CN114958881B - 一种大豆基因GmPP2C89及一种过表达载体与应用 - Google Patents
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Abstract
本发明涉及一种大豆基因GmPP2C89及一种过表达载体与应用，属于生物学技术领域。本发明涉及一种大豆基因GmPP2C89，所述大豆基因GmPP2C89的核苷酸序列如SEQIDNO.1所示。将本发明的该基因片段在拟南芥中过表达得到的转基因拟南芥可以提高拟南芥的耐盐性，在非生物胁迫条件下，转基因拟南芥还可以激活抗氧化酶活性，调节植株对非生物胁迫的适应能力，减少非生物胁迫的受损程度。为后期具有抗性的植物研究提供了科学依据。
Description
技术领域
本发明涉及生物学技术领域，尤其涉及一种大豆基因GmPP2C89及一种过表达载体与应用。
背景技术
植物响应非生物胁迫的基因表达调控是一个非常复杂的过程，可以发生在转录、翻译和翻译后修饰等多个层面，其中转录调控最为重要。在转录水平上，基因表达的调控主要通过启动子区的顺式作用元件控制。启动子是基因转录起始密码子上游的DNA序列，包含与转录因子相互作用的顺式作用元件，是转录因子启动或抑制基因表达的重要元件。顺式作用元件作为重要的分子开关，在植物生长发育的各个阶段和环境适应方面都发挥着重要的功能。植物启动子元件在非生物胁迫应答方面的功能也已有较多的报道。拟南芥bZIP转录因子AREB1、AREB2和ABF3通过结合DREB2A启动子中的ABRE元件，激活下游靶基因的表达，进而调控植物对渗透胁迫的响应。杨树ERF76基因启动子区含有多种非生物胁迫响应元件，如MYB、MYC和GT-1元件；并且在转基因拟南芥中ERF76启动子能够受盐胁迫诱导增强GUS报告基因的表达。梭梭树HaFT-3基因启动子，通过转基因拟南芥证明HaFT-3基因启动子受高温、低温、ABA、IAA诱导。番茄SlNAC1基因启动子上包含多种生物和非生物胁迫应答元件。
已有研究指出植物PP2C家族成员在植物盐、干旱等胁迫响应中发挥重要调控作用。拟南芥AtPP2CG1则以ABA依赖性方式对植物耐盐性进行正调控；小麦TaPP2C1过表达可增强转基因烟草的耐盐性。研究表明二穗短柄草BdPP2CA6在拟南芥中过表达导致其生长前期对ABA的超敏感，并通过ABA依赖途径增强拟南芥耐盐性。现有技术并未发现一种对于盐胁迫、渗透胁迫和干旱胁迫均有影响的基因，并将该基因应用于同时抵抗盐胁迫、渗透胁迫和干旱胁迫中的方案。
发明内容
本发明的目的在于提供一种大豆基因GmPP2C89及一种过表达载体与应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种大豆基因GmPP2C89，所述大豆基因GmPP2C89的核苷酸序列如SEQ ID NO.1所示。
本发明还提供了一种过表达载体，包括所述的大豆基因GmPP2C89以及空载载体pFGC5941。
本发明还提供了一种所述的大豆基因GmPP2C89在提高植物对于非生物胁迫抗性中的用途。
作为优选，所述大豆基因GmPP2C89在提高植物对于非生物胁迫抗性时的方法为：将大豆基因GmPP2C89在植物体内过表达。
作为优选，所述过表达为将含有大豆基因GmPP2C89的过表达载体转入植物体中；
所述过表达载体为所述的过表达载体；
所述转入方法为农杆菌介导的浸花法。
作为优选，所述植物为拟南芥。
作为优选，所述非生物胁迫为盐胁迫、干旱胁迫和渗透胁迫中的一种或几种。
本发明还提供了所述的过表达载体在提高植物对于非生物胁迫抗性中的用途。
作为优选，所述过表达载体在提高植物对于非生物胁迫抗性时的方法为：将过表达载体转入植物体中。
作为优选，所述转入的方法为农杆菌介导的浸花法；
所述非生物胁迫为盐胁迫、干旱胁迫和渗透胁迫中的一种或几种；
所述植物为拟南芥。
本发明提供了一种大豆基因GmPP2C89及一种过表达载体与应用。本发明的大豆基因GmPP2C89是通过对正常生长和盐处理的栽培大豆进行转录组测序，在转录组中筛选得到的一个平均FPKM值较对照上调倍数高达42倍的基因，在20％的聚乙二醇(PEG)和300mmol/L甘露醇溶液处理的大豆幼苗中该基因的表达水平也升高。说明本发明的GmPP2C89不仅与盐胁迫有关，还与干旱、渗透胁迫有关。
将本发明的基因制备成过表达载体，转入拟南芥植物中得到的转基因拟南芥在含100mmol/LNaCl的MS上的生长优势优于野生型拟南芥，转基因拟南芥在盐胁迫条件下较野生型拟南芥受伤害程度小，耐盐性更强。这说明本发明的基因与非生物胁迫有关，为抗性植物的筛选提供理论基础。
附图说明
图1为GmPP2C89基因在非生物胁迫下的表达水平(其中A图为盐处理后大豆GmPP2C89基因的FPKM值；B图为20％PEG处理后GmPP2C89基因的相对表达量；C图为300mmol/L甘露醇溶液处理后GmPP2C89基因的相对表达量)。
图2为GmPP2C89基因启动子区非生物胁迫响应元件的分布图。
图3为不同长度的GmPP2C89基因启动子克隆及GUS载体构建(其中A为不同长度启动子及顺式作用元件位置；B为不同片段启动子的电泳图；C为载体构建成功酶切电泳图)。
图4为转基因拟南芥筛选鉴定及不同胁迫处理后的GUS染色分析(其中A～D为不同长度的启动子融合GUS载体转化的拟南芥筛选和PCR鉴定；E为盐、PEG、甘露醇处理后的GUS染色结果图)。
图5为过表达大豆GmPP2C89基因增强转基因拟南芥的耐盐性(其中A～B为MS培养基和含100mmol/LNaCl的MS培养基上的根长表型和统计；C～E为4周的拟南芥经150mmol/LNaCl处理10d的表型和MDA含量、电解质渗透率)。
图6为拟南芥植株WT和GmPP2C89-OX中胁迫相关基因的表达模式。
具体实施方式
本发明提供了一种大豆基因GmPP2C89，所述大豆基因GmPP2C89的核苷酸序列如SEQ ID NO.1所示。
本发明还提供了一种过表达载体，包括所述的大豆基因GmPP2C89以及空载载体pFGC5941。在本发明中，所述空载载体pFGC5941的核苷酸序列如SEQ ID NO.2所示。在本发明中，所述过表达载体为pFGC5941::GmPP2C89；所述pFGC5941::GmPP2C89的序列为SEQ IDNO.3所示。在本发明中，所述pFGC5941::GmPP2C89的构建方法为：以大豆cDNA为模板，使用引物GmPP2C89F:CTCGAGATGTTCAGTTCGTTGCATGC和GmPP2C89R:TCTAGATCAATCAGTTGTTACTGATCCTCT克隆GmPP2C89基因的CDS片段，对PCR产物进行纯化后得到CDS片段，使用T4连接酶将目的片段连入pFGC5941载体的Xho I和Xba I酶切位点之间，即获得过表达载体pFGC5941::GmPP2C89。
本发明还提供了一种所述的大豆基因GmPP2C89在提高植物对于非生物胁迫抗性中的用途。
在本发明中，所述大豆基因GmPP2C89在提高植物对于非生物胁迫抗性时的方法为：将大豆基因GmPP2C89在植物体内过表达。在本发明中，所述过表达为将含有大豆基因GmPP2C89的过表达载体转入植物体中；所述过表达载体为上述所述的过表达载体；所述转入方法为农杆菌介导的浸花法。在本发明中，所述植物为拟南芥。在本发明中，所述非生物胁迫为盐胁迫、干旱胁迫和渗透胁迫中的一种或几种。
本发明还提供了所述的过表达载体在提高植物对于非生物胁迫抗性中的用途。
在本发明中，所述过表达载体在提高植物对于非生物胁迫抗性时的方法为：将过表达载体转入植物体中。在本发明中，所述转入的方法为农杆菌介导的浸花法；所述非生物胁迫为盐胁迫、干旱胁迫和渗透胁迫中的一种或几种；所述植物为拟南芥。
下面结合实施例对本发明提供的技术方案进行详细的说明，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
基因筛选
将大豆(Glycine max)播种于营养土和蛭石混合(1:1)基质中，用自来水浇灌，在温度为25℃，光照条件为16h昼/8h夜，空气湿度为65％的温室中生长，作为正常生长的大豆。
将大豆(Glycine max)播种于营养土和蛭石混合(1:1)基质中，浓度为150mmol/L的NaCl水溶液浇灌，在温度为25℃，光照条件为16h昼/8h夜，空气湿度为65％的温室中生长，作为盐处理的大豆。
处理10d后，提取正常生长的大豆和盐处理的大豆的总RNA，反转录获得cDNA后进行转录组测序，测序的结果数据已上传NCBI数据库(登陆号：PRJNA741583)中。对比测序结果的数据发现了一个盐处理后FPKM值上调42倍的基因为GmPP2C89。结果如图1所示。
利用20％PEG、300mmol/L甘露醇溶液分别处理生长10d的大豆幼苗，分别在0，6，12，24h时取叶片提取总RNA。利用反转录试剂盒SuperScript III First-StrandSynthesis SuperMix(ThermoFisher scientific)合成cDNA，使用荧光定量PCR仪(CFX96，Bio-Rad，美国)进行荧光定量PCR检测。利用2-△△CT法计算GmPP2C89基因的相对表达量，结果如图1所示。大豆UBI3基因和拟南芥ACTIN基因作为内参，该方法中的PCR引物如表1和SEQID NO.4～SEQ ID NO.26所示。
表1PCR引物
图1结果显示，正常生长的大豆中GmPP2C89基因的FPKM值分别为0.18713、0.296728、0.140107，盐处理的大豆中GmPP2C89基因的FPKM值分别为5.71465、10.0462、10.3251，计算各组中平均值后得出盐处理的大豆中GmPP2C89基因的FPKM值较正常生长即对照组大豆中GmPP2C89基因的FPKM值上调42倍。
20％PEG处理大豆幼苗12h后GmPP2C89基因的表达水平较0h时GmPP2C89基因的表达水平上调最高为25；甘露醇处理6h后GmPP2C89基因的表达水平较0h时上调倍数最高为28倍。
实施例2
GmPP2C89基因启动子区参与非生物胁迫的元件
根据大豆基因组数据库(https://soykb.org/)中启动子序列设计引物，引物如表1和SEQ ID NO.4～SEQ ID NO.26所示。以大豆基因组DNA为模板克隆启动子片段，得到2000bp的片段，并对其进行测序。将启动子序列提交到在线数据库PlantCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)进行顺式作用元件预测，统计非生物胁迫相关的元件位置和数量。结果如图2所示。
图2显示，利用PlantCARE数据库对GmPP2C89基因转录起始密码子ATG上游的2000bp序列进行顺式作用元件分析。发现该启动子区含有较多参与非生物胁迫响应的元件，包括ABRE、DRE、G-box、MBS、MYB、MYC和TC-rich repeats。其中ABRE元件数量最多，共4个；MYC元件3个；G-box、MBS、MYB和TC-rich repeats元件各2个；DRE元件1个。说明GmPP2C89基因可以参与非生物胁迫。
