Application of TaKNAT1D gene in regulating wheat ear traits

Abstract

The present invention discloses the application of wheat TaKNAT1D gene in regulating wheat ear traits, and belongs to the technical field of plant genetic engineering. The present invention discovers and confirms a new gene for regulating wheat ear traits, named TaKNAT1D gene, which is located on wheat chromosome 5D, and its nucleotide sequence is shown in SEQ ID NO.1. The present invention has found that overexpression of TaKNAT1D gene can increase the number of wheat spikelets, spike length and spikelet number, and the number of spikelets, spike length and spikelet number of wheat with TaKNAT1D gene knocked out are significantly reduced. Therefore, TaKNAT1D gene can be used as a regulatory gene for wheat ear traits, laying a foundation for increasing wheat yield and improving traits.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to application of TaKNAT1D gene in regulating wheat ear traits. Background Art

[0002] Wheat is the most widely planted and distributed crop in the world, covering an area of ​​about 200 million hectares. As one of the most important staple food crops, about 43 countries and about 40% of the population in the world rely on wheat as their staple food. my country is one of the world's largest wheat producers, with huge consumer demand and imports. However, affected by the adjustment of the agricultural industrial structure, the growth rate of wheat planting area has slowed down significantly. Although wheat production is still on the rise thanks to the continuous progress of my country's wheat breeding technology and planting methods and the development of planting facilities, in order to meet the needs of the growing global population, it is estimated that wheat production needs to increase from the current 3 tons per hectare to 5 tons per hectare. Therefore, it is crucial to increase wheat yield under the premise of stabilizing the planting area. Wheat yield is mainly affected by factors such as the number of spikes per unit area of ​​land, the number of grains per spike and the grain weight. Among them, the number of grains per spike can be increased by increasing the number of fertile spikelets and florets, which is closely related to the development of wheat spike meristems. Therefore, studying the genetic basis of wheat ear meristem development regulation and elucidating the molecular genetic network that controls the development of wheat ear meristem are crucial for understanding the mechanism of the formation of wheat grain number and yield traits and for breeding high-yield wheat varieties.

[0003] In recent years, although some genes and loci that regulate the development of wheat ear meristem have been identified through gene positioning and research, the number of functional genes discovered so far is far from sufficient to fully analyze the molecular regulatory mechanism of wheat ear meristem development. The exploration of genetic resources that play a significant role in yield improvement has not yet been fully launched. Therefore, it is of vital importance to analyze the molecular genetic basis of wheat ear meristem development and to explore more functional genes that regulate wheat ear meristem development and thus affect yield traits. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide the application of TaKNAT1D gene in regulating wheat ear traits. The present invention has found that overexpression of TaKNAT1D gene can increase the number of wheat spikelets, spike length and number of grains per spike, and the number of spikelets, spike length and number of grains per spike of wheat with TaKNAT1D gene knocked out are significantly reduced. Therefore, TaKNAT1D gene can be used as a regulatory gene for wheat ear traits, laying a foundation for increasing wheat yield and improving traits.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides the use of the TaKNAT1D gene in any one of the following (1)-(3):

[0007] (1) Regulating wheat ear traits;

[0008] (2) Regulating wheat yield;

[0009] (3) Wheat breeding;

[0010] The TaKNAT1D gene is a DNA molecule as shown in the following i) or ii) or iii):

[0011] i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0012] ii) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i);

[0013] iii) a DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii), and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.2.

[0014] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. Homology can be evaluated using computer software, such as the BLAST algorithm (Altschul et al. 1990. Journal of Molecular Biology 215: 403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90: 5873-5877).

[0015] In the above nucleic acid molecules, the 90% or more homology may be at least 90%, 92%, 93%, 95%, 96%, 98% or 99% homology.

[0016] In the above application, the wheat ear traits include: number of spikelets, ear length and number of grains per ear.

