GmbHLH25转录因子在丹波黑大豆响应答铝胁迫过程中的作用机理研究

Aluminum (Al) toxicity is a major limiting factor for crop production in acid soils. The key step to efficiently solve this problem is to deeply understand Al tolerance mechanism in plants. The preliminary results of this project showed that Glycine max Tamba (referred to as BS thereafter) is a kind of black soybean cultivars with strong Al tolerance. The transcription of a transcription factor GmbHLH25 gene, which was isolated and identified from BS roots, was upregulated after BS was stressed by Al. Overexpression of GmbHLH25 enhanced the Al resistance of transgenic tobacco. In this study, the subcellular localization and expression pattern of GmbHLH25 protein will be analyzed by the fusion of GmbHLH25 to reporter genes; its transactivation will be verified by the fusion of GmbHLH25 to the DNA-binding domain of GAL4 in a yeast system; the proteins binding with GmbHLH25 will be isolated from BS roots under Al stress conditions by co-immunoprecipitation (CoIP); the target genes regulated by GmbHLH25 will be identified using DNA micrray analysis; the binding of promoter sequences of target genes with GmbHLH25 will be validated by gel retardation analysis or yeast one hybridyation; the expression profiles of target genes in BS roots under Al stress will be analyzed and the pysiological functions of target gene encoding proteins will be investigated; the function of GmbHLH25 and its target genes will be further verified in transgenic model plants. The results obtained from this study will clarify the functional mechanism of GmbHLH25 in response to Al stress in BS. These results will not only allow us to further understand the moelcualr mechanism of transcriptional regulation in soybean in reseponse to Al stress, but also provide theoretical bases and gene resources for molecular breeding to improve the Al resistance of plants.

本项目的前期研究表明丹波黑大豆（简称BS）是一种耐铝能力很强的大豆栽培种，从BS根中分离鉴定的转录因子GmbHLH25在BS受铝胁迫后表达上调，过量表达GmbHLH25增加烟草对铝的抗性。本项目将应用报告基因与该转录因子的融合基因分析它的亚细胞定位和表达模式；通过它与酵母GAL4DNA结合域融合确认它的转录激活作用；应用免疫共沉淀技术分离铝胁迫BS根中与它互作的蛋白；应用DNA芯片技术鉴定它调控的靶基因；用凝胶阻滞法或酵母单杂交技术验证GmbHLH25与靶基因启动子DNA结合特性；分析铝胁迫下BS根中各靶基因的表达谱及靶基因编码蛋白的生理作用；在模式植物转基因体系中进一步验证GmbHLH25及其关键靶基因的功能，阐明GmbHLH25在BS响应铝胁迫过程中的作用机理，研究结果将使我们对大豆应答铝胁迫转录调控的分子机理有更进一步的认识和了解，为改良植物耐铝能力的分子育种提供理论依据和基因资源。

铝胁迫耐铝丹波黑大豆（RB）的SSH cDNA文库中发现GmbHLH30基因呈上调表达，推测该基因与RB的耐铝性相关。因此，我们开展铝胁迫下RB bHLH30的表达，bHLH30亚细胞定位，bHLH30的互作蛋白及其调控下游靶基因的分子机理研究，并通过转bHLH30基因烟草阐明RB bHLH30耐铝作用。研究结果表明50 µM Al处理RB，RB根中GmbHLH30的表达量随着50 µM Al处理时间的增加而增加，说明其表达受铝胁迫的诱导。用GmbHLH30特异的氨基酸序列（GGLNMYPGAEVSPIMN）合成多肽，并免疫新西兰兔获得GmbHLH30特异性抗体。通过Far-Western技术对铝胁迫处理0h和8h的RB根中的GmbHLH30表达水平分析表明GmbHLH30的表达水平受铝胁迫诱导。通过构建原核表达载体，纯化报告基因Egfp编码的绿色荧光蛋白，制备多克隆抗体，间接地在蛋白水平检测到GmbHLH30转录因子也受铝胁迫诱导表达。FITC法对GmbHLH30进行亚细胞定位分析表明铝胁迫处理8h的RB根根细胞核中观察到了强烈的绿色荧光，表明GmbHLH30转录因子定位在细胞核。同时构建GmbHLH30植物表达载体pKGWFS7-GmbHLH30，并转入野生型烟草获得了转GmbHLH30的烟草植株。50 µM Al处理转基因烟草8小时后，在荧光显微镜下观察到发绿色荧光的细胞核，这也证明GmbHLH30转录因子定位到细胞核。利用免疫共沉淀技术和蛋白质谱技术分离鉴定了与GmbHLH30结合的已知的三种靶蛋白，WD40蛋白、类ATP柠檬酸合成酶β链蛋白和类丙酮酸脱羧同工酶。利用染色质免疫共沉淀技术在铝胁迫下RB植物体内筛选GmbHLH30转录因子可能作用的下游靶基因。MATE基因启动子区域内一个片段能与GmbHLH30转录因子在铝胁迫下特异性的结合，该片段包含3个能与bHLH转录因子结合的顺式作用元件。通过构建验证转录因子转录活性的植物载体，在烟草体内再次验证这种结合作用。实验结果表明，铝胁迫下，编码GmbHLH30转录因子基因的转录和表达首先受到诱导，接着该转录因子与MATE基因的启动子区域的顺式作用元件结合，调控MATE蛋白的表达来应答铝胁迫。构建植物表达载体pK2-35S-GmbHLH30，转染烟草，筛选获得耐铝能力较强的阳性表达植株GmbHLH30-10和20