생명과학회지 (Journal of Life Science)

  • 제20권6호
  • /
  • Pages.929-933
  • /
  • 2010
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

누에에서 곰팡이(Aspergillus niger) 감염에 의해 유도 발현되는 유전자의 클로닝과 동정

Cloning and Identification of Differentially Expressed Genes Induced by Fungal Infection from Silkworm, Bombyx mori

  • 투고 : 2010.03.15
  • 심사 : 2010.04.23
  • 발행 : 2010.06.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

본 연구는 곤충으로부터 새로운 항 진균 단백질을 발굴하기 하기 위한 목적으로 누에를 대상으로 Aspergillus niger의 감염을 유도하였을 때 발현되는 유전자의 특성을 분석한 것이다. Annealing control primer 법에 기초한 GeneFishing Kit를 사용하여 A. niger를 약 $6{\times}10^8$ colony per unit로 5령기 누에 유충의 체강에 감염시킨 후, 6시간 경과한 다음에 유도 발현되는 유전자(differentially expressed genes, DEGs)를 분석 한 결과, 10개의 유도 발현되는 유전자를 분리하였고 RT-PCR을 통해서 lysozyme, enbocin 그리고 한 개의 기능이 알려지지 않는 유전자등 3개의 유전자가 A. niger의 감염에 의해서 유의하게 과 발현된다는 것을 검증하였다. 일반적으로 그람 음성 및 양성 세균의 감염에 의해 유도된다고 알려진 enbocin 유전자가 A. niger의 감염에서도 과 발현이 유도되는 본 연구의 결과는 앞으로 enbocin 유전자의 항 진균 활성 연구에 중요한 기초 자료로 활용될 수 있을 것이다.

We tried to identify differentially expressed genes (DEGs) from a silkworm, Bombyx mori, involved in fungal (Aspergillus niger) infection. A total RNA purified from fungal-induced and normal B. mori ($5^{th}$ instar larvae) was used for the cDNA synthesis. Differentially expressed genes were screened by annealing control primer (ACP)-based PCR technique. Comparing the gene expression profiles between fungal infection and control silkworm, we detected 10 genes that were differentially expressed in fungal induction and performed molecular cloning and nucleotide sequencing of the 10 genes. We confirmed the expression patterns of 3 DEGs by RT-PCR. The 3 DEGs over-expressed in fungal infection were identified as lysozyme, enbocin and an unknown gene. They were first identified to be genes induced by fungal infection. Although the detailed functions of 3 genes and their products remain to be determined, the genes will provide insight into the molecular mechanisms of insect-immune systems induced by fungal infection.

