CN111675756A - 花生开花习性基因AhFH1及其等位变异的克隆与应用 - Google Patents
花生开花习性基因AhFH1及其等位变异的克隆与应用 Download PDFInfo
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- CN111675756A CN111675756A CN202010631475.6A CN202010631475A CN111675756A CN 111675756 A CN111675756 A CN 111675756A CN 202010631475 A CN202010631475 A CN 202010631475A CN 111675756 A CN111675756 A CN 111675756A
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Abstract
本发明属于植物分子遗传学和基因工程技术领域,提供一种花生开花习性基因AhFH1及其等位变异的克隆与应用,通过实验确定花生开花习性基因AhFH1及其两种去功能性等位变异,去功能性等位变异能够引起花生从交替开花转变为连续开花;通过对基因AhFH1的过表达或自身启动子表达补充到连续开花花生品种中,可将其转变为交替开花;通过对AhFH1基因的敲除或抑制表达可将交替开花花生转变为连续开花;通过分子标记对该基因的等位变异实现分子标记辅助选择育种,通过分子生物技术手段利用该基因及其启动子,对花生开花习性相关的交替‑连续开花及由其引起的花生分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状进行生物技术的遗传改良或分子育种。
Description
技术领域:
本发明属于植物分子遗传学和基因工程技术领域,涉及花生开花习性基因AhFH1(Flowering Habit 1 of Arachis hypogaea L.)及其等位变异的克隆与应用,通过分子生物学手段利用该基因可以对花生开花习性相关的交替-连续开花及由其引起的花生分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状进行生物技术的遗传改良或分子育种。
背景技术:
花生(Arachis hypogaea L.)富含脂肪、蛋白质和多种维生素,营养价值极高,因此其成为了许多国家重要的经济作物(万书波,2003)。花生植株由一条直立的主茎、一对胚起源的第一侧枝和发育于主茎基部的多条一级侧枝和侧枝上又发育的二级、三级等次级侧枝构成的束状株型。花生的开花习性是花生株型相关的一个重要的性状,具体是由花生腋芽原基发育成花序或者枝条的分化问题,最终表现为花序和分枝在枝条上的排列方式问题,主要有两种典型的代表:连续开花型(如图1A)和交替开花型(如图1B)。连续开花型最典型的性状表现为:主茎开花同时侧枝上每一节(叶腋)均可着生花序,或在一级侧枝的基部1-2节或第一节上着生二次营养枝、花序,以后各节全部连续着生花序;在二级分枝上第一至第二节及以后各节均着生花序。这种模式限制了花生的分枝数,所以又称疏枝型花生。交替开花型的典型性状表现为:主茎不开花同时花序与营养枝在侧枝上交替出现,一般是侧枝基部1-3节或1-2节上生长营养枝,不长花序,其后的4-6节或3-4节长花序,不长营养枝,如此交替发生(常见2花:2枝交替出现)。这类花生从基部开始着生营养枝,且分枝占节位的近二分之一,有分枝多而密的株丛特点,所以又称密枝型花生。分类学上简单地根据主茎是否开花将花生栽培品种分为两个亚种:连续开花亚种(Arachis hypogaeasubsp.fastigiata)和交替开花亚种(Arachis hypogaea subsp.hypogaea)(KrapovickasA,et al.2007)。除此之外还有较少的非典型性或中间型品种,主要表现为连续开花偶有分枝和交替分枝同时所有叶腋均开花等类型。花生的开花习性通过影响花生的花序数目、分枝数目和荚果集中程度、成熟一致性等直接影响花生地上部株型,进而影响花生群体产量、种植方式和籽仁的品质。由于花生叶腋发育渐次进行,因而连续开花型花生开花结荚时间和空间上均比较集中、成熟较一致、荚果收获容易、品质一致。交替开花型花生开花结荚时间上前后差距较大,间隔较远,导致荚果分散、荚果成熟不一致、荚果的产量和品质均受到影响。
目前在拟南芥、番茄、大豆、水稻和玉米等植物中,针对花序分化与发育的遗传基础研究已经取得了较大的进展。TFL1同源基因和FT同源基因LFY/AP1之间的相互拮抗共同调节拟南芥花序分枝模式(Alvarez J,1992;Liljegren SJ,1999;Conti L,2007;HiraokaK,2013)。拟南芥tfl1突变体具有单花,并导致花序分枝中花序顶端分生组织终止为单独的花(Alvarez J,1992);TFL1过表达导致拟南芥晚开花(Benlloch R,2007)。大豆作为光周期敏感的短日照植物,研究发现大豆FT基因家族成员GmFT1a能够延迟大豆开花和成熟,与开花促进基因GmFT2a/GmFT5a相互拮抗,共同调节大豆的生长发育(Liu W,2017,Kong F.,2010,Sun H,2011,Cai Y,2018)。同样作为典型的短日照作物玉米,研究者通过图位克隆和关联分析克隆到一个控制玉米开花期的数量性状位点(ZmCCT9),该位点上游的一个Harbinger-like转座子抑制ZmCCT9基因的表达,从而促进玉米在长日照条件下开花(HuangC,2017,Hsiao-Yi H,2012,Yang Q,2013)。在番茄中研究发现局部FT(特别是SFT)和TFL1(SP)的比例控制初级枝条和次生枝条有限或无限生长的平衡。番茄的枝桠结构随成花素(sft)和成花素抑制基因(sp)增减而发生的变化,因此利用杂交和基因编辑手段培育出高产的番茄品种,为获得理想型植株提供了新的研究方向(Krieger U.,2010,Soyk S.,2017)。
在植物的分枝发育过程中,茎尖分生组织(SAM)分化成植物的主茎,而侧枝的发育则是由腋生分生组织分化而来,其中腋生分生组织分化为侧枝的过程受环境和植物内部因素共同调控。在拟南芥、水稻、番茄和玉米等植物的分枝模式研究中获得了很多与控制分枝发育相关的基因(Zhi WNT,2014,Soyk S.,2017)。根据植物的分枝表型可将这些基因分为两类,一类为控制叶腋分生组织形成的基因,如最早在番茄上发现的LS基因,该基因不仅可以控制腋生分生组织形成,ls突变体的番茄植株表现为几乎不形成分枝(Schumacher K,1999),在水稻中也发现了LS的同源基因OsMOC1基因,突变后导致水稻不能形成分蘖芽从而影响水稻的分蘖数目(Li X,2003),另外在番茄中调控腋生分生组织的BL基因(Gregor S,2002),并在拟南芥中发现了番茄BL的同源基因RAX(Keller T,2006);另一类是与腋生分生组织生长相关的基因,其中这些突变并不影响腋生分生组织的形成,其中包括玉米中与抑制腋芽生长的TB1基因(Doebley J,1997,Lauren H,2002),水稻中的TB1同源基因被称为OsTB1或FINECULM1,拟南芥、豌豆和番茄中被称为BRC1(Aguilar-Martínez JA,2007,NilsB,2012,Mar MT,2011,Minakuchi K,2010),在拟南芥中与TB1编码类似蛋白的BRC1基因,其调控拟南芥的腋芽发育(Aguilar-Martínez JA,2007),并在番茄中发现与拟南芥BRC1基因具有类似的功能的SlBRC1a和SlBRC1b基因(Mar MT,2011)等。最近也有报道独角金内酯通过BRC1调控拟南芥的侧枝发育(Wang,L.,et al.2020)。
目前对于花生开花习性基因的研究工作较少,虽然有对花生成花素基因家族开展的生物信息学分析,但栽培花生的成花素同源基因家族成员预测的至少有29个以上,具体是哪一个基因控制了花生的交替连续开花并不清楚(Jin,Tang et al.2019)。对花生开花习性基因的克隆和功能研究还未见报道,定位并克隆花生开花习性基因,将为针对花生的开花习性及其相关性状进行的遗传改良和基因工程或基因编辑育种提供目标基因。
发明内容:
为了克服上述缺陷,本发明提供一种花生开花习性基因AhFH1及其等位变异的克隆与应用。
为了达到上述目的,本发明的技术方案是:
本发明提供一种花生开花习性基因AhFH1及其等位变异的克隆与应用,利用交替开花花生品种与连续开花花生品种杂交构建的遗传分离群体,通过连锁定位和图位克隆,结合亲本间的候选基因序列差异分析,明确了候选基因AhFH1(如图2),对基因AhFH1在种质资源中的克隆、比较分析和关联验证等发现花生开花习性基因AhFH1至少存在三种等位变异:一种完整功能性等位变异AhFH1和两种去功能性的等位变异Ahfh1(同时包括去功能性等位变异Ahfh1-1和Ahfh1-2)。本发明提供对上述基因AhFH1及其等位变异和启动子在作物遗传改良中的应用,优选在花生的开花习性及由其引起的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状的分子遗传改良中的应用。
本发明所述的花生开花***的核苷酸序列为SEQ IDNO:1,其转录的mRNA对应的cDNA序列为SEQ ID NO:2,其编码的蛋白序列为SEQ ID NO:3。等位变异AhFH1的代表品种为花生基因组测序品种Tifrunner或浙江地方品种小红毛等,其对应花生的开花***的克隆引物对为FH1g-F/R,其核苷酸序列如SEQ ID NO:4-5,利用该引物对在代表品种中的克隆产物电泳图如图3;花生开花***的克隆引物对FH1cd-F/R,其核苷酸序列如SEQ ID NO:6-7,利用该引物对在代表品种的cDNA中克隆完整功能性等位变异AhFH1的完整编码框,其产物电泳图如图4。
