CN113930431B - SEC12-like蛋白基因CPU1及其在提高大豆磷效率方面的应用 - Google Patents
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Abstract
本发明公开了SEC12‑like蛋白基因CPU1及其在提高大豆磷效率方面的应用,属于生物技术领域。通过全基因组关联分析的正向遗传学方法,鉴定到一个影响大豆磷吸收效率的主效遗传位点,并鉴定到其中的候选基因CPU1。基因CPU1在大豆中存在着自然变异,包括2个等位基因,磷低效等位基因CPU1‑H1和磷高效等位基因CPU1‑ H2。基于大豆整株转化植株的研究显示,抑制等位基因CPU1‑H2的表达显著降低了大豆的磷吸收效率,最终降低了转基因植株的生物量和产量。本发明为包括大豆在内的作物自然变异的遗传基础研究提供新的科学见解,同时为磷高效分子育种提供优异等位基因资源。
Description
技术领域
本发明属于生物技术领域,具体涉及SEC12-like蛋白基因CPU1及其在提高磷效率方面的应用。
背景技术
大豆作为我国重要的粮油饲兼用作物,提供了丰富的蛋白和油分。我国虽然是大豆的起源地,也曾经很长一段时间是世界上最大的大豆生产国、消费国和出口国;但是从1996年以后,我国成为了大豆净进口国,每年需要从美洲进口大量的大豆,粮食安全问题存在严重隐患(石慧等,2018)。同时大豆作为生物固氮量最大的豆科作物,在减肥增效、降低环境污染方面具有突出的表现(李欣欣等,2016)。因此,提高我国大豆的生产能力,在保障粮食安全和可持续生态农业发展方面均具有重要的意义。
磷是植物必需的大量矿质营养元素,对植物正常的生长发育起着至关重要的作用。豆科植物一直以来存在着“以磷增氮”效应:磷素可以促进豆科作物结瘤固氮,从而提高氮效率(陆景陵,1994)。磷素的主要来源是土壤,土壤中的全磷含量较高,但是大部分是难溶性的无机磷和有机磷,难以被植物利用,且磷素在土壤中的移动性较差。在实际的农业生产中,为了获得高产往往要通过大量施肥来补充磷元素,这会带来严重的环境污染问题。因此,如何提高作物自身的磷效率,使作物在减少施肥的条件下获得稳定的产量或在相同施肥的条件下更高产,是当前发展资源节约型、环境友好型生态农业所面临的重要科学难题。
近些年关联分析受到越来越多研究者的关注,至少有两个原因:一是关联分析用到的自然群体经历了长期的重组事件,因此会有较高的作图精度;二是因为自然群体有丰富的遗传变异,有利于解析性状变异的遗传基础,有利于鉴定到优异等位基因 (Yu andBuckler, 2006)。随着大豆参考基因组序列的公布和近年来大豆自然群体重测序工作的完成 (Schmutz et al. 2010, Lam et al. 2010), 在大豆中已经成功开展了全基因组关联分析 (Zhou et al. 2015, Fang et al. 2017)。
然而,在大豆中还鲜有解析大豆田间磷效率自然变异遗传基础的报道,目前还没有通过正向遗传学克隆到大豆磷效率主效基因的报道。
发明内容
针对上述问题,本发明提供一种SEC12-like蛋白基因CPU1及其在提高大豆磷效率方面的应用。发明人利用大豆磷效率核心群体,在田间进行磷效率相关指标的表型鉴定,同时基于二代高通量测序获得高密度分子标记,开展全基因组关联分析,鉴定到了一个控制磷吸收效率的主效遗传位点,并鉴定到候选基因CPU1。
基于CPU1大豆整株转化植株的研究表明,抑制CPU1的表达显著降低大豆磷吸收效率,最终降低了转基因植株的生物量和产量,证实了该基因在磷吸收效率方面的功能。
发明人发现CPU1在大豆自然群体中存在序列变异,其5’UTR的一个碱基替换改变了CPU1的翻译效率,从而影响大豆的磷吸收效率;同时发明人鉴定出CPU1的磷高效等位基因CPU1-H2。
为实现上述目的,本发明采用如下技术方案:
一种SEC12-like蛋白基因CPU1,所述基因CPU1在大豆中存在着自然变异,包括2个等位基因,磷低效等位基因CPU1-H1和磷高效等位基因CPU1-H2;在基因CPU1的5’UTR存在着一个开放阅读框uORF,这个uORF内存在两个SNPs,磷高效等位基因CPU1-H2和磷低效等位基因CPU1-H1位于uORF第20位的碱基分别为A和G;位于uORF第83位的碱基分别为C和A;所述磷高效等位基因CPU1-H2的核苷酸序列如SEQ ID NO.1所示;所述磷低效等位基因CPU1-H1的核苷酸序列如SEQ ID NO.5所示。
上述一种SEC12-like蛋白基因CPU1,其两个等位基因CPU1-H1和CPU1-H2 的cDNA序列相同,如SEQ ID NO.2所示。
上述一种SEC12-like蛋白基因CPU1,其中磷高效等位基因CPU1-H2的uORF的核苷酸序列如SEQ ID NO.3所示。
一种植物表达载体,所述植物表达载体含有上述基因CPU1。
上述植物表达载体,包括重组转化所形成的转基因植物,以及由此产生的外源基因表达产物。
上述SEC12-like蛋白基因CPU1在提高大豆磷吸收效率方面的应用。
进一步的,上述应用中抑制等位基因CPU1-H2的表达能降低大豆的磷吸收效率。
进一步的,上述应用中抑制等位基因CPU1-H2的表达能降低大豆的生物量和产量。
本发明的有益之处在于:本发明提供了一个能够提高大豆磷效率的新基因SEC12-like蛋白基因CPU1。CPU1在大豆自然群体中存在序列变异,其5’UTR的一个碱基替换改变了CPU1的翻译效率,从而影响大豆的磷吸收效率;同时发明人鉴定出CPU1的磷高效等位基因CPU1-H2。此项研究有助于全面解析大豆磷效率的遗传基础,为了解作物自然变异的遗传基础提供新的科学见解,同时为磷高效分子育种提供优异等位基因资源,最终对发展环境友好型资源节约型可持续生态农业具有重要的意义。
附图说明:
图1为大豆磷吸收效率的全基因组关联分析结果。左上方是quantile-quantile图,显示群体结构控制效果;下方是Manhattan图,横纵坐标分别对应着SNP所在的物理位置和P值的负对数值,图中的水平线代表基因组水平的关联分析显著性阈值。
图2为干涉CPU1对大豆转基因植株磷吸收效率、生物量和产量的影响。A为3个独立RNAi干涉家系的CPU1相对表达量;B为RNAi干涉家系和野生型材料在苗期的生长状况;C为RNAi干涉家系和野生型材料在苗期的生物量;D为RNAi干涉家系和野生型材料在苗期的磷吸收量;E为RNAi干涉家系和野生型材料在苗期的总根长;F为RNAi干涉家系和野生型材料在苗期的磷吸收效率;G为RNAi干涉家系和野生型材料在成熟期的生长状况;H为RNAi干涉家系和野生型材料在成熟期的单株荚数;*表示显著水平0.01 < P≤ 0.05 时,差异显著;**表示显著水平0.001 < P ≤ 0.01时,差异显著性介于显著与极显著之间;***表示显著水平P ≤ 0.001 时,差异极显著。
图3为CPU1两种等位基因间氨基酸序列的比较。
图4为CPU1两种等位基因间表达量的比较。
图5为通过重组载体的构建和Western-blot实验确定CPU1功能变异区域。A为不同等位基因的启动子和5’UTR重组载体的示意图;B为分别转入A中6个重组载体的大豆毛根的Western-blot结果;在多重比较中,不同的英文字母之间代表显著性差异(P<0.05)。
图6为通过重组载体的构建和Western-blot实验确定CPU1功能变异位点。A为含有不同基因型的5’UTR的重组载体示意图;B为分别转入(A)中6个重组载体的大豆毛根的Western-blot结果;
在多重比较中,不同的英文字母代表显著性差异(P<0.05)。
具体实施方式
以下结合附图和具体实施例对本发明作具体的介绍。
实施例1、磷吸收效率的遗传定位和候选基因鉴定
本发明利用前期收集的一套大豆磷效率种质资源核心群体(包含274份大豆材料),在广东博罗(东经 113°50′, 北纬 23°07′)的试验田开展田间试验,采用完全随机区组设计(每小区1.5 m2),设4个区组,针对磷效率相关指标进行表型鉴定。
磷含量的测定:磷含量(mg/plant)= 磷浓度(mg/g)× 植株干重(g/plant),其中磷浓度采用比色法测量(Murphy and Riley, 1963)。
总根长的测定:为了获得完整的植株根系,使用铁锹等工具按照40 cm×40 cm 的方形区域(以植株为中心)往下挖,直达主根尖端;将获取的根系带到实验室用水清洗后利用扫描仪扫描,然后使用图像处理软件WinRhizo Pro(Régent Instruments, Québec,Canada)提取总根长(m/plant)。
磷吸收效率的计算:磷吸收效率(mg/m)= 磷含量(mg/plant)÷ 总根长(m/plant)。
将大豆苗期植株(播种后1个月)的地上部和根部在105°C烘箱中杀青30分钟,然后在75°C烘箱中烘至恒重并称重。
本发明基于二代测序平台(Illumina NovaSeq PE150) 对上述大豆磷效率种质资源核心群体进行了全基因组重测序, 共产生了135 亿reads。DNA提取、文库构建和测序均由诺禾致源公司(Novogene Bioinformatics Technology Co., Ltd, China)完成。
重测序数据分析流程如下:使用fastp软件对测序文件进行质控;使用bwa软件将测序reads比对到大豆Williams 82参考基因组(http://plants.ensembl.org/info/website/ftp/index.html);利用SAMtools和Qualimap软件对上述产生的bam文件进行质控;利用GATK软件提取SNP和INDEL变异并对产生的vcf变异文件进行质控;利用Beagle软件完成基因型填充;利用snpEff软件注释SNP和INDEL的变异效应。
本发明基于上述基因型分型结果开展了群体结构分析、主成分分析和系统发生树构建,了解群体材料的亚群分组和亲缘关系;利用VCFtools鉴定亚群间的分化区域;利用PopLDdecay软件对全基因组的LD程度进行评估。本发明基于上述表型数据、基因型数据和亲缘关系矩阵,去除次等位基因频率(MAF)<0.05的SNPs,利用混合线性模型开展了全基因组关联分析,利用GEC软件确定合适的显著性阈值。
