Induction method for antirrhinum majus l. hairy root producing anthocyanin and glycoside derivative thereof, method for screening inducing gene, and production and use

Abstract

An induction method for an Antirrhinum majus L. hairy root producing an anthocyanin and a glycoside derivative thereof, a method for screening an inducing gene, and production and use, specifically belonging to the technical field of plant tissue culturing. The induction method for an Antirrhinum majus L. hairy root producing an anthocyanin and a glycoside derivative thereof comprises the following steps: using Agrobacterium rhizogenes containing an inducing gene to infect an Antirrhinum majus L. explant, co-culturing the Agrobacterium rhizogenes and the Antirrhinum majus L. explant, and performing sterilization culture to obtain the Antirrhinum majus L. hairy root producing the anthocyanin and the glycoside derivative thereof. The inducing gene comprises a gene encoding an MYB transcription factor, or a mutant thereof. The induction method can quickly and effectively obtain the hairy root synthesizing the anthocyanin and the glycoside derivative thereof while realizing the acquisition of the anthocyanin and the glycoside derivative thereof in a short time without being affected by seasons in the case of limited germplasm resources.

Description

An induction method of Antirrhinum majus L. hairy roots for producing anthocyanins and glycoside derivatives thereof, an induction gene screening method and production application TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant tissue culture, and particularly relates to an induction method of Antirrhinum majus L. (Amas) hairy roots for producing anthocyanins and glycoside derivatives thereof, an induction gene screening method and production application. BACKGROUND

[0002] Anthocyanins are natural water-soluble pigments present in many plants, and have multiple biological activities such as antioxidant, anti-inflammatory and anti-tumor activities. With the increasing awareness of the health benefits of anthocyanins, the market demand has expanded dramatically. The content of anthocyanins from natural plants is limited and greatly affected by environmental factors, which cannot meet the market needs, and therefore new production technologies need to be developed.

[0003] The production of anthocyanins by relying on traditional agricultural planting methods has problems such as long cycle, high cost, large resource consumption, and is limited by season and climate, and the yield and quality are unstable. Hairy roots have the characteristics of fast growth, no need to add exogenous hormones, high genetic stability, and are suitable for producing functional metabolites as a biological reactor. However, the current method for producing anthocyanins using hairy roots still has the defects of long induction cycle and low efficiency.

[0004] SUMMARY

[0005] The present application provides an induction method of Antirrhinum majus L. hairy roots for producing anthocyanins and glycoside derivatives thereof, an induction gene screening method and production application. The induction method described in the present application can quickly and effectively obtain hairy roots that synthesize anthocyanins and glycoside derivatives thereof, and at the same time, in the case of limited germplasm resources, anthocyanins and glycoside derivatives thereof can be obtained in a short time and without being affected by seasons, i.e. the induction cycle is short and the efficiency is high.

[0006] The present application provides an induction method of Antirrhinum majus L. hairy roots for producing anthocyanins and glycoside derivatives thereof, comprising the following steps:

[0007] infesting Antirrhinum majus L. explants with Agrobacterium rhizogenes containing an induction gene, co-culturing, sterilizing and culturing to obtain Antirrhinum majus L. hairy roots for producing anthocyanins and glycoside derivatives thereof;

[0008] The induction gene comprises a gene encoding a MYB transcription factor or a mutant thereof.

[0009] Preferably, the Agrobacterium rhizogenes comprises Agrobacterium rhizogenes MSU440.

[0010] Preferably, the Antirrhinum majus L. explants comprise Antirrhinum majus L. stem segments.

[0011] Preferably, the gene encoding the MYB transcription factor comprises a MYBA1 gene.

[0012] Preferably, the nucleotide sequence of the MYBA1 gene is shown in SEQ ID NO. 1.

[0013] Preferably, the co-culture uses a co-culture medium containing acetosyringone; the bacteria-free culture uses a bacteria-free culture medium containing carbenicillin and timentin.

[0014] Preferably, the time of the infection is 3-8 min.

[0015] The application also provides an application of the Antirrhinum majus hairy roots induced by the induction method in the production of anthocyanins and glycoside derivatives thereof.

