大豆盐胁迫相关GmNAC基因的鉴定、表达及变异分析
大豆耐盐基因定位及耐盐基因克隆研究进展解读
78 江苏农业科学 2010年第5期陈华涛, 陈新, 喻德跃, 等. 大豆耐盐基因定位及耐盐基因克隆研究进展[J].江苏农业科学, 2010(5:78-80.大豆耐盐基因定位及耐盐基因克隆研究进展陈华涛1, 2, 陈新, 喻德跃, 顾和平, 张红梅, 袁星星12111(1. 江苏省农业科学院蔬菜研究所, 江苏南京210014; 2. 南京农业大学国家大豆改良中心, 江苏南京210095摘要:土壤的盐渍化和次生盐渍化是影响农业生产和生态环境的重要因素之一。
大豆作为油料作物和经济作物, 在食品工业和农业生产中占重要地位。
大豆品种资源丰富, 不同品种间耐盐性差异较大, 使得培育耐盐高产等优良性状聚合的大豆品种成为可能。
相对于常规育种改良大豆的耐盐性, 分子标记辅助选择和转基因等新技术为我们改良大豆的耐盐能力提供了新途径。
本文综述了大豆耐盐基因定位及耐盐基因克隆的研究进展, 并探讨了分子标记辅助育种及转基因育种在大豆耐盐育种中的潜力和作用。
关键词:大豆; 耐盐性; 基因定位; 基因克隆中图分类号:S565. 101 文献标志码:A 文章编号:1002-1302(2010 05-0078-03土壤的盐渍化和次生盐渍化是影响农业生产和生态环境的重要因素之一。
全世界至少有20%的耕地盐渍化。
在干旱和半干旱地区, 土壤次生盐渍化日趋严重。
我国约有盐渍2化和次生盐渍化土地4000万h m 以上, 成为制约农业发展的主要环境因素之一。
栽培大豆[G lycine max (L . M err . ]属于中等耐盐作物, 其土壤盐度阈值是5.0ds/m。
灌溉水电导[1]率超过6. 7ds/m时, 植株就会死亡。
大豆品种间耐盐性存在差异。
据Abel 等的研究, 当土壤盐度从5. 0ds /m提高到10. 2ds/m时, 大豆植株的死亡率和叶片坏死均增加, 叶片出现缺绿, 茎秆重和籽实产量降低, 盐敏感品种与耐盐品种相比受到盐胁迫的影响更大。
大豆GmGolS2-1基因高温胁迫诱导表达及转基因烟草鉴定
大豆GmGolS2-1基因高温胁迫诱导表达及转基因烟草鉴定作者：邱爽张军何佳琦李铭杨周雨明邬长乐袁洪淼刘嘉仪翟莹来源：《江苏农业学报》2021年第01期摘要：肌醇半乳糖苷合成酶（GolS）是棉籽糖系列寡糖（RFO）生物合成途径中的关键酶，在植物应对非生物胁迫过程中发挥重要作用。
实时荧光定量RT-PCR结果显示，高温胁迫可以诱导GmGolS2-1在大豆幼苗中的表达。
将GmGolS2-1基因构建到植物表达载体pRI101上并通过叶盘法转化烟草，经卡那霉素抗性筛选，PCR及qRT-PCR检测共获得6株阳性转基因烟草植株（OE1～OE6）。
对野生型烟草植株和GmGolS2-1转基因烟草植株进行高温胁迫处理，结果显示野生型烟草的电解质渗透率和丙二醛含量均高于转基因烟草。
由此推测GmGolS2-1可以提高轉基因烟草的耐热性。
关键词：大豆;肌醇半乳糖苷;GolS基因;高温胁迫;转基因烟草中图分类号： S565.1 文献标识码： A 文章编号： 1000-4440（2021）01-0038-06Expression of soybean GmGolS2-1 induced by heat stress and identification of GmGolS2-1 transgenic tobaccoQIU Shuang1， ZHANG Jun2， HE Jia-qi1， LI Ming-yang1， ZHOU Yu-ming3， WU Chang-le1， YUAN Hong-miao1， LIU Jia-yi1， ZHAI Ying1（1.College of Life Science and Agro-Forestry， Qiqihar University， Qiqihar 161006，China;2.Branch of Animal Husbandry and Veterinary of Heilongjiang Academy of Agricultural Sciences， Qiqihar 161005， China;3.Jilin Zhongzhi Jiufang Consulting Co.， Ltd.， Changchun 130000， China）Abstract： Galactinol synthase （GolS） is the key enzyme in the biosynthetic pathway of raffinose family oligosaccharides （RFOs）， which plays an important role in the response to abiotic stresses of plants. The results of real-time fluorescence quantitative RT-PCR showed that the expression of GmGolS2-1 could be induced by high temperature stress in soybean seedlings. The GmGolS2-1 gene was constructed into expression vector pRI101 in plants and was transformed into tobacco using leaf disc method. Six positive transgenic tobacco plants （OE1-OE6） were obtained by kanamycin resistance screening， PCR and qRT-PCR. The wild-type and GmGolS2-1 transgenic tobacco plants were treated with heat stress. The results showed that the electrolyte leakage and malondialdehyde content of wild-type tobacco were both higher than that of transgenic tobacco. These data indicate that GmGolS2-1 can increase the tolerance to heat stress of transgenic tobacco.Key words： soybean;galactinol;GolS gene;heat stress;transgenic tobacco植物在遭受不良环境条件后，可以诱导合成大量渗透调节物质来增加植物细胞的渗透压，提高植物抵抗胁迫的能力，从而维持植物自身的代谢和生长发育。
大豆TGA转录因子基因GmTGA26在盐胁迫中的功能分析
DOI: 10.3724/SP.J.1006.2022.14123大豆TGA转录因子基因GmTGA26在盐胁迫中的功能分析柯丹霞*霍娅娅刘怡李锦颖刘晓雪信阳师范学院生命科学学院/ 大别山农业生物资源保护与利用研究院，河南信阳464000摘要：TGA转录因子是bZIP的一个亚家族，在病原体和非生物胁迫反应中发挥重要作用。
本研究在大豆中筛选并克隆得到1个TGA转录因子家族基因GmTGA26，同源蛋白比对表明GmTGA26具有保守的亮氨酸拉链结构域，与野生大豆同源性最高。
基因表达特性分析表明，GmTGA26在大豆中受盐胁迫诱导表达。
此外，GmTGA26编码核定位蛋白并且具有转录激活活性。
通过发根农杆菌介导的大豆毛根转化，得到过表达GmTGA26的“复合体”大豆植株，在盐胁迫条件下，与空载体对照相比，“复合体”大豆植株生长状态更好，丙二醛含量和相对质膜透性明显降低(P < 0.05)，而叶绿素含量和根系活力则有显著的升高(P < 0.05)。
qRT-PCR结果表明，盐胁迫条件下在大豆毛状根中过表达GmTGA26可显著上调胁迫响应基因的表达。
以上结果表明，过表达GmTGA26显著增强了“复合体”大豆植株的耐盐能力。
推测GmTGA26通过调控下游一系列胁迫响应基因从而参与调控大豆盐胁迫应激反应过程。
关键词: 大豆；TGA转录因子；毛根转化；耐盐性Functional analysis of GmTGA26 gene under salt stress in soybeanKE Dan-Xia*, HUO Ya-Ya, LIU Yi, LI Jin-Ying, and LIU Xiao-XueCollege of Life Sciences, Xinyang Normal University / Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang 464000, Henan, ChinaAbstract: TGA transcription factors are a subfamily of bZIP, which play important roles in pathogen and abiotic stress responses. A TGA transcription factor family gene GmTGA26was screened and cloned from soybean in this study. Homologous protein comparison showed that GmTGA26 had a conserved leucine zipper domain and had the highest homology with wild soybean. The analysis of gene expression characteristics revealed that GmTGA26gene was induced by salt stress in soybean. In addition, GmTGA26gene encoded nuclear localization protein and had transcriptional activation activity. The “complex” soybean plants overexpressing GmTGA26were obtained through Agrobacterium rhizogenes-mediated hairy root transformation of soybean. The growth state of “complex” soybean plants was better t han the empty vector control under salt stress. Meanwhile, the MDA content and relative plasma membrane permeability decreased significantly (P < 0.05), while the chlorophyll content and root activity increased significantly (P < 0.05). The qRT-PCR results indicated that overexpression of GmTGA26 in soybean hairy roots under salt stress could significantly up-regulate the expression of stress response genes. The above results showed that overexpression of GmTGA26 significantly enhanced the salt tolerance of “complex” soybean plants. It is speculated that GmTGA26 participates in the regulation of soybean salt stress response by regulating a series of downstream stress response genes.Keywords: soybean;TGA transcription factor; hairy root transformation; saline tolerance本研究由国家自然科学基金项目(U1904102)，河南省高等学校青年骨干教师培养计划和信阳师范学院“南湖学者奖励计划”青年项目资助。
大豆耐盐相关基因的功能研究
大豆耐盐相关基因的功能研究随着全球气候变化和人们对食品质量的日益关注，对作物的适应能力和质量控制等方面的研究也日益重要。
大豆作为世界上最主要的粮食作物之一，在经济和食品质量方面发挥着重要作用。
然而，一些环境因素对大豆的生长和发展有很大影响，其中之一就是盐害。
因此，大豆耐盐基因的研究就变得非常重要。
在大豆的普通生长过程中，高浓度的盐会干扰大豆的细胞代谢，并阻碍其正常的生长和开花。
长期以来，从不同角度探索大豆耐盐基因的功能，为保证大豆的生长和食品质量做出了很大努力。
本文将讨论大豆耐盐基因的功能及相关研究。
大豆耐盐基因的功能1.抵御盐胁迫，提供营养物质大豆耐盐基因作为一种适应盐胁迫的物质，在外界环境的干扰下，会自动担负起维持大豆营养供应的任务。
其中，重要的一个功能就是降低盐环境对植物的毒性，保证营养成分和代谢产物的积累。
2.调节生长大豆耐盐基因在植物生长和发育阶段起着很大的作用。
例如，基因表达的增强会促进大豆生长和发育过程中的各个环节，有利于营养物质的吸收和利用，保证豆荚形成的同时，保持正常分泌生长激素的功能。
3.调节形态结构和新陈代谢大豆耐盐基因在大豆的形态结构和新陈代谢方面发挥着重要的调节作用。
通过稳定细胞氧化还原状态，大豆耐盐基因可以保证细胞内的基本代谢过程正常可靠的运行。
此外，在盐胁迫的情况下，大豆耐盐基因也能够调节大豆的形态结构，维持大豆细胞的正常构成。
相关研究1.基因筛选技术的发展借助最新的基因筛选技术，科学家们可以更准确地识别出对大豆生长影响最大的耐盐基因。
目前，通过基因芯片和基因定向测序技术等高通量测序方法，可以对上万个基因进行同时筛选，为大豆耐盐基因的鉴定和功能研究提供更强的支持。
2.转基因技术的应用转基因技术是最主要耐盐基因筛选与转移的手段之一。
大豆人工转染过程中，可通过基因导入等手段改变大豆的生长环境，增强其耐盐性。
此外，通过基因工程技术，科学家们可以对特定基因进行精准的调控，以提高耐盐性即受盐性，实现大豆生长环境的优化。
大豆转录因子NAC1耐低磷胁迫的功能研究
大豆转录因子NAC1耐低磷胁迫的功能研究熊楚雯;郭智滨;周强华;程艳波;马启彬;蔡占东;年海【期刊名称】《中国农业科学》【年(卷),期】2024(57)3【摘要】【目的】磷含量偏低是影响酸性土壤作物产量的重要因素。
大豆(Glycine max)是重要的粮食和油料作物,也为喜磷作物,缺磷则影响其产量与品质。
NAC(NAM,ATAF1/2,CUC2)转录因子家族参与多种植物对生物胁迫和非生物胁迫响应的调控,是否参与大豆低磷胁迫响应尚未深入研究。
以耐低磷野生大豆BW69为材料,克隆获得耐低磷基因GsNAC1并对其表达特性及功能进行分析,为深入解析GsNAC1调控大豆低磷胁迫及其机制奠定基础。
【方法】从野生大豆BW69克隆GsNAC1的全长序列,并通过生物信息学分析探究其编码氨基酸序列的特征。
随后,利用实时荧光定量PCR技术(qRT-PCR)对其组织表达模式进行分析,并通过激光共聚焦显微镜观察其编码蛋白的亚细胞定位。
此外,通过大豆遗传转化试验,获得转基因株系并进行表型分析。
最后,通过转录组联合分析来鉴定转基因植株中与低磷胁迫相关的差异表达基因(differentially expressed genes,DEGs)。
【结果】成功克隆获得GsNAC1,编码区全长876 bp,通过构建系统发育树发现GsNAC1与AtATAF1的序列相似性为62.46%,与Williams 82参考基因组的GmNAC1序列没有差异;进一步的亚细胞定位结果显示,GsNAC1定位于细胞核;基于qRT-PCR技术,发现GsNAC1在大豆的根、茎、叶、顶端、花和豆荚均有表达,在根部的相对表达量最高,且受到低pH和低磷诱导表达显著上调。
通过水培法和土培法进行表型试验,在低磷处理下,与野生型(WT)相比,转基因株系鲜重根冠比、总根长、根表面积、根体积和磷含量均显著高于WT。
结合转录组测序数据进行分析,发现GsNAC1可能通过促进GmALMT6、GmALMT27、GmPAP27和GmWRKY21等基因表达增强其对低磷胁迫的耐受性。
大豆转录因子基因家族的鉴定与功能研究
大豆转录因子基因家族的鉴定与功能研究大豆是世界上三大主要粮食作物之一，其质量和产量与转录因子(TF)密切相关。
近年来，随着基因工程技术和计算生物学的发展，对大豆转录因子基因家族的鉴定和功能研究也越来越受到重视。
一、大豆转录因子基因家族的鉴定转录因子是在调控基因表达过程中重要的一类蛋白质。
它能够结合到DNA特定序列上，从而控制与该序列相关联的基因的表达。
在研究大豆转录因子基因家族之前，首先需要了解转录因子在大豆中的基本情况。
目前已有多个研究对大豆的转录因子进行了初步的分类和鉴定。
2009年，Rocha等人通过对GenBank中已知的大豆EST序列的分析，鉴定出了大豆中可能涉及到转录因子的583个序列。
其中，大部分序列属于WRKY、MYB、bHLH、NAC和AP2/ERF等家族。
2011年，Song等人在GenBank中收集了所有大豆EST序列，结合了大豆基因组测序数据，将大豆的转录因子家族分为了72个家族，共计5940个基因。
其中，MYB和WRKY是最大的两个家族，分别包含了约900个和600个基因。
除此之外，还有bHLH、bZIP、GATA等家族。
另外，赵登荣等人也对大豆的转录因子进行了研究，利用BLAST和PFAM等工具，将大豆中所有含有转录因子结构域的蛋白质序列提取出来，并进行了系统的分类和鉴定。
最终确定了包含70个家族、3,812个基因的大豆转录因子基因家族。
这些研究为后续的大豆转录因子研究奠定了基础。
二、大豆转录因子基因家族的功能研究在大豆转录因子基因家族中，有一些基因已经被证明在不同生长发育阶段和环境逆境中具有关键的调控作用。
以下是一些具有代表性的例子。
1. GmWRKY20WRKY家族中的成员GmWRKY20在水稻中已被研究出具有控制花粉发育的能力。
而在大豆中，该基因也发挥着重要的作用。
研究表明该基因对干旱和盐碱逆境的响应具有重要影响。
转基因大豆中过表达GmWRKY20的植株在干旱环境下比野生型有更好的生长状态和生产力。
盐胁迫下大豆木质部溶液中Na+和K+含量变化分析
