CN1312174C - 一种钠/质子反向转运器及其编码基因与应用 - Google Patents
一种钠/质子反向转运器及其编码基因与应用 Download PDFInfo
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Abstract
本发明公开了一种钠/质子反向转运器及其编码基因与应用。本发明所提供的钠/质子反向转运器来源于玉米,名称为zmNHX1,是具有序列表中序列2氨基酸残基序列的蛋白质,或者是将序列2的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有与序列2的氨基酸残基序列相同活性的由序列2衍生的蛋白质。钠/质子反向转运器zmNHX1的编码基因,是下列核苷酸序列之一:1)序列表中序列1的DNA序列;2)与序列表中序列1限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA序列。本发明的基因对培育抗盐植物品种,提高农作物产量具有重要意义。
Description
技术领域
本发明涉及植物基因工程领域中的钠/质子反向转运器及其编码基因与应用。
背景技术
盐渍对农业生产有极其严重的影响。盐胁迫是抑制植物生长,降低农作物产量的主要环境因素之一。中国15亿亩耕地中盐碱地大约有一亿亩,中低产田约10亿亩,大部分是由于干旱盐碱所致,另外还有3亿亩盐碱荒地。而且,在灌溉地区次生盐渍化田地还在继续增加。
Na+/H+反向转运器(Na+/H+antiporter)在细胞膜和液泡膜上都有分布。Na+/H+反向转运器与盐生植物的抗盐性有很大关系,它保证了Na+在液泡中的区域化,大大降低了液泡的渗透势,又减轻了Na+对细胞质的伤害。Na+/H+反向转运器还可以将进入根中的Na+泵出,维持体内低浓度的离子环境,这种机制在许多农作物中普遍存在。高等植物中Na+流出的主要机制是质膜上的H+/ATPase把H+泵出细胞,产生的电化学势可激活质膜上的Na+/H+反向转运器,H+进入细胞,Na+逆电化学势流出。从生理生化角度,在许多高等植物及藻类上都证明了Na+/H+反向转运器的活动。
许多植物的Na+/H+反向转运器载体的活性很低,因此他们在高盐环境中会失水最终死亡。在高盐环境中,Na+/H+反向转运器的合成及其活动都会受到抑制,但是通过提高细胞膜上不饱和脂肪酸的含量,可以消除这种阻碍因素的影响或通过转基因的方法增加Na+/H+反向转运器在植株中的含量来达到抗盐的目的。
1999年Apse用农杆菌介导的方法将AtNHX1转入拟南芥中,得到抗盐植株。这一结果给培育抗盐品种带来了美好的前景。
发明内容
本发明的目的是提供钠/质子反向转运器及其编码基因。
本发明所提供的钠/质子反向转运器来源于玉米,名称为zmNHX1,是具有序列表中序列2氨基酸残基序列的蛋白质,或者是将序列2的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有与序列2的氨基酸残基序列相同活性的由序列2衍生的蛋白质。
序列表中序列2氨基酸残基序列是由539个氨基酸残基组成的蛋白质。
以玉米EST设计引物,通过5’、3’RACE方法克隆到zmNHX1基因的全长cDNA。钠/质子反向转运器zmNHX1的编码基因,是下列核苷酸序列之一:
1)序列表中序列1的DNA序列;
2)与序列表中序列1限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA序列。
序列表中序列1的DNA序列由2035个碱基组成,该基因的读码框为自5’端第1到第1617位碱基。
本发明所提供的钠/质子反向转运器及其编码基因,可以来自所有单子叶和双子叶植物,如烟草,拟南芥,草坪草,杨树,大豆,小麦,水稻,棉花和番茄等。利用任何一种可以引导外源基因在植物中表达的载体,将本发明所提供的编码钠/质子反向转运器zmNHX1的基因导入植物细胞,可获得对高盐胁迫耐受力增强的转基因细胞系及转基因植株。本发明的基因在构建到植物表达载体中时,在其转录起始核苷酸前可加上任何一种增强启动子或诱导型启动子,如花椰菜花叶病毒(CAMV)35S启动子,Ubiqutin启动子,增强子既可以是转录增强子,也可以是翻译增强子。为了便于对转基因植物细胞或植物进行鉴定及筛选,可对所使用的载体进行加工,如加入植物可选择性标记(GUS基因、萤光素酶基因等)或具有抗性的抗生素标记物(庆大霉素,卡那霉素等)。携带有本发明钠/质子反向转运器zmNHX1基因的表达载体可以是Ti(Tumor-induced-癌诱导)质粒、Ri(Root-induced-根诱导)质粒、植物病毒载体等,转化可通过农杆菌介导法、基因枪法、花粉管通道法等常规生物技术方法实现,被转化的植物宿主既可以是单子叶植物,也可以是双子叶植物,如:水稻,小麦,玉米,大豆,棉花,蔬菜,树木和草坪草等。本发明的基因对培育抗盐植物品种,提高农作物产量具有重要意义。
附图说明
图1为Northern杂交分析结果。
具体实施方式
实施例1、钠/质子反向转运器zmNHX1的克隆
