CN110903368B - 一种控制玉米雌性性状的基因以及用于创制玉米雌性不育系的试剂盒、突变基因型和方法 - Google Patents
一种控制玉米雌性性状的基因以及用于创制玉米雌性不育系的试剂盒、突变基因型和方法 Download PDFInfo
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Abstract
本发明公开了一种控制玉米雌性性状的基因、用于创制玉米雌性不育系的试剂盒、突变基因型及方法。本发明提供了Zm00001d000204基因核酸和氨基酸序列以及利用CRISPR‑Cas9方法编辑Zm00001d000204基因序列，实现玉米雌性不育创制的方法，同时公开了编辑后具有雌性不育性状的玉米基因型序列。本发明通过利用CRISPR/Cas9方法突变玉米硫酸盐转运蛋白编码基因Zm00001d000204，发现了Zm00001d000204基因对玉米雌性性状的调控功能。利用CRISPR/Cas9基因编辑的方法，可以创制玉米雌性不育系，从而用于玉米杂交制种并提高制种效率。本发明还提供了具有雌性不育性状的Zm00001d000204突变基因型，这些突变基因型序列能够造成玉米雌性不育，可以用来创制新的雌性不育系。
Description
技术领域
本发明属于分子遗传学领域，具体涉及一种控制玉米雌性性状的基因，创制玉米雌性不育系的方法、试剂盒、突变基因型。本发明提供了利用CRISPR-Cas9编辑Zm00001d000204基因序列创制玉米雌性不育系的方法，同时公开了编辑后具有玉米雌性不育性状的基因型序列。
背景技术
育性是作物的关键农艺性状,其发生机理和遗传基础是发育生物学的研究热点。不育可分为雄性不育和雌性不育。雌性不育由于性状难鉴定、突变体难获得及内部机制复杂等原因,其研究远远滞后于雄性不育。
雌性不育在遗传育种和杂交制种方面具有很大的应用价值。近年来，水稻中的一些雌性育性基因被相继发现(高荣村，姜程曦，魏晓星，等.水稻雌性不育突变体研究进展及应用展望[J].浙江农业科学，2009，(5)：853-855.云南农业大学.雌性不育基因FST用于杂交水稻育种的方法：CN200910094988.1[P].2010-03-03.；北京大学.水稻雌性不育基因及其在杂交水稻制种中的应用：CN201210096970.7[P].2012-10-17.)。利用水稻雌性不育系、水稻雌性可育基因以及花粉失活和荧光筛选标记基因可以开发适合大规模机械化制种的杂交水稻组合(湖南杂交水稻研究中心.一种利用基因组编辑技术快速创制适合机械化制种的工程雌性不育系的方法：CN201811388237.6[P].2019-03-19.)。
然而，玉米中控制雌性发育的基因以及关于雌性不育的基因表达与调控机制还不清楚，相关的雌性不育系也较少，这严重制约了玉米杂交制种的效率和应用范围。因此，玉米雌性育性相关基因和人工创制玉米雌性不育系的方法具有很大的应用前景。
发明内容
针对现有技术的不足，本发明的目的是提供一种控制玉米雌性性状的基因、用于创制玉米雌性不育系的试剂盒、突变基因型及方法，可以用来创制玉米雌性不育系，从而应用于玉米杂交制种。
为实现上述目的，本发明提供了一种蛋白质，其特征在于：所述蛋白质的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示；或SEQ ID NO.1或SEQ ID NO.2所示的序列经过取代和/或缺失和/或添加一个或多个氨基酸，且具有与SEQ ID NO.1或SEQ ID NO.2所示的序列相同功能的序列。一般可以预见这些来自不同植物或不同玉米材料中的同源蛋白具有相同或者相似的功能，因此同样可以利用这些基因改良植物的农艺性状。进一步，即使不能预见这些蛋白的功能，本领域的一般技术人员可以根据本发明提供的方法和现有技术测定它们是否具有控制植物雌性育性的功能。
在另一方面，本发明还提供了一种核酸，其特征在于：所述核酸编码上述的蛋白；在一些实施方案中，上述核酸的序列为SEQ ID NO.3-SEQ ID NO.6所示。
在另一方面，本发明还提供了一种创制玉米雌性不育系的方法，其特征在于：抑制玉米中上述蛋白的表达和/或活性，选择玉米雌性不育的植株。
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序列为SEQ ID NO.7或SEQ ID NO.8所示。
更进一步地，本发明还提供了一种用于创制玉米雌性不育系的试剂盒，其特征在于：包括如下任一种：
(1)sgRNA分子，其序列如SEQ ID NO.9或SEQ ID NO.10所示；
(2)所述sgRNA的编码DNA分子；
(3)表达所述sgRNA的载体。
应用上述方法或上述试剂盒能够在各种玉米材料甚至是其他植物材料中人工创制雌性不育系。
在另一方面，本发明还提供了一种玉米雌性不育突变基因型，基因型序列如SEQID NO.11-SEQ ID NO.15任意一个所示。上述基因型序列可以通过有性杂交的方式导入不同遗传背景的玉米材料中，从而创制新的玉米雌性不育系。
本发明的优点及有益效果如下：Zm00001d000204基因及编码的蛋白调控玉米雌性育性是之前没有报道过的。本发明通过利用CRISPR/Cas9方法突变玉米基因Zm00001d000204，发现了Zm00001d000204基因对玉米雌性性状的调控功能。利用CRISPR/Cas9基因编辑的方法和编辑后的突变基因型序列，可以创制玉米雌性不育系，从而可以应用于玉米杂交制种。
附图说明
图1为本发明p000204_1F表达载体的物理图谱。其中，各元件英文及各缩写含义列举如下：
RB T-DNA repeat T-DNA右边界重复序列
M13 fwd M13引物序列(正向)
p000204_1F 靶标gRNA序列
Ubi promoter 泛素启动子
3×FLAG 标签序列
SV40NLS 猿猴病毒40核定位信号
Cas9 cas9基因序列
Nucleoplasm in NLS 核定位信号
NOS terminator 胭脂碱合成酶终止子
lac promoter 乳糖启动子
M13 rev M13引物序列(反向)
lac operator 乳糖操纵子
CAP biding site CAP结合位点
CaMV35S promoter(enhanced) 增强的花椰菜花叶病毒35S启动子
BlpR 编码Bar蛋白赋予植物具有草铵膦耐性
CaMV35S polyA single 花椰菜花叶病毒35S多聚腺苷酸序列
LB T-DNA repeat T-DNA左边界重复序列
Kan R 卡那霉素抗性序列
Ori 起始区序列
Bom 骨架区序列
pVS1 RepA pVS1复制子
pVS1 StaA pVS1转录起始区
图2为本发明靶点1基因编辑植株的表型。
从左往右依次是：000204-01，000204-03，000204-05，000204-06，000204-04阳性转化体未编辑和野生型对照。照片摄于授粉后40天。
图3为本发明靶点2基因编辑植株的表型。
从左往右依次是：000204-2，000204-07阳性转化体未编辑和野生型对照。照片摄于授粉后40天。
图4为本发明24个中国玉米自交系材料和B73基因组序列进化树分析。
图5为本发明靶点1(加粗)在不同玉米材料中的序列情况。
图6为本发明靶点2(加粗)在不同玉米材料中的序列情况。
具体实施方式
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”、“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。
