CN111378684B - 一种热诱导的基因编辑系统CRISPR-Cas12b在陆地棉中的应用 - Google Patents
一种热诱导的基因编辑系统CRISPR-Cas12b在陆地棉中的应用 Download PDFInfo
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Abstract
本发明属于植物基因工程技术领域，具体涉及一种热诱导的基因编辑系统CRISPR‑Cas12b首次在陆地棉中的应用。对含有棉花内源启动子pGhU6‑7的pRGEB32‑GhU6.7‑NPT Ⅱ载体进行改造，以密码子优化合成的AaCas12b蛋白取代原Cas9蛋白，构建棉花中具有编辑能力的载体。选取GhCLA为目标基因验证载体在棉花中的应用。设计2个靶标，利用农杆菌介导的转化将Cas12b编辑系统导入棉花基因组，使用三种温度不同时间处理下胚轴，转基因植株进行相关测序，检测异源四倍体棉花基因组中的编辑效率和全基因组检测脱靶效应。本发明使用特定的温度和处理时间得到良好的编辑效率和特异性。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及一种热诱导的基因编辑系统CRISPR-Cas12b在陆地棉中的应用。包括陆地棉的转化载体的构建，通过探索特定的处理温度和时间，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
2013年，科学家们在细菌和古生菌中发现了成簇规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats，CRISPR)和CRISPR相关Cas蛋白质(CRISPR/Cas)构成适应性免疫系统来防止噬菌体和病毒入侵，细菌的CRISPR/Cas系统可降解入侵的病毒或质粒DNA来保护自己(Bolotin et al.,2005；Jansen et al.,2002；Mojica et al.,2005；Pourcel et al.,2005)。该系统已经在动植物微生物中得到广泛的应用(Jiang et al.,2013；Li et al.,2013；Wang et al.,2014)。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、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(Hsu et al.,2014)。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。
Cas12b属于2类V-B型CRISPR系统。与常用的Cas12b不同的是C2c1只有RuvC核酸内切酶，没有一个类似HNH的部分。Cas12b的RuvC结构域十分保守(Liu et al.,2017)。本发明使用的是AaCas12b，来自酸土脂环芽孢杆菌(Alicyclobacillus acidoterrestris)，PAM序列为5'-TTN-3'，靶标长度为20nt，PAM位于靶标上游(Shmakov et al.,2015)。和Cas9类似的是，Cas12b需要tracrRNA参与基因编辑的过程，能够绑定sgRNA以形成一个二元复合物和进一步靶向DNA，形成三元复合物(Liu et al.,2017)。切割位点发生在非目标链PAM区下游23碱基处和目标链14到17碱基之间。因此，Cas12b可以产生DNA双链断裂，并且带有一个6-8nt的粘性末端，这是目前所有基因组编辑的CRISPR-Cas系统所能产生的最长粘性末端，可促进非同源重组修复。而且，Cas12b是一个与温度有关的核酸内切酶，需要42℃以上的温度，才能发挥切割作用，这表明它可以通过温度诱导产生活性切割靶向基因组，由于这个性能，对高温作物棉花基因组编辑成为了一中优势。更重要的是，它对错配十分敏感，在gRNA上邻近PAM序列的前18个核苷酸，单核苷酸突变就能停止对靶DNA的切割，后两个核苷酸的突变能减少活性(Liu et al.,2017)，说明在基因编辑领域，CRISPR/Cas12b系统可能是目前脱靶率最低的编辑工具。
目前，Cas12b编辑系统在动物中有过研究，但是在植物领域中并没有报道。陆地棉(Gossypium hirsutum)是一种广泛种植的异源四倍体(At和Dt)棉花，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，从而造成传统的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甚至脱靶。另外，棉花也属于耐高温作物，能够在40℃以上生存数天，为Cas12b蛋白的激活提供了可能。本发明的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，也为其Cas12b热激活蛋白在植物领域中的应用提供了可行性的思路。
发明内容
本发明的目的在于克服现有技术存在的缺陷，提供一种通过热诱导酶活对陆地棉基因组的精确编辑方法，所述的方法特别适合构建一种适用于陆地棉的基因组编辑系统。本发明基于pRGEB32-GhU6.7-NPTⅡ(Wang et al.,2018；Xie et al.,2015)和密码子优化合成的Cas12b序列(NCBI ID:PDB:5WQE)(Liu et al.,2017)，构建了融合AaCas12b核酸酶的适用于棉花遗传转化系统的基因编辑系统，即载体GhC12B。
本发明技术方案如下所述：
申请人提供了一种能够对陆地棉基因组5'-TTN-3'碱基位点精确识别并能产生长黏性末端的高效转化载体GhC12B，该载体的核苷酸序列如序列表SEQ ID NO:3所示。
申请人提供了一种能够对陆地棉基因组5'-TTN-3'碱基位点精确识别并能产生长黏性末端的高效转化载体GhC12B的方法，所述的方法如下所述：
(1)获得目的序列AaCas12b-NLS-3xHA，其核苷酸序列如序列表SEQ ID NO：4所示，该序列通过NCBI上获得(ID:PDB:5WQE)，前后加上BstbⅠ和XbaⅠ两个酶切位点，利用密码子优化合成到pUC57载体上(载体合成公司为南京金斯瑞生物科技有限公司)。
(2)利用BstbⅠ、XbaⅠ对SEQ ID NO：1所示的pRGEB32-GhU6.7-NPTⅡ载体和SEQ IDNO：2所示的pUC57-Cas12b进行酶切，再将pUC57-Cas12b切下来的Cas12b序列与酶切后pRGEB32-GhU6.7-NPTⅡ的序列连接，通过测序验证，得到如SEQ ID NO：3所示序列，利用该序列构建得到适用于陆地棉的基因组编辑的转化载体GhC12B。
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)本发明载体在棉花中的编辑效率为20％左右。
(2)本发明的载体Cas12b酶为温度45℃，处理时间为4天，对陆地棉的转化效率和编辑效率最高。
(3)本发明的载体在靶标序列上的缺失片段大小主要是9bp-14bp。
(4)本发明的载体在棉花中有较高的特异性。
附图说明
图1：本发明载体pUC57-Cas12b和载体pRGEB32-GhU6.7-NPTⅡ的改造路线图。
图2：本发明所使用的载体pRGEB32-GhU6.7-NPTⅡ的图谱。
图3：本发明构建的表达载体GhC12B的图谱。
图4：本发明的AaCas12b-NLS-3xHA片段的电泳图。附图标记说明：泳道1AaCpf1片段的电泳图，泳道2是AaCas12b-NLS-3xHA片段的电泳图，泳道M是5K的Marker。
图5：拼接后GhC12B的电泳图。附图标记说明：泳道是GhRBE3构建完成检测，泳道1是阴性对照，泳道M是5K的marker。
图6：是本发明的目的片段的扩增产物的电泳图。附图标记说明：其中：第一次PCR电泳图，两个片段的分别扩增。泳道M是5K的marker，泳道1-1、1-2、1-3是第一个片段，泳道2-1、2-2是第二个片段
图7：第二次PCR电泳图，利用重叠延伸PCR将第一次PCR的两个片段拼接。附图标记说明：泳道3-1、3-2、3-3、3-4都是将第一次PCR的两个片段拼接，泳道M是5K的marker。
图8：本发明GhCLA的遗传转化示意图。
图9：不同条件下本发明的GhCLA基因编辑后代编辑数目。附图标记说明：T0 lines表示不同条件下的转基因植株数目。mutants表示不同条件下棉花GhCLA基因产生编辑的植株数。横坐标轴代表不同温度下的处理时间，纵坐标轴代表植株数。
图10：不同条件下本发明的棉花GhCLA基因产生编辑的类型。附图标记说明：方框中的序列代表PTM序列，有下划线的序列代表sgRNA序列，椭圆内的序列代表碱基突变。-14bp代表编辑类型为缺少14个碱基。
图11：是本发明的载体在棉花转化过程状态图(48℃处理时期下胚轴出现发黑和死亡，说明48℃处理时期不适合对棉花基因组编辑)。附图标记说明：图11的A图显示下胚轴在正常情况下的状态，图11的B图显示下胚轴在48℃处理时期下的状态。
图12：本发明的GhCLA基因在不同处理条件下sgRNA编辑对不同样品的编辑效率图。附图标记说明：横坐标代表不同样品名称，纵坐标代表不同样品的编辑效率，不同形状的标志代表不同的温度处理。
图13：本发明在两种条件下产生GhCLA基因编辑的植株中，缺失长度的频率统计图。附图说明：横坐标轴代表缺失的碱基长度，纵坐标轴代表缺失的碱基长度频率，图13的A图代表对象为42℃处理2天的编辑植株，图13的B图代表对象为45℃处理4天的编辑植株。
图14：本发明的GhCLA基因在T0代和其子代(T1代)中，通过Sanger测序检测其可稳定遗传性和对四倍体棉花基因组的两个亚组At和Dt的编辑偏好性。附图说明：框中的序列代表PTM序列，有下划线的序列代表sgRNA序列，椭圆内的序列代表两个亚组At和Dt之间的碱基差异，柱状图代表其编辑效率。
