A branched-chain amino acid synthesis gene and its uses

Abstract

This invention provides a branched-chain amino acid synthesis gene and its uses, belonging to the field of genetic engineering technology. The nucleotide sequence of the gene HvSAP73 (HOVUSG3688500) is shown in SEQ ID NO.1. This invention has found that the HvSAP73 gene can regulate the accumulation levels of branched-chain amino acids, including isoleucine (Ile), leucine (Leu), and valine (Val), in plants. Overexpression of this gene in tobacco significantly increased the content of branched-chain amino acids Ile, Leu, and Val in tobacco leaves. Therefore, the HvSAP73 gene plays an important role in plant branched-chain amino acid metabolism, and this gene, its recombinant vector, recombinant bacteria, and transgenic plants all have broad application prospects in crop quality improvement.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an auxin-responsive SAUR gene family protein gene and its uses. Background Technology

[0002] Barley, a vital food crop widely cultivated in many parts of the world, is not only highly nutritious but also attracts attention due to its adaptability to harsh environments. Barley grains are rich in essential amino acids, crucial nutrients that the human body cannot synthesize and must obtain from food, playing a vital role in human health. However, despite the abundant amino acid content in barley, research into the genes involved in amino acid synthesis and regulation is still in its early stages. This limits a deeper understanding of the barley amino acid metabolic network and the potential for further enhancing its amino acid content through genetic engineering.

[0003] In plant growth, development, and metabolic regulation, various transcription factors and metabolic enzymes play crucial roles. In recent years, genes related to branched-chain amino acid (BCAA) synthesis have gradually become a hot topic in plant science research. BCAAs are essential amino acids that cannot be directly synthesized in plants through transamination and must be synthesized de novo via specific branching pathways. These include valine, leucine, and isoleucine. They not only serve as basic building blocks of proteins but also participate in regulating various physiological processes such as plant growth and development, stress responses, and metabolic signal transduction. Their synthesis and accumulation levels directly affect the nutritional quality, stress resistance, and yield of crops. Therefore, cloning and functionally validating key genes involved in the synthesis and metabolic regulation of BCAAs in plants, and elucidating their molecular mechanisms in BCAA accumulation and homeostasis, has significant theoretical value and application prospects for improving crop amino acid composition, enhancing nutritional quality, and strengthening environmental adaptability through genetic engineering. Summary of the Invention

[0004] The purpose of this invention is to provide an HvSAP73 gene that regulates the biosynthesis pathway of branched-chain amino acids and its uses.

[0005] The present invention provides a gene fragment, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The present invention also provides a recombinant vector comprising a gene fragment with a nucleotide sequence as shown in SEQ ID NO.1.

[0007] Furthermore, the recombinant vector is recombinant pEAQ or recombinant pCXSN.

[0008] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the aforementioned recombinant vector.

[0009] Furthermore, the recombinant bacteria is Agrobacterium.

[0010] The present invention also provides an HvSAP73 protein, wherein the HvSAP73 protein is obtained by expressing a gene fragment with the sequence shown in SEQ ID NO.1.

[0011] The present invention also provides the use of the aforementioned gene fragment, the aforementioned recombinant vector, the aforementioned recombinant bacteria, or the aforementioned HvSAP73 protein for the production of plants with high branched-chain amino acid content. Preferably, the plant is barley or tobacco.

[0012] The present invention also provides a method for constructing a transgenic plant that highly expresses HvSAP73 protein: a gene fragment with a nucleotide sequence as shown in SEQ ID NO.1 is transferred into a plant to obtain a transgenic plant that highly expresses HvSAP73 protein.

[0013] Furthermore, the plant in question is barley or tobacco.

[0014] Furthermore, the method for transferring the substance into the plant is one of the following: Agrobacterium-mediated transformation, gene gun method, electroporation, PEG-mediated transformation, liposome method, and calcium phosphate-DNA coprecipitation method.

[0015] This invention, through combined metabolomics and transcriptomics analysis of 21 key developmental stages throughout the entire growth period of the barley variety 'Zangqing 2000', reveals for the first time a significant positive correlation between the expression pattern of the HvSAP73 gene and the accumulation of valine (Val), leucine (Leu), and isoleucine (Ile) (Pearson correlation coefficients: Ile r=0.88, Val r=0.88, Leu r=0.86, p<0.01). HvSAP73, a newly discovered branched-chain amino acid regulatory gene, has not yet been functionally studied in barley. To verify its function, this invention constructed an HvSAP73 overexpression vector and transformed it into tobacco. LC-MS metabolic analysis showed that the contents of Val, Leu, and Ile in the leaves of the transgenic lines were significantly increased compared to the wild type (p<0.01). These results fully demonstrate that the protein encoded by HvSAP73 plays a key catalytic role in the plant branched-chain amino acid biosynthesis pathway, providing an important target gene for crop quality improvement.

