A wheat TabZIP19-5B gene and its application in regulating wheat salt tolerance, antiviral ability and thousand-grain weight

Abstract

The present invention provides a wheat TabZIP19-5B gene and its application in regulating wheat salt tolerance, antiviral ability and thousand-grain weight, belonging to the technical field of plant genetic engineering. By using the existing plant genetic engineering technology, the present invention for the first time clones the gene TabZIP19-5B that regulates wheat salt tolerance, antiviral ability and thousand-grain weight, and transfers this gene into wheat by an Agrobacterium-mediated method. Through comparative test analysis, it is proved that the transgenic plants overexpressing TabZIP19-5B of the present invention have significantly improved salt tolerance and resistance to BYDV, and the grain width is greater than that of the wild type. The TabZIP19-5B gene provided by the present invention plays an important role in regulating salt tolerance, resistance to BYDV and thousand-grain weight, and can be used as an important gene resource for cultivating new crop varieties with salt tolerance, antiviral ability and high thousand-grain weight.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a wheat TabZIP19-5B gene and its application in regulating salt tolerance, antiviral ability and 1000-grain weight of wheat. Background Art

[0002] Salt stress has a serious impact on plant growth and development, and its harms mainly include osmotic stress, ion toxicity, etc. Under salt stress, the ion balance inside and outside plant cells is disrupted, resulting in plant water loss, stomatal closure, limited photosynthesis, and affecting the normal growth and metabolism of plants. In addition, salt stress can also lead to metabolic disorders in plants, the accumulation of substances such as reactive oxygen species, affect the photosynthesis of plants, and ultimately inhibit plant growth. Exploring and utilizing drought- and salt-tolerance related genes and cultivating new varieties of drought- and salt-tolerant crops are the most direct and effective ways to overcome drought and saline-alkali hazards.

[0003] Wheat yellow dwarf disease is a viral disease in which barley yellow dwarf viruses (BYDVs) are transmitted and infect host plants by the virus vector aphids in a persistent, circulative and non-proliferative manner. The typical symptoms of host plants infected by BYDVs are that the leaf tips begin to turn yellow and spread downward to the whole leaf, the spike grains decrease, the plants are dwarfed, the leaves are chlorotic, the growth is retarded, the heading is delayed, the leaves turn yellow and the leaf tips are purple, etc., thus reducing the crop yield and even causing serious yield reduction. Wheat yellow dwarf disease antiviral breeding is of great significance for improving crop yield, ensuring food security, promoting sustainable agricultural development and advancing agricultural science and technology.

[0004] As one of the largest transcription factor families in plants, bZIP plays an important role in plant stress response and growth and development. This type of transcription factor has a relatively conserved domain consisting of approximately 60 - 80 amino acids, including a highly conserved basic region and a relatively variable leucine zipper region. bZIP specifically binds to the DNA sequence containing the ACGT core in the promoter of target genes in the form of homo - or hetero - dimers, thereby regulating the expression of target genes. After plants are stimulated by signals such as hormones, upstream signal - responsive kinases will phosphorylate bZIP; bZIP also enhances its own stability through phosphorylation. Under stress conditions (such as drought, salinity, temperature, light, heavy metals, and pathogens), bZIP binds to the promoter region of stress - related genes and interacts with other proteins to promote or inhibit the expression of target genes, thereby positively or negatively regulating plant response to stress. In addition, bZIP is involved in the synthesis and metabolism of many substances (such as anthocyanins, terpenoids, flavonoids, alkaloids, etc.) during plant growth and development, and mediates the regulation of hormone signaling pathways such as abscisic acid, salicylic acid, and jasmonic acid. The above results indicate that bZIP plays an important role in regulating plant growth and development as well as stress resistance. However, there are few reports on bZIP genes that co - regulate plant stress resistance and high yield, especially regarding bZIP genes in wheat that simultaneously regulate plant stress resistance and high yield have not been reported yet. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a wheat TabZIP19 - 5B gene and its application in regulating wheat salt tolerance, antiviral ability, and 1000 - grain weight.

[0006] In order to achieve the above - mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a wheat TabZIP19 - 5B gene, and the nucleotide sequence of the wheat TabZIP19 - 5B gene is shown as SEQ ID NO.1.

[0008] The present invention also provides the protein encoded by the wheat TabZIP19 - 5B gene, and the amino acid sequence of the protein encoded by the wheat TabZIP19 - 5B gene is shown as SEQ ID NO.2.

[0009] The present invention also provides a recombinant vector for over - expressing or knocking down the wheat TabZIP19 - 5B gene. The recombinant vector for over - expressing the wheat TabZIP19 - 5B gene includes an initial expression vector and the wheat TabZIP19 - 5B gene, and the initial expression vector for over - expressing the wheat TabZIP19 - 5B gene is the Cub - 3×flag vector;

[0010] The recombinant vector for knocking down the wheat TabZIP19-5B gene includes an initial expression vector and sgRNAs targeting the wheat TabZIP19-5B gene. The initial expression vectors for knocking down the wheat TabZIP19-5B gene are the MT1T2 vector and the pBUE413 vector, and the sgRNAs targeting the wheat TabZIP19-5B gene are as shown in SEQ ID NO.15 and SEQ ID NO.16.

