CN111218456A - 14-3-3 h1蛋白在TMV侵染烟草叶片中的应用 - Google Patents
14-3-3 h1蛋白在TMV侵染烟草叶片中的应用 Download PDFInfo
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Abstract
本发明提出了烟草14‑3‑3 h1蛋白的编码基因14‑3‑3 h1在防御TMV侵染烟草中应用,14‑3‑3 h1基因序列为SEQ ID NO.1;烟草14‑3‑3 h1蛋白通过抑制Arf蛋白促进囊泡转运功能抑制TMV在细胞内的复制,在防御TMV侵染烟草中应用。本发明从分子学和基因工程上研究TMV侵染烟草的详细过程,在不依赖化控药物使用的情况下,寻求一种安全有效的防控感染方法,达到进一步对烟草花叶病进行早期预警和防治的目的。
Description
技术领域
本发明涉及基因工程领域,具体为烟草14-3-3 h1蛋白及其编码基因在TMV侵染烟草中的应用。
背景技术
14-3-3蛋白是所有真核细胞中均表达的一种高度保守的酸性蛋白家族,它通过与磷酸化的靶蛋白结合来行使功能。14-3-3蛋白在植物信号传递网络中扮演着重要的角色,不仅可以传递外界环境的信号,还能通过调节通路下游蛋白,引起一系列生理反应。植物14-3-3蛋白作为调控因子参与了植物的生物与非生物胁迫、初生代谢与营养代谢调控、激素信号传递和光信号应答等过程。
14-3-3蛋白的多样性,决定了其发挥功能的方式也多种多样,它不仅可参与调控蛋白在不同环境中的定位变化,还可作为激活因子或抑制因子调控靶蛋白的结构、活性以及稳定性;甚至还可以发挥与分子伴侣相似的功能协助蛋白跨膜转运。不同的14-3-3蛋白亚型具有不同的细胞特异性并通过识别特异的磷酸化序列与靶蛋白相互作用。当14-3-3蛋白与特异靶蛋白作用时,其构象发生变化,从而与靶蛋白紧密结合。总之这些植物14-3-3蛋白可以通过磷酸化模式与靶蛋白结合,还可以作为一种“支架”蛋白与靶蛋白的结构域相互作用,参与调控多种靶蛋白如转录因子、蛋白激酶和细胞调亡因子等的活性。从而在植物初级代谢、生长发育、生物和非生物的应答反应、气孔关闭的调控、油脂的合成等方面发挥着突出的作用。
烟草花叶病毒(Tobacco Mosaic Virus,TMV)是第一个被提取出来的植物病毒,属于烟草花叶病属。烟草花叶病毒在烟草栽培生产上普遍存在,并且是一种对植物危害很大的侵染性病毒,其引发的病害,在世界范围内普遍发生。烟草花叶病毒的寄主种类十分广泛,其可侵染藜科、茄科、葫芦科、豆科、商陆科和苋科等30多科植物,总共包括300多种植物。Arf蛋白是GTP结合蛋白Ras超家族的一个亚家族,高度保守。它在植物囊泡运输,内质网高尔基体形态维持,以及植物病毒RNA复制方面都发挥着重要的作用。
目前,烟草花叶病在我国大部分地区都有不同程度的发生,给国家和劳动者造成了巨大的收入损失。目前,对病毒病的控制和防治是一个一直不能突破的难题,虽然在基因工程和化学控制方面的研究有了一定的突破,但在生产实践中的应用还存在一定的瓶颈,尤其是化控造成的农业污染使得对化控药物的使用也有一定的限制。
因此,从分子学和基因工程上研究TMV侵染烟草的详细过程,达到进一步对烟草花叶病进行控制和防治的目的是本领域技术人员亟需解决的问题。
发明内容
本发明正是基于上述技术问题至少之一,从分子学和基因工程上研究TMV侵染烟草的详细过程,在不依赖化控药物使用的情况下,寻求一种安全有效的防控感染方法,达到进一步对烟草花叶病进行早期预警和防治的目的。
有鉴于此,根据本发明的第一个方向提出了烟草14-3-3 h1蛋白的编码基因14-3-3 h1在防御TMV侵染烟草中应用,14-3-3 h1基因序列为SEQ ID NO.1。
根据本发明的第二个方向提出了烟草14-3-3 h1蛋白抑制TMV在细胞内的复制,在防御TMV侵染烟草中应用,烟草14-3-3 h1蛋白抑制了Arf蛋白促进囊泡转运功能。
为实现上述目的,本发明采用的技术方案包括以下步骤:
(1)14-3-3 h1基因的克隆
(2)14-3-3 h1蛋白的亚细胞定位
(3)筛选14-3-3 h1蛋白的互作蛋白
(4)构建14-3-3 h1 RNAi转化植株检测靶蛋白表达
(5)检测感染TMV后对靶蛋白的影响
其中步骤(1)的具体方法如下:
利用从烟草叶片中提取的总RNA为模板进行反转录实验,根据烟草14-3-3 h1的编码区序列,设计特异性引物(引物的5’和3’端分别引入NdeI和SalI酶切位点)进行RT-PCR扩增,用高保真酶扩增出14-3-3 h1基因的目的片段,为777bp。将上述扩增得到的14-3-3 h1基因连接到PMD-19-T(simple)载体上,并将连接产物转入大肠杆菌DH5α感受态细胞中。其中14-3-3 h1基因序列如SEQ ID NO.1所示,引物如SEQ ID NO.2和SEQ ID NO.3所示。
