CN114990137A - 拟南芥钙结合蛋白基因AtCAREF及应用 - Google Patents
拟南芥钙结合蛋白基因AtCAREF及应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
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- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
本发明公开了一种拟南芥钙结合蛋白基因AtCAREF及应用，其外源DNA插入突变体具有在长日照和短日照条件下促进开花的表型，说明基因AtCAREF所编码的蛋白具有调节开花的作用。利用AtCAREF调节植物开花的特性，通过CRISPR‑Cas9技术，抑制植物该基因的表达，可以获得早开花早结果的园艺植物新品种，对农业及园艺生产并提高植物经济价值具有重要的意义，因此对提高农业经济生产也具有重要的指导作用。
Description
技术领域
本发明属于生物技术领域，具体涉及拟南芥钙结合蛋白基因AtCAREF及应用。
背景技术
开花是高等植物的一个重要特征，它不仅决定植物个体能否顺利繁衍后代，同时也与人类的生活紧密相关。高等植物的开花调控包括3个方面：成花诱导、花发育和开花。其中，成花诱导是植物开花的基础，作物开花的早晚深刻影响着作物的高产稳产、品质和区域分布，育种家们将适宜的开花时间作为关键农艺性状之一，一直期待着能高效获得具有理想开花时间的优异种质。尤其是在果树中的应用，缩短果树的开花时间，将会大大提高农业经济价值。
植物中钙在一系列生理和代谢过程中起第二信使作用，是一种在多种功能调控中刺激应答的媒介物。在植物中有三种具有螺旋-环-螺旋(EF手图象)的Ca2+感受器类型，它们是CaM、CDPK(钙依赖性蛋白激酶)和CBL(钙调磷酸酶B类似蛋白)。
钙调蛋白(calmodulin，CaM)是生物细胞内一种重要的调控蛋白，作为一种多功能钙离子传感蛋白存在于所有真核生物中，在植物生长发育过程中，钙离子作为第二信使扮演着至关重要的角色。细胞内的钙离子受激素、光照、机械损伤以及病虫害等许多信号的调控(Blume et al.，2000)，这些信息形成的瞬时钙离子信号又被多种细胞内钙离子传感蛋白所破译(Luan et al.，2002)。即通过自身特有的CaM结合结构域(CaM-binding domain，CaMBD)与其靶蛋白相互作用调节细胞的正常生长和发育以及生理功能，包括细胞分裂、细胞代谢、胁迫、花药和雌蕊以及胚胎的发育等。
许多调控拟南芥开花基因已被调控，目前共统计并鉴定了57个成花调控基因，分别涉及如下6个遗传调控途径：包括光周期途径(Photoperiod pathway)、春化途径(Vernalization pathway)、环境温度途径(Ambient temperature pathway)、赤霉素途径(Gibberellin pathway)、自主途径(Autonomous pathway)、年龄途径(Age pathway)，这些信号途径既彼此独立又相互交联，形成一个复杂的激活与抑制、正反馈与负反馈共存的调控网络。在有关植物成花调控的数据还十分有限，离全面揭示整个成花调控的遗传网络还有很长的路要走。
早在1989年，Friedman等就发现Ca2+参与牵牛花的成花调控，不过已鉴定的拟南芥成花调控基因中，涉及到Ca2+/CaM信号的只有AtCML24及其高度同源的AtCML23。这2个基因编码的钙调类似蛋白(calmoduolin-like sensor，CAL)均能在与Ca2+结合后发生构象变化；AtCML24表达下调突变体在长日照下延迟10-30天开花(Delk et al.，2005)，而单独AtCML23的功能缺失突变体无成花表型，只有在与同样没有明显成花表型的AtCML24的弱突变体组成双突变体时才表现延迟成花，说明二者在成花调控方面功能冗余且AtCML23的贡献较小，进一步分析发现，这两个蛋白调控包括CO和FLC等成花信号整合基因在内的表达以及调控成花的NO的产生(Tsai et al.，2007)；不过，与它们直接结合的下游靶标仍不清楚。除此之外，近有烟草的两个钙素结合蛋白激酶NtMCK1和NtCBK1被发现涉及成花调控(梁述平等，2001；Hua et al.，2004)。因此，寻找更多参与成花调控的Ca2+/CaM信号通路成员，研究其分子机理应用于植物开花时间的控制，具有重要的理论和实践意义。
发明内容
