NtERF4 gene and its application in the regulation of total flavonoids and chlorogenic acid synthesis

Abstract

The present application belongs to the technical field of tobacco genetic engineering, and specifically relates to a tobacco NtERF4 gene and its application in the regulation of total flavonoids and chlorogenic acid synthesis. The inventor cloned a transcription factor gene NtERF4 that can promote the synthesis of total flavonoids and chlorogenic acid in tobacco. After analyzing its structure, it belongs to the AP2 subfamily in the ERF transcription factor (a type of ethylene transcription factor). The expression level of this gene is positively correlated with the total flavonoids and chlorogenic acid content in tobacco leaves, that is, by overexpressing this gene, the total flavonoids and chlorogenic acid content in tobacco leaves can be increased, and by reducing the expression level of this gene by gene silencing, the total flavonoids and chlorogenic acid content in tobacco leaves can be reduced. At the same time, the results of the study on the expression pattern of related genes show that the regulation of the synthesis of total flavonoids and chlorogenic acid in tobacco leaves by the NtERF4 transcription factor is achieved through the NtPAL, NtGST and NtGT gene regulation pathways.

Description

Technical Field

[0001] The present application belongs to the technical field of tobacco genetic engineering, and specifically relates to a tobacco NtERF4 gene and its application in the regulation of total flavonoids and chlorogenic acid synthesis. Background Art

[0002] Tobacco is one of the important economic crops in my country. The polyphenols in tobacco mainly include tannins (chlorogenic acid), coumarins (scopoletin, scopoletin, esculetin) and flavonoids (rutin, flavonoids, rhamnose), and these polyphenols are highly correlated with the aroma quality, aroma volume, irritation and permeability of tobacco. Therefore, in-depth research on the metabolic regulation mechanism of polyphenols in tobacco is not only of great significance for improving tobacco quality, but also has good basic technical research significance for other plant improvements.

[0003] Generally speaking, the content of flavonoids and chlorogenic acid in plants is affected by both external environmental factors (such as abiotic factors such as light intensity, ultraviolet rays, temperature, nitrogen levels, or biotic factors such as pathogen infection) and the internal regulation of plant hormones. But in general, whether it is external environmental factors or internal developmental regulatory factors, the impact on the synthesis of flavonoids and chlorogenic acid is ultimately achieved by regulating the expression of transcription factor genes or functional genes related to polyphenol synthesis.

[0004] Studies have shown that the main synthesis pathway of total flavonoids involves the phenylpropanoid biosynthesis pathway, among which the functional genes involved in regulating total flavonoids are mainly: phenylalanine ammonia lyase (PAL), cinnamic acid-4-hydroxylase (C4H), 4-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), etc. The main synthesis pathway of chlorogenic acid is that hydroxycinnamoyl-CoA quinoline hydroxycinnamic transferase (HQT) catalyzes the synthesis of chlorogenic acid from caffeoyl-CoA and quinic acid. Among them, phenylalanine ammonia lyase is a key enzyme in the phenylpropanoid metabolic pathway, which affects the content of trans-cinnamic acid and thus affects the content of chlorogenic acid. Transcription factors are switch elements that regulate gene expression. By regulating the expression level of transcription factors, the activity of downstream target genes can be effectively controlled, thereby affecting the synthesis and accumulation of corresponding substances. At present, the transcription factors reported to be involved in regulating the synthesis of total flavonoids and chlorogenic acid include MYB, bHLH (basic helixloop-helix) and WD40. Based on the important role of transcription factors in regulating gene expression, it is obviously of great technical significance to explore transcription factors that have important regulatory effects on the synthesis of total flavonoids and chlorogenic acid in tobacco. Summary of the invention

[0005] The purpose of the present application is to provide a tobacco NtERF4 gene, which, as an AP2-type transcription factor in the ERF-type transcription factor family, plays an important regulatory role in the synthesis of total flavonoids and chlorogenic acid in tobacco leaves.