盐胁迫下大豆木质部溶液中Na+和K+含量变化分析1. 盐胁迫对大豆生长的影响盐胁迫会导致土壤盐分浓度增加，使植物根系吸收的盐分过多，导致植物体内离子平衡失调。
在这种情况下，大豆的生长发育将受到抑制，表现为株高减少、叶片变黄、叶片边缘焦枯等现象。
盐胁迫还会影响大豆的产量和品质，严重影响大豆的种植效益。
研究盐胁迫下大豆木质部溶液中Na+和K+含量变化对于了解大豆抗盐能力及调控机制具有重要意义。
在盐胁迫条件下，大豆木质部溶液中的Na+和K+含量会出现相应的变化。
一般情况下，盐胁迫会导致大豆根系吸收的Na+增加，导致Na+在木质部溶液中的含量增加。
盐胁迫还会影响大豆对K+的吸收与利用，导致木质部溶液中K+含量减少。
盐胁迫会影响大豆木质部溶液中Na+和K+的含量平衡，对大豆的生长和发育产生不利影响。
3. 大豆对盐胁迫的适应能力及机制虽然盐胁迫对大豆生长发育产生了负面影响，但大豆在长期的生长过程中也会逐渐适应盐胁迫的环境。
研究发现，大豆对盐胁迫的适应能力主要表现为根系调节、离子平衡和抗氧化能力的提高。
在盐胁迫条件下，大豆会通过调节根系结构和功能来减少对盐分的吸收，并通过增加Na+/K+离子选择性吸收来保持内外离子平衡。
大豆还会通过增加抗氧化酶活性、积累抗氧化物质等方式提高自身的抗氧化能力，减轻盐胁迫对植物的伤害。
4. 对策与展望针对盐胁迫对大豆的影响，为了提高大豆对盐胁迫的适应能力和抗性，可以从以下几个方面进行研究和应对：一是通过育种和遗传改良的方式培育出对盐胁迫具有高抗性的大豆品种；二是利用分子生物学和遗传工程等技术手段来提高大豆对盐胁迫的适应能力；三是通过土壤改良和耕作管理等措施来减轻盐胁迫对大豆的影响。
未来的研究还需要从植物生长调控、离子平衡和抗氧化等方面深入研究，为大豆抗盐育种和生产提供科学依据。
盐胁迫是大豆生长发育过程中的重要环境因素，盐胁迫会影响大豆木质部溶液中Na+和K+含量的平衡，导致植物生长发育受到抑制。
碱性盐胁迫对不同类型野生大豆种子萌发及生长的影响
碱性盐胁迫对不同类型野生大豆种子萌发及生长的影响摘要野生大豆具有许多优良形状，如耐盐碱、抗寒、抗病等，同时，因其营养价值高，又是优良牧草。
野生大豆与大豆是近缘种，而大豆是中国主要的油料及粮食作物、故在农业育种上可利用野生大豆进一步培育优良的大豆品种。
本试验，以两种野生大豆作为实验材料对其进行碱性盐胁迫，观察分析碱性盐胁迫对野生大豆萌发及生长的影响，实验证明Huinan06116对盐胁迫适应性非常弱，而Tongyu06311在Na+浓度为0到60mmol/L范围内对盐碱环境表现出一定的适应性，只在浓度超过60mmol/L时受到了较强的抑制，本实验结果为培育抗盐碱栽培大豆提供量化参数体系。
在探索野生大豆杂交培育栽培大豆过程中可以提高大豆的耐盐特性，从而扩大大豆的种植面积，在合理利用盐渍化弃耕地创造经济价值的同时，改善土壤质量。
关键词：碱性盐胁迫Huinan06116 Tongyu06311 萌发生长AbstractWild soybean has many excellent shape, such as salinity, cold, disease, etc., but, because of its high nutritional value, but also a good pasture. Wild soybean and soybean is closely related species, and soybeans are China's major oil and grain crops, it can be used in agriculture and breeding of wild soybeans further develop elite soybean varieties. The trial of two wild soybeans as experimental material its basic salt stress, observation and analysis of wild soybean alkaline salt stress on germination and growth, proved Huinan06116 adaptation to salt stress very weak, and the Na+concentration Tongyu06311 when 0 to 60mmol / L range in the saline environment showed some adaptability, only at concentrations exceeding 60mmol / L by the strong inhibition of the results for the cultivation of salt tolerance of soybean cultivation system to provide quantitative parameters. Exploring the wild soybean crossbreeding can improve the process of cultivated soybean soybeans tolerant characteristics, thereby expanding the soybean acreage in the rational use of Salinization Wasteland in creating economic value while improving soil quality.Keywords: alkaline salt stress Huinan06116 Tongyu06311 germination growth目录摘要 0Abstract 0第一节前言 (2)1.1 土壤盐渍化及其对植物的伤害 (3)1.2盐胁迫对豆科植物种子萌发特性的影响 (4)1.3选题的目的与意义 (4)第二节实验材料与方法 (4)2.1材料培养与胁迫处理 (4)2.2萌发率及生长指标的测定 (4)2.2.1萌发的测定 (4)2.2.2生长指标测定 (5)2.3数据统计与作图 (5)第三节结果分析 (5)3.1萌发率 (5)3.2萌发势 (7)3.3萌发指数 (8)3.4活力指数 (8)3.5下胚轴长度变化 (9)3.6下胚轴及子叶生物量 (10)3.7下胚轴及子叶含水量变化 (12)第四节讨论 (12)第五节结论 (13)参考文献 (14)第一节前言野生大豆（Glycine soja Sieb. et Zucc）是豆科大豆属。
毕业论文——盐胁迫下大豆不同部位锰元素富集能力的研究
毕业论文盐胁迫下大豆不同部位锰元素富集能力的研究学院生命科学学院专业生物科学（生物制药方向）年级2010级学号 2010136601姓名李亚男指导教师史忠勇二零一四年六月盐胁迫下大豆不同部位锰元素富集能力的研究摘要：为了研究大豆在盐胁迫下不同部位对锰元素的富集能力，本实验通过水培养法培养三种不同品种的大豆，在适宜阶段使用不同盐浓度培养液处理豆苗。
采收材料后使用原子吸收仪分别测量出三种大豆同一植株根、茎、叶中锰元素的含量。
结果表明为：大豆同一植株中各部位对锰元素富集能力为根＞叶＞茎；培养液盐浓度升高不会影响大豆茎对锰元素的富集能力，但会降低叶对锰元素的富集能力。
关键字：富集能力；大豆；水培养法；锰元素；原子吸收光谱法Studies of manganese enrichment capability in different positions of soybeanunder salt stressAbstract：In order to study themanganese enrichment capability in different positions of soybean under salt stress, this experiment makes use of hydroponic curing to develop three different varieties of soybeans,and deals plants with different salt concentrationmediumin the appropriate phase.After collecting materials we useatomic absorption spectrometerto measure content of manganese element of three soybean plant root, stem, and leaf.The results show that:the manganese enrichment capability in different positions of soybean under salt stress is root > leaf >stem;theincreased salt concentration of medium does not affect themanganese enrichment capability of stem, but will reduce the manganese enrichment capability of leaf. keyword：Enrichment ability; Soybean; hydroponic curing; Mn; Atomic absorption spectrometry;目录1 引言 (1)1.1锰元素在大豆生长过程中的生理作用 (5)1.2水培养法的概述 (5)1.3原子吸收光谱法的概述 (2)2 实验过程 (2)2.1实验仪器与试剂 (2)2.1.1实验仪器 (2)2.1.2实验试剂 (2)2.1.3实验材料 (3)2.2样品的培养、采集与处理 (3)2.2.1样品的培养 (3)2.2.2样品的采集与处理 (3)2.3原子吸收仪的操作过程 (4)2.3.1开机 (4)2.3.2锰元素含量测定 (4)3结果与分析 (4)3.1锰元素的测量结果 (4)3.1.1标准曲线的绘制 (4)3.1.2样品分析结果 (5)3.2对于数据的处理和分析 (7)3.2.1 并豆1928各部位锰元素含量分析 (7)3.2.2品豆18各部位锰元素含量分析 (8)3.2.307F572号大豆各部位锰元素含量分析 (100)4结论 (11)参考文献 (12)致谢 (13)1引言大豆作为一种果实含有大量蛋白质和脂质的豆科植物，在日常生活中提供给我们丰富的蛋白质营养[1]。
盐胁迫下大豆木质部溶液中Na+和K+含量变化分析
盐胁迫下大豆木质部溶液中Na+和K+含量变化分析近年来，研究表明，盐胁迫下大豆木质部溶液中Na+和K+含量发生显著变化。
本文将从这一方面进行分析，探讨盐胁迫对大豆木质部Na+和K+含量的影响，为后续研究提供参考。
1.1 盐胁迫对大豆木质部Na+含量的影响盐胁迫会导致土壤中Na+的积累，随着Na+的渗透进入植物细胞，造成大豆木质部中Na+含量的增加。
研究表明，盐胁迫下大豆木质部中Na+含量呈现出显著增加的趋势。
这主要是由于盐胁迫会导致植物根系吸收Na+的增加，从而使得Na+在大豆木质部中积累。
2.1 Na+和K+在盐胁迫适应中的作用在盐胁迫条件下，Na+和K+在大豆的适应过程中起着重要的作用。
Na+是一种不可避免的毒害离子，过量积累会影响植物细胞内的生理代谢过程。
而K+则是植物生长发育的必需元素，能够维持细胞内的渗透压和离子平衡。
盐胁迫下大豆木质部Na+和K+含量的变化对于大豆对盐胁迫的适应机制具有重要影响。
2.2 Na+和K+的积累与转运在盐胁迫条件下，植物根系对Na+和K+的吸收和转运会发生变化。
一方面，盐胁迫会导致土壤中Na+的积累，从而增加植物根系对Na+的吸收。
盐胁迫会影响大豆根系对K+的吸收和转运，导致K+难以在植物体内正常运输和分配。
这些变化会直接影响大豆木质部中Na+和K+含量的变化。
在盐胁迫条件下，Na+和K+的变化会影响大豆木质部的渗透调节和离子平衡，进而影响木质部的生物合成和代谢过程。
Na+的积累会导致细胞内Na+/K+比值的增加，从而影响细胞的渗透调节和离子平衡。
K+的减少会影响根系吸收和转运，导致细胞内K+含量的减少，进而影响大豆木质部中许多酶的活性和代谢过程。
3.1 离子通道和转运蛋白的调控在盐胁迫条件下，植物需要通过调控离子通道和转运蛋白来维持细胞内Na+和K+的稳定。
研究发现，一些离子通道和转运蛋白在盐胁迫适应过程中发挥重要作用，通过调控这些蛋白的表达和活性，可以影响大豆木质部中Na+和K+含量的变化。
大豆中盐胁迫相应转录因子的DNA甲基化和组蛋白修饰的动态变化
The Dynamic Changes of DNA Methylation and Histone Modifications of Salt Responsive Transcription Factor Genes in SoybeanYuguang Song,Dandan Ji,Shuo Li,Peng Wang,Qiang Li,Fengning Xiang*The Key Laboratory of Plant Cell Engineering and Germplasm Innovation,School of Life Sciences,Shandong University,Jinan,Shandong,ChinaAbstractEpigenetic modification contributes to the regulation of gene expression and plant development under salinity stress.Here we describe the identification of49soybean transcription factors by microarray analysis as being inducible by salinity stress.A semi-quantitative RT-PCR-based expression assay confirmed the salinity stress inducibility of45of these49transcriptionfactors,and showed that ten of them were up-regulated when seedlings were exposed to the demethylation agent5-aza-2-deoxycytidine.Salinity stress was shown to affect the methylation status of four of these ten transcription factors(one MYB, one b-ZIP and two AP2/DREB family members)using a combination of bisulfite sequencing and DNA methylation-sensitive DNA gel blot analysis.ChIP analysis indicated that the activation of three of the four DNA methylated transcription factors was correlated with an increased level of histone H3K4trimethylation and H3K9acetylation,and/or a reduced level of H3K9 demethylation in various parts of the promoter or coding regions.Our results suggest a critical role for some transcription factors’activation/repression by DNA methylation and/or histone modifications in soybean tolerance to salinity stress.Citation:Song Y,Ji D,Li S,Wang P,Li Q,et al.(2012)The Dynamic Changes of DNA Methylation and Histone Modifications of Salt Responsive Transcription Factor Genes in Soybean.PLoS ONE7(7):e41274.doi:10.1371/journal.pone.0041274Editor:Xiaoyu Zhang,University of Georgia,United States of AmericaReceived May7,2012;Accepted June19,2012;Published July18,2012Copyright:ß2012Song et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.Funding:This work was supported by the National Special Science Research Program of China(grant no.2007CB948203)/AreaMana.aspx;the National Natural Science Foundation of China(grant nos.30970243and30771116);Excellent Youth Foundation of Shandong Province of China(grant no.JQ200810)/portal/;Science&Technology Plan of Shandong Province(grant2009GG10002001).cn/index.jsp and the Chinese Natural Education Ministry Doctor Station Foundation Fellowship(grant no.913111006)/kyjh-bsd/1.htm.The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.Competing Interests:The authors have declared that no competing interests exist.