谷胱甘肽硫转移酶（glutathione S-transferase，GST）是一种广泛存在于动植物和微生物等生物体内的多功能酶。在胁迫条件下，GST可以将谷胱甘肽与羟基自由基、膜脂的氧化物以及其它的代谢物等结合起来，达到减少有害物质对植物伤害的目的。高温高湿胁迫是我国南方春大豆在种子生理成熟期（R7期）不可避免的灾害，易造成种子收获前发生劣变，进而影响大豆种子的活力。前期研究发现，从种子活力遗传性存在差异的抗劣变品种湘豆3号与不抗劣变品种宁镇1号中筛选发现响应高温高湿（HTH）胁迫的关键蛋白大豆膜联蛋白GmANN和大豆钙调蛋白GmCDPK SK5，在拟南芥中过表达GmANN和GmCDPK SK5，发现它们参与高温高湿胁迫下种子活力的形成，GmGSTa与这两个蛋白都存在互作关系，qRT-PCR（实时定量反转录PCR）结果表明GmGSTa与GmANN在转录水平上协同响应高温高湿胁迫。为进一步研究GmGSTa是如何响应高温高湿胁迫的，本研究通过对其启动子的活性分析，运用酵母双杂交技术筛选与GmGSTa存在相互作用的蛋白，分析其高温高湿胁迫和干旱胁迫下的功能，来解析GmGSTa应答胁迫的分子机理，为利用GmGSTa基因培育抗高温高湿和抗旱大豆新品种提供关键候选基因。主要研究结果如下：

1. GmGSTa启动子不仅对盐、干旱和高温高湿胁迫有响应，对外源施加的ABA也有响应。构建GmGSTaPRO::GUS融合表达载体并在烟草瞬时表达系统和转基因拟南芥中进行GUS染色，结果显示，在烟草瞬时表达系统中，GmGSTa启动子对盐，干旱和高温高湿胁迫均有不同程度的响应；在转基因拟南芥中，对GmGSTa启动子的表达模式进行分析，发现GmGSTa在根、叶、花的雄蕊以及发育阶段和成熟阶段的种子中均有不同程度的表达，随着盐、干旱和ABA处理浓度的增加，GUS酶活性逐渐增强，GmGSTa启动子活性显著增加。

2. 筛选出与GmGSTa启动子上的干旱应答元件DRE1结合的热激转录因子GmHSFA2和脱水元件结合蛋白GmDREB2A2，分别负调控和正调控GmGSTa的表达。本研究以GmGSTa启动子上DRE1元件为诱饵通过酵母单杂文库（高温高湿库）进行筛选，得到66个蛋白，从中选取与本实验相关的2个转录因子GmHSFA2，GmDREB2A2进行后续研究，通过酵母单杂交回补验证试验发现GmHSFA2和GmDREB2A2能够和GmGSTa启动子的顺式作用元件DRE1结合，双荧光素酶实验证明GmHSFA2抑制GmGSTa启动子的活性，GmDREB2A2促进GmGSTa启动子的活性。在大豆根中过表达GmHSFA2和GmDREB2A2，发现GmHSFA2对GmGSTa有负调控作用，GmDREB2A2对GmGSTa有正调控作用。

3. 筛选出与GmGSTa存在互作的蛋白GmGSTU18、GmHSP20和GmOLE1。本研究以GmGSTa为诱饵蛋白，从实验室构建好的大豆高温高湿膜酵母文库质粒中筛选互作蛋白，共筛选得到52个与GmGSTa存在相互作用的蛋白，对其进行功能注释发现，这些候选互作蛋白主要涉及种子成熟、储藏、逆境抵御、转运、代谢产物的合成调控以及蛋白质修饰等方面。从中挑选谷胱甘肽S-转移酶GSTs基因家族的GmGSTU18、小分子热激蛋白GmHSP20和油质蛋白GmOLE1用于开展进一步的验证工作，通过酵母双杂交回转验证以及荧光素酶互补实验分别在体外和体内验证了GmGSTa与GmGSTU18、GmHSP20和GmOLE1之间存在特异性互作。受到高温高湿胁迫后，GmGSTU18、GmHSP20和GmOLE1分别在大豆R7期种子中的表达量有不同程度的升高。GmGSTU18、GmHSP20和GmOLE1在两个大豆品种的五种处理中（盐、碱、盐碱、干旱、ABA）均有不同程度的响应。

