CN115125257A - 水稻a9ip2基因在提高水稻抗稻瘟病上的应用 - Google Patents
水稻a9ip2基因在提高水稻抗稻瘟病上的应用 Download PDFInfo
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- CN115125257A CN115125257A CN202210619583.0A CN202210619583A CN115125257A CN 115125257 A CN115125257 A CN 115125257A CN 202210619583 A CN202210619583 A CN 202210619583A CN 115125257 A CN115125257 A CN 115125257A
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Abstract
本发明公开了水稻A9IP2基因在提高水稻抗稻瘟病上的应用,属于基因工程领域。所述水稻A9IP2基因的CDS区核苷酸序列如SEQ ID No.2所示。转基因实验证明过表达A9IP2基因能够提高水稻对稻瘟病的抗性。因此,将A9IP2基因导入水稻植株,有利于提高水稻稻瘟病抗性,促进水稻品种的遗传改良。
Description
技术领域
本发明属于基因工程领域,具体涉及水稻A9IP2基因在提高水稻抗稻瘟病上的应用。
背景技术
植物在自然生态系统中面临各种各样的病原体的侵染威胁,导致农业生产的产量和品质下降。植物病害主要包括真菌病害,细菌病害,病毒病害等,以重要单子叶粮食作物水稻病害为例:最具代表性的真菌病害如稻瘟病,由稻瘟病菌(Magnaporthe oryzae)侵染引起;细菌病害如白叶枯病,由白叶枯病菌(Xanthomonas oryzae pv.oryzae)侵染引起;病毒病害如水稻条纹病毒病,由水稻条纹病毒(Rice stripe virus)侵染引起;而侵染双子叶作物或蔬菜的常见真菌病害有灰霉菌(B.cinerea)侵染引起的腐烂病等;这些病害对作物或蔬菜产量和品质的影响最为严重,每年造成的减产就相当于总产量的10-30%,而农药的使用减轻了病害的影响,但存在农药残留以及对自然环境有负面影响等不利因素。因此,抗病育种是目前最经济有效的抵御病原菌的方法。挖掘新的稻瘟病抗病基因、抗性资源、培育广谱抗病品种,是一种绿色、高效保障作物产量和品质的策略,对农业生产具有重要意义。
作为蛋白质翻译后修饰之一的泛素化是调控蛋白质功能和活性的重要途径,主要是导致蛋白质被动性降解。蛋白质的降解在细胞的生命活动全过程中起到非常关键的作用。
泛素是一种由76个氨基酸组成,且高度保守、广泛存在于多数真核细胞中的小分子蛋白质,能通过牢固的共价键与蛋白质相结合。蛋白质的泛素化修饰需要多种酶的参与完成,包括泛素活化酶(Ubiquitin Activating Enzyme,E1)、泛素结合酶(UbiquitinConjugating Enzyme,E2)和泛素连接酶(Ubiquintin Ligating Enzyme,E3)。目前还发现了去泛素化酶(deubiquitinase,E4),作为蛋白降解前将蛋白质上的泛素切掉以利于26S蛋白酶体降解的酶,而且同时也发现E4还可以挽救某些将要被降解的蛋白质的命运,具体作用正在被人们所揭示。泛素连接酶E3的种类较多,拟南芥中大概有1500多个E3。E3能够识别底物蛋白质N端的不稳定信号,并将泛素添加到底物蛋白上,通过26S蛋白酶体途径,最终将底物蛋白降解,从而参与调节植物的各种生理生化过程。植物体内的泛素连接酶根据亚基和作用机制可以分为四类:RING、HECT、U-box及cullin-RING(CRLs)。
