茶树网斑病毒的全基因序列及其检测方法与流程

本发明涉及分子生物工程技术领域，具体涉及一种茶树网斑病毒的全基因序列及其检测方法。

背景技术：

茶树为多年生常绿木本植物，地理分布广。作为一种重要的经济作物，为世界茶叶消费提供原材料。茶树在三千年前就已经被人类发现并利用。由于茶树中富含多酚、茶氨酸、咖啡碱等具有抗氧化和抗菌功能的代谢成分，因此长期以来人们认为茶树不会感染病毒。

利用宏基因组技术在茶树黄色变异枝条中检测了一种可能引起叶色黄花症状的茶树病毒—茶树网斑病毒（teaplantlinepatternvirus,tplpv），该病毒是在茶树中被首次发现和报道的病毒。以病毒感染的茶树叶片为植物材料，通过rna提取及race克隆的方法获得了该病毒的完整基因组信息，并进行了深入分析。根据该病毒的基因组序列特性，设计了3对引物，用于检测该病毒在茶树及其他有机体中的存在，引物信息及检测方法，可以为生产中快速鉴定存在叶色变异的茶树品种是否感染了茶树网斑病毒提供快速可靠的检测方法，也为科学研究中对病毒含量的准确检测提供了依据。

技术实现要素：

本发明的目的是提供一种茶树网斑病毒及其全基因序列。

本发明的另一目的是提供一种茶树网斑病毒的检测方法。

为了实现本发明的目的，本发明采用的技术方案如下：

茶树网斑病毒基因组序列及编码蛋白序列如seqidno:1所示，茶树网斑病毒基因组序列一共包含3段，对应为tplpv-rna1，tplpv-rna2和tplpv-rna3，每段末端均有长度不一的poly(a)结构，tplpv-rna1，tplpv-rna2，tplpv-rna3的长度分别为3373nt，2354nt，2142nt。

本发明提供了tplpv-rna1在第30-3158碱基位置存在完整的开放阅读框，编码一个1042个氨基酸残基的甲基转移酶和解旋酶复合体；tplpv-rna2在第30-2120碱基位置存在完整的开放阅读框，编码一个696个氨基酸残基的rna聚合酶；tplpv-rna3则包含2个开放阅读框，分别在第157-1026碱基位置和1024-1815碱基位置存在完整的开放阅读框，编码289和203个氨基酸长度的移动蛋白和外壳蛋白。

本发明提供了用于检测权利要求1所述茶树网斑病毒的特异性pcr引物，针对病毒基因组的3个片段对应设计了3对引物序列：

本发明还提供了基于普通pcr检测和荧光定量pcr检测茶树网斑病毒的方法，本发明还提供了利用上述引物进行普通定性pcr和荧光定量pcr检测。

本发明还提供了利用上述引物进行普通定性pcr和荧光定量pcr检测的步骤：

a：提取病毒感染的茶树叶片总rna；

b：利用反转录试剂盒以兼并引物n25为合成引物，按照试剂盒要求合成cdna模板；

c:将终产物稀释到800µl后用于普通定性pcr检测和荧光定量pcr检测。

本发明还提供了茶树网斑病毒的普通定性pcr检测方法，其中反应体系为：

反应程序为：95℃3min；95℃15s,52℃15s,72℃30s，共35个循环；72℃2min后结束程序；

检测方法为：向上述10µlpcr反应产物中加入2μl6xloadingbuffer，混匀后用1%凝胶电泳检测产物条带，用100-1000bp范围的ladder作为片段大小对照标准。

本发明还提供了茶树网斑病毒的荧光定量pcr检测方法，其中反应体系为：

反应程序为：95°c10min；95°c20s，52°c10s，70°c35s，35个循环；溶解曲线检测温度65°c-95°c。

适合工作平台：roche480，abi7500等所有需要引物检测的定量平台。

本发明具有以下有益效果：

1）本发明提供了茶树网斑病毒及其全基因序列；

2）本发明根据茶树网斑病毒的基因组序列特性，设计了3对引物，用于检测该病毒在茶树及其他有机体中的存在，本发明设计的引物具有特异性强、结果可靠，在普通pcr检测和荧光定量pcr检测中都有稳定可靠的表现；