[0011] The present invention also provides a recombinant bacterium for overexpressing or knocking down the wheat TabZIP19-5B gene, and the recombinant bacterium is transfected with the recombinant vector.

[0012] The present invention also provides a method for overexpressing the wheat TabZIP19-5B gene, which includes the following steps:

[0013] Connect the wheat TabZIP19-5B gene to the Cub-3×flag vector to obtain the recombinant vector Cub-TabZIP19-5B-3×flag;

[0014] After transforming the recombinant vector by Agrobacterium, introduce it into the recipient plant to obtain the TabZIP19-5B transgenic plant, and achieve the overexpression of the wheat TabZIP19-5B gene.

[0015] The present invention also provides a method for knocking down the wheat TabZIP19-5B gene, which includes the following steps:

[0016] Connect the sgRNAs targeting the wheat TabZIP19-5B gene to the MT1T2 vector and the pBUE413 vector to obtain the recombinant plasmid;

[0017] After transforming the recombinant plasmid by Agrobacterium, introduce it into the recipient plant to obtain the TabZIP19-5B transgenic plant, and achieve the knockdown expression of the wheat TabZIP19-5B gene.

[0018] The present invention also provides the application of the wheat TabZIP19-5B gene, the protein, the recombinant vector or the recombinant bacterium in regulating the salt tolerance of plants. Overexpressing the wheat TabZIP19-5B gene in plants can improve the salt tolerance of plants, and knocking down the wheat TabZIP19-5B gene in plants can reduce the salt tolerance of plants.

[0019] The present invention also provides the use of the wheat TabZIP19-5B gene, the protein, the recombinant vector or the recombinant bacterium in regulating the ability of plants to resist barley yellow dwarf virus. Overexpressing the wheat TabZIP19-5B gene in plants can improve the ability of plants to resist barley yellow dwarf virus, while knocking down the wheat TabZIP19-5B gene in plants can reduce the ability of plants to resist barley yellow dwarf virus.

[0020] The present invention also provides the use of the wheat TabZIP19-5B gene, the protein, the recombinant vector or the recombinant bacterium in regulating the thousand-grain weight of plants. Overexpressing the wheat TabZIP19-5B gene in plants can increase the thousand-grain weight of plants, while knocking down the wheat TabZIP19-5B gene in plants can reduce the thousand-grain weight of plants.

[0021] Preferably, the plant is wheat.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention discovers a wheat transcription factor TabZIP19-5B gene and its application in regulating wheat salt tolerance, virus resistance and thousand-grain weight. By using the existing plant genetic engineering technology, the gene TabZIP19-5B that regulates wheat salt tolerance, virus resistance and thousand-grain weight is cloned for the first time, and this gene is transferred into wheat by the Agrobacterium-mediated method. Through comparative test analysis, it is proved that the transgenic plants overexpressing TabZIP19-5B in the present invention have significantly improved salt tolerance and BYDV resistance, and the grain width is greater than that of the wild type. It is confirmed that the gene provided by the present invention plays an important role in regulating salt tolerance, BYDV resistance and thousand-grain weight, and can be used as an important gene resource for cultivating new crop varieties with salt tolerance, virus resistance and high thousand-grain weight. Description of the Drawings

[0024] Figure 1 is the cloning of the TabZIP19-5B gene;

[0025] Figure 2 is the spatio-temporal specific expression of TabZIP19-5B under salt stress (wherein, A is the expression level of TabZIP19-5B in leaves at different times under salt stress; B is the expression level of TabZIP19-5B in roots at different times under salt stress);

[0026] Figure 3 is the subcellular localization of the TabZIP19-5B gene;

[0027] Figure 4Establishment of TabZIP19-5B transgenic wheat plants (where A is the detection of the bar gene in wheat overexpressing TabZIP19-5B; B is the identification of the expression level of the overexpressing TabZIP19-5B lines; C is the detection of knockdown of TabZIP19-5B; D is the target of the homozygous lines of knockdown TabZIP19-5B wheat);

[0028] Figure 5 Salt tolerance analysis of TabZIP19-5B transgenic wheat lines (where A is the evaluation of the salt tolerance of the overexpressing TabZIP19-5B lines. The left figure is under normal conditions, and the right figure is under 250 mM salt treatment conditions; B is the expression level and protein content of the three lines in Figure A; C is the evaluation of the salt tolerance of the wheat lines with knockdown of TabZIP19-5B expression. The left figure is under normal conditions, and the right figure is under 250 mM salt treatment conditions; D is the phenotypic salt tolerance of the adult plants of TabZIP19-5B-OE#34 and TabZIP19-5B-KO#1);

[0029] Figure 6 Analysis of the resistance of TabZIP19-5B transgenic wheat lines to BYDV virus (where A is the identification result of the resistance of wheat overexpressing the TabZIP19-5B gene to BYDV virus, B is the RT-PCR identification of the BYDV virus content in wheat overexpressing the TabZIP19-5B gene, and C is the ear traits of wheat overexpressing and knockdown of the TabZIP19-5B gene);

