CN104630234B - 一种水稻穗发育基因rpd与应用 - Google Patents

Abstract

本发明属于分子生物学领域，涉及一种水稻穗发育基因RPD与应用。一种水稻穗发育基因RPD，其开放阅读框序列为：SEQ ID NO.1,其编码区的序列为：SEQ ID NO.2。本发明提供的水稻穗发育基因RPD基因是一个新的水稻穗发育基因，该基因可以调控水稻穗及颖花发育。为水稻育种及水稻穗发育机制研究提供了新的基因资源，可以在调控水稻穗发育方面得到应用。

Description

一种水稻穗发育基因RPD与应用

一、技术领域

本发明属于分子生物学领域，涉及一种水稻穗发育基因RPD与应用。

二、背景技术

水稻是我国最重要的粮食作物，稻米是我国65％以上人口的主食。水稻产量的高低直接影响我国的粮食安全。水稻穗部性状是决定其产量的核心因素之一，水稻穗部性状在产量结构中的作用已越来越得到重视。研究水稻穗发育机制将有助于提高水稻产量(Akter et al.2014)。

近期，由于分子生物学的发展，一批调控穗发育的基因已被克隆。水稻ASP1编码一个转录共抑子，能调控水稻发育过程及生理反应的多个方面，如穗、枝梗、小穗等发育(Yoshida et al.2012)。APO1编码一个F-box蛋白，参与分生组织属性的时间调控，正调节一级枝梗数目和小穗数目(Ikeda et al.2005；Ikeda et al.2007；Ikeda-Kawakatsu etal.2009)。APO2调控水稻从营养生长向生殖生长的转变，控制水稻的抽穗期以及穗部枝梗发育和分蘖的形成(Nagashree et al.2008)。LAX1编码bHLH转录因子，是水稻腋芽原基形成的主要调节因子(Komatsu et al.2003b)。LAX2是一个核蛋白，能与LAX1蛋白互作，并一起参与调控水稻腋生分生组织的形成(Tabuchi et al.2011)。FZP编码包含一个ERF结构域的转录因子，调控水稻小穗的发育(Komatsu et al.2003a；Kato and Horibata 2012)。LP编码一个富含Kelch的F-box蛋白，在调节水稻株型，尤其是改善穗型方面有重要作用(Liet al.2011)。SP1编码一个属于多肽转运蛋白家族的转运蛋白，)，决定水稻穗长(Li etal.2009)。TAW1基因编码一个功能未知的核蛋白，通过提高花序分生组织活性和抑制形成小穗分生组织的相变来调控花序发育(Yoshida et al.2013)。

整个穗子的大小、颖花数目的多少都是影响穗型的重要农艺性状。从这些可能对穗型产生影响的基因入手，不仅可以帮助我们理解这些突变体产生畸变穗形态的原因，而且对于运用分子育种技术定向改造控制穗发育的关键基因而改良穗型具有关键意义(卢寰和时振英，2013)。