实施例3
构建GUS融合载体
在克隆出2000bp的启动子片段基础上，根据顺式作用元件的功能和分布区域，将启动子片段分成3个不同长度的缺失片段，即本研究共包含4个启动子片段：启动子全长片段(P1)、3个5'端缺失片段(P2、P3和P4)。相比全长启动子，P2片段缺失了MYB、MYC和ABRE元件各1个；P3片段缺失了MYB、MYC和ABRE元件各2个，DRE和TC-rich repeats元件各1个；而P4片段缺失了全部的非生物胁迫响应元件，仅保留了核心启动子区。如图3A所示。
以大豆DNA为模板，使用高保真DNA聚合酶克隆得到长度分别为2000、1370、980、240bp的启动子片段如图3B。利用同源重组技术将启动子片段插入PBI101载体以构建重组载体，并进行酶切验证，结果如图3C所示。
通过图3可以看出，本发明得到的GUS融合载体扩增得到的产物的序列长度分别为2000、1370、980、240bp，说明本发明的GUS融合载体构建成功。
实施例4
转基因拟南芥的获得及GUS染色分析
将实施例3得到的GUS融合载体利用农杆菌介导的浸花法转化野生型拟南芥，利用卡那霉素初步筛选得到抗性植株，并进一步进行PCR鉴定获得转基因植株如图4A～D所示，得到的转基因植物用P1::GUS、P2::GUS、P3::GUS、P4::GUS表示。将转基因植株幼苗浸没到GUS染色液，用真空泵抽真空5min，然后置于37℃，避光12h后。将GUS染液回收后，加入75％乙醇脱色(除去叶绿素)，然后拍照观察GUS染色情况，结果如图4E所示。所述GUS染色液的配置方法为：1.44mL 1mol/LNa2HPO4、1.06mL 1mol/L NaH2PO4、1mL 100mmol/L K4Fe(CN)6·3H2O、1mL 100mmol/LK3Fe(CN)6、500μL 500mmol/L EDTA、50μL TritonX-100、20mg X-Gluc、用去离子水定容至50mL。
分别用150mmol/LNaCl、20％PEG以及300mmol/L甘露醇处理转基因拟南芥幼苗48h后，取叶片浸没到GUS染色液，用真空泵抽真空5min，然后置于37℃，避光12h后。将GUS染液回收后，加入75％乙醇脱色(除去叶绿素)，然后拍照观察GUS染色情况，结果如图4E所示。
图4显示，正常培养转基因拟南芥P1::GUS、P2::GUS、P3::GUS和P4::GUS叶片都有较浅的蓝色，说明截断的启动子序列也能在正常条件下启动基因的表达。在NaCl处理后，P1::GUS和P2::GUS植株叶片的GUS染色明显变深，P3::GUS叶片次之，P4::GUS叶片GUS表达则未被盐处理诱导。PEG模拟干旱处理也导致P1::GUS和P2::GUS叶片的GUS染色变深，P3::GUS和P4::GUS叶片中GUS都未被诱导。甘露醇处理则表现出与盐处理类似的现象，但是GUS染色相对较浅。说明GmPP2C89基因启动子区的非生物胁迫响应元件在盐、干旱和渗透胁迫中发挥特定功能。
实施例5
GmPP2C89基因过表达增强转基因拟南芥耐盐性
克隆GmPP2C89的CDS序列，构建过表达载体pFGC5941::GmPP2C89，利用农杆菌介导的浸花法转化野生型拟南芥。收获种子后，经过卡那霉素或草铵膦筛选并进行PCR鉴定后，得到转基因拟南芥GmPP2C89-OX。将转基因拟南芥GmPP2C89-OX的种子经75％酒精消毒后播种于MS培养基和含有100mmol/LNaCl的MS培养基上，在生长室垂直放置培养7d，统计根长，与野生型拟南芥WT种子分别在MS培养基和含有100mmol/LNaCl的MS培养基上的生长情况为对照。根长结果如图5A～B所示。拟南芥在营养土中正常生长4周后，用150mmol/LNaCl水溶液处理10d，测定拟南芥叶片中MDA含量和电解质渗透率。此外，提取拟南芥叶片RNA，利用荧光定量PCR检测抗氧化酶基因(SOD、POD和CAT)和ABA途径关键基因(RD26、RD29A和RD29B)的表达水平，结果如图5C～D所示。培养室条件：温度25℃，长日照(昼16h/夜8h)，50％～70％相对湿度。
图5显示，在正常MS培养基上，野生型拟南芥和GmPP2C89-OX拟南芥根长无明显差异，而在含100mmol/LNaCl的MS上GmPP2C89-OX根长显著高于野生型拟南芥。对生长4周的拟南芥植株进行盐处理，结果显示野生型拟南芥和GmPP2C89-OX的生长都受到抑制，但GmPP2C89-OX的长势明显优于野生型拟南芥。测定正常和盐处理条件下拟南芥叶片中的MDA含量和电解质渗透率，发现盐处理条件下拟南芥GmPP2C89-OX叶片MDA含量和电解质渗透率显著低于野生型，说明拟南芥GmPP2C89-OX在盐胁迫条件下受伤害较轻、其耐盐性增强。
实施例6
GmPP2C89过表达植株中胁迫相关基因表达发生改变
为了探究GmPP2C89介导的信号途径，利用荧光定量PCR技术检测了参与胁迫响应的抗氧化酶基因(SOD、POD和CAT)和ABA途径关键基因(RD26、RD29A和RD29B)的表达水平。PCR反应总体系为20μL：10μL SYBR Green Mix，8μL RNAase-free水，正向和反向引物各0.5μL，cDNA模板1μL。利用荧光定量PCR仪进行PCR扩增反应，反应程序：94℃，预变性32min；94℃变性10s，60℃退火10s，72℃延伸10s，共40个循环。利用2－ΔΔCT法计算基因相对表达水平，大豆UBI3基因作为内参。结果如图6所示。
图6显示，盐处理后这些基因在野生型拟南芥和GmPP2C89-OX中都明显上调；其中SOD、POD和RD29B在GmPP2C89-OX中的表达水平显著高于野生型拟南芥；而CAT、RD26和RD29A在野生型拟南芥和GmPP2C89-OX间无明显差异。这一结果证实GmPP2C89通过调控胁迫相关基因的表达，增强拟南芥对盐胁迫的适应能力。
由以上实施例可知，本发明提供了一种大豆基因GmPP2C89及一种过表达载体与应用。本发明的大豆基因GmPP2C89是通过对正常生长和盐处理的栽培大豆进行转录组测序，在转录组中筛选得到的一个平均FPKM值较对照上调倍数高达42倍的基因，在20％的聚乙二醇(PEG)和300mmol/L甘露醇溶液处理的大豆幼苗中该基因的表达水平也升高。说明本发明的GmPP2C89不仅与盐胁迫有关，还与干旱、渗透胁迫有关。将本发明的基因在拟南芥中过表达得到的转基因拟南芥可以提高拟南芥的耐盐性，在非生物胁迫条件下，转基因拟南芥还可以激活抗氧化酶活性，调节植株对非生物胁迫的适应能力，减少非生物胁迫的受损程度。为后期具有抗性的植物研究提供了科学依据。
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> 湖南科技学院
<120> 一种大豆基因GmPP2C89及一种过表达载体与应用
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1161
<212> DNA
<213> 人工序列(Artificial Sequence)
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atgttcagtt cgttgcatgc aaaaacgatt atgaagacgc caaaacgcca ctcgccgccg 60
ccaaattctt ccctgcgagt ggagtctgat tccggcgaaa tcgtcacgag gttcaagaac 120
ggtcgccgga gaagaattaa aatcaagcga atgaaataca cttgccagac gaagatccgc 180
gtcaggaatg acgccggcgc cggcggcgtg tctcaacttc tggcggtgga ttctgaagga 240
gagaagagag agattcacga gaacgtggag atatcgctgt cgctggcttc gtcgtcgtcg 300
gaagaggaag agcgattgtc gtcgaagcaa aacgacggcg ttttgtcgta cgggtcggca 360
tcggtgatag ggagcaggaa ggagatggag gacgcggtga gtgaggagat agggtttgct 420
gcgaagtgcg acttcttcgc ggtgtacgac ggccacggcg gagctcaggt ggcggaggcg 480
tgcagggaga ggctgtacag gctggtggcg gaggagatgg agaggagtgc gagtcacgtg 540
gaatgggact ggcggggagt gatggaaggg tgttttcgga aaatggactg cgaggttgcc 600
ggcaatgcgg ctgtcaggac ggtgggttcc acggctgttg tcgccgtcgt cgcggcggcg 660
gaggttgtcg ttgctaattg cggtgattgt agggccgtcc tgggaagagg aggtgaggcc 720
gtggacttgt ctagtgatca taagcccgac aggcccgatg agttgatacg aattgaagag 780
gctggtggaa gggtcattaa ctggaatggc cagcgcgtgc ttggtgttct tgccacttca 840
cgatccatag gagatcagta ccttcgacct tatgtaatat caaaaccaga agtgacagtg 900
accaagcgaa gcagcaagga tgaatttctc atactagcaa gtgatggatt gtgggatgtg 960
atgtcaagtg aagttgcgtg ccaagttgta aggaaatgct tccacgggca gattaggaga 1020
gtgtgcgatg gagttgggaa tcatcaaaac cgtgctaccg aggctgcagg cctcttggca 1080
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ttgaaaattc ataagaaaag caaacgttac atgaattgat gaaacaatac aaagacagat 180
aaagccacgc acatttagga tattggccga gattactgaa tattgagtaa gatcacggaa 240
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ccatatgcag gagcggatca ttcattgttt gtttggttgc ctttgccaac atgggagtcc 360
