Interest in and limits to the utilization of reporter genes for the analysis of transcriptional regulation of nitrate reductase
Interest in and limits to the utilization of reporter genes for the analysis of transcriptional regulation of nitrate reductase
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DOI：
10.1007/bf00279369
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发表时间：
1992-11
期刊：
影响因子：
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通讯作者：
H. Vaucheret;A. Marion-Poll;C. Meyer;J. Faure;E. Marín;M. Caboche
中科院分区：
文献类型：
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作者：
H. Vaucheret;A. Marion-Poll;C. Meyer;J. Faure;E. Marín;M. Caboche
报道基因技术和突变体分析被用来确定硝酸盐和硝酸盐或铵衍生代谢物（N-代谢物）在真二倍体种烟草（Nicotiana plumbaginifolia）和双二倍体种烟草（Nicotiana tabacum）中调节硝酸还原酶（NR）表达的分子基础。那白花丹叶突变体E23是由在编码硝酸还原酶的单拷贝基因的第一外显子中插入aTnt1样逆转录转座子（Tnp2）引起的。其中一个转录本终止于该逆转录转座子的5 ′ LTR（长末端重复）序列，另一个转录本终止于3 ′ LTR。硝酸盐和N-代谢产物调节这些截短的转录本的表达，表明内含子剪接和终止过程对这些调节事件不是必需的。AGUS报告基因序列与N.烟草这种融合是功能的瞬时表达试验与原生质体来自叶肉细胞的N。烟草然而，在这些实验条件下，已知的影响thenia-1转录物稳态水平的调节机制都不起作用。转基因植物携带这种融合或翻译融合的GUS连接到thenia-1ornia-2基因的启动子。通过农杆菌介导法获得了转基因烟草。低比例的转基因植株（105个独立转化体中的22个）表达GUS活性，尽管在低水平。只有4个植株的GUS基因表达量可检测到。这种mRNA的浓度显着增加，在NR-缺陷的背景下，表明N-代谢产物的调节。然而，只有2株植物表现出硝酸盐的调节（诱导）。尝试useaux2ornptII报告序列连接到thenia-1ornia-2启动子作为标记基因的硝酸盐同化途径的调节突变体的选择是不成功的，因为我们无法分离转基因植物，这些报告基因被适当地调节硝酸盐。这些结果的影响进行了讨论。
Reporter gene techniques and mutant analysis were used to identify the molecular basis of the regulation of the expression of nitrate reductase (NR) by nitrate and nitrate-, or ammonium-derived metabolites (N-metabolites), in the true diploïd speciesNicotiana plumbaginifoliaand in the amphidiploïd speciesNicotiana tabacum. TheN. plumbaginifoliamutant E23 results from the insertion of aTnt1-like retrotransposon (Tnp2) in the first exon of the single-copyniagene, which encodes nitrate reductase. One of the resulting transcripts ends in the 5′ LTR (long terminal repeat) sequence of this retrotransposon, and another one in the 3′ LTR. Nitrate and N-metabolites modulate the expression of these truncated transcripts, indicating that intron splicing and termination processes are not essential to these regulatory events. AGUSreporter sequence was transcriptionally linked to the promoter of thenia-1gene ofN. tabacum. This fusion was functional in transient expression assays done with protoplasts derived from mesophyll cells ofN. tabacum. However none of the regulatory mechanisms known to affect steady-state levels of thenia-1transcript were operative under these experimental conditions. Transgenic plants carrying either this fusion or translational fusions ofGUSlinked to the promoter of either thenia-1ornia-2gene ofN. tabacumwere obtained byAgrobacterium-mediated transfer. A low proportion of the transgenic plants (22 out of 105 independent transformants) expressed GUS activity although at a low level. Only 4 plants exhibited a detectable level ofGUSmRNA. The concentration of this mRNA increased significantly in an NR-deficient background, indicating regulation by N-metabolites. Only 2 plants, however, showed regulation (induction) by nitrate. Attempts to useaux2ornptIIreporter sequences linked to either thenia-1ornia-2promoter as marker genes for the selection of regulatory mutants of the nitrate assimilation pathway were unsuccessful because of our inability to isolate transgenic plants in which these reporter genes were properly regulated by nitrate. The implications of these results are discussed.