CN106868020B - 大豆辅助分子伴侣蛋白编码基因GmHSP40在调控植物开花上的应用 - Google Patents

大豆辅助分子伴侣蛋白编码基因GmHSP40在调控植物开花上的应用 Download PDF

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CN106868020B
CN106868020B CN201710172587.8A CN201710172587A CN106868020B CN 106868020 B CN106868020 B CN 106868020B CN 201710172587 A CN201710172587 A CN 201710172587A CN 106868020 B CN106868020 B CN 106868020B
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刘建中
徐辉杨
倪敏
许为
王志荣
张蕾
张驰
钟晨丽
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Abstract

本发明涉及一种源自大豆的辅助分子伴侣蛋白编码基因GmHSP40在控制植物开花时间上的应用。GmHSP40的cDNA核苷酸序列如SEQ.1所示;利用重组DNA技术构建了过表达GmHSP40的载体并通过转基因技术获得了无论是在长日照还是在短日照条件下开花期显著延迟的转基因拟南芥植株;本发明还证实过表达GmHSP40是通过诱导开花的负调控因子FLC及FLM编码基因的表达,而抑制了开花的正调控因子FT及SOC1编码基因的表达,从而导致拟南芥开花显著延迟。GmHSP40可用于调控植物开花时间及揭示调控植物开花的分子机理。

Description

大豆辅助分子伴侣蛋白编码基因GmHSP40在调控植物开花上 的应用
技术领域
本发明涉及植物基因工程领域,主要申请保护大豆辅助分子伴侣蛋白编码基因GmHSP40在调控植物开花上的应用。
背景技术
HSP40蛋白(heat shock protein 40),最初是在E.coli中发现的,其分子量大约为41kDa,它是HSP70蛋白共伴侣分子(Qiu等,2006.The diversity of the DnaJ/Hsp40family,the crucial partners for Hsp70chaperones[J].Cellular&MolecularLife Sciences Cmls,63(22)2560–2570.)。Hsp40蛋白与HSP70蛋白相互作用(Hennessy等,2005.Rational mutagenesis of a 40kDa heat shock protein from Agrobacteriumtumefaciens identifies amino acid residues critical to its in vivo function[J].International Journal of Biochemistry&Cell Biology,37(1):177-191.)。后来在大部分真核生物中,包括动植物、酵母以及人类等,都能找到它的同源蛋白,它们都含有一个非常保守的J结构域。大量的研究表明J蛋白在生物体的生长发育等各方面都发挥着重要的作用(Orme等,2001.A novel plastid-targeted J-domain protein in Arabidopsisthaliana[J].Plant Molecular Biology,46(5):615-626.)。
HSP70由ATPase活性结构域、C端多变结构域和多肽结合结构域组成,HSP70的ATP酶活性的增强对新生蛋白的折叠、蛋白质正确构象的维持、蛋白质的转运、组装和解离等过程都是必须的(Caplan等,1993.Eukaryotic homologues of Escherichia coli dnaJ:adiverse protein family that functions with hsp70stress proteins[J].MolecularBiology of the Cell,4(6):555-563.)。而HSP70蛋白的ATP酶活性较低,HSP40蛋白能调节HSP70的ATP酶活性从而促进HSP70蛋白完成其各种功能,这与HSP70自身的结构特点是相关的(Fan等,2003.Mechanisms for regulation of Hsp70function by Hsp40[J].CellStress&Chaperones,8(4):309-316.)。由于ATPase活性区不仅能够与ATP结合,同时也能与ADP结合。所以,Hsp70有ADP-Hsp70(ADP结合状态,底物亲和力较高)和ATP-Hsp70(ATP结合状态,后者的底物亲和力较低)两种状态(Mayer等,2005.Hsp70chaperones:Cellularfunctions and molecular mechanism[J].Cellular&Molecular Life Sciences,62:670~684.)。