Identification of promoter elements in a low-temperature-responsive gene (blt4.9) from barley (Hordeum vulgare L.)
Identification of promoter elements in a low-temperature-responsive gene (blt4.9) from barley (Hordeum vulgare L.)
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DOI：
10.1023/a:1006098132352
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发表时间：
1998-11-01
影响因子：
5.1
通讯作者：
Hughes, MA
中科院分区：
文献类型：
--
作者：
Dunn, MA;White, AJ;Hughes, MA
Blt4大麦基因家族编码非特异的脂转移蛋白，通过原位定位表明，该基因在叶片的表皮细胞中表达。该基因家族转录控制的低温反应成员blt4.9主要在茎分生组织中表达。Blt4.9的启动子区域(1938bp)包含与低温、脱落酸和其他环境因子相关的序列基序。缺失分析表明，在大麦枝条外植体瞬时表达系统中，CAAT和TATA盒附近的42bp序列能够增强对报告基因的低温响应。其他启动子区域显示包含负调控区域和正调控区域。对来自低温或对照植物的核蛋白进行了电泳迁移率改变分析(EMSA)，以进一步研究blt4.9启动子。人工合成的寡核苷酸被用来确定位于42bp低温响应启动子区域的六核苷酸CCGAAA是低迁移率核蛋白复合体的结合位点。这种复合体存在于低温处理工厂和对照工厂的核提取液中，是该区域内形成的唯一复合体。低温响应型42个碱基启动子序列中CCGAAA基序的突变使低温响应性降低到基础水平。在这项研究中，一个相关的上游元件CCGAC在其他植物中是已知的低温响应元件，不与核蛋白结合。推测来自第一个ATG的六核苷酸CCGAAA在-195处参与了大麦blt4.9的低温反应。
The blt4 barley gene family encodes non-specific lipid transfer proteins and has been shown, by in situ localisation, to be expressed in the epidermal cells of leaves. The transcriptionally controlled, low-temperature-responsive member of this gene family, blt4.9, is predominantly expressed in shoot meristems. The promoter region (1938 bp) of blt4.9 contains sequence motifs which have been implicated in responses to low temperature, abscisic acid and other environmental factors. Deletion analysis showed that a 42 bp sequence proximal to, but not including, the CAAT and TATA boxes, confers enhanced low-temperature response to a reporter gene in a barley shoot explant transient expression system. Other promoter regions were shown to contain negative and positive regulatory regions. Electrophoretic mobility shift analysis (EMSA) was used with nuclear proteins from either low-temperature or control-temperature-treated plants to further investigate the blt4.9 promoter. Synthetic oligonucleotides were used to identify a hexanucleotide, CCGAAA, within the 42 bp, low-temperature-responsive promoter region, as the binding site of a low-mobility nuclear protein complex. This complex was present in nuclear extracts from both low-temperature-treated and control plants and was the only complex formed within this region. Mutation of the CCGAAA motif within the low-temperature-responsive 42 bp promoter sequence reduced low-temperature responsiveness to basal levels. A related upstream element, CCGAC, known to be a low-temperature-responsive element in other plants, did not bind to nuclear proteins in this study. It is proposed that the hexanucleotide CCGAAA, at -195 from the first ATG, is involved in the low-temperature response of blt4.9 in barley.