A branched-chain amino acid transaminase gene hvbcat3 and use thereof

Abstract

This invention provides a branched-chain amino acid transaminase gene and its applications, belonging to the field of genetic engineering. This invention is the first to discover the branched-chain amino acid transaminase gene HvBCAT3, which shows a significant positive correlation with leucine (Leu) accumulation in crops. This invention provides an important target gene, HvBCAT3, for crop quality improvement, and is of great value in increasing the content of branched-chain amino acids in crops and improving dietary nutrition. The HvBCAT3 gene, its recombinant vectors, recombinant bacteria, and transgenic plants all have promising applications in the food and feed industries.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a branched-chain amino acid transaminase gene and its uses. Background Technology

[0002] Branched-chain amino acids (BCAAs), including valine, isoleucine, and leucine, are essential amino acids indispensable in plants and animals. They play crucial roles in protein synthesis, energy metabolism, and signal regulation. Valine and isoleucine participate in gluconeogenesis and energy supply, while leucine, as an activator of the mTOR signaling pathway, regulates cell growth and protein translation. In barley (Hordeum vulgare L. var. nudum), BCAAs not only affect grain protein quality but are also closely related to its stress resistance and nutritional function. As a staple crop in high-altitude areas, the BCAA content of barley directly affects the intake of essential amino acids, especially in plateau regions, where high-BCAA barley varieties are of significant value in improving dietary nutrition.

[0003] Branched-chain amino acid transaminase (BCAT) is an enzyme involved in amino acid metabolism. Its main function is to catalyze the transamination reaction between branched-chain amino acids (leucine, isoleucine, and valine) and α-ketoglutarate, playing a key role in the catabolic and anabolistic metabolism of amino acids.

[0004] Although the biological functions of BCAAs have been extensively studied, their biosynthetic pathways in barley remain incompletely understood. In microorganisms and model plants, BCAA synthesis is catalyzed by key enzymes such as acetolactate synthase (ALS), ketoacid reductase (KARI), and dihydroxyacid dehydratase (DHAD), but the gene regulatory network, metabolic flux allocation, and environmental response mechanisms of these enzymes in barley remain unclear. Elucidating the key genes and regulatory mechanisms of BCAA synthesis in barley will fill this research gap and provide a theoretical basis for subsequent metabolic engineering.

[0005] Since the content of BCAAs directly affects the nutritional quality and processing characteristics of highland barley, elucidating its synthesis mechanism will not only help in the breeding of high-quality highland barley varieties, but may also expand its application in functional foods and feed industries, and has broad commercial potential. Summary of the Invention

[0006] In order to solve the above-mentioned problems in the prior art, the purpose of this invention is to provide a branched-chain amino acid transaminase gene and its uses.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides a gene whose nucleotide sequence is shown in SEQ ID NO.1. This invention also provides a recombinant vector containing the gene whose nucleotide sequence is shown in SEQ ID NO.1.

[0009] Furthermore, the recombinant vector is recombinant pEAQ or recombinant pCXSN.

[0010] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the above-described recombinant vector.

[0011] Furthermore, the recombinant bacteria is recombinant Agrobacterium.

[0012] Furthermore, the recombinant Agrobacterium is recombinant EHA105.

[0013] The present invention also provides a method for producing transgenic plants that highly express branched-chain amino acid transaminase, wherein the method involves transferring the above-mentioned gene into a plant to obtain a transgenic plant that highly expresses branched-chain amino acid transaminase.

[0014] Furthermore, the method for introducing the plant is Agrobacterium-mediated transformation, gene gun method, electroporation, PEG-mediated transformation, liposome method, or calcium phosphate-DNA coprecipitation method; the plant is tobacco or barley.

[0015] Furthermore, the method for introducing the plant is Agrobacterium-mediated transformation.

[0016] This invention also provides the use of the above-mentioned genes, recombinant vectors, and recombinant bacteria in the preparation of transgenic plants that highly express branched-chain amino acid transaminases.

[0017] Furthermore, the plant is tobacco or barley.

