| The cotton bollworm, Helicoverpa armigera, which belongs to Lepidoptera Noctuidae, distributes broadly in the world and harms a lot of crops including cotton, cereal, corn, potato, bean and tobacco leaf. The cotton bollworm is one of the major pests of the world agriculture, which often brings great lost in economy. At the present time, the majority chemical pesticides used to prevent cotton bollworm are harmful to human and environment, and there is controversy on the security of the transgene crops such as the Bt cotton. Therefore, development of the biological pesticides based on the theory studies is always the focus of the pest prevention. For the holometabolous insects, periodically molting occurs during larval development. The hormones regulate many physiological events throughout the insect life cycle. Through the research of the molting related genes, especial the genes regulated by 20E, we may further understand the molecular mechanism of development and ecdysone regulation, and find the novel molecular targets to effectively control the pest. We identify some putative genes which might be involved in metamophosis, by Suppression subtractive hybridization (SSH) of tester cDNA from metamorphically committed larvae and driver cDNA from feeding 5th instar larvae and choose eukaryotic translation initiation factor 5C (eIF5C) as target to clone the full cDNA and analyze its character during the development of the cotton bollworm in the experiments.Molting is a physiological process common to all holometabolous insects, its life cycle is characterized by a series of molts: larval molts, during which the larva progresses from one instar to the next, and metamorphic molts, which lead to pupation and eclosion. Generally, molting consists of two phases, inactive phase and ecdysis. Subsequent metamorphosis, the transformation of larva to pupa to adult, includes metamorphic molting. During this process, more complicated physiological processes occur, including histolysis of larval tissues, remodeling and formation of adult tissues, in addition to a molting cascade similar to the larval molt. Insect larval molting and metamorphosis are governed by ecdysteroids (20-hydroxyecdysone, 20E) and juvenile hormone (JH), with 20E orchestrating the molting process and JH determining the nature of the molt. In the presence of JH, 20E directs larval molting. Otherwise, 20E directs metamorphosis. Increasing evidence indicates that other hormones and receptors may contribute to the complex developmental pathways associated with metamorphosis. Many genes have been shown to be involved in molting or metamorphosis, such as the transcription factors EcR, USP, HR3, Broad C, and the programmed cell death pathway genes and some key regulatory genes have been identified, such as Broad complex, E74B and E93, but very few genes downstream of Broad complex, E74B and E93 are identified. Therefore, the molecular mechanisms that lead to larval molt and metamorphosis are poorly understood. The lepidopteran, Helicoverpa armigera shares molting and metamorphosis features with Manduca sexta and Bombyx mori. Because no genomic sequence or commercial microarrays exist for H. armigera, large scale screening of genes expressed differentially expressed during molting and metamorphosis is a great challenge. We performed two-dimensional electrophoresis and identified 30 new proteins whose expression increased during larval molting. Here, we use SSH to identify more genes involved in larval molting and metamorphosis in H. armigera.We performed SSH between tissues from a variety of developmental stages, including metamorphically committed and feeding 5th instar larvae, and feeding 5th instar and metamorphically committed larvae including molting 5th and feeding 6th instar larvae which have been completed in previous study. One hundred expressed sequence tags (ESTs) were identified and included 73 putative genes with similarity to known genes, and 27 unknown ESTs. SSH results were further characterized by RT-PCR. The expression levels of eleven genes were found to change during larval molting or metamorphosis, suggesting a functional role during these processes. These results provide a new set of genes expressed specifically during larval molt or metamorphosis such as eukaryotic translation initiation factor 5C and nuclear transport factor and so on, which are candidates for further studies into the regulatory mechanisms of those stage-specific genes during larval molt and metamorphosis.Initiation phase of protein synthesis requires a large number of protein factors. To date, it is known that more than 12 translation initiation factors (eIFs) are required in eukaryotes. More and more evidence showed that eIFs is not only essential to the initiation of protein translation but also important in other life processes. Some eIFs is related to signal pathway such as a novel function of Drosophila eIF4A as a negative regulator of Dpp/BMP (decapentaplagic/bone morphogenetic protein) signalling that mediates SMAD (mother against dpp) degradation. Eukaryotic initiation factor 6 selectively regulates Wnt signaling andβ-catenin protein synthesis.eIF5C is a phylogenetically conserved protein predicted to contain an N-terminal leucine zipper motif and a C-terminal eIF5C domain. Few data show the function of eIF5C except it is associated with the ribosome through interaction with Drosophila ribosomal protein L5 (dRPL5) which suggest its possible role in protein synthesis.The orthologs of eIF5C containing eIF5C domain or leucine zipper motif play important roles in many species. Eukaryotic Translation Initiation Factor 5 functions as a GTPase-activating protein. Basic-leucine zipper proteins control molting and metamorphosis in Drosophila. In human, BZAP45 contributes to transcriptional control at the G1/S phase transition. Similar to it's homologs, eIF5C might enroll in the process of larval molting and metamorphosis.Based on the expressed sequence tags (EST) of eIF5C from SSH library of larval metamorphosis of Helicoverpa armigera, the full-length cDNA was 1675 bp with an open reading frame (ORF) of 1260 bp, which encodes a polypeptide of 419 amino acids (GenBank accession No.: EU526835). The gene with an N-terminal leucine zipper motif and a C-terminal eIF5C domain was denominated Ha-eIF5C. We further express Ha-eIF5C in E.coli and purified it using a Glutathione Sepharose 4B (GS4B) column. The rabbit antiserum against Ha-eIF5C was prepared.Ha-eIF5C transcript was detected by Quantitative real-time PCR (QRT-PCR). The result indicated that Ha-eIF5C transcript was detected a high level in the head-throax, integument, midgut and fat body but not in haemocytes in metamorphosis stage. QRT-PCR analyze the expression of Ha-eIF5C in developmental midgut and fat body show that there is a transient peak at the 5th head capsule splitted stage and increased during metamorphosis process. The expression of Ha-eIF5C protein agrees with the mRNA transcription.Immunocytochemical localization of eIF5C expression were demonstrated in the midgut, fat body and integument. A strong signal of Ha-eIF5C was detected at the border of the midgut suggesting that eIF5C localized at the muscle. Ha-eIF5C was distributed into cytoplasm in the midgut, however in the integument and fat body it was localized in both cytoplasm and nucleus. Compared with 5th feeding and molting stages, the expression level of eIF5C was higher during metamorphosis, which were consistent with the previous analyses.The expression level of Ha-eIF5C was correlated with the titer of 20E. It requires more efforts to demonstrate the function of Ha-eIF5C during larval molting and metamorphosis.
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