CN114736278B - 一种马铃薯花色素苷生物合成负调控基因、转录因子及应用 - Google Patents
一种马铃薯花色素苷生物合成负调控基因、转录因子及应用 Download PDFInfo
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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Abstract
本发明公开了一种马铃薯花色素苷生物合成负调控基因、转录因子及应用,涉及生物技术领域。马铃薯花色素苷生物合成负调控转录因子具有如SEQ ID NO.1所示的氨基酸序列;或具有如SEQ ID NO.1所示的氨基酸序列和如SEQ ID NO.3所示的氨基酸序列。本发明提供的马铃薯花色素苷生物合成负调控基因及转录因子,具有用于构建转化植物的表达载体、重组菌及培育花色素苷积累量可调控的新品种的应用潜力。在生产上对调控植物花色素苷积累、调控植物花色变浅、调控植物黄酮醇积累、调控植物黄酮醇合成中有着广泛的应用前景。
Description
技术领域
本发明涉及生物技术领域,具体而言,涉及一种铃薯花色素苷生物合成负调控基因、转录因子及应用。
背景技术
马铃薯是世界第三大重要的粮食作物,马铃薯产业的可持续发展对我国的农业生产和粮食安全具有重要意义。马铃薯种质资源丰富,其中彩色马铃薯不仅色彩绚丽,而且营养丰富,它具有较为优良的加工品质、较高的附加值,因此其市场发展潜力无限。其中,彩色马铃薯富含花色素苷,花色素苷除了赋予马铃薯黑红紫等丰富的色泽外,还作为植物中最主要的黄酮类物质。花色素苷已被证明具有超强抗氧化,能够有效抑制炎症反应、提高人体免疫力、防范心血管疾病、提高抗癌能力等。目前,花色素苷的医药价值受到人们广泛关注,因此对马铃薯块茎花色素苷调控机制开展研究,可以深入探索花色素苷的生物合成途径,为创制彩色马铃薯品种奠定基础。
目前,研究表明花色素苷的合成代谢主要受到MYB、bHLH和WD40三大类转录因子的调控,而MYB为最关键的转录因子。早期研究发现马铃薯块茎薯皮中的花色素苷积累主要受到三个基因位点:D、P和R的控制。Salaman首次发现了四倍体马铃薯R基因,编码二氢黄酮醇还原酶(DFR),是马铃薯植株产生红色色素所必需的,DFR不仅会增加马铃薯块茎中红色色素-天竺葵色素合成,也能促进紫色色素-矮牵牛色素的合成;P基因编码类黄酮3′5′羟化酶(F3′5′H)则控制紫色色素的产生;Jung等研究发现,D为MYB转录因子,编码R2R3 MYB–StAN1基因,在红皮和紫皮马铃薯中高表达,调控马铃薯薯皮花色素苷合成。进一步研究发现StAN1转录因子对马铃薯块茎和叶片花色素苷合成均有促进作用,StAN1基因的编码序列和预测的启动子在不同马铃薯基因型中存在广泛的核苷酸序列变异。StAN1在马铃薯红肉和紫肉品种中存在三种等位基因(基因家族):StAN1-R0,StAN1-R1和StAN1-R3,其中StAN1-R1调控花色素苷的能力最强,除StAN1之外,MYB转录因子StMYBA1、StMYB113也可以促进花色素苷的积累,同时在彩色块茎中含有3个功能StbHLH1转录因子,与StAN1具有不同的共调控能力。Payyavula等进一步研究发现StAN1基因受蔗糖诱导,不仅调控马铃薯薯皮花色素苷合成,而且调控苯丙氨酸代谢途径。高温会诱导StMYB44转录因子的表达,以不依赖于bHLH的方式下调花色素苷结构基因以及转录激活子的表达,从而抑制花色素苷生物合成分支,导致苯丙烷代谢途径转入绿原酸或木质素生物合成分支中。
总之,马铃薯块茎花色素苷调控机制还有待探明,特提出本发明。
发明内容
本发明的目的在于提供一种铃薯花色素苷生物合成负调控基因、转录因子及应用以解决上述技术问题。
本发明是这样实现的:
本发明提供了一种马铃薯花色素苷生物合成负调控转录因子,其具有如SEQ IDNO.1所示的氨基酸序列;
或同时具有如SEQ ID NO.1所示的氨基酸序列和如SEQ ID NO.3所示的氨基酸序列。
