CN111926014A - DNA插入片段下调番茄YFT1 allele表达及其在番茄品质改良中的应用 - Google Patents
DNA插入片段下调番茄YFT1 allele表达及其在番茄品质改良中的应用 Download PDFInfo
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Abstract
本发明一种公开了DNA插入片段下调番茄YFT1allele表达及其在番茄品质改良中的应用。通过功能互补遗传转化实验为YFT1在番茄果色形成过程中功能确认提供了坚实的遗传学证据。启动子活性研究进一步确认了YFT1基因启动子驱动能力显著降低,并且仅与573bp DNA片段插入有关,并进一步从分子水平揭示,YFT1调控区域(启动子)的573bp DNA片段插入导致基因显著下调表达是由于其含有2个负调控区段(‑272bp~‑173bp,‑172~‑73bp);同时该插入事件也导致了YFT1allele在yft1产生了可变剪接和5'UTR区域中的uORF被打破。
Description
技术领域
本发明属于分子生物学和遗传学领域,涉及一种影响番茄果色形成及乙烯信号途径核心组分基因YFT1转录调控及应用,尤其涉及一种DNA插入片段下调番茄YFT1 allele表达及其在番茄品质改良中的应用。
背景技术
番茄(Solanum lycopersicum,2n=24)属于茄科(Solanaceae)番茄属(Lycopersicon)植物,是世界上第二大蔬菜作物。番茄是研究浆果和茄科相关领域的重要模式植物。果色是番茄重要外观品质性状,市场主流的红果、黄果和红黄果色形成与其果实中所积累的类胡萝卜素多寡有关,特别是果实中番茄红素和β-胡萝卜素比例。因而,番茄的果色还关系到番茄品质、营养和生物学功能。番茄类胡萝卜素生物合成途径每一步从分子水平(基因)和蛋白水平(催化酶)均已说详细研究和确定,而对类胡萝卜素合成的分子调控机制揭示还较少,也将是今后该领域的研究重点和方向。
作为有呼吸跃高峰的番茄,乙烯在果实成熟过程中扮演着关键角色;因而,乙烯生物合成及其信号转导对番茄果实成熟和果色形成具有重要的调控作用。下调番茄乙烯合成关键基因ACS(ACC SYNTHASE)和ACO(ACC OXIDASE)表达水平,会影响到番茄果实的类胡萝卜素的累积,红果番茄有时表现出淡红色、橙色甚至黄色表型。乙烯信号转导也会对番茄果实成熟和颜色形成产生影响。下调乙烯受体基因LeETR4(ETHYLENE RESPONSE 4)、LeETR6(ETHYLENE RESPONSE 6)以及LeCTR1(CONSTITUTIVE TRIPLE RESPONSE 1)表达,促进了番茄果实成熟,果实提早着色。乙烯不敏感番茄突变体Nr(Never ripe)表现出难以成熟的表型,使番茄红素累积水平下降。下调乙烯信号途径下游组分LeEIL(ETHYLENE INSENSITIVE3-like)表达,与野生型相比表现出果实成熟延迟表型,并呈现出黄色或橘黄色。
定位于内质网膜YFT1/EIN2(ETHYLENE INSENSITIVE 2)是乙烯信号途径核心组分白,与受烯调控上游的乙烯受体和CTR1(CONSTITUTIVE TRIPLE RESPONSE1)互作,决定其羧基端(CEND)是否可从EIN2上切离,携带乙烯信号CEND在胞质中在蛋白水平调控EIN3-BINDING F-BOX 1/2(EBF1/2)表达,决定后者的入核量,或直接入核,与EIN3/EIL1形成复合体,调控下游乙烯应答基因表达。
EIN2功能丧失,植物丧失了对乙烯刺激的应答。拟南芥ein2突变体对乙烯完全不敏感,苗期“三重”反应消失。EIN2还是多种植物激素信号途径的交叉点,拟南芥ein2突变体可以通过脱落酸、生长素运输抑制剂及细胞分裂素和茉莉酸胁迫得以恢复,并延缓衰老。EIN2在盐胁迫、氧化应激及其他生物或非生物胁迫中扮演重要角色。EIN2还参与因衰老而引起的细胞死亡调控,NAC转录因子家族的ORE1(oresara 1),可以促进拟南芥叶片细胞衰老和死亡,EIN2通过正调控ORE1表达而促进细胞衰老。ein2突变体中细菌性青枯病的发病速度与野生型相比明显下降。EIN2还可可限制植物细胞扩增,拟南芥ein2突变体株高、叶面积、果荚长度等显著高于野生型植株。特别是,EIN2具有调控具有呼吸跃变高峰的果实成熟,番茄LeEIN2下调表达表出迟熟,类胡萝卜素累积下降和硬度增大。
综上所述,EIN2对植物生长发育调控特别是通过调控乙烯合成和信号转导途径研究的比较深入,而调控EIN2(YFT1)表达的研究鲜见报道。
发明内容
针对现有技术的缺陷,本发明提供了一种DNA插入片段下调番茄YFT1 allele表达及其在番茄品质改良中的应用。
本发明公开了一种与番茄果色形成相关基因YFT1(YELLOW FRUITED TOMATO 1)遗传损伤导致番茄果色改变的分子机制;具体涉及yft1番茄突变体的YFT1 allele基因调控序列即其起始密码子(ATG)上游-318bp处发生了573bp DNA片段插入(IF573)和13bp DNA片段缺失(Del 13)而导致YFT1 allele在番茄yft1中显著下调表达。功能互补遗传转化实验为YFT1在番茄果色形成过程中功能确认提供了坚实的遗传学证据。启动子活性研究进一步确认了YFT1基因启动子驱动能力显著降低,并且仅与573bp DNA片段插入有关,而不受13bpDNA片段缺失(Del 13)的影响。研究结果进一步从分子水平揭示,YFT1调控区域(启动子)的573bp DNA片段插入导致基因显著下调表达是由于其含有2个负调控区段(-272bp~-173bp,-172~-73bp,将插入片段3'末端指定为-1bp);同时该插入事件也导致了YFT1allele在yft1产生了可变剪接和5'UTR(untranslated region)中的uORF(upstream openreading frame)被打破。
本发明的目的是通过以下技术方案实现的:
本发明提供了一种573pb DNA插入片段在下调番茄YFT1 allele表达中应用,所述573pb DNA插入片段的序列如SEQ ID NO:2所示,所述573pb DNA插入片段导致YFT1 allele在转录加工水平改变。
本发明提供了一种uORF1序列在下调番茄YFT1 allele表达中的应用,所述uORF1序列如SEQ ID NO:7所示,由DNA插入片段导致uORF1序列变化而得到。
本发明提供了一种DNA插入片段在下调番茄YFT1 allele表达中的应用,所述DNA插入片段如SEQ ID NO:9和/或SEQ ID NO:10所示。
优选地,所述DNA插入片段的插入位点为YFT1 allele基因5'UTR区域的ATG上游-318bp。
本发明提供了一种下调YFT1 allele表达的方法,包括在YFT1 allele基因5'UTR区域的ATG上游-318bp插入如前述的DNA插入片段。
本发明提供了一种前述的DNA插入片段在调控番茄果色形成和果实成熟中的应用。
优选地,所述调控果实成熟的应用中,DNA插入片段调控番茄乙烯合成和释放。
优选地,所述调控果实成熟的应用中,DNA插入片段调控番茄成熟发育期间类胡萝卜素的合成。
本发明提供了一种前述的DNA插入片段在番茄遗传改良和育种中的应用。
本发明涉及黄果晚熟突变体yft1(yellow fruited tomato 1)和野生型红果cv.M82番茄,均由以色列希伯来大学Dani Zamir教授提供(http:// zamir.sgn.cornell.edu/mutateds)。yft1番茄突变体突变体(n3122)系利用快中子(60Coγ-射线)轰击番茄cv.M82种子而获得。并且经Gao et al.(2016)将YFT1 allele基因用图位克隆技术将其定位于番茄第9染色体,并初步确认其候选基因是EIN2(Solyc09g007870)。不过缺少最直接的遗传证据,而发明专利进一步揭示,导致YFT1遗传损伤包括2个分子生物学事件:1)在YFT1起始密码子(ATG)上游-318bp处的一个573bp DNA片段的插入(IF573)和2)一个13bp DNA片段的缺失。从而导致YFT1显著下调表达。通过对YFT1启动子(-1bp~-3000bp/-3564bp)通过逐步删除和功能区段分析发现,导致yft1番茄突变体中YFT1 allele基因下调表达的主要是由IF573所致,而与13bp DNA片段缺失(Del 13)无关(图2)。
对yft1番茄YFT1 allele调控区域的IF573事件的DNA序列提交番茄基因组(https://www.solgenomics.net/)分析发现,IF573的DNA序列位于番茄6号染色体的Solyc06g082410.1(48234430…48234807,Nodulin-like protein)和Solyc06g082420.2(48242060…48244274,Peroxidase 3)之间序列(48238759~48239331)相同,而进一步PCR扩增和测序发现,在yft1番茄突变体中第6染色体的相同DNA区段并未发现缺失(图1);同时,13bp DNA片段缺失(Del 13)并未影响YFT1启动子的强度(图2);暗示导致YFT1 allele下调表达是因IF573事件所致。并通过互补/敲低番茄转基因实验为YFT1在果色形成过程中功能提供了坚实的遗传学证据。用RACE技术分别在cv.M82/yft1中克隆YFT1/YFT1 allele全长的cDNAs,比对YFT1和YFT1 allele的5′UTR序列发现,IF573引入了异常剪切位点(-974bp,GT~-350bp,AG),致使yft1中YFT1 allele与cv.M82的YFT1相比,转录成3个不同转录本yft1.1、yft1.2、yft1.3,其中仅有yft1.2和yft1.3具有与YFT1相同的编码区。
分析yft1.2序列发现,5′UTR区域包含2个大小不同uORF1(105nt)和uORF2(99nt),而YFT1仅转录合成一条转录本(4858nt),其中其5′UTR区域仅包含一个与yft1的转录本(yft1.2和yft1.3)同源uORF1(99nt,其5'端78nt序列与yft1的相同)。用Luciferase活性分析检测YFT1 allele启动子能力发现,创制YFT1 allele uORF1(105nt)突变(ATG→AgG)启动子,可以显著地提高YFT1 allele启动子活性。将IF573不同DNA区段置于组成型CaMV 35S启动子下游,驱动Luciferase表达发现IF573含有2个负调控区域(-272bp~-173bp和-172bp~-73bp,这里IF573 3'末端指定为-1bp)。综上所述,IF573事件因引入异常剪接位点、负调控区域和uORF1(105nt)被打破而导致yft1中的YFT1 allele显著下调,并影响果实成熟发育和果色形成。在生产实践中,可以根据此研究结果调控番茄果色形成和进行品质改良。
本发明提供了一种iYFT1(为YFT1基因中的287bp特异片段)在下调番茄YFT1表达中的应用,所述iYFT1的序列如SEQ ID NO:17所示。
本发明还提供了一种前述的iYFT1在调控番茄果色形成和果实成熟中的应用。
优选地,所述调控果实成熟的应用中,iYFT1调控番茄乙烯合成和释放。
优选地,所述调控果实成熟的应用中,iYFT1调控番茄成熟发育期间类胡萝卜素的合成。
本发明提供了一种前述的iYFT1在番茄遗传改良和育种中的应用。
与现有技术相比,本发明具有如下有益效果:
1.首次揭示了番茄YFT1基因下调表达的分子机制;
2.基于研究成果,可能靶向地对番茄果实成熟调控和对番茄品质特别是果色形成的改良。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显:
图1 573bp插入片段的来源及缺失分析;
图2突变体yft1中引起YFT1下调表达的突变事件分析;
图3番茄果色在不同遗传背景下的颜色变化(功能互补实验);
图4YFT1在转基因株系中表达水平差异;
图5插入位点上下游表达水平分析;
图6野生型cv.M82和突变体yft1中YFT1/YFT1 allele结构示意图;
图7以突变的方式来分析uORF对启动子活性的影响;
图8通过与CaMV 35S启动子融合的方法检测573bp插入片段的功能区域;
图9不同遗传背景材料的果实乙烯释放量;
图10不同遗传背景材料ACS2、ACS4和ACO1在不同果实发育时期的表达水平;
图11不同遗传背景番茄乙烯信号途径关键基因的表达;
图12不同遗传背景材料的番茄幼苗在ACC作用下的表现;
图13番茄果实有色体超微结构观察;
图14不同遗传背景番茄果实类胡萝卜素含量;
图15类胡萝卜素合成途径关键基因在不同遗传背景材料中的表达水平。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明,但不以任何形式限制本发明。应当指出的是,对本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变化和改进。这些都属于本发明的保护范围。
实施例一:YFT1/YFT1 allele启动子活性分析
