CN110241131A - 拟南芥植保素转运蛋白pdr8基因的应用 - Google Patents
拟南芥植保素转运蛋白pdr8基因的应用 Download PDFInfo
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Abstract
本发明公开了拟南芥植保素转运蛋白PDR8基因的应用。本发明通过实时定量PCR技术发现灰霉菌处理后的野生型拟南芥中的PDR8表达水平显著上升。本发明进一步通过功能学实验发现,PDR8的缺失会增加拟南芥对灰霉菌的敏感性,PDR8的过表达会增加拟南芥对灰霉菌的抗性,表明拟南芥植保素转运蛋白PDR8能够提高植物的抗病性。本发明提供的拟南芥植保素转运蛋白PDR8能够应用于提高植物对灰霉菌的抗病能力以及培育抗灰霉菌的转基因植物新品种。同时PDR8基因的启动子序列与GUS报告基因融合表达后可显示植物是否受到灰霉菌侵染,可应用于培育能显示植物是否受到灰霉菌侵染的转基因植物新品种。此外,植物抗病性的提高可以减少化学农药的使用,降低环境污染,可以应用于环境保护工程领域。
Description
技术领域
本发明属于生物技术领域。本发明涉及一种拟南芥植保素转运蛋PDR8基因的应用。
背景技术
植物生长发育会受到外界生物因素(如病原微生物、害虫等)和非生物因素(如干旱、洪涝等)的影响,其中病原微生物的侵染是影响植物生长发育的主要生物因素。据报道,70%-80%的植物病害是由于植物被病原真菌侵染引起的,灰霉菌(Botrytis.cinerea)就是其中一类常见的植物致病病原真菌,属于腐生型真菌。已知灰霉菌可侵染包括重要的粮食作物和经济作物在内的1400多种植物,每年在全球范围内造成巨大的经济损失。因此,如何提高植物对灰霉菌的抗病性是亟待解决的问题。
化学农药在一定程度上可以控制病原微生物对农作物的侵染,但是起不到立竿见影的效果,尤其对灰霉菌的防治效果一般,而且化学农药的长期使用会导致灰霉菌产生耐药性。此外,化学农药的利用率约为10%,约90%的农药残留在环境中,因而化学农药的大量使用会对环境造成巨大的污染,进而影响人类健康。因此,提高植物对灰霉菌的抗病性可以减少化学农药的使用,降低环境污染,对保护环境和人类健康具有重要的意义。
当病原微生物侵染植物时,会诱发植物的先天免疫反应,从而激活一系列相关抗病性基因的表达,进而合成对病原微生物有杀灭或抑制作用的次生代谢物。植物的次生代谢物种类繁多、性质各异、功能也各不相同。如植保素是一类能够在病原微生物侵染部位及附近合成并积累的具有抗菌活性的低分子量的代谢化合物,主要含有酚类和萜类;ROS是活性氧分子簇,植物可利用自身产生的活性氧物质来抑制或杀死病原微生物,同时也可以通过ROS的大量积累来诱导细胞死亡,从而限制病原微生物进一步侵染植物;胼胝体是由无定形的多糖类物质积聚而成的一种聚合物,当植物感知到病原菌的侵染时,胼胝体就会在细胞壁处大量沉积,从而限制病原微生物进一步侵染植物。
ABC转运蛋白(ATP-binding cassette transporter protein)是一类跨膜蛋白质超家族,与植物激素、金属离子、脂质、代谢物和外源物质的运输密切相关,在植物的生长发育、抗重金属以及抗病过程中发挥着重要的调控作用。PDR家族是ABC超家族中的一个亚家族,在转运重金属以及提高植物对重金属的耐受性中发挥着重要的调控和运输作用,同时PDR家族的某些成员参与次生代谢物的转运,在植物的生长发育以及抗病过程中也发挥着重要的调控作用。如PDR1通过参与次生代谢物白藜芦醇的运输来提高植物的抗病性;PDR5通过参与一种抗虫害的次生代谢物的运输来避免该次生代谢物的大量积累对植物造成伤害;PDR8通过向质膜外运输Cd2+来提高植物对重金属Cd2+的耐受性,同时PDR8还可以通过参与生长素前体吲哚-3-丁酸的转运来调控植物生长发育。
目前有关拟南芥植保素转运蛋白PDR8在植物抗病过程中功能的研究还未见报道,因此本发明探寻到的拟南芥植保素转运蛋白PDR8对于提高植物对灰霉菌的抗病性将具有重要的意义。
发明内容
本发明的目的在于提供了一种拟南芥植保素转运蛋白PDR8基因在提高植物对灰霉菌的抗病性中的应用。本发明包括以下步骤:(1)构建含有拟南芥植保素转运蛋白PDR8基因的重组载体;(2)将所构建的重组载体转化到植物或植物细胞中;(3)筛选获得对灰霉菌抗病性提高的转基因植物。
本发明的目的在于提供了一种拟南芥植保素转运蛋白PDR8基因的启动子在显示植物是否受到灰霉菌侵染的应用。包括以下步骤:(1)构建含有拟南芥植保素转运蛋白PDR8基因的启动子序列与GUS报告基因融合表达的重组载体;(2)将所构建的重组载体转化到植物或植物细胞中;(3)筛选获得能显示植物是否受到灰霉菌侵染的转基因植物。