本发明的目的之一在于提供一种拟南芥基因AtCAREF，在拟南芥基因组数据库中编号为At2g34030，通过功能验证发现其是一个钙结合蛋白，具有调节植物开花的功能。
本发明的第二个目的是提供上述拟南芥钙结合蛋白基因AtCAREF的应用。
为了实现上述目的，本发明采用的技术方案概述如下：
一种拟南芥钙结合蛋白基因AtCAREF，其核苷酸序列如SEQ ID NO.1所示，所述的核苷酸序列由1701个碱基组成，共编码566个氨基酸，其序列如SEQ ID NO.2所示。
上述拟南芥钙结合蛋白基因AtCAREF编码的蛋白选自，
(1)其氨基酸序列如SEQ ID NO.2所示；
(2)将SEQ ID NO.2氨基酸序列经过一个或多个(如1-30个；较佳地1-20个；更佳地1-10个；如5个，3个)氨基酸残基的取代、缺失或添加而形成的，且具有(1)蛋白功能的由(1)衍生的蛋白；
(3)与(1)限定的蛋白序列有80％(较佳地90％以上，如95％，98％，99％或更高)以上同源性且具有(1)蛋白功能的由(1)衍生的蛋白。
也就是说本发明所保护的基因的功能，不仅包括上述拟南芥钙结合蛋白基因AtCAREF，还包括与SEQ ID NO.1具有较高同源性(如同源性高于40％；较佳地高于50％；较佳地高于60％；更佳地高于70％；更佳地高于80％；更佳地高于90％；更佳地高于95％；更佳地高于98％)的同源基因。
本发明最主要的目的是在分子水平上对拟南芥钙结合蛋白基因AtCAREF进行克隆和鉴定，从而解析控制植物开花时间的调控机理。本发明还公开了抑制上述拟南芥钙结合蛋白基因AtCAREF的表达在促进拟南芥提早开花中应用。
作为本发明的一种实施方式，本发明获得了基因AtCAREF功能缺失突变体caref并对其进行了鉴定，功能缺失突变体caref是AtCAREF基因因为外源DNA的插入而被破坏，成为功能缺失突变体，可以作为AtCAREF基因功能研究的材料。
另外，由于实验证明，通过抑制所述基因AtCAREF的表达后，植株的开花时间提早，可以得知，基因AtCAREF具有延迟植物开花的作用。根据该特性，可以通过转基因的方式来获得开花时间需要推迟的植株，具体地，可以通过将基因AtCAREF导入目的植物，得到转基因植物，该植株开花时间晚于目的植物的植株。
具体地，基因AtCAREF具体可通过所述重组表达载体导入所述目的植物。所述方法中，所述重组表达载体可通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导、农杆菌介导等常规生物学方法转化植物细胞或组织，并将转化的植物组织培育成植株。
具体地，为了提高植物的优良性状，本发明还保护一种新的植物育种方法，包括如下步骤(1)、(2)或(3)：
(1)通过增加目的植物中蛋白AtCAREF的活性，获得开花时间晚于目的植物的植株；
(2)通过促进目的植物中基因AtCAREF的表达，获得开花时间晚于目的植物的植株；
(3)通过抑制拟南芥中基因AtCAREF的表达，获得提早开花的拟南芥植株。
“促进目的植物中基因AtCAREF的表达”的实现方式可为如下(1)或(2)或(3)：
(1)将基因AtCAREF导入目的植物；
(2)引入强启动子和/或增强子；
(3)本领域内的其它常见方法。
抑制目的植物中的基因AtCAREF的表达可以采用敲除基因AtCAREF的方式，敲除基因AtCAREF的方式可以采用基因编辑技术。也可以直接购买基因AtCAREF功能缺失突变体caref。
其中，目的植物(target plant)，本发明所述目的植物是番茄。
目的基因(target gene)，也称靶标基因，在基因工程设计和操作中，被用于基因重组、改变受体细胞性状和获得预期表达产物的基因。可以是生物体本身的，也可以是来自不同生物体的。
本发明中，对于适用于本发明的植物没有特别的限制，只要其适合进行基因的转化操作，如各种农作物、花卉植物、或林业植物等。所述的植物比如可以是(不限于)：双子叶植物、单子叶植物或裸子植物。
作为一种优选方式，所述的“植物”包括但不限于：拟南芥，还包括樱桃、草莓、苹果、梨、枣、桃、猕猴桃、葡萄、橘子、柿子、李子、杏和香蕉等在内的水果；包括玫瑰、月季、菊花、百合、郁金香等在内的花卉；包括大白菜、番茄、辣椒、黄瓜、洋葱、胡萝卜等在内的蔬菜作物；包括小麦、大麦、大豆、棉花、水稻、马铃薯、高粱等在内的粮食作物。凡是具有该基因或者与之同源的基因均适用。
本发明中所说的“植物”包括整株植物，其亲本和子代植株以及植物的不同部位，包括种子、果实、芽、茎、叶、根(包括块茎)、花、组织和器官，在这些不同的部分均有我们目的基因或者核酸。这里所提及的“植物”也包括植物细胞、悬浮培养物、愈伤组织、胚、分生组织区、配子体、孢子体、花粉和小孢子，同样，其中每种前述对象包含目的基因/核酸。
