,3Screening and identification of CKI1 upstream transcription regulators in Arabidopsis
Zhenning Liu1,2,3, Li Yuan2,4, Venkatesan Sundaresan2, Xiaolin Yu,3通讯作者:
编委: 张宪省
收稿日期:2018-11-21修回日期:2019-02-28网络出版日期:2019-05-20
| 基金资助: |
Received:2018-11-21Revised:2019-02-28Online:2019-05-20
| Fund supported: |
作者简介 About authors
刘振宁,博士,讲师,研究方向：植物生殖发育E-mail:
摘要
关键词：
Abstract
Keywords：
PDF (589KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文
本文引用格式
刘振宁, 袁黎, VenkatesanSundaresan, 余小林. 拟南芥CKI1基因上游转录调控因子筛选及鉴定[J]. 遗传, 2019, 41(5): 430-438 doi:10.16288/j.yczz.18-314
Zhenning Liu, Li Yuan, Venkatesan Sundaresan, Xiaolin Yu.
双组分信号系统(two-component system, TCS)最初是在研究大肠杆菌(Escherichia coli)氮调节蛋白系统时发现的[1]。最简单的双组分信号系统包括用于感应输入信号的组氨酸激酶(histidine kinase, HK)和用于调节输出信号的反应调节因子(response regulator, RR)两种元件,原核生物常以简单的双组分信号系统形式存在。在细菌、酵母、粘液菌和植物中,双组分信号系统进化为包括组氨酸激酶(histidine kinase, HK)、组氨酸磷酸转移蛋白(histidine phosphotransfer, HP)和反应调节因子(response regulator, RR)的多步磷酸化传递的双组分信号系统。
CKI1(AT2G47430)是第一个被发现的具有细胞分裂素受体特征的组氨酸激酶蛋白,在不添加细胞分裂素的培养基上,CKI1的过量表达能够导致特异的细胞分裂素应答反应[2,3]。CKI1蛋白主要定位于内质网和细胞质膜上[3,4,5]。Deng等[6]采用qRT-PCR方法研究了CKI1基因的表达模式,发现CKI1主要在拟南芥(Arabidopsis thaliana)的开放花、角果和花蕾中表达。Hejatko等[7]利用原位杂交技术和启动子GUS融合蛋白分析表明,CKI1在胚囊发育的FG1 (female gametophyte 1)时期到成熟胚囊期FG7时期均稳定表达,尤其在成熟胚囊期的中央细胞中表达量比较高,在卵细胞和助细胞中的表达量比较低。另外,CKI1在拟南芥花序的维管组织中也有明显的表达信号[8]。CKI1功能缺失突变体(cki1/+)植株的营养生长阶段没有明显的表型,但雌配子体表现为50%败育的表型。遗传学实验证据表明,CKI1功能缺失的突变体其T-DNA插入位点不能通过母本传递到子代中,因而也无法获得纯合的功能缺失突变体。对cki1/+突变体胚囊的细胞学观察表明,cki1/+突变体胚囊的败育发生在FG4时期向FG5时期的转换 期[4,6,7,9]。水稻(Oryza sativa)中CKI1同源基因(OsHK1)的功能缺失突变体也表现出雌配子体败育表型[10]。本课题组前期研究发现,CKI1通过作用于下游AHP2、AHP3和AHP5调控中央细胞的命运分化[5,11,12],但是目前仍不清楚该基因的转录调控机制。Dobisova等[13]对ProCKI1:uidA转基因株系进行EMS诱变,对CKI1上游转录调控因子进行筛选,发现CKI1基因的表达受到光敏色素蛋白的调控,并且CKI1启动子区域存在大量G-box和GATA motif等光周期响应元件,进一步通过酵母单杂交实验也证明PIF3和CCA1等受光敏色素蛋白调控的转录因子能够与CKI1启动子区域互作。但是筛选到的光敏色素蛋白相关转录因子的突变体并没有表现出cki1/+突变体的表型,由此推测,在胚囊发育 的阶段还存在其他转录调控因子能够调控CKI1的表达。
酵母单杂交是根据在细胞内DNA结合蛋白与DNA顺式作用元件结合调控报告基因表达的原理,用来鉴定调控基因转录的上游转录因子基因的一种有效方法[14]。随着酵母单杂交系统的不断发展和完善,该方法近年来已被广泛用于克隆和鉴定各种动植物的转录因子。本研究利用酵母单杂交技术对CKI1上游转录调控因子进行了筛选和鉴定,为进一步揭示CKI1基因的转录调控机制提供了新的数据及重要信息。
1 材料与方法
1.1 材料
拟南芥Col-0野生型为本实验室保存。拟南芥种子先用含20% Bleach和0.1% Tween 20的消毒液进行表面消毒10 min,然后使用灭菌的双蒸水清洗3~5遍,置于MS培养基上培养。待幼苗两周大的时候,将幼苗转移到带有培养基质(草炭:蛭石:珍珠岩为6:3:2)的穴盘中培养,培养条件保持16 h光照/8 h黑暗周期,温度为22℃,相对湿度为60%。1.2 不同片段长度CKI1启动子载体的构建
因为酵母基因组比植物基因组小得多,酵母启动子一般都比较短,如果整合到酵母基因组中的外源启动子太长,可能会影响目的蛋白和启动子中顺式作用元件结合的效率,而且会造成更多的假阳性[15,16]。因此,酵母单杂交一般选用几到几十bp的特定顺式作用元件或者尽可能短的有启动活性序列来构建诱饵表达载体。首先将CKI1基因启动子平均分成若干区段,分别设计引物序列进行扩增,引物序列见表1。然后通过SacⅠ和KpnⅠ双酶切克隆到H2B-eGFP- NOST-pCAMBIA1300中间载体中,构建带有相应启动子片段的promoter-H2B-eGFP-NOST-pCAMBIA1300表达载体(图1)。Table 1