本发明所述的去功能性等位变异Ahfh1-1,其基因组水平的核苷酸序列如SEQ IDNO:8,去功能性等位变异Ahfh1-1是在基因组水平的基因末端存在一个以ATG为起始的+1872bp至+3273bp位置的1492bp的缺失(将该缺失命名为功能性分子标记InDel-1492bp),它包含最后一个外显子大部分及全部的3`UTR,该等位变异的代表品种为基因组测序品种狮头企和地方品种伏花生等,其对应花生的开花习性为连续开花型。
本发明所述的去功能性等位变异Ahfh1-2,其基因组水平的核苷酸序列如SEQ IDNO:11,去功能性等位变异Ahfh1-2编码的cDNA的+335bp位置的碱基C缺失,其编码的cDNA序列如SEQ ID NO:12,该碱基C缺失导致Ahfh1-2的翻译框移码,并提前形成终止子,翻译的蛋白不完整无功能。该等位变异的代表品种为花生品种云南七彩和龙花生559等,其对应花生的开花***的克隆引物对FH1g-F/R(SEQ ID NO:4-5)和cDNA水平的克隆引物对FH1cd-F/R(SEQ ID NO:6-7),同样可以分别实现对Ahfh1-2基因组水平和cDNA水平的克隆。基因AhFH1和等位变异Ahfh1-2间的单碱基差异可以通过对扩增产物的测序进行区分。
本发明还提供一种区分花生开花习性基因的交替开花型AhFH1和连续开花型Ahfh1-1等位变异的功能性分子标记InDel-1492bp,其对应的引物对为InDel-1492bp-F/R,其核苷酸序列如SEQ ID NO:9-10所示(该引物对为优选引物,其他可以扩增鉴别上述AhFH1和Ahfh1-1间1492bp缺失的引物对亦可选用)。结合测序确认AhFH1序列无变异的情况下,功能性分子标记InDel-1492bp扩增产物通过琼脂糖电泳检测可以区分AhFH1和Ahfh1-1两种等位变异,AhFH1的扩增产物为2556bp,Ahfh1-1的扩增产物为1064bp(如图5)。
本发明还提供一种调控花生开花习性基因AhFH1的启动子序列在作物遗传改良中的应用,优选在改良花生的开花习性及与其相关的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状中的应用;所述基因AhFH1/Ahfh1的启动子序列有两种主要分型,分别来自Tifrunner和狮头企,分别如SEQ ID NO:13-14所示,同时提供启动子克隆引物对为FH1p-F/R,其核苷酸序列为SEQ ID NO:15-16。利用该引物对可以对基因AhFH1的启动子进行克隆。比较Tifrunner和狮头企,后者主要存在一个214bp的***(将其命名为分子标记InDel-214bp),通过琼脂糖电泳可以检测这一差异(如图6)。代表品种可以扩增出三种带型,除了单条短带型的Tifrunner型和单条长带型的狮头企型,还有长短带都有的双带型Florunner,双带型为异源四倍体花生的A和B亚基因的两个亚基因组同源基因分别为无214bp***和有214bp***。分子标记InDel-214bp可以用于对双亲杂交后代的AhFH1基因位点的分子标记辅助选择。
本发明还提供了一种过表达重组构建体:利用烟草花叶病毒的35S启动子的过表达载体p35S::AhFH1,其载体骨架为植物过表达载体PHB,其含有所述花生开花习性基因AhFH1相关核苷酸序列,该过表达载体构建所需的引物对为OE-FH1-F和OE-FH1-R,其序列如SEQ ID NO:17-18所示;利用该引物对在交替开花花生的cDNA中或含有该基因完整编码框的质粒中扩增获得基因AhFH1,将扩增产物通过酶切连接或重组连接入过表达载体PHB(如图7)或其它植物过表达载体,构建成为一种过表达转基因载体p35S::AhFH1(如图7A)。将该过表达载体转化入连续开花型花生可以提高花生的分枝数目,进而影响与其相关的其他性状。
本发明还提供一种互补表达重组构建体:以上述构建的过表达转基因载体p35S::AhFH1为基础,利用基因AhFH1的自身启动子构建互补表达转基因载体:pFH1::AhFH1,其含有所述花生开花习性基因AhFH1相关核苷酸序列。所述互补表达载体构建所需的引物对为FH1pro-F/R,其序列如SEQ ID NO:19-20所示,上游引物FH1pro-F的EcoR I酶切位点为“gaattc”,下游引物FH1pro-R的Pst I酶切位点为“ctgcag”。利用该引物对交替开花花生品种的DNA进行启动子克隆扩增,将连入T载体的扩增产物或扩增产物直接通过EcoR I和PstI双酶切回收后的目的片段连接入利用同样酶切线性化的过表达转基因载体p35S::AhFH1回收的大片段,构建成为一种互补表达转基因载体pFH1::AhFH1(如图7B)。另外互补表达载体的构建也可以利用适当的引物直接扩增交替开花品种中功能性AhFH1包含启动子和编码区在内的基因组全长连入适当的植物转基因载体,此处不再赘述。将该互补表达载体转化入连续开花型花生可以将其改变为交替开花型花生,提高分枝数目,进而影响与其相关的其他性状。
本发明还提供一种基因编辑载体构建体,其含有本发明所述的AhFH1或等位基因Ahfh1的部分核苷酸序列,所述载体是基因编辑载体KO-AhFH1。用于构建基因AhFH1基因编辑的目标靶点的序列优选有两种,分别为:sgRNA1和sgRNA2,其序列如SEQ ID NO:21-22所示;将该两个片段分别连接进入CRISPR/Cas9载体BGK041(图8)的sgRNA区分别构建成针对目标基因AhFH1的基因编辑敲除载体KO-AhFH1。将该基因编辑载体转化入交替开花型花生品种可以将其AhFH1基因实现基因编辑而改变,进而挑选出去功能性编辑后代,而实现交替开花型花生转变为连续开花型,降低其分枝数目、增加其开花数目、荚果数目以及其相关的其他性状。所述sgRNA1和sgRNA2为优选靶标序列,根据不同的CRISPR/Cas9载体***或编辑效率,靶标序列可以不同。
本发明所述的花生开花习性基因AhFH1及其等位变异直接来源于花生,也可以来源于大豆、油菜、棉花、水稻、玉米、小麦等农作物的相似度足够高的同源基因。
本发明还提供改良花生开花习性性状的方法,该方法包括制备含有上述中AhFH1或等位基因Ahfh1相关核苷酸序列构建体的花生植株。
本发明具有以下有益效果:
本发明所提供的花生开花习性基因AhFH1的克隆与应用,具有以下有益效果:
(1)本发明提供的花生开花习性基因AhFH1及其等位变异,为探索花生开花习性基因AhFH1调控花生开花习性的分子机制和初步构建其参与开花分枝调控的分子网络,以及研究该基因功能在作物间的演化规律等提供重要的参考。
(2)本发明提供的花生开花习性基因AhFH1及其等位变异基因间的差异,可以开发为功能性分子标记,用于作物分子标记辅助选择育种,优选在改良花生的开花习性及与其相关的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状中起到关键性的作用。
(3)本发明提供的花生开花习性基因AhFH1的基因序列和氨基酸或多肽或蛋白质在作物遗传改良中,优选在改良花生的开花习性及与其相关的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状中起到关键性的作用。
(4)本发明提供的花生开花习性基因AhFH1的过表达载体、互补表达载体和基因编辑载体及含有所述载体的植株,优选在改良花生的开花习性及与其相关的分枝数目、荚果数目、荚果集中程度、成熟一致性和荚果产量等性状中起到关键性的作用。
附图说明:
图1为本发明涉及的花生开花习性模式图,A为连续开花型,B为交替开花型。
图2为本发明涉及的花生开花习性基因AhFH1的图位克隆过程图。
图3为本发明涉及的花生开花***基因全长的克隆电泳图(引物对FH1g-F/R)。
图4为本发明涉及的花生开花习性基因AhFH1的cDNA克隆电泳图(引物对FH1cd-F/R)。
图5为本发明涉及的鉴别AhFH1和Ahfh1-1两种等位变异的功能性分子标记InDel-1492bp的电泳图(引物对InDel-1492bp-F/R)。
图6为本发明涉及的花生开花习性基因AhFH1的两种启动子在基因组中克隆电泳图(引物对FH1p-F/R)。
图7为本发明涉及的构建体p35S::AhFH1和pFH1::AhFH1的结构图。
图8为本发明涉及的基因编辑构建体KO-AhFH1的结构图。
具体实施方式:
下面通过实施例结合附图对本发明技术做进一步说明。范例中所用的DNA提取、RNA提取、RNA反转录为cDNA、PCR扩增、酶切连接等分子生物学手段不做特殊说明均为常规分子生物学实验手段,可以查阅《分子生物学实验指南》。
实施例1:花生开花习性基因AhFH1的图位克隆
(1)花生品种材料和杂交群体的构建:268份花生品种种质资源由青岛农业大学花生中心保藏。从中挑选交替开花型栽培花生Florunner与连续开花型栽培花生平度9616杂交,种植杂交种F1,自交获得F2分离群体,连续多代株系自交繁育,最终形成F6代重组自交系PF-F6。交替开花型栽培花生小红毛与连续开花型栽培花生河南南阳杂交,种植杂交种F1,自交获得F2分离群体,连续多代株系自交繁育,最终形成F7代重组自交系HN-F7。
(2)植物DNA的提取:采用改良的CTAB法提取植物DNA。
(3)植物RNA的提取及反转录:采用Takara的RNA Extraction Kit提取RNA,反转录为cDNA采用的是Takara的PrimeScriptTM RT reagent Kit。
