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棉花学报, ):
doi: 10.11963/issn.1702006
陆地棉基因GhWRKY40-1的克隆及表达分析
Cloning and Expression Analysis of the GhWRKY40-1 Gene in Gossypium hirsutum L.
司爱君, 田琴, 陈红, 余渝
Si Aijun, Tian Qin, Chen Hong, Yu Yu
Cotton Research Institute, Xinjiang Academy of Agricultural and Reclamation Science/Key Laboratory of China Northwestern Inland Region, Ministry of Agriculture/Key Laboratory of Genetic Improvement and High Yield, Xinjiang Production and Construction Corps, Shihezi, Xinjiang 832000, China
基金资助: 国家自然科学基金();
中图分类号:
S562.035&&&&
文献标识码:
【目的】WRKY转录因子通过激活下游一系列抗逆应答基因的表达而提高植株的综合抗性。本研究旨在研究WRKY转录因子在陆地棉中的功能。【方法】通过基因芯片筛选鉴定出1个逆境诱导的陆地棉WRKY转录因子基因,由于此基因编码蛋白含有1个典型的WRKY结构域,且与拟南芥AtWRKY40具有较高相似性,故命名为GhWRKY40-1。采用电子克隆及反转录-聚合酶链反应技术获得GhWRKY40-1基因cDNA全长,并对其进行序列分析及结构与功能预测。【结果】GhWRKY40-1的开放阅读框为981 bp,编码326个氨基酸,预测蛋白相对分子质量为35.78×103,等电点为8.39。GhWRKY40-1在拟南芥原生质体中瞬时表达定位于细胞核,且具有转录激活活性。RT-PCR结果表明,GhWRKY40-1受甘露醇诱导上调表达。【结论】推测GhWYKY40-1可能参与干旱胁迫应答反应,并且在植物逆境胁迫中发挥重要作用。上述分析为进一步从分子水平上验证其抗逆生物学功能以及揭示棉花非生物胁迫抗逆机制奠定了基础。
[Objective] WRKY transcription factors enhance plant resistance by activating the expression of a series of stress-responsive downstream genes. The purpose of our research was to analyze functions of WRKY transcription factors in upland cotton. [Method] A stress-induced upland-cotton WRKY transcription factor gene was screened by cDNA microarray analysis. The gene, which was found to contain a typical WRKY domain and to share a highly similar amino acid sequence with AtWRKY40, was accordingly named GhWRKY40-1. The full-length cDNA of GhWRKY40-1 was obtained by electronic cloning and reverse transcription-polymerase chain reaction (RT-PCR) technology, with protein sequence analysis and structure-function prediction performed using bioinformatics software. [Result] According to our results, GhWRKY40-1 contains a 981-bp open reading frame encoding 326 amino acid residues, with a predicted relative molecular mass of 35.78 kDa and an isoelectric point of 8.39.In Arabidopsis protoplasts, GhWRKY40-1 was localized in the nucleusand was found to have transcriptional activation activity. RT-PCR analysis showed that GhWRKY40-1 was up-regulated under mannitol stress. [Conclusion] We hypothesize that GhWYKY40-1 is involved in drought stress response and plays an important role in plant stress responses. Our characterization and bioinformatics analyses have laid the foundation for future confirmation of the biological functions of resistance at the molecular level and elucidation of mechanisms of abiotic stress resistance in cotton.
Key words:
transcription factor
gene cloning
sequence analysis
WRKY转录因子是1类锌指型转录因子,成员均含有1~2个大约60个氨基酸残基所组成的保守的WRKY结构域,且含有7个绝对保守的氨基酸残基WRKYGQK,并由此得名为WRKY。
WRKY转录因子广泛参与植物的逆境应答,在拟南芥、小麦、大豆和水稻中已有大量的文献报道WRKY转录因子受环境因子（如高盐[-]、干旱[-]、低温[, ]、高温[, ]等）的诱导表达,通过特异地与靶基因调控区的顺式元件结合,调节靶基因表达的强度及时空特异性[],在抗逆信号传导及诱导下游功能基因的表达中起关键作用。WRKY基因首先克隆于甘薯（Impoea batatas）,随后在拟南芥、水稻和烟草等模式植物以及马铃薯[]、大麦[]、灌木[]、辣椒[]、遏蓝菜[]、葡萄[]、番茄[]、小麦[]等植物中均发现了参与逆境胁迫的WRKY基因。其中,在拟南芥和水稻中分别发现了70多个和100多个WRKY转录因子成员[-]。目前关于WRKY转录因子的研究在拟南芥、水稻和烟草等模式植物中居多且主要集中在抗病反应上,而我国重要经济作物棉花中WRKY转录因子的研究报道相对较少。本研究利用基因芯片从陆地棉中筛选到1个逆境诱导WRKY转录因子基因GhWRKY40-1,通过电子克隆及RT-PCR（Reverse transcription-polymerase chain reaction）技术获得基因全长,并采用生物信息学分析方法对其结构、功能等进行了预测,分析其在棉花不同组织中及不同非生物胁迫下的表达情况,并对其进行序列比对及系统进化树分析,初步明确该基因的功能,为今后深入研究该基因的分子生物学特性及遗传进化等奠定基础。
1 材料与方法
1.1 植物材料与处理
试验材料为陆地棉品种珂字棉312（Coker312）,由中国科学院遗传与发育生物学研究所杨维才研究员提供。挑选籽粒饱满、均匀的棉花种子种植于蛭石中,待幼苗第3片真叶完全展开时,将棉苗分别用甘露醇（Mannitol,Man,200 μmol·L-1）、脱落酸（Abscisic acid,ABA,1 μmol·L-1）、高pH（10.0）及低温（4 ℃）诱导处理24 h,然后将处理材料及对照（未经处理）整株用液氮速冻,于-80 ℃保存备用。
琼脂糖凝胶回收试剂盒、DNA Marker、Taq DNA聚合酶购自北京康为世纪生物技术公司;克隆载体pDM18-T载体购自TaKaRa公司;大肠杆菌菌株DH5α、农杆菌菌株GV3101、双元载体pBI12(R)由本实验室保存;反转录试剂盒（SuperScript R Ⅲ）购自Invitrogen公司,限制性内切酶EcoRⅠ、BamHⅠ、SacⅠ与T4 DNA ligase购自F引物合成、测序均由英俊生物技术有限公司完成;其他试剂均为国产或进口分析纯试剂。
1.3.1 陆地棉GhWRKY40-1基因的筛选及基因全长扩增。以上述低温、高pH、甘露醇及外源ABA胁迫处理24 h后的Coker312为材料,采用CTAB/酸酚法提取棉花整株总RNA[],交由博奥生物有限公司完成基因表达谱芯片。同时,采用CTAB法提取棉花DNA,经琼脂糖凝胶电泳检测其质量。对照样品的总RNA经DNaseⅠ消化后,取2~5 μg反转录合成cDNA用于基因克隆。
根据目的基因表达序列标签（Expressed sequence tag,EST）序列搜索UNIGENE数据库,聚类组装成1个叠连群,并利用VECTOR NTI 10.0软件将EST序列拼接装配。通过网上同源基因蛋白水平上的比对及基因非编码区（Untranslated region,UTR）序列特性确定全长,并通过DNAStar软件及开放阅读框（Open reading frame,ORF）finder进行开放阅读框预测。根据序列设计合成1对特异引物GhWRKY40-1-F（5'-CGGCACGAGGAAAGTTTCAGCTT-3'）和GhWRKY40-1-R（5'- TCGTTTTTTGCAAGGCTGTATTTAC-3'）,以对照样品cDNA为模板扩增GhWRKY40-1基因全长,将其连接到pMD18-T载体,选阳性克隆进行测序。
50 μL PCR反应体系中含cDNA（20 mg·L-1） 1 μL,10×PCR buffer 5 μL,dNTP（10 mmol·L-1）2 μL,引物（25 μmol·L-1）1 μL,Taq（5 U·μL-1）0.5 μL,用ddH2O补至50 μL。PCR程序为94 ℃预变性594 ℃变性30 s,56 ℃复性30 s,72 ℃延伸1 min,共32个循环;72 ℃延伸10 min。
1.3.2 陆地棉GhWRKY40-1基因的序列分析、同源比对及系统发育树的构建。将获得的目的片段克隆测序,测序结果通过DNAstar软件及ORF finder工具在线分析,采用MEGA7.0软件将GhWRKY40-1与拟南芥等物种的WRKY转录因子进行多序列比对及系统进化树构建[]。系统进化树构建步骤为：采用MEGA7.0软件内置的ClustalW程序默认的IUB记分矩阵进行核酸序列多重比对,根据多重比对结果构建系统发生树。参数设置：采取邻接法（Neighbor-joining method）的Complete Deletion模式构建系统发育树,采用随机逐步比较的方式搜索最佳系统树,对生成的系统发生树进行Bootstrap校正。利用ExPASy Proteomics tools中的ProtParam对GhWRKY40-1蛋白质的理化性质进行分析,并用Motif scan对GhWRKY40-1编码的蛋白质中潜在修饰位点进行预测,通过SOSUI在线预测蛋白的亲水性,通过SMART网站（http://smart.embl-heidelberg.de/）对GhWRKY40-1基因进行蛋白结构预测和功能分析。利用同源建模SWISS-MODEL程序,通过在ExPDB晶体图像数据库中搜索相似序列,进行优化比对后,对GhWRKY40-1蛋白质三维结构进行预测。生成的PDB（Protein data bank）结果在SWISS-pdb Viewer软件中观察蛋白质的结构。
1.3.3 GhWRKY40-1蛋白在拟南芥原生质体中亚细胞定位分析。经蛋白亚细胞定位预测网站PSORT (http://psort.hgc.jp/form.html)对转录因子进行电子定位,分析GhWRKY40-1基因序列中是否有核定位信号。用pBI221作为瞬时表达载体,BamHI/SalⅠ限制性内切酶酶切pMD 18T-GhWRKY40-1质粒,获得双酶切后的GhWRKY40-1片段,凝胶回收后连接到经过同样黏性末端的pBI221-GFP载体,与GFP基因的5'端融合,构建由35S启动子驱动的GhWRKY40-1- GFP表达载体。将GhWRKY40-1-GFP和pBI221- GFP载体质粒（作为正对照）转化到拟南芥原生质体中（方法参考http://genetics.mgh.arvard.edu/ sheenweb/protocols）,暗培养16~18 h后用ZEISS LSM 700激光共聚焦扫描显微镜观察。
1.3.4 GhWRKY40-1的转录激活活性分析。采用双荧光素酶报告基因测试系统,在拟南芥原生质体中检测GhWRKY40-1蛋白的转录调控功能。将GhWRKY40-1全长基因构建到效应基因载体上,构建GAL4DBD-GhWRKY40-1融合蛋白。以空GAL4DBD为负对照,以融合有VP16的载体为正对照。以2个报告基因表达后发出荧光量的比值（相对荧光素酶活性）反映测试蛋白的转录激活活性。
2 结果与分析
2.1 逆境应答40-1基因的筛选及序列分析
根据SAM软件的数据分析筛选出1个多逆境诱导基因DT467159（图1）,其编码的蛋白具有WRKY家族特有的WRKYGQK序列（图2加粗阴影处）。进化树分析显示,其与拟南芥AtWRKY40编码的蛋白具有较高相似性（图3）,且与GhWRKY11和GhWRKY40（2）的亲缘关系较近（图4）。此前,已有不同来源的陆地棉GhWRKY40[]在GenBank注册登记：注册登录号为KC414680的基因组DNA序列以及登录号为KC414679的mRNA序列,编码序列（Coding sequence,CDS）长度为945 bp[-];注册登录号为KF669767的mRNA序列,CDS为1458 bp[];注册登录号为KF031086的mRNA序列,CDS为873 bp[]。由于陆地棉材料的来源不同,各基因序列也略有不同。故暂将本研究发现的DT467159基因命名为GhWRKY40-1。经过半定量RT-PCR分析,发现GhWRKY40-1基因在甘露醇胁迫下上调表达（图5）,说明此基因可能与抗旱相关。
棉花基因芯片表达谱分析——多逆境诱导基因DT467159色块表示基因在样品中的表达值,红色表示表达值高,红色深浅反映了数据的绝对值大小,绿色表示表达值低,灰色表示表达值居中。结果中没有表达值低的样品,因此没有绿色色块出现。
Analysis of cotton expression spectrum—multiple stresses induced gene, DT467159 The small squares in the figure shows the expression quantity of the genes. Red color indicates that the expression quantity is high, its intensity reflects the absolu green means the expression quantity is low, and gray indicates the gene expression level is middle. There is no sample with low expression level in this figure, so no green square appears.