[0030] Figure 7 Traits of TabZIP19-5B transgenic grains (where A is the change in grain width of TabZIP19-5B-OE; B is the change in grain length of TabZIP19-5B-OE; C is the statistical analysis of the grain width of 20 seeds; D is the statistical analysis of the grain length of 20 seeds; E is the thousand-grain weight statistics of three TabZIP19-5B-OE lines). Detailed implementation

[0031] The present invention provides a wheat TabZIP19-5B gene. The nucleotide sequence of the wheat TabZIP19-5B gene is shown as SEQ ID NO.1, specifically: ATGGACGACGGGGACCTCGATTTCTCCAACCCGGAGGCGTACCTCTGCTCGGACACCGGCGCCGGCTGCTCCATGGACAGCTACTTCGACGGCATCCTCAACGACGCGGAGCACCTTGCGTGCACCCACACCCACACCTGCAACCCGCCCGTCGACGACAGCTCGCACACCCACACCTGCGTCCACGTCCACACCAAGATCGTCTCGGCGTCGTCGGATGGCGGCGCCGCCGACTCCCCGGCCGAGAACAGCGGCGCCTCCAAGAAGCGGCGGCCGTCCGGCAACCGCGCGGCCGTGAGGAAGTACCGGGAGAAGAAGAAGGCCCACACGGCGCTGCTGGAGGAAGAGGTGGTTCAGCTGAAGGCTCTGAACAAGCAGCTGCTGAAGAAGCTCCAGAATCACGCGGCGCTCGAGGCCGAGGCCGCCAGGCTCCGCTGCCTGCTCGTCGATGTCAGGGGGAGGATCGACGGGGAGATTGGCGCTTTCCCTTACCAGCGGCCTGTGAAGAACGTCGATTTGGTTTCTGGCGTTGATCAGGGGGGCTTTCTTGGCAGTGCCCAGGTTATGAACTCGTGTGATTTCAGATGCAACGATCAGATGTATTGCAATCCAGGAATGCAGATGAGAACTATCGGTGATGATGGTGCTATGAGTGGTCAGGTATTTGGGCAAGGCACTGGGGATATTGCAAACATCCAATGCATGGGGGGTGCGAAATCTGGGCTCACAATGCCCCCAGGCTGTGGGGGTATGGGGACAATGCCTTCTGGCTGTTTACCCAGTTCTGAAAAGCAGTGA。

[0032] The present invention also provides the protein encoded by the wheat TabZIP19-5B gene. The amino acid sequence of the protein encoded by the wheat TabZIP19-5B gene is shown in SEQ ID NO.2, specifically MDDGDLDFSNPEAYLCSDTGAGCSMDSYFDGILNDAEHLACTHTHTCNPPVDDSSHTHTCVHVHTKIVSASSDGGAADSPAENSGASKKRRPSGNRAAVRKYREKKKAHTALLEEEVVQLKALNKQLLKKLQNHAALEAEAARLRCLLVDVRGRIDGEIGAFPYQRPVKNVDLVSGVDQGGFLGSAQVMNSCDFRCNDQMYCNPGMQMRTIGDDGAMSGQVFGQGTGDIANIQCMGGAKSGLTMPPGCGGMGTMPSGCLPSSEKQ.

[0033] The present invention also provides a recombinant vector for overexpressing or knocking down the wheat TabZIP19-5B gene. The recombinant vector for overexpressing the wheat TabZIP19-5B gene includes an initial expression vector and the wheat TabZIP19-5B gene. The initial expression vector for overexpressing the wheat TabZIP19-5B gene is the Cub-3×flag vector;

[0034] The recombinant vector for knocking down the wheat TabZIP19-5B gene includes an initial expression vector and an sgRNA targeting the wheat TabZIP19-5B gene. The initial expression vectors for knocking down the wheat TabZIP19-5B gene are the MT1T2 vector and the pBUE413 vector. The sgRNAs targeting the wheat TabZIP19-5B gene are shown in SEQ ID NO.15 and SEQ ID NO.16, specifically 5’-CACCAAGATCGTCTCGGCGT-3’ and 5’-TGAGGAAGTACCGGGAGAAG-3’.

[0035] The present invention also provides a recombinant bacterium for overexpressing or knocking down the wheat TabZIP19-5B gene. The recombinant bacterium is transfected with the recombinant vector. In the present invention, the original bacterium of the recombinant bacterium is Agrobacterium, and the Agrobacterium is preferably Agrobacterium tumefaciens GV3101.

[0036] The present invention also provides a method for overexpressing the wheat TabZIP19-5B gene, comprising the following steps:

[0037] Connect the wheat TabZIP19-5B gene to the Cub-3×flag vector to obtain the recombinant vector Cub-TabZIP19-5B-3×flag;

[0038] The recombinant vector is transformed into a recipient plant through Agrobacterium tumefaciens to obtain TabZIP19-5B transgenic plants, achieving overexpression of the wheat TabZIP19-5B gene.

