CN113684188B

CN113684188B - 苦楝树缩叶病毒全基因组序列及其检测方法

Info

Publication number: CN113684188B
Application number: CN202110819767.7A
Authority: CN
Inventors: 孙凯; 周昕; 俞晓平; 叶子弘; 张蓬军; 尹传林
Original assignee: China Jiliang University
Current assignee: China Jiliang University
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2023-02-10
Anticipated expiration: 2041-07-20
Also published as: CN113684188A

Abstract

本发明公开了苦楝树缩叶病毒全基因组序列及其检测方法。该病毒全基因组序列如序列表SEQNO:1所示；该病毒的全基因组序列包含编码gp1、gp2、gp3以及gpY四个蛋白的核苷酸序列，四个核苷酸序列分别如序列表SEQ NO:2‑5所示，gp1、gp2、gp3以及gpY的氨基酸序列分别如SEQ NO:6‑9所示。本发明还提供了检测所述苦楝树缩叶病毒的特异性检测引物，本发明设计的引物具有特异性强、结果可靠，在普通PCR检测中都有稳定可靠的表现。

Description

苦楝树缩叶病毒全基因组序列及其检测方法

技术领域

本发明涉及测序和生物信息学技术领域，具体涉及一种苦楝树缩叶病毒全基因组系列及其检测方法

背景技术

苦楝树(Melia azedaeach L.)为楝科落叶乔木。原产于澳大利亚和东南亚，在我国常分布于四川、云南、贵州、江苏和浙江等省。该植物在湿润的沃土上生长迅速，对土壤要求不严，在酸性土、中性土与石灰岩地区均能生长，是平原及低海拔丘陵区的良好造林树种。苦楝树亦是药用植物，其全株或特定部位(叶、茎、根)均可入药，常用于治疗腹泻、糖尿病、风湿、高血压等疾病。目前苦楝的研究主要集中在栽培和药用活性方面，国内尚未发现感染苦楝的病毒。近期发现一种苦楝树病害普遍发生，呈爆发趋势，感病植株呈现症状植株矮小、叶片扭曲，防治形势不容乐观。如果任由该病害进一步传播蔓延，将会对我国林业带来很大影响。

发明内容

本发明的目的是针对现有技术暂无苦楝树病毒病害报道，以及现阶段缺失苦楝树缩叶病毒的检测手段的问题，提供了苦楝树缩叶病毒全基因组序列及其检测方法和相关引物。

本发明首先提供了苦楝树缩叶病毒全基因组序列，其全基因组序列如序列表SEQNO:1所示；该病毒的全基因组序列包含编码gp1、gp2、gp3以及gpY四个蛋白的核苷酸序列，四个核苷酸序列分别如序列表SEQ NO:2-5所示，gp1、gp2、gp3以及gpY的氨基酸序列分别如SEQ NO:6-9所示。

另一方面，本发明提供了一种用于扩增所述苦楝树缩叶病毒全基因组序列的扩增引物，所述扩增引物包括两对扩展引物对，其序列如下：

CLSV-1-F：CTGCTAACTGGTTCTCCTTTCC(SEQ NO:10)；

CLSV-1-R：GATCCTGCTACGGGAACAAC(SEQ NO:11)；

CLSV-2-F：GGGTAAGTCTTGTTGATTACATTACTG(SEQ NO:12)；

CLSV-2-R：CCAAATCTCTGTATGCCTGATC(SEQ NO:13)。

本发明还提供了一种所述苦楝树缩叶病毒全基因组序列获取方法，其使用所述扩增引物进行扩增，包括如下步骤：

1)抽提病毒侵染组织的总DNA；

2)采用所述扩增引物，以所述总DNA为模板，进行PCR扩增；

3)回收PCR扩增得到的DNA产物，并将其连接到pLB平末端载体，进行DNA测序，得到苦楝树缩叶病毒全基因组序列。

所述步骤(3)中，回收长度为3223bp和3931bp的DNA产物。

另一方面，本发明以后了一种检测所述苦楝树缩叶病毒的特异性检测引物，所述特异性检测引物如下：

CLSV-d1-F：GAGAATGATGTATCCCACGG(SEQ NO:14)；

CLSV-d1-R：CCTGATCCTGAATATGTGTTGT(SEQ NO:15)。

本发明还提供了一种检测所述苦楝树缩叶病毒的方法，其特征在于使用所述特异性检测引物进行PCR检测。

优选的，所述检测方法包括以下步骤：

(1)提取疑似感病的苦楝树总DNA；

(2)将步骤(1)终产物植物总DNA稀释到100ng/μl，用所述检测引物进行普通定性PCR扩增；

(3)检测，用1％琼脂糖凝胶电泳检测PCR产物条带，用1kb DNA marker作为片段大小对照标准，根据电泳图确定检测结果。

本发明利用siRNA测序和生物信息学技术，已明确引发该病害的为一种新的植物病毒，属于花椰菜花叶病毒科，杆状DNA病毒属(暂命名为苦楝树缩叶病毒，Chinaberryleaf shrinking virus，CLSV)，我们以病毒感染的苦楝树叶片为材料，经DNA提取、PCR克隆等方法获得了该病毒的完整基因组信息。利用生物信息学软件明确病毒蛋白编码情况和氨基酸序列根据该病毒的基因组序列特性，设计了检测引物，可用于检测该病毒在苦楝树及其他有机体中的存在。我们设计的引物具有特异性强、结果可靠，在普通PCR检测有稳定可靠的表现。

附图说明

图1为苦楝树缩叶病毒侵染症状。左为发病叶片，右为健康叶片；

图2为苦楝树缩叶病毒基因组结构简图；

图3为苦楝树缩叶病毒与其他杆状DNA病毒属成员的进化树分析；

图4为检测引物实际样品检测。

具体实施方式

下面结合具体实施方式对本发明做进一步阐述和说明。本发明中各个实施方式的技术特征在没有相互冲突的前提下，均可进行相应组合。

1.基于siRNA深度测序的植物病毒鉴定技术

(1)样品采集与RNA提取：将田间发现有病毒症状(花叶、卷叶、黄化、皱缩等)的苦楝树叶发病部位剪下，放入采集袋中，做好标记；回到实验室后，立即用液氮研磨，利用Trizol法提取植物总RNA，或者用液氮速冻后，立即保存于-80度冰箱备用。其中，苦楝树缩叶病毒侵染症状如图1所示。

植物总RNA提取

1)取0.1g植物叶片，液氮中快速研磨至粉末；

2)粉末转移至1.5ml离心管中，加入1ml Trizol，振荡混匀，室温静置5min；

3)向离心管中加入0.2mL氯仿，剧烈摇晃15sec后室温静置3min；

4)4℃12,000rpm离心15min，小心吸取上清转移至新的1.5mL离心管中，重复步骤3到4一到两遍；

5)将上清转移到新的1.5mL离心管中，加入等体积预冷的异丙醇后颠倒数次混匀，室温放置20～30min；

6)4℃12,000rpm离心10min，弃上清，用1mL 70％酒精洗涤沉淀两次；

7)4℃12,000rpm离心5min，弃上清，室温干燥沉淀；

8)用50 L DEPC处理水溶解沉淀；

9)取适量样品进行琼脂糖凝胶电泳检测提取RNA的质量；

10)将样品置于-80℃冰箱保存。

(2)siRNA深度测序：将提取的样品总RNA送至上海美吉生物科技有限公司进行siRNA建库与测序。

(3)siRNA深度测序数据处理与分析：利用生物信息学软件对测序数据进行原始数据过滤(去除原始数据中包含的接头信息、低质量序列)、序列拼接(根据小RNA具有overlap的特点，用Velvet软件对上述小RNA进行序列拼接，得到若干contigs)、序列比对(上述contigs在NCBI数据库中进行BLASTn和BLASTx比对，得到这些contigs在病毒基因组上的位置)。

