Application of NtKAT2.3 in regulation and control of tobacco ferulic acid synthesis

Abstract

The invention belongs to the field of tobacco gene engineering, and particularly relates to application of NtKAT2.3 in tobacco ferulic acid synthesis regulation. The nucleotide sequence of the tobacco gene NtKAT2.3 is as shown in SEQ ID No.1 (Sequence Identifier Number 1). The expression quantity of the NtKAT2.3 gene is in negative correlation with the content of ferulic acid in tobacco. Preliminary research results show that the NtKAT2.3 gene involved in the invention is highly related to the content of the ferulic acid in tobacco, and after the gene is silenced, the content of the ferulic acid in a tobacco strain after gene silencing is obviously improved. Based on the result, the inventor believes that on the basis of further deep research on the gene, a good technical foundation is expected to be laid for tobacco quality improvement and further new variety cultivation.

Description

Technical Field

[0001] The invention belongs to the field of tobacco genetic engineering, and specifically relates to the application of NtKAT2.3 in the regulation of tobacco ferulic acid synthesis. Background Art

[0002] Polyphenols are an important type of secondary metabolites in the growth process of tobacco, and they play a key role in the formation of tobacco aroma. Therefore, the type and content of tobacco polyphenols have a very important influence on the aroma quality of tobacco leaves.

[0003] As a member of the polyphenolic compounds, ferulic acid participates in the phenylpropanoid metabolic pathway in tobacco, which is also responsible for the synthesis of key components in tobacco aroma (including phenols, terpenoids, and alkaloids). In addition, ferulic acid not only participates in the synthesis of coumarin and chlorogenic acid as an intermediate, but can also be converted into CoA esters and caffeic acid, and then participate in the synthesis of compounds such as flavonoids. In addition, in the process of studying the biosynthesis of lignin, it was found that ferulic acid, as an intermediate metabolite, can be converted into the corresponding CoA ester under the catalysis of 4CL enzyme (4-coumaric acid: coenzyme A ligase), and then participate in the synthesis of lignin. Since lignin is very important for the strength and rigidity of plant cell walls, as well as for resisting environmental adversity, the regulation of ferulic acid metabolism is also of great significance to plant research. Summary of the invention

[0004] In view of the fact that ferulic acid metabolism regulation has very important technical significance in plant, especially tobacco quality regulation, the purpose of this application is to provide a method highly correlated with the 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 introduced as follows.

[0006] NtKAT2.3 is used in regulating tobacco ferulic acid synthesis, and the nucleotide sequence of NtKAT2.3 is shown in SEQ ID No. 1; Correspondingly, gene NtKAT2.3 The encoded ferulic acid biosynthesis protein NtKAT2.3 consists of 465 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2, wherein the amino acids at positions 55-310 and 318-441 are conserved Thiolase N and Thiolase C domains.

[0007] For silencing genes NtKAT2.3 The recombinant vector TRV2-NtKAT2.3, which uses TRV2 as a vector and recombines a gene for the synthesis of ferulic acid in tobacco NtKAT2.3 Targeting sequence.

[0008] The gene NtKAT2.3 It is used in tobacco to regulate the ferulic acid content in tobacco; in specific applications, NtKAT2.3 The gene expression level was negatively correlated with the ferulic acid content in tobacco (i.e., by reducing NtKAT2.3 After reducing the expression of NtKAT2.3 protein, the ferulic acid content in tobacco leaves increased significantly); using gene silencing technology or gene overexpression method, by reducing or increasing NtKAT2.3 The gene expression level then regulates the ferulic acid content in tobacco leaves by regulating the expression level of tobacco NtKAT2.3 protein.

[0009] Utilizing the tobacco ferulic acid synthesis gene NtKAT2.3 The method for breeding new tobacco varieties is based on transgenic technology, transient expression technology or genome editing technology. First, construct NtKAT2.3 Gene virus-induced silencing vectors, RNAi interference vectors, overexpression vectors or genome editing vectors are then used to transform tobacco, and new tobacco varieties with increased or decreased ferulic acid content are screened; For example, using virus-induced gene silencing (VIGS) technology to interfere with NtKAT2.3 The expression of the gene is silenced and then screened to obtain NtKAT2.3 A new gene-silencing variety (i.e., a new tobacco variety with increased ferulic acid content); NtKAT2.3 The ferulic acid content in gene-silenced tobacco new varieties was significantly increased.

