逆境下ERF转录因子调控基因表达所结合的关键顺式元件及抗逆调控机理研究

Our study showed that only 20% of the genes regulated by an ERF protein from Tamarix hispida contain GCC-box and DRE in their promoter regions, and similar results were also observed in studies of other ERF genes from T. hispida. Further studies showed that ERF may bind to other motif rather than GCC-box and DRE motifs to regulate gene expression in response to abiotic stresses. Based on this observation, further studies will be performed in this project. The ERF gene with high stress tolerance is transformed into T. hispida, and the transgenic T. hispida were treated with salt, drought, ABA or without treatment. After these treatments, ChIP libraries were respectively built from these samples, and ChIP-Seq were further performed to generate the DNA fragments bound by ERF under stress or normal growth conditions. The conserved motifs among these DNA fragments were identified, which may be recognized by ERF and will be further confirmed by yest one hybrid analysis. The frequencies of these motifs present in different ChIP libraries were analyezed to identify the motif bound by ERF with binding affinity in reponse to abiotic stress. The transcriptoms from wild type and transgenic T. hispida were built, and were assembled with the sequences generated from ChIP-Seq, and these motifs contained in which genes will be revealed by the assembled sequences. Based on these studies, the motifs recognzied by ERF protein and its binding affinities in response to abiotic stress will be identified and the stress tolerance pathways these motifs involved will be revealed.

我们对柽柳的一条ERF基因ThERF1研究发现，仅20%的ThERF1调控基因启动子中有GCC-box或DRE元件，对柽柳其他ERF研究也有类似现象。进一步研究显示逆境下ERF可能结合其他元件调控基因表达，GCC-box和DRE不起主要作用。有鉴于此，本研究拟将抗逆ERF转入柽柳，对转基因柽柳进行正常、盐、旱、ABA处理，分别建立ChIP文库，进行ChIP-Seq高通量测序，获得ERF在不同逆境及非逆境下结合的DNA片段序列，进行序列的保守元件分析，鉴定出胁迫及非胁迫下ERF可能结合的元件，酵母单杂交验证。分析这些元件在胁迫和非胁迫下出现频率，鉴定出逆境下ERF在表达调控中主要结合的元件。建立逆境和非逆境下柽柳转录组，通过转录组与ChIP序列的深度拼接，阐明这些元件可能存在于哪些基因启动子中，参与了何种抗逆代谢途径的调控，进而鉴定出逆境胁迫下ERF所调控的关键元件及其所参与的抗逆调控机理。

本项目克隆了柽柳 ERF家族基因16个，利用瞬时转化技术将其转化到柽柳中，获得转基因柽柳，对转基因柽柳进行逆境胁迫相关的生理指标测定，从中筛选出一个耐盐能力优良的ERF基因，其属于ERF/AP2 transcription factor family，命名为ThCRF1。ThCRF1 蛋白具有转录激活功能，其转录激活域位于N端，其可以与TTG、DRE 和 GCC-box 等顺式作用元件相结合。我们对ThCRF1 过表达及其RNAi抑制表达的柽柳及ThCRF1 过表达的转基因拟南芥进行了抗逆生理及分子生物学机制研究。发现ThCRF1主要通过两个途径调控柽柳的耐盐能力，一是其表达能够诱导海藻糖合成酶海藻糖-6-磷酸合成酶(TPS)和海藻糖-6-磷酸磷酸酶基因(TPPs)的表达，提高海藻糖的含量，进而提高其耐盐能力。海藻糖作为一种重要的渗透调节物质在植物耐盐中起重要渗透调控和保护作用。另一个途径是ThCRF1能够调控SOD和POD基因表达来提高活性氧清除能力，进而提高其耐盐能力。将ThCRF1与Flag融合瞬时转化到柽柳中，使之过表达，并对转基因植株分别进行盐、旱胁迫，然后利用Flag抗体进行ChIP。通过ChIP-seq分析获得peaks，对peaks进行MEME分析，获得了一个在ThCRF1结合片段中广泛分布的保守元件，其序列为“CCAAC(A)C(A)”，命名为CCAA-box，酵母单杂交及EMSA分析结果显示CCAA-box能够被ThCRF1结合，染色质免疫共沉淀(ChIP)分析说明ThCRF1能够在柽柳中结合CCAA-box元件来调控基因的表达。克隆了柽柳及拟南芥的ERF家族基因，研究发现柽柳和拟南芥的其他ERF基因也结合CCAA-box元件，因此，ERF结合CCAA-box具有普遍性。利用RNA-seq比较了ThCRF1过表达、RNAi抑制表达和对照柽柳在盐胁迫下的基因表达，进而鉴定出了ThCRF1直接和间接调控的基因，阐明了ThCRF1可能调控的耐盐分子机制，RNA-seq分析发现ThCRF1主要通过调控antioxidant活性相关的基因表达来提高detoxification途径的活性来提高耐盐能力，进一步证实了前面研究的结果，即ThCRF1能够提高活性氧清除能力来提高柽柳的耐盐能力。