CN110804623A

Movatterモバイル変換

Info

Publication number: CN110804623A
Application number: CN201911192165.2A
Authority: CN
Inventors: 李爱丽; 孔星辰; 陶姝; 邓中印; 耿帅锋; 毛龙
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2020-02-18

Abstract

Translated fromChinese

本发明涉及植物分子生物学技术领域，具体涉及小麦TaMADS6基因在调控植物穗和籽粒发育以及开花时间中的应用。本发明发现TaMADS6蛋白具有调控植物开花、穗和籽粒发育的功能，提高植物中TaMADS6蛋白的表达量，植物的小穗数、小穗轴数、穗籽粒数显著提高，开花时间明显延迟。TaMADS6具有较高的应用价值，可应用于小麦遗传育种和种质资源改良，为培育高产植物提供了基因资源和新思路，对农业生产具有重要意义。The invention relates to the technical field of plant molecular biology, in particular to the application of wheat TaMADS6 gene in regulating the development of plant ear and grain and flowering time. The invention finds that the TaMADS6 protein has the function of regulating plant flowering, panicle and grain development, increases the expression level of the TaMADS6 protein in the plant, significantly increases the number of spikelets, rachis and grains per ear, and significantly delays the flowering time. TaMADS6 has high application value and can be used in wheat genetic breeding and germplasm resource improvement, providing genetic resources and new ideas for cultivating high-yielding plants, and is of great significance to agricultural production.

Description

Translated fromChinese

小麦TaMADS6基因在调控植物穗和籽粒发育以及开花时间中的应用The wheat TaMADS6 gene regulates panicle and grain development and flowering timeApplications

技术领域technical field

本发明涉及植物分子生物学技术领域，具体涉及小麦TaMADS6基因在调控植物穗和籽粒发育以及开花时间中的应用。The invention relates to the technical field of plant molecular biology, in particular to the application of wheat TaMADS6 gene in regulating the development of plant ear and grain and flowering time.

背景技术Background technique

小麦作为一种重要的粮食作物，全世界约有40％的人以小麦为主要粮食。同时小麦作为三大谷物之一，产量几乎全做食用，是世界上总产量仅次于玉米的粮食作物。随着消费水平的提高，对小麦的产量和品质也提出了更高的要求。小麦产量构成的三要素为单位面积穗数、穗粒数和粒重，三者关系的相互协调，在提高小麦产量方面具有重要的意义。其中，穗粒数取决于小穗数和小花的发育，因此对小麦穗早期的发育的研究具有重要的意义。As an important food crop, wheat is the main food for about 40% of the world's population. At the same time, as one of the three major grains, wheat is almost entirely edible, and it is the world's largest grain crop after corn. With the increase of consumption level, higher requirements are also put forward for the yield and quality of wheat. The three elements of wheat yield are the number of panicles per unit area, the number of grains per panicle and the grain weight. The coordination of the three relationships is of great significance in improving wheat yield. Among them, the number of grains per ear depends on the number of spikelets and the development of florets, so it is of great significance to study the early development of wheat ears.

MADS-box基因家族是在进化上十分保守，在植物生长发育过程中起重要作用的一类基因。植物MADS-box家族主要包括I型和II型两种类型，其中，I型MADS-box蛋白根据MADS-box功能域的保守关系又可以分为Mα、Mβ和Mγ三类。目前为止，I型MADS-box基因的研究相对较少，它们的功能主要集中在雌配子体、胚和胚乳的发育。目前研究最多的是II型MADS-box蛋白，II型MADS-box蛋白又可以分为MIKCC型和MIKC*型。II型MADS-box基因有四个功能域：MADS(M)结构域、Intervening(I)结构域、Coiled-coil Keratin-like(K)结构域和C-terminus(C)结构域。其中，M结构域最保守，具有结合CArG-box并调控下游基因的作用。I结构域保守性较弱，能促进蛋白二聚体的形成和蛋白二聚体与DNA的结合。K结构域的保守性仅次于M结构域，蛋白间的互作主要通过K结构域，K结构域又可分为K1、K2和K3结构域，其中K3结构域是最重要的。C结构域保守性最弱，是MADS-box基因的转录激活区域。然而，目前为止，TaMADS6在小麦中的功能研究和生产应用研究却少有报道。The MADS-box gene family is a class of genes that are evolutionarily conserved and play an important role in plant growth and development. The plant MADS-box family mainly includes two types: type I and type II. Among them, type I MADS-box proteins can be divided into three types: Mα, Mβ and Mγ according to the conserved relationship of MADS-box functional domains. So far, relatively few type I MADS-box genes have been studied, and their functions are mainly focused on the development of the female gametophyte, embryo and endosperm. At present, the type II MADS-box protein is the most studied, and the type II MADS-box protein can be divided into MIKCC type and MIKC* type. Type II MADS-box gene has four functional domains: MADS (M) domain, Intervening (I) domain, Coiled-coil Keratin-like (K) domain and C-terminus (C) domain. Among them, the M domain is the most conserved and has the function of binding CArG-box and regulating downstream genes. The I domain is less conserved and can promote the formation of protein dimers and the binding of protein dimers to DNA. The conservation of the K domain is second only to the M domain. The interaction between proteins is mainly through the K domain. The K domain can be divided into K1, K2 and K3 domains, of which the K3 domain is the most important. The C domain is the least conserved and is the transcriptional activation region of the MADS-box gene. However, so far, the functional studies and production application studies of TaMADS6 in wheat are rarely reported.

