,, 马琳, 张锦锦, 王筱, 庞永珍, 王学敏,中国农业科学院北京畜牧兽医研究所,北京100193Gene Expression and Salt-Tolerance Analysis of MsDWF4 Gene from Alfalfa
CUI MiaoMiao,, MA Lin, ZHANG JinJin, WANG Xiao, PANG YongZhen, WANG XueMin,Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193通讯作者:
责任编辑: 李莉
收稿日期:2019-10-30接受日期:2020-03-13网络出版日期:2020-09-16
| 基金资助: |
Received:2019-10-30Accepted:2020-03-13Online:2020-09-16
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崔苗苗, 马琳, 张锦锦, 王筱, 庞永珍, 王学敏. 紫花苜蓿MsDWF4的表达特性及耐盐性效应[J]. 中国农业科学, 2020, 53(18): 3650-3664 doi:10.3864/j.issn.0578-1752.2020.18.003
CUI MiaoMiao, MA Lin, ZHANG JinJin, WANG Xiao, PANG YongZhen, WANG XueMin.
0 引言
【研究意义】紫花苜蓿（Medicago sativa L.）为豆科苜蓿属多年生牧草[1],茎叶中含有丰富的蛋白质、矿物质和维生素,因其高营养价值和高消化率等优点,被誉为“牧草之王”,是中国乃至世界上种植面积最大,种植范围最广的豆科牧草[1,2,3]。然而在中国,紫花苜蓿多种植在没有灌溉条件的瘠薄地和盐碱地[4],造成紫花苜蓿单位面积产量低、干草品质差、粗蛋白含量较低、种子细小和总量供应不足等问题[5],严重制约了苜蓿的产业化发展。海关统计数据显示中国苜蓿的进口量呈逐年上升趋势[6]。因此,加快培育高产抗逆紫花苜蓿是提高苜蓿产业化程度,尽快摆脱依赖进口局面的有效手段[7]。研究抗逆基因对紫花苜蓿抗逆性的调控作用,对于深入了解紫花苜蓿的抗逆机制,从而更好的指导紫花苜蓿抗逆育种具有非常重要的意义。油菜素内酯（brassionsterinds,BRs）,又称为芸苔素内酯,因其强大的生理活性,被列为第六大类植物激素[8],参与并促进植物种子发芽、根茎伸长生长、光形态建成和生殖发育等过程和增强植物对高温、低温和高盐等逆境的抗逆性[9]。近年来,对BRs的研究较多,但对BRs相关基因调控植物抗逆性的分子机制研究相对较少,在紫花苜蓿中更缺乏相关内容的报道。DWF4编码的细胞色素氧化酶CYP90B1,是BRs合成的关键限速酶[10],在BRs的生物合成中催化C-22羟基化反应。挖掘紫花苜蓿中的DWF4,研究其对紫花苜蓿抗逆性的调控作用,对于在油菜素内酯合成途径方面了解紫花苜蓿的抗逆应答机制具有重要的意义。【前人研究进展】油菜素内酯是1970年由MITCHELL等[11]从油菜花粉中筛选和分离出来的具有高生物活性的物质,并命名为油菜素。1979年,GROVE等[12]解析出其化学结构为甾醇内酯,迄今为止,已经分离出了70多种类似的化合物,统称为油菜素甾醇类化合物[13]。通过对外源施加的标记中间产物和BR突变体的研究,目前,BRs的合成途径已经日渐清晰：从起始物鲨烯,经过多个生物化学反应,最终合成BRs[14],这其中有众多的基因参与。DWF4编码的细胞色素P450家族的CYP90B1蛋白酶（一种细胞色素P450单加氧酶）,是甾醇到brs2生物合成途径分支点的第一限速酶,起C-22羟化酶的作用[10,15],该步骤（图1[10]）是油菜素内酯（BRS）生物合成和内源性油菜素内酯水平反馈调控的关键[13]。1998年,CHOE等[13]和SHIMADA等[16]从拟南芥中鉴定得到第一个DWF4,该基因的突变体生长缓慢,植株矮小,叶片小而多,且高度不育,而超表达DWF4促使植物中油菜素内酯合成的增加,进而促进植物的生长并增强植物的抗逆性。进一步研究表明,在促进植物生长方面,超表达DWF4主要通过促进植物细胞的伸长和分裂进而促进转植株的生长[17]。此外DWF4还参与调控植物的分枝、花序、种子产量等,与野生型相比,超表达DWF4的转基因拟南芥的株高和种子产量均被显著提高,且超表达DWF4的转基因植株的二级分枝数[13,18]、鲜重和生物量[19]等均显著增加。DWF4的生理功能极其广泛,除了在生长发育中起作用,还能够显著提高植物的抗逆性。研究显示,超表达DWF4可以增强烟草对非生物胁迫的抗性[19],提高马铃薯的耐盐性[20]和芥菜的抗寒能力[21]。在生理学水平上,DWF4主要通过增加渗透调节物质的积累和提高抗氧化酶的活性增强植物对干旱、冷害和高温的抗性[22,23],研究证明,DWF4通过调控相关抗氧化防御系统的活性,以保护作物抵御盐害[24,25,26,27]。【本研究切入点】迄今为止,紫花苜蓿中油菜素内酯合成酶基因DWF4的功能研究鲜见报道,其调控紫花苜蓿抗逆性的机制还不是很清楚。【拟解决的关键问题】本研究从紫花苜蓿中克隆得到紫花苜蓿油菜素内酯合成酶基因MsDWF4,分析其序列特征,调查该基因对多种逆境与激素的响应,构建基因超表达紫花苜蓿材料,并开展耐盐性研究,为探索DWF4调控紫花苜蓿抗逆性的分子机制奠定基础。图1
新窗口打开|下载原图ZIP|生成PPT图1油菜素甾醇的生物合成途径示意图[10]
Fig. 1A simplified brassinosteroid biosynthetic pathway [10]
1 材料与方法
1.1 试验材料
试验于2018年6月至2019年6月在中国农业科学院北京畜牧兽医研究所牧草资源实验室进行。试验所用植物材料为紫花苜蓿中苜1号（Medicago sativa L. cv. Zhongmu No.1）,由中国农业科学院北京畜牧兽医研究所杨青川研究员惠赠。1.2 植物的培养
将紫花苜蓿种子在预先铺好滤纸的培养皿中萌发,至种子露白后,移到1/2MS液体培养基中,在人工智能温室中培养。光照：16 h光/8 h黑暗,温度：25℃/23℃,光强400 μmol·m-2·s-2。1.3 组织表达特性
取紫花苜蓿根尖、花、嫩叶、茎、茎尖和子房等各组织,利用Eastep? Super总RNA提取试剂盒（北京华奥新创科技有限公司）提取各组织总RNA。利用Trans? Script All-in-One First-Strand cDNA Synthesis试剂盒（北京全式金生物技术有限公司）将RNA反转录为cDNA。1.4 逆境和激素处理
将紫花苜蓿中苜1号种子平铺于滤纸上,发芽4 d后移到1/2 MS营养液中,水培两周后,选取长势一致的幼苗进行各项处理。将紫花苜蓿幼苗材料分别移到含有15% PEG、150 mmol·L-1 NaCl、1 μmol·L-1生长素（IAA）、1 μmol·L-1油菜素甾醇（BR）、1 mmol·L-1脱落酸（ABA）和0.1 mmol·L-1的茉莉酸（JA）的1/2MS液体培养基中处理;高温处理时,将幼苗移入35℃培养箱;低温处理时,将幼苗放在4℃培养箱中。以上处理,分别在0、2、4、6、8、12、24和48 h取地上部和根部样品,提取RNA,并反转录为cDNA。每个处理3次生物学重复。1.5 DWF4的克隆
根据已有物种的DWF4序列信息,以Noble Foundation（https://www.noble.org）网站上公布的紫花苜蓿基因序列为参照,设计cDNA全长引物ORF-F/R。以紫花苜蓿植株cDNA为模板,利用KOD-Plus高保真PCR酶（北京百灵克生物科技有限公司）进行PCR扩增。反应条件为10×Buffer for KOD-Plus 5 μL、2 mmol·L-1 dNTP 5 μL、25 mmol·L-1 MgSO4 2 μL、ORF-F 1.5 μL、ORF-R 1.5 μL、cDNA 2 μL、KOD-Plus 1 μL和ddH2O 32 μL。PCR反应程序为94℃ 2 min;94℃ 15 s,56℃ 30 s,68℃ 2 min,共32个循环;68℃ 5 min,4℃保存。将扩增后的PCR产物用AxyPrep DNA凝胶回收试剂盒（购自北京京哲永兴生物技术责任有限公司）回收,与pEASY-T1载体（购自北京全式金生物技术有限公司）连接,将重组质粒转化大肠杆菌DH5α感受态细胞,经分子检测后,阳性克隆菌液送生工生物工程（上海）股份有限公司测序。1.6 生物信息学分析
对获得的序列进行生物信息学分析,生物信息学分析工具及用途见表1。Table 1
表1
表1本研究用到的生物信息学工具
Table 1
| 生物信息学工具 Bioinformatics tools | 网址 Website | 用途 Purpose |
|---|---|---|
| DNAMAN | https://www.lynnon.com/qa.html | 多序列比对Multiple sequence alignment |
| MEGAX | https://www.megasoftware.net/ | 系统发育进化树的建Establishment of phylogenetic tree |
| Evolview | https://www.evolgenius.info/evolview | 系统发育进化树的美Beautification of phylogenetic tree |
| ExPASy | https://web.expasy.org/protparam/ | 蛋白质一级结构分析Protein primary structure analysis |
| TMHMM2.0Server | http://www.cbs.dtu.dk/services/TMHMM | 蛋白跨膜结构域分析Analysis of protein transmembrane domain |
