Wheat TabZIP19-5B gene and application of wheat TabZIP19-5B gene in regulation and control of salt tolerance, virus resistance and thousand seed weight of wheat

Abstract

The invention provides a wheat TabZIP19-5B gene and application of the wheat TabZIP19-5B gene in regulating and controlling salt tolerance, virus resistance and thousand seed weight of wheat, and belongs to the technical field of plant genetic engineering. The gene TabZIP19-5B for regulating and controlling salt tolerance, virus resistance and thousand seed weight of wheat is obtained through cloning for the first time by utilizing an existing plant gene engineering technology, the gene is transferred into the wheat through an agrobacterium tumefaciens-mediated method, and comparative test analysis proves that the salt tolerance and BYDV resistance of a transgenic plant overexpressing the TabZIP19-5B are remarkably improved, so that the gene TabZIP19-5B can be used for regulating and controlling the salt tolerance, virus resistance and thousand seed weight of the wheat. And the grain width is larger than that of wild type. The TabZIP19-5B gene provided by the invention plays an important role in the process of regulating and controlling salt tolerance, BYDV resistance and thousand seed weight, and can be used as an important gene resource for cultivating new varieties of salt-tolerant, antiviral and thousand seed weight crops.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a wheat TabZIP19-5B gene and application thereof in regulating salt tolerance, virus resistance and thousand-grain weight of wheat. Background Art

[0002] Salt stress has a serious impact on plant growth and development, and its hazards mainly include osmotic stress and ion poisoning. Under salt stress, the ion balance inside and outside plant cells is destroyed, causing the plant to lose water, stomata to close, photosynthesis to be restricted, and affecting the normal growth and metabolism of the plant. In addition, salt stress can also cause metabolic disorders in the plant body, produce accumulation of substances such as reactive oxygen, affect plant photosynthesis, and ultimately inhibit plant growth. Exploring and utilizing drought-resistant and salt-tolerant genes and cultivating new varieties of drought-resistant and salt-tolerant crops are the most direct and effective ways to overcome the hazards of drought and salinity.

[0003] Wheat yellow dwarf disease is a viral disease that is transmitted by barley yellow dwarf viruses (BYDVs) through the virus vector aphids in a persistent, cyclical and non-proliferative manner and infects host plants. The typical symptoms of host plants infected by BYDVs are yellowing of the leaf tips and spreading downward to the entire leaf, reduced grains, dwarfing of plants, loss of leaf green, slow growth, delayed heading, yellowing and purple leaf tips, etc., which reduce crop yields and even cause serious yield reductions. Breeding for wheat yellow dwarf disease resistance is of great significance for increasing crop yields, ensuring food security, promoting sustainable agricultural development, and promoting agricultural scientific and technological progress.

[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 conservative domain consisting of approximately 60-80 amino acids, including a highly conserved alkaline region and a relatively variable leucine zipper region. bZIP specifically binds to the DNA sequence containing the ACGT core in the promoter of the target gene in the form of homologous or heterologous dimers, thereby regulating the expression of the target gene. After the plant is stimulated by signals such as hormones, the upstream signal response kinase will phosphorylate bZIP; bZIP also enhances its own stability through phosphorylation. Under adverse stress conditions (such as drought, salt, 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 the plant's response to adverse stress. In addition, bZIP participates in the synthesis and metabolism of many substances (such as anthocyanins, terpenes, flavonoids, alkaloids, etc.) during plant growth and development, and mediates and regulates hormone signaling pathways such as abscisic acid, salicylic acid and jasmonic acid. The above results show that bZIP plays an important role in regulating plant growth and development and stress resistance. However, there are few reports on bZIP genes that synergistically regulate plant stress resistance and high yield, especially for wheat. There is no report on bZIP genes that simultaneously regulate plant stress resistance and high yield. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a wheat TabZIP19-5B gene and its application in regulating wheat salt tolerance, virus resistance and thousand-grain weight.

[0006] In order to achieve the above-mentioned object of the invention, 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 in SEQ ID NO.1.

[0008] The present invention also provides a 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.

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

[0010] 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 vector for knocking down the wheat TabZIP19-5B gene is an MT1T2 vector and a pBUE413 vector. The sgRNA targeting the wheat TabZIP19-5B gene is 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, wherein the recombinant bacterium is transformed with the recombinant vector.

