CN110184267A - 割手密反转录转座子序列及其鉴定方法 - Google Patents

Abstract

本发明公开了割手密反转录转座子序列及其鉴定方法，利用割手密和热带种基因组数据聚类分析，筛选出4条割手密反转录转座子序列，根据该序列分别设计引物扩增获得其全长序列，然后制备成探针，在割手密和热带种的中期染色体上进行荧光原位杂交(FISH)鉴定，结果显示，这4条反转录转座子序列均仅在割手密染色体上产生清晰明亮的信号，本发明的反转录转座子序列可直接用于特异识别割手密染色体，为甘蔗栽培品种中割手密血缘鉴定提供更加准确的信息，也将为甘蔗染色体工程育种奠定基础。

Description

割手密反转录转座子序列及其鉴定方法

技术领域

本发明涉及生物信息学与分子细胞遗传学领域，具体涉及割手密反转录转座子序列及其鉴定方法。

背景技术

重复序列的含量是影响植物基因组大小的最主要因素之一。通常，植物的重复序列所占比例越大，其基因组也越大。例如，拟南芥基因组相对较小，仅为121Mb，其重复序列大约占25％；高达17Gb的小麦基因组含有大约90％的重复序列。反转录转座子是植物基因组中广泛存在的一类重复序列，它以RNA为中间体通过自身编码的反转录酶进行反转录，产生染色体外DNA插入到基因组新的靶点位点，在基因组中以“复制-粘贴”的方式移动，最终会造成反转录转座子拷贝数增加。在植物基因组中，不同类型的反转录转座子的丰度和分布特征不尽相同，它们的分布及其活动对植物基因组的大小、结构、功能以及表观遗传都有着重要的影响。

目前，已测序的甘蔗割手密单倍体AP85-441中含有1842Mbp的重复序列，占组装基因组的58.65％，反转录转座子占基因组的45.62％。因此，割手密基因组中含有大量可待开发的割手密染色体特异重复序列，尤其是以反转录转座子为主的重复序列，将为跟踪与鉴定整合到甘蔗栽培品种中的割手密染色体及染色体片段提供可用于特异识别割手密染色体的标记。

割手密是重要的甘蔗野生种质资源之一，广泛分布于热带和亚热带地区的不同生境中，该物种在不同的生境下具有丰富且可利用的遗传变异类型，兼具强抗逆性、长势旺、适应性广、早熟易开花、宿根性强等丰富的优良性状。因而，甘蔗育种家将割手密用于杂交育种，扩宽甘蔗抗逆性育种遗传基础。热带种具有茎秆粗壮、糖分高且纤维份低等特点，因此热带种又被称为高贵种。在甘蔗高贵化育种中担任轮回亲本的角色，热带种是甘蔗遗传改良育种中高糖基因的贡献种质资源，具有低纤维、高含糖量、大茎等多种优良性状，是现代甘蔗栽培品种中都存在并且所占比重最大的血缘种质。因此，割手密与热带种在甘蔗遗传育种中都发挥着极其重要的作用。

由于甘蔗属物种多为高度杂合的多倍体植物，其染色体数目众多、形态小且相似，并且至今仍没有能够可用于快速精确识别甘蔗栽培品种中割手密染色体的特异探针。此外，利用基因组原位杂交技术区分割手密染色体的效果不佳。因此，有必要开发能够快速精确识别割手染色体的特异探针，本发明利用割手密和热带种基因组数据进行聚类分析得到了4条割手密反转录转座子序列，并且通过FISH实验的进一步验证，明确了这些序列的反转录转座子仅在割手密染色体上产生清晰明亮的信号。同时，为了方便应用，本发明根据其序列开发了相应地特异引物，为其克隆及应用提供了有力的工具。

发明内容

本发明的目的是发掘特异识别割手密染色体的反转录转座子序列，并用于精确追踪甘蔗栽培品种中割手密染色体组成和遗传情况，将为甘蔗等复杂多倍体植物染色体研究提供一种经济而高效的鉴定方法。

为实现上述目的，本发明采用如下技术方案：

割手密反转录转座子序列，所述序列根据割手密和热带种基因组聚类数据分析得到，名称分别为：序列1-Cluster168Contig15、序列2-Cluster56Contig54、序列3-Cluster100Contig20、序列4-Cluster38Contig50，其核苷酸序列分别为SEQ ID NO.1-4所示。

所述序列1-Cluster168Contig15的引物序列为：

上游引物：5'-GTTCTCAGGATTCTTCAGTATTTCG-3'；

下游引物：5'-TCACATTGGATGCTAAGCCCTAAGA-3'；

所述序列2-Cluster56Contig54的引物序列为：

上游引物：5'-GGCAGGCAGAGCAACACTATTACAG-3'；

下游引物：5'-GTTCTCGTGGCTTCTGGACTCTTCT-3'；

所述序列3-Cluster100Contig20的引物序列为：

上游引物：5'-CATTGATGTTAGTAATCCCTTCCCA-3'；

下游引物：5'-GAGAAACATAGCAATCACTCCCCCG-3'；

所述序列4-Cluster38Contig50的引物序列为：

上游引物：5'-GATAGATTTTACCCCTGTTTTCGCT-3'；

下游引物：5'-TCGTCACACAGTCACTTGCTTTGGC-3'。

割手密反转录转座子序列的鉴定方法，包括如下步骤：

(1)根据割手密和热带种基因组聚类数据分析得到4条割手密反转录转座子序列；

(2)利用Primer Premier 5.0对上述4条反转录转座子序列进行引物设计；

(3)将上述4条割手密反转录转座子序列进行PCR扩增，得到PCR产物；

(4)采用OMEGA试剂盒进行PCR产物纯化回收，得到纯化PCR产物；

(5)将纯化PCR产物制备成探针，在割手密和热带种的中期染色体上进行荧光原位杂交鉴定。

上述步骤(3)中，所述PCR扩增反应体系为：1×ExTaq Buffer，0.2mM dNTPMixture，250nM上游引物，250nM下游引物，2.5ng/μl SES208基因组DNA，0.05U/μl ExTaq。

上述步骤(3)中，所述PCR扩增的条件为：95℃预变性3min；98℃变性30s，68℃退火及延伸6min-8min，35个循环；72℃终延伸10min，所述序列1的退火及延伸时间为8min，序列2的退火及延伸时间为6min，序列3的退火及延伸时间为6min，序列4的退火及延伸时间为8min。

本发明的优点在于：

本发明利用割手密和热带种基因组数据聚类分析得到高丰度的反转录转座子序列，这是挖掘可用于特异识别割手密染色体的反转录转座子序列的有效方法，通过生物信息学分析，得到具有高丰度的contigs，并通过FISH技术验证了4条反转录转座子序列均仅在割手密染色体上产生信号，而在热带种染色体上无信号，表明本发明所得的割手密反转录转座子可真实且可靠地特定识别割手密染色体。

本发明根据不同的序列设计相应的PCR引物，并且引物特异性良好，扩增的条带单一且明亮，故适合于纯化PCR产物，标记探针，用于FISH实验；PCR引物的设计为割手密反转录转座子序列的克隆和应用提供了重要工具；本发明为割手密反转录转座子序列在割手密染色体识别研究积累了宝贵材料，有利于甘蔗杂交品种中割手密染色体的鉴定研究。

附图说明

图1：割手密反转录转座子序列在割手密SES208和热带种LA Purple的杂交结果，A：有丝分裂中期染色体，B：反转录转座子信号，C：合成图。

具体实施方式

本发明所用试剂盒：OMEGA试剂盒的货号：D2500-01，名称：OMEGA Gel ExtractionKit；地高辛试剂盒：Digoxigenin-11-dUTP，罗氏Roche公司。

下面将结合本发明中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。

实施例1：根据割手密SES208和热带种LA Purple基因组聚类数据分析得到割手密反转录转座子序列，名称分别为：序列1-Cluster168Contig15、序列2-Cluster56Contig54、序列3-Cluster100Contig20、序列4-Cluster38Contig50，其核苷酸序列分别为SEQ IDNO.1-4所示。所述的序列1-Cluster168Contig15、序列2-Cluster56Contig54、序列3-Cluster100Contig20、序列4-Cluster38Contig50相应的分子标记引物序列如SEQ IDNO.5-12所示。

表1割手密反转录转座子序列扩增引物

利用Primer Premier 5.0对反转录转座子序列进行引物设计。

将上述4条割手密反转录转座子序列进行PCR扩增，PCR扩增的条件为：95℃预变性3min；98℃变性30s，68℃退火及延伸6min-8min，35个循环；72℃终延伸10min。需要注意的是，扩增序列长度不同，68℃退火及延伸时间有所区别：序列1为8min、序列2为6min、序列3为6min、序列4为8min。PCR扩增的条带单一而且明亮，采用OMEGA试剂盒进行PCR产物纯化回收，得到纯化PCR产物。