[0039] In the present invention, the Agrobacterium tumefaciens is preferably Agrobacterium tumefaciens GV3101; the preferred transformation method is: the Agrobacterium tumefaciens GV3101 competent cells and the recombinant vector Cub-TabZIP19-5B-3×flag are mixed and quickly frozen and heat shocked, and then cultured successively in a liquid LB medium without antibiotics and an LB plate medium containing rifampicin and kanamycin to obtain an Agrobacterium tumefaciens strain containing the recombinant expression vector Cub-TabZIP19-5B-3×flag. The quick freezing method is liquid nitrogen quick freezing, and the preferred quick freezing time is 3 to 8 minutes, more preferably 4 to 7 minutes; the heat shock method is water bath heat shock, and the preferred heat shock temperature is 34 to 40°C, more preferably 35 to 38°C; the preferred heat shock time is 3 to 7 minutes, more preferably 4 to 6 minutes; the concentration of rifampicin in the LB plate medium containing rifampicin and kanamycin is preferably 40 to 60 μg / mL, more preferably 45 to 55 μg / mL; the concentration of kanamycin in the LB plate medium containing rifampicin and kanamycin is preferably 40 to 60 μg / mL, more preferably 45 to 55 μg / mL; the preferred culture method is dark culture, the preferred culture temperature is 24 to 32°C, more preferably 25 to 30°C, and the preferred culture time is 1 to 3 days, more preferably 1.5 to 2.5 days.

[0040] In the present invention, the preferred method of introduction is as follows: inoculate the Agrobacterium strain containing the recombinant expression vector Cub-TabZIP19-5B-3×flag into MG liquid medium, culture, centrifuge, and collect the bacterial cells; suspend the bacterial cells in the infection solution, mix and incubate the immature embryos of wheat seeds with the infection solution containing the bacterial cells, and culture the incubated immature embryos successively in a co-culture solid medium, an induction solid medium, and a glyphosate screening medium, and then transfer them to a rooting medium for rooting culture. After rooting, transplant them into a substrate to obtain plants overexpressing the wheat TabZIP19-5B gene. The temperature of the incubation is preferably room temperature, and the time of the incubation is preferably 3-8 min, more preferably 4-7 min; the temperature of the culture in the co-culture solid medium is preferably 20-25 °C, more preferably 21-24 °C; the time of the culture in the co-culture solid medium is preferably 1-3 days, more preferably 1.5-2.5 days; the temperature of the culture in the induction solid medium is preferably 20-30 °C, more preferably 23-27 °C, and the time of the culture in the induction solid medium is preferably 3-8 days, more preferably 4-7 days.

[0041] The present invention also provides a method for knocking down the wheat TabZIP19-5B gene, comprising the following steps:

[0042] Link the sgRNA targeting the wheat TabZIP19-5B gene to the MT1T2 vector and the pBUE413 vector to obtain a recombinant plasmid;

[0043] After transforming the recombinant plasmid with Agrobacterium, introduce it into the recipient plant to obtain a TabZIP19-5B transgenic plant, and achieve the knockdown expression of the wheat TabZIP19-5B gene. In the present invention, the Agrobacterium is preferably Agrobacterium GV3101.

[0044] The present invention also provides the application of the wheat TabZIP19-5B gene, the protein, the recombinant vector or the recombinant bacterium in regulating the salt tolerance of plants. Overexpressing the wheat TabZIP19-5B gene in plants can improve the salt tolerance of plants, and knocking down the wheat TabZIP19-5B gene in plants can reduce the salt tolerance of plants.

[0045] The present invention also provides the application of the wheat TabZIP19-5B gene, the protein, the recombinant vector or the recombinant bacterium in regulating the resistance of plants to barley yellow dwarf virus. Overexpressing the wheat TabZIP19-5B gene in plants can improve the resistance of plants to barley yellow dwarf virus, and knocking down the wheat TabZIP19-5B gene in plants can reduce the resistance of plants to barley yellow dwarf virus.

[0046] The present invention also provides the use of the wheat TabZIP19-5B gene, the protein, the recombinant vector or the recombinant bacterium in regulating the thousand-grain weight of plants. Overexpressing the wheat TabZIP19-5B gene in plants increases the thousand-grain weight of plants, and knocking down the wheat TabZIP19-5B gene in plants reduces the thousand-grain weight of plants. In the present invention, the plant is wheat.

[0047] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] The experimental materials used in the present invention: the wheat variety of common wheat (Triticum aestivum L.) (Chinese Spring, English name Chinese Spring, is a very important local wheat variety, and Chinese Spring is widely used in wheat genetics research. See CN 118086326 A).

[0049] Example 1

[0050] Full-length CDS cloning of TabZIP19-5B genome

[0051] 1. Take Chinese Spring wheat seeds of uniform size, after disinfection and sterilization, sow them on filter paper, and treat them at 4°C for 3 days until they germinate. Then transfer the germinated seeds to nutrient soil and culture them at 22°C for two weeks to obtain wheat seedlings.

[0052] 2. Take wheat seedlings, quickly freeze them in liquid nitrogen, and store them at -80°C for later use. Extract the total RNA of wheat leaves by the Trizol method (TianGen), and then reverse transcribe it into cDNA using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara).

