CN106676112B - 桔小实蝇Taiman基因和其qRT-PCR检测方法以及其siRNA - Google Patents

桔小实蝇Taiman基因和其qRT-PCR检测方法以及其siRNA Download PDF

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CN106676112B
CN106676112B CN201710178061.0A CN201710178061A CN106676112B CN 106676112 B CN106676112 B CN 106676112B CN 201710178061 A CN201710178061 A CN 201710178061A CN 106676112 B CN106676112 B CN 106676112B
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豆威
刘鸿
王进军
岳勇
牛金志
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Abstract

本发明公开了一种桔小实蝇Taiman基因,其核苷酸序列如SEQ ID NO:15所示;提供如SEQ ID NO:15所示的桔小实蝇Taiman基因的siRNA,序列如SEQ ID NO:25所示;还提供对SEQ ID NO:15所示的基因进行qRT‑PCR检测的方法,qPCR中的使用的上、下游引物为序列分别如SEQ ID NO:37、38所示;还提供了如SEQ ID NO:25所示的siRNA在桔小实蝇发育、变态与生殖调节方面的应用,在桔小实蝇防治上的应用,或者在制备桔小实蝇杀虫剂方面的应用。解决了桔小实蝇目前没有有效的Taiman基因的siRNA的问题,在研发新型杀虫剂方面有很好的应用前景。

Description

桔小实蝇Taiman基因和其qRT-PCR检测方法以及其siRNA
技术领域
本发明属于基因工程领域,具体涉及桔小实蝇Taiman基因和其qRT-PCR检测方法以及其siRNA。
背景技术
桔小实蝇(Bactrocera dorsalis)作为我国重要的检疫性害虫,在水果和蔬菜产业方面的危害最为严重,也被认为是我国国际贸易中农产品出口和国内农业的可持续发展中重要的制约因子。桔小实蝇以其寄主范围广、繁殖能力强和世代重叠的特点,使防治的难度大大加大。近年来,主要使用化学防治的方法对于桔小实蝇的治理,虽然简单方便,但长期使用单一药剂,抗药性日益上升为亟待解决的问题。
RNA干扰(RNA interference,RNAi)是近年来发现的一种重要基因沉默手段,是指在进化过程中高度保守的、由双链RNA(dsRNA,Double-stranded RNA,双链核糖核酸)诱发的、同源RNA高效特异性降解的现象,是通过dsRNA的介导特异性的降解对应序列的mRNA。由于使用RNAi技术可以特异性抑制特定基因的表达,所以该技术已被广泛用于探索基因功能和基因治疗领域。
保幼激素(Juvenile hormone,JH)是调控昆虫发育、变态与生殖最重要的激素之一。在昆虫幼虫期,JH阻止由蜕皮激素(Ecdysone)引起的变态,从而使幼虫蜕皮后仍然维持幼虫形态。在幼虫最后一次蜕皮前,JH滴度降低或缺失,导致全变态昆虫化蛹和不完全变态昆虫羽化为成虫。保幼激素在全变态和不全变态昆虫生长发育变态过程中不可或缺,所以保幼激素信号通路Taiman基因的功能研究有利于我们了解保幼激素作用机理。由于人类、高等动物、植物并不含有保幼激素,故保幼激素信号通路基因作为害虫的靶标相对安全,基于该途径研发的新型杀虫剂,应用于害虫的防治具有很大的潜力,但是目前针对桔小实蝇保幼激素通路Taiman基因的相关研究较少,也未见针对桔小实蝇保幼激素信号通路Taiman基因的dsRNA的报道。
发明内容
本发明的目的是提供一种桔小实蝇Taiman基因,其核苷酸序列如SEQ ID NO:15所示。
在本发明的第一方面,提供一种如SEQ ID NO:15所示的桔小实蝇Taiman基因的siRNA,序列如SEQ ID NO:25所示。
在本发明的另一方面,提供一种对SEQ ID NO:15所示的桔小实蝇Taiman基因进行qRT-PCR检测的方法,qPCR中的使用的上、下游引物为BdTaiE-F、BdTaiE-R,其序列分别如SEQ ID NO:37、38所示。
在上述qRT-PCR检测的方法中,PCR反应体系为20μl,包括:cDNA模板1μl,上、下游qPCR引物各1μl,去核酸酶水7μl和荧光染料qPCR Master Mix 10μl。
在上述qRT-PCR检测的方法中,qRT-PCR反应条件为:95℃预变性2min;95℃变性30s、60℃退火30s,共40个循环。
本发明还提供了如SEQ ID NO:25所示的siRNA在桔小实蝇发育、变态与生殖调节方面的应用,在桔小实蝇防治上的应用,或者在制备桔小实蝇杀虫剂方面的应用。
本发明的有益效果是:本发明运用PCR技术克隆获得桔小实蝇保幼激素信号通路Taiman基因的可变剪切体,并根据该序列得到Taiman基因的siRNA,可以应用于对桔小实蝇进行RNA干扰从而起到防治桔小实蝇的效果。通过实验验证,siRNA-Tai-E基因沉默效率高,基因干扰后桔小实蝇有明显表型变化,解决了桔小实蝇目前没有有效的保幼激素信号Taiman基因的siRNA的问题,在研发新型杀虫剂方面有很好的应用前景。
附图说明
图1为扩增桔小实蝇Taiman基因得到的5个可变剪切体进行氨基酸序列相似性比对结果图。
图2为桔小实蝇的Taiman基因的氨基酸系统发育树。
图3为桔小实蝇注射dsRNA后Taiman基因或注射siTaiman-E后Taiman-E基因的qRT-PCR检测结果,其中A、B为注射dsRNA,C、D为注射siTaiman-E。
图4为桔小实蝇注射dsRNA或siTaiman-E后幼虫化蛹时间变化统计图,其中,A为注射dsRNA,B为注射siTaiman-E。
图5为桔小实蝇注射dsRNA或siTaiman-E后蛹的表型,其中,A、C、E为注射dsRNA后的蛹的表型,B、D、F为注射siTaiman-E后的蛹的表型。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
主要试剂及生产者:
PrimeSTAR Max Premix、PrimeSTAR Max Premix、胶回收纯化试剂盒、Perfectreal time RT reagent(Takara公司,日本)
TRIzol kit(Invitrogen公司,美国)
RNeasy Plus Micro Kit(QIAGEN公司,德国)
Transcript Aid T7 High Yield Transcription Kit、TranscriptAid EnzymeMix、ATP/CTP/GTP/UTP Mix、PrimeScriptRT Enzyme Mix I、Oligo dT Primer(Thermofisher Scientific公司,美国)
qPCR Master Mix(Promega公司,美国)
实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。
实施例一、桔小实蝇Taiman基因可变剪切体序列克隆
按照如下步骤操作:
1)利用RNA提取试剂盒RNeasy Plus Micro Kit按照使用说明提取桔小实蝇的总RNA,然后利用反转录试剂盒Perfectreal time RT reagent按照使用说明将1μg总RNA反转录成为cDNA。
2)以上述得到的cDNA为模板,根据NCBI上预测的桔小实蝇Taiman基因序列以及该基因保守结构序列,分段设计全长扩增引物,共设计了5对分段扩增引物进行全长扩增:BdTaiS1-F/BdTaiS1-R,BdTaiS2-F/BdTaiS2-R,BdTaiS3-F/BdTaiS3-R,BdTaiS4-F/BdTaiS4-R,BdTaiS5-F/BdTaiS5-R。PCR条件为:98℃预变性3min;98℃变性30s、60℃退火15s、72℃延伸2min,共35个循环;72℃条件下延伸10min。PCR反应体系共25μl,包括:浓度为1000ng/μl的cDNA模板1μl,浓度为0.15-0.25μM的前述上、下游引物各1μl,primeSTAR MaxPremix 12μl以及去核酸酶水10μl。前述5对分段扩增引物的序列如下:
BdTaiS1-F:5’-GTGTAATCCTGGGCAGAAACG-3’(如SEQ ID NO:1所示),
BdTaiS1-R:5’-ACCGCTACCACAACCAATGTC-3’(如SEQ ID NO:2所示),
BdTaiS2-F:5’-AATAATATGCCGTATGGAGTTAGCG-3’(如SEQ ID NO:3所示),
BdTaiS2-R:5’-CACTTATGCTGCTACTGCTGTTT-3’(如SEQ ID NO:4所示),
BdTaiS3-F:5’-CCTTCGCCTCAGTCAAATACA-3’(如SEQ ID NO:5所示),
BdTaiS3-R:5’-CAGTGGGAGTAGGTGCTGATACA-3’(如SEQ ID NO:6所示),
BdTaiS4-F:5’-GCGTTATGGACTATGTTTCAGACG-3’(如SEQ ID NO:7所示),
BdTaiS4-R:5’-GTTGAGACTTCGCCGCTGATT-3’(如SEQ ID NO:8所示),
BdTaiS5-F:5’-CCTATGTTAAGCGCGCAGTT-3’(如SEQ ID NO:9所示),
BdTaiS5-R:5’-CGACGGCCGTTAGTAGTGTA-3’(如SEQ ID NO:10所示)。
3)将PCR产物在1%琼脂糖凝胶电泳中进行分离,分别得到1600bp、1500bp、1300bp、1100bp、750bp左右的条带,将PCR产物纯化,随后将纯化产物进行加A反应,然后将加A后的产物与载体pGEM-T Easy连接,转化至感受态大肠杆菌DH5α中。转化后经蓝白斑筛选,选出白色菌落转入LB液体培养基中,在37℃恒温摇床震荡繁殖(4h),以菌液为模板进行PCR扩增,将阳性克隆菌液送往上海英骏公司进行测序。
4)将测序结果通过手工校对、序列拼接,发现BdTaiS5-F/BdTaiS5-R的扩增产物(750bp的产物)有5个可变剪切体,将5个可变剪切体对应的Taiman基因分别命名为Taiman-A、Taiman-B、Taiman-C、Taiman-D、Taiman-E基因,其核苷酸序列分别如序列表中SEQ IDNO:11~15所示。
5)用DNAMAN 6.0对引物BdTaiS5-F/BdTaiS5-R扩增的片段所获得的5个可变剪切体氨基酸序列进行相似性比对,比对结果如图1所示:Taiman-A、Taiman-B、Taiman-C、Taiman-D的氨基酸序列依次比Taiman-E的氨基酸序列缺失249、183、167、75个氨基酸。
6)构建氨基酸系统发育树,结果如图2所示:用前述SEQ ID NO:11~15所示的序列与双翅目的果蝇、埃及伊蚊聚到一枝,确定得到的序列为桔小实蝇Taiman基因序列。
实施例二、桔小实蝇Taiman基因的dsRNA
1)根据克隆获得的桔小实蝇Taiman基因序列,设计扩增Taiman基因的上、下游引物(含有T7启动子的序列):T7-Tai-core-F(如SEQ ID NO:16所示)、T7-Tai-core-R(如SEQID NO:17所示),引物序列分别为:
T7-Tai-core-F:5’-TAATACGACTCACTATAGGGGCTCTCAATTCGACGACACC-3’;
T7-Tai-core-R:5’-TAATACGACTCACTATAGGGACCTGACCGTTAAGCAGTGA-3’。
利用RNA提取试剂盒RNeasy Plus Micro Kit按照使用说明提取桔小实蝇的总RNA,然后利用反转录试剂盒Perfect real time RT reagent按照使用说明将1μg总RNA反转录成为cDNA,反转录体系为20μl,其中包括:total RNA 1μl(约1μg),5×PrimeScriptBuffer 4μl,PrimeScriptRT Enzyme Mix I 1μl,Oligo dT Primer(50μM)1μl,Random6mers(100μM)1μl,RNase Free ddH2O 12μl。
2)对步骤2)得到的cDNA进行PCR扩增,反应条件为:98℃预变性3min;98℃变性30s、60℃退火10s、72℃延伸5min,共35个循环;72℃条件下延伸10min;PCR反应体系共25μl,包括:浓度为1000ng/μl的cDNA模板0.5μl,浓度为0.15-0.25μM的上、下游引物(T7-Tai-core-F、T7-Tai-core-R)各1μl,PrimeSTAR Max Premix 12.5μl以及去核酸酶水10μl。
3)将步骤3)中的PCR产物用胶回收纯化试剂盒按照说明书纯化回收之后作为合成dsRNA的转录模板,用体外合成dsRNA试剂盒Transcript Aid T7 High YieldTranscription Kit按照使用说明转录合成并纯化得到桔小实蝇Taiman基因的dsRNA溶液,将该dsRNA命名为dsTaiman-core,为由序列如SEQ ID NO:18所示的核苷酸和与其序列互补的核苷酸组成的双链RNA,SEQ ID NO:18所示序列为:
GCUCUCAAUUCGACGACACCUAAUAACAACAAUAGCAGCUCAACUUCUAAAAGCAACGAUAAUCGAAAUAAUACAGCAUCUACACGUUGUUUACGAUGUGCAACUGACAAUUGCUCUAUCCAUCCAGUACAACAAGGUGACGUUUCAUCGACCGAACCCCCAUUGCCGGAGCCAUCACUGCUUAACGGUCAGGU。
将dsRNA溶液浓度稀释到200-300ng/μl备用。dsRNA合成反应体系为20μl,其中包括:胶回收片段模板4μl(约1μg)、5×TranscrptAid Reaction Buffer 4μl、浓度为100mM的ATP/CTP/GTP/UTP Mix 8μl、TranscriptAid Enzyme Mix 2μl和DEPC-treated水2μl;反应条件为:37℃孵育6h。