而HSP40能够导致Hsp70在以上两种状态之间转变(Laufen等,1999.Mechanism ofregulation of hsp70chaperones by DnaJ co-chaperones[J].Proceedings of theNational Academy of Sciences of the United States of America,96(10):5452-5457.),当HSP40与Hsp70的ATPase活性区作用,ATPase活性加强促进ATP水解形成ADP,使ATP-Hsp70转变为ADP-Hsp70形态。这种改变能影响Hsp70多肽结合区构象的变化,从而增强了其底物亲和力。J结构域,作为J蛋白的标志,由4个α螺旋和1个HPD模块(含有极为保守的His、Pro、Asp序列)组成(Rajan等,2009.Arabidopsis thalianaJ-class heat shockproteins:Cellular stress sensors[J].Functional&Integrative Genomics,9(4):433-446.)。按照蛋白结构域,J蛋白可分为Ⅰ、Ⅱ和Ⅲ三个亚型(Walsh等,2004.The J-proteinfamily:modulating protein assembly,disassembly and translocation[J].EmboReports,5(6):567-571.),Ⅰ型J蛋白具有4种结构域:螺旋状的J结构域、G/F(甘氨酸/苯丙氨酸)结构域、CXXCXGXG锌指结构域以及羧基末端区;II型J蛋白没有锌指结构域;而Ⅲ型J蛋白仅含有螺旋的J结构域。另外,IV型J蛋白最近也被发现,被称为J-like蛋白,它的结构和序列与J结构域相似,但它没有HPD模块,如拟南芥JLP2(J-like protein 2)的HPD模块被His、Val、Asp序列(HVD)所取代(Peng等,2010.Magmas gene structure and evolution[J].Silico Biology,5(3):251-263.)。
在拟南芥中,共发现有120个HSP40,这些蛋白已经被报道是定位在不同的亚细胞的隔间并参与各种生物过程(Rajan等,2009.Arabidopsis thalianaJ-class heat shockproteins:Cellular stress sensors[J].Functional&Integrative Genomics,9(4):433-446.)。在植物细胞内,HSP40广泛存在于线粒体、叶绿体、细胞质和内质网等细胞器中。根据生物信息学分析,预计有50个HSP40蛋白定位在细胞质中,19个位于线粒体内,12个在叶绿体中,9个在内质网上,3个位于细胞骨架上,1个位于质膜上,24个在细胞核中以及2个在液泡中(Rajan等,2009.Arabidopsis thalianaJ-class heat shock proteins:Cellularstress sensors[J].Functional&Integrative Genomics,9(4):433-446.)。但是,只有少数J结构域蛋白的功能被确定据报道,植物J结构域蛋白参与各种应激反应(Yang等,2010.The Arabidopsis chaperone J3 regulates the plasma membrane H(+)-ATPasethrough interaction with the PKS5kinase[J].Plant Cell,22(4):1313–1332.),以及叶绿体的运动(Noriyuki等,2005.An auxilin-like J-domain protein,JAC1,regulatesphototropin-mediated chloroplast movement in Arabidopsis[J].Plant Physiology,139(1):151-162.)。在寄主—病原体相互作用过程中,病毒外壳和运动蛋白与J蛋白相互作用,分别促进病毒的装配和运动(Shimizu等,2009.Identification of a novel tobaccoDnaJ-like protein that interacts with the movement protein of tobacco mosaicvirus[J].Archives of Virology,154(6):959-67.)。
控制拟南芥植株开花时间的信号途径主要有四条:春化途径(VernalizationPathway)、光周期途径(Photoperiodic Pathway)、赤霉素途径(Gibberellin Pathway)以及自发途径(Autonomous Pathway)(Song等,2013.Flowering time regulation:photoperiod-and temperature-sensing in leaves[J].Trends Plant Sci.18(10):575-83)。FLOWERING LUCUS(FT)和SUPPRESSOR OF OVEREXPRESSION OF CO I(SOC1)分别为光周期途径中CONSTANS(CO)和FLOWER LOCUS(FLC)调控因子的靶基因,在各个调控途径下游具有促进开花的作用,是这些开花途径共同的开化调控整合因子,抑制其表达可导致拟南芥推迟开花(Song等,2013.Flowering time regulation:photoperiod-and temperature-sensing in leaves[J].Trends Plant Sci.18(10):575-83)。