CN110283840A - 陆地棉基因组的精确高效编辑方法 - Google Patents

陆地棉基因组的精确高效编辑方法 Download PDF

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CN110283840A
CN110283840A CN201910290886.0A CN201910290886A CN110283840A CN 110283840 A CN110283840 A CN 110283840A CN 201910290886 A CN201910290886 A CN 201910290886A CN 110283840 A CN110283840 A CN 110283840A
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金双侠
李波
芮杭萍
李亚军
王琼琼
张献龙
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Huazhong Agricultural University
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Abstract

本发明属于植物基因工程技术领域，具体涉及陆地棉基因组的精确高效编辑方法。本发明对含有棉花内源启动子pGhU6‑7的pRGEB32‑GhU6.7‑NPTⅡ载体进行改造，以LbCpf1蛋白取代原有的Cas9蛋白，构建在棉花中具有编辑能力的载体GhRBE3。选取GhCLA为目标基因验证GhRBE3在棉花中的应用。设计1个靶标，利用农杆菌介导的遗传转化将Cpf1基因编辑系统导入棉花基因组，对转基因植株进行Sanger测序和高通量测序，检测本发明在异源四倍体棉花基因组中的编辑效率及脱靶效应。本发明具有良好的编辑效率和特异性。

Description

陆地棉基因组的精确高效编辑方法
技术领域
本发明属于基因工程技术领域，具体涉及一种陆地棉基因组的精确高效编辑方法。本发明包括构建陆地棉的高效转化载体，利用构建的载体在陆地棉功能基因组中进行精准编辑。
背景技术
CRISPR/Cas系统(Clustered regularlyinterspaced short palindromicrepeats/CRISPR-associated protein)是细菌和古菌在抵御外来病毒和噬菌体入侵的过程中不断迚化而来的一种获得性免疫防御机制。CRISPR/Cas9系统是目前应用最多最广泛的基因编辑系统，能够高效特异和灵活的识别靶基因中的特定位点，然而，随着研究的不断深入，CRISPR/Cas9也暴露了比较严重的脱靶效应(Hsu et al 2014)、PAM序列的限制导致其切割位点较少，基因编辑后后代不易纯合等缺陷。因此需要改造CRISPR/Cas9系统，降低其脱靶效应，提高基因编辑精准度，扩大靶标位点范围，寻找新的基因编辑系统。
Cpf1核酸酶来自毛螺科菌(Lachnospiraceae bacterium ND2006)，由1228个氨基酸组成。与CRISPR/Cas9不同的是，CRISPR/Cpf1核酸酶的PAM序列基序为5'-TTTV-3'，具备“T/A”富集区的PAM识别位点；向导RNA是结构简单的单一的crRNA分子，不需要tracrRNA参与编辑过程；切割位点发生在目标链PAM区下游23核苷酸处和非目标链18核苷酸处，使DNA双链断裂，产生粘性末端(Zetsche et al 2015)，除此之外，Cpf1的切割位点离PAM基序较远，允许先前已经突变的序列被反复切断，促进了同源重组修复(HDR)的产生。从而可以实现更简单，精准的基因组定向遗传修饰。张勇等研究团队构建了Cpf1核酸酶植物基因组编辑新系统构，并对其PAM识别位点进行了精确界定，实现了Cpf1核酸酶介导的水稻基因组有效编辑。同时，构建了LbCpf1核酸酶的RR及RVR变体表达系统，在水稻基因组中成功实现了以多种PAM位点的编辑，拓展了Cpf1可编辑位点数量，有效推进了Cpf1核酸酶在植物基因组编辑中的应用。
目前，Cpf1核酸酶编辑系统在很多物种中被报道，但是在异源四倍体棉花中尚无报道。陆地棉是一种广泛种植的异源四倍体(AtDt)，其基因组中的许多基因具有高度同源性，并且基因组比较复杂及庞大，传统的CRISPR-Cas9系统在需要对某些特定基因进行编辑及功能分析时无能为力。本发明的碱基编辑系统作为可行而又有效的精准编辑工具，为棉花基因组功能分析，作物遗传改良和新品种选育提供重要技术支持。
发明内容
本发明的目的在于提供一种陆地棉基因组的精准高效编辑方法，特别是构建一种适用于陆地棉的碱基编辑系统。本发明基于pRGEB32-GhU6.7-NPTⅡ(Xie et al 2015,Wanget al 2018)和pHUN611(安徽省农业科学院水稻研究所魏鹏程老师课题组提供)载体，构建了融合LbCpf1核酸酶的适用于棉花遗传转化系统的碱基编辑系统(即载体GhRBE3)。
本发明技术方案如下所述：
一种能够精确识别基因组“-TTTV-”碱基并能产生黏性末端的编辑陆地棉基因组高效转化载体GhRBE3，该载体的核苷酸序列如SEQ ID NO:3所示。
一种能够精确识别基因组“-TTTV-”碱基并能产生黏性末端的编辑陆地棉基因组高效转化载体GhRBE3，该载体通过下列步骤制备获得：
(1)获得目的序列LbCpf1-NLS-3xHA，其核苷酸序列如序列表SEQ ID NO：4所示，具体地，该目的序列通过以下步骤制备获得：
1)利用Primer5软件在pHUN611载体(其核苷酸序列如序列表SEQ ID NO：2所示)序列上设计引物，正向引物为：AAAAAGCAGGCTTCGAAATGTCCAAGCTGGAGAAGTTTACA；反向引物为：GAAAGCTGGGTCTAGATTAGGCATAGTCGGGGACATC；
2)以pHUN611载体为模板，进行PCR扩增，获得LbCpf1-NLS-3xHA目的序列，其核苷酸序列如序列表SEQ ID NO：4所示；
(2)利用BstbⅠ、XbaⅠ对pRGEB32-GhU6.7-NPTⅡ载体(其核苷酸序列序列表SEQ IDNO：1所示)进行酶切，与步骤(1)中获得的LbCpf1-NLS-3xHA目的序列进行连接，通过测序验证，得到如序列表SEQ ID NO：3所示的适用于陆地棉的基因编辑的高效转化载体GhRBE3。
本发明的转化载体GhRBE3可在陆地棉基因组编辑中应用。
本发明的效果为：
1、本发明在棉花中的编辑效率达87％。
2、本发明在靶标序列上的编辑窗口主要在PAM“-TTTV-”后第15个碱基到第23个碱基之间(即：N15-N23)。
3、本发明在靶标序列上的缺失片段大小主要是5bp-12bp。
4、本发明在棉花中有较高的特异性。
附图说明
图1：载体pHUN611和载体pRGEB32-GhU6.7改造的路线图。
图2：本发明构建的表达载体GhRBE3的载体的图谱。
图3：本发明的扩增后LbCpf1-NLS-3xHA片段的电泳图。附图标记说明：泳道1,2,3是LbCpf1-NLS-3xHA片段,泳道M是5K的marker。
图4：拼接后GhRBE3的电泳图。附图标记说明：泳道1，2是GhRBE3构建完成检测，泳道CK是阴性对照，泳道M是5K的marker。
图5：本发明crRNA连接到GhRBE3的电泳图。附图标记说明：泳道2-1，2-2，2-3，2-4，是目的片段连接到GhRBE3载体,泳道M是5K的marker。
图6：本发明GhCLA的遗传转化图。附图标记说明：图6中的字母编号分别是：A.共培养阶段。B.选择培养阶段。C.愈伤组织阶段。D.分化培养阶段。E.生根培养阶段。F.营养液培养。图G-图H.转基因植株在温室生长情况。
图7：本发明的GhCLA基因编辑后代表型图：附图标记说明：WT leaf.野生型棉花(即非转基因)叶片。T0plant.T0代植株发育图。T0chimetic leaf.T0代植株嵌合体叶表型图。WT plants.野生型(即非转基因)棉花植株图。T1chimetic plants.T0代植株嵌合体叶表型图。T1albino plants.T0代植株白化苗表型图。
图8：本发明的GhCLA基因crRNA编辑检测图。附图标记说明：图8中的A图是GhCLA基在棉花T0代植株At/Dt亚族的编辑情况；图8中的B图是GhCLA基在棉花T1代植株At/Dt亚族的编辑情况；图8中的A图,图8中的B图中下划线指的是该载体在基因组中能够识别的“-TTTV-”序列和所对应的切割位点序列。
图9：本发明的GhCLA基因crRNA编辑不同样品编辑效率图。附图标记说明：图9中星状标记代表不同的样品，横坐标代表总样品数，纵坐标代表不同样品的编辑效率。
图10：是本发明的GhCLA基因crRNA编辑靶标碱基的缺失频率图。附图标记说明：图10中横坐标代表靶标crRNA的不同碱基位点，纵坐标代表不同碱基每个碱基的缺失频率。
图11：是本发明的GhCLA基因crRNA编辑基因缺失片段大小图。附图标记说明：图10中横坐标代表靶标基因缺失的片段大小，纵坐标代表不同的缺失片段频率。
图12：是本发明的GhCLA基因crRNA潜在脱靶位点Sanger测序峰图。附图标记说明：利用Sanger测序检测T1代植株潜在脱靶位点，测序峰图和潜在脱靶位点对应图。图12中的A图代表预测潜在脱靶位点1和4的Sanger测序峰图；图12中的B图代表预测潜在脱靶位点2,5和6的Sanger测序峰图；图12中的C图代表预测潜在脱靶位点3的Sanger测序峰图。
具体实施方式
对序列表的说明：
序列表SEQ ID NO：1是本发明的载体pRGEB32-GhU6.7的核苷酸序列。序列长度为16241bp。
序列表SEQ ID NO：2是本发明的载体pHUN611的核苷酸序列。序列长度为18805bp。
序列表SEQ ID NO：3是陆地棉基因组高效转化载体GhRBE3的核苷酸序列。序列长度为15731bp。
序列表SEQ ID NO：4是LbCpf1-NLS-3xHA融合蛋白的核苷酸序列。序列长度为3822bp。
实施例1：LbCpf1-NLS-3xHA目的序列的扩增
以pHUN611载体(序列表如SEQ ID NO：2)为模板进行PCR扩增得到目的序列LbCpf1-NLS-3xHA(序列表如SEQ ID NO：4)。扩增的引物为：
LbCpf/F:AAAAAGCAGGCTTCGAAATGTCCAAGCTGGAGAAGTTTACA，
和LbCpf/R:GAAAGCTGGGTCTAGATTAGGCATAGTCGGGGACATC；
PCR反应体系见表1。
表1 LbCpf1-NLS-3xHA序列的PCR反应体系
实施例2：转化载体GhRBE3的构建
将pRGEB32-GhU6.7-NPTⅡ(序列表见SEQ ID NO：1)进行双酶切，酶切体系见表2。37℃酶切5小时，酶切产物凝胶电泳观察单酶切完全后再加4μL的BstbI，65℃酶切20min，凝胶电泳观察酶切条带是否正确，然后利用凝胶回收试剂盒(购自OMEGA公司,货号D2500-02)将酶切产物纯化。酶切体系见表2。
表2 pRGEB32-GhU6.7-NPTⅡ的酶切体系
将酶切后的pRGEB32-GhU6.7-NPTⅡ载体与LbCpf1-NLS-3xHA融合蛋白片段通过ClonExpress II One Step Cloning Kit(Vazyme C112-02)进行连接，转化到大肠杆菌感受态，挑取阳性克隆进行测序，将序列正确的质粒命名为GhRBE3质粒(见序列表SEQ ID NO：3)。其中In-fusion连接反应体系见表3。
表3 In-fusion连接反应体系
37℃水浴30min，冰上放置5min，可-20℃下保存。
实施例3：GhRBE3-crRNA载体的构建
1.GhCLA基因的crRNA设计
选择陆地棉1-脱氧木酮糖-5-磷酸合成酶(Cloroplasto alterados,CLA)Gh_A10G2292基因为验证基因。利用生物信息学软件sgRNAcas9_3.0.5(Xie et al 2014)在基因外显子区域设计PAM序列为“-TTTV”的crRNA靶标序列,选择了1个crRNA用来构建基因编辑系统植物表达载体。所述的crRNA的序列见表4。
表4 crRNA的序列
2.crRNA与GhRBE3载体的连接
靶标插入GhRBE3载体序列为tRNA-crRNA-gRNA的重复序列，采用基因合成方式获得靶标序列，之后PCR扩增后连接到GhRBE3载体上。现以crRNA为例进行说明。PCR的引物如下：pRGEB32-7/S:aagcatcagatgggcaAACAAAGCACCAGTGGTC，CLA1/AS:TCTAGCTCTAAAACTGCACCAGCCGGGAA，以合成基因模板进行PCR扩增，获得tRNA+crRNA+gRNA片段，将片段连接到pGREB32-GhU6-7载体的BsaI酶切位点处。
表5 PCR体系
表6 PCR条件
实施例4：农杆菌介导的遗传转化
具体步骤如下：
A.将剥好的棉花种子(品种为Jin668，专利申请号2015108336180专利文献中已报道)用0.1％升汞杀菌，无菌水清洗数次后放入无菌苗培养基中，28℃暗培养1d，挑去种皮，将苗扶正，在28℃，暗培养4-5d；
B.将下胚轴切成小茎段，用活化后的农杆菌侵染，弃菌液，并吹干；
C.将下胚轴平铺在放有滤纸的共培养培养基中，于20℃，暗培养1-2d；
D.将下胚轴转入到附加2,4-D的愈伤组织诱导培养基中，放入光照培养室，20-30d左右用新鲜愈伤组织诱导培养基继代培养一次；
E.当愈伤组织长成米粒状颗粒，转入分化培养基中，进一步分化成胚状体；
F.将分化出的小苗继代到生根培养基中，直至长成生根良好健康的小苗；
G.将小苗转到清水中，进行炼苗，一周左右后，转移到到温室。
转化所用的培养基组分及配比：
无菌苗萌发培养基：1/2MS大量元素，15g/L葡萄糖，2.5g/L的Phytagel；pH:6.1-6.2。
愈伤组织诱导培养基：MSB+24-D 0.1mg/L+KT 0.1mg/L+3％Glucose+0.3％Phytagel；pH:5.85-5.95。
农杆菌活化培养基：胰化蛋白胨5g/L+NaCl 5g/L+MgSO4.7H2O 0.1g/L+KH2PO4+0.25g/L+甘露醇5g/L+甘氨酸1.0g/L；pH:5.85-5.95。
共培养培养基：MSB+2，4-D 0.1mg/l+KT 0.1mg/l+50mg/l AS+3％Glucose+0.25％Phytagel，pH5.8。
选择培养基：MSB+2，4-D 0.1mg/L+KT 0.1mg/L+3％Glucose+0.3％Phytagel，卡那霉素50mg/L和头孢霉素400mg/L；pH:5.85-5.95。
分化培养基：分化培养基：MSB培养基中去掉NH4NO3,将KNO3用量加倍+Gln 1.0g/L+Asn 0.5g/L+IBA 0.5mg/L+KT 0.15mg/L+3％Glucose+0.25％Phytagel，pH:6.1-6.2。
生根培养基：1/2MS无机盐+B5有机物，15g/L葡萄糖，2.5g/L的Phytagel；pH:5.90-5.95；
MSB的成分：MS培养基+B5维生素。
实施例5：GhRBE3对在转基因棉花植株中基因编辑检测中的应用
(1)Sanger测序检测编辑效率
提取的棉花嫩叶阳性基因组DNA【使用天根生化(北京)科技有限公司生产的试剂盒，按照试剂盒说明书操作】，以阳性DNA为模板，扩增GhCLA(Gh_A10G2292)基因包含crRNA(TTTGCAATGGACAGAGCTGGCCTCGTT)的片段，然后将PCR片段连入pGEM-T easy载体(购自普洛麦格(北京)生物技术有限公司代理)，将连接产物热激转化大肠杆菌感受态TOP10，挑取的单克隆进行阳性检测并进行Sanger测序。将测序结果与靶标序列比对。每个样品送至少15个单克隆测序，统计每个靶点被编辑的效率。样品表型见图7，测序结果见图8。
(2)高通量Hi-Tom深测序检测编辑效率
首先在靶点位置前后设计引物，分别用Hi-Tom接头的引物以独立的单株DNA为模板扩增靶点序列(第一轮PCR)，再用前后引物上的一对不同的标签(Barcode)对应不同的样品扩增第二轮PCR(按常规方法),获得的PCR产物进行等量混合，然后用纯化试剂盒(OMEGA公司,货号D2500-02)纯化混合产物，最后进行双端150bp测序。通过标签寻找正确对应的样品，分析序列的编辑情况。深度测序的结果见图9,10,11。
实施例6：GhRBE3系统在转基因棉花植株中脱靶情况的检测中的应用
(1)脱靶位点
申请人利用CRISPR-P和OFFinder工具鉴定到与crRNA靶点存在5个错配碱基以内6个最可能的脱靶位点进行测序，结果见表7。
表7预测潜在脱靶位点
(2)Sanger测序检测脱靶效应
利用如实施例5中步骤(1)的方法设计引物，提取不同植株的DNA(见图7)，扩增靶标序列位点的片段，分别连接T载体热激转化后挑取单克隆进行Sanger测序，数据结果表明,在这6个最可能脱靶的靶标并没有发生脱靶效应(见图12)。
本发明在棉花中首次成功建立了适应于棉花基因组特性的基因编辑系统，该系统显示出较高的编辑效率和很高的特异性，它将成为棉花功能基因组研究新的重要的技术手段。
参考文献
1.Hsu PD,Lander ES,Zhang F.Development and applications of CRISPR-Cas9for genome engineering.Cell,2014,157(6):1262-1278；
2.Zetsche B,Gootenberg J,Abudayyeh O,Cpf1is a single RNA-guidedendonuclease of a class 2CRISPR-Cas system[J].Cell,2015,163(3):759-771；
3.Xie,K.,B.Minkenberg&Y.Yang.Boosting CRISPR/Cas9multiplex editingcapability with the endogenous tRNA-processing system.Proceedings of theNational Academy of Sciences of the United States of America,2015,112,3570-3575；
4.Wang P,Zhang J,Sun L,Ma Y,Xu J,Liang S,Deng J,Tan J,Zhang Q,Tu L,Daniell H,Jin S,Zhang X.High efficient multisites genome editing inallotetraploid cotton(Gossypium hirsutum)using CRISPR/Cas9 system.PlantBiotechnol Jouranl,2018,16:137-150；
5.Xie,S.,Shen,B.,Zhang,C.,Huang,X.and Zhang,Y,sgRNAcas9:a softwarepackage for designing CRISPR sgRNA and evaluating potential off-targetcleavage sites,2014,PLoS One 9,e100448。
序列表
<110> 华中农业大学
<120> 陆地棉基因组的精确高效编辑方法
