一个快速响应干旱的F-box基因的克隆和表达分析

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尹恒, 余琴鸯, 安利佳, 李文利. 一个快速响应干旱的F-box基因的克隆和表达分析. 作物学报, 2014, 40(6): 1027-1034[YIN Heng, YU Qin-Yang, AN Li-Jia, LI Wen-Li. Cloning and Expression Analysis of an F-box Gene (SiFBX) Rapidly Responsive to Drought Stress . Acta Agron Sin, 2014, 40(6): 1027-1034] 复制到剪切板

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作物学报编辑部

一个快速响应干旱的F-box基因的克隆和表达分析

尹恒, 余琴鸯, 安利佳, 李文利^*

大连理工大学生命科学与技术学院, 辽宁大连 116024

^*通讯作者(Corresponding author): 李文利, E-mail:biolwl@dlut.edu.cn

第一作者联系方式: E-mail:jiuxinguo@sina.com

收稿日期:2014-01-12

基金资助:本研究由辽宁省科技厅农业攻关项目(2011208001)资助;

摘要

F-box是Skp1-Cullin1-F-box (SCF)型泛素连接酶E3的重要组成部分, 在泛素化介导的蛋白质降解中选择性识别靶蛋白。本文从谷子苗期干旱胁迫条件下构建的转录组文库中克隆了与耐旱早期响应相关的F-box基因, 命名为SiFBX (GenBank登录号为KC252635.1)。该基因全长510 bp, 编码170个氨基酸。蛋白质结构预测表明, 该蛋白含有丰富的精氨酸、亮氨酸、丝氨酸, 缺少跨膜结构域及信号肽序列。系统进化分析表明, 该基因与已报道的EID1和FBW4亲缘关系较近。在该基因上游1.9 kb序列处, 预测到启动子的核心序列及与多种逆境胁迫相关的调控序列。荧光定量PCR分析表明, 该基因分别在正常干旱、PEG和ABA诱导下, 表达量出现显著变化。

关键词: 谷子; 干旱响应; F-box; gRT-PCR

Cloning and Expression Analysis of an F-box Gene (SiFBX) Rapidly Responsive to Drought Stress

YIN Heng, YU Qin-Yang, AN Li-Jia, LI Wen-Li^*

School of Life Science & Biotechnology, Dalian University of Technology, Dalian 116024, China

Fund:

Abstract

F-box proteins, components of the Skp1-Cullin1-F-box (SCF) protein E3 ubiquitin ligase complex, serve as the variable component responsible for substrate recognition and recruitment in SCF-mediated proteolysis. The anti-drought relative gene ofSiFBX (GenBank accession number KC252635.1) which belongs to the F-box super family was cloned from foxtail millet (Setaria italic). The full-length cDNA ofSiFBX was 510 bp, which encoded 170 amino acid residues. Protein analysis and structure predication showed that it has a higher proportion of arginine (R), leucine (L), and serine (S) and a lack of trans-membrane domains and signal peptide. Phylogenetic analysis demonstrated thatSiFBX has similarity withEID1 andFBW4. Many abiotic stress-relatedcis-acting elements and transcription factors were discovered in the 1.9 kb upstream region ofSiFBX. The results of real-time PCR showed that there were remarkable changes in the expectation level ofSiFBX for the treatments with PEG, water-withholding, and ABA.

Keyword: Setaria italica; Drought response; F-box protein; qRT-PCR

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0 引言

研究表明, 泛素化蛋白连接酶E3对植物生长发育和逆境胁迫响应等过程中的关键步骤具有重要的调控作用^{[ 1]}, Skp1-Cullin1-F-box (SCF)型蛋白复合物是E3中研究最深入的一类。F-box蛋白也是真核细胞中一大类蛋白质家族, 包含了一个35~60个氨基酸组成的F-box结构域, 在SCF型E3介导的蛋白降解中, 起着靶蛋白识别和稳定SCF复合物的作用。F-box蛋白结构域的N-端部分与SKP结合, 通过其C-端部分与靶蛋白结合发挥作用。在F-box蛋白结构域的下游, 常常伴随一些重要的次级元件, 如LRR (leucine-rich repeat)、WD repeat、亮氨酸拉链结构等^{[ 2]}。

Shinozaki等^{[ 3]}首先在拟南芥基因组序列中发现了近700个编码F-box蛋白的基因, 占基因组编码总蛋白的3%左右。Jain等^{[ 4]}也在水稻基因组中发现了687个F-box蛋白, 根据F-box蛋白C端的不同将其分为10大类亚家族。对功能已知的F-box蛋白深入研究表明, F-box蛋白几乎参与所有的植物生长发育、细胞分裂及植物激素的响应调控过程^{[ 5]}, 如 LKP、 ZTL、 FKF参与开花和对光周期的调控^{[ 6, 7]}; EID1参与光形态的建成^{[ 8]}; UFO影响植物花的发育^{[ 9, 10, 11, 12]}; ORE1和 MAX2调控叶片衰老和后期树冠的分支数^{[ 13]}。目前, 通过大规模测序和生物信息学等手段已经预测了大量的F-box蛋白, 除少部分蛋白功能得到验证外, 更多的F-box蛋白还处于功能未知状态, 这些蛋白在干旱胁迫中的作用则知之更少。

