CN114426975B - 番茄谷氧还蛋白SlGRXC9基因及应用 - Google Patents
番茄谷氧还蛋白SlGRXC9基因及应用 Download PDFInfo
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Abstract
本发明公开了一种番茄谷氧还蛋白SlGRXC9基因及应用,本发明首次从番茄中获得番茄谷氧还蛋白SlGRXC9基因,并且通过利用农杆菌介导的方法将SlGRXC9从番茄MT(micro‑Tom)中敲除进行功能验证,得出SlGRXC9基因缺失可诱导番茄植株对冷胁迫敏感,同时出现株高降低、叶长和叶宽降低等矮化症状,这也体现了SlGRXC9在调控番茄发育及响应低温胁迫中的具有重要的作用。
Description
技术领域
本发明属于生物技术领域,具体涉及番茄谷氧还蛋白SlGRXC9基因及应用。
背景技术
低温胁迫严重影响番茄等园艺作物的品质和产量,已成为限制我国设施农业发展的瓶颈问题之一。随着基因工程技术的不断发展,可通过基因工程技术对番茄响应低温胁迫相关基因进行精确编辑,研究基因编辑株系对低温处理的表型变化。如果基因编辑植株低温处理敏感,则在植物中过表达该基因;如果基因编辑植株对低温处理的耐受性增强,则直接应用于生产。本发明致力于通过基因工程手段来提高蔬菜作物在不良环境条件下的抗性,将对提高蔬菜产量、品质和经济效益,保障我国蔬菜均衡供应具有十分重要的科学和现实意义。
番茄是全世界非常重要的果实类作物以及模式作物,生长发育易受到不利环境条件的影响。随着2012年番茄的基因组测序,科学家采用反向遗传学研究手段,在番茄中报道了许多响应逆境胁迫的基因。活性氧(Reactive oxygen species,ROS)是植物体内正常代谢的信号小分子,在植物生长发育和抗逆反应的信号转导中具有重要作用。但是过多的活性氧积累会严重影响植物的生长发育,造成氧化胁迫。谷氧还蛋白(Glutaredoxins,GRXs)是一类小分子的氧化还原蛋白,能够调节蛋白质的氧化还原状态,从而维持蛋白质的活性,在植物抗氧化胁迫及生长发育反应中具有重要作用。GRX在早期也被称为巯基转移酶,最早在哺乳动物中发现,一般含有100个左右氨基酸残基。在大肠杆菌中发现了一个结构和功能类似的蛋白,由于它依赖于GSH的还原活性,所以被命名为GRX。GRXs是一个多基因家族,根据其活性位点序列可以将其分成CPYC型、CGFS型和CC型。本发明中的番茄谷氧还蛋白SlGRXC9属于CC型。目前对模式植物拟南芥的研究表明,GRXC9在调控叶柄发育、真菌抗性和防御响应方面发挥着重要作用。对于植物中的GRXC9是否还具有其它方面的应用目前还没有报道,尤其是有关番茄响应冷胁迫调控相关机理的深入研究却鲜有报道。因此,从基因层面研究蛋白氧化还原状态对番茄的生长发育及逆境抗性的影响具有重要意义。
发明内容
本发明的目的之一在于提供一种番茄谷氧还蛋白SlGRXC9基因。
本发明的第二个目的是提供上述番茄谷氧还蛋白SlGRXC9基因的应用。
为了实现上述目的,本发明采用的技术方案概述如下:
一种番茄谷氧还蛋白SlGRXC9基因,其核苷酸序列如SEQ ID NO.1 所示,所述的核苷酸序列由444个碱基组成,共编码147个氨基酸,其序列如SEQ ID NO.2 所示。
上述谷氧还蛋白SlGRXC9基因编码的蛋白选自,
(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,不仅包括上述番茄谷氧还蛋白SlGRXC9基因,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番茄谷氧还蛋白SlGRXC9进行克隆和鉴定,从而解析番茄冷胁迫响应和生长发育调控相关机理的研究提供理论基础。本发明还公开了抑制上述番茄谷氧还蛋白SlGRXC9基因的表达在如下方面的应用:
(1)矮化植物;
(2)冷胁迫处理敏感表型;
上述应用通过敲除上述基因的方式来获得矮化、冷胁迫敏感症状的植物。
其中,矮化表现包括平均株高的高度降低,小叶表型包括叶片的长和宽均变小,冷胁迫表型包括4℃处理敏感表型。
作为本发明的一种实施方式,将多核苷酸通过常规的方法克隆到CRISRP载体中,将所述的带有外源基因的重组载体导入到可表达所述SlGRXC9蛋白到植物细胞中,使所述的植物细胞中SlGRXC9蛋白缺失。可通过将所述植物细胞再生成植物,获得SlGRXC9基因缺失的突变体植物。并利用农杆菌转化法将重组质粒转入植物中。
由于实验证明,通过基因编辑技术抑制所述SlGRXC9基因的表达后,植株表现为在低温处理下敏感,也就是说耐寒性降低,可以得知,SlGRXC9基因具有增强植株耐寒性的作用。
根据该特性,可以通过转基因的方式来获得耐寒的植株,具体地,可以通过将SlGRXC9基因导入目的植物,得到转基因植物,该植株耐寒性高于目的植物。
具体地,SlGRXC9基因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SlGRXC9蛋白的活性,获得耐寒性强于目的植物的植株;
(2)通过促进目的植物中SlGRXC9基因的表达,获得耐寒性强于目的植物的植株;
(3)通过抑制目的植物中的SlGRXC9基因的表达,获得矮化和/或低温处理敏感症状的植株。
“促进目的植物中SlGRXC9基因的表达”的实现方式可为如下(1)、(2)或(3):
(1)将GmPCBER4基因导入目的植物;
(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SlGRXC9基因的表达可以采用敲除SlGRXC9基因的方式,敲除SlGRXC9基因的方式可以采用基因编辑技术。