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

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

[0014] The recombinant vector is introduced into a recipient plant after transformation with Agrobacterium to obtain a TabZIP19-5B transgenic plant, thereby achieving overexpression of the wheat TabZIP19-5B gene.

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

[0016] The sgRNA targeting the wheat TabZIP19-5B gene was connected to the MT1T2 vector and the pBUE413 vector to obtain a recombinant plasmid;

[0017] The recombinant plasmid was introduced into the recipient plant after Agrobacterium transformation to obtain TabZIP19-5B transgenic plants, thereby achieving knockdown expression of the wheat TabZIP19-5B gene.

[0018] The present invention also provides the use of the wheat TabZIP19-5B gene, the protein, the recombinant vector or the recombinant bacteria in regulating the salt tolerance of plants, overexpressing the wheat TabZIP19-5B gene in plants to improve the salt tolerance of plants, and knocking down the wheat TabZIP19-5B gene in plants to 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 bacteria in regulating the ability of plants to resist barley yellow dwarf virus, overexpressing the wheat TabZIP19-5B gene in plants to improve the ability of plants to resist barley yellow dwarf virus, and knocking down the wheat TabZIP19-5B gene in plants to 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 bacteria in regulating the thousand-grain weight of plants, overexpressing the wheat TabZIP19-5B gene in plants to increase the thousand-grain weight of plants, and knocking down the wheat TabZIP19-5B gene in plants to 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 has discovered a wheat transcription factor TabZIP19-5B gene and its application in regulating wheat salt tolerance, virus resistance and thousand-grain weight. The present invention uses existing plant genetic engineering technology to clone the gene TabZIP19-5B for regulating wheat salt tolerance, virus resistance and thousand-grain weight for the first time, and transfers the gene into wheat by an Agrobacterium-mediated method. Comparative test analysis proves that the transgenic plant overexpressing TabZIP19-5B in the present invention has significantly improved salt tolerance and BYDV resistance compared with the wild type, 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 varieties of salt-tolerant, virus-resistant and thousand-grain-weight crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the TabZIP19-5B gene clone;

[0025] Figure 2 is the spatiotemporal specific expression of TabZIP19-5B under salt stress (where 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 4is the establishment 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 strain overexpressing TabZIP19-5B; C is the detection of knocking down TabZIP19-5B; D is the knocking down of the target site of TabZIP19-5B homozygous wheat strain);

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

[0029] Figure 6 The analysis of BYDV resistance of TabZIP19-5B transgenic wheat lines (A is the identification result of BYDV resistance of wheat overexpressing TabZIP19-5B gene, B is the BYDV content of wheat overexpressing TabZIP19-5B gene identified by RT-PCR, and C is the spike traits of wheat overexpressing and knocking down TabZIP19-5B gene);

[0030] Figure 7 These are the grain traits of TabZIP19-5B transgenic plants (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 grain width of 20 seeds; D is the statistical analysis of grain length of 20 seeds; E is the thousand-grain weight statistics of the three strains of TabZIP19-5B-OE). DETAILED DESCRIPTION

[0031] 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, specifically ATGGACGACGGGGACCTCGATTTCTCCAACCCGGAGGCGTACCTCTGCTCGGACACCGGCGCCGGCTGCTCCATGGACAGCTACTTCGACGGCATCCTCAACGACGCGGAGCACCTTGCGTGCACCCACACCCACACCTGCAACCCGCCCGTCGACGACAGCTCGCACACCCACACCTGCGTCCACGTCCACACCAAGATCGTCTCGGCGTCGTCGGATGGCGGCGCCGCCGACTCCCCGGCCGAGAACAGCGGCGCCTCCAAGAAGCGGCGGCCGTCCGGCAACCGCGCGGCCGTGAGGAAGTACCGGGAGAAGAAGAAGGCCCACACGGCGCTGCTGGAGGAAGAGGTGGTTCAGCTGAAGGCTCTGAACAAGCAGCTGCTGAAGAAGCTCCAGAATCACGCGGCGCTCGAGGCCGAGGCCGCCAGGCTCCGCTGCCTGCTCGTCGATGTCAGGGGGAGGATCGACGGGGAGATTGGCGCTTTCCCTTACCAGCGGCCTGTGAAGAACGTCGATTTGGTTTCTGGCGTTGATCAGGGGGGCTTTCTTGGCAGTGCCCAGGTTATGAACTCGTGTGATTTCAGATGCAACGATCAGATGTATTGCAATCCAGGAATGCAGATGAGAACTATCGGTGATGATGGTGCTATGAGTGGTCAGGTATTTGGGCAAGGCACTGGGGATATTGCAAACATCCAATGCATGGGGGGTGCGAAATCTGGGCTCACAATGCCCCCAGGCTGTGGGGGTATGGGGACAATGCCTTCTGGCTGTTTACCCAGTTCTGAAAAGCAGTGA。

