技术领域technical field
本发明涉及农学领域，具体地，涉及一种GRF8基因及其应用。The invention relates to the field of agronomy, in particular to a GRF8 gene and its application.
背景技术Background technique
水稻作为举足轻重的粮食作物，为世界上超过半数的人口提供了粮食。随着人口的日趋增长和可耕地面积的逐渐减少，如何在有限的耕地上种出更多的粮食，一直是农业工作者的研究重心。籽粒大小是直接影响粮食产量的重要因素之一，大粒具有明显的提高产量的优势。As an important food crop, rice provides food for more than half of the world's population. With the increasing population and the gradual reduction of arable land, how to grow more food on limited arable land has always been the research focus of agricultural workers. Grain size is one of the important factors directly affecting grain yield, and large grain has the obvious advantage of increasing yield.
目前本领域稻类控制水稻粒形的基因的克隆和研究至今还没有获得突破。因此，本领域迫切需要开展控制水稻粒形等相关基因的功能研究，以便用于改善植物的性状。So far, no breakthroughs have been made in the cloning and research of rice genes controlling rice grain shape in this field. Therefore, there is an urgent need in this field to carry out functional research on genes related to controlling rice grain shape, so as to improve plant traits.
发明内容Contents of the invention
本发明的目的在于提供控制水稻等植物的粒形等相关基因及其应用。The object of the present invention is to provide related genes for controlling grain shape and the like in plants such as rice and applications thereof.
本发明第一方面提供了一种物质的用途，所述物质为GRF8基因或其编码蛋白、或其促进剂或抑制剂，用于调控植物的性状或制备调控植物性状的制剂或组合物，其中，所述植物的性状包括选自下组的一种或多种性状：The first aspect of the present invention provides a use of a substance, the substance is the GRF8 gene or its encoded protein, or its promoter or inhibitor, for regulating the traits of plants or preparing preparations or compositions for regulating plant traits, wherein , the traits of the plant include one or more traits selected from the group consisting of:
(i)粒形；(i) particle shape;
(iv)株型。(iv) Plant type.
在另一优选例中，所述粒形包括粒长、粒宽、和/或千粒重。In another preferred example, the grain shape includes grain length, grain width, and/or thousand grain weight.
在另一优选例中，所述组合物包括农用组合物。In another preferred example, the composition includes an agricultural composition.
在另一优选例中，所述组合物包含(a)GRF8基因或其编码蛋白、或其促进剂或抑制剂；和(b)农学上可接受的载体。In another preferred example, the composition comprises (a) the GRF8 gene or its encoded protein, or its promoter or inhibitor; and (b) an agriculturally acceptable carrier.
在另一优选例中，所述组合物中，含有0.0001-99wt％，较佳地0.1-90wt％的组分(a)，以所述组合物的总重量计。In another preferred embodiment, the composition contains 0.0001-99wt%, preferably 0.1-90wt%, of component (a), based on the total weight of the composition.
在另一优选例中，所述组合物或制剂的剂型选自下组：溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、或其组合。In another preferred example, the dosage form of the composition or preparation is selected from the group consisting of solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, or combinations thereof.
在另一优选例中，所述促进剂包括促进GRF8基因或其编码蛋白表达的小分子化合物。In another preferred example, the promoter includes a small molecular compound that promotes the expression of the GRF8 gene or its encoded protein.
在另一优选例中，所述的促进剂选自下组：小分子化合物、核酸分子、或其组合。In another preferred embodiment, the accelerator is selected from the group consisting of small molecule compounds, nucleic acid molecules, or combinations thereof.
在另一优选例中，所述抑制剂选自下组：反义核酸、抗体、小分子化合物、Crispr试剂、siRNA、shRNA、miRNA、小分子配体、或其组合。In another preferred embodiment, the inhibitor is selected from the group consisting of antisense nucleic acid, antibody, small molecule compound, Crispr reagent, siRNA, shRNA, miRNA, small molecule ligand, or a combination thereof.
在另一优选例中，所述植物包括农作物、林业植物、蔬菜、瓜果、花卉、牧草(包括草坪草)；优选地包括禾本科，更优选地包括水稻、玉米、高粱、小麦。In another preferred example, the plants include crops, forestry plants, vegetables, fruits, flowers, pastures (including lawn grasses); preferably include Gramineae, more preferably include rice, corn, sorghum, and wheat.
在另一优选例中，所述的植物包括：水稻、小麦、玉米、或高粱。In another preferred example, the plants include: rice, wheat, corn, or sorghum.
在另一优选例中，所述的水稻选自下组：籼稻、粳稻、或其组合。In another preferred embodiment, the rice is selected from the group consisting of indica rice, japonica rice, or a combination thereof.
在另一优选例中，所述的GRF8基因选自下组：cDNA序列、基因组序列、或其组合。In another preferred example, the GRF8 gene is selected from the group consisting of cDNA sequence, genome sequence, or a combination thereof.
在另一优选例中，所述GRF8基因来自禾本科植物。In another preferred example, the GRF8 gene is from a Poaceae plant.
在另一优选例中，所述的GRF8基因来自选自下组的一种或多种植物：水稻、玉米、高粱、小麦、谷子、二穗短柄草。In another preferred embodiment, the GRF8 gene is from one or more plants selected from the group consisting of rice, corn, sorghum, wheat, millet, and Brachypodium distachyon.
在另一优选例中，所述GRF8基因选自下组：水稻的GRF8基因(OsGRF8)、玉米的GRF8同源基因(ZmGRF8，EU954645.1)、高粱的GRF8同源基因(SbGRF8，XM_021461903)、小麦的GRF8同源基因(TaGRF8，KU748867.1)、或其组合。In another preferred example, the GRF8 gene is selected from the group consisting of rice GRF8 gene (OsGRF8), maize GRF8 homologous gene (ZmGRF8, EU954645.1), sorghum GRF8 homologous gene (SbGRF8, XM_021461903), Wheat GRF8 homologous gene (TaGRF8, KU748867.1), or a combination thereof.
在另一优选例中，所述GRF8蛋白的氨基酸序列选自下组：In another preferred example, the amino acid sequence of the GRF8 protein is selected from the following group:
(i)具有SEQ ID NO.:2所示氨基酸序列的多肽；(i) a polypeptide having the amino acid sequence shown in SEQ ID NO.:2;
(ii)将如SEQ ID NO.:2所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述调控植物性状功能的由(i)衍生的多肽；或(ii) The amino acid sequence shown in SEQ ID NO.: 2 is formed by substituting, deleting or adding one or several (such as 1-10) amino acid residues, and having the function of regulating plant traits by (i) a derivatized polypeptide; or
(iii)氨基酸序列与SEQ ID NO.:2所示氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有所述GRF8活性的多肽。(iii) The amino acid sequence has a homology ≥ 80% (preferably ≥ 90%, more preferably ≥ 95% or ≥ 98%) to the amino acid sequence shown in SEQ ID NO.: 2, a polypeptide having said GRF8 activity .