[0006] The technical solution adopted in this application is described in detail as follows.

[0007] The tobacco NtERF4 gene has a full length of 684 bp, has no introns, and has a base sequence as shown in SEQ ID No. 1, which is as follows:

[0008] .

[0009] The transcription factor NtERF4 protein encoded by the tobacco NtERF4 gene contains 227 amino acids, and the specific amino acid sequence is shown in SEQ ID No. 2; specifically, it is as follows:

[0010] MAVKNKVSNGDLKGGNVKTNGVKEVHYRGVRKRPWGRYAAEIRDPGKKSRVWLGTFDTAEEAAKAYDTAAREFRGPKAKTNFPLPSENQSTSHSSTMESSSGETGIHAPPHAPL ELDLTRRLGSVAADGGDHCRRSGEVGYPIFHQQPTVAVLPNGQPVLLFDSLWRPGVVNRPQPYHVPMMAGFNGVNAGVDPTVSDSSSVVEENQYDGKRGIDLDLNLAPPTEF*.

[0011] When the tobacco NtERF4 gene is prepared by PCR amplification, the primer sequences for PCR amplification are designed as follows:

[0012] NtERF4-F: 5'-ATGGCTGTCAAAAATAAGGTTA-3',

[0013] NtERF4-R: 5'-TCAAAATTCCGTAGGTGGAG-3'.

[0014] The recombinant overexpression vector containing the tobacco NtERF4 gene uses the pCAMBIA1301 plasmid as a vector and is recombined with the tobacco NtERF4 gene.

[0015] During the construction of the transient silencing VIGS vector for the tobacco NtERF4 gene, the nucleotide sequence at positions 18 to 488 of the tobacco NtERF4 gene is used as a guide sequence.

[0016] The tobacco NtERF4 gene or the transcription factor NtERF4 protein encoded by the tobacco NtERF4 gene is used in tobacco, and the gene or the protein encoded by it is related to the total flavonoids and / or chlorogenic acid content in tobacco leaves; specifically, the tobacco NtERF4 gene (i.e., the NtERF4 protein content) is positively correlated with the total flavonoids and chlorogenic acid content in tobacco leaves; after overexpressing the NtERF4 gene to increase the NtERF4 protein content, it is used to increase the total flavonoids and chlorogenic acid content in tobacco leaves; after reducing the NtERF4 gene by gene silencing technology (or gene editing technology) to reduce the NtERF4 protein content, it is used to reduce the total flavonoids and chlorogenic acid content in tobacco leaves.

[0017] The application of the tobacco NtERF4 gene in the breeding of new tobacco leaf varieties utilizes genetic engineering technology to prepare a recombinant overexpression vector containing the tobacco NtERF4 gene, or a silencing vector for silencing the tobacco NtERF4 gene. After the tobacco is transformed, new tobacco varieties with increased or decreased total flavonoids and chlorogenic acid content in the tobacco leaves can be screened.

[0018] In this application, the inventor cloned a transcription factor gene NtERF4 that can promote the synthesis of total flavonoids and chlorogenic acid in tobacco based on common tobacco (Nicotiana tabacum). After analyzing its structure, it belongs to the AP2 subfamily of ERF transcription factors (a type of ethylene transcription factor). Further, after overexpressing or silencing the gene, the results showed that the expression level of the gene was positively correlated with the total flavonoids and chlorogenic acid content in tobacco leaves, that is, by overexpressing the gene, the total flavonoids and chlorogenic acid content in tobacco leaves can be increased, and by reducing the gene expression level by gene silencing (or gene editing technology), the total flavonoids and chlorogenic acid content in tobacco leaves can be reduced. At the same time, the results of the study of related gene expression patterns showed that the regulation of the synthesis of total flavonoids and chlorogenic acid in tobacco leaves by the NtERF4 transcription factor is achieved through the NtPAL, NtGST and NtGT gene regulation pathways. Based on this result, a certain technical foundation can be laid for the cultivation of new tobacco varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of comparison and analysis of the amino acid sequence of tobacco NtERF4 and its homologous proteins in Arabidopsis thaliana;