本发明所述植物包括农作物,优选的所述植物包括:玉米、水稻、番茄、马铃薯、花生、大豆、棉花、烟草、黄瓜、甜瓜或西瓜中的任意一种或多种
为达以上目的,本发明通过下述方案实现:
(1)本发明通过RT-PCR(Real-Time PCR,实时定量PCR)技术检测野生型拟南芥幼苗经过灰霉菌处理后在0h、6h、12h和18h的PDR8表达情况,证明灰霉菌的侵染会导致拟南芥中PDR8的表达水平显著上升。
(2)本发明将PDR8基因的启动子序列与GUS报告基因融合表达,构建了PDR8pro:: GUS植物表达载体,通过农杆菌介导转化野生型拟南芥后,筛选获得PDR8pro::GUS转基因株系。通过GUS染色技术检测PDR8pro::GUS转基因株系的叶片经过灰霉菌处理后在48小时的染色情况。证明灰霉菌的侵染会诱导拟南芥中转运蛋白PDR8基因的启动子驱动的GUS报告基因的表达,从而显示拟南芥是否受到灰霉菌的侵染。
(3)本发明通过比较从拟南芥生物资源中心(Arabidopsis thaliana ResourceCenter(ABRC))获得的pdr8-3突变体和野生型植株的叶片经过灰霉菌处理后在60小时的叶片病斑大小,证明PDR8的缺失会增加拟南芥对灰霉菌的敏感性。
(4)本发明构建了35S::PDR8-HA植物表达载体,通过农杆菌介导转化野生型拟南芥后,筛选获得35S::PDR8-HA 转基因株系。通过比较35S::PDR8-HA 转基因株系和野生型植株的叶片经过灰霉菌处理后在60小时的叶片病斑大小,证明PDR8的过表达会增加拟南芥对灰霉菌的抗病性。
PDR8基因的启动子序列与GUS报告基因融合表达后可作为显示标志来显示植物是否受到灰霉菌侵染。同时,提高植物抗病性可以减少化学农药的使用,降低环境污染,可以应用于环境保护领域。
附图说明
图1显示了灰霉菌的侵染会诱导拟南芥中转运蛋白PDR8的表达。
A.灰霉菌的侵染会诱导拟南芥中PDR8的表达水平显著上升。通过RT-PCR技术检测野生型拟南芥幼苗经过灰霉菌处理后在0h、6h、12h和18h的PDR8表达情况,数据形式为PDR8的转录水平占EF1α转录水平的百分比(SD,n=3)。
B. 灰霉菌的侵染会诱导拟南芥中转运蛋白PDR8基因的启动子驱动的GUS报告基因的表达。通过GUS染色技术检测PDR8pro::GUS转基因株系的叶片经过灰霉菌处理后在48小时的染色情况(Bar=2mm)。
图2显示了PDR8的缺失会增加拟南芥对灰霉菌的敏感性。
A. pdr8-3突变体的叶片经过灰霉菌处理后形成的病斑明显大于野生型。pdr8-3突变体和野生型植株的叶片经过灰霉菌处理后在60小时的叶片病斑大小图像(Bar=5mm)。
B.pdr8-3突变体的叶片经过灰霉菌处理后形成的病斑面积明显大于野生型。pdr8-3突变体和野生型植株的叶片经过灰霉菌处理后形成的病斑面积的统计分析结果(P≤0.01,*表示两个样品之间差异性显著)。
图3显示了PDR8的过表达会增加拟南芥对灰霉菌的抗病性。
A.35S::PDR8-HA 转基因株系的叶片经过灰霉菌处理后形成的病斑明显小于野生型。35S::PDR8-HA 转基因株系和野生型植株的叶片经过灰霉菌处理后在60小时的叶片病斑大小图像(Bar=5mm)。
B. 35S::PDR8-HA 转基因株系的叶片经过灰霉菌处理后形成的病斑面积明显小于野生型。35S::PDR8-HA 转基因株系和野生型植株的叶片经过灰霉菌处理后形成的病斑面积的统计分析结果(P≤0.01,*表示两个样品之间差异性显著)。
具体实施方式
下面结合具体实施例来进一步阐述本发明。应理解,这些实例仅用于说明本发明而不用于限制本发明的范围。
实施例一:灰霉菌侵染的拟南芥中PDR8表达分析
(一)通过RT-PCR进行基因表达分析
将野生型拟南芥种子均匀涂布在固体1/2MS培养基平板上,置于22 ℃光照培养箱,16h光照/8 h黑暗。培养六天后将幼苗从平板上移到含有6 ml液体1/2MS培养基的GC小瓶中(瓶子规格为20 ml,10棵苗/瓶),移至全日照的22℃光照培养箱中培养七天。然后用灰霉菌的孢子悬液处理幼苗,孢子的终浓度为8×104个/ml,分别在0h、6h、12h、18h收取样品,液氮研磨后通过TRIzol试剂盒(Invitrogen)提取总RNA,利用RT-PCR技术分析拟南芥经过灰霉菌处理后在不同时间点的PDR8表达情况。结果如图1A所示,灰霉菌的侵染会导致拟南芥中PDR8的表达水平显著上升。
RT-PCR的引物序列如下:
PDR8–F-BamHI:cgcggatccatggattacaatccaaatcttcct
