野生大豆GsMYB7转录因子调控耐酸铝的分子机理研究

Soybean growth, development and response to external stresses are subjected to the regulation of transcription factor, which is a kind of important regulatory factors widely existing in soybean. GsMYB7 enhanced the plant resistance to acid aluminum stress of its overexpressed soybean, but the mechanism involved in regulating the stress of acid aluminum remained unclear. In present proposal, we aim to expound the molecular mechanism of GsMYB7 in regulating acid aluminum stress to soybean by identifying the downstream genes via chromatin immunoprecipitation sequencing (Chip-seq) or gene expression analysis and electrophoretic mobility shift assay (EMSA). Yeast two-hybrid is used to screen GsMYB7’s interaction proteins by yeast library of soybean root tip, which will be further identified by the technologies of bimolecular fluorescence complementation (BiFC), Co-Immunoprecipitation(Co-Ip), and GST Pull-down to confirm the interaction in vivo or vitro. The function of screened interaction protein is investigated using overexpression transgenic system in both Arabidopsis and soybean. Taken together, this proposal will uncover the molecular mechanism of GsMYB7 resistant to acid aluminum stress, enrich the understanding of how MYB transcription factors play roles in Al stress regulation, which will provide a theoretical basis for soybean molecular breeding and genetic resources. Therefore, the present proposal is of both theoretical and practical significance.

转录因子是一类重要的调控因子，调控大豆的生长发育过程及其对逆境胁迫的响应。前期研究发现，超表达GsMYB7可增强转基因大豆对酸铝胁迫的耐性，但其调控耐酸铝的具体机制尚不清楚。本研究拟通过GsMYB7转基因株系表达谱分析、染色质免疫共沉淀技术(ChIP)或者凝胶迁移实验（EMSA）鉴定其调控的下游基因；通过酵母双杂交进一步从大豆根尖酵母文库中筛选GsMYB7互作蛋白，并运用双分子荧光互补技术（BiFC）、GST Pull-down、Co-Ip技术进行验证，再通过转基因手段对筛选出的GsMYB7互作蛋白的基因进行功能鉴定。最终解析GsMYB7转录因子耐酸铝的分子机理。本研究既可丰富植物MYB转录因子调控酸铝胁迫的理论基础，又为大豆分子育种提供基因资源，具有重要科学意义和应用前景。

MYB转录因子广泛调控大豆的生长发育及其对逆境胁迫的响应，其中野生大豆GsMYB7转录因子具有增强耐酸铝功能。在此基础上，采用RNA-Seq测序技术、酵母双杂技术筛选与GsMYB7互作的蛋白、其调控的基因和途径，利用转基因技术研究其调控的关键基因的耐酸铝功能，以解析GsMYB7基因在转录及转录后水平参与大豆耐酸铝调控的分子机制。结果表明，GsMYB7过表达之后改变了547个基因的表达水平，其中267个基因的表达量显著上调；在酸铝胁迫下，获得了394个差异表达的基因，其中178个基因的表达量显著上调。GO(gene ontology)分析的结果表明，GsMYB7调控的基因主要与氧化还原过程、转录调控和代谢过程相关。在差异表达基因中，约410个基因与膜相关，其中390个基因膜组分关联、190个基因是核相关的。这些差异表达基因主要编码转移酶、重金属结合相关、水解和氧化还原相关蛋白。KEGG富集分析表明，这些基因主要参与植物不饱和脂肪酸的生物合成、半胱氨酸和蛋氨酸途径、内质网中的蛋白质加工和α-亚麻酸代谢。. 综合RNA-Seq测序结果的生物信息学分析和酵母双杂交筛库的结果，发现有9个候选基因(LOC100818413, LOC100817436、AOS2, LOC100786004, LOC547578, LOC100781111, bZIP73A, LOC100803789 and LOC100803787)在酸铝胁迫的条件下显著上调表达，有可能受GsMYB7基因调控并参与对酸铝胁迫的响应。将候选基因之一GmbZIP73A基因受酸铝胁迫诱导表达，该基因编码ZIP转录因子，GmbZIP73A增强了转基因拟南芥对酸铝胁迫的耐受性；GmbZIP73A基因的启动子含有MYB蛋白结合的特异序列，酵母单杂结果表明GsMYB7蛋白能够特异结合改特异基序。研究结果表明，GsMYB7转录因子可能直接激活GmbZIP73A转录因子，从而启动其下游基因的表达以增强对酸铝胁迫的耐受性。