本发明包括任何植物细胞，或任何由其中的方法获得或可获得的植物，以及所有的植物部分及其繁殖体。本专利也包含由任何前述方法所获得的转染细胞、组织、器官或完整植物。唯一的要求是子代表现出相同的基因型或表型特征，使用本专利中的方法获得的子代特性相同。
本发明还扩展到如上所述的植物的可收获的部分，但不限于种子、叶、果实、花、茎、根、根茎、块茎和球茎。同时进一步涉及植株收获后的其他衍生物，如干燥颗粒或粉末、油、脂肪和脂肪酸、淀粉或蛋白质。本发明还涉及由相关植物获得的食品或食品添加剂。
本发明的优点：
发明人通过对基因AtCAREF功能缺失突变体caref和野生型进行功能验证，有利于从分子机制上阐明AtCAREF在调控植株开花时间中的作用，对进一步阐明开花时间调控机制，培育提早开花的植物新品种具有积极的指导作用。因此，可通过基因工程手段，调节AtCAREF基因的表达，进而调节植物的开花时间。对于需要培育早开花早结果的果树，该基因对其同源基因的功能具有指导意义，因此对提高农业经济生产也具有重要的作用。
附图说明
图1是拟南芥钙结合蛋白基因AtCAREF家族进化树示意图。
图2是突变体拟南芥鉴定；图2A是突变体T-DNA插入位点；图2B突变体拟南芥PCR鉴定；
图中，P1表示正向引物，P2表示反向引物，Lbc1表示插入片段。
图3为野生型拟南芥和caref突变体在培养皿及土里的表型鉴定。
图4是野生型拟南芥和突变体拟南芥生长一个月的表型及种子鲜重对比实验结果。
图5是野生型拟南芥和突变体拟南芥生长过程表型实验结果。
图6是野生型拟南芥和突变体拟南芥生长曲线示意图。
具体实施方式
下面将通过具体实施例对本发明进行详细的描述。提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
若未特别指明，实施例中所用技术手段为本领域技术人员所熟知的常规手段。下述实施例中的试验方法，如无特别说明，均为常规方法。如无特殊说明，所采用的试剂及材料，均可以通过商业途径获得。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。
除非另有说明，本发明的实施将使用本领域技术人员显而易见的植物学常规技术、微生物、组织培养、分子生物学、化学、生物化学、DNA重组及生物信息学技术。这些技术均在已经公开的文献中进行了充分解释，另外，本发明所采用的DNA提取、系统发育树的构建、基因编辑方法、基因编辑载体的构建、基因编辑植物获得等方法，除了下述实施例采用的方法外，采用现有文献中已经公开的方法均能实现。
此处使用的“核酸”、“核酸序列”、“核苷酸”、“核酸分子”或“多聚核苷酸”术语意思是指包括分离的DNA分子(例如，cDNA或者基因组DNA)，RNA分子(例如，信使RNA)，自然类型，突变类型，合成的DNA或RNA分子，核苷酸类似物组成的DNA或RNA分子，单链或是双链结构。这些核酸或多聚核苷酸包括基因编码序列、反义序列及非编码区的调控序列，但不仅限于此。这些术语包括一个基因。“基因”或“基因序列”广泛用来指一有功能的DNA核酸序列。因此，基因可能包括基因组序列中的内含子和外显子，和/或包括cDNA中的编码序列，和/或包括cDNA及其调控序列。在特殊实施方案中，例如有关分离的核酸序列，优先默认其为cDNA。
另外，为了对本发明技术方案更直观的理解，对于本发明涉及到的一些专业术语解释如下：
“突变体”(Mutant)，是指发生突变的个体，具有与野生型不同的表型的特点。
“表达载体”(Expression vectors)，是指在克隆载体基本骨架的基础上增加表达元件(如启动子、RBS、终止子等)，使目的基因能够表达的载体。
一.基因AtCAREF的获取
利用转录组、基因组和蛋白组学等多种技术手段筛选得到基因AtCAREF，家族进化树示意图见图1，其核苷酸序列如SEQ ID NO.1所示，所述的核苷酸序列由1701个碱基组成，共编码566个氨基酸，其序列如SEQ ID NO.2所示。由于AtCAREF有CaM结合结构域，并且经实验鉴定，AtCAREF基因具有与其他钙调蛋白相同的调节植物开花的功能，因此命名为AtCAREF。
如图2A所示，T-DNA插入突变体caref(SAIL-341-D09)种子购于ABRC(theArabidopsis Biological Resource Center，Ohio State University，USA)，ABRC提供的Salk T-DNA插入资料显示，caref的T-DNA反向插入AtCAREF的外显子上。