[0016] The application also provides a method for producing anthocyanins and glycoside derivatives thereof by the Antirrhinum majus hairy roots induced by the induction method, comprising the following steps: liquid culture of the Antirrhinum majus hairy roots to obtain a product containing anthocyanins and glycoside derivatives thereof.

[0017] The application also provides a method for screening a gene for inducing the Antirrhinum majus hairy roots to produce anthocyanins and glycoside derivatives thereof, comprising the following steps:

[0018] Infection of the Antirrhinum majus explants with Agrobacterium rhizogenes containing different genes to be screened, co-culture, bacteria-free culture, and screening of the genes for producing anthocyanins and glycoside derivatives thereof as the genes for inducing the Antirrhinum majus hairy roots to produce anthocyanins and glycoside derivatives thereof.

[0019] The application provides an induction method for producing anthocyanins and glycoside derivatives thereof by the Antirrhinum majus hairy roots. The application proposes a method for inducing the hairy roots capable of biosynthesizing anthocyanins and glycoside derivatives thereof. In specific embodiments, the Antirrhinum majus, a plant chassis for producing anthocyanins and glycoside derivatives thereof, is obtained by inducing the hairy roots of the aseptic seedlings of different plants. Meanwhile, the induction conditions of the hairy roots are optimized and screened (comparison of different species, comparison of different explants of Amas aseptic seedlings, comparison of strains, and comparison of biomasses of engineering materials), to obtain the optimal induction method for the hairy roots to synthesize anthocyanins and glycoside derivatives thereof. The hairy roots have biochemical and genetic stability, can grow rapidly in a hormone-free medium, have similar biosynthetic capacity to the parent plants, and have a fast biomass growth rate. The method of the application has high induction efficiency and large biomass, can provide sufficient materials for the industrialization of anthocyanins and glycoside derivatives thereof in the later stage, and realizes the improvement of the content of anthocyanins and glycoside derivatives thereof and the high yield of anthocyanins. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed in the embodiments will be briefly introduced as follows. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without any creative effort.

[0021] FIG. 1 is a diagram of the results of bHLH and MYBA1 gene induction on Amas stem hairy roots provided by the present application;

[0022] FIG. 2 is a diagram of the results of species comparison (Antirrhinum majus and Antirrhinum majus) test provided by the present application;

[0023] FIG. 3 is a diagram of the results of species comparison (Arabidopsis thaliana and Antirrhinum majus) test provided by the present application; wherein, A: Arabidopsis thaliana leaf hairy root induction under strain A; B: Arabidopsis thaliana stem hairy root induction under strain E; C: GUS staining of Arabidopsis thaliana leaf hairy roots induced under strain A; D: GUS staining of Arabidopsis thaliana stem hairy roots induced under strain E; E: negative control (left) and positive control (right) of Arabidopsis thaliana leaves carrying GUS gene;

[0024] FIG. 4 is a diagram of the results of partial explant type comparison test provided by the present application;

[0025] FIG. 5 is a diagram of the results of strain induction efficiency comparison provided by the present application;

[0026] FIG. 6 is a diagram of the results of engineered material biomass comparison test provided by the present application. DETAILED DESCRIPTION

[0027] The present application provides a method for inducing Antirrhinum majus hairy roots for producing anthocyanins and glycoside derivatives thereof, comprising the following steps:

[0028] Infesting Antirrhinum majus explants with Agrobacterium rhizogenes containing an induction gene, co-culturing, and removing bacteria to obtain Antirrhinum majus (transgenic) hairy roots for producing anthocyanins and glycoside derivatives thereof;

[0029] The induction gene comprises a gene encoding a MYB transcription factor or a mutant thereof.

