Application of MhMYB1 gene in improving total flavonoids content of Himlaya Mirabilis

Abstract

The application discloses application of MhMYB1 gene in improving flavonoid content in Miraculum himalayanum, wherein the nucleotide sequence of the MhMYB1 gene is shown as SEQ ID NO. 1, the encoded amino acid sequence is shown as SEQ ID NO. 2, overexpression of the MhMYB1 gene can improve the content of flavonoids in Miraculum himalayanum hairy roots, therefore, the discovery of the MhMYB1 gene of Miraculum himalayanum provides relevant gene resources for molecular breeding aiming at improving flavonoid content in medicinal plants, and can be applied to large-scale production of flavonoids in Miraculum himalayanum, and has great application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of the MhMYB1 gene in increasing the total flavonoid content of Himalayan four o'clock flower. Background Technology

[0002] Himalayan Mirabilis jalapa is a top-grade root in Tibetan medicine. As a tonic, it is widely used in Tibetan medicinal preparations such as the Twenty-Five Flavor Catechu Pill and the Three Flavor Himalayan Mirabilis jalapa Decoction. Market demand is constantly increasing, making it one of the most promising specialty varieties among Tibetan medicinal materials. However, due to the extremely narrow habitat distribution of Himalayan Mirabilis jalapa, and the fact that only its roots are used medicinally, relying solely on wild harvesting would have a devastating impact on the already endangered species. Furthermore, artificially cultivated Himalayan Mirabilis jalapa has a lower content of flavonoids compared to wild resources. Flavonoids are the main active ingredients in Himalayan Mirabilis jalapa, and their content directly determines the quality of the plant. Therefore, cultivating Himalayan Mirabilis jalapa with high flavonoid content has been a long-term goal of the industry.

[0003] Existing research indicates that MYB transcription factors are key regulators of flavonoid biosynthesis, controlling the expression of multiple enzyme genes involved in the flavonoid synthesis pathway. This has significant research value in cultivating new medicinal plant varieties that yield high levels of flavonoids. By deeply analyzing the genome and transcriptome of *Mirabilis jalapa*, identifying the MYB transcription factors regulating flavonoid accumulation in *Mirabilis jalapa*, and utilizing genetic engineering techniques, it is possible to achieve large-scale flavonoid synthesis in *Mirabilis jalapa*. Therefore, identifying transcription factors that regulate the flavonoid biosynthesis pathway is crucial for cultivating *Mirabilis jalapa* varieties with high flavonoid yields. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide an application of overexpression of the MhMYB1 gene in increasing the total flavonoid content of Himalayan four o'clock flower; another objective of the present invention is to provide a method for increasing the total flavonoid content in Himalayan four o'clock flower.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] 1. Application of overexpression of MhMYB1 gene in increasing total flavonoid content in Himalayan four o'clock flower.

[0007] The MhMYB1 gene is derived from Himalayan four o'clocks, and the nucleotide sequence of the MhMYB1 gene is shown in SEQ ID NO.1. The amino acid sequence encoded by the MhMYB1 gene is shown in SEQ ID NO.2.

[0008] 2. A method for increasing the total flavonoid content in Himalayan four o'clock flower, comprising the following steps: overexpressing the MhMYB1 gene in Himalayan four o'clock flower, wherein the nucleotide sequence of the MhMYB1 gene is shown in SEQ ID NO.1 and the amino acid sequence encoded by the MhMYB1 gene is shown in SEQ ID NO.2.

[0009] Preferably, the method for overexpressing the MhMYB1 gene in this invention is as follows: cloning the MhMYB1 gene, then constructing a plant expression vector to obtain a recombinant plant expression vector containing the MhMYB1 gene, then transforming the obtained recombinant plant expression vector into Agrobacterium rhizogenes to obtain engineered bacteria, finally transforming Himalayan Mirabilis explants with the engineered bacteria, screening for Himalayan Mirabilis hairy roots, identifying positive hairy roots by PCR, and obtaining Himalayan Mirabilis hairy roots with increased total flavonoid content.

[0010] Preferably, the method for cloning the MhMYB1 gene in this invention is as follows: using Himalayan Mirabilis cDNA as a template, PCR amplification is performed using the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers, followed by electrophoresis detection and recovery to obtain the MhMYB1 gene with homologous arms.

[0011] Preferably, the recombinant plant expression vector of the present invention is prepared by replacing the GUS gene on the pBI121 vector with the MhMYB1 gene carrying a homologous arm through homologous recombination using the pBI121 vector as the backbone.

[0012] Preferably, the Agrobacterium rhizogenes of the present invention is C58C1.

