CN108977460A - 重组表达载体及其在增加水稻产量和降低镉浓度上应用 - Google Patents
重组表达载体及其在增加水稻产量和降低镉浓度上应用 Download PDFInfo
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Abstract
本发明公开了一种重组表达载体及其在增加水稻产量和降低镉浓度上应用,该重组表达载体含有水稻基因OsNAR2.1的启动子和水稻基因OsAMT1.1,所述水稻基因OsNAR2.1的启动子核苷酸序列如SEQ ID No.1所示,水稻基因OsAMT1.1的核苷酸序列如SEQ ID No.2所示。本发明的重组表达载体能显著提高水稻的生长,增加水稻的籽粒产量,降低水稻籽粒中镉浓度。
Description
技术领域
本发明属于基因工程领域,涉及一种含水稻基因OsNAR2.1的启动子和水稻基因OsAMT1.1的重组表达载体及其应用。
背景技术
氮素是作物重要的大量营养元素之一,参与生物体各种代谢过程。它是植物体中很多生命物质的组成成分,比如:氨基酸、蛋白质、核酸、酶、叶绿素等。氮分别占植物体干重的1.5-2%和植物总蛋白的16%(Frink CR.,Waggoner PE.and Ausubel JH.Nitrogenfertilizer:retrospect and prospect.Proc.Nati.Acad.Sci.USA.1999.96:1175~1180.)。目前,中国氮肥用量占全球氮肥用量的30%(彭少兵,黄见良,钟旭华,杨建昌,王光火,邹应斌,张福锁,朱庆森,Roland Buresh,Christian Witt.提高中国稻田氮肥利用率的研究策略.中国农业科学.2002,35(9):1095~1103.),成为世界第一大消费国。其中水稻田中氮肥的施用量超过其它任何农作物,氮肥的损失量占施肥总量的70%。我国普遍存在着由于氮肥利用率低和大量的氮素损失导致的一系列环境问题。
镉被认为是强致癌物质,它在人体的潜伏可以长达10-30年,甚至影响下一代的健康。近些年,市场上出现了大量的“镉米”,“镉米”之所以出现,一个重要的原因就是随着工业化的迅猛发展,含“镉”等重金属的污染物通过各种途径进入水稻生产区的土壤,造成大米重金属严重超标等现象(Clemens S.,Aarts MGM.,Thomine S.,Verbruggen N.Plantscience:The key to preventing slow cadmium poisoning.Trends PlantSci.2013.18:92–99;Sebastian A.,Prasad MNV.Cadmium minimization in rice.Areview.Agron.Sustain.Dev.2014.34:155–173)。
发明内容
本发明所要解决的技术问题为:如何提供一种重组表达载体,提高水稻产量。
本发明的技术方案为:一种重组表达载体,含有水稻基因OsNAR2.1的启动子和水稻基因OsAMT1.1,所述水稻基因OsNAR2.1的启动子核苷酸序列如SEQ ID No.1所示,水稻基因OsAMT1.1的核苷酸序列如SEQ ID No.2所示。
进一步地,重组表达载体中载体为pTCK303质粒载体。
进一步地,重组表达载体中含有增强子或报告基因。
进一步地,增强子为转录增强子或翻译增强子。
进一步地,报告基因为抗生素抗性基因、GUS报告基因或荧光素酶报告基因。
本发明的重组表达载体在提高水稻的生长或/和提高水稻产量或/和降低水稻镉浓度上的应用。
与现有技术相比,本发明具有以下有益效果:
1、通过系统研究,首次公开了日本晴水稻转水稻基因OsNAR2.1启动子启动水稻基因OsAMT1.1可以增加水稻苗期的生物量,与对照相比,转基因株系的根重和地上部干重分别增加了约42.1%和38.6%。
2、本发明的水稻基因OsNAR2.1启动子启动水稻基因OsAMT1.1的转基因植株,与对照相比,转基因水稻的总分蘖、有效分蘖、穗长、单穗重、结实率和单株穗重都显著增加,转基因水稻田间产量增加18.3%-22.7%,总生物量增加19.2%-21.2%。
3、本发明首次公开了日本晴水稻转水稻基因OsNAR2.1启动子启动水稻基因OsAMT1.1可以减少水稻中各个部位镉的浓度,其中糙米中镉的浓度减少约47.6%
附图说明
图1:水稻基因OsNAR2.1启动子启动水稻基因OsAMT1.1载体示意图。
图2:苗期水培条件下转基因株系的生长表型;a:水培四周时的生长表型。b:转基因株系OsAMT1.1的表达鉴定。c:转基因株系地上和地下部生物量统计。
图3:转基因株系T2代的产量统计;a:收获期的生长表型。b:转基因株系产量和生物量统计。
图4:转基因株系T3代的产量统计;a:收获期的生长表型。b:转基因株系产量和生物量统计。
图5:转基因株系各个部位镉浓度分析。
具体实施方式
一、水稻基因OsNAR2.1启动子序列的克隆
1)水稻基因组DNA的提取
