CN107012153B - 氮营养运输基因OsNPF8.1在提高水稻分蘖数中的应用 - Google Patents
氮营养运输基因OsNPF8.1在提高水稻分蘖数中的应用 Download PDFInfo
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Abstract
本发明公开了氮营养运输基因OsNPF8.1在提高水稻分蘖数中的应用，属于植物基因工程领域。OsNPF8.1基因编码蛋白质的氨基酸序列如SEQ ID NO.1所示，cDNA序列如SEQ ID NO.2所示。本发明通过构建水稻OsNPF8.1基因超表达植株、OsNPF8.1基因干扰植株，发现通过提高OsNPF8.1基因表达，可以使正常的水稻分蘖数和每株穗数增加，因此OsNPF8.1基因可用于水稻选育中以提高水稻产量。OsNPF8.1基因在阐述氮素影响植物生长及发育过程方面以及在水稻株型改良方面具有重要的应用价值。
Description
技术领域
本发明属于植物基因工程领域，具体涉及氮营养运输基因OsNPF8.1在提高水稻分蘖数中的应用。
背景技术
中国水稻种植面积占世界作物总种植面积的20%，但氮肥施用量却占到世界总施用量的37%；1995年中国氮肥生产量和使用量已达世界第一位，但氮肥使用效率较低，氮肥的施用量已较50年前增长20倍，按这种趋势，预计到2050年，将会再翻3倍。氮肥施用过量会导致水体富营养化等生态污染问题（徐国华，范晓荣．水稻硝转运蛋白基因OsNRT1.1a和OsNRT1.1b的功能研究．南京农业大学，2011：4-6）。更多的氮素营养通过反硝化作用、水土流失、自然挥发、微生物利用等途径遭到浪费。
如果将氮素的吸收效率提高1%，就相当于每年节约了十多亿美元的开支。从中国国情分析，扩大种植面积提高总产量的潜力已经很有限，唯一的出路是在有限的土地上生产出更多的稻谷，即提高单位面积产量。在过去的传统耕作中，通过选择氮利用效率更高的作物，来提高氮的利用效率；但与分子水平上的育种相比，这个过程显得缓慢而低效（张洪程，戴其根．水稻氮素利用的基因型差异与生理机理研究．扬州大学，2008：10-13）。要提高氮利用率，必须从氮的分子吸收机制中寻找突破口。硝酸根运输基因家族分为低亲和力硝酸根运输基因与高亲和力硝酸根运输基因两类（周诗毅．糖类和氨基酸对水稻诱导型高亲和力硝酸盐转运系统的影响．华中科技大学，2009：15-16）。通过氮同化作用，将硝态氮和铵态氮吸收并转化为氨基酸，称为氮的第一类吸收。通过对氮的运输使种子营养物质增加，增加饱满度，称为氮的第二类吸收，也就是氮的再利用（Kant S, Bi Y, Steven J, et al.Understanding plant response to nitrogen limitation for the improvement ofcrop nitrogen use efficiency . Journal of Experimental, 2011, 62(4): 1499-1509）。增加氮吸收积累量或氮转运量，都可以增产。因此，在现代化农业建设中，通过分子育种手段来提高水稻对氮肥的利用效率，可以减少氮肥污染，还能增加产量。
NRT1/PTR家族（NRT1/PTR family, NPF）是指能够介导2-3个氨基酸残基的小分子肽及硝酸根等物质进行跨膜运输的蛋白（Rentsch D, Schmidt S, Tegeder M.Transporters for uptake and allocation of organic nitrogen compounds inplants. FEBS Let, 2007, 581: 2281-2289）。NRT1/PTR家族成员参与了种子形成过程中蛋白质的积累和萌发中蛋白降解后小分子多肽形式转运（Martre P, Porter J R,Jamieson P D, et al. Modeling grain nitrogen accumulation and proteincomposition to understand the sink/source regulations of nitrogenremobilization for wheat. Plant Physiol, 2003, 133: 1959-1967）。目前关于NPF家族成员研究的报道很少，本发明涉及的OsNPF8.1基因是水稻NPF基因家族的一个基因。本发明发现OsNPF8.1基因对水稻分蘖调控有极其重要的作用，可应用于植物株型改良从而使水稻增产。
发明内容
本发明的目的在于解决现有技术中存在的问题，提供水稻NPF基因家族成员OsNPF8.1基因在提高水稻分蘖数中的应用。
本发明的目的通过下述技术方案实现：
本发明以水稻的NPF基因家族成员OsNPF8.1基因为对象，从水稻中花11中克隆了OsNPF8.1的cDNA序列。通过构建OsNPF8.1基因超表达载体，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OsNPF8.1基因超表达植株，其分蘖数与对照野生型中花11相比显著提高。通过RNAi技术构建OsNPF8.1基因干涉表达载体，将干扰表达载体导入中花11中，得到OsNPF8.1基因表达量下降的干扰植株，干扰植株的分蘖数与中花11相比显著降低。这些结果表明，通过提高OsNPF8.1基因的表达，可以使正常的水稻分蘖数增加，从而提高穗数和水稻产量。
基于本发明发现的OsNPF8.1基因的功能，OsNPF8.1基因可用于水稻选育中。所述的水稻选育为提高水稻分蘖数，从而提高穗数和水稻产量。具体可通过提高OsNPF8.1基因的表达使水稻分蘖数和每株穗数增加，达到提高水稻产量的目的。
OsNPF8.1基因也可用于提高其他植物的产量，如通过转基因使OsNPF8.1基因在植物中（超量）表达，来提高植物的分枝数量，进而使植物的产量得到提高。所述的植物是指单子叶植物或双子叶植物；如：小麦、番茄、草坪草或苜蓿等。