图15：本发明对产生编辑的陆地棉植株全基因组预测脱靶位点检测。附图标记说明：一共检测10个最有可能的脱靶位点。每一个饼图说明了脱靶位点的编辑效率。扇形图N代表参考基因组没有产生变化reads，另外一个扇形图代表产生碱基突变的reads数。
图16：本发明在4株编辑陆地棉植株中的独特的变异。附图标记说明：图16中的A图表示四种阳性转基因植株中去除参考基因组和阴性对照中的突变数(Unique)。图16中的B图表示四种阳性转基因植株中去除共享的突变剩下独有的突变(Individual)。
具体实施方式
对序列表的说明：
序列表SEQ ID NO：1是本发明涉及的载体pRGEB32-GhU6.7的核苷酸序列。序列长度为16241bp。
序列表SEQ ID NO：2是本发明涉及的载体pUC57-Cas12b的核苷酸序列。序列长度为6184bp。
序列表SEQ ID NO：3是本发明构建的陆地棉基因组转化载体GhC12b的核苷酸序列。序列长度为15333bp。
序列表SEQ ID NO：4是AaCas12b-NLS-3xFLAG融合蛋白基因的核苷酸序列。序列长度为3822bp。
序列表SEQ ID NO：5是AaCas12b-NLS-3xFLAG融合蛋白的蛋白质序列。编码1186个蛋白质序列。
实施例1：AaCas12b-NLS-3xFLAG目的序列的克隆
本发明克隆的AaCas12b-NLS-3xFLAG目的序列如序列表SEQ ID NO：4所示，该序列从NCBI上获得(登陆号为ID:PDB:5WQE)，在该序列的前后加上BstbI和XbaⅠ两个酶切位点，交南京金斯瑞生物科技有限公司，利用密码子优化合成到pUC57载体上，将得到的转化载体命名为pUC57-Cas12b。利用XbaⅠ内切酶对pUC57-Cas12b进行酶切(酶切体系见表1)。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，按试剂盒的说明书操作)将酶切产物纯化。
酶切反应体系见表1。
表1pUC57-Cas12b的酶切体系
实施例2：转化载体GhC12b的构建
将载体pRGEB32-GhU6.7-NPTⅡ(序列表见SEQ ID NO：1)进行双酶切，酶切体系见表1。
将酶切后的pRGEB32-GhU6.7-NPTⅡ载体与AaCas12b-NLS-3xFLAG基因片段通过载体
-T Easy Vector Systems(Promega A1380)进行T4 DNA连接，连接体系见表2。转化至大肠杆菌感受态，挑取阳性克隆进行测序，将序列正确的质粒命名为GhC12B(见序列表SEQ ID NO：3)。
表2T4 DNA连接反应体系
37℃水浴30min，冰上放置5min，可-20℃下保存。
实施例3：GhC12B-sgRNA载体的构建
1.GhCLA基因的sgRNA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在基因外显子区域设计PAM序列为5'-TTN-3'的sgRNA靶标序列,选择了2个sgRNA用来构建基因编辑系统植物表达载体(Xie et al.,2014)。所述的sgRNA的序列见表3。
表3sgRNA的序列
2.crRNA与GhC12B载体的连接
靶标插入GhC12B载体序列为tRNA-sgRNA-gRNA的重复序列，需要中间载体转换第一次PCR的引物如下：pRGEB32-7/S:AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG，将sgRNA1加到反向引物的接头上，CLA1/AS:
ATGTGTTGGACCATCTGCACTGCACCAGCCGGGAAT，下划线的碱基是sgRNA1反向序列。以PGTR载体为模板进行PCR扩增tRNA序列，获得tRNA+sgRNA1片段(Xie et al.,2015)。
CLA2/S:GTGCAGATGGTCCAACACATGTTTTAGAGCTAGAAATA下划线碱基是sgRNA1序列，将sgRNA2加到反向引物的接头上；
CLA2/AS:GAAGCTGCCTGTAAGATTTGTGCACCAGCCGGGAAT带下划线的碱基是sgRNA2反向序列，同样以PGTR载体为模板进行PCR扩增，获得gRNA+tRNA+sgRNA2片段。第二次PCR利用重叠PCR将两个片段拼成tRNA+sgRNA1+gRNA+tRNA+sgRNA2片段。引物序列如下：Inf CLA2/AS:TTCTAGCTCTAAAACGAAGCTGCCTGTAAGATTTG带下划线的碱基是sgRNA2反向序列，InfpRGEB32-7/S:AAGCATCAGATGGGCAAACAAA。利用一步克隆将tRNA+sgRNA1片段和gRNA+tRNA+sgRNA2片段分别连接到pGREB32-GhU6-7载体的BsaI酶切位点处。利用HpaI和SbfI双酶切pGREB32-GhU6-7，将目的片段使用infusion试剂盒试剂盒ClonExpress II One Step CloningKit(Vazyme C112-02)连接到GhC12B载体的HpaI和SbfI双酶切位点处。PCR体系体系见表4-7
表4PCR体系
表5第一次PCR条件
表6第二次PCR条件
表7In-fusion连接反应体系
实施例4：农杆菌介导的遗传转化
具体步骤如下：
A.将去壳的棉花种子(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，将苗子扶正，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+2,4-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+Asn0.5g/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：GhC12B对在转基因棉花植株中基因编辑检测中的应用
(1)高通量Hi-Tom深测序检测编辑效率
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】，在两个靶点位置前后设计引物，由于sgRNA1和sgRNA2相距很近(距离小于150bp),所以设计一对引物同时包含sgRNA1和sgRNA2两个靶标序列，同时加上Hi-Tom接头(序列见表8)。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8,9,11,12。
表8Hi-Tom扩增子引物
注：小写字母为Hi-Tom接头，大写字母为GhCLA(Gh_A10G2292)基因包含sgRNA1和sgRNA2片段的引物
(2)Sanger测序检测T1代的稳定遗传性
使用不含Hi-Tom接头的引物扩增GhCLA(Gh_A10G2292)基因包含sgRNA1和sgRNA2片段然后将PCR片段连入
-T Easy Vector Systems(Promega A1380)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。对比结果见图13。
实施例6：GhC12B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sgRNA靶点存在5个错配碱基以内10个最可能的脱靶位点进行测序，结果见表9。
表9预测潜在脱靶位点
注：表9中序列中小写字母代表与sgRNA错配的碱基
(2)全基因组分析GhC12B系统在棉花中的脱靶影响
为了评估GhC12B在棉花全基因组范围内的脱靶效应，本发明使用4个转基因T0代棉花植株(植株编号为a297、a158、b133、b157)与1个阴性棉花植株(PC)进行50×深度的全基因组测序(WGS)。通过计算确定了所有潜在脱靶位点，以Cas-OFFinder为基础，在全基因组上共找到不同分值的sgRNA1和sgRNA2的499和1001个脱靶位点。申请人使用华中农业大学作物遗传改良国家重点实验室发表一个野生型(WT)植株重测序数据作为对照，以减少背景或种系突变或者组织培养的变异(Li et al.,2019)，结果见图14。最后，在4株编辑的棉花植株，一株阳性对照和一株阴性对照中一共存在1 773 469,1 772 644,1 772 789,1773 068,1 773 889,1 774432indels和3 237 186,3 237 274,3 237 410,3 237 395,3237 136,3 237 453SNPs，其中4株编辑的棉花植株(植株编号为a297、a158、b133、b157)共有48644,95363,69546,43908indels和139595,179453,138425,159382SNPs被分享，这些变异可能是由于体细胞无性系变异引起的。对体细胞无性系变异进行过滤后，并对其余的变异进行过滤，进一步检测本发明的GhC12B诱导突变。申请人将这些变异与潜在脱靶进行重叠，结果显示在1500个潜在的脱靶位点(≤5mismatches)中没有检测到任何真正的脱靶突变。结果见图15。
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，该系统显示可以通过温度诱导酶活对陆地棉基因组的精确编辑和很高的特异性，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:
Bolotin,A.,Quinquis,B.,Sorokin,A.and Ehrlich,S.D.(2005)Clusteredregularly interspaced short palindrome repeats(CRISPRs)have spacers ofextrachromosomal origin Microbiology 151,2251-2261.
