植物在生长过程中通常会受到多种不利环境因素影响，如盐害、干旱、高温和低温胁迫等，其中盐胁迫是限制农业生产的主要因素之一。目前，全球耕地面积的20%受到盐胁迫的影响。大豆(Glycine max)是世界上种植最广泛的豆科植物，被认为是中度耐盐作物，但是在盐胁迫下其生长也被抑制、发芽率和结实率降低，进而导致减产。盐胁迫条件下形成的离子毒害对植物生长、发育和产量都会产生严重的不利影响，其中关于Na+毒害的研究较多，而以Cl-为代表的阴离子毒害仅有少量报道。就栽培大豆而言，在盐胁迫下，Na+和Cl-都可对生长中的大豆植株产生离子毒害，但是Cl-的毒害大于Na+毒害, 大豆受害程度与其叶片和茎中的Cl-含量呈正相关。因此，盐胁迫下大豆植株体内Cl-吸收、转运及其调控的分子机制已成为植物耐盐性研究的重要方面。转录因子在植物盐胁迫响应和适应过程发挥重要调控作用，在缓解植物盐离子毒害的基因转录调控方面，目前已被研究的转录因子主要通过调控以Na+、K+为代表的阳离子转运蛋白或通道基因的表达，进而调控上述阳离子在植株体内的吸收和分配及稳态维持来增强植物对盐逆境的适应能力。不过，介导以Cl-为代表的阴离子转运的氯离子通道蛋白家族基因(CLCs)的转录调控机制尚未见报道。

锚点本研究针对已被报道参与植物盐胁迫适应过程的大豆第I亚家族的8个CLCs基因，锚点建立基因共表达网络结合转录调控数据库筛选和鉴定其转录调控因子，并通过酵母单杂交、烟草瞬时表达、EMSA、qRT-PCR、转录激活活性、亚细胞定位、基因过表达大豆发根组合植株和转基因拟南芥的耐盐性分析等多种技术手段，着重对感兴趣的2类重要转录因子参与大豆耐盐性调控的生理功能机制进行了深入探究。主要研究结果如下：

利用植物基因共表达数据库Genevestigator构建栽培大豆第I亚家族8个CLCs基因的共表达网络，同时转录因子调控数据库Plant Reg Map预测大豆CLCs基因成员上游所有可能的转录调控因子。初步筛选到107个转录因子编码基因，其中，在GmCLC1基因上游预测到10个转录因子，在GmCLC-b1基因上游24个，GmCLC-b2基因上游8个，GmCLC-c1基因上游21个，GmCLC-c2基因上游7个，GmCLC-d1基因上游7个，GmCLC-d2基因上游22个和GmCLC-g基因上游8个。通过酵母单杂交实验和烟草叶片瞬时表达分析，最终共在GmCLC1、GmCLC-b1、GmCLC-c1、GmCLC-c2和GmCLC-d2上游鉴定到8个转录因子： Glyma.04G054200(WRKY)、Glyma.08G152500(bHLH)、Glyma.13G187500(MYB)、Glyma.10G257900(C2H2)、Glyma.04G044900(C2H2)、Glyma.05G234600(MYB)、Glyma.20G203700(ERF)、Glyma.04G170100(MYB)。

基于大豆GmCLC1和GsCLC-c2增强植物耐盐的功能，本研究首先对GmCLC1的候选转录因子GmbHLH3的功能进行深入探究。通过启动子序列点突变、酵母单杂交（yeast one-hybrid, Y1H）、烟草瞬时表达、GUS染色、EMSA实验发现GmbHLH3转录因子可结合GmCLC1启动子-550位点的G-box元件，激活下游基因表达。此外，GmbHLH3基因过表达的大豆发根组合植株根中GmCLC1的表达水平显著高于Vector植株。对大豆发根组合植株的耐盐性分析发现，从盐胁迫下植株的生长表型、株高、叶片死细胞数量、叶片MDA含量和根和叶REL值等指标都能显示OEGmbHLH3植株较Vector植株耐盐性明显增强。盐处理下OEGmbHLH3植株根中Cl-和NO3-的积累显著增加，并显著降低Cl-向地上部（茎和叶）的转运和积累，从而导致其根、茎和叶各器官Cl-/NO3-值显著降低。OEGmbHLH3发根植株根、茎和叶Na+含量及茎、叶Na+/K+值也呈类似变化。盐胁迫下转GmbHLH3基因拟南芥也表现出明显增强的耐盐性，其根中Cl-和Na+含量、根和地上部NO3-和K+含量均与大豆组合植株表现的变化趋势基本一致。在盐处理的转GmbHLH3拟南芥中AtCLCc、AtCLCg、AtNCED3和AtP5CS1被激活表达上调。而GmbHLH3基因沉默的RNAi大豆发根组合植株在盐胁迫下的表型与Vector植株一致，未出现耐盐性降低的表型。