[0010] Based on studies on wheat, Arabidopsis, and wheat, it is shown that ferulic acid has a very important use 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 NtKAT2.3 The gene is highly correlated with the ferulic acid content of tobacco. After silencing the gene, the ferulic acid content in the tobacco strain can increase by more than 129%, which is a very significant improvement. Based on this result, the inventors believe that further in-depth research on the gene is expected to lay a good technical foundation for improving tobacco quality and further breeding of new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a comparison diagram of typical plant phenotypes of the tobacco TRV2-PDS, TRV2-GFP and TRV2-NtKAT2.3 vector transformation groups of the present invention; Figure 2 Compared with the control plants NtKAT2.3 Relative expression level of NtKAT2.3 gene in gene silenced plants; Figure 3Comparison results of ferulic acid content in control tobacco leaves and virus-induced gene silencing tobacco leaves. DETAILED DESCRIPTION

[0012] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly introduced as follows.

[0013] Biomaterials: Nicotiana benthamiana is a common tobacco material in the prior art. The tobacco involved in the following embodiments was planted at the Zhengzhou Tobacco Institute planting base, and the seedlings were raised in seedling pots. The seedlings were transplanted two weeks after germination and planted in plastic pots (10 cm×10 cm). Daily fertilizer and water management was performed under the conditions of 22°C, 16h light / 8h dark. VIGS (Virus Induced Gene Silencing) is a commonly used virus-mediated gene silencing technology in the prior art. TRV2 (tobacco rattlevirus, TRV) involved in the following embodiments is a commonly used viral vector and can be obtained from public channels. As a professional research institution and professional scientific researcher, the applicant and the inventor have long-term preservation of the vector. The vector carries a kanamycin selection marker and a 35S promoter, as well as multiple cloning sites such as EcoR I and BamH I, and can be used to carry and transform exogenous genes.

[0014] Experimental reagents: LB liquid medium: each liter contains 10 g bacterial peptone, 10 g sodium chloride (NaCl), and 5 g yeast extract; YEB liquid medium: Each liter contains 5g beef extract, 5g bacterial peptone, 5g sucrose, 1g yeast extract, and 2mL 1M magnesium sulfate (MgSO4); 1M 2-(N-morpholino)ethanesulfonic acid (MES) stock solution: ddH 2 O, sterilize by filtration, and store at -20℃ until use; 200 mM acetosyringone (Acetosyringone, As) stock solution: dissolved in dimethyl sulfoxide (DSMO) and stored at -20°C for future use; MMA (100 mL): 1 mL (1 M) MgCl2, 1 mL (1 M, pH5.6) MES, 75 μL (200 mM) As.

[0015] Before use, the above culture medium was sterilized at 121°C for 15 min. Example

[0016] Based on the analysis of tobacco secondary metabolites in the early stage, 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 not disclosed by the applicant, the VIGS technology was used to analyze the ferulic acid synthesis-related genes in tobacco. NtKAT2.3 The gene function was further verified. The relevant experimental process is briefly described as follows.

[0017] 1. Targeted silencing sequence PCR Based on the previous studies on tobacco genome and related NtKAT2.3 Gene sequence analysis, select specific coding sequences as target fragments for subsequent gene silencing vector construction. The specific primer sequences for PCR amplification are designed as follows: NtKAT2.3-F:5'-TATTTCTTGGTCCTCTTT-3', NtKAT2.3-R:5'-GTGTTCATTCCTTCGT-3'; 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; Using the above designed primers and prepared cDNA template, PCR amplification is performed; 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; After the PCR amplification product is detected by agarose gel electrophoresis, the amplification product is recovered and purified for later use.

[0018] It should be noted that the tobacco NtKAT2.3 The gene comprises 1765 bases, the base sequence of which is shown in SEQ ID No. 1, and is as follows:

[0019] Correspondingly, tobacco NtKAT2.3 The tobacco ferulic acid synthesis protein NtKAT2.3 encoded by the gene includes 465 amino acids, and the amino acid sequence is shown in SEQ ID No. 2, which is as follows: MEKAIERQRVLLEHLQPIRSSSHHTHDQSSSLTPSICLAGDSAAYRRTAAFDDDVVIVAAYRTAICKAKRGGFKDTLADDDLLAPVLKAVVEKTNLSPNEVGDIVVGTVLAPGSLRA MECRMAAFYAGFPETVPIRTVNRQCSSGLQAVADVAASIKAGFYDIGIGAGLELMTVDNIGRVQQVNPKVDAFAQARDCLLPMGITSENVAQRFGVTRLEQDQAAVISHQRAAAATA SGKFKDEIIPVLTKIVDPQTRREKPVVISVDDGIRPNTNLTNLAKLKPAFKSDGTTTAGTASQVSDGAAAVLLMKRSVAMQKGLPILGVFRSFAAVGVDPAVMGIGPAVAIPAAVK SAGLELDNIDLFEINEAFASQFVYCRKKLNLDSEKVNVNGGAMALGHPLGATGARCVATLLHEMKRRGKDCRFGVISMCIGSGMGAAAVFERGDAVDDLCNARVNNNNNFLSKDAK.

