CN108424893B - 玉米磷高效基因ZmAPL9及其应用 - Google Patents
玉米磷高效基因ZmAPL9及其应用 Download PDFInfo
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- CN108424893B CN108424893B CN201810488315.3A CN201810488315A CN108424893B CN 108424893 B CN108424893 B CN 108424893B CN 201810488315 A CN201810488315 A CN 201810488315A CN 108424893 B CN108424893 B CN 108424893B
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Abstract
本发明属于植物基因工程技术领域，具体涉及玉米磷高效基因ZmAPL9及其应用。本发明要解决的技术问题是在玉米基因组中克隆控制玉米酸性磷酸酶活性基因，创制高酸性磷酸酶活性的转基因玉米材料，解决植物磷元素利用效率问题。本发明的技术方案是玉米酸性磷酸酶活性基因ZmAPL9在玉米育种中的用途。本发明为玉米磷高效材料的创制和新品种的选育提供了理论基础和应用参考。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及玉米磷高效基因ZmAPL9及其应用。
背景技术
磷素是影响玉米生长发育及稳产的重要元素。虽然土壤中磷的总量很高，但由于土壤对磷的强烈化学固定作用，大部分不能被植物直接利用。而过量施用磷肥导致江河、湖泊，造成水体富营养化，对环境造成严重污染。因此通过提高玉米对磷元素的吸收、转运具有重要意义。
酸性磷酸酶(acid phosphatase，APase)对磷的吸收利用具有重要作用。根尖分泌的APases能水解根际土壤有机磷，使之可被植物吸收利用。植株内部APases能促进磷的重新分布与再利用，使磷由衰老器官向生长器官转移，在低磷环境下促进植物生长。低磷诱导使植物根系的酸性磷酸酶活性显著提高，从而促进了有机磷的转化，因此根系分泌酸性磷酸酶活性的高低可作为筛选磷高效型植物的一个重要指标。因此研究控制玉米酸性磷酸酶活性的基因可为提高玉米磷素的吸收、转运，为培育耐低磷、资源节约型玉米新品种提供理论依据。
20世纪中叶以来，对控制植物酸性磷酸酶活性基因的研究一直持续进行，并取得了一定成绩。目前已从拟南芥基因组中鉴定出了44个酸性磷酸酶基因(Li D,Zhu H,Liu K,et al.Purple acid phosphatases of Arabidopsis thaliana.Journal of BiologicalChemistry,2002,277(31):27772-27781)，从白羽扇豆、番茄、大豆等植物中也克隆了酸性磷酸酶基因，并证明了它们在植物适应低磷环境中起着重要作用(Zinn KE,Liu J,AllanDL,Vance CP(2009)White Lupin(Lupinus albus)response to phosphorus stress:evidence for complex regulation of LaSAP1.Plant and Soil 322:1-15；Baldwin JC,Karthikeyan AS,Raghothama KG(2001)LEPS2,a phosphorus starvation-induced novelacid phosphatase from tomato.Plant Physiology 125:728-737；Tian J,Venkatachalam P,Liao H,Yan X,Raghothama K(2007)Molecular cloning andcharacterization of phosphorus starvation responsive genes in common bean(Phaseolus vulgaris L.).Planta 227:151-165)。超量表达MtPAP1的拟南芥转基因植株的生物学产量、植株无机磷含量和全磷含量明显高于野生种(Xiao K,Gu J-T,Harrison M,Wang Z-Y(2006)Expression characteristics of MtPAP1and its exotic expressionin Arabidopsis affecting organic phosphorus absorption of plants.Journal ofPlant Physiology and Molecular Biology 32:99-106)；在大豆中超量表达AtPAP15也同样显著提高了植株的磷利用效率(Wang X,Wang Y,Tian J,Lim BL,Yan X,Liao H(2009)Overexpressing