Name: potato virus x-based microrna silencing (vbms) in potato.
Uploaded: 2020-05-11T13:30:00.000+00:00
Duration: 11 min 51 s
Description: Watch this Scientific Journal Video about Potato Virus X-Based microRNA Silencing VbMS In Potato. at JoVE.com

我们感谢清华大学的廖玉乐博士提供PVX-LIC载体。这项工作得到了德克萨斯州A&M AgriLife Research的启动基金和美国农业部国家食品和农业研究所的Hatch Project TEX0-1-9675的支持。

1. 种植马铃薯植物。
<ol><li>用Murashige和Skoog（MS）培养基加上Gamborg维生素（MS基盐混合物，Gamborg维生素，30g / L蔗糖，3.5 g / L琼脂，pH = 5.7）在培养管（25 x 150 mm）中繁殖马铃薯植物。将试管置于生长室中，温度为20-22°C，16小时光照/ 8小时黑暗光周期，光强度为120μmol/ m2∙s1。 注意:新芽和根通常在植物的1-2周内发育。每月用新鲜的MS / Gamborg维生素培养基繁殖植物。</li>
	<li>四周后，将体外植物移植到土壤中，并在20-22°C，16小时光照/ 8小时黑暗光周期和光强度120μmol/ m2∙s1下的温室中生长。 注:根叶新长的植物适合移栽。在前3-4天内保持新鲜移植植物的水分。</li>
</ol>2. 构造 VbMS 向量。
<ol><li>设计并克隆短串联TM分子（STTM， 图1）22，53 用于感兴趣的miRNA。 注:根据实验数据或从miRbase数据库获取miRNA序列54，55，56，57，58，59。本研究中使用的miR166序列之前已经描述过60。<ol>
<li>通过将不匹配序列（通常为5'-CTA-3'）插入到miRNA的反向补体序列中来设计TM模块，该序列位于与miRNA的第10-11个 核苷酸相对应的位点。 注意:例如，Stu-miR160序列是5'-UGCCUGGCUCCCUGUAUGCC-3'61，其中第10-11个核苷酸以粗体显示。反向补体序列（在脱氧核苷酸中）为5'-GGCATACAGGGAGCCAGGCA-3'，不匹配凸起插入位点以粗体显示。TM分子序列（脱氧核苷酸）应为5'-GGCATACAGG-CTA-GAGCCAGGCA-3'。</li>
		<li>设计用于克隆STM片段的引物（图1）。使用具有 48-nt 间隔序列的 DNA 寡核苷酸作为 PCR 克隆的模板。正向引物由LIC1连接子（5'-CgACgACAAgACCgT-3'），上述为TM分子设计的正向序列以及48-nt间隔物的部分5'序列（5'-GTTTTGTTGTTATGGT-3'）组成。反向引物由LIC2连接子（5'-gAggAgAagAgCCgT-3'），TM分子的反向补体序列和48-nt间隔物（5'-ATTCTTCTTCTTTAGACCAT-3'）的3'序列的部分反向补体组成。 注意:48-nt间隔序列是5'-GTTGTTGTTGTGTTCTAATTTAAATAATATGGTCTAAGAAGAAGAAT-3'。例如，对于STTM-miR160，前向引物应为5'-CgACgACAAgACCgT-GGCATACAGG-CTA-GAGCCAGGCA-GTTGTTGTTGTTGTTGGT-3';反向引物应为5'-gAggAgAagAgCCgT-TGCCTGGCTC-TAG-CCTGTATGCC-ATTCTTCTTCTTTAGACCAT-3'（图1）。</li>