在另一优选例中，所述GRF8基因的核苷酸序列选自下组：In another preferred example, the nucleotide sequence of the GRF8 gene is selected from the following group:
(a)编码如SEQ ID NO.:2所示多肽的多核苷酸；(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.:2;
(b)序列如SEQ ID NO.:1所示的多核苷酸；(b) a polynucleotide whose sequence is shown in SEQ ID NO.:1;
(c)核苷酸序列与SEQ ID NO.:1所示序列的同源性≥75％(较佳地≥85％，更佳地≥90％或≥95％)的多核苷酸；(c) a polynucleotide whose nucleotide sequence is ≥75% (preferably ≥85%, more preferably ≥90% or ≥95%) homologous to the sequence shown in SEQ ID NO.:1;
(d)在SEQ ID NO.:1所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；(d) Truncating or adding 1-60 (preferably 1-30, more preferably 1-10) cores at the 5' end and/or 3' end of the polynucleotide shown in SEQ ID NO.:1 polynucleotides of nucleotides;
(e)与(a)-(d)任一所述的多核苷酸互补的多核苷酸。(e) A polynucleotide complementary to the polynucleotide described in any one of (a)-(d).
本发明第二方面提供了一种物质的用途，所述物质为GRF8基因或其编码蛋白、或其促进剂，用于调控植物的性状或制备调控植物性状的制剂或组合物，其中，所述植物的性状包括选自下组的一种或多种性状：The second aspect of the present invention provides a use of a substance, the substance is the GRF8 gene or its encoded protein, or its promoter, for regulating the traits of plants or preparing preparations or compositions for regulating plant traits, wherein, the Plant traits include one or more traits selected from the group consisting of:
(i)增加粒长；和/或(i) increase grain length; and/or
(ii)缩短粒宽；和/或(ii) shortened grain width; and/or
(iii)增加千粒重；和/或(iii) increased thousand kernel weight; and/or
(iv)株型松散；和/或(iv) loose plant type; and/or
(v)增加产量。(v) Increase production.
本发明第三方面提供了一种物质的用途，所述物质为GRF8蛋白、或其抑制剂，用于调控植物的性状或制备调控植物性状的制剂或组合物，其中，所述植物的性状包括选自下组的一种或多种性状：The third aspect of the present invention provides a use of a substance, the substance is a GRF8 protein, or an inhibitor thereof, for regulating plant traits or preparing a preparation or composition for regulating plant traits, wherein the plant traits include One or more traits selected from the group consisting of:
(i)减少粒长；和/或(i) reduce grain length; and/or
(ii)增加粒宽；和/或(ii) increase grain width; and/or
(iii)减少千粒重；和/或(iii) reduce thousand kernel weight; and/or
本发明第四方面提供了一种改良植物性状的方法，包括步骤：The fourth aspect of the present invention provides a method for improving plant traits, comprising the steps of:
调节所述植物中GRF8基因或其编码蛋白的表达量和/或活性，从而改良植物的性状。Regulating the expression level and/or activity of the GRF8 gene or its encoded protein in the plant, thereby improving the traits of the plant.
在另一优选例中，当所述植物中GRF8的活性E1与野生型同种类型的植物中的相同GRF8本底活性E0之比(E1/E0)≥2倍，较佳地≥5倍，更佳地≥10倍时，所述植物的性状改良选自下组：In another preferred example, when the ratio (E1/E0) of the activity E1 of GRF8 in the plant to the background activity E0 of the same GRF8 in the wild-type plant of the same type is ≥ 2 times, preferably ≥ 5 times, When more preferably ≥ 10 times, the trait improvement of the plant is selected from the following group:
(i)增加粒长；和/或(i) increase grain length; and/or
(ii)缩短粒宽；和/或(ii) shortened grain width; and/or
(iii)增加千粒重；和/或(iii) increased thousand kernel weight; and/or
(iv)株型松散；和/或(iv) loose plant type; and/or
(v)增加产量。(v) Increase production.
在另一优选例中，当所述植物中GRF8的活性E1与野生型同种类型的植物中的相同GRF8本底活性E0之比(E1/E0)≤1/2，较佳地≤1/5，更佳地≤1/10时，所述植物的性状改良选自下组：In another preferred example, when the ratio (E1/E0) of the activity E1 of GRF8 in the plant to the background activity E0 of the same GRF8 in the wild-type plant of the same type is ≤1/2, preferably ≤1/2 5. When more preferably ≤ 1/10, the trait improvement of the plant is selected from the following group:
(i)减少粒长；和/或(i) reduce grain length; and/or
(ii)增加粒宽；和/或(ii) increase grain width; and/or
(iii)减少千粒重。(iii) Reduce 1000-kernel weight.
在另一优选例中，所述方法包括步骤：In another preferred embodiment, the method includes the steps of:
(i)提供一植物或植物细胞；和(i) providing a plant or plant cell; and
(ii)将GRF8基因或其编码的多肽、或其促进剂或抑制剂导入所述植物或植物细胞，从而获得转基因的植物或植物细胞。(ii) introducing the GRF8 gene or its encoded polypeptide, or its promoter or inhibitor into the plant or plant cell, so as to obtain a transgenic plant or plant cell.
本发明第五方面提供了一种制备基因工程的植物组织或植物细胞的方法，包括步骤：A fifth aspect of the present invention provides a method for preparing genetically engineered plant tissue or plant cells, comprising the steps of:
调节植物组织或植物细胞中的GRF8基因或其编码蛋白的表达量和/或活性，从而获得基因工程的植物组织或植物细胞。Regulating the expression level and/or activity of the GRF8 gene or its encoded protein in the plant tissue or plant cell, so as to obtain the genetically engineered plant tissue or plant cell.
在另一优选例中，所述调节植物组织或植物细胞中的GRF8基因或其编码蛋白的表达量和/或活性包括(a)提高植物组织或植物细胞中的GRF8基因或其编码蛋白的表达量和/或活性；和/或(b)降低植物组织或植物细胞中的GRF8基因或其编码蛋白的表达量和/或活性。In another preferred example, said regulating the expression level and/or activity of the GRF8 gene or its encoded protein in plant tissues or plant cells comprises (a) increasing the expression of the GRF8 gene or its encoded proteins in plant tissues or plant cells and/or (b) reducing the expression level and/or activity of the GRF8 gene or its encoded protein in plant tissues or plant cells.
本发明第六方面提供了一种制备转基因植物的方法，包括步骤：The sixth aspect of the present invention provides a method for preparing transgenic plants, comprising the steps of:
将本发明第五方面所述方法制备的基因工程的植物组织或植物细胞再生为植物体，从而获得转基因植物。The genetically engineered plant tissue or plant cell prepared by the method according to the fifth aspect of the present invention is regenerated into a plant body, thereby obtaining a transgenic plant.
本发明第七方面提供了一种转基因植株，所述转基因植株中导入GRF8基因或其编码的多肽、或其促进剂或抑制剂。The seventh aspect of the present invention provides a transgenic plant into which the GRF8 gene or its encoded polypeptide, or its promoter or inhibitor, is introduced.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.
附图说明Description of drawings
图1显示了一些相关OsGRF基因的表达检测；其中，(a)MIM396转基因材料中靶基因OsGRF基因的表达检测；(b)OsGRF8OE转基因材料中OsGRF8基因的表达检测。统计数据经t-检验后呈现显著差异和极显著差异分别用*(P<0.05)和**(P<0.01)表示。Figure 1 shows the detection of the expression of some related OsGRF genes; among them, (a) the detection of the expression of the target gene OsGRF gene in the MIM396 transgenic material; (b) the detection of the expression of the OsGRF8 gene in the OsGRF8OE transgenic material. Statistical data showed significant difference and extremely significant difference after t-test, indicated by * (P<0.05) and ** (P<0.01), respectively.
图2显示了OsGRF8基因的两个转录本。Figure 2 shows two transcripts of the OsGRF8 gene.
