Application of phytoene synthetase gene NtPSY in tobacco aroma improvement

Abstract

The invention relates to application of a phytoene synthetase gene NtPSY of tobacco in tobacco aroma improvement, and belongs to the technical field of molecular biology and biology. The invention relates to application of a tobacco phytoene synthetase gene NtPSY in tobacco aroma improvement, in particular to application of a recombinant plant expression vector containing the tobacco phytoene synthetase gene NtPSY in tobacco aroma improvement. The plant expression vector has the beneficial effects that the plant expression vector is constructed by utilizing the nucleotide sequence of the phytoene synthetase gene NtPSY of the tobacco, the plant expression vector is transformed and introduced into the tobacco by utilizing an agrobacterium-mediated method, and a T3-generation tobacco transgenic plant is obtained through screening and molecular identification of the dipropylamidophos. Through sensory quality evaluation, it is found that the aroma amount and concentration of the single-material tobacco prepared from the transgenic tobacco are remarkably improved. Results show that overexpression of the phytoene synthetase gene NtPSY of the tobacco can effectively improve the aroma quality of the tobacco.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and biotechnology, and specifically relates to a tobacco phytoene synthase gene NtS Application in genetic improvement of tobacco aroma quality. Background Art

[0002] As an important economic crop, the quality and aroma characteristics of tobacco have an important impact on the market value of cigarette products. However, there is a certain degree of lack of tobacco aroma varieties in my country. Although many tobacco varieties have been approved in recent years, the number of varieties that can be planted is still far from enough compared with my country's huge planting area and complex ecological conditions. Among the tobacco varieties currently planted in my country, the three varieties of K326, Yunyan 85 and Yunyan 87 account for 78.13% of the planting area, and the planting area of ​​each variety exceeds 267,000 hectares. This phenomenon of over-reliance on a few varieties has limited the diversity and selectivity of tobacco aroma varieties. In addition, although the quantity and quality of my country's self-bred flue-cured tobacco varieties have been significantly improved, there is still a certain gap in aroma quality compared with some introduced varieties, and the aroma quality needs to be improved.

[0003] Traditional tobacco improvement methods mainly rely on breeding and cultivation techniques, but these methods have certain limitations in improving tobacco aroma quality. In recent years, with the rapid development of genetic engineering technology, improving tobacco quality through genetic engineering has become a new research direction.

[0004] Phytoene synthase (PSY) is a key rate-limiting enzyme in the carotenoid biosynthesis pathway. NtS It has important biological functions in tobacco. Studies have shown that NtS The expression level of the gene directly affects the synthesis of carotenoids, which in turn may affect the accumulation of aroma precursors in tobacco. Carotenoids are not only important pigment components in tobacco, but also produce a variety of aroma compounds through degradation, such as β-damascenone and γ-damascenone, which are important components of tobacco aroma. In addition, NtS Overexpression of the gene in rice, eggplant and apple increased the content of carotenoids, providing a theoretical basis for improving tobacco aroma through genetic engineering.

[0005] However, despite PSY Research on this gene in other plants has been reported, but there are currently no reports on its application in improving aroma in tobacco. Summary of the invention

[0006] The present invention provides a tobacco phytoene synthase gene NtSApplication in tobacco aroma improvement to play NtS Genes have important scientific significance and potential practical value in the application of tobacco aroma improvement.

[0007] The technical solution adopted by the present invention is that tobacco phytoene synthase gene NtS Application in tobacco aroma improvement.

[0008] The tobacco phytoene synthase gene of the present invention NtS The nucleotide sequence is shown in SEQ ID NO:1.

[0009] A recombinant plant expression vector containing tobacco phytoene synthase gene NtS .

[0010] The nucleotide sequence of the recombinant plant expression vector of the present invention is shown in SEQ ID NO:2.

[0011] The invention discloses an application of the recombinant plant expression vector in tobacco aroma improvement.

