CN118126148A - 调控番茄果实可溶性固形物基因SlC2H2-71的应用 - Google Patents
调控番茄果实可溶性固形物基因SlC2H2-71的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,尤其涉及调控番茄果实可溶性固形物基因SlC2H2‑71的应用。本发明提供的SlC2H2‑71基因编码一种C2H2型锌指转录因子,可以通过抑制细胞壁蔗糖转化酶基因SlLIN5的表达,减少蔗糖分解并负调控可溶性固形物的积累,在糖酸代谢通路中发挥着重要作用,为植物的风味品质育种提供了新的优良基因资源。而且,通过抑制或敲除等基因编辑手段降低SlC2H2‑71基因的表达水平,可提高番茄红熟果实可溶性固形物的含量,尤其是提高果糖、葡萄糖、苹果酸和柠檬酸的含量,降低蔗糖含量,为改善番茄风味品质、提高番茄果实可溶性固形物含量提供了新的育种思路。
Description
技术领域
本发明涉及生物技术领域,特别涉及调控番茄果实可溶性固形物基因SlC2H2-71的应用,尤其涉及转录因子SlC2H2-71作为靶点,在调控番茄果实可溶性固形物、调控可溶性糖或可溶性酸或者调控番茄细胞壁蔗糖转化酶基因SlLIN5表达中的应用。
背景技术
番茄(Solanum lycopersicum)因其具有丰富的营养价值及良好的风味品质深受消费者的喜爱,是蔬菜市场上主要果菜之一,也是一种浆果类模式作物。味觉甜度会显著影响消费者对番茄的偏好,改善风味是商业番茄生产的主要挑战。可溶性固形物(Solublesolids content,SSC)是衡量番茄果实风味的关键决定因素之一,SSC主要由可溶性糖和有机酸等营养物质组成,其含量高低还在作物的发育、产量和胁迫响应中起着关键作用。在柑橘中,果实发育前两个阶段中果汁囊泡的伸长与可溶性固体含量变化相关;生产者培育SSC含量更高的番茄时,产量通常会受到影响,导致番茄种植者的盈利阈值降低。SSC的积累也可以增加细胞渗透浓度,降低水势,增强保水能力,提高植物抗寒性。因此,调控番茄红熟果实可溶性固形物含量变化对改善番茄果实口感、风味、品质和产量等具有重要意义。
基于基因工程育种技术,通过挖掘调控SSC途径的关键基因,并将其应用到作物育种中去,是改良番茄果实品质和选育优良品种经济而有效的策略。蔗糖是番茄光合产物运输的主要物质,在淀粉和纤维素的合成、果实品质形成等过程发挥了重要调控作用。蔗糖分解过程主要依靠蔗糖合成酶(SUS)和蔗糖转化酶(INV),其中,SUS参与的反应是可逆的且以合成蔗糖为主,而INV则是不可逆地将蔗糖分解为葡萄糖和果糖,INV可分为液泡蔗糖转化酶(VIN)、细胞型蔗糖转化酶(CIN)和细胞壁蔗糖转化酶(CWIN),前两者为胞内酶,后者为胞外酶。番茄果实成熟期积累糖分的类型主要由蔗糖转化酶的活性决定。已有研究者利用近等基因系定位到一个SSC相关主效QTL位点,位于9号染色体上,是由Brix9-2-5编码的质外体蔗糖转化酶基因LIN5,其在果实和花中特异表达,并已被克隆和广泛应用。LIN5位点的碱基多态性变化使番茄果实中葡萄糖含量提高28%、果糖含量提高18%,并且不影响产量。采用RNAi抑制番茄SlLIN5的表达会导致番茄幼果的败育,而通过RNAi或CRISPR/Cas9抑制转化酶抑制因子基因SlINVINH1的表达,则导致细胞壁蔗糖转化酶活性提高,促进了番茄果实和种子的发育,提高了果实可溶性固形含量(SSC)。
尽管已有相应研究,但总体而言对SSC调控基因的克隆、功能鉴定等方面研究较少。用于番茄SSC改良方面的遗传资源仍相当匮乏,尤其是目前番茄栽培品种遗传背景越来越狭窄,传统常规育种手段以及种质资源很难在番茄SSC改良上取得很大突破。因此,迫切需要挖掘新的番茄SSC相关调控基因并加以利用,这可为改善果实风味品质等农艺性状提供新的育种方向,对番茄风味品质的遗传改良效益明显。
发明内容
针对现有技术中的上述技术问题,本发明提供了一种SlC2H2-71基因,其编码C2H2型锌指转录因子,可以通过抑制细胞壁蔗糖转化酶基因SlLIN5的表达负调控SSC的积累;通过降低C2H2-71基因的表达水平,可提高番茄红熟果实SSC的含量,由此本发明提供了转录因子SlC2H2-71作为靶点,在调控番茄果实可溶性固形物、调控可溶性糖或可溶性酸或者调控番茄细胞壁蔗糖转化酶基因SlLIN5表达中的应用,目的在于解决或至少部分解决现有技术中的问题。本发明具体通过以下方案实现:
本发明第一方面提供了转录因子SlC2H2-71在调控番茄果实可溶性固形物中的应用。
进一步地,所述转录因子SlC2H2-71的核苷酸序列如SEQ ID NO.1所示,或者,所述转录因子SlC2H2-71的蛋白序列如SEQ ID NO.2所示。
进一步地,所述调控包括正向调控或负向调控,其中,通过增强转录因子SlC2H2-71的表达量,实现负向调控番茄果实可溶性固形物的积累;或者,通过降低转录因子SlC2H2-71的表达量,实现正向调控番茄果实可溶性固形物的积累。
进一步地,所述负向调控番茄果实可溶性固形物的积累包括以下步骤:构建SlC2H2-71超量表达载体,转化野生型番茄植株,培养获得SlC2H2-71基因表达量提高的转基因番茄。
进一步地,所述正向调控番茄果实可溶性固形物的积累包括以下步骤:构建SlC2H2-71基因敲除载体,转化野生型植物,培养获得SlC2H2-71基因表达量降低的转基因番茄。
