Application of Wheat TaLAC129 Gene

Abstract

The present invention provides a wheat TaLAC129 gene. The nucleotide sequence of the wheat TaLAC129 gene is shown as SEQ ID NO.1; the amino acid sequence of the encoded protein is shown as SEQ ID NO.2. The present invention also provides the application of the wheat TaLAC129 gene in cultivating transgenic wheat plants. The method for cultivating transgenic wheat plants is as follows: introducing the TaLAC129 gene into wild-type wheat plants to overexpress the TaLAC129 gene, thereby obtaining transgenic plants. It is found through research that overexpressing the TaLAC129 gene can significantly increase the yield of wheat, and can also increase the laccase activity and lignin content of the roots, and resist the invasion of harmful bacteria or fungi. Therefore, the TaLAC129 gene can be used to increase the yield and regulate plant stress resistance, and has great application value for plant breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of a wheat TaLAC129 gene. Background Art

[0002] Wheat (Triticum aestivum L.) is a food crop widely cultivated and grown worldwide, and approximately 40% of the global population takes wheat as the main staple food. In China, the sown area of wheat accounts for about 20% - 30% of the national cultivated land area, which is particularly important in agricultural production. Therefore, wheat production is an important guarantee for maintaining China's livelihood plan, ensuring economic and social development, and social stability. With urbanization, land desertification and salinization, especially in the case of limited and shrinking arable land resources, it is impossible to significantly restore and increase the wheat planting area, and it is decreasing day by day. The contradiction between this and the continuously rigid growth of food consumption demand is becoming increasingly prominent and serious. Therefore, increasing wheat yield per unit is the ultimate shortcut to ensure the increase of the total wheat output and at the same time to cope with the increasingly severe food crisis.

[0003] Laccase (LAC, EC 1.10.3.2) belongs to the cupredoxin oxidase family and is a multifunctional copper-containing oxidase. LAC plays an irreplaceable role in plant growth and development and is a key factor in the formation of the plant secondary cell wall. It regulates the deposition of lignin, affects the development of vascular bundles, and is widely involved in the development of organs such as leaves, roots, flowers, and seeds. LAC affects the growth of cell walls, tissue development, and mechanical strength by regulating lignin synthesis, and also affects plant reproductive growth, being closely related to crop productivity traits. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a wheat TaLAC129 gene, its encoded protein, and application in view of the deficiencies of the above-mentioned prior art. By introducing this gene into a target plant to overexpress the TaLAC129 gene, a transgenic plant can be obtained, which can not only significantly increase the yield of the plant, but also increase the laccase activity and lignin content in the roots, resist the invasion of harmful bacteria or fungi, and can be used to regulate plant stress resistance.

[0005] To solve the above technical problem, the technical solution adopted by the present invention is: a wheat TaLAC129 gene, the nucleotide sequence of the wheat TaLAC129 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the wheat TaLAC129 gene is as shown in SEQ ID NO.2.

[0006] The present invention also provides a biological material containing the wheat TaLAC129 gene, and the biological material includes a recombinant expression vector and an engineered bacterium.

[0007] The present invention also provides an application of the wheat TaLAC129 gene, and the wheat TaLAC129 gene is used for cultivating transgenic wheat plants;

[0008] Preferably, the method for cultivating transgenic wheat plants is as follows: introducing the TaLAC129 gene into wild-type wheat plants to overexpress the TaLAC129 gene, thereby obtaining transgenic wheat plants;

[0009] Preferably, the method for overexpressing the TaLAC129 gene in wheat plants is as follows:

[0010] S1. Construction of TaLAC129 gene overexpression vector

[0011] S101. Linearized vector: The overexpression vector BL130035S-Flag is double digested with BamHI and XbaI to obtain a linearized vector;

[0012] S102. Obtaining the target fragment: According to the CDS sequence of the target gene TaLAC129, specific primers containing restriction enzyme sites and vector terminal homologous sequences are designed; using wheat cDNA as a template, high-fidelity enzyme is used for PCR amplification, detected by gel electrophoresis and recovered to obtain the target fragment of the TaLAC129 gene; the specific primers are pBL-TaLAC129-XbaI-F and pBL-TaLAC129-BamHI-R; the nucleotide sequence of the primer pBL-TaLAC129-XbaI-F is as shown in SEQ ID NO.3; the nucleotide sequence of the primer pBL-TaLAC129-BamHI-R is as shown in SEQ ID NO.4;

