Tobacco ferulic acid synthesis gene Ntkat2.1 and application thereof

Abstract

The present invention belongs to the field of tobacco genetic engineering, and specifically relates to a tobacco ferulic acid synthesis gene NtKAT2.1 The tobacco ferulic acid synthesis gene NtKAT2.1 The nucleotide sequence is shown in SEQ ID No.1. NtKAT2.1 The gene expression level is negatively correlated with the ferulic acid content in tobacco. Preliminary research results show that the NtKAT2.1 The gene is highly correlated with ferulic acid content in tobacco. Silencing the gene significantly increased ferulic acid content in the tobacco strain by 127.45%. Based on this result, the inventors believe that further research on this gene will lay a solid technical foundation for improving tobacco quality and developing new varieties.

Description

Technical Field

[0001] The present invention belongs to the field of tobacco genetic engineering, and specifically relates to a tobacco ferulic acid synthesis gene NtKAT2.1 and its applications. Background Art

[0002] Polyphenols are an important secondary metabolite produced during tobacco growth and play a key role in the formation of tobacco aroma. Therefore, the type and content of tobacco polyphenols have a significant impact on the aroma quality of tobacco leaves.

[0003] Ferulic acid, a member of the polyphenolic group, participates in the phenylpropanoid metabolic pathway in tobacco, which is also responsible for the synthesis of key components of tobacco aroma, including phenols, terpenoids, and alkaloids. Furthermore, ferulic acid not only serves as an intermediate in the synthesis of coumarins and chlorogenic acid but can also be converted into CoA esters and caffeic acid, thereby participating in the synthesis of compounds such as flavonoids. Furthermore, research on lignin biosynthesis has revealed that ferulic acid, as an intermediate metabolite, can be converted into its corresponding CoA ester under the catalysis of the enzyme 4CL (4-coumaric acid:coenzyme A ligase), thereby participating in the synthesis of lignin. Because lignin is crucial for plant cell wall strength and rigidity, as well as for resistance to environmental stress, the regulation of ferulic acid metabolism is of great significance to plant research. Summary of the Invention

[0004] In view of the important technical significance of ferulic acid metabolism regulation in plants, especially tobacco quality regulation, the present application aims to provide a method for regulating ferulic acid content in tobacco. NtKAT2.1 genes, thus laying a certain technical foundation for the cultivation of new tobacco varieties.

[0005] The technical solutions adopted in this application are briefly introduced as follows.

[0006] Tobacco ferulic acid synthesis gene NtKAT2.1 , the nucleotide sequence of which is shown in SEQ ID No. 1;

[0007] Correspondingly, ferulic acid synthesis gene NtKAT2.1 The encoded ferulic acid synthesis protein NtKAT2.1 consists of 464 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2, wherein the amino acids at positions 54-309 and 318-440 are conserved Thiolase N and Thiolase C domains.

[0008] Used to silence tobacco ferulic acid synthesis genes NtKAT2.1The recombinant vector TRV2-NtKAT2.1, which uses TRV2 as a vector and recombines the gene for tobacco ferulic acid synthesis NtKAT2.1 targeting sequence.

[0009] The tobacco ferulic acid synthesis gene NtKAT2.1 It is used in tobacco to regulate the content of ferulic acid in tobacco; in specific applications, NtKAT2.1 Gene expression levels were negatively correlated with ferulic acid content in tobacco (i.e., by decreasing NtKAT2.1 After the gene expression level was reduced and the expression of NtKAT2.1 protein was reduced, the ferulic acid content in tobacco leaves was significantly increased); by using gene silencing technology or gene overexpression method, by reducing or increasing NtKAT2.1 The gene expression level then regulates the ferulic acid content in tobacco leaves by regulating the expression level of tobacco NtKAT2.1 protein.

[0010] Utilizing the tobacco ferulic acid synthesis gene NtKAT2.1 The new tobacco variety breeding method is based on transgenic technology, transient expression technology or genome editing technology, first constructing NtKAT2.1 Gene virus-induced silencing vectors, RNAi interference vectors, overexpression vectors or genome editing vectors are then transformed into tobacco, and new tobacco varieties with increased or decreased ferulic acid content are screened;

[0011] For example, using virus-induced gene silencing (VIGS) technology to interfere with NtKAT2.1 The expression of the gene is silenced and then screened to obtain NtKAT2.1 A new gene-silenced tobacco variety (i.e., a new tobacco variety with increased ferulic acid content); NtKAT2.1 The ferulic acid content in gene-silenced tobacco varieties was significantly increased.