发明内容SUMMARY OF THE INVENTION

为解决现有技术中存在的技术问题，本发明的目的在于提供小麦TaMADS6基因在调控植物穗和籽粒发育以及开花时间中的应用。In order to solve the technical problems existing in the prior art, the purpose of the present invention is to provide the application of the wheat TaMADS6 gene in the regulation of plant ear and grain development and flowering time.

本发明发现小麦TaMADS6(氨基酸序列分别如SEQ ID NO.1-3所示，CDS序列分别如SEQ ID NO.4-6所示)与植物的花、穗和籽粒的发育相关，提高植物中TaMADS6的表达量，能够延迟小麦的开花时间，显著提高小麦的小穗数和籽粒数。The present invention finds that wheat TaMADS6 (the amino acid sequence is shown as SEQ ID NO.1-3 respectively, and the CDS sequence is shown as SEQ ID NO.4-6 respectively) is related to the development of flowers, ears and grains of plants, and increases TaMADS6 in plants. The expression level of wheat can delay the flowering time of wheat and significantly increase the number of spikelets and grains of wheat.

具体地，本发明的技术方案如下：Specifically, the technical scheme of the present invention is as follows:

第一方面，本发明提供TaMADS6蛋白或其编码基因或含有TaMADS6蛋白的编码基因的生物材料在调控植物穗发育中的应用。In a first aspect, the present invention provides the application of TaMADS6 protein or its encoding gene or biological material containing TaMADS6 protein encoding gene in regulating plant ear development.

第二方面，本发明提供TaMADS6蛋白或其编码基因或含有TaMADS6蛋白的编码基因的生物材料在调控植物籽粒发育中的应用。In a second aspect, the present invention provides the application of TaMADS6 protein or its encoding gene or biological material containing TaMADS6 protein encoding gene in regulating the development of plant grains.

第三方面，本发明提供TaMADS6蛋白或其编码基因或含有TaMADS6蛋白的编码基因的生物材料在调控植物开花时间中的应用。In a third aspect, the present invention provides the application of TaMADS6 protein or its encoding gene or biological material containing TaMADS6 protein encoding gene in regulating flowering time of plants.

第四方面，本发明提供TaMADS6蛋白或其编码基因或含有TaMADS6蛋白的编码基因的生物材料在植物遗传育种中的应用。In a fourth aspect, the present invention provides the application of TaMADS6 protein or its encoding gene or biological material containing TaMADS6 protein encoding gene in plant genetics and breeding.

第五方面，本发明提供TaMADS6蛋白或其编码基因或含有TaMADS6蛋白的编码基因的生物材料在植物种质资源改良中的应用In a fifth aspect, the present invention provides the application of TaMADS6 protein or its encoding gene or biological material containing the encoding gene of TaMADS6 protein in the improvement of plant germplasm resources

具体地，上述应用中，通过提高所述植物中TaMADS6蛋白的表达量和/或活性，提高植物的小穗数、小穗轴数或穗粒数，或者延迟植物的开花时间。Specifically, in the above application, by increasing the expression and/or activity of TaMADS6 protein in the plant, the number of spikelets, rachis or grains per ear of the plant is increased, or the flowering time of the plant is delayed.

本发明中，所述TaMADS6蛋白具有如下任一种氨基酸序列：In the present invention, the TaMADS6 protein has any of the following amino acid sequences:

(1)如SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示的氨基酸序列；(1) an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3;

(2)如SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列；(2) The amino acid sequence of a protein with the same function obtained by the substitution, insertion or deletion of one or more amino acids in the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3;

(3)在如SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示的氨基酸序列的N端和/或C端连接标签得到的融合蛋白；(3) a fusion protein obtained by linking a tag to the N-terminal and/or C-terminal of the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3;

(4)与如SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示的氨基酸序列具有至少90％同源性的氨基酸序列；优选地，所述同源性为至少95％；更优选为99％。(4) An amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; preferably, the homology is at least 95% ; more preferably 99%.

上述如SEQ ID NO.1所示的氨基酸序列为位于小麦6A染色体上的小麦TaMADS6蛋白(TaMADS6-A)的氨基酸序列，SEQ ID NO.2所示的氨基酸序列为位于小麦6B染色体上的小麦TaMADS6蛋白(TaMADS6-B)的氨基酸序列，SEQ ID NO.3所示的氨基酸序列为位于小麦6D染色体上的小麦TaMADS6蛋白(TaMADS6-D)的氨基酸序列，本领域技术人员可根据小麦TaMADS6蛋白的氨基酸序列以及氨基酸的保守性替换等本领域常规技术手段，在不影响其活性的前提下，取代、缺失和/或增加一个或几个氨基酸，得到与小麦TaMADS6蛋白具有相同功能的TaMADS6蛋白突变体。The above-mentioned amino acid sequence shown in SEQ ID NO.1 is the amino acid sequence of the wheat TaMADS6 protein (TaMADS6-A) located on the wheat 6A chromosome, and the amino acid sequence shown in SEQ ID NO.2 is the wheat TaMADS6 located on the wheat 6B chromosome The amino acid sequence of protein (TaMADS6-B), the amino acid sequence shown in SEQ ID NO.3 is the amino acid sequence of the wheat TaMADS6 protein (TaMADS6-D) located on the wheat 6D chromosome, those skilled in the art can Conventional technical means in the field such as sequence and conservative substitution of amino acids, without affecting its activity, replace, delete and/or add one or several amino acids to obtain TaMADS6 protein mutants with the same function as wheat TaMADS6 protein.

