CN114854787B - 一种植物重组表达载体及其构建方法和应用 - Google Patents
一种植物重组表达载体及其构建方法和应用 Download PDFInfo
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Abstract
本发明提供了一种植物重组表达载体及其构建方法和应用,属于基因工程技术领域。植物重组表达载体pGR20含有耐除草剂基因表达框、抗虫基因表达框和耐逆基因表达框。耐除草剂基因表达框包括CaMV35S启动子、优化密码子的g2m‑epsps基因和CaMVPolyA终止子,抗虫基因表达框包括CaMV35S启动子、优化密码子的cry1C基因和NOS终止子,耐逆基因表达框包括CaMV35S启动子、大豆GmNFYB1基因和NOS终止子。利用这一表达载体可获得耐除草剂抗虫耐逆复合性状的转基因植物,在培育复合性状植物品种中有良好的应用前景。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种植物重组表达载体及其构建方法和应用。
背景技术
草害、虫害和非生物逆境胁迫是影响作物生产的重要因素,尤其是农村劳动力转移及东北主产区人均耕地面积多,田间管理水平下降,在杂草防除方面主要依赖化学除草,辅以机械和人工除草。如果苗期田间管理期间,雨水集中或者由于化学除草失败,容易造成草荒,一般减产10%以上,严重时撂荒绝产。且传统除草剂的使用造成杂草危害普遍、土壤环境恶化和对作物药害严重等问题。病虫害的发生与存在直接影响着作物的产量与品质,通常需要喷施大量的杀虫剂来控制虫害。如果不喷药防治,因虫害造成的减产达到10%以上,严重的年份减产可达50%以上。此外,干旱和盐碱也是影响农作物生产的主要因素之一。
利用基因工程技术培育耐除草剂抗虫耐逆复合性状转基因作物为杂草、虫害的有效防治以及应对干旱、盐碱等逆境,提供了更好的解决方案。耐草甘膦除草剂特性使转基因作物种植时可采用残效期短、杀草谱广的高效除草剂,避免对下茬作物的危害,从而实现合理轮作。抗虫作物的培育和种植带来极大的经济和环境效益,大大减少了杀虫剂的使用量。培育转基因耐逆品种不但可以增加干旱、盐渍化土壤上大豆的产量,而且可以有效的利用这些闲置土地,为解决我国“人口多、耕地少”问题开辟新途径。
复合性状转基因作物的不断涌现是转基因产业化发展的趋势与特点,也是转基因作物未来发展的趋势,符合种植者和消费者的多元化需求。2018年具有复合抗虫性状的转基因作物种植面积增加4%,占全球种植面积的42%。常用于培育复合性状转基因作物的方法主要有3种,分别是转化现有的转基因作物,将多个基因连接到同一载体上再转入受体中以及利用常规的杂交育种技术将已有转基因性状进行聚合。转化现有转基因作物以及利用常规的杂交育种技术将不同性状进行聚合存在着费时、繁琐、转化效率低、需要不同的筛选标记基因等问题。将多个基因连接到同一载体上同时进行遗传转化是比较快速的方法,可以同时获得具有多个性状的转基因植株,但由于基因表达框较多、载体序列较长,载体构建过程中需要精准的设计。
发明内容
有鉴于此,本发明的目的在于提供一种植物重组表达载体及其构建方法和应用,将耐除草剂、抗虫和耐逆基因构建到同一个表达载体中,利用这一表达载体对植物进行遗传转化,可以培育出耐除草剂抗虫耐逆复合性状转基因植物,从而为复合性状转基因作物的开发和应用提供了快速的方法,具有良好的应用前景
本发明提供了一种植物重组表达载体pGR20,包含耐除草剂基因表达框、抗虫基因表达框和耐逆基因表达框。
优选的,所述耐除草剂基因表达框依次包含CaMV 35S启动子、优化密码子的耐草甘膦基因和CaMV PolyA终止子;
所述耐草甘膦基因为g2m-epsps;
所述耐除草剂基因表达框如以下一种DNA片段:
A.核苷酸序列如SEQ ID NO:1所示的DNA片段;
B.与SEQ ID NO:1互补的DNA片段;
C.在SEQ ID NO:1所示的DNA片段的基础上替换、缺失或***一个或几个碱基形成的DNA片段。
优选的,所述抗虫基因表达框依次包含CaMV 35S启动子、优化密码子的抗虫基因和NOS终止子;所述抗虫基因包括cry1C基因;
所述抗虫基因表达框的核苷酸序列如以下一种DNA片段:
1)核苷酸序列如SEQ ID NO:2所示的DNA片段;
2)与SEQ ID NO:2互补的DNA片段;
3)在SEQ ID NO:2所示的DNA片段的基础上替换、缺失或***一个或几个碱基形成的DNA片段。
优选的,所述耐逆基因表达框依次包含CaMV 35S启动子、大豆的耐逆基因和NOS终止子;所述耐逆基因包括GmNFYB1基因;
所述耐逆基因表达框如以下一种DNA片段:
I核苷酸序列如SEQ IDNO:3所示的DNA片段;
II与SEQ ID NO:3互补的DNA片段;
III在SEQ ID NO:3所示的DNA片段的基础上替换、缺失或***一个或几个碱基形成的DNA片段。
优选的,所述耐除草剂基因表达框的多克隆位点为XhoI;
所述抗虫基因表达框的多克隆位点为KpnI/HindIII;
所述耐逆基因表达框的多克隆位点为BglII/BstEII;
所述重组表达载体pGR20的骨架载体包括pCAMBIA3301。
优选的,所述pGR20的核苷酸序列如SEQ ID NO:4所示。
本发明提供了所述植物重组表达载体pGR20的构建方法,包括以下步骤:
含BglII和BstEII酶切位点的GmNFYB1基因片段和商用载体pCAMBIA3301分别用BglII和BstEII双酶切后连接,构建出p3301-NF载体;
将抗虫基因cry1C和商用载体pBI121分别用BamHI和SacI双酶切后连接,构建出pBI121-crylC载体;
以所述pBI121-crylC载体为模板,利用两端带有KpnI和HindIII酶切位点的特异性引物,扩增得到抗虫基因表达框的片段,与利用KpnI和HindIII酶切的p3301-NF载体连接,构建p3301-NF-cry1C植物表达载体;
利用序列合成的含有XhoI酶切位点的g2m-epsps片段和所述p3301-NF-cry1C载体用XhoI酶切后连接,形成包含耐除草剂基因表达框的植物重组表达载体pGR20。
本发明提供了所述植物重组表达载体pGR20或所述构建方法得到的植物重组表达载体pGR20在培育转基因植物中的应用。
优选的,所述转基因植物为耐除草剂抗虫耐逆复合性状的转基因植物。
本发明提供了一种耐除草剂、抗虫和耐逆的转基因植物的培育方法,包括如下步骤:将所述的植物重组表达载体pGR20导入受体植物中,得到转基因植物。
本发明提供了一种植物重组表达载体pGR20,包含耐除草剂基因表达框、抗虫基因表达框和耐逆基因表达框。本发明将耐除草剂、抗虫和耐逆基因构建到同一个表达载体中,利用这一表达载体对植物进行遗传转化,可以培育出耐除草剂抗虫耐逆复合性状转基因植物,从而为复合性状转基因作物的开发和应用提供了快速的方法,具有良好的应用前景。
附图说明
图1为本发明提供的植物表达载体pGR20示意图;
图2为T2代转基因大豆中g2m-epsps、cry1C和GmNFYB1基因的PCR检测;M:DNAmarker,泳道1:无模板对照,泳道2:质粒阳性对照;泳道3:非转基因受体对照;泳道4-13:不同株系的转基因大豆;
图3为转基因大豆的耐草甘膦除草剂特性鉴定;
图4为转基因大豆的抗虫性鉴定;
图5为转基因大豆的耐旱性鉴定。
具体实施方式
本发明提供了一种植物重组表达载体pGR20,包含耐除草剂基因表达框、抗虫基因表达框和耐逆基因表达框。
在本发明中,所述耐除草剂基因表达框优选依次包含CaMV 35S启动子、优化密码子的耐草甘膦基因和CaMV PolyA终止子。耐草甘膦基因优选为g2m-epsps。所述耐草甘膦基因表达框如以下一种DNA片段:
A.核苷酸序列如SEQ ID NO:1所示的DNA片段;
B.与SEQ ID NO:1互补的DNA片段;
C.在SEQ ID NO:1所示的DNA片段的基础上替换、缺失或***一个或几个碱基形成的DNA片段。
所述耐草甘膦基因表达框能够高效表达g2m-epsps基因,从而使遗传转化体系表现较强的耐除草剂特性。
在本发明中,所述抗虫基因表达框优选依次包含CaMV 35S启动子、优化密码子的抗虫基因和NOS终止子。所述抗虫基因优选包括cry1C基因。所述抗虫基因表达框的核苷酸序列如以下一种DNA片段:1)核苷酸序列如SEQ ID NO:2所示的DNA片段;
2)与SEQ ID NO:2互补的DNA片段;
3)在SEQ ID NO:2所示的DNA片段的基础上替换、缺失或***一个或几个碱基形成的DNA片段。
所述抗虫基因表达框能够高效表达cry1C基因,从而使遗传转化体系表现较强的抗虫特性。
在本发明中,所述耐逆基因表达框优选依次包含CaMV 35S启动子、大豆的基因和NOS终止子。所述耐逆基因优选包括GmNFYB1基因。所述耐逆基因表达框如以下一种DNA片段:I核苷酸序列如SEQ ID NO:3所示的DNA片段;
II与SEQ ID NO:3互补的DNA片段;
III在SEQ ID NO:3所示的DNA片段的基础上替换、缺失或***一个或几个碱基形成的DNA片段。
所述耐逆基因表达框能够高效表达GmNFYB1基因,从而使遗传转化体系表现出较强的耐逆特性。所述耐逆特性优选为耐旱特性。
在本发明中,所述耐草甘膦基因表达框的多克隆位点优选为XhoI。所述抗虫基因表达框的多克隆位点优选为KpnI/HindIII;所述耐逆基因表达框的多克隆位点优选为BglII/BstEII。所述重组表达载体pGR20的骨架载体包括pCAMBIA3301。所述pGR20的核苷酸序列优选如SEQ ID NO:4所示。
本发明提供了所述植物重组表达载体pGR20的构建方法,包括以下步骤:
含BglII和BstEII酶切位点的GmNFYB1基因片段和商用载体pCAMBIA3301分别用BglII和BstEII双酶切后连接,构建出p3301-NF载体;
将含有BamHI和SacI双酶切位点的抗虫基因cry1C和商用载体pBI121分别用BamHI和SacI双酶切后连接,构建出pBI121-crylC载体;
以所述pBI121-crylC载体为模板,利用两端带有KpnI和HindIII酶切位点的特异性引物,扩增得到抗虫基因表达框的片段,与KpnI和HindIII酶切后的p3301-NF载体连接,构建p3301-NF-cry1C植物表达载体;
利用序列合成的含有XhoI酶切位点的g2m-epsps片段和所述p3301-NF-cry1C载体用XhoI酶切后连接,形成包含耐除草剂基因表达框的植物重组表达载体pGR20。
