,1, 陈丽1,2, 陈磊1, 张盈川1, 文静1, 易斌1, 涂金星1, 傅廷栋1, 沈金雄,1,*Mechanism research of Bna-novel-miR311-HSC70-1 module regulating heat stress response in Brassica napus L.
LU Hai-Qin,1, CHEN Li1,2, CHEN Lei1, ZHANG Ying-Chuan1, WEN Jing1, YI Bin1, TU Jing-Xing1, FU Ting-Dong1, SHEN Jin-Xiong,1,*通讯作者:
收稿日期:2020-01-16接受日期:2020-06-2网络出版日期:2020-06-22
| 基金资助: |
Received:2020-01-16Accepted:2020-06-2Online:2020-06-22
| Fund supported: |
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鲁海琴, 陈丽, 陈磊, 张盈川, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄. Bna-novel-miR311-HSC70-1模块调控甘蓝型油菜响应热胁迫的机制[J]. 作物学报, 2020, 46(10): 1474-1484. doi:10.3724/SP.J.1006.2020.04014
LU Hai-Qin, CHEN Li, CHEN Lei, ZHANG Ying-Chuan, WEN Jing, YI Bin, TU Jing-Xing, FU Ting-Dong, SHEN Jin-Xiong.
近年来, 极端高温天气频繁发生, 导致农作物减产严重。高温诱导下, 植物产生抵抗高温的蛋白进行防御, 如热休克蛋白HSP70 (heat shock protein 70)等, 其作为分子伴侣协助植物抵抗高温胁迫的影响[1,2]。HSP70家族蛋白高度保守, 主要有2个功能区, 与ATP结合并使其水解的核酸结合区(nucleotide binding domain, NBD)和底物结合区(substrate binding domain, SBD), 其行使功能需DnaJ (DnaJ like protein)和NEF (nucleotide exchange factor) 2类蛋白参与。首先HSP40 (DnaJ)通过其J结构域识别底物, 并将底物传递到HSP70; HSP70水解ATP后, 与底物结合并对底物进行修饰; 随后NEF (HSP110)与HSP70的NBD区结合, 促进ADP释放; 当新的ATP与HSP70结合, HSP110从HSP70上脱离, 且释放加工好的底物[3,4,5,6]。
MicroRNA (miRNA)是一类长度为20~24 nt具有重要功能的单链小分子RNA, 其为基因表达负调控因子, 最终以RNA诱导沉默复合物(RNA-induced silencing complex, RISC)行使其对靶基因的切割和抑制翻译等功能[7]。直到2002年才首次在植物中发现miRNA[8], 后续深入研究发现, miRNA参与调控植物生命活动的多个过程, 包括植物的生长发育、激素应答、营养元素的摄取与利用、以及多种生物与非生物胁迫[9,10,11,12,13,14,15]。参与响应植物热胁迫的miRNA也相继被报道, 其中主要是植物热胁迫下鉴定响应高温的miRNA, 如小麦高温胁迫下利用高通量技术鉴定响应热胁迫的miRNA[16], 玉米和番茄高温胁迫下鉴定高温相关的miRNA[17,18,19]。只有少数报道具体某个miRNA如何响应高温胁迫, 如番茄中过表达sha-miR319d提高其耐热性[20]; 过量表达miR160和miR157都增加棉花对高温胁迫的敏感性[21]; 紫花苜蓿和拟南芥中超表达miR156, 其耐热性都获得提高[22,23]。油菜是我国重要的食用植物油来源之一, 市场需求巨大[24], 然而油菜中响应热胁迫的miRNA研究报道尚少, 其中主要是油菜热胁迫下鉴定响应高温的miRNA, 如芥菜型和白菜型油菜高温胁迫下鉴定响应热胁迫的miRNA[25,26], 其具体应对高温胁迫的机制尚不清楚。
本研究通过高通量测序技术获得甘蓝型油菜中新miRNA (novel-miR311), 揭示其在拟南芥和甘蓝型油菜热胁迫时通过切割HSC70-1, 使本应大量产生的HSC70-1蛋白不能大量产生, 从而导致拟南芥和甘蓝型油菜耐热性降低, 以期为油菜抗高温育种提供新的思路。
1 材料与方法
1.1 材料
测序用的材料来源于甘蓝型油菜正常株型品系08-8008和矮秆突变体4942C-5 (轮回亲本)构建的回交BC5群体(取其中的矮秆紧凑单株和正常株型单株各8株, 抽薹初期取茎顶端组织分别混样, 2次重复); 转基因受体材料为甘蓝型油菜品种J572和哥伦比亚型拟南芥(Col, Columbia); 5°-RACE文库的供试材料为矮秆紧凑突变体4942C-5。1.2 试验方法
1.2.1 5°-RACE文库构建和5°-RACE验证靶基因切割位点 利用GeneRacer kit (Invitrogen, USA)试剂盒构建5°-RACE文库。流程如下: 取矮秆紧凑突变体4942C-5茎尖提取总RNA, 取5 μg总RNA不经过脱磷酸化处理和去帽子处理直接连RNA接头, 用SuperScript III反转录合成cDNA。靶基因切割位点验证: 以5°-RACE文库为模板, 通过两轮Touchdown PCR扩增, 获得目标大小片段, 通过TA克隆把PCR产物连接到PGEM-Teasy载体上测序, 最终确定目标mRNA精确的剪切位点。具体步骤和PCR反应体系及反应条件参照陈丽[27]。1.2.2 novel-miR311超表达载体的构建 将novel-miR311前体与NCBI甘蓝型油菜全基因组数据库比对寻到前体的侧翼序列, 为保证miRNA前体茎环结构能够在体内正确转录并切割, 选取茎环结构前后各-200 bp左右侧翼序列设计引物。利用超表达载体pS2300 (由本实验室赵伦博士提供)连接novel-miR311前体序列转化大肠杆菌(DH5α)挑取单克隆测序, 以序列正确的单克隆质粒转化农杆菌(GV3101)。
1.2.3 拟南芥的遗传转化 利用农杆菌蘸花法进行拟南芥的遗传转化。将转化的农杆菌进行培养, 采用花序浸花法侵染野生型拟南芥植株。将所收种子干燥春化后消毒播于50 μg mL-1卡那霉素的1/2 MS培养基上, 置于光照培养室中培养10 d左右, 挑取生长嫩绿且有根的植株移栽于温室培养, 进行后续鉴定。
1.2.4 油菜的遗传转化 采用农杆菌介导的方法进行油菜遗传转化, 方法参考陈丽[27]。
1.2.5 阳性苗鉴定 取得到的转基因油菜和拟南芥幼苗叶片, 用CTAB (hexadecyl trimethyl ammoni um bromide, 十六烷基三甲基溴化铵)法提取基因组DNA。以提取的DNA为模板, 一条载体上的引物, 一条基因内部的引物, 本研究中目的片段长度为456 bp。PCR体系包含1 μL模板、3 μL ddH2O、引物各0.5 μL、5 μL PCR Mix (擎科)。PCR程序为94℃ 3 min; 94℃ 30s, 58℃ 30s, 72℃ 1 min, 共32个循环; 72℃ 5 min。使用1%琼脂糖凝胶电泳检测PCR产物。
1.2.6 RNA提取 采用TRIzol Reagent法[27]抽提总RNA。
1.2.7 miRNA和靶基因的反转录及定量PCR
参照Varkonyi-Gasic等[28]设计miRNA的RT-PCR (reverse transcription PCR)引物。在Bio-Rad CFX96仪器上进行miRNA和靶基因的qPCR (quantitative real-time PCR)反应, miRNA以U6作为内参, 靶基因以ACTIN2基因作为内参。miRNA和靶基因的RT-PCR和qPCR具体方法参照陈丽[27]。使用2 delta-delta Ct方法[29]计算miRNA和基因的相对表达倍数变化。
1.2.8 高温胁迫处理 参照Leng等[30]的方法并略作修改进行拟南芥高温胁迫处理。挑选健壮的拟南芥转基因阳性株系和野生型种子, 使用75%的酒精清洗1 min, 后以0.1%升汞洗8~10 min, 再以无菌水洗3~4遍, 每遍4~5 min, 将晾干的种子播到1/2 MS培养基上, 光照培养室(16 h光照/8 h黑暗, 温度23℃, 湿度60%)生长10 d左右后, 置于光照培养箱(只有温度改变, 其他条件不变)进行42℃ 4 h培养, 放回光照培养室培养7 d后, 观察表型并照相。
油菜种子高温胁迫后发芽试验: 分别挑取健壮圆润的转基因油菜阳性苗和对照J572的种子100粒, 用锡箔纸包好, 分别将种子进行4℃ 2 h、37℃ 2 h、4℃ 2 h-室温1 h-70℃ 2 h、37℃ 2 h-室温1 h-70℃ 2 h和70℃ 2 h处理, 不经高温处理为对照。将用锡箔纸包好的种子分别置于恒温培养箱进行37℃处理、4℃冰箱进行4℃处理以及烘箱中进行70℃处理。处理后的种子摆入装有2层滤纸的10×10的培养方皿中, 置于光照培养室(16 h光照/8 h黑暗, 温度23℃, 湿度60%)中进行发芽试验, 期间向培养方皿中不定时加水。
