本发明属于基因工程技术领域,具体涉及一种大通量筛选植物病毒复制必需寄主基因的方法。
背景技术
植物正义rna病毒的rna具有侵染性,进入植物细胞后可作为mrna被细胞的核糖体翻译产生病毒复制所需要的蛋白质(flasinskietal.,1996),进而启动病毒的复制、运动等生命过程。植物正义rna病毒可以通过构建侵染性克隆以方便长期保存,进行改造(flasinskietal.,1996,takahashietal.,1997)。目前有许多植物正义rna病毒的侵染性克隆被报道,总结方法可以归为两类:1)将病毒的全基因组的cdna插入至组成型启动子,如花棷菜花叶病毒(camv)35s启动子下游,通过农杆菌在植物中表达病毒基因组rna;2)将病毒的全基因组的cdna插入至原核转录启动子(如sp6和t7)下游,通过体外转录获得病毒全基因组rna,然后通过机械摩擦接种植物。上述两种方法存在着以下缺陷:1)组成型启动子一旦进入细胞即开始表达,因此不能条件性地控制表达的时间;2)由于rna的自然特征,原核转录启动子转录过程产生的病毒rna非常容易被环境中rna酶降解,造成病毒侵染效率低下。
筛选一种植物病毒复制必需寄主基因需要构建一个可以理论上可以覆盖植物基因组所有基因的突变体库,然后通过逐一接种病毒,筛选病毒不能复制的突变体获得。这种方法存在一个明显的缺陷,即需要大量种植突变体至一定株高并逐一进行病毒接种,需要非常大的人力、速度慢、温室以及实验材料。
技术实现要素:
针对目前侵染性克隆构建方法中病毒侵染效率低,基因表达难控制的问题,本发明提供了一种大通量筛选植物病毒复制必需寄主基因的方法,包括如下步骤:
1)诱导型病毒侵染性克隆的构建:所述表达载体包含农杆菌t-dna左边界序列、农杆菌t-dna右边界序列、诱导型启动子、植物正义rna病毒全长cdna、真核转录终止子、大肠杆菌和农杆菌复制必需元件和抗性筛选基因,其中,植物正义rna病毒全长cdna位于诱导型启动子和真核转录终止子序列之间,农杆菌t-dna右边界序列位于诱导型启动子上游,农杆菌t-dna左边界序列位于真核转录终止子下游;获得的诱导型病毒侵染性克隆转化大肠杆菌宿主菌中,获得阳性转化子;
2)诱导型病毒侵染性克隆的遗传转化:将步骤1)获得的阳性转化子导入农杆菌,然后将诱导型病毒侵染性克隆转入植物体内,获得转基因植物;
3)植物病毒复制必需基因的大通量筛选:用转基因植物构建突变体库,突变体发芽后,利用诱导剂诱导病毒侵染,进行突变体的筛选,得到病毒不能被诱导的突变体植物,结合图位克隆和全基因组测序的方法确定筛选的植物病毒复制必需基因。
优选地,所述大肠杆菌的宿主菌为大肠杆菌trans10,所述农杆菌为农杆菌gv3101。
优选地,所述农杆菌t-dna左边界序列如seqidno:1所示;所述农杆菌t-dna右边界序列如seqidno:2所示;所述的大肠杆菌和农杆菌复制必需元件的序列如seqidno:3所示。
优选地,步骤1)所述植物正义rna病毒为马铃薯y病毒属病毒或马铃薯x病毒属病毒;所述的马铃薯y病毒属病毒包括芜菁花叶病毒、马铃薯y病毒、马铃薯a病毒、李痘病毒、番木瓜环斑病毒、大豆花叶病毒和烟草蚀纹病毒;所述的马铃薯x病毒属病毒包括马铃薯x病毒、马铃薯奥古巴花叶病毒、凤果花叶病毒、车前草花叶病毒和狐尾草花叶病毒。
优选地,步骤1)所述植物正义rna病毒全长cdna为芜菁花叶病毒的全长cdna,其序列如seqidno:4所示。
优选地,步骤1)所述植物正义rna病毒全长cdna为马铃薯x病毒的全长cdna,其序列如seqidno:5所示。
优选地,步骤1)所述的诱导型启动子为雌激素诱导型启动子xve,其序列如seqidno:6所示。
优选地,步骤1)所述抗性筛选基因为潮霉素基因,序列如seqidno:7所示,所述潮霉素基因,其上游为雌激素诱导型启动子xve-1,其序列如seqidno:8所示,其下游为雌激素诱导型启动子xve-2,其序列如seqidno:9所示;步骤1)所述的植物正义rna病毒全长cdna,其上游的启动子为雌激素诱导型启动子xve-2。
优选地,步骤1)所述真核转录终止子为t3a终止子,其序列如seqidno:10所示。
优选地,步骤3)所述的突变体筛选采用雌激素诱导法。
本发明步骤1)所述的诱导型病毒侵染性克隆转入植物体内是利用农杆菌介导法或基因枪法实现的,步骤2)所述的转化植物为拟南芥col-0生态型或普通烟草;步骤3)所述的突变体库构建方法采用t-dna法或ems诱变法;所述的植物病毒复制必需寄主基因的筛选是结合图位克隆和全基因组测序法来确定目的基因,当转化的植物为拟南芥时,通过基因定位获得的基因,该基因中插入带有除草剂抗性基因的t-dna序列,则确定其为植物病毒复制必需寄主基因;当转化的植物为烟草时,通过基因定位获得的基因,该基因与参考基因(即未突变的基因)核苷酸序列相比带有可引起编码氨基酸发生变化或编码氨基酸提前终止的核苷酸突变,则确定其为植物病毒复制必需寄主基因。
有益效果
本发明涉及一种大通量筛选植物正义rna病毒复制、运动相关的必需寄主基因的方法。本方法可一次性进行大量突变体的筛选,因此可节省大量的时间、实验材料和经费,提高效率。
附图说明
图1诱导型tumv侵染性克隆载体xve::tumv-gfp载体图谱。
图2诱导型pvx侵染性克隆载体xve::pvx-gfp载体图谱。
具体实施方式
lb培养基:胰蛋白胨10g/l、酵母提取物5g/l、氯化钠10g/l,ph7.5;
lb固体培养基:每升lb液体培养基加15g~20g琼脂粉;
ms固体培养基:购自美国phytotech公司,货号:m401;
gibsonassemblymastermix试剂盒购买自nebbiolabs公司,货号:e2611;
大肠杆菌trans10感受态细胞购买自全式金生物技术有限公司,货号:cd101;
easypureplasmidminiprepkit试剂盒提取购买自全式金生物技术有限公司,货号:em101;
easypurepcrpurificationkit试剂盒提取购买自全式金生物技术有限公司,货号:ep101;
雌激素诱导剂购买自西格玛奥德里奇公司,货号:e8875;
除草剂购买自北京酷来搏科技有限公司,货号:cb2471;
植物生长箱购买自武汉瑞华仪器设备有限责任公司,货号:ar1200,其用来培养拟南芥的条件为:光周期为18小时光照,6小时黑暗;温度为23℃;
黑光灯购买自美国uvp公司,货号uvgl-55;
其他试剂或仪器如无特殊说明,均可通过现有的商业化途径购买获得。
实施例1:大通量筛选芜菁花叶病毒复制必需寄主基因的方法。
步骤1.诱导型芜菁花叶病毒(turnipmosaicvirus,tumv)侵染性表达载体构建。
1)为了更加直观简便对转化有诱导型病毒载体的转基因作物后代进行筛选,本实施例利用带有绿色荧光标记基因gfp标签的tumv侵染性质粒pcambia-tumv-gfp为模板,该pcambia-tumv-gfp质粒中包含了一个在编码p1和hcpro蛋白之间插入有编码绿色荧光蛋白(gfp)序列的tumv基因组(tumv-gfp),见seqidno:11所示,所述侵染性质粒pcambia-tumv-gfp记载在cottons,grangeonr,thiviergek,etal.,2009.turnipmosaicvirusrnareplicationcomplexvesiclesaremobile,alignwithmicrofilaments,andareeachderivedfromasingleviralgenome.jvirol83,10460-71。
不添加荧光标记基因,同样能够实现上述载体构建及载体功能的发挥,突变体的鉴定则在植物遗传转化后,利用相应的筛选压力或诱导条件(如除草剂的筛选或雌激素的诱导下)通过性状分析以及分子检测等方法来进行。
用下述引物对进行pcr反应,扩增tumv-gfp基因组1-6487nt之间的序列,记为序列a,包括雌激素诱导型启动子序列的片段总大小为6507bp。
所述引物对为:
引物1:5′-gttcatttcatttggagaggaaaaaatataaaaactcaacataac-3′(小写部分为雌激素诱导型启动子序列);
引物2:5′-ttgatggtgaactgcctcaagagc-3′;
用下述引物对进行pcr反应,扩增tumv基因组6464-10578nt以及polya序列,记为序列b,包括tumv-gfp基因组polya序列以及pmdc序列的片段总大小为4213bp。
所述引物对为:
引物3:5′-gctcttgaggcagttcaccatcaa-3′;
引物4:5′-gacgaaagctgggaggcctggtcgactctagggggatccga-3′(大写部分来自于pcambia-tumv-gfp质粒,小写部分来自于pmdc7);
上述pcr反应条件均为:95℃预变性3min,进入pcr循环:95℃变性15sec,65℃退火10sec,72℃延伸3.30min,30个循环;最后72℃延伸7min。
pcr反应后用1%的琼脂糖凝胶电泳检测并回收pcr产物。所述pcr产物回收采用全式金生物技术有限公司的easypurepcrpurificationkit试剂盒。
2)以带有雌激素诱导型启动子xve的植物双元表达载体pmdc7为模版,该载体记载在curtismd,grossniklausu,2003.agatewaycloningvectorsetforhigh-throughputfunctionalanalysisofgenesinplanta.plantphysiol133,462-9。
用下述引物对进行pcr反应,扩增pmdc7中包含xve诱导型启动子、植物潮霉素筛选基因、农杆菌t-dna左边界、大肠杆菌和农杆菌复制和抗性筛选序列、农杆菌tdna右边界以及t3a终止子之间序列,记为序列c,片段大小为11447bp。
所述引物对为:
引物5:5′-cctctccaaatgaaatgaacttc-3′;
引物6:5′-caggcctcccagctttcgtccgtatc-3′。
pcr反应条件:95℃预变性3min,进入pcr循环:95℃变性15sec,65℃退火10sec,72℃延伸6min,30个循环;最后72℃延伸7min。
3)参照gibsonassemblymastermix试剂盒说明书,利用同源重组的方法将序列a、序列b和序列c拼接为一个诱导型tumv侵染性克隆载体,序列d,命名为xve::tumv-gfp,见图1所示,其中,植物潮霉素筛选基因序列位于诱导型启动子xve内部,潮霉素抗性基因上游的启动子为雌激素诱导型启动子xve-1,其序列如seqidno:8所示;tumv全长cdna序列上游的启动子为雌激素诱导型启动子xve-2,其序列如seqidno:9所示;tumv的全长cdna位于xve诱导型启动子xve-2和t3a终止子之间,tumv-gfp的全长cdna中1229-1972nt,共744nt,为插入的gfp荧光标记基因,农杆菌t-dna右边界rb,位于雌激素诱导型启动子xve-1上游,农杆菌t-dna左边界lb,位于真核转录终止子t3a下游。
其中xve-1的258-1709bp编码一个人工转录因子,所述的人工转录因子由大肠杆菌lexa蛋白的dna结合结构域,vp16蛋白转录激活结构域和人雌激素受体结构域3部分组成。lexa蛋白的dna结合结构域具有识别xve-2序列的功能,vp16蛋白转录激活结构域和人雌激素受体结构域组成部分具有雌激素依赖性转录激活功能,即在结合雌激素时可以启始xve-2后tumv全长cdna基因的转录。
所述农杆菌t-dna左边界lb,其序列如seqidno:1所示;农杆菌t-dna右边界序列rb,其序列如seqidno:2所示;大肠杆菌和农杆菌复制必需元件,其序列如seqidno:3所示;tumv的全长cdna,其序列如seqidno:4所示;雌激素诱导型启动子xve,其序列如seqidno:6所示;抗性筛选基因潮霉素基因,其序列如seqidno:7所示;雌激素诱导型启动子xve-1,其序列如seqidno:8所示;雌激素诱导型启动子xve-2,其序列如seqidno:9所示;真核转录终止子t3a,其序列如seqidno:10所示。
将xve::tumv-gfp载体转化大肠杆菌trans10感受态细胞中,操作方法根据产品说明书进行。转化后,于37℃培养1小时后涂于含有卡那霉素(终浓度为50μg/ml)的lb固体培养基,37℃培养过夜培养。
用菌落pcr筛选阳性克隆。菌落pcr反应所用的引物对为:引物7:5′-ggctttcaagcgccagtgcgag-3′;引物8:5′-gaagtgctcttgcacaagggtg-3′;扩增的目的片段大小为824bp。
菌落pcr反应体系为50μl,其中2×taqpcrmix25μl,引物7和引物8各1μl,模版为转化的大肠杆菌克隆,其余为灭菌水。反应条件:94℃预变性3min,进入pcr循环:94℃变性30sec,60℃退火20sec,72℃延伸1min,30个循环;最后72℃延伸7min。
获得的阳性克隆,用含有卡那霉素(终浓度为50μg/ml)的lb液体培养基,37℃过夜培养,easypureplasmidminiprepkit试剂盒提取质粒,送上海生工生物技术有限公司进行序列测定,鉴定正确的质粒命名为xve::tumv-gfp,即诱导型芜菁花叶病毒(turnipmosaicvirus,tumv)侵染性表达载体。
步骤2.诱导型芜菁花叶病毒(tumv)侵染性表达载体的遗传转化
1)参照《分子克隆实验指南》,用电击法将xve::tumv-gfp导入农杆菌gv3101株系(购自北京华越洋生物),转化后涂布于含有终浓度为50μg/ml卡那霉素和终浓度为100μg/ml利福平的lb固体培养基上,28℃培养至长出克隆。随机挑取6个克隆进行pcr检测,反应体系、反应条件同诱导型芜菁花叶病毒(turnipmosaicvirus,tumv)侵染性克隆构建步骤1中3)所述的菌落pcr。
2)用含有xve::tumv-gfp质粒的农杆菌浸泡拟南芥哥伦比亚型生态型(col-0)花序10sec后,放回植物生长箱直至结实,收集经农杆菌处理的花序的种子。所述拟南芥蘸花转化方法参照cloughsj,bentaf,1998.floraldip:asimplifiedmethodforagrobacterium-mediatedtransformationofarabidopsisthaliana.theplantjournal:forcellandmolecularbiology16,735-43.。
3)参照harrisonsj,mottek,parsleyk,aspinalls,grayjc,cottagea,2006.arapidandrobustmethodofidentifyingtransformedarabidopsisthalianaseedlingsfollowingfloraldiptransformation.plantmethods2,19.takahashiy,takahashit,uyedai,1997.acdnaclonetocloveryellowveinpotyvirusgenomeishighlyinfectious.virusgenes14,235-43.提供的拟南芥种子消毒和筛选的方法,将收集的拟南芥种子用体积分数为10%双氧水进行消毒后,平铺至含有潮霉素终浓度为15mg/l的ms固体培养基上,放至4℃春化2天后在22℃植物培养箱培养进行6小时光照、2天黑暗、1天光照的光周期处理,筛选出抗性植株,获得转xve::tumv-gfp的拟南芥阳性植株。
4)将阳性植株转移至栽培土中,种植至结实,再进行第二代的筛选和第三代的筛选,直至获得不再分离的纯合的第三代转基因植物,所述第二代、第三代的筛选同第一代的筛选方法。
步骤3.拟南芥中tumv复制必需寄主基因的大通量筛选
1)用从拟南芥生物中心(abrc;https://www.arabidopsis.org)购买的pdap101质粒转化农杆菌gv3101株系,所述pdap101中带有除草剂抗性,位于d-dna左右边界序列之间,具体操作同参照《分子克隆实验指南》,电击法转化农杆菌。