2.苦楝树缩叶病毒的全基因组序列测定

(1)植物DNA提取

采用CTAB法抽提病毒侵染组织的总DNA，具体方法为：取0.2g植物组织叶片，在液氮中磨成粉末，将粉末转移至1.5ml离心管中，加入600μL 65℃预热的CTAB提取液，快速混匀，65℃条件下保温30min，不时颠倒混匀；待冷至室温后加入500μL氯仿/异戊醇(24：1)，颠倒混匀，12000rpm，离心5min；取上清液，加入600μL预冷的异丙醇，颠倒混匀，12000rpm，离心5min；弃去上清液，沉淀用800μL 70％的乙醇洗涤沉淀两次；稍干燥，将DNA沉淀溶解于30μL灭菌的双蒸水，4℃保存。

(2)病毒序列的扩增与测序

根据病毒来源的contigs设计2对特异性病毒全长基因组扩增引物，(CLSV-1-F：CTGCTAACTGGTTCTCCTTTCC和CLSV-1-R：GATCCTGCTACGGGAACAAC；以及CLSV-2-F：GGGTAAGTCTTGTTGATTACATTACTG和CLSV-2-R：CCAAATCTCTGTATGCCTGATC；)利用PCR技术获得片段1:3223bp；片段2:3931bp。利用snapgene软件进行拼接获得全长为7018bp的环状DNA病毒基因组。PCR反应体系如下：以发病植物总DNA作为模板，选择KOD-Plus-Neo高保真DNA聚合酶进行目的片段的扩增，反应体系如下：

PCR反应循环的设置：

回收长度为3223bp和3931bp的DNA产物，并将其连接到pLB平末端载体，终载体送杭州有康生物技术公司进行DNA测序。

pLB平末端连接反应体系如下：

轻弹离心管以混匀反应液，短暂离心3-5sec，将混合反应液放置室温(22℃)反应5min。反应结束后，将离心管置于冰上，进行后续的转化反应。

3.病毒基因组生物信息学分析

(1)病毒基因组结构分析：利用NCBI ORF finder软件以及SnapGene软件预测病毒编码ORF；利用NCBI保守区分析工具对病毒基因上的保守结构域进行分析。

病毒基因组核苷酸序列分析显示该病毒与可可枝肿病毒(Cacao swollen shootvirus，CSSV)基因组序列具有61.3％的相似性。

利用Snapgene软件对该病毒编码基因进行预测分析显示，该病毒的病毒链编码gp1、gp2、gp3以及gpY四个蛋白。氨基酸同源性比对发现病毒编码的gp1、gp2、gp3氨基酸序列与CSSV编码氨基酸列相似性在51％～67％之间；而gpY与CSSV编码蛋白无显著相似性。苦楝树缩叶病毒基因组结构简图如图2所示，苦楝树缩叶病毒与其他杆状DNA病毒属成员的进化树分析如图3所示。

4.苦楝树缩叶病毒检测方法

1、提取疑似感病的苦楝树总DNA(方法如上文所示)。

2、将终产物稀释到100ng/ul，后用普通定型PCR检测，引物为CLSV-d1-F：GAGAATGATGTATCCCACGG+CLSV-d1-R：CCTGATCCTGAATATGTGTTGT(产物143bp)。

以2xTaq PCR Mix含燃料产品为例(南京喏维赞Green Taq Mix)普通PCR体系如下：

PCR反应循环的设置

检测方法为：用1％琼脂糖凝胶电泳检测PCR产物条带，用1kb DNA marker(ThermoFisher GeneRuler 1kb DNA Ladder)作为片段大小对照标准。

本实施例对8个样品进行了检测，其中1,4和8为具有缩叶症状的植物样品，“+”表示阳性质控质粒，“-”代表未添加模版的阴性对照PCR反应，其与样品为无症状叶片。检测结果如图4所示，从结果可见，本发明的特异性检测引物特异性强、结果可靠，在普通PCR检测中有稳定可靠的表现

以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明的保护范围应以所附权利要求为准。

序列表

<110> 中国计量大学

<120> 苦楝树缩叶病毒全基因组序列及其检测方法

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7018

<212> DNA

<213> 苦楝树缩叶病毒(Chinaberry leaf shrinking virus)

