,1, 白晓璟1, 廉小平2, 张贺翠1, 罗绍兰1, 蒲敏1, 左同鸿1, 刘倩莹1, 朱利泉,1Cloning and Expression Analysis of BoSPx in Brassica oleracea
WANG YuKui,1, BAI XiaoJing1, LIAN XiaoPing2, ZHANG HeCui1, LUO ShaoLan1, PU Min1, ZUO TongHong1, LIU QianYing1, ZHU LiQuan,1通讯作者:
第一联系人:
收稿日期:2018-04-21接受日期:2018-07-21网络出版日期:2018-11-16
| 基金资助: |
Received:2018-04-21Accepted:2018-07-21Online:2018-11-16
摘要
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王玉奎, 白晓璟, 廉小平, 张贺翠, 罗绍兰, 蒲敏, 左同鸿, 刘倩莹, 朱利泉. 甘蓝BoSPx的克隆与表达分析[J]. 中国农业科学, 2018, 51(22): 4328-4338 doi:10.3864/j.issn.0578-1752.2018.22.011
WANG YuKui, BAI XiaoJing, LIAN XiaoPing, ZHANG HeCui, LUO ShaoLan, PU Min, ZUO TongHong, LIU QianYing, ZHU LiQuan.
0 引言
【研究意义】自交不亲和性（self-incompatibility,SI）是植物在长期的进化过程中为了防止自交衰退、保持遗传变异和促进杂种优势,形成的一种复杂而完善的重要遗传机制。芸薹属甘蓝属于典型的孢子体型自交不亲和植物（sporophytic self-incompatibility,SSI）,其分子机制主要集中于SI信号传导元件协同作用抑制自花花粉萌发或花粉管生长上[1],是基于磷酸化激活SI信号元件与泛素化降解花粉亲和因子的过程。GU等[2]以SRK（S-locus receptor kinase）胞内激酶域为诱饵,通过酵母双杂交技术从甘蓝型油菜柱头cDNA文库鉴定出与SRK结合的靶蛋白—ARC1（arm repeating containing）,ARC1在自交不亲和反应过程中起正调控作用。STONE等[3]和VANOOSTHUYSE等[4]利用反义抑制和RNAi技术分别抑制甘蓝型油菜（Brassica napus）和琴叶拟南芥（Arabidopsis lyrata）ARC1的表达,以及在甘蓝型油菜中过表达Exo70A1均只能部分打破材料的不亲和性。在拟南芥中,ARC1直系同源基因是假性遗传因子,向自交亲和的拟南芥中仅仅转入琴叶拟南芥的SCR-SRK也能表现出强自交不亲和性[5,6,7]。由此表明ARC1可能并非SRK唯一下游信号元件。因此,寻找其他参与调控自交不亲和反应的蛋白质元件的编码基因,对进行人工调控来改变芸薹属植物的自交育性和创造新的育种理论和方法具有重要意义。【前人研究进展】甘蓝SI信号传导过程还涉及很多其他功能分子[8],如钙和生长素响应蛋白等。钙与生长素在自交亲和性异花花粉萌发并穿越柱头的伸长过程中起关键作用[9,10]。IWANO等[11]通过双授粉试验和用单倍型SP11（SCR）处理共表达YC3.60和Sb-SRK的琴叶拟南芥和芜菁的乳突细胞,发现Ca2+浓度的增加是由于单倍型SCR与SRK相互特异性识别引起的,同时,乳突细胞中Ca2+浓度的增加最终导致花粉水化被抑制。迄今为止,胞质中Ca2+浓度的增加如何抑制花粉水化的分子机制尚不清楚。当雄蕊和雌蕊发育时,生长素（IAA）主要累积于花药和花萼中,在花药中,IAA随着花粉的发育不断积累,但其对雄蕊发育的影响仅局限于花丝变短。在授粉后相应器官的反应方面,在拟南芥成花诱导和花的发育过程中出现水溶性的IAA,而且IAA可以促进花粉在雌蕊上萌发和花粉管的生长[12]。梨自花授粉后柱头内的IAA含量变化较显著[13],IAA在烟草自交不亲和的花粉与柱头的识别过程中出现了相似的显著变化[14],而且自交不亲和性与柱头生长素含量之间存在负相关关系,表明柱头表面细胞内未知的生长素信号响应柱头对自花花粉的抑制作用[15]。【本研究切入点】除了ARC1,SRK（SI的关键元件）下游还可能存在其他未知信号元件。前期研究中,发现了一种花丝较短的SI型材料,其钙调素和生长素响应相关基因表达量明显较高,但其分子机理尚不清楚。【拟解决的关键问题】本研究拟通过克隆甘蓝BoSPx,并对其进行序列、启动子活性以及组织特异性表达分析,同时利用qRT-PCR检测自花授粉和异花授粉后的表达模式,探索该蛋白与信号传导元件SRK、ARC1等之间的相互作用,以期为甘蓝自交不亲和性的深入研究提供依据。1 材料与方法
1.1 试验材料及其处理
材料取于西南大学园艺园林学院十字花科蔬菜研究基地选育的甘蓝高代自交不亲和系A4和F1植株,2017年3月底对开花前1—2 d花蕾人工去雄,然后分别以F1和A4植株的花粉为父本,以A4柱头为母本进行授粉处理。用F1植株花粉给去雄的A4柱头进行0、15、30和60 min的异花授粉处理,简称CP（cross- pollination）组。用A4植株花粉给去雄A4柱头进行0、15、30和60 min的自花授粉处理,简称SP（self-pollination）组。授粉处理后用毛笔快速扫去柱头上的花粉,液氮速冻,-80℃保存备用。1.2 转录组测序及差异表达基因筛选
分别提取上述处理材料的RNA,送百迈克公司进行转录组测序,利用TopHat2软件将所得转录组数据与参考基因组序列比对[16],采用Trinity软件对样品数据进行Unigenes组装,通过Fold Change≥2且错误发现率FDR≤0.05来筛选候选差异基因。根据表达量的差异倍数、GO（Gene Ontology）富集分析以及KEGG（kyoto encyclopedia of gene and genomes）富集分析筛选表达差异显著的基因。1.3 目的基因的克隆
利用Primer Premer 5.0软件对上述筛选出的目的基因设计引物1300-GFP-F/1300-GFP-R（电子附表1）,以结球甘蓝A4柱头cDNA和gDNA为模板进行扩增,PCR反应体系为20 μL ddH2O、25 μL 2×PCR Mixster Mix混合酶、上/下游引物各1 μL和cDNA模板1 μL;反应程序为94℃ 3 min;94℃ 30 s,58℃ 30 s,72℃ 40 s,40个循环;72℃ 5 min。将目的片段与PCAMBIA1300载体连接,并转化。菌液PCR和双酶切鉴定阳性克隆,送至上海生物工程股份有限公司测序。1.4 生物信息学分析
通过NCBI网站在线查找目的基因的开放阅读框（ORF）、5′端非翻译区（5′UTR）和3′端非翻译区（3′UTR）。通过Bio-soft和DNAMAN8.0软件推导编码区的氨基酸序列;利用ExPASy-ProtParam tool（1.5 组织特异性表达分析
按照RNA提取试剂盒RNAprep pure Plant Kit说明书（天根）提取甘蓝柱头、叶片、花药等不同组织的RNA,并反转录合成第一链cDNA。以Actin3为内参基因,用Phanta Max Super-Fidelity DNA Polymerase 25 μL反应体系检测不同组织中目的基因的表达情况。反应程序为95℃ 3 min;95℃ 15 s,60℃ 15 s,72℃ 10 s,35个循环;72℃ 5 min。1.6 实时荧光定量PCR分析
提取不同授粉处理的柱头RNA,并进行反转录反应,合成单链cDNA,参考LIAO等[17]方法利用荧光定量PCR仪检测目的基因BoSPx的表达模式。以甘蓝Action3为内参基因,3次重复。利用2-ΔΔCT法分析BOSPx的相对表达量。1.7 亚细胞定位
将目的基因序列与绿色荧光基因GFP连接,构建PCAMBIA1300-BoSPx融合表达载体,以生长4周龄的拟南芥莲座叶为材料制备原生质体,利用瞬时转染法将PCAMBIA1300空载体和重组载体PCAMBIA1300-BoSPx分别转入原生质体,23℃黑暗培养16 h,利用激光共聚焦显微镜观察BoSPx蛋白的定位[18]。1.8 共转化酵母感受态细胞及酵母双杂互作的鉴定
参照MatchmakerTM Gold Yeast Two-Hybrid System操作说明,利用聚乙二醇/醋酸锂法（PEG/ LiAC）将构建好的重组质粒pGBKT7-BoSPx分别与pGADT7-SRK、pGADT7-ARC1、pGADT7- BoSAUR71和pGADT7-BoPID相应地转化到酵母感受态细胞Y2HGold。并设立阴性对照（pGADT7-T×pGBKT7- Lam）和阳性对照（pGADT7-T和pGBKT7- 53）,将共转化后的酵母感受态细胞涂布于营养缺陷型SD/-Leu -Trp固体培养基中,30℃倒置培养4 d。挑取白斑用无菌水稀释涂布于营养缺陷型SD/-Trp/- Leu/-His/-Ade固体培养基中。30℃倒置培养3—5 d,观察其生长情况。2 结果
2.1 根据转录组数据筛选差异表达基因
根据转录组文库中自花授粉与异花授粉不同时段表达量的差异,以P<0.05和|log2（fold change）|≥2作为显著差异表达阈值,筛选自花授粉不同时段差异显著的基因。对筛选出的自花授粉表达差异显著的基因进行GO和KEGG富集分析,发现1个与Ca2+和生长素信号途径相关的调控基因。该基因在未授粉时相对表达量为13,自花授粉15 min后达到最高,为221.98,而后急剧下调,其异花授粉表达量变化不明显（图1）,两者的表达量水平差异达4.5倍,此时正是SI相关基因表达时期。表明该基因可能是自交不亲和相关基因,命名为BoSPx。图1
新窗口打开|下载原图ZIP|生成PPT图1转录组数据分析BoSPx自花授粉和和异花授粉后的表达模式
SP：自花授粉;CP：异花授粉。下同
Fig. 1Transcriptome data were analyzed for expression patterns of BoSPx after self-pollination and cross- pollination
SP: Self-pollination; CP: Cross-pollination. The same as below
2.2 结球甘蓝BoSPx是不含内含子的单外显子基因
根据转录组数据获得BoSPx的部分编码区CDS序列,结合NCBI数据库和芸薹属数据库BLAST比对获得BoSPx全长cCDA序列。以甘蓝柱头cDNA和gDNA为模板,进行PCR扩增。获得BoSPx的cDNA和gDNA全长均为400 bp左右（图2）,经克隆测序与数据库获得的序列完全一致,该基因CDS序列为396 bp,说明该基因是不含内含子的单外显子基因。BoSPx编码一个具有131个氨基酸残基的蛋白质。图2
新窗口打开|下载原图ZIP|生成PPT图2甘蓝BoSPx的cDNA和gDNA序列的扩增
Fig. 2Amplification of BoSPx gene from cDNA and gDNA of the stigma of Brassica oleracea
2.3 BoSPx蛋白的结构分析
