Identification Of The Diapause-associated Genes Esr16 And FKBP12 In Helicoverpa Armigera

By using mRNA differential display PCR (DD-PCR), we have identified 56 cDNA fragments from Helicoverpa armigera brain that are expressed differentially between the diapausing pupae and the pupae of diapause termination by injecting 20-hydroxyecdysone. RT-PCR and Real-time PCR analysis revealed that mRNA levels of the FK506-binding protein 12 (FKBP12) and two unknown genes were higher in the diapausing pupae, whereas the ecdysteroid-regulated 16 kDa protein (esr16), NADH dehydrogenase 1 alpha subcomplex subunit 6, and two unknown genes were higher in the pupae of diapause termination. These results provide us an insight on elucidating the molecular mechanism of diapause.Using a strategy of rapid amplification of cDNA ends, the cDNA encoding esr16 and FKBP12 were cloned from the brain of H. armigera. The esr16 cDNA contains an open reading frame encoding a 146-amino acid protein, which shows high identity with the Manduca sexta (64%),Bombyx mori (60%)and Drosophila simulans (50%). Esr16 mRNA can be detected in all tissues, such as brain, suboesophageal ganglion, thoracic ganglion, abdominal ganglion, midgut and fat body. Northern blot and Western blot analysis show that the mRNA and protein in the brain of diapausing pupae were lower than the nondiapausing pupae.When the diapause was terminated, the level of esr16 mRNA was up-regulated. The results showed that the esr16 may play a core role in promoting the development of the pupae in Helicoverpa armigera.The FKBP12 cDNA contains an open reading frame encoding a 108-amino acid protein, which shows high identity with the Bombyx mori (96%),Manduca sexta (92%),Tribolium castaneum (81%) and Apis mellifera (81%). FKBP12 mRNA can be detected in all tissues, such as brain, suboesophageal ganglion, thoracic ganglion, abdominal ganglion, midgut and fat body. Northern blot and Western blot analysis show that the mRNA and protein in the brain of diapausing pupae were higher than the nondiapausing pupae.When the diapause was terminated, the level of FKBP12 mRNA was down-regulated. These results imply that the FKBP12 may play a role in maintaining the diapausing pupae in Helicoverpa armigera.