Hsu,Patrick D.,Lander,Eric S.and Zhang,F.(2014)Development andApplications of CRISPR-Cas9 for Genome Engineering.Cell 157,1262-1278.
Jansen,R.,Embden,J.D.A.v.,Gaastra,W.and Schouls,L.M.(2002)Identification of genes that are associated with DNA repeats inprokaryotes.Mol.Microbiol.43,1565-1575.
Jiang,W.,Zhou,H.,Bi,H.,Fromm,M.,Yang,B.and Weeks,D.P.(2013)Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification inArabidopsis,tobacco,sorghum and rice.Nucleic Acids Res.41,e188-e188.
Li,J.,Wang,M.,Li,Y.,Zhang,Q.,Lindsey,K.,Daniell,H.,Jin,S.and Zhang,X.(2019)Multi-omics analyses reveal epigenomics basis for cotton somaticembryogenesis through successive regeneration acclimation process.PlantBiotechnol.J.17,435-450.
Li,W.,Teng,F.,Li,T.and Zhou,Q.(2013)Simultaneous generation andgermline transmission of multiple gene mutations in rat using CRISPR-Cassystems.Nat.Biotechnol.31,684-686.
Liu,L.,Chen,P.,Wang,M.,Li,X.,Wang,J.,Yin,M.and Wang,Y.(2017)C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism.Mol.Cell65,310-322.
Mojica,F.J.M.,Díez-
C.s.,García-Martínez,J.and Soria,E.(2005)Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive fromForeign Genetic Elements.J.Mol.Evol.60,174-182.
Pourcel,C.,Salvignol,G.and Vergnaud,G.(2005)CRISPR elements inYersinia pestis acquire new repeats by preferential uptake of bacteriophageDNA,and provide additional tools for evolutionary studies.Microbiology151,653-663.
Shmakov,S.,Abudayyeh,Omar O.,Makarova,Kira S.,Wolf,Yuri I.,Gootenberg,Jonathan S.,Semenova,E.,Minakhin,L.,Joung,J.,Konermann,S.,Severinov,K.,Zhang,F.and Koonin,Eugene V.(2015)Discovery and FunctionalCharacterization of Diverse Class 2CRISPR-Cas Systems.Mol.Cell 60,385-397.
Wang,P.,Zhang,J.,Sun,L.,Ma,Y.,Xu,J.,Liang,S.,Deng,J.,Tan,J.,Zhang,Q.,Tu,L.,Daniell,H.,Jin,S.and Zhang,X.(2018)High efficient multisites genomeediting in allotetraploid cotton(Gossypium hirsutum)using CRISPR/Cas9system.Plant Biotechnol.J.16,137-150.
Wang,T.,Wei,J.J.,Sabatini,D.M.and Lander,E.S.(2014)Genetic Screens inHuman Cells Using the CRISPR-Cas9System.Science 343,80.
Xie,K.,Minkenberg,B.and Yang,Y.(2015)Boosting CRISPR/Cas9 multiplexediting capability with the endogenous tRNA-processing system.Proceedings ofthe National Academy of Sciences 112,3570.
Xie,S.,Shen,B.,Zhang,C.,Huang,X.and Zhang,Y.(2014)sgRNAcas9:ASoftware Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites.PLoS ONE 9,e100448。
序列表
<110> 华中农业大学
<120> 一种热诱导的基因编辑系统CRISPR-Cas12b在陆地棉中的应用
<141> 2020-03-12
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16240
<212> DNA
<213> 陆地棉(Gossypium hirsutum)
<220>
<221> gene
<222> (1)..(16240)
<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 16240
<210> 2
<211> 6184
<212> DNA
<213> 陆地棉(Gossypium hirsutum)
<220>
<221> gene
<222> (1)..(6184)
<400> 2
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtttcga aatggactat aaggaccacg 420
acggagacta caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa 480
agaagaagcg gaaggtcggt atccacggag tcccagcagc cgctgttaag tctattaagg 540
ttaaacttag attggatgat atgcctgaga tcagggctgg tctttggaag ttgcataaag 600
aagttaacgc tggagttaga tactacaccg agtggctttc acttttgagg caagaaaatt 660
tgtacagaag atctcctaac ggagatggag aacaagagtg tgataaaact gctgaagagt 720
gcaaggctga acttttggag aggcttagag ctagacaagt tgaaaacggt catagaggac 780
cagctggttc agatgatgag cttttgcaat tggctaggca actttacgaa cttttggttc 840
ctcaagctat tggagctaag ggagatgctc aacaaatcgc tagaaaattt ctttctccat 900
tggctgataa ggatgctgtt ggtggacttg gtattgctaa ggctggaaat aagcctagat 960
gggttagaat gagggaagct ggagagccag gttgggaaga ggaaaaggaa aaagctgaga 1020
ctaggaaatc agctgataga acagctgatg ttcttagagc tttggctgat tttggtctta 1080
agcctttgat gagggtttat actgattctg aaatgtcttc agttgagtgg aagccactta 1140
gaaaaggaca agctgttaga acatgggata gggatatgtt ccaacaagct atcgaaagaa 1200
tgatgtcatg ggagtcttgg aatcaaaggg ttggtcaaga atacgctaaa ttggttgagc 1260
aaaagaatag gtttgaacaa aagaatttcg ttggacaaga gcatcttgtt catttggtta 1320
accaacttca acaagatatg aaagaagctt cacctggttt ggaatctaag gagcaaactg 1380
ctcattatgt tacaggtaga gctcttaggg gatcagataa ggtttttgag aagtggggaa 1440
aacttgctcc agatgctcct ttcgatttgt acgatgctga aattaaaaac gttcaaagaa 1500
ggaacacaag aaggtttggt tctcatgatt tgttcgctaa gcttgctgaa ccagagtatc 1560
aagctctttg gagagaagat gcttcatttt tgaccagata tgctgtttac aactctatcc 1620
ttagaaaatt gaaccatgct aagatgtttg ctactttcac acttcctgat gctaccgctc 1680
atccaatctg gactaggttc gataagttgg gtggaaatct tcatcaatac actttccttt 1740
tcaacgaatt tggagagaga aggcatgcta tcagattcca taagcttttg aaggttgaga 1800
atggtgttgc tagagaagtt gatgatgtta cagttcctat ttctatgtca gagcaacttg 1860
ataatctttt gccaagagat cctaacgaac caatcgcttt gtattttagg gattacggtg 1920
ctgagcaaca ttttactgga gaattcggtg gagctaagat ccaatgtaga agggatcaac 1980
ttgctcatat gcatagaagg agaggtgcta gagatgttta tttgaacgtt tcagttagag 2040
ttcaatctca atcagaagct aggggtgaga gaagacctcc ttacgctgct gtttttagac 2100
ttgttggaga taaccatagg gctttcgttc atttcgataa gttgtcagat tatcttgctg 2160
agcatccaga tgatggaaag cttggttcag aaggactttt gtctggtttg agagttatgt 2220
ctgttgatct tggattgagg acatctgctt caatttctgt tttcagagtg gctaggaagg 2280
atgagcttaa acctaactct aagggtagag ttcctttctt tttcccaatc aagggaaatg 2340
ataacttggt tgctgttcat gaaaggtcac aacttttgaa acttccaggt gaaaccgagt 2400
ctaaggattt gagagctatt agggaggaaa gacaaaggac acttagacaa ttgaggaccc 2460
aacttgctta cttgagactt ttggttaggt gcggttcaga ggatgttgga aggagagaaa 2520
gatcttgggc taaacttatt gagcaacctg ttgatgctgc taatcatatg actccagatt 2580
ggagggaagc ttttgaaaac gagcttcaaa agttgaaatc acttcatggt atctgctctg 2640
ataaggagtg gatggatgct gtttatgaat cagttaggag agtttggaga catatgggaa 2700
aacaagttag agattggagg aaggatgtta gatcaggaga gaggcctaaa attagaggat 2760
acgctaagga tgttgttggt ggaaactcta tcgaacaaat cgagtatctt gaaaggcaat 2820
acaagttctt gaagtcatgg tctttcttcg gtaaagtttc aggacaagtt atcagggctg 2880
aaaagggttc taggttcgct attacactta gggagcatat cgatcatgct aaagaagata 2940