图3显示了OsGRF8OE转基因植株的粒形分析；(a)转基因植株的粒形变化；(b)转基因植株的粒长(seed length)和粒宽(seed width)统计；(c)转基因植株的千粒重(1000-grain-weight)统计。统计数据经t-检验后呈现显著差异和极显著差异分别用*(P<0.05)和**(P<0.01)表示。Figure 3 shows the grain shape analysis of OsGRF8OE transgenic plants; (a) grain shape change of transgenic plants; (b) statistics of grain length (seed length) and grain width (seed width) of transgenic plants; (c) thousand-grain weight of transgenic plants (1000-grain-weight) statistics. Statistical data showed significant difference and extremely significant difference after t-test, indicated by * (P<0.05) and ** (P<0.01), respectively.
图4显示了OsGRF8OE的株型。Figure 4 shows the strain type of OsGRF8OE.
具体实施方式Detailed ways
经过广泛而深入的研究，本发明人通过对大量的植物农艺性状位点的研究和筛选，首次意外地发现了一种植物(如水稻)的GRF8基因或其编码蛋白、或其促进剂或抑制剂，用于调控植物的性状，所述性状包括：(i)粒形；(iv)株型。此外，申请人还意外的发现，过表达GRF基因或其编码蛋白，可显著(i)增加粒长；和/或(ii)缩短粒宽；和/或(iii)增加千粒重；和/或(iv)株型松散；和/或(v)增加产量。在此基础上完成了本发明。After extensive and in-depth research, the inventors have unexpectedly discovered a plant (such as rice) GRF8 gene or its encoded protein, or its promoter or inhibitor for the first time by studying and screening a large number of plant agronomic traits. An agent for regulating the traits of plants, the traits include: (i) grain shape; (iv) plant type. In addition, the applicant also unexpectedly found that overexpressing the GRF gene or its encoded protein can significantly (i) increase the grain length; and/or (ii) shorten the grain width; and/or (iii) increase the thousand-grain weight; and/or ( iv) looser plant architecture; and/or (v) increased yield. The present invention has been accomplished on this basis.
GRF8基因GRF8 gene
如本文所用，术语“本发明的GRF8基因”、“GRF8基因”可互换使用，均指来源于植物(如水稻、小麦)的GRF8基因或其变体。在一优选实施方式中，本发明的GRF8基因的核苷酸序列如SEQ ID NO.:1所示。As used herein, the terms "GRF8 gene of the present invention" and "GRF8 gene" are used interchangeably, and both refer to the GRF8 gene or its variants derived from plants (such as rice, wheat). In a preferred embodiment, the nucleotide sequence of the GRF8 gene of the present invention is shown in SEQ ID NO.:1.
本发明还包括与本发明的优选基因序列(SEQ ID NO.:2)具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的核酸，所述核酸也能有效地调控植物(如水稻)的农艺性状。“同源性”是指按照位置相同的百分比，两条或多条核酸之间的相似水平(即序列相似性或同一性)。在本文中，所述基因的变体可以通过插入或删除调控区域，进行随机或定点突变等来获得。The present invention also includes 50% or more (preferably more than 60%, more than 70%, more than 80%, more preferably more than 90%, more preferably more than 95%) of the preferred gene sequence (SEQ ID NO.: 2) of the present invention, Most preferred are nucleic acids with a homology of more than 98%, such as 99%, which can also effectively regulate the agronomic traits of plants (such as rice). "Homology" refers to the level of similarity (ie, sequence similarity or identity) between two or more nucleic acids, in terms of percentage positions that are identical. Herein, variants of the gene can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutation, and the like.
在本发明中，SEQ ID NO.:1中的核苷酸序列可以经过取代、缺失或添加一个或多个，生成SEQ ID NO.:1的衍生序列，由于密码子的简并性，即使与SEQ ID NO.:1的同源性较低，也能基本编码出如SEQ ID NO.:2所示的氨基酸序列。另外，“在SEQ ID NO.:1中的核苷酸序列经过取代、缺失或添加至少一个核苷酸衍生序列”的含义还包括能在中度严谨条件下，更佳的在高度严谨条件下与SEQ ID NO.:1所示的核苷酸序列杂交的核苷酸序列。这些变异形式包括(但并小限于)：若干个(通常为1-90个，较佳地1-60个，更佳地1-20个，最佳地1-10个)核苷酸的缺失、插入和/或取代，以及在5’和/或3’端添加数个(通常为60个以内，较佳地为30个以内，更佳地为10个以内，最佳地为5个以内)核苷酸。In the present invention, the nucleotide sequence in SEQ ID NO.: 1 can undergo substitution, deletion or addition of one or more to generate a derivative sequence of SEQ ID NO.: 1. Due to the degeneracy of codons, even with The homology of SEQ ID NO.:1 is relatively low, and it can also basically encode the amino acid sequence shown in SEQ ID NO.:2. In addition, the meaning of "the nucleotide sequence in SEQ ID NO.:1 has been substituted, deleted or added at least one nucleotide derivative sequence" also includes the ability to perform under moderately stringent conditions, preferably under highly stringent conditions A nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO.:1. These variations include (but are not limited to): the deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , insertion and/or substitution, and addition of several (usually within 60, preferably within 30, more preferably within 10, and most preferably within 5) at the 5' and/or 3' end ) nucleotides.
应理解，尽管本发明的实例中提供的基因来源于水稻，但是来源于其它类似的植物(尤其是与水稻属于同一科或属的植物)的、与本发明的序列(优选地，序列如SEQ IDNO.:1所示)具有一定同源性(保守性)的GRF8的基因序列，也包括在本发明的范围内，只要本领域技术人员在阅读了本申请后根据本申请提供的信息可以方便地从其它植物中分离得到该序列。It should be understood that although the genes provided in the examples of the present invention are derived from rice, other similar plants (especially plants belonging to the same family or genus as rice) and sequences of the present invention (preferably, sequences such as SEQ The gene sequence of GRF8 with a certain homology (conservation) shown in ID NO.: 1 is also included in the scope of the present invention, as long as those skilled in the art can conveniently read the application according to the information provided by the application This sequence was isolated from other plants.
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括：DNA、基因组DNA或人工合成的DNA，DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO.:1所示的编码区序列相同或者是简并的变异体。A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include: DNA, genomic DNA, or synthetic DNA, and DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO.: 1 or a degenerate variant.
编码成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。A polynucleotide encoding a mature polypeptide includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and non-coding sequences.
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多苷或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences. The present invention also relates to variants of the above polynucleotides, which encode fragments, analogs and derivatives of polyglycosides or polypeptides having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酞胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。The present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, More preferably, hybridization occurs at 95% or more.
应理解，虽然本发明的GRF8基因优选来自水稻，但是来自其它植物的与水稻GRF8基因高度同源(如具有80％以上，如85％,90％,95％甚至98％序列相同性)的其它基因也在本发明考虑的范围之内。比对序列相同性的方法和工具也是本领域周知的，例如BLAST。It should be understood that although the GRF8 gene of the present invention is preferably from rice, other plants from other plants that are highly homologous to the rice GRF8 gene (such as having more than 80%, such as 85%, 90%, 95% or even 98% sequence identity) Genes are also contemplated by the present invention. Methods and tools for aligning sequence identities are also well known in the art, such as BLAST.