油菜幼苗高温试验: 挑取健壮的转基因油菜阳性苗和对照J572的种子, 同时播种于装有营养土10×10的钵子里, 播种完将其置于油菜温室(温室条件与上述光照培养室一致)培养10 d左右(长出2片小真叶), 随后在光照培养箱(除温度外, 其他条件与油菜温室一致)进行43℃ 5 h、44℃ 5 h和45℃ 5 h培养, 处理后再置于原油菜温室培养7 d后观察表型并拍照。
2 结果与分析
2.1 Bna-novel-miR311及其靶基因
通过回交群体中矮秆紧凑型单株和正常株型单株混池的小RNA高通量测序, 在2种材料池中都鉴定到novel-miR311和其前体序列, 与拟南芥基因组比较, 发现它们仅在油菜中存在。利用MFOLD软件对novel-miR311前体序列进行折叠发现, 其可形成完美的发夹结构, 利于被Dicer-like (DCL1)酶切割, 且其最小折叠自由能为dG = -65.61 kJ mol-1, 符合miRNA的特征小于-18 kJ mol-1。同时通过对上述2种材料池的降解组测序鉴定到被novel-miR311剪切的10个候选靶基因, 进一步分析发现, 其中8个靶基因为HSP70家族基因(5个为热应激同源蛋白基因HSC70-1, 3个为热诱导型蛋白基因HSP70-2); 另外2个靶基因为转导蛋白家族基因(表1)。5°-RACE验证BnaCnng03470D和BnaA10g27060D (HSC70-1)被novel-miR311切割(图1)。
Table 1
表1
表1novel-miR311降解组测序预测到的10个候选靶基因
Table 1
| miRNA 名称 miRNA name | 靶基因 Target gene | 拟南芥同源基因 Arabidopsis homologous gene | 靶基因功能 Function of target gene |
|---|---|---|---|
| novel miR311 | BnaC06g18840D | AT3G63460.1 | Transducin family protein |
| novel miR311 | BnaA07g19590D | AT3G63460.1 | Transducin family protein |
| novel miR311 | BnaC03g46700D | AT5G02490.1 | Heat shock protein 70 (Hsp 70) family protein |
| novel miR311 | BnaA03g39360D | AT5G02490.1 | Heat shock protein 70 (Hsp 70) family protein |
| novel miR311 | BnaA03g39350D | AT5G02490.1 | Heat shock protein 70 (Hsp 70) family protein |
| novel miR311 | BnaC04g42010D | AT5G02500.1 | Heat shock cognate protein 70-1 (HSC70-1) |
| novel miR311 | BnaA10g27080D | AT5G02500.1 | Heat shock cognate protein 70-1 (HSC70-1) |
| novel miR311 | BnaA09g05850D | AT5G02500.1 | Heat shock cognate protein 70-1 (HSC70-1) |
| novel miR311 | BnaCnng03470D | AT5G02500.1 | Heat shock cognate protein 70-1 (HSC70-1) |
| novel miR311 | BnaA10g27060D | AT5G02500.1 | Heat shock cognate protein 70-1 (HSC70-1) |
新窗口打开|下载CSV
图1
新窗口打开|下载原图ZIP|生成PPT图15°-RACE验证靶基因
冒号表示碱基配对, 点号表示错配, 箭头表示切割位点, 数字表示挑斑克隆。
Fig. 1Validation of target genes using 5°-RACE
“:” represents the miRNA and its targets are labeled on the right, “.” represents mismatch, the arrow indicates the cleavage site, and the numbers above sequences represent the detected cleavage site of independent clones.
2.2 Bna-novel-miR311候选靶基因与其在拟南芥中的同源基因
利用NCBI公共数据库分别对油菜中的10个候选靶基因和其在拟南芥基因组中的同源基因的序列相似度比较分析发现, novel-miR311在油菜中的所有靶基因与其在拟南芥中的同源基因都有85%以上的相似度, 且油菜中10个靶基因的靶标位点与拟南芥中的同源基因相差无几, 特别是HSC70-1基因的靶序列在油菜和拟南芥中毫无差别(图2)。这一结果说明novel-miR311存在结合拟南芥中靶基因并进行靶标作用的可能, 故构建novel-miR311超表达载体, 转化拟南芥和油菜。图2
新窗口打开|下载原图ZIP|生成PPT图2油菜和拟南芥靶基因作用位点序列对比
a: 转导蛋白基因; b: 热激蛋白HSP70-2基因; c: 热应激同源蛋白HSC70-1基因。红色部分为novel-miR311与靶基因结合部位。
Fig. 2Comparison of target gene sites between Brassica napus and Arabidopsis
a: transduction protein gene; b: heat shock protein HSP70-2 gene; c: heat shock cognate protein HSC70-1 gene. The red part is the binding site of novel-miR311 and target gene.
2.3 Bna-novel-miR311在拟南芥高温胁迫中的功能验证
2.3.1 Bna-novel-miR311及其靶基因在拟南芥组织中的表达分析 选取3个阳性拟南芥转基因株系, 分别检测novel-miR311及HSC70-1基因在其根、茎、叶、花和角果的表达量, 以野生型作为对照。野生型拟南芥中, 5个组织几乎全都检测不到novel-miR311的表达, 与拟南芥基因组中无novel-miR311前体序列相符合; 而HSC70-1基因的表达量在野生型中显著高于阳性株系(图3)。说明novel-miR311对拟南芥中与油菜的同源靶基因进行切割。图3
新窗口打开|下载原图ZIP|生成PPT图3野生型和阳性拟南芥中novel-miR311和HSC70-1分别在根、茎、叶、花和角果中的表达量
每次试验3次生物学重复。*和**分别表示P < 0.05和P < 0.01。
Fig. 3Expression levels of novel-miR311 and HSC70-1 in root, stem, leaf, flower, and pod of wild type and positive Arabidopsis thaliana
Data are shown as mean ± SD of three biological replicates. * and ** represent P < 0.05 and P < 0.01, respectively.
2.3.2 高温胁迫前后拟南芥阳性株系的表型观察
选取3个T3代拟南芥转基因株系和野生型进行高温胁迫试验, 经几批预试验发现, 42℃ 4 h处理下出现较明显的表型。图4-a方皿中拟南芥左半边为阳性苗, 右半边为野生型。novel-miR311 (N311)阳性苗的3个株系经42℃ 4 h处理后, 生长势和存活率显著低于野生型拟南芥。每个株系中热胁迫后阳性苗生长不受影响的百分比显著低于野生型, 而死亡百分比高于野生型(图4)。
图4
新窗口打开|下载原图ZIP|生成PPT图4野生型和阳性拟南芥在1/2MS上的高温胁迫实验
a: 表型分析; b: 统计学分析。
Fig. 4Response to high temperature stress on 1/2 MS in wild type and positive Arabidopsis thaliana
a: phenotypic analysis; b: statistical analysis.
