CN110862994B - 控制玉米株高和穗位高基因zkm76及其应用 - Google Patents
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Abstract
本发明属于分子遗传学领域。具体涉及控制玉米株高和穗位高的基因ZKM76及其在降低玉米株高和穗位高性状方面的应用。本发明提供了一个控制玉米株高和穗位高基因ZKM76的序列，且公开了利用基因工程手段突变ZKM76基因以降低玉米株高和穗位高的方法。
Description
技术领域
本发明属于分子遗传学领域。具体涉及控制玉米株高和穗位高的基因ZKM76及其在降低玉米株高和穗位高性状方面的应用。本发明提供了一个控制玉米株高和穗位高基因ZKM76的序列，且公开了利用基因工程手段突变ZKM76基因以降低玉米株高和穗位高的方法。
背景技术
矮化性状带来了作物产量的突破，降低作物株高的方法具有极大的应用潜力。在玉米的主要农艺性状中，株高和穗位高影响到玉米的抗倒性、光合效能、收获指数，与玉米产量密切相关。因此，在玉米育种实践与种质资源改良工作中，株高和穗位高性状具有重要的价值。
由于株高和穗位高受主效基因和微效多基因的共同控制，表现为典型的数量性状遗传。尽管有一些调控玉米株高和穗位高的基因被定位与克隆(华南农业大学.玉米ZmPIF3s突变型蛋白、其编码基因及其在育种上的应用:CN201910273522.1[P].2019-08-02.；杭州瑞丰生物科技有限公司.CYP78A基因在增加玉米株高和增强植株长势中的应用：CN201510230547.5[P].2016-12-07.；中国农业大学.与玉米株高相关基因及其编码蛋白与应用：CN200410037404.4[P].2005-01-26.；中国农业科学院作物科学研究所.利用基因编辑技术创制玉米矮化材料的方法：CN201910371358.8[P].2019-08-16.)，更多的株高和穗位高性状相关基因有待进一步克隆。
发明内容
本发明的目的之一在于提供一个控制玉米株高和穗位高性状的基因ZKM76的序列。
本发明的目的之二在于公开一种降低玉米株高和穗位高性状的方法。
为实现上述目的，本发明采用如下技术方案：
本发明提供了一种控制玉米株高和穗位高的基因ZKM76及其在降低玉米株高和穗位高性状中的应用，其特征在于：上述基因的核酸序列如SEQ ID NO.1-SEQ ID NO.3所示。其中，SEQ ID NO.1序列为ZKM76的基因组序列，SEQ ID NO.2序列和SEQ ID NO.3序列为该基因的两个转录本序列。
在另一方面，本发明还提供了一种降低玉米株高和穗位高的方法，其特征在于：抑制玉米中上述ZKM76基因编码蛋白的表达和/或活性，选择玉米株高和穗位高降低的植株。
在一些实施方案中，上述蛋白的氨基酸序列如SEQ ID NO.4或SEQ ID NO.5所示。
在一些实施方案中，上述抑制蛋白表达和/或活性的方法包括基因编辑、RNA干扰、T-DNA插入、物理或化学诱变中任一种。
在一些实施方案中，上述基因编辑采用CRISPR/Cas9方法。
在一些实施方案中，上述CRISPR/Cas9方法在玉米中的基因组靶标区域的DNA序列如SEQ ID NO.6所示。
在另一方面，本发明还提供了一种用于降低玉米株高和穗位高的试剂盒，其特征在于：包括如下任一种：
(1)sgRNA分子，其序列如SEQ ID NO.7所示；
(2)所述sgRNA的编码DNA分子；
(3)表达所述sgRNA的载体。
在另一方面，本发明还提供了用上述试剂盒得到的一种玉米突变基因型，其特征在于：所述突变基因型序列如SEQ ID NO.8所示。
本发明的优点及有益效果如下：控制玉米株高和穗位高基因ZKM76是之前没有报道过的。本发明利用多亲本高世代自交系群体定位到控制玉米株高和穗位高性状的基因组区域，并利用CRISPR/Cas9方法突变该区域中的功能基因，发现ZKM76基因能够控制玉米株高和穗位高性状。利用CRISPR/Cas9基因编辑的方法和编辑后的突变基因型序列，可以降低玉米株高和穗位高，创制矮化玉米品种，从而提高玉米产量，降低倒伏率，提高种植效率，方便机械化收获。
附图说明
图1株高QTL定位结果。纵轴表示每个标记关联分析检验的p-value，取-log10，横轴表示染色体的位置。
图2 ZKM76基因编辑载体图。各元件英文及缩写含义列举如下：
RB T-DNA右边界序列
gRNA 向导RNA
UBI 泛素启动子
Cas9 cas9基因序列
NOS terminator 胭脂碱合成酶终止子
35S 花椰菜花叶病毒35S启动子
Bar 草铵膦耐性的筛选标记基因
PolyA 花椰菜花叶病毒35S多聚腺苷酸序列
LB T-DNA左边界序列
Kan 卡那霉素抗性序列
pBR322 pBR322载体复制起始位点
Bom site 载体Bom基因位点
pVS1 pVS1 复制子
STA region 转录起始区
图3利用CRISPR-Cas9技术突变玉米ZKM76基因蛋白后的株高和穗位高表现。ZKM76表示基因编辑植株，CK表示未编辑植株。
具体实施方式
提供以下定义和方法用以更好地界定本申请以及在本申请实践中指导本领域普通技术人员。除非另作说明，术语按照相关领域普通技术人员的常规用法理解。本文所引用的所有专利文献、学术论文、行业标准及其他公开出版物等，其中的全部内容整体并入本文作为参考。
如本文所用，“玉米”是任何玉米植物并包括可以与玉米育种的所有植物品种，包括整株植物、植物细胞、植物器官、植物原生质体、植物可以从中再生的植物细胞组织培养物、植物愈伤组织、植物或植物部分中完整的植物细胞，所述植物部分例如胚、花粉、胚珠、种子、叶、花、枝、果实、茎杆、根、根尖、花药等。除非另有所指，核酸以5’至3’方向从左向右书写；氨基酸序列以氨基至羧基方向从左向右书写。氨基酸在本文可以用其通常所知的三字母符号或IUPAC-IUB生物化学命名委员会推荐的单字母符号来表示。同样地，可以用通常接受的单字母码表示核苷酸。数字范围包括限定该范围的数字。如本文所用，“核酸”包括涉及单链或双链形式的脱氧核糖核苷酸或核糖核苷酸多聚物，并且除非另有限制，包括具有天然核苷酸基本性质的已知类似物(例如，肽核酸)，所述类似物以与天然存在的核苷酸类似的方式与单链核酸杂交。如本文所用，术语“编码”或“所编码的”用于特定核酸的上下文时，指该核酸包含指导该核苷酸序列翻译成特定蛋白的必需信息。使用密码子表示编码蛋白的信息。如本文所用，涉及特定多核苷酸或其所编码的蛋白的“全长序列”指具有天然(非合成)内源序列的整个核酸序列或整个氨基酸序列。全长多核苷酸编码该特定蛋白的全长、催化活性形式。本文可互换地使用术语“多肽”和“蛋白”，以指氨基酸残基的多聚物。该术语用于氨基酸多聚物，其中一个或多个氨基酸残基是相应天然存在的氨基酸的人工化学类似物。该术语还用于天然存在的氨基酸多聚物。本文可互换地使用术语“残基”或“氨基酸残基”或“氨基酸”，以指被并入蛋白、多肽或肽(统称“蛋白”)的氨基酸。氨基酸可以是天然存在的氨基酸，并且除非另有限制，可以包括天然氨基酸的已知类似物，所述类似物可以与天然存在的氨基酸相似的方式起作用。