三、发明内容

技术问题

本发明目的在于探究影响水稻穗发育基因及其克隆该基因的方法，为人类进一步对水稻穗发育机制完全识别，并运用分子育种技术定向改造控制穗发育的关键基因而改良穗型。

技术方案

一种水稻穗发育基因RPD，其开放阅读框序列为：SEQ ID NO.1。

所述的水稻穗发育基因RPD，其编码区的序列为：SEQ ID NO.2。

所述的水稻穗发育基因RPD编码的蛋白质，其氨基酸序列为：SEQ ID NO.3。

所述的水稻穗发育基因RPD可以在调控水稻穗发育方面得到应用。

所述的水稻穗发育基因RPD编码的蛋白质可以在调控水稻穗发育方面得到应用。

水稻穗发育新基因RPD，其开放阅读框的序列为：

ATGGAATTGGATAATGCAAGACCACCAGAACTTGGAATGAAATTTGCAACACTTGAAGATGCACAGAGATTCTATGAGACACATGCTCTTAAAACTGGCTTTGTTGCAAAGCGGGGGACGAACTACAGGAGAAAAAAGTTTACCATAGAGTGCCACAAGACTGGCACATCAAAGCTAACTCCGAACCCACAGAGGAAGAGGAAGAAAAATATTATAGAGAGGACTCAATGCCAGGCAAAGGTGGTAGTGAAGCTCAATAAGGGACAATGGGAGTATGCAGCAGTCCGGAATGAACACAACCATCCATTGTGTCCTAGCTATTCGCTTAGATTCTCAAAGCGCAAACGCAGGCGAAATCCTCCAAGCCAGAAACAGCTGGATGTTCAGAGAAATAGTGACCAACTGACACAGGCAGATAATCTTGAGGAACGGTTGTCGCAACCTCTTATTTCAGCTGATTCAAATGAAGTAAGCTTGCTAACAAAGGTTCCCCAGATATTTTTGTTTTGTATACATGTGATGGAAGTGATTTTTTAATGATCTGTTATCCTGGTTGTACCTCAGGTAAACAACAAAAGGATGCCTAGACATGCAGAGATTAGTATATCTACTGTAGAGAAGGAACCTGTTGACAAACTACAGCGAGCTGGTGATTATCAGGTATGAAAAGGTGAAATCTAACAAGAGTACCAATAATAAGGTATCAAATAGTAGCTTAACGTAAGTTATTCTCTTGCATGCAGGAGCAATCTTCAAATGGCATAGACAAAAATACAGAACGACCAATTGTAGATAAATTGGGAGACCAAACAAGCTCGATAGACCATTCACTTCAGCATACTGAAGAGCATAACAGATCACATGATAATGTGGAAAGCAGTGAAGCGCCAAGCGAGGATACAAGCAGTGAAAGCAATTCAGACAGCAGCTCAGGTGATGAGTCGGATAAGGAGCTTGGGAAGTACTTCTATCCTAGTTTCGACGAACTGAAGAATTCAAGACCACCAGAACCCGGAATGAAATTTCCATCCCTTCAAGCCGCACAAAGATTCTATTACGCACATGCTCTCCTTACTGGTTTTGTAGGAAAGAGGGGAACCAACTACAAGAGAAAGAAGTTTCACTTAGAATGCAACAAGAGTGGCAAAATGAAGTTAACGAAGTCATCTGAAAACCCAATGAAGAAGAGGAGAAGCAATCTTGTAGAGAAGACACAATGCAAGGCAAGGGTGATAGTGAAACTCGATAAGGGAGAATGGGAGTTCACAGCAGTTCGCCATGAGCACAATCATCCGTTATGCCCAAGCCCTTTGCTTGCAAGATTCATAGTAGACCACAAACAAATGTCAACTGGAGAGAAGTCATTTTTAAGAGTTCTGCAACAAAATAGGGTACCTCCTAAGAAAATTATGAAGATTTTCAGGAAACTAAGAGTTTGTTTCGGAGACATACCATTTGAAAATAAAGATGAGCACAACATAGCACAGACAGAACATAGAAAAGCAAACTCAGATGTTGAAAGCGCATTGAAGCACTTCACAGAATTGCAGATTCAAAACCCAGAATTTCTGTACGTAATGCAAAAAGATGAGGACAACACAGTTACAAGCATCTTCTGGACTGATGCAAGATTGAGGATCGAGTATGATATTTTCGGAGATCTCATTATGTTTGATGCTGCCTATAGCACTGATATGTATAACATGCCTTTTGTGCCTATTATTGGAATAAATAGCCACGCGACACCCTTCCTGTTAGGATGTGCTTTGTTAAAAGATGAGAAAGTAGAAACCTTCGAATGGATGTTGCGTACATTTTTGCAAGTGATGGGAGGAAAAATGCCAAGAGCGGTCATAACAAACCAGGACACATCAATGGAGAAGGCATTTGCAGAACTCATGCCGCATGTAAGGCTTAGGTTTTGCAAGCGGCATGTTATGAGCAAAGCTCAGGAAAAGCTTGGAGACTTCATGACAGCAAGAGGTAACATAAATACAGATCTACATAACTTAGTAGGCAACTCGCTGGTAGAAACAGAATTTGAAGAAGGATGGGTTGAGCTTATTGAGAGATACAATGCAAGTGAAAACCAACACCTGCAACTCATGTGGCAAACAAGAAAAAGTTGGGCACCTGTCTATTTTAGAGAAGATCTCTACCCATTTATTGACTCAGTTGGTTCCAACGAGGGAATAAACTCATTATTTAAAGGTAATATGCTTCCAAAGGACACAATAGATAAGTTCATTGGGCAATTTAAGGAGATACAAGAGAATATAGCAAAAGTTCATGAAGAAGATAGATTTCAGTCAGGAGCTGACCTTAAATACATCTCAATGCAACCAATAGAACAACATGCAGCACATATTTACACAAGGGAAATATTTCTGAAAGTACAGGAAGAACTACTACATTCTACTGCATTCAACGTGCAGGAGATACAAATAGGGACAGTGTACAGACTTGAAAAGGTCTTCAACTATGAGAACCCAGAGTTTGATAGAAATTATTTTGAAGTGCTTGCTGAACCTGGCATCAATGCATTCAAGTGCCAATGTGCAAAATTTACAAGGGATGGAATACCTTGCTGCCACATATTCAGACTTTTCACTCAGTTTGGAATCAATGAAATACCAGAGCAGTACATAATGCCCAGATGGACTAAAAAATTCAGAGAGGAGCAGCTAAAACAGAACAAGGAAAAATTATTTGACAAGCATGGTATAAAGGATTCAGAAAATACATTGAGATATGCAATGCTAATGAGTAAAGCGGCTGAAATTTGCCCGAAAATTTGCCATGATGAAGCAAAATGCAGCATATTCATGCAGGAACTAGACAAGATTCAAGAGAAGTTGATAACGGAGAGCAGAGAAAATGCACAGAATGATAACCCATGTCGTGAACAAGGGGCCTTGCAACAGGAGCATCGTGGGAGCAGTAGCAATACAGAGCAAGAGCAACAGCATACAGGTGGCTTGTAA