*E-mail:xfn0990@IntroductionSoybean(Glycine max(L).Merr.)is an important source of protein and oil in both the human and domestic animal diet.As for most crop species,its productivity is significantly compromised by soil salinity[1],but,like most plants,it has evolved a variety of mechanisms to aid its survival under environmental stress.The expression of many plant genes is altered by salinity stress;some of these encode aspects of cellular metabolism and stress tolerance, while others are regulatory in nature[1,2].Transcription factors (TFs),which belong to the latter class,have been classified into a number of families on the basis of their sequence,and some members of the MYB,NAC,b-ZIP and AP2-DREB families have been shown to be intimately involved in the stress response [3,4,5,6,7,8].Such as,the heterologous expression of three soybean MYB and three b-ZIP TFs in Arabidopsis thaliana improved its response to salinity and freezing stress[9,10].Similarly the heterologous expression of GmDREB2was able to enhance the drought and salinity tolerance of tobacco[11],as did the over-expression of either GmNAC11or GmNAC20for soybean[12]. Once a plant detects the onset of stress,TFs characteristically respond by inducing the expression of a cascade of downstream targets.However,their activation is in part also dependent on their chromatin structure,which is largely determined by epigenetic means[13,14,15,16].Cytosine methylation within the promoter sequence has been shown to underlie numerous cases of gene down-regulation or silencing[17,18,19,20,21].DNA methylation in the plant genome mostly at CG dinucleotides and CNG trinucleotides,but also at an asymmetrical sequence contexts CNN (N is any nucleotide but G)[22,23,24].The N terminus of the histone molecule can be acetylated,phosphorylated,methylated, ubiquitinated or ribosylated[25].The presence of the trimethy-lated form of histone H3K4and of the acetylated form of H3K9in the promoter region have been frequently associated with transcriptional activation,while that of the dimethylated form of H3K9represses it[26,27,28].Sometimes,H3K9methylation can trigger cytosine methylation in both Neurospora crassa[29]and A. thaliana[30],while cytosine methylation at the CNG trinucleotide appears to be partially dependent on the activity of a histone methyltransferase[29,31,32].A number of examples where epigenetic modification has contributed to the regulation of gene expression during periods of environmental stress have been presented.In particular,the low temperature induced expression of the maize gene ZmMI1has been correlated with a reduction of DNA methylation in its nucleosome core[33].In tobacco,several stress agents are known to promote demethylation in the NtGPDL coding sequence,leading to alterations in its level of expression[34].The submergence of rice seedlings reduces histone H3K4trimethylation and acetyla-tion in genes encoding both alcohol dehydrogenase and pyruvate decarboxylase,leading to their up-regulation[35].In A.thaliana, the drought-induced expression of a number of stress-responsivegenes has been associated with an increase in H3K4trimethyla-tion and H3K9acetylation[36].Here,we set out to document the induction by salinity stress of DNA methylation and histone modification in a number of salinity responsive soybean TFs,and to identify what relationship there is,if any,between the expression of a TF and the epigenetic status of its promoter sequence.In addition,since to date no systematic attempt has been made to investigate the dynamics and reversibility of both DNA and histone modification over the course of a stress episode, we have explored this feature focusing on four salinity stress inducible soybean TFs.Materials and MethodsExposure of soybean seedlings to salinity stress and5-ADC treatmentSeedlings of the soybean cultivar Williams82were grown in vermiculite under a16h photoperiod at25u C for14days before being exposed to stress treatment.Once the seedlings had been removed from the vermiculite and their roots rinsed in water,they were then treated with either150mM NaCl for1h,3h,6h,12h or 24h,or with50m M5-aza-2-deoxycytidine(5-ADC)for12h,24h, 48h or72h.RNA and DNA was extracted from snap-frozen plants both before the stress treatment had begun and then at each time interval.Mock treatments(ddH2O only)were included as a control.RNA was prepared from0.2g plant material using the TRIzol(Invitrogen)reagent,following the manufacturer’s proto-col,and DNA was extracted from1g plant material using a DNeasy Plant Mini kit(Qiagen).Microarray analysisRNA were isolated from the mock(M0,M1,M3,M6,M12, M24)and salinity treated(S0,S1,S3,S6,S12,S24)seedlings.0.5m g RNA that extracted from each time point of the mock and salinity-stressed seedlings were mixed respectively to obtain the mock and salinity-stressed RNA pools,and then they were used to synthesize the cDNA.The cDNA was labeled with biotin,and then hybridized to an Affymetrix soybean Genome Array according to the manufacturer’s instructions(15h in a rotating hybridization oven set at45u C and60rpm).After the hybridiza-tion,the microarrays were scanned using a GeneChip H Scanner 3000(Affymetrix,P/N00-00212).Then the scaling factor, background,noise,and percentage presence were calculated according to the Affymetrix Data Mining Tool protocols (Affymetrix).All resulting datasets were filtered using the absolute call metric(present or absent)implemented within Microsoft Access(Microsoft Corporation,Redmond,WA)and the micro-array data were processed in an R(v2.7.0)environment,using the LIMMA package[37].Quantile normalization was performed.A single repeat microarray analysis for each group wasperformed. Figure1.The expression of the49TFs in mock-stressed and salinity-stressed seedlings.(a)GmAP2-DREBs,(b)GmMYBs,(c)GmNACs and (d)Gmb-ZIPs.M0-M24refer to seedlings exposed to just ddH2O for,respectively,0h,1h,3h,6h,12h and24h;S0–S24refer seedlings exposed to 150mM NaCl for0h,1h,3h,6h,12h and24h,respectively.Each gene-specific region was amplified by RT–PCR using the gene-specific primers (Table S2).The TUBULIN gene(Genbank accession AY907703)was used as an internal control.The experiment was repeated three times with similar result.doi:10.1371/journal.pone.0041274.g001Transcript level analysisSemi-quantitative RT-PCR (sqRT-PCR)and quantitative real-time RT-PCR (qRT-PCR)were employed to quantify transcript levels more precisely.RNA was extracted from 0.2g of seedling material ground in liquid nitrogen by the addition of 1ml TRIZOL reagent (Invitrogen)and treated with RNase-free DNase I.A 3m g aliquot of total RNA was used to generate the first cDNA strand with the SuperScript First-Strand Synthesis System (Invitrogen)according to the manufactuter’s instructions.A 1m l aliquot of this cDNA was used as the template for a 22–34cycle sqRT-PCR,where the cycling regime was 94u C/30s,55u C/30s,72u C/30s.A fragment of the soybean TUBULIN gene (Genbank accession AY907703)was used as a reference.Primer sequences are given in Table S2.Each 15m l qRT-PCR contained 7.5m l Maxima SYBR Green qPCR Master mix buffer (Roche),0.5m l 10m M specific primers,1.5m l of a 1:10dilution of cDNA and 5.5m l ddH 2O.The cycling regime consisted of a denaturation step (95u C/3min)followed by 18–35cycles of 95u C/30s,60u C/15s,72u C/15s,and a fragment of the soybean TUBULIN gene (GenBank accession AY907703)was used as a internal control.Primer sequences are given in Table S2.The relative expression level of the target sequence was determined using the 22DD Ct method [38].Each estimate was derived from the mean of three independent biological replicates.Bisulfite DNA sequencingA 2m g DNA aliquot was dissolved in 50m l ddH 2O and denatured by adding 5.5m l 3M NaOH and incubating for 30min at 42u C.Thereafter,510m l 2.3M sodium bisulfite (pH 5.0),30m l 10mM hydroquinone and 65m l ddH 2O were added,and the solution overlaid with mineral oil and held for 16h at 55u C.The DNA was recovered using a Wizard H DNA Clean-Up System kit (Promega A7280),and a 90m l aliquot treated with 10m l 3M NaOH for 15min at 37u C,then neutralized by adding 70m l 10M ammonium acetate.Finally,the DNA was precipitat-ed by adding 400m l ethanol and 10m l glycogen,and re-suspended in 50m l ddH 2O to provide the template for a series of PCRs based on the gene-specific primers listed in Table S2.A fragment of Glyma20g32730featuring many CG,CNG and CNN sites was amplified from genomic DNA,then inserted into pMD18-T vector and transferred into Dm-E.coli strain JM110.The plasmid was released from the bacterial cells by the plasmid extraction kit (TianGen.Cat.DP103-03)and treated with bisulfite in parallel with the soybean genomic DNA as a control to monitor the transformation efficiency of unmethylated cytosine to thymine.The subsequent PCR consisted of 34–37cycles of 94u C/30s,55u C/30s,and 72u C/40s.The resulting amplicons were purified with a Wizard H DNA Clean-Up System kit,ligated into the pMD18-T vector (TaKaRa)and transferred into E.coli for sequencing.Ten clones from each amplicon weresequenced.Figure 2.Expression of 45salinity inducible TFs in seedlings exposed to 5-ADC treatment.