谷子逆境应答相关的钙依赖蛋白激酶基因<italic>SiCDPK1</italic>的克隆与表达

引用本文

余琴鸯, 尹恒, 安利佳, 李文利. 谷子逆境应答相关的钙依赖蛋白激酶基因SiCDPK1的克隆与表达 . 作物学报, 2014, 40(9): 1531-1539[YU Qin-Yang, YIN Heng, AN Li-Jia, LI Wen-Li. Cloning and Expression Analysis of a Calcium-Dependent Protein Kinase GeneSiCDPK1 inSetaria italica . Acta Agronomica Sinica, 2014, 40(9): 1531-1539] 复制到剪切板

Permissions

作物学报编辑部

谷子逆境应答相关的钙依赖蛋白激酶基因SiCDPK1的克隆与表达

余琴鸯, 尹恒, 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 116023

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

收稿日期:2014-06-16

基金:本研究由辽宁省科技厅农业攻关项目(2011208001)资助;

摘要

CDPK是一类重要的钙信号感受蛋白和响应蛋白, 在植物非生物胁迫应答方面具有重要的作用。为探究耐旱作物谷子CDPK在抗逆胁迫中的应答机制, 利用RT-PCR技术从谷子幼苗cDNA中克隆到一个与逆境胁迫相关的CDPK基因, 命名为SiCDPK1 (GenBank登录号为KC249975.1)。以拟南芥CDPK基因序列为查询序列, 预测谷子基因组含有28个CDPK基因。其系统发育分析表明, 谷子CDPK基因家族由4个亚类组成, 其中SiCDPK1属于第II亚类, 其全长1596 bp, 编码531个氨基酸, 预测蛋白分子量为59.5 kD, 等电点pI为5.94, 含有典型CDPK的保守结构。启动子调控区含有与多种逆境胁迫相关的调控元件。实时定量结果显示,SiCDPK1基因受PEG、ABA、高盐、自然干旱胁迫诱导表达。本试验为谷子抗逆应答机制的深入研究奠定了良好的理论基础。

关键词: 谷子; CDPK; 逆境胁迫; 钙信号

Cloning and Expression Analysis of a Calcium-Dependent Protein Kinase GeneSiCDPK1 inSetaria italica

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

School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116023, China

Fund:

Abstract

CDPK (calcium-dependent protein kinase) is a kind of essential calcium sensors and calcium responders, which plays important roles in response to various abiotic stresses in plants. In this research, we cloned the abiotic stress response relatedCDPK gene with RT-PCR inSetaria italica, designated asSiCDPK1 (GenBank accession number KC249975.1). We predicted 28CDPKs in theSetaria italicagenome by using the knownArabidopsis CDPK sequences as query sequences. The phylogenetic analysis showed theSiCDPK gene family was divided into four subgroups, in whichSiCDPK1belongs to subgroup II. The ORF ofSiCDPK1contains 1596 bp, which encodes 531 amino acids. The predicted protein molecular weight is 59.5 kD and pI is 5.94. SiCDPK1 has conserved protein domains of CDPK. The regulatory element analysis of promoter inSiCDPK1 showed a lot ofcis-acting elements associated with different abiotic stresses. The RT-PCR results showed thatSiCDPK1 was induced by PEG, ABA, salinity and drought. The research onSiCDPK1paves a way for unraveling the mechanism of abiotic stresses in plants.

Keyword: Setaria italica; CDPK; Abiotic stress; Ca2+ signal

Show Figures

钙离子是真核细胞内重要的第二信使, 介导复杂的信号转导调控网络, 在植物生长、发育、免疫防御、激素调节、生物胁迫、非生物胁迫等各个方面起着重要的作用。目前, 主要有CaM (calmodulin), CBL (calcineurin B-like protein), CDPK (calcium-dependent protein kinase) 3类钙离子识别蛋白。其中, CDPK还是钙离子响应蛋白。CDPK是一类典型的Ser/Thr蛋白激酶, 结构较保守, 其蛋白主要包括4部分, 从N端到C端依次为可变区、蛋白激酶

区、自抑连接区、钙离子结合区, N端含有保守的MGXXXSXX区域, 能够被豆蔻酰化修饰, 从而将CDPK蛋白定位在细胞内膜系统上起重要的作用^{[ 1, 3, 4, 5, 6, 7, 8]}。除了豆蔻酰化, 有的CDPK还能被棕榈酰化^{[ 4, 9]}, 增强与膜质结合的能力。自抑连接区中有CDPK自抑制片段, 可作为CDPK的假底物。当假底物与蛋白激酶区结合时, CDPK处于未激活状态^{[ 2]}。钙离子结合区含有保守的钙离子结合EF手性结构域, 当钙离子与此结构结合后可以引起CDPK蛋白结构变化, 解除自抑, 从而激活CDPK^{[ 2]}。不同的CDPK蛋白具有不同的钙离子结合能力, 而对于单个CDPK而言, 钙离子是调节CDPK活性的重要因子^{[ 2, 10]}。

CDPKs为多基因家族, 拟南芥中含有34个^{[ 11, 12, 13]}, 水稻中含有31个^{[ 14, 15]}, 小麦中至少含有20个^{[ 16]}, 玉米中含有40个^{[ 17]}, 其他物种均包含多个 CDPKs基因。CDPK可在植物所有的组织和器官中表达, 但有的CDPK组织特异性表达, 如AtCPK17和AtCPK34主要在成熟的花粉中表达并调节花粉管的伸长^{[ 18]}。CDPK可以被定位于不同的亚细胞结构, 如细胞质基质、细胞核、细胞膜、内质网、液泡、线粒体、叶绿体、油体、过氧化物酶体等^{[ 3, 5, 7, 16, 18, 19, 20, 21, 22, 23, 24, 25]}。

从1991年, Harper等^{[ 26]}从大豆中克隆得到第一个 CDPK-α基因全序列开始, 不同物种、不同功能的 CDPK基因被不断研究。研究证实^{[ 15, 16, 27, 28]}, CDPK能够响应不同的外界刺激, 包括ABA、冷、干旱、高盐、热、病原菌入侵和机械损伤等。 CDPK启动子含有响应不同胁迫的顺式作用元件^{[ 28]}。CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[ 24, 29, 30, 31, 32, 33, 34, 35, 36, 37]}等。CDPK参与植物细胞内其他重要的信号通路, 包括ABA信号转导通路、ROS信号通路、茉莉酸信号通路、乙烯信号通路、水杨酸合成途径、非生物胁迫应答等。近年来, CDPK在抗逆应答方面的研究取得了一定的进展^{[ 1, 32]}。CDPK调节植物激素和非生物胁迫信号转导途径中的相关基因表达、代谢、离子通道、气孔运动等^{[ 1]}。CDPK是植物发育、免疫防御和胁迫应答的中心因子, 并参与其他重要的信号转导通路。不同的信号通路相互交错、互补、连接, 形成植物细胞内复杂而有序的调控网络。

谷子具有根系发达, 叶片细窄, 水分利用率高等耐旱性特征。谷子与粮食作物亲缘关系较近且基因组较小, 已经逐步成为未来作物遗传改良育种理想的模式作物^{[ 38]}, 将对未来禾谷类作物的品种改良、能源作物的遗传解析产生深远影响。目前, 已完成谷子全基因组测序, 同时谷子分子标记(SSR、RFLP、AFLP、SRAP等)与功能基因组学研究^{[ 48]}也取得了一定的进展, 必将带动谷子研究的高潮。国内, 在谷子抗锈基因、抗病基因、抗除草剂基因等方面取得了一定的进展, 如病程相关基因 SiSGT1、NBS类抗病基因、抗锈基因 RUS、抗锈基因 RLr1、抗除草剂基因 Srf^{[ 39]}、丝/苏氨酸蛋白激酶类抗病基因^{[ 40]}等。在抗旱方面, 国内学者对谷子 SiDnaJ基因^{[ 42]}、干旱应答元件结合蛋白基因 SiDREB^{[ 42]}、12-氧代植二烯酸还原酶基因 SiOPR1^{[ 43]}、3-磷酸甘油醛脱氢酶基因 GAPDH^{[ 44]}、磷脂酶D基因 SiPLDa1^{[ 45]}、 CBL家族^{[ 46]}、 PP2C家族^{[ 38]}和 ABF3基因^{[ 47]}等进行了研究。但目前未见关于谷子 CDPK基因家族研究的报道。谷子抗逆的分子机制的深入研究将为利用基因工程、分子生物学育种奠定良好的基础。

本研究以晋谷34为试验材料, 利用RT-PCR技术克隆得到耐旱作物谷子中第一个与植物非生物胁迫应答相关的钙依赖性蛋白激酶基因 SiCDPK1, 拟通过蛋白预测、系统发育分析、启动子调控元件分析等生物信息学分析和考察在ABA、高盐、PEG和自然干旱胁迫下的表达模式, 以期为揭示谷子 CDPK基因家族抗逆分子机制奠定基础。