14-3-3-F:CCCATATGGGAGAAACATCAATGGCGTCG;SEQ ID NO.2
14-3-3-R:GCGTCGACTCAATCCTAAAGAAATGTCACC;SEQ ID NO.3
将14-3-3 h1基因转化到毕赤酵母中获得14-3-3 h1蛋白。
其中步骤(2)的具体方法如下:
构建了14-3-3-GFP荧光表达载体,随后转入农杆菌EHA105,在本氏烟叶片中瞬时表达,48h后在激光共聚焦显微镜下观察。
根据14-3-3 h1蛋白的亚细胞定位,筛选与14-3-3 h1蛋白的互作蛋白,其中步骤(3)的具体方法如下:利用酵母双杂交筛选14-3-3 h1蛋白的互作蛋白
构建诱饵表达载体PGBKT7-14-3-3,转入酵母AH109感受态细胞,将已转入重组质粒PGBKT7-14-3-3的酵母AH109细胞与烟草cDNA文库融合培养(30℃,40rpm),20h后取一滴菌液在显微镜下观察,酵母AH109细胞发生融合繁殖后,继续培养4h,收集菌体并用0.5xYPDA液体培养基重悬,然后涂布SD/-His-Leu-Trp固体培养基,30℃培养7天。挑取培养基上形态良好的菌落溶解于0.9%的生理盐水中,吸取1μL点在SD/–Ade–His–Leu–Trp+X-α-Gal固体培养基上并在30℃下生长2天。将蓝色菌落用20μL0.9%的生理盐水溶解作为模板,随后用PGADT7载体通用引物3’AD和T7进行菌落PCR,并将没有杂带的PCR产物进行测序;测序结果在NCBI数据库中进行BLAST比对,挑选出四个克隆数目最多的基因进行后续实验。筛选出的四个阳性克隆分别为腺苷酸核糖基化作用因子(Arf),端锚聚合酶(Tankyrase),质体蓝素B'/B”(Pc),磷酸核酮糖羧化酶小亚基S41(RbCS)。
为了进一步验证四个筛选出的蛋白与烟草14-3-3 h1蛋白是否存在互作关系。采用Gateway技术分别构建了BIFC(14-3-3-cYFP,Arf-nYFP,Tankyrase-nYFP,Pc-nYFP和RbCS-nYFP)双分子荧光表达载体。将双分子荧光表达载体分别转化农杆菌EHA105,在本氏烟叶片中瞬时表达,48h后在激光共聚焦显微镜下观察荧光。
其中步骤(4)的具体方法如下:
采用LIC连接方式构建RNAi表达载体,将测序正确的pRNAi-14-3-3转化农杆菌EHA105,通过叶盘转化法进行烟草遗传转化,在黑暗条件下培养2-3天后转移至分化培养基,在光照条件下进行分化培养。待长出愈伤组织,直至出现幼芽;然后将幼芽切下转入生根培养基继续光照培养,直至生根完成移栽入土中成苗。
构建与烟草14-3-3 h1蛋白存在互作关系蛋白的瞬时表达载体,并将测序正确的瞬时表达载体转入农杆菌EHA105中,然后分别在野生型植株和RNAi转基因植株中瞬时表达,2天后取样并提取总蛋白进行Western Blot检测各蛋白表达情况。
其中步骤(5)的具体方法如下:
对正常生长的野生型和RNAi转基因植株中的与烟草14-3-3 h1蛋白存在互作关系靶蛋白基因表达进行检测。
后用TMV病毒提取液分别处理野生型植株和RNAi转基因植株,24h后取其与侵染TMV相邻上部叶作为样品,进行qPCR检测。
通过以上技术方案,本发明从分子学和基因工程上研究TMV侵染烟草的详细过程,在不依赖化控药物使用的情况下,寻求一种安全有效的防控感染方法,达到进一步对烟草花叶病进行控制和防治的目的。
附图说明
图1为14-3-3 h1蛋白的三维结构图;
图2为14-3-3 h1蛋白的亚细胞定位图;
图3为酵母AH109细胞融合繁殖图;
图4为菌落PCR电泳图;
图5为14-3-3 h1蛋白与Arf蛋白共定位图;
图6为RNAi转基因烟草PCR电泳图;
图7为瞬时表达载体菌液PCR电泳图;
其中M为BM2000 Marker;1、9和19为阴性对照;2-8为Arf-mCherry;10-18为Tankyrase-mCherry;20-26为RbCS-mCherry;
图8为Western Blot检测各蛋白表达图;
图9为野生型和RNAi转基因植株中的靶蛋白基因表达量;
图10为TMV侵染后的野生型和RNAi转基因植株中靶蛋白基因表达量。
具体实施方式
为了能够更清楚地理解本发明的上述目的为特征和优点,下面结合附图和具体实施方式对本发明进行进一步的详细描述。需要说明的是,在不冲突的情况下,本申请的实施例及实施例中的特征可以相互组合。
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是,本发明还可以采用其他不同于在此描述的其他方式来实施,因此,本发明的保护范围并不受下面公开的具体实施例的限制。
实施例1
14-3-3 h1基因的克隆