同时利用上述方法合成并纯化得到GFP(绿色荧光蛋白)的dsRNA溶液作为阴性对照,反转录得到的cDNA进行PCR扩增时使用的上下游引物序列分别为GFP-ds-T7F(如SEQ IDNO:19所示)、GFP-ds-T7R(如SEQ ID NO:20所示),序列为:
GFP-ds-T7F:5’-TAATACGACTCACTATAGGGCAGTTCTTGTTGAATTAGATG-3’;
GFP-ds-T7R:5’-TAATACGACTCACTATAGGGTTTGGTTTGTCTCCCATGATG-3’。
实施例三、桔小实蝇Taiman基因的siRNA
根据实施例一中得到的桔小实蝇Taiman基因的5个可变剪切体,针对5个可变剪切体对应的Taiman基因Taiman-A、Taiman-B、Taiman-C、Taiman-D、Taiman-E基因,相应地设计5条特异性siRNA序列送于广州瑞博生物有限公司合成,siRNA序列如下:
siTaiman-A:5’-GUGUAUCUGCACUACUUAAUA-3’,如SEQ ID NO:21所示;
siTaiman-B:5’-UCUAAUGUCGGUCCUGGCGGU-3’,如SEQ ID NO:22所示;
siTaiman-C:5’-AAUUCUUUUCAAGGUGGCGGC-3’,如SEQ ID NO:23所示;
siTaiman-D:5’-UCAACAUUUACCUGCAGGUAA-3’,如SEQ ID NO:24所示;
siTaiman-E:5’-GUAUUGUUGGCUUUGGUGGCA-3’,如SEQ ID NO:25所示;
siGFP:5’-GCCACAACGUCUAUAUCAUGG-3’,如SEQ ID NO:26所示。
实施例四、桔小实蝇Taiman基因的基因沉默验证
一、将桔小实蝇Taiman基因的dsRNA/siRNA导入桔小实蝇
将实施例二、三中的桔小实蝇Taiman基因的dsRNA/siRNA导入桔小实蝇:
利用Nanojiect II Auto-Nanoliter(Drummond Scientific)显微注射仪对桔小实蝇注射dsRNA/siRNA,按照如下步骤操作:
1)将玻璃毛细管(美国Drummond)固定于拉针仪(美国Sutter)指定位置,参数设置为HEAT=510,PULL=60,VEL=35,DEL=30的拉针程序,取下拉制的毛细管。
2)将拉制后的毛细管至于磨针仪(美国Sutter)指定位置,调整角度,将毛细管尖端磨成光滑表面,减少注射时对虫体的伤害。
3)将磨好的毛细管置于显微注射仪(美国Drummond),操作步骤严格遵循NanojectII/Auto Nanoliter Injector说明书。
4)挑取桔小实蝇体型大小一致的4日龄幼虫,置于装有清水的培养皿中洗去体表饲料残余,然后用滤纸吸干体表水分。
5)手持4日龄幼虫,将1μg Taiman基因dsRNA/80ng siRNA溶液注射至幼虫腹末倒数第三节间,以GFP的dsRNA/siRNA溶液的处理作为对照。
6)将注射后的幼虫放入幼虫人工饲料内,置于恒温箱正常饲养(温度27.5±1℃,相对湿度70±5%,无光照)。
每30头桔小实蝇为一个生物重复,设置3个Taiman基因dsRNA/siRNA溶液的生物重复作为实验组。同时设置注射GFP的dsRNA/siRNA溶液的处理作为对照组。
二、沉默效率以及表型的检测:
随机选取注射Taiman基因的dsRNA/siRNA后的24h和72h桔小实蝇幼虫,以及GFP的dsRNA/siRNA溶液处理的对照组的桔小实蝇,使用TriZol法分别提取RNA之后利用Perfectreal time RT reagent反转录得到相应的cDNA,反转录体系为20μl,其中包括:total RNA1μl(约1μg),5×PrimeScript Buffer 4μl,PrimeScriptRT Enzyme Mix I 1μl,Oligo dTPrimer(50μM)1μl,Random 6mers(100μM)1μl,RNase Free ddH2O 12μl。反应条件为:37℃15min,85℃5s。
利用qRT-PCR技术,检测Taiman基因以及5条可变剪切体在实验组、对照组中的相对表达量。qPCR中,针对5个可变剪切体的公共部分检测的上、下游引物为BdTai-core-F(如SEQ ID NO:27所示)、BdTai-core-R(如SEQ ID NO:28所示);针对Taiman-A基因检测的上、下游引物为BdTaiA-F(如SEQ ID NO:29所示)、BdTaiA-R(如SEQ ID NO:30所示);针对Taiman-B基因检测的上、下游引物为BdTaiB-F(如SEQ ID NO:31所示)、BdTaiB-R(如SEQ IDNO:32所示);针对Taiman-C基因检测的上、下游引物为BdTaiC-F(如SEQ ID NO:33所示)、BdTaiC-R(如SEQ ID NO:34所示);针对Taiman-D基因检测的上、下游引物为BdTaiD-F(如SEQ ID NO:35所示)、BdTaiD-R(如SEQ ID NO:36所示);针对Taiman-E基因检测的上、下游引物为BdTaiE-F(如SEQ ID NO:37所示)、BdTaiE-R(如SEQ ID NO:38所示)。各引物序列如下:
BdTaiA-F:5’-CCTATGTTAAGCGCGCAGTT-3’;
BdTaiA-R:5’-TAAGTAGTGCAGATACACCCC-3’;
BdTaiB-F:5’-CCTATGTTAAGCGCGCAGTT-3’;
BdTaiB-R:5’-GCCAGGACCGACATTAGTTC-3’;
BdTaiC-F:5’-CCTATGTTAAGCGCGCAGTT-3’;
BdTaiC-R:5’-GCCGCCACCTTGAAAAGAAT-3’;
BdTaiD-F:5’-AGGTGGTGAATCTGGTGGTT-3’;
BdTaiD-R:5’-CCTGCAGGTAAATGTTGGGT-3’;
BdTaiE-F:5’-GGAGTGTCGGTATTGTTGGC-3’;
BdTaiE-R:5’-CCTGAGCGCGACCATAAAAT-3’;
BdTai-core-F:5’-GCTCTCAATTCGACGACACC-3’;
BdTai-core-R:5’-ACCTGACCGTTAAGCAGTGA-3’。
qRT-PCR反应条件为:95℃预变性2min;95℃变性30s、60℃退火30s,共40个循环。PCR反应体系为20μl,包括:cDNA模板1μl(250ng),前述上、下游qPCR引物各1μl,去核酸酶水7μl和荧光染料qPCR Master Mix 10μl。选用桔小实蝇的α-Tubulin基因为内参基因进行相对定量分析,分析方法则采用国际上惯用的2-ΔΔCt法。内参基因α-Tubulin的qPCR引物序列:
α-Tubulin-F:5’-CGCATTCATGGTTGATAACG-3’,如SEQ ID NO:39所示;
α-Tubulin-R:5’-GGGCACCAAGTTAGTCTGGA-3’,如SEQ ID NO:40所示。
qRT-PCR检测结果显示,在RNA干扰4日龄幼虫24-72h后,桔小实蝇的Taiman基因5条可变剪切体在注射dsRNA的实验组中的相对表达量相对于对照组(GFP)均显著下调,而且下调高达50%左右,如图3A、3B所示。注射siTaiman-E的实验组中Taiman-E基因的相对表达量也显著下调,如图3C、3D所示。同时,通过观察发现,Taiman基因的dsRNA及Taiman-E基因的siRNA处理的实验组的桔小实蝇幼虫化蛹提前,提前约24h(结果如图4所示),同时实验组的蛹重和蛹长较对照组相比显著减小,图5中A、C、E为注射Taiman基因dsRNA后的蛹的表型,处理组与对照组相比蛹明显变小,图5中B、D、F为注射siRNA-Tai-E后的蛹的表型,Taiman-E基因被单独干扰之后的表型与dsRNA干扰的Taiman-core(前述5个可变剪切体的公共部分)被干扰后的表型一致,实验验证了实验组的桔小实蝇的Taiman基因的表达受到抑制,且Taiman基因参与昆虫的变态发育,也进一步证明了实施例一中的序列SEQ ID NO:11~15为桔小实蝇Taiman基因。实验结果也说明利用本发明方法对桔小实蝇进行Taiman基因的dsRNA和Taiman-E的siRNA,能够有效导入dsRNA和siRNA,达到RNA干扰的效果。
研究发现,同时分别注射针对5个可变剪切体对应的Taiman基因Taiman-A、Taiman-B、Taiman-C、Taiman-D、Taiman-E基因的siRNA进行功能验证,结果发现在5条可变剪切体中,Taiman-E基因被干扰后的表型与dsRNA处理后的表型一致,而其他4条可变剪切体被干扰后无明显表型,说明Taiman-E基因主要发挥该方面功能。
序列表
<110> 西南大学
<120> 桔小实蝇Taiman基因和其qRT-PCR检测方法以及其siRNA
<130> 1
<160> 40
<210> 1
<211> 21
<212> DNA
<213> 人工序列
<223> BdTaiS1-F
<400> 1
gtgtaatcct gggcagaaac g 21
<210> 2
<211> 21
<212> DNA
<213> 人工序列
<223> BdTaiS1-R
<400> 2
accgctacca caaccaatgt c 21
<210> 3
<211> 25
<212> DNA
<213> 人工序列
<223> BdTaiS2-F
<400> 3
aataatatgc cgtatggagt tagcg 25
<210> 4
<211> 23
<212> DNA
<213> 人工序列
<223> BdTaiS2-R
<400> 4
cacttatgct gctactgctg ttt 23
<210> 5
<211> 21
<212> DNA
<213> 人工序列
<223> BdTaiS3-F
<400> 5
ccttcgcctc agtcaaatac a 21
<210> 6
<211> 23
<212> DNA
<213> 人工序列
<223> BdTaiS3-R
<400> 6
cagtgggagt aggtgctgat aca 23
<210> 7
<211> 24
<212> DNA
<213> 人工序列
<223> BdTaiS4-F
<400> 7
gcgttatgga ctatgtttca gacg 24
<210> 8
<211> 21
<212> DNA
<213> 人工序列
<223> BdTaiS4-R
<400> 8
gttgagactt cgccgctgat t 21
<210> 9
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiS5-F
<400> 9
cctatgttaa gcgcgcagtt 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiS5-R
<400> 10
cgacggccgt tagtagtgta 20
<210> 11
<211> 5520
<212> DNA
<213> 人工序列
<223> Taiman-A
<400> 11
atgtcaattg ttgcagccga aaatgcaggg cttggcccat gtgatttacc agattcgtgg 60
gcaccaaaca caaataatag caacaacaat attactagta tcgttgcaaa tagtagagac 120
atctcatcgt tatcagcagt cacagaaaat tcatcaaaca cggctgtccc tcttaatatt 180
gacaacaata ttagctcgaa tatagtgttc ttcaagaacc aaagcagtcc aactacaact 240
gccattcaac cgaccacttc tttcggcttt tcaccaacat caacggcaag agcaacaaca 300
tcaaccactt ccagtcttat atacaaggtg cctctctcat cgccagccgc tgctacagcc 360
gctgttactg tttcaccagc cagcattggt agtctagtaa cgtctttagg tgtgggtctg 420
gtaccctcaa taccgacgtg tgtgtccgtt actggaacga gtaccggtgg aactggagcc 480
tttgctggtg tactgttatc aagcccaata tcagctggtg gtagtagcaa tagtatttcg 540
gctcaaaaaa ccattacaaa ttatcaagta aacaacgtca ataataacgc aaataacacc 600
ggcaatacca gcgttagaag tctaacactt ccattgcagc aacaacaact acaattgcaa 660
caacaaaacg gcttattgct acataacaaa cagagcaacc aaaacaacaa taacagctct 720
ttagtgaata ataaccacga taaaaatagc aacaatttta atttaatact acgacagaag 780
tctgcagcaa ctgtaagttt agccgccgca cttcaaaatt caatcaccat gaatgcggta 840
gcttcgccaa tctcaaacaa cccagcgacg ccgactcgaa aaattagaag aaaaactgat 900
ccgaaagcca atttgccaca atcacagatt aataaatgta ataacgagaa aaggcgtcgc 960
gaattggaaa acaattatat cgagcaattg tccgagtttt tacaactcaa caaacgcggt 1020
gatacagcct caacgaaacc agacaaggca gcaatactga accaagtagt gaaaacgtat 1080
cgtgagtttt gtgacaaggg ccaaagccgt gatatttctt cctcaacgtc gactaccgct 1140
tcttcagctc tcaattcgac gacacctaat aacaacaata gcagctcaac ttctaaaagc 1200