本发明的GRF8核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的DNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。The full-length GRF8 nucleotide sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available DNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
GRF8基因编码的多肽Polypeptide encoded by GRF8 gene
如本文所用，术语“本发明多肽”、“GRF8基因的编码蛋白”、可以互换使用，都是指来源于水稻的GRF8的多肽及其变体。在一优选实施方式中，本发明多肽的一种典型的氨基酸序列如SEQ ID NO.:2所示:As used herein, the terms "polypeptide of the present invention", "protein encoded by the GRF8 gene", can be used interchangeably, and all refer to the polypeptide of GRF8 and its variants derived from rice. In a preferred embodiment, a typical amino acid sequence of the polypeptide of the present invention is shown in SEQ ID NO.:2:
MLSSCGGHGHGNPRSLQEEHHGRCGEQQGGGGGGGQEQEQDGFLVREARASPPSPSSSSFMLSSCGGHGHGNPRSLQEEHHGRCGEQQGGGGGGGQEQEQDGFLVREARASPPPSSSSSF
LGSTSSSCSGGGGGGQMLSFSSPNGTAGLGLSSGGSMQGVLARVRGPFTPTQWMELEHQALGSTSSSCSGGGGGGQMLSFSSPNGTAGLGLSSGGSMQGVLARVRGPFTPTQWMELEHQA
LIYKHIAANVSVPSSLLLPIRRSLHPWGWGSFPPGCADVEPRRCRRTDGKKWRCSRDAVGLIYKHIAANVSVPSSLLLPIRRSLHPWGWGSFPPGCADAVEPRRCRRTDGKKWRCSRDAVG
DQKYCERHINRGRHRSRKHVEGRKATLTIAEPSTVIAAGVSSRGHTVARQKQVKGSAATVDQKYCERHINRGRHRSRKHVEGRKATLTIAEPSTVIAAGVSSRGHTVARQKQVKGSAATV
SDPFSRQSNRKFLEKQNVVDQLSPMDSFDFSSTQSSPNYDNVALSPLKLHHDHDESYIGHSDPFSRQSNRKFLEKQNVVDQLSPMDSFDFSSTQSSPNYDNVALSPLKLHHDHDESYIGH
GAGSSSEKGSMMYESRLTVSKETLDDGPLGEVFKRKNCQSASTEILTEKWTENPNLHCPSGAGSSSEKGSMMYESRLTVSKETLDDGPLGEVFKRKNCQSASTEILTEKWTENPNLHCPS
GILQMATKFNSISSGNTVNSGGTAVENLITDNGYLTARMMNPHIVPTLL*(SEQ ID NO.:2)。GILQMATKFNSISSGNTVNSGGTAVENLITDNGYLTARMMNPHIVPTLL* (SEQ ID NO.: 2).
本发明涉及一种调控植物性状的SDG40多肽及其变体，在本发明的一个优选例中，所述多肽的氨基酸序列如SEQ ID NO.:2所示。本发明的多肽能够有效调控植物(如水稻)的性状。The present invention relates to a SDG40 polypeptide and its variants for regulating plant traits. In a preferred example of the present invention, the amino acid sequence of the polypeptide is shown in SEQ ID NO.:2. The polypeptide of the present invention can effectively regulate the traits of plants (such as rice).
本发明还包括与本发明的SEQ ID NO.:2所示序列具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的具有相同或相似功能的多肽或蛋白。The present invention also includes 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably More than 98%, such as 99%) homologous polypeptides or proteins with the same or similar functions.
所述“相同或相似功能”主要是指：“调控植物(如水稻)的农艺性状”。The "same or similar function" mainly refers to: "regulating the agronomic traits of plants (such as rice)".
本发明的多肽可以是重组多肽、天然多肽、合成多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. Polypeptides of the present invention may be naturally purified, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may or may not include an initial methionine residue.
本发明还包括具有GRF8蛋白活性的GRF8蛋白片段和类似物。如本文所用，术语“片段”和“类似物”是指基本上保持本发明的天然GRF8蛋白相同的生物学功能或活性的多肽。The present invention also includes GRF8 protein fragments and analogs having GRF8 protein activity. As used herein, the terms "fragment" and "analogue" refer to polypeptides that substantially retain the same biological function or activity of the native GRF8 protein of the present invention.
本发明的多肽片段、衍生物或类似物可以是：(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的；或(ii)在一个或多个氨基酸残基中具有取代基团的多肽；或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽；或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或融合蛋白)。根据本文的定义这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。The polypeptide fragments, derivatives or analogs of the present invention may be: (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues The group may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent in one or more amino acid residues; or (iii) the mature polypeptide is combined with another compound (such as a compound that extends the half-life of the polypeptide, For example, a polypeptide formed by fusion of polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or fusion protein). These fragments, derivatives and analogs are within the purview of those skilled in the art as defined herein.
本发明中，所述的多肽变体是如SEQ ID NO.:1所示的氨基酸序列，经过若干个(通常为1-60个，较佳地1-30个，更佳地1-20个，最佳地1-10个)取代、缺失或添加至少一个氨基酸所得的衍生序列，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能，在C末端和/或\末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。这些保守性变异最好根据表1进行替换而产生。In the present invention, the polypeptide variant is the amino acid sequence shown in SEQ ID NO.: 1, after several (usually 1-60, preferably 1-30, more preferably 1-20 , preferably 1-10) the derivative sequence obtained by substituting, deleting or adding at least one amino acid, and adding one or several (usually within 20, preferably within 10) at the C-terminal and/or N-terminal , more preferably within 5) amino acids. For example, in the protein, when amino acids with similar or similar properties are used for substitution, the function of the protein will not generally be changed, and the addition of one or several amino acids at the C-terminal and/or \terminal will generally not change the function of the protein. These conservative variations are preferably produced by making substitutions according to Table 1.
表1Table 1
本发明还包括所要求保护的蛋白的类似物。这些类似物与天然SEQ ID NO.:2差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白的类似物包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分了生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述例举的代表性的蛋白。The invention also includes analogs of the claimed proteins. The difference between these analogues and the natural SEQ ID NO.: 2 may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis, or other techniques known to be divided into biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.
修饰(通常不改变一级结构)形式包括：体内或体外蛋白的化学衍生形式如乙酸化或羧基化。修饰还包括糖基化，如那些在蛋白质合成和加工中进行糖基化修饰。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。Modified (usually without altering primary structure) forms include: in vivo or in vitro chemical derivatization of proteins such as acetylation or carboxylation. Modifications also include glycosylation, such as those carried out in protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine).
在本发明中，GRF8的基因包括基因组基因以及cDNA基因。In the present invention, the gene of GRF8 includes genome gene and cDNA gene.
对于水稻而言，一种典型的野生型GRF8蛋白的氨基酸序列如SEQ ID NO.:2所示。一种典型的编码野生型GRF8蛋白的cDNA核苷酸序列如SEQ ID NO.:1所示，而水稻的GRF8基因组序列的登录号为LOC_Os11g35030。For rice, the amino acid sequence of a typical wild-type GRF8 protein is shown in SEQ ID NO.:2. A typical cDNA nucleotide sequence encoding wild-type GRF8 protein is shown in SEQ ID NO.: 1, and the accession number of the rice GRF8 genome sequence is LOC_Os11g35030.
