阅读说明：本技术 植物白粉病抗性相关蛋白Pm5e及其编码基因和应用 (Plant powdery mildew resistance related protein Pm5e, and coding gene and application thereof ) 是由 刘志勇 郭广昊 谢菁忠 王勇 吴秋红 陆平 陈永兴 董玲丽 于 2019-03-08 设计创作，主要内容包括：本发明公开了一种植物白粉病抗性相关蛋白Pm5e及其编码基因和应用。本发明提供的蛋白质,获自小麦,命名为Pm5e蛋白,为序列表中序列1所示的蛋白质。编码Pm5e蛋白的核酸分子也属于本发明的保护范围。本发明还保护Pm5e蛋白的应用,为提高植物的白粉病抗病性。本发明还保护一种制备转基因植物的方法,包括如下步骤：在受体植物中导入所述核酸分子,得到白粉病抗病性增强的转基因植物。本发明对于植物白粉病育种具有重大的应用推广价值。(The invention discloses a plant powdery mildew resistance related protein Pm5e, and a coding gene and application thereof. The protein provided by the invention is obtained from wheat, is named as Pm5e protein and is a protein shown in a sequence 1 in a sequence table. The nucleic acid molecule encoding the Pm5e protein also belongs to the protection scope of the invention. The invention also protects the application of the Pm5e protein, and aims to improve the disease resistance of plants to powdery mildew. The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the nucleic acid molecule is introduced into a receptor plant to obtain a transgenic plant with enhanced powdery mildew disease resistance. The method has great application and popularization values for breeding the powdery mildew of the plants.)

1. A protein which is (a1) or (a2) or (a3) or (a4) as follows:

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

(a2) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);

(a3) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) and is related to the disease resistance of the powdery mildew of plants;

(a4) a protein derived from wheat, having 98% or more identity to (a1) and involved in plant powdery mildew resistance.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is (b1) or (b2) or (b3) or (b4) as follows:

(b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(b2) DNA molecule shown in sequence 3 in the sequence table;

(b3) a DNA molecule derived from wheat and having 95% or more identity to (b1) or (b2) and encoding said protein;

(b4) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) under stringent conditions and encodes the protein.

4. An expression cassette, recombinant vector or recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.

5. The use of the protein of claim 1, which is (c1) or (c 2):

(c1) regulating and controlling powdery mildew disease resistance of plants;

(c2) improve the powdery mildew resistance of the plants.

6. The use of the nucleic acid molecule of claim 2 or 3 as follows (d1) or (d 2):

(d1) cultivating transgenic plants with changed powdery mildew resistance;

(d2) cultivating transgenic plants with enhanced powdery mildew resistance.

7. A method of making a transgenic plant comprising the steps of: introduction of the nucleic acid molecule of claim 2 or 3 into a recipient plant to obtain a transgenic plant with enhanced resistance to powdery mildew.

8. Use of the method of claim 7 in plant breeding.

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 improving the powdery mildew resistance of the target plant.

10. The method of claim 7, 8 or 9, wherein: the plant is a monocotyledon or a dicotyledon.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a plant powdery mildew resistance related protein Pm5e, and a coding gene and application thereof.

Background

Wheat is one of the world's most prominent food crops, providing approximately 20% of the heat required globally. Wheat powdery mildew is a main fungal disease caused by Blumeria graminis f.sp.tritici, Bgt, and can obviously affect the tiller number, ear number and thousand kernel weight of wheat and seriously reduce the yield and quality of wheat. Therefore, controlling the powdery mildew hazard has important significance for improving the wheat yield, reducing the pesticide and labor cost and protecting the environment. Chemical prevention and control not only harms the environment and damages human health, but also can cause pathogenic bacteria to generate drug resistance, thereby causing more serious loss. The most economical, environment-friendly and effective measures for preventing and treating the wheat powdery mildew are to excavate the disease-resistant genes and cultivate the disease-resistant varieties.

At present, the disease-resistant breeding of wheat powdery mildew in China faces the problems that the variation speed of powdery mildew is too high, the epidemic range of new highly toxic microspecies is continuously expanded and available resistance genes are limited, and the problem of narrowing of the genetic basis of bred varieties is increasingly serious, so that new disease-resistant genes need to be continuously excavated, the sources of the disease-resistant genes are expanded, and the existing disease-resistant genes are reasonably distributed, so that the multiple genes are effectively polymerized to breed broad-spectrum and durable-resistance varieties, pathogenic bacteria are difficult to break out and spread in a large scale, the harm of the wheat powdery mildew is effectively prevented and controlled, the investment is reduced, and the benefit is improved.