AtPAP15enhances phosphorus efficiency in soybean.PlantPhysiology 151:233-240)；Jun Wasaki等将白扇雨豆酸性磷酸酶基因LASAP2转入烟草，提高了烟草吸收有机磷的能力(Wasaki J,Maruyama H,Tanaka M,Yamamura T,Dateki H,Shinano T,Ito S,Osaki M(2009)Overexpression of the LASAP2gene for secretoryacid phosphatase in white lupin improves the phosphorus uptake and growth oftobacco plants.Soil Science and Plant Nutrition55:107-113)。邱红波等(Qiu H,MeiX,Liu C,Wang J,Wang G,Wang X,Liu Z,Cai Y(2013)Fine mapping of quantitativetrait loci for acid phosphatase activity in maize leaf under low phosphorusstress.Molecular Breeding 32:629-639；Qiu H,Liu C,Yu T,Mei X,Wang G,Wang J,CaiY(2014)Identification of QTL for acid phosphatase activity in root andrhizosphere soil of maize under low phosphorus stress.Euphytica 197:133-143)利用磷高效基因型自交系082与磷低效基因型自交系掖107对玉米的叶片、根系和根际土壤酸性磷酸酶活性3个性状进行了初步QTL分析。然而目前尚无关于控制玉米酸性磷酸酶活性基因克隆的报道。
发明内容
本发明要解决的技术问题是在玉米基因组中克隆控制玉米酸性磷酸酶活性基因，创制高酸性磷酸酶活性的转基因玉米材料，解决植物磷元素利用效率问题。
本发明的技术方案是玉米酸性磷酸酶活性基因ZmAPL9在玉米育种中的用途。
进一步的，所述的用途为玉米酸性磷酸酶活性基因ZmAPL9在培育磷高效利用玉米品种中的用途。
具体的，所述的玉米酸性磷酸酶活性基因ZmAPL9的编码蛋白质具有如SEQ IDNO.2所示的氨基酸序列。
具体的，所述的玉米酸性磷酸酶活性基因ZmAPL9具有如SEQ ID NO.1所示的核苷酸序列。
本发明还提供了提高玉米耐低磷环境的制剂，其主要活性成分为玉米酸性磷酸酶活性基因ZmAPL9的编码蛋白质或表达玉米酸性磷酸酶活性基因ZmAPL9的元件。
具体的，所述的玉米酸性磷酸酶活性基因ZmAPL9的编码蛋白质具有如SEQ IDNO.2所示的氨基酸序列。
具体的，所述的玉米酸性磷酸酶活性基因ZmAPL9具有如SEQ ID NO.1所示的核苷酸序列。
在前期研究工作中，以磷低效自交系掖107和磷高效自交系082为研究材料，对控制玉米酸性磷酸酶活性的遗传机进行了解析，把控制玉米酸性磷酸酶活性基因ZmAPL9定位到玉米9号染色体上，位于标记ac219和ac2096之间。
根据植物基因组数据库，段约为546kb的物理距离内共有12个具有EST支持的候选基因。其中有假设蛋白7个，功能注释基因5个。通过对定位区间内基因序列进行扩增、测序，克隆了082自交系ZmAPL9(GRMZM2G041022)如SEQ ID NO.1所示的核苷酸序列。
此外，比较了ZmAPL9基因在082和掖107亲本间的差异。具体表现在：在ZmAPL9CDS中141bp的位置，082自交系中为C碱基，掖107自交系中为G碱基(SEQ ID NO.19)；在574bp的位置，082自交系中为A碱基，掖107自交系中为T碱基；在582bp的位置，082自交系中为C碱基，掖107自交系中为G碱基；在621bp的位置，082自交系中为C碱基，掖107自交系中为G碱基；在622bp的位置，082自交系中为G碱基，掖107自交系中为A碱基；在624bp的位置，082自交系中为T碱基，掖107自交系中为C碱基；在733与734bp位置，掖107自交系中该基因有CCGTGT共6个碱基的***；在082ZmAPL9CDS中814bp位置为G碱基，在掖107相对应位置为A；在082ZmAPL9CDS中815bp位置为C碱基，在掖107相对应位置为G；在831与839bp位置，掖107自交系中该基因有GCAGTGGAG共9个碱基的缺失；在897bp位置，082自交系中为A碱基，掖107自交系中为T碱基。
082自交系ZmAPL9CDS全长984bp，没有内含子，编码一个其具有SEQ ID NO.2所示的氨基酸序列。
用引物ZmAPL9-GSP-S扩增ZmAPL9基因，构建了ZmAPL9的亚细胞定位载体，亚细胞定位结果表明该基因编码蛋白位于细胞膜和核膜。