aacgataatc gaaataatac agcatctaca cgttgtttac gatgtgcaac tgacaattgc 1260
tctatccatc cagtacaaca aggtgacgtt tcatcgaccg aacccccatt gccggagcca 1320
tcactgctta acggtcaggt gcccgaaata tctgcctatt ttgaggcgtt ggaacattac 1380
ctctcttcag tcgattgggt gcttttacaa gtcactgcag acgggataat cgagtcgtgt 1440
acgcagaata tacgagagct aatcggctat gacaaacagg aactgtttcg taaaccacta 1500
tatcagtacc tacactccga cgatcatgcg aaattaaatc caattataaa taatatgccg 1560
tatggagtta gcggaaatgc aactggtgac attggttgtg gtagcggtag ccaaagtggc 1620
tggaatgatt tggaagatgc gaatagtggt acaaattcgc aacattcaac agcttcgtta 1680
ggtccaggaa gtgctaaagc taaaagaaat atagcagcta aagtccgaat gctagtgaaa 1740
gatttgcgtt cggctactca aacatctatg acgcaccaaa acgatgcttt ggatcagaaa 1800
actgtcggac aacatataaa ccccgaaaaa tatgaggaag ttatgctcct agcaacaccg 1860
atgaaagacg atggtgatac aacgtcctcc atattgtgtt tgattacccg tcccgaagat 1920
gaaccggcca tacaacagat acaaccgcag gccatagagc atttaacctt taaacttgac 1980
gtacacggca agatactcaa tttggatgta accgcacttc gtcaacccta tcgtcaacat 2040
ttaagtagtt ggttgggacg ccttttgcag gatctttgtc atccgcaaga cttaaatgct 2100
ctgaaatcac acttacgcga ggttcaagag tcagctgctt cagtccattt gatgaactct 2160
ccatcatcac actcacctgg tggaacgcct gcaacactca ttcatcctcc cattgcgaat 2220
gtgatgtcac gtccatttag attgcgtctc ggcgcacccg atgtttatgt ccatgttaag 2280
gcgaattcac gacttttttt aaatcagtcg caaactgagg gcgattatat tatgtctctg 2340
caaacattac ttaacacgga taacgatata ggcggaggag gtataagtgg attgaacatc 2400
ggaggtatcg ggaacagtag tatgatgcca tctagcagtt tgtcaacaat gtcgcctagt 2460
ccatcgttag tttcgcccct ttctctgccc ttggactcac ttgttaataa caattcgtca 2520
tgctcctctt cgtcggcgtc agctttagca agtgtaagta gcggtgtaca tatgttagga 2580
ggactagttg gaggtggtgg tatgccacca atcaccacgc aaactactaa tgtgggtgga 2640
ccactgatga cttcagctgt tatcaatggt acagggagtg gtacacaacg taacaatcct 2700
gcagtggcaa catcggcatc gacttcgaat aattccaatc tggtgaattc gtttaccgca 2760
tcgcctgctg gacccgaagc aacgattttc tatagttccg acccttttga ttttgacttg 2820
gcacattcca gttttgaaat ggacgccact ggttggacgg attcgcgtcc aaactctcgc 2880
gcttctgtta ctaccccagt aagcactccg cgtccaccgt cagcgggaca tggtttcagt 2940
ccggcagtat gtgcttcacc atctacacca tatcaactgt cgtcacactc agctgcgagc 3000
ttgccttcgc ctcagtcaaa tacaagtcag ccgtcatctg ctggtcccgg ggttagtgga 3060
gggccgtttg ggtttggttt tccggcgttt gaaacaagtg ataaaaataa tgataaagac 3120
cacatgaacg ctagcataaa cagcagtagc agcataagtg gcaataattc tggacctcaa 3180
ggtggttcca gtggtgcgtt gagcggccct gcaaatttac caaatggtgg tccgccatta 3240
cttaccactg ttgctaacat gactcaacta tctgcacagc cgaatccacc ccaaccggaa 3300
tcagagcgtt tacggcactt attgacaaat aaatcgcatt caatgacctc aggaatgaac 3360
ccggccgatg gtgataaaga tcatcttagc ctgtgccata agctgcttaa cgaaaaatcg 3420
gaggatgatg atggaaaagg accttcgctt ggtcgacaaa gtgaattgat gcgtcaacta 3480
caaaaggacg atggtcatta tagtcattca cacggtcata gccacgggca acatcactcc 3540
aaggacatgt cgcaagagga tttactaaaa agtttaaagt accagggtga tcctacaact 3600
cgcaagcgtt cgctcaatga accggatgat ggcatattag caaaacgtga aagtgaccgc 3660
ccatccaagt tacgtgagag caataaaatg cttgcctcgc tgttagaaaa tccgccaaag 3720
aatcctatag tcactgtacc acctcaagtc aaaactatac ctgacattgc accttcagca 3780
agccgcgtca actcgacacc cggtgttatg agtgtttcag cgccaaatag tctaggtggc 3840
atgccaccaa aaagcggcat aatgacgacg acctcatcaa ttagtgcagg aggcagttct 3900
gtcggagccg gtggacggag caatagtatg cgcaaacaat tttccgatgc ctacctcacg 3960
ctgcaacagc agcaacatca gcaacaactc cacctacaac aacaacagca acaatgttta 4020
ggtaacactc aacgttcttc acatcagcag ccaacacaac tatctcaacc gcaaataaac 4080
tttgcttcat cggacgcctt cggaacatct tcacataaca ccacagctac ctcagggaca 4140
gccaacgtca cagcgtcaat tgtgacgtca caagcaaact cccaattagc cgctcctagc 4200
ggcgctgatg gcgattcaga attatctaag attttggata gcgttatgga ctatgtttca 4260
gacgatgggc catttgtatc agcacctact cccactgcct tggctggatt aacgcagcaa 4320
gagattaatg aacgcatggc tatcaacgct attcaaaatt cattgatggt cgagacatct 4380
caactgcaac agcagcaaca tcatcaacaa cagccgcaac cgccagccta tcctggcaac 4440
ataatgggga gtggcactgg agggaattta actcagcaac aacaacagct acagctccag 4500
catcacctgc agatgttgca aaggcagcag gctgcaattg gaggcaatca acagtcaact 4560
caggtacaac aaatgttgga ggtactacgt gtcaacgcca atcaaagttt ccaacgaccg 4620
ccacctatgt attcggcgtc gcgtaacagg ggcccaatga atactgttac gacacctgga 4680
ggctctgttt tgccagcgca tcagcagtac cgaattcgtc aacaacagca acagcaacaa 4740
caacagaaag agcgtcttct tcagcaacaa caaaagcaac agcttttggt tcctgagagt 4800
gcaacagcgc gcacagacca gttatgtcta aatccgaaca tcaacaacat tggttcgctg 4860
ttaaataaca cagtagcgcc aaatgtcgcc ttgtctcgta ccaacttgcc gcccgattcg 4920
caactgagtc cgaattttgc acagaattta cttcaacaac aacagttaag tccagggcaa 4980
cgtaatgcac catttagtcc acaaacgaat gcaggttatg caccgcaata ttcacaaagc 5040
ggacaacgtc tatcgcccca tcatcaacag catatttcac agcagcaaca acaagtgaat 5100
gctcagcaac aacaacagat ggcgtttcag gctgcccaag cagcaaacgc taatgtcact 5160
ccgcagctat caccacgcca accgcctttt ggtggaggag caggtggtgt acaatcacct 5220
acaggcatgc cgtcatcaca acaatggaat gcaagtggta atgttagtac gccaagtgta 5280
caactaagtg gaaatgtgac aactcaacgc ggtcattcat tacaacaaca taatcctatg 5340
ttaagcgcgc agttacaaca gggtgttagt ccttatacaa cacgtccgta tcaaaatcag 5400
cggcgaagtc tcaactctcc gaacggaggg ggtgtatctg cactacttaa tacaccagat 5460
ccttccatga attttagctt cgatgcacaa gatttctttg gcaacaatgc tacaaggtga 5520
<210> 12
<211> 5718
<212> DNA
<213> 人工序列
<223> Taiman-B
<400> 12
atgtcaattg ttgcagccga aaatgcaggg cttggcccat gtgatttacc agattcgtgg 60
gcaccaaaca caaataatag caacaacaat attactagta tcgttgcaaa tagtagagac 120
atctcatcgt tatcagcagt cacagaaaat tcatcaaaca cggctgtccc tcttaatatt 180
gacaacaata ttagctcgaa tatagtgttc ttcaagaacc aaagcagtcc aactacaact 240
gccattcaac cgaccacttc tttcggcttt tcaccaacat caacggcaag agcaacaaca 300
tcaaccactt ccagtcttat atacaaggtg cctctctcat cgccagccgc tgctacagcc 360
gctgttactg tttcaccagc cagcattggt agtctagtaa cgtctttagg tgtgggtctg 420
gtaccctcaa taccgacgtg tgtgtccgtt actggaacga gtaccggtgg aactggagcc 480
tttgctggtg tactgttatc aagcccaata tcagctggtg gtagtagcaa tagtatttcg 540
gctcaaaaaa ccattacaaa ttatcaagta aacaacgtca ataataacgc aaataacacc 600
ggcaatacca gcgttagaag tctaacactt ccattgcagc aacaacaact acaattgcaa 660
caacaaaacg gcttattgct acataacaaa cagagcaacc aaaacaacaa taacagctct 720
ttagtgaata ataaccacga taaaaatagc aacaatttta atttaatact acgacagaag 780
tctgcagcaa ctgtaagttt agccgccgca cttcaaaatt caatcaccat gaatgcggta 840
gcttcgccaa tctcaaacaa cccagcgacg ccgactcgaa aaattagaag aaaaactgat 900
