Becker T W, Caboche M, Carrayol E, Hirel B
Laboratoire de Biologie Cellulaire, INRA, Centre de Versailles, France.
Plant Mol Biol. 1992 Jun;19(3):367-79. doi: 10.1007/BF00023384.
A full-length cDNA encoding glutamine synthetase (GS) was cloned from a lambda gt10 library of tobacco leaf RNA, and the nucleotide sequence was determined. An open reading frame accounting for a primary translation product consisting of 432 amino acids has been localized on the cDNA. The calculated molecular mass of the encoded protein is 47.2 kDa. The predicted amino acid sequence of this precursor shows higher homology to GS-2 protein sequences from other species than to a leaf GS-1 polypeptide sequence, indicating that the cDNA isolated encodes the chloroplastic isoform (GS-2) of tobacco GS. The presence of C- and N-terminal extensions which are characteristic of GS-2 proteins supports this conclusion. Genomic Southern blot analysis indicated that GS-2 is encoded by a single gene in the diploid genomes of both tomato and Nicotiana sylvestris, while two GS-2 genes are very likely present in the amphidiploid tobacco genome. Western blot analysis indicated that in etiolated and in green tomato cotyledons GS-2 subunits are represented by polypeptides of similar size, while in green tomato leaves an additional GS-2 polypeptide of higher apparent molecular weight is detectable. In contrast, tobacco GS-2 is composed of subunits of identical size in all organs examined. GS-2 transcripts and GS-2 proteins could be detected at high levels in the leaves of both tobacco or tomato. Lower amounts of GS-2 mRNA were detected in stems, corolla, and roots of tomato, but not in non-green organs of tobacco. The GS-2 transcript abundance exhibited a diurnal fluctuation in tomato leaves but not in tobacco leaves. White or red light stimulated the accumulation of GS-2 transcripts and GS-2 protein in etiolated tomato cotyledons. Far-red light cancelled this stimulation. The red light response of the GS-2 gene was reduced in etiolated seedlings of the phytochrome-deficient aurea mutant of tomato. These results indicate a phytochrome-mediated light stimulation of GS-2 gene expression during greening in tomato.
从烟草叶片RNA的λgt10文库中克隆了一个编码谷氨酰胺合成酶（GS）的全长cDNA，并测定了其核苷酸序列。在该cDNA上定位了一个开放阅读框，其编码一个由432个氨基酸组成的初级翻译产物。计算出的编码蛋白质的分子量为47.2 kDa。该前体的预测氨基酸序列与其他物种的GS-2蛋白质序列的同源性高于与叶片GS-1多肽序列的同源性，表明分离得到的cDNA编码烟草GS的叶绿体同工型（GS-2）。GS-2蛋白质特有的C端和N端延伸的存在支持了这一结论。基因组Southern杂交分析表明，在番茄和林烟草的二倍体基因组中，GS-2由单个基因编码，而在双二倍体烟草基因组中很可能存在两个GS-2基因。Western杂交分析表明，在黄化和绿色番茄子叶中，GS-2亚基由大小相似的多肽代表，而在绿色番茄叶片中可检测到另一种表观分子量更高的GS-2多肽。相比之下，在所有检测的烟草器官中，GS-2由大小相同的亚基组成。在烟草或番茄的叶片中都能高水平检测到GS-2转录本和GS-2蛋白质。在番茄的茎、花冠和根中检测到较低量的GS-2 mRNA，但在烟草的非绿色器官中未检测到。GS-2转录本丰度在番茄叶片中呈现昼夜波动，但在烟草叶片中没有。白光或红光刺激黄化番茄子叶中GS-2转录本和GS-2蛋白质的积累。远红光消除了这种刺激。在番茄缺乏光敏色素的金黄色突变体的黄化幼苗中，GS-2基因的红光反应减弱。这些结果表明，在番茄绿化过程中，光敏色素介导了对GS-2基因表达的光刺激。
