CN113355352B - 一种以太子参TuMV-phe病毒基因为基础修饰改造病毒表达载体的方法 - Google Patents
一种以太子参TuMV-phe病毒基因为基础修饰改造病毒表达载体的方法 Download PDFInfo
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Abstract
本发明提供了一种以太子参TuMV‑phe病毒基因为基础修饰改造病毒表达载体的方法，利用太子参TuMV‑phe病毒基因组作为基础，将其改造成能够接收和携带外源基因的太子参病毒表达载体。将测试的指示性基因插入太子参特异TuMV‑phe载体，通过农杆菌介导方法侵染太子参和烟草，可以在2种植物体内快速表达外源基因蛋白产物。本发明方法规避了侵染、组织再生和阳性筛选等常规转化方法复杂冗余的流程，实现了在常规遗传转化体系难以进行的植物中高效表达基因瓶颈，所构建的病毒载体具有简单、快速、高效、特异性强、受体基因种类广等特点，为后续在太子参中高效表达具有药用和经济价值的蛋白提供了有效的技术平台。
Description
技术领域
本发明属于生物技术领域，具体涉及一种以太子参TuMV-phe病毒基因为基础修饰改造病毒表达载体的方法，主要适用于太子参TuMV类病毒（TuMV-phe）表达载体的快速改造和构建技术细节流程，同时，本发明所获取的太子参花叶病毒载体适用于以快速太子参和烟草植物作为底盘植物快速高效表达外源蛋白技术关键点和详细流程。
背景技术
植物病毒病虽然对作物产量和品质造成大量损失，但是病毒病在植物基因功能验证中却起着重要的作用。随着越来越多的动植物基因组序列完成测定，分子遗传学的研究重点逐步向基因功能的方向发展。同时，随着基因工程技术的发展，如反转录、体外转录等技术的研发为RNA类的病毒载体的构建提供了有效的技术支持。在对植物病毒深入研究中，研究者基于病毒基因组结构，通过基因替代、基因互补等方法已经构建了大量的植物病毒表达载体。目前，在病毒载体构建运用于基因功能研究的主要类型分为基因过表达和基因沉默两种。其中，以病毒基因组为基础的病毒外源表达载体已经广泛应用于不同病毒和不同的宿主植物。
近年来，多种植物病毒已被用于构建成各种基因表达载体，为植物中外源蛋白的表达提供了新的工具。相较于转基因技术下的基因功能研究，以植物病毒基因组作为自主复制的基因表达载体以下特点：①由于病毒基因组小且结构简单，在进行基因组修饰和构建过程中更易于操作；②构建一组基因验证的周期性较短，接种病毒载体后外源基因通常在2周内就会出现表达；③病毒载体可在植物体内进行大量增值，间接促使插入外源蛋白基因在植物中能大量表达等。随着不同植物中大量病毒基因组信息序列的测序，病毒基因组也相应被修饰改造成各种表达载体、RNAi载体，这些载体被广泛用于表达特定外源蛋白表达或验证植物本源基因的功能，极大的促进了非模式植物功能基因组学的研究。
太子参TuMV-phe病毒为申请人所鉴定和克隆的太子参特异病毒，属于TuMV类病毒，病毒全长9839bp（SEQ ID NO.1），可以转录为一个9,495bp的较长的转录本，进一步翻译成3164 aa的多聚蛋白，多聚蛋白进一步在NIa-Pro切割下形成P1, HC-Pro, P3, 6K1, CI,6K2, NIa-VPg, NIa-Pro, NIb 和 CP 等10个子蛋白，不同多聚蛋白首尾相接处均含有Nla-Pro蛋白酶切割位点。通过这些切割位点，不同蛋白均可以顺利从多聚蛋白集合体中释放出来成为一个单独游离蛋白。这些游离蛋白进一步用于病毒基因组的复制、病毒颗粒的装配以及病毒在植物体内移动。根据TuMV-phe病毒的多聚蛋白间的有秩序的排列规律和蛋白间保守的切割位点，可以人为定向插入一个外源蛋白，伪装成病毒蛋白，镶嵌进入病毒基因组内。同时，在目标插入外源蛋白的2端添加Nla-Pro酶切保守位点，则可以保证外源插入蛋白顺利从多聚蛋白上释放，从而实现目标蛋白的高效、快速合成。
发明内容
本发明的目的是提供一种以太子参TuMV-phe病毒基因为基础修饰改造病毒表达载体的方法，主要是在太子参花叶病核心病原病毒基因组的基础上的特异病毒表达载体改造、构建方法，同时，优化和建立太子参花叶病病毒表达载体在太子参和烟草植物中快速表达外源蛋白的技术流程。本发现所建立的太子参病毒表达载体可以在烟草、太子参2种植物底盘中高效的表达不同外源蛋白基因，无需复杂的组织培养、再生和侵染等复杂流程。
为实现上述目的，采用以下技术方案：
一种以太子参TuMV-phe病毒基因（SEQ ID NO.1）基础修饰改造病毒表达载体的构建方法，包括下述步骤：在太子参花叶病病毒基因组TuMV-phe的基础上，选择在病毒基因组编码蛋白基因NIb和CP间插入外源蛋白基因。同时，选择Rosea1（Ros1）（Bedoya等，2012）和EGFP作为候选外源插入基因，对花叶并病毒载体蛋白表达能力进行初步测定。
首先，在Ros1基因的5’端或3’端添加Nla-Pro酶切割识别位点，保证外源蛋白可以顺利从多聚蛋白上释放出来，并在候选插入外源基因2端加入常规限制性内切酶位点，以方便不同类型外源蛋白基因快速导入载体。其次，将添加有特定位点的序列克隆进入太子参TuMV-phe基因组内，构建形成含有特定外源片段TuMV-phe的病毒基因组克隆载体。然后，从上述修饰载体中克隆出含有外源片段TuMV-phe基因组，将其导入到表达载体，构建形成含有外源报告基因的TuMV-phe病毒表达载体。用上述含病毒表达载体的农杆菌侵染太子参和烟草植物，通过植株内的花青素累积量和EGFP的荧光强度，评断TuMV-phe的外源表达蛋白能力，将能够有效表达2个蛋白的载体作为最终太子参特异病毒表达载体。
本发明与现有技术相比具有以下优点和效果：
（1）本发明中所构建、优化的基于太子参TuMV-phe病毒基础上表达载体构建方法可以快速修饰TuMV类病毒基因组，形成特定植物特异病毒表达载体，可以为其它具有多聚蛋白转录特征的病毒表达载体构建提供参考信息。
（2）本发明中所构建的太子参特异病毒载体能够在太子参和烟草的多个受体植株体内快速表达目标蛋白。
（3）本发明所获取的太子参病毒特异表达载体直接侵染太子参幼苗植株即可在太子参植株体内快速表达蛋白，并随着植株生长蛋白丰度逐渐累积。省去了植物常规遗传转化方中组织培养、外植体再生、阳性筛选等复杂流程。
（4）本发明首次利用太子参特异植物病毒表达载体在太子参植株体内表达出Ros1和EGFP基因，实现了以药用植物太子参作为底盘植株表达外源蛋白的可能性。
（5）本发明创新性地提出的在药用植物中表达目标蛋白方法，解决了具有复杂遗传背景、次生代谢成分，外植体再生困难的植物难以进行遗传转化和蛋白表达的瓶颈。使这类植物的遗传转化和关键功能基因验证成为可能。同时，本发明病毒表达载体表达外源蛋白方法，仍属于瞬时表达范畴，发明中所用的病毒基因组信息，病毒编码蛋白以及外源蛋白均不能插入、替换、重组进入宿主基因组，不影响物种基因组的稳定性和关键基因信息。因此，本发明外源病毒表达方法规避了常规转基因的潜在危害性，具有安全、绿色和高效特定。
附图说明
图1为本发明所构建太子参病毒表达载体流程及对应载体图。其中，A为太子参TuMV-phe的克隆载体图，报告基因Ros1、2端酶切位点和EVYHQ/A已经插入到病毒基因组内；B为将位于克隆载体中TuMV-phe-Ros1片段转移至表达载体pCB301内，形成pCB301-TuMV-phece-Ros1病毒表达载体；C为太子参TuMV-phe侵染克隆；D为在TuMV-phe侵染克隆基础上所构建病毒表达载体，具体过程为将报告基因Ros1、2端酶切位点和CVYHQ/A插入病毒基因组内，形成另一种病毒表达载体pCB301-TuMV-phecc-Ros1；E为用EGFP作为报告基因替换pCB301-TuMV-phecc-Ros1病毒表达载体中的Ros1基因，形成pCB301-TuMV-phecc-EGFP病毒表达载体。
图2 为TuMV-phe病毒为基础所构建表达载体外源基因表达效果。其中，A表示pCB301-TuMV-phecc-Ros1所携带外源基因Ros1分别在本氏烟和太子参植株内的表达结果；B表示pCB301-TuMV-phecc-EGFP所携带外源基因EGFP在本氏烟和太子参植株表达结果；C表示用PCR方法检测不同病毒表达载体在太子参和本氏烟植株体内的侵染状况。
具体实施方式
太子参特异病毒表达载体的构建以及在太子参、烟草中进行外源蛋白表达技术，主要包括太子参病毒基因组的改造和修饰、病毒表达载体的构建和活性评测、农杆菌介导的病毒表达载体侵染技术等关键技术体系。太子参TuMV-phe外源表达载体主要是通过在TuMV-phe所编码的“多聚蛋白编码框”中的NIb和CP蛋白编码基因间插入外源蛋白，通过病毒转录、反应成含有外源蛋白的多聚病毒蛋白，同时，在目标蛋白的侧翼插入NIa-Pro酶感知位点实现目标蛋白从多聚蛋白中的有效释放。本实施案例选了金鱼草中的能高效积累花青素Ros1和常见报告基因EGFP基因，以确定TuMV-phe基础上的表达载体在模式植物和太子参中表达外源基因的能力。为了确保TuMV-phe外源蛋白能从多聚蛋白切割下来，在插入位点中的一端需要人为添加NIa-Pro酶识别切割位点。同时，为了方便其它外源基因插入，需要在病毒目标插入位点2端加入限制性内切酶位点。
附表1. 载体改造过程中所用的引物序列
实施例1
1.太子参TuMV-phe病毒基因组基础上的外源表达载体pCB301-TuMV-phece-Ros1的构建。
（1）为了将外源报告基因Ros1基因插入TuMV-phe基因组内，在pSMART-E-TuMV-phe（SEQ ID NO.1）的基础上，以NIb/CP基因间交界处作为分割点，将载体中TuMV-phe载体分成2段进行克隆，用引物TuMV-phe-Exp-P1-F和 TuMV-phe-Exp-P1-R（附表1）克隆第1段Exp-P1；用引物TuMV-phe-Exp-P1-F和TuMV-phe-Exp-P1-R（附表1）克隆第2段Exp-P2。以公布的Ros1基因（NCBI：DQ275529.1）为模板，人工合成其基因序列。然后，以人工合成的Ros1基因作为模板，用引物Ros1-NheI-F （添加NheI酶切位点，附表1）和Ros1-NcoI-Ps-R（添加NcoI酶切位点和Nla-pro酶识别切割位点，附表1）克隆报告基因Ros1。
上述片段克隆所用PCR程序为：95℃ 3 min→95℃ 15 sec→（95℃ 15 sec→60℃15 sec（根据片段长度决定，1000bp/min）→72℃ 1 min）30cycles→72℃ 5 min→4℃ ∞→END。克隆体系为：表2
| 组分 | 用量（μl） |
| H<sub>2</sub>O | 6.6 |
| 10×Buffer（Vayzme） | 10 |
| dNTP(10mmol/L) | 0.4 |
| 克隆片段正向引物（-F, 10μmol/L） | 0.8 |
| 克隆片段正向引物（-R, 10μmol/L） | 0.8 |
| 质粒模板（1ng/μL） | 1.0 |
| Phanta Max Super-Fidelity DNA Polymerase（Vayzme） | 0.4 |
3个片段产物需DpnI消化12 h后，消化体系为：
表3
消化产物分别切胶回收，用同源重组试剂盒（Vayzme公司，重组体系按照试剂盒说明书进行）将3个片段连接成pSMART-E-TuMV-phe-Ros1质粒（图1A，SEQ ID NO.2），重组体系见下表4，重组液转入大肠杆菌DH10B内挑选阳性质粒进行扩增。
表4
（2）为了将克隆载体中pSMART-E-TuMV-phe-Ros1中TuMV-Ros1转入表达载体内，以pSMART-E-TuMV-phe-Ros1模板，分别用引物TuMV-phe-InfeClo-P1-F和 TuMV-phe-Clo-P1-R克隆TuMV-phe-Ros1-P1段（附表1），TuMV-phe-Clo-P2-F 和 TuMV-phe-InfeClo-P2-R引物克隆TuMV-phe-Ros1-P2段（附表1）。同时，用pCB301-LinZ-F和 pCB 301-LinZ-R引物（附表1）线性化pCB301载体。克隆体系同表2，3个片段产物用DpnI酶消化12 h，消化体系同表3，通过同源重组方法将3个消化产物产物连接成pCB301-TuMV-phece-Ros1载体（图1B，SEQ IDNO. 3）。
2. 构建TuMV-phe基础上的外源表达载体pCB301-E-TuMV-phecc-Ros1和pCB301-E-TuMV-phecc-EGFP。
以pCB301-phe载体（图1C，SEQ ID NO.4）作为模板，分成3段进行克隆，TuMV-phe-InfeClo-P1-F和TuMV-phe-Clo-P1-R 克隆第一段Expcc-P1, 用TuMV-phe-Clo-P2-F和TuMV-phe-Expcc-P2-R 克隆第二段Expcc-P2, 用引物TuMV-phe-Expcc-P3-F 和pCB301-Linz-R克隆第三段Expcc-P3。以合成的Ros1基因为模板，用Ros1-cc-NheI-F （包含NheI酶切位点，附表1）和Ros1-cc-NheI-Ps-R （包含NheI酶切位点和另一个Nla-pro酶切识别位点，附表1）引物对克隆Ros1基因。同时，以EGFP基因为模板，用EGFP-cc-NheI-F （带着NheI酶切位点，附表1）和EGFP-cc-NheI-Ps-R （包含NheI酶切位点和另一个NIa-Pro酶切割位点，附表1）引物对克隆EGFP基因。Expcc-P1、Expcc-P2、Expcc-P3、Ros1-cc等4个片段均需用DpnI消化12 h，消化体系同表3。消化产物分别切胶回收，用同源重组试剂盒将Expcc-P1、Expcc-P1、Expcc-P2、Ros1-cc片段连接成pCB301-TuMV-phecc-Ros1质粒（图1D，SEQ IDNO.5），同时，利用NheI和NcoI对pCB301-TuMV-phecc-Ros1质粒进行双酶切，回收酶切产物后与EGFP进行同源重组连接成pCB301-TuMV-phecc-EFGP载体（图1E，SEQ ID NO. 6），重组液转入大肠杆菌挑选阳性质粒进行扩增。
3. TuMV-phe基础上的外源表达载体转化农杆菌。用根癌农杆菌冻融法进行
pCB301-TuMV-phece-Ros1、pCB301-TuMV-phecc-Ros1和pCB301-TuMV-phecc-EGFP载体的转化，在100μg/mL卡那霉素和50μg/mL利福平条件下筛选阳性菌落。
具体步骤如下：① 取GV3101感受态于冰水浴中融化。② 加入300-500 ng质粒于GV3101中，冰上静置5 min。③ 于液氮中处理5 min。④ 37℃水浴5 min。⑤ 于冰上5 min。⑥ 超净下加入700 μl-800 μl无抗性LB，28℃，180 rpm，2-3 h。⑦ 5000 rmp，1 min。留100μl于（卡那+利福平）LB上，28℃，培养48 h。⑧ 挑菌于5 mL（LB液体Kan+Rif）28℃，180 rmp，24 h。⑨液体于50μl液体LB（Kan+Rif）大瓶子，红色变橘色测菌液OD值。当农杆菌浓度（OD600）达到0.5到1.0时，以4000 rpm离心10 min收集农杆菌，然后将其重悬于含有10 mMMES、pH 5.6、10 mM MgCl2和200 μM AS的混合物中，获得侵染液。侵染液浓度调制OD=1.0后静置3 h。
4. 包含TuMV-phe的农杆菌侵染受体植物。
① 用本氏烟作为侵染对象。侵染模式植物本氏烟草采用的是传统的浸润法。选择生长至第4片叶长开的烟草幼苗，以第3片和第2片叶作为侵染对象。具体操作如下：将 1mL注射器去除针头，使用圆头摩擦叶片，施加压力通过叶脉渗方法，将侵染液浸润整个叶片。② 用太子参作为侵染对象当太子参子叶展开后，最上面的一对叶子被作为侵染叶。简单的步骤如下：将重悬液在室温下放置3小时，用1mL无针注射器擦伤叶子的背面，然后用浸润法将侵染液引入整个叶片内。在2种植物侵染14 DPI（侵染后天数）后，使用一对TuMV-phe特异性引物CP-Infec-Dect-F和CP-Infec-Dect-R（附表1）确证病毒载体在本氏烟和太子参中侵染率。