利用DNAMAN软件和ExPASy ProParam在线软件分析BoSPx蛋白的结构特征（图3-A）,在氨基酸残基组成上,Glu和Leu出现频率较高,分别占氨基酸总数的10.7%和9.9%,该蛋白相对分子质量为14.78 kD,理论等电点pI为4.54,带正电荷的氨基酸残基总数为25,带负电荷的氨基酸残基总数为16;不稳定指数为51.83,为短半衰期蛋白;总平均疏水指数（grand average of hyropathicity,GRAVY）为-0.378,推测BoSPx蛋白可能为亲水蛋白;BoSPx蛋白定位于细胞核和细胞质,该蛋白含有3个EF-Hand模体（第48—60、64—80和81—96位）,其两侧是由12个残基构成的α-螺旋结构域。通常组成EF-hand模体环的12个氨基酸残基以X、Y、Z、-X、-Y、-Z模式构成（图3-B）。分别位于第1、3、5、7、9、12位的氨基酸残基上,X、Y、Z、-X、-Y、-Z配体空间排列形成五边双锥结构参与金属配体配位,EF-Hand模体环中的第6个氨基酸残基在大多数情况下是Gly,位置1（X）、3(Y)和12（-Z）是最保守的[19,20,21]。1位上的氨基酸残基似乎总是疏水的,12位上的Glu或Asp提供了2个氧原子配位钙,EF-hand模体环以明显的几何图形容纳钙形成一个稳定的四螺旋束结构域。钙离子结合钙结合蛋白时可以诱导EF-hand模体环构象发生变化,导致靶蛋白激活或失活。图3
新窗口打开|下载原图ZIP|生成PPT图3甘蓝BoSPx蛋白的结构特征
A：BoSPx序列及其推导的氨基酸序列;B：同MARSDEN等[22]共有序列比对分析BoSPx的EF-Hand结构域特征,下划线部分为3个EF-hand结构域;C：BoSPx蛋白三维结构预测,黑色标记的为Ca2+结合的核心区域
Fig. 3Structural characteristics of BoSPx protein in Brassica oleracea
A:BoSPx gene sequence and deduced amino acid sequence; B: Analysis of EF-hand domain features of BoSPx using a consensus sequence analysis with MARSDEN et al[22]; C: BoSPx protein three-dimensional structure prediction
通过对BoSPx蛋白进行预测和分析,发现该蛋白二级结构主要由α螺旋（alpha helix,Hh）和loop（L）组成。其中Hh占58.02%,L占41.2%,为全α蛋白。BoSPx没有核定位信号（nuclear localization signals,NLS）,不存在跨膜螺旋区。BoSPx没有N-糖基化位点,其含有16个磷酸化位点,其磷酸化位点主要集中在第0—30、50—80、100—125位氨基酸残基处。BoSPx第101位甲硫氨酸疏水性最强,系数为1.8;第13位谷氨酰胺亲水性最强,系数为-2.667。疏水性分析显示该蛋白平均负峰值个数显著多于正峰数,说明它们亲水性较好,属于可溶性蛋白。利用Swiss-modle在线软件对BoSPx的三级结构进行预测,得到该蛋白的三维结构（图3-C）。
2.4 BoSPx蛋白系统进化分析
通过对BoSPx与其他物种氨基酸序列进行比对构建系统进化树（图4）。BoSPx与甘蓝型油菜BnSPx蛋白的同源性最近,其相似度达96%;与亚麻芥亲缘关系最远,为79%。按照亲缘关系远近的原则,选取同源性最近的4种植物进行多序列比对,并利用Smart预测可获得每个蛋白的EF-Hand模体,通过E-value值分析可知BoSPx蛋白的EF-hand模体具有相对较高的同源性。为了进一步比较各个蛋白之间的同源性,通过DNAMAN软件中的Multiple Sequence Alignment程序比较,并通过Graphic File输出结果,发现EF-Hand模体存在几个相对保守的氨基酸片段（图5）,不同功能的蛋白在此区域存在较高的同源性。图4
新窗口打开|下载原图ZIP|生成PPT图4BoSPx与其他物种BoSPx氨基酸序列的系统进化树
Fig. 4Phylogenetic tree of BoSPx and other species BoSPx amino acid sequence
图5
新窗口打开|下载原图ZIP|生成PPT图5甘蓝BoSPx与其他植物的同源蛋白氨基酸序列比对
BnSPx：XP_013685948;RsSPx：XP_018468047;BrSPx：XP_009127085;下划线代表EF-Hand结构域,同时也代表该蛋白在此区域保守性高
Fig. 5Multiple alignment of deduced amino acid sequence of BoSPx with homologous proteins of other species
BnSPx: XP_013685948; RsSPx: XP_018468047; BrSPx: XP_009127085; The underlined representation of the EF-Hand domain also indicates that the protein is highly conserved in this region
2.5 BoSPx蛋白功能分析
为了进一步解析该基因是否受自花授粉显著诱导,通过芸薹属数据库（http://brassicadb.org/brad/ index.php）及NCBI数据库选取BoSPx起始密码子上游2 000 bp的序列,利用PlantCARE在线软件分析该基因启动子的顺式作用元件,结果显示,BoSPx的启动子区含有多个元件,包括启动子基本元件TATA-box和CAAT-box等;还含有光、ABA、IAA和胁迫响应相关等10多种应答顺式元件（表1）,其中,IAA元件是一个TGACGTAA序列片段,位于起始密码子上游500 bp左右,推测BoSPx蛋白结合钙后,引起IAA浓度变化后,反馈调节BoSPx表达,形成一个正反馈过程,从而导致花器异常[23,24,25]。Table 1
表1
表1BoSPx上游调控区顺式作用元件
Table 1
| 相关功能预测 Associated putative function | 启动子顺式作用元件 Cis-elements in the promoter region |
|---|---|
| 脱落酸响应元件Abscisic acid responsiveness | ABRE |
| 光响应元件Light responsive element | ATCT-motif, Box4, G-Box, ACE, CATT-motif, GAG-motif Gap-box, TCCC-motif, I-box, MNF1, TCT- motif, as-2-box |
| 顺式作用调节元件Cis-acting regulatory element | ARE |
| 启动子和增强子区域Promoter and enhancer regions | CAAT-box |
| 热应激反应Heat stress responsiveness | HSE |
| 低温响应元件Low-temperature responsiveness | LTR |
| 干旱诱导反应元件Drought induced response element | MBS |
| 调控胚乳表达元件Regulatory element for endosperm expression | GCN4_motif, Skn-1_motif |
| 水杨酸响应元件Salicylic acid responsiveness | TCA-element |
| 茉莉酸响应元件MeJA-responsiveness | CGTCA-motif, TGACG-motif |
| 生长素响应元件Auxin-responsive element | TGA-element |
新窗口打开|下载CSV
2.6 BoSPx的表达分析
通过对萼片、花瓣、柱头、叶片和花药中BoSPx的表达进行RT-PCR分析（图6）。结果表明,BoSPx在开花前1—2 d的柱头、萼片、叶片、花药、花瓣中均有表达,且在柱头中表达量最高。通过对不同授粉处理后柱头中BoSPx的qRT-PCR分析（图7）,结果表明,BoSPx在自花授粉后表达趋势为先上调后下调,在异花授粉后下调。自花授粉15 min后相对表达量约为7.804,而异花授粉15 min后相对表达量约为0.223,两者相对表达量相差约35倍。此时正是SI相关基因表达时期,在自花授粉15 min时能够强烈诱导柱头BoSPx表达,说明BoSPx的表达对自交不亲和性产生反应。图6
新窗口打开|下载原图ZIP|生成PPT图6甘蓝BoSPx在不同组织中的RT-PCR分析
Fig. 6RT-PCR analysis of BoSPx gene in different tissues of Brassica oleracea
图7
新窗口打开|下载原图ZIP|生成PPT图7BoSPx在不同授粉处理的表达分析
Fig. 7Expression analysis of BoSPx in response to self and cross pollination
2.7 BoSPx的亚细胞定位
通过构建GFP-BoSPx融合表达载体,并转化拟南芥原生质体细胞。发现GFP蛋白分布在整个细胞中;而含GFP-BoSPx的蛋白主要分布在细胞质和细胞核中,表明BoSPx蛋白定位在细胞质和细胞核内（图8）。图8
新窗口打开|下载原图ZIP|生成PPT图8BoSPx蛋白在拟南芥原生质体中的亚细胞定位分析
Fig. 8Subcellular localization of BoSPx protein in Arabidopsis protoplasts
2.8 BoSPx与SRK、ARC1、BoSAUR71和BoPID之间的相互作用
将阴阳对照载体和重组载体组合（pGBKT7- BoSPx×(pGADT7-SRK、pGADT7-ARC1、pGADT7- SAUR71、pGADT7-PID)）共转化酵母感受态Y2HGold菌种中,分别涂布在SD/-Leu/-Trp（DDO）固体培养基上,30℃倒置培养3 d后,固体平板有菌落生成（图9）。通过PCR检测,均能扩增出目的条带,说明重组质粒同时转化进入酵母细胞中。图9
新窗口打开|下载原图ZIP|生成PPT图9共转化质粒在SD/-Leu/-Trp平板上的生长状况
A：阳性对照 Positive control(pGADT7-T+pGBKT7-53);B：阴性对照Negative control(pGADT7-T+ pGBKT7-Lam);C：pGADT7-SRK + pGBKT7-BoSPx;D：pGADT7-ARC1+pGBKT7-BoSPx;E：pGADT7-SAUR71+ pGBKT7-BoSPx;F：pGADT7-BoPID + pGBKT7-BoSPx
Fig. 9The Co-Transformed plasmid in yeast grown on SD/-Leu/-Trp plate
从DDO固体培养基上挑取阳性克隆悬浮于5 μL无菌水中,混匀后滴在营养缺陷型（SD/-Ade/-His/- Leu/-Trp）固体培养基上,30℃倒置培养3 d后,发现阴性对照不能生长,显红色（图10）;共转化质粒BoSPx/SRK、BoSPx/ARC1不能生长,无色;BoSPx/BoSAUR71、BoSPx/BoPID和阳性对照能够生长,显白色（表2）。说明BoSPx在酵母中与SRK（XP_013591187）和ARC1（XP_013636377）无相互作用,与BoPID（XP_013631900.1）和BoSAUR71（XP_013632246）之间存在相互作用[26]。
图10