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)。所述杂交探针可以是基因组DNA片段、cDNA片段、RNA片段或其它寡核苷酸，并且可以用诸如32P的可检测基团或其它可检测标志物来标记。因而，例如，可以通过标记基于实施方案序列的合成寡核苷酸制备杂交探针。制备杂交探针和构建cDNA及基因组文库的方法通常为本领域已知。可以在严紧条件下进行所述序列的杂交。如本文所用，术语“严紧条件”或“严紧杂交条件”表示如下条件，即在该条件下，相对于与其它序列杂交，探针将以可检测的更大程度(例如，背景的至少2倍、5倍或10倍)与其靶序列杂交。严紧条件是序列依赖性的并且在不同环境中有所不同。通过控制杂交严紧性和/或控制清洗条件，可以鉴定与所述探针100％互补的靶序列(同源探针法)。可选择地，可以调节严紧条件，以允许一些序列错配，以便检测较低的相似度(异源探针法)。通常，探针长度少于约1000或500个核苷酸。通常，严紧条件是如下的条件，即在该条件中，盐浓度为pH 7.0至8.3下，少于约1.5M Na离子，通常约0.01M至1.0M Na离子浓度(或其它盐)，并且温度条件为：当用于短探针时(例如10到50个核苷酸)，至少约30℃；当用于长探针时(例如大于50个核苷酸)，至少约60℃。还可以通过添加诸如甲酰胺的去稳定剂来实现严紧条件。示例性的低严紧条件包括37℃下使用30％至35％的甲酰胺缓冲液、1M NaCl、1％SDS(十二烷基硫酸钠)杂交，50℃至55℃下在1×至2×SSC(20×SSC＝3.0MNaCl/0.3M柠檬酸三钠)中清洗。示例性的中度严紧条件包括37℃下在40％至45％甲酰胺、1.0M NaCl、1％SDS中杂交，55℃至60℃下在0.5×至1×SSC中清洗。示例性的高严紧条件包括37℃下在50％甲酰胺、1M NaCl、1％SDS中杂交，60℃至65℃下在0.1×SSC中最后清洗至少约20分钟。任选地，清洗缓冲液可以包含约0.1％至约1％SDS。杂交持续时间通常少于约24小时，通常为约4小时至约12小时。特异性通常依赖杂交后的清洗，关键因素在于最后清洗溶液的离子强度和温度。DNA-DNA杂合体的Tm(热力学熔点)可以近似自Meinkoth andWahl(1984)Anal.Biochem.138:267-284的公式：Tm＝81.5℃+16.6(logM)+0.41(％GC)-0.61(％甲酰胺)-500/L；其中M是一价阳离子的克分子浓度，％GC是DNA中鸟苷和胞嘧啶核苷酸的百分数，“甲酰胺％”是杂交溶液的甲酰胺百分数，而L是杂合体的碱基对长度。Tm是(确定的离子强度和pH下)50％的互补靶序列与完全匹配的探针杂交时的温度。通常将清洗至少进行至达到平衡，并且达到低的杂交背景水平，诸如进行2小时、1小时或30分钟。每1％的错配对应使Tm降低约1℃；因而，可以调节Tm、杂交和/或清洗条件，从而与所需一致性的序列杂交。例如，如果需要≥90％一致性的序列，可以将Tm降低10℃。通常，将严紧条件选择为比确定离子强度和pH下的特异序列及其互补序列的Tm低约5℃。然而，在非常严紧的条件下，可以在比所述Tm低4℃下进行杂交和/或清洗；在中度严紧条件下，可以在比所述Tm低6℃下进行杂交和/或清洗；在低严紧条件下，可以在比所述Tm低11℃下进行杂交和/或清洗。
术语“性状”是指植物或特定的植物材料或细胞的生理、形态、生化或物理特性。在一些情况下，此特性对人眼是可见的，诸如种子或植株大小，或者可通过生物化学技术测量，诸如检测种子或叶的蛋白质、淀粉或油含量，或者通过观察代谢或生理过程，例如通过测量对水剥夺或特定盐或糖或氮浓度的耐受性，或者通过观察一个或多个基因的表达水平，或者通过农艺观察结果诸如渗透胁迫耐受性或收率。
“转基因”是指其基因组因异源核酸(诸如重组DNA构建体)的存在而发生改变的任何细胞、细胞系、愈伤组织、组织、植株部分或植株。本文所用的术语“转基因”包括那些最初的转基因事件以及从最初的转基因事件通过有性杂交或无性繁殖而产生的那些，并且不涵盖通过常规植物育种方法或通过自然发生的事件(诸如随机异花受精、非重组病毒感染、非重组细菌转化、非重组转座或自发突变)进行的基因组(染色体或染色体外)改变。
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。
阴性或对照植物可以包括，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。
以下结合附图和具体实施方式对本发明作进一步地详细描述。
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(Sambrook J&Russell DW,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。
实施例一 玉米Zm00001d000204基因序列和gRNA序列
在maizeGDB数据库(https://www.maizegdb.org/)中，查询到玉米Zm00001d000204(GRMZM2G444801)基因，在B73中的CDS核酸序列见SEQ ID NO.3所示，基因功能注释为硫酸盐转运蛋白(sulfate transporter，sfp5)，其编码蛋白包含681个氨基酸，序列如SEQ ID NO.1所示。
进一步查询了数据库中其他玉米材料(DK105、Mo17、EP1)以及自主选育的玉米材料KN5585中该基因的序列信息，发现该基因编码的氨基酸序列在这4份玉米材料中与B73的相似度极高，均编码681个氨基酸。其中，Mo17、DK105中的蛋白序列与B73相同，KN5585、EP1中的序列与B73相比有一个P31A的变异，序列如SEQ ID NO.2所示。
该基因的CDS核酸序列在这4份玉米材料中与B73的相似度也极高，其中DK105中的序列与B73相同，KN5585、EP1、Mo17序列分别如SEQ ID NO.4-SEQ ID NO.6所示，与B73中的基因序列(SEQ ID NO.3)相似度达到99％。
通常，硫酸盐转运蛋白在硫酸盐运输过程中具有功能。由于该基因在玉米中的实际功能还没有公开的研究资料。为了明确基因在玉米中的功能，本发明拟采用CRISPR/Cas9基因编辑方法突变Zm00001d000204基因序列，敲除该基因在玉米中的功能。
本发明选取玉米自交系KN5585(由未米生物科技(江苏)有限公司选育的自交系)作为基因编辑的受体。本发明分别选取基因保守区的SEQ ID NO.7和SEQ ID NO.8所示序列作为CRISPR/Cas9基因编辑的靶标区域，合成gRNA序列(序列见SEQ ID NO.9和SEQ IDNO.10)。
实施例二 玉米Zm00001d000204基因的功能以及利用CRISPR/Cas9方法创制玉米雌性不育系
1、CRISPR/Cas9基因编辑载体的构建。
本发明的基因编辑载体为GX-CPB-ZmUbi-hspCas9。该载体的基础载体为CPB-ZmUbi-hspCas9。本发明通过Overlap PCR获得U6-sgRNA进而通过同源重组克隆到基础载体中，具体的构建流程如下：
(1)U6启动子的克隆。从B73中克隆U6启动子，U6启动子的序列信息如SEQ IDNO.16所示。
(2)靶标gRNA的设计。将受体材料KN5585基因组序列输入http://cbi.hzau.edu.cn/crispr/进行靶标设计。本发明优选的两个靶标区域的DNA序列如SEQ IDNO.7或SEQ ID NO.8所示。本发明的sgRNA骨架序列通过人工合成得到，其序列信息如SEQID NO.9和SEQ ID NO.10所示。
(3)通过Overlap PCR获得U6-sgRNA。引物对pU6F1/pU6R用于扩增第一个靶标的U6启动子，产物长度为515bp；引物对p0204-1F(3F)/pgRR1用于扩增第一个靶标的sgRNA，产物长度为127bp；引物对pU6F1/gRR1用于进行Overlap PCR第2步扩增(U6-sgRNA)，产物长度为634bp。Overlap PCR第1步分别扩增U6和sgRNA，PCR产物分别稀释50倍后混合作为模板进行Overlap PCR第2步扩增。扩增产物电泳切胶回收并测序确认序列。Overlap PCR体系和条件如下：Overlap PCR第1步的15μL的反应体系如下，模板DNA(U6或sgRNA，≥30ng/μL)：0.5μL，Primer F/R：各1.2μL，灭菌ddH2O：3.7μL，2×phanta max Buffer：7.5μL，dNTP mix：0.6μL，Phanta酶(产品编号：P505-d1/d2/d3)：0.3μL。Overlap PCR第2步的反应体系为30μL体系。U6吸1μL，加49μL ddH2O稀释；sgRNA吸1μL，加49μL ddH2O稀释各吸10μL，混匀。具体如下：混合模板DNA(U6+sgRNA)：1.5μL，Primer F/R：各2.4μL，灭菌ddH2O：6.9μL，2×phanta maxBuffer：15μL，dNTP mix：1.2μL，Phanta酶：0.6μL。Overlap PCR程序如下：(1)94℃5分钟，(2)94℃30秒，(3)62℃35秒，(4)72℃30秒，第(5)步是从(2)步－(4)步循环32次，(6)72℃10分钟，(7)25℃5分钟。载体构建所需的引物序列见表1。