上述在N端和/或C端连接标签中，所述标签包括但不限于MYC标签、Flag标签、HA标签、His标签等。In the above-mentioned N-terminal and/or C-terminal linking tags, the tags include but are not limited to MYC tags, Flag tags, HA tags, His tags and the like.

本发明中，所述TaMADS6蛋白的CDS序列具有如下任一种核苷酸序列：In the present invention, the CDS sequence of the TaMADS6 protein has any of the following nucleotide sequences:

(1)如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸或其互补序列；(1) Nucleotides as shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 or their complementary sequences;

(2)如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列经一个或多个核苷酸的替换、插入或缺失得到的具有相同功能蛋白的核苷酸序列。(2) A nucleus with the same functional protein obtained by the substitution, insertion or deletion of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 nucleotide sequence.

上述如SEQ ID NO.4所示的核苷酸序列为位于小麦6A染色体上的TaMADS6蛋白的CDS序列，SEQ ID NO.5所示的核苷酸序列为位于小麦6B染色体上的TaMADS6蛋白的CDS序列，SEQ ID NO.6所示的核苷酸序列为位于小麦6D染色体上的TaMADS6蛋白的CDS序列。考虑到密码子的简并性，所有编码TaMADS6蛋白的核苷酸序列均在本发明的保护范围内。The above-mentioned nucleotide sequence shown in SEQ ID NO.4 is the CDS sequence of the TaMADS6 protein located on the wheat chromosome 6A, and the nucleotide sequence shown in SEQ ID NO.5 is the CDS of the TaMADS6 protein located on the wheat chromosome 6B Sequence, the nucleotide sequence shown in SEQ ID NO. 6 is the CDS sequence of TaMADS6 protein located on the wheat 6D chromosome. Considering the degeneracy of codons, all nucleotide sequences encoding TaMADS6 proteins are within the scope of the present invention.

本发明中，所述生物材料包括表达盒、载体、宿主细胞、工程菌、转基因植物细胞系、转基因植物组织、转基因植物器官、转基因植株、所述转基因植株的可再生细胞产生的组织培养物或所述组织培养物产生的原生质体。In the present invention, the biological material includes expression cassettes, vectors, host cells, engineered bacteria, transgenic plant cell lines, transgenic plant tissues, transgenic plant organs, transgenic plants, tissue cultures produced from regenerable cells of the transgenic plants, or Protoplasts produced by the tissue culture.

第六方面，本发明提供一种调控植物穗、籽粒发育或开花时间的方法，包括：调控所述植物中TaMADS6蛋白的表达量和/或活性；所述TaMADS6蛋白具有如下任一种氨基酸序列：In the sixth aspect, the present invention provides a method for regulating the development of plant ears, grains or flowering time, comprising: regulating the expression and/or activity of TaMADS6 protein in the plant; the TaMADS6 protein has any of the following amino acid sequences:

优选地，上述方法中，通过提高所述植物中所述TaMADS6蛋白的表达量和/或活性，提高植物的小穗数、小穗轴数或穗粒数，或者延迟所述植物的开花时间。Preferably, in the above method, by increasing the expression and/or activity of the TaMADS6 protein in the plant, the number of spikelets, rachis or grains per ear of the plant is increased, or the flowering time of the plant is delayed.

所述提高植物中所述TaMADS6蛋白的表达量可通过本领域常规技术手段实现，例如：在所述植物中导入携带所述TaMADS6蛋白的编码基因的表达载体。The increase in the expression level of the TaMADS6 protein in the plant can be achieved by conventional technical means in the art, for example, introducing an expression vector carrying the gene encoding the TaMADS6 protein into the plant.

所述携带TaMADS6蛋白的编码基因的表达载体可为携带TaMADS6蛋白的编码基因的pCAMBIA3300-Ubi-MCS载体(pCAMBIA3300-Ubi-MCS载体为以pCAMBIA3300载体为骨架，在其多克隆位点前加入玉米的Ubiquition启动子，抗除草剂Bar基因由35S启动子驱动)。The expression vector carrying the encoding gene of the TaMADS6 protein can be the pCAMBIA3300-Ubi-MCS vector carrying the encoding gene of the TaMADS6 protein (the pCAMBIA3300-Ubi-MCS vector is a skeleton with the pCAMBIA3300 vector added before its multi-cloning site. Ubiquition promoter, herbicide resistance Bar gene is driven by 35S promoter).

本发明中，所述植物为单子叶植物或双子叶植物。所述植物包括但不限于小麦、拟南芥、水稻、玉米等。In the present invention, the plant is a monocotyledonous plant or a dicotyledonous plant. The plants include, but are not limited to, wheat, Arabidopsis, rice, corn, and the like.

第七方面，本发明提供TaMADS6基因的特异性扩增引物，其序列如SEQ ID NO.7-8所示，或如SEQ ID NO.9-10所示。In a seventh aspect, the present invention provides specific amplification primers for the TaMADS6 gene, the sequences of which are shown in SEQ ID NO. 7-8, or as shown in SEQ ID NO. 9-10.