表1
表1 本研究使用的引物信息
Table 1
| 引物名称 | 引物序列(5'→3') | 用途 |
|---|---|---|
| CKI1pro-SacⅠ-F | CGAGCTCGATTGGGACTAAAGTCTTGT | 扩增CKI1启动子 |
| CKI1pro-SacⅠ-F1 | CGAGCTCATTTAAACCGGAAAGTTACATTGGA | 扩增CKI1启动子 |
| CKI1pro-SacⅠ-F2 | CGAGCTCTTATCGTCAAACAAGTTTATATCAT | 扩增CKI1启动子 |
| CKI1pro-SacⅠ-F3 | CGAGCTCAGATGTAAGTTTAAGTCGTGAATTT | 扩增CKI1启动子 |
| CKI1pro-SacⅠ-F4 | CGAGCTCAATTATTCACTCAAATTCACAAAG | 扩增CKI1启动子 |
| CKI1pro-SacⅠ-F5 | CGAGCTCAAACTAATATCCGAATGCCTGAGAT | 扩增CKI1启动子 |
| CKI1pro-KpnⅠ-R1 | GGGGTACCAATGTGAAATCGAGTGAACCATAAA | 扩增CKI1启动子 |
| CKI1pro-KpnⅠ-R2 | GGGGTACCTCATATTATCTTCTTCCTCGGAGCA | 扩增CKI1启动子 |
| CKI1pro-KpnⅠ-R3 | GGGGTACCATCTCAGGCATTCGGATATTAGTTT | 扩增CKI1启动子 |
| CKI1pro-KpnⅠ-F5 | GGGGTACCAAACTAATATCCGAATGCCTGAGAT | 构建诱饵表达载体 |
| CKI1pro-XhoⅠ-F5 | CCGCTCGAGAAACTAATATCCGAATGCCTGAGAT | 构建诱饵表达载体 |
| CKI1pro-XhoⅠ-R2 | CCGCTCGAGTCATATTATCTTCTTCCTCGGAGCA | 构建诱饵表达载体 |
| pGADT7-F | CACTACAGGGATGTTTAATACCACT | 阳性克隆菌落PCR和测序 |
| pGADT7-R | CATGGCCAAGATTGAAACTTAGAGG | 阳性克隆菌落PCR |
新窗口打开|下载CSV
图1
新窗口打开|下载原图ZIP|生成PPT图1不同长度的CKI1基因启动子活性分析载体构建示意图
CKI1启动子区域包括4段5′UTR(粉色区域)、3段内含子(蓝色区域)和5′UTR上游序列(紫色区域)。F、F1、F2、F3、F4、F5和R1、R2、R3分别为上、下游引物。
Fig. 1Graphic representation of vector constructions for CKI1 promoter activities with different lengths
1.3 转基因株系的筛选和观察
经过测序验证后,构建成功的表达载体通过电转化法转入农杆菌AGL1菌株中,并通过浸花转化法转入到拟南芥野生植株中[17,18]。待转化的拟南芥植株种子成熟,收集和干燥种子,然后通过上述拟南芥种子表面消毒和播种方法,将种子在含有25 mg/L潮霉素B的MS培养基中进行筛选,最后将筛选获得的幼苗转移到培养基质中正常培养。对拟南芥胚囊中荧光信号进行观察。首先在载玻片上滴几滴灭菌的双蒸水,然后在解剖镜下将选取的雌蕊在双蒸水中使用解剖针解剖开,仅留下胚珠,去除多余的组织,盖上盖玻片,立即在荧光显微镜(Zeiss, Axioskop 2 plus)下观察拍照。
1.4 诱饵表达载体的构建与酵母遗传转化
诱饵表达载体的构建和酵母遗传转化参照酵母单杂交试剂盒(Clontech公司,美国)进行。为提高转录因子和顺式作用元件的识别率和结合率,在诱饵表达载体构建中,本研究使用3个串联重复的F5/R2片段。首先,使用SacⅠ和KpnⅠ双酶切将第1个F5/R2片段克隆到pAbAi载体中,然后使用KpnⅠ和XhoⅠ双酶切将第2个F5/R2片段克隆到pAbAi载体中,最后使用XhoⅠ单酶切将第3个F5/R2片段克隆到pAbAi载体中,完成pBait-AbAi载体的构建。每一步载体构建均经过了酶切和测序验证。然后将诱饵表达载体用BstBⅠ限制性内切酶切成线性质粒,转入YIH酵母菌株感受态中。最后选取5个单菌落,使用Matchmarker Insert Check PCR Mix 1试剂盒(Clontech公司,美国)进行菌落PCR鉴定含有诱饵表达载体的阳性菌落,选取阳性菌落备用。1.5 抑制诱饵菌株生长的最低AbA浓度的筛选
选取阳性菌落活化,然后用适量的0.9%NaCl重悬菌液,调制OD600值约为0.002。分别在含0 ng/mL、100 ng/mL、150 ng/mL、200 ng/mL、250 ng/mL、300 ng/mL、400 ng/mL、500 ng/mL、600 ng/mL、800 ng/mL和1000 ng/mL等不同浓度AbA的SD/-Ura固体培养基涂布100 μL菌液,置于30℃培养2~3 d,观察酵母菌落的生长情况。1.6 酵母单杂交文库的构建与筛选
Smyth等[19]根据拟南芥花发育不同阶段的形态特征将其划分为12个时期。本研究选取拟南芥花发育阶段在10~12时期的雌蕊提取RNA,然后将所提取的RNA按照Clontech公司单杂交文库试剂盒的操作步骤进行反转录,合成含有SMARTⅢ和CDSⅢ锚定末端的双链DNA (double strand cDNA, dscDNA)。RNA提取参照Invitrogen公司RNA提取试剂TRIzol产品操作说明书进行,RNA纯化参照QIAGEN公司RNA纯化试剂盒操作说明书进行。第一、第二链cDNA的合成、dscDNA的纯化参照Clontech公司酵母单杂交试剂盒进行。首先将制备好的3 μg dscDNA、6 μL pGADT7- Rec载体(经SmaⅠ线性化处理)和20 μL鲑鱼精子DNA (10 mg/mL)转入酵母感受态中,转化液涂布在150 mm直径含250 ng/mL浓度AbA的SD/-Leu固体培养基平板上,每个板涂布大约150 μL,同时将100 μL稀释100倍的转化液涂布在SD/-Leu固体培养基平板上作为对照。将平板置于30℃培养箱中倒置培养3~5 d,观察酵母菌落的生长情况并计算转化效率。