本发明第二方面提供了转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用。
进一步地,所述可溶性糖包括蔗糖、果糖和/或葡萄糖,所述可溶性酸包括苹果酸和/或柠檬酸。
本发明第三方面提供了转录因子SlC2H2-71在调控番茄细胞壁蔗糖转化酶基因SlLIN5表达中的应用。
本发明的优点及积极效果为:本发明提供的SlC2H2-71转录因子可以通过抑制细胞壁蔗糖转化酶基因SlLIN5的表达,减少蔗糖分解并负调控可溶性固形物的积累,在糖酸代谢通路中发挥着重要作用,为植物的风味品质育种提供了新的优良基因资源。而且,通过抑制或敲除等基因编辑手段降低SlC2H2-71基因的表达水平,可提高番茄红熟果实可溶性固形物的含量,尤其是提高果糖、葡萄糖、苹果酸和柠檬酸的含量,降低蔗糖含量,为改善番茄风味品质、提高番茄果实可溶性固形物含量提供了新的育种思路。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面对实施例描述中所使用的附图作简单的介绍。
图1为本发明实施例的8种番茄材料可溶性固形物含量图;
图2为本发明实施例的8种番茄材料基于转录组数据的C2H2家族基因差异表达热图;
图3为本发明实施例的SlC2H2-71基因在8种番茄材料果实中的表达情况图;
图4为本发明实施例的SlC2H2-71基因结构和功能分析图;其中,图A为SlC2H2-71基因的蛋白结构域,图B为SlC2H2-71锌指蛋白转录因子的系统进化树;
图5为本发明实施例的SlC2H2-71基因的亚细胞定位图;
图6为本发明实施例的SlC2H2-71基因的组织表达情况图,其中,图A为组织特异性GUS染色图,图B为因组织特异性相对表达量图;
图7为本发明实施例转化有SlC2H2-71基因的酵母细胞在SD/-Trp和SD/-Trp-His-Ade培养基上的生长状态图;
图8为本发明实施例的SlC2H2-71基因的敲除原理图,其中,图A为SlC2H2-71基因的编辑靶点,图B为SlC2H2-71基因的编辑方式;
图9为本发明实施例SlC2H2-71基因敲除对番茄果实的影响图,其中,图A为突变体和野生型番茄果实实物图,图B为突变体和野生型番茄果实可溶性固形物、蔗糖、葡萄糖、果糖、苹果酸和柠檬酸含量图;
图10为本发明实施例RNA-seq数据分析开花后45天SlC2H2-71突变体和野生型番茄中糖和酸调节途径的基因表达差异图;
图11为本发明实施例通过qRT-PCR定量糖和酸调节途径基因的相对表达量图;
图12为本发明实施例转化含有SlC2H2-71基因的诱饵载体和含有SlLIN5基因启动子的猎物载体的酵母菌株在SD/-Leu-Ura培养基的生长状态图;
图13为本发明实施例利用35S::UAS-GUS报告系统表征SlC2H2-71转录因子的转录活性结果图;其中,图A为35S::UAS-GUS系统中效应器和报告载体的简化图谱,图B为效应器和报告载体共转化烟草叶片后的GUS染色结果图,图C为效应器和报告载体共转化烟草叶片后的GUS基因表达水平图;
图14为本发明实施例采用双荧光素酶测定SlC2H2-71和SlLIN5启动子之间的相互作用结果图;其中,图A为双荧光素酶系统中效应器和报告载体的简化图谱,图B为效应器和报告载体共转化烟草叶片后启动子的相对活性差异图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例对本发明进行进一步详细说明。此处所描述的实施例仅用以解释本发明,并不用于限定本发明。在本发明中所用的表示用量、百分比的所有数字以及其他数值,在所有情况下都应理解为以词语“大约”所修饰。因此,除非特别说明,否则在说明书和所附权利要求书中所列出的数字参数都是近似值,其可能会根据试图获得的理想性质的不同而加以改变。各个数字参数至少应被看作是根据所报告的有效数字和通过常规的四舍五入方法而获得的。“包括”、“包含”、“含有”、“具有”等类似词语的含义是非限制性的,即可加入不影响结果的其它步骤和其它成分。“和/或”应被视对在具有或不具有另一者的情况下两种指定特征或组分中的每一种的具体公开。例如,“A和/或B”将被视为包含以下情形:(i)A、(ii)B、以及(iii)A和B。
锌指(zinc finger)蛋白是迄今在真核生物基因组中分布最广的一类的蛋白,锌指蛋白根据半胱氨酸和组氨酸残基的数目和位置可以分为C2H2、C4、C6、C4HC3、C3HC4、C2HC、C3H以及联合型等多种类型。C2H2是一类具有指状结构域的转录因子家族,含有植物特有的QALGGH序列,在植物生长发育、生物或非生物胁迫过程中发挥着广泛调节作用。目前关于C2H2转录因子的相关研究主要集中在植物生长发育和抵御逆境方面,对可溶性固形物积累方面的研究还较少。番茄基因组中共发现了104个C2H2转录因子,其中约半数的基因在花、果实和种子中表达,可能参与了番茄果实的发育过程。可溶性固形物含量(SSC)是番茄果实风味的重要影响因素,果实可溶性固形物积累在糖代谢过程中受到转录调控。因此,本申请以C2H2型锌指蛋白作为研究对象,探讨C2H2锌指蛋白对果实可溶性固形物积累的调控机制,以期挖掘出新的SSC调控基因,为番茄风味品质的遗传改良提供优良的基因资源和新的育种方向。
