大豆[Glycine max (L.) Merr.]作为十分重要的粮食作物和油料作物，在我国农业生产、食品工业和国民经济发展中具有不可替代的战略地位。作为植物蛋白和食用油的主要来源，大豆不仅为人类提供丰富的营养物质，还在畜禽饲料、生物燃料和工业原料等领域发挥着重要作用。然而，盐胁迫作为影响植物生长和农业生产的主要非生物胁迫之一，严重制约大豆品质及产量。据统计，全球有10.7%的土壤盐碱化程度加剧，总面积高达13.81亿公顷，而我国盐碱土总面积约为3467万公顷，这对大豆生产构成了严峻挑战。盐胁迫通过渗透胁迫、离子毒害和氧化应激等影响大豆的生长发育，导致大豆植株矮小、叶片黄化和结荚减少，最终造成大豆品质和产量下降。 因此，挖掘大豆中的耐盐基因、解析耐盐分子机制并培育大豆耐盐品种，已成为大豆遗传改良和育种研究的重要内容。

烯醇化酶ENO2（Enolase2）在拟南芥、水稻等多种植物中的耐盐功能已经得到证实。然而，迄今为止除了GmENO2a（Glyma.09G153900）和GmENO2b（Glyma.16G204600），大豆GmENO基因家族的其他成员功能尚不清楚。本实验室前期通过酵母双杂交以盐胁迫响应蛋白GmHAL3b（Glyma.07G082800）为诱饵筛选大豆耐逆酵母核体系文库，一共筛选到包括GmENO2d（Glyma.19G190900.1）在内的21个候选互作蛋白。本研究以GmENO2d为研究对象，分析了其调控大豆耐盐性的功能，并且探究了GmENO2d分别与GmHAL3a（Glyma.03G020900）以及GmHAL3b之间的互作，同时调查了GmHAL3a和GmHAL3b双基因敲除株系与GmHAL3b敲除株系的田间农艺性状。研究结果如下：

1. 基因结构显示GmENO2d基因全长为5679 bp，CDS长度为1335 bp，包含17个外显子和16个内含子，5’端和3’端都含有UTR结构域。GmENO2d蛋白在N端和C端各包含一个典型的ENOLASE结构域，并且蛋白质二级结构中的Strand区域和Helix区域主要富集在ENOLASE结构域内。实时荧光定量PCR结果显示，GmENO2d在大豆根系、下胚轴、上胚轴、叶片、子叶五个组织中均有表达，在上胚轴中的相对表达量最高，而在子叶中最低。对大豆盐敏感品种天隆1号进行200 mM NaCl处理发现，叶片中GmENO2d受到盐胁迫的强烈诱导，GmENO2d的表达量在盐处理3 h和6 h显著下降，24 h 显著升高。GmENO2d的表达受GA、IAA、SA、ZT、MeJA和ABA多种植物激素诱导，其中对MeJA和ABA的响应最为显著。亚细胞定位显示GmENO2d是一个定位在植物细胞的细胞质、细胞膜和细胞核上的蛋白。

2. 通过发根农杆菌介导的大豆发状根转化技术获得了GmENO2d的过表达大豆发状根，用100 mM NaCl处理阳性大豆发状根发现，GmENO2d_OE大豆发状根的长度变化显著高于EV。利用烟草脆裂病毒（TRV）介导的基因沉默（VIGS）技术获得了GmENO2d沉默株系，发现GmENO2d基因沉默后降低了大豆幼苗的耐盐性。利用根癌农杆菌介导的大豆子叶节稳定遗传转化技术获得并通过检测Bar蛋白和GmENO2d转录水平鉴定到了3个GmENO2d的过表达株系（GmENO2d_OE1、GmENO2d_OE2和GmENO2d_OE3）。对GmENO2d过表达株系的不同生长阶段进行耐盐性分析发现，萌发期使用75 mM NaCl处理后，GmENO2d_OE1的胚根长显著高于野生型（WT）；幼苗期使用200 mM NaCl处理后，GmENO2d过表达株系的SPAD值、株高、K⁺运输系数显著高于WT，而MDA、H₂O₂和叶片Na⁺含量显著低于WT。以上对GmENO2d过表达发状根和过表达株系的耐盐性分析结果表明，GmENO2d正向调控大豆耐盐性。

3. 通过酵母回转验证了GmENO2d分别与GmHAL3a以及GmHAL3b在酵母中互作。通过BiFC验证了GmENO2d分别与GmHAL3a以及GmHAL3b在植物细胞的细胞质、细胞膜和细胞核互作，并且GmENO2d分别与GmHAL3a以及GmHAL3b互作不会导致GmENO2d的蛋白定位发生改变。与WT相比，盐处理3 h后盐胁迫响应基因GmHAL3a、GmCYP2、GmRbohB1和GmST1在GmENO2d_OE1叶片中的表达量显著下降，而GmHAL3b的表达量显著升高；盐处理24 h后这些盐胁迫响应基因以及GmNHX的表达量均显著升高。在田间环境下，与WT相比，GmHAL3a/b_KO株高升高，但分枝数、单株粒数、二粒荚数、三粒荚数、瘪荚数、单株产量、有效粒数和有效粒重降低；GmHAL3b_KO主茎节数、一粒荚数和瘪荚数升高，但单株粒数、二粒荚数、三粒荚数、单株产量、百粒重、有效粒数和有效粒重降低。GmENO2d进一步完善了GmHAL3介导的耐盐分子网络。