[0017] The second aspect of the present invention provides the use of a protein encoded by the TaKNAT1D gene in the following (1) or (2):

[0018] (1) Regulating wheat ear traits;

[0019] (2) Regulate wheat production.

[0020] In the above application, the amino acid sequence of the protein encoded by the TaKNAT1D gene is shown as SEQ ID NO.2.

[0021] In the above application, the wheat ear traits include: number of spikelets, ear length and number of grains per ear.

[0022] The third aspect of the present invention provides a recombinant expression vector or genetically engineered bacteria containing the TaKNAT1D gene for use in any one of the following (1)-(3):

[0023] (1) Regulating wheat ear traits;

[0024] (2) Regulating wheat yield;

[0025] (3) Wheat breeding.

[0026] In the above application, the wheat ear traits include: number of spikelets, ear length and number of grains per ear.

[0027] A fourth aspect of the present invention provides a method for regulating wheat ear traits, comprising the following steps:

[0028] Up-regulating the expression of TaKNAT1D gene in wheat plants, obtaining transgenic wheat plants with increased spikelet number, spike length and spikelet number;

[0029] Alternatively, the expression of the TaKNAT1D gene in wheat plants is inhibited or downregulated to obtain transgenic wheat plants with reduced spikelet number, spike length and spikelet number.

[0030] In the above method, up-regulating the expression of TaKNAT1D gene in wheat plants is achieved by the following means:

[0031] The TaKNAT1D gene is introduced exogenously; or a DNA fragment that can activate or increase the transcription level, translation level or protein activity of the wheat TaKNAT1D gene is introduced.

[0032] In the above method, mutation or inhibition of the function of TaKNAT1D protein in wheat plants can be achieved by gene knockout.

[0033] Among them, gene knockout is to inactivate a specific target gene by changing the DNA sequence. For example, the TaKNAT1D gene can be knocked out using the Crispr-Cas9 knockout vector.

[0034] A fifth aspect of the present invention provides a method for cultivating a high-yield wheat variety, comprising the following steps:

[0035] The TaKNAT1D gene is exogenously transferred into wheat starting plants to overexpress the TaKNAT1D gene, thereby obtaining transgenic wheat plants with increased spikelet number, spike length and spikelet number compared with the wheat starting plants;

[0036] The transgenic wheat plants are used as parents for self-pollination, or are hybridized with wheat plants of other plant types to obtain wheat varieties with high-yield traits.

[0037] Beneficial effects of the present invention:

[0038] After long-term research and exploration, the present invention has discovered the gene TaKNAT1 that can increase the number of grains per ear, which is the main yield factor of wheat. The inventors used the DNA sequence of the full-length CDS fragment of the above gene to construct an overexpression vector and a gene knockout vector using Crispr-Cas9 technology, and then introduced Agrobacterium, and transformed wheat embryos using Agrobacterium-mediated method, and finally obtained a transgenic wheat strain. The inventors obtained transgenic wheat overexpressing the TaKNAT1 gene and transgenic wheat plants with TaKNAT1 gene knockout by identification. Through phenotypic statistics and analysis, it was found that the number of spikelets, spike length and number of grains per ear of transgenic wheat overexpressing TaKNAT1 were significantly increased compared with the control variety Fielder, while the number of spikelets, spike length and number of grains per ear of wheat knocked out by TaKNAT1 were significantly reduced. Therefore, the overexpression of this gene applied to the main wheat varieties has a large yield-increasing potential, is an important gene resource for improving wheat yield, and has important economic value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Schematic diagram of the structure of the wheat overexpression vector pUbi::TaKNAT1-6MYC and the process of transformation of wheat immature embryos mediated by Agrobacterium; in the figure, (A) Schematic diagram of pUbi::TaKNAT1-6MYC and PC186 vectors; (B) co-culture of immature embryos with Agrobacterium; (C) CIM-induced callus regeneration; (D) SIM-induced seedling regeneration; (E) Rooting medium to regenerate roots. CIM: screening medium B, SIM: differentiation medium.