[0020] Figure 2 is the expression level of tobacco NtERF4 gene in different tissues;

[0021] Figure 3 It is the identification of the expression level of NtERF4 overexpressing transgenic plants and gene silencing plants; wherein: A is the gene expression level in the overexpressing strain; B is the gene expression level in the silencing strain;

[0022] Figure 4 qRT-PCR was used to detect the expression of genes related to the synthesis of total flavonoids and chlorogenic acid;

[0023] Figure 5 The total flavonoid content of NtERF4 overexpressing transgenic plants and gene silenced plants is detected; wherein: A is the flavonoid content in the overexpressing strain; B is the flavonoid content in the silenced strain;

[0024] Figure 6The chlorogenic acid content in NtERF4 overexpressing transgenic plants and gene silenced plants was detected; wherein: A is the chlorogenic acid content in the overexpressing strain; B is the chlorogenic acid content in the silenced strain. DETAILED DESCRIPTION

[0025] 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.

[0026] Biomaterials:

[0027] Common tobacco (Nicotiana tabacum) and Nicotiana benthamiana are both tobacco materials commonly used in tobacco research or actual tobacco cultivation and planting. As a professional tobacco research institution, the applicant has long-term preservation of relevant research materials;

[0028] In the research experiment, tobacco cultivation and management conditions were as follows: greenhouse cultivation and management, culture conditions: temperature 28°C, humidity 70%-75%, light 16h, dark 8h;

[0029] Plasmid pCAMBIA1301, Agrobacterium GV3101, and TRV vectors (TRV1 and TRV2) are all commonly used materials in molecular biology research and can be obtained from public channels.

[0030] Example 1

[0031] In view of the important role of transcription factors in regulating biological metabolites, combined with previous research and research on related homologous genes, the inventors attempted to clone related transcription factors in tobacco. The cloning process of the tobacco NtERF4 gene is briefly described as follows.

[0032] 1. Extract RNA and prepare cDNA by reverse transcription

[0033] Tobacco seedling leaf tissue was used as the sample source (common tobacco at the four-leaf stage). After being frozen in liquid nitrogen, it was fully ground and total RNA was extracted using a tobacco RNA rapid extraction kit (Gene Answer, RT0110) (refer to the kit instructions for related operations, which will not be repeated here);

[0034] The extracted total RNA was digested with DNaseⅠ (Omega, #E1091-01) to remove genomic DNA. After treatment, the quality of RNA was tested (agarose gel electrophoresis), and the concentration of RNA samples was measured using Nanodrop to ensure that it met the requirements for subsequent use.

[0035] The PrimeScript™ II 1st Strand cDNA Synthesis Kit (TAKARA, 6210A) was used to synthesize and reverse transcribe the first strand of cDNA using the total RNA extracted above (refer to the instructions of the kit for operation) to prepare cDNA, and the concentration of the obtained cDNA sample was measured to ensure that it met the needs of subsequent gene cloning.

[0036] 2. PCR amplification

[0037] The sequence for PCR amplification was designed as follows:

[0038] NtERF4-F: 5'-ATGGCTGTCAAAAATAAGGTTA-3',

[0039] NtERF4-R: 5'-TCAAAATTCCGTAGGTGGAG-3';

[0040] The cDNA prepared above was used as a template and PCR amplification was performed using high-fidelity PrimeSTAR® HS DNA Polymerase (TAKARA, R010A). The amplification system was designed as follows:

[0041] 2×PrimeSTAR GC Buffer (Mg2+ plus), 25μl;

[0042] dNTP Mixture (2.5 mM each), 4μl;

[0043] PrimeSTAR HS DNA Polymerase (2.5 U / μl), 0.5μl;

[0044] DNA, 1 μl;

[0045] Add ddH2O to 50 μl;

[0046] The amplification program was as follows: 98°C, 5 min; 98°C, 30 s; 58°C, 30 s; 68°C, 45 s, 30 cycles; 68°C, 10 min.