第八方面，本发明提供TaMADS6基因的检测引物，其序列如SEQ ID NO.12-13所示。如SEQ ID NO.12-13所示的引物可用于TaMADS6基因过表达转基因植株的鉴定。In an eighth aspect, the present invention provides detection primers for the TaMADS6 gene, the sequences of which are shown in SEQ ID NO. 12-13. The primers shown in SEQ ID NO. 12-13 can be used for the identification of TaMADS6 gene overexpressing transgenic plants.

本发明的有益效果在于：The beneficial effects of the present invention are:

本发明发现TaMADS6蛋白具有调控植物开花、穗和籽粒发育的功能，提高植物中TaMADS6蛋白的表达量，植物的小穗数、小穗轴数和穗籽粒数显著提高，开花时间明显延迟。本发明通过在小麦中过表达TaMADS6构建得到的3个转基因植株的开花时间分别推迟21天、16天和4天，小穗数分别提高27.72％、20.09％和8.81％，每穗籽粒数分别提高31.03％、26.47％和17.75％。本发明发现的TaMADS6的新功能具有较高的应用价值，为培育高产植物提供了基因资源和新思路，TaMADS6可应用于小麦遗传育种和种质资源改良，对农业生产具有重要意义。The present invention finds that the TaMADS6 protein has the functions of regulating plant flowering, ear and grain development, increases the expression of the TaMADS6 protein in the plant, significantly increases the number of spikelets, rachis and grains per ear, and significantly delays the flowering time. In the present invention, the flowering time of the three transgenic plants constructed by overexpressing TaMADS6 in wheat is respectively delayed by 21 days, 16 days and 4 days, the number of spikelets is increased by 27.72%, 20.09% and 8.81% respectively, and the number of grains per panicle is increased respectively 31.03%, 26.47% and 17.75%. The new function of TaMADS6 discovered in the invention has high application value, and provides genetic resources and new ideas for cultivating high-yield plants. TaMADS6 can be applied to wheat genetic breeding and germplasm resource improvement, and has great significance to agricultural production.

附图说明Description of drawings

图1为本发明实施例1中pAHC25中间载体的质粒图谱。Figure 1 is the plasmid map of the pAHC25 intermediate vector in Example 1 of the present invention.

图2为本发明实施例2中pCAMBIA3300-Ubi-MCS过表达载体的质粒图谱。Figure 2 is the plasmid map of the pCAMBIA3300-Ubi-MCS overexpression vector in Example 2 of the present invention.

图3为本发明实施例3中TaMADS6-D转化Fielder的T3代阳性植株的鉴定；其中，M代表Marker，WT代表野生型Fielder，1-10代表不同的T3代植株，所用模板为三叶期幼苗的DNA，扩增产物为约700bp的条带的转基因植株是阳性植株。Figure 3 is the identification of T3 generation positive plants transformed by TaMADS6-D into Fielder in Example 3 of the present invention; wherein, M represents Marker, WT represents wild-type Fielder, 1-10 represent different T3 generation plants, and the template used is the three-leaf stage Seedling DNA, transgenic plants whose amplified product is a band of about 700 bp are positive plants.

图4为本发明实施例3中TaMADS6-D过表达转基因植株中TaMADS6-D表达量检测结果；其中，Fielder代表野生型，OE-L1、OE-L22和OE-L6代表TaMADS6-D过表达小麦T3代植株；TaMADS6代表TaMADS6-D基因，TaTubulin代表内参基因。Figure 4 is the detection result of TaMADS6-D expression in TaMADS6-D overexpressed transgenic plants in Example 3 of the present invention; wherein, Fielder represents wild type, OE-L1, OE-L22 and OE-L6 represent TaMADS6-D overexpressed wheat T3 generation plants; TaMADS6 represents the TaMADS6-D gene, and TaTubulin represents the reference gene.

图5为本发明实施例3中Fielder和TaMADS6-D过表达转基因植株的开花表型及其统计结果；其中，A为生长表型；B为开花时间统计结果；C为抽穗期野生型和过表达植株中TaVRN3基因的表达量检测；Fielder表示野生型，OE-L1、OE-L22和OE-L6表示TaMADS6-D过表达小麦T3代植株；*代表差异显著，**代表差异极显著。Figure 5 is the flowering phenotype and statistical results of Fielder and TaMADS6-D overexpressed transgenic plants in Example 3 of the present invention; wherein, A is the growth phenotype; B is the statistical result of flowering time; The expression level of TaVRN3 gene in the expressing plants was detected; Fielder means wild type, OE-L1, OE-L22 and OE-L6 means TaMADS6-D overexpressing wheat T3 generation plants; * means significant difference, ** means extremely significant difference.

图6为本发明实施例3中Fielder和TaMADS6-D过表达转基因植株的小穗数及每穗籽粒表型结果，其中，A为小穗数表型；B为每穗小穗数统计结果，C为每穗小穗轴数统计结果；D为每穗籽粒数表型；E为每穗籽粒数统计结果；Fielder表示野生型，OE-L1、OE-L22和OE-L6表示TaMADS6-D过表达小麦T3代植株；*代表差异显著，**代表差异极显。Fig. 6 is the number of spikelets and the phenotype results of grains per spike of Fielder and TaMADS6-D overexpressed transgenic plants in Example 3 of the present invention, wherein, A is the number of spikelets phenotype; B is the statistical result of the number of spikelets per spike, C is the statistical result of the number of twigs per ear; D is the phenotype of the number of grains per ear; E is the statistical result of the number of grains per ear; Fielder means wild type, OE-L1, OE-L22 and OE-L6 means TaMADS6-D Expression of wheat T3 generation plants; * means significant difference, ** means extremely significant difference.