为了解析导致yft1中YFT1 allele基因下调表达的遗传损伤的2个事件——IF573(序列如SEQ ID NO:2所示)插入和Del 13删除所扮演的生物学角色,用PCR技术从cv.M82/yft1中分别克隆YFT1的启动子pYFT1和yft1的YFT1 allele的启动子pyft1,构建了系列启动子突变载体,以期分析明确两个突变事件对YFT1 allele下调表达的影响,具体步骤如下:
1.构建检测启动子活性载体
基于YFT1基因上游序列,设计并合成引物[pYFT1-F:5'-gatgttatcggggaggaagaaac-3'(SEQ ID NO:13),pYFT1-R:5'-tatttgatttaaatggttagcaagc-3'(SEQ ID NO:14)]扩增cv.M82和突变体yft1中YFT1基因上游启动子序列,并分别命名为pYFT1和pyft1,并将其构建至GUS基因(β-glucuronidase)上游(载体pCambia1391,本实验室保存)。
同时,鉴于突变体启动子区域发生了IF573插入和Del 13缺失两个突变事件,构下述突变启动子活性检测载体:pYFT1-Del13(在pYFT1删除Del 13 DNA片段)、pyft1+Del13d(将Del 13插入至pyft1的IF573的下游)、pyft1+Del13u(将Del 13插入至pyft1的IF573的上游),并分别将突变的启动子融合于GUS基因上游,获得含有不同突变启动子的质粒。
2.GUS酶活分析
将步骤1构建的含有不同突变启动子的质粒用冻融法转化农杆菌GV3101,并分别以含pYFT1和pyft1为正负对照,用注射法转化同一烟草叶片,并用记号笔在叶片上作好标记;转化完成后,将烟草置于暗处培养24h,然后再于光下培养24h;然后取样并用织破碎仪在液氮中研成粉末,取适当量加入GUS酶提取液,提取GUS粗蛋白。以MUG为底物,分别在酶促反应0min、15min、30min、45min和60min取样,加入0.2M Na2CO3为反应终止剂,利用荧光酶标仪(Biotek仪器有限公司,佛蒙特州,美国)测定酶促反应产物的荧光强度。蛋白的浓度用Braford法测定,并用下述下公式求得GUS酶活:
不同突变启动子驱动GUS表达的酶活的测定结果如图2所示。
实施例二:突变体yft1中YFT1 allele遗传损伤分析
yft1番茄突变体YFT1 allele表达水平显著下调是因该基因遗传损伤所致,所述的遗传损伤系该基因在起始密码子(ATG)上游-318bp发生了:1)一个573bp DNA片段(序列如SEQ ID NO:2所示)插入和2)一个13bp DNA序列缺失(Del 13)两个突变事件造成,并在Gao L,Zhao W,Qu H,Wang Q and Zhao L.The yellow-fruited tomato 1(yft1)mutanthas altered fruit carotenoid accumulation and reduced ethylene production asa result of a genetic lesion in ETHYLENE INSENSITIVE2.Theoretical and AppliedGenetics,2016,129:717-728进行了详细阐述,而YFT1 allele遗传损伤中IF573和Del 13所扮演的角色并未揭示;同时,IF573来源也缺乏实验证据,导致yft1突变体番茄的黄果表型是由于在YFT1 allele基因调控区域插入所致,还是因其从原基因组位置转移所致,也缺乏分子生物学和遗传学证据。为此,开展下述研究,以期揭示其中的科学问题,具体步骤如下:
1.IF573插入片段来源分析
基于茄科植物基因组学数据库(http://solgenomics.net/)的Blast工具,将IF573的DNA序列(SEQ ID NO:2)以fasta格式提交,选用Tomato Genome(Current version),数据库选择Tomato genome chromosomes(Build SL 3.0),与番茄的基因组数据库进行比对,以确定573bp插入片段所在染色体及位置。Blast结果分析发现,IF573 DNA片段被映射于番茄6号染色体Solyc06g082410.1(48234430…48234807,Nodulin-like protein)和Solyc06g082420.2(48242060…48244274,Peroxidase 3)之间的48238759~48239331。
2.番茄基因组DNA提取
1)田间选取番茄野生型cv.M82及突变体yft1植株的幼嫩叶片,放入事先加入400μL CTAB(加2%体积β-巯基乙醇)的1.5mL EP管中,带回实验室后,补加300μL CTAB(加2%体积β-巯基乙醇),并加入2粒干净的钢珠,组织研磨仪55Hz 90sec将样品粉碎,之后将样品放入65℃的水浴锅中加热30min,期间每隔5min颠倒混匀一次;
2)将加热的1.5mL EP管稍冷,在通风橱中用1mL Eppendorf移液器小心地吸取700μL氯仿:异戊醇(V:V=24:1)加入到1.5mL EP管中,上下颠倒混匀5min;
3)将1.5mL EP管放入离心机中,10000rpm离心10min;小心吸取400μL上层液体加入到新的1.5mL EP管中,注意一定不要吸到液体分层处的物质;
4)加入800μL-20℃预冷的无水乙醇,上下颠倒,12000rpm离心10min,倒掉离心管内液体,70%乙醇洗涤3次,通风橱内晾干;
5)在风干的1.5mL离心管中加入50μL ddH2O溶解DNA样品,加入1μL RNA水解酶A37℃ 30min去除其中的RNA;
6)溶解的DNA样品,取1μL用Nanodrop 2000测定A260/A230、A260/A280、浓度(ng/μL),取2μL加入6×loading buffer,琼脂糖凝胶电泳检测。
3.IF573 DNA片段扩增及测序
基于IF573片段的Blast结果,根据基侧翼DNA序列设计一对引物[Ch06-573-F:5′-gtaaataataacttcgtagatcatggc-3′(SEQ ID NO:11);Ch06-573-R:5′-tggtcctccatcaagaatttctcc-3′)(SEQ ID NO:12)],以上述所提取的cv.M82和yft1番茄基因组DNA为模板,在KOD FX酶催化下扩增,反应体系如下:
将上述PCR反应物充分混均后,T100TM PCR仪(Bio-Rad生命医学产品有限公司,加利福尼亚州,美国)中进行扩增反应,反应参数为:95℃预变性5min;95℃变性30sec,55℃退火30sec,68℃延伸(1kb/min),35个循环;最后68℃延伸10min。PCR产物与6×loadingbuffer混匀,用1%的琼脂糖凝胶在1×TAE(Tris-acetate-EDTA)进行电泳分离,在120V电泳20min后,在UVP凝胶成像系统(UVP公司,加利福尼亚州,美国)下检测并拍照,切取目标条带进行胶回收,送铂尚生物技术(上海)有限公司测序。将源自cv.M82和yft1番茄的扩增片段进行比对,未发现cv.M82和yft1番茄扩增的DNA序列间存在差异,亦即说yft1番茄中在第6染色体上并未发生缺失,暗示yft1番茄突变体所展示出的与cv.M82差异表型并非因第6染色体的IF573缺失所致,而进一步确认是由于其插入YFT1 allele基因上游调控区域导致其遗传损伤所致(图1)。
实施例三:YFT1基因功能互补实验
1.载体构建
1)过表达载体YFT1-CDS构建:用PCR技术克隆以YFT1的CDS序列[YFT1-CDS-F:5′-atggagtctgaaactctgactagag-3′(SEQ ID NO:15);YFT1-CDS-R:5′-caagacgaaagggggtgat-3′(SEQ ID NO:16)]
采用的扩增引物序列如和所示),将其构建于pHB质粒(由上海交通大学杨洪全教授实验室惠赠,Mao J,Zhang YC,Sang Y,Li QH,Yang HQ.2005.A role for Arabidopsiscryptochromes and COP1 in the regulation of stomatal opening.Proceedings ofthe National Academy of Sciences,USA 102,12270-12275.)的BamH I和Xba I位点,并命名为YFT1-CDS;
2)敲低载体YFT1-RNAi构建:基于YFT1的cDNA序列中一287bp特异序列(命名为iYFT1,序列如SEQ ID NO:17所示)设计引物(RNAi-F:5′-cacctaagccatccagatttatcaac-3′(SEQ ID NO:18);RNAi-R:5′-catgtccccttcaattctgag-3′(SEQ ID NO:19)),用高保真酶KOD plus扩增;将扩增片段(纯化后的PCR产物)与中间载体pTOPO-ENTR/D Vector(购自Invitrogen公司)连接,构建pTOPO-iYFT1,5min后用于转入大肠杆菌DH5α感受态,连接体系如下:
选用(菌落PCR和测序)阳性克隆提取质粒,通过LR重组反应将连入的特异性片段iYFT1置换至干扰载体pHELLSGATE12上,构建YFT1的RNAi干扰载体YFT1-RNAi,连接体系如下:
2.农杆菌介导的番茄转化
将YFT1-CDS和YFT1-RNAi质粒用冻融法转化EHA105,然后分别用于转化突变体yft1和野生型cv.M82。
1)播种:选取野生型cv.M82和突变体yft1的种子各约200粒,放入无菌150mL三角瓶中,加入25mL 70%的乙醇,轻轻摇荡三角瓶2min,倒掉瓶中液体,注意尽量不要倒出种子;加入无菌水漂洗1次,倒掉瓶中液体;加入25mL 30%的次氯酸钠溶液,轻轻摇荡三角瓶10min,倒掉瓶中废液,用无菌水漂洗5~6次,最后一次,将种子置于无菌滤纸上;用镊子将种子均匀地摆放在发苗培养基(1/2MS0固体培养基)上,每皿约放置种子20粒,置于25℃16h光照/8h黑暗的光周期条件下培养;
2)预培养:发苗约7天后,番茄苗长到子叶展开,未见生长点的状态时,用剪刀剪去子叶的叶尖,剩余的部分,每0.3cm剪下放入预培养的培养基MS1上,25℃避光培养24h;
3)摇菌:预培养前一天,从-80℃冰箱中取出保存的菌种(即前面所述“将YFT1-CDS和YFT1-RNAi质粒用冻融法转化EHA105”保存的菌),加入5mL三抗培养基(过表达载体YFT1-CDS的转化选用利福平50mg/L、链霉素50mg/L、卡那霉素100mg/L,干扰载体YFT1-RNAi的转化选用利福平50mg/L、链霉素50mg/L、壮观霉素100mg/L),28℃ 200rpm过夜培养;第二天下午,取过夜培养的菌液10μL加入25mL三抗培养基(与上面相对应)中扩大培养,28℃ 200rpm过夜培养,第三天上午,测定菌液的OD600值,直至OD600达到0.5~0.6之间;
4)侵染:4000rpm离心15min,收集菌体,去上清;加入等体积重悬培养基(1/2MS0液体)重悬,之后置入28℃ 180rpm摇床轻轻摇动30min;将菌液倒入放有外植体的MS1上,轻轻摇动,侵染8~10min;
5)共培养:用移液枪轻轻吸走全部侵染菌液,用无菌的镊子摆放好所有外植体后,避光培养36h;
6)筛选:共培养完成后,将外植体移动至筛选培养基MS2上,在25℃、16h光照/8h黑暗的光周期下培养,每隔10~12天更新一次培养基,期间用无菌手术刀片割去褐化的组织,约40天后,会有外植体分化长出含生长点的幼芽;
7)生根:待幼芽生长至3~5cm长时,用无菌的手术刀片切下,放入生根培养基MS3上,约7天后,再生苗开始长根;
以上步骤均需在无菌条件下进行。
8)驯化栽植:待再生苗的根系生成时,将苗从生根培养基中挪出,洗净其根上附着的培养基,栽植到装有湿润的、草炭与蛭石的比例为1:1的塑料花盆中,外面缠绕一层保鲜膜保湿,待7天后撤去保鲜膜。
3.转基因植株鉴定及果实表型观察
用PCR方法检测再生菌,将阳性苗栽植于转基因专用隔离温室,同时观察cv.M82、yft1、YFT1-CDS和YFT1-RNAi植株生长和果实表型:花后35dpa(days past anthesis)、47dpa和54dpa果实颜色(图3);并检测YFT1在各个材料中转录表达(图4)。结果显示:YFT1-CDS在47dpa时期,进入破色期,54dpa时期,果实进入红熟期;而YFT1-RNAi却在47dpa时期仍果实保持绿色,54dpa时期果实显黄色(图3)。分析YFT1的表达水平发现,YFT1-CDS中YFT1的表达水平恢复到与cv.M82无显著差异的水平,而YFT1-RNAi中YFT1的表达水平显著低于cv.M82,但高于yft1(图4)。
实施例四:YFT1/YFT1allele全长cDNA克隆