在本发明中,所述含BglII和BstEII酶切位点的GmNFYB1基因片段优选采用SEQ IDNO:11和SEQ ID NO:12所示的引物对扩增得到,所得片段的核苷酸序列如SEQ ID NO:13所示。扩增的体系优选如下10×Buffer 3μL、dNTP 3μL、MgCl23μL、上下游引物各0.5μL、KODPlus Neo 0.5μL、模板1μL和余量的ddH2O。扩增的程序优选如下:95℃预变性3min;95℃变性30s,58℃退火30s,68℃延伸1min,34个循环;72℃延伸5min。所述双酶切的反应体系优选为PCR回收产物10μL,10×Buffer 2μL,BglII 1μL,BstEII 1μL和余量的ddH2O。所述双酶切的反应条件优选为37℃酶切过夜。所述连接优选用T4连接酶进行。所述连接的条件优选为16℃连接4小时。所述连接后,优选进行重组载体的验证。本发明对所述验证的方法没有特殊限制,采用本领域所熟知的验证方法即可,例如转化大肠杆菌中培养,提取质粒,进行菌落PCR扩增或测序。本发明对商用载体pCAMBIA3301的来源没有特殊限制,采用本领域所熟知的商用载体pCAMBIA3301的来源即可。在本发明实施例中所述商用载体pCAMBIA3301购自Cambia公司。
在本发明中,含有BamHI和SacI双酶切位点的抗虫基因cry1C优选采用SEQ ID NO:14和SEQ ID NO:15所示的引物对扩增得到,所得片段的核苷酸序列如SEQ ID NO:16所示。扩增的体系优选如下10×Buffer 3μL、dNTP 3μL、MgCl23μL、上下游引物各0.5μL、KOD PlusNeo 0.5μL,模板1μL和余量的ddH2O。扩增的程序优选如下:95℃预变性3min;95℃变性30s,58℃退火30s,68℃延伸1min,34个循环;72℃延伸5min。所述双酶切的反应体系优选为PCR回收产物10μL,10×Buffer 2μL,BamHI 1μL,SacI 1μL和余量的ddH2O。所述双酶切的反应条件优选为37℃酶切过夜。所述连接优选用T4连接酶进行。所述连接的条件优选为16℃连接4小时。所述连接后,优选进行重组载体的验证。本发明对所述验证的方法没有特殊限制,采用本领域所熟知的验证方法即可,例如转化大肠杆菌中培养,提取质粒,进行菌落PCR扩增或测序。本发明对商用载体pBI121的来源没有特殊限制,采用本领域所熟知的商用载体pBI121的来源即可。在本发明实施例中所述商用载体pBI121购自Clontech公司。
在本发明中,所述两端带有KpnI和HindIII酶切位点的特异性引物如SEQ ID NO:17和SEQ ID NO:18所示。扩增得到抗虫基因表达框的片段与KpnI和HindIII酶切后的p3301-NF载体连接时,连接的条件优选为16℃连接4小时。所述连接后,优选进行重组载体的验证。本发明对所述验证的方法没有特殊限制,采用本领域所熟知的验证方法即可,例如转化大肠杆菌中培养,提取质粒,进行菌落PCR扩增或测序。
在本发明中,利用序列合成的含有XhoI酶切位点的g2m-epsps片段和所述p3301-NF-cry1C载体用XhoI酶切后连接,形成包含耐除草剂基因表达框的植物重组表达载体pGR20。本发明对含有XhoI酶切位点的g2m-epsps片段的序列合成没有特殊限制,采用本领域所熟知的序列合成技术即可。在本发明实施例中所述含有XhoI酶切位点的g2m-epsps片段由苏州泓迅生物科技股份有限公司合成。
本发明提供了所述植物重组表达载体pGR20或所述构建方法得到的植物重组表达载体pGR20在培育转基因植物中的应用。
在本发明中,所述转基因植物优选为耐除草剂抗虫耐逆复合性状的转基因植物。
本发明提供了一种耐除草剂、抗虫和耐逆的转基因植物的培育方法,包括如下步骤:将所述的植物重组表达载体pGR20导入受体植物中,得到转基因植物。
在本发明中,所述转基因植物优选包括筛选。所述筛选包括目标基因PCR检测。所述目标基因PCR检测用引物包括g2m-F/g2m-R、cry1C-F/cry1C-R、NFYB1-F/NFYB1-R。所述PCR反应条件:94℃变性3min,94℃变性30s,61~63℃退火30s,72℃延伸30s,35个循环,循环结束后72℃延伸8min。
在本发明中,筛选得到的转基因植物进行性状筛选,耐除草剂鉴定结果表明转基因大豆生长不受抑制,叶片不褪绿、不皱缩,无新叶黄化等草甘膦药害反应,转基因大豆对草甘膦表现为高抗;抗虫性鉴定结果表明,受体对照叶片损伤严重,表现为感虫;而转基因大豆叶片保持完整,对斜纹夜蛾表现高抗;耐逆性鉴定结果表明,转基因大豆苗期抗旱性显著高于受体。
下面结合实施例对本发明提供的一种植物重组表达载体及其构建方法和应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
植物表达载体pGR20的构建方法
(1)利用带有BglII和BstEII酶切位点酶切位点的基因特异性引物,从大豆品种Williams 82的cDNA中通过PCR扩增出GmNFYB1(Glyma.10G192000.1)基因。扩增出的GmNFYB1基因片段和商用载体pCAMBIA3301分别用BglII和BstEII双酶切后连接,构建出p3301-NF载体,形成包含CaMV35S-GmNFYB1-NOS元件的耐逆基因表达框,核苷酸序列见SEQID NO:3。
(2)将抗虫基因cry1C和商用载体pBI121分别用BamHI和SacI双酶切后连接,构建出pBI121-crylC载体,形成包含CaMV 35S-cry1C-NOS元件的抗虫基因表达框,核苷酸序列见SEQ ID NO:2。
(3)从构建的pBI121-crylC载体中,利用两端带有KpnI和HindIII酶切位点的特异性引物,扩增出大小为3.5kb,包含CaMV 35S-cry1C-NOS元件的抗虫基因表达框的片段,与利用KpnI和HindIII酶切的p3301-NF载体连接,构建p3301-NF-cry1C植物表达载体。
(4)利用序列合成的含有XhoI酶切位点的g2m-epsps片段和p3301-NF-cry1C载体用XhoI酶切后连接,形成包含CaMV 35S-cry1C-PolyA元件的耐除草剂基因表达框,核苷酸序列见SEQ ID NO:1。最终获得pGR20植物表达载体,载体示意图见图1,核苷酸序列见SEQID NO:4。
实施例2
植物表达载体在培育转基因植物中的应用
1、pGR20载体转化大豆
采用农杆菌介导大豆子叶节转化法,农杆菌菌株为Ag10。大豆子叶节消毒后诱导愈伤,然后与农杆菌共培养以感染愈伤,筛选被转化的愈伤,之后在选择培养上进行植株再生。
无菌外植体的获得:选用正常萌发无污染的幼苗制备外植体。在超净工作台中用手术刀沿下胚轴将大豆切下,保留3-4mm的下胚轴,置于无菌培养皿中,皿中添加适量的共培养液以方便剥离种皮,再沿子叶下胚轴垂直将胚轴切开,剔除干净真叶组织,在子叶和子叶下胚轴的连接处轴向作5-7个切口,切口约3-4mm长。每个外植体是由一片子叶连着一段下胚轴组成,一枚种子可以形成两个外植体。
农杆菌的准备:从超低温冰箱中取出低温保藏的农杆菌菌株于冰上冻融,用接种环或无菌枪头蘸取少量的菌种接种于LB平板上,LB固体培养基含50mg/L对应的抗生素。置于28℃恒温条件下倒置培养1-2d以获得单克隆。2d后,挑取单克隆接种于5mlYEP培养液中(含对应抗生素)于220rpm、28℃过夜活化培养(约12h)。当菌液第一次活化至饱和状态时,从中抽取1ml菌液接种至含100mlYEP(含对应抗生素)的三角摇瓶中,在28℃,220rpm条件下第二次活化。待农杆菌充分活化至OD600=1.0左右时,将菌液于4000rpm、4℃条件下离心10min,弃上清收集沉淀,用等体积共培养液悬浮沉淀于管底的菌体,此时OD600约为0.5~0.8左右,备用。
外植体与农杆菌共培养:外植体制备时,每40~60片外植体置于一个100ml的三角瓶中,每瓶添加约50ml重悬的农杆菌菌液,菌液需漠过外植体。于黑暗或弱光条件下共侵染30-35min,每隔5min轻摇三角瓶一次以使农杆菌和外植体充分接触。侵染完成后小心倒掉多余的农杆菌菌液,在共培养培养基上平铺一层无菌滤纸,将浸染后的外植体向轴一侧朝下平铺于滤纸上,24℃,黑暗或弱光条件下共培养3d。
抗性筛选与再生:共培养3d后,外植体经抗性丛生芽诱导、伸长芽诱导、生根等阶段获得再生植株;在丛生芽诱导和伸长阶段分别添加适宜的抗性筛选剂,生根阶段根据实验时的实际需求,添加适宜浓度的农杆菌抑菌剂及IBA以诱导生根,丛生芽诱导和伸长芽诱导阶段每两周继代外植体一次,继代时在外植体的背面重新制备一新的切口以使外植体能更好地吸收养分。伸长芽伸长至4-6cm,可诱导生根,生根后先揭开培养皿上的封口膜开口炼苗1-3天;再移栽至盆栽或大田中生长,获得转基因植株。
2、转基因大豆PCR检测
基因组DNA的提取:取0.1g匀碎的样品至1.5ml离心管中,加500μl CTAB提取液,65℃孵育30min;加入等体积酚-氯仿-异戊醇(25:24:1,V/V),混匀,13000r/min离心10min,取上清液,重复上述步骤1~2次;上清液加两倍体积的CTAB沉淀液,室温孵育30min;离心后用350μl NaCl(1.2mol/L)溶解沉淀,加入氯仿,混匀;离心后将上清转移到新的离心管;加0.6倍体积的异丙醇和1/10倍体积的3mol/L NH4Ac(pH 6.8),充分混匀,低温静置15min;离心后用70%乙醇洗涤沉淀,室温干燥;将DNA溶解在50μl TE缓冲液中,加入1μl的RNaseA(10mg/ml),37℃温育15min,-20℃保存;用Spectrophotometer ND-1000检测DNA浓度,每次上样量为1.5μl。琼脂糖电泳检测DNA浓度:配制0.8%琼脂糖电泳液,加适量EB。电泳条件U=120V,I=140mA。
转基因品系PCR检测:利用特异性引物检测转基因大豆品系的目的基因(表1)。PCR反应条件:94℃变性3min,94℃变性30s,61-63℃退火30s,72℃延伸30s,35个循环,循环结束后72℃延伸8min。取PCR扩增产物10μl,1.5%琼脂糖凝胶电泳检测(含溴化乙锭),5V/cm电泳40min,紫外灯下观察照相。
表1.转基因检测引物列表
提取转基因大豆植株的基因组DNA为模版,对g2m-epsps、cry1C和GmNFYB1目的基因进行PCR检测。检测结果如图2所示。结果表明在转基因材料中均可以检测到目的基因的存在,说明外源基因在已经稳定整合到大豆基因组中。
实施例3
转基因植物耐除草剂、抗虫、耐逆性功能检测
为了鉴定转基因植株的抗性,对PCR检测阳性的转基因植株开展了耐除草剂、抗虫和耐旱性鉴定。
1、耐除草剂鉴定
将PCR检测阳性的转基因大豆及其受体种子播种于盆栽中,待出土幼苗生长至2片真叶完全展开时期喷施草甘膦除草剂,草甘膦除草剂施用量为农药登记中剂量的2倍(有效成分1800g/公顷),用药2周后调查。结果表明在上述喷施剂量下,受体品种不具备抗性,2周后植株全部死亡;转基因大豆生长不受抑制,叶片不褪绿、不皱缩,无新叶黄化等草甘膦药害反应,表明这些转基因大豆对草甘膦表现为高抗(图3)。
2、抗虫性鉴定
采集转基因大豆和受体植株上健康、鲜嫩程度一致的展开叶片,用打孔器将叶片打成2.5cm圆片,选取6片放入铺有海绵培养皿内,以13mL无菌水保湿,按2头/皿的比例接入2龄斜纹夜蛾幼虫,7天后进行结果调查分析。结果表明受体对照叶片损伤严重,表现为感虫;而转基因大豆叶片保持完整,对斜纹夜蛾表现高抗(图4)。
3、耐逆性鉴定