图1为大豆磷吸收效率的全基因组关联分析结果。其中:左上方是quantile-quantile图,显示群体结构控制效果;下方是Manhattan图,横纵坐标分别对应着SNP所在的物理位置和P值的负对数值,图中的水平线代表基因组水平的关联分析显著性阈值。结果表明:在20号染色体鉴定到了一个显著的磷吸收效率的关联信号(参见图1),信号对应的区间内存在10个基因,根据这些基因在多个组织中的表达谱信息,锁定到一个在根中特异高表达的基因作为候选基因,命名为CPU1,注释信息显示该基因编码SEC12-like蛋白(鸟苷酸置换因子类似蛋白)。
实施例2、CPU1的克隆和功能验证
根据CPU1基因的cDNA序列(如SEQ ID NO.2所示)设计了特异引物F1/R1,以野生型大豆品种YC04-5根的cDNA样品为模板,扩增出147 bp的片段;使用SwaⅠ+ AscⅠ酶切上述147bp片段获得正向片段,然后将正向片段克隆到pFGC5941载体的SwaⅠ和AscⅠ之间;使用Sma I+ BamH I酶切上述147 bp片段获得反向片段,然后将反向片段克隆到已含有正向片段的pFGC5941载体的Sma I 和BamH I之间得到重组载体,将重组载体转化到农杆菌 EHA105感受态细胞中,摇菌备用。采用大豆根癌农杆菌介导的子叶节转化方法(Wang et al. 2009)获取CPU1整株干涉(CPU1-RNAi)材料,最终得到CPU1表达量受到显著抑制的三个独立的转基因株系(RNAi1,RNAi2,RNAi3)。
片段扩增引物序列如下:
F1:5'-TCAACCCGGGGGCGCGCCATGCTCTCATTTTCGTCTCTG-3';
R1:5'-TGCCGGATCCATTTAAATCGAAAGAGTTCGAAAATTG-3'。
将CPU1-RNAi材料与野生型材料(YC04-5)种植在生长室的蛭石中,每天浇营养液,营养液配方如表1:
表1
植物生长室的生长条件:13小时/26 °C光照和11小时/24°C黑暗;光照强度为400μmol photons m-2s-1;相对湿度为65%。
播种后18天,收获植株的地上部和根部,并对根部进行扫描。利用WinRHIZO软件对扫描图像进行分析获得植株的总根长。将植株的地上部和根部在65°C烘箱中烘两天,然后称干重。将磨好的烘干植物样品放入消煮管,加入3 mL浓硝酸在消煮炉上进行样品消煮,然后使用ICP-MS(Agilent 7900, Agilent Technologies, SantaClara, CA, USA)测量磷浓度并计算出磷吸收效率。
图2为干涉CPU1对大豆转基因植株磷吸收效率、生物量和产量的影响。其中:A为3个独立RNAi干涉家系的CPU1相对表达量;B为RNAi干涉家系和野生型材料在苗期的生长状况;C为RNAi干涉家系和野生型材料在苗期的生物量;D为RNAi干涉家系和野生型材料在苗期的磷吸收量;E为RNAi干涉家系和野生型材料在苗期的总根长;F为RNAi干涉家系和野生型材料在苗期的磷吸收效率;G为RNAi干涉家系和野生型材料在成熟期的生长状况;H为RNAi干涉家系和野生型材料在成熟期的单株荚数。*表示显著水平0.01 < P≤ 0.05 时,差异显著;**表示显著水平0.001 < P ≤ 0.01时,差异显著性介于显著与极显著之间;***表示显著水平P ≤ 0.001 时,差异极显著。
结果表明:在苗期,相比于野生型材料,CPU1-RNAi材料的磷吸收效率显著降低(参见图2(F)),导致RNAi材料的植株磷吸收量和生物量显著降低(参见图2(C-D)),但是总根长没有显著差异(参见图2(E));在成熟期,相比于野生型材料,CPU1-RNAi材料的产量显著降低(参见图2(G-H))。上述结果说明CPU1是通过提高大豆的磷吸收效率而不是根的长度来促进植物对磷的吸收。
实施例3、CPU1氨基酸序列和表达量的变异情况
通过全基因组关联分析鉴定到CPU1,说明CPU1自身存在着序列变异从而导致大豆群体磷吸收效率的表型变异,因此找到这个功能变异位点将为后期基因编辑育种和精准分子标记辅助选择育种提供宝贵的信息。
基于实施例1中的重测序结果和全基因组关联分析结果,发明人发现在大豆自然群体中主要存在两种CPU1的等位基因,CPU1-H1(核苷酸序列如SEQ ID NO.5所示)和CPU1- H2(核苷酸序列如SEQ ID NO.1所示),且与磷吸收效率显著关联的变异位点存在于启动子区和5’UTR,没有发现编码区的关联信号,暗示磷吸收效率的变异不是由编码区的变异引起。为了进一步确定功能变异所在区域,分别随机选取含有两种等位基因的大豆材料各5份,以这10份材料的cDNA为模板,利用引物F10/R10扩增出这10份材料的CDS序列并进行测序。同时检测了这10份材料根部CPU1的表达量(播种后18天)。
植物总RNA的提取和反转录: 参照Trizol(Takara, Japan)说明书的方法提取总RNA;参照 One Step gDNA Removal and cDNA Synthesis SuperMix反转录酶试剂盒(TransGen, China)说明书的方法合成 cDNA第一链。
CDS序列扩增引物:
F10:5'-CGAGGCTCAGCAGGAGAATTCATGGGGAATGATGCAGG GTC-3'
R10:5'-GCCCTTGCTCACCATCATATCTACTGGCCCCCAAA-3'
实时荧光定量PCR检测基因表达量:利用 Top Green qPCR SuperMix试剂盒(TransGen, China)进行实时荧光定量 PCR分析 。
10 μL反应体系为:
反应程序为 95℃,2分钟;95℃,15秒;60℃,15秒;72℃,30秒; 设置循环数40次。采用 2-△△Ct法,以大豆看家基因 GmEF-1α为参照,计算基因的相对表达量。
实时荧光定量PCR引物如下:
CPU1-F: 5'-TGGAAAAAGAAGCGAACTGGGT-3';
CPU1-R: 5'-GCTTCCAACACATAAGTGGTCA-3';
GmEF-1α-F: 5'-TGCAAAGGAGGCTGCTAACT-3';
GmEF-1α-R: 5'-CAGCATCACCGTTCTTCAAA-3'。
图3为CPU1两种等位基因间氨基酸序列的比较,其中序列比较是针对随机挑选的分别对应两种等位基因的各5份大豆材料。图4为CPU1两种等位基因间表达量的比较,其中用到的10份大豆材料与图3用到的材料相同。结果表明:两种等位基因间不存在氨基酸序列的差异(参见图3);两种等位基因间不存在表达量的差异(参见图4);因此CPU1的变异来源不是氨基酸序列和表达量的差异,说明功能变异位点不在编码区和启动子区。
实施例4、CPU1功能变异位点所在区域的确定
基于上述全基因组关联分析结果,CPU1两种等位基因在5’UTR存在两个碱基的差异,为了确定5’UTR是CPU1功能变异位点所在区域,发明人首先构建了6个重组载体(将CPU1不同等位基因(H1或H2)的启动子和5’UTR进行组合,并连接上CPU1-GFP),转入大豆毛根中,通过Western-blot实验对蛋白水平进行定量。Western-blot实验中加入一抗anti-GFPantibody (1:1,000; TransGen, Beijing, China)或anti H+-ATPase (1:2,000;Agrisera, Vännäs, Sweden)过夜;然后加入对应的二抗horseradish peroxidase (HRP)-conjugated anti-mouse IgG (TransGen, Beijing, China)或horseradish peroxidase(HRP)-conjugated anti-rabbit IgG (Biosharp, Hefei, China);使用SuperSignalWest Dura Trial Kit (Thermo Scientific, MA, USA)进行曝光显影并利用AmershamImager 600 System (GE Healthcare Bio-Sciences AB, Uppsala, Sweden)进行成像分析。
重组载体的构建:
(1)利用引物F10/R10将CPU1的CDS(如SEQ ID NO.4 所示 )扩增出来,克隆到pFGC5941-p35S-GFP载体的EcoRI和AscI酶切位点形成CPU1-GFP;
(2)利用引物F11/R11分别扩增出H1和H2等位基因的启动子-5’UTR,然后分别克隆到CPU1-GFP载体的EcoRI酶切位点,形成H2 promoter +H2 5'UTR :CPU1-GFP(图5A中的A载体)和H1 promoter + H1 5'UTR :CPU1-GFP (图5A中的B载体);
(3)分别利用引物F11/R12和F12/R11分别扩增出两种等位基因的启动子区和5’UTR,利用引物F11/R11通过重叠PCR将启动子区和5’UTR连接起来,形成H2 promoter +H1 5'UTR 和H1 promoter +H2 5'UTR 的PCR产物;分别将这两种PCR产物克隆到(2)中CPU1-GFP载体的EcoRI酶切位点,形成H2 promoter +H1 5'UTR :CPU1-GFP(图5A中的C载体)和H1 promoter +H2 5'UTR :CPU1-GFP(图5A中的D载体)。
(4)利用引物F13/R11扩增出两种等位基因的5’UTR,然后克隆到(2)中CPU1-GFP载体的EcoRI酶切位点形成H2 5'UTR :CPU1-GFP (图5A中的E载体)和H1 5'UTR :CPU1-GFP(图5A中的F载体)。
构建重组载体所用引物如下:
F10:5'-CGAGGCTCAGCAGGAGAATTCATGGGGAATGATGCAGGGTC-3'
F11:5'-CGAGGCTCAGCAGGAGGCGCGCCGGACATGTGCACCACGAGGAATATTAGG-3'
F12:5'-TCGCGCTAATGCCGCGGAATCTTAAGCG-3'
F13:5'-CGAGGCTCAGCAGGAGAATTCCGGAATCTTAAGCGAAT ATC-3'
R10:5'-GCCCTTGCTCACCATCATATCTACTGGCCCCCAAA-3'
R11:5'-TGCATCATTCCCCATCGAAAGTGTTCGAAAATTGGATAC CCAG-3'
R12:5'-CGCTTAAGATTCCGCGGCATTAGCGCGA-3'
