Application of LmbZIP11 gene in regulating and controlling content of chlorogenic acid and total flavonoids

Abstract

The invention relates to a method and application of an LmbZIP11 gene and a homologous NbbZIP11 gene of the LmbZIP11 gene in regulating and controlling the content of chlorogenic acid and total flavonoids in plants. The method comprises the following steps: extracting RNA (Ribonucleic Acid) of leaves of lonicera macranthoides, designing a specific primer, cloning to obtain an LmbZIP11 gene, further constructing a recombinant overexpression vector and a VIGS (Vinyl Insulin Growth Sequence) silencing vector of the LmbZIP11 gene, and respectively transforming arabidopsis thaliana and nicotiana benthamiana; experiments show that the content of total flavonoids and chlorogenic acid in arabidopsis thaliana is remarkably increased through overexpression of the LmbZIP11 gene, and the content of metabolites in tobacco is remarkably reduced through silence of the NbbZIP11 gene homologous with the LmbZIP11 in Benen tobacco; the invention provides a new strategy for regulating the synthesis of plant secondary metabolites by using the LmbZIP11 gene, and can be used for improving the quality of medicinal plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a method and application of an LmbZIP11 gene and a homologous NbbZIP11 gene in regulating the content of chlorogenic acid and total flavonoids in plants. Background Art

[0002] Lonicera japonica is the source plant of the important medicinal plant Lonicera japonica (Liquorice Flos) in southern China. It is rich in phenolic acids, including chlorogenic acid and flavonoids. These polyphenols are highly correlated with the medicinal quality of Lonicera japonica. Therefore, in-depth research on the metabolic regulation mechanisms of polyphenols in Lonicera japonica is not only important for the selection of high-quality Lonicera japonica germplasm resources, but also has fundamental technical significance for the construction of bioreactors based on Lonicera japonica.

[0003] Generally speaking, the chlorogenic acid and flavonoid content of plants in nature is affected by light intensity. Light not only regulates the synthesis of secondary metabolites but is also crucial for plant growth and environmental adaptation. However, overall, the effects of light on chlorogenic acid and flavonoid synthesis ultimately occur through the activation of plant light signaling components, thereby regulating the expression of transcription factor genes or functional genes involved in polyphenol synthesis.

[0004] Research has shown that the primary biosynthesis pathway of flavonoids involves the phenylpropanoid biosynthesis pathway, while the primary biosynthesis pathway for chlorogenic acid involves the synthesis of chlorogenic acid from caffeoyl-CoA and quinic acid, catalyzed by hydroxycinnamoyl-CoA-quinoline hydroxycinnamoyltransferase (HQT). The bZIP transcription factor family plays a key role in gene expression, metabolism, and growth and development during photomorphogenesis. bZIP transcription factors act as signaling elements involved in light-regulated transcriptional activation of G-box promoters. Modulating the expression levels of bZIP transcription factors can effectively regulate the function of downstream target genes, thereby affecting the synthesis and accumulation of the corresponding substances. Currently, bZIP transcription factors reported to regulate the phenylpropanoid biosynthesis pathway include HY5, bZIP1, bZIP2, bZIP3, and bZIP22. Although bZIP family genes have been reported to be associated with chlorogenic acid or flavonoid accumulation in plants, their expression is affected by light conditions, and no bZIP family genes have been reported to correlate with chlorogenic acid and flavonoid accumulation in different plant tissues. Based on the important role of the bZIP transcription factor family in gene expression regulation, the discovery of bZIP transcription factors that play an important role in regulating the accumulation of substances such as chlorogenic acid and total flavonoids in the light regulation of Lonicera japonica has very important technical significance for the subsequent efficient expression of phenolic acids and flavonoids through genetic engineering.

[0005] Currently, the genetic transformation system for Lonicera japonica is not yet mature, and the key bZIP transcription factors that regulate chlorogenic acid and flavonoid biosynthesis remain unknown. Existing technologies for regulating secondary metabolism in this species rely primarily on traditional breeding, which is inefficient and time-consuming, and lacks precise genetic engineering strategies. Summary of the Invention