A9IP2基因编码的蛋白是水稻上的一种具有RING结构域的蛋白,目前其功能未见有相关报道。
发明内容
本发明的目的是提供水稻A9IP2基因在提高水稻抗稻瘟病上的应用。
本发明提供了水稻A9IP2基因在提高水稻抗稻瘟病上的应用。
优选的,水稻A9IP2基因的CDS区核苷酸序列如SEQ ID No.2所示。
本发明还提供了水稻致病因子A9IP2蛋白在提高水稻抗稻瘟病上的应用。
优选的,水稻致病因子A9IP2蛋白的氨基酸序列如SEQ ID No.1所示。
本发明还提供了水稻A9IP2基因在水稻育种中的应用,通过筛选过表达水稻A9IP2基因的水稻植株获得抗稻瘟病菌的水稻株系。
本发明提供了水稻致病因子A9IP2蛋白在水稻育种中的应用,通过筛选水稻致病因子A9IP2蛋白表达量高的水稻植株获得抗稻瘟病菌的水稻株系。
本发明提供了一种抗稻瘟病菌的转基因水稻构建方法,方法如下:
将水稻A9IP2基因转入水稻植株内获得过表达水稻A9IP2基因的转基因水稻。
具体的,将水稻A9IP2基因克隆到载体中,先转入到农杆菌EHA105中,再转入水稻细胞内,获得过表达水稻A9IP2基因的转基因水稻。
优选的,载体为pUCBi1390载体。
与现有技术相比,本发明具有的有益效果为:转基因实验证明敲除A9IP2基因能够使水稻更感病,过表达A9IP2基因能够提高水稻对稻瘟病的抗性,将A9IP2基因导入水稻植株,有利于提高水稻稻瘟病抗性,促进水稻品种的遗传改良。
附图说明
图1为水稻A9IP2基因敲除pYLgRNA-OsU3载体图谱。
图2为水稻A9IP2基因敲除pYLCRISPR/Cas9Pubi-H载体图谱(A)及阳性转基因水稻家系图(B)。
图3为水稻A9IP2基因过表达载体图谱(A)及阳性转基因水稻家系图(B);其中,**p<0.01,***p<0.001。
图4为A9IP2基因敲除的突变体接种稻瘟病菌有毒生理小种KJ201菌株后的表型结果图(A:感病情况;B:相对菌丝生物量;C:相对病斑面积;D:荧光检测结果);其中,*p<0.05,**p<0.01。
图5为A9IP2过表达植株接种稻瘟病菌有毒生理小种KJ201后的表型结果图(A:感病情况;B:相对菌丝生物量;C:相对病斑面积);其中,**p<0.01。
具体实施方式
实施例1
水稻A9IP2基因敲除植株的创制及鉴定。
为了阐明A9IP2基因在水稻对稻瘟病抗性中的作用,我们采用了CRISPR/Cas9基因编辑技术构建了A9IP2基因的敲除突变体。根据水稻A9IP2的基因号,在网站“http://rice.uga.edu/#search”下查找到对应的CDS序列(如SEQ ID No.2所示)及gDNA序列(如SEQID No.3所示),A9IP2基因编码的蛋白对应的氨基酸序列如SEQ ID No.1所示。然后根据文献中方法(CRISPR-GE:A Convenient Software Toolkit for CRISPR-Based GenomeEditing,Mol.Plant,2017.)设计敲除引物并构建转基因载体,所用的载体为pYLgRNA-OsU3(addgene ID:66192)和pYLCRISPR/Cas9Pubi-H(addgene ID:66187),载体图谱如图1和图2A所示。引物序列如下所示:
A9IP2cas9-F:ggcaTTCTTCTTCATATGATCAT;
A9IP2cas9-R:aaacATGATCATATGAAGAAGAA。
将测序正确的转基因载体转入EHA105农杆菌菌株,送至武汉伯远生物科技有限公司转化水稻感病品种TP309。获得转基因苗后,用CTAB法提取对应单株的DNA,同时设计阳性转基因苗检测引物,用2×PCR Master Mix(With Dye)进行PCR扩增,跑胶确定有目的条带后将PCR产物送公司测序。