[0013] S103. Recombination reaction, transformation of the recombinant product and colony PCR identification, and the specific operation methods are as follows:

[0014] S10301. Recombination reaction: Prepare a reaction system on ice, 1 μL of linearized vector, 1 μL of target fragment, 2 μL of 5X Buffer, 1 μL of recombinase, ddH 2 O 5 μL; use a pipette to mix well, centrifuge to collect the reaction solution to the bottom of the tube, and react at 37 °C for 30 min to obtain a recombinant product;

[0015] S10302, Recombinant product transformation: Thaw the cloning-competent cells DH5α on ice. Take 10 μL of the recombinant product and add it to 100 μL of the competent cells, mix well, and let it stand on ice for 30 min; after heat shock in a 42°C water bath for 45 s, cool it on ice for 2 - 3 min; add 900 μL of LB medium, shake the bacteria at 37°C for 1 h at a rotation speed of 200 - 250 rpm; then centrifuge at 5000 rpm for 5 min, discard the supernatant; resuspend the bacterial pellet with LB medium, and spread it evenly on a preheated (to 37°C) resistant LB solid medium plate with a sterile spreading rod; incubate it upside down in a 37°C incubator for 12 - 16 h;

[0016] S10303, Colony PCR identification: Pick several colonies on the recombinant reaction transformation plate for colony PCR identification. The amplification primers use the universal sequencing primers on the vector. If the cloning is correct, a band of 2038 bp in size should appear; the universal sequencing primers are 35S-promoter and NOS-R; the nucleotide sequence of the primer 35S-promoter is as shown in SEQ ID NO.5; the nucleotide sequence of the primer NOS-R is as shown in SEQ ID NO.6; the colony PCR reaction system is: 1 μL of bacterial solution, 1 μL of primer 35S-promoter, 1 μL of primer NOS-R, 5 μL of enzyme, add sterile ddH 2 O to 10 μL; the PCR reaction program is: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 3 min, 35 cycles, and finally extension at 72°C for 5 min;

[0017] S104, Select the colonies with correct cloning for next-generation sequencing to obtain the TaLAC129 gene overexpression vector plasmid p35S:TaLAC129-flag;

[0018] S2, Genetic transformation of TaLAC129 gene overexpressing wheat

[0019] Transform the TaLAC129 gene overexpression vector plasmid p35S:TaLAC129-flag obtained in S1 into the competent cells of Agrobacterium tumefaciens GV3101, and spread and culture it on a double-antibiotic (kanamycin and rifampicin) plate; pick an Agrobacterium monoclonal for colony PCR identification, inoculate the clone with the correct target band size into the medium, and shake the bacteria for 12 h; streak the bacterial solution on a double-antibiotic plate, and select a monoclonal for colony PCR identification again. Send the correct Agrobacterium monoclonal to the transgenic platform for wheat genetic transformation, and integrate the transgenic vector into wheat immature embryos through the Agrobacterium infection method. The receptor material is the spring wheat variety Fielder, and finally obtain TaLAC129 transgenic plants;

[0020] S3. Identification of TaLAC129 Transgenic Positive Plants

[0021] The TaLAC129 transgenic plants obtained in S2 were detected for positive plants by bar test strips, and then subjected to PCR amplification with primers Bar-1300-F and Bar-1300-R to determine whether the plants contained the bar gene;

[0022] The nucleotide sequence of the primer Bar-1300-F is shown in SEQ ID NO.7;

[0023] The nucleotide sequence of the primer Bar-1300-R is shown in SEQ ID NO.8;

[0024] If the amplified product has a band of 689 bp in size, it is determined that the plant contains the bar gene, and it is judged as overexpression of the TaLAC129 gene and is a TaLAC129 transgenic positive plant; if the amplified product does not have a band of the above size, it is determined that the plant does not contain the bar gene and is judged as a negative plant.

