拟南芥冷胁迫响应双向启动子的功能分析及改良

Bidirectional promoters are defined as the regions that are shared between two divergent genes, when those two genes are transcribed away from one another. Computational analysis revealed that divergent gene pairs are mainly coexpressed, but some display opposite regulation. The mechanisms of anti-correlated regulation remain unclear. Based on the information previously reported by us and other groups, we hypothesize that the cis-acting el ement controls the negative correlation expression of a bidirectional gene pair in an orientation-dependent manner..To test this hypothesis, a 578-bp intergenic region of a protein-encoding gene pair(At1g71850 and At1g71860)was chosen for the present study. At1g71850 and At1g71860 loci encode a ubiquitin carboxyl-terminal hydrolase family protein(UCH), and a protein with tyrosine phosphatase(PTP1) activity that is down-regulated in response to cold and up-regulated in response to salt stress, respectively. .We have successfully tested this native Arabidopsis DNA sequence as a bidirectional promoter to drive GFP and GUS reporter genes. Additionally, down- regulation of GUS and up-regulation of GFP were observed under low-temperature stress conditions. In silico analysis of the divergent promoter was performed to look for cis-elements using the PlantCARE and PLACE web tools. An ocs-like element was identified in two positions. The ocs element, originally identified in the promoter region of the octopine synthase gene, was described by us and others as a position-dependent or orientation-dependent regulatory element. Taken together, the findings presented here provide the first evidence for the existence of orientation-dependent elements in the bidirectional promoter responsible for the negative correlation expression of divergent genes..To further verify this hypothesis, we will perform a comprehensive analysis of the tissue-specific expression and induction pattern of the bidirectional promoter, and then use the 5'-deletion and electrophoretic mobility shift assays to identify the regulatory regions controlling the induction pattern of the bidirectional promoter. Finally, we will characterize the orientation-dependence of the cold- responsive element and improve the natural bidirectional promoter by introducing this cis-element. In summary, these studies provide new insight into the mechanism of negative correlation expression of the divergent genes in higher plants, and establish a novel approach for constructing a new bidirectional promoter different from the earlier bi-directional promoter strategy in that the new bidirectional promoter is obtained by improvement of a single natural bidirectional promoter and not by bidirectionalization of polar promoters.

双向启动子是指位于相邻但转录方向相反的两个基因之间的DNA序列，这两个基因称为双向基因对。生物信息学研究表明双向基因对存在基因共表达现象，也有少量表现为表达负相关。双向基因对表达负相关的机制尚未阐明。我们推测方向敏感性调控元件的存在导致双向基因对表达负相关。为证实该假说，本项目以拟南芥泛素C末端水解酶基因和蛋白酪氨酸磷酸酶基因之间的序列为研究对象，首先证实其具有双向转录活性，并在冷胁迫时调控外源基因对表达负相关，是一个双向启动子，其序列含有类似于ocs元件的短序列。前期我们已发现ocs元件可能具有方向或位置敏感特性。接下来我们将对这个双向启动子进行详细的功能分析，明确冷胁迫响应元件的方向敏感特性，并利用该元件改造双向启动子使其两端均具有冷诱导高效表达活性。研究结果将从胁迫响应元件的方向敏感性这一新视角探讨双向启动子调控的基因对表达负相关的分子机制，并开辟一条获得新特性双向启动子的新途径。

生物信息学研究表明双向启动子广泛存在于动植物基因组中，预示着它们具有重要的生物学功能。目前，双向启动子的研究甚少。我们以拟南芥泛素C端水解酶基因和蛋白酪氨酸磷酸酶基因(AtUCH4/AtPTP1双向基因对)的间隔序列为研究对象进行分析，研究结果为双向启动子的调控机制阐释和双向启动子的利用改造提供依据和新思路。本研究主要结果和结论如下：.1 双向基因对AtUCH4/AtPTP1的间隔序列具有双向启动子活性.转基因拟南芥GUS染色和酶活分析结果表明，双向基因对AtUCH4/AtPTP1的间隔序列是一个具有不对称转录活性的双向启动子。瞬时表达活性检测结果也表明该双向启动子能够同时启动GUS和GFP基因在异源植物烟草中表达。.2 AtUCH4/AtPTP1双向启动子由不同的正、负调控区组成.启动子缺失实验分析表明，AtUCH4/AtPTP1双向启动子可分为三个亚结构区域。AtUCH4启动子的基础转录活性由一个弱的正调控区(region II)和一个弱的负调控区(region III)控制。而AtPTP1启动子的活性由一个强的正调控区(region I)和一个弱的负调控区(region II)控制。.3 AtUCH4/AtPTP1双向启动子具有非生物胁迫响应活性.Genevestigator和RT-PCR分析发现，内源基因对AtUCH4/AtPTP1在低温胁迫下表达变化呈负相关。GUS酶活分析表明内源基因对在响应低温时表达负相关是由AtUCH4/AtPTP1双向启动子调控的。另外，我们的实验结果表明该双向启动子也具有盐胁迫响应活性。.4 AtUCH4/AtPTP1双向启动子含有低温响应元件.通过分析as-1/ocs元件突变序列及不同长度的AtUCH4/AtPTP1双向启动子在冷处理后的活性变化，证实了位于region II区的as-1/ocs元件是一个方向敏感性低温响应元件。.5 AtUCH4/AtPTP1双向启动子的改良及低温诱导表达活性分析.以AtUCH4/AtPTP1双向启动子序列为基础，引入反向的含有低温响应元件的52 bp启动子序列，最终获得含有低温响应元件反向重复的双向启动子植物表达载体。QRT-PCR分析结果表明，改造后的双向启动子两端均具有低温高效诱导表达活性，由此我们建立了一条通过改造天然双向启动子来获得新特性双向启动子的途径。