GhWRKY40-1编码的氨基酸序列分析加粗阴影部分为WRKY结构域中的WRKYGQK序列。
Analysis of amino acid sequence encoded by GhWRKY40-1 The bold and shadowed part is conserved WRKYGQK in WRKY domain.
DT467159（GhWRKY40-1）与拟南芥WRKY转录因子的系统发育树
Phylogenesis analysis of DT467159 (GhWRKY40-1) and WRKY transcriptional factor of Arabidopsis thaliana
DT467159（GhWRKY40-1）与陆地棉WRKY转录因子的系统发育树
Phylogenesis analysis of DT467159 (GhWRKY40-1) and WRKY transcriptional factor of Gossypium hirsutum
不同胁迫条件下GhWRKY40-1基因的表达分析
Expression analysis of GhWRKY40-1 under different stress
2.2 GhWRKY40-1蛋白理化性质分析、结构域预测及系统发育树的构建
2.2.1 GhWRKY40-1编码的蛋白理化性质分析。蛋白质组成等的预测显示,GhWRKY40-1是1个由326个氨基酸残基组成,相对分子质量为35.78×103,等电点8.39的蛋白质。SOSUI在线预测显示,GhWRKY40-1是1个稳定的可溶性蛋白,其亲水性的总平均值为-0.658896。
利用Motif scan对GhWRKY40-1编码的蛋白质序列潜在修饰位点预测,结果发现,序列中含有1个WRKY保守域(174~234 aa),表明GhWRKY40-1是棉花WRKY转录因子的成员。序列中含有潜在的酰胺化位点1个（56~59 aa）;cAMP-cGMP依赖型蛋白激酶磷酸化位点3个（58~61,81~84,118~121 aa）;酪氨酸激酶Ⅱ磷酸化位点7个（23~26,61~64,109~112,138~141,215~218,236~239,274~277 aa）;肉豆蔻酰化位点2个（129~134,184~189 aa）;蛋白激酶C磷酸化位点8个（34~36,61~63,109~111,116~118,145~147,244~246,271~273, 317~319 aa）。这些磷酸化位点大多位于基因的激酶区和调控区,推测其对于GhWRKY40-1蛋白激酶的结构和功能有很重要的作用。以上表明,GhWRKY40-1转录因子可能存在多种翻译后水平的修饰,以调节其自身的表达水平。
2.2.2 GhWRKY40-1蛋白的结构预测。如图6所示,GhWRKY40-1为典型的WRKY转录因子,在其61~112个氨基酸处有1段复合螺旋区（Coiled coil region）,174~234个氨基酸处有典型的WRKY结构域,121~145个氨基酸处有1个低复杂性序列区域。GhWRKY40-1既不是膜蛋白,也不是分泌蛋白,蛋白预测未发现跨膜区（Transmembrane segment）和信号肽（Signal peptide）。
GhWRKY40-1蛋白的结构域分析数字表示氨基酸编号;WRKY代表WRKY结构域;绿色为复合螺旋区,紫色为低复杂性序列区域。
Conserved domain analysis of GhWRKY40-1 protein Figures are nu WRKY represents the WRKY green bars represents
purple bars represents a low complexity region.
GhWRKY40-1为亲水性蛋白,定位于细胞核,但由于其C端无明显的转录激活域,推测此蛋白可能具有较弱的转录激活功能。
GhWRKY40-1的蛋白质三维结构预测（图7）显示,GhWRKY40-1主要由β-折叠和无规则卷曲组成。
GhWRKY40-1蛋白的三维结构预测
Three-dimensional structure prediction of GhWRKY40-1 protein
2.2.3 蛋白多重序列比对。将推测的GhWRKY40-1基因编码的序列与油菜、菠菜、苜蓿、黄瓜、大豆、毛白杨等不同物种的WRKY转录因子进行多重序列比对和同源性分析,发现基因保守区域氨基酸具有较高相似性。GhWRKY40-1与不同种属WRKY蛋白序列均有1段大约由60个高度保守的氨基酸残基所组成的多肽构成的WRKY结构域,其中有1个绝对保守的7个“WRKYGQK”（图8,红色图框标记）氨基酸残基。在保守区域之外,氨基酸序列差异较大。通过Mega7.0软件构建系统发生树,并进行Bootstrap校正,结果见图8。
GhWRKY40-1的多序列比对及同源性分析序列名称前2个字母代表物种（Two letter of the factor names represent different plant species）：AA,Artemisia annua,青蒿; AT,Arabidopsis thaliana,拟南芥;BG, Bruguiera gymnorhiza,木榄;BN,Brassica napus, 甘蓝型油菜;CA,Capsicum annuum,辣椒; CS, Cucumis sativus,黄瓜;GA, Gossypium arboreum,亚洲棉;GH,Gossypium hirsutum,陆地棉;GM,Glycine max,大豆;GR,Gentiana rigescens,滇龙胆;LT,Larrea tridentata,石碳酸灌木;MS,Medicago sativa,紫花苜蓿;PT, Populus tomentosa,毛白杨;SO,Spinacia oleracea,菠菜;VV,Vitis vinifera,无核葡萄。
Multiple alignment and phylogenic analysis of GhWRKY40-1 transcription factor
GhWRKY40-1与不同物种WRKY转录因子的系统发育树分析序列名称前2个字母代表物种（Two letter of the factor names represent different plant species）：Di,Dioscorea ipomoea,甘薯;Nt,Nicotiana tabacum,烟草;Pc,Petroselinum crispum,欧芹;Aa,Artemisia annua,青蒿;Ms,Medicago sativa,紫花苜蓿;Vp,Vitis pseudoreticulata,华东葡萄;Bg,Bruguiera gymnorhiza,木榄;Ga,Gossypium arboreum,亚洲棉;Pt,Populus tomentosa,毛白杨;Cs,Cucumis sativus,黄瓜;Gm,Glycine max,大豆;Lt,Larrea tridentata,石碳酸灌木;Vv,Vitis vinifera,无核葡萄;Gh,Gossypium hirsutum,陆地棉;So,Spinacia oleracea,菠菜;At,Arabidopsis thaliana,拟南芥; Bn,Brassica napus,甘蓝型油菜。
Phylogenesis analysis of GhWRKY40-1 and other WRKY transcription factors
2.3 40-1基因的亚细胞定位分析
蛋白预测发现GhWRKY40-1蛋白包含了核定位信号,为了验证分析该结果和蛋白核定位特性,通过在拟南芥原生质体细胞中瞬时表达GhWRKY40-1基因检测其在胞内的定位情况。结果表明,转对照pBI221-GFP的拟南芥原生质体中,绿色荧光分布在细胞核和细胞质中,而转GhWRKY40-1-GFP的拟南芥原生质体中,绿色荧光富集在细胞核区域,红色荧光表示叶绿体的位置（图10）。这些结果表明GhWRKY40-1蛋白是核定位蛋白,这与转录因子核定位的特征是一致的。
GhWRK40-1在拟南芥原生质体中的亚细胞定位分析
Analysis of the subcellular localization of GhWYKY40-1 in protoplast of Arabidopsis
2.4 GhWRKY40-1的转录激活活性分析
如图11所示,阳性对照VP16调控的报告基因表达量与阴性对照相比,差异极显著。GAL4DBD-GhWRKY40-1调控的报告基因表达量明显高于阴性对照,说明GhWRKY40-1有转录激活活性。
GhWRK40-1在拟南芥原生质体中的转录激活活性分析 GAL4DBD为阴性对照,VP16为阳性对照。
Transcription activation analysis of GhWYKY40-1 in protoplast of Arabidopsis GAL4DBD as negative control, VP16 as a positive control.