[0047] The PCR amplification products were detected by 1% agarose gel electrophoresis (130V electrophoresis for 30 minutes), and the gel was cut and recovered (using AxyPrep DNA Gel Recovery Kit (Axygen, AP-GX-250G) for recovery, refer to its instructions for operation). The amplification products were connected to the T vector and sequenced for identification.

[0048] T vector connection reference reference is as follows:

[0049] In a microcentrifuge tube: pMDTM18-T Vector (Takara) 1 μl, PCR amplified fragment 0.2 pmol, Solution I 5 μl, dd-H2O added to 10 μl;

[0050] The ligation reaction was carried out at 16°C overnight;

[0051] Then, all 10 µl of the ligation product was added to the competent E. coli cells, heat-shocked and transformed, and finally the positive clones were selected for sequencing and identification.

[0052] 3. Gene sequence analysis

[0053] Based on the sequencing results, homology comparison and structural analysis were performed. The results showed that the cloned gene was closest to the Arabidopsis thaliana AtERF4 protein sequence (the comparison results are shown in Figure 1 The sequence similarity is 49.6% and the identity is 41.7%. Structural analysis shows that the two have large differences in N-terminal and C-terminal sequences, but are highly conserved in the key AP2 domain region. Therefore, the cloned gene was named NtERF4.

[0054] 4. Expression pattern of tobacco NtERF4 gene

[0055] Based on the preliminary study on the structure of NtERF4 gene, the inventors further studied the expression pattern of NtERF4 gene using qPCR quantitative method. The specific experimental results are briefly described as follows.

[0056] First, the leaves, stems, petals, roots, axillary buds, and terminal buds of tobacco at the flowering stage (about 110 days after planting) were used as samples, and total RNA was extracted and reverse transcribed to prepare cDNA as described above.

[0057] Subsequently, the primer sequences for qPCR detection were designed as follows:

[0058] The primer sequence of the internal reference gene (GAPDH) was designed as follows:

[0059] GAPDH-F: 5'-TGGGTGTCAACGAGAAGGAA-3',

[0060] GAPDH-R: 5'-TCTGGGTGGCAGTAAGGGA-3';

[0061] The target gene primer sequences are:

[0062] ERF4-RT-F: 5'-TCGTTGGACCCAAAGCTAAAA-3',

[0063] ERF4-RT-R: 5'-ATGCCAGTCTCTCCGCTAGA-3';

[0064] The reaction system is designed as follows:

[0065] 2×SYBR I Master, 5 μL;

[0066] Upstream and downstream primers, 0.5 μL each;

[0067] cDNA, 50 ng;

[0068] Add ddH2O to 10 μl;

[0069] The reaction program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 20 s, and extension at 72°C for 20 s, for 40 cycles;

[0070] Finally, according to the cycle threshold (CT value), 2 -ΔΔCT Methods The relative expression of genes was calculated.

[0071] Specific results such as Figure 2 The analysis showed that the expression level of tobacco NtERF4 gene was highest in leaves, followed by roots and flowers, while the expression level in other tissues was relatively low.

[0072] Example 2

[0073] On the basis of Example 1, in order to further study and clarify the function of NtERF4 gene, the inventors further constructed an overexpression vector containing the gene and further transformed tobacco. The specific experimental process and results are briefly described as follows.

