Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Analysis of an acid-responsive element in a promoter of laccase gene in the inky cap, Coprinellus congregatus

Coprinellus congregatus의 laccase 유전자 프로모터의 산성반응인자 분석

  • Kim, Su Yeon (Department of Biochemistry, Kangwon National University) ;
  • Nguyen, Linh (Department of Biochemistry, Kangwon National University) ;
  • Choi, Hyoung T. (Department of Biochemistry, Kangwon National University)
  • Received : 2016.06.14
  • Accepted : 2016.07.08
  • Published : 2016.09.30
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Abstract

The inky cap, Coprinellus congregatus, produces several laccase isozymes during its life cycle: both hyphal tip laccase and sclerotial laccase are involved in the fungal development. When this fungus was transferred to an acid liquid medium (pH 4.0-4.5), a new laccase was synthesized and secreted into the culture supernatant. In order to examine its regulation by external pH, green fluorescent protein gene was ligated at the downstream of the promoters having different lengths. These expression vectors having different promoter lengths were inserted into the fungal transformation vector, pBARGEM7-1. These expression vectors were introduced to the mating type a1 and a2 monokaryons, and the transformants were selected by the phosphinothricin resistance. Transformant a1 (a1TF) and transformant a2 (a2TF) were mated with each other to generate homozygotic dikaryon transformants. All these transformants were grown in neutral liquid medium for 5 days, and then the whole cell homogenates were transferred to the acidic liquid medium (pH 4.1). After 36 h incubation at $25^{\circ}C$, cells were harvested for the analysis of GFP expression. GFP expression was detected in the transformant having full-length promoter (2.0 kb), but other transformants having shorter length promoter (shorter than 1.29 kb) failed to show the fluorescence. Therefore, the acid-responsive element in the laccase promoter should be localized between -2.0 kb ~ -1.29 kb region.

먹물버섯의 하나인 Coprinellus congregatus는 생활사 동안 여러 종의 laccase 효소를 생성한다. 균사 끝 효소와 버섯시원체 효소 및 sclerotium (균핵) 효소들은 모두 이 균의 분화와 관련되었다. 이핵체 균사를 산성 액체배지(pH 4.0-4.5)에 접종하면 새로운 laccase가 합성되어 분비된다. 이 laccase 유전자의 프로모터의 어느 부분이 산 충격의 신호에 관련된 단백질이 결합하는가 분석하기 위하여 녹색형광단백질(green fluorescent protein, GFP) 유전자를 laccase 프로모터 2.0 kb 다음에 연결하고, 이를 형질전환 벡터인 pBARGEM7-1에 삽입함으로써 발현벡터를 구축하였다. 이 promoter-GFP 조합의 5'-region부터 차례로 제거한 짧은 길이의 이 발현벡터를 먹물버섯 교배형 a1균과 a2균에 형질전환 방법으로 도입시키고 phosphinothricin 저항성으로 형질전환체들을 선발하였다. 선발된 형질전환체 a1 (a1TF)과 a2 (a2TF)를 서로 교배하여 동형접합(homozygotic) 이핵체 형질전환체를 만들었다. 이들을 산성 액체배지에서 36시간 배양하고 균체를 모아 confocal microscope를 사용하여 형광을 분석하였다. Laccase 유전자의 전체 프로모터(2.0 kb)를 가진 발현벡터(F0-GFP)를 도입한 동형접합 형질전환체에서는 형광을 보였으나, 그 보다 짧은 길이(1.29 kb 이하)의 프로모터를 가진 형질전환체에서는 형광이 나타나지 않았다. 이 결과에 근거하여 먹물버섯의 산 충격에 대한 신호를 받는 부위가 laccase 유전자 프로모터의 -2.0 kb ~ -1.29 kb 사이에 있을 것으로 추정한다.

Keywords

References

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