[0032] The present invention also provides a 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, wherein the recombinant vector for overexpressing the wheat TabZIP19-5B gene comprises an initial expression vector and the wheat TabZIP19-5B gene, and the initial expression vector for overexpressing the wheat TabZIP19-5B gene is a 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 vector for knocking down the wheat TabZIP19-5B gene is an MT1T2 vector and a pBUE413 vector. The sgRNA targeting the wheat TabZIP19-5B gene is shown in SEQ ID NO.15 and SEQ ID NO.16, specifically 5'-CACCAAGATCGTCTCGGCGT-3', 5'-TGAGGAAGTACCGGGAGAAG-3'.

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

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

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

[0038] The recombinant vector is introduced into a recipient plant after transformation with Agrobacterium to obtain a TabZIP19-5B transgenic plant, thereby achieving overexpression of the wheat TabZIP19-5B gene.

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

[0040] In the present invention, the introduction method is preferably: inoculating the Agrobacterium strain containing the recombinant expression vector Cub-TabZIP19-5B-3×flag into MG liquid culture medium, culturing, centrifuging, and collecting bacteria; suspending the bacteria in an infection solution, mixing and incubating the immature embryos of wheat seeds and the infection solution containing the bacteria, culturing the incubated immature embryos in a co-culture solid culture medium, an induction solid culture medium, and a glyphosate screening culture medium in sequence, and then transferring to a rooting culture medium for rooting culture, and transplanting to a matrix after rooting to obtain plants overexpressing the wheat TabZIP19-5B gene. The incubation temperature is preferably room temperature, and the incubation time is preferably 3 to 8 minutes, and more preferably 4 to 7 minutes; the culturing temperature in the co-cultivation solid medium is preferably 20 to 25°C, and more preferably 21 to 24°C; the culturing time in the co-cultivation solid medium is preferably 1 to 3 days, and more preferably 1.5 to 2.5 days; the culturing temperature in the induction solid medium is preferably 20 to 30°C, and more preferably 23 to 27°C, and the culturing time in the induction solid medium is preferably 3 to 8 days, and more preferably 4 to 7 days.

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

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

[0043] The recombinant plasmid is transformed by Agrobacterium and then introduced into a recipient plant to obtain a TabZIP19-5B transgenic plant, thereby achieving 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 use of the wheat TabZIP19-5B gene, the protein, the recombinant vector or the recombinant bacteria in regulating the salt tolerance of plants, overexpressing the wheat TabZIP19-5B gene in plants to improve the salt tolerance of plants, and knocking down the wheat TabZIP19-5B gene in plants to reduce the salt tolerance of plants.

[0045] The present invention also provides the use of the wheat TabZIP19-5B gene, the protein, the recombinant vector or the recombinant bacteria in regulating the ability of plants to resist barley yellow dwarf virus, overexpressing the wheat TabZIP19-5B gene in plants to improve the ability of plants to resist barley yellow dwarf virus, and knocking down the wheat TabZIP19-5B gene in plants to reduce the ability of plants to resist 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 bacteria in regulating the thousand-grain weight of plants, overexpressing the wheat TabZIP19-5B gene in plants to increase the thousand-grain weight of plants, and knocking down the wheat TabZIP19-5B gene in plants to reduce the thousand-grain weight of plants. In the present invention, the plant is wheat.

[0047] The technical solutions provided by the present invention are described in detail below in conjunction with 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 are wheat varieties of common wheat (Triticum aestivum L.) (Chinese Spring, English name Chinese Spring, is a very important wheat landrace, and Chinese Spring is widely used in wheat genetics research. See CN 118086326 A).

[0049] Example 1

[0050] TabZIP19-5B genomic CDS full-length clone

[0051] 1. Take Chinese spring wheat seeds of uniform size, disinfect and sterilize them, sow them on filter paper, treat them at 4℃ for 3 days until they turn white, then transfer the germinated seeds to nutrient soil and culture them at 22℃ for two weeks to obtain wheat seedlings.