[0030] The application uses Agrobacterium rhizogenes containing an inducible gene to infect Antirrhinum majus explants. In the application, the inducible gene comprises a MYB transcription factor-encoding gene or a mutant thereof. In the application, the MYB transcription factor-encoding gene comprises a MYBA1 gene. In specific embodiments, the nucleotide sequence of the MYBA1 gene is shown in SEQ ID NO. 1. In the application, the construction method of the Agrobacterium rhizogenes containing the inducible gene comprises: constructing the inducible gene into a vector to obtain a recombinant vector, and transforming the recombinant vector into Agrobacterium rhizogenes to obtain Agrobacterium rhizogenes containing the inducible gene. The application does not have special limitations on the type of vector, and the vector can be a vector containing a reporter gene or not containing a reporter gene. The application does not have special limitations on the reporter gene, which can be a GUS gene. In specific embodiments, the vector can be pCambia1301 or pCambia1301-GUS. The application does not have special limitations on the source of pCambia1301 or pCambia1301-GUS, and a conventional commercially available pCambia1301-GUS can be used. In specific embodiments, the inducible gene comprises a MYBA1 gene. When the inducible gene is a MYBA1 gene, the construction method of the Agrobacterium rhizogenes containing the MYBA1 gene comprises: constructing the MYBA1 gene into a vector to obtain a recombinant vector, which is characterized by a transcription unit containing a strong promoter (such as a 35S promoter) and can overexpress MYBA1. Transform the recombinant vector into Agrobacterium rhizogenes to obtain Agrobacterium rhizogenes containing the MYBA1 gene. In specific embodiments, the MYBA1 gene is constructed into a pCambia1301 vector. The application screens an effective anthocyanin synthesis gene MYBA1, which can accelerate the synthesis of anthocyanins and their glycoside derivatives, and creates plant tissues that can realize the biosynthesis of anthocyanins and their glycoside derivatives. In the application, the Agrobacterium rhizogenes comprises Agrobacterium rhizogenes MSU440. In specific embodiments, the Agrobacterium rhizogenes is Agrobacterium rhizogenes MSU440. In the application, the explant comprises a stem segment. In specific embodiments, the explant is a stem segment. In the application, the length of the stem segment is 0.3-1.0 cm. In specific embodiments, the length of the stem segment can be 0.4 cm, 0.5 cm, 0.6 cm, or 0.8 cm. In the application, the explant is a sterile receptor material. The application uses sterile receptor material for hairy root induction, which can obtain hairy roots that can be directly used for the sterile production of anthocyanins in a bioreactor. Selecting a stem segment as an explant has a higher induction efficiency. The Agrobacterium rhizogenes engineering strain selects Agrobacterium rhizogenes MSU440, which has a higher induction efficiency.

[0031] The Antirrhinum majus explants are infected, co-cultured, and cultured without bacteria to obtain the Antirrhinum majus hairy roots for producing anthocyanins and glycoside derivatives thereof. In the present application, the co-culturing is performed using a co-culturing medium containing acetosyringone, and the culture without bacteria is performed using a culture medium without bacteria containing carbenicillin and timentin. In the present application, the concentration of acetosyringone in the co-culturing medium is 100 μmol / L. In the present application, the mass concentration of timentin in the culture medium without bacteria is 100-300 mg / L, and the mass concentration of carbenicillin is 100-300 mg / L. In specific embodiments, the co-culturing medium contains 4.74 g of MS medium, 30 g of sucrose, and 8 g of agar per liter, and the pH value is 5.8; and the concentration of acetosyringone in the co-culturing medium is 100 μmol / L. In specific embodiments, the culture medium without bacteria contains 4.74 g of MS medium, 30 g of sucrose, and 8 g of agar per liter, and the pH value is 5.8; and the mass concentration of timentin in the culture medium without bacteria is 200 mg / L, and the mass concentration of carbenicillin is 200 mg / L. In specific embodiments, the infection time is 3-8 min, and can also be 5 min. In specific embodiments, the co-culturing time is 2 days. In specific embodiments, the co-culturing temperature is 25°C. In specific embodiments, the co-culturing is dark culture. In specific embodiments, the hairy roots with a root length of 2-3 cm are selected for culture without bacteria. In specific embodiments, the culture without bacteria is replaced every other week until the culture is completely sterile.

[0032] The induction method described in the technical solutions of the present application can induce Antirrhinum majus hairy roots. The present application uses Antirrhinum majus hairy roots as plant tissues for culture, which can effectively obtain a large amount of hairy roots for synthesizing anthocyanins and glycoside derivatives thereof in a short time, and can be used for liquid culture to realize the efficient synthesis and production of anthocyanins and glycoside derivatives thereof.