将转基因大豆和受体在温室用盆栽的方式播种,保证每个盆内土量一致,每个材料10粒种子。出苗后进行间苗,保证每盆苗数相同。待植株长到两个三出复叶时期停止浇水,开始自然干旱处理。结果表明转基因大豆苗期抗旱性显著高于受体(图5)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国农业科学院作物科学研究所
<120> 一种植物重组表达载体及其构建方法和应用
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2610
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 60
acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 120
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 180
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg gctagagcag 240
cttgccaaca tggtggagca cgacactctc gtctactcca agaatatcaa agatacagtc 300
tcagaagacc aaagggctat tgagactttt caacaaaggg taatatcggg aaacctcctc 360
ggattccatt gcccagctat ctgtcacttc atcaaaagga cagtagaaaa ggaaggtggc 420
acctacaaat gccatcattg cgataaagga aaggctatcg ttcaagatgc ctctgccgac 480
agtggtccca aagatggacc cccacccacg aggagcatcg tggaaaaaga agacgttcca 540
accacgtctt caaagcaagt ggattgatgt gataacatgg tggagcacga cactctcgtc 600
tactccaaga atatcaaaga tacagtctca gaagaccaaa gggctattga gacttttcaa 660
caaagggtaa tatcgggaaa cctcctcgga ttccattgcc cagctatctg tcacttcatc 720
aaaaggacag tagaaaagga aggtggcacc tacaaatgcc atcattgcga taaaggaaag 780
gctatcgttc aagatgcctc tgccgacagt ggtcccaaag atggaccccc acccacgagg 840
agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat 900
atctccactg acgtaaggga tgacgcacaa tcccactatc cttcgcaaga ccttcctcta 960
tataaggaag ttcatttcat ttggagagga cacgctgaaa tcaccagtct ctctctacaa 1020
atctatctct atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc catctcttat 1080
ctccaatctc tcgaaatcca gtcaacgcaa atctccctta tcggtttctc tgaagacgca 1140
gcagcatcca cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac 1200
gttaattggc tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcatgcat 1260
ggcttgcctc ccagatgatt cggggcctca cgttgggcat agcacgccgc ctcgcctcga 1320
ccaggagcct tgcactctca gcagccagaa gaccgtgacg gtcacaccgc ccaacttccc 1380
actgacgggc aaggtggctc cacctgggtc gaagtctatc acaaataggg ctctcctgct 1440
cgctgctctc gctaagggca cttcaaggct gtccggggct ctcaagtctg acgatacccg 1500
gcacatgtca gtcgccctgc gccagatggg cgttaccatc gacgagcccg acgataccac 1560
gttcgtggtc acaagccagg gctcgctgca gctcccagct cagcctctgt tcctcggcaa 1620
cgctgggact gcgatgaggt tcctgaccgc tgctgtcgct actgttcagg gcaccgttgt 1680
gctcgacggg gatgagtaca tgcagaagag gcccatcggc ccactgctcg ctacgctcgg 1740
ccagaatggg attcaggtcg actccccaac gggctgccca ccagttacag tgcacggcat 1800
ggggaaggtg caggcgaagc gcttcgagat cgacggcggg ctgtccagcc agtacgtcag 1860
cgctctgctc atgctcgctg cttgcggcga ggctcctatc gaggtggctc tgacagggaa 1920
ggacattggc gctaggggct acgtcgacct gaccctcgat tgcatgaggg ctttcggcgc 1980
tcaggtggat gctgtcgacg atacaacttg gagggtggct ccaactggct acaccgctca 2040
tgactacctc atcgagcctg atgcttccgc tgctacttac ctgtgggctg ctgaggttct 2100
caccggcggg aggatcgaca ttggcgtggc tgctcaggac ttcacccagc cagatgctaa 2160
ggcccaggcg gtcatcgcgc agttccccaa catgcaggct actgtcgttg gctcccagat 2220
gcaggacgct attccaacgc tggccgtgct cgccgcgttc aacaatacgc ctgtccgctt 2280
cacagagctg gcgaacctcc gcgttaagga gtgcgacagg gtgcaggccc tgcatgatgg 2340
cctcaatgag atcaggccag gcctggctac gattgagggg gacgatctgc tcgtggcttc 2400
ggacccagct ctggctggca cggcgtgcac agctctcatc gacacccacg cggatcatcg 2460
gattgctatg tgcttcgcgc tggctggcct caaagtcagc gggatccgca ttcaggaccc 2520
ggattgcgtt gccaagactt accctgatta ctggaaggct ctggcgtccc tgggcgttca 2580
cctcaatgat tagcgatcga caagctcgag 2610
<210> 2
<211> 3075
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gagctcggta ccagattagc cttttcaatt tcagaaagaa tgctaaccca cagatggtta 60
gagaggctta cgcagcaggt ctcatcaaga cgatctaccc gagcaataat ctccaggaaa 120
tcaaatacct tcccaagaag gttaaagatg cagtcaaaag attcaggact aactgcatca 180
agaacacaga gaaagatata tttctcaaga tcagaagtac tattccagta tggacgattc 240
aaggcttgct tcacaaacca aggcaagtaa tagagattgg agtctctaaa aaggtagttc 300
ccactgaatc aaaggccatg gagtcaaaga ttcaaataga ggacctaaca gaactcgccg 360
taaagactgg cgaacagttc atacagagtc tcttacgact caatgacaag aagaaaatct 420
tcgtcaacat ggtggagcac gacacacttg tctactccaa aaatatcaaa gatacagtct 480
cagaagacca aagggcaatt gagacttttc aacaaagggt aatatccgga aacctcctcg 540
gattccattg cccagctatc tgtcacttta ttgtgaagat agtggaaaag gaaggtggct 600
cctacaaatg ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca 660
gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa 720
ccacgtcttc aaagcaagtg gattgatgtg atatctccac tgacgtaagg gatgacgcac 780
aatcccacta tccttcgcaa gacccttcct ctatataagg aagttcattt catttggaga 840
gaacacgggg gactctagag gatccaccat ggaggagaac aatcagaacc agtgtatccc 900
ttacaattgt ctttctaatc ctgaagaagt tcttttggat ggagaaagga tctcaactgg 960
taactcatca attgacatct ctctctcact tgttcagttc ttggtttcta actttgtgcc 1020
aggaggagga ttccttgttg gacttatcga cttcgtttgg ggaatcgttg gaccttctca 1080
atgggatgca tttctcgttc agatcgaaca gctcatcaac gaaagaatcg ctgagttcgc 1140
taggaatgct gctattgcta accttgaagg acttggaaac aacttcaaca tctacgtgga 1200