发明人发现StMYB3基因为转录抑制子,既可以单独负向调控花色素苷生物合成,也可以与另一种转录抑制子StMYBATV协同负向调控花色素苷生物合成。进一步通过双分子荧光互补技术BiFC发现StMYB3和StMYBATV会与花色素苷转录调控因子StbHLH1结合。说明在彩色块茎色泽积累过程中,转录激活子StAN1和StbHLH协同正向调控块茎花色素苷生物合成,转录抑制子StMYB3和StMYBATV竞争性的与StbHLH1结合,起到反馈调节的作用,以避免花色素苷的过度积累。基于此,发明人提出一种马铃薯花色素苷生物合成负调控转录因子StMYB3,其具有SEQ ID NO.1所示的氨基酸序列。
SEQ ID NO.3为StMYBATV的氨基酸序列。
本发明还提供了一种马铃薯花色素苷生物合成负调控基因,其编码上述的马铃薯花色素苷生物合成负调控转录因子;
在一种可选的实施方式中,负调控基因具有如SEQ ID NO.2所示的核苷酸序列(StMYB3);或具有如SEQ ID NO.2所示的核苷酸序列和SEQ ID NO.4所示的核苷酸序列。
SEQ ID NO.4所示的核苷酸序列为StMYBATV基因的序列。
发明人发现,将StMYB3和StMYBATV共转化植物后,通过过表达或激活表达,能与StbHLH1相互作用,与StAN1竞争性结合StbHLH,避免花色素苷的过度积累。
单纯的转化StMYB3和StMYBATV至植物后,通过过表达或激活表达,也能避免花色素苷的过度积累。
本发明还提供了一种表达盒或载体,其含有上述马铃薯花色素苷生物合成负调控基因。
本发明还提供了一种重组菌或重组细胞,其含有上述的马铃薯花色素苷生物合成负调控基因、或表达盒或载体。
如上述的马铃薯花色素苷生物合成负调控转录因子、上述的马铃薯花色素苷生物合成负调控基因、表达盒或载体、重组菌或重组细胞具有如下用途:
在调控植物花色素苷积累中的应用、在调控植物花色变浅中的应用、在调控植物黄酮醇积累中的应用、或者在调控植物黄酮醇合成中的应用。
上述调控植物花色变浅包括不限于马铃薯着色的改善、植物颜色培育。例如使得马铃薯的薯肉颜色从黄色变为浅黄甚至到白色,或从紫色变为浅紫色,或从深紫变为紫色乃至浅紫色,从红色变为浅黄乃至白色。在其他实施方式中,只要能使得植物的颜色或着色变浅均属于本发明的保护范围之内。
上述调控植物花色素苷积累、调控植物黄酮醇积累中的应用包括不限于:与调控前相比,植物花色素苷积累量、植物黄酮醇积累量略微或明显下降。
在本发明应用较佳的实施方式中,上述用途包括如下至少一种的应用方式:
将马铃薯花色素苷生物合成负调控基因送入目的植物细胞;例如通过基因枪法将目的基因送入目的植物细胞。
将载体转化目的植物,载体含有马铃薯花色素苷生物合成负调控基因;转化的方法包括不限于农杆菌介导基因转化法,基因枪转化法、花粉管通道法。
将重组菌或重组细胞导入目的植物,重组菌或重组细胞含有马铃薯花色素苷生物合成负调控基因;
目的植物选自马铃薯或园艺植物。
园艺作物选自活的栽培或产果农业和观赏作物,包括不限于:水果、蔬菜、树木、花卉、草、根、种子和景观及观赏植物。
在一种可选的实施方式中,园艺植物选自:烟草、樱桃、桃、梨、柑桔、葡萄、草莓、西番莲、五色梅、天竺葵、君子兰、红薯或芦荟。
在本发明应用较佳的实施方式中,上述应用包括:修饰目的植物的内源花色素苷生物合成负调控基因,使其编码马铃薯花色素苷生物合成负调控转录因子。
在本发明提供了花色素苷生物合成负调控基因的基础上,本领域技术人员容易想到通过本领域常规的转基因技术、基因编辑技术(如通过锌指核酸内切酶(ZFN,zinc-finger nucleases)技术、类转录激活因子效应物核酸酶(TALEN,transcriptionactivator-like effector nucleases)技术或CRISPR/Cas9)、诱变育种技术(如化学、辐射诱变等)等对目标植物进行改造,使其具有上述花色素苷生物合成负调控基因,进而获得植物花色素苷积累可控的并能够正常生长和发育的植物新品种。因此,无论采用何种技术,只要其利用了本发明提供的花色素苷生物合成负调控基因赋予植物花色素苷积累可控,均属于本发明的保护范围。
在一种可选的实施方式中,目的植物选自马铃薯、梨或荔枝。
如上述的马铃薯花色素苷生物合成负调控转录因子、上述的马铃薯花色素苷生物合成负调控基因、表达盒或载体、重组菌或重组细胞在制备用于改善马铃薯着色或园艺作物颜色的制剂中的应用。
本发明还提供了一种用于检测马铃薯花色素苷生物合成负调控基因的引物组合,其包括第一引物对,第一引物对具有如SEQ ID NO.5-6所示的序列;