2)以步骤2获得的转基因拟南芥为材料,同步骤2中2)所述的方法,将转化有pdap101质粒的农杆菌进行拟南芥col-0生态型的转化,培养至结实后,收集经农杆菌处理的花序的种子;
3)将2)中收集的拟南芥种子用水浸泡后,放置4℃春化2天后,直接播种至栽培土中,待全部发芽后用10mg/l的除草剂basta水溶液进行喷施(每隔一天喷施一次,共喷施3次),筛选带有抗除草剂basta抗性的转基因植株,获得大量抗转基因拟南芥一代种子;将所有一代种子进行种植,并收集第二代种子,构建拟南芥突变体库。
4)根据步骤2中3)所述的方法处理收集的第二代种子,平铺至ms固体培养基上,22℃光照培养至发芽,用10mg/l的雌激素水溶液诱导剂喷施1次/天,于每天同一时间喷施,连续2天,利用雌激素诱导剂诱导芜菁花叶病毒基因组表达,2周后用黑光灯在黑暗条件下筛选没有gfp表达的拟南芥突变体,转移至栽培土中,种植至结实。
5)将4)中获得的拟南芥突变体进行种植至开花,将露出花瓣的成熟花剪去,留下还未成熟的花,用尖头镊子将花萼、花瓣、雄蕊都去除,仅留下带有雌蕊的子房,小心在处理完的子房上扣上来自野生型拟南芥兰德伯格生态型(ler)的成熟花朵,让col-0拟南芥突变体的卵子和ler的花粉进行杂交,获得杂交种子;将所有第一代种子进行种植,并收集第二代种子,构建杂交后代群体。
6)将5)中获得的后代群体进行种植,每株单独取样,采用全式金生物技术easypureplantgenomicdnakit提取基因组dna,参照liq,shenwy,zhengq,tanyf,gaoj,shengjx,weiyd,kunstl,zoujt.2017.effectsofeifiso4g1mutationonseedoilbiosynthesis.plantj,90(5):966-978,通过图位克隆和全基因组测序的方法,进行目的基因的位置定位,当检测到插入了带有除草剂抗性基因的t-dna序列的基因,则确定其为植物病毒复制必需寄主基因。
实施例2.大通量筛选马铃薯x病毒复制必需寄主基因的方法。
重复实施例1,与实施例1的不同在于,本实施例中所述的诱导型病毒侵染性克隆为带有gfp标签的马铃薯x病毒(potatovirusx,pvx)侵染性克隆:以带有gfp标签的马铃薯x病毒侵染性克隆为模版(pvx.gfp-2a-cp),见seqidno:12所示,所述侵染性质粒记载在cruzss,chapmans,robertsag,robertsim,priordam,oparkakj.1996.assemblyandmovementofaplantviruscarryingagreenfluorescentproteinovercoat.proc.natl.acad.sci.usa,93:6286-6290.用引物9和引物10扩增1-3953nt序列,记为序列e(包括雌激素诱导型启动子部分序列,总长度3973nt),用引物11和引物12扩增3933-3987nt序列,记为序列f(包括polya以及pmdc7载体中t3a终止子部分序列,总长度3109nt)。
不添加荧光标记基因,同样能够实现上述载体构建及载体功能的发挥,突变体的鉴定则在植物遗传转化后利用相应的诱导条件(如雌激素的诱导下)通过性状分析以及分子检测等方法来进行。
引物9:5′-gttcatttcatttggagagggaaaactaaaccatacaccaccaacacaacc-3′(小写部分为雌激素诱导型启动子序列);
引物10:gataacgtgcctaggaaaatctg
引物11:gattttcctaggcacgttatc
引物12:5′-gacgaaagctgggaggcctgggaaattcgcggtacccactag-3′(大写部分来自于pvx.gfp-2a-cp质粒,小写部分来自于pmdc7)
参照gibsonassemblymastermix试剂盒说明书,利用同源重组的方法将序列e、序列f和序列c拼接为一个诱导型pvx侵染性克隆载体,命名为xve::pvx-gfp,见图2所示,与实施例1不同在于,本实施例中所构建的诱导型pvx侵染性克隆载体中,植物正义rna病毒全长cdna为马铃薯x病毒的全长cdna,其序列如seqidno:5所示。pvx-gfp的全长cdna中5671-6210nt,共540nt,为插入的gfp荧光标记基因。
转化的植物为烟草,步骤2中所述的xve::pvx通过农杆菌介导转化到烟草(nicotianatobacum)细胞中,具体方法参照赵莉,钟鸣,马慧,李浩戈,陈丽静,钟声.2011.农杆菌介导的烟草高效遗传转化体系的建立,江苏农业科学.39(3):67-68,74;步骤3)中进行,pvx复制必需基因的大通量筛选时,突变体库将采用ems诱导法;所述方法根据史跃伟,任学良,王轶,孙艳霞,邬晓勇.2009.γ射线与ems单一及复合处理对烤烟种子活力的诱变效应.贵州农业科学,12:62-65,描述的方法进行。构建的突变体库不进行除草剂筛选,直接喷施雌激素水溶液(15mg/l)诱导病毒表达,于每天相同的时间喷施一次,连续2天,2周后用黑光灯在黑暗条件下筛选没有gfp表达的烟草突变体,转移至栽培土中,种植至结实。杂交所用烟草为三生烟(nicotianatobaccicv.samsun)。其余方法参照实施例1。
通过图位克隆和全基因组测序的方法,进行目的基因的位置定位,当检测到该基因与参考基因(序列未变化之前的基因)核苷酸序列相比带有可引起编码氨基酸发生变化或编码氨基酸提前终止的核苷酸突变,则确定其为植物病毒复制必需寄主基因。
采用相同的策略和方法,本发明所述的大通量筛选植物病毒复制必需寄主基因的方法,适用于除芜菁花叶病毒(tumv)和马铃薯x病毒(pvx)以外的其他马铃薯y病毒属病毒和马铃薯x病毒属病毒。所述的马铃薯y病毒属病毒包括tumv、马铃薯y病毒(potatovirusy,pvy;genbank登录号:nc_001616.1)、马铃薯a病毒(potatovirusa,pva;genbank登录号:nc_004039.1)、李痘病毒(plumpoxvirus,ppv;genbank登录号:nc_001445.1)、番木瓜环斑病毒(papayaringspotvirus,prsv;genbank登录号:nc_001785.1)、大豆花叶病毒(soybeanmosaicvirus,smv;genbank登录号:nc_002634.1)和烟草蚀纹病毒(tobaccoetchvirus,tev;genbank登录号:nc_001555.1);所述的马铃薯x病毒属病毒包括pvx、马铃薯奥古巴花叶病毒(potatoaucubamosaicvirus,pamv;genbank登录号:nc_003632.1)、凤果花叶病毒(pepinomosaicvirus,pepmv;genbank登录号:nc_004067.1)、车前草花叶病毒(plantagoasiaticamosaicvirus,plamv;genbank登录号:nc_003849.1)和狐尾草花叶病毒(foxtailmosaicvirus,fomv;genbank登录号:nc_001483.1)。
通过pcr扩增获得上述病毒的基因组cdna全长,参照实施例1和实施例2中步骤1所述的方法,利用同源重组技术构建含有诱导型启动子、植物正义rna病毒cdna以及t3a终止子的诱导型病毒侵染性表达载体。
参照施例1和实施例2步骤2、3所述的方法筛选所述植物病毒复制必需寄主基因。
本发明方法提供了一种大通量筛选植物病毒复制必需寄主基因的方法,应用本方法可同时实现对大量(1万株以上植物)同时进行筛选。相对原来对突变体每株进行机械接种的方法,可极大地提高筛选效率。
序列表
<110>东北农业大学
<120>一种大通量筛选植物病毒复制必需寄主基因的方法
<130>
<160>24
<170>patentinversion3.5
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<213>农杆菌t-dna左边界
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<213>农杆菌t-dna右边界序列
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gtttacccgccaatatatcctgtcaa26
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<211>6510
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<213>大肠杆菌和农杆菌复制必需元件的序列
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<210>4
<211>9834
<212>dna
<213>芜菁花叶病毒tumv的全长cdna(不含polya尾巴)
<400>4
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aggtgagcacgaaggcaagttgtacgatgccaggatcaaagtcacgaggacaatgagtca1200
caagattgtgcactttagtgccgcgggagccaacttctggaaaggcttcgacagatgctt1260
tctcgcataccgtagtgacaatcgcgagcatacatgctattcagggctagatgtcactga1320
gtgcggcgaggtggcagcactgatgtgtttggctatgttcccatgcggaaagataacctg1380
ccctgactgtgtaacagatagtgagctatcccaaggacaagcaagcggaccatctatgaa1440
gcacaggttgacacagctacgcgatgtcatcaagtcaagctacccacgcttcaagcatgc1500
agtgcagatactagataggtatgagcaatcactgagcagtgcaaacgagaactaccaaga1560
tttcgcagaaatccagagcataagcgatggagttgaaaaagctgcattcccacacgtcaa1620
caagctaaacgcaatattgatcaaaggggccacagtaacaggagaggaattctcgcaggc1680
tacgaagcacttgctcgagatagcacgatacctgaagaacagaaccgagaacattgagaa1740
gggttcactgaagtcctttcgcaacaagatttcccagaaagcgcacatcaacccaacact1800
aatgtgtgacaaccagctcgatagaaatggaaatttcatatggggtgagagaggatacca1860
tgcaaaacgattcttcagcaactactttgaaataatcgatccaaagaaaggctacaccca1920
atacgagacaagagcggtaccaaatgggtcacggaaacttgcaatcggcaaactaatagt1980
cccaacgaacttcgaagttttaagggaacagatgaaaggcgaaccggtagaaccataccc2040
agtaacagtcgagtgtgtgagcaagttacagggtgacttcgtccatgcatgttgttgtgt2100
cacaacagaatcaggcgacccagtcttgtctgagatcaaaatgccaaccaaacaccatct2160
agtgattggtaacagcggtgatccaaagtacatagatctccctgagatcgaggagaataa2220
aatgtacatagcgaaagaaggttattgttacatcaatatcttcctagccatgttggtaaa2280
tgtcaaggagtcgcaggcaaaggagttcacgaaagttgttagggacaaactagttggcga2340
acttggcaagtggcccactctgttagatgtagcaaccgcttgttatttcctgaaagtatt2400
ttacccagacgttgctaacgccgaattgccacgcatgctagtggaccataagacaaagat2460
aattcatgtcgttgattcatatgggtcactgtcaactggatatcatgtccttaagacaaa2520
cactgtggaacaactcatcaaattcacgagatgtaatttggagtcaagcttgaaacacta2580
ccgcgttggaggaacagaatgggaggacactcatggatccagcaacatagataatccaca2640
gtggtgcatcaagaggctcataaaaggagtctacaaaccaaagcaactgaaagaagacat2700
gttggcaaaccctttcttaccactatatgctctactgtcaccaggtgtcatcctggcatt2760
ttacaatagtggctctctagagtacttgatgaaccattacatcagggtggacagcaacgt2820
cgccgttttgttggtcgttttgaaatctctagcgaagaaggtgtcaactagtcagagtgt2880
gttagcccagcttcaaatcattgaacgaagtctaccagaactcatcgaagcaaaggctaa2940
tgttaatgggccagatgacgcagccactcgcgcgtgtaacagattcatgggcatgcttct3000
gcatatggcagaaccaaactgggagcttgcggatggtggatacacaattctgagggatca3060
tagcatctccattttggaaaaaagttatctacaaatcttggacgaagcatggaacgagtt3120
aagttggtcggagcgctgtgctataagatactactcgtcaaagcaagcaatctttacaca3180
gaaagatttgccaatgaaaagcgaagccgatttaggcggcagatacagcgtgtcagtcat3240
gtcatcttacgaacggagtaagcaatgtatgaaaagcgtgcactctagtataggtaatag3300
attacgtagtagtatgtcttggactagtagcaaggtgtcgaatagtgtgtgtaggactat3360
taactatttagtaccagatgtgttcaagtttatgaatgtactcgtttgtatcagcttact3420
aatcaagatgactgccgaggcgaatcacatcgtcaccacgcaaagaaggctcaaactaga3480
tgtcgaggagacagagcgcaggaaaatagaatgggagcttgcattccaccatgccattct3540
gacgcagagtgcaggtcaacacccaacgatagacgagttcagagcgtacatcgccgacaa3600
ggcaccacatctaagtgagcatatcgagcctgaagaaaaggcggtggttcatcaagcgaa3660
gagacaatccgagcaagaactcgagcgtataatagcatttgttgcattggtgctcatgat3720
gttcgatgcagaacgaagcgactgtgtcacaaagattctcaacaagcttaagggactagt3780
cgccactgtggaacctacagtctaccatcagactctcaatgatatagaggatgacttgag3840
tgagaggaacctcttcgtcgattttgagcttagcagcgatggagatatgctccaacagct3900