用RACE技术克隆cv.M82中YFT1基因的全长cDNA,设计5′RACE和3′RACE特异性引物(gene-specific primers,GSP)[YFT1-5′GSP-R:5′-tggtgaatggtgcaactgagcttctg-3′(SEQID NO:24)和YFT1-3′GSP-F:5′-cttccaacaggaaacccggttctgc-3′(SEQ ID NO:25)],分别扩增YFT1 cDNA的5′末端和3′末端。参考5′RACE和3′RACE的测序结果,设计引物[YFT1-cDNA-F:5′-agctcacacaccataactctccatctctctaggatatg-3′(SEQ ID NO:26);YFT1-cDNA-R:5′-tcttgtaataacatcttttcccgccttttagcccg-3′(SEQ ID NO:27)),扩增YFT1基因的全长cDNA,结果发现yft1突变体中IF573(序列如SEQ ID NO:2所示)插入了YFT1 allele基因的5'UTR区域。分别设计插入位点上游定量引物[YFT1-uI-F:5′-agcttccttaaaagcttctgatctg-3′(SEQID NO:20);YFT1-uI-R:5′-caataatccgattgccgattctg-3′(SEQ ID NO:21)]和插入位点下游定量引物[YFT1-dI-F:5′-cttggattttgaaaaggtccatcag-3′(SEQ ID NO:22);YFT1-uI-R:5′-acacttttcacactgggacttgct-3′(SEQ ID NO:23)],用RT-qPCR技术分析果实不同发育期(35dpa,47dpa和54dpa)的YFT1和YFT1 allele插入位点(ATG上游-318bp)上、下游转录表达时发现,YFT1在插入位点(ATG上游-318bp)下游mRNA累积比上游的多,不过无显著差异;而YFT1 allele在插入位点(ATG上游-318bp)上游的mRNA累积显著高于下游;说明YFT1allele在yft1番茄中存在可变剪接mRNA转录后加工模式(图5)。
用RACE(Rapid amplification of cDNA ends)技术分析,确认yft1突变体中YFT1allele的可变剪接方式,在cv.M82中仅检测到一种成熟转录本(647nt 5′UTR,3951nt CDS和260nt 3'UTR,序列如SED ID NO:1所示);而在yft1中,检测到了yft1.1(SED ID NO:4所示DNA序列)、yft1.2(SED ID NO:5所示DNA序列)和yft1.3(SED ID NO:6所示DNA序列)。其中yft1.1转录本较短,终止于插入片段内部;yft1.2和yft1.3与cv.M82中的YFT1的转录本具有相同的编码区和3′UTR,差异仅发生在5′UTR区,相差625bp可变剪切序列(SED ID NO:3)(图6)。这与IF573引入的异常剪接位点有关。
实施例五:IF573在YFT1allele调控区引入了uORFs
对YFT1 allele基因启动子特别是IF573(序列如SEQ ID NO:2所示)分析发现,与YFT1启动子相比引入了2个uORFs(upstream open reading frames)——uORF1(105nt,序列如SED ID NO:7所示)和uORF2(99nt,序列如SED ID NO.8所示);对所引入的uORF突变对pyft1启动子活力影响(以pYFT1作参照),具体实验操作如下:
1.系列uORFs突变启动子构建
突变野生型cv.M82的YFT1和突变型yft1中YFT1 allele的5′UTR区uORFs,分别构建了pyft1-del625(删除yft1.3的5′UTR区的第二内含子,625bp)、pYFT1(uORF1m)(突变pYFT1的5′UTR区域的uORF1,AT-407G突变为Ag-407G,以ATG上游第一个核苷酸为-1,其位点标记作相应调整)、yft1(uORF1m)(突变pyft1 5′UTR区的uORF1,AT-967G突变为Ag-967G)、yft1(uORF2m)(突变pyft1 5′UTR区uORF2,A-614TG突变为c-614TG)、yft1(uORF1m+uORF2m)[同时突变pyft1 5′UTR区uORF1(AT-967G突变为Ag-967G)和uORF2(A-614TG突变为c-614TG)],以pYFT1和pyft1为参照,将其构建于载体pGreen II 0800LUC(由上海交通大学杨洪全教授惠赠)的LUCIFERASE之前。
2.番茄果实瞬时转化
选择测序正确的质粒,将其转入农杆菌GV3101感受态,挑选阳性单斑,将其加入到5mL LB抗性培养基(利福平50mg/L、庆大霉素50mg/L、卡那霉素100mg/L),摇床28℃200rpm过夜培养;
第二天,吸取过夜培养的菌液加入50mL加抗性(利福平50mg/L、庆大霉素50mg/L、卡那霉素100mg/L)的诱导培养基中,摇床28℃ 200rpm过夜培养;离心收集菌体,弃上清,用注射培养基重悬菌体,并将其OD600调至1.0,室温20rpm培养2h;
选择长势良好、大小一致的MicroTOM绿熟期果实,用带针头的1mL注射器吸取菌液,从果脐处将针头插入3~4mm,慢慢推动注射器使菌液缓缓进入到果实内部,直到菌液从萼片的顶端渗出为止;之后,将MicroTOM番茄株置于暗处培养24h,光下培养24h。
3.LUCIFERASE酶活测定
将转化的果实取下,弃掉种子和坏掉的部分,其余部分在液氮的作用下,用研钵研磨成粉末状;取约100mg粉末加入到1.5mL预冷EP管中,加入400μL 1×PLB裂解液(Passivelysis buffer),充分震荡混匀,12000rpm离心30sec;取上层清液8μL,加入到新的1.5mL EP管中,然后迅速加入40μL LAR II(Luciferase assay reagent II),轻轻点动EP管底部,混匀,点击测定键;再加入40μL Stop and Glo Buffer,轻轻混匀,点击测定键,记录测定数据。分析酶活发现,pyft1中的uORF1(序列如SED ID NO:7所示)在下调pyft1启动子活性扮演着重要角色(图7)。
实施例六:IF573中的负调控区域
为了进一步分析IF573 DNA片段在调控YFT1 allele在yft1中下调表达的功能区段,将IF573用PCR技术分割成系列区段(100bp逐步删除并与35S启动子融合),基于LUC表达水平确定IF573各功能区段具体操作如下:
将IF573(序列如SEQ ID NO:2所示)逐渐删除(每步100bp)区段构建于CaMV 35S启动子下游,形成融合启动子35S-IF573,、35S-IF473、35S-IF373、35S-IF273、35S-IF173、35S-IF73和以IF573为参照,并将这些融合启动子分子构建于LUC基因上游。LUC表达水平显示,35S-IF573、35S-IF473、35S-IF373、35S-IF273启动子活性与IF573无显著差异,并显著低于35S-IF173和35S-IF573启动子活性,说明IF573 3′端的-272bp~-173bp(序列如SED ID NO:9所示)和-172bp~-73bp存在负调控区域(序列如SED ID NO:10所示)(图8)。
实施例七:IF573调控番茄乙烯行为通过YFT1介导
YFT1为乙烯信号途径的核心组分,YFT1下调表达后,必然对乙烯合成和信号转导产生的影响,为了分析IF573 DNA片段调控番茄的乙烯行为,测定cv.M82、yft1、YFT1-CDS、YFT1-RNAi在乙烯合成、乙烯释放和乙烯信号方面的差异,分析IF573 DNA片段通过YFT1介导调控番茄的乙烯行为。
1.番茄果实乙烯释放量测定
选取cv.M82、yft1、YFT1-CDS、YFT1-RNAi在35dpa、47dpa、54dpa的果实,每个材料选取三个不同单株采集果实,记录果实重量;将果实先放入25℃恒温培养箱中,以消除采摘生理胁迫可能引起的果实乙烯释放水平变化,2h后,将果实小心地放入500mL气体收集瓶中,密封,放入25℃恒温培养箱中放置,4h后,收集气体;用20mL注射器从气体收集瓶的顶端缓慢地抽取气体,注入到50mL的铝箔采样袋(德霖气体包装有限公司,大连,中国)中保存。
用1mL注射器缓慢吸取铝箔采样袋中的气体,迅速地注入到GC-2010气相色谱仪(岛津仪器有限公司,日本)的进样口中,测定乙烯含量;样品乙烯的浓度通过标准乙烯气体(10ppm)标定,分别注入0.1mL、0.3mL、0.5mL、0.6mL、0.9mL标准乙烯气体(10ppm),然后以浓度为纵坐标,峰面积为横坐标建立标准曲线;根据乙烯标准曲线,计算样品乙烯浓度,通过以下公式计算番茄乙烯释放量。
测定结果显示,有IF573 DNA片段插入的yft1果实乙烯释放量显著下降,果实乙烯释放水平与YFT1表达水平相关(图9)。
2.乙烯合成关键基因表达分析
提取cv.M82、yft1、YFT1-CDS、YFT1-RNAi在35dpa、47dpa、54dpa的果实RNA,反转录为cDNA,作为定量模板,设计定量引物(如表1所示),利用Light Cycle 96型实时荧光定量PCR仪(Roche有限公司,瑞士)分析乙烯合成关键基因ACS2/4(ACC SYNTHASE 2/4)和ACO1(ACC OXIDASE 1)的表达量变化,结果分析认为,YFT1下调表达株yft1和YFT1-RNAi中ACS2/4和ACO1表达水平显著下降,即IF573 DNA片段可通过下调YFT1 allele表达,影响番茄果实乙烯合成关键基因表达(图10)。
表1
3.乙烯信号途径关键基因表达分析
设计定量引物(见表1),分析乙烯信号途径关键基因NR(NEVER RIPE)、ETR4(ETHYLENE RECEPTOR 4)、CTR1(CONSTITUTIVE TRIPLE RESPONSE 1)、EIL3/4(ETHYLENEINSENSITIVE 3-LIKE 3/4)、AP2a(APETALA 2a)、ERF4/6(ETHYLENE-RESPONSIVETRANSCRIPTION FACTOR 4/6)在cv.M82、yft1、YFT1-CDS、YFT1-RNAi四种不同遗传背景材料中表达水平差异,分析结果显示NR、ETR4、CTR1、EIL3/4、AP2a、ERF4/6在yft1和YFT1-RNAi中的表达水平显著低于cv.M82和YFT1-CDS,暗示IF573 DNA片段可通过下调YFT1 allele表达,影响番茄果实乙烯信号传导(图11)。
4幼苗乙烯“三重”反应分析
为分析IF573 DNA片段影响番茄对乙烯信号的响应,分析cv.M82、yft1、YFT1-CDS、YFT1-RNAi幼苗乙烯“三重”反应的差异。具体步骤如下:
1)选取cv.M82、yft1、YFT1-CDS、YFT1-RNAi各材料种子,消毒后,分别播种于ACC+/ACC-的MS培养基上,每个处理进行三次重复,放置于25℃条件下避光培养7天;
2)CD-15CPX型游标卡尺(三丰量具有限公司,日本)测定每个材料在ACC+/ACC-的MS培养基上幼苗的下胚轴和根长,并拍照。统计根长和下胚轴长发现,在ACC的作用下,yft1、YFT1-RNAi的根长和下胚轴长显著长于cv.M82和YFT1-CDS,即IF573 DNA片段下调YFT1allele表达后,导致番茄对乙烯信号的敏感度下降(图12)。
实施例八:IF573调控番茄果实有色体发育
为分析IF573 DNA片段对番茄果实有色发育的调控,选取cv.M82、yft1、YFT1-CDS、YFT1-RNAi在35dpa、47dpa、54dpa果实质体超微结构观察。具体操作如下:
1.制片与超微观察
1)取样和固定:利用锋利的手术刀片,迅速地在果实赤道面上切取约1mm3的果皮组织,放入2.5%的戊二醛固定液中,然后将样品放入连接有真空泵(Millipore有限公司,马萨诸塞州,美国)的真空干燥箱中进行抽真空处理,抽真空时间为30min,待组织块完全浸入固定液,将其放置于4℃的环境下继续固定24h;
2)漂洗与脱水:将固定液吸出,加入0.1M PB漂洗样品,之后将样品放入4℃冰箱放置15min,重复操作3次;吸出漂洗液,加入适量锇酸(中镜科仪技术有限公司,北京,中国),于4℃冰箱静置2h;之后吸出锇酸放入专用的垃圾瓶中;然后用0.1M PB漂洗样品,于4℃冰箱中放置15min,重复3次;随后在4℃的条件下进行梯度脱水,50%乙醇、70%乙醇、90%乙醇分别脱水15min,之后用90%乙醇:90%丙酮体积比为1:1的混合溶液置换20min,90%丙酮置换20min;
3)包埋与固化:随后的操作在室温下进行,每次置换完成之后,置于100rpm的水平摇床上;首先用100%丙酮置换20min,置换3次,之后用丙酮:环氧树脂(Ted Pella股份有限公司,加利福尼亚州,美国)体积比为1:1的混合溶液置换1h,丙酮:环氧树脂体积比为1:2的混合溶液置换过夜;第二天,用纯树脂置换7h,取出包埋板,加入适量树脂,小心地用牙签将样品摆到包埋板中,每个包埋块在两端分别放置一块样品,在记录本上标记样品的位置,包埋完成后,放入60℃的电热鼓风干燥箱中聚合48h;
4)样品切片、染色,电镜观察:固化好的包埋块,先进行修块,将样品面直接暴露,以利于后续操作,切片采用UC6-FC6型冷冻超薄切片机(Leica仪器有限公司,德国)进行,染色用2%的枸橼酸铅(中镜科仪技术有限公司,北京,中国),超微结构观察利用120kV生物型透射电镜(FEI公司,俄勒冈州,美国)进行。
超微结构观察发现,yft1和YFT1-RNAi叶绿体向有色体转化相对cv.M82和YFT1-CDS迟缓,这说明IF573 DNA片段可通过YFT1介导调控番茄果实有色体发育(图13A)。
2.脂质小球数量统计
质体超微结构观察过程中,选取3个不同视野统计脂质小球数目,统计三个生物学重复。结果发现,47dpa和54dpa时期,yft1和YFT1-RNAi的脂质小球数量显著少于cv.M82和YFT1-CDS,这说明IF573 DNA片段可通过下调YFT1 allele表达,调控脂质小球的数量(图13B)。