在一种可选的实施方式中,还包括第二引物对,第二引物对具有如SEQ ID NO.7-8所示的序列。
本发明还提供了一种试剂盒或试剂,其包括上述的引物组合。
本发明具有以下有益效果:
本发明提供的马铃薯花色素苷生物合成负调控基因及转录因子,具有用于构建转化植物的表达载体、重组菌及培育花色素苷积累量可调控的新品种的应用潜力。通过烟草等模式植物转化技术证明,大量表达该负调控基因能够抑制烟草的花色素苷的生物合成,改变花色,因而,在生产上对调控植物花色素苷积累、调控植物花色变浅、调控植物黄酮醇积累、调控植物黄酮醇合成中有着广泛的应用前景。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为StMYB3、AtMYB3、AtMYB4、PhMYB27、FaMYB1、AmMYB308、SlTHM27和PhMYB4的系统发育树和氨基酸序列比对结果图;
图2为马铃薯StMYB3和StMYBATV转录因子的功能分析结果图;
图3为转基因植株nptⅡ基因的PCR检测结果图;
图4为转基因植株目的基因StMYB3的PCR检测结果图;
图5为转StMYB3烟草花朵表型(以转空载体的植株为对照)图;
图6为马铃薯块茎薯肉花色素苷生物合成的基因调控网络示意图。
具体实施方式
现将详细地提供本发明实施方式的参考,其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上,对本领域技术人员而言,显而易见的是,可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如,作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中,来产生更进一步的实施方式。
除非另有定义,否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试,但现在描述一些方法和材料。除非另有说明,否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明,否则实践本发明将采用植物生理学、植物分子遗传学、细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术,所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术,如《分子克隆:实验室手册(Molecular Cloning:A Laboratory Manual)》,第二版(Sambrook等人,1989);《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编,1984);《植物生理学》(苍晶等人,2017);《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press,Inc.);《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编);《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编,1987);《植物分子遗传学》(Monica A.Hughes等人著);《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编,1994),所述文献中的每个文献均通过引用明确并入本文中。
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例选取三个不同颜色的马铃薯杂交子代在三个生育时期(块茎形成期-S1、膨大期-S2和成熟期-S3)的薯肉(Y:白-浅黄-黄,R:白-浅黄-红,P:浅紫-紫-深紫)进行转录组和代谢组测序,利用权重基因共表达网络WGCNA分析,确定了与花色素苷合成呈显著相关的7个核心模块,在1个显著正相关的核心模块中获得了2个核心MYB基因-StMYB3和StMYBATV。