[0053] 3. Using the cDNA of wheat as a template, perform PCR amplification with the primer pair composed of 5’-ATGGACGACGGGGACCTC-3’ (SEQ ID NO.3) and 5’-TCACTGCTTTTCAGAACTGG-3’ (SEQ ID NO.4) to obtain the PCR amplification product of the full-length CDS of TabZIP19-5B. Detect the PCR product by 1.0% agarose gel electrophoresis (as Figure 1 shown).

[0054] 4. The PCR reaction procedure is: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 5 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, cycle 35 times; extension at 72°C for 10 min.

[0055] 5. Ligate the PCR amplification product to a cloning vector, transform it into Escherichia coli, and pick monoclonal colonies for sequencing.

[0056] Experimental results: The sequencing results showed that the nucleotide sequence CDS of the PCR amplification product was as shown in SEQ ID NO.1. The gene shown in SEQ ID NO.1 was named the TabZIP19-5B gene. The TabZIP19-5B gene encodes the TabZIP19-5B protein, and the amino acid sequence of the TabZIP19-5B protein was as shown in SEQ ID NO.2.

[0057] Example 2

[0058] Expression analysis of the TabZIP19-5B gene

[0059] 1. Take Fileder wheat seeds of uniform size, after disinfection and sterilization, sow them on filter paper, treat them at 4°C for 3 days, and then transfer the germinated seeds to nutrient solution and culture them at 22°C for two weeks to obtain Fileder seedlings with two leaves and one heart.

[0060] 2. After completing step 1, transfer a part of the Fileder seedlings to nutrient solution containing 200 mM NaCl and treat them for 0 h, 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, and 48 h.

[0061] 3. After completing step 2, extract the RNA of each sample, and then reverse transcribe it to obtain cDNA of Fileder roots and leaves at different times.

[0062] 4. After completing step 3, using the cDNA of Fileder wheat as a template, detect the relative expression level of the TabZIP19-5B gene by real-time fluorescence quantitative PCR (using the TaEF gene as an internal reference gene).

[0063] The primer pair for detecting the expression level of the TabZIP19-5B gene was 5’-CCTGTGAAGAACGTCGATTTGGTTTCT-3’ (SEQ ID NO.5) and 5’-ACCACTCATAGCACCATCATCACC-3’ (SEQ ID NO.6).

[0064] The primer pair for detecting the expression level of the TaEF gene was 5’-GCTGACTGTGCTGTTCTCATCATC-3’ (SEQ IDNO.7) and 5’-GCGCCTTTGAGTACTTGGGAG-3’ (SEQ ID NO.8).

[0065] Experimental results: As Figure 2As shown in the figure. The results show that the TabZIP19-5B gene is induced by salt stress.

[0066] Example 3

[0067] Subcellular localization of the TabZIP19-5B-GFP fusion protein

[0068] Using the correctly sequenced recombinant cloning plasmid obtained in Example 1 as a template, 5’-CTGTACAAGGGTACCATGGACGACGGGGAC-3’ (SEQ ID NO.9) and 5’-TCATCTAGAGGATCCTCACTGCTTTTCAGAACT-3’ (SEQ ID NO.10) as primers for PCR product amplification, the CDS sequence of the target gene was ligated into the expression vector pBin-GFP to obtain the recombinant vector pBin-TabZIP19-5B-GFP, which was transferred into Agrobacterium tumefaciens GV3101. The transformation of the empty vector pBin-GFP was used as a control, and transient expression was carried out in tobacco leaves. Observation was performed under a laser confocal microscope after 48 h.

[0069] Experimental results: As Figure 3 shown, the transformed empty vector pBin-GFP was widely present in the whole cell, while the transformed pBin-TabZIP19-5B-GFP fusion protein was mainly localized in the nucleus and also present in small amounts in the cytoplasm.

[0070] Example 4

[0071] Establishment of TabZIP19-5B transgenic wheat plants

[0072] To verify the function of the TabZIP19-5B gene using transgenic wheat, the specific procedure is as follows:

[0073] I. Establishment of overexpressing TabZIP19-5B wheat plants

[0074] 1. Construction of the TabZIP19-5B overexpression vector

[0075] Design primers to perform PCR amplification of TabZIP19-5B from the plasmid. After digestion with enzymes and ligation by homologous recombination, the full-length cDNA of TabZIP19-5B was ligated to the BamHI-linearized Cub-3×flag vector (the Cub-3×flag vector is stored in our laboratory. For the specific construction method, refer to Tian et al., A viral movement protein targets host catalases for 26S proteasome-mediated degradation to facilitate viral infection and aphid transmission in wheat. Mol Plant, 2024, 17(4): 614-630.). Then, the ligated product was transformed into Escherichia coli. Positive clones were screened by colony PCR. The positive clones could specifically amplify a band of about 800 bp (including part of the vector backbone), which was consistent with the size of the full-length cDNA of TabZIP19-5B, 750 bp, indicating that the vector might have been successfully constructed. The results from the sequencing company (Beijing Tsingke Biotechnology Co., Ltd.) showed that the reading frame of the recombinant vector was correct and there was no frameshift mutation, indicating that the Cub-TabZIP19-5B-3×flag vector was successfully constructed and could be used for subsequent experiments.