实施例九:IF573调控番茄果色形成
番茄果色形成与类胡萝卜素的积累密切相关,为分析IF573 DNA片段对番茄果色形成的调控,观察基因互补实验YFT1-CDS、YFT1-RNAi果色变化,测定分析cv.M82、yft1、YFT1-CDS、YFT1-RNAi的类胡萝卜素含量,以及类胡萝卜素合成途径关键基因的表达,具体步骤如下:
1.果色变化观察
选取cv.M82、yft1以及YFT1-CDS、YFT1-RNAi在35dpa、47dpa、54dpa的番茄果实观察果色变化,并拍照,结果发现:YFT1-CDS在54dpa时,果色恢复为红色;而YFT1-RNAi在54dpa时呈黄色。这说明IF573 DNA片段可通过下调YFT1 allele表达,将番茄成熟果色改变为黄色(图3)。
2.类胡萝卜素含量测定
1)类胡萝卜素提取
(1)采集野生型cv.M82、yft1以及YFT1-CDS、YFT1-RNAi在35dpa、47dpa、54dpa的果实,选取外果皮部分,在避光的条件下,在液氮的作用下,用研钵迅速研磨成粉末状;
(2)称取约500mg粉末加入到RNA free的15mL离心管中,记录粉末加入质量;加入1.5mL甲醇,加入一定体积的60%KOH(W/V),使其终浓度变为6%(W/V),充分地颠倒混匀,将15mL离心管放入60℃水浴锅中,避光条件下,温浴30min;
(3)稍冷,吸取1.5mL Tris缓冲液,加入15mL离心管中,充分颠倒混匀,放入4℃冰箱放置10min;
(4)取出15mL离心管,置于冰上,加入4mL氯仿,颠倒混匀,静置10min,4℃ 4000rpm离心10min;
(5)用干净的5mL注射器,吸取下层有机相,加入新的15mL离心管中;剩余的水相加入4mL氯仿,重复一次步骤(4)的操作,用干净的5mL注射器吸取下层有机相,吸入相对应的15mL离心管中,提取液用氯仿补足至10mL;
(6)吸取1.5mL提取液,加入到2mL离心管中,放入SPD2010型离心浓缩仪(ThermoFisher科技有限公司,马萨诸塞州,美国)中干燥。
2)类胡萝卜素测定
(1)干燥后的样品加入50μL甲基叔丁基醚(MTBE)溶解样品,待样品彻底溶解后,12000rpm离心10min;
(2)吸取上层液体置入专用的上样瓶中,进样体积为1μL,利用高效合相色谱仪UPC2(Waters科技有限公司,马萨诸塞州,美国)测定类胡萝卜素,其参数设置参考Li等(2015)参数设计,具体为:进样室温度保持在10℃,柱子选用ACQUITY UPC2 HSS C18 SB(100mm×3.0mm,1.8μm),双相流动相由(A)CO2和(B)甲醇:乙醇(V:V)=1:2组成,线性洗脱梯度为:0.5min,95%A+5%B;2min,70%A+30%B;5min,70%A+30%B;5.5min,95%A+5%B;7min,95%A+5%B。系统流量设置为1.5mL/min,柱温设置为45℃,背压为22.9MPa;二极管阵列检测器(photo-diode array detector,PDA detector)的探测波长在210nm到500nm之间,补偿波长在210nm到280nm之间。
3)标准曲线的制作
(1)用甲基叔丁基醚(methyl tert-butyl ether,MTBE)溶解番茄红素、β-胡萝卜素、α-胡萝卜素、叶黄素标品,并将标品溶液用MTBE稀释为一系列浓度梯度(5μg/mL,10μg/mL,50μg/mL,250μg/mL,500μg/mL);
(2)将各标品按照浓度从低到高的顺序进样,测定完成后,建立浓度与峰面积相关的标准曲线,用于计算各成分的浓度。
测定类胡萝卜素结果显示,yft1和YFT1-RNAi的类胡萝卜素含量显著低于cv.M82和YFT1-CDS,这说明IF573 DNA片段可通过调控YFT1 allele表达调控类胡萝卜素合成(图14)。
3.类胡萝卜素途径关键基因表达
测定分析cv.M82、yft1、YFT1-CDS、YFT1-RNAi类胡萝卜素途径关键基因PSY1(PHYTOENE SYNTHASE 1)、ZDS(Z-CAROTENE DESATURASE)、CYCB(CHROMOPLAST SPECIFICLYCOPENE Β-CYCLASE)、LCYE(LYCOPENE Ε-CYCLASE)的表达水平,发现,PSY1、ZDS、CYCB在cv.M82和YFT1-CDS的表达水平显著高于yft1和YFT1-RNAi,而LCYE则相反,在yft1和YFT1-RNAi的表达水平相对较高(图15);这说明IF573 DNA片段可通过YFT1调控类胡萝卜素合成途径基因表达。
综上所述,本发明基于一种yft1番茄突变体公开了一种573bp DNA片段(SEQ IDNO:2),在yft1中下调YFT1 allele表达,其分子机制是由于IF573插入引入异常剪接位点导致YFT1 allele发生可变剪接、打破5'UTR的uORF和携带负调控序列。同时,IF573事件通过下调yft1中YFT1 allele表达而影响果实有色体发能、乙烯行为和果色。
以上对本发明的具体实施例进行了描述。需要理解的是,本发明并不局限于上述特定实施方式,本领域技术人员可以在权利要求的范围内做出各种变化或修改,这并不影响本发明的实质内容。在不冲突的情况下,本申请的实施例和实施例中的特征可以任意相互组合。
序列表
110 上海交通大学
120 DNA插入片段下调番茄YFT1 allele表达及其在番茄品质改良中的应用
130 KAG43895
160 49
170 PatentIn version 3.5
210 1
211 4858
212 DNA
213 Solanum lycopersicum
400 1
agctcacaca ccataactct ccatctctct aggatatgta ttatgtacag gttcctctct 60
ttatctctta gtagtatata tagatattag agcttcctta aaagcttctg atctgaactc 120
tgagcactga aatttataga gaagaagact gaagaaaacc catccagaaa aggaaggaaa 180
actatttgaa gaagaaatca gaatcggcaa tcggattatt gacggatgca aaggtgttaa 240
tgcggtgtat ttggttggag ttggtggatt tagcaactcg aaaagacttc catctttata 300
aggcgcactt ctcaaagtta ttgttcgaag ttggttgatt ttagcagctt gaaaagactc 360
ttaataaatt gcttttgtca agttcttcat gtccattgct tttgggtgca aacttgctca 420
aaattctcca gagataacga ggggttttgg tatcctgttc taaccgtgct acattgagct 480
acagtctaca gttggagctg cagctgctac atagaaaagc tgtgtggtcg gaacttggaa 540
cttcactggt tggattgtga gcttgttcat gtcaaatggg ttgctaagtg atgctgtagt 600
gctagtttac tgcgatctct gggcttgcta accatttaaa tcaaataatg gagtctgaaa 660
ctctgactag agaatatagg cagcccagca tgcttcagcg agtactttct gcttctgtgc 720
caatgctgtt gattgcagtt ggctatgttg atcctgggaa atgggctgca atggttgatg 780
gaggagcccg atttgggttt gatttggtca tgctagtact cttgttcaat tttgctgcca 840
ttctgtgcca gtatctgtct gcttgtatag ccttggttac agaccgagat cttgcgcaga 900
tttgcagtga agaatatgac aaagttacat gcatattcct aggaattcaa gctgaggttt 960
cgatgattgc tttggacctc acaatggttt tgggcactgc ccatgggctt aatgttgtgt 1020
ttggagttga cctgtttagc tgtgttttcc tgactgcaac cggtgccatt ttgtttccac 1080
tgcttgcttc tctcttggac aatggcagtg caaaattctt atgtattggc tgggcaagct 1140
ctgtactgct ctcttatgtt tttggagtgg ttataactct acctgaaact ccattctcca 1200
ttggtggtgt gctgaataag tttagtggag agagtgcatt tgcattgatg agtcttcttg 1260
gagcaagtat tatgcctcac aatttttacc tccattcttc tattgtacag caaggtaagg 1320
aatcaacaga gctttccagg ggagctctgt gtcaggacca tttttttgcc attgttttca 1380
tattcagtgg cattttcctg gtcaactatg ccgcgatgaa ttcagcagcg aatgtgtctt 1440
acagtactgg ccttttgttg ctgacatttc aggacacatt gtcattgctc gatcaggttt 1500
tcagaagctc agttgcacca ttcaccataa tgctggttac atttatttcc aatcaagtta 1560
caccactaac ttgggatctt ggtagacaag cagttgtgca tgacttattt ggaatggaca 1620
tcccaggctg gcttcatcat gtgacgatca gagttatttc cattgtccca gctctttatt 1680
gtgtatggag ttcaggagct gaaggcctat atcagttact tatactgaca caggttgtgg 1740
tggctcttgt ccttccatct tctgtcatac ccctgttcag agttgcttct tccagatcaa 1800
ttatgggtat ccacaaaatt tctcagttaa tggagttctt atctcttggc acatttattg 1860
gcttacttgg cctaaagatt atatttgtca tagagatgat atttggaaat agtgattggg 1920
ttaataattt gaagtggaat attgggagta gtgtgtctac tccatatttt tttctcctca 1980
tcgcagcctc tttatgtctt tgtctgatgc tgtggttagc agttactcct ctgaaatctg 2040
caagttccag gttcgatgct caggcgtttc tgcaaacgca tgtgcctgag ccatattcgg 2100
agtgtaatca acttggtgcg agtaatgcta tgtttggtct agtagaagga tcctcccaaa 2160
agcaagaagg tgcatttcat gtggaaaaat ccttggtaag ccatccagat ttatcaacta 2220
aagatcctga tcaactcttg ccagaatctc tcttggattt tgaaaaggtc catcagttgg 2280
ctactattga tgagagcaaa tctgaaacaa cattttcagc tcctgctgtc gttcatcctg 2340
aggtacctgt atcagcagga gcaagtccca gtgtgaaaag tgtttgtaat gaggtttctg 2400
gtgttgtatc agtggatacc agtgtcttca atactgaaac tgtggatgtc gcagagaaga 2460
ctctcagaat tgaaggggac atggcaaatg acagggatga tggagattcg tgggaagagc 2520
ctgaagaggc aatcaaagga gtatctgaga acgctcaatc ttttatttct gatggtccgg 2580
ggtcatacaa aagtctaagt ggaaaactag aggacacggg gagtggtaca ggaagtctat 2640
caagattagc aggtcttggt cgtgcagcta ggaggcagtt aacagaagct ctaaatgagt 2700
tttgggggca gctttttgat taccatggcg tggcaacagc agaagcgaag tccaagaaac 2760
tggatataat acttggtctg gattcaaaga tgaatccaaa acctgcccct gcatcattaa 2820
