β-카로틴 강화벼의 분자생물학적 특성과 안전성 평가
Molecular Biological Characteristics and Biosafety Assessment for β-carotene Biofortified Transgenic Rice
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초록
β-카로틴 강화벼 도입유전자의 도입 위치와 안정성을 도입위치 주변염기서열 분석과 서던 분석한 결과, 벼 염색체 2번 중 30,363,938번과 30,363,973번 사이의 단일 부위에 도입유전가 도입되었으며, T5-T7 세대 동안 도입된 모든 유전자들이 안정적으로 유지되고 있으며, 도입유전자의 운반체인 Backbone DNA (pSB11)는 β-카로틴 강화벼에 삽입되지 않았음을 확인하였다. 선발 마커로 도입된 PAT 단백질의 발현분석 결과에서도 T5-T7 세대별, 생육시기별로 안정적으로 발현됨을 검정하였으며, 목적 형질인 β-카로틴 분석 결과에서도 모품종인 낙동벼에 비해 9.4배 함량이 증가됨을 확인하였다. 이상의 분석기법을 통해 복수세대에서 β-카로틴 강화벼의 도입 유전자들이 안정적으로 유지되고 목적 단백질들이 안정적으로 발현되고 있음을 확인하였다.
Abstract
The β-carotene biofortified transgenic rice was developed by transforming rice cv. Nakdongbyeo with phytoenesynthase (Psy) and carotene desaturase (Crt I) genes isolated from Capsicum and Pantoea. The aim of this study was to performmolecular characterization of rice transformants of T5-T7 generation harboring Psy and Ctr I genes driven by endosperm specificglobulin promoter for biosafety evaluation of β-carotene biofortified transgenic rice. The structure and sequence of T-DNA inthe transformation vector and the insertion sites, flanking sequences and generational stability of inserted T-DNA in transgenicrice lines were analyzed. The transformation vector consisted of right border, MAR gene, carotenogenic genes unit, herbicideresistance selectable marker unit, MAR gene and left border in sequential order. T-DNA was introduced at the position of30,363,938-30,363,973 bp of chromosome No. 2 by adaptor-ligation PCR. Stable integration of T-DNA and stable expression ofbar gene was confirmed in T5 to T7 generations. It was also confirmed that the backbone DNA of transformation vectorcontaining antibacterial gene was not present in the genome of β-carotene biofortified transgenic rice. HPLC analysis confirmedthat carotenoids were consistently detected through T5-T7 generations.
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