阅读说明：本技术 植物抗旱相关蛋白TaCLE3B及其编码基因与应用 (Plant drought-resistant related protein TaCLE3B, and coding gene and application thereof ) 是由 辛明明 冯曼 孙其信 倪中福 彭惠茹 姚颖垠 胡兆荣 郭伟龙 杨光辉 于 2020-07-16 设计创作，主要内容包括：本发明公开了植物抗旱相关蛋白TaCLE3B及其编码基因与应用。本发明提供的蛋白质,来源于小麦(Triticum aestivum L.),命名为TaCLE3B蛋白,是序列表中序列1所示的蛋白质。编码TaCLE3B蛋白的基因也属于本发明的保护范围。编码TaCLE3B蛋白的基因命名为TaCLE3B基因。本发明还保护TaCLE3B蛋白在调控植物侧根数目和/或调控植物抗旱性中的应用。本发明还保护TaCLE3B基因的应用,为如下(b1)和/或(b2)：(b1)培育侧根数目增多的转基因植物；(b2)培育抗旱性增高的转基因植物。本发明对于植物侧根发育以及植物抗旱的研究和应用具有重要意义。(The invention discloses a plant drought-resistant related protein TaCLE3B, and a coding gene and application thereof. The protein provided by the invention is derived from wheat (Triticum aestivum L.), is named as TaCLE3B protein, and is a protein shown as a sequence 1 in a sequence table. The gene coding for the TaCLE3B protein also belongs to the protection scope of the invention. The gene encoding the TaCLE3B protein was designated as TaCLE3B gene. The invention also protects the application of the TaCLE3B protein in regulating the number of lateral roots of plants and/or regulating the drought resistance of plants. The invention also protects the application of the TaCLE3B gene, which is (b1) and/or (b 2): (b1) cultivating transgenic plants with an increased number of lateral roots; (b2) cultivating the transgenic plant with increased drought resistance. The invention has important significance for the development of the lateral roots of the plants and the research and the application of the drought resistance of the plants.)

1. A protein which is (a1) or (a2) or (a4) or (a4) or (a5) or (a 6):

(a1) protein shown as a sequence 1 in a sequence table;

(a2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein shown in the sequence 1 in the sequence table, is related to the number of plant lateral roots and is derived from the protein;

(a3) the protein shown in the sequence 1 in the sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and is related to plant drought resistance and derived from the protein;

(a4) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);

(a5) a protein derived from wheat and having 98% or more identity to (a1) and associated with the number of lateral roots of a plant;

(a6) a protein derived from wheat, having 98% or more identity to (a1) and associated with drought resistance of a plant.

2. A gene encoding the protein of claim 1.

3. The gene of claim 2, wherein: the gene is as follows (1) or (2) or (3) or (4):

(1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(2) DNA molecule shown in sequence 3 in the sequence table;

(3) a DNA molecule which hybridizes with the DNA molecule defined in (1) or (2) under stringent conditions and encodes the protein;

(4) a DNA molecule derived from wheat and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA molecule defined in (1) or (2) and encoding said protein.

4. A recombinant vector, expression cassette or recombinant bacterium comprising the gene of claim 2 or 3.

5. Use of the protein of claim 1 for modulating the number of lateral roots in a plant and/or modulating drought resistance in a plant.

6. The gene of claim 2 or 3, or the use of the recombinant vector of claim 4, or the use of the expression cassette of claim 4, is (b1) and/or (b 2):

(b1) cultivating transgenic plants with an increased number of lateral roots;

(b2) cultivating the transgenic plant with increased drought resistance.

7. A method of breeding a transgenic plant comprising the steps of: a transgenic plant having an increased number of lateral roots, which is obtained by introducing the gene according to claim 2 or 3 into a recipient plant.

8. A method of breeding a transgenic plant comprising the steps of: a transgenic plant having an increased drought resistance is obtained by introducing the gene according to claim 2 or 3 into a recipient plant.

9. A method of plant breeding comprising the steps of: increasing the content and/or activity of the protein of claim 1 in the target plant, thereby increasing the number of lateral roots in the plant.

10. A method of plant breeding comprising the steps of: increasing the content and/or activity of the protein according to claim 1 in the target plant, thereby increasing the drought resistance of the plant.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a plant drought-resistant related protein TaCLE3B, and a coding gene and application thereof.

Background

The lateral roots are generally originated from the pericycle built-up cells, are important components of a huge root system absorption network of plants, and play important roles in fixing plants, expanding the absorption area of the root system, enhancing the physiological function of the root system and the like. Along with the development of the root system, the internal structure of the root is continuously aged, the degree of cork is increased, the absorption capacity is rapidly reduced, and the supply of water and nutrients required by the growth of crops is mainly completed by the lateral roots.

Wheat has an important position in agricultural production as the second major grain crop in China, and the polypeptide hormone genes participating in the lateral root development and drought resistance mechanism of the wheat are not reported.

Disclosure of Invention

The invention aims to provide a plant drought-resistant related protein TaCLE3B, and a coding gene and application thereof.

The protein provided by the invention is derived from wheat (Triticum aestivum L.), is named as TaCLE3B protein, and is (a1) or (a2) or (a4) or (a4) or (a5) or (a 6):

(a1) protein shown as a sequence 1 in a sequence table;

(a2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein shown in the sequence 1 in the sequence table, is related to the number of plant lateral roots and is derived from the protein;

(a3) the protein shown in the sequence 1 in the sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and is related to plant drought resistance and derived from the protein;

(a4) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);

(a5) a protein derived from wheat and having 98% or more identity to (a1) and associated with the number of lateral roots of a plant;

(a6) a protein derived from wheat, having 98% or more identity to (a1) and associated with drought resistance of a plant.