| ExPASy-Protscale | https://web.expasy.org/protscale/ | 蛋白亲疏水性分析Protein hydrophobicity analysis |
| ExPASy-Swiss-model | https://swissmodel.expasy.org/interactive | 蛋白三级结构预测Protein tertiary structure prediction |
| CDD | https://www.ncbi.nlm.nih.gov/Structure/cdd | 蛋白保守结构域预测Protein conserved domain prediction |
| NetPhos3.1Server | http://www.cbs.dtu.dk/services/NetPhos/ | 蛋白激酶磷酸化修饰位点预测 Prediction of protein kinase phosphorylation modification sites |
新窗口打开|下载CSV
1.7 MsDWF4的表达特异性分析
根据得到的紫花苜蓿DWF4的CDS序列设计实时荧光定量PCR特异引物qDF/R（表2）,采用两步法,用ABI7500型荧光定量PCR仪（美国应用生物系统公司,美国）进行qRT-PCR反应扩增。反应体系为×SYBR qPCR Mix（Low Rox）5 μL、qDF（10 μmol·L-1）0.2 μL、qDR（10 μmol·L-1）0.2 μL、ddH2O 1.6 μL和cDNA3 μL。反应条件为95℃ 2 min;95℃ 15 s,60℃ 30 s,40个循环。根据得到的Ct值,将各处理0 h作为对照,用2-△△Ct法[28]计算MsDWF4的相对表达量。以MsActin2为内参基因,每个试验3个样品,每个样品3次重复。Table 2
表2
表2本试验所用引物
Table 2
| 引物名称 Primer name | 引物序列 Primer sequence (5′-3′) |
|---|---|
| ORF-F | CACCCTATTGGCTTCACA |
| ORF-R | GAAAGTGGTCCTATTGACA |
| qDF | AACAAAACATGCCAAACCCAA |
| qDR | CCATTTCCCAAGTATTCCACCA |
| Msactin-F | CAAAAGATGGCAGATGCTGAGGAT |
| Msactin-R | CATGACACCAGTATGACGAGGTCG |
| pMsDWF4-F | ACGGGGGACGAGCTCGGTACCATGTCTGACTCAGATGTAACTT |
| pMsDWF4-R | CTTGCTCACCATGTCGACTCTAGATATTATAGAGTGGGCTTGGACTCTAATTTGTAGG |
| MDF | CCACTGACGTAAGGGATGACG |
| MDR | TGTCGAAACCGATGATACGAACGAA |
新窗口打开|下载CSV
1.8 超表达载体的构建
利用ClonExpressⅡOne Step 克隆试剂盒（北京京哲永兴生物技术责任有限公司）,一步克隆法构建MsDWF4超表达载体。具体为：设计含有KpnⅠ和XbaⅠ酶切位点的引物pMsDWF4F/R（下划线部分表示酶切位点序列,表2）。用pMsDWF4-F/R引物扩增得到含有酶切位点的目的基因的开放阅读框,限制性内切酶KpnⅠ和XbaⅠ双酶切pCAMBIA1300植物表达载体质粒,将目的片段连接到载体上。在插入位点的上下游设计载体检测引物MDF/R,利用PCR和测序鉴定阳性载体质粒。1.9 农杆菌转化及转基因植株的检测
将构建好的载体质粒,采用冻融法转化入农杆菌菌株GV3101,然后用农杆菌介导法转化紫花苜蓿[29]。用CTAB法提取转基因植株基因组DNA,以基因组DNA为模板,以载体质粒为阳性对照（对照1）,以非转基因紫花苜蓿基因组DNA和去离子水为阴性对照（对照2和对照3）,MDF/R为上下游引物,进行PCR检测,筛选转基因阳性株系。1.10 转基因紫花苜蓿的耐盐性分析
提取转基因植株的RNA,逆转录为cDNA后,以qDF/R为引物,进行qRT-PCR检测,筛选出表达量高的株系。将筛选出的株系进行扦插培养,控制生长条件一致,每隔4周修剪一次,直至植株状态一致。12周后,用200 mmol·L-1 NaCl处理转基因和对照植株（扦插处理同时进行）,在处理0、6和12 h分别取各个材料的地上部组织,检测抗氧化酶过氧化氢酶（catalase,CAT,紫外分光光度法）、过氧化物酶（peroxidase,POD,可见分光光度法）和超氧化物歧化酶（superoxide dismutase,SOD,可见分光光度法）的活性,同时用qRT-PCR检测各材料中MsDWF4的表达水平。2 结果
2.1 MsDWF4序列与生物信息学分析
应用同源克隆技术从紫花苜蓿中扩增得到一个完整的开放阅读框（ORF）（图2）。该序列的CDS区长度为1 470 bp,编码489个氨基酸。蛋白保守结构域的预测结果分析发现该基因编码的蛋白在第1—486位氨基酸残基有一个特异性的保守结构域PLN02500（cytochrome P450 90B1）,该蛋白属于P450超家族。图2
新窗口打开|下载原图ZIP|生成PPT图2MsDWF4的扩增结果
Fig. 2Amplification results of MsDWF4
Marker：Trans2K?DNA marker
多序列比对结果表明,该序列编码的蛋白质与其他物种的DWF4高度同源,同源性为61.0%—95.5%。其中与蒺藜苜蓿DWF4（Medicago truncatula,MtDWF4）序列同源性高达95.5%（图3）。说明该序列可能是一个DWF4,将其命名为MsDWF4。
图3
新窗口打开|下载原图ZIP|生成PPT图3不同物种DWF4氨基酸序列比对结果
Fig. 3Amino acid sequence alignment results of DWF4 from different species
将MsDWF4与其他物种的DWF4蛋白,利用MEGA X构建系统发育树,结果表明,紫花苜蓿与豆科物种亲缘关系最近;与壳斗科、茜草科等的关系较远;而与玉米和普通小麦等禾本科物种的亲缘关系最远（图4）。
图4
新窗口打开|下载原图ZIP|生成PPT图4不同物种DWF4系统发育树分析
Fig. 4Phylogenetic tree analysis of DWF4 in different species
GsDWF4：野大豆Glycine soja,KHN36124.1;GmDWF4：大豆Glycine max,XP_003538851.1;CcDWF4：木豆Cajanus cajan,XP_020218337.1;VuDWF4：豇豆Vigna unguiculate,XP_027906963.1;VrDWF：绿豆Vigna radiate var. radiate,XP_014521401.1;AiDWF4：花生Arachis ipaensis,XP_016201854.1;AhDWF4：落花生Arachis hypogaea,XP_025698850.1;AdDWF4：蔓花生Arachis duranensis,XP_015964027.1;MpDWF4：刺毛黎豆Mucuna pruriens,RDX99099.1;MtDWF4：蒺藜苜蓿Medicago truncatula,XP_003611982.2;QlDWF4：大叶栎Quercus lobate,XP_030975180.1;QsDWF4：欧洲栓皮栎Quercus suber,XP_023894277.1;RaDWF4：玫瑰木Rhodamnia argentea,XP_030523810.1;SoDWF4：Syzygium oleosum,XP_030468974.1;CsDWF4：大麻Cannabis sativa,XP_030498974.1;MnDWF4：川桑Morus notabilis,XP_010091301.2;CeDWF4：香樱咖啡Coffea eugenioides,XP_027179795.1;CaDWF4：小果咖啡Coffea Arabica,XP_027074813.1;NaDWF4：野生烟草Nicotiana attenuate,OIT06386.1;SpDWF4：潘那利番茄Solanum pennelli,XP_015064267.1;StDWF4：马铃薯Solanum tuberosum,QAB35648.1;CrDWF4：白菜Capsella rubella,XP_006290928.1;AlDWF4：玉山筷子芥Arabidopsis lyrata subsp. Lyrata,EFH54017.1;ZmDWF4：玉米Zea mays,APQ46083.1;BdDWF4：二穗短柄草Brachypodium distachyon,XP_003558427.1;TaDWF4：普通小麦Triticum aestivum,AAR11387.1;TuDWF4：乌拉尔图小麦Triticum urartu,EMS59113.1。蓝色：豆科;黄色：壳斗科;红色：桃金娘科;绿色：桑科;橘红色：茜草科;粉色：茄科;黑色：十字花科;紫色：禾本科;红色圆圈：MsDWF4 Blue: Leguminosae; Yellow: Fagaceae; Red: Myrtle; Green: Moraceae; Orange: Rubiaceae; Pink: Solanaceae; Black: Cruciferae; Purple: Gramineae; Red circle: MsDWF4
蛋白质理化性质分析结果可得出,MsDWF4蛋白分子量为55.69 kD,理论等电点（pI）为9.00。跨膜结构域的预测结果（电子附图1）显示,在MsDWF4的N端有1个跨膜螺旋（trans-membrane helix,TMHs）结构,位于第7—25位氨基酸;MsDWF4整条链中亲水性氨基酸残基多于疏水性氨基酸残基,表明MsDWF4可能为一个跨膜亲水蛋白（电子附图2）。