ccgaaagcca atttgccaca atcacagatt aataaatgta ataacgagaa aaggcgtcgc 960
gaattggaaa acaattatat cgagcaattg tccgagtttt tacaactcaa caaacgcggt 1020
gatacagcct caacgaaacc agacaaggca gcaatactga accaagtagt gaaaacgtat 1080
cgtgagtttt gtgacaaggg ccaaagccgt gatatttctt cctcaacgtc gactaccgct 1140
tcttcagctc tcaattcgac gacacctaat aacaacaata gcagctcaac ttctaaaagc 1200
aacgataatc gaaataatac agcatctaca cgttgtttac gatgtgcaac tgacaattgc 1260
tctatccatc cagtacaaca aggtgacgtt tcatcgaccg aacccccatt gccggagcca 1320
tcactgctta acggtcaggt gcccgaaata tctgcctatt ttgaggcgtt ggaacattac 1380
ctctcttcag tcgattgggt gcttttacaa gtcactgcag acgggataat cgagtcgtgt 1440
acgcagaata tacgagagct aatcggctat gacaaacagg aactgtttcg taaaccacta 1500
tatcagtacc tacactccga cgatcatgcg aaattaaatc caattataaa taatatgccg 1560
tatggagtta gcggaaatgc aactggtgac attggttgtg gtagcggtag ccaaagtggc 1620
tggaatgatt tggaagatgc gaatagtggt acaaattcgc aacattcaac agcttcgtta 1680
ggtccaggaa gtgctaaagc taaaagaaat atagcagcta aagtccgaat gctagtgaaa 1740
gatttgcgtt cggctactca aacatctatg acgcaccaaa acgatgcttt ggatcagaaa 1800
actgtcggac aacatataaa ccccgaaaaa tatgaggaag ttatgctcct agcaacaccg 1860
atgaaagacg atggtgatac aacgtcctcc atattgtgtt tgattacccg tcccgaagat 1920
gaaccggcca tacaacagat acaaccgcag gccatagagc atttaacctt taaacttgac 1980
gtacacggca agatactcaa tttggatgta accgcacttc gtcaacccta tcgtcaacat 2040
ttaagtagtt ggttgggacg ccttttgcag gatctttgtc atccgcaaga cttaaatgct 2100
ctgaaatcac acttacgcga ggttcaagag tcagctgctt cagtccattt gatgaactct 2160
ccatcatcac actcacctgg tggaacgcct gcaacactca ttcatcctcc cattgcgaat 2220
gtgatgtcac gtccatttag attgcgtctc ggcgcacccg atgtttatgt ccatgttaag 2280
gcgaattcac gacttttttt aaatcagtcg caaactgagg gcgattatat tatgtctctg 2340
caaacattac ttaacacgga taacgatata ggcggaggag gtataagtgg attgaacatc 2400
ggaggtatcg ggaacagtag tatgatgcca tctagcagtt tgtcaacaat gtcgcctagt 2460
ccatcgttag tttcgcccct ttctctgccc ttggactcac ttgttaataa caattcgtca 2520
tgctcctctt cgtcggcgtc agctttagca agtgtaagta gcggtgtaca tatgttagga 2580
ggactagttg gaggtggtgg tatgccacca atcaccacgc aaactactaa tgtgggtgga 2640
ccactgatga cttcagctgt tatcaatggt acagggagtg gtacacaacg taacaatcct 2700
gcagtggcaa catcggcatc gacttcgaat aattccaatc tggtgaattc gtttaccgca 2760
tcgcctgctg gacccgaagc aacgattttc tatagttccg acccttttga ttttgacttg 2820
gcacattcca gttttgaaat ggacgccact ggttggacgg attcgcgtcc aaactctcgc 2880
gcttctgtta ctaccccagt aagcactccg cgtccaccgt cagcgggaca tggtttcagt 2940
ccggcagtat gtgcttcacc atctacacca tatcaactgt cgtcacactc agctgcgagc 3000
ttgccttcgc ctcagtcaaa tacaagtcag ccgtcatctg ctggtcccgg ggttagtgga 3060
gggccgtttg ggtttggttt tccggcgttt gaaacaagtg ataaaaataa tgataaagac 3120
cacatgaacg ctagcataaa cagcagtagc agcataagtg gcaataattc tggacctcaa 3180
ggtggttcca gtggtgcgtt gagcggccct gcaaatttac caaatggtgg tccgccatta 3240
cttaccactg ttgctaacat gactcaacta tctgcacagc cgaatccacc ccaaccggaa 3300
tcagagcgtt tacggcactt attgacaaat aaatcgcatt caatgacctc aggaatgaac 3360
ccggccgatg gtgataaaga tcatcttagc ctgtgccata agctgcttaa cgaaaaatcg 3420
gaggatgatg atggaaaagg accttcgctt ggtcgacaaa gtgaattgat gcgtcaacta 3480
caaaaggacg atggtcatta tagtcattca cacggtcata gccacgggca acatcactcc 3540
aaggacatgt cgcaagagga tttactaaaa agtttaaagt accagggtga tcctacaact 3600
cgcaagcgtt cgctcaatga accggatgat ggcatattag caaaacgtga aagtgaccgc 3660
ccatccaagt tacgtgagag caataaaatg cttgcctcgc tgttagaaaa tccgccaaag 3720
aatcctatag tcactgtacc acctcaagtc aaaactatac ctgacattgc accttcagca 3780
agccgcgtca actcgacacc cggtgttatg agtgtttcag cgccaaatag tctaggtggc 3840
atgccaccaa aaagcggcat aatgacgacg acctcatcaa ttagtgcagg aggcagttct 3900
gtcggagccg gtggacggag caatagtatg cgcaaacaat tttccgatgc ctacctcacg 3960
ctgcaacagc agcaacatca gcaacaactc cacctacaac aacaacagca acaatgttta 4020
ggtaacactc aacgttcttc acatcagcag ccaacacaac tatctcaacc gcaaataaac 4080
tttgcttcat cggacgcctt cggaacatct tcacataaca ccacagctac ctcagggaca 4140
gccaacgtca cagcgtcaat tgtgacgtca caagcaaact cccaattagc cgctcctagc 4200
ggcgctgatg gcgattcaga attatctaag attttggata gcgttatgga ctatgtttca 4260
gacgatgggc catttgtatc agcacctact cccactgcct tggctggatt aacgcagcaa 4320
gagattaatg aacgcatggc tatcaacgct attcaaaatt cattgatggt cgagacatct 4380
caactgcaac agcagcaaca tcatcaacaa cagccgcaac cgccagccta tcctggcaac 4440
ataatgggga gtggcactgg agggaattta actcagcaac aacaacagct acagctccag 4500
catcacctgc agatgttgca aaggcagcag gctgcaattg gaggcaatca acagtcaact 4560
caggtacaac aaatgttgga ggtactacgt gtcaacgcca atcaaagttt ccaacgaccg 4620
ccacctatgt attcggcgtc gcgtaacagg ggcccaatga atactgttac gacacctgga 4680
ggctctgttt tgccagcgca tcagcagtac cgaattcgtc aacaacagca acagcaacaa 4740
caacagaaag agcgtcttct tcagcaacaa caaaagcaac agcttttggt tcctgagagt 4800
gcaacagcgc gcacagacca gttatgtcta aatccgaaca tcaacaacat tggttcgctg 4860
ttaaataaca cagtagcgcc aaatgtcgcc ttgtctcgta ccaacttgcc gcccgattcg 4920
caactgagtc cgaattttgc acagaattta cttcaacaac aacagttaag tccagggcaa 4980
cgtaatgcac catttagtcc acaaacgaat gcaggttatg caccgcaata ttcacaaagc 5040
ggacaacgtc tatcgcccca tcatcaacag catatttcac agcagcaaca acaagtgaat 5100
gctcagcaac aacaacagat ggcgtttcag gctgcccaag cagcaaacgc taatgtcact 5160
ccgcagctat caccacgcca accgcctttt ggtggaggag caggtggtgt acaatcacct 5220
acaggcatgc cgtcatcaca acaatggaat gcaagtggta atgttagtac gccaagtgta 5280
caactaagtg gaaatgtgac aactcaacgc ggtcattcat tacaacaaca taatcctatg 5340
ttaagcgcgc agttacaaca gggtgttagt ccttatacaa cacgtccgta tcaaaatcag 5400
cggcgaagtc tcaactctcc gaacggaggg ggtgtatctg gcaatgcagt tacaagtaat 5460
atcggaacta atgtcggtcc tggcggtctg cgaataactt ccaccccgca aagtcccttg 5520
agcagtgctc aacagggacc aatgagtggt ggcaattctg gaggtttaaa tagtacaaat 5580
tcgcttagca tgcaacaaca tcaatctggg tcaataggaa ctggtgttag ccaaaatgca 5640
ctacttaata caccagatcc ttccatgaat tttagcttcg atgcacaaga tttctttggc 5700
aacaatgcta caaggtga 5718
<210> 13
<211> 5766
<212> DNA
<213> 人工序列
<223> Taiman-C
<400> 13
atgtcaattg ttgcagccga aaatgcaggg cttggcccat gtgatttacc agattcgtgg 60
gcaccaaaca caaataatag caacaacaat attactagta tcgttgcaaa tagtagagac 120
atctcatcgt tatcagcagt cacagaaaat tcatcaaaca cggctgtccc tcttaatatt 180
gacaacaata ttagctcgaa tatagtgttc ttcaagaacc aaagcagtcc aactacaact 240
gccattcaac cgaccacttc tttcggcttt tcaccaacat caacggcaag agcaacaaca 300
tcaaccactt ccagtcttat atacaaggtg cctctctcat cgccagccgc tgctacagcc 360
gctgttactg tttcaccagc cagcattggt agtctagtaa cgtctttagg tgtgggtctg 420