图5为通过重组载体的构建和Western-blot实验确定CPU1功能变异位点所在区域,其中:A为不同等位基因的启动子和5’UTR重组载体的示意图;B为分别转入A中6个重组载体的大豆毛根的Western-blot结果。在多重比较中,不同的英文字母之间代表显著性差异(P<0.05)。
结果表明:仅有5’UTR是无法启动CPU1-GFP的表达;不同等位基因的启动子对CPU1-GFP的蛋白含量没有影响,说明功能变异位点不在启动子区;不同等位基因的5’UTR对CPU1-GFP的蛋白含量有显著的影响,说明功能变异位点在5’UTR,影响了CPU1的翻译效率。
实施例5、CPU1的功能变异位点的确定
5’UTR存在两个SNPs,为探究哪一个SNP影响了CPU1的翻译效率以及机制,发明人发现CPU1的5’UTR存在着一个开放阅读框(uORF),且其中存在的两个SNPs正好位于这个uORF内,分别位于uORF的第20位(磷高效等位基因CPU1-H2中为A;磷低效等位基因CPU1-H1中为G)和第83位(磷高效等位基因CPU1-H2中为C;磷低效等位基因CPU1-H1中为A),导致氨基酸的变化和提前终止。
为了确定真正的功能变异位点,同时确定功能变异位点是否是通过uORF来影响CPU1的翻译效率,发明人构建了6个重组载体(将两个SNPs不同的基因型进行组合;将uORF的起始密码子进行突变ATG→AAA;然后连上CPU1-GFP),转入大豆毛根中,通过Western-blot实验对CPU1-GFP蛋白水平进行定量。Western-blot实验中加入一抗anti-GFPantibody (1:1,000; TransGen, Beijing, China)或anti H+-ATPase (1:2,000;Agrisera, Vännäs, Sweden)过夜;然后加入对应的二抗horseradish peroxidase (HRP)-conjugated anti-mouse IgG (TransGen, Beijing, China)或horseradish peroxidase(HRP)-conjugated anti-rabbit IgG (Biosharp, Hefei, China);使用SuperSignalWest Dura Trial Kit (Thermo Scientific, MA, USA)进行曝光显影并利用AmershamImager 600 System (GE Healthcare Bio-Sciences AB, Uppsala, Sweden)进行成像分析。
重组载体的构建:
(1)利用F14/R10引物扩增CPU1的CDS序列,然后克隆到pFGC5941 -p35S-GFP的AscI酶切位点产生p35S:CPU1-GFP重组载体;
(2)利用F15/R15引物分别扩增出两种等位基因的5’UTR;
(3)利用F16/F17/R15引物进行重叠PCR获得H1 SNP476 +H2 SNP413 基因型的5’UTR;
(4)利用F15/F18/R18/R15引物进行重叠PCR获得H2 SNP476 +H1 SNP413 基因型的5’UTR;
(5)利用F19/R15引物分别扩增出起始密码子突变(ATG→AAA)的两种等位基因的5’UTR;
(6)将上述(2)-(5)中的6个PCR产物分别克隆到(1)中p35S:CPU1-GFP载体的AscI酶切位点,构建出图(6)A中G-L重组载体。
所用引物:
F14:5'-TTACAATTACCATGGGGCGCGCCATGGGGAATGATGCAG GGTC-3'
F15:5'-TTACAATTACCATGGCGGAATCTTAAGCGAATATC-3'
F16:5'-TTACAATTACCATGGCGGAATCTTAAGCGAATATCTCCAT AGTTGCTAAT -3'
F17:5'-ATATCTCCATAGTTGCTAATATGTTTTGTTTCTTCCAGCGT TGTT-3'
F18:5'-CTTCAATTTTTTAAACCCTCAAAAT-3'
F19:5'-TTACAATTACCATGGCGGAATCTTAAGCGAATATCTCCAT AGTTGCTAATA AATTTTG-3'
R10:5'-GCCCTTGCTCACCATCATATCTACTGGCCCCCAAA-3'
R15:5'-TGCATCATTCCCCATCGAAAGTGTTCGAAAATT-3'
R18:5'-ATTTTGAGGGTTTAAAAAATTGAAG-3'
图6为通过重组载体的构建和Western实验确定CPU1功能变异位点,其中:A为含有不同基因型的5’UTR的重组载体示意图;B为分别转入(A)中6个重组载体的大豆毛根的Western-blot结果。
在多重比较中,不同的英文字母代表显著性差异(P<0.05)。
结果表明:(1)在不突变uORF起始密码子的条件下,导致提前终止的SNP413会显著改变CPU1-GFP的翻译效率,而引起氨基酸变化的SNP476对翻译效率没有显著影响;(2)当uORF的起始密码子被突变后,检测不到CPU1-GFP蛋白,表明uORF对于CPU1-GFP的翻译是必需的。已有的大部分报道uORF是抑制下游基因的翻译,发明人的研究是在植物中首次发现uORF也可以促进下游基因的翻译,同时也是第一次发现uORF的自然变异导致植物群体表型变异的报道。
综上所述,通过全基因组关联分析鉴定到一个在根特异高表达的SEC12-like蛋白基因CPU1,通过RNAi整株稳定转化材料验证了该基因在磷吸收效率方面的功能。在自然界,该基因存在两个主要的等位基因,其5’UTR存在一个促进CPU1翻译的uORF,其中磷低效等位基因CPU1-H1中一个SNP导致uORF长度的延长(第83位碱基由A变成C,延长后的uORF核苷酸序列如SEQ ID NO.3所示),提高了CPU1的翻译效率,形成了磷高效等位基因CPU1-H2,该等位基因将会成为后期磷高效分子育种的重要遗传资源,鉴定到的功能变异位点也会为后期基因编辑育种提供了精准的靶点。总之,该发明为提高作物磷效率和产量,发展资源节约型环境友好型的生态农业具有重要的应用意义。
需要说明的是,上述实施例不以任何形式限制本发明,凡采用等同替换或等效变换的方式所获得的技术方案,均落在本发明的保护范围内。
参考文献:
石慧,王思明. 相对优势地位的转变:中美大豆发展比较研究. 中国农史.(2018). 37(5):58-64.
李欣欣,许锐能,廖红. 大豆共生固氮在农业减肥增效中的贡献及应用潜力. 大豆科学. (2016). 35(4):531-535.
陆景陵. 植物营养学. 中国农业大学出版社. (1994).
Yu, J., and Buckler, E.S. Genetic association mapping and genomeorganization of maize. Current Opinion in Biotechnology. (2006). 17(2):155-160. (Yu, J., and Buckler, E.S. 玉米的遗传关联分析和基因组结构. 生物技术主流观点. (2006) . 17(2):155-160)
Schmutz, J., Cannon, S.B., Schlueter, J. et al. Genome sequence ofthe palaeopolyploid soybean. Nature. (2010). 463(7278):178-183. (Schmutz, J.,Cannon, S.B., Schlueter, J. et al. 古四倍体大豆的基因组序列. 自然. (2010).463(7278):178-183)
Lam, H.M., Xu, X., Liu, X. et al. Resequencing of 31 wild andcultivated soybean genomes identifies patterns of genetic diversity andselection. Nature Genetics. (2010). 42(12):1053-1059. (Lam, H.M., Xu, X.,Liu, X. et al. 重测序31份野生和栽培大豆基因组鉴定大豆遗传多样性和受选择模式.自然遗传学. (2010) . 42(12):1053-1059)
Zhou, Z., Jiang, Y., Wang, Z. et al. Resequencing 302 wild andcultivated accessions identifies genes related to domestication andimprovement in soybean. Nature biotechnology. (2015). 33(4):408-414. (Zhou,Z., Jiang, Y., Wang, Z. et al. 重测序302份野生和栽培大豆鉴定大豆驯化和改良相关的基因. 自然生物技术. (2015). 33(4):408-414)
Fang, C., Ma, Y., Wu, S. et al. Genome-wide association studiesdissect the genetic networks underlying agronomical traits in soybean. Genomebiology. (2017). 18. (Fang, C., Ma, Y., Wu, S. et al. 全基因组关联分析解析大豆农艺性状的遗传网络. Genome biology. (2017). 18)
Wang, X., Wang, Y., Tian, J. et al. Overexpressing AtPAP15 enhancesphosphorus efficiency in soybean. Plant Physiol. (2009) 151, 233-240. (Wang,X., Wang, Y., Tian, J. et al. 超表达AtPAP15增强大豆的磷效率. 植物生理.(2009). 151, 233-240)。
SEQUENCE LISTING
<110> 福建农林大学
<120> SEC12-like蛋白基因CPU1及其在提高大豆磷效率方面的应用
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<160> 26
<170> PatentIn version 3.3