The labels are specifically shown in table 1.

TABLE 1 sequences of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL
HA	9	YPYDVPDYA

The gene coding for the TaCLE3B protein also belongs to the protection scope of the invention.

The gene encoding the TaCLE3B protein was designated as TaCLE3B gene.

The TaCLE3B gene is (1) or (2) or (3) or (4) as follows:

(1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(2) DNA molecule shown in sequence 3 in the sequence table;

(3) a DNA molecule which hybridizes with the DNA molecule defined in (1) or (2) under stringent conditions and encodes the protein;

(4) a DNA molecule derived from wheat and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA molecule defined in (1) or (2) and encoding said protein.

The stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS and 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS.

The recombinant vector containing the gene TaCLE3B, the expression cassette containing the gene TaCLE3B or the recombinant bacterium containing the gene TaCLE3B belong to the protection scope of the invention.

The recombinant expression vector containing the gene of TaCLE3B can be constructed by using the existing plant expression vector.

When constructing a recombinant expression vector, any one of an enhanced, constitutive, tissue-specific or inducible promoter may be added in front of its transcription initiation nucleotide, either alone or in combination with other plant promoters. In addition, enhancers, including translational or transcriptional enhancers, may be used in the construction of recombinant expression vectors, and these enhancer regions may be ATG initiation codons or initiation codons in adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plants, the expression vector used may be processed, for example, by adding a gene expressing an enzyme or a luminescent compound which produces a color change in a plant, an antibiotic marker having resistance, or a chemical-resistant marker gene, etc. From the viewpoint of transgene safety, the transformed plants can be directly screened for phenotypes without adding any selectable marker gene.

The plant expression vector can be specifically a vector pMWB 111.

The recombinant vector containing the TaCLE3B gene can be specifically a recombinant plasmid pMWB111-TaCLE3B obtained by inserting a double-stranded DNA molecule shown in sequence 3 of a sequence table into Hind III enzyme cutting site of the vector pMWB 111.

The invention also protects the application of the TaCLE3B protein in regulating the number of lateral roots of plants and/or regulating the drought resistance of plants.

The invention also protects the application of the TaCLE3B gene, a recombinant vector containing the TaCLE3B gene or an expression cassette containing the TaCLE3B gene, which is (b1) and/or (b2) as follows:

(b1) cultivating transgenic plants with an increased number of lateral roots;

(b2) cultivating the transgenic plant with increased drought resistance.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: the TaCLE3B gene is introduced into a receptor plant to obtain a transgenic plant with increased lateral root number. The TaCLE3B gene is introduced into a receptor plant, and the recombinant plasmid pMWB111-TaCLE3B is specifically introduced into the receptor plant.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: the gene TaCLE3B is introduced into a receptor plant to obtain a transgenic plant with increased drought resistance. The TaCLE3B gene is introduced into a receptor plant, and the recombinant plasmid pMWB111-TaCLE3B is specifically introduced into the receptor plant.

The invention also provides a plant breeding method, which comprises the following steps: the content and/or activity of the TaCLE3B protein in the target plant is increased, so that the number of lateral roots of the plant is increased.

The invention also provides a plant breeding method, which comprises the following steps: the content and/or activity of the TaCLE3B protein in the target plant is increased, so that the drought resistance of the plant is increased.

Any of the above recipient plants is a monocot or a dicot. Any of the above recipient plants is a gramineae plant. Any one of the above recipient plants is a wheat plant. Any one of the above recipient plants is hexaploid wheat. Any one of the above recipient plants is wheat CB 037.

Any of the above target plants is a monocotyledon or dicotyledon. Any of the above target plants is a gramineae plant. Any one of the above target plants is a Triticum plant. Any of the above target plants is hexaploid wheat. Any one of the above plants of interest is wheat CB 037.

The invention discovers that the introduction of the gene TaCLE3B into wheat can obviously increase the lateral root number of wheat and obviously improve drought resistance. The invention has important significance for the development of the lateral roots of the plants and the research and the application of the drought resistance of the plants.

Drawings

FIG. 1 shows the result that the expression of the TaCLE3B gene is tissue-specific.

FIG. 2 is the electrophoresis chart of the PCR identification of the partial plant.

FIG. 3 shows the relative expression results of TaCLE3B gene in partial plants.

Fig. 4 shows the results of root scanning.

FIG. 5 shows the statistics of lateral root number.

FIG. 6 is a photograph of the phenotype of the drought-treated group.

Fig. 7 is a survival rate statistic result of the drought-treated group.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. Unless otherwise stated, the quantitative tests in the following examples were performed in triplicate, and the results were averaged.

Wheat CB037 belongs to the group of hexaploid wheat (referred to in the literature as "what cultivar, cvCB 037"), vector pMWB111 (referred to in the literature as "pMWB 111 vector"), both described in: ning Zhang, Yujing Yin, Xinye Liu, Shaming Tong, Jiewen Xing, Yuan Zhang, Ramesh N.Pudage, Edenys Miranda Izquierdo, Huiru Pen, Mingming Xin, Zhaorong Hu, Zhongfu Ni, QixinSun, Yingyin Yao, (2017). The E3 Ligase TaSAP5 alcohols bottom Stress modification The Degradation of P proteins plant Physiology 175, 1878-.