CN104277101B - 水稻硝酸盐转运蛋白nrt1.1b在提高植物氮利用效率中的应用 - Google Patents
水稻硝酸盐转运蛋白nrt1.1b在提高植物氮利用效率中的应用 Download PDFInfo
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Abstract
本发明公开了水稻硝酸盐转运蛋白NRT1.1B在提高植物氮利用效率中的应用。所述蛋白NRT1.1B为序列表序列2所示的蛋白，可用于提高植物硝酸盐含量或提高植物硝酸盐的转运能力，进而提高植物氮的利用效率。实验结果证明，过表达水稻中的硝酸盐转运蛋白NRT1.1B获得的转基因株系，其硝酸盐含量明显提高，表明硝酸盐转运蛋白NRT1.1B在提高植物氮利用效率中具有重要的应用潜能。本发明为培育高氮肥利用率的作物提供了一个新的途径。
Description
技术领域
本发明涉及生物技术领域，具体为一种水稻硝酸盐转运蛋白NRT1.1B在提高植物氮利用效率中的应用。
背景技术
在各种营养元素中，氮在植物生命活动过程中占据首要地位。氮是植物体内许多重要化合物，如核酸(DNA、RNA)、蛋白质(包括酶)、磷脂、叶绿素、光敏素、维生素(B1、B2、B6等)、植物激素(IAA、CTK)、生物碱等组成成分，所以氮素在维持和调节植物生理功能上具有多方面的作用。但在自然的土壤环境中，可以被植物直接吸收利用的氮源含量较低，所以氮是影响植物生长发育的一个重要限制因素。为促进作物增产，氮肥在农业中被大量使用。上世纪80年代以来，我国氮肥用量急剧增加，在占世界7％的耕地上消耗了全球35％的氮肥。中国粮食年产量从1981年(3.25亿吨)至2008年(5.29亿吨)增长了63％，而氮肥消费量却增长了近2倍。氮肥的大量使用不但增加了生产成本，而且还导致一系列严重的环境问题。在施加的氮肥中，只有不到30％可以被作物所吸收，而大部分残留的氮肥会导致严重的土壤酸化以及水体富营养化。而我们国家的土壤酸化以及水体富营养化的面积正呈现逐年增大的趋势。此外，氮肥的生产会消耗大量能源，据统计每生产1吨氮肥需要消耗2800千克的优质煤及1600度电能，由此会造成大量碳排放，对大气产生严重污染。此外，我们国家还面临粮食生产的巨大缺口，需要逐年提高粮食产量。因此，氮肥过量使用对农业可持续发展提出战略性的挑战。提高作物的氮肥利用效率，减少氮肥在农业中的使用将会是解决这一系列问题的关键。
水稻作为最重要的粮食作物之一，超过世界三分之一的人口以稻米为主食。水稻根系具有非常发达的通气组织，使其根际微环境中易发生硝化作用，因此水稻中大约40％的氮源是以硝酸盐的形式被吸收。籼稻(indica)与粳稻(japonica)是亚洲栽培稻的两个主要的亚群，而且籼稻表现出更强的硝酸盐利用能力。利用籼稻与粳稻硝酸盐利用能力的差异，对相关的基因位点进行分离鉴定，进而实现利用关键基因位点培育氮高效的水稻新品种。氯酸盐是硝酸盐的毒性类似物，可以被硝酸盐转运蛋白吸收，进而在硝酸还原酶的作用下转化为对植物具有毒害作用的次氯酸盐。在拟南芥中，利用氯酸盐进行毒性筛选，已获得多个与硝酸盐利用相关的突变体，并成功鉴定了相关的基因。氯酸盐处理后，植物会表现出明显的生长停滞及叶片黄化坏死等毒害表型，因此可以使用氯酸盐敏感性作为水稻硝酸盐利用能力的评价指标，对导致籼稻与粳稻氮利用效率差异相关基因位点进行鉴定。
发明内容
本发明的一个目的是提供一种水稻硝酸盐转运蛋白NRT1.1B及其编码基因在提高植物氮利用效率中的应用。
上述应用中，所述硝酸盐转运蛋白NRT1.1B的氨基酸序列如SEQ ID No.2所示。
上述应用中，所述硝酸盐转运蛋白NRT1.1B的编码基因序列如SEQ ID No.1第1-1791位核苷酸分子所示。
上述应用中，所述氮利用率的提高为硝酸盐含量或硝酸盐转运能力的提高。
上述应用中，所述植物为单子叶植物或双子叶植物；所述单子叶植物具体为水稻。
本发明的另一个目的是提供一种构建转基因植物的方法。