[0013] The beneficial effects of this invention are as follows:

[0014] This invention discloses the MhMYB1 gene from *Mirabilis jalapa*, the nucleotide sequence of which is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. Overexpression of the *Mirabilis jalapa* MhMYB1 gene can increase the content of flavonoids in the hairy roots of *Mirabilis jalapa*. Therefore, the discovery of the *Mirabilis jalapa* MhMYB1 gene provides a relevant gene resource for molecular breeding to increase the content of flavonoids in medicinal plants, and can be applied to the large-scale production of flavonoids in *Mirabilis jalapa*, thus having great application value. Attached Figure Description

[0015] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0016] Figure 1 A schematic diagram of the structure of the plant expression vector pBI121-MhMYB1;

[0017] Figure 2 The results of quantitative PCR detection of the relative expression level of MhMYB1 in the hairy roots of Mirabilis jalapa in a preferred embodiment of the present invention are as follows (WT: common Mirabilis jalapa hairy roots; MhMYB1-1, MhMYB1-4, MhMYB1-6, MhMYB1-7, MhMYB1-9: represent different transgenic MhMYB1 Mirabilis jalapa hairy root lines).

[0018] Figure 3 The results of spectrophotometric determination of total flavonoid content in the hairy roots of Mirabilis jalapa in a preferred embodiment of the present invention are shown below (WT: common Mirabilis jalapa hairy root; MhMYB1-1, MhMYB1-4, MhMYB1-6, MhMYB1-7, MhMYB1-9: represent different MhMYB1 transgenic Mirabilis jalapa hairy root lines). Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0020] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0021] Example 1: Obtaining and identifying hairy roots of Himalayan Mirabilis jalapa with MhMYB1 gene overexpression

[0022] The inventors of this application isolated and cloned a MYB-type transcription factor, named MhMYB1, through in-depth analysis of the genome and transcriptome information of Himalayan four o'clock. The coding sequence (CDS) of the MhMYB1 gene is shown in SEQ ID No. 1, with a length of 1122 bp, encoding 373 amino acids, and its encoded amino acid sequence is shown in SEQ ID No. 2.

[0023] I. Construction of the pBI121-MhMYB1 recombinant vector

[0024] A suitable amount of root, stem, and leaf tissue from *Mirabilis jalapa* was collected, ground in liquid nitrogen, and RNA was extracted using the Trizol method. The RNA was then reverse transcribed into cDNA using the StarScript II reverse transcription kit A234. Gene-specific primers were designed based on the sequence of the MhMYB1 gene. The specific primers are as follows:

[0025] MhMYB1-F:5'-acgggggactctaga ggatcc atgggccgagcaccgtgt-3' (SEQ ID No. 3);

[0026] MhMYB1-R:5'-cgatcggggaaattc gagctc ctaggatagaagccaagctaccatag-3' (SEQ ID No. 4).

[0027] The MhMYB1 gene was then amplified from total cDNA using high-fidelity DNA polymerase via PCR. The amplified fragments were detected by 1% agarose gel electrophoresis and purified for later use.

[0028] The pBI121 vector was digested with BamHI and SacI, and the linearized vector backbone was recovered by DNA purification and recovery kit after 1% agarose gel electrophoresis.

[0029] The recovered MhMYB1 amplified fragment and the linearized pBI121 vector were spliced ​​and cloned using the BM Seamless Cloning Kit at a molar ratio of 3:1. After sequencing, the expression vector pBI121-MhMYB1 was obtained. Figure 1 ).

[0030] In this embodiment, the MhMYB1 gene of Himalayan Mirabilis jalapa is operatively linked to the plant expression vector pBI121, and the resulting expression vector pBI121-MhMYB1 can be used to regulate the content of flavonoids in Himalayan Mirabilis jalapa through transcriptional regulation strategies.

[0031] II. Transformation of Agrobacterium rhizogenes

[0032] The successfully ligated vector plasmid pBI121-MhMYB1 was transformed into Agrobacterium rhizogenes C58C1 strain to obtain recombinant Agrobacterium C58C1 / pBI121-MhMYB1. After PCR verification showed the presence of the expected band (1122bp), the bacterial culture was mixed with 50% glycerol in an equal ratio and stored at -80℃ for later use.

[0033] III. Genetic transformation of Mirabilis jalapa into transgenic Mirabilis jalapa hairy roots using Agrobacterium rhizogenes-mediated MhMYB1 overexpression vector.

[0034] (1) Preparation of explants from Himalayan Mirabilis jalapa

[0035] Mature and plump seeds of Mirabilis jalapa were selected, rinsed with running water overnight, then soaked in 75% ethanol for 1 minute, followed by soaking in 2% NaClO for 10 minutes. After rinsing three times with sterile water, the seeds were dried and inoculated onto MS solid medium. The seedlings were then cultured in a 20℃ light incubator to obtain sterile Mirabilis jalapa seedlings. Once the seedlings had 5-6 true leaves, explants from the sterile seedling leaves were harvested for transformation.

[0036] (2) Co-culture of Agrobacterium and explants

[0037] The explants obtained in the previous step were added to the resuspension of Agrobacterium rhizogenes engineered bacteria containing the pBI121-MhMYB1 expression vector (1 / 2 MS + AS 100 μmol / L). After co-culturing the explants and bacterial solution for 15 minutes, they were transferred to co-culture medium and incubated in the dark at 28°C for 2 days.