FLC是春化途径中重要的负调控因子,在植物信号转导、生长发育尤其是调控开花的过程起着重要作用,与CO的促进作用刚好相反,它是一个成花抑制基因,主要通过抑制SOC1和FT的表达而延迟开花(Hepworth等,2015.Flowering Locus C's Lessons:Conserved Chromatin Switches UnderpinningDevelopmental Timing and Adaptation[J].Plant Physiol.168(4):1237-45)。
发明内容
本发明主要提供一种大豆辅助分子伴侣蛋白编码基因GmHSP40在调控植物开花上的应用。
本发明解决其技术问题采用的技术方案:
一种GmHSP40基因的cDNA核苷酸序列如SEQ ID NO:1所示。
一种GmHSP40基因编码的蛋白质,其氨基酸序列如SEQ ID NO:2所示。
通过RT-PCR从大豆Williams82植株叶片中提取的总RNA中扩增出GmHSP40全长编码序列(Glyma15g057880.1),所用引物为GmHSP40-F,GmHSP40-R。将上述扩增的编码序列首先克隆到pENTR/D入门载体(Invitrogen,http://www.invitrogen.com)中以产生pENTR/D/GmHSP40,随后通过LR反应将该插入片段重组至双元目的载体PB7WG2,0(Karimi等,2002GATEWAYTM vectors for Agrobacterium-mediated plant transformation.TrendsPlant Sci.7,193–195.)中产生PB7WG2,0-GmHSP40。将双元载体PB7WG2,0-GmHSP40转化至农杆菌(GV3101)菌株中,然后通过浸花法(Clough,S.J等,1998Floral dip:a simplifiedmethod for Agrobacterium-mediated transformation of Arabidopsisthaliana.Plant J.16,735–743.)转至拟南芥Col-0中得到GmHSP40-OE转基因拟南芥植株。通过表型观察,我们发现过表达GmHSP40的转基因植株抽苔及开花比同日龄的Col-0野生型植株显著延迟,说明过表达GmHSP40抑制了拟南芥植株的开花。利用上述特性,可将GmHSP40基因的cDNA核苷酸序列或含有过表达GmHSP40全长cDNA的载体应用在促进或延迟农作物及花卉等观赏植物的开花时间上以及研究植物开花的分子机制中。
发明有益的效果是:能利用编码基因GmHSP40的特性对植物开花时间进行调控并揭示调控植物开花的分子机理。
附图说明
图1是过表达大豆GmHSP40全长cDNA载体PB7WG2,0-GmHSP40的T-DNA区域结构示意图;
图2是基因组PCR证明GmHSP40-OE1及GmHSP40-OE2两个转基因拟南芥株系的基因组中携带大豆GmHSP40基因,拟南芥Actin基因作为内参基因对照;
图3是过表达GmHSP40的两个转基因拟南芥株系(GmHSP40-OE1及GmHSP40-OE2)晚开化表型观察;
图4是Col-0和GmHSP40-OE1以及GmHSP40-OE2转基因拟南芥植株长日照条件的莲座叶数和开花天数的比较;
图5是Col-0和GmHSP40-OE1以及GmHSP40-OE2转基因拟南芥植株短日照条件的莲座叶数和开花天数的比较;
图6是RT-PCR检测拟南芥开花相关基因在Col-0和GmHSP40-OE中的表达差异;
图7是qRT-PCR检测拟南芥开花相关基因在Col-0和GmHSP40-OE中的表达差异;
图8是miR156和miR172在Col-0和GmHSP40-OE1和GmHSP40-OE2植株中的积累水平比较。
具体实施方式
下面结合附图对本发明作进一步说明:
通过RT-PCR从大豆Williams82植株叶片中提取的总RNA中扩增出GmHSP40全长编码序列(Glyma15g057880.1),所用引物为GmHSP40-F,
5′-CACCAAAATGGATGGTCACGGAGGAG-3′;GmHSP40-R,5′-ATGACCCTTAACCTTATCATCTGCA-3′。
将上述扩增的编码序列首先克隆到pENTR/D入门载体(Invitrogen)中以产生pENTR/D/GmHSP40,随后通过LR反应将该插入片段重组至双元目的载体PB7WG2,0中产生PB7WG2,0-GmHSP40,如图1所示。将双元载体PB7WG2,0-GmHSP40转化至农杆菌(GV3101)菌株中,然后通过浸花法转至拟南芥Col-0中,具体操作如下:
农杆菌转化:加1μg质粒到100μl GV3101感受态中;液氮冰激5min,37℃水浴热击5min;加800μl LB液体培养基于步骤(3)中于28℃摇2-4h;离心4000rpm,1min收集菌体;弃除上清液,加入100μl LB培养基轻轻悬浮;将菌液涂在平板上,28℃培养2-3天。
