CN113999828A - 草莓甲基转移酶mta和mtb基因在控制草莓成熟中的应用 - Google Patents
草莓甲基转移酶mta和mtb基因在控制草莓成熟中的应用 Download PDFInfo
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- CN113999828A CN113999828A CN202110141699.3A CN202110141699A CN113999828A CN 113999828 A CN113999828 A CN 113999828A CN 202110141699 A CN202110141699 A CN 202110141699A CN 113999828 A CN113999828 A CN 113999828A
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Abstract
本发明提供草莓甲基转移酶基因在控制草莓成熟中的应用,所述甲基转移酶基因编码甲基转移酶。本发明的实施例证明,甲基转移酶MTA和MTB可以通过调控果实色泽和软化关键基因的表达,来改变果实成熟进程。抑制甲基转移酶MTA基因或MTB基因在草莓果实中的表达,能延迟果实成熟的进程,而超表达MTA基因或MTB基因会导致草莓果实提前成熟。因此,通过合理利用甲基转移酶可以调整草莓果实的上市时间,为草莓生产服务。
Description
技术领域
本发明涉及生物技术领域中草莓甲基转移酶MTA和MTB基因在控制草莓成熟中的应用。
背景技术
草莓果实营养丰富,味道鲜美,深受大众喜爱。然而,目前草莓在生产中存在供应季节集中、采后不易贮藏,以及品种过于单一等问题,使草莓的生产成本居高不下。因此,寻找草莓成熟调控关键基因,进而应用遗传工程选育优良草莓品种是未来解决草莓生产问题的重要途径。
N6-甲基腺嘌呤(m6A)是真核生物信使RNA(mRNA)最常见的化学修饰,可增强外显子剪切、促进小RNA(miRNA)加工、降低mRNA稳定性和提高mRNA的翻译效率,能在转录后水平调控基因的表达水平。m6A修饰是由RNA甲基转移酶和RNA去甲基转移酶协调控制的一个可逆过程。在哺乳动物中,m6A修饰复合体中甲基转移酶METTL3和METTL14作为mRNA结合和甲基催化的核心组分,在m6A修饰中起关键作用。迄今为止,还未有草莓甲基转移酶或去甲基转移酶基因功能的研究。
发明内容
本发明所要解决的技术问题是如何控制草莓成熟期。
为了解决以上技术问题,本发明提供了草莓甲基转移酶基因在控制草莓成熟中的应用,所述甲基转移酶基因编码甲基转移酶,所述甲基转移酶是如下A1或A2的蛋白质:
A1、氨基酸序列是序列表中序列2或序列4所示的氨基酸序列的蛋白质;
A2、在A1)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。
上述应用中,序列表中的序列2由741个氨基酸残基组成,序列表中的序列4由1152个氨基酸残基组成。
上述应用中,所述甲基转移酶可来源于草莓。
上述应用中,所述甲基转移酶基因具体可为如下D1或D2所示的基因:
D1、编码链的编码序列是序列表中序列1或序列3的DNA分子;
D2、核苷酸序列是序列表中序列1或序列3的DNA分子。
上述应用中,所述控制草莓成熟,可通过抑制或降低所述甲基转移酶基因的表达延缓草莓成熟。具体可为通过转入甲基转移酶基因干扰表达载体实现。所述甲基转移酶基因干扰表达载体可为将甲基转移酶基因连接到干扰表达载体中得到的重组表达载体。所述干扰表达载体可为pK7GWIWG2D或pH7GWIWG2D。
上述应用中,所述控制草莓成熟可通过促进或增强所述甲基转移酶基因的表达促进草莓成熟。具体可为通过转入甲基转移酶基因过表达载体实现。所述过表达载体可为将甲基转移酶基因连接到过表达载体中得到的重组表达载体。所述过表达载体可为pCambia2300、pCambia 1300、pBI121中的任一种。
为了解决上述技术问题,本发明提供了延缓草莓成熟的试剂,所述试剂的活性成分为抑制编码所述甲基转移酶的基因的表达、降低所述甲基转移酶的丰度、和/或敲除编码所述甲基转移酶的基因的物质。
上述试剂的活性成分还可含有其他生物成分或/和非生物成分,上述药剂的其他活性成分本领域技术人员可根据延缓草莓成熟的效果确定。
为了解决上述技术问题,本发明提供了促进草莓成熟的试剂,所述试剂的活性成分为增强编码所述甲基转移酶的基因的表达和/或增加所述甲基转移酶的丰度的物质。
上述试剂的活性成分还可含有其他生物成分或/和非生物成分,上述药剂的其他活性成分本领域技术人员可根据促进草莓成熟的效果确定。
为了解决上述技术问题,本发明的还提供一种延缓草莓成熟的方法,包括如下步骤:抑制受体草莓中所述甲基转移酶的表达、降低所述甲基转移酶的丰度、和/或敲除编码所述甲基转移酶的基因,得到成熟期晚于所述受体草莓的目的草莓。
为了解决上述技术问题,本发明的还提供一种促进草莓成熟的方法,包括如下步骤:增强受体草莓中所述甲基转移酶的表达和/或增加所述甲基转移酶的丰度,得到成熟期早于所述受体草莓的目的草莓。
本发明还提供蛋白质,为甲基转移酶,是如下A1或A2的蛋白质:
A1、氨基酸序列是序列表中序列2或序列4所示的氨基酸序列的蛋白质;
A2、在A1)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。
本发明还提供所述蛋白质的核酸分子,所述核酸分子具体为如下D1或D2所示的核酸分子:
D1、编码链的编码序列是序列表中序列1或序列3的DNA分子;
D2、核苷酸序列是序列表中序列1或序列3的DNA分子。
本发明中,所述控制草莓成熟期可为延缓草莓成熟或促进草莓成熟。
本发明提供调控草莓甲基转移酶基因的物质在控制草莓成熟期中的应用,所述甲基转移酶基因编码甲基转移酶。本发明的实施例证明,甲基转移酶MTA和MTB可以通过调控果实色泽和软化关键基因的表达,来改变果实成熟进程。抑制甲基转移酶MTA基因或MTB基因在草莓果实中的表达,能延迟果实成熟的进程,而超表达MTA基因或MTB基因会导致草莓果实提前成熟。因此,通过合理利用甲基转移酶可以调整草莓果实的上市时间,为草莓生产服务。
附图说明
图1是实施例1中草莓MTA和MTB基因的RT-PCR扩增产物的电泳图谱。其中,1-3为草莓MTA基因的扩增条带;4-6为草莓MTB基因的扩增条带;7-8为对照;M为DNA分子量标准(擎科生物,北京)。
图2是实施例2中草莓MTA和MTB基因在果实成熟中的表达情况。图2的A图为不同成熟期的二倍体草莓果实中MTA、MTB基因的表达。图中,S6为花后15-20天果;RS1为花后30天果;RS3为花后35天果。图2的B图,不同成熟期的八倍体草莓果实及MTA、MTB基因的表达。图中,SG为小绿果;Wt为白果;IR为转色果;FR为全熟果。
图3是实施例3中瞬时干扰八倍体草莓MTA或MTB表达的果实表型图。将分别含有对照载体、RNAi-MTA或RNAi-MTB载体的农杆菌注射草莓果实后(0天为注射当天),RNAi果实在第7天呈现延迟成熟表型。
图4是实施例4中在八倍体草莓中瞬时过表达MTA或MTB的果实表型图。将分别含有对照载体、OE-MTA或OE-MTB载体的农杆菌分别注射草莓果实后(0天为注射当天),OE果实在第5天呈现加速成熟表型。
图5是实施例5中MTA-RNAi果实和MTB-RNAi果实的基因表达情况。数据表示为平均值±标准差,重复数为3,*表示显著性分析结果为P≤0.05,**表示显著性分析结果为P≤0.01。
图6是实施例5中OE-MTA果实和OE-MTB果实的基因表达情况。数据表示为平均值±标准差,重复数为3,*表示显著性分析结果为P≤0.05,**表示显著性分析结果为P≤0.01。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均为常规生化试剂,可从商业途径得到。
1载体
下述实施例中载体pMDTM 19-T(货号:6013)为TaKaRa公司产品。
下述实施例中载体pK7GWIWG2D记载于文献“Cai J,Qin G,Chen T,et al.Themode of action of remorin1 in regulating fruit ripening at transcriptionaland post-transcriptional levels[J].NEW PHYTOLOGIST,2018,9”,公众可以从中国科学院植物研究所获得。
下述实施例中载体pCambia 2300记载于文献“Cai J,Qin G,Chen T,et al.Themode of action of remorin1 in regulating fruit ripening at transcriptionaland post-transcriptional levels[J].NEW PHYTOLOGIST,2018,9”,公众可以从中国科学院植物研究所获得。