用引物ZmAPL9-GSP-O扩增ZmAPL9基因，构建了ZmAPL9的过量表达载体，对玉米进行遗传转化。用硝基苯膦酸二钠法测定玉米叶片中的酸性磷酸酶的活性，发现转基因株系酸性磷酸酶活性极显著高于野生型。
用钼蓝显色定磷法测定玉米叶片中的无机磷含量。超表达玉米植株无机磷含量均显著或极显著高于野生型。
本申请中磷高效利用玉米品种，是指对磷的利用效率高，即使在土壤贫瘠可被植物利用的磷含量很低的情况下，也能有效利用磷。
本申请的有益效果：本申请提供的ZmAPL9是一个控制玉米酸性磷酸酶活性基因。该基因在玉米植株中的过量表达，可以显著提高植株叶片酸性磷酸酶活性，可以显著促进植株对无机磷的吸收与转运，为快速培育耐土壤瘠薄玉米新品种提供了一种新的途径。克隆了磷高效基因ZmAPL9，提供了该基因的核苷酸序列和蛋白质序列，为玉米磷高效材料的创制和新品种的选育提供了理论基础和应用参考。
附图说明
图1ZmAPL9的精细定位；黑色实线上方数字代表两标记间的物理距离(Mb)，标记下方括号内数字代表该标记在玉米染色体的物理位置，46、5、2、1、32代表在该标记位置筛选到的重组交换单株数目。
图2自交系082和掖107间ZmAPL9CDS序列比对。
图3ZmAPL9-GSP-S扩增ZmAPL9基因；泳道L1为DNA标准分子量，泳道L2为ZmAPL9基因PCR扩增片段。
图4亚细胞定位载体示意图；CaMV35s promoter：花椰菜花叶病毒35s启动子，GFP：绿色荧光蛋白基因；T-Border(right)：T-DNA右边界；STA region：稳定位点；pVS1rep：pVS1复制起始位点；pBR322ori：pBR322复制起始位点；pBR322-bom：pBR322框；kanamycin(R)：卡那霉素抗性基因；T-Border(L)：T-DNA左边界；CaMV35s polyA：花椰菜花叶病毒35s多聚腺苷酸；hptII：潮霉素抗性基因。
图5ZmAPL9-GSP-O扩增ZmAPL9基因；泳道L1为DNA标准分子量，泳道L2为ZmAPL9基因PCR扩增片段。
图6ZmAPL9过表达载体示意图；CaMV 35s promoter:花椰菜花叶病毒35s启动子，Nos-poly A：Nos多聚腺苷酸；T-Border(right)：T-DNA右边界；pVS1sta：pVS1稳定位点；pVS1rep：pVS1复制起始位点；pBR322ori：pBR322复制起始位点；pBR322-bom：pBR322框；kanamycin(R)：卡那霉素抗性基因；T-Border(L)：T-DNA左边界；CaMV 35s polyA：花椰菜花叶病毒35s多聚腺苷酸；phosphinothricin(R)：抗草铵膦基因。
图7ZmAPL9的亚细胞定位；A和B分别代表空载体转化玉米原生质体在暗场、明场下信号观察；C代表A和B图片合并后生成信号；D和E分别代表ZmAPL9::GFP转化玉米原生质体在暗场、明场下信号观察；F代表D和E图片合并后生成信号。
图8转基因植株PCR鉴定；泳道M代表DNA标准分子量，T0-1至T0-8代表T0代各转基因植株，CK+代表阳性对照，WT代表阴性对照。
图9转基因玉米阳性植株叶片酸性磷酸酶活性检测；Zm-WT为野生型；ZmOE-1至ZmOE-8为过表达转基因阳性植株。
图10转基因玉米阳性植株叶片无机磷含量分析；Zm-WT为野生型；ZmOE-1至ZmOE-8为过表达转基因阳性植株。
具体实施方式
为了详细阐释专利发明过程、发明专利用途，下述内容结合图表说明对发明实施例进行详细描述。本发明技术方案使用分子生物学实验方法均为常规操作技术，可参照《分子克隆实验指南(精编版)》(J.萨姆布鲁克、D.W.拉塞尔著，黄培堂等译，化学工业出版社2007)和《精编分子生物学实验指南》(F.M.奥斯伯、R.布伦特、R.E.金斯顿等著，金由辛、包慧中和赵丽云等译，科学出版社2008)。
本发明研究材料玉米自交系082为西南大学玉米研究所培育，掖107由河北农业大学提供；遗传转化玉米受体由中国种子集团有限公司提供。实验中所有试剂如反转录试剂盒、限制性内切酶购自Fermentas公司；质粒提取试剂盒、DNA回收试剂盒、普通Taq酶购自天根生化科技(北京)有限公司；高保真聚合酶PrimeSTAR、T4DNA连接酶购自TaKaRa公司；DNA测序在上海英骏生物技术有限公司完成；引物合成在生工生物工程(上海)股份有限公司完成；亚细胞定位载体和过表达载体由中国农业大学国家玉米改良中心提供。
实施例一 ZmAPL9的精细定位