gattgaagaa attggctgat aggattatca tggaggctct tggttatgtt tacgctttgg 3000
atgaaagagg aaagggaaaa tgggttgcta agtatcctcc atgtcaactt attcttttgg 3060
aggaattgtc tgagtaccaa ttcaataacg atagacctcc atcagaaaat aaccaactta 3120
tgcaatggtc acataggggt gttttccaag agttgattaa ccaagctcaa gttcatgatc 3180
ttttggttgg aaccatgtat gctgcttttt cttcaaggtt cgatgctaga actggtgctc 3240
ctggaatcag atgtaggaga gttccagcta ggtgcactca agaacataat cctgagccat 3300
ttccttggtg gcttaacaag ttcgttgttg aacatacatt ggatgcttgt cctcttagag 3360
ctgatgattt gattccaacc ggtgaaggag agatctttgt ttcacctttc tctgctgagg 3420
aaggagattt ccatcaaatc catgctgatt tgaatgctgc tcaaaacttg caacaaaggc 3480
tttggtcaga tttcgatatt tctcaaatca gacttaggtg cgattggggt gaagttgatg 3540
gagagcttgt tttgatccca aggttgacag gaaagagaac cgctgattca tattctaata 3600
aggttttcta taccaacact ggtgttactt attacgaaag agagagggga aagaaaagga 3660
gaaaagtttt cgctcaagag aagctttcag aggaagaggc tgaacttttg gttgaggctg 3720
atgaagctag agagaagtca gttgttttga tgagggatcc ttctggtatt atcaataggg 3780
gaaactggac cagacaaaaa gagttctggt ctatggttaa ccaaagaatc gaaggttacc 3840
ttgttaagca aatcagatca agggttccat tgcaagattc tgcttgcgaa aacactggag 3900
atattaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagtaatcta 3960
gagtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 4020
gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 4080
gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 4140
atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 4200
actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 4260
gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 4320
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 4380
ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 4440
ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 4500
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 4560
agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 4620
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 4680
aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 4740
gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 4800
agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 4860
ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 4920
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 4980
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 5040
aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 5100
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 5160
atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 5220
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 5280
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 5340
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 5400
tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 5460
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 5520
tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 5580
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 5640
atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 5700
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 5760
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 5820
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 5880
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 5940
gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 6000
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 6060
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 6120
taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 6180
cgtc 6184
<210> 3
<211> 15333
<212> DNA
<213> 陆地棉(Gossypium hirsutum)
<220>
<221> gene
<222> (1)..(15333)
<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 aggtctcgtt ttttttttcc tgcaggtcca caaattcggg 10020
tcaaggcgga agccagcgcg ccaccccacg tcagcaaata cggaggcgcg gggttgacgg 10080
cgtcacccgg tcctaacggc gaccaacaaa ccagccagaa gaaattacag taaaaaaaaa 10140
gtaaattgca ctttgatcca ccttttatta cctaagtctc aatttggatc acccttaaac 10200
ctatcttttc aatttgggcc gggttgtggt ttggactacc atgaacaact tttcgtcatg 10260
tctaacttcc ctttcagcaa acatatgaac catatataga ggagatcggc cgtatactag 10320
agctgatgtg tttaaggtcg ttgattgcac gagaaaaaaa aatccaaatc gcaacaatag 10380
caaatttatc tggttcaaag tgaaaagata tgtttaaagg tagtccaaag taaaacttat 10440
agataataaa atgtggtcca aagcgtaatt cactcaaaaa aaatcaacga gacgtgtacc 10500
aaacggagac aaacggcatc ttctcgaaat ttcccaaccg ctcgctcgcc cgcctcgtct 10560
tcccggaaac cgcggtggtt tcagcgtggc ggattctcca agcagacgga gacgtcacgg 10620
cacgggactc ctcccaccac ccaaccgcca taaataccag ccccctcatc tcctctcctc 10680
gcatcagctc cacccccgaa aaatttctcc ccaatctcgc gaggctctcg tcgtcgaatc 10740
gaatcctctc gcgtcctcaa ggtacgctgc ttctcctctc ctcgcttcgt ttcgattcga 10800
tttcggacgg gtgaggttgt tttgttgcta gatccgattg gtggttaggg ttgtcgatgt 10860
gattatcgtg agatgtttag gggttgtaga tctgatggtt gtgatttggg cacggttggt 10920
tcgataggtg gaatcgtggt taggttttgg gattggatgt tggttctgat gattgggggg 10980
aatttttacg gttagatgaa ttgttggatg attcgattgg ggaaatcggt gtagatctgt 11040
tggggaattg tggaactagt catgcctgag tgattggtgc gatttgtagc gtgttccatc 11100
ttgtaggcct tgttgcgagc atgttcagat ctactgttcc gctcttgatt gagttattgg 11160
tgccatgggt tggtgcaaac acaggcttta atatgttata tctgttttgt gtttgatgta 11220
gatctgtagg gtagttcttc ttagacatgg ttcaattatg tagcttgtgc gtttcgattt 11280
gatttcatat gttcacagat tagataatga tgaactcttt taattaattg tcaatggtaa 11340
ataggaagtc ttgtcgctat atctgtcata atgatctcat gttactatct gccagtaatt 11400
tatgctaaga actatattag aatatcatgt tacaatctgt agtaatatca tgttacaatc 11460
tgtagttcat ctatataatc tattgtggta atttcttttt actatctgtg tgaagattat 11520
tgccactagt tcattctact tatttctgaa gttcaggata cgtgtgctgt tactacctat 11580
ctgaatacat gtgtgatgtg cctgttacta tctttttgaa tacatgtatg ttctgttgga 11640
atatgtttgc tgtttgatcc gttgttgtgt ccttaatctt gtgctagttc ttaccctatc 11700
tgtttggtga ttatttcttg cagatagtta tcaacaagtt tgtacaaaaa agcaggcttc 11760
gaaatggact ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac 11820
gatgacgata agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca 11880