2植物品系
下述实施例中的草莓品种‘Hawaii 4’记载于非专利文献“Luo H,Dai C,Li Y,etal.Reduced Anthocyanins in Petioles codes for a GST anthocyanin transporterthat is essential forthe foliage and fruit coloration in strawberry.[J].JOURNAL OF EXPERIMENTAL BOTANY,2018,3”。公众可从中国科学院植物研究所获得,以重复本申请实验,不可作为其它用途使用。
下述实施例中的草莓品种‘Hongyan’记载于非专利文献“Riaz A,Aadil RM,Amoussa AMO,et al.Application ofchitosan-based apple peel polyphenols ediblecoating on the preservation ofstrawberry(Fragaria ananassa cv Hongyan)fruit.JOURNAL OF FOOD PROCESSING AND PRESERVATION,2021.DOI:10.1111/jfpp.15018.”。公众可从中国科学院植物研究所获得,以重复本申请实验,不可作为其它用途使用。
3试剂
下述实施例中,含100mg/L氨苄霉素的LB固体培养基的配制方法为(以200mL为例):固体LB培养基(200mL):酵母提取物1g,胰蛋白2g,氯化钠2g,3g琼脂粉,溶解于200mL去离子水中,120℃高压灭菌20min,在培养基温度降至50℃时,在超净台中加入200μL氨苄抗生素(100mg/mL),混匀后倒入培养皿中,凝固后4℃保存。
下述实施例中,含50mg/L卡那霉素、50mg/L利福平和50mg/L链霉素的LB液体培养基的配制方法为(以200mL为例):固体LB培养基(200mL):酵母提取物1g,胰蛋白2g,氯化钠2g,溶解于200mL去离子水中,120℃高压灭菌20min,在培养基温度降至50℃时,在超净台中加入100μL卡那霉素(50mg/mL)、100μL利福平(50mg/mL)、100μL链霉素(50mg/mL),混匀,4℃保存。
下述实施例中,转化液的配制方法为:将MgCl2,2-(N-吗啉)乙磺酸(MES),乙酰丁香酮溶解于去离子水中得到的pH值为5.6的溶液,该转化液中MgCl2的浓度为10mM,MES的浓度为10mM,乙酰丁香酮浓度为200μM。
下述实施例中,如无特殊说明,序列表中各核苷酸序列的第1位均为相应DNA的5′末端核苷酸,末位均为相应DNA的3′末端核苷酸。
实施例1、草莓MTA和MTB基因cDNA序列的克隆与序列测定
利用植物总RNA分离试剂盒(诺维赞生物公司,南京)提取野生草莓(Fragariavesca,‘Hawaii 4’)果实的总RNA,经过SimpliNano(thermo)定量后,各取1μg总RNA,采用反转录试剂盒(PrimerScriptRT reagentKit,Takara,大连)合成cDNA第一链。
根据已经公布的野生草莓基因组序列信息,设计MTA基因特异性的引物:
MTA-F:5′-ATGGAGGAGACCCAATCTGG-3′;
MTA-R:5′-TCCGGCCATCTCAACATCG-3′;
设计MTB基因特异性的引物:
MTB-F:5′-ATGGATTCGCCTGAACGTAG-3′;
MTB-R:5′-CAACAAATTCATGTGCCTGTGAG-3′。
引物由北京擎科生物科技有限公司合成。
利用高保真酶(I-5TM2x High-FidelityMasterMix,擎科生物)分别扩增MTA和MTB基因。
PCR反应体系为:cDNA模板100ng,正反向各引物(10μM)2μL,I-5TM2x High-FidelityMasterMix 25μL,水补齐至50μL。
PCR反应程序设置为:98℃2min;98℃10s,56℃15s,72℃15s,共35个循环;72℃终延伸5min。
将扩增产物在1%的琼脂糖凝胶上电泳检测,结果如图1所示,发现MTA-F和MTA-R的扩增产物在2000bp处有特异性目的条带,MTB-F和MTB-R的扩增产物在3000bp处有特异性目的条带。
用DNA胶回收试剂盒(诺维赞生物公司,南京)将上述扩增条带分别回收。
用DNA Ligation Kit试剂盒(TaKaRa,大连)将纯化后的MTA DNA片段和载体pMDTM19-T(TaKaRa,大连)按照TA克隆的原理进行连接,构建pMD19T-MTA重组质粒。将pMD19T-MTA质粒转化大肠杆菌TOP10菌株(擎科生物,北京)在含100mg/L氨苄霉素的LB固体培养基上筛选,挑取阳性单菌落,以M13-F通用测序引物检测pMD19T-MTA重组质粒序列。
M13-F:5′-ACTGGCCGTCGTTTTACAACG-3′。
用DNA Ligation Kit试剂盒(TaKaRa,大连)将纯化后的MTB DNA片段和载体pMDTM19-T(TaKaRa,大连)按照TA克隆的原理进行连接,构建pMD19T-MTB重组质粒。将pMD19T-MTA质粒转化大肠杆菌TOP10菌株(擎科生物,北京)在含100mg/L氨苄霉素的LB固体培养基上筛选,挑取阳性单菌落,以M13-F通用测序引物检测pMD19T-MTB重组质粒序列。
结果表明,我们获得了草莓的MTA和MTB基因的cDNA序列,因为该基因与已公布基因序列一致:MTA的cDNA序列如序列表中序列1所示,基因座位号是gene01911(ftp://ftp.bioinfo.wsu.edu/species/Fragaria_vesca/Fvesca-genome.v2.0.a2),其编码的蛋白含有741个氨基酸残基(如序列表序列2所示);MTB的cDNA序列如序列表中序列3所示,基因座位号是gene31441(ftp://ftp.bioinfo.wsu.edu/species/Fragaria_vesca/Fvesca-genome.v2.0.a2),其编码的蛋白含有1152个氨基酸残基(如序列表序列4所示)。
实施例2、甲基转移酶MTA和MTB基因在草莓果实中的表达特征
1、甲基转移酶MTA和MTB基因在二倍体草莓中表达情况
从实验温室采摘二倍体野生草莓(Fragaria vesca‘Hawaii 4’)果实,并根据草莓果距离开花后天数划分为三个成熟度(见图2的A图):绿果期(S6),大约为开花后15-20天的果;白果期(RS1),开花后30天的果;黄果期(RS6),开花后35天的果。果实采摘后,立即用液氮速冻运回实验室。
根据实施例1的方法,将三种不同成熟度的草莓样品提取RNA并反转录为cDNA,将cDNA浓度进一步稀释20倍用于荧光定量PCR反应的模板。利用荧光定量试剂(SYBRPremixEx TaqTM,TaKaRa)在荧光定量PCR仪上(STEPONE PLUS,ABI,美国)分别检测MTA和MTB基因的表达变化。
检测MTA基因所用的引物为MTA-qRT-PCR-F和MTA-qRT-PCR-R:
MTA-qRT-PCR-F:5′-GGTCGATGGCGGAGGAGAA-3′;
MTA-qRT-PCR-R:5′-GTCTTGTTTGCTCTCCGGCAAC-3′。
检测MTB基因所用的引物为MTB-qRT-PCR-F和MTB-qRT-PCR-R:
MTB-qRT-PCR-F:5′-GGTTCAACCCAGAAGCCTGAAGAC-3′;
MTB-qRT-PCR-R:5′-GTTCCGGATGTAAGCCTCGGT-3′。
反应体系为:模板2μL,SYBR Mix 10μL,正反向引物各0.8μL,Rox 0.8μL,灭菌双蒸水补齐至20μL。
反应程序为:98℃10min;95℃15S,60℃30S,40个循环;72℃5min;95℃1min,融解曲线分析为***默认设置。
结果如图2的A图所示,随着草莓成熟度的升高,MTA基因的表达逐渐升高而后下降,在RS1期表达量最高,而MTB基因随成熟度升高持续升高,说明MTA和MTB可能参与了草莓果实成熟调控。
2、甲基转移酶MTA和MTB基因在八倍体草莓中表达情况
从草莓基地(商用采摘园)大棚采摘草莓(Fragaria ananassa cultivar‘Hongyan’)果实,并根据以下特征划分果实成熟度(如图2的B图):小绿果(SG)特征为果皮深绿,种子绿色;白果(Wt)特征为果皮绿色转白,种子开始变褐色;转色果(IR)特征为三分之一果皮表面呈现红色,种子全部褐色;全熟果(FR)特征为果皮表面全部呈现红色,种子全部深褐色。果实采摘后,立即用液氮速冻运回实验室。
根据上述步骤1的方法,分别检测MTA和MTB基因在八倍体草莓中的表达变化。
结果如图2的B图所示,随着草莓成熟度的升高,MTA和MTB基因的表达逐渐升高而后下降,均在IR期表达量最高,说明MTA和MTB可能参与了草莓果实成熟调控。
