The analysis of differentially expressed novel transcripts in diapausing and diapause-activated eggs of Bombyx mori.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 59:197–201 (2005) The Analysis of Differentially Expressed Novel Transcripts in Diapausing and Diapause-Activated Eggs of Bombyx mori Jae-Sam Hwang,1* Hyun-Jeong Go,2 Tae-Won Goo,1 Eun-Young Yun,1 Kwang-Ho Choi,1 Su-Il Seong,2 Sang-Mong Lee,3 Bong-Hee Lee,4 Iksoo Kim,1 Taehoon Chun,5 and Seok-Woo Kang1 As an initial step to define the molecular mechanism of initiation and termination of diapause during the embryogenesis of silkworms, Bombyx mori, mRNA transcripts from maintained and activated diapause eggs were compared with differential expression using cDNA array. Twenty-four individual cDNA transcripts were expressed differentially in a total of 1,468 different cDNAs. Among those clones, mRNA transcript from cytochrome oxidase subunit I (COI), which was detected to be 2-kb transcripts, gradually increased in diapause-activated eggs during early embryogenesis. Further analysis revealed that mRNA transcripts from silkworm COI were highly expressed in testis, fat body, and midgut during the larval stage. These results may indicate that the expression of silkworm COI mRNA is regulated developmentally as well as tissue-specifically. Arch. Insect Biochem. Physiol. 59:197–201, 2005. © 2005 Wiley-Liss, Inc. KEYWORDS: DNA array; cytochrome oxidase subunit I; diapause; embryogenesis; insects; silkworm INTRODUCTION Most insects have evolved to enter a diapause at some stage of development to survive under unfavorable environmental conditions. Environmental factors such as temperature and photoperiod determine the proper moment of a diapause program (Denlinger, 1985). Also, depending on insect species, the embryonic diapause occurs either at an early embryonic stage or at a much later stage (Chapman, 1998). The silkworm, Bombyx mori, enters diapause at an early embryonic stage, before dermal differentiation is completed. Diapause of the silkworms continues as long as the eggs are kept at 25°C. Diapuse is terminated due to chilling at 5°C for about 3 months and when the eggs are transferred back to 25°C, embryonic development restarts. Alternatively, diapause is blocked when the eggs undergo HCl treatment at 47°C for 5 min after keeping them at 25°C for 20 h after oviposition. Diapause hormone (DH), one of the neurohormones, has been identified as a major factor of inducing diapause in the resulting embryos. The expression of DH mRNA in the early pupal stage correlates to the incidence of diapause (Sato et al., 1 Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA, Suwon, South Korea Department of Life Science, College of Natural Science, University of Suwon, Hwaseong, South Korea 3 Department of Sericulture and Entomology, Miryang National University, Miryang, Korea 4 Graduate School of Biotechnology, Korea University, Seoul, Korea 5 Department of Microbiology, School of Medicine, Hanyang University, Seoul, South Korea 2 Abbreviations used: CO = cytochrome c oxidase; COI = cytochrome oxidase subunit I; DH = diapause hormone. Contract grant sponsor: Biogreen 21 program, rural development administration, South Korea. *Correspondence to: Jae-Sam Hwang, Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA, Suwon 441-100, South Korea. E-mail: firstname.lastname@example.org Received 20 May 2004; Accepted 19 February 2005 © 2005 Wiley-Liss, Inc. DOI: 10.1002/arch.20057 Published online in Wiley InterScience (www.interscience.wiley.com) 198 Hwang et al. 1993; Xu et al., 1995). Although these findings clearly show that this hormone regulates in the induction of embryonic diapause, it is still unknown if the individual gene expression profile may regulate the stage of initiation or termination of diapause. Genomics, the study of genes and their function, holds the potential to resolve long-standing genetic questions. The systematic determination of the cDNA array by screening specific developmental stages of the insects is required to understand biological processes. As the first step to define the molecular mechanism of diapause during the embryogenesis of the silkworms, Bombyx mori, we carried out differential screening using cDNA expression array and Northern blot analysis. MATERIALS AND METHODS Experimental Animals Silkworm hybrids between Japanese strain 123 and Chinese strain 