[0076] 2. Transformation of wheat overexpressing TabZIP19-5B

[0077] Extract the plasmid of the constructed Cub-TabZIP19-5B-3×flag vector. Add 1 μl of the plasmid to 10 μl of the Agrobacterium tumefaciens competent cells of GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.). Then place it on ice for 5 min, in liquid nitrogen for 5 min, at 37 °C for 5 min, on ice for 5 min. Finally, add antibiotic-free LB for shaking culture. After 3 h, spread it on a solid LB plate containing kanamycin (50 mg / mL) and rifampicin (25 mg / mL) resistance. After culturing at 28 °C for 2 d, pick single colonies for PCR detection of the bacterial solution. Select single colonies with the correct band size and mark them. Then pick the marked single Agrobacterium colony and inoculate it into MG liquid medium for overnight culture. Centrifuge at 5000 rpm to collect the bacterial cells and resuspend them in the infection solution to a concentration of A 600=0.4 reserve; Collect immature wild-type wheat seeds 14 - 18 days after flowering, disinfect them, strip the immature embryos and rinse them in 2.0 mL of infection solution. Remove the infection solution, add 2.0 mL of fresh infection solution and reserve; Centrifuge at 7500 rpm for 10 min at 4 °C, remove the infection solution, then add 1.0 ml of infection solution, invert and mix well for 30 s, and incubate at room temperature for 5 min; Place the immature embryos with the scutellum facing up on the co-culture solid medium and culture them in the dark at 23 °C for 2 days; Cut off the hypocotyl, transfer the immature embryos to the induction solid medium and culture them in the dark at 25 °C for 5 days; Transfer to the glyphosate screening medium for screening culture; Transfer the normally growing ones to the rooting medium for rooting culture, and transplant them into the substrate after rooting to obtain the overexpressed T0 generation transgenic seedlings of TabZIP19 - 5B.

[0078] 3. Identification of positive seedlings and expression levels of overexpressed TabZIP19 - 5B wheat

[0079] (1) Identification of positive seedlings

[0080] Take samples of the overexpressed T0 generation transgenic seedlings of TabZIP19 - 5B, cut a wheat leaf and put it into a 2 ml centrifuge tube containing two steel beads, and quickly freeze it at low temperature; Shake and break it with a tissue disruptor; First add 600 μl of DS Buffer, then add 600 μl of DNA extraction solution (alkaline phenol: chloroform: isoamyl alcohol (25:24:1)), shake vigorously up and down and mix well, then place it in a 4 °C refrigerator for 25 min; Centrifuge at 12000 rpm for 15 min, transfer 600 μl of the supernatant to a 1.5 ml centrifuge tube, add 440 μl of isopropanol, invert and mix well multiple times up and down, and place it in a -20 °C refrigerator for 2.5 h; Centrifuge at 11400 rpm at room temperature for 15 min, discard the supernatant; Add 700 μl of absolute ethanol, invert and suspend the precipitate up and down, centrifuge at 12000 rpm for 4 min; Centrifuge at 12000 rpm for 4 min, discard the supernatant, open the lid and dry it in an oven, and add 45 μl of ddH2O to dissolve the extracted DNA.

[0081] The detection primers for the transgenic bar gene are 5’-CGTCAACCACTACATCGAGACAAG-3’ (SEQ ID NO.11) and 5’-GCTGAAGTCCAGCTGCCCAGAAAC-3’ (SEQ ID NO.12).

[0082] Experimental results: As shown in Figure 4 A in. Among the 40 lines of overexpressed TabZIP19 - 5B wheat, only 6 lines did not detect the bar gene, and 34 lines were positive seedlings.

[0083] (2) Identification of expression levels

[0084] Collect wheat leaf samples and put them into 2 ml RNase-free centrifuge tubes, quickly freeze them at low temperature, and break them using a tissue disruptor; isolate total RNA using the Trizol method (TianGen), immediately eliminate genomic contamination using the DNAseⅠ (Takara) method, then measure the concentration using Nanodrop1000 (Thermo Scientific product, USA), uniformly take 2 μl of the sample, and run on a 0.8% agarose gel. Take 1 μg of total RNA, use recombinant M-MLV reverse transcriptase, with Oligo(dT) as the primer, perform the synthesis of cDNAs, and conduct quantitative detection.

[0085] The primer pair for detecting the expression level of the TabZIP19-5B gene is 5’-CCTGTGAAGAACGTCGATTTGGTTTCT-3’ (SEQ ID NO.5) and 5’-ACCACTCATAGCACCATCATCACC-3’ (SEQ ID NO.6).

[0086] The primer pair for detecting the expression level of the TaActin1 gene is 5’-GCTGACTGTGCTGTTCTCATCATC-3’ (SEQ ID NO.13) and 5’-GCGCCTTTGAGTACTTGGGAG-3’ (SEQ ID NO.14).

