Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P
Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P
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DOI：
10.1105/tpc.12.1.65
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发表时间：
2000-01-01
期刊：
影响因子：
11.6
通讯作者：
Bowen, B
中科院分区：
文献类型：
--
作者：
Bruce, W;Folkerts, O;Bowen, B
为了确定玉米转录因子C1/R和P（负责激活类黄酮合成）控制的基因表达范围，我们使用GeneCalling（一种开放式、基于凝胶的mRNA分析技术）分析了玉米自交系黑墨西哥甜（BMS）的细胞悬浮系，该细胞悬浮系含有这些因子的雌二醇诱导型版本。EMS细胞用连续表达的雌激素受体/玉米C1激活剂结构域融合基因（ER-C1）和CI和R（CRC）、P或荧光素酶基因的融合物转化，所述荧光素酶基因由含有四个重复的雌激素受体结合位点的启动子调节。在加入雌二醇后，在相应的细胞系中检测到荧光素酶活性、花青素和黄烷-4-醇的量增加。通过分析雌二醇处理后0、6和24小时从重复样品中分离的mRNA，在这些EMS系中同时检测到已知和新基因的表达。鉴定了许多cDNA片段，其在6和24小时的丰度比0小时高两倍或更大。来自已知类黄酮基因的cDNA片段，除了查耳酮异构酶（chil），在激素诱导后在CRC表达系中被诱导，而在P表达系中仅查耳酮合酶（c2）和黄烷酮/二氢黄酮醇还原酶（a1）基因被诱导，正如所料。许多新的cDNA片段也诱导或抑制线表达CRC单独，P单独，或在独特的时间模式的转录因子。时间上的差异和证据的镇压表明一个更多样化的监管控制CRC或P比原来预期的。GeneCalling分析成功地检测了复杂代谢途径的成员，并发现了巧合调控或直接参与这些途径的新基因。
To determine the scope of gene expression controlled by the maize transcription factors C1/R and P, which are responsible for activating flavonoid synthesis, we used GeneCalling, an open-ended, gel-based, mRNA-profiling technology, to analyze cell suspension lines of the maize inbred Black Mexican Sweet (BMS) that harbored estradiol-inducible versions of these factors. EMS cells were transformed with a continually expressed estrogen receptor/maize C1 activator domain fusion gene (ER-C1) and either a fusion of CI and R (CRC), P, or luciferase genes regulated by a promoter containing four repeats of an estrogen receptor binding site. Increasing amounts of luciferase activity, anthocyanins, and flavan-4-ols were detected in the respective cell lines after the addition of estradiol, The expression of both known and novel genes was detected simultaneously in these EMS lines by profiling the mRNA isolated from replicate samples at 0, 6, and 24 hr after estradiol treatment. Numerous cDNA fragments were identified that showed a twofold or greater difference in abundance at 6 and 24 hr than at 0 hr, The cDNA fragments from the known flavonoid genes, except chalcone isomerase (chi1), were induced in the CRC-expressing line after hormone induction, whereas only the chalcone synthase (c2) and flavanone/dihydroflavonol reductase (a1) genes were induced in the P-expressing line, as was expected. Many novel cDNA fragments were also induced or repressed by lines expressing CRC alone, P alone, or both transcription factors in unique temporal patterns. The temporal differences and the evidence of repression indicate a more diverse set of regulatory controls by CRC or P than originally expected. GeneCalling analysis was successful in detecting members of complex metabolic pathways and uncovering novel genes that were either coincidentally regulated or directly involved in such pathways.