4. GmGSTa正向调控植株对干旱胁迫的耐受性。利用TRV-VIGS技术在大豆中沉默GmGSTa，干旱胁迫后，GmGSTa沉默植株相较于对照植株脱水速率变快，且更易积累H₂O₂，脯氨酸、可溶性糖含量下降，SOD活性降低，MDA含量和相对电导率升高，降低了植株对干旱胁迫的耐受性。在大豆根中过表达GmGSTa，阳性根中较空载根积累的H₂O₂更少。在拟南芥中过表达GmGSTa，过表达植株叶片离体失水率要明显低于野生型（WT），在干旱胁迫和ABA处理下过表达株系种子的发芽率、发芽势和发芽指数都要显著高于WT，且叶片中积累的O²⁻更少。过表达GmGSTa通过提高SOD活性、降低MDA含量、增加脯氨酸、可溶性糖和GSH含量，来提高细胞内氧化还原平衡和渗透压稳定，从而增强植株的耐旱性。

5. GmGSTa正向调控植株对HTH胁迫的耐受性及种子活力。沉默GmGSTa植株较对照植株在HTH胁迫后MDA含量升高，SOD活性、脯氨酸和可溶性糖含量降低，降低了植株对HTH胁迫的耐受性。过表达GmGSTa转基因拟南芥株系的种子在HTH处理后的发芽率、发芽势和发芽指数都高于WT种子。在正常条件下，过表达种子比WT种子POD活性和可溶性糖含量高，HTH胁迫后过表达种子较WT种子中SOD、POD活性升高，脯氨酸和可溶性糖含量升高，促进了种子的抗氧化能力和渗透调节能力，进而提高了拟南芥植株对HTH胁迫的耐受性及种子活力。

综上，通过对GmGSTa基因启动子的活性分析，并在基因上游找到能与DRE1元件结合的GmHSFA2，GmDREB2A2调控基因的表达，且找到与之存在特异性互作的GmGSTU18、GmHSP20和GmOLE1的蛋白，并解析GmGSTa正向调控耐高温高湿和耐旱性的功能，初步探究GmGSTa基因的分子机制，为大豆培育耐旱、耐高温高湿和高种子活力品种提供相关理论基础。

Glutathione S-transferase (GST) is a multifunctional enzyme widely found in organisms such as plants, animals and microorganisms. Under stress conditions, GST can combine glutathione with hydroxyl radicals, oxides of membrane lipids and other metabolites to reduce the damage of harmful substances to plants. High temperature and humidity（HTH） stress is an unavoidable disaster for spring soybean in the south of China during the physiological maturity of seeds (R7 stage), which is prone to cause the deterioration of seeds before harvest, and then affect the vigor of soybean seeds.Previous studies have found that the key proteins of soybean annexin GmANN and soybean calmodulin GmCDPK SK5 in response to high temperature and high humidity (HTH) stress were screened from the resistant variety Xiangdou NO.3 and the non-resistant variety Ningzhen NO.1 with different heritability of seed vigor, and overexpressed in Arabidopsis thaliana, and were found to be involved in the formation of seed vigor under high temperature and humidity stress, and GmGSTa interacts with these two proteins. qRT-PCR(Real-Time Quantitative Reverse Transcription PCR) results indicated that GmGSTa and GmANN synergized at the transcriptional level in response to high temperature and humidity stress.In order to further investigate how GmGSTa responds to high temperature and humidity stress, this study analyzed the activity of its promoter, screened for proteins that bind to the DRE1 element in the GmGSTa promoter, used the yeast two-hybrid system to identify proteins that interact with GmGSTa, and analyzed the functions of GmGSTa under high temperature and humidity stress as well as drought stress. The molecular mechanism of GmGSTa response to stress provides a key candidate gene for the use of GmGSTa gene to cultivate new soybean varieties with high temperature and high humidity resistance and drought resistance. The main results are as follows:

1. The GmGSTa promoter is not only responsive to salt, drought and high temperature and humidity stresses, but also to exogenously applied ABA. The GmGSTaPRO::GUS fusion expression vector was constructed and subjected to GUS staining in tobacco transient expression system and transgenic Arabidopsis thaliana, and the results showed that the GmGSTa promoter responded to salt, drought, high temperature and humidity stresses to varying extents in tobacco transient expression system. In transgenic Arabidopsis thaliana, the expression pattern of the GmGSTa promoter was analyzed, and it was found that GmGSTa was differentially expressed in roots, leaves, and stamens of flowers as well as seeds at developmental and maturation stages, and that the GUS enzyme activity was gradually enhanced and the GmGSTa promoter activity was significantly increased with the increase in the concentration of salt, drought, and ABA treatments.