本发明选取了8种不同可溶性固形物含量的番茄材料,通过对红熟果实转录组数据中SlC2H2家族基因表达分析,发现SlC2H2-71基因在高SSC材料中的表达量明显低于低SSC材料。SlC2H2-71是一种C2H2型锌指蛋白家族转录因子,进一步重点对SlC2H2-71进行了生物学功能和调控网络的全面分析。利用CRISPR-Cas9系统构建SlC2H2-71基因功能缺失突变系,发现转基因材料红熟果实中可溶性固形物、果糖、葡萄糖、苹果酸和柠檬酸的含量上升,而蔗糖含量却下降。对突变系转录组数据进行分析,表明突变系果实中糖酸代谢通路相关基因表达被调控,其中细胞壁蔗糖转化酶SlLIN5基因表达上调,并通过实时荧光定量PCR(qRT-PCR)证实了这一点。酵母单杂交(Y1H)、35S::UAS-GUS报告系统和双荧光素酶报告系统表明,SlC2H2-71转录因子可直接结合细胞壁蔗糖转化酶SlLIN5基因启动子并抑制其表达,继而影响糖代谢和番茄红熟果实可溶性固形物积累。
基于此,本发明一实施例提供了转录因子SlC2H2-71在调控番茄果实可溶性固形物中的应用。
SlC2H2-71是一个在番茄果实中高表达的转录抑制因子,其通过抑制细胞壁蔗糖转化酶基因SlLIN5(NM_001247864)的表达,以减少蔗糖分解并负调控可溶性固形物的积累,在糖酸代谢通路中发挥着重要作用。本发明为解析C2H2型锌指转录因子调控果实风味品质提供了理论基础,也为植物的风味品质育种提供了新的优良基因资源。而且,由于SlC2H2-71对番茄果实可溶性固形物的合成积累具有负调控作用,通过抑制或敲除等基因编辑手段下调SlC2H2-71基因的表达水平,以此上调SlLIN5基因的表达,进而提高番茄红熟果实可溶性固形物的含量,尤其是提高果糖、葡萄糖、苹果酸和柠檬酸的含量,降低蔗糖含量,为改善番茄风味品质、提高番茄果实可溶性固形物含量提供了新的育种思路。
可选地,所述转录因子SlC2H2-71的核苷酸序列如SEQ ID NO.1所示,或者,所述转录因子SlC2H2-71的蛋白序列如SEQ ID NO.2所示。
可选地,上述所述的调控作用包括正向调控或负向调控,通过增强转录因子SlC2H2-71的表达量,可以实现负向调控番茄果实SSC的积累;通过降低转录因子SlC2H2-71的表达量,可以实现正向调控番茄果实SSC的积累。需要说明的是,转录因子的表达量包括基因水平(mRNA)的表达量和/或蛋白水平的表达量,基因水平可通过RNA干扰技术、CRISPR-Cas9技术或超量表达载体等实现,蛋白水平可通过蛋白激活剂或抑制剂等实现,此为本领域常规技术手段,在此不再详细说明。
具体地,负向调控番茄果实SSC的积累包括以下步骤:构建SlC2H2-71超量表达载体,转化野生型番茄植株,培养获得SlC2H2-71基因表达量提高的转基因番茄。更具体地,构建SlC2H2-71超量表达载体包括以下步骤:利用SEQ ID NO.3-4所示的引物对,从番茄植株中扩增SlC2H2-71基因,将扩增得到的所述SlC2H2-71基因可操作地连接至pHELLSGATE8载体中,构建得到所述SlC2H2-71超量表达载体。
具体地,正向调控番茄果实SSC的积累包括以下步骤:构建SlC2H2-71基因敲除载体,转化野生型植物,培养获得SlC2H2-71基因表达量降低的转基因番茄。更具体地,构建SlC2H2-71基因敲除载体包括以下步骤:将靶向SlC2H2-71基因的gRNA编码基因可操作地连接至PTX载体中,构建得到所述SlC2H2-71基因敲除载体;所述gRNA编码基因的序列见SEQID NO.51和/或SEQ ID NO.52所示。
“可操作地连接”是指多个核酸片段在功能关系中的连接。当一个核酸与另一个核酸序列形成功能关系时,其为“可操作地连接”。例如,启动子与基因的“可操作地连接”是指以该启动子能够影响基因的转录的方式连接。可操作地连接意味着被连接的核苷酸序列可能是连续的。此为本领域常规技术手段,本发明对此不做特殊限定。
可选地,上述所述的可溶性固形物包括可溶性糖和可溶性酸,可溶性糖包括蔗糖、果糖和葡萄糖,可溶性酸包括苹果酸和柠檬酸。其中蔗糖的含量变化与果糖、葡萄糖、苹果酸和柠檬酸呈现相反趋势,例如,蔗糖含量降低时,果糖、葡萄糖、苹果酸和柠檬酸含量升高,总体上导致可溶性固形物含量升高。
基于与上述相同的发明构思,本发明实施例又一实施例提供了转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用。
具体地,可溶性糖包括蔗糖、果糖和/或葡萄糖,可溶性酸包括苹果酸和/或柠檬酸。
基于与上述相同的发明构思,本发明实施例再一实施例提供了转录因子SlC2H2-71在调控番茄细胞壁蔗糖转化酶基因SlLIN5表达中的应用。其调控方法与调控可溶性固形物含量基本相同,在此不再赘述。
下面结合具体实施例,进一步阐述本发明。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如冷泉港实验室出版的《分子克隆实验指南(第四版)》中所述的条件,或者厂商所建议的条件。
植物材料:番茄品种TS-638、TS-166、TS-210、TS-172、TS-20、TS-619、TS-280和TS-69来源于华中农业大学园艺林学学院番茄研究实验室,用于转化目的基因或载体的番茄材料为Ailsa Craig(AC)。所用番茄植株种植于湖北省武汉市华中农业大学果蔬园艺作物种质创新与利用全国重点实验室蔬菜种质创新研究基地。植株生长在25±2℃的温室中,光照周期为16h/8h,相对湿度为60%-70%。