具体实施方式Detailed ways

下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are given for illustrative purposes only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the spirit and spirit of the present invention.

下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.

下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

实施例1 pAHC25-6*MYC-TaMADS6-D中间载体的构建Example 1 Construction of pAHC25-6*MYC-TaMADS6-D intermediate vector

为构建小麦TaMADS6-D基因的过表达载体，首先将TaMADS6-D基因连接于pAHC25-6*MYC载体(pAHC25-6*MYC是通过pAHC25改造的载体，将pAHC25中的GUS序列替换为6*MYC序列，pAHC25的质粒图谱如图1所示)，构建中间载体pAHC25-6*MYC-TaMADS6-D，具体方法如下：In order to construct the overexpression vector of the wheat TaMADS6-D gene, the TaMADS6-D gene was first connected to the pAHC25-6*MYC vector (pAHC25-6*MYC is a vector transformed by pAHC25, and the GUS sequence in pAHC25 was replaced with 6*MYC. sequence, the plasmid map of pAHC25 is shown in Figure 1), and the intermediate vector pAHC25-6*MYC-TaMADS6-D was constructed. The specific method is as follows:

(1)使用序列如SEQ ID NO.7和SEQ ID NO.8所示的引物以小麦组织的cDNA为模板扩增带有Spe I和Sac I酶切位点的TaMADS6-D片段，使用胶回收试剂盒回收目的片段；(1) Use the primers shown in SEQ ID NO.7 and SEQ ID NO.8 to amplify the TaMADS6-D fragment with Spe I and Sac I restriction sites using the cDNA of wheat tissue as a template, and use gel to recover The kit recovers the target fragment;

(2)双酶切pAHC25-6*MYC和步骤(1)中的纯化产物，使用胶回收试剂盒回收线性化后的载体和目的片段；(2) Double enzyme digestion of pAHC25-6*MYC and the purified product in step (1), and use a gel recovery kit to recover the linearized vector and the target fragment;

(3)连接：反应体系为：目的片段2μL，线性化载体2μL，T4连接酶1μL，T4连接Buffer1μL，用ddH₂O补足至10μL，16℃过夜连接；(3) Ligation: The reaction system is: 2 μL of target fragment, 2 μL of linearized vector, 1 μL of T4 ligase, 1 μL of T4 ligation buffer, supplemented with ddH₂ O to 10 μL, and ligated at 16°C overnight;

(4)连接产物转化至宿主菌，挑选阳性克隆测序，将测序正确的载体命名为pAHC25-6*MYC-TaMADS6-D。(4) The ligation product was transformed into host bacteria, positive clones were selected for sequencing, and the sequenced vector was named pAHC25-6*MYC-TaMADS6-D.

实施例2 pCAMBIA3300-Ubi-MCS-6*MYC-TaMADS6-D过表达载体的构建Example 2 Construction of pCAMBIA3300-Ubi-MCS-6*MYC-TaMADS6-D overexpression vector

在小麦中过表达TaMADS6-D基因使用的过表达载体为pCAMBIA3300-UbiMCS(以pCAMBIA3300载体为骨架进行改造，在多克隆位点前加入玉米的Ubiquition启动子，抗除草剂Bar基因由35s启动子驱动，图谱如图2所示)。The overexpression vector used to overexpress the TaMADS6-D gene in wheat is pCAMBIA3300-UbiMCS (modified with the pCAMBIA3300 vector as the backbone, and the maize Ubiquition promoter is added before the multiple cloning site, and the herbicide-resistant Bar gene is driven by the 35s promoter. , the spectrum is shown in Figure 2).

使用序列如SEQ ID NO.9和SEQ ID NO.10所示的引物(如SEQ ID NO.9所示的引物带有BamH I酶切位点，如SEQ ID NO.10所示的引物带有Sma I酶切位点；)从实施例1构建的pAHC25-6*MYC-TaMADS6-D中间载体中扩增带有6*MYC和TaMADS6-D基因片段的融合基因片段,即6*MYC-TaMADS6-D。Use primers whose sequences are shown in SEQ ID NO.9 and SEQ ID NO.10 (the primer shown in SEQ ID NO.9 has a BamH I restriction site, and the primer shown in SEQ ID NO.10 has a Sma I restriction site;) Amplify a fusion gene fragment with 6*MYC and TaMADS6-D gene fragments from the pAHC25-6*MYC-TaMADS6-D intermediate vector constructed in Example 1, namely 6*MYC-TaMADS6 -D.

酶切、连接、转化及阳性克隆测序同实施例1的步骤(2)～(4)，测序引物为SEQ IDNO.11和SEQ ID NO.12，测序正确的即构建成pCAMBIA3300-Ubi-MCS-6*MYC-TaMADS6-D，此过表达载体的TaMADS6-D基因前面带有6*MYC标签。Enzyme digestion, ligation, transformation and sequencing of positive clones are the same as steps (2) to (4) of Example 1, and the sequencing primers are SEQ ID NO.11 and SEQ ID NO.12. 6*MYC-TaMADS6-D, the TaMADS6-D gene of this overexpression vector is preceded by a 6*MYC tag.

