Wheat stripe rust resistance related protein TaBURP1 and coding gene and application thereof

Abstract

The present application discloses a wheat stripe disease resistance related protein TaBURP1, a coding gene thereof and an application. The wheat stripe disease resistance related protein disclosed by the present application is A1) or A2): A1) a protein with an amino acid sequence of sequence 2; A2) a protein with an amino acid sequence shown in sequence 2 in the sequence table, which is subjected to substitution and / or deletion and / or addition of one or more amino acid residues and has the same function as the protein shown in sequence 2. Experiments prove that the coding gene of the stripe disease resistance related protein of the present application can reduce the disease resistance of plants to stripe diseases after interference, and a transgenic plant with reduced resistance to stripe diseases can be obtained to serve as a model plant for screening and preventing and controlling plant stripe diseases. The stripe disease resistance related protein and the gene thereof of the present application have important significance for cultivating plants resistant to stripe diseases, and are suitable for popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, this invention relates to the wheat stripe rust resistance-related protein TaBURP1, its encoding gene, and its applications. Background Technology

[0002] Wheat is a cereal grain belonging to the Poaceae family, and it is the world's second largest food crop after rice. my country is a major producer and consumer of wheat globally, with a perennial planting area of ​​approximately 24 million hectares and an annual output of nearly 130 million tons. Wheat stripe rust, caused by the wheat stripe rust fungus (Puccinia striiformis f. spritici), is a global airborne disease and one of the most widespread and damaging wheat diseases in my country, typically causing yield losses of 20%–30%, and in severe cases, almost complete crop failure. Breeding disease-resistant varieties is the most economical, safe, and effective method for controlling wheat stripe rust. Currently, obtaining disease-resistant crop varieties through genetic engineering breeding is an effective method. However, the most critical technical bottleneck in this method is the screening and functional discovery of effective disease-resistant genes.

[0003] The BURP protein domain is a conserved amino acid motif, named after four members of the family: BNM2, USP, RD22, and PG1β. Many genes in the BURP family respond to abiotic stresses; for example, RD22 is a drought-responsive gene, and its homologs respond to salt, ABA, and drought stress induction. The function of this family members in wheat disease resistance has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a wheat stripe rust resistance-related protein, its encoding gene, and its application.

[0005] The stripe rust resistance-related protein provided by this invention is derived from common wheat (Triticum aestivm L.) and is as follows: A1) or A2):

[0006] A1) The amino acid sequence is the same as sequence 2 of the protein;

[0007] A2) A protein that has undergone substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in Sequence 2 of the sequence listing and has the same function as the protein shown in Sequence 2.

[0008] Sequence 2 in the sequence listing consists of 506 amino acid residues.

[0009] To facilitate the purification of proteins in A1), tags as shown in Table 1 can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in Sequence 2 of the sequence listing.

[0010] Table 1. Sequence of Labels

[0011] Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0012] The proteins in A1) or A2) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically. The encoding genes of the proteins in A2) above can be obtained by deleting one or more amino acid residues from the DNA sequence shown in Sequence 1 from nucleotides 64-1584 at the 5' end of the sequence listing, and / or by performing a missense mutation on one or more nucleotide pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5' end and / or 3' end.

[0013] The genes encoding the aforementioned stripe rust-resistant proteins also fall within the scope of protection of this invention.

[0014] Preferably, the encoding gene is one of the following 1), 2), 3), or 4):

[0015] 1) The coding sequence is the cDNA or DNA molecule located at positions 64-1584 of sequence 1 in the sequence listing;

[0016] 2) The cDNA molecule or DNA molecule shown in sequence 1 of the sequence listing;

[0017] 3) A cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the stripe rust-associated protein;

[0018] 4) A cDNA molecule or DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) and encodes the stripe rust-associated protein.

[0019] The application of biomaterials related to the stripe rust resistance-associated protein in regulating plant disease resistance also falls within the scope of protection of this invention; the biomaterials are any one of B1) to B9) below:

[0020] B1) Nucleic acid molecules encoding the stripe rust-associated protein;

[0021] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0023] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0024] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0025] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0026] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2);

[0027] B8) Nucleic acid molecules that inhibit the expression of the gene encoding the stripe rust-related protein;

[0028] B9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B8).

[0029] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0030] In the above applications, the nucleic acid molecule described in B1) can be one of the following: 1), 2), 3), or 4):

[0031] 1) The coding sequence is the cDNA or DNA molecule located at positions 64-1584 of sequence 1 in the sequence listing;

[0032] 2) The cDNA molecule or DNA molecule shown in sequence 1 of the sequence listing;

[0033] 3) A cDNA molecule or DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the stripe rust-associated protein;

[0034] 4) A cDNA molecule or DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) and encodes the stripe rust-associated protein.