aagattctgc agttagatct cggtgacggg caggaccgga cggggcggta ccggcaggct 420
gaagtccagc tgccagaaac ccacgtcatg ccagttcccg tgcttgaagc cggccgcccg 480
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cagcccgatg acagcgacca cgctcttgaa gccctgtgcc tccagggact tcagcaggtg 600
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gacctcgccg tccacctcgg cgacgagcca gggatagcgc tcccgcagac ggacgaggtc 780
gtccgtccac tcctgcggtt cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat 840
gtagtggttg acgatggtgc agaccgccgg catgtccgcc tcggtggcac ggcggatgtc 900
ggccgggcgt cgttctgggc tcatcgattc gatttggtgt atcgagattg gttatgaaat 960
tcagatgcta gtgtaatgta ttggtaattt gggaagatat aataggaagc aaggctattt 1020
atccatttct gaaaaggcga aatggcgtca ccgcgagcgt cacgcgcatt ccgttcttgc 1080
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caaaagtcgt taatggctgc ggatcaagaa aaagttggaa tagaaacaga atacccgcga 1200
aattcaggcc cggttgccat gtcctacacg ccgaaataaa cgaccaaatt agtagaaaaa 1260
taaaaactag ctcagatact tacgtcacgt cttgcgcact gatttgaaaa atctcagaat 1320
tccaatccca caaaaatctg agcttaacag cacagttgct cctctcagag cagaatcggg 1380
tattcaacac cctcatatca actactacgt tgtgtataac ggtccacatg ccggtatata 1440
cgatgactgg ggttgtacaa aggcggcaac aaacggcgtt cccggagttg cacacaagaa 1500
atttgccact attacagagg caagagcagc agctgacgcg tacacaacaa gtcagcaaac 1560
agacaggttg aacttcatcc ccaaaggaga agctcaactc aagcccaaga gctttgctaa 1620
ggccctaaca agcccaccaa agcaaaaagc ccactggctc acgctaggaa ccaaaaggcc 1680
cagcagtgat ccagccccaa aagagactcc tttgccccgg agattacaat ggacgatttc 1740
ctctatcttt acgatctagg aaggaagttc gaaggtgaag gtgacgacac tatgttcacc 1800
actgataatg agaaggttag cctcttcaat ttcagaaaga atgctgaccc acagatggtt 1860
agagaggcct acgcagcagg tctcatcaag acgatctacc cgagtaacaa tctccaggag 1920
atcaaatacc ttcccaagaa ggttaaagat gcagtcaaaa gattcaggac taattgcatc 1980
aagaacacag agaaagacat atttctcaag atcagaagta ctattccagt atggacgatt 2040
caaggcttgc ttcataaacc aaggcaagta atagagattg gagtctctaa aaaggtagtt 2100
cctactgaat ctaaggccat gcatggagtc taagattcaa atcgaggatc taacagaact 2160
cgccgtgaag actggcgaac agttcataca gagtctttta cgactcaatg acaagaagaa 2220
aatcttcgtc aacatggtgg agcacgacac tctggtctac tccaaaaatg tcaaagatac 2280
agtctcagaa gaccaaaggg ctattgagac ttttcaacaa aggataattt cgggaaacct 2340
cctcggattc cattgcccag ctatctgtca cttcatcgaa aggacagtag aaaaggaagg 2400
tggctcctac aaatgccatc attgcgataa aggaaaggct atcattcaag atctctctgc 2460
cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 2520
tccaaccacg tcttcaaagc aagtggattg atgtgacatc tccactgacg taagggatga 2580
cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt 2640
ggagaggaca cgctcgagta taagagctct atttttacaa caattaccaa caacaacaaa 2700
caacaaacaa cattacaatt acatttacaa ttaccatggg gcgcgcccaa tcgatgattt 2760
aaatgtgtaa gaatttctta tgttacatta ttacattcaa cgttttatct taattggctc 2820
ttcatttgat tgaaatttga caattatttc ttgttttttt ttttgtcaca ctctttttgg 2880
gttggggtgg ccgacgaatt gtgggaaggt agaaagaggg gaggactttt gttatactcc 2940
attagtaatt actgtttccg tttcaattta tgtgacaata tttccttttt agtcggttcc 3000
aaaagaaaat gtcagcatta taaacaattt aattttgaaa ttacaatttt gccattaata 3060
aaatgattta caaccacaaa agtatctatg agcctgtttg ggtgggctta taagcagctt 3120
attttaagtg gcttataagt caaaaagtga cantttttga gaagttagaa aatcctaact 3180
tctcaaaaag tagcttttaa gccacttatg acttataagt ccaaaaattt ttaagttacc 3240
aaacatatat taatgggttt ataagcttat aagccacttt taagctcacc caaacgggtt 3300
ctatgtctca ctttagacta caaattttaa aagtcttcat ttatttctta atctccgtgg 3360
cgagtnaaac tataacacat aaagtgaaac ggagggaata agatggagtc ataaactaat 3420
ccaaatctat actctctccg ttaatttgtt ttttagtttg atttggtaca ttaataaaac 3480
agatttttcg aaggttataa acacagacag atgtttccca gcgagctagc aaaattccaa 3540
gatttctgtc gaaaattcgt gtgtttctag ctagtacttg atgttatctt taacctttta 3600
gtaatttttt gtccttttct ttctattttt catcttacaa tgaattatga gcaagttcct 3660
taagtagcat cacacgtgag atgtttttta tgatattgac taaatccaat ctttaccatt 3720
ccttaactag taaaatacaa cacatgttaa ttgatacatt gcttaacact gaggttagaa 3780
aattttagaa attagttgtc caaatgcttt gaaattagaa atctttaatc ccttattttt 3840
ttttaaaatg ttttttctca ctccaaagaa agagaaactg acatgaaagc tcaaaagatc 3900
atgaatctta ctaactttgt ggaactaaat gtacatcaga atgtttctga catgtgaaaa 3960
tgaaagctct taattttctt cttttattta ttgagggttt ttgcatgcta tgcattcaat 4020
ttgagtactt taaagcacct ataaacactt acttacactt gccttggagt ttatgtttta 4080
gtgttttctt cacatctttt ttggtcaatt tgcaggtatt tggatcctag gtgagtctag 4140
agagttaatt aagacccggg actagtccct agagtcctgc tttaatgaga tatgcgagac 4200
gcctatgatc gcatgatatt tgctttcaat tctgttgtgc acgttgtaaa aaacctgagc 4260
atgtgtagct cagatcctta ccgccggttt cggttcattc taatgaatat atcacccgtt 4320
actatcgtat ttttatgaat aatattctcc gttcaattta ctgattgtac cctactactt 4380
atatgtacaa tattaaaatg aaaacaatat attgtgctga ataggtttat agcgacatct 4440
