大豆盐碱胁迫响应基因GmLEA1的功能鉴定及其调控作用机制研究

Soybean [Glycine max (L.) Merr.], is one of the most economically important oilseed crops in China and worldwide. Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress that affects crop productivity. Late embryogenesis abundant (LEA) proteins are highly expressed at the late stages of embryo development in plant seeds. In many plant species, LEA proteins are also accumulated in vegetative tissues in response to abiotic stresses including drought, freezing, and salinity, as well as plant hormones such as abscisic acid and ethylene, and their overexpression in transgenic plants has resulted in increased tolerance to such abiotic stresses. For these reasons, it has been postulated that there is a positive correlation between the expression of LEAs and abiotic stress tolerance in plants. Although previous studies showed LEA proteins play important roles in stress tolerance, it still remains unclear about the potential function of LEA protein in regulating saline-alkaline stress. Previously, by using the transcriptome sequencing database of soybean subjected to saline-alkaline stress, a soybean LEA gene (GmLEA1) which is dramatically induced with saline-alkaline treatment, has been characterized and analyzed. In this project, the structure and property of GmLEA1 will be further illustrated. Meanwhile, the promoter sequence of GmLEA1 gene will be cloned and analyzed, and its core promoter region (CPR, including regulatory cis-elements), which is critical for the responsiveness of the gene to saline-alkaline stress, will be identified. Moreover, a yeast one-hybrid screen will be performed to isolate CPR binding proteins (such as transcription factors). The interaction between CPR and candidate transcription factor, as well as the protein binding capacity of CPR will then be confirmed by gel mobility shift assays (EMSA) and chromatin immunoprecipitation assay (ChIP). In addition, by using the transgenic technology such as gene overexpression, mutant replenishment, and CRISPR/Cas9 gene editing, the important role of GmLEA1 involved in saline-alkaline tolerance of soybean will be carried out. The results obtained in the study will contribute importantly to better understanding of the molecular mechanism of GmLEA1 in soybean under saline-alkaline stress, and provide the theoretical basis for increasing saline-alkaline tolerance of soybean by genetic engineering.

大豆是我国重要的粮食和油料作物，土壤盐碱化是限制其产量和品质的重要环境因子之一。植物胚胎发育晚期丰富（LEA）蛋白是一类逆境诱导表达蛋白，在非生物胁迫中起重要的保护作用，然而其在盐碱胁迫中的功能和调控机制还不是很清楚。申请人前期筛选到一个显著受盐碱胁迫诱导表达的LEA基因（GmLEA1）。本项目拟进一步研究GmLEA1基因的结构和蛋白生化特性；克隆GmLEA1基因启动子序列，结合GUS融合基因标记法分析其在盐碱胁迫下的诱导表达模式；利用酵母单杂交、凝胶阻滞和染色质免疫共沉淀等技术，发掘和鉴定GmLEA1基因响应盐碱胁迫调控的关键顺式作用元件和转录因子；通过超表达、突变体回补和基因编辑（CRISPR/Cas9系统）等转基因手段，明确GmLEA1在盐碱胁迫中的功能，解析GmLEA1调控植物耐盐碱的分子作用机理。本研究将有助于加深对GmLEA1蛋白功能的认识，并为大豆耐盐碱分子育种提供理论依据。

大豆（Glycine max（L.）Merr.）是我国乃至全球重要的油料作物，因其富含蛋白质和油脂而被广泛种植并用于食品、动物饲料和工业产品等。大豆产量受各种自然环境胁迫的影响而显著降低，其中，盐碱胁迫被认为是影响大豆产量的主要因素。因此，培育耐盐碱的大豆品种是保证油料作物安全的必要条件。目前，许多调控植物响应盐碱非生物胁迫关键过程（包括胁迫信号感知、信号转导和适应等）的基因已被鉴定。但大豆中耐盐碱相关基因的功能还有待深入研究。本研究利用大豆盐碱胁迫下的转录组数据，从大豆耐盐碱品种“N24852”中克隆得到一个胚胎发育晚期蛋白基因GmLEA1。GmLEA1基因具有大豆不同组织中表达具有特异性，且在大豆花和种子器官中具有较高的表达水平，在茎中的表达水平较低。生物信息学分析结果显示，GmLEA1基因启动子序列包含大量非生物胁迫相关的顺式作用元件，且其受盐碱、高温和外源植物激素脱落酸（ABA）处理显著诱导表达；此外，聚乙二醇（PEG）和冷胁迫处理显著抑制GmLEA1基因的表达。利用拟南芥原生质体、烟草叶片表皮瞬时表达体系和大豆毛状根表达系统，确定GmLEA1蛋白主要定位在细胞质和细胞核中；过表达GmLEA1基因提高了酵母、拟南芥对盐碱胁迫的耐受性。此外，利用过表达转基因手段，明确GmLEA1基因在大豆耐盐碱胁迫体内的功能，通过酵母双杂和转录本分析初步解析GmLEA1基因在大豆耐盐碱分子机制。本研究为抗盐碱性大豆分子育种在盐碱地合理有效利用提供理论依据，并为通过基因工程手段来提高大豆的品质和抗性奠定理论基础。