新窗口打开|下载原图ZIP|生成PPT图10酵母融合株在SD/-Ade/-His/-Leu/-Trp平板上的相互作用检测
1：阳性对照Positive control(pGADT7-T+pGBKT7-53);2：阴性对照Negative control(pGADT7-T+pGBKT7-Lam);3：pGADT7-SRK+pGBKT7- BoSPx;4：pGADT7-ARC1+pGBKT7-BoSPx;5：pGADT7-SAUR71+ pGBKT7-BoSPx;6：pGADT7-BoPID+pGBKT7-BoSPx
Fig. 10Test protein-protein interactions on SD/-Ade/-His/- Leu/-Trp plate
Table 2
表2
表2质粒共转化酵母的相互作用分析
Table 2
| 编号 No. | 酵母菌种（质粒） Mating strain (plasmid) | 培养基 Yeast medium | 菌斑 Colony | 颜色 Color |
|---|---|---|---|---|
| 1 | Y2HGold（pGADT7-T×pGBKT7-53） | SD/-Leu/-Trp | 是Yes | 白色White |
| 2 | Y2HGold(pGADT7-T×pGBKT7-Lam) | SD/-Leu/-Trp | 是Yes | 红色Red |
| 3 | Y2HGold (pGADT7-SRK×pGBKT7-BoSPx) | SD/-Leu/-Trp | 是Yes | 红色Red |
| 4 | Y2HGold (pGADT7-ARC1×pGBKT7-BoSPx) | SD/-Leu/-Trp | 是Yes | 红色Red |
| 5 | Y2HGold (pGADT7-SAUR71×pGBKT7-BoSPx) | SD/-Leu/-Trp | 是Yes | 白色White |
| 6 | Y2HGold (pGADT7-BoPID×pGBKT7-BoSPx) | SD/-Leu/-Trp | 是Yes | 白色White |
| 7 | Y2HGold（pGADT7-T×pGBKT7-53） | SD/-Ade/-His/-Leu/-Trp | 是Yes | 白色White |
| 8 | Y2HGold(pGADT7-T×pGBKT7-Lam) | SD/-Ade/-His/-Leu/-Trp | 是Yes | 红色Red |
| 9 | Y2HGold (pGADT7-SRK×pGBKT7-BoSPx) | SD/-Ade/-His/-Leu/-Trp | 无No | 无No |
| 10 | Y2HGold (pGADT7-ARC1×pGBKT7-BoSPx) | SD/-Ade/-His/-Leu/-Trp | 无No | 无No |
| 11 | Y2HGold (pGADT7-SAUR71×pGBKT7-BoSPx) | SD/-Ade/-His/-Leu/-Trp | 是Yes | 白色White |
| 12 | Y2HGold (pGADT7-BoPID×pGBKT7-BoSPx) | SD/-Ade/-His/-Leu/-Trp | 是Yes | 白色White |
新窗口打开|下载CSV
3 讨论
3.1 BoSPx受自花授粉诱导显著表达
本研究基于以自交不亲和甘蓝柱头经不同授粉处理后的转录组数据,筛选出1个受自花授粉诱导显著上调表达的新基因BoSPx,荧光定量分析表明BoSPx在自花授粉15 min后的相对表达量约为7.804,约为未授粉表达量的7.8倍,而异花授粉15 min后的相对表达量约为0.223,在自花授粉15 min时,两者的表达量水平差异约达35倍,此时正是SI相关基因表达时期。如此表达量差异和表达时期,说明BoSPx是响应甘蓝自交不亲和反应的基因。BoSPx启动子中含有ABA、IAA和胁迫响应相关等10多种应答顺式元件,并且BoSPx在甘蓝柱头的表达量明显高于其他部位,表明其可能通过某些未知信号传导途径参与调控自交不亲和反应。3.2 BoSPx可能是响应生长素的钙结合蛋白
BoSPx含有3个EF-hand模体,其能结合Ca2+后发生蛋白构象的改变传递钙信号。THOMAS等[27]通过研究罂粟SI过程中发现Ca2+浓度增加使肌动蛋白解聚最终导致下游细胞程序性死亡（programmed cell death,PCD）。GEITMANN等[28]也证实了在虞美人（Papaver rhoeas）自交不亲和反应过程中,花粉管中细胞骨架发生改变和依赖第二信使Ca2+启动细胞程序性死亡,最终抑制花粉管生长。IWANO等[11]通过双授粉试验和制备原生质体转化试验证实了在芸薹属自交不亲和反应过程Ca2+浓度增加是必须的,并且足以导致花粉水化被抑制。同时也证明了乳突细胞中钙离子浓度的增加是由同种单倍型SCR和SRK特异性识别相互作用引起的。且在自花授粉6 min后Ca2+浓度达到最高。这与BoSPx自花授粉后表达量水平基本一致。推测BoSPx可能作为Ca2+结合蛋白元件参与了甘蓝自花授粉后花粉与柱头的相互作用[29,30]。另外,NASRALLAH等[25]通过过量表达ARF3（Auxin Response Factor 3）下调生长素反应促进十字花科植物SI反应中“自我”花粉的抑制。BoSPx蛋白定位于细胞核和细胞质,这种定位与ARC1相同,研究表明ARC1在SUMO前后分别定位于细胞质和细胞核内[31,32],在SI反应中作为S受体激酶底物时,其定位于细胞质内。BoSPx相同于ARC1在参与SI反应中的定位变化进一步说明BoSPx蛋白的表达很可能是对自交不亲和反应的响应。以上结果表明,我们克隆到的基因BoSPx的表达产物是Ca2+响应的蛋白元件,BoSPx可能通过调节生长素的合成或分布来参与SI反应。3.3 BoSPx可能参与未知的SI过程
本研究中通过酵母双杂试验证明BoSPx不能与SRK、ARC1互作,但能与BoPID（PINOID）和BoSAUR71相互作用,说明BoSPx没有直接参与SRK- ARC1-Exo70A1途径,而很可能参与了另外一条导致SI的未知途径[33,34]。在拟南芥中,PID（PINOID）蛋白丝氨酸/苏氨酸蛋白激酶是生长素信号传导的关键组分,可作为细胞生长素流出的正调控因子,也可作为生长素信号传导的负调控因子;BENJAMINS等[35]通过过量表达PID和喷洒钙离子抑制剂证实了钙内流和钙离子抑制剂能够在体内增强PID激酶活性。CHRISTENSEN等[36]发现PID（PINOID）能够促进PINs（auxin efflux carrier component）蛋白家族磷酸化从而调节PINs蛋白的极性定位。这些结果进一步明确,BoSPx可能通过调节生长素的分布来参与SI反应,从而可能导致花器异常发育。转录组数据中BoPID在自花授粉后有差异表达,未授粉表达量为39,自花授粉15 min为13,在授粉60 min上调表达为59,与BoSPx的表达模式正好相反,说明BoSPx与BoPID蛋白互作可能会改变BoPID蛋白的激酶活性,从而促进PIN蛋白磷酸化进一步改变PIN的极性定位。BoSPx可能通过结合Ca2+激活BoPID激酶活性,BoPID磷酸化BoPINs蛋白改变BoPINs蛋白的极性定位[37,38,39]。BoPINs蛋白定位的改变可能会使植物体内生长素浓度梯度分布紊乱,已有研究表明自交不亲和性与生长素含量之间存在负相关关系[15]。进一步表明BoSPx蛋白可能通过调控BoPID磷酸化BoPINs家族蛋白使柱头乳突细胞内生长素浓度分布紊乱,导致花器异常甚至胚胎发育异常,不能正常授粉受精,最终导致不亲和反应[12,25]。BoSAUR71（small auxin-up RNA71）是一种生长素早期应答基因。SAUR蛋白能够体外与钙调蛋白结合,这表明它可能是钙调蛋白和生长素信号之间的桥梁和联系分子[40],生长素在对花器发育发挥生理作用的同时,又可能在分子水平上通过IAA响应元件,调节BoSPx的表达,如此形成反馈作用。KANT等[41]研究证明了在水稻中SAUR39在生长素的合成和运输中起着负调节作用。BoSPx通过与生长素家族蛋白SAUR71形成复合物可能负调控生长素的浓度和分布,既可能影响花柱或花粉管生长,造成花器异常,不能正常受精,又可能通过IAA响应元件,调节BoSPx的表达,形成反馈作用。这可能是BoSPx参与的甘蓝对钙和SI的共响应模式。4 结论
BoSPx受SI的自花授粉诱导显著表达,可能是响应生长素的钙结合蛋白,对SI和钙产生共响应;BoSPx不能与SRK、ARC1互作,但能与BoPID和BoSAUR71相互作用,BoSPx可能参与到SRK-ARC1-ExO70A1途径以外的未知信号通路中。（责任编辑 李莉）
参考文献 原文顺序
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被引期刊影响因子
DOI:10.1093/jxb/erq021URLPMID:20181663 [本文引用: 1]
Abstract In the Brassicaceae, the acceptance of compatible pollen and the rejection of self-incompatible pollen by the pistil involves complex molecular communication systems between the pollen grain and the female reproductive structures. Preference towards species related-pollen combined with self-recognition systems, function to select the most desirable pollen; and thus, increase the plant's chances for the maximum number of successful fertilizations and vigorous offspring. The Brassicaceae is an ideal group for studying pollen-pistil interactions as this family includes a diverse group of agriculturally relevant crops as well as several excellent model organisms for studying both compatible and self-incompatible pollinations. This review will describe the cellular systems in the pistil that guide the post-pollination events, from pollen capture on the stigmatic papillae to pollen tube guidance to the ovule, with the final release of the sperm cells to effect fertilization. The interplay of other recognition systems, such as the self-incompatibility response and interspecific interactions, on regulating post-pollination events and selecting for compatible pollen-pistil interactions will also be explored.