(a)GmAP2-DREBs ,(b)GmMYBs ,(c).GmNACs .and (d)Gmb-ZIPs .M0-M72refers to seedlings treated with water only for,respectively 0h,12h,24h,48h and 72h,while A0-A72refer to seedlings exposed to 50m M 5-ADC for 0h,12h,24h,48h and 72h,respectively.Each gene-specific region was amplified by RT–PCR using the gene-specific primers (Table S2).The TUBULIN gene (Genbank accession AY907703)was used as an internal control.The experiment was repeated three times with similar result.doi:10.1371/journal.pone.0041274.g002Figure3.Methylation status of the promoter region of four salinity-responsive TFs in untreated(S0)and salinity-stressed(S1–S24) seedlings(S1:1h,S3:3h,S6:6h,S12:12h,S24:24h).(a)The black and white boxes indicate,respectively,exon and untranslated regions.The short bars annotated with‘‘I,II,III’’,‘‘a’’or b’’indicate,respectively the sequences subjected to ChIP analysis,genomic bisulfite sequencing and those used as probes for Southern blotting.The long vertical bars marked‘‘c’’display the distribution of CG dinucleotides(marked with red vertical lines), and CNG(blue vertical lines)and CNN(black vertical lines)trinucleotides.The red vertical lines marked with a rectangular indicate CCGG sites analyzed by Southern blotting.The thick black vertical lines represent the proportion of methylated cytosine.Ten positive clones from each gene’s amplicon were sequenced.The data reflect the outcome of three independent experiments,and error bars represent standard error(SD).(b)The efficiency of the bisulfite treatment to transform unmethylated cytosine to thymine.A fragment of Glyma20g32730with numerous cytosines was cloned into Dm-E.coli cells and the plasmid was treated with bisulfite in parallel with the soybean genomic DNA.All clones processed showed a transformation rate.99.7%.(c)Methylation-sensitive DNA gel blot analysis of non-stressed(S0)and salinity-stressed seedlings(S1–S24).Genomic DNA was digested to generate large fragments,then with one or other of the schizomers Hpa II or Msp I.Hybridization probes indicated.A DNA fragment amplified from the probe sequence was used as a positive control(+),and ddH2O was used as a negative control(2).doi:10.1371/journal.pone.0041274.g003The process was repeated three times using biologically indepen-dent samples.Chromatin immunoprecipitation (ChIP)assayThe ChIP protocol was modified from that of Johnson et al .(2002).Briefly,1g of plant tissue was fixed by immersion in 1%v/v formaldehyde under vacuum for 10min.The extracted DNA/protein complex was then sheared by sonication to a size range of ,100–1000bp.After centrifugation,the complex was immuno-precipitated by challenging with H3K9ac,H3K4me3and H3K9me2antibodies (Millipore cat.07–392,07–473and 05–768R)at a titer of 1:100.The residual protein was degraded by the addition of 10m l (20mg/ml)proteinase K,followed by a phenol/chloroform extraction.A 2m l aliquot of the final solution was used as a template for qRT-PCR analysis as described above.A 1000x diluted input DNA (Input)obtained from 500m l of extract was purified in parallel with the immunoprecipitated samples as a control,and ChIP reactions were also performed in the absence of antibody (No AB)to detect the occurrence of any non-specific binding.Relative levels of H3K9acetylation,H3K9dimethylation and H3K4trimethylation were normalized to an internal control (GenBank accession AY907703).The sequences of all PCR primers used are given in Table S2.The mean and standard deviation are shown for three independent ChIP experiments and the significance of differences between means assessed with a t test.Methylation-sensitive Southern blot analysisGenomic DNA (100m g)extracted from both non-stressed and salinity-stressed seedlings was treated for at least 6h with 100U of the appropriate restriction enzymes (EcoR V and Nde I for Glyma11g02400;Sac I for Glyma16g27950;Bgl II for Glyma20g30840;Sac I and Bgl II for Glyma08g41450)(TaKaRa)to generate large fragments containing the target sequences.The digested DNA was extracted by phenol/chloroform and divided into two equal aliquots,one of which was treated with Hpa II and the other with its schizomer Msp I [39].The digested DNA was re-extracted,electrophoretically separated through an 0.8%agarose gel and transferred onto a Hybond N +membrane (Amersham).Probes for each gene were designed to detect the methylation status within the target sequence that analysed by genomic bisulfite sequencing.About 3m g of probe DNA was labeled using a DIG-High Prime kit (Roche),and the subsequent hybridization and detection procedure was performed using a DIG High Prime DNA Labeling and Detection Starter Kit I (Roche),according to the manufac-turer’s instructions.The positive control consisted of a 100x diluted DNA fragment that amplified from the genomic DNA in the same regions that prepared for probes,while the negative control was ddH 2O.ResultsThe identification of salinity stress responsive TFs in soybeanA set of differentially expressed genes were identified by comparing the soybean Affymetrix microarray profiles generated by probing with RNA extracted from salinity-stressed and non-stressed plants.We mainly focused on the four groups of AP2/EREB,bZIP,NAC and MYB transcription factors that have been verified for salt stress in Arabidopsis or other plants.Of the 1,335MYB,NAC,AP2/DREB and b-ZIP TFs represented on the microarray,49appeared to be up-regulated (fold change of hybridization signal .2,p ,0.01)by salinity stress.These consisted of 15(of 448)GmMYB s,9(of 226)GmNAC s,16(of 426)GmAP2/DREB s and 9(of 235)Gmb-ZIP s (Table S1).When the expression of these 49TFs was assayed by sqRT-PCR in both mock-stressed (M0–M24)and salinity-stressed soybean seedlings (S1–S24)with gene-specific primers (Table S2),14of the GmMYBs ,8of the GmNACs ,15of the GmAP2/DREBs and 8of the Gmb-ZIPs were confirmed to be markedly induced by salinity stress (Figure 1).Expression pattern analysis indicated that 19of them were strongly induced at a relatively early stage of exposure to salinity (1–3h),while the others were induced somewhat later (6–24h).Expression of the salinity induced TFs in the presence of 5-ADCThe expression of the 45salinity-induced TFs was then monitored in the mock treated (M0–M72)and the seedlings that exposed to 5-ADC for various periods (A0–A72).As a result,ten of the them showed higher levels of expression in treated (M0–M72)than in mock-treated (A0–A72)seedlings;these ten TFsconsistedFigure 4.DNA methylation patterns in Glyma11g02400,Glyma16g27950,Glyma08g41450and Glyma20g30840in non-stressed (S0)and salinity-stressed (S1S24)seedlings.The left axis shows the percentage of methylated cytosines at each site (present as CG,CNG and CNN).The data represent the mean of three biological replicates.Error bars represent standard errors.doi:10.1371/journal.pone.0041274.g004of four GmMYB s (Glyma11g02400,Glyma07g30860,Glyma12g34650,Glyma15g07230),one GmNAC (Glyma15g08480),four GmAP2/DREB s (Glyma20g32730,Glyma20g30840,Glyma16g27950,Gly-ma10g00980)and one Gmb-ZIP genes (Glyma8g41450)(Figure 2).The expression level of nine of these TFs was very low for the first12h of exposure,but thereafter rose substantially;the exception was the Gmb-ZIP Glyma08g41450,the expression of which was induced somewhatearlier.Figure 5.Promoter methylation status in four salinity-responsive TFs in non-treated (A0)and 5-ADC treated seedlings (A12–A72).For Figure legend please refer to Figure 3legend.doi:10.1371/journal.pone.0041274.g005Glyma20g30840in none treated(S0)and salinity-stressed(S1–S24)seedlings.