谷子( Setaria italic L.)是我国北方古老的谷类作物之一, 具有突出的耐旱性及水分利用效率。谷子具有基因组小(约500 Mb)、自交结实率高、具C₄合成途径、与目前主要粮食作物、饲料作物及能源作物亲缘关系较密切等特点, 已经逐步成为基因组学研究的理想的模式作物^{[ 14]}。由于谷子的良好耐旱性, 农艺性状改良方面表现出广阔的研究潜力。利用分子生物学技术, 已经从谷子中发现大量与耐旱相关的基因。目前, 国内学者已经在谷子中克隆到的基因有DnaJ蛋白基因( SiDnaJ)^{[ 15]}、干旱应答元件结合蛋白基因( SiDREB)^{[ 16]}、12-氧代植二烯酸还原酶基因( SiOPR1)^{[ 17]}、3-磷酸甘油醛脱氢酶基因( GAPDH)^{[ 18]}、磷脂酶D基因( SiPLDa1)^{[ 19]}、 CBL家族^{[ 20]}、 PP2C家族^{[ 14]}和 ABF3^{[ 21]}等。但与已知的拟南芥逆境相关的基因(8000个)和水稻干旱应激诱导基因(约5000个)数目相比, 在抗旱性极强的谷子中发现和鉴定的与干旱相关的基因屈指可数。因此, 继续加大谷子抗旱性研究, 发掘谷子抗旱基因, 深入了解谷子抗旱机制尤为重要。

本研究以干旱胁迫处理下的谷子幼苗为材料, 构建了耐旱相关转录组数据库, 克隆得到一个可能参与快速响应干旱胁迫的F-box基因, 命名为 SiFBX。利用生物信息学手段对该基因及其上游启动子序列进行分析, 并通过实时定量PCR初步验证其在干旱胁迫及ABA激素胁迫下的表达调控。该基因的发现, 为研究干旱胁迫下谷子转录后翻译水平的耐性机制提供了基础。

1 材料与方法

1.1 植物材料及胁迫处理

晋谷34由山西农业科学院作物遗传研究所提供。将种子播于苗床(草炭土、珍珠岩和蛭石的质量比为1∶1∶1)上, 在湿度为70%、温度22℃条件下培养。生长20 d后(三至四叶期), 取长势大小一致的幼苗分别进行模拟干旱(幼苗置于含有20% PEG的1/2 Hoagland营养液中, pH 7.0)、正常干旱(断水处理)和喷施ABA (100 μmol L^-1)胁迫处理。分别在PEG和ABA胁迫处理后0.5、1.0、2.0、4.0和8.0 h取样, 正常干旱处理为断水后每天9∶00取样, 分别断水1、2、3、4、5和6 d, 每处理每次取10株, 3次重复。样品于液氮速冻, -80℃保存备用。

1.2 SiFBX基因编码区全长cDNA的克隆

从注释的干旱诱导的谷子转录组数据库中, 筛选出F-box基因片段, 使用Trinity (release-20130225)进行序列的组装(默认参数), 利用DNAMAN软件(http://www.lynnon.com/)进行ORF搜索(ORF>50), 得到F-box基因编码序列。根据此序列, 设计并合成引物 Si FBX-FM和 SiFBX-RM (表1)。取正常干旱(断水)处理3 d的样品, 用RNAiso Plus (TaKaRa)分离mRNA, 使用PrimeScript First Strand cDNA Synthesis Kit (TaKaRa)反转录合成cDNA。利用 SiFBX-FM和 SiFBX-RM引物进行PCR扩增该基因编码区全长片段, 回收PCR产物, 连接到pMD-18T Simple载体(TaKaRa), 转化大肠杆菌(DH5α), 挑取阳性克隆, 经质粒酶切验证正确后测序。

表1 SiFBX基因克隆及Real-time PCR分析所需引物 Table 1 Primers used in SiFBX genes cloning and real-time PCR analysis

1.3 生物信息学分析

用ProParam (http://espasy.org/tools/protparam.html) 分析 SiFBX蛋白的物理和化学性质。用Predict Protein (http://www.Predictprotein.org/)和ProScale (http://expasy.org/tools/protscale.html)预测 SiFBX蛋白质的二级结构及疏水区域。利用TMpred (http://www.ch.embnet.org/software/TMPRED_form.html)和TMHMM (http://www.cbs.dtu.dk/services/TMHMM)分析 SiFBX的蛋白跨膜结构域。通过PROSITE (http://www.expasy.ch/Prosite)预测该蛋白质的结构域和功能位点。利用CCD数据库和PFAM数据库分析该蛋白的保守结构域和家族, 确定该基因所属的蛋白家族^{[ 22]}。利用ClastalX (默认参数)进行多重序列比对, 使用MEGA (重复次数1000)构建N-J系统进化树。使用在线预测工具PlantCARE^{[ 23]} (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)预测 SiFBX的启动子结构。

1.4 RNA提取和Real-time PCR分析

根据获得的 SiFBX基因序列, 设计并合成 Real-time PCR引物 SiFBX-RT-F和 SiFBX-RT-R。以 Actin基因作为内参, 设计引物SiActin-F和SiActin-R (表1)。