<141> 2019-04-11
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16241
<212> DNA
<213> 陆地棉(Gossypium hirsutum)
<220>
<221> gene
<222> (1)..(16241)
<400> 1
cttgtacaaa gtggttgata acagcgacta caaggatgac gatgacaagg cttagagctc 60
gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc 120
cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 180
catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata 240
catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc 300
ggtgtcatct atgttactag atcgggaatt cactggccgt cgttttacac tggccgtcgt 360
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 420
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 480
gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga ttgtcgtttc 540
ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga 600
aaagagcgtt tattagaata acggatattt aaaagggcgt gaaaaggttt atccgttcgt 660
ccatttgtat gtgcatgcca accacagggt tcccctcggg atcaaagtac tttgatccaa 720
cccctccgct gctatagtgc agtcggcttc tgacgttcag tgcagccgtc ttctgaaaac 780
gacatgtcgc acaagtccta agttacgcga caggctgccg ccctgccctt ttcctggcgt 840
tttcttgtcg cgtgttttag tcgcataaag tagaatactt gcgactagaa ccggagacat 900
tacgccatga acaagagcgc cgccgctggc ctgctgggct atgcccgcgt cagcaccgac 960
gaccaggact tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac caagctgttt 1020
tccgagaaga tcaccggcac caggcgcgac cgcccggagc tggccaggat gcttgaccac 1080
ctacgccctg gcgacgttgt gacagtgacc aggctagacc gcctggcccg cagcacccgc 1140
gacctactgg acattgccga gcgcatccag gaggccggcg cgggcctgcg tagcctggca 1200
gagccgtggg ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc 1260
attgccgagt tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc 1320
aaggcccgag gcgtgaagtt tggcccccgc cctaccctca ccccggcaca gatcgcgcac 1380
gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag aggcggctgc actgcttggc 1440
gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag 1500
gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc cctggcggcc 1560
gccgagaatg aacgccaaga ggaacaagca tgaaaccgca ccaggacggc caggacgaac 1620
cgtttttcat taccgaagag atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc 1680
cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct ggccggtttg tctgatgcca 1740
agctggcggc ctggccggcc agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa 1800
ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat 1860
gagtaaataa acaaatacgc aaggggaacg catgaaggtt atcgctgtac ttaaccagaa 1920
aggcgggtca ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg 1980
ggccgatgtt ctgttagtcg attccgatcc ccagggcagt gcccgcgatt gggcggccgt 2040
gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt 2100
gaaggccatc ggccggcgcg acttcgtagt gatcgacgga gcgccccagg cggcggactt 2160
ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc caagccctta 2220
cgacatatgg gccaccgccg acctggtgga gctggttaag cagcgcattg aggtcacgga 2280
tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg 2340
tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt cttgagtccc gtatcacgca 2400
gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc gttcttgaat cagaacccga 2460
gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa aactcatttg 2520
agttaatgag gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg 2580
agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc agccatgaag 2640
cgggtcaact ttcagttgcc ggcggaggat cacaccaagc tgaagatgta cgcggtacgc 2700
caaggcaaga ccattaccga gctgctatct gaatacatcg cgcagctacc agagtaaatg 2760
agcaaatgaa taaatgagta gatgaatttt agcggctaaa ggaggcggca tggaaaatca 2820
agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc 2880
aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc actggaaccc ccaagcccga 2940
ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg 3000
atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag 3060
aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc 3120
aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag 3180
attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg 3240
tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc 3300
ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt 3360
acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag 3420
ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct 3480
gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa 3540
acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg 3600
tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc 3660
cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga 3720
acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt 3780
ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga 3840
cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca 3900
agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg 3960
gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct 4020
aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaagcac 4080
tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc 4140
cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata 4200
taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta 4260
aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag 4320
cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg 4380
ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag 4440
ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg caccctgcct cgcgcgtttc 4500
ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 4560
taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 4620
cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg 4680
cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 4740
gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc 4800
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4860
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4920
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4980
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5040
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5100
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 5160
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5220
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5280
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5340
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5400
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5460
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5520
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5580
ggaacgaaaa ctcacgttaa gggattttgg tcatgcattc taggtactaa aacaattcat 5640
ccagtaaaat ataatatttt attttctccc aatcaggctt gatccccagt aagtcaaaaa 5700
atagctcgac atactgttct tccccgatat cctccctgat cgaccggacg cagaaggcaa 5760
tgtcatacca cttgtccgcc ctgccgcttc tcccaagatc aataaagcca cttactttgc 5820
catctttcac aaagatgttg ctgtctccca ggtcgccgtg ggaaaagaca agttcctctt 5880
cgggcttttc cgtctttaaa aaatcataca gctcgcgcgg atctttaaat ggagtgtctt 5940
cttcccagtt ttcgcaatcc acatcggcca gatcgttatt cagtaagtaa tccaattcgg 6000
ctaagcggct gtctaagcta ttcgtatagg gacaatccga tatgtcgatg gagtgaaaga 6060
gcctgatgca ctccgcatac agctcgataa tcttttcagg gctttgttca tcttcatact 6120
cttccgagca aaggacgcca tcggcctcac tcatgagcag attgctccag ccatcatgcc 6180
gttcaaagtg caggaccttt ggaacaggca gctttccttc cagccatagc atcatgtcct 6240
tttcccgttc cacatcatag gtggtccctt tataccggct gtccgtcatt tttaaatata 6300
ggttttcatt ttctcccacc agcttatata ccttagcagg agacattcct tccgtatctt 6360
ttacgcagcg gtatttttcg atcagttttt tcaattccgg tgatattctc attttagcca 6420
tttattattt ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa 6480
caagacgaac tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc 6540
tttttcaaag ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa 6600
accgcggtga tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg 6660
cgagatcatc cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt 6720
aacatgagca aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat 6780
gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg 6840
gctggtggca ggatatattg tggtgtaaac aaattgacgc ttagacaact taataacaca 6900
ttgcggacgt ttttaatgta ctgaattaac gccgaattaa ttcgggggat ctggatttta 6960
gtactggatt ttggttttag gaattagaaa ttttattgat agaagtattt tacaaataca 7020
aatacatact aagggtttct tatatgctca acacatgagc gaaaccctat aggaacccta 7080
attcccttat ctgggaacta ctcacacatt attatggaga aactcgagct cagaagaact 7140
cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca 7200
cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg 7260
ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc 7320
ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcat 7380
cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat 7440
gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct 7500
cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc 7560
gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga 7620
gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt 7680
cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt 7740
cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct 7800
gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat 7860
agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa 7920
tcatcccggg atctgcgaaa gctcgagaga gatagatttg tagagagaga ctggtgattt 7980
cagcgtgtcc tctccaaatg aaatgaactt ccttatatag aggaaggtct tgcgaaggat 8040
agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg 8100
aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt 8160
tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc 8220
atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc 8280
aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt 8340
ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca 8400
tgttatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga 8460
tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga 8520
tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc 8580
ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc 8640
tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg 8700
agtagacgag agtgtcgtgc tccaccatgt tggcaagctg ctctagccaa tacgcaaacc 8760
gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg 8820
gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 8880
ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 8940
tcacacagga aacagctatg accatgatta cgccaagctt ttaatctgat gctccacctg 9000
cttttgattt tctttattgg aagagtcttt aagagatatg ttaagtagca taacagtttc 9060
atcaaaaaca acatttctgt taatcacaac ttttctattt tcaggatacc ataacttata 9120
cacttttaca ctagctttat aaccaagaaa aacacattta atggtacaca attttaattt 9180
tccattatca gcatgagtat acgcaaaaca cccaaaaatc tttaaatcag aatcgtcagc 9240
aggattacta aaccatactt cttatggagt ctttttctca atagcaacga atagagacgg 9300
attgatcaaa aacatatagt tgacattgct ttggcccaaa ataactttga taagttgcca 9360
tttgacaaca tacatcgaac attctccatg atcgttctat tcattcgttc tacaatacct 9420
tttttaaaat gttcaggttc taaaatgaaa aacaatatga attgcatgaa ttgcttatat 9480
gtcctatgaa ttataaagga atgcggttga aatattccca tcgatacata catacatatt 9540
cgtgaagtat gttccaatat aatatcaata ttgggattta cgttttataa agcaacatta 9600
ttgattggta atatacatta attccaaggc aaacccaaat attttaaaat ttaacctaca 9660
actgtggtaa atcaaactta atagtaaccc gattgtaatg tgaagtcaaa tatgaaagta 9720
acattggttt atatatatat ttttctctaa attctaataa tcaagttggg ataagtgata 9780
aacactgagc ttgccacgtg tgttaacctc gttttcatca tgtgccactc caaagacatc 9840
aggcctctat tcaagctggc atggtcagga cgtggtagca tacttcaggg atctggttag 9900
aaaatatccc atatcgctaa agaactataa cacaggagcg tttatataag cgaaagaagc 9960
atcagatggg caggagaccg aggtctcggt tttagagcta gaaatagcaa gttaaaataa 10020
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc 10080
tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcatgc ctgcaggtcc 10140
acaaattcgg gtcaaggcgg aagccagcgc gccaccccac gtcagcaaat acggaggcgc 10200
ggggttgacg gcgtcacccg gtcctaacgg cgaccaacaa accagccaga agaaattaca 10260
gtaaaaaaaa agtaaattgc actttgatcc accttttatt acctaagtct caatttggat 10320
cacccttaaa cctatctttt caatttgggc cgggttgtgg tttggactac catgaacaac 10380
ttttcgtcat gtctaacttc cctttcagca aacatatgaa ccatatatag aggagatcgg 10440
ccgtatacta gagctgatgt gtttaaggtc gttgattgca cgagaaaaaa aaatccaaat 10500
cgcaacaata gcaaatttat ctggttcaaa gtgaaaagat atgtttaaag gtagtccaaa 10560
gtaaaactta tagataataa aatgtggtcc aaagcgtaat tcactcaaaa aaaatcaacg 10620
agacgtgtac caaacggaga caaacggcat cttctcgaaa tttcccaacc gctcgctcgc 10680
ccgcctcgtc ttcccggaaa ccgcggtggt ttcagcgtgg cggattctcc aagcagacgg 10740
agacgtcacg gcacgggact cctcccacca cccaaccgcc ataaatacca gccccctcat 10800
ctcctctcct cgcatcagct ccacccccga aaaatttctc cccaatctcg cgaggctctc 10860
gtcgtcgaat cgaatcctct cgcgtcctca aggtacgctg cttctcctct cctcgcttcg 10920
tttcgattcg atttcggacg ggtgaggttg ttttgttgct agatccgatt ggtggttagg 10980
gttgtcgatg tgattatcgt gagatgttta ggggttgtag atctgatggt tgtgatttgg 11040
gcacggttgg ttcgataggt ggaatcgtgg ttaggttttg ggattggatg ttggttctga 11100
tgattggggg gaatttttac ggttagatga attgttggat gattcgattg gggaaatcgg 11160
tgtagatctg ttggggaatt gtggaactag tcatgcctga gtgattggtg cgatttgtag 11220
cgtgttccat cttgtaggcc ttgttgcgag catgttcaga tctactgttc cgctcttgat 11280
tgagttattg gtgccatggg ttggtgcaaa cacaggcttt aatatgttat atctgttttg 11340
tgtttgatgt agatctgtag ggtagttctt cttagacatg gttcaattat gtagcttgtg 11400
cgtttcgatt tgatttcata tgttcacaga ttagataatg atgaactctt ttaattaatt 11460
gtcaatggta aataggaagt cttgtcgcta tatctgtcat aatgatctca tgttactatc 11520
tgccagtaat ttatgctaag aactatatta gaatatcatg ttacaatctg tagtaatatc 11580
atgttacaat ctgtagttca tctatataat ctattgtggt aatttctttt tactatctgt 11640
gtgaagatta ttgccactag ttcattctac ttatttctga agttcaggat acgtgtgctg 11700
ttactaccta tctgaataca tgtgtgatgt gcctgttact atctttttga atacatgtat 11760
gttctgttgg aatatgtttg ctgtttgatc cgttgttgtg tccttaatct tgtgctagtt 11820
cttaccctat ctgtttggtg attatttctt gcagatagtt atcaacaagt ttgtacaaaa 11880
aagcaggctt cgaaggagat agaaccaatt ctctaaggaa atacttaacc atggactata 11940
aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 12000
tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 12060
acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 12120
acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 12180
agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 12240
agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 12300
tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 12360
tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 12420
aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 12480
gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 12540
ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 12600
tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 12660
gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 12720
tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 12780
gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 12840
agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 12900
agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 12960
tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 13020
acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 13080
agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 13140
gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 13200
gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 13260
tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 13320
ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 13380
ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 13440
tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 13500
gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 13560
agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 13620
ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 13680
aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 13740
aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 13800
atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 13860
acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 13920
tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 13980
acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 14040
agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 14100
ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 14160
aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 14220
ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 14280
acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 14340
gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 14400
aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 14460
agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 14520
accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 14580
gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 14640
gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 14700
agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 14760
gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 14820
ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 14880
agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 14940
atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 15000
acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 15060
aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 15120
gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 15180
ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 15240
caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 15300
aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 15360
tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 15420
actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 15480
tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 15540
ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 15600
agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 15660
agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 15720
aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 15780
tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 15840
acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 15900
ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 15960
aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 16020
agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 16080
ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 16140
tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt 16200
aagaattcgc ggccgcactc gagatatcta gacccagctt t 16241
<210> 2
<211> 13401
<212> DNA
<213> 陆地棉(Gossypium hirsutum)
<220>
<221> gene
<222> (1)..(13401)
<400> 2
gtttacccgc caatatatcc tgtcaaacac tgatagttta aactgaaggc gggaaacgac 60
aatctgatcc aagctcaagc taagctcacg tgacggaatt aagcttaagg gatctttaaa 120
catacgaaca gatcacttaa agttcttctg aagcaactta aagttatcag gcatgcatgg 180
atcttggagg aatcagatgt gcagtcaggg accatagcac aagacaggcg tcttctactg 240
gtgctaccag caaatgctgg aagccgggaa cactgggtac gttggaaacc acgtgatgtg 300
aagaagtaag ataaactgta ggagaaaagc atttcgtagt gggccatgaa gcctttcagg 360
acatgtattg cagtatgggc cggcccatta cgcaattgga cgacaacaaa gactagtatt 420
agtaccacct cggctatcca catagatcaa agctgattta aaagagttgt gcagatgatc 480
cgtggcaaga gaccaaccca gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag 540
tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg tggtaacggc 600
gcagtggcgg ttttcatggc ttgttatgac tgtttttttg gggtacagtc tatgcctcgg 660
gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac 720
gatgttacgc agcagggcag tcgccctaaa acaaagttaa acatcatggg ggaagcggtg 780
atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa 840
ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac 900
agtgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct 960
ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct 1020
gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc 1080
gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc 1140
acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg 1200
gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg 1260
ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat 1320
gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag 1380
gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt 1440
gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag 1500
ttggaagaat ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg caaataatgt 1560
ctagctagaa attcgttcaa gccgacgccg cttcgcggcg cggcttaact caagcgttag 1620
atgcactaag cacataattg ctcacagcca aactatcagg tcaagtctgc ttttattatt 1680
tttaagcgtg cataataagc cggtctcatt ttttttagta gtagcatctg acggtgggga 1740
agcttgatat cgaattcctg cagtgcagcg tgacccggtc gtgcccctct ctagagataa 1800
tgagcattgc atgtctaagt tataaaaaat taccacatat tttttttgtc acacttgttt 1860
gaagtgcagt ttatctatct ttatacatat atttaaactt tactctacga ataatataat 1920
ctatagtact acaataatat cagtgtttta gagaatcata taaatgaaca gttagacatg 1980
gtctaaagga caattgagta ttttgacaac aggactctac agttttatct ttttagtgtg 2040
catgtgttct cctttttttt tgcaaatagc ttcacctata taatacttca tccattttat 2100
tagtacatcc atttagggtt tagggttaat ggtttttata gactaatttt tttagtacat 2160
ctattttatt ctattttagc ctctaaatta agaaaactaa aactctattt tagttttttt 2220
atttaataat ttagatataa aatagaataa aataaagtga ctaaaaatta aacaaatacc 2280
ctttaagaaa ttaaaaaaac taaggaaaca tttttcttgt ttcgagtaga taatgccagc 2340
ctgttaaacg ccgtcgacga gtctaacgga caccaaccag cgaaccagca gcgtcgcgtc 2400
gggccaagcg aagcagacgg cacggcatct ctgtcgctgc ctctggaccc ctctcgagag 2460
ttccgctcca ccgttggact tgctccgctg tcggcatcca gaaatgcgtg gcggagcggc 2520
agacgtgagc cggcacggca ggcggcctcc tcctcctctc acggcacggc agctacgggg 2580
gattcctttc ccaccgctcc ttcgctttcc cttcctcgcc cgccgtaata aatagacacc 2640
ccctccacac cctctttccc caacctcgtg ttgttcggag cgcacacaca cacaaccaga 2700
tctcccccaa atccacccgt cggcacctcc gcttcaaggt acgccgctcg tcctcccccc 2760
ccccccctct ctaccttctc tagatcggcg ttccggtcca tggttagggc ccggtagttc 2820
tacttctgtt catgtttgtg ttagatccgt gtttgtgtta gatccgtgct gctagcgttc 2880
gtacacggat gcgacctgta cgtcagacac gttctgattg ctaacttgcc agtgtttctc 2940
tttggggaat cctgggatgg ctctagccgt tccgcagacg ggatcgattt catgattttt 3000
tttgtttcgt tgcatagggt ttggtttgcc cttttccttt atttcaatat atgccgtgca 3060
cttgtttgtc gggtcatctt ttcatgcttt tttttgtctt ggttgtgatg atgtggtctg 3120
gttgggcggt cgttctagat cggagtagaa ttctgtttca aactacctgg tggatttatt 3180
aattttggat ctgtatgtgt gtgccataca tattcatagt tacgaattga agatgatgga 3240
tggaaatatc gatctaggat aggtatacat gttgatgcgg gttttactga tgcatataca 3300
gagatgcttt ttgttcgctt ggttgtgatg atgtggtgtg gttgggcggt cgttcattcg 3360
ttctagatcg gagtagaata ctgtttcaaa ctacctggtg tatttattaa ttttggaact 3420
gtatgtgtgt gtcatacatc ttcatagtta cgagtttaag atggatggaa atatcgatct 3480
aggataggta tacatgttga tgtgggtttt actgatgcat atacatgatg gcatatgcag 3540
catctattca tatgctctaa ccttgagtac ctatctatta taataaacaa gtatgtttta 3600
taattatttt gatcttgata tacttggatg atggcatatg cagcagctat atgtggattt 3660
ttttagccct gccttcatac gctatttatt tgcttggtac tgtttctttt gtcgatgctc 3720
accctgttgt ttggtgttac ttctgcagcc cgggggatcc ccaatacttg tatggccgcg 3780
gccgcatgtc caagctggag aagtttacaa actgttacag cctctccaaa accctcaggt 3840
ttaaagcgat cccggtgggc aagacccagg agaacatcga caacaagagg ctcctggtgg 3900
aagacgagaa gcgcgccgaa gactacaagg gcgtgaagaa gctgctcgat aggtactacc 3960
tcagctttat taacgacgtg ctgcacagca tcaaactcaa gaatctcaac aactacatct 4020
ccctcttccg caaaaagacc cgcaccgaga aggagaacaa ggagctggag aacctggaga 4080
tcaacctccg caaggaaatc gccaaagcgt tcaagggcaa tgaagggtac aagagcctct 4140
tcaagaaaga catcatcgaa actatcctcc cagagtttct cgatgacaag gacgagatcg 4200
cgctggtgaa ctcctttaac gggttcacaa ccgcgtttac cggcttcttt gataacaggg 4260
aaaatatgtt ctccgaggag gccaagtcca ccagcatcgc cttcaggtgt atcaacgaga 4320
acctcacccg ctacatttcc aatatggaca ttttcgagaa ggtggatgcg atcttcgata 4380
agcacgaggt gcaggagatc aaagagaaga ttctcaattc cgattatgac gtcgaggatt 4440
tcttcgaagg ggagttcttt aattttgtgc tcacacaaga gggcattgac gtgtacaacg 4500
cgattatcgg gggcttcgtc acagagtccg gggagaagat taaggggctg aatgagtaca 4560
tcaatctgta caatcagaag accaagcaga aactgccgaa attcaagccg ctctacaagc 4620
aagtcctgtc cgatagggaa agcctctcct tctacggcga gggctatacc agcgacgagg 4680
aggtgctgga agtcttccgc aacacactga ataagaatag cgagattttc tcctccatca 4740
agaagctcga gaagctcttt aagaactttg acgagtacag ctccgccggg attttcgtga 4800
agaacgggcc ggcgatcagc accatctcca aggacatctt tggcgagtgg aacgtcatca 4860
gggacaagtg gaacgccgag tacgacgaca tccacctgaa gaagaaggcg gtggtgaccg 4920
agaagtatga ggacgatcgc aggaagtcct tcaaaaaaat cggctccttc agcctcgaac 4980
agctccagga gtatgccgat gcggatctgt ccgtcgtcga gaagctgaag gaaatcatca 5040
ttcagaaggt cgacgagatc tataaagtgt acgggtccag cgagaagctg ttcgacgccg 5100
actttgtgct cgagaagtcc ctcaaaaaga atgacgccgt ggtggccatt atgaaagacc 5160
tgctcgactc cgtgaagtcc ttcgaaaatt acattaaagc gttctttggg gaggggaagg 5220
aaactaacag ggatgagtcc ttctatggcg actttgtcct cgcgtacgac atcctgctga 5280
aggtcgacca catttacgac gcgatccgca actacgtgac acagaagccg tactccaaag 5340
acaagttcaa gctgtacttc cagaacccgc aatttatggg gggctgggac aaggataaag 5400
agacagacta ccgcgcgaca attctccgct atggctccaa atactatctg gccatcatgg 5460
acaagaagta cgcgaagtgc ctgcagaaga tcgacaaaga cgacgtcaat ggcaactatg 5520
aaaagatcaa ctacaagctg ctgccgggcc cgaacaagat gctcccgaag gtgttcttca 5580
gcaagaagtg gatggcctac tacaatccaa gcgaggatat tcagaaaatc tataaaaacg 5640
ggaccttcaa gaagggggac atgtttaacc tcaacgactg ccacaagctc attgatttct 5700
tcaaggatag catttcccgc tacccgaaat ggtccaatgc gtacgatttt aacttctccg 5760
agacagaaaa gtacaaagac atcgcgggct tttacaggga ggtggaggag caagggtata 5820
aagtttcttt tgaatccgcg agcaagaagg aagtcgacaa gctcgtcgag gagggcaagc 5880
tctacatgtt ccaaatttat aacaaggact tttccgacaa gagccatggg accccaaacc 5940
