Application of wheat disease-resistant factor TatrxH4 in prevention and treatment of gibberellic disease

Abstract

The invention discloses application of a wheat disease-resistant factor TatrxH4 in prevention and treatment of gibberellic disease, and belongs to the technical field of genetic engineering, and a coding gene CDS sequence of the wheat disease-resistant factor TatrxH4 is shown as SEQ ID NO: 1; the amino acid sequence of the wheat disease-resistant factor TatrxH4 is as shown in SEQ ID NO: 2; the wheat disease-resistant factor TatrxH4 plays a positive regulation role in immune response of wheat to gibberellic disease, and over-expression of the wheat disease-resistant factor TatrxH4 improves the resistance of wheat to gibberellic disease. According to the invention, an agrobacterium tumefaciens-mediated method and an RNA interference technology are adopted to obtain a wheat strain of an overexpressed disease-resistant factor TatrxH4, and the disease-resistant factor TatrxH4 is identified according to the morbidity condition, disease index and expression condition of disease-resistant related marker genes of an overexpressed plant and common Fielder wheat infected with fusarium graminearum PH-1, so that the disease-resistant factor TatrxH4 is identified. The wheat disease-resistant factor TatrxH4 is known to play a positive regulation role in the immune response of wheat scab resistance, so that the wheat disease-resistant factor TatrxH4 can be used for creating a scab-resistant wheat germplasm material and plays a practical role in prevention and treatment of the wheat scab on the basis of the positive regulation role of the wheat disease-resistant factor TatrxH4.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and specifically to the application of the wheat disease resistance factor TaTrxH4 in the control of Fusarium head blight. Background Technology

[0002] Fusarium head blight (FHB) is a devastating disease of wheat caused primarily by Fusarium graminearum. It not only leads to reduced wheat yields but also poses a threat to human and animal health due to its toxins. Therefore, improving wheat's resistance and creating resistant varieties using molecular techniques have become crucial methods for controlling Fusarium head blight.

[0003] Fusarium graminearum exhibits strong environmental adaptability, overwintering or oversummering in soil and crop debris (such as straw and rachis) for 1-2 years, and tolerating a wide temperature range. It is primarily airborne, with its conidia and ascospores spreading several kilometers by wind and rain, settling on crop ears or leaves. High field humidity and continuous rainy weather greatly promote germination and infection of the host. When Fusarium graminearum infects wheat, it damages the spikelets, causing shriveled and wrinkled grains, and in severe cases, the entire ear withers, resulting in reduced wheat yield. In addition, Fusarium graminearum produces a variety of toxins during infection, among which deoxynivalenol (DON, commonly known as vomitoxin) is the most common and most harmful. DON is stable and difficult to completely remove even after grain processing (such as milling and cooking). After humans or livestock consume toxic grains or feed, they may experience symptoms such as vomiting, diarrhea, and loss of appetite. Long-term intake may also damage the immune and reproductive systems.

[0004] Promoting the planting of disease-resistant varieties is the most economical and effective way to control the spread of wheat diseases, and screening for superior disease-resistant genes is the foundation for creating disease-resistant varieties. However, because wheat resistance to Fusarium head blight is a complex quantitative trait with a complex genetic basis and is easily affected by the environment, there are certain difficulties in screening and utilizing resistance genes. Currently available germplasm resources and resistance genes are extremely scarce, and there is an urgent need to discover new disease-resistant genes, expand methods for wheat resistance to Fusarium head blight, and create new disease-resistant germplasm materials.