实施例3、草莓MTA和MTB干扰表达载体的构建及其应用
1、MTA干扰表达载体和MTB干扰表达载体的构建。
1.1、MTA干扰表达载体的构建
根据草莓MTA基因序列,选择去除甲基转移酶基因保守结构域的特异区域,设计可以扩增该区域的上下游引物MTA-RNAi-F和MTA-RNAi-R:
MTA-RNAi-F:5′-ATGGAGGAGACCCAATCTGGC-3′;
MTA-RNAi-R:5′-GTCCTCGTCCATTGAATCGGG-3′。
引物由北京擎科生物科技有限公司合成。
利用高保真酶(I-5TM2x High-Fidelity MasterMix,擎科生物)扩增pMD19T-MTA质粒。
PCR反应体系为:pMD19T-MTA质粒模板100ng,正反向各引物(10μM)2μL,I-5TM2xHigh-Fidelity Master Mix 25μL,水补齐至50μL。
PCR反应程序设置为:98℃2min;98℃10s,56℃15s,72℃15s,共35个循环;72℃终延伸5min。
回收和纯化目的条带,将DNA片段连入8/GW/Vector克隆载体(Invitrogen)。利用LR重组酶(LR Clonase II Ezyme mix,Invitrogen)通过GATEWAY克隆的方法连入pK7GWIWG2D载体。通过大肠杆菌转化、抗生素筛选、双酶切鉴定和序列测定,形成MTA干扰表达载体,命名为RNAi-MTA。
1.2、MTB干扰表达载体的构建
根据草莓MTB基因序列,选择去除甲基转移酶基因保守结构域的特异区域,设计可以扩增该区域的上下游引物
针对MTB基因所用的引物为MTB-RNAi-F和MTB-RNAi-R:
MTB-RNAi-F:5′-ATGGATTCGCCTGAACGTAG-3′;
MTB-RNAi-R:5′-TCCCCATCCTGATACCAGT-3′。
引物由北京擎科生物科技有限公司合成。
利用高保真酶(I-5TM2x High-Fidelity MasterMix,擎科生物)扩增pMD19T-MTB质粒。
PCR反应体系为:pMD19T-MTB质粒模板100ng,正反向各引物(10μM)2μL,I-5TM2xHigh-Fidelity Master Mix 25μL,水补齐至50μL。
PCR反应程序设置为:98℃2min;98℃10s,56℃15s,72℃15s,共35个循环;72℃终延伸5min。
回收和纯化目的条带,将DNA片段连入8/GW/Vector克隆载体(Invitrogen)。利用LR重组酶(LR Clonase II Ezyme mix,Invitrogen)通过GATEWAY克隆的方法连入pK7GWIWG2D载体。通过大肠杆菌转化、抗生素筛选、双酶切鉴定和序列测定,形成MTB干扰表达载体,命名为RNAi-MTB。
2、RNAi-MTA和RNAi-MTB农杆菌制备和草莓果实瞬时转化。
通过冻融法将RNAi-MTA和RNAi-MTB重组表达载体分别转化农杆菌GV3101,挑取阳性克隆接种至50mL含50mg/L卡那霉素、50mg/L利福平和50mg/L链霉素的LB液体培养基中,28℃在200rpm(转/分钟)摇菌至OD600=0.6,以5000g离心10min收集菌液。用50mL转化液悬浮菌体,在室温条件下,以5000g离心10min收集菌液;再用50mL转化液重新悬浮菌体,在室温静置2h以上,分别获得了转化RNAi-MTA的农杆菌菌液和转化RNAi-MTB的农杆菌菌液。
将上述转化RNAi-MTA的农杆菌菌液和转化RNAi-MTB的农杆菌菌液通过注射法分别侵染草莓‘Hongyan’的果实。具体方法为:利用1ml无菌微量注射器,在草莓绿果期于果实中轴处注射,直至果实呈现水渍状。其中,注射转化RNAi-MTA的农杆菌菌液的果实称为MTA-RNAi果实,注射转化RNAi-MTB的农杆菌菌液的果实称为MTB-RNAi果实。注射后果实放置在室温,相对湿度70-90%,每天观察。
结果如图3所示,相比于转入空载体pK7GWIWG2D的对照草莓‘Hongyan’果实,转入RNAi-MTA载体的草莓果实(MTA-RNAi果实)和转入RNAi-MTB载体的草莓果实(MTB-RNAi果实)注射后的着色均明显减速,说明瞬时MTA-RNAi或MTB-RNAi干扰可以延迟八倍体草莓果实的成熟。
实施例4、草莓MTA和MTB超表达载体的构建及其应用
1、MTA和MTB超表达载体的构建。
1.1、MTA超表达载体的构建
根据MTA的cDNA序列(序列表序列1)和植物表达载体pCambia 2300的序列,设计用于扩增MTA全长基因(不含终止密码子序列TAA)的特异性引物MTA-2300-F和MTA-2300-R:
MTA-2300-F:5′-GGTACCCGGGGATCCTCTAGAATGGAGGAGACCCAATCTGG-3′;
MTA-2300-R:5′-GCCCTTGCTCACCATTCTAGATCCGGCCATCTCAACATCG-3′。
以实施例1的pMD19T-MTA重组质粒为模板,扩增用于连接pCambia 2300载体的DNA片段。
PCR反应体系为:pMD19T-MTA质粒模板100ng,正反向各引物(10μM)2μL,I-5TM2xHigh-Fidelity Master Mix 25μL,水补齐至50μL。
PCR反应程序设置为:98℃2min;98℃10s,56℃15s,72℃15s,共35个循环;72℃终延伸5min。
扩增片段经纯化后,利用同源重组酶(In-Fusion HD Cloning,TaKaRa,大连)按照同源重组克隆原理连接于pCambia 2300载体,形成OE-MTA载体。
1.2、MTB超表达载体的构建
根据MTB的cDNA序列(序列表序列3)和植物表达载体pCambia 2300的序列,设计用于扩增MTB全长基因(不含终止密码子序列TAA)的特异性引物MTB-2300-F和MTB-2300-R:
MTB-2300-F:5′-GGTACCCGGGGATCCTCTAGAATGGATTCGCCTGAACGTAG-3′;
MTB-2300-R:5′-GCCCTTGCTCACCATTCTAGACAACAAATTCATGTGCCTGTGAG-3′。
以实施例1的pMD19T-MTB重组质粒为模板,扩增用于连接pCambia 2300载体的DNA片段。
PCR反应体系为:pMD19T-MTB质粒模板100ng,正反向各引物(10μM)2μL,I-5TM2xHigh-Fidelity Master Mix 25μL,水补齐至50μL。
PCR反应程序设置为:98℃2min;98℃10s,56℃15s,72℃15s,共35个循环;72℃终延伸5min。
扩增片段经纯化后,利用同源重组酶(In-Fusion HD Cloning,TaKaRa,大连)按照同源重组克隆原理连接于pCambia 2300载体,形成OE-MTB载体。
2、OE-MTA和OE-MTB农杆菌制备和草莓果实瞬时转化。
按照实施例3的农杆菌转化方法制备OE-MTA农杆菌菌液和OE-MTB农杆菌菌液。按照实施例3的草莓果实瞬时转化方法,将上述农杆菌菌液分别转化草莓‘Hongyan’的果实。其中,注射OE-MTA农杆菌菌液的果实称为OE-MTA果实,注射OE-MTB农杆菌菌液的果实称为OE-MTB果实。
结果如图4所示,相比于转入空载体(pCambia 2300)的对照草莓‘Hongyan’的果实,转入OE-MTA的草莓果实(OE-MTA果实)和转入OE-MTB载体的草莓果实(OE-MTB果实)在注射后着色均明显加速,说明瞬时超表达MTA或MTB基因可以加速八倍体草莓果实的成熟。
实施例5、MTA和MTB转基因果实的分子检测
草莓果实的感官品质是由色泽和质地来决定的,通常色泽鲜艳和质地软化的草莓品质较好,因此色泽和软化关键酶基因的表达是衡量草莓品质变化的重要分子特征。根据实施例1的方法,将在实施例3获得的MTA-RNAi果实和MTB-RNAi果实,以及实施例4获得的OE-MTA果实和OE-MTB果实提取RNA,并反转录为cDNA,按照实施例2的方法和引物,利用实时荧光定量RT-PCR检测MTA-RNAi果实、MTB-RNAi果实、OE-MTA果实和OE-MTB果实中MTA、MTB,以及花青苷合成关键酶基因(CHS)和细胞壁代谢关键酶基因(PG1)的表达情况。
CHS基因所用引物为CHS-qRT-PCR-F和CHS-qRT-PCR-R:
CHS-qRT-PCR-F:5′-CATACCCCGACTACTACTTTCGT-3′;
CHS-qRT-PCR-R:5′-CGCACATACTGGGATTCTCTT-3′。
PG1基因所用引物为PG1-qRT-PCR-F和PG1-qRT-PCR-R:
PG1-qRT-PCR-F:5′-GCAAGTAGAGTCGCACAGTTTT-3′;
PG1-qRT-PCR-R:5′-TCAGTATTAGGCTTCCCACCA-3′。