aagttgaaag cagtgcgtat attccatcgg ggagtgcaag gataccagag cctctgatca 2880
actcgcatgt gtactctccc aagcagcaat ttgcgtcaaa cattgtggac tctgcttata 2940
gagtcccaaa ggagccatct tcgacatctt ctatgtggtc taaccatatg aaattagtag 3000
gtgcatatgt gcaaagttcc aacagcaaca tgcttgactc aggggagagg cgctattcta 3060
gtatgcggat tccagcgact tctgctggct atgatcagca gcctgccact gtgcatggat 3120
atcagattac tgcttacctt aatcaacttg cgaaagaaag aggatctgat tatttaaatg 3180
ggcaactgga gtcaccatct cctcgttctg tatcatcact gacgtcaaac tatgcagaac 3240
cattggctcg tgtttcgggg caaaaacctc agagtggagt cagtagtcga gcaccacctg 3300
gttttggaaa tgtccctgta ggccgaaata attcgatgca gcccactaac actacttccg 3360
tcgaccatag ctctactgaa actgctgaaa gcgtggctgg ttcagccaac tctaagaagt 3420
actacagctt gcctgatatc tcagggcgct atgttcctcg ccaagattct atagtgtcag 3480
atgcgagagc tcaatggtac aattccatgg gattcggaca atctggtggt cgatctacat 3540
acgaacaagc ctatatgagt ggttcactaa gggcaggtgg tcctcagagg tatgaacatt 3600
ctcctaaagt ctgcagagat gcattctcct tgcagtacag ctccaattca gggactggat 3660
ccctgtggtc tagacagcct tttgagcaat ttggtgtagc tggtaagcca gatgttggta 3720
gtggcgatca tggaactgtg cagagttcct ctgctcaaga gagtacatct acggttgact 3780
tggaagctaa gctgcttcag tctttcagaa gttgtattgt gaaacttttg aaactggaag 3840
gatctgagtg gttatttagg caagatgatg gggctgatga ggatcttata ggtcggattg 3900
ctgcaagaga gaaatttctc tatgaagctg aaactaggga gataagtaga ttgaccaaca 3960
ttggtgaatc acacttctct tccaacagga aacccggttc tgccccaaaa cctgaagaga 4020
tggattacac caagttcttg gtgatgtcag ttccccactg cggagaaggt tgtgtttgga 4080
aagtagatct gattataagc ttcggtgtgt ggtgcattca cagaattctt gagctttcac 4140
ttatggaaag taggccagag ttgtggggca aatataccta tgttctcaac cgtcttcagg 4200
gcatagtaga tctggcattt tcaaagcccc attctccgac gagccattgt ttttgtcttc 4260
aaattccggc tggccgccag caaaaggcaa gcccccctcc aatttctaat ggaaacttgc 4320
cgccacaagc aaaacagggt cgaggaaaat gcacgactgc agcaatgctc ttagagatga 4380
tcaaagacgt ggagacagca atttcctgtc gaaagggacg aacgggcact gcagcagggg 4440
atgtagcctt tcctaaagga aaagagaacc tggcatccgt cctcaagcgc tataaacgtc 4500
gattatccaa taagccggta ggaaaccagg aggtggctgg agtcgctgga ccgcgcaaag 4560
taacgctgtc tgcctcatca ccccctttcg tcttgtaacg ctcttttctc agttcatagc 4620
aaatgactgg tgagatcacc attgttagac ttgttcttag tttctgtgaa ttatcccccc 4680
ctccccaact atacctccct tgcacctcat gtgtattttg aatctttgca gcttattcac 4740
ccccatccct tgtacctcaa tctgtataca taggataaaa tgttgtaacg aggttactgt 4800
aaaactgcaa tggtgaaatg aaacgggcta aaaggcggga aaagatgtta ttacaaga 4858
210 2
211 573
212 DNA
213 Solanum lycopersicum
400 2
agtgcaatta tgcaaacttt gttatagcat acaaatataa aatttttatt tgctatatat 60
gaaaattgtc cttttcaata tacacaatat ttttttttaa cgaaaaatat tcaataatat 120
aagctacatg ttgatacgcc attgattatg ttattatttt gatgtgattg ctatttactt 180
attagatcat gcattcaccc tctgaggact gtattacatt taatatatga tcttaaaaat 240
ctaattactt gtataaaaaa tgcatcatat acctacatga ttggacatca aaattatgtt 300
tttattttat ttaaaacaga tctaatgtca tataattggt attcgcgatc aaacaacaat 360
tggattatat attaataaac ctaaaagttc tcaaccacaa tgcatttgtc ttaggtagga 420
catgttttac cctaaaagac ttcctaactc cgaattaatt gaattccaat acatatattg 480
aatttgacaa ttagaataat tgtcgagtca tatataatat tttttttttg tttcaattta 540
ggtgatactt ttcattttgg agagtcaaac aat 573
210 3
211 625
212 DNA
213 Solanum lycopersicum
400 3
gtgttaatgc ggtgtatttg gttggagttg gtggatttag caactcgaaa agacttccat 60
ctttataagg cgcacttctc aaagagtgca attatgcaaa ctttgttata gcatacaaat 120
ataaaatttt tatttgctat atatgaaaat tgtccttttc aatatacaca atattttttt 180
ttaacgaaaa atattcaata atataagcta catgttgata cgccattgat tatgttatta 240
ttttgatgtg attgctattt acttattaga tcatgcattc accctctgag gactgtatta 300
catttaatat atgatcttaa aaatctaatt acttgtataa aaaatgcatc atatacctac 360
atgattggac atcaaaatta tgtttttatt ttatttaaaa cagatctaat gtcatataat 420
tggtattcgc gatcaaacaa caattggatt atatattaat aaacctaaaa gttctcaacc 480
acaatgcatt tgtcttaggt aggacatgtt ttaccctaaa agacttccta actccgaatt 540
aattgaattc caatacatat attgaatttg acaattagaa taattgtcga gtcatatata 600
atattttttt tttgtttcaa tttag 625
210 4
211 458
212 DNA
213 Solanum lycopersicum
400 4
agctcacaca ccataactct ccatctctct aggatatgta ttatgtacag gttcctctct 60
ttatctctta gtagtatata tagatattag agcttcctta aaagcttctg atctgaactc 120
tgagcactga aatttataga gaagaagact gaagaaaacc catccagaaa aggaaggaaa 180
actatttgaa gaagaaatca gaatcggcaa tcggattatt gacggatgca aaggtgttaa 240
tgcggtgtat ttggttggag ttggtggatt tagcaactcg aaaagacttc catctttata 300
aggcgcactt ctcaaagagt gcaattatgc aaactttgtt atagcataca aatataaaat 360
ttttatttgc tatatatgaa aattgtcctt ttcaatatac acaatatttt tttttaacga 420
aaaatattca ataatataag ctacatgttg atacgcca 458
210 5
211 5418
212 DNA
213 Solanum lycopersicum
400 5
agctcacaca ccataactct ccatctctct aggatatgta ttatgtacag gttcctctct 60
ttatctctta gtagtatata tagatattag agcttcctta aaagcttctg atctgaactc 120
tgagcactga aatttataga gaagaagact gaagaaaacc catccagaaa aggaaggaaa 180
actatttgaa gaagaaatca gaatcggcaa tcggattatt gacggatgca aaggtgttaa 240
tgcggtgtat ttggttggag ttggtggatt tagcaactcg aaaagacttc catctttata 300
aggcgcactt ctcaaagagt gcaattatgc aaactttgtt atagcataca aatataaaat 360
ttttatttgc tatatatgaa aattgtcctt ttcaatatac acaatatttt tttttaacga 420
aaaatattca ataatataag ctacatgttg atacgccatt gattatgtta ttattttgat 480
gtgattgcta tttacttatt agatcatgca ttcaccctct gaggactgta ttacatttaa 540
tatatgatct taaaaatcta attacttgta taaaaaatgc atcatatacc tacatgattg 600
gacatcaaaa ttatgttttt attttattta aaacagatct aatgtcatat aattggtatt 660
cgcgatcaaa caacaattgg attatatatt aataaaccta aaagttctca accacaatgc 720
atttgtctta ggtaggacat gttttaccct aaaagacttc ctaactccga attaattgaa 780
ttccaataca tatattgaat ttgacaatta gaataattgt cgagtcatat ataatatttt 840
ttttttgttt caatttaggt gatacttttc attttggaga gtcaaacaat ttggttgatt 900
ttagcagctt gaaaagactc ttaataaatt gcttttgtca agttcttcat gtccattgct 960
tttgggtgca aacttgctca aaattctcca gagataacga ggggttttgg tatcctgttc 1020
taaccgtgct acattgagct acagtctaca gttggagctg cagctgctac atagaaaagc 1080
