CN114181290B - 一个调控植物氮素利用效率和产量的蛋白质及其应用 - Google Patents
一个调控植物氮素利用效率和产量的蛋白质及其应用 Download PDFInfo
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
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- A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
- A01H6/4636—Oryza sp. [rice]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
本发明公开了一个调控植物氮素利用效率和产量的蛋白质及其应用。本发明通过转基因技术和CRISPR‑Cas9技术分别获得了转OsTCP19水稻和水稻OsTCP19突变体。实验证明:与野生型水稻相比,转OsTCP19水稻的分蘖数减少、氮响应能力下降,而与野生型水稻相比,水稻OsTCP19突变体的分蘖数增加。说明OsTCP19蛋白质具有调控植物产量和氮利用效率的功能,为培育高产量和高氮利用效率品种奠定基础。
Description
技术领域
本发明属于生物技术领域,具体涉及一个调控植物氮素利用效率和产量的蛋白质及其应用。
背景技术
氮素是植物需求最大的矿质营养元素。在农业生产中,以氮肥为主的化学肥料的持续投入带来了粮食产量的大幅度增加。然而,氮肥过量施用导致大量未被作物吸收利用的氮肥进入空气、水体或残留在土壤中,引起空气污染、地表水富营养化以及土壤酸化,造成严重的环境破坏。此外,随着世界人口的不断增长,未来对粮食的需求还会不断增加,因此,为保障农业可持续发展,实现“减肥增效”育种目标、提高农作物氮素利用效率(nitrogen-use efficiency,NUE)至关重要。水稻是世界上广泛种植的主粮作物,其氮肥施用量又远远超过其他农作物,在水稻中鉴定并利用NUE相关基因对于农业生产意义重大。
NUE受多种遗传因素和环境因素共同影响,是一个复杂的农艺性状,涉及到氮素吸收、转运、同化、再利用以及后续的生长发育过程,难以用单一指标对其进行表型鉴定。有鉴于此,相关的功能基因一直难以通过传统遗传学方法进行克隆鉴定,极大限制了氮高效育种工作的开展。
NUE可被定义为单位面积固定施氮量作物的产量,而水稻的产量由分蘖数、穗粒数和千粒重三要素决定,因此,利用这些产量要素的氮响应能力(也可理解为低氮(LN)到高氮(HN)下的增加比率,即(HN-LN)/LN)作为NUE的相对表型值,开展NUE相关基因位点的克隆更具实际应用价值。
发明内容
本发明的第一个目的是提供OsTCP19蛋白质的新用途。
本发明提供了OsTCP19蛋白质在调控植物分蘖和/或产量和/或品质和/或氮素利用率和/或氮响应能力中的应用。
所述OsTCP19蛋白质是如下A1)或A2)或A3)或A4)任一所示蛋白质:
A1)由序列表中序列3所示的氨基酸序列组成的蛋白质;
A2)在序列表中序列3所示的蛋白质的N端或/和C端连接标签得到的融合蛋白;
A3)将序列表中序列3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且功能相同的蛋白质;
A4)与A1)-A3)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质。
其中,序列表中序列3由387个氨基酸残基组成。
所述标签具体如表1所示。
表1标签的序列
标签 | 残基 | 序列 |
Poly-Arg | 5-6(通常为5个) | RRRRR |
Poly-His | 2-10(通常为6个) | HHHHHH |
FLAG | 8 | DYKDDDDK |
Strep-tag II | 8 | WSHPQFEK |
c-myc | 10 | EQKLISEEDL |
HA | 9 | YPYDVPDYA |
上述A1)-A4)任一所示的蛋白质可人工合成,也可先合成其编码基因,再进行生物表达得到。
本发明的第二个目的是提供与OsTCP19蛋白质相关的生物材料的新用途。
本发明提供了与OsTCP19蛋白质相关的生物材料在如下B1)-B4)任一种中的应用:
B1)调控植物分蘖和/或产量和/或品质和/或氮素利用率和/或氮响应能力;
B2)培育分蘖数增加和/或产量增加和/或品质提高和/或氮素利用率提高和/或氮响应能力提高的转基因植物;
B3)培育分蘖数减少和/或产量减少和/或品质下降和/或氮素利用率下降和/或氮响应能力下降的转基因植物;
B4)植物育种;
所述与OsTCP19蛋白质相关的生物材料为下述C1)至C8)中的任一种:
C1)编码OsTCP19蛋白质的核酸分子;
C2)含有C1)所述核酸分子的表达盒;
C3)含有C1)所述核酸分子的重组载体;
C4)含有C2)所述表达盒的重组载体;
C5)含有C1)所述核酸分子的重组微生物;
C6)含有C2)所述表达盒的重组微生物;
C7)含有C3)所述重组载体的重组微生物;
C8)含有C4)所述重组载体的重组微生物。
上述应用中,C1)所述核酸分子为如下1)或2)或3)或4)或5)的DNA分子:
1)序列表中序列1所示的基因组DNA分子;
2)序列表中序列2所示的cDNA分子;
3)来源于水稻的与1)或2)限定的DNA序列具有98%以上同源性且编码植物分蘖和/或产量和/或品质和/或氮素利用率和/或氮响应能力相关蛋白的DNA分子;
4)在严格条件下与1)或2)限定的DNA序列杂交且编码植物分蘖和/或产量和/或品质和/或氮素利用率和/或氮响应能力相关蛋白的DNA分子;
5)与1)或2)限定的DNA序列具有90%以上同源性且编码植物分蘖和/或产量和/或品质和/或氮素利用率和/或氮响应能力相关蛋白的DNA分子。
本领域普通技术人员可以很容易地采用已知的方法,例如定向进化和点突变的方法,对本发明的编码OsTCP19蛋白质的核苷酸序列进行突变。那些经过人工修饰的,具有编码OsTCP19蛋白质的核苷酸序列75%或者更高同一性的核苷酸,只要编码OsTCP19蛋白质且具有相同功能,均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列3所示的氨基酸序列组成的蛋白质的核苷酸序列具有75%或更高,或85%或更高,或90%或更高,或95%或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。