gccgctgtta agtctattaa ggttaaactt agattggatg atatgcctga gatcagggct 11940
ggtctttgga agttgcataa agaagttaac gctggagtta gatactacac cgagtggctt 12000
tcacttttga ggcaagaaaa tttgtacaga agatctccta acggagatgg agaacaagag 12060
tgtgataaaa ctgctgaaga gtgcaaggct gaacttttgg agaggcttag agctagacaa 12120
gttgaaaacg gtcatagagg accagctggt tcagatgatg agcttttgca attggctagg 12180
caactttacg aacttttggt tcctcaagct attggagcta agggagatgc tcaacaaatc 12240
gctagaaaat ttctttctcc attggctgat aaggatgctg ttggtggact tggtattgct 12300
aaggctggaa ataagcctag atgggttaga atgagggaag ctggagagcc aggttgggaa 12360
gaggaaaagg aaaaagctga gactaggaaa tcagctgata gaacagctga tgttcttaga 12420
gctttggctg attttggtct taagcctttg atgagggttt atactgattc tgaaatgtct 12480
tcagttgagt ggaagccact tagaaaagga caagctgtta gaacatggga tagggatatg 12540
ttccaacaag ctatcgaaag aatgatgtca tgggagtctt ggaatcaaag ggttggtcaa 12600
gaatacgcta aattggttga gcaaaagaat aggtttgaac aaaagaattt cgttggacaa 12660
gagcatcttg ttcatttggt taaccaactt caacaagata tgaaagaagc ttcacctggt 12720
ttggaatcta aggagcaaac tgctcattat gttacaggta gagctcttag gggatcagat 12780
aaggtttttg agaagtgggg aaaacttgct ccagatgctc ctttcgattt gtacgatgct 12840
gaaattaaaa acgttcaaag aaggaacaca agaaggtttg gttctcatga tttgttcgct 12900
aagcttgctg aaccagagta tcaagctctt tggagagaag atgcttcatt tttgaccaga 12960
tatgctgttt acaactctat ccttagaaaa ttgaaccatg ctaagatgtt tgctactttc 13020
acacttcctg atgctaccgc tcatccaatc tggactaggt tcgataagtt gggtggaaat 13080
cttcatcaat acactttcct tttcaacgaa tttggagaga gaaggcatgc tatcagattc 13140
cataagcttt tgaaggttga gaatggtgtt gctagagaag ttgatgatgt tacagttcct 13200
atttctatgt cagagcaact tgataatctt ttgccaagag atcctaacga accaatcgct 13260
ttgtatttta gggattacgg tgctgagcaa cattttactg gagaattcgg tggagctaag 13320
atccaatgta gaagggatca acttgctcat atgcatagaa ggagaggtgc tagagatgtt 13380
tatttgaacg tttcagttag agttcaatct caatcagaag ctaggggtga gagaagacct 13440
ccttacgctg ctgtttttag acttgttgga gataaccata gggctttcgt tcatttcgat 13500
aagttgtcag attatcttgc tgagcatcca gatgatggaa agcttggttc agaaggactt 13560
ttgtctggtt tgagagttat gtctgttgat cttggattga ggacatctgc ttcaatttct 13620
gttttcagag tggctaggaa ggatgagctt aaacctaact ctaagggtag agttcctttc 13680
tttttcccaa tcaagggaaa tgataacttg gttgctgttc atgaaaggtc acaacttttg 13740
aaacttccag gtgaaaccga gtctaaggat ttgagagcta ttagggagga aagacaaagg 13800
acacttagac aattgaggac ccaacttgct tacttgagac ttttggttag gtgcggttca 13860
gaggatgttg gaaggagaga aagatcttgg gctaaactta ttgagcaacc tgttgatgct 13920
gctaatcata tgactccaga ttggagggaa gcttttgaaa acgagcttca aaagttgaaa 13980
tcacttcatg gtatctgctc tgataaggag tggatggatg ctgtttatga atcagttagg 14040
agagtttgga gacatatggg aaaacaagtt agagattgga ggaaggatgt tagatcagga 14100
gagaggccta aaattagagg atacgctaag gatgttgttg gtggaaactc tatcgaacaa 14160
atcgagtatc ttgaaaggca atacaagttc ttgaagtcat ggtctttctt cggtaaagtt 14220
tcaggacaag ttatcagggc tgaaaagggt tctaggttcg ctattacact tagggagcat 14280
atcgatcatg ctaaagaaga tagattgaag aaattggctg ataggattat catggaggct 14340
cttggttatg tttacgcttt ggatgaaaga ggaaagggaa aatgggttgc taagtatcct 14400
ccatgtcaac ttattctttt ggaggaattg tctgagtacc aattcaataa cgatagacct 14460
ccatcagaaa ataaccaact tatgcaatgg tcacataggg gtgttttcca agagttgatt 14520
aaccaagctc aagttcatga tcttttggtt ggaaccatgt atgctgcttt ttcttcaagg 14580
ttcgatgcta gaactggtgc tcctggaatc agatgtagga gagttccagc taggtgcact 14640
caagaacata atcctgagcc atttccttgg tggcttaaca agttcgttgt tgaacataca 14700
ttggatgctt gtcctcttag agctgatgat ttgattccaa ccggtgaagg agagatcttt 14760
gtttcacctt tctctgctga ggaaggagat ttccatcaaa tccatgctga tttgaatgct 14820
gctcaaaact tgcaacaaag gctttggtca gatttcgata tttctcaaat cagacttagg 14880
tgcgattggg gtgaagttga tggagagctt gttttgatcc caaggttgac aggaaagaga 14940
accgctgatt catattctaa taaggttttc tataccaaca ctggtgttac ttattacgaa 15000
agagagaggg gaaagaaaag gagaaaagtt ttcgctcaag agaagctttc agaggaagag 15060
gctgaacttt tggttgaggc tgatgaagct agagagaagt cagttgtttt gatgagggat 15120
ccttctggta ttatcaatag gggaaactgg accagacaaa aagagttctg gtctatggtt 15180
aaccaaagaa tcgaaggtta ccttgttaag caaatcagat caagggttcc attgcaagat 15240
tctgcttgcg aaaacactgg agatattaaa aggccggcgg ccacgaaaaa ggccggccag 15300
gcaaaaaaga aaaagtaatc tagacccagc ttt 15333
<210> 4
<211> 3567
<212> DNA
<213> 陆地棉(Gossypium hirsutum)
<220>
<221> gene
<222> (1)..(3567)
<220>
<221> CDS
<222> (1)..(3567)
<400> 4
ttc gaa atg gac tat aag gac cac gac gga gac tac aag gat cat gat 48
Phe Glu Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp
1 5 10 15
att gat tac aaa gac gat gac gat aag atg gcc cca aag aag aag cgg 96
Ile Asp Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg
20 25 30
aag gtc ggt atc cac gga gtc cca gca gcc gct gtt aag tct att aag 144
Lys Val Gly Ile His Gly Val Pro Ala Ala Ala Val Lys Ser Ile Lys
35 40 45
gtt aaa ctt aga ttg gat gat atg cct gag atc agg gct ggt ctt tgg 192
Val Lys Leu Arg Leu Asp Asp Met Pro Glu Ile Arg Ala Gly Leu Trp
50 55 60
aag ttg cat aaa gaa gtt aac gct gga gtt aga tac tac acc gag tgg 240
Lys Leu His Lys Glu Val Asn Ala Gly Val Arg Tyr Tyr Thr Glu Trp
65 70 75 80
ctt tca ctt ttg agg caa gaa aat ttg tac aga aga tct cct aac gga 288
Leu Ser Leu Leu Arg Gln Glu Asn Leu Tyr Arg Arg Ser Pro Asn Gly
85 90 95
gat gga gaa caa gag tgt gat aaa act gct gaa gag tgc aag gct gaa 336
Asp Gly Glu Gln Glu Cys Asp Lys Thr Ala Glu Glu Cys Lys Ala Glu
100 105 110
ctt ttg gag agg ctt aga gct aga caa gtt gaa aac ggt cat aga gga 384
Leu Leu Glu Arg Leu Arg Ala Arg Gln Val Glu Asn Gly His Arg Gly
115 120 125
cca gct ggt tca gat gat gag ctt ttg caa ttg gct agg caa ctt tac 432
Pro Ala Gly Ser Asp Asp Glu Leu Leu Gln Leu Ala Arg Gln Leu Tyr
130 135 140
gaa ctt ttg gtt cct caa gct att gga gct aag gga gat gct caa caa 480
Glu Leu Leu Val Pro Gln Ala Ile Gly Ala Lys Gly Asp Ala Gln Gln
145 150 155 160
atc gct aga aaa ttt ctt tct cca ttg gct gat aag gat gct gtt ggt 528
Ile Ala Arg Lys Phe Leu Ser Pro Leu Ala Asp Lys Asp Ala Val Gly
165 170 175
gga ctt ggt att gct aag gct gga aat aag cct aga tgg gtt aga atg 576