一般而言，种子库中购买的种子有可能含有杂合子及野生型，所以需要对购买的种子进行分子标记鉴定，从中筛选纯合子。将突变体caref相应的野生型种子灭菌后分别种到1/2MS固体培养基上，光下培养两周，随机取10颗幼苗，利用Trizol试剂盒提取植物总RNA，然后用One-Step RT-PCR试剂盒进行扩增，所用引物如下：
正向引物：ATGGCAGGTGTTACTCTCTTCTTCT；
反向引物：TTAGTTCTTGTCTAGAAAATTTAAACGGA；
将RT-PCR扩增产物进行琼脂糖凝胶电泳，结果如图2B所示。图2B中的野生型可以检测出基因AtCAREF的表达产品-mRNA，而突变体caref检测不到到相关基因的表达，说明基因AtCAREF因为外源DNA的插入而被破坏，成为功能缺失突变体，可以作为基因AtCAREF功能研究的材料。
三.基因AtCAREF的功能鉴定
本发明发明人获得了AtCAREF的功能缺失突变体caref。为了进一步了解AtCAREF基因在植株生长发育过程中的作用，拟通过实验观察了突变体caref及野生型拟南芥Columbia的生长情况。
将前述2种拟南芥种子用75％酒精浸泡3s，再用适量蒸馏水浸泡后，在4℃冰箱处理48h，然后0.1％HgCl2消毒灭菌5min，最后用无菌水清洗5次，播种到1/2MS培养基和营养土与蛭石比例为1∶1的土壤中，置于16小时光照/8小时黑暗的光周期、22-24℃的温度条件下培养。逐日观察各基因型植株的生长发育情况(图5)，最典型的差异是突变体和野生型的开花时间不同(图3)。以本领域熟知的拟南芥花茎长度达1cm时为开花标志，与野生型Columbia植株相比，caref突变体早开花10天左右，开花时植株的莲座叶多1-2片(图6)，caref突变体在使拟南芥提前开花的情况下，两种拟南芥种子鲜重并未有明显差异(图4)，因此，说明AtCAREF基因所编码的蛋白AtCAREF具有抑制开花的功能，且不影响种子的鲜重。
以上所述之实施例，只是本发明的较佳实施例而已，仅仅用以解释本发明，并非限制本发明实施范围，对于本技术领域的技术人员来说，当然可根据本说明书中所公开的技术内容，通过置换或改变的方式轻易做出其它的实施方式，故凡在本发明的原理上所作的变化和改进等，均应包括于本发明申请专利范围内。
序列表
<110> 鲁东大学，山东百思禾未来农业科技发展有限公司，招远市盛慧农业技术发展有限公司
<120> 拟南芥钙结合蛋白基因AtCAREF及应用
<130> 2022
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1701
<212> DNA
<213> Arabidopsis thaliana
<400> 1
atggcaggtg ttactctctt cttctccctt ctctctatag ctctgattat ctctggtgtg 60
agctctcgtg tcctgatcag tcatgtccct ctgaataact cgatcttgat atctgatggg 120
atccatgacg ccttgaatca cgagtttctc accctcgatc ctccaaagag cttgtcaagg 180
accgcgtgtg ttcatgtcta cgggtttctt ccatgtgcag acaatgttga aggttatatc 240
ttccaagttt tttcctttgg gagtctcttg attatcggtg actatttctt gtccgaagga 300
agatctaaac tctttgtaat attcgaggtc gggttctatg gtggtatcat cttccctctt 360
cttacaatgt tcccaagaat cgctcttatg ctctcgaccg gactatcatt gagtagggat 420
gttgctagct cctttataga tgataacgtt ggacttactg tgggacacac tgtgttttct 480
ctcacaattc aatggggagc ttgtgttgtg tttagcatca ctggtccacg ttctgaccaa 540
gcagatggat tgatcgaaaa actaaagata ttgaaaggct ttgttgaagc aagagtagaa 600
gcggatccta agaataagaa agctgcaggg attatgctgt tgagtctatc tccctttctc 660