DOI:10.1073/pnas.95.1.382URLPMID:9419384 [本文引用: 1]
Screening of a yeast two-hybrid library for proteins that interact with the kinase domain of an S-locus receptor kinase (SRK) resulted in the isolation of a plant protein called ARC1 (Arm Repeat Containing). This interaction was mediated by the C-terminal region of ARC1 in which five arm repeat units were identified. Using the yeast two-hybrid system and in vitro binding assays, ARC1 was found to interact specifically with the kinase domains from SRK-910 and SRK-A14 but failed to interact with kinase domains from two different Arabidopsis receptor-like kinases. In addition, treatment with a protein phosphatase or the use of a kinase-inactive mutant reduced or abolished the binding of ARC1 to the SRK-910 kinase domain, indicating that the interaction was phosphorylation dependent. Lastly, RNA blot analysis revealed that the expression of ARC1 is restricted to the stigma, the site of the self-incompatibility response.
DOI:10.1105/tpc.009845URLPMID:12671085 [本文引用: 1]
Abstract ARC1 is a novel U-box protein required in the Brassica pistil for the rejection of self-incompatible pollen; it functions downstream of the S receptor kinase (SRK). Here, we show that ARC1 has E3 ubiquitin ligase activity and contains several motifs that influence its subcellular localization. ARC1 can shuttle between the nucleus, cytosol, and proteasome/COP9 signalosome (CSN) when expressed in tobacco BY-2 suspension-cultured cells. However, ARC1 localization to the proteasome/CSN occurs only in the presence of an active SRK. In the pistil, ubiquitinated protein levels increase specifically with incompatible pollinations, but they do not change in ARC1 antisense-suppressed pistils. In addition, inhibition of the proteasomal proteolytic activity disrupts the self-incompatibility response. We propose that ARC1 promotes the ubiquitination and proteasomal degradation of compatibility factors in the pistil, which in turn leads to pollen rejection.
[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
[M]
[本文引用: 1]
DOI:10.1007/978-3-540-68486-2_8URL [本文引用: 1]
ABSTRACT The sporophytic self-incompatibility (SI) system, which operates in the Brassicaceae is primarily controlled by two multi-allelic loci, encoding the SP11/SCR pollen ligand, and the stigma-specific S Receptor Kinase (SRK). Haplotypespecific recognition of SP11/SCR by SRK triggers the activation of SRK's intracellular kinase domain. This is predicted to cause the phosphorylation-mediated recruitment of the ARC1 E3 ubiquitin ligase. ARC1 is predicted to inhibit its substrate by ubiquitination, and recentwork suggests that Exo70A1 is a target of ARC1. Exo70A1 is predicted to regulate targeted secretion and is required in the stigma to promote compatible pollen hydration, germination and pollen tube growth. SRK is also known to interact with a number of other proteins, including the M locus protein kinase (MLPK), which may function with SRK to co-regulate ARC1. Here we review our present knowledge of the various cellular components that act in concert during the SI response.We also discuss the cellular mechanisms of how SI can cause pollen rejection through the inhibition of stigmatic factors that promote compatible pollen acceptance.
DOI:10.1104/pp.108.123380URLPMID:18552232 [本文引用: 1]
The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.
[本文引用: 2]
[本文引用: 2]
DOI:10.3969/j.issn.1000-1573.2007.01.008URL [本文引用: 1]
以自交不亲和梨品种鸭梨及其亲和变异品种金坠梨为材料,测定了鸭梨和金坠梨自花授粉、鸭梨异花授粉后花柱激素含量的变化,以期阐明内源激素与自交不亲和性的关系。结果表明：金坠梨大气球期花柱中促进生长的激素IAA、GA3、ZR含量均极显著高于鸭梨,而ABA含量极显著低于鸭梨。2个品种自花授粉后72 h内,花柱内IAA、ZR含量呈下降趋势;在授粉后24 h时内GA3含量下降,随后上升;金坠梨花柱ABA含量授粉后24h内上升,然后下降;鸭梨花柱ABA含量变化在自花授粉24 h内处于下降趋势,然后上升;鸭梨异花授粉的花柱内激素变化与自花授粉的不同,授粉24 h后花柱内IAA、GA3、ZR含量与金坠梨自花授粉的相近。
DOI:10.3969/j.issn.1000-1573.2007.01.008URL [本文引用: 1]
以自交不亲和梨品种鸭梨及其亲和变异品种金坠梨为材料,测定了鸭梨和金坠梨自花授粉、鸭梨异花授粉后花柱激素含量的变化,以期阐明内源激素与自交不亲和性的关系。结果表明：金坠梨大气球期花柱中促进生长的激素IAA、GA3、ZR含量均极显著高于鸭梨,而ABA含量极显著低于鸭梨。2个品种自花授粉后72 h内,花柱内IAA、ZR含量呈下降趋势;在授粉后24 h时内GA3含量下降,随后上升;金坠梨花柱ABA含量授粉后24h内上升,然后下降;鸭梨花柱ABA含量变化在自花授粉24 h内处于下降趋势,然后上升;鸭梨异花授粉的花柱内激素变化与自花授粉的不同,授粉24 h后花柱内IAA、GA3、ZR含量与金坠梨自花授粉的相近。
DOI:10.1023/A:1016355824539URL [本文引用: 1]
Transformation of Nicotiana silvestris Spegar et Comes plants by the ipt or iaaM + iaaH genes changed the hormonal status of plant reproductive organs. The total content of cytokinins and ABA increased, whereas IAA content in the pistils and anthers of the ipt -plants did not change. Reduced fertility of the ipt plants correlated with an elevated cytokinin and ABA contents of their reproductive organs. Pollen tubes of these plants showed defective growth in pistils in situ , and ovaries manifested a low metabolic activity. The transgenic ( iaaM + iaaH )-plants were characterized by an elevated IAA content and reduced ABA content, whereas the total content of cytokinins did not change. The fertility of these plants did not differ from that in the wild type.
