CN115215930B - 控制玉米种子总蛋白和类胡萝卜素含量的蛋白ptox1及其编码基因与应用 - Google Patents
控制玉米种子总蛋白和类胡萝卜素含量的蛋白ptox1及其编码基因与应用 Download PDFInfo
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Abstract
本发明公开了一种控制玉米种子总蛋白和类胡萝卜素含量的蛋白PTOX1及其编码基因与应用，属于植物基因工程技术领域。本发明首次研究发现，蛋白PTOX1及编码蛋白PTOX1的玉米PTOX1基因参与调控玉米种子的总蛋白质含量和类胡萝卜素含量，该基因的终止突变即可获得蛋白质含量提高的植株；超表达该基因可以提高玉米中的维生素A原含量。因此，蛋白PTOX1及编码蛋白PTOX1基因在植物育种和种质资源改良方面具有良好的应用前景。
Description
技术领域
本发明涉及植物基因工程技术领域，具体涉及一种控制玉米种子总蛋白和类胡萝卜素含量的蛋白PTOX1及其编码基因与应用。
背景技术
玉米是我国种植面积最大的作物，集粮、经、饲于一体，在我国粮食安全和经济发展中占有重要地位。玉米是重要的饲料原料，我国70％的玉米用于畜禽饲料加工，但在畜禽养殖过程中，饲料中蛋白需要达到11％到20％时才能满足营养需求，而目前普通玉米的蛋白含量只有7％到9％，因此必须在饲料中添加豆粕进行蛋白补充。但是我国的大豆和豆粕严重依赖于进口，极易受复杂的国际形势影响。因此，加快推动高蛋白玉米新品种选育，不仅具有重要的科学意义，还有重大的产业和经济价值，对保障我国畜牧业安全具有重要意义。
类胡萝卜素是人体必需的营养元素，是人体极易缺乏的维生素A的前体物质，与人类的健康密切相关。类胡萝卜素具有清除代谢过程中产生的自由基和活性氧，延缓衰老，提高机体的免疫力，抗癌防癌等活性。但人体自身不能合成类胡萝卜素，需要从日常饮食中获得。类胡萝卜素广泛存在于谷物、水果和蔬菜中，然而，大多数谷物中仅含有少量的类胡萝卜素，对于少食水果和蔬菜的人群，饮食中的类胡萝卜素含量远远不能满足人体需求，这一问题在发展中国家尤为普遍。因此，通过生物强化提高谷物中的类胡萝卜素含量是缓解维生素A匮乏的有效途径。玉米作为最广泛被食用的主要作物之一，培育高类胡萝卜素的玉米新品种对于维生素A缺乏人群膳食营养的改善，解决人体“隐性饥饿”的民生问题具有重要意义。
发明内容
针对上述现有技术，本发明的目的是提供一种控制玉米种子总蛋白和类胡萝卜素含量的蛋白PTOX1及其编码基因与应用。
为实现上述目的，本发明采用如下技术方案：
本发明的第一方面，提供一种蛋白，名称为PTOX1，来源于玉米属的玉米(Zea maysL.)。所述蛋白PTOX1为如下(A1)-(A3)任一所示的蛋白质：
(A1)由序列表中序列1所示的氨基酸序列组成的蛋白质；
(A2)将序列表中序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加，且与控制玉米种子总蛋白和类胡萝卜素含量相关的由序列1衍生的蛋白质；
(A3)在(A1)或(A2)中所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
其中，(A1)中序列1所示的氨基酸序列包括343个氨基酸残基，在该蛋白质序列中，疏水氨基酸占171个，亲水氨基酸占172个；碱性氨基酸占48个，酸性氨基酸占17个；该蛋白质的分子量为39.35KD，等电点为5.614。
(A2)中的蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。上述(b)中的蛋白质的编码基因可通过将序列表中序列6所示的cDNA序列中的编码区(第202-1233位)缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变。
为了使(A1)或(A2)中的蛋白质便于纯化，可在(A1)或(A2)的蛋白质的氨基末端或羧基末端连接上标签。所述标签可以为Poly-Arg(通常为6个RRRRR)，Poly-His(通常为6个HHHHHH)，FLAG(DYKDDDDK)，Strep-tag II(WSHPQFEK)或c-myc(EQKLISEEDL)。
本发明的第二方面，提供编码上述蛋白的核酸分子。
所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA、hnRNA或tRNA等。
优选的，所述核酸分子是如下(1)-(11)任一所示的DNA分子：
(1)序列表中序列2所示的DNA分子；
(2)序列表中序列3所示的DNA分子；
(3)序列表中序列4所示的DNA分子；
(4)序列表中序列5所示的DNA分子；
(5)序列表中序列6或编码区包括序列6第108-1139位所示的DNA分子；
(6)序列表中序列7或编码区包括序列7第108-665位所示的DNA分子；
(7)序列表中序列8或编码区包括序列8第108-1103位所示的DNA分子；
(8)序列表中序列9或编码区包括序列9第108-579位所示的DNA分子；
(9)除(1)或(5)以外的编码序列1所示蛋白的DNA分子；
(10)在严格条件下与(1)-(9)中任一限定的DNA分子杂交且编码与控制玉米种子总蛋白和类胡萝卜素含量相关的由序列1衍生的蛋白质的DNA分子；
(11)与(1)-(9)中任一限定的DNA序列具有80％以上同一性，且编码控制玉米种子总蛋白和类胡萝卜素含量相关的由序列1衍生的蛋白质的DNA分子。
其中，序列2为PTOX1基因在所述野生型玉米RP125基因组中的序列；序列3为PTOX1基因在突变体ptox1-1玉米基因组中的序列；序列4为PTOX1基因在突变体ptox1-2玉米基因组中的序列；序列5为PTOX1基因在突变体ptox1-3玉米基因组中的序列；序列6为PTOX1基因在所述野生型玉米中的cDNA序列(其中108-1139位为编码区序列)；序列7为PTOX1基因在所述突变体ptox1-1中的cDNA序列(其中第108-665位为编码区序列)；序列8为PTOX1基因在所述突变体ptox1-2中的cDNA序列(其中第108-1103位为编码区序列)；序列9为PTOX1基因在所述突变体ptox1-3中的cDNA序列(其中第108-579位为编码区序列)。
这里所述的“严格条件”是在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；或，0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码蛋白PTOX1的核苷酸序列具有80％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用计算机软件进行评价，例如可采用BLAST算法测定(Altschul et al.1990.Journal of Molecular Biology 215:403-410；Karlin andAltschul.1993.Proceedings of the National Academy of Sciences 90:5873-5877)。
本发明的第三方面，提供含有上述核酸分子的重组载体、表达盒、转基因细胞系或重组微生物。
所述重组载体可为重组表达载体，也可为重组克隆载体。
所述重组表达载体可用现有的植物表达载体构建。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等，如pGreen0029、pCAMBIA3301、pCAMBIA1300、pBI121、pBin19、pCAMBIA2301、pCAMBIA1301-UbiN或其它衍生植物表达载体。所述植物表达载体还可包含外源基因的3’端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3’端。使用所述基因构建重组表达载体时，在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子，例如花椰菜花叶病毒(CaMV)35S启动子、泛素基因Ubiquitin启动子(pUbi)、胁迫诱导型启动子rd29A等，它们可单独使用或与其它的植物启动子结合使用；此外，使用本发明的基因构建重组表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用重组表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因、具有抗性的抗生素标记物或是抗化学试剂标记基因等。也可不加任何选择性标记基因，直接以逆境筛选转化植株。
所述表达盒由能够启动所述基因表达的启动子，所述基因，以及转录终止序列组成。
所述转基因细胞系为转入所述基因的非繁殖材料。
本发明的第四方面，提供上述蛋白PTOX1、编码蛋白PTOX1的核酸分子、含有上述核酸分子的重组载体、表达盒、转基因细胞系或重组微生物在如下(1)或(2)中的应用：
(1)植物育种和/或制种；
(2)调控植物种子总蛋白和类胡萝卜素含量。
上述应用中，所述植物既可为单子叶植物，也可为双子叶植物。其中，所述单子叶植物可以为禾本科植物，具体如玉米。
优选的，所述类胡萝卜素包括：玉米素、玉米黄素、α-类胡萝卜素和β-类胡萝卜素。
本发明的第五方面，提供一种培育种子高蛋白含量的转基因植物的方法，包括以下步骤：
抑制受体植物中蛋白PTOX1的表达，得到转基因植物；所述转基因植物与所述受体植物相比种子总蛋白含量提高。
上述方法中，可以采用CRISPR/Cas9核酸酶对所述受体植物中所述编码基因的基因组DNA序列进行特异性剪切，使所述受体植物丧失表达有功能的所述蛋白的能力。
其中，所述CRISPR/Cas9核酸酶对所述受体植物中编码PTOX1蛋白的基因组DNA序列进行特异性剪切时的靶标片段为所述受体植物中编码PTOX1蛋白的基因组DNA序列中符合5’-NX-NGG-3’或5’-CCN-NX-3’序列排列规则的片段；N表示A、G、C和T中的任一种，14≤X≤30，且X为整数，NX表示X个连续的脱氧核糖核苷酸。更加具体的，所述靶标片段为所述受体植物中编码PTOX1蛋白的基因组DNA序列中的“5’-AGTCGGAGCTGACGACCCCATGG-3’”，即序列2的第439-461位。
本发明的第六方面，提供一种培育种子类胡萝卜素含量增加的转基因植物的方法，包括以下步骤：
向受体植物中导入蛋白PTOX1的编码基因，使蛋白PTOX1在受体植物中过表达，得到转基因植物；所述转基因植物与所述受体植物相比种子类胡萝卜素含量增加。