3）本发明所提供的引物信息及检测方法，可以为生产中快速鉴定存在叶色变异的茶树品种是否感染了茶树网斑病毒提供快速可靠的检测方法，也为科学研究中对病毒含量的准确检测提供了依据。

附图说明

图1为茶树网斑病毒基因组结构示意图；

图2为茶树网斑病毒基于rna2编码蛋白的系统发育树分析图；

图3为利用设计引物对含病毒材料进行普通pcr检测的电泳图（注：l,ladder;1-4,阴性对照;5,阳性样品；tplpv1-3,引物名称）；

图4为利用设计引物对多个含病毒材料进行荧光定量pcr时的扩增曲线图；

图5为荧光定量pcr产物的溶解曲线图。

具体实施方式

以下以具体实施例来说明本发明的技术方案，但本发明的保护范围不限于此。

实施例一病毒的基因组全长序列克隆

1.病毒感染的茶树叶片总rna提取

a.称取0.2g新鲜或-70℃保存植物组织样品置于预冷的研钵中。加入液氮，充分研磨成粉末状。将研磨碎的样品快速转移至事先预冷的1.5ml离心管中。加入600µl42℃预热ctab提取液（2%ctab,2%pvpp,20mmol/ledta,1.4mol/lnacl，100mmol/ltris-cl，ph8.0），立即用力上下颠倒并漩涡振荡30s，加入12µl2%pvpp，12µlβ-巯基乙醇和45µl蛋白酶k。使材料均匀分散于溶液中，于42℃水浴锅中90分钟。其间剧烈振荡3次。

b.稍冷却后，加入等体积的氯仿:异戊醇，漩涡振荡，13000rpm，4℃离心15min。

c．用移液枪小心吸取上清于另一试管中，加入等体积的氯仿：异戊醇，漩涡振荡，13000rpm，4℃离心15min。

d．取上清，加入等体积的4mlicl和1/2体积的无水乙醇，混匀。4℃放置3小时。

e.13000rpm，4℃离心15min。去上清,加入600-800ml2mlicl,洗涤离心1300rpm,4℃，15min。

f.吸上清，加入75%乙醇洗涤。1300rpm,4℃，5min

g.去上清，吹干后加入适量的depch2o溶解,加入1µldnasei室温处理20min后，-80℃保存。

2.病毒基因组序列的race扩增

利用上述提取的rna，经1%凝胶电泳检测为完整后，用nanodrop®2000c紫外分光光度计进行浓度检测。使用商品化的race(rapidamplificationofcdnaends)试剂盒（smarter®race5'/3'kit），依照试剂盒使用说明进行引物设计和pcr扩增。扩增片段经胶回收、克隆后送测序公司进行测序。序列片段利用dnastar软件进行拼接，获得完整基因组序列。

实施例二病毒基因组及编码蛋白序列信息

克隆获得的茶树网斑病毒（tplpv）基因组序列一共包含3段，每段末端（3’端）均有长度不一的poly(a)结构。除poly(a)结构外，3段序列（tplpv-rna1，tplpv-rna2，tplpv-rna3）的长度分别为3373nt，2354nt，2142nt。tplpv-rna1在第30-3158碱基位置存在完整的开放阅读框，编码一个1042个氨基酸残基的甲基转移酶和解旋酶复合体（methyltransferase-helicase）。tplpv-rna2在第30-2120碱基位置存在完整的开放阅读框，编码一个696个氨基酸残基的rna聚合酶（rnapolymerase）。tplpv-rna3则包含2个开放阅读框，分别在第157-1026碱基位置和1024-1815碱基位置存在完整的开放阅读框，编码289和203个氨基酸长度的移动蛋白（movementprotein）和外壳蛋白（coatprotein）。该病毒基因组序列及其编码蛋白序列见序列表。该病毒与其病毒间存在较远的进化关系。相比较而言，与美国李网斑病毒（americanplumlinepatternvirus,tplpv）的进化关系最近（图1-2）。

实施例三病毒检测引物信息

我们针对病毒基因组的3个片段分别设计一对引物。经验证，3对引物具有特异性强，扩增效率高的特点，可以用于普通pcr的定性检测及qrt-pcr（荧光定量pcr）的定量检测。应对该病毒的3个片段同时检测，具有稳定性好、准确率高的优点。