结果如图5所示,与转RNAi空载体pK7GWIWG2D的对照‘Hongyan’果实相比,抑制MTA的MTA-RNAi果实中MTA基因表达显著受到抑制,软化关键酶基因PG1和色泽关键酶基因CHS的表达显著降低,抑制MTB基因表达的MTB-RNAi果实中,MTB基因表达显著受到抑制,PG1和CHS的表达显著降低。
如图6所示,与转过表达空载体pCambia 2300的对照‘Hongyan’果实相比,OE-MTA果实中MTA基因的表达显著增加,软化关键酶基因PG1和色泽关键酶基因CHS的表达显著增强;OE-MTB果实中,MTB基因的表达显著增加,PG1和CHS的表达显著增强。
综上,甲基转移酶MTA和MTB可以通过调控果实色泽和软化关键基因的表达,来改变果实成熟进程。抑制MTA基因或MTB基因在草莓果实中的表达,能延迟果实成熟的进程,而超表达MTA基因或MTB基因导致草莓果实提前成熟。因此,通过合理利用该基因可以调整草莓果实的上市时间,为草莓生产服务。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
序列表
<110> 中国科学院植物研究所
<120> 草莓甲基转移酶MTA和MTB基因在控制草莓成熟中的应用
<130> GNCSY210098
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2226
<212> DNA
<213> 草莓(Fragaria vesca)
<400> 1
atggaggaga cccaatctgg cggcaaagac gacaccgccg tgtccgccgt caaagcaatg 60
cgccaagacc tcgaaacccg aatcgaaacc caacgcgcca cccagctcga actcctctct 120
agtctccaat ccgaactcga ccccgccatc gtccccacca tcgatctctc cctcaaggtc 180
ctctcctcct tcaaccaccg ccccttcact cccacccctc ctctacccga cccgaaaccc 240
aacccgaccc gaaaccccgc cgcccgcctc ccctcccctc aaccacccct ccctcctcct 300
ccgctgcagc ccgaaagccc taatctttct ccccgacccg acccgacccg ggagtcctca 360
cccgattcaa tggacgagga cgcgaacccg ctctcggtga tccgggccat gattgcggtc 420
cggtttctgg agagggttcc gttcatgctg gtggagtcgt cggagctcct gaagcagctc 480
gagaccgacg cgaaggcctc gccggaggag aaggcggcgc tgcgggaggt ggggggagag 540
aacgggggaa ttctggcggt ggagatggcg ctgcggtcga tggcggagga gaacggcggc 600
gttatactgg aggagtacgc ggtcaacggg aagtctaggg ttacggtgag ggatattgac 660
cggactaggc tgatgaagga gttgccggag agcaaacaag actcgatttt ggatgggaat 720
gggagtttta atcagatggt gagtggtggc atggatggtg ggaacaatgt gggagggttt 780
atgggccgag gtgggccctg gggggttgcg ccggagcatt acatgggcgg gttgccgccg 840
ttgtttccgg ggataggtcc gagaggagga agagggatga tgggaatgcc gagagggatg 900
ttagggtctc caatgccgag gcctaatatg gggcagaatg gaggaatggt ggttagtccc 960
aatggaatgc cgaataagaa catgaagagc gaagaggatg agatgaagga tcttgaggca 1020
ttgctgaata agaagagttt taaggagttg cagaagtcga aaactgggga ggagcttttg 1080
gacctcatcc acaggccaac tgcaaaggag actagaacgg caaacaagtt caaaagcaaa 1140
ggtggttcac gagtgaagga atactgcagc tccctaacaa aagaggactg ccgacgtcaa 1200
tctaattcgt tgctcgcttg tgagaaggtt cattttaggc gcataattgc tctacatact 1260
gatgtcaatt taggagactg ttcttttcta gatacttgcc gtcacatgaa gacatgcaag 1320
tatgtacatt atgagctcga ttcaacacca gatgtgtcaa acatgatggc tccccctaga 1380
ccattaaagc agcgtgctga atattgttct gaggtggaac tcggtcaacc acaatggatt 1440
aactgtgata tccgcaattt tagaatggat attttgggac agtttggagt cataatggcc 1500
gatccaccgt gggacattca tatggagttg ccttatggga ctatggctga tgatgaaatg 1560
cgcactctta atgttcctgc attgcagact gatggtctga ttttcctttg ggttactggg 1620
cgtgcgatgg agcttggccg tgagtgttta gaactttggg gatacaagcg tatcgaggag 1680
atgatttggg tgaaaactaa tcagcttcaa cgaattatta gaactgggcg cactggccac 1740
tggctaaatc atagcaaaga gcattgccta gttggaataa aaggggatcc attagtgaat 1800
aggaatattg atactgatgt cattgttgcc gaggtcagag agacaagtcg taagccagat 1860
gagatgtacc ctttgctgga gaggattagt ccaaggacaa ggaagctgga gctgtttgct 1920
cgtatgcaca acactcatgc agggtggatg tcacttggta atcaactaag tggtgtacga 1980
ttggttgatg aaggcttgcg tgcacggttc aaggctgcgt acccagaggt ggaggtgcag 2040
ccagcatccc cacccagagc ttctgccatg gaagctgatt caaatgctac tcaaaccaga 2100
agtccctttt ccgaagcaaa acccacagag gctgctgctc cacaacaagc agagcctgca 2160
gggcctgatg cgcctgcttc tgaggtgaag ccaacgctta gcaccgatgt tgagatggcc 2220
ggatga 2226
<210> 2
<211> 741
<212> PRT
<213> 草莓(Fragaria vesca)
<400> 2
Met Glu Glu Thr Gln Ser Gly Gly Lys Asp Asp Thr Ala Val Ser Ala
1 5 10 15
Val Lys Ala Met Arg Gln Asp Leu Glu Thr Arg Ile Glu Thr Gln Arg
20 25 30
Ala Thr Gln Leu Glu Leu Leu Ser Ser Leu Gln Ser Glu Leu Asp Pro
35 40 45
Ala Ile Val Pro Thr Ile Asp Leu Ser Leu Lys Val Leu Ser Ser Phe
50 55 60
Asn His Arg Pro Phe Thr Pro Thr Pro Pro Leu Pro Asp Pro Lys Pro
65 70 75 80
Asn Pro Thr Arg Asn Pro Ala Ala Arg Leu Pro Ser Pro Gln Pro Pro
85 90 95