[0006] The present invention addresses the technical problems to be solved and overcomes the shortcomings of the prior art by providing a method and application of the LmbZIP11 gene and its homologous NbbZIP11 gene in regulating the chlorogenic acid and total flavonoid content of plants. The chlorogenic acid and flavonoid contents of different tissues of Lonicera japonica (Lonicera grisea), the source plant of Lonicera japonica, vary significantly. Based on this, the expression sequence of the light-related transcription factor LmbZIP11 gene was discovered and cloned. Furthermore, an LmbZIP11 gene overexpression vector and a VIGS silencing vector were used in Arabidopsis thaliana and Nicotiana benthamiana. Overexpression of the LmbZIP11 gene increased metabolite content in Arabidopsis thaliana, while silencing the NbbZIP11 homologous gene in Nicotiana benthamiana reduced metabolite content. The chlorogenic acid and flavonoid contents of different tissues of Lonicera japonica vary significantly. The present invention addresses the technical challenges of how to regulate the LmbZIP11 gene in Lonicera japonica and how to efficiently express chlorogenic acid and flavonoid metabolites.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] The nucleotide sequence of the LmbZIP11 gene of Lonicera japonica is shown in SEQ ID NO: 1, and is as follows:

[0009] ATGGATTCGTCGAGTGGGACCACTTCAGGATCCACACTACTTCAGAGCTCTGGCTCAGATGAAGATCTGCTGCAACTAATGGAACAGAGGAAGAGGAGGAGGTTAATTTCGAATCGCGAGTCGGCTCGGCGATCCCGGATGAAGAAACAGAAAAGATTGAGCGAGTTGATGACTCAGCTGAGTCAACTCAGGAAGGAGAACAATCAGCTTATTGCGAGT TTGAGATTTACTAGTCAGCATTTTGTGAATGTGGAGGCAGAGAACTCTGTTTTGAGGGCTCAAGTGGGTGAACTTAGCCACAGATTGGAGTCTTTAAATGAAATCGTCAGTTTCTTGAGTGTCGGTGAACTTGGGGGTGAGTTTATCAATGAGTCTTTGAGTTATTTGTATGTGAATCAGCCTGTTATGGCCTCTGCTGCTGATATGTTGATGTACTGA.

[0010] Specifically, the amino acid sequence of the protein encoded by the LmbZIP11 gene of Lonicera grisea is shown in SEQ ID NO: 2, and is as follows:

[0011] MDSSSGTTSGSTLLQSSGSDEDLLQLMEQRKRRRLISNRESARRSRM KKQKRLSELMTQLSQLRKENNQLIASLRFTSQHFVNVEAENSVLRAQVG ELSHRLESLNEIVSFLSVGELGEFINESLSYLYVNQPVMASAADMLMY*.

[0012] Specifically, the nucleotide sequence of the Nicotiana benthamiana NbbZIP11 gene, which has a high homology to the LmbZIP11 of Lonicera japonica, is shown in SEQ ID NO: 3, as follows:

[0013] ATAATACTAGTAATAAAATAACCCTTCCCTTCTTCCTTCCACTCATCATCACAACCACCATTAATTTCAATTTACCACCATTAAAACAGTTAATGGTGATAATTCCTAATACTTTTTTATAAAAAAAAAAATTAATACTGATAAAGCATATCAGCAGAAGCCATAATTGGCTGATTAACATACATCAAATTCCATGGATTATTCATCATATCCTCCT CATAATGATGATGAGTTTCAAAAACTCCGTTATTATTGTTGTTGTTGTTGGAATTGATGTAACTGAGGATTTCATTAAGAGATTGCAACCTTTGACTAAGTTCCATCATCTGAGCTCTTAAAACAGAGTTCTCTGCTTCAACATTAGCGTACTGTTGACTTGTCAAGTTGATGTTGCTAAGGATGTTGCTGTTTTCCTTTTTCAGTTGCGCAACTTGAC.

[0014] The beneficial effect of the present invention is that the present invention adopts a genetic engineering method to solve the technical problems of how to regulate the LmbZIP11 gene in Lonicera japonica and how to efficiently express chlorogenic acid and flavonoids.

[0015] Furthermore, technical support is provided for exploring potential LmbZIP11 gene-responsive metabolites.

[0016] Furthermore, it provides a technical basis for using genetic engineering to transform Lonicera japonica to efficiently express metabolites.

[0017] Furthermore, technical support is provided for the quality improvement of honeysuckle.

[0018] Compared with the existing technology, the present invention provides a method and application of the LmbZIP11 gene and its homologous NbbZIP11 gene in regulating the content of chlorogenic acid and total flavonoids in plants. This method effectively solves the problem of the lack of genetic transformation technology in the study of gene regulation of Lonicera japonica, and is convenient and fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the result of homology alignment and structural analysis between LmbZIP11 gene of Lonicera japonica and LjbZIP11 gene of Lonicera japonica.