gtaccctcaa taccgacgtg tgtgtccgtt actggaacga gtaccggtgg aactggagcc 480
tttgctggtg tactgttatc aagcccaata tcagctggtg gtagtagcaa tagtatttcg 540
gctcaaaaaa ccattacaaa ttatcaagta aacaacgtca ataataacgc aaataacacc 600
ggcaatacca gcgttagaag tctaacactt ccattgcagc aacaacaact acaattgcaa 660
caacaaaacg gcttattgct acataacaaa cagagcaacc aaaacaacaa taacagctct 720
ttagtgaata ataaccacga taaaaatagc aacaatttta atttaatact acgacagaag 780
tctgcagcaa ctgtaagttt agccgccgca cttcaaaatt caatcaccat gaatgcggta 840
gcttcgccaa tctcaaacaa cccagcgacg ccgactcgaa aaattagaag aaaaactgat 900
ccgaaagcca atttgccaca atcacagatt aataaatgta ataacgagaa aaggcgtcgc 960
gaattggaaa acaattatat cgagcaattg tccgagtttt tacaactcaa caaacgcggt 1020
gatacagcct caacgaaacc agacaaggca gcaatactga accaagtagt gaaaacgtat 1080
cgtgagtttt gtgacaaggg ccaaagccgt gatatttctt cctcaacgtc gactaccgct 1140
tcttcagctc tcaattcgac gacacctaat aacaacaata gcagctcaac ttctaaaagc 1200
aacgataatc gaaataatac agcatctaca cgttgtttac gatgtgcaac tgacaattgc 1260
tctatccatc cagtacaaca aggtgacgtt tcatcgaccg aacccccatt gccggagcca 1320
tcactgctta acggtcaggt gcccgaaata tctgcctatt ttgaggcgtt ggaacattac 1380
ctctcttcag tcgattgggt gcttttacaa gtcactgcag acgggataat cgagtcgtgt 1440
acgcagaata tacgagagct aatcggctat gacaaacagg aactgtttcg taaaccacta 1500
tatcagtacc tacactccga cgatcatgcg aaattaaatc caattataaa taatatgccg 1560
tatggagtta gcggaaatgc aactggtgac attggttgtg gtagcggtag ccaaagtggc 1620
tggaatgatt tggaagatgc gaatagtggt acaaattcgc aacattcaac agcttcgtta 1680
ggtccaggaa gtgctaaagc taaaagaaat atagcagcta aagtccgaat gctagtgaaa 1740
gatttgcgtt cggctactca aacatctatg acgcaccaaa acgatgcttt ggatcagaaa 1800
actgtcggac aacatataaa ccccgaaaaa tatgaggaag ttatgctcct agcaacaccg 1860
atgaaagacg atggtgatac aacgtcctcc atattgtgtt tgattacccg tcccgaagat 1920
gaaccggcca tacaacagat acaaccgcag gccatagagc atttaacctt taaacttgac 1980
gtacacggca agatactcaa tttggatgta accgcacttc gtcaacccta tcgtcaacat 2040
ttaagtagtt ggttgggacg ccttttgcag gatctttgtc atccgcaaga cttaaatgct 2100
ctgaaatcac acttacgcga ggttcaagag tcagctgctt cagtccattt gatgaactct 2160
ccatcatcac actcacctgg tggaacgcct gcaacactca ttcatcctcc cattgcgaat 2220
gtgatgtcac gtccatttag attgcgtctc ggcgcacccg atgtttatgt ccatgttaag 2280
gcgaattcac gacttttttt aaatcagtcg caaactgagg gcgattatat tatgtctctg 2340
caaacattac ttaacacgga taacgatata ggcggaggag gtataagtgg attgaacatc 2400
ggaggtatcg ggaacagtag tatgatgcca tctagcagtt tgtcaacaat gtcgcctagt 2460
ccatcgttag tttcgcccct ttctctgccc ttggactcac ttgttaataa caattcgtca 2520
tgctcctctt cgtcggcgtc agctttagca agtgtaagta gcggtgtaca tatgttagga 2580
ggactagttg gaggtggtgg tatgccacca atcaccacgc aaactactaa tgtgggtgga 2640
ccactgatga cttcagctgt tatcaatggt acagggagtg gtacacaacg taacaatcct 2700
gcagtggcaa catcggcatc gacttcgaat aattccaatc tggtgaattc gtttaccgca 2760
tcgcctgctg gacccgaagc aacgattttc tatagttccg acccttttga ttttgacttg 2820
gcacattcca gttttgaaat ggacgccact ggttggacgg attcgcgtcc aaactctcgc 2880
gcttctgtta ctaccccagt aagcactccg cgtccaccgt cagcgggaca tggtttcagt 2940
ccggcagtat gtgcttcacc atctacacca tatcaactgt cgtcacactc agctgcgagc 3000
ttgccttcgc ctcagtcaaa tacaagtcag ccgtcatctg ctggtcccgg ggttagtgga 3060
gggccgtttg ggtttggttt tccggcgttt gaaacaagtg ataaaaataa tgataaagac 3120
cacatgaacg ctagcataaa cagcagtagc agcataagtg gcaataattc tggacctcaa 3180
ggtggttcca gtggtgcgtt gagcggccct gcaaatttac caaatggtgg tccgccatta 3240
cttaccactg ttgctaacat gactcaacta tctgcacagc cgaatccacc ccaaccggaa 3300
tcagagcgtt tacggcactt attgacaaat aaatcgcatt caatgacctc aggaatgaac 3360
ccggccgatg gtgataaaga tcatcttagc ctgtgccata agctgcttaa cgaaaaatcg 3420
gaggatgatg atggaaaagg accttcgctt ggtcgacaaa gtgaattgat gcgtcaacta 3480
caaaaggacg atggtcatta tagtcattca cacggtcata gccacgggca acatcactcc 3540
aaggacatgt cgcaagagga tttactaaaa agtttaaagt accagggtga tcctacaact 3600
cgcaagcgtt cgctcaatga accggatgat ggcatattag caaaacgtga aagtgaccgc 3660
ccatccaagt tacgtgagag caataaaatg cttgcctcgc tgttagaaaa tccgccaaag 3720
aatcctatag tcactgtacc acctcaagtc aaaactatac ctgacattgc accttcagca 3780
agccgcgtca actcgacacc cggtgttatg agtgtttcag cgccaaatag tctaggtggc 3840
atgccaccaa aaagcggcat aatgacgacg acctcatcaa ttagtgcagg aggcagttct 3900
gtcggagccg gtggacggag caatagtatg cgcaaacaat tttccgatgc ctacctcacg 3960
ctgcaacagc agcaacatca gcaacaactc cacctacaac aacaacagca acaatgttta 4020
ggtaacactc aacgttcttc acatcagcag ccaacacaac tatctcaacc gcaaataaac 4080
tttgcttcat cggacgcctt cggaacatct tcacataaca ccacagctac ctcagggaca 4140
gccaacgtca cagcgtcaat tgtgacgtca caagcaaact cccaattagc cgctcctagc 4200
ggcgctgatg gcgattcaga attatctaag attttggata gcgttatgga ctatgtttca 4260
gacgatgggc catttgtatc agcacctact cccactgcct tggctggatt aacgcagcaa 4320
gagattaatg aacgcatggc tatcaacgct attcaaaatt cattgatggt cgagacatct 4380
caactgcaac agcagcaaca tcatcaacaa cagccgcaac cgccagccta tcctggcaac 4440
ataatgggga gtggcactgg agggaattta actcagcaac aacaacagct acagctccag 4500
catcacctgc agatgttgca aaggcagcag gctgcaattg gaggcaatca acagtcaact 4560
caggtacaac aaatgttgga ggtactacgt gtcaacgcca atcaaagttt ccaacgaccg 4620
ccacctatgt attcggcgtc gcgtaacagg ggcccaatga atactgttac gacacctgga 4680
ggctctgttt tgccagcgca tcagcagtac cgaattcgtc aacaacagca acagcaacaa 4740
caacagaaag agcgtcttct tcagcaacaa caaaagcaac agcttttggt tcctgagagt 4800
gcaacagcgc gcacagacca gttatgtcta aatccgaaca tcaacaacat tggttcgctg 4860
ttaaataaca cagtagcgcc aaatgtcgcc ttgtctcgta ccaacttgcc gcccgattcg 4920
caactgagtc cgaattttgc acagaattta cttcaacaac aacagttaag tccagggcaa 4980
cgtaatgcac catttagtcc acaaacgaat gcaggttatg caccgcaata ttcacaaagc 5040
ggacaacgtc tatcgcccca tcatcaacag catatttcac agcagcaaca acaagtgaat 5100
gctcagcaac aacaacagat ggcgtttcag gctgcccaag cagcaaacgc taatgtcact 5160
ccgcagctat caccacgcca accgcctttt ggtggaggag caggtggtgt acaatcacct 5220
acaggcatgc cgtcatcaca acaatggaat gcaagtggta atgttagtac gccaagtgta 5280
caactaagtg gaaatgtgac aactcaacgc ggtcattcat tacaacaaca taatcctatg 5340
ttaagcgcgc agttacaaca gggtgttagt ccttatacaa cacgtccgta tcaaaatcag 5400
cggcgaagtc tcaactctcc gaacggaggg ggtgtatctg gcaatgcagt tacaagtaat 5460
atcggaacta atgtcggtct gcagcggcaa aattcttttc aaggtggcgg cggaggaaat 5520
gtcggtcctg gcggtctgcg aataacttcc accccgcaaa gtcccttgag cagtgctcaa 5580
cagggaccaa tgagtggtgg caattctgga ggtttaaata gtacaaattc gcttagcatg 5640
caacaacatc aatctgggtc aataggaact ggtgttagcc aaaatgcact acttaataca 5700
