CN116675754A - EMB1444-like在调控番茄成熟发育及育种中应用 - Google Patents
EMB1444-like在调控番茄成熟发育及育种中应用 Download PDFInfo
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Abstract
本发明公开了EMB1444‑like在调控番茄成熟发育及育种中应用,具体地涉及明确了EMB1444‑like特异地结合于YFT1启动子区域E‑box(CACTTG,‑1295bp~‑1290bp,SEQ ID NO.1)基序(YFT1起始密码子ATG上游第一个核苷酸定义为‑1bp)调控YFT1转录表达。创制EMB1444‑like下调表达突变体(EMB1444‑like‑RNAi,sledl),sledl突变体乙烯合成受限、类胡萝卜素合成受阻、叶绿体/有色体发育延迟;对通过调控转录因子基因EMB1444‑like表达对番茄品质和果实成熟期育种改良提供了坚实的遗传学证据。
Description
技术领域
本发明属于分子生物学和遗传学领域,具体涉及一种EMB1444-like在调控番茄成熟发育及育种中应用;尤其涉及一种EMB1444-like通过调控YFT1表达而影响番茄果色形成和果实成熟发育在育种应用;特别涉及一种番茄转录因子SlEMB1444-like(EMB1444-likein Solanum lycopersicum)通过调控乙烯信号核心组分基因YFT1/EIN2(YELLOW FRUITEDTOMATO 1/ETHYLENE INSENSITIVE 2)表达而控制番茄果色形成和果实成熟发育的应用。
背景技术
番茄(Solanum lycopersicum,2n=24)属于茄科(Solanaceae)茄属(Solanum)番茄族(Lycopersicon section)植物,是世界最大果、蔬兼用作物。番茄基因组小、生长周期短、易于遗传操作等优点,是浆果研究的重要模式植物。果色不仅关系到番茄外观品质,还与营养品质密切相关;因此,果色是番茄品质性状。市售番茄以红果和黄果为主,其果色形成与类胡萝卜素组分和累积有关。番茄类胡萝卜素生物合成途径在生物化学(催化酶)和分子水平(基因)研究的已比较深入,而对该途径分子机制仍不清晰。
番茄是典型的呼吸跃变型果实,有呼吸高峰,乙烯在番茄果实成熟过程中发挥关键作用。下调乙烯合成关键基因ACS(ACC SYNTHASE)和ACO(ACC OXIDASE)表达,乙烯信号转导被抑制,番茄果实成熟也将受到影响;抑制了番茄果实类胡萝卜素合成,番茄呈现橙或黄色。下调乙烯受体基因LeETR4(ETHYLENE RESPONSE 4)、LeETR6(ETHYLENE RESPONSE 6)和LeCTR1(CONSTITUTIVE TRIPLE RESPONSE 1)表达,番茄果实成熟延迟。乙烯不敏感突变体Nr(Never ripe)番茄表现迟熟表型,番茄红素累积下降。下调乙烯信号途径核心转录因子LeEIL(ETHYLENE INSENSITIVE 3-like)表达,果实成熟延迟,呈黄果表型。
我们从番茄中图位克隆的YFT1(YELLOW FRUITED TOMATO 1)基因编码SlEIN2(ETHYLENE INSENSITIVE 2in Solanum lycopersicum)是乙烯信号途径核心组分;受体荷载乙烯分子,使CTR1(CONSTITUTIVE TRIPLE RESPONSE1)失活,CTR1失去了磷酸化SlEIN2蛋白功能;羧基端(CEND)从EIN2上解离下来。一部分CEND入核与EIN3/EIL1结合,促进后者活性;一部分CEND在胞质中与P-bodies(processing body)形成复合体,降解EIN3-BINDINGF-BOX 1/2(EBF1/2)mRNA,抑制EBF1/2蛋白合成,入核量下降,保持了EIN3/EIL1稳态,促进下游乙烯应答基因表达。