Gly Leu Gly Ile Ala Lys Ala Gly Asn Lys Pro Arg Trp Val Arg Met
180 185 190
agg gaa gct gga gag cca ggt tgg gaa gag gaa aag gaa aaa gct gag 624
Arg Glu Ala Gly Glu Pro Gly Trp Glu Glu Glu Lys Glu Lys Ala Glu
195 200 205
act agg aaa tca gct gat aga aca gct gat gtt ctt aga gct ttg gct 672
Thr Arg Lys Ser Ala Asp Arg Thr Ala Asp Val Leu Arg Ala Leu Ala
210 215 220
gat ttt ggt ctt aag cct ttg atg agg gtt tat act gat tct gaa atg 720
Asp Phe Gly Leu Lys Pro Leu Met Arg Val Tyr Thr Asp Ser Glu Met
225 230 235 240
tct tca gtt gag tgg aag cca ctt aga aaa gga caa gct gtt aga aca 768
Ser Ser Val Glu Trp Lys Pro Leu Arg Lys Gly Gln Ala Val Arg Thr
245 250 255
tgg gat agg gat atg ttc caa caa gct atc gaa aga atg atg tca tgg 816
Trp Asp Arg Asp Met Phe Gln Gln Ala Ile Glu Arg Met Met Ser Trp
260 265 270
gag tct tgg aat caa agg gtt ggt caa gaa tac gct aaa ttg gtt gag 864
Glu Ser Trp Asn Gln Arg Val Gly Gln Glu Tyr Ala Lys Leu Val Glu
275 280 285
caa aag aat agg ttt gaa caa aag aat ttc gtt gga caa gag cat ctt 912
Gln Lys Asn Arg Phe Glu Gln Lys Asn Phe Val Gly Gln Glu His Leu
290 295 300
gtt cat ttg gtt aac caa ctt caa caa gat atg aaa gaa gct tca cct 960
Val His Leu Val Asn Gln Leu Gln Gln Asp Met Lys Glu Ala Ser Pro
305 310 315 320
ggt ttg gaa tct aag gag caa act gct cat tat gtt aca ggt aga gct 1008
Gly Leu Glu Ser Lys Glu Gln Thr Ala His Tyr Val Thr Gly Arg Ala
325 330 335
ctt agg gga tca gat aag gtt ttt gag aag tgg gga aaa ctt gct cca 1056
Leu Arg Gly Ser Asp Lys Val Phe Glu Lys Trp Gly Lys Leu Ala Pro
340 345 350
gat gct cct ttc gat ttg tac gat gct gaa att aaa aac gtt caa aga 1104
Asp Ala Pro Phe Asp Leu Tyr Asp Ala Glu Ile Lys Asn Val Gln Arg
355 360 365
agg aac aca aga agg ttt ggt tct cat gat ttg ttc gct aag ctt gct 1152
Arg Asn Thr Arg Arg Phe Gly Ser His Asp Leu Phe Ala Lys Leu Ala
370 375 380
gaa cca gag tat caa gct ctt tgg aga gaa gat gct tca ttt ttg acc 1200
Glu Pro Glu Tyr Gln Ala Leu Trp Arg Glu Asp Ala Ser Phe Leu Thr
385 390 395 400
aga tat gct gtt tac aac tct atc ctt aga aaa ttg aac cat gct aag 1248
Arg Tyr Ala Val Tyr Asn Ser Ile Leu Arg Lys Leu Asn His Ala Lys
405 410 415
atg ttt gct act ttc aca ctt cct gat gct acc gct cat cca atc tgg 1296
Met Phe Ala Thr Phe Thr Leu Pro Asp Ala Thr Ala His Pro Ile Trp
420 425 430
act agg ttc gat aag ttg ggt gga aat ctt cat caa tac act ttc ctt 1344
Thr Arg Phe Asp Lys Leu Gly Gly Asn Leu His Gln Tyr Thr Phe Leu
435 440 445
ttc aac gaa ttt gga gag aga agg cat gct atc aga ttc cat aag ctt 1392
Phe Asn Glu Phe Gly Glu Arg Arg His Ala Ile Arg Phe His Lys Leu
450 455 460
ttg aag gtt gag aat ggt gtt gct aga gaa gtt gat gat gtt aca gtt 1440
Leu Lys Val Glu Asn Gly Val Ala Arg Glu Val Asp Asp Val Thr Val
465 470 475 480
cct att tct atg tca gag caa ctt gat aat ctt ttg cca aga gat cct 1488
Pro Ile Ser Met Ser Glu Gln Leu Asp Asn Leu Leu Pro Arg Asp Pro
485 490 495
aac gaa cca atc gct ttg tat ttt agg gat tac ggt gct gag caa cat 1536
Asn Glu Pro Ile Ala Leu Tyr Phe Arg Asp Tyr Gly Ala Glu Gln His
500 505 510
ttt act gga gaa ttc ggt gga gct aag atc caa tgt aga agg gat caa 1584
Phe Thr Gly Glu Phe Gly Gly Ala Lys Ile Gln Cys Arg Arg Asp Gln
515 520 525
ctt gct cat atg cat aga agg aga ggt gct aga gat gtt tat ttg aac 1632
Leu Ala His Met His Arg Arg Arg Gly Ala Arg Asp Val Tyr Leu Asn
530 535 540
gtt tca gtt aga gtt caa tct caa tca gaa gct agg ggt gag aga aga 1680
Val Ser Val Arg Val Gln Ser Gln Ser Glu Ala Arg Gly Glu Arg Arg
545 550 555 560
cct cct tac gct gct gtt ttt aga ctt gtt gga gat aac cat agg gct 1728
Pro Pro Tyr Ala Ala Val Phe Arg Leu Val Gly Asp Asn His Arg Ala
565 570 575
ttc gtt cat ttc gat aag ttg tca gat tat ctt gct gag cat cca gat 1776
Phe Val His Phe Asp Lys Leu Ser Asp Tyr Leu Ala Glu His Pro Asp
580 585 590
gat gga aag ctt ggt tca gaa gga ctt ttg tct ggt ttg aga gtt atg 1824
Asp Gly Lys Leu Gly Ser Glu Gly Leu Leu Ser Gly Leu Arg Val Met
595 600 605
tct gtt gat ctt gga ttg agg aca tct gct tca att tct gtt ttc aga 1872
Ser Val Asp Leu Gly Leu Arg Thr Ser Ala Ser Ile Ser Val Phe Arg
610 615 620
gtg gct agg aag gat gag ctt aaa cct aac tct aag ggt aga gtt cct 1920
Val Ala Arg Lys Asp Glu Leu Lys Pro Asn Ser Lys Gly Arg Val Pro
625 630 635 640
ttc ttt ttc cca atc aag gga aat gat aac ttg gtt gct gtt cat gaa 1968
Phe Phe Phe Pro Ile Lys Gly Asn Asp Asn Leu Val Ala Val His Glu
645 650 655
agg tca caa ctt ttg aaa ctt cca ggt gaa acc gag tct aag gat ttg 2016
Arg Ser Gln Leu Leu Lys Leu Pro Gly Glu Thr Glu Ser Lys Asp Leu
660 665 670
aga gct att agg gag gaa aga caa agg aca ctt aga caa ttg agg acc 2064
Arg Ala Ile Arg Glu Glu Arg Gln Arg Thr Leu Arg Gln Leu Arg Thr
675 680 685
caa ctt gct tac ttg aga ctt ttg gtt agg tgc ggt tca gag gat gtt 2112
Gln Leu Ala Tyr Leu Arg Leu Leu Val Arg Cys Gly Ser Glu Asp Val
690 695 700
gga agg aga gaa aga tct tgg gct aaa ctt att gag caa cct gtt gat 2160
Gly Arg Arg Glu Arg Ser Trp Ala Lys Leu Ile Glu Gln Pro Val Asp
705 710 715 720
gct gct aat cat atg act cca gat tgg agg gaa gct ttt gaa aac gag 2208
Ala Ala Asn His Met Thr Pro Asp Trp Arg Glu Ala Phe Glu Asn Glu
725 730 735
ctt caa aag ttg aaa tca ctt cat ggt atc tgc tct gat aag gag tgg 2256
Leu Gln Lys Leu Lys Ser Leu His Gly Ile Cys Ser Asp Lys Glu Trp
740 745 750
atg gat gct gtt tat gaa tca gtt agg aga gtt tgg aga cat atg gga 2304
Met Asp Ala Val Tyr Glu Ser Val Arg Arg Val Trp Arg His Met Gly
755 760 765
aaa caa gtt aga gat tgg agg aag gat gtt aga tca gga gag agg cct 2352
Lys Gln Val Arg Asp Trp Arg Lys Asp Val Arg Ser Gly Glu Arg Pro
770 775 780
aaa att aga gga tac gct aag gat gtt gtt ggt gga aac tct atc gaa 2400
Lys Ile Arg Gly Tyr Ala Lys Asp Val Val Gly Gly Asn Ser Ile Glu
785 790 795 800
caa atc gag tat ctt gaa agg caa tac aag ttc ttg aag tca tgg tct 2448
Gln Ile Glu Tyr Leu Glu Arg Gln Tyr Lys Phe Leu Lys Ser Trp Ser
805 810 815
ttc ttc ggt aaa gtt tca gga caa gtt atc agg gct gaa aag ggt tct 2496