ATGCTGAGCTCTTGTGGTGGCCATGGCCATGGAAATCCAAGAAGCTTGCAAGAAGAACACATGCTGAGCTCTTGTGGTGGCCATGGCCATGGAAATCCAAGAAGCTTGCAAGAAGAACAC
CATGGCAGATGTGGTGAGCAGCAAGGTGGAGGAGGAGGAGGAGGGCAAGAGCAAGAGCAACATGGCAGATGTGGTGAGCAGCAAGGTGGAGGAGGAGGAGGAGGGCAAGAGCAAGAGCAA
GATGGGTTCTTGGTGAGAGAGGCAAGGGCATCCCCACCATCTCCATCTTCTTCATCATTTGATGGGTTCTTGGTGAGAGAGGCAAGGGCATCCCACCATCTCCATCTTCTTCATCATTT
CTTGGATCCACAAGCTCTTCTTGTTCTGGAGGAGGAGGAGGAGGGCAGATGTTGAGCTTCCTTGGATCCACAAGCTCTTCTTGTTCTGGAGGAGGAGGAGGAGGGCAGATGTTGAGCTTC
TCCTCCCCCAATGGAACAGCAGGGTTGGGCTTGAGCTCAGGAGGAAGCATGCAGGGGGTCTCCTCCCCCAATGGAACAGCAGGGTTGGGCTTGAGCTCAGGAGGAAGCATGCAGGGGGTC
TTGGCAAGGGTCAGGGGGCCGTTCACCCCAACACAGTGGATGGAGCTGGAGCACCAGGCATTGGCAAGGGTCAGGGGGCCGTTCACCCAACACAGTGGATGGAGCTGGAGCACCAGGCA
CTGATCTACAAGCACATTGCTGCAAATGTTTCTGTCCCTTCCAGCTTGCTCCTCCCCATCCTGATCTACAAGCACATTGCTGCAAATGTTTCTGTCCCTTCCAGCTTGCTCCTCCCCATC
AGGAGAAGCCTCCATCCATGGGGATGGGGATCATTCCCTCCTGGCTGTGCTGATGTAGAAAGGAGAAGCCTCCATCCATGGGGATGGGGATCATTCCCTCCTGGCTGTGCTGATGTAGAA
CCCAGAAGATGCCGCCGCACAGACGGCAAGAAGTGGCGGTGCTCCAGAGATGCTGTTGGGCCCAGAAGATGCCGCCGCACAGACGGCAAGAAGTGGCGGTGCTCCAGAGATGCTGTTGGG
GATCAGAAGTATTGTGAGCGACACATAAACCGTGGTCGCCATCGTTCAAGAAAGCATGTGGATCAGAAGTATTGTGAGCGACACATAAACCGTGGTCGCCATCGTTCAAGAAAGCATGTG
GAAGGCCGAAAGGCGACACTCACCATTGCAGAACCATCCACGGTTATTGCTGCTGGTGTAGAAGGCCGAAAGGCGACACTCACCATTGCAGAACCATCCACGGTTATTGCTGCTGGTGTA
TCATCTCGCGGCCACACTGTGGCTCGGCAGAAGCAGGTGAAAGGCTCAGCTGCTACTGTCTCATCTCGCGGCCACACTGTGGCTCGGCAGAAGCAGGTGAAAGGCTCAGCTGCTACTGTC
TCTGATCCTTTCTCGAGACAATCCAACAGGAAATTTCTGGAGAAACAGAACGTTGTCGACTCTGATCCTTTCTCGAGACAATCCAACAGGAAATTTCTGGAGAAACAGAACGTTGTCGAC
CAATTGTCTCCCATGGATTCATTTGATTTCTCATCCACACAATCTTCTCCAAACTATGACCAATTGTCTCCCATGGATTCATTTGATTTCTCATCCCACAATCTTCTCCAAACTATGAC
AATGTAGCATTGTCACCACTGAAGTTGCACCATGATCATGATGAATCTTACATCGGGCATAATGTAGCATTGTCACCACTGAAGTTGCACCATGATCATGATGAATCTTACATCGGGCAT
GGAGCAGGCAGTTCATCAGAAAAAGGCAGTATGATGTACGAAAGTCGGTTAACAGTCTCTGGAGCAGGCAGTTCATCAGAAAAAGGCAGTATGATGTACGAAAGTCGGTTAACAGTCTCT
AAGGAAACACTTGATGATGGACCTTTAGGTGAAGTTTTCAAAAGAAAGAATTGCCAATCAAAGGAAACACTTGATGATGGACCTTTAGGTGAAGTTTTCAAAAGAAAGAATTGCCAATCA
GCTTCTACAGAAATCTTAACTGAAAAATGGACTGAGAACCCCAACTTACATTGCCCATCTGCTTCTACAGAAATCTTAACTGAAAAATGGACTGAGAACCCCAACTTACATTGCCCATCT
GGAATCCTACAAATGGCTACTAAGTTCAATTCAATTTCCAGCGGCAACACAGTAAATAGTGGAATCCTACAAATGGCTACTAAGTTCAATTCAATTTCCAGCGGCAACACAGTAAATAGT
GGTGGCACCGCAGTGGAGAATCTTATCACTGATAATGGATATCTTACTGCAAGAATGATGGGTGGCACCGCAGTGGAGAATCTTATCACTGATAATGGATATCTTACTGCAAGAATGATG
AATCCTCATATTGTCCCAACACTTCTCTAA(SEQ ID NO.:1)。AATCCTCATATTGTCCCAACACTTCTCTAA (SEQ ID NO.: 1).
代表性的其他物种的GRF8同源基因包括(但并不限于)：玉米的GRF8同源基因(名称ZmGRF8，NCBI基因号：EU954645.1)、高粱的GRF8同源基因(名称SbGRF8，NCBI基因号：110435841，或XM_021461903)、小麦的GRF8同源基因(名称TaGRF8，NCBI基因号:KU748867.1)。Representative GRF8 homologous genes of other species include (but are not limited to): the GRF8 homologous gene of maize (name ZmGRF8, NCBI gene number: EU954645.1), the GRF8 homologous gene of sorghum (name SbGRF8, NCBI gene number : 110435841, or XM_021461903), the GRF8 homologous gene of wheat (name TaGRF8, NCBI gene number: KU748867.1).
表达载体Expression vector
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明突变蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。The present invention also relates to vectors containing the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors of the present invention or the mutant protein coding sequences of the present invention, and methods for producing the polypeptides of the present invention through recombinant techniques.
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的突变蛋白。一般来说有以下步骤：The polynucleotide sequences of the present invention can be used to express or produce recombinant muteins by conventional recombinant DNA techniques. Generally speaking, there are the following steps:
(1).用本发明的编码本发明突变蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；(1). Use the polynucleotide (or variant) encoding the mutein of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;
(2).在合适的培养基中培养的宿主细胞；(2). Host cells cultured in a suitable medium;
(3).从培养基或细胞中分离、纯化蛋白质。(3). Isolate and purify protein from culture medium or cells.