[0074] 1. Construction of recombinant overexpression vector

[0075] The pCAMBIA1301 plasmid was used as a vector to reconstruct the NtERF4 gene. The specific process is as follows:

[0076] First, design specific amplification primers containing restriction sites as follows:

[0077] NtERF4-XbaI-F:5'-ATACACCAAATTGACTCTAGAATGGCTGTCAAAAATAAGGTTA-3';

[0078] NtERF4-KpnI-R: 5'-GCCCTTGCTCACCATGGTACCTCAAAATTCCGTAGGTGGAG-3';

[0079] Subsequently, referring to the operation of the above-mentioned Example 1, PCR amplification was performed using the above-mentioned primers with cDNA as a template, and the amplified product was extracted;

[0080] Then, the plasmid pCAMBIA1301 was double-digested with XbaⅠ and KpnⅠ (digestion at 37℃ for 3 h), and the digestion products were detected and recovered by electrophoresis;

[0081] Then, the restriction digestion product of the plasmid pCAMBIA1301 was connected with the PCR amplification product. The 10 μl connection system was designed as follows:

[0082] pCAMBIA1301 digestion product, 1 μl;

[0083] PCR amplification product (NtERF4 gene fragment), 0.2 pmol;

[0084] MultiS, 1 μl;

[0085] 5x CE buffer (Vazyme, C113-01), 2 μl;

[0086] Add ddH2O to 10 μl;

[0087] Ligation at 37°C for 30 min;

[0088] Finally, the heat shock transformation method was used to transform the above-mentioned ligation products into competent E. coli cells, and the positive clones were screened for bacterial liquid PCR identification and sequencing identification to ensure that the transformation and recombination were correct, and finally the correct recombinant plasmid was extracted for use.

[0089] It should be explained that operations not described in detail can be referred to as conventional molecular biology operations and will not be repeated here.

[0090] 2. Preparation of infection solution

[0091] The recombinant overexpression plasmid prepared in step (I) is transformed into Agrobacterium, the correct transformed strain is screened, and the infection solution for transgenic is prepared. The specific operation steps are as follows:

[0092] Take 1 μl of the recombinant expression plasmid and add it to 100 μl of GV3101 Agrobacterium competent cells, let it stand for 15 min, transfer it to liquid nitrogen for quick freezing for 5 min, then warm it at 37°C for 5 min, and then place it on ice for 5 min;

[0093] Add 1 mL of LB liquid medium and culture at 28℃ with shaking for 3 h;

[0094] Take 200 μl of bacterial solution and spread it on LB solid culture plate containing 20 mg / L rifampicin (Rif) and 20 mg / L kanamycin (Kan), culture at 28°C for 2-3 days, and verify the correct transformation of the strain by colony PCR.

[0095] 3. Tobacco transformation

[0096] The infection solution prepared in step (ii) was used to transform tobacco using the leaf disc method. The specific operation is as follows.

[0097] First, the leaves of vigorous tobacco (K326) were sterilized and cut into 1 cm 2 The left and right pieces were placed in MS differentiation medium and pre-cultured for 2 days at 28°C, light intensity of 2000 Lx, and light duration of 16 h / d;

[0098] The MS differentiation medium: MS medium + 1.0 mg / L 6-BA + 0.1 mg / L NAA;

[0099] Subsequently, the pre-cultured leaf pieces were placed in the Agrobacterium solution containing the target plasmid (i.e., prepared in step (ii)) for infection for 15 minutes, during which the solution was shaken several times to ensure sufficient infection. After the infection was completed, the leaf pieces were removed and the excess solution around the leaf pieces was dried with sterile filter paper;

[0100] Afterwards, the infected leaf pieces were transferred into MS differentiation medium and cultured at 28°C in the dark for 4 days;

[0101] After that, the co-cultivated plants were washed three times with sterile water, dried with sterile paper, and transferred to MS differentiation medium containing kanamycin (50 mg / L) and carbenicillin (400 mg / L) and cultured at a constant temperature (28°C, light intensity 2000Lx, light duration 16 h / d); during the culture process, the medium was replaced every 10 days;

[0102] Afterwards, when the adventitious buds grow to 1-2 cm, the clustered adventitious buds are cut into individual buds and transferred to MS rooting medium containing kanamycin, carbenicillin and activated carbon to promote their rooting;

[0103] The MS rooting medium: MS medium + kanamycin (50 mg / L) + carbenicillin (250 mg / L);

[0104] Finally, after the root system has developed well, take out the tissue culture seedlings, wash the culture medium at the roots with clean water, cut off a small number of leaves at the bottom, transfer them to a pot filled with loose sterile soil, and manage and cultivate them as usual.