tgtgtggtcg gaacttggaa cttcactggt tggattgtga gcttgttcat gtcaaatggg 1140
ttgctaagtg atgctgtagt gctagtttac tgcgatctct gggcttgcta accatttaaa 1200
tcaaataatg gagtctgaaa ctctgactag agaatatagg cagcccagca tgcttcagcg 1260
agtactttct gcttctgtgc caatgctgtt gattgcagtt ggctatgttg atcctgggaa 1320
atgggctgca atggttgatg gaggagcccg atttgggttt gatttggtca tgctagtact 1380
cttgttcaat tttgctgcca ttctgtgcca gtatctgtct gcttgtatag ccttggttac 1440
agaccgagat cttgcgcaga tttgcagtga agaatatgac aaagttacat gcatattcct 1500
aggaattcaa gctgaggttt cgatgattgc tttggacctc acaatggttt tgggcactgc 1560
ccatgggctt aatgttgtgt ttggagttga cctgtttagc tgtgttttcc tgactgcaac 1620
cggtgccatt ttgtttccac tgcttgcttc tctcttggac aatggcagtg caaaattctt 1680
atgtattggc tgggcaagct ctgtactgct ctcttatgtt tttggagtgg ttataactct 1740
acctgaaact ccattctcca ttggtggtgt gctgaataag tttagtggag agagtgcatt 1800
tgcattgatg agtcttcttg gagcaagtat tatgcctcac aatttttacc tccattcttc 1860
tattgtacag caaggtaagg aatcaacaga gctttccagg ggagctctgt gtcaggacca 1920
tttttttgcc attgttttca tattcagtgg cattttcctg gtcaactatg ccgcgatgaa 1980
ttcagcagcg aatgtgtctt acagtactgg ccttttgttg ctgacatttc aggacacatt 2040
gtcattgctc gatcaggttt tcagaagctc agttgcacca ttcaccataa tgctggttac 2100
atttatttcc aatcaagtta caccactaac ttgggatctt ggtagacaag cagttgtgca 2160
tgacttattt ggaatggaca tcccaggctg gcttcatcat gtgacgatca gagttatttc 2220
cattgtccca gctctttatt gtgtatggag ttcaggagct gaaggcctat atcagttact 2280
tatactgaca caggttgtgg tggctcttgt ccttccatct tctgtcatac ccctgttcag 2340
agttgcttct tccagatcaa ttatgggtat ccacaaaatt tctcagttaa tggagttctt 2400
atctcttggc acatttattg gcttacttgg cctaaagatt atatttgtca tagagatgat 2460
atttggaaat agtgattggg ttaataattt gaagtggaat attgggagta gtgtgtctac 2520
tccatatttt tttctcctca tcgcagcctc tttatgtctt tgtctgatgc tgtggttagc 2580
agttactcct ctgaaatctg caagttccag gttcgatgct caggcgtttc tgcaaacgca 2640
tgtgcctgag ccatattcgg agtgtaatca acttggtgcg agtaatgcta tgtttggtct 2700
agtagaagga tcctcccaaa agcaagaagg tgcatttcat gtggaaaaat ccttggtaag 2760
ccatccagat ttatcaacta aagatcctga tcaactcttg ccagaatctc tcttggattt 2820
tgaaaaggtc catcagttgg ctactattga tgagagcaaa tctgaaacaa cattttcagc 2880
tcctgctgtc gttcatcctg aggtacctgt atcagcagga gcaagtccca gtgtgaaaag 2940
tgtttgtaat gaggtttctg gtgttgtatc agtggatacc agtgtcttca atactgaaac 3000
tgtggatgtc gcagagaaga ctctcagaat tgaaggggac atggcaaatg acagggatga 3060
tggagattcg tgggaagagc ctgaagaggc aatcaaagga gtatctgaga acgctcaatc 3120
ttttatttct gatggtccgg ggtcatacaa aagtctaagt ggaaaactag aggacacggg 3180
gagtggtaca ggaagtctat caagattagc aggtcttggt cgtgcagcta ggaggcagtt 3240
aacagaagct ctaaatgagt tttgggggca gctttttgat taccatggcg tggcaacagc 3300
agaagcgaag tccaagaaac tggatataat acttggtctg gattcaaaga tgaatccaaa 3360
acctgcccct gcatcattaa aagttgaaag cagtgcgtat attccatcgg ggagtgcaag 3420
gataccagag cctctgatca actcgcatgt gtactctccc aagcagcaat ttgcgtcaaa 3480
cattgtggac tctgcttata gagtcccaaa ggagccatct tcgacatctt ctatgtggtc 3540
taaccatatg aaattagtag gtgcatatgt gcaaagttcc aacagcaaca tgcttgactc 3600
aggggagagg cgctattcta gtatgcggat tccagcgact tctgctggct atgatcagca 3660
gcctgccact gtgcatggat atcagattac tgcttacctt aatcaacttg cgaaagaaag 3720
aggatctgat tatttaaatg ggcaactgga gtcaccatct cctcgttctg tatcatcact 3780
gacgtcaaac tatgcagaac cattggctcg tgtttcgggg caaaaacctc agagtggagt 3840
cagtagtcga gcaccacctg gttttggaaa tgtccctgta ggccgaaata attcgatgca 3900
gcccactaac actacttccg tcgaccatag ctctactgaa actgctgaaa gcgtggctgg 3960
ttcagccaac tctaagaagt actacagctt gcctgatatc tcagggcgct atgttcctcg 4020
ccaagattct atagtgtcag atgcgagagc tcaatggtac aattccatgg gattcggaca 4080
atctggtggt cgatctacat acgaacaagc ctatatgagt ggttcactaa gggcaggtgg 4140
tcctcagagg tatgaacatt ctcctaaagt ctgcagagat gcattctcct tgcagtacag 4200
ctccaattca gggactggat ccctgtggtc tagacagcct tttgagcaat ttggtgtagc 4260
tggtaagcca gatgttggta gtggcgatca tggaactgtg cagagttcct ctgctcaaga 4320
gagtacatct acggttgact tggaagctaa gctgcttcag tctttcagaa gttgtattgt 4380
gaaacttttg aaactggaag gatctgagtg gttatttagg caagatgatg gggctgatga 4440
ggatcttata ggtcggattg ctgcaagaga gaaatttctc tatgaagctg aaactaggga 4500
gataagtaga ttgaccaaca ttggtgaatc acacttctct tccaacagga aacccggttc 4560
tgccccaaaa cctgaagaga tggattacac caagttcttg gtgatgtcag ttccccactg 4620
cggagaaggt tgtgtttgga aagtagatct gattataagc ttcggtgtgt ggtgcattca 4680
cagaattctt gagctttcac ttatggaaag taggccagag ttgtggggca aatataccta 4740
tgttctcaac cgtcttcagg gcatagtaga tctggcattt tcaaagcccc attctccgac 4800
gagccattgt ttttgtcttc aaattccggc tggccgccag caaaaggcaa gcccccctcc 4860
aatttctaat ggaaacttgc cgccacaagc aaaacagggt cgaggaaaat gcacgactgc 4920
agcaatgctc ttagagatga tcaaagacgt ggagacagca atttcctgtc gaaagggacg 4980
aacgggcact gcagcagggg atgtagcctt tcctaaagga aaagagaacc tggcatccgt 5040
cctcaagcgc tataaacgtc gattatccaa taagccggta ggaaaccagg aggtggctgg 5100
agtcgctgga ccgcgcaaag taacgctgtc tgcctcatca ccccctttcg tcttgtaacg 5160
ctcttttctc agttcatagc aaatgactgg tgagatcacc attgttagac ttgttcttag 5220
tttctgtgaa ttatcccccc ctccccaact atacctccct tgcacctcat gtgtattttg 5280
aatctttgca gcttattcac ccccatccct tgtacctcaa tctgtataca taggataaaa 5340
tgttgtaacg aggttactgt aaaactgcaa tggtgaaatg aaacgggcta aaaggcggga 5400
aaagatgtta ttacaaga 5418
210 6
211 4793
212 DNA
213 Solanum lycopersicum
400 6
agctcacaca ccataactct ccatctctct aggatatgta ttatgtacag gttcctctct 60
ttatctctta gtagtatata tagatattag agcttcctta aaagcttctg atctgaactc 120
tgagcactga aatttataga gaagaagact gaagaaaacc catccagaaa aggaaggaaa 180
actatttgaa gaagaaatca gaatcggcaa tcggattatt gacggatgca aaggtgatac 240
ttttcatttt ggagagtcaa acaatttggt tgattttagc agcttgaaaa gactcttaat 300
aaattgcttt tgtcaagttc ttcatgtcca ttgcttttgg gtgcaaactt gctcaaaatt 360
ctccagagat aacgaggggt tttggtatcc tgttctaacc gtgctacatt gagctacagt 420
ctacagttgg agctgcagct gctacataga aaagctgtgt ggtcggaact tggaacttca 480