[0025] Preferably, the transgenic wheat plants satisfy at least one of the following phenotypes Ⅰ or Ⅱ:

[0026] Ⅰ: The laccase activity of the roots of the transgenic wheat plants is stronger than that of the wild-type wheat plants; the lignin content of the roots of the transgenic wheat plants is stronger than that of the wild-type wheat plants;

[0027] Ⅱ: The yield of the transgenic wheat plants is higher than that of the wild-type wheat plants.

[0028] The present invention also provides an application of the wheat TaLAC129 gene, and the wheat TaLAC129 gene is used for high-yield wheat breeding and high-stress-resistant wheat breeding.

[0029] The present invention has the following beneficial effects compared with the prior art:

[0030] The present invention provides a wheat TaLAC129 gene. By introducing this gene into the target plant to overexpress the TaLAC129 gene, transgenic plants are obtained. It is found that these transgenic plants can not only significantly increase the yield of wheat, but also improve the laccase activity and lignin content of the roots, resist the invasion of fungi, and can be used to regulate the stress resistance of plants. Introducing the TaLAC129 gene of the present invention into the target plant to obtain new transgenic plant varieties has great application value for plant breeding.

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Brief Description of the Drawings

[0032] Figure 1It is the map of the TaLAC129 gene expression vector in Example 2 of the present invention.

[0033] Figure 2 It is the gel electrophoresis map of the bar gene detection of the transgenic wheat lines OE7 and OE8 in Example 3 of the present invention.

[0034] Figure 3 It is the analysis of plant height, phenotype and biomass of the transgenic wheat OE7, OE8 and wild-type wheat (WT) in Example 5 of the present invention. Among them, Figure a is the comparison of plant height at different growth stages; Figure b is the comparison of growth phenotype maps; Figure c is the comparison of biomass of different organs (stem and leaf, glume, grain and root system) at the harvest stage.

[0035] Figure 4 It is the analysis map of the grain traits of the transgenic wheat OE7, OE8 and wild-type wheat (WT) in Example 5 of the present invention. Among them, Figure a is the phenotype map of grain length, and Figure b is the phenotype map of grain width.

[0036] Figure 5 It is the analysis map of the root laccase activity and lignin content of the transgenic wheat OE7, OE8 and wild-type wheat (WT) in Example 6 of the present invention. Detailed implementation mode

[0037] Example 1

[0038] This example provides a cloning method for the TaLAC129 gene, including the following steps:

[0039] 1. Use the RNAprepPure Plant Total RNA Extraction Kit (DP432) of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the total RNA of the root sample of the wheat variety "Chinese Spring". After detecting the purity and concentration of the RNA with a Nanodrop OneC meter, store it at -80 °C.

[0040] 2. Amplify the full-length sequence of the TaLAC129 gene

[0041] Dilute the extracted RNA, and then use the FastKing One-Step Genomic DNA Removal and cDNA Synthesis SuperMix Kit (KR118) of Tiangen Company to reverse-transcribe the RNA into cDNA. Use this cDNA as the PCR amplification template, and perform PCR amplification with the primers pBL-TaLAC129-XbaI-F and pBL-TaLAC129-BamHI-R;

[0042] The nucleotide sequence of the primer pBL-TaLAC129-XbaI-F is as shown in SEQ ID NO.3; the nucleotide sequence of the primer pBL-TaLAC129-BamHI-R is as shown in SEQ ID NO.4;

[0043] The PCR amplification reaction system is as follows: 1 μL of cDNA template, 1 μL of primer pBL-TaLAC129-BamHI-F, 1 μL of primer pBL-TaLAC129-XbaI-R, 12.5 μL of high-fidelity enzyme, add sterilized ddH 2 O to 25 μL;

[0044] The PCR reaction program is as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 2 min, 35 cycles, and finally extension at 72°C for 5 min.

[0045] The obtained product was ligated into Escherichia coli, and after PCR and restriction enzyme digestion identification, sequencing was performed, and the nucleotide sequence of the TaLAC129 gene was as shown in SEQ ID NO.1, and the product size was 1725 bp; the amino acid sequence of the encoded protein was as shown in SEQ ID NO.2.