2.4 Bna-novel-miR311在甘蓝型油菜高温胁迫中的功能验证
2.4.1 高温胁迫前后油菜阳性株系种子的发芽分析选取3个T1代油菜转基因株系与对照J572种子进行高温胁迫后的发芽试验。经4℃ 2 h、37℃ 2 h处理与不经高温处理在第2天的发芽率无显著差异; 经4℃ 2 h-室温1 h-70℃ 2 h、37℃ 2 h-室温1 h-70℃ 2 h和70℃ 2 h处理较不经高温处理在第2天的发芽率显著降低, 但对照J572的发芽率显著高于油菜转基因株系, 且经过一段时间低温训练后再进行70℃高温处理较直接进行70℃高温处理的发芽率稍高(图5) (正常发芽的种子: 双子叶圆粒种子幼根达种子直径长)。
图5
新窗口打开|下载原图ZIP|生成PPT图5甘蓝型油菜阳性苗与J572的种子高温胁迫后的发芽试验
a: 不同温度处理前后对比图; b: 2 d发芽率统计图。
Fig. 5Germination experiment under high temperature stress in Brassica napus positive seedlings and J572 seeds
a: comparison chart before and after treatment at different temperatures; b: germination rate chart for two days.
2.4.2 高温胁迫前后油菜阳性株系的表型观察
选取4个T1代novel-miR311 (N311)株系转基因油菜和对照J572进行热胁迫试验, 图5-a所有钵子中左边为油菜转基因阳性苗, 右边为J572。由于转基因油菜种子数量的原因, 43℃ 5 h处理只有2个重复, 44℃ 5 h处理和45℃ 5 h处理有4个重复。
处理前转基因植株与对照J572植株生长势并无差别(图6-a), 经43℃ 5 h处理温室再培养7 d后转基因植株较J572明显矮小; 经44℃ 5 h处理, 所有转基因植株全部死亡, 而在N311-6和N311-63的钵子中对应的J572分别存活2株和1株; 经45℃ 5 h处理, 所有油菜全部死亡。qPCR结果显示, 无论是热胁迫前还是热胁迫后, 油菜阳性苗体内novel-miR311的表达量较J572显著上调, 其靶基因HSC70-1显著下调, 而44℃处理后所有株系中novel-miR311的表达量较处理前略微下降, 而靶基因上升(图6-b)
图6
新窗口打开|下载原图ZIP|生成PPT图6甘蓝型油菜阳性苗与J572在土壤中的高温胁迫表型及表达分析
a: 热胁迫前后阳性苗与J572的对比图, 图中红色箭头指存活的油菜。b: 热胁迫前后novel-miR311和HSC70-1的表达量。每次试验3次生物学重复。**表示P < 0.01。
Fig. 6Phenotype and expression analysis under high temperature stress treatment of Brassica napus positive seedlings and J572 in soil
a: comparison of positive seedlings and J572 before and after heat stress, the red arrow in figures refers to the surviving rapeseed. b: expression of novel-miR311 and HSC70-1 before and after heat stress. Data are shown as mean ± SD of three biological replicates. ** P < 0.01.
3 讨论
3.1 Bna-novel-miR311及其靶基因
novel-miR311是利用高通量测序在油菜茎尖中发现的新miRNA, 降解组测序预测其靶基因为热激蛋白家族。序列比对结果发现, novel-miR311存在于油菜而不存在于拟南芥中, 且novel-miR311的10个靶基因与其在拟南芥中同源基因相似度非常高, 特别是在靶序列处相似度更高, 故而保证novel-miR311在拟南芥中切割其靶基因的可能。5°-RACE技术验证油菜中2个靶基因BnaCnng03470D和BnaA10g27060D (HSC70-1)被novel-miR311切割, 因此构建novel-miR 311超表达载体, 转化拟南芥和油菜。3.2 Bna-novel-miR311在响应拟南芥高温胁迫途径中的作用
qPCR分析发现, 拟南芥阳性苗中novel-miR311表达量均显著高于野生型, 而其靶基因显著低于野生型, 说明转基因拟南芥体内novel-miR311表达量水平上升, 且在体内对其靶基因切割。值得一提的是, 野生型拟南芥中几乎检测不到novel-miR311, 符合novel-miR311前体序列在拟南芥基因组中比对不上的结果。novel-miR311的靶基因为热激蛋白家族基因HSP70, 而HSP70蛋白作为分子伴侣在植物遭受逆境胁迫时协助产生的变性蛋白质再折叠, 防止变性蛋白质聚集, 并溶解或降解蛋白质聚体[31,32]。热激因子(heat shock factor, HSF)与热胁迫产生的变性蛋白竞争结合HSP70, 变性蛋白更易结合HSP70, 使得钝化态HSF单体解离, 解离的HSF单体形成活化态HSF三聚体, 再次结合到HSP70基因上游促进其大量产生[33,34]。
本研究利用拟南芥进行高温、干旱和盐胁迫的试验发现, 只有高温胁迫后出现较明显的转novel-miR311阳性苗存活率显著低于野生型的表型。同时有研究表明, 拟南芥中过量表达HSC70-1基因提高其耐热性[35,36], 而此结果与本研究中的拟南芥热胁迫试验结果正好对应。说明当转基因拟南芥遭受热胁迫时, 由于novel-miR311对HSC70-1基因的切割, 从而影响其翻译的蛋白修饰损伤蛋白的功能, 进一步导致拟南芥阳性苗热胁迫后死亡率升高。因此novel-miR311通过影响拟南芥中HSP70的作用途径, 使其不能大量产生HSP70蛋白, 从而影响因高温产生变性蛋白的修复, 进而造成转基因拟南芥耐热性降低(图7)。
图7
新窗口打开|下载原图ZIP|生成PPT图7HSP70作用途径
根据Jacob等[33]并略作修改, 虚框中为引入本文结果。
Fig. 7HSP70 action pathway
According to Jacob et al.[33] with slight modification, the results in the dashed box are for the introduction of this article.
