Localization of tobacco cytosolic glutamine synthetase enzymes and the corresponding transcripts shows organ- and cell-specific patterns of protein synthesis and gene expression
Localization of tobacco cytosolic glutamine synthetase enzymes and the corresponding transcripts shows organ- and cell-specific patterns of protein synthesis and gene expression
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DOI：
10.1007/bf00019468
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发表时间：
1996-07-01
影响因子：
5.1
通讯作者：
Hirel, B
中科院分区：
文献类型：
--
作者：
Dubois, F;Brugiere, N;Hirel, B
用免疫细胞化学和原位杂交技术研究了谷氨酰胺合成酶在烟草中的亚细胞定位和两个编码胞浆酶基因的差异表达。从烟草幼苗cDNA文库中分离到两个编码胞质GS的全长cDNA克隆(Gln1-3和Gln1-5)。这两个克隆的编码区有很强的同源性，而3‘-和5’-未翻译的序列不同。为了确定转录水平，在核糖核酸酶保护实验中使用了Gln1-3和Gln1-5的特定序列。本实验清楚地表明，编码Gln1-3的基因在根和花中表达，而编码Gln1-5的基因在茎中高水平转录，在根和花中低水平转录。用免疫金标记法研究了谷氨酰胺合成酶在烟草营养器官和生殖器官中的亚细胞和细胞分布。在成熟的叶片组织或花瓣和萼片中，仅在叶绿体和叶绿体的基质中可见可塑性GS。在叶和花等营养器官或生殖器官中检测到胞质GS。在叶片中，胞质GS优先定位于维管组织。利用烟草器官切片和编码Gln1-3和Gln1-5基因的特异性反义RNA探针进行原位杂交。Gln1-5转录本定位于茎和根的维管组织中，而Gln1-3转录本存在于所有根细胞和花器官中，包括花瓣、萼片和花药。
The subcellular localization of glutamine synthetase in tobacco and the differential expression of two genes encoding cytosolic enzyme was investigated using both immunocytochemistry and in situ hybridization. Two full length cDNA clones each encoding cytosolic GS (Gln1-3 and Gln1-5) were isolated from a tobacco seedling cDNA library. A strong homology was found in the coding region of the two clones whereas the 3'- and 5'-untranslated sequences were dissimilar. In order to determine the levels of transcription, specific sequences from Gln1-3 and Gln1-5 were used in an RNAse protection assay. This experiment clearly showed that the gene encoding Gln1-3 is expressed in roots and flowers whereas the gene encoding Gln1-5 is transcribed at a high level in stems and at a lower level in roots and flowers. Immunogold labelling was used to examine the subcellular and cellular distribution of glutamine synthetase in vegetative and reproductive organs of tobacco plants. In mature leaf tissue or petals and sepals, plastidic GS was visualised only in the stroma matrix of chloroplasts and plastids. Cytosolic GS was detected in a number of vegetative or reproductive organs including leaves and flowers. In leaves cytosolic GS was preferentially located in the vascular tissue. In situ hybridization was performed using sections of tobacco organs and specific antisense RNA probes to the genes encoding Gln1-3 and Gln1-5. Gln1-5 transcripts were localised in the vascular tissues of stems and roots whereas Gln1-3 transcripts were detected in all root cells and floral organs including petals, sepals and anthers.