植物的雄不稔常存在於自然界當中，其表現型通常為孢子發生時之異常，而無法產生花粉、產生花粉卻失去功能或花器結構改變等。而雄不稔性可大致分為可遺傳性和不具遺傳性。而遺傳性的雄不稔可依據雄不稔基因位置，將其分類為細胞核基因調控、細胞質基因調控或兩者互相調控。NADH脫氫酶是粒線體電子傳遞鏈中的第一個複合體 ( respiratory complex 1 ) 在植物是由至少49個蛋白質單元組成，其中有9個基因包含Nad1為粒線體編碼基因。
近年，TALEN基因編輯技術被用於植物的粒線體基因進行編輯。本研究使用TALEN技術編輯粒線體Nad1基因，藉由構築一個細胞核TALEN基因表現載體pTA29-Talen，再由農桿菌送至菸草的細胞核表現，再經由粒線體導引訊號 (Tm)引導至粒線體胞質內，進而辨識並編輯Nad1基因。藉由kanamycin篩選得到轉殖pTA29-TALEN至少43個菸草轉殖品系。
初步以解剖顯微鏡進行觀察，發現04號轉殖品系之花粉粒數量明顯低於未轉殖菸草植株。進一步使用光學顯微鏡觀測經I2-KI和TTC染色的花粉，評估各品系之花粉粒染色程度，依照染色深淺程度來判別各品系之花粉活力程度。結果顯示，在01、02、31轉殖品系並無發現花粉活力低下的表現，但在04轉殖品系發現花粉活力遠低於未轉殖菸草之花粉活力，推測大部分花粉已失去活性。此外，於花粉萌發實驗中比較各品系花粉管之生長情形，結果顯示01、02、31轉殖品系之花粉管長度和未轉殖菸草長度相似，但在04轉殖品系之花粉管長度遠低於未轉殖菸草長度，顯示其萌發能力亦受影響。綜合以上結果，推測 04 號轉殖品系中絨氈層細胞粒線體 Nad1 基因被 mitoTALEN 編輯後，導致線粒體功能異常進而引發細胞質雄性不稔（cytoplasmic male sterility, CMS）之表型。

Plant mitochondria play an important role in cellular biogenesis, such as respiration. The organelle and their genomes in angiosperms are typically inherited from maternal ancestors. Mitochondrial genomes contain around 57 genes, mainly encoding the protein subunits associated with translation and respiration processes. The mitochondrial NADH dehydrogenase complex (or respiratory complex I) consists of at least 49 protein subunits. Among them, nine subunits, including Nad1, are encoded by mitochondrial DNA (mtDNA) in plants. Currently, technologies for directly manipulating the mtDNA of land plants are unavailable. Alternatively, transcription activator-like effector (TALE) based technologies that could introduce changes into the mtDNA of land plants via Agrobacteria-mediated nuclear transformation have been recently explored in breeding new traits for both basic and applied research in agriculture. Cytoplasmic male sterility (CMS), arising from nuclear and mitochondrial genome incompatibility, is a factor contributing to the infertility of certain crops due to the failure of pollen germination or dehiscence in the anther. The CMS traits could facilitate the production of hybrid plants, which have great potential in breeding advantageous traits referred to as hybrid vigor in crops. In this study, mitochondria-targeted transcription activator-like effector nuclease (mitoTALEN) was applied to edit the Nad1 gene in tapetal cells of tobacco. The functional loss of tapetal cells is expected to cause the CMS phenotype. Previously, a nuclear expression vector in which TALENs driven by a tapetal cell-specific promoter TA29 was constructed and used to transform tobacco plants via the Agrobacterium-mediated method. Several transgenic events with the integration of TALEN gene into the nuclear genome were obtained. The abnormal development of anther, the significant reduction of pollen viability and germination, and the decrease in seed set were observed in at least one transgenic event, which suggested mitoTALEN-mediated disruption of the mitochondrial Nad1 gene in tapetal cells might cause CMS in tobacco plants.

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