[0016] 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.

[0017] 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

[0018] Figure 1 This study analyzed the expression levels of HvSAP73 and the accumulation levels of Ile, Leu, and Val in 21 tissues throughout the entire growth period of Zangqing 2000.

[0019] Figure 2 Functional analysis of HvSAP73. A. Expression level analysis of HvSAP73 in control and tobacco transgenic lines; B. Accumulation level analysis of the three branched-chain amino acids in control and tobacco transgenic lines. p <0.05, p <0.01 (t-test).

[0020] Figure 3 This is a diagram showing the secondary spectrum analysis results of three branched-chain amino acids. Detailed Implementation

[0021] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0022] The auxin-responsive SAUR gene family protein gene (HvSAP73 gene) in highland barley has the sequence shown in SEQ ID NO.1. This gene fragment can be obtained by direct synthesis or by other conventional preparation methods in the art.

[0023] SEQ ID NO.1: Example 1: Association analysis of HvSAP73 gene expression and the content of branched-chain amino acids Ile, Leu, and Val throughout the entire growth period of highland barley. 1. Method 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: (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℃. (2) Sowing: After the seeds show white sprouts, sow them in the seed trays. The substrate ratio is perlite: peat moss = 1:3 (by volume). Cover the surface with fine peat moss, water and cover with plastic wrap. Remove the film after the barley sprouts. (3) Sample collection (collecting samples from the following 21 tissues according to reproductive period): 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. (4) Multi-omics joint analysis Transcriptome sequencing: Three biological replicates were used for each sample, and sequencing was performed using the Illumina HiSeq platform; Metabolomics assay: The content of branched-chain amino acids (Leu, Val, and Ile) was detected by targeted LC-MS / MS. Correlation analysis: The correlation between HvSAP73 expression level and branched-chain amino acid content was analyzed using Pearson correlation coefficient.

[0024] 2. Results Correlation analysis showed that HvSAP73 expression was significantly positively correlated with the levels of Ile (r=0.88, p<0.01), Val (r=0.86, p<0.01), and Leu (r=0.88, p<0.01). Figure 1 ).

[0025] Example 2: Study on transient expression in tobacco 1. Transient expression of tobacco 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); ② Pick positive Agrobacterium clones and put them into 500 μl of LB containing the corresponding antibiotic (kn) and incubate for 20-24 hours; 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; 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; 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 by circling the area where Agrobacterium has penetrated on the leaf. Use tobacco injected with transformation buffer as a control; ⑥ After injecting Agrobacterium or transformation buffer, 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 injected tobacco plants should not be sprayed with water directly on the leaves).

[0026] 2. Product collection and purification Leaf fragments from the Agrobacterium-infiltrated region were cut and placed in pre-weighed EP tubes containing steel balls. These tubes were labeled and quickly placed in liquid nitrogen for lyophilization. The lyophilized samples were then ground using a grinder (MM 400, Retsch) at 30Hz for 60 seconds. The ground sample powder was then transferred to 2ml EP tubes. The weight of each EP tube was weighed using an electronic balance and recorded. An appropriate amount of the ground sample (range 30-60mg) 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, the formula V = net weight of sample (mg) was applied. Add 12 μL / mg of 70% MeOH solution to ice at 4°C. Mix well, vortex for 15 seconds, repeating this process every half hour for a total of 4 vortexes. Extract at 4°C for at least 12 hours. Then centrifuge. Pre-cool the centrifuge to 4°C, set the time to 10 minutes and the speed to 12000 rpm. Vortex the sample and then centrifuge, ensuring symmetrical balance. After centrifugation, aspirate the supernatant. Filter the supernatant through a microporous membrane (0.22 μm pore size) and transfer it to a sample vial for LC-MS analysis.

[0027] 3. Detection of target product 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.

[0028] 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.

[0029] 4. Results The results showed that the present invention transferred the HvSAP73 gene into tobacco, which resulted in tobacco plants expressing the HvSAP73 protein (15-30 times higher than wild-type tobacco) and induced the accumulation of Ile, Leu and Val in tobacco.

[0030] To further verify the stable expression of HvSAP73 protein and branched-chain amino acids in transgenic tobacco plants, this invention constructed T1 and T2 generation transgenic tobacco seeds.