本氏烟侵染结果如图2，pCB301-TuMV-phece-Ros1、pCB301-TuMV-phecc-Ros1二个载体转农杆菌后侵染本氏烟，在侵染后的第3天，侵染叶片相邻叶子开始逐渐褪绿、叶边缘扭曲。侵染后第7-9天，被侵染植株叶子严重扭曲。红色斑点开始在受感染的叶子上沉积，从老叶子扩散到新叶上。生长势较弱植株在侵染后9天才开始积累红色色素，侵染后第14天，其他叶片中开始逐渐出现红色色素。侵染后第22天，红色素开始在茎节，新叶，花瓣和花托等不同器官中广泛积。相比较而言，pCB301-TuMV-phece-Ros1在同一时期其侵染叶片的着色程度低于pCB301-TuMV-phecc-Ros1载体。如图2将pCB301-TuMV-phecc-EGFP载体导入到农杆菌中侵染烟草，在侵染后第9天，用荧光显微镜观察到侵染植物的叶脉中可以观察到微弱的绿色荧光，而对照植物的叶片中则未观察到荧光。侵染后19天，EGFP开始在受侵染的叶片中大量表达，并遍及整个植物。
太子参侵染结果如图2，用含有pCB301-TuMV-phece-Ros1、pCB301-TuMV-phecc-Ros1载体的农杆菌感染太子参后第7天，被侵染的太子参叶片中出现轻微的红色细丝，并向叶脉延伸。侵染后第14天，红色细丝开始从被侵染的叶子转移到其他叶子，但2个载体病毒在本氏烟中的着色的程度明显低于本氏烟。随着侵染时间的延长，叶片中红色细丝的积累水平逐渐增加，侵染后42天，植株死。如图2将pCB301-TuMV-phecc-EGFP载体农杆菌转化后侵染太子参，侵染后第9天进行观察，未发现有荧光反应，侵染后第20天进行观察，在侵染叶片的临叶的静脉上可以观察到的明显荧光。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福建农林大学
福建天人药业股份有限公司
焦作市玖道种苗繁育有限公司
泰宁县古农堂生物科技有限公司
<120> 一种以太子参TuMV-phe病毒基因为基础修饰改造病毒表达载体的方法
<130> 25
<160> 25
<170> PatentIn version 3.5
<210> 1
<211> 11822
<212> DNA
<213> pSMART-E-TuMV-phe
<400> 1
gacgaattct ctagatatcg ctcaatactg accatttaaa tcatacctga cctccatagc 60
agaaagtcaa aagcctccga ccggaggctt ttgacttgat cggcacgtaa gaggttccaa 120
ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat cgagattttc 180
aggagctatg agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa 240
catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc 300
gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa 360
aggtagcgtt gccaatgatg ttacagatga gatggtcagg ctaaactggc tgacggaatt 420
tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac 480
cactgcgatc ccagggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga 540
aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa 600
ttgtcctttt aacggcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa 660
cggtttggtt ggtgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt 720
ctggaaagaa atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga 780
tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg 840
acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga 900
gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat 960
gaataaattg cagtttcact tgatgctcga tgagtttttc taaatgacca aacaggaaaa 1020
aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact 1080
caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga 1140
tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgatgagg 1200
gcccaaagga ggatcaaggc aatgtaatca cctggctcac cttcgggtgg gcctttctgc 1260
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgatgctc 1320
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 1380
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 1440
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 1500
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 1560
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 1620
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 1680
gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 1740
gaagccagtt acctcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 1800
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 1860
gaagatcctt tgattttcta ccgaagaaag gcccacccgt gaaggtgagc cagtgagttg 1920
attgcagtcc agttacgctg gagtcaaaaa atataaaaac tcaacacaac atacacaaaa 1980
cgattaaagc aaacacaaat atttcaaagc attcaagcaa tcaaaatatt ttcttttaaa 2040
tctttcattg ttaccaaagc aatcaccaac aacgaatcaa atggcaacag ttacattcgc 2100
gtcagctatc accaacgcca ccaccaacaa accagcactc accggaatgg tacaatttgg 2160
gagtttccca ccagtgccat tgcgatccac caccgctatt acagttgcca cttcagtggc 2220
gcaacctaaa ctgtacacag tgcagtttgg aagccttgac tcagtagtcg tcaagggtgt 2280
agcagggtcc tttgccaagg caacacgcca gcagcctaac gttgaaatag acgttagcct 2340
cagtgaagcc gcagctctgg aggttgcgaa acctagatcg aatgccgtgt tgagaatgca 2400
cgaggaggca aacaaggaga gagcaatctt tttggactgg gaggctagtt tgaggagaag 2460
ctcgtatgga attgctgaga acgagaaggt tgtgatgaca actcgtggcg tcagcaagat 2520
agtgcctaga agttcaaggg caatgaagca aaagcgcgca agggagaggc gtagagcgca 2580
gcaaccaatt atactaaagt gggaacccaa actgagcggg atctcaatcg gaggagggcc 2640
ctccgcgagc gcgatcgaag tagaagaagt ccgcacaaag tggccgcttc acaagacacc 2700
gtcaatgaag aagaggatgg tgcacaaaac atgtaagatg aacgaccaag gaattgacat 2760
gttgacacga tcccttatta agattttcaa gactaagagt gccaacattg agtacatcgg 2820
gaagaaatcg atcaaggtcg atttcatcag aaaagagcgg acgaaattcg caagaatcca 2880
agtagcacac ctacttggga agcgagcaca gcgcgacttg ttagctggaa tggaagaaaa 2940
ccattttatt gacattctca gcgagtactc aggtaacaaa acaaccataa atccaggagt 3000
agtttgcgca ggttggagtg gcatagttgt tagaaatgga attctaaccc agaaacgaag 3060
caggagccca tcaaaggcct ttgtaattag aggtgagcac gaaggcaagt tgtatgacgc 3120
caggaccaaa atcacaagaa caatgagtca caaaattgtg cactttagtg cagcaggagc 3180
taacttctgg aaaggcttcg atagatgctt tctcgcatat cgtagtgaca atcgtgaaca 3240
cacatgctat tcagggctag acgtcactga atgtggtgag gtagcagcac tgatgtgttt 3300
ggccatgttc ccatgcggaa agataacctg ccctgactgc gtgacagaca gtgagttgtc 3360
ccaaggacaa gcaagcagac catctatgaa gcataggtta acacaattgc gcgatgtcat 3420
caaatcaagc tacccacgct tcaagcatgc agtgcagata ctagataggt atgagcaatc 3480
actgagcagt gcaaatgaga actatcagga tttcgcagaa atccagagca taagcgacgg 3540
agttgaaaaa gctgcattcc cacacatcaa caaactaaac gcaatattga tcaagggggc 3600
cacagcgaca ggagaggaat tctcgcaggc tacgaagcat ttgctcgaga tagcacgata 3660
cctgaagaac agaactgaga acatcgagaa gggttcacta aagtcctttc gtaataagat 3720
ttcccagaaa gcgcacatca acccaacatt aatgtgtgac aaccagctcg ataggaatgg 3780
aaatttcata tggggtgaga gagggtacca tgcaaaacga ttctttagca actattttga 3840
gataatcgat ccgaagaaag gctacaccca gtatgagaca agagcggtgc ctaatgggtc 3900
acggaaactt gcaatcggca aactaatagt cccaacaaac ttcgaagttt taagggaaca 3960
aatgaaaggc gaaccagtag aaccgtaccc agtaacagtc gaatgtgtga gtaaattaca 4020
gggtgacttc gtccacgcat gttgttgtgt cacaacagaa tcaggcgacc cagtcttgtc 4080
tgaaatcaaa atgccaacta aacaccacct agtgattggt aacagcggcg atccaaagta 4140
catagatctc cctgagatcg aggagaataa aatgtacata gcgaaagagg gttattgtta 4200
cattaacatc ttcctagcta tgttggtgaa tgtcaaggag tcgcaggcaa aggagttcac 4260
gaaagttgtt agggacaaac tagtcggcga gcttggcaag tggcccactc tactagatgt 4320
agcaaccgcc tgttatttct tgaaagtatt ttacccagac gttgccaacg ccgaattgcc 4380
acgcatgtta gtggatcata agacaaagat aattcatgtc gttgattcat atgggtcact 4440
gtcaactgga tatcacgtcc ttaagacaaa cactgtggaa caactcatta aattcacgag 4500
atgcaatttg gagtcaagct tgaaacacta ccgcgtcgga ggaacagagt gggaggacat 4560
tcatggagcc agcaacatag atgatccgca gtggtgcatc aagaggctca taagaggagt 4620
ttacagacca aagcaactga aagaagacat gttggcgaac cctttcttac cactatatgc 4680
cctattgtca ccaggtgtca tcctggcatt ttacaatagc ggctctctag agtgcttgat 4740
gaaccattac attagggttg atagcaatgt cgccgttttg ttggttgttt tgaaatctct 4800
agcgaagaag gtatcaacta gccagagtgt gttagcccag ctccaaatca ttgaacgaag 4860
tctacctgaa ctcgtcgaag caaaggctaa tgttaatggg tcaggtgacg cagcctcgcg 4920
cgcgtgtgac agattcatgg gcatgctttt gcacatggca gaaccaaact gggagcttgc 4980
ggatggtgga tacacaattc tgagagatca tagcatctcc attttagaaa aaagttatct 5040
acaaatcttg gacgaagcat ggaacgagtt aagttggtcg gagcgctgtg ctataagata 5100
ctactcgtca aagcaagcaa tttttacaca gaaagatttg ccaatgaaaa gcgacgtcga 5160
tttaggcggc agatacagcg tgtcagtcat gtcatcttac gaatggagta agcgatgtat 5220
gaaaagcgta tactctagaa taggcagtaa attacgtagt agtatgtctt ggactagtag 5280
caaggtatcg aatagtgtgt ataagactat aaattattta gtaccagatg tgctcaagtt 5340
tattaacgtg cttgtttgta tcggcatact aattacgatg gctgctgagg cgaatcgcat 5400
cgtcatcacg caaaggaggc tcaaactgga tgtcgaagag acagagcgca gaaaagcaga 5460
