Application of escherichia coli proB74 and proA genes in improvement of rice fragrance quality

Abstract

The invention discloses an application of escherichia coli proB74 and proA genes in improvement of rice fragrance quality. According to the invention, exogenous escherichia coli proB74 and proA genes are over-expressed by using a transgenic means, and agronomic traits such as proline and 2-AP contents, yield and the like of transgenic rice are analyzed. Preliminary research results show that overexpression of exogenous escherichia coli proB74 and proA genes can improve the proline content, greatly improve the 2-AP content and further improve the rice fragrance, but has no adverse effect on the agronomic traits of fragrant rice. The method provided by the invention can improve the fragrance quality of the rice variety, and is beneficial to increasing the market economic value of the rice variety.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to the application of Escherichia coli proB74 and proA genes in improving the aroma quality of rice. Background Art

[0002] Rice (Oryza sativa L.) is an important food crop worldwide. Aromatic rice, known as a treasure among rice varieties, exudes a unique and pleasant aroma during cooking. In recent years, demand for aromatic rice has continued to increase both domestically and internationally. Aroma is a key factor in determining the price of aromatic rice in international trade, with high-quality aromatic rice fetching three times the price of non-aromatic rice. Currently, aroma has become a key indicator of quality in the evaluation system for high-quality indica rice in southern my country. Therefore, breeding high-quality aromatic rice has important economic and social benefits for establishing premium rice brands, improving agricultural quality and efficiency, and increasing farmers' incomes.

[0003] 2-Acetyl-1-pyrroline (2AP) is a characteristic aroma component common to all fragrant rice varieties. Factors such as fragrant rice variety, cultivation method, climate conditions, and storage methods all influence 2AP accumulation. The 2AP content in rice grains is a key determinant of rice price. Therefore, research on increasing 2AP content in rice grains is crucial for enhancing the economic value of fragrant rice. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, one of the objectives of the present invention is to provide the use of Escherichia coli proB74 and proA genes in improving the aroma quality of rice.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] Application of Escherichia coli proB74 and proA genes in improving rice aroma quality.

[0007] Furthermore, the nucleotide sequence of the proB74 gene is shown as SEQ ID NO.2; the nucleotide sequence of the proA gene is shown as SEQ ID NO.3.

[0008] Furthermore, the application is: overexpressing proB74 and proA genes in rice to improve the aroma quality of rice.

[0009] Furthermore, the overexpression of proB74 and proA genes is achieved by a method comprising the following steps:

[0010] S1. Insert the chloroplast signal peptide RC2, proB74 and proA gene fragments into the downstream of the ubi promoter of the pRHVc-GFP vector in sequence to obtain the recombinant vector proAB74;

[0011] S2. The recombinant vector proAB74 is transformed into callus tissue through Agrobacterium-mediated transformation. After screening of resistant callus tissue and plant regeneration, rice overexpressing proB74 and proA genes is obtained.

[0012] Furthermore, the specific operations of step S1 are:

[0013] (1) Using rice cDNA as a template, PCR amplification was performed using primers proAB74-1F and proAB74-1R to obtain the chloroplast signal peptide RC2 gene fragment; using Escherichia coli as a template, PCR amplification was performed using primers proAB74-2F and proB-muR and primers proB-muF and proAB74-2R to obtain two fragments of the proB74 gene; and PCR amplification was performed using primers proAB74-3F and proAB74-3R to obtain the proA gene fragment;

[0014] (2) selecting the vector pRHVc-GFP as the backbone vector for vector construction, BamH I and Hind III as the restriction enzyme sites for vector construction, and connecting the chloroplast signal peptide RC2 gene fragment, the proB74 gene fragments, the proA gene fragment and the backbone vector obtained in step (1) by homologous recombination to obtain the recombinant vector proAB74;

[0015] Among them, the sequence of the primer pair for PCR amplification is:

[0016] proAB74-1F: 5'-tatccagatccagtgggatccATGGCCCCCTCCGTGATG-3';

[0017] proAB74-1R: 5'-ctggctgtcactcatCATACATCGTATTCTCCCTCCGT-3';

[0018] proAB74-2F: 5'-gtatgATGAGTGACAGCCAGACGCTG-3';