利用从烟草叶片中提取的总RNA为模板进行反转录实验,根据烟草14-3-3 h1的编码区序列,设计特异性引物(引物的5’和3’端分别引入NdeI和SalI酶切位点)进行RT-PCR扩增,用高保真酶扩增出14-3-3 h1基因的目的片段,为777bp。将上述扩增得到的14-3-3 h1基因连接到PMD-19-T(simple)载体上,并将连接产物转入大肠杆菌DH5α感受态细胞中。其中14-3-3 h1基因序列如SEQ ID NO.1所示,引物如SEQ ID NO.2和SEQ ID NO.3所示。
14-3-3-F:CCCATATGGGAGAAACATCAATGGCGTCG;SEQ ID NO.2
14-3-3-R:GCGTCGACTCAATCCTAAAGAAATGTCACC;SEQ ID NO.3
通过在线软件Sopma的预测分析得出,14-3-3 h1蛋白中含有α螺旋、无规则卷曲和β折叠延伸链结构。其中α螺旋结构占64.73%,相对较多;无规则卷曲占29.07%,次之;β折叠占6.20%,所占部分最少。由此可见α螺旋分布于整条肽链中,构成了14-3-3 h1蛋白的主要结构。通过在Swiss-Model中查找匹配,找到与14-3-3蛋白相似度最高的氨基酸序列,其PDB号为2o98A。然后在RCSB-PDB中搜索得到其三维结构如图1。
实施例2
14-3-3 h1蛋白的亚细胞定位
为了明确14-3-3 h1蛋白在细胞中的分布,以SpeI为酶切位点将载体线性化,与PCR产物进行37℃连接反应30min,连接产物转化大肠杆菌细胞,涂布抗性筛选培养基过夜培养。将构建好的14-3-3-GFP荧光表达载体,转入农杆菌EHA105中。本氏烟叶片在注射前光照5个小时,以使叶片气孔充分张开,利于注射。用无菌注射器针头轻轻划下表皮形成注射点。用2mL无菌注射器吸取适量菌液,以左手垫在注射部位下方,轻轻推进注射器进行注射。注射完的植株在黑暗环境中过夜培养后,再以16h光照,8h黑暗正常培养。处理48h后在激光共聚焦显微镜下观察。其中14-3-3 h1蛋白的亚细胞定位引物如SEQ ID NO.4和SEQ IDNO.5所示。
14-3-3-F:CCATGGTAGATCTGACTAGTATGGCGTCGCCACGCGAG;SEQ ID NO.4;
14-3-3-R:AAGTTCTTCTCCTTTACTAGTCTGCTGCTCATTATCTGG;SEQ ID NO.5。
结果显示如图2,由图2可知14-3-3 h1蛋白在细胞膜和细胞核上均有表达,体现了它在细胞中功能的多样性。
实施例3
筛选14-3-3 h1蛋白的互作蛋白
构建诱饵表达载体PGBKT7-14-3-3,转入酵母AH109感受态细胞,将已转入重组质粒PGBKT7-14-3-3的酵母AH109细胞与烟草cDNA文库融合培养(30℃,40rpm),20h后取一滴菌液在显微镜下观察,在视野中可以观察到三叶草结构如图3,由图3可知酵母AH109细胞已经发生融合繁殖。继续培养4h后,收集菌体并用0.5xYPDA液体培养基重悬,然后涂布SD/-His-Leu-Trp固体培养基,30℃培养7天。挑取培养基上形态良好的菌落溶解于0.9%的生理盐水中,吸取1μL点在SD/–Ade–His–Leu–Trp+X-α-Gal固体培养基上并在30℃下生长2天。将蓝色菌落用20μL0.9%的生理盐水溶解作为模板,随后用PGADT7载体通用引物3’AD和T7进行菌落PCR,结果如图4,将没有杂带的PCR产物进行测序;测序结果在NCBI数据库中进行BLAST比对,挑选出四个克隆数目最多的基因进行后续实验。比对结果见表1。
表1没有杂带的PCR产物测序比对表
由表1可知,筛选出的四个阳性克隆分别为腺苷酸核糖基化作用因子(Arf),端锚聚合酶(Tankyrase),质体蓝素B'/B”(Pc),磷酸核酮糖羧化酶小亚基S41(RbCS)。
为了进一步验证四个筛选出的蛋白与烟草14-3-3 h1是否存在互作关系。采用Gateway技术分别构建了BIFC(14-3-3-cYFP,Arf-nYFP,Tankyrase-nYFP,Pc-nYFP和RbCS-nYFP)双分子荧光表达载体。将双分子荧光表达载体分别转化农杆菌EHA105,在本氏烟叶片中瞬时表达,48h后在激光共聚焦显微镜下观察荧光。
其中引物如SEQ ID NO.6-SEQ ID NO.15所示。
14-3-3-F:CACCATGGCGTCGCCACGCGAG;SEQ ID NO.6;
14-3-3-R:TTACTGCTGCTCATTATCTGG;SEQ ID NO.7;
Arf-F:CACCATGGGTTTATCATTCGGG;SEQ ID NO.8;
Arf-R:TCAACCCTTGTTTGCAATG;SEQ ID NO.9;
Tankyrase-F:CACCATGGCAGTACAAAGGGG;SEQ ID NO.10;
Tankyrase-R:CTAACGGCCAAGTTCAGC;SEQ ID NO.11;
Pc-F:CACCATGGCCAGTGTAACCTCT;SEQ ID NO.12;
Pc-R:TAAAGAGGCTAATCAGAA;SEQ ID NO.13;