通过Swiss-model对MsDWF4进行三级结构同源建模,构建出MsDWF4的同源模型6a17.1.A（电子附图3）。6a17.1.A为拟南芥DWF4的三级结构。全局模型质量估计（global model quality estimation,GMQE）值为0.81,MsDWF4与模板相似性为76.29%,可见紫花苜蓿DWF4与拟南芥DWF4的蛋白三级结构高度相似,两者可能具有相似的蛋白功能。
激酶磷酸化修饰位点预测结果（图5）发现MsDWF4蛋白有32个丝氨酸（Serine）,18个位点较为活跃;有21个苏氨酸（Threonine）,12个位点较为活跃;有14个酪氨酸（Tyrosine）,4个位点较为活跃。
图5
新窗口打开|下载原图ZIP|生成PPT图5MsDWF4激酶磷酸化修饰位点的预测
Fig. 5Prediction of kinase-specific phosphorylation sites of MsDWF4
2.2 紫花苜蓿DWF4表达特性
2.2.1 紫花苜蓿DWF4组织表达特性分析 MsDWF4组织特性分析结果（图6）表明,MsDWF4在根尖组织中的相对表达量最高,在花、嫩叶、茎尖和子房等组织中的表达量次之,其次是子房中的表达量,在成熟的茎中表达丰度最低。图6
新窗口打开|下载原图ZIP|生成PPT图6MsDWF4组织特异性表达模式
Fig. 6Tissue-specific expression pattern of MsDWF4
2.2.2 MsDWF4表达对逆境和外源激素的响应 为了明确MsDWF4对多种非生物逆境（35℃、4℃、15% PEG、150 mmol·L-1 NaCl）和外源激素（1 μmol·L-1 BR、1 μmol·L-1 IAA、1 mmol·L-1 ABA和0.1 mmol·L-1 JA）的响应模式,利用qRT-PCR技术对MsDWF4在不同处理条件下的表达水平进行分析（图7和图8）。
图7
新窗口打开|下载原图ZIP|生成PPT图7不同逆境处理条件下MsDWF4的相对表达量
A、C、E和G分别为各处理下的MsDWF4在地上部表达量,B、D、F和H分别为各处理下MsDWF4在根部的表达量。*：在P<0.05差异显著,**：在P<0.01差异极显著。误差线为每组的标准误差SE（n=3）。下同
Fig. 7Relative expression of MsDWF4 gene under different stress treatments
A, C, E and G are the MsDWF4 expressions of the shoot part under corresponding treatment, and B, D, F and H are the MsDWF4 expressions in the root under corresponding treatment. *: Significant difference at P<0.05, **: Significant difference at P<0.01. Error bar is the standard error SE (n=3) for each group. The same as below
图8
新窗口打开|下载原图ZIP|生成PPT图8不同激素处理下MsDWF4的相对表达量
A、C、E和G分别为各处理下MsDWF4在地上部的表达量,B、D、F和H分别为各处理下MsDWF4在根部的表达量
Fig. 8Relative expression of MsDWF4 under different hormone treatments
A, C, E and G are the MsDWF4 expressions in the shoot under the corresponding treatment, and B, D, F and H are the MsDWF4 expressions in the root under the corresponding treatments
结果显示,NaCl显著诱导基因在地上部和根部的表达,MsDWF4对盐胁迫的响应迅速,在处理2 h后地上部（图7-A）的表达就达到极显著水平,然后在处理6 h后表达快速下降;根部（图7-B）的基因诱导表达水平整体上低于地上部。15% PEG处理下,MsDWF4在地上部（图7-C）被快速诱导,处理4 h表达水平达到峰值,随后表达丰度下降;在根部（图7-D）的表达呈先降低后升高的趋势,并在处理12 h时达到峰值。35℃处理条件下,MsDWF4的相对表达量总体呈上升趋势,地上部（图7-E）和根（图7-F）中MsDWF4的表达量分别在处理6和48 h时达到峰值。低温（4℃）胁迫同样诱导MsDWF4表达显著上调,处理6 h时地上部（图7-G）和根部（图7-H）MsDWF4的相对表达量达到最高值,分别为对照的7.7倍和6.5倍。
MsDWF4对多种激素处理均有响应。外源油菜素内酯处理下,MsDWF4在地上部（图8-A）和根（图8-B）中的表达被抑制,且随着BR处理时间的延长,基因被抑制的程度不断增强。IAA处理下,植株中MsDWF4的表达被诱导且其表达量在处理12 h（地上部）（图8-C）和4 h（根部）（图8-D）达到峰值,分别为对照的10.78倍和8.69倍。ABA诱导MsDWF4表达上调,地上部（图8-E）和根部（图8-F）MsDWF4的表达量在ABA处理2和6 h时达到最高,分别为对照的6.03倍和5.01倍。JA抑制MsDWF4在植株中的表达,MsDWF4的表达在处理4 h（地上部）（图8-G）和2 h（根部）（图8-H）最低,为对照的19.23%和33.21%。
2.3 超表达MsDWF4转基因紫花苜蓿的检测
通过遗传转化,共获得11个超表达MsDWF4的转基因紫花苜蓿株系,并对其进行PCR检测,结果表明,株系2、株系6、株系7、株系8、株系10与株系11的PCR扩增片段长度与目标条带一致（图9）,表明这些株系为阳性株系,超表达载体已经成功转化到紫花苜蓿中。图9
新窗口打开|下载原图ZIP|生成PPT图9转基因植株检测
Fig. 9Identification of positive transgenic lines
Marker：Direct-Load TM Star marker D5000
利用qRT-PCR检测这6个株系中MsDWF4的表达情况,结果显示,株系6和株系7表达量较高（图10）,命名为L6和L7。
图10
新窗口打开|下载原图ZIP|生成PPT图10超表达MsDWF4株系的qRT-PCR检测
Fig. 10Expression level analysis of MsDWF4 gene in overexpression lines by using qRT-PCR
2.4 转基因紫花苜蓿的耐盐性检测
为检测转基因紫花苜蓿的耐盐性,分析了高盐（200 mmol·L-1 NaCl）处理下转基因紫花苜蓿株系（L6和L7）及对照株系中MsDWF4的表达量和3种抗氧化酶（POD、CAT和SOD）的活性。结果显示,转MsDWF4和对照株系中MsDWF4的表达均被盐胁迫显著诱导,而且转基因株系的诱导水平显著高于对照植株。在正常条件下,转基因株系的CAT、POD和SOD酶活性均显著高于野生型;受高盐胁迫,转基因株系和对照株系中3种酶活性均显著增强,但2个转基因株系的抗氧化酶活性显著高于对照,增加速度更快。在盐处理12 h后,CAT和POD活性在转基因材料和对照中均下降,CAT活性仍高于对照植株。而SOD的活性在处理12 h仍然呈上升趋势,并且显著高于对照植株。图11
新窗口打开|下载原图ZIP|生成PPT图11200 mmol·L-1 NaCl处理条件下MsDWF4的表达及抗氧化酶的活性
不同小写字母表示差异达显著水平（P<0.05）
Fig. 11Expression level of MsDWF4 and antioxidant enzyme activity analysis in CK and transgenic lines under 200 mmol·L-1 NaCl treatment
Different small letters indicated significant difference at 5% level
3 讨论
DWF4是油菜素内酯合成的限速酶基因,已在多种植物中得到分离鉴定,但紫花苜蓿中DWF4的研究还未见报道。本研究利用同源克隆的方法分离得到紫花苜蓿油菜素内酯合成酶基因MsDWF4。对MsDWF4蛋白特性进行分析得知MsDWF4为不稳定的跨膜亲水蛋白,其不稳定性可能为后续蛋白质的功能研究增加难度[30]。MsDWF4蛋白的三级结构的预测分析表明,DWF4蛋白的三级立体结构形成一个中空的活性囊。研究表明,DWF4蛋白的活性囊结构是其与配合物的结合位置,活性囊的形状和体积会随着配合物种类的变化而变化[30],DWF4的特异性抑制剂BRZ（brassinazole）,与DWF4结合后,DWF4的空间构象发生改变,抑制BRs合成过程中的C-22位的羟基化反应,降低内源BRs水平[31,32,33,34]。因此,紫花苜蓿的MsDWF4蛋白可能也是通过这样的方式进行BR生物合成调控的。这些结构和理化性质的分析有利于加深对MsDWF4蛋白的了解,并且为之后MsDWF4的功能研究提供理论基础。蛋白质磷酸化是一种重要的翻译后修饰[35],主要集中在肽链中的酪氨酸、丝氨酸、苏氨酸残基上,磷酸化后,这些氨基酸带上电荷,导致蛋白质的结构发生变化,进一步引起蛋白质的活性发生变化[36],磷酸化修饰与激素反应,信号转导,细胞周期及生长发育等诸多的生物学问题密切相关[36]。本研究预测了MsDWF4的磷酸化位点以及活跃的磷酸化位点,为后期开展MsDWF4功能的研究奠定了基础。