3.3 Bna-novel-miR311在响应油菜高温胁迫途径中的作用
研究表明油菜受热胁迫时诱导HSP70大量产生[37], 烟草中超表达油菜HSP70基因提高其耐热性[38]。本研究发现热胁迫后, 转基因油菜的生长势和存活率显著低于J572, qPCR结果显示, 转基因油菜中novel-miR311表达量显著高于J572, 而HSC70-1基因的表达量整体上热胁迫后高于胁迫前, 但无论胁迫前还是胁迫后其阳性苗中HSC70-1基因的表达量都显著低于对照J572, 说明热胁迫后油菜中HSC70-1基因的表达量上升, 但因油菜阳性苗相对J572体内上升的novel-miR311对其靶基因真实切割, 导致热胁迫后HSC70-1基因大量产生, 结合转基因油菜种子高温处理后的2 d发芽率显著低于对照试验结果, 说明novel-miR311也可影响油菜热胁迫后HSP70的大量产生而影响HSP70的作用途径。该结果完善了高温胁迫下植物体内HSP70的作用途径, 也为油菜抗高温研究提供了新思路。本研究中转基因油菜和拟南芥热胁迫后的表型都不是非常明显, 可能是处理的条件需再探究; 高温胁迫时的幼苗大小也是影响最后结果的原因之一; 高温胁迫时植物大量产生的HSP70家族蛋白不仅有HSC70-1, 还有其他类型, 其他类型的HSP70蛋白也行使修饰损伤蛋白的功能。
4 结论
本研究鉴定到油菜novel-miR311, 其在拟南芥与油菜中真实切割HSC70-1。novel-miR311在拟南芥和油菜高温胁迫中因切割HSC70-1影响其翻译的蛋白行使修饰损伤蛋白的功能, 从而导致高温胁迫后拟南芥和油菜阳性苗耐热性降低。附图1
新窗口打开|下载原图ZIP|生成PPT附图1novel-miR311茎环结构
Supplementary Fig. 1novel-miR311 stem-loop structure
Supplementary table 1
附表1
附表1novel-miR311的前体和成熟体序列
Supplementary table 1
| 类别Type | 序列Sequence (5°-3°) |
|---|---|
| 成熟体 Mature sequence (5′-3′) | TTGGTGATAATTGGATTGGCA |
| 前体 Precursor sequence (5′-3′) | TATGTGGTTAGAGCCAATCCATTTATCACCAATTCGTTTAAGTTGGTATAAATGTCGGTTTAAGAGCGATCAGGCCTATCAAAAATAGGCCGGATCGAATTCTGTTCAGTTGGGCAAGGTGTGTTTGTGCTCTGATACCATGATAAATTTCTTAGTTTTACAATTAAAACTAATTGGTGATAATTGGATTGGCACGTCCCTAAC |
| 引物 Primer (5′-3′) | 5' primer: AACTGCAGTTGTAGTTTTGAGAGATTAGAAGTGG 3' primer: CGGGATCCCTTTTATTAATCCCTCAGTAATACACC |
新窗口打开|下载CSV
参考文献 原文顺序
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被引期刊影响因子
DOI:10.1104/pp.126.2.789URLPMID:11402207 [本文引用: 1]
We isolated cDNA clones for two nuclear-encoded, organellar members of the Arabidopsis hsp70 gene family, mtHsc70-2 (AF217458) and cpHsc70-2 (AF217459). Together with the completion of the genome sequence, the hsp70 family in Arabidopsis consists of 14 members unequally distributed among the five chromosomes. To establish detailed expression data of this gene family, a comprehensive reverse transcriptase-polymerase chain reaction analysis for 11 hsp70s was conducted including analysis of organ-specific and developmental expression and expression in response to temperature extremes. All hsp70s showed 2- to 20-fold induction by heat shock treatment except cpHsc70-1 and mtHsc70-1, which were unchanged or repressed. The expression profiles in response to low temperature treatment were more diverse than those evoked by heat shock treatment. Both mitochondrial and all cytosolic members of the family except Hsp70b were strongly induced by low temperature, whereas endoplasmic reticulum and chloroplast members were not induced or were slightly repressed. Developmentally regulated expression of the heat-inducible Hsp70 in mature dry seed and roots in the absence of temperature stress suggests prominent roles in seed maturation and root growth for this member of the hsp70 family. This reverse transcriptase-polymerase chain reaction analysis establishes the complex differential expression pattern for the hsp70s in Arabidopsis that portends specialized functions even among members localized to the same subcellular compartment.
DOI:10.1080/02648725.2017.1340546URLPMID:28649918 [本文引用: 1]
Studying the strategies of improving abiotic stress tolerance is quite imperative and research under this field will increase our understanding of response mechanisms to abiotic stress such as heat. The Hsp70 is an essential regulator of protein having the tendency to maintain internal cell stability like proper folding protein and breakdown of unfolded proteins. Hsp70 holds together protein substrates to help in movement, regulation, and prevent aggregation under physical and or chemical pressure. However, this review reports the molecular mechanism of heat shock protein 70 kDa (Hsp70) action and its structural and functional analysis, research progress on the interaction of Hsp70 with other proteins and their interaction mechanisms as well as the involvement of Hsp70 in abiotic stress responses as an adaptive defense mechanism.
URLPMID:8175707 [本文引用: 1]
The ATPase fragment of the bovine 70-kDa heat shock cognate protein is an attractive construct in which to study its mechanism of ATP hydrolysis. The three-dimensional structure suggests several residues that might participate in the ATPase reaction. Four acidic residues (Asp-10, Glu-175, Asp-199, and Asp-206) have been individually mutated to both the cognate amine (asparagine/glutamine) and to serine, and the effects of the mutations on the kinetics of the ATPase activity (Wilbanks, S. M., DeLuca-Flaherty, C., and McKay, D. B. (1994) J. Biol. Chem. 269, 12893-12898) and the structure of the mutant ATPase fragments have been determined, typically to approximately 2.4 A resolution. Additionally, the structures of the wild type protein complexed with MgADP and Pi, MgAMPPNP (5'-adenylyl-beta, gamma-imidodiphosphate) and CaAMPPNP have been refined to 2.1, 2.4, and 2.4 A, respectively. Combined, these structures provide models for the prehydrolysis, MgATP-bound state and the post-hydrolysis, MgADP-bound state of the ATPase fragment. These models suggest a pathway for the hydrolytic reaction in which 1) the gamma phosphate of bound ATP reorients to form a beta, gamma-bidentate phosphate complex with the Mg2+ ion, allowing 2) in-line nucleophilic attack on the gamma phosphate by a H2O molecule or OH- ion, with 3) subsequent release of inorganic phosphate.
DOI:10.1677/JME-08-0116URLPMID:18852216 [本文引用: 1]
Molecular chaperones are best recognized for their roles in de novo protein folding and the cellular response to stress. However, many molecular chaperones, and in particular the Hsp70 chaperone machinery, have multiple diverse cellular functions. At the molecular level, chaperones are mediators of protein conformational change. To facilitate conformational change of client/substrate proteins, in manifold contexts, chaperone power must be closely regulated and harnessed to specific cellular locales--this is controlled by cochaperones. This review considers specialized functions of the Hsp70 chaperone machinery mediated by its cochaperones. We focus on vesicular trafficking, protein degradation and a potential role in G protein-coupled receptor processing.
[本文引用: 1]
[本文引用: 1]
DOI:10.1093/carcin/bgt111URL [本文引用: 1]
The HSP70 family of heat shock proteins consists of molecular chaperones of approximately 70kDa in size that serve critical roles in protein homeostasis. These adenosine triphosphatases unfold misfolded or denatured proteins and can keep these proteins in an unfolded, folding-competent state. They also protect nascently translating proteins, promote the cellular or organellar transport of proteins, reduce proteotoxic protein aggregates and serve general housekeeping roles in maintaining protein homeostasis. The HSP70 family is the most conserved in evolution, and all eukaryotes contain multiple members. Some members of this family serve specific organellar- or tissue-specific functions; however, in many cases, these members can function redundantly. Overall, the HSP70 family of proteins can be thought of as a potent buffering system for cellular stress, either from extrinsic (physiological, viral and environmental) or intrinsic (replicative or oncogenic) stimuli. As such, this family serves a critical survival function in the cell. Not surprisingly, cancer cells rely heavily on this buffering system for survival. The overwhelming majority of human tumors overexpress HSP70 family members, and expression of these proteins is typically a marker for poor prognosis. With the proof of principle that inhibitors of the HSP90 chaperone have emerged as important anticancer agents, intense focus has now been placed on the potential for HSP70 inhibitors to assume a role as a significant chemotherapeutic avenue. In this review, the history, regulation, mechanism of action and role in cancer of the HSP70 family are reviewed. Additionally, the promise of pharmacologically targeting this protein for cancer therapy is addressed.
