CN114164212A - 玉米营养器官特异性表达启动子及其应用 - Google Patents
玉米营养器官特异性表达启动子及其应用 Download PDFInfo
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Abstract
本发明公开了一种玉米营养器官特异性表达启动子及其应用,主要是采用PCR技术从玉米(Zea mays)B73自交系中分离得到一段全长2288bp的启动子序列。将该启动子替换双元表达载体pCAMBIA1301上的CaMV35S启动子,从而驱动下游GUS报告基因的表达。本发明进一步涉及将含有该玉米营养器官特异性表达启动子的重组植物表达载体转化水稻(中花11)验证功能,确定该启动子能驱动GUS基因在水稻的根、茎、叶表达,在胚,胚乳中不表达或者低表达。本发明的启动子可用于转基因工程中对植物代谢调控、生长、发育、抗病或抗逆等相关的研究。
Description
技术领域
本发明涉及一种从植物中分离的启动子,具体涉及一种玉米营养器官特异性表达启动子及其应用。
背景技术
启动子是RNA聚合酶识别、结合和转录的一段DNA序列,它含有RNA聚合酶特异性结合和转录起始所需的保守序列,称为顺式作用原件,这些顺式作用原件可以与转录因子相结合从而调控基因的表达。根据启动子作用的方式以及功能的不同可以将启动子分为三种,主要包括组成型启动子、组织或器官特异型启动子和诱导型启动子。组成型启动子可以调控RNA和蛋白质的表达使其表达恒定在一定的水平上,不会因为不同的组织器官而发生改变。在植物转基因工程中最常用的启动子是组成型启动子。单子叶植物中主要使用水稻的 Actin和玉米的Ubiquitin启动子,在双子叶植物中一般使用的是花椰菜花叶病毒(CaMV)35S 启动子。诱导型启动子在正常情况下不能驱动基因的表达,而在某些特定的条件下会显著地提高基因的表达水平。诱导型启动子中含有多种顺式作用元件,可以根据植物的生长需要来有效的控制基因的表达。组织特异型启动子是指一类仅在特定组织和器官中才能驱动基因表达的启动子,通常含有一些特异性的调控元件。为了使外源基因在植物体内有效发挥作用, 同时减少其对植物的不利影响,人们对组织特异性表达启动子的研究和应用越来越重视。
组织特异型启动子发挥作用的位点一般存在于TATA-Box盒的上游,大小一般不超过 30bp。目前已经从植物中分离得到了不同类型的组织特异型启动子,如棉花种子特异性表达启动子LEA,玉米淀粉合成酶SSIIa是玉米胚乳特异性启动子,马铃薯块茎储藏蛋白patatin 基因启动子等。玉米营养器官特异性启动子就是这类启动子,可用于调控植株的表达系统,控制其只在营养器官中表达,也可以提高所需要元素的表达量。因此,利用植物营养器官特异性启动子控制外源基因在植物中特异表达具有重要的理论和实践意义。
玉米(Zea mays)是中国最大的粮食、饲料、能源和工业原料兼用的高产作物,研究玉米的相关基因对基因工程的发展具有重要的意义。从玉米中分离获得玉米营养器官特异性表达的启动子,利用启动子的特异性表达可以提高目的基因在营养器官中的表达水平,从而对玉米进行分子改良或者生产具有特殊用途的新玉米品种,将在分子育种方面发挥重要的作用。该实验克隆了玉米营养器官特异性表达启动子,将其与带有GUS报告基因的表达载体重组,利用农杆菌介导法转化水稻,并用PCR和GUS组织化学染色鉴定,从而鉴定了P6的全长启动子能驱动GUS基因在营养器官根茎叶中表达,而胚,胚乳等其它组织中低表达或者都不表达。
发明内容
本发明的目的是提供一种玉米营养器官特异性表达启动子及其表达载体,以及启动子和表达载体的用途。
为了实现上述目的,本发明通过如下技术方案实现:
本发明从玉米属玉米(Zea mays L.)B73中克隆得到一种玉米营养器官特异性表达启动子,将其命名为P6,该启动子片段大小为2288bp,其核苷酸序列如SEQ ID NO.1所示。
本发明提供了玉米营养器官特异性表达启动子在水稻营养器官特异性表达目的基因中的用途。具体为所述的启动子用于在水稻根茎叶中大量表达目的基因,在胚和胚乳中少量甚至不表达。
包含上玉米营养器官特异性表达启动子的生物材料也属于本发明保护的范围。