转化效率满足要求后,将线性化的融合表达载体pGADT7-Rec2和获得的SMARTⅢ和CDSⅢ锚定末端的dscDNA同时转入YIH Gold[Bait/AbAi]酵母感受态中,转化液涂布在含250 ng/mL浓度AbA的SD/-Leu固体培养基平板上,在30℃条件下培养3~5 d后观察统计培养基上的单菌落数量。
1.7 阳性克隆鉴定和测序
将能够在含250 ng/mL浓度AbA的SD/-Leu固体培养基上生长的单菌落进一步在含300 ng/mL浓度AbA的SD/-Leu固体培养基上进行划线,经过3次划线后均生长的克隆被认为是阳性克隆。选取阳性克隆,摇菌,提取酵母质粒。以酵母质粒为模板,使用Matchmaker Insert Check PCR Mix 2试剂盒(Clontech公司,美国)扩增目的片段,将凝胶回收的目的片段连接到T载体上,再进一步转化到大肠杆菌中,并选取阳性克隆进行测序。引物序列见表1。2 结果与分析
2.1 不同长度的CKI1基因启动子活性分析
根据前人对CKI1启动子活性的研究,FR2序列(1661 bp)是CKI1启动子启动活性所必需的[6,7],但对于酵母单杂交来说并不是特别理想的启动子长度。为进一步获得CKI1启动子尽可能短的功能序列,本研究在FR2启动子不同区间设计引物进行分段扩增。首先将FR2序列截短成F/R1 (1080 bp)、F/R2 (1661 bp)、F1/R1 (698 bp)、F1/R2 (1279 bp)、F2/R1 (488 bp)、F2/R2 (1069 bp)、F3/R1 (301 bp)和F3/R2 (882 bp)等8个片段(图1),并分别构建了promoter::H2B-eGFP表达载体,转化拟南芥,观察转基因阳性植株胚囊中的启动子活性。结果表明,只有F/R2、F1/R2、F2/R2和F3/R2这4段序列具有CKI1启动子活性。随后,将F3/R2这一段序列再次截短为F4/R3 (224 bp)、F4/R2 (581 bp)和F5/R2 (382 bp)等3个片段,分别构建promoter::H2B-eGFP表达载体,转化拟南芥,观察转基因阳性植株胚囊中的启动子活性。结果表明,只有F4/R2和F5/R2这两段序列具有CKI1启动子的活性(图2,A~P),其表达模式与CKI1启动子全长的表达模式一致[5]。研究结果表明,能够被转录因子识别的顺式作用元件位于F5/R2这个区间的内含子区域。进一步通过 PLACE和PlantCARE在线启动子预测工具对该内含子区域转录调控元件进行分析,结果表明该区域存在对光照、昼夜节律、厌氧感应,以及细胞分裂素和茉莉酸甲酯两种植物激素响应的元件(图3)。图2
新窗口打开|下载原图ZIP|生成PPT图2CKI1基因启动子片段F5/R2在胚囊中的启动活性
A和B：大孢子母细胞时期;C和D：功能大孢子时期~FG1时期;E和F：FG2~FG3时期;G和H：FG4时期;I和J：FG5时期;K和L：FG6时期;M~P：FG7时期。两个极核刚完成融合时,在助细胞和卵细胞中能检测到CKI1启动子活性,但随后助细胞和卵细胞中的信号消失,仅反足细胞和中央细胞中具有CKI1启动子活性。标尺为20 μm。
Fig. 2Promoter activities of the CKI1 promoter fragment F5/R2 in Arabidopsis embryo sacs
图3
新窗口打开|下载原图ZIP|生成PPT图3内含子F5/R2区域中转录调控元件的预测
Fig. 3Prediction of transcription regulation elements in F5/R2 intron regions
2.2 酵母单杂交文库的构建和筛选
2.2.1 诱饵表达载体的构建本研究选取F5/R2序列作为诱饵序列,并将3个串联重复的F5/R2片段克隆到pAbAi载体中,构建诱饵表达载体p3×F5/R2-AbAi。鉴于诱饵表达载体存在报告载体的本底表达,本文首先测试了含有诱饵表达载体的p3×F5/R2-AbAi的YIH Gold酵母菌株(YIH Gold [Bait/AbAi])在不同浓度AbA中生长的情况。与对照相比,AbA能明显地抑制含有诱饵表达载体的YIH Gold菌株的生长(图4,A~J)。在含有100 ng/mL、150 ng/mL和200 ng/mL浓度AbA的培养基中依然有少数菌落的生长,而250 ng/mL浓度以上的AbA能彻底抑制酵母的生长。因此,250 ng/mL浓度的AbA被确定为后续文库筛选最合适的浓度。
图4
新窗口打开|下载原图ZIP|生成PPT图4测试含诱饵表达载体的YIH Gold酵母菌株在含有不同浓度AbA的培养基上的生长情况
A：不含AbA;B：100 ng/mL AbA;C：150 ng/mL AbA;D：200 ng/mL AbA;E：250 ng/mL AbA;F：300 ng/mL AbA;G：400 ng/mL AbA;H：500 ng/mL AbA;I：600 ng/mL AbA;J：800 ng/mL AbA。
Fig. 4The growth of YIH Gold yeast strains was tested with bait expression vectors in medium with various concentrations of AbA
