Cloning and expression of the cDNA encoding the FXPRL family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera

Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are encoded by a single mRNA in the suboesophegeal ganglion (SG) and are responsible for induction of embryonic diapause in Bombyx mori and sex pheromone biosynthesis in lepidopteran insects. PBAN cDNA analyses revealed that the DH-like peptide is present in several species that have a pupal diapause. However, the function of the DH-like peptide remains unknown. In the present study, we cloned the cDNA encoding DH-PBAN in Helicoverpa armigera utilizing the rapid amplification of the cDNA ends method. The nucleotide se quence analysis revealed that the longest open reading frame of this cDNA encodes a 194-amino acid precursor protein that con tains a 33-aa PBAN, a 24-aa DH-like peptide, and three other neuropeptides, all of which have a common C-terminal pentapeptide motif FXPR/KL ( X=G, T, S). A homology search showed that H. armigera DH-like and PBAN are highly homologous to those from other insects. Northern blot analysis demonstrated a single message RNA corresponding to the size of Har-DH-PBAN cDNA from pupal SG with significantly higher expression in the SG of nondiapause pupae than diapausing pupae. Western blot analysis showed DH-like peptide expression from SG of both males and females. When DH-like peptide was injected into nondiapause larvae and pupae, it did not induce diapause, but rather efficiently broke pupal diapause in H. armigera. The ED(50) of DH to terminate pupal diapause is 20 pmol/pupae. The other four FXPRLamide neuropeptides from the DH-PBAN polyprotein precursor have cross activity for diapause termination. These observations therefore suggest a potential role for these FXPRL family peptides in promoting continuous development in several noctuid species. The high expression of this gene in pharate adults and adults indicates that the FXPRL family peptides may have multiple physiological functions.     

家蚕滞育的分子机理2.蛹期滞育激素基因的表达与滞育决定

滞育激素是由食道下神经节分泌的重要昆虫神经肽,诱导昆虫的滞育。选择具有ＲＮＡ聚合酶能够识别的启动子的质粒载体,将滞育激素ｃＤＮＡ克隆进去,在体外大量合成单一的滞育激素ｃＲＮＡ为参照,测定食道下神经节分泌滞育激素ｍＲＮＡ量来确定滞育激素的分泌量。结果证明食道下神经节分泌的滞育激素的数量决定昆虫的滞育。     

Structural characterization and expression analysis of prothoracicotropic hormone in the corn earworm, Helicoverpa zea

The cDNA encoding prothoracicotropic hormone (PTTH), the brain neuropeptide that stimulates the prothoracic glands to synthesize ecdysone, was cloned from the corn earworm Helicoverpa zea (Hez). The amino acid sequence deduced from the cDNA indicates a molecular structure that is distinct from the PTTH's reported in other Lepidoptera, but all contain an identical proteolytic cleavage site and the seven cysteine residues that are essential for activity. Northern hybridization shows a single mRNA present in the brain-subesophageal ganglion complex. Using RT-PCR, we observed constant amounts of PTTH mRNA during larval development but large fluctuations at pupation and prior to adult eclosion.     

亚洲玉米螟神经肽羽化激素基因cDNA的克隆及组织表达

羽化激素对调节昆虫的蜕皮和发育起关键作用。亚洲玉米螟Ostrinia furnacalis是亚洲农业重要害虫之一,本实验研究了亚洲玉米螟羽化激素基因cDNA的分子结构和表达模式。利用兼并性引物RT-PCR技术,克隆了亚洲玉米螟羽化激素基因cDNA的中间片段,然后再用RACE方法,获得羽化激素基因的 cDNA全长序列。结果表明： 亚洲玉米螟羽化激素基因cDNA全长986 bp（GenBank登录号： DQ668369）,开放阅读框为267 bp,编码88个氨基酸的前体蛋白,其中包括前26个氨基酸组成的信号肽和62个氨基酸的成熟肽。亚洲玉米螟羽化激素基因与烟草天蛾、棉铃虫和家蚕已报道同源基因的同源性较高,分别为79.5%、77.3%和67.0%,与黑腹果蝇同源基因的同源性最低,仅45.5%。亚洲玉米螟羽化激素基因mRNA只在脑中表达,在咽下神经节、胸神经节、腹神经节等神经组织中检测不到,在非神经组织如中肠、脂肪体和表皮中也不表达。     

An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas' disease

Triatoma infestans is a hemiptera, vector of Chagas' disease that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects' salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its SG was randomly sequenced. Also, salivary proteins were submitted to two-dimensional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results.     