(4)利用Advanced-BSR-Seq(Advanced Bulked Segregation RNA sequencing)的方法初定位花生开花习性基因AhFH1(引用专利“一种同时定位两个性状相关基因的方法CN110675915A”):
对平度9616和Florunner以及60个个体(30个交替开花型和30个连续开花型)分别进行转录组测序,获得62个样品的转录组测序数据。通过转录组数据表与栽培品种Tifrunner参考基因组序列比对筛选SNP结果,最终得到高质量的可信的SNP位点12,421个。对高质量的SNP进行交替开花池和连续开花池间的△SNP-index分析,将开花习性基因初定位在第12号染色体末端,即第12号染色体117 682 534bp到119 846 824bp之间,总长度约为2.16M(Tifrunner参考基因组,第一版)。
(5)花生开花习性基因AhFH1的精细定位和候选基因预测:
利用连续开花花生品种平度9616和交替开花花生品种Florunner构建的重组自交系的全群体,采用连锁定位的方式将控制花生开花习性的基因定位在第12染色体的末端InDel标记P-21和P-29之间(长度约0.89Mb)。进一步,针对连锁定位验证的标记P-21和P-29间筛选获得的25个重组个体(群体由445个株系构成),继续用内部的InDel标记进行基因型鉴定,结合表型分析将目标位点缩小在InDel标记P-21与SR-4之间的446kb区间内。根据亲本基因组重测序数据在该区间内设计了多个测序片段进行双亲测序,获取了亲本间的2个SNP标记,对内部的9个重组个体进行测序和表型对照,最终将这一位点缩小在P-21与SNP-6之间的387KB的区间内(如图2,标记的引物见表1)。在这个387KB区间里,参考基因组共预测注释有约44个基因,通过生物信息学分析,将区间内一个PEBP/CET基因家族(成花素FT基因家族)的基因Arahy.BBG51B初步选定为花生开花习性基因AhFH1的候选基因。
表1精细定位的标记的引物信息
(6)花生开花习性基因AhFH1的克隆及关联验证:
通过精细定位将Arahy.BBG51B初步选定为花生开花***的克隆引物FH1g-F/R(SEQ ID NO:4-5),使用该引物对以交替开花花生的基因组DNA为模板,通过PCR扩增克隆所述候选基因的基因组完整序列(如图3)。根据参考序列设计了从cDNA中克隆基因AhFH1的引物对FH1cd-F/R,其序列如SEQ ID NO:6-7所示,使用该引物对以交替开花花生的侧枝茎端或叶片组织的cDNA为模板,通过PCR扩增克隆所述候选基因的完整编码框(如图4)。本实施例涉及的代表性的栽培品种为小红毛、河南南阳、Florunner、平度9616、四粒红、鲁花11、马渐渐103、龙花生559、Tifrunner和狮头企。由于在基因全长的扩增中(引物对FH1g-F/R),确实存在代表性的栽培品种未克隆出基因组水平的基因全长,因此对候选基因下游继续设计跨度更大的引物对,其中引物对可以扩增出较小的片段对应的等位变异Ahfh1-1(SEQ ID NO:8),对扩增片段进行测序发现其存在一个1492bp的缺失,将这一引物对命名为InDel-1492bp-F/R,其序列如SEQ ID NO:9-10所示,可以直接鉴别AhFH1和Ahfh1-1两种等位变异,对应等位变异AhFH1扩增产物为2556bp,对应等位变异Ahfh1-1的扩增产物为1064bp,可以通过琼脂糖电泳检测其差异(如图5)。利用该分子标记可以在等位差异为AhFH1和Ahfh1-1的品种间杂交育种中,进行开花习性等位变异的分子标记辅助选择,或在种质资源中进行等位变异AhFH1和Ahfh1-1的鉴别。
根据参考序列设计针对启动子的克隆引物FH1p-F/R(SEQ ID NO:15-16),在代表品种中进行候选基因AhFH1启动子的克隆,并对目的条带进行测序比较分析。通过克隆和测序比较分析发现候选基因AhFH1的启动子序列有两种主要类型,分别来自Tifrunner和狮头企(SEQ ID NO:13-14),但在种质中的克隆条带有三种类型,除了单一的Tifrunner型短带和狮头企型的长带,还存在长短两条带的双带型(如图6),结合后期研究结果分析发现双带型为异源四倍体花生A和B两套亚基因中的两种启动子类型均存在。
通过对启动子区和基因末端的克隆发现,亲本小红毛(交替开花型)的A和B基因组上的序列是完整并且完全一致的,不存在启动子的***和基因末端的缺失。平度9616(连续开花型)和河南南阳(连续开花型)的A基因组和B基因组上该基因的序列也完全一致,但其启动子存在214bp的***和基因末端存在1492bp的缺失。然而亲本Florunner(交替开花型)在启动子和基因末端扩增中均出现两个条带,经测序分析发现两个条带都是目的条带,分析为A基因组和B基因组上存在等位差异,在A基因组上存在启动子的***和基因末端的缺失,在B基因组上不存在启动子的***和基因末端的缺失。由此可见:平度9616和Florunner之间B基因组上存在差异。这一结果,也验证了小红毛和河南南阳构建的RIL群体开花***度9616和Florunner构建的RIL群体的开花***的基因编码区的克隆和测序,发现该类种质的AhFH1基因的第4外显子上编码的cDNA的+335bp位置的碱基C缺失,导致提前形成终止子,翻译的蛋白缺失63个氨基酸,从而影响花生的开花习性,即为等位变异Ahfh1-2(SEQ ID NO:11)。
结合启动子区的***与否(214bp)和基因末端的缺失与否(1492bp)以及第4外显子上编码的cDNA的+335bp位置的碱基C缺失,这三个位点对268份种质资源进行关联分析发现268份种质资源表型和AhFH1的去功能与否的吻合度达到了百分之百,完整功能的AhFH1对应交替开花,去功能性Ahfh1-1或Ahfh1-2对应连续开花,A和B亚基因组杂合对应交替开花。至此,我们将这一候选基因Arahy.BBG51B确定为花生开花习性基因AhFH1(FloweringHabit 1 of Arachis hypogaea L.),基因AhFH1存在一种完整功能性的等位变异AhFH1和至少两种去功能性等位变异Ahfh1-1和Ahfh1-2。
通过分析交替开花型测序品种Tifrunner的参考序列,发现其不同套染色体的同源染色体A02和B02在这一候选基因的上下游约500kb的范围内几乎是完全相同的,可能是A、B亚基因组间易位导致的,同时对A02的基因组注释的Arahy.DYRS20与Arahy.BBG51B完全相同,故本实施例所述AhFH1包含有两个位点:A02染色体的Arahy.DYRS20(将其命名为AhFH1A)与B02染色体的Arahy.BBG51B(将其命名为AhFH1B)。结合基因的克隆和关联验证分析表明:花生开花习性基因AhFH1在A和B两套亚基因组构成的异源四倍体栽培花生中理论是存在四种基因型:AhFH1A/AhFH1B、Ahfh1a/Ahfh1b、Ahfh1a/AhFH1B和AhFH1A/Ahfh1b,而通常在同一个品种中,A完全等于B,a完全等于b,所以实际可以简单地将其分为三种基因型:AhFH1/AhFH1、Ahfh1/Ahfh1和AhFH1/Ahfh1,其中AhFH1/AhFH1和AhFH1/Ahfh1为交替开花基因型,只有Ahfh1/Ahfh1为连续开花型。对于交替型的两个亲本杂交分离出现连续开花型,理论上是Ahfh1a/AhFH1B和AhFH1A/Ahfh1b两种基因型品种杂交,后代出现重组型Ahfh1a/Ahfh1b,对应的表型为连续开花。
实施例2:花生开花习性基因AhFH1的过表达转基因应用
本实施例利用烟草花叶病毒的35S作为启动子构建过表达转基因载体p35S::AhFH1,通过花粉管导入法将花生开花***板,挑取单克隆通过PCR检测,将阳性克隆送去青岛擎科梓熙生物技术有限公司测序,选择测序正确的菌株摇菌,提取带有目的片段的质粒,即为AhFH1过表达转基因载体:p35S::AhFH1,其结构如图7A所示。将AhFH1过表达载体转化农杆菌GV3101感受态细胞,涂布卡那霉素和利福平双抗的YEB平板,挑取单克隆后通过PCR检测阳性后作转基因菌株备用。将该过表达转基因载体转化入连续开花型花生可以提高分枝数目,进而影响与其相关的其他性状。
实施例3:花生开花习性基因AhFH1自身启动子的互补表达应用
该互补表达转基因载体构建所需的引物对为FH1pro-F和FH1pro-R,其序列如SEQID NO:19-20所示,利用该引物对在交替开花花生的DNA模板中进行克隆扩增,将连入T载体的扩增产物或扩增产物直接通过EcoR I和Pst I双酶切回收后的目的片段连接入利用同样酶切线性化的过表达转基因载体p35S::AhFH1,构建成为一种互补表达转基因载体pFH1::AhFH1(如图7B)。
FH1pro-F:5`-CGGAATTCACGAAATCTCAACTTGTTTACGT-3`(SEQ ID NO:18)
FH1pro-R:5`-AACTGCAGTGTTAAAGAGAATGAAAGAGAA-3`(SEQ ID NO:19);(FH1pro引物:上游AhFH1pro-F的EcoR I酶切位点为“GAATTC”,下游FH1pro-R的Pst I酶切位点为“CTGCAG”)。
互补表达转基因载体的构建也可以利用适当的引物直接扩增交替开花品种中功能性AhFH1包含启动子和编码区在内的基因组全长连入适当的植物转基因载体,此处不再赘述。
以花生开花***双抗的YEB平板,挑取单克隆后通过PCR检测阳性后作转基因菌株备用。将该互补表达载体转化入连续开花型花生可以将其改变为交替开花型花生,提高分枝数目,进而影响与其相关的其他性状。