利用RNAiso试剂盒(TaKaRa, 辽宁大连)分别提取1.1中材料的总RNA, 分别以总RNA作为模板, 使用PrimerScript RT Master Mix (Perfect Real-time)试剂盒(TaKaRa, 大连)反转录合成cDNA。总RNA 200 ng左右加入5×PrimerScript RT Master Mix (for Real-time) 2 μL, RNase Free dH₂O补充至10 μL。于PCR仪(TaKaRa)设定37℃ 15 min; 85℃ 5 s; 4℃合成cDNA。使用One Step SYBR PrimerScript RT-PCR Kit (Perfect Real Time)试剂盒(TaKaRa, 辽宁大连)进行PCR, 反应体系包括2×SYBR Premix Ex Taq II (含荧光染料) 12.5 μL、10 μmol L^-1引物各1 μL、ddH₂O 8.5 μL和cDNA模板2 μL。以谷子 Actin基因作为内参基因, 采用两步法进行实时荧光定量PCR扩增(ABI7300, Applied Biosystems)。反应条件为95℃ 30 s; 95℃ 5 s, 61℃ 31 s (搜集信号), 40个循环。使用SDS软件分析实时定量PCR结果。

2 结果与分析

2.1 SiFBX基因全长cDNA的克隆和染色体定位

利用生物信息学方法和PCR技术克隆得到一个与耐旱相关的F-box基因(图1)。分析结果表明, 在其编码的蛋白氨基酸序列中, 第72、第73、第80和第96位的氨基酸分别为亮氨酸(L)、脯氨酸(P)、缬氨酸(V)和丝氨酸(S), 具有F-box家族蛋白的基本特征^{[ 24]}, 因此命名为 SiFBX, 并将该序列提交到GenBank (登录号为KC252635.1)。

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	图1 SiFBX 基因PCR扩增条带1~6: SiFBX在50.5、52.0、54.4、56.8、59.0和60.0℃下PCR结果; Marker: DL2000 (TaKaRa)。Fig. 1 PCR results of SiFBX gene1-6: PCR products of SiFBX gene at the temperature of 50.5, 52.0, 54.4, 56.8, 59.0, and 60.0℃ respectively; Marker: DL2000 (TaKaRa).

将序列与谷子基因组数据库(http://www.phyto-zome.net/)比对, 发现该基因位于谷子Scaffold_2区段的43 260 108~43 260 815位点。使用DNAMAN对该区域的序列进行预测分析, 发现编码 SiFBX的片段是由2个内含子和3个外显子组成(图2)。

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	图2 SiFBX在谷子基因组上的位置和结构Fig. 2 Location and structure of SiFBX in Setaria italic genome

2.2 SiFBX基因的生物信息学分析

SiFBX蛋白由170个氨基酸组成, 分子量为18.6 kD, 等电点p I为10.90 (图3)。在170个氨基酸残基中, 精氨酸(R, 10.0%), 亮氨酸(L, 11.2%), 丝氨酸(S, 12.9%)所占比例达到了34.1%。

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	图3 SiFBX基因序列及推导氨基酸序列分析Fig. 3 Analysis of SiFBX gene sequence and amino acid sequence deduced

二级结构预测发现, 在 SiFBX蛋白N端含有由48个氨基酸构成的螺旋结构, 在C端含有由12个氨基酸组成的螺旋结构。用ProScale进行疏水性分析显示, 该蛋白含有6个疏水区, 分别位于氨基酸序列的15~17、66~69、76~82、92~96、104~105和155~161区域内, 尤其在66~105处, 疏水区域较为集中, 可能为 SiFBX的作用位点。利用PROSITE进行 SiFBX蛋白质结构域、家族和功能位点分析发现, SiFBX含有一个F-box结构域(66~144)(图4-C)。在已知的SCF型E3复合物中, F-box蛋白结构域的N-端和C-端分别结合SKP蛋白和下游蛋白, 据此预测, 其中2个可能的结合位点, 一个为F-box蛋白与SKP结合部位(实线显示区域), 另一个为F-box蛋白与靶蛋白结合部位(虚线显示区域)(图3)。

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图4 植物所有非冗余F-box基因的家族分析以及 SiFBX、 EID1、 FBW4的保守结构域比较A: 所有植物非冗余F-box基因的亲缘关系分析; B: 树A显示的进化上的保守基因簇部分; C: SiFBX、 EID1和 FBW4蛋白的
结构域对比分析。Fig. 4 Analysis of all non-redundant F-box gene family in plants and the relatively conserved domains among SiFBX, EID1, and FBW4A: Phylogenetic relationship analysis for all non-redundant F-box gene in plants; B: A part of tree A on a evolution of conserved gene
cluster; C: Comparative analysis of protein domains among SiFBX, EID1, and FBW4.

利用TMpred和TMHMM对 SiFBX蛋白质进行跨膜结构分析, 结果均未发现跨膜结构, 表明 SiFBX可能不是一个跨膜蛋白。通过Signal IP预测, 未能够找到信号肽结构, 说明该蛋白在细胞中不是分泌蛋白, 可能位于细胞质基质或者细胞核基质中。

首先利用CCD数据库的CD-search工具, 确定该基因属于 pfam00646基因家族。其次利用NCBI搜索, 得到34 777条相关基因, 其中植物来源共 15 196条。然后结合UniportKB及Swiss-port数据库筛选得到563条非冗余序列。用MEGA4.0软件进行多重序列比对以及构建N-J系统进化树, 563条非冗余序列被分为6大类。 SiFBX与已报道的 EID1 (empfindlicher im dunkelroten Licht 1)和 FBW4 (F-box containing WD40 repeat-domain protein 4)等基因分为同一大类, 该大类共12条蛋白序列(图4-A)。在此大类中, SiFBX与FB220、FB84、FBL91以及SKI32相似度较高(图4-B), 进一步分析 SiFBX、 EID1和 FBW4的保守结构域发现, EID1可能含有一个未知的次级结构域, FBW4含有一个WD40-repeats次级结构域, 而 SiFBX下游没有发现次级结构域(图4-C), 说明 SiFBX是一个新发现的F-box基因。