[0052] 2. Take wheat seedlings, freeze them quickly with liquid nitrogen, and store them at -80℃ for later use. Use Trizol method (TianGen) to extract total RNA from wheat leaves, and then use PrimeScript TM cDNA was obtained by reverse transcription using II 1st Strand cDNA Synthesis Kit (Takara).

[0053] 3. Using wheat cDNA as template, PCR amplification was performed using a primer pair consisting 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. The PCR product was detected by 1.0% agarose gel electrophoresis ( Figure 1 shown).

[0054] 4. The PCR reaction program is: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1 minute, 35 cycles; 72℃ extension for 10 minutes.

[0055] 5. Connect the PCR amplification product to the cloning vector, transform it into E. coli, and pick out single clones for sequencing.

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

[0057] Example 2

[0058] Expression analysis of TabZIP19-5B gene

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

[0060] 2. After completing step 1, transfer some of the Fileder seedlings to a nutrient solution containing 200 mM NaCl 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, then reverse transcribe it to obtain cDNA of Fileder roots and leaves at different times.

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

[0063] The primer pair for detecting the expression level of 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 TaEF gene was 5'-GCTGACTGTGCTGTTCTCATCATC-3' (SEQ ID NO. 7) and 5'-GCGCCTTTGAGTACTTGGGAG-3' (SEQ ID NO. 8).

[0065] Experimental results: Figure 2The results showed that the expression of TabZIP19-5B gene was induced by salt stress.

[0066] Example 3

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

[0068] The recombinant cloning plasmid with the correct sequence obtained in Example 1 was used as a template, 5'-CTGTACAAGGGTACCATGGACGACGGGGAC-3' (SEQ ID NO.9) and 5'-TCATCTAGAGGATCCTCACTGCTTTTCAGAACT-3' (SEQ ID NO.10) were used as primers to amplify the PCR product, and the CDS sequence of the target gene was connected into the expression vector pBin-GFP to obtain the recombinant vector pBin-TabZIP19-5B-GFP, which was transformed into Agrobacterium GV3101, and the empty pBin-GFP was used as the control for transient expression in tobacco leaves, and observed under a laser confocal microscope for 48 hours.

[0069] Experimental results: Figure 3 As shown, the 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 existed in a small amount in the cytoplasm.

[0070] Example 4

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

[0072] The function of TabZIP19-5B gene was verified using transgenic wheat. The specific process is as follows:

[0073] 1. Establishment of wheat plants overexpressing TabZIP19-5B

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

[0075] Primers were designed to amplify TabZIP19-5B from the plasmid by PCR. After restriction digestion and homologous recombination, the full-length cDNA of TabZIP19-5B was connected to the BamHI linearized Cub-3×flag vector (the Cub-3×flag vector was preserved by this laboratory. The specific construction method refers to Tian et al., A viral movement protein targets hostcatalases for 26S proteasome-mediated degradation to facilitate viral infection and aphid transmission in wheat. Mol Plant, 2024, 17(4): 614-630.), and 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 full-length cDNA size of TabZIP19-5B of 750 bp, indicating that the vector may have been successfully constructed. The results from the sequencing company (Beijing Qingke Biotechnology Co., Ltd.) showed that the reading frame of the recombinant vector was correct and there was no frameshift mutation, indicating that the construction of the Cub-TabZIP19-5B-3×flag vector was complete and could be used for subsequent experiments.

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

[0077] The constructed Cub-TabZIP19-5B-3×flag vector was extracted with plasmid, and 1 μl of the plasmid was added to 10 μl of GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) Agrobacterium competent medium, then placed on ice for 5 min, liquid nitrogen for 5 min, 37°C for 5 min, and on ice for 5 min. Finally, LB without antibiotics was added for shaking culture. After 3 h, it was spread on a solid LB plate carrying kana (50 mg / mL) and rifampicin (25 mg / mL) resistance, and cultured at 28°C for 2 days. Single clones were selected for PCR bacterial liquid detection, and single colonies with the correct band size were selected for marking. Then, the marked single Agrobacterium colony was selected and inoculated into MG liquid culture medium for overnight culture. The bacteria were collected by centrifugation at 5000 rpm and resuspended with infection solution to a concentration of A. 600=0.4 for later use; collect immature wild-type wheat seeds that have been flowering for 14 to 18 days, remove the young embryos after disinfection, rinse them in 2.0 mL of infection solution, remove the infection solution, and add 2.0 mL of new infection solution for later use; centrifuge at 7500 rpm and 4°C for 10 min, remove the infection solution, then add 1.0 ml of infection solution, mix by inversion for 30 s, and incubate at room temperature for 5 min; place the young embryos on a co-culture solid medium with the scutellum facing up, and culture them in the dark at 23°C for 2 days; cut off the embryo axis, transfer the young embryos to an induction solid medium, and culture them in the dark at 25°C for 5 days; transfer them to a glyphosate screening medium for screening culture; transfer those with normal growth to a rooting medium for rooting culture, and transplant them to a matrix after rooting to obtain TabZIP19-5B overexpression T0 generation transformed seedlings.