tccagccgaaaagacatttgcctcatggtggagtcatcaactaagcagaggattcacaat3960
cccacactacaggacagaagggaagttcatgactttcaccagagcaactgccacggaagt4020
cgcgggtaaaatagcacacgagagtgacaaagacatattactaatgggagcagtaggatc4080
aggtaagtcaactggcttgccatatcatctctccagaaaagggaacgtattactccttga4140
gccgactcggccacttgcagaaaacgtacacaagcagttgtcgcaggcaccgttccatca4200
gaacacaactcttaggatgcgcggactaacagcattcgggtcggcaccaatctcagtgat4260
gaccagtggttttgcactcaattactttgcaaacaacagaatgcgaattgaagaatttga4320
ctttgtcatatttgatgaatgtcacgttcatgacgccaatgcaatggcgatgagatgttt4380
gctacatgagtgtgactattctggcaaaattatcaaagtttcagccacaccaccaggtcg4440
agaagttgagttctccactcaataccccgtgtcgataagcacagaagacacactatcgtt4500
tcaggattttgtgaacgcacagggtagtggaagcaattgtgatgtgatttcaaaaggaga4560
caatatcctcgtgtatgtagcaagctacaatgaggtagacgcgctttcaaaacttctaat4620
tgaaagagacttcaaagtcacgaaggttgatggaagaacgatgaaagttggaaacatcga4680
gatcaccacaagtggaacacctagtaagaagcacttcatagttgcaaccaacatcataga4740
gaacggtgttactctagacatcgatgtggttgctgattttggaacgaaggtactcccata4800
tcttgatacagacagcagaatgctgagcacaactaagacaagcatcaattatggggaacg4860
tatccaaaggctaggaagagtcggaaggcacaagccaggtcacgctctgcgaataggtca4920
cacagagaaggggttgagcgaagttccaagttgtattgcaacagaagcagctttaaagtg4980
cttcacttatgggcttccagtgatcaccaacaacgtctcgacaagtattcttggtaatgt5040
aacggtaaagcaggcacgaacaatgtctgtatttgagataacaccgttctacacaagcca5100
agtggtgagatatgatggctccatgcatccacaggtgcacgcactcttaaagagattcaa5160
actcagagactctgagattgttttgaataaattagccatacctcaccgaggagtgaacgc5220
ttggctcacagctagtgagtatgcacgacttggcgcgaatgttgaagataggcgtgacgt5280
tcgaattccttttatgtgtcgcgacatcccagaaaaacttcatctagacatgtgggatgt5340
gattgttaaattcaaaggtgatgcaggttttggtcggctttcaagcgccagtgcgagcaa5400
ggtagcttatactctacagacggacgtcaactccatacagcgaacagtcactatcataga5460
tacactaatcgctgaggagagaaggaagcaggaatacttcaagacggtaacctccaactg5520
tgtctcttcttcgaacttctcactgcagagcataacaaatgcgataaaatctcgtatgat5580
gaaagatcacacgtgcgagaacatatcagtgcttgaaggagcgaagtcacagttactcga5640
gtttagaaacctgaatgctgatcactcatttgctacaaaaaccgatggaatatctcggca5700
tttcatgagtgagtatggagctcttgaggcagttcaccatcaaaacaccagcgacatgag5760
caaattcctcaagcttaagggcaaatggaataaaacgctaatcacgcgagatgtgctggt5820
actttgtggagttcttggaggtggattgtggatggttattcagcacctgcggtcaaagat5880
gtccgaacccgtaacccatgaagcgaaaggtaagaggcaaaggcagaaactaaaatttcg5940
caatgcccgagacaacaaaatgggtagagaagtgtacggagatgatgataccatagagca6000
tttcttcggtgatgcctacacaaagaaagggaagagcaagggtaggacacgtggtatcgg6060
acacaaaaacaggaagttcatcaacatgtatgggtttgatcctgaagatttctctgcagt6120
tcgtttcgtggatccactcacaggagcgacgttggacgacaacccgctcacagacatcac6180
ccttgtgcaagagcacttcggcaacataagaatggacttactcggggaggatgagctgga6240
ctcaaatgaaatacgtgtgaataagactattcaagcctactacatgaacaataaaacagg6300
caaggctttgaaggtggatctgacaccacacatacctctcaaggtgtgtgatcttcacgc6360
aaccattgctggattcccagagcgagaaaacgagctgaggcagactggaaaggctcagcc6420
catcaacatagacgaagtgccaagagctaacaacgaactcgtcccagtggaccacgagag6480
taactccatgttcagagggttgcgtgactacaacccaatatcaaacaacatttgtcatct6540
cacaaatgtttcagatggagcatcaaactcgttatatggagtcggtttcggaccactcat6600
attaacgaaccgacacctctttgagcggaataacggtgaactcgtaataaaatcacgaca6660
tggtgagttcgtgattaaaaacacaactcagctacacttgctaccgattccagacagaga6720
tcttctgctaatccggttaccaaaggacgtcccaccctttccacagaaattgggtttcag6780
gcaacctgagaaaggtgaacgaatttgcatggtggggtccaatttccaaaccaagagcat6840
aacgagtatagtctctgagactagtacaataatgccagtggagaacagtcagttttggaa6900
acactggattagcactaaagacggccaatgcggaagtccaatggtgagcacgaaagacgg6960
gaaaatactcggattacacagcctagcgaacttccagaactccatcaattactttgctgc7020
tttcccagatgattttgccgagaagtatcttcataccattgaagcacacgagtgggtcaa7080
gcactggaagtataacactagcgccatcagttggggctctttgaatatacaagcatcgca7140
accgtccggcttgttcaaagtaagcaagctaatctcagacctcgacagcacggcagtcta7200
cgcacaaacccagcagaatcggtggatgttcgagcagctcaacgggaacctaaaagcgat7260
agcacactgccctagccagcttgtgacaaagcacacagttaaaggaaaatgtcagatgtt7320
tgacttgtatctcaagttgcatgatgaagcacgagagtatttccaaccgatgctgggcca7380
gtatcaaaagagcaaactcaatcgagaagcatatgcaaaggatcttctgaaatatgcaac7440
gccaatcgaagcaggaaacatcgactgtgatctgtttgaaaagacagttgaaatagtcgt7500
atcagatctgcgaggttatggtttcgaaacatgcaattatgtcactgatgagaatgacat7560
attcgaagctcttaacatgaaatccgcagttggagcgttgtataaaggaaagaagaagga7620
ttacttcgctgagttcacacccgagatgaaagaagaaatactgaaacaaagttgtgaacg7680
gctcttcctaggaaagatgggagtgtggaacggctcgctgaaggcagagttgcgaccact7740
agaaaaagtggaagcaaacaaaacacggacgtttactgccgcaccactagacacactgtt7800
gggtggaaaagtttgcgtggatgatttcaacaaccagttctatgatcacaaccttagagc7860
tccttggagcgttggcatgacaaagttttattgtggttgggatcgcttgttggagtcgtt7920
gccagatggttgggtgtattgcgatgctgatggctcacagttcgacagctcgctatcgcc7980
atacttgatcaacgcagtactcaacatccgcttaggattcatggaagagtgggacatagg8040
ggaggtaatgctgagaaatttgtacaccgaaatcgtgtatacccctatttctacaccaga8100
tggtacactcgtcaagaagttcaaaggaaacaatagcggacagccatcgactgttgtgga8160
caacacgctcatggtcatattggcagtcaactattcactcaagaaaagcggaattccaag8220
tgagttgcgcgacagcatcatcagattcttcgtcaacggagatgatttactgctaagcgt8280
acacccagagtatgagtatattcttgacactatggcagacaactttcgtgaactgggcct8340
gaagtatactttcgactcaagaaccagggaaaaaggagacctctggtttatgtcgcacca8400
ggggcacaaaagagagggaatctggattcccaagctcgagccagagcgaatagtatcgat8460
tctagaatgggatcggtcgaaagagccatgccatcgactagaggcaatctgcgcagcgat8520
gattgagtcgtggggatacgacaagttaactcacgagatacgcaagttctacgcgtggat8580
gattgaacaagctccatttagctccctagcacaagaagggaaagctccttacatagcgga8640
aacagcgctgaggaagctctaccttgataaggaaccagctcaagaggatctcacccatta8700
tttgcaagcaatctttgaggattatgaagatggtgctgaggcttgtgtttatcaccaggc8760
aggtgaaacgcttgatgcaggtttgacagacgagcaaaagcaggcagagaaggagaagaa8820
ggagagagagaaggcagaaaaggaacgagagaggcaaaagcagttggcactcaagaaagg8880
caaggatgttgcacaagaagagggaaaacgcgacaaggaagtaaacgctggaacctctgg8940
aactttcagtgtacccagactcaagagtctgacaagcaagatgcgcgtgccaagatacga9000
gaaaagagtggctctaaacctcgatcatctaatcctatacacgccggagcagacggatct9060
atccaacacacgttcaacgcgaaagcagtttgacacatggtttgaaggtgtaatggctga9120
ttacgaactgacggaggacaaaatgcaaatcattctcaatggtttaatggtctggtgcat9180
tgagaacggaaccttcccgaacataaacggaatgtgggtgatgatggacggcgacgatca9240
ggtggaattcccgatcaaaccgctcattgaccacgccaaacccacatttaggcagataat9300
ggcccatttcagtgacgtagctgaagcgtacattgaaaagcgtaaccaagaccgaccata9360
catgccacgatatggtcttcagcgcaatttaaccgacatgagcttagctcgatacgcatt9420
tgatttctatgaaatgacttctaggactccaatacgtgcgagagaggcacacatccagat9480
gaaagcagcagcactgcgtggcgcaaataataatttgttcggcttggatggaaacgttgg9540
tacaacggtagagaacacggagaggcatacgaccgaggacgttaatcggaacatgcataa9600
cttactgggcgttcaggggttgtgaagttgtatgctggtagactataagtatttaagttt9660
actcgttagtattctcgcttatgggaaatatgtaagtttgttaaagcagccagtgtgact9720
ttgtcatgtgtgttgttgttactttctgtattttcgccgaacattttattggtgttagcg9780
catgtagtgaggatcgtcctcgattgccttaacatttgataggatgcaagggac9834
<210>5
<211>6443
<212>dna
<213>马铃薯x病毒的全长cdna(不含polya尾巴)
<400>5
gaaaactaaaccatacaccaccaacacaaccaaacccaccacgcccaattgttacacacc60
cgcttgaaaaagaaagtttaacaaatggccaaggtgcgcgaggtttaccaatcttttaca120
gactccaccacaaaaactctcatccaagatgaggcttatagaaacattcgccccatcatg180
gaaaaacacaaactagctaacccttacgctcaaacggttgaagcggctaatgatctagag240
gggttcggcatagccaccaatccctatagcattgaattgcatacacatgcagccgctaag300
accatagagaataaacttctagaggtgcttggttccatcctaccacaagaacctgttaca360