124 were used in this study. The eggs after oviposition were preserved at 25°C for 2 months to obtain eggs in diapause stage. To obtain diapause-activated eggs, the eggs after oviposition were incubated at 25°C for 3 months and then were incubated at 5°C for 3 months, inducing the termination of diapause. After the termination of diapause, the eggs restarted their development by being incubated at 25°C. Construction of the Full-Length Enriched cDNA Library A cDNA Library from B. mori diapausing eggs and diapause-activated eggs was constructed by using a modification of Maruyama and Sugano’s method (Maruyama and Sugano, 1994). Briefly, 100 µg of total RNA was treated with 3 U of bacterial alkaline phosphatase (TaKaRa) in 100 µl of 100 mM Tris-HCl (pH 7.5), 2 mM DTT, and 80 U of RNasin (Promega, Madison, WI) at 37°C for 60 min. After phenol extraction and ethanol precipitation, the total RNA was treated with 100 U of tobacco acid pyrophosphatase (Waco) in 100 µl of 50 mM sodium acetate (pH 5.5), 5 mM EDTA, 10 mM 2-mercaptoethanol, and 80 U of RNasin at 37°C for 60 min. The pre-treated total RNA was then ligated with 0.4 µg of 5-oligoribonucleotide (5-oligo: 5-AGC AUC GAG UCG GCCC UUG UUG GCC UAC UGG-3) using 250 U of RNA ligase (TaKaRa) in 100 µl of 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 5 mM 2-mercaptoethanol, 0.5 mM ATP, 25% PEG 8000, and 100 U of RNasin at 20°C for 3 h. After completing these oligo-capping reactions, mRNA was isolated using a commercial kit, QIAGEN Oligotex™. The synthesis of first-strand cDNA from the purified mRNA and cDNA amplification was performed as described by Maruyama and Sugano (1994). The amplified PCR Products were then digested with SfiI, and cDNAs longer than 1.3 kb were ligated into DraIII-digested pCNSD2 in an orientation-defined manner. The pCNSD2 vector contains 5 EcoRI -DraIII- EcoRV- DraIII sites at multi-cloning sites, which were achieved by modifying pCNS vector (GenBank accession no. AF416744). The ligated cDNA was then transformed into Escherichia coli Top10F’ (Invitrogen, La Jolla, CA) by electroporation (Gene Pulser II, BioRad, Hercules, CA). Then, cDNA sequences were determined by the dideoxy-mediated chain termination method using a 377 automatic sequencer (ABI) and were analyzed by BLAST databases (http://www.ncbi.nlm.nih.gov/blastn). As a result, a total of 1,468 different cDNAs were obtained and used for cDNA expression arrays. cDNA Expression Array A total of 1,468 different cDNAs were arrayed onto Hybond-N membranes (Amersham Biosciences, Sweden) using 96-well format dot blotter (Bio-Rad) after denaturation. To prepare probes, mRNAs were isolated from diapausing eggs and diapause-activated eggs. Then, 32P-labeled cDNA probes were generated by reverse transcription of 0.5–1.0 µg of each poly(A)+ RNA sample in the presence of [α-32P]dATP. Each cDNA probe was then hybridized with the membrane at 65°C. A hybridization solution containing 50% formamide, 5 × SSC, 10 × Denhardt’s solution (0.2% each of bovine serum albumin, Ficoll, and polyvinylpyrArchives of Insect Biochemistry and Physiology cDNA Array in Silkworm Diapausing Eggs rolidone), 25 µg/ml sonicated salmon sperm DNA, and 50 mM sodium phosphate (pH 7.0) was used. After hybridization, membranes were washed for 30 min with increasing stringency, from 2 × SSC and 0.1% SDS to 0.1 × SSC and 0.1% SDS. After a high-stringency wash, membranes were then exposed to X-ray film (AGFA, Germany) for 1 or 3 days at –70°C. Northern Blot Analysis Total RNAs were extracted from developing eggs and five tissues from the 5th instar larva using SV total RNA isolation system (Promega). To obtain the developing eggs, diapause-programmed eggs were treated with hot HCl for 20 h after oviposition. Ten microgram of total RNA per sample was separated on 1.2% agarose/3% formaldehyde denaturing gel, and transferred onto a nylon membrane (Amersham, Arlington Heights, IL). The cDNA probe was labeled with [α-32P]dCTP using Prime-It II random primer labeling kit (Stratagene, La Jolla, CA), according to the manufacturer’s instructions. Membrane was hybridized with a cDNA probe at 65°C overnight, and analyzed by autoradiography. RESULTS AND DISCUSSION Differentially Expressed cDNAs From Silkworm Diapausing Eggs and Diapause-Activated Eggs High-quality mRNAs were isolated from silkworm diapausing eggs and diapause-activated eggs. The mRNAs were