CN116622648A - 一种紫藤花叶病毒WiMV及其侵染性克隆载体和应用 - Google Patents
一种紫藤花叶病毒WiMV及其侵染性克隆载体和应用 Download PDFInfo
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Abstract
本发明公开了一种紫藤花叶病毒WiMV及其侵染性克隆载体和应用，所述紫藤花叶病毒WiMV的全基因组序列如SEQ ID NO：1所示；其侵染性克隆载体具体为，将紫藤花叶病毒WiMV全基因组序列构建到pCB301载体而得，命名为pCB301‑WiMV载体；所述pCB301载体中含有35S启动子和核酸酶Rz。该侵染性克隆载体具有稳定的侵染性，在模式植物本氏烟上能够稳定侵染长达50天；而且，该载体不会造成本氏烟生长发育的严重表型，可以对载体加以改造利用。本发明还将GFP插入到WiMV侵染性克隆pCB301‑WiMV中得到pCB301‑WiMV‑GFP，GFP能随着WiMV的系统侵染而在系统叶中表达，因此，该载体能用于外源蛋白表达，作为生物反应器。
Description
技术领域
本发明涉及基因工程技术领域，特别是涉及一种紫藤花叶病毒及其侵染性克隆载体和应用。
背景技术
植物病毒在自然界普遍发生，危害植物的生长发育并造成无法逆转的损害。目前，对于植物病毒的研究主要集中在经济作物上的病毒，国内外对于木本植物病毒的研究非常有限，而且我国对于木本植物病毒的研究主要集中在果树病毒和经济树木病毒。
紫藤(Wisteria sinensis)属于豆科、紫藤属植物，具有较高的观赏性，在公园、校园等地常被作为园林美化的重要树种而广泛种植。此外，紫藤还在木材、医药方面也有应用。然而，紫藤常受到花叶病的侵害，发病的紫藤叶片出现畸形、缩小、褪绿、斑驳、花叶、黄化等症状，严重地危害紫藤的生长发育，开花受阻，这使得紫藤的观赏价值和经济价值受到显著的影响。
紫藤花叶病是一种世界性病害，新西兰、澳大利亚、美国、伊朗和一些欧洲国家都报道过该病害。目前，该病害被证实是由病毒侵染所致，随着对病毒的研究不断深入，目前对不同侵染紫藤病毒的寄主范围、传播特性、分子生物学特征等都有了进一步的了解。为了方便深入的开展紫藤花叶病毒的与寄主植物之间的相互作用机制、WiMV功能基因组、WiMV分子检测、WiMV的抗性分析、抗WiMV药剂和药效检测等方面的研究工作，构建和使用病毒侵染性克隆是推动这些研究工作的重要前提。然而到目前为止仍然缺乏有关紫藤花叶病毒侵染性克隆这种重要的工具。
发明内容
本发明的目的是提供一种紫藤花叶病毒WiMV及其侵染性克隆载体和应用。
具体的，本发明采用的技术方案具体如下：
在紫藤叶片中发现了一种马铃薯Y病毒属(Potyvirus)新病毒——紫藤花叶病毒WiMV，通过小RNA测序鉴定以及PCR扩增测序拼接首次获得了病毒的全核苷酸序列，其全基因组序列如SEQ ID NO：1所示。
一种紫藤花叶病毒WiMV的侵染性克隆载体，具体为：将紫藤花叶病毒WiMV全基因组序列构建到pCB301载体而得，命名为pCB301-WiMV载体；所述pCB301载体中含有35S启动子和核酸酶Rz。
其中，所述pCB301-WiMV载体的基因序列如SEQ ID NO：2所示。
本发明所述紫藤花叶病毒WiMV的侵染性克隆载体的构建方法，包括以下步骤：
(1)根据紫藤花叶病毒WiMV全基因序列设计三对特异性引物：WiMV-F1、WiMV-R1、WiMV-F2、WiMV-R2、WiMV-F3、WiMV-R3，引物序列如SEQ ID NO：3-8所示；所述紫藤花叶病毒WiMV全基因组序列如SEQ ID NO：1所示；
(2)分别以引物对WiMV-F1/R1，WiMV-F2/R2，WiMV-F3/R3，分三段扩增WiMV核苷酸全序列，分别得到三段特异的PCR条带，并回收特异的PCR条带，命名为WiMV-fragment1、WiMV-fragment2、WiMV-fragment3；