[0033] The present application also provides the use of the Antirrhinum majus hairy roots induced by the induction method described in the technical solutions of the present application in the production of anthocyanins and glycoside derivatives thereof. The hairy roots have biochemical and genetic stability, can grow rapidly in a hormone-free medium, have similar biosynthetic capacity to the parent plants, and have a fast biomass growth rate, which can provide sufficient materials for the industrialization of anthocyanins and glycoside derivatives thereof, and can quickly and effectively obtain hairy roots for synthesizing anthocyanins and glycoside derivatives thereof, and can obtain anthocyanins and glycoside derivatives thereof in a short time and without seasonal influence under the condition of limited germplasm resources. The use of the hairy roots of the present application in the production of anthocyanins and glycoside derivatives thereof can further improve the production efficiency and yield.

[0034] The application further provides a method for producing anthocyanins and glycoside derivatives thereof by the induced Antirrhinum majus hairy roots, comprising the following steps: liquid culturing the Antirrhinum majus hairy roots to obtain a product containing anthocyanins and glycoside derivatives thereof. In specific embodiments, the temperature of the liquid culturing can be 25-29°C, or 26°C, 27°C or 28°C. In specific embodiments, the rotation speed of the liquid culturing can be 90-120 rpm, or 100 rpm or 110 rpm. In specific embodiments, the culture medium used in the liquid culturing comprises an MS liquid culture medium. By liquid culturing the Antirrhinum majus hairy roots, high yield of anthocyanins and glycoside derivatives thereof can be achieved.

[0035] The application further provides a method for screening a gene for inducing the Antirrhinum majus hairy roots to produce anthocyanins and glycoside derivatives thereof based on the above-mentioned induction method, comprising the following steps:

[0036] Agrobacterium rhizogenes containing different genes to be screened are used to infect the Antirrhinum majus explants, co-cultured, and cultured in a sterile environment, and the genes capable of producing the Antirrhinum majus hairy roots producing anthocyanins and glycoside derivatives thereof are screened as the genes for inducing the Antirrhinum majus hairy roots to produce anthocyanins and glycoside derivatives thereof.

[0037] In order to further illustrate the application, the induction method of the Antirrhinum majus hairy roots for producing anthocyanins and glycoside derivatives thereof, the gene screening method and the production application provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the application.

[0038] Example 1

[0039] Induction of plant tissues.

[0040] 1. Experimental materials.

[0041] Amas aseptic seedlings.

[0042] The Amas seeds collected in the wild are washed with sterile water for 2 times, the upper layer of unsaturated seeds is discarded, 75% alcohol is used for 2 min, sterile water is washed for 3 times, 5% NaClO is vortexed for 5 min, sterile water is washed for 5 times, inoculated into an MS culture medium, cultured at 25°C for 16 / 8 h, and when the aseptic seedlings reach 6-8 leaves, they are used for hairy root induction.

[0043] 2. Experimental reagents.

[0044] MS medium, sucrose, agar, vector pCambia1301-GUS (purchased from Newphybio), Agrobacterium rhizogenes K599 (subsequent experiments are replaced by A), Agrobacterium rhizogenes MSU440 (subsequent experiments are replaced by B), Agrobacterium rhizogenes Ar.1193 (subsequent experiments are replaced by C), Agrobacterium rhizogenes Ar. Qual (subsequent experiments are replaced by D), Agrobacterium rhizogenes C58C1 (subsequent experiments are replaced by E); Agrobacterium rhizogenes is purchased from Weidi Biology.

[0045] 3Instrument

[0046] Balance (Sartorius Science Instrument, SQP), plant incubator (Shanghai Yiheng Science Instrument Co., Ltd.), clean bench (Suzhou Antai Air Technology Co., Ltd. SW-CJ-2FD), autoclave (Shanghai Boin Instrument Co., Ltd. CT-90B), alcohol lamp, tissue culture bottle, pipette, gun head, sealing film, rubber band, culture dish.

[0047] 4Experimental preparation

[0048] MS medium: weigh MS medium 4.74 g, sucrose 30 g, agar 8 g, add water to 1 L, adjust pH to 5.8, sterilize at 121℃ for 30 min.