[0020] Figure 2 This is the result of detecting the expression level of LmbZIP11 gene in Lonicera japonica. Different lowercase letters indicate significant differences, the same below.

[0021] Figure 3 The graph shows the expression level detection results of the LmbZIP11 gene in Arabidopsis positive plants and the expression level detection results of the NbbZIP11 gene in Nicotiana benthamiana after silencing; in the figure, WT represents the wild-type control group, OE represents the overexpression group, and TRV represents the silencing group.

[0022] Figure 4 This is the result of total flavonoids content detection in Arabidopsis positive plants expressing LmbZIP11 gene.

[0023] Figure 5 This is the result of detecting the chlorogenic acid content in Arabidopsis positive plants expressing the LmbZIP11 gene. DETAILED DESCRIPTION

[0024] To make the purpose, technical solutions and advantages of this application more clear, the embodiments of the present invention will be described in detail below. It should be noted that this embodiment is implemented under the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection rights of the present invention are not limited to the following embodiments.

[0025] Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly described as follows.

[0026] Biomaterials:

[0027] Gray Felt Honeysuckle (Lonicera macranthoides Hand-Mazz), Arabidopsis thaliana, and Nicotiana benthamiana. As a professional authentic medicinal plant genetic engineering research institution, the applicant has long-term preserved relevant research materials.

[0028] In the research experiment, the cultivation and management conditions of honeysuckle were as follows: management in an artificial plant culture room, cultivation conditions: temperature 25℃, humidity 50%-60%, 3000Lux light 16h, dark 8h;

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

[0030] In view of the important role of bZIP transcription factors in the regulation of biological metabolites, combined with previous preliminary research and research on related homologous genes, the inventors attempted to clone the bZIP transcription factor in Lonicera japonica.

[0031] Example 1: Cloning and expression analysis of the LmbZIP11 gene of Lonicera japonica

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

[0033] S1: Mature leaf tissue of Lonicera japonica provided by the laboratory was used as the sample source. After being frozen in liquid nitrogen, it was thoroughly ground and total RNA was extracted using the Polysaccharide and Polyphenol RNA Extraction Kit (TIANGEN, DP41) (refer to the kit instructions for relevant operations, which will not be repeated here);

[0034] S2: The extracted total RNA was digested with DNase I (TIANGEN, RT411) to remove genomic DNA. After treatment, the RNA quality was tested by agarose gel electrophoresis, and the concentration of the RNA sample was measured using a nucleic acid protein analyzer (OSE-260-05, TIANGEN) to ensure that it met the requirements for subsequent use.

[0035] S3: Use the FastKing one-step genomic cDNA first-strand synthesis premixed reagent reverse transcription kit (TIANGEN, KR118) to synthesize and reverse transcribe the first-strand cDNA using the total RNA extracted above (refer to the kit instructions for operation) to prepare cDNA, and use a nucleic acid protein analyzer (OSE-260-05, TIANGEN) to measure the concentration of the obtained cDNA sample to ensure that it meets the needs of subsequent gene cloning.

[0036] (2) PCR amplification

[0037] S1: Design PCR amplification primer sequences as follows:

[0038] LmbZIP11-F: 5'-ATGGATTCGTCGAGTGGGAC-3',

[0039] LmbZIP11-R: 5'-TCAGACATCAACATATCAGCAGC-3';

[0040] The cDNA prepared above was used as a template and PCR amplification was performed using High-Fidelity Gold Mix (green) (TSINGKE, TSE101). The amplification system was as follows:

[0041] Gold Mix (green), 44 μL;

[0042] LmbZIP11-F, 2 μL;

[0043] LmbZIP11-R, 2 μL;

[0044] cDNA, 2 μL;

[0045] S2: Amplification program was as follows: initial denaturation at 98°C for 2 min; denaturation at 98°C for 10 s; annealing at 57°C for 30 s; extension at 72°C for 30 s, 35 cycles; final extension at 72°C for 5 min;

[0046] S3: PCR amplification products were examined by electrophoresis on a 1% agarose gel containing a nucleic acid dye (120V for 30 minutes). The target fragments were then excised using a standard agarose gel DNA recovery kit (TIANGEN, DP209) (refer to the kit instructions for instructions). The amplified products were ligated into a T-vector (pClone007 Simple Vector Kit) using the pClone007 Simple Vector Kit (TIANGEN, TSV-007VS), transformed into E. coli DH5α, and sequenced for identification (refer to the kit instructions for instructions).