[0040] Figure 2:Comparison of Fielder transformation process of pUbi::TaKNAT1-6MYC and control pC186 empty vector; in the figure, (A, E) callus cultured to CIM 14d after transformation of PC186 empty vector / Ubi::TaKNAT1-6MYC vector; (B, F) callus cultured to CIM 42d after transformation of PC186 empty vector / Ubi::TaKNAT1-6MYC vector; (C, G) callus cultured to SIM 5d after transformation of PC186 empty vector / Ubi::TaKNAT1-6MYC vector; (D, H) callus cultured to SIM 20d after transformation of PC186 empty vector / Ubi::TaKNAT1-6MYC vector; (IP) corresponding stereomicroscope observation of callus or regenerated seedlings (AH). Scale bar = 0.5mm (IK, MO), 1cm (L, P). CIM: screening medium B, SIM: differentiation medium.

[0041] Figure 3 : Detection results of the T0 generation transgenic positive plants transformed with TaKNAT1 overexpression; in the figure, T0-#1-T0-#14 represent the numbers of the transgenic plants.

[0042] Figure 4 :Detection results of the positive transgenic plants of Fielder T0 generation transformed with TaKNAT1 knockout vector; (A) Target location and vector structure of Crispr-Cas9 knockout vector. (B) Test strip detection results. (C) Genome editing of the knockout strain, with the PAM region marked in red and the target sequence in yellow.

[0043] Figure 5 : Comparison of panicle traits between the T1 generation of Fielder transgenic plants transformed with overexpression of TaKNAT1 and the wild type of Fielder; in the figure, (A) panicle length phenotype of the control and overexpression lines; (B) panicle number phenotype of the control and overexpression lines. (C) Statistical data of panicle length, total number of spikelets per panicle, number of fertile spikelets, number of fertile florets per panicle, number of grains per panicle, and panicle density; the error value represents the standard error (SEM), which is calculated by at least three independent biological replicates. * represents P < 0.5, ** represents P < 0.01, and *** represents P < 0.001.

[0044] Figure 6:Comparison of panicle traits between TaKNAT1 knockout mutant plants and Fielder wild type plants; In the figure, (A) panicle length phenotype and statistics; (B) phenotype and statistics of grain number per panicle; (C) statistical data of total spikelet number per panicle, fertile spikelet number and fertile floret number per panicle. Error values ​​represent standard error (SEM), calculated by at least three independent biological replicates. * represents P < 0.5, ** represents P < 0.01, and *** represents P < 0.001. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0046] As mentioned above, wheat ear traits are one of the important agronomic traits of wheat. The yield of wheat can be increased by increasing the number of spikelets, ear length and number of grains per ear. However, due to the complex genome of wheat, the genetic resources with the potential to increase yield in wheat are currently scarce.

[0047] In view of this, the present invention has conducted an in-depth study on genes related to wheat ears in the wheat genome. The hypothetical protein CFC21_079689[Triticum aestivum](GenBank:KAF7074871.1) is a gene with unknown function located on chromosome 5D of wheat. In order to study its function, the present invention first cloned the gene from wheat and named the cloned gene TaKNAT1D gene. The nucleotide sequence of the TaKNAT1D gene is shown in SEQID NO.1, which is as follows:

[0048]

[0049] The amino acid sequence of the protein encoded by the TaKNAT1D gene is shown in SEQ ID NO.2, and is as follows:

[0050] MERFPNLGGGGSSGSSSASMASFLQLPPPSASSPSPELAGEQHGSRLALQQLLAAPPPSAQQQQQRREISPADVATIKAKIMAHPLYSPLLASYLDCQKVGAPPEVLERLSAVAAKLDAGHGRGKHESPRPDPELDQFMEAYCNMLAKYREELARPIQEATEFFKSVE TQLDSITFTDSTNCEGAGSSEDELDTSCVEEIDPSAEDKELKHQLLRKYGGYVGSLRQEFCKRRKKGKLPKEARQKLLHWWELHSKWPYPSETEKIALAESTGLDQKQINNWFINQRKRHWKPAPEDMPFSVMDGGVGVGVSFLPAPQGPALYMDRAPFMVDGMYRLGS.

