Abiotic stresses severely constrain soybean productivity worldwide. Here we demonstrate that gmeif2b5 (eukaryotic initiation factors) mutants confer dual stress tolerance through coordinated mechanisms. Integrative RNA-Seq and protein interaction analyses revealed that gmeif2b5 mutants increase lignin deposition to increase drought resilience and balanced Na+/K+ homeostasis to enhance salt tolerance; GmeIF2B5 physically interacts with GmPRX4, a plant heme peroxidase; GmPRX4 overexpression increases drought and salt resistance in soybean; GmeIF2B5 plays the predominant role in the GmeIF2B5-GmPRX4 module, and double mutants exhibiting synergistic stress tolerance improvements. Our work uncovers a 'GmeIF2B5-GmPRX4 regulatory axis' that: mobilises lignin-based structural fortification for enhanced drought resistance and orchestrates ionic equilibrium for increased salt tolerance. This study pioneers the role of eIF2B genes in soybean stress adaptation, establishing a multi-tiered regulatory node for precision molecular design of stress-resilient crops.

The degree of forage lignification is a key factor affecting its digestibility by ruminants such as cattle and sheep. Sudan grass (Sorghum sudanense S.) is a high-quality sorghum forage, and its lignocellulose is mostly stored in the secondary cell wall. However, the secondary cell wall synthesis mechanism of Sudan grass has not yet been studied in depth. To further study the secondary cell wall synthesis mechanism of Sudan grass using established transcriptome data, this study found that CcNAC6, a homologous gene of Arabidopsis AtSND2, is related to the secondary cell wall synthesis of Sudan grass. Accordingly, we constructed a CcNAC6-overexpressing line of Arabidopsis to investigate the function of the CcNAC6 gene in secondary cell wall synthesis. The results showed that the overexpression of the CcNAC6 gene could significantly increase the lignin content of Arabidopsis. Based on subcellular localization analysis, CcNAC6 is found in the nucleus. In addition, yeast two-hybridization screening showed that CcCP1, associated with secondary cell wall synthesis, can interact with CcNAC6. Therefore, the above results indicate that CcNAC6 has a positive regulatory effect on the secondary cell wall synthesis of Sudan grass, and it is speculated that CcNAC6 may be the main regulator of the secondary cell wall synthesis of Sudan grass through its interaction with another regulatory protein, CcCP1. This study provides a theoretical basis and new genetic resources for the creation of new Sudan grass germplasm with a low lignin content.

Sudan grass is a high-quality forage of sorghum. The degree of lignification of Sudan grass is the main factor affecting its digestibility in ruminants such as cattle and sheep. Almost all lignocellulose in Sudan grass is stored in the secondary cell wall, but the mechanism and synthesis of the secondary cell wall in Sudan grass is still unclear. In order to study the mechanism of secondary cell wall synthesis in Sudan grass, we used an in vitro induction system of Sudan grass secondary cell wall. Through transcriptome sequencing, it was found that the NAC transcription factor CcNAC1 gene was related to the synthesis of the Sudan grass secondary cell wall. This study further generated CcNAC1 overexpression lines of Arabidopsis to study CcNAC1 gene function in secondary cell wall synthesis. It was shown that the overexpression of the CcNAC1 gene can significantly increase lignin content in Arabidopsis lines. Through subcellular localization analysis, CcNAC1 genes could be expressed in the nucleus of a plant. In addition, we used yeast two-hybrid screening to find 26 proteins interacting with CcNAC1. GO and KEGG analysis showed that CcNAC1 relates to the metabolic pathways and biosynthesis of secondary metabolites. In summary, the synthesis of secondary cell wall of Sudan grass can be regulated by CcNAC1.

Seedling drought stress is one of the most important constraints affecting soybean yield and quality. To unravel the molecular mechanisms under soybean drought tolerance, we conducted comprehensive comparative transcriptome analyses of drought-tolerant genotype Jindou 21 (JD) and drought-sensitive genotype Tianlong No.1 (N1) seedlings that had been exposed to drought treatment. A total of 6038 and 4112 differentially expressed genes (DEGs) were identified in drought-tolerant JD and drought-sensitive N1, respectively. Subsequent KEGG pathway analyses showed that numerous DEGs in JD are predominately involved in signal transduction pathways, including plant hormone signaling pathway, calcium signaling pathway, and MAPK signaling pathway. Interestingly, JA and BR plant hormone signal transduction pathways were found specifically participating in drought-tolerant JD. Meanwhile, the differentially expressed CPKs, CIPKs, MAPKs, and MAP3Ks of calcium and MAPK signaling pathway were only identified in JD. The number of DEGs involved in transcription factors (TFs) is larger in JD than that of in N1. Moreover, some differently expressed transcriptional factor genes were only identified in drought-tolerant JD, including FAR1, RAV, LSD1, EIL, and HB-PHD. In addition, this study suggested that JD could respond to drought stress by regulating the cell wall remodeling and stress-related protein genes such as EXPs, CALSs, CBPs, BBXs, and RD22s. JD is more drought tolerant than N1 owing to more DEGs being involved in multiple signal transduction pathways (JA, BR, calcium, MAPK signaling pathway), stress-related TFs, and proteins. The above valuable genes and pathways will deepen the understanding of the molecular mechanisms under drought stress in soybean.