所述的生物材料为植物表达载体、表达盒、宿主细胞或宿主菌。
所述的植物表达载体转入了上述的玉米营养器官特异性表达的启动子。优选的,所述的植物表达载体是用上述的玉米营养器官特异性表达启动子取代植物双元表达载体pCAMBIA1301上的CaMV35S启动子,构建成在启动子的下游含有GUS报告基因的营养器官特异性表达载体,命名为pCAM–P6。
本发明包含所述玉米营养器官特异性表达启动子或所述植物表达载体的宿主细胞为根癌农杆菌宿主细胞。
上述的生物材料在水稻营养器官特异性表达目的基因中的应用。
作为一种技术方案,所述的生物材料为植物表达载体,所述的植物表达载体能作为一种转化体。所述的植物表达载体能以根癌农杆菌作为宿主细胞,利用农杆菌介导法转化水稻。
通过农杆菌介导法转化水稻,获得了13株转基因水稻苗子,通过PCR分子检测得到11 株阳性苗。并对阳性植株的不同时期的不同组织部位都进行了GUS组织化学染色。最终通过GUS组织化学染色鉴定P6启动子为玉米营养器官特异性表达启动子,pCAM-P6为一种营养器官特异性表达的载体。
本发明的有益效果:
本发明涉及一种玉米营养器官特异性表达启动子的研究。主要是采用PCR技术从玉米 (Zea mays)B73自交系中分离得到一段全长2288bp的启动子序列。将该启动子替换双元表达载体pCAMBIA1301上的CaMV35S启动子,从而驱动下游GUS报告基因的表达。本发明进一步涉及将含有该玉米营养器官特异性表达启动子的重组植物表达载体转化水稻(中花11)验证功能,确定该启动子能驱动GUS基因在水稻的根、茎、叶表达,在胚,胚乳中不表达或者低表达。本发明的启动子可用于转基因工程中对植物代谢调控、生长、发育、抗病或抗逆等相关的研究。
附图说明
图1为启动子分子检测结果。
其中,A为PCR结果;B为双酶切结果。M:DL-5000 1:条带大小为2288bp 2: XbaI和NcoI双酶切条带。
图2为转基因植株PCR检测结果。
其中,A为潮霉素基因PCR结果;B为目的基因PCR结果。M:DL-5000水:空对照;中花:阴性对照1-13:转基因株。
图3为本发明的GUS基因的组织化学染色结果图。
其中:A胚和胚乳;BC幼茎;D颖壳;E叶;F根。
具体实施方式
本发明结合附图和具体实施例作进一步说明。应该理解,这些实施例仅用于说明目的,而不用于限制本发明范围。
所用引物均由上海生工生物工程有限公司合成,上海生工生物工程有限公司测序; pTEAY-T1、Taq酶、Trans5α感受态、T4连接酶及相关的试剂盒购置北京全式金公司;限制性内切酶XbaI和NcoI购自TaKaRa公司;相应的抗生素来自上海生工和SIGMA公司;其余试剂均为国产分析纯。下述实施例中所用方法如无特别说明均为常规方法。
实施例1:玉米营养器官特异启动子的克隆
根据NCBI网站上公布的如SEQ ID NO.1所示的PZmP6启动子全长序列,设计PCR扩增该启动子的引物,所设计的克隆引物上下游分别加了酶切位点,上游为XbaI (TCTAGA)酶切位点,下游为NcoI(CCATGG)酶切位点。
引物序列如下:
引物1(上游引物):5’-GCTCTAGAAGGCGACTTTCAGCTACAC-3’
引物2(下游引物):5’-CATGCCATGGCATGGTTCCTTCCTTGCG-3’
用提取的玉米自交系B73基因组DNA(全式金公司植物基因组试剂盒提取)为模板,通过高保真DNA聚合酶Primer Star max扩增获得目的启动子克隆,PCR反应体系为:
PCR反应条件为:预变性:94℃10min;变性:94℃10s;退火:58℃5s;延伸:72℃1min,33个循环;总延伸:72℃10min。在总延伸之前,需要加0.5μLTaq酶,给3’端加上 PolyA尾巴,方便连接到pEASY T1 Cloning Vector载体进行测序。
上述PCR反应程序结束后,用1%的琼脂糖凝胶电泳对PCR产物进行检测,如图1A所示。切胶回收目的片段;将回收片段利用自连接的方法连接到pEASY T1 Cloning Vector上,热激法转化到大肠杆菌感受态Trans5α细胞中;通过菌落PCR和酶切检测筛选阳性克隆菌株;对检测结果初步判断正确的连接片段,送去测序(上海生工公司),测序结果如序列表中SEQ ID NO:1的DNA序列所示,由2288个碱基组成,将其与NCBI上报道的序列比对,测序结果完全一致。