将StMYB3与AtMYB3、AtMYB4、PhMYB27、FaMYB1、AmMYB308、S1THM27和PhMYB4的系统发育树和氨基酸序列比对,结果参照图1所示,A中系统进化树显示StMYB3、PhMYB27和FaMYB1具有较高的同源性。B中R2和R3结构域,分别用黑色和浅灰色表示。C端C1和C2的保守基序用红框表示。GenBank登录号如下:AmMYB308(P81393.1),AtMYB3(NP 564176.2),AtMYB4(NP 195574.1),PhMYB4(F1B281.1),SlTHM27(NP_001233975.1),FaMYB1(AAK84064.1),PhMYB27(AHX24372.1)和StMYB3(MW768000)。
图1序列比对发现,StMYB3与PhMYB27、FaMYB1、AtMYB3同源性很高,且在StMYB3中也发现了存在于第4亚组MYB蛋白C端的两个保守抑制结构域:C1(LlsrGIDPx T/N HR)和C2/EAR motif(LxLxL or DLNxxP)。
实施例2
为了验证StMYB3和StMYBATV的功能,现构建植物表达载体。具体如下:
以马铃薯红色品系的薯肉在块茎成熟时期的cDNA为模板,用两对引物(MYB3F:5’-ATGAGAAAGCCTTGTTGTGATAACA-3’,MYB3R:5’-CTATGGAAGTGAAT TGAGATCAAGCAA-3’;MYBATV:5’-ATGGCTGATTTGGATAGTTCAAGCA-3’,MYBATV:5’-TTATTGGCTGGTGGAATTTCTTGAGT-3’)分别对StMYB3(Soltu.DM.05G004700)和StMYBATV(Soltu.DM.12G023200)进行全长扩增。StMYB3基因组位置Scaffold:chr05,mRNA Genomic Coords(5'-3'):4046418–4048317。使用Platinum Taq DNA高保真聚合酶(Invitrogen,美国)进行PCR扩增。利用NC(nimblecloning)克隆,将StMYB3和StMYBATV的全长片段连接到植物表达载体pNC-Cam2304-MCS35S。
为了进一步研究StMYB3的功能,在烟草(N.tabacum)叶片中进行了瞬时表达试验。
烟草瞬时表达分析(Transient assays)方法如下:
根据已报道的试验方法,在烟草(N.benthamiana或N.tabacum)上进行了瞬时表达和双荧光素酶试验。利用pGreenII 0800-LUC中的马铃薯DFR基因启动子-prom-3-StDFR,pSAK277中的R2R3-MYB StAN1-R1,CaMV 35S启动子驱动的pNC-Cam2304-MCS35S中的StMYB3和StMYBATV进行烟草瞬时表达试验,并以空载体作为对照,分别转化到农杆菌菌株GV3101中。然后将含有报告基因、StMYB3/StMYBATV和StAN1-R1的农杆菌按1:4.5:4.5混合。农杆菌侵染的具体方法参照Hellens等人(2005)的报道。采用Varioskan Flash多功能酶标仪(Thermo Fisher Scientific,USA)测定LUC和REN的活性。StAN1-R1作为激活子。
为了进一步检测StMYB3和StMYBATV的功能,发明人进行了烟草叶片的显色试验。将StAN1-R1与StMYB3/StMYBATV等比例混合后,注入烟草幼苗的嫩叶中(叶龄约两周),或将StMYB3、StMYBATV单独注入烟草幼苗的嫩叶中,在注射后7天观察烟草叶片颜色,并拍摄照片。
烟草叶片显色试验结果参照图2所示,结果表明,单独注射StMYB3并没有诱导花色素苷的产生,而花色素苷激活因子StAN1-R1使烟草叶片呈现强烈的红色。StMYB3与StAN1-R1(1:1)同时注射4d后烟草叶片未观察到色素的积累(图2中A),;7d后,单独注射StAN-R1的叶片中由于花色素苷积累呈暗红色,而StAN-R1和StMYB3共同注射的叶片呈浅红色(图2中A)。试验结果说明,StMYB3具有抑制花色素苷积累的作用,使得植物叶片颜色变浅。
将StAN1-R1与StMYBATV等比混合,注射在烟草叶片左侧,在烟草叶片右侧注射StAN1-R1+EV。图2中B结果显示,叶片颜色比右侧的单独注射StAN-R1的叶片颜色要浅。