水稻(日本晴)幼苗长至3叶期,称取0.1g左右叶片,用液氮研碎,研磨充分加入2ml离心管,迅速加入1ml DNA提取液(购自TIANGEN,中国),充分摇匀振荡后,抽提水稻基因组DNA。
2)OsNAR2.1基因全长的克隆
通过NCBI网站(http://www.ncbi.nlm.nih.gov/)的基因数据库中检索到水稻的OsNAR2.1(AP004023.2)基因的启动子,用软件Primer 5.0设计引物序列(如下),从水稻基因组DNA中扩增NAR2.1基因的启动子序列。
P1:5’-TGCTGACAAACCAAACCGACT-3’(SEQ ID No.3)
P2:5’-CCCCACCTCTCCCACCTCAC-3’(SEQ ID No.4)
以步骤1)获得的水稻基因组DNA为模板,用高保真酶(Prime Star HS DNApolymerase购自Takara公司)进行PCR扩增,PCR程序如下:94℃预变性2min,94℃变性30s,53℃复性30s,72℃延伸30s,30个循环后,72℃5min,4℃恒温。琼脂糖电泳分离、切胶回收后克隆至pMD-19载体(购自Takara公司),测序正确后就获得具有完整编码区的水稻基因OsNAR2.1的启动子序列(SEQ ID No.1)。
二、水稻基因OsAMT1.1序列的克隆
1)总RNA的提取
水稻(日本晴)幼苗长至3叶期,用0.2mM硝态氮进行处理6小时后立即取根迅速置于液氮中冷冻保存,称取0.1g左右根,用液氮研碎,研磨充分加入1.5ml离心管,迅速加入1ml Trizol试剂(购自Invitrogen,USA),充分摇匀振荡后,抽提总RNA。
2)OsAMT1.1基因全长的克隆
利用水稻基因组注释计划网站(http://rice.plantbiology.msu.edu/)的基因数据库中检索到水稻的OsAMT1.1基因序列OsAMT1.1(LOC_Os04g43070)。设计引物(如下)从组织cDNA库中钓出OsAMT1.1的全长序列。
P3:5’-ATGGCGACGTGCGCGGCGGACC-3’(SEQ ID No.5)
P4:5’-TTACACTTGGTTGTTGCTGTTG-3’(SEQ ID No.6)
以步骤1)获得的总RNA为模板,经反转录合成cDNA第一链后,用高保真酶(PrimeStar HS DNA polymerase购自Takara公司)进行PCR扩增,PCR程序如下:94℃预变性2min,94℃变性30s,53℃复性30s,72℃延伸30s,30个循环后,72℃5min,4℃恒温。琼脂糖电泳分离、切胶回收后克隆至pMD-18载体(购自Takara公司),测序正确后就获得具有完整编码区的水稻高亲和硝酸盐运输蛋白基因OsAMT1.1的全长序列(SEQ ID No.2)。
三、表达水稻基因OsNAR2.1启动子启动水稻基因OsAMT1.1转基因株系的获得
1)水稻基因OsNAR2.1启动子启动OsAMT1.11载体的构建
根据步骤一中获得的水稻基因OsNAR2.1启动子序列,见SEQ ID No.1的序列,用软件Primer 5.0设计引物序列(如下):
P5:5’-GAGGCGCGCCTGCTGACAAACCAAACCGACT-3’(Asc I)(SEQ ID No.7)
P6:5’-CATTAATTAACCCCACCTCTCCCACCTCAC-3’(Pac I)(SEQ ID No.8)
以步骤一中获得的克隆OsNAR2.1启动子的pMD-18载体为模板,用高保真酶(PrimeStar HS DNA polymerase购自Takara公司)进行PCR扩增,PCR程序如下:94℃预变性2min,94℃变性30s,53℃复性30s,72℃延伸30s,30个循环后,72℃5min,4℃恒温。琼脂糖电泳分离、切胶回获得F端和R端分别添加酶切位点Asc I和Pac I的OsNAR2.1启动子的PCR产物。将回收产物用限制性内切酶Asc I和Pac I进行双酶切,同时用Asc I和Pac I双酶切植物过量表达载体pTCK303质粒,然后分别回收酶切过的PCR片段和载体。回收后通过T4连接酶将线性化的载体与酶切过的PCR片段在4℃下连接过夜,转化到大肠杆菌DH5a感受态细胞中,涂在含有卡那霉素50μgmL-1的LB固体培养基上生长12h后,挑取阳性菌落,提取质粒经Asc I和Pac I双酶切验证片段大小无误后,将该菌液进行DNA测序,将含有测序正确克隆命名为pOsNAR2.1-pTCK303。
根据步骤二中获得的水稻基因OsAMT1.1基因全长序列,见SEQ ID No.2的序列,用软件Primer 5.0设计引物序列(如下):
P7:5’-GAGGCGCGCCATGGCGACGTGCGCGGCGGACC-3’(Asc I)(SEQ ID No.9)
P8:5’-GAAAGCTTTTACACTTGGTTGTTGCTGTTG-3’(HindIII)(SEQ ID No.10)
