,1,**, 张贺翠1,**, 刘倩莹1, 廉小平2, 谢琴琴1, 胡燈科1, 张以忠1, 王玉奎1, 白晓璟1, 朱利泉,1,*Molecular cloning and expression analysis of BoGSTL21 in self-incompatibility Brasscia oleracea
ZUO Tong-Hong,1,**, ZHANG He-Cui1,**, LIU Qian-Ying1, LIAN Xiao-Ping2, XIE Qin-Qin1, HU Deng-Ke1, ZHANG Yi-Zhong1, WANG Yu-Kui1, BAI Xiao-Jing1, ZHU Li-Quan,1,*通讯作者:
收稿日期:2020-01-8接受日期:2020-07-2网络出版日期:2020-08-18
| 基金资助: |
Received:2020-01-8Accepted:2020-07-2Online:2020-08-18
| Fund supported: |
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左同鸿, 张贺翠, 刘倩莹, 廉小平, 谢琴琴, 胡燈科, 张以忠, 王玉奎, 白晓璟, 朱利泉. 甘蓝自交不亲和性相关基因BoGSTL21的克隆与表达分析[J]. 作物学报, 2020, 46(12): 1850-1861. doi:10.3724/SP.J.1006.2020.04004
ZUO Tong-Hong, ZHANG He-Cui, LIU Qian-Ying, LIAN Xiao-Ping, XIE Qin-Qin, HU Deng-Ke, ZHANG Yi-Zhong, WANG Yu-Kui, BAI Xiao-Jing, ZHU Li-Quan.
自交不亲和性(self-incompatibility, SI)是植物在长期进化过程中为限制自交衰退、促进杂交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自交不亲和(sporophytic self-incompatibility, SSI)植物, 其分子机制主要集中在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。花粉和柱头相互识别的过程包括花粉黏附、花粉外被释放、水合、花粉萌发及花粉管生长穿入柱头等[1]。目前研究认为SI信号传导途径是由SRK-ARC1-Exo70A1等介导的蛋白质泛素化降解途径。Gu等[2]以S-位点受体激酶(S-locus receptor kinase, SRK)的胞内激酶域为诱饵, 通过酵母双杂交技术从甘蓝型油菜柱头cDNA文库中鉴定出与SRK结合的靶蛋白——臂重复蛋白 1 (Arm repeat containing 1, ARC1), 其在自交不亲和信号传递过程中起正调控作用。Vanoosthuyse等[3]和Stone等[4]利用RNAi干扰(RNA interference, RNAi)和反义抑制技术分别抑制琴叶拟南芥(Arabidopsis lyrata)和甘蓝型油菜(Brassica napus)中ARC1的表达, 以及在甘蓝型油菜中过表达Exo70A1均只能部分打破自交不亲和性。拟南芥ARC1基因以假性遗传因子的形式存在, 向自交亲和拟南芥中仅转入琴叶拟南芥的S-位点半胱氨酸富集蛋白(S-locus cysteine-rich protein, SCR)和SRK也能表现出强自交不亲和性[5,6,7]。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, 对甘蓝SI自交不亲和分子机制的深入研究具有重要意义。
在植物中, 谷胱甘肽转移酶(Glutathione S- transferases, GSTs; EC 2.5.1.18)是一个多基因家族, 首先在玉米中被发现[8], 此后不断有GST的报道, 现已发现拟南芥有48个GST基因, 大豆有25个GST基因, 玉米有42个GST基因, 水稻有59个GST基因[9,10,11]。根据蛋白同源性和基因组织结构, 可将植物的GST分为φ、τ、ζ、θ、Lambda和脱氢抗坏血酸还原酶(DHARs) 6类[12,13]。后来又陆续发现一些小类如Iota、Hemerythrin和微粒体GST等。生物化学和免疫学研究表明, GST家族基因主要在胞质中表达[14,15]; 植物的φ、τ、ζ和θGST是蛋白二聚体, 是由约26 kD亚基组成的同源二聚体或异源二聚体, 形成疏水的50 kD的蛋白[16,17,18]; 但Lambda GST和DHAR与φ、τ、ζ和θ GST的结构不同, 它们是由一条多肽链组成的单体[12,18]。在植物中, 一些GST基因的表达具有组织特异性, 如在玉米(Zea mays)的花粉中包含单一GST, 而角质绒片中有相应的5个同功酶[19]。GST在植物的初级代谢和二级代谢、胁迫耐受、细胞信号等方面行使功能[18]。由于授粉过程中的自花花粉刺激柱头产生SI信号传导的过程, 类似于逆境刺激植物细胞产生的过程, 那么GST是否有可能通过某种分子过程与自交不亲和相联系呢?本研究通过转录组测序分析了甘蓝自花和异花授粉后基因表达情况, 首次筛选到1个受自花授粉诱导上调表达的基因BoGSTL21, 进而通过基因克隆、生物信息学分析、组织特异性分析、启动子活性分析、原核表达以及酵母双杂交等深入探索, 以期证明该蛋白参与甘蓝自交不亲和反应。
1 材料与方法
1.1 材料与菌种
甘蓝高代自交不亲和系‘A4’和‘F1’植株由西南大学十字花科蔬菜研究所选育, 于2018年3月底至4月初, 选取开花前1~2 d长势一致的‘A4’花蕾, 于开花当天人工去雄, 用成熟的‘A4’和‘F1’花粉对‘A4’进行自花和异花授粉。分别取未授粉柱头, 以及自花、异花授粉15、30和60 min的柱头, 立刻放入液氮速冻保存。同时取叶片、茎、花蕾、萼片、花瓣、花药和柱头于-80℃保存备用。本试验所用的拟南芥为哥伦比亚野生型拟南芥, 用于基因启动子的遗传转化试验。大肠杆菌DH5α和原核表达菌株E. coli BL21 (DE3)均购自上海唯地生物技术有限公司, 农杆菌GV3101购自北京全式金生物技术有限公司。
1.2 转录组测序及差异表达基因筛选
提取未授粉柱头、自花授粉(15、30、60 min)以及异花授粉(15、30、60 min)柱头的RNA, 送北京百迈克公司用Illumina HiSeq高通量测序平台测序, 测序类型为PE150, 构建转录组文库进行建库测序, 获得转录组数据。采用FPKM (Fragments Per Kilobase of transcript per Million fragments mapped)作为衡量转录本或基因表达水平的指标。在差异表达基因检测过程中, 将差异倍数Fold Change≥2且错误发现率(false discovery rate) FDR<0.01作为筛选标准。根据基因在不同授粉处理后的相对表达量, 筛选在自花授粉后表达差异明显、异花授粉后变化趋势不明显的基因。1.3 BoGSTL21基因的克隆
根据转录组测序获得的cDNA序列, 结合芸薹属数据库、同源重组酶原理以及原核表达载体pGEX-4T-1的序列特征, 用Primer Premier 6.0软件设计引物GST-F/R (表1), 分别以结球甘蓝‘A4’花蕾gDNA和cDNA为模板, 在50 μL PCR反应体系中依次加入20 μL ddH2O、25 μL 2×PCR Mixster Mix混合酶、上/下游引物各2 μL和cDNA模板1 μL; 反应程序为94℃ 3 min; 94℃ 30 s, 60℃ 30 s, 72℃ 55 s, 40个循环; 72℃ 5 min。PCR产物经1×104 mg L-1琼脂糖凝胶电泳, 回收目的片段, 利用clonExpress快速克隆技术将目的片段连接到pGEX-4T-1载体上, 后转入大肠杆菌感受态细胞DH5α, 经菌液PCR将筛选出的阳性克隆送上海生物工程股份有限公司测序。Table 1
表1
表1基因克隆及其荧光定量PCR分析所用引物
Table 1
| 引物名称 Primer name | 引物序列 Primer sequence (5°-3°) | 用途 Functions |
|---|---|---|
| BoGSTL21-PGEX-4T-1 | F: GATCTGGTTCCGCGTGGATCCATGAGTGCCGGAGTGAGAGTTAG | 基因的原核表达 |
| R: CTCGAGTCGACCCGGGAATTCGGGACGTGCTTCTGCTTGG | Prokaryotic expression | |
| qRT-PCR | F: TTCCTTTGCCGATTTAGTTTGG | 荧光定量PCR引物 |
| R: AGTGTTCATCTCCTTAAGCCAA | qRT-PCR | |
| dActin | F: GGCTGATGGTGAAGATATTCA | 内参引物 |
| R: CAAGCACAATACCAGTAGTAC | Internal reference primers | |
| BoGSTL21-BK | F: TCAGAGGAGGACCTGCATATGAGTGCCGGAGTGAGAGTTA | 酵母双杂交引物 Yeast two-hybrid primers |
| R: TCGACGGATCCCCGGGAATTCGGGACGTGCTTCTGCTTG | ||
| BoFAB1C-AD | F: GTACCAGATTACGCTCATATGGGGATGGTGAAGTTCTCTGTG | |
| R: ATGCCCACCCGGGTGGAATTCGTTCCATGGCTCAGGAACC | ||
| BoPATL2-AD | F: GTACCAGATTACGCTCATATGGCTCAAGAAGAGATACAGAAG | |
| R: ATGCCCACCCGGGTGGAATTCTTATGCTTGCGTTTTGAACC | ||
| BoF9N12_9-AD | F: GTACCAGATTACGCTCATATGGTGGTGTCGCCAAAGATAG | |