浸花法创制过表达GmHSP40的转基因拟南芥株系:将已经转化过的农杆菌在含抗生素筛选培养基上长出单克隆,对鉴定后确定转化成功的单克隆于5ml液体YEP培养基含(抗生素)28℃活化培养16h;取适量(1:50)活化培养的菌液于50ml液体YEP培养基(含抗生素)中28℃扩大培养6h;将200ml农杆菌细胞4000rpm、10min室温离心,悬浮于1倍体积的0.05%Silwet L-77,10mM MgCl2,5%蔗糖溶液(即100ml H2O加入Silwet L-775ml,MgCl2。6H2O 0.202g,蔗糖5g);侵染时将农杆菌悬浮液倒满50ml离心管的小盖子,将拟南芥横放着,使花序侵入小盖子中的悬浮液10s,轻微晃动小盖,200ml农杆菌悬浮液至少有效用于转化300株拟南芥,1500个花序以上,这一步骤要保证能在植株上看到菌液;塑料膜包滚植株让侵染后的植株保持16-24h高湿度黑暗24h;次日去掉包裹的塑料,将侵染后的拟南芥放回温室,然后收集拟南芥种子;春化收集到的种子,种在托盘里,等长出绿色幼苗后,喷洒basta除草剂,能够存活下来的即为所需要的转基因的拟南芥株系。通过农杆菌的转化及基因组PCR验证(图2)得到了两个独立的转基因株系GmHSP40-OE1和GmHSP40-OE2。
将春化好的Col-0、GmHSP40-OE1和GmHSP40-OE2种子均匀铺在1/2MS+1.5%Agar平板上,分别标记好各材料的名称和日期,用封口膜将平板封口,垂直放置于拟南芥人工光照培养箱(光照条件:22℃,16h;黑暗条件:20℃,8h;湿度85%)培养皿架上,待其培养一周后,将幼苗分别移栽到土里。长日照:将移栽至土里的Col-0、GmHSP40-OE1和GmHSP40-OE2幼苗(穴苗盘)置于拟南芥人工光照培养箱,其培养条件为:光照条件:22℃,16h;黑暗条件:20℃,8h;湿度85%。待拟南芥植株刚抽苔1-2cm时,分别统计各个植株的开花天数以及相应的莲座叶片数,如图4;短日照:将移栽至土里的Col-0、GmHSP40-OE1和GmHSP40-OE2幼苗(穴苗盘)置于拟南芥人工光照培养箱,其培养条件为:光照条件:22℃,8h;黑暗条件:20℃,16h;湿度85%。待拟南芥植株刚抽苔1-2cm时,分别统计各个植株的开花天数以及相应的莲座叶片数,如图5。
研究表明,叶片数是用来区分光周期对花发育及开花诱导的效应,这是由于一旦启动开花,拟南芥主茎上将不会再长出莲座叶(陈晓等,2006.光周期影响植物花时的分子机制[J].西北植物学报,26(7):1490-1499.)。通过表型观察,我们发现过表达GmHSP40的转基因植株抽苔及开花比同日龄的Col-0野生型植株显著延迟(图3所示)。我们统计比较了两者的莲座叶数和开花天数,结果表明,无论是在长日照还是短日照条件下,GmHSP40-OE转基因拟南芥植株的莲座比Col-0野生型植株的莲座叶片数要显著增多;并且在长日照条件下GmHSP40转基因拟南芥植株的开花天数要比Col-0延迟了至少15天,在短日照条件下GmHSP40-OE转基因拟南芥植株的开花天数要比Col-0延迟了至少30天。说明过表达GmHSP40抑制了拟南芥植株的开花,过表达大豆GmHSP40导致拟南芥植株开花显著延迟GmHSP40-OE转基因拟南芥植株的晚开花表型。
采用CTAB法提取基因组DNA:取1g拟南芥叶片,移入1.5mLEppendorf管中,用研钵研碎样品致浆状;加入400-500微升的CTAB提取缓冲液,混匀(CTAB在65℃水浴预热),每5分钟轻轻震荡几次,20min后12000r/min离心10min;小心吸取上清液,加入等体积的氯仿(各400微升)溶液,混匀(用震荡仪),4℃,12000r/min,离心10min;小心吸取上清液,加入等体积的异丙醇,用震荡仪混匀,4℃,12000r/min,离心10min;弃去上清液,用70%乙醇洗涤沉淀一次;室温下干燥后(一般干燥5-15min)溶于30-50微升去离子水中,混合,离心,于-20℃或-70℃下保存备用。通过基因组PCR证明GmHSP40-OE1及GmHSP40-OE2两个转基因拟南芥株系的基因组中携带大豆GmHSP40基因,如图2所示。
为了研究Col-0植株和GmHSP40-OE转基因植株中开花相关基因的表达差异,我们通过RT-PCR和qRT-PCR检测了Col-0和GmHSP40-OE转基因植株间开花相关基因的表达差异。具体方法如下:
首先,利用有机试剂Trizol粗提拟南芥RNA:取1g拟南芥叶片放入研钵(研钵使用前应先洗净并烘干,再加入少量无水乙醇并点燃对其进行消毒,之后再放入液氮中预先冷冻)中,向装有叶片的研钵中加入适量液氮(应浸没所有的叶片),在液氮中将叶片按一个方向快速的研磨至粉末,接着将粉末迅速的转移至1.5mL的离心管(提RNA专用)中(离心管在使用前最好先用液氮进行浸泡,且尽量使管内的粉末不超过0.5mL);打开通风厨,在通风厨中迅速向上述离心管内各加入1mL的Trizol试剂(Trizol有剧毒,加药品时应特别注意),立即放在涡旋振荡器上强力振荡,使其充分混匀,每振荡一会就揭开离心管盖放气,防止离心管会热胀冷缩;提前将离心机预冷至4℃,振荡后将离心管放到离心机中,在4℃12000r/min条件下离心10min。将离心后管中的上清液移至一个新的1.5mL离心管中,在室温下放置5min;向上述装有上清的新的离心管中加入200uL氯仿(三氯甲烷),盖紧管盖,涡旋振荡仪上强力振荡15s,使其充分的混匀后,在室温下静置2-3min,使溶液出现分层;然后在4℃12000r/min条件下离心15min。