上述方法中，为实现蛋白PTOX1在受体植物中过表达，可以采用35S和/或Ubiquitin和/或编码基因自身启动子对所述受体植物中所述编码基因的蛋白进行高表达，使所述受体植物高表达有功能的所述蛋白的能力。所述编码基因可通过重组表达载体pCAMBIA1300-PTOX1或pCAMBIA3301-PTOX1导入所述受体植物。
上述方法中，所述受体植物既可为单子叶植物，也可为双子叶植物。其中，所述单子叶植物可以为禾本科植物，具体如玉米。
本发明的第七方面，提供一种提高玉米种子总蛋白含量的方法，包括使玉米中序列1所示蛋白的表达量减少或不表达；和/或使玉米中序列1所示蛋白的活性降低或丧失的步骤。
本发明的第八方面，提供一种提高玉米种子类胡萝卜素含量的方法，包括使玉米中序列1所示蛋白的表达量增加和/或活性提高的步骤。
本发明的有益效果：
(1)本发明的玉米PTOX1基因分离自玉米，作为玉米的内源基因，对玉米的基因工程改造非常有利。
(2)玉米PTOX1基因参与调控玉米种子的总蛋白质含量和类胡萝卜素含量，该基因的终止突变即可获得蛋白质含量提高的植株，超表达该基因可以提高玉米中的维生素A原含量。PTOX1基因在植物育种和种质资源改良方面具有良好的应用前景。
附图说明
图1为野生型材料与突变体材料果穗表型对比以及类胡萝卜含量和蛋白含量比较。A：野生型材料和突变体Zmptox1-1的植株比较；B：野生型材料和突变体Zmptox1-1的果穗对比；C：野生型材料与突变体材料Zmptox1-1的类胡萝卜素含量比较；D：野生型材料与突变体材料Zmptox1-1的总蛋白含量比较。
图2为PTOX1基因的图位克隆及等位性验证。A：PTOX1基因的图位克隆；B：PTOX1基因基因结构示意图；C：野生型、等位突变体以及等位突变体材料杂交后代果穗表型比较；D：野生型、等位突变体以及等位突变体材料杂交后代叶黄素含量对比；E：野生型、等位突变体以及等位突变体材料杂交后代玉米黄素含量对比。
图3为转基因受体(B104)及超表达材料(OE-1、OE-2、OE-3)中ZmPTOX1基因的表达水平及种子类胡萝卜素含量对比关系。A:转基因受体(B104)及超表达材料(OE-1、OE-2、OE-3)中ZmPTOX1基因的表达水平差异；B：转基因受体(B104)及超表达材料(OE-1、OE-2、OE-3)种子中叶黄素含量比较；C：转基因受体(B104)及超表达材料(OE-1、OE-2、OE-3)种子中玉米黄素含量比较；D：转基因受体(B104)及超表达材料(OE-1、OE-2、OE-3)种子中α-胡萝卜素含量比较；E：转基因受体(B104)及超表达材料(OE-1、OE-2、OE-3)种子中β-胡萝卜素含量比较。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
正如背景技术部分所介绍的，玉米作为饲料原料时，选育高蛋白玉米新品种具有重大的产业和经济价值；提高玉米种子的类胡萝卜素含量则可以改善维生素A缺乏人群膳食营养。因此，开发控制玉米种子总蛋白和类胡萝卜素含量的新的基因资源将在植物育种领域发挥重要的作用。
基于此，本发明采用图位克隆的策略，用玉米白粒突变体Zmptox1-1与玉米自交系B73组配F1和F2群体，将控制这一突变性状的基因定位到玉米二号染色体2.69Mb到2.79Mb之间，以公布的B73基因组测序结果为参考物理距离约为0.10Mb，共包括2个基因。其中基因号为Zm00001d001909的序列在突变体与野生型之间存在差异，突变体有一个G到A的碱基替换，导致氨基酸由色氨酸(Trp)变为终止密码子。突变体Zmptox1-2中基因号为Zm00001d001909的第2个内含子第一个碱基由G突变成A，导致基因第2个外显子发生选择性剪切，跟PTOX1基因在野生型玉米自交系RP125中的cDNA序列相比其第396-431位共36个碱基丢失，致使12个编码氨基酸的丢失。突变体Zmptox1-3中基因号为Zm00001d001909的基因的618位碱基由G突变成A，导致Zm00001d001909的基因第3个内含子的48个碱基保留，致使编码PTOX1基因的提前终止。遗传分析表明，突变体Zmptox1-1、Zmptox1-2和Zmptox1-3互为等位突变体，等位材料及杂交后代均为白粒表型。因此Zm00001d001909基因即是控制玉米种子由黄变白的目的基因，命名为PTOX1。。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案，以下将结合具体的实施例详细说明本申请的技术方案。
本发明实施例和对比例中所用的试验材料均为本领域常规的试验材料，均可通过商业渠道购买得到。未注明详细条件的实验方法是按照常规试验方法或按照供应商所建议的操作说明书进行的。其中：
所述野生型玉米自交系RP125、突变体Zmptox1-1、Zmptox1-2、Zmptox1-3、常规材料B73、玉米转基因受体B104为本实验室收集和保存，公众可以从山东农业大学获得，以重复本实验。玉米基因组测序信息参考MaizeGDB数据库，该数据库链接如下：http：//www.maizegdb.org/。
实施例1：控制玉米种子总蛋白和类胡萝卜素含量基因PTOX1的图位克隆
一、玉米突变体Zmptox1-1的表型
与正常植株(Wild type)相比，玉米突变体Zmptox1-1在植株整体形态方面没有异常(图1中A)，但其种子由黄色变成白色(图1中B)，玉米黄素、叶黄素、α-胡萝卜素和β-胡萝卜素等多种类胡萝卜素含量极显著降低(表1，图1中C)，其中玉米黄素平均降低了4.4倍，叶黄素平均降低了7.1倍，α-胡萝卜素平均降低了10倍，β-胡萝卜素平均降低了16.7倍，但种子总蛋白质含量极显著增加(表1，图1中D)，由8.76％(Wild type)增加到13.32％(Zmptox1-1)百粒重没有明显差异。因此认为突变体种子由黄变白是由于种子中类胡萝卜素含量极显著降低导致的。
表1：黄粒野生型与白粒突变体种子类胡萝卜素及总蛋白含量的测定结果
二、遗传定位群体的构建
我们将白粒突变体Zmptox1-1与玉米自交系B73杂交组配F1，F1代种子种子为黄色、植株正常，F1代自交果穗上种子颜色出现黄粒、白粒分离，随机对1个果穗上的种子进行表型鉴定，利用适合性卡方测验分析黄色和白色种子表型分离比符合3∶1的孟德尔分离定律(表2)，说明Zmptox1-1种子变白表型由单隐性基因控制。我们将F2群体扩大并作为遗传定位群体用于目的基因的图位克隆。
表2：F2分离群体的适合性卡方测验
三、PTOX1基因的图位克隆
首先，以构建遗传定位群体的两个亲本，突变体Zmptox1-1和B73的基因组DNA为模板，用玉米全基因组引物筛选在突变体Zmptox1-1和B73之间有多态性的引物。然后，从F2群体中选取极端表型的白粒单株和黄粒正常单株各20株，验证多态性引物是否与粒色性状连锁。筛选出连锁引物，用于对群体中93个单株基因型的测定，结合种子颜色正常与否的表型筛选出基因型与表型不符的为交换单株，并根据不同引物筛选出的交换单株个数的不同，依照减少趋势确定定位区间，由此将PTOX1基因定位于玉米二号染色体的引物标记Ind-22和umc2245之间。在Ind-22和umc2245之间继续开发多态性分子标记，并用于检测F2群体的所有单株(850株)，最终将PTOX1基因定位在标记Ind-268(2.69Mb)和Ind-270(2.79Mb)之间，参考已公布的玉米自交系B73基因组测序结果，物理距离约为0.10Mb(图2中A)。其中，用于基因定位的分子标记引物序列如表3所示(序列表中序列10-序列31)。
表3：用于基因定位的分子标记引物及PTOX1基因扩增引物序列
四、PTOX1基因的克隆
参考玉米基因组测序信息，定位区间的0.1Mb范围内共包括2个基因，分别是Zm00001d001908和Zm00001d001909。以野生型和突变体的基因组DNA为模板，扩增这2个基因并比较其序列差异，发现只有基因号为Zm00001d001909的序列在突变体与野生型之间存在差异，突变体有一个G到A的碱基替换，导致氨基酸由色氨酸(Trp)变为终止密码子。因此推测Zm00001d001909基因可能就是Zmptox1-1的候选基因，图2中B展示了Zm00001d001909的基因结构，由9个外显子(黑色方框)和8个内含子(黑色线段)组成。
以所述野生型玉米自交系材料RP125的基因组DNA为模板，利用Zm00001d001909基因的引物primer_ptox1(表3)进行PCR扩增，所得PCR产物的序列为序列表中序列2，序列2即为PTOX1基因在野生型玉米自交系RP125基因组中的序列；以所述突变体Zmptox1-1材料的基因组DNA为模板，同样采用引物primer_ptox1进行PCR扩增，所得PCR产物的序列为序列表中序列3，序列3即为PTOX1基因在突变体Zmptox1-1基因组中的序列，具体为：跟序列2比其1207位碱基由G突变为A。提取野生型自交系材料的总RNA，反转录为cDNA，采用引物primer_ptox1进行PCR扩增，所得PCR产物的序列为序列表中序列6，序列6即为PTOX1基因在野生型玉米自交系RP125中的cDNA序列，其中第108-1139位为编码区序列；提取突变体Zmptox1-1材料的总RNA，反转录为cDNA，同样采用引物primer_ptox1进行PCR扩增，所得PCR产物的序列为序列表中序列7，序列7即为PTOX1基因在在突变体Zmptox1-1中的cDNA序列，其中第665位碱基G突变为A，跟野生型玉米自交系RP125相比，Zmptox1-1编码的PTOX1蛋白提前终止，编码蛋白的氨基酸由343个变为185个，致使蛋白功能丧失或/和减弱。序列2和序列6均编码序列表中序列1所示的PTOX1蛋白。
五、PTOX1基因的等位验证
