CN112898396A - OsWRKY53在正向调控BR信号中的应用 - Google Patents
OsWRKY53在正向调控BR信号中的应用 Download PDFInfo
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Abstract
OsWRKY53在正向调控BR信号中的应用,涉及OsWRKY53正向调控BR信号和水稻株型的分子机理解析。本发明的目的是阐明了OsWRKY53正向调控水稻株型的分子机理。BR信号负调控因子OsGSK2通过直接结合并磷酸化OsWRKY53,降低后者的蛋白稳定性,负向调控BR信号。BR信号正调控因子OsBZR1通过与OsWRKY53相互结合,以一种平行的遗传学关系,协同调控BR下游相关基因的表达,正向调控BR信号。本发明应用于水稻株型育种领域。
Description
技术领域
本发明涉及OsWRKY53正向调控BR信号和水稻株型的分子机理解析。
背景技术
水稻是一种重要的粮食作物,世界上一半以上的人口都以其为主食。近年来粮食需求不断增加,耕地面积却持续减少,提高作物产量是稳定经济发展的关键,也是现在农业面临的挑战。水稻株型是影响作物产量的重要指标,主要由叶倾角、粒型等组成。而植物激素BR,即油菜素内脂是影响水稻株型的重要植物激素。BR功能获得型突变体一般表现为叶倾角增大、粒型增大等特征;而BR功能缺陷型突变体一般表现为叶倾角变小、粒型变小等表型。说明可以通过调控BR信号或BR生物合成来调控水稻株型,进而达到提高作物产量的目的。水稻转录因子OsWRKY53被报道是BR信号的一个正调控因子,能够正向调控BR信号和水稻株型,但是其作用机理并不清楚。
发明内容
本发明的目的是首次解析OsWRKY53在BR信号转导通路中的作用机理,进而阐明OsWRKY53正向调控水稻株型的分子机理。为水稻株型育种提供重要理论价值,对筛选水稻高产新株型具有重要的应用价值。
本发明提供OsWRKY53在正向调控BR信号中的应用。
进一步的,所述OsWRKY53通过与OsBZR1相互结合,协同调控BR下游响应基因的表达。
所述OsBZR1为BR信号正调控因子。
本发明还提供OsWRKY53在负向调控BR信号中的应用。
进一步的,所述OsWRKY53通过被OsGSK2结合并磷酸化,而降低蛋白稳定性。
所述OsGSK2为BR信号负调控因子。
在BR含量低的情况下,BR信号负调控因子OsGSK2处于活性状态,通过结合并磷酸化下游的BR信号正调控因子OsWRKY53,降低其蛋白稳定性,负向调控BR信号,使水稻表现为叶倾角变小、粒型变小等表型。而当植物体内BR含量升高时,OsGSK2的活性被抑制,使OsWRKY53和OsBZR1的功能被完全释放,二者在BR信号的调控上处于一种平行的关系,二者通过相互结合,协同激活BR下游响应基因的表达,使水稻表现为叶倾角增大、粒型增大等特征。
本发明的有益效果:
本发明首次阐明OsWRKY53正向调控BR信号和水稻株型的分子机理。
本发明通过在OsGSK2-RNAi转基因水稻中,采用CRISPR/Cas9敲除技术,获得OsGSK2-RNAi oswrky53双突变体,双突变体能将OsGSK2-RNAi BR信号增强的表型恢复至野生型水平,说明在BR信号的调控上,OsWRKY53作用于OsGSK2的遗传学下游。
本发明通过双分子荧光互补、LUC互补成像系统、免疫共沉淀等技术手段证实OsWRKY53能够与OsGSK2相结合;体内磷酸化分析、蛋白稳定性分析等手段充分证实OsGSK2通过磷酸化OsWRKY53降低后者的蛋白稳定性。
本发明通过遗传转化手段,将OsWRKY53在Osbzr1-D中过量表达,获得Osbzr1-DOsWRKY53-OE双突变体,双突变体表现出更加显著的BR信号増强的表型,说明OsWRKY53与OsBZR1协同调控BR信号。同时,在Osbzr1-D中,采用CRISPR/Cas9敲除技术,获得Osbzr1-Doswrky53双突变体,发现在Osbzr1-D中敲除OsWRKY53基因并不影响Osbzr1-D的BR信号増强的表型,说明OsWRKY53与OsBZR1在遗传学上是一种平行的关系。
本发明通过双分子荧光互补、LUC互补成像系统、免疫共沉淀等技术手段证实OsWRKY53能够与OsBZR1相结合;转录调控能力分析发现,OsWRKY53与OsBZR1协同抑制BR生物合成基因OsD2的表达。说明二者通过相互结合,协同调控BR下游相关基因的表达。