实施例4:花生开花习性基因AhFH1的基因编辑的敲除应用
本实施例利用CRISPR/Cas9体系进行基因编辑敲除,具体操作步骤包括:在线设计并生成sgRNA靶点序列(http://www.biogle.cn/index/excrispr),选择两个得分最高的靶位点sgRNA1和sgRNA2(SEQ ID NO:21-22),将生成的sgRNA序列交由青岛擎科梓熙生物技术有限公司合成互补的两条单链Oligo,将合成的Oligo加水溶解至10μM,在200ul PCR管中加入18μl Buffer Anneal、1μl Up Oligo和1μl Low Oligo混合后,95℃加热3分钟,然后以约0.2℃/秒缓慢降至20℃,制备Oligo二聚体(具体参照百格载体说明书);将Oligo二聚体通过连接酶连接至线性化的CRISPR/Cas9载体即为KO-AhFH1载体;在200ul PCR管中加入2ulKO-AhFH1载体、1ul Oligo二聚体、1μl Enzyme Mix和16ul ddH2O,混匀后室温(20℃)反应1小时,将连接产物热激法分别转化大肠感菌DH5a感受态细胞,涂布卡那霉素抗性的LB平板,挑取单克隆,通过PCR检测,将阳性克隆送去青岛擎科梓熙生物技术有限公司测序,选择测序正确的菌株摇菌,提取质粒,即为AhFH1敲除质粒:KO-AhFH1-1/2。将AhFH1基因敲除质粒KO-AhFH1-1/2转化农杆菌感受态细胞,涂布卡那霉素和利福平双抗的YEB平板,挑取单克隆后通过PCR检测阳性,选择阳性克隆转化交替开花型花生(如小红毛或209小花生);所述CRISPR/Cas9载体选用BGK041(如图8),该载体采用大豆U6启动子驱动sgRNA序列,能够高效的用于双子叶植物,采用加强型CaMV 35S启动子高效表达Cas9蛋白。将该基因编辑载体转化入交替开花型花生品种可以将其AhFH1基因实现基因编辑而改变,进而挑选出去功能性编辑后代,而实现交替开花型花生转变为连续开花型,降低其分枝数目、增加其开花数目、荚果数目以及其相关的其他性状。
基因编辑所用的CRISPR/Cas9载体BGK041骨架购买自百格基因公司(http://www.biogle.cn/index/excrispr),仅用于范例说明,也可以采用其它植物CRISPR/Cas9基因编辑载体或其他单碱基编辑载体。
序列表
<110> 青岛农业大学
<120> 花生开花习性基因AhFH1及其等位变异的克隆与应用
<130> 20200703
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3812
<212> DNA
<213> 花生(Arachis hypogaea L.)
<400> 1
tggtcctgaa attaaaacat tatcgtttaa tttatacttg tgtcattccc tccaagactt 60
ctttcctcta tttatttatt ttttgaatat aaagattaaa gaacatgagc accacaaact 120
ttttaaagat aagaaaagaa aaataaaaaa taaaagtgac aagtgacctt taagagtatt 180
ataaatatgg atgtagagat cacactccca ttatacccaa caacaaacaa aattcacttc 240
atattcttct tcttctcttt cattctcttt aacaatggca aagatgtcat cttcagatcc 300
tttagttctt ggaagagtgg ttggagatgt catccactct ttcaatccaa gtgttcaaat 360
gtctgtcact tacaacacca aacaagtctt caatggccat gagttcttcc cttctgctct 420
taccactagg cctaaggttg ccattgatgg tggtgacatg aggacttttt acacactggt 480
aataatttca ttattcatac atatatatat atattatttg ttataataga agagaggcaa 540
aaaataatta ataaataata ttatggtaga tattaaaaaa aaaataaggg tgatcattgt 600
tgatgattgt gagtttgctt ttttttttcc tatttaattt tacactagat caattttact 660
tacaagattt cactactatt taatttgttg atcctttttc tctcccaaaa aagggggaaa 720
aacaaaaccc taggggttag ttcttacaaa atttggagct atttgagaaa aattaatgta 780
tcctcttgtt tcttttgtag atcatgacag atcctgatgt tcctggccca agtgatcctt 840
atctgagaga acatttgcac tggtaacttg atatatatga ttgaatttga acctaggttt 900
tttatttttt ttttatttta tttttttcta gtaaaattag gtttaattat ataactttca 960
tgtaatacaa aagaaaaatg tctgtttcat catatatact aaaagttgac cttcaaaaac 1020
aaattatgag aagagaaata gtgacaataa tcttatgatt aatacatgaa ctttggtaaa 1080
gttatactaa accttttttt ggctctcaaa tctaactagt gtatttagaa cttattagcc 1140
ttttcacttt tgaatacaaa ttttcttaat ttaattaaag cattagctat agataaatcc 1200
ccaatatgtt taatttatcc actaaaaata gtgatatcac atgaactatc ttataattaa 1260
gaagaagcct ttttcccctc tcttacacac acactctctt ttttttcctc ctaatattca 1320
cattttaaac aaatactaat ataattcttg tacaattatt gtaaatacat atacttggta 1380
tattacctct aacatatata tctatttata gcaattatac aaaaaattat acacaataat 1440
taattattta tataacattt ttgcacattt taatacacaa aaaataatta agtgtagata 1500
gagaggcttc tgatctaaca gtctactctt aattggttgc ttttgcagga tggttacaga 1560
cattcctggc acaacagatg ccacatttgg taggtttcat tgattcatag atctaattaa 1620
aatacaccat atatgtgtta ttctgtgtta ttcatttcta tatataattc aaattgacct 1680
accttattat tattaagtga aaatgtttta tacattatat agactaccca actatcaaat 1740
gactatacta attttttttt tatgtatctt tcaaaaataa aatagttaca ccaaatattt 1800
gcatgaactg aagtctagaa tttagttcat attgtactat attatgaata caattactaa 1860
aattatctac taataaaatg acaaagattc acatgtagtt atttttatgt caattatagt 1920
taatagttaa aaatcgttaa ataataattt agttaaatta atggttctta aatattaaca 1980
tcatgtgagt ttttaccgga taaaatatat atatttatat ataaatacat agtgactgat 2040
tttaataatt aatatatgtc tgataagtaa taaaaaagat aaatgaataa tagtaacaat 2100
tttgtttgat gtaaaatgga agggaaagag atagtgagct atgaaagccc taagccacag 2160
atagggatac acaggtatgt gtttgtgttg ttgaagcaaa agagaaggca gagtgtaacc 2220
ccaccaagtt caagggatca cttcaacact atcaacttct ctgctcactc tgacctttct 2280
cttcctgttg ctgctgtcta cttcaatgct cagagagaaa ccgccgctag aagacgctaa 2340
ttcatatata tgtagctgca tgcacaccta tatatatata tatttattat tagtaaataa 2400
agcaagaagc atgcgagcaa ggttaggtta tggctatata tcatcatcat catcatgaag 2460
aatgaaaagg gtatggcacg tggcttcttc tcataggtgg tgttaatctt ggatatatag 2520