| R: ATGCCCACCCGGGTGGAATTCTCAGGTGATGGGTTGGGC | ||
| 1391 | F: GAACTGATCGTTAAAACTGC | 通用引物 |
| R: TGGTCTTCTGAGACTGTATC | Universal primer | |
| BoGSTL21-GUS | F: CAAGCTTGGCTGCAGGTCGACATGTTATACGTTGCGAACGC | 基因的启动子活性分析 |
| R: GGTGGACTCCTCTTAGAATTCACTCAATCGTTCTTCTTCCGT | Promoter activity analysis |
新窗口打开|下载CSV
1.4 生物信息学分析
利用Bio-soft (1.5 BoGSTL21基因的表达分析
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、花药等组织的RNA和不同授粉处理的柱头RNA, 参照北京全式金生物技术有限公司反转录试剂盒TransScript First-Strand cDNA Synthesis SuperMix说明书反转录合成cDNA, 于-20℃保存备用。以叶片、花蕾、萼片、花瓣、花粉和柱头组织的cDNA为模板, 参照SYBR Premix EX Taq说明书, 以dActin作为内参(表1), 根据获得的BoGSTL21基因序列设计荧光定量PCR特异性引物qRP-F、qRP-R (表1), 用7500型实时荧光定量PCR仪扩增。同时以未授粉柱头、自花授粉(15、30和60 min)和异花授粉(15、30和60 min)的柱头cDNA为模板, dActin为内参, 对目的基因进行荧光定量PCR。反应体系为20 μL, 含2×SYBR Premix Ex Taq 10 μL、引物各1 μL、cDNA模板1 μL、超纯水7 μL。扩增程序为95℃ 1 min; 95℃ 15 s, 58℃ 15 s, 72℃ 30 s, 40个循环。每个试验设置3次重复, 采用2-ΔΔCT法计算目的基因的相对表达水平。
1.6 启动子活性分析
在NCBI选取BoGSTL21基因起始密码子上游1500 bp左右的核苷酸序列, 载体为pCAMBIA 1391, 酶切位点为Sal I和EcoR I, 引物为GUS-F/R (表1)。按照同源重组的方法构建BoGSTL21-GUS融合表达载体。将构建好的融合表达载体转化农杆菌GV3101感受态细胞, 用通用引物1391-F/R (表1)进行PCR检测, 并将阳性转化子单克隆按1:100扩大培养, 并通过遗传转化获得阳性拟南芥植株。通过筛选将得到的纯合转基因拟南芥种子播种, 分别取幼苗、茎、叶和花, 置GUS染色液中, 37℃培养箱中温育过夜。将侵染过的样品转入70%酒精中脱色2~3次, 然后于体视显微镜下观察照相。1.7 原核表达
选择测序正确的重组原核表达载体的菌液扩大培养, 抽提重组质粒转化表达菌株E. coli BL21 (DE3), PCR检测的引物为GST-F/GST-R, 将阳性转化子单克隆按1:100扩大培养, 取100 μL菌液接种于100 mL含100 μg mL-1氨苄抗性LB培养基中, 置于摇床37℃, 225转 min-1振荡培养至OD600达到0.6~0.8, 加入IPTG至终浓度为1 mmol L-1, 16℃过夜诱导, 诱导结束后, 收集其菌体, 再进行超声破碎, 然后离心收集上清液, 经海狸GST融合蛋白纯化磁珠纯化目的蛋白, 用SDS-PAGE电泳检测蛋白的表达, 同时在试验中设置pGEX-4T-1空载作为对照。1.8 甘蓝酵母双杂交筛选
1.8.1 构建诱饵载体及自激活验证 根据目的基因编码区序列以及诱饵载体pGBKT7的限制性酶切位点(EcoR I和Nde I)设计重组引物。利用同源重组的方法连接目的片段后转化大肠杆菌DH5α感受态细胞。按照LiAc法[20]制备酵母Y2Hgold感受态细胞。利用聚乙二醇/醋酸锂法(PEG/LiAc)将重组质粒pGBDT7-BoGSTL21与pGADT7空载共转化酵母Y2HGold感受态细胞, 转化后的菌液涂布在SD-Trp、DDO (SD/-Leu/-Trp)、QDO (SD/-Leu/-Trp/- His/-Ade/)平板上, 30℃倒置培养3~5 d, 以共转阴性对照pGBDT7-Lam×pGADT7-T平板作为对照, 验证pGBKT7-BoGSTL21重组质粒是否存在自激活。1.8.2 BoGSTL21互作蛋白筛选 根据制造商酵母方案手册(Invitrogen), 使用Clontech双杂交系统筛选酵母双杂交, 将共转化后的酵母感受态细胞涂布于营养缺陷型TDO (SD/-Leu/-Trp/-His)固体培养基上, 30℃倒置培养3~5 d, 观察菌落在营养缺陷型TDO固体培养基上的生长情况, 参照酵母质粒提取试剂盒说明书提取质粒pGADT7载体, 然后以提取的酵母质粒为模板, 以T7 5'-TAATACGACTCAC TATAGGG-3'和AD 5'-AGATGGTGCACGATGC ACAG-3'为引物进行RCR扩增, 将胶回收产物送上海生工生物工程有限公司测序。将获得的序列在NCBI数据库进行BLAST比对, 找到同源序列, 统计序列长度、基因登录号、基因注释等信息。
1.8.3 点对点回转阳性验证 将测序所得序列BLAST比对得到BoGSTL21互作候选蛋白, 构建pGADT7载体重组质粒, 利用PEG/LiAc法共转化Y2Hgold酵母感受态, 30℃恒温震荡90 min, 涂布于SD/-Trp-Leu固体培养基上, 平板倒置培养3~5 d, 观察是否有菌落生成, 后将菌斑稀释100倍点涂在营养缺陷型QDO固体培养基上, 观察菌落在QDO固体培养基上的生长情况。
2 结果与分析
2.1 BoGSTL21转录水平分析
根据甘蓝自花和异花授粉0~60 min的柱头转录组数据分析, 成功筛选到1个受自花授粉诱导上调表达的基因, 将该基因的cDNA序列在芸薹属数据库和NCBI数据库中进行Blast分析发现, 它与甘蓝中的谷胱甘肽-S-转移酶基因BoGSTL2 (Brasscia oleracea glutathione transferase lambda 2)同源, 故将该基因命名为BoGSTL21。BoGSTL21基因在柱头上的相对表达量随授粉时间变化, 从自花授粉(self-pollination, SP) 0 min开始持续上调表达, 授粉30 min时其在柱头上的相对表达量达到最大, 为3.77, 而后急剧下调; 而在异花授粉(cross-pollination, CP)过程中表达量变化不明显, 略微下调(图1)。经过反复核定, 在授粉30 min时, 两者的表达量水平差异达到4倍, 此时正是SI相关基因表达的关键时期。表明BoGSTL21基因的表达可能受自花授粉诱导。图1
新窗口打开|下载原图ZIP|生成PPT图1用柱头转录组数据分析BoGSTL21自花授粉和异花授粉后的表达模式
SP: 自花授粉; CP: 异花授粉。
Fig. 1Analysis of expression patterns of BoGSTL21 after self-pollination and cross-pollination using stigma transcriptome data
SP: self-pollination; CP: cross-pollination.
2.2 BoGSTL21基因的克隆
BoGSTL21基因gDNA PCR产物大小约1800 bp, cDNA PCR产物约1000 bp (图2)。经测序发现, BoGSTL21基因gDNA序列长度为1823 bp, cDNA序列长度为900 bp。由于使用同一对引物同源克隆得到cDNA和gDNA序列相差923 bp, 结合NCBI数据库并进一步对两者进行Clustal软件比对分析发现, 该基因的cDNA全长1119 bp, 开放阅读框长度为900 bp, 由9个外显子(分别为257、60、61、87、99、103、92、108和252 bp)和8个内含子(分别为103、82、81、85、72、98、89和94 bp)组成。图2
新窗口打开|下载原图ZIP|生成PPT图2甘蓝BoGSTL21的cDNA和gDNA序列扩增产物电泳图
Fig. 2Amplification product electrophoresis of BoGSTL21 cDNA and gDNA sequences in Brassica oleracea
2.3 BoGSTL21蛋白结构特征
BoGSTL21基因编码含299个氨基酸残基的蛋白质, 相对分子质量为33.96 kD, 理论等电点(pI)为8.49。在氨基酸组成中, 丝氨酸(Ser)出现频率最高, 占氨基酸总数的10.4%。带负电荷的氨基酸残基(Asp+Glu)总数为37个, 带正电荷的氨基酸残基(Arg+Lys)总数为40个。该蛋白的不稳定系数为55.77, 大于40, 因此为不稳定蛋白。总平均疏水性指数(grand average of hyropathicity, GRAVY)为-0.383, 推测为亲水性蛋白。BoGSTL21蛋白具有典型的植物GST基因的蛋白结构特点, 其中85~162位氨基酸为GST-N端结构域(图3, 实线框表示), 169~290位氨基酸为GST-C端结构域(图3, 下画虚线所示), 中间为连接区, 属于Lambda家族成员。图3
新窗口打开|下载原图ZIP|生成PPT图3甘蓝BoGSTL21 cDNA结构图及其推导的氨基酸序列
虚线框为N-糖基化位点, 实线框为GST-N结构域, 下画虚线为GST-C结构域。
Fig. 3Gene structure of Brassica oleracea BoGSTL21 and its deduced amino acid sequence
The dotted box denotes the N-glycosylation site, the solid line frame denotes the GST-N domain, and the underlined line denotes the GST-C domain.
