CN116716335A - Application of dandelion TmABI5 gene in promoting plant to synthesize chicoric acid - Google Patents

Abstract

The invention discloses an application of dandelion TmABI5 gene in promoting plants to synthesize chicoric acid, and belongs to the technical field of plant molecular biology. According to the invention, dandelion leaves are used as materials, dandelion TmABI5 genes are obtained through cloning, an over-expression vector pHellgate-TmABI 5-GFP is constructed on the basis, and the dandelion leaves are transferred into dandelion to obtain transgenic plants. The analysis result of the chicoric acid content shows that the chicoric acid content of the transgenic plant is obviously higher than that of the control group; the gene function identification result shows that the TmABI5 gene activates the expression of the Tm4CL1 gene in dandelion through transcription, and the content of chicoric acid is increased. The TmABI5 gene can be used as an important gene for carrying out genetic engineering and improved breeding of dandelion.

Description

Application of dandelion TmABI5 gene in promoting plant to synthesize chicoric acid

Technical Field

The invention belongs to the technical field of plant molecular biology, and particularly relates to an application of dandelion TmABI5 gene in promoting plant to synthesize chicoric acid.

Background

Dandelion (Taraxacum mongolicum hand-mazz.) originates in the middle east and spreads throughout the country, belonging to the genus Taraxacum (Taraxacum f.h.wigg.) of the family Asteraceae. The dandelion contains rich chicoric acid, chlorogenic acid, dandelionsterol and other components, and has the functions of resisting bacteria, diminishing inflammation, resisting oxidation, promoting urination, preventing cancer and other medicine effects. Phenolic acid materials are involved in the formation and development of flowers, fruits, seeds and other functions in plants, such as antioxidant activity, protection against biological and non-biological attack by plant pathogens. The phenolic acid compounds which are studied in the current stage are mainly chlorogenic acid, caffeic acid, chicoric acid, salicylic acid and the like. Therefore, the identification of transcription factors involved in the synthesis and regulation of phenolic acids is of great biological and industrial significance.

The phenolic acid synthesis pathway in plants mainly originates from the phenylalanine pathway, which involves multiple enzymes, but the last step of the enzymatic reaction in the first three steps of the phenylalanine pathway has the synthase gene of 4-coumaric acid: coenzyme A ligase (4 CL gene), 4CL gene is the key switch of phenolic acid synthesis downstream, 4CL gene has been cloned and well analyzed in Arabidopsis thaliana, tomato, potato, grape, echinacea and other multiple plants, and 4CL gene expression level is regulated by transcription factors. Transcription factors are typically capable of binding to cis-acting elements on structural gene promoters to regulate expression of structural genes. Wherein the bZIP transcription factor activates the phenolic acid multiple synthase gene promoters to regulate biosynthesis of phenolic acid compounds. However, the bZIP transcription factor in dandelion has not been reported to regulate the metabolic pathway of dandelion chicoric acid. The ABI5 transcription factor has been reported to bind to the 4CL gene promoter. Thus, it is presumed that there is a pathway in dandelion in which the ABI5 transcription factor activates the 4CL promoter and thus controls dandelion chicoric acid biosynthesis.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide the application of the dandelion TmABI5 gene in promoting the plant to synthesize chicoric acid, thereby improving the quality of dandelion.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the application of the dandelion TmABI5 gene in promoting dandelion to synthesize chicoric acid is provided, and the nucleotide sequence of the dandelion TmABI5 gene is shown in SEQ ID NO. 1.

The application of the dandelion TmABI5 gene in promoting dandelion to synthesize chicoric acid comprises the following steps:

(1) Constructing a carrier of a dandelion TmABI5 gene;

(2) Transforming the constructed dandelion TmABl5 gene vector into dandelion;

(3) And (5) culturing and screening to obtain the transgenic dandelion with increased chicoric acid content.

The transformation is genetic transformation mediated by agrobacterium.

The vector is a plant expression vector.

The plant expression vector is pHellgate-TmABI 5-GFP.

The application of the dandelion TmABI5 gene in promoting the expression of the Tm4CL1 gene is provided, and the nucleotide sequence of the dandelion TmABI5 gene is shown as SEQ ID NO. 1.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, dandelion leaves are used as materials, dandelion TmABI5 genes are obtained through cloning, an over-expression vector pHellgate-TmABI 5-GFP is constructed on the basis, and the dandelion leaves are transferred into dandelion to obtain transgenic plants. The analysis result of the chicoric acid content shows that the chicoric acid content of the transgenic dandelion is obviously higher than that of the control group; the gene function identification result shows that the TmABI5 gene activates the expression of the Tm4CL1 gene in dandelion through transcription, and the content of chicoric acid is increased.

