CN106929522B - 氨基酸转运基因OsAAP1在低氮下促进水稻生长的应用 - Google Patents
氨基酸转运基因OsAAP1在低氮下促进水稻生长的应用 Download PDFInfo
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- CN106929522B CN106929522B CN201710099941.9A CN201710099941A CN106929522B CN 106929522 B CN106929522 B CN 106929522B CN 201710099941 A CN201710099941 A CN 201710099941A CN 106929522 B CN106929522 B CN 106929522B
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Abstract
本发明公开了氨基酸转运基因OsAAP1在低氮下促进水稻生长的应用，属于植物基因工程领域。OsAAP1基因编码蛋白质的氨基酸及其cDNA序列分别如SEQ ID NO.1、2所示。本发明通过构建水稻OsAAP1基因的超表达植株，发现在低氮下提高OsAAP1基因的表达，可以使水稻根长、根数、株高、分蘖数增加，生物量、产量提高。通过构建干扰植株，发现在低氮下通过降低OsAAP1基因表达可以使水稻根长、根数、株高、分蘖数减少。因此OsAAP1基因可用于水稻在低氮下促进水稻生长，来提高水稻生物量、产量。OsAAP1基因在水稻耐受低肥或贫瘠土壤适应性方面、在减少农田化肥使用量及水稻品种改良中具有重要应用。
Description
技术领域
本发明属于植物基因工程领域，具体涉及氨基酸转运基因OsAAP1在低氮下促进水稻生长的应用。
背景技术
植物通过吸收土壤中的氨、硝酸根、氨基酸、可溶性肽等来获得氮素；氮的吸收和转运主要依靠铵根运输蛋白（AMT）、硝酸根运输蛋白（NRT）、氨基酸运输蛋白（AAT）、肽运输蛋白（PTR）等运输蛋白来完成（Williams L, Miller A. Transporters responsible forthe uptake and partitioning of nitrogenous solutes. Annu Rev Plant Biol andPlant Mol Biol, 2001, 52: 659-688.）。铵通过植物AMT吸收后再通过谷氨酰胺合成酶（GS）和谷氨酸合酶（GOGAT）合成谷氨酰胺和谷氨酸，后者再进一步形成其它氨基酸（SonodaY, Ikeda A, Saiki S, et al. Feedback regulation of the ammonium transportergene family AMT1 by glutamine in rice. Plant Cell Physiol, 2003, 44: 1396-1402.）。植物可通过高亲和转运系统（HATS）的NRT2和低亲和转运系统（LATS）的NRT1吸收环境中的硝酸盐，由硝酸还原酶（NR）和亚硝酸还原酶（NiR）还原形成铵，再进一步形成氨基酸（Paungfoo-Lonhienne C, Lonhienne T G, Rentsch D, et al. Plants can useprotein as a nitrogen source without assistance from other organisms. PNAS,2008, 105: 4524-4529.）。
在高等植物中，AAT是一类跨膜蛋白，将氨基酸从胞外运至胞内，同时还在氨基酸远距离运输、病原反应和非生物胁迫等方面发挥着重要作用（Tegeder M. Transportersfor amino acids in plant cells: some functions and many unknowns. Curr OpinPlant Biol, 2012, 15: 1-7.）。AAT基因被分为两个超家族：APC（氨基酸、多胺和胆碱转运）超家族和AAAP（氨基酸/生长素透性酶）超家族。APC超家族分为三个亚家族：CATs（阳离子氨基酸转运蛋白）家族、ACTs（氨基酸/胆碱转运蛋白）家族和PHSs（多胺、H+协同转运蛋白）家族。AAAP超家族分为六个亚家族：AAPs（氨基酸透性酶）家族、LHTs（赖氨酸和组氨酸转运蛋白）家族、ProTs（脯氨酸转运蛋白）家族、GATs（γ-氨基酸丁酸，GABA）家族、AUXs（生长素转运蛋白）家族和ANTs（芳香族和中性氨基酸转运蛋白）家族（Fischer WN, Andre B,Rentsch D, et al. Amino acid transport in plants. Trends Plant Sci, 1998, 3:188-195.）。