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

[0079] (1) Identification of positive seedlings

[0080] The TabZIP19-5B overexpression T0 generation transformed seedlings were sampled, and a wheat leaf was cut and placed in a 2ml centrifuge tube with two steel balls, and quickly frozen at low temperature; the tissue disruptor was shaken and broken; 600μl DS Buffer was added first, and then 600μl DNA extraction solution (alkaline phenol: chloroform: isoamyl alcohol (25:24:1)) was added, and the mixture was vigorously shaken up and down and placed in a 4°C refrigerator for 25min; 12000rpm, 15min, 600μl supernatant was transferred to a 1.5ml centrifuge tube, 440μl isopropanol was added, and the mixture was mixed by inversion several times, and placed in a -20°C refrigerator for 2.5h; centrifuged at 11400rpm at room temperature for 15min, and the supernatant was discarded; 700μl anhydrous ethanol was added, and the precipitate was suspended by inversion, 12000rpm, 4min; 12000rpm, 4min, the supernatant was discarded, the lid was opened and dried in an oven, and 45μl ddH2O was added to dissolve the extracted DNA.

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

[0082] Experimental results: Figure 4 As shown in A. Among the 40 wheat lines overexpressing TabZIP19-5B, only 6 lines had no detected bar gene, and 34 lines had positive seedlings.

[0083] (2) Expression level identification

[0084] Wheat leaf samples were collected and placed in 2 ml RNAse-free centrifuge tubes, frozen at low temperature, and broken using a tissue disruptor; total RNA was isolated using the Trizol method (TianGen), followed by the DNAse I (Takara) method to eliminate genomic contamination, and then the concentration was determined using Nanodrop 1000 (Thermo Scientific product, USA), and 2 μl of samples were uniformly taken and run on 0.8% agarose gel. 1 μg of total RNA was taken, and cDNAs were synthesized using recombinant M-MLV reverse transcriptase and Oligo (dT) as primers, and quantitative detection was performed.

[0085] The primer pair for detecting the expression level of TabZIP19-5B gene was 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 TaActin1 gene was 5'-GCTGACTGTGCTGTTCTCATCATC-3' (SEQ ID NO. 13) and 5'-GCGCCTTTGAGTACTTGGGAG-3' (SEQ ID NO. 14).

[0087] Experimental results: Figure 4 As shown in B. The expression level of TabZIP19-5B overexpressing wheat in T0 was quantitatively analyzed, and the results showed that OE#1, OE#11, and OE#34 lines were about 4 times that of the control group.

[0088] 2. Establishment of TabZIP19-5B knockdown wheat plants

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

[0090] Submit wheat exons to http: / / crispr.dbcls.jp / , select “Wheat (Triticum aestivum) genome, IWGSC1.0+popseq (Nov, 2014)” as the species, and after selecting the appropriate design target, obtain two sgRNA sequences targeting the TabZIP19-5B gene: sgRNA1 5'-CACCAAGATCGTCTCGGCGT-3' (SEQ ID NO.15), sgRNA2 5'-TGAGGAAGTACCGGGAGAAG-3' (SEQ ID NO.16), primer adapters need to be added to construct dual target sites, and PCR amplification is performed using the intermediate vector MT1T2 as a template, TabZIP19-5B-F0 / TabZIP19-5B-R0 and TabZIP19-5B-F / TabZIP19-5B-R as amplification primers to obtain a vector containing two target sites and one gRNA-Scfford. After gel recovery, it is digested with BsaI and the final vector pBUE413 is digested with BsaI at the same time. pBUE413 contains a gRNA-Scfford alone, and finally connected with T4 ligase, so that each target site is followed by a gRNA-Scfford, and the vector for knocking down the TabZIP19-5B gene can be constructed. The method for constructing the dual-target CRISPR / Cas9 knockout intermediate vector MT1T2 and the final vector pBUE413 is 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. The recombinant plasmid was transferred into EHA105, spread on a solid LB plate carrying kana (50 mg / mL) and rifampicin (25 mg / mL) resistance, and cultured at 28°C for 2 days. Single clones were selected for PCR detection, and single colonies with the correct band size were marked. The Agrobacterium plate was sent to the transgenic platform of the National Key Laboratory of Crop Stress Resistance and Efficient Production of Northwest Agriculture and Forestry University for transgenic wheat transformation.