tttatgtttcttaaacccagaaagctaaactacatgagaagaaacccgcggatcaaggac420
attttccaaaatgttgccattgaaccaagagacgtagccaggtaccccaaggaaacaata480
attgacaaactcacagagatcacaacggaaacagcatacattagtgacactctgcacttc540
ttggatccgagctacatagtggagacattccaaaactgcccaaaattgcaaacattgtat600
gcgaccttagttctccccgttgaggcagcctttaaaatggaaagcactcacccgaacata660
tacagcctcaaatacttcggagatggtttccagtatataccaggcaaccatggtggcggg720
gcataccatcatgaattcgctcatctacaatggctcaaagtgggaaagatcaagtggagg780
gaccccaaggatagctttctcggacatctcaattacacgactgagcaggttgagatgcac840
acagtgacagtacagttgcaggaatcgttcgcggcaaaccacttgtactgcatcaggaga900
ggagacttgctcacaccggaggtgcgcactttcggccaacctgacaggtacgtgattcca960
ccacagatcttcctcccaaaagttcacaactgcaagaagccgattctcaagaaaactatg1020
atgcagctcttcttgtatgttaggacagtcaaggtcgcaaaaaattgtgacatttttgcc1080
aaagtcagacaattaattaaatcatctgacttggacaaatactctgctgtggaactggtt1140
tacttagtaagctacatggagttccttgccgatttacaagctaccacctgcttctcagac1200
acactttctggtggcttgctaacaaagacccttgcaccggtgagggcttggatacaagag1260
aaaaagatgcagctgtttggtcttgaggactacgcgaagttagtcaaagcagttgatttc1320
cacccggtggatttttctttcaaagtggaaacttgggacttcagattccaccccttgcaa1380
gcgtggaaagccttccgaccaagggaagtgtcggatgtagaggaaatggaaagtttgttc1440
tcagatggggacttgcttgattgcttcacaagaatgccagcttatgcggtaaacgcagag1500
gaagatttagctgcaatcaggaaaacgcccgagatggatgtcggtcaagaagttaaagaa1560
cctgcaggagacagaaatcaatactcaaaccctgcagaaactttcctcaacaagctccac1620
aggaaacacagtagggaggtgaaacaccaggccgcaaagaaagctaaacgcctagctgaa1680
atccaggagtcaatgagagctgaaggtgatgccgaaccaaatgaaataagcgggggcatg1740
ggggcaatacccagcaacgccgaacttcctggcacgaatgatgccagacaagaactcaca1800
ctcccaaccactaaacctgtccctgcaaggtgggaagatgcttcattcacagattctagt1860
gtggaagaggagcaggttaaaccccttggaaaagaaaccgttgaaacagcgacgcaacaa1920
gtcatcgaaggacttccttggaaacactggattcctcaattaaatgctgttggattcaag1980
gcgctggaaattcagagggataggagtggaacaatgatcatgcccatcacagaaatggtc2040
tccgggctggaaaaagaggacttccctgaaggaactccaaaagagttggcacgagaattg2100
ttcgctatgaacagaagccctgccaccatccctttggacctgcttagagccagagactac2160
ggcagtgatgtaaagaacaagagaattggtgccatcacaaagacacaggcaacgagttgg2220
ggcgaatacttgacaggaaagatagaaagcttgactgagaggaaagttgcgacttgtgtc2280
attcatggagctggaggttctggaaaaagtcatgccatccagaaggcattgagagaaatt2340
ggcaagggctcggacatcactgtagtcctgccgaccaatgaactgcggctagattggagt2400
aagaaagtgcctaacactgagccctatatgttcaagacctatgaaaaggcgttaattggg2460
ggaacaggcagcatagtcatctttgacgattactcaaaacttcctcccggttacatagaa2520
gccttagtctgtttctactctaaaatcaagctaatcattctaacaggagatagcagacaa2580
agcgtctaccatgaaactgctgaggacgcctccatcaggcatttgggaccagcaacagag2640
tacttctcaaaatactgccgatactatctcaatgccacacaccgcaacaagaaagatctt2700
gcgaacatgcttggtgtctacagtgagagaacgggagtcaccgaaatcagcatgagcgcc2760
gagttcttagaaggaatcccaactttggtaccctcggatgagaagagaaagctgtacatg2820
ggcaccgggaggaatgacacgttcacatacgctggatgccaggggctaactaagccgaag2880
gtacaaatagtgttggaccacaacacccaagtgtgtagcgcgaatgtgatgtacacggca2940
ctttctagagccaccgataggattcacttcgtgaacacaagtgcaaattcctcggccttc3000
tgggaaaagttggacagcaccccttacctcaagactttcctatcagtggtgagagaacaa3060
gcactcagggagtacgagccggcagaggcagagccaattcaagagcctgagccccagaca3120
cacatgtgtgtcgagaatgaggagtccgtgctagaagagtacaaagaggaactcttggaa3180
aagtttgacagagagatccactctgaatcccatggtcattcaaactgtgtccaaactgaa3240
gacacaaccattcagttgttttcgcatcaacaagcaaaagatgagaccctcctctgggcg3300
actatagatgcgcggctcaagaccagcaatcaagagacaaacttccgagaattcctgagc3360
aagaaggacattggggacgttctgtttttaaactaccaaaaagctatgggtttacccaaa3420
gagcgtattcctttttcccaagaggtctgggaagcttgtgcccacgaagtacaaagcaag3480
tacctcagcaagtcaaagtgcaacttgatcaatgggactgtgagacagagcccagacttc3540
gatgaaaataagattatggtattcctcaagtcgcagtgggtcacaaaggtggaaaaacta3600
ggtctacccaagattaagccaggtcaaaccatagcagccttttaccagcagactgtgatg3660
ctttttggaactatggctaggtacatgcgatggttcagacaggctttccagccaaaagaa3720
gtcttcataaactgtgagaccacgccagatgacatgtctgcatgggccttgaacaactgg3780
aatttcagcagacctagcttggctaatgactacacagctttcgaccagtctcaggatgga3840
gccatgttgcaatttgaggtgctcaaagccaaacaccactgcataccagaggaaatcatt3900
caggcatacatagatattaagactaatgcacagattttcctaggcacgttatcaattatg3960
cgcctgactggtgaaggtcccacttttgatgcaaacactgagtgcaacatagcttacacc4020
catacaaagtttgacatcccagccggaactgctcaagtttatgcaggagacgactccgca4080
ctggactgtgttccagaagtgaagcatagtttccacaggcttgaggacaaattactccta4140
aagtcaaagcctgtaatcacgcagcaaaagaagggcagttggcctgagttttgtggttgg4200
ctgatcacaccaaaaggggtgatgaaagacccaattaagctccatgttagcttaaaattg4260
gctgaagctaagggtgaactcaagaaatgtcaagattcctatgaaattgatctgagttat4320
gcctatgaccacaaggactctctgcatgacttgttcgatgagaaacagtgtcaggcacac4380
acactcacttgcagaacactaatcaagtcagggagaggcactgtctcactttcccgcctc4440
agaaactttctttaaccgttaagttaccttagagatttgaataagatggatattctcatc4500
agtagtttgaaaagtttaggttattctaggacttccaaatctttagattcaggacctttg4560
gtagtacatgcagtagccggagccggtaagtccacagccctaaggaagttgatcctcaga4620
cacccaacattcaccgtgcatacactcggtgtccctgacaaggtgagtatcagaactaga4680
ggcatacagaagccaggacctattcctgagggcaacttcgcaatcctcgatgagtatact4740
ttggacaacaccacaaggaactcataccaggcactttttgctgacccttatcaggcaccg4800
gagtttagcctagagccccacttctacttggaaacatcatttcgagttccgaggaaagtg4860
gcagatttgatagctggctgtggcttcgatttcgagaccaactcaccggaagaagggcac4920
ttagagatcactggcatattcaaagggcccctactcggaaaggtgatagccattgatgag4980
gagtctgagacaacactgtccaggcatggtgttgagtttgttaagccctgccaagtgacg5040
ggacttgagttcaaagtagtcactattgtgtctgccgcaccaatagaggaaattggccag5100
tccacagctttctacaacgctatcaccaggtcaaagggattgacatatgtccgcgcaggg5160
ccataggctgaccgctccggtcaattctgaaaaagtgtacatagtattaggtctatcatt5220
tgctttagtttcaattacctttctgctttctagaaatagcttaccccacgtcggtgacaa5280
cattcacagcttgccacacggaggagcttacagagacggcaccaaagcaatcttgtacaa5340
ctccccaaatctagggtcacgagtgagtctacacaacggaaagaacgcagcatttgctgc5400
cgttttgctactgactttgctgatctatggaagtaaatacatatctcaacgcaatcatac5460
ttgtgcttgtggtaacaatcatagcagtcattagcacttccttagtgaggactgaacctt5520
gtgtcatcaagattactggggaatcaatcacagtgttggcttgcaaactagatgcagaaa5580
ccataagggccattgccgatctcaagccactctccgttgaacggttaagtttccattgat5640
actcgaaagaggtcagcaccagctagcatgcccgcgagcacaacacagcccatagggtca5700
actacctcaactaccacaaaaactgcaggcgcaactcctgccacagcttcaggcctgttc5760
actatcccggatggggatttctttagtacagcccgtgccatagtagccagcaatgctgtc5820
gcaacaaatgaggacctcagcaagattgaggctatttggaaggacatgaaggtgcccaca5880
gacactatggcacaggctgcttgggacttagtcagacactgtgctgatgtaggatcatcc5940
gctcaaacagaaatgatagatacaggtccctattccaacggcatcagcagagctagactg6000
gcagcagcaattaaagaggtgtgcacacttaggcaattttgcatgaagtatgccccagtg6060
gtatggaactggatgttaactaacaacagtccacctgctaactggcaagcacaaggtttc6120
aagcctgagcacaaattcgctgcattcgacttcttcaatggagtcaccaacccagctgcc6180
atcatgcccaaagaggggctcatccggccaccgtctgaagctgaaatgaatgctgcccaa6240
actgctgcctttgtgaagattacaaaggccagggcacaatccaacgactttgccagccta6300
gatgcagctgtcactcgaggtcgtatcactggaacaacaaccgctgaggctgttgtcact6360
ctaccaccaccataactacgtctacataaccgacgcctaccccagtttcatagtattttc6420
tggtttgattgtatgaataatat6443
<210>6
<211>2477
<212>dna
<213>雌激素诱导型启动子xve
<400>6
tagtttaaactgaaggcgggaaacgacaatctgatccaagctcaagctaagcttgcatgc60
ctgcaggatatcgtggatccaagcttgccacgtgccgccacgtgccgccacgtgccgcca120
cgtgcctctagaggatccatctccactgacgtaagggatgacgcacaatcccactatcct180
tcgcaagacccttcctctatataaggaagttcatttcatttggagaggacacgctgggat240
ccccaattccgggcggaatgaaagcgttaacggccaggcaacaagaggtgtttgatctca300