reverse-transcribed to 32P-labeled cDNAs and hybridized to a cDNA array blot that contains 1,468 Expressed Sequence Tag (EST) cDNA clones. After stringent washes and exposure to X-ray film, the expression profiles of 1,468 genes in diapausing eggs and diapause-activated eggs were obtained. By comparing duplicated hybridized blots, we identified 24 genes whose expression patterns were changed as summarized in Tables 1 and 2. Only genes with expression levels that were altered more than twofold in comparisons of diapausing eggs and diapause-activated eggs are included. Ten genes showed increased expression in diapause-activated eggs. Among them, four August 2005 199 TABLE 1. Summary of Highly Expressed Genes in Diapause-Activated Eggs of Silkworm* Clone no. 0440 0465 1103 1931 2065 2086 2451 2776 2823 4984 Gene bank no. AF399909 NM079488 NI NI NI NI NI NI AF083339 AB070579 Putative function Alcohol dehydrogenase Dead-box-1 NI NI NI NI NI NI Cytochrome oxides subunit 1 18 wheeler Species D. rerio D. melanogaster B. mori B. mori *NI, no identity. genes had functions and the other six genes were functionally unidentified (Table 1). Four functional genes were alcohol dehydrogenase, dead-box-1, cytochrome c oxidase subunit I (COI), and 18 wheeler (Bm18w). Bm18w is a Toll-like molecule that acts as an adhesion molecule during various processes of morphogenesis in Drosophila melanogaster (Eldon et al., 1994). Most of Bm18w mutants in fruit fly develop up to the third larval instar, but die prior to pupariation (Williams et al., 1997). Fourteen genes showed decreased expression patterns in diapause-activated eggs. Seven out of 14 genes have known functions (Table 2). Among them, embryonic abnormal visual system gene (elav) is a gene-specific regulator of alternative premRNA processing, which is important to generate TABLE 2. Summary of Highly Expressed Genes in Diapausing Eggs of Silkworm* Clone no. Gene bank no. 0330 Y12701 0893 1539 1624 1695 2220 2522 AF031652 AF429973 L08106 NI NI AF288217 2878 2912 2937 3104 4091 4605 4907 NI NI AF315319 NI NI NI U28654 *NI, no identity. Putative function ATP synthetase, gamma subunit Caspase-8/-10 homolog Ribosomal protein L8 Glycl-tRNA synthetase NI NI Translationally-controlled tumor protein NI NI Heat shock protein hsp 20.8A NI NI NI Retinol dehydratase Species D. melanogaster D. melanogaster S. frugiperda B. mori D. rerio B. mori S. frugiperda 200 Hwang et al. a embryonic neural system in Drosophila (Soller and Whiter, 2003). Caspase-8/-10 homolog (Dcp2) gene and translationally controlled tumor protein (tct1) gene were also down-regulated in diapause-activated eggs. DCP2 contains two N-terminal death effector domains fused to a caspase-like domain, which is the mediator of an apoptosis (Inohara et al., 1997). A protein encoded by tct1 is ubiquitously expressed and is present in evolutionarily diverse organisms. Recently, TCT1 was reported as a regulator of translation elongation factor eEF1A in yeast (Cans et al., 2003). Silkworm COI Expression Using cDNA expression array, we found a number of genes that show differential expression in silkworm diapausing eggs and diapause-activated eggs. We focused on the differential expression of cytochrome oxidase subunit I (COI), whose expression Fig. 1. Expression analyses of B. mori COI mRNA. A: Stage-specific expression of the B. mori COI mRNA during embryogenesis. Northern blot analysis was done with total RNA samples from diapausing eggs and HCl-activated eggs. The B. mori COI mRNA was detected as an approximately 2.0 kb single mRNA transcript (top). D: diapausing eggs; 0–10: days after HCl treatment. B: Tissue-specific expression of the B. mori COI mRNA at 5th instar larvae. was significantly increased in diapause-activated eggs. To validate this observation, we examined COI mRNA expression by Northern blot analysis. As shown in Figure 1A, COI mRNA was expressed at a low level in diapausing eggs, but began to remarkably increase the expression level at day 6 in developing eggs. Cytochrome c oxidase (CO) is a terminal enzyme in the energy transducing respiratory chain of eukaryotes (Babcock and Wikstrom, 1992). The increased expression of this electron transfer protein may be associated with oxygen consumption that accompanies the termination of diapause and the resumption of development. A silkworm embryo is completed to larval form between day 6 and 7 after