Wheat peel thickness related gene TaNUD and application thereof

Abstract

The invention discloses a wheat peel thickness related gene TaNUD and application thereof. The wheat TaNUD gene knockout and overexpression material is obtained by an applicant through a gene engineering technology, and an experiment proves that compared with a wild plant Fielder, strains of mutants aaddbb-1 (nudabd) and aabbdd-2 obtained by knocking out the gene TaNUD present a phenotype that the thickness of mature grain peel is reduced; and the overexpression system is just opposite. Therefore, the TaNUD gene not only can adjust the thickness of the seed peel, but also can provide important gene reserve for molecular design breeding, and lays a foundation for cultivating high-quality wheat varieties.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology and genetic engineering, and relates to a wheat pericarp thickness related gene TaNUD and application thereof. BACKGROUND

[0002] The pericarp is the outermost tissue of a wheat kernel, which wraps the whole kernel and protects the kernel from invasion. The pericarp thickness is an important factor affecting flour making, liquor brewing and sprout emergence resistance, and is regulated by some genes related to flavonoid biosynthesis and transcription factors of different families such as TT, MADS-box and Aux / IAA.

[0003] Transcription factors, also known as anti- / type action factors, can activate gene expression by specifically binding to the related cis-acting elements in the promoter region of the gene. At present, a variety of plant-related transcription factors have been reported, such as AP2 / ERF, bHLH, MYB and NAC, among which the AP2 / ERF transcription factor family is one of the largest transcription factor families in plants, which not only participates in the regulation of plant growth and development, but also plays an important role in response to stress. Therefore, in-depth research and understanding of the AP2 / ERF transcription factor family genes are of great significance for crop breeding improvement.

[0004] The applicant identifies an ERF-type transcription factor TaNUD (nudum) through bioinformatics analysis, and there is no related report on the application of the transcription factor in the regulation of wheat pericarp thickness. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a wheat pericarp thickness related gene TaNUD and application thereof.

[0006] To achieve the above purpose, the present application provides the following technical solutions.

[0007] 1. A wheat pericarp thickness related gene TaNUD, wherein the coding region (CDS) sequence of the gene is shown as SEQ ID NO. 1.

[0008] As one of the preferred technical solutions, the nucleotide sequence of the gene TaNUD is shown as SEQ ID NO. 2.

[0009] 2. A coding protein of the wheat pericarp thickness related gene TaNUD or a homologous protein thereof, wherein the amino acid sequence of the coding protein is shown as SEQ ID NO. 3.

[0010] 3. Application of knocking out the gene TaNUD in reducing the pericarp thickness of wheat.

[0011] 4. Application of overexpressing the gene TaNUD in increasing the pericarp thickness of wheat.

[0012] 5. Use of an inhibitor for reducing the expression of the aforementioned protein-encoding gene or its homologous gene in reducing the pericarp thickness of wheat.

[0013] 5. Use of an enhancer for increasing the expression of the aforementioned protein-encoding gene or its homologous gene in increasing the pericarp thickness of wheat.

[0014] 6. Use of a knockout of the aforementioned gene TaNUD in wheat breeding.

[0015] 7. Use of an overexpression of the aforementioned gene TaNUD in wheat breeding.

[0016] 8. Use of a reduction in the expression of the aforementioned protein-encoding gene or its homologous gene in wheat breeding.

[0017] 9. Use of an increase in the expression of the aforementioned protein-encoding gene or its homologous gene in wheat breeding.

[0018] The present application has the following beneficial effects:

[0019] The applicant identifies an ERF-type transcription factor TaNUD (nudum) through bioinformatics analysis. TaNUD has a high expression level in the pericarp and a low expression level in the roots, stems, leaves and endosperm. By knocking out TaNUD through gene editing, the mutant aaddbb-1 and aabbdd-2 are obtained, and the pericarp thickness of these mutants is significantly reduced. Meanwhile, overexpression lines are obtained through transgenesis, and it is observed that the pericarp thickness is increased.

[0020] The present application identifies a gene TaNUD that affects the pericarp thickness of wheat, which can provide gene resources for subsequent molecular design breeding to cultivate high-quality wheat varieties.