[0005] Thioredoxin (Trx) family members are low-molecular-weight (12-14 kDa) redox regulators widely distributed in prokaryotes and eukaryotes. Their active site contains a highly conserved WC[G / P]PC pentapeptide domain. This motif participates in the regulation of the electron transport chain and numerous intracellular biochemical processes through disulfide bond reduction. They are widely distributed in plants, animals, bacteria, and yeast. In plants, Trxes have various biological functions, including maintaining redox balance, participating in plant metabolism, gene transcription and translation, inhibiting apoptosis, and responding to biotic and abiotic stresses. Numerous studies have shown that most H-type thioredoxin members are involved in the salicylic acid (SA) signaling pathway-mediated defense responses. The salicylic acid signaling pathway genes NPR1 and NPR3 mediate antagonistic crosstalk between salicylic acid (SA) and jasmonic acid (JMA). Nuclear-localized NPR1 plays a role in the expression of SA-dependent defense genes (including PR-1), and it also appears to negatively regulate SA accumulation by inhibiting the expression of the salicylic acid synthesis pathway gene ICS1, thus playing a crucial role in plant growth, development, and stress responses. Therefore, elucidating the function of the disease resistance factor TrxH in wheat's resistance to Fusarium head blight infection is of great significance. Summary of the Invention

[0006] To clarify the role of wheat transcription factor TaTrxH4 in the interaction between wheat and pathogens, and to provide more superior disease-resistant genes, this invention provides the application of wheat disease resistance factor TaTrxH4 in the control of Fusarium head blight.

[0007] To solve the above problems, the present invention adopts the following technical solution.

[0008] Furthermore, in the above applications, the CDS sequence of the encoding gene of the wheat disease resistance factor TaTrxH4 is shown in SEQ ID NO:1; the amino acid sequence of the wheat disease resistance factor TaTrxH4 is shown in SEQ ID NO:2; and the wheat disease resistance factor TaTrxH4 plays a positive regulatory role in the immune response of wheat against Fusarium head blight.

[0009] Furthermore, in the above applications, overexpression of the wheat disease resistance factor TaTrxH4 enhances wheat's resistance to Fusarium head blight.

[0010] Based on this, the present invention provides a method for breeding wheat varieties resistant to Fusarium head blight, comprising: constructing transgenic wheat plants capable of overexpressing the wheat disease resistance factor TaTrxH4, wherein the amino acid sequence of the wheat disease resistance factor TaTrxH4 is shown in SEQ ID NO:2.

[0011] Furthermore, in the above-mentioned cultivation method, the method for constructing the transgenic wheat plant is as follows: constructing an overexpression recombinant vector and transferring it into wheat plants; the overexpression recombinant vector contains the encoding gene of the wheat disease resistance factor TaTrxH4.

[0012] Furthermore, in the above cultivation method, the upstream primer used to construct the overexpression recombinant vector is shown in SEQ ID NO:5, and the downstream primer is shown in SEQ ID NO:6.

[0013] Furthermore, in the above-mentioned cultivation method, the method of transferring the overexpression recombinant vector into wheat plants includes Agrobacterium-mediated transformation.

[0014] The present invention also claims protection for a wheat germplasm material resistant to Fusarium head blight obtained by the above method.

[0015] Compared with existing technologies, the advantages of this invention, "Application of wheat disease resistance factor TaTrxH4 in the control of Fusarium head blight," are as follows:

[0016] (1) This invention demonstrates the feasibility of using the wheat disease resistance factor TaTrxH4 to breed wheat varieties resistant to Fusarium head blight. This invention uses Agrobacterium-mediated transformation to initially obtain wheat lines overexpressing the disease resistance factor. Positive plants are then screened using Western blotting (WB) and RT-qPCR. By comparing the TaTrxH4-OE transgenic lines with ordinary Fielder wheat, the disease incidence, disease index, and expression levels of disease resistance-related marker genes after Fusarium graminearum infection were analyzed. This revealed that the wheat disease resistance factor TaTrxH4 plays a positive regulatory role in the immune response of wheat to Fusarium head blight, clarifying TaTrxH4 as a candidate gene with both disease resistance and agronomical safety, providing a theoretical basis and key target for green control and disease-resistant breeding of wheat Fusarium head blight.

