Molecular Cloning And Expression Analysis Of Met And Kr-h1 Genes In Sitodiplosis Mosellana (Gehin) (Diptera:Cecidomyiidae)

Juvenile hormone is an important insect hormone that regulates insect diapause,metamorphosis and reproduction.Juvenile hormone receptor Met and its downstream transcription factor Kr-h1 play a key role in the JH signaling pathway.The orange wheat blossom midge,Sitodiplosis mosellana（Gehin）,is one of the important wheat pests.Larvae feed on the developing kernels,which leads to shriveling,and pre-sprouting damage.To escape from adverse environment and maintain species survival,mature larvae falling from wheat ears to the ground can successfully enter diapause,in which they oversummer and overwintering.The role of Met and Kr-h1 in wheat midge larvae diapause and metamorphosis is unclear.In view of this,we cloned full-length cDNAs of Met and Kr-h1 from S.mosellana larvae by RACE,and examined their expression in response to diapause and metamorphosis using q PCR.The main results are as follows:1.The full-length cDNA sequence for Met（Gen Bank accession no.KY660529）obtained from S.mosellana larvae was 5,264 long and contained a 2,733 bp ORF starting at nucleotide1,231 and ending at nucleotide 3,963.5’-UTR and 3’-UTR were 1,230 bp and 1,301 bp,respectively.The polypeptide deduced from the ORF comprised 910 amino acid residues with a predicted molecular weight of 102.3 k Da and a putative p I of 5.51.The deduced protein sequence from SmMet contained four typical domains of b HLH,PAS-A,PAS-B and PAC.Sequence alignments analysis showed that the homology of SmMet protein with other insect Met was more than 50%,especially shared the highest identity with Anopheles gambiae,which was 55%.2.The full-length cDNA sequence for SmKr-h1（Gen Bank accession no.KY646174）was2,327 long and contained a 1,842 bp ORF.5’-UTR and 3’-UTR were 64 bp and 421 bp,respectively.The polypeptide deduced from the ORF comprised 613 amino acid residues with a predicted molecular weight of 67.7 k Da and a putative p I of 8.73.The deduced protein sequence from SmKr-h1 contained eight C₂H₂-type zinc-finger motifs.Sequence alignments analysis showed that the homology of SmKr-h1 protein with other insects was more than 70%,especially shared the highest identity with Aedes aegypti,which was 81%.3.The expression level of SmMet and SmKr-h1 were significantly different at different stages of diapause.They had similar change trends of the m RNA transcription levels in the process of diapause.Namely,the relative expression levels were basically constant from pre-diapause,then entering diapause,to October,and reached the highest in post-diapause quiescence（December）,then decreased gradually,and returned to pre-diapause level before recovery development.The level of expression remained unchanged after development.The results showed that SmMet and SmKr-h1 may play a role in wheat midge diapause induction and maintenance.4.SmMet and SmKr-h1 were expressed in the wheat midge larvae,pupa and adult.The expression levels of the two genes in the 3rd larvae were significantly higher than those in the2nd,and female adults were significantly higher than male adults.SmMet showed the highest expression level in the 3rd larvae of wheat,followed by the late stage.There was no significant difference among the 3 development period（pupae,1st and 2nd larvae）;The expression level of SmKr-h1 was highest in the late stage of 3rd larvae,followed by pre-pupae,and they were significantly higher than that of other stages.There was no significant difference between the 1st and 3rd larvae.It was speculated that SmMet and SmKr-h1 may have important function in larval development,metamorphosis from larvae to pupae,and adult reproduction of S.mosellana.