		<li>使用合成的通用48-nt垫片作为模板和高保真DNA聚合酶，通过PCR扩增50μL体积的STTM片段。<ol>
<li>通过将每个引物0.5 μL（40 μM）、0.5 μL48-nt间隔物（40 μM）、5 μL 10x PCR缓冲液、1 μL dNTP混合物（每个10 mM）、0.1 μL高保真DNA聚合酶（10 U/μL）和43 μL ddH2O混合至总体积为50 μL来设置PCR反应。 94°C 3分钟，32次循环94°C持续45秒，60°C循环45秒，72°C循环60秒。</li>
		</ol>
</li>
		<li>通过乙醇沉淀纯化STTM片段。向PCR产物中加入2.5体积的乙醇和1/10体积的3M乙酸钠（pH = 4.0）。剧烈混合并以14，000× g 离心10分钟。除去上清液并用1mL 70%乙醇冲洗沉淀。干燥沉淀并将其溶解在20μL的ddH 2 O中。 注:使用DNA电泳检查STTM PCR产物的扩增。</li>
		<li>通过将2.5μL纯化的STTM PCR产物，0.5μL10x T4 DNA聚合酶缓冲液，0.05μL1M二硫脑油醇（DTT），0.25μL100mM dATP，0.1μLT4 DNA聚合酶（3 U / μL）和1.6μLdH2O混合至总体积为5μL，在冰上建立T4 DNA聚合酶反应。 并在75°C下处理产物20分钟以灭活T4 DNA聚合酶。</li>
	</ol></li>
	<li>准备基于 PVX 的 VbMS 构造。<ol>
<li>在100μL的体积中消化5μgPVX-LIC质粒38和2.5μLSmaI（20 U / μL）。 </li>
		<li>向消化后的PVX-LIC产品中加入等体积的苯酚:氯仿:异丙醇（25:24:1，pH = 6.7/8.0）并剧烈混合。以14，000× g 离心10分钟，并将上清液转移到新的离心管中。加入等体积的氯仿:异丙醇（24:1）并剧烈涡旋。以14，000× g 离心10分钟。 注:PVX-LIC 载体包含用于克隆的 LIC 盒。PVX-LIC载体的LIC盒含有 ccdB 基因和耐氯霉素基因，需要使用含有卡那霉素（50μg/ L）和氯霉素（15μg/ L）的LB培养基在 大肠杆菌 菌株DB3.1中维持/繁殖。</li>
		<li>将上清液转移到新的离心管中。加入2.5体积的乙醇和1/10体积的3M乙酸钠（pH = 4.0）并剧烈混合。以14，000× g 离心10分钟，除去上清液。</li>
		<li>用1mL 70%乙醇冲洗沉淀并剧烈涡旋。以14，000× g 离心10分钟，除去上清液。干燥沉淀并用100μLddH 2O溶解消化的PVX-LIC质粒。</li>
		<li>通过将2.5μL消化的PVX-LIC载体DNA，0.5μL10x T4 DNA聚合酶缓冲液，0.05μL1 M DTT，0.25μL100mM dTTP和0.1μLT4 DNA聚合酶（3 U / μL）混合在冰上建立T4 DNA聚合酶反应。将混合物在37°C下孵育15分钟，并在75°C下处理产物20分钟至20分钟灭活T4 DNA聚合酶。</li>
	</ol></li>
	<li>使用 LIC 反应将 STTM 序列克隆到 PVX-LIC 载体中。混合T4 DNA聚合酶处理的STTM PCR产物（5μL）和T4 DNA聚合酶处理的PVX-LIC质粒（5μL）。在70°C下孵育5分钟，以0.1°C / s的斜坡冷却至22°C，并使用PCR机器在22°C下保持30分钟。 注意:将孵育时间延长至4°C过夜，以实现更高的LIC克隆效率。</li>
	<li>将5μL的LIC反应产物转化为 大肠杆菌 DH5α，并在含有50μg/ mL卡那霉素的LB板上生长62，63。通过PCR选择阳性菌落，并使用PVX-LIC载体的克隆引物和通用引物，然后进行测序。<ol>