atgatagagc gccacaataa caaacaattg cgttttatta ttacaaatcc aattttaaaa 4500
aaagcggcag aaccggtcaa acctaaaaga ctgattacat aaatcttatt caaatttcaa 4560
aagtgcccca ggggctagta tctacgacac accgagcggc gaactaataa cgctcactga 4620
agggaactcc ggttccccgc cggcgcgcat gggtgagatt ccttgaagtt gagtattggc 4680
cgtccgctct accgaaagtt acgggcacca ttcaacccgg tccagcacgg cggccgggta 4740
accgacttgc tgccccgaga attatgcagc atttttttgg tgtatgtggg ccccaaatga 4800
agtgcaggtc aaaccttgac agtgacgaca aatcgttggg cgggtccagg gcgaattttg 4860
cgacaacatg tcgaggctca gcaggacctg caggcatgca agcttggcac tggccgtcgt 4920
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 4980
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 5040
gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga ttgtcgtttc 5100
ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga 5160
aaagagcgtt tattagaata acggatattt aaaagggcgt gaaaaggttt atccgttcgt 5220
ccatttgtat gtgcatgcca accacagggt tcccctcggg atcaaagtac tttgatccaa 5280
cccctccgct gctatagtgc agtcggcttc tgacgttcag tgcagccgtc ttctgaaaac 5340
gacatgtcgc acaagtccta agttacgcga caggctgccg ccctgccctt ttcctggcgt 5400
tttcttgtcg cgtgttttag tcgcataaag tagaatactt gcgactagaa ccggagacat 5460
tacgccatga acaagagcgc cgccgctggc ctgctgggct atgcccgcgt cagcaccgac 5520
gaccaggact tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac caagctgttt 5580
tccgagaaga tcaccggcac caggcgcgac cgcccggagc tggccaggat gcttgaccac 5640
ctacgccctg gcgacgttgt gacagtgacc aggctagacc gcctggcccg cagcacccgc 5700
gacctactgg acattgccga gcgcatccag gaggccggcg cgggcctgcg tagcctggca 5760
gagccgtggg ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc 5820
attgccgagt tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc 5880
aaggcccgag gcgtgaagtt tggcccccgc cctaccctca ccccggcaca gatcgcgcac 5940
gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag aggcggctgc actgcttggc 6000
gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag 6060
gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc cctggcggcc 6120
gccgagaatg aacgccaaga ggaacaagca tgaaaccgca ccaggacggc caggacgaac 6180
cgtttttcat taccgaagag atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc 6240
cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct ggccggtttg tctgatgcca 6300
agctggcggc ctggccggcc agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa 6360
ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat 6420
gagtaaataa acaaatacgc aaggggaacg catgaaggtt atcgctgtac ttaaccagaa 6480
aggcgggtca ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg 6540
ggccgatgtt ctgttagtcg attccgatcc ccagggcagt gcccgcgatt gggcggccgt 6600
gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt 6660
gaaggccatc ggccggcgcg acttcgtagt gatcgacgga gcgccccagg cggcggactt 6720
ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc caagccctta 6780
cgacatatgg gccaccgccg acctggtgga gctggttaag cagcgcattg aggtcacgga 6840
tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg 6900
tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt cttgagtccc gtatcacgca 6960
gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc gttcttgaat cagaacccga 7020
gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa aactcatttg 7080
agttaatgag gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg 7140
agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc agccatgaag 7200
cgggtcaact ttcagttgcc ggcggaggat cacaccaagc tgaagatgta cgcggtacgc 7260
caaggcaaga ccattaccga gctgctatct gaatacatcg cgcagctacc agagtaaatg 7320
agcaaatgaa taaatgagta gatgaatttt agcggctaaa ggaggcggca tggaaaatca 7380
agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc 7440
aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc actggaaccc ccaagcccga 7500
ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg 7560
atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag 7620
aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc 7680
aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag 7740
attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg 7800
tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc 7860
ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt 7920
acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag 7980
ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct 8040
gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa 8100
acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg 8160
tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc 8220
cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga 8280
acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt 8340
ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga 8400
cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca 8460
agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg 8520
gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct 8580
aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaaggtc 8640
tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc 8700
cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata 8760
taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta 8820
aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag 8880
cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg 8940
ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag 9000
ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg caccctgcct cgcgcgtttc 9060
ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 9120
taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 9180
cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg 9240
cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 9300
gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc 9360
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 9420
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 9480
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 9540
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 9600
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 9660
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 9720
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 9780
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 9840
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 9900
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 9960
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 10020
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 10080
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 10140
ggaacgaaaa ctcacgttaa gggattttgg tcatgcattc taggtactaa aacaattcat 10200
ccagtaaaat ataatatttt attttctccc aatcaggctt gatccccagt aagtcaaaaa 10260
atagctcgac atactgttct tccccgatat cctccctgat cgaccggacg cagaaggcaa 10320
tgtcatacca cttgtccgcc ctgccgcttc tcccaagatc aataaagcca cttactttgc 10380
catctttcac aaagatgttg ctgtctccca ggtcgccgtg ggaaaagaca agttcctctt 10440
cgggcttttc cgtctttaaa aaatcataca gctcgcgcgg atctttaaat ggagtgtctt 10500
cttcccagtt ttcgcaatcc acatcggcca gatcgttatt cagtaagtaa tccaattcgg 10560
ctaagcggct gtctaagcta ttcgtatagg gacaatccga tatgtcgatg gagtgaaaga 10620
gcctgatgca ctccgcatac agctcgataa tcttttcagg gctttgttca tcttcatact 10680
cttccgagca aaggacgcca tcggcctcac tcatgagcag attgctccag ccatcatgcc 10740
gttcaaagtg caggaccttt ggaacaggca gctttccttc cagccatagc atcatgtcct 10800
tttcccgttc cacatcatag gtggtccctt tataccggct gtccgtcatt tttaaatata 10860
ggttttcatt ttctcccacc agcttatata ccttagcagg agacattcct tccgtatctt 10920
ttacgcagcg gtatttttcg atcagttttt tcaattccgg tgatattctc attttagcca 10980
tttattattt ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa 11040
caagacgaac tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc 11100
tttttcaaag ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa 11160
accgcggtga tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg 11220
cgagatcatc cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt 11280
aacatgagca aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat 11340
gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg 11400
gctgg 11405
<210> 3
<211> 11089
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 60
gacgttttta atgtactgaa ttaacgccga attaattcga gctcggatct gataatttat 120
ttgaaaattc ataagaaaag caaacgttac atgaattgat gaaacaatac aaagacagat 180
aaagccacgc acatttagga tattggccga gattactgaa tattgagtaa gatcacggaa 240
tttctgacag gagcatgtct tcaattcagc ccaaatggca gttgaaatac tcaaaccgcc 300
ccatatgcag gagcggatca ttcattgttt gtttggttgc ctttgccaac atgggagtcc 360
aagattctgc agttagatct cggtgacggg caggaccgga cggggcggta ccggcaggct 420
gaagtccagc tgccagaaac ccacgtcatg ccagttcccg tgcttgaagc cggccgcccg 480
cagcatgccg cggggggcat atccgagcgc ctcgtgcatg cgcacgctcg ggtcgttggg 540
cagcccgatg acagcgacca cgctcttgaa gccctgtgcc tccagggact tcagcaggtg 600
ggtgtagagc gtggagccca gtcccgtccg ctggtggcgg ggggagacgt acacggtcga 660
ctcggccgtc cagtcgtagg cgttgcgtgc cttccagggg cccgcgtagg cgatgccggc 720
gacctcgccg tccacctcgg cgacgagcca gggatagcgc tcccgcagac ggacgaggtc 780
gtccgtccac tcctgcggtt cctgcggctc ggtacggaag ttgaccgtgc ttgtctcgat 840
gtagtggttg acgatggtgc agaccgccgg catgtccgcc tcggtggcac ggcggatgtc 900
ggccgggcgt cgttctgggc tcatcgattc gatttggtgt atcgagattg gttatgaaat 960
tcagatgcta gtgtaatgta ttggtaattt gggaagatat aataggaagc aaggctattt 1020
atccatttct gaaaaggcga aatggcgtca ccgcgagcgt cacgcgcatt ccgttcttgc 1080
tgtaaagcgt tgtttggtac acttttgact agcgaggctt ggcgtgtcag cgtatctatt 1140
caaaagtcgt taatggctgc ggatcaagaa aaagttggaa tagaaacaga atacccgcga 1200
aattcaggcc cggttgccat gtcctacacg ccgaaataaa cgaccaaatt agtagaaaaa 1260