以步骤二中获得的克隆OsAMT1.1基因全长的pMD-18载体为模板,用高保真酶(Prime Star HS DNA polymerase购自Takara公司)进行PCR扩增,PCR程序如下:94℃预变性2min,94℃变性30s,53℃复性30s,72℃延伸30s,30个循环后,72℃5min,4℃恒温。琼脂糖电泳分离、切胶回收后获得F端和R端分别添加酶切位点HindIII和Asc I的OsAMT1.1基因全长的PCR产物。将回收产物用限制性内切酶EcoR I和Asc I进行双酶切,同时用EcoR I和AscI双酶切pOsNAR2.1-pTCK303质粒,然后分别回收酶切过的PCR片段和载体。回收后通过T4连接酶将线性化的载体与酶切过的PCR片段在4℃下连接过夜,转化到大肠杆菌DH5a感受态细胞中,涂在含有卡那霉素50μgmL-1的LB固体培养基上生长12h后,挑取阳性菌落,提取质粒经HindIII和Asc I双酶切验证片段大小无误后,将该菌液进行DNA测序,将含有测序正确克隆命名为pOsNAR2.1-OsAMT1.1-pTCK303。即为表达载体,载体示意图见图1。
2)转基因植株的获得
获得的转有上述载体的农杆菌,侵染水稻愈伤组织,共培养60小时,经过选择培养、分化、生根、炼苗得到T0代转基因植株。对所有转基因材料都进行了扩繁,得到了稳定遗传的T2代和T3代材料。
在含2.5mM氮源(NH4 +:NO3 -=4:1)的水培营养液培养四周,qRT-PCR鉴定发现,与对照(WT:日本晴野生型水稻;Vector:转空载的转基因株系)相比,水稻基因OsNAR2.1启动子启动水稻基因OsAMT1.1转基因株系(Tr1,Tr2,Tr3)根部OsAMT1.1的表达显著提高,而地上部提高不显著,根重和地上部干重分别增加了约42.1%和38.6%(图2)。
田间实验表明,与对照相比,水稻基因OsNAR2.1启动子启动水稻基因OsAMT1.1转基因株系的总分蘖、有效分蘖、穗长、单穗重、结实率和单株穗重都显著增加(表1),转基因水稻田间产量增加18.3%-22.7%,总生物量增加19.2%-21.2%(图3,图4)。
与对照相比,水稻基因OsNAR2.1启动子启动水稻基因OsAMT1.1转基因株系,可以减少水稻中各个部位镉的浓度,其中糙米中镉的浓度减少约47.6%(图5)
表1转基因株系T2代的农艺性状统计
综上所述,本发明发现表达水稻基因OsNAR2.1启动子启动水稻基因OsAMT1.1能显著增加水稻的生长,提高水稻的最终籽粒产量,降低水稻各个部位包括糙米中镉的浓度;可利用本发明OsNAR2.1启动子启动OsAMT1.1表达载体,该表达载体中必要时可包括增强子,不论是转录增强子或翻译增强子。为了简化转化细胞的鉴定可使用选择性标记包括对抗生素抗性的酶,也可利用颜色变化(例如B-葡糖醛酸糖苷酶GUS)或发光(例如荧光素酶)来识别的化合物的酶类,也可用无标记选择。所用的表达载体可使用Ti质粒,Ri质粒,植物病毒载体等。转化方法可用经农杆菌介导法、基因枪法、花粉管通道法或其它方法转化植物。表达该载体可以增加水稻的生长,提高水稻的最终籽粒产量,降低水稻籽粒中镉的浓度。
序列表
<110> 申请人名称 中国农业科学院深圳农业基因组研究所
<120> 重组表达载体及其在增加水稻产量和降低镉浓度上应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1698
<212> DNA
<213> 水稻(Oryza sativa)
<400> 1
gattccccac ctctcccacc tcactcctac ctcactccta gtcctctgcc gaaagtactt 60
cctccgtttc acaatgtaag tcattctaat atttttcaca ttcatattga tgtttgaatc 120
tagattgata tatatgttta gattcgttag catcgatatg aatatgggaa atgctagaat 180
gacttatatt gtgaaacaga gtgagtatca tgtaaaagtt agaaggaaaa aaatagagct 240
gtttgtgatg atatgggtgt ggttgtgttg tgtgagccga tgtccattgt actgtactca 300
ttttaaatgt acgtaccgtt aacttatata gttatatgcg tttgatcatt tgtcaaaatt 360
tagtgaaact ttaaaattta ttatacttaa agtatattta atgataaatt taaaataaaa 420
taaactttca gctgttatgt tcaaaatcaa catcgtcaga tattttaaat taaaggtagt 480
acttttaaaa aaaggatttt tgcggtgtgt cgtggcgaaa ctgctaccaa gtttcaatga 540
tcatatgcca tttcatagga taattactct catcgtggta agtaagaatc gattgcctat 600