[0019] proAB74-2R: 5'-ccatttgttccagcatGCCGCCGCCGCCGCCTGAGCCGCCACGGGTAATCATGTCAT-3';

[0020] proAB74-3F: 5'-cggcATGCTGGAACAAATGGGCAT-3';

[0021] proAB74-3R: 5'-agcggccgcactagtaagcttTTACGCACGAATGGTGTAATCAC-3';

[0022] proB-muF: 5'-ATGCTGCTGACCCGTGCTaATATGGAAGACCGTGAAC-3';

[0023] proB-muR: 5'-GTTCACGGTCTTCCATATtAGCACGGGTCAGCAGCAT-3';

[0024] The conditions for the PCR amplification reaction were: 95°C for 5 min; 35 cycles of 95°C for 40 s, 58°C for 50 s, and 72°C for 3 min; and 72°C for 10 min.

[0025] Furthermore, the Agrobacterium is EHA105 Agrobacterium.

[0026] Furthermore, the improved rice aroma quality includes: increasing the content of aroma substances in rice grains.

[0027] Furthermore, the rice grain aroma substance includes 2-acetyl-1-pyrroline (2AP).

[0028] Furthermore, the rice is fragrant rice.

[0029] Furthermore, the aromatic rice includes japonica aromatic rice.

[0030] Furthermore, the japonica aromatic rice includes Yunjingyou 14.

[0031] Application of the above method for overexpressing proB74 and proA genes in preparing transgenic plants.

[0032] Furthermore, the application comprises the following steps: cultivating the obtained rice overexpressing proB74 and proA genes into plants.

[0033] The present invention has the following advantages and effects compared to the prior art:

[0034] This study overexpressed the E. coli proB74 and proA genes in rice grains using transgenic methods. The transgenic rice was then analyzed for proline and 2-AP content, as well as agronomic traits such as yield and resistance. Preliminary results showed that proline content was increased, while 2-AP content was significantly increased, further enhancing the aroma of the rice, while having no adverse effects on the agronomic traits of the aromatic rice.

[0035] The method provided by the present invention can improve the aroma quality of rice varieties, which is beneficial to increasing their market economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the result of proline content test in brown rice of genetically modified material;

[0037] Figure 2 This is the result of the detection of pyrroline-5-carboxylic acid (P5C) content in brown rice of genetically modified material;

[0038] Figure 3 This is the result of the detection of 2-acetyl-1-pyrroline (2-AP) content in brown rice, a genetically modified material;

[0039] Figure 4 This is the result of agronomic traits testing of transgenic materials;

[0040] In the figure, * indicates significant difference compared with the control (P<0.05), and ** indicates extremely significant difference compared with the control (P<0.01). DETAILED DESCRIPTION

[0041] The present invention will be described in further detail below with reference to embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0042] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention relates.

[0043] The gene fragments and their sequences involved in the following examples are as follows:

[0044] The nucleotide sequence of chloroplast signal peptide RC2 (SEQ ID NO.1):

[0045] ATggCCCCCTCCGTGATGGCGTCGTCGGCCACCACCGTCGCTCCCTTCCAGGGGCTCAAGTCCACCGCCGGCATGCCCGTCGCCCGCCGCTCCGGCAACTCCAGCTTCGGCAACGTCAGCAATGGCGGCAGGATCAGGTGCATGCAGGTGTGGCCGATTGAGGGCATCAAGAAGTTCGAGACCCTCTCCTACCTGCCACCGCTCTCGAGCTCAGGAAATAGCAGCTTCGGAAATGTGTCCAACGGAGGGAGAATACGATGTATG。

[0046] Nucleotide sequence of proB74 gene (SEQ ID NO.2):