RbCS-F:CACCATGGCTTCCTCAGTTATG;SEQ ID NO.14;
RbCS-R:CGGAGATTTTAGTAGCCT;SEQ ID NO.15;
结果如图5,由图5可知腺苷酸核糖基化作用因子(Arf)与14-3-3 h1蛋白有荧光互补效应(黄色荧光);说明两个蛋白存在互作关系。
在本研究中,以ER/Golgi为Marker,当只有14-3-3 h1蛋白表达时,ER/GolgiMarker亚细胞定位形态较为稳定,且两个蛋白有共定位现象;当只与Arf蛋白共表达时,ER/Golgi Marker亚细胞定位形态较为活跃,出现弥散的点状结构;当14-3-3 h1蛋白与Arf蛋白发生互作,ER/Golgi Marker亚细胞定位形态也出现弥散状态,但弥散程度介于前两者之间。这一结果表明,14-3-3 h1蛋白的表达抑制了Arf蛋白对于细胞内囊泡运输的正向作用。烟草花叶病毒是一种正义链RNA病毒,它在宿主细胞的复制需要利用多种细胞器膜结合复合体,其中包含多种病毒自身和宿主的组分,而Arf蛋白对细胞内囊泡运输及蛋白转运的重要功能使其对TMV病毒在植物体内的复制也发挥着一定作用。14-3-3 h1蛋白抑制了Arf蛋白发挥其促进囊泡转运功能,即抑制了TMV在细胞内的复制。可见14-3-3 h1蛋白对抵抗TMV感染具有一定积极作用。对互作蛋白的深入研究,为解析14-3-3 h1蛋白发挥功能的机制提供了新的思路。
实施例4
构建14-3-3 h1 RNAi转化植株检测靶蛋白表达
采用LIC连接方式构建RNAi表达载体,将测序正确的pRNAi-14-3-3 h1转化农杆菌EHA105,通过叶盘转化法进行烟草遗传转化,在黑暗条件下培养2-3天后转移至分化培养基,在光照条件下进行分化培养。待长出愈伤组织,直至出现幼芽;然后将幼芽切下转入生根培养基继续光照培养,直至生根完成移栽入土中成苗。分别以转化株和野生株烟草叶片基因组DNA为模板,用带有载体片段和目标基因片段设计特异引物进行PCR检测。
引物如SEQ ID NO.16-SEQ ID NO.21所示;
LIC-1:CGACGACAAGACCCTAGAAACATCAATGGCGTCG;SEQ ID NO.16;
LIC-2:GAGGAGAAGAGCCCTCTGAGCGGATTTGTAAGC;SEQ ID NO.17;
LIC-3:CCAGCACGGAACCCTTGAGGAGAAGAGCCCT;SEQ ID NO.18;
LIC-4:AGAGCACACGACCCTTCGACGACAAGACCCT;SEQ ID NO.19;
Sequencing-F:TCTTCTTCGTCTTACACATC;SEQ ID NO.20;
Sequencing-R:AAGACCGGCAACAGGATTC;SEQ ID NO.21。
结果显示如图6,根据条带的亮度,得到15株RNAi转基因烟草。
构建Arf-mCherry,Tankyrase-mCherry,RbCS-mCherry瞬时表达载体,菌液PCR电泳结果如图7:由图7可知瞬时表达载体载体构建成功。将测序正确的瞬时表达载体转入农杆菌EHA105中,然后分别在野生型植株和RNAi转基因植株叶片中瞬时表达,2天后取样并提取总蛋白进行Western Blot检测各蛋白表达情况。结果显示如图8,由图8可知Arf和Tankyrase蛋白的表达量明显升高,说明14-3-3 h1蛋白对这两个靶蛋白是负调控;RbCS蛋白表达量并未发生显著变化。
实施例5
检测感染TMV后对靶蛋白的影响
对正常生长的野生型和RNAi转基因植株中的三个靶蛋白基因(Arf,Tankyrase,RbCS)表达进行检测。定量PCR检测引物如SEQ ID NO.22-SEQ ID NO.31所示;
14-3-3-F:GCTGCTACCGGCGATTCTAA;SEQ ID NO.22;
14-3-3-R:CTCGGCACCGGTCTTAAACT;SEQ ID NO.23;
Arf-F:TCTTCACTCTCTCCGCCAAC;SEQ ID NO.24;
Arf-R:TTTGAGAGCCAGTCAAGCCC;SEQ ID NO.25;
Tankyrase-F:TGGAGTTGGAGGAAGACCCT;SEQ ID NO.26;
Tankyrase-R:ATCTAGGGCAAGGCGAAAGG;SEQ ID NO.27;
RbCS-F:CCTGATTTGAGCCAGGAGCA;SEQ ID NO.28;
RbCS-R:AGACGAATCCGTGCTCAGTC;SEQ ID NO.29;
Actin-F:GGAAACATCGTCCTTAGTGGTG;SEQ ID NO.30;
Actin-R:AATCCAGACACTGAACTTGCG;SEQ ID NO.31。
结果如图9,由图9可知在转基因植株中,腺苷酸核糖基化作用因子(Arf)的表达量约为野生型的1.5倍,端锚聚合酶(Tankyrase)的表达量约为野生型的1.6倍,说明在抑制了烟草14-3-3 h1基因的表达以后,Arf和Tankyrase的基因表达量均有提高;而磷酸核酮糖羧化酶小亚基S41(RbCS)的表达量变化并不明显。
