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Construction of a tobacco master line to improve Rubisco engineering in chloroplasts

机译：建造烟草主生产线以改善Rubisco叶绿体工程

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摘要

The inability to assemble Rubisco from any photosynthetic eukaryote within Escherichia coli has hampered structure–function studies of higher plant Rubisco. Precise genetic manipulation of the tobacco chloroplast genome (plastome) by homologous recombination has facilitated the successful production of transplastomic lines that have either mutated the Rubisco large subunit (L) gene, rbcL, or replaced it with foreign variants. Here the capacity of a new tobacco transplastomic line, cmtrL, to augment future Rubisco engineering studies is demonstrated. Initially the rbcL was replaced with the selectable marker gene, aadA, and an artificial codon-modified cmrbcM gene that codes for the structurally novel Rubisco dimer (L2, ∼100 kDa) from Rhodosprillum rubrum. To obtain cmtrL, the aadA was excised by transiently introducing a T-DNA encoding CRE recombinase biolistically. Selection using aadA enabled transplantation of mutated and wild-type tobacco Rubisco genes into the cmtrL plastome with an efficiency that was 3- to 10-fold higher than comparable transformations into wild-type tobacco. Transformants producing the re-introduced form I tobacco Rubisco variants (hexadecamers comprising eight L and eight small subunits, ∼520 kDa) were identified by non-denaturing PAGE with fully segregated homoplasmic lines (where no L2 Rubisco was produced) obtained within 6–9 weeks after transformation which enabled their Rubisco kinetics to be quickly examined. Here the usefulness of cmtrL in more readily examining the production, folding, and assembly capabilities of both mutated tobacco and foreign form I Rubisco subunits in tobacco plastids is discussed, and the feasibility of quickly assessing the kinetic properties of those that functionally assemble is demonstrated.
机译：无法从大肠杆菌中的任何光合作用真核生物中组装Rubisco，阻碍了高等植物Rubisco的结构功能研究。通过同源重组对烟草叶绿体基因组（质体组）进行精确的遗传操作，已经成功地生产了使突变Rubisco大亚基（L）基因rbcL或被外源变体替代的转基因组。在此证明了一种新的烟草转基因系 cm trL可以增强今后的Rubisco工程研究的能力。最初，rbcL被选择标记基因aadA和人工密码子修饰的 cm rbcM基因替换，该基因编码结构上新颖的Rubisco二聚体（L 2 ，〜100 kDa）（来自红景天）。为了获得 cm trL，通过瞬时导入生物体枪法瞬时导入编码CRE重组酶的T-DNA来切除aadA。使用aadA进行选择可以将突变的野生型烟草Rubisco基因移植到 cm trL质体组中，其效率比向野生型烟草的转化高3到10倍。通过完全变性同质系的非变性PAGE（其中没有L 2 Rubisco）进行非变性PAGE，鉴定出产生了重新引入的I型烟草Rubisco变体（包含八个L和八个小亚基的六聚体，约520 kDa）的转化体。转化后的6-9周内获得），从而可以快速检查其Rubisco动力学。这里讨论了 cm trL在更容易地检查突变的烟草和烟草质体中异型I Rubisco亚基的生产，折叠和组装能力方面的实用性，以及快速评估动力学特性的可行性。在功能上进行了组装。

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    《Journal of Experimental Botany》 |2008年第7期|p.1909-1921|共13页
  • 作者

    Spencer M. Whitney* and Robert E. Sharwood;

  • 作者单位

    Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 0200, Australia;

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