实施例2：荧光原位杂交(FISH)鉴定

荧光原位杂交技术是一项检测目的序列在染色体分布的技术，该方法的检测结果真实且可靠，不仅适用于单拷贝基因在染色体上的鉴定研究，还适用于多拷贝重复序列在染色体上的鉴定研究。

割手密反转录转座子序列的荧光原位杂交鉴定包括以下步骤：

(1)分别取旺盛生长的割手密SES208和热带种LA Purple根尖，在室温下放入8-羟基喹啉溶液进行预处理；

(2)切除根冠和伸长区部分，留下根尖分生区，在37℃条件下用纤维素酶和果胶酶对根尖分生区细胞进行酶解去壁；

(3)吸取酶解后的根尖分生区组织块，用火焰干燥法制备中期染色体玻片；

(4)利用缺口平移法，将从割手密基因组中扩增得到的反转录转座子纯化PCR产物用地高辛试剂盒(Digoxigenin-11-dUTP，罗氏Roche公司)标记，具体的探针制备反应体系为：1μg纯化PCR产物，50mM Tris-HCl，50mM MgCl₂，0.05mM dATP，0.05mM dCTP，0.05mMdGTP，0.05mM dTTP，0.05mM Dig-dUTP，0.1U/μl Polymerase I，0.005U/μl DNase I；将上述反应体系于15℃水浴中反应1.5h，通过加入1μl 0.5M EDTA(pH8.0)或加热到65℃保持10min来终止反应，即获得含有地高辛半抗原标记物的探针；

(5)配制含50％甲酰胺(V/V)、10％硫酸葡聚糖、2×SSC、100ng地高辛标记探针的20μl杂交液，将杂交液于90℃变性5min后，迅速放于冰水中10min；染色体玻片在70℃变性1min后，将杂交液滴加到染色体制片上，放入湿盒中在37℃中杂交过夜；

(6)杂交后进行探针洗脱，在室温下依次用2×SSC洗脱3次，再用1×PBS洗脱1次，洗脱时间均为5min；

(7)探针洗脱后加入能与地高辛半抗原标记物特异结合的红色荧光抗体，37℃孵育1h；

(8)孵育后进行抗体洗脱，在室温下用1×PBS洗脱3次，洗脱时间均为5min；

(9)空气干燥玻片后，在玻片上加入含有DAPI的抗褪色剂进行染色体复染；

(10)利用荧光显微镜进行图像采集，鉴定出可特异识别割手密染色体的反转录转座子序列。

从图1中可以看到，本发明序列产生的信号均仅在割手密(SES208)染色体上，而在热带种(LA Purple)染色体上无信号，验证了这些反转录转座子序列可用于特异识别割手密染色体。

由于割手密和热带种基因组中存在大量同源序列，仅用传统的基因组原位杂交技术，无法清晰地识别甘蔗栽培品种中割手密染色体，本发明利用割手密和热带种基因组数据聚类分析得到的割手密反转录转座子序列，并利用FISH进行鉴定出4条特异识别割手密染色体的反转录转座子，证明了该方法是有效的。

序列表

<110> 福建农林大学

<120> 割手密反转录转座子序列

<130> 2019

<160> 12

<170> SIPOSequenceListing 1.0

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agttctttgt tttattgttc tttggtttat gagtttactt tgatctcttc gcgtagagtt 780

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tcgtctagcc ggaccgtggt agtagcgagg aacgtgacaa ttccgagtta cctttgtatt 900

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actctgtttt cggctactca cttcaagatc tcacaggaat cagtcctatg atttgtaccc 3060

atcgtattcc gacagatcct tctgttacac cctctcgaga gcctcaacgt agacttaaca 3120

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ttatggttag agaaggaata gtgctaggac acctagtgtc tgaaagaggt attgaggtag 3780

atagagctaa aattgaagta attgaacaac tacctccacc tgtgaatata aaaggaattc 3840

gaagttttct tggccatgct ggtttttatc gcagatttat aaaagacttt tcatttattg 3900

cgagaccact tactcttttg ctagccaagg atgctccttt cgaatttgat gatgcatgtc 3960

taaattcttt caatttatta aagcaagcac tcatctctgc accaatcatt caaccccctg 4020

attggtcgtt gccttttgaa attatgtgtg atgctagtga ttatgctgtg ggggcagttt 4080

tgggacaaac taaagataaa aagcatcatg caattgctta tgcaagtaaa actttgacag 4140

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ataaatttag atcttattta gttggagcta agataattgt ttacactgat catgctgcac 4260

