Molecular Cloning And Expression Of HSP90and HSP70in The Wheat Midge, Sitodiplosis Mosellana(GEHIN)

The wheat blossom midge, Sitodiplosis mosellana (Gehin), is an important insect pest of wheat. Their diapause divergence is one of the main reasons causing sporadic out-break. Heat shock proteins (Hsps) are very important in insect diapause. In this test, Hsp90and Hsp70genes of S. mosellana were cloned by using rapid-amplification of cDNA ends (RACE) and reverse transcription PCR (RT-PCR), and the expression was analyzed through real-time PCR. The main results are as follows:1. The full-length sequences obtained were named as SmHsp90and SmHsp70(GenBank accession number:KJ813012and KJ813012). The full-length cDNA of SmHsp90is2412bp, and the open reading frame (ORF) of SmHsp90was2148bp in length, encoding715amino acid residues, with a calculated molecular weight of81.98kD and the theoretical point of5.02. The full-length cDNA of SmHsp70is2199bp and the open reading frame (ORF) is1926bp in length, encoding641amino acid residues, with a calculated molecular weight of70.41kD and the theoretical point of5.48. Multiple sequence alignment revealed that deduced amino acid sequence of SmHsp90had a high degree of identity to Hsp from other insect species, such as Ceratitis capitata, Bactrocera oleae, liriomyza sativae, while SmHsp70had a high degree of identity to Rhagoletispomonella.2. The expression of SmHsp90and SmHsp70in different diapause stage of S. mosellana was analyzed using real-time PCR. The results showed that the transcripts of SmHsp90and SmHsp70were up-regulated in all diapause stages, while the expression levels were different. The expression level of SmHsp90reached the highest point in December, while the expression level of SmHsp70reached the highest point in August. The two genes reached the lowest point in March.In this paper the full-length cDNA of SmHsp90and SmHsp70was cloned. The real-time PCR analysis showed that the transcripts of SmHsp90and SmHsp70were related to different diapause stages. The results in this paper will be helpful for further study of the function of SmHsp90and SmHsp70m regulation of S. mosellana diapause.