表1 载体构建所需的引物序列
(4)通过重组克隆构建到骨架载体。将CPB-Ubi-hspcas9载体用Hind III消化，回收。通过同源重组将U6-gRNA和载体二者连接。配反应液前保证各Overlap产物浓度接近一致，20μL的同源重组体系如下：Cas Hind III：3μL，T-1F Overlap：1μL，灭菌ddH2O：10μL，5×CE MultiS buffer：4μL，Exnase MultiS(产品编号：C113-01/02)：2μL。
如图1所示为目的基因Zm00001d000204的单靶标gRNA序列000204_1F，标记基因Cas9和bar与骨架载体pCAMBIA构建的表达载体p000204_1F的物理图谱。单靶标gRNA序列000204_2F构建的表达载体p000204_2F的物理图谱除gRNA序列有差异外其余与p000204_1F相同。
2、农杆菌介导的玉米遗传转化和转化体的创制
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，管中余下玉米胚加入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/LBialaphos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中，25℃，5000lx，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、CRISPR/Cas9的突变结果检测
本项研究采用CTAB法提取玉米叶片DNA，具体方法如下：称取约0.1g叶片，放入离心管，加入600μL CTAB提取缓冲液，5μL RNase A，震荡分散，65℃水浴0.5hr，其间轻摇2-3次；加入等体积氯仿/Tris-饱和酚(1:1，v/v)，混匀，轻摇10min；4℃10000rpm离心20min；转移上清至新管，加入1/10体积的3M乙酸钠(pH值5.2)、0.6-1倍体积的冷异丙醇；轻摇混匀，至絮状沉淀出现；4℃10000rpm离心10min；弃去上清，用体积百分含量70％乙醇洗沉淀2次；风干，加入50μL 1×TE溶解沉淀，-20℃保存。用Nanodrop 2000检测DNA浓度，稀释至10ng/L用作PCR模板。
根据Zm00001d000204基因序列设计PCR引物。
(1)检测靶标：000204_1F；产物大小：999bp；引物序列为：
000204_1F:5’-GCCCAGAATATAGGCAGGCA-3’；
000204_1R:5’-GCCAAATCGTAAGCTCAACCT-3’。
(2)检测靶标：000204_2F；产物大小：899bp；引物序列为：
000204_2F:5’-GACCTTTGCGTCCATCAAC-3’；
000204_2R:5’-CTCCTGTTCATTCGTAACATGCA-3’。
提取基因组DNA，按照以下PCR参数进行扩增：
反应体系：15μL MIX常规PCR体系，1μL forward primer，1μL reverse primer,2μL DNA，3.5μL H2O，7.5μL vazyme，2×taq mix。
反应程序：常规PCR：58℃退火、延伸1分钟、35轮循环。
通过测序分析10个转化体靶标区域的DNA序列，明确靶标区域是否发生编辑突变。其中有5个转化体靶标区域序列发生了变化，编辑前后的序列如表2所示。000204-01，000204-03，000204-05，000204-06在靶标1区域发生缺失突变或单碱基插入突变，而000204-2在靶标2的DNA区域发生缺失突变。
表2 5个编辑转化体靶标区域DNA突变后的序列
下划线标注靶标序列，“-”表示碱基缺失，粗体表示碱基插入。
对5个阳性转化体的编辑后的氨基酸序列进行Clustal比对发现，与野生型和未发生编辑的WT相比，突变后的品系000204-01，000204-03，000204-05，000204-06其编码的核苷酸，在靶标1区域由于碱基的缺失或插入均发生了移码突变，并在其后发生氨基酸提前终止；突变后的品系000204-02靶标2区域的缺失使其氨基酸发生移码突变，且随后氨基酸发生提前终止。因此这些转化体中Zm00001d000204蛋白的功能均表现缺失。
4、基因编辑转化体的表型分析
选取基因序列发生变化且导致移码突变的植株进行表型观察，发现发生基因编辑的植株自交和开放授粉下均无法正常结实(生长地点：华中农业大学温室)，编辑植株的表型照片见图2和图3，这表明该基因控制玉米的雌性性状，基因编辑创制的突变体为雌性不育突变体。将突变体花粉给野生型玉米授粉，野生型玉米可以正常结实，表明雄穗育性不受影响。以000204-02品系为例，收获F1并播种，抽穗后均可正常散粉，套袋自交可正常结实，单穗收获F2种子。取一个单穗自交的F2种子穗行播种，自然授粉鉴定育性，总计28株，其中20株正常结实，8株不结实，基本符合3:1分离，表明该不育性状由单个隐性基因控制。
实施例三 玉米Zm00001d000204基因在不同玉米自交系中的序列以及在不同遗传背景下创制玉米雌性不育系
1、将突变基因型通过回交转育的方式导入其他玉米材料中。
本发明获得的突变体及上述突变基因的功能标记(SEQ ID NO.3-SEQ ID NO.8)可用于各种分子标记辅助选择的方法，将突变基因型通过杂交回交的方式转育到其它玉米遗传背景中，大概的流程为：
杂交：以突变株为父本，与受体玉米材料为母本杂交获得F1种子；第一轮回交后获得F1，播种后获得F1植株，将F1植株与轮回亲本进行杂交，获得BC1种子；BC1不育基因选择(前景选择)：播种BC1种子，获得不少于500株幼苗，在幼苗期采集各单株叶片，按实施例二所述方法提取DNA，利用相应引物对进行扩增，电泳和测序，选取基因型为杂合的单株继续种植，弃去纯合野生型的单株；
BC1背景选择：采用一组(例如100个，或200个等)在突变体和轮回亲本之间存在多态性的，且在基因组上均匀分布的分子标记(包括但不限于SSR、INDEL、SNP、EST、RFLP、AFLP、RAPD、SCAR等类型标记)，对从中选出的单株进行鉴定，选取与轮回亲本相似度高(例如大于88％相似度，或2％中选率等)的材料；后续每次回交均可以采用相同的方法进行分子标记辅助选择，。最终选出100％背景纯合的单株。如果中选单株的基因型为纯合突变体，则该单株为最终的目标材料，可进一步与轮回亲本或与其它玉米材料杂交保存材料。如果中选单株是杂合基因型，可直接用于保存种质，或通过自交获得不育株用于杂交育种或制种。
2、直接突变其他玉米材料中的Zm00001d000204基因。
本发明通过基因组重测序的方法获得了24个玉米骨干自交系中的Zm00001d000204基因组序列(Chr9_20343120...20348157区间)。这24个玉米骨干自交系分别为5237、E28、Q1261、昌7-2、丹340、黄C、黄野4、黄早四、天涯4、自330、综3、Lx9801、西502、81515、F349、H21、吉853、冀53、旅28、原辅黄、双741、K12、农系110和综31，通过Clustal比对分析，发现该基因在各玉米材料中相对保守，序列变化不大(图4)。此外，设计的两个基因编辑靶标位于序列的保守区(图5、图6)，可以利用这两个靶标以实施例二的方法编辑不同玉米材料中的Zm00001d000204基因，从而创制不同遗传背景的玉米雌性不育系。