[0046] Example 2

[0047] This example is the construction of the TaLAC129 gene expression vector, which includes the following steps:

[0048] S1. Linearize the vector: The overexpression vector BL130035S-Flag was double-digested with BamHI and XbaI to obtain a linearized vector;

[0049] S2. Obtain the target fragment: According to the CDS sequence of the target gene TaLAC129, specific primers containing restriction enzyme sites and vector terminal homologous sequences were designed; using the cDNA of "Chinese Spring" wheat as a template, PCR amplification was performed using high-fidelity enzyme, detected by gel electrophoresis and recovered to obtain the target fragment of the TaLAC129 gene; the specific primers were pBL-TaLAC129-XbaI-F and pBL-TaLAC129-BamHI-R; the nucleotide sequence of the primer pBL-TaLAC129-XbaI-F was as shown in SEQ ID NO.3; the nucleotide sequence of the primer pBL-TaLAC129-BamHI-R was as shown in SEQ ID NO.4;

[0050] S3. The recombinant reaction, transformation of the recombinant product and colony PCR identification were carried out according to the instructions of the Novoprotein C112 recombination kit. The specific operation method is as follows:

[0051] Recombinant reaction: Prepare the reaction system on ice, 1 μL of linearized vector, 1 μL of target fragment, 2 μL of 5X Buffer, 1 μL of recombinase, ddH 2O 5 μL; gently pipette and mix well, centrifuge to collect the reaction solution at the bottom of the tube, and react at 37 °C for 30 min to obtain the recombinant product;

[0052] Transformation of the recombinant product: Thaw the cloning competent cells DH5α on ice. Take 10 μL of the recombinant product and add it to 100 μL of the competent cells. Gently flick the tube wall to mix well and let it stand on ice for 30 min; Heat shock in a 42 °C water bath for 45 s, then immediately cool on ice for 2 - 3 min; Add 900 μL of LB medium (without antibiotics), shake the bacteria at 37 °C for 1 h at a rotation speed of 200 - 250 rpm; Then centrifuge at 5000 rpm (2400×g) for 5 min, discard the supernatant, and resuspend the bacteria with LB medium; Preheat the LB solid medium plate with the corresponding resistance in a 37 °C incubator, then use a sterile spreading rod to spread the bacterial solution on the LB solid medium plate and spread it evenly; Incubate upside down in a 37 °C incubator for 12 - 16 h;

[0053] Colony PCR identification: Pick several colonies on the recombinant reaction transformation plate for colony PCR identification. The amplification primers use the universal sequencing primers on the vector. If the cloning is correct, a band of 2038 bp in size should appear; The universal sequencing primers are 35S - promoter and NOS - R; The nucleotide sequence of the primer 35S - promoter is as shown in SEQ ID NO.5; The nucleotide sequence of the primer NOS - R is as shown in SEQ ID NO.6; The colony PCR reaction system is: 1 μL of bacterial solution, 1 μL of primer 35S - promoter, 1 μL of primer NOS - R, 5 μL of enzyme, add sterilized ddH 2 O to 10 μL; The PCR reaction program is: Pre - denature at 95 °C for 3 min, denature at 95 °C for 15 s, anneal at 58 °C for 15 s, extend at 72 °C for 3 min, 35 cycles, and finally extend at 72 °C for 5 min;

[0054] S4. Select the colonies with correct cloning for next - generation sequencing to obtain the TaLAC129 gene over - expression vector plasmid p35S:TaLAC129 - flag.

[0055] Figure 1 This is the map of the TaLAC129 gene expression vector in this example.

[0056] Example 3

[0057] This example is about the over - expression of the TaLAC129 gene in wheat genetic transformation.

[0058] The correctly sequenced overexpression vector plasmid was transformed into competent Agrobacterium (GV3101) cells by the "freeze-thaw method" and cultured by spreading on a double-antibiotic (kanamycin antibiotic and rifampicin antibiotic) plate. Single colonies were picked for colony PCR identification. The identification method and amplification procedure were the same as in Example 2. Clones with the correct target band size were inoculated into the medium and cultured with shaking for 12 h. The bacterial solution was streaked on a double-antibiotic plate, and single colonies were selected again for colony PCR identification. The correct Agrobacterium single colonies were sent to the transgenic platform for wheat genetic transformation. The transgenic vector was integrated into wheat immature embryos by the Agrobacterium infection method. The receptor material was the spring wheat variety Fielder, and finally TaLAC129 transgenic plants were obtained.