2.2.2 酵母单杂交文库的建立和筛选
dscDNA经检测满足建库要求,融合表达载体pGADT7-Rec2转入YIH Gold[Bait/AbAi]酵母感受态中的效率为1.125×106 cfu/μg,也高于1×105 cfu/μg的最低转化率要求。将含有250 ng/mL AbA的SD/-Leu培养基上筛选到的单克隆菌落接种到新的含有300 ng/mL AbA的SD/-Leu培养基上继续培养,此过程重复两次。经过3次划线接种均生长的克隆,被认为是阳性克隆。经过3次筛选最终获得226个阳性克隆。
2.2.3 目的序列的基因功能注释
将上述获得的226个阳性克隆进行测序,然后将测序结果与拟南芥基因数据库中的基因进行比对,去除低质量及冗余重复的序列,共获得66条可读序列。对这66条可读序列进行基因功能注释,发现8个具有DNA结合功能的蛋白,包括RING/ FYVE/PHD zinc finger家族蛋白、染色质组装修饰蛋白、组蛋白、AUX/IAA蛋白等(表2)。根据中央细胞转录组测序数据库[20],进一步分析了这8个候选基因在中央细胞中的表达情况,除了AT1G04550在中央细胞中不表达,其余7个基因在中央细胞中都有一定的表达量。因此,这7个基因可能是CKI1上游转录调控因子的候选基因。除了转录因子外,本研究还发现其中36个阳性克隆测序结果为一个功能未知基因—AT3G12965。该基因编码杂相RNA(miscellaneous RNA),是一类庞杂的小RNA分子,具有类酶催化、RNA加工和控制基因表达开关的作用[21]。目前对miscellaneous RNA研究极少,其具体的功能机制尚不清楚。
Table 2
表2
表2 目的基因的功能注释
Table 2
| 基因座 | 基因名称 | 所属基因家族 | 中央细胞中的转录组测序总reads数 |
|---|---|---|---|
| AT5G16680 | - | PHD-finger基因家族 | 434 |
| AT2G13370 | chromatin remodeling 5 | SWI2/SNF2基因家族 | 134 |
| AT5G10400 | HISTONE 3.1(HTR9) | 组蛋白基因家族 | 1205 |
| AT1G09200 | HISTONE 3.1 | 组蛋白基因家族 | 409 |
| AT4G21430 | B160 | JmjC基因家族 | 182 |
| AT5G57660 | ATCOL5(BBX6) | COL基因家族 | 38 |
| AT1G04550 | BDL(IAA12) | AUX/IAA基因家族 | 不表达 |
| AT4G20380 | LSD1 | LSD基因家族 | 350 |
新窗口打开|下载CSV
3 讨论
3.1 CKI1基因的内含子具有启动子功能
绝大多数真核生物的基因是断裂基因,由外显子和内含子间隔组成。外显子是真核生物基因中编码蛋白质的序列,而内含子为非编码蛋白序列。越来越多的研究发现,内含子并非垃圾序列,内含子对基因的表达存在调控作用。目前在很多基因的内含子中发现了基因表达的调控元件,内含子具有启动子的功能。Chang[22]发现猪MyHC基因内含子中存在重要的调控元件,可以调控转录的起始来增强基因的表达。Salgueino等[23]发现玉米泛素基因的第一个内含子能够启动gusA表达,具有类似于启动子的功能,对该内含子结构特征的进一步研究发现该启动子具有TATA box和CAAT box。谢先芝等[24]对番茄蛋白酶抑制剂Ⅱ基因的内含子TPI序列进行研究,发现TPI序列具有启动子活性,能够启动下游gusA基因的表达,TPI序列中也存在着类似于真核基因启动子TATA box的结构。小鼠Murrl基因第一个内含子中存在一个U2afbp-rs基因,该内含子能够启动U2afbp-rs基因的表达,具有类似启动子的功 能[25]。本研究通过对CKI1基因启动子的研究,发现CKI1基因第一个内含子也具有启动子活性,并且在该区域内鉴定到了能够响应光照、昼夜节律、厌氧反应与植物激素的转录调控元件。但具体哪些转录调控元件能够真正介导上游转录因子对CKI1基因的转录激活仍需要进一步研究。3.2 CKI1基因上游转录调控因子的候选基因
转录因子是调控真核生物基因表达的重要因子,典型的转录因子都有DNA结合区,不同的转录因子与靶基因启动子的顺式作用元件特异性结合并相 互作用,从而激活或抑制靶基因的表达。酵母单杂交技术是一种体内鉴定DNA与蛋白质相互作用的有效方法。该系统筛选到的蛋白质是在体内相对自然条件下有结合功能的蛋白质,比体外技术获得的结果更能体现真核基因表达调控的真实情况。通过酵母单杂交系统,本研究筛选出到7个在中央细胞中表达的转录因子基因。同时,考虑到酵母单杂交的假阳性问题,今后有必要对这7个候选基因进一步采用EMSA或者ChIP-qPCR验证酵母单杂交的筛选结果,以排除可能的假阳性。前期研究表明,CKI1基因功能缺失突变体表现出50%的雌配子体败育表型,由此推测,CKI1基因上游转录调控因子的突变体可能也具有雌配子体败育的表型。研究显示,只有AT1G09200[26]和AT4G20380[27]这两个基因的功能缺失突变体没有明显的雌配子发育异常表型,其余5个基因的突变体是否具有雌配子发育异常表型尚不清楚。因此,上述5个基因是今后需要重点关注的候选基因。3.3 转录调控因子基因的高通量筛选