Drawings

FIG. 1 is a pHelsgate-TmABI 5-GFP vector construction;

FIG. 2 shows a map of subcellular localization of the TmABI5 gene protein in tobacco leaf cells;

FIG. 3 shows a flow chart of genetic transformation of dandelion;

FIG. 4 is a PCR identification map of TmABI5 overexpressing transgenic dandelion;

FIG. 5 is a graph showing analysis of the expression level of TmABI5 gene in over-expressed dandelion;

FIG. 6 is a graph of the content analysis of TmABI5 transgenic dandelion chlorogenic acid, tartaric acid and chicoric acid;

FIG. 7 is a diagram showing the verification of TmABI5 binding to Tm4CL1 gene promoter by single hybridization of yeast and Dual-LUC test.

Detailed Description

The present invention will be further described with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present invention more apparent. Unless otherwise indicated, all technical means used in the following examples are conventional means well known to those skilled in the art.

The plant material adopted by the invention is dandelion with the size of 2 months, the illumination is 8/16h, and the illumination intensity is 2000-30001x.

Example 1: taraxacum mongolicum TmABI5 gene clone

1. Primer design

The present invention screens sequences for homology higher than 60% in existing transcriptome databases (NCBI accession number: PRJNA 861012) and genomic data (Lin, T et al 2022) based on reported AB15 gene sequences in other species using local blast alignment. According to the coding sequence of TRITY_DN 564_c0_g2, the Primer design is carried out by utilizing an online sequence Primer 5, wherein the Primer sequence is as follows:

TmABI5-F：5’-ATGAGTTCGTTTATCAACTTC-3’，

TmABI5-R：5’-CTACCATGGCCCTGTTAATG-3’。

2. gene cloning and recovery of target fragment

Total RNA of dandelion leaves was extracted using a polysaccharide polyphenol plant total RNA extraction kit produced by TARAKA company, and the specific method is referred to in the specification.

Taraxacum mongolicum leaf RNA was reverse transcribed into cDNA using an RNA reverse transcription kit manufactured by the full gold company.

Using the cDNA synthesized by reverse transcription as a template, the PCR system was carried out according to a conventional PCR system (12.5. Mu.L mix, 11.5. Mu. L H) ₂ O, 0.5. Mu.L of each of the upstream and downstream primers, 1. Mu.L of the template, 25. Mu.L in total) and a PCR reaction program (pre-denaturation 94℃for 3min;30 cycles: denaturation at 94℃for 30s, annealing at 52℃for 30s, and extension at 72℃for 30s; and finally, the time is prolonged for 10min at 72 ℃) to clone and obtain the target gene. The size of the target fragment is between 1000 and 1500 bp. After completion of the reaction, the amplified product was purified by 1Separating by electrophoresis with% agarose gel; the correct size strips were excised and recovered and purified using a TAKARA gel recovery kit.

3. Construction of cloning vector and transformation of E.coli

Recovery of the product the target gene was ligated into the cloning vector using a conventional Ta cloning vector kit and transformed into e.coli DH5a, and positive identification of the monoclonal strain was performed using primers M13F (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') and TmABI5R (5'-CTACCATGGCCCTGTTAATG-3'). The PCR identified correct (1000-1500 bp) strain was sent to Shanghai Biotechnology Co., ltd for sequencing, and the nucleotide sequence of the Taraxacum TmABI5 gene (NCBI accession number: 2706189) is shown in SEQ ID NO. 1.

Example 2: construction and function verification of dandelion TmABI5 gene expression vector

1. Construction of fusion expression vectors

And (3) using the constructed cloning vector as a template, redesigning a vector construction primer to amplify the target gene, so that the upper and lower streams of the target gene are provided with XhoI enzyme cutting sites, and performing single enzyme cutting on pHellgate-GFP stored in a laboratory by using XhoI enzyme to recover the target gene and a large vector fragment (the vector construction is shown in figure 1). The sequence of the desired primer is as follows:

G1-TmABI5-F：

5’-TTTGGAGAGGACACGCTCGAGATGAGTTCGTTTATCAACTTC-3’，

G1-TmABI5-R：

5’-GCTCACCATGAATTCCTCGAGCCATGGCCCTGTTAATG-3’。

TmABI5 was ligated into pHellgate-GFP vector using a noniprandial homologous recombination kit according to the instructions, and DH5a strain was transformed and then subjected to conventional positive identification to obtain a plant fusion expression vector pHellgate-TmABI 5-GFP. Positive identification and sequencing analysis were performed using 35S-F (5'-GACGCACAATCCCACTATCC-3') and the target gene downstream primer TmAB15-R (5'-CTACCATGGCCCTGTTAATG-3'). The strain with the correct sequencing result is subjected to plasmid extraction for standby.