(a)Relative H3K9demethylation,acetylation and H3K4Methylation status as affected by salinity stressTo investigate the DNA methylation status of above candidate genes under salinity stress,the sequence corresponding to the translation start codon and the promoter region of the ten TFs was subjected to bisulfite sequencing.First,the efficiency of the sodium bisulfite treatment to convert cytosine to thymine was estimated. The efficiency of the sodium bisulfite treatment to convert cytosine to thymine in the cytosine rich segment of Glyma20g32730was estimated to be99.7%.Bisulfite sequencing result indicated that the Glyma11g02400,Glyma08g41450,Glyma16g27950and Gly-ma20g30840promoters all appeared to be differentially methylated by the imposition of salinity stress(Figure3a,b,Figure S1),but those of the other six genes were largely non-methylated (Figure S2).In the Glyma11g02400promoter from position2518 to2274,most of the cytosines were demethylated following exposure to salinity stress for1–24h(Figure3a).In the immediate downstream region of the Glyma16g27950transcription start codon (+24to+233),about35%of the cytosines were methylated both before the salinity stress was imposed and for the first three hours of stress,but thereafter only few methylated cytosines remained (Figure3a).In the Glyma20g30840promoter region1(287to +163),51%of the cytosines were methylated prior to exposure to salinity stress,but this proportion fell to27%after1h,12%after 3h,and to even lower levels as the stress was prolonged further (Figure3a);meanwhile in region2of the same promoter(2163to 2405),42%of the cytosines were methylated at the start of the stress period,and this proportion hardly altered thereafter (Figure3a).In the Glyma08g41450region immediately downstream of the transcription start codon(+24to+233),35%of the cytosines were methylated prior to the imposition of stress,and the same proportion was maintained throughout(Figure3a).DNA methylation-sensitive Southern blotting was applied to verify these observations.The restriction fragments including Glyma11g02400,Glyma16g27950and Glyma20g30840were more readily digested by Hpa II after6h of salinity stress than the same samples obtained from non-stressed seedlings,which consistent with a reduction in global cytosine methylation caused by salinity stress analyzed by bisulfite sequencing(Figure3a,c).The sequence surrounding Glyma08g41450was not digestible by Hpa II,but several small restriction fragments were generated by Msp I digestion,suggesting that CCGG sites in the region of this TF were hypermethylated in both non-stressed and stressed seedlings (Figure3c).Thus the Southern blotting outcomes were in general consistent with the bisulfite sequence data.The analysis of DNA methylation pattern of them indicated that methylation was affecting either CG dinucleotides or CNG/CNN trinucleotides under salinity stressed process(Figure4).In the Glyma11g02400promoter,98%of the CG’s,60%of the CNG’s and6%of the CNN’s were methylated in the non-stressed seedlings,but almost all the CG’s,CNG’s and CNN’s were demethylated in plants exposed to salinity stress for more than6h (Figure4).For Glyma16g27950,some80%of the CG’s,72%of the CNG’s and4%of the CNN’s were methylated in non-stressed seedlings and those during the early phase(1–3h)of the salinity treatment,but by6h a significant fall in CG and CNG methylation was observed(Figure4).Within region1of the Glyma20g30840 promoter,95%of the CG’s,along with40%of the CNG’s and4%of the CNN’s,were methylated at3h after the imposition of stress, but by6h,a marked reduction in CG and CNG methylation had occurred.CG,CNG and CNN methylation in the Glyma08g41450 promoter was unaffected by salinity stress(Figure4).Clearly,DNA methylation in Glyma11g02400,Glyma16g27950and Gly-ma20g30840(region1)varied over the period of the salinity stress episode.Methylation status of Glyma11g02400,Glyma08g41450, Glyma16g27950and Glyma20g30840as affected by the presence of5-ADCTo identify whether the up-regulation of these four genes were related with cytosine demethylation under5-ADC treatment,the effect on the DNA methylation status of the four responsive TFs in plants treated with5-ADC was analyzed using genomic bisulfite sequencing.The relevant test for the transformation efficiency of unmethylated cytosine to thymine using Glyma20g32730is illustrated.As a result,all four TFs were hypermethylated in non-treated seedlings;after a24h exposure to5-ADC,some evidence of demethylation was obtained,but from48h onwards it was clear that a substantial level of demethylation had occurred (Figure5a,b).This observation was supported by a DNA methylation-sensitive DNA gel blot(Figure5c).All of the four TFs showed an increased digestion with HpaII after salinity stress for more than48h,suggesting a reduction of cytosine methylation in5-ADC stressed seedlings(Figure5c).Histone modification of hypermethylated genes induced by salinity stressThe histone content(H3K4me3,H3K9ace and the inactive H3K9me2)of the four TFs(Glyma11g02400,Glyma08g41450, Glyma16g27950and Glyma20g30840)which responded to salinity stress by altering their methylation status was then examined, using a combination of ChIP and qRT-PCR(Figure6a).An unmethylated gene Glyma20g32730was also analysed in parralle with them as a control(Figure S3).When Glyma11g02400was induced by salinity stress,a significant increase in H3K4me3 (regions I,II and III)and a decrease in H3K9me2(regions I and II)was observed,but the H3K9ace sites remained unmodified (Figure6a).Within Glyma20g30840(regions II and III)and Glyma08g41450(region III),a high level of H3K9me2and a low level of H3K4me3and H3K9ac was present in both non-stressed seedlings and those sampled during the early phase(1–3h)of salinity stress;at later time points(6–24h),a significant decrease in H3K9me2and increase of H3K4me3and H3K9ac content was observed(Figure6a).A similar H3K4me3,H3K9ac or H3K9me2 signal was detected in all three regions of the Glyma16g27950 promoter(Figure6a).Thus,like the DNA methylation,histone modification was also subject to dynamic change during the course of the salinity stress episode.Changes of epigenetic modification in regulating the TFs expression during salinity stressThe expression of Glyma11g02400was low in non-stressed seedlings,while its promoter was hypermethylated and was highly enriched for H3K9me2and depleted for H3K4me3(Figure6a,b,trimethylation content(ChIP assay).A1:1,000dilution of input DNA(Input)served as a control for PCR amplifications and the ChIP reactions carried out in the absence of antibody(N0AB).Relative H3K9acetylation,H3K9dimethylation and H3K4trimethylation were determined by qRT-PCR and normalized to an internal control TUBULIN gene(Genbank accession AY907703).Data represent the mean of three biological replicates.Asterisks indicate means differing significantly from the S0situation.Error bars represent standard errors.*P,0.05,**P,0.01.(b)Gene expression(qRT-PCR) profiles.