营养生长阶段的玉米(生长2周,4到5叶期)用150mmNaCl处理3小时。用TRIZLO(PROMAGA)提取根总RNA。根据玉米EST序列设计并合成基因特异引物
GSP1:5’agcgtgcacacagaatccgttgcag
GSP2:5’tgctgaatatcgctattgcgccgag
GSP3:5’ctcggcgcaatagcgatattcagca
GSP4:5’ctgcaacggattctgtgtgcacgct
具体操作按SMART RACE(CLONTECH)试剂盒进行。首先进行逆转录。反映体系为:1ul盐处理玉米根总RNA,1ul 5’-CDS或3’-CDS,1ul SMATII OLIGO引物(10um/L),2ul灭菌水。混匀后,于70℃加热2min。置冰上冷却2min,离心使液体沉底。加入2ul 5X第一链缓冲液,1ul DTT(20mm/L),1ul dNTP(10mm/L)和1ul MMLV逆转录酶(20U/ul),42℃逆转录1.5小时,加入100ul灭菌水,于72℃加热2min终止反应。
随后,以通用引物混合物(UPM)和GSP1,UPM和GSP3分别进行5’,3’“TOUCHDOWN”PCR扩增。在PERKIN-ELMOR(PE)DNA THERMAL CYCLER 9700启动PCR反应。条件为5X(94℃30S,72℃3min),5X(94℃30S,70℃10S,72℃2min),20X(94℃30S,63℃30S,72℃30S)72℃min。以该次反应为模板,分别使用通用引物(NUP)和GSP2,GSP4进行巢式PCR。5’RACE获得400bp大小的条带,3’RACE获得1.6Kb大小的条带。分别回收纯化后,5’RACE片段连在PGEM-T EASY载体上,3’RACE直接测序。测序结果和已知EST序列进行拼接,并结合测序峰图进行校正。获得拼接好的序列zmNHX1基因,如序列表中的序列1所示,全长共2035bp,其中3’非编码区为418bp,编码区为1617bp。编码产物为539个氨基酸,如序列表中的序列2所示。
实施例2、zmNHX1基因在逆境胁迫条件下的表达特征
玉米种于含蛭石的花盆中,待长到3叶一心时期进行不同浓度的盐胁迫处理:50mM,150mM NaCl进行浇灌至土壤饱和。3小时后收取,提取根、叶总RNA,进行Northern blot分析。结果如图3所示,图中R:对照根;L:对照叶;50mmR:50mMNaCl处理根;50mmL:50mM NaCl处理叶;150mmR:150mM NaCl处理根;150mmL:150mMNaCl处理叶。从图中可以看出,zmNHX1基因在根中表达,在叶中不表达。50mMNaCl处理后,zmNHX1表达量大幅度升高。在100mM NaCl处理时,zmNHX1转录水平高于50mM NaCl处理时的转录水平。表明zmNHX1在根中表达并受盐胁迫诱导。
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Trp His Asn Asp Ile Leu Leu His Ile Ser Leu Gly Ala Ile Ala
125 130 135
Ile Phe Ser Arg Met Asn Ile Gly Thr Leu Asp Val Gly Asp Phe
140 145 150
Leu Ala Ile Gly Ala Ile Phe Ser Ala Thr Asp Ser Val Cys Thr
155 160 165
Leu Gln Val Leu His Gln Asp Glu Thr Pro Phe Leu Tyr Ser Leu
170 175 180
Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser Val Val Leu
185 190 195
Phe Asn Ala Leu Gln Asn Phe Asp Leu Asn His Ile Asp Val Ala
200 205 210
Val Val Leu Asn Phe Leu Gly Asn Phe Cys Tyr Leu Phe Val Ser
215 220 225
Ser Thr Leu Leu Gly Val Phe Thr Gly Leu Leu Ser Ala Tyr Ile
230 235 240
Ile Lys Lys Leu Tyr Ile Gly Arg His Ser Thr Asp Arg Glu Val
245 250 255
Ala Leu Met Met Leu Met Ala Tyr Leu Ser Tyr Met Leu Ala Glu
260 265 270
Leu Leu Asp Leu Ser Gly Ile Leu Thr Val Phe Phe Cys Gly Ile
275 280 285
Val Met Ser His Tyr Thr Trp His Asn Val Thr Glu Ser Ser Arg
290 295 300
