Koop H U, Steinmüller K, Wagner H, Rössler C, Eibl C, Sacher L
Botanisches Institut der Universität München, Germany.
Planta. 1996;199(2):193-201. doi: 10.1007/BF00196559.
A new vector, pFaadAII, for transformation of plastids of Nicotiana tabacum L. has been developed. It harbours a chimeric gene consisting of the aadA coding region from Escherichia coli, the 16S rDNA promoter from tobacco combined with a synthetic ribosome-binding site, a 500-bp fragment containing the 3' untranslated transcript region (UTR) of the Chlamydomonas rbcL gene and 3.75-kb (5') and 0.95-kb (3') tobacco plastome sequences allowing for targeting the foreign sequences to the intergenic region between the rpl32 and trnL genes of the tobacco plastome. The vector thus targets foreign sequences to the small single-copy region of the plastome, which has so far not been modified by transformation. Leaf protoplasts of Nicotiana tabacum L. were treated with polyethylene glycol (PEG) in the presence of the vector. The protocol for PEG treatment aiming at plastome transformation was optimized. Cell lines were cultured in the presence of spectinomycin and streptomycin using a novel and efficient protoplast culture and selection system. Regenerants were characterized by polymerase chain reaction (PCR) analysis, Southern hybridization and reciprocal crossing. The transformation procedure is described in detail and parameters influencing its efficiency are presented. Special effort is placed on analyzing suitable selection conditions. Only a proportion of the cell lines with a resistant phenotype could be confirmed by molecular analysis and/or reciprocal crossings to represent plastome transformants. Integration of the plastome specific aadA cassette into the nuclear genome accounted for a fraction of the resistant cell lines. Still, as many as 20-40 plastome transformants can be expected from the treatment of 10(6) protoplasts. Therefore, the improved protocol for PEG-mediated plastome transformation in combination with the new aadA-vector supplies a simple, reproducible and cost-efficient alternative to the biolistic procedure.
已开发出一种用于转化烟草叶绿体的新型载体pFaadAII。它含有一个嵌合基因，该嵌合基因由来自大肠杆菌的aadA编码区、来自烟草的16S rDNA启动子与一个合成核糖体结合位点、一个包含衣藻rbcL基因3'非翻译转录区（UTR）的500 bp片段以及3.75 kb（5'）和0.95 kb（3'）烟草叶绿体基因组序列组成，这些序列可将外源序列靶向到烟草叶绿体基因组中rpl32和trnL基因之间的基因间隔区。因此，该载体将外源序列靶向到叶绿体基因组的小单拷贝区域，该区域迄今为止尚未通过转化进行修饰。在载体存在的情况下，用聚乙二醇（PEG）处理烟草叶原生质体。优化了旨在进行叶绿体基因组转化的PEG处理方案。使用一种新颖且高效的原生质体培养和选择系统，在壮观霉素和链霉素存在下培养细胞系。通过聚合酶链反应（PCR）分析、Southern杂交和正反交对再生植株进行鉴定。详细描述了转化过程，并给出了影响其效率的参数。特别致力于分析合适的选择条件。只有一部分具有抗性表型的细胞系能够通过分子分析和/或正反交被确认为叶绿体基因组转化体。叶绿体基因组特异性aadA盒整合到核基因组中占了一部分抗性细胞系。尽管如此，用10⁶个原生质体进行处理，预计仍可获得多达20 - 40个叶绿体基因组转化体。因此，改进后的PEG介导的叶绿体基因组转化方案与新的aadA载体相结合，为基因枪轰击法提供了一种简单、可重复且经济高效的替代方法。