实施例2:pZmP6基因启动子表达载体的建立
采用双酶切的方法获得已连接到pEASY T1 Cloning Vector上的p6片段和农杆菌双元载体 pCAMBIA1301的大片段,使用的双酶切位点为XbaI和NcoI,酶切体系如下,将加好的体系放于37℃金属浴中,酶切3h。
将上述大小片段用T4DNA连接酶进行连接,25℃连接3h,连接10μL体系如下:
将上述连接好的产物pCAM–pP6质粒轻轻打入到200μL EHA105农杆菌感受态细胞中,放入电激槽中,采用1800V,6us电激,加入200μL YEP液体培养基,28℃,220rpm 预表达4~5h;10000rpm离心30s,弃上清,加入100μL YEP液体培养基,重新悬浮细胞,涂布于含100μg/mL Kan和50μg/mL Rif的YEP固体平板上,28℃暗培养约24~48h;挑取平板上长出的微黄色的单菌落,接种于含100μg/mL Kan和50μg/mL Rif的YEP液体培养液中,摇菌24~48h;待菌液浑浊,提取质粒;分别用PCR和双酶切验证,见图1B。
实施例3:农杆菌介导水稻转化
通过根癌农杆菌(Agrobacterum-mediated)农杆菌介导法将含pCAM–P6表达载体的农杆菌导入水稻中。水稻遗传转化的过程为:诱导;侵染;选择;分化;生根和移栽。
1.水稻愈伤组织诱导培养基
将100mL N6大量;10mL N6微量;10mL Vitamin(所述10mL Vitamin为1L加0.1g肌醇粉末);10mL Fe2+-EDTA;300mg/L脯氨酸;600mg/L水解酪蛋白CH;30g/L蔗糖;加dd H2O 定容到700mL,用KOH调pH值至5.9,加3.0g/L琼脂粉,煮沸,加入2,4-D储备溶液2.5mL,用dd H2O定容至1000mL,再分装到组培瓶中,所述组培瓶为25mL/瓶,高压蒸汽灭菌;待用。
2.侵染培养基
将50mL l N6大量;5mL N6微量;10mL Fe2+-EDTA;10mL Vitamin(所述10mLVitamin 为1L加0.1g肌醇粉末);800mg水解酪蛋白CH;20g/L蔗糖;加dd H2O定容至1000mL,用 KOH调pH值至5.6,加3ml/L 2,4-D储备溶液,6.0g/L琼脂粉,高压蒸汽灭菌,待培养基冷却到40℃~60℃,加入20mL Glucose葡萄糖储备溶液(1g/ml)和1000mL AS储备溶液,倒平板。
3.选择培养基
将100mL N6大量;10mL N6微量;10mL Vitamin(所述10mL Vitamin为1L加0.1g肌醇粉末);10mL Fe2+-EDTA;600mg/L水解酪蛋白CH;30g/L蔗糖;加dd H2O定容到1000mL,用KOH调pH值至5.9,加3.0g/L琼脂粉,加入2,4-D储备溶液2.5mL,煮沸,再分装到组培瓶中,所述组培瓶为25mL/瓶,高压蒸汽灭菌;将培养基冷却到40℃~60℃,再加入5mL/L羧苄青霉素储备溶液、1mL/L潮霉素储备溶液(市售)、1mL/L头孢储备溶液。在超净工作台上分装到组培瓶中。
4.分化培养基
将100mL MSmax;10mL Msmin;10mL Fe2+-EDTA;10mL Vitamin(所述10mL Vitamin为1L加0.1g肌醇粉末);1000mg/L水解酪蛋白CH;30g/L蔗糖;2mL/L NAA储备溶液; 2mL/L6-BA储备溶液;0.200uL/L KT储备溶液;200uL/L IAA储备溶液加入dd H2O定容至 1000mL,用KOH调pH值至6.0,加3g/L琼脂粉,高压蒸汽灭菌;待培养基冷却到 40℃~60℃,加入5mL/L羧苄青霉素储备溶液和2.5mL/L潮霉素储备溶液,在超净工作台上分装到组培瓶中。
5.生根培养基
将50ml MSmax;5mL Msmin;10mL Fe2+-EDTA;10mLVitamin(所述10mL Vitamin为1L加0.1g肌醇粉末);20g/L蔗糖加入dd H2O定容至700mL,用KOH调pH值至5.8;煮沸,加0.2ml/L IBA储备溶液,再加入dd H2O定容至1000mL,倒入生根瓶。