基因和引物:涉及的C2H2型锌指蛋白SlC2H2-71及相关引物序列如表1所示。
表1本发明实施例SlC2H2-71及相关引物的DNA序列
1、C2H2型锌指蛋白SlC2H2-71是番茄果实可溶性固形物积累的潜在调控因子
C2H2型锌指蛋白成员众多,在番茄生长中发挥重要作用。根据已有研究,番茄基因组共编码104个SlC2H2基因。为了探讨SlC2H2锌指蛋白家族对番茄果实风味品质的影响,选取8份番茄材料,根据可溶性固形物(Soluble solids content,SSC)含量将它们分为高(TS-638、TS-166、TS-210和TS-172)和低(TS-20、TS-619、TS-280和TS-69)两种类型(见图1)。
对红熟番茄果实转录组数据(RPKM(RNA-Seq Data))中C2H2家族基因的表达情况进行分析,发现SlC2H2-71基因(Sol Genomics Network数据库基因编号为Solyc06g075250)表达存在显著差异(见图2);且该基因在低SSC含量品种中呈现高表达,在高SSC含量品种中呈现低表达(见图3)。其中,转录组数据为自测获得,获得方法:对8份高、低SSC番茄材料开花后45天的红熟果实提取总RNA进行转录组(RNA-Seq)测序。RNA-seq文库在DNBSEQ测序平台(BGI Genomics,中国)进行。
2、C2H2型锌指蛋白SlC2H2-71基因结构和功能分析
2.1、功能预测分析
使用HMMER网站(https://www.ebi.ac.uk/Tools/hmmer/search/phmmer)分析SlC2H2-71基因编码的蛋白结构域;基于蛋白序列,使用MEGA7软件进行物种间系统发育树分析;使用PlantPan网站(http://plantpan.itps.ncku.edu.tw/plantpan4/index.html)和PlantCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)网站进行启动子功能元件分析。
结果见图4,其中A图示出了SlC2H2-71基因的蛋白结构域,B图示出了SlC2H2-71锌指蛋白转录因子的系统进化树。SlC2H2-71属于Group IIC2H2型锌指蛋白转录因子,定位于6号染色体上,包含一个2236碱基的开放阅读框(ORF,序列见SEQ ID NO.1),编码528个氨基酸(蛋白序列见SEQ ID NO.2),具有一个C2H2结合域(62-86aa),与其他植物物种的对应序列具有高度同源性。
2.2、亚细胞定位和组织表达分析
构建CaMV35S:SlC2H2-71-GFP载体,方法包括:使用引物SlC2H2-71-GFP-Fw/Rv从番茄植株Ailsa Craig(AC)中扩增SlC2H2-71基因的编码区(CDS),使用II一步克隆试剂盒(购自Vazyme,中国,货号C112-02)克隆到GFP载体中,具体可参考文献[1]“SongJ,ShangL,Chen S,LuY,ZhangY,Ouyang B,Ye Z,Zhang J:Interactions between ShPP2-1,an F-box family gene,andACR11A regulate cold tolerance of tomato.Hortic Res2021,8(1):148.”。将CaMV35S:SlC2H2-71-GFP载体和细胞核标记CaMV35S:ERF-mCherry载体分别转化到农杆菌菌株GV3101中,菌液混合后注射烟草叶片。25℃条件下生长48h后,用Leica共聚焦软件对烟草叶片进行GFP和mCherry荧光信号观察。未插入SlC2H2-71基因的CaMV35S:GFP载体作为阴性对照,无SlC2H2-71基因表达,则无法产生GFP荧光信号。
亚细胞定位结果见图5(Bar=10μm),其中,红色荧光为细胞核marker蛋白H-mCherry,绿色荧光为转录因子SlC2H2-71-GFP蛋白,黄色荧光为SlC2H2-71-GFP蛋白和细胞核marker蛋白H-mCherry叠加产生,Bright为明场细胞核图。可以看到,转录因子SlC2H2-71与细胞核marker蛋白H-mCherry表达部位一致,确定转录因子SlC2H2-71在细胞核中表达。
进一步将SlC2H2-71的启动子扩增并克隆到含有GUS报告基因的pMV2载体中,构建SlC2H2-71pro::GUS载体,方法包括:使用引物SlC2H2-71-GUS-Fw/Rv从AC中扩增SlC2H2-71基因的启动子区域,使用II试剂盒克隆到pMV2载体中,具体可参考文献[1]。通过电击的方法将SlC2H2-71pro::GUS载体转化到农杆菌菌株GV3101中,然后采用农杆菌介导的方式将载体转化入番茄子叶叶盘中,诱导成苗。待番茄成熟后,取样对根、茎、叶、花和果实进行GUS染色。
另外,取样采用qRT-PCR方法测定SlC2H2-71基因在不同器官中的表达量,方法包括:采用Trizol试剂(购自Aidlab,中国,货号RN01-TRLpure Reagent)从番茄成熟果实中分离总RNA;利用HiScript II第一链cDNA合成试剂盒(购自Vazyme,中国,货号:R212-02)合成互补DNA;之后使用SlC2H2-71-qPCR-Fw/Rv引物对扩增,在SYBR Light Cycler 480仪器上鉴定基因的转录水平。以番茄Actin基因(Solyc11g005330)为内参对照,采用2-ΔΔCT法定量相对基因表达。前述过程具体可参考文献[2]“Livak,K.J.and T.D.Schmittgen.AnalysisofRelative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method.Methods.2001,25(4):402-408.”