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cgtgacatta attaataagg ctgtgccttg tttcattggt taattaatta atgattaaat 360
aaagcaaggc aaagctcttt ctatcttcct ttgacttttt ttttcagagg ctctattttt 420
cttctctgac atttctattt aaatttgccg aagaatccaa ttcaccgatc tccgaagagc 480
tccatttgga aaaagaagcg aactgggtat ccaattttcg aacactttcg atggggaatg 540
atgcagggtc acctcagggt ccggttacgt gtgggtcgtg gattcggagg cctgagaatt 600
tgaacttggt ggtgttagga aggtccagac gtggcaattc ttgtccttct ctcttggaga 660
ttttctcctt cgatcccaag accacttctc tgtctacctg tcctctggta ttcctctaaa 720
actctgaata tacatacacg tatcatgtgt gtgtgtgttg tgtttaagta tgcatgtgcg 780
tgtgtaattt attttatatt atgtatagag tgactcattt gtaacattaa tttgttttgt 840
gcagaccctt tttattgtat gttgaaaaac tgttgttttc tttgtgttat gtttgtgtat 900
gtctgagcat gtagattctg tggagtgagt catttgaaac acgagccttt ttgtgcatat 960
actttttgat tattggccga gaaactgttt actttttcct ctctgaagca gatggtgggt 1020
ggaagtagat attatgcaca aattctgttg ttgaaaagta tttttagtgt tgaaattctg 1080
ggttgctgaa tggaagcaaa gtttgaatgg gctatggctt tggttttaat gatgtttttg 1140
ttttgatatt tcagaccact tatgtgttgg aagcagagga aggtgatcct gttgctattg 1200
cagtccaccc aagtggggat gattttgtgt gcgctctcag caatggtagc tgcaagtaag 1260
tttcttttgt aagggcttcg agattgaagc gttcttttat atgtattcat cttttgaaat 1320
acttccgtga tgtgtctcaa cttgcatttc taaaattagc agttcacttg cgataatctc 1380
agaaacagac tccaacattt tatctttctt taaccgttca aagtacaaga taaaactgta 1440
ggctcagttc taccaaattt ctctctgaca gtttctcgtt cctttttttt ttttccctgg 1500
gaactaggga atgtttgaca taatagttat tgttgtttct taggtataga tagatgaatt 1560
ttgccttgag ttattttcgt tggatgattt gtgccatcct tggatagtta agatcctaca 1620
tcagttaggt atatggcaat agctttagag gtagagttag actcatttca ttctcaattc 1680
taatatgata tcaaagcgta ttcaggcctg atgtttgacc acctgcacat gtctggtgca 1740
gcctacaaac ttcatgctct agcctctaga tgtctagtcc tggacatgat atcctcccat 1800
gattcttatt tctaattgat actgaactga acatataata tagattgaag tatttctcca 1860
tggcttgtag attgtttgag ctgtatggtc gtgaaacaaa catgaagttg ttggctaagg 1920
aactggctcc tctacagggt attggtcctc agaaatgcat tgcttttagt gttgatgggt 1980
ctaaatttgc tgctggtggg ttggtaagca tcactttata tccaaccaat tgcttttatt 2040
ttctattcag cactttgagt ttttcctttt caagtttgat cttgtatgtt tgacttctgt 2100
ctttaacaag tgtaggatgg acatctcaga attatggagt ggcctagtat gcgcgtgatt 2160
ttggatgaac caagagcaca caaatcagtt cgggatatgg attttaggta ggtatagtaa 2220
acaaatctat ttggatcctt ctaaaggagg catcaatccc tacagctagt aaaattgtaa 2280
taaatagttg ataaagttgg ttactatagt aatgttattt cgagttctta caaccagata 2340
agataatttt tgctttgcat gttcatgcct gcaataactt gactgtgtag atatgatctt 2400
ttagaaaata aaagtatgtt acattgtaaa tattttaatc ctgaaacttt aatgatattg 2460
tacttactat attgtccttc attttttccc ttactttagt ctagactcag aatttctagc 2520
ttcaacttct actgatggtt cagcaagaat ctggaagatt gaagatggtg ttcctttgac 2580
tactttgtct cgcaactcgg tatggtgtat ttgatttaag aacctggggc aagatctgta 2640
tgcagtactt gtattgcttg atccaaatat ttccttttgt ctctttagga tgaaaagatt 2700
gaattatgtc gattttccat ggatggaacc aaaccatttt tattttgctc tgttcaaaaa 2760
ggtataagag tatcttgttt ctagtatatt ctatagtatt aatttgtata ttcttcaaat 2820
ctctttgacc agcaaagcat ggcctttata atagatactt atatctttta gcaggtgata 2880
cttctgtcac tgcggtttat gagattagca catggaataa aattgggcac aagaggctga 2940
ttagaaagtc tgcttcagta atgtccatta gccatgatgg gaaatacctt tctctgtaag 3000
aacctgcagt tatcttctga ctttttggct tatgtgtggt cattggtcaa cattcttcct 3060
ttatctttcg ttagttttga tttccaaatt ttatccagat agttttgtga ctattgtaag 3120
tcttgcatct taagcaagtg aataatttag aatttttatt tcttttgttt tgaccaatag 3180
aatttttatt caattgcctt ctgttatcct cagcagtctg catgcttgaa ggagtgcttg 3240
aatccccctc ccccatgcat tatctgatgt aggaatgtaa atatcccaat ctaaaaatgt 3300
tgaccaggag gtctttcgtt tacctgactt ctcccctggg taaacaaaca tctccatcat 3360
aatcgaaact aaaacttcaa tataagagtg gaagagattg aatagaggct gaaattgcat 3420
tcttcaatga atacctaagt gtaaaaaagt ttaattaagt ctctttgaaa attgaaatgt 3480
actcttacca taaatttcag atttccgtgt aagtccttct tattaataaa gccattcact 3540
ttcttaactg tcatagatct ccttgtctgt attaatatat aaatcatttg ggtaccaaag 3600
tgggattgtg attttggcca tttctccaaa attgtgaatg aatgaagaaa acaatgttag 3660
aattgatcat gtttttccat cttattactt tggctctttt tgatctatag cactacattt 3720
atgtttatgt ggctctagtt ccttctttga gtgtcttttc ttgtgaatca ttttttgacc 3780
tttgcacaca taagtcatct gggtgataga ctacctaatc attttcttct gcataactgc 3840
agagtttttt agtttgtgtt tactgtatct ccaatttaat gcataaaaaa gctgttgaaa 3900
agttgactgc agaatgcaca taaattaact tgtttaaact cattttgtcc gtcagctcga 3960
tcctatttcc ttttagatct gcataactgc agggtttttt agtttgtgta ttttactgta 4020
