식물에서 표적 유전자의 동시 과발현, 조직/발달 특이적 발현 및 스트레스 유도성 발현을 위한 binary 벡터의 제작과 분석 Construction and Analysis of Binary Vectors for Co-Overexpression, Tissue- or Development-Specific Expression and Stress-Inducible Expression in Plant원문보기
유전자를 이소성으로 발현하고 억제하는 것은 유전자의 기능 연구에 있어서 매우 유용하다. 본 연구에서는 표적 유전자의 동시 과발현, 조직/발달 단계 특이적 발현 및 스트레스 유도성 발현을 위해 pPZP를 골격으로 다양한 binary벡터를 제작하고 그 유용성을 검증하였다. 변형된 CaMV 35S 프로모터를 이용하여, 다른 두 개의 유전자를 동시 과발현시키는 binary 벡터를 제작하였고, 이 벡터가 동시에 그리고 같은 장소에서 다른 두 개의 표적 유전자를 과발현 하는데 효과적임을 확인하였다. At2S3, KNAT1 및 LFY 프로모터를 포함하는 조직 또는 발달 단계 특이적 발현 binary 벡터들을 제작하고 분석한 결과, 이 벡터들은 각각 배/유식물 시기, 새싹 끝의 분열조직 및 잎 원기 특이적 발현에 유용하였다. RD29A와 AtNCED3 프로모터를 포함하는 스트레스 유도성 발현 binary 벡터들은 고염, ABA, MV 또는 저온과 같은 비생물성 스트레스에 의한 유전자의 이소성 발현에 유용하였다. 본 연구에서 제작된 binary 벡터들은 표적 유전자의 이소성 발현을 통해 유전자의 생물학적 기능연구, 분자생물학적작용 기작 연구에 유용하게 사용될 것으로 사료된다.
유전자를 이소성으로 발현하고 억제하는 것은 유전자의 기능 연구에 있어서 매우 유용하다. 본 연구에서는 표적 유전자의 동시 과발현, 조직/발달 단계 특이적 발현 및 스트레스 유도성 발현을 위해 pPZP를 골격으로 다양한 binary 벡터를 제작하고 그 유용성을 검증하였다. 변형된 CaMV 35S 프로모터를 이용하여, 다른 두 개의 유전자를 동시 과발현시키는 binary 벡터를 제작하였고, 이 벡터가 동시에 그리고 같은 장소에서 다른 두 개의 표적 유전자를 과발현 하는데 효과적임을 확인하였다. At2S3, KNAT1 및 LFY 프로모터를 포함하는 조직 또는 발달 단계 특이적 발현 binary 벡터들을 제작하고 분석한 결과, 이 벡터들은 각각 배/유식물 시기, 새싹 끝의 분열조직 및 잎 원기 특이적 발현에 유용하였다. RD29A와 AtNCED3 프로모터를 포함하는 스트레스 유도성 발현 binary 벡터들은 고염, ABA, MV 또는 저온과 같은 비생물성 스트레스에 의한 유전자의 이소성 발현에 유용하였다. 본 연구에서 제작된 binary 벡터들은 표적 유전자의 이소성 발현을 통해 유전자의 생물학적 기능연구, 분자생물학적작용 기작 연구에 유용하게 사용될 것으로 사료된다.
In this study, we constructed various kinds of binary vectors with the pPZP backbone for co-overexpression, tissue- or development-specific expression and stress-inducible expression, and validated them for ectopic expression of target genes. Using a modified CaMV 35S promoter, a binary vector was g...
In this study, we constructed various kinds of binary vectors with the pPZP backbone for co-overexpression, tissue- or development-specific expression and stress-inducible expression, and validated them for ectopic expression of target genes. Using a modified CaMV 35S promoter, a binary vector was generated for co-overexpression of two different genes and was confirmed to be efficient for overexpressing two different target genes at the same time and place. Binary vectors containing At2S3, KNAT1 or LFY promoters were constructed for tissue-specific or development-specific gene expression, and the binary vectors were suited for embryo/young seedling stage-, shoot apical meristem- or leaf primordia-specific expressions. Furthermore, the binary vectors containing RD29A or AtNCED3 promoters were validated as suitable vectors for gene expression induced by abiotic stresses such as high salt, ABA, MV and low temperature. Taken together, the binary vectors constructed in this study would be very useful for analyzing the biological functions of target genes and molecular mechanisms through ectopic expression.