上述90%以上同源性,可为91%、92%、93%、94%、95%、96%、97%、98%或99%以上的同源性。
上述应用中,所述严格条件是在2×SSC,0.1%SDS的溶液中,在68℃下杂交并洗膜2次,每次5min,又于0.5×SSC,0.1%SDS的溶液中,在68℃下杂交并洗膜2次,每次15min;或,0.1×SSPE(或0.1×SSC)、0.1%SDS的溶液中,65℃条件下杂交并洗膜。
上述应用中,所述载体可为质粒、黏粒、噬菌体或病毒载体。
所述重组载体是将上述核酸分子插入表达载体中,得到表达上述蛋白质的重组载体。使用所述核酸分子构建重组载体时,可在其转录起始核苷酸前加上任何一种增强型、组成型、组织特异型或诱导型启动子,它们可单独使用或与其它的植物启动子结合使用;此外,使用所述核酸分子构建重组表达载体时,还可使用增强子,包括翻译增强子或转录增强子,这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等,但必需与编码序列的阅读框相同,以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的,可以是天然的,也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选,可对所用植物表达载体进行加工,如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、具有抗性的抗生素标记物(庆大霉素标记物、卡那霉素标记物等)或是抗化学试剂标记基因(如抗除莠剂基因)等。从转基因植物的安全性考虑,可不加任何选择性标记基因,直接以逆境筛选转化植株。在本发明的具体实施例中,所述重组载体为gOsTCP19。所述gOsTCP19为将序列1所示的DNA片段插入载体pCAMBIA2300-ocs的BamHI和HindIII酶切位点之间得到的重组载体。
上述应用中,所述微生物可为酵母、细菌、藻或真菌,如农杆菌。所述重组微生物为含有上述重组载体的微生物。本发明的具体实施例中,所述重组微生物为含有上述重组载体的农杆菌AGL1。
上述应用中,所述调控植物产量体现在调控植物分蘖数;所述调控植物氮素利用率体现在调控植物氮响应能力。所述调控植物分蘖和/或产量和/或品质和/或氮素利用率和/或氮响应能力具体体现在:当植物中的OsTCP19蛋白质的含量和/或活性降低时,所述植物分蘖数和/或产量和/或品质和/或氮素利用率和/或氮响应能力增加或提高;当植物中的OsTCP19蛋白质的含量和/或活性提高时,所述植物分蘖数和/或产量和/或品质和/或氮素利用率和/或氮响应能力减少或下降。
本发明的第三个目的是提供抑制OsTCP19蛋白质活性的物质或抑制OsTCP19蛋白质编码基因表达的物质或敲除OsTCP19蛋白质编码基因的物质的新用途。
本发明提供了抑制OsTCP19蛋白质活性的物质或抑制OsTCP19蛋白质编码基因表达的物质或敲除OsTCP19蛋白质编码基因的物质在培育分蘖数增加和/或产量增加和/或品质提高和/或氮素利用率提高和/或氮响应能力提高的转基因植物中的应用。
进一步的,所述物质为CRISPR-Cas9系统;所述CRISPR-Cas9系统包括Cas9蛋白质和sgRNA,所述sgRNA靶向OsTCP19蛋白质的编码基因序列或其上游启动子序列或其非编码区序列或其下游调控区序列。
更进一步的,所述sgRNA的靶序列为序列4所示的DNA分子和序列5所示的DNA分子。所述CRISPR-Cas9系统为OsTCP19 CRISPR/Cas9敲除载体。所述OsTCP19CRISPR/Cas9敲除载体含有两个sgRNA,分别记作sgRNA1和sgRNA2。所述sgRNA1的靶点序列为序列4所示的DNA分子;所述sgRNA2的靶点序列为序列5所示的DNA分子。
本发明的第四个目的是提供一种培育分蘖数增加和/或产量增加和/或品质提高和/或氮素利用率提高和/或氮响应能力提高的转基因植物的方法。
本发明提供的培育分蘖数增加和/或产量增加和/或品质提高和/或氮素利用率提高和/或氮响应能力提高的转基因植物的方法为如下D1)或D2):
D1)包括如下步骤:抑制目的植物中OsTCP19蛋白质的活性,得到分蘖数增加和/或产量增加和/或品质提高和/或氮素利用率提高和/或氮响应能力提高的转基因植物;
D2)包括如下步骤:抑制目的植物中OsTCP19蛋白质编码基因的表达或敲除目的植物中OsTCP19蛋白质编码基因,得到分蘖数增加和/或产量增加和/或品质提高和/或氮素利用率提高和/或氮响应能力提高的转基因植物。
进一步的,所述敲除目的植物中OsTCP19蛋白质编码基因的方法包括将上述CRISPR-Cas9系统导入目的植物的步骤。
更进一步的,所述CRISPR-Cas9系统为上述OsTCP19 CRISPR/Cas9敲除载体。
本发明的第五个目的是提供一种培育分蘖数减少和/或产量减少和/或品质下降和/或氮素利用率下降和/或氮响应能力下降的转基因植物的方法。
本发明提供的培育分蘖数减少和/或产量减少和/或品质下降和/或氮素利用率下降和/或氮响应能力下降的转基因植物的方法包括如下步骤:提高目的植物中OsTCP19蛋白质的活性和/或含量,得到分蘖数减少和/或产量减少和/或品质下降和/或氮素利用率下降和/或氮响应能力下降的转基因植物。
进一步的,所述氮响应能力下降具体体现在所述转基因植物的分蘖氮响应值低于所述目的植物。所述分蘖氮响应值的计算公式如下:(高氮处理组分蘖数-低氮处理组分蘖数)/低氮处理促分蘖数。所述高氮处理组为按照每100平方米1.5kg尿素施肥,所述低氮处理组为按照每100平方米0.5kg尿素施肥。
所述提高目的植物中OsTCP19蛋白质的活性和/或含量的方法为在目的植物中过表达OsTCP19蛋白质。所述过表达的方法为将OsTCP19蛋白质编码基因导入目的植物。
更进一步的,所述OsTCP19蛋白质编码基因为序列1所示的DNA分子。所述OsTCP19蛋白质编码基因通过上述重组载体gOsTCP19导入目的植物。
本发明的最后一个目的是提供上述CRISPR-Cas9系统。
上述CRISPR-Cas9系统在培育分蘖数增加和/或产量提高和/或品质提高和/或氮素利用率提高和/或氮响应能力提高的转基因植物或植物育种中的应用也属于本发明的保护范围。
上述任一所述应用或方法中,所述植物可为单子叶植物或双子叶植物。进一步的,所述单子叶植物可为禾本科植物。更进一步的,所述禾本科植物具体为水稻(如水稻品种中花11)。