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:茄科的番茄,凡是具有该基因或者与之同源的基因均适用。尤其适用于需要矮化的植物,比如蔷薇科苹果树、樱桃树,均可通过敲除SlGRXC9基因获得矮化植株。
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:
(1)发明人利用农杆菌介导的方法将SlGRXC9从番茄MT中敲除进行功能验证,有利于从分子机制上阐明SlGRXC9在调控株型和冷胁迫响应方面的作用,对进一步阐明番茄株型调控机制,培育低温耐受性番茄新品种具有积极的指导作用。
(2)SlGRXC9基因缺失可同时诱导整株番茄各器官的畸形发育,出现株高降低、小叶表型等症状,并且出现低温处理敏感症状,这也体现了SlGRXC9在植物发育的进程中扮演的关键作用。
(3)对于一些需要矮化的植物如果树和观赏植物(减少不必要的养分消耗,以便充分利用光能,地力,提早结果、提高产量或增加观赏效果),可以通过敲除SlGRXC9基因的方式,为植物矮化育种提供一种新的途径。
(4)可以通过转基因的方式来获得耐寒的植株,具体地,可以通过将SlGRXC9基因导入目的植物,得到转基因植物,该植株耐寒性高于目的植物,为植物耐寒育种提供一种新的途径。
附图说明
图1是2种不同编辑类型的SlGRXC9敲除系。
图2是野生型番茄WT、WT cas9、slgrxc9 #1和slgrxc9#2的发育表型。
图3为野生型番茄WT、WT cas9、slgrxc9 #1 和slgrxc9#2的冷胁迫处理表型;图中,WT:野生型;WT cas9:空载对照;slgrxc9 #1和slgrxc9#2:突变体植株。
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:
“基因编辑”(Gene editing),是一种新兴的精确对生物体基因组特定靶序列进行修饰的基因功能技术。
“基因敲除”(Gene knockout),是通过同源重组将外源基因定点整合入靶细胞基因组上某一确定的位点,以达到定点修饰改造染色体上某一基因的目的的一种技术。
“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。
发明人通过生物信息学技术在番茄基因组基础上得到番茄谷氧还蛋白SlGRXC9基因序列。本发明将其与植物编辑载体连接后导入野生型番茄进行表型鉴定。
与传统转基因技术相比,CRISPR/Cas9基因编辑技术存在许多优点。传统转基因技术是将外源目的基因导入目的生物体中,从而改变植物性状,目的基因在受体基因组中的插入位置是随机的,而CRISPR/Cas9基因编辑技术没有外源基因的导入,只对内源基因进行修饰,其更加安全、高效;其次,CRISPR/Cas9基因编辑技术可以实现同时对多个位点进行编辑,并能够对突变或插入位点实现精确调控,这是传统转基因技术所无法完成的;另外,传统转基因技术只能在RNA水平上下调目的基因的表达,并不能完全抑制其转录,且遗传不稳定,而CRISPR/Cas9基因编辑技术能够实现靶基因的突变,更加稳定可靠。
1、SlGRXC9基因的分离
MT番茄幼苗总RNA的提取:按照Trizol提取试剂(Takara)说明进行,第一链cDNA合成按照反转录试剂盒PrimerScriptTM II 1st Strand cDNA Synthesis Kit(Takara)说明进行。以番茄的cDNA为模板,使用Primer STAR Max高保真酶扩增(Takara),退火温度为58℃。
以下述序列为引物,通过PCR扩增获得SlGRXC9基因的全长序列。SlGRXC9基因的全长序列如序列表中SEQ ID NO.1所示,共444 bp,共编码蛋白的氨基酸序列如序列表中SEQID NO.2所示,共147个。
引物序列为:
SlGRXC9-F:5'- ATGCAACAAGCACTTCCTTAC -3';
SlGRXC9-R:5'- TCAAAGCCATAAGGCTCCAGC -3'。
2、SlGRXC9基因的功能鉴定试验
为研究SlGRXC9基因是否调控番茄对低温的耐受性,通过基因敲除系番茄来鉴定其功能。
2.1构建重组载体
在SlGRXC9基因CDS区域选取靶位点,即TAACGTTGTTAAGGATAACGCGG。回收纯化sgRNA-SlGRXC9片段并与酶切后的载体链接,从而获得SlGRXC9-CRISPR重组质粒。并通过农杆菌介导进行番茄遗传转化,获得番茄SlGRXC9-CRISPR的转化苗。
2.2转基因阳性株的筛选及表型分析
取抗性植株叶片100 mg,采用CTAB法提取基因组总DNA。并根据靶基因序列设计特异性引物,进行PCR扩增,并送样测序。
引物序列为:
SlGRXC9-g1-F: 5'- GTTGATCGACATCATCGTGTA -3';
SlGRXC9-g1-R: 5'- CGATTTCATAAATGGCAGGAT -3'。
经测序鉴定,共获得2种不同编辑类型的SlGRXC9敲除系,如slgrxc9 #1 (+1 bp)和slgrxc9#2(-5 bp)(图1)。
图2为野生型番茄WT(野生型)、WT cas9(空载对照)、slgrxc9 #1 (+1 bp)和slgrxc9#2(-5 bp)的表型观察图,从图2可以看出,SlGRXC9基因敲除系的平均株高均显著小于野生型以及空载对照,形成矮化表型;SlGRXC9基因敲除系叶片大小的生长显著受到抑制,与野生型以及空载对照相比,其叶长、叶宽显著降低。
