효모세포의 전기융합 및 융합세포의 RAPD-PCR을 이용한 유전적 분석

Electrofusion of Yeast Cells and Their Genetic Analysis Using RAPD-PCR

  • 김승 (조선대학교 유전자과학과) ;
  • 김재성 (조선대학교 유전자과학과) ;
  • ;
  • 박인성 (루크제약(주)) ;
  • 조문구 (우석대학교 식품생명공학과) ;
  • 박열 (조선대학교 유전자과학과) ;
  • 전홍성 (조선대학교 유전자과학과) ;
  • 최봉석 (조선대학교 유전자과학과) ;
  • 박세은 (조선대학교 유전자과학과) ;
  • 최한석 (익산대학 특용작물가공과) ;
  • 김명곤 (전북대학교 식품공학과) ;
  • 김성준 (조선대학교 유전자과학과)
  • Kim, Seung (Department of Genetic Engineering, Chosun University) ;
  • Kim, Jae-Sung (Department of Genetic Engineering, Chosun University) ;
  • Sapkota, Kumar (Department of Genetic Engineering, Chosun University) ;
  • Park, In-Sung (LUKE Pharmaceutical Co.) ;
  • Cho, Moon-Gu (Department of Food Biotechnology, Woosuk University) ;
  • Park, Yeal (Department of Genetic Engineering, Chosun University) ;
  • Chun, Hong-Sung (Department of Genetic Engineering, Chosun University) ;
  • Choi, Bong-Suk (Department of Genetic Engineering, Chosun University) ;
  • Park, Se-Eun (Department of Genetic Engineering, Chosun University) ;
  • Choi, Han-Suk (Department of Biotechnology, Iksan National College) ;
  • Kim, Myung-Kon (Department of Food Science and Technology, Chonbuk National University) ;
  • Kim, Sung-Jun (Department of Genetic Engineering, Chosun University)
  • 발행 : 2006.09.30

초록

본 연구는 전기적 세포융합방법인 electrofusion을 이용하여 우량균주의 육성을 목적으로 S. cerevisiae KCTC7904와 Z. rouxii KCTC7966 간의 전기융합을 실시하였고, 융합주의 내당성과 내염성을 확인하였으며, 융합주의 선별방법을 연구하였다. 또한 융합주의 유전안정성과 RAPD-PCR 분석을 통한 융합여부의 직접적인 증거를 확인하고자 실험하였다. S. cerevisiae KCTC7904와 Z. rouxii KCTC 7966를 각각 $12{\sim}36$시간 배양하여 분리 세척한 다음 1.5% 2-mercaptoethanol로 20분간 전 처리하여 lyticase(200 U/ml)로 $30^{\circ}C$에서 최종적으로 $60{\sim}90$분간 처리했을 때, 91% 이상의 원형질체를 얻을 수 있었다. 얻어진 원형질체를 $1.0{\sim}1.2\;M$ sorbitol 용액으로 세척 한 다음 각각 1 : 1의 비율로 혼합하여 1.5 MHz/50 pV의 고주파를 가하였을때 paired protoplast가 형성되었으며, 615 $V/256\;{\mu}sec$의 고전압을 가한 결과 약 25% 정도의 융합체를 얻을 수 있었다. 선별된 융합주의 내당성과 내염성을 각각의 모균주와 비교하여 실험한 결과 50%의 glucose와 15%의 NaCl을 함유한 배지에서 모두 생육이 가능하여 각각의 균주 특성을 가지고 있음을 확인하였고, 또한 융합주를 $4^{\circ}C$에서 5개월간 보관했을 때 약 28%정도가 모균주로 복귀되었지만, 72%의 융합안정성을 나타내므로 비교적 안정한 상태를 확인할 수 있었다. 융합의 진위 여부를 증명하기 위해 유전적인 분석방법인 RAPD-PCR 법을 사용하여 실험한 결과 각각의 균주가 agarose gel 상에서 보인 band의 패턴이 융합주에서도 모두 보여짐을 확인하였다.