[0021] The applicant obtains wheat TaNUD gene knockout and overexpression materials through genetic engineering technology, and experiments prove that, compared with wild-type plants, the mutant aaddbb-1 and aabbdd-2 obtained by knocking out the gene TaNUD show a phenotype of reduced pericarp thickness of mature grains, while the overexpression lines are just the opposite. Therefore, the TaNUD gene can regulate the pericarp thickness of grains, and can provide important gene reserves for molecular design breeding and lay a foundation for cultivating high-quality wheat varieties. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application provides the following drawings for illustration:

[0023] Figure 1 Evolutionary tree map of NUD genes in different species.

[0024] Figure 2Figure 6 is a diagram showing TaNUD expression pattern analysis, where A is the expression level of three alleles of NUD in different tissues, and B is the expression level of three alleles of NUD in seed pericarp at different developmental stages.

[0025] Figure 3 Figure 7 is a diagram showing TaNUD-D in situ hybridization results.

[0026] Figure 4 Figure 8 is a diagram showing gene editing knockout and overexpression line construction.

[0027] Figure 5 Figure 9 is a diagram showing pericarp thickness changes of knockout lines and overexpression lines. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present application will be described in detail below with reference to the attached drawings.

[0029] NUD gene evolution analysis

[0030] The barley pericarp thickness regulating gene HvNud was found in published articles (Taketa S, Amano S, Tsujino Y, et al. Barley grain with adhering hulls is controlled by an ERF family transcription factor gene regulating a lipid biosynthesis pathway. Proc Natl Acad Sci, 2008, 105(10):4062-4067), and three homologous genes TraesCS7A02G376300, TraesCS7B02G277800 and TraesCS7D02G372700 in wheat were obtained according to sequence similarity, and the amino acid sequences were obtained from the Ensembl Plants website according to the gene number. The amino acid sequences of nud homologous genes in barley (Hordeum vulgare), Arabidopsis thaliana, rice (Oryza sativa) and corn (Zea mays) were searched by the conserved domain AP2 of the protein encoded by TraesCS7D02G372700 through the gene number. Multiple alignment of the domains was performed by Clustal W, and phylogenetic tree analysis of the AP2 domain was performed using MEGA7 software. The phylogenetic tree was constructed by selecting the Poisson data model, the neighbor-joining method, the bootstrap method for phylogenetic test, and the number of repetitions was 1000. Through phylogenetic tree analysis, it was found that the TraesCS7D02G372700 gene had the closest genetic relationship with the barley nud gene; through amino acid sequence comparison, it was found that the AP2 domain of the barley nud gene and the three partial homologous genes were completely identical, and the sequence similarity was as high as 96%, so the three partial homologous genes were named NUD-A, NUD-B and NUD-D. Figure 1 )

[0031] TaNUD expression pattern analysis

[0032] With Fielder as the material, plant in different environments: 2022 Shangzhuang (22SZ), 2023 Shangzhuang (23SZ), take Fielder root, stem, leaf and wheat grain pericarp and endosperm tissue 5, 10, 15, 20, 25 days after pollination as the material pericarp, use polysaccharide polyphenol plant RNA extraction kit (Coolaber) to extract total RNA of the above tissue samples, reverse transcribe cDNA with HiScript II Q RTSuperMix for qPCR (+gDNA wiper), and detect the expression amount of TaNUD by real-time fluorescent quantitative PCR (qRT-PCR) with AceQ qPCR SYBR Green Master Mix (Without ROX). Among them, the internal reference gene is actin myosin, the upstream primer is 5'-GGAATCCATGAGACCACCTAC-3', and the downstream primer is 5'-GACCCAGACAACTCGCAAC-3'. The qRT-PCR detection of TaNUD-A uses the upstream primer 5'-TGCTGCAAGACGCCGTCC-3' and the downstream primer 5'-GTGGACGTGGACGAGCAA-3'; the qRT-PCR detection of TaNUD-B uses the upstream primer 5'-TGTAAGACACCATCCCCGTC-3' and the downstream primer 5'-CGTGGACGTCGACGAGGAA-3'; the qRT-PCR detection of TaNUD-D uses the upstream primer 5'-CCGAGAAGTCGCACATTG-3' and the downstream primer 5'-CTCTCCTCGTCTTCCATTGTC-3'. The PCR reaction system is as follows: SYBR Green Master Mix, 7.5 μl; upstream primer (10 μm), 0.3 μl; downstream primer (10 μm), 0.3 μl; ddH2O, 5.4 μl. The PCR reaction program is as follows: 95℃, 5 min; 40 cycles [95℃, 30 s; 60℃, 30 s; 72℃, 20 s]; melting curve 65℃ to 95℃, 5 s; increase by 0.5℃ each time.