上述步骤所用到的一些热不稳定性化合物如抗生素、AS、6-BA及KT细菌滤器除菌后于培养基温度降至50℃左右时加入。
6.培养基相关溶液的配制和抗生素溶液配制
(1)Msmax储备溶液(10X):
用ddH2O定容至1000mL,室温保存;
(2)MSmin储备溶液(100X):
Na2MoO4要单独溶解,再加入到混合液中,用ddH2O定容至1000mL,室温保存;
(3)N6大量储备溶液(10X):
用ddH2O定容至1000mL,室温保存;
(4)N6微量储备溶液(100X):
用ddH2O定容至1000mL,室温保存;
(5)Fe2+-EDTA(100X):
待两者全部溶化后,相混溶,70℃水浴中保温2h,用ddH2O定容至1000mL,4℃储存;
(6)Vitamin(100X):
用ddH2O定容至1000mL,4℃储存;
(7)1mg/mL的2,4-D储备溶液:
称取5.61g 2,4-D加入1.0mL 1N KOH,点振5min后加入10mL H2O,再晃动直至2,4-D全部溶解,再用ddH2O定容至100mL,室温保存;
(8)1mg/mL的6-BA储备溶液:
称取0.1g 6-BA,加入1.0mL 1N KOH,晃动直至6-BA全部溶解,用ddH2O定容至100mL,室温保存;
(9)1mg/mL的NAA储备溶液:
称取0.1g NAA萘乙酸,加入1.0mL 1N KOH,晃动直至NAA全部溶解,用ddH2O定容至100mL,室温保存;
(10)1mg/mL的IAA储备溶液:
称取0.1g IAA吲哚乙酸,加入1.0mL 1N KOH,晃动直至IAA全部溶解,用ddH2O定容至 100mL;
(11)抗生素储液配
(12)AS储备溶液:
称量0.1962g药品,用DMSO溶解,定容成5ml,浓度为200mM。
(13)IBA储备溶液:
称量IBA(吲哚乙酸)0.05g,用1M的NaOH溶解充分,定容成500ml,配制成最终使用浓度100mg/L。4℃保存,使用浓度为0.2mg/mL。
(14)KT储备液:
称量0.2ug的激动素KT,溶解于1mol/L的盐酸中。
实施例4.转基因水稻的的鉴定
1.转基因水稻的PCR分子检测
为了检测转基因水稻是否为阳性植株,以提取的转基因水稻总DNA为模板,用目的基因P6启动子片段和表达载体上所含的潮霉素和GUS基因为检测对象,设计引物,扩增片段,用以初步鉴定转基因植株,图2为13棵转基因阳性植株的PCR检测结果。
用于检测潮霉素抗性基因的引物序列如下:
引物5(上游引物):5’-TAGGAGGGCGTGGATATGGC-3’
引物6(下游引物):5’-TACACAGCCATCGGTCCAGA-3’
PCR反应体系参照PZmP6的克隆,反应条件如下:PCR反应条件为:预变性:94℃10min;变性:94℃30s;退火:62℃30s;延伸:72℃1min,34个循环;总延伸:72℃ 10min。
用目的基因检测的引物序列如下:
引物1(上游引物):5’-GCTCTAGAAGGCGACTTTCAGCTACAC-3’
引物2(下游引物):5’-CATGCCATGGCATGGTTCCTTCCTTGCG-3’
PCR反应体系和反应条件如上(玉米营养器官特异性启动子P6的克隆)。
2.GUS基因的组织化学染色
分子检测结果初步鉴定为转基因株后,分别对转P6启动子植株不同时期的不同部位进行GUS组织化学染色。具体操作步骤如下:
(1)取转基因水稻不同时期不同的组织:将各部位放入试管中,加入适量的GUS固定液,室温下温和摇动30~60min,用50nmol/L的磷酸钠缓冲液(pH7.0)洗10~15min,重复几次,以除去组织中残留的固定液;
(2)对染色液进行1min中真空抽滤,将各组织置于GUS染色液中保温4~12h;结束后,将染色组织先置于75%乙醇漂洗脱色,再用50%和20%乙醇各侵泡20min以上,直到材料呈白色;肉眼或显微镜下观察,组织上有蓝色小点即为GUS表达部位。如图3所示 GUS基因的组织化学染色结果。A胚和胚乳;BC幼茎;D颖壳;E叶;F根。结果显示: PZmP6启动子能驱动GUS基因在根茎叶中表达,在胚、胚乳和颖壳组织中不表达。从而证明了启动子PZmP6在水稻的营养器官中特异性表达。