酵母单杂交技术目前是鉴定DNA和蛋白质互作以及筛选转录因子常用的主流技术。利用特定序列的顺式作用元件或者一段启动子序列筛选目的转录因子需要构建cDNA文库,而cDNA文库存在基因冗余重复、基因序列不完整和非特异性的问题,容易产生很多的假阳性克隆和重复克隆,为后期的进一步鉴定和验证增加了大量的工作量。本研究利用酵母单杂交技术对拟南芥雌蕊的cDNA文库进行了筛选,获得了226个阳性克隆,仅获得66条可读序列,其中转录因子基因只有8个,上述阳性克隆存在着大量冗余重复的序列和低质量的序列。为提高酵母单杂交筛选转录因子的效率和特异性,转录调控因子基因的高通量筛选技术日益受到重视。Castrillo等[16]构建了包含1200个拟南芥转录因子的文库,在96孔板上通过传统的酵母配对的方式对脂肪酶基因LIP1的上游转录因子进行了高通量的筛选,找到了LIP1的上游调控基因。利用同样的方法,Ou等[28]构建了包含1589个拟南芥转录因子的文库,筛选到了8个能够调控Med25基因表达的转录因子。Pruneda-Paz等[15]构建了包含1956个拟南芥转录因子的文库,筛选到了能够结合到CCA1基因启动子区域的转录因子。这种转录因子高通量的筛选技术能够直接筛选到与目的DNA互作的转录因子,避免了cDNA文库筛选存在的过高的假阳性问题和冗余重复序列。这些拟南芥转录因子文库在ABRC网站上都可以订购获得,无疑对特定基因上游调控转录因子的筛选大有裨益。但上述转录因子筛选文库使用的是拟南芥各器官混合建库,针对器官或组织特异性的转录因子筛选存在一定的局限性,因而进一步完善针对特定发育时期或器官、组织特异性的转录因子文库具有重要的潜在应用价值。(责任编委: 张宪省)
参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
URLPMID:7961767 [本文引用: 1]
Abstract We examined the effects of mutations in glnL, encoding the signal-transducing kinase/phosphatase nitrogen regulator II (NRII), on the regulated phosphatase activity involved in nitrogen regulation. With wild-type NRII, this phosphatase activity was only observed in the presence of the signal transduction protein II (PII). Three different glnL mutations result in altered NRII proteins that had phosphatase activity in the absence of PII. The most active of these contained an alteration of the site of NRII autophosphorylation, histidine 139, to asparagine (H139N). The phosphatase activity of the NRII-H139N protein was further stimulated by the PII protein and by ATP. This suggests that the PII protein is not directly involved in a catalytic step of the regulated phosphatase activity but rather plays a regulatory role. We also measured the effect on the regulated phosphatase activity of alterations at conserved residues in the kinase/phosphatase domain of NRII and the effect of deleting the non-conserved N-terminal domain of NRII. For this we used fusion proteins containing the Escherichia coli maltose-binding protein (MBP) linked to the protein of interest. A protein consisting of MBP linked to wild-type NRII was a less active kinase than was wild-type NRII but in the presence of PII had wild-type phosphatase activity. A protein consisting of MBP linked to just the C-terminal domain of wild-type NRII had kinase activity but lacked phosphatase activity. Alterations at the highly conserved residues Asp-287, Gly-289, and Gly-291 in NRII affected both activities. A fusion of MBP to the NRII-H139N protein lacked kinase activity but had phosphatase activity in the absence of PII. Thus, while the kinase and phosphatase activities of NRII could be genetically separated, some of the highly conserved residues in the C-terminal domain of NRII (Asp-287, Gly-289, Gly-291) are apparently important for both activities.