[0051] The present invention selects TaKNAT1D as a research object, firstly amplifies the TaKNAT1 gene from the cDNA of the young spike of Chinese spring wheat, constructs an overexpression vector with a DNA sequence containing the full-length CDS fragment of the TaKNAT1 gene, then introduces the overexpression vector into Agrobacterium, and infects the young embryo of wheat by Agrobacterium-mediated method, and then performs subculture until a transgenic positive strain with overexpression of the TaKNAT1 gene is obtained. At the same time, a specific target is designed by predicting a website, a Crispr-Cas9 knockout vector is constructed, and a transgenic positive plant with TaKNAT1 gene knockout is obtained by the same transformation method. Through identification and spike phenotype statistical analysis, it is found that overexpression of the TaKNAT1 gene will increase the spike length, spikelet number and spikelet number; while TaKNAT1 gene mutation will cause a decrease in the spikelet number, spike length and spikelet number of Fielder wheat. Therefore, the TaKNAT1 gene has a certain yield-increasing potential and can be used for variety improvement of high-yield wheat.

[0052] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0053] The experimental materials used in the examples of the present invention that are not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels. If the specific experimental conditions and methods are not specified in the examples of the present invention, conventional conditions are usually followed, such as J. Sambrook et al., ed., Molecular Cloning Experiment Guide (3rd Edition), Science Press, 2002; DL Spector et al., ed., Cell Experiment Guide, Science Press, 2001; or the conditions recommended by the manufacturer.

[0054] The wheat materials used in the present invention are Chinese spring wheat and Fielder wheat.

[0055] The composition of the culture medium used for wheat genetic transformation is as follows:

[0056] Co-culture medium: MS powder 2.12 g, sucrose 10 g, 2,4-D 2 mg, glutamine 5 mg, hydrolyzed casein 0.5 g, silver nitrate 4 mg, agar 8 g, dilute to 1 L with sterile water; pH = 5.8.

[0057] Recovery medium: MS powder 2.12 g, sucrose 10 g, 2,4-D 2 mg, glycine 2 mg, silver nitrate 4 mg, vitamin B5 0.5 mg, agar 8 g, dilute to 1 L with sterile water; pH = 5.8.

[0058] Screening medium A: MS powder 2.12 g, sucrose 10 g, zeatin 5 mg, glycine 2 mg, IAA 0.5 mg, hygromycin 15 mg, silver nitrate 4 mg, vitamin B5 0.5 mg, agar 8 g, dilute to 1 L with sterile water; pH = 5.8.

[0059] Screening medium B: MS powder 2.12 g, sucrose 10 g, 6-BA 5 mg, IAA 0.5 mg, glycine 2 mg, hygromycin 30 mg, silver nitrate 4 mg, vitamin B5 0.5 mg, agar 8 g, dilute to 1 L with sterile water; pH = 5.8.

[0060] Differentiation medium: MS powder 2.12 g, sucrose 10 g, zeatin 5 mg, IAA 0.5 mg, glycine 2 mg, hygromycin 15 mg, vitamin B5 0.5 mg, silver nitrate 4 mg, asparagine 5 mg, glutamine 5 mg, agar 8 g, dilute to 1 L with sterile water; pH = 5.8.

[0061] Rooting medium: MS powder 2.12 g, sucrose 10 g, IAA 0.5 mg, paclobutrazol 0.5 mg, glycine 1 mg, hygromycin 15 mg, vitamin B5 0.5 mg, asparagine 5 mg, glutamine 5 mg, agar 8 g, dilute to 1 L with sterile water; pH = 5.8.