通过对MsDWF4表达模式的分析,发现MsDWF4主要在根尖、茎尖等生长旺盛的组织器官中表达,这与前人研究相似[16,37]。暗示MsDWF4可能与其他物种的DWF4一样,参与了紫花苜蓿的生长发育过程,对植株生长具有促进作用。具体功能还需要进一步的验证。
多项研究显示,DWF4参与了植物抗逆性的调控。SAHNI等[38]通过对植物的生长状态和应激反应的表型观测发现,与野生型相比,超表达拟南芥DWF4的转基因植物对脱水和热激的耐受性明显增加。低温环境下,转DWF4的植株相对电导率明显低于野生型,而脯氨酸积累量则明显高于对照野生型[21]。拟南芥AtDWF4过表达的幼苗相较于野生型幼苗低温耐受性明显提高[39],即超表达DWF4能够提高植株的抗寒性。本研究对MsDWF4在高温、低温和干旱胁迫下的表达进行了分析。结果显示,NaCl、15%PEG、35℃和4℃逆境处理下,MsDWF4的表达均可被不同程度的诱导,说明MsDWF4很可能也参与了这些逆境胁迫的响应。
自然环境中,当植物在生长发育过程中长时间受到非生物胁迫（如干旱、高盐、高温和低温等）时,植物细胞感知逆境信号并转换为胞内信号,进而通过调控相关基因表达和蛋白的合成,改变其细胞内部的生理代谢,启动各种防护机制以抵御和适应逆境胁迫[40]。其中抗氧化酶系统就是一个非常重要的防御机制。当植物遭受逆境胁迫时,细胞通过增加相关抗氧化防护酶（CAT、POD和SOD等）活性的和一些非酶类的小分子物质（还原性谷胱甘肽、抗坏血酸盐、生育酚等）的含量来清除逆境胁迫下细胞内积累的大量的活性氧（reactive oxygen species,ROS）以降低ROS对细胞器和各种生物大分子的破坏,从而维持细胞内环境的稳态及各种代谢活动的正常进行[41,42,43]。研究发现,盐胁迫下超表达DWF4的马铃薯植株中,渗透调节物质（如脯氨酸、可溶性蛋白和可溶性糖等）的含量和抗氧化酶（SOD、POD和APX等）的活性均高于对照组,渗透调节物质和抗氧化酶共同减轻植物盐胁迫伤害从而来提高马铃薯的抗盐性[13,20,24-27]。PeDWF4可促使盐胁迫下的植物细胞大量表达DWF4进而增加BR含量,从而提高转基因材料的抗盐性[44]。本研究中,盐胁迫下,MsDWF4的表达被显著诱导,且高盐胁迫下超表达MsDWF4的紫花苜蓿植株中MsDWF4的表达量和SOD、POD和CAT这些抗氧化酶的活性都较对照高,这些抗氧化酶可清除盐胁迫下植物体内增加的ROS,减轻紫花苜蓿因盐胁迫造成的伤害,从而使转基因植株具有更高的耐盐性。说明MsDWF4通过调节植物的抗氧化酶系统,正向调控了紫花苜蓿的耐盐性。
ABA被称为“逆境激素”[45],逆境胁迫条件下植株体内的脱落酸大量积累,并参与逆境胁迫应答。已有研究发现,ABA处理后玉米DWF4的相对表达量升高[46]。本研究中,MsDWF4受多种逆境胁迫的诱导表达上调,因此我们分析了ABA激素对MsDWF4表达的影响。结果显示,无论是在地上部还是根部,该基因表达都受ABA诱导上调。暗示MsDWF4对逆境胁迫的响应可能是依赖于或部分依赖于ABA途径的。
油菜素内酯（BRs）的动态平衡是高等植物正常生长发育的基础,植物内源油菜素内酯积累增加会导致油菜素内酯生物合成降低以及降解增加。DWF4是BR生物合成的第一限速酶基因,被认为是平衡BR浓度水平的目的调节基因[13]。外施油菜素内酯合成抑制剂Brz（brassinazole）可以增加DWF表达,而用表油菜素内酯（brassinolide,BL）处理植物,会使特异参与油菜素内酯生物合成途径的基因DWF4和BR6ox1等的表达降低,内源性BL含量增加时BR特异性生物合成基因,DWF4、CPD、BR6ox1和ROT3等基因的表达下调,而BR缺陷体中这些基因的表达上调[47,48,49]。本研究中用1 μmol·L-1 BR处理紫花苜蓿后,MsDWF4的表达被显著抑制。说明在紫花苜蓿中,外源BR对DWF4起到抑制作用,以维持植物体内油菜素内酯的动态平衡。
除了ABA,BR与其他激素也存在互作。REN等[50]通过对与JA信号转导相关基因的相对表达量的检测发现,当外界条件变化触发JA信号时,JA通过下调DWF4的表达来破坏BRs信号,从而实现植物的最佳生长。本研究中,0.1 μmol·L-1 JA处理下,根部和地上部MsDWF4的表达均被抑制,暗示JA是对MsDWF4进行负调控从而实现对紫花苜蓿生长的调控。KIM等[51]、CHUNG等[52]和YOSHIMITSU等[53]通过对启动子的分析和内源性BRs的测定证明生长素对BRs生物合成有刺激作用且直接调控BR的生物合成。本研究中用低浓度的生长素（1 μmol·L-1）处理紫花苜蓿后,根部和地上部中MsDWF4的表达均被显著上调。说明在紫花苜蓿中,生长素对BR的生物合成同样起到积极的促进作用。
4 结论
获得紫花苜蓿油菜素内酯合成的关键酶基因MsDWF4,该基因编码489个氨基酸,为亲水性跨膜蛋白,属于P450超家族。MsDWF4主要在生长活跃的组织和器官中表达,可能参与紫花苜蓿某些生长发育方面的调控,并对多种非生物逆境发生响应。MsDWF4正向调控紫花苜蓿的耐盐性。参考文献 原文顺序
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被引期刊影响因子
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DOI:10.3864/j.issn.0578-1752.2013.08.020URL [本文引用: 1]
紫花苜蓿（Medicago sativa L.）适应性强、生物质产量高，茎可用于生产酒精，叶片可用于家畜饲料，是最具开发潜力的能源植物之一。本文从紫花苜蓿生物能源利用角度出发，系统回顾了近年来紫花苜蓿作为能源植物在种质资源评价、细胞壁主要成分（纤维素、半纤维素、木质素）合成途径及其遗传调控以及栽培管理对酒精生产效率的影响等方面的研究进展，重点讨论了苜蓿细胞壁发育和木质素生物合成基因及其调控效应，并对紫花苜蓿作为能源植物的研发前景进行了展望。
DOI:10.3864/j.issn.0578-1752.2013.08.020URL [本文引用: 1]
紫花苜蓿（Medicago sativa L.）适应性强、生物质产量高，茎可用于生产酒精，叶片可用于家畜饲料，是最具开发潜力的能源植物之一。本文从紫花苜蓿生物能源利用角度出发，系统回顾了近年来紫花苜蓿作为能源植物在种质资源评价、细胞壁主要成分（纤维素、半纤维素、木质素）合成途径及其遗传调控以及栽培管理对酒精生产效率的影响等方面的研究进展，重点讨论了苜蓿细胞壁发育和木质素生物合成基因及其调控效应，并对紫花苜蓿作为能源植物的研发前景进行了展望。
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Plants unable to synthesize or perceive brassinosteroids (BRs) are dwarfs. Arabidopsis dwf4 was shown to be defective in a steroid 22alpha hydroxylase (CYP90B1) step that is the putative rate-limiting step in the BR biosynthetic pathway. To better understand the role of DWF4 in BR biosynthesis, transgenic Arabidopsis plants ectopically overexpressing DWF4 (AOD4) were generated, using the cauliflower mosaic virus 35S promoter, and their phenotypes were characterized. The hypocotyl length of both light- and dark-grown AOD4 seedlings was increased dramatically as compared to wild type. At maturity, inflorescence height increased >35% in AOD4 lines and >14% in tobacco DWF4 overexpressing lines (TOD4), relative to controls. The total number of branches and siliques increased more than twofold in AOD4 plants, leading to a 59% increase in the number of seeds produced. Analysis of endogenous BR levels in dwf4, Ws-2 and AOD4 revealed that dwf4 accumulated the precursors of the 22alpha-hydroxylation steps, whereas overexpression of DWF4 resulted in increased levels of downstream compounds relative to Ws-2, indicative of facilitated metabolic flow through the step. Both the levels of DWF4 transcripts and BR phenotypic effects were progressively increased in dwf4, wild-type and AOD4 plants, respectively. This suggests that it will be possible to control plant growth by engineering DWF4 transcription in plants.