Soybean [Glycine max (L.) Merr.], as a vital food crop and oil crop, holds an irreplaceable strategic position in China’s agricultural production, food industry and national economic development. It not only provides abundant nutrients for humans, but also plays a significant role in diverse fields such as livestock and poultry feed, biofuels, and industrial raw materials. However, salt stress, as one of the primary abiotic stresses affecting plant growth and agricultural production, severely constrains the quality and yield of soybean. According to statistics, soil salinization is intensifying on 10.7% of the global land area, encompassing a total of 1.381 billion hectares. In China, the total area of saline-alkali soils is approximately 34.67 million hectares, which poses a severe challenge to the production of soybean.Salt stress adversely affects the growth and development of soybean through osmotic stress, ion toxicity, and oxidative stress, resulting in stunted plants, leaf chlorosis, and reduced pod formation. Ultimately, this leads to a decrease in quality and yield of soybean. Therefore, mining salt tolerant genes in soybean, elucidating the molecular mechanisms of salt tolerance, and cultivating salt tolerant soybean varieties have emerged as crucial objectives in soybean genetic improvement and breeding research.

The salt tolerance function of ENO2 (Enolase2) has been confirmed in various plants including Arabidopsis thaliana and rice (Oryza sativa L.). However, apart from GmENO2a (Glyma.09G153900) and GmENO2b (Glyma.16G204600), the functions of other GmENOs remain unknown. In our laboratory’s previous study, a salt-responsive protein GmHAL3b (Glyma.07G082800), was employed as bait in a yeast two-hybrid (Y2H) screen against a stress-tolerant soybean cDNA library. A total of 21 candidate interacting proteins including GmENO2d (Glyma.19G1909000.1) were screened. In this study, the function of GmENO2d in regulating salt tolerance in soybean was analyzed, and the interactions between GmENO2d and GmHAL3a (Glyma.03G020900), as well as between GmENO2d and GmHAL3b were investigated. Additionally, the field agronomic traits of GmHAL3a and GmHAL3b double-gene knockout lines, along with GmHAL3b knockout lines were examined. Findings were as follows:

1. The gene structure revealed that GmENO2d has a total length of 5679 bp and its coding sequence (CDS) was 1335 bp, containing 17 exons and 16 introns. Both the 5' and 3' ends contain UTR domains. GmENO2d protein contains one typical ENOLASE domain at both its N-terminus and C-terminus. Within the secondary structure of the protein, the Strand region and Helix region are predominantly enriched within the ENOLASE domains. The results of qRT- PCR showed that GmENO2d was expressed in roots, hypocotyls, epicotyls, leaves and cotyledons, with the highest relative expression in epicotyls and the lowest in cotyledons. Treatment of soybean salt-sensitive variety Tianlong 1 with 200 mM NaCl revealed that GmENO2d was strongly induced by salt stress. The expression level of GmENO2d in the leaves significantly decreased at 3 h and 6 h of salt treatment, and significantly increased at 24 h. The expression of GmENO2d was induced by a variety of plant hormones, including GA, IAA, SA, ZT, MeJA and ABA, with the most significant response to MeJA and ABA. Subcellular localization revealed that GmENO2d is a protein localized in the cytoplasm, cell membrane and nucleus of plant cells.

2. We obtained GmENO2d overexpressing soybean hair roots by Agrobacterium rhizogenes mediated soybean hair root transformation technology. When positive soybean hair roots were treated with 100 mM NaCl, the length change of GmENO2d_OE was significantly higher than that of EV. GmENO2d silenced strains were obtained using tobacco crispness virus (TRV) - mediated gene silencing (VIGS) technology, and it was found that GmENO2d gene silencing reduced salt tolerance in soybean seedlings. Three GmENO2d-overexpressing lines (GmENO2d_OE1, GmENO2d_OE2 and GmENO2d_OE3) were obtained by Agrobacterium tumetionium-mediated stable genetic transformation of soybean cotyledons. Salt tolerance analysis of GmENO2d overexpression lines at different growth stages showed that GmENO2d_OE1 had higher radicle length after 75 mM NaCl treatment at germination stage than the wild type (WT), and after 200 mM NaCl treatment in the seedling stage, the GmENO2d overexpression lines had higher SPAD value, plant height, K⁺ selected coefficient, and lower MDA, H₂O₂ and leaf Na⁺ content than the WT. The above results indicated that GmENO2d positively regulated salt tolerance in soybean.

3. The interaction between GmENO2d and GmHAL3a, as well as between GmENO2d and GmHAL3b in yeast was verified by yeast rotation. BiFC was used to verify that GmENO2d interacted with GmHAL3a and GmHAL3b in the cytoplasm, cell membrane and nucleus of plant cells, respectively, which did not change the protein localization of GmENO2d. Compared with the WT, the expression levels of salt stress responsive genes  GmHAL3a, GmCYP2, GmRbohB1 and GmST1 in GmENO2d_OE1 leaves significantly decreased, while the expression level of GmHAL3b significantly increased after 3 h of salt treatment. The expression levels of these genes and GmNHX significantly increased after 24 h of salt treatment. In the field, compared with the WT, GmHAL3a/b_KO increased plant height, but decreased the number of branches, seeds per plant, two pods, three pods, dead pods, yield per plant, effective seeds number and effective grain weight. In GmHAL3b_KO, the number of main stem nodes, one pod and flat pod increased, but the number of seeds per plant, two pods, three pods, yield per plant, 100 seeds weight, effective seeds number and effective seeds weight decreased. GmENO2d further enhances the salt tolerant molecular network mediated by GmHAL3.