水稻基因组中共找到85个AAT家族成员（Zhao H, Ma H, Yu L, et al. Genome-wide survey and expression analysis of amino acid transporter gene family inrice (Oryza sativa L.). PLoS ONE, 2012, 7: e49210.）。研究发现OsAAT5、OsAAT7、OsAAT24、OsAAT49和OsAAT60的T-DNA插入突变体的稻米产量及植物体干重均下降，证明AAT对水稻的氮素积累及碳氮分配有着重要作用（Lu Y, Song Z, Lu K, et al. Molecularcharacterization, expression and functional analysis of the amino acidtransporter gene family (OsAATs) in rice. Acta physiol Plant, 2012, 34: 1943-1962.）。研究发现超量表达OsAAP6会增加水稻籽粒中储藏性蛋白和氨基酸含量，从而改善稻米营养和风味（Peng B, Kong H, Li Y, et al. OsAAP6 functions as an importantregulator of grain protein content and nutritional quality in rice. NatCommun, 2014, 5: doi:10.1038.）。氨基酸转运蛋白对水稻等各种植物体的氨基酸吸收、转运和储藏具有重要的作用。关于水稻AAT家族成员研究的报道很少，目前水稻氨基酸转运家族OsAAP1基因对水稻的生长发育目前未有任何研究。本发明发现OsAAP1基因在低氮下对水稻生物量影响有较为重要的作用，可应用于植物氮利用效率提高，减少化肥使用和水稻株型改良。
发明内容
本发明的目的在于解决现有技术中存在的问题，提供氨基酸转运基因OsAAP1在低氮下促进水稻生长的应用。
本发明的目的通过下述技术方案实现：
本发明以水稻的氨基酸转运基因OsAAP1为对象，从水稻中花11中克隆了OsAAP1的cDNA序列。通过构建OsAAP1基因的超表达载体，采用农杆菌EHA105介导的遗传转化方法，将超表达载体导入正常粳稻品种中花11中，得到OsAAP1基因的超表达植株，在低氮（氮浓度1mM以下）培养下，超表达植株的根长、根数、株高、分蘖数等与对照野生型中花11相比显著提高。同时构建了OsAAP1基因的干扰载体，将干扰载体导入中花11中，得到OsAAP1基因的干扰植株，在低氮培养下，其根长、根数、株高、分蘖数等与中花11相比显著降低。这些结果表明，通过提高OsAAP1基因表达，可以促进水稻生长，增加生物量和产量；OsAAP1基因在水稻耐受低肥或贫瘠土壤适应性方面有应用价值，另外在减少农田化肥使用量、减轻环境负面影响上也具有重要应用；还可以通过分子育种应用于水稻品种改良中。
基于本发明发现的OsAAP1基因的功能，其可用于低氮条件下促进水稻生长、提高水稻生物量、产量。具体可通过基因工程实现，即超表达提高OsAAP1基因的表达，使水稻根长、根数、株高、分蘖数等增加，达到提高水稻生物量、产量的目的。所述的OsAAP1基因编码的OsAAP1蛋白的氨基酸序列如SEQ ID NO.1所示；所述的OsAAP1基因的cDNA序列优选如SEQID NO.2所示。
应该理解为，在不影响OsAAP1蛋白活性的前提下（即不在蛋白的活性中心），本领域技术人员可对SEQ ID NO.1所示的氨基酸序列进行各种取代、添加和/或缺失一个或几个氨基酸获得具有同等功能的氨基酸序列。因此，OsAAP1蛋白还包括SEQ ID NO.1所示氨基酸序列经取代、替换和/或增加一个或几个氨基酸获得的具有同等活性的蛋白质。此外，应理解，考虑到密码子的简并性以及不同物种密码子的偏爱性，本领域技术人员可以根据需要使用适合特定物种表达的密码子。
本发明的优点和效果：
（1）本发明克隆的OsAAP1基因提高表达后可以在低氮下使水稻根长、根数、株高、分蘖数增加，说明OsAAP1基因对提高水稻生物量、产量有较明显作用，因此，通过基因工程技术提高OsAAP1基因的表达能够提高植物生物量、产量。这不仅有助于通过减少氮肥使用培育高产水稻，还可以通过分子育种进行植物的品种改良。
（2）OsAAP1基因的成功克隆，进一步证实了氨基酸转运家族在氮吸收过程中的重要作用，对阐明氨基酸转运家族的生物学功能有重要的意义，另外对进一步了解植物氮代谢途径，提高氮吸收效率有极大的推动作用。