[本文引用: 1]
[本文引用: 1]
DOI:10.1101/gad.1004402URLPMID:12101121 [本文引用: 1]
MicroRNAs (miRNAs) are an extensive class of ~22-nucleotide noncoding RNAs thought to regulate gene expression in metazoans. We find that miRNAs are also present in plants, indicating that this class of noncoding RNA arose early in eukaryotic evolution. In this paper 16 Arabidopsis miRNAs are described, many of which have differential expression patterns in development. Eight are absolutely conserved in the rice genome. The plant miRNA loci potentially encode stem-loop precursors similar to those processed by Dicer (a ribonuclease III) in animals. Mutation of an Arabidopsis Dicer homolog, CARPEL FACTORY, prevents the accumulation of miRNAs, showing that similar mechanisms direct miRNA processing in plants and animals. The previously described roles of CARPEL FACTORY in the development of Arabidopsis embryos, leaves, and floral meristems suggest that the miRNAs could play regulatory roles in the development of plants as well as animals.
DOI:10.1038/srep29938URLPMID:27444058 [本文引用: 1]
MicroRNAs (miRNAs) control gene expression as key negative regulators at the post-transcriptional level. MiR160 plays a pivotal role in Arabidopsis growth and development through repressing expression of its target AUXIN RESPONSE FACTOR (ARF) genes; however, the function of miR160 in monocots remains elusive. In this study, we found that the mature rice miR160 (OsmiR160) was mainly derived from OsMIR160a and OsMIR160b genes. Among four potential OsmiR160 target OsARF genes, the OsARF18 transcript was cleaved at the OsmiR160 target site. Rice transgenic plants (named mOsARF18) expressing an OsmiR160-resistant version of OsARF18 exhibited pleiotropic defects in growth and development, including dwarf stature, rolled leaves, and small seeds. mOsARF18 leaves were abnormal in bulliform cell differentiation and epidermal cell division. Starch accumulation in mOsARF18 seeds was also reduced. Moreover, auxin induced expression of OsMIR160a, OsMIR160b, and OsARF18, whereas expression of OsMIR160a and OsMIR160b as well as genes involved in auxin signaling was altered in mOsARF18 plants. Our results show that negative regulation of OsARF18 expression by OsmiR160 is critical for rice growth and development via affecting auxin signaling, which will advance future studies on the molecular mechanism by which miR160 fine-tunes auxin signaling in plants.
DOI:10.1093/jxb/ers239URL [本文引用: 1]
Iron (Fe) is an essential plant micronutrient, and its deficiency limits plant growth and development on alkaline soils. Under Fe deficiency, plant responses include up-regulation of genes involved in Fe uptake from the soil. However, little is known about shoot responses to Fe deficiency. Using microarrays to probe gene expression in Kas-1 and Tsu-1 ecotypes of Arabidopsis thaliana, and comparison with existing Col-0 data, revealed conserved rosette gene expression responses to Fe deficiency. Fe-regulated genes included known metal homeostasis-related genes, and a number of genes of unknown function. Several genes responded to Fe deficiency in both roots and rosettes. Fe deficiency led to up-regulation of Cu,Zn superoxide dismutase (SOD) genes CSD1 and CSD2, and down-regulation of FeSOD genes FSD1 and FSD2. Eight microRNAs were found to respond to Fe deficiency. Three of these (miR397a, miR398a, and miR398b/c) are known to regulate transcripts of Cu-containing proteins, and were down-regulated by Fe deficiency, suggesting that they could be involved in plant adaptation to Fe limitation. Indeed, Fe deficiency led to accumulation of Cu in rosettes, prior to any detectable decrease in Fe concentration. ccs1 mutants that lack functional Cu,ZnSOD proteins were prone to greater oxidative stress under Fe deficiency, indicating that increased Cu concentration under Fe limitation has an important role in oxidative stress prevention. The present results show that Cu accumulation, microRNA regulation, and associated differential expression of Fe and CuSOD genes are coordinated responses to Fe limitation.
DOI:10.1105/tpc.114.127340URL [本文引用: 1]
Light and copper are important environmental determinants of plant growth and development. Despite the wealth of knowledge on both light and copper signaling, the molecular mechanisms that integrate the two pathways remain poorly understood. Here, we use Arabidopsis thaliana to demonstrate an interaction between SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) and ELONGATED HYPOCOTYL5 (HY5), which mediate copper and light signaling, respectively. Through whole-genome chromatin immunoprecipitation and RNA sequencing analyses, we elucidated the SPL7 regulon and compared it with that of HY5. We found that the two transcription factors coregulate many genes, including those involved in anthocyanin accumulation and photosynthesis. Moreover, SPL7 and HY5 act coordinately to transcriptionally regulate MIR408, which results in differential expression of microRNA408 (miR408) and its target genes in response to changing light and copper conditions. We demonstrate that this regulation is tied to copper allocation to the chloroplast and plastocyanin levels. Finally, we found that constitutively activated miR408 rescues the distinct developmental defects of the hy5, spl7, and hy5 spl7 mutants. These findings revealed the existence of crosstalk between light and copper, mediated by a HY5-SPL7 network. Furthermore, integration of transcriptional and posttranscriptional regulation is critical for governing proper metabolism and development in response to combined copper and light signaling.
DOI:10.1093/jxb/ers333URLPMID:23162117 [本文引用: 1]
MicroRNAs (miRNAs) constitute a novel mechanism of gene regulation affecting plant development, growth, and stress response. To study the role of miRNAs in arsenic (As) stress, microarray profiling of miRNAs was performed in Brassica juncea using a custom Phalanx Plant OneArray containing 381 unique miRNA probes representing 618 miRNAs from 22 plant species. miRNA microarray hybridization of roots exposed to As for 1h and 4h revealed that a total of 69 miRNAs belonging to 18 plant miRNA families had significantly altered expression. The As-responsive miRNAs also exhibited a time- and organ-dependent change in their expression. Putative target prediction for the miRNAs suggested that they regulate various developmental processes (e.g. miR156, miR169, and miR172), sulphur uptake, transport, and assimilation (miR395, miR838, and miR854), and hormonal biosynthesis and/or function (e.g. miR319, miR167, miR164, and miR159). Notable changes were observed in the level of auxins [indole-3-acetic acid (IAA), indole-3- butyric acid, and naphthalene acetic acid], jasmonates [jasmonic acid (JA) and methyl jasmonate], and abscisic acid. The exogenous supply of JA and IAA improved growth of plants under As stress and altered expression of miR167, miR319, and miR854, suggesting interplay of hormones and miRNAs in the regulation of As response. In conclusion, the present work demonstrates the role of miRNAs and associated mechanisms in the plant's response towards As stress.
DOI:10.1007/s10534-016-9981-xURLPMID:27853903 [本文引用: 1]
The prevalence of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease are increased by age. Alleviation of their symptoms and protection of normal neurons against degeneration are the main aspects of the research to establish novel therapeutic strategies. Iron as the one of most important cation not only play important role in the structure of electron transport chain proteins but also has pivotal duties in cellular activities. But disruption in iron hemostasis can make it toxin to neurons which causes lipid peroxidation, DNA damage and etc. In patients with Alzheimer and Parkinson misbalancing in iron homeostasis accelerate neurodegeneration and cause neuroinflmmation. mTOR as the common signaling pathway between cancer and neurodegenerative disorders controls iron uptake and it is in active form in both diseases. Anti-cancer drugs which target mTOR causes iron deficiency and dual effects of mTOR inhibitors can candidate them as a therapeutic strategy to alleviate neurodegeneration/inflammation because of iron overloading.
DOI:10.1105/tpc.106.180960URL [本文引用: 1]
DOI:10.1016/j.plantsci.2018.01.010URLPMID:29606207 [本文引用: 1]
MicroRNAs (miRNAs) are a class of non-coding small RNAs (sRNAs) that play crucial regulatory roles in various developmental processes. Silique length indirectly influences seed yield in rapeseed (Brassica napus); however, the molecular roles of miRNAs in silique length are largely unknown. Here, backcross progenies of rapeseed with long siliques (LS) and short siliques (SS) were used to elucidate these roles. Four small RNA libraries from siliques in an early stage of development were sequenced, and a total of 814 non-redundant miRNA precursors were identified, representing 65 known and 394 novel miRNAs. Expression analyses revealed that 17 miRNAs were differentially expressed in LS and SS lines. Furthermore, through degradome sequencing, we identified 522 cleavage events. Correlation analysis of the differentially expressed miRNAs and their targets suggested that miR159 and miR319 represses cell proliferation and miR160 regulates auxin signal transduction to control silique length. Additionally, the upregulation of miR2111, miR399, miR827, and miR408 reflected restricted silique development due to inorganic phosphate/copper deficiency. More significantly, high expression of miR160 in rapeseed may repress auxin response factors and result in increased silique length, illustrating that silique length might be regulated via an auxin-response pathway.