[0047] ATGAGTGACAGCCAGACGCTGGTGGTAAAACTCGGCACCAGTGTGCTAACAGGCGGATCGCGCCGTCTGAACCGTGCCCATATCGTTGAACTTGTTCGCCAGTGCGCGCAGTTACATGCCGCCGGGCATCGGATTGTTATTGTGACGTCGGGCGCGATCGCCGCCGGACGTGAGCACCTGGGGTACCCGGAACTGCCAGCGACTATCGCCTCGAAACAACTGCTGGCGGCGGTAGGGCAGAGTCGACTGATTCAACTGTGGGAACAGCTGTTTTCGATTTATGGCATTCACGTCGGGCAGATGCTGCTGACCCGTGCTAATATGGAAGACCGTGAACGCTTCCTGAACGCCCGCGACACCCTGCGTGCGTTGCTCGATAACAATATCGTTCCGGTAATCAATGAGAACGATGCTGTCGCTACGGCAGAGATTAAGGTCGGCGATAACGATAACCTTTCTGCGCTGGCGGCGATTCTGGCGGGTGCCGATAAACTGTTGCTGCTGACCGATCAAAAAGGTTTGTATACCGCTGATCCGCGCAGCAATCCGCAggcagaactgattaaagatgtttacggcattgatgacgcactgcgcgcgattgccggtgacagcgtttcaggcctcggaactggcggcatgagtaccaaattgcaggccgctgacgtggcttgccgtgcgggtatcgacaccattattgccgcgggcagcaagccgggcgttattggtgatgtgatggaaggcatttccgtcggtacgctgttccatgcccaggcgactccgcttgaaaaccgtaaacgctggattttcggtgcgccgccggcgggtgaaatcacggtagatgaaggggcaactgccgccattcttgaacgcggcagctccctgttgccgaaaggcattaaaagcgtgactggcaatttctcgcgtggtgaagtcatccgcatttgcaacctcgaaggtcgcgatatcgcccacggcgtcagtcgttacaacagcgatgcattacgccgtattgccgggcaccactcgcaagaaattgatgcaatactgggatatgaatacggcccggttgccgttcaccgtgatgacatgattacccgtGGCGGCTCAGGCGGCGGCGGCGGC。

[0048] Nucleotide sequence of proA gene (SEQ ID NO.3):

[0049]

[0050] The japonica aromatic rice variety Yunjingyou 14 involved in the following embodiments has been disclosed in the document "Xie Wenjun. Effects of γ-aminobutyric acid (GABA) and light on aroma accumulation and yield quality of aromatic rice [D]. South China Agricultural University, 2020."

[0051] The plant overexpression vector pRHVc-GFP involved in the following examples has been disclosed in the supplementary data of the document "He F, Zhang F, Sun W, et al. A Versatile Vector Toolkit for Functional Analysis of Rice Genes [J]. Rice, 2018, 11 (1): 27.

[0052] Example 1

[0053] 1. We mutated position 319 of the wild-type proB74 gene of Escherichia coli, changing the aspartic acid (D) at position 107 of the encoded protein to asparagine (N), resulting in the proB74 gene with the nucleotide sequence shown in SEQ ID NO. 2. We also added a chloroplast signal peptide RC2 (nucleotide sequence shown in SEQ ID NO. 1) before the proB74 and proA genes (nucleotide sequence shown in SEQ ID NO. 3). This construct was then inserted downstream of the ubi promoter in the pRHVc-GFP vector. The specific method is as follows:

[0054] 1.1 Obtaining chloroplast signal peptide RC2, Escherichia coli proB74 and proA genes:

[0055] (1) Using rice cDNA as a template, PCR amplification was performed with primer pair proAB74-1F and proAB74-1R to obtain the chloroplast signal peptide RC2 gene fragment; using Escherichia coli as a template, PCR amplification was performed with primer pair proAB74-2F, proB-muR and primer pair proB-muF, proAB74-2R to obtain the proB74 gene fragment; and PCR amplification was performed with primer pair proAB74-3F, proAB74-3R to obtain the proA gene fragment.

[0056] The PCR amplification reaction conditions were as follows: 95°C for 5 min; 35 cycles of 95°C for 40 s, 58°C for 50 s, and 72°C for 3 min; 72°C for 10 min;

[0057] The sequences of the PCR amplification primers are:

[0058] proAB74-1F: 5'-tatccagatccagtgggatccATGGCCCCCTCCGTGATG-3';

[0059] proAB74-1R: 5'-ctggctgtcactcatCATACATCGTATTCTCCCTCCGT-3';

[0060] proAB74-2F: 5'-gtatgATGAGTGACAGCCAGACGCTG-3';