前期研究中，利用磷高效玉米自交系082为母本、磷低效自交系掖107为父本，构建了含有1441个单株的BC3F2群体对玉米叶片酸性磷酸酶活性QTL-ZmAPL9定位于SSR标记ac188和ac2128之间(图1)。在Gramene(http://www.gramene.org/)网站下载这两个标记之间的DNA序列，用SSRHunter搜索SSR，然后用Primer-BLAST(https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi？LINK_LOC＝BlastHome)设计引物，筛选出了在082和掖107之间有多态生的SSR标记umc1743、ac219、ac2096、ac2111(引物序列如表1所示)，并分别在这些标记位置筛选出交换单株5个、2个、1个和6个。最终将ZmAPL9定位于物理距离为546kb的SSR标记ac219和ac2096之间。图1中黑色实线上方数字代表两标记间的物理距离(Mb)，标记下方括号内数字代表该标记在玉米染色体的物理位置，红字数字代表在该标记位置筛选到的重组交换单株数目。
表1 ZmAPL9精细定位及克隆引物序列
实施例二 定位区间基因预测及克隆
在Gramene(http://www.gramene.org/)网站下载SSR标记ac219和ac2096之间的DNA序列，用softberry(http://linux1.softberry.com/berry.phtml？topic＝fgenesh&group＝programs&subgroup＝gfind)FGENESH预测基因，用Blastp(https://blast.ncbi.nlm.nih.gov/Blast.cgi？PROGRAM＝blastp&PAGE_TYPE＝BlastSearch&BLAST_SPEC＝&LINK_LOC＝blasttab)进行蛋白质注释。发现在546kb的区间内，共有基因12个，其中有假设基因7个，功能注释基因5个(表2)。设计引物，对定位区间内基因序列进行扩增、测序，发现GRMZM2G041022在082和掖107之间CDS序列差异较大，存在较多的***、缺失及SNP位点(图2)，因此将GRMZM2G041022确定为ZmAPL9的候选基因。
表2精细定位区间基因预测及功能注释
| 基因ID | 功能注释 |
| AC219190.3_FG002 | 伸展蛋白A20 |
| GRMZM2G334592 | 甘露糖结合凝集素家族受体蛋白激酶 |
| GRMZM2G122447 | VQ蛋白 |
| GRMZM2G122476 | 假设蛋白 |
| GRMZM2G104581 | 假设蛋白 |
| GRMZM2G145294 | 假设蛋白 |
| GRMZM2G704287 | 假设蛋白 |
| GRMZM2G305238 | 假设蛋白 |
| GRMZM2G305244 | 假设蛋白 |
| GRMZM2G151689 | 锌指蛋白 |
| GRMZM2G180863 | 核糖磷酸焦磷酸激酶 |
| GRMZM2G041022 | 假设蛋白 |
实施例三 载体构建
(1)亚细胞定位载体构建
用添加了BamHI和XbaI酶切位点的过表达载体引物ZmAPL9-GSP-S扩增ZmAPL9全长CDS(图3)，连接到双元载体pCAMBIA1300(国家玉米改良中心惠赠)中的GFP前构建成融合基因，ZmAPL9由35s启动子驱动(图4)。pCAMBIA1300在T-DNA区域带有一个选择标记基因HptII，该基因编码潮霉素磷酸转移酶，可使潮霉素磷酸化而失活。
(2)过表达载体构建
用添加了EcoRI和HindIII酶切位点的过表达载体引物ZmAPL9-GSP-O扩增ZmAPL9全长CDS(图5)，连接到双元载体pCAMBIA3301(国家玉米改良中心惠赠)中，ZmAPL9由35s启动子驱动(图6)。pCAMBIA3301载体在T-DNA区域带有一个选择标记基因bar，该基因编码草丁膦乙酞CoA转移酶(PAT)，可催化草丁膦的自由氨基乙酞化，从而使除草剂草丁膦失活。
实施例四 ZmAPL9亚细胞定位
培养黄化玉米苗(出芽后约1cm开始暗培养)，取叶片切成0.5mm细丝，加入酶解液，抽真空30min；28℃，50rpm黑暗消化3-4h后，80rpm，2min完全释放原生质体；然后用100目网筛过滤酶解液(纤维素酶R10 1.5％，果胶酶R10 0.5％，山梨糖醇0.645mol L-1，氯化钾10mmol L-1，2-(N-***啉)乙磺酸20mmol L-1，牛血清蛋白0.1％，聚乙烯吡咯烷酮K300.1％，氯化钙10mmol L-1，β-巯基乙醇5mmol L-1)，100×g，2min，移除上清；加入10mLW5(氯化钠154mmol L-1，氯化钙125mmol L-1，氯化钾5mmol L-1，2-(N-***啉)乙磺酸10mmolL-1)至滤渣中，手动温和摇晃5min；用100目过滤网筛过滤滤渣洗涤液，100×g，2min，移除上清；加入10mL W5洗涤原生质体1次，100×g，2min沉淀原生质体，移除上清；加入10mL MMg(甘露醇0.4mol L-1，氧化镁15mmol L-1，2-(N-***啉)乙磺酸10mmol L-1)重悬沉淀，100×g，2min，移除上清后加入适量MMg，镜检，计数；加入10μL DNA(10～20μg)，200μL原生质体(2mL离心管)，轻柔混匀；加入220μL PEG4000-Ca2+溶液(PEG400040％，甘露醇0.2mol L-1，氯化钙0.1mol L-1)，用1mL枪头轻柔混匀，25℃培养18min；加入880μL W5，轻柔混匀，110×g，1min，移去上清，重复该步骤；加入500μL W5(含50mg mL-1氨)温和重悬原生质体；23℃培养12～16h；最后在显微镜下观察，ZmAPL9亚细胞定位结果如图7所示，ZmAPL9蛋白定位于细胞质膜及核膜上。
