Hemon P, Robbins M P, Cullimore J V
Department of Biological Sciences, University of Warwick, Coventry, England.
Plant Mol Biol. 1990 Dec;15(6):895-904. doi: 10.1007/BF00039428.
Two transgenic tobacco lines were genetically engineered to contain chimaeric genes encoding the glutamine synthetase (GS) gamma polypeptide of Phaseolus vulgaris (French bean), expressed from the cauliflower mosaic virus 35S promoter. One (MIT-1) contained two copies of a construct including the first 60 amino acids of the Nicotiana plumbaginifolia beta-F1 ATPase to target the GS polypeptide to the mitochondrion. The other (CYT-4) contained a single copy of a cytosolic GS construct. Leaves of in vitro plantlets expressed the constructs and contained a novel GS polypeptide, which assembled into active GS isoenzymes constituting about 25% of the total GS activity. In in vitro plantlets of MIT-1, but not CYT-4, the novel polypeptide was found to be associated with the mitochondria. Moreover in MIT-1, the size of the novel polypeptide was not that predicted of the precursor (44.9 kDa) but was about 39 kDa, the same size as the authentic GS gamma polypeptide in CYT-4. These results are consistent with the precursor being imported into the mitochondria and cleaved near the fusion junction between the two sequences. These experiments have therefore shown that the presequence of the beta-F1 ATPase has successfully targeted the GS gamma polypeptide to the mitochondria of transgenic tobacco where it has assembled into an active isoenzyme. However, in fully regenerated plants growing photoautotrophically in growth-room conditions, although the constructs were still expressed, the gamma polypeptide did not accumulate to the same levels as in in vitro plantlets and new isoenzyme activities were now barely detectable. Moreover in leaves of the mature MIT-1 plants, the gamma polypeptide was found to be associated with the insoluble fraction of the mitochondria. The results of these experiments are discussed.
通过基因工程构建了两个转基因烟草株系，使其包含编码菜豆谷氨酰胺合成酶（GS）γ多肽的嵌合基因，该基因由花椰菜花叶病毒35S启动子驱动表达。其中一个株系（MIT-1）含有一个构建体的两个拷贝，该构建体包含烟草β-F1 ATP酶的前60个氨基酸，用于将GS多肽靶向线粒体。另一个株系（CYT-4）含有一个胞质GS构建体的单拷贝。离体苗的叶片表达了这些构建体，并含有一种新的GS多肽，该多肽组装成活性GS同工酶，约占总GS活性的25%。在MIT-1的离体苗中，而非CYT-4中，发现该新多肽与线粒体相关。此外，在MIT-1中，新多肽的大小并非前体预测的大小（44.9 kDa），而是约39 kDa，与CYT-4中天然GSγ多肽的大小相同。这些结果表明前体已被导入线粒体并在两个序列之间的融合连接处附近被切割。因此，这些实验表明β-F1 ATP酶的前导序列已成功将GSγ多肽靶向转基因烟草的线粒体，在那里它组装成了一种活性同工酶。然而，在生长室条件下自养生长的完全再生植株中，尽管构建体仍在表达，但γ多肽的积累水平不如在离体苗中，新的同工酶活性现在几乎检测不到。此外，在成熟的MIT-1植株的叶片中，发现γ多肽与线粒体的不溶性部分相关。讨论了这些实验的结果。
