CN114480476B - 一个可以用于改良木薯抗病性的蛋白及编码基因的应用 - Google Patents
一个可以用于改良木薯抗病性的蛋白及编码基因的应用 Download PDFInfo
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Abstract
本发明提出了一个可以用于改良木薯抗病性的蛋白及编码基因的应用,所述蛋白为胁迫相关蛋白,具体为MeSAP13蛋白。实验证明MeSAP13蛋白及编码基因可抑制地毯草黄单胞菌属木薯萎蔫致病变种生长数量,能够有效地提高木薯对细菌性枯萎病的抗性,可用于改良木薯抗细菌性枯萎病中的应用。
Description
技术领域
本发明涉及基因应用技术领域,特别涉及一个可以用于改良木薯抗病性的蛋白及编码基因的应用。
背景技术
木薯(Manihot esculenta Crantz)是世界三大薯类作物之一,全球第六大粮食作物,每年为热带地区7亿人口提供主粮。但在生产上,病害一直是限制木薯产业健康发展的重要制约因素之一。
细菌性枯萎病是由地毯草黄单胞菌木薯萎蔫致病变种引起的细菌性病害,最早在巴西发生,后来通过块根等繁殖体在世界范围内传播开来。先后给多个国家的木薯生产带来毁灭性打击,曾导致我国木薯产量损失高达30%以上,而且扩散为害的形势十分严峻,已成为国际上重要的检疫性病害之一。生产上主要通过苗期检疫、药物防治等措施进行防控,我国主栽的华南系列、桂热系列木薯品种均不抗该病。因此,创制抗细菌性枯萎病木薯新种质,选育抗病新品种,增强我国木薯主栽品种的抗病性是预防细菌性枯萎病同时又保护环境的根本出路。
MeSAP13蛋白是胁迫相关蛋白(SAPs)家族成员,具有典型A20/AN1锌指结构域。SAPs调控植物响应非生物胁迫的途径研究的较为清晰,当植物感受外界非生物胁迫刺激后,细胞膜蛋白接受信号并将信号传递至受体类细胞质蛋白激酶RLCKs,接着RLCKs活化SAPs。活化后的SAPs蛋白通过3种方式调控植物抵抗非生物胁迫:(1)通过泛素化途径降解抗逆相关蛋白;(2)通过蛋白间的相互作用激活抗逆基因表达;(3)通过与转录因子结合进入细胞核直接调控抗逆相关基因的表达。此信号通路的研究表明,SAPs蛋白的磷酸化是其参与植物抗逆反应的关键节点,但目前并没有MAPK参与SAPs蛋白的磷酸化报道,关于SAPs在植物抗病过程的功能研究较少。
发明内容
鉴于此,本发明的目的在于提出一个可以用于改良木薯抗病性的蛋白及编码基因的应用。
本发明的目的之一在于,提供一个可以用于改良木薯抗细菌性枯萎病的胁迫相关蛋白,该蛋白具体为MeSAP13蛋白。该MeSAP13蛋白的氨基酸序列如SEQ ID NO:1所示,核苷酸序列如SEQ ID NO:2所示。
本发明的目的之二在于,提供上述MeSAP13蛋白及编码基因用于改良木薯抗细菌性枯萎病的应用,具体为可抑制地毯草黄单胞菌属木薯萎蔫致病变种的生长数量方面的应用。
与现有技术相比,本发明的有益效果为:
本发明发现了一个来源于胁迫相关蛋白(SAPs)家族的MeSAP13蛋白,可用于改良木薯抗细菌性枯萎病中的应用,为提高木薯抗病性提供了新的理论和研究途径。
本发明通过构建VIGS沉默载体进行抗病性研究,MeSAP13基因沉默的实验结果表明,VIGS沉默载体pCsCMV-MeSAP13侵染的木薯苗,对细菌性枯萎病的抗病性减弱,叶片中地毯草黄单胞菌属木薯萎蔫致病变种(Xanthomonasaxo-nopodispv.manihotis,Xam)数量高,而含木薯普通花叶病毒(Cassava common mosaic virus,CsCMV)基因的质粒侵染获得的木薯苗,其叶片的病斑面积和地毯草黄单胞菌属木薯萎蔫致病变种的数量均小于VIGS沉默载体pCsCMV-MeSAP13侵染的木薯苗。实验证实了MeSAP13蛋白及编码基因可抑制地毯草黄单胞菌属木薯萎蔫致病变种生长数量,能够有效地提高木薯对细菌性枯萎病的抗性,可用于改良木薯抗细菌性枯萎病中的应用。
附图说明
图1为MeSAP13蛋白亲水性分析,注:负值表示亲水性,正值表示疏水性;
图2为MeSAP13基因序列内含子与外显子结构示意图;
图3为MeSAP13蛋白的保守结构的分析;
图4为MeSAP13蛋白高级结构预测模型图;
图5为MeSAP13蛋白在烟草中的亚细胞定位图;
图6为MeSAP13基因响应XamHN11胁迫的相对表达量图;
图7为VIGS沉默后目的基因表达水平检测,注control为负对照pCsCMV-A木薯,VIGS为pCsCMV-MeSAP13木薯;
图8为VIGS沉默载体的农杆菌侵染木薯的表型变化图;
图9为XamHN11侵染基因沉默后木薯的不同时间点的叶片背面的表型变化图;
图10为叶片背面的病斑面积柱状图;