SlC2H2-71基因的组织表达结果见图6,其中,图A为组织特异性GUS染色图,图B为基因表达量图,横坐标为不同器官((根(R)、茎(S)、叶(L)、花(F)、未成熟果(IM)、成熟青果(MG)、破色期果(BR)、成熟早期黄果(YR)、红熟果(RR)),纵坐标为相对表达量。图中可以看到SlC2H2-71在果实组织中表达较高,与Tomato eFP Browser网站(http://bar.utoronto.ca/efp_tomato/cgi-bin/efpWeb.cgi)基因表达数据相符。
2.3、酵母中转录激活分析
将SlC2H2-71基因的全长ORF克隆到pGBKT7载体,转化到酵母菌株AH109中。pGBKT7-SlC2H2-71载体构建方法包括:使用引物SlC2H2-71-BD-Fw/Rv(见SEQ ID NO.19-20)从AC材料中扩增SlC2H2-71基因的编码区,使用II试剂盒克隆到pGBKT7载体中。pGBKT7-P53+pGADT7-RecT载体作为阳性对照,pGBKT7和pGBKT7-lam+pGADT7-RecT载体作为阴性对照。其中,pGBKT7、pGBKT7-P53、pGADT7-RecT、pGBKT7-lam载体为商业载体。载体及其构建过程具体参考文献[1]。之后使用YeastmakerTMYeastTransformation System2试剂盒(购自Takara,日本,货号Cat#630439)进行酵母双杂交(Y2H)检测,方法参考文献[3]“Ying Wang,Wenxian Gai,LiangdanYuan,Lele Shang,Fangman Li,Zhao Gong,Pingfei Ge,Yaru Wang,Jinbao Tao,Xingyu Zhang,Haiqiang Dong,Yuyang Zhang,Heat-inducible SlWRKY3 confers thermotolerance by activating the SlGRXS1 genecluster in tomato,Horticultural Plant Journal,2022,ISSN 2468-0141.”将转化的酵母菌体在SD/-Trp培养基(色氨酸缺陷型培养基)30℃培养3d。用ddH2O稀释阳性克隆,在SD/-Trp和SD/-Trp-His-Ade培养基上进行点斑。在30℃培养3d后观察结果。
酵母转录激活实验用于验证转录因子自身是否具有转录激活活性。将需要验证的转录因子SlC2H2-71构建到BD载体上,不需要构建AD载体,如果SlC2H2-71具有转录激活活性,那么载体下游的报告基因就能被激活表达从而在筛选培养基SD/-Trp和SD/-Trp-His-Ade上正常长斑。转录激活实验结果见图7,显示含有pGBKT7-SlC2H2-71载体的酵母细胞在SD/-Trp和SD/-Trp-His-Ade培养基上均能正常生长,表明SlC2H2-71具有转录激活活性。确定SlC2H2-71符合转录因子的特性。在后续实验中进一步证实SlC2H2-71对下游靶基因LIN5具有转录抑制作用。
3、SlC2H2-71负调控番茄红熟果实可溶性固形物积累
为了探究SlC2H2-71转录因子对番茄红熟果实可溶性固形物(SSC)含量的影响,本实施例采用CRISPR-Cas9系统在AC材料中对SlC2H2-71基因进行敲除,方法包括:1)靶点设计:使用targetDesign网站(http://skl.scau.edu.cn/targetdesign/)设计2个靶位点,第一个靶位点位于从SlC2H2-71基因起始密码子ATG开始的第464-486个碱基位置,第二个靶位点位于起始密码子ATG开始的第595-617个碱基位置(见图8中的A图);2)敲除载体构建:将包含靶位点片段的PCR产物(gRNA编码基因序列分别为:T1:CATGATCCATCGCGAGCTC;T2:TACTAAGATTTGTGGCACA)构建到PTX载体中,具体可参考文献[4]“Xie K,Zhang J,YangY.Genome-Wide Prediction of Highly Specific GRNA Spacers for Crispr-Cas9-Mediated Genome Editing in Model Plants andMajor Crops.Mol Plant,2014,7(5):923~926.”。同时构建SlC2H2-71-pHELLSGATE8超量表达载体,方法包括:使用引物SlC2H2-71-OE-Fw/Rv从AC材料中扩增SlC2H2-71基因的编码区(见SEQ ID NO.1),使用II一步克隆试剂盒(C112-02,Vazyme,中国)克隆到pHELLSGATE8载体中。使用农杆菌介导的方法将超量表达载体转化入番茄材料AC中。开花后45天收获红熟果实用于表型测定。需要说明的是,由于SlC2H2-71超量表达系材料无法正常开花结果,后续未对其进行深入研究。