[0033] The results of fluorescent real-time quantification show that the expression amount of TaNUD gene in pericarp tissue is high, and the expression amount in root, stem, leaf and endosperm tissue is very low. From the pericarp tissue of different pollination days, the expression patterns of A copy and B copy are similar, and the expression amount of both is the highest in the pericarp tissue 5 days after pollination, and then decreases in turn with the development of wheat pericarp; the expression of D copy in the pericarp tissue 10 days after pollination is higher than that 5 days after pollination, and then decreases. Therefore, the high expression of TaNUD gene in the pericarp tissue 5 and 10 days after pollination indicates that TaNUD gene may be involved in the formation of early pericarp. Figure 2 ​

[0034] DNA sequence of TaNUD:

[0035] ATGGTACAGTCCAAGAAGAAGTTTCGCGGCGTCAGGCAGCGCCACTGGGGCTCCTGGG

[0036] TCTCCGAGATCAGGCACCCTCTCCTGTAAGCCTCACTACTCCCTAGCTCTCTCTCTCACT

[0037] CTCACTAGCTGCTCTACTCCTACTCGCCGGCCTACTCGTGTAGCTATCCCGCTGTGTCCTG

[0038] AGAGATAGATACATTGATTCCTGTTTGAATTGCTACGTACTTTAAGGTTGCTTTCGTCGAT

[0039] TTTCTCTTGATGCGTTCCAAATGCATGCATGTGTGTTGTGTATGGTTTGCAGGAAGAGGA

[0040] GGGTGTGGCTGGGCACCTTTGAGACGGCGGAGGAGGCGGCGCGGGCGTACGACGAGG

[0041] CCGCCATCCTGATGAGCGGGCGCAACGCCAAGACCAACTTCCCCGTGCCAAGGAGCGC

[0042] CAACGGGGAGATCATCGTCGCCCCAGCAGCGGCGCGGGACGGCCGCGGTGGCGTCGGC

[0043] TCGTCGTCCTCTGGCGCGGCCGGCGCCAGCAGCCTGTCACAGATCCTCAGCGCCAAGCT

[0044] CCGCAAGTGCTGCAAGACACCGTCCCCGTCCCTCACCTGCCTCCGCCTCGACACCGAG

[0045] AAGTCGCACATTGGCGTCTGGCAGAAGCGCGCGGGTGCCCGCGCCGACTCCAGCTGGG

[0046] TCATGACCGTCGAGCTCAACAAGGAGCCGGCGACAGCGGCAGCGGCACCAACGCCCA

[0047] GTGACAGCACGGTGTCGGCGACTCCTTCCTCGTCCACTTCCACGTCCACAACGGGCTCG

[0048] CCGCCGGAGACAATGGAAGACGAGGAGAGGATCGCGCTGCAGATGATCGAGGAGCTGC

[0049] TGAGCAGGAGCAGCCCGGCCTCGCCGTCACATGGGCTGCTGCACGGTGAAGAAGGCAG

[0050] CCTCGTCATCTGA

[0051] CDS sequence of TaNUD:

[0052] ATGGTACAGTCCAAGAAGAAGTTTCGCGGCGTCAGGCAGCGCCACTGGGGCTCCTGGG

[0053] TCTCCGAGATCAGGCACCCTCTCCTGAAGAGGAGGGTGTGGCTGGGCACCTTTGAGAC

[0054] GGCGGAGGAGGCGGCGCGGGCGTACGACGAGGCCGCCATCCTGATGAGCGGGCGCAA

[0055] CGCCAAGACCAACTTCCCCGTGCCAAGGAGCGCCAACGGGGAGATCATCGTCGCCCCA

[0056] GCAGCGGCGCGGGACGGCCGCGGTGGCGTCGGCTCGTCGTCCTCTGGCGCGGCCGGCG

[0057] CCAGCAGCCTGTCACAGATCCTCAGCGCCAAGCTCCGCAAGTGCTGCAAGACACCGTC

[0058] CCCGTCCCTCACCTGCCTCCGCCTCGACACCGAGAAGTCGCACATTGGCGTCTGGCAGA

[0059] AGCGCGCGGGTGCCCGCGCCGACTCCAGCTGGGTCATGACCGTCGAGCTCAACAAGGA

[0060] GCCGGCGACAGCGGCAGCGGCACCAACGCCCAGTGACAGCACGGTGTCGGCGACTCCT

[0061] TCCTCGTCCACTTCCACGTCCACAACGGGCTCGCCGCCGGAGACAATGGAAGACGAGG

[0062] AGAGGATCGCGCTGCAGATGATCGAGGAGCTGCTGAGCAGGAGCAGCCCGGCCTCGCC

[0063] GTCACATGGGCTGCTGCACGGTGAAGAAGGCAGCCTCGTCATCTGA

[0064] Amino acid sequence of TaNUD:

[0065] MDDIAEPGSPTSPTATSSSSSSSSSSSVAANKRPRKDSRHPTYHGVRMRSWGKWVSEIREPR

[0066] KKSRIWLGTFATAEMAARAHDVATLAIKGRAAHLNFPDLAHLLPRPASAAPKDVQAAAILA

[0067] ASADFPCGASANAKSPDSASDASAVSPPPPPAPHAEPVQDPEDALFDLPDLLLDLRYEASAG

[0068] LPCASSWVVDEDIVGAGVFRLEEPLLWEY

[0069] In situ hybridization to verify the expression position of TaNUD gene

[0070] In order to determine the expression position of TaNUD in the grain, the applicant selected the in situ hybridization specific probe of TaNUD-D with the highest expression amount to conduct in situ hybridization experiment on the grain of wild type wheat Fielder 5DAF. The in situ hybridization result shows that TaNUD-D is specifically expressed in the inner pericarp. This result shows that TaNUD-D may play an important role in the early development process of the grain. Figure 3

[0071] ​Gene editing knock-out of TaNUD

[0072] Target design and genetic transformation. There are three homologous genes of TaNUD in the ABD subgenome of wheat. The sgRNA for knocking out the three homologous genes of TaNUD was designed by using the E-CRISPR online tool (http: / / www.e-crisp.org / E-CRISP / designcrispr.html) with the following parameters: 1. Select organism: Triticum aestivum IWGSC1+popseq.31; 2. Select target region by gene symbol or sequence: Input is FASTA sequence; 3. Start application: medium; Single design. Two sgRNAs were designed, sgRNA1: GGCGTCAGGCAGCGCCACTGG, sgRNA2: GTGGCTGGGCACCTTTGAGA( Figure 4 ). The sgRNAs were amplified by PCR with the following conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min, purification of the target fragment, Bsa I enzyme digestion, T4 ligase ligation, and finally the two sgRNAs were ligated to the gene editing vector pBUE414 (Xing HL, Dong L, Wang Z P, et al. A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol, 2014, 14(1): 327). The recombinant vector pBUE414 was transformed into Agrobacterium EHA105, and then the Agrobacterium EHA105 containing the recombinant vector was used to dip the young embryos of the wheat variety Fielder. After herbicide (BAR) resistance screening, the resistant callus was subcultured, and the resistant callus was cultured for differentiation until young seedlings were differentiated.

[0073] Strain identification. A total of 5 T0 regenerated plants were obtained. The whole genome DNA of the 5 regenerated plants was extracted, and primers were designed according to the flanking sequences of the target site (upstream primer 5'-CACTGCTCCCCCCACTCTT-3', downstream primer 5'-ACGGTGTCTTGCAGCACTT-3'), and PCR amplification was performed: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 30 s, 35 cycles; 72 °C extension for 5 min; sequencing of the target fragment; identification of the gene editing site type. At the same time, T1 generation seeds were harvested. Then, T1 generation families were planted, 12 plants were planted for each family, the whole genome DNA of the plants was extracted, PCR amplification was performed using the above specific primers, the target fragment was sequenced, and the gene editing site type was identified again. After gene editing site identification, the family TaNUD-abd1 and TaNUD-abd-2 in which the homologous genes on the ABD subgenome were all mutated were screened out.

[0074] TaNUD-abd-2. T2 generation seeds were harvested.