（3）尽管目前已克隆到了一些氮营养途径中影响植物生长的基因，但对植物生长发育的分子机制仍不清楚。而本发明克隆的OsAAP1基因能够提高水稻的生物量，对确定植物增产的关键因素有极大的推动作用。
附图说明
图1是低氮（0.5mM硝酸铵）培养下对照中花11、OsAAP1基因超表达植株3个株系和OsAAP1基因干扰植株3个株系的整株表型图。
图2是对照中花11（WT）、OsAAP1基因超表达植株3个株系（OE1-3）和OsAAP1基因干扰植株3个株系（Ri1-3）在不同氮水平培养条件下根长的统计柱状图，数据采用SPSS软件进行变量分析（ANOVA），使用Duncan’s在0.05水平上进行差异显著性分析，同一种浓度培养的水稻指标在此浓度下均与对照相比，具有差异显著的表为*。低氮浓度为硝酸铵0.5mM，中氮浓度为硝酸铵2mM，高氮浓度为硝酸铵8mM。
图3是对照中花11（WT）、OsAAP1基因超表达植株3个株系（OE1-3）和OsAAP1基因干扰植株3个株系（Ri1-3）在不同氮水平培养条件下根数的统计柱状图，数据采用SPSS软件进行变量分析（ANOVA），使用Duncan’s在0.05水平上进行差异显著性分析，同一种浓度培养的水稻指标在此浓度下均与对照相比，具有差异显著的表为*。低氮浓度为硝酸铵0.5mM，中氮浓度为硝酸铵2mM，高氮浓度为硝酸铵8mM。
图4是对照中花11（WT）、OsAAP1基因超表达植株3个株系（OE1-3）和OsAAP1基因干扰植株3个株系（Ri1-3）在不同氮水平培养条件下株高的统计柱状图，数据采用SPSS软件进行变量分析（ANOVA），使用Duncan’s在0.05水平上进行差异显著性分析，同一种浓度培养的水稻指标在此浓度下均与对照相比，具有差异显著的表为*。低氮浓度为硝酸铵0.5mM，中氮浓度为硝酸铵2mM，高氮浓度为硝酸铵8mM。
具体实施方式
下面结合实施例对本发明做进一步详细的说明，但本发明的实施方式不限于此。若未特别指明，下述实施例所用的技术手段为本领域技术人员所熟知的常规手段；所用的实验方法均为常规方法，并可按照已描述的重组技术（参见分子克隆，实验室手册，第2版，冷泉港实验室出版社，冷泉港，纽约）完成；所用的材料、试剂等，均可从商业途径得到。
实施例1 OsAAP1基因超表达植株的构建
提取水稻中花11的RNA，并将其反转录成cDNA，利用引物对：
F1：5'-AGATCTATGGGGATGGAGAGGCCGCAAGAG-3'（BglII），
R1：5'-CTTAAGTCATGAGGAGACGCTGAATGG-3'（AflII）；
通过PCR扩增OsAAP1基因的cDNA后，通过BglII和AflII酶切后连入pCAMBIA-1301载体（pCAMBIA-1301载体购自Cambia公司），构建出OsAAP1基因的超表达载体OsAAP1-p1301。采用农杆菌EHA105介导的遗传转化方法，将超表达载体导入正常水稻品种中花11中。
将得到的所有转基因小苗移栽于带泥土的筐中，定期浇水，施肥，待小苗长高约10cm时，将50株水稻转基因小苗浸泡于500mL蒸馏水制备的浓度为50mg/L的潮霉素溶液48小时，之后叶子为绿色并舒张、生长状态良好的植株为阳性转基因植株；而叶子枯黄并卷曲的植株为阴性植株，随即死亡。阳性植株单株种植并收种，直至T2代鉴定出在上述潮霉素溶液中无任何叶子枯黄并卷曲的为纯合的转基因植株，即得到OsAAP1基因超表达植株。将超表达植株、中花11的种子在培养皿上用蒸馏水浸种3天并培养7天后，转入水稻营养液培养，营养液配方参考国际水稻所配方，但将硝酸铵调成0.5mM（低氮）、2mM（中氮）和8mM（高氮），分别培养40天，观察表型，统计根长、根数和株高。植株表型、根长、根数和株高的统计结果见图1-4，可见在低氮培养下，OsAAP1基因超表达植株与对照中花11植株相比根长、根数和株高增加，并达到差异显著。而在中氮时，超表达植株根长、根数和株高并没有比对照显著增加。在高氮时，超表达植株只有根数比对照增加达到显著差异，而根长和株高没有比对照显著增加。后期，OsAAP1基因超表达植株的分蘖数、氨基酸含量及最后的产量也均显著高于对照中花11。
实施例2 OsAAP1基因干扰植株的构建
提取水稻中花11的RNA，并将其反转录成cDNA，利用引物对：
F2：5'-GGTACCTGGGGATGGAGAGGCCGCAA-3'（KpnI），
R2：5'-GGATCCTTGCGCTTGCCATGGACGGGGT-3'（BamH I）；
F3：5'-ACTAGTTGGGGATGGAGAGGCCGCAA-3'（SpeI），
R3：5'-GAGCTCTTGCGCTTGCCATGGACGGGGT-3'（Sac I）；