PDR8–B-SmaI:tcccccgggtctggtctggaagttgagagt
(二)通过GUS染色进行基因表达及定位分析
1. PDR8pro::GUS转基因株系的获得
以野生型拟南芥的基因组DNA为模板,通过PCR扩增出PDR8基因启动子,为了构建克隆的需要,借助引物引入法,在靶序列5’端加上BamHI酶切位点,在靶序列3’端加上SmaI酶切位点,扩增PDR8基因的启动子的引物序列如下:
PDR8pro–F-BamHI:cgcggatccatgtatggtggagaaatgtgtg
PDR8pro–B-SmaI:tcccccgggcgttcaccaagaaagcttgtga
通过BamHI和SmaI酶切位点将载体pBI121中的35S启动子替换成PDR8基因的启动子,构建pBI121-PDR8pro::GUS植物表达载体,通过农杆菌介导转化野生型拟南芥后,筛选获得PDR8pro::GUS转基因株系。
2. GUS染色
将野生型拟南芥和PDR8pro::GUS转基因株系的种子直接播种在培养土中,置于湿度为65%的22℃植物培养间中,12 h光照/12 h黑暗。培养至四周左右,将灰霉菌的孢子悬液(终浓度为8×104个/ml)注射到野生型拟南芥和PDR8pro::GUS转基因植株叶片内,48小时后将被灰霉菌侵染的叶片置于GUS染色液(50mM NaH2PO4,50mM Na2HPO4,10mM EDTA(pH=8.0),2mM K3[Fe(CN)6],2mM K4[Fe (CN)6],2mM X-Gluc,1% Silwet L-77)中,37℃孵育2小时。乙醇脱色后观察染色情况及染色部位,结果如图1B所示,叶片上灰霉菌的侵染部位明显染成蓝色,即灰霉菌的侵染诱导拟南芥中转运蛋白PDR8基因的启动子驱动的GUS报告基因的表达。
实施例二:PDR8的缺失对灰霉菌的抗病性分析
(一)pdr8-3突变体的获得
pdr8-3突变体是从ABRC(拟南芥资源中心)获得的一个T-DNA插入的功能缺失性突变体。通过RT-PCR技术鉴定出pdr8-3突变体确实为功能缺失性突变体。RT-PCR引物序列如实施例一所示。
(二)pdr8-3突变体对灰霉菌的抗病性分析
将野生型拟南芥和pdr8-3突变体的种子直接播种在培养土中,置于湿度为65%的22℃植物培养间中,12 h光照/12 h黑暗。培养至四周左右,取合适大小的叶片置于培养皿内的湿滤纸上,将5µl的灰霉菌的孢子悬液(1.5×105个/ml)分别滴在野生型拟南芥和pdr8-3突变体叶片上,60小时后观察并统计分析被灰霉菌侵染的叶片菌斑面积,结果如图2所示,pdr8-3突变体的菌斑面积明显大于野生型,PDR8的缺失会增加拟南芥对灰霉菌的敏感性。
实施例三:PDR8的过表达对灰霉菌的抗病性分析
(一)35S::PDR8-HA 转基因株系的获得
以野生型拟南芥的cDNA为模板,通过PCR扩增出PDR8基因片段,为了构建克隆的需要,借助引物引入法,在靶序列5’端加上BamHI酶切位点,在靶序列3’端加上SmaI酶切位点,扩增PDR8基因的引物序列如下:
PDR8–F-BamHI:cgcggatccatggattacaatccaaatcttcct
PDR8–B-SmaI:tcccccgggtctggtctggaagttgagagtt
通过BamHI和SmaI酶切位点将扩增出的PDR8基因片段插入到35S启动子驱动的pCAMBIA1300载体上,并与HA标签蛋白融合表达,构建出的pCAMBIA1300-35S::PDR8-HA植物表达载体,通过农杆菌介导转化野生型拟南芥后,筛选获得35S::PDR8-HA转基因株系。
(二)35S::PDR8-HA 转基因株系对灰霉菌的抗病性分析
将野生型拟南芥和35S::PDR8-HA 转基因株系的种子直接播种在培养土中,置于湿度为65%的22℃ 植物培养间中,12 h光照/ 12 h黑暗。培养至四周左右,取合适大小的叶片置于培养皿内的湿滤纸上,将5µl的灰霉菌的孢子悬液(1.5×105个/ml)分别滴在野生型拟南芥和35S::PDR8-HA 转基因株系叶片上,60小时后观察并统计分析被灰霉菌侵染的叶片菌斑面积,结果如图3所示,35S::PDR8-HA 转基因株系的菌斑面积明显小于野生型,PDR8的过表达会增加拟南芥对灰霉菌的抗病性。
综上所述,灰霉菌的侵染会诱导拟南芥中PDR8的表达,PDR8的缺失会增加拟南芥对灰霉菌的敏感性,PDR8的过表达会增加拟南芥对灰霉菌的抗病性。因此,拟南芥植保素转运蛋白PDR8能够应用于提高植物对灰霉菌的抗病能力以及培育抗灰霉菌的转基因植物新品种。同时PDR8基因启动子序列与GUS报告基因融合表达后可显示植物是否受到灰霉菌侵染,可应用于培育能显示植物是否受到灰霉菌侵染的转基因植物新品种。此外,植物抗病性的提高可以减少化学农药的使用,降低环境污染,可以应用于环境保护工程领域。