키워드

참고문헌

  1. Alcouloumre, M. S., M. A. Ghannoum, A. S. Ibrahim, M. E. Sested, and J. E. Edwards. 1993. Fungicidal properties of defensin NP-1 and activity against Cryotococcus neoformans in vitro. Antimicrobial Agents and Chemotherapy 37, 2628-2632. https://doi.org/10.1128/AAC.37.12.2628
  2. Boman, H. G., I. Faye, G. H. Gudmundsson, J. Y. Lee, and D. A. Lidholm. 1991. Cell free immunity in cecropia : A model system for antibacterial proteins. Eur. J. Biochem. 201, 23-31. https://doi.org/10.1111/j.1432-1033.1991.tb16252.x
  3. Bradbury, A. F. and D. G. Smyth. 1991. Peptide amidation. Tips Biochem. Sci. 16, 112-115. https://doi.org/10.1016/0968-0004(91)90044-V
  4. Brehelin, M. and N. Boemare. 1988. Immune recognition in insects: conflicting effect of autologous plasma and serum. J. Compara. Physiol. Biochem. System. Environ. Physiol. 157, 759-764. https://doi.org/10.1007/BF00691006
  5. Casteels, P., C. Ampe, L. Riviere, J. V. Damme, C. Elicone, M. Fleming, F. Jacops, and F. Tempst. 1990. Isolation and Characterization of abaecin, a major antibacterial response peptide in the honeybee (Apis mellipera). Eur. J. Biochem. 187, 381-386. https://doi.org/10.1111/j.1432-1033.1990.tb15315.x
  6. Cociancich, S., A. Ghazi, C. Hetru, J. A. Hoffmann, and L. Leteliers. 1993. Insect defensin, an inducible antibacterial peptide forms voltage-dependent channels in Microccus luteus. J. Bio. Chem. 268, 19239-19245.
  7. Cociancich, S., M. Goyffon, F. Bontms, P. Bulet, F. Bouet, A. Menez, and J. Hoffman. 1993. Purification and characterization of a scorpion defensin, a 4 kDa antibacterial peptide presenting structural similarities with insect defencin and scorpion toxins. Biophy. Biochem. Res. Commun. 194, 17-22. https://doi.org/10.1006/bbrc.1993.1778
  8. Cui, X. S., M. R. Shin, K. A. Lee, and N. H. Kim. 2005. Identification of differentially expressed genes in murine embryos at the blastcyst stage using annealing control primer system. Molecular Reproduction and development 70, 278-287. https://doi.org/10.1002/mrd.20210
  9. Dimarcq, J. L., E. Keppi, B. Dunbar, J. Lambert, J. M. Reichhart, D. Hoffman, S. M. Rankine, J. E. Fothergil, and J. A. Hoffman. 1988. Purification and characterization of a family of novel inducible antibacterial proteins from immunized level of the dipteran Phormia terranovae and complete amino acid sequence of the predominant member, Diptericin A. Eur. J. Biochem. 171, 17-22. https://doi.org/10.1111/j.1432-1033.1988.tb13752.x
  10. Haltmark, D., A. Engstrom, K. Anderson, H. Steiner, H. Bennich, and H. G. Bomam. 1983. Attacins, a family of antibacterial proteins from Hyalophora cecropia. EMBO J. 2, 571-576.
  11. Hara, S., and M. Yamakawa. 1995. Moricin, a novel antibacterial peptide, isolated from the silkworm, Bombyx mori. J. Biol. Chem. 270, 29923-29927. https://doi.org/10.1074/jbc.270.50.29923
  12. Hoffman, J. A., F. C. Kafatos, and C. A. Janeway. 1999. Phylogenetic perspectives in innate immunity. Science 284, 1313-1318. https://doi.org/10.1126/science.284.5418.1313
  13. Hwang, I. T., Y. J. Kim, S. H. Kwak, Y. Y. Gu, and J. Y. Chun. 2003. Annealing control primer system for improving specificity of PCR amplification. Biotechniques 35, 1180-1184.
  14. Jolles, P. and J. Jolles. 1984. What’s new in lysozme research? Always a model system. Mole. Cell Biochem. 63, 165-189.
  15. Kaneko, Y., S. Furukawa, H. Tanaka, and M. Yamakawa. 2007. Expression of antimicrobial peptide genes encoding enbocin and gloverin isoforms in the silkworm, Bombyx mori. Biosci. Biotechnol. Biochem. 71, 2233-2241. https://doi.org/10.1271/bbb.70212
  16. Kim S. H., B. S. Park, E. Y. Yun, Y. H. Je, S. D. Woo, S. W. Kang, K. Y. Kim, and S. K. Kang. 1998. Cloning and expression of a novel gene encoding a new antibacterial peptide from silkworm, Bombyx mori. BBRC. 246, 388-392.
  17. Mulnix, A. B. and P. E. Dunn. 1994. Structure and induction of a lysozyme gene from the tobacco hornworm, Manduca sexta. Insect Biochem. Mole. Biol. 24, 271-281. https://doi.org/10.1016/0965-1748(94)90007-8
  18. Qu, X. M., H. L. Tang, and S. Hakan. 1989. The effect of cecropins B and D from the Chinese oak silkmoth(Antheraea pernyi) on liposomes. Biophys. Biochem. Acta. 21, 35-42.
  19. Schmidt, O., I. Faye, I. Lindstrom-Dinnetz, and S. C. Sun. 1993. Specific immune recognition of insect hemoline. Develop. and Compara. Immunol. 17, 195-200. https://doi.org/10.1016/0145-305X(93)90038-R
  20. Shalon, D. 1998. Gene expression microarrays: a new tool or genomic research. Pathol. Biol. 243, 107-109.
  21. Yoon, S. J., H. M. Chung, and K. Y. Cha. 2005. Identification of differentially expressed gene expression in germinal vesicle vs metaphase II mouse oocytes by using annealing control primers. Fertility and Sterility 83, 1293-1296. https://doi.org/10.1016/j.fertnstert.2004.09.037
  22. Yum, S. S., S. O. Woo, and E. S. Choi. 2005. Analysis of gene expression in benzopyrene-exposed Sebastes schlegeli using differential display polymerase chain reaction. Journal of Environmental Toxicology 20, 67-73.