Functional analysis of four members of the PsbP family in photosystem II in Nicotiana tabacum using differential RNA interference.
Functional analysis of four members of the PsbP family in photosystem II in Nicotiana tabacum using differential RNA interference.
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DOI：
10.1093/pcp/pci207
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发表时间：
2005-12
影响因子：
4.9
通讯作者：
Seiko Ishihara;Yumiko Yamamoto;K. Ifuku;F. Sato
中科院分区：
文献类型：
--
作者：
Seiko Ishihara;Yumiko Yamamoto;K. Ifuku;F. Sato
基因冗余常见于高等植物中，使遗传分析复杂化。本研究利用差异RNA干扰(DRNAi)技术对烟草PSBP基因家族进行了研究。PSBP是PSII的膜-外源性亚基，在裂水反应中起重要作用。烟草有四个PSBP同源基因，每个同源基因在体内的功能尚未确定。为了获得具有不同数量和组成的PSBP成员的转基因烟草植株，利用3‘-非翻译区作为沉默触发器(DRNAi)的RNA干扰(RNAi)对PSBP同源基因进行了差异沉默。此外，不含3‘-UTR的额外PSBP基因被互补地转化到上述沉默的植株中，积累了来自外源基因的PSBP，同时维持了内源靶基因的差异沉默。通过使用dRNAi和随后在dRNAi中的互补(替换)，我们清楚地证明，无论PSBP成员的积累如何，PSII活性与PSBP总量呈线性相关。因此，我们得出的结论是，在体内，烟草PSBP成员的蛋白质功能是等价的，而四个同源基因的充分表达是PSII活性最佳所必需的。这些结果表明，dRNAi的使用及其随后的互补/替代将为研究植物多基因家族的功能提供一种新的实验途径。
Gene redundancy is frequently found in higher plants and complicates genetic analysis. In this study, a method referred to as 'differential RNA interference (dRNAi)' was used to investigate the psbP gene family in Nicotiana tabacum. PsbP is a membrane-extrinsic subunit of PSII and plays important roles in the water splitting reaction. N. tabacum has four psbP isogenes and the function of each isogene has not yet been characterized in vivo. To obtain transgenic tobacco plants with various amounts and compositions of PsbP members, the psbP isogenes were differentially silenced by RNA interference (RNAi) using the 3'-untranslated region (UTR) as a silencing trigger (dRNAi). In addition, the extra psbP genes without the 3'-UTR were complementarily transformed into the above silenced plants, which accumulated PsbP originating from the exogenous gene while differential silencing of the endogenous target was maintained. By using dRNAi and subsequent complementation (substitution) in dRNAi, we clearly demonstrated that, regardless of the of PsbP members that were accumulated, PSII activity was linearly correlated with the total amount of PsbP. Therefore, we concluded that the protein functions of the PsbP members in N. tabacum are equivalent in vivo, whereas full expression of the four isogenes is required for optimum PSII activity. These results demonstrate that the use of dRNAi and subsequent complementation/substitution in dRNAi would provide a new experimental approach for studying the function of multigene families in plants.