2.3 SiFBX基因的上游启动子分析

从 SiFBX基因的ATG开始, 截取 S iFBX基因5° 区域上游1925 bp的核苷酸序列, 使用PlantCARE在线预测 SiFBX的结构(图5)。

在ATG上游759 bp处发现TATA-box区域, 转录起始位点位于上游739 bp处, 在上游738 bp至上游1039 bp区域预测到多种逆境胁迫相关的顺式作用元件(表2), 包括与光、温度、激素、厌氧、干旱、细胞周期响应及组织特异性响应元件等。

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	图5 SiFBX启动子结构示意图Fig. 5 Structure diagram of SiFBX promoter

表2 Si FBX基因上游调控区胁迫相关的启动子元件 Table 2 Stress-regulated element in upstream regions of SiFBX gene

2.4 SiFBX基因的表达模式分析

在用20% PEG胁迫处理谷子幼苗后, SiFBX在谷子叶片呈现显著上调表达的趋势, 在2.0 h表达量达到最大, 为对照组的30.9倍。根部和茎部均在1.0 h达到最大, 分别为对照组的4.1倍和1.5倍。当用100 μmol L^-1ABA处理后, SiFBX基因在0.5 h叶片中的表达量达到最大, 为对照组的1.2倍, 在1 h表达量最低, 仅为对照组的0.07倍, 随后开始出现上升趋势。茎部在4 h和8 h显著上调表达, 分别为对照组的1.5倍和1.4倍。根部在各个时间点的表达量显著低于对照组, 一直处于抑制状态。在正常干旱处理下, SiFBX基因在第2天和第5天的茎中表达显著下调, 分别为对照组的0.41倍和0.57倍, 而在第6天的叶片中则显著上调表达, 根中没有明显变化(图6)。

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	图6 SiFBX基因在谷子中的表达模式分析Fig. 6 Expression pattern analysis of SiFBX gene in Setaria itallica L.

3 讨论

F-box蛋白在植物的生长发育和激素响应中具有重要作用^{[ 5]}。我们从谷子中克隆了一个快速响应干旱的F-box基因, 命名为 SiFBX。通过对所有植物F-box家族的分析发现该基因与目前报道的 EID1以及 FBW4同源关系接近。EID1是一个光敏色素A依赖的HIR-信号通路的负调控元件, 参与植物光形态的建成, 具有核定位信号肽^{[ 25]}, 能够特异地与 ASK ( Arabidopsis Skp1-like proteins)和 Cullin1相互作用形成稳定的二聚体和三聚体^{[ 8]}。 EDL3 ( EID1-like protein 3)是与 E ID1高度相似的F-box家族蛋白, 在拟南芥中参与依赖ABA的种子萌发、根系生长、黄化苗绿化以及开花等过程的调控, 同时也参与响应温度、干旱、渗透以及盐胁迫^{[ 26]}。 FBW4是一个编码WD40结构域的F-box蛋白, 在氧化胁迫诱导的细胞凋亡途径中, 尽管与 RCAN1相互作用, 但也能够独立应对H₂O₂刺激^{[ 27]}。本研究中, SiFBX与 EID1和 FBW4被聚为一类, 说明 SiFBX可能具有与 EID1和 FBW4相似的功能。对比 SiFBX、 EID1和 FBW4的结构域发现, SiFBX不仅缺少占据了EID1和FBW4蛋白很大比重的次级结构域, 而且还缺少 EID1和 FBW4所特有的核定位信号肽。在SiFBX蛋白的C端, 仅有一个由12个氨基酸组成的小型双螺旋结

柱上小写字母不同表示在0.05的水平上差异显著。

Bars superscripted by different letters are significantly different at the 0.05 probability level.

构, 说明SiFBX在结合下游靶蛋白时, 蛋白复合物的结合程度可能不如EID1和FBW4稳定。由于缺少可能的细胞定位和跨膜结构域, SiFBX在细胞质中处于游离状态, 加之 SiFBX含有较高比例的精氨酸、亮氨酸和丝氨酸组成, 很容易被丝氨酸/苏氨酸蛋白激酶激活, 参与信号的转导途径。以上原因或许导致SiFBX对于干旱、渗透以及ABA激素胁迫的快速而强烈的响应。

本研究中, 实时定量结果也显示了 SiFBX对干

旱、渗透和ABA胁迫的响应, 与 EDL3的功能比较类似。叶片和根部是ABA的主要合成器官, ABA在叶绿体及其他质体中合成后, 双向运输, 叶片合成的ABA沿着韧皮部向根部运输, 运输速度慢, 而根部合成的ABA沿着导管向叶片运输, 运输速度快。受到胁迫时, ABA会在植物体叶片中迅速积累, 参与对逆境的响应。研究表明, EDL3甚至能够响应轻微胁迫, 低浓度ABA (5 μmol L^-1)处理下, 90 min转录水平就能增加4倍, 高浓度的ABA处理反而会抑制 EDL3的转录^{[ 25]}。与该结果相比较, 我们推测 SiFBX可能参与ABA信号分子的转导, ABA的浓度影响 SiFBX的表达。正常干旱处理下, 随胁迫程度逐渐加剧, 叶片中积累的ABA含量在不断增加, 增强了 SiFBX基因的表达, 因此在叶片中的表达量呈现上调趋势。在PEG的处理下, 胁迫程度剧烈而突然, 叶片合成的ABA快速增加, SiFBX的表达量也快速增加, 当ABA的浓度积累到一定程度时, 抑制了 SiFBX信号的转导, 表达量开始呈下降趋势(图6)。而当用100 μmol L^-1 ABA处理叶片时, 高浓度的ABA迅速抑制 SiFBX在根和叶中的表达。随着ABA途径的信号分子的降解, SiFBX受到的抑制作用开始缓慢解除, SiFBX在根、茎和叶中表达量呈现上升趋势(图6)。