Molecular cloning of Bombyx cerebral opsin (Boceropsin) and cellular localization of its expression in the silkworm brain

We have cloned a cDNA for a novel opsin from the larval brain of the silkworm Bombyx mori in which the photoperiodic photoreceptor had been supposed to reside in the cephalic central nervous system (CNS). Its deduced amino acid sequence was composed of 381 amino acids and included amino acid residues highly conserved in insect visual pigments. This opsin belonged to the long wavelength photoreceptor group of insect opsins and showed the greatest degree of homology (84%) with the green visual photoreceptor in the sphingid moth. We have designated this Bombyx cerebral opsin as Boceropsin. Southern blotting experiments indicated that the Boceropsin gene is present in a single copy, and RT-PCR analysis revealed that Boceropsin mRNA is expressed in the larval brain but not in the subesophageal ganglion (Sg) or thoracic ganglion (Tg). Immunohistochemical analyses demonstrated that Boceropsin protein is present bilaterally in some defined cells localized in the brain of Bombyx larvae. This is the first report of expression of an opsin-based protein in CNS of an insect. The possibility that the Boceropsin functions as the photoperiodic receptive pigment in the silkworm is also discussed.     

Effect of the brain and suboesophageal ganglion on pupal development in Helicoverpa armigera through regulation of FXPRLamide neuropeptides

Recent studies in Helicoverpa armigera report a novel role for diapause hormone (DH), pheromone biosynthesis activating neuropeptide (PBAN) and three other FXPRLamide neuropeptides secreted from suboesophageal ganglion (SG) in terminating pupal diapause. In the present paper, we investigated the role of these five FXPRLamide family neuropeptides on pupal development. Although removal of SG could not make nondiapause-destined pupae enter diapause-like status, it did make them eclose approximately 0.6-1.2 days later when compared with the controls. The results of competitive ELISAs showed a high level of FXPRLamide titer in the hemolymph of the SG-removed pupae and this may be due to the expression of the DH-PBAN gene in tissues other than SG. DH-PBAN mRNA and peptides were also detected in the thoracic ganglia (TGs) by RT-PCR and immunocytochemistry. The expression of DH-PBAN gene in the TGs of the SG-removed pupae is significantly higher than that in normal pupae by quantitative PCR and immunocytochemistry. Decerebration experiments proved that the decerebrated pupae could enter diapause-like status through down-regulation of FXPRLamide titer in hemolymph. Our studies confirm that the brain plays an important role in the determination of pupal development by regulating the synthesis and release of FXPRLamide neuropeptides in H. armigera. Thus, the function of FXPRLamide peptides in H. armigera is closely correlated with pupal development.     

Expression and distribution of phocein and members of the striatin family in neurones of rat peripheral ganglia

Phocein and members of the striatin family (striatin, SG2NA and zinedin) are intracellular proteins, mainly expressed in neurones of the mammalian central nervous system where they are thought to be involved in vesicular traffic and Ca(2+) signalling. Here, we have investigated whether these proteins are also present in the peripheral nervous system, by analysing their expression and distribution within sensory neurones of the vagal (nodose and jugular) ganglia, the petrosal ganglion, the dorsal root ganglion, and also in the sympathetic neurones of the superior cervical ganglion. RT-PCR experiments showed that mRNAs of phocein, striatin, SG2NA and zinedin are present in all studied peripheral ganglia. Immunocytochemical detections demonstrate that phocein, striatin and SG2NA are expressed in neurones of vagal, petrosal and dorsal root ganglia. Immunoblotting experiments confirm these data and in addition demonstrate that: (1) the proteins phocein, striatin and SG2NA are also present in the superior cervical ganglion and (2) zinedin is detected in all studied ganglia. The distribution appears to differ: immunoreactivity for striatin and SG2NA is found only in soma of sensory neurons, whereas immunoreactivity for phocein is observed in both soma and processes. Our study thus demonstrates that phocein and the members of the striatin family are expressed not only in central nervous system but also in the peripheral nervous system and, in particular, in afferent sensory neurones.     