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DOI:10.1104/pp.008722URLPMID:12376657 [本文引用: 1]
The natural occurrence of 22-hydroxylated steroids in cultured Catharanthus roseus cells and in Arabidopsis seedlings was investigated. Using full-scan gas chromatography-mass spectrometry analysis, (22S)-22-hydroxycampesterol (22-OHCR), (22S,24R)-22-hydroxyergost-4-en-3-one (22-OH-4-en-3-one), (22S,24R)-22-hydroxy-5alpha-ergostan-3-one (22-OH-3-one), 6-deoxocathasterone (6-deoxoCT), 3-epi-6-deoxoCT, 28-nor-22-OHCR, 28-nor-22-OH-4-en-3-one, 28-nor-22-OH-3-one, 28-nor-6-deoxoCT, and 3-epi-28-nor-6-deoxoCT were identified. Metabolic experiments with deuterium-labeled 22-OHCR were performed in cultured C. roseus cells and Arabidopsis seedlings (wild type and det2), and the metabolites were analyzed by gas chromatography-mass spectrometry. In both C. roseus cells and wild-type Arabidopsis seedlings, [(2)H(6)]22-OH-4-en-3-one, [(2)H(6)]22-OH-3-one, [(2)H(6)]6-deoxoCT, and [(2)H(6)]3-epi-6-deoxoCT were identified as metabolites of [(2)H(6)]22-OHCR, whereas the major metabolite in det2 seedlings was [(2)H(6)]22-OH-4-en-3-one. Analysis of endogenous levels of these brassinosteroids revealed that det2 accumulates 22-OH-4-en-3-one. The levels of downstream compounds were remarkably reduced compared with the wild type. Exogenously applied 22-OH-3-one and 6-deoxoCT were found to rescue det2 mutant phenotypes, whereas 22-OHCR and 22-OH-4-en-3-one did not. These results substantiate the existence of a new subpathway (22-OHCR --> 22-OH-4-en-3-one --> 22-OH-3-one --> 6-deoxoCT) and reveal that the det2 mutant is defective in the conversion of 22-OH-4-en-3-one to 22-OH-3-one, which leads to brassinolide biosynthesis.
DOI:10.1104/pp.013029URLPMID:12529536 [本文引用: 2]
Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. There is only limited information on where BRs are synthesized and used. We studied the organ specificity of BR biosynthesis in Arabidopsis, using two different approaches: We analyzed the expression of BR-related genes using real-time quantitative reverse transcriptase-polymerase chain reaction, and analyzed endogenous BRs using gas chromatography-mass spectrometry. Before starting this study, we cloned the second BR-6-oxidase (BR6ox2) gene from Arabidopsis and found that the encoded enzyme has the same substrate specificity as the enzyme encoded by the previously isolated 6-oxidase gene (BR6ox1) of Arabidopsis. Endogenous BRs and the expression of BR-related genes were detected in all organs tested. The highest level of endogenous BRs and the highest expression of the BR6ox1, BR6ox2, and DWF4 genes were observed in apical shoots, which contain actively developing tissues. These genes are important in BR biosynthesis because they encode the rate-limiting or farthest downstream enzyme in the BR biosynthesis pathway. The second highest level of endogenous BRs and expression of BR6ox1 and DWF4 were observed in siliques, which contains actively developing embryos and seeds. These findings indicate that BRs are synthesized in all organs tested, but are most actively synthesized in young, actively developing organs. In contrast, synthesis was limited in mature organs. Our observations are consistent with the idea that BRs function as the growth-promoting hormone in plants.
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[本文引用: 1]
DOI:10.1007/s00299-007-0418-4URL [本文引用: 1]
DWF4 encodes a rate-limiting mono-oxygenase that mediates 22α-hydroxylation reactions in the BR biosynthetic pathway and it is the target gene in the BR feedback loop. Knockout of DWF4 results in a dwarfed phenotype and other severe defects in Arabidopsis. Here we report on the isolation of the ZmDWF4 gene in maize. Sequence analysis revealed that the open reading frame of ZmDWF4 was 1,518bp, which encodes a protein composed of 505 amino acid residues with a calculated molecular mass of 57.6kD and a predicated isoelectric point (pI) of 9.54. Phylogenetic analysis indicated that ZmDWF4 was very close to the Arabidopsis DWF4. In young maize seedlings, the expression of ZmDWF4 in shoots was much higher than that in roots. The highest expression of ZmDWF4 was observed in husk leaves and the lowest in silks during flowering stage. The expression of ZmDWF4 in maize was significantly down regulated by exogenous brassinolide. A heterogeneous complementary experiment demonstrated that the defects of three Arabidopsis DWF4 mutants could be rescued by constitutive expression of ZmDWF4, with leaf expandability, inflorescence stem heights and fertile capabilities all restored to normal levels. Increases in seed and branch number as well as the height of florescence stem were observed in the over-expressed transformants. These findings suggest that ZmDWF4 may be an ortholog gene of Arabidopsis DWF4 and responsible for BR biosynthesis in maize.
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DOI:10.1007/s11738-010-0661-0URL [本文引用: 1]
We conducted pot experiments to investigate the effects of brassinolide on 1-year-old Xanthoceras sorbifolia B. seedlings. In the experiment, roots were soaked in 0-0.4 mg/l brassinolide. After the seedlings were established, the soil water content in the pots was regulated to simulate drought conditions and various physiological parameters were measured. The results showed that the treatment with 0.2 mg/l brassinolide decreased the malondialdehyde content and electrolyte leakage of seedlings growing under moderate or severe water stress when compared with untreated seedlings. Leaf water content, relative water content, soluble sugar content, soluble protein content, free proline content, ascorbic acid content, glutathione content and superoxide dismutase, peroxidase, catalase and ascorbate peroxidase activities were all greater in water-stressed seedlings in the 0.2 mg/l brassinolide treatment as compared to the control. The results indicate that the application of brassinolide can ameliorate the effects of water stress and enhance drought resistance of Xanthoceras sorbifolia seedlings.
