Expression of a conifer glutamine synthetase gene in transgenic poplar
Expression of a conifer glutamine synthetase gene in transgenic poplar
复制标题
DOI：
10.1007/s004250050649
复制
发表时间：
1999-11-01
期刊：
影响因子：
4.3
通讯作者：
Kirby, EG
中科院分区：
文献类型：
--
作者：
Gallardo, F;Fu, JM;Kirby, EG
谷氨酰胺合成酶（GS; EC 6.3.1.2）催化的铵同化为有机氮已被认为是植物氮利用的限制步骤（H-M. Lam et al. 1995，Plant Cell 7. 887-898）。我们已经开发了一种分子方法，以增加谷氨酰胺生产的转基因杨树过表达的针叶树GS基因。将花椰菜花叶病毒35 S启动子与松树胞质GS cDNA和胭脂碱合成酶多聚腺苷酸化区融合构建的嵌合体转入pBin 19，通过根癌农杆菌转化杂种白杨无性系（INRA 7171-B4，美洲山杨×银白杨）。转化的白杨品系通过它们在含有卡那霉素的选择性培养基上生长的能力来选择。通过Southern杂交和聚合酶链反应分析，证实了导入基因在白杨基因组的存在。转基因在所有白杨品系中均在mRNA水平上表达。相应的多肽（41 kDa）和GS活性增加的转基因检测表明，松树成绩单正确加工的被子植物翻译机器和GSI亚基组装在功能全酶。在白杨中的松树GS 1基因的表达与总可溶性蛋白质的水平的增加和转化树的叶片中的叶绿素含量的增加。此外，平均净增长的GS-过表达克隆的高度显着大于非转化的控制，范围从76%的高度增加在2个月至21.3%的增加在6个月。我们的研究结果表明，可以通过对谷氨酰胺生物合成的遗传操作来设计树木的氮利用效率。
The assimilation of ammonium into organic nitrogen catalyzed by the enzyme glutamine synthetase (GS; EC 6.3.1.2) has been suggested to be the limiting step for plant nitrogen utilization (H-M. Lam et al. 1995, Plant Cell 7. 887-898). We have developed a molecular approach to increase glutamine production in transgenic poplar by the overexpression of a conifer GS gene. A chimeric construct consisting of the cauliflower mosaic virus 35S promoter fused to pine cytosolic GS cDNA and nopaline synthetase polyadenylation region was transferred into pBin19 for transformation of a hybrid poplar clone (INRA 7171-B4, Populus tremula x P. alba) via Agrobacterium tumefaciens. Transformed poplar lines were selected by their ability to grow on selective medium containing kanamycin. The presence of the introduced gene in the poplar genome was verified by Southern blotting and polymerase chain reaction analysis. Transgene expression was detected in all selected poplar lines at the mRNA level. The detection of the corresponding polypeptide (41 kDa) and increased GS activity in the transgenics suggest that pine transcripts are correctly processed by the angiosperm translational machinery and that GSI subunits are assembled in functional holoenzymes. Expression of the pine GS1 gene in poplar was associated with an increase in the levels of total soluble protein and an increase in chlorophyll content in leaves of transformed trees. Furthermore, the mean net growth in height of GS-overexpressing clones was significantly greater than that of non-transformed controls, ranging from a 76% increase in height at 2 months to a 21.3% increase at 6 months. Our results suggest that the efficiency of nitrogen utilization may be engineered in trees by genetic manipulation of glutamine biosynthesis.