2. Tobacco subcellular localization

Dividing the constructed recombinant plasmid and the control plasmidAnd (3) transforming the agrobacterium GV3101 strain, and selecting a positive strain after colony PCR verification is correct, and placing the correct positive strain in a refrigerator at 4 ℃ for later use. Preparation of Agrobacterium solution culturing according to conventional culture conditions to OD ₆₀₀ ＝0.8-1.0。

Adding 100 mu M acetosyringone into the cultured bacterial liquid, and after light shielding for 2 hours, injecting the bacterial liquid into the surface skin of the back of the tobacco leaf by using a 1mL syringe, and observing 35S after light shielding for 48-72 hours: tmABI5: GFP fusion protein.

The results are shown in FIG. 2, and the laser confocal microscope observation is carried out at 35S: tmABI5: GFP fusion proteins localize to the nucleus consistent with other members of the bZIP family of other subfamilies a.

3. Agrobacterium-mediated transformation of dandelion

Sterilizing herba Taraxaci seeds stored in laboratory with 75% ethanol for 5-10min, washing with sterile water for 3 times, sucking dry filter paper, and placing on MS solid culture medium plate with sterilized filter paper; growing for 1 week to root length of 2-3cm, transferring 2-4 leaves to a culture flask (250 mL), and continuing to grow for 2 months; cutting the healthy dandelion leaves with a sterile scalpel to a leaf size of 0.5-1cm ² Pre-culturing for 48-72h, wherein the pre-culturing culture medium is MS solid culture medium; the GV3101 strain containing pHellsgate-TmABI5-GFP fusion expression vector is subjected to conventional activation culture, and the bacterial solution is cultured to OD ₆₀₀ =0.8-1.0; suspending the pre-cultured dandelion leaves for 3-5min by using agrobacterium tumefaciens bacteria solution, sucking the leaves to dryness by using sterile filter paper, transferring the leaves to a co-culture medium (MS solid medium), and culturing the leaves in darkness for 48-72h; leaf placement callus induction (MS+0.1 mg/L6-BA+2 mg/L NAA+30.0g/L sucrose+7.0 g/L agar+50 mg/L Kan+500mg/L cefotaxime) after co-cultivation for about 2-3 weeks; transferring the induced callus to bud induction culture medium (MS+2mg/L6-BA+0.2 mg/L NAA+30.0g/L sucrose+7.0 g/L agar+50 mg/L Kan+500mg/L cefotaxime), growing for about 4-6 weeks, and decreasing the concentration of cefotaxime antibiotics every week (gradient is 400, 300, 200, 100 in sequence); transferring to rooting culture medium (1/2MS+2 mg/L6-BA+0.2 mg/L NAA+30.0g/L sucrose+7.0 g/L agar+50 mg/L Kan+100mg/L cefotaxime) after the buds grow to 3-5cm for about 4-5 weeks; hardening seedlings of dandelion with good rooting for 3-5 days, and transferring to a soil matrix(humus/vermiculite = 3/1). And finally, obtaining 19 transgenic lines to be identified, wherein the 19 transgenic lines grow well.

4. Transgenic plant detection

According to the instruction of the root plant genome DNA extraction kit, extracting the DNA of 19 dandelion leaves with good growth obtained in the above way, and carrying out gene detection. Primers Kan-F (5'-ATGGCAATTACCTTATCCGC-3') and Kan-R (5'-TCAGAAGAACTCGTCAAGAAG-3') carried by the strain and upstream and downstream of the Kan+ gene are used; the GFP-F (5'-ATGGTGAGCAAGGGCGAGGAG-3') and GFP-R (5'-TTACTTGTACAGCTCGTCCATG-3') primers were used to identify the presence of Kan+ and GFP genes in wild type plants, empty control plants and positive plants, respectively. The results show that the wild type does not contain Kan+ gene and GFP gene; the empty control plants and the positive plants contain Kan+ genes and GFP genes.

Positive plants were further screened using vector 35S promoter primer 35S-F (5'-GACGCACAATCCCACTATCC-3') and target gene downstream primer TmABI5-R (5'-CCATGGCCCTGTTAATG-3').

As a result, as shown in FIG. 3, 4 positive TmABI5 transgenic lines (OE 1-4) and 1 Empty Vector (EV) control lines were obtained by screening.