(3)构建pCB301-WiMV重组载体质粒：酶切pCB301-Rz载体质粒，并切胶回收纯化，将三个目的PCR片段与经双酶切的pCB301载体进行重组反应，然后将连接产物转入大肠杆菌DH5α，PCR筛选阳性克隆并扩繁培养，提取质粒，通过酶切验证、测序验证正确后，转化到农杆菌中。
其中，所述步骤(1)具体为，将WiMV全基因组序列均分为三段，分别设计三对特异性引物：WiMV-F1，WiMV-R1；WiMV-F2，WiMV-R2；WiMV-F3，WiMV-R3，每一段的下游引物和下一段的上游引物有15-20bp的重叠，便于后续的片段重组。
其中，所述步骤(2)具体为，分别以引物对WiMV-F1/R1，WiMV-F2/R2，WiMV-F3/R3扩增WiMV核苷酸全序列，分别得到三段特异的PCR条带，经过琼脂糖凝胶电泳，并割胶回收纯化特异的PCR目的条带。
其中，所述步骤(3)具体为，通过两个限制性内切酶StuI和Smal酶切pCB301-Rz载体质粒，经过琼脂糖凝胶电泳，并割胶回收纯化，将三个目的PCR片段WiMV-fragment1、WiMV-fragment2、WiMV-fragment3等比例混合与经StuI和SmaI双酶切的pCB301载体通过In-fusion进行重组反应，然后将连接产物转入大肠杆菌DH5α，PCR筛选阳性克隆并扩繁培养，提取质粒，通过StuI和Smal酶切酶切验证、测序验证正确后，转化到农杆菌GV3101中。
本发明含有pCB301-WiMV载体质粒的农杆菌菌株可用于研究WiMV分子检测、WiMV的抗性分析、抗WiMV药剂药效检测、WiMV编码蛋白的功能、WiMV病毒与寄主因子之间的相互作用、理解WiMV的致病机制并挖掘寄主植物针对WiMV侵染的抗性因子或感病因子，为WiMV病毒病害的防治提供新策略。
另外，本发明还在pCB301-WiMV载体的基础上，构建了插入GFP的pCB301-WiMV-GFP表达载体，可以在本氏烟中表达外源蛋白GFP。所述pCB301-WiMV-GFP载体的序列如SEQ IDNO：31所示。
本发明所述pCB301-WiMV-GFP侵染性克隆载体的构建方法，包括以下步骤：
(1)选用NcoI、KpnI两个酶酶切pCB-WiMV载体，将该区域内的序列进行改造替换，插入新的目的序列；
(2)以pCB301-WiMV载体质粒为模板用WiMV-P1-F/R、WiMV-HC-F/R引物对扩增DNA片段，引物对序列如SEQ ID NO：25-26、29-30所示；以含有GFP序列的载体质粒为模板用WiMV-GFP-F/R引物对扩增DNA片段，引物对序列如SEQ ID NO：27-28所示；
(3)用NcoI、KpnI两个酶酶切pCB301-WiMV载体；
(4)将步骤(2)获得的PCR片段与经NcoI和KpnI双酶切的pCB301-WiMV载体利用同源重组酶进行In-fusion重组反应；
(5)转化大肠杆菌，筛斑鉴定阳性克隆。经过酶切验证、测序验证后转化农杆菌；
(6)接种植物测试病毒侵染性以及是否表达绿色荧光蛋白GFP。
本发明将GFP插入到WiMV侵染性克隆pCB301-WiMV中得到pCB301-WiMV-GFP，GFP能随着WiMV的系统侵染而在系统叶中表达，因此，pCB301-WiMV-GFP可以指示WiMV在植物体内分布和蔓延的进程，并且还能够用于外源蛋白的表达，作为生物反应器。
同现有技术相比，本发明的突出效果在于：
(1)本发明在紫藤叶片中发现了一种马铃薯Y病毒属(Potyvirus)新病毒，通过小RNA测序鉴定以及PCR扩增测序拼接首次获得了病毒的全核苷酸序列，并首次构建了具有侵染活性的WiMV的侵染性克隆，将其接种于健康本氏烟植物，能引起稳定、长期的病毒症状，并且从接种的本氏烟植物上检测到了WiMV的存在。
