CN109182357B - 玉米促分裂原活化蛋白激酶基因ZmMPK20在调控气孔运动及植物耐热中的应用 - Google Patents
玉米促分裂原活化蛋白激酶基因ZmMPK20在调控气孔运动及植物耐热中的应用 Download PDFInfo
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Abstract
本发明公开了一种玉米促分裂原活化蛋白激酶基ZmMPK20在调控气孔运动及植物耐热中的应用。经综合实验证实:ZmMPK20在高温调控气孔运动中起到正调控因子的作用,同时在植物应对高温胁迫中有调控作用。预示本发明的应用将有助于改良植物的抗高温胁迫能力,对培育抗高温高产的作物新品种有重要的理论指导意义,对我国农业生产具有巨大应用价值。
Description
技术领域
本发明涉及一种玉米促分裂原活化蛋白激酶基因的应用,尤其涉及一种玉米促分裂原活化蛋白激酶基因ZmMPK20(Gene ID:100191429)在调控气孔运动及植物耐热中的应用,属于分子生物学、基因工程技术领域。
背景技术
通过生物信息学对已公布的ZmMPK20基因序列(Gene ID:100191429)分析,表明其编码一种促分裂原活化蛋白激酶(Mitogen-activated protein kinase,MPK)。其属于MAPK激酶家族,参与MAPKKK级联反应。
根据玉米研究网站(http://www.maizegdb.org)提供的eFP Blowser结果显示,ZmMPK20基因在植物叶片等部位大量表达。
植物表皮的气孔是植物进行蒸腾作用和水分散失的主要通道,气孔的张开和闭合,直接影响植物保水性及正常的生理活动。随着温度的上升植物气孔先张开增加蒸腾速率和光合作用效率,当到达临界温度时植物气孔逐渐关闭以减少水分散失。因此,对玉米基因ZmMPK20在调控气孔运动及植物耐热中的深入研究,在改良作物,尤其是玉米的抗逆能力方面有重要的价值。经检索,玉米促分裂原活化蛋白激酶基因ZmMPK20在调控气孔运动及植物耐热中的应用目前还未见报道。
发明内容
针对现有研究的不足,本发明的目的是提供一种玉米促分裂原活化蛋白激酶基因ZmMPK20在调控气孔运动及植物耐热中的应用。
本发明所述玉米促分裂原活化蛋白激酶基因ZmMPK20在调控气孔运动及植物耐热中的应用;其中,所述玉米促分裂原活化蛋白激酶基因ZmMPK20是玉米促分裂原活化蛋白激酶基因Zm00001d039141,其核苷酸序列如SEQ ID NO.1所示。
其中:所述植物优选是拟南芥和玉米。
申请人前期以玉米EMS诱变突变体1c5为实验对象,发现在高温胁迫下突变体1c5叶片卷曲相对野生型B73更为严重,持续的高温胁迫下突变体出现花粉量减少和减产的表型。高温胁迫下1c5叶片失水率更高,植株叶片含水量更少进而导致叶片卷曲。统计气孔和表皮细胞密度发现野生型B73和突变体之间没有显著差别,但是高温处理下开放气孔占的比例有明显差别:高温下突变体的更多的气孔处于开放的状态,导致植株体内水分散失加快,叶片卷曲。利用重测序技术测定大量群体确定点突变位点位于ZmMPK20基因内部的激酶结构域,点突变导致翻译后蛋白质第307位氨基酸由脯氨酸变为亮氨酸。进一步通过RT-PCR技术克隆玉米促分裂原活化蛋白激酶基因ZmMPK20基因。利用克隆到的基因片段ZmMPK20构建亚细胞定位载体,经过瞬时转化拟南芥叶肉细胞原生质体确定ZmMPK20表达的蛋白和MitoTracker共定位于线粒体。利用克隆到的基因片段ZmMPK20构建植物表达载体pCM1307,经过分子学和遗传学操作转化拟南芥,得到ZmMPK20过表达植株(OE1、OE2)。通过气孔运动、植株高温处理等实验方法对上述植株进行生理性状分析,确定ZmMPK20过表达植株(OE1、OE2)相对对照组(拟南芥野生型Col-0)植物的耐热性有所提高,证实了ZmMPK20在植物耐热中的应用。
本发明首次阐明了玉米促分裂原活化蛋白激酶基因ZmMPK20在调控气孔运动及植物耐热中的应用,为利用ZmMPK20基因改良植物体内的保水性及抗热能力奠定了基础,预示本发明的应用将有助于提高植物的耐热胁迫能力,对培育抗热高产的作物新品种有重要的理论指导意义,对我国农业生产具有巨大应用价值。