[0087] Experimental results: As shown in B of Figure 4 . Quantitative analysis was performed on the expression level of TabZIP19-5B overexpressed wheat T0 generation, and the results showed that the OE#1, OE#11, and OE#34 lines were about 4 times that of the control group.

[0088] II. Establishment of wheat plants with knocked-down TabZIP19-5B

[0089] 1. Construction of the gene vector for knocking down TabZIP19-5B

[0090] Submit the wheat exons on the website http: / / crispr.dbcls.jp / . Select "Wheat (Triticum aestivum) genome, IWGSC1.0 + popseq (Nov, 2014)" for the species. After selecting the appropriate designed target sites, two sgRNA sequences targeting the TabZIP19-5B gene are obtained: sgRNA1 5’-CACCAAGATCGTCTCGGCGT-3’ (SEQ ID NO.15), sgRNA2 5’-TGAGGAAGTACCGGGAGAAG-3’ (SEQ ID NO.16). To construct a dual-target, primer adapters need to be added. Using the intermediate vector MT1T2 as a template, TabZIP19-5B-F0 / TabZIP19-5B-R0 and TabZIP19-5B-F / TabZIP19-5B-R are used as amplification primers for PCR amplification to obtain a product containing two target sites and a gRNA-Scfford. After gel recovery, it is digested with BsaI. At the same time, the final vector pBUE413 is digested with BsaI. pBUE413 alone contains a gRNA-Scfford. Finally, it is ligated with T4 ligase so that each target site is followed by a gRNA-Scfford, and thus the vector for knocking down the TabZIP19-5B gene can be constructed. The methods for constructing the dual-target CRISPR / Cas9 knockout intermediate vector MT1T2 and the final vector pBUE413 are specifically referred to Sun GL et al. Matrilineal empowers wheat pollen with haploid induction potency by triggering postmitosis reactive oxygen species activity. The New phytologist, 2022, 233(6), 2405–2414. https: / / doi.org / 10.1111 / nph.17963. Then, the recombinant plasmid is transferred into EHA105 and spread on a solid LB plate containing kanamycin (50 mg / mL) and rifampicin (25 mg / mL) resistance. After culturing at 28 °C for 2 days, single colonies are picked for PCR detection, and the single colonies with the correct band size are marked. The Agrobacterium plate is sent to the transgenic platform of the National Key Laboratory of Crop Stress Resistance and High-Efficiency Production, Northwest A&F University for transgenic wheat transformation.

[0091] TabZIP19-5B-F: aataatggtctcaggcCACCAAGATCGTCTCGGCGT (SEQ ID NO.17)

[0092] TabZIP19-5B-R: attattggtctctaaacCTTCTCCCGGTACTTCCTCA(SEQ ID NO.18)

[0093] TabZIP19-5B-F0: CACCAAGATCGTCTCGGCGTgttttagagctagaaatagc(SEQ ID NO.19)

[0094] TabZIP19-5B-R0: CTTCTCCCGGTACTTCCTCAcgcttcttggtgcc(SEQ ID NO.20)

[0095] (2) Screening of homozygous lines with knocked-down TabZIP19-5B gene

[0096] The T0 generation transgenic seedlings with knocked-down expression of TabZIP19-5B obtained from the transgenic platform of the National Key Laboratory of Crop Stress Resistance and High-Efficiency Production, Northwest A&F University were planted in the soil. After the wheat took root in the soil for a period of time, a leaf segment was taken for DNA extraction, and primers 5'-GGAGTGAGTACGGTGTGCACACCCACACCTGCGTCCAC-3' (SEQ ID NO.21) and 5'-GAGTTGGATGCTGGATGGTGAACCACCTCTTCCTCCAG-3' (SEQ ID NO.22) were designed near the two target sites to amplify the DNA templates of wheat from different lines, and the PCR products were sequenced.

[0097] Experimental results: As shown in C and D of Figure 4 . The PCR results of the Cas9 element showed that only 12 lines were positive seedlings, and sequencing showed that only KO#1, KO#2, and KO#3 successfully edited ABD.

[0098] Example 5

[0099] Regulation of wheat salt tolerance by TabZIP19-5B gene

[0100] 1. Identification of salt tolerance at the seedling stage: The lines with relatively high expression levels identified from the overexpressed T0 generation transgenic seedlings (1 plant, 11 plants, and 34 plants) of TabZIP19-5B in Example 4 were advanced in the greenhouse, and identified at the transcriptional level. Then, the seeds of T2 were planted to obtain the seeds of T3 generation (three overexpressed wheat seeds: OE#1, OE#11, OE#34).

[0101] The T0 generation transgenic plants with knockdown expression of TabZIP19-5B in Example 4 (1 plant, 2 plants, and 3 plants) were identified to obtain lines with relatively high expression levels, which were advanced in the greenhouse, and identified at the transcriptional level. Then, the seeds of T2 were planted to obtain the seeds of T3 generation (three knockdown expression wheat seeds: KO#1, KO#2, KO#3).