1 材料与方法

1.1 植物材料及胁迫处理

晋谷34由山西省农业科学院作物遗传研究所提供, 将种子播种于苗床(草炭土、珍珠岩和蛭石的质量比为1∶1∶1)上, 控制适宜谷子生长的空气湿度、温度、光照等条件。幼苗长至三至四叶, 取长势一致的谷子幼苗分别设PEG模拟干旱处理(含20% PEG的1/2 Hoagland营养液, pH 7.0)、高盐处理(含100 mmol L^-1 NaCl的1/2 Hoagland营养液, pH 7.0)、正常干旱处理(断水)和外部喷施ABA (100 μmol L^-1)胁迫处理。取PEG和ABA胁迫处理0、0.5、1、2、4和8 h后的样品, 正常干旱处理断水0、1、2、3、4、5和6 d后每天9:00的样品, 每个样品10株, 3次重复。将样品于液氮速冻, -80℃冰箱保存待用。

1.2 SiCDPK1基因编码区ORF全长cDNA克隆

从谷子转录组数据库中, 筛选出 SiCDPK1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 SiCDPK1基因编码序列。根据此序列, 设计并合成引物 SiCDPK1-F和 SiCDPK1-R (表1)。取不同时期正常干旱(断水)处理、高盐处理、外部喷施ABA处理、PEG模拟干旱处理的植物材料混合样品, 用RNAiso Plus (TaKaRa)提取mRNA, 使用PrimeScript First Strand cDNA Synthesis Kit (TaKaRa)反转录合成cDNA。利用SiCDPK1-F和SiCDPK1-R引物进行PCR扩增 SiCDPK1基因编码区ORF全长cDNA片段, 回收特异性PCR扩增产物, 连接到pMD-18T Simple载体(TaKaRa)上, 转化到大肠杆菌(DH5α)感受态细胞中, 挑取抗性培养基上生长的阳性单菌落, 经质粒酶切验证后送华大基因测序, 得到目的基因 SiCDPK1全长cDNA。

表1 SiCDPK1基因克隆及Real-time PCR引物 Table 1 Primers used in SiCDPK1 gene 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SiCDPK1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SiCDPK1的二级结构及疏水区域。利用TMpred (http://www.ch.embnet.org/software/TMPRED_form.html)和TMH MM (http://www.cbs.dtu.dk/services/TMHMM)分析SiCDPK1的跨膜结构域。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。以拟南芥 CDPK基因家族序列为查询序列, 与谷子基因组比对预测谷子CDPK基因家族成员, 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Neighbor- Joinig系统进化树。使用在线预测工具PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/ html/)预测 SiCDPK1的启动子结构。

1.4 RNA提取和Real-time PCR分析

根据克隆得到的 SiCDPK1序列, 设计并合成Real-time PCR引物SiCDPK1-RT-F和SiCDPK1- RT-R。以 SiActin基因作为Real-time PCR内参, 设计引物SiActin-F和SiActin-R (表1)。

利用RNAiso试剂盒(TaKaRa, 大连)分别提取PEG模拟干旱处理、高盐处理、正常干旱处理和外部喷施ABA胁迫处理后材料的根、茎、叶的总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。取总RNA约200 ng于200 μL离心管中, 加入5× PrimerScript RT Master Mix (for Real-time) 2 μL, 用RNase Free dH₂O将反应体系补充至10 μL。将PCR仪(TaKaRa)设定为37℃保温15 min, 85℃保温5 s, 降温至4℃保存。使用One Step SYBR PrimerScript RT-PCR Kit (Perfect Real- time)试剂盒(TaKaRa, 大连)进行PCR, 反应体系为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 SiActin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。

	Figure Option View Download New Window
	图1 SiCDPK1 基因PCR扩增条带1~4: SiCDPK1在53.0、54.8、56.6和58.4℃下PCR结果; M: DL2000(TaKaRa)。1~4: SiCDPK1在53.0、54.8、56.6和58.4℃下PCR结果; M: DL2000(TaKaRa)。Fig. 1 PCR results of SiCDPK1 gene1-4: PCR products of SiCDPK1 gene at the temperature of 53.0, 54.8, 56.6, and 58.4℃ respectively; M: DL2000 (TaKaRa).

2 结果与分析

2.1 SiCDPK1基因全长cDNA的克隆和染色体定位

利用生物信息学和PCR技术克隆得到一个谷子抗逆应答相关的钙依赖蛋白激酶基因 SiCDPK1(GenBank登录号为KC249975.1)。通过NCBI (http://www.ncbi.nlm.nih.gov/) Blastn比对结果可知, SiCDPK1与玉米( Zea mays)、水稻( Oryza sativa)、小麦( Triticum aestivum)、大麦( Hordeum vulgare)、二穗短柄草( Brachypodium distachyon)等单子叶植物部分 CDPK基因具有高度的同源性, 具有 CDPK基因家族的基本特性。

将测序得到的 SiCDPK1序列与谷子基因组数据库(http://www.phytozome.net/)比对, 发现该基因位于谷子基因组Scaffold_2区段的41 740 734~ 41 743 777。使用DNAMAN对该区域的序列进行结构化展示(图2), SiCDPK1是由7个内含子和8个外显子组成的。

	Figure Option View Download New Window
	图2 SiCDPK1在谷子基因组上的位置和结构Fig. 2 Location and structure of SiCDPK1 in Setaria italic genome

2.2 SiCDPK1基因的生物信息学分析

ProParam预测结果显示SiCDPK1蛋白由531个氨基酸组成(图3), 分子量为59.5 kD, 等电点p I为5.94。SiCDPK1蛋白含谷氨酸(E, 8.3%)、丙氨酸(A, 7.3%)、甘氨酸(G, 7.3%)、亮氨酸(L, 7.2%)和赖氨酸(K, 7.0%), 这5种氨基酸所占比例达到了37.1%。

Figure Option
View Download New Window

图3 SiCDPK1基因序列及推导氨基酸序列分析阴影部分为蛋白激酶区, 加粗部分为钙离子结合区, 两者中间为自抑连接区, 其中虚线框内为自抑片段, 剩余部分为可变区。下画线显示的是EF hand结构域。序列开头框内为豆蔻酰化保守序列MGXXXSXX, 其中G为豆蔻酰化位点, C为棕榈酰化位点。Fig. 3 Analysis of SiCDPK1 gene sequence and deduced amino acid sequenceThe shadow shows the protein kinase domain, and the bold shows the calmodulin-like domain. The junction domain is between the protein kinase domain and the calmodulin-like domain, in which the autoinhibitory domain is shown by dashed box. The rest is variable domain. EF hand is underlined. The myristoylation conserved domain is MGXXXSXX, which is shown by the box at the beginning of the text. G is myristoylation site and C is palmitoylation site.

Predict Protein二级结构预测显示, SiCDPK1蛋白含有15个蛋白结合位点, 17个螺旋结构helix, 11个串strand, 主要以螺旋结构为主。疏水区主要分布在蛋白N端的蛋白激酶区。用ProScale进行疏水性分析显示, 150~300氨基酸部分疏水性较集中, 与Predict Protein预测结果相符。利用TMpred和TMHMM分析, 均未发现明显跨膜结构, 表明SiCDPK1不是跨膜蛋白。PROSITE分析结果显示SiCDPK1蛋白含有丰富的可磷酸化位点(14个丝氨酸S、11个苏氨酸T、6个酪氨酸Y)。通过Signal IP预测发现SiCDPK1蛋白不含有信号肽结构, 说明该蛋白不是分泌蛋白。

通过NCBI BlastP直接比对或利用CCD数据库的CD-search工具分析表明, SiCDPK1蛋白具有典型的CDPK保守结构, N端的蛋白激酶结构域属于PKc_like superfamily, C端含有4个EF hand结构域, 可形成2个钙离子结合loop。以已知的拟南芥 CDPK基因序列(TAIR,http://www.Arabidopsis.org/, release 10.0)为查询或搜索序列, 在谷子基因组数据库(http://www.phytozome.net/)中通过Blast找到预测的CDPK基因成员, 并通过谷子基因组数据库或NCBI数据库手动分析初步确定候选的谷子 CDPK基因家族成员有28个(表2)。从NCBI上下载拟南芥的34个 CDPK基因序列, 并结合预测到的谷子CDPK基因家族成员序列, 用ClastalX和MEGA4.0软件进行多重序列比对以及构建Neighbor-Joining系统进化树(图4)。从图4中, 我们看出, 谷子的 CDPK基因家族与拟南芥 CDPK基因家族一样, 可以分成4个亚类, 第IV亚类的基因数较少。 SiCDPK1/ SiCPK6属于 CDPK基因家族的第II亚类, 与 AtCPK9和 AtCPK33亲缘关系较近。目前, AtCPK9和 AtCPK33基因的功能并未被确定^{[ 1]}, 因此探讨 SiCDPK1基因的功能将推动 CDPK基因家族的研究进展。

表2 预测SiCDPK蛋白信息 Table 2 Information of predicted SiCDPKs protein

	Figure Option View Download New Window
	图4 SiCDPK1蛋白的系统发育分析Fig. 4 Phylogenetic analysis of SiCDPK1 protein

2.3 SiCDPK1基因的上游启动子分析

从 SiCDPK1基因的ATG开始, 截取 SiCDPK1基因5°上游1969 bp的核苷酸序列, 使用PlantCARE在线预测 SiCDPK1基因的启动子及调控元件。ATG上游177 bp处发现TATA-box区域, 转录起始位点于上游212 bp处, 在上游210 bp至上游1487 bp区域预测到多种逆境胁迫相关的顺式作用元件(图5), 包括与光、温度、激素、厌氧、干旱、细胞周期及发育相关顺式调控元件。

	Figure Option View Download New Window
	图5 SiCDPK1启动子顺式调控元件示意图直线上方为(+)链调控元件, 下方为(-)链调控元件。Fig. 5 Schematic diagram of cis-acting element in the promoter region of SiCDPK1The element above the line is in (+)-sense strand, and the element below the line is in (-)-sense strand.