Phe Phe Gly Lys Val Ser Gly Gln Val Ile Arg Ala Glu Lys Gly Ser
820 825 830
agg ttc gct att aca ctt agg gag cat atc gat cat gct aaa gaa gat 2544
Arg Phe Ala Ile Thr Leu Arg Glu His Ile Asp His Ala Lys Glu Asp
835 840 845
aga ttg aag aaa ttg gct gat agg att atc atg gag gct ctt ggt tat 2592
Arg Leu Lys Lys Leu Ala Asp Arg Ile Ile Met Glu Ala Leu Gly Tyr
850 855 860
gtt tac gct ttg gat gaa aga gga aag gga aaa tgg gtt gct aag tat 2640
Val Tyr Ala Leu Asp Glu Arg Gly Lys Gly Lys Trp Val Ala Lys Tyr
865 870 875 880
cct cca tgt caa ctt att ctt ttg gag gaa ttg tct gag tac caa ttc 2688
Pro Pro Cys Gln Leu Ile Leu Leu Glu Glu Leu Ser Glu Tyr Gln Phe
885 890 895
aat aac gat aga cct cca tca gaa aat aac caa ctt atg caa tgg tca 2736
Asn Asn Asp Arg Pro Pro Ser Glu Asn Asn Gln Leu Met Gln Trp Ser
900 905 910
cat agg ggt gtt ttc caa gag ttg att aac caa gct caa gtt cat gat 2784
His Arg Gly Val Phe Gln Glu Leu Ile Asn Gln Ala Gln Val His Asp
915 920 925
ctt ttg gtt gga acc atg tat gct gct ttt tct tca agg ttc gat gct 2832
Leu Leu Val Gly Thr Met Tyr Ala Ala Phe Ser Ser Arg Phe Asp Ala
930 935 940
aga act ggt gct cct gga atc aga tgt agg aga gtt cca gct agg tgc 2880
Arg Thr Gly Ala Pro Gly Ile Arg Cys Arg Arg Val Pro Ala Arg Cys
945 950 955 960
act caa gaa cat aat cct gag cca ttt cct tgg tgg ctt aac aag ttc 2928
Thr Gln Glu His Asn Pro Glu Pro Phe Pro Trp Trp Leu Asn Lys Phe
965 970 975
gtt gtt gaa cat aca ttg gat gct tgt cct ctt aga gct gat gat ttg 2976
Val Val Glu His Thr Leu Asp Ala Cys Pro Leu Arg Ala Asp Asp Leu
980 985 990
att cca acc ggt gaa gga gag atc ttt gtt tca cct ttc tct gct gag 3024
Ile Pro Thr Gly Glu Gly Glu Ile Phe Val Ser Pro Phe Ser Ala Glu
995 1000 1005
gaa gga gat ttc cat caa atc cat gct gat ttg aat gct gct caa aac 3072
Glu Gly Asp Phe His Gln Ile His Ala Asp Leu Asn Ala Ala Gln Asn
1010 1015 1020
ttg caa caa agg ctt tgg tca gat ttc gat att tct caa atc aga ctt 3120
Leu Gln Gln Arg Leu Trp Ser Asp Phe Asp Ile Ser Gln Ile Arg Leu
1025 1030 1035 1040
agg tgc gat tgg ggt gaa gtt gat gga gag ctt gtt ttg atc cca agg 3168
Arg Cys Asp Trp Gly Glu Val Asp Gly Glu Leu Val Leu Ile Pro Arg
1045 1050 1055
ttg aca gga aag aga acc gct gat tca tat tct aat aag gtt ttc tat 3216
Leu Thr Gly Lys Arg Thr Ala Asp Ser Tyr Ser Asn Lys Val Phe Tyr
1060 1065 1070
acc aac act ggt gtt act tat tac gaa aga gag agg gga aag aaa agg 3264
Thr Asn Thr Gly Val Thr Tyr Tyr Glu Arg Glu Arg Gly Lys Lys Arg
1075 1080 1085
aga aaa gtt ttc gct caa gag aag ctt tca gag gaa gag gct gaa ctt 3312
Arg Lys Val Phe Ala Gln Glu Lys Leu Ser Glu Glu Glu Ala Glu Leu
1090 1095 1100
ttg gtt gag gct gat gaa gct aga gag aag tca gtt gtt ttg atg agg 3360
Leu Val Glu Ala Asp Glu Ala Arg Glu Lys Ser Val Val Leu Met Arg
1105 1110 1115 1120
gat cct tct ggt att atc aat agg gga aac tgg acc aga caa aaa gag 3408
Asp Pro Ser Gly Ile Ile Asn Arg Gly Asn Trp Thr Arg Gln Lys Glu
1125 1130 1135
ttc tgg tct atg gtt aac caa aga atc gaa ggt tac ctt gtt aag caa 3456
Phe Trp Ser Met Val Asn Gln Arg Ile Glu Gly Tyr Leu Val Lys Gln
1140 1145 1150
atc aga tca agg gtt cca ttg caa gat tct gct tgc gaa aac act gga 3504
Ile Arg Ser Arg Val Pro Leu Gln Asp Ser Ala Cys Glu Asn Thr Gly
1155 1160 1165
gat att aaa agg ccg gcg gcc acg aaa aag gcc ggc cag gca aaa aag 3552
Asp Ile Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys
1170 1175 1180
aaa aag taa tct aga 3567
Lys Lys Ser Arg
1185
<210> 5
<211> 1186
<212> PRT
<213> 陆地棉(Gossypium hirsutum)
<400> 5
Phe Glu Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp
1 5 10 15
Ile Asp Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg
20 25 30
Lys Val Gly Ile His Gly Val Pro Ala Ala Ala Val Lys Ser Ile Lys
35 40 45
Val Lys Leu Arg Leu Asp Asp Met Pro Glu Ile Arg Ala Gly Leu Trp
50 55 60
Lys Leu His Lys Glu Val Asn Ala Gly Val Arg Tyr Tyr Thr Glu Trp
65 70 75 80
Leu Ser Leu Leu Arg Gln Glu Asn Leu Tyr Arg Arg Ser Pro Asn Gly
85 90 95
Asp Gly Glu Gln Glu Cys Asp Lys Thr Ala Glu Glu Cys Lys Ala Glu
100 105 110
Leu Leu Glu Arg Leu Arg Ala Arg Gln Val Glu Asn Gly His Arg Gly
115 120 125
Pro Ala Gly Ser Asp Asp Glu Leu Leu Gln Leu Ala Arg Gln Leu Tyr
130 135 140
Glu Leu Leu Val Pro Gln Ala Ile Gly Ala Lys Gly Asp Ala Gln Gln
145 150 155 160
Ile Ala Arg Lys Phe Leu Ser Pro Leu Ala Asp Lys Asp Ala Val Gly
165 170 175
Gly Leu Gly Ile Ala Lys Ala Gly Asn Lys Pro Arg Trp Val Arg Met
180 185 190
Arg Glu Ala Gly Glu Pro Gly Trp Glu Glu Glu Lys Glu Lys Ala Glu
195 200 205
Thr Arg Lys Ser Ala Asp Arg Thr Ala Asp Val Leu Arg Ala Leu Ala
210 215 220
Asp Phe Gly Leu Lys Pro Leu Met Arg Val Tyr Thr Asp Ser Glu Met
225 230 235 240
Ser Ser Val Glu Trp Lys Pro Leu Arg Lys Gly Gln Ala Val Arg Thr
245 250 255
Trp Asp Arg Asp Met Phe Gln Gln Ala Ile Glu Arg Met Met Ser Trp
260 265 270
Glu Ser Trp Asn Gln Arg Val Gly Gln Glu Tyr Ala Lys Leu Val Glu
275 280 285
Gln Lys Asn Arg Phe Glu Gln Lys Asn Phe Val Gly Gln Glu His Leu
290 295 300
Val His Leu Val Asn Gln Leu Gln Gln Asp Met Lys Glu Ala Ser Pro
305 310 315 320
Gly Leu Glu Ser Lys Glu Gln Thr Ala His Tyr Val Thr Gly Arg Ala
325 330 335
Leu Arg Gly Ser Asp Lys Val Phe Glu Lys Trp Gly Lys Leu Ala Pro
340 345 350
Asp Ala Pro Phe Asp Leu Tyr Asp Ala Glu Ile Lys Asn Val Gln Arg
355 360 365
Arg Asn Thr Arg Arg Phe Gly Ser His Asp Leu Phe Ala Lys Leu Ala
370 375 380
Glu Pro Glu Tyr Gln Ala Leu Trp Arg Glu Asp Ala Ser Phe Leu Thr
385 390 395 400
Arg Tyr Ala Val Tyr Asn Ser Ile Leu Arg Lys Leu Asn His Ala Lys
405 410 415
Met Phe Ala Thr Phe Thr Leu Pro Asp Ala Thr Ala His Pro Ile Trp
420 425 430
Thr Arg Phe Asp Lys Leu Gly Gly Asn Leu His Gln Tyr Thr Phe Leu
435 440 445
Phe Asn Glu Phe Gly Glu Arg Arg His Ala Ile Arg Phe His Lys Leu
450 455 460
Leu Lys Val Glu Asn Gly Val Ala Arg Glu Val Asp Asp Val Thr Val
465 470 475 480
Pro Ile Ser Met Ser Glu Gln Leu Asp Asn Leu Leu Pro Arg Asp Pro
485 490 495
Asn Glu Pro Ile Ala Leu Tyr Phe Arg Asp Tyr Gly Ala Glu Gln His
500 505 510