附图说明
图1:ZmMPK20参与温度调控气孔运动过程。
结果显示在升温促进气孔开放和高温抑制气孔关闭过程中,ZmMPK20突变体在高温下开放气孔比例比野生型B73大,说明其参与温度调控气孔运动过程,突变体植株对高温弱敏感。
图2:ZmMPK20影响植物相对含水量。
在42℃高温处理下统计离体叶片相对含水量。与野生型B73对比,ZmMPK20突变体由于气孔开度大失水快,相对含水量低,说明ZmMPK20可能通过参与温度调控气孔运动影响植物离体叶片含水量,影响植物体内水分散失。
图3:ZmMPK20过表达在拟南芥参与温度调控气孔运动过程
高温处理下拟南芥气孔运动实验,与野生型(Col-0)对比,ZmMPK20过表达株系(OE1、OE2)气孔开度变小,说明其参与温度调控气孔运动过程,过表达植株在温度调控气孔运动过程对高温过敏感。
图4:ZmMPK20过表达在拟南芥参与植物抗热胁迫过程。
正常温度条件培养下(左图所示),野生型与过表达株系生长状态良好,没有明显表型差异;热处理下(右图所示),与野生型(Col-0)对比,由于ZmMPK20过表达株系(OE1、OE2)气孔开度变小,耐热能力增强,导致持续高温处理下ZmMPK20过表达株系(OE1、OE2)成活率高于野生型(Col-0),说明ZmMPK20在植物耐热胁迫应答中起到重要的调节作用。
具体实施方式
以下实施例用于阐明本发明,但不用来限制本发明的范围。下列实例中未注明具体的实验方法,均可按照常规方法进行,或按照产品制造生产厂商的使用说明。
实施例1玉米促分裂原活化蛋白激酶基因ZmMPK20功能缺失突变体的获得和分子鉴定
1,EMS诱变获得玉米热胁迫下叶片卷曲突变体
EMS诱变后对三叶期植株42℃热处理,发现1c5表现出对热胁迫敏感的表型,叶片严重卷曲。在正常情况下1c5与野生型B73叶片没有差异。
2,利用F2代进行重测序确定有效SNP
突变体1c5与野生型回交获得F1代B73/1c5,自交后获得F2代B73/1c5,F2代出现野生型性状和突变体性状比例为3:1的分离比,说明此性状是隐形性状并且符合孟德尔遗传定律。选择F2代具有突变体性状的植株群体50棵,提取基因组DNA进行重测序。根据重测序结果获得有效SNP所在基因位置并进行下一步验证。
3,SNP测序验证
获得有效SNP所在基因位置,在SNP前后500bp设计引物扩增片段进行测序,在F2代50株具有突变体性状的植株中都具有相同的点突变(由碱基G变为A),点突变导致翻译后蛋白由脯氨酸变为亮氨酸,由此确定ZmMPK20基因内部点突变导致突变体出现热胁迫下叶片卷曲的表型,ZmMPK20基因与突变体性状连锁。
实施例2玉米促分裂原活化蛋白激酶基因ZmMPK20转基因植株获得
1,PCR方法克隆ZmMPK20基因片段
以玉米B73的cDNA为模板,以下面上下游引物组合进行PCR扩增:
ZmMPK20-OE-F:5'-GCTCTAGAATGTCACGTTTCATGTTCCTTC-3'
ZmMPK20-OE-R:5'-GGGGTACCCTAATACATCCTTGACATGCCAG-3'
PCR产物跑电泳后,切胶回收,纯化得到ZmMPK20基因片段。
2,载体连接和转化
首先用纯化后的ZmMPK20基因片段连接中间克隆载体Blunt(北京全式金公司产品),转化大肠杆菌DH5α,通过抗生素筛选和菌落PCR得到阳性克隆;连接后的Blunt载体进行测序,证明得到的ZmMPK20核苷酸序列与SEQ ID NO.1完全相同,测序正确后进行下一步酶切。
通过酶切组合XbaⅠ/KpnⅠ将ZmMPK20核苷酸序列从Blunt载体中切下,再次胶回收连接植物表达载体pCM1307。
3,转基因植株的筛选和获得
连接正确的植物表达载体pCM1307-ZmMPK20转化农杆菌EHA105,通过农杆菌浸染的方法进行转基因操作,转入野生型植株,获得过表达株系。具体实施方法如下:
农杆菌感受态转化步骤:
1)-80℃冰箱中取出农杆菌感受态,冰浴融化,加入目的DNA,轻弹均匀,冰浴30分钟;
2)冰浴结束后,液氮速冻1分钟,紧接着37℃水浴3-5分钟;
3)加入800μl无抗生素LB液体培养基,28℃、振荡培养2-4小时;