在 SiFBX的启动子区域发现6类分别与光、温度、激素、厌氧、干旱、细胞周期响应及组织特异性响应相关的顺式作用元件, 推测 SiFBX可能通过这些元件对外界逆境做出应答。 MYB15与 MYB84参与了 SiFBX基因的转录, 过量表达 MYB15基因可以增强拟南芥对于ABA和干旱的耐性^{[ 28]}, 而 MYB84基因被报道具有增强水稻抵抗盐害的作用^{[ 29]}, 说明 SiFBX在应对逆境胁迫中发挥潜在作用。转录组数据表明干旱胁迫条件下, 谷子中 SiFBX的表达量大幅度上升(结果未列出), 说明干旱应答相关顺式作用元件可能与信号分子结合, 激活该基因的转录。荧光定量PCR结果显示, 干旱和PEG诱导条件下该基因在谷子叶片中表达量显著上调(图6), 这可能与启动子区域的干旱应答顺式作用元件有关。我们推论 SiFBX启动子区域多种逆境应答相关作用元件可能调控该基因在不同逆境条件下的表达。 SiFBX启动子区域中存在ABA、乙烯、茉莉酸应答相关顺式作用元件, 表明该基因参与了信号相关转导途径。进一步表明该基因表达产物SiFBX蛋白是通过上游激素相关顺式作用元件响应激素信号, 而激素信号转导途径与植物的耐逆性相关^{[ 30, 31]}。

4 结论

从谷子幼苗中克隆获得 SiFBX基因, 编码一个F-box蛋白, 通过启动子区域的逆境及激素应答相关顺式作用元件调控其转录水平, 实现对干旱逆境的响应。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。The authors have declared that no competing interests exist.

参考文献

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2012

2.506

0.0

. 2012, 39:9681-9696 DOI:10.1007/s11033-012-1833-3

Wheat F-box protein recruits proteins and regulates their abundance during wheat spike development

1.Division of Biotechnology, Korea University, Anam-Dong, Seongbuk-Gu, Seoul, 136-701, Republic of Korea

2.Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 1266 Sinjeong, Jeongeup, 580-185, Republic of Korea

Abstract
F-box proteins, components of the Skp1–Cullin1–F-box (SCF) protein E3 ubiquitin ligase complex, serve as the variable component responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins interact with Skp1 through the F-box motif and with ubiquitination substrates through C-terminal protein interaction domains. F-box proteins regulate plant development, various hormonal signal transduction processes, circadian rhythm, and cell cycle control. We isolated an F-box protein gene from wheat spikes at the onset of flowering. The Triticum aestivum cyclin F-box domain (TaCFBD) gene showed elevated expression levels during early inflorescence development and under cold stress treatment. TaCFBD green fluorescent protein signals were localized in the cytoplasm and plasma membrane. We used yeast two-hybrid screening to identify proteins that potentially interact with TaCFBD. Fructose bisphosphate aldolase, aspartic protease, VHS, glycine-rich RNA-binding protein, and the 26S proteasome non-ATPase regulatory subunit were positive candidate proteins. The bimolecular fluorescence complementation assay revealed the interaction of TaCFBD with partner proteins in the plasma membranes of tobacco cells. Our results suggest that the TaCFBD protein acts as an adaptor between target substrates and the SCF complex and provides substrate specificity to the SCF of ubiquitin ligase complexes.

... 0 引言研究表明, 泛素化蛋白连接酶E3对植物生长发育和逆境胁迫响应等过程中的关键步骤具有重要的调控作用^[1], Skp1-Cullin1-F-box (SCF)型蛋白复合物是E3中研究最深入的一类 ...

2011

3.73

0.0

... 在F-box蛋白结构域的下游, 常常伴随一些重要的次级元件, 如LRR (leucine-rich repeat)、WD repeat、亮氨酸拉链结构等^[2] ...

2002

4.134

0.0

... Shinozaki等^[3]首先在拟南芥基因组序列中发现了近700个编码F-box蛋白的基因, 占基因组编码总蛋白的3%左右 ...

2007

6.555

0.0

... Jain等^[4]也在水稻基因组中发现了687个F-box蛋白, 根据F-box蛋白C端的不同将其分为10大类亚家族 ...

2006

8.455

0.0

. 2006, 9:631-638 DOI:10.1016/j.pbi.2006.09.003

F-box proteins everywhere

The ubiquitin proteasome system is a key regulator of many biological processes in all eukaryotes. This mechanism employs several types of enzymes, the most important of which are the ubiquitin E3 ligases that catalyse the attachment of polyubiquitin chains to target proteins for their subsequent degradation by the 26S proteasome. Among the E3 families, the SCF is the best understood; it consists of a multi-protein complex in which the F-box protein plays a crucial role by recruiting the target substrate. Strikingly, nearly 700 F-box proteins have been predicted in Arabidopsis, suggesting that plants have the capacity to assemble a multitude of SCF complexes, possibly controlling the stability of hundreds of substrates involved in a plethora of biological processes. Interestingly, viruses and even pathogenic bacteria have also found ways to hijack the plant SCF and to reprogram it for their own purposes.

