초록 ▼

- BAC 클론선발을 자체적으로 수행할 수 있는 시스템을 구축하여 선발 뒤 염기서열들을 결정하였다.
불마름병 저항성 유전자에 상당히 근접해 있으며, VIGS을 이용하여 기능을 검증할 예정이다. 또한, 불마름병 저항성 유전자가 위치한 지역과 리폭시게나아제 유전자가 위치한 지역의 염기서열을 각각 분석한 결과, 전형적인 콩 유전체의 중복현상이 고찰되었다.
- 뿌리혹 감염시 콩 뿌리에서 발현되는 유전자 β-carotene hydroxylase1 (bCH1, Antioxidant 합성효소)를 이용하여 형질전환벡터를 작성하고 형질전

- BAC 클론선발을 자체적으로 수행할 수 있는 시스템을 구축하여 선발 뒤 염기서열들을 결정하였다.
불마름병 저항성 유전자에 상당히 근접해 있으며, VIGS을 이용하여 기능을 검증할 예정이다. 또한, 불마름병 저항성 유전자가 위치한 지역과 리폭시게나아제 유전자가 위치한 지역의 염기서열을 각각 분석한 결과, 전형적인 콩 유전체의 중복현상이 고찰되었다.
- 뿌리혹 감염시 콩 뿌리에서 발현되는 유전자 β-carotene hydroxylase1 (bCH1, Antioxidant 합성효소)를 이용하여 형질전환벡터를 작성하고 형질전환 담배 식물체를 이용하여 유전자의 기능을 확인하였다. 주요 작물인 벼와 콩에도 도입하여 형질전환 식물체를 작성하여, 염과 건조스트레스에 내성이 증진되면서 포장조건하에서 농업적 형질 또한 개선된 형질전환 벼 2계통 및 6파생계통을 선발하였다.
- 항암, 항산화 기능이 있는 아이소플라본을 고함유한 나물콩 품종개발을 위하여 여교배 집단 유래 8계통을 사용하였으며, 외관품질, 다수, 내도복, 고아이소플라본 함량을 분석하였고, 지역적응시험 선발 후 품종(신강콩)으로 출원하였다.

Abstract ▼

1. Soybean genome analysis
1) Sequence level analysis of recently duplicated regions in soybean [Glycine max (L.) Merr.]genome
A single recessive gene, rxp, on linkage group (LG) D2 controls bacterial leaf pustule resistance in soybean. We identified two homoeologous contigs (GmA and GmA’) com