应理解所述实施例仅是范例性的,不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改或替换均落入本发明的保护范围。
序列表
<110> 上海大学
<120> 拟南芥植保素转运蛋PDR8基因的应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4410
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 1
atggattaca atccaaatct tcctccttta ggaggaggtg gtgttagtat gagaagaagc 60
ataagtcgaa gtgtaagcag agcaagtagg aacattgaag atatcttctc atctggttca 120
agaagaacac aatcagtcaa cgacgatgaa gaagctctta aatgggctgc cattgagaag 180
ctaccaactt acagtcgtct ccgaaccact ctcatgaacg ctgtagtcga agacgatgtt 240
tacggtaacc agctcatgag caaggaggtt gatgtaacca agcttgatgg tgaagatcgt 300
cagaagttta ttgacatggt tttcaaagta gctgagcaag ataatgaaag gatcttgact 360
aagctaagaa acaggatcga tagagttggt atcaaacttc caactgttga agtcaggtac 420
gagcatttga cgattaaagc tgattgttac actggtaata gatctcttcc tacacttttg 480
aatgttgtga ggaacatggg agagtctgct ttaggtatga ttggtattca atttgctaag 540
aaagctcagc ttacgattct taaagatatc tctggggtta ttaaacctgg aaggatgaca 600
cttttgttgg gtcctccttc ttctggtaag accactcttt tgttggcttt agctgggaaa 660
cttgataaat ctctacaagt cagtggtgat attacttaca atggttacca actcgatgag 720
tttgttccga gaaagacctc tgcttacatt agtcagaacg atcttcatgt tggtatcatg 780
actgttaagg agactcttga cttctctgct aggtgtcaag gtgttggtac tcgttatgat 840
ctgttgaatg agcttgcgag gagagaaaag gacgctggta tattcccgga agccgatgtt 900
gatctcttca tgaaagcttc tgctgctcaa ggtgttaaga acagtctcgt cactgattat 960
actctcaaaa ttttggggct tgacatttgc aaagacacaa tagttggaga tgacatgatg 1020
agaggtatat ctggaggtca gaagaaacgt gtcacaactg gtgagatgat tgttggacct 1080
actaagacac tcttcatgga cgaaatatcc actggtcttg acagttccac tactttccaa 1140
atcgtcaagt gtctgcaaca aatcgttcac ctcaatgaag ccacggtgct catgtctctc 1200
ctccagcctg ctcctgagac ttttgattta ttcgatgata tcatcttggt gtcggaaggt 1260
cagatcgtgt accaaggacc gagagacaac attcttgagt tctttgagag ctttgggttc 1320
aagtgtcctg agagaaaagg aacagctgat ttcctgcaag aggttacttc caagaaagat 1380