各自PCR扩增出OsAAP1基因的cDNA片段，通过上述相应的限制性内切酶酶切后连入pTCK303载体，构建出OsAAP1基因的干扰表达载体OsAAP1-pTCK303。采用农杆菌EHA105介导的遗传转化方法，将干扰表达载体导入正常粳稻品种中花11中。
将得到的所有转基因小苗移栽于带泥土的筐中，定期浇水，施肥，待小苗长高约10cm时，将50株水稻转基因小苗浸泡于500mL蒸馏水制备的浓度为50mg/L的潮霉素溶液48小时，之后叶子为绿色并舒张、生长状态良好的植株为阳性转基因植株；而叶子枯黄并卷曲的植株为阴性植株，随即死亡。阳性植株单株种植并收种，直至T2代鉴定出在上述潮霉素溶液中无任何叶子枯黄并卷曲的为纯合的转基因植株，即得到OsAAP1基因干扰植株。将干扰植株、中花11的种子在培养皿上用蒸馏水浸种3天并培养7天后，转入水稻营养液培养，营养液配方参考国际水稻所配方，但将硝酸铵调成0.5mM（低氮）、2mM（中氮）和8mM（高氮），分别培养40天，观察表型，统计根长、根数和株高。植株表型、根长、根数和株高的统计结果见图1-4，可见在低氮、中氮和高氮培养下，OsAAP1基因干扰植株与对照中花11植株相比根长、根数和株高均减少，并达到差异显著。后期，OsAAP1基因干扰植株的分蘖数、氨基酸含量及最后的产量也均显著低于对照中花11。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 武汉生物工程学院
<120> 氨基酸转运基因OsAAP1在低氮下促进水稻生长的应用
<130> 1
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 487
<212> PRT
<213> Oryza sativa
<400> 1
Met Gly Met Glu Arg Pro Gln Glu Lys Val Ala Thr Thr Thr Thr Ala
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Ala Phe Asn Leu Ala Glu Ser Gly Tyr Ala Asp Arg Pro Asp Leu Asp
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Asp Asp Gly Arg Glu Lys Arg Thr Gly Thr Leu Val Thr Ala Ser Ala
35 40 45
His Ile Ile Thr Ala Val Ile Gly Ser Gly Val Leu Ser Leu Ala Trp
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Ala Ile Ala Gln Leu Gly Trp Val Ile Gly Pro Ala Val Leu Val Ala
65 70 75 80
Phe Ser Val Ile Thr Trp Phe Cys Ser Ser Leu Leu Ala Asp Cys Tyr
85 90 95
Arg Ser Pro Asp Pro Val His Gly Lys Arg Asn Tyr Thr Tyr Gly Gln
100 105 110
Ala Val Arg Ala Asn Leu Gly Val Ala Lys Tyr Arg Leu Cys Ser Val
115 120 125
Ala Gln Tyr Val Asn Leu Val Gly Val Thr Ile Gly Tyr Thr Ile Thr
130 135 140
Thr Ala Ile Ser Met Gly Ala Ile Lys Arg Ser Asn Trp Phe His Arg
145 150 155 160
Asn Gly His Asp Ala Ala Cys Leu Ala Ser Asp Thr Thr Asn Met Ile
165 170 175
Ile Phe Ala Gly Ile Gln Ile Leu Leu Ser Gln Leu Pro Asn Phe His
180 185 190
Lys Ile Trp Trp Leu Ser Ile Val Ala Ala Val Met Ser Leu Ala Tyr
195 200 205
Ser Thr Ile Gly Leu Gly Leu Ser Ile Ala Lys Ile Ala Gly Gly Ala
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His Pro Glu Ala Thr Leu Thr Gly Val Thr Val Gly Val Asp Val Ser