... 对功能已知的F-box蛋白深入研究表明, F-box蛋白几乎参与所有的植物生长发育、细胞分裂及植物激素的响应调控过程^[5], 如LKP、ZTL、FKF参与开花和对光周期的调控^[6,7] ...

... 3 讨论F-box蛋白在植物的生长发育和激素响应中具有重要作用^[5] ...

2004

9.251

0.0

2005

0.0

2006

6.582

0.0

... EID1参与光形态的建成^[8] ...

... EID1是一个光敏色素A依赖的HIR-信号通路的负调控元件, 参与植物光形态的建成, 具有核定位信号肽^[25], 能够特异地与ASK (ArabidopsisSkp1-like proteins)和Cullin1相互作用形成稳定的二聚体和三聚体^[8] ...

2008

0.0

... UFO影响植物花的发育^[9,10,11,12] ...

2003

9.251

0.0

... UFO影响植物花的发育^[9,10,11,12] ...

2003

9.737

0.0

... UFO影响植物花的发育^[9,10,11,12] ...

2008

9.251

0.0

... UFO影响植物花的发育^[9,10,11,12] ...

2007

3.137

0.0

. 2007, 66:816-823 DOI:10.1016/j.chemosphere.2006.06.041

Cyanide phytoremediation by water hyacinths (Eichhornia crassipes)

Abstract

Although cyanide is highly toxic, it is economically attractive for extracting gold from ore bodies containing only a few grams per 1000 kg. Most of the cyanide used in industrial mining is handled without observable devastating consequences, but in informal, small-scale mining, the use is poorly regulated and the waste treatment is insufficient. Cyanide in the effluents from the latter mines could possibly be removed by the water hyacinth Eichhornia crassipes because of its high biomass production, wide distribution, and tolerance to cyanide (CN) and metals. We determined the sodium cyanide phytotoxicity and removal capacity of E. crassipes. Toxicity to 5–50 mg CN L⁻¹ was quantified by measuring the mean relative transpiration over 96 h. At 5 mg CN L⁻¹, only a slight reduction in transpiration but no morphological changes were observed. The EC₅₀ value was calculated by probit analysis to be 13 mg CN L⁻¹. Spectrophotometric analysis indicated that cyanide at 5.8 and 10 mg L⁻¹ was completely eliminated after 23–32 h. Metabolism of K¹⁴CN was measured in batch systems with leaf and root cuttings. Leaf cuttings removed about 40% of the radioactivity from solution after 28 h and 10% was converted to ¹⁴CO₂; root cuttings converted 25% into ¹⁴CO₂ after 48 h but only absorbed 12% in their tissues. The calculated K_m of the leaf cuttings was 12 mg CN L⁻¹, and the V_max was 35 mg CN (kg fresh weight)⁻¹ h⁻¹. Our results indicate that E. crassipes could be useful in treating cyanide effluents from small-scale gold mines.

... ORE1和MAX2调控叶片衰老和后期树冠的分支数^[13] ...

2013

0.0

... 谷子具有基因组小(约500 Mb)、自交结实率高、具C₄合成途径、与目前主要粮食作物、饲料作物及能源作物亲缘关系较密切等特点, 已经逐步成为基因组学研究的理想的模式作物^[14] ...

... 目前, 国内学者已经在谷子中克隆到的基因有DnaJ蛋白基因(SiDnaJ) ^[15]、干旱应答元件结合蛋白基因(SiDREB) ^[16]、12-氧代植二烯酸还原酶基因(SiOPR1)^[17]、3-磷酸甘油醛脱氢酶基因(GAPDH) ^[18]、磷脂酶D基因(SiPLDa1) ^[19]、CBL家族^[20]、PP2C家族^[14]和ABF3^[21]等 ...

2007

0.0

0.951

. 2007, 22(4):9-13

Cloning of DnaJ-like protein gene from foxtail millet

DnaJ蛋白是近年研究较多的同热激等胁迫反应相关的基因.本研究用0.8%NaCl胁迫下的谷子幼苗提取RNA,用反转录酶M-MLV扩增得到第一链cDNA,再以第一链cDNA为模板进行PCR扩增,克隆获得了完整的谷子DnaJ蛋白基因,该基因cDNA全长1 260 bp,编码419个氨基酸,具有DnaJ蛋白分子伴侣系统的3个保守结构域.将谷子DnaJ蛋白基因构建入表达载体pGFP,获得了谷子DnaJ基因的表达载体.该基因的克隆和表达载体构建,为谷子DnaJ蛋白基因的功能分析以及谷子耐热抗旱机理研究有重要意义.

2011

0.0

2007

0.0

2009

0.0

0.951

. 2009, 24(3):10-14

Cloning and structure analysis of foxtail millet APDH gene

3-磷酸甘油醛脱氢酶基因(GAPDH)在谷子盐旱胁迫早期高频率表达,为研究该基因在胁迫反应及其他代谢中的详细功能,本研究利用RACE技术克隆了谷子的GAPDH基因.该基因cDNA全长1328 bp,包括1008bp的开放阅读框,共编码338个氨基酸,肽段的分子量为35911.03Da,极性氨基酸占29.12%.序列比较分析发现,谷子的GAPDH同玉米的同源序列表现了最高的相似性,同水稻和小麦同源序列的相似性次之,同双子叶植物的同源序列则相差较远.

