한국콩연구회

ORE7 유전자 도입을 통한 초다수확성 GM 콩 품종 개발

Development of GM soybean cultivar with super-yields by the introduction of ORE7

사업명 : 차세대바이오그린21

주관연구기관 : 전주생물소재연구소

발행년월 : 2015-02

주관부처 : 농촌진흥청

원문

http://www.ndsl.kr/ndsl/commons/util/ndslOriginalView.do?dbt=TRKO&cn=TRKO201500010421&rn=&url=&pageCode=PG18

초록

It has been known that ORE7 encoding AT-hook DNA-binding protein extends leaf longevity and increases the post-harvest storage life of Arabidopsis through the regulation of chromatin architecture. To elucidate the role of ORE7 on the yield increase and senescence delay of Korean soybean variety Kwangan, we have transformed with ORE7 using highly efficient soybean transformation system, and generated transgenic soybeans expressing the gene. ORE7-introduced soybeans showed phenotypic characterizations of leaf senescence delay and oxidative stress tolerance. Interestingly, the regulation of ORE7 expression conferred the trait alterations of soybean. The higher level of ORE7 transcripts induced a delayed senescence trait whereas the lower level of those induced a highly yield trait such as increases of plant size and seed production. We have studied the expression of gene families encoding for histidine kinase(HK) functioned as cytokinin receptor and histidine phosphotransfer proteins(HP) to elucidate the role of ORE7 in the cytokinin signalling of soybean. ORE7 induced proportionally the transcript increase of GmHK13, GmHK14, GmHK15, GmHK16, and GmHK17 in the HK gene family and GmHP04 in the HP gene family.

On the other hand, the higher level of ORE7 transcripts induced the reduced transcripts of GmHP10 in the HP gene family whereas the lower level of those induced the increased transcripts of GmHP10. Therefore, we suggest that ORE7 confers yield increase and/or senescence delay through the regulation of HK and HP genes in the cytokinin signalling of the soybean. In order to develop GM soybean cultivar with agricultural traits of highly-yields, we have constructed newly vector systems, pCKLSL-35SP:ORE7, pCKLSL-TPP:ORE7 and pCSENIF-ORE7, to solve the regulation of ORE7 expression level as well as the problem of intellectual property rights. we have transformed with 3 newly vector systems in Korean soybean variety Kwangan, generated 39 T0 transgenic ORE7-soybeans and harvested the transgenic T1 seeds. And we have generated 23 T2 transgenic ORE7-soybean lines through generation progress and harvested the seeds of transgenic T3 lines. We have selected potential 1 copy clean T0 transgenic soybeans through the verification of copy number of T0 transgenic lines by qRT-PCR, and then analyzed flanking DNA of inserted-vector sequences in the selected 1 copy transgenic plants. As the results, we have currently selected 3 T0 transgenic lines with intergenic region integration of 1 copy vector in the soybean genome. In progress, selected transgenic soybeans have been verified by the analyses of objective and agricultural traits for the development of GM soybean cultivar with highly-yields.

연구개발 결과

콩 형질전환용 벡터 개발: constitute promoter인 CaMV 35S promoter와 애기장대 TCTP promoter, 그리고 inducible promoter인 SEN1 promoter를 적용한 특허 회피/최소화 벡터 pCKLSL-35SP:ORE7, pCKLSL-TPP:ORE7 그리고 pCSENIF-ORE7을 개발하여 GM 콩 작물 개발에 적용 (생물정보 및 생명자원 등록).

형질전환체 생산: 상기 3가지 벡터를 이용하여 현재 T0 형질전환체 생산 형질전환체의 분자유전학적 분석: 생산된 형질전환체 중 39종의 T0 형질전환체를 대상으로 재 조합 유전자 도입, 발현 그리고 copy수를 확인한 결과, 26종이 1 copy 형질전환 라인인 것으로 판단됨.

GM 예비 계통 선발: 39종의 T0 형질전환체를 대상으로 flanking sequence를 분석한 결과, 3라인이 1 copy 재조합 DNA 삽입 그리고 재조합 DNA의 삽입이 콩 유전체의 intergenic region에 이루어 진 것으로 나타났으며, 이를 우리는 GM 예비 계통으로 선별하여 추후 포장에서 다수성 농업형질 분석 및 세대 진전을 거쳐 우수 계통(이벤트)으로 육성하고자 함