tccgtgatcacatcagccagacaggtatgccgccgacgcgtgcggaaatcgcgcagcgtt360
tggggttccgttccccaaacgcggctgaagaacatctgaaggcgctggcacgcaaaggcg420
ttattgaaattgtttccggcgcatcacgcgggattcgtctgttgcaggaagaggaagaag480
ggttgccgctggtaggtcgtgtggctgccggtgaaccgtcgagcgcccccccgaccgatg540
tcagcctgggggacgagctccacttagacggcgaggacgtggcgatggcgcatgccgacg600
cgctagacgatttcgatctggacatgttgggggacggggattccccgggtccgggattta660
ccccccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcaga720
tgtttaccgatgcccttggaattgacgagtacggtggggatccgtctgctggagacatga780
gagctgccaacctttggccaagcccgctcatgatcaaacgctctaagaagaacagcctgg840
ccttgtccctgacggccgaccagatggtcagtgccttgttggatgctgagccccccatac900
tctattccgagtatgatcctaccagacccttcagtgaagcttcgatgatgggcttactga960
ccaacctggcagacagggagctggttcacatgatcaactgggcgaagagggtgccaggct1020
ttgtggatttgaccctccatgatcaggtccaccttctagaatgtgcctggctagagatcc1080
tgatgattggtctcgtctggcgctccatggagcacccagtgaagctactgtttgctccta1140
acttgctcttggacaggaaccagggaaaatgtgtagagggcatggtggagatcttcgaca1200
tgctgctggctacatcatctcggttccgcatgatgaatctgcagggagaggagtttgtgt1260
gcctcaaatctattattttgcttaattctggagtgtacacatttctgtccagcaccctga1320
agtctctggaagagaaggaccatatccaccgagtcctggacaagatcacagacactttga1380
tccacctgatggccaaggcaggcctgaccctgcagcagcagcaccagcggctggcccagc1440
tcctcctcatcctctcccacatcaggcacatgagtaacaaaggcatggagcatctgtaca1500
gcatgaagtgcaagaacgtggtgcccctctatgacctgctgctggagatgctggacgccc1560
accgcctacatgcgcccactagccgtggaggggcatccgtggaggagacggaccaaagcc1620
acttggccactgcgggctctacttcatcgcattccttgcaaaagtattacatcacggggg1680
aggcagagggtttccctgccacagtctgagagctccctggcgaattcccagagatgttag1740
ctgaaatcatcactaatcagataccaaaatattcaaatggaaatatcaaaaagcttctgt1800
ttcatcaaaaatgactcgacctaactgagtaagctagcttgttcgagtattatggcattg1860
ggaaaactgtttttcttgtaccatttgttgtgcttgtaatttactgtgttttttattcgg1920
ttttcgctatcgaactgtgaaatggaaatggatggagaagagttaatgaatgatatggtc1980
cttttgttcattctcaaattaatattatttgttttttctcttatttgttgtgtgttgaat2040
ttgaaattataagagatatgcaaacattttgttttgagtaaaaatgtgtcaaatcgtggc2100
ctctaatgaccgaagttaatatgaggagtaaaacatcccaaacagcttgggctgcaggtc2160
gaggctaaaaaactaatcgcattatcatcccctcgacgtactgtacatataaccactggt2220
tttatatacagcagtactgtacatataaccactggttttatatacagcagtcgacgtact2280
gtacatataaccactggttttatatacagcagtactgtacatataaccactggttttata2340
tacagcagtcgaggtaagattagatatggatatgtatatggatatgtatatggtggtaat2400
gccatgtaatatgctcgactctaggatcttcgcaagacccttcctctatataaggaagtt2460
catttcatttggagagg2477
<210>7
<211>1637
<212>dna
<213>潮霉素基因
<400>7
aggcgggaaacgacaatctgatcatgagcggagaattaagggagtcacgttatgaccccc60
gccgatgacgcgggacaagccgttttacgtttggaactgacagaaccgcaacgattgaag120
gagccactcagccgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccat180
tattgcgcgttcaaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaa240
atgctccactgacgttccataaattcccctcggtatccaattagagtctcatattcactc300
tcaatccaaataatctgcaccggatcccctagaatgaaaaagcctgaactcaccgcgacg360
tctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcg420
gagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgg480
gtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttgcatcg540
gccgcgctcccgattccggaagtgcttgacattggggaattcagcgagagcctgacctat600
tgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgccc660
gctgttctgcagccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccag720
acgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgat780
ttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacacc840
gtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgcccc900
gaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggc960
cgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtc1020
gccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttc1080
gagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcatt1140
ggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcg1200
cagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatc1260
gcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtgga1320
aaccgacgccccagcactcgtccgagggcaaaggaatagcgatcgttcaaacatttggca1380
ataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttct1440
gttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatg1500
ggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatata1560
gcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcggggaat1620
tgatcccccctcgacag1637
<210>8
<211>2143
<212>dna
<213>诱导型启动子xve-1
<400>8
tagtttaaactgaaggcgggaaacgacaatctgatccaagctcaagctaagcttgcatgc60
ctgcaggatatcgtggatccaagcttgccacgtgccgccacgtgccgccacgtgccgcca120
cgtgcctctagaggatccatctccactgacgtaagggatgacgcacaatcccactatcct180
tcgcaagacccttcctctatataaggaagttcatttcatttggagaggacacgctgggat240
ccccaattccgggcggaatgaaagcgttaacggccaggcaacaagaggtgtttgatctca300
tccgtgatcacatcagccagacaggtatgccgccgacgcgtgcggaaatcgcgcagcgtt360
tggggttccgttccccaaacgcggctgaagaacatctgaaggcgctggcacgcaaaggcg420
ttattgaaattgtttccggcgcatcacgcgggattcgtctgttgcaggaagaggaagaag480
ggttgccgctggtaggtcgtgtggctgccggtgaaccgtcgagcgcccccccgaccgatg540
tcagcctgggggacgagctccacttagacggcgaggacgtggcgatggcgcatgccgacg600
cgctagacgatttcgatctggacatgttgggggacggggattccccgggtccgggattta660
ccccccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcaga720
tgtttaccgatgcccttggaattgacgagtacggtggggatccgtctgctggagacatga780
gagctgccaacctttggccaagcccgctcatgatcaaacgctctaagaagaacagcctgg840
ccttgtccctgacggccgaccagatggtcagtgccttgttggatgctgagccccccatac900
tctattccgagtatgatcctaccagacccttcagtgaagcttcgatgatgggcttactga960
ccaacctggcagacagggagctggttcacatgatcaactgggcgaagagggtgccaggct1020
ttgtggatttgaccctccatgatcaggtccaccttctagaatgtgcctggctagagatcc1080
tgatgattggtctcgtctggcgctccatggagcacccagtgaagctactgtttgctccta1140
acttgctcttggacaggaaccagggaaaatgtgtagagggcatggtggagatcttcgaca1200
tgctgctggctacatcatctcggttccgcatgatgaatctgcagggagaggagtttgtgt1260
gcctcaaatctattattttgcttaattctggagtgtacacatttctgtccagcaccctga1320
agtctctggaagagaaggaccatatccaccgagtcctggacaagatcacagacactttga1380
tccacctgatggccaaggcaggcctgaccctgcagcagcagcaccagcggctggcccagc1440
tcctcctcatcctctcccacatcaggcacatgagtaacaaaggcatggagcatctgtaca1500
gcatgaagtgcaagaacgtggtgcccctctatgacctgctgctggagatgctggacgccc1560
accgcctacatgcgcccactagccgtggaggggcatccgtggaggagacggaccaaagcc1620
acttggccactgcgggctctacttcatcgcattccttgcaaaagtattacatcacggggg1680
aggcagagggtttccctgccacagtctgagagctccctggcgaattcccagagatgttag1740
ctgaaatcatcactaatcagataccaaaatattcaaatggaaatatcaaaaagcttctgt1800
ttcatcaaaaatgactcgacctaactgagtaagctagcttgttcgagtattatggcattg1860
ggaaaactgtttttcttgtaccatttgttgtgcttgtaatttactgtgttttttattcgg1920
ttttcgctatcgaactgtgaaatggaaatggatggagaagagttaatgaatgatatggtc1980
cttttgttcattctcaaattaatattatttgttttttctcttatttgttgtgtgttgaat2040
ttgaaattataagagatatgcaaacattttgttttgagtaaaaatgtgtcaaatcgtggc2100
ctctaatgaccgaagttaatatgaggagtaaaacatcccaaac2143
<210>9
<211>334
<212>dna
<213>雌激素诱导型启动子xve-2
<400>9
agcttgggctgcaggtcgaggctaaaaaactaatcgcattatcatcccctcgacgtactg60
tacatataaccactggttttatatacagcagtactgtacatataaccactggttttatat120
acagcagtcgacgtactgtacatataaccactggttttatatacagcagtactgtacata180
taaccactggttttatatacagcagtcgaggtaagattagatatggatatgtatatggat240