阳性转基因苗检测及测序引物为:
A9IP2-cas9-seq-F:AGGGAATGTGGGGTGCTAGA;
A9IP2-cas9-seq-R:CGCCCGCAAAGCAAATATCA。
PCR反应体系(50μL)如下所示:
PCR反应条件:
94℃预变性5min;94℃变性30s,56℃退火30s,72℃延伸,延伸时间根据扩增片段大小确定(30s/kb),共32-35个循环;72℃延伸5min;12℃保存。
经比对序列发现A9IP2-54和A9IP2-62为纯合突变体植株(图2B)。
实施例2
A9IP2过表达植株的创制及鉴定。
(1)水稻A9IP2基因的克隆及植物表达载体的构建:
选用水稻感病品种TP309种子,浸种催芽后待幼苗长至1cm左右后点播于直径约17cm的花盆中,每盆约5-7粒,置于温室进行正常的水肥管理,期间注意适时喷施农药,防止虫害。待约10天左右时剪取长势正常的幼嫩幼苗,锡箔纸包裹后迅速放入液氮中冷冻。用TRIZOL(TAKARA)提取总RNA,再取总RNA 1μg,利用M-MLV逆转录酶和Olig(dT)18进行反转录合成cDNA。用A9IP2-OX-F/R进行PCR扩增目的片段,跑胶纯化后回收目的片段,然后用KpnI、BamHI双酶切线性化本实验保存的pUCBi1390载体(图3A),再将目的片段用无缝克隆的方式连接到载体上。
A9IP2过表达载体引物:
A9IP2-OX-F:gttacttctgcactaggtaccATGGGGGCAAAGGACTCCA;
A9IP2-OX-R:tcttagaattcccggggatccTCAGTACAGCCTGATCCTTGTGG。
PCR反应体系(50μL)如下所示:
PCR反应条件:
94℃预变性2min;94℃变性30s,56℃退火30s,68℃延伸,延伸时间根据扩增片段大小确定(30s/kb),共32-35个循环;68℃延伸5min;12℃保存。
(2)转基因植株的获得及鉴定:
将步骤1获得的表达载体pUCBi1390-A9IP2转入农杆菌EHA105,然后再转入水稻感病品种TP309中,该过程委托武汉伯远生物科技有限公司(http://www.biorun.com/index.html)完成,获得潮霉素阳性的转基因植株,之后进行qRT-PCR,水稻Ubiquitin基因(LOC_Os03g13170)为内参,引物设计使用在线软件GenScript qRT-PCR(www.genscript.com/tools/real-time-pcr-tagman-primer-design-tool),用2-ΔΔCT法计算基因的相对表达量。
qRT-PCR引物:
A9IP2基因检测引物:
qRT-A9IP2-F:CTGACAGGAAGGAGGCAGAA;
qRT-A9IP2-R:GACGGCCTAGAAGTTGGAGA;
内参Ubiquitin基因检测引物:
qRT-OsUBQ-F:AAGAAGCTGAAGCATCCAGC;
qRT-OsUBQ-R:CCAGGACAAGATGATCTGCC。
qRT-PCR反应体系(10μL):
qRT-PCR反应条件:
95℃预变性30s;95℃变性20s,60℃延伸30s,共40个循环。
检测后发现A9IP2-3(#3)、A9IP2-7(#7)、A9IP2-15(#15)和A9IP2-16(#16)为过表达阳性单株(图3B)。
实施例3
A9IP2基因敲除植株稻瘟病抗性鉴定。
(1)离体打孔接种:
选取5-8周叶龄的水稻幼苗,剪取野生型和敲除突变体水稻植株相同部位的叶片(倒二叶),用稻瘟病有毒生理小种KJ201菌株进行打孔接种,然后置于光照培养箱,5-9天左右调查发病情况,提取对应部位的DNA,进行相对菌丝生物量的统计计算。