[0035] The term "identity" used here refers to sequence similarity to a natural nucleic acid sequence. "Identity" can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0036] The above stringent conditions can be hybridization and washing of the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0037] B8) The nucleic acid molecule may specifically be a DNA molecule that is reverse complementary to any segment of the DNA molecule represented by nucleotides 89-2021 of sequence 1 in the sequence listing, such as a DNA molecule that is reverse complementary to the DNA segment represented by nucleotides 2132-2204 of sequence 1 in the sequence listing.

[0038] (B9) The recombinant vector may be pCaBS-γ-BURP1VIGS, which is a recombinant vector obtained by reverse insertion of the DNA fragment shown in positions 31-204 of sequence 1 in the sequence listing into the multiple cloning site of the pCaBS-γ vector using a restriction endonuclease. The restriction endonuclease may be ApaI.

[0039] The microorganisms may specifically be yeast, bacteria, algae, or fungi. The bacteria may be Agrobacterium, such as Agrobacterium EHA105.

[0040] B9) The recombinant microorganism can be obtained by introducing the pCaBS-γ-BURP1VIGS into the microorganism.

[0041] The transgenic cell lines, transgenic plant tissues, and transgenic plant organs mentioned do not include plant propagation material.

[0042] In the above applications, the plant can be a monocotyledonous plant or a dicotyledonous plant. The monocotyledonous plant can be a grass (Poaceae). The grass can be wheat, such as Wheat Harvest No. 3.

[0043] The disease resistance mentioned can be resistance to stripe rust. The stripe rust can be a disease caused by wheat stripe rust fungus, such as the disease caused by stripe rust pathotype CYR32.

[0044] The present invention also provides a method for cultivating susceptible transgenic plants, the method comprising: inhibiting the expression of the gene encoding the stripe rust-related protein in the target plant to obtain a susceptible transgenic plant with lower resistance than the target plant.

[0045] The target plant can be a monocotyledonous plant or a dicotyledonous plant. The monocotyledonous plant can be a grass (Poaceae). The grass can be wheat, such as Wheat Harvest No. 3.

[0046] The reduction of the expression of the gene encoding the stripe rust-related protein in the target plant is achieved by introducing a DNA molecule that is reverse complementary to the DNA fragment represented by nucleotides 31-204 of sequence 1 in the sequence listing into the target plant.

[0047] DNA molecules that are reverse complementary to the DNA fragment represented by nucleotides 31-204 of sequence 1 in the sequence listing were introduced into the target plant via BMSV virus.

[0048] The disease resistance mentioned refers to resistance to stripe rust. Stripe rust can be a disease caused by wheat stripe rust fungus, such as the disease caused by stripe rust race CYR32.

[0049] Experiments have shown that interfering with (i.e., suppressing gene expression) the encoding gene of the stripe rust-resistant protein of this invention can reduce plant resistance to stripe rust, such as significantly shortening the incubation period of stripe rust and increasing the length and number of stripe rust spore masses. Transgenic plants with reduced resistance to stripe rust can be obtained and used as model plants for screening drugs to prevent stripe rust in plants. Therefore, the stripe rust-resistant protein and its gene of this invention are of great significance for breeding stripe rust-resistant plants and are suitable for widespread application. Attached Figure Description

[0050] Figure 1 This report presents the results of identifying stripe rust resistance in virus-mediated gene silencing wheat. It includes three plant types: uninoculated (Mock, i.e., blank control), inoculated with empty vector virus (BSMV), and inoculated with recombinant virus with an interference fragment inserted into the viral sequence (BSMV-TaBURP1 or vTaBURP1). A shows the phenotype 16 days after inoculation with the skin-friendly stripe rust race CYR32; B shows the spore mass count and differential analysis 16 days after inoculation with the skin-friendly stripe rust race CYR32. Detailed Implementation

[0051] Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0052] Taichung29*6 / Yr5: This is recorded in the literature “Xu Shichang, Wu Liren, Wan Anmin, Wang Fengle, Zhao Wensheng, Niu Yongchun. Progress in the breeding of wheat stripe rust resistant near-isogenic lines based on Taichung29 [J]. Plant Protection, 2004, 30(02):19-22.”

[0053] The pCaBS-γ vector in the following examples is the pCa-γbLIC vector from the literature “Yuan, C., C. Li, L. Yan, A.O. Jackson, Z. Liu, C. Han, J. Yu and D. Li (2011). A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS One 6(10):e26468”.

[0054] pCaBS-α vector: Described in the document "Yuan, C., C. Li, L. Yan, AO Jackson, Z. Liu, C. Han, J. Yu and D. Li (2011). A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS One 6(10):e26468".