所述的OsNPF8.1基因编码的OsNPF8.1蛋白的氨基酸序列如SEQ ID NO.1所示；所述的OsNPF8.1基因的cDNA序列优选如SEQ ID NO.2所示。
应该理解为，在不影响OsNPF8.1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，OsNPF8.1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OsNPF8.1基因超表达后使水稻分蘖能力增强，说明OsNPF8.1基因对提高水稻产量较明显，因此，通过基因工程技术提高OsNPF8.1基因的表达能够提高植物产量。这不仅有助于通过正常施氮条件下培育高产水稻，还可以通过分子育种进行植物的品种改良。
（2）OsNPF8.1基因的成功克隆，进一步证实了NPF家族在氮吸收过程中的重要作用，对阐明NPF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提高植物产量的基因，但对植物增产的分子机制仍不清楚。而本发明克隆的OsNPF8.1基因能够提高水稻的产量，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是对照中花11、OsNPF8.1基因超表达植株2个株系和OsNPF8.1基因干扰植株2个株系的整株表型图。
图2是对照中花11、OsNPF8.1基因超表达植株2个株系和OsNPF8.1基因干扰植株2个株系分蘖数的统计柱状图，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，不同组别小写字母（a、b、c）表示差异显著。
图3是对照中花11、OsNPF8.1基因超表达植株2个株系和OsNPF8.1基因干扰植株2个株系中OsNPF8.1基因相对表达量的统计柱状图，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，不同组别小写字母（a、b、c）表示差异显著。
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 OsNPF8.1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'-GGTACCATGGGAGAGGTTGCAGAAGACATC-3'（kpnI），
R1：5'-TCTAGAGTTTGCTCCGGCGTGCTCGGCCTT-3'（XbaI）；
通过PCR扩增OsNPF8.1基因的cDNA后，再通过kpnI、XbaI酶切后连入pCAMBIA-1306载体（pCAMBIA-1306载体购自Cambia公司），构建出OsNPF8.1基因的超表达载体OsNPF8.1-p1306。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，种于大田中，待苗长大后，提取基因组DNA通过PCR对转基因植株进行检测，检测引物对为：
F2：5'-GATGTTGGCGACCTCGTATT-3'，
R2：5'-TCGTTATGTTTATCGGCACTTT-3'。
若扩增出517bp的片段，则说明转基因植株为阳性植株。阳性植株单株收种并种植，直至T2代鉴定出纯合的转基因植株，即得到OsNPF8.1基因超表达植株。OsNPF8.1基因超表达植株的分蘖数远多于对照中花11植株，差异显著，如图1、2所示。
取OsNPF8.1基因超表达植株叶片，提取RNA并将其反转录成cDNA，通过实时荧光定量PCR检测OsNPF8.1基因的表达量，结果显示（图3）超表达植株OsNPF8.1基因的表达量与对照中花11相比显著升高。实时荧光定量PCR所用引物对为：
F3：5'-GCTGCACGAGACGCTCGACA-3'，
R3：5'-AGCAGCCGCACCACGCTCTT-3'。
实施例2 OsNPF8.1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：
F4：5'-GGTACCGGGGTGGTGTTCTTGGCGCTGTAC-3'（KpnI），
R4：5'-GGATCCACGAGCACCTGCGCGATCCT-3'（BamH I）；
F5：5'-ACTAGTGGGGTGGTGTTCTTGGCGCTGTAC-3'（SpeI），
R5：5'-GAGCTCACGAGCACCTGCGCGATCCT-3'（Sac I）；
各自PCR扩增出OsNPF8.1基因的cDNA片段后，通过相应的限制性内切酶酶切后连入pTCK303载体，构建出OsNPF8.1基因的干扰表达载体OsNPF8.1-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，种于大田中，待苗长大后，提取基因组DNA通过PCR对转基因植株进行检测，检测引物对为：
F2：5'-GATGTTGGCGACCTCGTATT-3'，
R2：5'-TCGTTATGTTTATCGGCACTTT-3'。
若扩增出517bp的片段，则说明转基因植株为阳性植株。阳性植株单株收种并种植，直至T2代鉴定出纯合的转基因植株，即得到OsNPF8.1基因干扰植株。OsNPF8.1基因干扰植株的分蘖数远少于对照中花11植株，差异显著，如图1、2所示。
取OsNPF8.1基因干扰植株叶片，提取RNA并将其反转录成cDNA，通过实时荧光定量PCR检测 OsNPF8.1基因的表达量，结果显示（图3）干扰植株OsNPF8.1基因的表达量与对照中花11相比显著降低，如图3所示。实时荧光定量PCR所用引物同实施例1。
上述结果表明，通过提高OsNPF8.1基因的表达，可以增加水稻的分蘖数，进而提高穗数和水稻产量。
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> 氮营养运输基因OsNPF8.1在提高水稻分蘖数中的应用
