Artificial Mutation for Silkworm Molecular Breeding Using Gene Scissors

다국어 초록 (Multilingual Abstract)

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9을 이용하는 유전자 가위 기술은 미래 육종 기술로서 주목받고 있다. 본 연구에서는 3세대 유전자 가위 CRISPR/Cas9을 이용한 누에 kynurenine 3-monooxygenase (KMO) 유전자 편집을 통한 돌연변이 유발 및 선택 교배를 통하여 유전자의 세대간 전달을 분석하고자 하였다. 유전자 편집을 위하여 누에의 KMO 유전자에 대한 3종의 가이드 RNA를 제작하고, 제작된 gRNA는 Cas9 단백질과 복합체를 형성시켜 누에 세포주(BM-N)에 도입 후 T7 endonucleaseI 분석을 수행하여 최적의 gRNA를 선발하였다. 선발된 K1N gRNA는 누에 유전자를 편집하기 위하여 Cas9 단백질과 복합체를 형성시킨 후 누에 초가 배아에 미세주사하고 사육하였다. 미세주사 후 부화율은 18% 가량으로 낮게 나타났으나 생존한 개체 중 돌연변이 발생율은 60% 이상으로 비교적 높게 나타났다. 돌연변이가 발생된 G0세대의 KMO 유전자는 이형접합자 형태로 나타났으며, 표현형의 변화는 관찰되지 않았다. 하지만 형접합자들 사이의 근친 교배에 의해 탄생한 G1세대 돌연변이에서는 일부에서 알과 눈의 색 변화가 확인되었으며, 변이가 확인된 개체들사이의 근친교배를 통해 생산된 G2세대에서는 모든 개체에서 표현형의 변화가 나타났다. 이러한 결과에 비추어 볼 때, 유전자 가위를 이용한 돌연변이 육종에는 한계가 있으나 전통 교배 육종과 융합을 통하여 육종 기간을 비약적으로 단축시킬 수 있는 곤충 육종 기술로 발전가능성이 높다고 판단된다.

다국어 초록 (Multilingual Abstract)

Gene editing technology using the clustered regularly interspaced short palindromic repeat (CRISPR) and the CRISPR associated protein (Cas)9 has been highly anticipated in developing breeding techniques. In this study, we discuss gene scissors as a tool for silkworm molecular breeding through analysis of Bombyx mori Kynurenine 3-Monooxygenase (BmKMO) gene editing using the CRISPR/Cas9 system and analysis of generational transmission through mutagenesis and selective crossing. The nucleotide sequence of the BmKMO gene was analyzed, and three guide RNAs (gRNAs) were prepared. Each synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA and Cas9 complexes were subsequently microinjected into the silkworm embryos; the hatching rate was 18% and the incidence of mutation was 60%. The gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed. In homozygotes generated by self-crossing, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/ Cas9 system is possible and could be an effective way of shortening the time required.

목차 (Table of Contents)

• 서론
• 재료 및 방법
• 결과 및 고찰
• References
• 초록

참고문헌 (Reference)

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