实施例四病毒的定性及定量检测方法

利用上述rna提取方法获得植物样品完整的总rna，利用反转录试剂盒（superscript®iiifirst-strandsynthesissystem）以兼并引物n25（5'-nnnnnnnnnnnnnnnnnnnnnnnnn-3'）为合成引物，按照试剂盒要求合成cdna模板。将终产物稀释到800µl（约40倍）后用于普通pcr检测和qrt-pcr检测。

1.普通定性pcr检测方法及检测效果

pcr反应体系：

pcr反应程序：95℃3min；95℃15s,52℃15s,72℃30s，共35个循环；72℃2min后结束程序。

检测方法：向上述10µlpcr反应产物中加入2μl6xloadingbuffer，混匀后用1%凝胶电泳检测产物条带，用100-1000bp范围的ladder作为片段大小对照标准。

检测结果如图三所示，其中：l代表ladder;1-4代表阴性对照;5代表阳性样品；tplpv1-3代表引物名称。

2.定量的荧光定量pcr检测方法及效果图

定量反应体系：

反应程序：95°c10min；95°c20s，52°c10s，70°c35s，35个循环；溶解曲线检测温度65°c-95°c。

适合工作平台：roche480，abi7500等所有需要引物检测的定量平台。

检测效果如图4-5所示。

最后应当说明的是，以上实施仅用以说明本发明的技术方案而非限制本发明，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围内。

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valpheileaspgluglyphemetglnhisserglycysiletyrala

820825830

alaleuthrleualalysalalysglucyspheilepheglyaspthr

835840845

gluglnilepropheileserargcysproleumetargleuarghis

850855860

glnmetilevalglyserlysthrvalargcysleuaspthrhisarg

865870875880

serprometaspalathrcysleuleuasnlysleutyrlysglnlys

885890895

argprovallysthrthrserlysvalalaargservalservalhis

900905910

proileserserasnglnglnileprosergluproalaleutyrleu

915920925

cystyrthrglnalaglulysmetaspleuleuserthrserhisleu

930935940

lysglyargargvalleuthrvalhisglualaglnglygluserval

945950955960

aspasnvalilephecysargleuserargthrserthraspleuthr

965970975

serglylyshisproilemetglyprocyshisalaleuvalalaleu

980985990

serarghislyslysserleulysvaltyrserlysalaserthrleu

99510001005

aspmetasnaspileleutyrasnleucysserglulysileserglu

101010151020

arggluleuthrglnalaphethrilesergluargleulysleuasp

1025103010351040

leuser

<210>3

<211>2381

<212>rna

<213>茶树网斑病毒(teaplantlinepatternvirus)

<400>3

ucagauuaucacaagguauuguguuaaucauggaugucaaaagcugguugucuucuuuau60

caucuucaucuuuggauccucuucgugaggcuuuauugugugaugaugauaugccgauug120

auaaggucgaagagaacacccauauauccagauuuauugguugggugauucugaggaaag180

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cuacgaaugugguuuuuguagaauccgaugcaucuuauuauaaggaaauggaggauuuag360

aucuugcguuuucuuuuccgucggaagauaagcgaggggaugugaaugauucaucuccug420

auucaguugaauugacugaaguuucccagguggaagucgaaccguaucucgauuucaaac480

uggaaguuuguacucuaccagaugacgagggauugugccgucaacuggauuggaaagcuc540

agacccauguggucguugagaauguucugaggaacucuuccauugagauuauuaacucaa600

ugaggacuuacaaugccaauugcauucaagaugcaauugaugaaguauuuccccaucacu660

ucucugucgaugauagauauuuucagacuuuugucgaaacuuccgacauugauuuagaau720

uggaugcugcucauauagauaugaguaaguuucccaagaaguuugacauccacuucgauu780

gggaacccaaguuguccacuggacuaacuucuacuagguauaauaguuuucgcgaggcug840

cguuagcuauuaagaagagaaaucuuaacguuccagauuugacugcggauuuggacgugg900

acucucucgugccuaaagugauggagagguuuuuaaagauccuugauuuagguaaacucu960

ccggauuccccgaugauucucaugacgucggaugcuuccugcuagauuauuugaguagga1020

aaggauugaguuaugacguguuggaaccaggguguaugguuuccuugaacaaauaccguc1080

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ugaguuuggcggcgaaguuguuguguguuuuguccgacaaagucgucauaccaaugggaa1260

