Branched chain amino acid transaminase gene and application thereof

Abstract

The invention provides a branched chain amino acid transaminase gene and application thereof, and belongs to the field of gene engineering. According to the invention, the branched chain amino acid transaminase gene HvBCAT5 is found for the first time, and the gene has obvious positive correlation with the accumulation of valine (Val), isoleucine (Ile) and leucine (Leu) of crops. The invention provides an important target gene HvBCAT5 for crop quality improvement, and has important value for increasing the content of branched chain amino acid of crops and improving dietary nutrition. The HvBCAT5 gene as well as a recombinant vector, a recombinant bacterium and a transgenic plant thereof have good application prospects in the fields of food and feed industry.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a branched-chain amino acid transaminase gene and its uses. Background Art

[0002] Branched-chain amino acids (BCAAs), including valine (Val), isoleucine (Ile), and leucine (Leu), are essential amino acids indispensable in plants and animals. They play key roles in protein synthesis, energy metabolism, and signal regulation. Valine and isoleucine are involved in gluconeogenesis and energy supply, while leucine, as an activator of the mTOR signaling pathway, regulates cell growth and protein translation. In hulless barley (Hordeum vulgare L. var. nudum), BCAAs not only affect the protein quality of grains but are also closely related to its stress resistance and nutritional functions. As a staple food crop in high-altitude regions, the BCAA content of hulless barley directly affects the intake of essential amino acids by the human body. Especially in plateau regions, hulless barley varieties with high BCAA content have important value for improving dietary nutrition.

[0003] Branched-chain amino acid transaminase (BCAT) is an enzyme involved in amino acid metabolism, mainly catalyzing the transamination reaction between branched-chain amino acids (leucine, isoleucine, and valine) and α-ketoglutaric acid, and playing a key role in the catabolism and anabolism of amino acids.

[0004] Although the biological functions of BCAAs have been widely studied, their biosynthetic pathways in hulless barley have not been fully elucidated. In microorganisms and model plants, the synthesis of BCAAs is catalyzed by key enzymes such as acetolactate synthase (ALS), keto acid reductoisomerase (KARI), and dihydroxy acid dehydratase (DHAD). However, the gene regulatory network, metabolic flux distribution, and environmental response mechanism of these enzymes in hulless barley are still unclear. Analyzing the key genes for BCAA synthesis in hulless barley will fill the research gap in this field and provide a theoretical basis for subsequent metabolic engineering.

[0005] Since the content of BCAAs directly affects the nutritional quality and processing characteristics of hulless barley, clarifying its synthesis mechanism not only helps in the breeding of high-quality hulless barley varieties but may also expand its applications in the functional food and feed industries, with broad commercial potential. Summary of the Invention

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

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a gene, and the nucleotide sequence of the gene is as shown in SEQ ID NO.1.

[0009] The present invention also provides a recombinant vector, and the recombinant vector contains the gene with the nucleotide sequence as shown in SEQ ID NO.1.

[0010] Further, the recombinant vector is recombinant pEAQ or recombinant pCXSN.

[0011] The present invention also provides a recombinant bacterium, and the recombinant bacterium contains the above-mentioned recombinant vector.

[0012] Further, the recombinant bacterium is recombinant Agrobacterium.

[0013] Further, the recombinant Agrobacterium is recombinant EHA105.

[0014] The present invention also provides a method for producing a transgenic plant with high expression of branched-chain amino acid transaminase. The method is to transfer the above gene into a plant to obtain a transgenic plant with high expression of branched-chain amino acid transaminase.

[0015] Further, the method for transferring into the plant is the Agrobacterium method, the gene gun method, the electrotransformation method, the PEG-mediated method, the liposome method or the calcium phosphate-DNA coprecipitation method; the plant is tobacco or hulless barley.

[0016] Further, the method for transferring into the plant is the Agrobacterium method.

[0017] The present invention also provides the use of the above gene, recombinant vector and recombinant bacterium in the preparation of a transgenic plant with high expression of branched-chain amino acid transaminase.

[0018] Further, the plant is tobacco or hulless barley.