[0018] The present invention has achieved the following beneficial effects:

[0019] This invention, through combined metabolomics and transcriptomics analysis of 21 key developmental stages throughout the entire growth period of the barley variety "Zangqing 2000," has for the first time discovered a significant positive correlation between the expression pattern of the branched-chain amino acid transaminase gene HvBCAT3 and the accumulation of leucine (Leu) (Pearson correlation coefficient r>0.6, p<0.01). To verify its function, this invention further constructed an HvBCAT3 overexpression vector and transformed it into tobacco. LC-MS metabolic analysis showed that the Leu content in the leaves of the transgenic lines was significantly increased compared to the wild type (p<0.01). These results fully demonstrate that the branched-chain amino acid transaminase encoded by HvBCAT3 plays a key catalytic role in the biosynthesis pathway of branched-chain amino acids in plants, providing an important target gene for crop quality improvement. This gene, its recombinant vector, recombinant bacteria, and transgenic plants all have promising application prospects.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1 The expression level of HvBCAT3 and the accumulation level of Leu in 21 tissues throughout the entire growth period of Zangqing 2000 are shown as trends.

[0023] Figure 2 Functional analysis of HvBCAT3. A. Expression levels of HvBCAT3 in control and transgenic tobacco lines; B. Accumulation levels of the three branched-chain amino acids in control and transgenic tobacco lines. *p<0.05, **p<0.01 (t-test).

[0024] Figure 3 This is a secondary spectrum of three branched-chain amino acids. Detailed Implementation

[0025] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0026] The branched-chain amino acid transaminase gene (HvBCAT3 gene) in highland barley, the gene sequence of which is shown in SEQ ID NO.1, can be obtained by direct synthesis or by other conventional preparation methods in the art.

[0027] SEQ ID NO.1:

[0028]

[0029] Example 1: Association analysis between HvBCAT3 gene expression and branched-chain amino acid Leu content throughout the entire growth period of highland barley.

[0030] 1. Method

[0031] Transcriptomic and metabolomic analyses were performed on 21 key tissues throughout the entire growth period of the highland barley variety "Zangqing 2000". The specific steps are as follows:

[0032] (1) Seed treatment: Soak the seeds in 0.5% potassium permanganate for disinfection, rinse them clean after 10 minutes, place them in a petri dish with filter paper moistened with distilled water on both sides, and place them in a constant temperature incubator overnight for dark cultivation to promote germination at 26℃.

[0033] (2) Sowing: After the seeds show white sprouts, sow them in the seed trays. The substrate ratio is perlite: peat moss = 1:3 (volume ratio). Cover the surface with fine peat moss, water and cover with plastic wrap. Remove the film after the barley sprouts.

[0034] (3) Sample collection (21 tissue samples were collected according to the reproductive period):

[0035] Germination stage: germinating seeds, plumules, and radicles; Tillering stage: leaves and roots; Jointing stage: leaves and roots; Young spikelet differentiation stage (10-20cm): leaves and leaf sheaths; Young spikelet differentiation stage (40-50cm): leaves, leaf sheaths, and young spikelets; Heading stage: leaves and stems 5 days before heading, and stems and spikelets during heading; Grain filling stage: flag leaves and endosperm 10 days after pollination; Maturity stage: endosperm 20, 30, and 40 days after pollination.

[0036] (4) Multi-omics joint analysis

[0037] Transcriptome sequencing: Three biological replicates were used for each sample, and sequencing was performed using the Illumina HiSeq platform;

[0038] Metabolomics assay: The content of branched-chain amino acids (Leu) was detected by targeted LC-MS / MS.

[0039] Correlation analysis: The correlation between HvBCAT3 expression level and branched-chain amino acid content was analyzed using Pearson correlation coefficient.

[0040] 2. Results

[0041] The expression levels of HvBCAT3 and the accumulation levels of Leu in 21 tissues throughout the entire growth period of the Tibetan Qinghai 2000 seedlings are shown in the figure below. Figure 1 As shown. Correlation analysis showed that HvBCAT3 expression level was related to Leu (r = 0.63, p = 1.4 × 10⁻⁶). -3 (The content is significantly positively correlated).