<130> 1
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 580
<212> PRT
<213> Oryza sativa
<400> 1
Met Gly Glu Val Ala Glu Asp Ile Tyr Thr Gln Asp Gly Thr Val Asp
1 5 10 15
Val Lys Gly Asn Pro Ala Thr Lys Lys Asn Thr Gly Asn Trp Arg Ala
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Cys Pro Tyr Ile Leu Ala Asn Glu Cys Cys Glu Arg Leu Ala Tyr Tyr
35 40 45
Gly Met Ser Thr Asn Leu Val Asn Tyr Met Lys Thr Arg Leu Gly Gln
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Glu Ser Ala Ile Ala Ala Asn Asn Val Thr Asn Trp Ser Gly Thr Cys
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Tyr Ile Thr Pro Leu Leu Gly Ala Phe Leu Ala Asp Ala Tyr Met Gly
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Arg Phe Trp Thr Ile Ala Ser Phe Met Ile Ile Tyr Ile Leu Gly Leu
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Ala Leu Leu Thr Met Ala Ser Ser Val Lys Gly Leu Val Pro Ala Cys
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Asp Gly Gly Ala Cys His Pro Thr Glu Ala Gln Thr Gly Val Val Phe
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Leu Ala Leu Tyr Leu Ile Ala Leu Gly Thr Gly Gly Ile Lys Pro Cys
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Val Ser Ser Phe Gly Ala Asp Gln Phe Asp Glu Asn Asp Glu Gly Glu
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Lys Arg Ser Lys Ser Ser Phe Phe Asn Trp Phe Tyr Phe Ser Ile Asn
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Ile Gly Ala Leu Val Ala Ser Ser Val Leu Val Tyr Val Gln Thr His
195 200 205
Val Gly Trp Gly Trp Gly Phe Gly Ile Pro Ala Val Val Met Ala Val
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Ala Val Ala Ser Phe Phe Val Gly Thr Pro Leu Tyr Arg His Gln Arg
225 230 235 240
Pro Gly Gly Ser Pro Leu Thr Arg Ile Ala Gln Val Leu Val Ala Ser
245 250 255
Ala Arg Lys Trp Gly Val Glu Val Pro Ala Asp Gly Ser Arg Leu His
260 265 270
Glu Thr Leu Asp Arg Glu Ser Gly Ile Glu Gly Ser Arg Lys Leu Glu
275 280 285
His Thr Gly Gln Phe Ala Cys Leu Asp Arg Ala Ala Val Glu Thr Pro
290 295 300
Glu Asp Arg Ser Ala Ala Asn Ala Ser Ala Trp Arg Leu Cys Thr Val
305 310 315 320
Thr Gln Val Glu Glu Leu Lys Ser Val Val Arg Leu Leu Pro Ile Trp
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Ala Ser Gly Ile Val Phe Ala Thr Val Tyr Gly Gln Met Ser Thr Met
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Phe Val Leu Gln Gly Asn Thr Leu Asp Ala Ser Met Gly Pro His Phe
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Ser Ile Pro Ala Ala Ser Leu Ser Ile Phe Asp Thr Leu Ser Val Ile