如本文所用，可以互换地使用术语“分离的”和“纯化的”，以涉及核酸或多肽或其生物学活性部分，其基本上或本质上不含如在其天然存在的环境中所发现的通常伴随或反应于该核酸或多肽的组分。因而，用重组技术产生分离的或纯化的核酸或多肽时，分离的或纯化的核酸或多肽基本上不含其它细胞物质或培养基，或者化学合成分离的或纯化的核酸或多肽时，基本上不含化学前体或其它化学品。“分离的”核酸通常不含在该核酸所衍生自的生物体的基因组DNA中天然侧翼于该核酸(即位于该核酸5’和3’端的序列)的序列(诸如，编码蛋白的序列)。例如，在各种实施方案中，所分离的核酸可以包含在该核酸所衍生自的细胞的基因组DNA中天然侧翼于该核酸的少于约0.5kb的核苷酸序列。
在本申请中，将词语“包括”、“包含”或其变体应理解为除所描述的元素、数或步骤外，还包含其它元素、数或步骤。“受试植物”或“受试植物细胞”是指遗传改造已经生效的植物或植物细胞，或者如此改造的植物或细胞的子代细胞，该子代细胞包含所述改造。“对照”或“对照植物”或“对照植物细胞”提供用于测量受试植物或植物细胞表型改变的参考点。对照植物或植物细胞可以包括，例如：(a)野生型植物或细胞，即与遗传改造起始材料具有相同基因型的植物或细胞，所述遗传改造产生受试植物或细胞；(b)与所述起始材料具有相同基因型但已用空构建体(即用对目的性状无已知效果的构建体，诸如包含标物基因的构建体)转化的植物或植物细胞；(c)是受试植物或植物细胞的非转化分离子的植物或植物细胞；(d)与所述受试植物或植物细胞在遗传上一致但未暴露于会诱导目的基因表达的条件或刺激物的植物或植物细胞；或(e)受试植物或植物细胞自身，其处于目的基因不被表达的条件下。
本领域技术人员会容易地认同，诸如位点特异性诱变和随机诱变、聚合酶链式反应方法和蛋白工程化技术的分子生物学领域的进步提供了广泛的适当的工具和操作步骤，以用于改造或者工程化农业上感兴趣的蛋白的氨基酸序列和潜在的基因序列。
在一些实施方案中，可以对本申请的核苷酸序列进行改变，以进行保守氨基酸替换。保守氨基酸替换的原则和实例在下文中进一步描述。在某些实施方案中，可以依照公开的单子叶密码子偏好性对本申请的核苷酸序列进行不改变氨基酸序列的替换，例如可以用单子叶植物偏好的密码子替换编码同一氨基酸序列的密码子，而不改变该核苷酸序列所编码的氨基酸序列。在一些实施方案中，以编码同一氨基酸序列的不同密码子替换本申请中的部分核苷酸序列，从而在改变核苷酸序列的同时不改变其编码的氨基酸序列。保守变体包括由于遗传密码子简并性而编码实施方案的蛋白中的一种的氨基酸序列的那些序列。在一些实施方案中，根据单子叶植物偏好密码子替换本申请中的部分核苷酸序列。本领域技术人员会认识到氨基酸添加和/或取代通常基于氨基酸侧链取代基的相对相似性，例如，所述取代基的疏水性、电荷、大小等等。具有各种前述所考虑性质的示例性氨基酸取代基团为本领域技术人员所公知，并且包括精氨酸与赖氨酸；谷氨酸和天门冬氨酸；丝氨酸和苏氨酸；谷氨酰胺和天冬酰胺；以及缬氨酸、亮氨酸和异亮氨酸。关于不影响目的蛋白生物学活性的适当氨基酸取代的指南可以在Dayhoff等人(1978)Atlas of Protein Sequence andStructure(蛋白序列和结构图集)(Natl.Biomed.Res.Found.,Washington,D.C)(通过引用并入本文)的模型中找到。可以进行诸如将一个氨基酸换作具有相似性质的另一个氨基酸的保守性取代。序列一致性的鉴定包括杂交技术。例如，将已知核苷酸序列的全部或部分用作与其它相应核苷酸序列选择性杂交的探针，所述其它相应核苷酸序列存在于来自所选生物体的已克隆基因组DNA片段或cDNA片段群(即基因组文库或cDNA文库)。
在一些实施方案中，还包括核苷酸序列及其编码的氨基酸序列的片段。如本文所用，术语“片段”指实施方案的多核苷酸的核苷酸序列的一部分或者多肽的氨基酸序列的一部分。核苷酸序列的片段可以编码蛋白片段，所述蛋白片段保留天然或相应全长蛋白的生物学活性，并因而具有蛋白活性。突变体蛋白包括天然蛋白的生物活性片段，其包含保留天然蛋白生物学活性的连续氨基酸残基。一些实施方案还包括转化的植物细胞或转基因植物，其包含至少一种实施方案的核苷酸序列。在一些实施方案中，使用表达载体转化植物，所述表达载体包含至少一种实施方案的核苷酸序列以及与其可操作地连接的在植物细胞中驱动表达的启动子。转化的植物细胞和转基因植物表示基因组内包含异源多核苷酸的植物细胞或植物。一般来说，所述异源多核苷酸在转化的植物细胞或转基因植物的基因组内稳定地整合，以致将所述多核苷酸传递给后代。可以将所述异源多核苷酸单独地或作为表达载体的一部分整合进基因组。在一些实施方案中，本申请涉及的植物包括植物细胞、植物原生质体、可以再生出植物的植物细胞组织培养物、植物愈伤组织、植物团块和植物细胞，其为完整的植物或者植物的部分，诸如胚胎，花粉，胚珠，种子，叶，花，枝，果实，果仁，穗，穗轴，壳，秸秆，根，根尖，花药等等。本申请还包括源于本申请的转基因植物或其子代、并因而至少部分地包含本申请的核苷酸序列的植物细胞、原生质体、组织、愈伤组织、胚胎以及花、茎、果实、叶以及根。
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本申请的范围。若无特别指明，实施例按照常规实验条件，如Sambrook等人的分子克隆实验手册(SambrookJ&Russell D W,Molecular cloning:a laboratory manual,2001)，或按照制造厂商说明书建议的条件。若未特别指明，实施例中所用的化学试剂均为常规市售试剂，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例
实施例1玉米株高和穗位高QTL的定位过程
1、群体构建
本发明将24个中国的优良玉米自交系，通过两代双列杂交、六代开放授粉及六代自交的方式获得了1404个子代组成的多亲本高世代自交系群体(CUBIC)。这24个中国优良玉米自交系包括LV28(旅28)、E28、DAN340(丹340)、F349、ZI330(自330)、ZONG3(综3)、ZONG31(综31)、HUANGC(黄C)、HZS(黄早四)、HYS(黄野四)、TY4(天涯4)、YUANGFH(原辅黄)、CHANG7-2(昌7-2)、K12、XI502(西502)、LX9801、H21、SHUANG741(双741)、Q1261、JI853(吉853)、JI53(冀53)、5237、81515、NX110(农系110)，该群体的24个亲本及1404子代自交系全部进行基因组测序，获得了超过14M的SNP和InDel变异。
2、表型分析
所有24个亲本和1404子代自交系在黄淮海及东北5个地点种植，表型变异丰富。所考查农艺表型包括植株高度、穗位高、开花期、穗重等产量性状等。将所有环境重复计算出每个自交系的最佳线性无偏预测因子(BLUP)值用于后续分析，包括基本表型统计、相关性分析和GWAS。通过线性回归估算由给定性状的QTL解释的表型方差或遗传力，并联合估算解释的总方差。
3、基于单变量的GWAS分析(sGWAS)
