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甘藍葉綠體基因轉殖載體開發之建立

By 劉程煒, 曾夢蛟, Cheng-Wei Liu and Menq-Jiau Tseng

Abstract

葉綠體的基因屬母系遺傳,不會隨著花粉散播,且植物細胞含多套的葉綠體基因組,有助於外來基因大量表現,因此開發葉綠體基因轉殖技術將可避免基因轉殖作物造成的基因汙染及破壞生物平街。本研究利用PCR的方式,大量增幅並分離出初秋'甘藍葉綠體基因粗中invert repeat (IR)區域中trnV到rrn23S約4.lkb的核酸序列片段,經核酸定存與NCBI核酸資料庫比對之結果顯示所分離片段之基因序列與已發表的阿拉伯芥等葉綠體基因組序列有99%的相似性,包括有IR中完整的trnV、16S ribosomal RNA、trnl、trnA以及部分 23S ribosomal RNA的基因序列片段。將此基因片段均分為二個部分,同時構築到pBluScript II(SK-)載體上成為蕓苔屬葉綠體之通用轉殖載體(universal vector)─pASCC2O1。此載體帶有蕓苔屬葉綠體之lRA區域之trnV-rrn16S(左)及trnI-trnA-rrn23S(右)核酸序列,作為葉綠體基因轉移之重組位置,並含有以prrn為啟動子的aadA基因作為抗生素篩選基因。Expression of foreign genes via plastid genomes not only dramatically enhances the level of expression(5,000-10,000 copies of prokaryotic chloroplasts per plant cell), but also prevents out cross of the introduced foreign genes via pollen grains (their maternal inheritance in most crops). The objectives of the current research are to isolate plastid gene sequences from cabbage(Brassica oleracea L. var. capitata L.), and to construct a universal transplastomic vector for the gene transformation of Brassica vegetables. A 4.1 kb of DNA fragment of cabbage chloroplast between trnV and rrn 23S of invert repeat (IR) was amplified with PCR. This fragment contains trnV, rrn 16S, trnI, trnA, and a part of rrn23S. A universal transformation vector (pASC201) for Brassica chloroplast was constructed with trnV-rrn16S (left) and trnI-trnA-rrn 23S(right) of IRA rergion as recombination site for the insertion of transgene, and a chimeric aadA marker gene (spectinomycin resistance) was also inserted between rrn16S and rrn23S plastid gene sequences

Topics: Cabbage, Chloroplast transformation, GUS gene, 甘藍, 葉綠體基因轉移, GUS基因
Year: 2014
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/81762
Journal:

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