内含子中启动子元件调控菜豆PvSR2基因转录的机制

Intron plays important roles in a gene expression. Recent studies have shown that the alternative intronic promoter usage is an important source for generating protein and regulatory diversity from the same gene. Although several intronic promoters are found in a few plant genes including our previously reported bean PvSR2 gene, the transcriptional regulation of these plant genes by their intronic promoters remain largely unknown. To provide further evidence for this, we characterized the roles of intronic promoter on the PvSR2 transcription. We revealed that PvSR2 generated a shorter PvSR2 transcript (S-PvSR2) through the use of intronic promoter. S-PvSR2 was predominantly expressed in aerial organs (leaf and stem). Immunoblot assay confirmed that S-PvSR2 encodes a novel 60-anino acid protein distinct from PvSR2, which named as NP2 (novel PvSR2). NP2 seemed to function as a transcription factor, because it was localized to the nucleus and showed the transcriptional activation activity in yeast. Interestingly, NP2 bound to the PvSR2 upstream promoter and thereby increased the PvSR2 expression, thus constituting a positive autoregulatory loop. .We apply this proposal based on above results. The overall objective of this proposal is to further understand the intronic promoter-mediated transcriptional regulation of PvSR2. In the next four years, we wish to achieve the following three specific objectives: (1) Identification of NP2-binding sites within the PvSR2 upstream promoter and DNA-binding domain of NP2; (2) Isolation and characterization of transcriptional regulatory factors of PvSR2 interacting with NP2; (3) Identification of the PvSR2 intronic promoter sequences involved in organ-specific response and its binding transcription factors. Our results will further our understanding about the intron-mediated PvSR2 transcription and provide a new insight into the molecular mechanism underlying intronic promoter-mediated transcription of its own gene, i.e. the intronic promoter-mediated autoregulatory loop controlling gene transcription.

内含子中启动子(intronic promoter)是内含子调控基因转录的重要元件，在基因表达多样性方面发挥着重要作用。植物内含子中启动子研究还处于初级阶段，仅在几个基因（包括我们报道的菜豆PvSR2基因）内含子中发现启动子，这些启动子调控基因转录的机制还不清楚。近期工作表明PvSR2内含子中启动子产生一个在茎、叶特异表达的转录本，赋予PvSR2器官特异表达模式。该转录本编码一个能与上游启动子结合并激活PvSR2表达的转录因子NP2，形成一个自调控环路。在此基础上，本项目综合应用细胞、分子和遗传学技术，解析NP2识别的启动子元件，筛选NP2互作的PvSR2转录调控因子，鉴定内含子中启动子器官特异响应区段及其结合转录因子。通过这些研究最终阐明内含子中启动子调控PvSR2转录两种方式（自调控环路和器官特异表达）的分子机制。研究结果对揭示内含子中启动子调控植物基因转录方式及其机制具有一定意义。

PvSR2基因是从法国菜豆分离到的重金属（包括镉）应答的基因，异源表达该基因能够提高转基因烟草镉抗性。项目主要关注内含子中启动子调控菜豆PvSR2基因镉应答的机制，揭示了一个新颖的基因表达调控方式---内含子中启动子反式激活宿主基因表达，对深入了解内含子启动子介导宿主基因的表达调控机制具有一定理论意义。. 取得如下结果：.1.PvSR2内含子中启动子，驱动产生一个短的PvSR2转录本(S-PvSR2)。S-PvSR2编码一个金属响应元件(metal-responsive element, MRE)结合转录因子PvMTF-1。PvMTF-1在mRNA水平和蛋白质水平应答镉胁迫。同时，它还是一个镉抗性的转录因子，因为PvMTF-1通过直接调控色氨酸合成相关基因ASA2表达提高烟草Cd抗性。.2. PvMTF-1也可以特异结合PvSR2基因上游启动子中MRE,并激活PvSR2基因表达，构成一个自调控环路。.3.菜豆乙烯响应因子家族成员PvERF15能够识别内含子中启动子富含AC的元件（ACE）调控PvMTF-1应答镉胁迫。. 此外， 因为课题组关心植物应答镉和热逆境胁迫基因表达调控机制，还在拟南芥热激转录因子HsfA2的可变剪接和剪接因子SR34b参与镉胁迫应答方面展开了拓展性工作: 1)研究发现热诱导的可变剪接可以调控HsfA2表达; 2)SR34b通过调控铁转运蛋白质IRT1基因的表达模式和mRNA稳定性应答镉胁迫。. 以上研究结果已发表SCI收录研究论文4篇,其中植物学著名期刊Plant Physiology 2篇(其中1篇为ESI高被引前3%), 培养毕业硕士研究生6人。. 发表论文如下：.① Sun et al., Plant Physiology 167 (2015): 1136-1148..② Yang et al., BBRC 463(2015): 1097-1101..③ Zhang et al., BBRC 455(2014): 312-317..④ Liu et al., Plant Physiology 162(2013): 512-521.（ESI top 3%）

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