根据最小等位基因频率(MAF)过滤后，共计1180万个高质量SNP用于下游分析。前十个主成分(PC)共解释了8.76％的遗传方差，同时整合了亲缘关系K矩阵作为随机效应一起用于sGWAS分析。关联的显着性阈值设置为1.23E-8，等于0.05/Ne，其中Ne是根据所有变异计算的独立检验的有效数量。确定QTL区间的标准为：每个性状的显著SNP被首先提取，只保留至少两个连续SNP小于20Kb的位点同时合并作为基本区间单元，20KB以内只有单独显著位点的SNP在下一步分析中被排除；如果相邻区间单元之间的任何显著SNP有较高连锁不平衡关系(LD，r2≥0.2)，则将相邻区间单元进一步合并为QTL候选区间。为了减少由较小的QTL效应而获得的QTL间隔较小而未包含功能基因的可能性，对于那些间隔小于50Kb且最显著SNP的显著性低于阈值100倍的QTL(即1.23E-10)，将对QTL区间向两侧继续延伸25Kb获得最终QTL区间。将区间内最显著的SNP作为sQTL的显著性，将区间间隔内所有基因作为候选基因。
4、获得QTL区间
利用上述的定位方法，本发明找到了玉米株高和穗位高性状的3组最显著SNP，分别位于第2染色体13886872-14540646区间内、第5染色体7231744-7873497区间内和10染色体146705700-150358009区间内。
其中，第2染色体13886872-14540646区间内经MaizeGDB数据库(https://www.maizegdb.org/)的查询包含12个有注释的基因(图1)，编号分别为GRMZM2G032554、GRMZM2G032583、GRMZM2G163067、GRMZM2G098099、GRMZM2G014009、GRMZM2G014187、GRMZM2G066489、GRMZM2G405064、GRMZM2G010011、GRMZM2G146847、GRMZM2G146866、GRMZM2G016705。
实施例2基因编辑敲除候选基因分析基因功能
本发明利用CRISPR-Cas9基因编辑技术对上述区间内的12个基因进行定点突变。实施方式包括基因编辑载体的构建、玉米的遗传转化以及编辑效果的功能验证。具体如下：
1.基因编辑载体的构建
本发明通过BsaI酶切，切去骨架载体pCXB053(未米生物科技(江苏)有限公司构建)中的ccdB序列，把靶标序列(如SEQ ID NO.6所示)通过T4连接酶连接到U6和gRNA之间。具体的构建流程如下：
1)合成引物
Primer TF：ATTGGGGCTCAGAGTGAACCTCC；Primer TR:AAACGGAGGTTCACTCTGAGCCC。在上海生工合成，用超纯水溶解后混合均匀。
2)配Annealing Buffer TE
Tris-Cl PH8.0 10mM
EDTA 0.1mM
NaCl： 50mM
3)退火连接
连接体系：
Annealing Buffer 50μL
Mix Primer 5μL
连接程序：
95℃ 3min
0.1℃/s匀速下降
20℃ 1min
储存在-20℃
4)骨架载体用BsaI限制酶酶切
酶切体系：
37度酶切5h后，直接回收(使用quangen回收试剂盒)。
回收产物加水稀释到50ng/μL，加等量的T4 Buffer稀释，存放于-20℃。
5)T4酶连
酶连体系：
酶连程序：
25℃ 2h(PCR仪中)
6)转化大肠杆菌
酶连反应液10μL与100μL大肠杆菌5a感受态混合，严格静置冰浴30min，42℃热激35s，冰浴2min，加入500L无抗生素LB，37℃震荡复苏1h。3000g离心1分钟，吸走上清液，留下100μL液体吹打混匀细菌细胞后涂皿(含50mg/L卡那霉素的固体培养基)，37℃倒置培养12h。
挑取阳性克隆测序和抽提质粒。测序验证引物为PUV3-R：CTGGCGAAAGGGGGATGTGCTGCAA。
2.玉米遗传转化
将载体通过电击法转入农杆菌EHA105中，PCR进行鉴定。以新鲜剥离的1mm左右的玉米自交系KN5585(未米生物科技(江苏)有限公司选育的自交系)的幼胚为材料，将剥取的玉米胚放入含有1.8mL悬浮液的2mL塑料离心管中，30min内大约处理未成熟幼胚150个；吸去悬浮液，余下玉米胚在管中然后加入1.0mL农杆菌悬浮液，放置5min。将离心管中的幼胚悬浮后倒入共培养基上，并用移液器吸去表面多余的农杆菌菌液，于23℃黑暗共培养3天。共培养后，将幼胚转移到休息培养基中，于28℃黑暗培养6天后，放至含5mg/L Bialaphos的筛选培养基上，开始筛选培养2周，然后转到含8mg/L Bialaphos筛选培养基上筛选培养2周。将抗性愈伤组织转移至分化培养基1中，25℃，5000lx，光照培养1周。再将愈伤转移至分化培养基2中，光照培养2周；将分化生出的小苗转移至生根培养基上，25℃，5000lx，光照培养直到生根；将小苗转入小盆中生长，一定生长阶段后移栽于温室中，3-4个月后收获后代种子。
3.基因编辑植株的性状评价
对T1代材料提取苗期DNA检测基因编辑情况，设计引物扩增靶标编辑区段，扩增体系为：DNA：3μL，双向引物各1μL，2×TaqMix：7.5μL，ddH2O：2.5μL，总体积10μL。PCR反应条件如下：(1)94℃5分钟，(2)94℃40秒，(3)57℃30秒，(4)72℃60秒，(5)从(2)步－(4)步循环35次，(6)72℃7分钟，(7)4℃保存。PCR产物交由武汉擎科生物科技有限公司进行Sanger测序。将转化株测序结果与野生型KN5585的基因组比对，发生碱基替换、插入或缺失的材料即为阳性编辑材料，否则为阴性材料。经过对各基因编辑材料的表型鉴定，发现GRMZM2G010011基因的编辑植株株高和穗位高发生了变化(表1)。
表1各基因编辑玉米材料的株高性状数据
数据以“平均值±标准差”表示，不同字母表示各材料在p＝0.05水平的显著性差异。
4.株高和穗位高变化植株的深入分析
进一步对株高和穗位高性状有变化的GRMZM2G010011基因(命名为ZKM76)编辑植株进行深入分析。该基因共获得3个独立转化事件。设计引物5’-AGAATGCATCAAGTGATATGGTTACTCTTG-3’和5’-ACTAAAAGCTGTTGGTGCAGAACT-3’，PCR扩增靶标编辑区段并对PCR产物进行测序。在3个独立转化事件中，1个事件在基因中发生了编辑(具体为一个碱基C的缺失，序列如SEQ ID NO.8所示)，为阳性植株；2个没有编辑，为阴性植株。调查阳性转化事件T1代材料授粉后期的株高，发现阳性突变体和分离出的阴性植株之间株高和穗位高具有显著差异，表明该基因控制株高和穗位高性状。数据见表2。
表2 ZKM76基因编辑玉米材料的株高和穗位高性状数据
数据以“平均值±标准差”表示，不同字母表示各材料在p＝0.05水平的显著性差异。“-”表示碱基缺失。
ZKM76基因，位于Chr2:14,450,181-14,459,904区间内，基因组序列如SEQ IDNO.1所示，全长11,131bp。包含2个转录本，两个转录本的cDNA序列如SEQ ID NO.2和SEQ IDNO.3所示，编码的氨基酸序列如SEQ ID NO.4和SEQ ID NO.5所示。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 未米生物科技（江苏）有限公司，华中农业大学
<120> 控制玉米株高和穗位高基因ZKM76及其应用
<130> 1
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11131
<212> DNA
<213> 玉米(Zea mays L.)