caagaacagt actgggtgaa cccgaacaga ccttatcact acattccggt ttcagagttt 1440
gccagtagat acaagagttt ccatgttggg acgaagatgt ctaacgaact tgcagtaccg 1500
ttcgataagt ctcgcggcca caaagcagct cttgtgttcg ataagtactc tgtctcaaag 1560
agggagcttc tcaagagctg ttgggacaaa gagtggctgc ttatgcagcg aaacgcgttc 1620
ttctatgttt tcaagactgt ccagatcgtc atcattgctg caatcacgtc tacactcttc 1680
ctgagaaccg aaatgaacac aagaaacgag ggtgatgcta atctctacat aggagcattg 1740
ctatttggaa tgatcatcaa catgtttaat gggtttgcgg agatggctat gatggtttca 1800
agactccctg tgttctacaa acagagggat ctcttgtttt atccatcctg gaccttctca 1860
cttcccactt tcttgcttgg gattccaagc tcaatattag aatcgacggc ttggatggtg 1920
gtgacttatt actccattgg ttttgcacct gacgccagcc gcttcttcaa gcagtttctt 1980
ctggtgtttc tgattcaaca aatggctgca tccctcttta ggttgattgc ttctgtgtgc 2040
agaaccatga tgattgctaa tactggtggt gctctcactc tacttcttgt gttcttgctc 2100
ggaggcttcc ttcttccgaa aggcaagatt cctgactggt ggggttgggc ttactgggta 2160
tctcctctca cctatgcttt caacggtcta gtagtcaatg aaatgtttgc tcccagatgg 2220
atgaacaaaa tggcttcttc taacagcaca ataaagcttg gaactatggt gcttaatact 2280
tgggatgtct accatcaaaa gaactggtac tggatttcag ttggagcctt gctttgtttc 2340
acagccctct tcaacattct attcaccttg gcacttacct atctcaaccc tcttgggaag 2400
aaggcaggtt tacttccaga agaagaaaat gaagacgctg atcaggggaa agatccaatg 2460
cgtagatctt tgtctactgc agatgggaac agaagaggag aggtcgcaat ggggagaatg 2520
agtagggact ctgcggctga agcatcaggt ggtgcaggca ataagaaagg aatggttctt 2580
cctttcactc ctttagctat gtcctttgac gacgtcaaat actttgttga catgcctggg 2640
gaaatgagag accaaggagt tacagaaaca agactccaac tgcttaaagg tgtgactggt 2700
gcatttaggc caggagtttt gactgcgctt atgggagtga gtggtgccgg taagactacg 2760
cttatggacg ttttggccgg aaggaaaaca ggtggataca ttgaaggaga tgtgagaata 2820
tcaggattcc caaaggttca agaaacattt gctagaatct caggatattg tgagcagacc 2880
gatattcact ccccgcaagt aacagtcaga gaatctctga ttttctctgc tttccttcgt 2940
cttcctaaag aagtcggcaa agatgaaaaa atgatgtttg tggatcaagt gatggaattg 3000
gtagagctgg acagtcttag ggactccatt gttggtttac cgggtgtcac ggggctttcc 3060
