臺灣博碩士論文加值系統

食物及激素對東方果實蠅卵黃原蛋白表現之影響
張梅君
中文摘要
本研究在探討青春激素、脫皮激素以及食物中蛋白質成份的有無對東方果實蠅（Bactrocera dorsalis（Hendel））卵黃原蛋白基因以及其蛋白表現之影響。以RT-PCR觀察正常情況下東方果實蠅雌成蟲兩種卵黃原蛋白基因Bdvg1 （accession no. AF368053）及Bdvg2 (accession no. AF368054)之表現情形，由結果得知羽化後1日齡雌成蟲卵黃原蛋白基因Bdvg1及Bdvg2即有微量的表現，並隨著日齡增加，基因表現量也逐漸增加。經早熟素（precocene Ⅱ）處理之東方果實蠅雌成蟲，其Bdvg1及Bdvg2有延遲表現的現象，分別為第5和6日齡才開始表現，但進一步施用青春激素類似物（juvenile hormone, JHA; pyriproxyfen），可使之回復原本的表現情形，而施用20-hydroxyecdysone，則無法回復表現。食物中缺乏蛋白質（餵食糖水）可以抑制兩個卵黃原蛋白基因的表現，進一步以pyriproxyfen以及20-hydroxyecdysone處理，前者可刺激卵黃原蛋白基因48小時後回覆表現，而後者則無。利用西方墨點法（Western blot）觀察青春激素類似物處理餵食糖水的東方果實蠅雌成蟲卵黃原蛋白的表現，發現無論是否有再施用青春激素類似物，只要是未攝食蛋白質的蟲體皆無法正常表現卵黃原蛋白。卵巢摘除處理後卵黃原蛋白僅有微弱的表現，若再施打20-hydroxyecdysone，則有微弱的回復。由此可推測沒有提供蛋白質的來源，無法使卵黃原蛋白合成，而食物中的蛋白質可能間接刺激青春激素的產生，青春激素除了刺激脂肪體分泌卵黃原蛋白外，另一方面刺激卵巢分泌脫皮激素，脫皮激素可能刺激脂肪體提昇卵黃原蛋白表現的含量。

Effects of diet and hormones on the expression of vitellogenins in the oriental fruit fly, Bactrocera dorsalis (Hendel)
Mie-Chun Chang
ABSTRACT
The effects of diet and insect hormones on the expression of vitellogenin (Vg) mRNA and proteins in Bactrocera dorsalis (Hendel) were investigated in this study. Using RT-PCR, we demonstrated that the Bdvg1 (accession no. AF368053) and Bdvg2 (accession no. AF368054) start expression on the first day after eclosion. Treatment of the newly emerged females with 20 g/l precocene II, the Bdvg1 and Bdvg2 were delayed their expression about two to three days. Replenishment of precocene-treated females with juvenile hormone analog (JHA, pyriproxyfen), the Bdvg1 and Bdvg2 expression were recovered; however, replenishment with 20-hydroxyecdysone resulted no increase in the Bdvg1 and Bdvg2 expression. The vitellogenin genes were not detected under dietary protein deficiency, i.e. sugar-fed insects. Replenishment with JHA to the sugar-fed females caused slightly increase on the levels of the Vg mRNAs; however, it could not induce their protein synthesis. Finally, removal of the ovaries (i.e. ovariotomy) of newly emerged females obstructed the expression of the Bdvg1 and Bdvg2; however, the gene expression were rescued when replenished with 20-hydroxecdysone. In conclusion, these data suggest that both dietary protein and hormones are required for the vitellogenin synthesis in female B. dorsalis. Possibly, dietary protein can induce of juvenile hormone release, and then, to stimulate the ovaries to synthesize ecdysteroid; subsequently, ecdysteroids further stimulate the fat body to synthesize vitellogenins.