功能丧失EIN2突变体ein2植物对乙烯不敏感,苗期“三重”反应消失。同时,EIN2还是多种植物激素信号途径交叉点;EIN2在盐胁迫、氧化应激及其他生物或非生物胁迫中还扮演着重要角色。NAC转录因子家族ORE1(oresara 1),具有促进拟南芥叶片细胞衰老和死亡的功能,EIN2正调控ORE1表达促进细胞衰老。EIN2还有限制植物细胞扩增的功能,拟南芥ein2突变体株高、叶面积、果荚长度显著增大。特别是,EIN2可以调控具有呼吸跃变高峰的果实成熟发育,番茄中EIN2的同源基因YFT1调控乙烯合成、乙烯信号转导和类胡萝卜素合成、有色体发育(Zhao,W.H.,et al.Yellow-fruited phenotype is caused by 573bpinsertion at 5'UTR of YFT1 allele in yft1 mutant tomato.Plant Science,2020,300:110637)。尽管EIN2在植物特别是番茄果色形成和果实发育中占据重要地位,而如何调控YFT1/SlEIN2的研究却鲜见报道,特别是通过调控YFT1表达而改进番茄果色形成和果实成熟发育在番茄改良中的应用还不清楚。本发明专利就是在揭示通过调控YFT1表达控制番茄果色形成和果实成熟发育在育种中的应用提供理论依据和实际路径。
发明内容
本发明的目的在于提供一种EMB1444-like在调控番茄成熟发育及育种中应用,以期达到对番茄品质改良培育优质品种的目的。
本发明的技术方案如下:
本发明提供一种bHLH家族的转录因子EMB1444-like在调控番茄果实成熟发育中的应用,所述EMB1444-like的氨基酸序列如SEQ ID NO.3所示。
所述EMB1444-like的CDS序列如SEQ ID NO.4所示。
所述转录因子EMB1444-like通过调控乙烯合成、乙烯信号转导、有色体发育、类胡萝卜累积、果色形成来调控番茄果实成熟发育和番茄果色。
所述EMB1444-like正调控乙烯信号转导关键组分SlEIN2编码基因YFT1的转录表达。
所述EMB1444-like特异地结合于乙烯信号途径关键基因YFT1上游启动子pYFT1的上游调控区域E-box基序(CACTTG,-1295bp~-1290bp,SEQ ID NO.1)及其侧翼而调控YFT1表达;所述E-box基序及其侧翼序列如SEQ ID NO.2所示。
本发明还提供一种下调转录因子EMB1444-like基因表达的RNA干扰序列,所述RNA干扰序列的DNA序列如SEQ ID NO.5所示。
本发明还提供一种EMB1444-like下调表达载体,所述表达载体的构建方法包括如下步骤:
用特异引物EMB1444-like-RNAi-F:5′-cgcggatccgctccaggagttgatgacgag-3′(SEQ ID NO.44)和EMB1444-like-RNAi-R:5′-ccggaattcggtgttttataatggaactttcac-3′(SEQ ID NO.45)扩增EMB1444-like的350bp特异DNA片段(SEQ ID NO.5),构建至RNAi载体pHELLSGATE 12,获得EMB1444-like下调表达载体EMB1444-like-RNAi。
如权利7所述的表达载体在番茄果实成熟中的应用。
本发明还提供一种EMB1444-like下调表达突变体sledl,将所述的EMB1444-like下调表达载体转化农杆菌,选取阳性转化农杆菌,转入野生型番茄M82(Solanumlycopersicum),获得突变体sledl。
所述野生型番茄为野生型番茄M82。
本发明提供了一种转录因子EMB1444-like在调控番茄果色形成中的应用。EMB1444-like下调表达突变体sledl(EMB1444-like-RNAi)改变了类胡萝卜素合成途径相关基因表达,限制了番茄果实类胡萝卜素合成,改变了果实颜色。
本发明提供了一种转录因子EMB1444-like调控番茄果实成熟发育中的应用。其通过反馈调控番茄果实乙烯合成途径关键基因表达和乙烯合成,从而影响番茄果实成熟发育。
本发明提供了一种转录因子EMB1444-like,其下调表达突变体sledl改变了乙烯信号途径相关基因表达而扰乱了乙烯信号转导,影响了番茄果实成熟进程。
本发明提供了一种转录因子EMB1444-like在番茄遗传改良和育种中的应用。包括:通过改变EMB1444-like表达而控制类胡萝卜素合成、乙烯合成和信号转导和有色体发育,从而实现对番茄果色形成和果实成熟发育调控,可以应用于番茄育种对番茄品质和成熟期的改良。
本发明还提供了一种转录因子EMB1444-like通过特异地调控乙烯信号核心组分YFT1/EIN2(YELLOW FRUITED TOMATO 1/ETHYLENE INSENSITIVE 2)表达而控制番茄果色形成和果实成熟发育的分子机制阐释及其在指导番茄育种实践中的应用。
与现有技术相比,本发明具有如下有益效果:
1.首次揭示了独立于乙烯信号的转录因子EMB1444-like正调控番茄YFT1基因表达;
2.基于EMB1444-like对乙烯合成、类胡萝卜素积累和有色体发育调控,可以更加精准地实现对番茄果色形成和果实成熟遗传改良,培育品质、货架日期和成熟期合适的番茄新品种。
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双分子荧光素酶实验筛选YFT1候选转录因子;
图2EMB1444-like在YFT1启动子上结合区段确定;
图3明确EMB1444-like在YFT1启动子p7区域结合位点;
图4不同遗传背景番茄材料YFT1和EMB1444-like表达;
图5番茄M82、yft1和sledl(EMB1444-like-RNAi)突变体果色动态变化(标尺:1cm;Scale bars=1cm);
图6PSY1、CRTISO和CYCB在番茄M82、yft1和sledl中表达;
图7有色体超微结构观察(标尺:500nm;Scale bars=500nm);
图8番茄M82、yft1和sledl乙烯合成释差异;
图9基因ACO1和ACS2/4在番茄M82、yft1和sledl果实表达;
图10番茄M82、yft1和sledl乙烯信号途径关键基因表达。
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前提下,可以做出若干变化和改进。这些都属于本发明的保护范围。
本发明涉及黄果迟熟突变体yft1(yellow fruited tomato 1,n3122)和野生型(wild type,WT)红果番茄cv.M82,均由以色列希伯来大学Dani Zamir教授提供(http://zamir.sgn.cornell.edu/mutateds)。yft1突变体是快中子(60Coγ-射线)辐射番茄cv.M82种子筛选获得。本发明所用TEA数据库(http://tea.solgenomics.net/),相似系数阈值设定为0.7;共检测到2386个与YFT1(Solyc09g007870)表达模式相似基因;其中包含85个转录因子。将3,000bp YFT1启动子上游序列(pYFT1)提交PLACE(https://www.dna.affrc.go.jp/PLACE/?action=newplace)去预测pYFT1所含顺式调控元件,在3,000bp的pYFT1预测有23个转录因子靶向pYFT1功能元件。双分子荧光素酶活实验(dualluciferase assay)结果显示,Solyc09g066280编码转录因子EMB1444-like使pYFT1强度提高4.2倍(图1)。用酵母单杂交和EMSA(electrophoretic mobility shift assay)分析进行三轮筛选及点突变分析(第一轮,pYFT1分为p1~p12片段,长约300bp,相邻段重叠50bp;第二轮和第三轮片段长度分别为120bp和50bp,重叠区域20bp),明确了EMB1444-like特异地结合于pYFT1的p7-3-2(-1319bp~-1270bp)(定义YFT1起始密码子ATG上游第一个核苷酸为-1bp),其中含有E-box(CACTTG,-1295bp~-1290bp,SEQ ID NO.1)基序(图2和图3)。
所述EMB1444-like的氨基酸序列如SEQ ID NO.3所示。所述EMB1444-like的cDNA序列如SEQ ID NO.4所示。
构建了M82背景下的EMB1444-like下调表达突变体(EMB1444-like-RNAi,sledl)。RT-qPCR分析EMB1444-like和YFT1表达发现,EMB1444-like在M82和yft1突变体果实中表达水平无显著差异;而YFT1在EMB1444-like下调表达sledl突变体果实表达显著低于M82;EMB1444-like是YFT1上游调控因子(图4)。sledl突变体番茄果实成熟发育延迟,开花后54天(54days post anthesis,54dpa)果实呈现黄色(图5)。同时,也发现sledl突变体有色体发育相比M82延迟,累积番茄红素质体小球数量也显著下降(图6)。