水稻穗发育新基因RPD，其编码区的序列为：

ATGGAATTGGATAATGCAAGACCACCAGAACTTGGAATGAAATTTGCAACACTTGAAGATGCACAGAGATTCTATGAGACACATGCTCTTAAAACTGGCTTTGTTGCAAAGCGGGGGACGAACTACAGGAGAAAAAAGTTTACCATAGAGTGCCACAAGACTGGCACATCAAAGCTAACTCCGAACCCACAGAGGAAGAGGAAGAAAAATATTATAGAGAGGACTCAATGCCAGGCAAAGGTGGTAGTGAAGCTCAATAAGGGACAATGGGAGTATGCAGCAGTCCGGAATGAACACAACCATCCATTGTGTCCTAGCTATTCGCTTAGATTCTCAAAGCGCAAACGCAGGCGAAATCCTCCAAGCCAGAAACAGCTGGATGTTCAGAGAAATAGTGACCAACTGACACAGGCAGATAATCTTGAGGAACGGTTGTCGCAACCTCTTATTTCAGCTGATTCAAATGAAGTAAACAACAAAAGGATGCCTAGACATGCAGAGATTAGTATATCTACTGTAGAGAAGGAACCTGTTGACAAACTACAGCGAGCTGGTGATTATCAGGAGCAATCTTCAAATGGCATAGACAAAAATACAGAACGACCAATTGTAGATAAATTGGGAGACCAAACAAGCTCGATAGACCATTCACTTCAGCATACTGAAGAGCATAACAGATCACATGATAATGTGGAAAGCAGTGAAGCGCCAAGCGAGGATACAAGCAGTGAAAGCAATTCAGACAGCAGCTCAGGTGATGAGTCGGATAAGGAGCTTGGGAAGTACTTCTATCCTAGTTTCGACGAACTGAAGAATTCAAGACCACCAGAACCCGGAATGAAATTTCCATCCCTTCAAGCCGCACAAAGATTCTATTACGCACATGCTCTCCTTACTGGTTTTGTAGGAAAGAGGGGAACCAACTACAAGAGAAAGAAGTTTCACTTAGAATGCAACAAGAGTGGCAAAATGAAGTTAACGAAGTCATCTGAAAACCCAATGAAGAAGAGGAGAAGCAATCTTGTAGAGAAGACACAATGCAAGGCAAGGGTGATAGTGAAACTCGATAAGGGAGAATGGGAGTTCACAGCAGTTCGCCATGAGCACAATCATCCGTTATGCCCAAGCCCTTTGCTTGCAAGATTCATAGTAGACCACAAACAAATGTCAACTGGAGAGAAGTCATTTTTAAGAGTTCTGCAACAAAATAGGGTACCTCCTAAGAAAATTATGAAGATTTTCAGGAAACTAAGAGTTTGTTTCGGAGACATACCATTTGAAAATAAAGATGAGCACAACATAGCACAGACAGAACATAGAAAAGCAAACTCAGATGTTGAAAGCGCATTGAAGCACTTCACAGAATTGCAGATTCAAAACCCAGAATTTCTGTACGTAATGCAAAAAGATGAGGACAACACAGTTACAAGCATCTTCTGGACTGATGCAAGATTGAGGATCGAGTATGATATTTTCGGAGATCTCATTATGTTTGATGCTGCCTATAGCACTGATATGTATAACATGCCTTTTGTGCCTATTATTGGAATAAATAGCCACGCGACACCCTTCCTGTTAGGATGTGCTTTGTTAAAAGATGAGAAAGTAGAAACCTTCGAATGGATGTTGCGTACATTTTTGCAAGTGATGGGAGGAAAAATGCCAAGAGCGGTCATAACAAACCAGGACACATCAATGGAGAAGGCATTTGCAGAACTCATGCCGCATGTAAGGCTTAGGTTTTGCAAGCGGCATGTTATGAGCAAAGCTCAGGAAAAGCTTGGAGACTTCATGACAGCAAGAGGTAACATAAATACAGATCTACATAACTTAGTAGGCAACTCGCTGGTAGAAACAGAATTTGAAGAAGGATGGGTTGAGCTTATTGAGAGATACAATGCAAGTGAAAACCAACACCTGCAACTCATGTGGCAAACAAGAAAAAGTTGGGCACCTGTCTATTTTAGAGAAGATCTCTACCCATTTATTGACTCAGTTGGTTCCAACGAGGGAATAAACTCATTATTTAAAGGTAATATGCTTCCAAAGGACACAATAGATAAGTTCATTGGGCAATTTAAGGAGATACAAGAGAATATAGCAAAAGTTCATGAAGAAGATAGATTTCAGTCAGGAGCTGACCTTAAATACATCTCAATGCAACCAATAGAACAACATGCAGCACATATTTACACAAGGGAAATATTTCTGAAAGTACAGGAAGAACTACTACATTCTACTGCATTCAACGTGCAGGAGATACAAATAGGGACAGTGTACAGACTTGAAAAGGTCTTCAACTATGAGAACCCAGAGTTTGATAGAAATTATTTTGAAGTGCTTGCTGAACCTGGCATCAATGCATTCAAGTGCCAATGTGCAAAATTTACAAGGGATGGAATACCTTGCTGCCACATATTCAGACTTTTCACTCAGTTTGGAATCAATGAAATACCAGAGCAGTACATAATGCCCAGATGGACTAAAAAATTCAGAGAGGAGCAGCTAAAACAGAACAAGGAAAAATTATTTGACAAGCATGGTATAAAGGATTCAGAAAATACATTGAGATATGCAATGCTAATGAGTAAAGCGGCTGAAATTTGCCCGAAAATTTGCCATGATGAAGCAAAATGCAGCATATTCATGCAGGAACTAGACAAGATTCAAGAGAAGTTGATAACGGAGAGCAGAGAAAATGCACAGAATGATAACCCATGTCGTGAACAAGGGGCCTTGCAACAGGAGCATCGTGGGAGCAGTAGCAATACAGAGCAAGAGCAACAGCATACAGGTGGCTTGTAA

水稻穗发育的一种新基因RPD编码的蛋白，其编码的氨基酸序列为：

MELDNARPPELGMKFATLEDAQRFYETHALKTGFVAKRGTNYRRKKFTIECHKTGTSKLTPNPQRKRKKNIIERTQCQAKVVVKLNKGQWEYAAVRNEHNHPLCPSYSLRFSKRKRRRNPPSQKQLDVQRNSDQLTQADNLEERLSQPLISADSNEVNNKRMPRHAEISISTVEKEPVDKLQRAGDYQEQSSNGIDKNTERPIVDKLGDQTSSIDHSLQHTEEHNRSHDNVESSEAPSEDTSSESNSDSSSGDESDKELGKYFYPSFDELKNSRPPEPGMKFPSLQAAQRFYYAHALLTGFVGKRGTNYKRKKFHLECNKSGKMKLTKSSENPMKKRRSNLVEKTQCKARVIVKLDKGEWEFTAVRHEHNHPLCPSPLLARFIVDHKQMSTGEKSFLRVLQQNRVPPKKIMKIFRKLRVCFGDIPFENKDEHNIAQTEHRKANSDVESALKHFTELQIQNPEFLYVMQKDEDNTVTSIFWTDARLRIEYDIFGDLIMFDAAYSTDMYNMPFVPIIGINSHATPFLLGCALLKDEKVETFEWMLRTFLQVMGGKMPRAVITNQDTSMEKAFAELMPHVRLRFCKRHVMSKAQEKLGDFMTARGNINTDLHNLVGNSLVETEFEEGWVELIERYNASENQHLQLMWQTRKSWAPVYFREDLYPFIDSVGSNEGINSLFKGNMLPKDTIDKFIGQFKEIQENIAKVHEEDRFQSGADLKYISMQPIEQHAAHIYTREIFLKVQEELLHSTAFNVQEIQIGTVYRLEKVFNYENPEFDRNYFEVLAEPGINAFKCQCAKFTRDGIPCCHIFRLFTQFGINEIPEQYIMPRWTKKFREEQLKQNKEKLFDKHGIKDSENTLRYAMLMSKAAEICPKICHDEAKCSIFMQELDKIQEKLITESRENAQNDNPCREQGALQQEHRGSSSNTEQEQQHTGGL。