[0062] Example 1: Cloning of wheat TaKNAT1 gene

[0063] 1. Extraction and Purification of Total RNA from Wheat

[0064] The RNA extraction kit used in this experiment is the UltraPureRNA Extraction Kit provided by Beijing Kangwei Reagent Biotechnology Co., Ltd. The specific experimental steps are as follows:

[0065] (1) Sample processing: Weigh 30-50 mg of Chinese spring wheat spikelet tissue material and grind it thoroughly in liquid nitrogen. Transfer the ground powder to a centrifuge tube containing 1 mL of TRIzon Reagent and shake it thoroughly to fully lyse the tissue sample. Place it at room temperature for 5 min to completely separate the protein-nucleic acid complex.

[0066] (2) Add chloroform at a ratio of 200 μL chloroform per 1 mL TRIzon Reagent, cover the tube, shake vigorously for 15 seconds, and then let stand at room temperature for 2 minutes.

[0067] (3) Precool the centrifuge at 4°C and centrifuge at 12,000 rpm / min for 10 min. After centrifugation, transfer the upper aqueous phase in the centrifuge tube containing the three layers to a new RNase-Free centrifuge tube. The RNA is mainly concentrated in the upper aqueous phase.

[0068] (4) Add an equal volume of 70% ethanol to the centrifuge tube. The ethanol should be prepared freshly and the water used for preparation should be RNase-free water. Invert and mix thoroughly.

[0069] (5) Transfer the solution obtained in the previous step into the adsorption column equipped with a collection tube once or multiple times, centrifuge at 12000 rpm / min at room temperature for 30 seconds, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0070] (6) Add 700 μL of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm / min at room temperature for 30 s, discard the waste liquid, and return the adsorption column to the collection tube.

[0071] (7) Add 500 μL of Buffer RW2 containing anhydrous ethanol to the adsorption column, centrifuge at 12,000 rpm at room temperature for 30 s, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube.

[0072] (8) Repeat step (7).

[0073] (9) Centrifuge the empty adsorption column at 12000 rpm / min at room temperature for 2 min, pour out the liquid in the collection tube, and dry it with a vacuum drying pump for about 5 min. The purpose of this step is to remove the residual ethanol in the adsorption column to avoid affecting the subsequent enzymatic reaction (enzyme digestion, PCR, etc.).

[0074] (10) Place the dried adsorption column in a new RNase-free centrifuge tube, add 30-50 μL RNase-Free Water to the middle of the adsorption column, let stand at room temperature for 1-2 min, and centrifuge at 12000 rpm / min for 2 min. The resulting solution is the RNA solution. Take a small amount of RNA solution for concentration determination and electrophoresis detection. The remaining RNA solution is quickly frozen with liquid nitrogen and stored in a -80°C refrigerator to prevent RNA degradation.

[0075] To ensure that the RNA quality meets the requirements, the purity and concentration of the purified RNA samples were detected by spectrophotometer and agarose gel electrophoresis, respectively. The purity and concentration standards are as follows: RNA purity is OD 260 / 280 and OD 260 / 230 All were within the range of 1.8-2.0, and the RNA concentration was within the range of 1.0-2.0 μg / μL.

[0076] 2. Synthesis of the First Strand of cDNA

[0077] The first-chain cDNA synthesis kit used in this experiment was completed by FastQuantRT Kit (with gDNase) from Tiangen Biotechnology Co., Ltd. The specific experimental steps are as follows:

[0078] (1) Take the template RNA out of the -80°C freezer and place it on ice to thaw. Take the kit out in advance and thaw it at room temperature. After thawing, quickly place it on ice. Prepare the genomic DNA removal system reaction solution. The preparation method is as follows:

[0079]

[0080] (2) Add the various components as shown in the table above, mix thoroughly, centrifuge briefly, and place in a preheated 42°C metal bath for incubation for 3 min.

[0081] (3) Prepare the reverse transcription reaction mixture (10 μL system). The preparation method is shown in the following table:

[0082]

[0083] (4) Add the components as shown in the table above to the centrifuge tube, mix well, centrifuge briefly, add to the genomic DNA removal system reaction solution, and shake thoroughly to mix well.