2.2 SiCDPK1基因的表达模式分析

在用20%的PEG胁迫处理谷子幼苗后, SiCDPK1在谷子根和叶中表达量呈现显著上调的趋势, 分别在1 h和2 h时明显上调, 并有持续上调的趋势, 在8 h时表达量达到最大, 分别为对照组的9.5倍和46.2倍。而在茎部表达量则未出现明显的变化(图6)。高盐处理时, SiCDPK1在根中除了1 h出现上调外, 其余各点表达量下调。而在茎和叶中, 各个处理时间点 SiCDPK1表达量均呈现上调趋势。相对于PEG处理, 较晚响应高盐胁迫, 在茎和叶中分别在4 h和8 h表达量最高, 峰值分别为16.1倍和17.6倍(图6)。高盐处理时, SiCDPK1在不同组织中表达趋势的不同, 显示了 SiCDPK1复杂表达模式, 表达量的变化, 显示其在不同胁迫时期的响应。当用100 μmol L^-1ABA处理后, SiCDPK1基因的表达量在根茎叶中变化不及PEG和高盐处理明显, 但仍出现变化, 显示其参与ABA信号通路(图6)。在正常干旱处理下, SiCDPK1基因在根茎叶中表达量先升高后下降。根和茎中, 均在第1天表达量达到最高, 分别为6.5倍和13.1倍。叶中, SiCDPK1基因在第2天表达量才明显上升, 于第5天达到最高值(图6)。

	Figure Option View Download New Window
	图6 SiCDPK1基因表达模式分析柱上小写字母不同表示在0.05的水平上差异显著。Fig. 6 Expression pattern analysis of SiCDPK1 geneBars superscripted by different letters are significantly different at 0.05 probability level.

3 讨论

克隆得到的 SiCDPK1具有 CDPK家族的典型特征^{[ 13, 34]}, 谷子CDPK基因家族成员含有的EF hand结构域数量不同(表2中EF hand结构域数由谷子基因组数据库注释得到), 以3个EF hand结构域为主, 显示了不同SiCDPK活性受钙离子结合的调节^{[ 13, 35]}。通过系统发育分析可知, 其属于 CDPK基因家族的第II亚类, 与其同源性较高的拟南芥CDPK家族成员有 AtCPK9、 AtCPK33、 AtCPK15、 AtCPK21、 AtCPK23等。其中 AtCPK9、 AtCPK33和 AtCPK15这3个基因的功能并未被确定, 而 AtCPK21已被证实参与植物渗透胁迫应答和气孔运动调控, AtCPK23被证实参与干旱和高盐胁迫应答、气孔运动调控。从本文实时定量数据分析可知, SiCDPK1/SiCPK6基因受PEG渗透胁迫的诱导, 对高盐胁迫产生应答, 从而表达量变化, 然而在不同的组织中其变化不同, 显示了 SiCDPK1基因具有组织特异性, 在不同组织中的功能也不尽相同。 SiCDPK1基因受ABA诱导表达, 与其启动子调控区域含有ABRE顺式作用元件相呼应。在自然干旱中, 我们可以看到 SiCDPK1表达量在最初2 d明显上升, 说明 SiCDPK1在谷子干旱胁迫应答中起一定作用。

AtCPK9、 AtCPK33、 AtCPK15、 AtCPK21和 AtCPK23基因, N端均能被豆蔻酰化和棕榈酰化^{[ 35]}, SiCDPK1蛋白N端同样具有豆蔻酰化和棕榈酰化的修饰位点(图3)。由于豆蔻酰化和棕榈酰化的修饰, AtCPK9、 AtCPK21和 AtCPK23被定位于细胞膜上, 推测 SiCDPK1也被定位于细胞膜上。

从 SiCDPK1启动子调控元件的分析可知, SiCDPK1基因很可能参与激素调节(GA、ABA)、光感应、厌氧应答、病原菌免疫、周期调控、胁迫应答和调控植物生长发育(胚乳、分生组织)等。推测 SiCDPK1与bZIP、WRKY等转录因子相关, 调控下游基因表达。由此可见, SiCDPK1在谷子的生长发育及逆境应答中具有重要的功能。

目前, 对谷子抗逆基因研究还处于初步阶段, SiCDPK1基因为证实的一个谷子抗逆相关的钙离子依赖蛋白激酶基因。植物的生长发育、对外界刺激的响应是一个复杂而综合的网络, 是CDPK、MAPK、CBL-CIPK、SnRK等通路^{[ 36, 37]}相互作用、相互联系的结果。对CDPK生理功能和底物的研究, 有利于揭示植物抗逆胁迫的应答机制, 为提高植物的抗逆性奠定坚实的理论基础。目前, 较常用的研究方法和技术有基因敲除、酵母双杂、Microarray和BiFC等技术。同一物种中的不同CDPK具有不同的生理功能, 然而它们之间又存在着功能冗余的现象, 使得单独敲除或干扰单个CDPK并不能取得理想的试验效果, 双缺或三缺突变体成为研究的必要, 如 cpk4cpk11、 cpk3cpk6、 cpk7cpk8cpk32^{[ 33]}。CDPK新的作用底物不断被发现, 然而大多数都是体外实验得到的^{[ 13]}, 而在体内筛选CDPK作用底物目前还不是很成熟。发现更多的CDPK特异性底物能够加速CDPK功能的鉴定, 系统和全面地对 CDPK基因家族进行分析、研究和整理, 合理综合使用生物化学、细胞生物学、遗传学、生物信息学等方法, 必将更好地推动CDPK信号转导系统的研究。

4 结论

从谷子中克隆到一个与逆境胁迫应答相关的钙依赖蛋白激酶基因 SiCDPK1, 含有 CDPK基因家族典型的结构特征, N端的蛋白激酶结构域属于PKc_like superfamily, C端含有4个EF hand结构域。N端含有豆蔻酰化和棕榈酰化修饰位点, 推测 SiCDPK1定位于细胞膜上。 SiCDPK1含有多个与植物逆境胁迫相关和响应激素的调控元件, 如ABRE、ARE、Box-W1等, 推测SiCDPK1可能作用于bZIP、WRKY等转录因子, 从而调控下游基因表达。 SiCDPK1受PEG、ABA、高盐、自然干旱的诱导表达, 在谷子逆境胁迫应答中起重要作用。