acggagcaga gaaagagact gacaatcgcg gtggagcttg tagccaaccc ttccatcatc 3120
tttatggatg agccaacttc agggctagac gctagagcag cggctattgt gatgagggcg 3180
gtaaggaaca cagtggacac tggaagaacc gtggtctgca ccattcatca gcctagcatt 3240
gatatctttg aagcatttga tgaattgatg ctgatgaaga gaggaggaca agtgatttac 3300
gcgggtccat tgggtcaaaa ctctcacaag gtggttgagt actttgaatc tttccccgga 3360
gtgtccaaga ttccagaaaa gtataacccg gccacttgga tgctcgaagc tagctcactc 3420
gccgctgagc taaagcttag tgttgacttt gctgagttat acaatcaatc agcattgcac 3480
cagcgaaaca aagcgttggt aaaagaacta agtgtaccac cagcaggagc atcagatctt 3540
tactttgcta cacaattctc acaaaacaca tggggacagt tcaaatcatg cttatggaaa 3600
caatggtgga cgtattggag atctccagac tacaatcttg tccgtttcat cttcacattg 3660
gcaacatctc tcttgattgg tacagtcttc tggcaaatcg gaggtaacag gtcgaacgca 3720
ggggatctaa caatggtgat aggagcattg tatgccgcga ttatcttcgt gggaatcaac 3780
aactgttcaa cagtacaacc gatggttgca gtggaaagaa cagtgttcta cagagaaaga 3840
gcagcaggaa tgtactcagc catgccatat gccatctctc aagtcacttg tgagcttccc 3900
tatgtcctta ttcaaaccgt ttactactca ctcatcgtct acgccatggt tggtttcgaa 3960
tggaaagccg aaaagttctt ctggttcgtc ttcgttagct acttctcatt cctctactgg 4020
acttactacg gcatgatgac tgtttccctc acaccaaacc aacaagtcgc ttcgattttc 4080
gcctcagcgt tttacggtat tttcaacctc ttctctggtt tcttcattcc aagacccaaa 4140
atcccaaaat ggtggatttg gtactactgg atctgccctg ttgcatggac cgtgtatgga 4200
ttgatagtgt cgcagtacgg tgatgtggag acacgtatcc aagtccttgg tggtgctcct 4260
gacttaaccg tcaagcaata cattgaggac cattatggtt tccaatctga ctttatggga 4320
ccagtggcgg ctgtactcat cgctttcacc gtcttcttcg ccttcatctt cgccttctgc 4380
atcagaactc tcaacttcca gaccagataa 4410
<210> 2
<211> 2585
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 2
atgtatggtg gagaaatgtg tgtatttgga ttataataga aatacctatt ttcatctaga 60
agctcaggat ctagttattg ataattagag ctcgaaactg agatggatct tattgtttgg 120
atagttgttt taactattca ggatcttatt gtttggatag ttgtttaact atttataact 180
agagatcgaa attgagatgg atcatgaatt tcattgaaaa ttaaggctaa actgtttggc 240
