Department of Molecular Biology and Microbiology, College of Medicine, University of Central Florida, Orlando, Florida 32816-2364, USA.
Plant Physiol. 2010 Apr;152(4):2088-104. doi: 10.1104/pp.109.152017. Epub 2010 Feb 3.
Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5' untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5' UTR and 3' UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5' UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5' UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.
异源调控元件和侧翼序列已被用于几种作物的叶绿体转化，但它们的作用和机制尚未得到研究。在所有分类单元中，光系统 II 蛋白 D1（psbA）上游区域的核苷酸序列同一性为 59％；在所有研究的基因和分类单元中都存在类似的变化。不同家族之间 psbA 5'非翻译区（UTR）的二级结构和预测 Gibbs 自由能值反映了这种变化。因此，为烟草（Nicotiana tabacum）和生菜（Lactuca sativa）制作了叶绿体转化载体，具有内源性（Nt-Nt、Ls-Ls）或异源（Nt-Ls、Ls-Nt）psbA 启动子、5'UTR 和 3'UTR，调节炭疽保护性抗原（PA）或与人胰岛素原（Pins）融合的霍乱毒素 B 亚基（CTB）的表达。在转基因烟草基因组中的同源重组过程中，独特的生菜侧翼序列完全被消除，但独特的烟草序列没有被消除。与内源性 psbA 调控元件相比，Nt-Ls 或 Ls-Nt 转基因株系中 PA 和 97%的 CTB-Pins 的表达减少了 80%，而内源性 psbA 调控元件的 PA 和 CTB-Pins 的积累量高达 29.6%总可溶性蛋白和 72.0%总叶蛋白，比 Rubisco 高 2 倍。Ls-Nt-CTB-Pins 中的转基因转录物减少了 84%，Nt-Ls-PA 株系中的转录物减少了 72%。含有内源性 5'UTR 的转录物在非多核糖体部分稳定。基质 RNA 结合蛋白优先与内源性 psbA 5'UTR 结合。通过优化植物生长调节剂，为生菜商业品种开发了一种快速且可重复的再生系统。这些发现强调了测序完整作物叶绿体基因组、利用内源性调控元件和侧翼序列以及优化植物生长调节剂以实现有效叶绿体转化的必要性。