上述所述构建转基因植物的方法包括如下步骤：使权利要求1所述蛋白质在受体植物中过表达，获得氮利用率提高的转基因植株。
上述方法中，所述使权利要求1所述蛋白质在受体植物中过表达的方法为：向受体植物中导入SEQ ID No.1所示的硝酸盐转运蛋白NRT1.1B的编码基因序列，得到转基因植物；转基因植物与受体植物相比，氮利用效率提高。
上述方法中，所述氮利用效率的提高为硝酸盐含量的提高。
本发明最后一个目的是提供一种构建近等基因系植物的方法。
上述所述构建近等基因系植物的方法包括如下步骤：将供体亲本与受体亲本进行杂交，再与受体亲本继续多次回交，得到近等基因系植物；近等基因系植物与受体亲本和供体亲本相比，氮利用率提高。
上述方法中，所述的氮利用率提高体现在如下方面中的至少一种：
(1)硝酸盐含量；
(2)氯酸盐敏感性；
(3)植物的株型；
(4)单株的产量；
(5)单株的分蘖数；
(6)小区产量；
(7)氮利用效率。
上述方法中，所述回交的次数具体为5次。
上述方法中，所述的供体亲本为籼稻品种IR24，所述的受体亲本为粳稻品种日本晴。
实验证明，过表达蛋白NRT1.1B的转基因水稻株系中，随着蛋白NRT1.1B编码基因相对表达量的提高，其硝酸盐含量也明显提高。结果表明蛋白NRT1.1B在提高植物对硝酸盐利用中具有重要的应用潜能。本发明为培育高氮肥利用率的作物提供了一个新的途径。
附图说明
图1中a为NRT1.1B在950份水稻品种中的进化分析；b为18份籼粳稻品种中NRT1.1BCDS分析；c为NRT1.1B基因结构示意图。
图2中a为34份籼粳稻品种的硝酸盐含量比较；b为134份籼粳稻品种的的氯酸盐敏感性比较。
图3为注射NRT1.1B-japonica(NBjap)及NRT1.1B-indica(NBind)cRNA的爪蟾卵母细胞15N-KNO3吸收活性比较；CHL1为阳性对照。
图4中a为受体亲本日本晴(Nip)，供体亲本IR24及NIL的氯酸盐敏感性分析，标尺为3cm；b为受体亲本日本晴(Nip)，供体亲本IR24及NIL硝酸盐含量分析。
图5中a为NRT1.1B-japonica过表达转基因株系(Jap-2/3/7)及NRT1.1B-indica过表达转基因株系(Ind-1/3/6)的硝酸盐转运蛋白的表达量分析；b为NRT1.1B-indica过表达转基因株系中和NRT1.1B-japonica过表达转基因株系的硝酸盐含量分析。
图6中a为受体亲本日本晴(Nip)与NIL在不同硝酸盐浓度下的生长比较；b为受体亲本日本晴与NIL在不同硝酸盐浓度下的叶绿素含量，光合速率及生物量分析。
图7中a为低氮条件下日本晴及NIL的株型比较；b为高氮条件下日本晴及NIL的株型比较；c为低氮/高氮条件下日本晴及NIL的单株产量比较；d为低氮条件下日本晴及NIL的分蘖数，单株产量，小区产量及氮利用效率比较；e为高氮条件下日本晴及NIL的分蘖数，单株产量，小区产量及氮利用效率比较。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、籼稻和粳稻NRT1.1B的CDS区核苷酸变异情况
通过对大规模水稻品种进行进化分析，结果表明：硝酸盐转运蛋白编码基因NRT1.1B(LOC_Os10g40600)在籼稻与粳稻品种间呈现显著的分化(图1a)。序列分析的结果表明，籼稻与粳稻的NRT1.1B的CDS区存在两个单核苷酸变异(SNP1，SNP2)，如图1b和图1c所示，而且CDS区第980位的SNP1(T<C)导致了籼粳稻亚种间氨基酸的变异(Met<Thr)。
实施例2、籼稻和粳稻的15NO3 -含量和氯酸盐敏感性测定
改良木村营养液的成分包括：2mM KNO3、0.36mM Ca(Cl)2·4H2O、0.54mMMgSO4·7H2O、0.18mM KH2PO4、40μM Fe(II)-EDTA、18.8μM H3BO3、13.4μMMnCl2·4H2O、0.32μMCuSO4·5H2O、0.3μM ZnSO4·4H2O以及0.03μM Na2MoO4·4H2O。