URL [本文引用: 1]
油菜素内酯化合物（Brassinosteroids，BRs）是国际上公认的一类高效、广谱、无毒的新型植物甾醇类生长激素。近年来研究发现，BRs 的使用可显著提高植物对生物和非生物逆境的抗性，从而促进植物生长发育，大幅度提高作物产量。本文综述了BRs 的类型、应用方式以及在提高蔬菜抵抗干旱、低氧、温度、盐离子、病害、重金属等环境胁迫方面的研究成果，并对未来的研究方向进行了展望。
URL [本文引用: 1]
油菜素内酯化合物（Brassinosteroids，BRs）是国际上公认的一类高效、广谱、无毒的新型植物甾醇类生长激素。近年来研究发现，BRs 的使用可显著提高植物对生物和非生物逆境的抗性，从而促进植物生长发育，大幅度提高作物产量。本文综述了BRs 的类型、应用方式以及在提高蔬菜抵抗干旱、低氧、温度、盐离子、病害、重金属等环境胁迫方面的研究成果，并对未来的研究方向进行了展望。
[本文引用: 2]
URL [本文引用: 1]
研究了2,4-表油菜素内酯(EBR)对氯化钠胁迫下黄瓜幼苗叶片光合作用及抗氧化系统的影响．结果表明： 与对照相比，氯化钠胁迫导致黄瓜幼苗叶片超氧阴离子产生速率、过氧化氢和丙二醛含量、细胞膜透性显著升高，净光合速率、气孔导度、蒸腾速率、胞间CO2浓度显著下降，幼苗生长显著受抑．EBR可提高黄瓜幼苗叶片超氧化物歧化酶、过氧化物酶、过氧化氢酶等抗氧化酶活性，降低超氧阴离子产生速率、过氧化氢和丙二醛含量及细胞膜透性，维持良好的光合性能，从而促进幼苗生长，有效缓解氯化钠胁迫造成的伤害.
URL [本文引用: 1]
研究了2,4-表油菜素内酯(EBR)对氯化钠胁迫下黄瓜幼苗叶片光合作用及抗氧化系统的影响．结果表明： 与对照相比，氯化钠胁迫导致黄瓜幼苗叶片超氧阴离子产生速率、过氧化氢和丙二醛含量、细胞膜透性显著升高，净光合速率、气孔导度、蒸腾速率、胞间CO2浓度显著下降，幼苗生长显著受抑．EBR可提高黄瓜幼苗叶片超氧化物歧化酶、过氧化物酶、过氧化氢酶等抗氧化酶活性，降低超氧阴离子产生速率、过氧化氢和丙二醛含量及细胞膜透性，维持良好的光合性能，从而促进幼苗生长，有效缓解氯化钠胁迫造成的伤害.
[本文引用: 1]
[本文引用: 1]
DOI:10.1007/s10535-012-0108-0URL [本文引用: 2]
The effects of exogenous 24-epibrassinolide (EBR) on the growth, oxidative damage, antioxidant system and ion contents in eggplant (Solanum melongena L.) seedlings under salt stress were investigated. Eggplant seedlings were exposed to 90 mM NaCl with 0, 0.025, 0.05, 0.10 and 0.20 mg dm(-3) EBR for 10 d. EBR, especially at concentration 0.05 mg dm(-3), alleviated growth suppression caused by NaCl stress, decreased electrolyte leakage, superoxide production and content of malondialdehyde and H2O2 in NaCl-treated plants. EBR also increased activities of superoxide dismutase, guaiacol peroxidase, catalase and ascorbate peroxidase and the contents of ascorbic acid and reduced glutathione. Furthermore, we also found that Na+, Cl- contents were decreased, K+, Ca2+ contents and K+/Na+, Ca2+/Na+ ratios were increased in the presence of EBR under salt stress.
DOI:10.1006/meth.2001.1262URL [本文引用: 1]
DOI:10.1101/sqb.1985.050.01.054URLPMID:3868487 [本文引用: 1]
[本文引用: 2]
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DOI:10.1038/s41477-019-0436-6URLPMID:31182839 [本文引用: 1]
Brassinosteroids (BRs) are essential plant steroid hormones that regulate plant growth and development(1). The most potent BR, brassinolide, is produced by addition of many oxygen atoms to campesterol by several cytochrome P450 monooxygenases (CYPs). CYP90B1 (also known as DWF4) catalyses the 22(S)-hydroxylation of campesterol and is the first and rate-limiting enzyme at the branch point of the biosynthetic pathway from sterols to BRs(2). Here we show the crystal structure of Arabidopsis thaliana CYP90B1 complexed with cholesterol as a substrate. The substrate-binding conformation explains the stereoselective introduction of a hydroxy group at the 22S position, facilitating hydrogen bonding of brassinolide with the BR receptor(3-5). We also determined the crystal structures of CYP90B1 complexed with uniconazole(6,7) or brassinazole(8), which inhibit BR biosynthesis. The two inhibitors are structurally similar; however, their binding conformations are unexpectedly different. The shape and volume of the active site pocket varies depending on which inhibitor or substrate is bound. These crystal structures of plant CYPs that function as membrane-anchored enzymes and exhibit structural plasticity can inform design of novel inhibitors targeting plant membrane-bound CYPs, including those involved in BR biosynthesis, which could then be used as plant growth regulators and agrochemicals.
DOI:10.1007/s00344-003-0065-0URL [本文引用: 1]
When exogenous chemicals allow rapid, conditional, reversible, selective, and dose-dependent control of biological functions, they act like conditional mutations, either inducing or suppressing the formation of a specific phenotype of interest. Exploration of the small molecules that induce the brassinosteroid (BR) deficient-like phenotype in Arabidopsis led us to identify brassinazole as the first candidate for a BR biosynthesis inhibitor. Brassinazole treatment reduced BR content in plant cells. Investigation of target site(s) of brassinazole revealed that the compound directly binds to the DWF4 protein, a cytochrome P450 monooxygenase that catalyzes 22-hydroxylation of the side chain of BRs. These results suggest that brassinazole is a BR biosynthesis inhibitor. There are currently at least two BR biosynthesis inhibitors that act like conditional mutations in BR biosynthesis. They allow the investigation of the functions of BRs in a variety of plant species. Application of BR biosynthesis inhibitors to a standard genetic screen to identify mutants that confer resistance to these inhibitors allowed the identification of new components working in BR signal transduction. This method has advantages over mutant screens using BR-deficient mutants as a background. Development of chemicals that induce phenotypes of interest is now emerging as a useful way to study biological systems in plants and this would be a complement to classical biochemical and genetic methods.
DOI:10.1074/jbc.M103524200URLPMID:11319239 [本文引用: 1]
Brassinazole, a synthetic chemical developed in our laboratory, is a triazole-type brassinosteroid biosynthesis inhibitor that induces dwarfism in various plant species. The target sites of brassinazole were investigated by chemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treated Catharanthus roseus cells. The levels of castasterone and brassinolide in brassinazole-treated plant cells were less than 6% of the levels in untreated cells. In contrast, campestanol and 6-oxocampestanol levels were increased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position. DWF4, which is an Arabidopsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylase, was expressed in Escherichia coli, and the binding affinity of brassinazole and its derivatives to the recombinant DWF4 were analyzed. Among several triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity. The binding affinity and the activity for inhibiting hypocotyl growth were well correlated among the derivatives. In brassinazole-treated A. thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs. These results indicate that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.
DOI:10.1089/adt.2012.478URL [本文引用: 1]
In small-molecule/protein interaction studies, technical difficulties such as low solubility of small molecules or low abundance of protein samples often restrict the progress of research. Here, we describe a quartz-crystal microbalance (QCM) biosensor-based T7 phage display in combination use with a receptor-ligand contacts (RELIC) bioinformatics server for application in a plant Brz2001/DWARF4 system. Brz2001 is a brassinosteroid biosynthesis inhibitor in the less-soluble triazole series of compounds that targets DWARF4, a cytochrome P450 (Cyp(450)) monooxygenase containing heme and iron. Using a Brz2001 derivative that has higher solubility in 70% EtOH and forms a self-assembled monolayer on gold electrode, we selected 34 Brz2001-recognizing peptides from a 15-mer T7 phage-displayed random peptide library using a total of four sets of one-cycle biopanning. The RELIC/MOTIF program revealed continuous and discontinuous short motifs conserved within the 34 Brz2001-selected 15-mer peptide sequences, indicating the increase of information content for Brz2001 recognition. Furthermore, an analysis of similarity between the 34 peptides and the amino-acid sequence of DWARF4 using the RELIC/MATCH program generated a similarity plot and a cluster diagram of the amino-acid sequence. Both of these data highlighted an internally located disordered portion of a catalytic site on DWARF4, indicating that this portion is essential for Brz2001 recognition. A similar trend was also noted by an analysis using another 26 Brz2001-selected peptides, and not observed using the 27 gold electrode-recognizing control peptides, demonstrating the reproducibility and specificity of this method. Thus, this affinity-based strategy enables high-throughput detection of the small-molecule-recognizing portion on the target protein, which overcomes technical difficulties such as sample solubility or preparation that occur when conventional methods are used.