In this study, we constructed various kinds of binary vectors with the pPZP backbone for co-overexpression, tissue- or development-specific expression and stress-inducible expression, and validated them for ectopic expression of target genes. Using a modified CaMV 35S promoter, a binary vector was generated for co-overexpression of two different genes and was confirmed to be efficient for overexpressing two different target genes at the same time and place. Binary vectors containing At2S3, KNAT1 or LFY promoters were constructed for tissue-specific or development-specific gene expression, and the binary vectors were suited for embryo/young seedling stage-, shoot apical meristem- or leaf primordia-specific expressions. Furthermore, the binary vectors containing RD29A or AtNCED3 promoters were validated as suitable vectors for gene expression induced by abiotic stresses such as high salt, ABA, MV and low temperature. Taken together, the binary vectors constructed in this study would be very useful for analyzing the biological functions of target genes and molecular mechanisms through ectopic expression.
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제안 방법
In this study, various binary vectors with pPZP backbone were constructed and tested for ectopic expression of target genes. For ectopic expression, binary vectors were generated for co-overexpression, tissue-specific, development-specific and stress-inducible expression.
1. Schematic maps of binary vectors constructed in this study. (A) T-DNA regions of pFGL1217, pFGL1092, pFGL1093, pFGL1167, pFGL722 and pFGL1213.
1A). To confirm that the construct is able to overexpress two different target genes properly, RFP and sGFP reporter genes were expressed under the control of each modified CaMV 35S promoter (Fig. 1B). Using the RFP and sGFP signal, it was confirmed that the RFP and sGFP were overexpressed in the cytoplasm and the nucleus of the Arabidopsis protoplasts containing modified CaMV 35S::RFP:modified CaMV 35S::sGFP (Fig.
대상 데이터
Each of the three promoters was cloned into pFGL75 (pPZP211-Tnos). Binary vectors containing At2S3, KNAT1 and LFY promoters were named as pFGL1092, pFGL1093 and pFGL1167, respectively (Fig. 1A). In order to confirm that the constructs were able to express target genes properly in plants, GUS reporter gene was expressed under the control of each promoter (Fig.
For the tissue-specific or development-specific expression, the promoters of Arabidopsis thaliana 2S ALBUMIN 3 (At2S3), KNOTTED-LIKE FROM ARABIDOPSIS THALIANA 1 (KNAT1) and LEAFY (LFY) were used. At2S3 is expressed in the early developmental stage [10,24].
A modified CaMV 35S promoter, combination of domain B and minimal promoter, was used for the co-overexpression of target genes in this study [18]. To overexpress two different genes concomitantly, a binary vector containing two modified CaMV 35S promoters was constructed and named as pFGL1217 (Fig. 1A). To confirm that the construct is able to overexpress two different target genes properly, RFP and sGFP reporter genes were expressed under the control of each modified CaMV 35S promoter (Fig.
성능/효과
In this study, co-overexpression, tissue-/development-specific expression and stress-inducible expression binary vectors were generated for the ectopic expression of target genes.
Our results showed that the magnitude of stress-inducibility of each vector was different, suggesting that each vector can be used to express target genes differently depending on the level of expression; binary vector with RD29A promoter showed strong induction but AtNCED3 promoter was moderate. Taken together, our binary vectors for stress-inducible expression may be suitable to engineer stress-tolerant transgenic plants by genetic modification.
2). These results suggest that the method of generating transgenic plants overexpressing two different genes using this vector might reduce cost and time compared with existing methods such as cross-fertilization and two transformations which are difficult to select the transgenic plants that overexpress both genes.
4B and 4C). Using GUS assay and RT-PCR analysis, it was confirmed that GUS expression was induced by NaCl, ABA and MV treatments in the cotyledons of the AtNCED3::GUS plants (Fig. 5A and 5B).
1B). Using the RFP and sGFP signal, it was confirmed that the RFP and sGFP were overexpressed in the cytoplasm and the nucleus of the Arabidopsis protoplasts containing modified CaMV 35S::RFP:modified CaMV 35S::sGFP (Fig. 2).
후속연구
For ectopic expression, binary vectors were generated for co-overexpression, tissue-specific, development-specific and stress-inducible expression. Our results indicate that binary vectors for ectopic expression constructed in this study would be beneficial to investigate the biological functions of target genes.
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