<400> 1
ttgagacagt tgatggtttg tccgaagcat tggaaatctg agagtgagct cccttgcatg 60
attgctttgg ctaatttggt tttgatatgc ccgcgactgt aggtgttgtt atgctcttgt 120
ttatttattg tgttttctgt tatagctata gagtttgata ctagatcatt tagttcagtg 180
tattccctga tgagctaagg gcgccttcct tccatattta cttcttattt caaccaaacc 240
aagatgttag gttttaggcc cagaaccatg catatgactg attgtaagct gtaggcacag 300
atgccaatgt gtttttttct gttttcaact tttaatgact acatctctgt tgagggtggc 360
cggtgatgag caacctgtct ctttatttat aacaaactca taccaagaat gacaggtgct 420
tttgcttcta tatttgttga tctactatat ttgcttctat attgggagtt caatataatg 480
attaagtaat ataagtctcg ggatatattt tagttgatct actatatttg ttgtctgatt 540
ggaatcctaa tacttttgtt aaattatttt tgttgaccta atactttcaa gtaactgcag 600
attacttctg tgaaaattat acggaacaag cagactgggc actcagaagg ctatggtttt 660
attgagtttt cctctcgagc tgctgcagaa catactctga taaacttcaa tgggcagatg 720
atgtcgaatg ttgagatgac ttttaagctg aactgggctt ctgctagcac tggtgataag 780
cgtggagata gtggttctga tcacacaata tttgttggtg atttggctca tggtgttact 840
gactccatgt tggaagatgt gttcagagct aagtaccctt cagttaggga gctaatgttg 900
ttgttgatag gatgattgga tggcccaaag gatatggttt cgtgcgtttt ggagatctga 960
ataatgcatt gtgaagatgt atatttgtgc ttgtttaatc tgaaaagggg acaactttta 1020
gatatagcat aataggatat gaagatgaaa gatctgtact tagaaatttg ttcatgaatg 1080
ctttgtttga catttcactt cgtatgctgg catctcagtg tattaagtgt taatgttaca 1140
gttattcttg gatcaacatc attcgagtat ttgactattg tttgtcacgt gcacttatta 1200
ttcttgcatc actattttga accaattttt aagctatcgt agcaacgcac gggcacgcac 1260
ctagtctata atgattcatc ggagacaacc taagacaaat ataattataa aaaacaaaaa 1320
aacggataaa atggggaaaa tatcgtttgc agatttggag tgaaaacacc aactgcaact 1380
gcaagtggcc gggcggcccc agccgacaga aagaaggaat ccaaagccgt tgcgcatcgc 1440
ccatcgccgc ccacacctct tctgcagttc tacttcacgt cttgaccacg tccgcctcgg 1500
cccctggtct cctactctcc tccccactcc tccctcggcc tggagtccag tcctcgactc 1560
cgcctgccgc ctgctgcctg acaagccacg cgctgcggca gccgtacggg cactagggtt 1620
ttcttccact tcgaccgcgg gctcgggatc tcgcgccgca ccgcaatgca gcacacgtgc 1680
cgctgatccc gtggccgcct ccttcctttc ctcctcctgc ggtcctgcgc cgcctctgct 1740
gcagcgggca gccgccatgg cgcggcgcca cgggtggcag ctccccgccc acaccctgca 1800
ggttcgcctg cggttctcct tgttccgcac catgagttgt cctccgctct gtatgtgcta 1860
gttgagcgcg taatgtggtc tcggatcccg tcgcgcgtgc cgtcgcccgc tcggttttcc 1920
gctttcggtc ggtgttcgaa cgattttttc tgttgctatg cttacctttt gacgaggaat 1980
gctatggcca gcctaatttg atgcgatccg atgcgatgtg atgtgttttg ctcttctcta 2040
aacattattt tcccgatgcg tctgcgcggc tctgtatact ctgaaaagcg tgcgagaatt 2100
tggtcacttc tgctacttct ctagtttgac gcgaagcagt agaacatgga tttggtttgg 2160
ttaaggtttt atcctaacgg caggtttacc tgaattttat gcagagctac atcctgatct 2220
tctgcggcat agaaggtgtc ttgtttttta tctggcattt cgctttctcc aatagaagtg 2280
tcactttttg ttggtattct catgggccgt tctgcacttt cgtctagcat gtgatgtcgg 2340
acttgaccta caaatttgtt cgctgccgcg ctattatttt gtctccttgc gctttcacct 2400
tattccgtaa tatgtctaac ttcatttttg caaggtctaa tatagttatg ctgtgttaaa 2460
ctgaatgttg tattcgattc atgatggacc ctatttttcg gggttgctgt tgaaatgtgt 2520
gaactctact ttcctttgcg ttaagcctca caatggaagc ttcagtgttc ctaaacaata 2580
accgatgcgt tctgtttgtt gtcattcatt tggatctggt catacccact agcatgttcc 2640
ttagctttat gccactgtgt cagcaaatta atagttgttt gtgctacagc cagacgccca 2700
aaataaaaca tgctacactg aattaaaaag ttgttaatgt atcggtgagt ttgaatgtcg 2760
tactttgtcg tgtcctattt gagcgtcaat gcccagctat tagcttaggc agctctgtga 2820
agcgtgttgt tatgaagcct ttttctggat tcattgaaga attgaattgt aatatcctac 2880
ttctgttgat gcagtatcag agaacagcgg tatccccgtg aggtcagagt ggatcgtgga 2940
gtagtgacta gtgaccaaga ttttttaagc atgaactatg agtataatat tagtttttgt 3000
cttaggaaca atgttgatta tgcgatagta tcaagtaggt atttccattg ttttgaattt 3060
tctggtgaac attcagttag cagtagcctg gatccattca cgagccaaat catcatgctt 3120
actttcattt gccccttctc tataaagtct aaatgcaagt gcaagatttg agggaaaggc 3180
tatgaattct gagctgctat gactttacga gtgtcctgta gaagaattat gaaaaaccta 3240
agctaatgag attgtcttat aaaaaaatat tttctcttgg aagtgattat aattttttga 3300
gtccatactt gtaaacttga cagtataatc aatttccatg cttgtaagct catcgtttag 3360
taatatctct taggagcttc ccgtcttaga tagggattgt tatgtacatt atgggagtca 3420
tggccttcag ttctgttgaa tttgtggccc ccttacattt catttgtgct tcctcctaaa 3480
gtctatcctt gctggcaaca catccttatc tatgccctat tggtcctatc agtggaagag 3540
tatgattgta tgaataacat tttttatata tttcttttca ggtggtggct ataacagtgt 3600
actctgtgct ttgtatagca ttctatgctt tcttctctcc atttcttggg aaggacttgt 3660
atcaatatat cgctgtcggt atttatagtt ttttggtaag gttccttcct accatgcttg 3720
gatctaatat ctgagtaaat gatcacttct ttgatactcg ttttctgttt agccaggctt 3780
tatctgtgct gattctatat gtcagatgta ctgcgattga tcctgctgac tctggcatcc 3840
tgatttccat ggatgacata ctaatttata aatcagaagg tatatcttgc ttattaacaa 3900
tgtttctgta gtattttgta ccttatcact ttatttacta atacttcttc atcttctgtt 3960
ttaaagcaca tgtagacact caagatgaag ctggtaagcc aggattgagg aacgatgagg 4020
atatccgaaa gcataaatca tgctttggaa gggtttgttt ttgttgcgct atttttacaa 4080
ctggggactg tcgcggaggg gatgaagcta accatcaaga agattatggt gaagaggcac 4140
tcttttgcac cctttgcaac gcagaggtac tttatgctga gccccaaccg tagtcgatgt 4200
gcatagtgtg ccatatttct gtttgaccca atttctcttt atcttaccaa gattatattg 4260
ttgaattctt tccttccagt ttaccaattt attaagtacc catgtacatt aaaactaaaa 4320
ctgttggttg cagcataaca gatgctaaga gttggtgcct aaaaaatatt agctatcttt 4380
tgtattcatt tagttctagg tactctcaat acggaaccgt ttggaatgtt gttagctaga 4440
tattttttat gcaacaaagt ttttttattt aagcataaga aaatgactag tttatatatt 4500
ttcacgacta ttcggtgctt ctactttttg gaagccagca gctggcatct tcatttgcca 4560
tgatgcttgc ttatcttttc tttgtattaa ttttcagtgc actttatgtt tggacacatg 4620