SEQ ID NO.1:
AGGCGACTTTCAGCTACACCCAATGGGTGCACTTTGCAAGAAAGTGCACACCATTA GAATAATATTCAAAGAGCACTTCATGGCTTCTCTCATGAAGACTATTCGAATGCTAC TTGATTCTACTAGATGATGTTCTGGAGATTCAAGAGAACTTCGAAGTTCAACCATG AAGACACACCATAGGGCCAACTATCTGACATGTAGATGGGCCAATACACGAAGCTA TATAATGTCATAGAACAAAGTGGTGTGTTCTCCAAGGCAAAACATTACTAACCTAGT AGGATCAAAGGGAAGAATGTTTCTATAATAGGATTAGAAATCTTCTACCTTAGCTAA CTATGACTTGATTTGTACACCTACTATCTGATACTTCCCTTGTAACACACCATAATTA GTAATCCAATGTTTGATATCATGCCATACATGACATAGGGTATTATGTTAGACTATGC ACCCCTCTAGCCATCCATAGATCCTTCCATATTATAGGCCATTCAATACTTAGATTGC TTCATGGTTATCTTCGGATACAATCTATGTTGGATTAATATGTCACAACACTTCATG GAATGTTTCCTTGCATGTGTATGAAGTGTATTATCTTTGGGCTTTTGGCTAGATCGA GCGACCTAAGGTGAAGTCAACCAACCTCCCCTTACAGACTTTGTGACAAAACCAA CTTGCCTTGGGCTTCATGATAAGTAAAAAAGCTTTGGACCAAATTTGAGGGCCGTT GGGCATGCCCATAGGTTGCCCAATCCTTCTATCTGAGCTTCAGCCCGGCACAATCT TCCAGCAAGCATAGCTTTTGAGTCTAAACCAAGAATTAGGTGAACCAAAAAAAACT TCATTCCATTCATCTTGATGAGTCCTTCAACATGATGTAGTCAAATGTGATTTGGTC AAGTATTTCACCATGTACAAAGTCACATTTGTTCCTCACTTGGAAGCATGTCGTAATGCCCAGATAAACCCTTGTTTCAGTACCATAGGTCCTTTGGATGAAGGGATGTTGCT TTTCCACCATTATTCATGGTATTTGTACTATAAATAACCTATGTTCCTTTCAAAACAT GTTTGGACACAACTTACGGAACTTGTTAAATTTAAACCTTCTAGCTAAGATGTTGCT TAAGTGATATATGGAAGTTGTATTCTGGTAGTCAAGTTAGTGGAGTTAAAGACCAC CAACATGCATAGTGGAATATGTAGTGGGTGTGACTCCAGGGATAACCAAGTTATTA GGTTACTAAACTAAGATGGCCAATAAGTTAATATTTTTGTTTACCTTAGTTAACTATA GGGGTATTTGTGTCAAGGACAAGAGGCTTGCGTTGCAAGACGAGTGTGAGGTCTG CACCGTTGAGCTTGTCTTAATTGTCTTACTCTGCTGTTTCTGTTGCATGCGAACGG AAGAGAAGACCTGCAAGGCATGTCTCGTTAGGCATGCACCGTCCTCAGGATGATG GTTCTCATGTGTGCCTGCATGCATCATTGTTTACCTGTCGTACCTCGTGTTTTCTTC ACGGGAAATCGGTTCCGTTCCTAGCTCACCGTCCGCTAGCTGGCTCTCCATCCATC CAGCCCACCGTATATATGCCAAGCCCTCACACCAACCGTCAGGCCATCTCCTTCCG CTATCCCCTACCCCCTTCCCTTTCTCTATATACAAACTAAAGAGGCACCGTCTCCCA