有益效果

1.本发明提供的水稻穗发育基因RPD基因是一个新的水稻穗发育基因，该基因可以调控水稻穗及颖花发育。

2.本发明提供的水稻穗发育基因RPD基因为水稻育种及水稻穗发育机制研究提供了新的基因资源，可以在调控水稻穗发育方面得到应用

四、附图说明

图1基因定位和候选基因分析

(a：rpd基因初步定位；b：覆盖目标基因区域的跨跌克隆群：PAC1：P0521F09，PAC2：P0413A11，PAC3：P0017G06，BAC1：OSJNBa0055M07，PAC4：P0444A09，PAC5：P0669G10，BAC2：OSJNBa0063E14，BAC3：OSJNBa0018M09，BAC4：OJ1115_D03，BAC5：OJ1756_H07，BAC6：OJ1086_G08，BAC7：OJ1124_E11，BAC8：OJ1057_D08，BAC9：OSJNBa0073G17；c：rpd基因精细定位；d：rpd基因突变位点。)

图2 rpd基因氨基酸序列改变位点

图3 rpd和RPD蛋白结合位点

(a：RPD蛋白结合位点；b：rpd蛋白结合位点。)

图4 rpd和RPD三级结构

(a：RPD蛋白三级结构；b：rpd蛋白三级结构；c：a图和b图重叠比对。)

图5 F₂群体分子标记鉴定

(L：DNA ladder；1：糯稻-7；2：南粳41；3：IR28；4：9311；5：RPD/rpd杂合株；6：rpdrpd纯合株；7-18：RPD/rpd×IR28F₂群体中具有rpd表型的植株。)

图6转基因植株表型

(a：突变体；b：转基因阳性植株)

生物保藏

RPD/rpd杂合体，分类命名水稻(Oryza sativa),于2014年11月25在中国微生物菌种保藏管理委员会普通微生物中心进行保藏，地址北京市朝阳区北辰西路1号院中科院微生物研究所，保藏号是CGMCC NO：10010。

五、具体实施方式

1.定位群体构建

利用保藏号是CGMCC NO：10010的RPD/rpd杂合体(从南粳41(江苏省农业种质资源中期库，保存号：M3A00405736)和糯稻-7(江苏省农业种质资源中期库，保存号：M3A00402380)杂交F₆代中自然突变获得水稻穗发育突变体rpd及其杂合体RPD/rpd)分别与IR28(江苏省农业种质资源中期库，保存号：M3A00401612)和9311(江苏省农业种质资源中期库，保存号：M3A00407616)杂交后自交，获得F₂群体，用于RPD基因定位。

2.基因组DNA提取

抽穗后，调查F₂群体各单株表型，取rpd表型的个体，用TPS法提取基因组DNA(Miura et al.,2008)。

3.多态筛选

从Gramene(http://www.gramene.org/)选取452对均匀分布在水稻12条染色体上的SSR引物对rpd、IR28、9311及其F₁进行多态性筛选，获得280个多态性较好的共显性标记。

PCR采用10μL反应体系，体系如下：1μL模板DNA,1μL 10×PCR buffer，0.8μL2.5mmol/L dNTP，0.8μL 2.5mmol/L引物，0.25U Taq DNA polymerase。PCR反应条件如下：94℃预变性2min，94℃变性30s，55℃退火30s，72℃延伸1min，30次循环，72℃最后延伸7min，4℃保存。

PCR产物加1.5μL 6×Loading buffer，在8％非变性聚丙烯酰胺凝胶(100mL聚丙烯酰胺中含7.6g丙烯酰胺和0.4g甲叉双丙烯酰胺)，200V电泳2h，银染，拍照，统计结果。

4.基因初步定位

从rpd/IR28F₂群体中随机选取30个隐性单株作为定位群体，将rpd定位在第2染色体上，与RM13061、RM13046和RM5356连锁。接下来，随机选取228个隐性单株为定位群体，将目标基因定位在RM12933和RM324之间，距离目标基因分别是1.5cM和0.7cM。

5.标记开发

InDel标记开发：从GRAMENE中查询目标基因两侧分子标记所在的BAC，根据IRGSP中的BAC顺序，构建了目标基因区域的跨叠克隆群。从NCBI(http://www.ncbi.nlm.nih.gov)下载相应的Nipponbare BAC序列，并进行拼接。同时，从BGI(http://rice.genomics.org.cn/rice/index2.jsp)下载相应的9311序列。用DANMAN对Nipponbare和9311序列进行比对，找出插入和缺失位点，用Primer premier 5.0设计InDel引物。

dCAPS标记开发：在候选区域，每隔10kb用Primer premier 5.0设计一对引物，扩增片段长度为1kb左右。以rpd、9311和IR28基因组DNA为模板，用上述引物分别进行扩增和测序，用DANMAN进行比对，找出rpd与IR28或9311间存在的SNP。用dCAPS Finder 2.0(http://helix.wustl.edu/dcaps/dcaps.html)将SNP转化为dCAPS标记。

6.基因精细定位

以7307个隐性个体为定位群体，用SSR标记和Indel标记将目标基因定位在RM12988和InD97之间108kb范围内。最后，用新开发的dCAPS标记将目标基因定位在dC7和dC8之间的14.6Kb范围内(图1)。