在我们构建的突变体库中，另找到2份跟Zmptox1-1表型相似的突变体材料，其种子也是由黄粒变成白粒，分别命名为Zmptox1-2和Zmptox1-3(图2中C)，分别构建Zmptox1-1×Zmptox1-2、Zmptox1-1×Zmptox1-3、Zmptox1-2×Zmptox1-3杂交的遗传材料，杂交当代种子及自交后代种子均为白粒表型，说明这三份材料为同一个基因的等位变异材料，提取Zmptox1-2和Zmptox1-3的基因组DNA，并进行ZmPTOX1的基因扩增，测序后数据分析发现：以所述突变体Zmptox1-2材料的基因组DNA为模板，同样采用引物primer_ptox1进行PCR扩增，所得PCR产物的序列为序列表中序列4，序列4即为PTOX1基因在突变体Zmptox1-2基因组中的序列，具体为：跟序列2比其524位碱基由G突变为A，该突变是基因号为Zm00001d001909的第2个内含子第一个碱基由G突变成A，推测该突变可能导致基因的选择性剪切，致使编码蛋白功能发生改变。提取突变体Zmptox1-2材料的总RNA，反转录为cDNA，同样采用引物primer_ptox1进行PCR扩增，所得PCR产物的序列为序列表中序列8，序列8即为PTOX1基因在在突变体Zmptox1-2中的cDNA序列，跟PTOX1基因在野生型玉米自交系RP125中的cDNA序列相比其第396-431位共36个碱基丢失，丢失的序列位于基因号为Zm00001d001909的第2个外显子上，导致编码的PTOX1蛋白缺失12个氨基酸。以所述突变体Zmptox1-3材料的基因组DNA为模板，同样采用引物primer_ptox1进行PCR扩增，所得PCR产物的序列为序列表中序列5，序列5即为PTOX1基因在突变体Zmptox1-3基因组中的序列，具体为：跟序列2比其751位碱基由G突变为A，该突变是基因号为Zm00001d001909的第3个内含子第一个碱基由G突变成A，推测该突变可能导致基因的选择性剪切，致使编码蛋白功能发生改变。提取突变体Zmptox1-3材料的总RNA，反转录为cDNA，同样采用引物primer_ptox1进行PCR扩增，所得PCR产物的序列为序列表中序列9，序列9即为PTOX1基因在在突变体Zmptox1-3中的cDNA序列，跟PTOX1基因在野生型玉米自交系RP125中的cDNA序列相比，其中第526-573位共48个碱基为内含子保留序列，保留的序列位于基因号为Zm00001d001909的第3个内含子上，导致编码的PTOX1蛋白提前终止，致使蛋白功能丧失或/和减弱。
实施例2：玉米ZmPTOX1基因调控类胡萝卜含量
(1)野生型材料与突变体材料ptox1-1类胡萝卜素含量检测
突变体ptox1-1以及等位材料ptox1-2、ptox1-3的胚乳淀粉层较野生型胚乳淀粉层的颜色浅(图2中C)，而影响种子淀粉层颜色最主要的色素为类胡萝卜素。利用高效液相色谱分析和测定野生型种子、ptox1突变体种子(Zmptox1-1、Zmptox1-2和Zmptox1-3)和ptox1双突变体种子(Zmptox1-1×Zmptox1-2、Zmptox1-1×Zmptox1-3、Zmptox1-2×Zmptox1-3)中的类胡萝卜素含量，ptox1突变体中不同种类胡萝卜素含量均低于野生型对照，同时发现双突变体材料中类胡萝卜素含量均低于野生型，并且均低于ptox1单突变体(图2中D和E)。
(2)野生型材料和超表达材料种子类胡萝卜素含量检测
构建ZmPTOX1基因超表达载体，将其转化到野生型B104的愈伤组织中，再继续培养获得超表达ZmPTOX1基因的植株OE，用qRT-ptox1引物测定超表达植株中ZmPTOX1基因相对表达水平，OE超表达材料ZmPTOX1基因相对表达量均高于野生型B104(图3中A)。利用高效液相色谱分析和测定野生型B104和超表达材料OE-1、OE-2和OE-3种子中的类胡萝卜素含量，过表达材料中叶黄素、玉米黄素、α-胡萝卜素和β-胡萝卜素等四种类胡萝卜素含量均高于野生型(图3中B、C、D和E)。三个独立转化转基因植株OE-1、OE-2和OE-3种子中玉米黄素含量比受体材料B104平均增加2.6倍，叶黄素平均增加2.1倍，α-胡萝卜素平均增加2.5倍，β-胡萝卜素平均增加5.3倍(表4)。
表4：过表达转基因T1代受体材料B104成熟种子类胡萝卜素含量测定
以上证明，玉米PTOX1基因是一种控制玉米种子总蛋白和类胡萝卜素含量的功能基因。
综合以上各实施例的研究结果，可见：通过图位克隆和转基因功能验证，本发明克隆的PTOX1基因是一种控制种子总蛋白和类胡萝卜素含量的功能基因，该基因编码的蛋白突变以后，会导致玉米种子总蛋白显著增加、类胡萝卜素显著降低，超表达PTOX1基因，可显著提高玉米种子中类胡萝卜素含量。本发明为植物特别是玉米的品质性状研究提供了新的基因资源，选择具有ptox1/ptox1或PTOX1/PTOX1优良等位基因型的自交系作为供体用于玉米的遗传改良，将在玉米育种领域的应用中发挥重要作用。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
SEQUENCE LISTING
<110> 山东农业大学
<120> 控制玉米种子总蛋白和类胡萝卜素含量的蛋白PTOX1及其编码基因与应用
<130> 2021
<160> 31
<170> PatentIn version 3.5
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<400> 2
gactctccac catcactcgc gcgccggcgc cgctcacacg cccatttcca ccccacaaga 60
cccacacgga atcccggcac cctacctctc caacacgcac cagcatcatg gcggtggctt 120
cgacctcgcc gctatccgcc aagcccgcca cggccccctc gccgcccgct ccggtgtccg 180
ggttcctcgc tctccccgcc cgccgcggcc gcgcaacgcg cctcggctcc gccgccgcgt 240
ggtacgccgc gcgaagccta gcccgcctgc tccttctatc tccccccaca ccgtccctgt 300
gctgttctcc cgcaataacc cggacctgtg caggaggagg cttcgcgtgg aggcgatctg 360
gaagcagcag gagaagcggg cggaggtgtc cgtcgaggaa tccgcccccg tcagggaggc 420
cgccgcgccc ctggacggag tcggagctga cgaccccatg gttccttcct cggacgagag 480
ctgggtggtc aggctcgagc agtcggtcaa cattttcctc acggtgattc agcaacgcac 540
aattcacccc gatcccatcc cccacccccc acccacccac cccccaccca cgcacacaac 600
tgcaaattcc tgtagccaaa catctcgatg ttctcacgtg ctattccttt ccacaggaat 660
cggtgattat agtactcaat accgtgtacc gtgatcggaa ctacgccagg ttttttgtgc 720
tggagacgat tgccagggtg ccgtatttcg gtgagggctc cgttttgtca tatgcttgca 780
gttgcggtgt atgaaaaggt ccctactgag ttttccagcg ttggtttcgg tcgtgccgtt 840
gctgttcttt cttttttact ctgcttggtt atgaacagcg ttcatatcgg tgcttcacat 900
gtatgaaacc tttggctggt ggagacgagc tgattatcta aaagttcact ttgcgcagag 960
cttgaacgag tttcatcatc tcttgatcat ggaagtacgc atctttccag taaaatccat 1020
gtgtgccaga tcttcctttc agtgctatta tgtcaaagtt atatcacggc ttaattttat 1080
tccgttctgt tggtttgact aattaatttt ccatattaat tatcagttca tttctttaca 1140
tgaattcatc ctacttatgg attcagccct tggctcatat caggaattgg gtggcaacgc 1200
tatatggatt gatcgtttcc ttgctcggtt tatggcgttt ttttactact tcatgactgt 1260
tgcgatgtac atgttgagcc cacgaatggc atgtaagtac tcttaacaat tatgaagtaa 1320
atgtttgttt tctattacca tgcccttttg tgatgataac ccttgcatct acgaatggag 1380
tacagatcac ttctctgaat gtgtggagag acatgcgtac tccacctatg ataagttcct 1440
caagctccat gaaggtaaag ctaacttaat ggccttctat tatttactgg gcaagtcagc 1500
tttgctactg tttttatata ccaaataaat agatattaag atatttattc aaatatgatt 1560
attgtttatt tctaaacact aagatacgtg tggtctagtg gttagatttc tggagctgag 1620
ggtgtggatt caagtgctca ctctgcactt ttttgtgtgg tgtggtagct gcgtgggtgg 1680
ggatgggtgc tgagcagacg cagtagctga ctacgtgggc gctagggtcc acagtgcagt 1740
agctgagaga agggcgagag aaccaatcag agagagtggg tgggcctaga gtgtcagcgt 1800
gtccacaggg cagtagttga gaggggcggg agaaccagtc ggggcgcgtg ggataggctt 1860
agagtgtcag cgtggagggt gaagttgtgg tagcaccagt tgcctacatt aagttattaa 1920
tagagtagta tagataaata ggtattgaga tatttattca aatataatta ttgtttattt 1980
ataaacacta agatatgtgt ggtctggtgg ttacactcta atgtttggag caaggggttg 2040
tgggttcgag tgctcgcttt gcactattat ttacgcggtg tggtagcacg gagggtgaag 2100
ccgtggtagc accaggtgtt cacattaggt ttttattctt aatagattag tatagattta 2160
tatgatttct ggttgttgaa cctggcttag gatttttatc catgtaaggc agtgagctag 2220