[0059] A total of 22 transgenic T0 seedlings were obtained through the transgenic platform. The T0 seedlings were continuously planted and cultured to obtain T1 generation plants, and then a bar detection test strip was used to detect positive seedlings; then the identified T1 generation positive plants were continuously cultured to obtain T2 generation plants, a total of 17 plants. The bar gene of the wheat plants was extracted for DNA to confirm the detection of positive plants. The PCR amplification primers were Bar-1300-F and Bar-1300-R;

[0060] The nucleotide sequence of the primer Bar-1300-F is shown in SEQ ID NO.7;

[0061] The nucleotide sequence of the primer Bar-1300-R is shown in SEQ ID NO.8;

[0062] If the amplification product has a band of 689 bp in size, the tested plant contains the bar gene, and it is determined to be overexpression of the TaLAC129 gene and is a TaLAC129 transgenic positive plant; if the amplification product does not have a band of the above size, the tested plant does not contain the bar gene and is determined to be a negative plant.

[0063] The results showed that two positive transgenic wheat lines OE7 and OE8 were obtained from the genetic transformation of TaLAC129 gene overexpressing wheat in this example. The gel electrophoresis diagrams of the bar gene detection of the OE7 and OE8 lines are shown in Figure 2 , where the M lane in the figure is the Marker, and 1-17 are the PCR samples of wheat plants respectively.

[0064] Example 4

[0065] This example is a pot experiment of TaLAC129 gene overexpressing wheat.

[0066] 1. Experimental design

[0067] The wild-type spring wheat variety Fielder (WT) and two positive transgenic wheat lines (OE7 and OE8) obtained after screening were planted in flower pots, with 2.5 kg of soil filled in each pot and two wheat seedlings in each pot. Before planting, urea calculated according to pure N 0.2 g·kg -1 of the soil was applied as the basal fertilizer for wheat growth. The experiment was designed to sample at the harvest stage of wheat growth, with four replicates set up.

[0068] The soil used in the pot experiment was taken from the field soil of Majiadi Village, Yangling District, Shaanxi Province. Soil was randomly collected from multiple points and mixed for later use. The soil type is Lou soil, and the basic physical and chemical properties are shown in Table 1:

[0069] Table 1 Physical and chemical properties of the soil

[0070]

[0071] 2. Sample collection and determination

[0072] At the wheat harvest stage, the plant heights of two wheat plants in each pot of transgenic plants and wild-type plants were measured, and the spike lengths and spike numbers of wheat in the pots were manually recorded. The above-ground plant samples of each pot of wheat in each treatment were completely harvested, dried, threshed, and then the biomasses of stems and leaves, glumes, as well as the yield (grain biomass), grain length, grain width, spike grain number, and 1000-grain weight were measured respectively.

[0073] Example 5

[0074] This example is the analysis of the effect of overexpressing the TaLAC129 gene on wheat yield.

[0075] Figure 3 It is the analysis of the plant heights, phenotypes, and biomasses of transgenic wheat OE7 and OE8 and wild-type wheat (WT) in Example 5 of the present invention. Among them, Figure 3 a is the comparison of plant heights at different growth stages; Figure 3 b is the comparison of growth phenotype diagrams; Figure 3 c is the comparison of biomasses of different organs (stems and leaves, glumes, grains, and roots) at the harvest stage. As Figure 3 shown in a, there were no significant differences in the plant heights of the two transgenic wheat lines OE7 and OE8 at the jointing stage and heading stage; the plant height of wheat at the harvest stage with overexpressed TaLAC129 gene increased significantly (P<0.05). Compared with WT, the plant height of OE7 line increased by 6.8%, and the plant height of OE8 plant increased by 8.6%. As Figure 3Comparison of the biomass of different organs (stem and leaf, glume, grain and root system) of wheat at the harvest stage found that overexpression of the TaLAC129 gene could reduce the biomass of wheat stems and leaves to a certain extent, but only the OE7 line reached a significant level (P<0.05) compared with the WT; there was no significant difference in the glume biomass among different lines; compared with the WT, the grain biomass (grain yield) of transgenic wheat increased significantly, and OE7 and OE8 increased by 92.72% and 61.26% respectively; while the root biomass of transgenic wheat decreased significantly, and OE7 and OE8 decreased by 53.40% and 41.93% respectively.

