National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, No, 35, Tsinghua East Road, Beijing, PR China.
BMC Plant Biol. 2010 Jan 29;10:18. doi: 10.1186/1471-2229-10-18.
CBL1 is a calcium sensor that regulates drought, cold and salt signals in Arabidopsis. Overexpression of CBL1 gene in Arabidopsis and in Ammopiptanthus mongolicus showed different tolerant activities. We are interested in understanding the molecular mechanism of the upstream region of the CBL1 gene of A. mongolicus (AmCBL1). We investigated and characterized the promoter of the AmCBL1 gene, for promoters play a very important role in regulating gene expression in eukaryotes.
A 1683-bp 5' flanking region was isolated from A. mongolicus. The sequence was identified as AmCBL1 promoter. Analysis of the promoter sequence indicated a 690-bp intron and some basic cis-acting elements were related to various environmental stresses and plant hormones. To identify the functional region of the AmCBL1 promoter, five plant expression vectors fused with the GUS (beta-glucuronidase) gene, driven by series deleted fragments of AmCBL1 promoter at different lengths from -1659, -1414, -1048, -296 to -167 bp relative to the transcriptional start site were constructed and transformed into Nicotiana tabacum L. cv. 89. Functional properties of each promoter segment were examined by GUS staining and fluorescence quantitative analyses using at least three single-copy PCR-positive plants of transgenic tobacco, treated with various environmental stresses and plant hormones for different times. We demonstrated that the AmCBL1 promoter was a vascular-specific and multiple-stress-inducible promoter. Our results further imply that the promoter fragment B1S3 possessed sufficient essential cis-acting elements, accounting for vascular-specific and stress-induced expression patterns. It may also indicate that for response to some stresses certain cis-elements are required in tissues outside the region of the B1S3 construct.
To help resolve uncertainties about the upstream regulatory mechanism of the CBL1 gene in desert plants, we suggest that the function of the AmCBL1 promoter, particularly under conditions of abiotic stress, to be examined for possible usefulness in molecular breeding. Regardless of the outcome, the allocation and relative quantification of the GUS-fusion AmCBL1 promoter segments at transcriptional levels in different tissues under various stresses across separate promoter segments suggests that the AmCBL1 promoter is a phloem-specific and multiple-stress-inducible promoter. These data coupled with the ongoing AmCBL1 5' UTR intron analyses provide a solid foundation for their future use in molecular breeding as new promoters of stress-resistance genes from desert plants.
CBL1 是一种钙传感器，可调节拟南芥中的干旱、寒冷和盐信号。在拟南芥和柠条中过表达 CBL1 基因表现出不同的耐受活性。我们有兴趣了解柠条 CBL1 基因（AmCBL1）上游区域的分子机制。我们研究并表征了 AmCBL1 基因的启动子，因为启动子在真核生物中对调节基因表达起着非常重要的作用。
从柠条中分离出 1683bp 的 5'侧翼区。该序列被鉴定为 AmCBL1 启动子。启动子序列分析表明，存在一个 690bp 的内含子，一些基本的顺式作用元件与各种环境胁迫和植物激素有关。为了鉴定 AmCBL1 启动子的功能区域，构建了五个植物表达载体，分别与 AmCBL1 启动子的不同长度的系列缺失片段融合，从转录起始位点上游-1659、-1414、-1048、-296 至-167bp。通过 GUS 染色和荧光定量分析，至少对三个转 AmCBL1 基因烟草的单拷贝 PCR 阳性植株进行了每个启动子片段的功能特性研究，用不同的环境胁迫和植物激素处理不同时间。结果表明，AmCBL1 启动子是一种血管特异性和多胁迫诱导启动子。我们的结果进一步表明，启动子片段 B1S3 具有足够的必需顺式作用元件，负责血管特异性和胁迫诱导的表达模式。这也可能表明，对于某些胁迫的反应，某些顺式元件在 B1S3 构建体区域之外的组织中是必需的。
为了帮助解决沙漠植物中 CBL1 基因上游调控机制的不确定性，我们建议在非生物胁迫条件下检查 AmCBL1 启动子的功能，以便在分子育种中可能有用。无论结果如何，在不同组织中不同胁迫下转录水平上分配和相对定量 GUS 融合的 AmCBL1 启动子片段，以及不同启动子片段之间的相对定量，表明 AmCBL1 启动子是一种韧皮部特异性和多胁迫诱导启动子。这些数据加上正在进行的 AmCBL1 5'UTR 内含子分析，为它们在未来作为沙漠植物中抗逆基因的新启动子在分子育种中的应用提供了坚实的基础。