图11为XamHN11侵染木薯叶片中病原菌的数量图。
具体实施方式
为了更好理解本发明技术内容,下面提供具体实施例,对本发明做进一步的说明。
本发明实施例所用的实验方法如无特殊说明,均为常规方法。
本发明实施例所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实验材料如下:
植物材料:木薯品种SC8、烟草品种本氏烟。
菌株:大肠杆菌菌株Trans5α;农杆菌菌株GV3101;木薯细菌性枯萎病菌(Xanthomonas axonopodis pv.Manihotis HN11,XamHN11)。
质粒载体:亚细胞定位载体pCAMBIA1300-GFP-MeSAP13、空载pCAMBIA1300-GFP;VIGS沉默载体pCsCMV-B、pCsCMV-A、pCsCMV-MeSAP13。
实施例1-MeSAP13基因及蛋白生物信息学分析
MeSAP13蛋白(Manes.14G001000.1),该蛋白的N端含有A20锌指基序:CX(3)CX(11)CX(2)CX(2),C端含有AN1锌指基序CX(2)CX(10)CX(1)CX(4)CX(2)HX(5)HXC,属于I型(即A20+AN1)锌指蛋白。MeSAP13蛋白的氨基酸序列如SEQ ID NO:1所示,利用在线网站ExPASy分析木薯MeSAP13蛋白的生理生化性质,结果显示MeSAP13蛋白理论分子式:C793H1275N235O247S15,理论相对分子质量:18.5KDa,理论等电点pI:8.44。该蛋白富含:丙氨酸Ala(9.8%)、赖氨酸Lys(8.7%)、缬氨酸Val(8.7%)、半胱氨酸Cys(6.9%)、甘氨酸Gly(6.9%)、丝氨酸Ser(6.9%)、天冬酰胺Asn(6.4%)、脯氨酸Pro(6.4%)、谷氨酸Glu(5.2%)。亲水指数从-2.744到2.222(负值表示亲水性,正值表示疏水性),亲水性平均指数-0.349,表明该蛋白水溶性较好(如图1所示)。理论不稳定系数:26.36,属于稳定蛋白质。利用NCBI站点ConservedDomain Database及SWISS-MODEL分析MeSAP13蛋白保守结构域(如图3所示)及预测MeSAP13蛋白高级结构(如图4所示),发现MeSAP13蛋白N端具有典型的A20锌指结构域,C端具有AN1锌指结构域,是典型的锌指蛋白。利用在线网站Plant-mPLoc SEfvEf预测MeSAP13蛋白可能定位于细胞核。
根据测序结果,利用NCBI在线网站进行序列比对,并分析其开放阅读框,显示MeSAP13基因全长522bp,由一个外显子编码,核苷酸序列如SEQ ID NO:2所示。利用GSDS在线网站分析MeSAP13基因的结构特点,结果显示,该基因含有1个外显子和2个内含子(如图2所示),其中CDS序列核苷酸序列如SEQ ID NO:3所示。
实施例2-MeSAP13基因的亚细胞定位
将重组载体pCAMBIA1300-GFP-MeSAP13、pCAMBIA1300-GFP空载以及35S-H2B-mCherry细胞核mark转化农杆菌感受态细胞GV3101。挑取单菌落于10mL含有卡那和利福平抗生素的LB液体培养基中,28℃,200rpm培养24h。取50μL过夜培养菌液于50mL含有卡那和利福平抗生素的LB液体培养基中,28℃,200rpm培养至OD600值为0.5~0.6左右,约为12h。4000rpm离心5min收集菌体,弃去上清。加入20mL缓冲液(10mmol/L MgCl2,10mmol/L MES)重悬菌体,4000rpm离心5min,弃去上清。再次加入20mL缓冲液(10mmol/L MgCl2,10mmol/LMES)重悬菌体,4000rpm离心5min,弃去上清。加入植物注射液(10mmol/L MgCl2,10mmol/LMES,150μmol/L乙酰丁香酮)重悬菌体,调OD600值为0.6~0.8左右,将35S-H2B-mCherry细胞核mark分别与pCAMBIA1300-GFP-MeSAP13和pCAMBIA1300-GFP空载按照1:1比例混合,室温静置2-3h。选取生长4-5周大的长势良好的烟草本生烟,用1mL无菌注射器去掉针头后取适量菌液,于烟草叶片背面轻柔注射菌液,标注注射区域。注射过的烟草置于24℃培养48h,剪取注射区域烟草叶片,将叶片置于载玻片上,滴加少于清水制片,激光共聚焦显微镜下观察有无荧光。
结果如图5所示,图5中第一列是明场,第二列是pCAMBIA1300-GFP-MeSAP13融合蛋白表达发出的绿色荧光,第三列是35S-H2B-mCherry细胞核mark发出的红色荧光,第四列是叠加场。结果显示:在烟草中,pCAMBIA1300-GFP空载可以被诱导表达,而且荧光扩散在整个细胞中;pCAMBIA1300-GFP-MeSAP13融合蛋白表达的GFP在细胞核和细胞膜都有,说明MeSAP13蛋白定位在细胞核和细胞膜中。