BoGSTL21蛋白二级结构是由α-螺旋(alpha helix) 39.13%、β折叠(beta turn) 3.01%、无规则卷曲(random coil) 41.81%和延伸链(extended strand) 16.05%组成。BoGSTL21蛋白不含信号肽, 不存在跨膜结构域, 含有1个N-糖基化位点(图3, 虚线框所示), 36个磷酸化位点, 主要集中在5~40、45~90、145~180、205~220位氨基酸处。氨基酸序列第228位亮氨酸(Leu)亲水性最强, 系数为-2.778; 第280位谷氨酰胺(Gln)疏水性最强, 系数为2.856。疏水性分析显示, 该蛋白平均负峰值个数显著多于正峰个数, 说明它们亲水性较好, 属于可溶性蛋白。
2.4 BoGSTL21蛋白系统进化分析
对BoGSTL21与其他物种GSTL2同源蛋白的系统进化树分析发现, BoGSTL21与甘蓝型油菜BnGSTL2亲缘关系最近, 其相似度达97.02%; 与荠菜CrGSTL2亲缘关系最远, 为70.23% (图4)。结合NCBI数据库将BoGSTL21进化关系最近的甘蓝BoGSTL2、白菜BrGSTL2、甘蓝型油菜BnGSTL2和萝卜RsGSTL2进行多序列比对分析发现, 它们的序列在GST-N和GST-C区域高度保守(图5)。表明GSTL2蛋白均具有功能一致的细胞响应保守蛋白元件(包括花粉刺激等外界非生物胁迫)。图4
新窗口打开|下载原图ZIP|生成PPT图4BoGSTL21与其他物种GSTL2氨基酸序列的系统进化树
Fig. 4Phylogenetic tree of BoGSTL21 and other species GSTL2 amino acid sequences
图5
新窗口打开|下载原图ZIP|生成PPT图5甘蓝BoGSTL21与其他物种的同源蛋白氨基酸序列比对
黑色: 氨基酸一致性为100%; 深灰色: 氨基酸一致性为75%; 白色: 氨基酸一致性为50%。三角形标记BoGSTL21和BoGSTL2的不同之处; 下画直线为GST-N端结构域, 下画虚线为GST-C端结构域, 同时也代表该蛋白在这一区段保守性高。
Fig. 5Alignment of BoGSTL21 of Brassica oleracea with homologous proteins of other species
Black: amino acid identity 100%; Dark gray: amino acid identity 75%; White: amino acid identity 50%. The triangle marks the difference between BoGSTL21 and BoGSTL2; the underlined line denotes the GST-N terminal domain, and the underlined line denotes the GST-C terminal domain, which also indicates that the protein is highly conserved in this segment.
2.5 BoGSTL21启动子的特异性分析
对BoGSTL21基因启动子(起始密码子上游2000 bp的核苷酸序列)分析发现, 其包含了光响应、茉莉酸响应、水杨酸反应、生长素应答、赤霉素反应、脱落酸反应、低温和干旱诱导的反应等多种顺式作用元件(表2), 表明该基因除了响应授粉刺激, 还可能响应多种信号。Table 2
表2
表2BoGSTL21基因上游调控区顺式作用元件
Table 2
| 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(ABA)应答 Abscisic acid response | ABRE |
| 光响应 Light response | G-Box, GT1-motif, TCT-motif |
| 低氧诱导 Anaerobic induction response | ARE |
| 促进和增强基因转录 Promote and enhance gene transcription | CAAT-box |
| 赤霉素应答 Gibberellin-response | GARE-motif, P-box |
| MYBHv1结合位点 MYBHv1 binding site | CCAAT-box |
| 涉及光响应的MYBHv1结合位点 MYB binding site involved in light response | MRE |
| 转录启动约-30的核心启动子 Core promoter element around -30 of transcription start | TATA-box |
| 低温响应 Low-temperature response | LTR |
| 干旱诱导 Drought induced response | MBS |
| 水杨酸(SA)反应 Salicylic acid response | TCA-element |
| MeJA茉莉酸响应 MeJA-response | CGTCA-motif, TGACG-motif |
| 生长素(IAA)应答 Auxin-response | TGA-element |
新窗口打开|下载CSV
2.6 BoGSTL21基因的表达模式分析
BoGSTL21基因在甘蓝的不同组织中均有表达, 且表达量有差异(图6)。在萼片中的表达量最高, 柱头、叶片表达量次之, 且它们之间表达量相差不大, 而在花瓣、花药、茎和花蕾中表达相对较低。图6
新窗口打开|下载原图ZIP|生成PPT图6BoGSTL21基因在不同组织中的表达分析
Fig. 6Expression analysis of BoGSTL21 in different organs
本研究从甘蓝基因组gDNA中扩增BoGSTL21起始密码子上游1.5 kb的启动子区域, 将其与β-葡糖醛酸糖苷酶(GUS)报告基因融合后转化野生型拟南芥植株, 筛选获得了转基因T2代阳性植株。将阳性植株的拟南芥种子、幼苗、莲座期叶片、花期花蕾和果荚等浸没到GUS染液中进行GUS染色分析发现, 在种子的发育阶段, BoGSTL21基因在刚刚萌发的种子中有表达, 但表达量不高(图7-a, c); 在幼苗的发育阶段, BoGSTL21基因主要在子叶中表达(图7-d); 在叶片中, BoGSTL21基因主要在成熟叶的叶脉中表达(图7-e, f); 在整个花的发育过程中, BoGSTL21基因在柱头和萼片中均有表达, 柱头中的表达量随着发育时间而变化, 并且主要在成熟的柱头中高表达(图7-g~i); BoGSTL21基因在花粉中有低表达(图7-g~i)。在果荚发育阶段, BoGSTL21基因在未成熟的果荚中高表达, 随着果荚发育表达量降低, 在果荚后期集中在顶部和基部表达(图7-i, j)。
图7
新窗口打开|下载原图ZIP|生成PPT图7GUS染色分析
a~c: 种子不同发育时期; d, e: 幼苗不同发育时期; f: 叶的不同发育时期; g~i: 花的不同发育时期; j, k: 果荚的不同发育时期。
Fig. 7GUS staining analysis
a-c: different stages of seed development; d, e: different stages of seedling development; f: different stages of leaf development; g-i: different stages of flower development; j, k: different stages of fruit pod development.
对不同授粉处理后柱头中BoGSTL21基因的qRT-PCR分析发现, BoGSTL21基因在自花授粉后表达趋势为先上调后下调, 异花授粉后变化不大。由图8可知, 自花授粉30 min后相对表达量约为6.2, 而异花授粉30 min后相对表达量约为0.73, 前者明显高于后者且相差8.5倍。此时正是SI反应的关键时期, 在自花授粉30 min时能显著诱导柱头BoGSTL21表达, 该结果与转录组分析结果一致, 表明BoGSTL21基因响应甘蓝自花授粉后的反应。
图8
新窗口打开|下载原图ZIP|生成PPT图8BoGSTL21基因在不同授粉处理后柱头组织中的表达分析
SP: 自花授粉; CP: 异花授粉。
Fig. 8Expression analysis of BoGSTL21 in stigma past pollination under bots self and cross pollination conditions
SP: self-pollination; CP: cross-pollination.