本发明阐明了OsWRKY53正向调控BR信号和水稻株型的分子机理,为水稻株型育种提供重要线索和理论基础,对改造水稻株型,进而提高作物产量提供重要理论依据,具有广阔的应用前景。
附图说明
图1为OsGSK2-RNAi oswrky53双突变体总体形态图;
图2为OsGSK2-RNAi oswrky53双突变体剑叶叶倾角形态图;
图3为OsGSK2-RNAi oswrky53双突变体剑叶叶倾角大小统计结果;
图4为OsGSK2-RNAi oswrky53双突变体粒型形态图;
图5为OsGSK2-RNAi oswrky53双突变体粒长统计结果;
图6为OsGSK2-RNAi oswrky53双突变体粒宽统计结果;
图7为外源BR对OsGSK2-RNAi oswrky53双突变体叶倾角的敏感性实验结果;
图8为外源BR对OsGSK2-RNAi oswrky53双突变体叶倾角的敏感性实验的统计结果;
图9为BR生物合成基因在OsGSK2-RNAi oswrky53双突变体及对照中的表达特征;
图10为OsGSK2与OsWRKY53的双分子荧光互补的成像结果
图11为OsGSK2与OsWRKY53的LUC互补成像结果
图12为OsGSK2与OsWRKY53的染色体免疫共沉淀的结果
图13为OsGSK2磷酸化OsWRKY53的体外磷酸化结果
图14为OsGSK2降低OsWRKY53蛋白稳定性的结果
图15为Osbzr1-D OsWRKY53-OE双突变体总体形态图
图16为Osbzr1-D OsWRKY53-OE双突变体剑叶叶倾角形态图
图17为Osbzr1-D OsWRKY53-OE双突变体剑叶叶倾角大小统计结果
图18为外源BR对Osbzr1-D OsWRKY53-OE双突变体叶倾角的敏感性实验结果
图19为外源BR对Osbzr1-D OsWRKY53-OE双突变体叶倾角的敏感性实验的统计结果
图20为Osbzr1-D oswrky53双突变体总体形态图
图21为Osbzr1-D oswrky53双突变体剑叶叶倾角形态图
图22为Osbzr1-D oswrky53双突变体剑叶叶倾角统计结果
图23为BR生物合成基因在Osbzr1-D oswrky53双突变体及对照中的表达特征
图24为OsBZR1与OsWRKY53双分子荧光互补的成像结果
图25为OsBZR1与OsWRKY53的LUC互补成像结果
图26为OsBZR1与OsWRKY53的染色体免疫共沉淀的结果
图27为双元荧荧光素酶植物表达载体及效应载体对应的示意图
图28为OsBZR1与OsWRKY53协同抑制OsD2表达的实验结果
具体实施方式
下面对本发明的实施例做详细说明,以下实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方案和具体的操作过程,但本发明的保护范围不限于下述的实施例。
实施例一、OsGSK2-RNAi oswrky53双突变体的获得
1、将OsWRKY53基因的CDS序列输入CRISPR Primer Designer软件,设计2对靶位点引物(F1和R1;F2和R2),构建敲除载体CRISPR/Cas9-OsWRKY53。
正向引物F1:5'-GGCATTCCAGTCGTACCTCTGAGC-3'
反向引物R1:5'-AAACGCTCAGAGGTACGACTGGAA-3'
正向引物F2:5'-GCCGAGCTGGAGGACGGGTACAAC-3'
反向引物R2:5'-AAACGTTGTACCCGTCCTCCAGCT-3'
2、以OsGSK2-RNAi转基因水稻为实验材料,采用农杆菌介导的遗传转化手段获得OsGSK2-RNAi oswrky53双突变体。
3、设计涵盖靶点序列的一对测序引物(F3和R3),对靶点序列进行扩增,并对PCR产物进行测序,直到获得纯合的OsGSK2-RNAi oswrky53双突变体。
正向引物F3:5'-CGGGGTGCCCAAGTTCAAGTC-3'
反向引物R3:5'-ATGGAGCAGCCGTTGTAGGTG-3'