ggcattcaag gaatgggaag aagatcctaa caacccagca accaggacca gagtgatcga 1260
taggttccgt atccttgatg gacttcttga aagggacatt cctagcttta ggatctctgg 1320
atttgaagtt ccacttctct ctgtttacgc tcaagctgct aatctccatc ttgctatcct 1380
tagagattct gtgatcttcg gagaaagatg gggattgaca accatcaacg tgaacgagaa 1440
ctacaacaga ctcatcaggc acatcgatga gtacgctgat cactgtgcta acacttacaa 1500
ccgtggactc aacaaccttc ctaagtctac ctatcaagat tggatcacat acaaccgact 1560
taggagagac cttacattga ctgttcttga tatcgctgct ttctttccaa actatgacaa 1620
taggagatat ccaattcagc cagttggtca acttacaagg gaagtttaca ctgacccact 1680
catcaacttc aacccacagc ttcagtctgt tgctcagctt cctaccttca acgttatgga 1740
gagcagcgca atcagaaatc ctcacctctt cgacatcttg aacaacctta caatctttac 1800
cgattggttt agtgttggac gtaacttcta ctggggagga catcgagtga tctctagcct 1860
catcggaggt ggtaacatca catctcctat ctacggaaga gaggctaacc aggagcctcc 1920
aagatcattc actttcaacg gacctgtgtt caggactctt tcaaatccta ctcttcgact 1980
tcttcagcaa ccttggccag ctccaccatt caaccttcgt ggtgttgaag gagttgagtt 2040
ctctacacct acaaacagct tcacctatcg tggaagaggt actgttgatt ctcttactga 2100
acttccacct gaggaaacag tgtgccacct cgtgaaggat acagtcatcg tctttgtcat 2160
gcaaccttcg ttcaaagatc tggaacacct ttccttacaa ctggtgttgt gttctcttgg 2220
actcatcgta gtgcaactct taccaacaca attgatccag agaggatcaa ccagatccct 2280
cttgtgaaag gattcagagt ttggggagga acctctgtga ttacaggacc aggattcaca 2340
ggaggtgata tccttcgaag aaacaccttt ggtgacttcg tttctcttca agtgaacatc 2400
aactcaccaa tcacccaaag ataccgtctt agatttcgtt acgcttctag tagggatgca 2460
cgagttatcg ttcttacagg agctgcatct acaggagtgg gaggtcaagt tagtgtgaac 2520
atgcctcttc agaaaactat ggagatcgga gagaacctca catctagaac attcagatac 2580
accgacttca gtaatccttt ctcattcaga gctaatccag acatcatcgg tatcagtgaa 2640
caacctctct tcggtgcagg ttctatcagt agcggtgaac tttacatcga caagatcgag 2700
atcatccttg cagatgcaac atttgaagca gaatctgacc ttgaaagagc acaaaagtag 2760
tgaccaacgt atttatatca gaaaatagat gagtcgaaat taaaagctta tacccgttaa 2820
tgagatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt 2880
gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa 2940
tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa 3000
tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca 3060
tctatgttac tagat 3075
<210> 3
<211> 1319
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccctaggcat ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg 60
gcgaacagtt catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca 120
tggtggagca cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc 180
aaagggcaat tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt 240
gcccagctat ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat 300
gccatcattg cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca 360
aagatggacc cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt 420
caaagcaagt ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact 480
atccttcgca agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg 540
ggactatgtc ggatgcaccg gcgagtccga gtcacgagag tggtggcgag cagagccctc 600
gcggctcgtt gtccggcgcg gctagagagc aggaccggta ccttcccatt gccaacatca 660
gccgcatcat gaagaaggct ctgcctccca atggcaagat tgcgaaggat gcaaaagaca 720
caatgcaaga atgcgtttct gaattcatca gcttcattac cagcgaggcg agtgagaaat 780
gccagaagga gaagagaaag acaatcaatg gagacgattt actatgggcc atggcaactt 840
tagggtttga agactacatt gagccgctta aggtgtacct ggctaggtac agagaggcgg 900
agggtgacac taaaggatct gctagaagtg gtgatggatc tgctagacca gatcaagttg 960
gccttgcagg tcaaaatgct cagcttgttc atcagggttc gctgaactat attggtttgc 1020
aggtgcaacc acaacatctg gttatgcctt caatgcaagg ccatgaatag cgttcaaaca 1080
tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat 1140
aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg acgttattta 1200
tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg atagaaaaca 1260
aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg ttactagat 1319
<210> 4
<211> 13578
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct 60
atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca 120
agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt 180
gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca 240
agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga 300
ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca 360
ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg 420
acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca 480
ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg 540
acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg 600
agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg 660
tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga 720
tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga 780
ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg 840
gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac 900
gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac 960
cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt 1020
ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg 1080
gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt 1140
tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca 1200
aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc 1260
aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg 1320
ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa 1380
ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc 1440
cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg 1500
atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc 1560
accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa 1620
gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag 1680
gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac 1740
ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc 1800
cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta 1860
aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca 1920
gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc 1980
agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca 2040
ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa 2100
atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc 2160
accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc 2220
tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga 2280
cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga 2340
gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg 2400
tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc 2460
cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc 2520
gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg 2580
tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca 2640
cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact 2700
gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa 2760
gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga 2820
tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt 2880
tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga 2940
agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga 3000
gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct 3060
gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct 3120
ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg 3180
cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc 3240
aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt 3300
catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca 3360
gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga 3420
tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa 3480
cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa 3540
aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc 3600
ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg 3660
gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc 3720
aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc 3780
actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg 3840
aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3900
ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3960
tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 4020
gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 4080
ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260
ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440
ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4500
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560
ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680
gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860
atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920
acgttaaggg attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata 4980
atattttatt ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata 5040
ctgttcttcc ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt 5100
gtccgccctg ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa 5160
gatgttgctg tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt 5220
ctttaaaaaa tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc 5280
gcaatccaca tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc 5340
taagctattc gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc 5400
cgcatacagc tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag 5460
gacgccatcg gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag 5520
gacctttgga acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac 5580
atcataggtg gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc 5640
tcccaccagc ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta 5700
tttttcgatc agttttttca attccggtga tattctcatt ttagccattt attatttcct 5760
tcctcttttc tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc 5820
