Hormone Regulation And Expression Pattern Of Hsp90, Thymosin And Serpin And During Development Of Helicoverpa Armigera

Molting and metamorphosis are important biological processes of the growth and development of insects, regulated by a variety of hormones including 20-hydroxyecdysone(20E) secreted by prothoracic glands.20E regulates the expression of molting and metamorphosis-related late genes by 20E signal pathway mediated by its receptor and ultimately control the physiological process, such as the degradation of the old cuticle. Innate immune response are important for defending foreign microorganism in insects and other invertebrates. Studies have shown that some innate immune-related genes are also regulated by 20E.Here we identified three genes highly expressed during metamorphosis from Helicoverpa armigera, heat shock protein HaHsp90, thymosin gene HaTHY1 and HaTHY2 and serine protease inhibitor HaSerpin.1. HaHsp90To investigate the role of highly conserved Hsp90 in insect hormone cascades, We cloned full length cDNA sequence of HaHsp90. It contains 2154 bp open reading frame(ORF) and encodes 717 amino acids. This protein has a HATPase-c (Histidine kinase-like ATPases) domain. BLAST analysis revealed that the similarity between HaHsp90 and Hsp83 from Spdoptera frugiperda reached 99%.Northern Blotting analysis of the expression pattern and hormone regulation showed that the expression level of HaHsp90 was higher in epidermis, midgut and heamocytes at metamorphosis stage 6th 96 h and 120 h, and up-regulated by both 20E and methoprene. Cellular RNAi analysis revealed that silence of ecdysone receptor EcRB1 and its partner USP1 had no effect on the expression of HaHsp90. However, knocking down HaHsp90 down-regulated the expression of USP1 HHR3 and E75. These results revealed that HaHSP90 was involved in 20E sigal pathway. Meanwhile, Hsp90 was likely participated in methoprene signal transduction.2. HaTHYl and HaTHY2Studies on thymosin mainly focuse on veterbrates, to investigate its roles in insects, we cloned two different cDNA sequence of thymosin gene for the first time, named HaTHY1 and HaTHY2. Their ORF is 510 bp and 396 bp respectively, and the predicted molecular weight of the deduced protein is 19 kDa and 14.5 kDa respectively. Genomic sequence analysis revealed that HaTHY1 and HaTHY2 were transcript variants of a single gene.RT-PCR analysis revealed that HaTHY1 was dominantly expressed in epidermis and midgut while HaTHY2 was mainly expressed in fat body during metamorphosis and heamocytes. Additionally, the expression level of HaTHY1 was up-regulated by 20E, Both were upregulated by immune chanllege. These results revealed that the two isoforms had different function.3. HaSerpinSerine protease inhibitor(serpin) is involved in serine protease cascade in prophenoxidase activation pathway in insect innate immune, to further investigate the role of serpin in 20E cascade,, we cloned serpin from H. armigera for the first time, named HaSerpin. The full length cDNA of HaSerpin is 1441 bp. It was evolutionary closer to serpin-1 from S. frugiperda.RT-PCR analysis revealed that the mRNA level of HaSerpin was much higher at metamorphosis stage than other stages and it was up-regulated by 20E, suggesting that serpin was possibly involved in insect metamorphosis.