atggtgacat tttcggctat atttgattca cattcttgga gtcacatcat tgtgttgatc 720
acactcataa tttcttcttc ttcaaccgtt gtctactttg tttactcgta tcttgatacg 780
gctaaccaag agaagagctt agatcacgcg aggtttgagc tcatgtcaga agttcataag 840
cacttaaaga ggttttcacc gaaacacctt ataaaagatg gagaactaag caaagagagc 900
ttgaaaagtt tgttcaagaa aactgataag aacaaagatg ggaagataca aatatctgaa 960
ctaaaagact taaccataga gctttccaac tttgggagaa tgagatatga catcaatgag 1020
cttgctaaag cttttcttga ggattttgat ggagacaacg acggcgaatt agaagagaat 1080
gaattcgagg aagggattgc gagacttctc aaacaataca aattcaacgt tgaggatcaa 1140
agagagaatc agactgagga aaatggagtt cttaagctgg aaattaaacc gaagaaaaca 1200
cttgttacta agttgctatc tatggaaaca ttaatagcta cgactgaagt catcgtaggg 1260
atcctaattg tactctttct tgcaaaacca tttatgctga acatccagct cttatccatc 1320
tcagctggaa ttccttcttt ctacatcgtg ttcgcgatga tccctttcgc tagaaactta 1380
aagaacactc tatctacacg cttctgtcgt ggcaaagaca agaaaagagt ctcgtccaat 1440
actttctccg agatctacaa agatattaca atgaacaatc tcttgggaat gtcggtaata 1500
ttggcaattg tgtatacaag gggcttgact tggaaatact cggtagaaac tcttattatg 1560
gtgatcgttg gcctcataat cggcttacca atctatataa gatcgactta cccgttctgg 1620
atgtgtgtct tggcctttgc tatgtacttc ttttctcttc tccttatcta tatccgttta 1680
aattttctag acaagaacta a 1701
<210> 2
<211> 566
<212> PRT
<213> Arabidopsis thaliana
<400> 2
Met Ala Gly Val Thr Leu Phe Phe Ser Leu Leu Ser Ile Ala Leu Ile
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Ile Ser Gly Val Ser Ser Arg Val Leu Ile Ser His Val Pro Leu Asn
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Asn Ser Ile Leu Ile Ser Asp Gly Ile His Asp Ala Leu Asn His Glu
35 40 45
Phe Leu Thr Leu Asp Pro Pro Lys Ser Leu Ser Arg Thr Ala Cys Val
50 55 60
His Val Tyr Gly Phe Leu Pro Cys Ala Asp Asn Val Glu Gly Tyr Ile
65 70 75 80
Phe Gln Val Phe Ser Phe Gly Ser Leu Leu Ile Ile Gly Asp Tyr Phe
85 90 95
Leu Ser Glu Gly Arg Ser Lys Leu Phe Val Ile Phe Glu Val Gly Phe
100 105 110
Tyr Gly Gly Ile Ile Phe Pro Leu Leu Thr Met Phe Pro Arg Ile Ala