本发明提供了一种与植物产量和氮利用效率相关的OsTCP19蛋白,并通过转基因技术和CRISPR-Cas9技术分别获得了转OsTCP19水稻和水稻OsTCP19突变体。实验证明:与野生型水稻相比,转OsTCP19水稻的分蘖数减少、氮响应能力下降,而与野生型水稻相比,水稻OsTCP19突变体的分蘖数增加。说明OsTCP19蛋白质具有调控植物产量和氮利用效率的功能,为培育高产量和高氮利用效率品种奠定基础。
附图说明
图1为转OsTCP19水稻和野生型水稻的分蘖数。a为转OsTCP19水稻和野生型水稻的表型。b为转OsTCP19水稻和野生型水稻中OsTCP19表达量的检测结果。c为转OsTCP19水稻和野生型水稻的分蘖数。
图2为转OsTCP19水稻和野生型水稻的氮响应能力检测。a为转OsTCP19水稻和野生型水稻在低氮处理条件下的分蘖数。b为转OsTCP19水稻和野生型水稻在高氮处理条件下的分蘖数。c为转OsTCP19水稻和野生型水稻的分蘖氮响应值。
图3为水稻OsTCP19突变体和野生型水稻的分蘖数检测。a为水稻OsTCP19突变体和野生型水稻的表型。b为水稻OsTCP19突变体和野生型水稻的分蘖数。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
中花11记载于文献:Ma Y,Liu L,Zhu C,Sun C,Xu B,Fang F,Tang J,Luo A,CaoS,Li G,Qian Q,Xue Y,Chu C(2009)Molecular analysis of rice plants harboring amulti-functional T-DNA tagging system,J.Genet.Genomics 36(5):267-276中,公众可从中国科学院遗传与发育生物学研究所获得,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
pCAMBIA2300-ocs记载于文献Wang W,Hu B,Yuan D,Liu Y,Che R,Hu Y,Ou S,Zhang Z,Wang H,Li H,Jiang Z,Zhang Z,Gao X,Qiu Y,Meng X,Liu Y,Bai Y,Liang Y,Wang Y,Zhang L,Li L,Mergen S,Jing H,Li J,and Chu C(2018)Expression of thenitrate transporter OsNRT1.1A/OsNPF6.3 confers high yield and earlymaturation in rice.Plant Cell,30(3):638-651中,公众可从中国科学院遗传与发育生物学研究所获得,该载体只可重复本发明的相关实验所用,不可作为其它用途使用。
pYLsgRNA-U3、pYLsgRNA-U6a和pYLCRISPR/Cas9Pubi-H载体均记载于文献:Ma X,Zhang Q,Zhu Q,et al.A Robust CRISPR/Cas9 System for Convenient,High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants.Mol.Plant.2015;8(8):1274-1284中,公众可从中国科学院遗传与发育生物学研究所获得,该载体只可重复本发明的相关实验所用,不可作为其它用途使用。
农杆菌(Agrobacterium tumefaciens)株系AGL1记载于文献:Wang W,Hu B,YuanD,Liu Y,Che R,Hu Y,Ou S,Zhang Z,Wang H,Li H,Jiang Z,Zhang Z,Gao X,Qiu Y,MengX,Liu Y,Bai Y,Liang Y,Wang Y,Zhang L,Li L,Mergen S,Jing H,Li J,and Chu C(2018)Expression of the nitrate transporter OsNRT1.1A/OsNPF6.3 confers highyield and early maturation in rice.Plant Cell,30(3):638-651中,公众可从中国科学院遗传与发育生物学研究所获得,该载体只可重复本发明的相关实验所用,不可作为其它用途使用。
实施例1、转OsTCP19水稻的获得及其分蘖检测
一、转OsTCP19水稻的获得
1、重组表达载体的构建
1)基因的克隆
以中花11(Zhonghua 11,ZH11)基因组DNA为模板,利用gOsTCP19-F/gOsTCP19-R引物组合(该gOsTCP19-F/gOsTCP19-R引物组合含有BamHI和HindIII两个酶切位点接头及用于后续同源重组的载体序列)进行PCR扩增,得到大小为3844bp的DNA片段,其核苷酸序列如序列表中序列1所示,其中,序列1中第1-1944位为OsTCP19基因启动子,第1945-2240位为OsTCP19基因5’UTR序列,第2241-3404位为OsTCP19基因编码区序列,第3405-3654位为OsTCP19基因3’UTR序列,第3655-3844位为OsTCP19基因下游序列。引物序列具体如下:
gOsTCP19-F:
gOsTCP19-R:
gOsTCP19-F引物序列的5’端加BamHI酶切位点,gOsTCP19-R引物序列的5’端加HindIII酶切位点(下划线所示)。gOsTCP19-F/gOsTCP19-R引物序列中的加粗序列为载体上的序列,用于后续同源重组构建载体。gOsTCP19-F引物序列中的斜体序列为序列1第1-20位核苷酸序列;gOsTCP19-R引物序列中的斜体序列为序列1第3825-3844位核苷酸序列的反向互补序列。
2)构建表达载体
将步骤1)得到的包含OsTCP19基因全长的大小为3844bp的DNA片段插入载体pCAMBIA2300-ocs的BamHI和HindIII酶切位点之间,得到重组表达载体gOsTCP19。
2、转基因水稻的获得
将步骤1获得的重组表达载体gOsTCP19通过热击法转入农杆菌(Agrobacteriumtumefaciens)株系AGL1中,筛选得到含有gOsTCP19的重组农杆菌菌株。用含有gOsTCP19的重组农杆菌菌株侵染中花11愈伤组织,具体的转化筛选方法参见文献“易自力,曹守云,王力,何锶洁,储成才,唐祚舜,周朴华,田文忠.提高农杆菌转化水稻频率的研究.遗传学报,2001,28(4):352-358”一文,最终获得转基因水稻,获得的转基因水稻为T0代转基因水稻。收获T0代株系的种子,获得T1代转基因水稻。