2.7 BoGSTL21与BoFAB1C、BoPATL2和BoF9N12_9之间的相互作用
将成功构建pGBDT7-BoGSTL21重组质粒与pGADT7空载体共转化酵母Y2Hgold感受态细胞发现, 融合蛋白pGBDT7-BoGSTL21在DDO (SD/-Leu/ -Trp)平板上有单菌落长出, 在QDO (SD/-Leu/-Trp/ -His/-Ade/)平板上无菌落长出, 与阴性对照结果一致。表明诱饵蛋白BoGSTL21没有自激活活性。进而以BoGSTL21为诱饵蛋白进行酵母文库筛选, 经测序比对得到3个潜在的候选蛋白(表3)。Table 3
表3
表3候选蛋白的功能注释分析
Table 3
| 候选蛋白Candidate protein | 蛋白名称 Protein name | 功能注释 Functional annotations |
|---|---|---|
| BoFAB1C | 1-磷脂酰肌醇-3-磷酸5-激酶 1-phosphatidylinositol-3- phosphate 5-kinase | 具有1-磷脂酰肌醇-3-磷酸5-激酶活性, ATP结合,参与磷脂酰肌醇磷酸化, 花粉发育, 气孔关闭的生物过程。 It has 1-phosphatidylinositol-3-phosphate 5-kinase activity, ATP binding, participates in the biological processes of phosphatidylinositol phosphorylation, pollen development, and stomatal closure. |
| BoPATL2 | patellin-2 | PATL属于具有高尔基动力学(GOLD)结构域和Sec14p-like结构域串联的蛋白质家族。PATL受生长素调节。 PATL belongs to a family of proteins with a Golgi dynamics (GOLD) domain and a Sec14p-like domain tandem. PATL is regulated by auxin. |
| BoF9N12_9 | 磷酸核糖3-差向异构酶 Ribulose-phosphate 3-epimerase | 属于醛缩酶型TIM桶家族蛋白, 参与碳水化合物代谢过程, 磷酸戊糖途径, 氨基酸的生物合成。 Belongs to the aldolase type TIM barrel family protein, involved in carbohydrate metabolism, pentose phosphate pathway, amino acid biosynthesis. |
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将得到的候选蛋白连接pGADT7构建重组质粒, 以阴阳对照载体和重组载体组合pGADT7 (BoFAB1C、BoPATL2、BoF9N12_9)与目的质粒pGBKT7- BoGSTL21共转化酵母感受态细胞, 涂布于DDO固体平板上, 30℃倒置培养3~5 d, 观察发现DDO平板上有菌落生成(图9)。通过PCR检测, 均能扩增出目的条带, 说明重组质粒成功转入酵母细胞。为进一步检测蛋白质之间的相互作用, 将DDO平板上的单菌落转移至QDO平板上, 30℃倒置培养3 d后发现, 阴性对照在QDO平板上不能生长, 颜色显无色(图9), BoFAB1C/BoGSTL21、BoPATL2/BoGSTL21、BoF9N12_ 9/BoGSTL21和阳性对照均能在平板上生长, 并出现白色反应(表4)。表明BoGSTL21与BoFAB1C、BoPATL2、BoF9N12_9之间存在相互作用。
Table 4
表4
表4质粒共转化酵母的相互作用分析
Table 4
| 编号 No. | 酵母菌种(质粒) Yeast 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-BoFAB1C×pGBKT7-BoGSTL21) | SD/-Leu/-Trp | 是Yes | 白色White |
| 4 | Y2HGold (pGADT7-BoPATL2×pGBKT7-BoGSTL21) | SD/-Leu/-Trp | 是Yes | 白色White |
| 5 | Y2HGold (pGADT7-BoF9N12×pGBKT7-BoGSTL21) | SD/-Leu/-Trp | 是Yes | 白色White |
| 6 | Y2HGold (pGADT7-T×pGBKT7-53) | SD/-Ade/-His/-Leu/-Trp | 是Yes | 白色White |
| 7 | Y2HGold (pGADT7-T×pGBKT7-Lam) | SD/-Ade/-His/-Leu/-Trp | 是Yes | 无色No |
| 8 | Y2HGold (pGADT7-BoFAB1C×pGBKT7-BoGSTL21) | SD/-Ade/-His/-Leu/-Trp | 是Yes | 白色White |
| 9 | Y2HGold (pGADT7-BoPATL2×pGBKT7-BoGSTL21) | SD/-Ade/-His/-Leu/-Trp | 是Yes | 白色White |
| 10 | Y2HGold (pGADT7-BoF9N12×pGBKT7-BoGSTL21) | SD/-Ade/-His/-Leu/-Trp | 是Yes | 白色White |
新窗口打开|下载CSV
图9
新窗口打开|下载原图ZIP|生成PPT图9酵母双杂交验证BoFAB1C/BoGSTL21、BoPATL2/ BoGSTL21、BoF9N12_9/BoGSTL21之间的相互作用
第1~3排表示pGADT7-BoFAB1C/BoPATL2/BoF9N12_9× pGBDT7- BoGSTL21; 第4排表示阳性对照(pGADT7-T× pGBDT7-p53); 第5排表示阴性对照(pGADT7-T×pGBDT7-Lam)。
Fig. 9Interaction of BoFAB1C/BoGSTL21, BoPATL2/BoGSTL 21, and BoF9N12_9/BoGSTL21 in yeast two-hybrid assay
The first to third row indicates pGADT7-BoFAB1C/BoPATL2/ BoF9N12_9×pGBDT7-BoGSTL21; the fourth row indicates positive control (pGADT7-T×pGBDT7-p53); the fifth row indicates negative control (pGADT7-T×pGBDT7-Lam). DDO: SD/-Leu/-Trp; QDO: SD/-Leu/-Trp/-His/-Ade/.
2.8 BoGSTL21原核表达
将BoGSTL21-pGEX-4T-1重组菌株扩大培养, 并在16℃过夜诱导表达, 经海狸GST融合蛋白纯化磁珠纯化目的蛋白, SDS-PAGE电泳, 所得结果如图10所示; 与未诱导的BoGSTL21-pGEX-4T-1 (泳道1)、诱导的BoGSTL21-pGEX-4T-1 (泳道2)、纯化的pGEX-4T-1空载(泳道3)相比, 泳道4在60 kD左右具有单一纯化条带, 其蛋白质相对分子质量与BoGSTL21-pGEX-4T-1估计值相符。其中pGEX-4T-1空载体表达约26 kD的蛋白, BoGSTL21蛋白预测的分子大小约为34 kD, 二者融合约为60 kD的蛋白, 表明BoGSTL21-pGEX-4T-1融合蛋白在原核细胞中诱导表达成功。图10
新窗口打开|下载原图ZIP|生成PPT图10BoGSTL21蛋白的原核表达
M: 蛋白分子量标准; 1: BoGSTL21-pGEX-4T-1未诱导蛋白; 2: BoGSTL21-pGEX-4T-1诱导蛋白; 3: pGEX-4T-1蛋白; 4: BoGSTL21-pGEX-4T-1纯化蛋白。
Fig. 10Prokaryotic expression of BoGSTL21 protein
M: molecular weight standard of protein; 1: BoGSTL21-pGEX- 4T-1 is an uninduced control; 2: BoGSTL21-pGEX-4T-1 is the purified fusion protein; 3: pGEX-4T-1 protein; 4: BoGSTL21- pGEX-4T-1 purified protein.
3 讨论
本研究基于自交不亲和甘蓝柱头不同授粉处理后的转录组数据, 成功筛选到1个受自花授粉诱导的上调表达基因BoGSTL21。利用qRT-PCR验证了该基因在授粉后表达模式与转录组结果基本一致; 组织定量结果和GUS染色结果也基本一致, 表明BoGSTL21基因响应甘蓝自花授粉后的反应。BoGSTL21基因启动子分析发现, 其含有胁迫反应、生长素(IAA)、脱落酸(ABA)和代谢调节等多种顺式作用元件, 表明BoGSTL21基因可能参与调控复杂的生物反应过程。植物的谷胱甘肽转移酶(GSTs)是一个基因超家族, 分为φ、τ、ζ、θ、Lambda和脱氢抗坏血酸还原酶(DHARs) 6类[12,13]。BoGSTL21基因编码蛋白质具有典型的植物GST基因的蛋白结构特点, 其中85~162位氨基酸为GST-N端结构域, 169~290位氨基酸为GST-C端结构域, 属于Lambda类GST。Zeng 等[21]通过对甘蓝自花和异花授粉不同时间处理后的花柱提取总蛋白, 再通过蛋白质双向电泳技术及质谱分析发现, GST家族基因在自花授粉后差异表达, 被认为是一种SI相关基因。本研究中通过转录组数据得到的BoGSTL21也是一种GST基因, 说明GST作为SI相关基因很可能参与自交不亲和反应。GST在植物的初级代谢、二级代谢、胁迫耐受和细胞信号转导中行使功能, 从而影响植物的生长发育[18]。许多研究表明, GST也可以解除外界毒素以及内源有毒代谢物的侵害[9-10,12]。此外, GST家族蛋白还参与了植物中类黄酮物质的积累和转运[22]。例如玉米Bronze2是最早发现与花青素苷这一类黄酮物质转运有关的GST家族成员, 其编码谷胱甘肽转移酶GSTIII [23], 能与花青素形成复合物并参与其转运。牵牛花中An9基因编码谷胱甘肽转移酶GSTI, 也可以结合花青素苷并参与其转运[24,25]。类黄酮物质除了具有保护植物免受紫外线伤害、抵抗病原菌的侵害等功能外, 对植物生殖发育也具有重要的影响[26], 如参与花粉-柱头的相互作用的过程[27], 促进花粉萌发及影响花粉管的极性生长中起作用[28,29]。而本研究中自交不亲和反应的本质是花粉与柱头识别及信号传导的过程, 表明BoGSTL21基因也可能是通过参与类黄酮物质的积累和转运, 影响类黄酮物质参与花粉-柱头的相互作用的过程, 从而参与自交不亲和反应。
本研究以BoGSTL21为诱饵蛋白, 利用甘蓝酵母文库筛选到3个互作蛋白, 分别为BoFAB1C、BoF9N12_9、BoPATL2。FAB1C编码1-磷脂酰肌醇- 3-磷酸5-激酶, 具有1-磷脂酰肌醇-3-磷酸5-激酶活性, FAB1在拟南芥花粉管生长和受精中发挥作用[30], FAB1A/B的功能丧失和功能获得突变会损害内膜稳态, 从而使拟南芥的多效性发育异常[31], 拟南芥FAB1/PIKfyve蛋白对于存活花粉的发育至关重要[32]。F9N12_9属于醛缩酶型TIM桶家族蛋白, 参与碳水化合物代谢过程、磷酸戊糖途径、氨基酸的生物合成。表明BoGSTL21参与多种复杂的生物反应过程。PATL2属于具有高尔基动力学(GOLD)结构域和Sec14p-like结构域串联的蛋白质家族。四重突变体(patl2456)在根内胚层细胞中显示PIN1侧向改变, PATELLINS是拟南芥中生长素介导PIN1重定位和植物发育的调节剂[33]。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, 自交不亲和性与生长素含量之间存在负相关关系[34]。通过改变可可树中生长素浓度梯度可能会影响或控制自交不亲和性[35]。表明BoGSTL21蛋白可能通过与PATL2蛋白发生相互作用, 进而调控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[36,37]。植物中GSTs可作为IAA的载体, 与之形成植物激素结合蛋白[38,39,40]。表明也可能是BoGSTL21蛋白作为IAA的载体, 形成植物激素结合蛋白, 引起生长素浓度变化, 从而参与自交不亲和反应。
BoGSTL21基因在自花授粉过程中0~60 min上调表达, 在异花授粉过程中变化不大。而甘蓝自交不亲和反应主要在开花后30~60 min之内完成[41], 表明该基因可能参与自花授粉后花粉与柱头相互作用的复杂反应过程。本试验成功克隆了甘蓝的GSTL21基因, 通过分析其氨基酸序列的生物信息学、表达特性及酵母互作, 为了解GSTL21基因与自交不亲和性的联系奠定了基础, 为SI的深入研究提供了新内容。
4 结论
从高代自交不亲和甘蓝‘A4’材料自花和异花授粉处理的柱头转录组数据中筛选到1个差异表达的基因BoGSTL21, 该基因的开放阅读框长度为900 bp, 编码299个氨基酸, 含有GST-N和GST-C结构域, 其编码蛋白大小为34 kD。BoGSTL21基因在不同组织中均有表达, 在花的发育阶段, 柱头中的表达量随发育时间而变化, 且在成熟的柱头中高表达。该基因0~60 min上调表达, 在异花授粉过程中表达量变化并不明显。BoGSTL21蛋白与BoFAB1C、BoF9N12_9、BoPATL2相互作用, 参与多种复杂的生物过程, 包含SI反应过程。BoGSTL21可能通过多种途径如参与黄酮类物质的积累和运转或作为IAA的载体或与生长素相关蛋白PATL2互作, 参与柱头响应自花花粉刺激的分子过程, 推测可能是实现参与SI相关过程的新蛋白。参考文献 原文顺序
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被引期刊影响因子
DOI:10.1016/j.pbi.2011.11.001URLPMID:22153653 [本文引用: 1]
Double fertilization is a flowering plant mechanism whereby two immotile sperm cells fertilize two different female gametes. One of the two sperm cells fertilizes the egg cell to produce the embryo and the other fertilizes the central cell to produce the endosperm. Despite the biological and agricultural significance of double fertilization, the mechanism remains largely unknown owing to difficulties associated with the embedded structure of female gametes in the maternal tissue. However, molecular genetic approaches combined with novel live-cell imaging techniques have begun to clarify the actual behavior of the sperm cells, which is different from that described by previous hypotheses. In this review article, we discuss the mechanism of double fertilization based on the dynamics of the two sperm cells in Arabidopsis.