[0012] Studies based on wheat, Arabidopsis, and wheat have shown that ferulic acid plays a very important 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. The preliminary research results show that the NtKAT2.1 The gene is highly correlated with ferulic acid content in tobacco. Silencing the gene significantly increased ferulic acid content in the tobacco strain by 127.45%. Based on this result, the inventors believe that further research on this gene will lay a solid technical foundation for improving tobacco quality and developing new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a comparison diagram of typical plant phenotypes of the tobacco TRV2-PDS, TRV2-GFP and TRV2-NtKAT2.1 vector transformation groups of the present invention;

[0014] Figure 2 Compared with the control plants NtKAT2.1 Relative expression level of NtKAT2.1 gene in gene silenced plants;

[0015] Figure 3 Comparison results of ferulic acid content in control tobacco leaves and virus-induced gene-silencing tobacco leaves. DETAILED DESCRIPTION

[0016] The present application will be further explained below with reference to the following examples. Before introducing the specific examples, a brief description of the experimental background of some of the following examples is given below.

[0017] Biomaterials:

[0018] Nicotiana benthamiana is a commonly used tobacco material in the prior art. The tobacco involved in the following examples was planted at the Zhengzhou Tobacco Institute planting base. The seedlings were raised in seedling pots, transplanted two weeks after germination, and planted in plastic pots (10 cm × 10 cm). Daily fertilizer and water management was carried out under the conditions of 22°C, 16h light / 8h dark.

[0019] VIGS (Virus Induced Gene Silencing) is a commonly used virus-mediated gene silencing technology in the prior art. TRV2 (Tobacco rattle virus viral vector, TRV) used in the following examples is a commonly used viral vector and is publicly available. As professional research institutions and researchers, the applicants and inventors have long-term access to this vector. This vector carries a kanamycin selection marker and a 35S promoter, as well as multiple cloning sites such as EcoRI and BamHI, making it suitable for carrying and transforming exogenous genes.

[0020] Experimental reagents:

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

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

[0023] 1M 2-(N-morpholino)ethanesulfonic acid (MES) stock solution: Dissolve in ddH2O, filter sterilize, and store at -20°C until use;

[0024] 200 mM acetosyringone (As) stock solution: Dissolve in dimethyl sulfoxide (DSMO) and store at -20°C until use;

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

[0026] Before use, the above culture medium was sterilized at 121°C for 15 min. Example

[0027] Based on the analysis of tobacco secondary metabolites in the early stage, it was considered that ferulic acid plays a very important role in tobacco quality improvement. Therefore, referring to the ferulic acid regulatory gene sequences in other plants in the prior art and based on the tobacco genome database that the applicant has not disclosed, the VIGS technology was used to analyze the ferulic acid synthesis-related genes in tobacco. NtKAT2.1 The gene function was further verified. The relevant experimental process is briefly described below.

[0028] (I) Targeted silencing sequence PCR

[0029] Based on the previous research on tobacco genome and related NtKAT2.1 Gene sequence analysis was performed to select specific coding sequences as target fragments for subsequent gene silencing vector construction. The specific primer sequences for PCR amplification were designed as follows:

[0030] NtKAT2.1-F:5'-ACGAAGTAGAAAACTAGC- 3',

[0031] NtKAT2.1-R:5'-TATAAAGCATACCATGAA- 3';

[0032] During PCR amplification, tobacco K326 leaves were used as samples, total RNA was extracted and reverse transcribed to prepare cDNA as a template for PCR amplification;

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

[0034] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 2 min, and after 36 cycles, complete extension at 72°C for 5 min.

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

[0036] It should be noted that the tobacco NtKAT2.1 The gene comprises 1768 bases, the base sequence of which is shown in SEQ ID No. 1, as follows:

[0037]

[0038] Correspondingly, tobacco NtKAT2.1 The tobacco ferulic acid synthesis protein NtKAT2.1 encoded by the gene comprises 464 amino acids, and the amino acid sequence is shown in SEQ ID No. 2, which is as follows:

[0039] MEKAIERQRVLLQHLRPSQTSSSLENIESSIAASVCSAGDSAAYQRTSVFGDDVVIVAAYRTPLCKAKRGGFKDTYPDDLLAPVLKALMEKTNVSPSEIGDIVVGTVLAPGSQRAS ECRMAAFYAGFPETVPVRTVNRQCSSGLQAVADVAAAIKAGFYDIGIGAGLESMTTNPMAWEGSVNPKVKMMAQAQDCLLPMGITSENVAHRFGVTRQEQDQAAVDSHRKAAAASA SGKFKDEIIPVPTKIVDPKTGDETPVTISVDDGIRPNASVSDLAKLKPVFKKSGTTTAGNSSQVTDGAGAVLLMKRSIAMQKGLPILGVFRTFAAVGVDPAIMGIGPAVAIPAAVK SAGLELEDIDLFEINEAFASQYVYCRKKLELDPEKINVNGGAMALGHPLGATGARCVATLLHEMKRRGKDCRFGVVSMCIGTGMGAAAVFERGDSCDELCNARKIGSHNLLSKDAL.