7.候选基因分析

用FGENESH对这14.6kb范围内的基因数目和结构进行了预测和分析，共发现2个基因(图1)。用Primer premier 5.0设计3对跨叠引物对这14.6kb范围进行扩增和测序。3对跨叠引物序列为：P-Part I-F:5’-TCTACTACGCCCACCGTCAA-3’；P-Part I-R:5’-TTGTGCGGCTTGAAGGGATG-3’；P-Part II-F:5’-AGCAGCTCAGGTGATGAGTC-3’；P-Part II-R:5’-TGGGATTGTGGTTTGGCTGT-3’；P-Part III-F:5’-AGAAGGATCGAAATCCGGGC-3’；P-PartIII-R:5’-CTCGGCTCGGCTCATGTATT-3’。PCR扩增用 GXL DNA聚合酶进行，采用2步PCR法，反应条件为：98℃ 10s，68℃ 6min，30个循环，最后68℃延伸10min。PCR产物胶回收，用Taq DNA Polymerase加A后，克隆到pEASY-T5Zero Cloning vector(TransGen,Beijing)载体上，挑取阳性克隆送Invitrogen测序，用DANMAN进行序列比对。

比对结果表明：在这14.6kb范围内，仅存在第1个预测基因中存在突变(图1)，即其第3个外显子中的第389个碱基由G变为T，导致其编码蛋白第318个AA由Cys变为Phe(图2)。

设计了一对引物分别从野生型(南粳41(江苏省农业种质资源中期库，保存号：M3A00405736)和糯稻-7(江苏省农业种质资源中期库，保存号：M3A00402380))和突变体(从南粳41(江苏省农业种质资源中期库，保存号：M3A00405736)和糯稻-7(江苏省农业种质资源中期库，保存号：M3A00402380)杂交F₆代中自然突变获得水稻穗发育突变体rpd)中扩增了RPD完整的编码区。正向引物和反向引物分别为：P-CDS-F:5’-TAAAGGGCTTGGAGCACACTAT-3’和P-CDS-R:5’-ATCAGCTTCCCATACTGCCG-3’。PCR扩增用 GXL DNA聚合酶进行，采用2步PCR法，反应条件为：98℃ 10s，68℃ 4min，30个循环，最后68℃延伸10min。PCR产物胶回收，用Taq DNA Polymerase加A后，克隆到pEASY-T5Zero Cloning vector(TransGen,Beijing)载体上，挑取阳性克隆送Invitrogen测序。测序结果证实了rpd第3个外显子中G389T突变位点的真实性。

8.蛋白结构分析

rpd和RPD蛋白二级结构，用PredictProtein(https:// www.predictprotein.org/)软件分析，结果表明：较之RPD，rpd中螺旋比例由24.30％变为25.91％，折叠的比例由12.85％变为12.63％，环的比例由62.85％变为61.46％。rpd中蛋白质与蛋白质相互作用的结合位点亦发生了改变(图3)。

rpd和RPD蛋白三级结构，用SWISS-MODEL(http://swissmodel.expasy.org/)软件分析，并用UCSF Chimera(Pettersen et al.2004)软件比对，结果表明：其3级结构发生改变(图4)。

9.突变位点分析

根据rpd变异位点，用PRIMER1(http://primer1.soton.ac.uk/primer1.html)设计了ARMS-PCR引物：P-inner-F:5’-CTACAAGAGAAAGAAGTTTCACTTAGAGTG-3’；P-inner-R:

5’-TTAACTTCATTTTGCCACTCTTGTCGA-3’；P-outer-F 5’-GGATAAGGAG

CTTGGGAAGTACTTCTAT-3’；P-outer-R 5’-GTTTGTGGTCTACTATGAATCTTGCAAG-3’。PCR反应条件为：94℃预变性5min，94℃变性30s，58℃退火30s，72℃延伸60s，35个循环，最后72℃延伸10min。用该引物对7307个隐性个体进行检测，均表现为rpd带型(398bp和215bp)，表明该突变表型是RPD基因变异导致的(图5)。

10.互补载体构建和转化

以基因组DNA为模板，用两步法从中扩增出完整的目标基因。用TaKaRa MiniBESTDNA Fragment Purification Kit Ver.4.0纯化PCR产物。用 Seamless PLUSCloning and Assembly Kit将目标基因连接到经BamH I酶切的pCAMBIA1305.1线性载体上。用农杆菌转化突变体成熟胚，在含有50mg/L潮霉素筛选剂的筛选培养基上，32℃培养14天，再转到新鲜配制的筛选培养基上继续筛选14天，将具有抗性的胚性愈伤组织分化培养基上，32℃光照培养，分化成苗。获得42株转基因后代，25株转基因阳性植株穗部颖花和枝梗退化表型得到恢复，表现为正常表型(图6)，证实了RPD基因是控制水稻穗发育基因，可以在调控水稻穗发育方面得到应用。

SEQUENCE LISTING

<110> 江苏省农业科学院

<120> 一种水稻穗发育基因RPD与应用

<130> 0

<160> 5

<170> PatentIn version 3.1

<210> 1

<211> 2984

<212> DNA

<213> Oryza sativa

<220>

<221> 水稻穗发育基因RPD开放阅读框

<222> (1)..(2984)