[0084] (5) Place in a 42°C metal bath and incubate for 15 min.

[0085] (6) Incubate at 95°C for 3 min, then place on ice and store in a -20°C refrigerator for the next experimental step.

[0086] 3. Cloning of TaKNAT1 gene

[0087] The reverse transcribed cDNA was used as template and PCR amplification was performed using the following primer pairs:

[0088] Upstream primer: 5'-ATGGAGAGGTTCCCTAATCTTGGG-3', as shown in SEQ ID NO.3;

[0089] Downstream primer: 5'-TCACGACCCGAGGCGGTA-3', as shown in SEQ ID NO.4;

[0090] The PCR amplification system was 2 μL upstream primer (50 pmol / μL), 2 μL downstream primer (50 pmol / μL), 5 μL 10×PCR buffer, 2 μL dNTP mixture (10 mmol / L), 0.5 μL EVO DNA polymerase (5U), 1 μL cDNA template, and DEPC·H2O was added to make the total volume 25 μL;

[0091] The amplification conditions were as follows: pre-denaturation at 94°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, 32 cycles, and extension at 72°C for 7 min.

[0092] The CDS region sequence of the cloned TaKNAT1 gene is shown in SEQ ID NO.1.

[0093] Example 2: Construction of TaKNAT1 gene plant overexpression vector and gene knockout vector

[0094] 1. Construction of plant expression vector pUbi::TaKNAT1-6MYC:

[0095] (1) Take 4 μL of the amplified product of TaKNAT1 gene cloned in Example 1 and mix with pENTR TM / D-TOPO Vector vector connection, the operation steps are the same as pENTR TM / D-TOPO Vector product manual.

[0096] (2) The ligation product was transformed into Escherichia coli Top10 and cultured overnight on LB solid medium containing kanamycin (100 mg / L).

[0097] (3) Pick a single colony and culture it overnight in LB liquid medium containing kanamycin (100 mg / L). Extract plasmid DNA using the alkaline method and perform sequence determination.

[0098] (4) The amplified product was sequenced and analyzed, and its sequence was shown in SEQ ID NO.1, indicating that the TaKNAT1 gene had been connected to pENTR TM / D-TOPO Vector, the cloning vector construction is completed.

[0099] (5) The cloning vector was digested with restriction endonuclease Apa I, and the target fragment was detected and recovered by electrophoresis.

[0100] (6) The cloning vector after restriction digestion was connected to the pC186 vector through LR reaction. The operation steps were carried out according to the LR instructions of the product of Life Technologies Company.

[0101] (7) The ligation product was transformed into Escherichia coli Top10 and cultured overnight on LB solid medium containing kanamycin (50 mg / L).

[0102] (8) Pick a single colony and culture it overnight in LB liquid medium containing kanamycin (50 mg / L). Extract plasmid DNA using alkaline method and perform sequencing analysis to obtain the expression vector pUbi::TaKNAT1-6MYC ( Figure 1 ).

[0103] 2. Construction of TaKNAT1 gene knockout vector:

[0104] (1) sgRNA was designed in E-CRISP (http: / / www.e-crisp.org / E-CRISP / ), and the target site specific to the ABD locus of the TaKNAT1 gene was screened by analyzing the blast results of the target site on the wheatomics website.

[0105] Target 1: GACGCTCTTGAAGAACTCCG NGG; (SEQ ID NO.5)

[0106] Target 2: GCCTCTCCAGCACCTCCGGC NGG. (SEQ ID NO.6)

[0107] "N" in the target sequence represents any nucleotide of A, T, C, or G.

[0108] (2) Construction of Crispr-Cas9 gene knockout vector.