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> 华中农业大学，未米生物科技（江苏）有限公司
<120> 一种控制玉米雌性性状的基因以及用于创制玉米雌性不育系的试剂盒、突变基因型和方法
<130> 1
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 681
<212> PRT
<213> 玉米(Zea mays L.)
<400> 1
Met Val Val Asn Asn Lys Val Asp Ser Leu Ser Tyr Asp Val Glu Ala
1 5 10 15
Pro Pro Pro Ser Ala Ala Gly Ala Ala Ala Gly Gln Ala Pro Pro Pro
20 25 30
Ala Ser Pro Ala Pro Ala Pro Ala Pro Ala Thr His Ala Pro Val Thr
35 40 45
Arg Glu Gly Gly Ala Ala Ser Val Leu Glu Leu His Lys Val Ser Leu
50 55 60
Pro Glu Arg Arg Thr Thr Ala Lys Ala Leu Arg Gln Arg Leu Ala Glu
65 70 75 80
Val Phe Phe Pro Asp Asp Pro Leu His Gln Phe Lys Asn Gln Ser Ser
85 90 95
Ala Arg Arg Leu Val Leu Ala Leu His Tyr Phe Phe Pro Ile Phe Gln
100 105 110
Trp Gly Ser Ala Tyr Ser Pro Arg Leu Leu Arg Ser Asp Leu Val Ala
115 120 125
Gly Leu Thr Ile Ala Ser Leu Ala Ile Pro Gln Gly Ile Ser Tyr Ala
130 135 140
Lys Leu Ala Asn Leu Pro Pro Ile Val Gly Leu Tyr Ser Ser Phe Val
145 150 155 160
Pro Pro Leu Ile Tyr Ala Leu Leu Gly Ser Ser Arg Asp Leu Ala Val
165 170 175
Gly Pro Val Ser Ile Ala Ser Leu Val Met Gly Ser Met Leu Arg Asp
180 185 190
Ala Val Ser Pro Asp Glu Gln Pro Leu Leu Tyr Leu Gln Leu Ala Phe
195 200 205
Thr Ala Thr Phe Phe Ala Gly Val Phe Gln Ala Ser Leu Gly Phe Leu
210 215 220
Arg Leu Gly Phe Ile Val Asp Phe Leu Ser Lys Ala Thr Leu Thr Gly
225 230 235 240
Phe Met Gly Gly Ala Ala Val Ile Val Ser Leu Gln Gln Leu Lys Gly
245 250 255
Leu Leu Gly Ile Ser His Phe Thr Ser His Met Gly Phe Leu Asp Val
260 265 270
Met Arg Ser Val Val Asn Arg His Asp Glu Trp Lys Trp Gln Thr Ile
275 280 285
Val Met Gly Ser Ala Phe Leu Ala Ile Leu Leu Leu Thr Arg Gln Ile
290 295 300
Ser Ala Arg Asn Pro Lys Leu Phe Trp Val Ser Ala Gly Ala Pro Leu
305 310 315 320
Ala Ser Val Ile Ile Ser Thr Ile Leu Ser Phe Ile Trp Lys Ser Pro
325 330 335
Ser Ile Ser Val Ile Gly Ile Leu Pro Arg Gly Val Asn Pro Pro Ser
340 345 350
Ala Asn Met Leu Ser Phe Ser Gly Ser Tyr Val Ala Leu Thr Ile Lys
355 360 365
Thr Gly Ile Met Thr Gly Ile Leu Ser Leu Thr Glu Gly Ile Ala Val
370 375 380
Gly Arg Thr Phe Ala Ser Ile Asn Asn Tyr Gln Val Asp Gly Asn Lys
385 390 395 400
Glu Met Met Ala Ile Gly Leu Met Asn Met Ala Gly Ser Cys Ala Ser
405 410 415
Cys Tyr Val Thr Thr Gly Ser Phe Ser Arg Ser Ala Val Asn Tyr Ser
420 425 430
Ala Gly Cys Arg Thr Ala Leu Ser Asn Val Val Met Ala Ala Ala Val
435 440 445
Leu Val Thr Leu Leu Phe Leu Met Pro Leu Phe His Tyr Thr Pro Asn
450 455 460
Val Ile Leu Ala Ala Ile Ile Ile Thr Ala Val Val Gly Leu Val Asp
465 470 475 480
Val Arg Gly Ala Ala Arg Leu Trp Lys Val Asp Lys Leu Asp Phe Leu
485 490 495
Ala Cys Val Ala Ala Phe Leu Gly Val Leu Leu Val Ser Val Gln Thr
500 505 510
Gly Leu Gly Val Ala Val Gly Ile Ser Leu Phe Lys Val Leu Leu Gln
515 520 525
Val Thr Arg Pro Asn Val Val Val Glu Gly Leu Val Pro Gly Thr Gln
530 535 540
Ser Tyr Arg Ser Val Ala Gln Tyr Arg Glu Ala Val Arg Val Pro Gly
545 550 555 560
Phe Leu Val Val Gly Val Glu Ser Ala Val Tyr Phe Ala Asn Ser Met
565 570 575
Tyr Leu Val Glu Arg Val Met Arg Tyr Leu Arg Asp Glu Glu Glu Arg
580 585 590
Ala Leu Lys Ser Asn His Pro Ser Ile Arg Cys Val Val Leu Asp Met
595 600 605
Gly Ala Val Ala Ala Ile Asp Thr Ser Gly Leu Asp Ala Leu Ser Glu
610 615 620
Leu Lys Lys Val Leu Asp Lys Arg Asn Ile Glu Leu Val Leu Ala Asn
625 630 635 640
Pro Val Gly Ser Val Ala Glu Arg Met Phe Asn Ser Ala Val Gly Glu
645 650 655
Ser Phe Gly Ser Gly Arg Leu Phe Phe Ser Val Ala Glu Ala Val Ala
660 665 670
Ala Gly Ala Cys Lys Ala Ala Gln Pro
675 680
<210> 2
<211> 681
<212> PRT
<213> 玉米(Zea mays L.)