图3A是为野生型番茄WT(野生型)、WT cas9(空载对照)、slgrxc9 #1 (+1 bp)和slgrxc9#2(-5 bp)分别在4度低温冷室下进行处理,从图3A可以看出,SlGRXC9敲除系与野生型和空载对照相比,对低温处理更敏感,因此生理表型电解质渗透率数值更大 (图3B),另外,图3C表明SlGRXC9的基因表达水平受低温处理的显著诱导(Actin7作为内参)。
整体看来,与野生型相比,基因敲除系植株表现出株高降低、叶长和叶宽降低,形成矮化表型。除此之外,SlGRXC9基因对番茄低温耐受性存在明显影响。
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。
SEQUENCE LISTING
<110> 河南大学三亚研究院
<120> 番茄谷氧还蛋白SlGRXC9基因及应用
<130> 2022
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 444
<212> DNA
<213> Lycopersicon esculentum
<400> 1
atgcaacaag cacttcctta caagtcatca tgtatatctc taacaccaag agttgatcga 60
catcatcgtg taagtaatat caattcatta ttatacgtta aaggttcaaa agaagaattg 120
aataacgttg ttaaggataa cgcggttatc gttgtgggaa gacgaggttg ttgtatgagc 180
catgttgtga aacgtttact tcattgtctc ggagcaaatc ctgccattta tgaaatcgag 240
gaagacgatg aaaacgaagt ggttgatgag ttggagaata ttatcgtcgc cggaggtagt 300
gatcggaaag acaccggacg gttgcaattt ccggcggtgt tcgtcggagg ggagctgttt 360
ggtggattgg atcggattat ggcggctcat attaccggcg agttgactcc tgtgttgaaa 420
aaggctggag ccttatggct ttga 444
<210> 2
<211> 147
<212> PRT
<213> Lycopersicon esculentum
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Arg Val Asp Arg His His Arg Val Ser Asn Ile Asn Ser Leu Leu Tyr
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Val Lys Gly Ser Lys Glu Glu Leu Asn Asn Val Val Lys Asp Asn Ala
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Val Ile Val Val Gly Arg Arg Gly Cys Cys Met Ser His Val Val Lys
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Arg Leu Leu His Cys Leu Gly Ala Asn Pro Ala Ile Tyr Glu Ile Glu
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Ala Gly Gly Ser Asp Arg Lys Asp Thr Gly Arg Leu Gln Phe Pro Ala
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Val Phe Val Gly Gly Glu Leu Phe Gly Gly Leu Asp Arg Ile Met Ala
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Ala His Ile Thr Gly Glu Leu Thr Pro Val Leu Lys Lys Ala Gly Ala
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Leu Trp Leu
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Claims (3)
1.抑制番茄谷氧还蛋白SlGRXC9基因表达的试剂在矮化番茄方面的应用,其特征在于,所述SlGRXC9基因的核苷酸序列如SEQ ID NO.1所示,所述矮化表现包括平均株高降低,叶长和叶宽降低,所述试剂通过敲除SlGRXC9基因的方式来获得矮化的番茄。
2.根据权利要求1所述的应用,其特征在于,敲除SlGRXC9基因采用基因编辑技术。
3.一种植物育种方法,其特征在于,所述方法为通过抑制目的植物中的SlGRXC9基因的表达,获得矮化的植株;所述SlGRXC9基因的核苷酸序列如SEQ ID NO.1所示,所述SlGRXC9蛋白的氨基酸序列如SEQ ID NO.2所示,所述矮化表现包括平均株高降低,叶长和叶宽降低,所述目的植物为番茄,所述抑制目的植物中SlGRXC9基因的表达的方式为敲除SlGRXC9基因。
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