植物葉綠體轉殖為現今植物基因轉殖的重要方法，目前最常使用的轉殖方法為基因槍轟擊法，將基因轉入植物葉綠體的基因中，再經由植株的篩選以及再生培養，來獲得轉基因植株。而利用基因槍轟擊法的轉殖成功率遠低於核轉殖法，因此本研究嘗試利用Transcription activator-like effector nuclease(TALEN)技術來改善葉綠體基因轉殖之轉殖效率。利用 XVE 誘導表現系統將含有DNA 結合區LexA(X)、轉錄活化區位 VP16(V)以及 β-estradiol 接受器的調節區(E)(XVE system)之誘導載體與 TALEN 基因重組構築成為植物細胞核表現載體。轉殖植物表現出重組 TALEN 蛋白可經由位於 N 端的 Transit peptide(TP)將 TALEN 蛋白攜帶至葉綠體中，結合至目標基因的辨識位，而造成特定位點的雙股 DNA 斷裂。利用植物細胞中葉綠體的 DNA 的自然修復機制如：同源重組(Homologous recombination)、非同源末端連接(Nonhomologous end joining)，可能造成葉綠體 DNA 突變，另外，再額外提供 Donor DNA 的狀態下，可以促進同源重組提升葉綠體轉殖的效率。本研究已建立菸草重組 TALEN 基因的 XVE 誘導表現系統，並嘗試將外源 DNA 轟擊進入誘導的葉片組織，以促進同源重組過程，並進而促進葉綠體重組效率，但尚未得到明確之結果。

To edit the plastid DNA, chloroplast transformation by biolistic bombardment is the most common method that used to deliver expression vectors into plastid. Subsequently, the transplastomic plant could be obtainedunder long selection and regeneration process. However, biolistic bombardment selection and regeneration process is time consuming. Recently, the transcription activator like effector nuclease (TALENs) technology has been widely applied to edit nuclear genome in plants. In this study, we try to apply the inducible TALEN expression technology to site-specifically edit the plastid DNA. A pair of nuclear TALENs expression vectors carrying either hptII or nptII gene as selection marker were constructed. The recombinant TALEN were designed to recognize the specific nucleotides in rbcL-accD intergenic spacer. In addition, the synthetic TALEN genes were fused with the transit peptide sequence, and were driven by chemical-inducible promoter. The expressed recombinant TALEN proteins induced by chemicals were expected to target to chloroplasts through TOC-TIC translocons. Subsequently, the recombinant TALEN proteins would bind to the specific target sites, and cause cpDNA double-strand break. Agrobacteria-mediated transformation was carried out to generate transgenic tobacco. Transgenic tobacco lines that are resistant to both hygromycin and kanamycin have been obtained. Transgene integration has been confirmed by PCR analysis. Western detection of recombinant TALEN in transgenic plant is still on-going. Phenotypic change such as chlorosis was observed in some transgenic lines under chemical induction. In addition, taking advantage of inducible TALEN expression system in transgenic plants, an effort to increase the chloroplast transformation efficiency by providing donor vector through bombardment is on-going.

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Chemical-Inducible Gene Expression Systems for
Plants—a Review
¤
Ian Jepson,* Alberto Martinez & Justin P. Sweetman
Chemical-Inducible Gene Expression Systems for
Plants—a Review
¤
Ian Jepson,* Alberto Martinez & Justin P. Sweetman
Chemical-Inducible Gene Expression Systems for
Plants—a Review
¤
Ian Jepson,* Alberto Martinez & Justin P. Sweetman
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