离心后,离心管中出现分层(液相和有机相),轻轻的取出离心管,不要让分层的部分弄混,将离心管倾斜45°,用移液枪轻轻吸出最上层液体放入一个新的1.5mL离心管中,注意一定不能吸到中间层和下层液体;往上述离心管中加入与上清液相同体积的异丙醇溶液,在室温下放置10min,使RNA沉降,然后在4℃12000r/min条件下离心10min;离心后去除上清液(小心吸取,不要对准沉淀吸取),保留沉淀。若不能彻底吸取上清,可以用离心机再稍微离心2min,继续吸取上清;另取一新的1.5mL的离心管,分别加入750ul100%的酒精和250ul RNase-free water,充分吹打混匀;向装有沉淀的离心管中加入1mL配制好的75%的乙醇,用移液枪轻轻吹打,使沉淀悬浮,然后在4℃7500r/min条件下离心5min;去除上清,留沉淀,室温放置2-3min。再向管中加入30uL RNase-free water以溶解RNA,用枪头轻轻吹打,使其均匀溶解。
按RNA的提取试剂盒(RNeasy Plant Mini Kit.QIAGEN)说明书上给出的步骤对RNA进行纯化。试验前需要做的准备工作:β-ME(β-巯基乙醇)在使用前应加入Buffer RLT,每1mL Buffer RLT中应加入10uLβ-ME(1mL RLT/10uLβ-ME);第一次打开Buffer RPE时,应根据上面的标签提示,向内加入4倍体积(44mL)的乙醇(100%),上下摇晃混合均匀;配制DNA消化酶DNase I Stock Solution向DNase I瓶中加入550uL去RNA酶的水(RNase-freewater),轻轻来回晃动3-5次,混匀,使粉末充分溶解在RNase-free water中,然后取70uLBuffer RDD与10uL DNase I Stock Solution吹打混匀后放置冰上;于-20℃冰箱内保存DNase I Stock Solution。提取具体步骤:(1)将70uL RNase-free water加入到上述粗提取到的30uL RNA,再向其中加入350uL的Buffer RLT,使其混合均匀;(2)向(1)中离心管内加入250uL的100%的乙醇溶液,用枪头轻轻吹打使其完全混匀后,迅速将管内700uL溶液转移到2ml粉色的RNeasy spin column中,盖上盖子后,在4℃12000rpm条件下离心15s,待离心后,弃掉管中液体;(3)在上述粉色RNeasy spin column中加入350uL Buffer RW1溶液,在4℃12000rpm条件下离心15s,待离心后,弃掉管中液体;(4)新取一个1.5mL去RNA酶的离心管,向其加入70uL的Buffer RDD,再加入10uL的DNase I Stock Solution,使其混合均匀;(5)将(4)混匀的80uL DNase I Stock Solution混合液转移至(3)中粉色的RNeasyspin column中,室温放置15min;(6)将350uL Buffer RW1加至(5)中粉色的RNeasy spincolumn中,在4℃12000rpm条件下离心15s,待离心后,弃掉管中液体;(7)向(6)中粉色的RNeasy spin column加入500uL Buffer RPE,在4℃12000rpm条件下离心15s,待离心后,弃掉管中液体;(8)继续加入500uL的Buffer RPE溶液到(7)中粉色的RNeasy spin column中,在4℃12000rpm条件下离心2min,待离心后,弃掉管中液体;(9)取一1.5ml RNase-free的离心管,将管盖剪掉,然后将(8)中粉色的RNeasy spin column放置其上,并向柱子内加入30-50uL的RNase-free water,在4℃12000rpm条件下离心1min,待离心后,弃掉管中液体;(10)收集管底的液体部分,即为纯化的RNA,小心吸取置于1.5ml新的RNase-free的离心管中。取1uL溶液利用NANoDROP 2000C测定其RNA浓度并记录,保存于-80℃备用。
cDNA的合成:逆转录之前,应先进行RNA变性处理:从-80℃冰箱取出用RNA置于冰盒中,用移液枪枪头吸出适量的RNA(RNA含量参照下表1中体系)于200uL离心管内,设置PCR仪器的条件为65℃,5min,再将离心管放入PCR仪内,进行热变处理;热变性处理后,从PCR仪中取出离心管,并立即放入冰内(防止其复性),参照下表1中的反应体系,向离心管内添加相应量的各个成分;逆转录反应:设置PCR仪的条件为37℃,15min;98℃,5min;进行逆转录处理。反应结束后,将所得cDNA放入-20℃冰箱内保存。
表1 cDNA合成体系
Figure GDA0002561724100000091
PCR扩增:PCR程序设置为“预变性95℃3min;变性95℃30s;退火55℃30s;延伸72℃1min;充分延伸72℃5min;保存4℃24h”,循环34次。退火温度和延伸时间根据表2各引物来调节;根据2xTaqPCR Master Mix说明书中PCR反应体系(表3),配制反应液;加入相应引物及以上体系进行PCR扩增;制胶,电泳。
表2引物序列
Figure GDA0002561724100000092
Figure GDA0002561724100000101
表3 PCR扩增反应体系