如图1所示,是OsGSK2-RNAi oswrky53双突变体的总体形态图,在OsGSK2-RNAi中敲除OsWRKY53基因能够将其叶倾角增大的表型完全恢复到野生型水平(图2、3)。粒型较GSK2-RNAi相比显著变小(图4、5、6)。叶倾角对外源BR的敏感性恢复至野生型水平(图7、8,图8中■表示ZH11,▲表示GSK2-RNAi,●表示GSK2-RNAi wrky53),BR生物合成基因的表达量显著降低(图9)。以上结果均表明,在BR信号的调控上,OsWRKY53位于BR信号负调控因子OsGSK2的遗传学下游。
二、OsWRKY53与OsGSK2之间双分子荧光互补分析
1、将OsWRKY53(引物:F4和R4)和OsGSK2(引物:F5和R5)分别连入入门载体pENTR中,获得pENTR-OsWRKY53和pENTR-OsGSK2。随后,通过LR的方式,LR至植物表达载体c-GFP和n-GFP上,获得cGFP-OsWRKY53和nGFP-OsGSK2。
2、采用农杆菌介导的遗传转化方法转化烟草叶片
(1)目的载体转化农杆菌GV3101,将GV3101感受态从-80℃冰箱取出,置于冰上融化;将500ng~1μg的目的质粒加入100ul GV3101感受态中,冰上放置30min;迅速置于液氮中5min;从液氮中取出,迅速置于37℃水预锅中水浴5min;冰上2min;加入800μl液体LB培养基,置于全温震荡器(购自MKN公司)中,28℃,120rpm孵育4~5h;离心,弃大部分上清,将剩余菌液涂抹于含有壮观霉素(100ug/ml)(购自Amresco)和利福平(50ug/ml)(购自Amresco)的LB固体培养基上,28℃培养3天左右。
(2)待长出菌落后,挑取单克隆于相应抗生素的液体培养基中,160rpm,28℃培养,过夜。
(3)将以上菌液按照1:100的比例加入含有相应抗生素及终浓度为10mM MES(pH=5.6)和40μM乙酰丁香酮的液体培养基中,28℃,160rpm振荡培养14h。
(4)3200g离心10min,收集菌体,弃上清,用10mM MgCl2重悬菌体,并用分光光度计调整菌液浓度至OD600=1.5,注射所用的携带病毒PTGS抑制子的P19农杆菌浓度调为OD600=1.0,所有重悬菌液中加入乙酰丁香酮至终浓度为200μM,室温静置3小时。
(5)各组合2种菌液与P19菌液按1:1:2的体积比混合至10ml离心管中。
(6)选择烟草植株上部生长良好的叶片,一般以叶片较厚,维管束不明显的为好,用去掉针的注射器将菌液注射到烟草叶片背面。
3、注射好的烟草叶片,暗培养12h,正常光照条件培养2~3天,剪取叶片进行激光共聚焦显微镜观察。
正向引物F4:5'-caccATGGCGTCCTCGACGGGG-3'
反向引物R4:5'-CTAGCAGAGGAGCGACTCGACG-3'
正向引物F5:5'-caccATGGACCAGCCGGCGCC-3'
反向引物R5:5'-TTAGCTCCCAGTATTGAAGAAGTT-3'
如图10所示,只有将OsWRKY53和OsGSK2共同注射的烟草叶片,才能检测到绿色荧光信号,并且互作复合物定位在细胞核内;单独转入其中一方的烟草叶片,检测不到绿色荧光信号;说明OsWRKY53和OsGSK2在植物体内能够相互结合。
三、OsWRKY53与OsGSK2的LUC互补成像系统分析
1、将OsWRKY53(引物:F6和R6)和OsGSK2(引物:F7和R7)分别连入植物表达载体pCAMBIA1300-cLUC和pCAMBIA1300-nLUC中,获得cLUC-OsWRKY53和nLUC-OsGSK2。
2、采用农杆菌介导的遗传转化方法,注射烟草叶片(方法同上)。
3、培养3天后,剪取待观察叶片,在表面涂抹终浓度为1mM的Beetle luciferinpotassium salt(Promega,E1605),暗处理10min,于化学发光成像仪(Tanon 5200)上进行显像。
正向引物F6:5'-TACGCGTCCCGGGGCGGTACCATGGCGTCCTCGACGGGGGG-3'
反向引物R6:5'-ACGAAAGCTCTGCAGGTCGACGCAGAGGAGCGACTCGACGA-3'
正向引物F7:5'-ACGGGGGACGAGCTCGGTACCATGGACCAGCCGGCGCC-3'
反向引物R7:5'-CGCGTACGAGATCTGGTCGACGCTCCCAGTATTGAAGAAGTT-3'
