Horticulture, Environment, and Biotechnology : HEB

Korean Society of Horticultural Science (한국원예학회)
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Development of Rescue Cloning Vector with Phosphinothricin Resistant Gene for Effective T-DNA Tagging

T-DNA Tagging을 위한 포스피노트리신 저항성 유전자를 포함하는 Rescue Cloning 벡터의 개발

Kim, Hyoung-Seok;Kim, Sung-Hoon;Park, Young-Doo
김형석;김성훈;박영두

  • Published : 20030000
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Abstract

This study was carried out to develop an effective rescue cloning vector, pRCV3, for T-DNA tagging. In phosphinothricin (PPT) herbicide added medium, the newly developed vector pRCV3 possesed PPT resistant genes which were capable of selecting a characteristic transformation system. It also contained within T-DNA, ampicillin resistant gene and bacterial replication origin for accomplishing plasmid rescue. To isolate both left and right insert junctions of a T-DNA tag, one unique restriction enzyme site within the vector was removed. Moreover, to detect various unpredictable integration patterns, PCR confirmation primer sets, which can be amplified in several important regions of the vector, were prepared. Based on these data, sequencing primers were designed to use directly for analysis of flanking plant DNA. To confirm an applicability of developed rescue cloning vector, tobacco (Nicotiana tabaccum cv. Havana SR1) was transformed with Agrobacterium tumerfaciens LBA4404 harboring pRCV3, and then plasmid rescue technique was applied to transformants which were confirmed by PCR

본 연구는 T-DNA tagging을 위한 rescue cloning 벡터(pRCV3)를 개발하기 위하여 수행되었다. 개발된 rescue cloning 벡터는 제초제인 phosphinothricin(PPT)가 첨가된 배지에서 형질 전환체를 선발할 수 있는 PPT 저항성 유전자를 포함하고 있으며 이와 더불어 T-DNA 내에 플라스미드 rescue를 수행할 수 있도록 암피실린 저항성 유전자와 박테리아 복제점을 포함하고 있다. 또한 T-DNA 좌우측의 식물 DNA를 분리하기 위하여 벡터 내에 존재하지 않는 제한 효소 인지 위치를 제거하였으며 T-DNA의 다양한 삽입 양상을 확인하기 위해 벡터 내 여러 중요한 부분에서 증폭될 수 있는 PCR 확인 용 primer 세트가 작성되었다. 그리고, 이때의 자료를 기본으로 인접된 식물 DNA의 염기 서열 분석을 위해 sequencing primer를 제작하였다. 개발된 rescue cloning용 벡터의 적용 가능성을 확인하기 위하여 pRCV3를 포함하는 Agrobacterium tumerfaciens로 담배를 형질 전환시켰고, plasmid rescue 기술은 PCR에 의해 확인된 형질 전환체들에 적용되었다.

Keywords

References

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