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<400> 1

atggaattgg ataatgcaag accaccagaa cttggaatga aatttgcaac acttgaagat 60

gcacagagat tctatgagac acatgctctt aaaactggct ttgttgcaaa gcgggggacg 120

aactacagga gaaaaaagtt taccatagag tgccacaaga ctggcacatc aaagctaact 180

ccgaacccac agaggaagag gaagaaaaat attatagaga ggactcaatg ccaggcaaag 240

gtggtagtga agctcaataa gggacaatgg gagtatgcag cagtccggaa tgaacacaac 300

catccattgt gtcctagcta ttcgcttaga ttctcaaagc gcaaacgcag gcgaaatcct 360

ccaagccaga aacagctgga tgttcagaga aatagtgacc aactgacaca ggcagataat 420

cttgaggaac ggttgtcgca acctcttatt tcagctgatt caaatgaagt aagcttgcta 480

acaaaggttc cccagatatt tttgttttgt atacatgtga tggaagtgat tttttaatga 540

tctgttatcc tggttgtacc tcaggtaaac aacaaaagga tgcctagaca tgcagagatt 600

agtatatcta ctgtagagaa ggaacctgtt gacaaactac agcgagctgg tgattatcag 660

gtatgaaaag gtgaaatcta acaagagtac caataataag gtatcaaata gtagcttaac 720

gtaagttatt ctcttgcatg caggagcaat cttcaaatgg catagacaaa aatacagaac 780

gaccaattgt agataaattg ggagaccaaa caagctcgat agaccattca cttcagcata 840

ctgaagagca taacagatca catgataatg tggaaagcag tgaagcgcca agcgaggata 900

caagcagtga aagcaattca gacagcagct caggtgatga gtcggataag gagcttggga 960

agtacttcta tcctagtttc gacgaactga agaattcaag accaccagaa cccggaatga 1020

aatttccatc ccttcaagcc gcacaaagat tctattacgc acatgctctc cttactggtt 1080

ttgtaggaaa gaggggaacc aactacaaga gaaagaagtt tcacttagaa tgcaacaaga 1140

gtggcaaaat gaagttaacg aagtcatctg aaaacccaat gaagaagagg agaagcaatc 1200

ttgtagagaa gacacaatgc aaggcaaggg tgatagtgaa actcgataag ggagaatggg 1260

agttcacagc agttcgccat gagcacaatc atccgttatg cccaagccct ttgcttgcaa 1320

gattcatagt agaccacaaa caaatgtcaa ctggagagaa gtcattttta agagttctgc 1380

aacaaaatag ggtacctcct aagaaaatta tgaagatttt caggaaacta agagtttgtt 1440

tcggagacat accatttgaa aataaagatg agcacaacat agcacagaca gaacatagaa 1500

aagcaaactc agatgttgaa agcgcattga agcacttcac agaattgcag attcaaaacc 1560

cagaatttct gtacgtaatg caaaaagatg aggacaacac agttacaagc atcttctgga 1620

ctgatgcaag attgaggatc gagtatgata ttttcggaga tctcattatg tttgatgctg 1680

cctatagcac tgatatgtat aacatgcctt ttgtgcctat tattggaata aatagccacg 1740

cgacaccctt cctgttagga tgtgctttgt taaaagatga gaaagtagaa accttcgaat 1800

ggatgttgcg tacatttttg caagtgatgg gaggaaaaat gccaagagcg gtcataacaa 1860

accaggacac atcaatggag aaggcatttg cagaactcat gccgcatgta aggcttaggt 1920

tttgcaagcg gcatgttatg agcaaagctc aggaaaagct tggagacttc atgacagcaa 1980

gaggtaacat aaatacagat ctacataact tagtaggcaa ctcgctggta gaaacagaat 2040

ttgaagaagg atgggttgag cttattgaga gatacaatgc aagtgaaaac caacacctgc 2100

aactcatgtg gcaaacaaga aaaagttggg cacctgtcta ttttagagaa gatctctacc 2160

catttattga ctcagttggt tccaacgagg gaataaactc attatttaaa ggtaatatgc 2220

ttccaaagga cacaatagat aagttcattg ggcaatttaa ggagatacaa gagaatatag 2280

caaaagttca tgaagaagat agatttcagt caggagctga ccttaaatac atctcaatgc 2340

aaccaataga acaacatgca gcacatattt acacaaggga aatatttctg aaagtacagg 2400

aagaactact acattctact gcattcaacg tgcaggagat acaaataggg acagtgtaca 2460

gacttgaaaa ggtcttcaac tatgagaacc cagagtttga tagaaattat tttgaagtgc 2520

ttgctgaacc tggcatcaat gcattcaagt gccaatgtgc aaaatttaca agggatggaa 2580

taccttgctg ccacatattc agacttttca ctcagtttgg aatcaatgaa ataccagagc 2640

agtacataat gcccagatgg actaaaaaat tcagagagga gcagctaaaa cagaacaagg 2700

aaaaattatt tgacaagcat ggtataaagg attcagaaaa tacattgaga tatgcaatgc 2760

taatgagtaa agcggctgaa atttgcccga aaatttgcca tgatgaagca aaatgcagca 2820

tattcatgca ggaactagac aagattcaag agaagttgat aacggagagc agagaaaatg 2880

cacagaatga taacccatgt cgtgaacaag gggccttgca acaggagcat cgtgggagca 2940

gtagcaatac agagcaagag caacagcata caggtggctt gtaa 2984

<210> 2

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<221> 水稻穗发育基因RPD编码区

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<220>

<221> 水稻穗发育基因RPD编码区

<222> (1)..(2805)

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atggaattgg ataatgcaag accaccagaa cttggaatga aatttgcaac acttgaagat 60