aagaacaucaauuauuugacaucaauguugauguauuuagaaaaauaaaauucuggaagg1320

aaaucgacuauucuaaguucgauaaaucucaagguaagcugcaccaugagauucagagag1380

ccauuuucagagaccuucagugugaugaaggauuccugaguacuugguuccauucccaug1440

aauuuuccacuaucuuugauagugaagcagggauagguuuuuccacugauuuucaacguc1500

guacuggagaugcguguacguaucucgguaacacucuuguuacuuuagcgacguuaagcu1560

uuguauaugauuuggauucucccgauguaggacugauuguuguguccggagaugauucuc1620

uaauugguacauuucauucagaacuacccagggaaagagagauuuuaugcucuacuuugu1680

uuaauuuugagacaaaguuuccucacaaucaaccauuuauuugcaguaaauuucugguug1740

aaauugagacgacggauggucguggaaaaguggucgcugucccaucaccaauuaaauugc1800

ugaugaaguuaggaccuaagucgaugaacaagcaauuguuuccggcuuggguggagagca1860

uuaaggacuacgcugcuccauuuaggaaugcguucguugcugaacagguguguaauuugg1920

ccaguuauagauuugguagaagauguuuuacauuccuacuaccugcuauaucugaagugc1980

ucuggauguugaguaaucccaacguuaaacuuaaggaaaaacuuuuccuucagaauuccu2040

cuuaucuuucuaaucauugucaugauaggagagaaaaagagaagaaaagaaaucguccua2100

gggcgagaucuaaacguugacuguuguuaugaauuuuaaugguuauuucgcuuuggcgaa2160

ggucauuaaugcuaacucuuugaguuaaugguuagucacugugugcgacugcuacgagau2220

gaaccuauggggauucaaauaguggugagauaucuuaaucugucgguugcccgaagaucg2280

uaugauaucgacuccagauuaguauggagauucuacauuuaacuuacguuaaauguaaag2340

uagcuaaggcuaccaaaaaaaaaaaaaaaaaaaaaaaaaaa2381

<210>4

<211>696

<212>prt

<213>茶树网斑病毒(teaplantlinepatternvirus)