[0020] (II) Recombinant construction of TRV2-NtKAT2.3 vector The PCR amplification product and TRV2 empty vector recovered in step (i) are double-digested with EcoRI and BamHI, respectively, and the digestion products are recovered; Then, the recovered digestion products were ligated using T4 DNA ligase; Then, the ligation product was transformed into E. coli competent DH5α cells; after the transformation was completed, the transformation product was spread on LB solid medium containing 50 mg / L Kan and cultured at 37°C overnight for screening; 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, and the correctly identified plasmid vector was named: TRV2-NtKAT2.3.

[0021] For related operations, please refer to the existing conventional molecular biology techniques or the instructions of related reagents and kits, which will not be described in detail.

[0022] It should also be noted that, referring 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.

[0023] 3. Transformation of Agrobacterium and preparation of infection fluid The positive clone plasmids of TRV2-GFP (vector control), TRV2-PDS (VIGS silencing efficiency control) and TRV2-NtKAT2.3 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 YEB plates (containing 50 mg / L Kan + 50 mg / L Rif) and inverted culture at 28°C for 2 days (colony PCR detection was also used to verify the correct transformation).

[0024] 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 50uL of the overnight culture was transferred to 50 mL of YEB liquid medium (containing 50 mg / L Kan) and cultured until OD 600 = 1.0, centrifuge at 4000g for 5 min, collect the cells, and then wash the precipitated cells with MMA (1 mL (1 M) MgCl 2 , 1 mL (1 M, pH 5.6) MES, 75 μL (200 mM) As) and resuspend. 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.

[0025] 4. Instantaneous conversion and detection 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 (iii) into the tobacco leaves. After injection, continue to culture the tobacco leaves in an artificial incubator to observe phenotypic changes.

[0026] The phenotypic changes of tobacco 3 weeks after injection are shown in Figure 2. Figure 1 As shown in the figure, it can be seen that the new leaves of the plants infected with TRV2-PDS by Agrobacterium tumefaciens have bleaching phenomenon, indicating that the infection is successful. In other words, this result shows that transient transformation can successfully achieve corresponding gene silencing.

[0027] Further qRT-PCR was used to NtKAT2.3 The gene expression was detected. Figure 2 As shown in Figure 2, compared with the control plants, the NtKAT2.3 gene silenced plants NtKAT2.3The expression level of α-glucose was significantly decreased, down by about 56.80%. This result also shows that the gene silencing effect is good.

[0028] Further testing of ferulic acid content showed that Figure 3 As shown, it can be seen that: NtKAT2.3 The ferulic acid content in gene-silenced plants increased by 129.32%. This result indicates that the NtKAT2.3 gene is a key negative regulatory gene for ferulic acid synthesis in tobacco. Further in-depth research based on this gene can lay a good genetic resource foundation for the breeding of new tobacco varieties.

Claims

1. Tobacco Gene NtKAT2.3 , characterized in that, The tobacco gene NtKAT2.3 The nucleotide sequence is shown in SEQ ID No.

1.

2. The tobacco gene of claim 1 NtKAT2.3 The encoded protein NtKAT2.3 is characterized by: The amino acid sequence of protein NtKAT2.3 is shown in SEQ ID NO.

2.

3. The tobacco gene according to claim 1 NtKAT2.3 The invention is used in regulating tobacco ferulic acid synthesis, characterized in that: NtKAT2.3 The gene expression level is negatively correlated with the ferulic acid content in tobacco; when applied, gene silencing technology or gene overexpression method is used to reduce or increase NtKAT2.3 The gene expression level then regulates the ferulic acid content in tobacco leaves by regulating the expression level of tobacco NtKAT2.3 protein.

4. Utilize the tobacco gene of claim 1 NtKAT2.3 The tobacco variety breeding method is characterized in that: First construct a NtKAT2.3 The virus-induced silencing vector of the gene is then used to transform tobacco, and tobacco varieties with increased ferulic acid content are screened.

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