[0105] During the screening and cultivation process, when the transgenic seedlings obtained through the preliminary screening grew to 8 true leaves, DNA was extracted from the leaves of the T0 generation plants and PCR verification was performed to identify positive transgenic plants.

[0106] The T0 transgenic plants that were initially identified as positive were planted and cultivated until seeds were harvested, and the seeds were further planted to obtain T1 plants. Subsequently, PCR amplification was performed using the vector-specific upstream primer NtERF4-1300-F and the gene-specific downstream primer NtERF4-1300-R using the genomic DNA of the T1 plants as a template. Plants that could amplify a specific single band were overexpression-positive plants.

[0107] The upstream primer NtERF4-1300-F and the gene-specific downstream primer NtERF4-1300-R are specifically designed as follows:

[0108] NtERF4-1300-F: 5'- ACCAATACATACACTAGCATC -3',

[0109] NtERF4-1300-R: 5'-AATCGGGTACCCAACTTCAC-3'.

[0110] Example 3

[0111] The research purpose is the same as that of Example 2. To further study and clarify the function of the NtERF4 gene, the inventors further constructed a silencing vector for the gene and further transformed tobacco. The specific experimental process and results are briefly described as follows.

[0112] 1. Construction of gene silencing vector

[0113] A specific nucleic acid fragment in the NtERF4 gene (nucleotide sequence from position 18 to position 488 in SEQ ID NO.1 in the sequence list) was selected as a guide sequence to construct a transient silencing VIGS vector for silencing the NtERF4 gene. The specific process is as follows:

[0114] First, the primer sequences for PCR amplification were designed as follows:

[0115] NtERF4-VIGS-F:

[0116] 5'- ATCGACGACAAGACCCTGCAGGGTTAGTGATGGCGATCTGA -3',

[0117] NtERF4-VIGS-R:

[0118] 5'-CTGAGGAGAAGAGCCCTGCAGTCAAAGAGCAAAACCGGCTG -3';

[0119] Referring to the above, cDNA was used as a template and the above primer sequences were used for PCR amplification (amplification length: 471 bp) to obtain the guide sequence of VIGS.

[0120] Subsequently, the amplified guide sequence was connected to the TRV2 vector by homologous recombination (connection at 37°C for 30 min), and further screening and sequencing were performed to construct a correctly connected TRV2-NtERF4 vector.

[0121] The homologous recombination connection system is designed as follows:

[0122] TRV2 vector (enzyme digestion product), 1 μl;

[0123] PCR amplification product, 0.2 pmol;

[0124] MultiS, 1 μl;

[0125] 5x CE buffer (Vazyme, C113-01), 2 μl;

[0126] Add ddH2O to 10 μl;

[0127] Ligate at 37°C for 30 min.

[0128] 2. Preparation of transfection solution

[0129] First, the correctly connected TRV-NtERF4 vector was transformed into Agrobacterium GV3101 by freeze-thaw method, and positive monoclonal colonies were selected for bacterial liquid PCR verification to ensure that the transformation was correct, and the correctly transformed strain (bacterial liquid) was stored for later use;

[0130] As controls, TRV1, TRV2, and TRV2-PDS (positive control, PDS, phytoene dehydrogenase) were transformed into Agrobacterium GV3101 under the same operating conditions, and control transfection solutions were prepared.