实施例五 转基因玉米酸性磷酸酶活性的测定方法
按照图6所示构建的过表达载体，对玉米进行遗传转化。利用引物ZmAPL9-S对转基因植株进行鉴定，获得了共8株阳性转基因株系(图8)。取转基因阳性植株新鲜叶片0.05g，放入2mL离心管中用冷冻磨样机磨成细粉；向离心管中加入2mL醋酸-醋酸钠缓冲液(0.2molL-1，pH5.8)并迅速用力摇匀，放置冰上；4℃条件下，6000rpm离心10min后放置冰上；吸取0.1mL上清液(酶液)加入到用醋酸-醋酸钠缓冲液配制的8mL 4-硝基苯磷酸二钠溶液中(0.5g/L)；反应液置于37℃恒温水浴锅孵育30min后立即加入0.5mL的氢氧化钠(6mol/L)溶液终止反应；把0.1mL醋酸-醋酸钠缓冲液加入到用醋酸-醋酸钠缓冲液配制的8mL 4-硝基苯磷酸二钠溶液中，混匀后做空白对照，405nm波长测定反应液吸光度；最后根据以下公式计算玉米叶片酸性磷酸酶活性：
OD：吸光度；V：酶制剂总体积；Vt：反应混合液总体积；T：反应时间；FW(鲜重)：0.05g；V1：酶液体积；A：pH14条件下，对硝基苯酚的吸光系数。
根据上述方法对玉米转基因株系叶片酸性磷酸酶活性测定结果表明，所有8个株系均极显著上调(图9)。
实施例六 转基因玉米叶片无机磷的含量的测定方法
转基因玉米叶片无机磷含量检测分为标准曲线绘制及叶片无机磷含量测定两个步骤，具体方法如下：
(1)标准曲线绘制
将适量磷酸二氢钾样品在105℃烘干至恒重，精确称取0.447g加水溶解并定容，配置成100mL磷酸二氢钾溶液，作为储备液。临用前，精密量取1mL储备液并定容至100mL作为对照品容液(每1mL含10.05μg)
准确量取对照品容液0mL、0.3mL、0.6mL、0.9mL、1.2mL、1.5mL分别置于试管中，加水至3mL，摇匀；分别加入定磷试剂3mL，37℃水浴孵育1h；孵育结束将反应液放置于4℃放置5min；以0号管作为空白对照，820nm处测定各管吸光度；以吸光度为横坐标，含磷量为纵坐标，绘制标准曲线。
(2)转基因玉米叶片无机磷的含量的测定
配制10％(w/v)的抗坏血酸及以0.5mol/L的硫酸为溶剂浓度为0.4％(w/v)的钼酸铵溶液，把抗坏血酸溶液和钼酸铵溶液以1:6的比例混合成工作液；然后液氮研磨0.05g样品并放置于2mL离心管；向离心管中加入2mL 2％的醋酸，42℃孵育30min；12000rpm离心2min得到上清液；分别把1mL抗坏血酸溶液和钼酸铵混合液加入到1mL上清液中(检测溶液)和1mL 2％的醋酸中37℃水浴孵育1h(对照)；孵育结束将反应液放置于4℃放置5min；将反应液820nm处测定光吸收值；最后根据标准曲线计算Pi含量。
按照此方法，检测得出在转基因玉米株系叶片中，有6个株系叶片无机磷含量呈极显著上调，有2个株系叶片无机磷含量呈显著上调(图10)。
磷素是影响玉米生长发育及稳产的重要元素。我国西南地区土壤贫瘠，土壤中磷素缺乏；北方地区虽然土壤中磷的总量很高，但由于土壤对磷素强力固化作用，大部分不能被植物直接利用。酸性磷酸酶对磷的吸收与利用具有重要促进作用。玉米叶片的酸性磷酸酶能够促进磷的重新分布与再利用。通过构建ZmAPL9过表达载体转化玉米，可显著提高玉米叶片酸性磷酸酶活性，从而提高玉米叶片磷元素的含量，因此通过转基因手段可快速培育出耐低磷胁迫、磷素利用效率高的玉米新品种。
虽然，上述内容已经对发明内容及实施例作了翔实的描述，但相关研究领域专业人员均知道，在本发明基础上可对形式和细节做出各式各样的修改和改进。因此，在不偏离本发明精神的基础上所做的一切修改和改进，均属于本发明保护的范围。
序列表
<110> 西南大学
<120> 玉米磷高效基因ZmAPL9及其应用
<130> 201801t
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 984
<212> DNA
<213> 082自交系ZmAP9 CDS序列(Artificial Sequence)
<400> 1
atggccacca aagcaggctc ctcactgtcg ccatgcatca ggctcgtgga ggaatccctg 60
ctcctcccga cgcaagacgc cggcctcttc gcaaaggcct tcgtgctcat cttcgcccac 120
accttcctct tcatcgcggt cgccgtccac ttcgctcacc cactcgccac ggacatcctc 180
gccgacatca aagcacaaag gagcagcagc aggaccacta ccactaccgg cgccgcgagc 240