[0061] proAB74-2R: 5'-ccatttgttccagcatGCCGCCGCCGCCGCCTGAGCCGCCACGGGTAATCATGTCAT-3';

[0062] proAB74-3F: 5'-cggcATGCTGGAACAAATGGGCAT-3';

[0063] proAB74-3R: 5'-agcggccgcactagtaagcttTTACGCACGAATGGTGTAATCAC-3';

[0064] proB-muF: 5'-ATGCTGCTGACCCGTGCTaATATGGAAGACCGTGAAC-3';

[0065] proB-muR: 5'-GTTCACGGTCTTCCATATtAGCACGGGTCAGCAGCAT-3'.

[0066] (2) Electrophoresis detection: After PCR amplification, gel electrophoresis detection is performed, and the results show the target band.

[0067] (3) Purification of target fragment: After electrophoresis, the PCR amplification product is purified.

[0068] 1.2 Construction of proAB74 vector

[0069] (1) Based on the cloned chloroplast signal peptide RC2, proB74 gene fragments, and proA gene fragments, the vector pRHVc-GFP was selected, BamH I and Hind III were used as restriction enzyme sites for vector construction, and the purified vector backbone and target fragments were ligated using homologous recombination;

[0070] (2) Transformation: Heat shock transform the DH5α strain, screen it with kanamycin-containing LB plates, and verify it with colony PCR; select the positive plasmid with the correct sequencing result, name it proAB74, and store it at -20℃ for future use.

[0071] 1.3 Transformation of proAB74 vector into EHA105 Agrobacterium:

[0072] (1) Place 5 μL of plasmid (about 500 ng) and EHA105 competent Agrobacterium cells on ice for 5 min, freeze in liquid nitrogen for 5 min, heat shock in a 37°C water bath for 5 min, and finally place on ice for 5 min.

[0073] (2) Activate in a shaker at 200 rpm for 2 h at 28°C;

[0074] (3) The transformed bacterial solution was spread on an LB plate containing spectinomycin and rifampicin antibiotics to screen and obtain positive strains.

[0075] 1.4 Transformation of callus tissue

[0076] (1) Obtaining rice callus from mature embryos:

[0077] 1) Take mature Yunjing You 14 seeds that are well-filled and free of mildew and lesions, and use a small rice huller to remove the husks, leaving intact rice kernels.

[0078] 2) In a clean bench, add an appropriate amount of rice to a sterile Erlenmeyer flask. Sterilize the surface with 75% alcohol for 1 minute, then rinse 3-5 times with sterile distilled water.

[0079] 3) Add 10% sodium hypochlorite solution, add sealing film, and sterilize in a shaker at room temperature at 160 rpm for 30 minutes;

[0080] 4) Discard the sodium hypochlorite solution, rinse the seeds 3-5 times with sterile distilled water, transfer to sterile filter paper, and air-dry until the surface turns white.

[0081] 5) Transfer the seeds to callus induction medium plates and culture at 30°C in the light for 4-6 days;

[0082] (2) Agrobacterium-mediated transformation of rice callus:

[0083] 1) Streak a plate containing EHA105 Agrobacterium tumefaciens strains containing the proAB74 vector plasmid stored at -80°C (containing 50 mg / L kanamycin and 50 mg / L rifampicin) and incubate at 28°C for two days.

[0084] 2) Pick a single colony and incubate in 5 mL of LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin) at 28°C and 200 rpm for 24 hours.

[0085] 3) Add 50 μL of the bacterial suspension from the previous step to 50 mL of LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin) and incubate at 28°C, 200 rpm, until the absorbance OD600 reaches 0.5;

[0086] 4) Centrifuge the culture at 6000 rpm for 10 minutes at room temperature, discard the supernatant, and collect the precipitated cells. Resuspend the cells in AAM liquid medium (containing 150 μM acetosyringone) and adjust the OD600 to 0.1.

[0087] 5) In a laminar flow hood, select pale yellow, compact, healthy rice callus tissue (1-2 mm in size) and immerse it in the prepared bacterial resuspension solution. Gently shake for 1 minute.