taaaatattt gctcactaag aaagatgcta aacctcgctt aatcagatgg attttattac 4320

tccaagaatt tgacttagaa ataaaagata aaaagggagt agaaaattct gttgctgatc 4380

acttgtctag aatgtacttt aagaattcac aggaaccccc cattaatgac tcactccggg 4440

acgacatgct ttacgggatt aacagatctg acccctggta tgcagatatt gttaatttta 4500

tggtttcagg ttatgtacca cccggagcaa acaagaagaa gcttattcag gaaagtcgtt 4560

cacatatatg ggatgagcca tatctcttcc gagtatgcgc tgatggccta cttagaagat 4620

gtgtgaccac tgaggaagga ttgaagatca tcgacagatg tcactcatca ccatacggag 4680

gtcactatgg agcattccgt acacattcaa agatctggca atgtggattc tactggccta 4740

cgatgtacga tgacacgaag caatatatca gaagatgtgg gccatgtcaa aggcacggaa 4800

acataaatac aagggatgcc atgccactca ccaacaacct tcagattgaa ctctttgatg 4860

tctggggaat agactacatg ggtccatttc ccccatctaa gaagtgtgag ttcatcttgg 4920

tggcggttga ttacgtctcc aagtgggtag aggcactacc ttgcaacatg ccgacaatat 4980

cagttcgaag aggatgtttg aggaaatcat atttccaaga tttggagtcc ccaggatagt 5040

gataagtgat ggaggatcac acttcattga caagcgcttc gagcactatc tatcaagaca 5100

tggaatccgt cacaacgtcg ctactcccta tcatcctcag acaagtggcc aagcagagac 5160

ttctaacaag caaatcaaga acattcttca gaagacggtg aacgagatgg gaacggcatg 5220

gaaggacaag ttacccgatg cactctgggc ttaccggaca gcatacaaga ccccaattgg 5280

aatgtctcca taccaattgg tatacggaaa gacctgtcac ctacctgttg aacttgagtt 5340

caaggcacac tgggccataa aaagatggaa tatggaccta gatatcgccg gaaaacatag 5400

aagaatgcaa ttatcggagt tagaagaatg gcgggagaaa gcatatcaca attcaaagat 5460

ctacaaagaa agagtcaaga gatggcatga caagaggatc aagaagaagg agttctcacc 5520

cggagataag gtattacttt ttaattccag ggtgaagctt ttcgggcatg gaaagctccg 5580

gagcaaatgg gaaggaccat tcaaggtaat tcattcatca tcccacggag ctatcacact 5640

tcaaaatgac gaaggtacgt tattcaaggt aaatggtcaa cgtcttaaat tatttttaga 5700

gcccaataaa gaattagaag aaatagacgt gatcaatttt taccttccaa ttaaaaatta 5760

aagcccgacg cttttaattt gacgtttttg ggccaagtat atatttttcg ggataaaaac 5820

acggtgagaa acacgctcga gaaagcaggc ttgtagagga gccggacaca gggcgggcgc 5880

cctgaggatg agggcgggcg ccctgcccct gtctcccctc ggccccagac ttctcccacg 5940

cgataacctc gcccgttgct atctggaaaa ttccgtcctc ggtttgtttc gcaaacatga 6000

cggcgggaag agtcccgacc atgcacgaga gcagttttta ccccctatat aaacagaccc 6060

ccaacgtcag cttttagcac caattcattc aagccttctc tccttcctca aattagatct 6120

tgcttagttc ctagctgctc caatggccga gaagttccac gtcgattggg aagtcgtccc 6180

ctacgacctc aacaagaagc ccaaggagga tcccgacgcc tacgctctcg tcccggccaa 6240

cacagagcga cagctagaag ccatgccacc acgtcaacgc agctctgccc aatcctactt 6300

tgctcgccca gttcttaccg cacctacgca gcccctattg ctcgaaggct cgccatcatc 6360

gtcgaagggg aaggagatca tcaaggtacc agccggaaca aagatcctgc cgccaaaacc 6420

caatgagcgg atcatcggag tgaagaccaa ccggagcggc gagatctcta gtgtccgcta 6480

taccacggag gagagacctt acttcgaggg tgttagggca gctaaggtcc gcttcgttcc 6540

tccgaaggaa gcacccaagc atgctcttaa tgctttcgag acacctccca agcgccgcag 6600

gaccattgca gatgtggatg aggagctacg ggtgatcaag aacagcatta tagaaattca 6660

gaactccaat atctctatgg atcggaggac cttcaaccat aacactacga tcctaaagct 6720

acgggatgac cttgccgacg ccaacaagag gatcgatgag ttagagcata gtcttaggag 6780

gcgtgagcgt cgctgagcta tctagattag atcttggggc tatatatgtc ttagttatta 6840

ttagattcga ttaggttcgc ttataatcag tttagatcta ttatattcgg ttattattat 6900

tattcggttg taataaatgc ctcaagatta ataaagatta ttattagtat gtcttgtgtg 6960

tctctacttt acttttgtgc aagaaagcag aaaacaagta tgggggagat tccctgacat 7020

gtcacacaca cttcgatcgc accactccat gaaccaggta cacactctgc acacttttat 7080

tacacactta cacactcacc ttagtttgtg cagaatttta tctctctaaa catgataaat 7140

taaaaggata aaaatgctta taatcatgat caatctcact ctgtgatatt tcctggaaac 7200

ctgcaattat taaaattatt tcaaaaccct gtgttgctaa aagtcattgt ggaataagag 7260

atggtaaggg tatgagtacc ttattcttag tatctttatt gcttggagaa tttgttttaa 7320

aaatctcaaa attatagcta ccatcctcaa gttttatatg cctgctaaac atgaaaatat 7380

taattaaagc tatctgcttt gtattgagtt tgttcaaaac aagttagacc cttgttgaga 7440

gatttatcat actcctaaga tcaagacatt tttattcaga aagattcact cttccgaagt 7500

attattgcgt tagaggcatg ggctatgcaa aattatgaat atatcgagga aataaaaagg 7560

agcaagtgct cgataacctc gtggaaaaaa atggacaagt gtccggcagt agaattaggg 7620

gtacctcggt atccacccaa aatgaaaaaa aatgagatat gataaaaata tgatggaaag 7680

aaaatgatag cccatggtcc ctctaataag caatatgcca gtaagagtga caagttttta 7740

attttcaaaa tctttgatct aagagtatgg cattcttctc ctcggatccg gttttgacca 7800

tacaataaat gcaaggtatg tatgcttaaa gaattatttt tgcaaaatca aaacagcctc 7860

agagagaaat ataaaagata atgagtgact ctgagagcac ctatgaggat aaaggtatgc 7920

taagtttttc ttttcaaaaa tatgtaaaaa ctccaagtga tagggattaa gaagaagaaa 7980

ggctctttac tctgaccata tatttccctg actataagtg cacagtggat ttttacaaca 8040

ccctgcaggt atgaaagaat gtttcaaccc cagatgtttt attaaccaac cttttctcga 8100

ggacgagtaa aagcctaagt atgggggtgt ttgttgacgg ttcttaagta tcaattataa 8160

ccatcaaata aataaagaaa aggacctata tgcaagcaac acctagaatt agggtttgat 8220

ctaacagaat tccacgagtt ttgctgttta tctatttctg cagggggtta tcaggaaata 8280

cggaagaaag gcccacatgt cggattaatg acgggatatt aaccgaacac gtaattatct 8340

tacatctaga agattccaga agccacggga acgaacggga gccgtaacgg gccaggacac 8400

agggcgcccg ccctgtgccc tagggcgccc gccctgcccc cgggaccaat caggtcgagt 8460

ctcgcggatt atgctccacc gcctttgagg atcaaggaaa accgtaggat gaaggtcggt 8520

ttgatccgac ggtccagatt catccaaaag ggctatataa gcaaggcccc tgacccctgg 8580

tggagaagac cccaattcat tattcagttg catatctcta gttagggttt agagagagag 8640

gttccctcta gggttcccac ctcttagggc ttagcatcca atgtgaaagt agaattagtt 8700

ctactagatt gagagagata gagtggaggt gtagatcgga ggaagccggc ctgtcggtgt 8760

ctactccgag gttgtacctg cgggagcaag ttcttctaac ccgaggcttg ttctcaggat 8820

tcttcagtat ttcgacttct aaattctagt aagttctttg ttttattgtt ctttggttta 8880

tgagtttact ttgatctctt cgcgtagagt ttagagtaat catctctagc gtaaacgtgg 8940

tgtttaagct aggatactca tagatatccc ctcgtctagc cggaccgtgg tagtagcgag 9000

gaacgtgaca attccgagtt acctttgtat tccatatccc gttagcagga tcgatagggt 9060

ttataggtgc gggttgaaca tcctttgtgg tgtctagatt ccgtaaacct ccccaataga 9120

acagtagatc atccttacca aggttagaag aagagtgcgg ttgtagtctt ctctatacat 9180

cactcacatc gaatcatagt ggttgtagcc taaaggttag tagtaataga tttggttagt 9240

cagatgcact ctttctccta aaggtaaaaa tataaatacg atacccagga taacatctcg 9300

ggtgaagtgc tcaccgatat ccgtgcgctt gcggatccta tttcctaatt gcgttaccaa 9360

atatcaaca 9369

<210> 2

<211> 8351

<212> DNA

<213> Artificial

<400> 2

tgttgacggt ccttaagtat caattataat tatcaaataa atagagaaaa ggatccaaat 60

gaaaccaaca cctagactta gggttttatc tgacagaatt ccacgagttt tgctgtttat 120

ctatttctgc agggggttat caggaaatac ggaagaaagg cccacatgtc ggattaatga 180

cgggatatta accgaacacg taattatctt acatctagaa gagtccagaa gccacgagaa 240