tgcctagtgc acttctgaga gttgctctcc actagtgcgg gagcctccga caatggatct 2280
gccctttaaa tcagtgggcc agtaaaggtc aaatcaattt attttatgct attgctgtca 2340
tgtttcaaaa ttttcagttt ctagttctca gaaaccatcc agaactctaa aatcatttcc 2400
tatcataaat tcttattcaa acatttttat gtcttaatag tgttgagacc caacttgtaa 2460
ttacagaagc tgctaattca ttttacctgc acttccactt ggatgatgta atttttctgt 2520
ttcctttcaa cagaggaatt gaaaacacta ccagctccag aggcagcatt gaactattac 2580
ctgaatgagg acctttactt atttggtatt cgcttatgtt tcttaaaatc ttaaagttat 2640
acatgaacag ttgataccta ttctaattgt ttacatacct cttttgcccc tcagatgagt 2700
ttcagacaac aagaattcca tgttctagga ggcctaaaat aggtaaactt gctgttctct 2760
cttttgtccc ctcaataggt gtgtattcct tttttttgta cttcttcgga aggttggaac 2820
taaacatgtt agctgcgttt gtgaaattta actggctgct ccatcatgga gtcgtaaaat 2880
ctcgattttg attccttgtt tactggattc tggcctcctg acgtgaacca tttgtaccgc 2940
ctatctattt ctctgccata agactgttgt aagtactcac tgattcccat atgcagataa 3000
cttgtatgat gtattcgtca atatacgaga tgacgaggca gagcactgca agacaatgaa 3060
ggcatgtcaa acacatggaa ctcttcgttc tcctcactca atgccgaact gcttagaagc 3120
tgatacagaa tgtgtaatac ctgaaaacga ttgtgaaggt attgtggact gtgtcaaaaa 3180
gtcccttaca aagtaaatag tatgtagaat cctctttcgt gtgttttcag gcatcatagt 3240
atacaaaagt acaaattctg gatgatatcc tctctttttt cctgtgtatc tttgttaaag 3300
caaaatccgg gtca 3314
<210> 3
<211> 3314
<212> DNA
<213> Zea mays L
<400> 3
gactctccac catcactcgc gcgccggcgc cgctcacacg cccatttcca ccccacaaga 60
cccacacgga atcccggcac cctacctctc caacacgcac cagcatcatg gcggtggctt 120
cgacctcgcc gctatccgcc aagcccgcca cggccccctc gccgcccgct ccggtgtccg 180
ggttcctcgc tctccccgcc cgccgcggcc gcgcaacgcg cctcggctcc gccgccgcgt 240
ggtacgccgc gcgaagccta gcccgcctgc tccttctatc tccccccaca ccgtccctgt 300
gctgttctcc cgcaataacc cggacctgtg caggaggagg cttcgcgtgg aggcgatctg 360
gaagcagcag gagaagcggg cggaggtgtc cgtcgaggaa tccgcccccg tcagggaggc 420
cgccgcgccc ctggacggag tcggagctga cgaccccatg gttccttcct cggacgagag 480
ctgggtggtc aggctcgagc agtcggtcaa cattttcctc acggtgattc agcaacgcac 540
aattcacccc gatcccatcc cccacccccc acccacccac cccccaccca cgcacacaac 600
tgcaaattcc tgtagccaaa catctcgatg ttctcacgtg ctattccttt ccacaggaat 660
cggtgattat agtactcaat accgtgtacc gtgatcggaa ctacgccagg ttttttgtgc 720
tggagacgat tgccagggtg ccgtatttcg gtgagggctc cgttttgtca tatgcttgca 780
gttgcggtgt atgaaaaggt ccctactgag ttttccagcg ttggtttcgg tcgtgccgtt 840
gctgttcttt cttttttact ctgcttggtt atgaacagcg ttcatatcgg tgcttcacat 900
gtatgaaacc tttggctggt ggagacgagc tgattatcta aaagttcact ttgcgcagag 960
cttgaacgag tttcatcatc tcttgatcat ggaagtacgc atctttccag taaaatccat 1020
gtgtgccaga tcttcctttc agtgctatta tgtcaaagtt atatcacggc ttaattttat 1080
tccgttctgt tggtttgact aattaatttt ccatattaat tatcagttca tttctttaca 1140
tgaattcatc ctacttatgg attcagccct tggctcatat caggaattgg gtggcaacgc 1200
tatatgaatt gatcgtttcc ttgctcggtt tatggcgttt ttttactact tcatgactgt 1260
tgcgatgtac atgttgagcc cacgaatggc atgtaagtac tcttaacaat tatgaagtaa 1320
atgtttgttt tctattacca tgcccttttg tgatgataac ccttgcatct acgaatggag 1380
tacagatcac ttctctgaat gtgtggagag acatgcgtac tccacctatg ataagttcct 1440
caagctccat gaaggtaaag ctaacttaat ggccttctat tatttactgg gcaagtcagc 1500
tttgctactg tttttatata ccaaataaat agatattaag atatttattc aaatatgatt 1560
attgtttatt tctaaacact aagatacgtg tggtctagtg gttagatttc tggagctgag 1620
ggtgtggatt caagtgctca ctctgcactt ttttgtgtgg tgtggtagct gcgtgggtgg 1680
ggatgggtgc tgagcagacg cagtagctga ctacgtgggc gctagggtcc acagtgcagt 1740
agctgagaga agggcgagag aaccaatcag agagagtggg tgggcctaga gtgtcagcgt 1800
gtccacaggg cagtagttga gaggggcggg agaaccagtc ggggcgcgtg ggataggctt 1860
agagtgtcag cgtggagggt gaagttgtgg tagcaccagt tgcctacatt aagttattaa 1920
tagagtagta tagataaata ggtattgaga tatttattca aatataatta ttgtttattt 1980
ataaacacta agatatgtgt ggtctggtgg ttacactcta atgtttggag caaggggttg 2040
tgggttcgag tgctcgcttt gcactattat ttacgcggtg tggtagcacg gagggtgaag 2100
ccgtggtagc accaggtgtt cacattaggt ttttattctt aatagattag tatagattta 2160
tatgatttct ggttgttgaa cctggcttag gatttttatc catgtaaggc agtgagctag 2220
tgcctagtgc acttctgaga gttgctctcc actagtgcgg gagcctccga caatggatct 2280
gccctttaaa tcagtgggcc agtaaaggtc aaatcaattt attttatgct attgctgtca 2340
tgtttcaaaa ttttcagttt ctagttctca gaaaccatcc agaactctaa aatcatttcc 2400
tatcataaat tcttattcaa acatttttat gtcttaatag tgttgagacc caacttgtaa 2460
ttacagaagc tgctaattca ttttacctgc acttccactt ggatgatgta atttttctgt 2520
ttcctttcaa cagaggaatt gaaaacacta ccagctccag aggcagcatt gaactattac 2580
ctgaatgagg acctttactt atttggtatt cgcttatgtt tcttaaaatc ttaaagttat 2640
acatgaacag ttgataccta ttctaattgt ttacatacct cttttgcccc tcagatgagt 2700