[0088] 6) Discard the culture medium, rinse 2-3 times with sterile distilled water, and then air-dry on sterile filter paper;

[0089] 7) Place a piece of filter paper soaked in liquid co-cultivation medium on the surface of the solid co-cultivation medium, spread the callus tissue on it, and culture in a dark room at 25-26°C for 2 days;

[0090] (3) Screening of resistant callus and plant regeneration

[0091] 1) Transfer the dark-cultured callus to a sterile Erlenmeyer flask, rinse five times with sterile distilled water, add sterile distilled water containing 500 mg / L cephalosporin, and shake at 160 rpm for 20 minutes. Remove the callus and air-dry it on sterile filter paper.

[0092] 2) Callus tissue was transferred to selective medium and cultured in the dark at 30°C, with fresh selective medium replaced every two weeks;

[0093] 3) After the callus on the selective medium develops bright yellow granular resistant callus, it is transferred to the selective differentiation medium and cultured at 30°C under light. The differentiation medium is replaced every two weeks.

[0094] 4) After the callus tissue grows into resistant green shoots, it is transferred to rooting medium and cultured at 30°C with 16 hours / day light intensity;

[0095] 5) After 3-4 weeks, when the resistant seedlings have taken root, select the plants with well-developed root systems, open the top cover and add distilled water to harden the seedlings;

[0096] 6) PCR identification of transgenic plants, PCR primer sequences are as follows:

[0097] ProAB74-F: 5'-gtatgATGAGTGACAGCCAGACGCTG-3';

[0098] ProAB74-R: 5'-agcggccgcactagtaagcttTTACGCACGAATGGTGTAATCAC-3'.

[0099] Finally, the transgenic strain proAB74-OE overexpressing proB74 and proA genes was successfully obtained.

[0100] 2. Test materials and sample collection

[0101] The transgenic plants were grown in a transgenic experimental field at South China Agricultural University and cultivated using conventional water and fertilizer management methods. At harvest, seeds were collected from individual plants, and the grains were naturally air-dried.

[0102] 3. Determination of proline content in aromatic brown rice

[0103] The proline content in aromatic brown rice was determined according to the method of Bates et al. (1973). The specific steps are as follows: First, weigh 0.3 g of hulled aromatic brown rice, grind it into a powder using liquid nitrogen, and add 4 mL of 3% sulfosalicylic acid solution. Transfer the rice to a glass test tube and heat it in a 100°C water bath for 30 minutes. Then remove the rice and allow it to cool naturally to room temperature. Aspirate 2 mL of the supernatant, then add 2 mL of glacial acetic acid and 2 mL of 3% ninhydrin solution, respectively. Heat the test tube in a 100°C water bath for 30 minutes. After cooling naturally, add 4 mL of toluene, shake thoroughly, and let it stand for 10 minutes to allow for liquid phase separation. Pipette the colored upper layer into a centrifuge tube and centrifuge it at 10,000 rpm for 5 minutes at room temperature. Measure the absorbance at 520 nm, and calculate the proline content using a standard curve in μg / g fresh weight (FW).

[0104] 4. Determination of Pyrroline-5-carboxylic Acid (P5C) Content in Fragrant Brown Rice

[0105] The pyrroline-5-carboxylic acid (P5C) content in aromatic brown rice was determined according to the method of Wu et al. (2009). The specific steps were as follows: First, 0.3 g of dehulled aromatic brown rice was weighed and ground into a powder using liquid nitrogen. Next, the ground powder was transferred to a 15 mL centrifuge tube and 8 mL of 50 mM Tris-HCl buffer (pH 7.6) was added. The tube was centrifuged at 8000 rpm for 20 minutes at 4°C. Subsequently, 375 μL of the supernatant was aspirated, and 500 μL of 10% trichloroacetic acid solution and 125 μL of 0.25% 2-aminobenzaldehyde solution were added in that order. After standing at room temperature for 30 minutes, the tube was centrifuged at 8000 rpm for 10 minutes at room temperature, and the supernatant was aspirated. Finally, the absorbance was measured at 440 nm, and the P5C content was calculated using a standard curve and expressed in μmol / gFW.