[0012] The beneficial effects of the present invention are: Phytoene synthase gene NtS The nucleotide sequence was sequenced and a plant expression vector was constructed. The vector was transformed into tobacco using Agrobacterium-mediated method. After screening with bialaphos and molecular identification, T 3 The transgenic tobacco plants were first generated. Sensory quality evaluation revealed that the aroma and concentration of the single-ingredient tobacco produced by the transgenic tobacco were significantly improved. NtS The overexpression of can effectively improve the aroma quality of tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Yes NtS Phenotypic comparison of transgenic plants and non-transgenic plants, with non-transgenic tobacco on the left and transgenic tobacco on the right. PSY Genetic tobacco; Figure 2 Transformed plants NtS Gene PCR detection chart; Figure 3 It is for transfer NtS Real-time fluorescence quantitative PCR results of transgenic plants and non-transgenic plants. DETAILED DESCRIPTION

[0014] Example 1: Tobacco Phytoene Synthase Gene NtS Synthesis Tobacco database obtained through NCBI PSYGene sequence information (NM_001325140.1), the nucleotide sequence of which is shown in SEQ ID NO: 1. The intermediate vector pUC- PSY According to the analysis of the target vector and target gene restriction sites, Nco I and Bst EII has two restriction endonuclease sites, which can be used with restriction endonucleases Nco I and Bst EII to pUC- PSY The plasmid was double-digested with the following reaction system: 10 μL 10×NEBuffer, 4 μL NcoI, 4 μL BstEII, 22 μL plasmid (500 ng / μL), and water was added to 100 μL. The plasmid was first incubated at 37°C for 15 min, then at 60°C for 15 min, and finally at 80°C for 20 min to inactivate the plasmid. The plasmid was then recovered by gel cutting. PSY Gene fragment, tobacco phytoene synthase gene NtS The total length is 1323 bp, the start codon is ATG, the stop codon is TGA, and the nucleotide sequence is shown in SEQ ID NO: 1.

[0015] Embodiment 2: NtS Construction of plant expression vectors for genes according to NtS The ORF sequence of the intermediate vector pUC and the related information of the multiple cloning site are added when designing primers to amplify NtS Gene. NtS The gene was digested with the corresponding restriction endonuclease to recover the small fragment (gene). At the same time, the expression vector pGM3301-CAM was digested with the same restriction endonuclease to recover the large fragment (vector). The recovered large and small fragments were connected using T4 ligase to construct NtS The plant expression vector of the gene has a nucleotide sequence as shown in SEQ ID NO: 2. The recombinant vector was transformed into Escherichia coli, and the plasmid was extracted for PCR and enzyme digestion identification.

[0016] 1. Pick the pUC- NtS DH5α single colony was cultured in 20 mL liquid LB containing 50 mg / L Kan at 37°C and 150 rpm overnight for no more than 16 h. The plasmid was extracted using the SanPrep Column Plasmid DNA Miniprep Kit from Sangon.

[0017] 2. Use restriction endonucleases Nco I and Bst EII to pUC- NtS The plasmid was double-digested, and the 100 μL reaction system was as shown in Table 1: Table 1. 100μL reaction system Reagents volume 10×NEBuffer 10μl I 4μl EII 4 μg Plasmid (500ng / μL) 22μl RNase Free Water up to 100μl First, warm it at 37℃ for 15min, then warm it at 60℃ for 15min, and finally warm it at 80℃ for 20min to inactivate it. Then, cut the gel and recover it. NtS Fragment.

[0018] 3. Use restriction enzymes Nco I and Bst The pGM3301-CAM plasmid was double-digested by EII, and the 100 μL reaction system was as shown in Table 2: Table 2. 100μL reaction system Reagents volume 10×NEBuffer 10μl I 5μl EII 5μg Plasmid (500ng / μL) 33μl RNase Free Water up to 100μl First, incubate at 37°C for 15 min, then at 60°C for 15 min, and finally at 80°C for 20 min to inactivate the fragment. Then, perform gel excision to recover the pGM3301 fragment.