实施例3 过表达TaMADS6-D基因的转基因小麦的构建Example 3 Construction of transgenic wheat overexpressing TaMADS6-D gene

1、TaMADS6-D基因过表达载体转化农杆菌1. Transformation of Agrobacterium with TaMADS6-D gene overexpression vector

(1)从-80℃冰箱取出农杆菌感受态C58C1于冰上融化；(1) Take out Agrobacterium-competent C58C1 from the -80°C refrigerator and thaw on ice;

(2)每管感受态均分两管，加入2μL pCAMBIA3300-Ubi-MCS-6*MYC-TaMADS6-D质粒，轻弹混匀，冰上放置30min；(2) Divide each tube of competence into two tubes, add 2 μL of pCAMBIA3300-Ubi-MCS-6*MYC-TaMADS6-D plasmid, flick and mix, and place on ice for 30 minutes;

(3)液氮5min；(3) Liquid nitrogen 5min;

(4)37℃水浴5min；(4) 37℃ water bath for 5min;

(5)冰上5min；(5) 5min on ice;

(6)在超净台里，每管加400μL无抗LB培养基，轻弹混匀，28℃，200r/min，震荡培养3-4h；(6) In the ultra-clean bench, add 400 μL of anti-LB medium to each tube, flick and mix, 28 ° C, 200 r/min, and shake for 3-4 hours;

(7)菌液涂于含Rif和Kan固体平板上，封口膜封好，28℃培养箱中倒置培养2d；(7) The bacterial liquid was coated on the solid plate containing Rif and Kan, sealed with parafilm, and cultured upside down in a 28°C incubator for 2 days;

(8)挑取单克隆于LB培养基(含Rif和载体抗性抗生素)中，28℃震荡培养12-16h，PCR检测筛选阳性克隆；(8) Picking single clones in LB medium (containing Rif and carrier-resistant antibiotics), cultivating with shaking at 28°C for 12-16 hours, and screening positive clones by PCR detection;

(9)加甘油后-80℃冰箱保菌。(9) After adding glycerol, keep the bacteria in the refrigerator at -80°C.

2、农杆菌转化小麦2. Agrobacterium transformation of wheat

选取温室内开花后14-20天的籽粒，提前查看幼胚的状态，选取生长状态良好的穗子取材料。取籽粒放入干净的三角瓶中用70％乙醇洗1min，然后用15％次氯酸钠清洗15min，中间每隔1min晃一下，最后用无菌水清洗4-5次，每次1min。参照日本烟草公司的技术进行农杆菌转化小麦。当生根培养基中的小麦幼苗长到8-10cm高时即可以移栽幼苗。洗净幼苗根部的培养基，将其转移到网室栽培。Select the seeds 14-20 days after flowering in the greenhouse, check the status of the young embryos in advance, and select the ears with good growth conditions to take materials. Take the seeds and put them into a clean conical flask, wash with 70% ethanol for 1min, then wash with 15% sodium hypochlorite for 15min, shake every 1min in the middle, and finally wash with sterile water 4-5 times for 1min each time. Agrobacterium-transformation of wheat was carried out with reference to the technique of Japan Tobacco Company. When the wheat seedlings in the rooting medium grow to a height of 8-10cm, the seedlings can be transplanted. The medium at the roots of the seedlings was washed and transferred to a net room for cultivation.

采用序列如SEQ ID NO.13和SEQ ID NO.12所示的引物对TaMADS6-D转化FielderT3代植株进行阳性植株鉴定，结果如图3所示，其中扩增产物为约700bp条带的植株代表阳性植株。The primers whose sequences are shown in SEQ ID NO.13 and SEQ ID NO.12 were used to identify the positive plants of TaMADS6-D transformed FielderT3 generation plants. The results are shown in Figure 3, wherein the amplified products are representative of plants with a band of about 700bp. positive plants.

选取上述鉴定正确的转基因植株OE-L1、OE-L22和OE-L6进行后续的基因表达和植株表型分析。取抽穗期叶子进行半定量检测，以小麦Tubulin作为内参基因。野生型Fielder和转基因植株中TaMADS6-D基因表达量的检测结果如图4所示，结果表明，TaMADS6-D在转基因植株中的表达量远高于野生型植株的表达量，说明TaMADS6-D已在转基因植株中过表达。The above-identified transgenic plants OE-L1, OE-L22 and OE-L6 were selected for subsequent gene expression and plant phenotype analysis. The leaves at heading stage were used for semi-quantitative detection, and wheat Tubulin was used as the internal reference gene. The detection results of TaMADS6-D gene expression in wild-type Fielder and transgenic plants are shown in Figure 4. The results show that the expression level of TaMADS6-D in transgenic plants is much higher than that in wild-type plants, indicating that TaMADS6-D has been Overexpressed in transgenic plants.