如图11所示,只有将WRKY53和GSK2同时转入的烟草叶片,才能检测到LUC信号;而单独将其中一方与空载体转入的烟草叶片,检测不到LUC信号;说明在植物体内,OsWRKY53与OsGSK2存在互作关系。
四、OsWRKY53与OsGSK2的免疫共沉淀分析
1、将OsWRKY53(引物:F8和R8)和OsGSK2(引物:F9和R9)分别连入植物表达载体HBT-FLAG和PRT107中,获得mOsGSK2-FLAG和MYC-OsWRKY53。
2、转化水稻原生质体
(1)预先配制0.6M甘露醇溶液
(2)将培养好的水稻苗从茎基部截断后,用刀片切成0.5mm薄片,将切好的水稻细条迅速转移入0.6M甘露醇溶液中,在28℃,60-80rpm条件下培养30min。
(3)用Miracloth过滤后,将Miracloth上的水稻薄片转移入含有酶液的250ml三角瓶中,用锡箔纸包住,避免见光,放入28℃摇床,在黑暗条件下60-80rpm酶解4-5h。
Enzyme solution:0.6M甘露醇,10mM MES(pH=5.7),1.5%Cellulase R-10(Yakult),0.75%Macerozyme R-10(Yakult),0.1%BSA,3.4mM CaCl2,50mMβ-巯基乙醇,50μg/ml羧苄青霉素。
(4)加入与酶液等体积的W5溶液,用力摇晃15s,充分释放原生质体。
W5 solution:154mM NaCl,125mM CaCl2,5mM KCl,2mM MES
(5)用W5溶液润洗灭菌过的网筛,将酶解的原生质体通过网筛,过滤到50ml离心管中,水平转子450g离心3min,离心机升降速调整为3,小心去除上清。
(6)用15ml左右W5溶液重悬原生质体,450g,离心3min。
(7)弃上清,用适量MMG溶液重悬原生质体。
MMG solution:0.6M甘露醇,15mM MgCl2,4mM MES
(8)根据实验目的,混合质粒,每种质粒转化的量为10μg,加入100μl上述制备好的水稻原生质体,再加入110μl 40%PEG,轻轻混匀,28℃转化15min。
40%PEG solution:0.6M甘露醇,100mM CaCl2,40%PEG 4000(Fluka,Sigma-Aldrich)
(9)加入1.8ml W5溶液终止反应,450g,离心3min,弃上清。用750μl W5溶液重悬原生质体,转移至24孔培养板中,28℃培养12-16h。
3、CO-IP实验
(1)取40μl proteinA/G beads,incubation buffer洗beads 3次,加入400μlincubationbuffer(加入相应体积的PMSF和PI),用移液器吹打混匀,平均分配至1.5ml EP管中,各加入2μl FLAG标签抗体,置于静音混合器孵育2~3h。
(2)收集水稻原生质体
(3)加入Extraction buffer裂解目的蛋白,具体视蛋白量而定;冰上裂解30min,每隔5min涡旋震荡,使原生质体充分裂解。
(4)4℃,12000rpm高速离心10min,将上清液转移至步骤1中的EP管中,加入相应体积预冷的水或Incubation buffer至TritionX-100浓度小于2%,置于静音混合器上混合2~3h。
(5)吸取50~100μl混合后的蛋白提取液作为Input;用Wash buffer洗beads 3~4次,最终加入50μl Wash buffer洗脱beads,加入10μl loading buffer煮沸5~10min,4℃,12000rpm高速离心5min。
(6)上样,进行SDS-PAGE电泳。
(7)Western blot分析,用FLAG抗体(Abmart,M20008)检测IP结果,用MYC抗体(ThermoFisher,9E10)检测CO-IP结果。
Incubation buffer:50mM Tris-HC(l PH7.5),150mM NaCl,0.5M EDTA,1mM PI,1mM PMSF,20μM MG132。
Extraction buffer:50mM Tris-HCl(PH7.5),150mM NaCl,0.5M EDTA,1mM PI,1mM PMSF,20μM MG132,10%甘油,0.5%Trition X-100。
Wash buffer:50mM Tris-HCl(PH7.5),250mM NaCl,0.1%TritionX-100,1mM PI,1mM PMSF,20μM MG132。