DOI:10.1111/ppl.2008.134.issue-1URL [本文引用: 2]
DOI:10.1186/gb-2013-14-4-r36URL [本文引用: 1]
[本文引用: 1]
DOI:10.3864/j.issn.0578-1752.2017.12.019URL [本文引用: 1]
【目的】高温胁迫已经成为威胁作物生长发育的主要非生物胁迫因素之一,转录因子在植物非生物胁迫响应中起着重要作用。通过对大豆锌指转录因子基因Gm Di19-5在高温胁迫下的响应和功能鉴定,以及利用酵母双杂交技术从大豆c DNA文库筛选与其互作的候选蛋白,研究Gm Di19-5对高温胁迫的响应机制。【方法】以大豆c DNA为模板,利用实时荧光定量PCR检测Gm Di19-5在高温处理不同时间段的表达模式;通过Plant CARE和PLACE数据库预测Gm Di19-5启动子元件,并对Gm Di19-5启动子转基因拟南芥在高温处理下进行的组织化学染色,分析Gm Di19-5启动子在高温胁迫下的活性;构建p GBKT7-Gm Di19-5诱饵载体,验证自激活活性;利用酵母双杂交技术,以p GBKT7-Gm Di19-5为诱饵筛选大豆c DNA文库,筛选与其互作的候选蛋白;进一步用酵母双杂交系统验证Gm Di19-5与候选蛋白的互作;利用实时荧光定量PCR检测候选蛋白基因在对高温处理的响应情况;构建融合表达载体,用GFP-Gm Di19-5融合表达载体转化拟南芥原生质体,检测Gm Di19-5蛋白的亚细胞定位情况。【结果】实时荧光定量PCR结果显示,Gm Di19-5在高温胁迫下上调表达;启动子元件分析表明,Gm Di19-5包含多种与胁迫应答相关的顺式作用元件,包括热响应元件HSE;组织化学染色分析发现,经高温处理后,过表达拟南芥幼苗的地上部分和地下部分均有报告基因的表达;亚细胞定位结果表明,Gm Di19-5蛋白定位于拟南芥原生质体的细胞核中;通过酵母双杂交技术筛选到了与Gm Di19-5互作的候选蛋白,试验进一步验证了Gm Di19-5与Gm Dna J在酵母细胞中互作;另外,实时荧光定量PCR结果显示,互作蛋白基因Gm Dna J受高温胁迫诱导表达。【结论】Gm Di19-5受高温诱导表达,Gm Di19-5可能与Gm Dna J互作,表明Gm Di19-5功能的发挥可能需要Gm Dna J的参与。
DOI:10.3864/j.issn.0578-1752.2017.12.019URL [本文引用: 1]
【目的】高温胁迫已经成为威胁作物生长发育的主要非生物胁迫因素之一,转录因子在植物非生物胁迫响应中起着重要作用。通过对大豆锌指转录因子基因Gm Di19-5在高温胁迫下的响应和功能鉴定,以及利用酵母双杂交技术从大豆c DNA文库筛选与其互作的候选蛋白,研究Gm Di19-5对高温胁迫的响应机制。【方法】以大豆c DNA为模板,利用实时荧光定量PCR检测Gm Di19-5在高温处理不同时间段的表达模式;通过Plant CARE和PLACE数据库预测Gm Di19-5启动子元件,并对Gm Di19-5启动子转基因拟南芥在高温处理下进行的组织化学染色,分析Gm Di19-5启动子在高温胁迫下的活性;构建p GBKT7-Gm Di19-5诱饵载体,验证自激活活性;利用酵母双杂交技术,以p GBKT7-Gm Di19-5为诱饵筛选大豆c DNA文库,筛选与其互作的候选蛋白;进一步用酵母双杂交系统验证Gm Di19-5与候选蛋白的互作;利用实时荧光定量PCR检测候选蛋白基因在对高温处理的响应情况;构建融合表达载体,用GFP-Gm Di19-5融合表达载体转化拟南芥原生质体,检测Gm Di19-5蛋白的亚细胞定位情况。【结果】实时荧光定量PCR结果显示,Gm Di19-5在高温胁迫下上调表达;启动子元件分析表明,Gm Di19-5包含多种与胁迫应答相关的顺式作用元件,包括热响应元件HSE;组织化学染色分析发现,经高温处理后,过表达拟南芥幼苗的地上部分和地下部分均有报告基因的表达;亚细胞定位结果表明,Gm Di19-5蛋白定位于拟南芥原生质体的细胞核中;通过酵母双杂交技术筛选到了与Gm Di19-5互作的候选蛋白,试验进一步验证了Gm Di19-5与Gm Dna J在酵母细胞中互作;另外,实时荧光定量PCR结果显示,互作蛋白基因Gm Dna J受高温胁迫诱导表达。【结论】Gm Di19-5受高温诱导表达,Gm Di19-5可能与Gm Dna J互作,表明Gm Di19-5功能的发挥可能需要Gm Dna J的参与。
URL [本文引用: 1]
自交不亲和性是甘蓝在长期进化过程中形成的防止自交衰败、促进杂交优势的一种复杂而完善的遗传机制。克隆自交不亲和性相关基因对甘蓝自交不亲和性的深入研究和利用有重要意义。本研究通过挖掘0~60 min自花和异花授粉的甘蓝柱头转录组数据,筛选到一个受自花授粉诱导上调表达的基因,命名为BoSPI。BoSPI开放阅读框534bp,编码177个氨基酸,理论等电点为4.21,不包含信号肽和跨膜区,含有4个保守的EF-hand结构域。BoSPI基因起始密码子上游2000 bp的核苷酸序列中含有真菌诱导响应、代谢调节以及器官形成等应答元件。BoSPI基因在大肠杆菌中可诱导表达为17 k D的蛋白。BoSPI在柱头中表达量最高,在花瓣、萼片、叶片、雄蕊表达量较低。BoSPI蛋白被定位在细胞膜和细胞质。自花授粉30 min后对BoSPI基因的诱导表达显著增强。表明BoSPI参与了柱头响应自花花粉刺激的分子过程,可能是实现甘蓝自交不亲和性相关的某种新功能基因。
URL [本文引用: 1]
自交不亲和性是甘蓝在长期进化过程中形成的防止自交衰败、促进杂交优势的一种复杂而完善的遗传机制。克隆自交不亲和性相关基因对甘蓝自交不亲和性的深入研究和利用有重要意义。本研究通过挖掘0~60 min自花和异花授粉的甘蓝柱头转录组数据,筛选到一个受自花授粉诱导上调表达的基因,命名为BoSPI。BoSPI开放阅读框534bp,编码177个氨基酸,理论等电点为4.21,不包含信号肽和跨膜区,含有4个保守的EF-hand结构域。BoSPI基因起始密码子上游2000 bp的核苷酸序列中含有真菌诱导响应、代谢调节以及器官形成等应答元件。BoSPI基因在大肠杆菌中可诱导表达为17 k D的蛋白。BoSPI在柱头中表达量最高,在花瓣、萼片、叶片、雄蕊表达量较低。BoSPI蛋白被定位在细胞膜和细胞质。自花授粉30 min后对BoSPI基因的诱导表达显著增强。表明BoSPI参与了柱头响应自花花粉刺激的分子过程,可能是实现甘蓝自交不亲和性相关的某种新功能基因。
DOI:10.1139/o90-084URLPMID:2198059 [本文引用: 1]
This paper describes the sequence homology of belonging to the superfamily. Specifically, this similarity has been examined for 276 twelve-residue -loops. It has been found that, in the -loop, several residues appear invariant, regardless of the species of origin or the affinity of the . These residues are Asp at position 1 (+X of the coordinating position of the ), Asp or at position 3 (+Y), at position 6, Ile at position 8, and Glu at position 12 (-Z). It has also been found that conservation of certain residues can vary in similar sites in similar . For example, position 3 (+Y) in site 3 of is always an , whereas in the residue is always Asp. This study also examined the -affinities of fragments comprising the loop, helix-loop, loop-helix, and helix-loop-helix. These were compared with larger enzymatic or chemically generated fragments in an effort to understand the various contributions to the -affinity of a single-site versus a two-site domain as found in and . Based on free energy differences, it was found that a 34-residue helix-loop-helix represents about 60% of the affinity found in the intact . Cooperativity with a second site accounted for the remaining 40% of the affinity.
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[本文引用: 1]
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DOI:10.1139/o90-084URLPMID:2198059 [本文引用: 2]
This paper describes the sequence homology of belonging to the superfamily. Specifically, this similarity has been examined for 276 twelve-residue -loops. It has been found that, in the -loop, several residues appear invariant, regardless of the species of origin or the affinity of the . These residues are Asp at position 1 (+X of the coordinating position of the ), Asp or at position 3 (+Y), at position 6, Ile at position 8, and Glu at position 12 (-Z). It has also been found that conservation of certain residues can vary in similar sites in similar . For example, position 3 (+Y) in site 3 of is always an , whereas in the residue is always Asp. This study also examined the -affinities of fragments comprising the loop, helix-loop, loop-helix, and helix-loop-helix. These were compared with larger enzymatic or chemically generated fragments in an effort to understand the various contributions to the -affinity of a single-site versus a two-site domain as found in and . Based on free energy differences, it was found that a 34-residue helix-loop-helix represents about 60% of the affinity found in the intact . Cooperativity with a second site accounted for the remaining 40% of the affinity.
DOI:10.1007/s12374-011-9197-0URL [本文引用: 1]
Abstract2+ sensors and relays them to the downstream target effectors in cells. During the post-embryonic developmental process, auxin plays a critical role in maintaining the mitotically inactive status of the quiescent center (QC) and the root growth and development that follows. In this report, we demonstrate that Ca plays an important role in the maintenance of the QC, probably by regulating PIN1-mediated auxin transport. Perturbation of the intracellular Ca levels with chemicals that modify the Ca level decreases the endogenous auxin level and the size of the auxin maximum in the root tip and, at the same time, activates QC cell division and expansion. This decreased level of auxin is almost completely restored to the control level by the treatment of exogenous auxin. Interestingly, treatment with Ca level modifying chemicals significantly decreased the PIN1 expression in the root vasculature. Taken together, we suggest that balancing Ca homeostasis is one of contributing factors in establishing the proper auxin maximum in the root tip and maintaining the QC identity.