(2)活性氧(ROS)测定:
剪取长势良好的野生型TP309和A9IP2基因的突变体水稻叶片,用直径为0.5cm打孔器在对应的主叶脉两侧打孔,然后将其放入装有ddH2O的2.0mL的离心管中,室温下避光处理过夜。次日每个单株随机挑取完整的3个叶盘置于1.5mL的离心管,然后快速加入100μL鲁米诺、1μL几丁质和1μL辣根过氧化物酶,再立刻放入Glomax 20/20Luminometer仪器中,动态测量20min,每隔10s检测一次荧光,每个样品重复5次。
结果发现接种稻瘟病有毒生理小种KJ201菌株后,A9IP2基因敲除的突变体植株较野生型更感病(图4A)。计算相对菌丝生物量(图4B)及相对病斑面积(图4C)发现,A9IP2基因敲除的突变体水稻植株的两个家系接种后的菌丝生物量和相对病斑面积均显著大于野生型TP309水稻植株。鲁米诺化学发光法检测结果显示发现当用Chitin同时处理野生型TP309和A9IP2基因的敲除株系后,突变体的ROS进发较野生型低,起峰速率及荧光峰值均明显低于野生型(图4D)。
实施例4
A9IP2基因过表达植株稻瘟病抗性鉴定。
离体打孔接种方法如实施例3。结果显示当接种稻瘟病有毒生理小种KJ201菌株后,A9IP2基因过表达植株表现为比野生型TP309植株更抗病(图4A),A9IP2基因过表达突变体的两个家系接种后的菌丝生物量和相对病斑面积均显著小于野生型TP309植株(图5B和图5C)。
序列表
<110> 中国水稻研究所
<120> 水稻A9IP2基因在提高水稻抗稻瘟病上的应用
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ggacaggtta ctcgaagtgt tgatacacag tctgggcagt taagcccgca ggagcgtgat 720
acgattgatg ctatagtaaa agctagccag ttccccttgt ctattgttct tgttggggtg 780
ggtgatggac catgggacat gatgcatcag tttgatgaca atatacctgc tcgctcattt 840
gacaatttcc agtttgtgaa tttcacggac atcatgtcaa agagcatagc agctgacagg 900
aaggaggcag aatttgcact atccgccttg atggaaatcc caacacaata caaggcaaca 960
cttgatctcc aacttctagg ccgtcgccaa agaatacagc caagaattcc tctgccgcca 1020
cctatgagga atgcttattc gaggtctact agctttgacc aacattctgg tgtttattca 1080
cgatctagta gcttcggccc acaaacaagt ggctttcagc aatctgaaag cttcaaacag 1140
agacagcctg tggctaccac tgcacctgac acttatacct cggaaagttc tttagaagga 1200
agacttttgt gcgctatctg tatggataaa tcaaaggatc ttgcatttgg atgtggacat 1260
cagacctgtt atgaatgcgg gaagaatttg gtgcgctgcc ctatgtgcca gcagcatata 1320
accacaagga tcaggctgta ctga 1344
<210> 3
<211> 5488
<212> DNA
<213> 水稻(Oryza sativa L.)