Figure GDA0002561724100000102
荧光定量PCR分析:参照THUNDERIRD qPCR Mix试剂盒(TOYOBO)的说明书中反应体系(表4),配制反应液;荧光定量PCR参数设置为“预变性95℃5min;变性95℃5s;延伸60℃45s”35个循环;加入引物及以上体系进行荧光定量PCR反应。
表4荧光定量PCR反应体系
Figure GDA0002561724100000103
RT-PCR结果显示,与Col-0植株相比,在GmHSP40-OE转基因植株中,调控开花的两个主要正调控因子编码基因FT和SOC1的表达量下调;而开花负调控基因FLC和FLM表达量在GmHSP40-OE转基因植株中明显上调(图6)。与RT-PCR结果一致,qRT-PCR结果也表明在GmHSP40-OE转基因植株中,FT和SOC1的表达量显著下调,而FLM和FLC表达量显著上调(图7)。这些结果表明过表达大豆GmHSP40诱导了开花相关的负调控基因的表达,而抑制了开花相关的正调控基因的表达,导致拟南芥推迟开花。
FLOWERING LOCUS T(FT)基因编码的蛋白是一种长距离运转的开花素信号分子,在成花启动上发挥着重要作用;FT能直接促进拟南芥开花,是开花调控的关键基因之一(Noriko等,2011.Expression of FLOWERING LOCUS T from Arabidopsis thalianainduces precocious flowering in soybean irrespective of maturity group andstem growth habit[J].Planta,233(3):561-568.),在拟南芥中,FT过表达会使植株表现出早花表型,而其突变体ft出现晚花表型(Kardailsky等,2000.Activation tagging ofthe floral inducerFT[J].Science,286(5446):1962-1965.)。FT和SOC1作为开花途径最下游的关键整合因子,它们的表达量的显著降低;在开花途径中,FT与SOC1是直接促进植物开花的基因。过表达GmHSP40显著抑制了FT与SOC1表达,导致GmHSP40-OE植株延迟开花;而FLC和FLM分别作为FT和SOC1上游最直接的负调控基因,它们的表达量的升高进一步的抑制了FT和SOC1的表达,所以这也是其晚开花最直接的原因;这些结果表明过表达大豆GmHSP40诱导了开花的负调控因子编码基因的表达,而抑制了开花的正调控因子编码基因的表达,从而导致开花显著延迟。过表达GmHSP40对FT表达的抑制是直接效应还是通过诱导FLC的表达而间接实现的还有待于进一步研究。
另有研究表明,miR156对维持植物的营养生长起了重要作用,它与靶基因SQUAMOSA PROMOTER BINDING PROTEIN LIKE(SPL)共同组成了一条控制开花的新途径(Fu等,2012.Overexpression of miR156in switchgrass(Panicum virgatum L.)results invarious morphological alterations and leads to improved biomass production[J].Plant Biotechnology Journal,10(4):443-452.),SPL能促进FT和SOC1的表达,而过表达miR156能抑制相关SPL基因的表达进而抑制FT和SOC1的表达,使植株开花延迟(Spanudakis等,2014.The role of microRNAs in the control of flowering time[J].Journal of Experimental Botany,65(2):365-380.);而miR172对植物开花过程具有促进作用,它可抑制其靶基因TARGET OF EAT(TOE)的表达来控制开花时间;在拟南芥中,TOE的过表达会抑制FT和SOC1的表达而使植株晚开花(Glazinska等,2009.The putativemiR172 target gene In APETALA2-like is involved in the photoperiodic flowerinduction of Ipomoea nil[J].Plant Physiol,166(16):1801-1813.)。而我们的实验证实miR156和miR172的积累水平在Col-0和GmHSP40-OE植株中的积累无显著差异(图8),说明GmHSP40-OE植株晚开花并不是由这两个miRNA的积累差异引起的,而是由其它机制造成的。
除上述实施例外,本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案,均落在本发明要求的保护范围。
<110>浙江师范大学
<120>大豆辅助分子伴侣蛋白编码基因GmHSP40在调控植物开花上的应用
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atggatggtc acggaggagg aggaggaggg agcagaggag aagcggagct ctggctttac 60
acagcgaaca aggttttaag cgcacgtgac ctgcacgggg cccgctcatt tgcgatccgg 120