<li>使用 PVX-LIC 正向引物 （5'-GTGTTGGCTTGCAAACTAGAT-3'） 结合用于 STTM 克隆的反向引物进行菌落 PCR，以鉴定阳性克隆。PCR条带的大小应为~300 bp。 注:通过终止器循环测序验证STTM片段的序列64，65。</li>
	</ol></li>
	<li>从经过验证的克隆中分离PVX-STTM质粒，并将其转化为农杆菌菌株GV3101，GV2260或EHA10562，63。通过 PCR 验证农杆菌菌落。 注:使用 PVX-LIC 的正向引物和 STTM 克隆的反向引物通过 PCR 确认 农杆菌菌 落。</li>
</ol>3. 在马铃薯植物中执行基于 PVX 的 VbMS 测定。
<ol><li>将4周龄的体外马铃薯植物移植到土壤中。移植的植物将在3-4天后进行VbMS测定。</li>
	<li>用含有PVX-STTM质粒的 农杆菌 接种马铃薯植物。 注意:对于马铃薯中的VbMS测定，同时进行 农杆菌介导的浸润和牙签刮擦接种。<ol>
<li>将含有 PVX-STTM 载体的阳性转化剂选取并接种到含有 50 μg/mL 卡那霉素和 50 μg/mL 利福平的 50 mL 液体 LB 中。在28°C培养箱中以220rpm生长16小时，直到OD 600 = 1.0。</li>
		<li>同时，将阳性的农杆菌菌落划到至少两个含有50μg/ mL卡那霉素和50μg/ mL利福平的新LB板上，并在28°C下生长1天。包括 PVX-LIC38，66 载体作为对照，将 PVX-GFP67，68 作为监测病毒传播。</li>
		<li>通过在室温下以3，400×g离心10分钟收集农杆菌液体培养物。重悬具有等体积浸润缓冲液（10 mM MgCl2，10 mM MES和200μM乙酰汗根，pH = 5.6）的农杆菌细胞，并调节至OD 600 = 1.0。在室温下孵育6小时。</li>
		<li>用1mL无针注射器将 农杆菌 培养物浸润到完全膨胀的叶子的轴侧。<ol>
<li>翻转并用一只手握住叶子，然后用一根手指从浸润部位的轴侧支撑叶片。用另一只手将注射器垂直到叶表面，并将 农杆菌 培养物浸入层的轴侧。</li>
		</ol>
</li>
		<li>从LB板中刮下 农杆菌培养物 ，并用牙签刮擦浸润的马铃薯植物的前一个或两个节间的茎表面。轻轻划伤茎的表皮。避免刺穿茎，这可能会对植物造成严重损害。</li>
	</ol>
</li>
	<li>在温室中以16小时光照/ 8小时黑暗光周期和120μmol/ m2∙s1 的光强度在22°C下生长浸润的植物。 注意:由miRNA沉默引起的表型通常出现在接种后2-4周（图2， 图3）。浸润后 VbMS 表型通常需要 10–20 天才能出现。VbMS表型取决于特定miRNA和靶基因的特性、生长条件和马铃薯品种。</li>
</ol>4. 执行表达式分析。
<ol><li>当表型出现在接种后2-4周时，用剪刀从VbMS植物中收集具有表型的组织，例如芽，叶，花或根，以及来自对照植物的组织。从收集的组织中分离总RNA。</li>
	<li>通过电泳检查RNA质量62，63 ，并通过用分光光度计测量OD260 吸光度来量化RNA浓度。</li>
	<li>使用茎环实时逆转录聚合酶链反应 （RT-PCR） 分析 miRNA 表达。<ol>
<li>对于特定的miRNA，设计一个茎环逆转录引物。茎环RT引物包含一个通用的5'主链和一个3'6-nt的特异性miRNA延伸。设计一个5'通用骨架（5'-GTCTCCTCTGGTGCagggtccgaggtattcGCACCAGAGGAGAC-3'），形成茎环结构。（大写对应于反向重复序列，小写对应于循环区域）。</li>