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

View Option

[1]	Boudsocq M, Sheen J. CDPKs in immune and stress signaling. Trends Plant Sci, 2013, 18: 30-40 [本文引用:4] [JCR: 11.808]
[2]	Rutschmann F, Stalder U, Piotrowski M, Oeckinq C, Schaller A. LeCPK1, a calcium-dependent protein kinase from tomato. Plasma membrane targeting and biochemical characterization. Plant Physiol, 2002, 129: 156-168 [本文引用:3] [JCR: 6.555]
[3]	Lu S X, Hrabak E M. An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum. Plant Physiol, 2002, 128: 1008-1021 [本文引用:2] [JCR: 6.555]
[4]	Martin M L, Busconi L. Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation. Plant J, 2000, 24: 429-435 [本文引用:2] [JCR: 6.582]
[5]	Benetka W, Mehlmer N, Maurer-Stroh S, Sammer M, Korand a M, Neumüller R, Betschinqer J, Knoblich J A, Teiqe M, Eisenhaber F. Experimental testing of predicated myristoylation targets involved in asymmetric cell division and calcium-dependent signaling. Cell Cycle, 2008, 7: 3709-3719 [本文引用:2] [JCR: 5.243]
[6]	Chehab E W, Patharkar O R, Heqeman A D, Taybi T, Cushman J C. Autophosphorylation and subcellular localization dynamics of a salt- and water defict-induced calcium-dependent protein kinase from ice plant. Plant Physiol, 2004, 135: 1430-1446 [本文引用:1] [JCR: 6.555]
[7]	Mehlmer N, Wurzinqer B, Stael S, Hofmann-Rodriques D, Csaszar E, Pfister B, Bayer R, Teiqe M. The Ca²+-dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis. Plant J, 2010, 63: 484-498 [本文引用:2] [JCR: 6.582]
[8]	Stael S, Bayer R G, Mehlmer N, Teiqe M. Protein N-acylation overrides differing targeting signals. FEBS Lett, 2011, 585: 517-522 [本文引用:1] [JCR: 3.582]
[9]	Witte C P, Keinath N, Dubiella U, Demoulière R, Seal A, Romeis T. Tobacco calcium-dependent protein kinases are differentially phosphorylated in vivo as part of a kinase cascade that regulates stress response. J Biol Chem, 2010, 285: 9740-9748 [本文引用:1] [JCR: 4.651]
[10]	Christodoulou J, Malmendal A, Harper J F, Chazin W J. Evidence for differing roles for each lobe of the calmodulin-like domain in a calcium-dependent protein kinase. J Biol Chem, 2004, 279: 29092-29100 [本文引用:1] [JCR: 4.651]
[11]	Harper J F, Breton G, Harmon A. Decoding Ca²+ signals through plant protein kinases. Annu Rev Plant Biol, 2004, 55: 263-288 [本文引用:1] [JCR: 25.962]
[12]	Harper J F, Harmon A. Plants, symbiosis and parasites: a calcium signaling connection. Nat Rev Mol Cell Biol, 2005, 6: 555-566 [本文引用:1]
[13]	Cheng S H, Willmann M R, Chen H C, Sheen J. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol, 2002, 129: 469-485 [本文引用:4] [JCR: 6.555]
[14]	Asano T, Tanaka N, Yang G, Hayashi N, Komatsu S. Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: comprehensive analysis of the CDPKs gene family in rice. Plant Cell Physiol, 2005, 46: 356-366 [本文引用:1] [JCR: 4.134]
[15]	Ray S, Aqarwal P, Arora R, Kapoor S, Tyaqi A K. Expression analysis of calcium-dependent protein kinase gene family during reproductive development and abiotic stress conditions in rice (Oryza sativa L. ssp. indica). Mol Genet Genomics, 2007, 278: 493-505 [本文引用:2] [JCR: 2.881]
[16]	Li A L, Zhu Y F, Tan X M, Wang X, Wei B, Guo H Z, Zhang Z L, Chen X B, Zhao G Y, Kong X Y, Jia J Z, Mao L. Evolutionary and functional study of the CDPK gene family in wheat (Triticum aestivum L. ). Plant Mol Biol, 2008, 66: 429-443 [本文引用:3] [JCR: 3.518]
[17]	Ma P, Liu J, Yang X, Ma R. Genome-wide identification of the maize calcium-dependent protein kinase gene family. Appl Biochem Biotechnol, 2013, 169: 2111-2125 [本文引用:1] [JCR: 1.348]
[18]	Myers C, Romanowsky S M, Barron Y D, Garq S, Azuse C L, Curran A, Davis R M, Hatton J, Harmon A C, Harper J F. Calcium-dependent protein kinase regulate polarized tip growth in pollen tubes. Plant J, 2009, 59: 528-539 [本文引用:2] [JCR: 6.582]
[19]	Boudsocq M, Willmann M R, McCormack M, Lee H, Shan L, He P, Bush J, Cheng S H, Sheen J. Differential innate immune signaling via Ca²+ sensor protein kinase. Nature, 2010, 464: 418-422 [本文引用:1] [JCR: 38.597]
[20]	Zou J J, Wei F J, Wang C, Wu J J, Ratnasekera D, Liu W X, Wu W H. Arabidopsis calcium-dependent protein kinase CPK10 functions in abscisic acid- and Ca²+-mediated stomatal regulation in response to drought stress. Plant Physiol, 2010, 154: 1232-1243 [本文引用:1] [JCR: 6.555]
[21]	Yu X C, Li M J, Gao G F, Feng H Z, Geng X Q, Peng C C, Zhu S Y, Wang X J, Shen Y Y, Zhang D P. Abscisic acid stimulates a calcium-dependent protein kinase in grape berry. Plant Physiol, 2006, 140: 558-579 [本文引用:1] [JCR: 6.555]
[22]	Zhu S Y, Yu X C, Wang X J, Zhao R, Li Y, Fan R C, Shang Y, Du S Y, Wang X F, Wu F Q, Xu Y H, Zhang X Y, Zhang D P. Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis. Plant Cell, 2007, 19: 3019-3036 [本文引用:1] [JCR: 9.251]
[23]	Dammann C, Ichida A, Honq B, Romanowsky S M, Hrabak E M, Harmon A C, Pickard B G, Harper J F. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis. Plant Physiol, 2003, 132: 1840-1848 [本文引用:1] [JCR: 6.555]
[24]	Choi H I, Park H J, Park J H, Kim S, Im M Y, Seo H H, Kim Y W, Hwang I, Kim S Y. Arabidopsis calcium-dependent protein kinase AtCPK32 interacts with ABF4, a transcriptional regulator of abscisic acid-responsive gene expression, and modulates its activity. Plant Physiol, 2005, 139: 1750-1761 [本文引用:2] [JCR: 6.555]
[25]	Coca M, San Segundo B. AtCPK1 calcium-dependent protein kinase mediates pathogen resistance in Arabidopsis. Plant J, 2010, 63: 526-540 [本文引用:1] [JCR: 6.582]
[26]	Harper J F, Sussman M R, Schaller G E, Putnam-Evans C, Charbonneau H, Harmon A C. A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science, 1991, 252: 951-954 [本文引用:1]
[27]	Romeis T, Ludwig A A, Martin R, Jones J D G. Calcium- dependent protein kinases play an essential role in a plant defence response. EMBO J, 2001, 20: 5556-5567 [本文引用:1] [JCR: 9.822]
[28]	Wan B, Lin Y, Mou T. Expression of rice Ca²+-dependent protein kinases (CDPKs) genes under different environmental stresses. FEBS Lett, 2007, 581: 1179-1189 [本文引用:2] [JCR: 3.582]
[29]	Kanchiswamy C N, Takahashi H, Quadro S, Maffei M E, Bossi S, Bertea C, Zebelo A S, Muroi A, Ishihama N, Yoshioka H, Boland W, Takabayashi J, Endo Y, Sawasaki T, Arimura G. Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling. BMC Plant Biol, 2010, 10-97 [本文引用:1] [JCR: 4.354]
[30]	Ishida S, Yuasa T, Nakata M, Takahashi Y. A tobacco calcium- dependent protein kinase, CDPK1, regulates the transcription factor REPRESSION OF SHOOT GROWTH in response to gibberellins. Plant Cell, 2008, 20: 3273-3288 [本文引用:1] [JCR: 9.251]
[31]	Ito T, Nakata M, Fukazawa J, Ishida S, Takahashi Y. Alteration of substrate specificity: the variable N-terminal domain of tobacco Ca²+-dependent protein kinase is important for substrate recognition. Plant Cell, 2010, 22: 1592-1604 [本文引用:1] [JCR: 9.251]
[32]	Schulz P, Herde M, Romeis T. Calcium-dependent protein kinases: hubs in plant stress signaling and development. Plant Physiol, 2013, 163: 523-530 [本文引用:2] [JCR: 6.555]
[33]	Hubbard K E, Sieqel R S, Valerio G, Brand t B, Schroeder J I. Absicsic acid and CO₂ signalling via calcium sensitivity priming in guard cells, new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analysis. Ann Bot, 2012, 109: 5-17 [本文引用:2] [JCR: 0.657]
[34]	Harmon A C, Gribskov M, Harper J F. CDPKs-a kinase for every Ca²+ signal?Trends Plant Sci, 2000, 5: 154-159 [本文引用:2] [JCR: 11.808]
[35]	Liese A, Romeis T. Biochemical regulation of in vivo function of plant calcium-dependent protein kinase (CDPK). Biochim Biophy Acta, 2013, 1833: 1582-1589 [本文引用:3]
[36]	Hrabak E M, Chen C W, Gribskov M, Harper J F, Choi J H, Halford N, Kudla J, Luan S, Nimmo H G, Sussman M R, Thomas M, Walker-Simmons K, Zhu J K, Harmon A C. The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol, 2003, 132: 666-680 [本文引用:2] [JCR: 6.555]
[37]	Ludwiq A A, Romeis T, Jones J D. CDPK-mediated signaling pathways: specificity and cross-talk. J Exp Bot, 2004, 55: 181-188 [本文引用:2] [JCR: 5.242]
[38]	Lata C, Gupta S, Prasad M. Foxtail millet: a model crop for genetic and genomic studies in bioenergy grasses. Crit Rev Biotechnol, 2013, 33: 328-343 [本文引用:2] [JCR: 5.095]
[39]	李志江. 谷子抗除草剂基因的发现及其应用. 基因组学与应用生物学, 2010, 29: 768-774 Li Z J. Discovery and application of herbicide resisitant gene in foxtail millet. Genom Appl Biol, 2010, 29: 768-774 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.7323]
[40]	瓮巧云, 宋晋辉, 张爱香. 谷子丝/苏氨酸蛋白激酶类抗病基因同源序列的克隆与分析. 河南农业科学, 2012, 41: 106-108 Weng Q Y, Song J H, Zhang A X. Cloning and analysis of STK disease resistant gene analogs in millet. J Henan Agric Sci, 2012, 41: 106-108 (in Chinese with English abstract) [本文引用:1]
[41]	崔润丽, 智慧, 王永芳, 李伟, 李海权, 黄占景, 刁现民. 谷子DnaJ蛋白基因的克隆. 华北农学报, 2007, 22(4): 9-13 Cui R L, Zhi H, Wang Y F, Li W, Li H Q, Huang Z J, Diao X M. Cloning of DnaJ-like protein gene from foxtail millet. Acta Agric Boreali-Sin, 2007, 22(4): 9-13 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.951]
[42]	杨希文, 胡银岗. 谷子DREB转录因子基因的克隆及其在干旱胁迫下的表达模式分析. 干旱地区农业研究. 2011, 29(5): 69-74 Yang X W, Hu Y G. Cloning of a DREB gene from foxtail millet (Setaria italica L) and its expression during drought stress. Agric Res Arid Areas, 2011, 29(5): 69-74 (in Chinese with English abstract) [本文引用:2]
[43]	Zhang J P, Zheng J, Zhu Y, Guo J F, Wang G Y. Cloning and characterization of a putative 12-oxophytodienoic acid reductase cDNA induced by osmotic stress in roots of foxtail millet. DNA Seq, 2007, 18: 138-144 [本文引用:1]
[44]	崔润丽, 智慧, 王永芳, 李伟, 李海权, 黄占景, 刁现民. 谷子3-磷酸甘油醛脱氢酶基因的克隆与结构分析. 华北农学报, 2009, 24(3): 10-14 Cui R L, Zhi H, Wang Y F, Li W, Li H Q, Huang Z J, Diao X M. Cloning and structure analysis of Foxtail Millet APDH gene. Acta Agric Boreali-Sin, 2009, 24(3): 10-14 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.951]
[45]	Peng Y L, Zhang J P, Cao G Y. Overexpression of a PLDα1 gene from Setaria italica enhances the sensitivity of Arabidopsis to abscisic acid and improves its drought tolerance. Plant Cell Rep, 2010, 29: 793-802 [本文引用:1] [JCR: 2.509]
[46]	赵晋锋, 余爱丽, 田岗, 杜艳伟, 郭二虎, 刁现民. 谷子CBL基因鉴定及其在干旱、高盐胁迫下的表达分析. 作物学报, 2013, 39: 360-367 Zhao J F, Yu A L, Tian G, Du Y W, Guo E H, Diao X M. Identification of CBL genes from foxtail millet (Setaria italica [L. ] Beauv. ) and its expression under drought and salt stresses. Acta Agron Sin, 2013, 39: 360-367 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.667]
[47]	张雁明, 王莉, 张彬, 王海岗, 彭锁堂, 李萍, 韩渊怀. 谷子ABF3基因对PEG胁迫的响应. 山西农业大学学报(自然科学版), 2013, 33: 191-196 Zhang Y M, Wang L, Zhang B, Wang H G, Peng S T, LI P, Han Y H. The response of ABF3 Gene to PEG stress in foxtail millet (Setaria italica L. ). J Shanxi Agric Univ (Nat Sci Edn), 2013, 33: 191-196 [本文引用:1]
[48]	李志江, 刁现民. 谷子分子标记与功能基因组研究进展. 中国农业科技导报, 2009, 11(4): 16-22 Li Z J, Diao X M. Research progress on molecular marker and functional genomic of foxtail millet (Setaria italic Beauv. ). J Agric Sci Technol, 2009, 11(4): 16-22 (in Chinese with English abstract) [本文引用:1]