Two type I crustacean hyperglycemic hormone (CHH) genes in Morotoge shrimp (Pandalopsis japonica): cloning and expression of eyestalk and pericardial organ isoforms produced by alternative splicing and a novel type I CHH with predicted structure shared with type II CHH peptides

Crustacean hyperglycemic hormone (CHH) peptide family members play critical roles in growth and reproduction in decapods. Three cDNAs encoding CHH family members (Pj-CHH1ES, Pj-CHH1PO, and Pj-CHH2) were isolated by a combination of bioinformatic analysis and conventional cloning strategies. Pj-CHH1ES and Pj-CHH1PO were products of the same gene that were generated by alternative mRNA splicing, whereas Pj-CHH2 was the product of a second gene. The Pj-CHH1 and Pj-CHH2 genes had four exons and three introns, suggesting the two genes arose from gene duplication. The three cDNAs were classified in the type I CHH subfamily, as the deduced amino acid sequences had a CHH precursor-related peptide sequence positioned between the N-terminal signal sequence and C-terminal mature peptide sequence. The Pj-CHH1ES isoform was expressed at a higher level in the eyestalk X-organ/sinus gland (XO/SG) complex and at a lower level in the gill. The Pj-CHH1PO isoform was expressed at higher levels in the XO/SG complex, brain, abdominal ganglion, and thoracic ganglion and at a lower level in the epidermis. Pj-CHH2 was expressed at a higher level in the thoracic ganglion and at a lower level in the gill. Real-time polymerase chain reaction was used to quantify the effects of eyestalk ablation on the mRNA levels of the three Pj-CHHs in the brain, thoracic ganglion, and gill. Eyestalk ablation reduced expression of Pj-CHH1ES in the brain and Pj-CHH1PO and Pj-CHH2 in the thoracic ganglion. Sequence alignment of the Pj-CHHs with CHHs from other species indicated that Pj-CHH2 had an additional alanine at position #9 of the mature peptide. Molecular modeling showed that the Pj-CHH2 mature peptide had a short alpha helix (α1) in the N-terminal region, which is characteristic of type II CHHs. This suggests that Pj-CHH2 differs in function from other type I CHHs.     

Cloning and characterization of a cDNA coding for the alpha-subunit of a stimulatory G protein from Schistosoma mansoni.

Guanine nucleotide-binding proteins (G proteins) mediate signals between serotonin receptors and adenylate cyclase in Schistosoma mansoni. A bovine Gs alpha cDNA probe was used to isolate a cDNA clone, SG12, encoding the entire alpha-subunit of a G protein of S. mansoni. The cDNA is 1897 base pairs long, contains an open reading frame of 1137 base pairs, and codes for a deduced protein of 379 amino acids. The putative protein encoded by the clone has an exact amino acid match with bovine Gs alpha of 65% and a 78% match when conserved amino acid substitutions are considered. In contrast, the exact and conserved matches of the schistosome alpha-subunit with bovine Gi are 41 and 61%, respectively. A comparison of the deduced amino acid sequence of SG12 with a variety of different G alpha proteins indicates that all the major structural features characteristic of a Gs alpha protein are present in the S. mansoni gene. The schistosome clone contains the putative site for ADP-ribosylation by cholera toxin found in Gs alpha but does not contain the ADP-ribosylation site for pertussis toxin present in Gi alpha. The amino acids are completely conserved at the GTP-binding sites. On a Northern blot, the cDNA hybridizes to a major band of 3.1 kilobases in RNA from adult schistosomes. The message appears to be absent in miracidia and cercariae, but a faint 3.1-kilobase band is visible in the early schistosomule stage preceding adulthood. This evidence, when added to previous biochemical data, indicates that the expression of this gene is developmentally controlled.     

Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids.

Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids. Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R. serpentina cell suspension cultures. The active enzyme was expressed in Escherichia coli and purified to homogeneity. Analysis of its deduced amino-acid sequence assigned the SG from R. serpentina to family 1 of glycosyl hydrolases. In contrast to the SG from C. roseus, the enzyme from R. serpentina is predicted to lack an uncleavable N-terminal signal sequence, which is believed to direct proteins to the endoplasmic reticulum. The temperature and pH optimum, enzyme kinetic parameters and substrate specificity of the heterologously expressed SG were studied and compared to those of the C. roseus enzyme, revealing some differences between the two glucosidases. In vitro deglucosylation of strictosidine by R. serpentina SG proceeds by the same mechanism as has been shown for the C. roseus enzyme preparation. The reaction gives rise to the end product cathenamine and involves 4,21-dehydrocorynantheine aldehyde as an intermediate. The enzymatic hydrolysis of dolichantoside (Nbeta-methylstrictosidine) leads to several products. One of them was identified as a new compound, 3-isocorreantine A. From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in R. serpentina and C. roseus cell suspension cultures occurs at a later stage than strictosidine deglucosylation.