<400>4

metaspvallyssertrpleuserserleuserserserproleuasp

151015

proleuargglualaleuleucysaspaspaspmetproileasplys

202530

valglugluasnthrhisileserargpheileglytrpvalileleu

354045

arglysvalphepheserglugluphealaseraspglutyrtyrphe

505560

tyrseraspcysleuargleuargphephegluleupheservalpro

65707580

metgluleutyraspalaasnaspserglyserthrasnvalvalphe

859095

valgluseraspalasertyrtyrlysglumetgluaspleuaspleu

100105110

alapheserpheprosergluasplysargglyaspvalasnaspser

115120125

serproaspservalgluleuthrgluvalserglnvalgluvalglu

130135140

protyrleuaspphelysleugluvalcysthrleuproaspaspglu

145150155160

glyleucysargglnleuasptrplysalaglnthrhisvalvalval

165170175

gluasnvalleuargasnserserilegluileileasnsermetarg

180185190

thrtyrasnalaasncysileglnaspalaileaspgluvalphepro

195200205

hishispheservalaspaspargtyrpheglnthrphevalgluthr

210215220

seraspileaspleugluleuaspalaalahisileaspmetserlys

225230235240

pheprolyslyspheaspilehispheasptrpgluprolysleuser

245250255

thrglyleuthrserthrargtyrasnserpheargglualaalaleu

260265270

alailelyslysargasnleuasnvalproaspleuthralaaspleu

275280285

aspvalaspserleuvalprolysvalmetgluargpheleulysile

290295300

leuaspleuglylysleuserglypheproaspaspserhisaspval

305310315320

glycyspheleuleuasptyrleuserarglysglyleusertyrasp

325330335

valleugluproglycysmetvalserleuasnlystyrarghismet

340345350

ilelysserglnleulysprovalglugluaspserleuhisvalglu

355360365

argproleualaalathrilethrtyrhisglylysglulysvalser

370375380

valthrthrprophepheleuserleualaalalysleuleucysval

385390395400

leuserasplysvalvalileprometglylysgluhisglnleuphe

405410415

aspileasnvalaspvalphearglysilelysphetrplysgluile

420425430

asptyrserlyspheasplysserglnglylysleuhishisgluile

435440445

glnargalailepheargaspleuglncysaspgluglypheleuser

450455460

thrtrpphehisserhisglupheserthrilepheaspsergluala

465470475480

glyileglypheserthrasppheglnargargthrglyaspalacys

485490495

thrtyrleuglyasnthrleuvalthrleualathrleuserpheval

500505510

tyraspleuaspserproaspvalglyleuilevalvalserglyasp

515520525

aspserleuileglythrphehissergluleuproarggluargglu

530535540

ileleucysserthrleupheasnphegluthrlyspheprohisasn

545550555560

glnpropheilecysserlyspheleuvalgluilegluthrthrasp

565570575

glyargglylysvalvalalavalproserproilelysleuleumet

580585590

lysleuglyprolyssermetasnlysglnleupheproalatrpval

595600605

gluserilelysasptyralaalapropheargasnalaphevalala

610615620

gluglnvalcysasnleualasertyrargpheglyargargcysphe

625630635640

thrpheleuleuproalailesergluvalleutrpmetleuserasn

645650655

proasnvallysleulysglulysleupheleuglnasnsersertyr

660665670

leuserasnhiscyshisaspargargglulysglulyslysargasn

675680685

argproargalaargserlysarg

690695

<210>5

<211>2172

<212>rna

<213>茶树网斑病毒(teaplantlinepatternvirus)

<400>5

uucaaaucaauaccaacugcuugagaucuuauuacguguugauuuaucaccguuuaaacu60

caagaauuguugaacgacgaaccugaaacguagauuguuggauuaccacuuuacguuuca120

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ucgaacucaggcgcuggucauguugcgaauguuuauucacguuuacaacuggcugcggua240