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:10.1104/pp.103.023846URLPMID:14555783 [本文引用: 1]
Recognition of self-pollen during the self-incompatibility response in Brassica oleracea is mediated by the binding of a secreted peptide (the S locus cysteine-rich protein) to the S locus receptor kinase (SRK), a member of the plant receptor kinase (PRK) superfamily. Here, we describe the characterization of three proteins that interact with the cytosolic kinase domain of SRK. A B. oleracea homolog of Arabidopsis kinase-associated protein phosphatase was shown to interact with and dephosphorylate SRK and was itself phosphorylated by SRK. Yeast (Saccharomyces cerevisiae) two-hybrid screens identified two additional interactors, calmodulin and a sorting nexin, both of which have been implicated in receptor kinase down-regulation in animals. A calmodulin-binding site was identified in sub-domain VIa of the SRK kinase domain. The binding site is conserved and functional in several other members of the PRK family. The sorting nexin also interacted with diverse members of the PRK family, suggesting that all three of the interacting proteins described here may play a general role in signal transduction by this family of proteins.
DOI:10.1105/tpc.009845URLPMID:12671085 [本文引用: 1]
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.
DOI:10.1126/science.1072205URLPMID:12114625 [本文引用: 1]
Transitions from cross-fertilizing to self-fertilizing mating systems have occurred frequently in natural and domesticated plant populations, but the underlying genetic causes are unknown. We show that gene transfer of the stigma receptor kinase SRK and its pollen-borne ligand SCR from one S-locus haplotype of the self-incompatible and cross-fertilizing Arabidopsis lyrata is sufficient to impart self-incompatibility phenotype in self-fertile Arabidopsis thaliana, which lacks functional orthologs of these genes. This successful complementation demonstrates that the signaling cascade leading to inhibition of self-related pollen was maintained in A. thaliana. Analysis of self-incompatibility will be facilitated by the tools available in this species.
DOI:10.1073/pnas.0406970101URLPMID:15505209 [本文引用: 1]
The switch from an out-crossing to a self-fertilizing mating system is one of the most prevalent evolutionary trends in plant reproduction and is thought to have occurred repeatedly in flowering plants. However, little is known about the evolution of self-fertility and the genetic architecture of selfing. Here, we establish Arabidopsis thaliana as a model for genetic analysis of the switch to self-fertility in the crucifer family, where the ancestral out-crossing mode of mating is determined by self-incompatibility (SI), a genetic system controlled by the S locus. We show that A. thaliana ecotypes exhibit S-locus polymorphisms and differ in their ability to express the SI trait upon transformation with S-locus genes derived from the obligate out-crosser Arabidopsis lyrata. Remarkably, at least one ecotype was reverted to a stable, self-incompatible phenotype identical to that of naturally self-incompatible species. These ecotype differences are heritable and reflect the fixation in different A. thaliana populations of independent mutations that caused or enforced the switch to self-fertility. Their continued analysis promises to identify the loci that were the targets of natural selection for selfing and to contribute to a mechanistic understanding of the SI response.
DOI:10.1105/tpc.114.129387URL [本文引用: 1]
Self-incompatibility (SI) is the primary determinant of the outbreeding mode of sexual reproduction in the Brassicaceae. All Arabidopsis thaliana accessions analyzed to date carry mutations that disrupt SI functions by inactivating the SI specificity-determining S locus or SI modifier loci. S-locus genes isolated from self-incompatible close relatives of A. thaliana restore robust SI in several accessions that harbor only S-locus mutations and confer transient SI in accessions that additionally harbor mutations at modifier loci. Self-incompatible transgenic A. thaliana plants have proved to be valuable for analysis of the recognition and signaling events that underlie SI in the Brassicaceae. Here, we review results demonstrating that S-locus genes are necessary and sufficient for SI signaling and for restoration of a strong and developmentally stable SI phenotype in several accessions of A. thaliana. The data indicate that introduction of a functional E3 ligase-encoding ARC1 gene, which is deleted in all accessions that have been analyzed to date, is not required for SI signaling leading to inhibition of self pollen or for reversion of A. thaliana to its fully self-incompatible ancestral state.
DOI:10.1104/pp.46.1.103URLPMID:16657398 [本文引用: 1]
Glutathione conjugation (GS-atrazine) of the herbicide, 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) is another major detoxication mechanism in leaf tissue of corn (Zea mays, L.). The identification of GS-atrazine is the first example of glutathione conjugation as a biotransformation mechanism of a pesticide in plants. Recovery of atrazine-inhibited photosynthesis was accompanied by a rapid conversion of atrazine to GS-atrazine when the herbicide was introduced directly into leaf tissue. N-De-alkylation pathway is relatively inactive in both roots and shoots. The nonenzymatic detoxication of atrazine to hydroxyatrazine is negligible in leaf tissue. The hydroxylation pathway contributed significantly to the total detoxication of atrazine only when the herbicide was introduced into the plant through the roots. The metabolism of atrazine to GS-atrazine may be the primary factor in the resistance of corn to atrazine.
DOI:10.1016/S0076-6879(05)01011-6URLPMID:16399386 [本文引用: 2]
Soluble plant glutathione transferases (GSTs) consist of seven distinct classes, six of which have been functionally characterized. The phi and tau class GSTs are specific to plants and the most numerous and abundant of these enzymes. Both have classic conjugating activities toward a diverse range of xenobiotics, including pesticides, where they are major determinants of herbicide selectivity in crops and weeds. In contrast, the zeta and theta class GSTs are conserved in animals and plants and have very restricted activities toward xenobiotics. Theta GSTs function as glutathione peroxidases, reducing organic hydroperoxides produced during oxidative stress. Zeta GSTs act as glutathione-dependent isomerases, catalyzing the conversion of maleylacetoacetate to fumarylacetoacetate, the penultimate step in tyrosine degradation. The other two classes of plant GSTs, the dehydroascorbate reductases (DHARs) and lambda GSTs, differ from phi, tau, zeta, and theta enzymes in being monomers rather than dimers and possessing a catalytic cysteine rather than serine in the active site. Both can function as thioltransferases, with the DHARs having a specialized function in reducing dehydroascorbate to ascorbic acid. The determination of the diverse plant-specific functions of the differing GST classes is described.
DOI:10.1016/s0014-5793(03)01077-9URLPMID:14572664 [本文引用: 2]
Glutathione S-transferases (GSTs) have poorly understood roles in plant responses to environmental stresses. A polyethylene glycol (PEG)-induced tau class GST was identified in rice roots by protein microsequencing. PEG and the heavy metals Cd (20 microM), Zn (30 microM), Co and Ni rapidly and markedly induced osgstu4 and osgstu3 in rice seedling roots. Osgstu4 and osgstu3 were also induced in roots by hypoxic stress but not by cold nor heat shock. Salt stress and abscisic acid (ABA) rapidly induced osgstu3 in rice roots, whereas osgstu4 exhibited a late salt stress and no ABA response. Salicylic acid, jasmonic acid and the auxin alpha-naphthalene acetic acid triggered osgstu4 and osgstu3 expression. Osgstu4 and osgstu3 were rapidly and markedly induced by the antioxidant dithiothreitol and the strong oxidant hydrogen peroxide, which suggested that redox perturbations and reactive oxygen species are involved in their stress response regulations.