CAACATCTCTCTTTCTCTTCCTCCTTCCCCAAGTCGGCGCGGTAGCTTCCCGTGTA CCGTACACAGCAGGATCCTCCGCCTTCCTTGTGTTCCGCGCTTCCACAACAGGTTC GACTCATCGATGACCCAGATTTATAAGATTGTGATGGGTCTTTTGCTTGGGTCGAT TTTGCGGGCGCATCTAGGCACATGTTCATGTATTTGTGCTATATTTTTCTACAGTTT ACCGCAAGGAAGGAACCATGGACATGAACTCCAACGCCAACAACAGCACTGCCGC AGCAGCATCGGCTCCCATCAACAACCAGCAGGAGGCTGTGGTGTCATCCCCAACC AGAAAGGAGCAAG。
序列表
<110> 连云港市农业科学院
<120> 玉米营养器官特异性表达启动子及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2045
<212> DNA
<213> 玉米(Zea mays L.)
<400> 1
aggcgacttt cagctacacc caatgggtgc actttgcaag aaagtgcaca ccattagaat 60
aatattcaaa gagcacttca tggcttctct catgaagact attcgaatgc tacttgattc 120
tactagatga tgttctggag attcaagaga acttcgaagt tcaaccatga agacacacca 180
tagggccaac tatctgacat gtagatgggc caatacacga agctatataa tgtcatagaa 240
caaagtggtg tgttctccaa ggcaaaacat tactaaccta gtaggatcaa agggaagaat 300
gtttctataa taggattaga aatcttctac cttagctaac tatgacttga tttgtacacc 360
tactatctga tacttccctt gtaacacacc ataattagta atccaatgtt tgatatcatg 420
ccatacatga catagggtat tatgttagac tatgcacccc tctagccatc catagatcct 480
tccatattat aggccattca atacttagat tgcttcatgg ttatcttcgg atacaatcta 540
tgttggatta atatgtcaca acacttcatg gaatgtttcc ttgcatgtgt atgaagtgta 600
ttatctttgg gcttttggct agatcgagcg acctaaggtg aagtcaacca acctcccctt 660
acagactttg tgacaaaacc aacttgcctt gggcttcatg ataagtaaaa aagctttgga 720
ccaaatttga gggccgttgg gcatgcccat aggttgccca atccttctat ctgagcttca 780
gcccggcaca atcttccagc aagcatagct tttgagtcta aaccaagaat taggtgaacc 840
aaaaaaaact tcattccatt catcttgatg agtccttcaa catgatgtag tcaaatgtga 900
tttggtcaag tatttcacca tgtacaaagt cacatttgtt cctcacttgg aagcatgtcg 960
taatgcccag ataaaccctt gtttcagtac cataggtcct ttggatgaag ggatgttgct 1020
tttccaccat tattcatggt atttgtacta taaataacct atgttccttt caaaacatgt 1080
ttggacacaa cttacggaac ttgttaaatt taaaccttct agctaagatg ttgcttaagt 1140
