Inducible trans-activation of plastid transgenes:: Expression of the R. eutropha phb operon in transplastomic tobacco
Inducible trans-activation of plastid transgenes:: Expression of the R. eutropha phb operon in transplastomic tobacco
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DOI：
10.1093/pcp/pci157
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发表时间：
2005-09-01
影响因子：
4.9
通讯作者：
Koop, HU
中科院分区：
文献类型：
--
作者：
Lössl, A;Bohmert, K;Koop, HU
如果基因产物对转基因植物有害，就可能发生结构性转基因表达的有害影响。在含有PHB操纵子的叶绿体转化体中观察到了生长抑制和雄性不育等限制因素，这些操纵子编码了生产聚酯多羟基丁酸(PHB)所需的基因。为了适时地诱导烟草中PHB的合成，我们构建了一个反式激活系统来调控叶绿体中PHB操纵子的转录。该系统由一个定位于核的乙醇诱导的T7RNA聚合酶(T7RNAP)组成，该聚合酶在T7调控元件的控制下靶向于含有PHB操纵子的质体。用5%乙醇处理后，可适度诱导PHB的合成。PHB的含量达到了1383ppm的干重，在未诱导的组织中测得总的本底活性为171ppm。在转录水平上，证明了T7RNAP的诱导，我们发现PHB操纵子至少转录成两个mRNAs。没有乙醇诱导，花和可育种子的发育是可能的。从而解决了抑制转基因表达的主要问题。我们的结果表明，这个可诱导的反式激活系统可以作为外源基因在质体内结构性表达的替代方案。
Deleterious effects of constitutive transgene expression can occur if gene products are harmful to the transformed plant. Constraints such as growth inhibition and male sterility have been observed in plastid transformants containing the phb operon encoding the genes required for the production of the polyester polyhydroxybutyric acid (PHB). In order to induce PHB synthesis in tobacco in a well-timed manner, we have constructed a trans-activation system to regulate transcription of the phb operon in plastids. This system consists of a nuclear-located, ethanol-inducible T7RNA polymerase (T7RNAP) which is targeted to plastids harboring the phb operon under control of T7 regulatory elements. Following treatment with 5% ethanol, moderate induction of PHB synthesis was found. PHB amounts reached 1,383 ppm in dry weight, and an overall background activity of 171 ppm was measured in uninduced tissues. On the transcriptional level, T7RNAP induction was proven and we found that the phb operon is transcribed into at least two mRNAs. Without ethanol induction, development of flowers and fertile seeds was possible. Thus, the main problem of inhibitory transgene expression was solved. Our results show that this inducible trans-activation system could serve as an alternative to constitutive expression of transgenes in the plastome.