ttcagacaac aagaattcca tgttctagga ggcctaaaat aggtaaactt gctgttctct 2760
cttttgtccc ctcaataggt gtgtattcct tttttttgta cttcttcgga aggttggaac 2820
taaacatgtt agctgcgttt gtgaaattta actggctgct ccatcatgga gtcgtaaaat 2880
ctcgattttg attccttgtt tactggattc tggcctcctg acgtgaacca tttgtaccgc 2940
ctatctattt ctctgccata agactgttgt aagtactcac tgattcccat atgcagataa 3000
cttgtatgat gtattcgtca atatacgaga tgacgaggca gagcactgca agacaatgaa 3060
ggcatgtcaa acacatggaa ctcttcgttc tcctcactca atgccgaact gcttagaagc 3120
tgatacagaa tgtgtaatac ctgaaaacga ttgtgaaggt attgtggact gtgtcaaaaa 3180
gtcccttaca aagtaaatag tatgtagaat cctctttcgt gtgttttcag gcatcatagt 3240
atacaaaagt acaaattctg gatgatatcc tctctttttt cctgtgtatc tttgttaaag 3300
caaaatccgg gtca 3314
<210> 4
<211> 3314
<212> DNA
<213> Zea mays L
<400> 4
gactctccac catcactcgc gcgccggcgc cgctcacacg cccatttcca ccccacaaga 60
cccacacgga atcccggcac cctacctctc caacacgcac cagcatcatg gcggtggctt 120
cgacctcgcc gctatccgcc aagcccgcca cggccccctc gccgcccgct ccggtgtccg 180
ggttcctcgc tctccccgcc cgccgcggcc gcgcaacgcg cctcggctcc gccgccgcgt 240
ggtacgccgc gcgaagccta gcccgcctgc tccttctatc tccccccaca ccgtccctgt 300
gctgttctcc cgcaataacc cggacctgtg caggaggagg cttcgcgtgg aggcgatctg 360
gaagcagcag gagaagcggg cggaggtgtc cgtcgaggaa tccgcccccg tcagggaggc 420
cgccgcgccc ctggacggag tcggagctga cgaccccatg gttccttcct cggacgagag 480
ctgggtggtc aggctcgagc agtcggtcaa cattttcctc acgatgattc agcaacgcac 540
aattcacccc gatcccatcc cccacccccc acccacccac cccccaccca cgcacacaac 600
tgcaaattcc tgtagccaaa catctcgatg ttctcacgtg ctattccttt ccacaggaat 660
cggtgattat agtactcaat accgtgtacc gtgatcggaa ctacgccagg ttttttgtgc 720
tggagacgat tgccagggtg ccgtatttcg gtgagggctc cgttttgtca tatgcttgca 780
gttgcggtgt atgaaaaggt ccctactgag ttttccagcg ttggtttcgg tcgtgccgtt 840
gctgttcttt cttttttact ctgcttggtt atgaacagcg ttcatatcgg tgcttcacat 900
gtatgaaacc tttggctggt ggagacgagc tgattatcta aaagttcact ttgcgcagag 960
cttgaacgag tttcatcatc tcttgatcat ggaagtacgc atctttccag taaaatccat 1020
gtgtgccaga tcttcctttc agtgctatta tgtcaaagtt atatcacggc ttaattttat 1080
tccgttctgt tggtttgact aattaatttt ccatattaat tatcagttca tttctttaca 1140
tgaattcatc ctacttatgg attcagccct tggctcatat caggaattgg gtggcaacgc 1200
tatatggatt gatcgtttcc ttgctcggtt tatggcgttt ttttactact tcatgactgt 1260
tgcgatgtac atgttgagcc cacgaatggc atgtaagtac tcttaacaat tatgaagtaa 1320
atgtttgttt tctattacca tgcccttttg tgatgataac ccttgcatct acgaatggag 1380
tacagatcac ttctctgaat gtgtggagag acatgcgtac tccacctatg ataagttcct 1440
caagctccat gaaggtaaag ctaacttaat ggccttctat tatttactgg gcaagtcagc 1500
tttgctactg tttttatata ccaaataaat agatattaag atatttattc aaatatgatt 1560
attgtttatt tctaaacact aagatacgtg tggtctagtg gttagatttc tggagctgag 1620
ggtgtggatt caagtgctca ctctgcactt ttttgtgtgg tgtggtagct gcgtgggtgg 1680
ggatgggtgc tgagcagacg cagtagctga ctacgtgggc gctagggtcc acagtgcagt 1740
agctgagaga agggcgagag aaccaatcag agagagtggg tgggcctaga gtgtcagcgt 1800
gtccacaggg cagtagttga gaggggcggg agaaccagtc ggggcgcgtg ggataggctt 1860
agagtgtcag cgtggagggt gaagttgtgg tagcaccagt tgcctacatt aagttattaa 1920
tagagtagta tagataaata ggtattgaga tatttattca aatataatta ttgtttattt 1980
ataaacacta agatatgtgt ggtctggtgg ttacactcta atgtttggag caaggggttg 2040
tgggttcgag tgctcgcttt gcactattat ttacgcggtg tggtagcacg gagggtgaag 2100
ccgtggtagc accaggtgtt cacattaggt ttttattctt aatagattag tatagattta 2160
tatgatttct ggttgttgaa cctggcttag gatttttatc catgtaaggc agtgagctag 2220
tgcctagtgc acttctgaga gttgctctcc actagtgcgg gagcctccga caatggatct 2280
gccctttaaa tcagtgggcc agtaaaggtc aaatcaattt attttatgct attgctgtca 2340
tgtttcaaaa ttttcagttt ctagttctca gaaaccatcc agaactctaa aatcatttcc 2400
tatcataaat tcttattcaa acatttttat gtcttaatag tgttgagacc caacttgtaa 2460
ttacagaagc tgctaattca ttttacctgc acttccactt ggatgatgta atttttctgt 2520
ttcctttcaa cagaggaatt gaaaacacta ccagctccag aggcagcatt gaactattac 2580
ctgaatgagg acctttactt atttggtatt cgcttatgtt tcttaaaatc ttaaagttat 2640
acatgaacag ttgataccta ttctaattgt ttacatacct cttttgcccc tcagatgagt 2700
ttcagacaac aagaattcca tgttctagga ggcctaaaat aggtaaactt gctgttctct 2760
cttttgtccc ctcaataggt gtgtattcct tttttttgta cttcttcgga aggttggaac 2820
taaacatgtt agctgcgttt gtgaaattta actggctgct ccatcatgga gtcgtaaaat 2880
ctcgattttg attccttgtt tactggattc tggcctcctg acgtgaacca tttgtaccgc 2940
ctatctattt ctctgccata agactgttgt aagtactcac tgattcccat atgcagataa 3000
cttgtatgat gtattcgtca atatacgaga tgacgaggca gagcactgca agacaatgaa 3060
ggcatgtcaa acacatggaa ctcttcgttc tcctcactca atgccgaact gcttagaagc 3120
tgatacagaa tgtgtaatac ctgaaaacga ttgtgaaggt attgtggact gtgtcaaaaa 3180
gtcccttaca aagtaaatag tatgtagaat cctctttcgt gtgttttcag gcatcatagt 3240