Leu Pro Pro Pro Pro Leu Gln Pro Glu Ser Pro Asn Leu Ser Pro Arg
100 105 110
Pro Asp Pro Thr Arg Glu Ser Ser Pro Asp Ser Met Asp Glu Asp Ala
115 120 125
Asn Pro Leu Ser Val Ile Arg Ala Met Ile Ala Val Arg Phe Leu Glu
130 135 140
Arg Val Pro Phe Met Leu Val Glu Ser Ser Glu Leu Leu Lys Gln Leu
145 150 155 160
Glu Thr Asp Ala Lys Ala Ser Pro Glu Glu Lys Ala Ala Leu Arg Glu
165 170 175
Val Gly Gly Glu Asn Gly Gly Ile Leu Ala Val Glu Met Ala Leu Arg
180 185 190
Ser Met Ala Glu Glu Asn Gly Gly Val Ile Leu Glu Glu Tyr Ala Val
195 200 205
Asn Gly Lys Ser Arg Val Thr Val Arg Asp Ile Asp Arg Thr Arg Leu
210 215 220
Met Lys Glu Leu Pro Glu Ser Lys Gln Asp Ser Ile Leu Asp Gly Asn
225 230 235 240
Gly Ser Phe Asn Gln Met Val Ser Gly Gly Met Asp Gly Gly Asn Asn
245 250 255
Val Gly Gly Phe Met Gly Arg Gly Gly Pro Trp Gly Val Ala Pro Glu
260 265 270
His Tyr Met Gly Gly Leu Pro Pro Leu Phe Pro Gly Ile Gly Pro Arg
275 280 285
Gly Gly Arg Gly Met Met Gly Met Pro Arg Gly Met Leu Gly Ser Pro
290 295 300
Met Pro Arg Pro Asn Met Gly Gln Asn Gly Gly Met Val Val Ser Pro
305 310 315 320
Asn Gly Met Pro Asn Lys Asn Met Lys Ser Glu Glu Asp Glu Met Lys
325 330 335
Asp Leu Glu Ala Leu Leu Asn Lys Lys Ser Phe Lys Glu Leu Gln Lys
340 345 350
Ser Lys Thr Gly Glu Glu Leu Leu Asp Leu Ile His Arg Pro Thr Ala
355 360 365
Lys Glu Thr Arg Thr Ala Asn Lys Phe Lys Ser Lys Gly Gly Ser Arg
370 375 380
Val Lys Glu Tyr Cys Ser Ser Leu Thr Lys Glu Asp Cys Arg Arg Gln
385 390 395 400
Ser Asn Ser Leu Leu Ala Cys Glu Lys Val His Phe Arg Arg Ile Ile
405 410 415
Ala Leu His Thr Asp Val Asn Leu Gly Asp Cys Ser Phe Leu Asp Thr
420 425 430
Cys Arg His Met Lys Thr Cys Lys Tyr Val His Tyr Glu Leu Asp Ser
435 440 445
Thr Pro Asp Val Ser Asn Met Met Ala Pro Pro Arg Pro Leu Lys Gln
450 455 460
Arg Ala Glu Tyr Cys Ser Glu Val Glu Leu Gly Gln Pro Gln Trp Ile
465 470 475 480
Asn Cys Asp Ile Arg Asn Phe Arg Met Asp Ile Leu Gly Gln Phe Gly
485 490 495
Val Ile Met Ala Asp Pro Pro Trp Asp Ile His Met Glu Leu Pro Tyr
500 505 510
Gly Thr Met Ala Asp Asp Glu Met Arg Thr Leu Asn Val Pro Ala Leu
515 520 525
Gln Thr Asp Gly Leu Ile Phe Leu Trp Val Thr Gly Arg Ala Met Glu
530 535 540
Leu Gly Arg Glu Cys Leu Glu Leu Trp Gly Tyr Lys Arg Ile Glu Glu
545 550 555 560
Met Ile Trp Val Lys Thr Asn Gln Leu Gln Arg Ile Ile Arg Thr Gly
565 570 575
Arg Thr Gly His Trp Leu Asn His Ser Lys Glu His Cys Leu Val Gly
580 585 590
Ile Lys Gly Asp Pro Leu Val Asn Arg Asn Ile Asp Thr Asp Val Ile
595 600 605
Val Ala Glu Val Arg Glu Thr Ser Arg Lys Pro Asp Glu Met Tyr Pro
610 615 620
Leu Leu Glu Arg Ile Ser Pro Arg Thr Arg Lys Leu Glu Leu Phe Ala
625 630 635 640
Arg Met His Asn Thr His Ala Gly Trp Met Ser Leu Gly Asn Gln Leu
645 650 655
Ser Gly Val Arg Leu Val Asp Glu Gly Leu Arg Ala Arg Phe Lys Ala
660 665 670
Ala Tyr Pro Glu Val Glu Val Gln Pro Ala Ser Pro Pro Arg Ala Ser
675 680 685
Ala Met Glu Ala Asp Ser Asn Ala Thr Gln Thr Arg Ser Pro Phe Ser
690 695 700
Glu Ala Lys Pro Thr Glu Ala Ala Ala Pro Gln Gln Ala Glu Pro Ala
705 710 715 720
Gly Pro Asp Ala Pro Ala Ser Glu Val Lys Pro Thr Leu Ser Thr Asp
725 730 735
Val Glu Met Ala Gly
740
<210> 3
<211> 3459
<212> DNA
<213> 草莓(Fragaria vesca)
<400> 3
atgaagagtg atagggcggg ggacgacgag gagtgggagg ggagtgataa gaggaagcac 60
aggtcgagca ggtcaaggaa gtcggggaac ggagaggacg tggatggtgg tggaaggaga 120
agaagtcatg gggataggag tgagagccgt aaacggtcag gtggctctag caatgcggat 180
agcgaggagg aggactatga cttgagaaag gagtcgcggt ctaagatgat gaagaagaag 240
caagaggaga gtagtttgga gaagttgagc aactggtatc aggatgggga atttgataat 300
aggcaagatg gtggagataa gtcagggggt agagggctgg ttagagctga agaaaatgaa 360
agaaggaaac tggcttcaaa gctcgcacag cacgagattt cccagactaa aagtaagagc 420
aaagaagaaa agtctcatga tggagaacat gaaaagacac tggacagaga ttctaagtat 480
tcggacagga aagaaagcat tcgagagaag actcatgggt cttctgaaca ggtgaggact 540
tcaagaagaa aatgggatga atcagatggt ggcaagaaag cagaagaaat ttataatgaa 600