gtatcaacac aaagttttaa tcgatactaa gtgcagttcc ctttgtattg tgatacaggt 4680
gcgcaaacat agcaagcact gtcgaagttg cgacaagtgt gttgatgggt ttgatcatca 4740
ctgccgggta ttatatcaaa tcttctctag cttgtaatct tctacttgac agcgtgcctt 4800
gtcggagacg aagaagatga tccctggatc acccaaatag cagcacaaac actacaagaa 4860
cacattgatg ccctgtttgg gagtaaatta tttttgtagt ttctaggcaa taccatggtt 4920
tttagtaata ccatagtatt ttatgctata gggtgtttgg ttgcattcct gaaaactatg 4980
tttttaaaac catggtattc taggtaccat gatatttttg gagtattgta tcctgtttgg 5040
aaccaaagta tttcaaaact atggttttta aatactgtgg tttataattg tcatgacata 5100
aataccatgg ttttcaaata ccaaggtttc ttcacagttt ttattggaac cggtgcttgt 5160
ttggttccac ggtacgcatc ttatattaat taatcaatta attagataag gcaaagataa 5220
aggggcgcgg gtttgagtac tcgattcaca ctattttttg cgcctgcgca cgcgcgggcg 5280
ctaggggcgt ggggaaggtg aagccgtggt atcaacaggt gcccacatta ggttcttaat 5340
agattagtat agattattct atagattgat taatttagca tgtatggcat agctcgcgag 5400
tcgaacgagc cagttcggct cagctcgctc caccaacgag gccaaaaaat aggctcagtt 5460
tagctcgctt gaggctcgtg agccactcca agctcgagcc agctcgcgag cctccagctt 5520
attttccagc cctacctttg agccgcaaca agcatcatcc acacataaac tgtgtgtgcg 5580
accagagcaa atgcctcatc gttttctatt cccgactgct ggatctaccc cttggcgaca 5640
agctgacaat ctgggccttg tgcttgacga tgatgcctgc tcattacgct ttagtttgta 5700
aacccacttc atgtcaattg ggtgatggct tggaggatgg tcgacatgag cccacgtctt 5760
gttctcctca attgatgtca tctcctctgt ggctctgtcg tcaccttgag tcagcgggca 5820
ttgccttgag cctccacgaa catgttcggt tcatcatcat cggtggtaag gagcaagtcc 5880
tcatccacca tgaactcgtt ctgccaagcc aagaatttcc accgacttga gcacattttc 5940
tagcgtcctg aaccggagag gcgacttaac gtcgtggttg gcgtcgaggt ctcgagtagt 6000
gttaggtggt cgatgggaac aaacatcgtc gttgctttct ggcctagcag ctctcttaga 6060
tctggtcgac gtcgttgttc gatagtagtt tgcacaccat gtcaccctct gacatttccc 6120
taagccccta gaacccgagg tgtctgaacc tggcgtgcca ttgctagggc acattcaagc 6180
ttcaggcaac gacgcatgca atctgagtga tgtcaatgtt gagggcgtcc acgactcatg 6240
ctcgacaatg ttcaattgcc gctcgagtag tcgagcttcc actgccagag cggtggagcc 6300
atgacacaac atgcaccgcc aactcatcct tggtcaagtt cgagaaggat tttgatcgcc 6360
atcacgatat gcgagtacct tggaggagtg gcctatagga atttgcgcat gatatttgaa 6420
tcttggtacc tagtgtcgag gacctccagt tgatttacta cgtcagtgat gcgcatgctg 6480
aagtcgttgg catcttaccg ttgttgaacc tatgcgagtc gaacttgcgt tgtagcgtgt 6540
tcgccttcac ttggcgcatt cggtccatgg tgaggtgcat tttttgagtg cgactagcaa 6600
tcttagcggt tgccttgctc gcgatcaccc cttgcatctt cggaggtaca ccaactagtc 6660
accaagggtg atggcttcta gagcattgcg attgtcagtg tagttgacgg tgctgatgtt 6720
cactgccatc cacatgccat gaatagtagt tggtcttggt caacaacgag tactgcctac 6780
tgtcgccact ctctcatgac ccggtagatc accgggttgt agtcgtcatt gctagagcca 6840
ctcctcatct cctgtcggca atgcggtgcc gaggcagttc gttccgctgc atgactgcta 6900
gcacggcctt gacaacatag tggctctgat accaaatcta atcatctact tgtcggcatg 6960
ccttgttgga gacaaagaag atgatccgtg gataacccaa atgctcacaa aaaaactcaa 7020
catgaacact gagttttgga gatgatccgt ggataaccca aatgctcaca aaaaaactca 7080
acatgaacac tgagttttgg accttttggt gctgccaaaa tatactctac ctttttatat 7140
tctattgatc tgattaaata caattacaaa gtctcacagc cacaaatcag gcatcaatat 7200
cttctactgg accatgctat cccacagcca atagcttgtc atatcgtgtt acttgggtct 7260
aaaaacagga acagaaaaca aggaaagaca gccattgtat ggtgcatgca tctagcgcac 7320
tataggtacg cccaaacaaa gggctcaatc aattagctaa gaaagaaaac taaccaatca 7380
gccaaggaaa caagaaaccg agctggatta tgcaacatag ctaatttggc tttcttcgtc 7440
ttttctaatt tttgttgatt tttcccttct gttactactc aaacctgtgt tttatctttt 7500
gatgcagtgg ctgaacaatt gtgttggaag aaaaaattat atcacatttt tatgccttat 7560
ggctgtgagt ctggcttggg tatgttaaaa atcatggaac actaatattt ccaactgtca 7620
acattacatc attttaaacg ccaacaagct tgtatcttgt ggattattcc aaaagcattt 7680
ttgtgattag ttgctatttt cttaaattca gcttgctgct gagtgtggag ttggtattgc 7740
tgtttttgtt cgttgcttca ctgacaagac agtcatagaa gatcagattg gggaaaagct 7800
gggttacggt ctttcacgtg caccctttgc aaccattgta gtaagttata aattattatt 7860
actttgatgt attagtctag tttctcttta caatttgagt ttttttctgt tatatagtta 7920
acagattctt tcatgtacat ttttattaat tttgcaaact tcatcatctc tatttcagga 7980
tccttgttaa tcaaactggt aggtactaac tttaattagg ctgttgagat aagacaagat 8040
atatagttca atacattagc atgaaaaggg gccaatctta tctagaagtg aaaataagga 8100
attgctaact tttagaagca gaaattgtaa cccagcacaa ccaatgctga gcgtattgca 8160
catctgctac aatccacatt aagggctgat ttggtggaca agatcacgag gggatccatg 8220
ggggaggaat ccccttgcta attgaatagc aaggagattc ctccccatgg atcccctcgt 8280
gatcactatc caccaaatca gccctaatag tctttgtcca aacaagacat tttacttgac 8340
ctttagtcca attgaggttt aggtcttagc tgaaaataca aggtccatgc tgactacgta 8400
cacaagagaa atattatcat agccgaggaa gttaatactg gcagtaattt aaactgtcaa 8460
attgagcgga cataaatgtt ctgtaaactg cggtgactct gagatagtca atcgtttatg 8520
aattacgata tggcatggct agtaaggtac cacaatcttg acaaagaaac ctttctgact 8580
ccaggccctg gctacagctc tttcaatgct tgcttcagta ccactaggag aactattttt 8640
cttccatatg atattaatcc ggaaggttag tcacttgtca ttatttgtct cattttacta 8700
tatcgtggcc aagaatgcat caagtgatat ggttactctt gttgcacttt tttttttcag 8760
ggcataacaa cttatgaata tgttgtggcc atgagggctc agagtgaacc tcctggacct 8820
tctgtcaatg atgaccaaca aagcttggca tcttctccaa tgagttctgc accaacagct 8880
tttagtggga gctcgtttgc acggcactac aaaggcgcat ggtgcactcc cccacgtatc 8940
ttcattgacc aggtactatg taatgtcatc tttccttagt atcattgtct tgtgtttttg 9000
ttctactccc tgcagtcaca ttttgggttg ccccaagtaa atcattttaa attttattta 9060
caaattagaa attgctctct atgtgttgag ttcaatgaca tgagtagatt tgtctcgaaa 9120
tatattttca taaaaacgta cttttgctat gtttcataat atcttgtaat aaaaaaaata 9180
ccagtcaaag ttgcattctg aagattgtag agtccaagac gtcacttatt tgtgactgga 9240