本发明还提供了一种包括本发明的基因的重组载体。作为一种优选的方式，重组载体的启动子下游包含多克隆位点或至少一个酶切位点。当需要表达本发明目的基因时，将目的基因连接入适合的多克隆位点或酶切位点内，从而将目的基因与启动子可操作地连接。作为另一种优选方式，所述的重组载体包括(从5'到3'方向)：启动子，目的基因，和终止子。如果需要，所述的重组载体还可以包括选自下组的元件：3'多聚核苷酸化信号；非翻译核酸序列；转运和靶向核酸序列；抗性选择标记(二氢叶酸还原酶、新霉素抗性、潮霉素抗性以及绿色荧光蛋白等)；增强子；或操作子。The present invention also provides a recombinant vector comprising the gene of the present invention. As a preferred manner, the downstream of the promoter of the recombinant vector contains multiple cloning sites or at least one restriction site. When the target gene of the present invention needs to be expressed, the target gene is linked into a suitable multiple cloning site or restriction site, so that the target gene is operably linked to the promoter. As another preferred mode, the recombinant vector includes (from 5' to 3' direction): a promoter, a target gene, and a terminator. If necessary, the recombinant vector may also include elements selected from the group consisting of: 3' polynucleotide signal; non-translated nucleic acid sequence; transport and targeting nucleic acid sequence; resistance selectable marker (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); enhancers; or operons.
在本发明中，编码突变蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。In the present invention, the polynucleotide sequence encoding the mutant protein can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.
本领域的技术人员熟知的方法能用于构建含本发明突变蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding muteins of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
本领域普通技术人员可以使用熟知的方法构建含有本发明所述的基因的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。使用本发明的基因构建重组表达载体时，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。Those of ordinary skill in the art can use well-known methods to construct expression vectors containing the genes described in the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. When using the gene of the present invention to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before the transcription initiation nucleotide.
包括本发明基因、表达盒或载体可以用于转化适当的宿主细胞，以使宿主表达蛋白质。宿主细胞可以是原核细胞，如大肠杆菌，链霉菌属、农杆菌；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物(如大肠杆菌)时，可以用CaCl2法处理，也可用电穿孔法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法(如显微注射、电穿孔、脂质体包装等)。转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法、幼胚转化法、花芽浸泡法等。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得转基因的植物。An expression cassette or vector comprising the gene of the present invention can be used to transform an appropriate host cell such that the host expresses the protein. The host cells can be prokaryotic cells, such as Escherichia coli, Streptomyces, and Agrobacterium; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Those of ordinary skill in the art will know how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism (such as Escherichia coli), it can be treated with CaCl2 or electroporation. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Transformation of plants can also use methods such as Agrobacterium transformation or gene gun transformation, such as leaf disk method, immature embryo transformation method, flower bud soaking method and the like. Transformed plant cells, tissues or organs can be regenerated into plants by conventional methods, so as to obtain transgenic plants.
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、植物细胞(如水稻细胞)。The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces sp.; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (eg rice cells).
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with theCaCl2 method using procedures well known in the art. Another method is to useMgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
本发明的主要优点包括：The main advantages of the present invention include:
(1)本发明首次筛选到一种可调控植物性状的GRF8基因。(1) The present invention screens for the first time a GRF8 gene that can regulate plant traits.
(2)本发明首次发现，提高GRF8基因或其编码蛋白的表达量和/或活性，可(i)增加粒长；和/或(ii)缩短粒宽；和/或(iii)增加千粒重；和/或(iv)株型松散；和/或(v)增加产量。(2) The present invention finds for the first time that increasing the expression and/or activity of the GRF8 gene or its encoded protein can (i) increase the grain length; and/or (ii) shorten the grain width; and/or (iii) increase the thousand-grain weight; And/or (iv) loose plant type; and/or (v) increased yield.
(3)本发明首次发现，GRF8基因是调控水稻粒长、粒宽和粒形的一个重要基因，具有增加产量的潜能。(3) The present invention finds for the first time that the GRF8 gene is an important gene regulating rice grain length, grain width and grain shape, and has the potential to increase yield.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor LaboratoryPress,1989)中所述的条件，或按照制造厂商所建议的条件。除非有特别说明，否则实施例中所用的材料和试剂均为市售产品。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions. Unless otherwise specified, the materials and reagents used in the examples are all commercially available products.
实施例1OsGRF8基因信息的初步分析及超表达转基因植株的获得Example 1 Preliminary analysis of OsGRF8 gene information and acquisition of overexpression transgenic plants
OsGRF基因是miR396的靶基因。为对此信息进行验证，我们通过实时定量PCR(real-time PCR)技术检测了MIM396转基因植株中的12个OsGRF基因的表达情况，结果显示MIM396材料中，12个靶基因的表达均有不同程度的明显下调(图1a)。在一定程度上说明这些OsGRF基因是miR396的靶基因，选取了其中的OsGRF8基因进行进一步的遗传功能研究。OsGRF gene is the target gene of miR396. In order to verify this information, we detected the expression of 12 OsGRF genes in MIM396 transgenic plants by real-time quantitative PCR (real-time PCR). significantly down-regulated (Figure 1a). To a certain extent, these OsGRF genes are the target genes of miR396, and the OsGRF8 gene is selected for further genetic function research.
研究表明，水稻OsGRF8基因(LOC_Os11g35030)具有两个转录本(图2)。分别扩增了这两个不同长度的转录本的cDNA，并克隆到超表达载体p130135SNOS中，由35S驱动表达。并分别将质粒通过农杆菌介导的方法遗传转化到野生型水稻品种中花11(ZH11)中。Studies have shown that the rice OsGRF8 gene (LOC_Os11g35030) has two transcripts (Fig. 2). The cDNAs of these two transcripts of different lengths were amplified separately and cloned into the overexpression vector p130135SNOS for expression driven by 35S. And the plasmids were genetically transformed into the wild-type rice variety Zhonghua 11 (ZH11) through the Agrobacterium-mediated method.
转基因植株的表型分析表明，较长的一个转录本和较短的转录本的超表达的转基因植株，均呈现出粒形变化，其中较长的转录本的粒形变化更显著。将较长的转录本的转基因植株命名为OsGRF8OE，随机选择4株呈现明显粒形变化的T0代单株，通过实时定量PCR(real-time PCR)技术检测其中OsGRF8基因的表达变化，结果显示其中OsGRF8基因均被不同程度明显超表达(图1b)。The phenotypic analysis of the transgenic plants showed that the transgenic plants overexpressing the longer transcript and the shorter transcript both showed changes in grain shape, and the longer transcript showed more significant changes in grain shape. The transgenic plants with longer transcripts were named OsGRF8OE, and 4 T0 generation single plants with obvious grain shape changes were randomly selected, and the expression changes of OsGRF8 gene were detected by real-time quantitative PCR (real-time PCR), and the results showed that OsGRF8 genes were significantly overexpressed to varying degrees (Figure 1b).
实施例2OsGRF8超表达转基因植株呈现出增大的粒形和增加的千粒重Example 2 OsGRF8 overexpression transgenic plants present increased grain shape and increased thousand-grain weight
OsGRF8OE转基因植株呈现出明显的粒形变化(图3a)，主要表现为粒长加长，粒宽变短(图3b)，因此，千粒重也明显增加(图3c)，千粒重的增加幅度在15-17.4％之间。OsGRF8OE transgenic plants showed obvious changes in grain shape (Fig. 3a), mainly manifested as longer grain length and shorter grain width (Fig. 3b). Therefore, the thousand-grain weight also increased significantly (Fig. 3c), and the increase range of thousand-grain weight was 15-17.4 %between.