Val Thr Thr Lys His Ala Phe Ala Thr Leu Ser Phe Ile Ala Glu
305 310 315
Thr Phe Leu Phe Leu Tyr Val Gly Met Asp Ala Leu Asp Ile Glu
320 325 330
Lys Trp Glu Phe Ala Ser Asp Ser Pro Gly Lys Ser Ile Gly Ile
335 340 345
Ser Ser Ile Leu Leu Gly Leu Val Leu Leu Gly Arg Ala Ala Phe
350 355 360
Val Phe Pro Leu Ser Phe Leu Ser Asn Leu Thr Lys Lys Ser Pro
365 370 375
Leu Glu Lys Ile Thr Phe Arg Gln Gln Ile Val Ile Trp Trp Ala
380 385 390
Gly Leu Met Arg Gly Ala Val Ser Ile Ala Leu Ala Tyr Asn Lys
395 400 405
Phe Thr Arg Ser Gly His Thr Glu Leu His Gly Asn Ala Ile Met
410 415 420
Ile Thr Ser Thr Ile Thr Val Val Leu Phe Ser Thr Met Val Phe
425 430 435
Gly Met Met Thr Lys Pro Leu Ile Arg Leu Leu Leu Pro Ala Cys
440 445 450
Ser Asn Thr Ala Thr Ser Glu Pro Ser Ser Pro Lys Ser Leu His
455 460 465
Ser Pro Leu Leu Thr Ser Met Gln Gly Ser Asp Ile Glu Thr Gly
470 475 480
Ser Ala Gln Ile Val Arg Pro Ser Ser Leu Arg Met Leu Leu Ser
485 490 495
Lys Pro Thr His Thr Val His Tyr Tyr Trp Arg Lys Phe Asp Asp
500 505 510
Ala Leu Met Arg Pro Met Phe Gly Gly Arg Gly Phe Val Pro Phe
515 520 525
Ser Pro Gly Ser Pro Thr Glu Gln Ser Val His Asp Gly Arg
530 535 539
Claims (7)
1、钠/质子反向转运器,是具有序列表中序列2氨基酸残基序列的蛋白质。
2、权利要求1所述钠/质子反向转运器的编码基因,是下列核苷酸序列之一:
1)序列表中序列1的DNA序列;
2)与序列表中序列1限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA序列。
3、根据权利要求2所述的基因,其特征在于:所述钠/质子反向转运器的编码基因是序列表中序列1的DNA序列。
4、根据权利要求3所述的基因,其特征在于:该基因的读码框为自5’端第1到第1617位碱基。
5、含有权利要求3所述基因的表达载体。
6、含有权利要求3所述基因的转基因细胞系。
7、权利要求3所述基因在培育抗盐植物品种中的应用。
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CNB021463107A CN1312174C (zh) | 2002-10-18 | 2002-10-18 | 一种钠/质子反向转运器及其编码基因与应用 |
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CN1312174C true CN1312174C (zh) | 2007-04-25 |
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN1204364A (zh) * | 1995-10-12 | 1999-01-06 | 康乃尔研究基金会有限公司 | 抗旱或抗盐胁迫转基因谷类植物的生产 |
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN1204364A (zh) * | 1995-10-12 | 1999-01-06 | 康乃尔研究基金会有限公司 | 抗旱或抗盐胁迫转基因谷类植物的生产 |
Non-Patent Citations (1)
Title |
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植物盐胁迫应答的分子机制 沈义国,陈受宜,遗传,第23卷第4期 2001 * |
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