tccacaccat gtacttcaaa ctgctctttg acgagaacaa ccacgggcaa atcaggctga 6000
gcggcggcgc cgaattattc atgcgcaggg cctccctcaa gaaggaagag ctggtcgtcc 6060
atccagccaa ttccccgatc gcgaacaaga acccggacaa tccgaaaaag accaccaccc 6120
tgtcctacga cgtctacaag gacaaacgct tcagcgaaga ccagtacgaa ttacacatcc 6180
caattgcgat taataagtgc ccaaagaata tcttcaaaat taatacagag gtcagggtgc 6240
tgctcaaaca cgacgacaat ccgtatgtca tcggcattga caggggcgag cgcaatctgc 6300
tctatatcgt ggtcgtggat gggaagggca atattgtgga gcagtactcc ctgaacgaga 6360
ttatcaacaa cttcaatggg attaggatta agaccgacta tcacagcctg ctcgacaaga 6420
aagaaaaaga gaggtttgag gcccgccaaa actggacctc cattgagaat atcaaagaat 6480
taaaggccgg ctatatttcc caagtcgtcc acaagatctg cgagctggtg gagaaatatg 6540
acgccgtgat tgcgctcgaa gacttaaatt ctgggttcaa gaactcccgc gtgaaggtgg 6600
aaaaacaggt gtatcagaaa ttcgagaaaa tgctgatcga caaactcaat tatatggtgg 6660
ataagaagtc caacccgtgt gccacagggg gcgcgctgaa gggctatcag atcaccaaca 6720
agttcgagag cttcaagagc atgagcaccc agaacgggtt tattttctac atcccggcgt 6780
ggctcacctc caagattgac ccgagcaccg gcttcgtgaa cctcctgaag acaaagtata 6840
cctccattgc cgacagcaag aagtttatct cctccttcga ccgcattatg tatgtgccgg 6900
aggaggacct cttcgagttc gccctcgact acaaaaactt cagccgcaca gatgcggatt 6960
acatcaagaa gtggaagctg tactcctacg ggaacaggat ccgcatcttc aggaatccaa 7020
aaaaaaataa cgtctttgac tgggaggaag tgtgcctgac atccgcctac aaggaactgt 7080
tcaataaata cggcatcaat taccagcagg gcgacattcg cgccctcctc tgtgagcagt 7140
ccgacaaagc gttttactcc agcttcatgg ccctcatgtc cctgatgctc caaatgagga 7200
atagcatcac agggcgcacc gacgtcgact tcctcatcag cccggtgaag aactccgacg 7260
ggatctttta cgactcccgc aactatgagg cgcaagagaa tgcgatcctc ccgaagaacg 7320
ccgatgcgaa cggggcctat aatatcgcca ggaaagtgct ctgggccatc gggcagttca 7380
aaaaggcgga ggatgagaag ctcgacaagg tgaaaattgc catttccaac aaggagtggc 7440
tggagtacgc gcagacctcc gtgaagcaca aaaggccggc ggccacgaaa aaggccggcc 7500
aggcaaaaaa gaaaaaggga tcctacccat acgatgttcc agattacgct tatccctacg 7560
acgtgcctga ttatgcatac ccatatgatg tccccgacta tgcctaagag ctcagggtgg 7620
gcgcgccgac ccagctttct tgtacaaagt ggtgatatcc cgcggccatg gcggccggga 7680
gcatgcgacg tcgatctaac tgactagccg cggccatgct agagtccgca aaaatcacca 7740
gtctctctct acaaatctat ctctctctat ttttctccag aataatgtgt gagtagttcc 7800
cagataaggg aattagggtt cttatagggt ttcgctcatg tgttgagcat ataagaaacc 7860
cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac 7920
caaaatccag tgacctgcag aattccggcc gggagcatgc gacgtcgatc taactgacta 7980
gccgcggcca tgctagagtc cgcaaaaatc accagtctct ctctacaaat ctatctctct 8040
ctatttttct ccagaataat gtgtgagtag ttcccagata agggaattag ggttcttata 8100
gggtttcgct catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa 8160
tacttctatc aataaaattt ctaattccta aaaccaaaat ccagtgacct gaattcgagg 8220
cggtttgcgt attggctaga gcagcttgcc aacatggtgg agcacgacac tctcgtctac 8280
tccaagaata tcaaagatac agtctcagaa gaccaaaggg ctattgagac ttttcaacaa 8340
agggtaatat cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcaaa 8400
aggacagtag aaaaggaagg tggcacctac aaatgccatc attgcgataa aggaaaggct 8460
atcgttcaag atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc 8520
atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgataac 8580
atggtggagc acgacactct cgtctactcc aagaatatca aagatacagt ctcagaagac 8640
caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct cggattccat 8700
tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg cacctacaaa 8760
tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga cagtggtccc 8820
aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct 8880
tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc acaatcccac 8940
tatccttcgc aagaccttcc tctatataag gaagttcatt tcatttggag aggacacgct 9000
gaaatcacca gtctctctct acaaatctat ctctctcgag ctttcgcaga tcccgggggg 9060
caatgagata tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa 9120
aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc 9180
agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc 9240
tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg 9300
cttgacattg gggagtttag cgagagcctg acctattgca tctcccgccg tgcacagggt 9360
gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctacaacc ggtcgcggag 9420
gctatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga 9480
ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc 9540
catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct 9600
ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg 9660
gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg 9720
agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg 9780
tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca 9840
ggatcgccac gactccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc 9900
ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc 9960
cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg 10020
accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg 10080
agggcaaaga aatagagtag atgccgaccg gatctgtcga tcgacaagct cgagtttctc 10140
cataataatg tgtgagtagt tcccagataa gggaattagg gttcctatag ggtttcgctc 10200
atgtgttgag catataagaa acccttagta tgtatttgta tttgtaaaat acttctatca 10260
ataaaatttc taattcctaa aaccaaaatc cagtactaaa atccagatcc cccgaattaa 10320
ttcggcgtta attcagtaca ttaaaaacgt ccgcaatgtg ttattaagtt gtcactagtc 10380
aggttaactc aattcggcgt taattcagta cattaaaaac gtccgcaatg tgttattaag 10440
ttgtctaagc gtcaatttgt ttacaccaca atatatcctg ccaccagcca gccaacagct 10500
ccccgaccgg cagctcggca caaaatcacc actcgataca ggcagcccat cagtccggga 10560
cggcgtcagc gggagagccg ttgtaaggcg gcagactttg ctcatgttac cgatgctatt 10620
cggaagaacg gcaactaagc tgccgggttt gaaacacgga tgatctcgcg gagggtagca 10680
tgttgattgt aacgatgaca gagcgttgct gcctgtgatc acttaagtaa ctaactaaca 10740
ggaagagttt gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga 10800
tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg 10860
ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag 10920
ataaaacgaa aggcccagtc ttccgactga gcctttcgtt ttatttgatg cctggcagtt 10980
ccctactctc gcttagtagt tagacgtccc cgagatccat gctagaccat gaatccagaa 11040
gcccgagagg ttgccgcctt tcgggctttt tctttttcaa aaaaaaaaat ttataaaacg 11100
atctgttgcg gccggccgcc gggttgtggg caaaggcgct cgacggtggg caaccgcttg 11160
cggttgtcca cgggcggagc cggtgcgcgt agcgcattgt ccacaagcca agggcgacca 11220
ataattgata tatatattca taattgaaaa gctaattgaa catactactt gctgtaacta 11280
cttgccggag cgaggggtgt ttgcaagctg ttgatctgaa agggctatta gcgttctcac 11340
gtgccttttt gattagcgat ttcacgtgac cttattagcg atttcacgta ctccgattag 11400
cgatttcacg taccctgatt agcgatttca cgtggatagt ttttggagcg ggccggaaag 11460
ccccgtgaat caaggctttg cggggcatta gcggtttcac gtggataact accctctatc 11520
cacaggcttc cggggataaa aaagcccgct cgacggcggg ctgttggatg gggatctagc 11580
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 11640
ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 11700
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 11760
actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 11820
cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 11880
tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 11940
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 12000
caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 12060
agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 12120
tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 12180
tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 12240
gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctaccgg 12300
gtctgacgct cagtggaacg gggcccaatc tgaataatgt tacaaccaat taaccaattc 12360
tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc 12420
aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt 12480
ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca 12540
acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac 12600
gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg 12660
ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga 12720
ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat 12780
cgaatgcaac cggcgcaggg acactgccag cgcatcaaca atattttcac ctgaatcagg 12840
atattcttct aatacctgga atgctgtttt tccggggatc gcagtggtga gtaaccatgc 12900
atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca 12960
gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag 13020
aaacaactct ggcgcatcgg gcttcccata caagcgatag attgtcgcac ctgattgccc 13080
gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg 13140
cggcctcgac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta 13200
agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag 13260
attttgagac acgggccaga gctgcagttt gatcccgagg ggaaccctgt ggttgacatg 13320
cacatacaaa tggacgaacg gataaacctt ttcacgccct tttaaatatc cgttattcta 13380
ataaacgctc ttttctctta g 13401
<210> 3
<211> 15731
<212> DNA
<213> 陆地棉(Gossypium hirsutum)
<220>
<221> gene
<222> (1)..(15731)
<400> 3
cttgtacaaa gtggttgata acagcgacta caaggatgac gatgacaagg cttagagctc 60
gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc 120
cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 180
catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata 240
catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc 300
ggtgtcatct atgttactag atcgggaatt cactggccgt cgttttacac tggccgtcgt 360
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 420
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 480
gttgcgcagc ctgaatggcg aatgctagag cagcttgagc ttggatcaga ttgtcgtttc 540
ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa acctaagaga 600
aaagagcgtt tattagaata acggatattt aaaagggcgt gaaaaggttt atccgttcgt 660
ccatttgtat gtgcatgcca accacagggt tcccctcggg atcaaagtac tttgatccaa 720
cccctccgct gctatagtgc agtcggcttc tgacgttcag tgcagccgtc ttctgaaaac 780
gacatgtcgc acaagtccta agttacgcga caggctgccg ccctgccctt ttcctggcgt 840
tttcttgtcg cgtgttttag tcgcataaag tagaatactt gcgactagaa ccggagacat 900
tacgccatga acaagagcgc cgccgctggc ctgctgggct atgcccgcgt cagcaccgac 960
gaccaggact tgaccaacca acgggccgaa ctgcacgcgg ccggctgcac caagctgttt 1020
tccgagaaga tcaccggcac caggcgcgac cgcccggagc tggccaggat gcttgaccac 1080
ctacgccctg gcgacgttgt gacagtgacc aggctagacc gcctggcccg cagcacccgc 1140
gacctactgg acattgccga gcgcatccag gaggccggcg cgggcctgcg tagcctggca 1200
gagccgtggg ccgacaccac cacgccggcc ggccgcatgg tgttgaccgt gttcgccggc 1260
attgccgagt tcgagcgttc cctaatcatc gaccgcaccc ggagcgggcg cgaggccgcc 1320
aaggcccgag gcgtgaagtt tggcccccgc cctaccctca ccccggcaca gatcgcgcac 1380
gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag aggcggctgc actgcttggc 1440
gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg aggaagtgac gcccaccgag 1500
gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg aggccgacgc cctggcggcc 1560
gccgagaatg aacgccaaga ggaacaagca tgaaaccgca ccaggacggc caggacgaac 1620
cgtttttcat taccgaagag atcgaggcgg agatgatcgc ggccgggtac gtgttcgagc 1680
cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct ggccggtttg tctgatgcca 1740
agctggcggc ctggccggcc agcttggccg ctgaagaaac cgagcgccgc cgtctaaaaa 1800
ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt cgctgcgtat atgatgcgat 1860
gagtaaataa acaaatacgc aaggggaacg catgaaggtt atcgctgtac ttaaccagaa 1920
aggcgggtca ggcaagacga ccatcgcaac ccatctagcc cgcgccctgc aactcgccgg 1980
ggccgatgtt ctgttagtcg attccgatcc ccagggcagt gcccgcgatt gggcggccgt 2040
gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc ccgacgattg accgcgacgt 2100
gaaggccatc ggccggcgcg acttcgtagt gatcgacgga gcgccccagg cggcggactt 2160
ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt ccggtgcagc caagccctta 2220
cgacatatgg gccaccgccg acctggtgga gctggttaag cagcgcattg aggtcacgga 2280
tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc aaaggcacgc gcatcggcgg 2340
tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt cttgagtccc gtatcacgca 2400
gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc gttcttgaat cagaacccga 2460
gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa attaaatcaa aactcatttg 2520
agttaatgag gtaaagagaa aatgagcaaa agcacaaaca cgctaagtgc cggccgtccg 2580
agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg cagacacgcc agccatgaag 2640
cgggtcaact ttcagttgcc ggcggaggat cacaccaagc tgaagatgta cgcggtacgc 2700
caaggcaaga ccattaccga gctgctatct gaatacatcg cgcagctacc agagtaaatg 2760
agcaaatgaa taaatgagta gatgaatttt agcggctaaa ggaggcggca tggaaaatca 2820
agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt ggaggaacgg gcggttggcc 2880
aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc actggaaccc ccaagcccga 2940
ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca aatcggcgcg gcgctgggtg 3000
atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc atcgaggcag 3060
aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa gaatcccggc 3120
aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac gagcaaccag 3180
attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc atcatggacg 3240
tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc cgctacgagc 3300
ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt gtgtgggatt 3360
acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga taccgggaag 3420
ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta ctcaagttct 3480
gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc attcggttaa 3540
acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc ctggtgacgg 3600
tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa accgggcggc 3660
cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca gaaggcaaga 3720
acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc atcggccgtt 3780
ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg ttgttcaaga 3840
cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc accgtgcgca 3900
agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg gggcaggctg 3960
gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc gccggttcct 4020
aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt cgaaaagcac 4080
tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg aaccggaacc 4140
cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa gtgactgata 4200
taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt aaaactctta 4260
aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag ctgcaaaaag 4320
cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg cctatcgcgg 4380
ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg cgcggacaag 4440
ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg caccctgcct cgcgcgtttc 4500
ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 4560
taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 4620
cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg 4680
cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 4740
gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc 4800
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4860
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4920
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4980
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5040
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5100
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 5160
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5220
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5280
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5340
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5400
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5460
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5520
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5580
ggaacgaaaa ctcacgttaa gggattttgg tcatgcattc taggtactaa aacaattcat 5640
ccagtaaaat ataatatttt attttctccc aatcaggctt gatccccagt aagtcaaaaa 5700
atagctcgac atactgttct tccccgatat cctccctgat cgaccggacg cagaaggcaa 5760
tgtcatacca cttgtccgcc ctgccgcttc tcccaagatc aataaagcca cttactttgc 5820
catctttcac aaagatgttg ctgtctccca ggtcgccgtg ggaaaagaca agttcctctt 5880
cgggcttttc cgtctttaaa aaatcataca gctcgcgcgg atctttaaat ggagtgtctt 5940
cttcccagtt ttcgcaatcc acatcggcca gatcgttatt cagtaagtaa tccaattcgg 6000
ctaagcggct gtctaagcta ttcgtatagg gacaatccga tatgtcgatg gagtgaaaga 6060
gcctgatgca ctccgcatac agctcgataa tcttttcagg gctttgttca tcttcatact 6120
cttccgagca aaggacgcca tcggcctcac tcatgagcag attgctccag ccatcatgcc 6180
gttcaaagtg caggaccttt ggaacaggca gctttccttc cagccatagc atcatgtcct 6240
tttcccgttc cacatcatag gtggtccctt tataccggct gtccgtcatt tttaaatata 6300
ggttttcatt ttctcccacc agcttatata ccttagcagg agacattcct tccgtatctt 6360
ttacgcagcg gtatttttcg atcagttttt tcaattccgg tgatattctc attttagcca 6420
tttattattt ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa 6480
caagacgaac tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc 6540
tttttcaaag ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa 6600
accgcggtga tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg 6660
cgagatcatc cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt 6720
aacatgagca aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat 6780
gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg 6840