使用便携糖度计(PAL-BX|ACID 3,ATAGO CO.,LTD.,Japan)测定番茄红熟果实的可溶性固形物含量;采用气相色谱质谱联用仪测定糖酸代谢物含量。样品处理及测定方法参考文献[5]“许让伟,程运江.(2018).柑橘果实糖酸含量测定.Bio-101e1010211.”,具体而言,番茄红熟果实使用液氮研磨,用80%甲醇(购自Sigma Aldrich,货号CAS#A452-4)进一步提取,进行真空浓缩,用盐酸羟胺(购自Sigma Aldrich,货号CAS#55460)、六甲基二硅烷(HMDS,购自Sigma Aldrich,货号CAS#52619)和三甲基氯硅烷(TMCS,购自SigmaAldrich,货号CAS#92361)进行衍生化。衍生化后的上清液加入自动进样瓶中,使用AgilentTechnologies 7890B GC System(Agilent Technologies,美国)进行GC-FID分析。
SlC2H2-71-CR1突变系在第一个和第二个靶位点之间发生130bp大片段缺失,SlC2H2-71-CR2突变系在两个靶位点分别发生1bp的碱基插入(见图8中的B图);这两个突变系均表现为SlC2H2-71基因功能的缺失。在AC材料生长过程中,观察发现SlC2H2-71功能缺失并未影响番茄果实生长和外观表型,但红熟果实的可溶性固形物含量显著提高,与对照系相比,Slc2h2-71突变系中果糖、葡萄糖、苹果酸和柠檬酸的含量升高,但蔗糖含量下降。图9中A图示出了开花后45天的SlC2H2-71基因突变体和野生型AC材料果实的实物图(Bar=1cm),B图示出了Slc2h2-71基因突变体CR1、CR2和野生型AC材料(CK)果实中可溶性固形物(Soluble solids content)、蔗糖(Sucrose)、葡萄糖(Glucose)、果糖(Fructose)、苹果酸(Malic acid)和柠檬酸(Citric acid)的含量,星号表示单因素方差分析显著性:*P≥0.05,**P≥0.01。
4、SlC2H2-71调控糖酸代谢相关基因表达
为了进一步探索SlC2H2-71基因对番茄红熟果实可溶性固形物及糖酸积累的调控机制,本实施例对SlC2H2-71突变体开花后45天的番茄红熟果实进行了转录组(RNA-Seq)分析。选取AC和SlC2H2-71突变体开花后45d的红熟果实提取总RNA,采用3个独立的生物重复进行RNA测序。RNA-seq文库在DNBSEQ测序平台(BGI Genomics,中国)进行。使用STAR将测序序列与番茄参考基因组(Version SL2.50)进行比对(文献[6]“Dobin A,Davis C A,Schlesinger F,Drenkow J,Zaleski C,Jha S,Batut P,Chaisson M,Gingeras T R.Star:Ultrafast Universal Rna-Seq Aligner.Bioinformatics,2013,29(1):15~21.”)。将reads组装成转录本,使用特征计数计算每个转录本的读取计数,采用edgeR筛选差异表达基因(DEGs),参数设置log2FC≥1.0、FDR<0.01。使用clusterProfiler注释基因本体(GO)功能。热图绘制使用TBtools软件。
通过比较SlC2H2-71突变体(CR1和CR2)和野生型AC红熟果实的DEGs,共鉴定出1686个DEGs。分析发现SlC2H2-71突变体果实中有690个基因上调,996个基因下调。GO富集分析显示,“分子功能”模块与氧气结合、催化活性和信号受体活性等有关,“细胞成分”模块与转移酶的活动、类囊体和膜等相关,“生物过程”模块包括对生物刺激的反应、对非生物刺激的反应、分解代谢的过程、碳水化合物代谢过程等。KEGG分析显示,光合作用、生物合成代谢、能量代谢、碳固定和碳水化合物代谢等通路注释丰富。
为了进一步寻找番茄果实可溶性固形物积累相关基因,筛选出糖酸代谢通路中DEGs,结果见图10,方格将基因表达模式表示为FPKM值,热图显示基因表达量的高低,包括12个与糖代谢有关的基因,27个与酸代谢有关的基因。通过qRT-PCR定量糖酸调节途径基因的相对转录水平,结果见图11,SlLIN5、SlVI、SlPK-8、SlACO-2和SlALMT-4基因的扩增引物见SEQ ID NO.9-10和SEQ ID NO.43-50。qRT-PCR和RNA-Seq数据的结果相互一致,显示细胞壁蔗糖转化酶(酸性转化酶)在SlC2H2-71突变体果实中显著上调。因为SlC2H2-71突变体果实中蔗糖含量降低,猜测SlC2H2-71可能通过调控细胞壁转化酶基因的表达来调节蔗糖的积累。同样,在超量材料中检测到SlLIN5基因(NCBI GenBank编号:NM_001247864)表达量降低。因此,推测SlC2H2-71可能作为转录因子调控SlLIN5的表达和番茄果实中SSC的积累。
5、SlC2H2-71转录因子抑制SlLIN5基因表达
5.1、酵母单杂交实验分析SlC2H2-71转录因子与SlLIN5基因启动子结合情况