tttaggtgtc acttattatt atatcatgta tgtggctaat taatgtattg tgttagttgt 2580
atgtgttgtt tgtgagttat gtaatataat gttatgaact tgagaattaa gaataaataa 2640
atatcttttt attactacag taacctagtt taatttccct atatatatat atatatatat 2700
atatatatat atatatatat atatatatat atatatatat atatatatat atatataaca 2760
ataaaaaata gtcatgttta caagccaaga aatgatagaa attaataaca ataataatga 2820
acttattaga aaatatttaa gatttgatcg atcatttatt catgaaaaaa tataggaaac 2880
taagtttcag ataattaata ttgtttaatg ttttttattg taaaattttt tctttaacct 2940
ttattttgaa aaatttagga tttagaatta aaatataaca tataaaatta aggatttaaa 3000
atttaaaatt aaaaaaatta actgatcttg gctaaaaaaa ttagctcctt agttgaactc 3060
attcatgcat cctaattgat gcttttagtt tcgtttattg ttaggaatga aataaataga 3120
cataggtgtg tttgcaatta ttataaccaa ttttctaatt ttcaagctta aaataatata 3180
tatatttttt tcatataatc acagatgttc gtgtatcatt taactgccat aactttttag 3240
tttcaacttt cagcattcaa attaagccaa aatgtaatat gcattagtta ggttattagt 3300
gtgagagaaa actcaaaatt tgagttataa taataataac aattgataaa tcataaagtt 3360
caatctgttg ttaaagaaaa ttcagcaaca ctcatatata agggtatgtt atgtattata 3420
aaataaatca ttttggaatt gttatatata ttaattaata ttaatacaaa agatcgaata 3480
catttttatc aaaaagttca atttctgttt atttttccga aagatgatat gacttctaac 3540
aaaaataaat cacaaaggct cttaattatt cactttttga gacaatttga tcttcttctt 3600
tttttttttt tacctaaaac aatgttatat tgaggaattg aatagtttgt attttaataa 3660
aaacccgatg taatttggaa ttaaaaatat cggggatgtc ttaaacaagg taaataatta 3720
aaaaaaaatt cagtttattt aacaaaaata aatattaatg attattttat ttattttaaa 3780
tttataagat taaactatct gatattaaaa at 3812
<210> 2
<211> 1116
<212> DNA
<213> 花生(Arachis hypogaea L.)
<400> 2
tggtcctgaa attaaaacat tatcgtttaa tttatacttg tgtcattccc tccaagactt 60
ctttcctcta tttatttatt ttttgaatat aaagattaaa gaacatgagc accacaaact 120
ttttaaagat aagaaaagaa aaataaaaaa taaaagtgac aagtgacctt taagagtatt 180
ataaatatgg atgtagagat cacactccca ttatacccaa caacaaacaa aattcacttc 240
atattcttct tcttctcttt cattctcttt aacaatggca aagatgtcat cttcagatcc 300
tttagttctt ggaagagtgg ttggagatgt catccactct ttcaatccaa gtgttcaaat 360
gtctgtcact tacaacacca aacaagtctt caatggccat gagttcttcc cttctgctct 420
taccactagg cctaaggttg ccattgatgg tggtgacatg aggacttttt acacactgat 480
catgacagat cctgatgttc ctggcccaag tgatccttat ctgagagaac atttgcactg 540
gatggttaca gacattcctg gcacaacaga tgccacattt gggaaagaga tagtgagcta 600
tgaaagccct aagccacaga tagggataca caggtatgtg tttgtgttgt tgaagcaaaa 660
gagaaggcag agtgtaaccc caccaagttc aagggatcac ttcaacacta tcaacttctc 720
tgctcactct gacctttctc ttcctgttgc tgctgtctac ttcaatgctc agagagaaac 780
cgccgctaga agacgctaat tcatatatat gtagctgcat gcacacctat atatatatat 840
atttattatt agtaaataaa gcaagaagca tgcgagcaag gttaggttat ggctatatat 900
catcatcatc atcatgaaga atgaaaaggg tatggcacgt ggcttcttct cataggtggt 960
gttaatcttg gatatatagt ttaggtgtca cttattatta tatcatgtat gtggctaatt 1020
aatgtattgt gttagttgta tgtgttgttt gtgagttatg taatataatg ttatgaactt 1080
gagaattaag aataaataaa tatcttttta ttacta 1116
<210> 3
<211> 174
<212> PRT
<213> 花生(Arachis hypogaea L.)
<400> 3
Met Ala Lys Met Ser Ser Ser Asp Pro Leu Val Leu Gly Arg Val Val
1 5 10 15
Gly Asp Val Ile His Ser Phe Asn Pro Ser Val Gln Met Ser Val Thr
20 25 30
Tyr Asn Thr Lys Gln Val Phe Asn Gly His Glu Phe Phe Pro Ser Ala
35 40 45
Leu Thr Thr Arg Pro Lys Val Ala Ile Asp Gly Gly Asp Met Arg Thr
50 55 60
Phe Tyr Thr Leu Ile Met Thr Asp Pro Asp Val Pro Gly Pro Ser Asp
65 70 75 80
Pro Tyr Leu Arg Glu His Leu His Trp Met Val Thr Asp Ile Pro Gly
85 90 95
Thr Thr Asp Ala Thr Phe Gly Lys Glu Ile Val Ser Tyr Glu Ser Pro
100 105 110
Lys Pro Gln Ile Gly Ile His Arg Tyr Val Phe Val Leu Leu Lys Gln
115 120 125
Lys Arg Arg Gln Ser Val Thr Pro Pro Ser Ser Arg Asp His Phe Asn
130 135 140
Thr Ile Asn Phe Ser Ala His Ser Asp Leu Ser Leu Pro Val Ala Ala
145 150 155 160
Val Tyr Phe Asn Ala Gln Arg Glu Thr Ala Ala Arg Arg Arg
165 170
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggatgtag agatcacact cccat 25
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tagccataac ctaaccttgc 20
<210> 6
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aacaaaattc acttcatatt cttct 25
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cataacctaa ccttgctcgc 20
<210> 8
<211> 2666
<212> DNA
<213> 花生(Arachis hypogaea L.)