sledl突变体果实类胡萝卜素合成途径关键基因PSY1(PHYTOENE SYNTHASE 1)、CRTISO(CAROTENOID ISOMERASE)和CYCB(CHROMOPLAST-SPECIFIC LYCOPENEΒ-CYCLASE)表达与野生型M82相比显著下调(图7)。在54dpa,番茄红素和β-类胡萝卜素积累量也显著低于M82(表5)。分析发现,sledl突变体番茄的乙烯合成关键基因ACS2/4(ACC SYNTHESIS 2/4)和ACO1(ACC OXIDASE 1)和乙烯信号途径基因NR(NEVER RIPE)、ETR4(ETHYLENE RECEPTOR4)、AP2a(APETALA 2a)和ERF4/6(ETHYLENE RESPONSIVE FACTOR 4/6)表达显著下调,抑制了乙烯合成,干扰了乙烯信号转导(图8,图9和图10)。综上所述,EMB1444-like通过调控YFT1表达,抑制了乙烯和类胡萝卜素合成,延迟了有色体发育,从而影响果色形成和果实成熟发育。为番茄果色形成和果实成熟发育遗传改良和育种提供了重要的理论依据。
实施例1:YFT1高效转录因子筛选
为了筛选获得调控YFT1表达的转录因子,本发明基于Tomato Expression Atlas(http://tea.solgenomics.net/)转录组数据,确定与YFT1(Solyc09g007870)表达模式相近转录因子。提交YFT1起始密码子ATG上游3000bp序列(pYFT1,SEQ ID NO.6)至PLACE网站(https://www.dna.affrc.go.jp/PLACE/?action=newplace),预测pYFT1顺式调控元件(表1),选取与pYFT1结合位点转录因子作为候选转录因子。用双分子荧光素酶(dualluciferase assay)技术筛选YFT1的高效转录因子。具体步骤如下:
1.载体构建
用特异引物[pYFT1-F:5'-tccatcgatatattgatacgatatc-3'(SEQ ID NO.7),pYFT1-R:5'-tatttgatttaaatggttagcaagc-3'(SEQ ID NO.8)],PCR扩增3,000bp的pYFT1,克隆至pGreen II 0800-LUC载体(上海泽叶生物科技有限公司产品,上海,中国)的Sal I和Nco I位点间,Firefly Luciferase(LUC)上游,构建pYFT1::LUC。
基于TEA(http://tea.solgenomics.net/)转录组数据库,选取番茄果实中与YFT1(Solyc09g007870)表达模式相似系数高于0.70转录因子(Trancription factors,TFs)。用PLACE网站(https://www.dna.affrc.go.jp/PLACE/?action=newplace)预测pYFT1顺式作用元件(表1)。
表1YFT1启动子序列中的主要顺式作用元件*
*YFT1起始密码子ATG上游第一个核苷酸定义为-1bp。
共筛选23个与pYFT1序列中顺式元件结合候选转录因子(TFs)(表2)。将编码确定的23个候选转录因子基因PCR扩增,其中用特异引物(E80-BamH I-F:5′-ctctctctcaagcttggatccatggcaagccaactgcaac-3′(SEQ ID NO.76)和E80-Sac I-R:5′-gctcaccatactagtgagctccaagttgatctttgcctgcag-3′(SEQ ID NO.77)扩增EMB1444-like CDS序列(SEQ IDNO.4),其并克隆至pHB载体(本实验保存)BamH I和Xba I位点,构建表达载体pHB::TFs。
表2,23个在YFT1启动子序列中预测有结合位点的候选转录因子
2.烟草叶片瞬时转化
重组载体pHB::TFs和pYFT1::LUC分别转化携带pSoup-p19农杆菌GV3101感受态(购自上海语纯生物科技有限公司,上海,中国)。用1/2MS0液体培养基将菌体稀释至OD600=0.6,并1:1(v:v)混合pHB::TFs和pYFT1::LUC农杆菌(GV3101),以pHB(空载)和pYFT1::LUC混合菌液作阴性对照。用一次性去掉针头注射器吸取混合菌液,缓慢注射烟草叶片;完成后,吸取多余菌液,转移至暗处培养24h,然后再置于光下培养24h后打孔器取样。
3.Luciferase酶活测定