[0019] The present invention has achieved the following beneficial effects:

[0020] Through the combined analysis of metabolome and transcriptome at 21 key developmental stages during the whole growth period of the highland barley variety "Tibetan Blue 2000", the present invention first found that the expression pattern of the branched-chain amino acid transaminase gene HvBCAT5 was significantly positively correlated with the accumulation of valine (Val), leucine (Leu), and isoleucine (Ile) (Pearson correlation coefficient r > 0.6, p < 0.01). To verify its function, we constructed an overexpression vector of HvBCAT5 and transformed tobacco. LC-MS metabolic detection showed that the contents of Leu, Val, and Ile in the leaves of transgenic lines were significantly increased compared with those of the wild type (p < 0.01). These results fully demonstrated that the branched-chain amino acid transaminase encoded by HvBCAT5 plays a key catalytic role in the branched-chain amino acid biosynthesis pathway in plants, providing an important target gene for crop quality improvement. The gene, its recombinant vector, recombinant bacteria, and transgenic plants all have good application prospects.

[0021] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions, or changes can be made.

[0022] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0023] Figure 1 Shows the change trends of the expression level of HvBCAT5 and the accumulation levels of Ile, Val, and Leu in 21 tissues during the whole growth period of Tibetan Blue 2000.

[0024] Figure 2 Shows the functional analysis of HvBCAT5. A. Expression levels of HvBCAT5 in control and tobacco transgenic lines; B. Accumulation levels of three branched-chain amino acids in control and tobacco transgenic lines. *p < 0.05, **p < 0.01 (t-test).

[0025] Figure 3 Shows the secondary spectra of three branched-chain amino acids. Detailed Description of the Invention

[0026] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0027] The branched-chain amino acid transaminase gene (HvBCAT5 gene) in highland barley, the gene sequence of which is shown in SEQ ID NO.1. This gene fragment can be obtained by direct synthesis or by other conventional preparation methods in the art.

[0028] SEQ ID NO.1:

[0029]

[0030] Example 1. Correlation analysis of HvBCAT5 gene expression and branched-chain amino acid contents of Ile, Val, and Leu during the whole growth period of hulless barley

[0031] 1. Method

[0032] A combined transcriptome and metabolome analysis was performed on 21 key tissues during the whole growth period of the hulless barley variety "Tibetan Blue 2000". The specific steps are as follows:

[0033] (1) Seed treatment: Seeds were soaked and disinfected with 0.5% potassium permanganate, washed clean after 10 min, placed in a petri dish with filter paper moistened with distilled water on both the top and bottom, and placed in a constant-temperature incubator for overnight dark cultivation for germination at a temperature of 26°C;

[0034] (2) Sowing: After the seeds showed white tips, they were sown into a plug tray. The substrate ratio was perlite:peat moss = 1:3 (volume ratio). The surface was covered with fine peat moss, watered, and covered with a plastic film. The plastic film was removed after the hulless barley germinated;

[0035] (3) Sample collection (collect the following 21 tissue samples according to the growth period):

[0036] Germination stage: germinated seeds, plumules, radicles; tillering stage: leaves, roots; jointing stage: leaves, roots; young spike differentiation stage (10 - 20 cm): leaves, leaf sheaths; young spike differentiation stage (40 - 50 cm): leaves, leaf sheaths, young spikes; heading stage: leaves and stems 5 days before heading, stems and spikes at the heading stage; filling stage: flag leaves and endosperms 10 days after pollination; maturity stage: endosperms 20, 30, and 40 days after pollination.

[0037] (4) Multi-omics combined analysis Transcriptome sequencing: Three biological replicates of each sample were sequenced; Metabolome detection: LC-MS / MS was used for targeted detection of the contents of branched-chain amino acids (Val, Ile, and Leu); Correlation analysis: Pearson correlation coefficient was used to analyze the correlation between the expression level of HvBCAT5 and the contents of branched-chain amino acids.

[0041] 2. Results

[0042] The changing trends of the expression level of HvBCAT5 and the accumulation levels of Ile, Val, and Leu in 21 tissues during the whole growth period of Tibetan Blue 2000 are as Figure 1 shown. Correlation analysis showed that the expression level of HvBCAT5 was correlated with Val (r = 0.85, p = 5.6×10 -6)、Ile (r = 0.81, p = 6.2×10 -4 ) and Leu (r = 0.89, p = 2.5×10 -5 ), showing a significant positive correlation).