3 讨论与结论
近年来的研究发现,与单抗基因相比,由于转录因子可通过激活下游多个功能基因的表达来提高植物综合抗逆能力,因此成为作物改良的研究热点;尤其是WRKY转录因子,因其在植物抗胁迫反应中起重要作用而备受关注。大部分WYKY类转录因子基因并非组成型表达,而是受到干旱、高盐、低温、机械损伤、细菌、病毒、真菌等多种非生物和生物胁迫以及乙烯（Ethylene,ET）、ABA、水杨酸（Salicylic acid,SA）等植物信号分子的诱导,且该类因子的表达具有组织特异性。在复杂的抗逆调控网络中,WYKY转录因子主要参与转录水平和翻译水平的调控。
张娜等[]从陆地棉中克隆得到3个WRKY基因GhWRKY4、GhWRKY5、GhWRKY6,可能在抗旱和耐盐中起作用。史伟娜[]从鲁棉22中通过同源克隆方法分离到GhWRKY39基因,能够被病原菌和高盐诱导表达。杨淑巧等[]从棉花中克隆得到GhWRKY25基因,对干旱和盐碱都有应答,在植物生长发育及胁迫应答过程中发挥重要作用。赵曾强等[]从高抗枯萎病品种中棉所12中克隆得到GhWRKY44,可能参与棉花对病原菌和激素的应答反应。戴鹤[]从Pima90-53和农大601中分别克隆了WRKY46,结果表明WRKY46参与棉花抗病反应及SA、茉莉酸和ET信号转导网络。Cai等从雷蒙德氏棉中分离了116个WRKY基因,从陆地棉中克隆了102 个WRKY基因,发现棉花WRKY基因可能根据自我调整产生进化,并参与了各种胁迫反应[]。Dou等从雷蒙德氏棉中分离了120个候选WRKY基因,并将其分为3组,第1组有20个成员,第2组有88个成员,第3组有12个成员;第2组的88个基因又可分为5个亚组;WRKY基因的表达模式分析表明,其可能在棉花不同发育阶段中发挥重要作用[]。Chu等[-]从陆地棉中分离出GhWRKY41并在本生烟中的过表达,研究表明GhWRKY41可能通过ABA信号途径调节气孔关闭和ROS水平参与植物对干旱、高盐胁迫的响应过程。Shi等[]对GhWRKY39-1基因的过表达能增强转基因本生烟对病原体感染和高盐的抗性及氧化应激耐受性,表明GhWRKY39-1作为植物防御病原体感染、盐胁迫和活性氧反应的正向调节器。Yan等[]发现GhWRKY17在干旱、盐、H2O2和ABA处理下被诱导,并且在烟草中的组成型表达显著降低植物对干旱和盐胁迫的耐受性,表明GhWRKY17通过ABA信号传导和植物中细胞ROS产生的调节对干旱和盐胁迫起反应。Jia等[]的研究表明,GhWRKY68的表达受干旱、盐、ABA及H2O2的诱导,且在过表达烟草中对干旱和盐的抗性降低,推测GhWRKY68是通过调节ABA信号和细胞ROS响应干旱和盐胁迫的转录因子。Yan等[]在本生烟中对GhWRKY27a过表达及病毒诱导基因沉默的研究表明,GhWRKY27a的表达受非生物胁迫、病原体感染和多个防御相关的信号分子的诱导,且在对干旱耐受性和丝核菌属丝核菌感染的抗性中起负调控作用。Ding等[]在烟草中的的研究表明,TaWRKY1通过一系列ABA依赖途径的渗透胁迫防御过程以及调节气孔运动和植物保水能力来调控耐旱性,ABA受体基因NtPYL8在干旱引发的ABA信号传导中发挥重要作用。Fan等[]从菊花中分离CmWRKY1,与野生型植物相比,过表达CmWRKY1增强了植株对聚乙二醇处理的脱水耐受性,初步证明CmWRKY1通过ABA介导的途径在菊花干旱响应中发挥作用。由于WYKY转录因子广泛参与植物各种胁迫反应,近3 a来关于WYKY转录因子如何参与非生物胁迫应答反应的研究也已陆续开展,但棉花WRKY转录因子家族中仅有少数被深入研究,还有许多家族成员功能尚需验证。
本研究利用基因芯片及电子克隆技术,从陆地棉中筛选并克隆到1个逆境应答基因,由于它含有典型的WRKY家族特有的结构域特征,且与拟南芥AtWRKY40具有较高相似性,因此命名为GhWYKY40-1。GhWRKY40-1编码326个氨基酸,推测其相对分子质量为35.78×103。GhWRKY40-1转录因子具有典型的WRKY结构域,没有信号肽及跨膜区域,说明GhWRKY40-1既不是膜蛋白,也不是分泌蛋白。GhWRKY40-1序列中存在糖基化、磷酸化、豆蔻酰化和酰胺化等各种修饰位点,说明其可能存在多种翻译后水平的修饰作用。系统发育树分析表明,在棉花WRKY家族中,GhWRKY40-1与GhWRKY11聚在一起;不同物种来源的WRKY基因同源分析表明,GhWRKY40-1与油菜、菠菜、苜蓿、黄瓜、大豆、毛白杨等WRKY基因具有高度同源性,其中与菠菜SoWRKY1同源性最高。在拟南芥原生质体中的亚细胞定位及转录激活活性研究表明,正如大多数转录因子一样,GhWRKY40-1定位于细胞核,且具有较强的转录激活功能。通过利用生物信息学软件对GhWYKY40-1蛋白质的结构与功能进行初步预测结果发现,GhWYKY40-1可能参与多种逆境胁迫反应。
由于GhWRKY40受生物胁迫及非生物胁迫多种逆境诱导发挥重要作用,Wang等通过qPCR分析GhWRKY40在棉花中的表达模式表明,GhWRKY40受SA、茉莉酸及ET信号的调控,过表达GhWRKY40的烟草对创伤和细菌病原体做出协调反应[]。张坤对GhWRKY40-like在抗病中的功能进行了初步鉴定,研究发现GhWRKY40- like受SA、茉莉酸甲酯及病原菌诱导后呈上调表达,RNAi抑制表达转基因棉花表现出明显抗性[]。虽然已有不同来源的GhWYKY40注册,但其基因序列及编码框都略有不同,且对其相关功能的研究主要侧重于在生物胁迫中的作用[-]。鉴于WRKY转录因子对多种非生物胁迫都有响应的特性,作者将进一步研究GhWYKY40-1转录因子在干旱、低温等非生物逆境胁迫中的功能。RT-PCR研究结果表明 GhWRKY40-1基因受甘露醇诱导上调表达,推测此基因可能在植物耐旱反应中发挥重要作用。目前作者已通过烟草的遗传转化研究GhWRKY40-1基因功能,下一步将通过分析GhWRKY40-1基因与其他生物大分子（DNA、RNA和蛋白质等）之间的相互作用等,进一步揭示GhWRKY40-1基因在植物响应干旱胁迫过程中的调节途径,为今后深入研究该转录因子的分子生物学特性及遗传进化等提供依据,并为转录因子GhWYKY40-1在棉花育种中的应用奠定基础。
The authors have declared that no competing interests exist.
Jiang Yuanqing, Deyholos M K.Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses[J]. Plant Molecular Biology, 2009, 69(1): 91-105.
Previous microarray analyses of Arabidopsis roots identified two closely related WRKY transcription factors ( WRKY25
and WRKY33 ) among the transcripts that increased in abundance following treatment with NaCl. Here, we report further characterization of these genes, which we found to be inducible by a variety of abiotic stresses in an SOS-pathway independent manner, although WRKY33
induction was dependent on ABA signaling. Transcripts of both genes were detected in roots and leaves, while specific patterns of enrichment were observed in stems and floral buds for WRKY25
and WRKY33 , respectively. We also identified upstream intergenic regions from each gene that were sufficient to confer stress-inducible expression on a reporter gene. However, the stress sensitivity of wrky25
null mutants did not differ from wild-type under any assay condition, while wrky33
null mutants and wrky25wrky33
double mutants showed only a moderate increase in NaCl-sensitivity, suggesting functional redundancy with other transcription factors. Nevertheless, overexpression of WRKY25
was sufficient to increase Arabidopsis NaCl tolerance, while increasing sensitivity to ABA. Through microarray analyses of relevant genotypes, we identified 31 and 208 potential downstream targets of WRKY25 and WRKY33, respectively, most of which contained a W-box in their upstream regions.
[本文引用:1]
Zhou Qiyun, Tian Aiguo, Zou Hongfeng, et al.Soybean WRKY type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants[J]. Plant Biotechnology Journal, 2008, 6(5): 486-503.
