Anti-powdery mildew protein and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a powdery mildew resistant protein and application thereof. The protein comprises an amino acid sequence which has at least 98%, at least 99% or at least 100% identity with the amino acid sequence as shown in SEQ ID NO: 1. According to the application, candidate genes of pumpkin powdery mildew are positioned, and functional verification is carried out, so that a molecular mechanism of powdery mildew resistance of cucurbitaceae is expected to be analyzed, and technical support is provided for breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a powdery mildew resistant protein and application thereof. Background Art

[0002] Pumpkin is an annual herbaceous plant of the genus Cucurbita (Cucurbita L.) of the Cucurbitaceae family. It is an important melon crop and vegetable crop in my country. Pumpkins are mainly divided into three categories: Chinese pumpkin (Cucurbita moschata), Indian pumpkin (Cucurbita.maxima) and American pumpkin (Cucurbita pepo). With the increase in pumpkin planting area, various diseases are gradually becoming more serious. Powdery mildew is a major disease in pumpkin production. When it occurs, it will seriously affect its yield and quality, causing huge losses to pumpkin production.

[0003] Powdery mildew is a fungal disease that is an obligate parasite. There are many types of powdery mildew fungi, and the pathogenic strains of different crops are also different. Among them, the powdery mildew strains that harm pumpkin and other cucurbits are mainly from the genus Podosphaera. At present, the prevention and control of powdery mildew still mainly rely on the use of chemical pesticides, but long-term use of chemical fungicides can easily make pathogens resistant to drugs, and will cause pesticide residues and ecological environment pollution. Through the means of modern biotechnology, the exploration of pumpkin powdery mildew resistance genes, the study of molecular mechanisms, and the cultivation of new disease-resistant pumpkin varieties through gene editing or transgenic means are becoming the main solutions for the prevention and control of powdery mildew.

[0004] Current studies have shown that powdery mildew resistance may be determined by quantitative traits controlled by multiple gene loci. There are many reports on genetic analysis and related molecular markers of powdery mildew resistance in Cucurbitaceae, but there are very few reports on the actual cloning and identification of resistance genes. Brief description of the invention

[0006] In order to solve the above problems existing in the prior art, the present invention provides a powdery mildew resistant protein and application thereof.

[0007] The technical solution adopted by the present invention is as follows:

[0008] The present invention provides a powdery mildew resistance protein, which comprises an amino acid sequence having at least 98%, at least 99% or at least 100% identity with the amino acid sequence shown in SEQ ID NO:1.

[0009] In a specific embodiment, the amino acid sequence of the protein is shown in SEQ ID NO:1.

[0010] The present invention also provides a powdery mildew resistance gene having a nucleic acid sequence selected from the following:

[0011] (1) a nucleic acid sequence encoding the protein or its complementary sequence;

[0012] (2) the nucleic acid sequence shown in SEQ ID NO: 2 or its complementary sequence;

[0013] (3) a nucleic acid sequence that hybridizes to the sequence shown in (1) or (2) under stringent conditions; or

[0014] (4) A nucleic acid sequence that encodes the same protein as the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or its complementary sequence.

[0015] The invention also provides a gene expression box, which comprises the resistance gene.

[0016] The present invention also provides an expression vector, which comprises the resistance gene, or the gene expression box, or expresses the protein.

[0017] The present invention also provides a host cell, which comprises the resistance gene, or the gene expression cassette, or the expression vector.

[0018] In one embodiment, the host cell is a plant cell.

[0019] The present invention also provides a method for cultivating transgenic plants resistant to powdery mildew, which comprises transferring the gene or the vector into plants for expression, or regenerating the plant cells into plants to produce resistance to powdery mildew.

[0020] The present invention also provides plants produced by the method.

[0021] The present invention also provides the use of the protein or the resistance gene or the gene expression box or the expression vector in resistance breeding, wherein the breeding is for selecting plants with powdery mildew resistance.

[0022] This application locates the candidate genes of pumpkin powdery mildew and conducts functional verification, in order to analyze the molecular mechanism of Cucurbitaceae's resistance to powdery mildew and provide technical support for breeding. DETAILED DESCRIPTION OF THE INVENTION

[0024] Some of the terms used in this specification are defined below.

[0025] In the present invention, "plant" is understood to be any differentiated multicellular organism capable of photosynthesis, in particular monocotyledonous or dicotyledonous plants. The plants of the present invention include inbred lines, hybrids, open-pollinated varieties, doubled haploid plants or plants of segregating populations.

[0026] In a specific embodiment, the transgenic plant is pumpkin, wheat or Arabidopsis thaliana.