[0106] 5. Determination of 2-AP Content in Fragrant Brown Rice

[0107] The 2-AP content of aromatic brown rice was determined according to the method of Huang Zhonglin et al. (2012) with slight modifications. The specific steps are as follows: First, weigh approximately 0.5 g of hulled aromatic brown rice and grind it into a powder using liquid nitrogen. Place the powder in a glass bottle, add 10 mL of chromatographic-grade dichloromethane, seal the bottle, tighten the cap, and shake briefly to mix well before labeling. Place the sample bottle in a water-filled ultrasonic cleaner and sonicate at 40 kHz and 45°C for 40 minutes. After treatment, remove the sample bottle and allow it to cool to room temperature. Add the sample to an appropriate amount of anhydrous sodium sulfite solution, and immediately remove the supernatant using a 1 mL sterile syringe. The supernatant is then filtered through a 0.22 μm organic needle filter (13 mm diameter) into a headspace sample vial. Simultaneously, add 2 μL of 0.0917 g / L 2,4,6-trimethylpyrimidine (TMP) as an internal standard. Gas chromatography-mass spectrometry analysis is then performed immediately. The GC-MS method for determining 2-AP utilizes a Shimadzu GC-MS QP2010 Plus gas chromatography-mass spectrometer, using 2,4,6-trimethylpyrimidine (TMP) as the internal standard. Detailed measurement conditions are as follows: Chromatographic conditions: SH-Rxi-5Sil MS column, 30 m long, 0.25 mm inner diameter, 0.25 μm film thickness. The column temperature program was: 40°C for 1 min, then ramped to 65°C at 2°C / min and held for 1 min; then ramped to 220°C at 10°C / min and held for 5 min. High-purity helium (≥99.999%) was used as the carrier gas. Constant pressure, splitless injection was used, and the injection volume was 2 μL. Mass spectrometry conditions: electron impact (EI) ion source, ion source temperature of 200°C; ionization energy of 70 eV; interface temperature of 250°C; quadrupole temperature of 150°C; full scan mode, scanning mass range of m / z 30-350.

[0108] 6. Data acquisition of particle shape

[0109] Seeds from transgenic and control plants were harvested individually and sun-dried under the same conditions. Fifty full seeds were collected from each plant, and the seed length and width were measured using a multifunction scanner. The thousand-seed weight (the total weight of 1,000 seeds) was also measured. The above measurements were repeated three times, and the data were statistically analyzed using SAS (Statistical Analysis System) software.

[0110] 7. Experimental Results

[0111] 7.1 Analysis of Proline Content in Aromatic Brown Rice

[0112] Since the gene we regulated is closely related to the synthesis of proline, we tested the proline content of proAB74-OE. Figure 1 As shown in the figure, the proline content of brown rice in proAB74-OE was significantly increased compared with that in the control brown rice.

[0113] 7.2 Analysis of Pyrroline-5-carboxylic Acid (P5C) Content in Brown Rice

[0114] By regulating P5C and its related metabolites, we explored their effects on the quality of fragrant rice. Therefore, we tested the P5C content in proAB74-OE. Figure 2 As shown in the figure, compared with the control brown rice, the proAB74-OE brown rice contained higher levels of P5C.

[0115] 7.3 Analysis of 2-acetyl-1-pyrroline (2-AP) Content in Aromatic Brown Rice

[0116] The aroma of rice is a characteristic of the taste quality of rice and an important factor in determining its price. Among them, 2-acetyl-1-pyrroline (2-AP) is considered to be the main aroma component of fragrant rice. Therefore, we tested the 2AP content in proAB74-OE. The results are as follows Figure 3 As shown, compared with the control brown rice, the proAB74-OE brown rice contained higher levels of 2AP.

[0117] 7.4 Seed trait determination

[0118] In order to determine whether these transgenic materials have an impact on the main agronomic traits of rice seeds, we conducted a systematic investigation of the main agronomic traits of seeds of different transgenic lines. The key agronomic traits investigated included grain length, grain width, and 1000-grain weight. Figure 4As shown in the results, there was no significant negative effect on grain length, grain width, and 1000-grain weight of the proAB74-OE line compared to the control brown rice. A comprehensive analysis of these results indicates that transgenic rice has no adverse effects on the agronomic traits of aromatic rice.

