Abstract
The objectives of this study were to analyze mutant lines of Chinese cabbage ($Brassica$ $rapa$ ssp. $pekinensis$) using gene tagging system (plasmid rescue and inverse polymerase chain reaction) and to observe the phenotypic characteristics. Insertional mutants were derived by transferring DNA (T-DNA) of $Agrobacterium$ for functional genomics study in Chinese cabbage. The hypocotyls of Chinese cabbage 'Seoul' were used to obtain transgenic plants with $Agrobacterium$ $tumefaciens$ harboring pRCV2 vector. To tag T-DNA from the Chinese cabbage genomic DNA, plasmid rescue and inverse PCR were applied for multiple copies and single copy insertional mutants. These techniques were successfully conducted to Chinese cabbage plant with high efficiency, and as a result, T-DNA of pRCV2 vector showed distinct various integration patterns in the transgenic plant genome. The polyploidy level analysis showed the change in phenotypic characteristics of 13 mutant lines was not due to variation in somatic chromosome number. Compared with wild type, the $T_1$ progenies showed varied phenotypes, such as decreased stamen numbers, larger or smaller flowers, upright growth habit, hairless leaves, chlorosis symptoms, narrow leaves, and deeply serrated leaves. The polyploidy level analysis showed the change in phenotypic characteristics of 13 mutant lines was not due to variation in somatic chromosome number. To tag T-DNA from the Chinese cabbage genomic DNA, plasmid rescue and inverse PCR were applied for multiple copies and single copy insertional mutants. Mutants that showed distinct phenotypic difference compared to wild type with 1 copy of T-DNA by Southern blot analysis, and with 2n = 20 of chromosome number were selected. These selected mutant lines were sequenced flanking DNA, mapped genomic loci, and the genome information of the lines is being recorded in specially developed database.
본 연구는 gene tagging system(plasmid rescue와 inverse polymerase chain reaction)을 사용하여 얻은 배추($Brassica$ $rapa$ ssp. $pekinensis$) 형질전환체 계통을 분석하고 이들의 표현형을 관찰하고자 수행되었다. 배추의 기능유전체 연구를 위해 $Agrobacterium$의 전이 DNA(T-DNA)를 사용하여 삽입돌연변이체를 유기하였다. 형질전환체는 '서울' 배추 품종의 pRCV2 vector를 가진 $Agrobacterium$ $tumefaciens$을 접종하여 얻었다. 형질전환 $T_1$ 세대는 비형질전환체와 비교하여, 수술 수의 감소, 크거나 작은 꽃, 직립생장형, 잎에 털이 없는 것, 잎의 황백화 현상, 잎이 좁거나 결각이 깊은 것과 같은 다양한 표현형을 보였다. 형질전환 계통 중에서 발견된 13개의 돌연변이 계통의 표현형 변화는 체세포의 배수성 분석을 통하여 염색체 수의 변화에 기인하지 않은 것을 확인하였다. 배추 genomic DNA에서 T-DNA의 위치 확인을 위해 multiple copy와 single copy 형질전환체에 plasmid rescue와 inverse PCR 방법을 각각 적용하였다. 비형질전환체와 비교하여 뚜렷한 표현형적 차이를 보이고 Southern blot 분석 결과 1 copy의 T-DNA를 가지며 염색체 수가 20(2n)인 돌연변이체를 선발하여, flanking DNA 염기서열을 확인하고 배추 염색체내에서 각각의 유전자좌를 표시하였으며 이들 계통들에 대하여 데이터베이스를 작성 중에 있다.