atgtatatggtggtaatgccatgtaatatgctcgactctaggatcttcgcaagacccttc300
ctctatataaggaagttcatttcatttggagagg334
<210>10
<211>480
<212>dna
<213>t3a终止子
<400>10
caggcctcccagctttcgtccgtatcatcggtttcgacaacgttcgtcaagttcaatgca60
tcagtttcattgcccacacaccagaatcctactaagtttgagtattatggcattggaaaa120
gctgttttcttctatcatttgttctgcttgtaatttactgtgttctttcagtttttgttt180
tcggacatcaaaatgcaaatggatggataagagttaataaatgatatggtccttttgttc240
attctcaaattattattatctgttgtttttactttaatgggttgaatttaagtaagaaag300
gaactaacagtgtgatattaaggtgcaatgttagacatataaaacagtctttcacctctc360
tttggttatgtcttgaattggtttgtttcttcacttatctgtgtaatcaagtttactatg420
agtctatgatcaagtaattatgcaatcaagttaagtacagtataggctttttgtgtcgag480
<210>11
<211>10578
<212>dna
<213>tumv-gfp序列(1229-1972nt为gfp基因)
<400>11
aaaaaatataaaaactcaacataacatacacaaaacgattaaagcaaacacaaatctttc60
aaagcattcaagcaatcaaagattctcaaatctttcatcgttatcaaagcaatcaccaac120
agcaaaccaaatggcagcagttacattcgcatcagctatcaccaacgccatcaccagcaa180
accagcactcaccggaatggtgcagtttgggagtttcccaccaatgccattgcgatccac240
caccgtcaccacagtcgccacttcagtggcgcaacctaaactgtacacagtgcagtttgg300
aagccttgacccagtagtcgtcaagagtggagcagggtcccttgctaaggcaacacgcca360
gcagcctaacgttgaaatagacgttagcctcagtgaagccgcagctctggaggttgcgaa420
acctagatcgaatgccgtgttgaggatgcacgaggaagcaaacaaggagagagcactctt480
tttggactgggaggctagtttgaagagaagctcgtatggaattgctgaggacgagaaggt540
tgtaatgacaactcatggcgtcagcaagatagtgcccagaagttcaagggcaatgaagct600
aaagcgcgcaagggagaggcgtagagcgcagcaaccaattatattaaagtgggagcccaa660
attgagcgggatctcaatcggaggagggctctctgcgagcgtaatcgaagcagaagaggt720
tcgcacaaagtggccgcttcataagacaccgtcaatgaagaagaggacggtgcacagaat780
atgcaagatgaacgaccaaggagttgacatgttgacacgatccctggttaagattttcaa840
gactaagagtgccaacattgaatacatcggaaagaagtcgattaaggtcgatttcatcag900
aaaagaacgaacgaaattcgcaagaatccaagtagcacacttactcgggaagagagcaca960
gcgcgacttgttaactggaatggaagaaaaccattttattgacattctcagtaagtactc1020
aggtaacaaaacaaccataaatcctggagtagtttgcgcaggttggagtggcatagtcgt1080
tggaaatggaattctaacccagaaacgaagcagaagtccatcagaggcctttgtaattag1140
aggtgagcacgaaggcaagttgtacgatgccaggatcaaagtcacgaggacaatgagtca1200
caagattgtgcactttagtgccgcgggaatgagtaaaggagaagaacttttcactggagt1260
tgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtgg1320
agagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactgg1380
aaaactacctgttccatggccaacacttgtcactactttctcttatggtgttcaatgctt1440
ttcaagatacccagatcatatgaagcggcacgacttcttcaagagcgccatgcctgaggg1500
atacgtgcaggagaggaccatcttcttcaaggacgacgggaactacaagacacgtgctga1560
agtcaagtttgagggagacaccctcgtcaacaggatcgagcttaagggaatcgatttcaa1620
ggaggacggaaacatcctcggccacaagttggaatacaactacaactcccacaacgtata1680
catcatggccgacaagcaaaagaacggcatcaaagccaacttcaagacccgccacaacat1740
cgaagacggcggcgtgcaactcgctgatcattatcaacaaaatactccaattggcgatgg1800
ccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcc1860
caacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacaca1920
tggcatggatgaactatacaaagcttgtgtctatcaccaggcagccgcgggagccaactt1980
ctggaaaggcttcgacagatgctttctcgcataccgtagtgacaatcgcgagcatacatg2040
ctattcagggctagatgtcactgagtgcggcgaggtggcagcactgatgtgtttggctat2100
gttcccatgcggaaagataacctgccctgactgtgtaacagatagtgagctatcccaagg2160
acaagcaagcggaccatctatgaagcacaggttgacacagctacgcgatgtcatcaagtc2220
aagctacccacgcttcaagcatgcagtgcagatactagataggtatgagcaatcactgag2280
cagtgcaaacgagaactaccaagatttcgcagaaatccagagcataagcgatggagttga2340
aaaagctgcattcccacacgtcaacaagctaaacgcaatattgatcaaaggggccacagt2400
aacaggagaggaattctcgcaggctacgaagcacttgctcgagatagcacgatacctgaa2460
gaacagaaccgagaacattgagaagggttcactgaagtcctttcgcaacaagatttccca2520
gaaagcgcacatcaacccaacactaatgtgtgacaaccagctcgatagaaatggaaattt2580
catatggggtgagagaggataccatgcaaaacgattcttcagcaactactttgaaataat2640
cgatccaaagaaaggctacacccaatacgagacaagagcggtaccaaatgggtcacggaa2700
acttgcaatcggcaaactaatagtcccaacgaacttcgaagttttaagggaacagatgaa2760
aggcgaaccggtagaaccatacccagtaacagtcgagtgtgtgagcaagttacagggtga2820
cttcgtccatgcatgttgttgtgtcacaacagaatcaggcgacccagtcttgtctgagat2880
caaaatgccaaccaaacaccatctagtgattggtaacagcggtgatccaaagtacataga2940
tctccctgagatcgaggagaataaaatgtacatagcgaaagaaggttattgttacatcaa3000
tatcttcctagccatgttggtaaatgtcaaggagtcgcaggcaaaggagttcacgaaagt3060
tgttagggacaaactagttggcgaacttggcaagtggcccactctgttagatgtagcaac3120
cgcttgttatttcctgaaagtattttacccagacgttgctaacgccgaattgccacgcat3180
gctagtggaccataagacaaagataattcatgtcgttgattcatatgggtcactgtcaac3240
tggatatcatgtccttaagacaaacactgtggaacaactcatcaaattcacgagatgtaa3300
tttggagtcaagcttgaaacactaccgcgttggaggaacagaatgggaggacactcatgg3360
atccagcaacatagataatccacagtggtgcatcaagaggctcataaaaggagtctacaa3420
accaaagcaactgaaagaagacatgttggcaaaccctttcttaccactatatgctctact3480
gtcaccaggtgtcatcctggcattttacaatagtggctctctagagtacttgatgaacca3540
ttacatcagggtggacagcaacgtcgccgttttgttggtcgttttgaaatctctagcgaa3600
gaaggtgtcaactagtcagagtgtgttagcccagcttcaaatcattgaacgaagtctacc3660
agaactcatcgaagcaaaggctaatgttaatgggccagatgacgcagccactcgcgcgtg3720
taacagattcatgggcatgcttctgcatatggcagaaccaaactgggagcttgcggatgg3780
tggatacacaattctgagggatcatagcatctccattttggaaaaaagttatctacaaat3840
cttggacgaagcatggaacgagttaagttggtcggagcgctgtgctataagatactactc3900
gtcaaagcaagcaatctttacacagaaagatttgccaatgaaaagcgaagccgatttagg3960
cggcagatacagcgtgtcagtcatgtcatcttacgaacggagtaagcaatgtatgaaaag4020
cgtgcactctagtataggtaatagattacgtagtagtatgtcttggactagtagcaaggt4080
gtcgaatagtgtgtgtaggactattaactatttagtaccagatgtgttcaagtttatgaa4140
tgtactcgtttgtatcagcttactaatcaagatgactgccgaggcgaatcacatcgtcac4200
cacgcaaagaaggctcaaactagatgtcgaggagacagagcgcaggaaaatagaatggga4260
gcttgcattccaccatgccattctgacgcagagtgcaggtcaacacccaacgatagacga4320
gttcagagcgtacatcgccgacaaggcaccacatctaagtgagcatatcgagcctgaaga4380
aaaggcggtggttcatcaagcgaagagacaatccgagcaagaactcgagcgtataatagc4440
atttgttgcattggtgctcatgatgttcgatgcagaacgaagcgactgtgtcacaaagat4500
tctcaacaagcttaagggactagtcgccactgtggaacctacagtctaccatcagactct4560
caatgatatagaggatgacttgagtgagaggaacctcttcgtcgattttgagcttagcag4620
cgatggagatatgctccaacagcttccagccgaaaagacatttgcctcatggtggagtca4680
tcaactaagcagaggattcacaatcccacactacaggacagaagggaagttcatgacttt4740
caccagagcaactgccacggaagtcgcgggtaaaatagcacacgagagtgacaaagacat4800
attactaatgggagcagtaggatcaggtaagtcaactggcttgccatatcatctctccag4860
aaaagggaacgtattactccttgagccgactcggccacttgcagaaaacgtacacaagca4920
gttgtcgcaggcaccgttccatcagaacacaactcttaggatgcgcggactaacagcatt4980
cgggtcggcaccaatctcagtgatgaccagtggttttgcactcaattactttgcaaacaa5040
cagaatgcgaattgaagaatttgactttgtcatatttgatgaatgtcacgttcatgacgc5100
caatgcaatggcgatgagatgtttgctacatgagtgtgactattctggcaaaattatcaa5160
agtttcagccacaccaccaggtcgagaagttgagttctccactcaataccccgtgtcgat5220
aagcacagaagacacactatcgtttcaggattttgtgaacgcacagggtagtggaagcaa5280
ttgtgatgtgatttcaaaaggagacaatatcctcgtgtatgtagcaagctacaatgaggt5340
agacgcgctttcaaaacttctaattgaaagagacttcaaagtcacgaaggttgatggaag5400
aacgatgaaagttggaaacatcgagatcaccacaagtggaacacctagtaagaagcactt5460
catagttgcaaccaacatcatagagaacggtgttactctagacatcgatgtggttgctga5520
ttttggaacgaaggtactcccatatcttgatacagacagcagaatgctgagcacaactaa5580
gacaagcatcaattatggggaacgtatccaaaggctaggaagagtcggaaggcacaagcc5640
aggtcacgctctgcgaataggtcacacagagaaggggttgagcgaagttccaagttgtat5700
tgcaacagaagcagctttaaagtgcttcacttatgggcttccagtgatcaccaacaacgt5760