<400> 8
tggtcctgaa attaaaacat tatcgtttaa tttatacttg tgtcattccc tccaagactt 60
ctttcctcta tttatttatt ttttgaatat aaagattaaa gaacatgagc accacaaact 120
ttttaaagat aagaaaagaa aaataaaaaa taaaagtgac aagtgacctt taagagtatt 180
ataaataggt ggatactaca atgaagatgg cataattgtc ttcatatgag tatatttctt 240
tttgaccttt ggatgatgga ttgtatggtt agattttgat atttataaat gtgttgtttt 300
tgtttaaagt gtggctaaat aaataaacca cacttttaac taaagcatct tcatgagaag 360
atatttttgc catcttcatt gtagtatcca ccaaataaat atggatgtag agatcacact 420
cccattatac ccaacaacaa acaaaattca cttcatattc ttcttcttct ctttcattct 480
ctttaacaat ggcaaagatg tcatcttcag atcctttagt tcttggaaga gtggttggag 540
atgtcatcca ctctttcaat ccaagtgttc aaatgtctgt cacttacaac accaaacaag 600
tcttcaatgg ccatgagttc ttcccttctg ctcttaccac taggcctaag gttgccattg 660
atggtggtga catgaggact ttttacacac tggtaataat ttcattattc atacatatat 720
atatatatta tttgttataa tagaagagag gcaaaaaata attaataaat aatattatgg 780
tagatattaa aaaaaaaata agggtgatca ttgttgatga ttgtgagttt gctttttttt 840
ttcctattta attttacact agatcaattt tacttacaag atttcactac tatttaattt 900
gttgatcctt tttctctccc aaaaaagggg gaaaaacaaa accctagggg ttagttctta 960
caaaatttgg agctatttga gaaaaattaa tgtatcctct tgtttctttt gtagatcatg 1020
acagatcctg atgttcctgg cccaagtgat ccttatctga gagaacattt gcactggtaa 1080
cttgatatat atgattgaat ttgaacctag gttttttatt ttttttttat tttatttttt 1140
tctagtaaaa ttaggtttaa ttatataact ttcatgtaat acaaaagaaa aatgtctgtt 1200
tcatcatata tactaaaagt tgaccttcaa aaacaaatta tgagaagaga aatagtgaca 1260
ataatcttat gattaataca tgaactttgg taaagttata ctaaaccttt ttttggctct 1320
caaatctaac tagtgtattt agaacttatt agccttttca cttttgaata caaattttct 1380
taatttaatt aaagcattag ctatagataa atccccaata tgtttaattt atccactaaa 1440
aatagtgata tcacatgaac tatcttataa ttaagaagaa gcctttttcc cctctcttac 1500
acacacactc tctttttttt cctcctaata ttcacatttt aaacaaatac taatataatt 1560
cttgtacaat tattgtaaat acatatactt ggtatattac ctctaacata tatatctatt 1620
tatagcaatt atacaaaaaa ttatacacaa taattaatta tttatataac atttttgcac 1680
attttaatac acaaaaaata attaagtgta gatagagagg cttctgatct aacagtctac 1740
tcttaattgg ttgcttttgc aggatggtta cagacattcc tggcacaaca gatgccacat 1800
ttggtaggtt tcattgattc atagatctaa ttaaaataca ccatatatgt gttattctgt 1860
gttattcatt tctatatata attcaaattg acctacctta ttattattaa gtgaaaatgt 1920
tttatacatt atatagacta cccaactatc aaatgactat actaattttt tttttatgta 1980
tctttcaaaa ataaaatagt tacaccaaat atttgcatga actgaagtct agaatttagt 2040
tcatattgta ctatattatg aatacaatta ctaaaattat ctactaataa aatgacaaag 2100
attcacatgt agttattttt atgtcaatta tagttaatag ttaaaaatcg ttaaataata 2160
atttagttaa attaatggtt cttaaatatt aacatcatgt gagtttttac cgaataaaat 2220
atatatattt atatataaat acatagtgac tgattttaat aattaatata tgtctgataa 2280
gtaataaaaa agataaatga ataatagtaa caattttgtt tgatgtaaaa tggaagggaa 2340
agagatagtg agctatgaat tgaatagttt gtattttaat aaaaacccga tgtaatttgg 2400
aattaaaaat atcggggatg tcttaaacaa ggtaaataat taaaaaaaaa ttcagtttat 2460
ttaacaaaaa taaatattaa tgattatttt atttatttta aatttataag attaaactat 2520
ctgatattaa aaatttttaa gaataattaa atttgagtaa ttattcatta cttttttctc 2580
ttgtgatgtc aattagtgaa taatgcatga atttatttta aaatacgaga ctataagaaa 2640
aaaaactgaa atagtttatt ttttaa 2666
<210> 9
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aacttattag ccttttcact tttg 24
<210> 10
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ttaattccaa attacatcgg gtt 23
<210> 11
<211> 2654
<212> DNA
<213> 花生(Arachis hypogaea L.)
<400> 11
tggtcctgaa attaaaacat tatcgtttaa tttatacttg tgtcattccc tccaagactt 60
ctttcctcta tttatttatt ttttgaatat aaagattaaa gaacatgagc accacaaact 120
ttttaaagat aagaaaagaa aaataaaaaa taaaagtgac aagtgacctt taagagtatt 180
ataaatatgg atgtagagat cacactccca ttatacccaa caacaaacaa aattcacttc 240
atattcttct tcttctcttt cattctcttt aacaatggca aagatgtcat cttcagatcc 300
tttagttctt ggaagagtgg ttggagatgt catccactct ttcaatccaa gtgttcaaat 360
gtctgtcact tacaacacca aacaagtctt caatggccat gagttcttcc cttctgctct 420
taccactagg cctaaggttg ccattgatgg tggtgacatg aggacttttt acacactggt 480
aataatttca ttattcatac atatatatat atattatttg ttataataga agagaggcaa 540
aaaataatta ataaataata ttatggtaga tattaaaaaa aaaataaggg tgatcattgt 600
tgatgattgt gagtttgctt ttttttttcc tatttaattt tacactagat caattttact 660
tacaagattt cactactatt taatttgttg atcctttttc tctcccaaaa aagggggaaa 720
aacaaaaccc taggggttag ttcttacaaa atttggagct atttgagaaa aattaatgta 780
tcctcttgtt tcttttgtag atcatgacag atcctgatgt tcctggccca agtgatcctt 840
atctgagaga acatttgcac tggtaacttg atatatatga ttgaatttga acctaggttt 900
tttatttttt ttttatttta tttttttcta gaaaaattag gtttaattat ataactttca 960
tgtaatacaa aagaaaaatg tctgtttcat catatatact aaaagttgac cttcaaaaac 1020
aaattatgag aagagaaata gtgacaataa tcttatgatt aatacatgaa ctttggtaaa 1080
gttatactaa accttttttt ggctctcaaa tctaactagt gtatttagaa cttattagcc 1140
ttttcacttt tgaatacaaa ttttcttaat ttaattaaag cattagctat agataaatcc 1200
ccaatatgtt taatttatcc actaaaaata gtgatatcac atgaactatc ttataattaa 1260
gaagaagcct ttttcccctc tcttacacac acactctctt ttttttcctc ctaatattca 1320
cattttaaac aaatactaat ataattcttg tacaattatt gtaaatacat atacttggta 1380
tattacctct aacatatata tctatttata gcaattatac aaaaaattat acacaataat 1440
taattattta tataacattt ttgcacattt taatacacaa aaaataatta agtgtagata 1500
gagaggcttc tgatctaaca gtctactctt aattggttgc ttttgcagga tggttacaga 1560
cattcctggc acaacagatg ccacatttgg taggtttcat tgattcatag atctaattaa 1620
aatacaccat atatgtgtta ttctgtgtta ttcatttcta tatataattc aaattgacct 1680
accttattat tattaagtga aaatgtttta tacattatat agactaccca actatcaaat 1740
gactatacta attttttttt tatgtatctt tcaaaaataa aatagttaca ccaaatattt 1800
gcatgaactg aagtctagaa tttagttcat attgtactat attatgaata caattactaa 1860
aattatctac taataaaatg acaaagattc acatgtagtt atttttatgt caattatagt 1920
taatagttaa aaatcgttaa ataataattt agttaaatta atggttctta aatattaaca 1980
tcatgtgagt ttttaccgga taaaatatat atatttatat ataaatacat agtgactgat 2040
tttaataatt aatatatgtc tgataagtaa taaaaaagat aaatgaataa tagtaacaat 2100
tttgtttgat gtaaaatgga agggaaagag atagtgagct atgaaagcct aagccacaga 2160
tagggataca caggtatgtg tttgtgttgt tgaagcaaaa gagaaggcag agtgtaaccc 2220
caccaagttc aagggatcac ttcaacacta tcaacttctc tgctcactct gacctttctc 2280
ttcctgttgc tgctgtctac ttcaatgctc agagagaaac cgccgctaga agacgctaat 2340
tcatatatat gtagctgcat gcacacctat atatatatat atttattatt agtaaataaa 2400
gcaagaagca tgcgagcaag gttaggttat ggctatatat catcatcatc atcatgaaga 2460
atgaaaaggg tatggcacgt ggcttcttct cataggtggt gttaatcttg gatatatagt 2520
ttaggtgtca cttattatta tatcatgtat gtggctaatt aatgtattgt gttagttgta 2580
tgtgttgttt gtgagttatg taatataatg ttatgaactt gagaattaag aataaataaa 2640
tatcttttta ttac 2654
<210> 12
<211> 1105
<212> DNA
<213> 花生(Arachis hypogaea L.)
<400> 12
attaaaacat tatcgtttaa tttatacttg tgtcattccc tccaagactt ctttcctcta 60
tttatttatt ttttgaatat aaagattaaa gaacatgagc accacaaact ttttaaagat 120
aagaaaagaa aaataaaaaa taaaagtgac aagtgacctt taagagtatt ataaatatgg 180
atgtagagat cacactccca ttatacccaa caacaaacaa aattcacttc atattcttct 240
tcttctcttt cattctcttt aacaatggca aagatgtcat cttcagatcc tttagttctt 300
ggaagagtgg ttggagatgt catccactct ttcaatccaa gtgttcaaat gtctgtcact 360
tacaacacca aacaagtctt caatggccat gagttcttcc cttctgctct taccactagg 420
cctaaggttg ccattgatgg tggtgacatg aggacttttt acacactgat catgacagat 480
cctgatgttc ctggcccaag tgatccttat ctgagagaac atttgcactg gatggttaca 540
gacattcctg gcacaacaga tgccacattt gggaaagaga tagtgagcta tgaaagccta 600
agccacagat agggatacac aggtatgtgt ttgtgttgtt gaagcaaaag agaaggcaga 660
gtgtaacccc accaagttca agggatcact tcaacactat caacttctct gctcactctg 720
acctttctct tcctgttgct gctgtctact tcaatgctca gagagaaacc gccgctagaa 780
gacgctaatt catatatatg tagctgcatg cacacctata tatatatata tttattatta 840
gtaaataaag caagaagcat gcgagcaagg ttaggttatg gctatatatc atcatcatca 900
tcatgaagaa tgaaaagggt atggcacgtg gcttcttctc ataggtggtg ttaatcttgg 960
atatatagtt taggtgtcac ttattattat atcatgtatg tggctaatta atgtattgtg 1020
ttagttgtat gtgttgtttg tgagttatgt aatataatgt tatgaacttg agaattaaga 1080
ataaataaat atctttttat tacta 1105
<210> 13
<211> 2256
<212> DNA
<213> 花生(Arachis hypogaea L.)