oviposition. At this point, a large amount of CO may be required because of the increase in energy and oxygen consumption that accompanies tissue formation. COI mRNA was expressed at a low level in diapausing eggs because of the developmental arrest. Northern analysis was done with total RNA samples from testis, ovary, fat body, posterior silk gland, and midgut. Each lane contained 20 µg total RNA. An approximately 2.0-kb single mRNA transcript was detected (top). Lanes 1: Testis; 2: ovary; 3: fat body; 4: posterior silk gland; 5: midgut. As an internal control, the amount of 18S RNA is shown (bottom). Archives of Insect Biochemistry and Physiology cDNA Array in Silkworm Diapausing Eggs Previously, rates of oxygen consumption in eggs were monitored during the silkworm diapause (Yaginuma and Yamashita, 1999). Oxygen consumption increased slowly in the course of termination of diapause and blastokinesis. Oxygen consumption then increased rapidly until the stage of larval hatching. Oxygen consumption also increased in HCl-treated eggs, similar to developing eggs. In HCl-treated eggs, oxygen consumption increased gradually for 5 days and then increased markedly toward larval hatching. This is consistent with our observation that the expression of COI mRNA was markedly increased on 6 days after HCl treatment (Fig. 1A). We further examined tissue expression of COI mRNA in silkworm larva by Northern blot analysis (Fig. 1B). As a result, silkworm COI mRNA was expressed highly in testis and midgut of the 5th instar larva. This result is very similar to a previous report that COI mRNA expression is correlated to histogenesis and organogenesis in cockroaches (Martinez-Gonzalez and Hegardt, 1994). Thus, the COI mRNA expression of silkworm might be correlated to the increase of oxygen consumption during embryogenesis and organogenesis. ACKNOWLEDGMENTS This work was supported by a grant from Biogreen 21 program, Rural Development, Administration, Republic of Korea. LITERAURE CITED Babcock GT, Wikstrom M. 1992. Oxygen activation and the conservation of energy in cell respiration. Nature 356:301–309. Cans C, Passer BJ, Shalak V, Nancy-Portebois V, Crible V, Amzallag N, Allanic D, Tufino R, Argentini M, Moras D, Fiucci G, Goud B, Mirande M, Amson R, Telerman A. 2003. Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A. Proc Natl Acad Sci USA 100:13892–13897. Chapman RF. 1998. The Insects: structure and function, 4th ed. Cambridge: Cambridge University Press. Denlinger DL. 1985. Comprehensive insect physiology, bio- August 2005 201 chemistry and pharmacology, 1st ed. New York: Pergamon Press. Eldon E, Kooyer S, D’Evelyn D, Duman M, Lawinger P, Botas J, Bellen H. 1994. The Drosophila 18 wheeler is required for morphogenesis and has striking similarities to Toll. Development 120:885–899. Inohara N, Koseki T, Hu Y, Chen S, Núñez G. 1997. Interaction and regulation of the Caenorhabditis elegans death protease CED-3 by CED-4 and CED-9. Proc Natl Acad Sci USA 94:10717–10722. Martinez-Gonzalez J, Hegardt FG. 1994. Cytochrome c oxidase subunit I from the cockroach Blattella germanica: cloning, developmental pattern and tissue expression. Insect Biochem Mol Biol 24:619–626. Maruyama K, Sugano S. 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138:171–174. Sato Y, Oguchi M, Menjo N, Imai K, Saito H, Ikeda M, Isobe M, Yamashita O. 1993. Precursor polyprotein for multiple neuropeptides secreted from the suboesophageal ganglion of the silkworm Bombyx mori: characterization of the cDNA encoding the diapause hormone precursor and identification of additional peptides. Proc Natl Acad Sci USA 90:3251–3255. Soller M, Whiter K. 2003. ELAV inhibits 3'-end processing to promote neural splicing of ewg pre-mRNA. Genes Dev 17:2526–2538. Williams MJ, Rodriguez A, Kimbrell DA, Eldon ED. 1997. The 18-wheeler mutation reveals complex antibacterial gene regulation in Drosophila host defense. EMBO J 16:6120–6130. Xu WH., Sato Y, Ikeda M, Yamashita O. 1995. Molecular characterization of the gene encoding the precursor protein of diapause hormone and pheromone biosynthesis activating neuropeptide (DH-PBAN) of the silkworm, Bombyx mori and its distribution in some insects. Biochem Biophys Acta 1261:83–89. Yaginuma T, Yamashita O. 1999. Oxygen consumption in relation to sorbitol utilization at the termination of diapause in eggs of the silkworm, Bombyx mori. J Insect Physiol 45:621–627.