[0042] Example 2: Study on transient expression in tobacco

[0043] 1. Transient expression of tobacco

[0044] ① Transient expression vector (transient expression vector pEAQ, from John Innes Centre) containing the target gene (gene sequence as shown in SEQ ID NO.1) was transformed into Agrobacterium (EHA105);

[0045] ② Pick positive Agrobacterium clones and put them into 500 μl of LB containing the corresponding antibiotic (kn) and culture for 20-24 hours;

[0046] ③ Transfer 200 μl to 5 ml of LB containing the corresponding antibiotic (kn), and shake at 220 rpm on a 28°C incubator until OD is reached. 600 = Around 1.0.

[0047] ④ Collect bacterial cells by centrifuging at 10,000 rpm at room temperature for 15 min, and resuspend the bacterial cells in pre-prepared transformation buffer to OD. 600 =1.0, shake on a shaker for 3 hours; conversion buffer contains 10 mmol / L MgCl2, 10 mmol / L MES, 150 μmol / L acetylsylgenone, pH=5.6;

[0048] ⑤ Take a prepared 1ml syringe, remove the needle, select a syringe with a smooth nozzle, draw in the bacterial solution, take a 1-month-old Nicotiana benthamiana plant, hold the leaf with your hand, and inject from the underside of the leaf to allow Agrobacterium to penetrate. Mark each injected tobacco plant, circling the area where Agrobacterium penetrates on the leaf, and use a conversion buffer to inject tobacco as a control.

[0049] ⑥ After injecting Agrobacterium, tobacco plants are cultured in the dark for 24 hours, and then transferred to a tobacco incubator for 24-48 hours of light culture before sampling (note that water should not be sprayed directly onto the leaves of the injected tobacco plants).

[0050] 2. Product collection and purification

[0051] Leaf fragments from the Agrobacterium-infiltrated region were cut and placed in pre-weighed EP tubes containing steel balls. The tubes were labeled and quickly placed in liquid nitrogen for lyophilization. After lyophilization, the samples were ground using a grinder (MM 400, Retsch) at 30 Hz for 60 seconds. The ground sample powder was then transferred to 2 ml EP tubes. The weight of each EP tube was weighed using an electronic balance and recorded. An appropriate amount of the ground sample (range 30-60 mg) was added to each EP tube, weighed, and recorded. The net weight of all samples in the EP tubes was calculated. Given the net weight of each sample, 70% MeOH solution was added at 4°C on ice according to the formula: V = net sample weight (mg) * 12 μL / mg. The mixture was thoroughly mixed, vortexed for 15 seconds, and repeated every half hour for a total of 4 vortexes. The mixture was then stored at 4°C for at least 12 hours for extraction. Finally, the mixture was centrifuged. First, pre-cool the centrifuge to 4°C, set the time to 10 minutes and the speed to 12000 rpm. Vortex the sample and then place it in the centrifuge. When using the centrifuge, pay attention to symmetrical balance. After centrifugation, aspirate the supernatant. Filter the supernatant through a microporous membrane (0.22 μm pore size) and put it into a sample vial to obtain the sample extract, ready for LC-MS detection.

[0052] 3. Detection of target product

[0053] Place the vials containing the sample extract into the sample tray of the autosampler, and record the position of the injection port corresponding to each vial number. Simultaneously, open Analyst Software, double-click Hardware Configuration, select LCMS-V (with switching valve mode), click Activate Profile, select Acquire Mode, click Acquire, and then click the Equilibrate button above the image. The time is generally set to 3 minutes. This operation is to preheat the instrument, ensuring that the high-pressure pump, column, column oven, and ion source temperatures reach the conditions set in the method. Once all instrument components are in the Ready state, the Start Sample button in the function area will become clickable, indicating that the instrument and analytical conditions are normal. Then click Start Sample to begin the sample run. Before the first run, submit four blank samples.