2013

11.808

0.0

Boudsocq

, Sheen

. CDPKs in immune and stress signaling. Trends Plant Sci, 2013, 18: 30-40

Ca 2+ has long been recognized as a conserved second messenger and principal mediator in plant immune and stress responses. How Ca 2+ signals are sensed and relayed into diverse primary and global signaling events is still largely unknown. Comprehensive analyses of the plant-specific multigene family of Ca 2+ -dependent protein kinases (CDPKs) are unraveling the molecular, cellular and genetic mechanisms of Ca 2+ signaling. CDPKs, which exhibit overlapping and distinct expression patterns, sub-cellular localizations, substrate specificities and Ca 2+ sensitivities, play versatile roles in the activation and repression of enzymes, channels and transcription factors. Here, we review the recent advances on the multifaceted functions of CDPKs in the complex immune and stress signaling networks, including oxidative burst, stomatal movements, hormonal signaling and gene regulation.

... 区、自抑连接区、钙离子结合区, N端含有保守的MGXXXSXX区域, 能够被豆蔻酰化修饰, 从而将CDPK蛋白定位在细胞内膜系统上起重要的作用^{[1,3,4,5,6,7,8]} ...

... 近年来, CDPK在抗逆应答方面的研究取得了一定的进展^[1,32] ...

... CDPK调节植物激素和非生物胁迫信号转导途径中的相关基因表达、代谢、离子通道、气孔运动等^[1] ...

... 目前, AtCPK9和AtCPK33基因的功能并未被确定^[1], 因此探讨SiCDPK1基因的功能将推动CDPK基因家族的研究进展 ...

2002

6.555

0.0

Rutschmann

, Stalder

, Piotrowski

, Oeckinq

, Schaller

. LeCPK1, a calcium-dependent protein kinase from tomato. Plasma membrane targeting and biochemical characterization. Plant Physiol, 2002, 129: 156-168

The cDNA of Le CPK1, a calcium-dependent protein kinase, was cloned from tomato ( Lycopersicon esculentum Mill.). Le CPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, K m = 85 μ m ). Autophosphorylation of Le CPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca 2+ dependent and required the C-terminal, calmodulin-like domain of Le CPK1. Two classes of high- and low-affinity Ca 2+ -binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μ m , respectively. Le CPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H + -ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H + -ATPase and Le CPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of Le CPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the Le CPK1 N terminus was found to be required for plasma membrane targeting.

... 当假底物与蛋白激酶区结合时, CDPK处于未激活状态^[2] ...

... 钙离子结合区含有保守的钙离子结合EF手性结构域, 当钙离子与此结构结合后可以引起CDPK蛋白结构变化, 解除自抑, 从而激活CDPK^[2] ...

... 不同的CDPK蛋白具有不同的钙离子结合能力, 而对于单个CDPK而言, 钙离子是调节CDPK活性的重要因子^[2,10] ...

2002

6.555

0.0

S X

, Hrabak

E M

. An Arabidopsis calcium-dependent protein kinase is associated with the endoplasmic reticulum. Plant Physiol, 2002, 128: 1008-1021

Arabidopsis contains 34 genes that are predicted to encode calcium-dependent protein kinases (CDPKs). CDPK enzymatic activity previously has been detected in many locations in plant cells, including the cytosol, the cytoskeleton, and the membrane fraction. However, little is known about the subcellular locations of individual CDPKs or the mechanisms involved in targeting them to those locations. We investigated the subcellular location of one Arabidopsis CDPK, AtCPK2, in detail. Membrane-associated AtCPK2 did not partition with the plasma membrane in a two-phase system. Sucrose gradient fractionation of microsomes demonstrated that AtCPK2 was associated with the endoplasmic reticulum (ER). AtCPK2 does not contain transmembrane domains or known ER-targeting signals, but does have predicted amino-terminal acylation sites. AtCPK2 was myristoylated in a cell-free extract and myristoylation was prevented by converting the glycine at the proposed site of myristate attachment to alanine (G2A). In plants, the G2A mutation decreased AtCPK2 membrane association by approximately 50%. A recombinant protein, consisting of the first 10 amino acids of AtCPK2 fused to the amino-terminus of β-glucuronidase, was also targeted to the ER, indicating that the amino terminus of AtCPK2 can specify ER localization of a soluble protein. These results indicate that AtCPK2 is localized to the ER, that myristoylation is likely to be involved in the membrane association of AtCPK2, and that the amino terminal region of AtCPK2 is sufficient for correct membrane targeting.

... 区、自抑连接区、钙离子结合区, N端含有保守的MGXXXSXX区域, 能够被豆蔻酰化修饰, 从而将CDPK蛋白定位在细胞内膜系统上起重要的作用^{[1,3,4,5,6,7,8]} ...

... CDPK可以被定位于不同的亚细胞结构, 如细胞质基质、细胞核、细胞膜、内质网、液泡、线粒体、叶绿体、油体、过氧化物酶体等^{[3,5,7,16,18,19,20,21,22,23,24,25]} ...

2000

6.582

0.0

Martin

M L

, Busconi

. Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation. Plant J, 2000, 24: 429-435

... 区、自抑连接区、钙离子结合区, N端含有保守的MGXXXSXX区域, 能够被豆蔻酰化修饰, 从而将CDPK蛋白定位在细胞内膜系统上起重要的作用^{[1,3,4,5,6,7,8]} ...

... 除了豆蔻酰化, 有的CDPK还能被棕榈酰化^[4,9], 增强与膜质结合的能力 ...

2008

5.243

0.0

... 区、自抑连接区、钙离子结合区, N端含有保守的MGXXXSXX区域, 能够被豆蔻酰化修饰, 从而将CDPK蛋白定位在细胞内膜系统上起重要的作用^{[1,3,4,5,6,7,8]} ...

2004

6.555

0.0

... 区、自抑连接区、钙离子结合区, N端含有保守的MGXXXSXX区域, 能够被豆蔻酰化修饰, 从而将CDPK蛋白定位在细胞内膜系统上起重要的作用^{[1,3,4,5,6,7,8]} ...

2010

6.582

0.0

... 区、自抑连接区、钙离子结合区, N端含有保守的MGXXXSXX区域, 能够被豆蔻酰化修饰, 从而将CDPK蛋白定位在细胞内膜系统上起重要的作用^{[1,3,4,5,6,7,8]} ...