实施例3OsGRF8超表达转基因植株的株型变得松散Example 3 The plant type of OsGRF8 overexpression transgenic plants becomes loose
除了粒形的表型，转基因植株OsGRF8OE还呈现出明显的株型变化。跟野生型ZH11相比较，OsGRF8OE呈现出明显的松散的株型(图4)。针对株型的具体分析以及其它的表型分子正在进行中。OsGRF8基因功能的多样性，可能与OsGRF类基因编码转录因子蛋白有关。In addition to the grain shape phenotype, the transgenic plant OsGRF8OE also showed obvious changes in plant shape. Compared with the wild-type ZH11, OsGRF8OE showed an obvious loose plant type (Fig. 4). Specific analyzes for strain types and other phenotypic molecules are in progress. The diversity of OsGRF8 gene functions may be related to the transcription factor proteins encoded by OsGRF-like genes.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
序列表 sequence listing
<110> 中国科学院上海生命科学研究院<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
<120> 一种GRF8基因及其应用<120> A kind of GRF8 gene and application thereof
<130> P2018-1311<130> P2018-1311
<160> 2<160> 2
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 1230<211> 1230
<212> DNA<212>DNA
<213> 水稻(Oryza sativa)<213> Rice (Oryza sativa)
<400> 1<400> 1
atgctgagct cttgtggtgg ccatggccat ggaaatccaa gaagcttgca agaagaacac 60atgctgagct cttgtggtgg ccatggccat ggaaatccaa gaagcttgca agaagaacac 60
catggcagat gtggtgagca gcaaggtgga ggaggaggag gagggcaaga gcaagagcaa 120catggcagat gtggtgagca gcaaggtgga ggaggaggag gagggcaaga gcaagagcaa 120
gatgggttct tggtgagaga ggcaagggca tccccaccat ctccatcttc ttcatcattt 180gatgggttct tggtgagaga ggcaagggca tccccaccat ctccatcttc ttcatcattt 180
cttggatcca caagctcttc ttgttctgga ggaggaggag gagggcagat gttgagcttc 240cttggatcca caagctcttc ttgttctgga ggaggaggag gagggcagat gttgagcttc 240
tcctccccca atggaacagc agggttgggc ttgagctcag gaggaagcat gcagggggtc 300tcctccccca atggaacagc agggttgggc ttgagctcag gaggaagcat gcagggggtc 300
ttggcaaggg tcagggggcc gttcacccca acacagtgga tggagctgga gcaccaggca 360ttggcaaggg tcagggggcc gttcacccca acacagtgga tggagctgga gcaccaggca 360
ctgatctaca agcacattgc tgcaaatgtt tctgtccctt ccagcttgct cctccccatc 420ctgatctaca agcacattgc tgcaaatgtt tctgtccctt ccagcttgct cctccccatc 420
aggagaagcc tccatccatg gggatgggga tcattccctc ctggctgtgc tgatgtagaa 480aggagaagcc tccatccatg gggatgggga tcattccctc ctggctgtgc tgatgtagaa 480
cccagaagat gccgccgcac agacggcaag aagtggcggt gctccagaga tgctgttggg 540cccagaagat gccgccgcac agacggcaag aagtggcggt gctccagaga tgctgttggg 540
gatcagaagt attgtgagcg acacataaac cgtggtcgcc atcgttcaag aaagcatgtg 600gatcagaagt attgtgagcg acacataaac cgtggtcgcc atcgttcaag aaagcatgtg 600
gaaggccgaa aggcgacact caccattgca gaaccatcca cggttattgc tgctggtgta 660gaaggccgaa aggcgacact caccattgca gaaccatcca cggttatgc tgctggtgta 660
tcatctcgcg gccacactgt ggctcggcag aagcaggtga aaggctcagc tgctactgtc 720tcatctcgcg gccaacactgt ggctcggcag aagcaggtga aaggctcagc tgctactgtc 720
tctgatcctt tctcgagaca atccaacagg aaatttctgg agaaacagaa cgttgtcgac 780tctgatcctt tctcgagaca atccaacagg aaatttctgg agaaacagaa cgttgtcgac 780
caattgtctc ccatggattc atttgatttc tcatccacac aatcttctcc aaactatgac 840caattgtctc ccatggattc atttgatttc tcatccaacac aatcttctcc aaactatgac 840
aatgtagcat tgtcaccact gaagttgcac catgatcatg atgaatctta catcgggcat 900aatgtagcat tgtcaccact gaagttgcac catgatcatg atgaatctta catcgggcat 900
ggagcaggca gttcatcaga aaaaggcagt atgatgtacg aaagtcggtt aacagtctct 960ggagcaggca gttcatcaga aaaaggcagt atgatgtacg aaagtcggtt aacagtctct 960
aaggaaacac ttgatgatgg acctttaggt gaagttttca aaagaaagaa ttgccaatca 1020aaggaaacac ttgatgatgg acctttaggt gaagttttca aaagaaagaa ttgccaatca 1020
gcttctacag aaatcttaac tgaaaaatgg actgagaacc ccaacttaca ttgcccatct 1080gcttctacag aaatcttaac tgaaaaatgg actgagaacc ccaacttaca ttgcccatct 1080
ggaatcctac aaatggctac taagttcaat tcaatttcca gcggcaacac agtaaatagt 1140ggaatcctac aaatggctac taagttcaat tcaatttcca gcggcaacac agtaaatagt 1140
ggtggcaccg cagtggagaa tcttatcact gataatggat atcttactgc aagaatgatg 1200ggtggcaccg cagtggagaa tcttatcact gataatggat atcttactgc aagaatgatg 1200
aatcctcata ttgtcccaac acttctctaa 1230aatcctcata ttgtcccaac acttctctaa 1230
<210> 2<210> 2
<211> 409<211> 409
<212> PRT<212> PRT
<213> 水稻(Oryza sativa)<213> Rice (Oryza sativa)
<400> 2<400> 2
Met Leu Ser Ser Cys Gly Gly His Gly His Gly Asn Pro Arg Ser LeuMet Leu Ser Ser Cys Gly Gly His Gly His Gly Asn Pro Arg Ser Leu
1 5 10 151 5 10 15
Gln Glu Glu His His Gly Arg Cys Gly Glu Gln Gln Gly Gly Gly GlyGln Glu Glu His His Gly Arg Cys Gly Glu Gln Gln Gly Gly Gly Gly Gly
20 25 30 20 25 30
Gly Gly Gly Gln Glu Gln Glu Gln Asp Gly Phe Leu Val Arg Glu AlaGly Gly Gly Gln Glu Gln Glu Gln Asp Gly Phe Leu Val Arg Glu Ala
35 40 45 35 40 45
Arg Ala Ser Pro Pro Ser Pro Ser Ser Ser Ser Phe Leu Gly Ser ThrArg Ala Ser Pro Pro Ser Pro Ser Ser Ser Ser Ser Phe Leu Gly Ser Thr
50 55 60 50 55 60
Ser Ser Ser Cys Ser Gly Gly Gly Gly Gly Gly Gln Met Leu Ser PheSer Ser Ser Cys Ser Gly Gly Gly Gly Gly Gly Gly Gln Met Leu Ser Phe
65 70 75 8065 70 75 80
Ser Ser Pro Asn Gly Thr Ala Gly Leu Gly Leu Ser Ser Gly Gly SerSer Ser Pro Asn Gly Thr Ala Gly Leu Gly Leu Ser Ser Gly Gly Ser
85 90 95 85 90 95
Met Gln Gly Val Leu Ala Arg Val Arg Gly Pro Phe Thr Pro Thr GlnMet Gln Gly Val Leu Ala Arg Val Arg Gly Pro Phe Thr Pro Thr Gln
100 105 110 100 105 110
Trp Met Glu Leu Glu His Gln Ala Leu Ile Tyr Lys His Ile Ala AlaTrp Met Glu Leu Glu His Gln Ala Leu Ile Tyr Lys His Ile Ala Ala
115 120 125 115 120 125