DOI:10.1007/s10142-014-0421-0URLPMID:25480755 [本文引用: 1]
MicroRNAs (miRNAs) are small endogenous RNAs of ~22 nucleotides that have been shown to play regulatory role by negatively affecting the expression of genes at the post-transcriptional level. Information of miRNAs on some important crops like soybean, Arabidopsis, and rice, etc. are available, but no study on heat-responsive novel miRNAs has yet been reported in wheat (Triticum aestivum L.). In the present investigation, a popular wheat cultivar HD2985 was used in small RNA library construction and Illumina HiSeq 2000 was used to perform high-throughput sequencing of the library after cluster generation; 110,896,604 and 87,743,861 reads were generated in the control (22 degrees C) and heat-treated (42 degrees C for 2 h) samples, respectively. Forty-four precursor and mature miRNAs were found in T. aestivum from miRBase v 19. The frequencies of the miRNA families varied from 2 (tae-miR1117) to 60,672 (tae-miR159b). We identify 1052 and 902 mature miRNA sequences in HD2985 control and HS-treated samples by mapping on reference draft genome of T. aestivum. Maximum identified miRNAs were located on IWGSC_CSS_3B_scaff (chromosome 3B). We could identify 53 and 46 mature miRNA in the control and HS samples and more than 516 target genes by mapping on the reference genome of Oryza sativa, Zea mays, and Sorghum bicolor. Using different pipelines and plant-specific criteria, 37 novel miRNAs were identified in the control and treated samples. Six novel miRNA were validated using qRT-PCR to be heat-responsive. A negative correlation was, however, observed between the expression of novel miRNAs and their targets. Target prediction and pathway analysis revealed their involvement in the heat stress tolerance. These novel miRNAs are new additions to miRNA database of wheat, and the regulatory network will be made use of in deciphering the mechanism of thermotolerance in wheat.
DOI:10.1186/s12864-017-4238-9URLPMID:29096602 [本文引用: 1]
BACKGROUND: Autogamy in cultivated tomato varieties is a derived trait from wild type tomato plants, which are mostly allogamous. However, environmental stresses can cause morphological defects in tomato flowers and hinder autogamy. Under elevated temperatures, tomato plants usually exhibit the phenotype of stigma exsertion, with severely hindered self-pollination and fruit setting, whereas the inherent mechanism of stigma exsertion have been hitherto unknown. Numerous small RNAs (sRNAs) have been shown to play significant roles in plant development and stress responses, however, none of them have been studied with respect to stamen and pistil development under high-temperature conditions. We investigated the associations between stigma exsertion and small RNAs using high-throughput sequencing technology and molecular biology approaches. RESULTS: Sixteen sRNA libraries of Micro-Tom were constructed from plants stamen and pistil samples and sequenced after 2 d and 12 d of exposure to heat stress, respectively, from which a total of 110 known and 84 novel miRNAs were identified. Under heat stress conditions, 34 known and 35 novel miRNAs were differentially expressed in stamens, and 20 known and 10 novel miRNAs were differentially expressed in pistils. GO and KEGG pathway analysis showed that the predicted target genes of differentially expressed miRNAs were significantly enriched in metabolic pathways in both stamen and pistil libraries. Potential miRNA-target cleavage cascades that correlated with the regulation of stigma exsertion under heat stress conditions were found and validated through qRT-PCR and RLM-5' RACE. CONCLUSION: Overall, a global spectrum of known and novel miRNAs involved in tomato stigma exsertion and induced by high temperatures were identified using high-throughput sequencing and molecular biology approaches, laying a foundation for revealing the miRNA-mediated regulatory network involved in the development of tomato stamens and pistils under high-temperature conditions.
DOI:10.3390/ijms20071754URL [本文引用: 1]
DOI:10.1038/srep33777URLPMID:27653374 [本文引用: 1]
MicroRNAs (miRNAs) are 19-24 nucleotide (nt) noncoding RNAs that play important roles in abiotic stress responses in plants. High temperatures have been the subject of considerable attention due to their negative effects on plant growth and development. Heat-responsive miRNAs have been identified in some plants. However, there have been no reports on the global identification of miRNAs and their targets in tomato at high temperatures, especially at different elevated temperatures. Here, three small-RNA libraries and three degradome libraries were constructed from the leaves of the heat-tolerant tomato at normal, moderately and acutely elevated temperatures (26/18 degrees C, 33/33 degrees C and 40/40 degrees C, respectively). Following high-throughput sequencing, 662 conserved and 97 novel miRNAs were identified in total with 469 conserved and 91 novel miRNAs shared in the three small-RNA libraries. Of these miRNAs, 96 and 150 miRNAs were responsive to the moderately and acutely elevated temperature, respectively. Following degradome sequencing, 349 sequences were identified as targets of 138 conserved miRNAs, and 13 sequences were identified as targets of eight novel miRNAs. The expression levels of seven miRNAs and six target genes obtained by quantitative real-time PCR (qRT-PCR) were largely consistent with the sequencing results. This study enriches the number of heat-responsive miRNAs and lays a foundation for the elucidation of the miRNA-mediated regulatory mechanism in tomatoes at elevated temperatures.
URLPMID:31122194 [本文引用: 1]
DOI:10.1111/tpj.13620URLPMID:28635129 [本文引用: 1]
Male sterility caused by long-term high-temperature (HT) stress occurs widely in crops. MicroRNAs (miRNAs), a class of endogenous non-coding small RNAs, play an important role in the plant response to various abiotic stresses. To dissect the working principle of miRNAs in male sterility under HT stress in cotton, a total of 112 known miRNAs, 270 novel miRNAs and 347 target genes were identified from anthers of HT-insensitive (84021) and HT-sensitive (H05) cotton cultivars under normal-temperature and HT conditions through small RNA and degradome sequencing. Quantitative reverse transcriptase-polymerase chain reaction and 5'-RNA ligase-mediated rapid amplification of cDNA ends experiments were used to validate the sequencing data. The results show that miR156 was suppressed by HT stress in both 84021 and H05; miR160 was suppressed in 84021 but induced in H05. Correspondingly, SPLs (target genes of miR156) were induced both in 84021 and H05; ARF10 and ARF17 (target genes of miR160) were induced in 84021 but suppressed in H05. Overexpressing miR160 increased cotton sensitivity to HT stress seen as anther indehiscence, associated with the suppression of ARF10 and ARF17 expression, thereby activating the auxin response that leads to anther indehiscence. Supporting this role for auxin, exogenous Indole-3-acetic acid (IAA) leads to a stronger male sterility phenotype both in 84021 and H05 under HT stress. Cotton plants overexpressing miR157 suppressed the auxin signal, and also showed enhanced sensitivity to HT stress, with microspore abortion and anther indehiscence. Thus, we propose that the auxin signal, mediated by miRNAs, is essential for cotton anther fertility under HT stress.