4)培养结束后收集菌体,100μl LB液体培养基重悬,菌液涂布在YEP固体培养基(加相对应的抗生素),28℃倒置培养2-3天;
5)挑取单克隆菌斑做菌落PCR鉴定。
农杆菌浸染拟南芥:培养约4周的拟南芥,待抽薹后,将主花序顶端减去,促进侧生花序生长。将已转化好的农杆菌菌液在转化前一天放大培养(按照2%接种),28℃振荡培养过夜,至OD600=1.0-1.2,6000rpm离心15分钟收集菌体,用转化介质将菌液稀释至OD600=0.8-0.9,加入Sillwet至终浓度为0.02%。将花盆倒置,花序浸入转染液中20秒,花序上覆盖薄薄一层转染液,将花盆水平放置,在黑暗中培养24小时,随后22℃,长日照(16小时光照,8小时黑暗)培养,收取种子。通过抗性筛选标记和PCR鉴定得到阳性转基因植株。
实施例3玉米促分裂原活化蛋白激酶基因ZmMPK20在调控气孔运动及植物耐热中的应用
利用ZmMPK20过表达植株(OE1、OE2)、进行下面生理性状实验,分析ZmMPK20在拟南芥或其他经济作物中对气孔运动调控方面的应用。
1,拟南芥气孔运动实验
不同温度下气孔运动实验。
1)生长四周的拟南芥,放在20℃培养箱生长3h,室温光照培养(450mol.m-2.s-1),取莲座叶叶片撕表皮,制作水压片,显微镜400放大倍数下拍照;
2)之后转移至42℃培养箱中继续培养,分别在1,2,3h取叶片莲座叶叶片撕表皮,
制作水压片,显微镜400放大倍数下拍照;
3)用统计软件ImageJ统计气孔开度。
结果显示在高温促进气孔关闭过程中ZmMPK20过表达株系(OE1、OE2)的气孔开度比对照组Col-0小,说明过表达ZmMPK20基因后植株对温度过敏感(结果如图3所示)。说明ZmMPK20在植物耐热胁迫应答早期气孔关闭过程起到重要的调节作用。
2,拟南芥高温处理实验
生长3-4周的植株,在温室42℃处理,植株开始萎蔫至死亡时移至20℃培养箱,3天后观察植株存活状态。在热处理前和恢复3天后分别拍照。
植株高温处理实验结果如图4所示,高温处理下,与野生型(Col-0)对比,ZmMPK20过表达(OE1、OE2)耐热能力增强,说明ZmMPK20在植物抗高温胁迫应答中起到重要的调节作用,在培育抗高温作物新品种方面有潜在的应用价值。
序 列 表
<110>山东大学
<120>玉米促分裂原活化蛋白激酶基因ZmMPK20在调控气孔运动及植物耐热中的应用
<141> 2018-9-14
<160> 1
<210> 1
<211> 1902
<212> DNA
<213> 人工序列
<221> 玉米促分裂原活化蛋白激酶基因Zm00001d039141
<222>(1)…(1902)
<400>
atgtcacgtt tcatgttcct tccgtttcat gtcccgtttc ggtttcccaa actgagcatt 60
caagtagtct gcagtggggg tttctcgaaa cagctcattg atctaacagt cgtgttgcag 120
acttctgcag aggtcgactt cttcacagag tacggggatg cgaaccggta caagatccaa 180
gaggtcatcg gcaagggaag ctacggggtc gtctgctctg ccatcgatct ccacactcgg 240
cagagggtgg cgatcaagaa gatacatggc atcttcgagc acgtctccga tgccgcgagg 300
atcctccgcg agatcaagct tctgaggctc ctgaggcacc ctgacattgt cgagatcaag 360
cacattatgc tgcctccctc gagaaaggac ttcaaggaca ttttcgttgt ttttgagctc 420
atggagtccg acctccacca agttataaag gccaacgatg acttgaccaa ggagcattac 480
cagttctttc tctatcagtt acttcgggcc ctcaaataca ttcatactgc taatgtttac 540