		<li>形成环路的5'骨架序列的一部分用作后续PCR扩增的反向引物（骨架序列中的粗体斜体序列）。向茎环引物添加一个与感兴趣的miRNA的3'端反向互补的6-nt延伸序列，以提供逆转录的特异性（补充图1A）。 注意:设计Chen等人69和Erika Varkonyi-Gasic等人描述的茎环逆转录引物，如70，71，72所述。例如，Stu-miR160的茎环逆转录引物被设计为5'-GTCTCCTCTGGTGCagggtccggtattcGCACCAGAGGAGACGGCATA-3'。马铃薯miR165/166家族的茎环逆转录引物被设计为5'-GTCTTCTGGTGCagggtccgaggtattcGCACCAGAGGAGACGGGGGG（A/G）A-3'。（粗体大写序列提供了特定miRNA逆转录的特异性）。</li>
		<li>通过在冰上混合50-200ng总RNA，1μL茎环逆转录引物（100μM），2μL10x缓冲液，0.2μLRNase抑制剂（40 U / μL），0.25μLdNTPs（每个10mM）和1μL逆转录酶（200 U / μL）来设置逆转录反应。将不含核酸酶的ddH 2 O加入到20μL的总体积中。</li>
		<li>使用脉冲逆转录程序执行逆转录。将逆转录反应混合物在16°C孵育30分钟，进行30°C的温度循环30秒，42°C30 s和50°C共1 s的温度循环，共60个循环，并通过在85°C下孵育5分钟来灭活逆转录酶。</li>
		<li>对于miRNA表达的实时PCR分析，请根据miRNA序列设计正向引物，但不包括与上述设计的茎环逆转录引物重叠的序列。在前向引物的5'处添加3-7-nt延伸，以优化长度，熔化温度和GC含量（补充图1）。 注意:通用反向引物是5'-GTGCAGGGTCCGAGGT-3'。例如，Stu-miR160的茎环逆转录引物被设计为5'-CGGCTGCCTGGCTCC-3'。miR165/166家族的茎环逆转录引物是5'-CGGCTCGGACCAGGCTT-3'。粗体序列用作引物优化的5'扩展。用于miRNA的茎环逆转录引物和实时荧光定量PCR引物可以使用miRNA引物设计工具73进行设计。</li>
	</ol></li>
	<li>通过标准逆转录聚合酶链反应 （RT-PCR） 合成靶标 mRNA 的 cDNA。将逆转录反应混合物在37°C下孵育60分钟，并通过加热至85°C灭活逆转录酶5分钟。 注意:（1）如果靶标mRNA未知，则使用psRNA靶向程序74 预测靶标mRNA。（2）mRNA的通用逆转录引物是锚定引物5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT</li>
	<li>为目标 miRNA 和靶 mRNA 设置实时荧光定量 PCR 反应。在冰上混合0.5μL模板cDNA，5μL2x实时PCR缓冲液与SYBR绿色，0.05μL每个正向和反向引物（40μM）和4.4μLdH 2 O，总体积为5μL。</li>
	<li>在95°C下孵育3分钟，在95°C下循环40次3秒，60°C孵育30秒。执行随后的熔融曲线分析，如下所示:在95°C下孵育15 s，在20°C / s的斜坡下冷却至60°C，在60°C下保持60 s，在0.2°C / s的斜坡上加热至95°C，并在95°C下保持15 s。使用ΔΔCt方法分析Ct值76，77并绘制具有标准误差的均值。 注:（1）靶标mRNA的实时荧光定量PCR引物可以按所述设计75。（2）马铃薯 多泛素10 基因可作为马铃薯正常化的内部对照。马铃薯 多泛素10 基因的前向引物为5'-ATGTTGCCTTTTTTGTGTGGTTG-3'，反向引物为5'-TTATTTATTCACATAACACGACAGTC-3'。（3）对于实时荧光定量PCR分析，污染和引物二聚体的形成可能会产生假阳性结果。为了监测非特异性扩增并增加实时荧光定量 PCR 分析的责任，建议在实时荧光定量 PCR 测定中包括不带模板和逆转录酶的对照。</li>
</ol>