225 230 235 240
Ala Ser Glu Lys Ile Trp Arg Thr Phe Gln Ser Leu Gly Asp Ile Ala
245 250 255
Phe Ala Tyr Ser Tyr Ser Asn Val Leu Ile Glu Ile Gln Asp Thr Leu
260 265 270
Arg Ser Ser Pro Ala Glu Asn Glu Val Met Lys Lys Ala Ser Phe Ile
275 280 285
Gly Val Ser Thr Thr Thr Thr Phe Tyr Met Leu Cys Gly Val Leu Gly
290 295 300
Tyr Ala Ala Phe Gly Asn Arg Ala Pro Gly Asn Phe Leu Thr Gly Phe
305 310 315 320
Gly Phe Tyr Glu Pro Phe Trp Leu Val Asp Val Gly Asn Val Cys Ile
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Val Val His Leu Val Gly Ala Tyr Gln Val Phe Cys Gln Pro Ile Tyr
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Gln Phe Ala Glu Ala Trp Ala Arg Ser Arg Trp Pro Asp Ser Ala Phe
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Val Asn Gly Glu Arg Val Leu Arg Leu Pro Leu Gly Ala Gly Asp Phe
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Pro Val Ser Ala Leu Arg Leu Val Trp Arg Thr Ala Tyr Val Val Leu
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Thr Ala Val Ala Ala Met Ala Phe Pro Phe Phe Asn Asp Phe Leu Gly
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Leu Ile Gly Ala Val Ser Phe Trp Pro Leu Thr Val Tyr Phe Pro Val
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Gln Met Tyr Met Ser Gln Ala Lys Val Arg Arg Phe Ser Pro Thr Trp
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Thr Trp Met Asn Val Leu Ser Leu Ala Cys Leu Val Val Ser Leu Leu
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Ala Ala Ala Gly Ser Ile Gln Gly Leu Ile Lys Ser Val Ala His Tyr
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Lys Pro Phe Ser Val Ser Ser
485
<210> 2
<211> 1464
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atggggatgg agaggccgca agagaaggtg gccaccacca ccaccgccgc cttcaacctc 60
gccgagtccg gctacgccga ccgccccgac ctcgacgacg acggccgcga gaagcgcaca 120
gggacgctgg tgacggcgag cgcgcacata ataacggcgg tgatcggctc cggcgtgctg 180
tcgctggcgt gggcgatagc gcagctgggg tgggtgatcg ggccggccgt gctggtggcg 240