2010

2.509

0.0

. 2010, 29:793-802 DOI:10.1007/s00299-010-0865-1

Overexpression of a PLD alpha 1 gene from Setaria italica enhances the sensitivity of Arabidopsis to abscisic acid and improves its drought tolerance

1.China Agricultural University State Key Laboratory of Agrobiotechnology Beijing 100193 China
2.Gansu Agricultural University College of Agronomy Lanzhou 730070 China
3.China Agricultural University College of Agronomy and Biotechnology Beijing 100193 China
4.Chinese Academy of Agricultural Sciences Institute of Crop Sciences Beijing 100081 China

Phospholipase D (PLD) plays an important role in various physiological processes in plants, including drought tolerance. Here, we report the cloning and characterization of the full-length cDNA of PLDα1 from foxtail millet, which is a cereal crop with high water use efficiency. The expression pattern of the SiPLDα1 gene in foxtail millet revealed that it is up-regulated under dehydration, ABA and NaCl treatments. Heterologous overexpression of SiPLDα1 in Arabidopsis can significantly enhance their sensitivity to ABA, NaCl and mannitol during post-germination growth. Under water deprivation, overexpression of SiPLDα1 in Arabidopsis resulted in significantly enhanced tolerance to drought stress, displaying higher biomass and RWC, lower ion leakage and higher survival percentages than the wild type. Further analysis indicated that transgenic plants showed increased transcription of the stress-related genes, RD29A, RD29B, RAB18 and RD22, and the ABA-related genes, ABI1 and NCED3 under dehydration conditions. These results demonstrate that SiPLDα1 is involved in plant stress signal transduction, especially in the ABA signaling pathway. Moreover, no obvious adverse effects on growth and development in the 35S::SiPLDα1 transgenic plants implied that SiPLDα1 is a good candidate gene for improving crop drought tolerance.

2013

0.0

1.667

. 2013, 39:360-367 DOI:10.3724/SP.J.1006.2013.00360

Identification of CBL genes from foxtail millet (Setaria italica [L.] Beauv.) and its expression under drought and salt stresses

1 Millet Research Institute, Shanxi Academy of Agricultural Sciences, Changzhi 046011, China; 2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

CBL/CIPK signal network system plays an important role in plant stress response. In this study, we identified seven candidate CBL genes designated as SiCBL1–7 from foxtail millet (Setaria italica [L.] Beauv.) genome using bioinformatics methods. The distributions of the predicted foxtail millet CBL genes were uneven in the nine chromosomes. Sequence analysis showed that the protein sequences and structure of foxtail millet CBL genes were very conservative. All the putative CBL genes contained 7–8 introns. Most exons of those SiCBL genes contained the same base number and shared highly similar sequence identity. All foxtail millet CBL genes consisted of four EF-Hand functional domains and the distance between the EF-hand motifs was conservative. Evolutionary analysis revealed that the CBL genes existed in the early evolutionary stage of terrestrial plants and all CBL genes were divided into four subgroups. The expression patterns of those SiCBL genes under drought and salt stresses, were displayed by RT-PCR and the results showed that four SiCBL genes (SiCBL1, SiCBL2, SiCBL3,and SiCBL5) were strongly induced by drought and three SiCBL genes (SiCBL1, SiCBL3,and SiCBL7) were induced by salt stress. SiCBL3 maybe takes an important role in foxtail millet under drought because of its constitutive high level expression. Foxtail millet CBL genes reported in this study would enrich CBL members in plant kingdom and lay a foundation for studying the function and mechanisms of the CBL/CIPK network system response to stresses.

CBL/CIPK信号网络系统在植物对逆境应答过程中起重要作用。本文利用生物信息学方法从谷子基因组中鉴定出7个候选CBL基因，命名为SiCBL1~SiCBL7。分析表明谷子CBL基因在蛋白质序列和结构上非常保守，所有预测SiCBL基因都含有7~8个内含子，而且其外显子序列同源性很高并且大部分外显子含有相同的碱基数目。预测SiCBL基因均含有4个EF-Hand功能域而且相邻EF-Hand功能域之间的氨基酸数目非常保守。进化和聚类分析结果表明, CBL基因在陆生植物早期的进化过程中就已经存在，所有CBL基因被分为4个亚组。7个候选SiCBL基因中, 4个基因(SiCBL1、SiCBL2、SiCBL3和SiCBL5)受干旱胁迫诱导表达，3个(SiCBL1、SiCBL3和SiCBL7)受高盐胁迫诱导表达。SiCBL3基因在干旱胁迫下被强烈诱导可能意味着其在干旱应答中起重要作用。本文报道的谷子CBL基因丰富和完善了植物CBL成员, 为进一步研究CBL/CIPK网络系统在抗逆作物谷子逆境响应中的功能、机制奠定了基础。

2013

0.0

2009

9.737

0.0

... 利用CCD数据库和PFAM数据库分析该蛋白的保守结构域和家族, 确定该基因所属的蛋白家族^[22] ...

2002

0.0

... 使用在线预测工具PlantCARE^[23] (http://bioinformatics ...

1994

9.822

0.0

... 分析结果表明, 在其编码的蛋白氨基酸序列中, 第72、第73、第80和第96位的氨基酸分别为亮氨酸(L)、脯氨酸(P)、缬氨酸(V)和丝氨酸(S), 具有F-box家族蛋白的基本特征^[24], 因此命名为SiFBX, 并将该序列提交到GenBank (登录号为KC252635 ...

2000

9.251

0.0

... mol L^-1)处理下, 90 min转录水平就能增加4倍, 高浓度的ABA处理反而会抑制EDL3的转录^[25] ...