[0027] In the present invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue culture, plant callus, plant pieces, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0028] "Phenotype" refers to a detectable characteristic of a cell or organism that is a manifestation of gene expression.

[0029] A "phenotypic trait index" refers to a composite value of at least two phenotypic traits, wherein each phenotypic trait may be assigned a weight to reflect relative importance for selection.

[0030] A "vector" is any of a variety of nucleic acids containing a desired sequence or sequences to be delivered to a cell and / or expressed in a cell. The vector is usually composed of DNA, but RNA vectors are also available. Vectors include, but are not limited to, plasmids, F cosmids (fosmid), phagemids, viral genomes, synthetic chromosomes, and the like. As used herein, the phrase "engineering vector" includes a coding sequence for a nuclease in a nucleic acid-guided nuclease system and method for the present disclosure. In bacterial systems, the engineering vector may also include a λ Red recombineering system or its equivalent. The engineering vector also typically includes a selection marker. As used herein, the phrase "editing vector" includes a donor nucleic acid and a gRNA coding sequence, the donor nucleic acid optionally including a change to the target sequence, the change preventing the nuclease from binding at a PAM or spacer in the target sequence after editing occurs. The editing vector may also include a selection marker and / or a barcode. In some embodiments, the engineering vector and the editing vector may be combined; that is, the contents of the engineering vector may be found on the editing vector. In addition, engineering and editing vectors contain control sequences operably linked to, for example, a nuclease encoding sequence, a recombineering system encoding sequence (if present), a donor nucleic acid, a guide nucleic acid, and one or more selectable markers.

[0031] In the present invention, "plant cell" is understood to be any cell from or found in a plant, which is capable of forming, for example, undifferentiated tissue such as callus, differentiated tissue such as embryos, plant components, plants or seeds.

[0032] In the present invention, "host cell" should be understood as any unicellular or multicellular organism into which mutant protein encoding nucleic acid can be introduced, including, for example, bacteria such as Escherichia coli, fungi such as yeast (e.g., Saccharomyces cerevisiae), molds (e.g., Aspergillus), plant cells and plants, etc.

[0033] The term "mutation" refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the canonical or wild-type sequence or a reference sequence.

[0034] The terms "polynucleotide", "nucleic acid", "nucleic acid molecule" or "nucleic acid sequence" are used interchangeably and refer to oligonucleotides, nucleotides or polynucleotides and fragments or portions thereof, which may be single-stranded or double-stranded and represent sense or antisense strands. Nucleic acids include DNA, RNA or hybrids thereof, and may be of natural or synthetic origin. For example, nucleic acids may include mRNA or cDNA. Nucleic acids may include nucleic acids that have been amplified (e.g., using polymerase chain reaction). The nucleotide designations "R" mean purines such as guanine or adenine; "Y" means pyrimidines such as cytosine or thymine (uracil if RNA); "M" means adenine or cytosine; "K" means guanine or thymine; and "W" means adenine or thymine.

[0035] It is well known to those skilled in the art that, due to the degeneracy of the genetic code, there are a variety of different nucleic acid sequences that can encode the amino acid sequences disclosed herein. It is within the capabilities of those of ordinary skill in the art to generate other nucleic acid sequences encoding the same protein, and thus the present invention encompasses nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code. For example, in order to achieve high expression of a heterologous gene in a target host organism such as a plant, the gene can be optimized using codons preferred by the host organism to allow for better expression.

[0036] The term "transgenic" plant refers to a plant comprising a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated in the genome so that the polynucleotide is passed to successive generations. The heterologous polynucleotide can be integrated into the genome alone or as a part of a recombinant expression cassette. "Transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, whose genotype is changed due to the presence of heterologous nucleic acids, including those initially changed transgenic organisms or cells, and those produced from initial transgenic organisms or cell hybridization or asexual reproduction. As used herein, the term "transgenic" is not intended to include changing genomes (chromosomes or chromosomes outside) by conventional plant breeding methods (e.g., hybridization) or by naturally occurring events (e.g., self-fertilization, random cross fertilization, non-recombinant virus infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation).

[0037] As used herein, the terms "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences may refer to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0038] Methods of alignment of sequences for comparison are well known to those of skill in the art and can be determined using the BLAST algorithm (Altschul et al., 1990, Mol. Biol. 215:403-10) available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ) using default parameters.

[0039] Various programs and alignment algorithms are described, for example, in Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. Detailed considerations of sequence alignment methods and homology calculations can be found, for example, in Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0040] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is accessible at the National Center for Biotechnology Information (Bethesda, MD), as well as on the Internet, and is used in conjunction with several sequence analysis programs. A description of how to use this program to determine sequence identity is available in the "Help" section of BLAST™ on the Internet. To compare nucleic acid sequences, the "Blast 2 Sequences" function of the BLAST™ (Blastn) program using default parameters can be used. When evaluated in this way, nucleic acid sequences that have greater similarity to a reference sequence will show an increase in the percent identity.