[0017] (2) Based on the characteristics of the wheat disease resistance factor TaTrxH4, it can be used to create wheat germplasm materials resistant to Fusarium head blight and play a practical role in the prevention and control of wheat Fusarium head blight. The present invention provides a preferred method: constructing a recombinant overexpression vector containing the gene encoding the wheat disease resistance factor TaTrxH4, and then transferring it into wheat plants, and obtaining stable wheat lines capable of overexpressing the disease resistance factor through multiple generations of culture.

[0018] (3) This invention provides a technical approach for the breeding of wheat varieties resistant to Fusarium head blight, and at the same time provides a new method for the prevention and control of wheat Fusarium head blight, thereby reducing the pathogen resistance and environmental pollution caused by the overuse of pesticides. Attached Figure Description

[0019] Figure 1 Technical roadmap for constructing TaTrxH4 gene overexpression lines;

[0020] Figure 2 A comparison diagram of TaTrxH4 high expression lines and Fielder lines;

[0021] Figure 3 A comparison of SA content between TaTrxH4 gene overexpression lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) and control (Fielder) plants;

[0022] Figure 4 A comparison of the expression levels of the (TaNPR1, TaNPR3, TaICS1) genes between the (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) transgenic lines and the control (Fielder) plants;

[0023] Figure 5 A comparison of the resistance to Fusarium head blight between transgenic lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) and the control group (Fielder).

[0024] Figure 6 A comparison of the severity of Fusarium head blight in transgenic lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) and control (Fielder) plants;

[0025] Figure 7 A comparison of DON toxin levels in transgenic lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) and control (Fielder) plants after infection with Fusarium head blight.

[0026] Figure 8 A comparison of mature wheat ears between transgenic lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) and the control group (Fielder);

[0027] Figure 9 A comparison of plant height between transgenic lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) and the control group (Fielder);

[0028] Figure 10 A comparison of the thousand-grain weight of mature wheat between transgenic lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) and the control group (Fielder);

[0029] Figure 11 A comparison of grain traits between transgenic lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) and the control group (Fielder); Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] This embodiment provides the method for obtaining the gene encoding the wheat disease resistance factor TaTrxH4.

[0033] The nucleotide sequence of the gene encoding the wheat disease resistance factor TaTrxH4 was obtained from the website https: / / www.ncbi.nlm.nih.gov / search / all / ?term=XP_044318847.1. Primers for amplifying the full length of the TaTrxH4 gene were designed based on this nucleotide sequence. The primers used are:

[0034] TaTrxH4-F: CATTTACAATTATCGATATGGGGGGCTGTGTGGGCAA (SEQ ID NO: 3)

[0035] TaTrxH4-R:CTCACCCTAGGACTAGTACTGCCATCACCAGAGCTT(SEQ ID NO:4)

[0036] (1) Using cDNA from Fielder wheat plants as a template, the TaTrxH4 gene was amplified and sequenced. The CDS sequence of the TaTrxH4 gene is shown in SEQ ID NO:1, and the encoded amino acid sequence is shown in SEQ ID NO:2. The primers used were:

[0037] SEQ ID NO:1:

[0038] MVSLSFTSSLQLGAQAIRTRRDESGWSTFYPPWIFVKASELKKMGLSSSLVPMASALLLLCC

[0039] FTAWNAAA

[0040] AAASGGGGDGLRLNYYSESCPRAEEIVKEQVRRLYEEHGNTAVSWLRALFHDCTVKSCDASL

[0041] LLETDAAT

[0042] GLVSEQASPRSFGMRNFKYVGAIKSALERECPGTVSCADVLALAARDGAAMLGGPAAIPMRT

[0043] GRRDATES

[0044] RYGEVERYVPNHNDTVSAVLSRFAAMGLDAEAVVALLGAHSVGRVHCNNLVARLYPAVDGGM

[0045] EPAYGAYL

[0046] RGRCPTADAREDTRDVAYARNDRATPMVLDNMYHKNLLKGRGLLLVDQRLASDPRTAPFVKK

[0047] MAADNGYF

[0048] RETFAAALVRMSENGPLTGGQGEVRKDCRFVNAK

[0049] SEQ ID NO:2

[0050] ATGGAGCGGAGATGATCGTGAGTAACATTGTAAAGGAGG

[0051] AGGTGAACCGCGACCGTGGCATCGGCGCCGGGCTCATCCGCCTCTTGTTCCACGACTGCTTC

[0052] GTCCAGGG

[0053] TTGCGATGGGTCTGTCCTCCTTGACATTAGCGCCACGCCCAACGAGCCGACCGAGAAGGACG

[0054] GCATCCCC

[0055] AACAGGCGCAGCCTCCGCGGCTTCGAGGTGATCGACAGGATCAAGGACGCGCTGGAGGCCAC

[0056] GCCCGGGT

[0057] GCGAGCGTGTCGTCTCGTGCGCGGACATCGTCGCCTTTGCTGCGCGCGACGCCACCTACTTC

[0058] CTCAGCAA

[0059] CGAGACGATGTACTTCGAAATGCCGTCGGGCCGCTACGACGGGAACATGTCCCTCGCGAGCG

[0060] AGACCCTC

[0061] CCCAACCTGCCCCCTCCCTTCGCCGACATCACGATGCTCGAGGGTTTGTTCGCGAACAAGGG

[0062] CCTCAGCC

[0063] TCGACGACATGGTCACCCTCTCCGGCGCGCACTCCGTCGGCGTCTCCCACTGCTCGTCCTTC

[0064] CGTGACCG

[0065] CCTGCCGCCCAACCCTTCTTCGGATCCCATGGCCATGAACTCTACGATGGCCAACTTGGTGA

[0066] CAAGTCAG

[0067] TGCAGCAGAGGCGACAACCCTACGGTGGATCAGGACATCTACACCCCTGGATACCTGGACAA

[0068] CCAATACT

[0069] ACAAGAACGTGATTAGCCACGAAGTGTTGTTGAAATCAGATGCCGCGCTCGAGTCGCCCAAG

[0070] ACACTTGA

[0071] ATCCGTGAAACAAAATGCTAAGTTCTCTGTGGACTGGGAGTTAAAGTTCGGGGAAGCCATGG

[0072] TGAAGATG

[0073] GGCAACATCGACGTGAAGACCAGCAAGAATGGGGAGATCAGACACAAGTGCTGGTCCATCAA

[0074] CAAGAACT

[0075] ACTCCTAAGTGACTAG

[0076] Example 2

[0077] This embodiment provides the identification of disease resistance of TaTrxH4 and the construction of transgenic lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3).

[0078] Specific quantitative PCR primers were designed based on the sequences of disease resistance-related marker genes TaPR1, TaPR2, and the elongation factor gene TaActin. The qRT-PCR primer sequences are as follows:

[0079] RT-actin-F:ATCCACGTCACCACTTTCAA(SEQ ID NO:7)

[0080] RT-actin-R: TGCCTTGAGATCCACATTTG (SEQ ID NO:8)

[0081] TaNPR1-qPCR-F: ACGACCTGCAAGATACGCTTC (SEQ ID NO:9)

[0082] TaNPR1-qPCR-R:GTTCGACCTGCCAAGTTCC (SEQ ID NO:10)

[0083] TaNPR3-qPCR-F:ATGGAGACGTCGACCGTCAC(SEQ ID NO:11)

[0084] TaNPR3-qPCR-R: TCACCGCGACAGCCTCGCCTTCTTG (SEQ ID NO:12)

[0085] TaICS1-qPCR-F: AGAAATGAGGACGACGAGTTTGAC (SEQ ID NO: 13)

[0086] TaICS1-qPCR-R:CCAAGTAGTGCTGATCTAATTCCAA(SEQ ID NO:14)

[0087] According to such Figure 1The technical route shown describes how, by means of existing technology, those skilled in the art can construct the full-length TaTrxH4 gene sequence (SEQ ID NO:1) amplified in Example 1 into the overexpression vector CUB via a homologous recombination gateway reaction, forming the recombinant overexpression vector TaTrxH4-CUB. The successfully constructed recombinant overexpression vector TaTrxH4-CUB was then transfected into Fielder wheat plants using Agrobacterium (EHA105)-mediated transformation to obtain transgenic plants capable of overexpressing the TaTrxH4 gene.