将籼稻和粳稻的幼苗培养2周后转移至含有5mM 15N-KNO3的改良木村营养液中，吸收24h，然后使用0.1mm CaSO4处理1min，去离子水冲洗3次，取地上部分于70℃烘干。将烘干后的样品研磨至粉末，使用同位素比质谱仪(Thermo Finnigan Delta plus XP；Flash EA1112)进行15N含量测定。结果表明，籼稻品种较粳稻品种表现出更高的硝酸盐含量，如图2a所示。
氯酸盐敏感性测定使用2mM KClO3。高浓度氯酸盐处理时，先将萌发后的水稻种子培养于含有2mM KNO3的改良木村培养液中生长3d，然后转移至含有2mM KClO3及2mM KNO3的木村培养液中处理5d，然后进行表型观察。氯酸盐敏感性的定量计算方法为：(正常培养高度–氯酸盐处理高度/正常培养高度)×100。结果表明，籼稻品种较粳稻品种表现出更高的氯酸盐敏感性，如图2b所示。
实施例3、籼稻和粳稻NRT1.1B在爪蟾卵母细胞中的硝酸盐转运能力测定
分别提取籼稻IR24和粳稻日本晴的RNA，反转录为cDNA。分别以获得的cDNA为模板，采用下述引物序列分别对籼稻和粳稻的NRT1.1B CDS进行PCR扩增。扩增采用的引物两端分别引入限制性内切酶BamH I和EcoR I的识别位点(如下划线所示)，引物序列：F：5’-GGATCCATGGCGATGGTGTTGCCG-3’；R：5’-GAATTCTTAGTGGCCGACGGCGATGGT-3’。
将扩增的目的片段分别连接入爪蟾卵母细胞表达载体pCS2+，并使用mMESSAGEmMACHINE(Ambion；AM1340)试剂盒进行体外转录。按照报道的方法进行cRNA的注射及培养(Zhang et al.,1998)。将培养的卵母细胞进行硝酸盐(15N-KNO3)吸收实验及15N含量测定(Liu et al.,1999)。结果表明，在爪蟾卵母细胞系统中，NRT1.1B-indica与NRT1.1B-japonica都表现出硝酸盐的转运能力，且NRT1.1B-indica较NRT1.1B-japonica表现出更高的硝酸盐转运活性(图3)。
实施例4、NRT1.1B-indica近等基因系(NIL)的构建及氯酸盐敏感性及硝酸盐含量的测定
以籼稻品种IR24为供体亲本，以粳稻品种日本晴为受体亲本进行近等基因系的构建。将IR24与日本晴连续回交5次，得到BC5F2，使用分布于12条染色体上的PCR多态标记对回交后代进行鉴定，以得到含有NRT1.1B-indica的近等基因系。
对受体亲本日本晴(Nip)、供体亲本IR24及得到含有NRT1.1B-indica的近等基因系NIL的15NO3-含量和氯酸盐敏感性进行测定和比较。结果表明：NRT1.1B-indica近等基因系(NIL)相比受体亲本(日本晴)和供体亲本IR24，表现出更高的氯酸盐敏感性及硝酸盐含量(图4a和图4b)。
实施例5、NRT1.1B-indica近等基因系(NIL)和受体亲本(日本晴)氮利用效率的比较
(一)将NRT1.1B-indica近等基因系(NIL)和受体亲本(日本晴)分别处于不同浓度的(400μM、1mM、2mM)硝酸盐的水培条件下，对NRT1.1B-indica近等基因系(NIL)相比受体亲本(日本晴)的生长状态、叶绿素含量、光合速率及生物量进行测定。结果表明：NIL相比受体亲本(日本晴)表现出更好的生长状态，更高的叶绿素含量，光合速率及生物量(图6)。
(二)在大田种植条件下，比较低氮(LN)和高氮(HN)情况下NRT1.1B-indica近等基因系(NIL)和受体亲本(日本晴)的株型、单株产量、单株分蘖数、小区产量及氮利用效率。结果表明：无论在低氮(LN)还是高氮(HN)条件下，NIL较日本晴在株型、单株产量、单株分蘖数、小区产量及氮利用效率等多个方面均表现出显著的增加(图7)。
实施例6、NRT1.1B-indica/japonica过表达转基因株系的获得及其硝酸盐含量的测定