为了检测StMYB3和StMYBATV对花色素苷生物合成途径中结构基因转录水平的影响,我们进一步分析了StMYB3和StMYBATV与DFR启动子(prom-3-StDFR)的作用关系。
图2中C通过双荧光素酶检测到StMYBATV/StMYB3与StAN-R1和StbHLH1共同作用,抑制了启动子StDFR-LUC的活性。误差线为四个生物学重复的标准差。采用单因素方差分析确定的显著性差异,用小写字母(a,b,c等)表示(P<0.05)。结合图2中A-C可知,StMYBATV和/或StMYB3在StAN-R1激活下,可以和StbHLH1共同作用,抑制了启动子StDFR-LUC的活性。
试验结果参照图2中C图表明,StAN1-R1与StbHLH1可激活prom-3-StDFR,而StMYB3或StMYBATV则可抑制promo-3-StDFR活性。该试验结果进一步证实了StMYB3和StMYBATV是花色素苷生物合成的负向调控因子。
双分子荧光互补检测(BiFC assays)方法如下:
为了深入研究StMYB3、StMYBATV与StbHLH1在植物体内的相互作用关系,本实施例利用pNC-BiFC-Enn和pNC-BiFC-Ecn载体进行了BiFC实验。将StAN1、StMYB3、StMYBATV的ORF分别克隆到pNC-BiFC-Enn中,将StbHLH1-2克隆到pNC-BiFC-Ecn中。通过电穿孔转化将以上质粒转化到农杆菌GV3101菌株中。将含有StMYB3/StMYBATV的农杆菌与含有StbHLH的农杆菌等比例混合后,注入本氏烟草(N.benthamiana)叶片中。注射后48h,使用激光共聚焦扫描显微镜(LSCM 800,Carl Zeiss,Germany)检测荧光信号,激发波长为488nm。
已有研究表明,MYB StAN1与StbHLH1结合,共同调节马铃薯块茎中花色素苷的生物合成。本实施例发现,StMYB3和StMYBATV中也存在bHLH结合区域。为了进一步研究StMYB3和StMYBATV是否与StbHLH1相互作用,我们构建了StMYB3/StMYBATV和StbHLH1重组载体,并通过农杆菌介导分别转化到本氏烟草(N.benthamiana)叶片中。利用激光共聚焦显微镜发现,在注射了StMYB3-StbHLH1和StMYBATV-StbHLH1的本氏烟草表皮细胞中存在荧光信号(图2中D)。
结果表明,StMYB3和StMYBATV均可与StbHLH1相互作用,推测其与StAN1竞争性结合StbHLH,StMYB3和StMYBATV起反馈调节的作用,以避免花色素苷的过度积累。
图2中D图,(i)阳性和阴性对照。(ii)StMYB3和StbHLH1蛋白的相互作用。(iii)StMYBATV与StbHLH1蛋白的相互作用。
实施例3
本实施例使用农杆菌介导叶盘法将StMYB3基因转入野生型烟草中,在含有3%(w/v)蔗糖、0.7%(w/v)琼脂、1.0mg l-1 6-苄氨基嘌呤(BAP)、1.0mg l-1萘乙酸(NAA)、300mgl-1特美汀和150mg l-1卡那霉素(Kan)的选择培养基上获得阳性抗性植株,待阳性植株生根后转移到长日照(16小时光照/8小时黑暗)下的温室中培养。
转化后六到八周后,在含有Kan和Timentin的MS培养基上获得再生芽,大约一个月后生根,获得阳性植株。对转基因植株进行PCR检测,抗性植株DNA扩增到676bp的nptII特异性片段(图3)和537bp的目的基因片段(图4),与预期片段大小吻合。非转化植株无相应条带出现,证实获得了转基因植株。
图3和图4的图例:M:DNA Marker D;1:阳性对照;2:阴性对照;3~7:再生植株。
对转基因植株进行表型分析发现:转StMYB3的烟草植株花朵变为浅粉色,而对照的花朵为深红色(图5),进一步证明了StMYB3抑制花色素苷合成,与瞬时表达实验结果一致。
图6为发明人推测的马铃薯块茎薯肉花色素苷生物合成的基因调控网络示意图,(A)在红色品系R中,S1阶段激活R2R3-MYB和StAN1,S3阶段激活bHLH1,并与StAN1结合激活花色素苷生物合成基因的表达。在S3阶段,R2R3-MYB抑制因子StMYB3和R3-MYB抑制因子StMYBATV被激活,并与bHLH1结合,抑制花色素苷生物合成基因(如DFR)的表达。MBS表示MYB结合位点。(B)在黄色品系Y中,转录因子StAN1、StbHLH1、StMYB3和StMYBATV在三个阶段均未被激活。