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

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

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

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

[0095] (2) Screening of homozygous strains with knockdown of TabZIP19-5B gene

[0096] The TabZIP19-5B knockdown expression T0 generation transgenic seedlings obtained from the transgenic platform of the National Key Laboratory of Crop Resistance and Efficient Production of Northwest Agriculture and Forestry University were planted in the soil. After the wheat had rooted in the soil for a period of time, a leaf section was taken for DNA extraction, and primers 5'-GGAGTGAGTACGGTGTGCACACCCACACCTGCGTCCAC-3' (SEQ ID NO.21) and 5'-GAGTTGGATGCTGGATGGTGAACCACCTCTTCCTCCAG-3' (SEQID NO.22) were designed near the two target sites to amplify the DNA templates of different wheat strains, and the PCR products were sequenced.

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

[0098] Example 5

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

[0100] 1. Identification of salt tolerance at the seedling stage: The TabZIP19-5B overexpressing T0 generation transformed seedlings (1, 11 and 34) in Example 4 were identified to obtain strains with relatively high expression levels. They were further grown in a greenhouse and identified at the transcriptional level. Then, the T2 seeds were planted to obtain T3 generation seeds (three overexpressing wheat seeds: OE#1, OE#11, and OE#34).

[0101] The TabZIP19-5B knockdown expression T0 generation transformed seedlings (1, 2 and 3) in Example 4 were identified to obtain strains with relatively high expression levels, which were further generations in the greenhouse and identified at the transcription level. The T2 seeds were then planted to obtain T3 generation seeds (three knockdown expression wheat seeds: KO#1, KO#2, and KO#3).

[0102] The surface of TabZIP19-5B overexpression and knockdown T3 transgenic wheat seeds and Fileder seeds were disinfected and then placed in a petri dish filled with sterile water for germination. When the sprouts were 3 cm long, uniform seeds were selected and planted in a nutrient solution prepared with Hogland and calcium salt. When the wheat grew to more than two leaves and one heart, it was treated with 250mM NaCl for 14 days and photographed.

[0103] Salt tolerance identification in adult stage: Three overexpression wheat seeds (OE#34), knockdown expression wheat seeds (KO#1) and control Fileder seeds were planted in nutrient soil. When the seeds grew to 1 cm, uniform seeds were selected and planted in flower pots (the culture soil was: nutrient soil: vermiculite: field soil 2:1:1). When they grew to 4 weeks old, they were treated with 100mM, 300mM, 500mM and 700mM NaCl solutions every other week for 4 weeks, and photos were taken at the heading stage.

[0104] Experimental results: Figure 5 As shown in the figure, wheat overexpressing the TabZIP19-5B gene had reduced leaf death and better growth, while homozygous wheat with knockdown of the TabZIP19-5B gene had stunted growth and weak growth.

[0105] Example 6

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

[0107] Aphids growing uniformly on the leaves of infected black oats were selected and inoculated into overexpression (OE#34), knockdown expression wheat (KO#1) and control WT wheat at the two-leaf one-heart stage. Five aphids were inoculated on each wheat plant and isolated and raised in a plastic cover with air holes. Aphids were removed after 3 days, and samples were taken after 10 days and stored at -80℃. Wheat RNA was extracted (Biospin polysaccharide and polyphenol plant total RNA extraction kit (BSC65) was purchased from Hangzhou Biori Technology Co., Ltd.), the concentration of the extracted RNA was measured, 1000ng of RNA from each plant was reverse transcribed (RTmix with DNase (DN2211-01) was purchased from Beijing Dining 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: Figure 6 The virus content of wheat overexpressing the TabZIP19-5B gene decreased by 14.88 times compared with the WT control group. The antiviral ability of the TabZIP19-5B overexpressing strain was significantly improved, and the yellowing was less. The ear length of the TabZIP19-5B overexpressing strain became longer and the number of grains per ear increased, while the ear length of the TabZIP19-5B knockdown expression strain became shorter and the number of grains per ear decreased.