agatctgatt caagaagtag taagccttct gatcctaagt atgagccttc taaagagaaa 660
actgtgctag caaaaaatga accgagcgaa agtaaaatca ggggattaga ttcaagcatt 720
gaaaggggta ctaaatctaa taacaaggaa gagagaaaag ctgatgcgga aaagagtaag 780
agcaaaagca ggggagaaat acttgaagaa gataacaggg gcagtcctat cactcgtgaa 840
gacagatctg gcaaggagaa agctgaaaag catagacagc agagaactcc cactgctcgc 900
gatgctgctg agggcaggga gaggttgtct aatgcagatg atgatgcaag tgcagggatg 960
aatgataagg gtgctagaga atttggaaat accaccaggt caaggacacc tgagaggact 1020
gggaggcgat atcaggattc agaacacttt gagacggatt atgaccgaaa ttttaatctc 1080
aagcgaaaag aactggaaaa agatggctat agggacgacc gatctaaagg cagagatgat 1140
aactatagtg acaggagtag ggaccgggaa gtccccaaag aaaaaagacg gcaaccccca 1200
agtaatgaca aggattccaa aaatggggat atcagttatg atcatagcag ggaatggccg 1260
agatatggtc gtgagagggg tgacaacgag aggcctcatg gtcggtctgg taataggaaa 1320
gatggaaacc ggggtgaagc tgtgaaaact tcatcaaact ttggaatttc aaatgaaaac 1380
tatgatgtga ttgagatcca aactaagcca gattttgtaa gggcagagtt gggacccaac 1440
tttcctagga gaaacgaagt tggtcaacag tctgatggaa aatcagcacc aaatgatgaa 1500
gaatgtacaa ggaagagtga tatgtatggg tctggaccac ctagggaaga ttcaaaggaa 1560
agatatacag atgatactac ctcgcgagat cagagttcat ggaaggatga ctttgatgcc 1620
catggagtga agggaagggg acaaagaggt tctatgcctg gccgcagtgc tggtggccag 1680
agttctagtg gtggttcaca acctccatat ggaaatgcag agcagggacc cttcaacaga 1740
aatgcttctc aaggagtgaa aggaggtaga ggtgggagag gaggaagggg taggcctact 1800
ggaagagaca gccagcagat ggcaatccca attccgatga tgggatcacc atttggacct 1860
attggaatgc ctccgcctgg acccatgcag ccacttactc ctagcatgtc accagctcca 1920
ggtccgccaa tgtttccgtt ttctccacct gtgtggccgg gggctcgagg tgttgacatc 1980
agcatgttaa ccatcccacc tgttatgcct catggatcat ctggcccaag atttcctcct 2040
aatatggtga ctccaacaaa tccttccatg ttttgtggcc aatctggacc tggaagagga 2100
ggtcctccaa gcatatctag ccctggcttt aatccttctg ggccaatggg acgaggaact 2160
cctgctgata aaagtcaagg tggttgggtt cctcataaaa gtagtggacc tcctggtaaa 2220
gctccttcta gaggagagca gaatgattac tctcaaaatt ttgttgatac tggtatgcga 2280
ccccaaaatt tcatcaggga gctagagcta acaaatgttg tggaggatta ccccaagctt 2340
agggagctta tacagaaaaa agatgagatc gtggaaaaag ctgcttctaa tcccatgtat 2400
tataaatgca acctgaaaga gtttgagctg tctccagagt tctttggaac caagtttgat 2460
gtgattcttg ttgatccacc atgggaggaa tacgttcatc gtgctcctgg tgttgctgac 2520
catacagagt attggacatt tgaagaaata atgaatctta agatcgaggc aatagcagac 2580
acaccttcct ttatttttct ttgggtgggt gatggcatgg gtcttgagca gggccgtcaa 2640
tgtctgaaga agtggggatt tcgtagatgt gaagatatat gttgggtgaa gacaaacaaa 2700
actaatccga ctcctggttt gcggcatgat tctcatacat tgtttcagca ctcaaaggaa 2760
cattgcttga tgggaataaa gggtaccgtt cgtcgcagca ctgatggcca tataatccat 2820
gccaacattg atactgatgt aataatagct gaggaacccc catatggttc aacccagaag 2880
cctgaagaca tgtacaggat aattgagcat tttgccctcg gtcgcaggag gcttgagctc 2940
tttggtgaag accacaacat tcgggctggt tggctgactg ttggtaatgg attatcctca 3000
tcaaacttta ataccgaggc ttacatccgg aactttgctg acaaggatgg caaagtttgg 3060
caaggagggg gtggacgaaa tccacctccg gaagcacctc atctggttgt aactactcca 3120
gacatagaag cccttcgtcc caagtcacca atgaaaaacc agcagcagat gcagcagcag 3180
caatctgcat ctatttctct gacatcagta aattcctcca acagaaggcc tggaaactcc 3240
ccacaaaatc caaccggtct aagtatgaac caagaagctt caagttccaa tccatcaact 3300
ccagctcctt gggcggcttc accactggac ggctataaag ggagagaggg cagcattatg 3360
ccttcagatg ataagatttt tgatatgtat ggttacagtg gacagggaaa tggagactat 3420
atagattttg aggctcacag gcacatgaat ttgttgtaa 3459
<210> 4
<211> 1152
<212> PRT
<213> 草莓(Fragaria vesca)
<400> 4
Met Lys Ser Asp Arg Ala Gly Asp Asp Glu Glu Trp Glu Gly Ser Asp
1 5 10 15
Lys Arg Lys His Arg Ser Ser Arg Ser Arg Lys Ser Gly Asn Gly Glu
20 25 30
Asp Val Asp Gly Gly Gly Arg Arg Arg Ser His Gly Asp Arg Ser Glu
35 40 45
Ser Arg Lys Arg Ser Gly Gly Ser Ser Asn Ala Asp Ser Glu Glu Glu
50 55 60
Asp Tyr Asp Leu Arg Lys Glu Ser Arg Ser Lys Met Met Lys Lys Lys
65 70 75 80
