Kanevski I, Maliga P
Waksman Institute, Rutgers, State University of New Jersey, Piscataway 08855-0759.
Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1969-73. doi: 10.1073/pnas.91.5.1969.
The conserved plastid localization of rbcL suggests that biosynthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase [Rubisco; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] in chloroplasts is required to obtain functional enzyme. To examine the validity of this hypothesis, we relocated the plastid rbcL gene to the nucleus. First, we deleted the rbcL gene from the tobacco plastid genome by targeted insertion of a selectable aadA gene encoding spectinomycin resistance. The rbcL coding region was then inserted into an expression cassette and introduced into the nuclear genome of these plants by Agrobacterium-mediated transformation. We report that the nuclear rbcL functionally complements the defective plastids when the Rubisco large subunit is targeted to chloroplasts by a transit peptide. Therefore, the evolutionary process that relocates functional plastid genes to the nucleus has not yet occurred in the case of the rbcL gene. Targeted deletion of plastid genes, combined with their allotopic expression, will provide opportunities for studying the function of plastid enzyme complexes.
rbcL保守的质体定位表明，叶绿体中1,5-二磷酸核酮糖羧化酶[Rubisco；3-磷酸-D-甘油酸羧化裂解酶（二聚化），EC 4.1.1.39]大亚基的生物合成是获得功能性酶所必需的。为了检验这一假设的正确性，我们将质体rbcL基因转移到细胞核中。首先，通过靶向插入编码壮观霉素抗性的可选aadA基因，从烟草质体基因组中删除rbcL基因。然后将rbcL编码区插入表达盒，并通过农杆菌介导的转化引入这些植物的核基因组。我们报道，当Rubisco大亚基通过转运肽靶向叶绿体时，核rbcL在功能上补充了有缺陷的质体。因此，就rbcL基因而言，将功能性质体基因转移到细胞核的进化过程尚未发生。质体基因的靶向缺失及其异位表达，将为研究质体酶复合物的功能提供机会。
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