<400> 13
aaccaccttc ttgaaaagct ctgttgccta attaattagt cttaattaaa tttcaccatg 60
tcaactaagc atttaaaatt taaaacaatt taaattgatc gaataattaa cttatttatt 120
tatttatgta aatattaaga ttcaaatttt gttttatata tataataatt tattgattta 180
atgacaaatt cttaaataaa atttaaatct atttatgata aattattttt acgataatac 240
tagaaaaaaa acaattagaa catactttat ttaatattta ttaattattg caacaattga 300
taaatactaa ataatataaa tttttactgt atttgactaa ttttttttat tactgaatat 360
ttttgttatt tttacttacg ttaccatagt ttgcatgcgc acacacatgt atatatatgg 420
tgaaaactca agtgaagttg atacctgaga gccgttaaat gatttgactg atttgattag 480
attttcatcc aacaactctt aggtatcaac tttacgtaaa gtcgacttcg cctgagtttt 540
cactcatata tatacttgct aagcattatt aatttttttt catttttata tatatacact 600
ttccatataa ttttgcgcac ttgttttttc ttttttgaca tgatatacat aggtacattt 660
tgagaaaaat aaggagtttt gattagtgta tataaaagag gccttcgtcg tatgcaaaga 720
atatatatat gtgagcgagc caacatgtaa acagtggtga atgacaaact tttttcaaag 780
aaaagagaga ggacccgcat ggcaatagca cgcaaattaa ataatgatgc cttcaattct 840
gatttctgag tacaaaagaa aaattagcca attaataaga accaattcag atggagacaa 900
tattgtacct tgtgagaatt agagggatat gatttatttg cgtcataaat aacaacatct 960
tcaacttata tatattaaag tttatacacg aaatctcaac ttgtttacgt aataatgtat 1020
taattattta tatattttat caaatatatt aaattattaa aatcatttta aacttgaaag 1080
atcacttttg tttgagttat tttaaagaaa gaatgtatct tttattgaaa aattttaaat 1140
aaattaaaat aaaataaaat aacttctatt ttaaaaaatt aaatttaaaa tttaaattca 1200
ttataaaata ataaactcaa caaaaaatca atatttttaa taagtaaaaa gggtcattat 1260
gaaaataata ataaatccta tatgataaaa ttgaagtgtg aaaaaagaaa atctttgaag 1320
aagaagacga tgatgatatc atttaggttg taaagagtaa aaagatgaaa aaagatattt 1380
gtggtggtga cgatgatagc aataataaat taataataat agcaaattaa atagtaactt 1440
aaaaaaaatt tcaaaaaatg aagagataag aaaaaaaaaa cataaaaaat tcagaagggt 1500
tttagtggca ttttatttat aattgatcaa cgtcttatta gttttgtcaa atatattgta 1560
ttttttatga atattacatt agtttaattt attatagtta aatttgtcag aaataaaatt 1620
tttcatggtt aaaacttaga aacggtaatt tatctattta ttcatgcact actaatctac 1680
tagctagtat cctttttagc aaaagtttct tttcttgtag cttcatttgt ttaaatagtt 1740
tgtttttcct atagatagtt ttttttttgt taccatcact agatagagaa aatataaata 1800
ttgtaaacag attaaaaaaa taaatattca aaaaaagcag ataaacaact taaaattatt 1860
attattaaaa ttaataatat aaaaaagtct ttattattat tattattatt attattatta 1920
ttattattat tattattatt attattatta ttattattat tattattatt attattatta 1980
tttggtcctg aaattaaaac attatcgttt aatttatact tgtgtcattc cctccaagac 2040
ttctttcctc tatttattta ttttttgaat ataaagatta aagaacatga gcaccacaaa 2100
ctttttaaag ataagaaaag aaaaataaaa aataaaagtg acaagtgacc tttaagagta 2160
ttataaatat ggatgtagag atcacactcc cattataccc aacaacaaac aaaattcact 2220
tcatattctt cttcttctct ttcattctct ttaaca 2256
<210> 14
<211> 2479
<212> DNA
<213> 花生(Arachis hypogaea L.)
<400> 14
aaccaccttc ttgaaaagct ctgttgccta attaattagt cttaattaaa tttcaccatg 60
tcaactaagc atttaaaatt taaaacaatt taaattgatc gaataattaa cttatttatt 120
tatttatgta aatattaaga ttcaaatttt gttttatata tataataatt tattgattta 180
atgacaaatt cttaaataaa atttaaatct atttatgata aattattttt acgataatac 240
tagaaaaaaa acaattagaa catactttat ttaatattta ttaattattg caacaattga 300
taaatactaa ataatataaa tttttactgt atttgactaa ttttttttat tactgaatat 360
ttttgttatt tttacttacg ttaccatagt ttgcatgcgc acacacatgt atatatatgg 420
tgaaaactca agtgaagttg atacctgaga gccgttaaat gatttgactg atttgattag 480
attttcatcc aacaactctt aggtatcaac tttacgtaaa gtcgacttcg cctgagtttt 540
cactcatata tatacttgct aagcattatt aatttttttt catttttata tatatacact 600
ttccatataa ttttgcgcac ttgttttttc ttttttgaca tgatatacat aggtacattt 660
tgagaaaaat aaggagtttt gattagtgta tataaaagag gccttcgtcg tatgcaaaga 720
atatatatat gtgagcgagc caacatgtaa acagtggtga atgacaaact tttttcaaag 780
aaaagagaga ggacccgcat ggcaatagca cgcaaattaa ataatgatgc cttcaattct 840
gatttctgag tacaaaagaa aaattagcca attaataaga accaattcag atggagacaa 900
tattgtacct tgtgagaatt agagggatat gatttatttg cgtcataaat aacaacatct 960
tcaacttata tatattaaag tttatacacg aaatctcaac ttgtttacgt aataatgtat 1020
taattattta tatattttat caaatatatt aaattattaa aatcatttta aacttgaaag 1080
atcacttttg tttgagttat tttaaagaaa gaatgtatct tttattgaaa aattttaaat 1140
aaattaaaat aaaataaaat aacttctatt ttaaaaaatt aaatttaaaa tttaaattca 1200
ttataaaata ataaactcaa caaaaaatca atatttttaa taagtaaaaa gggtcattat 1260
gaaaataata ataaatccta tatgataaaa ttgaagtgtg aaaaaagaaa atctttgaag 1320
aagaagacga tgatgatatc atttaggttg taaagagtaa aaagatgaaa aaagatattt 1380
gtggtggtga cgatgatagc aataataaat taataataat agcaaattaa atagtaactt 1440
aaaaaaaatt tcaaaaaatg aagagataag aaaaaaaaaa cataaaaaat tcagaagggt 1500
tttagtggca ttttatttat aattgatcaa cgtcttatta gttttgtcaa atatattgta 1560
ttttttatga atattacatt agtttaattt attatagtta aatttgtcag aaataaaatt 1620
tttcatggtt aaaacttaga aacggtaatt tatctattta ttcatgcact actaatctac 1680
tagctagtat cctttttagc aaaagtttct tttcttgtag cttcatttgt ttaaatagtt 1740
tgtttttcct atagatagtt ttttttttgt taccatcact agatagagaa aatataaata 1800
ttgtaaacag attaaaaaaa taaatattca aaaaaagcag ataaacaact taaaattatt 1860
attattaaaa ttaataatat aaaaaagtct ttattattat tattattatt attattatta 1920
ttattattat tattattatt attattatta ttattattat tattattatt attattatta 1980
ttattattat ttggtcctga aattaaaaca ttatcgttta atttatactt gtgtcattcc 2040
