Application of wheat transcription factor TaHRP1 in wheat disease resistance breeding

Abstract

The present invention discloses the application of wheat transcription factor TaHRP1 in wheat disease resistance breeding, belongs to the field of gene engineering technology, and discloses the application of wheat transcription factor TaHRP1 in improving wheat stripe rust resistance, regulating wheat stripe rust resistance, cultivating wheat stripe rust resistant varieties or wheat disease resistance breeding. The amino acid sequence of the wheat transcription factor TaHRP1 is shown in SEQ ID NO: 2. The present invention finds that the TaHRP1 gene plays a positive regulatory role in the defense response of wheat against stripe rust, that is, the TaHRP1 gene can improve the ability of wheat to resist stripe rust. When the TaHRP1 gene is overexpressed in the plant body, it can transcriptionally activate the expression of a series of downstream disease resistance functional genes, giving the transgenic plant a good disease resistance effect. The present invention confirms that the TaHRP1 gene can be used as a target gene to improve wheat disease resistance, and has good application prospects in breeding disease-resistant wheat varieties.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of wheat transcription factor TaHRP1 in wheat disease resistance breeding. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the most widely distributed, planted, and commercially available food crops in the world. Wheat stripe rust, caused by Puccinia striiformis f.sp.tritici, is an extremely serious epidemic fungal disease that seriously affects wheat yields. Planting disease-resistant varieties is currently the most environmentally friendly and effective disease prevention and control measure, but variety resistance can be overcome by toxic mutations in pathogenic stripe rust species, leading to another outbreak of the disease. Therefore, continuously exploring new wheat stripe rust resistance genes and creating durable disease-resistant varieties are the fundamental ways to prevent and control wheat stripe rust.

[0003] In response to the invasion of pathogens, plants have evolved sophisticated defense mechanisms. The first layer of plant immunity is triggered when conserved molecular structures called microbe / pathogen-associated molecular patterns (MAMPs / PAMPs) are sensed by plasma membrane-localized pattern recognition receptors (PRRs). Immunity triggered by this pattern is called PAMP / pattern-triggered immunity (PTI); adapted pathogens have acquired many virulence mechanisms that suppress plant immunity, such as by secreting effector proteins. As a countermeasure, plants have evolved a second layer of the innate immune system to directly or indirectly recognize such effector proteins, thereby initiating effector-triggered immunity (ETI).

[0004] In the process of mutual game between plants and pathogens, there is a complex regulatory network and its signal transduction, and transcription factors (TF) play a very important role. Transcription factors, also known as trans-acting factors, activate or inhibit the transcriptional activity of target genes by binding to specific DNA elements (cis-acting elements) upstream of target genes, thereby regulating the spatiotemporal specific expression of target genes.

[0005] Therefore, studying the transcription factor-regulated disease resistance signaling pathway during the interaction between wheat and stripe rust and revealing the molecular mechanism of transcription factor resistance to stripe rust are of great significance for the breeding of excellent resistant varieties, the rational use of resistant varieties and the long-term control of wheat stripe rust. Summary of the invention

[0006] The purpose of the present invention is to provide an application of wheat transcription factor TaHRP1 in wheat disease resistance breeding to solve the problems existing in the above-mentioned prior art. The present invention finds that wheat transcription factor TaHRP1 and TaHRP1 gene play a positive regulatory role in the defense response of wheat to stripe rust, can improve the ability of wheat to resist stripe rust, have good application prospects in breeding disease-resistant wheat varieties, and lay an important foundation for wheat disease resistance breeding.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides the use of wheat transcription factor TaHRP1 in any of the following items:

[0009] (1) Application in improving wheat stripe rust resistance;

[0010] (2) Application in regulating wheat stripe rust resistance;

[0011] (3) Application in breeding wheat varieties resistant to stripe rust;

[0012] (4) Application in wheat disease resistance breeding;

[0013] The amino acid sequence of the wheat transcription factor TaHRP1 is shown in SEQ ID NO:2.

[0014] The present invention also provides the use of the coding gene of the wheat transcription factor TaHRP1 in any of the following items:

[0015] (1) Application in improving wheat stripe rust resistance;

[0016] (2) Application in regulating wheat stripe rust resistance;

[0017] (3) Application in breeding wheat varieties resistant to stripe rust;

[0018] (4) Application in wheat disease resistance breeding;

[0019] The nucleotide sequence of the coding gene is shown in SEQ ID NO:1.