液氮中将样品研磨成粉末,用Dual-Luciferase Reporter Assay System(Promega生物技术有限公司,威斯康辛州,美国)测定样品Luciferase酶活。取约100mg样品粉末置于1.5mL预冷离心管中,加400μL 1×PLB(passive lysis buffer)裂解液,充分震荡抽提;9600g离心30sec,取8μL上层清液至新1.5mL离心管,迅速加入40μL LAR II(luciferase assay reagent II)混匀,点击测定键;再加入40μL Stop&Glo buffer,轻轻混匀,再次点击测定键;结果为前后两次测定值的比值。
通过分析Luciferase酶活性,发现EMB1444-like(Solyc09g066280)所编码转录因子EMB1444-like调控pYFT1转录表达可提高至4.35倍(图1)。
实施例2:EMB1444-like在pYFT1结合位点确定
为了确定EMB1444-like在YFT1启动子上结合位点,将pYFT1(YFT1起始密码子ATG上游3000bp序列,其上游第一个核苷酸定义为-1bp)进行三轮逐步分割,并通过酵母单杂交和EMSA(electrophoretic mobility shift assay)实验验证。具体实验步骤如下:
1.酵母单杂交载体构建
用特异引物EMB1444-like-EcoRI-F:5′-gattatgcctctcccgaattcatggcaagccaactgcaac-3′(SEQ ID NO.22)和EMB1444-like-XhoI-R:5′-agaagtccaaagcttctcgagctacaagttgatctttgcctgc-3′(SEQ ID NO.23)扩增EMB1444-like的CDS序列,并克隆至载体pB42AD载体(购自上海泽叶生物科技有限公司,上海,中国)EcoRI和XhoI两位点间,构建重组质粒:pB42AD-EMB1444-like。用PCR对pYFT1进行三轮扩增。第一轮,将pYFT1(SEQ ID NO.5)分为12段,p1~p12(约300bp,相邻片段有50bp重叠)(表3),分别克隆至pLacZi载体(北京科瑞思博生物科技有限公司,北京,中国)EcoR I和Xho I位点间,构建pLacZi-ΔpYFT1重组载体系列,进行酵母单杂交筛选。第一轮筛选阳性片段,依次进行第二、第三轮分割,将目的DNA片段继续分割至120bp和50bp,同样分别克隆至pLacZi载体(表2)。
2.酵母单杂交分析
将构建好的pB42AD-EMB1444-like和pLacZi-ΔpYFT1重组质粒共同转化酵母感受态EGY48a(购自上海唯地生物技术有限公司),灭菌涂布棒涂抹菌液至SD(-Leu/-Ura)缺陷培养基,28℃倒置培养48h;选择生长状态良好且大小一致菌斑,60μL无菌ddH2O重悬,滴加6μL菌液至SD(-Leu/-Ura)+X-gal(20mg/mL)定性培养基,28℃倒置培养72h,观察菌斑颜色变化(图2)。
表3YFT1启动子序列三轮分段*
*定义YFT1起始密码子ATG上游第一个核苷酸为-1bp。
结果显示,EMB1444-like可以结合于YFT1启动子p7-3-2(-1319bp~-1270bp)区段。p7-3-2区域内含有E-box(CACTTG,-1295bp~-1290bp)序列,预测其可能为EMB1444-like转录因子结合基序。为了证实这一推测,将p7(-1549bp~-1250bp)的E-box基序突变成Δp7(CACTTG→ACACCT,SEQ ID NO.42),用酵母单杂交验证(图3)。EMSA检测发现,EMB1444-like蛋白仅与探针probe(5′-cctaatggtctacacaccCACTTGggtaaattttatcatttttttttaaattttac-3′,SEQ ID NO.43)结合,而不能与含突变E-box(CACTTG→ACACCT)探针结合(图3)。证明,EMB1444-like蛋白特异结合pYFT1的E-box(CACTTG,-1295bp~-1290bp,SEQ IDNO.1)。
进一步,EMB1444-like特异地结合于乙烯信号途径关键基因YFT1上游启动子pYFT1的上游调控区域E-box基序(CACTTG,-1295bp~-1290bp,SEQ ID NO.1)及其侧翼而调控YFT1表达;所述E-box基序及其侧翼序列如SEQ ID NO.2所示。
实施例3:EMB1444-like调控番茄果实成熟发育和果色形成