[0075] Overexpression of TaNUD

[0076] The CDS of the above TaNUD gene was amplified by PCR: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 15 s, 60 °C annealing for 15 s, 72 °C extension for 15 s, 35 cycles; 72 °C extension for 5 min; purification of the target fragment; homologous recombination and connection to the overexpression vector plasmid pWMB003 (Guo G, Liu X, Sun F, et al. Wheat miR9678 Affects Seed Germination by Generating Phased siRNAs and Modulating Abscisic Acid / Gibberellin Signaling. The Plant Cell, 2018, 30(4): 796-814) in the laboratory, and the UBI promoter was used to express the TaNUD gene. The genetic transformation process was the same as that of gene knockout.

[0077] Strain identification. Two T0 regenerated plants were obtained. The whole genome DNA of the two regenerated plants was extracted, and primers were designed according to the vector sequence (upstream primer 5'-TCGATGCTCACCCTGTTGTTTG-3', downstream primer 5'-GGCGGACTTGAAGAAGTCGTG-3'), and PCR amplification was performed: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 15 s, 60 °C annealing for 15 s, 72 °C extension for 60 s, 35 cycles; 72 °C extension for 5 min, and the target fragment was sequenced to identify whether it was a positive overexpression strain. At the same time, T1 generation seeds were harvested. Then, T1 generation families were planted, 12 plants were planted in each family, the whole genome DNA of the plants was extracted, PCR amplification was performed using the above specific primers, and the overexpression positive strain was identified again, and finally the overexpression strain NUD-OE was obtained.

[0078] Gene editing knockout and overexpression strain construction is shown in Figure 4 .

[0079] Measurement of mature grain pericarp thickness

[0080] 1) Take 50 grains of mature dry seeds of wild type Fielder and TaNUD gene knockout mutant (nudabd) and overexpression strain NUDO E of uniform size, put them into 10 mL centrifuge tubes, add 5 mL of deionized water, soak at room temperature for 2 h. Then take out the seeds, dry the surface water with toilet paper, and place them with their backs up. Cut vertically along the position of the wheat shield bottom cut surface, discard the shield end; then cut the wheat seeds vertically again at an interval of about 1 mm, so that they are in the form of uniform slices.

[0081] 2) Staining

[0082] Put the cut seeds in 5 ml centrifuge tubes, add 0.1% Evans Blue dye just covering the mass of all the grains, shake up and down every 10 seconds to ensure thorough mixing. After 2 min, take out the cut slices, rinse with deionized water, soak for 15 min, pour out the water and place the slices on the water absorption paper to dry naturally.

[0083] 3) Measurement of grain pericarp thickness

[0084] The pericarp thickness was measured, and it was found that the pericarp thickness of the gene edited family was significantly reduced. The pericarp thickness of the unedited family was 15.76-22.86 μm, and the pericarp thickness of the mutant families TaNUD-abd1 (nudabd) and TaNUD-abd2 was 12.21-19.98 μm and 13.33-19.57 μm, respectively.

[0085] All the sections were observed under microscope and photographed at the same magnification, and then imported into image J software to measure the thickness of the pericarp. When measuring, 6-8 pericarp cells at the same position on the section were selected for determination. Analysis found that the variation ranges of pericarp thickness of wild type Fielder, knockout mutant nudabd and overexpression line NUDOE were 18.59±2.367 μm, 16.27±1.796 μm and 21.78±1.853 μm, respectively. Figure 5

[0086] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.​

Claims

1. A wheat grain pericarp thickness associated gene, TaNUD, characterized in that, The coding region sequence is shown as SEQ ID NO.

1.

2. A protein encoded by the wheat kernel pericarp thickness-related gene TaNUD or a homologous protein thereof, characterized in that, The amino acid sequence of the encoded protein is shown as SEQ ID NO.

3.

3. Application of knocking out the gene TaNUD in claim 1 in reducing the thickness of wheat pericarp.

4. Application of overexpressing the gene TaNUD in claim 1 in increasing the thickness of wheat pericarp.

5. Application of an inhibitor for reducing the expression amount of the encoded protein in claim 2 or a homologous protein thereof in reducing the thickness of wheat pericarp.

6. Application of an enhancer for increasing the expression amount of the encoded protein in claim 2 or a homologous protein thereof in increasing the thickness of wheat pericarp.

7. Application of knocking out the gene TaNUD in claim 1 in wheat breeding.

8. Application of overexpressing the gene TaNUD in claim 1 in wheat breeding.

9. Application of reducing the expression amount of the encoded protein in claim 2 or a homologous protein thereof in wheat breeding.

10. Application of increasing the expression amount of the encoded protein in claim 2 or a homologous protein thereof in wheat breeding.

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