atgggagctt gcattccacc acgccattct gacacagagt gcaggtcaac acccaacgat 5520
agatgagttc agagcataca tcgctgacaa agcaccacat ctaagtaagc atatcgagcc 5580
tgaagaaaag gtagtagttc atcaagcgaa gagacaatcc gagcaagagc tcgagcgcat 5640
aatagcattt gttgcattgg tgctcatgat gtttgatgca gaacgaagcg attgtgtcac 5700
aaagatcctc aacaagctta agggactagt tgccactgtg gaacctacag tctaccatca 5760
gactcttaat gatatagaag atgatttgaa tgagaggaac ctcttcatcg attttgaact 5820
tagcagcgac ggcgacatgc tccaacagct tccagccgaa aagacatttg cctcatggtg 5880
gaatcatcaa ctaagtagag gattcacaat cccacattac agaacagaag ggaagttcat 5940
gacttttact agagcaactg ccacggaagt cgcaggtaaa atagcacacg agagtgacaa 6000
agatatattg ctaatgggag cagtaggatc aggtaagtca actggcttgc cttatcatct 6060
ctccagaaaa gggaatgtat tgctccttga gccgactcgg ccacttgcag aaaacgtaca 6120
caagcagttg tcgcaggcgc catttcatca gaacacaact cttaggatgc gcggactaac 6180
ggcatttgga tcggcaccaa tttcagtgat gactagtggt tttgcactca attactttgc 6240
aaacaacaga atgcgaatta aagagtttga ctttgtcata tttgatgaat gtcacgttca 6300
tgacgccaac gcaatggcga tgagatgttt gctacatgaa tgtgattatt ctggcaaaat 6360
tatcaaagtt tcagccacac caccaggtcg agaagttgag ttctctactc aataccctgt 6420
atcgataagc acagaagaca cactatcgtt tcagaatttt gtgaacgcac agggtagtgg 6480
aagcaattgt gatgtaattt caaaaggaga caatatcctc gtgtatgtag caagctacaa 6540
tgaggtagat gcgctttcaa aacttctaat tgaaagagac ttcaaagtca cgaaggttga 6600
cggaagaacg atgaaagttg gaaacatcga gatcaccaca agtggaacac ctagcaagaa 6660
gcacttcata gttgcaacca atatcattga gaacggtgtt actctagaca tcgatgtggt 6720
tgctgatttt ggaacgaagg tactcccata ccttgataca gacagcagaa tgcttagcac 6780
aacaaagaca agcatcaatt atggggagcg tatccaaaga ctaggaagag tcggacggca 6840
caaaccaggt cacgctctgc gaataggtca cacagagaag gggttgagcg aagttccaag 6900
ttgtattgca acagaagcag ctttgaagtg cttcacttat gggcttccag taatcaccaa 6960
caacgtctcg acaagcattc ttggtaatgt aacggtaaag caggcacgaa caatgtctgt 7020
gtttgagata acaccgttct acacaagcca agtggtgaga tatgatggct ccatgcaccc 7080
acaggtgcac gcacttttaa agaggttcaa actcagagac tctgagattg ttttgaataa 7140
attagccata cctcaccgag gagtgaatgc ttggctcaca gctagtgagt atgcacgact 7200
tggcgcgaat gttgaagata ggcgtgacgt tcgaatccct tttatgtgtc gcgacatccc 7260
agaaaaactt catctagaca tgtgggatgt gattgtcaaa tttaaaggtg atgcgggttt 7320
tggtcggctt tcaagcgcca gtgcgagcaa ggtagcttat actctacaga cggacgtcaa 7380
ctccatacag cgaacagtca ccatcataga tacactaatc gctgaggaga gaaggaagca 7440
ggaatacttc aagacggtaa cctccaactg cgtttcttct tcgaacttct cactgcagag 7500
cataacaaat gcgataaaat ctcgtatgat gaaagatcac acgtgcgaga acatatcagt 7560
acttgaagga gcgaagtcac agctactcga gtttagaaac ctgaatgctg atcactcatt 7620
tgccactaaa accgatggaa tatctcggca tttcatgagt gagtttggag ctcttgaggc 7680
agtccaacat caaaacacca gtgacatgag caaattcctc aagcttaagg gcaaatggaa 7740
caaaacacta gtcacgcgag atgtgttggt gctttgtgga gttcttggag gtggattgtg 7800
gatggttatt cagcacctgc ggtcaaagat ttccgaaccc gtaacccacg aagcaaaagg 7860
caagaggcaa agacagaaac taaagtttcg caatgcccga gacaacaaga tgggtagaga 7920
agtgtacgga gacgatgata ccatagagca tttctttggt gatgcctaca caaagaaagg 7980
gaagagtaag ggcaggacac gtggtattgg acacaaaaac aggaagttca tcaacatgta 8040
tgggtttgat cctgaagatt tctctgcagt tcgtttcgtg gatccactca caggagcgac 8100
attggatgac aacccgctca cagacatcac ccttgtgcaa gagcactttg gtaacataag 8160
aatgaacttg ctcggggagg atgagctgga tccaaatgaa ttacgtgtga ataagacaat 8220
tcaggcctac tacatgaaca ataaaacagg caaggctttg agggtggatc tgacaccaca 8280
catacctctc aaggtgtgtg atcttcacgc aaccattgct ggattcccag agcgagaaaa 8340
cgaactgagg cagactggaa aagctcagcc cattaacata gacgaagtgc caagagctaa 8400
taatgaactc gtcctagtag accacgagag taactccatg ttcagagggt tgcgtgacta 8460
caacccaata tcaaacaaca tttgtcatct cacaaatgtt tcagatggag catcaaactc 8520
gttgtatgga gtcggtttcg gaccactcat attaacgaac cgacacctct ttgagcggaa 8580
taacggtgaa ctcgtaataa aatcacgaca tggtgagttc gtgattaaaa acacaaccca 8640
gctatatttg ctaccgattc cagacagaga tcttctgcta atccgtctac caaaggatgt 8700
cccacccttt ccacagaaat tgggtttcag gcaacctgag aaaggtgaac gaatttgcat 8760
ggtggggtcc aacttccaaa ccaagagcat aacgagtata gtctccgaga ctagcacaat 8820
aatgccagtg gaaaacagtc agttttggaa acactggatc agcacgaaag atggccaatg 8880
cggaagtcca atggtgagca cgaaagacgg gaaaataatt ggattgcaca gtctagcaaa 8940
cttccagaac tccatcaatt acttcgctgc tttcccagac ggttttgccg agaagtatct 9000
tcataccatt gaagcacacg agtgggtcaa gcattggaaa tataacacta gtgctattag 9060
ttggggctct ttgaatatac aagcatcgca accgtcaggc ttgttcaaag taagcaagct 9120
aatctcagac ctcgacagca cagcagtcta cgcacaaacc cagcagaatc ggtggatgtt 9180
cgagcagctc aatgggaacc tgaaagcgat agcacactgc cctagccagc ttgtgacaaa 9240
gcacacagtc aaaggaaaat gtcagatgtt tgacttgtat ctcaagctgc atgatgaagc 9300
acgagagtat ttccaaccga tgctgggcca gtatcaaaag agcaaactca atcgagaagc 9360
atatgcaaag gatcttttga aatatgcaac gccaatcgaa gcaggaaaca tcgactgcga 9420
tttgtttgaa aagacagttg aaatagtcgt atcagatttg cgaggttatg gttttgaaac 9480
atgcaattat gtcactgatg agaatgacat attcgaagct cttaacatga aatccgcagt 9540
tggagcgttg tataaaggaa agaagaggga ttacttcgct gagttcacac ccgagatgaa 9600
agaagaaata ctgaaacaaa gttgtgaacg gctcttctta ggaaagatgg gagtgtggaa 9660
tggctcgttg aaggcagagt tacgaccact agaaaaagtg gaagcaaaca aaacacggac 9720
gtttactgcc gcaccgctag acacactatt gggtggaaag gtttgcgtgg atgatttcaa 9780
caaccagttc tatgatcaca accttagagc tccttggagc gttggcatga caaagtttta 9840
ttgtggttgg gatcacttgt tggagtcgtt gccagatggt tgggtgtatt gcgatgctga 9900
tggctcacag tttgacagct cgctatcacc atatttgatc aatgcagtgc tcaacatccg 9960
cttaggattc atggaagagt gggacgtagg ggaggtaatg ctgagaaatt tgtacaccga 10020
aattgtgtat acccctatct ctacaccaga tggtacactc gtcaagaaat tcaaaggaaa 10080
caatagcgga cagccatcga ccgttgtaga caacacgctc atggtcatac tggcagtcaa 10140
ctattcactc aagaaaagtg gaattccaag tgagctgcgc gacagtatta tcagattctt 10200
cgtcaacgga gatgatttac tgctaagcgt acacccagag tatgagtata tccttgacac 10260
tatgacagac aactttcatg aactgggcct gaagtatgct ttcgactcaa ggaccaggga 10320
aaaaggagac ctttggttta tgtcgcacca agggcacaaa agggagggaa tctggattcc 10380
caagcttgaa ccagagcgaa tagtatcgat tctagaatgg gatcggtcga aagagccatg 10440
ccatcgacta gaagcaatct gcgcagcgat gattgagtcg tggggatacg acaagctgac 10500
tcacgagata cgcaagtttt acgcatggat gattgaacaa gctccattta gctccctagc 10560
acaagaaggg aaagctcctt acatagcgga aacagcgctg aggaagctct accttgataa 10620
ggaaccagct caagaggatc tcacccatta cttgcaagca atctttgagg attatgaaga 10680
tggtgatgag gcttgtgttt atcaccaggc aggtgaaacg cttgatgcag gtttgacaga 10740
cgagcaaaag caggcggaga aggagaagaa ggagagagag aaggcagaaa aggaacgaga 10800
gaggcagaag cagttggcac tcaataaagg caaggatgtt gcacaagaag agggaaaacg 10860
cgacaaggaa gtaaacgctg gaacttctgg aactttcagt gtacccagac ttaagagtct 10920
gacaagcaag atgcgtgtgc caagatacga gcaaagagtg gctctaaacc tcaatcacct 10980
aatcctatac acgccagagc agacggatct atccaacaca cgttcaacgc gaaagcagtt 11040
taacacatgg tttgaaggtg taatggctga ttacgaactg acggaggaca aaatgcaaat 11100
cattctcaat ggtttaatgg tctggtgcat tgagaacgga acctccccga acataaacgg 11160
aatgtgggtg atgatggacg gcgatgatca ggtggaattc ccgatcaaac cgctcattga 11220
ccacgccaaa cccacattta ggcagataat ggcccatttc agtgacgtag ctgaagcgta 11280
cattgaaaag cgtaaccaag accgaccata catgccacga tatggtcttc agcgcaattt 11340
aaccgacatg agcttagctc gatacgcgtt tgatttctat gaaatgactt ctagaactcc 11400
aatacgtgcg agagaagcac acatccagat gaaagcagca gcactgcgtg gcgcaaataa 11460