用特异引物EMB1444-like-RNAi-F:5′-cgcggatccgctccaggagttgatgacgag-3′(SEQ ID NO.44)和EMB1444-like-RNAi-R:5′-ccggaattcggtgttttataatggaactttcac-3′(SEQ ID NO.45)扩增EMB1444-like的350bp特异性片段(SEQ ID NO.5),克隆至RNAi载体pHELLSGATE 12(Invitrogen,CA,USA),构建EMB1444-like下调表达载体EMB1444-like-RNAi,转化农杆菌EHA105感受态细胞用于转化野生型番茄M82,创制了M82背景下的EMB1444-like下调表达突变体sledl。对开花后35dpa(days post anthesis)、47dpa和54dpa番茄果实样品提取总RNA,用RT-qPCR分析EMB1444-like[EMB1444-like-qPCR-F:5′-cttcctcccatgaggttggt-3′,SEQ ID NO.46;EMB1444-like-qPCR-R:5′-cattctgcccctcaaccacaa-3′,SEQ ID NO.47]和YFT1[YFT1-F:5′-actgcggagaaggttgtg-3′,SEQ ID NO.48;YFT1-R:5′-atggctcgtcggagaatg-3′,SEQ ID NO.49]在不同遗传背景番茄M82、yft1和sledl表达和果色变化。结果发现,EMB1444-like转录表达在M82和yft1突变体果实间无显著差异;而YFT1在sledl果实表达水平显著低于M82(图4)。这暗示EMB1444-like是独立于乙烯信号途径正向调控YFT1表达转录因子。
sledl番茄呈现黄果,果实成熟发育延迟在54dpa,野生型M82果实达到红熟期;而sledl突变体果色却仅达到转色期,与47dpa的M82在果色类似(图5)。
实施例4:EMB1444-like对番茄果实类胡萝卜素合成和有色体发育影响
为了明确EMB1444-like对番茄果实类胡萝卜素合成调控方式,测定M82、yft1和sledl突变体果实不同发育期(35dpa、47dpa和54dpa)的总类胡萝卜素,番茄红素、β-胡萝卜素、α-胡萝卜素和叶黄素含量;并观察果实有色体超微结构和质体小球数量差异。
具体实验操作如下:
1.类胡萝卜素途径关键基因表达
取不同发育期(35dpa、47dpa和54dpa)M82、yft1和sledl赤道面果皮样品提取总RNA,特异性引物(表4)在Light Cycle 96型实时荧光定量PCR仪(Roche有限公司,瑞士)进行RT-qPCR分析,结果显示,PSY1(PHYTOENE SYNTHASE 1)、CRTISO(CAROTENOID ISOMERASE)和CYCB(CHROMOPLAST-SPECIFIC LYCOPENEΒ-CYCLASE)在M82果实中的表达水平均显著高于yft1和sledl,不过35dpa除外(图6);说明EMB1444-like通过调控类胡萝卜素合成途径相关基因表达而影响类胡萝卜素合成和累积,从而影响番茄果色形成。
表4分析类胡萝卜素合成途径关键基因表达引物序列
2.类胡萝卜素含量分析
2.1类胡萝卜素提取
(1)取不同发育期(35dpa、47dpa和54dpa)M82、yft1和sledl果实赤道面外果皮,避光下在液氮中研磨成粉末;
(2)称取约500mg粉末至15mL干净离心管,记录准确粉末质量(用减重法);添加1.5mL KOH和甲醇和混合液(w/v=6%),颠倒混匀,60℃水浴30min;
(3)冷却至室温,加入1.5mL Tris-HCl缓冲液(50mM Tris-HCl,1M NaCl,pH 7.5),颠倒混匀,4℃冰箱静置10min;
(4)加入4mL氯仿,颠倒混匀,冰上静置10min,4℃4000rpm离心10min;
(5)用干净5mL注射器吸取下层有机相至新15mL离心管;水相中再添加4mL氯仿重复抽提,将两次萃取的有机相合并,用氯仿补足至9mL;
(6)取1.5mL提取液至2mL离心管,SPD2010型离心浓缩仪(Thermo Fisher科技有限公司,马萨诸塞州,美国)浓缩干燥。
2.2类胡萝卜素测定
(1)加50μL甲基叔丁基醚(MTBE)溶解样品,充分震荡后在12000rpm离心10min;
(2)转移上层液体至上样瓶,取1μL进样,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。具体操作为: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,设置柱温45℃,背压22.9MPa;二极管阵列检测器探测波长在210nm到500nm之间,补偿波长在210nm到280nm之间。