tctccaattt aatgcatttt agctgttgaa aagttgactg cagcacataa attaacttgt 4080
ttaaactcat tttgtctgtc agcttgatcc tatttccttt tagaatcata atagccccaa 4140
aactcatgac tgtaatgcat ttcccaggaa acagcataac ctaaaataac atatcttatt 4200
ctgtttttct tcaattgtag cttgccacta ggcatggaca cctattgggg gggggggggg 4260
ggatgtctaa tttttaataa ttaataattt taaaaaatat ttatttttac acataaaatt 4320
gaaactaatt tttattttaa atgataataa ctttaatcat tatcataaaa acaacaaaca 4380
caaattagtt tttcacaatt ttattcaagt aatcacctta accattacag taataataac 4440
aagcacaact aattttatat aattttacac taactaactt taatcattat tataataata 4500
acatagataa ttcgttttta atagttttaa attaaccaac ttaaaaatat atatctatgt 4560
acatgagaag tgccaaggga gggggggggt agctgttaaa gtaagtcata gcttgtttaa 4620
ttataactat aaaaaaatgt ttaaatatgt tgtggtgaag taactatagc acacttgtaa 4680
accatattag cggagtctgg ggtacatcct ctataaaatt actataatat attcaccaaa 4740
caaattacta aaatattttg attaaaacat ttgaaggcct gtaataagtt cgtgatctga 4800
tttgcacttc acttgtatat cacataacaa tctatgataa tatgtcccca gcatttcttc 4860
tgctcatcgg acttctgtaa tttcaggggc agtaaagatg gagacatatg tgtagttgaa 4920
gtaaagaaaa tgcagatata ccattatagc aagagattgc acctgggtac aaatattgca 4980
tatctggagt tctgtcccgg ggaaaggtaa tttctatgct ctattggttt aatttggcac 5040
ctctgataaa tatcaatgta tgcagaattt tagtaattgc tgaaacctcc tcctttttga 5100
atattggaca cagttgggat taagctattc atttgaatat tggaacatgc attgggtaca 5160
aaaccttggt gttagcaatg aatttatatt agcaattgat tttttctcat cagatcatta 5220
gccagagtaa atgtggattt ttgaaattga accttggtgt tagagaacca atctgacctg 5280
aaagcttaag tcatttataa tggaagttaa gtcgtttttt ttaataaatt atagctaaca 5340
tgcctctgca gattaccttt tagtattgga ttctgattct gtgatcatac atagtaattt 5400
ctcattttaa aaaaaataca ttcagttaat aaatctattc ttttggtctt gcctactcac 5460
ccaggctttt tttgttcagg gttttactta caacctcagt agaatgggga gcgctggtca 5520
ccaagctgac tgtacctaaa gattggaaag gttctctctc tcttacacgc acacacttgc 5580
atgcatccct tcttcattct aacgccttac aataatgtct attcaatttg acattttcaa 5640
tatcctttca aacctgcaga gtggcagatc tatttggtgc tattgggact atttttagca 5700
tcagctgttg cattttacat attctttgag aactctgatt cattctggaa ctttcccatg 5760
ggcaaagacc aaccagcaag accaaggttt aaacctgtgt taaaagatcc ccagtcttat 5820
gatgaccaaa atatttgggg gccagtagat atgtgatcac attaacattc ttgatttagt 5880
cttcggtgct gttttggaag cagtatcagt agctgtaact ggtatcaata tttatttaag 5940
cccttataga gttaggcact tgactggtat tacaaacatt tacttctatt tttttggggt 6000
gaaaattctg agccaaaggc catgattggt atgtaatttt aatagaaact ttaggaataa 6060
tcaaatagct tccttaaatt tacaagttac acgcaaggct gctttgtagc tatgtgatgg 6120
gatccattga agaggcacgt ctttggatat ctttccattt ttcttatttt gtttcttgtt 6180
ttaatgataa cctcttacat tggttttatg cctttggtta gagaaaaata aa 6232
<210> 2
<211> 1915
<212> DNA
<213> SEQ ID NO.2
<400> 2
cggaatctta agcgaatatc tccatagttg ctaatatgtt ttgtttcttc cagcattgtt 60
gcatttactg gacccatctc tcccttcttt ctattaaaca aatcgcttca attttttcaa 120
ccctcaaaat taatcaactt tcattttttt tataaatcca accccctaaa catattttca 180
cattgcgttc aagcaacagt tgcatcatcc taataaaacc ctgtgatcat atacattcat 240
actcagcaac cttaaaacac aatatcacgt aaaaaagaat ccaattcacc gatctccgaa 300
gagctccatt tggaaaaaga agcgaactgg gtatccaatt ttcgaacact ttcgatgggg 360
aatgatgcag ggtcacctca gggtccggtt acgtgtgggt cgtggattcg gaggcctgag 420
aatttgaact tggtggtgtt aggaaggtcc agacgtggca attcttgtcc ttctctcttg 480
gagattttct ccttcgatcc caagaccact tctctgtcta cctgtcctct gaccacttat 540
gtgttggaag cagaggaagg tgatcctgtt gctattgcag tccacccaag tggggatgat 600
tttgtgtgcg ctctcagcaa tggtagctgc aaattgtttg agctgtatgg tcgtgaaaca 660
aacatgaagt tgttggctaa ggaactggct cctctacagg gtattggtcc tcagaaatgc 720
attgctttta gtgttgatgg gtctaaattt gctgctggtg ggttggatgg acatctcaga 780
attatggagt ggcctagtat gcgcgtgatt ttggatgaac caagagcaca caaatcagtt 840
cgggatatgg attttagtct agactcagaa tttctagctt caacttctac tgatggttca 900
gcaagaatct ggaagattga agatggtgtt cctttgacta ctttgtctcg caactcggat 960
gaaaagattg aattatgtcg attttccatg gatggaacca aaccattttt attttgctct 1020
gttcaaaaag gtgatacttc tgtcactgcg gtttatgaga ttagcacatg gaataaaatt 1080
gggcacaaga ggctgattag aaagtctgct tcagtaatgt ccattagcca tgatgggaaa 1140
tacctttctc tgggcagtaa agatggagac atatgtgtag ttgaagtaaa gaaaatgcag 1200
atataccatt atagcaagag attgcacctg ggtacaaata ttgcatatct ggagttctgt 1260
cccggggaaa gggttttact tacaacctca gtagaatggg gagcgctggt caccaagctg 1320
actgtaccta aagattggaa agagtggcag atctatttgg tgctattggg actattttta 1380
gcatcagctg ttgcatttta catattcttt gagaactctg attcattctg gaactttccc 1440
atgggcaaag accaaccagc aagaccaagg tttaaacctg tgttaaaaga tccccagtct 1500
tatgatgacc aaaatatttg ggggccagta gatatgtgat cacattaaca ttcttgattt 1560