cgaacgggag gcgtaacaga gccgggacag ggcgcccgcc ctagtcctag ggcgcccgcc 300

ctgctggagc caatcaggct ccgcctcgag gattatgctc caccgaccta gaggatcaag 360

gaaaaccgca cgatcaatgt cggtttgatc caacggccca gattcatccg aaagggctat 420

ataagcaagg cccctgccct ggaggagagg ccctcgtctc atacttcaaa ccctaattca 480

ggaggagagc ctctgatcaa gccctagagc caccacatca actagatctc tagttagcat 540

agctacatag gattagaact agaaggagtc aatcttcgat tggttcccgg atctgtcaag 600

aggattcttg gtaattcctc tcttgttctt caattgttca tcattgttct tcaatattat 660

gaatatgact ttgttctatt tcaatatatt ggttatgact ttgctctact tgattatatt 720

tgcaattata ttgttcttag tttatcatag ttatatgctt ggcttagtta gattggaatt 780

atatacatgc ctaggatcgt atagcgttta tccatgtgta cagtgggtga atgataatta 840

ttgtgtagac gtggtgtcta taccgtattt atctgcgatt gcaccctata tgccggattg 900

tggggtagtt cgcgatagtg acagcttcgt tgattcttat atagtccccc tctcgtgtat 960

agggcaggca gagcaacact attacagggg agtgattgct atgttcttca tcttccttgc 1020

taatattcac tatgcatgga tatagtcttt tctcaccatg attgccaagt ataattgcac 1080

taactatgat atgctagact ttatagttaa taataactta gggaatatct ttgtagttca 1140

tcctaattcc atgctaatga cttgctagaa tatctgttga ggtgcttatc attattatat 1200

gtggctagct gatcagatta attatctttg tcaccattat cactttacct ttacttaatg 1260

tgacatttat acctgtataa agagattgat aaatactctc ggttatacat gcaatgatgt 1320

gtactcagtt ccatattctc attccattat caaccatgat acttagaaat cccttcccag 1380

tggtaaaaat ataaataacg atacctggaa tacttcccgg ttaaaatgct acatcggtat 1440

taatctgtgc gcttgcagat cccttttatt atttatcttg atgagcaaat gcatatttca 1500

ataccgcgtc tctcatgtca tgctggggat gacaacttgg cttaagtggc atgagggata 1560

ggtttggcat ttttggcgcc gttatcagaa ttagaaaact aagtctactt ttggtagtga 1620

cgttaagaat gcccaacaag catttttggc gccgttgccg ggaaggttga ttactaagta 1680

ggaatgaata cggaactttg agtcatcatt tgcatcacta atctgatcga gcttatcaat 1740

tctcttatac agttttaccc ctgtattttc cattttgatt atttattgca gggtgatgca 1800

tgaatagaag acatcttcca gacaactttg ttgacgatcc cgaagcctta ttcagaagaa 1860

caagagccaa gctcaagaag acatcgtcaa cacttcagca caaagcttca tccaaatccg 1920

aagaccgccg aagtttcatc cggaatttgt cgactgaatt cgaagccatg gcgaacaagt 1980

cgatccgcga gttctcagct cccactacgg acaacatccg cactggacct gctgggagat 2040

cgaccgcaac ttcgagctca agcctgggct catcaacatg gtgcaagcta accagttctg 2100

tgggaagcca catgaagacg ctagtgctca tcttcaacac ttcctggaga tctgcagcac 2160

tttcaccatg gctgaagtcc ccagagacgc catactactt cgccttttcc cattctcact 2220

gttggggaga gcgaagcagt ggttctacgc tacaaaggag aagaacacta cgtgggcact 2280

ctgctccacg aactttctgg ccaaattctt tcccatgggc aagaccaatg ctctccgtgg 2340

gaagattaca agttttcagc aacaacatga tgaatccgtt ccagaagcat gggagcgttt 2400

ccaagactac atcctagaat gtccccatca tggaatggaa agctggctac tgatgcagac 2460

attttatcat gggctcatta acagtgcccg tgaaaccatg gatgctgcag ctggaggagc 2520

attcctatca ctcaccatac ctcaagccac agctcttgtg gagaagatgg catccaacca 2580

aggttggaat gaagaaagaa ctcagacacg caagagaggt ggaggtatgc accagctcaa 2640

ggaggtagac atgctgtctg ccaagctaga cctactcatg aagaagctcg atgatcgagc 2700

tggagaaaag aaagaagtca tgcacgtcta cgactcccac atgacttgtg aggagtgtgg 2760

aggtactgga cactcgggca atcactgtcc tgagttgctg gaggacgtga actacatcaa 2820

caataacaac aactactaca accgtcctca gcagaatcaa ggttggaatc aacagaggcc 2880

taactactca ggtaattacc aaggtaacaa ttctttcaat aataataata attatccacc 2940

tttgagagag ttagtatcca accaaggaaa gctaatggat aacctgtcta agaaattggc 3000

atctaatgat aaaatactag aaaatataaa taatagaatg gataatttct ctactgccat 3060

caagaaccag attagcttta ataaaatgat tgaatctcag ttgaatcaaa tagctgctgc 3120

tgttcctgct actaaccccg gtataccatc acaaccggaa ggactagaat ctgcaaatct 3180

tgtagacatg tttgatgcag gagattactg gagtaaccct atcgtggaag taagtactga 3240

ccgtctgccg gtcaagagag gcgatccagg acgccccgtc atcccgatct ccatcggcat 3300

gcgagacttc ccagaagcac tctgtgactt tggctccagc gtcaacatca tgcccagggt 3360

actctatgaa aaactctttt cacaaccatt attagaaaca accatgtgtt tgcagcttgc 3420

agataggaca ctgagtttcc cgagaggaat attgaagaac atctgtgtcc gagtgggttc 3480

ctcgtacgct ccagcagact tcgtagtgat agagaccggt tctgatgaga gggctcccgt 3540

catcctaggg agaccattcc tgaacaccgc gggagctgtc atctatgcta gtgctgccaa 3600

gatcagtttc tacatcaagg ggaggaagga aacgttttcc ttcaagaaca agaccgcaca 3660

aatcccagag caaccccaat atgaaccaag gaagaggacc aacaggagga acaagagcaa 3720

gaagcaagta tggaccgaga cagctaagat ggtcactgcc gtgcacaaag gtcaagatcg 3780

ccaactcaag tcaccgttct tgcctaagaa ggacgaccca ggtatgccaa gcatttattg 3840

ctccattaat gggtcccact tctacaagac actttgcgac actgggtcgg gcgtcaacat 3900

aatggccaag gtcacctatg aacttctgtt cggaaccatg cccttaaacc caacatatat 3960

tcagctccag atggcagatc agacattccg acaggtcgaa ggtacagtaa ctgacgtccc 4020

tgtcaagata gacgatcact ttgtccatac agactttcag gttattgaca tgggagaaga 4080

tgaatacgat ccacccatca tccttggaag accattcctt agtactgtca aagcaatcat 4140

ttacattgga actggagaag tccacatgca cttcccctct gagaaggtac gtctttactt 4200

tactgaccct aactatgtat ttgaagaatc caagcaggtc aggacaagaa gaaggcggcg 4260

taaccacaat cagaagcaac aggtcatcaa ggacggatgg gcagattatg aaggagaagt 4320

ggtaagatct gaagacatac cactcaacca acactgtcct gaggagacca aagcaccgag 4380

acaggtatgg aaagaaaaga cagttgtaca tgaagaagag gcgccgccgg aaccaccgac 4440

tacgccatcc accaagtccc aggacgactg aacgaataag agtcccgttc ggaggactta 4500

aaaacaccga acgccttgcc aagaggtaac ttggtagtta tcctttcctt tttaattatt 4560

tactttatct taaatagttt gcttagttaa tcatattcat actatcttaa aaagaaaata 4620

aaaatgttaa aaaccctaaa gccccatgtg agtatgcgag tggcataaaa cccataagta 4680

cattcactgt ggtggcataa aaaatatata tatatataat aaatacataa atattttctt 4740

ctgctttata aaaaaaaaaa aaaacataaa aaaatcaagg aggctcagca tgataaaggc 4800

tagatattta tgctaacact taatcagttc cacaaagctt tgttgtctat ttgagcttca 4860

cagaattcaa ggagactagc agacagagga cattctaatc gctgtcaggg tgctgccgac 4920

tttcaaatac acctccgcca tctgctagct acatcagaag agattatgtc aagatccagc 4980

ttgggggaga gcacctccat tatcttgcta agtatttcta tctttatctt tatatttata 5040

tatacttcta ccctataaaa ataaaaatat acataactat gaaaaaacca aataaagatt 5100

ttgtgcttat atatatatac ctatatcttt tgcttagtgt gttttaataa ataaataaag 5160

tggctatgct aaactgaatc taataataat aaaactctag catggatatg atgaatagtt 5220

gctttgccta actttcaaat ttgaagtcct ctctctaagt ttagacataa ctgttatcat 5280

ttaaagcttg ctctaaacct gaacttgtgg gaagagaact tgatctaaag tctaagttgt 5340

taacggatat gatatgggaa ggttgagctg ctgtttatct attcctagag atgctagaat 5400

tctggagaat tttatctttg aaaatcttaa aatgctacat gatgagttcc tgtatgatga 5460

gagtttaagt tcctaccaca gccatacata catgcttgct agactttaag ccataccttt 5520

actttttact gcttatgagc attgagtgta gtcaagctgt gtagaccctt aggagcttgt 5580

catgcggtta aaatcaagat tcacttgcac gatcactcat acatgctgct tctactccgg 5640