ctcgacaagtattcttggtaatgtaacggtaaagcaggcacgaacaatgtctgtatttga5820
gataacaccgttctacacaagccaagtggtgagatatgatggctccatgcatccacaggt5880
gcacgcactcttaaagagattcaaactcagagactctgagattgttttgaataaattagc5940
catacctcaccgaggagtgaacgcttggctcacagctagtgagtatgcacgacttggcgc6000
gaatgttgaagataggcgtgacgttcgaattccttttatgtgtcgcgacatcccagaaaa6060
acttcatctagacatgtgggatgtgattgttaaattcaaaggtgatgcaggttttggtcg6120
gctttcaagcgccagtgcgagcaaggtagcttatactctacagacggacgtcaactccat6180
acagcgaacagtcactatcatagatacactaatcgctgaggagagaaggaagcaggaata6240
cttcaagacggtaacctccaactgtgtctcttcttcgaacttctcactgcagagcataac6300
aaatgcgataaaatctcgtatgatgaaagatcacacgtgcgagaacatatcagtgcttga6360
aggagcgaagtcacagttactcgagtttagaaacctgaatgctgatcactcatttgctac6420
aaaaaccgatggaatatctcggcatttcatgagtgagtatggagctcttgaggcagttca6480
ccatcaaaacaccagcgacatgagcaaattcctcaagcttaagggcaaatggaataaaac6540
gctaatcacgcgagatgtgctggtactttgtggagttcttggaggtggattgtggatggt6600
tattcagcacctgcggtcaaagatgtccgaacccgtaacccatgaagcgaaaggtaagag6660
gcaaaggcagaaactaaaatttcgcaatgcccgagacaacaaaatgggtagagaagtgta6720
cggagatgatgataccatagagcatttcttcggtgatgcctacacaaagaaagggaagag6780
caagggtaggacacgtggtatcggacacaaaaacaggaagttcatcaacatgtatgggtt6840
tgatcctgaagatttctctgcagttcgtttcgtggatccactcacaggagcgacgttgga6900
cgacaacccgctcacagacatcacccttgtgcaagagcacttcggcaacataagaatgga6960
cttactcggggaggatgagctggactcaaatgaaatacgtgtgaataagactattcaagc7020
ctactacatgaacaataaaacaggcaaggctttgaaggtggatctgacaccacacatacc7080
tctcaaggtgtgtgatcttcacgcaaccattgctggattcccagagcgagaaaacgagct7140
gaggcagactggaaaggctcagcccatcaacatagacgaagtgccaagagctaacaacga7200
actcgtcccagtggaccacgagagtaactccatgttcagagggttgcgtgactacaaccc7260
aatatcaaacaacatttgtcatctcacaaatgtttcagatggagcatcaaactcgttata7320
tggagtcggtttcggaccactcatattaacgaaccgacacctctttgagcggaataacgg7380
tgaactcgtaataaaatcacgacatggtgagttcgtgattaaaaacacaactcagctaca7440
cttgctaccgattccagacagagatcttctgctaatccggttaccaaaggacgtcccacc7500
ctttccacagaaattgggtttcaggcaacctgagaaaggtgaacgaatttgcatggtggg7560
gtccaatttccaaaccaagagcataacgagtatagtctctgagactagtacaataatgcc7620
agtggagaacagtcagttttggaaacactggattagcactaaagacggccaatgcggaag7680
tccaatggtgagcacgaaagacgggaaaatactcggattacacagcctagcgaacttcca7740
gaactccatcaattactttgctgctttcccagatgattttgccgagaagtatcttcatac7800
cattgaagcacacgagtgggtcaagcactggaagtataacactagcgccatcagttgggg7860
ctctttgaatatacaagcatcgcaaccgtccggcttgttcaaagtaagcaagctaatctc7920
agacctcgacagcacggcagtctacgcacaaacccagcagaatcggtggatgttcgagca7980
gctcaacgggaacctaaaagcgatagcacactgccctagccagcttgtgacaaagcacac8040
agttaaaggaaaatgtcagatgtttgacttgtatctcaagttgcatgatgaagcacgaga8100
gtatttccaaccgatgctgggccagtatcaaaagagcaaactcaatcgagaagcatatgc8160
aaaggatcttctgaaatatgcaacgccaatcgaagcaggaaacatcgactgtgatctgtt8220
tgaaaagacagttgaaatagtcgtatcagatctgcgaggttatggtttcgaaacatgcaa8280
ttatgtcactgatgagaatgacatattcgaagctcttaacatgaaatccgcagttggagc8340
gttgtataaaggaaagaagaaggattacttcgctgagttcacacccgagatgaaagaaga8400
aatactgaaacaaagttgtgaacggctcttcctaggaaagatgggagtgtggaacggctc8460
gctgaaggcagagttgcgaccactagaaaaagtggaagcaaacaaaacacggacgtttac8520
tgccgcaccactagacacactgttgggtggaaaagtttgcgtggatgatttcaacaacca8580
gttctatgatcacaaccttagagctccttggagcgttggcatgacaaagttttattgtgg8640
ttgggatcgcttgttggagtcgttgccagatggttgggtgtattgcgatgctgatggctc8700
acagttcgacagctcgctatcgccatacttgatcaacgcagtactcaacatccgcttagg8760
attcatggaagagtgggacataggggaggtaatgctgagaaatttgtacaccgaaatcgt8820
gtatacccctatttctacaccagatggtacactcgtcaagaagttcaaaggaaacaatag8880
cggacagccatcgactgttgtggacaacacgctcatggtcatattggcagtcaactattc8940
actcaagaaaagcggaattccaagtgagttgcgcgacagcatcatcagattcttcgtcaa9000
cggagatgatttactgctaagcgtacacccagagtatgagtatattcttgacactatggc9060
agacaactttcgtgaactgggcctgaagtatactttcgactcaagaaccagggaaaaagg9120
agacctctggtttatgtcgcaccaggggcacaaaagagagggaatctggattcccaagct9180
cgagccagagcgaatagtatcgattctagaatgggatcggtcgaaagagccatgccatcg9240
actagaggcaatctgcgcagcgatgattgagtcgtggggatacgacaagttaactcacga9300
gatacgcaagttctacgcgtggatgattgaacaagctccatttagctccctagcacaaga9360
agggaaagctccttacatagcggaaacagcgctgaggaagctctaccttgataaggaacc9420
agctcaagaggatctcacccattatttgcaagcaatctttgaggattatgaagatggtgc9480
tgaggcttgtgtttatcaccaggcaggtgaaacgcttgatgcaggtttgacagacgagca9540
aaagcaggcagagaaggagaagaaggagagagagaaggcagaaaaggaacgagagaggca9600
aaagcagttggcactcaagaaaggcaaggatgttgcacaagaagagggaaaacgcgacaa9660
ggaagtaaacgctggaacctctggaactttcagtgtacccagactcaagagtctgacaag9720
caagatgcgcgtgccaagatacgagaaaagagtggctctaaacctcgatcatctaatcct9780
atacacgccggagcagacggatctatccaacacacgttcaacgcgaaagcagtttgacac9840
atggtttgaaggtgtaatggctgattacgaactgacggaggacaaaatgcaaatcattct9900
caatggtttaatggtctggtgcattgagaacggaaccttcccgaacataaacggaatgtg9960
ggtgatgatggacggcgacgatcaggtggaattcccgatcaaaccgctcattgaccacgc10020
caaacccacatttaggcagataatggcccatttcagtgacgtagctgaagcgtacattga10080
aaagcgtaaccaagaccgaccatacatgccacgatatggtcttcagcgcaatttaaccga10140
catgagcttagctcgatacgcatttgatttctatgaaatgacttctaggactccaatacg10200
tgcgagagaggcacacatccagatgaaagcagcagcactgcgtggcgcaaataataattt10260
gttcggcttggatggaaacgttggtacaacggtagagaacacggagaggcatacgaccga10320
ggacgttaatcggaacatgcataacttactgggcgttcaggggttgtgaagttgtatgct10380
ggtagactataagtatttaagtttactcgttagtattctcgcttatgggaaatatgtaag10440
tttgttaaagcagccagtgtgactttgtcatgtgtgttgttgttactttctgtattttcg10500
ccgaacattttattggtgttagcgcatgtagtgaggatcgtcctcgattgccttaacatt10560
tgataggatgcaagggac10578
<210>12
<211>6987
<212>dna
<213>pvx-gfp(5671-6210nt为gfp基因)
<400>12
gaaaactaaaccatacaccaccaacacaaccaaacccaccacgcccaattgttacacacc60
cgcttgaaaaagaaagtttaacaaatggccaaggtgcgcgaggtttaccaatcttttaca120
gactccaccacaaaaactctcatccaagatgaggcttatagaaacattcgccccatcatg180
gaaaaacacaaactagctaacccttacgctcaaacggttgaagcggctaatgatctagag240
gggttcggcatagccaccaatccctatagcattgaattgcatacacatgcagccgctaag300
accatagagaataaacttctagaggtgcttggttccatcctaccacaagaacctgttaca360
tttatgtttcttaaacccagaaagctaaactacatgagaagaaacccgcggatcaaggac420
attttccaaaatgttgccattgaaccaagagacgtagccaggtaccccaaggaaacaata480
attgacaaactcacagagatcacaacggaaacagcatacattagtgacactctgcacttc540
ttggatccgagctacatagtggagacattccaaaactgcccaaaattgcaaacattgtat600
gcgaccttagttctccccgttgaggcagcctttaaaatggaaagcactcacccgaacata660
tacagcctcaaatacttcggagatggtttccagtatataccaggcaaccatggtggcggg720
gcataccatcatgaattcgctcatctacaatggctcaaagtgggaaagatcaagtggagg780
gaccccaaggatagctttctcggacatctcaattacacgactgagcaggttgagatgcac840
acagtgacagtacagttgcaggaatcgttcgcggcaaaccacttgtactgcatcaggaga900
ggagacttgctcacaccggaggtgcgcactttcggccaacctgacaggtacgtgattcca960
ccacagatcttcctcccaaaagttcacaactgcaagaagccgattctcaagaaaactatg1020
atgcagctcttcttgtatgttaggacagtcaaggtcgcaaaaaattgtgacatttttgcc1080
aaagtcagacaattaattaaatcatctgacttggacaaatactctgctgtggaactggtt1140
tacttagtaagctacatggagttccttgccgatttacaagctaccacctgcttctcagac1200
acactttctggtggcttgctaacaaagacccttgcaccggtgagggcttggatacaagag1260
aaaaagatgcagctgtttggtcttgaggactacgcgaagttagtcaaagcagttgatttc1320
cacccggtggatttttctttcaaagtggaaacttgggacttcagattccaccccttgcaa1380
gcgtggaaagccttccgaccaagggaagtgtcggatgtagaggaaatggaaagtttgttc1440