[0055] pCaBS-β vector: described in the document "Yuan, C., C. Li, L. Yan, AO Jackson, Z. Liu, C. Han, J. Yu and D. Li (2011). A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots. PLoS One 6(10):e26468".

[0056] The information is described in the literature “Yuan,C.,C.Li,L.Yan,AO Jackson,Z.Liu,C.Han,J.Yu andD.Li (2011).A high throughput barley stripe mosaic virus vector for virus-induced gene silencing in monocots and dicots.PLoS One 6(10):e26468”.

[0057] The wheat stripe rust fungus is physiological race CYR32, which is recorded in the literature "Wan Anmin, Wu Liren, Jin Shelin, Yao Ge, Wang Baotong. Nomenclature and characteristics of wheat stripe rust fungus CYR32 in China [J]. Acta Plant Protection Sinica, 2003, 30(04):347-352".

[0058] Wheat Fengchan No. 3: It is recorded in the literature "Gan Aihua, Lin Ruiming, Xu Shichang, Wan Anmin, Ma Zhanhong. Genetic analysis of stripe rust resistance of wheat stripe rust identification host Fengchan No. 3 in China [J]. Journal of Plant Protection, 2006, 33(04):369-373.

[0059] Example 1: TaBURP1, a protein associated with stripe rust resistance in wheat, can regulate wheat resistance to stripe rust.

[0060] This embodiment provides a stripe rust-resistant protein (named TaBURP1) derived from the wheat stripe rust-resistant near-isogenic line Taichung29*6 / Yr5. Its sequence is sequence 2 in the sequence listing. In the wheat stripe rust-resistant near-isogenic line Taichung29*6 / Yr5, the cDNA sequence of the TaBURP1 gene is sequence 1 in the sequence listing. Positions 64-1584 of sequence 1 encode the TaBURP1 protein.

[0061] The sequence of sequence 1 is shown below:

[0062]

[0063]

[0064] Nucleotides 64-1584 of sequence 1 in the sequence listing are the coding sequence, which encodes the protein shown in sequence 2 in the sequence listing.

[0065] The sequence of sequence 2 is shown below:

[0066]

[0067] Construction of gene interference vectors:

[0068] Using cDNA from the near-isogenic wheat line Taichung29*6 / Yr5 resistant to stripe rust as a template, PCR amplification was performed using primers VIGS-BURP1F1 / VIGS-BURP1R1, and the target fragment BURP1VIGS was recovered and purified. The BURP1VIGS fragment was digested with ApaI restriction endonuclease, and the digested product was recovered. The pCaBS-γ vector was linearized using ApaI restriction endonuclease, and the vector backbone was recovered and purified. 10 ng of the vector backbone and 100 ng of the BURP1VIGS digested product were mixed thoroughly, and ligation was performed using T4 DNA polymerase. The ligation product was then transformed into competent *E. coli* cells. The correct recombinant vector was extracted from positive clones, and the recombinant vector with the correct sequence was designated pCaBS-γ-BURP1VIGS. pCaBS-γ-BURP1VIGS is a recombinant vector obtained by reverse insertion of the DNA fragment shown in positions 31-204 of sequence 1 in the sequence listing into the pCaBS-γ vector using ApaI restriction endonuclease. The primer sequences used are as follows:

[0069] VIGS-BURP1F1: 5'-AAGGAAGTTTAAGAGCTGTCGTCAGTTTCCTCA-3' (Italics indicate the connector sequence);

[0070] VIGS-BURP1R1:5'-AACCACCACCACCGTTTGAGAGAGGGAGCTTGGCA-3 (Italics indicate the connector sequence).

[0071] Wheat inoculation with virus for gene interference:

[0072] pCaBS-α, pCaBS-β, pCaBS-γ, and pCaBS-γ-BURP1VIGS vectors were introduced into EHA105 Agrobacterium cells, and the resulting recombinant bacteria were designated as EHA105 / pCaBS-α, EHA105 / pCaBS-β, EHA105 / pCaBS-γ, and EHA105 / pCaBS-γ-BURP1VIGS, respectively.

[0073] EHA105 / pCaBS-α, EHA105 / pCaBS-β, and EHA105 / pCaBS-γ-BURP1VIGS were mixed at equal concentrations and injected into well-grown Nicotiana Bunsenata as a transitional host to establish a BSMV-mediated gene interference wheat system. 7-10 days after injection, 1-2 leaves from the injected Nicotiana Bunsenata leaves and the uppermost leaf of the injected leaf were taken. Each gram of leaf was mixed with 3 mL of 50 mM PB (pH 7.0) and a small amount of purified diatomaceous earth, ground, and the juice was used to inoculate wheat variety Fengchan 3. Virus symptoms were observed 7 days after inoculation. The virus obtained from EHA105 / pCaBS-α, EHA105 / pCaBS-β, and EHA105 / pCaBS-γ-BURP1VIGS was designated BSMV-TaBURP1.