[0109] Four-primer PCR amplification was performed using pCBC-DT1T2 diluted 100 times as a template. -BsF / -BsR is the normal primer concentration; -F0 / -R0 was diluted 20 times for PCR amplification:

[0110] MT1T2-F:aataatGGTCTCAAGCgACGCTCTTGAAGAACTCCG; (SEQ ID NO.7)

[0111] MT1T2-F0:gACGCTCTTGAAGAACTCCGgttttagagctagaaatagc; (SEQ ID NO.8)

[0112] MT1T2-R0:GCCGGAGGTGCTGGAGAGGCGCTTCTTGGTGCC; (SEQ ID NO.9)

[0113] MT1T2-R:attattGGTCTCTAAACGCCGGAGGTGCTGGAGAGG. (SEQ IDNO.10)

[0114] The PCR amplification system was 1 μL F primer (50 pmol / μL), 1 μL diluted F0 primer (2.5 pmol / μL), 1 μL R primer (50 pmol / μL), 1 μL diluted R0 primer (2.5 pmol / μL), 5 μL 10×PCR buffer, 2 μL dNTP mixture (10 mmol / L), 0.5 μL EVO DNA polymerase (5U), 1 μL pCBC-DT1T2 template, and DEPC·H2O was added to make the total volume up to 25 μL.

[0115] The amplification conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 1 min, 32 cycles; and extension at 72°C for 5 min.

[0116] Recover the PCR amplification product and establish an enzyme digestion and ligation system as follows:

[0117]

[0118] The ligation product was transformed into E. coli Top10 and cultured overnight on LB solid medium containing kanamycin (100 mg / L). A single colony was picked and cultured overnight in LB liquid medium containing kanamycin (100 mg / L). Plasmid DNA was extracted by alkaline method and sequenced. The amplified product was sequenced and analyzed, and its sequence was shown as the target site, indicating that the target site sequence had been connected to the pUBE413 Vector vector, and the TaKNAT1 gene knockout vector was constructed.

[0119] Example 3: Agrobacterium-mediated transformation of wheat immature embryos and acquisition of positive transgenic plants

[0120] Wheat genetic transformation uses the Agrobacterium-mediated method. The genetic transformation process is as follows:

[0121] (1) The plant expression vector pUbi::TaKNAT1-6MYC and the TaKNAT1 gene knockout vector constructed in Example 2 were transformed into Agrobacterium EHA105, respectively, and Agrobacterium infection fluid for transformation was obtained.

[0122] (2) Pipette the Agrobacterium infection solution from step (1) into a 2 mL centrifuge tube containing Fielder wheat immature embryos and gently invert for 45 seconds to immerse the immature embryos in the bacterial solution. If the immature embryos cannot be immersed in the bacterial solution, centrifuge briefly.

[0123] (3) After standing for 5 minutes, pour the bacterial solution and immature embryos into a sterile disposable culture dish and aspirate half of the bacterial solution.

[0124] (4) Use a sterilized and cooled scalpel to pick up the young embryos and place them on the co-culture medium. The scalpel should be sterilized several times to prevent contamination that may affect subsequent experiments.

[0125] (5) Seal the culture dish with sealing film and place it in a dark incubator at 23°C for co-cultivation.

[0126] (6) After 2 days of co-culture, the embryonic axes were cut off and placed on recovery medium at 25°C in the dark.

[0127] (7) After 5 days, the immature embryonic callus began to swell and callus tissue began to form. The swollen callus tissue was transferred to screening medium A, and the browned and dead immature embryonic tissue was discarded. The culture was continued at 25°C in the dark.

[0128] (8) After 14 days, a relatively large callus was formed. The callus was cut into two halves and the cut surface in contact with the culture medium was transferred to screening medium B and cultured in the dark at 25°C.

[0129] (9) After 21 days, the callus tissue was transferred to the differentiation medium. The callus tissue began to differentiate and sprout. The callus tissue was placed in a light incubator at a temperature of 25°C and a light intensity of 2000 lx for cultivation.

[0130] (10) After the callus tissue differentiates into green seedlings, it is transferred to the rooting medium until the green seedlings grow 4-5 leaves and then transplanted to a greenhouse or artificial climate chamber for seedling cultivation.