aagtacgcat ccacatatat ccactcattc tcatctccag atccacataa aattattcta 5700

ctcctaatcc gggagagaat agccaaaaat attttcccat tcttgttatc ccctgtgaaa 5760

taaatgctca agctattttg gttactacca cttgctatat tgttctaagg agatgagtgc 5820

tctatacgag gaaataaaaa ggggcaagtg cccggaacct cgaaaagaaa aagatacgag 5880

gaaataaaaa ggagcaagtg ctcggaacct cgatgaaaaa aaagaaaaag tgtgagaaga 5940

gaggtaaaaa tggacaagtg tccgacagta gaattagggg tacaagatac ccacctgaga 6000

gaaaaaaaaa tatagagcac ctcattctcc tcaagagctt taaaagcaag aaaggtacgt 6060

atcccctcaa aaagagcaaa agtagaatta gactttcatc attgttatca tcactaccac 6120

catacaccat ttattcgcca cacatgcaca tcttgatttg acttattggc ttgtttctct 6180

ggatccatgg tttgactatg caataaatgt cttgtaagta tgtatacttt atctcccacc 6240

gatgagctcc agatatcaaa agccttatta gaataggatg agagagaagg caatgtcact 6300

ctgccttata ccacaaatac tacatacttt gctttgagag aaggcagaca tcattactgc 6360

cttggtgagg atccagaaat accacaaaag agagacctga gagagtcata caaggaatct 6420

ctgagtttta tttgaaaatc tgcaaaaaac tccaagagct atagctaatc aagaataaga 6480

gacatggcgc ttgactagac tgttctatct tttaaccgct caagacaaag gtgacggttg 6540

caagccccat ggtgaaaggt ataatgagta agttttaagt cttgacagtt tactttaact 6600

cagagatgag actctatttg aaagcatgtg tacgtcaaaa ttcaaaggca tttcagcaac 6660

tactgagtct ctccttgctc agggacgagc aagaggtaag cttgggggag tttgttgacg 6720

gtccttaagt atcaattata actatcaaat aaatagagaa aaggatccaa atgaaaccaa 6780

cacctagact tagggtttta tctgacagaa ttccacgagt tttgctgttt atctatttct 6840

gcaggggtta tcaggaaata cggaagaaag gcccacatgt cggattaatg acgggatatt 6900

aaccgaacac gtaattatct tacatctaga agagtccaga agccacgaga acgaacggga 6960

ggcgtaacgg agccggacac agggcgcccg ccctggtcct tagggcgccc gccctggccc 7020

cggagccaat caggctccgc ctcgaggatt atgctccacc gacctagagg atcaaggaaa 7080

accgtccgtt caatgtcggt ttgatccaac ggcccagatt catccgaaag ggctatataa 7140

gcaaggcccc tgcccctgga ggagaggccc tcgtctcata cttcaaaccc taattcagga 7200

ggagagcctc tgatcaagcc ctagagccac cacatcaact agatctctag ttagcatagc 7260

tacataggat tagaactaga aggagtcaat cttcgattgg ttcccggatc tgtcaagagg 7320

attcttggta attcctctat tgttcttcaa ttgttcatca ttgttcttca atattatgaa 7380

tatgactttg ttctatttca atatattggt tatgactttg ctctacttga ttatatttgc 7440

aattatattg ttcttagttt atcatagtta tatgcttggc ttagttagat tggaattata 7500

tacatgccta ggatcgtata gcgtttatcc atgtgtacag tgggtgaatg ataattattg 7560

tgtagacgtg gtgtctatac cgtatttatc tgcgattgca ccctatatgc cggattgtgg 7620

ggtagttcgc gatagtgaca gcttcgttga ttcttatata gtccccctct cgtgtatagg 7680

gcaggcagag caacactatt acaggggagt gattgctatg ttcttcatct tccttgctaa 7740

tattcactat gcatggatat agtcttttct caccatgatt gccaagtata attacactaa 7800

ctatgatatg ctagacttta tagttaataa taacttaggg aatatctttg tagttcatcc 7860

taattccatg ctaatgactt gctagaatat ctgttgaggt gcttatcatt attatatgtg 7920

gctgatcaga ttaattatct ttgtcaccat tatcacttta cctttactta atgtgacatt 7980

tatacctgta taaagagatt gataaatact ctcggttata catgcaatga tgtgtactca 8040

gttccatatt ctcattccat tatcaaccat gatacttaga aatcccttcc cagtggtaaa 8100

aatataaata acgatacctg gaatacttcc cggttaaaat gctacatcgg tattaatctg 8160

tgcgcttgca gatccctttt attatttatc ttgatgagca aatgcatatt tcaataccgc 8220

gtctctcatg tcatgctggg gatgacaact tggcttaagt ggcatgaggg ataggtttgg 8280

catttttggc gccgttatca gaattagaaa actaagtcta cttttggtag tgacgttaag 8340

aatgcccaac a 8351

<210> 3

<211> 8217

<212> DNA

<213> Artificial

<400> 3

tgttgacggt ccttaagtac caaatatagt tatcaaataa ataaagaaaa ggatccaaat 60

gcaaccaaca cctagactta gggttttatc tgacagaatt ccacgagttt tggtgtttgt 120

ctgtttctgc agggggttat cagaaaatac ggaggaaagg cccacacgtc gggtttacat 180

agagataata acgtgttcac cgattttcta tcatctagaa gactccagaa gccacgagat 240

cgaacgggag gccgaacggg cccggaggca gggcgcccgc cctgctccct agggcgcccg 300

ccctggcctg agagccaatc aggctccgtc tcgcggatta tgctccaccg acctaaggga 360

tcaaggaaaa ccgtgcgatt aatgtcggtt tgatccgacg gcccacgttc acttgagggg 420

gctatataag caggacccct ggcccctgga ggaggcactc cctcattctc aattcctcaa 480

accctaatct caggaggaga gtctctgatc aagccctaga gccaccacat caactagatc 540

tctagtatag catagctaca taggattaga actagaagga gtcaatcttc gattggtttc 600

cggatctgtc aagaggattc ttggtaattc ctttactgtt cttcattgtt catctttgtt 660

cttcaatatt atgaatacaa ctttgttcta tttcaatata ttgattatga ctatgctcta 720

cttgtttatg tttgcgatta tattgttctt agtttatcgt agttatacgc ttggcttagt 780

tagattggaa ttatatacat gtttaggatc gtatagcgtt tatccatcgg atccatgggt 840

aaatgataaa tattgtgtag gcgtggtgct tagaccatat ttatctgcga ttgcacctta 900

tatgccggat cgtggggtgg tccgcgatgg tgacagcttc gttggttctt atatagtccc 960

cctcccgtgt gtaaggcagg cagagcaaca ttattacggg ggagtgattg ctatgtttct 1020

catcttcctt gataatatca ctatgcatgg gcgtagtcct ttctcgcaat gattgccaag 1080

tgtacttgca ctaactatga tatgctagac tttatagtta agaataactt aggaaatatc 1140

cttgtagttc gtcctaatac catgctaatg acttgctaga atatctgttg aggtgcttat 1200

cattattata tgtggctagc tgatcagatt aattatcttt gtcaccattc atactttatc 1260

tatattttat gtgacactta cccctgtatg caagagatag atgaatgctc tcacttatac 1320

atgcaatgat tgatactcat tcctatattc cattccataa tcaacattga tgttagtaat 1380

cccttcccag tggtaaaaat ataaataacg atacctggaa tacttcccgg ttaaaatgct 1440

acatcggtat taatctgtgc gcttgcagat cttatttatt atttatttag aagagcagtt 1500

gcatatttca ataccgcgtc tctcatgtca tgctggggat gacaacttgg cttaagtggc 1560

atgagggata ggttcggcat ttttggcgcc gttatcagaa ttagaaaact aagtctactt 1620

ttggtaatga cgttaagaat gcccaacaag catttttggc gccgttgccg gggaaggttg 1680

atttactaac aaggaatgaa tacggaattt gagtcatcat tcgcatcatt aagtgattga 1740

gatcatcaat tctcccatac agatttaccc ctgtattttt ccattcttat tgttttgcag 1800

ggtgatgtat gaatagagga catcttccag aaaattttgt tgacaacccc gaagcattaa 1860

tcagaggggc aagagccaag ctcaagaagt caacacttcg acgcaacact tcatccaatc 1920

cagaagaccg ccgaagtttc atccggaatt tgtcaacaga gttcgcagcc atggcgaaca 1980

agacgatccg cgagttctca gctcccacta cggacaacat ccgcactgga cctgccgcgg 2040

ccatcgacaa gaactttgag ctcaagccag ggctcatcaa catggtacaa gccaaccagt 2100

tttgtgggaa gccgcacgaa gatgcaagtg ctcatctcca acacttcctg gagatttgca 2160

gcacattcac tttattagat gttcccagag acgccatact acttcgcctt ttcccattct 2220

cactattggg gagagcgaag cagtggttct acgcgacgaa ggataagaac actacgtggg 2280

cactctgctc tacgaacttt ctggctaagt tctttcccat gggcaagacc aatgctctcc 2340

gtgggaagat tacaagtttt cagcaacaac atgatgaatc cgttccagaa gcatgggagc 2400

gctttcaaga ttacatccta gaatgtcccc atcatggaat ggagagctgg ctacttatgc 2460

agactttcta tcatgggctc atcactagtg ctcgtgagac catggatgct gcagctggag 2520

gcgcatttct atcactcacc ataccacaag ctacagccct tgtggagaag atggcgtcca 2580