正向引物F8:5'-CTCCCCTTGCTCCGTGGATCCATGGACCAGCCGGCGCC-3'
反向引物R8:5'-GTCGTCCTTGTAGTCAGGCCTGCTCCCAGTATTGAAGAAGTT-3'
正向引物F9:
5'-CGCTCTAGAACTAGTGGATCCGGGTTAATTAACGGTGAACAAGTGAACAA-3'
反向引物R9:5'-TTTGCGGAGTACCCGGGTACCCTAGCAGAGGAGCGACTCGACG-3'
如图12所示,只有加入mOsGSK2-FLAG的泳道,才能检测到MYC-OsWRKY53的杂交信号;未加入mOsGSK2-FLAG的泳道,不能将MYC-OsWRKY53的蛋白免疫沉淀下来;说明在植物体内,OsWRKY53与OsGSK2能够相互结合。
五、OsGSK2对OsWRKY53的体外磷酸化分析
1、将Gateway系统的pENTR-OsWRKY53和pENTR-OsGSK2通过LR的方式,LR至原核诱导表达载体pVP13和pDEST15上,获得MBP-OsWRKY53和GST-OsGSK2。
2、原核诱导表达纯化蛋白
(1)将pVP13-OsWRKY53和pDEST15-OsGSK2转化大肠杆菌BL21
(2)挑取单克隆于含有相应抗生素的液体LB培养基中,37℃,160rpm过夜培养。
(3)按1:100的比例,将菌液加入到新鲜的液体培养基中,37℃,160rpm孵育3~4h,至菌液OD值为0.5左右。
(4)将步骤3的菌液转移至冰上,待菌液冷却下来,加入IPTG至终浓度为1mM,18℃,120rpm培养14~18h。
(5)收集菌体,加入裂解buffer,重悬菌体,冰上放置30min。
(6)超声破碎,4℃,12000rpm,高速离心1h。
(7)将上清转移至新的50ml离心管中,加入相应的结合beads,4℃,静音混合器上孵育3~4h。
(8)收集beads,弃上清,用wash buffer洗8~10次。
(9)加入相应洗脱buffer洗脱目的蛋白。
MBP裂解buffer:200mM NaCl;20mM Tris-HCl,pH=7.4;1mM EDTA;1mM DTT;1mMPMSF。
MBP wash buffer:200mM NaCl;20mM Tris-HCl,pH=7.4;1mM EDTA;1mM DTT;1mMPMSF。
MBP洗脱buffer:200mM NaCl;20mM Tris-HCl,pH=7.4;1mM EDTA;1mM DTT;1mMPMSF;10mM maltose。
GST裂解buffer:140mM NaCl;2.7mM KCl;10mM Na2HPO4;1.8mM KH2PO4;1mM PMSF。
GST wash buffer:140mM NaCl;2.7mM KCl;10mM Na2HPO4;1.8mM KH2PO4;1mMPMSF。
GST洗脱buffer:100mM NaCl;50mM Tris-HCl,pH=10;1mM PMSF。
3、体外磷酸化分析
(1)体外磷酸化反应:
MBP-OsWRKY53(适量);GST-GSK2(适量);1M Tris-HCl(PH7.5)0.5μl;
2M MgCl2 0.1μl;0.1M DTT 0.33μl;60mM ATP 1μl;H2O补齐到20μl。30℃条件下反应45min。
(2)加Loading buffer,煮沸5~10min。
(3)上样,进行SDS-PAGE电泳。
(4)转膜后,根据Phos-tag Biotin BTL-104反应试剂盒进行化学发光检测(APExBIO,F4001)。
如图13所示,GST-OsGSK2蛋白能够磷酸化MBP-OsWRKY53蛋白;并且,在GST-OsGSK2蛋白量足够的情况下,加入MBP-OsWRKY53蛋白量越多,OsWRKY53的磷酸化信号越强。为验证OsGSK2对OsWRKY53磷酸化的特异性,加入OsGSK2的竞争性抑制剂bikinin,发现随着bikinin加入量的增多,OsWRKY53的磷酸化信号逐渐减弱。以上实验充分表明,OsGSK2能够特异的磷酸化OsWRKY53。
五、OsGSK2降低OsWRKY53的蛋白稳定性分析
1、制备水稻原生质体(方法同上)