gtgagtataa ttgaaggcac cacaagttga tgtgcgttct atcatttcaa cttcaacaaa 9300
tcttatcaga aactcacaca ttatcccaga caatctatca aaattagtgc tgttcgttct 9360
gtcacagttg tctgcaatta taagttgcac ctatcttttc cttctgagcg agtcatcttg 9420
catatatttg atcctgtctg catcacattg caggacgaaa tcatcccaca cctggagcct 9480
ggccgagttc cttcaactgt tgatcctgat accacagacc caatggaaag aaccaaaacc 9540
catcctaaac ggccagtccg catcagtgcg tggaagcttg cgaagctcga ttcaaatgag 9600
gccatgaaag cagcagcgaa agccagagcc tcttcgtccg tgctgaaacc gatcaatact 9660
cgcaaccagt atgaggcaga cagtgatagc ctcagtagca gaaacagtgt cataagtgct 9720
gataccggac accataggta ccctcggtct tgcgggaatt cgcagtacaa gccctcttat 9780
ccaccaagca gagcaagtgc agatgacatc gagttgtacc cccagacacc gagcagcttc 9840
cagagcaact cacggacttc aactcccata gccgagcacc atccatccaa gcatttcaac 9900
ccaatctacc agacatcagc aaacaggtct ccattctcag caaaagcaag tgtcagtgaa 9960
gctcctgtct ctgaaatcac caatgcaggg aggtcctacc ccccaccaca agcggacaga 10020
tcctcccggt catctgttta ctgggatcag gaagctggca ggttcgtgtc tgctcaggca 10080
aaccagggat ctagctctcg ccctgcctat cctgatcttc tatacaccgg gcagaatata 10140
ttctttggtg gacctctgat tgcagatccc gcagcaagaa gcttcagaga tcctggtggc 10200
tcgagccaaa ggtctgccgg gcctcggtcg caccagctcc cggtgttcgt tcccagtgac 10260
ccccagaaag atcagctgtc caggttaccg tgaaatgcta tatctttttt ttttgggggg 10320
gggggggggt agtagtcttc cgaaacacat gttgccctgc cgggttatta atgtatagtt 10380
attagctctt agacctgaca gaatagttca tgactttgta tcgggttcat ggaaattaag 10440
acctatttag gcactatcaa ccataagcac tgaagttatg gaagtgctgc ggagtagtag 10500
tgaagtgaaa ctgacaagga tgttctgtca ccaaacctaa ttagcttcac ctcgccatat 10560
actcaaaaca aggcgataac taccaacgtg ctccttatta aaaaaaacta caaatgtgtt 10620
aaatctattc gctaatctgg atccattttc cctatctgtt ctcccggtgg aatgacctgt 10680
cgatatttgg ataatcttgt attatatgtc aactgtccaa gctatctcaa atccgcacga 10740
ggcatcacgc actcgtcggc tgggatgccc atgagtttat ctcggtctga gtagctgcaa 10800
ttatatttta ctccactttt tgcacgagga ataactaagt gggtggttgt gactccgcct 10860
ccgcggaatt gcgcttctgt gctccctagt ccctaatgat gttaaagtga aaagtgtttt 10920
tttctttcca tattttttgt tcttcgtgat ttaaaagtgg aagtgggtgt aactctccga 10980
ccgattcatc cacattccag agagggaggg agggagagag agcgagcacc atttcaggtc 11040
tagagactag agatatgtat atctctccgc gtccaaactg agaaactgtc atggccagtg 11100
tacagacatc gctcaaaatg tgcgtgggat t 11131
<210> 2
<211> 2292
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 2
ggcccctggt ctcctactct cctccccact cctccctcgg cctggagtcc agtcctcgac 60
tccgcctgcc gcctgctgcc tgacaagcca cgcgctgcgg cagccgtacg ggcactaggg 120
ttttcttcca cttcgaccgc gggctcggga tctcgcgccg caccgcaatg cagcacacgt 180
gccgctgatc ccgtggccgc ctccttcctt tcctcctcct gcggtcctgc gccgcctctg 240
ctgcagcggg cagccgccat ggcgcggcgc cacgggtggc agctccccgc ccacaccctg 300
caggtggtgg ctataacagt gtactctgtg ctttgtatag cattctatgc tttcttctct 360
ccatttcttg ggaaggactt gtatcaatat atcgctgtcg gtatttatag ttttttggct 420
ttatctgtgc tgattctata tgtcagatgt actgcgattg atcctgctga ctctggcatc 480
ctgatttcca tggatgacat actaatttat aaatcagaag cacatgtaga cactcaagat 540
gaagctggta agccaggatt gaggaacgat gaggatatcc gaaagcataa atcatgcttt 600
ggaagggttt gtttttgttg cgctattttt acaactgggg actgtcgcgg aggggatgaa 660
gctaaccatc aagaagatta tggtgaagag gcactctttt gcaccctttg caacgcagag 720
gtgcgcaaac atagcaagca ctgtcgaagt tgcgacaagt gtgttgatgg gtttgatcat 780
cactgccggt ggctgaacaa ttgtgttgga agaaaaaatt atatcacatt tttatgcctt 840
atggctgtga gtctggcttg gcttgctgct gagtgtggag ttggtattgc tgtttttgtt 900
cgttgcttca ctgacaagac agtcatagaa gatcagattg gggaaaagct gggttacggt 960
ctttcacgtg caccctttgc aaccattgta gccctggcta cagctctttc aatgcttgct 1020
tcagtaccac taggagaact atttttcttc catatgatat taatccggaa gggcataaca 1080
acttatgaat atgttgtggc catgagggct cagagtgaac ctcctggacc ttctgtcaat 1140
gatgaccaac aaagcttggc atcttctcca atgagttctg caccaacagc ttttagtggg 1200
agctcgtttg cacggcacta caaaggcgca tggtgcactc ccccacgtat cttcattgac 1260
caggacgaaa tcatcccaca cctggagcct ggccgagttc cttcaactgt tgatcctgat 1320
accacagacc caatggaaag aaccaaaacc catcctaaac ggccagtccg catcagtgcg 1380
tggaagcttg cgaagctcga ttcaaatgag gccatgaaag cagcagcgaa agccagagcc 1440
tcttcgtccg tgctgaaacc gatcaatact cgcaaccagt atgaggcaga cagtgatagc 1500
ctcagtagca gaaacagtgt cataagtgct gataccggac accataggta ccctcggtct 1560
tgcgggaatt cgcagtacaa gccctcttat ccaccaagca gagcaagtgc agatgacatc 1620
gagttgtacc cccagacacc gagcagcttc cagagcaact cacggacttc aactcccata 1680
gccgagcacc atccatccaa gcatttcaac ccaatctacc agacatcagc aaacaggtct 1740
ccattctcag caaaagcaag tgtcagtgaa gctcctgtct ctgaaatcac caatgcaggg 1800
aggtcctacc ccccaccaca agcggacaga tcctcccggt catctgttta ctgggatcag 1860
gaagctggca ggttcgtgtc tgctcaggca aaccagggat ctagctctcg ccctgcctat 1920
cctgatcttc tatacaccgg gcagaatata ttctttggtg gacctctgat tgcagatccc 1980
gcagcaagaa gcttcagaga tcctggtggc tcgagccaaa ggtctgccgg gcctcggtcg 2040
caccagctcc cggtgttcgt tcccagtgac ccccagaaag atcagctgtc caggttaccg 2100
tgaaatgcta tatctttttt ttttgggggg gggggggggg tagtagtctt ccgaaacaca 2160
tgttgccctg ccgggttatt aatgtatagt tattagctct tagacctgac agaatagttc 2220
atgactttgt atcgggttca tggaaattaa gacctattta ggcactatca accataagca 2280
ctgaagttat gg 2292
<210> 3
<211> 2323
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 3
ggcccctggt ctcctactct cctccccact cctccctcgg cctggagtcc agtcctcgac 60
tccgcctgcc gcctgctgcc tgacaagcca cgcgctgcgg cagccgtacg ggcactaggg 120
ttttcttcca cttcgaccgc gggctcggga tctcgcgccg caccgcaatg cagcacacgt 180
gccgctgatc ccgtggccgc ctccttcctt tcctcctcct gcggtcctgc gccgcctctg 240
ctgcagcggg cagccgccat ggcgcggcgc cacgggtggc agctccccgc ccacaccctg 300
