified as the main quarantine objects in China.Codling moth larvaes do great damage to apple, pear, crabapple, apricot and peach. And causeenormous losses to fruit production in Xinjiang and Gansu. Codling moth has been detectedin Heilongjiang, Inner Mongolia and Ningxia. And there is a great risk of codling mothproliferating.Odrant receptors plays an important role in olfactory system of insects, and may provevaluable as a behaviour regulation target. We extracted the total RNA of Cydia pomonellaantenna and inverse transcribed the RNA into first strand cDNA. Using a pair of specificprimers with restrict enzyme sites in the5’ end, we obtained the open-reading fragment ofCydia pomonella odrant receptor2(CpomOR2) by polymerase chain reaction with the cDNAof Cydia pomonella antennae. This open-reading fragment was analysised by TMPREDsoftware, it encoded a membrane protein that had7transmembrane zone,3intracellular loops,3xetacelluar loops,1intracelluar N terminus and1extracellular C terminus.The open-reading fragment was digested by restrict enzyme and cloned into pET-32avector. The expression vector pET-CpomOR2was integrated into E. coli BL21(DE3), andinduced by IPTG for fusion protein expression. Detected by SDS-PAGE, we found a specificprotein of110kD. Further detection with Western blot showed that recombinant CpomOR2protein was exprssed successfully in the bacteria.We Induced the recombinant bacteria to expresse fusion protein by IPTG with a finalconcentration gradient. The bacteria had a high expression level when the final concentrationof IPTG was1mmol/L, and the expression level stoped raising with the final concentrationincreasing. Induced by IPTG (final concentarion:1mmol/L) with a time gradient, we foundthe expression level was considerable when the bacteria was induceing for2h. Induceing for6h, the expression rose to the highest level, and reached a lower level when the bacteria wasinduced more than8h. Induced by IPTG (final concentration:1mmol/L) for6h is the optimalinduce condition for the expression of this fusion protein.