<400> 2
Met Val Val Asn Asn Lys Val Asp Ser Leu Ser Tyr Asp Val Glu Ala
1 5 10 15
Pro Pro Pro Ser Ala Ala Gly Ala Ala Ala Gly Gln Ala Pro Ala Pro
20 25 30
Ala Ser Pro Ala Pro Ala Pro Ala Pro Ala Thr His Ala Pro Val Thr
35 40 45
Arg Glu Gly Gly Ala Ala Ser Val Leu Glu Leu His Lys Val Ser Leu
50 55 60
Pro Glu Arg Arg Thr Thr Ala Lys Ala Leu Arg Gln Arg Leu Ala Glu
65 70 75 80
Val Phe Phe Pro Asp Asp Pro Leu His Gln Phe Lys Asn Gln Ser Ser
85 90 95
Ala Arg Arg Leu Val Leu Ala Leu His Tyr Phe Phe Pro Ile Phe Gln
100 105 110
Trp Gly Ser Ala Tyr Ser Pro Arg Leu Leu Arg Ser Asp Leu Val Ala
115 120 125
Gly Leu Thr Ile Ala Ser Leu Ala Ile Pro Gln Gly Ile Ser Tyr Ala
130 135 140
Lys Leu Ala Asn Leu Pro Pro Ile Val Gly Leu Tyr Ser Ser Phe Val
145 150 155 160
Pro Pro Leu Ile Tyr Ala Leu Leu Gly Ser Ser Arg Asp Leu Ala Val
165 170 175
Gly Pro Val Ser Ile Ala Ser Leu Val Met Gly Ser Met Leu Arg Asp
180 185 190
Ala Val Ser Pro Asp Glu Gln Pro Leu Leu Tyr Leu Gln Leu Ala Phe
195 200 205
Thr Ala Thr Phe Phe Ala Gly Val Phe Gln Ala Ser Leu Gly Phe Leu
210 215 220
Arg Leu Gly Phe Ile Val Asp Phe Leu Ser Lys Ala Thr Leu Thr Gly
225 230 235 240
Phe Met Gly Gly Ala Ala Val Ile Val Ser Leu Gln Gln Leu Lys Gly
245 250 255
Leu Leu Gly Ile Ser His Phe Thr Ser His Met Gly Phe Leu Asp Val
260 265 270
Met Arg Ser Val Val Asn Arg His Asp Glu Trp Lys Trp Gln Thr Ile
275 280 285
Val Met Gly Ser Ala Phe Leu Ala Ile Leu Leu Leu Thr Arg Gln Ile
290 295 300
Ser Ala Arg Asn Pro Lys Leu Phe Trp Val Ser Ala Gly Ala Pro Leu
305 310 315 320
Ala Ser Val Ile Ile Ser Thr Ile Leu Ser Phe Ile Trp Lys Ser Pro
325 330 335
Ser Ile Ser Val Ile Gly Ile Leu Pro Arg Gly Val Asn Pro Pro Ser
340 345 350
Ala Asn Met Leu Ser Phe Ser Gly Ser Tyr Val Ala Leu Thr Ile Lys
355 360 365
Thr Gly Ile Met Thr Gly Ile Leu Ser Leu Thr Glu Gly Ile Ala Val
370 375 380
Gly Arg Thr Phe Ala Ser Ile Asn Asn Tyr Gln Val Asp Gly Asn Lys
385 390 395 400
Glu Met Met Ala Ile Gly Leu Met Asn Met Ala Gly Ser Cys Ala Ser
405 410 415
Cys Tyr Val Thr Thr Gly Ser Phe Ser Arg Ser Ala Val Asn Tyr Ser
420 425 430
Ala Gly Cys Arg Thr Ala Leu Ser Asn Val Val Met Ala Ala Ala Val
435 440 445
Leu Val Thr Leu Leu Phe Leu Met Pro Leu Phe His Tyr Thr Pro Asn
450 455 460
Val Ile Leu Ala Ala Ile Ile Ile Thr Ala Val Val Gly Leu Val Asp
465 470 475 480
Val Arg Gly Ala Ala Arg Leu Trp Lys Val Asp Lys Leu Asp Phe Leu
485 490 495
Ala Cys Val Ala Ala Phe Leu Gly Val Leu Leu Val Ser Val Gln Thr
500 505 510
Gly Leu Gly Val Ala Val Gly Ile Ser Leu Phe Lys Val Leu Leu Gln
515 520 525
Val Thr Arg Pro Asn Val Val Val Glu Gly Leu Val Pro Gly Thr Gln
530 535 540
Ser Tyr Arg Ser Val Ala Gln Tyr Arg Glu Ala Val Arg Val Pro Gly
545 550 555 560
Phe Leu Val Val Gly Val Glu Ser Ala Val Tyr Phe Ala Asn Ser Met
565 570 575
Tyr Leu Val Glu Arg Val Met Arg Tyr Leu Arg Asp Glu Glu Glu Arg
580 585 590
Ala Leu Lys Ser Asn His Pro Ser Ile Arg Cys Val Val Leu Asp Met
595 600 605
Gly Ala Val Ala Ala Ile Asp Thr Ser Gly Leu Asp Ala Leu Ser Glu
610 615 620
Leu Lys Lys Val Leu Asp Lys Arg Asn Ile Glu Leu Val Leu Ala Asn
625 630 635 640
Pro Val Gly Ser Val Ala Glu Arg Met Phe Asn Ser Ala Val Gly Glu
645 650 655
Ser Phe Gly Ser Gly Arg Leu Phe Phe Ser Val Ala Glu Ala Val Ala
660 665 670
Ala Gly Ala Cys Lys Ala Ala Gln Pro
675 680
<210> 3
<211> 2046
<212> DNA
<213> unkown
<400> 3
atggtcgtga acaacaaggt ggacagcctg tcgtacgacg tcgaggcgcc gccgccctct 60
gccgccggcg ccgccgcggg gcaagcgcca ccgccggcgt cgccagctcc ggcgccggca 120
ccggcaaccc acgcgcccgt gacgcgggaa ggcggcgcgg cgtcggtgct ggagctgcac 180
aaggtgtcgc tgccggagcg gcggacgacg gcgaaggcgc tgcggcagcg cctggccgag 240
gtgttcttcc cggacgaccc gctgcaccag ttcaagaacc agtcgtcggc gcggcgcctc 300
gtgctggcgc tgcactactt cttccccatc ttccagtggg ggtccgccta cagcccgcgc 360
ctcctgcgct ccgacctcgt cgccggcctc accattgcca gcctcgccat cccgcaggga 420
atcagctacg ccaagctcgc caacctgccg ccaatcgttg gcctatattc cagcttcgtg 480