[0019] 4. Ligation of target gene and expression vector The target gene was ligated using T4 ligase NtS The fragment was connected to the expression vector pGM3301, and the 10 μL reaction system was as shown in Table 3: Table 3. 10μL reaction system Reagents volume Carrier 1.5μl Target gene 5.5μl 5×T4 DNA Ligase Buffer 2μg T4 DNA Ligase 1μl RNase Free Water 0μl Incubate at 25°C for 1 hour. After the reaction is complete, store at 4°C.

[0020] Example 3: Application of tobacco phytoene synthase gene NtPSY in tobacco aroma improvement genetic engineering 1. Preparation of genetically transformed engineered bacteria 1. DH5α Escherichia coli competent transformation and PCR detection (1) Thaw 50 μl of DH5α competent E. coli on ice; (2) Use a pipette to draw up 2 μl of the ligation product or recombinant plasmid and add it to 50 μl of DH5α E. coli competent cells; (3) After a 30-min ice bath, heat shock at 42°C for 45 seconds, and an ice bath for 2 minutes. Then, add 500 μl of LB liquid culture medium under sterile conditions; (4) Incubate at 37°C, 200 rpm for 1 hour to allow the cells to recover. Use a pipette to apply 50 μl of the culture medium to LB solid culture medium containing the corresponding antibiotics, and incubate at 37°C for 12-16 hours. (5) Pick a single colony for colony PCR and use Taq enzyme to perform PCR molecular detection on the bacterial solution.

[0021] NtSGene-specific cloning primers are as follows: F: 5'-TGTGGTGCAGGAGAACAGAT-3' R: 5'-TCCTCGCCCTCTGAATTTGT-3' The primer amplification length is 543 bp; the PCR reaction system is shown in Table 4, and the reaction procedure is shown in Table 5: Table 4. PCR reaction system Components volume 10×Taq PCR Buffer 10μl -F 0.5μl -R 0.5μl DNA (single colony) Dipping RNase Free Water up to 20μl Table 5. Reaction procedure

[0022] The bacterial solution with positive PCR results was propagated and mixed with 60% glycerol at a ratio of 1:1 and stored at -80°C for later use.

[0023] 2. Preparation of Agrobacterium ① Take the EHA105 recipient bacteria stored at -70℃ and spread them on the YEP plate containing rifampicin, and invert them at 28℃ for 2-3 days; spread the Helper bacteria on the LB plate without antibiotics, and spread the DH5α containing the recombinant vector on the LB plate containing kanamycin, and invert them at 37℃ overnight.

[0024] ② Pick a single colony of EHA105 and inoculate it into liquid YEP without antibiotics and culture it overnight at 28℃ and 150 rpm; pick a single colony of Help and DH5α containing the recombinant vector and inoculate them into liquid LB without antibiotics and culture them overnight at 37℃ and 150 rpm.

[0025] ③ Pipette 0.4 μL each of Help, DH5α containing the recombinant vector, and 0.6 μL of EHA105 into a centrifuge tube and mix well. Centrifuge at 5000 rpm for 1 min. Discard the supernatant. Pipette 200 μL of liquid YEP without antibiotics to break up the bacteria and mix well. Spread on a solid YEP plate containing rifampicin and kanamycin, and culture upside down at 28°C for 1-2 days.

[0026] ④PCR verification: Take two colonies and culture them at 28℃ overnight with shaking at 150 rpm for no more than 16 hours. Use the "SanPrep Column Plasmid DNA Mini-Extraction Kit" of Sangon to extract the plasmid and perform PCR detection. The reaction system is shown in Table 6, and the reaction procedure is shown in Table 5: Table 6. Reaction system Components volume 10×Taq PCR Buffer 10μl -F 0.5μl -R 0.5μl DNA 2μl RNase Free Water up to 20μl Pick the correct colony and place it in 20 mL YEP liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and culture it overnight at 28°C and 150 rpm. Mix the bacterial solution with 60% glycerol at a ratio of 1:1 and store it at -80°C for later use.