Abstract WRKY-type transcription factors have multiple roles in the plant defence response and developmental processes. Their roles in the abiotic stress response remain obscure. In this study, 64 GmWRKY genes from soybean were identified, and were found to be differentially expressed under abiotic stresses. Nine GmWRKY proteins were tested for their transcription activation in the yeast assay system, and five showed such ability. In a DNA-binding assay, three proteins (GmWRKY13, GmWRKY27 and GmWRKY54) with a conserved WRKYGQK sequence in their DNA-binding domain could bind to the W-box (TTGAC). However, GmWRKY6 and GmWRKY21, with an altered sequence WRKYGKK, lost the ability to bind to the W-box. The function of three stress-induced genes, GmWRKY13, GmWRKY21 and GmWRKY54, was further investigated using a transgenic approach. GmWRKY21-transgenic Arabidopsis plants were tolerant to cold stress, whereas GmWRKY54 conferred salt and drought tolerance, possibly through the regulation of DREB2A and STZ/Zat10. Transgenic plants over-expressing GmWRKY13 showed increased sensitivity to salt and mannitol stress, but decreased sensitivity to abscisic acid, when compared with wild-type plants. In addition, GmWRKY13-transgenic plants showed an increase in lateral roots. These results indicate that the three GmWRKY genes play differential roles in abiotic stress tolerance, and that GmWRKY13 may function in both lateral root development and the abiotic stress response.
[本文引用:3]
Wu Xiaolan, Shiroto Y, Kishitani S, et al.Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter[J]. Plant Cell Report, 2009, 28(1): 21-30.
Abstract An OsWRKY11 gene, which encodes a transcription factor with the WRKY domain, was identified as one of the genes that was induced by both heat shock and drought stresses in seedlings of rice (Oryza sativa L.). To determine if overexpression of OsWRKY11 confers heat and drought tolerance, OsWRKY11 cDNA was fused to the promoter of HSP101 of rice and introduced into a rice cultivar Sasanishiki. Overexpression of OsWRKY11 was induced by heat treatment. After heat pretreatment, the transgenic lines showed significant heat and drought tolerance, as indicated by the slower leaf-wilting and less-impaired survival rate of green parts of plants. They also showed significant desiccation tolerance, as indicated by the slower water loss in detached leaves. Our results indicate that the OsWRKY11 gene plays a role in heat and drought stress response and tolerance, and might be useful for improvement of stress tolerance.
[本文引用:1]
Qiu Yuping, Yu Diqiu.Over-expression of the stress-induced OsWRKY45 enhances disease resistance and drought tolerance in Arabidopsis[J]. Environmental and Experimental Botany, 2009, 65(1): 35-47.
The WRKY transcriptional factor superfamily regulates diverse functions, including processes such as plant development and stress response. In this study, we have shown that the rice WRKY45
( OsWRKY45 ) expression is markedly induced in response to stress-related hormone abscisic acid (ABA) and various stress factors, e.g., application of NaCl, PEG, mannitol or dehydration, treatment with 0 C and 42 C as well as infection by Pyricularia oryzae
Cav. and Xanthomonas oryzae
pv. oryzae . Together, these results indicate that the OsWRKY45
may be involved in the signal pathways of both biotic and abiotic stress response. Further analyses of 35S: OsWRK45 Arabidopsis
plants have shown that ectopic, constitutive over-expression of the OsWRKY45
transgene confers a number of properties to transgenic plants. These properties include significantly increased expression of PR
genes, enhanced resistance to the bacterial pathogen Pseudomonas syringae tomato
DC3000, enhanced tolerance to salt and drought stresses, decreased sensitivity toward ABA signalling during seed germination and post-germination processes, and modulation of ABA/stress-regulated genes during drought induction. In addition, higher levels of OsWRKY45
expression in transgenic plants correlate positively with the strength of the abiotic and biotic responses mentioned above. More specifically, the decreased ABA sensitivities, the enhanced disease resistance and drought tolerances may be attributed, in part, to stomatal closure and induction of stress-related genes during drought induction. The relationship between OsWRKY45
expression and ABA signalling is discussed.
[本文引用:]
Wang Zhi, Zhu Yan, Wang Lili, et al.A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter[J]. Planta, 2009, 230(6): 1155-1166.
&a name="Abs1"&&/a&Accumulation of compatible osmolytes, such as soluble sugars, in plants is an important osmoprotective mechanism. Sugars play
a role in osmotic adjustment and are associated with stabilization of proteins and cell structures, reactive oxygen species
scavenging, signaling functions or induction of adaptive pathways. Galactinol is the galactosyl donor for the synthesis of
raffinose family oligosaccharides (RFOs) and its synthesis by galactinol synthase (GolS) is the first committed step of the
RFOs biosynthetic pathway. GolS genes are induced by a variety of stresses in both stress-sensitive and tolerant-
however, the mechanism of transcriptional regulation is not fully established. In this paper, we characterized a GolS gene
(&i&BhGolS1&/i&) that was dehydration and ABA-inducible in the resurrection plant &i&Boea hygrometrica&/i& and conferred dehydration tolerance in a transgenic tobacco system. Four W-box &i&cis&/i&-elements were identified in the &i&BhGolS1&/i& promoter and shown to be bound by an early dehydration and ABA-inducible WRKY gene (&i&BhWRKY1&/i&). These data suggest a mechanism where &i&BhWRKY1&/i& is likely to function in an ABA-dependent signal pathway to regulate &i&BhGolS1&/i& expression, which leads to the accumulation of RFOs in desiccation-tolerant &i&B. hygrometrica&/i& leaves.
[本文引用:1]
Talanova V V, Titov A F, Topchieva L V, et al.Expression of WRKY transcription factor and stress protein genes in wheat plants during cold hardening and ABA treatment[J]. Plant Physiology, 2009, 56(5): 702-708.
Changes in expression of WRKY transcription factor and stress protein genes ( Wcor15, Wrab17, Wrab19 , and Wcs120 ) were studied on wheat ( Triticum aestivum
L., cv. Moskovskaya 39) seedlings exposed to cold hardening for 7 days at 4 C. The high level of WRKY
gene expression was noticed already in 15 min after the beginning of cold treatment, but the expression level lowered during longer treatments. Exogenous ABA (0.1 mM) suppressed the WRKY
gene expression. The level of Wcor15
gene expression increased gradually, reaching the peak on the second day, and then decreased. Gene expression of Wrab17
remained elevated throughout the period of cold exposure (7 days), and expression of Wrab19
was promoted within the first two days. Exogenous ABA induced expression of Wcor15, Wrab17 , and Wrab19
genes both at cold-hardening (4 C) and normal (22 C) temperatures. A significant increase in Wcs120
gene expression during cold hardening was ABA-independent. It is concluded that the increase of wheat plant resistance at the initial stage of cold hardening is related to gene expression of WRKY transcription factor and of stress proteins ( Wcor15, Wrab17, Wrab19 , and Wcs120 ), while the resistance increase during prolonged adaptation is related to gene expression of Wcor15
and Wrab17 .
[本文引用:1]
Li Shujia, Fu Qiantang, Huang Weidong, et al.Functional analysis of an Arabidopsis transcription factor WRKY25 in heat stress[J]. Plant Cell Report, 2009, 28(4): 683-693.
The WRKY family is one of the major groups of plant-specific transcriptional regulators. Arabidopsis
WRKY25, which is induced by heat stress, is one of the group I WRKY proteins and responds to both abiotic and biotic stress. This study has examined the regulatory role of WRKY25 using wrky25
mutant and over-expressing WRKY25
transgenic A. thaliana . After 45 C for different time periods, wrky25
null mutants showed a moderate increase in thermosensitivity with decreased germination, reduced hypocotyl and root growth, and enhanced conductivity compared to those of wide-type, while WRKY25
over-expressed transgenic seeds exhibited enhanced thermotolerance. Northern blot analysis of wrky25
mutants and WRKY25
over-expressing plants identified putative genes regulated by WRKY25. In consistence with the implication of WRKY25 in heat tolerance, the expression level of six heat-inducible genes and two oxidative stress-responsive genes was more or less down-regulated in wrky25
mutants during heat stress. Among them, heat shock protein
Hsp101 , heat shock transcription factor HsfB2a , and cytosolic ascrobate peroxidase APX1
were reduced more obviously than other detected genes. Meanwhile, over-expression of WRKY25
increased the expression of HsfA2 , HsfB1 , HsfB2a , and Hsp101
slightly or moderately. Together, these findings reveal that WRKY25 plays a partial role in thermotolerance.
[本文引用:1]
陈俊, 王宗阳. 植物MYB类转录因子研究进展[J]. 植物生理与分子生物学学报, 2002, 28(2): 81-88.
植物MYB转录因子以含有保守的MYB结构域为共同特征 ,广泛参与植物发育和代谢的调节。含单一MYB结构域的MYB转录因子在维持染色体结构和转录调节上发挥着重要作用 ,是MYB转录因子家族中较为特殊的一类。含两个MYB结构域的MYB转录因子成员众多 ,在植物体内主要参与次生代谢的调节和控制细胞的形态发生。含 3个MYB结构域的MYB蛋白与c MYB蛋白高度同源 ,可能在调节细胞周期中起作用
[本文引用:1]
Chen Jun, Wang Zongyang.Progress in the study of plant MYB transcription factors[J]. Acta Photophysiologica Sinica, 2002, 28(2): 81-88.
李立芹, 黄玉碧, 王西瑶. 马铃薯WRKY6基因的克隆、序列分析与原核表达研究[J]. 草业学报, 2011(2): 177-183.
在高等植物中WRKY转录因子家族成员众多,它广泛参与植物对生物胁迫、非生物胁迫、生长发育和代谢过程的调控。本研究采用同源序列克隆的方法获得马铃薯WRKY6基因,序列分析表明该蛋白属于WRKY家族第3组成员,锌指结构为C-X7-C-X23-H-X-C。构建系统发育树结果表明它与拟南芥WRKY70亲缘关系较近,相似性达58%。然后将其完整编码区构建到大肠杆菌表达载体。在培养温度18℃条件下添加0.2 mmol/L IPTG（异丙基硫代半乳糖苷）,诱导培养8 h可获得高表达融合蛋白。进一步实验获得特异性和纯度非常高的纯化蛋白。本研究为进一步确定该蛋白的体外活性及生物学功能奠定了坚实基础。
[本文引用:1]
Li Liqin, Huang Yubi, Wang Xiyao.Cloning, sequence analysis and prokaryotic expression of the WRKY6 of potato[J]. Acta Prataculturae Sinica, 2011(2): 177-183.