ccgccgctca tctacgcgct gctggggagc tcgcgggacc tggcggtggg gccggtgtcc 540
atcgcgtcgc tggtgatggg gtccatgctc cgggacgccg tgtcgccgga cgagcagccg 600
ctcctctacc tgcagctggc cttcaccgcc accttcttcg ccggcgtctt ccaggcgtcc 660
ctgggattcc tcaggctggg cttcatcgtg gacttcctgt ccaaggcgac gctgacgggc 720
ttcatgggcg gcgccgccgt catcgtgtcg ctgcagcagc tcaagggcct gctcggcatc 780
tcccacttca cctcccacat gggattcctc gacgtcatgc gctccgtcgt caaccgccac 840
gacgagtgga agtggcagac gatcgtcatg ggctccgcct tcctcgccat cctcctcctc 900
acgcgccaaa tcagcgccag gaatccaaag cttttctggg tatcagcagg tgctcccctg 960
gcgtcggtga tcatctccac catcctctcc ttcatctgga aatcccccag catcagtgtt 1020
atcggcatcc tccccagggg agtgaaccct ccttcggcga acatgctcag cttcagcggc 1080
tcctacgtgg cgctgacgat caaaaccggg atcatgacag gcatcctgtc cttaacagaa 1140
gggatcgcag tgggcaggac ctttgcgtcc atcaacaact accaggtgga cgggaacaag 1200
gagatgatgg cgatcgggct gatgaacatg gcgggctcct gcgcctcctg ctacgtgacg 1260
acggggtcct tctcccggtc ggcggtgaac tacagcgcgg gctgcaggac ggcgttgtcc 1320
aacgtcgtga tggcggcggc ggtgctggtg acgctgctgt tcctcatgcc gctgttccac 1380
tacaccccga acgtgatcct ggcggcgatc atcatcacgg cggtggtggg gctggtggac 1440
gtgcgcggcg ccgccaggct gtggaaggtg gacaagctgg acttcctggc gtgcgtggcg 1500
gcgttcctcg gcgtgctgct ggtgtccgtg cagacgggcc tgggcgtcgc cgtcggcatc 1560
tcgctcttca aggtcctgct gcaggtcacc cgccccaacg tcgtggtgga gggcctcgtc 1620
ccggggacgc agagctaccg cagcgtggcg cagtaccgcg aggccgtccg cgtgccgggc 1680
ttcctcgtcg tcggcgtcga gtccgccgtc tacttcgcca actccatgta cctggtggag 1740
cgggtcatgc gctacctccg cgacgaggag gagcgcgcgc tcaagtccaa ccacccctcc 1800
atccgatgcg tcgtcctcga catgggcgcc gtcgcggcga tcgacaccag cggtctagac 1860
gcgctgtccg agctcaagaa agtcctggac aaaagaaaca tcgagctggt gcttgccaac 1920
ccggtggggt cggtggcgga gaggatgttc aactcggcgg tgggcgagag cttcgggtcg 1980
ggccgcctct tcttcagcgt agcggaggcc gtcgcggcgg gggcgtgcaa agcggcgcag 2040
ccctga 2046
<210> 4
<211> 2046
<212> DNA
<213> unkown
<400> 4
atggtcgtga acaacaaggt ggacagcctg tcgtacgacg tcgaggcgcc gccgccctct 60
gccgccggcg ccgccgcggg gcaagcgcca gcgccggcgt cgccagcgcc ggcgccggct 120
ccggcaaccc acgcgcccgt gacgcgggag ggcggcgcgg cgtcggtgct ggagctgcac 180
aaggtgtcgc tgccggagcg gcggacgacg gcgaaggcgc tgcggcagcg cctggccgag 240
gttttcttcc cggacgaccc gctgcaccag ttcaagaacc agtcgtcggc gcggcgcctc 300
gtgctggcgc tgcactactt cttccccatc ttccagtggg ggtccgccta cagcccgcgc 360
ctcctgcgct ccgacctcgt cgccggcctc accattgcca gcctcgccat cccgcaggga 420
atcagctacg ccaagctcgc caacctgccg ccaatcgttg gcctatattc cagcttcgtg 480
ccgccgctca tctacgcgct gctggggagc tcgcgggacc tggcggtggg gccggtgtcc 540
atcgcgtcgc tggtgatggg gtccatgctc cgggacgccg tgtcgccgga cgagcagccg 600
ctcctctacc tgcagctggc cttcaccgcc actttcttcg ccggcgtctt ccaggcgtcc 660
ctgggattcc tcaggctggg cttcatcgtg gacttcctgt ccaaggcgac gctgacgggc 720
ttcatgggcg gcgccgccgt catcgtgtcg ctacagcagc tcaagggcct gctcggcatc 780
tcccacttca cctcccacat gggattcctc gacgtcatgc gctccgtcgt caaccgccac 840
gacgagtgga agtggcagac gatcgtcatg ggctccgcct tcctcgccat cctcctcctc 900
acgcgccaaa tcagcgccag gaatccaaag cttttctggg tatcagcagg tgctcccctg 960
gcgtcggtga tcatctccac catcctctcc ttcatctgga aatcccctag catcagtgtt 1020
attggcatcc tccccagggg agtgaaccct ccttcggcga acatgctcag cttcagcggc 1080
tcctacgtgg cgctgacgat caaaaccggg atcatgacag gcatcctgtc cttaacagaa 1140
gggattgcag tgggcaggac cttcgcgtcc atcaacaact accaggtgga cgggaacaag 1200
gagatgatgg cgatcgggct gatgaacatg gcgggctcct gcgcctcctg ctacgtgacg 1260
acggggtcct tctcccggtc ggcggtgaac tacagcgcgg gctgcaggac ggcgctgtcc 1320
aacgtcgtga tggcggcggc ggtgctggtg acgctgctgt tcctcatgcc gctgttccac 1380
tacaccccga acgtgatcct ggcggcgatc atcatcacgg cggtggtggg gctggtggac 1440
gtgcgaggcg ccgccaggct gtggaaggtg gacaagctgg acttcctggc gtgcgtggcg 1500
gcgttcctcg gcgtgctgct ggtgtccgtg cagacgggcc tgggcgtcgc cgtcggcatc 1560
tcgctcttca aggtcctgct gcaggtcacc cgccccaacg tcgtggtgga gggcctcgtc 1620
ccggggacgc agagctaccg cagcgtggcg cagtaccgcg aggccgtccg cgtgccgggc 1680