caatttgttc ggcttggatg gaaacgttgg tacaacggta gagaacacgg agaggcatac 11520
gaccgaggac gttaatcgga acatgcataa cttacttggc gttaaggggt tatgaagttg 11580
tatgctggta gactataagt atttaagttt actcgttagt attctcgctt atgggaaata 11640
tgtaagtttg ttaaagcagc cagtgtgact ctgtcatgtg tgttgttctt actttctata 11700
ttttcgccga acattttatt ggtgttagcg catgtggtga ggattgtcct cgattgcctt 11760
aacatttgat aggatgcaag ggactgaggc tcgtcctgaa tgatatcaag cttgaattcg 11820
tt 11822
<210> 2
<211> 690
<212> DNA
<213> pSMART-E-TuMV-phe-Ros1
<400> 2
gctagcatgg aaaagaattg tcgtggagtg agaaaaggta cttggaccaa agaagaagac 60
actctcttga ggcaatgtat agaagagtat ggtgaaggga aatggcatca agttccacac 120
agagcagggt tgaaccggtg taggaagagt tgcaggctga ggtggttgaa ttatctgagg 180
ccaaatatca aaagaggtcg gttttcgaga gatgaagtgg acctaattgt gaggcttcat 240
aagctgttgg gtaacaaatg gtcgctgatt gctggtagaa ttcctggaag gacagctaat 300
gacgtgaaga acttttggaa tactcatgtg gggaagaatt taggcgagga tggagaacga 360
tgccggaaaa atgttatgaa cacaaaaacc attaagctga ctaatatcgt aagaccccga 420
gctcggacct tcaccggatt gcacgttact tggccgagag aagtcggaaa aaccgatgaa 480
ttttcaaatg tccggttaac aactgatgag attccagatt gtgagaagca aacgcaattt 540
tacaatgatg ttgcgtcgcc acaagatgaa gttgaagact gcattcagtg gtggagtaag 600
ttgctagaaa caacggagga tggggaatta ggaaacctat tcgaggaggc ccaacaaatt 660
ggaaatccat gggaggtata tcatcaggca 690
<210> 3
<211> 10524
<212> DNA
<213> pCB301-TuMV-phece-Ros1
<400> 3
aaaaaatata aaaactcaac acaacataca caaaacgatt aaagcaaaca caaatatttc 60
aaagcattca agcaatcaaa gacttttaaa tctttcatcg ttaccaaagc aatcaccaac 120
aacgaatcaa atggcaacag ttacattcgc gtcagctatc accaacgcca ccaccaacaa 180
accagcactc accggaatgg tacaatttgg gagtttccca ccagtgccat tgcgatccac 240
caccgctatt acagttgcca cttcagtggc gcaacctaaa ctgtacacag tgcagtttgg 300
aagccttgac tcagtagtcg tcaagggtgt agcagggtcc tttgccaagg caacacgcca 360
gcagcctaac gttgaaatag acgttagcct cagtgaagcc gcagctctgg aggttgcgaa 420
acctagatcg aatgccgtgt tgagaatgca cgaggaggca aacaaggaga gagcaatctt 480
tttggactgg gaggctagtt tgaggagaag ctcgtatgga attgctgaga acgagaaggt 540
tgtgatgaca actcgtggcg tcagcaagat agtgcccaga agttcaaggg caatgaagca 600
aaagcgcgca agggagaggc gtagagcgca gcaaccaatt atactaaagt gggaacccaa 660
attgagcggg atcccaattg gaggagggct ctccgcgagc gcgatcgaag tagaagaagt 720
ccgcacaaag tggccgcttc acaagacacc gtcaatgaag aagaggatgg tgcacaaaac 780
atgtaagatg aacgaccaag gaattgacat gttgacacga tccctgatta agattttcaa 840
gactaagagt gccaacattg agtacatcgg gaagaaatcg atcaaggtcg atttcatcag 900
aaaagagcgg acgaaattcg caagaatcca agtagcacac ctacttggga agcgagcaca 960
gcgcgacttg ttaactggaa tggaagaaaa ccattttatt gacattctca gcgagtactc 1020
aggtaacaaa acaaccataa atccaggagt agtttgcgca ggttggagtg gcatagttgt 1080
tagaaatgga attctaaccc agaaacgaag caggagccca tcaaaggcct ttgtaattag 1140
aggtgagcac gaaggcaagt tgtatgacgc caggatcaaa atcacaagaa caatgagtca 1200
caaaattgta cactttagtg cagcaggagc taacttctgg aaaggcttcg atagatgctt 1260
tctcgcatat cgtagtgaca atcgtgaaca cacatgctat tcagggctag acgtcactga 1320
atgtggtgag gtggcagcac tgatgtgttt ggccatgttc ccatgcggaa agataacctg 1380
ccctgactgc gtgacagaca gtgagttatc ccaaggacaa gcaagcagac catctatgaa 1440
gcataggtta acacaactgc gcgatgtcat caaatcaagc tacccacgct tcaagcatgc 1500
agtgcagata ctagataggt atgagcaatc actgagcagt gcaaatgaga actatcagga 1560
tttcgcagaa atccagagca taagcgacgg agttgaaaaa gctgcattcc cacacatcaa 1620
caaactaaac gcaatattga tcaagggggc cacagcgaca ggagaggaat tctcgcaggc 1680
tacgaagcat ttgctcgaga tagcacgata cctgaagaac agaactgaga acatcgagaa 1740
gggttcacta aagtcctttc gtaataagat ttcccagaaa gcgcacatca acccaacatt 1800
aatgtgtgac aaccagctcg ataggaatgg aaatttcata tggggtgaga gagggtacca 1860
tgcaaaacga ttctttagca actattttga gataatcgat ccgaagaaag gctacaccca 1920
gtatgagaca agagcggtgc ctaatgggtc acggaaactt gcaatcggca aactaatagt 1980
cccaacaaac ttcgaagttt taagggaaca aatgaaaggc gaaccagtag aaccgtaccc 2040
agtaacagtc gaatgtgtga gtaaattaca gggtgacttc gtccacgcat gttgttgtgt 2100
cacaacagaa tcaggcgacc cagtcttgtc tgaaatcaaa atgccaacta aacaccacct 2160
agtgattggt aacagcggcg atccaaagta catagatctc cctgagatcg aggagaataa 2220
aatgtacata gcgaaagagg gttattgtta catcaacatc ttcctagcta tgttggtaaa 2280
tgtcaaggag tcgcaggcaa aggagttcac gaaagttgtt agggacaaac tagtcggcga 2340
acttggcaag tggcccactc tattagatgt agcaaccgcc tgttatttct tgaaagtatt 2400
ttacccagac gttgccaacg ccgaattgcc acgcatgtta gtggatcata agacaaagat 2460
aattcatgtc gttgattcat atgggtcact gtcaactgga tatcacgtcc ttaagacaaa 2520
cactgtggaa caactcatta aattcacgag atgcaatttg gagtcaagct tgaaacacta 2580
ccgcgtcgga ggaacagagt gggaggacac tcatggagcc agcaacatag atgatccgca 2640
gtggtgcatc aagaggctca taagaggagt ttacagacca aagcaactga aagaagacat 2700
gttggcgaac cctttcttac cactatatgc cctattgtca ccaggtgtca tcctggcatt 2760
ttacaatagc ggctctctag agtgcttgat gaaccattac attagggttg atagcaatgt 2820
cgccgttttg ttggttgttt tgaaatctct agcgaagaag gtatcaacta gccagagtgt 2880
gttagcccag ctccaaatca ttgaacgaag tctacctgaa ctcgtcgaag caaaggctaa 2940
tgttaatggg tcaggtgacg cagcctcgcg cgcgtgtgac agattcatgg gcatgctttt 3000
gcacatggca gaaccaaact gggagcttgc ggatggtgga tacacaattc tgagagatca 3060
tagcatctcc attttagaaa aaagttatct acaaatcttg gacgaagcat ggaacgagtt 3120
aagttggtcg gagcgctgtg ctataagata ctactcgtca aagcaagcaa tttttacaca 3180
gaaagatttg ccaatgaaaa gcgacgtcga tttaggcggc agatacagcg tgtcagtcat 3240
gtcatcttac gaatggagta agcgatgtat gaaaagcgta tactctagaa taggcagtaa 3300
attacgtagt agtatgtctt ggactagtag caaggtatcg aatagtgtgt ataagactat 3360
aaattattta gtaccagatg tgctcaagtt tattaacgtg cttgtttgta tcggcttact 3420
aattacgatg gctgctgagg cgaatcgcat cgtcatcacg caaaggaggc tcaaactgga 3480
tgtcgaagag acagagcgca gaaaagcaga atgggagctt gcattccacc acgccattct 3540
gacacagagt gcaggtcaac acccaacgat agatgagttc agagcataca tcgctgacaa 3600
agcaccacat ctaagtaagc atatcgagcc tgaagaaaag gtagtagttc atcaagcgaa 3660
gagacaatcc gagcaagagc tcgagcgcat aatagcattt gttgcattgg tgctcatgat 3720
gtttgatgca gaacgaagcg attgtgtcac aaagatcctc aacaagctta agggactagt 3780
tgccactgtg gaacctacag tctaccatca gactctcaat gatatagaag atgatttgaa 3840
tgagaggaac ctcttcatcg attttgaact tagcagcgac ggcgacatgc tccaacagct 3900
tccagccgaa aagacatttg cctcatggtg gagtcatcaa ctaagtagag gattcacaat 3960
cccacattac agaacagaag ggaagttcat gacttttact agagcaactg ccacggaagt 4020
cgcaggtaaa atagcacacg agagtgacaa agatatattg ctaatgggag cagtaggatc 4080
aggtaagtca actggcttgc cttatcatct ctccagaaaa gggaatgtat tgctccttga 4140
gccgactcgg ccacttgcag aaaacgtaca caagcagttg tcgcaggcgc catttcatca 4200
gaacacaact cttaggatgc gcggactaac ggcatttgga tcggcaccaa tttcagtgat 4260
gactagtggt tttgcactca attactttgc aaacaacaga atgcgaatta aagagtttga 4320
ctttgtcata tttgatgaat gtcacgttca tgacgccaac gcaatggcga tgagatgttt 4380
gctacatgag tgtgattatt ctggcaaaat tatcaaagtt tcagccacac caccaggtcg 4440
agaagttgag ttctctactc aataccctgt atcgataagc acagaagaca cactatcgtt 4500
tcaggatttt gtgaacgcac agggtagtgg aagcaattgt gatgtaattt caaaaggaga 4560
caatatcctc gtgtatgtag caagctacaa tgaggtagat gcgctttcaa aacttctaat 4620
tgaaagagac ttcaaagtca cgaaggttga cggaagaacg atgaaagttg gaaacatcga 4680
gatcaccaca agtggaacac ctagcaagaa gcacttcata gttgcaacca atatcattga 4740
gaacggtgtt actctagaca tcgatgtggt tgctgatttt ggaacgaagg tactcccata 4800
ccttgataca gacagcagaa tgcttagcac aacaaagaca agcatcaatt atggggagcg 4860