accaaggttg gaacgaagag aggactcaaa cacgcaagaa gggtggaggt atgcaccagc 2640

tcaaggaggt agacatgctg tctgcaaagt tagacctact catgaaaaag ctcgatgaca 2700

aagctggaga caaaagagaa gtcatgaacg tctacgactc tcacatgact tgtgaggaat 2760

gtggagacac tggacactca ggcaatcact gccctgagat gcttgaggat gcaaggtaca 2820

tcaacaacaa caacactaca accgtcctca acaaaatcaa ggttggaatc aacagaggcc 2880

taactactca ggtaactact caggtaatta tcaaggtaat aactcttaca acaacaataa 2940

taattttcca ccctgagaga gttagtgtct aatcaaggaa agctaatgga taacttgtct 3000

aagaaattgg catccaatga taaaatacta gaaaacataa ataatagaat ggataatttc 3060

tctactgcca tcaaaaacca aattagcttt aataaaatga ttgaatctca gttaaatcaa 3120

atagctgctg ctgttcctgc tactaacccc ggtataccat cacaaccgga aggattagaa 3180

tctgcaaatc ttgtagacat gtttgatgca ggtaactatt ggagtaatcc cgctgtcgga 3240

gtacataatg accttctgcc agtcaagaga ggcgatccag gacgccccgt catcccgatc 3300

tccatcggca tggtggactt cccagaagca ctctgtgact ttggctccag cgtcaacatt 3360

atgcccaggg tactctatga aaaattcttt acatatcctt tatcagaaac aactatgtgt 3420

ttgcagcttg cagatcggac actaagcttt cctaaaggaa tattaaagaa catgtgtgtc 3480

cgagttggta cctcgtatgc tccagctgac ttcgtggtga tagagaccgg gtccgatgag 3540

aggtcaccag ttattctggg aaggccattc ctgaacacct cgggagctgt catctacgcc 3600

agcgctgcga agatcaactt caacatcaag gggaggaagg agacgttttc cttcaagaac 3660

aagattacac aaatcccaga gcaaccccaa catgaaccaa ggaagaggac caacaggagg 3720

aacaagcaga acaagaacaa ccaaggatgg accgaatcag ctaagatggt cactgcagtt 3780

caaggaggtc aagatggtcg actcaagtcg ccgttcctaa tcaagaagga cgacccaggt 3840

atgccaagca ttgagtgctc aatcaatgga tactcctttc agaaggcgct ctgcgacact 3900

ggatcaggcg tcaacataat ggccacagtc acctatcagc tcttgtacgg gaccatgccc 3960

ttaaaaccaa catacactca gctccagatg gcagatcaga catcccgaaa ggtcgaaggt 4020

atagtaaccg atgtccctgt taaaattaac gatcattttg tccatacaga ctttcaggtc 4080

attgacatgg gagacgacga gtacgatcca cccatcatcc taggaagacc gttcctaggc 4140

accgtcaaag caatcatcta cattggaacc ggagaagtcc atatgcattt cccctctgag 4200

aaggtacgcc gctactttaa tgaccctaac tatatagttg aagattctaa gcaggtcagg 4260

acaagaagaa ggcgacgtaa ccgtaaccaa aggaggcaaa ccatcaagga cgtatgggca 4320

gactatgaag gagaggtcat aaggcccgag gatacacaac aggagaccga agcaccaagt 4380

cgggtatgga aagcgaagac agttacacaa gaagaggagg cgctgccgga accaccgtct 4440

acgccaccca aatcccagga caactaagaa gaaaagaagt cctgttcgga ggacttaaaa 4500

acaccgaacg ccgtgccaag aggtaaactt ggtagttatc attttccctt taattattgc 4560

tcagttaatc atgttcattc tgtctaaaaa aatgttgaaa acagtaagcc ccatgtgagt 4620

atgcgagtgg cataaaaccc ataagtacat tcactgtggt ggcataaaaa aatataataa 4680

taatatatat tttttctgtt ctataaaaat gaaaataaaa atagagagtg acatttatca 4740

aggaagctca aacatgataa aggctagata tttatgctaa cgcttaatca agttccacga 4800

agctttgttg tctatttgag ctccacagaa ttcaggaaga ctagcagatg gaggacatcc 4860

taatcgctgt cagggtgctg ccgacattca aatacacctc tgcacctgct agctacatca 4920

gaagaaatta cgtcaagatc cagcttgggg gaagcacccc catttatccc gataagtatt 4980

tttatctaca tttataccta tgctttatta aaataaaaag atgcataatc atgaaaaccc 5040

aaataaatat tttgtgttta tatatatttg cttagtttaa taaataaata aagtagctat 5100

gctaaactga atcttgaaaa taaaactcta gcatggatat gatgaatagt tgctctgcct 5160

aattttcaaa tttgttctct ctctagttta gacataactg ttataattta aaactggctc 5220

taaacctgaa ctagtgggaa gagaacttga tctgaagtct aagttgttag cggatatgat 5280

atgggaaggt tgagctgctg tttatctgtt cctagagatg ctagaattct ggagaatttt 5340

atctttgaaa atctttaaaa tgttgcatga tgagttcctg tatgatgaga gtttaaattc 5400

ctaccacagc catatataca tgcttgttag attaagagcc gcacttttac tacttactgc 5460

ttatgggcat tgagtgtggt caagctgtgt agacccttag gaacttgtca tgtggttaaa 5520

atcaagattc acttgcacgt tcactcatac atgctgcttc tactccggaa gtacgcatcc 5580

acatatatcc actcatttcc atctccagat tcacccaaaa ttattctact cctgacccgg 5640

gagagaatag ccaaaaacat tatcccattc ctgttattcc ctgtgaagtt aatgctcaag 5700

tcatttctac taccacttgc tatattttca aaagagggag attgctctaa aaaaaataaa 5760

acgaggaaat aaaaaggagc aagtgctcgg aacctcgaag aaagaaaaag tgagacgaga 5820

ggtaaaaatg gacaagtgtc cgacagtaga attaggggta caagataccc acctgagaga 5880

aaaaaaatag agcatctcat tcccctcaaa agttttaaaa agcaagaaag gtatgtattc 5940

cctcaaaaag agcattagac ttttgttatc accatacacc actcattcac cacacatgca 6000

catcttgatt tgacttattg acttgtttct ctggatccat ggtttgacta tgcaataaat 6060

gttctatgag tatgtatact gtatctccca cctatgagct ccagatatca aagccttatt 6120

agagtagggt gagagagaag gcaatgtcac tacgccttat accataaata ccacatactt 6180

tgagagagaa ggcatatacc attactgcct tggtaaggat ccagaaatac cacaaaagag 6240

agacccgaga aagtcataca aggaatctct gagttttatt tgaaaaattt gcaaaaactc 6300

cagagctata gctgatcaag aataagagac atggcgtttg actagaccgt tctatctttt 6360

aaccactcaa gatgcaggtg acggttgcaa gccccatggt gaaaggtaaa atgagtaagt 6420

tttaagtctt gacagtttac tctaactcag ggatgagacc ttatttgaaa gcatatgtac 6480

cgtcaacgtt caaaggcgtt gcagcaactt ctgatccata atgagtctat ccttgctcag 6540

ggacgagcaa gaggtaagct tgggggagtt tgttgacggt ccttaagtac caaatatagt 6600

tatcaaataa ataaagaaaa ggatccaaat gcaaccaaca cctagactta gggttttatc 6660

tgacagaatt ccacgagttt tggtgtttgt ctgtttctgc agggggttat cagaaaatac 6720

ggaggaaagg cccacacgtc gggtttacat agagataata acgtgttcac cgattttcta 6780

tcatctagaa gactccagaa gccacgagat cgaacgggag gccgaacggg cccggaggca 6840

gggcgcccgc cctgctccct agggcgcccg ccctggcctg agagccaatc aggctccgtc 6900

tcgcggatta tgctccaccg acctaaggga tcaaggaaaa ccgtgcgatt aatgtcggtt 6960

tgatccgacg gcccacgttc acttgagggg gctatataag caggacccct ggcccctgga 7020

ggaggcactc cctcattctc aattctcaaa ccctaatctc aggaggagag tctctgatca 7080

agccctagag ccaccacatc aactagatct ctagtatagc atagctacat aggattagaa 7140

ctagaaggag tcaatcttcg attggtttcc ggatctgtca agaggattct tggtaattcc 7200

tttactgttc ttcattgttc atctttgttc ttcaatatta tgaatacaac tttgttctat 7260

ttcaatatat tgattatgac tatgctctac ttgtttatgt ttgcgattat attgttctta 7320

gtttatcgta gttatacgct tggcttagtt agattggaat tatatacatg tttaggatcg 7380

tatagcgttt atccatcgga tccatgggta aatgataaat attgtgtagg cgtggtgctt 7440

agaccatatt tatctgcgat tgcaccttat atgccggatc gtggggtggt ccgcgatggt 7500

gacagcttcg ttggttctta tatagtcccc ctcccgtgtg taaggcaggc agagcaacat 7560

tattacgggg gagtgattgc tatgtttctc atcttccttg ataatatcac tatgcatggg 7620

cgtagtcctt tctcgcaatg attgccaagt gtacttgcac taactatgat atgctagact 7680

ttatagttaa gaataactta ggaaatatcc ttgtagttcg tcctaatacc atgctaatga 7740

cttgctagaa tatctgttga ggtgcttatc attattatat gtggctagct gatcagatta 7800

attatctttg tcaccattca tactttatct atattttatg tgacacttac ccctgtatgc 7860

aagagataga tgaatgctct cacttataca tgcaatgatt gatactcatt cctatattcc 7920

attccataat caacattgat gttagtaatc ccttcccagt ggtaaaaata taaataacga 7980