DOI:10.3390/plants2040650URL [本文引用: 1]
DOI:10.1073/pnas.1217343109URLPMID:23129621 [本文引用: 3]
In many angiosperms, outcrossing is enforced by genetic selfincompatibility (SI), which allows cells of the pistil to recognize and specifically inhibit "self" pollen. SI is often associated with increased stigma-anther separation, a morphological trait that promotes crosspollen deposition on the stigma. However, the gene networks responsible for coordinate evolution of these complex outbreeding devices are not known. In self-incompatible members of the Brassicaceae (crucifers), the inhibition of "self"-pollen is triggered within the stigma epidermal cell by allele-specific interaction between two highly polymorphic proteins, the stigma-expressed S-locus receptor kinase (SRK) and its pollen coat-localized ligand, the S-locus cysteinerich (SCR) protein. Using Arabidopsis thaliana plants that express SI as a result of transformation with a functional SRK-SCR gene pair, we identify Auxin Response Factor 3 (ARF3) as a mediator of crosstalk between SI signaling and pistil development. We show that ARF3, a regulator of pistil development that is expressed in the vascular tissue of the style, acts non-cell-autonomously to enhance the SI response and simultaneously down-regulate auxin responses in stigma epidermal cells, likely by regulating a mobile signal derived from the stylar vasculature. The inverse correlation we observed in stigma epidermal cells between the strength of SI and the levels of auxin inferred from activity of the auxin-responsive reporter DR5:: GUS suggests that the dampening of auxin responses in the stigma epidermis promotes inhibition of "self" pollen in crucifer SI.
DOI:10.16420/j.issn.0513-353x.2015-0352URLMagsci [本文引用: 1]
<p>扩增了从甘蓝柱头蛋白质谱鉴定中获得的泛素蛋白BoSU03 基因的编码序列。序列分析表明<br>BoSU03 与甘蓝泛素蛋白Bol003815 的核苷酸序列相似性最高，达98%，含有臂重复结构域，属于植物泛<br>素蛋白家族。为探究BoSU03 是否为自交不亲和S 位点受体激酶（SRK）下游的互作蛋白，以SRK 与ARC1<br>相互作用为对照，利用GAL 酵母双杂交系统对SRK 与BoSU03 进行了相互作用验证，结果表明，<br>BoSRK/BoSU03 的转化酵母在四缺平板SD/-Ade/-His/-Leu/-Trp/x-α-gal/25 mmol 3-AT 上长势较好，显色较<br>深，进一步检测发现BoSRK/BoSU03 转化酵母的β–半乳糖苷酶活性值大于BoSRK/BoARC1 转化酵母的<br>活性值，这为进一步分析鉴定BoSU03 是否参与SRK 下游信号传导过程提供了依据。</p>
DOI:10.16420/j.issn.0513-353x.2015-0352URLMagsci [本文引用: 1]
<p>扩增了从甘蓝柱头蛋白质谱鉴定中获得的泛素蛋白BoSU03 基因的编码序列。序列分析表明<br>BoSU03 与甘蓝泛素蛋白Bol003815 的核苷酸序列相似性最高，达98%，含有臂重复结构域，属于植物泛<br>素蛋白家族。为探究BoSU03 是否为自交不亲和S 位点受体激酶（SRK）下游的互作蛋白，以SRK 与ARC1<br>相互作用为对照，利用GAL 酵母双杂交系统对SRK 与BoSU03 进行了相互作用验证，结果表明，<br>BoSRK/BoSU03 的转化酵母在四缺平板SD/-Ade/-His/-Leu/-Trp/x-α-gal/25 mmol 3-AT 上长势较好，显色较<br>深，进一步检测发现BoSRK/BoSU03 转化酵母的β–半乳糖苷酶活性值大于BoSRK/BoARC1 转化酵母的<br>活性值，这为进一步分析鉴定BoSU03 是否参与SRK 下游信号传导过程提供了依据。</p>
DOI:10.1083/jcb.200604011URLPMID:16831890 [本文引用: 1]
Self-incompatibility (SI) prevents inbreeding through specific recognition and rejection of incompatible pollen. In incompatible Papaver rhoeas pollen, SI triggers a Ca2+signaling cascade, resulting in the inhibition of tip growth, actin depolymerization, and programmed cell death (PCD). We investigated whether actin dynamics were implicated in regulating PCD. Using the actin-stabilizing and depolymerizing drugs jasplakinolide (Jasp) and latrunculin B, we demonstrate that changes in actin filament levels or dynamics play a functional role in initiating PCD in P. rhoeas pollen, triggering a caspase-3-like activity. Significantly, SI-induced PCD in incompatible pollen was alleviated by pretreatment with Jasp. This represents the first account of a specific causal link between actin polymerization status and initiation of PCD in a plant cell and significantly advances our understanding of the mechanisms involved in SI.
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DOI:10.1016/S1360-1385(96)80028-8URL [本文引用: 1]
Plant sexual reproduction relies on intimate interactions between the pollen and the pistil. These largely extracellular interactions lead to cellular activities within the pollen, enabling the transport of the male gametes by the pollen-tube growth process to the ovules for fertilization. Recent analyses of pollen surface molecules and extracellular matrix molecules along the pollen-tube growth pathway have produced insights into the molecular and biochemical bases of some of these interactions. These studies, together with the genetic analysis of sexual reproduction, are elucidating the molecular mechanisms underlying pollination and fertilization.
DOI:10.1111/j.1469-8137.2008.02457.xURL [本文引用: 1]
Siphonogamy, the delivery of nonmotile sperm to the egg via a pollen tube, was a key innovation that allowed flowering plants (angiosperms) to carry out sexual reproduction on land without the need for water. This process begins with a pollen grain (male gametophyte) alighting on and adhering to the stigma of a flower. If conditions are right, the pollen grain germinates to produce a pollen tube. The pollen tube invades the stigma and grows through the style towards the ovary, where it enters an ovule, penetrates the embryo sac (female gametophyte) and releases two sperm cells, one of which fertilizes the egg, while the other fuses with the two polar nuclei of the central cell to form the triploid endosperm. The events before fertilization (pollen-pistil interactions) comprise a series of complex cellular interactions involving a continuous exchange of signals between the haploid pollen and the diploid maternal tissue of the pistil (sporophyte). In recent years, significant progress has been made in elucidating the molecular identity of these signals and the cellular interactions that they regulate. Here we review our current understanding of the cellular and molecular interactions that mediate the earliest of these interactions between the pollen and the pistil that occur on or within the stigma - the 'pollen-stigma interaction'.