按照获得T1代转基因水稻的方法,将空载体pCAMBIA2300-ocs转化中花11,得到空载体对照植株。
3、转基因水稻的鉴定
1)PCR鉴定
提取步骤2获得的T1代转基因水稻的基因组DNA。用针对NptII基因的引物F1和R1进行PCR鉴定,经鉴定表明含有NptII基因的植株(PCR产物大小约为500bp)即为阳性转基因水稻,命名为转OsTCP19水稻。引物序列具体如下:
F1:5’-TCCGGCCGCTTGGGTGGAGAG-3’;
R1:5’-CTGGCGCGAGCCCCTGATGCT-3’。
2)转录水平分析(RNA表达量)
以步骤1)获得的所有T1代阳性转基因水稻株系以及野生型水稻品种中花11为实验材料。提取各材料的总RNA并反转录得到cDNA。进而以所得cDNA为模板,针对OsTCP19基因进行实时定量荧光PCR,检测各材料中OsTCP19基因在转录水平上的表达量。实验重复3次,结果取平均值。以OsActin1作为内参基因。用于检测OsTCP19基因和OsActin1基因的引物序列如下:
OsTCP19-qF:5’-GACAGTGTACCGTGGCGT-3’;
OsTCP19-qR:5’-CGCCGGGAAGTTCATGAAAT-3’;
OsActin1-F:5’-ACCATTGGTGCTGAGCGTTT-3’;
OsActin1-R:5’-CGCAGCTTCCATTCCTATGAA-3’。
OsTCP19-qF为序列2第864-881位核苷酸序列,OsTCP19-qR为序列2第896-915位核苷酸序列的反向互补序列。
根据各实验材料中OsTCP19基因表达量的实时定量荧光PCR检测结果,选取中等表达和高表达共两个T1代株系,分别命名为TO1和TO2进行后续研究,TO1和TO2表达量检测结果如图1b所示(空载体对照与中花11对照表型一致,图1b中省略)。相比未转基因的野生型水稻品种中花11,T1代转OsTCP19水稻株系TO1和TO2中OsTCP19基因的表达量在转录水平上显著提高。
二、转OsTCP19水稻的分蘖检测
1、转OsTCP19水稻的分蘖数目检测
在正常条件下于生殖生长时期分别对T1代转OsTCP19水稻株系TO1和TO2、中花11对照植株和空载体对照植株进行分蘖的统计,每种材料统计16个单株。
结果如图1a和图1c所示(空载体对照与中花11对照表型一致,图1a和图1c中省略)。与中花11对照植株以及空载体对照相比,T1代转OsTCP19水稻株系TO1和TO2分蘖数目显著降低。其中TO1与对照中花11相比,平均分蘖数从6.3个降到4.2个。TO2与对照中花11相比,平均分蘖数从6.3个降到3.3个。
2、转OsTCP19水稻的分蘖氮响应检测
进一步在高低氮条件下(低氮:50kg ha-1,高氮:150kg ha-1)对T1代转OsTCP19水稻株系TO1和TO2、中花11对照植株和空载体对照进行分蘖数统计,并计算分蘖氮响应值。具体处理步骤:田间试验设置两个氮肥梯度(高氮和低氮)。其中高氮处理组按照每100平方米1.5kg尿素施肥,低氮处理组按照每100平方米0.5kg尿素施肥。分蘖氮响应值=(高氮处理组分蘖数-低氮处理组分蘖数)/低氮处理促分蘖数,每种材料两个条件下均统计16个单株。
结果如图2所示。与中花11对照植株以及空载体对照相比,T1代转OsTCP19水稻株系TO1和TO2在低氮和高氮条件下的分蘖数均显著降低(图2a和2b)。其中,在低氮条件下,与对照中花11相比,TO1平均分蘖数从5.1个降到3.4个,TO2平均分蘖数从5.1个降到2个。在高氮条件下,与对照中花11相比,TO1平均分蘖数从9.5个降到5.5个,TO2平均分蘖数从9.5个降到2.5个。进一步地,与中花11对照植株以及空载体对照相比,T1代转OsTCP19水稻株系TO1和TO2的氮响应能力显著降低。其中,与对照中花11相比,TO1平均分蘖氮响应值从0.8降到0.4,TO2平均分蘖氮响应值从0.8降到0.3(图2c)。
实施例2、水稻OsTCP19突变体的获得及其分蘖检测
一、水稻OsTCP19突变体的获得
1、sgRNA的设计
利用序列1作为参考序列,分别在OsTCP19编码区设计两对sgRNA引物序列,分别为OsTCP19-U3F/OsTCP19-U3R、OsTCP19-U6aF/OsTCP19-U6aR。引物序列具体如下:
OsTCP19-U3F:5’-ggcAGAGTAGCCATGGATGTCAC-3’;
OsTCP19-U3R:5’-aaacGTGACATCCATGGCTACTCT-3’;
OsTCP19-U6aF:5’-gccGAGCTCGGGCACAAGACCGA-3’;
OsTCP19-U6aR:5’-aaacTCGGTCTTGTGCCCGAGCT-3’。
为方便后续连接,OsTCP19-U3F引物序列的5’端加ggc接头,OsTCP19-U3R引物序列的5’端加aaac接头,OsTCP19-U6aF引物序列的5’端加gcc接头,OsTCP19-U6aR引物序列的5’端加aaac接头。
OsTCP19-U3F为序列1第2232-2251位核苷酸序列,OsTCP19-U3R为序列1第2232-2251位核苷酸序列的反向互补序列。OsTCP19-U6aF为序列1第2505-2524位核苷酸序列,OsTCP19-U3R为序列1第2505-2524位核苷酸序列的反向互补序列。
2、敲除载体的构建
1)将引物对OsTCP19-U3F/OsTCP19-U3R变性、退火得到OsTCP19-U3F/R的二聚体产物。将引物对OsTCP19-U6aF/OsTCP19-U6aR变性、退火得到OsTCP19-U6aF/R的二聚体产物。
2)将OsTCP19-U3F/R的二聚体产物连入pYLsgRNA-U3载体,同时将OsTCP19-U6aF/R的二聚体产物连入pYLsgRNA-U6a载体,分别得到两个中间载体。然后根据参考文献:Ma X,Zhang Q,Zhu Q,et al.A Robust CRISPR/Cas9 System for Convenient,High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.MolPlant.2015;8(8):1274-1284.中的步骤利用边切边连的方法将两个中间载体一起连入pYLCRISPR/Cas9Pubi-H终载体,得到双靶点的OsTCP19 CRISPR/Cas9敲除载体。双靶点的OsTCP19 CRISPR/Cas9敲除载体包含两个sgRNA,分别记作sgRNA 1和sgRNA2。sgRNA 1的靶点序列为AGAGTAGCCATGGATGTCAC(序列4);sgRNA2的靶点序列为GAGCTCGGGCACAAGACCGA(序列5)。