agtcttcggt gctgttttgg aagcagtatc agtagctgta actggtatca atatttattt 1620
aagcccttat agagttaggc acttgactgg tattacaaac atttacttct atttttttgg 1680
ggtgaaaatt ctgagccaaa ggccatgatt ggtatgtaat tttaatagaa actttaggaa 1740
taatcaaata gcttccttaa atttacaagt tacacgcaag gctgctttgt agctatgtga 1800
tgggatccat tgaagaggca cgtctttgga tatctttcca tttttcttat tttgtttctt 1860
gttttaatga taacctctta cattggtttt atgcctttgg ttagagaaaa ataaa 1915
<210> 3
<211> 120
<212> DNA
<213> SEQ ID NO.3
<400> 3
atgttttgtt tcttccagca ttgttgcatt tactggaccc atctctccct tctttctatt 60
aaacaaatcg cttcaatttt ttcaaccctc aaaattaatc aactttcatt ttttttataa 120
<210> 4
<211> 1185
<212> DNA
<213> SEQ ID NO.4
<400> 4
atggggaatg atgcagggtc acctcagggt ccggttacgt gtgggtcgtg gattcggagg 60
cctgagaatt tgaacttggt ggtgttagga aggtccagac gtggcaattc ttgtccttct 120
ctcttggaga ttttctcctt cgatcccaag accacttctc tgtctacctg tcctctgacc 180
acttatgtgt tggaagcaga ggaaggtgat cctgttgcta ttgcagtcca cccaagtggg 240
gatgattttg tgtgcgctct cagcaatggt agctgcaaat tgtttgagct gtatggtcgt 300
gaaacaaaca tgaagttgtt ggctaaggaa ctggctcctc tacagggtat tggtcctcag 360
aaatgcattg cttttagtgt tgatgggtct aaatttgctg ctggtgggtt ggatggacat 420
ctcagaatta tggagtggcc tagtatgcgc gtgattttgg atgaaccaag agcacacaaa 480
tcagttcggg atatggattt tagtctagac tcagaatttc tagcttcaac ttctactgat 540
ggttcagcaa gaatctggaa gattgaagat ggtgttcctt tgactacttt gtctcgcaac 600
tcggatgaaa agattgaatt atgtcgattt tccatggatg gaaccaaacc atttttattt 660
tgctctgttc aaaaaggtga tacttctgtc actgcggttt atgagattag cacatggaat 720
aaaattgggc acaagaggct gattagaaag tctgcttcag taatgtccat tagccatgat 780
gggaaatacc tttctctggg cagtaaagat ggagacatat gtgtagttga agtaaagaaa 840
atgcagatat accattatag caagagattg cacctgggta caaatattgc atatctggag 900
ttctgtcccg gggaaagggt tttacttaca acctcagtag aatggggagc gctggtcacc 960
aagctgactg tacctaaaga ttggaaagag tggcagatct atttggtgct attgggacta 1020
tttttagcat cagctgttgc attttacata ttctttgaga actctgattc attctggaac 1080
tttcccatgg gcaaagacca accagcaaga ccaaggttta aacctgtgtt aaaagatccc 1140
cagtcttatg atgaccaaaa tatttggggg ccagtagata tgtga 1185
<210> 5
<211> 6232
<212> DNA
<213> SEQ ID NO.5
<400> 5
cggaatctta agcgaatatc tccatagttg ctaatatgtt ttgtttcttc cagcgttgtt 60
gcatttactg gacccatctc tcccttcttt ctattaaaca aatcgcttca attttttaaa 120
ccctcaaaat taatcaactt tcattttttt tataaatcca accccctaaa catattttca 180
cattgcgttc aagcaacagt tgcatcatcc taataaaacc ctgtgatcat atacattcat 240
actcagcaac cttaaaacac aatatcacgt aaaaaaggtg agacatgtct ttttcgaacg 300
cgtgacatta attaataagg ctgtgccttg tttcattggt taattaatta atgattaaat 360
aaagcaaggc aaagctcttt ctatcttcct ttgacttttt ttttcagagg ctctattttt 420
cttctctgac atttctattt aaatttgccg aagaatccaa ttcaccgatc tccgaagagc 480
tccatttgga aaaagaagcg aactgggtat ccaattttcg aacactttcg atggggaatg 540
atgcagggtc acctcagggt ccggttacgt gtgggtcgtg gattcggagg cctgagaatt 600
tgaacttggt ggtgttagga aggtccagac gtggcaattc ttgtccttct ctcttggaga 660
ttttctcctt cgatcccaag accacttctc tgtctacctg tcctctggta ttcctctaaa 720
actctgaata tacatacacg tatcatgtgt gtgtgtgttg tgtttaagta tgcatgtgcg 780
tgtgtaattt attttatatt atgtatagag tgactcattt gtaacattaa tttgttttgt 840
gcagaccctt tttattgtat gttgaaaaac tgttgttttc tttgtgttat gtttgtgtat 900
gtctgagcat gtagattctg tggagtgagt catttgaaac acgagccttt ttgtgcatat 960
actttttgat tattggccga gaaactgttt actttttcct ctctgaagca gatggtgggt 1020
ggaagtagat attatgcaca aattctgttg ttgaaaagta tttttagtgt tgaaattctg 1080
ggttgctgaa tggaagcaaa gtttgaatgg gctatggctt tggttttaat gatgtttttg 1140
ttttgatatt tcagaccact tatgtgttgg aagcagagga aggtgatcct gttgctattg 1200
cagtccaccc aagtggggat gattttgtgt gcgctctcag caatggtagc tgcaagtaag 1260
tttcttttgt aagggcttcg agattgaagc gttcttttat atgtattcat cttttgaaat 1320
acttccgtga tgtgtctcaa cttgcatttc taaaattagc agttcacttg cgataatctc 1380
agaaacagac tccaacattt tatctttctt taaccgttca aagtacaaga taaaactgta 1440
ggctcagttc taccaaattt ctctctgaca gtttctcgtt cctttttttt ttttccctgg 1500
gaactaggga atgtttgaca taatagttat tgttgtttct taggtataga tagatgaatt 1560
ttgccttgag ttattttcgt tggatgattt gtgccatcct tggatagtta agatcctaca 1620
tcagttaggt atatggcaat agctttagag gtagagttag actcatttca ttctcaattc 1680
taatatgata tcaaagcgta ttcaggcctg atgtttgacc acctgcacat gtctggtgca 1740
gcctacaaac ttcatgctct agcctctaga tgtctagtcc tggacatgat atcctcccat 1800
gattcttatt tctaattgat actgaactga acatataata tagattgaag tatttctcca 1860
tggcttgtag attgtttgag ctgtatggtc gtgaaacaaa catgaagttg ttggctaagg 1920
aactggctcc tctacagggt attggtcctc agaaatgcat tgcttttagt gttgatgggt 1980
