KOREAN JOURNAL OF CROP SCIENCE (한국작물학회지)

The Korean Society of Crop Science (한국작물학회)
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Inhibition of SKTI Synthesis in Agrobacterium rhizogenes-induced Hairy Root Reduces the Number of Nodule in Soybean

Kunitz Trypsin Inhibitor 발현 억제에 의한 콩 뿌리혹 수의 감소

  • Kim, Sun-Hyung (Department of Crop Science & Biotechnology, Dankook University) ;
  • Lim, Chae-Woo (Department of Crop Science & Biotechnology, Dankook University) ;
  • Park, Ji-Young (Department of Crop Science & Biotechnology, Dankook University) ;
  • Hwang, Cheol-Ho (Department of Crop Science & Biotechnology, Dankook University)
  • 김선형 (단국대학교 식량생명공학과) ;
  • 임채우 (단국대학교 식량생명공학과) ;
  • 박지영 (단국대학교 식량생명공학과) ;
  • 황철호 (단국대학교 식량생명공학과)
  • Published : 2009.09.30
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Abstract

In nitrogen-limited conditions, rhizobia lead to formation of nitrogen-fixing nodules on the roots of leguminous plants. The process of nodulation is autoregulated by pre-existing nodules in the same root system. The altered profile of sap proteins by inoculation with B. japonicum may indicate presence of a signal responsible for autoregulation transferred through stem. The 20 kDa protein enhanced by innoculation significantly decreased in intensity from 2.5 to 7 days after inoculation (DAI). However 6 kDa protein did increase during such a transition period. Western blot analysis showed that both 20 kDa and 6 kDa were cross-reacted with the SKTI antiserum. This suggests that SKTI may be involved in soybean nodulation by specific induction and degradation in stem sap during early stage of nodulation. RNAi technique and Agrobacterium rhizogenes-mediated transformation were applied to investigate the function of SKTI in nodulation. We have found that the number of rhizobium-induced nodule was much less in SKTIi-silenced hairy roots than the non-silenced. Indeed the quantitative RT-PCR showed that the expression level of SKTI gene was reduced over 40% in the transgenic hairy roots compared to the non-transgenic. It appears that the observed early induction of SKTI and degradation into small peptide in a specific time manner may be involved in autoregulation of nodulation in soybean and the specific mechanism of such regulation remains to be investigated.

콩과식물의 뿌리혹 형성을 조절하는 신호물질의 확인을 위해 신팔달콩2호의 줄기 수액 단백질 중에서 B. japonicum USDA110의 접종 후 2.5일(DAI)에 20 kDa의 SKTI 단백질이 증가하였다가 7 DAI에는 감소되면서 6 kDa의 작은 크기의 단백질이 증가되었다. 이러한 단백질의 차등발현은 조사한 3종의 콩에서 모두 유사하게 나타났으며 특히 대원콩에서 가장 두드러졌다. Western 분석으로 7 DAI에서 증가하는 6 kDa 단백질이 SKTI 항체와 특이적 반응을 하는 것으로 확인하여 SKTI가 절단되어 생긴 펩타이드로 추정되었다. 이러한 결과를 통해 20 KDa의 SKTI단백질이 콩의 뿌리혹 착생 초기단계인 2.5 DAI에 영향을 주고, 7 DAI로 진행되면서 6 kDa의 작은 크기의 단백질로 분해되어 그 양이 감소하는 것으로 생각된다. RNAi를 이용하여 유전자 기능이 억제된 형질전환된 모상근의 뿌리혹을 실제 형질전환이 확인된 모상근에 착생된 뿌리혹의 수를 비교한 결과 비재조합 A. rhizogenes을 접종시킨 대조구에 비해 SKTI RNAi 유전자를 형질전환한 모상근에서 모상근 당 착생된 뿌리혹 수가 감소되었다. 실시간 PCR 방법으로 형질전환된 모상근의 SKTI 전사체 수준에서도 상응하는 차이를 확인하였다. 이에 정확한 기작을 알 수 없지만 SKTI유전자가 뿌리혹 형성 초기에 뿌리혹 형성과정에 직간접적으로 관련하고 있음을 확인하였다. Sesbania rostrata의 뿌리혹 발생과정의 Protease 저해제와 같이 뿌리 혹 내의 감염세포 대 비감염세포의 비율을 조절하는 SKTI 발현 억제는 이러한 균형을 교란하여 뿌리혹의 생성을 억제하는 것으로 추정된다.

Keywords

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