tatccaaaga ctaggaagag tcggacggca caaaccaggt cacgctctgc gaataggtca 4920
cacagagaag gggttgagcg aagttccaag ttgtattgca acagaagcag ctttgaagtg 4980
cttcacttat gggcttccag tgatcaccaa caacgtctcg acaagcattc ttggtaatgt 5040
aacggtaaag caggcacgaa caatgtctgt gtttgagata acaccgttct acacaagcca 5100
agtggtgaga tatgatggct ccatgcaccc acaggtgcac gcacttttaa agaggttcaa 5160
actcagagac tctgagattg ttttgaataa attagccata cctcaccgag gagtgaatgc 5220
ttggctcaca gctagtgagt atgcacgact tggcgcgaat gttgaagata ggcgtgacgt 5280
tcgaatccct tttatgtgtc gcgacatccc agaaaaactt catctagaca tgtgggatgt 5340
gattgtcaaa ttcaaaggtg atgcgggttt tggtcggctt tcaagcgcca gtgcgagcaa 5400
ggtagcttat actctacaga cggacgtcaa ctccatacag cgaacagtca ccatcataga 5460
tacactaatc gctgaggaga gaaggaagca ggaatacttc aagacggtaa cctccaactg 5520
cgtttcttct tcgaacttct cactgcagag cataacaaat gcgataaaat ctcgtatgat 5580
gaaagatcac acgtgcgaga acatatcagt acttgaagga gcgaagtcac agctactcga 5640
gtttagaaac ctgaatgctg atcactcatt tgccactaaa accgatggaa tatctcggca 5700
tttcatgagt gagtttggag ctcttgaggc agtccaccat caaaacacca gtgacatgag 5760
caaattcctc aagcttaagg gcaaatggaa caaaacacta gtcacgcgag atgtgttggt 5820
gctttgtgga gttcttggag gtggattgtg gatggttatt cagcacctgc ggtcaaagat 5880
ttccgaaccc gtaacccacg aagcaaaagg caagaggcaa agacagaaac taaagtttcg 5940
caatgcccga gacaacaaga tgggtagaga agtgtacgga gacgatgata ccatagagca 6000
tttctttggt gatgcctaca caaagaaagg aaagagtaag ggcaggacac gtggtattgg 6060
acacaaaaac aggaagttca tcaacatgta tgggtttgat cctgaagatt tctctgcagt 6120
tcgtttcgtg gatccactca caggagcgac attggatgac aacccgctca cagacatcac 6180
ccttgtgcaa gagcactttg gtgacataag aatgaacttg ctcggggagg atgagctgga 6240
tccaaatgaa ttacgtgtga ataagacaat ccaggcctac tacatgaaca ataaaacagg 6300
caaggctttg aaggtggatc tgacaccaca catacctctc aaggtgtgtg atcttcacgc 6360
aaccattgct ggattcccag agcgagaaaa cgaactgagg cagactggaa aagctcagcc 6420
cattaacata gacgaagtgc caagagctaa taatgaactc gtcctagtgg accacgagag 6480
taactccatg ttcagagggt tgcgtgacta caacccaata tcaaacaaca tttgtcatct 6540
cacaaatgtt tcagatggag catcaaactc gttgtatgga gtcggtttcg gaccactcat 6600
attaacgaac cgacacctct ttgagcggaa taacggtgaa ctcgtaataa aatcacgaca 6660
tggtgagttc gtgattaaaa acacaactca gctatatttg ctaccgattc cagacagaga 6720
tcttttgcta atccgtttac caaaggatgt cccacccttt ccacagaaat tgggtttcag 6780
gcaacctgag aaaggtgaac gaatttgcat ggtggggtcc aatttccaaa ccaagagcat 6840
aacgagtata gtctctgaga ctagcacaat aatgccagtg gaaaacagtc agttttggaa 6900
acactggatt agcacgaaag acggccaatg cggaagtcca atggtgagca cgaaagacgg 6960
gaaaataatt ggattgcaca gcctagcaaa cttccagaac tccatcaatt acttcgctgc 7020
tttcccagac ggttttgccg agaagtatct tcataccatt gaagcacacg agtgggtcaa 7080
gcattggaaa tataacacta gtgccattag ttggggctct ttgaatatac aagcatcgca 7140
accgtcaggc ttgttcaaag taagcaagct aatctcagac ctcgacagca cagcagtcta 7200
cgcacaaacc cagcagaatc ggtggatgtt cgagcagctc aatgggaacc tgaaagcgat 7260
agcacactgc cctagccagc ttgtgacaaa gcacacagtc aaaggaaaat gtcagatgtt 7320
tgacttgtat ctcaagctgc atgatgaagc acgagagtat ttccaaccga tgctgggcca 7380
gtatcaaaag agcaaactca atcgagaagc atatgcaaag gatcttttga aatatgcaac 7440
gccaatcgaa gcaggaaaca tcgactgtga tctgtttgaa aagacagttg aaatagtcgt 7500
atcagatttg cgaggttatg gtttcgaaac atgcaattat gtcactgatg agaatgacat 7560
attcgaagct cttaacatga aatccgcagt tggagcgttg tataaaggaa agaagagaga 7620
ttacttcgct gagttcacac ccgagatgaa agaagaaata ctgaaacaaa gttgtgaacg 7680
gctcttctta ggaaagatgg gagtgtggaa tggctcgttg aaggcagagt tacgaccact 7740
ggaaaaagtg gaagcaaaca aaacacggac gtttactgcc gcaccactag acacactatt 7800
gggtggaaag gtttgcgtgg atgatttcaa caaccagttc tatgatcaca accttagagc 7860
tccttggagc gttggcatga caaagtttta ttgtggttgg gatcgcttgt tggagtcgtt 7920
gccagatggt tgggtgtatt gcgatgctga tggctcacag tttgacagct cgctatcacc 7980
atatttgatc aatgcagtgc tcaacatccg cttagaattc atggaagagt gggacgtagg 8040
cgaggtaatg ctgagaaatt tgtacaccga aatcgtgtat acccctatct ctacaccaga 8100
tggtacactc gtcaagaaat tcaaaggaaa caatagcgga cagccatcaa ccgttgtaga 8160
caacacgctc atggtcatac tggcagtcaa ctattcactc aagaaaagtg gaattccaag 8220
tgagctgcgc gacagtatta tcagattctt cgtcaacgga gatgatttac tgctaagcgt 8280
acacccagag tatgagtata tccttgacac tatgacagac aactttcatg aactgggcct 8340
gaagtatgct ttcgactcaa ggaccaggga aaaaggagac ctttggttta tgtcgcacca 8400
agggcacaaa agagagggaa tctggattcc caagcttgaa ccagagcgaa tagtatcgat 8460
tctagaatgg gatcggtcga aagagccaag ccatcgacta gaagcaatct gcgcagcgat 8520
gattgagtcg tggggatacg acaagttgac tcacgagata cgcaagtttt acgcatggat 8580
gattgaacaa gctccattta gctccctagc acaagaaggg aaagctcctt acatagcgga 8640
aacagcgctg aggaagctct accttgataa ggaaccagct caagaggatc tcacccatta 8700
tttgcaagca atctttgagg attatgaaga tggtgatgag gcttgtgttt atcaccaggc 8760
agctagcatg gaaaagaatt gtcgtggagt gagaaaaggt acttggacca aagaagaaga 8820
cactctcttg aggcaatgta tagaagagta tggtgaaggg aaatggcatc aagttccaca 8880
cagagcaggg ttgaaccggt gtaggaagag ttgcaggctg aggtggttga attatctgag 8940
gccaaatatc aaaagaggtc ggttttcgag agatgaagtg gacctaattg tgaggcttca 9000
taagctgttg ggtaacaaat ggtcgctgat tgctggtaga attcctggaa ggacagctaa 9060
tgacgtgaag aacttttgga atactcatgt ggggaagaat ttaggcgagg atggagaacg 9120
atgccggaaa aatgttatga acacaaaaac cattaagctg actaatatcg taagaccccg 9180
agctcggacc ttcaccggat tgcacgttac ttggccgaga gaagtcggaa aaaccgatga 9240
attttcaaat gtccggttaa caactgatga gattccagat tgtgagaagc aaacgcaatt 9300
ttacaatgat gttgcgtcgc cacaagatga agttgaagac tgcattcagt ggtggagtaa 9360
gttgctagaa acaacggagg atggggaatt aggaaaccta ttcgaggagg cccaacaaat 9420
tggaaatcca tgggaggtat atcatcaggc aggtgaaacg cttgatgcag gtttgacaga 9480
cgagcaaaag caggcggaga aggagaagaa ggagagagag aaggcagaaa aggaacgaga 9540
gaggcagaag cagttggcac tcaataaagg caaggatgtt gcacaagaag agggaaaacg 9600
cgacaaggaa gtaaacgctg gaacctctgg aactttcagt gtacccagac ttaagagtct 9660
gacaagcaag atgcgtgtgc caagatacga gcaaagagtg gctctaaacc tcaatcacct 9720
aatcctatac acgccagagc agacggatct atccaacaca cgttcaacgc gaaagcagtt 9780
taacacatgg tttgaaggtg taatggctga ttacgaactg acggaggaca aaatgcaaat 9840
cattctcaat ggtttaatgg tctggtgcat tgagaacgga acctccccga acataaacgg 9900
aatgtgggtg atgatggacg gcgatgatca ggtggaattc ccgatcaaac cgctcattga 9960
ccacgccaaa cccacattta ggcagataat ggcccatttc agtgacgtag ctgaagcgta 10020
cattgaaaag cgtaaccaag accgaccata catgccacga tatggtcttc agcgcaattt 10080
aaccgacatg agcttagctc gatacgcgtt tgatttctat gaaatgactt ctagaactcc 10140
aatacgtgcg agagaagcgc acatccagat gaaagcagca gcactgcgtg gcgcaaataa 10200
caatttgttc ggcttggatg gaaacgttgg tacaacggta gagaacacgg agaggcatac 10260
gaccgaggac gttaatcgga acatgcataa cttacttggc gttaaggggt tatgaagttg 10320
tatgctggta gactataagt atttaagttt actcgttagt attctcgctt atgggaaata 10380
tgtaagtttg ttaaagcagc cagtgtgact ttgtcatgtg tgttgttgtt actttctatg 10440
ttttcgccga acattttatt ggtgttagcg catgtggtga ggattgtcct cgattgcctt 10500
aacatttgat aggatgcaag ggac 10524
<210> 4
<211> 14540
<212> DNA
<213> pCB301-TuMV-phe
<400> 4
actgacgagt ccctaaagga cgaaacggta cccggatgtg ttttccgggc tgatgagtcc 60
gtgaggacga aacctggctg caggcatgca agcttggcgt aatcatggtc atagcctaga 120
gtccgcaaaa atcaccagtc tctctctaca aatctatctc tctctatttt tctccagaat 180
aatgtgtgag tagttcccag ataagggaat tagggttctt atagggtttc gctcatgtgt 240
tgagcatata agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa 300