tccaaccact ccaaggagct gtcgttacta gcgatctacc tctgctacct cgcgagcaaa 300
ctcgccaccc agctcgtcac cgccctcgcc accgccacga cctactccgg cgagcgcctc 360
acgcgtaagg tggtcatgga gaggatcgcc ggaggcggcg gccttctcgg cacggctgcc 420
gtcgccggcg cgctggagct gtcgttcacg gctctactcg cggcgctact cgtatccacg 480
tggacctacg ctgacgactc ggtcgtaaag tctgtgtgtg gctactcgat gttctcggtg 540
gcgcttctcg tctacatcta cctcgccacg gtgatcccgg tcagcgtcgg cgtgtcggcg 600
gtggacagag gctgccacgg cgtttgggcg ctccgccggg cgtggcggct cttgagagca 660
agggggaagg aggctgccgt gctcgtgttc gtcgtcaaca tcctgcctgc tttcatctac 720
ccagcccccg tgtacgcgtt ctcgtacatg tacccggcgg acgagtactc cctctactac 780
ggacaggaca tggcggtcag gttttctctc cacgccccgg cgatgtggag gcagtggagc 840
cagggcgtgt gctggttcat gggcgtagtt ttcggatctg gtcttccctc ggttggagcg 900
cagctgttct ccatggtggc agccacggtg ttctgctgcc tctccataga aaccaacgat 960
gatgcagcgc gcgcagtagt ataa 984
<210> 2
<211> 327
<212> PRT
<213> 082自交系ZmAP9氨基酸序列(Artificial Sequence)
<400> 2
Met Ala Thr Lys Ala Gly Ser Ser Leu Ser Pro Cys Ile Arg Leu Val
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Glu Glu Ser Leu Leu Leu Pro Thr Gln Asp Ala Gly Leu Phe Ala Lys
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Ala Phe Val Leu Ile Phe Ala His Thr Phe Leu Phe Ile Ala Val Ala
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Val His Phe Ala His Pro Leu Ala Thr Asp Ile Leu Ala Asp Ile Lys
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Ala Gln Arg Ser Ser Ser Arg Thr Thr Thr Thr Thr Gly Ala Ala Ser
65 70 75 80
Ser Asn His Ser Lys Glu Leu Ser Leu Leu Ala Ile Tyr Leu Cys Tyr
85 90 95
Leu Ala Ser Lys Leu Ala Thr Gln Leu Val Thr Ala Leu Ala Thr Ala
100 105 110
Thr Thr Tyr Ser Gly Glu Arg Leu Thr Arg Lys Val Val Met Glu Arg
115 120 125
Ile Ala Gly Gly Gly Gly Leu Leu Gly Thr Ala Ala Val Ala Gly Ala
130 135 140
Leu Glu Leu Ser Phe Thr Ala Leu Leu Ala Ala Leu Leu Val Ser Thr
145 150 155 160
Trp Thr Tyr Ala Asp Asp Ser Val Val Lys Ser Val Cys Gly Tyr Ser
165 170 175
Met Phe Ser Val Ala Leu Leu Val Tyr Ile Tyr Leu Ala Thr Val Ile
180 185 190
Pro Val Ser Val Gly Val Ser Ala Val Asp Arg Gly Cys His Gly Val
195 200 205