ttcctcgtcg tcggcgtcga gtccgccgtc tacttcgcca actccatgta cctggtggag 1740
cgggtcatgc gctacctccg cgacgaggag gagcgcgcgc tcaagtccaa ccacccctcc 1800
atccgatgcg tcgtcctcga catgggcgcc gtcgcggcga tcgacacgag cggtctagac 1860
gcgctgtccg agctcaagaa agtcctggac aaaagaaaca tcgagctggt gcttgccaac 1920
ccggtggggt cggtggcgga gaggatgttc aactcggcgg tgggcgagag cttcgggtcg 1980
ggccgcctct tcttcagcgt agcggaggcc gtcgcggcgg gggcgtgcaa agcggcgcag 2040
ccctga 2046
<210> 5
<211> 2046
<212> DNA
<213> unkown
<400> 5
atggtcgtga acaacaaggt ggacagcctg tcgtacgacg tcgaggcgcc gccgccctct 60
gccgccggcg ccgccgcggg gcaagcgcca gcgccggcgt cgccagcgcc ggcgccggct 120
ccggcaaccc acgcgcccgt gacgcgggag ggcggcgcgg cgtcggtgct ggagctgcac 180
aaggtgtcgc tgccggagcg gcggacgacg gcgaaggcgc tgcggcagcg cctggccgag 240
gttttcttcc cggacgaccc gctgcaccag ttcaagaacc agtcgtcggc gcggcgcctc 300
gtgctggcgc tgcactactt cttccccatc ttccagtggg ggtccgccta cagcccgcgc 360
ctcctgcgct ccgacctcgt cgccggcctc accattgcca gcctcgccat cccgcaggga 420
atcagctacg ccaagctcgc caacctgccg ccaatcgttg gcctatattc cagcttcgtg 480
ccgccgctca tctacgcgct gctggggagc tcgcgggacc tggcggtggg gccggtgtcc 540
atcgcgtcgc tggtgatggg gtccatgctc cgggacgccg tgtcgccgga cgagcagccg 600
ctcctctacc tgcagctggc cttcaccgcc accttcttcg ccggcgtctt ccaggcgtcc 660
ctgggattcc tcaggctggg cttcatcgtg gacttcctgt ccaaggcgac gctgacgggc 720
ttcatgggcg gcgccgccgt catcgtgtcg ctgcagcagc tcaagggcct gctcggcatc 780
tcccacttca cctcccacat gggattcctc gacgtcatgc gctccgtcgt caaccgccac 840
gacgagtgga agtggcagac gatcgtcatg ggctccgcct tcctcgccat cctcctcctc 900
acgcgccaaa tcagcgccag gaatccaaag cttttctggg tatcagcagg tgctcccctg 960
gcgtcggtga tcatctccac catcctctcc ttcatctgga aatcccccag catcagtgtt 1020
atcggcatcc tccccagggg agtgaaccct ccttcggcga acatgctcag cttcagcggc 1080
tcctacgtgg cgctgacgat caaaaccggg atcatgacag gcatcctgtc cttaacagaa 1140
gggatcgcag tgggcaggac ctttgcgtcc atcaacaact accaggtgga cgggaacaag 1200
gagatgatgg cgatcgggct gatgaacatg gcgggctcct gcgcctcctg ctacgtgacg 1260
acggggtcct tctcccggtc ggcggtgaac tacagcgcgg gctgcaggac ggcgctgtcc 1320
aacgtcgtga tggcggcggc ggtgctggtg acgctgctgt tcctcatgcc gctgttccac 1380
tacaccccga acgtgatcct ggcggcgatc atcatcacgg cggtggtggg gctggtggac 1440
gtgcgcggcg ccgccaggct gtggaaggtg gacaagctgg acttcctggc gtgcgtggcg 1500
gcgttcctcg gcgtgctgct ggtgtccgtg cagacgggcc tgggcgtcgc cgtcggcatc 1560
tcgctcttca aggtcctgct gcaggtcacc cgccccaacg tcgtggtgga gggcctcgtc 1620
ccggggacgc agagctaccg cagcgtggcg cagtaccgcg aggccgtccg cgtgccgggc 1680
ttcctcgtcg tcggcgtcga gtccgccgtc tacttcgcca actccatgta cttggtggag 1740
cgggtcatgc gctacctccg cgacgaggag gagcgcgcgc tcaagtccaa tcacccctcc 1800
atccgatgcg tcgtcctcga catgggcgcc gtcgcggcga tcgacacgag cggtctagac 1860
gcgctgtccg agctcaagaa agtcctggac aaaagaaaca tcgagctggt gctcgccaac 1920
ccggtggggt cggtggcgga gaggatgttc aactcggcgg tgggcgagag cttcgggtcg 1980
ggccgcctct tcttcagcgt agcggaggcc gtcgcggcgg gggcgtgcaa agcggcgcag 2040
ccctga 2046
<210> 6
<211> 2046
<212> DNA
<213> unkown
<400> 6
atggtcgtga acaacaaggt ggacagcctg tcgtacgacg tcgaggcgcc gccgccctct 60
gccgccggcg ccgccgcggg gcaagcgcca ccgccggcgt cgccagctcc ggcgccggca 120
ccggcaaccc acgcgcccgt gacgcgggaa ggcggcgcgg cgtcggtgct ggagctgcac 180
aaggtgtcgc tgccggagcg gcggacgacg gcgaaggcgc tgcggcagcg cctggccgag 240
gtgttcttcc cggacgaccc gctgcaccag ttcaagaacc agtcgtcggc gcggcgcctc 300
gtgctggcgc tgcactactt cttccccatc ttccagtggg ggtccgccta cagcccgcgc 360
ctcctgcgct ccgacctcgt cgccggcctc accattgcca gcctcgccat cccgcaggga 420
atcagctacg ccaagctcgc caacctgccg ccaatcgttg gcctatattc cagcttcgtg 480
ccgccgctca tctacgcgct gctggggagc tcgcgggacc tggcggtggg gccggtgtcc 540
atcgcgtcgc tggtgatggg gtccatgctc cgggacgccg tgtcgccgga cgagcagccg 600
ctcctctacc tgcagctggc cttcaccgcc accttcttcg ccggcgtctt ccaggcgtcc 660
ctgggattcc tcaggctggg cttcatcgtg gacttcctgt ccaaggcgac gctgacgggc 720
ttcatgggcg gcgccgccgt catcgtgtcg ctgcagcagc tcaagggcct gctcggcatc 780
tcccacttca cctcccacat gggattcctc gacgtcatgc gctccgtcgt caaccgccac 840