DOI:10.1007/s00438-004-1006-8URLPMID:15069639 [本文引用: 1]
Glutathione S-transferases (GSTs) comprise a large family of key defence enzymes against xenobiotic toxicity. Here we describe the comprehensive characterisation of this important multigene family in the model monocot species rice [ Oryza sativa(L.)]. Furthermore, we investigate the molecular evolution of the family based on the analysis of (1) the patterns of within-genome duplication, and (2) the phylogenetic relationships and evolutionary divergence among rice, Arabidopsis, maize and soybean GSTs. By in-silico screening of the EST and genome divisions of the Genbank/EMBL/DDBJ database we have isolated 59 putative genes and two pseudogenes, making this the largest plant GST family characterised to date. Of these, 38 (62%) are represented by genomic and EST sequences and 23 (38%) are known only from their genomic sequences. A preliminary survey of EST collections shows a large degree of variability in gene expression between different tissues and environmental conditions, with a small number of genes (13) accounting for 80% of all ESTs. Rice GSTs are organised in four main phylogenetic classes, with 91% of all rice genes belonging to the two plant-specific classes Tau (40 genes) and Phi (16 genes). Pairwise identity scores range between 17 and 98% for proteins of the same class, and 7 and 21% for interclass comparisons. Rapid evolution by gene duplication is suggested by the discovery of two large clusters of 7 and 23 closely related genes on chromosomes 1 and 10, respectively. A comparison of the complete GST families in two monocot and two dicot species suggests a monophyletic origin for all Theta and Zeta GSTs, and no more than three common ancestors for all Phi and Tau genes.
DOI:10.1074/jbc.M202919200URLPMID:12077129 [本文引用: 4]
Searches with the human Omega glutathione transferase (GST) identified two outlying groups of the GST superfamily in Arabidopsis thaliana which differed from all other plant GSTs by containing a cysteine in place of a serine at the active site. One group consisted of four genes, three of which encoded active glutathione-dependent dehydroascorbate reductases (DHARs). Two DHARs were predicted to be cytosolic, whereas the other contained a chloroplast targeting peptide. The DHARs were also active as thiol transferases but had no glutathione conjugating activity. Unlike most other GSTs, DHARs were monomeric. The other class of GST comprised two genes termed the Lambda GSTs (GSTLs). The recombinant GSTLs were also monomeric and had glutathione-dependent thiol transferase activity. One GSTL was cytosolic, whereas the other was chloroplast-targeted. When incubated with oxidized glutathione, the putative active site cysteine of the GSTLs and cytosolic DHARs formed mixed disulfides with glutathione, whereas the plastidic DHAR formed an intramolecular disulfide. DHAR S-glutathionylation was consistent with a proposed catalytic mechanism for dehydroascorbate reduction. Roles for the cytosolic DHARs and GSTLs as antioxidant enzymes were also inferred from the induction of the respective genes following exposure to chemicals and oxidative stress.
DOI:10.1016/S0083-6729(05)72005-7URLPMID:16492471 [本文引用: 2]
Plant glutathioneS-transferases (GSTs) are a heterogeneous superfamily of multifunctional proteins, grouped into six classes. The tau (GSTU) and phi (GSTF) class GSTs are the most represented ones and are plant-specific, whereas the smaller theta (GSTT) and zeta (GSTZ) classes are also found in animals. The lambda GSTs (GSTL) and the dehydroascorbate reductases (DHARs) are more distantly related. Plant GSTs perform a variety of pivotal catalytic and non-enzymatic functions in normal plant development and plant stress responses, roles that are only emerging. Catalytic functions include glutathione (GSH)-conjugation in the metabolic detoxification of herbicides and natural products. GSTs can also catalyze GSH-dependent peroxidase reactions that scavenge toxic organic hydroperoxides and protect from oxidative damage. GSTs can furthermore catalyze GSH-dependent isomerizations in endogenous metabolism, exhibit GSH-dependent thioltransferase safeguarding protein function from oxidative damage and DHAR activity functioning in redox homeostasis. Plant GSTs can also function as ligandins or binding proteins for phytohormones (i.e., auxins and cytokinins) or anthocyanins, thereby facilitating their distribution and transport. Finally, GSTs are also indirectly involved in the regulation of apoptosis and possibly also in stress signaling. Plant GST genes exhibit a diversity of expression patterns during biotic and abiotic stresses. Stress-induced plant growth regulators (i.e., jasmonic acid [JA], salicylic acid [SA], ethylene [ETH], and nitric oxide [NO] differentially activate GST gene expression. It is becoming increasingly evident that unique combinations of multiple, often interactive signaling pathways from various phytohormones and reactive oxygen species or antioxidants render the distinct transcriptional activation patterns of individual GSTs during stress. Underestimated post-transcriptional regulations of individual GSTs are becoming increasingly evident and roles for phytohormones (i.e., ABA and JA) in these processes are being anticipated as well. Finally, indications are emerging that NO may regulate the activity of specific plant GSTs. In this review, the current knowledge on the regulatory and functional interactions of phytohormones and plant GSTs are covered. We refer to a previous extensive review on plant GSTs (Marrs, 1996) for most earlier work. An introduction on the classification and roles of plant GSTs is included here, but these topics are more extensively discussed in other reviews (Dixon et al., 2002a; Edwards et al., 2000; Frova, 2003).
DOI:10.1146/annurev.arplant.47.1.127URLPMID:15012285 [本文引用: 1]
Glutathione S-transferases (GSTs) play roles in both normal cellular metabolism as well as in the detoxification of a wide variety of xenobiotic compounds, and they have been intensively studied with regard to herbicide detoxification in plants. A newly discovered plant GST subclass has been implicated in numerous stress responses, including those arising from pathogen attack, oxidative stress, and heavy-metal toxicity. In addition, plant GSTs play a role in the cellular response to auxins and during the normal metabolism of plant secondary products like anthocyanins and cinnamic acid. This review presents the current knowledge about the functions of GSTs in regard to both herbicides and endogenous substrates. The catalytic mechanism of GST activity as well as the fate of glutathione S-conjugates are reviewed. Finally, a summary of what is known about the gene structure and regulation of plant GSTs is presented.
DOI:10.1016/s1360-1385(00)01601-0URLPMID:10785664 [本文引用: 1]
Glutathione S-transferases (GSTs) are abundant proteins encoded by a highly divergent, ancient gene family. Soluble GSTs form dimers, each subunit of which contains active sites that bind glutathione and hydrophobic ligands. Plant GSTs attach glutathione to electrophilic xenobiotics, which tags them for vacuolar sequestration. The role of GSTs in metabolism is unclear, although their complex regulation by environmental stimuli implies that they have important protective functions. Recent studies show that GSTs catalyse glutathione-depend-ent isomerizations and the reduction of toxic organic hydroperoxides. GSTs might also have non-catalytic roles as carriers for phytochemicals.
DOI:10.1023/a:1006257305725URLPMID:10527424 [本文引用: 1]
Two cDNAs encoding novel type III maize (Zea mays) GST subunits, ZmGST VI and ZmGST VII, have been cloned in addition to the previously described ZmGST V. Together with the type I GSTs ZmGST I and ZmGST III, these subunits were expressed in Escherichia coli, both individually and in tandem combinations using a customised pET vector. The GST dimers formed were then characterised. When type I GSTs were co-expressed only the respective homodimers were formed rather than the ZmGST I-III heterodimer. The failure to form this heterodimer, together with the negligible herbicide-detoxifying activity associated with recombinant ZmGST III-III, suggests that the identity of herbicide-detoxifying isoenzymes described in maize as being composed of ZmGST III subunits requires re-evaluation. In contrast, co-expression of the type III GSTs ZmGST V and ZmGST VI resulted in the formation of ZmGST V-V, ZmGST VI-VI and ZmGST V-VI dimers in the ratio 1:1:2 as predicted for random subunit association. ZmGST V-VI had kinetic characteristics intermediate between those of the two homodimers, indicating that the subunits were catalytically independent of one another. Co-expression of ZmGST V and ZmGST VII resulted in the formation of ZmGST V-VII and this isoenzyme was subsequently identified in maize plants. Attempts to dimerise type I GST subunits with type III GST subunits proved unsuccessful. These results demonstrate the utility of co-expressing recombinant GSTs to explore the potential of subunit-subunit associations and to help unravel the complexity of homodimeric and heterodimeric GSTs in plants.
DOI:10.1006/pest.1999.2396URL [本文引用: 1]
[本文引用: 4]
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DOI:10.1007/BF00040624URL [本文引用: 1]
DOI:10.1038/nprot.2007.17URLPMID:17401330 [本文引用: 1]
Here we describe a protocol for the production of frozen competent yeast cells that can be transformed with high efficiency using the lithium acetate/single-stranded carrier DNA/PEG method. This protocol allows the production of highly competent yeast cells that can be frozen and used at a later date and is especially useful for laboratories using one or two strains repeatedly. The production of yeast cells for freezing takes only approximately 30 min, once the yeast culture has grown up. Transformation with frozen competent yeast cells will take approximately 30 min, depending on the heat shock used.