2.3标准曲线制作
(1)用MTBE(Thermo Fisher Scientific Inc.,MA,USA)溶解番茄红素、β-胡萝卜素、α-胡萝卜素和叶黄素标品(Sigma Chemical Co.CA,USA),制备浓度梯度(5μg/mL,10μg/mL,50μg/mL,250μg/mL和500μg/mL);
(2)将各标品按照浓度从低到高顺序进样,建立浓度与峰面积的标准曲线,用于估测样品胡萝卜素组分浓度。
类胡萝卜素组分分析结果显示,yft1和sledl的总类胡萝卜素含量显著低于M82,说明EMB1444-like通过调控YFT1控制番茄类胡萝卜素累积(表5)。
表5番茄M82、yft1和sledl在不同发育时期果实类胡萝卜素含量(μg·g-1FW)*
FW(fresh weight):鲜重。
3.有色体发育与超微结构
3.1制片与超微结构观察
(1)取样和固定:取不同发育期(35dpa、47dpa和54dpa)的M82、yft1和sledl番茄果实赤道果皮,用手术刀片切成约1mm3组织,在2.5%戊二醛中固定,用真空泵(Millipore有限公司,马萨诸塞州,美国)真空处理30min至组织块完全浸入固定液,4℃继续固定24h。
(2)漂洗与脱水:吸掉固定液,用0.1M PB(phosphate buffer)漂洗样品,4℃放置15min,重复操作3次。弃漂洗液,加适量锇酸(中镜科仪技术有限公司,北京,中国),4℃静置2h,弃锇酸至专用垃圾瓶,然后用0.1M PB漂洗样品,4℃放置15min,重复3次。4℃条件下用50%乙醇、70%乙醇、90%乙醇分别脱水15min;90%乙醇:90%丙酮(v:v=1:1)置换20min,再用90%丙酮置换20min。
(3)包埋与固化:用100%丙酮置换20min,置换3次;丙酮:环氧树脂(Ted Pella股份有限公司,加利福尼亚州,美国)(v:v=1:1)混合溶液置换1h,丙酮:环氧树脂(v:v=1:2)过夜置换;纯树脂置换7h;取出包埋板,加适量树脂,用牙签小心地将样品摆放于包埋板合适位置上(记录样品位置),放入60℃电热鼓风干燥箱中聚合48h。
(4)样品切片、染色和电镜观察:对固化包埋块修块,样品面暴露出来后,用UC6-FC6型冷冻超薄切片机(Leica仪器有限公司,德国)切片,用2%枸橼酸铅(中镜科仪技术有限公司,北京,中国)染色;120kV生物型透射电镜(FEI公司,俄勒冈州,美国)观察有色体超微结构。
观察有色体发育进程发现,yft1和sledl有色体发育相对于野生型M82延迟;说明EMB1444-like通过调控YFT1表达影响番茄果实有色体发育(图7)。
3.2脂质体小球数量统计
在透射电镜下每个样品选取3个不同视野统计脂质体小球数量(三个生物学重复,n=3)。结果显示,在54dpa时期,yft1和sledl脂质小球数量显著低于野生型M82,同时,sledl果实脂质体小球数量显著高于yft1;EMB144-like可通过调控YFT1表达,影响番茄果实中脂质体小球数量(图7)。
实施例5:EMB1444-like调控乙烯生物合成
为了明确EMB1444-like调控YFT1对番茄果实乙烯合成和信号转导的影响,分析了M82、突变体yft1和sledl果实乙烯合成和乙烯信号转导关键基因表达差异,具体步骤如下:
1.番茄果实乙烯释放量测定
取不同发育期(35dpa、47dpa和54dpa)番茄M82、yft1和sledl果实(n=3),记录单果实重。25℃恒温放置2h以消除采摘生理胁迫可能引起果实乙烯释放水平变化。将果实小心密封于500mL气体收集瓶中,25℃恒温放置4h收集气体。用1mL一次性注射器缓慢吸取气体,迅速地注入GC-2010气相色谱仪(岛津仪器有限公司,日本),测定乙烯含量。样品乙烯浓度根据标准乙烯气体(10ppm)计算,标准曲线制备方法:分别注入0.1mL、0.3mL、0.5mL、0.6mL、0.9mL标准乙烯气体(10ppm),以浓度为纵坐标,峰面积为横坐标建立标准曲线。用下述公式计算番茄乙烯释放量。
检测结果显示,sledl突变体果实乙烯释放高峰比M82推迟,释放量也显著下降;而sledl突变体果实乙烯释放量显著高于yft1番茄(图8)。
2.EMB1444-like调控乙烯合成关键基因表达
