Construction of a Pure Cryparin-null Mutant for the Promoter Analysis of Cryparin Gene

Cryparin 유전자의 promoter 분석을 위한 cryparin 유전자 치환체의 순수 제조

  • Kim, Myoung-Ju (Faculty of Biological Sciences, Institute of Molecular Biology and Genetics, Chonbuk National University) ;
  • Yang, Moon-Sik (Faculty of Biological Sciences, Institute of Molecular Biology and Genetics, Chonbuk National University) ;
  • Kim, Dae-Hyuk (Faculty of Biological Sciences, Institute of Molecular Biology and Genetics, Chonbuk National University)
  • 김명주 (전북대학교 생물과학부 및 유전공학연구소) ;
  • 양문식 (전북대학교 생물과학부 및 유전공학연구소) ;
  • 김대혁 (전북대학교 생물과학부 및 유전공학연구소)
  • Published : 1998.12.30

Abstract

The cryparin of Cryphonectria parasitica belongs to a cell wall associated fungal hydrophobin. The cryparin, though it is encoded by a single copy gene, is known for the high expression during the liquid culture of C. parasitica, and it turns out that 22% of total mRNA was transcribed for cryparin at 48hr after the liquid culture. In addition, it is also known as one of down-regulated fungal proteins by the presence of double stranded RNA virus, Cryphonectria hypovirus 1. In previous studies (Kim et al., 1999), we have constructed a cryparin-null mutant by replacing the cryparin gene with hygromycin B resistance gene due to site directed homologous recombination. In order for the promoter analysis of cryparin which seems to be very strong as well as mycoviral specific, it is preferable to have a strain with only a target promoter replaced and a discernable target site for incoming vectors. However, the cryparin-null mutant revealed the presence of an additional copy of transforming vector except the one which replaced the cryparin gene. In addition, the cryparin-null mutant did not contain any markers for targeted integration of incoming vectors. This prompts us to design an experiment to obtain a strain for promoter analysis of cryparin gene. A different mating type strain EP6(Mata, $met^-$) was mated with the cryparin-null mutant ${\triangle}$Crp194-7(MatA, Crp${\triangle}$::hph) to make the progenies with only a single replacement vector and $met^-$ characteristic remained. Nutritional assay as well as Southern blot analysis revealed that the progeny, ${\triangle}$Crp194-a6, was the methionine auxotroph with a single replacing vector in genome. Northern blot analysis and PAGE showed that there was no cryparin produced in this bred strain either.

Cryparin은 Cryphonectria parasitica의 세포벽에 풍부한 소수성 단백질에 속한다. cryparin은 비록 하나의 유전자에 의해 발현되지만 액체배양 후 48시간이 지나면 발현된 전체 유전자중에서 22%를 차지할 정도의 높은 발현 양상을 나타낸다. 또한 cryparin은 RNA mycovirus인 Cryphonectria hypovirus 1의 감염에 의해 발현이 현저히 억제되는 유전자로 알려졌다. 이미 지난 실험(Kim et al., 1999)에서 상동염색체간의 재조합을 이용하여 cryparin 유전자를 항생제 hygromycin B 저항성 유전자로 치환한 치환체를 제조하였다. 발현율이 매우 높으면서도 virus에 의해 밀접하게 영향받는 cryparin 유전자의 promoter 분석을 위하여서는 대상이 되는 유전자 치환을 위한 vector만을 포함하며, 분석에 이용될 여러 유전자 운반체들이 어느 한곳에만 삽입되도록 하는 성질을 가진 균주의 개발이 필요하다. 그러나 지난번 실험의 결과 얻어진 cryparin 치환체는 치환용 vector외에도 무작위로 삽입된 vector가 존재하고 나아가 새로운 vector들이 어느 한곳에만 삽입되도록 하는 성질을 갖지 못하였다. 따라서 본 실험에서는 cryparin 유전자 치환체와 영양요구성 돌연변이체인 균주간의 교잡을 이용하여 분석 대상이 되는 유전자의 치환에 이용된 vector만을 포함하며, 분석에 이용될 여러 유전자 운반체들이 genome내의 어느 한곳에만 삽입되도록 하는 성질을 가진 균주를 제조하였다.

Keywords

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