Beet Armyworm Diapause Hormone - Sex Pheromone Biosynthesis Activating Peptide Gene (dh-pban, The) And Prothoracicotropic Hormone (ptth) Cloning And Expression Analysis

Two cDNAs encoding a putative precursor of prothoracicotropic hormone (PTTH) and diapause hormone (DH) from the beet armyworm, Spodoptera exigua (Spe), were isolated and sequenced, respectively. The PTTH cDNA is 1156 bp in length, containing an open reading frame encoding a 226-amino acid prepropeptide hormone. The deduced amino acid sequence is composed of a signal sequence, a precursor domain and a mature hormone including seven conserved cysteine residues and hydrophobic regions. Northern blot analysises demonstrate that the Spe-PTTH mRNA is about 1.2 kb expressed only in the brain Whole-mount immunocytochemistry by using an antiserum against Helicoverpa armigera (Har) re-PTTH showed that PTTH expression is in two pairs of neurosecretory cells in the S. exigua brain.The DH cDNA is 798 bp encoding a 197-amino acid precursor protein including DH, pheromone biosynthesis activating neuropeptide (PBAN), and three suboesophageal ganglion (SG) neuropeptides (SGNP), all of which share a conservative C-terminal pentapeptide motif FXPR/KL (X=G, T or S). The Spe-DH mRNA is about 800 bp expressed in the SG, and Spe-DH-like immunoreactivity is detected in the SG by using a H. armigera DH antibody. It is well known that both PTTH and DH are related with termination of pupal diapause. In the nondiapause species S. exigua, the two genes show the expression pattern associated with development, and suggesting that PTTH and DH have more functions other than regulating diapause in S. exigua.We investigated the difference of the content of polyhydric compounds in the hemolymph between diapause and nondiapause H. armigera individuals. The results show the trohalose content is at a higher level in diapause-destined individuals than those in nondiapause. Then, the cDNA encoding trehalose-6-phosphate synthase (TPS), which converts uridine-5'-diphosphoglucose and glucose-6-phosphate to trehalose-6-phosphate, was cloned from the fat body of H. armigera, using a strategy of rapid amplification of cDNA ends. The Har-TPS cDNA contains an open reading frame encoding a 536-amino acid, which shows 72-76% identity with other TPS reported so far. The TPS activity is higher in diapause type pupae in according with the content of trehalose. Northern blot analysis demonstrates the presence of a 2.0 kb transcript in the fat body and ovary. The Har-TPS mRNA is detected at high levels at early stage of diapause-destined pupae which are most likely to respond the changes of TPS activity and trehalose in hemolymph. The TPS protein was overexpressed in Bombyx mori nucleopolyhedrovirus expression system successfully, and showed that the catalytic activaty of TPS is approximately 5-fold higher in B. mori blood infected by the recombined-virus than by the mock inserted with another foreign fragment.