用TMV病毒提取液分别处理野生型植株和RNAi转基因植株,24h后取其与侵染TMV相邻上部叶作为样品,进行qPCR检测。结果如图10,由图10可知:在RNAi转基因植株中,Arf基因表达量约为2.0倍,Tankyrase基因表达量约为1.3倍,RbCS基因表达量约为2.0倍。14-3-3 h1 RNAi转化植株在受到TMV侵染24h以后三个靶蛋白基因的表达量均有上升。在14-3-3h1 RNAi转基因植株中,Arf蛋白的表达量上升,使得TMV更易在细胞内进行复制,植株更易感病。
Rubisco是植物C3途径中的关键酶,同时也是植物光呼吸的关键酶。它包含8个大亚基(RbCLS)和8个小亚基(RbCSs)。小亚基RbCS在细胞质中被翻译,随后转入到叶绿体中。在感病的本氏烟植株中,RbCS的沉默会导致叶片的坏死,从而使局部抗病性得到了提高,然而整体的病毒症状的发展是延迟的;且RbCS可以与烟草花叶病毒的运动蛋白(MP)在细胞质内发生互作,而在叶绿体中没有,因此RbCS与MP蛋白在细胞质内形成了蛋白复合物,从而阻断其进入叶绿体,阻碍光合作用的进行。在本研究中,用TMV病毒提取液分别对野生型植株和14-3-3 h1 RNAi转基因植株进行了处理,24h后取样进行荧光定量PCR检测,结果发现:病毒处理的RNAi转基因植株中该基因的表达量更高,推测其更利于病毒的感染。
而Tankyrase蛋白是一种位于端粒的聚腺苷二磷酸核糖聚合酶,与细胞的衰老,死亡密切相关。在结构上,一般由四部分组成:氨基端为HPS结构域;中心区域含24个锚蛋白重复区;之后有一个与SAM同源的区域;羧基端与聚腺苷二磷酸核糖聚合酶PARP。PARP是真核细胞中针对环境中和细胞内基因毒素引发DNA链断裂损伤的一个重要感受分子。而在对筛选出的Tankyrase蛋白序列结果分析过程中未发现PARP结构区域,却包含多个锚蛋白重复区,即ANK序列。ANK序列可以通过介导蛋白质间的相互作用来参与和细胞内信号传导、转录调控和发育调控等有关的相互作用。在ANK过表达的植株中,烟草花叶病毒的运动蛋白(MP)的移动得到了促进;然而在ANK抑制表达的植株中,MP蛋白的移动受到了抑制。并且含有ANK序列的蛋白质可以与MP蛋白互作,它的存在可以促进TMV通过胞间连丝在细胞间的转运,协助病毒在细胞间的扩散。在本研究中,14-3-3 h1 RNAi转基因植株中,Tankyrase的表达量上升,说明TMV在RNAi转基因更易利用ANK的协助来完成在细胞间的迁移,病毒更容易在RNAi转基因植株中扩散。
以上仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改为等同替换为改进等,均应包含在本发明的保护范围之内。
序列表
<110> 河南农业大学
<120> 14-3-3 h1蛋白在TMV侵染烟草叶片中的应用
<160> 31
<170> SIPOSequenceListing 1.0
<210> 1
<211> 521
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gcttacaaat ccgctcagga tattgcaaat accgagcttg ctcctacgca tccaattcga 60
ttgggacttg ctctcaattt ctctgtattc tactacgaga ttttgaattc acctgatcgt 120
gcttgtaatc tcgccaaaca ggcctttgat gaggccattg ctgagctgga caccttgggc 180
gaagagtcct acaaggatag cactctgatc atgcagcttc ttcgcgataa cctcacttta 240
tggacttcag atatgcagga tgatggaact gatgagatca aagaagcagc aaaaccagat 300
aatgagcagc agtaaactgg tgacatttct ttaggattga actgccatgt tgtaactttt 360
tatttttcaa ttgtctgagt tcagctcttt tagttctaga tcttatgatt tgtaacacct 420
aaaacaactg tttcttgtta tttgttgctt ttgtttgttt ctttgtggat ttatcttgta 480
tttggataat ttcctttttc tcaaaggaat gacttattgg g 521
<210> 2
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cccatatggg agaaacatca atggcgtcg 29
<210> 3
