Application of NtKAT2.3 in the Regulation of Ferulic Acid Synthesis in Tobacco

Abstract

This invention belongs to the field of tobacco genetic engineering, specifically involving the application of NtKAT2.3 in the regulation of ferulic acid synthesis in tobacco. The tobacco gene... NtKAT2.3 The nucleotide sequence is shown in SEQ ID No.1. NtKAT2.3 Gene expression levels are negatively correlated with ferulic acid content in tobacco. Preliminary research results indicate that the gene involved in this application... NtKAT2.3 The gene is highly correlated with ferulic acid content in tobacco. Silencing this gene significantly increased ferulic acid content in the resulting tobacco lines. Based on these results, the inventors believe that further in-depth research on this gene could lay a solid technical foundation for improving tobacco quality and developing new varieties.

Description

Technical Field

[0001] This invention belongs to the field of tobacco genetic engineering, specifically involving the application of NtKAT2.3 in the regulation of ferulic acid synthesis in tobacco. Background Technology

[0002] Polyphenolic compounds are important secondary metabolites produced during tobacco growth, playing a crucial role in the formation of tobacco aroma. Therefore, the type and content of tobacco polyphenolic compounds have a significant impact on the aroma quality of tobacco leaves.

[0003] Ferulic acid, as a member of the polyphenolic class, participates in the phenylpropanoid metabolic pathway in tobacco, which is also responsible for the synthesis of key components in tobacco aroma (including phenols, terpenes, and alkaloids). Furthermore, ferulic acid not only participates as an intermediate in the synthesis of coumarin and chlorogenic acid, but can also be converted into CoA esters and caffeic acid, thereby participating in the synthesis of flavonoids and other compounds. In addition, research on lignin biosynthesis has revealed that ferulic acid, as an intermediate metabolite, can be converted into the corresponding CoA esters under the catalysis of 4CL enzyme (4-coumaric acid: coenzyme A ligase), thus participating in lignin synthesis. Since lignin is crucial for the strength and rigidity of plant cell walls and for resisting environmental stressors, the regulation of ferulic acid metabolism is of great significance for plant research. Summary of the Invention

[0004] Given the significant technical importance of ferulic acid metabolism regulation in plant, especially tobacco, quality control, this application aims to provide a method that is highly correlated with ferulic acid content in tobacco. NtKAT2.3 Genes, thus laying a certain technical foundation for the breeding of new tobacco varieties.

[0005] The technical solution adopted in this application is briefly described below.

[0006] The application of NtKAT2.3 in the regulation of ferulic acid synthesis in tobacco, the nucleotide sequence of which is shown in SEQ ID No. 1;

[0007] Correspondingly, genes NtKAT2.3 The encoded ferulic acid synthesis protein NtKAT2.3 consists of 465 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2, wherein amino acids 55-310 and 318-441 are conserved Thiolase N and Thiolase C domains.

[0008] Used for silencing genes NtKAT2.3 The recombinant vector TRV2-NtKAT2.3, using TRV2 as a vector, contains recombinant genes targeting the tobacco ferulic acid synthesis gene. NtKAT2.3 The target sequence.

[0009] The gene NtKAT2.3 In tobacco, it is used to regulate the ferulic acid content; in specific applications... NtKAT2.3 Gene expression levels are negatively correlated with ferulic acid content in tobacco (i.e., by reducing...). NtKAT2.3 (After reducing gene expression levels, NtKAT2.3 protein expression was subsequently reduced, and ferulic acid content in tobacco leaves significantly increased). Gene silencing technology or gene overexpression methods can be used to reduce or increase ferulic acid levels. NtKAT2.3 Gene expression levels, in turn, regulate the ferulic acid content in tobacco leaves by modulating the expression level of the NtKAT2.3 protein.

[0010] Using the tobacco ferulic acid synthesis gene NtKAT2.3 The method for breeding new tobacco varieties, based on transgenic technology, transient expression technology, or genome editing technology, first constructs a system containing... NtKAT2.3 Viral silencing vectors, RNAi interference vectors, overexpression vectors, or genome editing vectors were used to transform tobacco, and new tobacco varieties with increased or decreased ferulic acid content were screened for.

[0011] For example, using virus-induced gene silencing (VIGS) technology to interfere with... NtKAT2.3 Gene expression is silenced, thus allowing for selection to obtain... NtKAT2.3 Gene-silencing new varieties (i.e., new tobacco varieties with increased ferulic acid content); the aforementioned NtKAT2.3 The ferulic acid content was significantly increased in the new gene-silenced tobacco varieties.

[0012] Studies on wheat, Arabidopsis thaliana, and other plants have shown that ferulic acid plays a vital role in plant growth. Therefore, the inventors conducted preliminary research on regulatory genes in tobacco that are associated with the regulation of ferulic acid synthesis. Preliminary results indicate that the genes involved in this application... NtKAT2.3 The gene is highly correlated with ferulic acid content in tobacco. Silencing this gene resulted in a significant increase of over 129% in ferulic acid content in the resulting tobacco lines. Based on these results, the inventors believe that further in-depth research on this gene could lay a solid technical foundation for improving tobacco quality and developing new varieties. Attached Figure Description

[0013] Figure 1 This is a comparative phenotype of typical plants transformed with the TRV2-PDS, TRV2-GFP, and TRV2-NtKAT2.3 vectors of this invention.