我们提出了一种基于PVX的miRNA沉默系统，通过将STTM设计整合到PVX载体中来表征马铃薯中内源性miRNA的功能。VbMS系统被证明可以有效地沉默马铃薯中的miRNA165/166，马铃薯是跨植物物种的高度保守的miRNA家族。
TM方法已被开发用于干扰基于人工miRNA靶标模拟的miRNA的表达，该拟态旨在在miRNA互补序列内的预期切割位点处创建不匹配环，从而导致靶向miRNA的封存并阻止其活性<sup class="xr...

植物微RNA （miRNA） 的特征是 20–24 个核苷酸长、核编码的调节性 RNA1 ，在植物生物过程的几乎每个方面都起着重要作用，包括生长和发育2、3、光合作用和代谢4、5、6、7、激素合成和信号传导8、9、生物和非生物反应10，11，12，13，以及营养和能量调节14，15。植物miRNA的调节作用是良好编程的，通常在转录后水平上通过切割或翻译抑制靶标mRNA来实现。
在马铃薯中miRNA的鉴定、转录谱分析和靶标预测方面取得了巨大进展16、17、18、19、20、21。然而，由于缺乏高效和高通量的遗传方法，包括马铃薯在内的植物中miRNA的功能表征落后于其他生物体。通过标准功能丧失分析对单个miRNA进行功能分析具有挑战性，因为大多数miRNA属于具有相当遗传冗余的家族22。此外，单个miRNA可以控制多个靶基因23 ，几个不同的miRNA可以协同调节相同的分子途径24，25。这些特性使得难以表征特定miRNA或miRNA家族的功能。
miRNA的大部分功能分析在很大程度上依赖于具有明显局限性的功能增益方法。人工miRNA（amiRNA）方法利用内源性原代转录本（pri-miRNA）在高水平上产生miRNA，从而抑制靶基因表达26，27，28，29。然而，使用强本构35S启动子的活化标记和miRNA过表达通常会导致miRNA的表达升高，这些表达不代表体内条件，因此可能无法反映miRNA30的内源性功能。已经开发了一种替代方法，涉及表达在结合和/或切割位点中含有不易脱位突变的靶基因的miRNA抗性形式31，32，33。但是，由于转基因伪影，这种方法也可能导致对源自miRNA耐药靶基因的表型的误解。因此，应谨慎从这些功能获得研究中得出结论34。上述方法的另一个主要局限性是它们需要转换，这是劳动密集型和耗时的。此外，转基因依赖性方法几乎不适用于顽固的植物物种。因此，必须开发一种快速有效的功能丧失方法来解开miRNA的功能。
为了绕过转化过程的先决条件，通过将靶向模拟（TM）策略与病毒衍生载体相结合，建立了基于病毒的microRNA沉默（VbMS）。在VbMS系统中，人工设计的TM分子从病毒骨架瞬时表达，为解剖植物内源性miRNA的功能提供了一种功能强大，高通量和省时的工具35，36。VbMS最初是在边 楸 和番茄中与烟草拨浪鼓病毒（TRV）35，36，37 一起开发的，并且已经扩展到拟南芥，棉花，小麦和玉米，使用各种其他病毒表达系统，包括马铃薯病毒X（PVX）38，棉花屑病毒（ClCrV）39，黄瓜花叶病毒（CMV）40，41，42，中国小麦花叶病毒（CWMV）43和大麦条纹花叶病毒（BSMV）44，45。
马铃薯（Solanum tuberosum）是世界上第四大粮食作物，也是种植最广泛的非藜麦作物，主要是因为它具有高营养价值、高能量产量和相对较低的投入要求46。马铃薯的几个特点使其成为一种有吸引力的双子叶植物模型植物。它是一种营养繁殖的多倍体作物，具有高越交率，杂合性和遗传多样性。然而，迄今为止，还没有报告使用VbMS表征马铃薯中miRNA的功能。在这里，我们提出了一种基于结扎独立克隆（LIC）的马铃薯PVX的VbMS方法来评估miRNA在马铃薯植物中的功能38。我们选择了miR165/166家族来说明VbMS测定，因为miR165/166家族及其靶标mRNA和III类同源域/Leu拉链（HD-ZIP III）转录因子已被广泛表征22，47，48。 HD-ZIP III 基因是分生组织发育和器官极性的关键调节因子，miR165/166功能的抑制导致 HD-ZIP III 基因表达增加，导致顶端优势降低和叶极性异常模式等多向性发育缺陷22，35，38，41.与miRNA165/166沉默相关的易于评分的发育表型能够准确评估基于PVX的VbMS测定的有效性。
在这项研究中，我们证明了基于PVX的VbMS系统可以有效地阻断马铃薯中miRNA的功能。由于基于PVX的病毒诱导基因沉默（VIGS）系统已经在许多马铃薯品种中建立49，50，51，52，因此这种基于PVX的VbMS方法可能适用于广泛的二倍体和四倍体马铃薯品种。