[0119] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Application of Escherichia coli proB74 and proA genes in improving the aroma quality of rice, characterized by: The nucleotide sequence of the proB74 gene is shown in SEQ ID NO.2; the nucleotide sequence of the proA gene is shown in SEQ ID NO.

3.

2. The use of the Escherichia coli proB74 and proA genes in improving the aroma quality of rice according to claim 1, characterized in that: The application is: over-expressing proB74 and proA genes in rice to improve the aroma quality of rice.

3. Use of the Escherichia coli proB74 and proA genes in improving the aroma quality of rice according to claim 1 or 2, characterized in that: The method of improving the aroma quality of rice includes increasing the content of aroma substances in rice grains.

4. The use of the Escherichia coli proB74 and proA genes in improving the aroma quality of rice according to claim 3, characterized in that: The rice grain aromatic substance includes 2-acetyl-1-pyrroline.

5. Use of the Escherichia coli proB74 and proA genes in improving the aroma quality of rice according to claim 1 or 2, characterized in that: The rice is aromatic rice; further, the aromatic rice is japonica aromatic rice; further, the japonica aromatic rice is Yunjingyou 14.

6. The use of the Escherichia coli proB74 and proA genes in improving the aroma quality of rice according to claim 2, characterized in that: The overexpression of proB74 and proA genes is achieved by a method comprising the following steps: S1. Insert the chloroplast signal peptide RC2, proB74 and proA gene fragments into the downstream of the ubi promoter of the pRHVc-GFP vector in sequence to obtain the recombinant vector proAB74; S2. The recombinant vector proAB74 is transformed into callus tissue through Agrobacterium-mediated transformation. After screening of resistant callus tissue and plant regeneration, rice overexpressing proB74 and proA genes is obtained.

7. Use of the Escherichia coli proB74 and proA genes in improving the aroma quality of rice according to claim 6, characterized in that: The specific operations of step S1 are: (1) Using rice cDNA as a template, PCR amplification was performed using primers proAB74-1F and proAB74-1R to obtain the chloroplast signal peptide RC2 gene fragment; using Escherichia coli as a template, PCR amplification was performed using primers proAB74-2F and proB-muR and primers proB-muF and proAB74-2R to obtain two fragments of the proB74 gene; and PCR amplification was performed using primers proAB74-3F and proAB74-3R to obtain the proA gene fragment; (2) selecting the vector pRHVc-GFP as the backbone vector for vector construction, BamH I and Hind III as the restriction enzyme sites for vector construction, and connecting the chloroplast signal peptide RC2 gene fragment, the proB74 gene fragments, the proA gene fragment and the backbone vector obtained in step (1) by homologous recombination to obtain the recombinant vector proAB74; The sequences of the primer pairs for PCR amplification are: proAB74-1F: 5'-tatccagatccagtgggatccATGGCCCCCTCCGTGATG-3'; proAB74-1R: 5'-ctggctgtcactcatCATACATCGTATTCTCCCTCCGT-3'; proAB74-2F: 5'-gtatgATGAGTGACAGCCAGACGCTG-3'; proAB74-2R: 5'-ccatttgttccagcatGCCGCCGCCGCCGCCTGAGCCGCCACGGGTAATCAT GTCAT-3'; proAB74-3F: 5'-cggcATGCTGGAACAAATGGGCAT-3'; proAB74-3R: 5'-agcggccgcactagtaagcttTTACGCACGAATGGTGTAATCAC-3'; proB-muF: 5'-ATGCTGCTGACCCGTGCTaATATGGAAGACCGTGAAC-3'; proB-muR: 5'-GTTCACGGTCTTCCATATtAGCACGGGTCAGCAGCAT-3'; The conditions for the PCR amplification reaction were: 95°C for 5 min; 35 cycles of 95°C for 40 s, 58°C for 50 s, and 72°C for 3 min; and 72°C for 10 min.

8. The use of the Escherichia coli proB74 and proA genes in improving the aroma quality of rice according to claim 7, characterized in that: The Agrobacterium is EHA105 Agrobacterium.

9. Use of the method for overexpressing proB74 and proA genes according to any one of claims 6 to 8 in preparing transgenic plants.

10. The use according to claim 9, characterized in that: The application comprises the following steps: cultivating the obtained rice over-expressing proB74 and proA genes into plants.

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