(2)本发明通过多片段重组的方法首次将WiMV病毒的基因组构建到高效植物表达载体pCB301上，并获得了具有系统侵染能力的病毒载体。
(3)本发明提供的WiMV农杆菌菌株使得WiMV长期大量保存。
(4)本发明WiMV病毒的侵染性克隆方便了后续的WiMV与寄主植物之间的相互作用、WiMV功能基因组、WiMV分子检测、抗WiMV药剂药效检测等研究。
(5)本发明构建的携带GFP的WiMV-GFP病毒能够在紫外灯下显示病毒的侵染进程和分布。此外，也说明PCB301-WiMV还能够用于表达外源蛋白，作为生物反应器。
(6)本发明WiMV的侵染性克隆的构建成功为全面了解该病毒编码蛋白的特性、遗传变异、发生规律以及致病机制，设计WiMV病毒病害的防控策略提供便利条件，对园艺观赏植物紫藤的生产具有重要的意义。
下面结合附图说明和具体实施例对本发明所述的紫藤花叶病毒WiMV及其侵染性克隆载体和应用作进一步说明。
附图说明
图1为侵染性克隆pCB301-WiMV载体构建示意图。
图2为pCB301-WiMV-GFP载体构建示意图。
图3为接种侵染性克隆pCB301-WiMV载体到本氏烟上15天后，植物感染病毒的症状特征；其中，对照(Mock)：接种含空载体的农杆菌；1-2：代表性的WiMV致病植物；比例尺＝2.5厘米。
图4为接种侵染性克隆pCB301-WiMV载体到本氏烟上21天后，植物感染病毒的症状特征；其中，对照(Mock)：接种含空载体的农杆菌；1-2：代表性的WiMV致病的植物；比例尺＝2.5厘米。
图5为接种侵染性克隆pCB301-WiMV载体到本氏烟上50天后，植物感染病毒的症状特征；其中，对照(Mock)：接种含空载体的农杆菌；图示为两株代表性的Mock侵染的植物、两株代表性的WiMV侵染的植物；比例尺＝6厘米。
图6为WiMV接种本氏烟15天的RT-PCR检测结果；其中，M：DNA Marker；1-2：Mock侵染的本氏烟；3-6：WiMV侵染的本氏烟；+：pCB301-WiMV载体质粒作为阳性对照。Nbactin扩增片段作为PCR扩增时等量模板的内参对照。
图7为WiMV接种本氏烟50天的RT-PCR检测结果；其中，M：DNA Marker；1-2：Mock侵染的本氏烟；3-4：WiMV侵染的本氏烟。Nbactin扩增片段作为PCR扩增时等量模板的内参对照。
图8为接种侵染性克隆pCB301-WiMV-GFP载体(简称WiMV-GFP)到本氏烟上10天和30天后，植物感染病毒的症状特征；其中，对照(Mock)：接种含空载体的农杆菌；WiMV-GFP：接种含有pCB301-WiMV-GFP的农杆菌。照片分别在自然光和紫外光下，拍摄植物病毒症状和GFP荧光。比例尺＝2.5厘米。
图9为WiMV-GFP接种本氏烟10天的Western blot检测结果；其中，M：蛋白质Marker；1-2：Mock侵染的本氏烟；3-4：WiMV-GFP侵染的本氏烟。丽春红染色的植物内源蛋白Rubisco大亚基(RbcL)作为上样量的参照。
图10为WiMV-GFP接种本氏烟30天的Western blot检测结果；其中，M：蛋白质Marker；1-2：Mock侵染的本氏烟；3-4：WiMV-GFP侵染的本氏烟。Bottom:植物底部的叶位；Up:植物顶部的叶位。用丽春红染色的植物内源蛋白Rubisco大亚基(RbcL)作为上样量的参照。
具体实施方式
本发明所提供的实施例如无特殊说明，均按照常规实验条件进行。
一、WiMV病毒基因组全序列的克隆
利用TRIzol提取感染病毒的紫藤叶片的总RNA，一部分送诺禾致源生物公司建库进行小RNA测序，另一部分反转录为cDNA用于后续试验。