[0047] (3) Gene sequence analysis

[0048] Based on the sequencing results, DNAMAN was used for homology comparison, and MEGA 6.0 was used for phylogenetic tree analysis, with default parameters. The results showed that the cloned gene was most closely related to Lonicera japonica LjbZIP11 (the results of the phylogenetic tree analysis of homologous genes are shown in Figure 2). Figure 1 As shown), the gene sequence similarity is 98.17% (the comparison results are shown in Figure 1 Therefore, the cloned gene was named LmbZIP11.

[0049] (IV) Expression pattern of the LmbZIP11 gene in Lonicera japonica

[0050] Based on the preliminary study on the structure of LmbZIP11 gene, the expression pattern of LmbZIP11 gene was further studied using qPCR quantitative method.

[0051] S1: Using old leaves, young leaves, and flowers (young bud stage, green bud stage, second white stage, large white stage, silver flower stage, and golden flower stage) of mature (laboratory-grown) Lonicera japonica as sample sources, total RNA was extracted and reverse transcribed to prepare cDNA, as described above.

[0052] S2: Design the primer sequences for qPCR detection as follows:

[0053] The primer sequence of the internal reference gene (LmActin) was designed as follows:

[0054] LmActin-F: 5'-GTTGCCATTCAGGCCGTTCTTTC-3',

[0055] LmActin-R: 5'-CAGAATCGAGCACAATACCGGTTG-3';

[0056] The target gene primer sequences are:

[0057] LmbZIP11-RT-F: 5'-TGAGGGCTCAAGTGGGTGAACT-3',

[0058] LmbZIP11-RT-R: 5'-CAGCAGCAGAGGCCATAACAGG-3';

[0059] The reaction system is designed as follows:

[0060] 2×Talent SYBR qPCR PreMix, 10 μL;

[0061] Upstream and downstream primers, 0.5 μL each;

[0062] cDNA template 50ng;

[0063] Add ddH2O to 20 μL;

[0064] S3: The reaction program was as follows: pre-denaturation at 95°C for 180 s; denaturation at 95°C for 5 s, annealing at 60°C for 15 s, and extension at 72°C for 20 s, for 45 cycles;

[0065] S4: Based on the cycle threshold (CT value), the relative expression of genes was calculated using the 2-ΔΔCT method; the specific results are as follows Figure 2 As shown, it can be seen that the expression level of the LmbZIP11 gene of Lonicera japonica is highest in the flowers of the second white stage, followed by the golden flower stage, while the expression level in the leaves is relatively low.

[0066] Example 2: Construction of LmbZIP11 gene overexpression vector and Arabidopsis transformation

[0067] Based on Example 1, the function of the LmbZIP11 gene was further studied and clarified, an overexpression vector containing the gene was constructed, and Arabidopsis thaliana was further transformed. The specific experimental process and results are briefly described below.

[0068] (1) Construction of recombinant overexpression vector

[0069] The LmbZIP11 gene was recombined using the pRHVnGFP plasmid as a vector. The specific process is as follows:

[0070] S1: Design specific amplification primers containing restriction enzyme cutting sites as follows:

[0071] LmbZIP11-BamH IF: 5'

[0072] -ACGAGCTGTACAAGGATGGATTCGTCGAGTGGGAC-3',

[0073] LmbZIP11-BamH IR:5'

[0074] -CCGAGCTCACCCGGGTCAGTACATCAACATATCAGCAGCA-3';

[0075] S2: Referring to the operation of Example 1 above, PCR amplification is performed using the above primers with the cDNA as a template, and the amplified product is detected and recovered by electrophoresis;

[0076] S3: The plasmid pRHVnGFP was digested with BamH I (37°C for 10 h), and the digestion products were detected and recovered by electrophoresis;

[0077] S4: Ligate the enzyme digestion product of the plasmid pRHVnGFP with the PCR amplification product. The 10 μL ligation system is designed as follows:

[0078] pRHVnGFP digestion product, 1 μL;

[0079] PCR amplification product (LmbZIP11 gene fragment), 0.2 pmol;

[0080] 2× SoSo Mix, 5 μL;

[0081] Add ddH2O to 10 μL;

[0082] Connect at 50°C for 15 minutes;

[0083] S5: Use the heat shock transformation method to transform the above ligation products into Escherichia coli DH5α competent cells, and screen the positive clones for bacterial liquid PCR identification and sequencing identification to ensure that the transformation and recombination are correct, and finally extract the recombinant correct plasmid for use.