3、水稻OsTCP19突变体的获得
将步骤2获得的敲除载体OsTCP19-CRISPR通过热击法转入农杆菌(Agrobacteriumtumefaciens)株系AGL1中,筛选得到含有OsTCP19-CRISPR的重组农杆菌菌株。用含有OsTCP19-CRISPR的重组农杆菌菌株侵染中花11愈伤组织,具体的转化筛选方法参见文献“易自力,曹守云,王力,何锶洁,储成才,唐祚舜,周朴华,田文忠.提高农杆菌转化水稻频率的研究.遗传学报,2001,28(4):352-358”一文,最终获得T0代转基因水稻。
4、水稻OsTCP19突变体的鉴定
通过PCR及测序检测步骤3获得的T0代转基因水稻。具体步骤如下:提取步骤3获得的T0代转基因水稻的基因组DNA。用针对OsTCP19基因的引物OsTCP19-CRF和OsTCP19-CRR进行PCR鉴定,并将PCR产物进行测序,获得OsTCP19基因发生突变的转基因株系。引物序列具体如下:
OsTCP19-CRF:5’-TCTTTCTAGCTCTACCGGCG-3’;
OsTCP19-CRR:5’-CGCCGGGAAGTTCATGAAAT-3’。
其中,OsTCP19-CRF为序列1第2052-2071位核苷酸序列,OsTCP19-CRR为序列1第3136-3155位核苷酸序列的反向互补序列。
经测序鉴定:在T0代转基因水稻中共获得两个OsTCP19基因突变纯合株系,分别命名为水稻OsTCP19突变株系T-cr1和水稻OsTCP19突变株系T-cr2。
与野生型水稻中花11的基因组DNA相比,T0代水稻OsTCP19突变株系T-cr1的差异仅在于在编码OsTCP19蛋白的基因中,两条染色体均发生了1个碱基的缺失突变,该缺失突变位于序列1第2521位,导致OsTCP19功能丧失或减弱。
与野生型水稻中花11的基因组DNA相比,T0代水稻OsTCP19突变株系T-cr2的差异仅在于在编码OsTCP19蛋白的基因中,两条染色体均发生了1个碱基的插入突变和204个碱基的缺失突变,该插入突变的碱基插入位置位于序列1第2248和2249位之间,该缺失突变位于序列1第2319-2522位,导致OsTCP19功能丧失或减弱。
收获T0代水稻OsTCP19突变株系T-cr1和T-cr2的种子,获得T1代水稻OsTCP19突变株系T-cr1和T-cr2,并用于下述分蘖检测。
二、水稻OsTCP19突变体的分蘖检测
在正常条件下于生殖生长时期对T1代水稻OsTCP19突变株系T-cr1和T-cr2、中花11对照植株进行分蘖数的统计,每种材料统计24个单株。
结果如图3所示,与中花对照植株相比,水稻OsTCP19突变株系T-cr1和T-cr2的分蘖数均显著增加。其中,水稻OsTCP19突变株系T-cr1平均从6.8个增加到8.8个,平均从6.8个增加到9.2个。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,如针对OsTCP19启动子调控序列或OsTCP19基因本身进行的基因编辑优化,这些改进和润饰也应视为本发明的保护范围。
序列表
<110>中国科学院遗传与发育生物学研究所
<120>一个调控植物氮素利用效率和产量的蛋白质及其应用
<160>5
<170>PatentIn version 3.5
<210>1
<211>3844
<212>DNA
<213>Artificial Sequence
<400>1
atctatgtcg agaggtgcgg agatcagcaa tggagtggct gatggcacgt gtaatggagt 60
gtgtgagtaa ctcttcaggg ttcttgccga agccctttat ataacgttag ggcttccggc 120
ggcatgcatg tcacaatatc ccttataatc tgggtacgtg caatctattt acatgggatt 180
ggataatatt tcttatctct tatcccttcc tatacatttt tttttgtggg gacccttcct 240
atacattaag gatagccgca tggacctggc tactcggcct gctctatgtg acacggcaca 300
aaggccttcc gtgaggtacc cgggtatcgt gccctccaca ggtgttgatc gacttttaag 360
tccacagtgg tgcaagggat atttcattta ctttgagtcg ttatatgcat gtggtagctt 420
gatagctact tagttaaagc agtttttatc catatcatct cggaacctcc ggagaattga 480
ctgaaaccta aggttccaaa caattgaccg gagactttga gaaataattt aatccgctac 540
aaaagtttgt ttaaatttca gttttcgcct attcactcct ctctaggctt tacaagggac 600
tttcagcatc cacacaataa aaggtagagt tgagcctatc ggttatatgg agaaaacatt 660
aaaaaattga ggatgatgta gagaatataa acaaaagaca aggtagaaac taactgcaag 720
tttatatgca aatatagcat gtctaattcc ctcctcatat gtgcaccaca cattataatc 780
aacatccaac ttggtatgat catccaatct tggttcgcaa acaccagaag agattgtgca 840
tacatgagcg ttttataagt ttgtacacta aattacatcc aactaacaaa ttcatatgcc 900
atcaatgcaa tttgctaaca atgagtttaa tagcaaaatg ctatactttc tatgggtaaa 960
ttgacactca ttttgatgtt tgtccattga caatacagga aactattcta aactctcgag 1020