综上,本发明通过烟草瞬时表达和稳定遗传转化实验发现StMYB3和StMYBATV均为转录抑制子,负向调控花色素苷生物合成,进一步通过双分子荧光互补技术BiFC发现StMYB3和StMYBATV会与花色素苷转录调控因子StbHLH1结合。说明在彩色块茎色泽积累过程中,转录激活子StAN1和StbHLH协同正向调控块茎花色素苷生物合成,转录抑制子StMYB3和/或StMYBATV竞争性的与StbHLH1结合起反馈调节的作用,以避免花色素苷的过度积累。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 甘肃农业大学
<120> 一种马铃薯花色素苷生物合成负调控基因、转录因子及应用
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Claims (4)
1.马铃薯花色素苷生物合成负调控转录因子、马铃薯花色素苷生物合成负调控基因、表达盒或载体、重组菌或重组细胞在如下任意一种用途中的应用,所述应用包括过表达或激活表达马铃薯花色素苷生物合成负调控转录因子,所述用途包括:
在降低烟草花色素苷积累中的应用、在调控烟草花色变浅中的应用、在降低烟草黄酮醇积累中的应用、或者在调控烟草黄酮醇合成中的应用;
所述马铃薯花色素苷生物合成负调控转录因子具有如SEQ ID NO.1所示的氨基酸序列;
所述具有如 SEQ ID NO.1 所示的氨基酸序列的负调控转录因子为 StMYB3;
或同时具有如 SEQ ID NO.1 所示的氨基酸序列和如 SEQ ID NO.3 所示的氨基酸序列;
所述具有如 SEQ ID NO.3 所示的氨基酸序列的负调控转录因子为 StMYBATV;
所述马铃薯花色素苷生物合成负调控基因编码所述的马铃薯花色素苷生物合成负调控转录因子;
所述表达盒或载体含有所述马铃薯花色素苷生物合成负调控基因;所述负调控基因
的核苷酸序列如 SEQ ID NO.2 所示;或所述负调控基因具有如 SEQ ID NO.2 所示和SEQ ID NO.4 所示的核苷酸序列;
所述重组菌或重组细胞含有所述马铃薯花色素苷生物合成负调控基因、或所述表达盒或载体。
2.根据权利要求 1 所述的应用,其特征在于,所述用途包括如下至少一种的应用方式:
将马铃薯花色素苷生物合成负调控基因送入目的植物细胞;
将所述载体转化目的植物,所述载体含有马铃薯花色素苷生物合成负调控基因;
将所述重组菌或重组细胞导入目的植物,所述重组菌或重组细胞含有马铃薯花色素
苷生物合成负调控基因;
所述目的植物选自烟草。
3.根据权利要求 1 所述的应用,其特征在于,所述用途包括:修饰烟草的内源花色素苷生物合成负调控基因,使其编码所述马铃薯花色素苷生物合成负调控转录因
子。
4.马铃薯花色素苷生物合成负调控转录因子、马铃薯花色素苷生物合成负调控基因、表达盒或载体、重组菌或重组细胞在制备用于改善烟草着色的制剂中的应用;
所述马铃薯花色素苷生物合成负调控转录因子具有如SEQ ID NO.1所示的氨基酸序列;
或同时具有如 SEQ ID NO.1 所示的氨基酸序列和如 SEQ ID NO.3 所示的氨基酸序列;
所述马铃薯花色素苷生物合成负调控基因编码所述的马铃薯花色素苷生物合成负调控转录因子;
所述表达盒或载体含有所述马铃薯花色素苷生物合成负调控基因;所述负调控基因
的核苷酸序列如 SEQ ID NO.2 所示;或所述负调控基因具有如 SEQ ID NO.2 所示和SEQID NO.4 所示的核苷酸序列;
所述重组菌或重组细胞含有所述马铃薯花色素苷生物合成负调控基因、或所述表达
盒或载体。
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NCBI Reference Sequence: XM_006344162.2,PREDICTED: Solanum tuberosum transcription factor MYB3-like (LOC102578287),mRNA;unknown;《NCBI》;参见CDS和ORIGIN部分 * |
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