URL [本文引用: 1]
[本文引用: 2]
URLPMID:3778002 [本文引用: 2]
In angiosperms, the egg cell forms within the multicellular, haploid female gametophyte. Female gametophyte and egg cell development occurs through a unique process in which a haploid spore initially undergoes several rounds of synchronous nuclear divisions without cytokinesis, resulting in a single cell containing multiple nuclei. The developing gametophyte then forms cell walls (cellularization) and the resulting cells differentiate to generate the egg cell and several accessory cells. The switch between free nuclear divisions and cellularization-differentiation occurs during developmental stage FG5 in Arabidopsis, and we refer to it as the FG5 transition. The molecular regulators that initiate the FG5 transition during female gametophyte development are unknown. In this study, we show using mutant analysis that two closely related MYB transcription factors, MYB64 and MYB119, act redundantly to promote this transition. MYB64 and MYB119 are expressed during the FG5 transition, and most myb64 myb119 double mutant gametophytes fail to initiate the FG5 transition, resulting in uncellularized gametophytes with supernumerary nuclei. Analysis of cell-specific markers in myb64 myb119 gametophytes that do cellularize suggests that gametophytic polarity and differentiation are also affected. We also show using multiple-mutant analysis that MYB119 expression is regulated by the histidine kinase CKI1, the primary activator of two-component signaling (TCS) during female gametophyte development. Our data establish a molecular pathway regulating the FG5 transition and implicates CKI1-dependent TCS in the promotion of cellularization, differentiation, and gamete specification during female gametogenesis.
URLPMID:27046830 [本文引用: 3]
Seeds of flowering plants contain embryos and nutritive endosperm arising from double fertilization of two dimorphic female gametes. Li et al. now show that the key to specification of only one female gamete for endosperm formation is the regulated localization of the CKI1 histidine kinase by expression control and protein translocation.
URLPMID:20363773 [本文引用: 3]
Cytokinin signaling is mediated by a multiple-step phosphorelay. Key components of the phosphorelay consist of the histidine kinase (HK)-type receptors, histidine phosphotransfer proteins (HP), and response regulators (RRs). Whereas overexpression of a nonreceptor-type HK gene CYTOKININ-INDEPENDENT1 (CKI1) activates cytokinin signaling by an unknown mechanism, mutations in CKI1 cause female gametophytic lethality. However, the function of CKI1 in cytokinin signaling remains unclear. Here, we characterize a mutant allele, cki1-8, that can be transmitted through female gametophytes with low frequency (鈭0.17%). We have recovered viable homozygous cki1-8 mutant plants that grow larger than wild-type plants, show defective megagametogenesis and rarely set enlarged seeds. We found that CKI1 acts upstream of AHP (Arabidopsis HP) genes, independently of cytokinin receptor genes. Consistently, an ahp1,2-2,3,4,5 quintuple mutant, which contains an ahp2-2 null mutant allele, exhibits severe defects in megagametogenesis, with a transmission efficiency of <3.45% through female gametophytes. Rarely recovered ahp1,2-2,3,4,5 quintuple mutants are seedling lethal. Finally, the female gametophytic lethal phenotype of cki1-5 (a null mutant) can be partially rescued by IPT8 or ARR1 (a type-B Arabidopsis RR) driven by a CKI1 promoter. These results define a genetic pathway consisting of CKI1, AHPs, and type-B ARRs in the regulation of female gametophyte development and vegetative growth.
[本文引用: 3]
[本文引用: 1]
URLPMID:12426401 [本文引用: 1]
Cytokinin-Independent 1 (CKI1) belongs to a group of putative plant histidine kinases whose members do not appear to act as ethylene receptors. The deduced protein structure, combined with the observation that Arabidopsis callus cultures overexpressing CKI1 exhibit a "cytokinin-independent" cell division and greening phenotype, led to the hypothesis that CKI1 is involved in cytokinin signaling, perhaps acting as a cytokinin receptor. To test the function of CKI1, we used a reverse-genetic approach to identify plants carrying T-DNA insertions in CKI1. Two independent alleles were identified, which produce the same developmental phenotype. Analyses of populations segregating for the cki1-5 or cki1-6 T-DNA insertion alleles failed to reveal any homozygous cki1 plants, indicating that the homozygous mutant condition was lethal. Based on segregation distortion, transmission studies, a microscopy-based examination of developing female gametophytes, and mRNA expression data, we suggest that CKI1 function is required for megagametophyte development. Our work with CKI1 mutants indicates that signal transduction by means of a His/Asp phosphorelay system may play an important and previously unsuspected role in female gametophyte development in Arabidopsis.