tagttgttta actcttaagc atgtatctgg agatatcttt attcattgtt tgattttgac 300
attgtggttc atcttgtttg atggagactc tttgtaggaa atcgctttgg ccaacaccaa 360
tgtgaactag tctggccaat ttagttaatt gattgatcat catattgttt ttggcatttt 420
tcagaacgtg taataacagt gaaggaattt tgaggtacct gctcaactga atcagattca 480
ggagggtttt aggagaggta atatggaagt agcaatgctt aagaaagata aagctagagc 540
tcttgatgat ctgctgaaaa tcgactaagg aagccaatga caaactcaga caggaaaaaa 600
ctgtaagaag aagaagaaaa taagagaaaa gcaacaaagc tatggaaagc ttaactttgg 660
atttgcaagt taagtatttt taaagcaagg gaagcattgg agaagatctt gttgatatgt 720
caaccatagc ttgaacattg tggagcccaa atgaaaaggt ataagttggc tgcaaaagat 780
acaaatgaaa actgtggaaa aattcttgaa gactgagata agtacacact caaaatgagt 840
ttttcaattc taagactatg tgggaacagc gagaacttca gttgtgtgga aaattgggaa 900
cttggcaaag tgaatttgtc tgaccttcag gaaaatgaga agaagaagaa gcatgtatga 960
aaaatacact taaggaactt gaggagaaga gtgaggattt gtaggaaaga gttggagaag 1020
cggagactgg aagcatgaaa gtcgaggcaa agaaggttgc tttagaaaac agaaagctca 1080
gagcaatgaa ggttttttct cttgaaaaac tcgataactt gtcaaaggta tatgaaagct 1140
tggtggatac aagtgaagcg tttttaaagc aaggcaagga ttggagaagg tcttgttgat 1200
atgtcaacca tagcttgaac attgtggagc ccaaattaaa agttataagt tggctgcaaa 1260
atatacaagt gaaaagtaag gaaaaattct tgaagactga gataagtaaa cacccaaaat 1320
gagtttgtcc attctaagac tgtgtagaga cagcgagaac ttcatttgcg cggaaaagtc 1380
gggaacttgg caaggtaaat gagaataaac tgaaggaact tgaggagaag aatgaggatc 1440
tgtaggaaag agttagagaa gcagaaactg gaagcatgaa atttaaagga agcttgttgg 1500
tcaacgaagt caagccaaag aacattgctt tagaaaactc agagcaatgt aggtttgttc 1560
tcttgaaaaa atcgatgagt cgtcctcaaa ggtaaatgaa aacttggagt ataaactgca 1620
gactgaagaa gaccgagagc ttaattgaca aagaaaccaa cctatagagc attactaaag 1680
aagctgagaa gagctcagga aaatgaaatt tgctggctga aggaatgaat tagacattgg 1740
ttaatacaca aaatgcagag cactaatcaa agattagttg agaaaatagc caaacgcaga 1800
gtttaagagg aagtcaatgg agtcttctta tcataacaac attgaggagt tgtcactaaa 1860
acttcttatt ccgggtattt aaatggcttc ttcctccgca acttctcact ttccagaaca 1920