DOI:10.1105/tpc.114.123851URL [本文引用: 1]
Plants are sessile organisms that gauge stressful conditions to ensure survival and reproductive success. While plants in nature often encounter chronic or recurring stressful conditions, the strategies to cope with those are poorly understood. Here, we demonstrate the involvement of ARGONAUTE1 and the microRNA pathway in the adaptation to recurring heat stress (HS memory) at the physiological and molecular level. We show that miR156 isoforms are highly induced after HS and are functionally important for HS memory. miR156 promotes sustained expression of HS-responsive genes and is critical only after HS, demonstrating that the effects of modulating miR156 on HS memory do not reflect preexisting developmental alterations. miR156 targets SPL transcription factor genes that are master regulators of developmental transitions. SPL genes are posttranscriptionally downregulated by miR156 after HS, and this is critical for HS memory. Altogether, the miR156-SPL module mediates the response to recurring HS in Arabidopsis thaliana and thus may serve to integrate stress responses with development.
DOI:10.1111/ppl.12787URLPMID:29923601 [本文引用: 1]
Heat stress and extreme temperatures negatively affect plant development by disrupting regular cellular and biochemical functions, ultimately leading to reduced crop production. Alfalfa (Medicago sativa) is an important forage crop grown worldwide as forage for livestock feed. Limiting the effects of abiotic stress by developing alfalfa cultivars that are stress tolerant would help mitigate losses to crop production. Members of the microRNA156 (miR156) family regulate the Squamosa Promoter-Binding Protein-Like (SPL) genes that in turn impact plant growth and development by regulating downstream genes in response to various abiotic stresses. In this study, alfalfa with miR156 overexpression and SPL13 RNAi knockdown show increased tolerance to heat stress (40 degrees C). Transgenic plants show high water potential and increased non-enzymatic antioxidant content under heat stress. Moreover, anthocyanin content and chlorophyll abundance were increased under stress. Expression of some important transcription factors and downstream genes involved in abiotic stress response were altered in miR156-overexpressing genotypes under heat. Taken together, our results demonstrate that the miR156/SPL13 network contributes to improving heat stress tolerance in alfalfa.
DOI:10.3724/SP.J.1006.2015.01481URL [本文引用: 1]
基于前期研究中构建的Sollux/Gaoyou DH群体在9个环境中的表型数据和新版遗传图谱，对油菜角果长度进行QTL定位，估测QTL的加性、上位性和环境互作效应。并通过条件QTL方法，解析角果长度与角果粒数和粒重之间的遗传关系，以期利用标记辅助，探讨通过选择角果长度基因型以增加角果粒数、提高千粒重，最终达到增加产量的可能性。结果共检测到在3个环境以上稳定表达的控制角果长度QTL 8个，加性效应值在0.09~0.26 cm之间，效应总和解释群体遗传总变异的60%。8对上位性QTL效应值在0.035~0.075 cm之间，效应总和为加性总效应的38%。QTL与环境互作效应只在少数位点和个别环境中显著。条件QTL研究表明，qSLA2、qSLC1-2和qSLC8-1位点，角果长度的变化对角果粒数影响较大；而通过选择qSLA7、qSLC1-2、qSLC8-1和qSLC8-2长角果标记基因型，可望同时提高角果粒数和千粒重。6个主效QTL 11个连锁标记基因型和表现型的关联分析，验证了条件QTL分析结果，表明通过对qSLA2、qSLA7、qSLC8-1和qSLC8-2位点6个连锁标记(ZAAS423、SUC1-3、ZAAS12a、ZAASA7-28、ZAAS433和ZAAS437)长角果基因型的聚合，可增长角果约2 cm，间接增加角果粒数2粒，同时提高千粒重0.4 g，从而可望实质性地提高油菜产量水平。]]>
[本文引用: 1]
[本文引用: 1]
DOI:10.1093/jxb/err337URL [本文引用: 1]
The species Brassica rapa includes various vegetable crops. Production of these vegetable crops is usually impaired by heat stress. Some microRNAs (miRNAs) in Arabidopsis have been considered to mediate gene silencing in plant response to abiotic stress. However, it remains unknown whether or what miRNAs play a role in heat resistance of B. rapa. To identify genomewide conserved and novel miRNAs that are responsive to heat stress in B. rapa, we defined temperature thresholds of non-heading Chinese cabbage (B. rapa ssp. chinensis) and constructed small RNA libraries from the seedlings that had been exposed to high temperature (46 degrees C) for 1 h. By deep sequencing and data analysis, we selected a series of conserved and novel miRNAs that responded to heat stress. In total, Chinese cabbage shares at least 35 conserved miRNA families with Arabidopsis thaliana. Among them, five miRNA families were responsive to heat stress. Northern hybridization and real-time PCR showed that the conserved miRNAs bra-miR398a and bra-miR398b were heat-inhibitive and guided heat response of their target gene, BracCSD1; and bra-miR156h and bra-miR156g were heat-induced and its putative target BracSPL2 was down-regulated. According to the criteria of miRNA and miRNA* that form a duplex, 21 novel miRNAs belonging to 19 miRNA families were predicted. Of these, four were identified to be heat-responsive by Northern blotting and/or expression analysis of the putative targets. The two novel miRNAs bra-miR1885b.3 and bra-miR5718 negatively regulated their putative target genes. 5'-Rapid amplification of cDNA ends PCR indicated that three novel miRNAs cleaved the transcripts of their target genes where their precursors may have evolved from. These results broaden our perspective on the important role of miRNA in plant responses to heat.
华中农业大学博士学位论文,
[本文引用: 4]
PhD Dissertation of Huazhong Agricultural University,
[本文引用: 4]
DOI:10.1186/1746-4811-3-12URLPMID:17931426 [本文引用: 1]
MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 mul of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.
DOI:10.1006/meth.2001.1262URLPMID:11846609 [本文引用: 1]
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
DOI:10.1007/s10265-016-0900-6URLPMID:28004282 [本文引用: 1]
Members of the HSP70 family function as molecular chaperones to maintain cellular homeostasis and help plants cope with environmental stimuli. However, due to functional redundancy and lack of effective chemical inhibitors, our knowledge of functions of individual HSP70s has remained limited. Here, we confirmed a subclass of HSP70s, including HSP70-1, -2, -3, -4, and -5, localized to the cytosol and nucleus in Arabidopsis thaliana. Histochemical analyses of promoter:GUS reporter lines showed that HSP70-1, -2, -3, and -4 genes were widely expressed, but HSP70-5 was not. In addition, individual HSP70 showed not only similar but also distinct transcriptions when treated by different abiotic stresses and phytohormones. No apparent phenotype was observed when individual HSP70 genes were overexpressed or knocked-out/down, but the double mutant hsp70-1 hsp70-4 and triple mutant hsp70-2 hsp70-4 hsp70-5 plants exhibited developmental phenotypes with shortened specific growth periods, curly and round leaves, twisted petioles, thin stems, and short siliques. Moreover, both mutants were hypersensitive to heat, cold, high glucose, salt and osmotic stress, but hyposensitive to abscisic acid. Genes related to flowering, and the cytokinin, brassinosteroid, and abscisic acid signaling pathways were differentially expressed in both mutants. Our studies suggest that, the individual HSP70 possibly performs both redundant and specific functions with the other members in the cytosolic/nuclear HSP70 subclass, and apart from enabling plants to cope with abiotic stresses, this subclass of cytosolic/nuclear HSP70 proteins also participates in diverse developmental processes and signaling pathways.