取不同发育期(35dpa、47dpa和54dpa)的M82、yft1和sledl果实赤道面果皮样品提取总RNA(n=3),反转录制备cDNA文库,作为RT-qPCR模板,用基因特异引物(表6)在LightCycle 96型实时荧光定量PCR仪(Roche有限公司,瑞士)对乙烯合成关键基因ACS2/4(ACCSYNTHASE 2/4)和ACO1(ACC OXIDASE 1)进行RT-qPCR分析结果显示,sledl突变体的ACS2/4和ACO1表达水平低于M82,而显者高于yft1的。说明EMB1444-like通过调控YFT1表达而影响番茄果实乙烯合成关键基因表达(图9)。
3.乙烯信号转导途径关键基因表达分析
用2.制备的cDNA文库作模板,分析乙烯信号途径关键基因NR(NEVER RIPE)、ETR4(ETHYLENE RECEPTOR 4)、AP2a(APETALA 2a)、ERF4/6(ETHYLENE-RESPONSIVETRANSCRIPTION FACTOR 4/6)表达发现,sledl突变体的NR、ETR4、AP2a、ERF4/6在yft1和EMB1444-like-RNAi表达水平显著低于M82,而显著高于yft1突变体,说明EMB1444-like通过调控YFT1表达而影响番茄果实乙烯信号传导(图10)。
表6用于乙烯合成和乙烯信号转导关键基因RT-qPCR分析引物序列
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综上所述,本发明基于一种转录因子EMB1444-like,通过特异地结合一个乙烯信号途径核心组分SlEIN2编码基因YFT1上游启动子pYFT1调控区的E-box(CACTTG,-1295bp~-1290bp)功能元件,正向调控YFT1表达,控制番茄果实乙烯合成、类胡萝卜素合成和叶绿体/有色体发育,从而影响番茄果色形成和果实成熟发育。本发明可以对番茄品质和成熟期的改良提供理论依据,基于此开展番茄育种培育优质和货架期适于生产应用的品种。
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。
Claims (10)
1.一种bHLH家族的转录因子EMB1444-like在调控番茄果实成熟发育中的应用,其特征在于,所述EMB1444-like的氨基酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的应用,其特征在于,所述EMB1444-like的CDS序列如SEQ IDNO.4所示。
3.根据权利要求1所述的应用,其特征在于,所述转录因子EMB1444-like通过调控乙烯合成、乙烯信号转导、有色体发育、类胡萝卜累积、果色形成来调控番茄果实成熟发育和番茄果色。
4.根据权利要求1所述的应用,其特征在于,所述EMB1444-like正调控乙烯信号转导关键组分SlEIN2编码基因YFT1的转录表达。
5.根据权利要求4所述的应用,其特征在于,所述EMB1444-like特异地结合于乙烯信号途径关键基因YFT1上游启动子pYFT1的E-box基序及其侧翼而调控YFT1表达;所述E-box基序如SEQ ID NO.1所示;所述E-box基序及其侧翼序列如SEQ ID NO.2所示。
6.一种下调转录因子EMB1444-like基因表达的RNA干扰序列,其特征在于,所述RNA干扰序列的DNA序列如SEQ ID NO.5所示。
7.一种EMB1444-like下调表达载体,其特征在于,所述表达载体的构建方法包括如下步骤:
用特异引物EMB1444-like-RNAi-F和EMB1444-like-RNAi-R扩增EMB1444-like特异DNA片段,如SEQ ID NO.5所示,构建至RNAi载体pHELLSGATE 12,获得EMB1444-like下调表达载体,命名为EMB1444-like-RNAi;所述EMB1444-like-RNAi-F的序列如SEQ ID NO.44所示;所述EMB1444-like-RNAi-R的序列如SEQ ID NO.45。
8.如权利7所述的表达载体在番茄果实成熟中的应用。
9.一种EMB1444-like下调表达突变体sledl,其特征在于,将权利要求7所述的EMB1444-like下调表达载体转化农杆菌,选取阳性转化农杆菌,转入野生型番茄M82,获得突变体sledl。
10.根据权利要求9所述的突变体sledl,其特征在于,所述野生型番茄为野生型番茄M82。
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