[0020] The present invention also provides the use of a recombinant vector comprising the encoding gene in any of the following:

[0021] (1) Application in improving wheat stripe rust resistance;

[0022] (2) Application in regulating wheat stripe rust resistance;

[0023] (3) Application in breeding wheat varieties resistant to stripe rust;

[0024] (4) Application in disease-resistant wheat breeding.

[0025] The present invention also provides the use of an engineered bacterium comprising the recombinant vector in any of the following:

[0026] (1) Application in improving wheat stripe rust resistance;

[0027] (2) Application in regulating wheat stripe rust resistance;

[0028] (3) Application in breeding wheat varieties resistant to stripe rust;

[0029] (4) Application in disease-resistant wheat breeding.

[0030] Furthermore, the resistance of wheat to stripe rust is improved by up-regulating the expression level of the wheat transcription factor TaHRP1 or the encoding gene.

[0031] The present invention also provides a method for creating a stripe rust-resistant transgenic wheat variety, comprising the step of introducing a biological material containing a wheat transcription factor TaHRP1 encoding gene into a recipient plant; the nucleotide sequence of the wheat transcription factor TaHRP1 encoding gene is shown in SEQ ID NO: 1.

[0032] Furthermore, the biological material comprises a recombinant vector, an expression cassette and an engineered bacterium.

[0033] The present invention discloses the following technical effects:

[0034] The present invention has found through gene function research that the TaHRP1 gene plays a positive regulatory role in the defense response of wheat against stripe rust, that is, the TaHRP1 gene can improve the ability of wheat to resist stripe rust. When the TaHRP1 gene is overexpressed in the plant, it can transcriptionally activate the expression of a series of downstream disease resistance functional genes, giving the transgenic plant a good disease resistance effect. The present invention confirms that the TaHRP1 gene can be used as a target gene to improve the disease resistance of wheat, has a good application prospect in cultivating disease-resistant wheat varieties, and lays an important foundation for wheat disease resistance breeding.

[0035] Compared with traditional disease-resistant breeding technology, the plant disease-resistant genetic engineering technology adopted in the present invention can break through the reproductive isolation and distant hybridization incompatibility between species, achieve targeted improvement of target traits in a shorter period of time, and provide more comprehensive, continuous, broad-spectrum and specific protection for crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 Figure 1 is a diagram for identifying TaHRP1 transgenic wheat materials; wherein (a) tahrp1 edited plants are schematic diagrams of editing TaHRP1 genes on chromosomes A, B, and D; (b) a diagram showing the results of determining the expression level of TaHRP1 in TaHRP1 overexpressing transgenic wheat materials; (c) a diagram showing the results of determining the protein level of TaHRP1 in TaHRP1 overexpressing transgenic wheat materials;

[0038] Figure 2 The results show that the T2 generation tahrp1-edited plants have weakened the ability of wheat to resist stripe rust; (a) is the phenotype of the leaves of the tahrp1 gene-edited plants infected with stripe rust; (b) is the biomass level of stripe rust on the leaves of the gene-edited plants; (c) is the histological observation at 24h and 48h after stripe rust infection; (d) and (e) are the statistical graphs of hyphae length (d) and hyphae area (e) at 24h and 48h after stripe rust infection;

[0039] Figure 3 The results show that TaHRP1 overexpression transgenic materials have improved wheat resistance to stripe rust; (a) is the phenotype of TaHRP1 overexpression transgenic plants after leaves are infected with stripe rust; (b) is the level of stripe rust biomass on the leaves of TaHRP1 overexpression transgenic plants; (c) and (d) are the expression levels of disease resistance-related genes TaPR1 (c) and TaPR2 (d) after TaHRP1 overexpression transgenic plants are inoculated with stripe rust. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1 Creation and identification of TaHRP1 transgenic edited plants

[0046] The CDS sequence of the wheat TaHRP1 gene has a nucleotide sequence as shown in SEQ ID NO: 1, and the amino acid sequence of the transcription factor TaHRP1 is shown in SEQ ID NO: 2:

[0047] ATGGATCCGCCGCACCCTCCACACCACCACCACCACCACCGGCTCCACCTCCGCCTCGACCCCGGCCACCACCACCGCATCCACATCCACCTCCGCCACCACGCCGCCCACCTCCTGCCCGCCGCCCGCTCCCTGTGCCCACCACCACCACACATCGGTTCCTCTTCCTCCTCCCCACACGCTTCCCCACCCGCCGACCGCGGCGGAAGGACCA CTCCCCGGGCCGCAGGGAGAGGCGGCGGGGGACGCGGATGAGCCGGCGCCGCAGGCGGAGCGAGGAGAGGAAGTTTACCTCGGGCAGGAAGAAGAGTGGGGGGAGGAGGAGGAGGAGCCGGTGTTCGTTCTGACGGAGGAGTGGGCCGAGTTCTTCGCCAAGGCCGACGCCAAGAGGAGGCTGGCCAAGCAGCAGAGGAAGAACCGGAAGAAATAG(SEQ ID NO: 1);

[0048] MDPPHPPHHHHHHHRLHLRLDPGHHHRIHIHLRHHAAHLLPAAAPCAHHHHHTSVPL PPPHTLPHPPTAAEGPLPGPQGEAAGDADEPAPQAERGEEVYLGQEEEWGEEEEEPVFVLTEEWAEFFAKADAKRRLAKQQRKNRKK* (SEQ ID NO: 2).

[0049] 1sgRNA target design

[0050] According to the genomic sequence of wheat TaHRP1 gene and the requirements of CRISPR / Cas9 technology target design, a guide RNA (gRNA) sequence contained on chromosomes A, B and D of wheat TaHRP1 gene was designed, and its sequence was GAGGAGAGGAAGTTTACCT, and the gRNA was constructed into the gene editing vector.

[0051] The construction method of CRISPR-Cas9-gRNA vector is as follows: First, take 100 μL of MT1T2-F and MT1T2-R primers, and take 6 μL of MT1T2-F0 and MT1T2-R0 and mix them. Configure the following system: 2 μL of mixed primers, 1 μL of pCBC-DT1T2 vector, 17 μL of ddH2O, and 23 μL of Mix enzyme. The PCR reaction program is 98℃45s; 98℃15s; 60℃30s; 72℃1min; 35 cycles, 72℃5min. The four primers are: TaHRP1-MT1T2-F: ATATATGGTCTCTGGCGACTCAGGACCACTCCCCGGGCCCGCAGG; TaHRP1-MT1T2-F0: gACTCAGGACCACTCCCCGGGCCGCAGGGTTTTTAGAGCTAGA AATAGC; TaHRP1-MT1T2-R0: CCCGAGGTAAACTTCCTCTCCTCGAGTCGCTTCTTGGTGCC; TaHRP1-MT1T2-R: ATTATTGGTCTCTAAACCCCGAGGTAAACTTCCTCTCCTCGAGT.

[0052] After the amplified product was cut and recovered, the following system was used for ligation: 2 μL of PCR product, 2 μL of pBUE411 (stored by the Wheat Institute of Sichuan Agricultural University), 1.5 μL of 10x NEB buffer, 1.5 μL of 10x BSA, 1 μL of BsaI, 1 μL of T4 ligase, and 6 μL of ddH2O. The reaction procedure was: 37°C, 5 h; 50°C, 5 min; 50°C, 10 min.

[0053] 2. Agrobacterium-mediated genetic transformation of wheat callus

[0054] The recipient material wheat variety Fielder was planted with a light cycle of 24°C and 16h; the dark cycle was 20°C and 8h. Seeds were taken 14-16 days after the recipient material bloomed, and the immature embryos were separated. The constructed CRISPR-Cas9-gRNA vector was transformed into the EHA105 Agrobacterium strain, and the positive single spot was picked and inoculated into the MGL liquid culture medium, and cultured in the dark at 28°C overnight. The collected bacterial liquid was resuspended and mixed with the infection solution and added to the immature embryos of the recipient material for infection. After two days of co-culture, the hypocotyls of the immature embryos were removed. After recovery culture, screening culture, differentiation culture and rooting culture, positive plants transferred with CRISPR-Cas9-gRNA were obtained. The offspring of the transgenic positive plants were tested and sequenced, and finally the transgenic plants (tahrp1) with successful editing of the TaHRP1 gene on chromosomes A, B and D were obtained (see Figure 1 (a)

[0055] 3. Identification of disease resistance of tahrp1 transgenic plants

[0056] To explore whether TaHRP1 is involved in the immune response of host wheat during stripe rust infection, tahrp1 transgenic plants and the control variety fielder were cultured in an incubator at 16°C and a light / dark cycle of 16h / 8h. When wheat grew to the two-leaf and one-heart stage, it was inoculated with stripe rust CYR23. Use a brush to dip the stripe rust in an appropriate amount of sterile water, evenly apply it on the wheat leaves, and culture it in a dark incubator at 10°C for 24h, and then culture it normally. The second leaf was inoculated with stripe rust CYR23 and samples were taken at different time points (0h, 24h, 48h, 120h) for observation.