1. Soybean genome analysis
1) Sequence level analysis of recently duplicated regions in soybean [Glycine max (L.) Merr.]genome
A single recessive gene, rxp, on linkage group (LG) D2 controls bacterial leaf pustule resistance in soybean. We identified two homoeologous contigs (GmA and GmA’) composed of five bacterial artificial chromosomes (BACs) during the selection of BAC clones around Rxp region. With the RIL population from the cross of Pureunkong and Jinpumkong 2, SNP and SSR marker genotyping was able to locate GmA’ on LG A1. Based on information in the Soybean Breeders Toolbox and our results, parts of LG A1 and LG D2 share duplicated regions. Alignment and annotation revealed that many homoeologous regions contained kinases and proteins related to signal transduction pathway. Interestingly, inserted sequences from GmA and GmA’ had homology with transposase and integrase. Estimation of evolutionary events revealed that speciation of soybean from Medicago and the recent divergence of two soybean homoeologous regions occurred at 60 and 12 million years ago, respectively. Distribution of synonymous substitution patterns, Ks, yielded a first secondary peak (mode Ks = 0.10 to 0.15) followed by two smaller bulges were displayed between soybean homologous regions. Thus, diploidized paleopolyploidy of soybean genome was again supported by our study.
2) The lipoxygenase gene family: a genomic fossil of shared polyploidy between Glycine max and Medicago truncatula
Soybean lipoxygenases (Lxs) play important roles in plant resistance and in conferring the distinct bean flavor. Lxs comprise a multi-gene family that includes GmLx1, GmLx2 and GmLx3, and many of these genes have been characterized. We were interested in investigating the relationship between the soybean lipoxygenase isozymes from an evolutionary perspective, since soybean has undergone two rounds of polyploidy. Here we report the tetrad genome structure of soybean Lx regions produced by ancient and recent polyploidy. Also, comparative genomics with Medicago truncatula was performed to estimate Lxs in the common ancestor of soybean and Medicago.
Two Lx regions in Medicago truncatula showing synteny with soybean were analyzed. Differential evolutionary rates between soybean and Medicago were observed and the median Ks values of Mt-Mt, Gm-Mt, and Gm-Gm paralogs were determined to be 0.75, 0.62, and 0.46, respectively. Thus, the comparison of Gm-Mt paralogs (Ks=0.62) and Gm-Mt orthologs (Ks=0.45) supports the ancient duplication of Lx regions in the common ancestor prior to the Medicago-Glycine split. After speciation, no Lx regions generated by another polyploidy were identified in Medicago. Instead tandem duplication of Lx genes was observed. On the other hand, a lineage-specific duplication occurred in soybean resulting in two pairs of Lx regions. Each pair of soybean regions was co-orthologous to one Lx region in Medicago. A total of 34 Lx genes (15 MtLxs and 19 GmLxs) were divided into two groups by phylogenetic analysis. Our study shows that the Lx gene family evolved from two distinct Lx genes in the most recent common ancestor.
This study analyz ed two pairs of Lx regions generated by two rounds of polyploidy in soybean. Each pair of soybean homeologous regions is co-orthologous to one region of Medicago, demonstrating the quartet structure of the soybean genome. Differential evolutionary rates between soybean and Medicago were observed; thus optimized rates of Ks per year should be applied for accurate estimation of coalescence times to each case of comparison: soybean-soybean, soybean-Medicago, or Medicago-Medicago. In conclusion, the soybean Lx gene family expanded by ancient polyploidy prior to taxon divergence, followed by a soybean-specific duplication and tandem duplications, respectively.
3) Chromosomal rearrangement and conservation of bacterial disease resistance among homeologous loci in soybean
Bacterial leaf pustule (BLP), caused by Xanthomonas axonopodis pv. glycines (Xag), is a prevalent bacterial disease of soybean [Glycine max (L.) Merr.] that has been reported in many soybean-growing regions of the world where high temperature and humidity prevail. Rangeand severity of the disease are expected to increase due to global warming and climate change. Soybean is an ancient tetraploid whose genome has gone through at least two rounds of polyploidy events. Many studies revealed the structure produced by a relatively recent polyploidy, but still few are reported regarding an ancient polyploidy due to high frequency of gene loss after duplication events. In our previous study, we reported two homeologous regions of Rxp locus produced by recent duplication and their paralogous region in M. truncatula. Here we report more comprehensive soybean genome structure anchored by Rxp genes and evolutionary history of Rxp regions from the most recent common ancestor legume through comparative genomics with M. truncatula. Our data show that the ancestral genome of soybean had experienced dynamic genome rearrangement followed by recent polyploidy event. Moreover, multiple Rxp quantitative trait loci (QTLs) in different soybean LGs are actually comprised of homeologous regions produced by two rounds of polyploidy events.
2. Identification of useful genes from soybean
To investigate the relationship between nodule development and xanthophyll cycle enzymes, the expression level of genes encoding xanthophyll cycle enzymes was examined using RT-PCR and real time PCR. The full-length cDNA of the putative β-carotene hydroxylase, GmBCH2, showing increased expression in leaves, was then isolated using RACE technique. To check whether GmBCH1 and GmBCH2 actually have the enzymatic activity of β-carotene hydroxylase, HPLC analysis was performed. To ascertain the subcellular localization of both β-carotene hydroxylases of Glycine max in plants, GmBCH1 and GmBCH2 genes were fused to GFP reporter gene under the control of CaMV35S promoter, respectively and then the resulting plasmids were transfected into arabidopsis protoplasts. Development of transgenic nodules containing GmBCH RNAi constructs targeting both GmBCH1 and GmBCH2 was observed to investigate the role of GmBCH in nodule development.