tttctaattc ctaaaaccaa aatccagtga cctgcagccc ggccggggga tccactagca 360
gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat attggcgggt 420
aaacctaaga gaaaagagcg tttattagaa taatcggata tttaaaaggg cgtgaaaagg 480
tttatccgtt cgtccatttg tatgtgcatg ccaaccacag gagatctcag taaagcgctg 540
gctgctgaac ccccagccgg aactgacccc acaaggccct agcgtttgca atgcaccagg 600
tcatcattga cccaggcgtg ttccaccagg ccgctgcctc gcaactcttc gcaggcttcg 660
ccgacctgct cgcgccactt cttcacgcgg gtggaatccg atccgcacat gaggcggaag 720
gtttccagct tgagcgggta cggctcccgg tgcgagctga aatagtcgaa catccgtcgg 780
gccgtcggcg acagcttgcg gtacttctcc catatgaatt tcgtgtagtg gtcgccagca 840
aacagcacga cgatttcctc gtcgatcagg acctggcaac gggacgtttt cttgccacgg 900
tccaggacgc ggaagcggtg cagcagcgac accgattcca ggtgcccaac gcggtcggac 960
gtgaagccca tcgccgtcgc ctgtaggcgc gacaggcatt cctcggcctt cgtgtaatac 1020
cggccattga tcgaccagcc caggtcctgg caaagctcgt agaacgtgaa ggtgatcggc 1080
tcgccgatag gggtgcgctt cgcgtactcc aacacctgct gccacaccag ttcgtcatcg 1140
tcggcccgca gctcgacgcc ggtgtaggtg atcttcacgt ccttgttgac gtggaaaatg 1200
accttgtttt gcagcgcctc gcgcgggatt ttcttgttgc gcgtggtgaa cagggcagag 1260
cgggccgtgt cgtttggcat cgctcgcatc gtgtccggcc acggcgcaat atcgaacaag 1320
gaaagctgca tttccttgat ctgctgcttc gtgtgtttca gcaacgcggc ctgcttggcc 1380
tcgctgacct gttttgccag gtcctcgccg gcggtttttc gcttcttggt cgtcatagtt 1440
cctcgcgtgt cgatggtcat cgacttcgcc aaacctgccg cctcctgttc gagacgacgc 1500
gaacgctcca cggcggccga tggcgcgggc agggcagggg gagccagttg cacgctgtcg 1560
cgctcgatct tggccgtagc ttgctgggcc atcgagccga cggactggaa ggtttcgcgg 1620
ggcgcacgca tgacggtgcg gcttgcgatg gtttcggcat cctcggcgga aaaccccgcg 1680
tcgatcagtt cttgcctgta tgccttccgg tcaaacgtcc gattcattca ccctccttgc 1740
gggattgccc cgactcacgc cggggcaatg tgcccttatt cctgatttga cccgcctggt 1800
gccttggtgt ccagataatc caccttatcg gcaatgaagt cggtcccgta gaccgtctgg 1860
ccgtccttct cgtacttggt attccgaatc ttgccctgca cgaataccag cgaccccttg 1920
cccaaatact tgccgtgggc ctcggcctga gagccaaaac acttgatgcg gaagaagtcg 1980
gtgcgctcct gcttgtcgcc ggcatcgttg cgccacatct aggtactaaa acaattcatc 2040
cagtaaaata taatatttta ttttctccca atcaggcttg atccccagta agtcaaaaaa 2100
tagctcgaca tactgttctt ccccgatatc ctccctgatc gaccggacgc agaaggcaat 2160
gtcataccac ttgtccgccc tgccgcttct cccaagatca ataaagccac ttactttgcc 2220
atctttcaca aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc 2280
gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg gggtgtcttc 2340
ttcccagttt tcgcaatcca catcggccag atcgttattc agtaagtaat ccaattcggc 2400
taagcggctg tctaagctat tcgtataggg acaatccgat atgtcgatgg agtgaaagag 2460
cctgatgcac tccgcataca gctcgataat cttttcaggg ctttgttcat cttcatactc 2520
ttccgagcaa aggacgccat cggcctcact catgagcaga ttgctccagc catcatgccg 2580
ttcaaggtgc aggacctttg gaacaggcag ctttccttcc agccatagca tcatgtcctt 2640
ttcccgttcc acatcatagg tggtcccttt ataccggctg tccgtcattt ttaaatatag 2700
gttttcattt tctcccacca gcttatatac cttagcagga gacattcctt ccgtatcttt 2760
tacgcagcgg tatttttcga tcagtttttt caattccggt gatattctca ttttagccat 2820
ttattatttc cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac 2880
aagacgaact ccaattcact gttccttgca ttctaaaact ttaaatacca gaaaacagct 2940
ttttcaaagt tgttttcaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa 3000
ccacaattat gggtgatgct gccaactcga gagcgggccg ggagggttcg agaagggggg 3060
gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt aaaaacaagg 3120
tttataaata ttggtttaaa agcaggttaa aagacaggtt agcggtggcc gaaaaacggg 3180
cggaaaccct tgcaaatgct ggattttctg cctgtggaca gcccctcaaa tgtcaatagg 3240
tgcgcccctc atctgtcagc actctgcccc tcaagtgtca aggatcgcgc ccctcatctg 3300
tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac ttatccccag gcttgtccac 3360
atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga ggctggccag 3420
ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc gccgggtgag 3480
tcggcccctc aagtgtcaac gtccgcccct catctgtcag tgagggccaa gttttccgcg 3540
aggtatccac aacgccggcg gccggccgcg gtgtctcgca cacggcttcg acggcgtttc 3600
tggcgcgttt gcagggccat agacggccgc cagcccagcg gcgagggcaa ccagcccggt 3660
gagcgtctct agtggactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc 3720
cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa caaattgacg 3780
cttagacaac ttaataacac attgcggacg tttttaaagt actggggtgg ttttggtacc 3840
gggccccccc tcgaggtcga cggtatcgat aagcttgata tcgaattcct gcaggtcaac 3900
atggtggagc acgacacact tgtctactcc aaaaatatca aagatacagt ctcagaagac 3960
caaagggcaa ttgagacttt tcaacaaagg gtaatatccg gaaacctcct cggattccat 4020
tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg cacctacaaa 4080
tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga cagtggtccc 4140
aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct 4200
tcaaagcaag tggattgatg tgataacatg gtggagcacg acactctcgt ctactccaag 4260
aatatcaaag atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta 4320
atatccggaa acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata 4380
gtggaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt 4440
gaagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg 4500
gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact 4560
gacgtaaggg atgacgcaca atcccactat ccttcgcaag accttcctct atataaggaa 4620
gttcatttca tttggagagg aaaaaatata aaaactcaac acaacataca caaaacgatt 4680
aaagcaaaca caaatatttc aaagcattca agcaatcaaa atattttctt ttaaatcttt 4740
cattgttacc aaagcaatca ccaacaacga atcaaatggc aacagttaca ttcgcgtcag 4800
ctatcaccaa cgccaccacc aacaaaccag cactcaccgg aatggtacaa tttgggagtt 4860
tcccaccagt gccattgcga tccaccaccg ctattacagt tgccacttca gtggcgcaac 4920
ctaaactgta cacagtgcag tttggaagcc ttgactcagt agtcgtcaag ggtgtagcag 4980
ggtcctttgc caaggcaaca cgccagcagc ctaacgttga aatagacgtt agcctcagtg 5040
aagccgcagc tctggaggtt gcgaaaccta gatcgaatgc cgtgttgaga atgcacgagg 5100
aggcaaacaa ggagagagca atctttttgg actgggaggc tagtttgagg agaagctcgt 5160
atggaattgc tgagaacgag aaggttgtga tgacaactcg tggcgtcagc aagatagtgc 5220
ctagaagttc aagggcaatg aagcaaaagc gcgcaaggga gaggcgtaga gcgcagcaac 5280
caattatact aaagtgggaa cccaaactga gcgggatctc aatcggagga gggccctccg 5340
cgagcgcgat cgaagtagaa gaagtccgca caaagtggcc gcttcacaag acaccgtcaa 5400
tgaagaagag gatggtgcac aaaacatgta agatgaacga ccaaggaatt gacatgttga 5460
cacgatccct tattaagatt ttcaagacta agagtgccaa cattgagtac atcgggaaga 5520
aatcgatcaa ggtcgatttc atcagaaaag agcggacgaa attcgcaaga atccaagtag 5580
cacacctact tgggaagcga gcacagcgcg acttgttagc tggaatggaa gaaaaccatt 5640
ttattgacat tctcagcgag tactcaggta acaaaacaac cataaatcca ggagtagttt 5700
gcgcaggttg gagtggcata gttgttagaa atggaattct aacccagaaa cgaagcagga 5760
gcccatcaaa ggcctttgta attagaggtg agcacgaagg caagttgtat gacgccagga 5820
ccaaaatcac aagaacaatg agtcacaaaa ttgtgcactt tagtgcagca ggagctaact 5880
tctggaaagg cttcgataga tgctttctcg catatcgtag tgacaatcgt gaacacacat 5940
gctattcagg gctagacgtc actgaatgtg gtgaggtagc agcactgatg tgtttggcca 6000
tgttcccatg cggaaagata acctgccctg actgcgtgac agacagtgag ttgtcccaag 6060
gacaagcaag cagaccatct atgaagcata ggttaacaca attgcgcgat gtcatcaaat 6120
caagctaccc acgcttcaag catgcagtgc agatactaga taggtatgag caatcactga 6180
gcagtgcaaa tgagaactat caggatttcg cagaaatcca gagcataagc gacggagttg 6240
aaaaagctgc attcccacac atcaacaaac taaacgcaat attgatcaag ggggccacag 6300