<400> 1

tggtatcaga gcagccagat ttaagctttt tatctggttg ctttctatgt taaagaagac 60

aaccagaaga atcttcatat ctgtaagttt cgctcaacta cttttaaatt tcttagataa 120

tttgtgcaag tttgatgatg ttttattaat gtacaggctg taaaagttat ctagaggagg 180

attctctttt ctttgtcaga ggtgaataga aaataaagaa gaaaagaaga aggtttctga 240

aagaaatatt aatgtctgcg caagaagaga actcgatcag acagtggtct cacaagtctc 300

atacagctaa cttggaggtc atcgatttgg aagatacgag aaagatcctc aaccataccc 360

tcgcgcacaa cattgcggtt acggcagatc gtgtgtcctt gagtgaacag gtgtttatca 420

agaacctcaa acaaatacac gaggatatac tccaactgat aagagaagtg gagtctaagt 480

ctatgtcttt gaagcgttgc tctcagccct tgcgtatcaa caaacctctc accgaggatt 540

tgtgcaagat actggtcaag ctcttggcat cccagccgaa actcgttgag gaaagggctt 600

tgtcactgac gcaagaagtt gaatgtaagt tgaatcgtgt ggagcaactt ctccatgagt 660

tggaaatttg tgttggacaa tgactaaaca gcctatgtac ccaaaagctg tgcgtgctgc 720

gctgagagtt aagccgcaaa tgcttaagca cactgctatt ggtggaatta cggctattat 780

actattatta aacaaaataa tgtgcagata cgtccagatc cttctcctcg ctcttatcgc 840

taagaagctg gacgatgaaa gcgtcagagc actgacaccg aagtcaaaac acgtttacca 900

gaaaaactta tcgacaagct ttcacgtctt cagattcaga aaggggagca aaggtgaaca 960

gcaggatacc aaacaaagag agggtaaagg cacacttatc ggtgacaagg atccgtataa 1020

aatcctggaa gaagaacgcg caaaactcgg attgcagcag agccaaaagg atgaatcaaa 1080

ggggaagcga acagccgcag agacatagtg acaccaccac aggctccgaa cctgaaacag 1140

caagcgttca gactcttacc gaagataaca ttagtggtcc aacacaaccg caagaacgaa 1200

tccaaaaggt gcgcaggcgc aagcacagtc tgacccgata cttatcccat gagccaaaac 1260

cacactacag acacggaaga aggaaggata cactggaaga ggagaagggc caccaggcaa 1320

ccataaggct ttacatgcaa gaaagcccac agcttgtgct ctcagaagta ttacaactgt 1380

ccagagggga ttttgtaaaa ctttacgttt ataccccacc gtcagaagaa tcggttctag 1440

taactgacgg aaaccagctg gacaggacat tcatacagcc agaaagcctc gagcaattac 1500

aacgatctgg ctactcattc atccacgtgg gaatcctaca atttagggtt caaatactgc 1560

atagagctga agaagggacc atggcaatgg tcgtattcag agataacaga tggcaaggag 1620

atcaagccat ctttgcaacc atggagattg atttgacaaa aggatatctc atggtgtatg 1680

tcatccctga cacaatgatg acagttgggg actttgcccg aaatattggt atttcagtgc 1740

aaacgcgggg atacagcaat tggcagaacg gagaagccaa cttgctaata accaggggca 1800

tgacagggag attatcaaat actcccaacg tggcatttgc ttatcaggta gacggagcag 1860

ttgactacct aatcagccat ggagttaaag ccatagcagg aaagaaatac gaccctagca 1920

gattaagggg tcaacaatgg gtgatgcgac caccgagact ggacatcaca cccatgcagc 1980

cacaaacagc agaaacaaga accttaatag acggctctgt gtctattagg tttggagatt 2040

atcaggcatc aacatcaagg ccaccgagat acaatgaaga ggatgaagag gcagcttcaa 2100

cagaagaaga gattatacct gtctggatga ttggcccaga agagcagcct gatgatacag 2160

acagagtcaa agtctgggaa gagagttgca gtggcaatgg cagattctac agatactaca 2220

ctgcaccccc acagcatgtt ggcgaaatca tagccacagg atggggctca gatgatgatt 2280

ctgacgagct agaactggaa gtccaacagt ttcttgagac tctggagcat gaggatgatg 2340

ctgacatgca cccagaacat caagaatttc ttgatggact caccgatggg ctcactacat 2400

ggtccattga ttcctatacg gaaggtggtg gggaggaaca gttaatcgca acatttggcg 2460

aaatggaata cccaacactt agaagaatcg ttgaggaaaa taggaatcag gcccaagatg 2520

aaggcccaag cccaagttta aaaagggacc agcaggtgta ttcacactca gaagtggtgg 2580

actacaaccc accagcggat atagccatga caccagtagg ctacccacct gctacaaatt 2640

tggaaatacc agcccagcca cagtatgatg gagcaagacc cagaggctca tttaagggat 2700

ccaaatacaa tgaattgtgg caactacctt cggcccaaac caacactggg gccatatttg 2760

taatgccaag gcagattgaa ctattccatg acgtgtttga cagatgggag tctataacaa 2820

agaactatgt gaccatgcaa ggtattactg atgcaacaga gaaggtagaa tttattgaaa 2880

atctactggg agaaatggag aaacaaacgt ggatacagtg gagaatgatg tatcccacgg 2940

agtttgaaac aatcataaca agggctgagg gaagagaagg gacacagaat attctgtccc 3000

agataagaag agtgttctcg ctagaggatc ctgctacggg aacaacacat attcaggatc 3060

aggcatacag agatttggaa aggttgactt gcaataacgt aaaggacctt gtccaatatc 3120

taaatgattt tggaaggctt gcagcaaaaa caggaaggat gtttctgaac actgaattaa 3180

gtgataagct gtggttgaaa atgcctggcg atattggcca gagaatgaaa atggctttca 3240

atgccaaatt cccggggaac cagataggcg tcttcccgag aatcctttat gcttatcagt 3300

tcatggaaca ggaatgcaag aacgcagctt tccagagaag cttgaagaac ctggccttct 3360

gcaaagacat tccttttcca ggatattatt ctgagtcttc aaagagcaga tacggggcta 3420

gaaagtctac aacctacaaa gggaaggctc actccacaca cgccagaatt gaaaagaaaa 3480

agcacctggt cagaaacagg aggtgtaaat gttatctgtg tggtgatgaa ggccccttcg 3540

ctagagaatg ccctaatgaa agaaggaatg taaggagggt tgctgttttt gaaaaccttg 3600

ccttaccaga cgactatgag attgtatctg tgcaggacgg agaagaagaa tctgatgcca 3660

tatacagtct atcggaaaac gagcacgaga atggggtgga ccttgaacca gtggtgggag 3720

cagcaacaga agagacttgc ttcatgttca gggaagaaga caactcgtat tgggtaggaa 3780

aaggaggcta cttagcctat aaaagagtaa caaaggccca gtacgaatgc aagcacactt 3840

gggctcataa caaagaagtt gaggcccaat ggataaactg cagcttctgc aagaggccca 3900

ctaacagaaa atgtagggct cattgtccaa aatgcaggct tactacttgc gaaatgtgtg 3960

ctgacccatt ctgtgagatt aatgtagtca gagaaccaga gatttacaag ccttatgatc 4020