[0111] Example 7

[0112] Identification of agronomic traits of wheat overexpressing TabZIP19-5B

[0113] Three high expression (OE#1, OE#11, OE#34) overexpression wheat seeds and Fileder wheat seeds of uniform size were selected, and after disinfection and sterilization, they were sown on filter paper, treated at 4°C for 3 days, and then taken out and placed in a 20°C incubator for 3 days, and transferred to a 18.8cm*18.8 square culture pot filled with a 1:1 mixture of Pindstrup imported matrix and V9 domestic matrix. The overexpression wheat was placed in a 22°C generation room for about three months, and when the wheat was fully mature, the individual plants were threshed and the grain length, grain width and grain weight were counted.

[0114] Experimental results: Figure 7 The grain width of the TabZIP19-5B overexpression strain increased significantly, the grain length of the TabZIP19-5B overexpression strain had no significant difference, and the thousand-grain weight of the TabZIP19-5B overexpression strain increased significantly.

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

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A wheat TabZIP19-5B gene, characterized in that The nucleotide sequence of the wheat TabZIP19-5B gene is shown in SEQ ID NO.

1.

2. The protein encoded by the wheat TabZIP19-5B gene according to claim 1, characterized in that The amino acid sequence of the protein encoded by the wheat TabZIP19-5B gene is shown in SEQ ID NO.

2.

3. A recombinant vector for overexpressing or knocking down wheat TabZIP19-5B gene, characterized in that: The recombinant vector for overexpressing wheat TabZIP19-5B gene comprises an initial expression vector and wheat TabZIP19-5B gene, and the initial expression vector for overexpressing wheat TabZIP19-5B gene is Cub-3×flag vector; 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 vector for knocking down the wheat TabZIP19-5B gene is an MT1T2 vector and a pBUE413 vector. The sgRNA targeting the wheat TabZIP19-5B gene is shown in SEQ ID NO.15 and SEQ ID NO.

16.

4. A recombinant bacterium for overexpressing or knocking down the wheat TabZIP19-5B gene, characterized in that: The recombinant bacteria are transformed with the recombinant vector according to claim 3.

5. A method for overexpressing wheat TabZIP19-5B gene, characterized in that: The following steps are involved: The wheat TabZIP19-5B gene was connected to the Cub-3×flag vector to obtain the recombinant vector Cub-TabZIP19-5B-3×flag; The recombinant vector is introduced into a recipient plant after transformation with Agrobacterium to obtain a TabZIP19-5B transgenic plant, thereby achieving overexpression of the wheat TabZIP19-5B gene.

6. A method for knocking down the wheat TabZIP19-5B gene, characterized in that: The following steps are involved: The sgRNA targeting the wheat TabZIP19-5B gene was connected to the MT1T2 vector and the pBUE413 vector to obtain a recombinant plasmid; The recombinant plasmid was introduced into the recipient plant after Agrobacterium transformation to obtain TabZIP19-5B transgenic plants, thereby achieving knockdown expression of the wheat TabZIP19-5B gene.

7. Use of the wheat TabZIP19-5B gene according to claim 1, the protein according to claim 2, the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in regulating the salt tolerance of plants, characterized in that: Overexpressing the wheat TabZIP19-5B gene in plants improves the salt tolerance of plants, while knocking down the wheat TabZIP19-5B gene in plants reduces the salt tolerance of plants.

8. Use of the wheat TabZIP19-5B gene according to claim 1, the protein according to claim 2, the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in regulating the ability of plants to resist barley yellow dwarf virus, characterized in that: Overexpressing the wheat TabZIP19-5B gene in plants improves the plants' resistance to barley yellow dwarf virus, while knocking down the wheat TabZIP19-5B gene in plants reduces the plants' resistance to barley yellow dwarf virus.

9. Use of the wheat TabZIP19-5B gene according to claim 1, the protein according to claim 2, the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in regulating plant thousand-grain weight, characterized in that: Overexpression of the wheat TabZIP19-5B gene in plants increased the thousand-grain weight of the plants, and knocking down the wheat TabZIP19-5B gene in plants reduced the thousand-grain weight of the plants.

10. The use according to any one of claims 7 to 9, characterized in that: The plant is wheat.

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