实施例3-MeSAP13基因响应XamHN11胁迫的相对表达量
用木薯细菌性枯萎病菌XamHN11侵染45日苗龄木薯SC8,分别取0h、3h、6h、1d、3d、6d叶片,通过qRT-PCR检测木薯MeSAP13基因的表达量变化。XamHN11侵染3h后,MeSAP13基因表达量显著升高,3d后MeSAP13基因表达量下降,推测MeSAP13基因确实能够响应Xam侵染(如图6所示)。
实施例4-VIGS沉默载体的构建
载体依据文献(Tuo Decai,Zhou Peng;et al.A cassava common mosaic virusvector for virus-induced gene silencing in cassava.[J].Plant methods,2021,17(1)),获得VIGS沉默载体pCsCMV-MeSAP13。采用引物MeSAP13-F和MeSAP13-R进行PCR扩增,利用海南壹田生物科技有限公司的Nimble Cloning试剂盒进行NC克隆反应,将反应产物转化DH5a感受态细胞,采用引物CsCMV-F和CsCMV-R进行PCR菌落筛选阳性克隆,获得的VIGS沉默载体命名为pCsCMV-MeSAP13。相关引物如下:
表1
实施例5-含VIGS沉默载体的农杆菌以及侵染木薯
将实施例3获得的VIGS沉默载体pCsCMV-MeSAP13、正对照pCsCMV-B和负对照pCsCMV-A分别转化农杆菌GV3101(pSoup-p19)感受态细胞,侵染木薯后分别获得pCsCMV-MeSAP13木薯、pCsCMV-B木薯和pCsCMV-A木薯。其中,正对照pCsCMV-B为含有病毒基因与指示基因的重组质粒,负对照pCsCMV-A为仅含有病毒基因的质粒。
浸染方法:挑取菌落PCR鉴定出的阳性克隆至5mL LB(50μg/mL卡那霉素+25μg/mL利福平)液体培养基中,28℃,220rpm过夜培养。取1mL过夜培养菌液于50mL LB(50μg/mL卡那霉素+25μg/mL利福平)液体培养基中,28℃,220rpm培养至OD600值为1.0。将菌液移至50mL离心管中,4000rpm离心5min,弃去上清液。加入适量10mM MES和10mM MgCl2混合液重悬菌体,4000rpm离心5min,弃去上清液,重复两次。加入25mL植物注射液(10mM MES+10mM MgCl2+200μM乙酰丁香酮)重悬菌体,在室温、黑暗条件下静置3h。选取长势良好、状态一致的木薯SC8,将菌液用1mL注射器压渗至木薯叶片背面,30d后开始观察表型变化。
MeSAP13基因沉默效果验证:取VIGS沉默40d后的pCsCMV-MeSAP13木薯和负对照pCsCMV-A木薯的新叶片,提取RNA,反转录得到cDNA,以此为模板,利用qRT-PCR技术检测MeSAP13基因的表达量。实验结果显示,目的基因的相对表达量下降显著,基因的表达量普遍在0.4-0.6左右,说明MeSAP13基因受到了有效的沉默(如图7所示)。
实施例6-木薯植物接种细菌性枯萎病病原菌
将保存的木薯细菌性枯萎病病原菌XamHN11在含有氯霉素的LPGA固体培养基中28℃倒置培养2d。挑取单克隆菌落于5mL含有氯霉素LPGA液体培养基中,28℃,220rpm培养约16h。吸取200μL菌液涂布于含有氯霉素的LPGA固体培养基上,28℃培养24h左右。用10mMMgCl2洗下菌体,4000rpm离心5min收集菌体,并用10mM MgCl2清洗菌体两到三次,调整细菌浓度为1×108CFU/mL(OD600≈0.1)。阴性对照为10mM MgCl2,用去除针头的1mL注射器吸取菌液,用手指抵住叶片正面,将菌液用注射器压渗到叶片背面,接种完毕后,观察不同时间点的木薯叶片水渍状病斑扩散情况。
叶片病斑的细菌生长计数:用直径为1cm的打孔器取下接种XamHN11后的0d、3d、6d木薯叶片,置于已灭菌的研钵中,加入1mL MgCl2充分研磨,稀释不同浓度。取3个连续稀释的浓度梯度的100μL上清涂布于LPGA固体培养基上,28℃倒置培养24h后统计菌落数量,根据平板上的菌落数计算叶片中的活菌数。
(1)VIGS沉默效果的表型分析
含有指示基因的正对照会使植物合成叶绿素的能力受阻,导致新叶部分出现褪绿白化的现象(如图8所示),受到pCsCMV-B侵染大约40d后的木薯新叶出现了明显的白化表型,而不含指示基因的负对照与接种pCsCMV-MeSAP13的木薯新叶则无明显变化。以正对照沉默指示基因产生的表型来指示MeSAP13基因的沉默效果,可以初步认为MeSAP13基因已经发生了有效的沉默,产生了表型变化。