ctaaatttgc tgctggtggg ttggtaagca tcactttata tccaaccaat tgcttttatt 2040
ttctattcag cactttgagt ttttcctttt caagtttgat cttgtatgtt tgacttctgt 2100
ctttaacaag tgtaggatgg acatctcaga attatggagt ggcctagtat gcgcgtgatt 2160
ttggatgaac caagagcaca caaatcagtt cgggatatgg attttaggta ggtatagtaa 2220
acaaatctat ttggatcctt ctaaaggagg catcaatccc tacagctagt aaaattgtaa 2280
taaatagttg ataaagttgg ttactatagt aatgttattt cgagttctta caaccagata 2340
agataatttt tgctttgcat gttcatgcct gcaataactt gactgtgtag atatgatctt 2400
ttagaaaata aaagtatgtt acattgtaaa tattttaatc ctgaaacttt aatgatattg 2460
tacttactat attgtccttc attttttccc ttactttagt ctagactcag aatttctagc 2520
ttcaacttct actgatggtt cagcaagaat ctggaagatt gaagatggtg ttcctttgac 2580
tactttgtct cgcaactcgg tatggtgtat ttgatttaag aacctggggc aagatctgta 2640
tgcagtactt gtattgcttg atccaaatat ttccttttgt ctctttagga tgaaaagatt 2700
gaattatgtc gattttccat ggatggaacc aaaccatttt tattttgctc tgttcaaaaa 2760
ggtataagag tatcttgttt ctagtatatt ctatagtatt aatttgtata ttcttcaaat 2820
ctctttgacc agcaaagcat ggcctttata atagatactt atatctttta gcaggtgata 2880
cttctgtcac tgcggtttat gagattagca catggaataa aattgggcac aagaggctga 2940
ttagaaagtc tgcttcagta atgtccatta gccatgatgg gaaatacctt tctctgtaag 3000
aacctgcagt tatcttctga ctttttggct tatgtgtggt cattggtcaa cattcttcct 3060
ttatctttcg ttagttttga tttccaaatt ttatccagat agttttgtga ctattgtaag 3120
tcttgcatct taagcaagtg aataatttag aatttttatt tcttttgttt tgaccaatag 3180
aatttttatt caattgcctt ctgttatcct cagcagtctg catgcttgaa ggagtgcttg 3240
aatccccctc ccccatgcat tatctgatgt aggaatgtaa atatcccaat ctaaaaatgt 3300
tgaccaggag gtctttcgtt tacctgactt ctcccctggg taaacaaaca tctccatcat 3360
aatcgaaact aaaacttcaa tataagagtg gaagagattg aatagaggct gaaattgcat 3420
tcttcaatga atacctaagt gtaaaaaagt ttaattaagt ctctttgaaa attgaaatgt 3480
actcttacca taaatttcag atttccgtgt aagtccttct tattaataaa gccattcact 3540
ttcttaactg tcatagatct ccttgtctgt attaatatat aaatcatttg ggtaccaaag 3600
tgggattgtg attttggcca tttctccaaa attgtgaatg aatgaagaaa acaatgttag 3660
aattgatcat gtttttccat cttattactt tggctctttt tgatctatag cactacattt 3720
atgtttatgt ggctctagtt ccttctttga gtgtcttttc ttgtgaatca ttttttgacc 3780
tttgcacaca taagtcatct gggtgataga ctacctaatc attttcttct gcataactgc 3840
agagtttttt agtttgtgtt tactgtatct ccaatttaat gcataaaaaa gctgttgaaa 3900
agttgactgc agaatgcaca taaattaact tgtttaaact cattttgtcc gtcagctcga 3960
tcctatttcc ttttagatct gcataactgc agggtttttt agtttgtgta ttttactgta 4020
tctccaattt aatgcatttt agctgttgaa aagttgactg cagcacataa attaacttgt 4080
ttaaactcat tttgtctgtc agcttgatcc tatttccttt tagaatcata atagccccaa 4140
aactcatgac tgtaatgcat ttcccaggaa acagcataac ctaaaataac atatcttatt 4200
ctgtttttct tcaattgtag cttgccacta ggcatggaca cctattgggg gggggggggg 4260
ggatgtctaa tttttaataa ttaataattt taaaaaatat ttatttttac acataaaatt 4320
gaaactaatt tttattttaa atgataataa ctttaatcat tatcataaaa acaacaaaca 4380
caaattagtt tttcacaatt ttattcaagt aatcacctta accattacag taataataac 4440
aagcacaact aattttatat aattttacac taactaactt taatcattat tataataata 4500
acatagataa ttcgttttta atagttttaa attaaccaac ttaaaaatat atatctatgt 4560
acatgagaag tgccaaggga gggggggggt agctgttaaa gtaagtcata gcttgtttaa 4620
ttataactat aaaaaaatgt ttaaatatgt tgtggtgaag taactatagc acacttgtaa 4680
accatattag cggagtctgg ggtacatcct ctataaaatt actataatat attcaccaaa 4740
caaattacta aaatattttg attaaaacat ttgaaggcct gtaataagtt cgtgatctga 4800
tttgcacttc acttgtatat cacataacaa tctatgataa tatgtcccca gcatttcttc 4860
tgctcatcgg acttctgtaa tttcaggggc agtaaagatg gagacatatg tgtagttgaa 4920
gtaaagaaaa tgcagatata ccattatagc aagagattgc acctgggtac aaatattgca 4980
tatctggagt tctgtcccgg ggaaaggtaa tttctatgct ctattggttt aatttggcac 5040
ctctgataaa tatcaatgta tgcagaattt tagtaattgc tgaaacctcc tcctttttga 5100
atattggaca cagttgggat taagctattc atttgaatat tggaacatgc attgggtaca 5160
aaaccttggt gttagcaatg aatttatatt agcaattgat tttttctcat cagatcatta 5220
gccagagtaa atgtggattt ttgaaattga accttggtgt tagagaacca atctgacctg 5280
aaagcttaag tcatttataa tggaagttaa gtcgtttttt ttaataaatt atagctaaca 5340
tgcctctgca gattaccttt tagtattgga ttctgattct gtgatcatac atagtaattt 5400
ctcattttaa aaaaaataca ttcagttaat aaatctattc ttttggtctt gcctactcac 5460
ccaggctttt tttgttcagg gttttactta caacctcagt agaatgggga gcgctggtca 5520
ccaagctgac tgtacctaaa gattggaaag gttctctctc tcttacacgc acacacttgc 5580
atgcatccct tcttcattct aacgccttac aataatgtct attcaatttg acattttcaa 5640
tatcctttca aacctgcaga gtggcagatc tatttggtgc tattgggact atttttagca 5700
tcagctgttg cattttacat attctttgag aactctgatt cattctggaa ctttcccatg 5760