DOI:10.1016/j.tplants.2004.03.006URLPMID:15130550 [本文引用: 1]
DOI:10.3724/SP.J.1006.2010.00636URL [本文引用: 1]
为确定热休克蛋白70 (HSP70)提高作物耐干旱高温复合胁迫的机制，对干旱、高温反应不同的4个玉米品种的生理特性进行了研究。结果显示：(1) 在干旱、高温、干旱高温复合胁迫条件下，叶片丙二醛(MDA)增加量以隆玉602最低，驻玉309最高；在干旱胁迫条件下，郑单958叶片MDA含量低于浚单20，而在高温胁迫条件下，浚单20叶片MDA含量低于郑单958。(2) 在干旱、高温、干旱高温复合胁迫条件下，隆玉602、郑单958、浚单20三个品种叶片抗氧化防护系统如抗坏血酸过氧化物酶(APX)、谷胱苷肽还原酶(GR)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性增加量均高于驻玉309；在干旱条件下，郑单958叶片APX、GR、SOD、CAT活性增加量显著高于浚单20，在高温条件下，两个品种的表现则相反；在干旱高温复合胁迫条件下，隆玉602 叶片APX、GR、SOD活性增加量显著高于郑单958和浚单20。(3) HSP70抑制剂斛皮素(Quercetin, Q)预处理显著抑制了3种胁迫诱导的4个品种叶片抗氧化酶活性的增加。这些研究结果暗示HSP70提高了干旱、高温、干旱高温复合胁迫诱导的抗氧化防护酶活性，且APX、GR、SOD 这3个抗氧化防护酶活性可以作为评价作物耐干旱、高温和干旱高温复合胁迫的生化指标。]]>
[本文引用: 1]
DOI:10.1111/pbi.12659URLPMID:27860233 [本文引用: 3]
Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multistress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat-shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone 'client proteins', many are primary metabolism enzymes and signal transduction components with essential roles for the proper functioning of a cell. HSPs/chaperones are controlled by the action of diverse heat-shock factors, which are recruited under stress conditions. In this review, we give an overview of the regulation of the HSP/chaperone network with a focus on Arabidopsis thaliana. We illustrate the role of HSPs/chaperones in regulating diverse signalling pathways and discuss several basic principles that should be considered for engineering multiple stress resistance in crops through the HSP/chaperone network.
DOI:10.1016/j.bbagrm.2011.10.002URLPMID:22033015 [本文引用: 1]
Ten years after the first overview of a complete plant Hsf family was presented for Arabidopsis thaliana by Nover et al. [1], we compiled data for 252 Hsfs from nine plant species (five eudicots and four monocots) with complete or almost complete genome sequences. The new data set provides interesting insights into phylogenetic relationships within the Hsf family in plants and allows the refinement of their classification into distinct groups. Numerous publications over the last decade document the diversification and functional interaction of Hsfs as well as their integration into the complex stress signaling and response networks of plants. This article is part of a Special Issue entitled: Plant gene regulation in response to abiotic stress.
DOI:10.1104/pp.102.019398URLPMID:12805626 [本文引用: 1]
Hsp70s function as molecular chaperones. The protective chaperone activities of hsp70 help to confer tolerance to heat, glucose deprivation, and drought. Overexpression of hsp70s in many organisms correlates with enhanced thermotolerance, altered growth, and development. To better understand the roles of hsp70 proteins in Arabidopsis, the molecular and physiological consequences of altered expression of the major heat shock cognate, Hsc70-1, were analyzed. Extensive efforts to achieve underexpression of Hsc70-1 mRNA using a full-length antisense cDNA resulted in no viable transgenic plants, suggesting that reduced expression is lethal. Constitutive overexpression of Hsc70-1 also appeared to be deleterious to viability, growth, and development because fewer transformants were recovered, and most were dwarfed with altered root systems. Despite being dwarfed, the overexpression plants progressed normally through four selected developmental stages. Heat treatment revealed that Hsc70-1 overexpression plants were more tolerant to heat shock (44 degrees C for 10 min). The elevated basal levels of HSC70-1 in transgenic plants led to delayed heat shock response of several heat shock genes. The data in this study suggest that tight regulation of Hsc70-1 expression is critical for the viability of Arabidopsis and that the functions of HSC70-1 contribute to optimum growth, development, thermotolerance, and regulation of the heat shock response.
DOI:10.1093/jxb/erp109URLPMID:19443614 [本文引用: 1]
Molecular chaperones of the heat shock cognate 70 kDa (HSC70) family are highly conserved in all living organisms and assist nascent protein folding in normal physiological conditions as well as in biotic and abiotic stress conditions. In the absence of specific inhibitors or viable knockout mutants, cytosolic/nuclear HSC70-1 overexpression (OE) and mutants in the HSC70 co-chaperone SGT1 (suppressor of G(2)/M allele of skp1) were used as genetic tools to identify HSC70/SGT1 functions in Arabidopsis development and abiotic stress responses. HSC70-1 OE caused a reduction in root and shoot meristem activities, thus explaining the dwarfism of those plants. In addition, HSC70-1 OE did not impair auxin-dependent phenotypes, suggesting that SGT1 functions previously identified in auxin signalling are HSC70 independent. While responses to abiotic stimuli such as UV-C exposure, phosphate starvation, or seedling de-etiolation were not perturbed by HSC70-1 OE, it specifically conferred gamma-ray hypersensitivity and tolerance to salt, cadmium (Cd), and arsenic (As). Cd and As perception was not perturbed, but plants overexpressing HSC70-1 accumulated less Cd, thus providing a possible molecular explanation for their tolerance phenotype. In summary, genetic evidence is provided for HSC70-1 involvement in a limited set of physiological processes, illustrating the essential and yet specific functions of this chaperone in development and abiotic stress responses in Arabidopsis.
DOI:10.1093/jxb/erh038URLPMID:14739270 [本文引用: 1]
High temperature stress (HTS), during flowering, decreases seed production in many plants. To determine the effect of a moderate HTS on flowering, fruit and seed set in Brassica napus, plants were exposed to a HTS (8/16 h dark/light, 18 degrees C night, ramped at 2 degrees C h-1, over 6 h, to 35 degrees C for 4 h, ramped at 2 degrees C h-1 back to 23 degrees C for 6 h) for 1 or 2 weeks after the initiation of flowering. Although flowering on the HTS-treated plants, during both the 1 week and 2 week HTS treatments, was equal to that of control-grown plants, fruit and seed development, as well as seed weight, were significantly reduced. Under HTS, flowers either developed into seedless, parthenocarpic fruit or aborted on the stem. At the cessation of the HTS, plants compensated for the lack of fruit and seed production by increasing the number of lateral inflorescences produced. During the HTS, pollen viability and germinability were slightly reduced. In vitro pollen tube growth at 35 degrees C, from both control pollen and pollen developed under a HTS, appeared abnormal, however, in vivo tube growth to the micropyle appeared normal. Reciprocal pollination of HTS or control pistils with HTS or control pollen indicated that the combined effects of HTS on both micro- and megagametophytes was required to knock out fruit and seed development. Expression profiles for a subset of HEAT SHOCK PROTEINs (HSP101, HSP70, HSP17.6) showed that both micro- and megagametophytes were thermosensitive despite HTS-induced expression from these genes.
DOI:10.1007/s00709-015-0867-5URLPMID:26298102 [本文引用: 1]
Heat shock proteins (HSPs) exist extensively in eukaryotes and are conserved molecular chaperones with important contribution to plant's survival under environmental stresses. Here, the cloning and characterization of one complementary DNA (cDNA) designated as BcHSP70 from young seedlings of Brassica campestris were reported in the present work. Bioinformatic analysis revealed that BcHSP70 belongs to the plant HSP gene family and had the closest relationship with HSP70-4 from Arabidopsis thaliana. Constitutive overexpression of BcHSP70 in tobacco obviously conferred tolerance to heat stress by affecting different plant physiological parameters. In our study, transgenic tobaccos exhibited higher chlorophyll content than wild-type control when exposed to heat stress. Superoxide dismutase (SOD) and peroxidase (POD) activities, which were helpful to decrease the damage to the membrane system, were significantly higher in transformants compared to wild-type lines. Meanwhile, lower comparative electrical conductivity and malondialdehyde (MDA) content and higher proline and soluble sugar accumulation were found in transgenic tobaccos than in wild-type lines. All these above results indicated that this isolated BcHSP70 cDNA owned the ability to improve the tolerance to heat stress in transgenic tobacco, which provides helpful information and good basement to culture new robust B. campestris variety resistant to high-temperature stress by molecular breeding in the future.