[0043] Example 2. Study on Tobacco Transient Expression

[0044] 1. Tobacco Transient Expression

[0045] ① Transform the transient expression vector (transient expression vector pEAQ, from John Innes Centre) containing the target gene (gene sequence shown in SEQ ID NO.1) into Agrobacterium tumefaciens (EHA105);

[0046] ② Pick positive Agrobacterium clones into 500 μl of LB containing the corresponding antibiotic (kn) and culture for 20 - 24 hours;

[0047] ③ Transfer 200 μl to 5 ml of LB containing the corresponding antibiotic (kn) and shake at 28°C and 220 rpm until OD 600 ≈1.0.

[0048] ④ Centrifuge at 10,000 rpm at room temperature for 15 min to collect the bacteria, and resuspend the bacteria with the pre - prepared transformation buffer until OD 600 = 1.0, and shake on a shaker for 3 h; the transformation buffer contains 10 mmol / L MgCl2, 10 mmol / L MES, 150 μmol / L acetosyringone, pH = 5.6;

[0049] ⑤ Take a prepared 1 - ml syringe, remove the needle, select a syringe with a smooth outlet, suck in the bacterial solution, take 1 - month - old Nicotiana benthamiana, hold the leaf with your hand, inject from the back of the leaf to allow the Agrobacterium to penetrate. Mark each tobacco plant that has been injected, and circle the area where the Agrobacterium has penetrated on the leaf. Select tobacco plants injected with the transformation buffer as a control.

[0050] ⑥ Incubate the tobacco plants injected with Agrobacterium in the dark for 24 h, then transfer them to a tobacco incubator for light culture for 24 - 48 hours and then samples can be taken (note that the injected tobacco plants should not be directly sprayed with water on the leaves).

[0051] 2. Product Collection and Purification

[0052] Cut the leaves in the Agrobacterium infiltration area, place them in a pre-weighed EP tube containing steel beads, make marks, quickly place them in liquid nitrogen, and perform freeze-drying. For the freeze-dried samples, use a grinder (MM 400, Retsch) to grind them for 60 s under the condition of 30 Hz, and put the ground sample powder into a 2 ml EP tube. Weigh each EP tube with an electronic balance and record; take an appropriate amount (range 30 - 60 mg) of the ground sample into the EP tube, weigh and record, and calculate the net weight of the samples in all EP tubes. Given the net weight of each sample, add 70% MeOH solution according to the volume V = sample net weight (mg) * 12 μL / mg and operate on ice at 4 °C. Mix well, vortex for 15 s, vortex once every half hour for a total of 4 times, and place in a 4 °C refrigerator for extraction for more than 12 h. Then centrifuge. First, turn on the centrifuge and pre-cool it to 4 °C, set the time to 10 min and the rotation speed to 12000 rpm, vortex the sample and then centrifuge it. Pay attention to symmetric balance when using the centrifuge. After centrifugation, aspirate the supernatant. Filter the supernatant with a microporous membrane (0.22 μm pore size) and load it into a sample vial to obtain the sample extract, and prepare for LC-MS detection.

[0053] 3. Detection of target product

[0054] Place the sample vial containing the sample extract into the sample tray in the autosampler, and record the position of the injection hole corresponding to each sample vial number. At the same time, open the software Analyst Software, double-click Hardware Configuration, select LCMS-V (with switching valve mode), click Activate Profile, and select the Acquire Mode mode, click Acquire, click the Equilibrate key above the figure, and generally set the time to 3 min. The purpose of this operation is to preheat the instrument to make the high-pressure infusion pump, chromatographic column, column oven, ion source temperature, etc. reach the conditions set in the method. After the status of each instrument component is Ready, the Start Sample key in the function area becomes clickable. At this time, it indicates that the instrument is normal and the analysis conditions are normal. Then click Start Sample to start running the sample. Submit 4 blank samples before the first run.

[0055] Take three samples of transiently expressed tobacco, namely OX-1, OX-2, and OX-3; wild-type (CK) Nicotiana benthamiana is used as a control.