ttctcggtca taacctggtt ctgctccagc ctcctcgccg actgctaccg atctcccgac 300
cccgtccatg gcaagcgcaa ctacacctac ggccaagccg tcagggccaa cctaggtgtg 360
gccaagtaca ggctctgctc ggtggcacag tacgtcaatc tcgtcggcgt caccattggc 420
tacaccatca ctacggccat cagcatgggt gcgatcaaac ggtccaactg gttccatcgc 480
aacggccacg acgcagcctg cttggcatct gacacgacca acatgatcat atttgctggc 540
atccaaatcc tcctctcgca gctgccgaat tttcacaaaa tttggtggct ctccattgtc 600
gctgctgtca tgtcactggc ctactcaacc attggccttg gcctctccat tgcaaaaatt 660
gcaggtgggg cccaccccga ggcaaccctc acaggggtga ctgttggagt ggatgtgtct 720
gcaagtgaga aaatctggag aacttttcag tcacttggtg acattgcctt tgcatactcc 780
tactccaatg tcctcataga aattcaggac acgctgcggt cgagcccggc ggagaacgag 840
gtgatgaaga aggcgtcgtt catcggagtc tcgacgacga cgacgttcta catgctgtgc 900
ggcgtgctcg gctacgcggc gttcggcaac cgcgcgccgg ggaacttcct caccggcttc 960
ggcttctacg agcccttctg gctcgtcgac gtcggcaacg tctgcatcgt cgtccacctc 1020
gtcggcgcct accaggtctt ctgccagccc atctaccagt tcgccgaggc ctgggcgcgc 1080
tcgcggtggc cggacagcgc cttcgtcaac ggcgagcgcg tgctccggct gccgctcggc 1140
gccggcgact tccccgtcag cgcgctccgc ctcgtctggc gcacggccta cgtcgtgctc 1200
accgccgtcg ccgccatggc gttccccttc ttcaacgact tcctcggcct catcggcgcc 1260
gtctccttct ggccgctcac cgtctacttc cccgtccaga tgtacatgtc tcaggccaag 1320
gtccggcgat tctcgccgac gtggacgtgg atgaacgtgc tcagcctcgc ctgcctcgtc 1380
gtctccctcc tcgccgccgc cggctccatc cagggcctca tcaaatccgt cgcacattac 1440
aagccattca gcgtctcctc atga 1464
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Claims (4)
1.OsAAP1基因在低氮下促进水稻生长中的应用；其特征在于：所述的OsAAP1基因编码的OsAAP1蛋白的氨基酸序列如SEQ ID NO.1所示。
2.OsAAP1基因在低氮下提高水稻生物量中的应用，其特征在于：所述的水稻生物量包括水稻根长、根数、株高；所述的OsAAP1基因编码的OsAAP1蛋白的氨基酸序列如SEQ IDNO.1所示。
3.OsAAP1基因在低氮下提高水稻产量中的应用，其特征在于：所述的提高水稻产量包括通过提高水稻分蘖数达到提高水稻产量的目的；所述的OsAAP1基因编码的OsAAP1蛋白的氨基酸序列如SEQ ID NO.1所示。
4.根据权利要求1-3任一项所述的应用，其特征在于：所述的OsAAP1基因的cDNA序列如SEQ ID NO.2所示。
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| CN114532338B (zh) * | 2022-02-16 | 2023-08-22 | 贵州大学 | 含γ-氨基丁酸的水稻培养液以及其在水稻抗氮胁迫中的应用和水稻培养方法 |
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