<400> 3
acaccacaca attcaagtat tcaacctcct tttcataagc agaagtatct ttgtcacact 60
ctactactac tagtctacta gcactcctaa tgatcgatcc ttcccacgtt cgaagcttcc 120
cctgtcactg tccatccatg gtggtggtca gacaacccca gcacacaaga aatctgccat 180
ttgcccagtc caccgctggg ttgcagtttt tgatgaagag attgacttga cctgagcacc 240
cttccgattg taaatagcag tttttgtatt gctgttggtt gagtgattgg gcaatcatcc 300
aatacatatc ttggcttctt ggctggactc cagtaaccga ggtcgcgtgc ctccgctcgc 360
tcgctcgctc atcgtcgtcg tcttcctcgg ggcctctctc gtcgtggagc ggaggcgtgc 420
cgcgtagcag gggtgagccc atgtgggtgg attgatttgg ggggatttcg tgctgttttg 480
gtggaagagc tctcttctgt acggtttctt ggttgcgctc tcgcaccaag aacatctcct 540
ttttccttct tgctcctctc tctctctctc tctctctctc tctctctctc tctctctctc 600
tctctctctc tctctctctc tctctctctg ggtctcggac tctcggctct tgattcttgg 660
ttcttggtga gtgtaggcga gaataaggcg ggttcttggt gggtttataa ggtattattt 720
ggatctaatc cggcgaagaa aagaattcgt tgtttttttt ttctcatagg catttgatat 780
gtttggtagg tcgcattctg gtgttcttgg acgcacttcg attgttcgat atgttcgatg 840
cttacatggg gattgggttg tttcgattgt taatcttgct aattgttcgt gagaagggaa 900
tgtggggtgc tagattgttc ttgggggtgg ggaattttac ctgccatgga cagtaattca 960
cccccaccct caattaggat ttcctcaaat tatctgcatg atcagtcaga acctgatggt 1020
ttcttgagat ttgtcagaaa agtctctgct tcttgtgcag ttattttatt aaaagtactg 1080
atcttttttg ttcccttttt tatcagggaa ttcaactgag gtgagaaatg ggggcaaagg 1140
actccaagcc ctcatacagc tattcttctt catatgatca tgggaattca tcatcgggat 1200
ataactcgag ataccctgca taccctgcaa atgcatcttc cagtcagaac acaaggtatg 1260
ctccttctat ggagaactat gtgcagccag aaacgcatgc caggttgcag aggaagtact 1320
cgaggattgg tgatgactac cgctctctga atcaagtatg tttttgatga catgaccttg 1380
tttaacccat gaattttgat atttgctttg cgggcgtttg gtatgcgtgc tatcgattac 1440
tagtagctta gttgaatggc atcagtatca ttcatctttg atgatatggc atagggaaat 1500
gtttgaataa gctatccttt tctaggatat tttcgaggag ggttccaaac aagagttgta 1560
ttctcttatg gaaaccacta tatatattgc attaacaaag aaaccactat agattctcaa 1620
tggagctttc tttgactatg gtattcatct cagtcagttt tttgtacgct ggaattcacc 1680
ctttttagta cacactttta aatgatttat aagatatcca gctgctattt attttccagt 1740
acattttcag ttaatggctt gccatcttta atcatatatg tgttgtttga gccaaagact 1800
ataacaagct ttccatttcc tgttatgtct cctacaaaag ataattcctt gctataatat 1860
ttatgtgctg ttgatgtgct tatcattgtg ttcaatttat gtatttttct tagtttcatt 1920
tccttcatag tgcaccagag tatacgtact actttactag agtcttgaac aatctcttcc 1980
ttttgagtac tgtgaacttg agattggaag tacacgtttt catttttatc gatatgtact 2040
gttacaggtg actgaagctt tggcacaagc aggtcttgaa tcatcaaatc ttattgtagg 2100
cattgatttt acaaagagca atgaatggac aggttaacag accacttctt gttttgaatt 2160
ctgtttcttt atatttgctt tgtgctgtgc tggtagccta ttacttgcga attagtagtg 2220
ttattatata catcttgatt tacctattat taggacaaac atccttttct accttagttg 2280
cgtatgatgc aaatgagaaa tggcatgtca acttctctta attctttgtt ataaatgagc 2340
tctcactagg gctgctgtct ggaaggaggt tggactttct tgaaaaaagt ttattgaggg 2400