cagagctaca tcctgatctt ctgcggcata gaaggtggtg gctataacag tgtactctgt 360
gctttgtata gcattctatg ctttcttctc tccatttctt gggaaggact tgtatcaata 420
tatcgctgtc ggtatttata gttttttggc tttatctgtg ctgattctat atgtcagatg 480
tactgcgatt gatcctgctg actctggcat cctgatttcc atggatgaca tactaattta 540
taaatcagaa gcacatgtag acactcaaga tgaagctggt aagccaggat tgaggaacga 600
tgaggatatc cgaaagcata aatcatgctt tggaagggtt tgtttttgtt gcgctatttt 660
tacaactggg gactgtcgcg gaggggatga agctaaccat caagaagatt atggtgaaga 720
ggcactcttt tgcacccttt gcaacgcaga ggtgcgcaaa catagcaagc actgtcgaag 780
ttgcgacaag tgtgttgatg ggtttgatca tcactgccgg tggctgaaca attgtgttgg 840
aagaaaaaat tatatcacat ttttatgcct tatggctgtg agtctggctt ggcttgctgc 900
tgagtgtgga gttggtattg ctgtttttgt tcgttgcttc actgacaaga cagtcataga 960
agatcagatt ggggaaaagc tgggttacgg tctttcacgt gcaccctttg caaccattgt 1020
agccctggct acagctcttt caatgcttgc ttcagtacca ctaggagaac tatttttctt 1080
ccatatgata ttaatccgga agggcataac aacttatgaa tatgttgtgg ccatgagggc 1140
tcagagtgaa cctcctggac cttctgtcaa tgatgaccaa caaagcttgg catcttctcc 1200
aatgagttct gcaccaacag cttttagtgg gagctcgttt gcacggcact acaaaggcgc 1260
atggtgcact cccccacgta tcttcattga ccaggacgaa atcatcccac acctggagcc 1320
tggccgagtt ccttcaactg ttgatcctga taccacagac ccaatggaaa gaaccaaaac 1380
ccatcctaaa cggccagtcc gcatcagtgc gtggaagctt gcgaagctcg attcaaatga 1440
ggccatgaaa gcagcagcga aagccagagc ctcttcgtcc gtgctgaaac cgatcaatac 1500
tcgcaaccag tatgaggcag acagtgatag cctcagtagc agaaacagtg tcataagtgc 1560
tgataccgga caccataggt accctcggtc ttgcgggaat tcgcagtaca agccctctta 1620
tccaccaagc agagcaagtg cagatgacat cgagttgtac ccccagacac cgagcagctt 1680
ccagagcaac tcacggactt caactcccat agccgagcac catccatcca agcatttcaa 1740
cccaatctac cagacatcag caaacaggtc tccattctca gcaaaagcaa gtgtcagtga 1800
agctcctgtc tctgaaatca ccaatgcagg gaggtcctac cccccaccac aagcggacag 1860
atcctcccgg tcatctgttt actgggatca ggaagctggc aggttcgtgt ctgctcaggc 1920
aaaccaggga tctagctctc gccctgccta tcctgatctt ctatacaccg ggcagaatat 1980
attctttggt ggacctctga ttgcagatcc cgcagcaaga agcttcagag atcctggtgg 2040
ctcgagccaa aggtctgccg ggcctcggtc gcaccagctc ccggtgttcg ttcccagtga 2100
cccccagaaa gatcagctgt ccaggttacc gtgaaatgct atatcttttt tttttggggg 2160
gggggggggg gtagtagtct tccgaaacac atgttgccct gccgggttat taatgtatag 2220
ttattagctc ttagacctga cagaatagtt catgactttg tatcgggttc atggaaatta 2280
agacctattt aggcactatc aaccataagc actgaagtta tgg 2323
<210> 4
<211> 614
<212> PRT
<213> 玉米(Zea mays L.)
<400> 4
Met Ala Arg Arg His Gly Trp Gln Leu Pro Ala His Thr Leu Gln Val
1 5 10 15
Val Ala Ile Thr Val Tyr Ser Val Leu Cys Ile Ala Phe Tyr Ala Phe
20 25 30
Phe Ser Pro Phe Leu Gly Lys Asp Leu Tyr Gln Tyr Ile Ala Val Gly
35 40 45
Ile Tyr Ser Phe Leu Ala Leu Ser Val Leu Ile Leu Tyr Val Arg Cys
50 55 60
Thr Ala Ile Asp Pro Ala Asp Ser Gly Ile Leu Ile Ser Met Asp Asp
65 70 75 80
Ile Leu Ile Tyr Lys Ser Glu Ala His Val Asp Thr Gln Asp Glu Ala
85 90 95
Gly Lys Pro Gly Leu Arg Asn Asp Glu Asp Ile Arg Lys His Lys Ser
100 105 110
Cys Phe Gly Arg Val Cys Phe Cys Cys Ala Ile Phe Thr Thr Gly Asp
115 120 125
Cys Arg Gly Gly Asp Glu Ala Asn His Gln Glu Asp Tyr Gly Glu Glu
130 135 140
Ala Leu Phe Cys Thr Leu Cys Asn Ala Glu Val Arg Lys His Ser Lys
145 150 155 160
His Cys Arg Ser Cys Asp Lys Cys Val Asp Gly Phe Asp His His Cys
165 170 175
Arg Trp Leu Asn Asn Cys Val Gly Arg Lys Asn Tyr Ile Thr Phe Leu
180 185 190
Cys Leu Met Ala Val Ser Leu Ala Trp Leu Ala Ala Glu Cys Gly Val
195 200 205
Gly Ile Ala Val Phe Val Arg Cys Phe Thr Asp Lys Thr Val Ile Glu
210 215 220
Asp Gln Ile Gly Glu Lys Leu Gly Tyr Gly Leu Ser Arg Ala Pro Phe
225 230 235 240
Ala Thr Ile Val Ala Leu Ala Thr Ala Leu Ser Met Leu Ala Ser Val
245 250 255
Pro Leu Gly Glu Leu Phe Phe Phe His Met Ile Leu Ile Arg Lys Gly
260 265 270
Ile Thr Thr Tyr Glu Tyr Val Val Ala Met Arg Ala Gln Ser Glu Pro
275 280 285
Pro Gly Pro Ser Val Asn Asp Asp Gln Gln Ser Leu Ala Ser Ser Pro
290 295 300
Met Ser Ser Ala Pro Thr Ala Phe Ser Gly Ser Ser Phe Ala Arg His
305 310 315 320
Tyr Lys Gly Ala Trp Cys Thr Pro Pro Arg Ile Phe Ile Asp Gln Asp
325 330 335
Glu Ile Ile Pro His Leu Glu Pro Gly Arg Val Pro Ser Thr Val Asp
340 345 350
Pro Asp Thr Thr Asp Pro Met Glu Arg Thr Lys Thr His Pro Lys Arg
355 360 365
Pro Val Arg Ile Ser Ala Trp Lys Leu Ala Lys Leu Asp Ser Asn Glu
370 375 380
Ala Met Lys Ala Ala Ala Lys Ala Arg Ala Ser Ser Ser Val Leu Lys
385 390 395 400
Pro Ile Asn Thr Arg Asn Gln Tyr Glu Ala Asp Ser Asp Ser Leu Ser
405 410 415
Ser Arg Asn Ser Val Ile Ser Ala Asp Thr Gly His His Arg Tyr Pro
420 425 430
Arg Ser Cys Gly Asn Ser Gln Tyr Lys Pro Ser Tyr Pro Pro Ser Arg
435 440 445
Ala Ser Ala Asp Asp Ile Glu Leu Tyr Pro Gln Thr Pro Ser Ser Phe
450 455 460
Gln Ser Asn Ser Arg Thr Ser Thr Pro Ile Ala Glu His His Pro Ser
465 470 475 480
Lys His Phe Asn Pro Ile Tyr Gln Thr Ser Ala Asn Arg Ser Pro Phe
485 490 495
Ser Ala Lys Ala Ser Val Ser Glu Ala Pro Val Ser Glu Ile Thr Asn
500 505 510
Ala Gly Arg Ser Tyr Pro Pro Pro Gln Ala Asp Arg Ser Ser Arg Ser
515 520 525
Ser Val Tyr Trp Asp Gln Glu Ala Gly Arg Phe Val Ser Ala Gln Ala
530 535 540
Asn Gln Gly Ser Ser Ser Arg Pro Ala Tyr Pro Asp Leu Leu Tyr Thr
545 550 555 560
Gly Gln Asn Ile Phe Phe Gly Gly Pro Leu Ile Ala Asp Pro Ala Ala
565 570 575
Arg Ser Phe Arg Asp Pro Gly Gly Ser Ser Gln Arg Ser Ala Gly Pro
580 585 590
Arg Ser His Gln Leu Pro Val Phe Val Pro Ser Asp Pro Gln Lys Asp
595 600 605
Gln Leu Ser Arg Leu Pro
610
<210> 5
<211> 537
<212> PRT
<213> 玉米(Zea mays L.)