Trp Ala Leu Arg Arg Ala Trp Arg Leu Leu Arg Ala Arg Gly Lys Glu
210 215 220
Ala Ala Val Leu Val Phe Val Val Asn Ile Leu Pro Ala Phe Ile Tyr
225 230 235 240
Pro Ala Pro Val Tyr Ala Phe Ser Tyr Met Tyr Pro Ala Asp Glu Tyr
245 250 255
Ser Leu Tyr Tyr Gly Gln Asp Met Ala Val Arg Phe Ser Leu His Ala
260 265 270
Pro Ala Met Trp Arg Gln Trp Ser Gln Gly Val Cys Trp Phe Met Gly
275 280 285
Val Val Phe Gly Ser Gly Leu Pro Ser Val Gly Ala Gln Leu Phe Ser
290 295 300
Met Val Ala Ala Thr Val Phe Cys Cys Leu Ser Ile Glu Thr Asn Asp
305 310 315 320
Asp Ala Ala Arg Ala Val Val
325
<210> 3
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ccactgataa tcccgctatg ct 22
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gtctgaactg aaatgtgcag cg 22
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ccaacaaagc aagggcagaa ag 22
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tcgtgttaat cctcaccaca cc 22
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<213> ac2096反向引物(Artificial Sequence)
<400> 10
aagttggcta ttgggaaggc at 22
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<400> 11
accaagccct actcctctac tt 22
<210> 12
<211> 23
<212> DNA
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<400> 12
ccggtcagtc atatcagaac act 23
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ggcagcagta cagtgaagag at 22
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<211> 22
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cggaattcat ggccaccaaa gcaggctcct ca 32
<210> 16
<211> 32
<212> DNA
<213> ZmAPL9-GSP-O反向引物(Artificial Sequence)
<400> 16
ccaagcttta ctactgcgcg cgctgcatca tc 32
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<211> 32
<212> DNA
<213> ZmAPL9-GSP-S正向引物(Artificial Sequence)
<400> 17
cgggatccat ggccaccaaa gcaggctcct ca 32
<210> 18
<211> 32
<212> DNA
<213> ZmAPL9-GSP-S反向引物(Artificial Sequence)
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gctctagata ctactgcgcg cgctgcatca tc 32
<210> 19
<211> 981
<212> DNA
<213> 掖107自交系ZmAP9 CDS序列(Artificial Sequence)
<400> 19
atggccacca aagcaggctc ctcactgtcg ccatgcatca ggctcgtgga ggaatccctg 60
ctcctcccga cgcaagacgc cggcctcttc gcaaaggcct tcgtgctcat cttcgcccac 120