tcagatggggacttgcttgattgcttcacaagaatgccagcttatgcggtaaacgcagag1500
gaagatttagctgcaatcaggaaaacgcccgagatggatgtcggtcaagaagttaaagaa1560
cctgcaggagacagaaatcaatactcaaaccctgcagaaactttcctcaacaagctccac1620
aggaaacacagtagggaggtgaaacaccaggccgcaaagaaagctaaacgcctagctgaa1680
atccaggagtcaatgagagctgaaggtgatgccgaaccaaatgaaataagcgggggcatg1740
ggggcaatacccagcaacgccgaacttcctggcacgaatgatgccagacaagaactcaca1800
ctcccaaccactaaacctgtccctgcaaggtgggaagatgcttcattcacagattctagt1860
gtggaagaggagcaggttaaaccccttggaaaagaaaccgttgaaacagcgacgcaacaa1920
gtcatcgaaggacttccttggaaacactggattcctcaattaaatgctgttggattcaag1980
gcgctggaaattcagagggataggagtggaacaatgatcatgcccatcacagaaatggtc2040
tccgggctggaaaaagaggacttccctgaaggaactccaaaagagttggcacgagaattg2100
ttcgctatgaacagaagccctgccaccatccctttggacctgcttagagccagagactac2160
ggcagtgatgtaaagaacaagagaattggtgccatcacaaagacacaggcaacgagttgg2220
ggcgaatacttgacaggaaagatagaaagcttgactgagaggaaagttgcgacttgtgtc2280
attcatggagctggaggttctggaaaaagtcatgccatccagaaggcattgagagaaatt2340
ggcaagggctcggacatcactgtagtcctgccgaccaatgaactgcggctagattggagt2400
aagaaagtgcctaacactgagccctatatgttcaagacctatgaaaaggcgttaattggg2460
ggaacaggcagcatagtcatctttgacgattactcaaaacttcctcccggttacatagaa2520
gccttagtctgtttctactctaaaatcaagctaatcattctaacaggagatagcagacaa2580
agcgtctaccatgaaactgctgaggacgcctccatcaggcatttgggaccagcaacagag2640
tacttctcaaaatactgccgatactatctcaatgccacacaccgcaacaagaaagatctt2700
gcgaacatgcttggtgtctacagtgagagaacgggagtcaccgaaatcagcatgagcgcc2760
gagttcttagaaggaatcccaactttggtaccctcggatgagaagagaaagctgtacatg2820
ggcaccgggaggaatgacacgttcacatacgctggatgccaggggctaactaagccgaag2880
gtacaaatagtgttggaccacaacacccaagtgtgtagcgcgaatgtgatgtacacggca2940
ctttctagagccaccgataggattcacttcgtgaacacaagtgcaaattcctcggccttc3000
tgggaaaagttggacagcaccccttacctcaagactttcctatcagtggtgagagaacaa3060
gcactcagggagtacgagccggcagaggcagagccaattcaagagcctgagccccagaca3120
cacatgtgtgtcgagaatgaggagtccgtgctagaagagtacaaagaggaactcttggaa3180
aagtttgacagagagatccactctgaatcccatggtcattcaaactgtgtccaaactgaa3240
gacacaaccattcagttgttttcgcatcaacaagcaaaagatgagaccctcctctgggcg3300
actatagatgcgcggctcaagaccagcaatcaagagacaaacttccgagaattcctgagc3360
aagaaggacattggggacgttctgtttttaaactaccaaaaagctatgggtttacccaaa3420
gagcgtattcctttttcccaagaggtctgggaagcttgtgcccacgaagtacaaagcaag3480
tacctcagcaagtcaaagtgcaacttgatcaatgggactgtgagacagagcccagacttc3540
gatgaaaataagattatggtattcctcaagtcgcagtgggtcacaaaggtggaaaaacta3600
ggtctacccaagattaagccaggtcaaaccatagcagccttttaccagcagactgtgatg3660
ctttttggaactatggctaggtacatgcgatggttcagacaggctttccagccaaaagaa3720
gtcttcataaactgtgagaccacgccagatgacatgtctgcatgggccttgaacaactgg3780
aatttcagcagacctagcttggctaatgactacacagctttcgaccagtctcaggatgga3840
gccatgttgcaatttgaggtgctcaaagccaaacaccactgcataccagaggaaatcatt3900
caggcatacatagatattaagactaatgcacagattttcctaggcacgttatcaattatg3960
cgcctgactggtgaaggtcccacttttgatgcaaacactgagtgcaacatagcttacacc4020
catacaaagtttgacatcccagccggaactgctcaagtttatgcaggagacgactccgca4080
ctggactgtgttccagaagtgaagcatagtttccacaggcttgaggacaaattactccta4140
aagtcaaagcctgtaatcacgcagcaaaagaagggcagttggcctgagttttgtggttgg4200
ctgatcacaccaaaaggggtgatgaaagacccaattaagctccatgttagcttaaaattg4260
gctgaagctaagggtgaactcaagaaatgtcaagattcctatgaaattgatctgagttat4320
gcctatgaccacaaggactctctgcatgacttgttcgatgagaaacagtgtcaggcacac4380
acactcacttgcagaacactaatcaagtcagggagaggcactgtctcactttcccgcctc4440
agaaactttctttaaccgttaagttaccttagagatttgaataagatggatattctcatc4500
agtagtttgaaaagtttaggttattctaggacttccaaatctttagattcaggacctttg4560
gtagtacatgcagtagccggagccggtaagtccacagccctaaggaagttgatcctcaga4620
cacccaacattcaccgtgcatacactcggtgtccctgacaaggtgagtatcagaactaga4680
ggcatacagaagccaggacctattcctgagggcaacttcgcaatcctcgatgagtatact4740
ttggacaacaccacaaggaactcataccaggcactttttgctgacccttatcaggcaccg4800
gagtttagcctagagccccacttctacttggaaacatcatttcgagttccgaggaaagtg4860
gcagatttgatagctggctgtggcttcgatttcgagaccaactcaccggaagaagggcac4920
ttagagatcactggcatattcaaagggcccctactcggaaaggtgatagccattgatgag4980
gagtctgagacaacactgtccaggcatggtgttgagtttgttaagccctgccaagtgacg5040
ggacttgagttcaaagtagtcactattgtgtctgccgcaccaatagaggaaattggccag5100
tccacagctttctacaacgctatcaccaggtcaaagggattgacatatgtccgcgcaggg5160
ccataggctgaccgctccggtcaattctgaaaaagtgtacatagtattaggtctatcatt5220
tgctttagtttcaattacctttctgctttctagaaatagcttaccccacgtcggtgacaa5280
cattcacagcttgccacacggaggagcttacagagacggcaccaaagcaatcttgtacaa5340
ctccccaaatctagggtcacgagtgagtctacacaacggaaagaacgcagcatttgctgc5400
cgttttgctactgactttgctgatctatggaagtaaatacatatctcaacgcaatcatac5460
ttgtgcttgtggtaacaatcatagcagtcattagcacttccttagtgaggactgaacctt5520
gtgtcatcaagattactggggaatcaatcacagtgttggcttgcaaactagatgcagaaa5580
ccataagggccattgccgatctcaagccactctccgttgaacggttaagtttccattgat5640
actcgaaagaggtcagcaccagctagcatgaaacggcatgactttttcaagagtgccatg5700
cccgaaggttatgtacaggaaagaactatatttttcaaggatgacgggaactacaagaca5760
cgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtatt5820
gattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacac5880
aatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattaga5940
cacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaatt6000
ggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcg6060
aaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctggg6120
attacacatggcatggatgaactatacaaatccggatctagaaattttgaccttcttaag6180
cttgcgggagacgtcgagtccaaccctgggcccgcgagcacaacacagcccatagggtca6240
actacctcaactaccacaaaaactgcaggcgcaactcctgccacagcttcaggcctgttc6300
actatcccggatggggatttctttagtacagcccgtgccatagtagccagcaatgctgtc6360
gcaacaaatgaggacctcagcaagattgaggctatttggaaggacatgaaggtgcccaca6420
gacactatggcacaggctgcttgggacttagtcagacactgtgctgatgtaggatcatcc6480
gctcaaacagaaatgatagatacaggtccctattccaacggcatcagcagagctagactg6540
gcagcagcaattaaagaggtgtgcacacttaggcaattttgcatgaagtatgccccagtg6600
gtatggaactggatgttaactaacaacagtccacctgctaactggcaagcacaaggtttc6660
aagcctgagcacaaattcgctgcattcgacttcttcaatggagtcaccaacccagctgcc6720
atcatgcccaaagaggggctcatccggccaccgtctgaagctgaaatgaatgctgcccaa6780
actgctgcctttgtgaagattacaaaggccagggcacaatccaacgactttgccagccta6840
gatgcagctgtcactcgaggtcgtatcactggaacaacaaccgctgaggctgttgtcact6900
ctaccaccaccataactacgtctacataaccgacgcctaccccagtttcatagtattttc6960
tggtttgattgtatgaataatataaat6987
<210>13
<211>45
<212>dna
<213>引物1
<400>13
gttcatttcatttggagaggaaaaaatataaaaactcaacataac45
<210>14
<211>24
<212>dna
<213>引物2
<400>14
ttgatggtgaactgcctcaagagc24
<210>15
<211>24
<212>dna
<213>引物3
<400>15
gctcttgaggcagttcaccatcaa24
<210>16
<211>41
<212>dna
<213>引物4
<400>16
gacgaaagctgggaggcctggtcgactctagggggatccga41
<210>17
<211>23
<212>dna
<213>引物5
<400>17
cctctccaaatgaaatgaacttc23
<210>18
<211>26
<212>dna
<213>引物6
<400>18
caggcctcccagctttcgtccgtatc26
<210>19
<211>22
<212>dna
<213>引物7
<400>19
ggctttcaagcgccagtgcgag22
<210>20
<211>22
<212>dna
<213>引物8
<400>20
gaagtgctcttgcacaagggtg22
<210>21
<211>51
<212>dna
<213>引物9
<400>21
gttcatttcatttggagagggaaaactaaaccatacaccaccaacacaacc51
<210>22
<211>23
<212>dna
<213>引物10
<400>22
gataacgtgcctaggaaaatctg23
<210>23
<211>21
<212>dna
<213>引物11
<400>23
gattttcctaggcacgttatc21
<210>24
<211>42
<212>dna
<213>引物12
<400>24
gacgaaagctgggaggcctgggaaattcgcggtacccactag42