ctggttggat tgtgagcttg ttcatgtcaa atgggttgct aagtgatgct gtagtgctag 540
tttactgcga tctctgggct tgctaaccat ttaaatcaaa taatggagtc tgaaactctg 600
actagagaat ataggcagcc cagcatgctt cagcgagtac tttctgcttc tgtgccaatg 660
ctgttgattg cagttggcta tgttgatcct gggaaatggg ctgcaatggt tgatggagga 720
gcccgatttg ggtttgattt ggtcatgcta gtactcttgt tcaattttgc tgccattctg 780
tgccagtatc tgtctgcttg tatagccttg gttacagacc gagatcttgc gcagatttgc 840
agtgaagaat atgacaaagt tacatgcata ttcctaggaa ttcaagctga ggtttcgatg 900
attgctttgg acctcacaat ggttttgggc actgcccatg ggcttaatgt tgtgtttgga 960
gttgacctgt ttagctgtgt tttcctgact gcaaccggtg ccattttgtt tccactgctt 1020
gcttctctct tggacaatgg cagtgcaaaa ttcttatgta ttggctgggc aagctctgta 1080
ctgctctctt atgtttttgg agtggttata actctacctg aaactccatt ctccattggt 1140
ggtgtgctga ataagtttag tggagagagt gcatttgcat tgatgagtct tcttggagca 1200
agtattatgc ctcacaattt ttacctccat tcttctattg tacagcaagg taaggaatca 1260
acagagcttt ccaggggagc tctgtgtcag gaccattttt ttgccattgt tttcatattc 1320
agtggcattt tcctggtcaa ctatgccgcg atgaattcag cagcgaatgt gtcttacagt 1380
actggccttt tgttgctgac atttcaggac acattgtcat tgctcgatca ggttttcaga 1440
agctcagttg caccattcac cataatgctg gttacattta tttccaatca agttacacca 1500
ctaacttggg atcttggtag acaagcagtt gtgcatgact tatttggaat ggacatccca 1560
ggctggcttc atcatgtgac gatcagagtt atttccattg tcccagctct ttattgtgta 1620
tggagttcag gagctgaagg cctatatcag ttacttatac tgacacaggt tgtggtggct 1680
cttgtccttc catcttctgt catacccctg ttcagagttg cttcttccag atcaattatg 1740
ggtatccaca aaatttctca gttaatggag ttcttatctc ttggcacatt tattggctta 1800
cttggcctaa agattatatt tgtcatagag atgatatttg gaaatagtga ttgggttaat 1860
aatttgaagt ggaatattgg gagtagtgtg tctactccat atttttttct cctcatcgca 1920
gcctctttat gtctttgtct gatgctgtgg ttagcagtta ctcctctgaa atctgcaagt 1980
tccaggttcg atgctcaggc gtttctgcaa acgcatgtgc ctgagccata ttcggagtgt 2040
aatcaacttg gtgcgagtaa tgctatgttt ggtctagtag aaggatcctc ccaaaagcaa 2100
gaaggtgcat ttcatgtgga aaaatccttg gtaagccatc cagatttatc aactaaagat 2160
cctgatcaac tcttgccaga atctctcttg gattttgaaa aggtccatca gttggctact 2220
attgatgaga gcaaatctga aacaacattt tcagctcctg ctgtcgttca tcctgaggta 2280
cctgtatcag caggagcaag tcccagtgtg aaaagtgttt gtaatgaggt ttctggtgtt 2340
gtatcagtgg ataccagtgt cttcaatact gaaactgtgg atgtcgcaga gaagactctc 2400
agaattgaag gggacatggc aaatgacagg gatgatggag attcgtggga agagcctgaa 2460
gaggcaatca aaggagtatc tgagaacgct caatctttta tttctgatgg tccggggtca 2520
tacaaaagtc taagtggaaa actagaggac acggggagtg gtacaggaag tctatcaaga 2580
ttagcaggtc ttggtcgtgc agctaggagg cagttaacag aagctctaaa tgagttttgg 2640
gggcagcttt ttgattacca tggcgtggca acagcagaag cgaagtccaa gaaactggat 2700
ataatacttg gtctggattc aaagatgaat ccaaaacctg cccctgcatc attaaaagtt 2760
gaaagcagtg cgtatattcc atcggggagt gcaaggatac cagagcctct gatcaactcg 2820
catgtgtact ctcccaagca gcaatttgcg tcaaacattg tggactctgc ttatagagtc 2880
ccaaaggagc catcttcgac atcttctatg tggtctaacc atatgaaatt agtaggtgca 2940
tatgtgcaaa gttccaacag caacatgctt gactcagggg agaggcgcta ttctagtatg 3000
cggattccag cgacttctgc tggctatgat cagcagcctg ccactgtgca tggatatcag 3060
attactgctt accttaatca acttgcgaaa gaaagaggat ctgattattt aaatgggcaa 3120
ctggagtcac catctcctcg ttctgtatca tcactgacgt caaactatgc agaaccattg 3180
gctcgtgttt cggggcaaaa acctcagagt ggagtcagta gtcgagcacc acctggtttt 3240
ggaaatgtcc ctgtaggccg aaataattcg atgcagccca ctaacactac ttccgtcgac 3300
catagctcta ctgaaactgc tgaaagcgtg gctggttcag ccaactctaa gaagtactac 3360
agcttgcctg atatctcagg gcgctatgtt cctcgccaag attctatagt gtcagatgcg 3420
agagctcaat ggtacaattc catgggattc ggacaatctg gtggtcgatc tacatacgaa 3480
caagcctata tgagtggttc actaagggca ggtggtcctc agaggtatga acattctcct 3540
aaagtctgca gagatgcatt ctccttgcag tacagctcca attcagggac tggatccctg 3600
tggtctagac agccttttga gcaatttggt gtagctggta agccagatgt tggtagtggc 3660
gatcatggaa ctgtgcagag ttcctctgct caagagagta catctacggt tgacttggaa 3720
gctaagctgc ttcagtcttt cagaagttgt attgtgaaac ttttgaaact ggaaggatct 3780
gagtggttat ttaggcaaga tgatggggct gatgaggatc ttataggtcg gattgctgca 3840
agagagaaat ttctctatga agctgaaact agggagataa gtagattgac caacattggt 3900
gaatcacact tctcttccaa caggaaaccc ggttctgccc caaaacctga agagatggat 3960
tacaccaagt tcttggtgat gtcagttccc cactgcggag aaggttgtgt ttggaaagta 4020
gatctgatta taagcttcgg tgtgtggtgc attcacagaa ttcttgagct ttcacttatg 4080
gaaagtaggc cagagttgtg gggcaaatat acctatgttc tcaaccgtct tcagggcata 4140
gtagatctgg cattttcaaa gccccattct ccgacgagcc attgtttttg tcttcaaatt 4200
ccggctggcc gccagcaaaa ggcaagcccc cctccaattt ctaatggaaa cttgccgcca 4260
caagcaaaac agggtcgagg aaaatgcacg actgcagcaa tgctcttaga gatgatcaaa 4320
gacgtggaga cagcaatttc ctgtcgaaag ggacgaacgg gcactgcagc aggggatgta 4380
gcctttccta aaggaaaaga gaacctggca tccgtcctca agcgctataa acgtcgatta 4440
tccaataagc cggtaggaaa ccaggaggtg gctggagtcg ctggaccgcg caaagtaacg 4500
ctgtctgcct catcaccccc tttcgtcttg taacgctctt ttctcagttc atagcaaatg 4560
actggtgaga tcaccattgt tagacttgtt cttagtttct gtgaattatc cccccctccc 4620
caactatacc tcccttgcac ctcatgtgta ttttgaatct ttgcagctta ttcaccccca 4680
tcccttgtac ctcaatctgt atacatagga taaaatgttg taacgaggtt actgtaaaac 4740
tgcaatggtg aaatgaaacg ggctaaaagg cgggaaaaga tgttattaca aga 4793
210 7
211 105
212 DNA
213 Solanum lycopersicum
400 7
atgcggtgta tttggttgga gttggtggat ttagcaactc gaaaagactt ccatctttat 60
aaggcgcact tctcaaagag tgcaattatg caaactttgt tatag 105
210 8
211 100
212 DNA
213 Solanum lycopersicum
400 8
atgattggac atcaaaatta tgtttttatt ttatttaaaa cagatctaat gtcatataat 60
tggtattcgc gatcaaacaa caattggatt atatattaa 99
210 9
211 100
212 DNA
213 Solanum lycopersicum
400 9
ttattttatt taaaacagat ctaatgtcat ataattggta ttcgcgatca aacaacaatt 60
ggattatata ttaataaacc taaaagttct caaccacaat 100
210 10
211 100
212 DNA
213 Solanum lycopersicum
400 10
gcatttgtct taggtaggac atgttttacc ctaaaagact tcctaactcc gaattaattg 60
aattccaata catatattga atttgacaat tagaataatt 100