[0038] (3) Screening and detection of resistant hairy roots

[0039] Explants of Mirabilis jalapa, co-cultured for 2 days, were transferred to selection medium supplemented with 200 mg / L cephalosporin and 100 mg / L kanamycin and cultured in the dark until resistant hairy roots appeared. After the well-grown hairy roots were cultured to complete sterility, genomic DNA was extracted from them using the CTAB method. Using the extracted genomic DNA as a template, PCR identification was performed using primer pair (35S promoter-F and pBI121-MhMYB1-R) to obtain transgenic hairy roots overexpressing the MhMYB1 gene.

[0040] 35S promoter-F: 5'-gacgcacaatcccactatcc-3' (SEQ ID No. 5);

[0041] pBI121-MhMYB1-R: 5'-ctaggatagaagccaagctaccatag-3' (SEQ ID No. 6).

[0042] (4) Detection of MhMYB1 gene expression in transgenic hairy roots using qRT-PCR

[0043] A suitable amount of transgenic hairy roots with uniform growth were collected, RNA was extracted, and then reverse transcribed into cDNA. The expression level of the MhMYB1 gene in the sampled hairy roots was detected by qRT-PCR using primers MhMYB12-eq-F and MhMYB12-eq-R. The control internal reference gene was 18S rRNA, and the primers were 18SQ-F and 18SQ-R.

[0044] MhMYB12-eq-F: 5'-gaatggcaatccaagcgga-3' (SEQ ID No. 7);

[0045] MhMYB12-eq-R: 5'-tgcattgcagctcggctag-3' (SEQ ID No. 8).

[0046] 18SQ-F: 5'-atgataactcgacggatcgc-3' (SEQ ID No. 9);

[0047] 18SQ-R: 5'-cttggatgtggtagccgttt-3' (SEQ ID No. 10).

[0048] The results are as follows Figure 2 As shown, the expression level of MhMYB1 in the hairy roots of transgenic Four O'Clock Himalayan was significantly higher than that in ordinary hairy roots, indicating that the MhMYB1 gene was successfully transferred into Four O'Clock Himalayan and obtained hairy roots of Four O'Clock Himalayan with overexpression of the MhMYB1 gene.

[0049] (5) Determination of total flavonoid content in the hairy roots of transgenic Himalayan four o'clock flower

[0050] a. Pretreatment of plant materials: Dry the plant materials at 60℃, grind them thoroughly, pass them through a No. 4 sieve, and accurately weigh 0.1g.

[0051] b. Extraction of flavonoids: Add 2 mL of 60% ethanol to each sample as the extraction solvent, extract by sonication for 30 min, centrifuge at 12000g for 15 min, collect the supernatant in a centrifuge tube and make up to 2 mL with 60% ethanol; during the color development process, add 0.4 mL of 10% sodium nitrite to the extract, shake well and let stand for 6 min, add 0.4 mL of 10% aluminum nitrate, shake well and let stand for another 6 min, add 4 mL of 1 mmol / L sodium hydroxide, make up to 10 mL with 30% ethanol, mix thoroughly and let stand for 15 min.

[0052] c. Detection of flavonoids: The OD values ​​of all samples were measured at 510 nm using ultraviolet spectrophotometry, with each experiment repeated three times. The total content of flavonoids was calculated based on the regression curve of the standard solution.

[0053] The results are as follows Figure 3 As shown, in this invention, overexpression of the MhMYB1 gene in Himalayan Mirabilis jalapa significantly increased the total flavonoid content. In the hairy roots of Himalayan Mirabilis jalapa with MhMYB1 overexpression, the relative content of total flavonoids was 1.41-2.11 times that of ordinary hairy roots at the same stage.

[0054] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for increasing the total flavonoid content in the hairy roots of Himalayan Mirabilis jalapa, characterized in that, The method includes the following steps: overexpressing the MhMYB1 gene in the hairy roots of Mirabilis jalapa, the nucleotide sequence of which is shown in SEQ ID NO.1 and the amino acid sequence encoded by which is shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that, The method for overexpressing the MhMYB1 gene is as follows: cloning the MhMYB1 gene, then constructing a plant expression vector to obtain a recombinant plant expression vector containing the MhMYB1 gene, transforming the obtained recombinant plant expression vector into Agrobacterium rhizogenes to obtain engineered bacteria, finally transforming Himalayan Mirabilis explants with the engineered bacteria, screening for Himalayan Mirabilis hairy roots, identifying positive hairy roots by PCR, and obtaining Himalayan Mirabilis hairy roots with increased total flavonoid content.

3. The method according to claim 2, characterized in that, The method for cloning the MhMYB1 gene is as follows: using Himalayan Mirabilis cDNA as a template, PCR amplification is performed using the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers, followed by electrophoresis detection and recovery to obtain the MhMYB1 gene with homologous arms.

4. The method according to claim 2, characterized in that, The recombinant plant expression vector was prepared by replacing the GUS gene on the pBI121 vector with the MhMYB1 gene carrying a homologous arm through homologous recombination, using the pBI121 vector as the backbone.

5. The method according to claim 2, characterized in that, The Agrobacterium rhizogenes is C58C1.

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