DOI QR코드

DOI QR Code

Functional Analysis of Aspergillus nidulans Genes Selected by Proteomic Analysis under Conditions Inducing Asexual Development

Aspergillus nidulans 무성분화 촉진 조건의 단백체 및 해당 유전자 기능분석

  • Lim, Joo-Yeon (Department of Bioscience and Biotechnology, Graduate School, Chungnam National University) ;
  • Kang, Eun-Hye (Department of Bioscience and Biotechnology, Graduate School, Chungnam National University) ;
  • Jung, Bo Ri (Department of Bioscience and Biotechnology, Graduate School, Chungnam National University) ;
  • Park, Hee-Moon (Department of Bioscience and Biotechnology, Graduate School, Chungnam National University)
  • 임주연 (충남대학교 대학원 생명과학과) ;
  • 강은혜 (충남대학교 대학원 생명과학과) ;
  • 정보리 (충남대학교 대학원 생명과학과) ;
  • 박희문 (충남대학교 대학원 생명과학과)
  • Received : 2017.07.28
  • Accepted : 2017.08.30
  • Published : 2017.09.01

Abstract

Despite the significance of external environmental factors in differentiation, putative factors involved in differentiation of Aspergillus nidulans have not yet been fully understood. A sporulation-specific proteome analysis of A. nidulans in the present study revealed that the expression levels of more than 2,400 proteins were affected under conditions inducing sporulation (0.6 M KCl) compared with normal conditions. Among the proteins with predicted functions, two targets, AN1342 and AN9419, were functionally analyzed using targeted deletion strains and phenotypic observations. For AN1342, because the deletion of the corresponding open reading frame caused a reduction in stalk length during asexual development and in pigment production in liquid culture, the gene was designated as sspA ($\underline{s}hort$ $\underline{s}talk$ & $\underline{p}igment$). Deletion of the AN9419 gene, which is predicted to encode alanyl-tRNA synthetase, led to severe growth defects due to alanine auxotrophy and abolishment of asexual reproduction and thus, the gene was designated as alaA.

Aspergillus nidulans의 포자형성 및 무성분화에 관여하는 새로운 인자를 찾고자, 포자형성 촉진 조건인 0.6 M KCl이 첨가된 배지와 첨가되지 않은 배지에서 자란 균사체의 단백질체 분석을 시도하였다. 2DE 분석을 통해 2,400여개의 spot을 확인하였고, 무성분화 유도 9시간 및18시간 별로 생성양의 변화양상을 기준으로 총 5개의 그룹으로 나눌 수 있었다. 기능 분석이 아직까지 이루어지지 않은 단백질을 암호화하고 있는 유전자들 가운데 DU 그룹에 속하는 AN1342와 DD 그룹에 속하는 AN9419 두 개의 유전자의 기능을 알아보고자 유전자결손 돌연변이주를 제작하고 표현형을 관찰하였다. Alanine-glyoxylate aminotransferase의 기능을 할 것으로 예측되는 AN1342을 결손 시키면 무성분화 기관인 stalk의 길이가 짧아졌고, 액체 배양 시 야생형과 달리 배지로 분홍색 색소가 분비되어 sspA라 명명하였다. AN9419 결손균주는 균사생장이 심각하게 저해되고 알라닌이 첨가되어야 균사생장이 가능하였으나 무성포자 형성은 거의 이루어지지 않았으며 GO분석을 통하여 alanyl-tRNA synthetase의 기능을 할 것으로 유추된 점에 근거하여 alaA라 명명하였다.

Keywords

References

  1. Pontecorvo G, Roper JA, Hemmons LM, Macdonald KD, Bufton AW. The genetics of Aspergillus nidulans. Adv Genet 1953;5:141-238.
  2. Adams TH, Wieser JK, Yu JH. Asexual sporulation in Aspergillus nidulans. Microbiol Mol Biol Rev 1998;62:35-54.
  3. Busby TM, Miller KY, Miller BL. Suppression and enhancement of the Aspergillus nidulans medusa mutation by altered dosage of the bristle and stunted genes. Genetics 1996;143:155-63.
  4. Palmer JM, Theisen JM, Duran RM, Grayburn WS, Calvo AM, Keller NP. Secondary metabolism and development is mediated by LlmF control of VeA subcellular localization in Aspergillus nidulans. PLoS Genet 2013;9:e1003193. https://doi.org/10.1371/journal.pgen.1003193
  5. Kim H, Han K, Kim K, Han D, Jahng K, Chae K. The veA gene activates sexual development in Aspergillus nidulans. Fungal Genet Biol 2002;37:72-80. https://doi.org/10.1016/S1087-1845(02)00029-4
  6. Kang EH, Song EJ, Kook JH, Lee HH, Jeong BR, Park HM. Depletion of TEX>${\varepsilon}$-COP in the COPI vesicular coat reduces cleistothecium production in Aspergillus nidulans. Mycobiology 2015;43:31-6. https://doi.org/10.5941/MYCO.2015.43.1.31
  7. Han KH, Lee DB, Kim JH, Kim MS, Han KY, Kim WS, Park YS, Kim HB, Han DM. Environmental factors affecting development of Aspergillus nidulans. J Microbiol 2003;41:34-40.
  8. Oh YT, Ahn CS, Kim JG, Ro HS, Lee CW, Kim JW. Proteomic analysis of early phase of conidia germination in Aspergillus nidulans. Fungal Genet Biol 2010;47:246-53. https://doi.org/10.1016/j.fgb.2009.11.002
  9. Kim Y, Nandakumar MP, Marten MR. Proteome map of Aspergillus nidulans during osmoadaptation. Fungal Genet Biol 2007;44:886-95. https://doi.org/10.1016/j.fgb.2006.12.001
  10. Shimizu M, Fujii T, Masuo S, Fujita K, Takaya N. Proteomic analysis of Aspergillus nidulans cultured under hypoxic conditions. Proteomics 2009;9:7-19. https://doi.org/10.1002/pmic.200701163
  11. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. https://doi.org/10.1016/0003-2697(76)90527-3
  12. Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci USA 1996;93:14440-5. https://doi.org/10.1073/pnas.93.25.14440
  13. Harsanyi Z, Granek IA, Mackenzie DW. Genetic damage induced by ethyl alcohol in Aspergillus nidulans. Mutat Res 1977;48:51-73. https://doi.org/10.1016/0027-5107(77)90190-7
  14. Kang EH, Kim JA, Oh HW, Park HM. LAMMER Kinase LkhA plays multiple roles in the vegetative growth and asexual and sexual development of Aspergillus nidulans. PLoS One 2013;8:e58762. https://doi.org/10.1371/journal.pone.0058762
  15. Glisin V, Crkvenjakov R, Byus C. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry 1974;13:2633-7. https://doi.org/10.1021/bi00709a025