[0027] (II) Transfer NtS Acquisition and Molecular Detection of Genetic Tobacco The gene was transformed into tobacco using Agrobacterium-mediated method as follows: 1. One day before genetic transformation, take the top stretched leaves of tobacco tissue culture seedlings that have been cultured for 2-3 months and cut them into 0.5 cm thick pieces with sterile scissors in a clean bench. 3 The leaf disks were inoculated with the leaves facing upwards in the pre-culture medium and cultured in the dark at 25°C for 24 h; 2. Simultaneously, the activated Agrobacterium was spread on YEP solid medium containing kanamycin and rifampicin resistance and cultured in the dark at 19°C for 24 hours; 3. On the day of genetic transformation, scrape the cultured Agrobacterium, resuspend the Agrobacterium with resuspension solution, and adjust the OD using a spectrophotometer. 600 The concentration of 1 was 1, and the pre-cultured tobacco leaf disc was immersed in the bacterial solution and infected for 10 minutes; 4. Take out the infected leaf disc, place it on sterile filter paper to absorb the excess bacterial solution, and inoculate it on the co-culture medium with the leaf facing up. The culture condition is 20℃ dark culture for 3 days; 5. Transfer the leaf disc after co-cultivation to screening culture 1, replace the fresh culture medium every 14 days, and place it in screening culture medium 2 after green buds grow; 6. After the green seedlings grow out, transfer them to the meso-rooting medium for cultivation. When the transgenic seedlings grow good roots and have 3-4 leaves, plant them in a seedling pot filled with vermiculite and place them in a greenhouse for growth; 7. There are no significant differences in agronomic traits between transformed and non-transformed plants, e.g. Figure 1 As shown, after the transgenic tobacco blooms and bears fruit, the seeds can be harvested when the siliques are completely yellow and about to crack; 8. Harvested Part T 0 The seeds of the first generation were tested by test strips and PCR identification to obtain T 1 The molecular detection results of transgenic plants were as follows Figure 2 As shown, after two generations, we get T 3 The tobacco plants can be used for subsequent aroma identification.

[0028] Embodiment 4: NtS Gene in T 3 Expression in transgenic tobacco and evaluation of tobacco sensory quality During the flowering period of tobacco in the field, the middle leaves of non-transgenic plants and transgenic plants were collected and tested using qRT-PCR technology. NtS Gene expression. The internal reference gene is EF1a , the amplification primers are EF1a-F: 5'-AGCTTCACCTCCCAGGTCATC-3' and EF1a -R: 5'-AGAACGCCTGTCAATCTTGG-3'. NtS The gene-specific detection primers were NtS -F:5'-AGGCAGAGCTTTCCGATGAA-3', NtS -R: 5'-CAATGGGCAAGGTCAGAAGC-3'. -△△Ct Calculation NtS The relative expression of genes. Figure 3 As shown, the leaves of transgenic tobacco plants PSY The gene expression level was significantly upregulated compared with the non-transgenic plants. The aroma volume and concentration of the overexpressed tobacco material were increased by artificial smoking. The sensory quality evaluation is shown in Table 7, the aroma quality evaluation is shown in Table 8, and the tobacco NtS Overexpression of the gene can improve the aroma quality of tobacco.