为了探究SlC2H2-71与相关靶基因SlLIN5启动子之间的相互作用,使用YeastmakerTM Yeast Transformation System 2试剂盒(购自Takara,日本)进行酵母单杂交(Y1H)检测。将SlC2H2-71全长ORF克隆到pGADT7中,生成猎物载体,扩增引物见SEQ IDNO.17-18。然后转化入含有线性化诱饵载体SlLIN5-pAbai的酵母菌株Y1HGold中,在SD/-Leu-Ura培养基上培养。其中,SlLIN5-pAbai载体为自行构建,方法包括:使用引物SlLin5pro-pAbai-Fw/Rv从AC的材料中扩增SlLIN5基因的启动子区域,使用II一步克隆试剂盒(C112-02,Vazyme,中国)克隆到pAbai载体中。前述过程具体参考文献[1]和文献[7]“Ouwerkerk,P.B.F.and Meijer,A.H.(2001),Yeast One-Hybrid Screeningfor DNA-Protein Interactions.CurrentProtocols in Molecular Biology,55:12.12.1-12.12.12.”。阳性克隆用灭菌蒸馏水稀释至OD值600为0.1,然后分别滴3μL在含或不含AbA的SD/-Leu-Ura培养基上并置于30℃培养箱中培养3天。SD/-Leu-Ura培养基的AbA浓度分别为0、10、20、30、40、50、60、70、80、90、100ng·mL-1。
转化诱饵载体和猎物载体的酵母菌株在SD/-Leu-Ura培养基的生长情况见图12。在30ng/mLAbA的培养基中,SlC2H2-71-AD+SlLIN5pro-pAbai可以正常长斑,说明SlC2H2-71可以结合SlLIN5基因启动子。
5.2、35S::UAS-GUS报告系统表征SlC2H2-71的转录活性
将SlC2H2-71的ORF序列连接到pHELLSGATE8载体上构建pHELLSGATE8-SlC2H2-71效应器1(Effector1,E1),将pHELLSGATE8空载体作为效应器2(Effector2,E2,空白对照)。然后将效应器(1或2)和SlLIN5pro-GUS报告载体(Reporter,R)转化到农杆菌GV3101。利用农杆菌介导将报告载体分别与两种效应物混合后注入本式烟草叶片,生长48h后剪下烟草叶片进行GUS染色。具体操作参考文献[8]“Wang Y,Wang L,Zou Y,Chen L,Cai Z,Zhang S,Zhao F,Tian Y,Jiang Q,Ferguson B J,Gresshoff P M,Li X.Soybean Mir172C Targetsthe Repressive Ap2 Transcription Factor Nnc1 to Activate Enod40 Expressionand Regulate Nodule Initiation.Plant Cell,2014,26(12):4782~4801.”。
图13示出了35S::UAS-GUS报告系统表征SlC2H2-71转录因子的转录活性结果,其中,图A为效应器和报告载体的简化图谱,35S和SlLIN5pro为启动子,分别用于驱动表达SlC2H2-71基因和GUS基因,图B示出了效应器和报告载体共转化烟草叶片后的GUS染色结果,其中左侧为正常叶片,右侧为proSlLIN5::GUS和pro35S::SlC2H2-71共侵染叶片,图C示出了效应器和报告载体共转化烟草叶片后的GUS基因表达水平图,星号表示差异有统计学意义(**P≤0.01)。结果表明proSlLIN5::GUS和pHELLSGATE8共侵染的烟草叶片呈现出蓝色GUS活性,而proSlLIN5::GUS和pro35S::SlC2H2-71共侵染后未表现出蓝色GUS活性,同时GUS基因表达显著降低。因此,SlC2H2-71可以与SlLIN5启动子相互作用并负调控其表达。
5.3、双荧光素酶活性分析SlC2H2-71转录因子与SlLIN5启动子的结合区域
为了探究SlC2H2-71与SlLIN5启动子结合的具体区域,对SlLIN5启动子的1.5Kb片段进行了功能元件分析,共发现3个可能的结合元件位点,分别命名为C1(69-78bp)、C2(938-947bp)、C3(1484-1493bp)。为了验证SlC2H2-71是否能够识别并结合这些顺式元件,根据这三个顺式元件的位置,将启动子片段截断分为8种,共构建了9个proSlLIN5::PGreen0800重组载体作为报告载体,SlLIN5基因的9种不同长度的启动子片段(I-IX)扩增用引物见SEQ ID NO.25-42。将SlC2H2-71全长序列(扩增用引物见SEQ ID NO.23-24)作为效应器(Effector)构建到pGreenII 62-SK载体中,载体pGreenII 62-SK为效应器的空白阴性对照。载体构建方法参考文献[9]“Shang L,Chen S,Lu Y,Zhang Y,Ouyang B,Ye Z,Zhang J:Interactions between ShPP2-1,an F-box family gene,and ACR11A regulatecold tolerance of tomato.Hortic Res 2021,8(1):148.”,双荧光素酶实验方法参考文献[10]“王磊,鄢文豪,欧阳亦聃.(2018).荧光素酶酶活恢复法检测体内蛋白质互作.Bio-101,e1010134.Doi:10.21769/BioProtoc.1010134”。将报告载体和效应器共转入农杆菌GV3101中,随后用IM缓冲液(10mmol/L MES、10mmol/LMgCl2、150μmol/LAS)活化农杆菌细胞。将不同载体的农杆菌混合注射到烟草叶片中,2天后测定酶活。使用Infinite M200 Pro仪器(Tecan,瑞士)检测荧光素酶报告基因,酶活试剂盒使用Dual-LuciferasereporterAssay System(购自Promega,货号E1960)。