gctggtggca ggatatattg tggtgtaaac aaattgacgc ttagacaact taataacaca 6900
ttgcggacgt ttttaatgta ctgaattaac gccgaattaa ttcgggggat ctggatttta 6960
gtactggatt ttggttttag gaattagaaa ttttattgat agaagtattt tacaaataca 7020
aatacatact aagggtttct tatatgctca acacatgagc gaaaccctat aggaacccta 7080
attcccttat ctgggaacta ctcacacatt attatggaga aactcgagct cagaagaact 7140
cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca 7200
cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg 7260
ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc 7320
ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcat 7380
cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat 7440
gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct 7500
cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc 7560
gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga 7620
gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt 7680
cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt 7740
cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct 7800
gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat 7860
agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa 7920
tcatcccggg atctgcgaaa gctcgagaga gatagatttg tagagagaga ctggtgattt 7980
cagcgtgtcc tctccaaatg aaatgaactt ccttatatag aggaaggtct tgcgaaggat 8040
agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg 8100
aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt 8160
tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc 8220
atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc 8280
aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt 8340
ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca 8400
tgttatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga 8460
tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga 8520
tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc 8580
ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc 8640
tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg 8700
agtagacgag agtgtcgtgc tccaccatgt tggcaagctg ctctagccaa tacgcaaacc 8760
gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg 8820
gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 8880
ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 8940
tcacacagga aacagctatg accatgatta cgccaagctt ttaatctgat gctccacctg 9000
cttttgattt tctttattgg aagagtcttt aagagatatg ttaagtagca taacagtttc 9060
atcaaaaaca acatttctgt taatcacaac ttttctattt tcaggatacc ataacttata 9120
cacttttaca ctagctttat aaccaagaaa aacacattta atggtacaca attttaattt 9180
tccattatca gcatgagtat acgcaaaaca cccaaaaatc tttaaatcag aatcgtcagc 9240
aggattacta aaccatactt cttatggagt ctttttctca atagcaacga atagagacgg 9300
attgatcaaa aacatatagt tgacattgct ttggcccaaa ataactttga taagttgcca 9360
tttgacaaca tacatcgaac attctccatg atcgttctat tcattcgttc tacaatacct 9420
tttttaaaat gttcaggttc taaaatgaaa aacaatatga attgcatgaa ttgcttatat 9480
gtcctatgaa ttataaagga atgcggttga aatattccca tcgatacata catacatatt 9540
cgtgaagtat gttccaatat aatatcaata ttgggattta cgttttataa agcaacatta 9600
ttgattggta atatacatta attccaaggc aaacccaaat attttaaaat ttaacctaca 9660
actgtggtaa atcaaactta atagtaaccc gattgtaatg tgaagtcaaa tatgaaagta 9720
acattggttt atatatatat ttttctctaa attctaataa tcaagttggg ataagtgata 9780
aacactgagc ttgccacgtg tgttaacctc gttttcatca tgtgccactc caaagacatc 9840
aggcctctat tcaagctggc atggtcagga cgtggtagca tacttcaggg atctggttag 9900
aaaatatccc atatcgctaa agaactataa cacaggagcg tttatataag cgaaagaagc 9960
atcagatggg caggagaccg aggtctcggt tttagagcta gaaatagcaa gttaaaataa 10020
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc 10080
tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcatgc ctgcaggtcc 10140
acaaattcgg gtcaaggcgg aagccagcgc gccaccccac gtcagcaaat acggaggcgc 10200
ggggttgacg gcgtcacccg gtcctaacgg cgaccaacaa accagccaga agaaattaca 10260
gtaaaaaaaa agtaaattgc actttgatcc accttttatt acctaagtct caatttggat 10320
cacccttaaa cctatctttt caatttgggc cgggttgtgg tttggactac catgaacaac 10380
ttttcgtcat gtctaacttc cctttcagca aacatatgaa ccatatatag aggagatcgg 10440
ccgtatacta gagctgatgt gtttaaggtc gttgattgca cgagaaaaaa aaatccaaat 10500
cgcaacaata gcaaatttat ctggttcaaa gtgaaaagat atgtttaaag gtagtccaaa 10560
gtaaaactta tagataataa aatgtggtcc aaagcgtaat tcactcaaaa aaaatcaacg 10620
agacgtgtac caaacggaga caaacggcat cttctcgaaa tttcccaacc gctcgctcgc 10680
ccgcctcgtc ttcccggaaa ccgcggtggt ttcagcgtgg cggattctcc aagcagacgg 10740
agacgtcacg gcacgggact cctcccacca cccaaccgcc ataaatacca gccccctcat 10800
ctcctctcct cgcatcagct ccacccccga aaaatttctc cccaatctcg cgaggctctc 10860
gtcgtcgaat cgaatcctct cgcgtcctca aggtacgctg cttctcctct cctcgcttcg 10920
tttcgattcg atttcggacg ggtgaggttg ttttgttgct agatccgatt ggtggttagg 10980
gttgtcgatg tgattatcgt gagatgttta ggggttgtag atctgatggt tgtgatttgg 11040
gcacggttgg ttcgataggt ggaatcgtgg ttaggttttg ggattggatg ttggttctga 11100
tgattggggg gaatttttac ggttagatga attgttggat gattcgattg gggaaatcgg 11160
tgtagatctg ttggggaatt gtggaactag tcatgcctga gtgattggtg cgatttgtag 11220
cgtgttccat cttgtaggcc ttgttgcgag catgttcaga tctactgttc cgctcttgat 11280
tgagttattg gtgccatggg ttggtgcaaa cacaggcttt aatatgttat atctgttttg 11340
tgtttgatgt agatctgtag ggtagttctt cttagacatg gttcaattat gtagcttgtg 11400
cgtttcgatt tgatttcata tgttcacaga ttagataatg atgaactctt ttaattaatt 11460
gtcaatggta aataggaagt cttgtcgcta tatctgtcat aatgatctca tgttactatc 11520
tgccagtaat ttatgctaag aactatatta gaatatcatg ttacaatctg tagtaatatc 11580
atgttacaat ctgtagttca tctatataat ctattgtggt aatttctttt tactatctgt 11640
gtgaagatta ttgccactag ttcattctac ttatttctga agttcaggat acgtgtgctg 11700
ttactaccta tctgaataca tgtgtgatgt gcctgttact atctttttga atacatgtat 11760
gttctgttgg aatatgtttg ctgtttgatc cgttgttgtg tccttaatct tgtgctagtt 11820
cttaccctat ctgtttggtg attatttctt gcagatagtt atcaacaagt ttgtacaaaa 11880
aagcaggctt cgaaatgtcc aagctggaga agtttacaaa ctgttacagc ctctccaaaa 11940
ccctcaggtt taaagcgatc ccggtgggca agacccagga gaacatcgac aacaagaggc 12000
tcctggtgga agacgagaag cgcgccgaag actacaaggg cgtgaagaag ctgctcgata 12060
ggtactacct cagctttatt aacgacgtgc tgcacagcat caaactcaag aatctcaaca 12120
actacatctc cctcttccgc aaaaagaccc gcaccgagaa ggagaacaag gagctggaga 12180
acctggagat caacctccgc aaggaaatcg ccaaagcgtt caagggcaat gaagggtaca 12240
agagcctctt caagaaagac atcatcgaaa ctatcctccc agagtttctc gatgacaagg 12300
acgagatcgc gctggtgaac tcctttaacg ggttcacaac cgcgtttacc ggcttctttg 12360
ataacaggga aaatatgttc tccgaggagg ccaagtccac cagcatcgcc ttcaggtgta 12420
tcaacgagaa cctcacccgc tacatttcca atatggacat tttcgagaag gtggatgcga 12480
tcttcgataa gcacgaggtg caggagatca aagagaagat tctcaattcc gattatgacg 12540
tcgaggattt cttcgaaggg gagttcttta attttgtgct cacacaagag ggcattgacg 12600
tgtacaacgc gattatcggg ggcttcgtca cagagtccgg ggagaagatt aaggggctga 12660
atgagtacat caatctgtac aatcagaaga ccaagcagaa actgccgaaa ttcaagccgc 12720
tctacaagca agtcctgtcc gatagggaaa gcctctcctt ctacggcgag ggctatacca 12780
gcgacgagga ggtgctggaa gtcttccgca acacactgaa taagaatagc gagattttct 12840
cctccatcaa gaagctcgag aagctcttta agaactttga cgagtacagc tccgccggga 12900
ttttcgtgaa gaacgggccg gcgatcagca ccatctccaa ggacatcttt ggcgagtgga 12960
acgtcatcag ggacaagtgg aacgccgagt acgacgacat ccacctgaag aagaaggcgg 13020
tggtgaccga gaagtatgag gacgatcgca ggaagtcctt caaaaaaatc ggctccttca 13080
gcctcgaaca gctccaggag tatgccgatg cggatctgtc cgtcgtcgag aagctgaagg 13140
aaatcatcat tcagaaggtc gacgagatct ataaagtgta cgggtccagc gagaagctgt 13200
tcgacgccga ctttgtgctc gagaagtccc tcaaaaagaa tgacgccgtg gtggccatta 13260
tgaaagacct gctcgactcc gtgaagtcct tcgaaaatta cattaaagcg ttctttgggg 13320
aggggaagga aactaacagg gatgagtcct tctatggcga ctttgtcctc gcgtacgaca 13380
tcctgctgaa ggtcgaccac atttacgacg cgatccgcaa ctacgtgaca cagaagccgt 13440
actccaaaga caagttcaag ctgtacttcc agaacccgca atttatgggg ggctgggaca 13500
aggataaaga gacagactac cgcgcgacaa ttctccgcta tggctccaaa tactatctgg 13560
ccatcatgga caagaagtac gcgaagtgcc tgcagaagat cgacaaagac gacgtcaatg 13620
gcaactatga aaagatcaac tacaagctgc tgccgggccc gaacaagatg ctcccgaagg 13680
tgttcttcag caagaagtgg atggcctact acaatccaag cgaggatatt cagaaaatct 13740
ataaaaacgg gaccttcaag aagggggaca tgtttaacct caacgactgc cacaagctca 13800
ttgatttctt caaggatagc atttcccgct acccgaaatg gtccaatgcg tacgatttta 13860
acttctccga gacagaaaag tacaaagaca tcgcgggctt ttacagggag gtggaggagc 13920
aagggtataa agtttctttt gaatccgcga gcaagaagga agtcgacaag ctcgtcgagg 13980
agggcaagct ctacatgttc caaatttata acaaggactt ttccgacaag agccatggga 14040