[0057] On the 14th day after inoculation, the spore production of leaves of tahrp1 transgenic plants was significantly increased compared with the control group ( Figure 2 Middle a). 120h after inoculation, the biomass of spore-forming leaves was analyzed, and it was found that the relative biomass of stripe rust on the leaves of tahrp1 transgenic plants was significantly increased compared with the control ( Figure 2 b). The biomass detection method is as follows:

[0058] The double standard curves were drawn with plasmids containing wheat EF and stripe rust EF, and then the DNA of the leaves was extracted 120 hours after inoculation for quantification. The detection primers were TaEF-F and TaEF-R; PstEF1-F and PstEF1-R. The copies of wheat EF and stripe rust EF in the sample were calculated based on the results of the double standard curves, and the calculation was performed relative to the control group. The reaction system used was: 2μL template product, 0.5μL forward and reverse primers, 10μL quantitative MIX, 7μL ddH2O. The amplification program used was: 95℃, 10min; 40 cycles (95℃, 15s; 55℃, 20s); 95℃, 15s; 60℃, 1min; 95℃, 15s. The primers used were as follows: TaEF-F: TGGTGTCATCAAGCCTGGTATGGT, TaEF-R: ACTCATGGTGCATCTCAACGGACT, PstEF1-F: TTCGCCGTCCGTGATATGAGACAA, PstEF1-R: ATGCGTATCATGGTGGTGGAGTGA.

[0059] 24h and 48h after inoculation, the infected wheat leaves were cut into leaf segments of about 2cm, placed in a decolorizing solution, and decolorized in a 37℃ water bath until the leaf segments completely turned green. Rinse with 50mM Tris-HCl (pH=7.5) buffer 3 times, 10min each time. Add 20μg / mL concentration of wheat germ agglutinin fluorescent dye solution and stain in the dark for 24h. Observe and count the length and area of ​​hyphae under a fluorescence microscope. The results are as follows Figure 2 As shown in middle c, substomatal vesicles and invading hyphae can be observed in the leaves of tahrp1 transgenic plants and the control group, but the substomatal vesicles of tahrp1 transgenic plants are larger and the invading hyphae are longer than those of the control group ( Figure 2 d, e). These results indicate that the knockout of TaHRP1 gene affects the growth and development of stripe rust hyphae and may promote the expansion of invasive hyphae in the early stage of stripe rust infection, suggesting the important role of this transcription factor in wheat resistance to stripe rust.

[0060] Example 2 Creation and identification of TaHRP1OE overexpressing transgenic plants

[0061] 1. Creation of TaHRP1OE overexpressing transgenic wheat materials

[0062] In order to further clarify the role of TaHRP1 in stripe rust resistance, the CDS sequence of TaHRP1 was used to construct the overexpression vector pEarleyGate101 (stored in the Wheat Research Institute of Sichuan Agricultural University). The construction process is as follows: 2.5 μL of TaHRP1 CDS gel recovery product, 0.3-0.4 μL of intermediate vector pDONR 221 (100 ng), 0.2 μL of BP reaction enzyme, 2 μL of 1×TE Buffer. Incubate at 25°C overnight. Then transform E. coli to obtain the BP recombinant vector. Then perform LR recombination reaction. 2.5 μL of BP recombinant vector, 0.3 μL of pEarleyGate101, 0.2 μL of LR reaction enzyme, 2 μL of 1×TE Buffer. Incubate at 25°C overnight, then transform E. coli to obtain the TaHRP1 overexpression vector pEarleyGate101.

