Studies on Biosynthesis of a Type II Sex Pheromone, Transportation of the Hydrophobic Precursor in a Body, and Mechanism of Their Hormonal Regulation

日本大环鱼(Ascotis selenaria cretacea，ASSC)的处女雌体分泌C_19 3，4-环氧基-6，9-二烯来吸引雄性。以前的实验表明，环氧烯基信息素是由信息素腺体外产生的相应的3，6，9-三烯生物合成的，并在与脂蛋白结合后通过血淋巴运输到信息素腺体。为了弄清这种转运，用KBrR平衡密度梯度超速离心法纯化了雌蛾体内的高密度脂蛋白(HDLp)，并通过对分离蛋白的溶剂提取物的GC-MS分析证实了三烯的结合。下一步，HDLp的作用是通过在雌性的腹部局部应用氢化三烯基前体来揭示的。三烯基前驱体与HDLp有关。在它们的信息素腺体中，三烯和氚环氧信息素WE…检测到更多，表明三烯通过血淋巴移动。对克氏桑蛾雄蛾和家蚕雌蛾的实验表明，HDLp与局部使用的三烯具有相同的联系。这一结果表明，与许多鳞翅目物种的雌性鳞翅目昆虫一样，成虫在食道下神经节(SG)中产生信息素生物合成激活神经肽(PBAN)，以调节其在信息素腺体中的生物合成。我们克隆了一个编码181个氨基酸的ASSC-PBAN基因，其中包括一个PBAN同源物和另外四个可能的多肽：滞育激素同系物、α-SG神经肽、β-SGNP和γ-SGNP，它们的C末端都有一个FXPR(K)L基序。虽然含有30-35个氨基酸的PBAN已从其他15个物种中鉴定出来，但ASSC-PBAN同源物由28个氨基酸组成，与其他物种相比同源性较低(&lt；46%)。ASSC-β-SGNP含有8个氨基酸，也比其他β-SGNP(16-22个氨基酸)短。此外，所有已知的PBAN cDNAs在β-SGNP和PBAN之间都有一个GRR序列作为切割位点，但Asc-PBAN的cDNA在相应的位置显示了一个不寻常的GR序列，这表明β-SGNP和PBAN之间可能没有切割。天然信息素多肽的层析行为揭示了包括βSGNP在内的ASSC-PBAN的独特结构，即SVDFTPRLGRQLVDDVPQRQIEEDRLGSRTRFFSPRL-NH_2，这是首次从产生环氧烯基性信息素的昆虫中确定PBAN的。较少

Virgin females of the Japanese giant looper (Ascotis selenaria cretacea, Assc) in the family of Geometridae secrete C_19 3,4-epoxy-6,9-diene to attract males. Previous experiments have suggested that the epoxyalkenyl pheromone is biosynthesized from the corresponding 3,6,9-triene produced outside of a pheromone gland and transported to it via hemolymph after association with lipophorin. In order to clarify this transport, high-density lipophorin (HDLp) in the female moths showing two bands (apoLp I with ca.250 kDa and apoLp II with ca.80 kDa) on an SDS-PAGE was purified by KBr equilibrium density-gradient ultracentrifugation, and the association of the triene was confirmed by GC-MS analysis of a solvent extract from the isolated protein. Next, the role of HDLp was revealed by a topical application of the deuterated trienyl precursor to the abdomens of the females. The trienyl precursor was associated with HDLp. In their pheromone glands, the triene and the deuterated epoxy pheromone we … More re detected, indicating movement of the triene via the hemolymph. Experiments with male moths of A.s.cretacea and female moths of Bombyx moil showed the same association of HDLp with the triene topically applied. This result suggested that the adult females of A.s.cretacea did not develop HDLp specialized in the triene transport.To regulate its biosynthesis in the pheromone gland, Assc females produce a pheromone biosynthesis-activating neuropeptide (PBAN) in the suboesophageal ganglion (SG), as do females in many lepidopteran species. We have isolated Assc-PBAN cDNA, which encodes 181 amino acids, including a PBAN homologue and four other putative peptides : a diapause hormone homologue, α-SG neuropeptide (SGNP), β-SGNP, and γ-SGNP, all of which shared an FXPR(K)L motif on their C-termini. Although PBANs with 30-35 amino acids have been characterized from 15 other species, the Assc-PBAN homologue consisted of 28 amino acids and showed low homology (< 46 %) compared with the others. Assc-β-SGNP with 8 amino acids was also shorter than the other β-SGNPs (16-22 amino acids). Furthermore, all of the known PBAN cDNAs have a GRR sequence between β-SGNP and PBAN as a cleavage site, but the Assc-PBAN cDNA showed an unusual GR sequence at the corresponding position, indicating the possibility of non-cleavage between the β-SGNP and PBAN. The chromatographic behavior of the natural pheromonotoropic peptide revealed the unique structure of Assc-PBAN including βSGNP, i.e., SVDFTPRLGRQLVDDVPQRQQIEEDRLGSRTRFFSPRL-NH_2, as the first determination of PBAN from the insects producing an epoxyalkenyl sex pheromone. Less