agttagctac tcctaataag ctggttattt ttctgtatct gaagtgcctg tgatgcaaat 2460
ggcatgtcaa cttctcttaa ttctttgtta taaaccccta ataagctggt tatttttctg 2520
tatctgaagt ttatgtgaac tacctgttat cttttgacat aaggagtgaa cctgattccg 2580
taacttcaca acaaatgcaa tgtgcaatct tttgccaaac aaaaccattt aaagtaaaga 2640
aatgtgaaat ggggcaatta tgcatgtagc aaggatgcct ctcatgttgc ttgcatatcc 2700
tacagtcatg ctatgtcaca aatcatagca ctctcattta acctgctgag cttttctcca 2760
ttaggtaaac tttccttcaa tcgccgatgt ctgcatgata ttggaaacac tccaaatcca 2820
tatgagcaag caatatctat tattggaagg acactttcag cttttgatga agataatttg 2880
attccttgct ttggatttgg tgacggtaag ttcattgttc ccattttcca ttttgatcac 2940
ttggtgtgtg ctcagtagca tatttaaaga acaatgctga tctaatgcct cctttgtggc 3000
tttgtctact ggcagcatca actcatgatc aggaggtatt cagcttttat ccagagaacc 3060
gcccatgcaa cggatttgaa gaggcattag aaagatacag agaaattgtt ccgactcttc 3120
gattagctgg ttcgtttcag taatttcaca atttagatgt ttattcttcc tttatttttt 3180
taaagaagga tcttgaattt agctggttat atataagatt gcagtagtaa ctacagatct 3240
tcagaaaaat aatgtatgca aaaattgcaa caggaccaac atcttttgcc ccgatgattg 3300
aaacagcaat tggtattgta gacagcactg gtggtcagta tcatgttctt ctgataatag 3360
ccgatggaca ggtctgtcac cattttctct tgttttatcc cttttcttta tttcttgtct 3420
ttctaagtaa ttgtgatcag cagaactctt ctctagatta tagttgatta ttttttttgt 3480
tcaaatcata tttttacagt ggctaaaact tctgctaaca ggttactcga agtgttgata 3540
cacagtctgg gcagttaagc ccgcaggagc gtgatacgat tgatgctata gtaaaagcta 3600
ggtacttgat cgagtcccta atttaatatc ggtatattct gcccaaatca aatgtcccga 3660
atcacacgtg ttaacatttt tttcgaaata aatttccagc cagttcccct tgtctattgt 3720
tcttgttggg gtgggtgatg gaccatggga catgatgcat cagtttgatg acaatatacc 3780
tgctcgctca tttgacaatt tccaggtgac tgttctacat ttaatttcca gtcattggct 3840
tatttttgtg tggttttcac aataatagag acatgtaaaa atgtttgctt tcatgcagtt 3900
tgtgaatttc acggacatca tgtcaaagag catagcagct gacaggaagg aggcagaatt 3960
tgcactatcc gccttgatgg aaatcccaac acaatacaag gcaacacttg atctccaact 4020
tctagggtaa tccatgtttt cccaggactg cactgggtca aatgtactag agtttgtgct 4080
tgcagcaatt gtatccattt gaacattttc ttcacgtttt ctttcgtttc agccgtcgcc 4140
aaagaataca gccaagaatt cctctgccgc cacctatgag gaatgcttat tcgaggtcta 4200
ctagctttga ccaacattct ggtgtttatt cacgatctag tagcttcggc ccacaaacaa 4260
gtggctttca gcaatctgaa agcttcaaac agagacagcc tgtggctacc actgcacctg 4320
acacttatac ctcggaaagt tctttagaag gaagacttgt aagtattttt gtttatattc 4380
acaccatgtt gaatagataa agtttaagat atatactgca acactgtagc attggtaaaa 4440
taaagtttaa gctatatact gcaagactac attggtgtca ttatcgtaca ccaccaacaa 4500
atagtctccc ttttattttg ttcgtactgt ggcatttcgg gaggctaatt gaaacagggg 4560
aagttgcatt gcattgctct atctgttctg accaccctgc ctgcgttcat ttggcttatt 4620
acttatttca cggatgccct ttatgaaaaa aaaaatatcg tgccttcaaa tggcccattt 4680
gatcttgatc tgataactac attatgctga atattgctat tgcatttcac attcttatgt 4740
catttgggca tactgttaat ccgcagatca tggaaagttc ctacacctct agtgttaagt 4800
