Molecular Cloning And Expression Analysis Of EcR Homologue In The Wheat Midge, Sitodiplosis Mosellana (Gehin)

Wheat blossom midge, Sitodiplosis mosellana (Géhin), is an important insect pest ofwheat in Europe, Asian and North America. The larvae feed on developing seeds, resulting insignificant yield losses, the yield is reduced by10to20percent, and sometime30-50percent,even total loss of the yield when outbreak. There are two outbreaks in China in last century(the1950s and1980s, respectively), caused serious economic losses. Since the feeding of S.mosellana is very difficult indoor the research about it was seldom, and the mechanism ofdiapause of S. mosellana is not well development.Ecdyson receptor (EcR), which belongs to the superfamily of nuclear receptor (NR), isthe main target of ecdyson, and EcR has important regulatory function. It is well known thatEcR has great effects on reproduction, behavior, memory, study and life span of insect.Besides, since ecdyson has important regulating role of insect diapause, EcR plays animportant role in diapause of insect.In this paper, the ecdyson receptor gene of S. mosellana was cloned by using reversetranscription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE), and theexpression was analyzed through real-time PCR. The main results are as follows:1. The ecdyson receptor gene was cloned by using of RT-PCR and RACE. Thefull-length sequence obtained was named as SmEcR (GenBank accession number: KC491135),and the open reading frame (ORF) of SmEcR was1386bp in length, encoding461aminoacid residues, with a calculated molecular weight of52.90kD and the theoretical point of6.24.Multiple sequence alignment revealed that deduced amino acid sequence of SmEcR had a highdegree of identity to EcRs from other insect species, such as Bradysia coprophila, Aedesaegypti, Aedes albopictus, and Culex quinquefasciatus, especially to the EcR of Bradysiacoprophila (92%). Sequence analysis by bioinformatics showed that SmEcR sequencecontains many conservative motifs, like ligand binding domain (LBD) and DNA-bindingdomain (DBD). Besides, a part cDNA sequence (744bp) of β-actin gene was cloned,encoding181amino acid residues.2. The expression of SmEcR in different diapause stage of S. mosellana was analyzed byusing real-time PCR. The result showed that the transcripts of SmEcR were detected in all diapause stages, while the expression levels were different, the expression level reach thehighest point in November, while reaching the lowest point in December.3. The expression of SmEcR in different development stages of S. mosellana wasanalyzed by using real-time PCR. The result showed that in larvae, diapause larvae(December), pupa and adult the transcripts of SmEcR can be detected. The expression ofSmEcR was fluctuated, and the larvae in wheat had lowest expression level while the adultsexpress highest SmEcR transcript, which was about100times of larvae in wheat.In this paper the full-length cDNA of SmEcR was cloned, and the real-time PCR analysisshowed that the transcripts of SmEcR were detected in all diapause stages and developmentstages. The results in this paper will be helpful for further study of the function of SmEcR inthe regulation of S. mosellana diapause.