图14示出了采用双荧光素酶测定SlC2H2-71和SlLIN5启动子之间的相互作用结果,其中,图A为双荧光素酶系统中效应器和报告载体的简化图谱,图B示出了效应器和报告载体共转化烟草叶片后启动子的相对活性差异,采用LUC/REN比值表征,其中,双荧光素酶报告基因检测系统包括萤火虫荧光素酶(Firefly luciferase,Luc)和海参荧光素酶(Renilla luciferase,Ren)。Luc值代表报告基因酶活,Ren值代表内参基因酶活,通过计算每管的Luc/Ren的比值,再以对照组的比值作为单位1,获得实验组的相对活性。双荧光素酶活性测定发现,共表达pGreenII 62-SK-SlC2H2-71和proSlLIN5::PGreen0800的叶片的LUC/REN比值显著低于对照叶片的LUC/REN比值,表明,SlC2H2-71可以直接与SlLIN5的启动子结合并抑制其表达,与35S::UAS-GUS报告系统结果一致。8个分段载体的酶活测定结果显示,与对照组相比,含有C2和C3两个顺式元件以及只含有C3顺式元件的SlLIN5启动子的LUC/REN比率显著降低;而单独含有C1或C2,以及不含顺式元件的启动子片段与对照组相比,LUC/REN比率无显著差异。综上结果表明,SlC2H2-71主要通过与SlLIN5启动子的C3顺式元件区域结合来调控其表达。
综合上述结果可知,SlC2H2-71是一个在番茄果实中高表达的转录抑制因子,其通过抑制细胞壁蔗糖转化酶基因SlLIN5的表达,减少蔗糖分解,在糖酸代谢通路中发挥着重要作用。通过抑制或敲除该SlC2H2-71基因,可提高番茄红熟果实中可溶性固形物、果糖、葡萄糖、苹果酸和柠檬酸的含量,降低蔗糖含量。本发明为解析C2H2型锌指转录因子调控果实风味品质提供了理论基础,也为植物的风味品质育种提供了新的优良基因资源和新的育种思路。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用。
2.根据权利要求1所述的转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用,其特征在于,所述转录因子SlC2H2-71的核苷酸序列如SEQ ID NO.1所示;
或者,所述转录因子SlC2H2-71的蛋白序列如SEQ ID NO.2所示。
3.根据权利要求1所述的转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用,其特征在于,所述调控包括正向调控或负向调控,其中,通过增强转录因子SlC2H2-71的表达量,实现负向调控番茄果实可溶性固形物的积累;
或者,通过降低转录因子SlC2H2-71的表达量,实现正向调控番茄果实可溶性固形物的积累。
4.根据权利要求3所述的转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用,其特征在于,所述负向调控番茄果实可溶性固形物的积累包括以下步骤:
构建SlC2H2-71超量表达载体,转化野生型番茄植株,培养获得SlC2H2-71基因表达量提高的转基因番茄。
5.根据权利要求4所述的转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用,其特征在于,构建所述SlC2H2-71超量表达载体包括以下步骤:
利用如SEQ ID NO.3-4所示的引物对,从番茄植株中扩增SlC2H2-71基因,将扩增得到的所述SlC2H2-71基因可操作地连接至pHELLSGATE8载体中,构建得到所述SlC2H2-71超量表达载体。
6.根据权利要求3所述的转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用,其特征在于,所述正向调控番茄果实可溶性固形物的积累包括以下步骤:
构建SlC2H2-71基因敲除载体,转化野生型植物,培养获得SlC2H2-71基因表达量降低的转基因番茄。
7.根据权利要求6所述的转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用,其特征在于,构建所述SlC2H2-71基因敲除载体包括以下步骤:
将靶向SlC2H2-71基因的gRNA编码基因可操作地连接至PTX载体中,构建得到所述SlC2H2-71基因敲除载体;所述gRNA编码基因的序列如SEQ ID NO.51和/或SEQ ID NO.52所示。
8.转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用。
9.根据权利要求8所述的转录因子SlC2H2-71在调控番茄果实可溶性糖或可溶性酸含量中的应用,其特征在于,所述可溶性糖包括蔗糖、果糖和/或葡萄糖,所述可溶性酸包括苹果酸和/或柠檬酸。
10.转录因子SlC2H2-71在调控番茄细胞壁蔗糖转化酶基因SlLIN5表达中的应用。
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