(一)NRT1.1B-indica/japonica过表达转基因株系的获得
分别提取籼稻IR24和粳稻日本晴的RNA，反转录为cDNA。分别以获得的cDNA为模板，采用下述引物序列分别对籼稻和粳稻的NRT1.1B CDS进行PCR扩增。扩增采用的引物两端分别引入限制性内切酶Xma I和Xba I的识别位点(如下划线所示)，引物序列：F：5’-CCCGGGATGGCGATGGTGTTGCCG-3’；R：5’-TCTAGATTAGTGGCCGACGGCGATGGT-3’。
用NcoI和PstI分别对花椰菜花叶病毒(CAMV)35S启动子和pCambia2300(购自Cambia)进行双酶切，将酶切后的花椰菜花叶病毒(CAMV)35S启动子连入pCambia2300载体中，获得双元表达载体pCambia2300-35S。用限制性内切酶Xma I和Xba I分别对扩增得到的目的片段和双元表达载体pCambia2300-35S进行双酶切，将酶切后的目的片段连接入双元表达载体pCambia2300-35S，获得重组表达载体。将上述重组表达载体进行测序验证，结果表明：在pCambia2300-35S载体的Xma I和Xba I酶切位点间插入的籼稻的基因序列如序列1中第1-1791位核苷酸所示，表明载体正确；在pCambia2300-35S载体的Xma I和Xba I酶切位点间插入的粳稻的基因序列如序列3中第1-1791位核苷酸所示，表明载体正确。序列1中第1-1791位核苷酸所编码的蛋白如序列2中第1-596位的氨基酸所示，序列3中第1-1791位核苷酸所编码的蛋白如序列4中第1-596位的氨基酸所示。
将上述获得的重组表达载体转入农杆菌AGL1(购自ATCC)，在分别转化粳稻品种中花11的愈伤组织，获得了过表达序列1中第1-1791位核苷酸所编码的蛋白的NRT1.1B-indica过表达转基因株系和过表达序列3中第1-1791位核苷酸所编码的蛋白的NRT1.1B-japonica过表达转基因株系。
对上述获得的NRT1.1B-indica过表达转基因株系和NRT1.1B-japonica过表达转基因株系进行荧光实时定量PCR。鉴定转基因株系的引物序列是F:5’-GGCAGGCTCGACTACTTCTA-3’；R:5’-AGGCGCTTCTCCTTGTAGAC-3’。结果表明，过表达NRT1.1B的籼稻和粳稻的表达量相差不多(图5a)。
(二)NRT1.1B-indica/japonica过表达转基因株系硝酸盐含量的测定
对上述获得的NRT1.1B-indica过表达转基因株系和NRT1.1B-japonica过表达转基因株系的硝酸盐含量进行测定。结果表明：NRT1.1B-indica过表达转基因株系中的硝酸盐含量显著高于NRT1.1B-japonica过表达转基因株系(图5b)。
Claims (6)
1.水稻硝酸盐转运蛋白NRT1.1B及其编码基因在提高植物氮利用率中的应用；
所述硝酸盐转运蛋白NRT1.1B的序列如SEQ ID No.2所示；
所述硝酸盐转运蛋白NRT1.1B的编码基因序列如SEQ ID No.1中第1-1791位核苷酸分子所示；
所述植物为水稻。
2.根据权利要求1所述的应用，其特征在于：所述氮利用率的提高为硝酸盐含量或硝酸盐转运能力的提高。
3.一种构建转基因植物的方法，包括如下步骤：向受体植物中导入硝酸盐转运蛋白NRT1.1B的编码基因序列，使硝酸盐转运蛋白NRT1.1B在受体植物中过表达，得到转基因植物；转基因植物与受体植物相比，氮利用效率提高；
所述植物为水稻；
所述硝酸盐转运蛋白NRT1.1B的序列如SEQ ID No.2所示；所述硝酸盐转运蛋白NRT1.1B的编码基因序列如SEQ ID No.1中第1-1791位核苷酸分子所示。
4.根据权利要求3所述的方法，其特征在于：所述氮利用效率的提高为硝酸盐含量的提高。