cacatggctt gatacaacaa caaagggact acatcgcgtg gtgtgaagga gagatgagta 4080

gactccggag ggaagtggag ttctacaaga aaaaggaaga agacaggctg gctaaagacc 4140

ttgaaaagga gagagcaaga caagaaaaaa ttgatgcaga gagagaagca tccagctcca 4200

aaggaaaaga agtgctagag gaaataaagg aagaatcaag tgatggagag atttccgcct 4260

attgggaatc tgaaagcact gtcaaggtca gaaccatagc agctgctgca ggagaatctg 4320

agcagaaagt caaagaagag aagccagtac gagagaacaa caatctctac aacatgaccg 4380

tggaagttga tattccagga gtgcctaaat tttcattaag ggcaatcctt gataccggag 4440

caacggtatg ttgtgtagat tcgaaagctc tgccaaagga agctattgaa gacaacacct 4500

ttgaagttca tttcagaggt atcaattcaa agcagagtgt gaacaaaaaa atgaaatatg 4560

ggaggatgtc agtaggaggg cataatttca gaatcccata tacgtatact ttccctatgg 4620

atcttggagg gaaaatacag tttattcttg gttgtaattt catcagaagc atgtacggag 4680

gagtaaggct agaaggtaac acagtaacct tctacaaaaa tattacagcc attgatacca 4740

agaccgtggc agttctggaa gaactagaag aagaagattt acagctgtat aatctagctg 4800

tactgccaca aaagggagga gaagcctttg aaagaaaaaa cagaaggctg atcgaggagt 4860

tgaaaaacca gggttacata ggcaatgagc ctatgaaata ttggattaaa aatcaagtta 4920

catgcaaact tgatatcaaa aaccctgaga tggtaataga agacaagcct ctgaaacatg 4980

tgacaccagc aatggaggaa caattcagaa gtcacatcaa ggctttgata gacctgaagg 5040

tgataaggcc aagtaaaagt cgtcacagaa caacggcttt tatggtaaat tcgggaacaa 5100

ccatcgaccc gattacaaag aaagtggtgc atggaaagga aagaatggta ttcaactata 5160

aaaggctgaa tgatctaacc cacaaggatc agtatagtct accaggaatc cacaccatca 5220

tgaggagaat tggccaagcc cagatatttt ccaagtttga tctgaaatcg ggctttcacc 5280

aagtagccat ggacccggag tctgtagaat ggacagcatt ttgggttcca gatgggcttt 5340

atgaatggct ggttattcca ttcgggttaa aaaatgctcc agctatattc caaagaaaga 5400

tggaccaatg cttcaaggga acagagaagt tcattgcggt ctacatagat gacatcttgg 5460

tcttttccaa gaacgaagag gagcatgcag gccatgtaag aaaaatgttg gaaatagtaa 5520

agtctaatgg actaatccta agcccaacaa agatgtgcct ggcccaacga tcggttgaat 5580

ttctaggggc aattatcaca ggaggaaagc tccaattaca gccccacatc atcaagaagg 5640

tggtccagtt cgataagccc agcttagaga ccactaaagg actaagagga tttcttgggc 5700

tcctcaacta tgcaagggcg tatatcccta atttgggccg actgctaagc ccattatacg 5760

ccaaaacaag cccaacagga gaaagaaggt tcagcaaaga agactggaag cttgtcagca 5820

acatccagac agaggtacag aagctaccgg acctgaacct accaccggag gagtgctgta 5880

taatcatcga aacagatggt tgtatagacg gatggggagc tgtctgcaaa tggaagaaga 5940

agagagcaga tagcagaaat acagaaatgg tctgcgccta tggtagtgga aagtttgaaa 6000

atcccaaatc caccatagat gctgaaataa cagccgtgat taaagcactg gagtctttta 6060

agatttatta tttagataaa cagtgtttag tcattagaac agattgtcag gccattgtat 6120

cgtttttcaa caagtcctgt aaccataaac cgtctagggt taggtgggta agtcttgttg 6180

attacattac tgggatggga ttagaagtca agttcgagca tattgatgga aaggagaacc 6240

agttagcaga cgtcctttca aggctaatct tccaaacatg gaaccaatct cctcaacaga 6300

caagtcagca gcttcttcaa gattgtctgc acagaaactc tttgatgtca attatccatg 6360

ttgctttacc tatgaaaaaa tatgtttcca acattcgtca gccggttccg gctgtaccag 6420

ttttgagcgc ccctacggct ccatttctaa tcaattccac tgggaagaaa atccaggagg 6480

agaaaagact ggagcttctt tacgctcact acaaagcatt atacggcatt ctgcgctacg 6540

cgtggtggga tctcacaacc tcagcggcca gcgactactg gaccaaacag ctttcagaaa 6600

gagttcctca aatatggacc ttcattcaac caccgagccc cctctaaaga cacagaagcc 6660

tatcgactaa ctcattggga tctgttcacc ttctccgtgt cttgttcaca taaacgtcat 6720

ctagccctca tctgccgaat cagaaaacaa ctaactcata agatcaataa cgcctccctt 6780

ttcatatttc tttcgtagaa agacatttgt acgccatttt caatattaac tgcgtgtagt 6840

ccaataaagc ttaaattgtt ttctaatgtg ctactgaccc tcaatcatat catacagaga 6900

gggttccaag ttagaacatt gaagcacccg gattatttac aatctatatt ggattaagtt 6960

ttgtgaacta agtctcgagt atagtcatat cccgcaaaat tttccacgac actaacaa 7018

<210> 2

<211> 432

<212> DNA

<213> 苦楝树缩叶病毒(Chinaberry leaf shrinking virus)

<400> 2

atgtctgcgc aagaagagaa ctcgatcaga cagtggtctc acaagtctca tacagctaac 60

ttggaggtca tcgatttgga agatacgaga aagatcctca accataccct cgcgcacaac 120

attgcggtta cggcagatcg tgtgtccttg agtgaacagg tgtttatcaa gaacctcaaa 180

caaatacacg aggatatact ccaactgata agagaagtgg agtctaagtc tatgtctttg 240

aagcgttgct ctcagccctt gcgtatcaac aaacctctca ccgaggattt gtgcaagata 300

ctggtcaagc tcttggcatc ccagccgaaa ctcgttgagg aaagggcttt gtcactgacg 360

caagaagttg aatgtaagtt gaatcgtgtg gagcaacttc tccatgagtt ggaaatttgt 420

gttggacaat ga 432

<210> 3

<211> 429

<212> DNA

<213> 苦楝树缩叶病毒(Chinaberry leaf shrinking virus)

<400> 3

atgactaaac agcctatgta cccaaaagct gtgcgtgctg cgctgagagt taagccgcaa 60

atgcttaagc acactgctat tggtggaatt acggctatta tactattatt aaacaaaata 120

atgtgcagat acgtccagat ccttctcctc gctcttatcg ctaagaagct ggacgatgaa 180

agcgtcagag cactgacacc gaagtcaaaa cacgtttacc agaaaaactt atcgacaagc 240

tttcacgtct tcagattcag aaaggggagc aaaggtgaac agcaggatac caaacaaaga 300

gagggtaaag gcacacttat cggtgacaag gatccgtata aaatcctgga agaagaacgc 360

gcaaaactcg gattgcagca gagccaaaag gatgaatcaa aggggaagcg aacagccgca 420

gagacatag 429

<210> 4

<211> 5577

<212> DNA

<213> 苦楝树缩叶病毒(Chinaberry leaf shrinking virus)