In the present study, an attempt has been made to produce hybrid yeast strains of different useful and dominant characteristics. The hybrid yeast strains were produced by electrofusion and their genetic analysis were performed by RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction). The protoplast of Saccharomyces cerevisiae KCTC 7904 and Zygosaccharomyces rouxii KCTC 7966 were obtained above 92% when treated with lyticase at $30^{\circ}C$ for $60{\sim}90$ min after the pretreatment of $1{\sim}2%$ 2-mercaptoethanol at $30^{\circ}C$ for $15{\sim}20$ min. The fusant was produced from paired protoplast stage under the electric pulse at high frequency conditions (1.5 MHz/50 pV, 615 $V/256\;{\mu}sec$) within glass-platinum made electrofusion chamber. Changes in RAPD patterns in mother cells and hybrid cells proved that the fusant contains two types of yeast gene originated from its parent. Furthermore, fermentation characters exhibits by the fusant cell confirmed its genetic changes. These results suggest that genetically stable hybrid yeast strains of economic importance can be produced by electrofusion technique and these electrofused yeast cells have an enormous impact in biotechnology and biomedicine.

키워드

참고문헌

  1. Randez-Gil F., Sanz P. and Prieto J. A. (1999) Engineering baker's yeast: room for improvement. Trends Biotechnol. 17, 237-244 https://doi.org/10.1016/S0167-7799(99)01318-9
  2. Stanton C., Ross R. P., Fitzgerald G. F. and Sinderen D. V. (2005) Fermented functional foods based on probiotics and their biogenic metabolites. Curr. Opin. Biotechnol. 16, 198-203 https://doi.org/10.1016/j.copbio.2005.02.008
  3. Mukai N., Nishimori C., Fujishige I. W., Mizuno A., Takahashi T. and Sato K. (2001) Beer brewing using a fusant between a sake yeast and a brewer's yeast. J. Biosci. Bioeng. 91, 482-486 https://doi.org/10.1263/jbb.91.482
  4. Kavanagh K. and Whittaker P. A. (1996) Application of protoplast fusion to the nonconventional yeast. Enzyme Microb. Technol. 18, 45-51 https://doi.org/10.1016/0141-0229(96)00072-5
  5. Thompson J. K., McConville K. J., Nicholson C. and Collins M. A. (2001) DNA Cloning in Lactobacillus helveticus by the Exconjugation of Recombinant mob-Containing Plasmid Constructs from Strains of Transformable Lactic Acid Bacteria. Plasmid. 46, 188-201 https://doi.org/10.1006/plas.2001.1540
  6. Seo, J. H., Kim Y. H., Jun D. Y. and Lee J. T. (1986) A study on strain improvement by protoplast fusion between amylase secreting yeast and alcohol fermenting yeast (I, II). J. Microbiol. Biotechnol. 14, 305-318
  7. Zachrisson, A. and Bornman C. H. (1986) Electromanipulation of plant protoplasts. Physiol. Plant. 67, 507-516 https://doi.org/10.1111/j.1399-3054.1986.tb05771.x
  8. Choi S. H., Sung C., Oh M. J. and Kim C. J. (1997) Intergeneric protoplast fusion in Saccharomycopsis fibuligera and Saccharomyces cerevisiae. J. Ferment. Bioeng. 84, 158-161 https://doi.org/10.1016/S0922-338X(97)82547-0
  9. Urano N., Higashikawa R. and Hirai H. (1998) Effect of mitochondria on electrofusion of yeast protoplasts. Enzyme Microb. Technol. 23, 107-112 https://doi.org/10.1016/S0141-0229(98)00021-0
  10. Finaz, C., Lefevre A. and Teissie J. (1984) Electrofusion: A new highly efficient technique for generating somatic cell hybrids. Exp. cell Res. 150, 477-482 https://doi.org/10.1016/0014-4827(84)90592-5