aattcactgt tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg 5880
ttttcaaagt tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca 5940
caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt 6000
gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag 6060
tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat 6120
cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga 6180
tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt 6240
taatgtactg aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg 6300
gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat acatactaag 6360
ggtttcttat atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg 6420
ggaactactc acacattatt atggagaaac tcgagcttgt cgatcgctaa tcattgaggt 6480
gaacgcccag ggacgccaga gccttccagt aatcagggta agtcttggca acgcaatccg 6540
ggtcctgaat gcggatcccg ctgactttga ggccagccag cgcgaagcac atagcaatcc 6600
gatgatccgc gtgggtgtcg atgagagctg tgcacgccgt gccagccaga gctgggtccg 6660
aagccacgag cagatcgtcc ccctcaatcg tagccaggcc tggcctgatc tcattgaggc 6720
catcatgcag ggcctgcacc ctgtcgcact ccttaacgcg gaggttcgcc agctctgtga 6780
agcggacagg cgtattgttg aacgcggcga gcacggccag cgttggaata gcgtcctgca 6840
tctgggagcc aacgacagta gcctgcatgt tggggaactg cgcgatgacc gcctgggcct 6900
tagcatctgg ctgggtgaag tcctgagcag ccacgccaat gtcgatcctc ccgccggtga 6960
gaacctcagc agcccacagg taagtagcag cggaagcatc aggctcgatg aggtagtcat 7020
gagcggtgta gccagttgga gccaccctcc aagttgtatc gtcgacagca tccacctgag 7080
cgccgaaagc cctcatgcaa tcgagggtca ggtcgacgta gcccctagcg ccaatgtcct 7140
tccctgtcag agccacctcg ataggagcct cgccgcaagc agcgagcatg agcagagcgc 7200
tgacgtactg gctggacagc ccgccgtcga tctcgaagcg cttcgcctgc accttcccca 7260
tgccgtgcac tgtaactggt gggcagcccg ttggggagtc gacctgaatc ccattctggc 7320
cgagcgtagc gagcagtggg ccgatgggcc tcttctgcat gtactcatcc ccgtcgagca 7380
caacggtgcc ctgaacagta gcgacagcag cggtcaggaa cctcatcgca gtcccagcgt 7440
tgccgaggaa cagaggctga gctgggagct gcagcgagcc ctggcttgtg accacgaacg 7500
tggtatcgtc gggctcgtcg atggtaacgc ccatctggcg cagggcgact gacatgtgcc 7560
gggtatcgtc agacttgaga gccccggaca gccttgaagt gcccttagcg agagcagcga 7620
gcaggagagc cctatttgtg atagacttcg acccaggtgg agccaccttg cccgtcagtg 7680
ggaagttggg cggtgtgacc gtcacggtct tctggctgct gagagtgcaa ggctcctggt 7740
cgaggcgagg cggcgtgcta tgcccaacgt gaggccccga atcatctggg aggcaagcca 7800
tgcatgccgt ggaaacagaa gacatgacct taagaggacg aagctcagag ccaattaacg 7860
tcatcccact cttcttcaat ccccacgacg acgaaatcgg ataagctcgt ggatgctgct 7920
gcgtcttcag agaaaccgat aagggagatt tgcgttgact ggatttcgag agattggaga 7980
taagagatgg gttctgcaca ccattgcaga ttctgctaac ttgcgccata gagatagatt 8040
tgtagagaga gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac ttccttatat 8100
agaggaaggt cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc agtggagata 8160
tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc 8220
ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg aacgatagcc 8280
tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg 8340
atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt 8400
tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag 8460
acgagagtgt cgtgctccac catgttatca catcaatcca cttgctttga agacgtggtt 8520
ggaacgtctt ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg 8580
tcggcagagg catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg 8640
ccaccttcct tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg 8700
aggaggtttc ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag 8760
actgtatctt tgatattctt ggagtagacg agagtgtcgt gctccaccat gttggcaagc 8820
tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 8880
ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 8940
agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 9000
gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattcg 9060
agctcggtac cagattagcc ttttcaattt cagaaagaat gctaacccac agatggttag 9120
agaggcttac gcagcaggtc tcatcaagac gatctacccg agcaataatc tccaggaaat 9180
caaatacctt cccaagaagg ttaaagatgc agtcaaaaga ttcaggacta actgcatcaa 9240
gaacacagag aaagatatat ttctcaagat cagaagtact attccagtat ggacgattca 9300
aggcttgctt cacaaaccaa ggcaagtaat agagattgga gtctctaaaa aggtagttcc 9360
cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt 9420
aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt 9480
cgtcaacatg gtggagcacg acacacttgt ctactccaaa aatatcaaag atacagtctc 9540
agaagaccaa agggcaattg agacttttca acaaagggta atatccggaa acctcctcgg 9600
attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc 9660
ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag 9720
tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac 9780
cacgtcttca aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca 9840
atcccactat ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag 9900
aacacggggg actctagagg atccaccatg gaggagaaca atcagaacca gtgtatccct 9960