[0041] It is also clear to those skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions can be introduced into a protein amino acid sequence without adversely affecting the activity and / or three-dimensional configuration of the protein molecule. Examples and embodiments of conservative amino acid substitutions are clear to those skilled in the art. Specifically, the amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., another non-polar amino acid residue can be substituted with a non-polar amino acid residue, another polar non-charged amino acid residue can be substituted with a polar uncharged amino acid residue, another basic amino acid residue can be substituted with a basic amino acid residue, and another acidic amino acid residue can be substituted with an acidic amino acid residue. As long as the substitution does not damage the biological activity of the protein, conservative substitutions in which one amino acid is replaced by other amino acids belonging to the same group fall within the scope of the present invention. In addition, the present invention also encompasses mutant proteins that also include one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the protein of the present invention.

[0042] As is well known in the art, one or more amino acid residues can be deleted from the N and / or C terminus of a protein while retaining its functional activity. Therefore, on the other hand, the present invention also relates to fragments in which one or more amino acid residues are deleted from the N and / or C terminus of a mutant protein while retaining its desired functional activity, which are also within the scope of the present invention and are referred to as biologically active fragments. In the present invention, "biologically active fragments" refer to a portion of a mutant protein of the present invention, which retains the biological activity of the mutant protein of the present invention. For example, a biologically active fragment of a mutant protein can be a portion in which one or more (e.g., 1-50, 1-25, 1-10 or 1-5, such as 1, 2, 3, 4 or 5) amino acid residues are deleted from the N and / or C terminus of the protein, but it still retains the biological activity of the full-length protein.

[0043] The term "gene editing" generally refers to a technique for inserting, deleting, modifying or replacing DNA in a genome. Many gene editing systems suitable for use in the methods of the present invention are known in the art, including but not limited to zinc finger nuclease (ZFNs) systems, transcription activator-like effector nuclease (TALEN) systems, and CRISPR / Cas systems.

[0044] The terms used herein are intended to describe specific embodiments and are not intended to set limitations. Unless otherwise expressly specified herein, the terms "a", "an" and "the" used in the English version of the above content also include their plural forms. The terms "comprises" and / or "comprising," or "includes" and / or "including" used herein specifically refer to the presence of the features, factors and / or components described herein, and do not exclude the presence and addition of one or more other features, factors and components. The term "and / or" used in the above content includes all items in the one or more combination lists.

[0045] The present invention has been described in detail through a series of embodiments, but the present invention is not limited to the disclosed embodiments. Any quantitative changes, substitutions, replacements, etc. within the scope of the present invention are not described herein, or can be modified according to public needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 pQYI0007 vector map.

[0047] Figure 2 pQYI0009 vector map.

[0048] Figure 3 PCR identification results of transgenic materials (Markers from top to bottom are 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp respectively).

[0049] Figure 4 Peak graph results of sequencing identification of gene knockout materials.

[0050] Figure 5 Leaf phenotype of transgenic pumpkin material (transfected with pQYI0007 vector) after inoculation.

[0051] Figure 6 Leaf phenotype of homozygous knockout pumpkin plants (transformed with pQY10009 vector) after inoculation.

[0052] Sequence Description

[0053]

[0054] DETAILED DESCRIPTION

[0055] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and the following examples are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all or the only experiments performed. It should be understood by those skilled in the art that many changes and / or modifications may be made to the invention shown in the specific aspects without departing from the spirit or scope of the present invention as broadly described. Therefore, the aspects herein are considered to be illustrative and not restrictive in all respects.

[0056] Example 1 Resistance gene localization

[0057] A large-scale EMS mutagenesis was carried out on Chinese pumpkin seeds, and the mutated offspring materials were tested and compared for powdery mildew resistance in the field. A powdery mildew-resistant material was found. Then, high-throughput sequencing combined with hybrid group analysis (BSA-seq) was used to analyze the sensitive wild-type pumpkin and the resistant materials after EMS mutagenesis, and finally the candidate gene NBLRR1 (amino acid sequence SEQ ID NO:1, nucleotide sequence SEQ ID NO:2) for pumpkin powdery mildew resistance was located.

[0058] Example 2 Construction of transgenic and gene editing vectors

[0059] In order to further verify the functions of the above genes, pumpkin overexpression vectors and knockout vectors were constructed, and pumpkin genetic transformation experiments were carried out.