<400> 4

atgaatcaaa ggggaagcga acagccgcag agacatagtg acaccaccac aggctccgaa 60

cctgaaacag caagcgttca gactcttacc gaagataaca ttagtggtcc aacacaaccg 120

caagaacgaa tccaaaaggt gcgcaggcgc aagcacagtc tgacccgata cttatcccat 180

gagccaaaac cacactacag acacggaaga aggaaggata cactggaaga ggagaagggc 240

caccaggcaa ccataaggct ttacatgcaa gaaagcccac agcttgtgct ctcagaagta 300

ttacaactgt ccagagggga ttttgtaaaa ctttacgttt ataccccacc gtcagaagaa 360

tcggttctag taactgacgg aaaccagctg gacaggacat tcatacagcc agaaagcctc 420

gagcaattac aacgatctgg ctactcattc atccacgtgg gaatcctaca atttagggtt 480

caaatactgc atagagctga agaagggacc atggcaatgg tcgtattcag agataacaga 540

tggcaaggag atcaagccat ctttgcaacc atggagattg atttgacaaa aggatatctc 600

atggtgtatg tcatccctga cacaatgatg acagttgggg actttgcccg aaatattggt 660

atttcagtgc aaacgcgggg atacagcaat tggcagaacg gagaagccaa cttgctaata 720

accaggggca tgacagggag attatcaaat actcccaacg tggcatttgc ttatcaggta 780

gacggagcag ttgactacct aatcagccat ggagttaaag ccatagcagg aaagaaatac 840

gaccctagca gattaagggg tcaacaatgg gtgatgcgac caccgagact ggacatcaca 900

cccatgcagc cacaaacagc agaaacaaga accttaatag acggctctgt gtctattagg 960

tttggagatt atcaggcatc aacatcaagg ccaccgagat acaatgaaga ggatgaagag 1020

gcagcttcaa cagaagaaga gattatacct gtctggatga ttggcccaga agagcagcct 1080

gatgatacag acagagtcaa agtctgggaa gagagttgca gtggcaatgg cagattctac 1140

agatactaca ctgcaccccc acagcatgtt ggcgaaatca tagccacagg atggggctca 1200

gatgatgatt ctgacgagct agaactggaa gtccaacagt ttcttgagac tctggagcat 1260

gaggatgatg ctgacatgca cccagaacat caagaatttc ttgatggact caccgatggg 1320

ctcactacat ggtccattga ttcctatacg gaaggtggtg gggaggaaca gttaatcgca 1380

acatttggcg aaatggaata cccaacactt agaagaatcg ttgaggaaaa taggaatcag 1440

gcccaagatg aaggcccaag cccaagttta aaaagggacc agcaggtgta ttcacactca 1500

gaagtggtgg actacaaccc accagcggat atagccatga caccagtagg ctacccacct 1560

gctacaaatt tggaaatacc agcccagcca cagtatgatg gagcaagacc cagaggctca 1620

tttaagggat ccaaatacaa tgaattgtgg caactacctt cggcccaaac caacactggg 1680

gccatatttg taatgccaag gcagattgaa ctattccatg acgtgtttga cagatgggag 1740

tctataacaa agaactatgt gaccatgcaa ggtattactg atgcaacaga gaaggtagaa 1800

tttattgaaa atctactggg agaaatggag aaacaaacgt ggatacagtg gagaatgatg 1860

tatcccacgg agtttgaaac aatcataaca agggctgagg gaagagaagg gacacagaat 1920

attctgtccc agataagaag agtgttctcg ctagaggatc ctgctacggg aacaacacat 1980

attcaggatc aggcatacag agatttggaa aggttgactt gcaataacgt aaaggacctt 2040

gtccaatatc taaatgattt tggaaggctt gcagcaaaaa caggaaggat gtttctgaac 2100

actgaattaa gtgataagct gtggttgaaa atgcctggcg atattggcca gagaatgaaa 2160

atggctttca atgccaaatt cccggggaac cagataggcg tcttcccgag aatcctttat 2220

gcttatcagt tcatggaaca ggaatgcaag aacgcagctt tccagagaag cttgaagaac 2280

ctggccttct gcaaagacat tccttttcca ggatattatt ctgagtcttc aaagagcaga 2340

tacggggcta gaaagtctac aacctacaaa gggaaggctc actccacaca cgccagaatt 2400

gaaaagaaaa agcacctggt cagaaacagg aggtgtaaat gttatctgtg tggtgatgaa 2460

ggccccttcg ctagagaatg ccctaatgaa agaaggaatg taaggagggt tgctgttttt 2520

gaaaaccttg ccttaccaga cgactatgag attgtatctg tgcaggacgg agaagaagaa 2580

tctgatgcca tatacagtct atcggaaaac gagcacgaga atggggtgga ccttgaacca 2640

gtggtgggag cagcaacaga agagacttgc ttcatgttca gggaagaaga caactcgtat 2700

tgggtaggaa aaggaggcta cttagcctat aaaagagtaa caaaggccca gtacgaatgc 2760

aagcacactt gggctcataa caaagaagtt gaggcccaat ggataaactg cagcttctgc 2820

aagaggccca ctaacagaaa atgtagggct cattgtccaa aatgcaggct tactacttgc 2880

gaaatgtgtg ctgacccatt ctgtgagatt aatgtagtca gagaaccaga gatttacaag 2940

ccttatgatc cacatggctt gatacaacaa caaagggact acatcgcgtg gtgtgaagga 3000

gagatgagta gactccggag ggaagtggag ttctacaaga aaaaggaaga agacaggctg 3060

gctaaagacc ttgaaaagga gagagcaaga caagaaaaaa ttgatgcaga gagagaagca 3120

tccagctcca aaggaaaaga agtgctagag gaaataaagg aagaatcaag tgatggagag 3180

atttccgcct attgggaatc tgaaagcact gtcaaggtca gaaccatagc agctgctgca 3240

ggagaatctg agcagaaagt caaagaagag aagccagtac gagagaacaa caatctctac 3300

aacatgaccg tggaagttga tattccagga gtgcctaaat tttcattaag ggcaatcctt 3360

gataccggag caacggtatg ttgtgtagat tcgaaagctc tgccaaagga agctattgaa 3420

gacaacacct ttgaagttca tttcagaggt atcaattcaa agcagagtgt gaacaaaaaa 3480

atgaaatatg ggaggatgtc agtaggaggg cataatttca gaatcccata tacgtatact 3540

ttccctatgg atcttggagg gaaaatacag tttattcttg gttgtaattt catcagaagc 3600

atgtacggag gagtaaggct agaaggtaac acagtaacct tctacaaaaa tattacagcc 3660

attgatacca agaccgtggc agttctggaa gaactagaag aagaagattt acagctgtat 3720

aatctagctg tactgccaca aaagggagga gaagcctttg aaagaaaaaa cagaaggctg 3780

atcgaggagt tgaaaaacca gggttacata ggcaatgagc ctatgaaata ttggattaaa 3840

aatcaagtta catgcaaact tgatatcaaa aaccctgaga tggtaataga agacaagcct 3900

ctgaaacatg tgacaccagc aatggaggaa caattcagaa gtcacatcaa ggctttgata 3960

gacctgaagg tgataaggcc aagtaaaagt cgtcacagaa caacggcttt tatggtaaat 4020

tcgggaacaa ccatcgaccc gattacaaag aaagtggtgc atggaaagga aagaatggta 4080

ttcaactata aaaggctgaa tgatctaacc cacaaggatc agtatagtct accaggaatc 4140

cacaccatca tgaggagaat tggccaagcc cagatatttt ccaagtttga tctgaaatcg 4200

ggctttcacc aagtagccat ggacccggag tctgtagaat ggacagcatt ttgggttcca 4260

gatgggcttt atgaatggct ggttattcca ttcgggttaa aaaatgctcc agctatattc 4320

caaagaaaga tggaccaatg cttcaaggga acagagaagt tcattgcggt ctacatagat 4380

gacatcttgg tcttttccaa gaacgaagag gagcatgcag gccatgtaag aaaaatgttg 4440

gaaatagtaa agtctaatgg actaatccta agcccaacaa agatgtgcct ggcccaacga 4500

tcggttgaat ttctaggggc aattatcaca ggaggaaagc tccaattaca gccccacatc 4560

atcaagaagg tggtccagtt cgataagccc agcttagaga ccactaaagg actaagagga 4620

tttcttgggc tcctcaacta tgcaagggcg tatatcccta atttgggccg actgctaagc 4680

ccattatacg ccaaaacaag cccaacagga gaaagaaggt tcagcaaaga agactggaag 4740

cttgtcagca acatccagac agaggtacag aagctaccgg acctgaacct accaccggag 4800

gagtgctgta taatcatcga aacagatggt tgtatagacg gatggggagc tgtctgcaaa 4860

tggaagaaga agagagcaga tagcagaaat acagaaatgg tctgcgccta tggtagtgga 4920

aagtttgaaa atcccaaatc caccatagat gctgaaataa cagccgtgat taaagcactg 4980

gagtctttta agatttatta tttagataaa cagtgtttag tcattagaac agattgtcag 5040

gccattgtat cgtttttcaa caagtcctgt aaccataaac cgtctagggt taggtgggta 5100

agtcttgttg attacattac tgggatggga ttagaagtca agttcgagca tattgatgga 5160

aaggagaacc agttagcaga cgtcctttca aggctaatct tccaaacatg gaaccaatct 5220

cctcaacaga caagtcagca gcttcttcaa gattgtctgc acagaaactc tttgatgtca 5280

attatccatg ttgctttacc tatgaaaaaa tatgtttcca acattcgtca gccggttccg 5340

gctgtaccag ttttgagcgc ccctacggct ccatttctaa tcaattccac tgggaagaaa 5400

atccaggagg agaaaagact ggagcttctt tacgctcact acaaagcatt atacggcatt 5460

ctgcgctacg cgtggtggga tctcacaacc tcagcggcca gcgactactg gaccaaacag 5520

ctttcagaaa gagttcctca aatatggacc ttcattcaac caccgagccc cctctaa 5577

<210> 5

<211> 393

<212> DNA

<213> 苦楝树缩叶病毒(Chinaberry leaf shrinking virus)

<400> 5

atggaaccaa tctcctcaac agacaagtca gcagcttctt caagattgtc tgcacagaaa 60

ctctttgatg tcaattatcc atgttgcttt acctatgaaa aaatatgttt ccaacattcg 120

tcagccggtt ccggctgtac cagttttgag cgcccctacg gctccatttc taatcaattc 180

cactgggaag aaaatccagg aggagaaaag actggagctt ctttacgctc actacaaagc 240

attatacggc attctgcgct acgcgtggtg ggatctcaca acctcagcgg ccagcgacta 300

ctggaccaaa cagctttcag aaagagttcc tcaaatatgg accttcattc aaccaccgag 360

ccccctctaa agacacagaa gcctatcgac taa 393

<210> 6

<211> 143

<212> PRT

<213> 苦楝树缩叶病毒(Chinaberry leaf shrinking virus)

<400> 6

Met Ser Ala Gln Glu Glu Asn Ser Ile Arg Gln Trp Ser His Lys Ser

1 5 10 15

His Thr Ala Asn Leu Glu Val Ile Asp Leu Glu Asp Thr Arg Lys Ile

20 25 30

Leu Asn His Thr Leu Ala His Asn Ile Ala Val Thr Ala Asp Arg Val

35 40 45

Ser Leu Ser Glu Gln Val Phe Ile Lys Asn Leu Lys Gln Ile His Glu

50 55 60

Asp Ile Leu Gln Leu Ile Arg Glu Val Glu Ser Lys Ser Met Ser Leu

65 70 75 80

Lys Arg Cys Ser Gln Pro Leu Arg Ile Asn Lys Pro Leu Thr Glu Asp

85 90 95

Leu Cys Lys Ile Leu Val Lys Leu Leu Ala Ser Gln Pro Lys Leu Val

100 105 110

Glu Glu Arg Ala Leu Ser Leu Thr Gln Glu Val Glu Cys Lys Leu Asn

115 120 125

Arg Val Glu Gln Leu Leu His Glu Leu Glu Ile Cys Val Gly Gln

130 135 140

<210> 7

<211> 142

<212> PRT

<213> 苦楝树缩叶病毒(Chinaberry leaf shrinking virus)