[0014] Figure 2 Compared with control plants NtKAT2.3The relative expression level of the NtKAT2.3 gene in gene-silenced plants;

[0015] Figure 3 This is a comparison of ferulic acid content in control tobacco leaves and virus-induced gene-silenced tobacco leaves. Detailed Implementation

[0016] The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the experimental background of some embodiments is briefly described below.

[0017] Biomaterials:

[0018] Tobacco Benedict, a commonly used tobacco material in the prior art, was planted at the Zhengzhou Tobacco Institute planting base in the following examples. Seedlings were raised in seedling pots and transplanted two weeks after germination. They were then planted in plastic pots (10cm×10cm) and managed with fertilizer and water at 22℃ under 16h light / 8h dark conditions.

[0019] VIGS (Virus Induced Gene Silencing) is a commonly used virus-mediated gene silencing technique in the prior art. TRV2 (tobacco rattlevirus, TRV), involved in the following examples, is a commonly used viral vector that is publicly available. The applicant and inventors, as professional research institutions and researchers, have long maintained a copy of the vector. This vector carries a kanamycin selection marker and a 35S promoter, as well as multiple cloning sites such as EcoRI and BamHI, and can be used to carry and transform exogenous genes.

[0020] Experimental reagents:

[0021] LB liquid medium: Each liter contains 10 g of bacterial peptone, 10 g of sodium chloride (NaCl), and 5 g of yeast extract.

[0022] YEB liquid medium: Each liter contains 5g beef extract, 5g bacterial peptone, 5g sucrose, 1g yeast extract, and 2 mL 1M magnesium sulfate (MgSO4).

[0023] 1M 2-(N-morpholine) ethanesulfonic acid (MES) stock solution: Dissolve in ddH2O, filter and sterilize, store at -20℃ for later use;

[0024] 200 mM Acetosyringone (As) stock solution: dissolved in dimethyl sulfoxide (DSMO), stored at -20°C for later use;

[0025] MMA (100 mL): 1 mL (1 M) MgCl2, 1 mL (1 M, pH5.6) MES, 75 μL (200 mM) As.

[0026] Before using the above culture medium, sterilize it at 121°C for 15 minutes. Example

[0027] Based on previous analysis of tobacco secondary metabolites, and considering the crucial role of ferulic acid in tobacco quality improvement, this study referenced ferulic acid regulatory gene sequences in other plants and used the applicant's unpublished tobacco genome database as a foundation. VIGS technology was then employed to analyze ferulic acid synthesis-related genes in tobacco. NtKAT2.3 The gene function was further validated. A brief summary of the relevant experimental procedures is as follows.

[0028] (I) Targeted silencing sequence PCR

[0029] Based on previous research on the tobacco genome and related... NtKAT2.3 Gene sequence analysis was performed, and specific coding sequences were selected as target fragments for subsequent gene silencing vector construction. The primer sequences for PCR amplification were designed as follows:

[0030] NtKAT2.3-F:5'-TATTTCTTGGTCCTCTTT-3',

[0031] NtKAT2.3-R:5'-GTGTTCATTCCTTCGT-3';

[0032] During PCR amplification, tobacco K326 leaves were used as samples to extract total RNA and reverse transcribe it to prepare cDNA as a template for PCR amplification.

[0033] PCR amplification was performed using the primers designed above and the prepared cDNA template;

[0034] The PCR amplification program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 2 min, 36 cycles, and 72℃ final extension for 5 min.

[0035] After detecting the PCR amplification products by agarose gel electrophoresis, the amplification products were recovered and purified for later use.

[0036] It should be noted that the tobacco mentioned NtKAT2.3The gene consists of 1765 bases, and its base sequence is shown in SEQ ID No. 1, as follows:

[0037]

[0038] Correspondingly, tobacco NtKAT2.3 The gene encodes the tobacco ferulic acid synthesis protein NtKAT2.3, which consists of 465 amino acids. The amino acid sequence is shown in SEQ ID No. 2, and is as follows:

[0039] MEKAIERQRVLLEHLQPIRSSSHHTHDQSSSLTPSICLAGDSAAYRRTAAFDDDVVIVAAYRTAICKAKRGGFKDTLADDDLLAPVLKAVVEKTNLSPNEVGDIVVGTVLAPGSLRA MECRMAAFYAGFPETVPIRTVNRQCSSGLQAVADVAASIKAGFYDIGIGAGLELMTVDNIGRVQQVNPKVDAFAQARDCLLPMGITSENVAQRFGVTRLEQDQAAVISHQRAAAATA SGKFKDEIIPVLTKIVDPQTRREKPVVISVDDGIRPNTNLTNLAKLKPAFKSDGTTTAGTASQVSDGAAAVLLMKRSVAMQKGLPILGVFRSFAAVGVDPAVMGIGPAVAIPAAVK SAGLELDNIDLFEINEAFASQFVYCRKKLNLDSEKVNVNGGAMALGHPLGATGARCVATLLHEMKRRGKDCRFGVISMCIGSGMGAAAVFERGDAVDDLCNARVNNNNNFLSKDAK.