2011

3.582

0.0

... 区、自抑连接区、钙离子结合区, N端含有保守的MGXXXSXX区域, 能够被豆蔻酰化修饰, 从而将CDPK蛋白定位在细胞内膜系统上起重要的作用^{[1,3,4,5,6,7,8]} ...

2010

4.651

0.0

... 除了豆蔻酰化, 有的CDPK还能被棕榈酰化^[4,9], 增强与膜质结合的能力 ...

2004

4.651

0.0

... 不同的CDPK蛋白具有不同的钙离子结合能力, 而对于单个CDPK而言, 钙离子是调节CDPK活性的重要因子^[2,10] ...

2004

25.962

0.0

... CDPKs为多基因家族, 拟南芥中含有34个^[11,12,13], 水稻中含有31个^[14,15], 小麦中至少含有20个^[16], 玉米中含有40个^[17], 其他物种均包含多个CDPKs基因 ...

2005

0.0

... CDPKs为多基因家族, 拟南芥中含有34个^[11,12,13], 水稻中含有31个^[14,15], 小麦中至少含有20个^[16], 玉米中含有40个^[17], 其他物种均包含多个CDPKs基因 ...

2002

6.555

0.0

Cheng

S H

, Willmann

M R

, Chen

H C

, Sheen

. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol, 2002, 129: 469-485

In plants, numerous Ca 2+ -stimulated protein kinase activities occur through calcium-dependent protein kinases (CDPKs). These novel calcium sensors are likely to be crucial mediators of responses to diverse endogenous and environmental cues. However, the precise biological function(s) of most CDPKs remains elusive. The Arabidopsis genome is predicted to encode 34 different CDPKs. In this Update , we analyze the Arabidopsis CDPK gene family and review the expression, regulation, and possible functions of plant CDPKs. By combining emerging cellular and genomic technologies with genetic and biochemical approaches, the characterization of Arabidopsis CDPKs provides a valuable opportunity to understand the plant calcium-signaling network.

... CDPKs为多基因家族, 拟南芥中含有34个^[11,12,13], 水稻中含有31个^[14,15], 小麦中至少含有20个^[16], 玉米中含有40个^[17], 其他物种均包含多个CDPKs基因 ...

... 3 讨论克隆得到的SiCDPK1具有CDPK家族的典型特征^[13,34], 谷子CDPK基因家族成员含有的EF hand结构域数量不同(表2中EF hand结构域数由谷子基因组数据库注释得到), 以3个EF hand结构域为主, 显示了不同SiCDPK活性受钙离子结合的调节^[13,35] ...

... CDPK新的作用底物不断被发现, 然而大多数都是体外实验得到的^[13], 而在体内筛选CDPK作用底物目前还不是很成熟 ...

2005

4.134

0.0

... CDPKs为多基因家族, 拟南芥中含有34个^[11,12,13], 水稻中含有31个^[14,15], 小麦中至少含有20个^[16], 玉米中含有40个^[17], 其他物种均包含多个CDPKs基因 ...

2007

2.881

0.0

... CDPKs为多基因家族, 拟南芥中含有34个^[11,12,13], 水稻中含有31个^[14,15], 小麦中至少含有20个^[16], 玉米中含有40个^[17], 其他物种均包含多个CDPKs基因 ...

... 研究证实^{[15,16,27,28]}, CDPK能够响应不同的外界刺激, 包括ABA、冷、干旱、高盐、热、病原菌入侵和机械损伤等 ...

2008

3.518

0.0

A L

, Zhu

Y F

, Tan

X M

, Wang

, Wei

, Guo

H Z

, Zhang

Z L

, Chen

X B

, Zhao

G Y

, Kong

X Y

, Jia

J Z

, Mao

. Evolutionary and functional study of the CDPK gene family in wheat (Triticum aestivum L. ). Plant Mol Biol, 2008, 66: 429-443

Calcium-dependent protein kinases (CDPKs) are crucial sensors of calcium concentration changes in plant cells under diverse endogenous and environmental stimuli. We identified 20 CDPK genes from bread wheat and performed a comprehensive study on their structural, functional and evolutionary characteristics. Full-length cDNA sequences of 14 CDPKs were obtained using various approaches. Wheat CDPKs were found to be similar to their counterparts in rice in genomic structure, GC content, subcellular localization, and subgroup classification. Divergence time estimation of wheat CDPK gene pairs and wheat–rice orthologs suggested that most duplicated genes already existed in the common ancestor of wheat and rice. The number of CDPKs in diploid wheat genome was estimated to be at least 26, a number close to that in rice, Arabidopsis, and poplar. However, polymorphism among EST sequences uncovered transcripts of all three homoeologous alleles for 13 out of 20 CDPKs. Thus, the hexaploid wheat should have 2–3 fold more CDPK genes expressing in their cells than the diploid species. Wheat CDPK genes were found to respond to various biotic and abiotic stimuli, including cold, hydrogen peroxide (H 2 O 2 ), salt, drought, powdery mildew ( Blumeria graminis tritici , Bgt ), as well as phytohormones abscisic acid (ABA) and gibberellic acid (GA). Each CDPK gene often responded to multiple treatments, suggesting that wheat CDPKs are converging points for multiple signal transduction pathways. The current work represents the first comprehensive study of CDPK genes in bread wheat and provides a foundation for further functional study of this important gene family in Triticeae .

1.Institute of Crop Sciences, Chinese Academy of Agriculture Sciences (CAAS) National Key Facility of Crop Gene Resources and Genetic Improvement (NFCRI), Key Laboratory of Crop Germplasm & Biotechnology Beijing 100081 P.R. China 2.Sichuan Agricultural University Colleage of Life Sciences Ya-an Sichuan 625014 P.R. China 3.Chinese Academy of Agricultural Sciences Institute of Crop Sciences & National Key Facility for Crop Gene Resources and Genetic Improvement 12 Zhongguancun South Street Beijing 100081 P.R. China

... CDPKs为多基因家族, 拟南芥中含有34个^[11,12,13], 水稻中含有31个^[14,15], 小麦中至少含有20个^[16], 玉米中含有40个^[17], 其他物种均包含多个CDPKs基因 ...

... 研究证实^{[15,16,27,28]}, CDPK能够响应不同的外界刺激, 包括ABA、冷、干旱、高盐、热、病原菌入侵和机械损伤等 ...

2013

1.348

0.0

... CDPKs为多基因家族, 拟南芥中含有34个^[11,12,13], 水稻中含有31个^[14,15], 小麦中至少含有20个^[16], 玉米中含有40个^[17], 其他物种均包含多个CDPKs基因 ...

2009

6.582

0.0

Myers

, Romanowsky

S M

, Barron

Y D

, Garq

, Azuse

C L

, Curran

, Davis

R M

, Hatton

, Harmon

A C

, Harper

J F

. Calcium-dependent protein kinase regulate polarized tip growth in pollen tubes. Plant J, 2009, 59: 528-539

Calcium signals are critical for the regulation of polarized growth in many eukaryotic cells, including pollen tubes and neurons. In plants, the regulatory pathways that code and decode Ca 2+ signals are poorly understood. In Arabidopsis thaliana, genetic evidence presented here indicates that pollen tube tip growth involves the redundant activity of two Ca 2+ -dependent protein kinases (CPKs), isoforms CPK17 and -34. Both isoforms appear to target to the plasma membrane, as shown by imaging of CPK17–yellow fluorescent protein (YFP) and CPK34–YFP in growing pollen tubes. Segregation analyses from two independent sets of T-DNA insertion mutants indicate that a double disruption of CPK17 and -34 results in an approximately 350-fold reduction in pollen transmission efficiency. The near sterile phenotype of homozygous double mutants could be rescued through pollen expression of a CPK34–YFP fusion. In contrast, a transgene rescue was blocked by mutations engineered to disrupt the Ca 2+ -activation mechanism of CPK34 (CPK34–YFP–E465A,E500A), providing in vivo evidence linking Ca 2+ activation to a biological function of a CPK. While double mutant pollen tubes displayed normal morphology, relative growth rates for the most rapidly growing tubes were reduced by more than three-fold compared with wild type. In addition, while most mutant tubes appeared to grow far enough to reach ovules, the vast majority (>90%) still failed to locate and fertilize ovules. Together, these results provide genetic evidence that CPKs are essential to pollen fitness, and support a mechanistic model in which CPK17 and -34 transduce Ca 2+ signals to increase the rate of pollen tube tip growth and facilitate a response to tropism cues.

... CDPK可在植物所有的组织和器官中表达, 但有的CDPK组织特异性表达, 如AtCPK17和AtCPK34主要在成熟的花粉中表达并调节花粉管的伸长^[18] ...