URLPMID:16919402 [本文引用: 1]
We identified four histidine kinase (HK) genes of a cytokinin receptor family, two histidine-containing phosphotransmitter (HPt) genes, thirteen A-type response regulator (RR) genes and six B-type RR genes in the rice genome. The HK genes ( OHK2, OHK3, OHK4 and OHK5 for Oryza sativa HK), the HPt genes ( OHP1 and OHP2 for O. sativa HPt) and the B-type RR genes ( ORR1, ORR2, ORR3, ORR4 and ORR6 for O. sativa RR) except one ( ORR5) showed expression in various organs. ORR5 was expressed in callus and flower. Three A-type RR genes ( OsRR4, OsRR9 and OsRR10 for O. sativa RR) showed cytokinin-induced expression, and three ( OsRR8, OsRR12 and OsRR13) showed expression in flower. We also identified two other genes named OHK1 and CHARK ( CHASE domain Receptor-like serine/threonine Kinase). OHK1 encodes an HK similar to Arabidopsis CKI1, which is involved in female gametophyte development. CHARK encodes a protein with an extracellular cytokinin-perceiving CHASE domain and a cytoplasmic serine/threonine kinase domain which are connected with a single transmembrane domain. The presence of all four gene families and CHARK in the rice genome suggests that a cytokinin signal is transduced by the phosphotransfer mechanism as is the case in Arabidopsis, and that rice may have an additional novel signalling pathway involving serine/threonine phosphorylation.
URLPMID:28633292 [本文引用: 1]
Only AHP2, AHP3, and AHP5 are downstream targets of CKI1 in regulating Arabidopsis female gametophyte development. Histidine phosphotransfer proteins (HPs) are key elements of the two-component signaling system, which act as a shuttle to transfer phosphorylation signals from histidine kinases (HKs) to response regulators (RRs).CYTOKININ INDEPENDENT 1(CKI1), a key regulator of central cell specification in the Arabidopsis female gametophyte, activates the cytokinin signaling pathway through the Arabidopsis histidine phosphotransfer proteins (AHPs). There are fiveHPgenes in Arabidopsis,AHP1 HP5, but it remains unknown whichAHPgenes act downstream ofCKI1in Arabidopsis female gametophyte development. Promoter activity analysis ofAHP1 HP5in embryo sacs revealedAHP1,AHP2,AHP3, andAHP5expression in the central cell. Phenotypic studies of various combinations ofahpmutants showed that triple mutations inAHP2,AHP3, andAHP5resulted in defective embryo sac development. Using cell-specific single and double markers in the female gametophyte, theahp2-2 ahp3 ahp5-2/+triple mutant ovules showed loss of central cell and antipodal cell fates and gain of egg cell or synergid cell attributes, resembling thecki1mutant phenotypes. These data suggest thatAHP2,AHP3, andAHP5are the major factors acting downstream ofCKI1in the two-component cytokinin signaling pathway to promote Arabidopsis female gametophyte development.
URL [本文引用: 1]
URLPMID:28292856 [本文引用: 1]
In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL (LPH) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 (CKI1), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 (HY1) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph/hy1-7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development.
[本文引用: 1]
URLPMID:4125603 [本文引用: 2]
Transcription factor (TF)-promoter interactions constitute the molecular wiring of transcriptional regulatory networks. In plants, discovery of the functional roles of TFs is limited by the redundant nature of network circuitry. Pruneda-Paz et al. now report the construction of a comprehensive and versatile Arabidopsis TF clone collection. Using this collection, they implement a high-throughput DNA binding assay suitable for investigating the regulatory landscape of each gene. The resources presented here provide a critical community platform for uncovering TF functions
[本文引用: 2]
[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
URLPMID:3266888 [本文引用: 1]
The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq). As a model we used the central cell of theArabidopsis thalianafemale gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed ade novoassembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms.
URL [本文引用: 1]
URLPMID:11129436 [本文引用: 1]
The porcine sarcomeric fast 2a myosin heavy chain (MyHC) gene was previously found to require a region extending 3′ from the transcriptional start site for high levels of expression. Here we established the existence of two novel opposing regulatory domains in intron 2. A positive regulatory element, defined to a 75bp region, resembles a TATA-less intronic promoter, with a consensus transcription initiation element. It can up-regulate its endogenous or a heterologous muscle promoter in a position specific manner, and on its own drive a reporter gene. In tandem with it is a dominant negative regulatory element, localised to a 81bp region, which can down-regulate its native gene and a heterologous muscle promoter. Bandshift and DNase I footprinting assays demonstrated that specific nuclear factors bound to both regulatory elements are distinctly different. Both elements appear to have no counterpart in intron 2 of the porcine fast 2x and 2b MyHC genes. Taken together, we demonstrate for the first time that a 5′-end terminal intron of a sarcomeric MyHC gene contains two critical regulatory domains, which may be involved in the complexity of temporo-spatial expression.
URLPMID:10809007 [本文引用: 1]
The promoterless maize ubiquitin first exon and intron fragment can drive gusA expression in immature tritordeum inflorescences and immature wheat scutella. In fluorescence assays, this fragment induces gusA expression in tritordeum inflorescences to 50 times higher than background. The activity of the complete promoter, exon and intron cassette was up to 20000-fold higher than background but the maize ubiquitin promoter in isolation had very low activity. A construct with the maize alcohol dehydrogenase first exon and intron had low activity, visible in histochemical assays. Both intron sequences have promoter-like features and in the ubiquitin intron there is a sequence homologous to the opaque-2-binding box. We suggest that the combination of these elements may explain the promoter activity detected in these introns.