caccgtgacc tcaagcccaa gaatatttta gcaaactcta actgcaaatt gaaaatatgt 600
gactttggac tagcccgagt cgcattcaat gataccccaa caacagtctt ctggacggat 660
tatgttgcaa caaggtggta cagagctccg gagctctgtg gatccttctt caccaagtat 720
acaccagcta ttgacatttg gagcattgga tgcatatttg ctgaggtgtt gacagggaag 780
cctttatttc ctggtaaaaa tgttgttcac cagctagatt tgatgactga tcttctaggt 840
acaccatcaa cggacacaat ttctcgggtt cggaatgaga aagcaagaag gtacttgagc 900
agcatgagaa agaaggaccc tgttccattt tcccagaagt ttcccagtgc agatcctttg 960
gcacttaaac tgttagaaaa actattagcg tttgatccaa aggaccgtct gacagcagaa 1020
gaggcattgc gtgatccata cttcaaaggt cttgccaggg ctgaaagaga accatcctgt 1080
cagccaatca gaaaagtgga atttgacttt gagcacaaaa gaatgtcaaa ggaagagata 1140
agagagttga tattccgcga gatactggaa tatcacccac aactgctgag tagctacatt 1200
aatggcacag agaggacaac ctttctctac ccaagtgctg ttgatcaatt taagaagcaa 1260
ttttctcatc ttgaagagag tggtggtaac ggtccatcag ttccaacgga caggaaacat 1320
gcatcccttc ccaggaccac tgtggttcac tcgaatccaa ttcctgccaa agaacaacct 1380
cttggtgcct catcaagggt tagaccagtc tctgatgatt catgtaagaa cccttgggag 1440
aaaggaagtg gtcctggaaa tgttcccagg acatctctga ctccacaagg gctgcaagca 1500
caagcaggat cagtaagagt taatggccca gtgacggatt caaggtatcc tcctcaccaa 1560
cagatcccac aagcatacgg ttaccgccaa atgcctgcaa ggttggacag taccaaccca 1620
tcgcaggcca tgggaggtta cacgctgcag tcgcagaagg cctatgcttg tgcaaacggc 1680
aaaggctcgc ctgatgtggc cgtgaacatg agagctcccc ccttccatct cccagctgga 1740
ccaaagaaga acccattaga taggatagca cctgacacca ccgacatata cacaagatcc 1800
ctgaacggca tcgtcgccgc cgccgctgca tcagtgggcg ctggcgctgg tactcaccga 1860
aacatcggcg ttgtgccatc tggcatgtca aggatgtatt ag 1902
Claims (1)
1.玉米促分裂原活化蛋白激酶基因ZmMPK20在调控气孔运动及植物耐热中的应用;其中,所述玉米促分裂原活化蛋白激酶基因ZmMPK20是玉米促分裂原活化蛋白激酶基因Zm00001d039141,其核苷酸序列如SEQ ID NO.1所示;所述植物是拟南芥和玉米。
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