对TaMADS6-D过表达的转基因植株的表型进行分析，开花表型如图5所示，结果显示，TaMADS6-D过表达能使Fielder表现出不同程度的生长迟缓的表型(图5的A)，最后开花期也晚于野生型，OE-L1相比野生型开花推迟约21天，OE-L22开花推迟约16天，OE-L6推迟开花约4天(图5的B)。以序列如SEQ ID NO.14和SEQ ID NO.15所示的引物检测抽穗期野生型和过表达植株中TaVRN3的表达，发现转基因植株中TaVRN3的表达呈现不同程度的降低(图5的C)，表明转基因植株开花推迟。The phenotype of TaMADS6-D overexpressed transgenic plants was analyzed, and the flowering phenotype was shown in Figure 5. The results showed that TaMADS6-D overexpression could make Fielder show different degrees of growth retardation phenotype (A of Figure 5). ), the last flowering period was also later than that of the wild type. Compared with the wild type, the flowering of OE-L1 was delayed by about 21 days, the flowering of OE-L22 was delayed by about 16 days, and the flowering of OE-L6 was delayed by about 4 days (Fig. 5B). The expression of TaVRN3 in the wild-type and overexpressed plants at the heading stage was detected with primers whose sequences are shown in SEQ ID NO.14 and SEQ ID NO.15, and it was found that the expression of TaVRN3 in the transgenic plants was reduced to varying degrees (C of Figure 5 ). , indicating that the flowering of transgenic plants was delayed.

穗和籽粒发育的表型结果如图6所示，结果显示，TaMADS6-D过表达植株中小穗数明显增多(图6的A和B)，与野生型相比，OE-L1、OE-L22和OE-L6的小穗数分别增加27.72％、20.09％和8.81％；且小穗数增多主要是小穗轴数增多导致的(图6的C)。进一步对籽粒数统计发现每穗的粒数明显增多(图6的D和E)，与野生型相比，OE-L1、OE-L22和OE-L6的每穗的粒数分别增加31.03％、26.47％和17.75％。The phenotypic results of panicle and grain development are shown in Figure 6. The results show that the number of spikelets in TaMADS6-D overexpressing plants is significantly increased (A and B in Figure 6). Compared with wild type, OE-L1, OE-L22 and OE-L6 spikelet numbers increased by 27.72%, 20.09% and 8.81%, respectively; and the increase in spikelet number was mainly caused by the increase in the number of spikelets (C in Figure 6). Further statistics on the number of grains found that the number of grains per panicle increased significantly (D and E in Figure 6). Compared with the wild type, the number of grains per panicle of OE-L1, OE-L22 and OE-L6 increased by 31.03%, 26.47% and 17.75%.

虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。Although the present invention has been described in detail above with general description, specific embodiments and tests, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art . Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

序列表sequence listing

<110> 中国农业科学院作物科学研究所<110> Institute of Crop Science, Chinese Academy of Agricultural Sciences

<120> 小麦TaMADS6基因在调控植物穗和籽粒发育以及开花时间中的应用<120> Application of wheat TaMADS6 gene in regulation of panicle and grain development and flowering time

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gccgaggtgg cgctcatcat cttctccagc cgcggcaagc tctacgagtt cggcagcgcc 180gccgaggtgg cgctcatcat cttctccagc cgcggcaagc tctacgagtt cggcagcgcc 180

ggggcactat ctgaaactca gagctggtac caggaaatgt caaagctaaa ggcaaaattc 300ggggcactat ctgaaactca gagctggtac caggaaatgt caaagctaaa ggcaaaattc 300

gaagcgttgc agcgaactca gagacacttg cttggggagg accttggacc gctcagcgtg 360gaagcgttgc agcgaactca gagacacttg cttggggagg accttggacc gctcagcgtg 360

aagacacaac ttatgatgga gcaggtggag gaactttgca ggaaggagcg tcagctggga 480aagacacaac ttatgatgga gcaggtggag gaactttgca ggaaggagcg tcagctggga 480

gacatcaaca ggcaactcaa gcacaagctc gatgctgaag gcagcaacag caacaactac 540gacatcaaca ggcaactcaa gcacaagctc gatgctgaag gcagcaacag caacaactac 540

agggccatgc agcagatctc ctgggctgcc ggcaccgtcg tggacgaggg cgccgccgca 600agggccatgc agcagatctc ctgggctgcc ggcaccgtcg tggacgaggg cgccgccgca 600

tatcacatgc agcagcatca gcaacaccct aatcattccg ctgctatgga ctgtgaaccc 660tatcacatgc agcagcatca gcaacaccct aatcattccg ctgctatgga ctgtgaaccc 660

actctgcaaa ttgggtacca tcatcagttc acggctcctg atcagccagc caataatatt 720actctgcaaa ttgggtacca tcatcagttc acggctcctg atcagccagc caataatatt 720

ccacggagca gcgcccccgg aggggagaac aacttcatgc tggggtggat tctctga 777ccacggagca gcgcccccgg aggggagaac aacttcatgc tggggtggat tctctga 777

<210> 6<210> 6

<211> 780<211> 780

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 6<400> 6

atggggaggg gaagggtcga gctgaagcgc atcgagaaca agatcaatcg gcaggtcacc 60atggggaggg gaagggtcga gctgaagcgc atcgagaaca agatcaatcg gcaggtcacc 60

gaagctttgc agcgaactca gagacacttg cttggggagg accttggacc gctcagcgtg 360gaagctttgc agcgaactca gagacacttg cttggggagg accttggacc gctcagcgtg 360

aagacacaac ttatgatgga gcaggtggag gaacttcgca ggaaggagcg tcagctggga 480aagacacaac ttatgatgga gcaggtggag gaacttcgca ggaaggagcg tcagctggga 480

agggccatgc agcaaatcac ctgggctgcc ggcaccgtcg tggatgaagg cgccgccgca 600agggccatgc agcaaatcac ctgggctgcc ggcaccgtcg tggatgaagg cgccgccgca 600

attccacgga gcagcggccc cggaggggag aacaacttca tgctcgggtg ggttctttga 780attccacgga gcagcggccc cggaggggag aacaacttca tgctcgggtg ggttctttga 780