DOI:10.3981/j.issn.1000-7857.2012.31.011URL [本文引用: 1]
蛋白质磷酸化是由蛋白质激酶催化的磷酸基转移反应,是最常见、最重要的蛋白质翻译后修饰方式之一,是一种普遍的生命活动调节方式,在细胞信号转导过程中起重要作用。本文介绍了蛋白质磷酸化修饰的主要类型与功能、磷酸化蛋白的鉴定及磷酸化位点的预测等方面研究进展,并着重介绍了一些灵敏度高、特异性强的以同位素标记、免疫印迹-化学发光法等作为核心的磷酸化蛋白质分析方案。Western blot方法被证明是鉴别磷蛋白的灵敏、特异方法,而NanoPro100/1000超微量蛋白分析系统等又在此基础上加以改善。蛋白磷酸化分析工具和软件的发展也很迅猛。
DOI:10.3981/j.issn.1000-7857.2012.31.011URL [本文引用: 1]
蛋白质磷酸化是由蛋白质激酶催化的磷酸基转移反应,是最常见、最重要的蛋白质翻译后修饰方式之一,是一种普遍的生命活动调节方式,在细胞信号转导过程中起重要作用。本文介绍了蛋白质磷酸化修饰的主要类型与功能、磷酸化蛋白的鉴定及磷酸化位点的预测等方面研究进展,并着重介绍了一些灵敏度高、特异性强的以同位素标记、免疫印迹-化学发光法等作为核心的磷酸化蛋白质分析方案。Western blot方法被证明是鉴别磷蛋白的灵敏、特异方法,而NanoPro100/1000超微量蛋白分析系统等又在此基础上加以改善。蛋白磷酸化分析工具和软件的发展也很迅猛。
DOI:10.1016/j.neuroscience.2009.11.066URLPMID:19961907 [本文引用: 2]
Post-translational modifications of proteins are a major determinant of biological function. Phosphorylation of proteins involved in signal transduction contributes to the induction and maintenance of several examples of cellular and synaptic plasticity. In this study we have identified phosphoproteins regulated by Pavlovian conditioning in lysates of Hermissenda nervous systems using two-dimensional electrophoresis (2DE) in conjunction with (32)P labeling, fluorescence based phosphoprotein in-gel staining, and mass spectrometry. Modification of protein phosphorylation regulated by conditioning was first assessed by densitometric analysis of (32)P labeled proteins resolved by 2DE from lysates of conditioned and pseudorandom control nervous systems. An independent assessment of phosphorylation regulated by conditioning was obtained from an examination of 2D gels stained with Pro-Q Diamond phosphoprotein dye. Mass spectrometric analysis of protein digests from phosphoprotein stained analytical gels or Coomassie Blue stained preparative gels provided for the identification of phosphoproteins that exhibited statistically significant increased phosphorylation in conditioned groups as compared to pseudorandom controls. A previously identified cytoskeletal related protein, Csp24 (24 kDa conditioned stimulus pathway phosphoprotein), involved in intermediate-term memory exhibited significantly increased phosphorylation detected 24 h post-conditioning. Our results show that proteins involved in diverse cellular functions such as transcriptional regulation, cell signaling, cytoskeletal regulation, metabolic activity, and protein degradation contribute to long-term post-translational modifications associated with Pavlovian conditioning.
DOI:10.1104/pp.005439URLPMID:12226529 [本文引用: 1]
Cytochrome P450 enzymes of the closely related CYP90 and CYP85 families catalyze essential oxidative reactions in the biosynthesis of brassinosteroid (BR) hormones. Arabidopsis CYP90B1/DWF4 and CYP90A1/CPD are responsible for respective C-22 and C-23 hydroxylation of the steroid side chain and CYP85A1 catalyzes C-6 oxidation of 6-deoxo intermediates, whereas the functions of CYP90C1/ROT3, CYP90D1, and CYP85A2 are still unknown. Semiquantitative reverse transcriptase-polymerase chain reaction analyses show that transcript levels of CYP85 and CYP90 genes are down-regulated by brassinolide, the end product of the BR biosynthesis pathway. Feedback control of the CYP90C1, CYP90D1, and CYP85A2 genes by brassinolide suggests that the corresponding enzymes might also participate in BR synthesis. CYP85 and CYP90 mRNAs show strong and transient accumulation during the 1st week of seedling development, as well as characteristic organ-specific distribution. Transcripts of CYP90A1 and CYP85A2 are preferentially represented in shoots and CYP90C1, CYP90D1, and CYP85A1 mRNAs are more abundant in roots, whereas CYP90B1 is ubiquitously expressed. Remarkably, the spatial pattern of CYP90A1 expression is maintained in the BR-insensitive cbb2 mutant, indicating the independence of organ-specific and BR-dependent regulation. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in shoots and roots of Arabidopsis, pea (Pisum sativum), and tomato (Lycopersicon esculentum) reveal similar partitioning patterns of BR intermediates in these species. Inverse correlation between CYP90A1/CPD transcript levels and the amounts of the CYP90A1 substrate 6-deoxocathasterone in shoots and roots suggests that transcriptional regulation plays an important role in controlling BR biosynthesis.
DOI:10.1038/srep28298URLPMID:27324083 [本文引用: 1]
As a resource allocation strategy, plant growth and defense responses are generally mutually antagonistic. Brassinosteroid (BR) regulates many aspects of plant development and stress responses, however, genetic evidence of its integrated effects on plant growth and stress tolerance is lacking. We overexpressed the Arabidopsis BR biosynthetic gene AtDWF4 in the oilseed plant Brassica napus and scored growth and stress response phenotypes. The transgenic B. napus plants, in comparison to wild type, displayed increased seed yield leading to increased overall oil content per plant, higher root biomass and root length, significantly better tolerance to dehydration and heat stress, and enhanced resistance to necrotrophic fungal pathogens Leptosphaeria maculans and Sclerotinia sclerotiorum. Transcriptome analysis supported the integrated effects of BR on growth and stress responses; in addition to BR responses associated with growth, a predominant plant defense signature, likely mediated by BES1/BZR1, was evident in the transgenic plants. These results establish that BR can interactively and simultaneously enhance abiotic and biotic stress tolerance and plant productivity. The ability to confer pleiotropic beneficial effects that are associated with different agronomic traits suggests that BR-related genes may be important targets for simultaneously increasing plant productivity and performance under stress conditions.
DOI:10.1007/s00344-010-9150-3URL [本文引用: 1]
Brassinosteroids (BRs) are essential for proper plant growth and development and also protect plants from a variety of environmental stresses. Seeds contain relatively high levels of BRs, and BRs have been implicated in embryonic patterning and germination. How BR levels in seeds impact germination, growth, and stress tolerance in early seedlings is currently not known. To assess this, the BR biosynthetic gene AtDWF4 was overexpressed in Arabidopsis under the control of a seed-specific oleosin promoter. The resulting transgenic seedlings could overcome inhibition of germination caused by exogenous abscisic acid (ABA) and the seedlings were more tolerant to cold stress compared to wild-type and vector control seedlings. Transcript levels of COR15A, a cold-responsive gene with an established function in cold tolerance, were approximately twofold higher in transgenic seedlings than in control seedlings under cold conditions. These results establish a role for BRs in opposing the inhibitory effects of ABA in seed germination and in promoting cold stress tolerance in early Arabidopsis seedlings.
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DOI:10.1104/pp.005439URLPMID:12226529 [本文引用: 1]
Cytochrome P450 enzymes of the closely related CYP90 and CYP85 families catalyze essential oxidative reactions in the biosynthesis of brassinosteroid (BR) hormones. Arabidopsis CYP90B1/DWF4 and CYP90A1/CPD are responsible for respective C-22 and C-23 hydroxylation of the steroid side chain and CYP85A1 catalyzes C-6 oxidation of 6-deoxo intermediates, whereas the functions of CYP90C1/ROT3, CYP90D1, and CYP85A2 are still unknown. Semiquantitative reverse transcriptase-polymerase chain reaction analyses show that transcript levels of CYP85 and CYP90 genes are down-regulated by brassinolide, the end product of the BR biosynthesis pathway. Feedback control of the CYP90C1, CYP90D1, and CYP85A2 genes by brassinolide suggests that the corresponding enzymes might also participate in BR synthesis. CYP85 and CYP90 mRNAs show strong and transient accumulation during the 1st week of seedling development, as well as characteristic organ-specific distribution. Transcripts of CYP90A1 and CYP85A2 are preferentially represented in shoots and CYP90C1, CYP90D1, and CYP85A1 mRNAs are more abundant in roots, whereas CYP90B1 is ubiquitously expressed. Remarkably, the spatial pattern of CYP90A1 expression is maintained in the BR-insensitive cbb2 mutant, indicating the independence of organ-specific and BR-dependent regulation. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in shoots and roots of Arabidopsis, pea (Pisum sativum), and tomato (Lycopersicon esculentum) reveal similar partitioning patterns of BR intermediates in these species. Inverse correlation between CYP90A1/CPD transcript levels and the amounts of the CYP90A1 substrate 6-deoxocathasterone in shoots and roots suggests that transcriptional regulation plays an important role in controlling BR biosynthesis.