(c)Cytosine methylation level(bisulfite sequencing).doi:10.1371/journal.pone.0041274.g006c).When the seedlings were exposed to salinity stress,its expression rose rapidly,while its promoter became gradually demethylated,the level of H3K9me2fell and that of H3K4me3 increased(Figure6a,b,c).Glyma20g30840behaved similarly,but the establishment of H3K4me3,DNA demethylation and gene expression were not contemporaneous.Demethylation was noticeable within1h of the imposition of salinity stress,but the establishment of H3K4me3did not occur until3h and the up-regulation of expression only at6h(Figure6a,b,c).Similarly, Glyma08g41450was up-regulated by12h after the start of the stress episode,but its promoter was hypermethylated throughout. Between6h and24h after the stress had begun,the level of H3K9me2fell and that of H3K4me3and H3K9ac rose(Figure6a, b,c).Glyma16g27950expression was repressed in non-stressed seedlings and during the early phase of the stress episode,when its promoter was ter the TF was gradually induced and its promoter demethylated,while the level of enrichment of H3K9me2,H3K4me3and H3K9ac did not change.A correlation analysis of their expression,methylation levels and histone modifications indicated that up-regulation of Glyma11g02400was associated with a decreased level of DNA methylation(r=20.89),H3K9me2(r=20.965,on average of regions II and III)and an increased level of H3K4me3(r=0.7,on average of regions I and II)(Figure6;Table S3).The expression of Glyma20g30840correlated negatively with DNA methylaion (r=20.78),H3K9me2(r=20.93on average of regions II and III)and positively with H3K9ac(r=0.96)and H3K4me3(r=0.93 on average)(Figure6;Table S3).Up-regulation of Glyma16g27950 just correlated negatively with DNA methylation(r=20.97),did not with histone modifications,while the up-regulation of Glyma08g41450correlated negatively with H3K9me2(r=20.67) and positively with H3K9ac(r=0.84)and H3K4me3(r=0.78), did not with DNA methylation during salinity stress(Figure6; Table S3).Therefore,the histone accumulation/depletion and/or cytosine methylation appears to underlie the activation by salinity stress of these four TFs.DiscussionTF transcription can be influenced by both DNA methylation and/or histone modification in a region specific mannerThe regulation of genes via cytosine methylation and histone modification is a well recognized component of the plant stress response[14,34,40,41].Both these epigenetic modifications are region-specific,and can be dynamic over time[42,43,44,45].Here we have identified a set of ten salinity-induced,cytosine methylation-dependent TFs,three of which displayed the expected relationship between promoter cytosine methylation and gene expression during the salinity stress process,but one of which (Glyma08g41450)remained up-regulated even though it was in a highly methylated state(Figure1,Figure3a).A similar unexpected relationship has been noted for an embryogenesis-related gene in carrot[42].Many genes are expressed despite their promoter region being highly methylated,so it seems probable that for Glyma08g41450,its up-regulation in plants exposed to salinity stress is independent of DNA methylation.One region of the Glyma20g30840promoter was hypermethylated in both stressed and non-stressed plants,while its neighbouring region responded to the stress by a reduction in methylation(Figure3a).This behaviour provides an example of region-specific regulation of methylation.The gradual up-regulation of Glyma11g02400in salinity-stressed seedlings was accompanied by a decrease in CG, CNG and CNN methylation(Figure4),while the rapid up-regulation of Glyma20g30840and Glyma16g27950was accompa-nied by a decrease in only CG and CNG methylation(Figure4), suggesting heterogeneity in the genome for gene expression regulation via DNA methylation.Histone modification provides a second major mechanism of epigenetic control over gene expression[46].In a range of A. thaliana stress-responsive genes,salinity stress has been shown to increase trimethylation at H3K4and decrease H3K9demethyl-ation[28].The up-regulation of Glyma20g30840and Gly-ma08g41450during the course of the salinity stress episode may have been achieved by the depletion of H3K9me2and the enrichment of H3K4me3and H3K9ac in regions II and III of Glyma20g30840and in region III of Glyma08g41450(Figure6a,b). The up-regulation of Glyma11g02400may have been brought about by the depletion of H3K9me2and the enrichment of H3K4me3in regions I,II and III(Figure6a,b).Thus,as for DNA methylation,the effect of salinity stress on histone modification appears to be heterogeneous across the genome. Transcriptional activation,DNA methylation and histone modification are not simultaneous eventsThere was a distinct time lag between transcriptional activation, DNA methylation and/or histone modification of Glyma11g02400, Glyma20g30840and Glyma08g41450.Thus,most of the cytosine content of the Glyma11g02400promoter was demethylated very soon(within1h)of the imposition of stress,but there was no evidence for the TF’s activation before3h(Figure6b,c).Similarly, Glyma20g30840was progressively demethylated over the full24h of the stress episode,but its up-regulation was complete within6h (Figure6b,c).In Glyma11g02400,Glyma20g30840and Glym08g41450,the H3K4me3content was already increasing by 1h,but the up-regulation of TF expression occurred substantially later(Figure6a,b).Similar time lags have been noted for the build-up of H3K4me3within the coding regions of the A.thaliana drought-related genes RD29A and RAP2.4during drought stress [36].Furthern more,the DNA demethylation of Glyma20g30840 was prior to the erasure or establishment of H3K9me2,H3K4me3 and H3K9ac(Figure6a,c)and all the three genes(Gly-ma11g02400,Glyma20g30840and Glym08g41450)show an ealier establishment of H3K4me3than H3K9me2and H3K9ac (Figure6a).Suggesting that there were also a time lage between the DNA demethylation and establishment of histone modifaci-tions.The interplay between DNA methylation and histone modification in the context of patterns of gene expressionIn A.thaliana,it has been demonstrated that the loss of CG methylation in met1plants has a large effect on H3K9me2content, leading to the idea that cytosine methylation can influence H3K9 modification[17,47,48,49].H3K9me2,mediated by KYP/ SUVH4and SUVH2,is also known to direct non-CG methylation [39,50].In the‘two-step’hypothesis for the regulation of transcription,CG methylation directs H3K9methylation and H3K9methylation recruits non-CG methylation[51].Moreover, hypermethylation of the stress inducible genes in Arabidopsis correlated with the enrichment of H3K9me2and depletion of H3K9ac histones under salt stress conditions[16].In this study, the behaviour of Glyma11g02400and Glyma20g30840was consis-tent with this model;as CG was progressively demethylated in these TFs,the content of H3K9me2,H3K4me3and/or H3K9ac rose and meanwhile a lower level of non-CG methylation was observed(Figure6a,Figure4).Note,however,that for。
栽培大豆GRAS转录因子家族基因鉴定及其盐胁迫下表达模式分析
江苏农业学报(Jiangsu J.qfAgr.Sci.),2021,37(2)：296-309http：// 296张斌，陈丽娟,李其华，等.栽培大豆GRAS转录因子家族基因鉴定及其盐胁迫下表达模式分析［J］.江苏农业学报,2021, 37（2）：296-309.doi：10.3969/j.issn.l000-4440.2021.02.004栽培大豆GRAS转录因子家族基因鉴定及其盐胁迫下表达模式分析张斌，陈丽娟，李其华，唐满生（湖南科技学院化学与生物工程学院，湖南省银杏工程技术研究中心，湖南永州425199）摘要：利用生物信息学手段及转录组测序方法对大豆78个GRAS家族基因进行系统分析。
染色体定位结果表明78个GRAS基因不均匀地分布在20条染色体上。
通过系统进化分析将大豆GRAS家族分为11个亚族。
基因结构和保守基序分布分析结果表明GRAS家族成员在进化上具有保守性，尤其是进化关系较近的成员多具有类似的基因结构和蛋白质结构。
转录组数据及qRT-PCR结果显示,5个基因受盐胁迫诱导上调,5个基因受盐胁迫诱导下调，其中GmGRAS14、GmGRAS33和GmGRAS69在盐处理12h时上调倍数最高，而GmGRAS17、GmGRAS54和GmGRAS57在盐处理12h时表达量下调倍数最高，说明这些基因可能在大豆响应盐胁迫方面发挥重要功能。
关键词：大豆；GRAS家族；盐胁迫；表达模式中图分类号：S565.101文献标识码：A文章编号：1000-4440(2021)02-0296-14 Identification of gene of GRAS transcription factor family in cultivated soybean(Glycine max L.)and expression pattern analysis under salt stress ZHANG Bin，CHEN Li-juan，LI Qi-hua，TANG Man-sheng(Hunan Provincial Engineering Research Center f or Ginkgo,College of Chemistry and Bioengineering,Hunan University of Science and Engineering,Yong-zhou425199,China)Abstract：In this study，78genes of GRAS family in soybean were systematically investigated using bioinformatics and RNA-seq.The chromosomal distribution map showed that78GRAS genes were randomly located in20chromosomes.Phylogenetic analysis showed that the soybean GRAS family could be divided into11subfamilies.The gene structure and conserved motif distribution analysis suggested that the GRAS family members were consertive in evolution，and most of the GRAS members with close evolutionary relationship had similar gene and protein structures.Transcriptomal and qRT-PCR results showed that five genes were up-regulated and five genes were down-regulated under salt stress.Among them，GmGRAS14,GmGRAS33and GmGRAS69had the highest fold of up-regulation at12h of salt treatment.GmGRAS17, GmGRAS54and GmGRAS57showed the highest down-regulation multiple at12h of salt treatment,suggesting that these genes may play an important role in the response to salt stress of soybean.Key words:soybean；GRAS family；salt stress；expression pattern收稿日期：2020-05-11基金项目：湖南科技学院重点项目（17XKY012）；湖南省大学生研究性学习和创新性实验计划项目［湘教通（2018）255号］作者简介：张斌（1981-）,男，湖南永州人，博士，讲师，主要从事植物发育生物学研究。