2010

38.597

0.0

2010

6.555

0.0

2006

6.555

0.0

2007

9.251

0.0

2003

6.555

0.0

2005

6.555

0.0

Choi

H I

, Park

H J

, Park

J H

, Kim

, Im

M Y

, Seo

H H

, Kim

Y W

, Hwang

, Kim

S Y

. Arabidopsis calcium-dependent protein kinase AtCPK32 interacts with ABF4, a transcriptional regulator of abscisic acid-responsive gene expression, and modulates its activity. Plant Physiol, 2005, 139: 1750-1761

The phytohormone abscisic acid (ABA) regulates stress-responsive gene expression during vegetative growth. The ABA regulation of many genes is mediated by a subfamily of basic leucine zipper class transcription factors referred to as ABFs (i.e. ABF1–ABF4), whose transcriptional activity is induced by ABA. Here we show that a calcium-dependent protein kinase is involved in the ABA-dependent activation process. We carried out yeast two-hybrid screens to identify regulatory components of ABF4 function and isolated AtCPK32 as an ABF4-interacting protein. AtCPK32 has autophosphorylation activity and can phosphorylate ABF4 in vitro. Mutational analysis indicated that serine-110 of ABF4, which is highly conserved among ABF family members, may be phosphorylated by AtCPK32. The serine-110 residue is essential for ABF4-AtCPK32 interaction, and transient expression assay revealed that it is also required for the normal transcriptional function of ABF4. The expression patterns and subcellular localization of AtCPK32 are similar to those of ABF4. Furthermore, its overexpression affects both ABA sensitivity and the expression of a number of ABF4-regulated genes. Together, our data demonstrate that AtCPK32 is an ABA signaling component that regulates the ABA-responsive gene expression via ABF4.

... CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[24,29,30,31,32,33,34,35,36,37]}等 ...

2010

6.582

0.0

1991

0.0

... 从1991年, Harper等^[26]从大豆中克隆得到第一个CDPK-#cod#x003b1 ...

2001

9.822

0.0

... 研究证实^{[15,16,27,28]}, CDPK能够响应不同的外界刺激, 包括ABA、冷、干旱、高盐、热、病原菌入侵和机械损伤等 ...

2007

3.582

0.0

Wan

, Lin

, Mou

. Expression of rice Ca²+-dependent protein kinases (CDPKs) genes under different environmental stresses. FEBS Lett, 2007, 581: 1179-1189

Ca 2+ -dependent protein kinases (CDPKs) play an essential role in plant Ca 2+ -mediated signal transduction. Twenty-nine CDPK genes have been identified in the rice genome through a complete search of genome and full-length cDNA databases. Eight of them were reported previously to be inducible by different stress stimuli. Sequence comparison revealed that all 29 CDPK genes ( OsCPK1-29 ) contain multiple stress-responsive cis -elements in the promoter region (1 kb) upstream of genes. Analysis of the information extracted from the Rice Expression Database indicates that 11 of the CDPK genes are regulated by chilling temperature, dehydration, salt, rice blast infection and chitin treatment. RT-PCR and RNA gel blot hybridization were performed in this study to detect the expression 19 of the CDPK genes. Twelve CDPK genes exhibited cultivar- and tissue-specific expression; four CDPK genes ( OsCPK6 , OsCPK13 , OsCPK17 and OsCPK25 ) were induced by chilling temperature, dehydration and salt stresses in the rice seedlings. While OsCPK13 ( OsCDPK7 ) was already known to be inducible by chilling temperature and high salt, this is the first report that the other three genes are stress-regulated. OsCPK6 and OsCPK25 are up-regulated by dehydration and heat shock, respectively, while OsCPK17 is down-regulated by chilling temperature, dehydration and high salt stresses. Based on this evidence, rice CDPK genes may be important components in the signal transduction pathways for stress responses. Findings from this research are important for further dissecting mechanisms of stress response and functions of CDPK genes in rice.

... 研究证实^{[15,16,27,28]}, CDPK能够响应不同的外界刺激, 包括ABA、冷、干旱、高盐、热、病原菌入侵和机械损伤等 ...

... CDPK启动子含有响应不同胁迫的顺式作用元件^[28] ...

2010

4.354

0.0

... CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[24,29,30,31,32,33,34,35,36,37]}等 ...

2008

9.251

0.0

... CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[24,29,30,31,32,33,34,35,36,37]}等 ...

2010

9.251

0.0

... CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[24,29,30,31,32,33,34,35,36,37]}等 ...

2013

6.555

0.0

Schulz

, Herde

, Romeis

. Calcium-dependent protein kinases: hubs in plant stress signaling and development. Plant Physiol, 2013, 163: 523-530

Calcium ions (Ca 2+ ) have long been recognized as a major, conserved second messenger in eukaryotic signal transduction. In plants, the multigene family of calcium-dependent protein kinases (CDPKs) encodes structurally conserved, unimolecular calcium sensor/protein kinase effector proteins. CDPKs are ideal candidates for perceiving intracellular changes in Ca 2+ concentration and translating them into specific phosphorylation events to initiate further downstream signaling processes. Accordingly, CDPKs have been predominantly characterized in rapid abiotic stress and immune signaling responses, with little focus upon CDPK function in long-term adaptive processes or plant development. The long-awaited identification of in vivo/in planta CDPK targets, including their respective phosphorylation sites, allows the elucidation of CDPK function on a molecular and biochemical level. Moreover, the resolution of kinase-specific phosphorylation patterns within a target protein provides mechanistic evidence suggesting that CDPKs act as signaling hubs in plant stress signaling and development. The CDPK gene family comprises Ser/Thr protein kinases organized in four subgroups ( Harmon et al., 2000 ; Cheng et al., 2002 ); families of varying size have been characterized in Arabidopsis ( Arabidopsis thaliana ; 34), rice ( Oryza sativa ; 29), wheat ( Triticum aestivum ; 20), maize ( Zea mays ; 35), and poplar ( Populus trichocarpa ; 20; Harmon et al., 2000 ; Asano et al., 2005 ; Li et al., 2008 ; Ma et al., 2013 ; Zuo et al., 2013 ). CDPK s have a conserved molecular structure, consisting of a variable N-terminal domain, fused to a Ser/Thr kinase domain, and a CDPK activation domain ( CAD ). The CAD is formed from a pseudosubstrate region (sometimes referred to as the “inhibitory junction domain”) and a Ca 2+ -binding domain, highly homologous to calmodulin (also described as a calmodulin-like domain; Fig. 1A ). The calcium-binding domain of a canonical CDPK contains up to four elongation factor ( EF ) hand motifs. These are organized in N-terminal and C-terminal EF lobes and may display different Ca 2+ -binding affinities ( Harper et al., 2004 ; Harper and Harmon, 2005 ; Boudsocq and Sheen, 2013 ; Liese and Romeis, 2013 ).

... CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[24,29,30,31,32,33,34,35,36,37]}等 ...

... 近年来, CDPK在抗逆应答方面的研究取得了一定的进展^[1,32] ...

2012

0.657

0.0

Hubbard

K E

, Sieqel

R S

, Valerio

, Brand t

, Schroeder

J I

. Absicsic acid and CO₂ signalling via calcium sensitivity priming in guard cells, new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analysis. Ann Bot, 2012, 109: 5-17

Background Stomatal guard cells are the regulators of gas exchange between plants and the atmosphere. Ca 2+ -dependent and Ca 2+ -independent mechanisms function in these responses. Key stomatal regulation mechanisms, including plasma membrane and vacuolar ion channels have been identified and are regulated by the free cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ). Scope Here we show that CO 2 -induced stomatal closing is strongly impaired under conditions that prevent intracellular Ca 2+ elevations. Moreover, Ca 2+ oscillation-induced stomatal closing is partially impaired in knock-out mutations in several guard cell-expressed Ca 2+ -dependent protein kinases (CDPKs) here, including the cpk4cpk11 double and cpk10 mutants; however, abscisic acid-regulated stomatal movements remain relatively intact in the cpk4cpk11 and cpk10 mutants. We further discuss diverse studies of Ca 2+ signalling in guard cells, discuss apparent peculiarities, and pose novel open questions. The recently proposed Ca 2+ sensitivity priming model could account for many of the findings in the field. Recent research shows that the stomatal closing stimuli abscisic acid and CO 2 enhance the sensitivity of stomatal closing mechanisms to intracellular Ca 2+ , which has been termed ‘calcium sensitivity priming’. The genome of the reference plant Arabidopsis thaliana encodes for over 250 Ca 2+ -sensing proteins, giving rise to the question, how can specificity in Ca 2+ responses be achieved? Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca 2+ signal transduction, a topic of central interest in cell signalling research. In this article we further propose an individual stomatal tracking method for improved analyses of stimulus-regulated stomatal movements in Arabidopsis guard cells that reduces noise and increases fidelity in stimulus-regulated stomatal aperture responses ( Box 1 ). This method is recommended for stomatal response research, in parallel to previously adopted blind analyses, due to the relatively small and diverse sizes of stomatal apertures in the reference plant Arabidopsis thaliana .

... CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[24,29,30,31,32,33,34,35,36,37]}等 ...

... 同一物种中的不同CDPK具有不同的生理功能, 然而它们之间又存在着功能冗余的现象, 使得单独敲除或干扰单个CDPK并不能取得理想的试验效果, 双缺或三缺突变体成为研究的必要, 如cpk4cpk11、cpk3cpk6、cpk7cpk8cpk32^[33] ...