2. The heat shock transcription factor GmHSFA2 and the dehydration element binding protein GmDREB2A2 that bind to the drought response element DRE1 on the GmGSTa promoter were screened, which negatively regulated and positively regulated the expression of GmGSTa, respectively. In this study, the DRE1 element on the GmGSTa promoter was used as a bait to screen the yeast one-hybrid library (high temperature and humidity library), and 66 proteins were obtained. Two transcription factors GmHSFA2 and GmDREB2A2 related to this experiment were selected for further research. Yeast one-hybrid complementation verified that GmHSFA2 and GmDREB2A2 could bind to the cis-acting element DRE1 of the GmGSTa promoter. Dual luciferase assay showed that GmHSFA2 repressed the activity of the GmGSTa promoter, and GmDREB2A2 promoted the activity of the GmGSTa promoter. Moreover, GmHSFA2 and GmDREB2A2 were overexpressed in soybean hairy roots, and it was found that GmHSFA2 had a negative regulatory effect on GmGSTa, and GmDREB2A2 had a positive regulatory effect on GmGSTa.

3. The proteins GmGSTU18, GmHSP20 and GmOLE1 that interact with GmGSTa were screened. In this study, GmGSTa was used as the bait protein to screen the interacting proteins from the high temperature and humidity membrane yeast library plasmid of soybean constructed in the laboratory. A total of 52 proteins interacting with GmGSTa were screened. Functional annotation showed that these candidate interacting proteins were mainly involved in seed maturation, storage, stress resistance, transport, synthesis and regulation of metabolites, and protein modification. From them, GmGSTU18 of the glutathione S-transferase GSTs gene family, GmHSP20, a small molecule heat stress protein, and GmOLE1, an oleosin protein, were selected for carrying out further validation, and the existence of specific interactions between GmGSTa and GmGSTU18, GmHSP20, and GmOLE1 was verified in vitro and in vivo by yeast two-hybrid rotary validation as well as luciferase complementation assay, respectively. After high temperature and humidity stress, the expression levels of GmGSTU18, GmHSP20 and GmOLE1 in soybean R7 seeds increased to varying degrees. GmGSTU18, GmHSP20 and GmOLE1 had different degrees of response in five (salt, alkali, saline-alkali, drought, ABA) treatments of two soybean varieties.

4. GmGSTa positively regulates plant tolerance to drought stress. GmGSTa was silenced in soybean using TRV-VIGS technology, and after drought stress, GmGSTa-silenced plants became faster in dehydration rate and were more likely to accumulate H₂O₂, decreased proline and soluble sugar content, decreased SOD activity, and increased MDA content and relative conductivity compared with control plants, which reduced the tolerance of plants to drought stress. Overexpression of GmGSTa in soybean roots resulted in less H₂O₂ accumulation in the positive roots than in the null-loaded roots. Overexpression of GmGSTa in Arabidopsis thaliana resulted in a significantly lower rate of leaf water loss in leaves of overexpression plants than that of WT, and the germination rate, germination potential, and germination index of seeds of overexpression lines were significantly higher than those of WT under both drought stress and ABA treatments, and less O^2- was accumulated in the leaves. Overexpression of GmGSTa enhanced intracellular redox balance and osmotic pressure stabilization by increasing SOD activity, decreasing MDA content, and increasing proline, soluble sugar, and GSH content to enhance the drought tolerance of the plants.

5. GmGSTa positively regulated plant tolerance to HTH stress and seed vigor. Silencing of GmGSTa plants resulted in higher MDA content and lower SOD activity, proline, and soluble sugar content than that of control plants after HTH stress, which reduced the tolerance of the plants to HTH stress.Seeds overexpressing GmGSTa transgenic Arabidopsis lines showed higher germination rate, germination potential, and germination index than WT seeds after HTH treatment. Under normal conditions, overexpressed seeds showed higher POD activity and soluble sugar content than WT seeds, and the elevated SOD and POD activities, proline and soluble sugar contents in overexpressed seeds compared with WT seeds after HTH stress promoted the antioxidant and osmoregulatory capacities of the seeds, which in turn improved the tolerance to HTH stress and seed vigor of Arabidopsis plants.

In summary, through the analysis of the activity of the GmGSTa gene promoter, the expression of GmHSFA2 and GmDREB2A2 regulatory genes that can bind to the DRE1 element was found in the upstream of the gene, and the proteins of GmGSTU18, GmHSP20 and GmOLE1 that specifically interact with it were found, and the function of GmGSTa in positively regulating high temperature, high humidity and drought tolerance was analyzed. The molecular mechanism of GmGSTa gene was preliminarily explored, which provided a theoretical basis for soybean cultivation of drought tolerance, high temperature, high humidity and high seed vigor varieties.

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