[0088] The primers used to construct the recombinant overexpression vector TaTrxH4-CUB were:

[0089] Cub-TaTrxH4-F:caggtcgactctagaggatccATGGGGGGCTGTGTGGGCAA(5)SEQ ID NO:5

[0090] Cub-TaTrxH4-R:gagctcggtacccggggatccTCACTTATCATCATCATCCTTATAATC TCCCTTATCATCATCATCCTTATAATCTCCCTTATCATCATCCTTATAATCACTGCCATCACC AAGAGCTT(6) SEQ ID NO: 6.

[0091] Transgenic plants were subjected to PCR and Western blot detection (WB and RT-qPCR techniques). Based on the detection results, T0 generation positive lines with excellent expression (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) were selected, and high-generation wheat lines that could stably overexpress the TaTrxH4 gene were obtained through extended culture.

[0092] Example 3

[0093] This embodiment provides the identification of disease resistance and agronomic trait characterization of transgenic lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3).

[0094] The resistance of the above-mentioned high-generation wheat lines was determined by inoculating them with Fusarium graminearum PH-1, using Fielder wheat plants as a control. The average disease severity and disease index of the transgenic wheat spikes in the T3 generation TaTrxH4 gene overexpression lines (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) and the control (Fielder) wheat plants (TaTrxH4-OE-1, TaTrxH4-OE-2, TaTrxH4-OE-3) were 1.50. Compared to PH-1, the average severity decreased by 1.80, the disease index decreased by 32.50%, and the yield of DON, an important virulence factor of Fusarium graminearum, was also significantly reduced. Furthermore, agronomic traits were analyzed. Compared to the control plant Fielder, there were no significant differences in ear morphology, grain traits (thousand-grain weight, grain length, and grain width), plant height, and grain weight per ear in the transgenic wheat plants. In conclusion, overexpression of TaTrxH4 in wheat enhanced resistance to Fusarium graminearum without significant changes in agronomic traits.

[0095] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. The application of wheat disease resistance factor TaTrxH4 in the control of Fusarium head blight, including wheat disease resistance factor TaTrxH4, characterized by: The aforementioned prevention and control method involves overexpressing the wheat disease resistance factor TaTrxH4 to enhance wheat's resistance to Fusarium head blight. The amino acid sequence of the wheat disease resistance factor TaTrxH4 is: SEQ ID NO:1; The pathogen causing wheat scab is Fusarium graminearum. The CDS sequence of the gene encoding the wheat disease resistance factor TaTrxH4 is SEQ ID NO:

2.

2. The application of the wheat disease resistance factor TaTrxH4 according to claim 1 in the control of Fusarium head blight, characterized in that: When the pathogen of wheat scab was used to infect the TaTrxH4 gene overexpressing lines, the disease resistance marker genes TaNPR1, TaNPR3 and TaICS1 were all upregulated in the TaTrxH4 gene overexpressing lines.

3. A method for breeding a wheat variety resistant to Fusarium head blight, characterized in that: Overexpression of the wheat disease resistance gene TaTrxH4 as described in claim 1 in wheat plants.

4. The method for breeding a wheat variety resistant to Fusarium head blight according to claim 3, characterized in that: An overexpression recombinant vector was constructed and transformed into wheat plants; the overexpression recombinant vector contained the encoding gene of the wheat disease resistance factor TaTrxH4.

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