gctcgagact ccgacccgag atacgagccc actgagctcc ttttagcggt gattgatacc 180
ctcatggccg gcgaggcccg gatcaacgac caactggact ggtacgctat tttgcaagtc 240
ctccgttaca ctcagaacat cgactacatc gccgcccagt accgccgcct cgccacccaa 300
ctcgaccctc accacaaccc cttcgccttc gccgctcatg ccttcacgct cgtccacgac 360
gcgtggaccg tactctccaa cccaactaaa aagactttct acgacaacca actccggctc 420
ctcactcaac cccctcctcc tcagccacca ccaccacctc ccgctcctcc tgccccggtg 480
gccttctttc ctattcagcc gccgcaaccg aacctaaacc ccaacccgat tccgaaccta 540
gtccctccaa gggaaagccc taggcctagg cctagggtag aggtggagcc gccaccaccg 600
gcaccgccgc catcgagtca gctggacaat gcgaccgaat tgactcgggc gagtgaggcc 660
gagagcgaag gggcgagttt ctggaccgcg tgcccttact gttacgttat gtacgagtat 720
ccgaaggtgt acgaggattg tactttgcgg tgccagaatt gtcggagggg gtttcatgcg 780
atggttgtac gttctccgcc aaaagatggt acttttggct cgttttgcag ttggggtttt 840
ttccctgtgg gtttttctgg ggattttaag gacattaacg ggtcttcttc caagtggaac 900
cctttctccc ctttgtttcc ttgcgccttg aagggggccg agcagaatag taggtaccag 960
aaggggcctt gggtttttta cgatgatgac gcgtctgcgg agttcgtcga ggctggttcc 1020
gacacgacgg aagatgattc cgatgatgat gactggcgtg gcgggaatca gaaggggacc 1080
acgaggagga ggaaaagaag gaagaggaga agcaatgctg gtggtgatgt tagaagggta 1140
cctacaattg agaggcctag aagacgggtt cagaatagtg acggtaatga cagtgtgggg 1200
aatggtgagg ctgtggatgg tggtgccctt gccgtgccgg tggcaccgga gtctagcaag 1260
aaggctgtgg cgttgggtgg atcaaggagg aggagtgaga ggaacttggg gaagttggat 1320
ttaaatgttg agtttagcaa tgaggtggag gagcctgtgc atggagcagg tgaagggaat 1380
gggaatgctg aggataatat tgaagggatt gggttttttg aggggctgga tgagttcctt 1440
agtagcttgc ctattctcaa tgttgttgca gatgataagg ttaagggtca ttag 1494
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Met Asp Gly His Gly Gly Gly Gly Gly Gly Ser Arg Gly Glu Ala Glu
1 5 10 15
Leu Trp Leu Tyr Thr Ala Asn Lys Val Leu Ser Ala Arg Asp Leu His
20 25 30
Gly Ala Arg Ser Phe Ala Ile Arg Ala Arg Asp Ser Asp Pro Arg Tyr
35 40 45
Glu Pro Thr Glu Leu Leu Leu Ala Val Ile Asp Thr Leu Met Ala Gly
50 55 60
Glu Ala Arg Ile Asn Asp Gln Leu Asp Trp Tyr Ala Ile Leu Gln Val
65 70 75 80
Leu Arg Tyr Thr Gln Asn Ile Asp Tyr Ile Ala Ala Gln Tyr Arg Arg
85 90 95
Leu Ala Thr Gln Leu Asp Pro His His Asn Pro Phe Ala Phe Ala Ala
100 105 110
His Ala Phe Thr Leu Val His Asp Ala Trp Thr Val Leu Ser Asn Pro
115 120 125