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcgtcgactc aatcctaaag aaatgtcacc 30
<210> 4
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccatggtaga tctgactagt atggcgtcgc cacgcgag 38
<210> 5
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aagttcttct cctttactag tctgctgctc attatctgg 39
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
caccatggcg tcgccacgcg ag 22
<210> 7
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttactgctgc tcattatctg g 21
<210> 8
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caccatgggt ttatcattcg gg 22
<210> 9
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tcaacccttg tttgcaatg 19
<210> 10
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caccatggca gtacaaaggg g 21
<210> 11
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ctaacggcca agttcagc 18
<210> 12
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
caccatggcc agtgtaacct ct 22
<210> 13
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
taaagaggct aatcagaa 18
<210> 14
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caccatggct tcctcagtta tg 22
<210> 15
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cggagatttt agtagcct 18
<210> 16
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
cgacgacaag accctagaaa catcaatggc gtcg 34
<210> 17
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gaggagaaga gccctctgag cggatttgta agc 33
<210> 18
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ccagcacgga acccttgagg agaagagccc t 31
<210> 19
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
agagcacacg acccttcgac gacaagaccc t 31
<210> 20
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tcttcttcgt cttacacatc 20
<210> 21
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
aagaccggca acaggattc 19
<210> 22
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gctgctaccg gcgattctaa 20
<210> 23
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ctcggcaccg gtcttaaact 20
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tcttcactct ctccgccaac 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tttgagagcc agtcaagccc 20
<210> 26