tttcggcagg ctgttgtttc aaagcatcga tctgcttgga caacttgagc aaagctagct 660
agaactgggt cgatataatt gcagcactag gcaatcaaga gacggagctg ccaccagcta 720
gctgagctga gctgatatga tcaacacagt gcagacttgg tcgtgttcga gttcgatcga 780
cggatggctg tcctgctctt gcgctcatgc atgtcatctc ttcggaagta ggagtacagc 840
agtacttgag gaatattatt agagagtaag ttgaactgtt ttcaatagtt cagggtgtaa 900
actaagctga ggaattgtta ggaggttaaa tgctgtggca aaatagtttg gaggagcgaa 960
atgatttttt tttcatatga aaaacatcta aatttatttt ttgccaaaac actagtatat 1020
catcaaattt tcatccatta agaacgcctt ctcaatatta ataattccaa tgtgatatct 1080
taatgctcaa tgaacctaaa atagtttgga tgagtgaaat ggactctttt tgagtttttt 1140
tccatatgaa aacatctaaa tttatttttt tttgccaaaa cactggtata tcatcaaatt 1200
ttcctccatt aagaacgcct tctcaacgtt aataactcca atgttattat cttaatgcca 1260
aatgaaccta ccatgaacgt catgctcaca atttaattaa caacaaccgg gcactcaaga 1320
tcattcgcgg ttgccgcttc tcaccggttg cctgaaccct tgggacccct ccaaaagctt 1380
aattaccccc aaaaccgcat gatctctctc ttctcttctc ttctcacacg tcgtcaaagc 1440
ctctgacttt ggatatcccc gaccccacta aacttaatca acttgatcat tacaacaatt 1500
aagttgcctc ttgaatccaa cgaagtagct ggtcaactct ccgagctcta gcctcgctct 1560
cccgcctata aattcaccga tcgatcgatc gatcgatctc agcatcagca gcagcagcag 1620
attcatttct tggtcttcgt ctccgtctcc gtccttgggt tgatatccag aatcagtcgg 1680
tttggtttgt cagcaatg 1698
<210> 2
<211> 1497
<212> DNA
<213> 水稻(Oryza sativa)
<400> 2
atggcgacgt gcgcggcgga cctggcgccg ctgctggggc cggtggcggc gaacgcgacg 60
gactacctgt gcaaccggtt cgccgacacg acgtcggcgg tggacgcgac gtacctgctc 120
ttctcggcgt acctcgtgtt cgccatgcag ctcgggttcg cgatgctctg cgccgggtcg 180
gtgcgggcca agaacacgat gaacatcatg ctcaccaacg tgctcgacgc cgcggccggg 240
gcgctcttct actacctctt cggcttcgcc ttcgccttcg gcacgccgtc caacggcttc 300
atcgggaagc agttcttcgg cctcaagcac atgccgcaga ccgggttcga ctacgacttc 360
ttcctcttcc agtgggcctt cgccatcgcc gccgccggga tcacgtcggg ctccatcgcc 420
gagaggacgc agttcgtcgc ctacctcatc tactccgcct tcctcaccgg gttcgtctac 480
ccggtggtgt cccactggat ctggtccgcc gatgggtggg cctctgcctc ccgcacgtcc 540
ggacctctgc tgttcggctc cggtgtcatc gacttcgccg gctccggcgt cgtccacatg 600
gtcggcggtg tcgccgggct ctggggcgcg ctcatcgagg gcccccgcat cgggaggttc 660