2000

11.808

0.0

Harmon

A C

, Gribskov

, Harper

J F

. CDPKs-a kinase for every Ca²+ signal?Trends Plant Sci, 2000, 5: 154-159

Numerous stimuli can alter the Ca 2+ concentration in the cytoplasm, a factor common to many physiological responses in plant and animal cells. Calcium-binding proteins decode information contained in the temporal and spatial patterns of these Ca 2+ signals and bring about changes in metabolism and gene expression. In addition to calmodulin, a calcium-binding protein found in all eukaryotes, plants contain a large family of calcium-binding regulatory protein kinases. Evidence is accumulating that these protein kinases participate in numerous aspects of plant growth and development.

... CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[24,29,30,31,32,33,34,35,36,37]}等 ...

2013

0.0

Liese

, Romeis

. Biochemical regulation of in vivo function of plant calcium-dependent protein kinase (CDPK). Biochim Biophy Acta, 2013, 1833: 1582-1589

Calcium (Ca 2 + ) is a major second messenger in plant signal transduction mediating stress- and developmental processes. Plant Ca 2 + -dependent protein kinases (CDPKs) are mono-molecular Ca 2 + -sensor/protein kinase effector proteins, which perceive Ca 2 + signals and translate them into protein phosphorylation and thus represent an ideal tool for signal transduction. This review focuses on recent developments in CDPK structural analysis and CDPK in vivo phosphorylation substrate identification. We discuss mechanisms implicated in the in vivo regulation of CDPK activity including Ca 2 + binding to the CDPK EF-hands, Ca 2 + -triggered intra-molecular conformation changes, and CDPK (auto)-phosphorylation. Moreover, we address regulation and integration into signaling cascades of selected members of the plant CDPK family, for which in vivo function and phosphorylation in abiotic and biotic stress signaling have been demonstrated. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

... CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[24,29,30,31,32,33,34,35,36,37]}等 ...

... AtCPK9、AtCPK33、AtCPK15、AtCPK21和AtCPK23基因, N端均能被豆蔻酰化和棕榈酰化^[35], SiCDPK1蛋白N端同样具有豆蔻酰化和棕榈酰化的修饰位点(图3) ...

2003

6.555

0.0

Hrabak

E M

, Chen

C W

, Gribskov

, Harper

J F

, Choi

J H

, Halford

, Kudla

, Luan

, Nimmo

H G

, Sussman

M R

, Thomas

, Walker-Simmons

, Zhu

J K

, Harmon

A C

. The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol, 2003, 132: 666-680

The CDPK-SnRK superfamily consists of seven types of serine-threonine protein kinases: calcium-dependent protein kinase (CDPKs), CDPK-related kinases (CRKs), phospho enol pyruvate carboxylase kinases (PPCKs), PEP carboxylase kinase-related kinases (PEPRKs), calmodulin-dependent protein kinases (CaMKs), calcium and calmodulin-dependent protein kinases (CCaMKs), and SnRKs. Within this superfamily, individual isoforms and subfamilies contain distinct regulatory domains, subcellular targeting information, and substrate specificities. Our analysis of the Arabidopsis genome identified 34 CDPKs, eight CRKs, two PPCKs, two PEPRKs, and 38 SnRKs. No definitive examples were found for a CCaMK similar to those previously identified in lily ( Lilium longiflorum ) and tobacco ( Nicotiana tabacum ) or for a CaMK similar to those in animals or yeast. CDPKs are present in plants and a specific subgroup of protists, but CRKs, PPCKs, PEPRKs, and two of the SnRK subgroups have been found only in plants. CDPKs and at least one SnRK have been implicated in decoding calcium signals in Arabidopsis. Analysis of intron placements supports the hypothesis that CDPKs, CRKs, PPCKs and PEPRKs have a common evolutionary origin; however there are no conserved intron positions between these kinases and the SnRK subgroup. CDPKs and SnRKs are found on all five Arabidopsis chromosomes. The presence of closely related kinases in regions of the genome known to have arisen by genome duplication indicates that these kinases probably arose by divergence from common ancestors. The PlantsP database provides a resource of continuously updated information on protein kinases from Arabidopsis and other plants.

... CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[24,29,30,31,32,33,34,35,36,37]}等 ...

... 植物的生长发育、对外界刺激的响应是一个复杂而综合的网络, 是CDPK、MAPK、CBL-CIPK、SnRK等通路^[36,37]相互作用、相互联系的结果 ...

2004

5.242

0.0

... CDPK的作用底物具有多样性, 包括酶、离子通道、水通道、转录因子(ABF4、RSG和HsfB2a)^{[24,29,30,31,32,33,34,35,36,37]}等 ...

... 植物的生长发育、对外界刺激的响应是一个复杂而综合的网络, 是CDPK、MAPK、CBL-CIPK、SnRK等通路^[36,37]相互作用、相互联系的结果 ...

2013

5.095

0.0

Lata

, Gupta

, Prasad

. Foxtail millet: a model crop for genetic and genomic studies in bioenergy grasses. Crit Rev Biotechnol, 2013, 33: 328-343

Foxtail millet is one of the oldest domesticated diploid C 4 Panicoid crops having a comparatively small genome size of approximately 515 Mb, short life cycle, and inbreeding nature. Its two species, Setaria italica (domesticated) and Setaria viridis (wild progenitor), have characteristics that classify them as excellent model systems to examine several aspects of architectural, evolutionary, and physiological importance in Panicoid grasses especially the biofuel crops such as switchgrass and napiergrass. Foxtail millet is a staple crop used extensively for food and fodder in parts of Asia and Africa. In its long history of cultivation, it has been adapted to arid and semi-arid areas of Asia, North Africa, South and North America. Foxtail millet has one of the largest collections of cultivated as well as wild-type germplasm rich with phenotypic variations and hence provides prospects for association mapping and allele-mining of elite and novel variants to be incorporated in crop improvement programs. Most of the foxtail millet accessions can be primarily abiotic stress tolerant particularly to drought and salinity, and therefore exploiting these agronomic traits can enhance its efficacy in marker-aided breeding as well as in genetic engineering for abiotic stress tolerance. In addition, the release of draft genome sequence of foxtail millet would be useful to the researchers worldwide in not only discerning the molecular basis of biomass production in biofuel crops and the methods to improve it, but also for the introgression of beneficial agronomically important characteristics in foxtail millet as well as in related Panicoid bioenergy grasses.

... 谷子与粮食作物亲缘关系较近且基因组较小, 已经逐步成为未来作物遗传改良育种理想的模式作物^[38], 将对未来禾谷类作物的品种改良、能源作物的遗传解析产生深远影响 ...

... 在抗旱方面, 国内学者对谷子SiDnaJ基因^[42]、干旱应答元件结合蛋白基因SiDREB^[42]、12-氧代植二烯酸还原酶基因SiOPR1^[43]、3-磷酸甘油醛脱氢酶基因GAPDH^[44]、磷脂酶D基因SiPLDa1^[45]、CBL家族^[46]、PP2C家族^[38]和ABF3基因^[47]等进行了研究 ...

2010

0.0

0.7323

... 国内, 在谷子抗锈基因、抗病基因、抗除草剂基因等方面取得了一定的进展, 如病程相关基因SiSGT1、NBS类抗病基因、抗锈基因RUS、抗锈基因RLr1、抗除草剂基因Srf^[39]、丝/苏氨酸蛋白激酶类抗病基因^[40]等 ...

2012

0.0

2007

0.0

0.951

崔润丽, 智慧, 王永芳, 李伟, 李海权, 黄占景, 刁现民. 谷子DnaJ蛋白基因的克隆. 华北农学报, 2007, 22(4): 9-13

Cui

R L

, Zhi

, Wang

Y F

, Li

H Q

, Huang

Z J

, Diao

X M

. Cloning of DnaJ-like protein gene from foxtail millet. Acta Agric Boreali-Sin, 2007, 22(4): 9-13 (in Chinese with English abstract)

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

崔润丽, 智慧, 王永芳, 李伟, 李海权, 黄占景, 刁现民. 谷子3-磷酸甘油醛脱氢酶基因的克隆与结构分析. 华北农学报, 2009, 24(3): 10-14

Cui

R L

, Zhi

, Wang

Y F

, Li

H Q

, Huang

Z J

, Diao

X M

. Cloning and structure analysis of Foxtail Millet APDH gene. Acta Agric Boreali-Sin, 2009, 24(3): 10-14 (in Chinese with English abstract)

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

Peng

Y L

, Zhang

J P

, Cao

G Y

. Overexpression of a PLDα1 gene from Setaria italica enhances the sensitivity of Arabidopsis to abscisic acid and improves its drought tolerance. Plant Cell Rep, 2010, 29: 793-802

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.

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

2013

0.0

1.667

赵晋锋, 余爱丽, 田岗, 杜艳伟, 郭二虎, 刁现民. 谷子CBL基因鉴定及其在干旱、高盐胁迫下的表达分析. 作物学报, 2013, 39: 360-367

Zhao

J F

, Yu

A L

, Tian

, Du

Y W

, Guo

E H

, Diao

X M

. Identification of CBL genes from foxtail millet (Setaria italica [L. ] Beauv. ) and its expression under drought and salt stresses. Acta Agron Sin, 2013, 39: 360-367 (in Chinese with English abstract)

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.

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

0.0

... 目前, 已完成谷子全基因组测序, 同时谷子分子标记(SSR、RFLP、AFLP、SRAP等)与功能基因组学研究^[48]也取得了一定的进展, 必将带动谷子研究的高潮 ...