atacaaaagt acaaattctg gatgatatcc tctctttttt cctgtgtatc tttgttaaag 3300
caaaatccgg gtca 3314
<210> 5
<211> 3314
<212> DNA
<213> Zea mays L
<400> 5
gactctccac catcactcgc gcgccggcgc cgctcacacg cccatttcca ccccacaaga 60
cccacacgga atcccggcac cctacctctc caacacgcac cagcatcatg gcggtggctt 120
cgacctcgcc gctatccgcc aagcccgcca cggccccctc gccgcccgct ccggtgtccg 180
ggttcctcgc tctccccgcc cgccgcggcc gcgcaacgcg cctcggctcc gccgccgcgt 240
ggtacgccgc gcgaagccta gcccgcctgc tccttctatc tccccccaca ccgtccctgt 300
gctgttctcc cgcaataacc cggacctgtg caggaggagg cttcgcgtgg aggcgatctg 360
gaagcagcag gagaagcggg cggaggtgtc cgtcgaggaa tccgcccccg tcagggaggc 420
cgccgcgccc ctggacggag tcggagctga cgaccccatg gttccttcct cggacgagag 480
ctgggtggtc aggctcgagc agtcggtcaa cattttcctc acggtgattc agcaacgcac 540
aattcacccc gatcccatcc cccacccccc acccacccac cccccaccca cgcacacaac 600
tgcaaattcc tgtagccaaa catctcgatg ttctcacgtg ctattccttt ccacaggaat 660
cggtgattat agtactcaat accgtgtacc gtgatcggaa ctacgccagg ttttttgtgc 720
tggagacgat tgccagggtg ccgtatttcg atgagggctc cgttttgtca tatgcttgca 780
gttgcggtgt atgaaaaggt ccctactgag ttttccagcg ttggtttcgg tcgtgccgtt 840
gctgttcttt cttttttact ctgcttggtt atgaacagcg ttcatatcgg tgcttcacat 900
gtatgaaacc tttggctggt ggagacgagc tgattatcta aaagttcact ttgcgcagag 960
cttgaacgag tttcatcatc tcttgatcat ggaagtacgc atctttccag taaaatccat 1020
gtgtgccaga tcttcctttc agtgctatta tgtcaaagtt atatcacggc ttaattttat 1080
tccgttctgt tggtttgact aattaatttt ccatattaat tatcagttca tttctttaca 1140
tgaattcatc ctacttatgg attcagccct tggctcatat caggaattgg gtggcaacgc 1200
tatatggatt gatcgtttcc ttgctcggtt tatggcgttt ttttactact tcatgactgt 1260
tgcgatgtac atgttgagcc cacgaatggc atgtaagtac tcttaacaat tatgaagtaa 1320
atgtttgttt tctattacca tgcccttttg tgatgataac ccttgcatct acgaatggag 1380
tacagatcac ttctctgaat gtgtggagag acatgcgtac tccacctatg ataagttcct 1440
caagctccat gaaggtaaag ctaacttaat ggccttctat tatttactgg gcaagtcagc 1500
tttgctactg tttttatata ccaaataaat agatattaag atatttattc aaatatgatt 1560
attgtttatt tctaaacact aagatacgtg tggtctagtg gttagatttc tggagctgag 1620
ggtgtggatt caagtgctca ctctgcactt ttttgtgtgg tgtggtagct gcgtgggtgg 1680
ggatgggtgc tgagcagacg cagtagctga ctacgtgggc gctagggtcc acagtgcagt 1740
agctgagaga agggcgagag aaccaatcag agagagtggg tgggcctaga gtgtcagcgt 1800
gtccacaggg cagtagttga gaggggcggg agaaccagtc ggggcgcgtg ggataggctt 1860
agagtgtcag cgtggagggt gaagttgtgg tagcaccagt tgcctacatt aagttattaa 1920
tagagtagta tagataaata ggtattgaga tatttattca aatataatta ttgtttattt 1980
ataaacacta agatatgtgt ggtctggtgg ttacactcta atgtttggag caaggggttg 2040
tgggttcgag tgctcgcttt gcactattat ttacgcggtg tggtagcacg gagggtgaag 2100
ccgtggtagc accaggtgtt cacattaggt ttttattctt aatagattag tatagattta 2160
tatgatttct ggttgttgaa cctggcttag gatttttatc catgtaaggc agtgagctag 2220
tgcctagtgc acttctgaga gttgctctcc actagtgcgg gagcctccga caatggatct 2280
gccctttaaa tcagtgggcc agtaaaggtc aaatcaattt attttatgct attgctgtca 2340
tgtttcaaaa ttttcagttt ctagttctca gaaaccatcc agaactctaa aatcatttcc 2400
tatcataaat tcttattcaa acatttttat gtcttaatag tgttgagacc caacttgtaa 2460
ttacagaagc tgctaattca ttttacctgc acttccactt ggatgatgta atttttctgt 2520
ttcctttcaa cagaggaatt gaaaacacta ccagctccag aggcagcatt gaactattac 2580
ctgaatgagg acctttactt atttggtatt cgcttatgtt tcttaaaatc ttaaagttat 2640
acatgaacag ttgataccta ttctaattgt ttacatacct cttttgcccc tcagatgagt 2700
ttcagacaac aagaattcca tgttctagga ggcctaaaat aggtaaactt gctgttctct 2760
cttttgtccc ctcaataggt gtgtattcct tttttttgta cttcttcgga aggttggaac 2820
taaacatgtt agctgcgttt gtgaaattta actggctgct ccatcatgga gtcgtaaaat 2880
ctcgattttg attccttgtt tactggattc tggcctcctg acgtgaacca tttgtaccgc 2940
ctatctattt ctctgccata agactgttgt aagtactcac tgattcccat atgcagataa 3000
cttgtatgat gtattcgtca atatacgaga tgacgaggca gagcactgca agacaatgaa 3060
ggcatgtcaa acacatggaa ctcttcgttc tcctcactca atgccgaact gcttagaagc 3120
tgatacagaa tgtgtaatac ctgaaaacga ttgtgaaggt attgtggact gtgtcaaaaa 3180
gtcccttaca aagtaaatag tatgtagaat cctctttcgt gtgttttcag gcatcatagt 3240
atacaaaagt acaaattctg gatgatatcc tctctttttt cctgtgtatc tttgttaaag 3300
caaaatccgg gtca 3314
<210> 6
<211> 1257
<212> DNA
<213> Zea mays L
<400> 6
gactctccac catcactcgc gcgccggcgc cgctcacacg cccatttcca ccccacaaga 60
cccacacgga atcccggcac cctacctctc caacacgcac cagcatcatg gcggtggctt 120
cgacctcgcc gctatccgcc aagcccgcca cggccccctc gccgcccgct ccggtgtccg 180
ggttcctcgc tctccccgcc cgccgcggcc gcgcaacgcg cctcggctcc gccgccgcgt 240
ggaggaggct tcgcgtggag gcgatctgga agcagcagga gaagcgggcg gaggtgtccg 300
tcgaggaatc cgcccccgtc agggaggccg ccgcgcccct ggacggagtc ggagctgacg 360
accccatggt tccttcctcg gacgagagct gggtggtcag gctcgagcag tcggtcaaca 420