[0131] Subsequently, the correctly transformed TRV1, TRV2, TRV2-PDS (positive control), and TRV2-NtERF4 Agrobacterium single colonies were inoculated into LB (5 mL) medium (kanamycin, 50 μg / mL), and cultured at 28°C and 250 r / min overnight with shaking for about 48 h; and then transferred to 50 mL LB again and cultured at 28°C overnight with shaking; after the culture was completed, centrifuged at 8000 r / min for 8 minutes, and the Agrobacterium precipitate was collected into a 50 mL centrifuge tube;

[0132] Then, the bacterial pellet was resuspended in a mixed solution containing 10 mmol / L 2-N-morpholinylethanesulfonic acid (MES), 20 μl / L acetosyringone (As) and 10 mmol / L MgCl2, and the OD600 was adjusted to about 1.0;

[0133] Finally, add an equal volume of the TRV1 Agrobacterium suspension to the above Agrobacterium suspensions containing TRV2, TRV2-PDS, and TRV-NtERF4, mix well, and place at room temperature for 3 to 6 hours as transfection solution.

[0134] 3. Transient infection

[0135] Use Nicotiana benthamiana seedlings with consistent growth that have been cultivated in advance (grown for 2 to 3 weeks) as transformants. For instantaneous infection, select about 3 to 4 leaves with consistent growth. Use a 1 mL needle-free sterile syringe to press the Agrobacterium suspension containing different TRV recombinant plasmids from the back of the leaves into all the unfolded leaves through the filter press method, so that the bacterial solution fills the entire leaf, and culture at 22°C and 75% humidity.

[0136] Detection experiment

[0137] For the overexpression plants constructed in Example 2 and the transiently transformed leaves in Example 3, the inventors detected and analyzed the expression level of the NtERF4 gene and the content of related secondary metabolites, and the specific situation is briefly described as follows.

[0138] (1) Gene expression

[0139] For the overexpression positive plants, the leaves (the 8th leaf position of tobacco maturity stage) were used as samples, and the expression level was detected by q-PCR method with reference to the above operation. The results are as follows Figure 3 As shown, it can be seen that although the expression levels of NtERF4 gene in different strains are quite different, the expression levels of NtERF4 gene are significantly increased compared with the untransformed wild type.

[0140] The results of the detection of NtERF4 gene expression in infected plants using leaves from gene-silenced plants 10 days later showed that ( Figure 3 As shown in the figure, the gene silencing effect in different strains was not much different, and compared with the wild type without silencing, the expression level of NtERF4 gene in the plants after gene silencing was significantly reduced. This result showed that the transient infection experiment was relatively successful.

[0141] (2) Changes in related gene expression

[0142] Based on the transcriptome sequencing data of overexpressed plants and gene silenced plants and the applicant's existing tobacco genome database, the inventors used the qPCR method to verify the relevant transcriptome data (chlorogenic acid and total flavonoids synthesis related genes). Figure 4 As shown. The analysis shows that:

[0143] The expression levels of NtPAL1, NtPAL2, NtPAL3, NtPAL4, NtGST1, NtGST2, NtGT1 and NtGT2 genes were significantly increased in NtERF4 overexpression plants and significantly decreased in NtERF4 silenced plants, indicating that NtERF4 promotes the expression of these genes.

[0144] (3) Total flavonoid content

[0145] When determining the total flavonoid content of secondary metabolites in leaves, the specific operation is as follows:

[0146] Weigh about 0.1 g of fresh leaf sample (8th leaf position of tobacco at maturity), grind it, add 1.5 mL of 60% ethanol, shake and extract at 60°C for 2 h, then centrifuge at 25°C and 12000 rpm for 10 min, and take the supernatant for testing;

[0147] In alkaline nitrite solution, since flavonoids can form red complexes with aluminum ions with a characteristic absorption peak at 510 nm, the absorbance of the reaction product at 510 nm is measured to ultimately calculate the total flavonoid content in the sample.

[0148] The results are as follows Figure 5 As shown, it can be seen that in tobacco plants overexpressing the NtERF4 gene, the content of total flavonoids was significantly increased. Compared with the control plants, the total flavonoid content of the overexpressed strains increased by about 42.5%~87.2%, while the total flavonoid content in NtERF4 silenced plants decreased significantly. Compared with the control plants, the total flavonoid content of the gene silenced plants decreased by 11.1%~36.9%.