[0074] Following the above method, EHA105 / pCaBS-γ-BURP1VIGS was replaced with EHA105 / pCaBS-γ, with all other steps remaining unchanged. An experiment was conducted as a control. The viruses obtained from EHA105 / pCaBS-α, EHA105 / pCaBS-β, and EHA105 / pCaBS-γ were denoted as BSMV.

[0075] Following the above method, wheat was inoculated with uninoculated *Nicotiana bungeana* as a blank control.

[0076] Identification of stripe rust resistance in plants with gene interference:

[0077] Wheat materials inoculated with BSMV, BSMV-TaBURP1, and a blank control were inoculated with a compatible wheat stripe rust fungus using the "sweeping inoculation method" 14 days after wheat inoculation. The inoculation amount for each wheat species was equal. After inoculation, the materials were kept in the dark and moist at 10±1℃ for 24 hours, and then transferred to an incubator (22-24℃) for incubation until the disease developed and urediniospores were produced. Biomass was measured and the density of stripe rust spores was calculated.

[0078] Detection of TaBURP1 gene expression level after gene interference: Wheat leaves were sampled 8 days after inoculation with stripe rust. Total RNA was extracted from wheat using TRIZOL, and first-strand cDNA was synthesized using the MMLV reverse transcription kit. The expression level of TaBURP1 gene was detected using real-time quantitative PCR (qRT-PCR). Primers for qRT-PCR were designed using constitutively expressed genes as internal controls. The internal control gene was wheat ADP-RF (ADP-RIBOSYLATION FACTOR, Ta54227), and the primer sequences were TaADP-RF1: 5'-CAGCTGCTGACTGAGATGGA-3', TaADP-RF2: 5'-ATGTCTGGCCTGTTGGTAGC-3'. The primer sequences for the TaBURP1 gene were qBURP1f: 5'-GGCACGAGGAGCAGATCATCC-3', qBURP1r: 5'-GAGCTTGGCATAGGGGTGTT-3'.

[0079] The experiment was repeated in triplicate, and the results were averaged. The results showed that the relative expression level of the TaBURP1 gene in wheat materials inoculated with BSMV-TaBURP1 was significantly lower than that in wheat materials inoculated with BSMV and the blank control. There was no significant difference in the relative expression level of the TaBURP1 gene between wheat materials inoculated with BSMV and the blank control.

[0080] The results of the interference plant resistance test to wheat stripe rust are as follows: Figure 1 As shown, spore masses in wheat materials inoculated with BSMV-TaBURP11 ruptured and became visible 11 days after inoculation with stripe rust, with a significantly shorter incubation period compared to wheat materials inoculated with BSMV and the control. At 16 days post-inoculation, there was no significant difference in spore mass density among the three wheat materials, but the length and number of spore masses in wheat materials inoculated with BSMV-TaBURP1 were significantly higher than those in wheat materials inoculated with BSMV and the control, indicating a higher severity of stripe rust in these materials. This suggests that inhibiting TaBURP1 gene expression significantly reduces wheat stripe rust resistance, and that TaBURP1 and its encoding gene can regulate wheat stripe rust resistance.

Claims

1. The application of suppressing the expression of the gene encoding the stripe rust resistance-related protein shown in SEQ ID No. 2 in the sequence listing in reducing the resistance of wheat to stripe rust; the gene encoding the stripe rust resistance-related protein is as follows: 1) or 2): 1) The coding sequence is the cDNA molecule located at positions 64-1584 of sequence 1 in the sequence listing; 2) The DNA molecule shown in sequence 1 of the sequence listing; The expression of the gene encoding the stripe rust-related protein shown in SEQ ID No. 2 of the sequence listing is inhibited by introducing a DNA molecule that is reverse complementary to the DNA fragment shown in nucleotides 31-204 of sequence 1 in the sequence listing into wheat.

2. Methods for cultivating susceptible transgenic plants, including: Suppressing the expression of the gene encoding the stripe rust-resistant protein shown in SEQ ID No. 2 in wheat sequence listing yields susceptible transgenic wheat with reduced resistance to stripe rust; stripe rust is a disease caused by wheat stripe rust fungus; the suppression of the expression of the gene encoding the stripe rust-resistant protein in wheat is achieved by introducing a DNA molecule that is reverse complementary to the DNA fragment shown in nucleotides 31-204 of sequence 1 in sequence listing into wheat.

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