DOI:10.1007/s10930-017-9697-yURLPMID:28299594 [本文引用: 1]
Angiosperms have developed self-incompatibility (SI) systems to reject self-pollen, thereby promoting outcrossing. The Brassicaceae belongs to typical sporophytic system, having a single S-locus controlled SI response, and was chosen as a model system to study SI-related intercellular signal transduction. In this regard, the downstream factor of EXO70A1 was unknown. Here, protein two-dimensional electrophoresis (2-DE) method and coupled with matrix-assisted laser desorption ionization/time of flight of flight mass spectrometry (MALDI-TOF -MS) and peptide mass fingerprinting (PMF) was used to further explore the mechanism of SI responses in Brassica oleracea L. var. capitata L. at protein level. To further confirm the time point of protein profile change, total proteins were collected from B. oleracea pistils at 0 min, 1 h, and 2 h after self-pollination. In total 902, 1088 and 1023 protein spots were separated in 0 min, 1 h and 2 h 2-DE maps, respectively. Our analyses of self-pollination profiles indicated that proteins mainly changed at 1 h post-pollination in B. oleracea. Moreover, 1077 protein spots were separated in cross-pollinated 1 h (CP) pistil 2-DE map. MALDI-TOF-MS and PMF successfully identified 34 differentially-expressed proteins (DEPs) in SP and CP 1 h 2-DE maps. Gene ontology and KEGG analysis revealed an array of proteins grouped in the following categories: stress and defense response (35%), protein metabolism (18%), carbohydrate and energy metabolism (12%), regulation of translation (9%), pollen tube development (12%), transport (9%) and cytoskeletal (6%). Sets of DEPs identified specifically in SP or only up-regulated expressed in CP pistils were chosen for funther investigating in floral organs and during the process of self- and cross-pollination. The function of these DEPs in terms of their potential involvement in SI in B. oleracea is discussed.
[本文引用: 1]
DOI:10.1105/tpc.10.7.1135URLPMID:9668133 [本文引用: 1]
Glutathione S-transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 (Bz2) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1.
DOI:10.1104/pp.123.4.1561URLPMID:10938372 [本文引用: 1]
AN9 is a glutathione S-transferase from petunia (Petunia hybrida) required for efficient anthocyanin export from the site of synthesis in the cytoplasm into permanent storage in the vacuole. For many xenobiotics it is well established that a covalent glutathione (GSH) tag mediates recognition of molecules destined for vacuolar sequestration by a tonoplast-localized ATP-binding cassette pump. Here we inquired whether AN9 catalyzes the formation of GSH conjugates with flavonoid substrates. Using high-performance liquid chromatography analysis of reaction mixtures containing enzyme, GSH, and flavonoids, including anthocyanins, we could detect neither conjugates nor a decrease in the free thiol concentration. These results suggest that no conjugate is formed in vitro. However, AN9 was shown to bind flavonoids using three assays: inhibition of the glutathione S-transferase activity of AN9 toward the common substrate 1-chloro 2,4-dinitrobenzene, equilibrium dialysis, and tryptophan quenching. We conclude that AN9 is a flavonoid-binding protein, and propose that in vivo it serves as a cytoplasmic flavonoid carrier protein.
DOI:10.1105/tpc.110.080804URL [本文引用: 1]
The majority of flavonoids, such as anthocyanins, proanthocyanidins, and isoflavones, are stored in the central vacuole, but the molecular basis of flavonoid transport is still poorly understood. Here, we report the functional characterization of a multidrug and toxin extrusion transporter (MATE2), from Medicago truncatula. MATE 2 is expressed primarily in leaves and flowers. Despite its high similarity to the epicatechin 3'-O-glucoside transporter MATE1, MATE2 cannot efficiently transport proanthocyanidin precursors. In contrast, MATE2 shows higher transport capacity for anthocyanins and lower efficiency for other flavonoid glycosides. Three malonyltransferases that are coexpressed with MATE2 were identified. The malonylated flavonoid glucosides generated by these malonyltransferases are more efficiently taken up into MATE2-containing membrane vesicles than are the parent glycosides. Malonylation increases both the affinity and transport efficiency of flavonoid glucosides for uptake by MATE2. Genetic loss of MATE2 function leads to the disappearance of leaf anthocyanin pigmentation and pale flower color as a result of drastic decreases in the levels of various flavonoids. However, some flavonoid glycoside malonates accumulate to higher levels in MATE2 knockouts than in wild-type controls. Deletion of MATE2 increases seed proanthocyanidin biosynthesis, presumably via redirection of metabolic flux from anthocyanin storage.
DOI:10.1046/j.1365-313x.2003.01943.xURLPMID:14675436 [本文引用: 1]
Flavonoid compounds such as anthocyanins and proanthocyanidins (PAs; so-called condensed tannins) have a multitude of functions in plants. They must be transported from the site of synthesis in the cytosol to their final destination, the vacuoles. Three models have been proposed for sequestering anthocyanins in vacuoles, but the transport machinery for PAs is poorly understood. Novel Arabidopsis mutants, transparent testa 19 (tt19), which were induced by ion beam irradiation, showed a great reduction of anthocyanin pigments in the vegetative parts as well as brown pigments in the seed coat. The TT19 gene was isolated by chromosome walking and a candidate gene approach, and was shown to be a member of the Arabidopsis glutathione S-transferase (GST) gene family. Heterologous expression of a putative ortholog, petunia anthocyanin 9 (AN9), in tt19 complemented the anthocyanin accumulation but not the brown pigmentation in the seed coat. This suggests that the TT19 gene is required for vacuolar uptake of anthocyanins into vacuoles, but that it has also a function different from that of AN9. The depositional pattern of PA precursors in the mutant was different from that in the wild type. These results indicate that TT19 participates in the PA pathway as well as the anthocyanin pathway of Arabidopsis. As involvement of GST in the PA pathway was previously considered unlikely, the function of TT19 in the PA pathway is also discussed in the context of the putative transporter for PA precursors.
DOI:10.1146/annurev.pp.40.060189.002023URL [本文引用: 1]
DOI:10.1073/pnas.89.15.7213URLPMID:11607312 [本文引用: 1]
Chalcone synthase catalyzes the initial step of that branch of the phenylpropanoid pathway that leads to flavonoids. A lack of chalcone synthase activity has a pleiotropic effect in maize and petunia mutants: pollen fertility as well as flavonoid synthesis is disrupted. Both maize and petunia mutants are self-sterile due to a failure to produce a functional pollen tube. The finding that the mutant pollen is partially functional on wild-type stigmas led to the isolation and identification of kaempferol as a pollen germination-inducing constituent in wild-type petunia stigma extracts. We show that adding micromolar quantities of kaempferol to the germination medium or to the stigma at pollination is sufficient to restore normal pollen germination and tube growth in vitro and full seed set in vivo. Further we show that the rescue ability resides in particular structural features of a single class of compounds, the flavonol aglycones. This finding identifies another constituent of plant reproduction and suggests that addition or removal of the flavonol signal during pollen germination and tube growth provides a feasible way to control plant fertility.
DOI:10.1016/j.pbi.2005.03.005URLPMID:15860429 [本文引用: 1]
Flavonoids, usually regarded as dispensable phytochemicals derived from plant secondary metabolism, play important roles in the biology of plants by affecting several developmental processes. Bioactive flavonoids also signal to microbes, serve as allelochemicals and are important nutraceuticals in the animal diet. Despite the significant progress made in identifying flavonoid pathway genes and regulators, little is currently known about the protein targets of flavonoids in plant or animal cells. Recently, there have been advances in our understanding of the roles that flavonoids play in developmental processes of plants. The multiple cellular roles of flavonoids can reflect their chemical diversity, or might suggest the existence of cellular targets shared between many of these seemingly disparate processes.
DOI:10.1038/nplants.2015.25URLPMID:27247031 [本文引用: 1]
The concept that proteins and small RNAs can move to and function in distant body parts is well established. However, non-cell-autonomy of small RNA molecules raises the question: To what extent are protein-coding messenger RNAs (mRNAs) exchanged between tissues in plants? Here we report the comprehensive identification of 2,006 genes producing mobile RNAs in Arabidopsis thaliana. The analysis of variant ecotype transcripts that were present in heterografted plants allowed the identification of mRNAs moving between various organs under normal or nutrient-limiting conditions. Most of these mobile transcripts seem to follow the phloem-dependent allocation pathway transporting sugars from photosynthetic tissues to roots via the vasculature. Notably, a high number of transcripts also move in the opposite, root-to-shoot direction and are transported to specific tissues including flowers. Proteomic data on grafted plants indicate the presence of proteins from mobile RNAs, allowing the possibility that they may be translated at their destination site. The mobility of a high number of mRNAs suggests that a postulated tissue-specific gene expression profile might not be predictive for the actual plant body part in which a transcript exerts its function.
DOI:10.1104/pp.110.167981URL [本文引用: 1]
In eukaryotic cells, PtdIns 3,5-kinase, Fab1/PIKfyve produces PtdIns (3,5) P-2 from PtdIns 3-P, and functions in vacuole/lysosome homeostasis. Herein, we show that expression of Arabidopsis (Arabidopsis thaliana) FAB1A/B in fission yeast (Schizosaccharomyces pombe) fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations of FAB1A and FAB1B fused with green fluorescent protein revealed that FAB1A/B-green fluorescent proteins localize to the endosomes in root epidermal cells of Arabidopsis. Furthermore, reduction in the expression levels of FAB1A/B by RNA interference impairs vacuolar acidification and endocytosis. These results indicate that Arabidopsis FAB1A/B functions as PtdIns 3,5-kinase in plants and in fission yeast. Conditional knockdown mutant shows various phenotypes including root growth inhibition, hyposensitivity to exogenous auxin, and disturbance of root gravitropism. These phenotypes are observed also in the overproducing mutants of FAB1A and FAB1B. The overproducing mutants reveal additional morphological phenotypes including dwarfism, male-gametophyte sterility, and abnormal floral organs. Taken together, this evidence indicates that imbalanced expression of FAB1A/B impairs endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification, which causes pleiotropic developmental phenotypes mostly related to the auxin signaling in Arabidopsis.