NAC transcription factors (TFs) could regulate drought stresses in plants; however, the function of NAC TFs in soybeans remains unclear. To unravel NAC TF function, we established that GmNAC12, a NAC TF from soybean (Glycine max), was involved in the manipulation of stress tolerance. The expression of GmNAC12 was significantly upregulated more than 10-fold under drought stress and more than threefold under abscisic acid (ABA) and ethylene (ETH) treatment. In order to determine the function of GmNAC12 under drought stress conditions, we generated GmNAC12 overexpression and knockout lines. The present findings showed that under drought stress, the survival rate of GmNAC12 overexpression lines increased by more than 57% compared with wild-type plants, while the survival rate of GmNAC12 knockout lines decreased by at least 46%. Furthermore, a subcellular localisation analysis showed that the GmNAC12 protein is concentrated in the nucleus of the tobacco cell. In addition, we used a yeast two-hybrid assay to identify 185 proteins that interact with GmNAC12. Gene ontology (GO) and KEGG analysis showed that GmNAC12 interaction proteins are related to chitin, chlorophyll, ubiquitin–protein transferase, and peroxidase activity. Hence, we have inferred that GmNAC12, as a key gene, could positively regulate soybean tolerance to drought stress.

NAC proteins represent one of the largest transcription factor (TF) families involved in the regulation of plant development and the response to abiotic stress. In the present study, we elucidated the detailed role of GmNAC8 in the regulation of drought stress tolerance in soybean. The GmNAC8 protein was localized in the nucleus, and expression of the GmNAC8 gene was significantly induced in response to drought, abscisic acid (ABA), ethylene (ETH) and salicylic acid (SA) treatments. Thus, we generated GmNAC8 overexpression (OE1 and OE2) and GmNAC8 knockout (KO1 and KO2) lines to determine the role of GmNAC8 in drought stress tolerance. Our results revealed that, compared with the wild type (WT) plant, GmNAC8 overexpression and GmNAC8 knockout lines exhibited significantly higher and lower drought tolerance, respectively. Furthermore, the SOD activity and proline content were significantly higher in the GmNAC8 overexpression lines and significantly lower in the GmNAC8 knockout lines than in the WT plants under drought stress. In addition, GmNAC8 protein was found to physically interact with the drought-induced protein GmDi19-3 in the nucleus. Moreover, the GmDi19-3 expression pattern showed the same trend as the GmNAC8 gene did under drought and hormone (ABA, ETH and SA) treatments, and GmDi19-3 overexpression lines (GmDi19-3-OE9, GmDi19-3-OE10 and GmDi19-3-OE31) showed enhanced drought tolerance compared to that of the WT plants. Hence, the above results indicated that GmNAC8 acts as a positive regulator of drought tolerance in soybean and inferred that GmNAC8 probably functions by interacting with another positive regulatory protein, GmDi19-3.

大豆(Glycine max(L.)Merr.)起源于中国,可以为人类提供主要的植物蛋白和油脂.随着环境条件的日渐恶劣,中国干旱与半干旱的地区发生干旱、高温、盐渍等非生物胁迫的频率日渐增高,严重影响了中国大豆的生产.转基因技术的出现使培育植物新品种的效率大大提高.利用转基因技术提高大豆对非生物胁迫耐受性的报道已经日渐增多.因此,对利用转基因技术来提高大豆对非生物胁迫耐受性的研究进行综述对于大豆生物工程育种具有十分重要的意义.

转基因大豆油是否可以安全食用,目前仍困扰着大部分中国消费者.鉴于此,文中从大豆的重要性、什么是转基因大豆、抗草甘膦大豆品种的创制及对根际微生物群落的影响、中美两国对转基因大豆的标识问题等方面进行了科普.目前中国已经建立一套适合中国国情的转基因生物安全法律法规和管理条例,为转基因大豆的研究和应用保驾护航.