accttcctct tcatcgcggt ggccgtccac ttcgctcacc cactcgccac ggacatcctc 180
gccgacatca aagcacaaag gagcagcagc aggaccacta ccactaccgg cgccgcgagc 240
tccaaccact ccaaggagct gtcgttacta gcgatctacc tctgctacct cgcgagcaaa 300
ctcgccaccc agctcgtcac cgccctcgcc accgccacga cctactccgg cgagcgcctc 360
acgcgtaagg tggtcatgga gaggatcgcc ggaggcggcg gccttctcgg cacggctgcc 420
gtcgccggcg cgctggagct gtcgttcacg gctctactcg cggcgctact cgtatccacg 480
tggacctacg ctgacgactc ggtcgtaaag tctgtgtgtg gctactcgat gttctcggtg 540
gcgcttctcg tctacatcta cctcgccacg gtgttcccgg tgagcgtcgg cgtgtcggcg 600
gtggacagag gctgccacgg gatctgggcg ctccgccggg cgtggcggct cttgagagca 660
agggggaagg aggctgccgt gctcgtgttc gtcgtcaaca tcctgcctgc tttcatctac 720
ccagcccccg tgtccgtgta cgcgttctcg tacatgtacc cggcggacga gtactccctc 780
tactacggac aggacatggc ggtcaggttt tctctccaca gcccggcgat gtggagccag 840
ggcgtgtgct ggttcatggg cgtagttttc ggatctggtc ttccctcggt tggtgcgcag 900
ctgttctcca tggtggcagc cacggtgttc tgctgcctct ccatagaaac caacgatgat 960
gcagcgcgcg cagtagtata a 981
Claims (3)
1.玉米酸性磷酸酶活性基因ZmAPL9在玉米育种中的用途，其特征在于：所述的玉米酸性磷酸酶活性基因ZmAPL9的编码蛋白质的氨基酸序列如SEQ ID NO.2所示。
2.如权利要求1所述的玉米酸性磷酸酶活性基因ZmAPL9在玉米育种中的用途，其特征在于：所述的玉米酸性磷酸酶活性基因ZmAPL9的核苷酸序列如SEQ ID NO.1所示。
3.玉米酸性磷酸酶活性基因ZmAPL9的编码蛋白质或表达玉米酸性磷酸酶活性基因ZmAPL9的元件在制备提高玉米耐低磷环境制剂中的用途，其特征在于：所述的玉米酸性磷酸酶活性基因ZmAPL9的编码蛋白质的氨基酸序列如SEQ ID NO.2所示；所述的玉米酸性磷酸酶活性基因ZmAPL9的核苷酸序列如SEQ ID NO.1所示。
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| CN102206617A (zh) * | 2011-03-30 | 2011-10-05 | 清华大学 | 一种酸性磷酸酶及其编码基因与应用 |
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| CN102206617A (zh) * | 2011-03-30 | 2011-10-05 | 清华大学 | 一种酸性磷酸酶及其编码基因与应用 |
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| Title |
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| Identification of QTL for acid phosphatase activity in root and rhizosphere soil of maize under low phosphorus stress;Hongbo Qiu等;《Euphytica》;20140117;全文 * |
| Localization of Acid Phosphatase Activity in Maize Root under Phosphorus Deficiency;MARIE KUMMEROVA;《BIOLOGIA PLANTARUM (PRAHA)》;19861231;第28卷(第4期);270-274 * |
| 玉米自交系苗期耐低磷的根系生理特性研究;黄爱缨 等;《中国生态农业学报》;20081130;第16卷(第6期);1419-1422 * |
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