NUCLEOTIDE-SEQUENCE OF THE TRYPTOPHAN DECARBOXYLASE GENE OF CATHARANTHUS-ROSEUS AND EXPRESSION OF TDC-GUSA GENE FUSIONS IN NICOTIANA-TABACUM
NUCLEOTIDE-SEQUENCE OF THE TRYPTOPHAN DECARBOXYLASE GENE OF CATHARANTHUS-ROSEUS AND EXPRESSION OF TDC-GUSA GENE FUSIONS IN NICOTIANA-TABACUM
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DOI：
10.1007/bf00391016
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发表时间：
1994-01-01
期刊：
影响因子：
--
通讯作者：
HOGE, JHC
中科院分区：
文献类型：
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作者：
GODDIJN, OJM;LOHMAN, FP;HOGE, JHC
色氨酸脱羧酶（TDC; EC 4.1.1.28）将色氨酸转化为色胺。在Catharanthus I roseus和其他能够产生萜类吲哚生物碱（TIAs）的植物中，TDC将初级代谢与参与这些化合物生物合成的次级代谢途径联系起来。tdc mRNA的积累受诱导子（诱导）和生长素（抑制）的发育调控和转录影响。在这里，我们报道了TDC是由一个单拷贝基因编码的玫瑰丁香基因组。对该基因进行分离和测序，未发现内含子。为了研究由tdc启动子控制的基因表达，一个2 kb的启动子片段和一些5'缺失的启动子衍生物通过翻译融合连接到一个β - d -葡糖醛酸酶报告基因（gusA）。在稳定转化的烟草植株和瞬时转染的烟草原生质体中监测了嵌合构建体的表达。转基因植物的组织化学和荧光分析表明，tdc启动子的1938 bp（相对于翻译起始密码子）在根、茎和叶中引起GUS活性。没有组织或细胞类型特异性。高达-398核苷酸的启动子缺失导致gusA表达水平降低，但与-1938构建的GUS活性染色模式相同。进一步缺失高达-232核苷酸的tdc启动子导致转基因植株所有部位GUS活性水平急剧降低和GUS染色丧失。与稳定转化相比，-232 tdc-gusA构建体在原生质体中瞬时表达的测定系统中产生的GUS活性水平与-398构建体相当。在该体系中测定的GUS活性不受合成生长素萘乙酸（NAA）存在与否的影响。
The enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) converts tryptophan into tryptamine. In Catharanthus I roseus and other plants capable of producing terpenoid indole alkaloids (TIAs) TDC links primary metabolism to the secondary metabolic pathway involved in the biosynthesis of these compounds. The accumulation of tdc mRNA in C. roseus cells is developmentally regulated and transcriptionally influenced by elicitors (induction) and auxins (repression). Here we report that TDC is encoded by a single copy gene in the C. roseus genome. No introns were observed upon isolation and sequencing of this gene. To study gene expression controlled by the tdc promoter, a 2 kb promoter fragment and a number of 5' deleted promoter derivatives were joined in translational fusion to a beta-D-glucuronidase reporter gene (gusA). Expression of the chimaeric constructs was monitored in stably transformed tobacco plants and in transiently transfected tobacco protoplasts. Histochemical and fluorimetric analysis of transgenic plants revealed that 1938 bp of the tdc promoter (with respect to the translational start codon) give rise to GUS activity in roots, stems and leaves. No tissue or cell type specificity was noted. Promoter deletions up to nucleotide -398 directed lower levels of gusA expression but conferred the same pattern of staining for GUS activity as the -1938 construct. Further deletion of the tdc promoter up to nucleotide -232 resulted in drastically reduced GUS activity levels and loss of GUS staining in all parts of the transgenic plants. In contrast to stable transformation, the -232 tdc-gusA construct gave rise to GUS activity levels comparable to those of the -398 construct in an assay system for transient expression in protoplasts. In this system the GUS activities measured were not affected by the presence or absence of the synthetic auxin naphthalene acetic acid (NAA).