<400> 7

Met Thr Lys Gln Pro Met Tyr Pro Lys Ala Val Arg Ala Ala Leu Arg

1 5 10 15

Val Lys Pro Gln Met Leu Lys His Thr Ala Ile Gly Gly Ile Thr Ala

20 25 30

Ile Ile Leu Leu Leu Asn Lys Ile Met Cys Arg Tyr Val Gln Ile Leu

35 40 45

Leu Leu Ala Leu Ile Ala Lys Lys Leu Asp Asp Glu Ser Val Arg Ala

50 55 60

Leu Thr Pro Lys Ser Lys His Val Tyr Gln Lys Asn Leu Ser Thr Ser

65 70 75 80

Phe His Val Phe Arg Phe Arg Lys Gly Ser Lys Gly Glu Gln Gln Asp

85 90 95

Thr Lys Gln Arg Glu Gly Lys Gly Thr Leu Ile Gly Asp Lys Asp Pro

100 105 110

Tyr Lys Ile Leu Glu Glu Glu Arg Ala Lys Leu Gly Leu Gln Gln Ser

115 120 125

Gln Lys Asp Glu Ser Lys Gly Lys Arg Thr Ala Ala Glu Thr

130 135 140

<210> 8

<211> 1858

<212> PRT

<213> 苦楝树缩叶病毒(Chinaberry leaf shrinking virus)