Mare C, Mazzucotelli E, Crosatti C, et al.HvWRKY38: a new transcription factor involved in cold and drought response in barley[J]. Plant Molecular Biology, 2004, 55(3): 399-416.
WRKY proteins constitute a large family of plant specific transcription factors implicated in many different processes. Here we describe Hv-WRKY38, a barley gene coding for a WRKY protein, whose expression is involved in cold and drought stress response. Hv-WRKY38 was early and transiently expressed during exposure to low non-freezing temperature, in ABA-independent manner. Furthermore, it showed a continuous induction during dehydration and freezing treatments. A WRKY38:YFP fusion protein was found to localise into the nucleus upon introduction into epidermal onion cells. Bacterially expressed Hv-WRKY38 was able to bind in vitro to the W-box element (T)TGAC(C/T) also recognisable by other WRKY proteins. Hv-WRKY38 genomic DNA was sequenced and mapped onto the centromeric region of the barley chromosome 6H. Arabidopsis and rice sequences homologous to Hv-WRKY38 were also identified. Our results indicate that Hv-WRKY38 transcription factor may play a regulatory role in abiotic stress response.
[本文引用:1]
Zou Xiaolu, Seemann J R, Neuman D, et al.A WRKY gene from creosote bush encodes an activator of the abscisic acid signaling pathway[J]. Journal of Biological Chemistry, 2004, 279(53): 55770-55779.
The creosote bush () is a xerophytic evergreen C3 shrub thriving in vast arid areas of North America. As the first step toward understanding the molecular mechanisms controlling the drought tolerance of this desert plant, we have isolated a dozen genes encoding transcription factors, including that encodes a protein of 314 amino acid residues. Transient expression studies with the fusion construct indicate that the LtWRKY21 protein is localized in the nucleus and is able to activate the promoter of an abscisic acid (ABA)-inducible gene, , in a dosage-dependent manner. The transactivating activity of LtWRKY21 relies on the C-terminal sequence containing the WRKY domain and a N-terminal motif that is essential for the repression activity of some regulators in ethylene signaling. LtWRKY21 interacts synergistically with ABA and transcriptional activators VP1 and ABI5 to control the expression of the promoter. Co-expression of , and leads to a much higher expression of the promoter than does the ABA treatment alone. In contrast, the Lt-WRKY21-mediated transactivation is inhibited by two known negative regulators of ABA signaling: 1-butanol, an inhibitor of phospholipase D, and abi1-1, a dominant negative mutant protein phosphatase. Interestingly, abi1-1 does not block the synergistic effect of , and co-expression, indicating that LtWRKY21, VP1, and ABI5 may form a complex that functions downstream of ABI1 to control ABA-regulated expression of genes.
[本文引用:1]
张春秋, 茆振川, 杨宇红, 等. 辣椒WRKY转录因子基因CaRKNIF1植物表达载体构建及番茄转化[J]. 分子植物育种, 2010, 8(4): 713-718.
WRKY转录因子广泛参与植物对生物和非生物的胁迫应答反应,在 植物防卫反应中起着重要作用.CaRKNIF1是我们从辣椒HDA149中分离得到的新WRKY转录因子基因(GenBank登陆号DQ180348), 本研究通过RT-PCR扩增得到CaRKNIF1基因的开放阅读框,经由中间载体pEASY-T1 Simple,将其定向连接到表达载体pCHF3上,得到植物表达载体pCHF3-CaRKNIF1.采用电击法将pCHF3-CaRKNIF1导入农杆 菌菌株LBA4404,农杆菌介导法获得了抗性番茄植株.PCR、Southern杂交和RT-PCR检测结果表明,CaRKNIF1基因已经成功整合到 番茄基因组中,并正常表达.此研究为进一步通过CaRKNIF1基因提高转基因番茄的抗逆性奠定了基础.
[本文引用:1]
Zhang Chunqiu, Mao Zhenchuan, Yang Yuhong, et al.Construction of plant expression vector for WRKY transcription factor CaRKNIF1 gene from pepper and its transformation to tomato[J]. Molecular Plant Breeding, 2010, 8(4): 713-718.
Wei Wei, Zhang Yuxiu, Han Lu, et al.A novel WRKY transcriptional factor from Thlaspi caerulescens negatively regulates the osmotic stress tolerance of transgenic tobacco[J]. Plant Cell Report, 2008, 27(4): 795-803.
[本文引用:1]
Liu Huaying, Yang Wenlong, Liu Dongcheng, et al.Ectopic expression of a grapevine transcription factor VvWRKY11 contributes to osmotic stress tolerance in Arabidopsis[J]. Molecular Biology Reports, 2011, 38(1): 417-427.
Plant WRKY transcriptional factors play an important role in response to biotic and abiotic stresses. In this study, a WRKY transcription factor was isolated from grapevine. This transcription factor showed 66% and 58% identity at the DNA and amino acid sequence levels, respectively, with Arabidopsis AtWRKY11 genes, and was therefore designated VvWRKY11. Phylogenetic analysis and structure comparison indicated that VvWRKY11 protein belongs to group IIc. The VvWRKY11 protein was shown to be located in the nucleus based on green fluorescent protein analysis. Yeast one-hybrid analysis further indicated that VvWRKY11 protein binds specifically to the W-box element. The expression profile of VvWRKY11 in response to treatment with phytohormone salicylic acid or pathogen Plasmopara viticola is rapid and transient. Transgenic Arabidopsis seedlings overexpressing VvWRKY11 showed higher tolerance to water stress induced by mannitol than wild-type plants. These results clearly demonstrated that the VvWRKY11 gene is involved in the response to dehydration stress. In addition, the role of VvWRKY11 protein in regulating the expression of two stress response genes, AtRD29A and AtRD29B, is also discussed.
[本文引用:1]
金慧, 栾雨时. 番茄WRKY基因的克隆与分析[J]. 西北农业学报, 2011, 20(4): 96-101.
WRKY转录因子可调控下游抗 逆基因的表达,进而在植物生物和非生物胁迫中起重要作用。以水杨酸诱导的番茄为材料,设计简并引物后进行RT-PCR扩增,得到4个含有WRKY结构域的 EST。选择其中之一,采用同源克隆的方法扩增出WRKY基因的全长。半定量PCR分析结果表明,400 mmol/L NaCl、4℃低温胁迫后该基因表达量发生变化,说明其可能与番茄的耐盐性和耐低温性有关。
[本文引用:1]
Jin Hui, Luan Yushi.Isolation and analysis of WRKY gene from tomato[J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2011, 20(4): 96-101.
史红飞, 高翔, 陈其皎, 等. 小麦NAC转录因子的基因克隆与序列分析[J]. 麦类作物学报, 2011, 31(3): 395-401.
为了深入研究小麦中NAC家族转录因子基因，针对NAC基因家族成员，设计了覆盖其全长编码区的1对特异引物，从陕253小麦品种中克隆了2条大小分别为1 463、1 549 bp的片段，命名为TaNAC2、TaNAC4( GenBank登录号为HQ872050 HQ872051)。序列分析表明，这2个序列包含典型NAC的完整编码序列，包括两个内含子，具有完整的开放阅读框；推导的氨基酸序列分别为383、405个，这2个基因在N 端均具有NAC基因的典型DNA结合结构域，即 NAC结构域，且氨基酸序列在该结构域的 A、B、C、D、E 5个亚区高度保守，仅在C亚区出现一个氨基酸的差异L M，而且在C D区出现罕见的半胱氨酸变异，此发现对于小麦品质的研究非常重要。同时发现这 2个NAC类转录因子都不含有核定位信号（NLS），但是有相关的转录调控功能区域。通过系统进化树分析，证实克隆序列属于NAC基因家族成员,并且发现的TaNAC4属于NAC转录因子家族的NAM亚组，TaNAC2属于NAC转录因子家族的CUC亚组。
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Shi Hongfei, Gao Xiang, Chen Qijiao, et al.Genes cloning and sequences analysis of NAC transcription factor in wheat[J]. Journal of Triticeae Crops, 2011, 31(3): 395-401.
李蕾, 谢丙炎, 戴小枫, 等. WRKY转录因子及其在植物防御反应中的作用[J]. 分子植物育种, 2005, 3(3): 401-408.
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Li Lei, Xie Bingyan, Dai Xiaofeng, et al.WRKY transcription factors and their roles in plant defense responses[J]. Molecular Plant Breeding, 2005, 3(3): 401-408.
田云, 卢向阳, 彭丽莎, 等. 植物WRKY转录因子结构特点及其生物学功能[J]. 遗传, 2006, 28(12): 1607-1612.
WRKY转录因子是近年来在植物中发现的N-端含有 WRKYGQK高度保守氨基酸序列的新型转录调控因子, 它能够与(T)(T)TGAC(C/T)序列(W-box)发生特异性作用, 调节启动子中含W-box元件的调节基因和/或功能基因的表达, 从而参与植物的各种防卫反应, 调节植物的生长发育等.文章主要论述了植物WRKY转录因子的基本结构及其生物学功能.
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Tian Yun, Lu Xiangyang, Peng Lisha, et al.The structure and function of plant WRKY transcription factors[J]. Hereditas, 2006, 28(12): 1607-1612.
蒋建雄, 张天真. 利用CTAB/酸酚法提取棉花组织总RNA[J]. 棉花学报, 2003, 15(3): 166-167.
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Jiang Jianxiong, Zhang Tianzhen.Extraction of total RNA in cotton tissues with CTAB/acidic phenolic method[J]. Cotton Science, 2003, 15(3): 166-167.
薛庆中. DNA和蛋白质序列数据分析工具[M]. 2版. 北京: 科学出版社, 2010: 58-109.
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Xue Qingzhong.Data analysis tool of DNA and protein sequences[M]. 2nd ed. Beijing: Science Press, 2010: 58-109.