agaatatcct ctcattgttt gcatgtcatc caaatgatta taattttctt tttaaaaaat 1080
aagcaagata aattaatatg tgataaatca ctccacaagc atgcaaggtt aaatttaact 1140
tcaacatgtt gcaatgagaa aaaaaacaaa tttgactatg aatatatgtt aactagcttt 1200
tttttttgca gattgtggaa gttaaaattt aacttacatg tttatgcagt gatatatcat 1260
atattaatca attttcttaa tttttcataa ctatttgtat gacatgcaaa caataatggg 1320
atatcctttg gagagtttaa aatccactcc attgacgata tattgtaata ctagcataat 1380
gctcatgctt tgctccatgt aaaggaatat atattatgac agtattatag ataaaacata 1440
ttttttatcg aaaatatatc agtatcactt gtttgcttat tatttacata tatggtattc 1500
acttatttaa aacgagaacc ctagccctag caaaaccggg ccgggctggg ccgggcttca 1560
ctgctttaca agagccagct tagtgaacct aataattctg ctcctgtcct ccttatcctg 1620
atcagtcctc ggtcggcttt tcctctctct ctctctctct ctcataaaac agagaccttt 1680
aatgcgtcgt gatctctccc ctccaatccc gacaaagtac acacaccatc gtctccccct 1740
cctgcctcga tgctagctct cccattccca tctctccact ctccctccta tcatatgctc 1800
ctcccttctc cactcttctt ctcaaatcct ctctcttcct cttcgtcctc ctcctcttgc 1860
aaccaaaaaa caccataaaa tagtaatccc aatccacaag ctcacctctc tcttgcacac 1920
acacacacct gacacaacac ttacacatca taccagaaga aaaaccaatt cgattttgcc 1980
aaattaccta gctatagcat ataggtaggt agctaggtag cttggttttc cctaatagct 2040
ctagcttgat ctctttctag ctctaccggc ggcgtggcgg cggcggcggc ggccgcacca 2100
ctctacatca tcacgtacac ctgaccagct tagcgggaag ccccacaaga agagtttgta 2160
ggtcaccaat cagatcatca gttcatcttt gtggttgtgt gggtgtgtgt gtatatatat 2220
accatggcgt gagagtagcc atggatgtca ccggagacgg cggaggagga gggcaacggc 2280
ccaatttccc cctgcagctc ctcgggaaga aggaggagca gacgtgctcg acgtcgcaga 2340
ctgccggggc gggcggcggc ggcgtcgtgg gcgcgaatgg gtcggcggcg gcggcgccgc 2400
cgaagcggac gtcgacgaag gaccggcaca cgaaggtgga cgggcggggg cggcgcatcc 2460
ggatgccggc gatctgcgcc gcgcgggtgt tccagctgac gcgggagctc gggcacaaga 2520
ccgacggcga gaccatcgag tggctgctgc agcaggcgga gccggcggtg atcgcggcga 2580
ccgggacggg caccatcccg gccaacttca cctccctcaa catctccctc cgctcctccg 2640
gctcgtcgct ctccatccct tctcacctcc gccttgccgg cttggctggc cctcgcttcg 2700
gcggcggcgc gcgggcggcg gacgcgtggg accgcgtcgt cggcctcggg ttcggcggtg 2760
cggccgacgc cccgtcctcc gccacctcct cctcctcgtc gccgcttctg ctgagcttcc 2820
actccggtag cgtcggcctt gacgtgtcgc cgccgtcggc gtcgacctcc ccggccgccg 2880
ccgacctctc ccggaagcgg cggtgggagc aagaaatgca gcagcagcag cagtaccagc 2940
agcagatggc cgggtacacg cagagccaaa ttcctgcggg cacggtgtgg atggtgccga 3000
gcagcaacgc gcaggccgcc ggtggcggcg ctccgccggg aggcggcggc gagtcgattt 3060
ggacgttccc gcagtcaggg agcggcggcg gcggcggcgc ggcgacagtg taccgtggcg 3120
tgccaagcgg actacatttc atgaacttcc cggcgacacc aatggcgctg ctccccggcg 3180
ggcagcagct cggcctcgcc ggcgccggcg ggggtggcga ggggcacccg gggatcctcg 3240
ccgcgctcaa tgcctaccgc gcacaggccg cgcagccgga cgccggcgcg gcggcgcaga 3300
atggagcgca aggctcaagt cagcatcgtc agcatcagca tcacggcggc ggcggcggcg 3360