Asn Val Ser Val Pro Ser Ser Leu Leu Leu Pro Ile Arg Arg Ser LeuAsn Val Ser Val Pro Ser Ser Leu Leu Leu Pro Ile Arg Arg Ser Leu
130 135 140 130 135 140
His Pro Trp Gly Trp Gly Ser Phe Pro Pro Gly Cys Ala Asp Val GluHis Pro Trp Gly Trp Gly Ser Phe Pro Pro Gly Cys Ala Asp Val Glu
145 150 155 160145 150 155 160
Pro Arg Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg Cys Ser ArgPro Arg Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg Cys Ser Arg
165 170 175 165 170 175
Asp Ala Val Gly Asp Gln Lys Tyr Cys Glu Arg His Ile Asn Arg GlyAsp Ala Val Gly Asp Gln Lys Tyr Cys Glu Arg His Ile Asn Arg Gly
180 185 190 180 185 190
Arg His Arg Ser Arg Lys His Val Glu Gly Arg Lys Ala Thr Leu ThrArg His Arg Ser Arg Lys His Val Glu Gly Arg Lys Ala Thr Leu Thr
195 200 205 195 200 205
Ile Ala Glu Pro Ser Thr Val Ile Ala Ala Gly Val Ser Ser Arg GlyIle Ala Glu Pro Ser Thr Val Ile Ala Ala Gly Val Ser Ser Arg Gly
210 215 220 210 215 220
His Thr Val Ala Arg Gln Lys Gln Val Lys Gly Ser Ala Ala Thr ValHis Thr Val Ala Arg Gln Lys Gln Val Lys Gly Ser Ala Ala Thr Val
225 230 235 240225 230 235 240
Ser Asp Pro Phe Ser Arg Gln Ser Asn Arg Lys Phe Leu Glu Lys GlnSer Asp Pro Phe Ser Arg Gln Ser Asn Arg Lys Phe Leu Glu Lys Gln
245 250 255 245 250 255
Asn Val Val Asp Gln Leu Ser Pro Met Asp Ser Phe Asp Phe Ser SerAsn Val Val Asp Gln Leu Ser Pro Met Asp Ser Phe Asp Phe Ser Ser
260 265 270 260 265 270
Thr Gln Ser Ser Pro Asn Tyr Asp Asn Val Ala Leu Ser Pro Leu LysThr Gln Ser Ser Pro Asn Tyr Asp Asn Val Ala Leu Ser Pro Leu Lys
275 280 285 275 280 285
Leu His His Asp His Asp Glu Ser Tyr Ile Gly His Gly Ala Gly SerLeu His His Asp His Asp Glu Ser Tyr Ile Gly His Gly Ala Gly Ser
290 295 300 290 295 300
Ser Ser Glu Lys Gly Ser Met Met Tyr Glu Ser Arg Leu Thr Val SerSer Ser Glu Lys Gly Ser Met Met Tyr Glu Ser Arg Leu Thr Val Ser
305 310 315 320305 310 315 320
Lys Glu Thr Leu Asp Asp Gly Pro Leu Gly Glu Val Phe Lys Arg LysLys Glu Thr Leu Asp Asp Gly Pro Leu Gly Glu Val Phe Lys Arg Lys
325 330 335 325 330 335
Asn Cys Gln Ser Ala Ser Thr Glu Ile Leu Thr Glu Lys Trp Thr GluAsn Cys Gln Ser Ala Ser Thr Glu Ile Leu Thr Glu Lys Trp Thr Glu
340 345 350 340 345 350
Asn Pro Asn Leu His Cys Pro Ser Gly Ile Leu Gln Met Ala Thr LysAsn Pro Asn Leu His Cys Pro Ser Gly Ile Leu Gln Met Ala Thr Lys
355 360 365 355 360 365
Phe Asn Ser Ile Ser Ser Gly Asn Thr Val Asn Ser Gly Gly Thr AlaPhe Asn Ser Ile Ser Ser Ser Gly Asn Thr Val Asn Ser Gly Gly Thr Ala
370 375 380 370 375 380
Val Glu Asn Leu Ile Thr Asp Asn Gly Tyr Leu Thr Ala Arg Met MetVal Glu Asn Leu Ile Thr Asp Asn Gly Tyr Leu Thr Ala Arg Met Met
385 390 395 400385 390 395 400
Asn Pro His Ile Val Pro Thr Leu LeuAsn Pro His Ile Val Pro Thr Leu Leu
405 405
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811150632.0ACN110982825B (en) | 2018-09-29 | 2018-09-29 | A kind of GRF8 gene and its application |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811150632.0ACN110982825B (en) | 2018-09-29 | 2018-09-29 | A kind of GRF8 gene and its application |
| Publication Number | Publication Date |
|---|---|
| CN110982825A CN110982825A (en) | 2020-04-10 |
| CN110982825Btrue CN110982825B (en) | 2023-07-18 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811150632.0AActiveCN110982825B (en) | 2018-09-29 | 2018-09-29 | A kind of GRF8 gene and its application |
| Country | Link |
|---|---|
| CN (1) | CN110982825B (en) |
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| CN101855355A (en)* | 2007-09-14 | 2010-10-06 | 巴斯夫植物科学有限公司 | Plants having increased yield-related traits and a method for making the same |
| CN103388004A (en)* | 2012-05-08 | 2013-11-13 | 中国科学院植物研究所 | Application of OsGRF6 protein in regulation of plant height |
| CN104341509A (en)* | 2013-07-31 | 2015-02-11 | 中国农业科学院作物科学研究所 | Application of oryza sativa transcription factor Os11g35030.1 gene sequence |
| CN107253980A (en)* | 2017-07-24 | 2017-10-17 | 武汉大学 | Application of the OsGRF7 genes in plant type of rice regulation and control |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101855355A (en)* | 2007-09-14 | 2010-10-06 | 巴斯夫植物科学有限公司 | Plants having increased yield-related traits and a method for making the same |
| CN103388004A (en)* | 2012-05-08 | 2013-11-13 | 中国科学院植物研究所 | Application of OsGRF6 protein in regulation of plant height |
| CN104341509A (en)* | 2013-07-31 | 2015-02-11 | 中国农业科学院作物科学研究所 | Application of oryza sativa transcription factor Os11g35030.1 gene sequence |
| CN107253980A (en)* | 2017-07-24 | 2017-10-17 | 武汉大学 | Application of the OsGRF7 genes in plant type of rice regulation and control |
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| Molecular Mechanism of microRNA396 Mediating Pistil Development in Arabidopsis;Liang et al.;《Plant Physiology》;20131127;第164卷;第249-258页* |
| Phosphate diVerentially regulates 14-3-3 family members and GRF9 plays a role in Pi-starvation induced responses;Aiqin Cao et al.;《Planta》;20070628;第226卷;第1219-1230页* |
| PREDICTED: Oryza sativa Japonica Group growth-regulating factor 8-like (LOC4350711)，mRNA，NCBI Reference Sequence: XM_015760217.2;genbank;《GenBank》;20180807;第1-2页* |
| 热胁迫下水稻miR396家族及靶基因OsGRFs的表达研究;叶超楠等;《农业生物技术学报》;20180331;第26卷(第3期);第393-400页* |
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| CN110982825A (en) | 2020-04-10 |
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