ccagatcctt ccatgaattt tagcttcgat gcacaagatt tctttggcaa caatgctaca 5760
aggtga 5766
<210> 14
<211> 6045
<212> DNA
<213> 人工序列
<223> Taiman-D
<400> 14
atgtcaattg ttgcagccga aaatgcaggg cttggcccat gtgatttacc agattcgtgg 60
gcaccaaaca caaataatag caacaacaat attactagta tcgttgcaaa tagtagagac 120
atctcatcgt tatcagcagt cacagaaaat tcatcaaaca cggctgtccc tcttaatatt 180
gacaacaata ttagctcgaa tatagtgttc ttcaagaacc aaagcagtcc aactacaact 240
gccattcaac cgaccacttc tttcggcttt tcaccaacat caacggcaag agcaacaaca 300
tcaaccactt ccagtcttat atacaaggtg cctctctcat cgccagccgc tgctacagcc 360
gctgttactg tttcaccagc cagcattggt agtctagtaa cgtctttagg tgtgggtctg 420
gtaccctcaa taccgacgtg tgtgtccgtt actggaacga gtaccggtgg aactggagcc 480
tttgctggtg tactgttatc aagcccaata tcagctggtg gtagtagcaa tagtatttcg 540
gctcaaaaaa ccattacaaa ttatcaagta aacaacgtca ataataacgc aaataacacc 600
ggcaatacca gcgttagaag tctaacactt ccattgcagc aacaacaact acaattgcaa 660
caacaaaacg gcttattgct acataacaaa cagagcaacc aaaacaacaa taacagctct 720
ttagtgaata ataaccacga taaaaatagc aacaatttta atttaatact acgacagaag 780
tctgcagcaa ctgtaagttt agccgccgca cttcaaaatt caatcaccat gaatgcggta 840
gcttcgccaa tctcaaacaa cccagcgacg ccgactcgaa aaattagaag aaaaactgat 900
ccgaaagcca atttgccaca atcacagatt aataaatgta ataacgagaa aaggcgtcgc 960
gaattggaaa acaattatat cgagcaattg tccgagtttt tacaactcaa caaacgcggt 1020
gatacagcct caacgaaacc agacaaggca gcaatactga accaagtagt gaaaacgtat 1080
cgtgagtttt gtgacaaggg ccaaagccgt gatatttctt cctcaacgtc gactaccgct 1140
tcttcagctc tcaattcgac gacacctaat aacaacaata gcagctcaac ttctaaaagc 1200
aacgataatc gaaataatac agcatctaca cgttgtttac gatgtgcaac tgacaattgc 1260
tctatccatc cagtacaaca aggtgacgtt tcatcgaccg aacccccatt gccggagcca 1320
tcactgctta acggtcaggt gcccgaaata tctgcctatt ttgaggcgtt ggaacattac 1380
ctctcttcag tcgattgggt gcttttacaa gtcactgcag acgggataat cgagtcgtgt 1440
acgcagaata tacgagagct aatcggctat gacaaacagg aactgtttcg taaaccacta 1500
tatcagtacc tacactccga cgatcatgcg aaattaaatc caattataaa taatatgccg 1560
tatggagtta gcggaaatgc aactggtgac attggttgtg gtagcggtag ccaaagtggc 1620
tggaatgatt tggaagatgc gaatagtggt acaaattcgc aacattcaac agcttcgtta 1680
ggtccaggaa gtgctaaagc taaaagaaat atagcagcta aagtccgaat gctagtgaaa 1740
gatttgcgtt cggctactca aacatctatg acgcaccaaa acgatgcttt ggatcagaaa 1800
actgtcggac aacatataaa ccccgaaaaa tatgaggaag ttatgctcct agcaacaccg 1860
atgaaagacg atggtgatac aacgtcctcc atattgtgtt tgattacccg tcccgaagat 1920
gaaccggcca tacaacagat acaaccgcag gccatagagc atttaacctt taaacttgac 1980
gtacacggca agatactcaa tttggatgta accgcacttc gtcaacccta tcgtcaacat 2040
ttaagtagtt ggttgggacg ccttttgcag gatctttgtc atccgcaaga cttaaatgct 2100
ctgaaatcac acttacgcga ggttcaagag tcagctgctt cagtccattt gatgaactct 2160
ccatcatcac actcacctgg tggaacgcct gcaacactca ttcatcctcc cattgcgaat 2220
gtgatgtcac gtccatttag attgcgtctc ggcgcacccg atgtttatgt ccatgttaag 2280
gcgaattcac gacttttttt aaatcagtcg caaactgagg gcgattatat tatgtctctg 2340
caaacattac ttaacacgga taacgatata ggcggaggag gtataagtgg attgaacatc 2400
ggaggtatcg ggaacagtag tatgatgcca tctagcagtt tgtcaacaat gtcgcctagt 2460
ccatcgttag tttcgcccct ttctctgccc ttggactcac ttgttaataa caattcgtca 2520
tgctcctctt cgtcggcgtc agctttagca agtgtaagta gcggtgtaca tatgttagga 2580
ggactagttg gaggtggtgg tatgccacca atcaccacgc aaactactaa tgtgggtgga 2640
ccactgatga cttcagctgt tatcaatggt acagggagtg gtacacaacg taacaatcct 2700
gcagtggcaa catcggcatc gacttcgaat aattccaatc tggtgaattc gtttaccgca 2760
tcgcctgctg gacccgaagc aacgattttc tatagttccg acccttttga ttttgacttg 2820
gcacattcca gttttgaaat ggacgccact ggttggacgg attcgcgtcc aaactctcgc 2880
gcttctgtta ctaccccagt aagcactccg cgtccaccgt cagcgggaca tggtttcagt 2940
ccggcagtat gtgcttcacc atctacacca tatcaactgt cgtcacactc agctgcgagc 3000
ttgccttcgc ctcagtcaaa tacaagtcag ccgtcatctg ctggtcccgg ggttagtgga 3060
gggccgtttg ggtttggttt tccggcgttt gaaacaagtg ataaaaataa tgataaagac 3120
cacatgaacg ctagcataaa cagcagtagc agcataagtg gcaataattc tggacctcaa 3180
ggtggttcca gtggtgcgtt gagcggccct gcaaatttac caaatggtgg tccgccatta 3240
cttaccactg ttgctaacat gactcaacta tctgcacagc cgaatccacc ccaaccggaa 3300
tcagagcgtt tacggcactt attgacaaat aaatcgcatt caatgacctc aggaatgaac 3360
ccggccgatg gtgataaaga tcatcttagc ctgtgccata agctgcttaa cgaaaaatcg 3420
gaggatgatg atggaaaagg accttcgctt ggtcgacaaa gtgaattgat gcgtcaacta 3480
caaaaggacg atggtcatta tagtcattca cacggtcata gccacgggca acatcactcc 3540
aaggacatgt cgcaagagga tttactaaaa agtttaaagt accagggtga tcctacaact 3600
cgcaagcgtt cgctcaatga accggatgat ggcatattag caaaacgtga aagtgaccgc 3660
ccatccaagt tacgtgagag caataaaatg cttgcctcgc tgttagaaaa tccgccaaag 3720
aatcctatag tcactgtacc acctcaagtc aaaactatac ctgacattgc accttcagca 3780
agccgcgtca actcgacacc cggtgttatg agtgtttcag cgccaaatag tctaggtggc 3840
atgccaccaa aaagcggcat aatgacgacg acctcatcaa ttagtgcagg aggcagttct 3900
gtcggagccg gtggacggag caatagtatg cgcaaacaat tttccgatgc ctacctcacg 3960
ctgcaacagc agcaacatca gcaacaactc cacctacaac aacaacagca acaatgttta 4020
ggtaacactc aacgttcttc acatcagcag ccaacacaac tatctcaacc gcaaataaac 4080
tttgcttcat cggacgcctt cggaacatct tcacataaca ccacagctac ctcagggaca 4140
gccaacgtca cagcgtcaat tgtgacgtca caagcaaact cccaattagc cgctcctagc 4200
ggcgctgatg gcgattcaga attatctaag attttggata gcgttatgga ctatgtttca 4260
gacgatgggc catttgtatc agcacctact cccactgcct tggctggatt aacgcagcaa 4320
gagattaatg aacgcatggc tatcaacgct attcaaaatt cattgatggt cgagacatct 4380
caactgcaac agcagcaaca tcatcaacaa cagccgcaac cgccagccta tcctggcaac 4440
ataatgggga gtggcactgg agggaattta actcagcaac aacaacagct acagctccag 4500
catcacctgc agatgttgca aaggcagcag gctgcaattg gaggcaatca acagtcaact 4560
caggtacaac aaatgttgga ggtactacgt gtcaacgcca atcaaagttt ccaacgaccg 4620
ccacctatgt attcggcgtc gcgtaacagg ggcccaatga atactgttac gacacctgga 4680
ggctctgttt tgccagcgca tcagcagtac cgaattcgtc aacaacagca acagcaacaa 4740
caacagaaag agcgtcttct tcagcaacaa caaaagcaac agcttttggt tcctgagagt 4800
gcaacagcgc gcacagacca gttatgtcta aatccgaaca tcaacaacat tggttcgctg 4860
ttaaataaca cagtagcgcc aaatgtcgcc ttgtctcgta ccaacttgcc gcccgattcg 4920
caactgagtc cgaattttgc acagaattta cttcaacaac aacagttaag tccagggcaa 4980
cgtaatgcac catttagtcc acaaacgaat gcaggttatg caccgcaata ttcacaaagc 5040
ggacaacgtc tatcgcccca tcatcaacag catatttcac agcagcaaca acaagtgaat 5100
gctcagcaac aacaacagat ggcgtttcag gctgcccaag cagcaaacgc taatgtcact 5160