Thr Lys Lys Thr Phe Tyr Asp Asn Gln Leu Arg Leu Leu Thr Gln Pro
130 135 140
Pro Pro Pro Gln Pro Pro Pro Pro Pro Pro Ala Pro Pro Ala Pro Val
145 150 155 160
Ala Phe Phe Pro Ile Gln Pro Pro Gln Pro Asn Leu Asn Pro Asn Pro
165 170 175
Ile Pro Asn Leu Val Pro Pro Arg Glu Ser Pro Arg Pro Arg Pro Arg
180 185 190
Val Glu Val Glu Pro Pro Pro Pro Ala Pro Pro Pro Ser Ser Gln Leu
195 200 205
Asp Asn Ala Thr Glu Leu Thr Arg Ala Ser Glu Ala Glu Ser Glu Gly
210 215 220
Ala Ser Phe Trp Thr Ala Cys Pro Tyr Cys Tyr Val Met Tyr Glu Tyr
225 230 235 240
Pro Lys Val Tyr Glu Asp Cys Thr Leu Arg Cys Gln Asn Cys Arg Arg
245 250 255
Gly Phe His Ala Met Val Val Arg Ser Pro Pro Lys Asp Gly Thr Phe
260 265 270
Gly Ser Phe Cys Ser Trp Gly Phe Phe Pro Val Gly Phe Ser Gly Asp
275 280 285
Phe Lys Asp Ile Asn Gly Ser Ser Ser Lys Trp Asn Pro Phe Ser Pro
290 295 300
Leu Phe Pro Cys Ala Leu Lys Gly Ala Glu Gln Asn Ser Arg Tyr Gln
305 310 315 320
Lys Gly Pro Trp Val Phe Tyr Asp Asp Asp Ala Ser Ala Glu Phe Val
325 330 335
Glu Ala Gly Ser Asp Thr Thr Glu Asp Asp Ser Asp Asp Asp Asp Trp
340 345 350
Arg Gly Gly Asn Gln Lys Gly Thr Thr Arg Arg Arg Lys Arg Arg Lys
355 360 365
Arg Arg Ser Asn Ala Gly Gly Asp Val Arg Arg Val Pro Thr Ile Glu
370 375 380
Arg Pro Arg Arg Arg Val Gln Asn Ser Asp Gly Asn Asp Ser Val Gly
385 390 395 400
Asn Gly Glu Ala Val Asp Gly Gly Ala Leu Ala Val Pro Val Ala Pro
405 410 415
Glu Ser Ser Lys Lys Ala Val Ala Leu Gly Gly Ser Arg Arg Arg Ser
420 425 430
Glu Arg Asn Leu Gly Lys Leu Asp Leu Asn Val Glu Phe Ser Asn Glu
435 440 445
Val Glu Glu Pro Val His Gly Ala Gly Glu Gly Asn Gly Asn Ala Glu
450 455 460
Asp Asn Ile Glu Gly Ile Gly Phe Phe Glu Gly Leu Asp Glu Phe Leu
465 470 475 480
Ser Ser Leu Pro Ile Leu Asn Val Val Ala Asp Asp Lys Val Lys Gly
485 490 495
His
497
<210>3
<211>26
<212>DNA
<213>人工序列
<400>3
caccaaaatg gatggtcacg gaggag 26
<210>4
<211>25
<212>DNA
<213>人工序列
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atgaccctta accttatcat ctgca 25

Claims (2)

1.一种通过转基因延迟农作物开花时间的方法,其特征是:将一种GmHSP40基因的cDNA序列克隆至植物过表达载体pB7WG2.0中,通过转基因获得过表达GmHSP40的转基因株系,所述GmHSP40基因的cDNA核苷酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的GmHSP40基因在延迟农作物的开花时间上的应用。
CN201710172587.8A 2017-03-21 2017-03-21 大豆辅助分子伴侣蛋白编码基因GmHSP40在调控植物开花上的应用 Active CN106868020B (zh)

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