ccccaaacct ccacaccatg tacttcaaac tgctctttga cgagaacaac cacgggcaaa 14100
tcaggctgag cggcggcgcc gaattattca tgcgcagggc ctccctcaag aaggaagagc 14160
tggtcgtcca tccagccaat tccccgatcg cgaacaagaa cccggacaat ccgaaaaaga 14220
ccaccaccct gtcctacgac gtctacaagg acaaacgctt cagcgaagac cagtacgaat 14280
tacacatccc aattgcgatt aataagtgcc caaagaatat cttcaaaatt aatacagagg 14340
tcagggtgct gctcaaacac gacgacaatc cgtatgtcat cggcattgac aggggcgagc 14400
gcaatctgct ctatatcgtg gtcgtggatg ggaagggcaa tattgtggag cagtactccc 14460
tgaacgagat tatcaacaac ttcaatggga ttaggattaa gaccgactat cacagcctgc 14520
tcgacaagaa agaaaaagag aggtttgagg cccgccaaaa ctggacctcc attgagaata 14580
tcaaagaatt aaaggccggc tatatttccc aagtcgtcca caagatctgc gagctggtgg 14640
agaaatatga cgccgtgatt gcgctcgaag acttaaattc tgggttcaag aactcccgcg 14700
tgaaggtgga aaaacaggtg tatcagaaat tcgagaaaat gctgatcgac aaactcaatt 14760
atatggtgga taagaagtcc aacccgtgtg ccacaggggg cgcgctgaag ggctatcaga 14820
tcaccaacaa gttcgagagc ttcaagagca tgagcaccca gaacgggttt attttctaca 14880
tcccggcgtg gctcacctcc aagattgacc cgagcaccgg cttcgtgaac ctcctgaaga 14940
caaagtatac ctccattgcc gacagcaaga agtttatctc ctccttcgac cgcattatgt 15000
atgtgccgga ggaggacctc ttcgagttcg ccctcgacta caaaaacttc agccgcacag 15060
atgcggatta catcaagaag tggaagctgt actcctacgg gaacaggatc cgcatcttca 15120
ggaatccaaa aaaaaataac gtctttgact gggaggaagt gtgcctgaca tccgcctaca 15180
aggaactgtt caataaatac ggcatcaatt accagcaggg cgacattcgc gccctcctct 15240
gtgagcagtc cgacaaagcg ttttactcca gcttcatggc cctcatgtcc ctgatgctcc 15300
aaatgaggaa tagcatcaca gggcgcaccg acgtcgactt cctcatcagc ccggtgaaga 15360
actccgacgg gatcttttac gactcccgca actatgaggc gcaagagaat gcgatcctcc 15420
cgaagaacgc cgatgcgaac ggggcctata atatcgccag gaaagtgctc tgggccatcg 15480
ggcagttcaa aaaggcggag gatgagaagc tcgacaaggt gaaaattgcc atttccaaca 15540
aggagtggct ggagtacgcg cagacctccg tgaagcacaa aaggccggcg gccacgaaaa 15600
aggccggcca ggcaaaaaag aaaaagggat cctacccata cgatgttcca gattacgctt 15660
atccctacga cgtgcctgat tatgcatacc catatgatgt ccccgactat gcctaatcta 15720
gacccagctt t 15731
<210> 4
<211> 3822
<212> DNA
<213> 陆地棉(Gossypium hirsutum)
<220>
<221> gene
<222> (1)..(3822)
<400> 4
atgtccaagc tggagaagtt tacaaactgt tacagcctct ccaaaaccct caggtttaaa 60
gcgatcccgg tgggcaagac ccaggagaac atcgacaaca agaggctcct ggtggaagac 120
gagaagcgcg ccgaagacta caagggcgtg aagaagctgc tcgataggta ctacctcagc 180
tttattaacg acgtgctgca cagcatcaaa ctcaagaatc tcaacaacta catctccctc 240
ttccgcaaaa agacccgcac cgagaaggag aacaaggagc tggagaacct ggagatcaac 300
ctccgcaagg aaatcgccaa agcgttcaag ggcaatgaag ggtacaagag cctcttcaag 360
aaagacatca tcgaaactat cctcccagag tttctcgatg acaaggacga gatcgcgctg 420
gtgaactcct ttaacgggtt cacaaccgcg tttaccggct tctttgataa cagggaaaat 480
atgttctccg aggaggccaa gtccaccagc atcgccttca ggtgtatcaa cgagaacctc 540
acccgctaca tttccaatat ggacattttc gagaaggtgg atgcgatctt cgataagcac 600
gaggtgcagg agatcaaaga gaagattctc aattccgatt atgacgtcga ggatttcttc 660
gaaggggagt tctttaattt tgtgctcaca caagagggca ttgacgtgta caacgcgatt 720
atcgggggct tcgtcacaga gtccggggag aagattaagg ggctgaatga gtacatcaat 780
ctgtacaatc agaagaccaa gcagaaactg ccgaaattca agccgctcta caagcaagtc 840
ctgtccgata gggaaagcct ctccttctac ggcgagggct ataccagcga cgaggaggtg 900
ctggaagtct tccgcaacac actgaataag aatagcgaga ttttctcctc catcaagaag 960
ctcgagaagc tctttaagaa ctttgacgag tacagctccg ccgggatttt cgtgaagaac 1020
gggccggcga tcagcaccat ctccaaggac atctttggcg agtggaacgt catcagggac 1080
aagtggaacg ccgagtacga cgacatccac ctgaagaaga aggcggtggt gaccgagaag 1140
tatgaggacg atcgcaggaa gtccttcaaa aaaatcggct ccttcagcct cgaacagctc 1200
caggagtatg ccgatgcgga tctgtccgtc gtcgagaagc tgaaggaaat catcattcag 1260
aaggtcgacg agatctataa agtgtacggg tccagcgaga agctgttcga cgccgacttt 1320
gtgctcgaga agtccctcaa aaagaatgac gccgtggtgg ccattatgaa agacctgctc 1380
gactccgtga agtccttcga aaattacatt aaagcgttct ttggggaggg gaaggaaact 1440
aacagggatg agtccttcta tggcgacttt gtcctcgcgt acgacatcct gctgaaggtc 1500
gaccacattt acgacgcgat ccgcaactac gtgacacaga agccgtactc caaagacaag 1560
ttcaagctgt acttccagaa cccgcaattt atggggggct gggacaagga taaagagaca 1620
gactaccgcg cgacaattct ccgctatggc tccaaatact atctggccat catggacaag 1680
aagtacgcga agtgcctgca gaagatcgac aaagacgacg tcaatggcaa ctatgaaaag 1740
atcaactaca agctgctgcc gggcccgaac aagatgctcc cgaaggtgtt cttcagcaag 1800
aagtggatgg cctactacaa tccaagcgag gatattcaga aaatctataa aaacgggacc 1860
ttcaagaagg gggacatgtt taacctcaac gactgccaca agctcattga tttcttcaag 1920
gatagcattt cccgctaccc gaaatggtcc aatgcgtacg attttaactt ctccgagaca 1980
gaaaagtaca aagacatcgc gggcttttac agggaggtgg aggagcaagg gtataaagtt 2040
tcttttgaat ccgcgagcaa gaaggaagtc gacaagctcg tcgaggaggg caagctctac 2100
atgttccaaa tttataacaa ggacttttcc gacaagagcc atgggacccc aaacctccac 2160
accatgtact tcaaactgct ctttgacgag aacaaccacg ggcaaatcag gctgagcggc 2220
ggcgccgaat tattcatgcg cagggcctcc ctcaagaagg aagagctggt cgtccatcca 2280
gccaattccc cgatcgcgaa caagaacccg gacaatccga aaaagaccac caccctgtcc 2340
tacgacgtct acaaggacaa acgcttcagc gaagaccagt acgaattaca catcccaatt 2400
gcgattaata agtgcccaaa gaatatcttc aaaattaata cagaggtcag ggtgctgctc 2460
aaacacgacg acaatccgta tgtcatcggc attgacaggg gcgagcgcaa tctgctctat 2520
atcgtggtcg tggatgggaa gggcaatatt gtggagcagt actccctgaa cgagattatc 2580
aacaacttca atgggattag gattaagacc gactatcaca gcctgctcga caagaaagaa 2640
aaagagaggt ttgaggcccg ccaaaactgg acctccattg agaatatcaa agaattaaag 2700
gccggctata tttcccaagt cgtccacaag atctgcgagc tggtggagaa atatgacgcc 2760
gtgattgcgc tcgaagactt aaattctggg ttcaagaact cccgcgtgaa ggtggaaaaa 2820
caggtgtatc agaaattcga gaaaatgctg atcgacaaac tcaattatat ggtggataag 2880
aagtccaacc cgtgtgccac agggggcgcg ctgaagggct atcagatcac caacaagttc 2940
gagagcttca agagcatgag cacccagaac gggtttattt tctacatccc ggcgtggctc 3000
acctccaaga ttgacccgag caccggcttc gtgaacctcc tgaagacaaa gtatacctcc 3060
attgccgaca gcaagaagtt tatctcctcc ttcgaccgca ttatgtatgt gccggaggag 3120
gacctcttcg agttcgccct cgactacaaa aacttcagcc gcacagatgc ggattacatc 3180
aagaagtgga agctgtactc ctacgggaac aggatccgca tcttcaggaa tccaaaaaaa 3240
aataacgtct ttgactggga ggaagtgtgc ctgacatccg cctacaagga actgttcaat 3300
aaatacggca tcaattacca gcagggcgac attcgcgccc tcctctgtga gcagtccgac 3360
aaagcgtttt actccagctt catggccctc atgtccctga tgctccaaat gaggaatagc 3420
atcacagggc gcaccgacgt cgacttcctc atcagcccgg tgaagaactc cgacgggatc 3480
ttttacgact cccgcaacta tgaggcgcaa gagaatgcga tcctcccgaa gaacgccgat 3540
gcgaacgggg cctataatat cgccaggaaa gtgctctggg ccatcgggca gttcaaaaag 3600
gcggaggatg agaagctcga caaggtgaaa attgccattt ccaacaagga gtggctggag 3660
tacgcgcaga cctccgtgaa gcacaaaagg ccggcggcca cgaaaaaggc cggccaggca 3720
aaaaagaaaa agggatccta cccatacgat gttccagatt acgcttatcc ctacgacgtg 3780
cctgattatg catacccata tgatgtcccc gactatgcct aa 3822

Claims (4)

1.一种能够精确识别基因组“-TTTV-”碱基并能产生黏性末端的编辑陆地棉基因组高效转化载体GhRBE3，其特征在于，该载体的核苷酸序列如SEQ ID NO:3所示。
2.一种能够精确识别基因组“-TTTV-”碱基并能产生黏性末端的编辑陆地棉基因组高效转化载体GhRBE3的方法，其特征在于，所述的GhRBE3载体通过下列步骤制备获得：
(1)获得目的序列LbCpf1-NLS-3xHA，其核苷酸序列如序列表SEQ ID NO：4所示，该目的序列通过以下步骤制备获得：
1)在SEQ ID NO：2所示序列的pHUM611载体上设计引物，所述引物的序列如下所示：
正向引物：AAAAAGCAGGCTTCGAAATGTCCAAGCTGGAGAAGTTTACA，
反向引物为：GAAAGCTGGGTCTAGATTAGGCATAGTCGGGGACATC；
2)以pHUN611载体为模板，进行PCR扩增，获得LbCpf1-NLS-3xHA目的序列，该目的序列如SEQ ID NO：4所示；
(2)利用BstbⅠ、XbaⅠ对SEQ ID NO：1所示的pRGEB32-GhU6.7-NPTⅡ载体进行酶切，再与LbCpf1-NLS-3xHA目的序列连接，通过测序验证，得到如SEQ ID NO：3所示序列的适用于陆地棉的单碱基编辑的高效转化载体GhRBE3。
3.权利要求1所述的载体GhRBE3在陆地棉基因组编辑中的应用。
4.权利要求2所述的载体GhRBE3的构建方法在陆地棉基因组编辑中的应用。
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CN108486146A (zh) * 2018-03-16 2018-09-04 中国农业科学院作物科学研究所 LbCpf1-RR突变体用于CRISPR/Cpf1系统在植物基因编辑中的应用
CN110760538A (zh) * 2019-11-18 2020-02-07 江苏省农业科学院 一种创制枯萎病抗性西瓜种质材料的方法
CN111378684A (zh) * 2020-03-15 2020-07-07 华中农业大学 一种热诱导的基因编辑系统CRISPR-Cas12b在陆地棉中的应用
CN111575311A (zh) * 2020-04-15 2020-08-25 南京农业大学 一种基于基因枪介导的棉花基因编辑方法及应用
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CN111575311A (zh) * 2020-04-15 2020-08-25 南京农业大学 一种基于基因枪介导的棉花基因编辑方法及应用
CN112359057A (zh) * 2020-10-23 2021-02-12 浙江大学 CRISPR/Cas12a基因编辑系统在84K杨树基因编辑中的应用
CN113278647A (zh) * 2021-05-25 2021-08-20 华中农业大学 一种陆地棉基因组的高效定向基因调控的编辑方法
CN113174401A (zh) * 2021-06-01 2021-07-27 华中农业大学 基因编辑介导的外源基因敲入到棉花基因组的方法及载体
CN113832180A (zh) * 2021-08-03 2021-12-24 华中农业大学 CRISPR/Cas13b介导的棉花RNA转录调控方法
CN115820691A (zh) * 2022-07-25 2023-03-21 安徽农业大学 一种基于LbCpf1变体的水稻碱基编辑系统和应用
CN115820691B (zh) * 2022-07-25 2023-08-22 安徽农业大学 一种基于LbCpf1变体的水稻碱基编辑系统和应用

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