[0060] The overexpression vector uses the pCambia1300 basic vector as the backbone. The modified vector expresses the target gene NBLRR1. The final vector number is pQYI0007. The vector map is shown in Figure 1 .

[0061] The vector for knocking out NBLRR1 was modified based on pBSE401. The final vector number is pQYI0009. The vector map is shown in Figure 2 .

[0062] Example 3 Genetic transformation and molecular identification of pumpkin

[0063] The overexpression and knockout vectors in Example 2 were transformed into Agrobacterium by electroporation, and then genetic transformation of pumpkin was performed using Agrobacterium-mediated method.

[0064] After obtaining the T0 generation of transgenic pumpkin, specific primers were designed on the target gene to perform PCR detection on positive plants. The PCR detection results of transgenic plants transformed with pQYI0007 vector are shown in Figure 3 , transgenic positive has obvious bands, while negative has none.

[0065] The gene knockout T0 plants transformed with the pQYI0009 vector were first preliminarily judged by eYGFPuv fluorescence whether the transgenic gene was positive or not, and then specific primers were designed at the target site to perform PCR amplification and sequencing to identify the genotype. The plants with successful gene editing were planted and self-pollinated to obtain the next generation of seeds. Then, the NBLRR1 gene of the obtained T1 generation pumpkin plants was sequenced and identified, and the T1 plants with homozygous knockout of NBLRR1 were found. The peak diagram of the identification of the homozygous T1 generation pumpkin plants is shown in Figure 4 Finally, the homozygous pumpkin plant materials cmnblrr1-2 and cmnblrr1-3 with NBLRR1 knockout were obtained.

[0066] Example 4 Powdery mildew resistance test of transgenic plants

[0067] According to the method provided by the National Crop Germplasm Resources Platform and the National Crop Science Data Center "Pumpkin Germplasm Resources Quality Control Standards", the transgenic plants were tested for powdery mildew. Among them, the leaf phenotype of the transgenic pumpkin material (transfected with pQYI0007 vector) after inoculation is shown in Figure 5 The transgenic positive plants showed obvious resistance to powdery mildew, which indicated that the overexpression of the candidate gene NBLRR1 played a role in resistance to powdery mildew.

[0068] Example 5 Powdery Mildew Resistance Test of Gene Knockout Pumpkin Plants

[0069] According to the method of Example 4, the powdery mildew test was performed on homozygous T1 generation pumpkin seedlings with NBLRR1 knockout. The powdery mildew incidence of gene knockout pumpkin plants cmnblrr1-2 and cmnblrr1-3 was shown in Figure 6 The leaves of gene knockout pumpkin plants cmnblrr1-2 and cmnblrr1-3 showed obvious powdery mildew spots, indicating that pumpkin plants with NBLRR1 gene knockout were susceptible to powdery mildew.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A protein for resisting powdery mildew, characterized in that: The protein comprises an amino acid sequence that is at least 98%, at least 99% or at least 100% identical to the amino acid sequence shown in SEQ ID NO:

1.

2. The protein according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

1.

3. A powdery mildew resistance gene, characterized in that: It has a nucleic acid sequence selected from the group consisting of: (1) a nucleic acid sequence encoding the protein as claimed in claim 1 or 2 or its complementary sequence; (2) the nucleic acid sequence shown in SEQ ID NO: 2 or its complementary sequence; (3) a nucleic acid sequence that hybridizes to the sequence shown in (1) or (2) under stringent conditions; or (4) A nucleic acid sequence that encodes the same protein as the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or its complementary sequence.

4. A gene expression cassette comprising the resistance gene according to claim 3.

5. An expression vector comprising the resistance gene according to claim 3, or the gene expression cassette according to claim 4, or expressing the protein according to claim 1 or 2.

6. A host cell comprising the resistance gene according to claim 3, or the gene expression cassette according to claim 4, or the expression vector according to claim 5; preferably, the host cell is a plant cell.

7. A method for cultivating transgenic plants resistant to powdery mildew and the plants produced by the method, comprising transferring the gene as claimed in claim 3 or the vector as claimed in claim 5 into a plant for expression, or regenerating the plant cell as claimed in claim 6 into a plant to produce resistance to powdery mildew; preferably, the transgenic plant is pumpkin, wheat or Arabidopsis thaliana.

8. Use of the protein according to claim 1 or 2, or the resistance gene according to claim 3, or the gene expression cassette according to claim 4, or the expression vector according to claim 5 in resistance breeding, wherein the breeding is for breeding plants with powdery mildew resistance; preferably, the plant is pumpkin, wheat or Arabidopsis thaliana.

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