[0102] The surfaces of the overexpressed and knockdown expressed T3 generation transgenic wheat seeds of TabZIP19-5B and Fileder seeds were disinfected, and then placed in a petri dish filled with sterile water for germination. When the bud length reached 3 cm, uniformly consistent seeds were selected and planted in the nutrient solution prepared with Hoagland and calcium salts. When the wheat grew to two leaves and one heart stage, it was treated with 250 mM NaCl for 14 days, and photographed for record.

[0103] Salt tolerance identification at adult stage: The overexpressed wheat seeds (OE#34), knockdown expressed wheat seeds (KO#1), and control Fileder seeds were planted in nutrient soil. When the seeds grew to 1 cm, uniformly consistent seeds were selected and planted in flower pots (the culture soil was: nutrient soil: vermiculite: field soil = 2:1:1). When it grew to 4 weeks old, it was treated with 100 mM, 300 mM, 500 mM, and 700 mM NaCl solutions for 4 weeks in turn every week, and photographed for record at the heading stage.

[0104] Experimental results: As Figure 5 shown. The degree of leaf withering of wheat overexpressing the TabZIP19-5B gene was reduced, and the growth condition was better, while the growth of homozygous wheat with knockdown expression of TabZIP19-5B was hindered and the growth was weak.

[0105] Example 6

[0106] Regulation of TabZIP19-5B gene on wheat resistance to BYDV

[0107] Select aphids with uniform growth on the leaves of virus-infected Avena strigosa and inoculate them onto the overexpressed (OE#34), knockdown expressed wheat (KO#1), and control WT wheat at the two-leaf and one-heart stage. 5 aphids were inoculated on each wheat plant, and they were isolated and reared with a plastic cover with air holes. After 3 days, the aphids were removed, and samples were taken after 10 days and stored at -80°C. Wheat RNA was extracted (the Biospin Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (BSC65) was purchased from Hangzhou Bioer Technology Co., Ltd.). After the extraction of RNA was completed, the concentration was measured. 1000 ng of RNA from each plant was reverse transcribed (RTmix with DNase (DN2211-01) was purchased from Beijing Dinning Biotechnology Co., Ltd.), and the BYDV content was detected by qRT-PCR.

[0108] qMP-F 5’-GCGCAGTTCGGAGAATGGT-3’(SEQ ID NO.23)

[0109] qMP-R 5’-CTGAAAGCGCTGGGCATTG-3’(SEQ ID NO.24)

[0110] Experimental results: As Figure 6 shown. The virus content of wheat overexpressing the TabZIP19-5B gene decreased by 14.88 times compared with the control group WT. The antiviral ability of the TabZIP19-5B overexpressing lines was significantly improved, with less chlorosis; the spike length of the TabZIP19-5B overexpressing lines became longer and the number of grains per spike increased, while the spike length of the TabZIP19-5B knockdown lines became shorter and the number of grains per spike decreased.

[0111] Example 7

[0112] Identification of Agronomic Traits of Wheat Overexpressing TabZIP19-5B

[0113] Select three overexpressing wheat seeds with uniform size (OE#1, OE#11, OE#34) and Fileder wheat seeds. After disinfection and sterilization, they are sown on filter paper, treated at 4°C for 3 days, then taken out and placed in an incubator at 20°C for 3 days, and transferred to a 18.8 cm * 18.8 square cultivation pot filled with a 1:1 mixture of Pindstrup imported substrate and V9 domestic substrate. The overexpressing wheat plants are cultivated in a generation-adding room at 22°C for about three months. When the wheat is completely mature, the single plants are threshed and the grain length, grain width and 1000-grain weight are statistically analyzed.

[0114] Experimental results: As Figure 7 shown. The grain width of the TabZIP19-5B overexpressing lines increased significantly, there was no significant difference in grain length of the TabZIP19-5B overexpressing lines, and the 1000-grain weight of the TabZIP19-5B overexpressing lines increased significantly.

[0115] As can be seen from the above examples, overexpressing the TabZIP19-5B gene can significantly improve the salt tolerance and BYDV resistance of wheat, and significantly increase the 1000-grain weight of wheat.

[0116] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of the wheat TabZIP19-5B gene in regulating the salt tolerance of wheat, characterized in that, Overexpressing the wheat TabZIP19-5B gene in wheat improves the salt tolerance of wheat, and knocking down the wheat TabZIP19-5B gene in wheat reduces the salt tolerance of wheat; The nucleotide sequence of the wheat TabZIP19-5B gene is shown as SEQ ID NO.

1.

2. Application of wheat TabZIP19-5B gene in regulating wheat resistance to barley yellow dwarf virus, characterized in that, Overexpressing the wheat TabZIP19-5B gene in wheat improves the resistance of wheat to barley yellow dwarf virus; The nucleotide sequence of the wheat TabZIP19-5B gene is shown as SEQ ID NO.

1.

3. Use of the wheat TabZIP19-5B gene in increasing the thousand-kernel weight of wheat, characterized in that, Overexpressing the wheat TabZIP19-5B gene in wheat increases the thousand-grain weight of wheat; The nucleotide sequence of the wheat TabZIP19-5B gene is shown as SEQ ID NO. 1.

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