ttttcctcac ggaatcggtg attatagtac tcaataccgt gtaccgtgat cggaactacg 480
ccaggttttt tgtgctggag acgattgcca gggtgccgta tttcgcgttc atatcggtgc 540
ttcacatgta tgaaaccttt ggctggtgga gacgagctga ttatctaaaa gttcactttg 600
cgcagagctt gaacgagttt catcatctct tgatcatgga agaattgggt ggcaacgcta 660
tatggattga tcgtttcctt gctcggttta tggcgttttt ttactacttc atgactgttg 720
cgatgtacat gttgagccca cgaatggcat atcacttctc tgaatgtgtg gagagacatg 780
cgtactccac ctatgataag ttcctcaagc tccatgaaga ggaattgaaa acactaccag 840
ctccagaggc agcattgaac tattacctga atgaggacct ttacttattt gatgagtttc 900
agacaacaag aattccatgt tctaggaggc ctaaaataga taacttgtat gatgtattcg 960
tcaatatacg agatgacgag gcagagcact gcaagacaat gaaggcatgt caaacacatg 1020
gaactcttcg ttctcctcac tcaatgccga actgcttaga agctgataca gaatgtgtaa 1080
tacctgaaaa cgattgtgaa ggtattgtgg actgtgtcaa aaagtccctt acaaagtaaa 1140
tagtatgtag aatcctcttt cgtgtgtttt caggcatcat agtatacaaa agtacaaatt 1200
ctggatgata tcctctcttt tttcctgtgt atctttgtta aagcaaaatc cgggtca 1257
<210> 7
<211> 1257
<212> DNA
<213> Zea mays L
<400> 7
gactctccac catcactcgc gcgccggcgc cgctcacacg cccatttcca ccccacaaga 60
cccacacgga atcccggcac cctacctctc caacacgcac cagcatcatg gcggtggctt 120
cgacctcgcc gctatccgcc aagcccgcca cggccccctc gccgcccgct ccggtgtccg 180
ggttcctcgc tctccccgcc cgccgcggcc gcgcaacgcg cctcggctcc gccgccgcgt 240
ggaggaggct tcgcgtggag gcgatctgga agcagcagga gaagcgggcg gaggtgtccg 300
tcgaggaatc cgcccccgtc agggaggccg ccgcgcccct ggacggagtc ggagctgacg 360
accccatggt tccttcctcg gacgagagct gggtggtcag gctcgagcag tcggtcaaca 420
ttttcctcac ggaatcggtg attatagtac tcaataccgt gtaccgtgat cggaactacg 480
ccaggttttt tgtgctggag acgattgcca gggtgccgta tttcgcgttc atatcggtgc 540
ttcacatgta tgaaaccttt ggctggtgga gacgagctga ttatctaaaa gttcactttg 600
cgcagagctt gaacgagttt catcatctct tgatcatgga agaattgggt ggcaacgcta 660
tatgaattga tcgtttcctt gctcggttta tggcgttttt ttactacttc atgactgttg 720
cgatgtacat gttgagccca cgaatggcat atcacttctc tgaatgtgtg gagagacatg 780
cgtactccac ctatgataag ttcctcaagc tccatgaaga ggaattgaaa acactaccag 840
ctccagaggc agcattgaac tattacctga atgaggacct ttacttattt gatgagtttc 900
agacaacaag aattccatgt tctaggaggc ctaaaataga taacttgtat gatgtattcg 960
tcaatatacg agatgacgag gcagagcact gcaagacaat gaaggcatgt caaacacatg 1020
gaactcttcg ttctcctcac tcaatgccga actgcttaga agctgataca gaatgtgtaa 1080
tacctgaaaa cgattgtgaa ggtattgtgg actgtgtcaa aaagtccctt acaaagtaaa 1140
tagtatgtag aatcctcttt cgtgtgtttt caggcatcat agtatacaaa agtacaaatt 1200
ctggatgata tcctctcttt tttcctgtgt atctttgtta aagcaaaatc cgggtca 1257
<210> 8
<211> 1221
<212> DNA
<213> Zea mays L
<400> 8
gactctccac catcactcgc gcgccggcgc cgctcacacg cccatttcca ccccacaaga 60
cccacacgga atcccggcac cctacctctc caacacgcac cagcatcatg gcggtggctt 120
cgacctcgcc gctatccgcc aagcccgcca cggccccctc gccgcccgct ccggtgtccg 180
ggttcctcgc tctccccgcc cgccgcggcc gcgcaacgcg cctcggctcc gccgccgcgt 240
ggaggaggct tcgcgtggag gcgatctgga agcagcagga gaagcgggcg gaggtgtccg 300
tcgaggaatc cgcccccgtc agggaggccg ccgcgcccct ggacggagtc ggagctgacg 360
accccatggt tccttcctcg gacgagagct gggtggaatc ggtgattata gtactcaata 420
ccgtgtaccg tgatcggaac tacgccaggt tttttgtgct ggagacgatt gccagggtgc 480
cgtatttcgc gttcatatcg gtgcttcaca tgtatgaaac ctttggctgg tggagacgag 540
ctgattatct aaaagttcac tttgcgcaga gcttgaacga gtttcatcat ctcttgatca 600
tggaagaatt gggtggcaac gctatatgga ttgatcgttt ccttgctcgg tttatggcgt 660
ttttttacta cttcatgact gttgcgatgt acatgttgag cccacgaatg gcatatcact 720
tctctgaatg tgtggagaga catgcgtact ccacctatga taagttcctc aagctccatg 780
aagaggaatt gaaaacacta ccagctccag aggcagcatt gaactattac ctgaatgagg 840
acctttactt atttgatgag tttcagacaa caagaattcc atgttctagg aggcctaaaa 900
tagataactt gtatgatgta ttcgtcaata tacgagatga cgaggcagag cactgcaaga 960
caatgaaggc atgtcaaaca catggaactc ttcgttctcc tcactcaatg ccgaactgct 1020
tagaagctga tacagaatgt gtaatacctg aaaacgattg tgaaggtatt gtggactgtg 1080
tcaaaaagtc ccttacaaag taaatagtat gtagaatcct ctttcgtgtg ttttcaggca 1140
tcatagtata caaaagtaca aattctggat gatatcctct cttttttcct gtgtatcttt 1200
gttaaagcaa aatccgggtc a 1221
<210> 9
<211> 1305
<212> DNA
<213> Zea mays L
<400> 9
gactctccac catcactcgc gcgccggcgc cgctcacacg cccatttcca ccccacaaga 60
cccacacgga atcccggcac cctacctctc caacacgcac cagcatcatg gcggtggctt 120
cgacctcgcc gctatccgcc aagcccgcca cggccccctc gccgcccgct ccggtgtccg 180
ggttcctcgc tctccccgcc cgccgcggcc gcgcaacgcg cctcggctcc gccgccgcgt 240
ggaggaggct tcgcgtggag gcgatctgga agcagcagga gaagcgggcg gaggtgtccg 300
tcgaggaatc cgcccccgtc agggaggccg ccgcgcccct ggacggagtc ggagctgacg 360
accccatggt tccttcctcg gacgagagct gggtggtcag gctcgagcag tcggtcaaca 420