Gln Glu Glu Ser Ser Leu Glu Lys Leu Ser Asn Trp Tyr Gln Asp Gly
85 90 95
Glu Phe Asp Asn Arg Gln Asp Gly Gly Asp Lys Ser Gly Gly Arg Gly
100 105 110
Leu Val Arg Ala Glu Glu Asn Glu Arg Arg Lys Leu Ala Ser Lys Leu
115 120 125
Ala Gln His Glu Ile Ser Gln Thr Lys Ser Lys Ser Lys Glu Glu Lys
130 135 140
Ser His Asp Gly Glu His Glu Lys Thr Leu Asp Arg Asp Ser Lys Tyr
145 150 155 160
Ser Asp Arg Lys Glu Ser Ile Arg Glu Lys Thr His Gly Ser Ser Glu
165 170 175
Gln Val Arg Thr Ser Arg Arg Lys Trp Asp Glu Ser Asp Gly Gly Lys
180 185 190
Lys Ala Glu Glu Ile Tyr Asn Glu Arg Ser Asp Ser Arg Ser Ser Lys
195 200 205
Pro Ser Asp Pro Lys Tyr Glu Pro Ser Lys Glu Lys Thr Val Leu Ala
210 215 220
Lys Asn Glu Pro Ser Glu Ser Lys Ile Arg Gly Leu Asp Ser Ser Ile
225 230 235 240
Glu Arg Gly Thr Lys Ser Asn Asn Lys Glu Glu Arg Lys Ala Asp Ala
245 250 255
Glu Lys Ser Lys Ser Lys Ser Arg Gly Glu Ile Leu Glu Glu Asp Asn
260 265 270
Arg Gly Ser Pro Ile Thr Arg Glu Asp Arg Ser Gly Lys Glu Lys Ala
275 280 285
Glu Lys His Arg Gln Gln Arg Thr Pro Thr Ala Arg Asp Ala Ala Glu
290 295 300
Gly Arg Glu Arg Leu Ser Asn Ala Asp Asp Asp Ala Ser Ala Gly Met
305 310 315 320
Asn Asp Lys Gly Ala Arg Glu Phe Gly Asn Thr Thr Arg Ser Arg Thr
325 330 335
Pro Glu Arg Thr Gly Arg Arg Tyr Gln Asp Ser Glu His Phe Glu Thr
340 345 350
Asp Tyr Asp Arg Asn Phe Asn Leu Lys Arg Lys Glu Leu Glu Lys Asp
355 360 365
Gly Tyr Arg Asp Asp Arg Ser Lys Gly Arg Asp Asp Asn Tyr Ser Asp
370 375 380
Arg Ser Arg Asp Arg Glu Val Pro Lys Glu Lys Arg Arg Gln Pro Pro
385 390 395 400
Ser Asn Asp Lys Asp Ser Lys Asn Gly Asp Ile Ser Tyr Asp His Ser
405 410 415
Arg Glu Trp Pro Arg Tyr Gly Arg Glu Arg Gly Asp Asn Glu Arg Pro
420 425 430
His Gly Arg Ser Gly Asn Arg Lys Asp Gly Asn Arg Gly Glu Ala Val
435 440 445
Lys Thr Ser Ser Asn Phe Gly Ile Ser Asn Glu Asn Tyr Asp Val Ile
450 455 460
Glu Ile Gln Thr Lys Pro Asp Phe Val Arg Ala Glu Leu Gly Pro Asn
465 470 475 480
Phe Pro Arg Arg Asn Glu Val Gly Gln Gln Ser Asp Gly Lys Ser Ala
485 490 495
Pro Asn Asp Glu Glu Cys Thr Arg Lys Ser Asp Met Tyr Gly Ser Gly
500 505 510
Pro Pro Arg Glu Asp Ser Lys Glu Arg Tyr Thr Asp Asp Thr Thr Ser
515 520 525
Arg Asp Gln Ser Ser Trp Lys Asp Asp Phe Asp Ala His Gly Val Lys
530 535 540
Gly Arg Gly Gln Arg Gly Ser Met Pro Gly Arg Ser Ala Gly Gly Gln
545 550 555 560
Ser Ser Ser Gly Gly Ser Gln Pro Pro Tyr Gly Asn Ala Glu Gln Gly
565 570 575
Pro Phe Asn Arg Asn Ala Ser Gln Gly Val Lys Gly Gly Arg Gly Gly
580 585 590
Arg Gly Gly Arg Gly Arg Pro Thr Gly Arg Asp Ser Gln Gln Met Ala
595 600 605
Ile Pro Ile Pro Met Met Gly Ser Pro Phe Gly Pro Ile Gly Met Pro
610 615 620
Pro Pro Gly Pro Met Gln Pro Leu Thr Pro Ser Met Ser Pro Ala Pro
625 630 635 640
Gly Pro Pro Met Phe Pro Phe Ser Pro Pro Val Trp Pro Gly Ala Arg
645 650 655
Gly Val Asp Ile Ser Met Leu Thr Ile Pro Pro Val Met Pro His Gly
660 665 670
Ser Ser Gly Pro Arg Phe Pro Pro Asn Met Val Thr Pro Thr Asn Pro
675 680 685
Ser Met Phe Cys Gly Gln Ser Gly Pro Gly Arg Gly Gly Pro Pro Ser
690 695 700
Ile Ser Ser Pro Gly Phe Asn Pro Ser Gly Pro Met Gly Arg Gly Thr
705 710 715 720
Pro Ala Asp Lys Ser Gln Gly Gly Trp Val Pro His Lys Ser Ser Gly
725 730 735
Pro Pro Gly Lys Ala Pro Ser Arg Gly Glu Gln Asn Asp Tyr Ser Gln
740 745 750
Asn Phe Val Asp Thr Gly Met Arg Pro Gln Asn Phe Ile Arg Glu Leu
755 760 765
Glu Leu Thr Asn Val Val Glu Asp Tyr Pro Lys Leu Arg Glu Leu Ile
770 775 780
Gln Lys Lys Asp Glu Ile Val Glu Lys Ala Ala Ser Asn Pro Met Tyr
785 790 795 800
Tyr Lys Cys Asn Leu Lys Glu Phe Glu Leu Ser Pro Glu Phe Phe Gly
805 810 815
Thr Lys Phe Asp Val Ile Leu Val Asp Pro Pro Trp Glu Glu Tyr Val
820 825 830
His Arg Ala Pro Gly Val Ala Asp His Thr Glu Tyr Trp Thr Phe Glu
835 840 845
Glu Ile Met Asn Leu Lys Ile Glu Ala Ile Ala Asp Thr Pro Ser Phe
850 855 860
Ile Phe Leu Trp Val Gly Asp Gly Met Gly Leu Glu Gln Gly Arg Gln