盐胁迫下大豆木质部溶液中Na+和K+含量变化分析
盐胁迫下大豆木质部溶液中Na+和K+含量变化分析1. 引言1.1 背景介绍大豆作为世界主要粮食作物之一，其生长受到各种环境因素的影响。
盐胁迫是影响作物生长和产量的主要因素之一，特别是在盐碱地区的种植中更为突出。
盐胁迫会导致作物细胞内外Na+和K+浓度失衡，从而影响作物的生长发育和产量。
目前，有关盐胁迫对大豆木质部溶液中Na+和K+含量的影响的研究相对较少，尤其是对其变化规律和机制的研究还不够深入。
本研究旨在通过盐胁迫处理大豆植株，测定其木质部溶液中Na+和K+的含量变化，探讨盐胁迫对大豆木质部溶液中Na+和K+含量的影响规律，并对其机制进行初步分析，为深入研究盐胁迫下大豆生长和产量的影响提供理论依据。
通过对大豆木质部溶液中Na+和K+含量变化的研究，可以为优化土壤调理和肥料施用提供科学依据，促进大豆的高产高效栽培，为解决盐碱地区大豆生产中的问题提供理论支持。
1.2 研究意义大豆作为重要的粮食和油料作物，在生长过程中受到各类胁迫的影响，其中盐胁迫是影响大豆生长与产量的重要因素之一。
随着全球土地盐碱化程度的加剧，盐胁迫对大豆生长的影响将愈发凸显。
研究盐胁迫对大豆木质部溶液中Na+和K+含量的变化规律及机制，对揭示大豆耐盐性的分子调控机制，为进一步培育抗盐性大豆品种提供科学依据，具有重要的理论和实际意义。
由于盐胁迫会导致大豆木质部溶液中Na+和K+含量的不断变化，从而影响大豆的生长、发育和产量，因此通过深入研究盐胁迫下大豆木质部溶液中Na+和K+含量的变化规律，可以为制定合理的土壤改良措施和施肥管理策略提供参考，为提高大豆的抗逆性和产量提供科学依据。
本研究的开展具有重要的现实意义，有望为大豆的生产提供新的科学理论支持和技术指导。
1.3 研究目的研究目的是为了探究盐胁迫对大豆木质部溶液中Na+和K+含量的影响，分析其变化规律以及机制。
在全球范围内，盐胁迫是一种严重影响植物生长和产量的重要因素，尤其对大豆这种盐敏感植物更为显著。
大豆耐盐相关基因GmNcl1功能标记的开发及验证
大豆耐盐相关基因GmNcl1功能标记的开发及验证宁丽华;何晓兰;张大勇【摘要】土壤盐渍化严重影响大豆生产,因而鉴定大豆耐盐种质的分子标记对大豆耐盐新品种的培育具有重要意义.本研究分析了大豆耐盐相关基因GmNcl1等位变异位点的限制性酶切位点,通过酶切PCR产物,琼脂糖凝胶电泳和酶切片段分析,开发建立了分子标记CAPS/Xba I.利用该标记对10份不同耐盐大豆种质进行酶切分型鉴定,然后通过测序试验进行验证.结果表明,用所开发的共显性标记CAPS/Xba I 对10份大豆种质进行耐盐性鉴定,鉴定结果与依据表型进行鉴定的结果一致.由此可见,CAPS/Xba I可用于大豆品种的耐盐性鉴定.%Soil salinization will lead to serious reduction of yield and affect the quality of soybean. Therefore, identif-ying molecular markers linked to the function gene of salt tolerance will be helpful to soybean breeding. On the basis of the allelic variations of GmNcl1, which was a salt tolerance gene, a pair of primers was designed, and the method of restriction analysis was used to develop function marker. Finally, a cleaved amplified polymorphic sequences( CAPS) marker named CAPS/Xba I was developed. Ten different salt tolerance characteristic soybean cultivars have been screened by this marker. The result of salt tolerance soybean identified by the codominant marker was consisted with the result of that identified ac-cording to phenotype. So, CAPS/Xba I could be used in the identification of soybean salt tolerance.【期刊名称】《江苏农业学报》【年(卷),期】2017(033)006【总页数】8页(P1227-1234)【关键词】大豆;耐盐性;GmNcl1;酶切扩增多态性序列(CAPS)标记【作者】宁丽华;何晓兰;张大勇【作者单位】江苏省农业科学院种质资源与生物技术研究所/江苏省农业生物学重点实验室,江苏南京 210014;江苏省农业科学院种质资源与生物技术研究所/江苏省农业生物学重点实验室,江苏南京 210014;江苏省农业科学院种质资源与生物技术研究所/江苏省农业生物学重点实验室,江苏南京 210014【正文语种】中文【中图分类】S565.1土壤盐渍化和次生盐渍化是一个全球性的问题，严重影响农业生产并造成生态问题。
野生大豆转录因子GsNA C20基因的分离及胁迫耐性分析
野生大豆转录因子GsNA C20基因的分离及胁迫耐性分析才华;朱延明;李勇;柏锡;纪巍;千冬冬;孙晓丽【期刊名称】《作物学报》【年(卷),期】2011(37)8【摘要】NAC (NAM,ATAF1/2,CUC2)转录因子作为一类新型转录因子已成为非生物胁迫基因工程领域的研究热点.本研究以野生大豆(Glycine soja)为材料,利用酵母单杂交的方法筛选到一个能够与MYB1AT元件(核心序列为AAACCA)结合的转录因子基因,该基因与大豆NAC20 (EU440353.1)基因具有99%的相似性,命名为GsNAC20.GsNAC20蛋白含有典型的NAC结构域和转录激活区.酵母试验表明,GsNA C20转录因子能够与耐逆相关顺式元件MYB1AT特异结合,但不具有自激活功能.细胞定位分析证明该基因位于细胞核中,符合转录因子的特征.GsNAC20能够响应高盐、干旱和低温胁迫,并且在根和叶中具有不同的表达模式.超量表达GsNAC20基因的拟南芥对盐胁迫的敏感性提高.以上结果表明GsNAC20参与植物非生物胁迫反应过程,该基因在非生物胁迫基因工程研究领域具有良好的理论研究和实际应用价值.%Abiotic stresses, such as salt and drought, affect plant growth, development and reduce crop yield. Isolation of a key regulatory gene linked to response to abiotic-stress and identification of the genes function are urgently needed. Glycine soja is an excellent material to isolate abiotic stress-related genes because of its high stress tolerance. Plant-specific transcription factor NAC (NAM, ATAF1/2, CUC2) proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. Ithas become a new research focus in the abiotic-stress field. Based on that, we screened a new NAC gene from Glycine soja by yeast one hybrid, which has 99% similarity with NAC20 of Glycine max (EU44O353.1), named as GsNAC20. GsNAC20 had typical NAC DNA-binding domain at the N-terminal and transcription activation region at the C-terminal. It can bind to MYB1AT element (the core sequence: AAACCA) in vitro, but no transcriptional activation activity in the yeast assay system, which was consistent with GmNAC20. Localization of GsNAC20 protein was analyzed by transient expression in tobacco epidermis cells and the result showed that GsNAC20 was localized in nucleus. Semi-quantitative RT-PCR showed the expression level of GsNAC20 was induced by drought, low temperature and salt stresses, but there existed difference between leaf and root in G soja. Arabidopsis thaliana plants overexpressing GsNAC20 showed higher sensitivity under salt stress. All results showed that GsNAC20 perhaps is a new member of NAC family in G soja, and is closely related to salt and drought stresses, so it can either be used as a new resource in gene engineering on stress tolerance or be further studied to provide more information for the researches on the mechanism of stress tolerance in plant.【总页数】9页(P1351-1359)【作者】才华;朱延明;李勇;柏锡;纪巍;千冬冬;孙晓丽【作者单位】东北农业大学生命科学学院,黑龙江哈尔滨150030;东北农业大学生命科学学院,黑龙江哈尔滨150030;东北农业大学生命科学学院,黑龙江哈尔滨150030;东北农业大学生命科学学院,黑龙江哈尔滨150030;东北农业大学生命科学学院,黑龙江哈尔滨150030;东北农业大学生命科学学院,黑龙江哈尔滨150030;东北农业大学生命科学学院,黑龙江哈尔滨150030【正文语种】中文【相关文献】1.谷子MYB类转录因子SiMYB42提高转基因拟南芥低氮胁迫耐性 [J], 丁庆倩;刁现民;闵东红;马有志;陈明;王小婷;胡利琴;齐欣;葛林豪;徐伟亚;徐兆师;周永斌;贾冠清2.大豆转录因子基因GmNF-YCa可提高转基因拟南芥渗透胁迫的耐性 [J], 李敏;于太飞;徐兆师;张双喜;闵东红;陈明;马有志;柴守诚;郑炜君3.野生大豆胁迫应答膜联蛋白基因的克隆及胁迫耐性分析 [J], 王希;李勇;朱延明;柏锡;才华;纪巍4.野生大豆转录因子GsWRKY57基因的克隆与抗旱性功能分析 [J], 王岩岩;张永兴;郭葳;代文君;周新安;矫永庆;沈欣杰5.野生大豆GsbZIP33基因的分离及胁迫耐性分析 [J], 才华;朱延明;柏锡;纪巍;李勇;王冬冬;孙晓丽因版权原因，仅展示原文概要，查看原文内容请购买。
大豆盐胁迫相关GmNAC基因的鉴定、表达及变异分析
通讯作者 (Corresponding author): 徐冉 , E-mail: soybeanxu@
第一作者联系方式： E-mail: 13854198480@, Tel: 0531-83179348 Received(收稿日期 ): 2015-12-16; Accepted(接受日期 ): 2016-03-14; Published online(网络出版日期 ): 2016-03-28. URL: /kcms/detail/11.1809.S.20160328.1116.008.html
Abstract: NAC genes play an important role in plant stress tolerance. In this study, bioinformatic method was used to identify the stress related GmNAC gene in soybean. The expression of candidated GmNAC genes in root and leaf was analyzed in soybean with NaCl treatment by Real-time PCR. Reverse transcription PCR was performed to clone genes with significant difference in expression. The results showed that there were 175 genes in soybean GmNAC gene family. There were 11 GmNAC proteins with highly conserved NAC located on the same evolutionary branch with the stress related NAC proteins in rice and Arabidopsis. The expression of 11 GmNAC genes in soybean root was higher than that in leaf. The GmNAC genes were all induced by NaCl stress, but part of the GmNAC genes showed different expression levels between root and leaf in soybean varieties with different salt tolerances. There were three synonymous mutations and one non-synonymous mutation on the CDS region of Glyma06g11970.1 and one synonymous mutation on the CDS region of Glyma06g16440.2 in Qihuang 34, Xudou 10, and Fendou 95. Keywords: Soybean; GmNAC; Phylogenetic tree; NaCl treatment; Expression analysis; Sequence variation
盐胁迫下苗期栽培大豆生理响应及Na+动态平衡关键基因的表达