ttttatgtac catcaaattt cgtgctaaac cctgagaatc ttactggatt acaaactaaa 4860
tgcagctttt ttttttgtgt gtggtttcag ttgtgcgcta tctgtatgga taaatcaaag 4920
gatcttgcat ttggatgtgg acatcaggtg cagaaaattt ccccttattg ttctctattt 4980
ctacataatt tcttgttata aatggcagca tgcatgataa aatgattcgc agacctgtta 5040
tgaatgcggg aagaatttgg tgcgctgccc tatgtgccag cagcatataa ccacaaggat 5100
caggctgtac tgagaaaaca aaaatggata tcctgaagtt tctgaagcct gcagaaacag 5160
aatctccaac atcagaagaa actcctgtgc aaaatttatg aaccagccta aattgtactg 5220
ttctttcgtc ttgtaattgg tggtttacta ttattattgc agtgagaaat gtttaggcag 5280
atagtgatcc atataaaaag ctaaggcaca cactgtagta ttaactacag tagttatgtg 5340
aagaagaggt acactaaata atgtacagta attagcttct ctattataca ttctcctttc 5400
tttcccttgg taaataagat aaaaggttgc aaaatttcca ggaattagtg aacaagcaga 5460
tatgcagtgt gttaaagctg ttagcact 5488
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggcattcttc ttcatatgat cat 23
<210> 5
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aaacatgatc atatgaagaa gaa 23
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agggaatgtg gggtgctaga 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cgcccgcaaa gcaaatatca 20
<210> 8
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gttacttctg cactaggtac catgggggca aaggactcca 40
<210> 9
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tcttagaatt cccggggatc ctcagtacag cctgatcctt gtgg 44
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctgacaggaa ggaggcagaa 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gacggcctag aagttggaga 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aagaagctga agcatccagc 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccaggacaag atgatctgcc 20
Claims (9)
1.水稻A9IP2基因在提高水稻抗稻瘟病上的应用。
2.如权利要求1所述的应用,其特征在于,水稻A9IP2基因的CDS区核苷酸序列如SEQ IDNo.2所示。
3.水稻致病因子A9IP2蛋白在提高水稻抗稻瘟病上的应用。
4.如权利要求3所述的应用,其特征在于,水稻致病因子A9IP2蛋白的氨基酸序列如SEQID No.1所示。
5.水稻A9IP2基因在水稻育种中的应用,通过筛选过表达水稻A9IP2基因的水稻植株获得抗稻瘟病菌的水稻株系。
6.水稻致病因子A9IP2蛋白在水稻育种中的应用,通过筛选水稻致病因子A9IP2蛋白表达量高的水稻植株获得抗稻瘟病菌的水稻株系。
7.一种抗稻瘟病菌的转基因水稻构建方法,其特征在于,将水稻A9IP2基因转入水稻植株内获得过表达水稻A9IP2基因的转基因水稻。
8.如权利要求7所述抗稻瘟病菌的转基因水稻构建方法,其特征在于,将水稻A9IP2基因克隆到载体中,先转入到农杆菌EHA105中,再转入水稻细胞内,获得过表达水稻A9IP2基因的转基因水稻。
9.如权利要求8所述抗稻瘟病菌的转基因水稻构建方法,其特征在于,载体为pUCBi1390载体。
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CN117051015A (zh) * | 2023-10-11 | 2023-11-14 | 中国农业科学院植物保护研究所 | 水稻OsFbx156基因在提高水稻抗稻瘟病上的应用 |
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"Accession No. XM_015787675.2", GENBANK * |
刘鑫等: "水稻泛素结合酶基因家族的生物信息学与表达分析", 中国水稻科学, vol. 30, no. 3, pages 223 - 231 * |
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Cited By (2)
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CN117051015A (zh) * | 2023-10-11 | 2023-11-14 | 中国农业科学院植物保护研究所 | 水稻OsFbx156基因在提高水稻抗稻瘟病上的应用 |
CN117051015B (zh) * | 2023-10-11 | 2023-12-12 | 中国农业科学院植物保护研究所 | 水稻OsFbx156基因在提高水稻抗稻瘟病上的应用 |
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