β-카로틴 강화벼 도입유전자의 도입 위치와 안정성을 도입 위치 주변염기서열 분석과 서던 분석한 결과, 벼 염색체 2번 중 30,363,938번과 30,363,973번 사이의 단일 부위에 도입유 전가 도입되었으며, T5-T7 세대 동안 도입된 모든 유전자들 이 안정적으로 유지되고 있으며, 도입유전자의 운반체인 Backbone DNA (pSB11)는 β-카로틴 강화벼에 삽입되지 않 았음을 확인하였다. 선발 마커로 도입된 PAT 단백질의 발현 분석 결과에서도 T5-T7 세대별, 생육시기별로 안정적으로 발 현됨을 검정하였으며, 목적 형질인 β-카로틴 분석 결과에서 도 모품종인 낙동벼에 비해 9.4배 함량이 증가됨을 확인하였 다. 이상의 분석기법을 통해 복수세대에서 β-카로틴 강화벼 의 도입 유전자들이 안정적으로 유지되고 목적 단백질들이 안정적으로 발현되고 있음을 확인하였다.

β-carotene hydroxylase (BCH) has been implicated as a key enzyme conferring a stress tolerant mechanism in plants by production of carotenoids, which serve as protectants against photoinhibition and precursor of ABA biosynthesis. We previously cloned a gene encoding a novel cytosolic form of BCH (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In the present work we extended our study to three GmBCHs as soybean is an allotetraploid, and examined their possible role(s) in nodule development. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in E. coli. Localization of GmBCHs by transfecting Arabidopsis protoplasts with GFP fusions and by EM immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids while GmBCH1 appeared to be cytosolic. RNAi of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids, in nodules. We therefore examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules, and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.

In this study, β -carotene concentrations was determined in soybean cultivar according to seed size, usage, seed coat color and cotyledon color as well as the process of seed germination. The total average concentration of β -carotene was 6.6~mug/g in soybean seed, 33.3~mug/g in soybean sprout. According to seed size, the total β -carotene concentration of soybean was 6.9~mug/g in large soybean seed, 6.7~mug/g in medium soybean seed, and 6.31~mug/g in small soybean seed. In soybean sprout, the total β -carotene concentration was 21.4~mug/g in large soybean sprout, 30.5~mug/g in medium soybean sprout, and 43.5~mug/g in small soybean sprout. According to the utilization of seed, the total β -carotene concentration of soybean seed was 7.2~mug/g in cooked with rice soybean seed, 6.1~mug/g in paste and curd soybean seed, and 6.3~mug/g in sprout soybean seed. In soybean sprout, the total β -carotene concentration was 25.9~mug/g in cooked with rice soybean sprout, 32.4~mug/g in paste and curd soybean sprout, and 41.9~mug/g in sprout soybean sprout. When comparison with seed coat color, the total β -carotene concentration of soybean with brown seed coat (8.8~mug/g ) was slightly higher than those of soybean with yellow (6.1~mug/g ). In soybean sprout, the total β -carotene concentration was 21.8~mug/g in black seed coat sprout, 38.7~mug/g in brown seed coat sprout, 34.1~mug/g in green seed coat sprout, 39.5~mug/g in yellow seed coat sprout, and 30.5~mug/g in mottle seed coat sprout. The results of this study suggested the functional characteristics of soybean through quantitative analysis of β -carotene.

β-carotene producing transformants were produced in the background of "Nagdong", a japonica rice cultivar. Introgression of the caroteniod locus into the elite cultivar "Ilpum". Was started to initiate a backcrossing program, we surveyed 220 SSR markers and found that 38% of them were polymorphic between the “Ilpum” as the recurrent parent and "Nagdong" transfomed as a recipient parent. First, backcross progenies have been produced and genotyped by the transgene specific PCR-based marker. Blast search indicated that the transgene PAC4-2 was located between SSR markers, RM5631 and RM5916. We produced 240 BC3F2 plants derived from thirteen BC3F1 plants containing the caroteniod gene. To develop near isogenic line, additional experiments including target and background selection are being performed using SSR markers. Traits of agronomic importance such as heading date, panicle number and culm length will also be evaluated. The results will be discussed.