<table><tbody><tr><td>100 &amp;micro;M dATP and 100 &amp;micro;M dTTP</td><td>Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA</td><td>TQAC136</td><td></td></tr><tr><td>3 M Sodium acetate, pH 4.0.</td><td>Teknova, Hollister, CA 95023, USA</td><td>#S0297</td><td></td></tr><tr><td>Acetosyringone</td><td>TCI America, Portland, OR 97203, USA</td><td>D2666-25G</td><td></td></tr><tr><td>Agrobacterium tumefaciens strains: GV3101, GV2260 or EHA105.</td><td></td><td></td><td></td></tr><tr><td>Chloroform</td><td>VWR Corporate, Radnor, PA 19087-8660, USA</td><td>VWRV0757-950ML</td><td></td></tr><tr><td>Dimethyl sulfoxide, DMSO</td><td>TCI America, Portland, OR 97203, USA</td><td>D0798-25G</td><td></td></tr><tr><td>DTT</td><td>VWR Corporate, Radnor, PA 19087-8660, USA</td><td>VWRV0281-25G</td><td></td></tr><tr><td>E. coli DB3.1</td><td></td><td></td><td>for maintenance of PVX-LIC and pTRV2e containing the ccdB gene</td></tr><tr><td>E. coli DH5&amp;alpha;</td><td></td><td></td><td>for the destination constructs generated by LIC cloning</td></tr><tr><td>Fertilizer: Peters Peat Lite Special 15-0-15 Dark Weather Feed</td><td>ICL Specialty Fertilizers, Summerville, SC 29483, USA</td><td>G99260</td><td></td></tr><tr><td>High fidelity PCR reagents: KAPA HiFi DNA Polymerase with dNTPs</td><td>Roche Sequencing and Life Science, Kapa Biosystems,&lt;br/&gt; Wilmington, MA, USA</td><td>7958960001</td><td></td></tr><tr><td>Isoamyl alcohol</td><td>VWR Corporate, Radnor, PA 19087-8660, USA</td><td>VWRV0944-1L</td><td></td></tr><tr><td>Koptec Pure Ethanol &amp;ndash; 200 Proof</td><td>Decon Labs, King of Prussia, PA 19406 , USA</td><td>V1005M</td><td></td></tr><tr><td>MES</td><td>TCI America, Portland, OR 97203, USA</td><td>M0606-250G</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>ThermoFisher, Waltham, MA 02451, USA</td><td>MFCD00149781</td><td></td></tr><tr><td>M-MuLV Reverse Transcriptase</td><td>New England BioLabs, Ipswich, MA 01938-2723 USA</td><td>M0253L</td><td></td></tr><tr><td>Nano-drop spectrometer: NanoDrop One&lt;sup&gt;C&lt;/sup&gt; Microvolume UV-Vis Spectrophotometer with Wi-Fi</td><td>ThermoFisher, Waltham, MA 02451, USA</td><td>ND-ONEC-W</td><td></td></tr><tr><td>PCR machine: Bio-Rad MyCycler PCR System</td><td>Bio-Rad, Hercules, California 94547, USA</td><td>170-9703</td><td></td></tr><tr><td>PCR machine: Eppendorf Mastercycler pro</td><td>Eppendorf, Hauppauge, NY 11788, USA</td><td>950030010</td><td></td></tr><tr><td>pH meter</td><td>Sper Scientific, Scottsdale, AZ 85260, USA</td><td>Benchtop pH / mV Meter - 860031</td><td></td></tr><tr><td>Phenol:chloroform:isoamyl alcohol (25:24:1), pH 6.7/8.0.</td><td>VWR Corporate, Radnor, PA 19087-8660, USA</td><td>VWRV0883-400ML</td><td></td></tr><tr><td>Phytagel: Gellan Gum</td><td>Alfa Aesar, Tewksbury, MA 01876, USA</td><td>J63423-A1</td><td></td></tr><tr><td>PVX VIGS vector: PVX-LIC</td><td></td><td></td><td>Zhao et al., 2016</td></tr><tr><td>Real-time PCR machine: QuantStudio 6 Flex Real-Time PCR System</td><td>ThermoFisher, Waltham, MA 02451, USA</td><td>4485697</td><td></td></tr><tr><td>Real-time PCR reagent: KAPA SYBR&amp;reg; FAST qPCR Master Mix (2x) Kit</td><td>Roche Sequencing and Life Science, Kapa Biosystems,&lt;br/&gt; Wilmington, MA 01887, USA</td><td>7959389001</td><td></td></tr><tr><td>Restriction enzyme: SmaI</td><td>New England BioLabs, Ipswich, MA 01938-2723 USA</td><td>R0141S</td><td></td></tr><tr><td>Reverse transcription reagents: qScript cDNA SuperMix</td><td>Quanta BioSciences, Gaithersburg, MD 20877 , USA</td><td>95107-100</td><td></td></tr><tr><td>RNA extraction Kit: E.Z.N.A. Plant RNA Kit</td><td>Omega Bio-tek, Inc., Norcross, Norcross, GA 30071 , USA</td><td>SKU: D3485-01</td><td></td></tr><tr><td>RNase Inhibitor Murine</td><td>New England BioLabs, Ipswich, MA 01938-2723 USA</td><td>M0314L</td><td></td></tr><tr><td>RNAzol RT</td><td>Sigma-Aldrich, St. Louis, MO 63103, USA</td><td>R4533</td><td></td></tr><tr><td>Soil: Metro-Mix 360</td><td>Sun Gro Horticulture, Agawam, MA 01001-2907, USA</td><td>Metro-Mix 360</td><td></td></tr><tr><td>T4 DNA polymerase and buffer</td><td>New England BioLabs, Ipswich, MA 01938-2723 USA</td><td>M0203S</td><td></td></tr></tbody></table>

