Grimmig B, Matern U
Lehrstuhl für Biochemie der Pflanzen, Albert-Ludwigs-Universität, Freiburg, Germany.
Plant Mol Biol. 1997 Jan;33(2):323-41. doi: 10.1023/a:1005780529457.
The sequence of the S-adenosyl-L-methionine:trans-caffeoyl-CoA O-methyltransferase (CCoAOMT, EC2.1.1.104) gene, including the 5'-flanking region of 5 kb, was determined from parsley (Petroselinum crispum) plants. The enzyme appears to be encoded by one or two genes, and the ORF is arranged in five exons spaced by introns from 107 to 263 bp in length. The genomic sequence matches the ORF of the cDNA previously reported from elicited parsley cell cultures, showing only three base changes that do not affect the enzyme polypeptide sequence. S1 nuclease protection assays and primer extension analyses with genomic and cDNA templates revealed the transcription start site 67 bp upstream of the translation start codon, indicating a shorter 5'-UTR than reported previously for the transcript. Promoter regulatory consensus elements such as two 'CAAT' boxes and one 'TATA' box were identified at -196, -127 and -31, respectively, relative to the transcription start site, and an SV 40-like enhancer element is located 347 bp upstream. Most notably, three putative cis-regulatory elements were recognized by sequence alignments, which represent motifs recurring in the promoters of several genes of the stress-inducible phenylpropanoid pathway (boxes P, A and L). Transient expression assays with a set of 5'-truncated promoter-GUS fusions show that significant promoter activity is retained in a 354 bp promoter fragment. In vitro DNase 1 footprint experiments and electrophoretic mobilty shift assays (EMSA) identified in this fragment a unique sequence motif with elicitor-inducible trans-factor binding activity, which was unrelated to boxes P, A, or L. This novel cis-regulatory element, designated box E, appears to be conserved in the TATA-proximal regions of other stress-inducible phenylpropanoid genes, and in vitro binding of nuclear protein was confirmed in EMSA assays for such an element from the PAL-1 promoter (-54 to -45). Moreover, the deletion of box E reduced the activity and erased the elicitor-responsiveness of the CCoAOMT promoter in transient expression assays. The results corroborate the proposed physiological function of CCoAOMT in elicited plant cells and may shed new light on the sequential action of trans-active factors in the regulation of phenylpropanoid genes.
从欧芹（Petroselinum crispum）植株中测定了S-腺苷-L-甲硫氨酸：反式咖啡酰辅酶A O-甲基转移酶（CCoAOMT，EC2.1.1.104）基因的序列，包括5 kb的5'-侧翼区域。该酶似乎由一个或两个基因编码，其开放阅读框（ORF）由五个外显子组成，外显子之间被长度为107至263 bp的内含子隔开。基因组序列与先前从诱导的欧芹细胞培养物中报道的cDNA的ORF匹配，仅显示三个不影响酶多肽序列的碱基变化。用基因组和cDNA模板进行的S1核酸酶保护试验和引物延伸分析揭示了翻译起始密码子上游67 bp处的转录起始位点，表明5'-非翻译区（UTR）比先前报道的转录本短。相对于转录起始位点，分别在-196、-127和-31处鉴定到启动子调控共有元件，如两个“CAAT”框和一个“TATA”框，并且一个类SV 40增强子元件位于上游347 bp处。最值得注意的是，通过序列比对识别出三个推定的顺式调控元件，它们代表了应激诱导的苯丙烷类途径的几个基因的启动子中反复出现的基序（框P、A和L）。用一组5'-截短的启动子-GUS融合体进行的瞬时表达试验表明，在一个354 bp的启动子片段中保留了显著的启动子活性。体外DNase 1足迹试验和电泳迁移率变动分析（EMSA）在该片段中鉴定出一个具有诱导子诱导的反式因子结合活性的独特序列基序，它与框P、A或L无关。这个新的顺式调控元件，命名为框E，似乎在其他应激诱导的苯丙烷类基因的TATA近端区域中是保守的，并且在EMSA试验中证实了来自PAL-1启动子（-54至-45）的这种元件的核蛋白体外结合。此外，在瞬时表达试验中，框E的缺失降低了CCoAOMT启动子的活性并消除了其诱导子反应性。这些结果证实了CCoAOMT在诱导的植物细胞中的拟生理功能，并可能为反式激活因子在苯丙烷类基因调控中的顺序作用提供新的线索。