[0040] (II) Recombination and construction of the TRV2-NtKAT2.3 vector

[0041] The PCR amplification products and TRV2 empty vector recovered in step (I) were digested with EcoRI and BamHI, respectively, and the digestion products were recovered.

[0042] Then, the recovered enzyme digestion products were ligated using T4 DNA ligase;

[0043] Next, the ligation product was transformed into competent E. coli DH5α cells; after transformation, the transformation product was plated on LB solid medium containing 50 mg / L Kan and incubated overnight at 37°C for screening.

[0044] Positive transformed colonies were selected for amplification, identification by bacterial PCR, and sequencing verification to ensure correct plasmid recombination. The correctly identified plasmid vector was named TRV2-NtKAT2.3.

[0045] For related procedures, please refer to the existing routine molecular biology techniques or the instructions for relevant reagents and kits; further details will not be provided.

[0046] It should also be noted that, referring to existing technologies and the above process, the inventors simultaneously prepared recombinant plasmid vectors TRV2-GFP and TRV2-PDS as controls for subsequent experimental verification.

[0047] (III) Transformation of Agrobacterium and preparation of infection solution

[0048] The positive clone plasmids TRV2-GFP (vector control), TRV2-PDS (VIGS silencing efficiency control), and TRV2-NtKAT2.3 prepared in step (II) above were transformed into Agrobacterium GV3101 competent cells using electroporation transformation. The correctly transformed recombinant strains were screened by YEB plates (containing 50 mg / L Kan + 50 mg / L Rif) and incubated upside down at 28°C for 2 days (colony PCR was used to verify that the transformation was correct).

[0049] The correctly transformed positive recombinant Agrobacterium was inoculated into 5 mL of YEB liquid medium (containing 50 mg / L Kan and 50 mg / L Rif) and cultured overnight at 28°C and 250 rpm. Then, 50 μL of the overnight culture was transferred to 50 mL of YEB liquid medium (containing 50 mg / L Kan) and cultured until OD... 600 =Approximately 1.0, centrifuged at 4000g for 5 min, collected the bacterial cells, and resuspended the precipitated bacterial cells in MMA (1 mL (1 M) MgCl2, 1 mL (1 M, pH 5.6) MES, 75 μL (200 mM) As), and adjusted the OD. 600 =Approximately 1.0; after standing at room temperature (approximately 25℃) for 3 hours, use this as the inoculum for subsequent conversion.

[0050] (iv) Instantaneous conversion and detection

[0051] Using leaves of 3-4 weeks old tobacco plants as experimental material, the transfection solution (infection solution) prepared in step (III) was injected into the tobacco leaves using a 1 mL syringe. The injected tobacco plants were then cultured in an artificial incubator to observe phenotypic changes.

[0052] Changes in tobacco phenotype 3 weeks after injection are as follows Figure 1 As shown, Agrobacterium containing TRV2-PDS caused bleaching in the new leaves of the plant, indicating successful infection. This result demonstrates that transient transformation can successfully silence the corresponding gene.

[0053] Further utilize qRT-PCR to NtKAT2.3Gene expression levels were detected. The results are as follows: Figure 2 As shown in the figure, it can be seen that compared to the control plants, the NtKAT2.3 gene-silenced plants showed higher levels of... NtKAT2.3 The expression level of [a gene] decreased significantly, by approximately 56.80%. This result also indicates that the gene silencing effect was good.

[0054] Further analysis of the ferulic acid content showed that the results were as follows: Figure 3 As shown, it can be seen that: NtKAT2.3 Ferulic acid content increased by 129.32% in gene-silenced plants. This result indicates that the NtKAT2.3 gene is a key negative regulator of ferulic acid synthesis in tobacco. Further in-depth research based on this gene can lay a solid genetic resource foundation for the breeding of new tobacco varieties.

Claims

1. Tobacco genes NtKAT2.3 Its application in the regulation of ferulic acid synthesis in tobacco is characterized by... In application, gene silencing technology is used to reduce... NtKAT2.3 Gene expression levels, in turn, regulate the expression of NtKAT2.3 protein in tobacco, thereby increasing and controlling the ferulic acid content in tobacco leaves; The tobacco gene NtKAT2.3 The nucleotide sequence is shown in SEQ ID No.

1.

2. Utilizing tobacco genes NtKAT2.3 The method for cultivating tobacco varieties is characterized by, First, construct a structure containing NtKAT2.3 A viral silencing vector for the gene was then used to transform tobacco, and tobacco varieties with increased ferulic acid content were screened for. The tobacco gene NtKAT2.3 The nucleotide sequence is shown in SEQ ID No. 1.

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