gcacagagat tctatgagac acatgctctt aaaactggct ttgttgcaaa gcgggggacg 120

aactacagga gaaaaaagtt taccatagag tgccacaaga ctggcacatc aaagctaact 180

ccgaacccac agaggaagag gaagaaaaat attatagaga ggactcaatg ccaggcaaag 240

gtggtagtga agctcaataa gggacaatgg gagtatgcag cagtccggaa tgaacacaac 300

catccattgt gtcctagcta ttcgcttaga ttctcaaagc gcaaacgcag gcgaaatcct 360

ccaagccaga aacagctgga tgttcagaga aatagtgacc aactgacaca ggcagataat 420

cttgaggaac ggttgtcgca acctcttatt tcagctgatt caaatgaagt aaacaacaaa 480

aggatgccta gacatgcaga gattagtata tctactgtag agaaggaacc tgttgacaaa 540

ctacagcgag ctggtgatta tcaggagcaa tcttcaaatg gcatagacaa aaatacagaa 600

cgaccaattg tagataaatt gggagaccaa acaagctcga tagaccattc acttcagcat 660

actgaagagc ataacagatc acatgataat gtggaaagca gtgaagcgcc aagcgaggat 720

acaagcagtg aaagcaattc agacagcagc tcaggtgatg agtcggataa ggagcttggg 780

aagtacttct atcctagttt cgacgaactg aagaattcaa gaccaccaga acccggaatg 840

aaatttccat cccttcaagc cgcacaaaga ttctattacg cacatgctct ccttactggt 900

tttgtaggaa agaggggaac caactacaag agaaagaagt ttcacttaga atgcaacaag 960

agtggcaaaa tgaagttaac gaagtcatct gaaaacccaa tgaagaagag gagaagcaat 1020

cttgtagaga agacacaatg caaggcaagg gtgatagtga aactcgataa gggagaatgg 1080

gagttcacag cagttcgcca tgagcacaat catccgttat gcccaagccc tttgcttgca 1140

agattcatag tagaccacaa acaaatgtca actggagaga agtcattttt aagagttctg 1200

caacaaaata gggtacctcc taagaaaatt atgaagattt tcaggaaact aagagtttgt 1260

ttcggagaca taccatttga aaataaagat gagcacaaca tagcacagac agaacataga 1320

aaagcaaact cagatgttga aagcgcattg aagcacttca cagaattgca gattcaaaac 1380

ccagaatttc tgtacgtaat gcaaaaagat gaggacaaca cagttacaag catcttctgg 1440

actgatgcaa gattgaggat cgagtatgat attttcggag atctcattat gtttgatgct 1500

gcctatagca ctgatatgta taacatgcct tttgtgccta ttattggaat aaatagccac 1560

gcgacaccct tcctgttagg atgtgctttg ttaaaagatg agaaagtaga aaccttcgaa 1620

tggatgttgc gtacattttt gcaagtgatg ggaggaaaaa tgccaagagc ggtcataaca 1680

aaccaggaca catcaatgga gaaggcattt gcagaactca tgccgcatgt aaggcttagg 1740

ttttgcaagc ggcatgttat gagcaaagct caggaaaagc ttggagactt catgacagca 1800

agaggtaaca taaatacaga tctacataac ttagtaggca actcgctggt agaaacagaa 1860

tttgaagaag gatgggttga gcttattgag agatacaatg caagtgaaaa ccaacacctg 1920

caactcatgt ggcaaacaag aaaaagttgg gcacctgtct attttagaga agatctctac 1980

ccatttattg actcagttgg ttccaacgag ggaataaact cattatttaa aggtaatatg 2040

cttccaaagg acacaataga taagttcatt gggcaattta aggagataca agagaatata 2100

gcaaaagttc atgaagaaga tagatttcag tcaggagctg accttaaata catctcaatg 2160

caaccaatag aacaacatgc agcacatatt tacacaaggg aaatatttct gaaagtacag 2220

gaagaactac tacattctac tgcattcaac gtgcaggaga tacaaatagg gacagtgtac 2280

agacttgaaa aggtcttcaa ctatgagaac ccagagtttg atagaaatta ttttgaagtg 2340

cttgctgaac ctggcatcaa tgcattcaag tgccaatgtg caaaatttac aagggatgga 2400

ataccttgct gccacatatt cagacttttc actcagtttg gaatcaatga aataccagag 2460

cagtacataa tgcccagatg gactaaaaaa ttcagagagg agcagctaaa acagaacaag 2520

gaaaaattat ttgacaagca tggtataaag gattcagaaa atacattgag atatgcaatg 2580

ctaatgagta aagcggctga aatttgcccg aaaatttgcc atgatgaagc aaaatgcagc 2640

atattcatgc aggaactaga caagattcaa gagaagttga taacggagag cagagaaaat 2700

gcacagaatg ataacccatg tcgtgaacaa ggggccttgc aacaggagca tcgtgggagc 2760

agtagcaata cagagcaaga gcaacagcat acaggtggct tgtaa 2805

<210> 3

<211> 934

<212> PRT

<213> Oryza sativa

<220>

<221> 水稻穗发育基因RPD编码的蛋白

<222> (1)..(934)

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Met Glu Leu Asp Asn Ala Arg Pro Pro Glu Leu Gly Met Lys Phe Ala

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Thr Leu Glu Asp Ala Gln Arg Phe Tyr Glu Thr His Ala Leu Lys Thr

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Gly Phe Val Ala Lys Arg Gly Thr Asn Tyr Arg Arg Lys Lys Phe Thr

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Ile Glu Cys His Lys Thr Gly Thr Ser Lys Leu Thr Pro Asn Pro Gln

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Arg Lys Arg Lys Lys Asn Ile Ile Glu Arg Thr Gln Cys Gln Ala Lys

65 70 75 80

Val Val Val Lys Leu Asn Lys Gly Gln Trp Glu Tyr Ala Ala Val Arg

85 90 95

Asn Glu His Asn His Pro Leu Cys Pro Ser Tyr Ser Leu Arg Phe Ser

100 105 110

Lys Arg Lys Arg Arg Arg Asn Pro Pro Ser Gln Lys Gln Leu Asp Val

115 120 125

Gln Arg Asn Ser Asp Gln Leu Thr Gln Ala Asp Asn Leu Glu Glu Arg

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Leu Ser Gln Pro Leu Ile Ser Ala Asp Ser Asn Glu Val Asn Asn Lys

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Arg Met Pro Arg His Ala Glu Ile Ser Ile Ser Thr Val Glu Lys Glu

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Pro Val Asp Lys Leu Gln Arg Ala Gly Asp Tyr Gln Glu Gln Ser Ser

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Asn Gly Ile Asp Lys Asn Thr Glu Arg Pro Ile Val Asp Lys Leu Gly

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Asp Gln Thr Ser Ser Ile Asp His Ser Leu Gln His Thr Glu Glu His

210 215 220

Asn Arg Ser His Asp Asn Val Glu Ser Ser Glu Ala Pro Ser Glu Asp

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Thr Ser Ser Glu Ser Asn Ser Asp Ser Ser Ser Gly Asp Glu Ser Asp

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Lys Glu Leu Gly Lys Tyr Phe Tyr Pro Ser Phe Asp Glu Leu Lys Asn

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Ser Arg Pro Pro Glu Pro Gly Met Lys Phe Pro Ser Leu Gln Ala Ala