[0076] Figure 4 Figure showing the analysis of grain traits of transgenic wheat OE7, OE8 and wild-type wheat (WT). Among them, Figure a is the phenotypic map of grain length, Figure b is the phenotypic map of grain width, bar = 2 cm. It can be seen from the figure that overexpression of the TaLAC129 gene significantly reduced the grain length of wheat, but increased the grain width, and the grains of transgenic wheat lines were more plump; among them, the grain length showed WT > OE8 > OE7; the grain width showed WT < OE8 < OE7.

[0077] After wheat harvest, agronomic traits related to grains were measured, including the number of spikes, grains per spike, 1000-grain weight, spike length, grain length and grain width, and the harvest index HI was calculated (see Table 2). The results showed that there was no significant difference in the number of spikes and spike length between transgenic wheat lines and the WT, but there was a downward trend; the grains per spike of transgenic wheat lines all increased to varying degrees, among which the OE7 line had a significant difference, and the average number of grains per spike was 27.83; the 1000-grain weight also increased significantly (P<0.05), and OE7 and OE8 increased by 55.72% and 36.93% respectively compared with the WT; compared with the WT, overexpression of TaLAC129 increased the wheat harvest index, and among them, OE7 had a significant difference, and its average harvest index was 0.41.

[0078] Table 2 Agronomic traits related to grains of transgenic wheat and wild-type wheat

[0079] Trait WT OE7 OE8 Number of ears 5.50±0.5a 4.50±0.5a 5.25±0.25a Number of grains per ear 17.73±3.02b 27.83±1.48a 21.04±1.98ab 1000-grain weight (g) 15.43±2.05b 24.03±1.53a 21.13±0.98a Ear length (cm) 9.24±0.11a 8.82±0.35a 8.86±0.25a Grain length (cm) 7.15±0.10a 6.55±0.13b 6.58±0.08b Grain width (cm) 5.10±0.04c 6.23±0.06a 5.80±0.04b Harvest index 0.19±0.03b 0.41±0.02a 0.27±0.03b

[0080] The above results indicate that overexpression of the TaLAC129 gene increases the grain width of wheat, makes the grains more plump, affects both the grains per spike and the 1000-grain weight among the three elements of yield, and makes them increase significantly. Therefore, overexpressing TaLAC129 transgenic plants can effectively increase wheat yield.

[0081] Example 6

[0082] This example is the analysis of the effect of overexpressing the TaLAC129 gene on the stress resistance of wheat, including the analysis of root laccase activity and lignin content. The measurement methods are as follows:

[0083] Determination of laccase activity: It was determined by visible spectrophotometry according to the instructions of the kit (BC1630). Weigh 0.1 g of wheat roots at different sampling times, add 1 mL of extraction solution, and perform homogenization in an ice bath; centrifuge at 10000 g for 10 min at 4 °C, take the supernatant, and place it on ice for later measurement; preheat the spectrophotometer for more than 30 min, adjust the wavelength to 420 nm, and zero with distilled water; in a 1 mL glass cuvette, add 150 μL of the sample in the measurement tube and 850 μL of the working solution of the kit; add 150 μL of distilled water and 850 μL of the working solution of the kit to the blank tube respectively. After thorough mixing, measure the absorbance value A1 at 420 nm for 10 s, quickly place it in a 45 °C water bath for 3 min, take it out, quickly dry it, and measure the absorbance value A2 at 190 s. Calculate ΔA measurement tube = A2 measurement - A1 measurement, ΔA blank tube = A2 blank - A1 blank, and ΔA = ΔA measurement tube - ΔA blank tube. Calculate according to the formula LAC enzyme activity (U·g -1 ) = 61.7×ΔA÷W, where W is the root mass of 0.1 g.