[目的]本文旨在研究MYB类转录因子基因GmMYB46的结构特征和定位,并阐述其对不同胁迫的响应,为明确其在逆境胁迫下的作用奠定基础.[方法]从大豆品种‘晋豆21’中克隆出GmMYB46的CDS序列,采用生物信息学方法对该基因及其编码蛋白进行分析,并利用PlantCARE软件分析GmMYB46基因启动子元件.通过洋葱表皮细胞瞬时表达系统对GmMYB46蛋白质全长及不同结构域M1 (aa1 ～ 123)和M2(aa124～334)进行亚细胞定位分析.采用实时荧光定量PCR检测GmMYB46在不同逆境处理下的表达情况.[结果]GmMYB46 CDS序列全长为1 005 bp,编码334个氨基酸,蛋白质相对分子质量为82.53×103,氨基酸序列中含有2个高度保守的SANT结构域.进化树分析表明该基因编码的蛋白与野生大豆GsMYB46亲缘关系最近.GmMYB46包含MBS、ABRE、GARE、TCA、LTR等逆境胁迫应答元件.亚细胞定位结果显示,pBIN-GmMYB46-GFP及pBIN-GmMYB46M1-GFP融合蛋白在细胞核中表达,pBIN-GmMYB46M2-GFP绿色荧光遍布整个细胞.实时荧光定量PCR分析表明,在干旱、盐(200 mmol· L-1 NaCl)、低温(4℃)、ABA(200 μmol·L-1)、SA(500 μmol· L-1)、GA(100 μmol·L-1)处理下均能诱导GmMYB46基因在大豆根、茎、叶中的上调表达.[结论]GmMYB46基因的保守结构域对亚细胞定位起决定性作用,该基因可能参与大豆对非生物胁迫的响应.

Heat shock protein 90 s (Hsp90s), one of the most conserved and abundant molecular chaperones, is an essential component of the protective stress response. A previous study reported at least 12 genes in the GmHsp90s family in soybean and that GmHsp90A2 overexpression enhanced thermotolerance in Arabidopsis thaliana. Here, we investigate the roles of GmHsp90A2 in soybean by utilizing stable transgenic soybean lines overexpressing GmHsp90A2 and mutant lines generated by the CRISPR/Cas9 system. The results showed that compared with wild-type plants (WT) and empty vector control plants (VC), T3 transgenic soybean plants overexpressing GmHsp90A2 exhibited increased tolerance to heat stress through higher chlorophyll and lower malondialdehyde (MDA) contents in plants. Conversely, reduced chlorophyll and increased MDA contents in T2 homozygous GmHsp90A2-knockout mutants indicated decreased tolerance to heat stress. GmHsp90A2 was found to interact with GmHsp90A1 in yeast two-hybrid assays. Furthermore, subcellular localization analyses revealed that GmHsp90A2 was localized to the cytoplasm and cell membrane; as shown by bimolecular fluorescence complementation (BiFC) assays, GmHsp90A2 interacted with GmHsp90A1 in the nucleus and cytoplasm and cell membrane. Hence, we conclude that GmHsp90A1 is able to bind to GmHsp90A2 to form a complex and that this complex enters the nucleus. In summary, GmHsp90A2 might respond to heat stress and positively regulate thermotolerance by interacting with GmHsp90A1.

转基因技术的突破打开了分子生物学的大门,为人类破译生物界遗传信息和定向改造生物提供了可能.转基因技术是利用自然界中现实存在的农杆菌侵染植物的现象,加以改造从而演化出来可以被科研实际应用的技术.自转基因技术可以在植物中实际应用以来,转基因植物在农业生产中产生了巨大的效益,降低了农药的施用量,降低了人力成本,改善了作物本身营养物质不均衡的缺点,为人类带来了巨大的效益.其实转基因技术并不可怕,可怕的是人们不能用理性的视角来看待转基因问题.

[目的]本研究通过对大豆水通道蛋白基因GmTIP1-1的克隆及其在不同胁迫处理下的差异表达分析,探究水通道蛋白在大豆耐受非生物胁迫中的作用机制。[方法]从大豆品种‘天隆1号’中克隆出GmTIP1-1的CDS序列,采用生物信息学方法对该基因及其编码蛋白进行分析。采用实时荧光定量PCR检测在不同逆境处理下GmTIP1-1在大豆根、茎、叶等组织的表达情况。利用基因枪轰击法对GmTIP1-1蛋白全长及不同跨膜结构域进行亚细胞定位分析。通过酵母双杂试验确定GmTIP1-1蛋白的互作蛋白,并利用实时荧光定量PCR检测在干旱处理下与GmTIP1-1互作蛋白基因的表达情况。[结果]GmTIP1-1的CDS序列全长为753 bp,编码250个氨基酸,等电点为6.51。系统进化分析发现,GmTIP1-1与苜蓿（Medicago truncatula）和黄瓜（Cucumis sativus）的亲缘关系十分相近。亚细胞定位结果显示,GmTIP1-1定位在细胞膜上。实时荧光定量PCR结果显示:GmTIP1-1在根中的表达量最高,在茎中次之,叶中最低;在干旱、ABA处理下均能诱导GmTIP1-1基因在大豆根、茎、叶中的表达;在干旱和ABA处理下,GmTIP1-1基因在根中的表达水平均高于在茎和叶中的表达水平。酵母双杂试验表明,GmTIP1-1与参与非生物胁迫调节的Gm SNARE蛋白以及响应逆境胁迫相关蛋白Gm F-box均存在互作。在干旱处理下,大豆根和茎中Gm SNARE和Gm F-box基因都会受到干旱诱导表达。[结论]GmTIP1-1蛋白定位于细胞膜,通过与参与非生物胁迫调节的Gm SNARE蛋白以及响应逆境胁迫相关蛋白Gm F-box互作来增强大豆对非生物胁迫的抗性。