通过Y1H、烟草瞬时表达和GUS活性测定证明GmERF160通过结合GmCLC-c2的启动子激活报告基因。进一步对野生大豆GsCLC-c2上游转录因子预测，也鉴定到1个ERF家族成员并且与GmERF160高度同源，命名为GsERF160。Gs/GmERF160基因在盐胁迫诱导下表达上调，尤其在根中更为明显。利用大豆发根组合植株和转基因拟南芥对Gs/GmERF160基因的功能进行分析，发现Gs/GmERF160基因过表达发根组合植株和转Gs/GmERF160基因拟南芥在盐胁迫下均表现出受盐害较轻，说明Gs/GmERF160能够正调控植物的耐盐性，并且能够提高大豆发根植株根中GmCLC-c2的表达水平。分析大豆发根组合植株根、茎和叶中的离子含量，发现盐处理下Gs/GmERF160基因过表达大豆组合植株根中Cl-显著增加，茎和叶Cl-含量显著降低；NO3-则在过表达植株根、茎和叶中的含量明显升高，最终导致茎和叶Cl-/NO3-值显著降低；而Na+/K+值在茎和叶中显著降低。转Gs/GmERF160基因拟南芥植株耐盐性也明显增强，这与盐处理下其地上部Cl-/NO3-和Na+/K+值显著低于野生型（WT）有关。在此基础上，鉴定到一个与GmERF160产生互作效应的GmEIN3蛋白。

综上所述，GmbHLH3转录因子在大豆中可通过上调GmCLC1的表达水平，介导盐胁迫下大豆植株根对Cl-、NO3-等的吸收和向地上部的转运过程以保持相对较低的Cl-/NO3-和Na+/K+值，使得GmbHLH3过表达大豆发根组合植株的耐盐性明显增强， ABA介导的信号途径或脯氨酸代谢途径也可能参与了上述盐害缓解过程。锚点在野生和栽培大豆Gs/GmCLC-c2基因上游鉴定到的转录因子Gs/GmERF160也参与盐胁迫下植株体内的阴离子稳态调节，其主要通过激活并增强GmCLC-c2的表达，调节Cl-、NO3-等在Gs/GmERF160过表达大豆发根组合植株或转基因拟南芥植株体内的分布，以维持地上部明显较低的Cl-/NO3-值，进而增强耐盐性。

Plants are usually affected by a variety of unfavorable environmental factors during their growth, such as salt, drought, high and low temperature stresses, among them salt stress is one of the main factors limiting agricultural production. Currently, 20% of the arable land is affected by salt stress. Soybean (Glycine max), the most widely planted cereal legume in the world, is considered to be a moderately salt-tolerant crop, but its growth was also inhibited under salt stress, germination, and seed setting rates are reduced, resulting in reduced yields. The ion toxicity formed under salt stress will have serious adverse effects on plant growth, development, and yield. Among them, there are many studies on Na+ toxicity, but only a few reports on anion toxicity represented by Cl-. In terms of cultivated soybean, under salt stress, both Na+ and Cl- could produce ion toxicity to growing soybean plants, but the toxicity of Cl- was greater than that of Na+, and the degree of soybean injury was positively correlated with the content of Cl- in leaves and stems. Therefore, the molecular mechanism of Cl- uptake, transport, and regulation in soybean plants under salt stress has become an important aspect of plant salt tolerance research. Transcription factors play an important regulatory role in the response and adaptation of plants to salt stress. In terms of transcriptional regulation of genes alleviating salt ion toxicity in plants, transcription factors have been reported to enhance the adaptability of plants to salt stress by regulating the expression of cation (Na+ and K+) transporter or channel genes, and then regulating the absorption, distribution, and homeostasis of these cations in plants. However, the transcriptional regulation mechanism of chloride channel protein family genes (CLCs) mediating anion (Cl-) transport has not been reported yet.

In this study, 8 CLCs of soybean subfamily I have been reported to be involved in the adaptation process of plants to salt stress. Gene co-expression network and transcriptional regulatory database was used to identify upstream transcriptional regulators; and Y1H, transient expression in tobacco leaves, EMSA, qRT-PCR, transcriptional activation activity, subcellular localization, salt tolerance identification of gene-overexpressed soybean hairy root-composite plants and transgenic Arabidopsis were performed, focused on the exploration of the physiological functions and mechanisms of two types important transcription factors involved in the regulation of soybean salt tolerance. The main results are as follows:

The plant gene co-expression database Genevestigator was used to construct the co-expression network of 8 CLCs of cultivated soybean subfamily I, and the transcription factor regulation database Plant Reg Map predicted all possible transcriptional regulators upstream of soybean CLCs. 107 transcription factor encoding genes were preliminarily screened, among which, 10 transcription factors predicted upstream of GmCLC1, 24 upstream of GmCLC-b1, 8 upstream of GmCLC-b2, 21 upstream of GmCLC-c1, and 7 upstream of GmCLC-c2, 7 upstream of GmCLC-d1, 22 upstream of GmCLC-d2 and 8 upstream of GmCLC-g. Yeast one-hybrid experiments and transient expression analysis in tobacco leaves were performed and 8 transcription factors were identified upstream of GmCLC1, GmCLC-b1, GmCLC-c1, GmCLC-c2, and GmCLC-d2: Glyma.04G054200(WRKY), Glyma.08G152500(bHLH), Glyma.13G187500(MYB), Glyma.10G257900(C2H2), Glyma.04G044900(C2H2), Glyma.05G234600(MYB), Glyma.20G203700(ERF), and Glyma.04G170100(MYB).

Based on the salt tolerance function of soybean GmCLC1 and GsCLC-c2, this study firstly explored the function of GmbHLH3, a candidate transcription factor of GmCLC1. Through point mutation of promoter sequence, Y1H, transient expression in tobacco, GUS activity assay, and EMSA, it was found that GmbHLH3 transcription factor could bind to the G-box element at the promoter of GmCLC1 gene to activate the expression of the downstream gene. In addition, the expression level of GmCLC1 in the roots of GmbHLH3-overexpressing soybean hairy root-composite plants was significantly higher than that of Vector plants. The salt tolerance of OEGmbHLH3 composite plants was significantly enhanced in terms of growth phenotype, plant height, number of dead cells in leaves, MDA content in leaves, and REL values in roots and leaves of plants. The accumulation of Cl- and NO3- in roots of OEGmbHLH3 plants significantly increased under salt treatment, and the transport and accumulation of Cl- to shoots (stems and leaves) were significantly decreased, resulting in a significant decrease of Cl-/NO3- values in roots, stems and leaves. The Na+ content in roots, stems and leaves and Na+/K+ values in stems and leaves of OEGmbHLH3 plants also showed similar changes. GmbHLH3-transgenic Arabidopsis also showed significantly enhanced salt tolerance, and the changes of Cl- and Na+ contents in roots, NO3- and K+ contents in roots and shoots were consistent with those of soybean composite plants. In addition, AtCLCc, AtCLCg, AtNCED3 and AtP5CS1 were activated and up-regulated in salt-treated GmbHLH3-transgenic Arabidopsis. The phenotype of the GmbHLH3-RNAi soybean hairy root-composite plants was consistent with that of the Vector plants under salt stress, and did not show reduced salt tolerance.

GmERF160 activates the reporter gene by binding to the promoter of GmCLC-c2 as demonstrated by Y1H, tobacco transient expression and GUS activity assay. Further prediction of the upstream transcription factor of wild soybean GsCLC-c2, a gene with high homology to GmERF160 was identified and named GsERF160. GsERF160 and GmERF160 genes were up-regulated under salt stress, especially in roots. The functions of Gs/GmERF160 genes were analyzed by soybean hair-root composite plants and transgenic Arabidopsis. It was found that soybean composite plants and transgenic Arabidopsis showed less salt damage under salt stress, indicating that Gs/GmERF160 can positively regulate the salt tolerance of plants and activate the expression of GmCLC-c2 in the roots of soybean composite plants. Under salt treatment, the content of Cl- in roots of Gs/GmERF160-overexpressing soybean composite plants was significantly increased, while the content of Cl- in stems and leaves was significantly decreased, the content of NO3- in roots, stems and leaves of overexpressed plants was significantly increased, resulting in a significant decrease in the value of Cl-/NO3- in stems and leaves. The salt tolerance of Gs/GmERF160-transgenic Arabidopsis plants was also significantly enhanced, which was related to the significantly lower Cl-/NO3- and Na+/K+ values in shoots under salt treatment than those in wild type (WT). On this basis, a GmEIN3 protein that interacted with GmERF160 was identified.

In summary, the transcription factor GmbHLH3 can confer enhanced salt tolerance in GmbHLH3-overexpressing soybean hairy root-composite plants by upregulating the GmCLC1 expression, mediating the Cl- and NO3- absorption and their transportation to shoots to maintain relatively low Cl-/NO3- and Na+/K+ values, and ABA-mediated signaling pathway or proline metabolism pathway may also be involved in the process of above-mentioned salt damage alleviation. 锚点The transcription factor Gs/GmERF160, identified upstream of Gs/GmCLC-c2 gene in wild and cultivated soybeans, also participates in the regulation of anion homeostasis in plants under salt stress. It mainly regulates the distribution of Cl-, NO3-, etc. in Gs/GmERF160-overexpressing hairy root-composite plants or transgenic Arabidopsis by activating and enhancing the expression of GmCLC-c2, and maintaining significantly lower Cl-/NO3- value in shoots, thereby enhancing salt tolerance.

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