[0084] It should be explained that operations not described in detail can be referred to existing routine molecular biology operations and will not be repeated here.

[0085] (2) Preparation of Agrobacterium infection solution

[0086] The recombinant overexpression plasmid prepared in step (1) is transformed into Agrobacterium, the correct transformed strain is screened, and the transgenic infection solution is prepared. The specific operation steps are as follows:

[0087] S1: Add 5 μL of recombinant overexpression plasmid to 50 μL of GV3101 Agrobacterium competent cells, incubate on ice for 30 minutes, then incubate at 42°C for 5 minutes, and then place on ice for 5 minutes;

[0088] S2: Add 700 μL of antibiotic-free LB liquid medium and culture at 28°C and 200 rpm for 3 h;

[0089] S3: Centrifuge the culture medium at 8000 rpm for 2 min, discard part of the supernatant, mix the remaining precipitate with the liquid culture medium by pipetting, spread it on a solid LB medium containing resistance (Rif, Kan), and culture it upside down in a constant temperature incubator at 28°C for 36-48 hours to obtain the transformed Agrobacterium liquid.

[0090] (3) Arabidopsis transformation

[0091] The infection solution prepared in step (2) was used to transform Arabidopsis thaliana by the inflorescence infection method. The specific operation is as follows.

[0092] S1: Select 25-day-old Arabidopsis plants in good growth condition and remove fruiting pods before infection;

[0093] S2: Take 1 mL of transformed Agrobacterium culture solution and transfer it into 150 mL of LB liquid medium containing Rif and Kan resistance. Incubate in a shaker at 28°C and 250 rpm for 16-20 hours.

[0094] S3: Prepare the resuspension as follows: dissolve 50 g sucrose and 0.5 g MES in 1 L of pure water, autoclave, cool, and add 200 μL of Silwet L-77.

[0095] S4: Centrifuge the cultured Agrobacterium at 6000 rpm for 10 min, discard the supernatant, collect the bacteria into a 50 mL centrifuge tube, resuspend them in the prepared resuspension solution, and adjust the OD600 of the bacteria to 0.8;

[0096] S5: Place the Arabidopsis thaliana pot on its side and pour the infection solution into the sterilized infection tray. Use tweezers to immerse the Arabidopsis inflorescence in the infection solution for 2 minutes, ensuring that the buds are completely immersed. For buds that are difficult to immerse, use a pipette to draw up the infection solution for immersion.

[0097] S6: The infected Arabidopsis plants were kept in the dark for 1 day, and then transferred to normal culture conditions for further cultivation. The seeds were collected after the Arabidopsis plants set fruit.

[0098] During the screening and cultivation process, when the transgenic seedlings obtained by the preliminary screening grew to 8 true leaves, genomic DNA of the T0 generation Arabidopsis leaves was extracted and PCR verification was performed to confirm the positive transgenic plants.

[0099] T0-generation transgenic Arabidopsis plants that were initially positive were grown until seeds were harvested, and these seeds were then used to produce T1-generation plants. Subsequently, PCR amplification was performed using genomic DNA from T1-generation plants as a template using the vector-specific upstream primer pRHVnGFP-LmbZIP11-F and the gene-specific downstream primer pRHVnGFP-LmbZIP11-R. Plants that amplified a specific single band were considered overexpression-positive plants.

[0100] The specific designs of the vector-specific upstream primer pRHVnGFP-LmbZIP11-F and the gene-specific downstream primer pRHVnGFP-LmbZIP11-R are as follows:

[0101] pRHVnGFP-LmbZIP11-F:5'-GCGATCACATGGTCCTGC-3',

[0102] pRHVnGFP-LmbZIP11-R:5'

[0103] -TCAGTACATCAACATATCAGCAGCA-3'.

[0104] (IV) Expression analysis of LmbZIP11 overexpressed in Arabidopsis

[0105] S1: Using the stem leaves of the Arabidopsis wild-type group and the UBI::LmbZIP11 experimental group (25 days after planting) as samples, total RNA was extracted and reverse transcribed to prepare cDNA as described above;

[0106] S2: Design the primer sequences for qPCR detection of the internal reference gene (AtActin) as follows:

[0107] AtActin-F: 5'-AGCACTTGCACCAAGCAGCATG-3',

[0108] AtActin-R: 5'-ACGATTCCTGGACCTGCCTCATC-3';

[0109] The target gene and reaction system are as described above.