5.一种构建近等基因系植物的方法，包括如下步骤：将供体亲本与受体亲本进行杂交，再与受体亲本继续多次回交，得到近等基因系植物；近等基因系植物与受体亲本和供体亲本相比，氮利用率提高；所述的供体亲本为籼稻品种IR24，受体亲本为粳稻品种日本晴。
6.根据权利要求5所述的方法，其特征在于：所述的氮利用率提高体现在如下方面中的至少一种：
(1)硝酸盐含量；
(2)氯酸盐敏感性；
(3)植物的株型；
(4)单株的产量；
(5)单株的分蘖数；
(6)小区产量；
(7)氮利用效率。
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| CN105481955B (zh) * | 2015-12-10 | 2018-11-13 | 中国农业科学院生物技术研究所 | 速生水生植物硝酸盐转运蛋白GeNRT2.1及其编码基因与应用 |
| CN106222262B (zh) * | 2016-07-27 | 2019-10-18 | 浙江省嘉兴市农业科学研究院（所） | 引物对及其在鉴别水稻氮高效利用基因nrt1.1b基因型中的应用 |
| CN107779468B (zh) * | 2016-08-17 | 2020-04-28 | 中国科学院遗传与发育生物学研究所 | 水稻nrt1.1a基因及其编码蛋白在提高植物产量育种中的应用 |
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| CN109811081A (zh) * | 2019-04-03 | 2019-05-28 | 江苏徐淮地区淮阴农业科学研究所 | 一种利用分子标记辅助选择氮高效粳稻的选育方法 |
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| CN110129480A (zh) * | 2019-06-13 | 2019-08-16 | 江苏徐淮地区淮阴农业科学研究所 | 检测水稻硝酸盐转运蛋白基因的引物组、试剂盒及其检测方法和应用 |
| CN110734484B (zh) * | 2019-11-26 | 2021-04-13 | 中国农业大学 | Nrt2_5蛋白在调控植物苞叶宽度中的应用 |
| CN110982828B (zh) * | 2020-01-02 | 2022-08-30 | 南京农业大学 | 一种水稻丛枝菌根特异诱导的硝酸盐转运蛋白基因及其应用 |
| CN113336835A (zh) * | 2020-03-02 | 2021-09-03 | 中国科学院分子植物科学卓越创新中心 | 参与硝酸根转运的蛋白、其编码基因和用途 |
| CN112695119A (zh) * | 2020-12-29 | 2021-04-23 | 华智生物技术有限公司 | 一种水稻硝酸盐转运蛋白基因nrt1.1b的snp分子标记及其应用 |
| CN114920810B (zh) * | 2021-02-01 | 2023-02-21 | 中国农业大学 | 硝酸盐吸收相关蛋白在调控玉米对硝酸盐吸收中的应用 |
| CN112795693B (zh) * | 2021-03-24 | 2022-02-18 | 湖南农业大学 | 与玉米叶片叶绿素含量相关的分子标记及其应用 |
| CN114921460B (zh) * | 2022-05-27 | 2023-11-17 | 广东省农业科学院水稻研究所 | 一种氮高效基因nrt1.1b的功能标记引物及其应用 |
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| US20170260538A1 (en) | 2017-09-14 |
| CN104277101A (zh) | 2015-01-14 |
| CN107208100A (zh) | 2017-09-26 |
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