(2)MeSAP13基因沉默木薯的抗病性情况
对侵染后的负对照和pCsCMV-MeSAP13木薯苗,再在其新叶接种OD600=0.1的病原菌XamHN11,10mM MgCl2为对照。水渍状病斑是接种病原菌XamHN11后产生的表型,病斑扩散面积的大小可以指示植株受感染的程度。取接种后的幼嫩叶片观察水渍状病斑的扩散情况,发现所有叶片的接种部位均产生了水渍状病斑,说明所有木薯都已经发病。结果如图9所示,当XamHN11侵染3d后,叶片背面逐渐开始出现水渍状病斑,侵染6d后,可以明显地看到病斑面积开始逐渐扩大,且沉默后的pCsCMV-MeSAP13木薯植株产生的水渍状病斑大于负对照pCsCMV-A,说明沉默MeSAP13基因后植株的抗病性减弱。
利用ImageJ软件计算叶片背面的病斑面积大小(如表1),并计算平均值(单位为mm2)。取各植株不同时间点的病斑面积大小绘制成柱状图,并进行显著性分析,结果如图10所示,侵染6d后的病斑面积明显大于侵染3d后的病斑面积,且沉默后的pCsCMV-MeSAP13植株的病斑面积高于负对照pCsCMV-A,说明沉默MeSAP13基因后的植株使得抗病性降低。
表1病斑面积与平均值
通过分析处理XamHN11侵染0d、3d、6d后木薯叶片中病原菌的繁殖情况发现,侵染0d时,差异并不显著,但在侵染3d和6d后,沉默MeSAP13基因的木薯叶片中含有的细菌数量高于负对照(如图11所示)。
以上研究结果表明,MeSAP13基因的沉默降低了木薯对细菌性枯萎病的抗性,因此,MeSAP13基因可用于改良木薯对细菌性枯萎病的抗性。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 海南大学
<120> 一个可以用于改良木薯抗病性的蛋白及编码基因的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 173
<212> PRT
<213> 木薯(Manihot esculenta)
<400> 1
Met Asp His Asp Glu Thr Gly Cys Gln Ala Pro Pro Glu Arg Pro Ile
1 5 10 15
Leu Cys Val Asn Asn Cys Gly Phe Phe Gly Ser Ala Ala Thr Met Asn
20 25 30
Leu Cys Ser Lys Cys His Lys Asp Met Leu Leu Lys Lys Glu Gln Ala
35 40 45
Lys Leu Ala Ala Thr Pro Thr Gly Asn Ile Val Asn Gly Ser Ala Ser
50 55 60
Asn Asn Val Glu Gln Pro Val Val Val Val Glu Ala Val Asp Val His
65 70 75 80
Val Asn Thr Val Gln Pro Asn Thr Ile Ser Val Gln Pro Ser Cys Ala
85 90 95
Ser Gly Leu Gly Glu Ser Val Glu Ala Lys Pro Lys Glu Gly Pro Ser
100 105 110
Arg Cys Gly Thr Cys Lys Lys Arg Val Gly Leu Thr Gly Phe Lys Cys
115 120 125
Arg Cys Gly Asn Phe Phe Cys Ala Ser His Arg Tyr Ser Asp Lys His
130 135 140
Asp Cys Pro Phe Asp Tyr His Ser Ala Ala Arg Gln Ala Ile Ala Lys
145 150 155 160
Ala Asn Pro Ile Val Lys Ala Glu Lys Leu Asp Lys Ile
165 170
<210> 2
<211> 3094
<212> DNA/RNA
<213> 木薯(Manihot esculenta)
<400> 2
acctttcctt tcaaaaatca aaagggaagc gttagagaga gaaagctttg gaaatctctc 60
tctctctctt atagagaaag ggaagtcacc aaacgcctct ctctctctat ctctctggct 120
ttgctcctct ctctctctct ccccctcctg cggtccctcc tctgcgatct ggtaagcatc 180