URLMagsci [本文引用: 1]
<p>应用PCR技术, 从番茄中分离了具有一个内含子的蛋白酶抑制剂Ⅱ基因的核基因. 该内含子序列(TPI)的长度为109 bp, 两端存在着典型的GT/AG双核苷酸结构, 其AT碱基对的含量非常高, 约占核苷酸对总数的80.7%. DNA重组实验表明, TPI序列能够有效地促进报告基因<em>gusA </em>的表达水平, 而且这种促进作用与该序列的插入位置及方向均无关系, 呈现出明显的增强子特性. 另外, GUS酶组织化学染色、荧光检测以及凝胶阻滞实验等均证明TPI序列可能还具有启动子的活性. 番茄蛋白酶抑制剂Ⅱ基因的核基因序列在GenBank中的注册序号为 AY007240.</p>
URLMagsci [本文引用: 1]
<p>应用PCR技术, 从番茄中分离了具有一个内含子的蛋白酶抑制剂Ⅱ基因的核基因. 该内含子序列(TPI)的长度为109 bp, 两端存在着典型的GT/AG双核苷酸结构, 其AT碱基对的含量非常高, 约占核苷酸对总数的80.7%. DNA重组实验表明, TPI序列能够有效地促进报告基因<em>gusA </em>的表达水平, 而且这种促进作用与该序列的插入位置及方向均无关系, 呈现出明显的增强子特性. 另外, GUS酶组织化学染色、荧光检测以及凝胶阻滞实验等均证明TPI序列可能还具有启动子的活性. 番茄蛋白酶抑制剂Ⅱ基因的核基因序列在GenBank中的注册序号为 AY007240.</p>
URLPMID:303337 [本文引用: 1]
The mouse Murr1 gene contains an imprinted gene, U2af1-rs1, in its first intron. U2af1-rs1 shows paternal allele-specific expression and is transcribed in the direction opposite to that of the Murr1 gene. In contrast to a previous report of biallelic expression of Murr1 in neonatal mice, we have found that the maternal allele is expressed predominantly in the adult brain and also preferentially in other adult tissues. This maternal-predominant expression is not observed in embryonic and neonatal brains. In situ hybridization experiments that used the adult brain indicated that Murr1 gene was maternally expressed in neuronal cells in all regions of the brain. We analyzed the developmental change in the expression levels of both Murr1 and U2af1-rs1 in the brain and liver, and we propose that the maternal-predominant expression of Murr1 results from transcriptional interference of the gene by U2af1-rs1 through the Murr1 promoter region.
[本文引用: 1]
URLPMID:24471507 [本文引用: 1]
Abstract As obligate photoautotrophs, plants are inevitably exposed to ultraviolet (UV) radiation. Because of stratospheric ozone depletion, UV has become more and more dangerous to the biosphere. Therefore, it is important to understand UV perception and signal transduction in plants. In the present study, we show that LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) are antagonistic regulators of UV-C-induced programmed cell death (PCD) in A rabidopsis thaliana . This regulatory dependence is manifested by a complex deregulation of photosynthesis, reactive oxygen species homeostasis, antioxidative enzyme activity and UV-responsive genes expression. We also prove that a UV-C radiation episode triggers apoptotic-like morphological changes within the mesophyll cells. Interestingly, chloroplasts are the first organelles that show features of UV-C-induced damage, which may indicate their primary role in PCD development. Moreover, we show that A rabidopsis Bax inhibitor 1 (AtBI1), which has been described as a negative regulator of plant PCD, is involved in LSD1-dependent cell death in response to UV-C. Our results imply that LSD1 and EDS1 regulate processes extinguishing excessive energy, reactive oxygen species formation and subsequent PCD in response to different stresses related to impaired electron transport.
URLPMID:21343311 [本文引用: 1]
The activities of transcription factors (TFs) require interactions with specific DNA sequences and other regulatory proteins. To detect such interactions in Arabidopsis, we developed a high-throughput screening system with a Gateway-compatible Gal4-AD–TF library of 1589 Arabidopsis TFs, which can be easily screened by mating-based yeast-one-hybrid (Y1H) and yeast-two-hybrid (Y2H) methods. The efficiency of the system was validated by examining two well-characterized TF–DNA and TF–protein interactions: the CHE–CCA1 promoter interaction by Y1H and NPR1–TGAs interactions by Y2H. We used this system to identify eight TFs that interact with a Mediator subunit, Med25, a key regulator in JA signaling. We identified five TFs that interacted with the GCC-box cis-element in the promoter of PDF1.2, a downstream gene of Med25. We found that three of these TFs, all from the AP2-EREBP family, interact directly both with Med25 and the GCC-box of PDF1.2, suggesting that Med25 regulates PDF1.2 expression through these three TFs. These results demonstrate that this high-throughput Y1H/Y2H screening system is an efficient tool for studying transcriptional regulation networks in Arabidopsis. This system will be available for other Arabidopsis researchers, and thus it provides a vital resource for the Arabidopsis community.