2、根据实验目的,混合质粒,转化水稻原生质体,过夜培养14h。
3、收集水稻原生质体,加入SDS裂解液(含1mM PMSF,1mM PI,20μM MG132),冰上裂解30min。
4、加入Loading buffer,煮沸5~10min。
5、上样,进行SDS-PAGE电泳。
6、转膜后,用MYC抗体检测MYC-OsWRKY53的表达特征。
如图14所示,加入OsGSK2的泳道,MYC-OsWRKY53蛋白水平的表达量显著降低,说明OsGSK2能够降低OsWRKY53的蛋白稳定性。
六、Osbzr1-D OsWRKY53-OE双突变体的获得
1、载体构建:以日本晴cDNA为模板,参照TaKaRa公司的HS DNAPolymerase操作说明书,以正向引物F10与反向引物R10为扩增引物,扩增OsWRKY53基因的编码区,并将扩增片段克隆进入植物过表达载体PC1390U,形成一个Ubiquitin启动子驱动的OsWRKY53基因过表达载体。
正向引物F10:5'-GTTACTTCTGCACTAGGTACCATGGCGTCCTCGACGGGG-3'
反向引物R10:
5'-TCTTAGAATTCCCGGGGATCCCTAGCAGAGGAGCGACTCGACG-3'
2、以BR功能获得型突变体Osbzr1-D为实验材料,采用农杆菌介导的遗传转化手段获得Osbzr1-D OsWRKY53-OE双突变体。
3、通过qRT-PCR以及Western blot的方式对双突变体进行鉴定。
如图15所示,Osbzr1-D OsWRKY53-OE双突变体表现出较单突变体更加显著的BR信号增强的表型,双突变体叶倾角大于单突变2倍左右,说明OsBZR1和OsWRKY53在叶倾角的调控面上存在协同作用(图16,17)。叶倾角对外源BR的敏感性实验分析发现,Osbzr1-DOsWRKY53-OE双突变体叶倾角对外源BR的敏感性显著强于单突变体,说明OsBZR1和OsWRKY53协同调控BR信号反应(图18,19,图19中■表示WT,●表示WRKY53-OE,▲表示bzr1-D,◆表示bzr1-DWRKY53-OE)。
七、Osbzr1-D oswrky53双突变体的获得
1、以Osbzr1-D转基因水稻为实验材料,采用农杆菌介导的遗传转化手段获得Osbzr1-Doswrky53双突变体。
2、通过测序以及Western blot的方式对双突变体进行鉴定。
如图20所示,是Osbzr1-D oswrky53双突变体的总体形态图,从图中可以发现,在Osbzr1-D中敲除OsWRKY53基因后,并不影响Osbzr1-D在BR表型方面的表现,主要体现在叶倾角大小的调控以及BR生物合成基因的表达上,双突变体与Osbzr1-D的表现都无显著差异(图21,22,23)。说明在在BR信号的调控上,OsWRKY53与OsBZR1是一种平行的关系,二者协同调控BR下游相关基因的表达。
八、OsWRKY53与OsBZR1的双分子荧光互补结果
1、将OsWRKY53和OsBZR1(引物:F11和R11)分别连入入门载体pENTR中,获得pENTR-OsWRKY53和pENTR-OsBZR1。随后,通过LR的方式,LR至植物表达载体c-GFP和n-GFP上,获得cGFP-OsWRKY53和nGFP-OsBZR1。
2、采用农杆菌介导的遗传转化方法转化烟草叶片(方法同上)
3、注射好的烟草叶片,暗培养12h,正常光照条件培养2~3天,剪取叶片进行激光共聚焦显微镜观察。
正向引物F11:5'-caccATGACGTCCGGGGCGGCGG-3'
反向引物R11:5'-TCATTTCGCGCCGACGCCGAG-3'
如图24所示,只有将OsWRKY53和OsBZR1共同注射的烟草叶片,才能检测到绿色荧光信号,并且互作复合物定位在细胞核内;单独转入其中一方的烟草叶片,检测不到绿色荧光信号;说明OsWRKY53和OsBZR1在植物体内能够相互结合。
九、OsWRKY53与OsBZR1的LUC互补成像系统分析
1、将OsWRKY53和OsBZR1(引物:F12和R12)分别连入植物表达载体pCAMBIA1300-cLUC和pCAMBIA1300-nLUC中,获得cLUC-OsWRKY53和nLUC-OsBZR1。
2、采用农杆菌介导的遗传转化方法,注射烟草叶片(方法同上)。