<400> 5
Met Asp Asp Ile Leu Ile Tyr Lys Ser Glu Ala His Val Asp Thr Gln
1 5 10 15
Asp Glu Ala Gly Lys Pro Gly Leu Arg Asn Asp Glu Asp Ile Arg Lys
20 25 30
His Lys Ser Cys Phe Gly Arg Val Cys Phe Cys Cys Ala Ile Phe Thr
35 40 45
Thr Gly Asp Cys Arg Gly Gly Asp Glu Ala Asn His Gln Glu Asp Tyr
50 55 60
Gly Glu Glu Ala Leu Phe Cys Thr Leu Cys Asn Ala Glu Val Arg Lys
65 70 75 80
His Ser Lys His Cys Arg Ser Cys Asp Lys Cys Val Asp Gly Phe Asp
85 90 95
His His Cys Arg Trp Leu Asn Asn Cys Val Gly Arg Lys Asn Tyr Ile
100 105 110
Thr Phe Leu Cys Leu Met Ala Val Ser Leu Ala Trp Leu Ala Ala Glu
115 120 125
Cys Gly Val Gly Ile Ala Val Phe Val Arg Cys Phe Thr Asp Lys Thr
130 135 140
Val Ile Glu Asp Gln Ile Gly Glu Lys Leu Gly Tyr Gly Leu Ser Arg
145 150 155 160
Ala Pro Phe Ala Thr Ile Val Ala Leu Ala Thr Ala Leu Ser Met Leu
165 170 175
Ala Ser Val Pro Leu Gly Glu Leu Phe Phe Phe His Met Ile Leu Ile
180 185 190
Arg Lys Gly Ile Thr Thr Tyr Glu Tyr Val Val Ala Met Arg Ala Gln
195 200 205
Ser Glu Pro Pro Gly Pro Ser Val Asn Asp Asp Gln Gln Ser Leu Ala
210 215 220
Ser Ser Pro Met Ser Ser Ala Pro Thr Ala Phe Ser Gly Ser Ser Phe
225 230 235 240
Ala Arg His Tyr Lys Gly Ala Trp Cys Thr Pro Pro Arg Ile Phe Ile
245 250 255
Asp Gln Asp Glu Ile Ile Pro His Leu Glu Pro Gly Arg Val Pro Ser
260 265 270
Thr Val Asp Pro Asp Thr Thr Asp Pro Met Glu Arg Thr Lys Thr His
275 280 285
Pro Lys Arg Pro Val Arg Ile Ser Ala Trp Lys Leu Ala Lys Leu Asp
290 295 300
Ser Asn Glu Ala Met Lys Ala Ala Ala Lys Ala Arg Ala Ser Ser Ser
305 310 315 320
Val Leu Lys Pro Ile Asn Thr Arg Asn Gln Tyr Glu Ala Asp Ser Asp
325 330 335
Ser Leu Ser Ser Arg Asn Ser Val Ile Ser Ala Asp Thr Gly His His
340 345 350
Arg Tyr Pro Arg Ser Cys Gly Asn Ser Gln Tyr Lys Pro Ser Tyr Pro
355 360 365
Pro Ser Arg Ala Ser Ala Asp Asp Ile Glu Leu Tyr Pro Gln Thr Pro
370 375 380
Ser Ser Phe Gln Ser Asn Ser Arg Thr Ser Thr Pro Ile Ala Glu His
385 390 395 400
His Pro Ser Lys His Phe Asn Pro Ile Tyr Gln Thr Ser Ala Asn Arg
405 410 415
Ser Pro Phe Ser Ala Lys Ala Ser Val Ser Glu Ala Pro Val Ser Glu
420 425 430
Ile Thr Asn Ala Gly Arg Ser Tyr Pro Pro Pro Gln Ala Asp Arg Ser
435 440 445
Ser Arg Ser Ser Val Tyr Trp Asp Gln Glu Ala Gly Arg Phe Val Ser
450 455 460
Ala Gln Ala Asn Gln Gly Ser Ser Ser Arg Pro Ala Tyr Pro Asp Leu
465 470 475 480
Leu Tyr Thr Gly Gln Asn Ile Phe Phe Gly Gly Pro Leu Ile Ala Asp
485 490 495
Pro Ala Ala Arg Ser Phe Arg Asp Pro Gly Gly Ser Ser Gln Arg Ser
500 505 510
Ala Gly Pro Arg Ser His Gln Leu Pro Val Phe Val Pro Ser Asp Pro
515 520 525
Gln Lys Asp Gln Leu Ser Arg Leu Pro
530 535
<210> 6
<211> 19
<212> DNA
<213> 玉米(Zea mays L.)
<400> 6
gggctcagag tgaacctcc 19
<210> 7
<211> 102
<212> RNA
<213> unkown
<400> 7
gggcucagag ugaaccuccg uuuuagagcu agaaauagca aguuaaaaua aggcuagucc 60
guuaucaacu ugaaaaagug gcaccgaguc ggugcuuuuu uu 102
<210> 8
<211> 18
<212> DNA
<213> unkown
<400> 8
gggctcagag tgaactcc 18
Claims (6)
1.ZKM76基因在降低玉米株高和穗位高性状中的应用，其特征在于：所述应用为ZKM76基因SEQ ID NO. 1所示序列第8810位C碱基缺失。
2.一种降低玉米株高和穗位高的方法，其特征在于：抑制玉米中SEQ ID NO. 1所示序列基因编码蛋白的表达，选择玉米株高和穗位高降低的植株。
3.根据权利要求2所述降低玉米株高和穗位高的方法，其特征在于：所述抑制蛋白表达的方法包括基因编辑、RNA干扰或T-DNA插入中任一种。
4.根据权利要求3所述降低玉米株高和穗位高的方法，其特征在于：所述基因编辑采用CRISPR/Cas9方法。
5.根据权利要求4所述降低玉米株高和穗位高的方法，其特征在于：所述CRISPR/Cas9方法在玉米中的基因组靶标区域的DNA序列如SEQ ID NO. 6所示。
6.一种用于降低玉米株高和穗位高的试剂盒，其特征在于：包括如下任一种：
（1）sgRNA分子，其序列如SEQ ID NO. 7所示；
（2）编码（1）所述sgRNA的DNA分子；
（3）表达（1）所述sgRNA的载体。
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| CN103451200A (zh) * | 2012-05-29 | 2013-12-18 | 华中农业大学 | 一种控制玉米株高的分子标记及应用 |
| CN109206494A (zh) * | 2018-10-29 | 2019-01-15 | 中国农业大学 | ZmRPH1基因在调控植物株高及抗倒伏能力中的应用 |
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| CN103451200A (zh) * | 2012-05-29 | 2013-12-18 | 华中农业大学 | 一种控制玉米株高的分子标记及应用 |
| CN109206494A (zh) * | 2018-10-29 | 2019-01-15 | 中国农业大学 | ZmRPH1基因在调控植物株高及抗倒伏能力中的应用 |
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