目 錄
中文摘要----------------------------------------------------------------------1
英文摘要----------------------------------------------------------------------2
前言----------------------------------------------------------------------------3
前人研究----------------------------------------------------------------------5
材料與方法------------------------------------------------------------------10
結果---------------------------------------------------------------------------18
討論---------------------------------------------------------------------------22
參考文獻---------------------------------------------------------------------26
圖------------------------------------------------------------------------------33
表------------------------------------------------------------------------------47
附錄---------------------------------------------------------------------------48

參考文獻
Adams, T. S. and Filipi P. A. 1988. Interaction between juvenile hormone, 20-hydroxyecdysone, the corpus cardiacum─allatum complex, and the ovaries in regulating vitellogenin levels in the housefly, Musca domestica. J. Insect Physiol. 34: 11-19.
Adams, T. S. and J. W. Gerst. 1992. Interaction between diet and hormones on vitellogenin levels in housefly, Musca domestica. Invertebr. Reprod. Dev. 21: 91-98.
Adams, T. S. and J. W. Gerst. 1993. Effect of diet on vitellogenin, vitellin and ecdysteroid levels during the second cycle of oogenesis in the housefly, Musca domestica. J. Insect Physiol. 39: 835-843.
Adams, T. S. and D. R. Nelson. 1990. The influences of diet on ovarian maturation, mating, and pheromone production in the housefly, Musca domestica. Invertebr. Reprod. Dev. 17: 193-201.
Agui, N., T. Shimada, S. Izumi and S. Tomino. 1991. Hormone control of vitellogenin mRNA levels in the male and female housefly, Musca domestica. J. Insect Physiol. 37: 383-390.
Applebaum, S. W., E. Avisar and Y. Heifetz. 1997. Juvenile hormone and locust phase. Arch. Insect Biochem. Physiol. 35: 375-391.
Barchuk, A. R., M. M. G. Bitondi and Z. L. P. Simoes. 2002. Effects of juvenile hormone and ecdysone on the timing of vitellogenin appearance in hemolymph of queen and worker pupae of Apis mellifera. J. Insect Sci. 2: 1-8.
Belles, X., J. Casas, A. Messeguer and M. D. Piulachs. 1987. In vitro biosynthesis of JH III by the corpora allata of Blattella germanica (L.) adult females. Insect Biochem. 17: 1007-1010.
Bownes, M. 1986. Expression of the genes coding for vitellogenin (yolk protein). Annu. Rev. Entomol. 31: 507-531.
Bownes, M. 1994. The regulation of yolk protein genes, a family of sex differentiation genes in Drosophila melanogaster. BioEssays 16: 745-752.
Bownes, M., E. Ronaldson, D. Mauchline and A. Martinez. 1993. Regulation of vitellogenin in Drosophila. Int. J. Insect Morphol. Embryol. 22: 349-367.
Burns, S. N., P. E. A. Teal, R. K. Vander Meer, J. L. Nation and J. T. Vogt. 2002. Identification and action of juvenile hormone III from sexually mature alate females of the red imported fire ant, Solenopsis invicta. J. Insect Physiol. 48: 357-365.
Chalaye, D. 1979. Immunochimique des proteins hemolymphatiques et ovocytaires de Rhodnius prolixus (Stal.). Can. J. Zool. 57: 329-336.
Chen, S. L. 2001. Cloning and structural analysis of vitellogenin genes of the oriental fruit fly, Bactrocera dorsalis (Hendel). Master’s Thesis. Department of Entomology, National Chung Hsing University, 87 pp. (in Chinese)
Chiu, H. T. 1978. Studies on the improvement of mass rearing for oriental fruit flies. Plant Prot. Bull. 20: 87-92 .(in Chinese)
Cole, T. J., S. B. Ramaswamy, A. Srinivasan and S. Dorn. 2002. Juvenile hormone catabolism and oviposition in the codling moth, Cydia pomonella, as functions of age, mating status, and hormone treatment. Arch. of Insect Biochem. Physiol. 49: 10-21.
Comas, D., M. D. Piulachs and X. Belles. 1999. Fast induction of vitellogenin gene expression by juvenile hormone III in the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae). Insect Biochem. Molec. Biol. 29: 821-827.
Cusson, M., C. G. Yu, K. Carruthers, G. R. Wyatt, S. S. Tobe and J. N. McNeil. 1994. Regulation of vitellogenin production in armyworm moths, Pseudaletia unipuncta. J. Insect Physiol. 40: 129-136.