acctggggtt agggtttatg attcccgtac ctcgattttt ttatcttttn nnnnnnnnnn 240
nnnnnnntgg gagggggtgt tttgagtctg ttgatttatt atgtagttgt tctatttgat 300
taaagcaggt cttacctgat gcagttttga gctaagagat cggcttaagg aatcatttcc 360
aattacgaat aactttcagg ttcgtgtaca tatgtgcttg tttgtttgta tttccttttt 420
tggtatttac aaattttcgc cttttattta attttggcac cataaagaga atgatataaa 480
aaaattagcg tcttctgtac attgtcagac tgccctgatg ggtgttgtat tagtagagtt 540
gtcttttcgt ggtgaccgat gattgtagtt caatatagat tttagcttgg ttttttggtt 600
tggttatgga ttgggtgaga tttgagaatg tatggttcag tatagatttt ggcttagttt 660
tgttactcga tttggttatg ggttgggtga gatttaagaa ttggaataat aattataaag 720
ttgagatttg agattttagc tttgttaggt gagtgattgt tgatgaatag cgtattacac 780
catttgattg gtgttactcg tctggctact cagggcaatt aagttggcaa agatgagttt 840
gattacatat atagtgggta ggagaaataa acctacaaat gtggaaattt gttaatgttg 900
gcctggatct ttctaaaaat gtagccctaa acagatggaa aggaagaaat ggattcatat 960
agcccattca actagtttct atttggtttt atttggactg gaataatttt gttgctcctt 1020
ggcatgcgtt gtttgctttt gtttttctat ggagtgaaat acttattcct gcttcacaag 1080
caatgccatc ctatttggca tacagctttc acatcaatct agtgtaagga gatgttagga 1140
agtgggcttg tatcatattt tgtcatgaaa gctgctgttt caaattccat caaaattatt 1200
agttgtttca aatgcattgt ataagcaatt ctgtgaaatt cagtgtaaga taatgtttga 1260
tatgcagaga aaccgaaaaa atgtaaagaa aaacataaac agaaaagagc accattatta 1320
tataaccaag ctgctgttta tcttttctga atctcttgtc caatcactga attccaaatc 1380
ttttgctgcc actactagaa attagtcaag tggtcaggta aacaagacta caaaataggc 1440
tgaagcttta tacttgtttt tcttgactat actaggatat tatcaacttg tgtcatgaat 1500
ttcctttctt tttggatttt tataatgaat ttaaggacat atggataaat tttatccttt 1560
ctcactgttt tgtttcctcg catgagtctc actgttttga gttatgtttc ctattctctt 1620
ttgagtacta tacaacagcc tctgtttatc tcacaaacca acctcactga acagctttgg 1680
aaattaaatc caacttcagt ccaatcacaa cctgtgagag atacaaaaat tgtgctcatc 1740
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ttgtgttttg aatcgaaact tatatcttct cctgtcaatt tttatttcat atttctggta 1920
aatgtatttt atggatgtgg attagttatt gcacattctc tttaaggatt gctaataagc 1980