[0031] Example 3: Construction of transgenic tobacco The method for obtaining HvSAP73 genetically modified tobacco is as follows: First, a recombinant expression vector pCXSN containing the HvSAP73 gene was constructed. Then, the recombinant expression vector was transformed into *Nicotiana benthamiana* (WT) using Agrobacterium-mediated transformation. Explant leaves soaked in Agrobacterium solution were transferred to MS+As medium and cultured in the dark for 3 days. The culture medium was then changed to differentiation medium and cultured until adventitious shoots reached 1-2 cm in length, after which they were transferred to growth medium. Finally, the obtained T0 generation seeds were sown in 1 / 2 MS medium (containing 25 mg·L⁻¹ Hyg) for selection. The growth of tobacco was observed under hygromycin selection. Transgenic positive seeds were selected for planting, and the expression level of HvSAP73 in T1 generation transgenic tobacco was identified using RT-PCR. Positive T2 generation transgenic tobacco seeds were then obtained.

[0032] Example 4: Metabolic Analysis of Transgenic Tobacco 1. Method 1.1 Plant materials This study used *Tobacco Bunge* (… Nicotiana benthamiana Wild-type (WT) and HvSAP73 overexpression lines (OX1, OX2, OX3, T2 generation).

[0033] 1.2 Metabolic Analysis of Transgenic Tobacco (1) Plant culture: After sterilization, the positive T2 generation transgenic tobacco seeds obtained by WT and Example 3 were sown on MS medium and cultured under continuous light at 25°C for 4 weeks. Fully unfolded functional leaves were taken for analysis.

[0034] (2) The expression level of HvSAP73 in leaves was identified by RT-PCR.

[0035] (3) Metabolite detection: Leaf samples were quick-frozen in liquid nitrogen and targeted metabolomics analysis was performed using LC-MS / MS. Detection parameters: Column: ACQUITY UPLC BEH C18 (2.1×100 mm, 1.7 μm), mobile phase: 0.1% formic acid aqueous solution and acetonitrile, mass spectrometry conditions: ESI positive ion mode, MRM scan.

[0036] 2. Results (1) Expression level verification ( Figure 2 A): qRT-PCR confirmed that the expression level of HvSAP73 in the OX strain was 15-30 times that in the WT strain.

[0037] (2) The metabolic analysis results showed that ( Figure 2 B): In the overexpression lines OX1-OX3: the content of Ile increased by 824.8%±56.2% (p<0.01), the content of Val increased by 647.8%±62.5% (p<0.01), and the content of Leu increased by 332.4%±64.1% (p<0.01). Figure 3 Secondary spectral analysis of the overexpressed metabolites revealed that the significantly enriched compounds were Ile, Leu, and Val.

[0038] The above results indicate that transgenic tobacco overexpressing HvSAP73 can stably overexpress the HvSAP73 protein and significantly increase the accumulation levels of Ile, Leu, and Val.

[0039] In summary, this study found that the HvSAP73 gene can significantly increase the accumulation levels of Ile, Leu, and Val in tobacco, thereby enhancing the accumulation of branched-chain amino acids. Overexpression of the HvSAP73 gene in tobacco significantly increased the content of Ile, Leu, and Val in tobacco leaves, and improved the accumulation of branched-chain amino acids. Therefore, HvSAP73 provides an important target gene for crop quality improvement, and this gene, along with its recombinant vectors, recombinant bacteria, and transgenic plants, all show promising application prospects.

Claims

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

1.

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

1.

3. The recombinant vector according to claim 2, characterized in that: The recombinant vector is recombinant pEAQ or recombinant pCXSN.

4. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the recombinant vector as described in claim 2 or 3.

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

6. An HvSAP73 protein, characterized in that: The HvSAP73 protein is obtained by expressing a gene fragment with the sequence shown in SEQ ID NO.

1.

7. The use of the gene fragment of claim 1, the recombinant vector of claim 2 or 3, the recombinant bacteria of claim 4, or the HvSAP73 protein of claim 6 for the production of plants with high branched-chain amino acid content; Preferably, the plant is barley or tobacco.

8. A method for constructing a transgenic plant that highly expresses HvSAP73 protein, characterized in that: The construction method includes: transferring a gene fragment with a nucleotide sequence as shown in SEQ ID NO.1 into a plant to obtain a transgenic plant that highly expresses the HvSAP73 protein.

9. The construction method according to claim 8, characterized in that: The plants mentioned are barley and tobacco.

10. The construction method according to claim 8, characterized in that: The method for transferring the substance into the plant is one of the following: Agrobacterium-mediated transformation, gene gun method, electroporation, PEG-mediated transformation, liposome method, and calcium phosphate-DNA coprecipitation method.

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