gagagagagaaggaggcguacauuaaagccuuauugaccaacccuauuaaagcuaggguu300

ugcguaccuauuccacucguaaagagugaagacguugugaugugucgacucagaagugag360

gaaauuaauuuccuuaccagggcauuuagaucgaggauuuuaaagucguccucuuucauu420

aaucaugccaauauuaguaucuguuacuguccaaauguauugaaaucccauucgggaaca480

uuuucaguaucgauaucuaaucccgacacuacugacguuaaguuguuggucgaaaaugcu540

ccuauaucugaggggagauuucuuuugacuaacuggccuaggucugucccuagugcccaa600

aacuuauaccugaacaucgaaugugaugguucagaugcgaccaauggaacucaacuuggu660

cuguuuaagauucuuugggacgagaaaccgucuuucaaaaugauccuggaaaaggauaug720

caggucauaaaugagaucauacccgaaacagacgcuguggguaagauguuuuccaaccaa780

acuuuggaacaauuucuaaauagguugaugcugaacgguucugagaagaacucccucacc840

cguuccaugucauaucacauuaacaguaaucuaagaauuacugaacuugaugagagcgcu900

gaaaggauuagcacaggcuuaggcucugauaagacuaagaucgguugcucaaaucgagau960

gauacaacguccagcgugcuggagguuaagauaggugacggguucaccgaauccgacgcu1020

ucuuaguuagucuugagaggauuagcgcgggcuacuucguaguaccggugagacuaagau1080

cgagacaaaucuggauguccaaugcaagugaaacuuaagcugaaaucaacuucuuugauu1140

cagguuguauuaacaauuuugauaccuaucaacuuugugauagaugcuucgugaaaaucg1200

aaaaugucaaccaauacucaacagaaaaagacucgacagaacaagagaucaacucaguuc1260

ggucaaaauagggcuaaacagagagcccagucuacuggugaaagcucaaccgucccuaau1320

uuugggacucuuagguuggagugggucaggcgagguccuggugaccuagacauccugcag1380

uucccauaucaauggguugcaaaggccgagaguucucucaaggcaacuagcaaugauauc1440

ugguauaucauugaucuccaugaauaugucaaagacaugauggaguuucccaccucggug1500

aaggguuucgucuuuuuauuugaggcgaacuuaucaggccaggcuugcuuaguuacuaag1560

aagacugguuccuucaaagaugcguucucaucguugaacgcauuuaaauuugaaaagggg1620

ugccauacggcaguucaacuguuggccccaucaauucuaaccuucgccacguuuucggcu1680

ggugaaguugcgcugguuuuuaaguucgaaggaacuuuaacagcaggugcugaauucaug1740

acccggaaaguuuggggucaaucaucaaugcugcccaaaguugaaauuaauaagaauuua1800

cuacgugggcaaugacuacacucgggagauuaccugagugagcuugaguacgacccgaug1860

ucggguugguuucucaauagaggaguuagaugguauuacuguccaauuccugcuuguguu1920

uuuaccaacgguuaucgccuuggcgaggaccauugaugcuaacucuuuugaguuaauggu1980

uagucacuaugugcgacuacuacgagaugaaccuauggggauucgaauaguagugagaua2040

ucuuaaucugucgguugcccgaagaucguaugauaucgacuccagauuaguauggagauu2100

cuacauuuaacuuacguuaaauguaaaguagcuaaggcuaccaaaaaaaaaaaaaaaaaa2160

aaaaaaaaaaaa2172

<210>6

<211>289

<212>prt

<213>茶树网斑病毒(teaplantlinepatternvirus)

<400>6

metalathrlysglualavalthrserasnserglyalaglyhisval

151015

alaasnvaltyrserargleuglnleualaalavalgluargglulys

202530

glualatyrilelysalaleuleuthrasnproilelysalaargval

354045

cysvalproileproleuvallyssergluaspvalvalmetcysarg

505560

leuargserglugluileasnpheleuthrargalapheargserarg

65707580

ileleulysserserserpheileasnhisalaasnileserilecys

859095

tyrcysproasnvalleulysserhisserglythrpheservalser

100105110

ileserasnproaspthrthraspvallysleuleuvalgluasnala

115120125

proilesergluglyargpheleuleuthrasntrpproargserval

130135140

proseralaglnasnleutyrleuasnileglucysaspglyserasp

145150155160

alathrasnglythrglnleuglyleuphelysileleutrpaspglu

165170175

lysproserphelysmetileleuglulysaspmetglnvalileasn

180185190

gluileileprogluthraspalavalglylysmetpheserasngln

195200205

thrleugluglnpheleuasnargleumetleuasnglyserglulys

210215220

asnserleuthrargsermetsertyrhisileasnserasnleuarg

225230235240

ilethrgluleuaspgluseralagluargileserthrglyleugly

245250255

serasplysthrlysileglycysserasnargaspaspthrthrser

260265270

servalleugluvallysileglyaspglyphethrgluseraspala

275280285

ser

<210>7

<211>203

<212>prt

<213>茶树网斑病毒(teaplantlinepatternvirus)

<400>7

metserthrasnthrglnglnlyslysthrargglnasnlysargser

151015

thrglnpheglyglnasnargalalysglnargalaglnserthrgly

202530

gluserserthrvalproasnpheglythrleuargleuglutrpval

354045

argargglyproglyaspleuaspileleuglnpheprotyrglntrp

505560

valalalysalagluserserleulysalathrserasnaspiletrp

65707580

tyrileileaspleuhisglutyrvallysaspmetmetgluphepro

859095

thrservallysglyphevalpheleupheglualaasnleusergly

100105110

glnalacysleuvalthrlyslysthrglyserphelysaspalaphe

115120125

serserleuasnalaphelyspheglulysglycyshisthralaval

130135140

glnleuleualaproserileleuthrphealathrpheseralagly

145150155160

gluvalalaleuvalphelysphegluglythrleuthralaglyala

165170175

gluphemetthrarglysvaltrpglyglnsersermetleuprolys

180185190

valgluileasnlysasnleuleuargglygln

195200