ccgcagctat caccacgcca accgcctttt ggtggaggag caggtggtgt acaatcacct 5220
acaggcatgc cgtcatcaca acaatggaat gcaagtggta atgttagtac gccaagtgta 5280
caactaagtg gaaatgtgac aactcaacgc ggtcattcat tacaacaaca taatcctatg 5340
ttaagcgcgc agttacaagg tgttagtcct tatacaacac gtccgtatca aaatcagcgg 5400
cgaagtctca actctccgaa cggagggggt gtatctggca atgcagttac aagtaatatc 5460
ggaactaatg tcggtctgca gcggcaaaat tcttttcaag gtggtgaatc tggtggtttt 5520
agtggtggta cctcaaattc cccatctcca cagtcaccat atggtgggcc cagctcaaac 5580
gtattccagc aacaacaaat gcaacgaatg caacggcaga gtagcgttcc gcaaactacc 5640
caacatttac ctgcaggtaa tgcagcaaac tcttctgatt tcgtaaaaca ggaattgagg 5700
gctatagtaa gcggacgtgc tcaacaacag gctgctgccg ctgggggagc tggtgcttct 5760
tctggtggaa acggagtgcc aggtggcggc ggaggaaatg tcggtcctgg cggtctgcga 5820
ataacttcca ccccgcaaag tcccttgagc agtgctcaac agggaccaat gagtggtggc 5880
aattctggag gtttaaatag tacaaattcg cttagcatgc aacaacatca atctgggtca 5940
ataggaactg gtgttagcca aaatgcacta cttaatacac cagatccttc catgaatttt 6000
agcttcgatg cacaagattt ctttggcaac aatgctacaa ggtga 6045
<210> 15
<211> 6273
<212> DNA
<213> 人工序列
<223> Taiman-E
<400> 15
atgtcaattg ttgcagccga aaatgcaggg cttggcccat gtgatttacc agattcgtgg 60
gcaccaaaca caaataatag caacaacaat attactagta tcgttgcaaa tagtagagac 120
atctcatcgt tatcagcagt cacagaaaat tcatcaaaca cggctgtccc tcttaatatt 180
gacaacaata ttagctcgaa tatagtgttc ttcaagaacc aaagcagtcc aactacaact 240
gccattcaac cgaccacttc tttcggcttt tcaccaacat caacggcaag agcaacaaca 300
tcaaccactt ccagtcttat atacaaggtg cctctctcat cgccagccgc tgctacagcc 360
gctgttactg tttcaccagc cagcattggt agtctagtaa cgtctttagg tgtgggtctg 420
gtaccctcaa taccgacgtg tgtgtccgtt actggaacga gtaccggtgg aactggagcc 480
tttgctggtg tactgttatc aagcccaata tcagctggtg gtagtagcaa tagtatttcg 540
gctcaaaaaa ccattacaaa ttatcaagta aacaacgtca ataataacgc aaataacacc 600
ggcaatacca gcgttagaag tctaacactt ccattgcagc aacaacaact acaattgcaa 660
caacaaaacg gcttattgct acataacaaa cagagcaacc aaaacaacaa taacagctct 720
ttagtgaata ataaccacga taaaaatagc aacaatttta atttaatact acgacagaag 780
tctgcagcaa ctgtaagttt agccgccgca cttcaaaatt caatcaccat gaatgcggta 840
gcttcgccaa tctcaaacaa cccagcgacg ccgactcgaa aaattagaag aaaaactgat 900
ccgaaagcca atttgccaca atcacagatt aataaatgta ataacgagaa aaggcgtcgc 960
gaattggaaa acaattatat cgagcaattg tccgagtttt tacaactcaa caaacgcggt 1020
gatacagcct caacgaaacc agacaaggca gcaatactga accaagtagt gaaaacgtat 1080
cgtgagtttt gtgacaaggg ccaaagccgt gatatttctt cctcaacgtc gactaccgct 1140
tcttcagctc tcaattcgac gacacctaat aacaacaata gcagctcaac ttctaaaagc 1200
aacgataatc gaaataatac agcatctaca cgttgtttac gatgtgcaac tgacaattgc 1260
tctatccatc cagtacaaca aggtgacgtt tcatcgaccg aacccccatt gccggagcca 1320
tcactgctta acggtcaggt gcccgaaata tctgcctatt ttgaggcgtt ggaacattac 1380
ctctcttcag tcgattgggt gcttttacaa gtcactgcag acgggataat cgagtcgtgt 1440
acgcagaata tacgagagct aatcggctat gacaaacagg aactgtttcg taaaccacta 1500
tatcagtacc tacactccga cgatcatgcg aaattaaatc caattataaa taatatgccg 1560
tatggagtta gcggaaatgc aactggtgac attggttgtg gtagcggtag ccaaagtggc 1620
tggaatgatt tggaagatgc gaatagtggt acaaattcgc aacattcaac agcttcgtta 1680
ggtccaggaa gtgctaaagc taaaagaaat atagcagcta aagtccgaat gctagtgaaa 1740
gatttgcgtt cggctactca aacatctatg acgcaccaaa acgatgcttt ggatcagaaa 1800
actgtcggac aacatataaa ccccgaaaaa tatgaggaag ttatgctcct agcaacaccg 1860
atgaaagacg atggtgatac aacgtcctcc atattgtgtt tgattacccg tcccgaagat 1920
gaaccggcca tacaacagat acaaccgcag gccatagagc atttaacctt taaacttgac 1980
gtacacggca agatactcaa tttggatgta accgcacttc gtcaacccta tcgtcaacat 2040
ttaagtagtt ggttgggacg ccttttgcag gatctttgtc atccgcaaga cttaaatgct 2100
ctgaaatcac acttacgcga ggttcaagag tcagctgctt cagtccattt gatgaactct 2160
ccatcatcac actcacctgg tggaacgcct gcaacactca ttcatcctcc cattgcgaat 2220
gtgatgtcac gtccatttag attgcgtctc ggcgcacccg atgtttatgt ccatgttaag 2280
gcgaattcac gacttttttt aaatcagtcg caaactgagg gcgattatat tatgtctctg 2340
caaacattac ttaacacgga taacgatata ggcggaggag gtataagtgg attgaacatc 2400
ggaggtatcg ggaacagtag tatgatgcca tctagcagtt tgtcaacaat gtcgcctagt 2460
ccatcgttag tttcgcccct ttctctgccc ttggactcac ttgttaataa caattcgtca 2520
tgctcctctt cgtcggcgtc agctttagca agtgtaagta gcggtgtaca tatgttagga 2580
ggactagttg gaggtggtgg tatgccacca atcaccacgc aaactactaa tgtgggtgga 2640
ccactgatga cttcagctgt tatcaatggt acagggagtg gtacacaacg taacaatcct 2700
gcagtggcaa catcggcatc gacttcgaat aattccaatc tggtgaattc gtttaccgca 2760
tcgcctgctg gacccgaagc aacgattttc tatagttccg acccttttga ttttgacttg 2820
gcacattcca gttttgaaat ggacgccact ggttggacgg attcgcgtcc aaactctcgc 2880
gcttctgtta ctaccccagt aagcactccg cgtccaccgt cagcgggaca tggtttcagt 2940
ccggcagtat gtgcttcacc atctacacca tatcaactgt cgtcacactc agctgcgagc 3000
ttgccttcgc ctcagtcaaa tacaagtcag ccgtcatctg ctggtcccgg ggttagtgga 3060
gggccgtttg ggtttggttt tccggcgttt gaaacaagtg ataaaaataa tgataaagac 3120
cacatgaacg ctagcataaa cagcagtagc agcataagtg gcaataattc tggacctcaa 3180
ggtggttcca gtggtgcgtt gagcggccct gcaaatttac caaatggtgg tccgccatta 3240
cttaccactg ttgctaacat gactcaacta tctgcacagc cgaatccacc ccaaccggaa 3300
tcagagcgtt tacggcactt attgacaaat aaatcgcatt caatgacctc aggaatgaac 3360
ccggccgatg gtgataaaga tcatcttagc ctgtgccata agctgcttaa cgaaaaatcg 3420
gaggatgatg atggaaaagg accttcgctt ggtcgacaaa gtgaattgat gcgtcaacta 3480
caaaaggacg atggtcatta tagtcattca cacggtcata gccacgggca acatcactcc 3540
aaggacatgt cgcaagagga tttactaaaa agtttaaagt accagggtga tcctacaact 3600
cgcaagcgtt cgctcaatga accggatgat ggcatattag caaaacgtga aagtgaccgc 3660
ccatccaagt tacgtgagag caataaaatg cttgcctcgc tgttagaaaa tccgccaaag 3720
aatcctatag tcactgtacc acctcaagtc aaaactatac ctgacattgc accttcagca 3780
agccgcgtca actcgacacc cggtgttatg agtgtttcag cgccaaatag tctaggtggc 3840
atgccaccaa aaagcggcat aatgacgacg acctcatcaa ttagtgcagg aggcagttct 3900
gtcggagccg gtggacggag caatagtatg cgcaaacaat tttccgatgc ctacctcacg 3960
ctgcaacagc agcaacatca gcaacaactc cacctacaac aacaacagca acaatgttta 4020
ggtaacactc aacgttcttc acatcagcag ccaacacaac tatctcaacc gcaaataaac 4080
tttgcttcat cggacgcctt cggaacatct tcacataaca ccacagctac ctcagggaca 4140
gccaacgtca cagcgtcaat tgtgacgtca caagcaaact cccaattagc cgctcctagc 4200
ggcgctgatg gcgattcaga attatctaag attttggata gcgttatgga ctatgtttca 4260
gacgatgggc catttgtatc agcacctact cccactgcct tggctggatt aacgcagcaa 4320
gagattaatg aacgcatggc tatcaacgct attcaaaatt cattgatggt cgagacatct 4380