<210> 7<210> 7

<211> 29<211> 29

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 7<400> 7

ccactagtat ggggagggga agggtcgag 29ccactagtat ggggagggga agggtcgag 29

<210> 8<210> 8

<211> 29<211> 29

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 8<400> 8

ccgagctctc aaagaaccca cccgagcat 29ccgagctctc aaagaaccca ccgagcat 29

<210> 9<210> 9

<211> 26<211> 26

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 9<400> 9

ccggatccac gtatcgattt aaagct 26ccggatccac gtatcgattt aaagct 26

<210> 10<210> 10

<211> 29<211> 29

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 10<400> 10

ggcccgggtc aaagaaccca cccgagcat 29ggcccgggtc aaagaaccca cccgagcat 29

<210> 11<210> 11

<211> 22<211> 22

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 11<400> 11

tttagccctg ccttcatacg ct 22tttagccctg ccttcatacg ct 22

<210> 12<210> 12

<211> 23<211> 23

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 12<400> 12

tgtataattg cgggactcta atc 23tgtataattg cgggactcta atc 23

<210> 13<210> 13

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 13<400> 13

gaaatgtcaa agctaaaggc 20gaaatgtcaa agctaaaggc 20

<210> 14<210> 14

<211> 19<211> 19

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 14<400> 14

gcggcaatga gatgaggac 19gcggcaatga gatgaggac 19

<210> 15<210> 15

<211> 19<211> 19

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 15<400> 15

gcgaagtccc tggtgttga 19gcgaagtccc tggtgttga 19

Claims

Translated fromChinese

1.TaMADS6蛋白或其编码基因或含有TaMADS6蛋白的编码基因的生物材料在调控植物穗发育中的应用。1. Application of TaMADS6 protein or its encoding gene or biological material containing TaMADS6 protein encoding gene in regulating plant ear development.

2.TaMADS6蛋白或其编码基因或含有TaMADS6蛋白的编码基因的生物材料在调控植物籽粒发育中的应用。2. The application of TaMADS6 protein or its coding gene or biological material containing TaMADS6 protein coding gene in regulating plant grain development.

3.TaMADS6蛋白或其编码基因或含有TaMADS6蛋白的编码基因的生物材料在调控植物开花时间中的应用。3. Application of TaMADS6 protein or its coding gene or biological material containing TaMADS6 protein coding gene in regulating flowering time of plants.

4.TaMADS6蛋白或其编码基因或含有TaMADS6蛋白的编码基因的生物材料在植物遗传育种中的应用。4. Application of TaMADS6 protein or its coding gene or biological material containing TaMADS6 protein coding gene in plant genetics and breeding.

5.TaMADS6蛋白或其编码基因或含有TaMADS6蛋白的编码基因的生物材料在植物种质资源改良中的应用。5. The application of TaMADS6 protein or its encoding gene or biological material containing TaMADS6 protein encoding gene in plant germplasm resource improvement.

6.根据权利要求1～5任一项所述的应用，其特征在于，通过提高所述植物中TaMADS6蛋白的表达量和/或活性，提高植物的小穗数、小穗轴数或穗粒数，或者延迟植物的开花时间。6. The application according to any one of claims 1 to 5, characterized in that, by increasing the expression and/or activity of TaMADS6 protein in the plant, the number of spikelets, the number of spikelets or the number of spikelets in the plant is improved number, or delay the flowering time of the plant.

7.根据权利要求1～6任一项所述的应用，其特征在于，所述TaMADS6蛋白具有如下任一种氨基酸序列：7. The application according to any one of claims 1 to 6, wherein the TaMADS6 protein has any of the following amino acid sequences:

8.根据权利要求1～7任一项所述的应用，其特征在于，所述生物材料包括表达盒、载体、宿主细胞、工程菌、转基因植物细胞系、转基因植物组织、转基因植物器官、转基因植株、所述转基因植株的可再生细胞产生的组织培养物或所述组织培养物产生的原生质体。8. The application according to any one of claims 1 to 7, wherein the biological material comprises expression cassettes, vectors, host cells, engineered bacteria, transgenic plant cell lines, transgenic plant tissues, transgenic plant organs, transgenic plants Plants, tissue cultures produced by regenerable cells of said transgenic plants, or protoplasts produced by said tissue cultures.

9.一种调控植物穗、籽粒发育或开花时间的方法，其特征在于，包括：调控所述植物中TaMADS6蛋白的表达量和/或活性；9. A method for regulating and controlling plant ear, grain development or flowering time, comprising: regulating the expression and/or activity of TaMADS6 protein in the plant;

所述TaMADS6蛋白具有如下任一种氨基酸序列：The TaMADS6 protein has any of the following amino acid sequences:

10.根据权利要求9所述的方法，其特征在于，通过提高所述植物中所述TaMADS6蛋白的表达量和/或活性，提高植物的小穗数、小穗轴数或穗粒数，或者延迟所述植物的开花时间。10. method according to claim 9, is characterized in that, by improving the expression amount and/or activity of described TaMADS6 protein in described plant, improve the number of spikelets, rachis or grains per ear of plant, or Delay the flowering time of the plants.