[0040] (II) Recombinant construction of TRV2-NtKAT2.1 vector

[0041] The PCR amplification product and TRV2 empty vector recovered in step (1) were double-digested with EcoRI and BamHI, and the digestion products were recovered;

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

[0043] Then, the ligation product was transformed into E. coli competent DH5α cells. After transformation, the transformation product was spread on LB solid medium containing 50 mg / L Kan and cultured at 37°C overnight for screening.

[0044] Positive transformed colonies were selected for amplification and bacterial liquid PCR identification, as well as sequencing verification to ensure that the plasmid recombination was correct. The correctly identified plasmid vector was named: TRV2-NtKAT2.1.

[0045] For related operations, please refer to existing conventional molecular biology techniques or the instructions of related reagents and kits, and no further details will be given.

[0046] It should also be noted that, with reference to the prior art 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 of TRV2-GFP (vector control), TRV2-PDS (VIGS silencing efficiency control) and TRV2-NtKAT2.1 prepared in the above step (ii) were transformed into Agrobacterium GV3101 competent cells respectively by electroporation transformation method, and the correctly transformed recombinant strains were screened by inverted culture on YEB plates (containing 50 mg / L Kan + 50 mg / L Rif) at 28°C for 2 days (colony PCR detection was also used to verify the correct transformation).

[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), cultured overnight at 28°C and 250 rpm, and then 50 μL of overnight culture was transferred to 50 mL of YEB liquid medium (containing 50 mg / L Kan) and cultured until the OD 600 = 1.0, centrifuge at 4000g for 5 min, collect the cells, and resuspend the precipitated cells in MMA (1 mL (1 M) MgCl2, 1 mL (1 M, pH 5.6) MES, 75 μL (200 mM) As) to adjust the OD 600 = about 1.0; after standing at room temperature (about 25℃) for 3 hours, use this as the infection solution for subsequent transformation.

[0050] (IV) Instantaneous conversion and detection

[0051] Use 3-4 week-old Nicotiana benthamiana leaves as experimental materials. Use a 1 mL syringe to inject the transfection solution (infection solution) prepared in step (3) into the tobacco leaves. After injection, continue to culture the tobacco leaves in an artificial incubator to observe phenotypic changes.

[0052] The phenotypic changes of tobacco 3 weeks after injection are as follows Figure 1 As shown in Figure 2, the new leaves of the plants infected with TRV2-PDS showed bleaching, indicating successful infection. This result also indicates that transient transformation can successfully achieve corresponding gene silencing.

[0053] Further qRT-PCR was used to NtKAT2.1The gene expression levels were detected. Figure 2 As shown. It can be seen that compared with the control plants, the NtKAT2.1 gene silenced plants NtKAT2.1 The expression level of α-terminal locus was significantly decreased, by about 37.83%. This result also indicates that the gene silencing effect is good.

[0054] Further test results of ferulic acid content showed that Figure 3 As shown, it can be seen that: NtKAT2.1 Ferulic acid content increased by 127.45% in gene-silenced plants. This result indicates that the NtKAT2.1 gene is a key negative regulator of ferulic acid biosynthesis in tobacco. Further research on this gene will lay a solid foundation for the development of new tobacco varieties.

Claims

1. Tobacco ferulic acid synthesis gene NtKAT2.1 The invention is applied in tobacco, characterized in that: When applied, gene silencing technology is used to reduce NtKAT2.1 The gene expression level then increases the ferulic acid content in tobacco leaves by regulating the expression level of tobacco NtKAT2.1 protein; The tobacco ferulic acid synthesis gene NtKAT2.1 The nucleotide sequence is shown in SEQ ID No.

1.

2. Utilizing tobacco ferulic acid synthesis genes NtKAT2.1 The tobacco variety breeding method is characterized in that: First build the NtKAT2.1 The virus-induced silencing vector of the gene is then used to transform tobacco, and tobacco varieties with increased ferulic acid content are screened; The tobacco ferulic acid synthesis gene NtKAT2.1 The nucleotide sequence is shown in SEQ ID No.1.

Images