  11. Lee D. W., Koh Y. S., Kim K. J., Kim B. C., Choi H. J., Kim D. S., Suhartono M. T. and Pyun Y. R. (1999) Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1. FEMS Microbiol. Lett. 179, 393-400 https://doi.org/10.1111/j.1574-6968.1999.tb08754.x
  12. Lee J. S. (1988) Strain improvement by protoplast fusion between thermophilic yeast and osmotolerant yeast, In Ph. D. thesis of Chungnam Univ. Republic of Korea, pp. 1-11
  13. Fadda M. E., Mossa V., PisanoM. B., Deplano M. and Cosentino S. (2004) Occurrence and characterization of yeasts isolated from artisanal Fiore Sardo cheese. Int. J. Food Microbiol. 95, 51-59 https://doi.org/10.1016/j.ijfoodmicro.2004.02.001
  14. Theelen B., Silvestri M., Guého E., Belkum A. V. and Boekhout T. (2001) Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLPTm), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE). FEMS Yeast Res. 1, 79-86 https://doi.org/10.1111/j.1567-1364.2001.tb00018.x
  15. Barszczewski W. and Robak M. (2004) Differentiation of contaminating yeasts in brewery by PCR-based techniques. Food Microbiol. 21, 227-231 https://doi.org/10.1016/S0740-0020(03)00071-6
  16. Couto B., Vogels M. M., Hofstra J., Huis-in't-Veld H., Vossen J. and Van-der J. (1995) Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts. J. Appl. Bacteriol. 79, 525-535 https://doi.org/10.1111/j.1365-2672.1995.tb03173.x
  17. Gomes L. H., Duarte K. M. R., Argueso J. L., Echeverrigaray S. and Tavares F. C. A. (2000) Methods for yeast characterization from industrial products. Food Microbiol. 17, 217-223 https://doi.org/10.1006/fmic.1999.0306
  18. Couto B., Vossen M. M., Van-der. J., Hofstra J. and Huis-in't- Veld H. (1994) RAPD analysis: a rapid technique for differentiation of spoliage yeasts. Int. J. Food. Microbiol. 24,249-260 https://doi.org/10.1016/0168-1605(94)90123-6
  19. Grando M. S., Ubeda J. and Briones A. I. (1994) RAPD analysis of wine Saccharomyces cerevisiae strains differentiated by pulsed field gel electrophoresis. Biotechnol. Tech. 8, 51-560
  20. Park I. S. (1999) Studies on the protoplast formation and electrofusion for the improvement of yeast strains. In M.S. thesis of Woosuk Univ. Republic of Korea, pp. 18-19
  21. Zimmermann U. and Vienken J. (1982) Electric field-induced cell-to-cell fusion. In J. Membrane Biol. 67, 165-182 https://doi.org/10.1007/BF01868659
  22. Zimmermann U. (1983) Electrofusion of cell: principles and industrial potent. Trends Biotechnol. 1, 149-155 https://doi.org/10.1016/0167-7799(83)90006-9
  23. Seo J. H., Kim Y. H., Jun D. Y. and Lee J. T. (1986) A study on strain improvement by protoplast fusion between amylase secreting yeast and alcohol fermenting yeast (Part1) Isolation and characterization of fusant between S. cerevisiae and S. diastaticus. J. Microbiol. Biotechnol. 14, 305
  24. Seo J. H., Kim Y. H., Jun D. Y. and Yi C. H. (1986) A study on strain improvement by protoplast fusion between amylase secreting yeast and alcohol fermenting yeast (Part 2) alcohol and glucoamylase productivities of fusant between S. cerevisiae and S. diastaticus. J. Microbiol. Biotechnol. 14, 311
  25. Lin C. C., Hsieh P. C., Mau J. L.and Teng D. F. (2005) Construction of an intergeneric fusion from Schizosaccharomyces pombe and Lentinula edodes for xylan degradation and polyol production. Enzyme Microb. Technol. 36, 107-117 https://doi.org/10.1016/j.enzmictec.2004.07.007