[0110] Example 3: Construction of VIGS silencing vector and transient transformation of the NbbZIP11 gene homologous to LmbZIP11 in Nicotiana benthamiana

[0111] Similar to the study objectives in Example 2, the function of the LmbZIP11 gene was further investigated and clarified. A VIGS transient silencing system targeting the NbbZIP11 homologous gene in Nicotiana benthamiana was constructed, predicting a silencing effect on NbbZIP11 in tobacco, and further transformation of tobacco was performed. The specific experimental procedures and results are briefly described below.

[0112] (1) Construction of VIGS silencing vector

[0113] Through sequence alignment, based on the specific sequence of tobacco NbbZIP11, which is homologous to LmbZIP11, a specific 300bp nucleic acid fragment was selected as the guide sequence to construct a transient silencing VIGS vector for silencing the tobacco NbbZIP11 gene. The specific process is as follows:

[0114] S1: Design the primer sequences for PCR amplification as follows:

[0115] NbbZIP11-VIGS-F:5'

[0116] -GCGGTTACCGAATTCATGGATTCGTCGAGTGGGAC-3',

[0117] NbbZIP11-VIGS-R:5'

[0118] -GGAGGCCTTCTAGAGAATTCGTGGCTAAGTTCACCCACTTGA-3';

[0119] Referring to the operation of Example 1 above, the cDNA was used as a template and the above primer sequences were used for PCR amplification (amplification length: 300 bp) to obtain the guide sequence of VIGS;

[0120] S2: Using seamless cloning and homologous recombination ligation, the amplified guide sequence was ligated to the pTRV2 vector at the EcoR I restriction site, and further screening and sequencing were performed to confirm the correct connection, thereby constructing the pTRV2-LmbZIP11 vector.

[0121] The connection system is designed as follows:

[0122] pTRV2 digestion product, 1 μL;

[0123] PCR amplification product (LmbZIP11-VIGS gene fragment), 0.2 pmol;

[0124] 2× SoSo Mix, 5 μL;

[0125] Add ddH2O to 10 μL;

[0126] Connect at 50℃ for 15 minutes.

[0127] (2) Preparation of transfection solution

[0128] S1: The correctly connected pTRV2-LmbZIP11 vector was transformed into Agrobacterium tumefaciens GV3101 using the freeze-thaw method. Positive monoclonal colonies were selected for bacterial liquid PCR verification to ensure correct transformation. The correctly transformed strains were stored for future use. As controls, pTRV1 and pTRV2-PDS (positive control, phytoene dehydrogenase, PDS) were transformed into Agrobacterium tumefaciens GV3101 under the same operating conditions, and control transfection solutions were prepared.

[0129] S2: A single Agrobacterium colony of correctly transformed pTRV1, pTRV2-PDS (positive control), or pTRV2-LmbZIP11 was inoculated into 700 μL of LB liquid medium (Rif, Kan) and cultured overnight with shaking at 28°C and 200 rpm for approximately 20 h. The colony was then transferred to 50 mL of LB liquid medium (Rif, Kan) and cultured overnight with shaking at 28°C. After the culture was completed, the colony was centrifuged at 6000 rpm for 10 min, and the Agrobacterium pellet was collected into a 50 mL centrifuge tube.

[0130] S3: Resuspend the bacterial pellet using the resuspension solution in Example 2 and adjust OD600 to about 1.0.

[0131] S4: Add an equal volume of the Agrobacterium resuspension containing pTRV2-PDS and pTRV2-LmbZIP11 to the Agrobacterium resuspension containing pTRV1, mix well, and let stand at room temperature for 3-6 hours as the transfection solution.

[0132] (3) Transient infection

[0133] Pre-cultivated (20 days after planting) Nicotiana benthamiana seedlings with consistent growth were used as transformation plants. For transient infection, select leaves with consistent growth. Using a 2 mL sterile syringe, draw up the mixture and place the rounded tip of the syringe against the lower back of the tobacco leaf. Press the corresponding position on the front with your hand and inject the bacterial solution until the entire leaf is water-soaked. Incubate each group of injected Nicotiana benthamiana plants in the dark for 2 days before returning to normal culture. Subsequent experiments were conducted 30 days after infection, after the positive control pTRV2-PDS exhibited a leaf albino phenotype.