[目的]WRKY转录因子与植物的生物和非生物胁迫应答密切相关.通过对大豆WRKY转录因子基因GmWRKY148的克隆及在大豆发状根中过表达后对疫霉根腐病抗性的分析,探究大豆与大豆疫霉菌互作的作用机理.[方法]以拟南芥的AtWRKY44序列为探针,利用同源克隆的方法在大豆Williams 82的根部组织中得到其同源基因Glyma.14G199800,命名为GmWRKY148.对GmWRKY148蛋白进行系统进化树分析;利用qRT-PCR方法分析该基因在大豆的根、茎、叶、子叶和接种大豆疫霉菌不同时间点的转录水平;利用双酶切的方法将GmWRKY148完整的CDS序列连接到植物过表达载体pBinGFP2中,利用基因枪法将重组质粒转化到洋葱表皮细胞中,进行亚细胞定位分析.利用发根农杆菌K599介导的遗传转化体系,经过GFP荧光筛选和qRT-PCR检测,获得大豆过表达GmWRKY148的阳性发状根(OE-GmWRKY148)和转入pBinGFP2空载体(EV)的阴性对照发状根.对过表达GmWRKY148发状根和阴性对照发状根接种大豆疫霉菌,统计病斑长度、疫霉积累量和卵孢子萌发情况.[结果]GmWRKY148的CDS序列全长为999 bp,编码332个氨基酸,等电点为7.61.系统进化分析发现,GmWRKY148与菜豆(Phaseolus vulgaris)、蒺藜苜蓿(Medicago truncatula)的WRKY转录因子亲缘关系十分相近,且与菜豆的亲缘关系最近.亚细胞定位结果显示,GmWRKY148定位在细胞核中.组织表达分析显示,GmWRKY148在根中的表达量最高,在茎和叶中次之,在子叶中最低.荧光定量PCR结果表明,接种大豆疫霉菌P6497后,在感病品种Williams和抗病品种Williams 82(含有Rps1k)中,GmWRKY148受诱导逐渐上调表达,在侵染24 h后表达水平均达到最高,但在Williams 82中的上调倍数更高.对过表达GmWRKY148和转空载体的大豆阳性发状根分别接种大豆疫霉菌P6497的菌丝块,比较接种24 h后的病斑长度和疫霉积累量,结果显示,与对照EV相比,过表达GmWRKY148大豆阳性发状根的病斑长度显著变短,疫霉积累量显著降低.对过表达GmWRKY148和EV的大豆阳性发状根分别接种疫霉菌P6497的游动孢子,并在接种后24、36和48 h用显微镜观察菌丝的侵染及卵孢子萌发数量,统计结果显示,过表达GmWRKY148的大豆阳性发状根与对照EV相比,菌丝的侵染率及卵孢子萌发率均显著降低.[结论]GmWRKY148参与调控大豆与大豆疫霉的互作,能够增强大豆对大豆疫霉菌的抗性.

miRNA (microRNA) are a class of endogenous and noncoding small molecular RNA with approximately 18-25 nucleotides (nt) in length.They can regulate gene expression through mRNA cleavage or translation inhibition.We analyzed the mature sequence,stem-loop structure and promoter sequence of gmamiR1507a.The level of gma-miR1507a in soybean tissue under stress was tested by stem-loop RT-PCR method.The target genes of gma-miR1507a were predicted by the online software psRNATarget.Then,we constructed artificial miRNA expression vectors-miRNA1507a-pCAMBIA3301.Furthermore,amiRNA1507a gene was overexpressed in soybean cotyledon and the transgenic hairy roots were obtained.The results showed that there were a number of cis-acting elements related to drought stress and pathogen infection in the promoter sequence of precursor gma-miR1507a and 9 target genes of gma-miR1507a were predicted.We obtained the transgenic hairy roots by hairy root-mediated genetic transformation system and GFP staining.The expression level of gmamiR1507a in the transgenic hairy roots significantly increased compared with that in empty vector.The expression of seven predicted target genes significantly reduced in transgenic hairy roots.Based on the results,the transgenic hairy roots of amiRNA 1507a overexpression could increase the expression of gma-miR1507a which could be used to analyze the function of gma-miR1507a.In addition,grma-miR1507a may involve in biotic stresses responses in soybean.