[0063] Referring to Example 1, the constructed overexpression vector pEarleyGate101 was transformed into EHA105 Agrobacterium strains, and then Agrobacterium was used to transform the TaHRP1OE overexpression transgenic wheat material. The second leaf of the T2 transgenic plant was used for quantitative detection of the expression level of TaHRP1 ( Figure 1 b). At the same time, plant total protein was extracted for TaHRP1 protein detection to identify positive plants of TaHRP1OE overexpressing transgenic wheat materials ( Figure 1c). The quantitative primers used to detect the expression of TaHRP1 are: TaHRP1-qRT-F: AGAGGAAGTTTACCTCGGGCA; TaHRP1-qRT-R: CGAAGAACTCGGCCCACTC. The system and procedures used refer to the above biomass detection method. The steps for TaHRP1 protein detection are as follows: 1) Grind 0.5g wheat leaf sample, add 2mL lysis buffer (10mM MgCl2, 1mM PMSF, 0.5% Triton X-100, 20mM HEPES-KOH, pH7.4, 5mM DTT, 1mM PMSF, 1% protease inhibitor cocktail), vortex vigorously, and place on ice. (2) Then centrifuge at 4℃ 12000g for 10min, take the supernatant and add an equal volume of 2x protein loading buffer, boil in a boiling water bath for 10 minutes, and then perform SDS-PAGE experiment.

[0064] Identification of disease resistance of 2TaHRP1OE overexpressing transgenic plants

[0065] On the 14th day after inoculation, the sporulation of leaves of TaHRP1OE transgenic plants was significantly reduced compared with the control group, and the hypersensitive necrosis was significantly increased ( Figure 3 After inoculation, the biomass of spore-forming leaves was analyzed and it was found that the relative biomass of stripe rust on the leaves of TaHRP1OE transgenic plants was significantly reduced compared with that of the control ( Figure 3 (b)

[0066] In order to further clarify the immune response of the host wheat during the stripe rust infection of the TaHRP1 gene, referring to Example 1, wheat leaves were taken 24 hours after the stripe rust inoculation, and the changes in the transcription levels of disease-resistant related genes were detected by qRT-PCR. The quantitative analysis procedures and detection steps of the disease-resistant related genes TaPR1 and TaPR2 were referred to the above-mentioned biomass detection method. The quantitative primers used are: TaPR1-F: GAGAATGCAGACGCCCAAGC; TaPR1-R: CTGGAGCTTGCAGTCGTTGATC; TaPR2-F: AGGATGTTGCTTCCATGTTTGCCG; TaPR2-R: AAGTAGATGCGCATGCCGTTGATG. The results are as follows. Figure 3 As shown in c and d, compared with the control, the expression levels of TaPR1 and TaPR2 in TaHRP1OE transgenic plants increased significantly, indicating that overexpression of the TaHRP1 gene enhanced the resistance of wheat to stripe rust. This further clarified the importance of the TaHRP1 gene in the resistance of wheat to stripe rust. In summary, the experimental results confirmed that TaHRP1 positively regulates the resistance of wheat to stripe rust and can be used as a target gene for molecular breeding to improve plant disease resistance.

[0067] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of wheat transcription factor TaHRP1 in any of the following: (1) Application in improving wheat stripe rust resistance; (2) Application in breeding wheat varieties resistant to stripe rust; The amino acid sequence of the wheat transcription factor TaHRP1 is shown in SEQ ID NO:

2.

2. Use of the gene encoding the wheat transcription factor TaHRP1 as claimed in claim 1 in any of the following: (1) Application in improving wheat stripe rust resistance; (2) Application in breeding wheat varieties resistant to stripe rust; The nucleotide sequence of the coding gene is shown in SEQ ID NO:

1.

3. Use of the recombinant vector comprising the coding gene according to claim 2 in any of the following: (1) Application in improving wheat stripe rust resistance; (2) Application in breeding of wheat varieties resistant to stripe rust.

4. Use of the engineered bacteria comprising the recombinant vector according to claim 3 in any of the following: (1) Application in improving wheat stripe rust resistance; (2) Application in breeding of wheat varieties resistant to stripe rust.

5. The use according to any one of claims 1 to 4, characterized in that: The wheat stripe rust resistance is improved by up-regulating the expression level of the wheat transcription factor TaHRP1 or the encoding gene.

6. A method for creating a transgenic wheat variety resistant to stripe rust, characterized in that: The method comprises the steps of introducing biological materials containing a wheat transcription factor TaHRP1 coding gene into a recipient plant; the nucleotide sequence of the wheat transcription factor TaHRP1 coding gene is shown in SEQ ID NO:

1.

7. The method according to claim 6, characterized in that The biological material comprises a recombinant vector, an expression box and an engineering bacterium.

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