[0134] (IV) LmbZIP11 expression analysis in tobacco VIGS system

[0135] S1: Using leaves of Nicotiana benthamiana wild-type group, pTRV2-PDS positive silenced group, and pTRV2-LmbZIP11 experimental group (30 days after infection) as samples, total RNA was extracted and reverse transcribed to prepare cDNA according to the procedure described in Example 1 above;

[0136] S2: Design the primer sequences for qPCR detection as follows:

[0137] The primer sequence of the internal reference gene (NbActin) was designed as follows:

[0138] NbActin-F: 5'-AAGGATGCCGTGAAGAAGATGT-3',

[0139] NbActin-R: 5'-GCATCGTAGTCAGGAGTCAACC-3';

[0140] The target gene primer sequences are:

[0141] LmbZIP11-RT-F: 5'-CTGGGGCTGGGTTCTATCAC-3',

[0142] LmbZIP11-RT-R: 5'-GCCAGCTGAAGCAAACACAT-3';

[0143] The reaction system is as mentioned above.

[0144] Verification example: Verification of the regulation of chlorogenic acid and total flavonoids by LmbZIP11 gene and its NbbZIP11 gene

[0145] For the overexpression Arabidopsis plants constructed in Example 2 and the transiently transformed tobacco leaves in Example 3, the inventors detected and analyzed the expression level of the bZIP11 gene and the content of related secondary metabolites, and the details are briefly described below.

[0146] (1) Gene expression

[0147] For the overexpression Arabidopsis positive plants, the leaves (25 stem leaves after planting) were used as samples, and the expression level was detected by qPCR method with reference to the above operation. The results are as follows Figure 3 As shown, it can be seen that the gene expression levels of Arabidopsis plants overexpressing LmbZIP11 are significantly different. Compared with the untransformed wild type, the gene expression level of LmbZIP11 is significantly increased.

[0148] The results of the detection of the expression level of NbbZIP11, which is homologous to the LmbZIP11 gene, in the leaves of Nicotiana benthamiana plants 30 days after gene silencing showed that (e.g. Figure 3 There was little difference in the gene silencing effect among different strains, but the expression level of NbbZIP11 gene in the plants after gene silencing was significantly reduced compared with the wild type. This result indicated that the transient infection experiment was relatively successful.

[0149] (2) Total flavonoid content

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

[0151] S1: Weigh approximately 0.1 g of fresh sample and grind it with liquid nitrogen. After the liquid nitrogen evaporates completely, add 1.5 mL of 60% ethanol and extract at 60°C with shaking for 2 h. Then, centrifuge at 12,000 rpm for 10 min at room temperature and collect the supernatant for analysis.

[0152] S2: In alkaline nitrite solution, flavonoids can form red complexes with aluminum ions with a characteristic absorption peak at 510 nm. Therefore, the absorbance value of the reaction product at 510 nm is measured to ultimately calculate the total flavonoid content in the sample.

[0153] The results are as follows Figure 4 As shown in the figure, the total flavonoid content in Arabidopsis plants overexpressing the LmbZIP11 gene increased significantly, by 63.8% compared to the control plants. In contrast, the total flavonoid content in Nicotiana benthamiana plants silenced for the homologous LmbZIP11 gene decreased significantly, by 31.3% to 37.4% compared to the control plants.

[0154] (3) Chlorogenic acid content

[0155] When determining the content of chlorogenic acid, a secondary metabolite in a sample, the specific operation is as follows:

[0156] S1: Grind the collected samples in liquid nitrogen, place them back in a -80°C refrigerator for 30 minutes, and then freeze-dry them in a vacuum freeze dryer for 2 days until they are completely dry. Then, pass the dried samples through a 60-mesh sieve for later use.

[0157] S2: Weigh approximately 0.1 g of freeze-dried sample, dissolve in 10 mL of 70% methanol, and extract by ultrasonication at 40 kHz and 250 W for 60 min. After ultrasonication, make up the volume to 10 mL.

[0158] S3: After standing at room temperature, centrifuge at 12000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm syringe filter for testing.

[0159] S4: Determination of chlorogenic acid content by liquid chromatography:

[0160] An Agilent 1100 series high performance liquid chromatography system was used, with a Pt-C18 column (250 × 4.6 nm, 5 μm); mobile phase A: 100% acetonitrile; mobile phase B: 0.5% acetic acid in water;

[0161] The gradient elution program is as follows:

[0162] 0-10min, 12%-20%A; 10-20min, 20%A; 20-25min, 20%-12%A.