通过小RNA测序拼接鉴定病叶中的植物病毒，获得25条拼接序列，拼接的片段在NCBI中blast比对，发现该紫藤叶片中的病毒属于马铃薯Y病毒属(Potyvirus)新病毒，结合拼接的已知病毒序列，设计引物Virus-F1/R1、Virus-F2/R2、Virus-F3/R3、Virus-F4/R4、Virus-F5/R5，如SEQ ID NO：9-18所示。
以反转录获得的cDNA为模板，用全式金公司的FastPfu DNA Polymerase(全式金)聚合酶，以引物对Virus-F1/R1、Virus-F2/R2、Virus-F3/R3、Virus-F4/R4、Virus-F5/R5分段扩增病毒序列。
体系如下：
PCR反应程序设定：
95℃预变性2min后开始以下循环反应：
4℃ 5min。
程序完成后取PCR产物3μl，电泳检测。
将扩增的PCR产物经过电泳，将目的片段割胶切下，用OMEGA公司的胶回收试剂盒。具体步骤参见试剂盒说明书，不在此赘述。
将纯化回收的产物纯化后，连入全式金的Blunt-Zero克隆载体(全式金生物，北京)，用M13F/M13R，如SEQ ID NO：23-24通用测序目的片段的序列，获得扩增的病毒序列信息。
将获得的5个片段的序列进行拼接，从而获得WiMV全基因组序列。
根据拼接获得的序列，将WiMV全基因组序列均分为三段，分别设计三对特异性引物WiMV-F1，WiMV-R1；WiMV-F2，WiMV-R2；WiMV-F3，WiMV-R3，如SEQ ID NO：3-8所示。每一段的下游引物和下一段的上游引物有15-20bp的重叠，便于后续的片段重组。
分别以引物对WiMV-F1/R1，WiMV-F2/R2，WiMV-F3/R3，分别扩增WiMV核苷酸全序列，分别得到三段特异的PCR条带。经过琼脂糖凝胶电泳，并割胶回收纯化特异的PCR目的条带，称为WiMV-fragment1、WiMV-fragment2、WiMV-fragment3。
二、构建重组的pCB301-WiMV载体质粒
(1)通过两个限制性内切酶StuI和Smal酶切pCB301-Rz载体质粒，准备一PCR管，向管子中加入如下反应体系：
(2)在PCR仪中37℃，孵育15分钟，然后将反应产物进行琼脂糖凝胶电泳，并割胶回收载体大片段并纯化，测定回收的浓度。
(3)将PCR片段WiMV-fragment1、WiMV-fragment2、WiMV-fragment3等比例混合(各60ng)与经StuI和SmaI双酶切的pCB301载体(100ng)利用One Step Cloning Kit同源重组酶(南京诺唯赞生物科技有限公司)进行In-fusion重组反应，体系如下：
在PCR仪中37℃，孵育30分钟，然后立即放于冰上。
(4)然后将连接产物转入大肠杆菌DH5α，涂布于卡那霉素抗性的平板上，37℃过夜培养。
(5)PCR筛选阳性克隆并扩繁培养，并提取质粒，序列如SEQ ID NO：2所示。侵染性克隆pCB301-WiMV载体构建示意图如图1所示。
(6)质粒通过StuI和Smal酶切酶切验证、测序验证正确后，转化到农杆菌GV3101中，-80℃保存菌株。
三、pCB301-WiMV载体质粒的转化与接种
(1)鉴定载体质粒成功转入到农杆菌后，过夜扩繁培养农杆菌单克隆。
(2)1500g，离心10min，获得菌体，用含终浓度为10mM MgCl2，10mM MES(pH5.6)和100μM乙酰丁香酮的植物接种缓冲液重悬菌体，OD600调整为1.0，室温静置1小时。
(3)将此含有pCB301-WiMV载体质粒的农杆菌悬浮液注射到4-5叶期的野生型本氏烟叶片，接种一片叶子，分别接种了4棵植物，接种后的植物放于温室中(16小时白昼/8小时黑暗，25℃，相对湿度60％)生长，并持续观察植物症状，判断构建的病毒是否具有侵染性。
四、本氏烟感染WiMV不同时期的症状以及WiMV病毒检测。
(1)定期观察接种植株的症状变化，拍照记录，结果所示，pCB-WiMV接种本氏烟15天后系统叶表现发现15天均能系统发病，症状为褪绿，叶片向内微卷(图3)。WiMV接种本氏烟15天的RT-PCR检测结果如图6所示。到21天时，症状更为明显(图4)，并持续观察后期的症状。