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
tggagttgga ggaagaccct 20
<210> 27
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
atctagggca aggcgaaagg 20
<210> 28
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
cctgatttga gccaggagca 20
<210> 29
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
agacgaatcc gtgctcagtc 20
<210> 30
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ggaaacatcg tccttagtgg tg 22
<210> 31
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
aatccagaca ctgaacttgc g 21
Claims (2)
1.烟草14-3-3h1蛋白的编码基因14-3-3h1在防御TMV侵染烟草中应用,其特征在于,所述基因序列为SEQ ID NO.1。
2.烟草14-3-3h1蛋白通过抑制TMV在细胞内的复制在防御TMV侵染烟草中应用,其特征在于,烟草14-3-3h1蛋白抑制了Arf蛋白促进囊泡转运功能。
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Citations (4)
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WO2009037329A2 (en) * | 2007-09-21 | 2009-03-26 | Basf Plant Science Gmbh | Plants with increased yield |
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CN103194456A (zh) * | 2013-04-24 | 2013-07-10 | 昆明理工大学 | 岷江百合抗真菌基因Lr14-3-3及其应用 |
CN107699575A (zh) * | 2017-09-04 | 2018-02-16 | 沈阳农业大学 | 番茄14‑3‑3蛋白tft6基因的酵母双杂交载体的构建 |
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2020
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WO2009037329A2 (en) * | 2007-09-21 | 2009-03-26 | Basf Plant Science Gmbh | Plants with increased yield |
US20120198585A1 (en) * | 2010-12-31 | 2012-08-02 | Shunyuan Xiao | Enhancing drought tolerance and bacterial resistance of crop species by functional interference of 14-3-3 |
CN103194456A (zh) * | 2013-04-24 | 2013-07-10 | 昆明理工大学 | 岷江百合抗真菌基因Lr14-3-3及其应用 |
CN107699575A (zh) * | 2017-09-04 | 2018-02-16 | 沈阳农业大学 | 番茄14‑3‑3蛋白tft6基因的酵母双杂交载体的构建 |
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KONAGAYA,K ET AL.: "Nicotiana tabacum mRNA for 14-3-3 h-2 protein, complete cds,GenBank: AB119479.1", 《GENBANK》 * |
PREM PRAKASH DAS ET AL.: "Comparative proteomics of Tobacco mosaic virus-infected Nicotiana tabacum plants identified major host proteins involved in photosystems and plant defence", 《JOURNAL OF PROTEOMICS》 * |
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