tacctggaat acttcccggt taaaatgcta catcggtatt aatctgtgcg cttgcagatc 8040

ttatttatta tttatttaga agagcagttg catatttcaa taccgcgtct ctcatgtcat 8100

gctggggatg acaacttggc ttaagtggca tgagggatag gttcggcatt tttggcgccg 8160

ttatcagaat tagaaaacta agtctacttt tggtaatgac gttaagaatg cccaaca 8217

<210> 4

<211> 9148

<212> DNA

<213> Artificial

<400> 4

tgttgacgat ccttaagtat caaatttaat tgtcaactaa acatggaaaa ggatcaatat 60

gcactagaca tctagaatta gggttttatc tgacagaatt ccacgagttt tggtgtttgt 120

ctatttctgc agggggttat cagaaaatac ggagagaagg cccacacgtc gggtttacat 180

agagatatta acatgtgcgc taattttcta tcatctagaa gactccagaa gacacgtgaa 240

cgaacgggag gccgaacggg gcccagccca gggcgcccgc cctagggttt ggggcggccg 300

ccctactccc gtggccaatc agcgtcaact tcgcggatta tgctccaccg acctaaagga 360

tcaaggaaaa ccgtgcgatt aatgtcggtt tgatccgacg gcccacgatc atttgagggg 420

gctatataag cagggcctct ccaccccagg ggaggaggag aaatcattat cagaggaagc 480

catcaagttt agggtttaga aactctctct cccgcagaga attagattta gctactccca 540

attctttcaa gttttatagg attgattaga tagaattaga gaagtagggc ctagcgctct 600

ggatttcgga tcttcgtcaa taaagattgg tattatttca tatctttctc tacgacttta 660

ttctaattgc attatgtctt tatttattat gttcctagtt tgctctagtt ctataagtga 720

tatagttatg attgatgatg agttcatgca tgagtttgca aagcgcttag ctcttttcac 780

gtgggagtta agtggtagat cacatgtagg cgtggtgctt agatgttatt tacctgcaaa 840

tgtatcctat tggccgggtc gtgtggtagt tcgcgatggt gacagcttcg ttgattctta 900

tatagtccac cctccgttga taggacaggc agaatttgta ttgcggagta agtcttgcta 960

tgttctgatt tactttagca atgttcctta tacatgaatg aagagtcttt tgtgctatat 1020

atgatcttgt agatgcttag agtagattat gacttagtaa atagtagata cttagaatcc 1080

attctcttgc tagtccgacg tcaccttaca tttatgtgga gtagtctatt tctaatcgct 1140

gtgttattta cccatgagct tatatttcat tatctttatt attatggctt accccctgcc 1200

aaagcaagtg actgtgtgac gagtttctca gtagtaatca tgttcttgca agtttatctc 1260

tagtctaagc cttgatagat tttacccctg ttttcgcttt cgccgttctc ttaagcaaaa 1320

ttataaataa cgatacctgg aatacttatc ctggtgaaat gctacaatga ggtattttat 1380

ctgtgcgctt gcggatagaa tagattattt tctagagagc cttatgttta taaatacctt 1440

agtacgctct agcaccatgc tagggatgac aacctagtat tcaagtggtg ttagctagtg 1500

tcaacaagca tttctggcgc cgttgccggg gaacggtaag gaaagtcagg aagtcggtca 1560

aggttattca aataaaattt tagactagac tattgagaaa taattgcata acagctacta 1620

tataaatgag aaatcatagc aaggcacctc tgctttggca ggttcaccct gttgttttcc 1680

tatgtttata ttttttacag ggtatatcag gattgacttt ggtgaattaa attcttcatc 1740

atcagaacca aagcaatcaa ggaagaagaa gctagctacc aattgttgaa gtgatggcac 1800

agaagacctt acaggaattt tctgccccaa gtcttgagaa cattcttact ggtccaagat 1860

ttgaagtaga agaaggagta cctgagttcg agctcaagtc aaacctcatc aacttggtgc 1920

aagctacaca attcagtggg aaggcacacg aagatgctag tgcacatttg cagaacttct 1980

tggagattgg aagcacaatt agcatcaacg gagttgacaa agacgtcata ctacttcgcc 2040

tctttccatt ttcactagaa gggaaggcga ggaagtggtt ctacaccaat caagcaaaca 2100

tcaaaaattg gacgaacctg tcagatgcct ttctatcaaa gtttttccct ataggcaaaa 2160

cagctgcctt aagaggaaat attgtcagtt tccaacagca gaagacagaa accattccag 2220

aagcatggga gcgttttcaa ggatacatat cagattgtcc tcaccatgga atggccaaat 2280

ggttacttat gcagaccttt tatcatggat taacccaaaa gtctcgtgag tgcctagatg 2340

catctgctga aggatcattc ttggagttta caattggaaa agcagagaca cttttggata 2400

agatagcaga aaaccaaagc tggttccaag acaagactca acattgtcat caaactgaag 2460

aaataccaga agaagtaaaa gcactatcaa ctaagatgga agatttgctc cattggattg 2520

accagagggc caagttcaaa gaagatcaaa gggctataga gacagtatac aaatatcaaa 2580

ccacctcaag tcaacccaat agcaaaggta tgaattcagg taatattctc aaacaacctt 2640

cattaaagga gataattgct caacaaacta aaactaatga tgaagtcaaa caaaggctag 2700

atacaaatga atcattttta aaagatatac acaataaaat ggattttcta ttaactgcct 2760

ttgatgagca aaacactctt aataagaggg tagagcttaa gctagctgct gtcttgcctg 2820

ttgccactaa ccttgagcag gtaaagaaca taactactag aggagggaga tctaccagag 2880

atcccccaca cccaagagag aagcaaaaaa caccagctcc agtgcaacca gcaatgatag 2940

aagaagagag accagttgaa gcagaagatc tgctacaacc atcaagaact ggagaaatga 3000

ggaaagattt tcacgacacc aactatttgc catttcccag aagaaacaga ggactacagt 3060

cggatgagca gtttggtaag tttgtagagg tcattcaaaa gttatatgtc aacatacctc 3120

tacttgatgc catacaggta cccacatatg cgaagtacat tagagatatt cttaacaaaa 3180

agaggccact gcccaccact gaggtaatca agctgacaga agaatgtagt gcggccatcc 3240

tcaaccaacc actaaggaag aagaaagatc caggatgtcc taccattgat tgctcaatcg 3300

gagaccagca ctttaacaat gcactttgtg atctcggagc aagtgtcagt gtgatgccag 3360

catcagtcta caagaagctt gaacacacaa ccctagaacc aacatcaatg tgcctacaac 3420

tagcagatca atcagttcga cacccgatgg gcatcgcaga aaatatccca gtcagaataa 3480

gagatttcct tgtgccagta gacttcgtgg tactggacat gaaccccgac tcaaaagtgt 3540

ccatcatcct tggaaggcca tttctgagca ccgccaatgc ccacattgat gtcagtaagg 3600

gagaaatcaa gttcagcata aacggacaag aagaacactt cacattcaag cccagaccag 3660

agagagactc tacagtggag gaggttcacg aagagaaacc actggagaca ccatctccag 3720

aggaaggcaa ttcagaagtt taaaaagatt tggaggtcca gcttggggga cctaaaattc 3780

ccaaaccctc gccgggaggt aattcggtat ttatccacat catttaattt tttgcataat 3840

taattcttgc attagtcata ctcatccata gcattattat aaaaatcaaa agtcccatat 3900

aaataatatt tgtggtgtgt aaaaacccat aattattaat tattgtggag gcacaaaaat 3960

atttttccat gatcattttt tagtttcaat tctcataatt tttcctgcat tatatttatt 4020

tatagcaatc ttctagaagc atgacccaca tcctttggtc ccatatgtca tacactacac 4080

ctcacataca tcataacaca taatttcacc caccaactca tctccactca accagacaac 4140

ttccaccgac caaccaccac ctatgcagca ttatttggcg taggagtgaa gcatgtgagg 4200

gagtgggaaa gtttcagcca cagagggcac tcaagtgggg cgcccgccct gttggccagg 4260

gcgcccgccc tgcccccagc tccaactata aaaggccacc tctctacctc attctcatcc 4320

cacacacatt ccagaaaaca tatgcaagct tgagctctcg actttcagaa gaagtgatct 4380

agtgggagag tgagaaggag agtggaaagg gaaagaaaga gtggaagaga gtttggaaaa 4440

tttttgagat agagagtgag atcacctagt aattagtgtt cccgctgtcc caagcggaat 4500

taaaagttgt ttagggggaa gtgctgctaa aatttataat tctctcggac gactaacccc 4560

tggacgactg acttctcgga cagctaacca ctaacatatt tttctcacat atttccacga 4620

gtttctgttt gtccctatta ttctgcagga tgtttaagaa agtaaagagc gcggccaagt 4680

ctctcaggag tggtacgagg agttcgtctc gactctcctc acgccagtcg gagatgagtg 4740

tcgatccggc acctccgcaa gctccatcgt cttcgtcggg tgcaccaaac aaggtcctac 4800

tcaagacagg agaccttggg ctcaggaatc gcagggagaa ggagattctc cagcagttga 4860

aaaacaagac attcattcac actcccacca tcgatttcgc cttactccaa gagacaggta 4920

tggctgctga atttgactta atttttcaaa tgataggatg gacggacttt tggaatatca 4980

ctgagcatgg ttcccgtctc cttactattg aatttctttg cacgttacaa tattgtgagg 5040

ggggaatttc ttttcggatg ttcaagcagg acattatgct gtcctggaga gagctgagta 5100