Dhadialla, T. S., K. E. Cook and G. R. Wyatt. 1987. Vitellogenin mRNA in locust fat body: coordinate induction of two genes by a juvenile hormone analog. Dev. Biol. 123: 108-114.
Edwards, G. C., R. P. Braun and G. R. Wyatt. 1993. Induction of vitellogenin synthesis in Locusta migratoria by the juvenile hormone analog, pyriproxyfen. J. Insect Physiol. 39: 609-614.
Engelmann, F. 1979. Insect vitellogenin: identification, biosynthesis and role in vitellogenesis. Adv. Insect Physiol. 14: 49-108.
Glinka, A. V. and G. R. Wyatt. 1996. Juvenile hormone activation of gene transcription in locust fat body. Insect Biochem. Molec. Biol. 26: 13-18.
Hagedorn, H. H., 1994. The endocrinology of adult female mosquito. Adv. Disease Vector Res. 10: 109-148.
Handler, A. M. 1997. Developmental regulation of yolk protein gene expression in Anastrepha suspense. Arch. Insect Biochem. Physiol. 36: 25-35.
Harnish, D. G. and B. N. White. 1982. Insect vitellins: identification, purification, and characterization from eight orders. J. Exp. Zool. 220: 1-10.
Izumi, S., K. Yano, Y. Yamamoto and S. Y. Takahashi. 1994. Yolk protein from insect eggs: structure, biosynthesis and programmed degradation during embryogenesis. J. Insect Physiol. 40: 735-746.
Kelly,T. J. and W. H. Telfer. 1977. Antigenic and electrophoretic variants of vitellogenin in Oncopoltus blood and their control by juvenile hormone. Dev. Biol. 61: 58-69.
Koeppe, J. K., K. M. Fuchs, T. T. Chen, L. M. Hunt, G. E. Kovalich and T. Briers. 1985. The role of juvenile hormone in reproduction. pp165-203. In: Kerbut, G. A. and L. I. Gilbert, eds. Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon Press, Oxford.
Kunkel, J. G. and J. H. Nordin. 1985. Yolk protein. pp 83-111. In: Kerbut, G. A. and L. I. Gilbert, eds. Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon Press, Oxford.
Lu, H. L. 2002. Immunohistochemical analysis of vitellogenesis in the oriental fruit fly, Bactrocera dorsalis (Hendel). Master’s Thesis. Department of Entomology, National Chung Hsing University, 60 pp. (in Chinese)
Lu, H. L. and K. H. Lu. 2003. Purification, characterization, and fat body preparation of vitellins of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae). Formosan Entomol. 23: 87-99. (in Chinese)
Martinez, A. and M. Bownes. 1994. The sequence and expression pattern of the Calliphora erythrocephala yolk protein A and B genes. J. Mol. Evol. 38: 336-351.
Nijhout, H. F. 1994. Insect hormones. Princeton University Press, Princeton, New Jersey USA. 276 pp.
Onishi, E. 1987. Growth and maturation of ovaries in isolated abdomens of Bomnyx mori: response to ecdysteroid and other steroids. Zool. Sci. 4: 315-321.
Pan, M. L., W. J. Bell and W. H. Telfer. 1969. Vitellogenic blood protein synthesis by insect fat body. Science 165: 393-394.
Racioppi J. V., R. M. Gemmill, P. H. Kogan, J. M. Calvo and H. H. Hagedorn. 1986. Expression and regulation of vitellogenin messenger RNA in the mosquito Ades aegypti. Insect Biochem. 16: 255-262.
Raikhel, A. S. and T. S. Dhadialla. 1992. Accumulation of yolk protein in insect oocytes. Annu. Rev. Entomol. 37: 217-251.
Raikhel, A. S. and S. Snigirevskaya. 1998. Vitellogenesis. pp. 933-955. In: Harrison, F. W. and M. Locke eds. Microscopic Anatomy of Invertebrates. Wiley-Liss Press. New Yolk.
Ramaswamy, S. B., S. Shengqiang, Y. I. Park and F. Zeng. 1990. Dynamics of juvenile hormone-mediated gonadotropism in the Lepidoptera. Arch. Insect Biochem. Physiol. 35: 539-558.