(2)进一步提取接种Mock和WiMV的植物总RNA进行RT-PCR检测，利用WiMV特异性检测引物WiMV-detect-F/R(如SEQ ID NO：19-20所示)检测WiMV是否存在，以本氏烟NbActin2-F/R(如SEQ ID NO：21-22所示)为对照。结果发病的4棵植物均检测到了WiMV特异性病毒条带(图5)。WiMV接种本氏烟50天的RT-PCR检测结果如图7所示。证实该病毒侵染性克隆可以有效侵染本氏烟。
上述实验结果表明：本发明构建的pCB301-WiMV侵染性克隆具有侵染性，能成功侵染本氏烟。
五、构建重组的pCB301-WiMV-GFP载体质粒
(1)PCR扩增各个片段，以pCB301-WiMV载体质粒为模板用WiMV-P1-F/R、WiMV-HC-F/R引物对(如SEQ ID NO：25-26、29-30所示)扩增DNA片段，标记P1(635bp)、HC(975bp)，以含有GFP序列的载体质粒为模板用WiMV-GFP-F/R引物对(如SEQ ID NO：27-28所示)扩增DNA片段，标记GFP(755bp)，琼脂糖凝胶电泳切胶回收各个片段。
(2)用NcoI、KpnI两个酶酶切pCB301-WiMV载体。准备一PCR管，向管子中加入如下反应体系，在PCR仪中37℃，孵育15分钟，然后将反应产物进行琼脂糖凝胶电泳，并割胶回收载体大片段并纯化，测定回收的浓度。
(3)将PCR片段P1、GFP、HC等比例混合(各20ng)与经NcoI和KpnI双酶切的pCB301-WiMV载体(100ng)利用One Step Cloning Kit同源重组酶(南京诺唯赞生物科技有限公司)进行In-fusion重组反应，体系如下：
| 试剂 | 用量 |
| NcoI和KpnI双酶切pCB301-WiMV质粒 | 100ng |
| P1 | 20ng |
| GFP | 20ng |
| HC | 20ng |
| 重组酶 | 2μL |
| 5*buffer | 4μL |
| ddH2O | 补足到20μL |
在PCR仪中37℃，孵育30分钟，然后立即放于冰上。
(4)然后将连接产物转入大肠杆菌DH5α，涂布于卡那霉素抗性的平板上，37℃过夜培养。
(5)PCR筛选阳性克隆并扩繁培养，并提取质粒，质粒通过经NcoI和KpnI双酶切验证、测序验证正确后，转化到农杆菌GV3101中，-80℃保存菌株。侵染性克隆pCB301-WiMV-GFP载体构建示意图如图2所示。所述pCB301-WiMV-GFP载体的序列如SEQ ID NO：31所示。
(6)接种植物测试病毒侵染性以及是否表达绿色荧光蛋白GFP。
六、pCB301-WiMV-GFP载体质粒的转化与接种
具体步骤同“步骤三”所示。
七、本氏烟感染WiMV-GFP不同时期的症状以及GFP蛋白的检测。
(1)在pCB301-WiMV-GFP接种本氏烟10天后系统叶表现病毒症状，并在紫外灯下可见GFP绿色荧光，到30天时，系统叶仍然有GFP不断表达(图8)。
(2)进一步提取接种Mock和WiMV-GFP的植物总蛋白进行western blot检测，利用GFP特异性检测抗体(品牌：Roche工作浓度1:10000)检测GFP是否存在，以丽春红染色的本氏烟Rubisco大亚基(rbcL)作为上样量的参照。结果在WiMV-GFP侵染的植物中能够检测到GFP的特异性条带(图9-10)。
上述实验结果表明：本发明构建的pCB301-WiMV-GFP侵染性克隆具有侵染性，并能在本氏烟中表达外源蛋白。
以上所述的实施例仅仅是对本发明的优选实施方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案作出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (10)