caactgcaac agcagcaaca tcatcaacaa cagccgcaac cgccagccta tcctggcaac 4440
ataatgggga gtggcactgg agggaattta actcagcaac aacaacagct acagctccag 4500
catcacctgc agatgttgca aaggcagcag gctgcaattg gaggcaatca acagtcaact 4560
caggtacaac aaatgttgga ggtactacgt gtcaacgcca atcaaagttt ccaacgaccg 4620
ccacctatgt attcggcgtc gcgtaacagg ggcccaatga atactgttac gacacctgga 4680
ggctctgttt tgccagcgca tcagcagtac cgaattcgtc aacaacagca acagcaacaa 4740
caacagaaag agcgtcttct tcagcaacaa caaaagcaac agcttttggt tcctgagagt 4800
gcaacagcgc gcacagacca gttatgtcta aatccgaaca tcaacaacat tggttcgctg 4860
ttaaataaca cagtagcgcc aaatgtcgcc ttgtctcgta ccaacttgcc gcccgattcg 4920
caactgagtc cgaattttgc acagaattta cttcaacaac aacagttaag tccagggcaa 4980
cgtaatgcac catttagtcc acaaacgaat gcaggttatg caccgcaata ttcacaaagc 5040
ggacaacgtc tatcgcccca tcatcaacag catatttcac agcagcaaca acaagtgaat 5100
gctcagcaac aacaacagat ggcgtttcag gctgcccaag cagcaaacgc taatgtcact 5160
ccgcagctat caccacgcca accgcctttt ggtggaggag caggtggtgt acaatcacct 5220
acaggcatgc cgtcatcaca acaatggaat gcaagtggta atgttagtac gccaagtgta 5280
caactaagtg gaaatgtgac aactcaacgc ggtcattcat tacaacaaca taatcctatg 5340
ttaagcgcgc agttacaagg tgttagtcct tatacaacac gtccgtatca aaatcagcgg 5400
cgaagtctca actctccgaa cggagggggt gtatctggca atgcagttac aagtaatatc 5460
ggaactaatg tcggtctgca gcggcaaaat tcttttcaag gtggtgaatc tggtggtttt 5520
agtggtggta cctcaaattc cccatctcca cagtcaccat atggtggccc aagctcaaac 5580
gtattccagc aacaacaaat gcaacgaatg caacgccaga gtagcgttcc gcaaactacc 5640
caacatttac ctggctctcc acgccctttc attgcgaatg taaatcacga aaatattgta 5700
ggtacaagcg gcaatggcgc cgccacaagc ctcggcgtaa atggcggtgt cagcgtcggc 5760
gggagtgtcg gtattgttgg ctttggtggc atgatgtaca ataatatgca gcatgctgct 5820
catgctgctc atgcgacctc acaacaaaca caaaacgaat tttatggtcg cgctcaggcc 5880
gcaggtaatg cagcaaactc ttctgatttc gtaaaacagg aattgagggc tatagtaagc 5940
ggacgtgctc aacaacaggc tgctgccgct gggggagctg gtgcttcttc tggtggaaac 6000
ggagtgccag gtggcggcgg aggaaatgtc ggtcctggcg gtctgcgaat aacttccacc 6060
ccgcaaagtc ccttgagcag tgctcaacag ggaccaatga gtggtggcaa ttctggaggt 6120
ttaaatagta caaattcgct tagcatgcaa caacatcaat ctgggtcaat aggaactggt 6180
gttagccaaa atgcactact taatacacca gatccttcca tgaattttag cttcgatgca 6240
caagatttct ttggcaacaa tgctacaagg tga 6273
<210> 16
<211> 40
<212> DNA
<213> 人工序列
<223> T7-Tai-core-F
<400> 16
taatacgact cactataggg gctctcaatt cgacgacacc 40
<210> 17
<211> 40
<212> DNA
<213> 人工序列
<223> T7-Tai-core-R
<400> 17
taatacgact cactataggg acctgaccgt taagcagtga 40
<210> 18
<211> 194
<212> RNA
<213> 人工序列
<223> dsTaiman-core
<400> 18
gcucucaauu cgacgacacc uaauaacaac aauagcagcu caacuucuaa aagcaacgau 60
aaucgaaaua auacagcauc uacacguugu uuacgaugug caacugacaa uugcucuauc 120
cauccaguac aacaagguga cguuucaucg accgaacccc cauugccgga gccaucacug 180
cuuaacgguc aggu 194
<210> 19
<211> 41
<212> DNA
<213> 人工序列
<223> GFP-ds-T7F
<400> 19
taatacgact cactataggg cagttcttgt tgaattagat g 41
<210> 20
<211> 41
<212> DNA
<213> 人工序列
<223> GFP-ds-T7R
<400> 20
taatacgact cactataggg tttggtttgt ctcccatgat g 41
<210> 21
<211> 21
<212> RNA
<213> 人工序列
<223> siTaiman-A
<400> 21
guguaucugc acuacuuaau a 21
<210> 22
<211> 21
<212> RNA
<213> 人工序列
<223> siTaiman-B
<400> 22
ucuaaugucg guccuggcgg u 21
<210> 23
<211> 21
<212> RNA
<213> 人工序列
<223> siTaiman-C
<400> 23
aauucuuuuc aagguggcgg c 21
<210> 24
<211> 21
<212> RNA
<213> 人工序列
<223> siTaiman-D
<400> 24
ucaacauuua ccugcaggua a 21
<210> 25
<211> 21
<212> RNA
<213> 人工序列
<223> siTaiman-E
<400> 25
guauuguugg cuuugguggc a 21
<210> 26
<211> 21
<212> RNA
<213> 人工序列
<223> siGFP
<400> 26
gccacaacgu cuauaucaug g 21
<210> 27
<211> 20
<212> DNA
<213> 人工序列
<223> BdTai-core-F
<400> 27
gctctcaatt cgacgacacc 20
<210> 28
<211> 20
<212> DNA
<213> 人工序列
<223> BdTai-core-R
<400> 28
acctgaccgt taagcagtga 20
<210> 29
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiA-F
<400> 29
cctatgttaa gcgcgcagtt 20
<210> 30
<211> 21
<212> DNA
<213> 人工序列
<223> BdTaiA-R
<400> 30
taagtagtgc agatacaccc c 21
<210> 31
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiB-F
<400> 31
cctatgttaa gcgcgcagtt 20
<210> 32
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiB-R
<400> 32
gccaggaccg acattagttc 20
<210> 33
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiC-F
<400> 33
cctatgttaa gcgcgcagtt 20
<210> 34
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiC-R
<400> 34
gccgccacct tgaaaagaat 20
<210> 35
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiD-F
<400> 35
aggtggtgaa tctggtggtt 20
<210> 36
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiD-R
<400> 36
cctgcaggta aatgttgggt 20
<210> 37
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiE-F
<400> 37
ggagtgtcgg tattgttggc 20
<210> 38
<211> 20
<212> DNA
<213> 人工序列
<223> BdTaiE-R
<400> 38
cctgagcgcg accataaaat 20
<210> 39
<211> 20
<212> DNA
<213> 人工序列
<223> α-Tubulin-F
<400> 39
cgcattcatg gttgataacg 20
<210> 40
<211> 20
<212> DNA
<213> 人工序列
<223> α-Tubulin-R
<400> 40
gggcaccaag ttagtctgga 20

Claims (6)

1.一种桔小实蝇Taiman基因,其特征在于,其核苷酸序列如SEQ ID NO:15所示。
2.一种权利要求1所述的桔小实蝇Taiman基因的siRNA,其特征在于,序列如SEQ IDNO:25所示。
3.一种对SEQ ID NO:15所示的桔小实蝇Taiman基因进行qRT-PCR检测的方法,其特征在于,qPCR中的使用的上、下游引物为BdTaiE-F、BdTaiE-R,其序列分别如SEQ ID NO:37、38所示。
4.如权利要求3所述的对SEQ ID NO:15所示的桔小实蝇Taiman基因进行qRT-PCR检测的方法,其特征在于,PCR反应体系为20μl,包括:cDNA模板1μl,上、下游qPCR引物各1μl,去核酸酶水7μl和荧光染料qPCRMaster Mix 10μl。
5.如权利要求3所述的对SEQ ID NO:15所示的桔小实蝇Taiman基因进行qRT-PCR检测的方法,其特征在于,qRT-PCR反应条件为:95℃预变性2min;95℃变性30s、60℃退火30s,共40个循环。
6.权利要求2所述的siRNA在桔小实蝇发育、变态与生殖调节方面的应用,在桔小实蝇防治上的应用,或者在制备桔小实蝇杀虫剂方面的应用。
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CN111378670B (zh) * 2020-03-01 2021-07-20 华中农业大学 一种分离的中黑盲蝽Taiman基因及其编码的蛋白
CN111606989B (zh) * 2020-06-15 2022-04-08 华中农业大学 生殖发育相关基因UCP及其dsRNA在防治桔小实蝇中的应用
CN115976033B (zh) * 2022-12-14 2024-05-17 西南大学 瓜实蝇tssk1和tssk3基因及其应用

Non-Patent Citations (3)

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A Role for Taiman in Insect Metamorphosis;Jesus Lozano等;《PLoS Genet》;20141031;第10卷(第10期);第e1004769页 *
GenBank: JQ266268.1:Bactrocera dorsalis Taiman mRNA, complete cds;GenBank;《GenBank》;20130528;第1-3页 *
The alternative splicing of BdTai and its involvement in the development of Bactrocera dorsalis;HongLiu等;《Journal of Insect Physiology》;20170831;第101卷;第132-141页 *

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