ttttcctcac ggaatcggtg attatagtac tcaataccgt gtaccgtgat cggaactacg 480
ccaggttttt tgtgctggag acgattgcca gggtgccgta tttcggtgag ggctccgttt 540
tgtcatatgc ttgcagttgc ggtgtatgaa aagcgttcat atcggtgctt cacatgtatg 600
aaacctttgg ctggtggaga cgagctgatt atctaaaagt tcactttgcg cagagcttga 660
acgagtttca tcatctcttg atcatggaag aattgggtgg caacgctata tggattgatc 720
gtttccttgc tcggtttatg gcgttttttt actacttcat gactgttgcg atgtacatgt 780
tgagcccacg aatggcatat cacttctctg aatgtgtgga gagacatgcg tactccacct 840
atgataagtt cctcaagctc catgaagagg aattgaaaac actaccagct ccagaggcag 900
cattgaacta ttacctgaat gaggaccttt acttatttga tgagtttcag acaacaagaa 960
ttccatgttc taggaggcct aaaatagata acttgtatga tgtattcgtc aatatacgag 1020
atgacgaggc agagcactgc aagacaatga aggcatgtca aacacatgga actcttcgtt 1080
ctcctcactc aatgccgaac tgcttagaag ctgatacaga atgtgtaata cctgaaaacg 1140
attgtgaagg tattgtggac tgtgtcaaaa agtcccttac aaagtaaata gtatgtagaa 1200
tcctctttcg tgtgttttca ggcatcatag tatacaaaag tacaaattct ggatgatatc 1260
ctctcttttt tcctgtgtat ctttgttaaa gcaaaatccg ggtca 1305
<210> 10
<211> 20
<212> DNA
<213> 人工序列
<400> 10
agccaggctg taggaacaga 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列
<400> 11
acactcgaaa aaggctgcca 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列
<400> 12
gggttgctgt gatgacgttt 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列
<400> 13
tggatgtgga cgagctacca 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列
<400> 14
gatgtcgtag gccagcatgt 20
<210> 15
<211> 21
<212> DNA
<213> 人工序列
<400> 15
caaggttcac tgctcatccc t 21
<210> 16
<211> 20
<212> DNA
<213> 人工序列
<400> 16
agggagcttg taccagtgct 20
<210> 17
<211> 22
<212> DNA
<213> 人工序列
<400> 17
acaatggtgg agcctgtatg aa 22
<210> 18
<211> 20
<212> DNA
<213> 人工序列
<400> 18
gtatcccagg ggcaccattc 20
<210> 19
<211> 25
<212> DNA
<213> 人工序列
<400> 19
tccatgcaat aatctgtact ttcca 25
<210> 20
<211> 20
<212> DNA
<213> 人工序列
<400> 20
tcgtgccaca aacacctaaa 20
<210> 21
<211> 20
<212> DNA
<213> 人工序列
<400> 21
ctcatctcta tcctgcgggc 20
<210> 22
<211> 24
<212> DNA
<213> 人工序列
<400> 22
cgtcgtcttc gacatgtact tcac 24
<210> 23
<211> 24
<212> DNA
<213> 人工序列
<400> 23
gccctgttat tggaacagtt tacg 24
<210> 24
<211> 20
<212> DNA
<213> 人工序列
<400> 24
ccctggaatc tcacacatga 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列
<400> 25
ccgtcacttc cactcctacc 20
<210> 26
<211> 21
<212> DNA
<213> 人工序列
<400> 26
aggcaactcc tgtgtctgtg t 21
<210> 27
<211> 18
<212> DNA
<213> 人工序列
<400> 27
catgatcgcc cactcctt 18
<210> 28
<211> 20
<212> DNA
<213> 人工序列
<400> 28
gactctccac catcactcgc 20
<210> 29
<211> 22
<212> DNA
<213> 人工序列
<400> 29
tgacccggat tttgctttaa ca 22
<210> 30
<211> 20
<212> DNA
<213> 人工序列
<400> 30
gactctccac catcactcgc 20
<210> 31
<211> 19
<212> DNA
<213> 人工序列
<400> 31
gagcgaggaa cccggacac 19
Claims (5)
1.蛋白PTOX1在调控玉米种子类胡萝卜素含量中的应用；
所述蛋白PTOX1的氨基酸序列如序列表中序列1所示；
所述类胡萝卜素包括：玉米素、玉米黄素、α-类胡萝卜素和β-类胡萝卜素。
2.核酸分子在调控玉米种子类胡萝卜素含量中的应用；所述核酸分子是序列表中序列6或编码区包括序列6第108-1139位所示的DNA分子；
所述类胡萝卜素包括：玉米素、玉米黄素、α-类胡萝卜素和β-类胡萝卜素。
3.含有权利要求2所述核酸分子的重组载体、表达盒、转基因细胞系或重组微生物在调控玉米种子类胡萝卜素含量中的应用；
所述类胡萝卜素包括：玉米素、玉米黄素、α-类胡萝卜素和β-类胡萝卜素。
4.一种培育种子类胡萝卜素含量增加的转基因植物的方法，其特征在于，包括以下步骤：
向受体植物中导入蛋白PTOX1的编码基因，使蛋白PTOX1在受体植物中过表达，得到转基因植物；所述转基因植物与所述受体植物相比种子类胡萝卜素含量增加；
所述蛋白PTOX1的氨基酸序列如序列表中序列1所示；
所述受体植物为玉米；所述类胡萝卜素包括：玉米素、玉米黄素、α-类胡萝卜素和β-类胡萝卜素。
5.一种提高玉米种子类胡萝卜素含量的方法，其特征在于，包括使玉米中序列1所示蛋白的表达量增加的步骤。
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6989472B1 (en) * | 1998-10-20 | 2006-01-24 | Universite Joseph Fourier | cDNA sequence transcribing an mRNA encoding the terminal oxidase associated with carotenoid biosynthesis, and uses thereof |
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| BRPI0615066A2 (pt) * | 2005-09-02 | 2011-05-03 | Univ Cornell | polinucleotìdeos que codificam enzimas do caminho biossintético de carotenóides e apocarotenóides em cafeeiro |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6989472B1 (en) * | 1998-10-20 | 2006-01-24 | Universite Joseph Fourier | cDNA sequence transcribing an mRNA encoding the terminal oxidase associated with carotenoid biosynthesis, and uses thereof |
Non-Patent Citations (4)
| Title |
|---|
| "Characterization of the plastid terminal oxidase gene in carrot-involvement in carotenoids accumulation during storage root development";M.D.Campos等;《International Symposium on Carrot and Other Apiaceae》;1;全文 * |
| "Eleven biosynthetic genes explain the majority of natural variation in carotenoid levels in maize grain";Christine H. Diepenbrock等;《The Plant Cell》;摘要、图1-2 * |
| "NM_001365750";佚名;《GENBANK》;全文 * |
| "植物质体末端氧化酶的分子特性与生理学功能";钱昱琪等;《植物生理学报》;第52卷(第11期);全文 * |
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