Xu Xinping, Chen Chunhong, Fan Baofang, et al.Physical and functional interactions between pathogen induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors[J]. Plant Cell, 2006, 18(5): 1310-1326.
Limited information is available about the roles of specific WRKY transcription factors in plant defense. We report physical and functional interactions between structurally related and pathogen-induced WRKY18, WRKY40, and WRKY60 transcription factors in Arabidopsis thaliana. The three WRKY proteins formed both homocomplexes and heterocomplexes and DNA binding activities were significantly shifted depending on which WRKY proteins were present in these complexes. Single WRKY mutants exhibited no or small alterations in response to the hemibiotrophic bacterial pathogen Pseudomonas syringae and the necrotrophic fungal pathogen Botrytis cinerea. However, wrky18 wrky40 and wrky18 wrky60 double mutants and the wrky18 wrky40 wrky60 triple mutant were substantially more resistant to P. syringae but more susceptible to B. cinerea than wild-type plants. Thus, the three WRKY proteins have partially redundant roles in plant responses to the two distinct types of pathogens, with WRKY18 playing a more important role than the other two. The contrasting responses of these WRKY mutants to the two pathogens correlated with opposite effects on pathogen-induced expression of salicylic acid-regulated PATHOGENESIS-RELATED1 and jasmonic acid-regulated PDF1.2. While constitutive expression of WRKY18 enhanced resistance to P. syringae, its coexpression with WRKY40 or WRKY60 made plants more susceptible to both P. syringae and B. cinerea. These results indicate that the three WRKY proteins interact both physically and functionally in a complex pattern of overlapping, antagonistic, and distinct roles in plant responses to different types of microbial pathogens.
[本文引用:1]
Wang Xiuling, Yan Yan, Li Yuzhen, et al. GhWRKY40, a multiple stress-responsive cotton WRKY gene, plays an important role in the wounding response and enhances susceptibility to Ralstonia solanacearum infection in transgenic Nicotiana benthamiana[J/OL]. PLoS ONE, 2014, 9(4): e93577 [2016-04-29]. .
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王秀玲. 棉花GhWRKY40基因的分离及其功能研究[D]. 泰安: 山东农业大学, 2014.
[本文引用:1]
Wang Xiuling.Identification and function analysis of GhWRKY40 in cotton (Gossypium hirsutum L.)[D]. Tai’an: Shandong Agricultural University, 2014.
Dou Lingling, Zhang Xiaohong, Pang Chaoyou, et al.Genome-wide analysis of the WRKY gene family in cotton[J]. Molecular Genetics and Genomics, 2014, 289(6): 1103-1121.
WRKY proteins are major transcription factors involved in regulating plant growth and development. Although many studies have focused on the functional identification of WRKY genes, our knowledge concerning many areas of WRKY gene biology is limited. For example, in cotton, the phylogenetic characteristics, global expression patterns, molecular mechanisms regulating expression, and target genes/pathways of WRKY genes are poorly characterized. Therefore, in this study, we present a genome-wide analysis of the WRKY gene family in cotton ( Gossypium raimondii and Gossypium hirsutum). We identified 116 WRKY genes in G. raimondii from the completed genome sequence, and we cloned 102 WRKY genes in G. hirsutum. Chromosomal location analysis indicated that WRKY genes in G. raimondii evolved mainly from segmental duplication followed by tandem amplifications. Phylogenetic analysis of alga, bryophyte, lycophyta, monocot and eudicot WRKY domains revealed family member expansion with increasing complexity of the plant body. Microarray, expression profiling and qRT-PCR data revealed that WRKY genes in G. hirsutum may regulate the development of fibers, anthers, tissues (roots, stems, leaves and embryos), and are involved in the response to stresses. Expression analysis showed that most group II and III GhWRKY genes are highly expressed under diverse stresses. Group I members, representing the ancestral form, seem to be insensitive to abiotic stress, with low expression divergence. Our results indicate that cotton WRKY genes might have evolved by adaptive duplication, leading to sensitivity to diverse stresses. This study provides fundamental information to inform further analysis and understanding of WRKY gene functions in cotton species.
[本文引用:2]
Cai Caiping, Niu Erli, Du Hao, et al.Genome-wide analysis of the WRKY transcription factor gene family in Gossypium raimondii and the expression of orthologs in cultivated tetraploid cotton[J]. The Crop Journal, 2014(Z1): 87-101.
WRKY proteins are members of a family of transcription factors in higher plants that function in plant responses to various physiological processes. We identified 120 candidate WRKY
genes from Gossypium raimondii
with corresponding expressed sequence tags in at least one of four cotton species, Gossypium hirsutum , Gossypium barbadense , Gossypium arboreum , and G. raimondii . These WRKY members were anchored on 13 chromosomes in G. raimondii
with uneven distribution. Phylogenetic analysis showed that WRKY candidate genes can be classified into three groups, with 20 members in group I, 88 in group II, and 12 in group III. The 88 genes in group II were further classified into five subgroups, groups IIa , containing 7, 16, 37, 15, and 13 members, respectively. We characterized diversity in amino acid residues in the WRKY domain and/or other zinc finger motif regions in the WRKY proteins. The expression patterns of WRKY
genes revealed their important roles in diverse functions in cotton developmental stages of vegetative and reproductive growth and stress response. Structural and expression analyses show that WRKY proteins are a class of important regulators of growth and development and play key roles in response to stresses in cotton.
[本文引用:3]
张娜, 赵佩, 沈法富. 陆地棉三个WRKY基因的克隆及表达分析[J]. 分子植物育种, 2012, 10(2): 169-173.
WRKY转录因子可调控下游抗逆基因的表达,进而在植物生长发育以及对外界环境的反应中起着重要的调控作用,目前对棉花WRKY家族基因的研究比较少.本研究利用电子克隆的方法,从陆地棉品种山农圣杂3号中克隆得到了3个具有完整开放阅读框的棉花WRKY基因,聚类分析表明它们同属于WRKY家族中的第Ⅱ类.利用RT-PCR结果表明:250 mmol/L NaC1盐胁迫和20％ PEG6000干旱胁迫下同时诱导GhWRKY4的基因表达,GhWRKY5仅受干旱胁迫下的诱导,而GhWRKY6对这两种逆境胁迫都没有变化.
[本文引用:1]
Zhang Na, Zhao Pei, Shen Fafu.Cloning and expression analysis of 3 WRKY genes from upland cotton[J]. Molecular Plant Breeding, 2012, 10(2): 169-173.
史伟娜. 棉花WRKY转录因子GhWRKY39的分离及功能研究[D]. 泰安: 山东农业大学, 2014.
[本文引用:1]
Shi Weina.Isolation and functional analyses of GhWRKY39 in cotton[D]. Tai’an: Shandong Agricultural University, 2014.
杨淑巧, 王志安, 张安红, 等. 棉花WRKY基因GhWRKY25的克隆和表达分析[J]. 生物技术进展, 2014, 4(4): 274-279.
棉花是一种重要的经济作物，在国民生产中起着重要的作用。然而干 旱盐碱等不良环境严重地制约着棉花的种植区域及其产量和品质。近年来，利用分子育种改良棉花对这些逆境的耐性的研究成为热点。本文从棉花中克隆了一个对干 旱和盐碱具有应答的WRKY基因，定名为GhWRKY25。该基因编码的蛋白包含两个典型的WRKY结构域，每个WRKY结构域的 C端含有一个 C2H2类型的锌指蛋白结构，进化树分析表明其属于Ⅰ类 WRKY蛋白，与可可的WRKY3同源性最高。利用qPCR分析发现该基因在棉花里为组成型表达，但在花中表达量较高。 GhWRKY25在施用SA、JA、ABA、GA 中表达表现为上调。干旱和NaCl处理时发现GhWRKY25基因表达均显著上调。通过上述结果可以推测GhWRKY25在棉花对于逆境的适应中具有重要 的作用，为该基因功能的进一步研究提供基础。
[本文引用:1]
Yang Shuqiao, Wang Zhi’an, Zhang Anhong, et al.Cloning and expression analysis of a WRKY gene GhWRKY25 in upland cotton[J]. Current Biotechnology, 2014, 4(4): 274-279.
赵曾强, 韩泽刚, 李会会, 等. 棉花GhWRKY44基因的克隆与表达分析[J]. 西北植物学报, 2015, 35(1): 10-15.
该研究以枯萎病菌诱导棉花根部基因表达谱中筛选得到的WRKY基因片段为探针,通过电子克隆结合RT-PCR技术,从高抗枯萎病品种‘中棉所12’中克隆到1个与抗枯萎病相关的WRKY转录因子基因GhWRKY44（GenBank登录号KJ801807）。序列分析表明,GhWRKY44基因开放阅读框1 197bp,编码398个氨基酸,含有2个WRKY结构域及C2H2型锌指结构,属于棉花WRKY转录因子家族第Ⅰ类;系统进化分析显示,GhWRKY44基因与拟南芥AtWRKY44亲缘关系最近。实时荧光定量PCR（qRT-PCR）检测该基因表达特性,结果发现枯萎病菌诱导后,GhWRKY44基因在抗病品种中优势表达,随处理后时间的推移,其表达量呈先增加后降低再增加的变化趋势,处理后3h时GhWRKY44基因表达量达到最大;而在感病品种中,GhWRKY44基因表达水平明显低于抗病品种,对病原菌响应时间也晚于抗病品种,处理后6h时GhWRKY44基因表达量才达到最大。水杨酸和茉莉酸均能诱导GhWRKY44基因的表达,水杨酸诱导后,GhWRKY44基因表达量迅速增加,且其表达量一直维持在一个较高水平;而茉莉酸诱导后,GhWRKY44基因表达量呈先增加后降低的变化趋势,且其表达水平明显低于水杨酸诱导。研究表明,GhWRKY44基因可能参与了棉花对病原菌和激素胁迫的应答反应。
[本文引用:1]
Zhao Zengqiang, Han Zegang, Li Huihui, et al.Cloning and expression analysis of GhWRKY44 in cotton[J]. Acta Botanica Boreali-Occidentalia Sinica, 2015, 35(1): 10-15.