tacttgtgag ggtagttaca catccgttaa gaaatttgca aggtttgtct tattagtatt 2040
tggatgaggc cttcagacat tctctctggc atgtttgttt tcgtatgtgc ttgatattca 2100
ttattctatg gttttggtgc aggacaacta aaagatggac catgatgaga caggatgtca 2160
agctcctcct gaacgcccga ttttgtgtgt taacaattgt ggcttctttg gaagtgcagc 2220
cacgatgaac ttgtgttcaa agtgccacaa agatatgctg ttgaaaaagg agcaggctaa 2280
gctcgctgca acacctactg gaaatatagt taatggatca gcaagcaaca atgtggaaca 2340
acctgttgtt gttgttgaag ctgttgatgt tcacgtcaac acagtgcagc caaataccat 2400
ctctgtgcag ccttcttgtg cttctgggtt gggggagagt gttgaggcaa agccaaagga 2460
gggtccaagt cggtgcggca cttgcaagaa acgagttggt ttaacagggt tcaagtgtcg 2520
atgtggcaac tttttctgtg catctcatcg ctactcggac aaacatgact gcccatttga 2580
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gcttgataag atctgaagat caaactgagt gaagtttcat gtcctggaag ttgatgatca 2700
ttttgtcttt gggggttctg ggttggccaa gtatcttagc aggtgtccat ctgcattgtt 2760
gttatgggag aagcaaggca gcatcggaca tctctctgca atatgaagaa ctctatgtct 2820
tgtgattggc gagagtttat atgttggctc cagtgttttt gagtctaaat ctgtgctggt 2880
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gcagtttgct ggtaatcgtc caaaatggct tgttggaatt cttagataat atatggaagt 3000
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<212> DNA/RNA
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atgctgttga aaaaggagca ggctaagctc gctgcaacac ctactggaaa tatagttaat 180
ggatcagcaa gcaacaatgt ggaacaacct gttgttgttg ttgaagctgt tgatgttcac 240
gtcaacacag tgcagccaaa taccatctct gtgcagcctt cttgtgcttc tgggttgggg 300
gagagtgttg aggcaaagcc aaaggagggt ccaagtcggt gcggcacttg caagaaacga 360
gttggtttaa cagggttcaa gtgtcgatgt ggcaactttt tctgtgcatc tcatcgctac 420
tcggacaaac atgactgccc atttgattat cacagtgctg cacgtcaggc tatagctaaa 480
gccaacccca ttgtcaaggc agagaagctt gataagatct ga 522
Claims (2)
1. 一种沉默胁迫相关蛋白在减弱植物抗细菌性枯萎病中的应用,其特征在于,所述胁迫相关蛋白为MeSAP13蛋白,所述MeSAP13蛋白的氨基酸序列如SEQ ID NO:1所示,所述植物为木薯。
2. 如权利要求1所述的胁迫相关蛋白在减弱植物抗细菌性枯萎病中的应用,其特征在于,所述MeSAP13蛋白的核苷酸序列如SEQ ID NO:2所示。
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