tacaattgtc tttctaatcc tgaagaagtt cttttggatg gagaaaggat ctcaactggt 10020
aactcatcaa ttgacatctc tctctcactt gttcagttct tggtttctaa ctttgtgcca 10080
ggaggaggat tccttgttgg acttatcgac ttcgtttggg gaatcgttgg accttctcaa 10140
tgggatgcat ttctcgttca gatcgaacag ctcatcaacg aaagaatcgc tgagttcgct 10200
aggaatgctg ctattgctaa ccttgaagga cttggaaaca acttcaacat ctacgtggag 10260
gcattcaagg aatgggaaga agatcctaac aacccagcaa ccaggaccag agtgatcgat 10320
aggttccgta tccttgatgg acttcttgaa agggacattc ctagctttag gatctctgga 10380
tttgaagttc cacttctctc tgtttacgct caagctgcta atctccatct tgctatcctt 10440
agagattctg tgatcttcgg agaaagatgg ggattgacaa ccatcaacgt gaacgagaac 10500
tacaacagac tcatcaggca catcgatgag tacgctgatc actgtgctaa cacttacaac 10560
cgtggactca acaaccttcc taagtctacc tatcaagatt ggatcacata caaccgactt 10620
aggagagacc ttacattgac tgttcttgat atcgctgctt tctttccaaa ctatgacaat 10680
aggagatatc caattcagcc agttggtcaa cttacaaggg aagtttacac tgacccactc 10740
atcaacttca acccacagct tcagtctgtt gctcagcttc ctaccttcaa cgttatggag 10800
agcagcgcaa tcagaaatcc tcacctcttc gacatcttga acaaccttac aatctttacc 10860
gattggttta gtgttggacg taacttctac tggggaggac atcgagtgat ctctagcctc 10920
atcggaggtg gtaacatcac atctcctatc tacggaagag aggctaacca ggagcctcca 10980
agatcattca ctttcaacgg acctgtgttc aggactcttt caaatcctac tcttcgactt 11040
cttcagcaac cttggccagc tccaccattc aaccttcgtg gtgttgaagg agttgagttc 11100
tctacaccta caaacagctt cacctatcgt ggaagaggta ctgttgattc tcttactgaa 11160
cttccacctg aggaaacagt gtgccacctc gtgaaggata cagtcatcgt ctttgtcatg 11220
caaccttcgt tcaaagatct ggaacacctt tccttacaac tggtgttgtg ttctcttgga 11280
ctcatcgtag tgcaactctt accaacacaa ttgatccaga gaggatcaac cagatccctc 11340
ttgtgaaagg attcagagtt tggggaggaa cctctgtgat tacaggacca ggattcacag 11400
gaggtgatat ccttcgaaga aacacctttg gtgacttcgt ttctcttcaa gtgaacatca 11460
actcaccaat cacccaaaga taccgtctta gatttcgtta cgcttctagt agggatgcac 11520
gagttatcgt tcttacagga gctgcatcta caggagtggg aggtcaagtt agtgtgaaca 11580
tgcctcttca gaaaactatg gagatcggag agaacctcac atctagaaca ttcagataca 11640
ccgacttcag taatcctttc tcattcagag ctaatccaga catcatcggt atcagtgaac 11700
aacctctctt cggtgcaggt tctatcagta gcggtgaact ttacatcgac aagatcgaga 11760
tcatccttgc agatgcaaca tttgaagcag aatctgacct tgaaagagca caaaagtagt 11820
gaccaacgta tttatatcag aaaatagatg agtcgaaatt aaaagcttat acccgttaat 11880
gagatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 11940
cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 12000
gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 12060
acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 12120
ctatgttact agatccctag gcatggagtc aaagattcaa atagaggacc taacagaact 12180
cgccgtaaag actggcgaac agttcataca gagtctctta cgactcaatg acaagaagaa 12240
aatcttcgtc aacatggtgg agcacgacac acttgtctac tccaaaaata tcaaagatac 12300
agtctcagaa gaccaaaggg caattgagac ttttcaacaa agggtaatat ccggaaacct 12360
cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg 12420
tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc 12480
cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 12540
tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg taagggatga 12600
cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt 12660
ggagagaaca cgggggacta tgtcggatgc accggcgagt ccgagtcacg agagtggtgg 12720
cgagcagagc cctcgcggct cgttgtccgg cgcggctaga gagcaggacc ggtaccttcc 12780
cattgccaac atcagccgca tcatgaagaa ggctctgcct cccaatggca agattgcgaa 12840
ggatgcaaaa gacacaatgc aagaatgcgt ttctgaattc atcagcttca ttaccagcga 12900
ggcgagtgag aaatgccaga aggagaagag aaagacaatc aatggagacg atttactatg 12960
ggccatggca actttagggt ttgaagacta cattgagccg cttaaggtgt acctggctag 13020
gtacagagag gcggagggtg acactaaagg atctgctaga agtggtgatg gatctgctag 13080
accagatcaa gttggccttg caggtcaaaa tgctcagctt gttcatcagg gttcgctgaa 13140
ctatattggt ttgcaggtgc aaccacaaca tctggttatg ccttcaatgc aaggccatga 13200
atagcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 13260
gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 13320
catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 13380
cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 13440
tatgttacta gatcgtttaa actatcagtg tttgacagga tatattggcg ggtaaaccta 13500
agagaaaaga gcgtttatta gaataacgga tatttaaaag ggcgtgaaaa ggtttatccg 13560
ttcgtccatt tgtatgtg 13578
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgtaagggat gacgcacaat 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cgaggaacag aggctgagct 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttctactggg gaggacatcg 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cggtatcttt gggtgattgg 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cgtaagggat gacgcacaat 20
<210> 10
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caatatagtt cagcgaaccc tga 23
<210> 11
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gaagatctat gtcggatgca ccggcga 27