β-carotene hydroxylase catalyzes the formation of zeaxanthin from β-carotene. Carotenoids act as antioxidants in biological systems. Carotenoids are the most effective quenchers of the reactive oxygen species (ROS) found in nature. In addition, carotenoids an inhibit lipid peroxidation and thus stablize membranes. The expression level of β -carotene hydroxylase was induced during nodulation in response to rhizobial inoculation, implying that antioxidants may be important for proper development of root nodules. The expression level of a gene encoding zeaxanthin epoxidase (ZEP), an enzyme that promotes the conversion of zeaxanthin to violaxanthin via antheraxanthin, was found to be decreased during nodulation. This indicates that zeaxanthin may play a certain role in nodulation. Recently, whole genome sequence of Glycine max has been identified. From the results of BLAST search with GmBCH1 cDNA sequence, sequences of another isoform, GmBCH3, with highest similarity could be found. Consistent with expression pa ttern of GmBCH1, expression level of GmBCH3 was also increased in mature nodules compared with root. To investigate the roles of zeaxanthin in nodulation, transgenic hairy roots containing construct for GmBCH RNAi were used. Nodulation of these transgenic hairy roots was poor compared to that of control. This result suggests that these genes may be associated with nodule development. Further study will find the mechanism related to zeaxanthin in nodule development.
We isolated 26 fragments of differentially expressed genes during soybean seed formation and examined their functions. Of the genes, the full-length cDNAs of the useful gene were isolated and studied further possible functions.
GmLRK1, identified to be a genuine kinase by phosphorylation assay, and its Arabidopsis ortholog At2g36570 may be essential for all the stages of soybean development, rather than in a specialized developmental procedure such as seed development. Together with the observations made from the mutant phenotypes for the Arabidopsis ortholog At2g36570, we propose that the function of GmLRK1 may be involved in the regulation of cell elongation/expansion. Thioredoxins (Trxs) are redox-active small proteins forming a disulfide bridge with targets. We identified the targets of Trx from soybean nodules and then examined the its interaction with them. Interaction between the two proteins suggest a putative function during nitrogen fixation, assimilation and nodule development.
3. Development of function added crops using useful gene from soybean
1) Development of value added crops by transformation using useful gene from soybean
We used a beta-carotene hydroxylase1 gene (GmbCH1) from Glycine max for developing of value added crop, and investigated novel GmbCH1 function in relation to tolerance to abiotic and biotic stress. We constructed two transformation vectors under the control of constitutive (CaMV 35S promoter) and seed specific/inducible promoter (globulin promoter). We produced transgenic plants using colored rice (Heuknambyeo), rice (Ilmibyeo) and soybean (Iksannamulkong).
In abiotic stress assay, we found that homozygous T2 Heuknambyeo transgenic lines had novel functions of enhanced tolerance to salt and drought, whereas those of Ilmibyeo transgenic rice did not show any clear effect on abiotic stress. These results may implicate that abiotic functions of GmbCH1 gene is likely to be associated with the pathway of anthocyanin synthesis and further study should be needed to verify the relationship between anthocyanin pathway and GmbCH1 gene expression. When GmbCH1 gene was expressed under the control of globulin promoter, transgenic rice plants showed a significant increase in biomass under salt and drought stress. In addition, agronomic trait of the transgenic rice in the filed condition showed that the number of panicles was increased while plant height and the length of panicle were similar to wild type plants. We selected two promising lines of transgenic GmbCH1 colored rice, Heuknambyeo, they could provide a useful genetic resource for developing stress tolerant rice varieties.
2) A new sprout-soybean cultivar "Shingang (Suwon 242)" with high isoflavone content
Isoflavone, an important nutraceutical molecule, has a potential function of anti-cancer and anti-oxidant effects. To release sprout-soybean varieties enriched for isoflavone levels, four cultivars or breeding lines of soybeans were chosen to develop three segregating populations. These populations were field tested for yield across five locations in Korea from 2006 to 2008. "Shingang(Suwon 242)" was selected from crossing between Sowon/V94-5152, Sowon/PI96983, Sowon/L29 BC₃F₄ backcross population. This cultivar has a determinate growth trait with seed size (11.0 g/100 seeds). It has good seed quality for sprout soybean. The average yield of “Shingang” was 2.78 ton per hectare in the regional yield trials (RYT) for three years from 2006 to 2008. The isoflavone content of “Shingang” was 2,921 ㎍/g in sprout seedling.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 11
• CONTENTS ... 16
• 목차 ... 18
• 제 1 장 연구개발과제의 개요 ... 20
• 제1절 연구개발의필요성 ... 20
• 제2장 국내외기술개발현황 23 ... 23
• 제1절 콩유전체연구 ... 23
• 제2절 유용유전자발굴연구 ... 25
• 제3절 형질전환및품종개발연구 ... 25
• 제3장 연구개발수행내용및결과 ... 27
• 제 1 세부과제 : 콩 유전체 분석(서울대 이석하) ... 27
• 1. 불마름병 저항성 유전자(rxp) 염기서열 분석 ... 27
• 2.불마름병저항성유전자(rxp) ... 43
• 3. Lipoxygenase염기서열분석 ... 52
• 제 2 세부과제: 콩 유용 유전자 발굴 (숙명여대 천충일) ... 67
• 1. β-carotene hydroxylase와 동일 대사경로상의 효소 연구 ... 67
• 2.실용가능성 유전자의분리 및기능조사 ... 72
• 제 3 세부과제: 콩 유용유전자를 이용한 신기능성 작물 개발 (국립식량과학원 김율호) ... 83
• 1.이론적,실험적접근방법 ... 83
• 2.연구내용 ... 84
• 3.연구결과 ... 85
• 가. 콩 유용 유전자 형질전환을 위한 기반 구축 및 작물 형질전환 ... 85
• 나. 콩 유용유전자 bCH1 유전자 도입 형질전환 벼의 세대진전 및 고정계통 선발 ... 88
• 다. 신기능성 형질전환 작물 개발을 위한 내재해성 기능 평가 ... 89
• 라. 콩 유용유전자 도입 신기능성 형질전환 작물 개발을 위한 내병성 기능 평가 ... 96
• 마. 아이소플라본 고함유 콩 계통의 특성 평가 및 품종 출원 ... 101
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 110
• 제 5장 연구개발 성과 및 성과활용 계획 ... 111
• 제1절연구개발성과 ... 111
• 제2절성과활용계획 ... 111
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 113
• 제 1 절 GS FLX (GS 20 sequencer, 454 sequencing) ... 113
• 제 2 절콩유전학및유전체학 ... 114
• 제 3 절SOY2008학회참석및발표 ... 115
• 제 4 절 Plant Biology 2005 학회 참석 및 발표 ... 116
• 제 5 절 Plant & Animal Genome Conference 참석 및 세계 생명 공학연구 동향 정보 수집 ... 119
• 제 6 절 환경스트레스에 대한 식물반응의 분자생물학적 해석 방법 연수 ... 121
• 제 7 장 참고문헌 ... 126
• 끝페이지 ... 127