DOI:10.1093/abbs/gmw075URL [本文引用: 1]
自我障碍(SI ) 是一个重要交配系统阻止使近交并且支持 outcrossing。ARC1 和 Exo70A1 在 Brassica SI 发信号作为 S 地点受体 kinase 和戏保守人士角色的下游的目标工作。基于顺序相同， Exo70A1 被划分成四子域：白氨酸拉链(列伊 128 -Leu 149 ), hypervariable 区域(重量的单位 172 -Leu 197 ), 相扑修正主题(Glu 260 -Ile 275 ), 和 pfamExo70 领域(他的 271 -Phe 627 ) 。ARC1 如下包含四个领域：白氨酸拉链(列伊 116 -Leu 137 ), 卷卷领域(Thr 210 -Val 236 ),U 盒子(毒蛇 282 -Trp 347 ) 主题，和手臂(翼 415 -Thr 611 ) 领域。生物信息学分析，酵母二混血儿的屏蔽和 ARC1 116-236 的白氨酸拉链和卷卷主题为和 Exo70A1 的相互作用被要求的下拉试金表演，当手臂主题的增加导致和 Exo70A1 的相互作用的损失时。同时，没有任何域的 Exo70A1 的N终端与 ARC1 显示出一个弱相互作用，并且 LacZ 表示的水平与白氨酸拉链的增加增加并且与 hypervariable 区域和相扑修正主题到达最大的值，显示那个 hypervariable 区域和 Exo70A1 172-275 的相扑修正主题为有 ARC1 的绑定主要负责，而 pfamExo70 领域为 ARC1 有很少亲密关系。位于 Exo70A1 hypervariable 区域的 Lys 181 可以是调停的 ubiquitination 地点在 ARC1 和 Exo70A1 之间的相互作用。因此，两有 ARC1 116-236 , 和 hypervariable 区域和 Exo70A1 172-275 的相扑修正主题的卷卷结构的白氨酸拉链是在 ARC1 和 Exo70A1 之间的核心相互作用领域。影响这些核心领域的任何因素将是调停的 ARC1 的管理者在自我不兼容的系统的 ubiquitin 降级。
DOI:10.3864/j.issn.0578-1752.2011.09.024URL [本文引用: 1]
【目的】利用酵母双杂交系统鉴定甘蓝自交不亲和决定因子S位点富含半胱氨酸蛋白/S位点蛋白11(SCR/SP11)与S位点受体激酶(SRK)胞外域(eSRK)片段间可能的相互作用区域。【方法】以结球甘蓝B3为材料,利用分子克隆技术,分别构建以pGBKT7为载体的全长SCRB3、SCRB3-1、SCRB3-2的重组诱饵质粒;以pGADT7为载体的全长eSRKB3、eSRKB3-1、eSRKB3-2的重组激活域(AD)质粒。用PEG/LiAc法将上述重组诱饵质粒转化感受态酵母菌Y2HGold,重组AD质粒转化感受态酵母菌Y187;Y2HGold[pGBKT7-SCRB3-s]、Y187[pGADT7-eSRKB3-s]两两融合培养,观察交配菌在SD/-Trp-Leu/x-α-gal/AbA(DDO/x/A)、SD/-Trp-Leu-Ade-His/x-α-gal/AbA(QDO/x/A)平板上的生长情况。【结果】酵母双杂交重组表达载体构建成功,且无毒性和自激活作用产生;9个试验组中只有SCRB3-1、SCRB3-2与eSRKB3-1、eSRKB3-2重组表达质粒转化子两两组合的4个融合株在QDO/x/A平板上出现蓝色克隆,激活了报告基因AUR1-C、MEL1、HIS3、ADE2。【结论】酵母双杂交系统适用于SCR与SRK蛋白相互作用的研究,初步确定了SCR与eSRK存在相互作用,SRK跨膜域的存在与否对其相互作用的研究没有影响。
DOI:10.3864/j.issn.0578-1752.2011.09.024URL [本文引用: 1]
【目的】利用酵母双杂交系统鉴定甘蓝自交不亲和决定因子S位点富含半胱氨酸蛋白/S位点蛋白11(SCR/SP11)与S位点受体激酶(SRK)胞外域(eSRK)片段间可能的相互作用区域。【方法】以结球甘蓝B3为材料,利用分子克隆技术,分别构建以pGBKT7为载体的全长SCRB3、SCRB3-1、SCRB3-2的重组诱饵质粒;以pGADT7为载体的全长eSRKB3、eSRKB3-1、eSRKB3-2的重组激活域(AD)质粒。用PEG/LiAc法将上述重组诱饵质粒转化感受态酵母菌Y2HGold,重组AD质粒转化感受态酵母菌Y187;Y2HGold[pGBKT7-SCRB3-s]、Y187[pGADT7-eSRKB3-s]两两融合培养,观察交配菌在SD/-Trp-Leu/x-α-gal/AbA(DDO/x/A)、SD/-Trp-Leu-Ade-His/x-α-gal/AbA(QDO/x/A)平板上的生长情况。【结果】酵母双杂交重组表达载体构建成功,且无毒性和自激活作用产生;9个试验组中只有SCRB3-1、SCRB3-2与eSRKB3-1、eSRKB3-2重组表达质粒转化子两两组合的4个融合株在QDO/x/A平板上出现蓝色克隆,激活了报告基因AUR1-C、MEL1、HIS3、ADE2。【结论】酵母双杂交系统适用于SCR与SRK蛋白相互作用的研究,初步确定了SCR与eSRK存在相互作用,SRK跨膜域的存在与否对其相互作用的研究没有影响。
DOI:10.3864/j.issn.0578-1752.2016.01.002URL [本文引用: 1]
[目的]深入研究甘蓝自交不亲和信号传导关键元件S-位点受体激酶SRK与臂重复蛋白ARC1及ARC1与Exo70A1间相互识别的分子机理,鉴定SRK-ARC1及ARC1-Exo70A1之间的互作区段,并分析其作用强度,明确蛋白间互作功能域.[方法]通过生物信息学分析得到蛋白功能域,根据分析结果以典型的自交不亲和结球甘蓝E1为材料分别扩增SRK、ARC1和Exo70A1含不同功能域的截短体片段,利用分子克隆技术将SRK激酶域(SRKj)及其截短体SRKjΔ1—SRKjΔ4,Exo70A1全长及其截短体Exo70h1Δ1—Exo70h1Δ3的编码序列分别亚克隆至pGADT7(AD)质粒,将ARC1及其截短体ARC1Δ1—ARC1Δ8的编码序列分别亚克隆至载体pGBKT7(BD)质粒.用PEG/LiAc法将获得的AD和BD重组质粒两两组合分别共转化到酵母AH109感受态中,观察融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 mM 3-AT平板上的菌落生长情况和颜色变化情况,进一步测定其β-半乳糖苷酶活性.最后通过原核表达体外孵育检测蛋白质相互作用的方法对SRK-ARC1及ARC1-Exo70A1的相互作用进行验证.[结果]DNA测序和内切酶分析显示成功构建18个酵母双杂交表达载体,且无自激活能力.在SRK-ARC1的1 0个试验组合中,只有ARC1Δ4、ARC1Δ8、ARC1与SRKj组合的融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 mM 3-AT培养基上长出蓝色菌落,激活报告基因HIS3、ADE2和MEL1.随着SRKj或ARC1截短体片段的延长,二者的β-半乳糖苷酶活性逐渐增加,其中,ARC1Δ4与SRKj组合的β-半乳糖苷酶活性最高(酶活为15.98).在ARC1-Exo70A1 16个试验组合中,Exo70A1Δ3与ARC1Δ1Δ3都相互作用,其融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 mM 3-AT培养基上长出蓝色菌落,激活报告基因HIS3、ADE2和MEL1.随着ARC1或Exo70A1截短体片段的延长,二者的β-半乳糖苷酶活性呈现先增加后降低的趋势,其中ARC1Δ2与Exo70A1Δ3组合的β-半乳糖苷酶活性最大(酶活性为25.07).说明ARC1的N端和Exo70A1的N端发生了互作,而ARC1的C端、全长与Exo70A1都不发生互作.体外表达检测蛋白相互作用发现,SRKj与ARC1Δ4、ARC1Δ2与Exo70A1Δ3均可以直接发生相互作用.[结论]SRK的激酶域(SRKj)与ARC1的C端臂重复区发生互作,缩短SRK激酶域中的任何结构域或者缩短ARC1的臂重复区,二者都不会发生互作.ARC1的亮氨酸拉链和蜷曲螺旋与Exo70h1的N端结构域(去除pfamExo70A1域)介导了二者的相互作用.SRK-ARC1的作用力强度小于ARC1-Exo70A1的作用力强度.
DOI:10.3864/j.issn.0578-1752.2016.01.002URL [本文引用: 1]
[目的]深入研究甘蓝自交不亲和信号传导关键元件S-位点受体激酶SRK与臂重复蛋白ARC1及ARC1与Exo70A1间相互识别的分子机理,鉴定SRK-ARC1及ARC1-Exo70A1之间的互作区段,并分析其作用强度,明确蛋白间互作功能域.[方法]通过生物信息学分析得到蛋白功能域,根据分析结果以典型的自交不亲和结球甘蓝E1为材料分别扩增SRK、ARC1和Exo70A1含不同功能域的截短体片段,利用分子克隆技术将SRK激酶域(SRKj)及其截短体SRKjΔ1—SRKjΔ4,Exo70A1全长及其截短体Exo70h1Δ1—Exo70h1Δ3的编码序列分别亚克隆至pGADT7(AD)质粒,将ARC1及其截短体ARC1Δ1—ARC1Δ8的编码序列分别亚克隆至载体pGBKT7(BD)质粒.用PEG/LiAc法将获得的AD和BD重组质粒两两组合分别共转化到酵母AH109感受态中,观察融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 mM 3-AT平板上的菌落生长情况和颜色变化情况,进一步测定其β-半乳糖苷酶活性.最后通过原核表达体外孵育检测蛋白质相互作用的方法对SRK-ARC1及ARC1-Exo70A1的相互作用进行验证.[结果]DNA测序和内切酶分析显示成功构建18个酵母双杂交表达载体,且无自激活能力.在SRK-ARC1的1 0个试验组合中,只有ARC1Δ4、ARC1Δ8、ARC1与SRKj组合的融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 mM 3-AT培养基上长出蓝色菌落,激活报告基因HIS3、ADE2和MEL1.随着SRKj或ARC1截短体片段的延长,二者的β-半乳糖苷酶活性逐渐增加,其中,ARC1Δ4与SRKj组合的β-半乳糖苷酶活性最高(酶活为15.98).在ARC1-Exo70A1 16个试验组合中,Exo70A1Δ3与ARC1Δ1Δ3都相互作用,其融合菌株在SD/-Leu-Trp-His-Ade/X-α-gal/25 mM 3-AT培养基上长出蓝色菌落,激活报告基因HIS3、ADE2和MEL1.随着ARC1或Exo70A1截短体片段的延长,二者的β-半乳糖苷酶活性呈现先增加后降低的趋势,其中ARC1Δ2与Exo70A1Δ3组合的β-半乳糖苷酶活性最大(酶活性为25.07).说明ARC1的N端和Exo70A1的N端发生了互作,而ARC1的C端、全长与Exo70A1都不发生互作.体外表达检测蛋白相互作用发现,SRKj与ARC1Δ4、ARC1Δ2与Exo70A1Δ3均可以直接发生相互作用.[结论]SRK的激酶域(SRKj)与ARC1的C端臂重复区发生互作,缩短SRK激酶域中的任何结构域或者缩短ARC1的臂重复区,二者都不会发生互作.ARC1的亮氨酸拉链和蜷曲螺旋与Exo70h1的N端结构域(去除pfamExo70A1域)介导了二者的相互作用.SRK-ARC1的作用力强度小于ARC1-Exo70A1的作用力强度.