cgacaggaga ggaattctcg caggctacga agcatttgct cgagatagca cgatacctga 6360
agaacagaac tgagaacatc gagaagggtt cactaaagtc ctttcgtaat aagatttccc 6420
agaaagcgca catcaaccca acattaatgt gtgacaacca gctcgatagg aatggaaatt 6480
tcatatgggg tgagagaggg taccatgcaa aacgattctt tagcaactat tttgagataa 6540
tcgatccgaa gaaaggctac acccagtatg agacaagagc ggtgcctaat gggtcacgga 6600
aacttgcaat cggcaaacta atagtcccaa caaacttcga agttttaagg gaacaaatga 6660
aaggcgaacc agtagaaccg tacccagtaa cagtcgaatg tgtgagtaaa ttacagggtg 6720
acttcgtcca cgcatgttgt tgtgtcacaa cagaatcagg cgacccagtc ttgtctgaaa 6780
tcaaaatgcc aactaaacac cacctagtga ttggtaacag cggcgatcca aagtacatag 6840
atctccctga gatcgaggag aataaaatgt acatagcgaa agagggttat tgttacatta 6900
acatcttcct agctatgttg gtgaatgtca aggagtcgca ggcaaaggag ttcacgaaag 6960
ttgttaggga caaactagtc ggcgagcttg gcaagtggcc cactctacta gatgtagcaa 7020
ccgcctgtta tttcttgaaa gtattttacc cagacgttgc caacgccgaa ttgccacgca 7080
tgttagtgga tcataagaca aagataattc atgtcgttga ttcatatggg tcactgtcaa 7140
ctggatatca cgtccttaag acaaacactg tggaacaact cattaaattc acgagatgca 7200
atttggagtc aagcttgaaa cactaccgcg tcggaggaac agagtgggag gacattcatg 7260
gagccagcaa catagatgat ccgcagtggt gcatcaagag gctcataaga ggagtttaca 7320
gaccaaagca actgaaagaa gacatgttgg cgaacccttt cttaccacta tatgccctat 7380
tgtcaccagg tgtcatcctg gcattttaca atagcggctc tctagagtgc ttgatgaacc 7440
attacattag ggttgatagc aatgtcgccg ttttgttggt tgttttgaaa tctctagcga 7500
agaaggtatc aactagccag agtgtgttag cccagctcca aatcattgaa cgaagtctac 7560
ctgaactcgt cgaagcaaag gctaatgtta atgggtcagg tgacgcagcc tcgcgcgcgt 7620
gtgacagatt catgggcatg cttttgcaca tggcagaacc aaactgggag cttgcggatg 7680
gtggatacac aattctgaga gatcatagca tctccatttt agaaaaaagt tatctacaaa 7740
tcttggacga agcatggaac gagttaagtt ggtcggagcg ctgtgctata agatactact 7800
cgtcaaagca agcaattttt acacagaaag atttgccaat gaaaagcgac gtcgatttag 7860
gcggcagata cagcgtgtca gtcatgtcat cttacgaatg gagtaagcga tgtatgaaaa 7920
gcgtatactc tagaataggc agtaaattac gtagtagtat gtcttggact agtagcaagg 7980
tatcgaatag tgtgtataag actataaatt atttagtacc agatgtgctc aagtttatta 8040
acgtgcttgt ttgtatcggc atactaatta cgatggctgc tgaggcgaat cgcatcgtca 8100
tcacgcaaag gaggctcaaa ctggatgtcg aagagacaga gcgcagaaaa gcagaatggg 8160
agcttgcatt ccaccacgcc attctgacac agagtgcagg tcaacaccca acgatagatg 8220
agttcagagc atacatcgct gacaaagcac cacatctaag taagcatatc gagcctgaag 8280
aaaaggtagt agttcatcaa gcgaagagac aatccgagca agagctcgag cgcataatag 8340
catttgttgc attggtgctc atgatgtttg atgcagaacg aagcgattgt gtcacaaaga 8400
tcctcaacaa gcttaaggga ctagttgcca ctgtggaacc tacagtctac catcagactc 8460
ttaatgatat agaagatgat ttgaatgaga ggaacctctt catcgatttt gaacttagca 8520
gcgacggcga catgctccaa cagcttccag ccgaaaagac atttgcctca tggtggaatc 8580
atcaactaag tagaggattc acaatcccac attacagaac agaagggaag ttcatgactt 8640
ttactagagc aactgccacg gaagtcgcag gtaaaatagc acacgagagt gacaaagata 8700
tattgctaat gggagcagta ggatcaggta agtcaactgg cttgccttat catctctcca 8760
gaaaagggaa tgtattgctc cttgagccga ctcggccact tgcagaaaac gtacacaagc 8820
agttgtcgca ggcgccattt catcagaaca caactcttag gatgcgcgga ctaacggcat 8880
ttggatcggc accaatttca gtgatgacta gtggttttgc actcaattac tttgcaaaca 8940
acagaatgcg aattaaagag tttgactttg tcatatttga tgaatgtcac gttcatgacg 9000
ccaacgcaat ggcgatgaga tgtttgctac atgaatgtga ttattctggc aaaattatca 9060
aagtttcagc cacaccacca ggtcgagaag ttgagttctc tactcaatac cctgtatcga 9120
taagcacaga agacacacta tcgtttcaga attttgtgaa cgcacagggt agtggaagca 9180
attgtgatgt aatttcaaaa ggagacaata tcctcgtgta tgtagcaagc tacaatgagg 9240
tagatgcgct ttcaaaactt ctaattgaaa gagacttcaa agtcacgaag gttgacggaa 9300
gaacgatgaa agttggaaac atcgagatca ccacaagtgg aacacctagc aagaagcact 9360
tcatagttgc aaccaatatc attgagaacg gtgttactct agacatcgat gtggttgctg 9420
attttggaac gaaggtactc ccataccttg atacagacag cagaatgctt agcacaacaa 9480
agacaagcat caattatggg gagcgtatcc aaagactagg aagagtcgga cggcacaaac 9540
caggtcacgc tctgcgaata ggtcacacag agaaggggtt gagcgaagtt ccaagttgta 9600
ttgcaacaga agcagctttg aagtgcttca cttatgggct tccagtaatc accaacaacg 9660
tctcgacaag cattcttggt aatgtaacgg taaagcaggc acgaacaatg tctgtgtttg 9720
agataacacc gttctacaca agccaagtgg tgagatatga tggctccatg cacccacagg 9780
tgcacgcact tttaaagagg ttcaaactca gagactctga gattgttttg aataaattag 9840
ccatacctca ccgaggagtg aatgcttggc tcacagctag tgagtatgca cgacttggcg 9900
cgaatgttga agataggcgt gacgttcgaa tcccttttat gtgtcgcgac atcccagaaa 9960
aacttcatct agacatgtgg gatgtgattg tcaaatttaa aggtgatgcg ggttttggtc 10020
ggctttcaag cgccagtgcg agcaaggtag cttatactct acagacggac gtcaactcca 10080
tacagcgaac agtcaccatc atagatacac taatcgctga ggagagaagg aagcaggaat 10140
acttcaagac ggtaacctcc aactgcgttt cttcttcgaa cttctcactg cagagcataa 10200
caaatgcgat aaaatctcgt atgatgaaag atcacacgtg cgagaacata tcagtacttg 10260
aaggagcgaa gtcacagcta ctcgagttta gaaacctgaa tgctgatcac tcatttgcca 10320
ctaaaaccga tggaatatct cggcatttca tgagtgagtt tggagctctt gaggcagtcc 10380
aacatcaaaa caccagtgac atgagcaaat tcctcaagct taagggcaaa tggaacaaaa 10440
cactagtcac gcgagatgtg ttggtgcttt gtggagttct tggaggtgga ttgtggatgg 10500
ttattcagca cctgcggtca aagatttccg aacccgtaac ccacgaagca aaaggcaaga 10560
ggcaaagaca gaaactaaag tttcgcaatg cccgagacaa caagatgggt agagaagtgt 10620
acggagacga tgataccata gagcatttct ttggtgatgc ctacacaaag aaagggaaga 10680
gtaagggcag gacacgtggt attggacaca aaaacaggaa gttcatcaac atgtatgggt 10740
ttgatcctga agatttctct gcagttcgtt tcgtggatcc actcacagga gcgacattgg 10800
atgacaaccc gctcacagac atcacccttg tgcaagagca ctttggtaac ataagaatga 10860
acttgctcgg ggaggatgag ctggatccaa atgaattacg tgtgaataag acaattcagg 10920
cctactacat gaacaataaa acaggcaagg ctttgagggt ggatctgaca ccacacatac 10980
ctctcaaggt gtgtgatctt cacgcaacca ttgctggatt cccagagcga gaaaacgaac 11040
tgaggcagac tggaaaagct cagcccatta acatagacga agtgccaaga gctaataatg 11100
aactcgtcct agtagaccac gagagtaact ccatgttcag agggttgcgt gactacaacc 11160
caatatcaaa caacatttgt catctcacaa atgtttcaga tggagcatca aactcgttgt 11220
atggagtcgg tttcggacca ctcatattaa cgaaccgaca cctctttgag cggaataacg 11280
gtgaactcgt aataaaatca cgacatggtg agttcgtgat taaaaacaca acccagctat 11340
atttgctacc gattccagac agagatcttc tgctaatccg tctaccaaag gatgtcccac 11400
cctttccaca gaaattgggt ttcaggcaac ctgagaaagg tgaacgaatt tgcatggtgg 11460
ggtccaactt ccaaaccaag agcataacga gtatagtctc cgagactagc acaataatgc 11520
cagtggaaaa cagtcagttt tggaaacact ggatcagcac gaaagatggc caatgcggaa 11580
gtccaatggt gagcacgaaa gacgggaaaa taattggatt gcacagtcta gcaaacttcc 11640
agaactccat caattacttc gctgctttcc cagacggttt tgccgagaag tatcttcata 11700
ccattgaagc acacgagtgg gtcaagcatt ggaaatataa cactagtgct attagttggg 11760
gctctttgaa tatacaagca tcgcaaccgt caggcttgtt caaagtaagc aagctaatct 11820
cagacctcga cagcacagca gtctacgcac aaacccagca gaatcggtgg atgttcgagc 11880
agctcaatgg gaacctgaaa gcgatagcac actgccctag ccagcttgtg acaaagcaca 11940
cagtcaaagg aaaatgtcag atgtttgact tgtatctcaa gctgcatgat gaagcacgag 12000
agtatttcca accgatgctg ggccagtatc aaaagagcaa actcaatcga gaagcatatg 12060
caaaggatct tttgaaatat gcaacgccaa tcgaagcagg aaacatcgac tgcgatttgt 12120
ttgaaaagac agttgaaata gtcgtatcag atttgcgagg ttatggtttt gaaacatgca 12180
attatgtcac tgatgagaat gacatattcg aagctcttaa catgaaatcc gcagttggag 12240
cgttgtataa aggaaagaag agggattact tcgctgagtt cacacccgag atgaaagaag 12300
