배추 유래 phytocystatin 유전자, Brcpi1의 분리 및 발현특성 분석
Isolation and characterization of Brcpi1 gene encoding phytocystatinfrom chinese cabbage (Brassica rapa L.) seedlings
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식물 유래 cystein protease 저해제는 phytocystatin 으로 분류되어, 식물의 생장과 발육과정상에서 상이한 발현 양 상을 보이고 있다. 본 연구에서는 배추 유묘로부터 phytocystatin 유전자를 분리하여 Brcpi1 (GenBank accession no.: EF079953)으로 명명하고, 분자적 특성 및 기능 분석 을 수행하였다. Brcpi1 유전자는 전장이 881 bp이고, ORF가 609 bp으로 203개의 아미노산으로 구성되었다. BrCPI1 단 백질은 아미노산 motif로써 G와 PW 및 LARFAV가 있고, 식물 유래 phytocystatin 활성부위인 QVVAG가 잘 보존되 었다. 또한 BrCPI 단백질은 Brassica oleracea (BoCPI-1), Arabidopsis thaliana (AtCYSB), Glycine max (GmCPI), Oryza sativa (OsCYS-2) 및 Zea may (ZmCPI)와의 아미노산 서열 에서 각각 95, 94, 81, 80 및 78 %의 상동성을를 보였다. Brcpi1 유전자의 계통수를 분석한 결과 기존에 알려진 6 개의 단백질에서 3개의 group으로 나눌 수 있으며, 애기 장대 및 양배추와 같은 cluster을 보였다. 또한 Southern blot 분석한 결과 Brcpi1 유전자는 배추 게놈에 낮은 copy 수로 존재하였다. 기관별 Brcpi1 유전자의 발현양상은 수 술이 가장 높게 나타났으며, 꽃받침, 주두 및 꽃잎 등의 순으로 나타났다. Brcpi1 유전자의 기능을 알아보기 위 해, 형질전환 애기장대를 이용하여 염, 저온 및 ABA 처 리에 따른 도입 유전자의 발현양상을 살펴본 결과 고염 및 저온 하에서 뿌리의 생장이 대조 구에 비해 크게 나타 났으며, 유전자의 발현에서도 강하게 나타났다. 그러나 ABA처리에서는 큰 변화를 볼 수 없었다. 따라서 본 연구 에서 분리한 Brcpi1 유전자는 염 및 저온에 민감하게 반 응하며, 조직 및 기관 특이적으로 기능하여 다양한 환경 stress에 저항할 수 있는 방어기작을 갖고 있는 것으로 예 측된다.
Abstract
A cDNA clone encoding phytocystatin was isolated from Brassica rapa seedlings, through rapid amplification of cDNA ends (RACE). This gene (name as Brcpi1; GenBank accession no.: EF079953) had a total length of 881 bp with an open reading frame of 609 bp, and encoded predicted polypeptide of 203 amino acid (aa) residues including a putative N-terminal signal peptide. Other relevant regions found its sequence included the G and PW conserved aa motifs, and the consensus LARFAV sequence for phytocystatins and the reactive site QVVAG. The BrCPI1 protein shared 95, 94, 81, 80 and 78% identity with other CPI proterins isolated from Brassica oleracea (BoCPI-1), Arabidopsis thaliana (AtCY SB), Glycine max (GmCPI), Oryza sativa (OsCYS-2) and Zea may (ZmCPI) at amino acid level, respectively. Southern blot analysis showed that Brcpi1 was a low copy gene. Expression pattern analysis revealed that Brcpi1 was a tissue-specific expressing gene during reproductive growth and strongly expressed at mature seedling stages. Furthermore, overexpression of Brcpi1 in transgenic Arabidopsis was enhanced tolerance to salt and cold stresses. Meanwhile the juvenile seedling of Brcpi1 transgenic plants was not affected by various concentrations ABA in MS medium. Taken together, the results showed that Brcpi1 functioned as a cysteine protease inhibitor and it exhibited a protective agent against diverse types of abiotic stress, which induced this gene in a tissue- and stress-specific manner.
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