[0084] Determination of lignin content: It was determined by ultraviolet spectrophotometry according to the instructions of the kit (BC4200). Weigh 5 mg of root samples that have been dried to constant weight at 80 °C and ground through a 30 - 50 mesh sieve into a 2 mL EP tube; preheat the spectrophotometer for more than 30 min, adjust the wavelength to 280 nm, and zero with glacial acetic acid; then proceed with acetylation: (1) React the measurement tube (5 mg of sample + 500 μL of reagent one) and the blank tube in a 65 °C water bath for 30 min at the same time; then centrifuge at 3000 g at room temperature for 5 min, discard the supernatant, and keep the precipitate; (2) Add 500 μL of reagent two respectively in the next step, then vortex for 5 min, centrifuge at 3000 g at room temperature for 5 min, discard the supernatant, and keep the precipitate; (3) Add 500 μL of reagent three respectively in the next step, then vortex for 5 min, centrifuge at 3000 g at room temperature for 5 min, discard the supernatant, and keep the precipitate; (4) Add 500 μL of reagent four and 20 μL of reagent five respectively, mix thoroughly (seal with a sealing film to prevent water loss), and place it in an 80 °C water bath for 40 min for acetylation. Shake for 30 s every 10 min, and then cool naturally to room temperature; (5) Finally, add 500 μL of reagent six respectively, mix thoroughly, centrifuge at 8000 g at room temperature for 10 min, and take the supernatant for later measurement. Determination: Take 20 μL of the supernatant from the previous step and 980 μL of glacial acetic acid in the measurement tube and the blank tube respectively, mix thoroughly, pipette the reaction solution into a 1 mL quartz cuvette, and measure the absorbance value A at 280 nm, denoted as A measurement tube and A blank tube, and calculate ΔA = A measurement tube - A blank tube. The blank tube is only measured 1 - 2 times. Calculate according to the formula: lignin content (mg·g -1 ) = 2.184×ΔA÷W, where W is the root mass of 5 mg.

[0085] The measurement results are as Figure 5 shown. As can be seen from Figure 5 a, overexpression of TaLAC129 can improve the LAC activity in wheat roots to a certain extent; when wheat grows for 60 days, compared with the wild type (WT), the LAC activities in the roots of the two overexpression lines OE7 and OE8 increase by 29.03% and 53.92% respectively (P>0.05); when wheat grows for 90 days, compared with the WT, the LAC activities in the roots of the two overexpression lines OE7 and OE8 increase significantly by 39.67% and 48.35% respectively (P<0.05); with the growth of wheat, the overall LAC activity in the roots at 90 days is lower than that at 60 days. As can be seen from Figure 5 b, overexpression of TaLAC129 can increase the lignin content in the roots of wheat growing for 90 days, and the overall change trend is consistent with the LAC activity in the roots. When wheat grows for 60 days, compared with the WT, the lignin contents in the roots of the two overexpression lines OE7 and OE8 increase by 7.91% and 16.12% respectively. When wheat grows for 90 days, compared with the WT, the lignin contents in the roots of the two overexpression lines OE7 and OE8 increase significantly by 16.38% and 21.67% respectively (P<0.05); similarly, with the growth of wheat, the overall lignin content in the roots at 90 days is lower than that at 60 days. The results show that overexpression of the TaLAC129 gene can improve the laccase activity and lignin content in wheat roots; combined with previous studies, it can be found that the increase in the lignin content in the roots can resist the invasion of harmful pathogens and can be used to improve plant stress resistance.

[0086] As mentioned above, it is only a preferred embodiment of the present invention and does not impose any limitation on the present invention. Any simple modification, change and equivalent change made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. Application of a wheat TaLAC129 gene Characterized in that, The wheat TaLAC129 gene is used for cultivating transgenic wheat plants. The wheat TaLAC129 gene has a nucleotide sequence as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is as shown in SEQ ID NO.2; The transgenic wheat plants satisfy at least one of the following phenotypes I or II: I: The laccase activity of the roots of the transgenic wheat plants is stronger than that of the wild-type wheat plants; the lignin content of the roots of the transgenic wheat plants is higher than that of the wild-type wheat plants; II: The yield of the transgenic wheat plants is higher than that of the wild-type wheat plants.

2. The application according to claim 1, Characterized in that, The method for cultivating transgenic wheat plants is as follows: introducing the TaLAC129 gene into wild-type wheat plants to cause TaLAC129 the gene to be overexpressed, thereby obtaining transgenic wheat plants.

3. Application of a wheat TaLAC129 gene Characterized in that, The wheat TaLAC129 gene is used for high-yield wheat breeding. The wheat TaLAC129 gene has a nucleotide sequence as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is as shown in SEQ ID NO.2.

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