210 11
211 27
212 DNA
213 Artificial Sequence
220
223 573 bp片段源位置扩增正向引物
400 11
gtaaataataacttcgtagatcatggc 27
210 12
211 24
212 DNA
213 Artificial Sequence
220
223 573 bp片段源位置扩增反向引物
400 12
tggtcctccatcaagaatttctcc 24
210 13
211 23
212 DNA
213 Artificial Sequence
220
223 扩增YFT1启动子正向引物
400 13
gatgttatcggggaggaagaaac 23
210 14
211 25
212 DNA
213 Artificial Sequence
220
223 扩增YFT1启动子反向引物
400 14
tatttgatttaaatggttagcaagc 25
210 15
211 25
212 DNA
213 Artificial Sequence
220
223 YFT1 CDS扩增正向引物
400 15
atggagtctgaaactctgactagag 25
210 16
211 19
212 DNA
213 Artificial Sequence
220
223 YFT1 CDS扩增反向引物
400 16
caagacgaaagggggtgat 19
210 17
211 287
212 DNA
213 Solanum lycopersicum
220
223 YFT1干扰序列
400 17
taagccatcc agatttatca actaaagatc ctgatcaact cttgccagaa tctctcttgg 60
attttgaaaa ggtccatcag ttggctacta ttgatgagag caaatctgaa acaacatttt 120
cagctcctgc tgtcgttcat cctgaggtac ctgtatcagc aggagcaagt cccagtgtga 180
aaagtgtttg taatgaggtt tctggtgttg tatcagtgga taccagtgtc ttcaatactg 240
aaactgtgga tgtcgcagag aagactctca gaattgaagg ggacatg 287
210 18
211 21
212 DNA
213 Artificial Sequence
220
223 干扰载体构建正向引物
400 18
cacctaagccatccagatttatcaac 26
210 19
211 21
212 DNA
213 Artificial Sequence
220
223 干扰载体构建反向引物
400 19
catgtccccttcaattctgag 21
210 20
211 25
212 DNA
213 Artificial Sequence
220
223 插入位点上游表达量分析定量PCR正向引物
400 20
agcttccttaaaagcttctgatctg 25
210 21
211 25
212 DNA
213 Artificial Sequence
220
223 插入位点上游表达量分析定量PCR反向引物
400 21
caataatccgattgccgattctg 25
210 22
211 25
212 DNA
213 Artificial Sequence
220
223 插入位点下游表达量分析定量PCR正向引物
400 22
cttggattttgaaaaggtccatcag 25
210 23
211 24
212 DNA
213 Artificial Sequence
220
223 插入位点下游表达量分析定量PCR反向引物
400 23
acacttttcacactgggacttgct 24
210 24
211 26
212 DNA
213 Artificial Sequence
220
223 5′ RACE GSP引物
400 24
tggtgaatggtgcaactgagcttctg 26
210 25
211 25
212 DNA
213 Artificial Sequence
220
223 3′ RACE GSP引物
400 25
cttccaacaggaaacccggttctgc 25
210 26
211 38
212 DNA
213 Artificial Sequence
220
223 YFT1 cDNA全长扩增正向引物
400 26
agctcacacaccataactctccatctctctaggatatg 38
210 27
211 35
212 DNA
213 Artificial Sequence
220
223 YFT1 cDNA全长扩增反向引物
400 27
tcttgtaataacatcttttcccgccttttagcccg 35
210 28
211 20
212 DNA
213 Artificial Sequence
220
223 ACO1定量正向引物
400 28
tggtgaccaacttgaggtga 20
210 29
211 23
212 DNA
213 Artificial Sequence
220
223 ACO1定量反向引物
400 29
caattggatcactttccattgcc 23
210 30
211 20
212 DNA
213 Artificial Sequence
220
223 ACS2定量正向引物
400 30
gtggtgccactggagctaat 20
210 31
211 20
212 DNA
213 Artificial Sequence
220
223 ACS2定量反向引物
400 31
gtccaaagtggtgcccaatg 20
210 32
211 20
212 DNA
213 Artificial Sequence
220
223 ACS4定量正向引物
400 32
atgggtctcgcggaaaatca 20
210 33
211 20
212 DNA
213 Artificial Sequence
220
223 ACS4定量反向引物
400 33
aagcatcaccaggatcagcc 20
210 34
211 20
212 DNA
213 Artificial Sequence
220
223 NR定量正向引物
400 34
gcagcggaacaatccctttg 20
210 35
211 20
212 DNA
213 Artificial Sequence
220
223 NR定量反向引物
400 35
acaacgttcatcggtcacct 20
210 36
211 20
212 DNA
213 Artificial Sequence
220
223 ETR4定量正向引物
400 36
tgctgttgcgagtagtggag 20
210 37
211 20
212 DNA
213 Artificial Sequence
220
223 ETR4定量反向引物
400 37
tgatcagccacaaccctgac 20
210 38
211 20
212 DNA
213 Artificial Sequence
220
223 EIL3定量正向引物
400 38
ttgatcgaaatggccctgct 20
210 39
211 20
212 DNA
213 Artificial Sequence
220
223 EIL3定量反向引物
400 39
gggtggagatacccccttct 20
210 40
211 20
212 DNA
213 Artificial Sequence
220
223 EIL4定量正向引物
400 40
tctatgcagccacaaggcaa 20
210 41
211 20
212 DNA
213 Artificial Sequence
220
223 EIL4定量反向引物
400 41
atcttgcttcggtgtggtgt 20
210 42
211 20
212 DNA
213 Artificial Sequence
220
223 CTR1定量正向引物
400 42
gcagcagacggaagagagtt 20
210 43
211 20
212 DNA
213 Artificial Sequence
220
223 CTR1定量反向引物
400 43
ctgagcagagagcccaaaca 20
210 44
211 20
212 DNA
213 Artificial Sequence
220
223 APa定量正向引物
400 44
gctgctttaggcttattcttccg 23
210 45
211 21
212 DNA
213 Artificial Sequence
220
223 APa定量反向引物
400 45
actccattgtctgatcagggg 21
210 46
211 20
212 DNA
213 Artificial Sequence
220
223 ERF4定量正向引物
400 46
tccgatgacatctcccctgt 20
210 47
211 20
212 DNA
213 Artificial Sequence
220
223 ERF4定量反向引物
400 47
atggccttctccttacccct 20
210 48
211 25
212 DNA
213 Artificial Sequence
220
223 ERF6定量正向引物
400 48
tcgtctctttcaattactttcactc 25
210 49
211 20
212 DNA
213 Artificial Sequence
220
223 ERF6定量反向引物
400 49
cgacagagcggaatcaggtt 20
Claims (9)
1.一种573bp DNA插入片段在下调番茄YFT1 allele表达中应用,其特征在于,所述573bp DNA插入片段的序列如SEQ ID NO:2所示,所述573pb DNA插入片段导致YFT1 allele在转录加工水平改变。
2.一种uORF1序列在下调番茄YFT1 allele表达中的应用,其特征在于,所述uORF1序列如SEQ ID NO:7所示,由DNA插入片段导致uORF1序列变化而得到。
3.一种DNA插入片段在下调番茄YFT1 allele表达中的应用,其特征在于,所述DNA插入片段如SEQ ID NO:9和/或SEQ ID NO:10所示。
4.根据权利要求3所述的应用,其特征在于,所述DNA插入片段的插入位点为YFT1allele基因5'UTR区域的ATG上游-318bp。
5.一种下调番茄YFT1 allele表达的方法,其特征在于,包括在YFT1 allele基因5'UTR区域的ATG上游-318bp插入如权利要求3所述的有功能的DNA插入片段。
6.一种根据权利要求3所述的DNA插入片段在调控番茄果色形成和果实成熟中的应用。
7.根据权利要求6所述的应用,其特征在于,所述调控果实成熟的应用中,DNA插入片段调控番茄乙烯合成和释放。
8.根据权利要求6所述的应用,其特征在于,所述调控果实成熟的应用中,DNA插入片段调控番茄成熟发育期间类胡萝卜素的合成。
9.一种根据权利要求3所述的DNA插入片段在番茄遗传改良和育种中的应用。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107384927A (zh) * | 2017-08-15 | 2017-11-24 | 上海交通大学 | 调控番茄果色基因yft1的启动子及其应用 |
CN109468408A (zh) * | 2019-01-02 | 2019-03-15 | 华中农业大学 | 一种与番茄耐旱基因紧密连锁的分子标记及应用 |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107384927A (zh) * | 2017-08-15 | 2017-11-24 | 上海交通大学 | 调控番茄果色基因yft1的启动子及其应用 |
CN109468408A (zh) * | 2019-01-02 | 2019-03-15 | 华中农业大学 | 一种与番茄耐旱基因紧密连锁的分子标记及应用 |
Non-Patent Citations (2)
Title |
---|
WEIHUA ZHAO等: "Yellow-fruited phenotype is caused by 573 bp insertion at 5’UTR of YFT1 allele in yft1 mutant tomato", 《PLANT SCIENCE》 * |
WEIHUA ZHAO等: "Yellow-fruited phenotype is caused by 573 bp insertion at 5’UTR of YFT1 allele in yft1 mutant tomato", 《PLANT SCIENCE》, vol. 300, 16 August 2020 (2020-08-16), pages 1 - 13, XP086310436, DOI: 10.1016/j.plantsci.2020.110637 * |
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