<400> 8

Met Asn Gln Arg Gly Ser Glu Gln Pro Gln Arg His Ser Asp Thr Thr

1 5 10 15

Thr Gly Ser Glu Pro Glu Thr Ala Ser Val Gln Thr Leu Thr Glu Asp

20 25 30

Asn Ile Ser Gly Pro Thr Gln Pro Gln Glu Arg Ile Gln Lys Val Arg

35 40 45

Arg Arg Lys His Ser Leu Thr Arg Tyr Leu Ser His Glu Pro Lys Pro

50 55 60

His Tyr Arg His Gly Arg Arg Lys Asp Thr Leu Glu Glu Glu Lys Gly

65 70 75 80

His Gln Ala Thr Ile Arg Leu Tyr Met Gln Glu Ser Pro Gln Leu Val

85 90 95

Leu Ser Glu Val Leu Gln Leu Ser Arg Gly Asp Phe Val Lys Leu Tyr

100 105 110

Val Tyr Thr Pro Pro Ser Glu Glu Ser Val Leu Val Thr Asp Gly Asn

115 120 125

Gln Leu Asp Arg Thr Phe Ile Gln Pro Glu Ser Leu Glu Gln Leu Gln

130 135 140

Arg Ser Gly Tyr Ser Phe Ile His Val Gly Ile Leu Gln Phe Arg Val

145 150 155 160

Gln Ile Leu His Arg Ala Glu Glu Gly Thr Met Ala Met Val Val Phe

165 170 175

Arg Asp Asn Arg Trp Gln Gly Asp Gln Ala Ile Phe Ala Thr Met Glu

180 185 190

Ile Asp Leu Thr Lys Gly Tyr Leu Met Val Tyr Val Ile Pro Asp Thr

195 200 205

Met Met Thr Val Gly Asp Phe Ala Arg Asn Ile Gly Ile Ser Val Gln

210 215 220

Thr Arg Gly Tyr Ser Asn Trp Gln Asn Gly Glu Ala Asn Leu Leu Ile

225 230 235 240

Thr Arg Gly Met Thr Gly Arg Leu Ser Asn Thr Pro Asn Val Ala Phe

245 250 255

Ala Tyr Gln Val Asp Gly Ala Val Asp Tyr Leu Ile Ser His Gly Val

260 265 270

Lys Ala Ile Ala Gly Lys Lys Tyr Asp Pro Ser Arg Leu Arg Gly Gln

275 280 285

Gln Trp Val Met Arg Pro Pro Arg Leu Asp Ile Thr Pro Met Gln Pro

290 295 300

Gln Thr Ala Glu Thr Arg Thr Leu Ile Asp Gly Ser Val Ser Ile Arg

305 310 315 320

Phe Gly Asp Tyr Gln Ala Ser Thr Ser Arg Pro Pro Arg Tyr Asn Glu

325 330 335

Glu Asp Glu Glu Ala Ala Ser Thr Glu Glu Glu Ile Ile Pro Val Trp

340 345 350

Met Ile Gly Pro Glu Glu Gln Pro Asp Asp Thr Asp Arg Val Lys Val

355 360 365

Trp Glu Glu Ser Cys Ser Gly Asn Gly Arg Phe Tyr Arg Tyr Tyr Thr

370 375 380

Ala Pro Pro Gln His Val Gly Glu Ile Ile Ala Thr Gly Trp Gly Ser

385 390 395 400

Asp Asp Asp Ser Asp Glu Leu Glu Leu Glu Val Gln Gln Phe Leu Glu

405 410 415

Thr Leu Glu His Glu Asp Asp Ala Asp Met His Pro Glu His Gln Glu

420 425 430

Phe Leu Asp Gly Leu Thr Asp Gly Leu Thr Thr Trp Ser Ile Asp Ser

435 440 445

Tyr Thr Glu Gly Gly Gly Glu Glu Gln Leu Ile Ala Thr Phe Gly Glu

450 455 460

Met Glu Tyr Pro Thr Leu Arg Arg Ile Val Glu Glu Asn Arg Asn Gln

465 470 475 480

Ala Gln Asp Glu Gly Pro Ser Pro Ser Leu Lys Arg Asp Gln Gln Val

485 490 495

Tyr Ser His Ser Glu Val Val Asp Tyr Asn Pro Pro Ala Asp Ile Ala

500 505 510

Met Thr Pro Val Gly Tyr Pro Pro Ala Thr Asn Leu Glu Ile Pro Ala

515 520 525

Gln Pro Gln Tyr Asp Gly Ala Arg Pro Arg Gly Ser Phe Lys Gly Ser

530 535 540

Lys Tyr Asn Glu Leu Trp Gln Leu Pro Ser Ala Gln Thr Asn Thr Gly

545 550 555 560

Ala Ile Phe Val Met Pro Arg Gln Ile Glu Leu Phe His Asp Val Phe

565 570 575

Asp Arg Trp Glu Ser Ile Thr Lys Asn Tyr Val Thr Met Gln Gly Ile

580 585 590

Thr Asp Ala Thr Glu Lys Val Glu Phe Ile Glu Asn Leu Leu Gly Glu

595 600 605

Met Glu Lys Gln Thr Trp Ile Gln Trp Arg Met Met Tyr Pro Thr Glu

610 615 620

Phe Glu Thr Ile Ile Thr Arg Ala Glu Gly Arg Glu Gly Thr Gln Asn

625 630 635 640

Ile Leu Ser Gln Ile Arg Arg Val Phe Ser Leu Glu Asp Pro Ala Thr

645 650 655

Gly Thr Thr His Ile Gln Asp Gln Ala Tyr Arg Asp Leu Glu Arg Leu

660 665 670

Thr Cys Asn Asn Val Lys Asp Leu Val Gln Tyr Leu Asn Asp Phe Gly

675 680 685

Arg Leu Ala Ala Lys Thr Gly Arg Met Phe Leu Asn Thr Glu Leu Ser

690 695 700

Asp Lys Leu Trp Leu Lys Met Pro Gly Asp Ile Gly Gln Arg Met Lys

705 710 715 720

Met Ala Phe Asn Ala Lys Phe Pro Gly Asn Gln Ile Gly Val Phe Pro

725 730 735

Arg Ile Leu Tyr Ala Tyr Gln Phe Met Glu Gln Glu Cys Lys Asn Ala

740 745 750

Ala Phe Gln Arg Ser Leu Lys Asn Leu Ala Phe Cys Lys Asp Ile Pro

755 760 765

Phe Pro Gly Tyr Tyr Ser Glu Ser Ser Lys Ser Arg Tyr Gly Ala Arg

770 775 780

Lys Ser Thr Thr Tyr Lys Gly Lys Ala His Ser Thr His Ala Arg Ile

785 790 795 800

Glu Lys Lys Lys His Leu Val Arg Asn Arg Arg Cys Lys Cys Tyr Leu

805 810 815

Cys Gly Asp Glu Gly Pro Phe Ala Arg Glu Cys Pro Asn Glu Arg Arg

820 825 830

Asn Val Arg Arg Val Ala Val Phe Glu Asn Leu Ala Leu Pro Asp Asp

835 840 845

Tyr Glu Ile Val Ser Val Gln Asp Gly Glu Glu Glu Ser Asp Ala Ile

850 855 860

Tyr Ser Leu Ser Glu Asn Glu His Glu Asn Gly Val Asp Leu Glu Pro

865 870 875 880

Val Val Gly Ala Ala Thr Glu Glu Thr Cys Phe Met Phe Arg Glu Glu

885 890 895

Asp Asn Ser Tyr Trp Val Gly Lys Gly Gly Tyr Leu Ala Tyr Lys Arg

900 905 910

Val Thr Lys Ala Gln Tyr Glu Cys Lys His Thr Trp Ala His Asn Lys

915 920 925

Glu Val Glu Ala Gln Trp Ile Asn Cys Ser Phe Cys Lys Arg Pro Thr

930 935 940

Asn Arg Lys Cys Arg Ala His Cys Pro Lys Cys Arg Leu Thr Thr Cys

945 950 955 960

Glu Met Cys Ala Asp Pro Phe Cys Glu Ile Asn Val Val Arg Glu Pro

965 970 975

Glu Ile Tyr Lys Pro Tyr Asp Pro His Gly Leu Ile Gln Gln Gln Arg

980 985 990

Asp Tyr Ile Ala Trp Cys Glu Gly Glu Met Ser Arg Leu Arg Arg Glu

995 1000 1005

Val Glu Phe Tyr Lys Lys Lys Glu Glu Asp Arg Leu Ala Lys Asp Leu

1010 1015 1020

Glu Lys Glu Arg Ala Arg Gln Glu Lys Ile Asp Ala Glu Arg Glu Ala

1025 1030 1035 1040

Ser Ser Ser Lys Gly Lys Glu Val Leu Glu Glu Ile Lys Glu Glu Ser

1045 1050 1055

Ser Asp Gly Glu Ile Ser Ala Tyr Trp Glu Ser Glu Ser Thr Val Lys

1060 1065 1070

Val Arg Thr Ile Ala Ala Ala Ala Gly Glu Ser Glu Gln Lys Val Lys

1075 1080 1085

Glu Glu Lys Pro Val Arg Glu Asn Asn Asn Leu Tyr Asn Met Thr Val

1090 1095 1100

Glu Val Asp Ile Pro Gly Val Pro Lys Phe Ser Leu Arg Ala Ile Leu

1105 1110 1115 1120

Asp Thr Gly Ala Thr Val Cys Cys Val Asp Ser Lys Ala Leu Pro Lys

1125 1130 1135

Glu Ala Ile Glu Asp Asn Thr Phe Glu Val His Phe Arg Gly Ile Asn

1140 1145 1150

Ser Lys Gln Ser Val Asn Lys Lys Met Lys Tyr Gly Arg Met Ser Val

1155 1160 1165

Gly Gly His Asn Phe Arg Ile Pro Tyr Thr Tyr Thr Phe Pro Met Asp

1170 1175 1180

Leu Gly Gly Lys Ile Gln Phe Ile Leu Gly Cys Asn Phe Ile Arg Ser

1185 1190 1195 1200

Met Tyr Gly Gly Val Arg Leu Glu Gly Asn Thr Val Thr Phe Tyr Lys

1205 1210 1215

Asn Ile Thr Ala Ile Asp Thr Lys Thr Val Ala Val Leu Glu Glu Leu

1220 1225 1230