3、培养3天后,剪取待观察叶片,在表面涂抹终浓度为1mM的Beetle luciferinpotassium salt(Promega,E1605),暗处理10min,于化学发光成像仪(Tanon 5200)上进行显像。
正向引物F12:5'-ACGGGGGACGAGCTCGGTACCATGACGTCCGGGGCGGCGG-3'
反向引物R12:5'-CGCGTACGAGATCTGGTCGACTTTCGCGCCGACGCCGAG-3'
如图25所示,只有将OsWRKY53和OsGSK2同时转入的烟草叶片,才能检测到LUC信号;而单独将其中一方与空载体转入的烟草叶片,检测不到LUC信号;说明在植物体内,OsWRKY53与OsBZR1存在互作关系。
十、OsWRKY53与OsBZR1的免疫共沉淀分析
1、将OsWRKY53和OsBZR1(引物:F13和R13)分别连入植物表达载体HBT-FLAG和1300-221-Flag中,获得MYC-OsWRKY53和FLAG-OsBZR1。
2、转化水稻原生质体(方法同上)
3、CO-IP实验(方法同上)
正向引物F13:5'-ACGATGATAAGGGCGGTACCATGACGTCCGGGGCGGCGG-3'
反向引物R13:5'-AGGCTACGTAGGATCCTTTCGCGCCGACGCCGAG-3'
如图26所示,只有加入OsBZR1-FLAG的泳道,才能检测到MYC-OsWRKY53的杂交信号;未加入OsBZR1-FLAG的泳道,不能将MYC-OsWRKY53的蛋白免疫沉淀下来;说明在植物体内,OsWRKY53与OsBZR1能够相互结合。
十一、OsWRKY53与OsBZR1协同抑制OsD2转录表达分析
1、将OsWRKY53(引物:F14和R14)和OsBZR1(引物:F15和R15)分别连入植物表达载体PRT107中,获得效应载体PRT107-OsWRKY53和PRT107-OsBZR1。将OsD2(引物:F16和R16)连入pGreenII 0800-LUC,获得报告载体pGreenII 0800-LUC-OsD2。
2、转化水稻原生质体(方法同上)
3、采用碧云天的植物双荧光素酶报告基因检测试剂盒(RG027)检测相对LUC活性。
正向引物F14:5'-CGCTCTAGAACTAGTGGATCCATGGCGTCCTCGACGGGG-3'
反向引物R14:
5'-TTTGCGGAGTACCCGGGTACC CTAGCAGAGGAGCGACTCGACG-3'
正向引物F15:5'-CGCTCTAGAACTAGTGGATCCATGACGTCCGGGGCGGCGG-3'
反向引物R15:5'-TTTGCGGAGTACCCGGGTACCTCATTTCGCGCCGACGCCGAG-3'
正向引物F16:5'-GGGCCCCCCCTCGAGGTCGACTTACTTGTTTTCTTTTCTGT-3'
反向引物R16:5'-CGCTCTAGAACTAGTGGATCCCCTTTTCTACCCCTCGAG-3'
如图28所示,OsBZR1和OsWRKY53都降低OsD2启动子驱动的LUC基因的表达,而将两者共同转入后,其LUC基因的表达受到更加显著的抑制(图27,28)。以上结果充分说明,OsWRKY53和OsBZR1通过相互结合协同调控BR下游响应基因的表达。
序 列 表
<110> 中国科学院东北地理与农业生态研究所
<120> OsWRKY53 在正向调控BR信号中的应用
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Claims (5)
1.OsWRKY53在正向调控BR信号中的应用。
2.根据权利要求1所述的应用,其特征在于所述OsWRKY53作用于OsGSK2的遗传学下游。
3.根据权利要求1或2所述的应用,其特征在于所述OsWRKY53作为OsGSK2的磷酸化底物,OsGSK2通过磷酸化OsWRKY53,降低OsWRKY53蛋白稳定性。
4.根据权利要求3所述的应用,其特征在于所述OsGSK2活性被抑制后,OsWRKY53恢复转录调控功能,进而正向调控BR信号和水稻株型。
5.根据权利要求1所述的应用,其特征在于所述OsWRKY53通过与OsBZR1相互结合,协同调控BR下游响应基因的表达。
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