Richard, D. S., N. L. Watkins, R. B. Serafin and L. I. Gilbert. 1998. Ecdysteroid regulate yolk protein uptake by Drosophila melanogaster oocytes. J. Insect Physiol. 44: 637-644.
Richard, D. S., M. Gilbert, B. Crum, D. M. Hollinshead, S. Schelble and D. Scheswohl. 2001. Yolk protein endocytosis by oocytes in Drosophila melanogaster : immunofluorescent localization of clathrin, adaptin and the yolk protein receptor. J. Insect Physiol. 47: 715-923.
Riddiford, L. M. 1993. Cellular and molecular actions of juvenile hormone I. General considerations and premetamorphic actions. Adv. Insect Physiol. 24: 213-274.
Sambrook, J. and E. F. Fritsch. 2001. Moleucular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New Yolk.
Sankhon, N., T. Lockey, R. C. Rosell, M. Rothschild and L. Coons. 1999. Effect of methoprene and 20-hydroxyecdysone on vitellogenin production in cultured fat bodies and backless explants from unfed female Dermacentor variabilis. J. Insect Physiol. 45: 755-761.
Sappington, T. W. and A. S. Raikhel. 1998. Molecular characteristics of insect vitellogenins and vitellgenin receptor. Insect Biochem. Molec. Biol. 28: 277-300.
Satyanarayana, K., J. H. Yu, G. Bhaskaran, K. H. Dahm and R. Meola. 1992. Regulation of vitellogenin synthesis by juvenile hormone in the corn earworm, Helicoverpa zea. Invertebr. Reprod. Dev. 21: 169-178.
Shirk, P. D., D. W. Bean and V. J. Brookes. 1990. Ecdysteroids control vitellogenesis and egg maturation in pharate adult females of the Indianmeal moth, Plodia interpunctella. Arch. Insect Biochem. Molec. Biol. 15: 183-199.
Shirk, P. D., P. Minoo and J. H. Postlethwait. 1983. 20-hydroxyecdysone stimulates the accumulation of translatable yolk polypeptide gene transcript in adult male Drosophila melanogaster. Proc. Natl. Acad. Sci. USA. 80: 186-190.
Sorge, D., R. Nauen, S. Range and K. H. Hoffmann. 2000. Regulation of vitellogenesis in the fall armyworm, Spodoptera frugiperda (Lepodoptera: Noctuidae). J. Insect Physiol. 46: 969-976.
Telfer, W. H., E. Huebner and D. S. Smith. 1982. The cell biology of vitellogenic follicles in Hyalophora and Rhodnius. pp. 118-149. 1. In: King, R. C., and H. Akai eds. Insect Ultrastructure. Vol. Plenum Press, New Yolk.
Trougakos, I. P., K. Lamnissou and L. H. Margaritis. 1999. Biochemical and immunocytochemical analysis of vitellogenesis in the olive fruit fly Dacus (Bactrocera) oleae (Diptera: Tephritidae). Cell Biol. Intl. 23: 417-429.
Tsai, Y. T. 1997. Effects of physiological factors on vitellogenesis and ovarin development in the oriental fruit fly, Bactrocera dorsalis (Hendel). Mastter’s Thesis. Department of Entomology, National Chung Hsing University, 93 pp. (in Chinese)
Tufail, M., M. Hatakeyama and M. Takeda. 2001. Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana. Arch. Insect Biochem. Physiol. 48: 72-80.
Wyatt, G. R. 1988. Vitellogenin synthesis and analysis of juvenile hormone action in locust fat body. Can. J. Zool. 66: 2600-2610.
Yan, Y. L., C. J. Kunert and J. H. Postlethwait. 1987. Sequence homologies among the three yolk polypeptide (Yp) genes in Drosophila melanogaster. Nucleic Acids Res. 15: 67-85.
Zeng, F., S. Shu, Y. I. Park and S. B. Ramaswamy. 1997. Vitellogenin and egg production in the moth, Helicoverpa zea. Arch. Insect Biochem. Physiol. 34: 287-300.