The gene expression levels of TmABI5 in the different strains were further analyzed using qRT-PCR using qRT-TmABI5-F (5'-TTCAACCGCAAATCACAGAG-3') and qRT-TmABI5-R (5'-TGGAGATCCAGCACCAAGAC-3'), internal reference genes are Actin-F (5'-AGCAGCTTCCATTCCGATCA-3') and Actin-R (5'-GGTTACATGTTCACCACCAC-3'), quantitative test methods reference (Liu et al, 2021).

As a result, as shown in FIG. 4, the expression level of the TmABI5 gene was significantly higher in each of the 4 over-expressed strains (OE 1-4) than in the empty vector control strain (EV 1).

Example 3

10mL of dry samples (OE 1-4 and EV 1-3) were weighed and extracted in an ultrasonic apparatus (Qsonic 700, USA) with 1mL of 70% methanol (v/v) at an amplitude of 50%,40s/20s at 4℃for a total of 15min. The extract was centrifuged at 5000 Xg for 10min at 4℃and the supernatant was collected. The residue was extracted again using the same conditions. Both supernatants were combined and diluted to 2.5mg/mL (dry matter and solvent). Agilent 1100 high performance liquid chromatography was used with Phenomenex Gemoni u C18 110A (250X 4.6mm,5 μm) as a liquid chromatography column. The flow rate was kept at 1.0mL/min and absorbance was measured by a UV detector at a wavelength of 254 nm. Three replicates were performed. The chlorogenic acid, monocaffeoyltartaric acid and chicoric acid contents in the obtained 4 transgenic lines were examined.

The liquid phase conditions are as follows: the mobile phase was 0.1% formic acid water (a) and acetonitrile (B). The gradient is as follows: 2% B,0.5min;2-14% B,1min;14-18% B,5.5min;18-80% B,2min;80% B,1min, and then the column was rebalanced with 2% B for 3min.

The results are shown in FIG. 5, where the content of chicoric acid in the transgenic line OE4 is significantly higher than in the other transgenic lines; the content of chicoric acid in the empty control strain EV1 is 16.68+/-2.85 mg/g, which is obviously higher than that of other empty control strains.

Example 4

And (3) carrying out chicoric acid synthetase gene expression analysis on the OE4 strain and the EV1 strain obtained by screening.

Results as shown in fig. 6, PAL1, C4H1, 4CL1, HCT2 and HQT/HTT1 gene expression levels were significantly higher in OE4 lines than in EV1 lines, thus concluding that: the TmABI5 gene promotes the expression of the genes so as to improve the content of chicoric acid and precursor compounds thereof, the expression pattern of the TmABI5 gene is consistent with that of the Tm4CL1 gene, and the TmABI5 gene can promote the expression of the Tm4CL1 gene.

Example 5

The forward 1564bp region of the Tm4CL1 promoter was obtained using chromosome walking (RACE technique) and was found to contain 1 ABRE cis-motif and 2G-box like motif, respectively. Further validation was performed using a yeast single hybridization experiment.

As a result, as shown in FIG. 7A, the TmABI5 gene is capable of binding to ABRE cis-motif; this suggests that the TmABI5 gene is capable of binding to the Tm4CL1 promoter; the results are shown in FIG. 7B, and the Dual-LUC results show that the TmABI5 gene promotes the expression of the Tm4CL1 gene.

Based on the above analysis, it was concluded that: the TmABI5 gene activates the expression of the Tm4CL1 gene in dandelion through transcription, so that the content of chicoric acid is increased.

Claims (6)

1. The application of the dandelion TmABI5 gene in promoting dandelion to synthesize chicoric acid is provided, and the nucleotide sequence of the dandelion TmABI5 gene is shown in SEQ ID NO. 1.

2. The use according to claim 1, characterized by the steps of:

(1) Constructing a carrier of a dandelion TmAB15 gene;

(2) Transforming the constructed dandelion TmABI5 gene vector into dandelion;

(3) And (5) culturing and screening to obtain the transgenic dandelion with increased chicoric acid content.

3. The use according to claim 2, wherein said transformation is transient transformation by agrobacterium.

4. The use according to claim 2, wherein said vector is a plant expression vector.

5. The use according to claim 4, wherein the plant expression vector is pHellgate-TmABI 5-GFP.

6. The application of the dandelion TmABI5 gene in promoting the expression of the Tm4CL1 gene is provided, and the nucleotide sequence of the dandelion TmABI5 gene is shown as SEQ ID NO. 1.