葉綠體基因轉殖大多是利用基因槍轟擊（Bombardment）的方式將外源基因導入植物的葉綠體中，接著由同源重組的方式準確地將外源基因插入植物綠體 DNA 中。但是要獲得葉綠體基因轉殖植株需要花費較多的時間來進行篩選及再生，且葉綠體基因轉殖的成功率並不高。近年來，基因編輯技術-轉錄似活化因子蛋白核酸酶（TALEN）已被廣泛應用於植物細胞核的編輯上。在本實驗室先前的研究中，已構築分別由 rbcS 啟動子和 β-estradiol 誘導的 TALEN 細胞核表現載體，並利用農桿菌感染的方式將該載體送入菸草的細胞核中，來對葉綠體 rpoB 基因和 rbcL-accD 之間進行編輯。而由於 TALEN 的作用，會對葉綠體的 DNA 造成雙股斷裂（Double strand break），進而增進葉綠體 DNA 的同源重組機制。因此，為了提高葉綠體基因轉殖效率，在本研究中，我們分別使用 rbcS 啟動子和 β-estradiol 誘導的 TALEN 細胞核表現轉殖植株作為宿主，其產生的重組 TALEN 蛋白可進入菸草葉綠體 DNA進行作用，同時利用基因槍轟擊的方式分別將pRpoB 或 pCpNluc06 葉綠體表現載體導入葉片中，期望能提升葉綠體基因轉殖之效率。將轟擊後的組織，使用抗生素進行篩選及再生後，已經獲得5個可能帶有外源基因的轉殖植株，目前正在進行分析。

Chloroplast transformation via biolistic bombardment is the most commonly used method to deliver expression vectors into plastid, and subsequently the transgene could integrate into plastid genome by homologous recombination. Under time-consuming process of selection and regeneration, then transplastomic plants could be generated. However, the efficiency of plastid transformation is very low. Recently, the transcription activator like effector nuclease (TALENs) technology has been widely applied to edit nuclear genome of plants for various biotechnological applications. In the previous study of this Lab., both leaf-specific and β-estradiol-inducible nuclear TALEN expression vectors have been constructed and transformed into the nuclear genomes of tobacco to edit the rpoB gene in chloroplasts. Transgenic plants conferred various degrees of phenotypic effects; because of the integrity of chloroplast DNA might be damaged. To improve the chloroplast transformation efficiency by taking advantage of recombinant TALEN protein which can cause the double strand break (DSB) of tobacco chloroplast DNA, in the present study, we used the transgenic tobacco plants which the nuclear TALEN gene expression is leaf-specific or β-estradiol-inducible as hosts, and then deliver the pRpoB or pCpNluc06 chloroplast expression vectors as donor DNA into leaf tissues via biolistic bombardment, respectively, to facilitate homologous recombination. Subsequently, under the process of antibiotic selection and regeneration, five putative transplastomic plants were generated. Molecular analysis of putative transplastomic plants is currently on-going.

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