865 870 875 880
Cys Leu Lys Lys Trp Gly Phe Arg Arg Cys Glu Asp Ile Cys Trp Val
885 890 895
Lys Thr Asn Lys Thr Asn Pro Thr Pro Gly Leu Arg His Asp Ser His
900 905 910
Thr Leu Phe Gln His Ser Lys Glu His Cys Leu Met Gly Ile Lys Gly
915 920 925
Thr Val Arg Arg Ser Thr Asp Gly His Ile Ile His Ala Asn Ile Asp
930 935 940
Thr Asp Val Ile Ile Ala Glu Glu Pro Pro Tyr Gly Ser Thr Gln Lys
945 950 955 960
Pro Glu Asp Met Tyr Arg Ile Ile Glu His Phe Ala Leu Gly Arg Arg
965 970 975
Arg Leu Glu Leu Phe Gly Glu Asp His Asn Ile Arg Ala Gly Trp Leu
980 985 990
Thr Val Gly Asn Gly Leu Ser Ser Ser Asn Phe Asn Thr Glu Ala Tyr
995 1000 1005
Ile Arg Asn Phe Ala Asp Lys Asp Gly Lys Val Trp Gln Gly Gly Gly
1010 1015 1020
Gly Arg Asn Pro Pro Pro Glu Ala Pro His Leu Val Val Thr Thr Pro
1025 1030 1035 1040
Asp Ile Glu Ala Leu Arg Pro Lys Ser Pro Met Lys Asn Gln Gln Gln
1045 1050 1055
Met Gln Gln Gln Gln Ser Ala Ser Ile Ser Leu Thr Ser Val Asn Ser
1060 1065 1070
Ser Asn Arg Arg Pro Gly Asn Ser Pro Gln Asn Pro Thr Gly Leu Ser
1075 1080 1085
Met Asn Gln Glu Ala Ser Ser Ser Asn Pro Ser Thr Pro Ala Pro Trp
1090 1095 1100
Ala Ala Ser Pro Leu Asp Gly Tyr Lys Gly Arg Glu Gly Ser Ile Met
1105 1110 1115 1120
Pro Ser Asp Asp Lys Ile Phe Asp Met Tyr Gly Tyr Ser Gly Gln Gly
1125 1130 1135
Asn Gly Asp Tyr Ile Asp Phe Glu Ala His Arg His Met Asn Leu Leu
1140 1145 1150
Claims (10)
1.草莓甲基转移酶基因在控制草莓成熟中的应用,其特征在于,所述甲基转移酶是如下A1或A2的蛋白质:
A1、氨基酸序列是序列表中序列2或序列4所示的氨基酸序列的蛋白质;
A2、在A1的N末端或/和C末端连接蛋白标签得到的融合蛋白质。
2.根据权利要求1所述的应用,其特征在于,所述甲基转移酶基因具体为如下D1或D2所示的基因:
D1、编码链的编码序列是序列表中序列1或序列3的DNA分子;
D2、核苷酸序列是序列表中序列1或序列3的DNA分子。
3.根据权利要求1或2所述的应用,其特征在于,所述控制草莓成熟,为通过抑制或降低权利要求1所述甲基转移酶基因的表达延缓草莓成熟。
4.根据权利要求1或2所述的应用,其特征在于,所述控制草莓成熟,为通过促进或增强权利要求1所述甲基转移酶基因的表达促进草莓成熟。
5.延缓草莓成熟的试剂,其特征在于,所述试剂的活性成分为抑制编码权利要求1所述甲基转移酶的基因的表达、降低权利要求1所述甲基转移酶的丰度、和/或敲除编码权利要求1所述甲基转移酶的基因的物质。
6.促进草莓成熟的试剂,其特征在于,所述试剂的活性成分为增强编码权利要求1所述甲基转移酶的基因的表达和/或增加权利要求1所述甲基转移酶的丰度的物质。
7.一种延缓草莓成熟的方法,其特征在于,包括如下步骤:抑制受体草莓中权利要求1所述甲基转移酶的表达、降低权利要求1所述甲基转移酶的丰度、和/或敲除编码权利要求1所述甲基转移酶的基因,得到成熟期晚于所述受体草莓的目的草莓。
8.一种促进草莓成熟的方法,其特征在于,包括如下步骤:增强受体草莓中权利要求1所述甲基转移酶的表达和/或增加权利要求1所述甲基转移酶的丰度,得到成熟期早于所述受体草莓的目的草莓。
9.蛋白质,其特征在于,其为如下A1或A2的蛋白质:
A1、氨基酸序列是序列表中序列2或序列4所示的氨基酸序列的蛋白质;
A2、在A1的N末端或/和C末端连接蛋白标签得到的融合蛋白质。
10.编码权利要求9所述蛋白质的核酸分子,其特征在于,所述核酸分子具体为如下D1或D2所示的核酸分子:
D1、编码链的编码序列是序列表中序列1或序列3的DNA分子;
D2、核苷酸序列是序列表中序列1或序列3的DNA分子。
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CN114350686A (zh) * | 2022-02-24 | 2022-04-15 | 华中农业大学 | 草莓sl基因及其应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1997027295A1 (en) * | 1996-01-23 | 1997-07-31 | Horticulture Research International | Fruit ripening-related genes |
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WO1997027295A1 (en) * | 1996-01-23 | 1997-07-31 | Horticulture Research International | Fruit ripening-related genes |
Non-Patent Citations (5)
Title |
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"PREDICTED: Fragaria vesca subsp. vesca methyltransferase-like protein 1 (LOC101310233), mRNA, NCBI Reference Sequence: XM_004302180.2", 《NCBI数据库》 * |
"PREDICTED: Fragaria vesca subsp. vesca N6-adenosine-methyltransferase MTA70-like (LOC101294526), transcript variant X2, mRNA, NCBI Reference Sequence: XM_004309537.2", 《NCBI数据库》 * |
LEILEI ZHOU等: "N6-methyladenosine RNA modification regulates strawberry fruit ripening in an ABA-dependent manner", 《GENOME BIOLOGY》, vol. 22 * |
LEILEI ZHOU等: "RNA methylomes reveal the m6A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening", 《GENOME BIOLOGY》, vol. 20 * |
TINGTING GU等: "Characterization of DNA methyltransferase and demethylase genes in Fragaria vesca", 《MOL GENET GENOMICS》 * |
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CN114350686A (zh) * | 2022-02-24 | 2022-04-15 | 华中农业大学 | 草莓sl基因及其应用 |
CN114350686B (zh) * | 2022-02-24 | 2024-05-10 | 华中农业大学 | 草莓sl基因及其应用 |
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