gcggcgacga gcggcatgag agcatgagcg ccagcgactc gtagggcttc cgttccgtcc 3420
ggtgtgtgcc ccgcattttc tctggttttg atctgttctt ctttaatttt ctccgttttt 3480
gtttgagttg ttgtctacta atgtctatgg ctttcttcct cgacccatca tatattcatg 3540
caaggatcat acagttgcat tccatgccat ctgcataaaa atatcttatt gtgtgctctt 3600
aagaatggat caccaatttc accatgataa agctcttgca agttgcaagt ggtagggttt 3660
ggttagaatg ctagagaacc ttttttttaa aaaaaatgaa aatgagaatg ctagagaacc 3720
tagaatcatt aatcaataaa tcattgcttt ggtatattga tcgatcgatg aggattatcc 3780
accaactaat cctcatccaa tgcatgcatg caagctagct agcatccatt cgatctgcca 3840
ctct 3844
<210>2
<211>1164
<212>DNA
<213>Artificial Sequence
<400>2
atggatgtca ccggagacgg cggaggagga gggcaacggc ccaatttccc cctgcagctc 60
ctcgggaaga aggaggagca gacgtgctcg acgtcgcaga ctgccggggc gggcggcggc 120
ggcgtcgtgg gcgcgaatgg gtcggcggcg gcggcgccgc cgaagcggac gtcgacgaag 180
gaccggcaca cgaaggtgga cgggcggggg cggcgcatcc ggatgccggc gatctgcgcc 240
gcgcgggtgt tccagctgac gcgggagctc gggcacaaga ccgacggcga gaccatcgag 300
tggctgctgc agcaggcgga gccggcggtg atcgcggcga ccgggacggg caccatcccg 360
gccaacttca cctccctcaa catctccctc cgctcctccg gctcgtcgct ctccatccct 420
tctcacctcc gccttgccgg cttggctggc cctcgcttcg gcggcggcgc gcgggcggcg 480
gacgcgtggg accgcgtcgt cggcctcggg ttcggcggtg cggccgacgc cccgtcctcc 540
gccacctcct cctcctcgtc gccgcttctg ctgagcttcc actccggtag cgtcggcctt 600
gacgtgtcgc cgccgtcggc gtcgacctcc ccggccgccg ccgacctctc ccggaagcgg 660
cggtgggagc aagaaatgca gcagcagcag cagtaccagc agcagatggc cgggtacacg 720
cagagccaaa ttcctgcggg cacggtgtgg atggtgccga gcagcaacgc gcaggccgcc 780
ggtggcggcg ctccgccggg aggcggcggc gagtcgattt ggacgttccc gcagtcaggg 840
agcggcggcg gcggcggcgc ggcgacagtg taccgtggcg tgccaagcgg actacatttc 900
atgaacttcc cggcgacacc aatggcgctg ctccccggcg ggcagcagct cggcctcgcc 960
ggcgccggcg ggggtggcga ggggcacccg gggatcctcg ccgcgctcaa tgcctaccgc 1020
gcacaggccg cgcagccgga cgccggcgcg gcggcgcaga atggagcgca aggctcaagt 1080
cagcatcgtc agcatcagca tcacggcggc ggcggcggcg gcggcgacga gcggcatgag 1140
agcatgagcg ccagcgactc gtag 1164
<210>3
<211>387
<212>PRT
<213>Artificial Sequence
<400>3
Met Asp Val Thr Gly Asp Gly Gly Gly Gly Gly Gln Arg Pro Asn Phe
1 5 10 15
Pro Leu Gln Leu Leu Gly Lys Lys Glu Glu Gln Thr Cys Ser Thr Ser
20 25 30
Gln Thr Ala Gly Ala Gly Gly Gly Gly Val Val Gly Ala Asn Gly Ser
35 40 45
Ala Ala Ala Ala Pro Pro Lys Arg Thr Ser Thr Lys Asp Arg His Thr
50 55 60
Lys Val Asp Gly Arg Gly Arg Arg Ile Arg Met Pro Ala Ile Cys Ala
65 70 75 80
Ala Arg Val Phe Gln Leu Thr Arg Glu Leu Gly His Lys Thr Asp Gly
85 90 95
Glu Thr Ile Glu Trp Leu Leu Gln Gln Ala Glu Pro Ala Val Ile Ala
100 105 110
Ala Thr Gly Thr Gly Thr Ile Pro Ala Asn Phe Thr Ser Leu Asn Ile
115 120 125
Ser Leu Arg Ser Ser Gly Ser Ser Leu Ser Ile Pro Ser His Leu Arg
130 135 140