[0131] The process of Agrobacterium-mediated transformation of wheat embryos Figure 1BE. Comparison of the Fielder transformation process of pUbi::TaKNAT1-6MYC and the control pC186 empty vector Figure 2 The results showed that pUbi::TaKNAT1-6MYC could significantly promote the induction and differentiation of wheat immature embryos.

[0132] The results of the detection of the positive transgenic plants of the Fielder T0 generation transformed by overexpressing TaKNAT1 are as follows Figure 3 As shown, three TaKNAT1 gene overexpressing wheat transgenic plants T0-#9, T0-#11 and T0-#12 were obtained.

[0133] The results of the positive transgenic plants of the Fielder T0 generation transformed with the TaKNAT1 knockout vector are as follows Figure 4 As shown, CR-#4T0 TaKNAT1 gene knockout wheat transgenic plants were obtained.

[0134] Example 4: Investigation of ear traits of transgenic wheat plants

[0135] 1. Test method:

[0136] The T1 generation plants of the wheat transgenic plants with overexpression of the TaKNAT1 gene obtained in Example 3 (T1-#9-4, T1-#11-1) and the T1 generation plants of the wheat transgenic plants with TaKNAT1 gene knockout (#4-1, #4-2, #4-3, #4-4, #4-5) were cultured in a culture room with 25°C-16h light / 22°C-8h darkness; the wild-type wheat (Triticum aestivum) variety Fielder was used as a control.

[0137] When the ear of the plant matures, 10-15 ears of each strain are cut with scissors to count the ear length, number of spikelets and number of grains per ear. The statistical data are analyzed for significance to compare the differences in ear traits between wild-type and transgenic plants.

[0138] 2. Test results:

[0139] The results of comparison of ear traits between the T1 transgenic plants transformed with overexpression of TaKNAT1 and the wild-type Fielder are shown in Figure 5 The results of the comparison of ear traits between TaKNAT1 knockout mutant plants and Fielder wild type plants are shown in Figure 6 The results showed that overexpression of TaKNAT1 gene would increase spike length, spikelet number and grain number per spike, while TaKNAT1 gene mutation would reduce spikelet number, spike length and grain number per spike in Fielder wheat.

[0140] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of TaKNAT1D gene in regulating wheat ear traits, wherein the TaKNAT1D gene is a DNA molecule as shown in i) or ii) below: i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

2. The use according to claim 1, characterized in that The wheat ear traits include: spikelet number, spike length and grain number per spike.

3. Use of the protein encoded by the gene according to claim 1 in regulating wheat ear traits, wherein the amino acid sequence of the protein is shown in SEQ ID NO.

2.

4. The use according to claim 3, characterized in that The wheat ear traits include: spikelet number, spike length and grain number per spike.

5. A method for regulating wheat ear traits, characterized in that: The following steps are involved: Up-regulating the expression of TaKNAT1D gene in wheat plants, obtaining transgenic wheat plants with increased spikelet number, spike length and spikelet number; Alternatively, the function of TaKNAT1D protein in wheat plants is mutated or inhibited to obtain transgenic wheat plants with reduced spikelet number, spike length and spikelet number; The TaKNAT1D gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

6. The method according to claim 5, characterized in that The expression of TaKNAT1D gene in wheat plants was upregulated by exogenous introduction of TaKNAT1D gene; Alternatively, a DNA fragment capable of activating or increasing the transcription level, translation level or protein activity of the wheat TaKNAT1D gene is introduced to upregulate the expression of the TaKNAT1D gene in wheat plants.

7. A method for breeding high-yield wheat varieties, characterized in that: The following steps are involved: The TaKNAT1D gene is exogenously transferred into wheat starting plants to overexpress the TaKNAT1D gene, thereby obtaining transgenic wheat plants with increased spikelet number, spike length and spikelet number compared with the wheat starting plants; Using transgenic wheat plants as parents for self-pollination, or hybridizing with wheat plants of other plant types to obtain wheat varieties with high-yield traits; The TaKNAT1D gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

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