盐胁迫下苗期栽培大豆生理响应及Na+动态平衡关键基因的表达宁丽华;张大勇;刘佳;何晓兰;万群;徐照龙;黄益洪;邵宏波【期刊名称】《中国农业科学》【年(卷),期】2016(049)024【摘要】[目的]研究耐盐栽培大豆和盐敏感栽培大豆对盐胁迫的响应,特别是盐胁迫对大豆幼苗光合特性、离子含量及Na+动态平衡相关基因表达的影响,通过比较盐胁迫下不同大豆品种的响应差异,揭示不同基因型大豆耐盐机制,为大豆栽培管理、耐盐品种的选育及人工调控提供理论参考.[方法]以耐盐栽培大豆(Y8D6008、Y8D6013)和盐敏感栽培大豆(Y8D6132、Y8D6136)为材料,选取长势一致的大豆幼苗于1/2×Hoagland营养液中培养,待第一片复叶完全展开时,营养液中加入NaCl,每天递增50 mmol·L-1到达处理浓度150 mmol·L-1,处理持续7d.以不加NaCl的1/2×Hoagland营养液作为对照,研究盐胁迫下大豆幼苗的光合特性、离子含量及Na+动态平衡相关基因表达变化.[结果]150mmol·L-1 NaCl不同程度地抑制了4种大豆幼苗生长,同时显著降低SPAD值、净光合速率、气孔导度和蒸腾速率,但是NaCl胁迫对盐敏感大豆影响程度显著高于耐盐品种;盐胁迫显著降低耐盐大豆的胞间CO2浓度,而盐敏感大豆与之相反,说明150 mmol·L-1 NaCl处理下气孔限制是引起耐盐品种光合速率下降主要因素,而盐敏感品种光合速率下降主要因素是非气孔限制.对大豆植株的不同离子含量进行测定,发现盐胁迫下4种大豆叶片中Na+积累均显著升高,盐敏感品种上升幅度显著高于耐盐品种,而K+含量与Na+含量的变化规律相反.盐敏感大豆叶片中磷含量(P)均受盐胁迫显著下降,而耐盐大豆叶片P在胁迫后略有增加.相关分析表明净光合速率变化幅度与叶片中Na+、K+和P含量变化幅度存在显著的相关性.对6个参与大豆植株体内Na+动态平衡相关基因GmSOS1、GmNcl1、GmSALT3、GmNHX1(离子通道基因)、GmCIPK1(信号转导基因)和GmAVP1(能量运输相关基因)相对表达量进行分析,发现盐胁迫后4种大豆的GmNcl1表达量均显著上调,盐敏感品种上调倍数高于耐盐大豆品种,这种表达交化与大豆的耐盐性具有一定的关联性,而其他5个基因表达量与大豆的耐盐性没有明显的关联性.[结论]与盐敏感大豆相比,耐盐大豆在盐胁迫环境条件下减少Na+在叶片中的积累,保持相对较高的K+和P含量,并维持相对较高的光合速率,这是耐盐大豆比盐敏感大豆具有较强耐盐特性的因素之一,另外Na+动态平衡相关基因GmNcl1可能与大豆耐盐特性有一定关联性.【总页数】12页(P4714-4725)【作者】宁丽华;张大勇;刘佳;何晓兰;万群;徐照龙;黄益洪;邵宏波【作者单位】江苏省农业科学院农业生物技术研究所/江苏省农业生物学重点实验室,南京210014;江苏省农业科学院农业生物技术研究所/江苏省农业生物学重点实验室,南京210014;江苏省农业科学院农业生物技术研究所/江苏省农业生物学重点实验室,南京210014;江苏省农业科学院农业生物技术研究所/江苏省农业生物学重点实验室,南京210014;江苏省农业科学院农业生物技术研究所/江苏省农业生物学重点实验室,南京210014;江苏省农业科学院农业生物技术研究所/江苏省农业生物学重点实验室,南京210014;江苏省农业科学院农业生物技术研究所/江苏省农业生物学重点实验室,南京210014;江苏省农业科学院农业生物技术研究所/江苏省农业生物学重点实验室,南京210014【正文语种】中文【相关文献】1.14个多年生黑麦草品种幼苗期对盐胁迫的生理响应 [J], 魏晓艳;梁丹妮;庞丁铭;兰剑2.番茄苗期响应盐胁迫的生理特性研究 [J], 王柏柯;唐亚萍;李宁;赛里克;罗淑萍;余庆辉3.饲用黑麦、小黑麦品种苗期耐盐性评价及盐胁迫下的生理响应 [J], 谢楠;赵海明;李源;游永亮;李伟;刘贵波;刘宏彩4.轻度盐胁迫下施氮量对小麦苗期的生理响应 [J], 杨柳;李絮花;胡斌;刘敏;刘文博;李金鑫;张静;王子凤5.OsWD40过表达水稻在盐胁迫下的生理响应 [J], 柏华美;黄梓轩;郭敏;鲍聆然;沈波因版权原因，仅展示原文概要，查看原文内容请购买。
盐胁迫下大豆木质部溶液中Na+和K+含量变化分析
盐胁迫下大豆木质部溶液中Na+和K+含量变化分析盐胁迫是指盐分浓度对植物生长与发育产生不良影响的现象，其中Na+和K+是两种主要的离子物质。
大豆作为我国的主要粮食作物之一，对盐胁迫的响应机制一直备受研究者的关注。
在盐胁迫下，植物木质部溶液中Na+和K+含量的变化对于揭示植物盐胁迫响应机制具有重要意义。
本文将针对盐胁迫下大豆木质部溶液中Na+和K+含量的变化进行分析。
一、盐胁迫对大豆Na+和K+含量的影响盐胁迫条件下，土壤中的盐分浓度增加，会导致盐分通过植物根系进入植物体内。
Na+是盐分中的主要成分之一，它进入植物体内后会影响植物的正常生长发育。
研究表明，盐胁迫条件下，大豆体内Na+含量明显增加。
这主要是因为盐胁迫条件下，土壤中的Na+含量增加，大豆通过根系吸收了大量的Na+。
过量的Na+进入植物细胞内后，会影响植物细胞的渗透调节，导致细胞内外离子平衡紊乱，进而影响植物的正常生长发育。
在盐胁迫条件下，大豆木质部溶液中Na+含量显著增加。
研究表明，盐胁迫条件下，大豆木质部溶液中Na+含量呈现出逐渐增加的趋势。
这是因为盐胁迫条件下，土壤中的Na+含量增加，大豆吸收了大量的Na+，导致大豆木质部溶液中的Na+含量明显增加。
这种情况表明，盐胁迫条件下大豆对Na+的排斥效应较弱，大豆木质部对Na+的排斥能力不足，从而导致Na+大量积累在大豆木质部溶液中。
这些结果为研究者揭示植物对盐胁迫的响应机制提供了重要线索。
在今后的研究中，研究者可以进一步探究盐胁迫下大豆木质部Na+和K+的转运机制，以及相关调节蛋白和基因的表达情况，从而揭示大豆对盐胁迫的生理响应机制。
这些研究成果也为进一步培育耐盐性大豆品种提供了理论依据和实验基础。
通过对盐胁迫下大豆木质部溶液中Na+和K+含量变化的深入研究，可以为我国的大豆生产提供更多的科学技术支持，从而提高大豆的产量和质量。
盐胁迫下大豆木质部溶液中Na+和K+含量变化分析
盐胁迫下大豆木质部溶液中Na+和K+含量变化分析研究表明，盐胁迫对大豆木质部溶液中Na+和K+含量产生了显著影响。
在盐胁迫条件下，大豆根系吸收的Na+含量明显增加，而K+则明显减少。
这是因为盐胁迫会破坏大豆根系的离子平衡，使得根系失去对K+选择性的吸收能力，同时增加对Na+的吸收。
由于Na+和K+具有相似的离子半径和电荷，它们在根系内竞争吸收，而盐分中高浓度的Na+会抑制K+的吸收，进而导致大豆木质部溶液中K+含量的降低。
与此盐胁迫还会引起大豆木质部溶液中Na+和K+的运输和分配变化。
研究发现，盐胁迫会增加Na+和K+的向上运输速率，以减少其在根部的积累。
这是因为过量的Na+和减少的K+会引起细胞内渗透调节紊乱，从而导致细胞失水、质膜破裂和离子平衡紊乱等不良反应。
为了减轻这些不利影响，大豆会通过提高Na+和K+的向上运输速率来减少根部离子积累，从而维持离子平衡。
研究还发现，在盐胁迫条件下，大豆会通过调节根系和茎叶部位离子渗透调节物质的表达来降低Na+和提高K+含量。
大豆根系中的Na+/H+和K+/H+交换体能够将过量的Na+向外排泄，并吸收更多的K+，从而降低Na+含量并提高K+含量。
大豆还会增加Na+和K+的累积载体的表达，以增加Na+和K+的吸收能力。
通过这些机制，大豆能够在一定程度上减轻盐胁迫带来的不良影响，提高耐盐性。
盐胁迫下大豆木质部溶液中Na+和K+含量的变化分析是了解大豆耐盐性和对盐胁迫响应机制的重要途径。
研究发现，盐胁迫会导致大豆根系吸收Na+增加、K+减少，同时增加Na+和K+的向上运输速率，并通过调节离子渗透调节物质的表达来降低Na+和提高K+含量，从而提高大豆的耐盐性。
这些研究结果对于筛选耐盐性大豆品种、改良耐盐性和利用盐碱地生产大豆具有重要的意义。
大豆耐盐相关基因的分离及其功能鉴定
大豆耐盐相关基因的分离及其功能鉴定余玉雯;孙海丹;郑易之;兰英;胥顺;唐玉林;麻晓亮【期刊名称】《深圳大学学报（理工版）》【年(卷),期】2004(021)004【摘要】利用大肠杆菌功能表达体系,从大豆开花40 d未成熟种子的cDNA表达文库中筛选获得了11个耐盐相关cDNA克隆.对其中3个克隆的插入片段进行测序. 结果显示,其中的Gm1013为一尚未报道的新基因片段;GmRPS25-2编码的蛋白质序列与西红柿40S 核糖体蛋白S25有91%的同源性;GmAIP-2编码的蛋白质序列与大豆根铝诱导蛋白SALI3-2有97%的同源性.其中GmAIP-2可表达分子量为30.8 kD的多肽.pET-GmAIP-2转化后的大肠杆菌在含800 mmol/L NaCl和700 mmol/L KCl的液体培养基中生长状况明显好于对照菌,即前者表现出较短的生长迟滞期和较高的生长量.【总页数】7页(P324-330)【作者】余玉雯;孙海丹;郑易之;兰英;胥顺;唐玉林;麻晓亮【作者单位】深圳大学生命科学学院,深圳市微生物基因工程重点实验室,深圳,518060;深圳大学生命科学学院,深圳市微生物基因工程重点实验室,深圳,518060;深圳大学生命科学学院,深圳市微生物基因工程重点实验室,深圳,518060;深圳大学生命科学学院,深圳市微生物基因工程重点实验室,深圳,518060;深圳大学生命科学学院,深圳市微生物基因工程重点实验室,深圳,518060;深圳大学生命科学学院,深圳市微生物基因工程重点实验室,深圳,518060;深圳大学生命科学学院,深圳市微生物基因工程重点实验室,深圳,518060【正文语种】中文【中图分类】Q781【相关文献】1.大豆耐盐相关基因GmNcl1功能标记的开发及验证 [J], 宁丽华;何晓兰;张大勇2.大豆耐盐相关基因GmNcl1的序列单倍型及表达分析 [J], 赫卫;刘林;关荣霞;邱丽娟3.大豆PM2蛋白11氨基酸结构域的耐盐功能鉴定 [J], 叶展辉;郑易之;刘昀4.大豆耐盐相关基因研究进展 [J], 方义生;曹东;杨红丽;刘小荣;张恒斌;陈李淼;周新安5.大豆耐盐相关基因STL的克隆与分析 [J], 李富鹏;张凌;王国丰;曹又方;王绛;唐克轩因版权原因，仅展示原文概要，查看原文内容请购买。
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Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250131, China; 2 Key Laboratory of Soybean Biology of Ministry of
Education, Northeast Agricultural University, Harbin 150001, China
[23-24]
1
1.1
材料与方法
试验材料
选用本课题组前期鉴定的耐盐品种徐豆 10、齐
黄 34 和盐敏感品种汾豆 95。
1.2
NAC 蛋白质序列的获得 从 植 物 转 录 因 子 数 据 库 PlantTFDB (http:// /)下载大豆 NAC 转录因子序 列 , 对去冗余后的蛋白序列根据 Phytozome (http:// /) 的 功 能 注 释 并 利 用 SMART (http:// smart.embl-heidelberg.de/) 分 析 氨 基 酸 结 构 域 , 获 得 具有 NAC 保守结构域的候选基因 ; 查询文献获得拟 南芥和水稻逆境胁迫相关的 NAC 蛋白质序列。 NAC 蛋白家族系统进化树分析 利用 MEGA 5.05 软件内置的 Clustal W 程序对
高温、低温、干旱和盐碱等非生物逆境胁迫严 重影响作物的生长 , 造成作物大幅度减产 。 NAC 蛋白是一类植物特异的参与多种植物生理生化过程 的转录因子 [2], 广泛参与植物生长发育以及逆境应 答。 NAC (NAM, ATAF1-2, CUC2) 最初起源于 NAM
[1]
(no apical meristem), ATAF1-2 和 CUC2 (cup-shaped cotyledon)
作物学报
ACTA AGRONOMICA SINICA 2016, 42(7): 990999 ISSN 0496-3490; CODEN TSHPA9
/ E-mail: xbzw@
DOI: 10.3724/SP.J.1006.2016.00990
[3]
。 NAC 蛋白包含一个保守的 N- 端 DNA
结合域和一个多样化的转录调控域 [4]。 NAC家族成员 能够激活植物的逆境应答基因 , 提高植物对逆境的 耐受性。 AtNAC072和 AtN究 由 东北 农业 大 学 大豆 生物 学 教 育部 重点 实 验 室开 放基 金 项 目 (SB14A04), 国 家现 代农 业 产 业技 术体 系 建 设专 项 (CARS-04CES18), 国家自然科学基金项目 (31501329)和山东省自然科学基金项目 (ZR2015YL070)资助。 The study was supported by the Open Foundation of Key Laboratory of Soybean Biology of Ministry of Education, Northeast Agricultural University (SB14A04), the China Agriculture Research System (CARS-04-CES18), the National Natural Science Foundation of China (31501329), and the Natural Science Foundation of Shandong Province (ZR2015YL070).
通讯作者 (Corresponding author): 徐冉 , E-mail: soybeanxu@
第一作者联系方式： E-mail: 13854198480@, Tel: 0531-83179348 Received(收稿日期 ): 2015-12-16; Accepted(接受日期 ): 2016-03-14; Published online(网络出版日期 ): 2016-03-28. URL: /kcms/detail/11.1809.S.20160328.1116.008.html
Abstract: NAC genes play an important role in plant stress tolerance. In this study, bioinformatic method was used to identify the stress related GmNAC gene in soybean. The expression of candidated GmNAC genes in root and leaf was analyzed in soybean with NaCl treatment by Real-time PCR. Reverse transcription PCR was performed to clone genes with significant difference in expression. The results showed that there were 175 genes in soybean GmNAC gene family. There were 11 GmNAC proteins with highly conserved NAC located on the same evolutionary branch with the stress related NAC proteins in rice and Arabidopsis. The expression of 11 GmNAC genes in soybean root was higher than that in leaf. The GmNAC genes were all induced by NaCl stress, but part of the GmNAC genes showed different expression levels between root and leaf in soybean varieties with different salt tolerances. There were three synonymous mutations and one non-synonymous mutation on the CDS region of Glyma06g11970.1 and one synonymous mutation on the CDS region of Glyma06g16440.2 in Qihuang 34, Xudou 10, and Fendou 95. Keywords: Soybean; GmNAC; Phylogenetic tree; NaCl treatment; Expression analysis; Sequence variation
Identification, Expression and Variation Analysis of Salt Tolerance Related GmNAC Genes in Soybean
ZHANG Yan-Wei1,2, ZHANG Li-Feng1, LI Wei1, WANG Cai-Jie1, ZHANG Jun1, and XU Ran1,
信息学方法鉴定了大豆逆境相关 GmNAC 基因 , 利用荧光定量 PCR 技术分析了 GmNAC 基因在耐盐差异的大豆品种 根部、 叶片的表达及其对 NaCl 胁迫的应答 , 采用反转录 PCR 技术克隆了表达差异显著的 GmNAC 基因。 结果表明 , 大 豆 GmNAC 基因家族包含 175 个基因 , 其中 11 个基因所编码的 GmNAC 蛋白与水稻和拟南芥的逆境相关 NAC 蛋白 位于同一进化分支 , 这些蛋白具有高度保守的 NAC 结构域 ; 这 11 个 GmNAC 基因在大豆根部的表达均高于在叶片 , 而且在叶片和根部均受 NaCl 诱导 , 部分基因在根部和叶片以及品种间表现出不同的表达规律 ; 在大豆品种齐黄 34、 徐豆 10 和汾豆 95 中 , Glyma06g11970.1 存在 3 个同义突变和 1 个非同义突变 , Glyma06g16440.2 存在 1 个同义突变。 关键词 : 大豆 ; GmNAC; 进化树 ; NaCl 处理 ; 表达分析 ; 序列变异
第7期
张彦威等 : 大豆盐胁迫相关 GmNAC 基因的鉴定、表达及变异分析
991
通路的关键基因 , AtNAC072的表达受高盐、脱落酸 (ABA)、生长素、乙烯、茉莉酸 (JA)、脱水、损伤等 多种因素诱导并参与植物的信号传导[5], AtNAC019的 表达 受ABA、 热、 高盐、 JA、 干旱应答等因素诱导, 并 调节植物生长发育[6-8]。AtNAC055是拟南芥JA信号通 路的关键基因, 调控JA的生物合成, 并能对高盐、 真菌 等多种逆境应答 [7-8]。 AtNAC102能够参与拟南芥的洪 涝 胁 迫 应 答 [9] 。 OsNAC002[10-13] 、 OsNAC048[14-15] 和 OsNAC068[16]参与水稻的耐旱、耐盐等多种逆境胁迫。 随着对大豆 NAC 基因家族研究的深入 , 越来越 多的大豆 NAC 类转录因子得到了鉴定。孟庆长等首 次在大豆中克隆得到 6 个 GmNAC 基因 [17-18]; 韩巧玲 等 [19]利用 NTT (核蛋白筛选系统 )从大豆耐盐品种铁 丰 8 号中克隆得到 GmNAC2a 基因 , 该基因对干旱、 高温、低温、高盐、 ABA、乙烯等多种途径均有响 应 ; 金 杭 霞 等 [20] 发 现 GmNAC2 参 与 逆 境 调 控 , GmNAC5 参与大豆发育调控 ; 才华等 [21]利用酵母单 杂交的方法从野生大豆中克隆得到能与耐逆相关顺 式元件 MYB1AT 特异结合的 GsNAC20 基因 , 该基因 能够响应高盐、干旱和低温胁迫 , 并且在根和叶中 具有不同的表达模式 , 超量表达 GsNAC20 的拟南芥 提高盐胁迫的敏感性 ; Tran 等 [22]在大豆中克隆了 31 个 GmNAC 基因 , 发现 9 个 NAC 基因能同时响应干 旱、高盐、冷、 ABA 等胁迫应答 , 聚类分析发现 9 个 GmNAC 基因与前人报道的逆境相关 NAC 基因位 于同一分支 ; Le 等