aaatactgaa acaaagttgt gaacggctct tcttaggaaa gatgggagtg tggaatggct 12360
cgttgaaggc agagttacga ccactagaaa aagtggaagc aaacaaaaca cggacgttta 12420
ctgccgcacc gctagacaca ctattgggtg gaaaggtttg cgtggatgat ttcaacaacc 12480
agttctatga tcacaacctt agagctcctt ggagcgttgg catgacaaag ttttattgtg 12540
gttgggatca cttgttggag tcgttgccag atggttgggt gtattgcgat gctgatggct 12600
cacagtttga cagctcgcta tcaccatatt tgatcaatgc agtgctcaac atccgcttag 12660
gattcatgga agagtgggac gtaggggagg taatgctgag aaatttgtac accgaaattg 12720
tgtatacccc tatctctaca ccagatggta cactcgtcaa gaaattcaaa ggaaacaata 12780
gcggacagcc atcgaccgtt gtagacaaca cgctcatggt catactggca gtcaactatt 12840
cactcaagaa aagtggaatt ccaagtgagc tgcgcgacag tattatcaga ttcttcgtca 12900
acggagatga tttactgcta agcgtacacc cagagtatga gtatatcctt gacactatga 12960
cagacaactt tcatgaactg ggcctgaagt atgctttcga ctcaaggacc agggaaaaag 13020
gagacctttg gtttatgtcg caccaagggc acaaaaggga gggaatctgg attcccaagc 13080
ttgaaccaga gcgaatagta tcgattctag aatgggatcg gtcgaaagag ccatgccatc 13140
gactagaagc aatctgcgca gcgatgattg agtcgtgggg atacgacaag ctgactcacg 13200
agatacgcaa gttttacgca tggatgattg aacaagctcc atttagctcc ctagcacaag 13260
aagggaaagc tccttacata gcggaaacag cgctgaggaa gctctacctt gataaggaac 13320
cagctcaaga ggatctcacc cattacttgc aagcaatctt tgaggattat gaagatggtg 13380
atgaggcttg tgtttatcac caggcaggtg aaacgcttga tgcaggtttg acagacgagc 13440
aaaagcaggc ggagaaggag aagaaggaga gagagaaggc agaaaaggaa cgagagaggc 13500
agaagcagtt ggcactcaat aaaggcaagg atgttgcaca agaagaggga aaacgcgaca 13560
aggaagtaaa cgctggaact tctggaactt tcagtgtacc cagacttaag agtctgacaa 13620
gcaagatgcg tgtgccaaga tacgagcaaa gagtggctct aaacctcaat cacctaatcc 13680
tatacacgcc agagcagacg gatctatcca acacacgttc aacgcgaaag cagtttaaca 13740
catggtttga aggtgtaatg gctgattacg aactgacgga ggacaaaatg caaatcattc 13800
tcaatggttt aatggtctgg tgcattgaga acggaacctc cccgaacata aacggaatgt 13860
gggtgatgat ggacggcgat gatcaggtgg aattcccgat caaaccgctc attgaccacg 13920
ccaaacccac atttaggcag ataatggccc atttcagtga cgtagctgaa gcgtacattg 13980
aaaagcgtaa ccaagaccga ccatacatgc cacgatatgg tcttcagcgc aatttaaccg 14040
acatgagctt agctcgatac gcgtttgatt tctatgaaat gacttctaga actccaatac 14100
gtgcgagaga agcacacatc cagatgaaag cagcagcact gcgtggcgca aataacaatt 14160
tgttcggctt ggatggaaac gttggtacaa cggtagagaa cacggagagg catacgaccg 14220
aggacgttaa tcggaacatg cataacttac ttggcgttaa ggggttatga agttgtatgc 14280
tggtagacta taagtattta agtttactcg ttagtattct cgcttatggg aaatatgtaa 14340
gtttgttaaa gcagccagtg tgactctgtc atgtgtgttg ttcttacttt ctatattttc 14400
gccgaacatt ttattggtgt tagcgcatgt ggtgaggatt gtcctcgatt gccttaacat 14460
ttgataggat gcaagggaca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 14520
gtctgtactt atatcagtac 14540
<210> 5
<211> 684
<212> DNA
<213> pCB301-TuMV-phecc-Ros1
<400> 5
tgcgtatacc atcaagccat ggaaaagaat tgtcgtggag tgagaaaagg tacttggacc 60
aaagaagaag acactctctt gaggcaatgt atagaagagt atggtgaagg gaaatggcat 120
caagttccac acagagcagg gttgaaccgg tgtaggaaga gttgcaggct gaggtggttg 180
aattatctga ggccaaatat caaaagaggt cggttttcga gagatgaagt ggacctaatt 240
gtgaggcttc ataagctgtt gggtaacaaa tggtcgctga ttgctggtag aattcctgga 300
aggacagcta atgacgtgaa gaacttttgg aatactcatg tggggaagaa tttaggcgag 360
gatggagaac gatgccggaa aaatgttatg aacacaaaaa ccattaagct gactaatatc 420
gtaagacccc gagctcggac cttcaccgga ttgcacgtta cttggccgag agaagtcgga 480
aaaaccgatg aattttcaaa tgtccggtta acaactgatg agattccaga ttgtgagaag 540
caaacgcaat tttacaatga tgttgcgtcg ccacaagatg aagttgaaga ctgcattcag 600
tggtggagta agttgctaga aacaacggag gatggggaat taggaaacct attcgaggag 660
gcccaacaaa ttggaaatgc tagc 684
<210> 6
<211> 741
<212> DNA
<213> pCB301-TuMV-phecc-EFGP
<400> 6
tgcgtatacc atcaagccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 60
atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 120
gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 180
cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 240
taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 300
caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 360
ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 420
ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 480
gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 540
ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 600
ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 660
aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 720
gacgagctgt acaaggctag c 741
<210> 7
<211> 24
<212> DNA
<213> TuMV-phe-Exp-P1-F
<400> 7
acgtgcttgt ttgtatcggc ttac 24
<210> 8
<211> 24
<212> DNA
<213> TuMV-phe-Exp-P1-R
<400> 8
ctggtgataa acacaagcct catc 24
<210> 9
<211> 22
<212> DNA
<213> TuMV- phe-Exp-P2-R
<400> 9
gccgatacaa acaagcacgt ta 22
<210> 10
<211> 21
<212> DNA
<213> TuMV- phe-Exp-P2-F
<400> 10
gcaggtgaaa cgcttgatgc a 21
<210> 11
<211> 53
<212> DNA
<213> Ros1-NheI-F
<400> 11
aggcttgtgt ttatcaccag gcagctagca tggaaaagaa ttgtcgtgga gtg 53
<210> 12
<211> 63
<212> DNA
<213> Ros1-NcoI-Ps-R
<400> 12
gcatcaagcg tttcacctgc ctgatgatat acctcccatg gatttccaat ttgttgggcc 60
tcc 63
<210> 13
<211> 51
<212> DNA
<213> TuMV-phe-InfeClo-P1-F
<400> 13
gttcatttca tttggagagg aaaaaatata aaaactcaac acaacataca c 51
<210> 14
<211> 24
<212> DNA
<213> TuMV-phe-Clo-P1-R
<400> 14
tgtgtgacct attcgcagag cgtg 24
<210> 15
<211> 27
<212> DNA
<213> TuMV-phe-Clo-P2-F
<400> 15
ctctgcgaat aggtcacaca gagaagg 27
<210> 16
<211> 95
<212> DNA
<213> TuMV-phe-infeClo-P2-R
<400> 16
gactcgtcag tgtactgata taagtacaga cttttttttt tttttttttt tttttttttt 60
tttttttttt ttgtcccttg catcctatca aatgt 95
<210> 17
<211> 56
<212> DNA
<213> pCB301-Linz-F
<400> 17
tatcagtaca ctgacgagtc cctaaaggac gaaacggtac ccggatgtgt tttccg 56
<210> 18
<211> 24
<212> DNA
<213> pCB301-Linz-R
<400> 18
cctctccaaa tgaaatgaac ttcc 24
<210> 19
<211> 25
<212> DNA
<213> TuMV-phe-Expcc-P2-R
<400> 19
agcctcatca ccatcttcat aatcc 25
<210> 20
<211> 62
<212> DNA
<213> Ros1-cc-NcoI-F
<400> 20
atgaagatgg tgatgaggct tgcgtatacc atcaagccat ggaaaagaat tgtcgtggag 60
tg 62
<210> 21
<211> 63
<212> DNA
<213> Ros1-cc-NheI-Ps-R
<400> 21
gcatcaagcg tttcacctgc ctggtgataa acacagctag catttccaat ttgttgggcc 60
tcc 63
<210> 22
<211> 58
<212> DNA
<213> EGFP-F
<400> 22
atgaagatgg tgatgaggct tgcgtatacc atcaagccat ggtgagcaag ggcgagga 58
<210> 23
<211> 63
<212> DNA
<213> EGFP-R
<400> 23
gcatcaagcg tttcacctgc ctggtgataa acacagctag ccttgtacag ctcgtccatg 60
ccg 63
<210> 24
<211> 24
<212> DNA
<213> CP-Infec-Dect-F
<400> 24
cacctaatcc tatacacgcc agag 24
<210> 25
<211> 20
<212> DNA
<213> CP-Infec-Dect-R
<400> 25
tccatccaag ccgaacaaat 20
Claims (2)
1.一种以太子参TuMV-phe病毒基因为基础修饰改造病毒表达载体的方法，其特征在于：包括以下步骤：
（1）选择太子参TuMV-phe病毒基因组作为改造源，在TuMV-phe病毒基因组内编码NIb和CP蛋白基因间添加NIa-Pro酶切识别位点和限制性酶切位点，将修饰后的TuMV-phe克隆至植物表达载体pCB301内，构建获得TuMV-phe基础上的病毒表达载体TuMV-phece和TuMV-phecc；
（2）将TuMV-phe基础上的病毒表达载体TuMV-phecc转入农杆菌GV3101内，筛选阳性GV3101并扩大培养，收集GV3001菌体，用农杆菌侵染液将其稀释到OD600=0.8；用浸润法将配置好的农杆菌对4叶期的烟草幼苗侵染，同步对经过炼苗训练的太子参脱毒苗进行侵染；
所述TuMV-phece和TuMV-phecc表达载体为先在TuMV-phe的NIb和CP间5’位置插入Nie酶切位点和限制性酶切位点NheI，3’加入了NcoI限制性酶切位点；所述的太子参TuMV-phe病毒基因其序列如SEQ ID NO.1所示。
2.根据权利要求1所述的一种以太子参TuMV-phe病毒基因为基础修饰改造病毒表达载体的方法，其特征在于：选择Ros1基因和EGFP基因作为报告基因来评测TuMV-phe病毒基础上表达载体的外源表达蛋白能力。
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