taaaaactag ctcagatact tacgtcacgt cttgcgcact gatttgaaaa atctcagaat 1320
tccaatccca caaaaatctg agcttaacag cacagttgct cctctcagag cagaatcggg 1380
tattcaacac cctcatatca actactacgt tgtgtataac ggtccacatg ccggtatata 1440
cgatgactgg ggttgtacaa aggcggcaac aaacggcgtt cccggagttg cacacaagaa 1500
atttgccact attacagagg caagagcagc agctgacgcg tacacaacaa gtcagcaaac 1560
agacaggttg aacttcatcc ccaaaggaga agctcaactc aagcccaaga gctttgctaa 1620
ggccctaaca agcccaccaa agcaaaaagc ccactggctc acgctaggaa ccaaaaggcc 1680
cagcagtgat ccagccccaa aagagactcc tttgccccgg agattacaat ggacgatttc 1740
ctctatcttt acgatctagg aaggaagttc gaaggtgaag gtgacgacac tatgttcacc 1800
actgataatg agaaggttag cctcttcaat ttcagaaaga atgctgaccc acagatggtt 1860
agagaggcct acgcagcagg tctcatcaag acgatctacc cgagtaacaa tctccaggag 1920
atcaaatacc ttcccaagaa ggttaaagat gcagtcaaaa gattcaggac taattgcatc 1980
aagaacacag agaaagacat atttctcaag atcagaagta ctattccagt atggacgatt 2040
caaggcttgc ttcataaacc aaggcaagta atagagattg gagtctctaa aaaggtagtt 2100
cctactgaat ctaaggccat gcatggagtc taagattcaa atcgaggatc taacagaact 2160
cgccgtgaag actggcgaac agttcataca gagtctttta cgactcaatg acaagaagaa 2220
aatcttcgtc aacatggtgg agcacgacac tctggtctac tccaaaaatg tcaaagatac 2280
agtctcagaa gaccaaaggg ctattgagac ttttcaacaa aggataattt cgggaaacct 2340
cctcggattc cattgcccag ctatctgtca cttcatcgaa aggacagtag aaaaggaagg 2400
tggctcctac aaatgccatc attgcgataa aggaaaggct atcattcaag atctctctgc 2460
cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 2520
tccaaccacg tcttcaaagc aagtggattg atgtgacatc tccactgacg taagggatga 2580
cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt 2640
ggagaggaca cgctcgagat gttcagttcg ttgcatgcaa aaacgattat gaagacgcca 2700
aaacgccact cgccgccgcc aaattcttcc ctgcgagtgg agtctgattc cggcgaaatc 2760
gtcacgaggt tcaagaacgg tcgccggaga agaattaaaa tcaagcgaat gaaatacact 2820
tgccagacga agatccgcgt caggaatgac gccggcgccg gcggcgtgtc tcaacttctg 2880
gcggtggatt ctgaaggaga gaagagagag attcacgaga acgtggagat atcgctgtcg 2940
ctggcttcgt cgtcgtcgga agaggaagag cgattgtcgt cgaagcaaaa cgacggcgtt 3000
ttgtcgtacg ggtcggcatc ggtgataggg agcaggaagg agatggagga cgcggtgagt 3060
gaggagatag ggtttgctgc gaagtgcgac ttcttcgcgg tgtacgacgg ccacggcgga 3120
gctcaggtgg cggaggcgtg cagggagagg ctgtacaggc tggtggcgga ggagatggag 3180
aggagtgcga gtcacgtgga atgggactgg cggggagtga tggaagggtg ttttcggaaa 3240
atggactgcg aggttgccgg caatgcggct gtcaggacgg tgggttccac ggctgttgtc 3300
gccgtcgtcg cggcggcgga ggttgtcgtt gctaattgcg gtgattgtag ggccgtcctg 3360
ggaagaggag gtgaggccgt ggacttgtct agtgatcata agcccgacag gcccgatgag 3420
ttgatacgaa ttgaagaggc tggtggaagg gtcattaact ggaatggcca gcgcgtgctt 3480
ggtgttcttg ccacttcacg atccatagga gatcagtacc ttcgacctta tgtaatatca 3540
aaaccagaag tgacagtgac caagcgaagc agcaaggatg aatttctcat actagcaagt 3600
gatggattgt gggatgtgat gtcaagtgaa gttgcgtgcc aagttgtaag gaaatgcttc 3660
cacgggcaga ttaggagagt gtgcgatgga gttgggaatc atcaaaaccg tgctaccgag 3720
gctgcaggcc tcttggcaga gatagcattg gcaaagggaa gtagagataa caccagtgtc 3780
attgtagttg agctaagagg atcagtaaca actgattgat ctagagagtt aattaagacc 3840
cgggactagt ccctagagtc ctgctttaat gagatatgcg agacgcctat gatcgcatga 3900
tatttgcttt caattctgtt gtgcacgttg taaaaaacct gagcatgtgt agctcagatc 3960
cttaccgccg gtttcggttc attctaatga atatatcacc cgttactatc gtatttttat 4020
gaataatatt ctccgttcaa tttactgatt gtaccctact acttatatgt acaatattaa 4080
aatgaaaaca atatattgtg ctgaataggt ttatagcgac atctatgata gagcgccaca 4140
ataacaaaca attgcgtttt attattacaa atccaatttt aaaaaaagcg gcagaaccgg 4200
tcaaacctaa aagactgatt acataaatct tattcaaatt tcaaaagtgc cccaggggct 4260
agtatctacg acacaccgag cggcgaacta ataacgctca ctgaagggaa ctccggttcc 4320
ccgccggcgc gcatgggtga gattccttga agttgagtat tggccgtccg ctctaccgaa 4380
agttacgggc accattcaac ccggtccagc acggcggccg ggtaaccgac ttgctgcccc 4440
gagaattatg cagcattttt ttggtgtatg tgggccccaa atgaagtgca ggtcaaacct 4500
tgacagtgac gacaaatcgt tgggcgggtc cagggcgaat tttgcgacaa catgtcgagg 4560
ctcagcagga cctgcaggca tgcaagcttg gcactggccg tcgttttaca acgtcgtgac 4620
tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 4680
tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 4740
ggcgaatgct agagcagctt gagcttggat cagattgtcg tttcccgcct tcagtttaaa 4800
ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag cgtttattag 4860
aataacggat atttaaaagg gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat 4920
gccaaccaca gggttcccct cgggatcaaa gtactttgat ccaacccctc cgctgctata 4980
gtgcagtcgg cttctgacgt tcagtgcagc cgtcttctga aaacgacatg tcgcacaagt 5040
cctaagttac gcgacaggct gccgccctgc ccttttcctg gcgttttctt gtcgcgtgtt 5100
ttagtcgcat aaagtagaat acttgcgact agaaccggag acattacgcc atgaacaaga 5160
gcgccgccgc tggcctgctg ggctatgccc gcgtcagcac cgacgaccag gacttgacca 5220
accaacgggc cgaactgcac gcggccggct gcaccaagct gttttccgag aagatcaccg 5280