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Gln Arg Phe Tyr Tyr Ala His Ala Leu Leu Thr Gly Phe Val Gly Lys

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Arg Gly Thr Asn Tyr Lys Arg Lys Lys Phe His Leu Glu Cys Asn Lys

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Ser Gly Lys Met Lys Leu Thr Lys Ser Ser Glu Asn Pro Met Lys Lys

325 330 335

Arg Arg Ser Asn Leu Val Glu Lys Thr Gln Cys Lys Ala Arg Val Ile

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Val Lys Leu Asp Lys Gly Glu Trp Glu Phe Thr Ala Val Arg His Glu

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His Asn His Pro Leu Cys Pro Ser Pro Leu Leu Ala Arg Phe Ile Val

370 375 380

Asp His Lys Gln Met Ser Thr Gly Glu Lys Ser Phe Leu Arg Val Leu

385 390 395 400

Gln Gln Asn Arg Val Pro Pro Lys Lys Ile Met Lys Ile Phe Arg Lys

405 410 415

Leu Arg Val Cys Phe Gly Asp Ile Pro Phe Glu Asn Lys Asp Glu His

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Asn Ile Ala Gln Thr Glu His Arg Lys Ala Asn Ser Asp Val Glu Ser

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Ala Leu Lys His Phe Thr Glu Leu Gln Ile Gln Asn Pro Glu Phe Leu

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Tyr Val Met Gln Lys Asp Glu Asp Asn Thr Val Thr Ser Ile Phe Trp

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Thr Asp Ala Arg Leu Arg Ile Glu Tyr Asp Ile Phe Gly Asp Leu Ile

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Met Phe Asp Ala Ala Tyr Ser Thr Asp Met Tyr Asn Met Pro Phe Val

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Pro Ile Ile Gly Ile Asn Ser His Ala Thr Pro Phe Leu Leu Gly Cys

515 520 525

Ala Leu Leu Lys Asp Glu Lys Val Glu Thr Phe Glu Trp Met Leu Arg

530 535 540

Thr Phe Leu Gln Val Met Gly Gly Lys Met Pro Arg Ala Val Ile Thr

545 550 555 560

Asn Gln Asp Thr Ser Met Glu Lys Ala Phe Ala Glu Leu Met Pro His

565 570 575

Val Arg Leu Arg Phe Cys Lys Arg His Val Met Ser Lys Ala Gln Glu

580 585 590

Lys Leu Gly Asp Phe Met Thr Ala Arg Gly Asn Ile Asn Thr Asp Leu

595 600 605

His Asn Leu Val Gly Asn Ser Leu Val Glu Thr Glu Phe Glu Glu Gly

610 615 620

Trp Val Glu Leu Ile Glu Arg Tyr Asn Ala Ser Glu Asn Gln His Leu

625 630 635 640

Gln Leu Met Trp Gln Thr Arg Lys Ser Trp Ala Pro Val Tyr Phe Arg

645 650 655

Glu Asp Leu Tyr Pro Phe Ile Asp Ser Val Gly Ser Asn Glu Gly Ile

660 665 670

Asn Ser Leu Phe Lys Gly Asn Met Leu Pro Lys Asp Thr Ile Asp Lys

675 680 685

Phe Ile Gly Gln Phe Lys Glu Ile Gln Glu Asn Ile Ala Lys Val His

690 695 700

Glu Glu Asp Arg Phe Gln Ser Gly Ala Asp Leu Lys Tyr Ile Ser Met

705 710 715 720

Gln Pro Ile Glu Gln His Ala Ala His Ile Tyr Thr Arg Glu Ile Phe

725 730 735

Leu Lys Val Gln Glu Glu Leu Leu His Ser Thr Ala Phe Asn Val Gln

740 745 750

Glu Ile Gln Ile Gly Thr Val Tyr Arg Leu Glu Lys Val Phe Asn Tyr

755 760 765

Glu Asn Pro Glu Phe Asp Arg Asn Tyr Phe Glu Val Leu Ala Glu Pro

770 775 780

Gly Ile Asn Ala Phe Lys Cys Gln Cys Ala Lys Phe Thr Arg Asp Gly

785 790 795 800

Ile Pro Cys Cys His Ile Phe Arg Leu Phe Thr Gln Phe Gly Ile Asn

805 810 815

Glu Ile Pro Glu Gln Tyr Ile Met Pro Arg Trp Thr Lys Lys Phe Arg

820 825 830

Glu Glu Gln Leu Lys Gln Asn Lys Glu Lys Leu Phe Asp Lys His Gly

835 840 845

Ile Lys Asp Ser Glu Asn Thr Leu Arg Tyr Ala Met Leu Met Ser Lys

850 855 860

Ala Ala Glu Ile Cys Pro Lys Ile Cys His Asp Glu Ala Lys Cys Ser

865 870 875 880

Ile Phe Met Gln Glu Leu Asp Lys Ile Gln Glu Lys Leu Ile Thr Glu

885 890 895

Ser Arg Glu Asn Ala Gln Asn Asp Asn Pro Cys Arg Glu Gln Gly Ala

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Leu Gln Gln Glu His Arg Gly Ser Ser Ser Asn Thr Glu Gln Glu Gln

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Gln His Thr Gly Gly Leu

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<210> 4

<211> 22

<212> DNA

<213> 人工

<220>

<221> P-CDS-F

<222> (1)..(22)

<223>

<400> 4

taaagggctt ggagcacact at 22

<210> 5

<211> 20

<212> DNA

<213> 人工

<220>

<221> P-CDS-R

<222> (1)..(20)

<223>

<400> 5

atcagcttcc catactgccg 20

Claims (2)

1.一种水稻穗发育基因RPD的应用，其特征在于，是指该水稻穗发育基因RPD在调控水稻穗发育方面的应用，该水稻穗发育基因RPD开放阅读框序列为：SEQ ID NO.1。

2.权利要求1所述水稻穗发育基因RPD编码的蛋白质的应用，其特征在于，是指该水稻穗发育基因RPD编码的蛋白质在调控水稻穗发育方面的应用。