[0149] (4) Chlorogenic acid content

[0150] When determining the total flavonoid content of secondary metabolites in leaves, the specific operation is as follows:

[0151] Weigh 100 mg of crushed leaf sample (8th leaf position of tobacco during maturity), add 2 mL of 80% methanol aqueous solution, extract at 60℃ for 30 min, heat at 80℃ for 1.5 h, take out and cool to room temperature;

[0152] Centrifuge at 4°C and 12,000 rpm for 10 min, take the supernatant, filter it with a syringe filter and prepare it for testing.

[0153] Determination of chlorogenic acid by liquid chromatography:

[0154] A Wufeng LC-100 high performance liquid chromatography system and a Yuexu C18 reverse phase column (150 mm*4.6 mm, 5 μm) were used; mobile phase A: 100% methanol; mobile phase B: 0.1% acetic acid aqueous solution, A:B=55:45;

[0155] Injection volume 10 μL, flow rate 0.8 mL / min;

[0156] The column temperature was 30°C and the UV wavelength was 320 nm.

[0157] The results are as follows Figure 6 As shown in the figure, it can be seen that in tobacco plants overexpressing the NtERF4 gene, the chlorogenic acid content increased significantly. Compared with the control plants, the chlorogenic acid content of the overexpression lines increased by about 4.8%~19.3%, while the chlorogenic acid content in the NtERF4 silenced plants decreased significantly. Compared with the control plants, the chlorogenic acid content in the silenced plants decreased significantly by 6.8%~36.2%.

Claims

1. Application of tobacco NtERF4 gene in tobacco, characterized in that: The gene is related to the total flavonoids and / or chlorogenic acid content in tobacco leaves; specifically, the tobacco NtERF4 gene is positively correlated with the total flavonoids and / or chlorogenic acid content in tobacco leaves; after the NtERF4 gene is overexpressed to increase the NtERF4 protein content, it is used to increase the total flavonoids and chlorogenic acid content in tobacco leaves; after the NtERF4 gene is reduced by gene silencing technology to reduce the NtERF4 protein content, it is used to reduce the total flavonoids and chlorogenic acid content in tobacco leaves; The base sequence of the tobacco NtERF4 gene is shown in SEQ ID No.

1.

2. The use of the transcription factor NtERF4 protein encoded by the tobacco NtERF4 gene in tobacco, characterized in that: The transcription factor NtERF4 protein content is positively correlated with the total flavonoids and chlorogenic acid content in tobacco leaves; after the NtERF4 gene is overexpressed to increase the NtERF4 protein content, it is used to increase the total flavonoids and chlorogenic acid content in tobacco leaves; after the NtERF4 gene is reduced by gene silencing technology to reduce the NtERF4 protein content, it is used to reduce the total flavonoids and chlorogenic acid content in tobacco leaves; The amino acid sequence of the transcription factor NtERF4 protein encoded by the tobacco NtERF4 gene is shown in SEQ ID No.

2.

3. Application of tobacco NtERF4 gene in tobacco variety breeding, characterized in that: By using genetic engineering technology, a recombinant overexpression vector containing the tobacco NtERF4 gene is prepared, or a transient silencing VIGS vector for silencing the tobacco NtERF4 gene is prepared, and after tobacco is transformed, tobacco varieties with increased or decreased total flavonoids and chlorogenic acid content in tobacco leaves are screened; The tobacco NtERF4 gene has a base sequence as shown in SEQ ID No. 1; The recombinant overexpression vector containing the tobacco NtERF4 gene uses the pCAMBIA1301 plasmid as a vector and is recombinant with the tobacco NtERF4 gene; The transient silencing VIGS vector for silencing the tobacco NtERF4 gene uses the nucleotide sequence at positions 18 to 488 of the tobacco NtERF4 gene as a guide sequence.

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