ctccaagact tctttcctct atttatttat tttttgaata taaagattaa agaacatgag 2100
caccacaaac tttttaaaga taagaaaaga aaaataaaaa ataaaagtga caagtgacct 2160
ttaagagtat tataaatagg tggatactac aatgaagatg gcataattgt cttcatatga 2220
gtatatttct ttttgacctt tggatgatgg attgtatggt tagattttga tatttataaa 2280
tgtgttgttt ttgtttaaag tgtggctaaa taaataaacc acacttttaa ctaaagcatc 2340
ttcatgagaa gatatttttg ccatcttcat tgtagtatcc accaaataaa tatggatgta 2400
gagatcacac tcccattata cccaacaaca aacaaaattc acttcatatt cttcttcttc 2460
tctttcattc tctttaaca 2479
<210> 15
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
acgaaatctc aacttgttta cgt 23
<210> 16
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tcttccaaga actaaaggat ctg 23
<210> 17
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ctcgagctgc aggagctcat ggcaaagatg tcatcttcag a 41
<210> 18
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cgaacgaaag ctctagactt agcgtcttct agcggcggtt tct 43
<210> 19
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cggaattcac gaaatctcaa cttgtttacg t 31
<210> 20
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aactgcagtg ttaaagagaa tgaaagagaa 30
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ggcctagtgg taagagcaga 20
<210> 22
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tgtaacccca ccaagttcaa 20
Claims (6)
1.一种花生开花***的基因末端存在一个以ATG为起始的+1872bp至+3273bp位置的包含最后一个外显子及3`UTR的1492bp的缺失,其对应花生的开花习性为连续开花型;所述去功能性等位变异Ahfh1-2,其核苷酸序列如SEQ ID NO:11所示,其编码的cDNA序列如SEQ IDNO:12所示,其编码的cDNA的+335bp位置的碱基C缺失,导致cDNA翻译框移码提前形成终止子,翻译的蛋白不完整,其对应花生的开花习性为连续开花型。
2.花生开花***的克隆引物对为FH1g-F/R,其核苷酸序列如SEQ ID NO:4和SEQ ID NO:5所示;基因AhFH1在cDNA水平的克隆引物对为FH1cd-F/R,其核苷酸序列如SEQ ID NO:6和SEQ ID NO:7所示;基因AhFH1启动子的克隆引物对为FH1p-F/R,其核苷酸序列如SEQ ID NO:15和SEQ ID NO:16所示,引物对FH1p-F/R同时可以用于对花生开花习性基因AhFH1启动子的两种不同类型的鉴别。
3.一种区分花生开花习性基因完整功能型AhFH1和去功能性Ahfh1-1的功能性分子标记InDel-1492bp,其特征在于,其对应的引物对InDel-1492bp-F/R的核苷酸序列分别如SEQID NO:9和SEQ ID NO:10所示,结合测序确认AhFH1序列无变异的情况下,标记InDel-1492bp可以通过PCR和电泳检测直接判断两种等位变异。
4.一种含有花生开花习性基因AhFH1的过表达转基因载体构建方法及应用,其特征在于,利用烟草花叶病毒的35S启动子构建过表达转基因载体p35S::AhFH1,其载体骨架为植物过表达载体PHB,其含有所述花生开花习性基因AhFH1相关核苷酸序列,所述过表达载体构建所需的引物对为OE-FH1-F和OE-FH1-R,其序列如SEQ ID NO:17-18所示;利用该引物对在交替开花花生的cDNA中或含有该基因完整编码框的质粒中扩增获得基因AhFH1,将扩增产物通过酶切连接或重组连接入过表达载体PHB或其它植物过表达载体,构建成为一种过表达转基因载体p35S::AhFH1;将该过表达载体转化入连续开花型花生可以提高花生的分枝数目,进而影响与其相关的其他性状。
5.一种含有所述花生开花习性基因AhFH1相关核苷酸序列的互补表达转基因载体构建方法及应用,其特征在于,过表达转基因载体p35S::AhFH1为基础,利用基因AhFH1的自身启动子构建互补表达载体:pFH1::AhFH1,其含有所述花生开花习性基因AhFH1相关核苷酸序列;所述互补表达载体构建所需的引物对为FH1pro-F/R,其序列如SEQ ID NO:19-20所示,上游引物FH1pro-F的EcoR I酶切位点为“gaattc”,下游引物FH1pro-R的Pst I酶切位点为“ctgcag”。利用该引物对交替开花花生品种的DNA进行启动子克隆扩增,将连入T载体的扩增产物或扩增产物直接通过EcoR I和Pst I双酶切回收后的目的片段连接入利用同样酶切线性化的过表达转基因载体p35S::AhFH1回收的大片段,构建成为一种互补表达转基因载体pFH1::AhFH1,将该互补表达载体转化入连续开花型花生可以将其改变为交替开花型花生,提高分枝数目,进而影响与其相关的其他性状。
6.一种含有所述基因AhFH1或等位基因Ahfh1的基因编辑载体构建方法及应用,其特征在于,所述载体是基因编辑载体KO-AhFH1;用于构建基因AhFH1基因编辑的目标靶点的序列优选有两种,分别为:sgRNA1和sgRNA2,其序列如SEQ ID NO:21-22所示;将该两个片段分别连接进入CRISPR/Cas9载体BGK041的sgRNA区分别构建成针对目标基因AhFH1的基因编辑敲除载体KO-AhFH1;将该基因编辑载体转化入交替开花型花生品种可以将其AhFH1基因实现基因编辑而改变,进而挑选出去功能性编辑后代,实现交替开花型花生转变为连续开花型,降低其分枝数目、增加其开花数目、荚果数目以及其相关的其他性状;所述sgRNA1和sgRNA2为优选靶标序列,根据不同的CRISPR/Cas9载体***或编辑效率,靶标序列可以不同。
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CN116286849A (zh) * | 2022-08-17 | 2023-06-23 | 广东省农业科学院作物研究所 | 调控花生含油率的基因AhWRI1及其应用 |
CN116334127A (zh) * | 2023-03-29 | 2023-06-27 | 青岛农业大学 | 花生籽仁可溶性糖含量调控基因AhSS1的克隆方法及应用 |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107613761A (zh) * | 2015-04-20 | 2018-01-19 | 孟山都技术有限公司 | 用于改变开花和植物构造以提高产量潜力的组合物和方法 |
CN110592264A (zh) * | 2019-10-17 | 2019-12-20 | 青岛农业大学 | 花生株型相关基因位点的分子标记方法及其应用 |
CN110592102A (zh) * | 2019-10-18 | 2019-12-20 | 青岛农业大学 | 调控花生侧枝角度、生长习性和株型的基因lba5及其应用 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AUPR087300A0 (en) * | 2000-10-19 | 2000-11-16 | Agresearch Limited | Manipulation of flowering and plant architecture |
CN102021199A (zh) * | 2010-08-26 | 2011-04-20 | 北京农业生物技术研究中心 | 一种调控百合花期的方法 |
CN110675915B (zh) * | 2019-10-24 | 2022-08-16 | 青岛农业大学 | 一种同时定位两个性状相关基因的方法 |
CN111675756B (zh) * | 2020-07-03 | 2022-06-28 | 青岛农业大学 | 花生开花习性基因AhFH1及其等位变异的克隆与应用 |
-
2020
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107613761A (zh) * | 2015-04-20 | 2018-01-19 | 孟山都技术有限公司 | 用于改变开花和植物构造以提高产量潜力的组合物和方法 |
CN110592264A (zh) * | 2019-10-17 | 2019-12-20 | 青岛农业大学 | 花生株型相关基因位点的分子标记方法及其应用 |
CN110592102A (zh) * | 2019-10-18 | 2019-12-20 | 青岛农业大学 | 调控花生侧枝角度、生长习性和株型的基因lba5及其应用 |
Non-Patent Citations (2)
Title |
---|
夏晗: "花生开花相关基因的克隆及表达研究", 《2011年中国作物学会学术年会论文摘要集》 * |
葛姗姗: "花生AhFT基因的克隆与序列分析", 《花生学报》 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022000835A1 (zh) * | 2020-07-03 | 2022-01-06 | 青岛农业大学 | 花生开花习性基因AhFH1及其等位变异的克隆与应用 |
CN113234851A (zh) * | 2021-06-30 | 2021-08-10 | 山东省农业科学院 | 一种与花生分枝角度紧密连锁的分子标记AhyBA1及其应用 |
CN116286849A (zh) * | 2022-08-17 | 2023-06-23 | 广东省农业科学院作物研究所 | 调控花生含油率的基因AhWRI1及其应用 |
CN116286849B (zh) * | 2022-08-17 | 2024-04-12 | 广东省农业科学院作物研究所 | 调控花生含油率的基因AhWRI1及其应用 |
CN116334127A (zh) * | 2023-03-29 | 2023-06-27 | 青岛农业大学 | 花生籽仁可溶性糖含量调控基因AhSS1的克隆方法及应用 |
CN116334127B (zh) * | 2023-03-29 | 2024-01-26 | 青岛农业大学 | 花生籽仁可溶性糖含量调控基因AhSS1的克隆方法及应用 |
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