<210> 12
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gggttaccct attcatggcc ttgcattga 29
<210> 13
<211> 541
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gaagatctat gtcggatgca ccggcgagtc cgagtcacga gagtggtggc gagcagagcc 60
ctcgcggctc gttgtccggc gcggctagag agcaggaccg gtaccttccc attgccaaca 120
tcagccgcat catgaagaag gctctgcctc ccaatggcaa gattgcgaag gatgcaaaag 180
acacaatgca agaatgcgtt tctgaattca tcagcttcat taccagcgag gcgagtgaga 240
aatgccagaa ggagaagaga aagacaatca atggagacga tttactatgg gccatggcaa 300
ctttagggtt tgaagactac attgagccgc ttaaggtgta cctggctagg tacagagagg 360
cggagggtga cactaaagga tctgctagaa gtggtgatgg atctgctaga ccagatcaag 420
ttggccttgc aggtcaaaat gctcagcttg ttcatcaggg ttcgctgaac tatattggtt 480
tgcaggtgca accacaacat ctggttatgc cttcaatgca aggccatgaa tagggtaacc 540
c 541
<210> 14
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cgggatccat ggaggagaac aatcaga 27
<210> 15
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cgagctccta cttttgtgct ctttcaa 27
<210> 16
<211> 1907
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
cgggatccat ggaggagaac aatcagaacc agtgtatccc ttacaattgt ctttctaatc 60
ctgaagaagt tcttttggat ggagaaagga tctcaactgg taactcatca attgacatct 120
ctctctcact tgttcagttc ttggtttcta actttgtgcc aggaggagga ttccttgttg 180
gacttatcga cttcgtttgg ggaatcgttg gaccttctca atgggatgca tttctcgttc 240
agatcgaaca gctcatcaac gaaagaatcg ctgagttcgc taggaatgct gctattgcta 300
accttgaagg acttggaaac aacttcaaca tctacgtgga ggcattcaag gaatgggaag 360
aagatcctaa caacccagca accaggacca gagtgatcga taggttccgt atccttgatg 420
gacttcttga aagggacatt cctagcttta ggatctctgg atttgaagtt ccacttctct 480
ctgtttacgc tcaagctgct aatctccatc ttgctatcct tagagattct gtgatcttcg 540
gagaaagatg gggattgaca accatcaacg tgaacgagaa ctacaacaga ctcatcaggc 600
acatcgatga gtacgctgat cactgtgcta acacttacaa ccgtggactc aacaaccttc 660
ctaagtctac ctatcaagat tggatcacat acaaccgact taggagagac cttacattga 720
ctgttcttga tatcgctgct ttctttccaa actatgacaa taggagatat ccaattcagc 780
cagttggtca acttacaagg gaagtttaca ctgacccact catcaacttc aacccacagc 840
ttcagtctgt tgctcagctt cctaccttca acgttatgga gagcagcgca atcagaaatc 900
ctcacctctt cgacatcttg aacaacctta caatctttac cgattggttt agtgttggac 960
gtaacttcta ctggggagga catcgagtga tctctagcct catcggaggt ggtaacatca 1020
catctcctat ctacggaaga gaggctaacc aggagcctcc aagatcattc actttcaacg 1080
gacctgtgtt caggactctt tcaaatccta ctcttcgact tcttcagcaa ccttggccag 1140
ctccaccatt caaccttcgt ggtgttgaag gagttgagtt ctctacacct acaaacagct 1200
tcacctatcg tggaagaggt actgttgatt ctcttactga acttccacct gaggaaacag 1260
tgtgccacct cgtgaaggat acagtcatcg tctttgtcat gcaaccttcg ttcaaagatc 1320
tggaacacct ttccttacaa ctggtgttgt gttctcttgg actcatcgta gtgcaactct 1380
taccaacaca attgatccag agaggatcaa ccagatccct cttgtgaaag gattcagagt 1440
ttggggagga acctctgtga ttacaggacc aggattcaca ggaggtgata tccttcgaag 1500
aaacaccttt ggtgacttcg tttctcttca agtgaacatc aactcaccaa tcacccaaag 1560
ataccgtctt agatttcgtt acgcttctag tagggatgca cgagttatcg ttcttacagg 1620
agctgcatct acaggagtgg gaggtcaagt tagtgtgaac atgcctcttc agaaaactat 1680
ggagatcgga gagaacctca catctagaac attcagatac accgacttca gtaatccttt 1740
ctcattcaga gctaatccag acatcatcgg tatcagtgaa caacctctct tcggtgcagg 1800
ttctatcagt agcggtgaac tttacatcga caagatcgag atcatccttg cagatgcaac 1860
atttgaagca gaatctgacc ttgaaagagc acaaaagtag gagctcg 1907
<210> 17
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ctcggtacca gattagcctt ttcaatttca 30
<210> 18
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gtataagctt ttaatttcga ctcatcta 28
Claims (7)
1.一种植物重组表达载体pGR20,其特征在于,包含耐除草剂基因表达框、抗虫基因表达框和耐逆基因表达框;
所述耐除草剂基因表达框依次包含CaMV 35S启动子、优化密码子的耐草甘膦基因和CaMV PolyA终止子;
所述耐草甘膦基因包括g2m-epsps;
所述耐除草剂基因表达框的核苷酸序列如SEQ ID NO:1所示的DNA片段;
所述抗虫基因表达框依次包含CaMV 35S启动子、优化密码子的抗虫基因和NOS终止子;所述抗虫基因包括cry1C;
所述抗虫基因表达框的核苷酸序列如SEQ ID NO:2所示的DNA片段;
所述耐逆基因表达框依次包含CaMV 35S启动子、大豆耐逆基因和NOS终止子;所述耐逆基因包括GmNFYB1;
所述耐逆基因表达框的核苷酸序列如SEQ ID NO:3所示的DNA片段。
2.根据权利要求1所述植物重组表达载体pGR20,其特征在于,所述耐除草剂基因表达框的多克隆位点为XhoI;
所述抗虫基因表达框的多克隆位点为KpnI/HindIII;
所述耐逆基因表达框的多克隆位点为BglII/BstEII;
所述重组表达载体pGR20的骨架载体包括pCAMBIA3301。
3.根据权利要求2所述植物重组表达载体pGR20,其特征在于,所述pGR20的核苷酸序列如SEQ ID NO:4所示。
4.权利要求1~3任意一项所述植物重组表达载体pGR20的构建方法,其特征在于,包括以下步骤:
含BglII和BstEII酶切位点的GmNFYB1基因片段和商用载体pCAMBIA3301分别用BglII和BstEII双酶切后连接,构建出p3301-NF载体;
将抗虫基因cry1C和商用载体pBI121分别用BamHI和SacI双酶切后连接,构建出pBI121-crylC载体;
以所述pBI121-crylC载体为模板,利用两端带有KpnI和HindIII酶切位点的特异性引物,扩增得到抗虫基因表达框的片段,与利用KpnI和HindIII酶切的p3301-NF载体连接,构建p3301-NF-cry1C植物表达载体;
利用序列合成的含有XhoI酶切位点的g2m-epsps片段和所述p3301-NF-cry1C载体用XhoI酶切后连接,形成包含耐除草剂基因表达框的植物重组表达载体pGR20。
5.权利要求1~3任一项所述植物重组表达载体pGR20或权利要求4所述构建方法得到的植物重组表达载体pGR20在培育转基因植物中的应用,所述转基因植物为大豆。
6.根据权利要求5所述的应用,其特征在于,所述转基因植物为耐除草剂抗虫耐逆复合性状的转基因植物。
7.一种耐除草剂、抗虫和耐逆的转基因植物的培育方法,包括如下步骤:将权利要求1~3任一项所述的植物重组表达载体pGR20导入受体植物中,得到转基因植物。
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