[0029] Table 7. Sensory quality evaluation Material Name Comprehensive evaluation of sensory quality OE1 The volume and richness of the aroma are increased; the style characteristics are obvious; OE2 The amount of aroma increases, the concentration increases; the strength, concentration, and aroma are all improved OE3 The aroma volume and concentration increase; the smoke characteristics in the middle OE4 The aroma increases, with a sun-dried smoke smell; like drying in the sun, cigar smell Table 8. Aroma quality rating table Material Momentum (20%) Concentration (20%) Aroma volume (20%) Richness (20%) Irritation (10%) Style characteristics (10%) Total score OE1 15 16 17 16 12 14 90 OE2 14 17 18 17 13 15 92 OE3 13 16 17 16 14 16 90 OE4 12 15 16 15 11 17 86 Note: 1. Evaluation indicators and weights Strength (20%): refers to the intensity and strength of tobacco stimulation to the mouth, throat and other parts, reflecting the impact of tobacco; Concentration (20%): describes the intensity of tobacco aroma, which is divided into light, medium, strong, and rich levels; Aroma volume (20%): Indicates the richness and volume of tobacco aroma, divided into different levels such as little, medium, much, and rich; Richness (15%): refers to the layering and diversity of tobacco aroma, reflecting the complexity of the aroma; Irritation (10%): describes the degree of irritation of tobacco to the mouth, throat and other parts, and is divided into different levels such as none, slight, moderate, strong and strong; Style characteristics (15%): refers to the specific aroma style characteristics of tobacco, such as the characteristics of central tobacco, Hunan tobacco feeling, sun-dried tobacco, air-dried tobacco, cigar flavor, etc.

[0030] 2. Scoring Criteria Strength: None (0 points), Slight (1-2 points), Moderate (3-4 points), Strong (5-6 points), Strong (7-8 points), Very Strong (9-10 points), Extremely Strong (11-12 points), Very Strong (13-14 points), Extremely Strong (15-20 points); Strength: light (0 points), relatively light (1-4 points), medium (5-8 points), relatively strong (9-12 points), strong (13-16 points), very strong (17-20 points); Aroma quantity: little (0 points), less (1-4 points), medium (5-8 points), more (9-12 points), more (13-16 points), rich (17-20 points); Richness: single (0 points), relatively simple (1-3 points), with certain levels (4-7 points), relatively rich levels (8-12 points), very rich (13-16 points), extremely rich (17-20 points); Irritation: None (20 points), Mild (15-18 points), Moderate (10-14 points), Strong (5-9 points), Strong (1-4 points), Very strong (0 points); Style characteristics: no obvious characteristics (0 points), with certain characteristic tendencies (1-4 points), with obvious characteristics (5-8 points), with prominent characteristics (9-12 points), highly representative (13-16 points), unique characteristics (17-20 points).

[0031] 3. Scoring Rules (1) An evaluation team consisting of 5 to 7 professional aroma assessors will evaluate the aroma quality of the materials in accordance with the standards such as YC / T 138-1998 “Sensory Evaluation Methods for Tobacco and Tobacco Products”. (2) In a standard fragrance evaluation environment, fragrance evaluators evaluate and score each indicator of the material one by one according to the prescribed steps and requirements. (3) The score of each indicator is assessed by the perfume evaluator within the corresponding score range according to the actual situation, and the average value is taken as the score of the indicator. (4) The total score is 100 points, which is obtained by adding the scores of each indicator multiplied by its weight.

[0032] According to the above technology, the present invention utilizes tobacco phytoene synthase gene NtPSY, Through the Agrobacterium-mediated method, NtS The expression vector was successfully transformed into tobacco and T 3 The results showed that the transgenic tobacco NtS The overexpression of the gene increased significantly, and the results of artificial smoking showed that the aroma volume and concentration of the single-material smoke made from transgenic tobacco were higher than those of non-transgenic tobacco. The above results indicate that the tobacco phytoene synthase gene NtS Overexpression in tobacco can improve the aroma of tobacco.

Claims

1. Tobacco phytoene synthase gene NtS Application in tobacco aroma improvement.

2. The use according to claim 1, wherein the tobacco phytoene synthase gene NtS The nucleotide sequence is shown in SEQ ID NO:

1.

3. A recombinant plant expression vector, characterized in that Tobacco phytoene synthase gene NtS .

4. The nucleotide sequence of the recombinant plant expression vector according to claim 3 is shown in SEQ ID NO:

2.

5. Use of the recombinant plant expression vector as claimed in claim 3 or 4 in improving tobacco aroma.

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