[0163] Injection volume 10 μL, flow rate 1 mL / min;

[0164] The column temperature was 30°C and the UV wavelength was 330 nm.

[0165] The results are as follows Figure 5 As shown in the figure, chlorogenic acid content was significantly increased in Arabidopsis plants overexpressing the LmbZIP11 gene, with a 90.8% increase compared to the control plants. In contrast, chlorogenic acid content was significantly decreased in Nicotiana benthamiana plants silenced for the homologous LmbZIP11 gene, with a 33.1% to 46.3% decrease compared to the control plants.

[0166] The present invention clearly discovered a new gene that regulates plant chlorogenic acid by measuring the content of various plants.

[0167] The above experimental results show that the method and application of the LmbZIP11 gene and its homologous NbbZIP11 gene provided by the present invention in regulating the content of chlorogenic acid and total flavonoids in plants are positively correlated. That is, by overexpressing the gene, the content of total flavonoids and chlorogenic acid in Arabidopsis thaliana can be increased, and by reducing the expression of the gene by gene silencing, the content of total flavonoids and chlorogenic acid in Nicotiana benthamiana can be reduced. Compared with the existing technology, it can comprehensively and objectively evaluate how the LmbZIP11 gene regulates the content of chlorogenic acid and total flavonoids in plants, and will provide technical support for quality innovation of Lonicera japonica.

Claims

1. A LmbZIP11 gene of Lonicera japonica, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2; the gene can increase the content of chlorogenic acid and total flavonoids in Arabidopsis thaliana.

2. A Nicotiana benthamiana NbbZIP11 gene, characterized in that The NbbZIP11 gene is a homologous gene of the LmbZIP11 gene according to claim 1, and its nucleotide sequence is shown in SEQ ID NO: 3; the gene can reduce the content of chlorogenic acid and total flavonoids in Nicotiana benthamiana.

3. A use of the Lombokinesia grisea LmbZIP11 gene according to any one of claims 1 to 2 in regulating the content of chlorogenic acid and total flavonoids in plants, characterized in that: The gene is transformed into Arabidopsis thaliana by constructing an overexpression vector, thereby increasing the content of chlorogenic acid and total flavonoids in Arabidopsis thaliana; The overexpression vector construction method comprises: using pRHVnGFP plasmid as a vector, connecting LmbZIP11 gene via BamH I restriction site; transforming Agrobacterium with the recombinant vector to prepare infection solution; transforming Arabidopsis thaliana by inflorescence infection method, and screening positive transgenic plants.

4. An application of the Nicotiana benthamiana NbbZIP11 gene in regulating the content of chlorogenic acid and total flavonoids in plants, characterized in that: The gene is transformed into Nicotiana benthamiana by constructing a VIGS silencing vector, thereby reducing the content of chlorogenic acid and total flavonoids in Nicotiana benthamiana; The method for constructing the VIGS silencing vector includes: designing specific primers for the LmbZIP11 gene and amplifying a guide sequence with a length of 300bp; connecting the guide sequence to the pTRV2 vector to construct the pTRV2-LmbZIP11 silencing vector; transforming the recombinant vector into Agrobacterium to prepare a transfection solution; and transforming Nicotiana benthamiana using a transient infection method to screen plants with significant silencing effects.

5. The use of the Lombok LmbZIP11 gene of claim 3 in regulating the content of chlorogenic acid and total flavonoids in plants, characterized in that: The construction primers of the overexpression vector are: LmbZIP11-BamH IF: 5' -ACGAGCTGTACAAGGATGGATTCGTCGAGTGGGAC-3', LmbZIP11-BamH IR:5' -CCGAGCTCACCCGGGTCAGTACATCAACATATCAGCAGCA-3'.

6. The use of the Nicotiana benthamiana NbbZIP11 gene in regulating the content of chlorogenic acid and total flavonoids in plants according to claim 4, characterized in that: The construction primers of the VIGS silencing vector are: NbbZIP11-VIGS-F:5' -GCGGTTACCGAATTCATGGATTCGTCGAGTGGGAC-3', NbbZIP11-VIGS-R:5' -GGAGGCCTTCTAGAGAATTCGTGGCTAAGTTCACCCACTTGA-3'.

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