atcacctcgg ttttcctcca cggagcattc tggaccttga ctccggcttg cccaattttg 5160

agaaacatca gttttggaga gaaatctcta gggacgaact cttttaccaa ccccgaacca 5220

gcgacatgga gcatcctaca ctccggatgt tccacaaatg gctcgggtac aatttttctt 5280

tcgtgatgac ttgagaaagg tgcgtgtggg agatctacaa ctcatttatg ccgccataaa 5340

caaaatccaa gtttcacctg ttactctttt agttgcccat tggcttggca cacctactct 5400

tcagggacct gtcgggtgta cttcactcat aactcgttta gccgttagcc ttaagttgtt 5460

agaaaattca tcgttagaat tcattgagga acctagattt tatcacggct acgacacttt 5520

tagatacgca cggatgttaa aaagggaagc ggggataatg tacatgctgt atgacaacaa 5580

caccaaggtt cggttaccta acccggacct tggcatatat tctgttcgaa attatttgat 5640

tgagactgca gcaccggtga accggagagc tccacagcgc gcggcatccg caaggatggc 5700

cacccatcag gaacatacat ggcaaggagc tgatcccggg ccagaagagg cagcacatct 5760

gcactataat gattacaacc ccagagttct tcgggaccca tgggcgcgac acgtccagcc 5820

agaagaacca ccacaagaga catggccgga gggacaatat caccagtggg agaacccgcc 5880

ttttacaaga aggtactcca ctgacccata tggagcttca gggtctaggc cccagcccca 5940

gtttgataca ggaagatact ccgacgcctc ctacgccttc tcgggggact actaccaaga 6000

gactgccgcc ttctacaccc gcaccgacaa cactctcctc gacatccgca ctacgcaagc 6060

agagcacgga agactcctgg aggagcaaca aaaatggaac caggagcaag ccactagagt 6120

gcaagcaata agggaagaca ccacaacctt gaacaacaac gtcacgacca tgctgcgcta 6180

cttcaacatt gagtgaagag agccacgaca acaaccagct tgggggagtt cctccccagt 6240

taccgagtga gttttaattg tttttcctat ttacttttca gtttttcttt ttctgtttta 6300

gatatttata tcttaaaaaa aacttagaaa acccaataaa tattttcttt cttaatttac 6360

tgcattccta aaaaatgaaa accaaataaa aagagtgtgt agataagtgt gctttatttt 6420

cctgtttagt tcaatctcta gacataaatg aagaaaatcc aaaaatatgt atgatggaaa 6480

tgatgaacag ttgctctgtt tacttacctt caagtgccta gcttttatat tagagttctc 6540

ccaagacttg ctaaaatctg aaatttacta tctgtgggaa catgagccta aaactaaagt 6600

ctaggtaaaa gataagacat gatataaagt ttgagctgct gtttatcttg ttcttacaac 6660

actaagttct ggaaatttat tttgaaaaaa aaacctgcaa atcacatgat gagttcctag 6720

cataacattc ttaccacagc catacttgct ataattgctg tatctttcga gtttcattga 6780

gctgtgtaga tccttaggag cttttcatgt ggttaaatca agatactcac ttgcacacat 6840

ctattacacc aatcattaaa aatctgttaa aaatattgtt atcactcact gtcccaaagt 6900

attgttattc tctctctcct aaaagatcaa atgcagaaag agacatgggc tatgcaaaaa 6960

tatgcgagga aataaaaagg ggcaagtgcc cggaacctcg aaaaagaaaa gaaaaagagt 7020

gagacgagag gtaaaaatgg acaagtgtcc gacagtagaa ttaggggtac aaagataccc 7080

acctgagcgg aaaaaatgga caagtgtccg acagtagaat taggggtatt tactacccac 7140

ctgaaaaaaa agaagaaaaa gagatagccc atgttctctc ccaataaaag atcaagagag 7200

gaggagagat agcaatatga ggaacagtga gcaataagtt ttatcatcac catcactatt 7260

atttactcca ccacacatgc acatcttgat ttaattgtat gttgagttcc tttggatccg 7320

cagttcgatt aaacatatgt atgggctgtt gaagtgaatc atgtctagga actctgagct 7380

ttattttgaa aacttatgca aaactccaga acaaaggtga ggtatagaca ggaggaatag 7440

tgcttggctt aattattttg tctttcaatt acctaaggct taagtacagg tgctaatccc 7500

caagacactt cactctaatc tgggagaatt ttatatgaaa gcatgtgtgc ctgtcaggaa 7560

agaaaacatc aaagcaactc ctgatccatc tgagttttag ttgtttgctc agggacgagc 7620

aaagggtaag cttgggggag tttgttgacg atccttaagt atcaaattta attgtcaact 7680

aaacatggaa aaggatcaat atgcactaga catctagaat tagggtttta tctgacagaa 7740

ttccacgagt tttggtgttt gtctatttct gcagggggtt atcagaaaat acggagagaa 7800

ggcccacacg tcgggtttac atagagatat taacatgtgc gctaattttc tatcatctag 7860

aagactccag aagacacgtg aacgaacggg aggccgaacg gggcccagcc cagggcgccc 7920

gccctagggt ttggggcggc cgccctactc ccgtggccaa tcagcgtcaa cttcgcggat 7980

tatgctccac cgacctaaag gatcaaggaa aaccgtgcga ttaatgtcgg tttgatccga 8040

cggcccacga tcatttgagg gggctatata agcagggcct ctccacccca ggggaggagg 8100

agaaatcatt atcagaggaa gccatcaagt ttagggttta gaaactctct ctcccgcaga 8160

gaattagatt tagctactcc caattctttc aagttttata ggattgatta gatagaatta 8220

gagaagtagg gcctagcgct ctggatttcg gatcttcgtc aataaagatt ggtattattt 8280

catatctttc tctacgactt tattctaatt gcattatgtc tttatttatt atgttcctag 8340

tttgctctag ttctataagt gatatagtta tgattgatga tgagttcatg catgagtttg 8400

caaagcgctt agctcttttc acgtgggagt taagtggtag atcacatgta ggcgtggtgc 8460

ttagatgtta tttacctgca aatgtatcct attggccggg tcgtgtggta gttcgcgatg 8520

gtgacagctt cgttgattct tatatagtcc accctccgtt gataggacag gcagaatttg 8580

tattgcggag taagtcttgc tatgttctga tttactttag caatgttcct tatacatgaa 8640

tgaagagtct tttgtgctat atatgatctt gtagatgctt agagtagatt atgacttagt 8700

aaatagtaga tacttagaat ccattctctt gctagtccga cgtcacctta catttatgtg 8760

gagtagtcta tttctaatcg ctgtgttatt tacccatgag cttatatttc attatcttta 8820

ttattatggc ttaccccctg ccaaagcaag tgactgtgtg acgagtttct cagtagtaat 8880

catgttcttg caagtttatc tctagtctaa gccttgatag attttacccc tgttttcgct 8940

ttcaccgttc tcttaagcaa aattataaat aacgatacct ggaatactta tcctggtgaa 9000

atgctacaat gaggtatttt atctgtgcgc ttgcggatag aatagattat tttctagaga 9060

gccttatgtt tataaatacc ttagtacgct ctagcatcat gctagggatg acaacctagt 9120

attcaagtgg tgttagctag tgtcaaca 9148

<210> 5

<211> 25

<212> DNA

<213> Artificial

<400> 5

gttctcagga ttcttcagta tttcg 25

<210> 6

<211> 25

<212> DNA

<213> Artificial

<400> 6

tcacattgga tgctaagccc taaga 25

<210> 7

<211> 25

<212> DNA

<213> Artificial

<400> 7

ggcaggcaga gcaacactat tacag 25

<210> 8

<211> 25

<212> DNA

<213> Artificial

<400> 8

gttctcgtgg cttctggact cttct 25

<210> 9

<211> 25

<212> DNA

<213> Artificial

<400> 9

cattgatgtt agtaatccct tccca 25

<210> 10

<211> 25

<212> DNA

<213> Artificial

<400> 10

gagaaacata gcaatcactc ccccg 25

<210> 11

<211> 25

<212> DNA

<213> Artificial

<400> 11

gatagatttt acccctgttt tcgct 25

<210> 12

<211> 25

<212> DNA

<213> Artificial

<400> 12

tcgtcacaca gtcacttgct ttggc 25

Claims (5)

1.割手密反转录转座子序列，其特征在于，所述序列根据割手密和热带种基因组数据聚类分析得到，名称分别为：序列1-Cluster168Contig15、序列2-Cluster56Contig54、序列3-Cluster100Contig20、序列4-Cluster38Contig50，其核苷酸序列分别为SEQ ID NO.1-4所示。

2.如权利要求1所述的割手密反转录转座子序列，其特征在于：所述序列1-Cluster168Contig15、序列2-Cluster56Contig54、序列3-Cluster100Contig20、序列4-Cluster38Contig50相应的反转录转座子序列扩增引物如SEQ ID NO.5-12所示。

3.割手密反转录转座子序列的鉴定方法，其特征在于，包括如下步骤：

(1)根据割手密和热带种基因组聚类数据分析得到4条割手密反转录转座子序列；

(2)利用Primer Premier 5.0对上述4条反转录转座子序列进行引物设计；

(3)将上述4条割手密反转录转座子序列进行PCR扩增，得到PCR产物；

(4)采用OMEGA试剂盒进行PCR产物纯化回收，得到纯化PCR产物；

(5)将纯化PCR产物制备成探针，在割手密和热带种的中期染色体上进行荧光原位杂交鉴定。

4.如权利要求3所述的割手密反转录转座子序列的鉴定方法，其特征在于，上述步骤(3)中，所述PCR扩增反应体系为：1×ExTaq Buffer，0.2mM dNTP Mixture，250nM上游引物，250nM下游引物，2.5ng/μl SES208基因组DNA，0.05U/μl ExTaq。

5.如权利要求3所述的割手密反转录转座子序列的鉴定方法，其特征在于，上述步骤(3)中，所述PCR扩增的条件为：95℃预变性3min；98℃变性30s，68℃退火及延伸6min-8min，35个循环；72℃终延伸10min，所述序列1的退火及延伸时间为7min，序列2的退火及延伸时间为5min，序列3的退火及延伸时间为4min，序列4的退火及延伸时间为6min。