ggcaaagacc aaccagcaag accaaggttt aaacctgtgt taaaagatcc ccagtcttat 5820
gatgaccaaa atatttgggg gccagtagat atgtgatcac attaacattc ttgatttagt 5880
cttcggtgct gttttggaag cagtatcagt agctgtaact ggtatcaata tttatttaag 5940
cccttataga gttaggcact tgactggtat tacaaacatt tacttctatt tttttggggt 6000
gaaaattctg agccaaaggc catgattggt atgtaatttt aatagaaact ttaggaataa 6060
tcaaatagct tccttaaatt tacaagttac acgcaaggct gctttgtagc tatgtgatgg 6120
gatccattga agaggcacgt ctttggatat ctttccattt ttcttatttt gtttcttgtt 6180
ttaatgataa cctcttacat tggttttatg cctttggtta gagaaaaata aa 6232
<210> 6
<211> 39
<212> DNA
<213> F1
<400> 6
tcaacccggg ggcgcgccat gctctcattt tcgtctctg 39
<210> 7
<211> 37
<212> DNA
<213> R1
<400> 7
tgccggatcc atttaaatcg aaagagttcg aaaattg 37
<210> 8
<211> 41
<212> DNA
<213> F10
<400> 8
cgaggctcag caggagaatt catggggaat gatgcagggt c 41
<210> 9
<211> 35
<212> DNA
<213> R10
<400> 9
gcccttgctc accatcatat ctactggccc ccaaa 35
<210> 10
<211> 22
<212> DNA
<213> CPU1-F
<400> 10
tggaaaaaga agcgaactgg gt 22
<210> 11
<211> 22
<212> DNA
<213> CPU1-R
<400> 11
gcttccaaca cataagtggt ca 22
<210> 12
<211> 20
<212> DNA
<213> GmEF-1α-F
<400> 12
tgcaaaggag gctgctaact 20
<210> 13
<211> 20
<212> DNA
<213> GmEF-1α-R
<400> 13
cagcatcacc gttcttcaaa 20
<210> 14
<211> 51
<212> DNA
<213> F11
<400> 14
cgaggctcag caggaggcgc gccggacatg tgcaccacga ggaatattag g 51
<210> 15
<211> 28
<212> DNA
<213> F12
<400> 15
tcgcgctaat gccgcggaat cttaagcg 28
<210> 16
<211> 41
<212> DNA
<213> F13
<400> 16
cgaggctcag caggagaatt ccggaatctt aagcgaatat c 41
<210> 17
<211> 43
<212> DNA
<213> R11
<400> 17
tgcatcattc cccatcgaaa gtgttcgaaa attggatacc cag 43
<210> 18
<211> 28
<212> DNA
<213> R12
<400> 18
cgcttaagat tccgcggcat tagcgcga 28
<210> 19
<211> 43
<212> DNA
<213> F14
<400> 19
ttacaattac catggggcgc gccatgggga atgatgcagg gtc 43
<210> 20
<211> 35
<212> DNA
<213> F15
<400> 20
ttacaattac catggcggaa tcttaagcga atatc 35
<210> 21
<211> 50
<212> DNA
<213> F16
<400> 21
ttacaattac catggcggaa tcttaagcga atatctccat agttgctaat 50
<210> 22
<211> 45
<212> DNA
<213> F17
<400> 22
atatctccat agttgctaat atgttttgtt tcttccagcg ttgtt 45
<210> 23
<211> 25
<212> DNA
<213> F18
<400> 23
cttcaatttt ttaaaccctc aaaat 25
<210> 24
<211> 58
<212> DNA
<213> F19
<400> 24
ttacaattac catggcggaa tcttaagcga atatctccat agttgctaat aaattttg 58
<210> 25
<211> 33
<212> DNA
<213> R15
<400> 25
tgcatcattc cccatcgaaa gtgttcgaaa att 33
<210> 26
<211> 25
<212> DNA
<213> R18
<400> 26
attttgaggg tttaaaaaat tgaag 25
Claims (8)
1.一种SEC12-like蛋白基因CPU1,其特征在于:所述基因CPU1在大豆中存在着自然变异,该基因座上存在2个等位基因,磷低效等位基因CPU1-H1和磷高效等位基因CPU1-H2;在基因CPU1的5’UTR存在着一个开放阅读框uORF,这个uORF内存在两个SNPs,磷高效等位基因CPU1-H2和磷低效等位基因CPU1-H1位于uORF第20位的碱基分别为A和G;位于uORF第83位的碱基分别为C和A;所述磷高效等位基因CPU1-H2的核苷酸序列如SEQ ID NO.1所示;所述磷低效等位基因CPU1-H1的核苷酸序列如SEQ ID NO.5所示。
2.根据权利要求1所述的一种SEC12-like蛋白基因CPU1,其特征在于:所述SEC12-like蛋白基因CPU1的2个等位基因的cDNA序列相同,如SEQ ID NO.2所示。
3.根据权利要求1所述的一种SEC12-like蛋白基因CPU1,其特征在于:所述磷高效等位基因CPU1-H2 uORF的核苷酸序列如SEQ ID NO.3所示。
4.一种植物表达载体,其特征在于:所述植物表达载体含有权利要求1所述基因CPU1。
5.根据权利要求4所述的表达载体,其特征在于:包括重组转化所形成的转基因植物,以及由此产生的外源基因表达产物。
6.权利要求1所述SEC12-like蛋白基因CPU1在提高大豆磷吸收效率方面的应用。
7.根据权利要求6所述的应用,其特征在于:抑制等位基因CPU1-H2的表达能降低大豆的磷吸收效率。
8.根据权利要求7所述的应用,其特征在于:抑制等位基因CPU1-H2的表达能降低大豆的生物量和产量。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2010087888A2 (en) * | 2009-01-28 | 2010-08-05 | The United States Of America, As Represented By The Secretary Of Agriculture | Development of low allergen soybean seeds using molecular markers for the p34 allele |
CN101921758A (zh) * | 2010-08-20 | 2010-12-22 | 南京农业大学 | 大豆耐低磷基因GmAPt的分子标记方法 |
CN102154322A (zh) * | 2011-03-14 | 2011-08-17 | 华南农业大学 | 基因GmEXPB2在提高大豆植株磷吸收效率方面的应用 |
CN108841832A (zh) * | 2018-06-12 | 2018-11-20 | 福建农林大学 | 磷酸盐转运子的转运协助因子GmPHF1b的应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010087888A2 (en) * | 2009-01-28 | 2010-08-05 | The United States Of America, As Represented By The Secretary Of Agriculture | Development of low allergen soybean seeds using molecular markers for the p34 allele |
CN101921758A (zh) * | 2010-08-20 | 2010-12-22 | 南京农业大学 | 大豆耐低磷基因GmAPt的分子标记方法 |
CN102154322A (zh) * | 2011-03-14 | 2011-08-17 | 华南农业大学 | 基因GmEXPB2在提高大豆植株磷吸收效率方面的应用 |
CN108841832A (zh) * | 2018-06-12 | 2018-11-20 | 福建农林大学 | 磷酸盐转运子的转运协助因子GmPHF1b的应用 |
Non-Patent Citations (1)
Title |
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A natural uORF variant confers phosphorus acquisition diversity in soybean;Zilong Guo;NATURE COMMUNICATIONS;第13卷(第3796期);第1-14页 * |
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