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Val Trp Val Pro Val Tyr Asp Arg Leu Ile Val Pro Ala Val Arg Ala
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Val Thr Gly Arg Pro Arg Gly Phe Thr Gln Leu Gln Arg Met Gly Ile
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Gly Leu Val Ile Ser Val Phe Ser Met Leu Ala Ala Gly Val Leu Asp
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Val Val Arg Leu Arg Ala Ile Ala Arg His Gly Leu Tyr Gly Asp Lys
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Asp Val Val Pro Ile Ser Ile Phe Trp Gln Val Pro Gln Tyr Phe Ile
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Ile Gly Ala Ala Glu Val Phe Thr Phe Val Gly Gln Leu Glu Phe Phe
465 470 475 480
Tyr Asp Gln Ala Pro Asp Ala Met Arg Ser Met Cys Ser Ala Leu Ser
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Leu Thr Thr Val Ala Leu Gly Asn Tyr Leu Ser Thr Leu Leu Val Thr
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Ile Val Thr His Val Thr Thr Arg Asn Gly Ala Val Gly Trp Ile Pro
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Asp Asn Leu Asn Arg Gly His Leu Asp Tyr Phe Phe Trp Leu Leu Ala
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Val Leu Ser Leu Ile Asn Phe Gly Val Tyr Leu Val Ile Ala Ser Trp
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Ala Gly Ala Asn
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<210> 2
<211> 1743
<212> DNA
<213> Oryza sativa
<400> 2
atgggagagg ttgcagaaga catctacacc caagatggca ccgtggacgt caagggcaac 60
ccggccacca agaagaacac cggcaactgg cgtgcctgcc cctacatcct cgctaacgag 120
tgctgcgaga ggctggccta ctatggcatg agcaccaacc tcgtgaacta catgaagacg 180
cggctcgggc aggagagcgc cattgccgcc aacaatgtca ccaactggtc ggggacttgc 240
tacatcaccc cccttctcgg tgccttcttg gctgatgcct acatgggcag gttctggacc 300
attgccagct tcatgatcat ctacatcctg ggtttggcgt tgctgacaat ggcgtcgtcg 360
gtgaaggggc tggtgccggc gtgcgacgga ggagcgtgtc acccgacgga ggcgcagacg 420
ggggtggtgt tcttggcgct gtacctgata gcgctgggca ccggcgggat caagccgtgc 480
gtgtcgtcgt ttggcgcgga ccagttcgac gagaacgacg agggggagaa gcggagcaag 540
agcagcttct tcaactggtt ctacttctcc atcaacatcg gcgcgctggt ggcgtcgtcg 600
gtgctggtgt acgtgcagac gcacgtcggg tgggggtggg ggttcggcat cccggccgtc 660
gtcatggccg tcgccgtcgc cagcttcttc gtcggcacgc cgctgtacag gcaccagcgg 720
cccgggggca gcccgctgac gaggatcgcg caggtgctcg tcgcgtcggc gaggaagtgg 780
ggcgtcgagg tccccgccga cgggtcgcgg ctgcacgaga cgctcgacag ggagtccggc 840
atcgagggca gccgcaagct ggagcacacc gggcagttcg cgtgcctcga cagggcggcg 900
gtggagacgc cggaggacag gtcggcggcg aacgcgtcgg cgtggcggct gtgcacggtg 960