1.一种紫藤花叶病毒WiMV，其特征在于：其全基因组序列如SEQ ID NO：1所示。
2.权利要求1所述紫藤花叶病毒WiMV的侵染性克隆载体，其特征在于：将紫藤花叶病毒WiMV全基因组序列构建到pCB301载体而得，命名为pCB301-WiMV载体；所述pCB301载体中含有35S启动子和核酸酶Rz。
3.根据权利要求2所述的紫藤花叶病毒WiMV的侵染性克隆载体，其特征在于：所述pCB301-WiMV载体的基因序列如SEQ ID NO：2所示。
4.权利要求2-3任一所述紫藤花叶病毒WiMV的侵染性克隆载体的构建方法，其特征在于，包括以下步骤：
(1)根据紫藤花叶病毒WiMV全基因序列设计三对特异性引物：WiMV-F1、WiMV-R1、WiMV-F2、WiMV-R2、WiMV-F3、WiMV-R3，引物序列如SEQ ID NO：3-8所示；所述紫藤花叶病毒WiMV全基因组序列如SEQ ID NO：1所示；
(2)分别以引物对WiMV-F1/R1，WiMV-F2/R2，WiMV-F3/R3，分三段扩增WiMV核苷酸全序列，分别得到三段特异的PCR条带，并回收特异的PCR条带，命名为WiMV-fragment1、WiMV-fragment2、WiMV-fragment3；
(3)构建pCB301-WiMV重组载体质粒：酶切pCB301-Rz载体质粒，并切胶回收纯化，将三个目的PCR片段与经双酶切的pCB301载体进行重组反应，然后将连接产物转入大肠杆菌DH5α，PCR筛选阳性克隆并扩繁培养，提取质粒，通过酶切验证、测序验证正确后，转化到农杆菌中。
5.一种含有权利要求2-3任一所述的紫藤花叶病毒WiMV的侵染性克隆载体的农杆菌菌株。
6.权利要求1所述的紫藤花叶病毒WiMV或权利要求2-3任一所述的侵染性克隆载体或权利要求5所述的农杆菌菌株在WiMV与寄主植物之间的相互作用、WiMV功能基因组、WiMV分子检测、抗WiMV药剂药效检测中的应用。
7.权利要求2-3任一所述的紫藤花叶病毒WiMV的侵染性克隆载体的应用，其特征在于：将外源蛋白GFP序列插入到pCB301-WiMV载体中P1和HC-Pro之间，得到表达GFP的pCB301-WiMV-GFP载体；所述pCB301-WiMV-GFP载体的序列如SEQ ID NO：31所示。
8.权利要求7所述pCB301-WiMV-GFP载体的构建方法，其特征在于，包括以下步骤：
(1)选用NcoI、KpnI两个酶酶切pCB-WiMV载体，将该区域内的序列进行改造替换，插入新的目的序列；
(2)以pCB301-WiMV载体质粒为模板用WiMV-P1-F/R、WiMV-HC-F/R引物对扩增DNA片段，引物对序列如SEQ ID NO：25-26、29-30所示；以含有GFP序列的载体质粒为模板用WiMV-GFP-F/R引物对扩增DNA片段，引物对序列如SEQ ID NO：27-28所示；
(3)用NcoI、KpnI两个酶酶切pCB301-WiMV载体；
(4)将步骤(2)获得的PCR片段与经NcoI和KpnI双酶切的pCB301-WiMV载体利用同源重组酶进行In-fusion重组反应；
(5)转化大肠杆菌，筛斑鉴定阳性克隆。经过酶切验证、测序验证后转化农杆菌；
(6)接种植物测试病毒侵染性以及是否表达绿色荧光蛋白GFP。
9.权利要求7制备的pCB301-WiMV-GFP重组载体在观察植物症状，示踪WiMV病毒在植物中分布和蔓延程度的应用。
10.权利要求2-3任一所述的紫藤花叶病毒WiMV的侵染性克隆载体和权利要求7制备的pCB301-WiMV-GFP重组载体作为生物反应器，表达外源蛋白的应用。
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