115 120 125
Leu Met Leu Ser Thr Gly Leu Ser Leu Ser Arg Asp Val Ala Ser Ser
130 135 140
Phe Ile Asp Asp Asn Val Gly Leu Thr Val Gly His Thr Val Phe Ser
145 150 155 160
Leu Thr Ile Gln Trp Gly Ala Cys Val Val Phe Ser Ile Thr Gly Pro
165 170 175
Arg Ser Asp Gln Ala Asp Gly Leu Ile Glu Lys Leu Lys Ile Leu Lys
180 185 190
Gly Phe Val Glu Ala Arg Val Glu Ala Asp Pro Lys Asn Lys Lys Ala
195 200 205
Ala Gly Ile Met Leu Leu Ser Leu Ser Pro Phe Leu Met Val Thr Phe
210 215 220
Ser Ala Ile Phe Asp Ser His Ser Trp Ser His Ile Ile Val Leu Ile
225 230 235 240
Thr Leu Ile Ile Ser Ser Ser Ser Thr Val Val Tyr Phe Val Tyr Ser
245 250 255
Tyr Leu Asp Thr Ala Asn Gln Glu Lys Ser Leu Asp His Ala Arg Phe
260 265 270
Glu Leu Met Ser Glu Val His Lys His Leu Lys Arg Phe Ser Pro Lys
275 280 285
His Leu Ile Lys Asp Gly Glu Leu Ser Lys Glu Ser Leu Lys Ser Leu
290 295 300
Phe Lys Lys Thr Asp Lys Asn Lys Asp Gly Lys Ile Gln Ile Ser Glu
305 310 315 320
Leu Lys Asp Leu Thr Ile Glu Leu Ser Asn Phe Gly Arg Met Arg Tyr
325 330 335
Asp Ile Asn Glu Leu Ala Lys Ala Phe Leu Glu Asp Phe Asp Gly Asp
340 345 350
Asn Asp Gly Glu Leu Glu Glu Asn Glu Phe Glu Glu Gly Ile Ala Arg
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Leu Leu Lys Gln Tyr Lys Phe Asn Val Glu Asp Gln Arg Glu Asn Gln
370 375 380
Thr Glu Glu Asn Gly Val Leu Lys Leu Glu Ile Lys Pro Lys Lys Thr
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Leu Val Thr Lys Leu Leu Ser Met Glu Thr Leu Ile Ala Thr Thr Glu
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Val Ile Val Gly Ile Leu Ile Val Leu Phe Leu Ala Lys Pro Phe Met
420 425 430
Leu Asn Ile Gln Leu Leu Ser Ile Ser Ala Gly Ile Pro Ser Phe Tyr
435 440 445
Ile Val Phe Ala Met Ile Pro Phe Ala Arg Asn Leu Lys Asn Thr Leu
450 455 460
Ser Thr Arg Phe Cys Arg Gly Lys Asp Lys Lys Arg Val Ser Ser Asn
465 470 475 480
Thr Phe Ser Glu Ile Tyr Lys Asp Ile Thr Met Asn Asn Leu Leu Gly
485 490 495
Met Ser Val Ile Leu Ala Ile Val Tyr Thr Arg Gly Leu Thr Trp Lys