[0056] 4. Results

[0057] The experimental results show that: (1) Expression level verification: qRT-PCR confirmed that the expression level of HvBCAT5 in the OX line is 50 - 80 times that of the WT ( Figure 2 A). (2) The metabolic analysis results show that ( Figure 2B): The Ile content in the overexpression lines OX1 - OX3 was 2.42 ± 0.42 times that of the WT (p < 0.01), the Val content was 3.17 ± 0.89 times that of the WT (p < 0.01), and the Leu content was 7.23 ± 0.64 times that of the WT (p < 0.01).

[0058] In the present invention, the gene HvBCAT5 was transferred into tobacco, enabling tobacco plants to highly express branched-chain amino acid transaminase (the expression level was increased by 50 - 80 times compared with wild-type tobacco), and inducing the accumulation of Ile, Val, and Leu in tobacco. The prepared tobacco had an increased accumulation of branched-chain amino acids, improving the application value of tobacco. Figure 3 Through the secondary spectrum analysis of the overexpressed metabolites, it was demonstrated that the significantly enriched compounds were Ile, Val, and Leu.

[0059] Example 3: Construction of transgenic tobacco

[0060] The method for obtaining HvBCAT5 transgenic tobacco is as follows:

[0061] First, construct the recombinant expression vector pCXSN:HvBCAT5 containing the HvBCAT5 gene. Second, use the Agrobacterium-mediated transformation method to transform the aforementioned recombinant expression vector into Nicotiana benthamiana (WT). Transfer the explant leaves soaked in Agrobacterium liquid onto the medium (MS + As) for dark culture for 3 days, then change to the differentiation medium for culture until adventitious buds grow to 1 - 2 cm, and then transfer to the growth medium. Finally, sow the obtained T0 generation seeds on the medium of 1 / 2MS (containing 25 mg·L -1 Hyg) for screening. Observe the growth of tobacco under hygromycin screening, select transgenic positive seeds for planting, and identify the expression level of HvBCAT5 in the T1 generation transgenic tobacco by RT-PCR technology to obtain positive T2 generation transgenic seeds.

[0062] In summary, through the combined analysis of the metabolome and transcriptome of 21 key developmental stages during the whole growth period of the highland barley variety "Zangqing 2000", the present invention first found that the expression pattern of the branched-chain amino acid transaminase gene HvBCAT5 was significantly positively correlated with the accumulation of valine (Val), isoleucine (Ile), and leucine (Leu). The present invention further constructed an overexpression vector of HvBCAT5 and transformed tobacco, and the contents of Val, Ile, and Leu in the leaves of transgenic lines were significantly higher than those of the wild type. The HvBCAT5 discovered by the present invention provides an important target gene for crop quality improvement, and the gene, its recombinant vector, recombinant bacterium, and transgenic plant all have good application prospects.

Claims

1. A gene, characterized in that: The nucleotide sequence of the said gene is as shown in SEQ ID NO.

1.

2. A recombinant vector, characterized in that: The said recombinant vector contains the gene with the nucleotide sequence as shown in SEQ ID NO.

1.

3. The recombinant vector according to claim 2, wherein: The said recombinant vector is recombinant pEAQ or recombinant pCXSN.

4. A recombinant bacterium, characterized in that: The said recombinant bacterium contains the recombinant vector described in claim 2 or 3.

5. The recombinant bacterium according to claim 4, wherein: The said recombinant bacterium is recombinant Agrobacterium.

6. The recombinant bacterium according to claim 5, wherein: The said recombinant Agrobacterium is recombinant EHA105.

7. A method for producing transgenic plants with high expression of branched-chain amino acid transaminase, characterized in that: The said method is to transfer the gene described in claim 1 into a plant to obtain a transgenic plant with high expression of branched-chain amino acid transaminase.

8. The method according to claim 7, characterized in that: The method of transferring into the plant is the Agrobacterium method, the gene gun method, the electroporation method, the PEG-mediated method, the liposome method or the calcium phosphate-DNA coprecipitation method; the plant is tobacco or hulless barley.

9. The method according to claim 8, characterized in that: The method of transferring into the plant is the Agrobacterium method.

10. Use of the gene described in claim 1, the recombinant vector described in claim 2 or 3, and the recombinant bacterium described in any one of claims 4 to 6 in the preparation of a transgenic plant with high expression of branched-chain amino acid transaminase; the plant is preferably tobacco or hulless barley.

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