gcaccaggcg cgaccgcccg gagctggcca ggatgcttga ccacctacgc cctggcgacg 5340
ttgtgacagt gaccaggcta gaccgcctgg cccgcagcac ccgcgaccta ctggacattg 5400
ccgagcgcat ccaggaggcc ggcgcgggcc tgcgtagcct ggcagagccg tgggccgaca 5460
ccaccacgcc ggccggccgc atggtgttga ccgtgttcgc cggcattgcc gagttcgagc 5520
gttccctaat catcgaccgc acccggagcg ggcgcgaggc cgccaaggcc cgaggcgtga 5580
agtttggccc ccgccctacc ctcaccccgg cacagatcgc gcacgcccgc gagctgatcg 5640
accaggaagg ccgcaccgtg aaagaggcgg ctgcactgct tggcgtgcat cgctcgaccc 5700
tgtaccgcgc acttgagcgc agcgaggaag tgacgcccac cgaggccagg cggcgcggtg 5760
ccttccgtga ggacgcattg accgaggccg acgccctggc ggccgccgag aatgaacgcc 5820
aagaggaaca agcatgaaac cgcaccagga cggccaggac gaaccgtttt tcattaccga 5880
agagatcgag gcggagatga tcgcggccgg gtacgtgttc gagccgcccg cgcacgtctc 5940
aaccgtgcgg ctgcatgaaa tcctggccgg tttgtctgat gccaagctgg cggcctggcc 6000
ggccagcttg gccgctgaag aaaccgagcg ccgccgtcta aaaaggtgat gtgtatttga 6060
gtaaaacagc ttgcgtcatg cggtcgctgc gtatatgatg cgatgagtaa ataaacaaat 6120
acgcaagggg aacgcatgaa ggttatcgct gtacttaacc agaaaggcgg gtcaggcaag 6180
acgaccatcg caacccatct agcccgcgcc ctgcaactcg ccggggccga tgttctgtta 6240
gtcgattccg atccccaggg cagtgcccgc gattgggcgg ccgtgcggga agatcaaccg 6300
ctaaccgttg tcggcatcga ccgcccgacg attgaccgcg acgtgaaggc catcggccgg 6360
cgcgacttcg tagtgatcga cggagcgccc caggcggcgg acttggctgt gtccgcgatc 6420
aaggcagccg acttcgtgct gattccggtg cagccaagcc cttacgacat atgggccacc 6480
gccgacctgg tggagctggt taagcagcgc attgaggtca cggatggaag gctacaagcg 6540
gcctttgtcg tgtcgcgggc gatcaaaggc acgcgcatcg gcggtgaggt tgccgaggcg 6600
ctggccgggt acgagctgcc cattcttgag tcccgtatca cgcagcgcgt gagctaccca 6660
ggcactgccg ccgccggcac aaccgttctt gaatcagaac ccgagggcga cgctgcccgc 6720
gaggtccagg cgctggccgc tgaaattaaa tcaaaactca tttgagttaa tgaggtaaag 6780
agaaaatgag caaaagcaca aacacgctaa gtgccggccg tccgagcgca cgcagcagca 6840
aggctgcaac gttggccagc ctggcagaca cgccagccat gaagcgggtc aactttcagt 6900
tgccggcgga ggatcacacc aagctgaaga tgtacgcggt acgccaaggc aagaccatta 6960
ccgagctgct atctgaatac atcgcgcagc taccagagta aatgagcaaa tgaataaatg 7020
agtagatgaa ttttagcggc taaaggaggc ggcatggaaa atcaagaaca accaggcacc 7080
gacgccgtgg aatgccccat gtgtggagga acgggcggtt ggccaggcgt aagcggctgg 7140
gttgtctgcc ggccctgcaa tggcactgga acccccaagc ccgaggaatc ggcgtgacgg 7200
tcgcaaacca tccggcccgg tacaaatcgg cgcggcgctg ggtgatgacc tggtggagaa 7260
gttgaaggcc gcgcaggccg cccagcggca acgcatcgag gcagaagcac gccccggtga 7320
atcgtggcaa gcggccgctg atcgaatccg caaagaatcc cggcaaccgc cggcagccgg 7380
tgcgccgtcg attaggaagc cgcccaaggg cgacgagcaa ccagattttt tcgttccgat 7440
gctctatgac gtgggcaccc gcgatagtcg cagcatcatg gacgtggccg ttttccgtct 7500
gtcgaagcgt gaccgacgag ctggcgaggt gatccgctac gagcttccag acgggcacgt 7560
agaggtttcc gcagggccgg ccggcatggc cagtgtgtgg gattacgacc tggtactgat 7620
ggcggtttcc catctaaccg aatccatgaa ccgataccgg gaagggaagg gagacaagcc 7680
cggccgcgtg ttccgtccac acgttgcgga cgtactcaag ttctgccggc gagccgatgg 7740
cggaaagcag aaagacgacc tggtagaaac ctgcattcgg ttaaacacca cgcacgttgc 7800
catgcagcgt acgaagaagg ccaagaacgg ccgcctggtg acggtatccg agggtgaagc 7860
cttgattagc cgctacaaga tcgtaaagag cgaaaccggg cggccggagt acatcgagat 7920
cgagctagct gattggatgt accgcgagat cacagaaggc aagaacccgg acgtgctgac 7980
ggttcacccc gattactttt tgatcgatcc cggcatcggc cgttttctct accgcctggc 8040
acgccgcgcc gcaggcaagg cagaagccag atggttgttc aagacgatct acgaacgcag 8100
tggcagcgcc ggagagttca agaagttctg tttcaccgtg cgcaagctga tcgggtcaaa 8160
tgacctgccg gagtacgatt tgaaggagga ggcggggcag gctggcccga tcctagtcat 8220
gcgctaccgc aacctgatcg agggcgaagc atccgccggt tcctaatgta cggagcagat 8280
gctagggcaa attgccctag caggggaaaa aggtcgaaaa ggtctctttc ctgtggatag 8340
cacgtacatt gggaacccaa agccgtacat tgggaaccgg aacccgtaca ttgggaaccc 8400
aaagccgtac attgggaacc ggtcacacat gtaagtgact gatataaaag agaaaaaagg 8460
cgatttttcc gcctaaaact ctttaaaact tattaaaact cttaaaaccc gcctggcctg 8520
tgcataactg tctggccagc gcacagccga agagctgcaa aaagcgccta cccttcggtc 8580
gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc gcggccgctg gccgctcaaa 8640
aatggctggc ctacggccag gcaatctacc agggcgcgga caagccgcgc cgtcgccact 8700
cgaccgccgg cgcccacatc aaggcaccct gcctcgcgcg tttcggtgat gacggtgaaa 8760
acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 8820
gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga 8880
cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat 8940
tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata 9000
ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 9060
gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 9120
taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 9180
cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 9240
ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 9300
aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 9360
tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 9420
gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 9480
cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 9540
ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 9600
cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 9660
gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 9720
cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 9780
tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 9840
ttaagggatt ttggtcatgc attctaggta ctaaaacaat tcatccagta aaatataata 9900
ttttattttc tcccaatcag gcttgatccc cagtaagtca aaaaatagct cgacatactg 9960
ttcttccccg atatcctccc tgatcgaccg gacgcagaag gcaatgtcat accacttgtc 10020
cgccctgccg cttctcccaa gatcaataaa gccacttact ttgccatctt tcacaaagat 10080
gttgctgtct cccaggtcgc cgtgggaaaa gacaagttcc tcttcgggct tttccgtctt 10140
taaaaaatca tacagctcgc gcggatcttt aaatggagtg tcttcttccc agttttcgca 10200
atccacatcg gccagatcgt tattcagtaa gtaatccaat tcggctaagc ggctgtctaa 10260
gctattcgta tagggacaat ccgatatgtc gatggagtga aagagcctga tgcactccgc 10320
atacagctcg ataatctttt cagggctttg ttcatcttca tactcttccg agcaaaggac 10380
gccatcggcc tcactcatga gcagattgct ccagccatca tgccgttcaa agtgcaggac 10440
ctttggaaca ggcagctttc cttccagcca tagcatcatg tccttttccc gttccacatc 10500
ataggtggtc cctttatacc ggctgtccgt catttttaaa tataggtttt cattttctcc 10560
caccagctta tataccttag caggagacat tccttccgta tcttttacgc agcggtattt 10620
ttcgatcagt tttttcaatt ccggtgatat tctcatttta gccatttatt atttccttcc 10680
tcttttctac agtatttaaa gataccccaa gaagctaatt ataacaagac gaactccaat 10740
tcactgttcc ttgcattcta aaaccttaaa taccagaaaa cagctttttc aaagttgttt 10800
tcaaagttgg cgtataacat agtatcgacg gagccgattt tgaaaccgcg gtgatcacag 10860
gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc tccgcgagat catccgtgtt 10920
tcaaacccgg cagcttagtt gccgttcttc cgaatagcat cggtaacatg agcaaagtct 10980
gccgccttac aacggctctc ccgctgacgc cgtcccggac tgatgggctg cctgtatcga 11040
gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg gctggctgg 11089
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gatagggttt gctgcgaagt g 21
<210> 5
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctccgccacc agcctgta 18
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tggaactgcc accttcacaa 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttccgaggtc atcagggtct 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
taaccaacca gacgagacgc 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tcgaatcttg caggagaggc 20
<210> 10
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caccctgaaa gtttgaacat gttcac 26
<210> 11
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tgagctttgc cagctttgtt g 21
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aggtcttaat ccaattccag 20
<210> 13
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gagttctgct gccgattc 18
<210> 14
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cacaatcact tggctccact gttg 24
<210> 15
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
acctagtagc tggtatggag gaact 25
<210> 16
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gttgaagagt ctccacaatc acttg 25
<210> 17
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
atacaaaaac acaaacattc aaaag 25
<210> 18
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
caactatgtt ctcaggtatt gcaga 25
<210> 19
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gtcatggaaa cgatgtctct ttagt 25
<210> 20
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gtgtaatgtt ggatgtgttc cc 22
<210> 21
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
acacaattga gttcaacaca aaccg 25
<210> 22
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tgcctgcagg tcgactctag atactctctg gtaatcgaat aca 43
<210> 23
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tgcctgcagg tcgactctag aagggtgtgg ccgaaccagt 40
<210> 24
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tgcctgcagg tcgactctag atatgtcttt ttgatggaaa ctc 43
<210> 25
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
tgcctgcagg tcgactctag accctcagta gaatccaact ca 42
<210> 26
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
accacccggg gatcctctag aaaacacaac agaaccacag aaa 43
Claims (4)
1.一种大豆基因GmPP2C89在提高植物对于非生物胁迫抗性中的用途；
所述大豆基因GmPP2C89的核苷酸序列如SEQ ID NO.1所示；
所述植物为拟南芥；
所述非生物胁迫为盐胁迫。
2.根据权利要求1所述的用途，其特征在于，所述大豆基因GmPP2C89在提高植物对于非生物胁迫抗性时的方法为：将大豆基因GmPP2C89在植物体内过表达。
3.根据权利要求2所述的用途，其特征在于，所述过表达为将含有大豆基因GmPP2C89的过表达载体转入植物体中；
所述过表达载体包括所述的大豆基因GmPP2C89以及空载载体pFGC5941；
所述转入方法为农杆菌介导的浸花法。
4.过表达载体在提高植物对于非生物胁迫抗性中的用途；
所述过表达载体包括所述的大豆基因GmPP2C89以及空载载体pFGC5941；
所述大豆基因GmPP2C89的核苷酸序列如SEQ ID NO.1所示；
所述过表达载体在提高植物对于非生物胁迫抗性时的方法为：将过表达载体转入植物体中；
所述转入的方法为农杆菌介导的浸花法；
所述非生物胁迫为盐胁迫；
所述植物为拟南芥。
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