500 505 510
Tyr Ser Val Glu Thr Leu Ile Met Val Ile Val Gly Leu Ile Ile Gly
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Leu Pro Ile Tyr Ile Arg Ser Thr Tyr Pro Phe Trp Met Cys Val Leu
530 535 540
Ala Phe Ala Met Tyr Phe Phe Ser Leu Leu Leu Ile Tyr Ile Arg Leu
545 550 555 560
Asn Phe Leu Asp Lys Asn
565
Claims (10)
1.拟南芥钙结合蛋白基因AtCAREF，其特征在于，所述基因AtCAREF的核苷酸序列如SEQID NO.1所示。
2.抑制权利要求1所述拟南芥钙结合蛋白基因AtCAREF的表达在促进植物提早开花中应用。
3.根据权利要求2所述的应用，其特征在于，抑制拟南芥钙结合蛋白基因AtCAREF的表达的方法是通过获得基因AtCAREF的功能缺失突变体caref。
4.一种植物育种方法，其特征在于，所述方法为通过抑制权利要求1所述拟南芥钙结合蛋白基因AtCAREF的表达，获得提早开花的植物。
5.根据权利要求4所述的方法，其特征在于，所述植物为拟南芥。
6.权利要求1所述的拟南芥钙结合蛋白基因AtCAREF在控制植物开花时间中的应用。
7.根据权利要求6所述的应用，其特征在于，控制植物开花时间表现为延后植物的开花时间。
8.根据权利要求6或7所述的应用，其特征在于，所述植物包括拟南芥、樱桃、草莓、苹果、梨、枣、桃、猕猴桃、葡萄、橘子、柿子、李子、杏和香蕉、玫瑰、月季、菊花、百合、郁金香、大白菜、番茄、辣椒、黄瓜、洋葱、胡萝卜、小麦、大麦、大豆、棉花、水稻、马铃薯、高粱。
9.一种植物育种方法，其特征在于，所述方法为(1)、(2)或(3)：
(1)通过增加目的植物中蛋白AtCAREF的活性，获得开花时间晚于目的植物的植株；
(2)通过促进目的植物中基因AtCAREF的表达，获得开花时间晚于目的植物的植株；
(3)通过抑制拟南芥中基因AtCAREF的表达，获得提早开花的拟南芥植株；
所述基因AtCAREF的核苷酸序列如SEQ ID NO.1所示，所述蛋白AtCAREF的氨基酸序列如SEQ ID NO.2所示。
10.根据权利要求9所述的方法，其特征在于，所述目的植物包括拟南芥、樱桃、草莓、苹果、梨、枣、桃、猕猴桃、葡萄、橘子、柿子、李子、杏和香蕉、玫瑰、月季、菊花、百合、郁金香、大白菜、番茄、辣椒、黄瓜、洋葱、胡萝卜、小麦、大麦、大豆、棉花、水稻、马铃薯、高粱。
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| CN117210490B (zh) * | 2023-11-08 | 2024-03-05 | 中国农业大学 | 一种调控苹果属植物自花结实的pchr基因及其应用 |
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Effective date of registration: 20231008 Address after: 264025 No. 184 Hongqi Middle Road, Zhifu District, Shandong, Yantai Patentee after: LUDONG University Patentee after: BSH (Shandong) Stem Cell Engineering Co.,Ltd. Patentee after: Zhaoyuan Shenghui Agricultural Technology Development Co.,Ltd. Address before: 264025 No. 184 Hongqi Middle Road, Zhifu District, Shandong, Yantai Patentee before: LUDONG University Patentee before: Shandong baisihe future agricultural technology development Co.,Ltd. Patentee before: Zhaoyuan Shenghui Agricultural Technology Development Co.,Ltd. |