戴鹤. 棉花WRKY转录因子家族基因鉴定、表达分析和WRKY46功能研究[D]. 保定: 河北农业大学, 2015.
[本文引用:1]
Dai He.Identification, expression analysis of cotton WRKY family of transcription factors and function analysis of WRKY46 in Gossypium barbadense[D]. Baoding: Hebei Agricultural University, 2015.
褚晓茜. 棉花GhWRKY41基因的分离与功能分析[D]. 泰安: 山东农业大学, 2014.
[本文引用:1]
Chu Xiaoqian.Identification and function analysis of GhWRKY41, a novel WRKY gene from cotton (Gossypium hirsutum L.)[D]. Tai’an: Shandong Agricultural University, 2014.
Chu Xiaoqian, Wang Chen, Chen Xiaobo, et al. The cotton WRKY gene GhWRKY41 positively regulates salt and drought stress tolerance in transgenic Nicotiana benthamiana[J/OL]. PLoS ONE, 2015, 10(11): e0143022 []. DOI: <ext-link ext-link-type="doi" xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="10.1371/journal.pone..1371/journal.pone.0157026.
WRKY transcription factors constitute a very large family of proteins in plants and participate in modulating plant biological processes, such as growth, development and stress responses. However, the exact roles of WRKY proteins are unclear, particularly in non-model plants. In this study, Gossypium hirsutum WRKY41 (GhWRKY41) was isolated and transformed into Nicotiana benthamiana. Our results showed that overexpression of GhWRKY41 enhanced the drought and salt stress tolerance of transgenic Nicotiana benthamiana. The transgenic plants exhibited lower malondialdehyde content and higher antioxidant enzyme activity, and the expression of antioxidant genes was upregulated in transgenic plants exposed to osmotic stress. A 尾-glucuronidase (GUS) staining assay showed that GhWRKY41 was highly expressed in the stomata when plants were exposed to osmotic stress, and plants overexpressing GhWRKY41 exhibited enhanced stomatal closure when they were exposed to osmotic stress. Taken together, our findings demonstrate that GhWRKY41 may enhance plant tolerance to stress by functioning as a positive regulator of stoma closure and by regulating reactive oxygen species (ROS) scavenging and the expression of antioxidant genes.
[本文引用:1]
Shi Weina, Hao Lili, Li Jing, et al.The Gossypium hirsutum WRKY gene GhWRKY39-1 promotes pathogen infection defense responses and mediates salt stress tolerance in transgenic Nicotiana benthamiana[J]. Plant Cell Reports, 2014, 33(3): 483-498.
Abstract KEY MESSAGE: Our results indicate that overexpression of the GhWRKY39 - 1 gene enhances resistance to pathogen infection and tolerance to high salt and oxidative stress in transgenic Nicotiana benthamiana. ABSTRACT: WRKY transcription factor genes play significant roles in the response to biotic and abiotic stresses. Cotton (Gossypium hirsutum) is an important fiber and oil crop worldwide. We isolated and characterized GhWRKY39-1, which is a group IId WRKY gene that is present as a single copy in the cotton genome. Quantitative PCR analyses indicated that GhWRKY39-1 was induced by pathogen infection, defense-related signaling molecules, and abiotic stresses, such as NaCl and methyl viologen. An analysis of the subcellular localization of the GhWRKY39-1 protein indicated that it localized to the nucleus. Furthermore, constitutive overexpression of GhWRKY39-1 in Nicotiana benthamiana conferred a greater resistance to infection by both the bacterial pathogen Ralstonia solanacearum and the fungal pathogen Rhizoctonia solani. The transgenic plants also exhibited elevated mRNA levels of several pathogen-related (PR) genes, including PR1c, PR2 and PR4. Moreover, transgenic plants displayed an enhanced tolerance to salt and oxidative stress and elevated expression of several oxidation-related genes, including APX, CAT, GST and SOD. Overall, these results indicate that GhWRKY39-1 functions as a positive regulator of plant defense against pathogen infection and responses to salt stress and reactive oxygen species.
[本文引用:1]
Yan Huiru, Jia Haihong, Chen Xiaobo, et al.The cotton WRKY transcription factor GhWRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production[J]. Plant & Cell Physiology, 2014, 55(12): 2060-2076.
[本文引用:1]
Jia Haihong, Wang Chen, Wang Fang, et al. GhWRKY68 reduces resistance to salt and drought in transgenic Nicotiana benthamiana[J/OL]. PLoS ONE, 2015, 10(3): e0120646 []. . DOI: 10.1371/journal.pone.0120646.
[本文引用:1]
Yan Yan, Jia Haihong, Wang Fang, et al.Overexpression of GhWRKY27a reduces tolerance to drought stress and resistance to Rhizoctonia solani infection in transgenic Nicotiana benthamiana[J]. Frontiers in Physiology, 2015, 6: 265.
WRKY proteins constitute transcriptional regulators involved in various biological processes, especially in coping with diverse biotic and abiotic stresses. However, in contrast to other well-characterized WRKY groups, the functions of group III WRKY transcription factors are poorly understood in the economically important crop cotton (Gossypium hirsutum). In this study, a group III WRKY gene from cotton, GhWRKY27a, was isolated and characterized. Our data indicated that GhWRKY27a localized to the nucleus and that GhWRKY27a expression could be strongly induced by abiotic stresses, pathogen infection, and multiple defense-related signaling molecules. Virus-induced gene silencing (VIGS) of GhWRKY27a enhanced tolerance to drought stress in cotton. In contrast, GhWRKY27a overexpression in Nicotiana benthamiana markedly reduced plant tolerance to drought stress, as determined through physiological analyses of leaf water loss, survival rates and the stomatal aperture. This susceptibility was coupled with reduced stomatal closure in response to abscisic acid and decreased expression of stress-related genes. In addition, GhWRKY27a-overexpressing plants exhibited reduced resistance to Rhizoctonia solani infection, mainly demonstrated by the transgenic lines exhibiting more severe disease symptoms, accompanied by attenuated expression of defense-related genes in Nicotiana benthamiana. Taken together, these findings indicated that GhWRKY27a functions in negative responses to drought tolerance and in resistance to Rhizoctonia solani infection.
[本文引用:1]
Ding Weiwei, Fang Weibo, Shi Shuya, et al.Wheat WRKY type transcription factor gene TaWRKY1, is essential in mediating drought tolerance associated with an ABA-dependent pathway[J]. Plant Molecular Biology Reporter, 2016, 34(6): 1111-1126.
Abstract WRKY-type transcription factors (TFs) are important regulators in plant growth, development and responses to diverse biotic and abiotic stresses. In this study, we characterized the function of TaWRKY1, a wheat (Triticum aestivum) WRKY family gene, in mediating plant tolerance to drought. TaWRKY1 bears a conserved WRKY motif and a C2H2 domain and targets onto the nucleus under both normal and simulated drought conditions. On exposure to simulated drought and ABA, the TaWRKY1 expression levels were significantly up-regulated. Ectopic overexpression of TaWRKY1 in tobacco conferred improved
the transgenic plants exhibited more biomass, slower leaf water loss rate (WLR), more osmolyte accumulation, and higher antioxidant enzyme activities than wild type (WT) plants after drought treatments. In addition, the stomata closure rate was promoted in the transgenic plants upon exposure to drought and -exogenous ABA with respect to that in WT plants, suggesting that TaWRKY1 mediates the stomata movement and impacts the leaf water retention capacity. The transcripts of NtPYL8, an ABA receptor gene in tobacco, were shown to be induced in the TaWRKY1-overexpressing plants under drought and ABA stresses. Moreover, overexpression and knockdown of NtPYL8 modified the stomata movement characterization, leaf WLR, and phenotype of the seedlings upon exposure to the drought stress. These findings suggest that NtPYL8 acts as one of the critical players in transducing ABA signaling initiated by osmotic stress, contributing to the TaWRKY1-mediated drought tolerance via regulating stomata movement and plant water retention capacity. Our findings together reveal that TaWRKY1 plays an important role in mediating the plant tolerance to drought through modification of a set of osmotic stress-defensive processes connecting an ABA-dependent pathway. NtPYL8 and its homolog in wheat are crucial members in transducing the drought-initiated ABA signaling and are functional in modulating stomata movement.
[本文引用:1]
Fan Qingqing, Song Aiping, Jiang Jiafu, et al. CmWRKY1 enhances the dehydration tolerance of chrysanthemum through the regulation of ABA-associated genes[J/OL]. PLoS ONE, 2016, 11(3): e0150572 [2016-04-29]. . DOI: 10.1371/journal. pone.0150572.
[本文引用:1]
张坤. 棉花转录因子GhWRKY40-like和GhWRKY70在抗黄萎病中的功能研究[D]. 武汉: 华中农业大学, 2015.
[本文引用:1]
Zhang Kun.Function analysis of GhWRKY40-like and GhWRKY70 in cotton responsive to Verticilium dahllae[D]. Wuhan: Huazhong Agricultural University, 2015.
Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses
... WRKY转录因子广泛参与植物的逆境应答,在拟南芥、小麦、大豆和水稻中已有大量的文献报道WRKY转录因子受环境因子（如高盐[1-2]、干旱[2-5]、低温[2, 6]、高温[3, 7]等）的诱导表达,通过特异地与靶基因调控区的顺式元件结合,调节靶基因表达的强度及时空特异性[8],在抗逆信号传导及诱导下游功能基因的表达中起关键作用.WRKY基因首先克隆于甘薯（Impoea batatas）,随后在拟南芥、水稻和烟草等模式植物以及马铃薯[9]、大麦[10]、灌木[11]、辣椒[12]、遏蓝菜[13]、葡萄[14]、番茄[15]、小麦[16]等植物中均发现了参与逆境胁迫的WRKY基因.其中,在拟南芥和水稻中分别发现了70多个和100多个WRKY转录因子成员[17-18].目前关于WRKY转录因子的研究在拟南芥、水稻和烟草等模式植物中居多且主要集中在抗病反应上,而我国重要经济作物棉花中WRKY转录因子的研究报道相对较少.本研究利用基因芯片从陆地棉中筛选到1个逆境诱导WRKY转录因子基因GhWRKY40-1,通过电子克隆及RT-PCR（Reverse transcription-polymerase chain reaction）技术获得基因全长,并采用生物信息学分析方法对其结构、功能等进行了预测,分析其在棉花不同组织中及不同非生物胁迫下的表达情况,并对其进行序列比对及系统进化树分析,初步明确该基因的功能,为今后深入研究该基因的分子生物学特性及遗传进化等奠定基础. ...
Soybean WRKY type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants
... WRKY转录因子广泛参与植物的逆境应答,在拟南芥、小麦、大豆和水稻中已有大量的文献报道WRKY转录因子受环境因子（如高盐[1-2]、干旱[2-5]、低温[2, 6]、高温[3, 7]等）的诱导表达,通过特异地与靶基因调控区的顺式元件结合,调节靶基因表达的强度及时空特异性[8],在抗逆信号传导及诱导下游功能基因的表达中起关键作用.WRKY基因首先克隆于甘薯（Impoea batatas）,随后在拟南芥、水稻和烟草等模式植物以及马铃薯[9]、大麦[10]、灌木[11]、辣椒[12]、遏蓝菜[13]、葡萄[14]、番茄[15]、小麦[16]等植物中均发现了参与逆境胁迫的WRKY基因.其中,在拟南芥和水稻中分别发现了70多个和100多个WRKY转录因子成员[17-18].目前关于WRKY转录因子的研究在拟南芥、水稻和烟草等模式植物中居多且主要集中在抗病反应上,而我国重要经济作物棉花中WRKY转录因子的研究报道相对较少.本研究利用基因芯片从陆地棉中筛选到1个逆境诱导WRKY转录因子基因GhWRKY40-1,通过电子克隆及RT-PCR（Reverse transcription-polymerase chain reaction）技术获得基因全长,并采用生物信息学分析方法对其结构、功能等进行了预测,分析其在棉花不同组织中及不同非生物胁迫下的表达情况,并对其进行序列比对及系统进化树分析,初步明确该基因的功能,为今后深入研究该基因的分子生物学特性及遗传进化等奠定基础. ...
... [2-5]、低温[2, 6]、高温[3, 7]等）的诱导表达,通过特异地与靶基因调控区的顺式元件结合,调节靶基因表达的强度及时空特异性[8],在抗逆信号传导及诱导下游功能基因的表达中起关键作用.WRKY基因首先克隆于甘薯（Impoea batatas）,随后在拟南芥、水稻和烟草等模式植物以及马铃薯[9]、大麦[10]、灌木[11]、辣椒[12]、遏蓝菜[13]、葡萄[14]、番茄[15]、小麦[16]等植物中均发现了参与逆境胁迫的WRKY基因.其中,在拟南芥和水稻中分别发现了70多个和100多个WRKY转录因子成员[17-18].目前关于WRKY转录因子的研究在拟南芥、水稻和烟草等模式植物中居多且主要集中在抗病反应上,而我国重要经济作物棉花中WRKY转录因子的研究报道相对较少.本研究利用基因芯片从陆地棉中筛选到1个逆境诱导WRKY转录因子基因GhWRKY40-1,通过电子克隆及RT-PCR（Reverse transcription-polymerase chain reaction）技术获得基因全长,并采用生物信息学分析方法对其结构、功能等进行了预测,分析其在棉花不同组织中及不同非生物胁迫下的表达情况,并对其进行序列比对及系统进化树分析,初步明确该基因的功能,为今后深入研究该基因的分子生物学特性及遗传进化等奠定基础. ...
... [2, 6]、高温[3, 7]等）的诱导表达,通过特异地与靶基因调控区的顺式元件结合,调节靶基因表达的强度及时空特异性[8],在抗逆信号传导及诱导下游功能基因的表达中起关键作用.WRKY基因首先克隆于甘薯（Impoea batatas）,随后在拟南芥、水稻和烟草等模式植物以及马铃薯[9]、大麦[10]、灌木[11]、辣椒[12]、遏蓝菜[13]、葡萄[14]、番茄[15]、小麦[16]等植物中均发现了参与逆境胁迫的WRKY基因.其中,在拟南芥和水稻中分别发现了70多个和100多个WRKY转录因子成员[17-18].目前关于WRKY转录因子的研究在拟南芥、水稻和烟草等模式植物中居多且主要集中在抗病反应上,而我国重要经济作物棉花中WRKY转录因子的研究报道相对较少.本研究利用基因芯片从陆地棉中筛选到1个逆境诱导WRKY转录因子基因GhWRKY40-1,通过电子克隆及RT-PCR（Reverse transcription-polymerase chain reaction）技术获得基因全长,并采用生物信息学分析方法对其结构、功能等进行了预测,分析其在棉花不同组织中及不同非生物胁迫下的表达情况,并对其进行序列比对及系统进化树分析,初步明确该基因的功能,为今后深入研究该基因的分子生物学特性及遗传进化等奠定基础. ...
Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter
... WRKY转录因子广泛参与植物的逆境应答,在拟南芥、小麦、大豆和水稻中已有大量的文献报道WRKY转录因子受环境因子（如高盐[1-2]、干旱[2-5]、低温[2, 6]、高温[3, 7]等）的诱导表达,通过特异地与靶基因调控区的顺式元件结合,调节靶基因表达的强度及时空特异性[8],在抗逆信号传导及诱导下游功能基因的表达中起关键作用.WRKY基因首先克隆于甘薯（Impoea batatas）,随后在拟南芥、水稻和烟草等模式植物以及马铃薯[9]、大麦[10]、灌木[11]、辣椒[12]、遏蓝菜[13]、葡萄[14]、番茄[15]、小麦[16]等植物中均发现了参与逆境胁迫的WRKY基因.其中,在拟南芥和水稻中分别发现了70多个和100多个WRKY转录因子成员[17-18].目前关于WRKY转录因子的研究在拟南芥、水稻和烟草等模式植物中居多且主要集中在抗病反应上,而我国重要经济作物棉花中WRKY转录因子的研究报道相对较少.本研究利用基因芯片从陆地棉中筛选到1个逆境诱导WRKY转录因子基因GhWRKY40-1,通过电子克隆及RT-PCR（Reverse transcription-polymerase chain reaction）技术获得基因全长,并采用生物信息学分析方法对其结构、功能等进行了预测,分析其在棉花不同组织中及不同非生物胁迫下的表达情况,并对其进行序列比对及系统进化树分析,初步明确该基因的功能,为今后深入研究该基因的分子生物学特性及遗传进化等奠定基础. ...
Over-expression of the stress-induced OsWRKY45 enhances disease resistance and drought tolerance in Arabidopsis
A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter
... WRKY转录因子广泛参与植物的逆境应答,在拟南芥、小麦、大豆和水稻中已有大量的文献报道WRKY转录因子受环境因子（如高盐[1-2]、干旱[2-5]、低温[2, 6]、高温[3, 7]等）的诱导表达,通过特异地与靶基因调控区的顺式元件结合,调节靶基因表达的强度及时空特异性[8],在抗逆信号传导及诱导下游功能基因的表达中起关键作用.WRKY基因首先克隆于甘薯（Impoea batatas）,随后在拟南芥、水稻和烟草等模式植物以及马铃薯[9]、大麦[10]、灌木[11]、辣椒[12]、遏蓝菜[13]、葡萄[14]、番茄[15]、小麦[16]等植物中均发现了参与逆境胁迫的WRKY基因.其中,在拟南芥和水稻中分别发现了70多个和100多个WRKY转录因子成员[17-18].目前关于WRKY转录因子的研究在拟南芥、水稻和烟草等模式植物中居多且主要集中在抗病反应上,而我国重要经济作物棉花中WRKY转录因子的研究报道相对较少.本研究利用基因芯片从陆地棉中筛选到1个逆境诱导WRKY转录因子基因GhWRKY40-1,通过电子克隆及RT-PCR（Reverse transcription-polymerase chain reaction）技术获得基因全长,并采用生物信息学分析方法对其结构、功能等进行了预测,分析其在棉花不同组织中及不同非生物胁迫下的表达情况,并对其进行序列比对及系统进化树分析,初步明确该基因的功能,为今后深入研究该基因的分子生物学特性及遗传进化等奠定基础. ...
Expression of WRKY transcription factor and stress protein genes in wheat plants during cold hardening and ABA treatment
... WRKY转录因子广泛参与植物的逆境应答,在拟南芥、小麦、大豆和水稻中已有大量的文献报道WRKY转录因子受环境因子（如高盐[1-2]、干旱[2-5]、低温[2, 6]、高温[3, 7]等）的诱导表达,通过特异地与靶基因调控区的顺式元件结合,调节靶基因表达的强度及时空特异性[8],在抗逆信号传导及诱导下游功能基因的表达中起关键作用.WRKY基因首先克隆于甘薯（Impoea batatas）,随后在拟南芥、水稻和烟草等模式植物以及马铃薯[9]、大麦[10]、灌木[11]、辣椒[12]、遏蓝菜[13]、葡萄[14]、番茄[15]、小麦[16]等植物中均发现了参与逆境胁迫的WRKY基因.其中,在拟南芥和水稻中分别发现了70多个和100多个WRKY转录因子成员[17-18].目前关于WRKY转录因子的研究在拟南芥、水稻和烟草等模式植物中居多且主要集中在抗病反应上,而我国重要经济作物棉花中WRKY转录因子的研究报道相对较少.本研究利用基因芯片从陆地棉中筛