Leu Ala Gly Leu Ala Gly Pro Arg Phe Gly Gly Gly Ala Arg Ala Ala
145 150 155 160
Asp Ala Trp Asp Arg Val Val Gly Leu Gly Phe Gly Gly Ala Ala Asp
165 170 175
Ala Pro Ser Ser Ala Thr Ser Ser Ser Ser Ser Pro Leu Leu Leu Ser
180 185 190
Phe His Ser Gly Ser Val Gly Leu Asp Val Ser Pro Pro Ser Ala Ser
195 200 205
Thr Ser Pro Ala Ala Ala Asp Leu Ser Arg Lys Arg Arg Trp Glu Gln
210 215 220
Glu Met Gln Gln Gln Gln Gln Tyr Gln Gln Gln Met Ala Gly Tyr Thr
225 230 235 240
Gln Ser Gln Ile Pro Ala Gly Thr Val Trp Met Val Pro Ser Ser Asn
245 250 255
Ala Gln Ala Ala Gly Gly Gly Ala Pro Pro Gly Gly Gly Gly Glu Ser
260 265 270
Ile Trp Thr Phe Pro Gln Ser Gly Ser Gly Gly Gly Gly Gly Ala Ala
275 280 285
Thr Val Tyr Arg Gly Val Pro Ser Gly Leu His Phe Met Asn Phe Pro
290 295 300
Ala Thr Pro Met Ala Leu Leu Pro Gly Gly Gln Gln Leu Gly Leu Ala
305 310 315 320
Gly Ala Gly Gly Gly Gly Glu Gly His Pro Gly Ile Leu Ala Ala Leu
325 330 335
Asn Ala Tyr Arg Ala Gln Ala Ala Gln Pro Asp Ala Gly Ala Ala Ala
340 345 350
Gln Asn Gly Ala Gln Gly Ser Ser Gln His Arg Gln His Gln His His
355 360 365
Gly Gly Gly Gly Gly Gly Gly Asp Glu Arg His Glu Ser Met Ser Ala
370 375 380
Ser Asp Ser
385
<210>4
<211>20
<212>DNA
<213>Artificial Sequence
<400>4
agagtagcca tggatgtcac 20
<210>5
<211>20
<212>DNA
<213>Artificial Sequence
<400>5
gagctcgggc acaagaccga 20
Claims (9)
1.OsTCP19蛋白质在调控水稻分蘖和/或氮响应能力中的应用;
所述OsTCP19蛋白质是如下A1)或A2)任一所示蛋白质:
A1)由序列表中序列3所示的氨基酸序列组成的蛋白质;
A2)在序列表中序列3所示的蛋白质的N端或/和C端连接标签得到的融合蛋白。
2.与OsTCP19蛋白质相关的生物材料在如下B1)或B2)任一种中的应用:
B1)调控水稻分蘖和/或氮响应能力;
B2)培育分蘖数减少和/或氮响应能力下降的转基因水稻;
所述与OsTCP19蛋白质相关的生物材料为下述C1)至C8)中的任一种:
C1)编码权利要求1中所述的OsTCP19蛋白质的核酸分子;
C2)含有C1)所述核酸分子的表达盒;
C3)含有C1)所述核酸分子的重组载体;
C4)含有C2)所述表达盒的重组载体;
C5)含有C1)所述核酸分子的重组微生物;
C6)含有C2)所述表达盒的重组微生物;
C7)含有C3)所述重组载体的重组微生物;
C8)含有C4)所述重组载体的重组微生物。
3.根据权利要求2所述的应用,其特征在于:C1)所述核酸分子为如下1)或2)所示的DNA分子:
1)序列表中序列1所示的基因组DNA分子;
2)序列表中序列2所示的cDNA分子。
4.CRISPR-Cas9系统在培育分蘖数增加和/或氮响应能力提高的转基因水稻中的应用;所述CRISPR-Cas9系统包括Cas9蛋白质和sgRNA,所述sgRNA的靶序列为序列4所示的DNA分子和序列5所示的DNA分子。
5.一种培育分蘖数增加和/或氮响应能力提高的转基因水稻的方法,包括如下步骤:抑制或敲除目的水稻中OsTCP19蛋白质编码基因,得到分蘖数增加和/或氮响应能力提高的转基因水稻;所述OsTCP19蛋白质的氨基酸序列如序列表中序列3所示。
6.根据权利要求5所述的方法,其特征在于:所述敲除目的水稻中OsTCP19蛋白质编码基因的方法包括将CRISPR-Cas9系统导入目的水稻的步骤;
所述CRISPR-Cas9系统包括Cas9蛋白质和sgRNA,所述sgRNA靶向OsTCP19蛋白质的编码基因序列。
7.根据权利要求6所述的方法,其特征在于:所述sgRNA的靶序列为序列4所示的DNA分子和序列5所示的DNA分子。
8.一种培育分蘖数减少和/或氮响应能力下降的转基因水稻的方法,包括如下步骤:提高目的水稻中OsTCP19蛋白质的含量,得到分蘖数减少和/或氮响应能力下降的转基因水稻;所述OsTCP19蛋白质的氨基酸序列如序列表中序列3所示。
9.一种CRISPR-Cas9系统,所述CRISPR-Cas9系统包括Cas9蛋白质和sgRNA;
所述sgRNA的靶序列为序列4所示的DNA分子和序列5所示的DNA分子。
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CN101541970A (zh) * | 2006-11-24 | 2009-09-23 | 克罗普迪塞恩股份有限公司 | 包含作为转基因的a类itcp或clavata1(clv1)或cah3多肽、具有增加的种子产量的转基因植物以及用于制备该植物的方法 |
CN109912702B (zh) * | 2017-12-13 | 2021-03-16 | 中国科学院遗传与发育生物学研究所 | 蛋白质OsARE1在调控植物抗低氮性中的应用 |
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