DOI:10.3864/j.issn.0578-1752.2016.04.016URL [本文引用: 1]
【目的】以自交不亲和材料甘蓝为研究对象,分析ROH1的组织表达、ROH1与EXO70A1的相互作用及其是否参与甘蓝生殖发育。【方法】从甘蓝花蕾中克隆出ROH1和EXO70A1,应用半定量RT-PCR检测ROH1的表达特性;应用实时荧光定量PCR进一步分析甘蓝授粉后1 h内ROH1和EXO70A1的表达量和二者的相关性;分别构建以p GBKT7为载体的EXO70A1重组"诱饵"质粒和以p GADT7为载体ROH1的重组猎物质粒,并将重组质粒转化酵母菌株,利用缺陷型培养基进行蛋白质相互作用检测,确定ROH1和EXO70A1是否存在相互作用。【结果】在甘蓝中ROH1为单外显子基因,编码含398个氨基酸残基的蛋白质,与拟南芥ROH1对比发现,甘蓝ROH1序列内存在一个连续16个氨基酸残基的缺失;它在甘蓝的雄蕊(花药)、花柱、幼茎、幼根及叶片中均有表达但表达量差异明显,其中,在幼叶、花柱和雄蕊(花药)中的表达量较高,在幼根和幼茎中的表达量较低;甘蓝授粉后柱头中ROH1的表达量在1 h内呈现出"上升-下降-上升"趋势,其中,以授粉后30 min的表达量最低,授粉后1 h的表达量达到最高值;而EXO70A1在授粉后1 h内的表达呈现出"下降-上升"的趋势,并以授粉15 min时表达量最低,授粉后1 h时的表达量最高;二者表达的动态变化说明ROH1和EXO70A1参与了甘蓝的生殖发育;ROH1与EXO70A1的表达量趋势线呈现出负相关性,并在30 min时出现了重叠区域,预示ROH1与EXO70A1之间可能存在相互作用;成功构建p GADT7-ROH1和p GBKT7-EXO70A1酵母表达载体,通过酵母双杂交试验,发现载体p GBKT7-EXO70A1无自激活能力,融合菌株同时激活4个报告基因(AUR1-C、MEL1、HIS3和ADE2)的表达,表明花药中ROH1和花柱中的EXO70A1之间存在较强的相互作用。【结论】甘蓝的ROH1和EXO70A1之间存在较强的相互作用并呈现出负相关性,ROH1可能通过调节EXO70A1在柱头的分泌影响生殖发育。
DOI:10.3864/j.issn.0578-1752.2016.04.016URL [本文引用: 1]
【目的】以自交不亲和材料甘蓝为研究对象,分析ROH1的组织表达、ROH1与EXO70A1的相互作用及其是否参与甘蓝生殖发育。【方法】从甘蓝花蕾中克隆出ROH1和EXO70A1,应用半定量RT-PCR检测ROH1的表达特性;应用实时荧光定量PCR进一步分析甘蓝授粉后1 h内ROH1和EXO70A1的表达量和二者的相关性;分别构建以p GBKT7为载体的EXO70A1重组"诱饵"质粒和以p GADT7为载体ROH1的重组猎物质粒,并将重组质粒转化酵母菌株,利用缺陷型培养基进行蛋白质相互作用检测,确定ROH1和EXO70A1是否存在相互作用。【结果】在甘蓝中ROH1为单外显子基因,编码含398个氨基酸残基的蛋白质,与拟南芥ROH1对比发现,甘蓝ROH1序列内存在一个连续16个氨基酸残基的缺失;它在甘蓝的雄蕊(花药)、花柱、幼茎、幼根及叶片中均有表达但表达量差异明显,其中,在幼叶、花柱和雄蕊(花药)中的表达量较高,在幼根和幼茎中的表达量较低;甘蓝授粉后柱头中ROH1的表达量在1 h内呈现出"上升-下降-上升"趋势,其中,以授粉后30 min的表达量最低,授粉后1 h的表达量达到最高值;而EXO70A1在授粉后1 h内的表达呈现出"下降-上升"的趋势,并以授粉15 min时表达量最低,授粉后1 h时的表达量最高;二者表达的动态变化说明ROH1和EXO70A1参与了甘蓝的生殖发育;ROH1与EXO70A1的表达量趋势线呈现出负相关性,并在30 min时出现了重叠区域,预示ROH1与EXO70A1之间可能存在相互作用;成功构建p GADT7-ROH1和p GBKT7-EXO70A1酵母表达载体,通过酵母双杂交试验,发现载体p GBKT7-EXO70A1无自激活能力,融合菌株同时激活4个报告基因(AUR1-C、MEL1、HIS3和ADE2)的表达,表明花药中ROH1和花柱中的EXO70A1之间存在较强的相互作用。【结论】甘蓝的ROH1和EXO70A1之间存在较强的相互作用并呈现出负相关性,ROH1可能通过调节EXO70A1在柱头的分泌影响生殖发育。
DOI:10.1104/pp.103.019943URL [本文引用: 1]
DOI:10.1016/S0092-8674(00)80682-0URLPMID:10693763 [本文引用: 1]
Arabidopsis plants carrying mutations in the PINOID ( PID) gene have a pleiotropic shoot phenotype that mimics that of plants grown on auxin transport inhibitors or of plants mutant for the auxin efflux carrier PINFORMED ( PIN), with defects in the formation of cotyledons, flowers, and floral organs. We have cloned PID and find that it is transiently expressed in the embryo and in initiating floral anlagen, demonstrating a specific role for PID in promoting primordium development. Constitutive expression of PID causes a phenotype in both shoots and roots that is similar to that of auxin-insensitive plants, implying that PID, which encodes a serine-threonine protein kinase, negatively regulates auxin signaling.
DOI:10.1126/science.1100618URL [本文引用: 1]
DOI:10.1073/pnas.0510283103URLPMID:16601102 [本文引用: 1]
Activity of the serine-threonine protein kinase PINOID (PID) has been implicated in the asymmetrical localization of the membraneassociated PINFORMED (PIN) family of auxin transport facilitators. However, the means by which PID regulates PIN protein distribution is unknown. We have used recombinant PID protein to dissect the regulation of PID activity in vitro. We demonstrate that intramolecular PID autophosphorylation is required for the ability of PID to phosphorylate an exogenous substrate. PID-like mammalian AGC kinases act in a phosphorylation cascade initiated by the phospholipid-associated kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1), which binds to the C-terminal hydrophobic PDK1-interacting fragment (PIF) domain found in PDK1 substrates. We find that Arabidopsis PDK1 interacts with PID, and that transphosphorylation by PDK1 increases PID autophosphorylation. We show that a PID activation loop serine is required for PDK1dependent PID phosphorylation. This activation is rapid and requires the PIF domain. Cell extracts from flowers and seedling shoots dramatically increase PID phosphorylation in a tissuespecific manner. A PID protein variant in which the PIF domain was mutated failed to be activated by the seedling shoot extracts. PID immunoprecipitated from Arabidopsis cells in which PDK1 expression was inhibited by RNAi showed a dramatic reduction in transphosphorylation of myelin basic protein substrate. These results indicate that AtPDK1 is a potent enhancer of PID activity and provide evidence that phospholipid signaling may play a role in the signaling processes controlling polar auxin transport.
DOI:10.1016/j.cell.2007.07.033URLPMID:17889649 [本文引用: 1]
In plants, cell polarity and tissue patterning are connected by intercellular flow of the phytohormone auxin, whose directional signaling depends on polar subcellular localization of PIN auxin transport proteins. The mechanism of polar targeting of PINs or other cargos in plants is largely unidentified, with the PINOID kinase being the only known molecular component. Here, we identify PP2A phosphatase as an important regulator of PIN apical-basal targeting and auxin distribution. Genetic analysis, localization, and phosphorylation studies demonstrate that PP2A and PINOID both partially colocalize with PINs and act antagonistically on the phosphorylation state of their central hydrophilic loop, hence mediating PIN apical-basal polar targeting. Thus, in plants, polar sorting by the reversible phosphorylation of cargos allows for their conditional delivery to specific intracellular destinations. In the case of PIN proteins, this mechanism enables switches in the direction of intercellular auxin fluxes, which mediate differential growth, tissue patterning, and organogenesis.
DOI:10.1016/j.plantsci.2006.08.005URL [本文引用: 1]
Auxin functions, at least in part, by regulating a set of early auxin response genes: Aux/ IAAs, GH3s and small auxin-up RNAs ( SAURs). The SAUR genes encode short transcripts that accumulate rapidly after auxin treatment. There are 72 SAUR genes in Arabidopsis. However, none of these genes have been functionally characterized, although some members have been implicated in calcium/calmodulin-mediated auxin responses and etiolated seedling growth. Here, we investigated the function of a SAUR gene, abolished apical hook maintenance 1 ( AAM1), through molecular genetic and transgenic studies. Transgenic Arabidopsis seedlings overexpressing AAM1 exhibited short, hookless hypocotyls with partially open cotyledons in darkness. The hookless phenotype was completely rescued by exogenous auxin. In contrast, hypocotyls of a T-DNA insertion mutant aam1 were slightly longer than wild-type hypocotyls and exhibited normal hook curvature. Notably, AAM1 is predominantly expressed on the inner side of the apical hook, similar to the DR5::GUS reporter. However, on illumination, the AAM1 transcripts were evenly distributed in the apical region, demonstrating that the apical hook development is intimately related to the localized expression of AAM1.
[本文引用: 1]