DOI:10.1104/pp.109.146159URLPMID:19846542 [本文引用: 1]
Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)] is a phospholipid that has a role in controlling membrane trafficking events in yeast and animal cells. The function of this lipid in plants is unknown, although its synthesis has been shown to be up-regulated upon osmotic stress in plant cells. PtdIns(3,5)P(2) is synthesized by the PIKfyve/Fab1 family of proteins, with two orthologs, FAB1A and FAB1B, being present in Arabidopsis (Arabidopsis thaliana). In this study, we attempt to address the role of this lipid by analyzing the phenotypes of plants mutated in FAB1A and FAB1B. It was not possible to generate plants homozygous for mutations in both genes, although single mutants were isolated. Both homozygous single mutant plant lines exhibited a leaf curl phenotype that was more marked in FAB1B mutants. Genetic transmission analysis revealed that failure to generate double mutant lines was entirely due to inviability of pollen carrying mutant alleles of both FAB1A and FAB1B. This pollen displayed severe defects in vacuolar reorganization following the first mitotic division of development. The presence of abnormally large vacuoles in pollen at the tricellular stage resulted in the collapse of the majority of grains carrying both mutant alleles. This demonstrates a crucial role for PtdIns(3,5)P(2) in modulating the dynamics of vacuolar rearrangement essential for successful pollen development. Taken together, our results are consistent with PtdIns(3,5)P(2) production being central to cellular responses to changes in osmotic conditions.
DOI:10.1242/jcs.204198URLPMID:28687624 [本文引用: 1]
Coordinated cell polarization in developing tissues is a recurrent theme in multicellular organisms. In plants, a directional distribution of the plant hormone auxin is at the core of many developmental programs. A feedback regulation of auxin on the polarized localization of PIN auxin transporters in individual cells has been proposed as a self-organizing mechanism for coordinated tissue polarization, but the molecular mechanisms linking auxin signalling to PIN-dependent auxin transport remain unknown. We used a microarray-based approach to find regulators of the auxin-induced PIN relocation in Arabidopsis thaliana root, and identified a subset of a family of phosphatidylinositol transfer proteins (PITPs), the PATELLINs (PATLs). Here, we show that PATLs are expressed in partially overlapping cell types in different tissues going through mitosis or initiating differentiation programs. PATLs are plasma membrane-associated proteins accumulated in Arabidopsis embryos, primary roots, lateral root primordia and developing stomata. Higher order patl mutants display reduced PIN1 repolarization in response to auxin, shorter root apical meristem, and drastic defects in embryo and seedling development. This suggests that PATLs play a redundant and crucial role in polarity and patterning in Arabidopsis.
DOI:10.1111/ppl.2008.134.issue-1URL [本文引用: 1]
DOI:10.3724/SP.J.1006.2019.84129URL [本文引用: 1]
BoPINs家族参与甘蓝自交不亲和性的成员数目与参与方式, 本文通过转录组分析获得BoPINs家族在甘蓝自花和异花授粉后的表达情况, 利用分子生物学技术和生物信息学方法对该家族的基因结构、蛋白进化亲缘关系和表达模式等特征进行分析。结果表明, 甘蓝BoPINs基因家族包含8个成员, 含有5波浪线9个外显子和4~8个内含子; 其编码的蛋白质的氨基酸残基数在350波浪线650之间, 相对分子质量为38波浪线70 kD; 除了BoPIN5和BoPIN8不含中间亲水区以外, 其余6个BoPINs家族成员都含有位于两端的疏水区和中间亲水环, 它们可能以膜锚定蛋白的形式发挥作用; 甘蓝BoPINs与芜菁BrPINs、拟南芥AtPINs基因家族亲缘关系较近; 染色体定位分析表明, BoPIN1-1、BoPIN3-1、BoPIN3-2和BoPIN6与S位点之间发生不同程度的连锁; 启动子活性分析表明, BoPINs家族蛋白参与甘蓝SI反应, 可能受IAA、ABA等激素相互交叉影响; BoPIN1-1、BoPIN1-2、BoPIN2、BoPIN3-1、BoPIN3-2、BoPIN4、BoPIN6、BoPIN7-1和BoPIN7-2在柱头中表达量均较高; 数据表达谱和荧光定量分析表明, 8个家族成员中的6个BoPINs基因在自花授粉后下调表达; 自花授粉后柱头生长素含量降低, 与SI反应呈负相关。因此, 在甘蓝BoPINs家族的8个成员中有6个BoPINs基因家族成员可能在膜上以负调节方式调控自交不亲和反应。]]>
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DOI:10.1034/j.1399-3054.2001.1120115.xURLPMID:11319022 [本文引用: 1]
The time course and control of floral abscission and fruit set in Theobroma cacao were studied after spray application of growth regulators. 1-Naphthaleneacetic acid (NAA) prevented flower abscission in a concentration dependent manner and induced the early stages of fruit development. The cytokinin benzylaminopurine (BAP) counteracted NAA but resulted in longer fruit retention. Measurements of endogenous levels of indole-3-acetic acid showed an inverse correlation between the number of flowers per plant and auxin content. The results suggest that the genetic control of self-incompatibility in T. cacao may be modulated by the hormonal content of the flower.
DOI:10.1007/s00425-005-0088-9URL [本文引用: 1]
To elucidate the role of auxin in flower morphogenesis, its distribution patterns were studied during flower development in Arabidopsis thaliana (L.) Heynh. Expression of DR5::GUS was regarded to reflect sites of free auxin, while immunolocalization with auxin polyclonal antibodies visualized conjugated auxin distribution. The youngest flower bud was loaded with conjugated auxin. During development, the apparent concentration of free auxin increased in gradual patterns starting at the floral-organ tip. Anthers are major sites of high concentrations of free auxin that retard the development of neighboring floral organs in both the acropetal and basipetal directions. The IAA-producing anthers synchronize flower development by retarding petal development and nectary gland activity almost up to anthesis. Tapetum cells of young anthers contain free IAA which accumulates in pollen grains, suggesting that auxin promotes pollen-tube growth towards the ovules. High amounts of free auxin in the stigma induce a wide xylem fan immediately beneath it. After fertilization, the developing embryos and seeds show elevated concentrations of auxin, which establish their axial polarity. This developmental pattern of auxin production during floral-bud development suggests that young organs which produce high concentrations of free IAA inhibit or retard organ-primordium initiation and development at the shoot tip.]]>
DOI:10.1073/pnas.1217343109URLPMID:23129621 [本文引用: 1]
In many angiosperms, outcrossing is enforced by genetic self-incompatibility (SI), which allows cells of the pistil to recognize and specifically inhibit
DOI:10.1073/pnas.91.2.689URLPMID:8290582 [本文引用: 1]
We used 5-azido-[7-3H]indole-3-acetic acid (5-azido-[7-3H]IAA), a photoaffinity analogue of the plant hormone indole-3-acetic acid (IAA), to search for auxin-binding proteins in Arabidopsis thaliana membranes. We identified an auxin-binding protein with a molecular mass of 24 kDa (Atpm24) in microsomes as well as in plasma membrane vesicles. Atpm24 was solubilized by 1% Triton X-100 and partially purified. A cDNA clone (Atpm24.1) corresponding to Atpm24 was isolated. The amino acid sequence predicted from the Atpm24.1 cDNA contains 212 amino acid residues with a relative molecular mass of 24,128 Da. Data base searches revealed that the predicted protein has homology to glutathione S-transferases (GSTs; EC 2.5.1.18). When Atpm24.1 was expressed in Escherichia coli, we found a high level of GST activity in the bacterial extracts. We have analyzed the substrate specificity of this protein and found that cumene hydroperoxide and trans-stilbene oxide but not trans-cinnamic acid or IAA-CoA were substrates. A role for this GST in physiological processes of plants is discussed.
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DOI:10.1139/gen-2012-0088URLPMID:23379337 [本文引用: 1]
Fertilization is controlled by a complex gene regulatory network. To study the fertilization mechanism, we determined time courses of the four developmental stages of fertilization in Chinese cabbage pak-choi (Brassica campestris subsp. chinensis) by cytological observation. We then used the Arabidopsis ATH1 microarray to characterize the gene expression profiles of pollinated and unpollinated pistils in B. campestris subsp. chinensis. The result showed 44 up-regulated genes and 33 down-regulated genes in pollinated pistils compared with unpollinated pistils. Gene ontology analysis identified 20% of the up-regulated genes as belonging to the category of cell wall metabolism. We compared the up-regulated genes in pollinated pistils with previously identified pollen development related genes. Ten genes were found to be in common, which were termed as continuously expressed genes, in the two processes in the present article. Their expression patterns during pollen development and fertilization processes were then verified by RT-PCR. One of the continuously expressed genes, the homologous gene of At3g01270 in B. campestris subsp. chinensis, was confirmed as specifically expressed in microspores and pollinated pistils by using in situ hybridization. The potential biological functions of the other continuously expressed genes were also discussed.