2011

5.242

0.0

... EDL3 (EID1-like protein 3)是与EID1高度相似的F-box家族蛋白, 在拟南芥中参与依赖ABA的种子萌发、根系生长、黄化苗绿化以及开花等过程的调控, 同时也参与响应温度、干旱、渗透以及盐胁迫^[26] ...

2008

0.0

... FBW4是一个编码WD40结构域的F-box蛋白, 在氧化胁迫诱导的细胞凋亡途径中, 尽管与RCAN1相互作用, 但也能够独立应对H₂O₂刺激^[27] ...

2009

2.076

1.323

. 2009, 36:17-29

Transgenic expression of MYB15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana

① State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China ② Graduate School of Chinese Academy of Sciences, Beijing 100039, China

Abiotic stresses cause serious crop losses. Knowledge on genes functioning in plant responses to adverse growth conditions is essen-tial for developing stress tolerant crops. Here we report that transgenic expression of MYB15, encoding a R2R3 MYB transcription factor in Arabidopsis thaliana, conferred hypersensitivity to exogenous abscisic acid (ABA) and improved tolerance to drought and salt stresses. The promoter of MYB15 was active in not only vegetative and reproductive organs but also the guard cells of stomata. Its transcript level was substantially upregulated by ABA, drought or salt treatments. Compared with wild type (WT) control, MYB15 overexpression lines were hypersensitive to ABA in germination assays, more susceptible to ABA-elicited inhibition of root elongation, and more sensitive to ABA-induced stomatal closure. In line with the above findings, the transcript levels of ABA biosynthesis (ABA1, ABA2), signaling (ABI3), and responsive genes (AtADH1, RD22, RD29B, AtEM6) were generally higher in MYB15 overexpression seedlings than in WT controls after treatment with ABA. MYB15 overexpression lines displayed improved survival and reduced water loss rates than WT control under water deficiency conditions. These overexpression lines also displayed higher tolerance to NaCl stress. Collectively, our data suggest that overexpression of MYB15 improves drought and salt tolerance in Arabidopsis possibly by enhancing the expression levels of the genes involved in ABA biosynthesis and signaling, and those encoding the stress-protective proteins.

非生物胁迫可造成作物严重减产。深入认识调控植物对不良环境反应的基因对于培育耐胁迫作物是必需的。在本文中，我们报道MYB15（编码一个可能的R2R3类转录因子）的转基因表达显著增强拟南芥对脱落酸的敏感性和抗旱性。MYB15的启动子在拟南芥营养和生殖器官以及气孔的保卫细胞中都可以指导标记基因的转录，MYB15的转录水平受脱落酸（ABA）、干旱和盐处理上调。与野生型对照相比，MYB15过量表达株系对ABA诱导的种子萌发延迟、根生长抑制和气孔关闭更加敏感。ABA处理后，MYB15过量表达株系中ABA合成（ABA1，ABA2）、信号传递（ABI3）以及响应基因（AtADH1，RD22，RD29B，AtEM6）的表达水平高于野生型对照。在干旱处理试验中，MYB15过量表达株系比野生型对照表现出更低的失水率和较高的成活率，这些株系的幼苗对盐处理也表现出较高的耐性。总之，本研究表明MYB15基因的过量表达增强拟南芥对干旱和盐处理的耐性，其机制可能与在过量表达株系中参与ABA合成和信号传递以及ABA响应基因的表达水平较高有关。

... MYB15与MYB84参与了SiFBX基因的转录, 过量表达MYB15基因可以增强拟南芥对于ABA和干旱的耐性^[28], 而MYB84基因被报道具有增强水稻抵抗盐害的作用^[29], 说明SiFBX在应对逆境胁迫中发挥潜在作用 ...

2012

0.0

2009

4.134

0.0

... 进一步表明该基因表达产物SiFBX蛋白是通过上游激素相关顺式作用元件响应激素信号, 而激素信号转导途径与植物的耐逆性相关^[30,31] ...

2009

3.518

0.0

. 2009, 71:291-305 DOI:10.1007/s11103-009-9524-8

Identification and expression profiling analysis of TIFY family genes involved in stress and phytohormone responses in rice

1.Huazhong Agricultural University National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan) 430070 Wuhan China

The TIFY family is a novel plant-specific gene family involved in the regulation of diverse plant-specific biologic processes, such as development and responses to phytohormones, in Arabidopsis. However, there is limited information about this family in monocot species. This report identifies 20 TIFY genes in rice, the model monocot species. Sequence analysis indicated that rice TIFY proteins have conserved motifs beyond the TIFY domain as was previously shown in Arabidopsis. On the basis of their protein structures, members of the TIFY family can be divided into two groups. Transcript level analysis of OsTIFY genes in tissues and organs revealed different tempo-spatial expression patterns, suggesting that expression and function vary by stage of plant growth and development. Most of the OsTIFY genes were predominantly expressed in leaf. Nine OsTIFY genes were responsive to jasmonic acid and wounding treatments. Interestingly, almost all the OsTIFY genes were responsive to one or more abiotic stresses including drought, salinity, and low temperature. Over-expression of OsTIFY11a, one of the stress-inducible genes, resulted in significantly increased tolerance to salt and dehydration stresses. These results suggest that the OsTIFY family may have important roles in response to abiotic stresses. The data presented in this report provide important clues for further elucidating the functions of the genes in the OsTIFY family.

... 进一步表明该基因表达产物SiFBX蛋白是通过上游激素相关顺式作用元件响应激素信号, 而激素信号转导途径与植物的耐逆性相关^[30,31] ...