caaagactta taaacaaacc atccttcacg tatgtgttga attgaaatgt gaatgtgact 1980
tcatcaacac aatgacttca aaacccaacg acggtcttcc tttcttttct caccaataaa 2040
atgtagaaaa gaaacaaagt acacaagaaa gacttttcat ttaacgatat ctccactagt 2100
agtagctata actaaatggg gtcatcgtta ttgactttga ataagaaatg ttcaaacact 2160
tcaattatta cataatatct ttgcaataaa ataacaaacg ttaaagagcc gtcgcattat 2220
tagcacaaaa aatatctttt gtaaataaaa aaacaacgta aaagagccgt cacatttgaa 2280
taataaaacc catcttgcac ataaaaaaca cgacttttta ttatttttga ttatttcaac 2340
aaagtatgaa tagtcaacat aaagaaaagt ccgccttgat gttccttcct cactccctat 2400
ttatccattt tctaatccca ccctcttctc ttcattcatc atcttcctcc tctctctctt 2460
ctctgtatca cccaactaaa tcctcacggg attagatcca aagtctcaaa ctttgatcca 2520
aaaacacaaa ctttgttcaa aagacataaa ctttgagcca aagtcacaag ctttcttggt 2580
gaacg 2585
<210> 3
<211> 33
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 3
cgcggatcca tggattacaa tccaaatctt cct 33
<210> 4
<211> 30
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 4
tcccccgggt ctggtctgga agttgagagt 30
<210> 5
<211> 31
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 5
cgcggatcca tgtatggtgg agaaatgtgt g 31
<210> 6
<211> 31
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 6
tcccccgggc gttcaccaag aaagcttgtg a 31
<210> 7
<211> 33
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 7
cgcggatcca tggattacaa tccaaatctt cct 33
<210> 8
<211> 31
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 8
tcccccgggt ctggtctgga agttgagagt t 31
Claims (5)
1.一种拟南芥植保素转运蛋白PDR8在提高植物对灰霉菌的抗病性中的应用。
2.一种拟南芥植保素转运蛋白PDR8基因的启动子在显示植物是否受到灰霉菌侵染中的应用。
3.根据权利要求1所述的应用,其特征在于具体步骤为:
构建含有上述PDR8基因的重组载体;
将所构建的重组载体转化到植物或植物细胞中;
筛选获得对灰霉菌抗病性提高的转基因植物。
4.根据权利要求2所述的应用,其特征在于具体步骤为:
构建含有权利要求2所述PDR8基因的启动子序列与GUS报告基因融合表达的重组载体;
将所构建的重组载体转化到植物或植物细胞中;
筛选获得能显示植物是否受灰霉菌侵染的转基因植物。
5.根据权利要求1、2、3或4所述的应用,其特征在于所述植物为:玉米、水稻、番茄、马铃薯、花生、大豆、棉花、烟草、黄瓜、甜瓜或西瓜。
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CN112458110A (zh) * | 2020-11-30 | 2021-03-09 | 浙江大学 | 植物抗病基因AtIQD1的应用 |
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