DOI:10.1016/j.devcel.2010.10.010URL [本文引用: 1]
Summary
Brassinosteroids (BRs) regulate a wide range of developmental and physiological processes in plants through a receptor-kinase signaling pathway that controls the BZR transcription factors. Here, we use transcript profiling and chromatin-immunoprecipitation microarray (ChIP-chip) experiments to identify 953 BR-regulated BZR1 target (BRBT) genes. Functional studies of selected BRBTs further demonstrate roles in BR promotion of cell elongation. The BRBT genes reveal numerous molecular links between the BR-signaling pathway and downstream components involved in developmental and physiological processes. Furthermore, the results reveal extensive crosstalk between BR and other hormonal and light-signaling pathways at multiple levels. For example, BZR1 not only controls the expression of many signaling components of other hormonal and light pathways but also coregulates common target genes with light-signaling transcription factors. Our results provide a genomic map of steroid hormone actions in plants that reveals a regulatory network that integrates hormonal and light-signaling pathways for plant growth regulation.Graphical Abstract
View high quality image (437K)
DOI:10.1046/j.1365-313x.1998.00158.xURLPMID:9675902 [本文引用: 1]
The Arabidopsis CPD gene encodes a cytochrome P450 steroid side-chain hydroxylase (CYP90) that plays an essential role in the biosynthesis of the plant hormone brassinolide. Expression of the CPD gene is confined to cotyledons and leaf primordia in etiolated seedlings and detectable in the adaxial parenchyma of expanding leaves in light-grown plants. Transcription of the CPD gene is not affected by the plant growth factors auxin, ethylene, gibberellin, cytokinin, jasmonic acid and salicylic acid, but is specifically down-regulated by brassinolide in both dark and light. Steady-state mRNA levels of a CPD promoter-driven uidA reporter gene correlate with the expression of resident CPD gene in transgenic plants. Intermediates of the early and late C-6 oxidation pathways of brassinolide, carrying C-22 and C-23 side-chain hydroxyls, efficiently inhibit the activity of the CPD promoter. Repression of CPD transcription by brassinosteroids is sensitive to the protein synthesis inhibitor cycloheximide, indicating a requirement for de novo synthesis of a regulatory factor.
DOI:10.1104/pp.109.140202URLPMID:19741050 [本文引用: 1]
The F-box protein CORONATINE INSENSITIVE1 (COI1) plays a central role in jasmonate (JA) signaling and is required for all JA responses in Arabidopsis (Arabidopsis thaliana). To dissect JA signal transduction, we isolated the partially suppressing coi1 (psc1) mutant, which partially suppressed coi1 insensitivity to JA inhibition of root growth. The psc1 mutant partially restored JA sensitivity in coi1-2 background and displayed JA hypersensitivity in wild-type COI1 background. Genetic mapping, sequence analysis, and complementation tests revealed that psc1 is a leaky mutation of DWARF4 (DWF4) that encodes a key enzyme in brassinosteroid (BR) biosynthesis. Physiological analysis showed that an application of exogenous BR eliminated the partial restoration of JA sensitivity by psc1 in coi1-2 background and the JA hypersensitivity of psc1 in wild-type COI1 background. Exogenous BR also attenuated JA inhibition of root growth in the wild type. In addition, the expression of DWF4 was inhibited by JA, and this inhibition was dependent on COI1. These results indicate that (1) BR is involved in JA signaling and negatively regulates JA inhibition of root growth, and (2) the DWF4 is down-regulated by JA and is located downstream of COI1 in the JA-signaling pathway.
DOI:10.1007/s00299-012-1381-2URL [本文引用: 1]
Key message
Arabidopsisgulliver3-D/dwarf4-D displays growth-promoting phenotypes due to activation tagging of a key brassinosteroid biosynthetic geneDWARF4.In gul3-D/dwf4-D, the Jasmonate and Salicylate signaling pathways were relatively activated and suppressed, respectively.Energy allocation between growth and defense is elegantly balanced to achieve optimal development in plants. Brassinosteroids (BRs), steroidal hormones essential for plant growth, are regulated by other plant hormones, including auxin and jasmonates (JA); auxin stimulates the expression of a key brassinosteroid (BR) biosynthetic gene, DWARF4 (DWF4), whereas JA represses it. To better understand the interaction mechanisms between growth and defense, we isolated a fast-growing mutant, gulliver3-D (gul3-D), that resulted from the activation tagging of DWF4, and examined the response of this mutant to defense signals, including JA, Pseudomonas syringae pv. tomato (Pst DC3000) infection, and wounding. The degree of root growth inhibition following MeJA treatment was significantly decreased in gul3-1D/dwf4-5D relative to the wild type, suggesting that JA signaling is partially desensitized in gul3-1D. Quantitative RT-PCR analysis of the genes involved in JA and salicylic acid (SA) responses, including MYC2, PDF1.2, CORI3, PR1, and PR2, revealed that JA signaling was preferentially activated in gul3-1D, whereas SA signaling was suppressed. As a result, gul3-1D was more susceptible to a biotrophic pathogen, Pst DC3000. Based on our results, we propose a model in which BR and JA cooperate to balance energy allocation between growth and defense responses. In ambient conditions, BRs promote plant growth; however, when stresses trigger JA signaling, JA compromises BR signaling by downregulating DWF4 expression.
DOI:10.1111/j.1365-313X.2011.04513.xURLPMID:21284753 [本文引用: 1]
Brassinosteroids (BRs) are growth-promoting steroidal hormones. Despite the importance of BRs in plant biology, the signal that initiates BR biosynthesis remains unknown. Among the enzymes involved in BR biosynthesis in Arabidopsis (Arabidopsis thaliana), DWARF4 catalyzes the rate-determining step. Through both the histochemical analysis of DWF4pro:GUS plants and the direct measurement of endogenous BR content, we discovered that BR biosynthesis is stimulated by auxin. When DWF4pro:GUS was subjected to auxin dose-response tests and a time-course analysis, GUS activity started to increase at an auxin concentration of 10 nm, rising noticeably after 1 h of auxin treatment. In addition, the analysis of the DWF4pro:GUS line in BR- and auxin-mutant backgrounds revealed that the induction by auxin requires auxin-signaling pathways but not BRs, which implies that auxin signaling directly controls BR biosynthesis. Furthermore, chromatin immunoprecipitation assays confirmed that auxin inhibits the binding of the transcriptional repressor, BZR1, to the DWF4 promoter. A microarray analysis that was designed to examine the transcriptomes after treatment with auxin alone or auxin plus brassinazole (a BR biosynthetic inhibitor) revealed that genes previously characterized as being auxin responsive are not properly regulated when BR biosynthesis is disrupted by brassinazole. Therefore, our results support the idea that auxin regulates BR biosynthesis, and that auxin thus relies on synthesized BRs for some of its growth-promoting effects in Arabidopsis.
DOI:10.1371/journal.pone.0023851URLPMID:21909364 [本文引用: 1]
The expression of DWARF4 (DWF4), which encodes a C-22 hydroxylase, is crucial for brassinosteroid (BR) biosynthesis and for the feedback control of endogenous BR levels. To advance our knowledge of BRs, we examined the effects of different plant hormones on DWF4 transcription in Arabidopsis thaliana. Semi-quantitative reverse-transcriptase PCR showed that the amount of the DWF4 mRNA precursor either decreased or increased, similarly with its mature form, in response to an exogenously applied bioactive BR, brassinolide (BL), and a BR biosynthesis inhibitor, brassinazole (Brz), respectively. The response to these chemicals in the levels of beta-glucuronidase (GUS) mRNA and its enzymatic activity is similar to the response of native DWF4 mRNA in DWF4::GUS plants. Contrary to the effects of BL, exogenous auxin induced GUS activity, but this enhancement was suppressed by anti-auxins, such as alpha-(phenylethyl-2-one)-IAA and alpha-tert-butoxycarbonylaminohexyl-IAA, suggesting the involvement of SCF(TIR1)-mediated auxin signaling in auxin-induced DWF4 transcription. Auxin-enhanced GUS activity was observed exclusively in roots; it was the most prominent in the elongation zones of both primary and lateral roots. Furthermore, auxin-induced lateral root elongation was suppressed by both Brz application and the dwf4 mutation, and this suppression was rescued by BL, suggesting that BRs act positively on root elongation under the control of auxin. Altogether, our results indicate that DWF4 transcription plays a novel role in the BR-auxin crosstalk associated with root elongation, in addition to its role in BR homeostasis.