potato virus x-based microrna silencing (vbms) in potato.

基于病毒的microRNA沉默（VbMS）是一种快速有效的工具，用于植物中microRNA（miRNA）的功能表征。VbMS系统已被开发并应用于各种植物物种，包括 Nicotiana benthamiana，番茄，拟南芥，棉花和单子叶植物，如小麦和玉米。在这里，我们描述了一个详细的方案，使用基于PVX的VbMS载体来沉默马铃薯中的内源性miRNA。为了降低特定miRNA的表达，设计了目标miRNA的靶向模拟（TM）分子，将其整合到植物病毒载体中，并通过 农杆菌 浸润在马铃薯中表达，以直接与感兴趣的内源性miRNA结合并阻断其功能。

图2 显示了PVX-STTM165/166马铃薯植株（Katahdin），其叶组织从叶片的轴侧沿叶脉异位生长。还观察到更严重的表型，如喇叭形叶的形成。相比之下，在PVX控制工厂中没有观察到表型异常。这些结果表明，VbMS系统可有效抑制四倍体马铃薯植株的内源性miRNA功能，PVX-VbMS系统是确定特定miRNA或miRNA家族功能的强大遗传工具。
图3 显示了PVX-STTM...

Watch this Scientific Journal Video about Potato Virus X-Based microRNA Silencing VbMS In Potato. at JoVE.com

Potato Virus X-Based microRNA Silencing VbMS In Potato.

我们提出了基于马铃薯病毒X（PVX）的microRNA沉默（VbMS）系统的详细方案，以功能性地表征马铃薯中的内源性microRNA（miRNA）。将目标miRNA的靶向拟态（TM）分子整合到PVX载体中，并在马铃薯中瞬时表达，以沉默靶miRNA或miRNA家族。

马铃薯病毒基于马铃薯的X型微RNA沉默（VbMS）。

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

potato-virus-x-based-microrna-silencing-vbms-in-potato

Research

JoVE Journal

Environment

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

3.1K 次观看.  Texas A&M University System. 我们提出了基于马铃薯病毒X（PVX）的microRNA沉默（VbMS）系统的详细方案，以功能性地表征马铃薯中的内源性microRNA（miRNA）。将目标miRNA的靶向拟态（TM）分子整合到PVX载体中，并在马铃薯中瞬时表达，以沉默靶miRNA或miRNA家族。

视频: Potato Virus X-Based microRNA Silencing VbMS In Potato.

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

科研

生物工程

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

农杆菌和根瘤菌--马铃薯介导的马铃薯转化与葡萄染色诱导素基因的启动子活性

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

环境科学

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

植物病原体分泌效应器中RNA沉默抑制器的筛选和鉴定

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

遗传学

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

免疫与感染

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

生物学

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

生物化学

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

小核糖核酸病毒取向的微小RNA基于调控

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

马铃薯和尼科蒂亚娜本塔米亚纳的农业渗透和PVX农业感染

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

优化和利用<em&gt;农杆菌</em&gt;介导的瞬态蛋白生产中<em&gt;烟草</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22)&#160;or&#160;phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus&#160;and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.

马铃薯病毒基于马铃薯的X型微RNA沉默（VbMS）。

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