[0054] Three samples of tobacco were used for transient expression: OX-1, OX-2, and OX-3; wild-type (CK) tobacco was used as a control.

[0055] 4. Results

[0056] Experimental results show that: (1) Expression level verification: qRT-PCR confirmed that the expression level of HvBCAT3 in the OX strain was 40-70 times that in the WT strain. Figure 2 A). (2) The metabolic analysis results showed that ( Figure 2B): In the overexpression lines OX1-OX3, the Leu content in the overexpression lines was 6.12 ± 0.53 times that of WT (p < 0.01).

[0057] This invention involves transferring the HvBCAT3 gene into tobacco, enabling the tobacco plant to highly express branched-chain amino acid transaminases (expression levels increased by 40-70 times compared to wild-type tobacco) and inducing Leu accumulation in tobacco. The resulting tobacco exhibits enhanced branched-chain amino acid accumulation, thereby improving the application value of tobacco. Figure 3 Secondary spectral analysis of the overexpressed metabolites confirmed that the significantly enriched compound was Leu.

[0058] Example 3: Construction of transgenic tobacco

[0059] The method for obtaining HvBCAT3 transgenic tobacco is as follows:

[0060] First, a recombinant expression vector pCXSN:HvBCAT3 containing the HvBCAT3 gene was constructed. Second, the recombinant expression vector was transformed into *Nicotiana benthamiana* (WT) using the *Agrobacterium* transformation method. Explant leaves soaked in *Agrobacterium* solution were transferred to MS+As medium and cultured in the dark for 3 days. The medium was then changed to differentiation medium and cultured until adventitious shoots of 1-2 cm appeared, after which they were transferred to growth medium. Finally, the obtained T0 generation seeds were sown on 1 / 2 MS (containing 25 mg·L⁻¹) medium. -1 Hygromycin (HvBCAT3) culture medium screening was used. The growth of tobacco was observed under hygromycin screening, and transgenic positive seeds were selected for planting. The expression level of HvBCAT3 in T1 generation transgenic tobacco was identified by RT-PCR technology, thereby obtaining positive T2 generation transgenic seeds.

[0061] In summary, this invention, through combined metabolomics and transcriptomics analysis of 21 key developmental stages throughout the entire growth period of the barley variety "Zangqing 2000," has for the first time discovered a significant positive correlation between the expression pattern of the branched-chain amino acid transaminase gene HvBCAT3 and the accumulation of leucine (Leu). Furthermore, this invention constructed an HvBCAT3 overexpression vector and transformed it into tobacco, resulting in a significant increase in Leu content in the leaves of the transgenic lines compared to the wild type. The HvBCAT3 discovered in this invention provides an important target gene for crop quality improvement, and this gene, along with its recombinant vector, recombinant bacteria, and transgenic plants, all show promising application prospects.

Claims

1. A method for producing transgenic plants that highly express leucine, characterized in that: The method involves transferring a gene with a nucleotide sequence as shown in SEQ ID NO.1 into a plant to obtain a transgenic plant that highly expresses leucine; the transgenic plant is tobacco or barley.

2. The method according to claim 1, characterized in that: The methods for introducing the substance into the plant include Agrobacterium-mediated transformation, gene gun method, electroporation, PEG-mediated transformation, liposome method, or calcium phosphate-DNA coprecipitation method.

3. The method according to claim 2, characterized in that: The method used to transfer the organism into the plant is Agrobacterium-mediated transformation.

4. Use of a gene, recombinant vector, or recombinant bacterium in the preparation of a transgenic plant that highly expresses leucine, wherein the transgenic plant is tobacco or barley; the nucleotide sequence of the gene is shown in SEQ ID NO.1, the recombinant vector contains the gene, and the recombinant bacterium contains the recombinant vector.

5. The use according to claim 4, characterized in that: The recombinant bacteria is recombinant Agrobacterium.

6. The recombinant bacteria according to claim 5, characterized in that: The recombinant Agrobacterium is recombinant EHA105.

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