gatatatgga agttgtattc tggtagtcaa gttagtggag ttaaagacca ccaacatgca 1200
tagtggaata tgtagtgggt gtgactccag ggataaccaa gttattaggt tactaaacta 1260
agatggccaa taagttaata tttttgttta ccttagttaa ctataggggt atttgtgtca 1320
aggacaagag gcttgcgttg caagacgagt gtgaggtctg caccgttgag cttgtcttaa 1380
ttgtcttact ctgctgtttc tgttgcatgc gaacggaaga gaagacctgc aaggcatgtc 1440
tcgttaggca tgcaccgtcc tcaggatgat ggttctcatg tgtgcctgca tgcatcattg 1500
tttacctgtc gtacctcgtg ttttcttcac gggaaatcgg ttccgttcct agctcaccgt 1560
ccgctagctg gctctccatc catccagccc accgtatata tgccaagccc tcacaccaac 1620
cgtcaggcca tctccttccg ctatccccta cccccttccc tttctctata tacaaactaa 1680
agaggcaccg tctcccacaa catctctctt tctcttcctc cttccccaag tcggcgcggt 1740
agcttcccgt gtaccgtaca cagcaggatc ctccgccttc cttgtgttcc gcgcttccac 1800
aacaggttcg actcatcgat gacccagatt tataagattg tgatgggtct tttgcttggg 1860
tcgattttgc gggcgcatct aggcacatgt tcatgtattt gtgctatatt tttctacagt 1920
ttaccgcaag gaaggaacca tggacatgaa ctccaacgcc aacaacagca ctgccgcagc 1980
agcatcggct cccatcaaca accagcagga ggctgtggtg tcatccccaa ccagaaagga 2040
gcaag 2045
Claims (10)
1.如SEQ ID NO.1所示的玉米营养器官特异性表达启动子在水稻营养器官特异性表达目的基因中的应用。
2.根据权利要求1所述的应用,其特征在于:所述的启动子用于在水稻根茎叶中大量表达目的基因,在胚和胚乳中少量甚至不表达。
3.包含如SEQ ID NO.1所示的玉米营养器官特异性表达启动子的生物材料。
4.根据权利要求3所述的生物材料,其特征在于:该生物材料为植物表达载体、表达盒、宿主细胞或宿主菌。
5.根据权利要求4所述的生物材料,其特征在于:所述的植物表达载体是用如SEQ IDNO.1所示的玉米营养器官特异性表达启动子取代植物双元表达载体pCAMBIA1301上的CaMV35S启动子,重新构建得到的植物表达载体。
6.包含如SEQ ID NO.1所示的玉米营养器官特异性表达启动子的生物材料在水稻营养器官特异性表达目的基因中的应用。
7.根据权利要求6所述的应用,其特征在于:所述的生物材料为植物表达载体、表达盒、宿主细胞或宿主菌。
8.根据权利要求6所述的应用,其特征在于:所述的植物表达载体是用如SEQ ID NO.1所示的玉米营养器官特异性表达启动子取代植物双元表达载体pCAMBIA1301上的CaMV35S启动子,重新构建得到的植物表达载体。
9.根据权利要求7或8所述的应用,其特征在于:所述的植物表达载体能作为一种转化体。
10.根据权利要求7、8或9所述的应用,其特征在于:所述的植物表达载体能以根癌农杆菌作为宿主细胞,利用农杆菌介导法转化水稻。
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