gaccacgccg gccgatcggt ggcgctcaag ggccacagcg cgtcgctcgt cgtgcttggc 720
accttcctgc tgtggttcgg ctggtacgga ttcaaccccg ggtcgttcac caccatcctc 780
aagacgtacg gcccggccgg cggcatcaac gggcagtggt ccggagtcgg ccgcaccgcc 840
gtgacgacga ccctggccgg cagcgtggcg gcgctcacca cgctgttcgg gaagcggctc 900
cagacggggc actggaacgt ggtcgacgtc tgcaacggcc tcctcggcgg gttcgccgcc 960
atcaccgccg ggtgcagcgt cgtcgacccg tgggccgcga tcatctgcgg gttcgtctcg 1020
gcgtgggtgc tcatcggcct caacgcgctc gccgcgcgcc tcaagttcga cgacccgctc 1080
gaggccgccc agctccacgg cgggtgcggc gcgtggggga tcctcttcac cgcgctcttc 1140
gcgaggcaga agtacgtcga ggagatctac ggcgccggcc ggccgtacgg cctgttcatg 1200
ggcggcggcg gcaagctgct cgccgcgcac gtcatccaga tcctggtcat cttcgggtgg 1260
gtcagctgca ccatgggacc tctcttctac gggctcaaga agctcggcct gctccgcatc 1320
tccgccgagg acgagacgtc cggcatggac ctgacacggc acggcgggtt cgcgtacgtc 1380
taccacgacg aggacgagca cgacaagtct ggggttggtg ggttcatgct ccggtccgcg 1440
cagacccgcg tcgagccggc ggcggcggct gcctccaaca gcaacaacca agtgtaa 1497
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgctgacaaa ccaaaccgac t 21
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ccccacctct cccacctcac 20
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggcgacgt gcgcggcgga cc 22
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ttacacttgg ttgttgctgt tg 22
<210> 7
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gaggcgcgcc tgctgacaaa ccaaaccgac t 31
<210> 8
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cattaattaa ccccacctct cccacctcac 30
<210> 9
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gaggcgcgcc atggcgacgt gcgcggcgga cc 32
<210> 10
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gaaagctttt acacttggtt gttgctgttg 30
Claims (6)
1.一种重组表达载体,含有水稻基因OsNAR2.1的启动子和水稻基因OsAMT1.1,所述水稻基因OsNAR2.1的启动子核苷酸序列如SEQ ID No.1所示,水稻基因OsAMT1.1的核苷酸序列如SEQ ID No.2所示。
2.根据权利要求1所述的一种重组表达载体,其特征在于,重组表达载体中载体为pTCK303质粒载体。
3.根据权利要求1所述的一种重组表达载体,其特征在于,重组表达载体中含有增强子或报告基因。
4.根据权利要求3所述的一种重组表达载体,其特征在于,增强子为转录增强子或翻译增强子。
5.根据权利要求3所述的一种重组表达载体,其特征在于,报告基因为抗生素抗性基因、GUS报告基因或荧光素酶报告基因。
6.根据权利要求1-5任一项所述的重组表达载体在提高水稻的生长或/和提高水稻产量或/和降低水稻镉浓度上的应用。
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