acgcaggtgg aggagctgaa gagcgtggtg cggctgctgc cgatctgggc gagcgggatc 1020
gtgttcgcga cggtgtacgg gcagatgagc accatgttcg tcctccaggg gaacacgctc 1080
gacgccagca tggggccgca cttctccatc ccggcggcgt cgctctccat cttcgacacc 1140
ctcagcgtca tcgtctgggt gccggtgtac gaccgcctca tcgtgccggc ggtgcgcgcc 1200
gtgacggggc gcccacgcgg gttcacccag ctgcagcgga tgggcatcgg cctcgtcatc 1260
tccgtcttct ccatgctcgc cgccggcgtg ctcgacgtcg tcaggctgcg cgccatcgct 1320
cgccacgggc tctacggcga caaggacgtc gtgcccatct ccatcttctg gcaggtgccg 1380
cagtacttca tcatcggcgc cgccgaggtg ttcacgttcg tcgggcagct ggagttcttc 1440
tacgaccagg ctcccgacgc catgcgcagc atgtgctcgg cgctgtcgct caccaccgtc 1500
gcgctaggca actacctcag cacgctgctc gtgaccatcg ttacccatgt caccacccgg 1560
aacggcgccg tcgggtggat cccggacaac ctcaaccgcg gccacctcga ctacttcttc 1620
tggctgctcg ccgtgctcag cctcatcaac ttcggcgtct acctcgtcat cgccagctgg 1680
tacacctaca agaagacggc ggattcaccg gacgacaagg ccgagcacgc cggagcaaac 1740
tga 1743
<210> 3
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<212> DNA
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<220>
<223> 引物F1
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ggtaccatgg gagaggttgc agaagacatc 30
<210> 4
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<220>
<223> 引物R1
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tctagagttt gctccggcgt gctcggcctt 30
<210> 5
<211> 20
<212> DNA
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<220>
<223> 引物F2
<400> 5
gatgttggcg acctcgtatt 20
<210> 6
<211> 22
<212> DNA
<213> Artificial
<220>
<223> 引物R2
<400> 6
tcgttatgtt tatcggcact tt 22
<210> 7
<211> 20
<212> DNA
<213> Artificial
<220>
<223> 引物F3
<400> 7
gctgcacgag acgctcgaca 20
<210> 8
<211> 20
<212> DNA
<213> Artificial
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<400> 8
agcagccgca ccacgctctt 20
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<211> 30
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<220>
<223> 引物F4
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ggtaccgggg tggtgttctt ggcgctgtac 30
<210> 10
<211> 26
<212> DNA
<213> Artificial
<220>
<223> 引物R4
<400> 10
ggatccacga gcacctgcgc gatcct 26
<210> 11
<211> 30
<212> DNA
<213> Artificial
<220>
<223> 引物F5
<400> 11
actagtgggg tggtgttctt ggcgctgtac 30
<210> 12
<211> 26
<212> DNA
<213> Artificial
<220>
<223> 引物R5
<400> 12
gagctcacga gcacctgcgc gatcct 26
Claims (2)
1.OsNPF8.1基因在提高水稻分蘖数中的应用，其特征在于：通过提高OsNPF8.1基因的表达使水稻分蘖数增加；所述的OsNPF8.1基因编码的OsNPF8.1蛋白的氨基酸序列如SEQ IDNO.1所示。
2.根据权利要求1所述的应用，其特征在于：所述的OsNPF8.1基因的cDNA序列如SEQ IDNO.2所示。
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