Glu Glu Glu Asp Leu Gln Leu Tyr Asn Leu Ala Val Leu Pro Gln Lys

1235 1240 1245

Gly Gly Glu Ala Phe Glu Arg Lys Asn Arg Arg Leu Ile Glu Glu Leu

1250 1255 1260

Lys Asn Gln Gly Tyr Ile Gly Asn Glu Pro Met Lys Tyr Trp Ile Lys

1265 1270 1275 1280

Asn Gln Val Thr Cys Lys Leu Asp Ile Lys Asn Pro Glu Met Val Ile

1285 1290 1295

Glu Asp Lys Pro Leu Lys His Val Thr Pro Ala Met Glu Glu Gln Phe

1300 1305 1310

Arg Ser His Ile Lys Ala Leu Ile Asp Leu Lys Val Ile Arg Pro Ser

1315 1320 1325

Lys Ser Arg His Arg Thr Thr Ala Phe Met Val Asn Ser Gly Thr Thr

1330 1335 1340

Ile Asp Pro Ile Thr Lys Lys Val Val His Gly Lys Glu Arg Met Val

1345 1350 1355 1360

Phe Asn Tyr Lys Arg Leu Asn Asp Leu Thr His Lys Asp Gln Tyr Ser

1365 1370 1375

Leu Pro Gly Ile His Thr Ile Met Arg Arg Ile Gly Gln Ala Gln Ile

1380 1385 1390

Phe Ser Lys Phe Asp Leu Lys Ser Gly Phe His Gln Val Ala Met Asp

1395 1400 1405

Pro Glu Ser Val Glu Trp Thr Ala Phe Trp Val Pro Asp Gly Leu Tyr

1410 1415 1420

Glu Trp Leu Val Ile Pro Phe Gly Leu Lys Asn Ala Pro Ala Ile Phe

1425 1430 1435 1440

Gln Arg Lys Met Asp Gln Cys Phe Lys Gly Thr Glu Lys Phe Ile Ala

1445 1450 1455

Val Tyr Ile Asp Asp Ile Leu Val Phe Ser Lys Asn Glu Glu Glu His

1460 1465 1470

Ala Gly His Val Arg Lys Met Leu Glu Ile Val Lys Ser Asn Gly Leu

1475 1480 1485

Ile Leu Ser Pro Thr Lys Met Cys Leu Ala Gln Arg Ser Val Glu Phe

1490 1495 1500

Leu Gly Ala Ile Ile Thr Gly Gly Lys Leu Gln Leu Gln Pro His Ile

1505 1510 1515 1520

Ile Lys Lys Val Val Gln Phe Asp Lys Pro Ser Leu Glu Thr Thr Lys

1525 1530 1535

Gly Leu Arg Gly Phe Leu Gly Leu Leu Asn Tyr Ala Arg Ala Tyr Ile

1540 1545 1550

Pro Asn Leu Gly Arg Leu Leu Ser Pro Leu Tyr Ala Lys Thr Ser Pro

1555 1560 1565

Thr Gly Glu Arg Arg Phe Ser Lys Glu Asp Trp Lys Leu Val Ser Asn

1570 1575 1580

Ile Gln Thr Glu Val Gln Lys Leu Pro Asp Leu Asn Leu Pro Pro Glu

1585 1590 1595 1600

Glu Cys Cys Ile Ile Ile Glu Thr Asp Gly Cys Ile Asp Gly Trp Gly

1605 1610 1615

Ala Val Cys Lys Trp Lys Lys Lys Arg Ala Asp Ser Arg Asn Thr Glu

1620 1625 1630

Met Val Cys Ala Tyr Gly Ser Gly Lys Phe Glu Asn Pro Lys Ser Thr

1635 1640 1645

Ile Asp Ala Glu Ile Thr Ala Val Ile Lys Ala Leu Glu Ser Phe Lys

1650 1655 1660

Ile Tyr Tyr Leu Asp Lys Gln Cys Leu Val Ile Arg Thr Asp Cys Gln

1665 1670 1675 1680

Ala Ile Val Ser Phe Phe Asn Lys Ser Cys Asn His Lys Pro Ser Arg

1685 1690 1695

Val Arg Trp Val Ser Leu Val Asp Tyr Ile Thr Gly Met Gly Leu Glu

1700 1705 1710

Val Lys Phe Glu His Ile Asp Gly Lys Glu Asn Gln Leu Ala Asp Val

1715 1720 1725

Leu Ser Arg Leu Ile Phe Gln Thr Trp Asn Gln Ser Pro Gln Gln Thr

1730 1735 1740

Ser Gln Gln Leu Leu Gln Asp Cys Leu His Arg Asn Ser Leu Met Ser

1745 1750 1755 1760

Ile Ile His Val Ala Leu Pro Met Lys Lys Tyr Val Ser Asn Ile Arg

1765 1770 1775

Gln Pro Val Pro Ala Val Pro Val Leu Ser Ala Pro Thr Ala Pro Phe

1780 1785 1790

Leu Ile Asn Ser Thr Gly Lys Lys Ile Gln Glu Glu Lys Arg Leu Glu

1795 1800 1805

Leu Leu Tyr Ala His Tyr Lys Ala Leu Tyr Gly Ile Leu Arg Tyr Ala

1810 1815 1820

Trp Trp Asp Leu Thr Thr Ser Ala Ala Ser Asp Tyr Trp Thr Lys Gln

1825 1830 1835 1840

Leu Ser Glu Arg Val Pro Gln Ile Trp Thr Phe Ile Gln Pro Pro Ser

1845 1850 1855

Pro Leu

<210> 9

<211> 130

<212> PRT

<213> 苦楝树缩叶病毒(Chinaberry leaf shrinking virus)

<400> 9

Met Glu Pro Ile Ser Ser Thr Asp Lys Ser Ala Ala Ser Ser Arg Leu

1 5 10 15

Ser Ala Gln Lys Leu Phe Asp Val Asn Tyr Pro Cys Cys Phe Thr Tyr

20 25 30

Glu Lys Ile Cys Phe Gln His Ser Ser Ala Gly Ser Gly Cys Thr Ser

35 40 45

Phe Glu Arg Pro Tyr Gly Ser Ile Ser Asn Gln Phe His Trp Glu Glu

50 55 60

Asn Pro Gly Gly Glu Lys Thr Gly Ala Ser Leu Arg Ser Leu Gln Ser

65 70 75 80

Ile Ile Arg His Ser Ala Leu Arg Val Val Gly Ser His Asn Leu Ser

85 90 95

Gly Gln Arg Leu Leu Asp Gln Thr Ala Phe Arg Lys Ser Ser Ser Asn

100 105 110

Met Asp Leu His Ser Thr Thr Glu Pro Pro Leu Lys Thr Gln Lys Pro

115 120 125

Ile Asp

130

<210> 10

<211> 22

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 10

ctgctaactg gttctccttt cc 22

<210> 11

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 11

gatcctgcta cgggaacaac 20

<210> 12

<211> 27

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 12

gggtaagtct tgttgattac attactg 27

<210> 13

<211> 22

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 13

ccaaatctct gtatgcctga tc 22

<210> 14

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 14

gagaatgatg tatcccacgg 20

<210> 15

<211> 22

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 15

cctgatcctg aatatgtgtt gt 22

Claims

1.一种扩增引物在扩增苦楝树缩叶病毒全基因组序列中的应用，其特征在于，所述苦楝树缩叶病毒全基因组序列如序列表SEQNO:1所示，所述扩增引物包括两对扩展引物对，其序列如下：

CLSV-1-F：CTGCTAACTGGTTCTCCTTTCC；

CLSV-1-R：GATCCTGCTACGGGAACAAC；

CLSV-2-F：GGGTAAGTCTTGTTGATTACATTACTG；

CLSV-2-R：CCAAATCTCTGTATGCCTGATC。

2.一种苦楝树缩叶病毒全基因组序列获取方法，其特征在于，所述苦楝树缩叶病毒全基因组序列如序列表SEQNO:1所示，所述方法使用权利要求1所述扩增引物进行扩增，包括如下步骤：

1）抽提病毒侵染植物组织的总DNA；

2）采用所述扩增引物，以所述总DNA为模板，进行PCR扩增；

3）回收PCR扩增得到的DNA产物，并将其连接到pLB平末端载体，进行DNA测序，得到苦楝树缩叶病毒全基因组序列。

3.如权利要求2所述的苦楝树缩叶病毒全基因组序列获取方法，其特征在于，步骤2）PCR扩增的反应体系如下：

10×KOD-Plus-Neo buffer 5μL

2 mM dNTPs 5μL

25 mM MgSO₄ 3μL

上游引物10μM 1.5μL

下游引物10μM 1.5μL

KOD-Plus-Neo DNA 1.0μL

模板DNA 0.5-2μL

加dd H₂O至总体积为50μL；

所述PCR 的反应循环的设置如下：

98 ℃ 3 min

98 ℃ 10 sec

56℃ 10 sec

68 ℃ 2 min

30循环数

68 ℃ 5 min。

4.如权利要求2所述的苦楝树缩叶病毒全基因组序列获取方法，其特征在于，所述步骤（3）中，回收长度为3223 bp和3931bp的DNA产物；pLB平末端载体连接反应体系如下：

目的PCR片段 2μL

pLB Vector 1μL

2×Reaction Solution 5μL

T4 DNA Ligase 1μL

ddH₂O 1μL。

5.一种特异性检测引物在检测苦楝树缩叶病毒中的应用，其特征在于，所述苦楝树缩叶病毒全基因组序列如序列表SEQNO:1所示，该病毒的全基因组序列包含编码gp1、gp2、gp3以及gpY四个蛋白的核苷酸序列，四个核苷酸序列分别如序列表SEQ NO:2-5所示，gp1、gp2、gp3以及gpY的氨基酸序列分别如SEQ NO:6-9所示；

所述特异性检测引物包括：

CLSV-d1-F：GAGAATGATGTATCCCACGG；

CLSV-d1-R：CCTGATCCTGAATATGTGTTGT。

6.一种检测苦楝树缩叶病毒的方法，其特征在于使用权利要求5所述检测引物进行PCR检测；所述苦楝树缩叶病毒全基因组序列如序列表SEQNO:1所示，该病毒的全基因组序列包含编码gp1、gp2、gp3以及gpY四个蛋白的核苷酸序列，四个核苷酸序列分别如序列表SEQNO:2-5所示，gp1、gp2、gp3以及gpY的氨基酸序列分别如SEQ NO:6-9所示。

7.如权利要求6所述检测方法，其特征在于，包括以下步骤：

(1)提取疑似感病的苦楝树总DNA；

(2)将步骤(1)终产物植物总DNA稀释到100ng/μl，用所述特异性检测引物进行普通定性PCR扩增；

8.如权利要求7所述检测方法，其特征在于，步骤（2）中的PCR体系如下：

2×Mix 10 μL

上游引物10 μM 1μL

下游引物10 μM 1 μL

DNA 1 μL

dd H₂O至总体积为 7 μL

PCR 反应循环的设置如下：

98℃ 3 min

98℃ 10 sec

56℃ 10 sec

72℃ 30 sec

30循环数

72℃ 5 min。