Phe Thr Gly Glu Phe Gly Gly Ala Lys Ile Gln Cys Arg Arg Asp Gln
515 520 525
Leu Ala His Met His Arg Arg Arg Gly Ala Arg Asp Val Tyr Leu Asn
530 535 540
Val Ser Val Arg Val Gln Ser Gln Ser Glu Ala Arg Gly Glu Arg Arg
545 550 555 560
Pro Pro Tyr Ala Ala Val Phe Arg Leu Val Gly Asp Asn His Arg Ala
565 570 575
Phe Val His Phe Asp Lys Leu Ser Asp Tyr Leu Ala Glu His Pro Asp
580 585 590
Asp Gly Lys Leu Gly Ser Glu Gly Leu Leu Ser Gly Leu Arg Val Met
595 600 605
Ser Val Asp Leu Gly Leu Arg Thr Ser Ala Ser Ile Ser Val Phe Arg
610 615 620
Val Ala Arg Lys Asp Glu Leu Lys Pro Asn Ser Lys Gly Arg Val Pro
625 630 635 640
Phe Phe Phe Pro Ile Lys Gly Asn Asp Asn Leu Val Ala Val His Glu
645 650 655
Arg Ser Gln Leu Leu Lys Leu Pro Gly Glu Thr Glu Ser Lys Asp Leu
660 665 670
Arg Ala Ile Arg Glu Glu Arg Gln Arg Thr Leu Arg Gln Leu Arg Thr
675 680 685
Gln Leu Ala Tyr Leu Arg Leu Leu Val Arg Cys Gly Ser Glu Asp Val
690 695 700
Gly Arg Arg Glu Arg Ser Trp Ala Lys Leu Ile Glu Gln Pro Val Asp
705 710 715 720
Ala Ala Asn His Met Thr Pro Asp Trp Arg Glu Ala Phe Glu Asn Glu
725 730 735
Leu Gln Lys Leu Lys Ser Leu His Gly Ile Cys Ser Asp Lys Glu Trp
740 745 750
Met Asp Ala Val Tyr Glu Ser Val Arg Arg Val Trp Arg His Met Gly
755 760 765
Lys Gln Val Arg Asp Trp Arg Lys Asp Val Arg Ser Gly Glu Arg Pro
770 775 780
Lys Ile Arg Gly Tyr Ala Lys Asp Val Val Gly Gly Asn Ser Ile Glu
785 790 795 800
Gln Ile Glu Tyr Leu Glu Arg Gln Tyr Lys Phe Leu Lys Ser Trp Ser
805 810 815
Phe Phe Gly Lys Val Ser Gly Gln Val Ile Arg Ala Glu Lys Gly Ser
820 825 830
Arg Phe Ala Ile Thr Leu Arg Glu His Ile Asp His Ala Lys Glu Asp
835 840 845
Arg Leu Lys Lys Leu Ala Asp Arg Ile Ile Met Glu Ala Leu Gly Tyr
850 855 860
Val Tyr Ala Leu Asp Glu Arg Gly Lys Gly Lys Trp Val Ala Lys Tyr
865 870 875 880
Pro Pro Cys Gln Leu Ile Leu Leu Glu Glu Leu Ser Glu Tyr Gln Phe
885 890 895
Asn Asn Asp Arg Pro Pro Ser Glu Asn Asn Gln Leu Met Gln Trp Ser
900 905 910
His Arg Gly Val Phe Gln Glu Leu Ile Asn Gln Ala Gln Val His Asp
915 920 925
Leu Leu Val Gly Thr Met Tyr Ala Ala Phe Ser Ser Arg Phe Asp Ala
930 935 940
Arg Thr Gly Ala Pro Gly Ile Arg Cys Arg Arg Val Pro Ala Arg Cys
945 950 955 960
Thr Gln Glu His Asn Pro Glu Pro Phe Pro Trp Trp Leu Asn Lys Phe
965 970 975
Val Val Glu His Thr Leu Asp Ala Cys Pro Leu Arg Ala Asp Asp Leu
980 985 990
Ile Pro Thr Gly Glu Gly Glu Ile Phe Val Ser Pro Phe Ser Ala Glu
995 1000 1005
Glu Gly Asp Phe His Gln Ile His Ala Asp Leu Asn Ala Ala Gln Asn
1010 1015 1020
Leu Gln Gln Arg Leu Trp Ser Asp Phe Asp Ile Ser Gln Ile Arg Leu
1025 1030 1035 1040
Arg Cys Asp Trp Gly Glu Val Asp Gly Glu Leu Val Leu Ile Pro Arg
1045 1050 1055
Leu Thr Gly Lys Arg Thr Ala Asp Ser Tyr Ser Asn Lys Val Phe Tyr
1060 1065 1070
Thr Asn Thr Gly Val Thr Tyr Tyr Glu Arg Glu Arg Gly Lys Lys Arg
1075 1080 1085
Arg Lys Val Phe Ala Gln Glu Lys Leu Ser Glu Glu Glu Ala Glu Leu
1090 1095 1100
Leu Val Glu Ala Asp Glu Ala Arg Glu Lys Ser Val Val Leu Met Arg
1105 1110 1115 1120
Asp Pro Ser Gly Ile Ile Asn Arg Gly Asn Trp Thr Arg Gln Lys Glu
1125 1130 1135
Phe Trp Ser Met Val Asn Gln Arg Ile Glu Gly Tyr Leu Val Lys Gln
1140 1145 1150
Ile Arg Ser Arg Val Pro Leu Gln Asp Ser Ala Cys Glu Asn Thr Gly
1155 1160 1165
Asp Ile Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys
1170 1175 1180
Lys Lys
1185
Claims (4)
1.一种能够对陆地棉基因组5'-TTN-3'碱基位点精确识别并能产生长黏性末端的高效转化载体GhC12B，其特征在于，该载体的核苷酸序列如SEQ ID NO:3所示。
2.一种能够对陆地棉基因组5'-TTN-3'碱基位点精确识别并能产生长黏性末端的高效转化载体GhC12B的构建方法，其特征在于，所述的GhC12B载体通过下列步骤制备获得：
(1)获得目的序列AaCas12b-NLS-3xFLAG，其核苷酸序列如序列表SEQ ID NO：4所示，该序列通过NCBI上获得(ID:PDB:5WQE)，前后加上BstbⅠ和XbaⅠ两个酶切位点，通过密码子优化合成到pUC57载体上得到新的载体pUC57-Cas12b，序列见SEQ ID NO：2；
(2)利用BstbⅠ、XbaⅠ对SEQ ID NO：1所示的pRGEB32-GhU6.7-NPTⅡ载体和SEQ ID NO：2所示的pUC57-Cas12b进行酶切，再将pUC57-Cas12b切下来的Cas12b序列与酶切后pRGEB32-GhU6.7-NPTⅡ的序列连接，通过测序验证，得到如SEQ ID NO：3所示的序列，利用所述SEQID NO：3所示的序列构建得到陆地棉的基因组编辑的转化载体GhC12B。
3.权利要求1所述的载体GhC12B在陆地棉基因组编辑中的应用。
4.权利要求2所述的构建方法在陆地棉基因组编辑中的应用。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108203714A (zh) * | 2016-12-20 | 2018-06-26 | 华中农业大学 | 一种棉花基因的编辑方法 |
| CN109112149A (zh) * | 2018-02-12 | 2019-01-01 | 华中农业大学 | 调控棉花黄萎病抗性的棉花钙依赖蛋白激酶GhCPK33基因及应用 |
| CN109112139A (zh) * | 2018-03-12 | 2019-01-01 | 华中农业大学 | 棉花基因GbTSA1和GbTSB1及其在抗黄萎病中的应用 |
| WO2019126709A1 (en) * | 2017-12-22 | 2019-06-27 | The Broad Institute, Inc. | Cas12b systems, methods, and compositions for targeted dna base editing |
| CN109983122A (zh) * | 2016-09-23 | 2019-07-05 | 巴斯夫农业种子解决方案美国有限责任公司 | 植物中的靶向基因组优化 |
| WO2019150200A2 (en) * | 2018-01-30 | 2019-08-08 | G+Flas Life Sciences | Dna free crispr plant transformation |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007098001A2 (en) * | 2006-02-16 | 2007-08-30 | The Texas A & M University System | Cotton plant with seed-specific reduction in gossypol |
| CN109593781B (zh) * | 2018-12-20 | 2021-02-23 | 华中农业大学 | 陆地棉基因组的精准高效编辑方法 |
| CN110283840B (zh) * | 2019-04-11 | 2021-04-13 | 华中农业大学 | 陆地棉基因组的精确高效编辑方法 |
-
2020
- 2020-03-15 CN CN202010179130.1A patent/CN111378684B/zh active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109983122A (zh) * | 2016-09-23 | 2019-07-05 | 巴斯夫农业种子解决方案美国有限责任公司 | 植物中的靶向基因组优化 |
| CN108203714A (zh) * | 2016-12-20 | 2018-06-26 | 华中农业大学 | 一种棉花基因的编辑方法 |
| WO2019126709A1 (en) * | 2017-12-22 | 2019-06-27 | The Broad Institute, Inc. | Cas12b systems, methods, and compositions for targeted dna base editing |
| WO2019150200A2 (en) * | 2018-01-30 | 2019-08-08 | G+Flas Life Sciences | Dna free crispr plant transformation |
| CN109112149A (zh) * | 2018-02-12 | 2019-01-01 | 华中农业大学 | 调控棉花黄萎病抗性的棉花钙依赖蛋白激酶GhCPK33基因及应用 |
| CN109112139A (zh) * | 2018-03-12 | 2019-01-01 | 华中农业大学 | 棉花基因GbTSA1和GbTSB1及其在抗黄萎病中的应用 |
Non-Patent Citations (2)
| Title |
|---|
| CRISPR-Cas nucleases and base editors for plant genome editing;Filiz Gurel等;aBIOTECH;第2020卷(第1期);74-87 * |
| 两种常用激素组合下棉花体细胞胚胎发生过程的组织学观察;朱华国等;棉花学报;第24卷(第2期);159-166 * |
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