gacgagtgga agtggcagac gatcgtcatg ggctccgcct tcctcgccat cctcctcctc 900
acgcgccaaa tcagcgccag gaacccaaag cttttctggg tatcagcagg tgctcccctg 960
gcgtcggtga tcatctccac catcctctcc ttcatctgga aatcccccag catcagtgtt 1020
attggcatcc tccccagggg agtgaaccct ccttcggcga acatgctcag cttcagcggc 1080
tcctatgtgg cgctgacgat caaaaccggg atcatgacag gcatcctgtc cttaacagaa 1140
gggatcgcag tgggcaggac cttcgcgtcc atcaacaact accaggtgga cgggaacaag 1200
gagatgatgg cgatcgggct gatgaacatg gcgggctcct gcgcctcctg ctacgtgacg 1260
acggggtcct tctcccggtc ggcggtgaac tacagcgcgg gctgcaggac ggcgctgtcc 1320
aacgtcgtga tggcggcggc ggtgctggtg acgctgctgt tcctcatgcc gctgttccac 1380
tacaccccga acgtgatcct ggcggcgatc atcatcacgg cggtggtggg gctggtggac 1440
gtgcgcggcg ccgccaggct gtggaaggtg gacaagctgg acttcctggc gtgcgtggcg 1500
gcgttcctcg gcgtgctgct ggtgtccgtg cagacgggcc tgggcgtcgc cgtcggcatc 1560
tcgctcttca aggtcctgct gcaggtcacc cgccccaacg tcgtggtgga gggcctcgtc 1620
ccggggacgc agagctaccg cagcgtggcg cagtaccgcg aggccgtccg cgtgccgggc 1680
ttcctcgtcg tcggcgtcga gtccgccgtc tacttcgcca actccatgta cctggtggag 1740
cgggtcatgc gctacctccg cgacgaggag gagcgcgcgc tcaagtccaa ccacccctcc 1800
atccgatgcg tcgtcctcga catgggcgcc gtcgcggcga tcgacacgag cggtctagac 1860
gcgctgtccg agctcaagaa agtcctggac aaaagaaaca tcgagctggt gcttgccaac 1920
ccggtggggt cggtggcgga gaggatgttc aactcggcgg tgggcgagag cttcgggtcg 1980
ggccgcctct tcttcagcgt agcggaggcc gtcgcggcgg gggcgtgcaa agcggcgcag 2040
ccctga 2046
<210> 7
<211> 20
<212> DNA
<213> 玉米(Zea mays L.)
<400> 7
gactcacata ggccaacgat 20
<210> 8
<211> 20
<212> DNA
<213> 玉米(Zea mays L.)
<400> 8
gaagtggcag acgatcgtca 20
<210> 9
<211> 103
<212> RNA
<213> unkown
<400> 9
gacucacaua ggccaacgau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103
<210> 10
<211> 103
<212> RNA
<213> unkown
<400> 10
gaaguggcag acgaucguca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103
<210> 11
<211> 32
<212> DNA
<213> unkown
<400> 11
gccgccaatc ggttggccta tgtgagtccc tc 32
<210> 12
<211> 32
<212> DNA
<213> unkown
<400> 12
gccgccaatc gtttggccta tgtgagtccc tc 32
<210> 13
<211> 29
<212> DNA
<213> unkown
<400> 13
gccgccaatc tggcctatgt gagtccctc 29
<210> 14
<211> 21
<212> DNA
<213> unkown
<400> 14
gccggcctat gtgagtccct c 21
<210> 15
<211> 22
<212> DNA
<213> unkown
<400> 15
ggcgtgctgc tgtcggcatc tc 22
<210> 16
<211> 500
<212> DNA
<213> 玉米(Zea mays L.)
<400> 16
gctgtttttg ttagccccat cgaatccttg acataatgat cccgcttaaa taagcaacct 60
cgcttgtata gttccttgtg ctctaacaca cgatgatgat aagtcgtaaa atagtggtgt 120
ccaaagaatt tccaggccca gttgtaaaag ctaaaatgct attcgaattt ctactagcag 180
taagtcgtgt ttagaaatta tttttttata tacctttttt ccttctatgt acagtaggac 240
acagtgtcag cgccgcgttg acggagaata tttgcaaaaa agtaaaagag aaagtcatag 300
cggcgtatgt gccaaaaact tcgtcacaga gagggccata agaaacatgg cccacggccc 360
aatacgaagc accgcgacga agcccaaaca gcagtccgta ggtggagcaa agcgctgggt 420
aatacgcaaa cgttttgtcc caccttgact aatcacaaga gtggagcgta ccttataaac 480
cgagccgcaa gcaccgaatt 500
Claims (8)
1.一种蛋白质在控制玉米雌性性状中的应用，其特征在于：所述蛋白质的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.一种核酸在控制玉米雌性性状中的应用，其特征在于：所述核酸编码权利要求1所述的蛋白；所述核酸的序列如SEQ ID NO.3-SEQ ID NO.6所示。
3.一种创制玉米雌性不育系的方法，其特征在于：抑制玉米中权利要求1所述蛋白的表达和/或活性，选择玉米雌性不育的植株。
4.根据权利要求3所述创制玉米雌性不育系的方法，其特征在于：所述抑制蛋白表达和/或活性的方法包括基因编辑、RNA干扰、T-DNA插入中任一种。
5.根据权利要求4所述创制玉米雌性不育系的方法，其特征在于：所述基因编辑采用CRISPR/Cas9方法。
6.根据权利要求5所述创制玉米雌性不育系的方法，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.7或SEQ ID NO.8所示。
7.一种用于创制玉米雌性不育系的试剂盒，其特征在于：包括如下任一种：
(1)sgRNA分子，其序列如SEQ ID NO.9或SEQ ID NO.10所示；
(2)所述sgRNA的编码DNA分子；
(3)表达所述sgRNA的载体。
8.一种玉米雌性不育突变基因，其特征在于：所述突变基因序列为SEQ ID NO.4所示序列第1510-1562位序列突变为SEQ ID NO.15所示。
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| CN113005128B (zh) * | 2021-03-12 | 2023-06-30 | 北京科技大学 | 雄性不育基因ZmMYB84及其在创制玉米雄性不育系中的应用 |
| CN115820720B (zh) * | 2022-07-25 | 2025-03-21 | 海南睿伦科技有限责任公司 | 一种制备玉米雄性不育材料的方法及相关基因 |
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