Under the influence of long-term wheat cultivation process, different geographical environments and complex natural conditions, valuable local wheat variety germplasm resources are enriched, and excellent genes which are lacked by the bred varieties are hidden. Wheat in China has various local varieties and is a huge treasury of excellent genetic resources.

Disclosure of Invention

The invention aims to provide a plant powdery mildew resistance related protein Pm5e, and a coding gene and application thereof.

The protein provided by the invention is obtained from wheat (Triticum aestivum L.), is named as Pm5e protein, and is (a1) or (a2) or (a3) or (a4) as follows:

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

(a2) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);

(a3) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) and is related to the disease resistance of the powdery mildew of plants;

(a4) a protein derived from wheat, having 98% or more identity to (a1) and involved in plant powdery mildew resistance.

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 protein can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The nucleic acid molecule encoding the Pm5e protein also belongs to the protection scope of the invention.

The nucleic acid molecule is (b1) or (b2) or (b3) or (b4) as follows:

(b1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(b2) DNA molecule shown in sequence 3 in the sequence table;

(b3) a DNA molecule derived from wheat and having 95% or more identity to (b1) or (b2) and encoding said protein;

(b4) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) under stringent conditions and encodes the 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.

Expression cassettes, recombinant vectors or recombinant microorganisms containing the nucleic acid molecules are within the scope of the invention.

The recombinant expression vector containing the nucleic acid molecule can be constructed using existing expression vectors. When the nucleic acid molecule is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added in front of the transcription initiation nucleotide, and can be used alone or combined with other plant promoters; in addition, when recombinant expression vectors are constructed using the nucleic acid molecules, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or adjacent regions initiation codons, 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 identification and screening of the transgenic plant or the transgenic microorganism, an expression vector to be used may be processed, for example, a gene for expressing an enzyme or a luminescent compound which produces a color change in the plant or the microorganism, a gene for an antibiotic marker having resistance or a chemical-resistant agent marker, etc. From the viewpoint of safety of transgenes, the transformed plants or microorganisms can be directly screened phenotypically without adding any selectable marker gene.

The recombinant vector may specifically be a recombinant expression vector. The recombinant expression vector can be specifically a recombinant plasmid obtained by inserting the nucleic acid molecule into an existing plant expression vector. The existing plant expression vector can be a pCAMBIA1300 vector or a pTCK303 vector.

The invention also protects the application of the Pm5e protein, which is (c1) or (c 2):

(c1) regulating and controlling powdery mildew disease resistance of plants;

(c2) improve the powdery mildew resistance of the plants.

The invention also protects the application of the nucleic acid molecule, which is (d1) or (d 2):

(d1) cultivating transgenic plants with changed powdery mildew resistance;

(d2) cultivating transgenic plants with enhanced powdery mildew resistance.

The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the nucleic acid molecule is introduced into a receptor plant to obtain a transgenic plant with enhanced powdery mildew disease resistance.

The invention also protects the application of the method in plant breeding. The purpose of the plant breeding is to obtain plants with enhanced powdery mildew disease resistance.

The invention also provides a plant breeding method, which comprises the following steps: the content and/or the activity of the Pm5e protein in the target plant are/is increased, so that the powdery mildew resistance of the target plant is improved.

Any of the above plants may be a monocot. Any of the above plants may be a graminaceous plant. Any of the above plants may be a plant of the genus Triticum. Any of the above plants may be wheat Fielder.

Any one of the above powdery mildews can be powdery mildews caused by powdery mildew pathogenic bacteria. The powdery mildew pathogenic bacteria can be Blumeria brucei wheat specialization type. The powdery mildew pathogenic bacteria can be E20 physiological race.

The inventor obtains the powdery mildew resistance gene Pm5e contained in local wheat varieties in China, and provides richer powdery mildew resistance gene resources for wheat breeding work. The Pm5e protein coding gene is introduced into powdery mildew disease resistant wheat, so that the disease resistance of a transgenic plant is remarkably improved, and the powdery mildew resistance is realized. The method has great application and popularization values for breeding the powdery mildew of the plants.

Drawings

FIG. 1 shows the results of expression analysis of Pm5e gene at different inoculation times.

FIG. 2 shows the results of the identification of powdery mildew resistance in step one of example 3.

FIG. 3 shows the results of the identification of powdery mildew resistance in step two of example 3.

FIG. 4 is a photograph of a phenotype of disease resistance identification in example 4.

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. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The wheat variety rejuvenation 30, referred to as wheat rejuvenation 30 for short, is a local variety of Chinese wheat with high powdery mildew resistance. The wheat variety Nongda 015, called Nongda wheat 015 for short, is a variety with high susceptibility to powdery mildew. Wheat variety Fielder, abbreviated as wheat Fielder, is a variety of powdery mildew disease. The wheat variety Xue Zao, short for Xue Zao, is a variety of powdery mildew.