Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems.
Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems.
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DOI：
10.1111/j.1365-313x.1993.00729.x
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发表时间：
1993-05
期刊：
影响因子：
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通讯作者：
C. O’neill;G. Horváth;E. Horvàth;P. Dix;P. Medgyesy
中科院分区：
文献类型：
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作者：
C. O’neill;G. Horváth;E. Horvàth;P. Dix;P. Medgyesy
利用从烟草双抗突变体中克隆的16S rRNA基因，通过聚乙二醇法直接转化白叶烟原生质体。在细胞培养中，通过对壮观霉素的抗性进行筛选，并通过未选择的链霉素抗性来鉴定可能的叶绿体转化子。或者，在两种抗生素存在的情况下选择细胞系。单独选择壮观霉素抗性的细胞系(及其再生植株)在随后的测试中显示出广泛的链霉素抗性分离，而双重选择的细胞系对两种抗生素都表现出稳定的抗性。抗性标记为母系遗传。通过对供体烟草叶绿体基因(壮观霉素抗性位点)中缺失AatII酶切位点、野生型白花烟中缺失的AatII酶切位点以及两个抗性位点核苷酸变化的序列分析，初步确定了抗性突变的来源。对其中一个转化子进行的ptDNA限制性内切酶分析和片段的重新克隆及全序列分析表明，整合过程中没有DNA重排。序列分析表明，引入的DNA片段是有针对性的同源整合，但该区域中亲本ptDNA序列出人意料的完全同源性阻碍了边界的定位。尽管叶绿体转化菌落的频率(2×10(-5))仍有待提高，但这种稳定转化叶绿体DNA的方法与基因枪法相当或更有效。
Nicotiana plumbaginifolia protoplasts were directly transformed by PEG treatment with a cloned 16S rRNA gene isolated from a double antibiotic-resistant Nicotiana tabacum plastid mutant. Putative plastid transformants were selected in cell culture by their spectinomycin resistance and identified by their unselected streptomycin resistance. Alternatively, cell lines were selected in the presence of both antibiotics. The cell line (and its regenerated plants) selected solely for spectinomycin resistance demonstrated an extensive segregation of streptomycin resistance in subsequent tests, while the double-selected line showed stable resistance for both antibiotics. The resistance markers were inherited maternally. In the putative plastid transformants the origin of the resistance mutations was identified by the absence of an AatII site, missing in the donor N. tabacum plastid gene (spectinomycin resistance site) but present in that of wild-type N. plumbaginifolia, and a sequence analysis of the particular nucleotide changes in both resistance sites. Restriction enzyme analysis of total plastid DNA (ptDNA), and the recloning and full sequencing of the fragment introduced, investigated in one of the plastid transformants, showed no DNA rearrangements accompanied with the integration process. Sequence analysis indicated a targeted, homologous integration of the DNA fragment introduced but an unexpectedly complete homology of the parental ptDNA sequences in this region prevented the location of borders. Although the frequency of plastid transformant colonies (2 x 10(-5)) should still be improved, this method for stable chloroplast DNA transformation is comparable with or more efficient than the particle bombardment techniques.