[0049] YEB medium: weigh beef extract 5 g, peptone 5 g, yeast extract powder 1 g, sucrose 5 g, MgSO4·7H2O 4 g, add water to 1 L, adjust pH to 7.4, sterilize at 121℃ for 30 min.

[0050] Carbenicillin stock solution (CB): prepare 100 mg / mL stock solution, weigh 5 g of carbenicillin powder, sterilize with water to 50 mL, filter sterilization with 0.22 μm water filter membrane, after dispensing, store at 4℃.

[0051] Time stock solution (Time): prepare 100 mg / mL stock solution, weigh 5 g of Time powder, sterilize with water to 50 mL, filter sterilization with 0.22 μm organic filter membrane, after dispensing, store at 4℃.

[0052] Acetyl-syringone (AS) stock solution: prepare 100 mmol / L stock solution, weigh 0.981 g of AS powder, dissolve with DMSO, sterilize with water to 50 mL, filter sterilization with 0.22 μm organic filter membrane, after dispensing, store at 4℃.

[0053] Co-culture medium: weigh MS medium 4.74 g, sucrose 30 g, agar 8 g, add water to 1 L, adjust pH to 5.8, sterilize at 121℃ for 30 min. After sterilization, add 100 μmol / L AS and mix well when the medium temperature is appropriate.

[0054] Bacteria-free medium: MS medium 4.74 g, sucrose 30 g, agar 8 g, add water to 1 L, adjust pH to 5.8, sterilize at 121 ℃ for 30 min. After sterilization, add 200 mg / L Time and 200 mg / L CB when the medium temperature is appropriate, mix well and pour into a flat plate.

[0055] 5 Test design.

[0056] The Amas aseptic seed stem segments were infected with five Agrobacterium rhizogenes strains to induce hairy roots, and the anthocyanin-producing hairy root induction conditions were screened.

[0057] 6 Test method steps.

[0058] 6.1 Strain activation.

[0059] The five Agrobacterium rhizogenes strains stored in the laboratory at -80 ℃ in the refrigerator were inoculated in YEB liquid medium in a clean bench, the shaking speed was 200 r / min, the temperature was 28 ℃, and the dark culture was carried out for 16 h. After the strain was cultured for 16 h, 1 mL was inoculated in YEB liquid medium in a clean bench, and the dark culture was carried out for 4-6 h at 200 r / min and 28 ℃. When the bacterial solution was in the logarithmic growth phase (OD 600 = 0.2-0.8), it was used for infection.

[0060] 6.2 Infection

[0061] In the clean bench, sterile scissors were used to cut the cultured aseptic seed stem segments into 0.5 cm, and the wound was treated with a needle. The five different Agrobacterium rhizogenes strains that had been activated were used to infect different explants for 5 min, during which shaking was not stopped to make the Agrobacterium completely contact with the stem segment wound. The excess bacterial solution was absorbed with sterile filter paper, and then inoculated on the co-culture medium at 25 ℃ in the dark for 2 d.

[0062] 6.3 Bacteria-free culture

[0063] The co-cultured explants were transferred to bacteria-free medium and cultured in the dark at 25 ℃ for 10 d to induce hairy roots. The induced hairy roots (2-3 cm long) were cut with sterile scissors and inoculated on bacteria-free medium. Every other week, they were transferred to fresh bacteria-free medium until they were completely sterile (1-2 months), and then the hairy roots were transferred to MS liquid medium for proliferation culture.

[0064] 6.4 Screening and overexpression of anthocyanin induction factors

[0065] The bHLH and MYBA1 genes were used as overexpression genes to construct vectors (pCambia1301), and the constructed vectors were respectively transferred into the optimal Agrobacterium rhizogenes and infected plant explants. The infection method was the same as the hairy root induction of Amas sterile seedlings. The stable bHLH-expressing Antirrhinum majus hairy roots and the stable MYBA1 gene-expressing Antirrhinum majus hairy roots were obtained.

[0066] The nucleotide sequence of the MYBA1 gene is shown in SEQ ID NO. 1:

[0067] The nucleotide sequence of the bHLH gene is shown in SEQ ID NO. 2:

[0068] 7 Test results.

[0069] 7.1 Amas sterile seedling hairy root induction.

[0070] Table 1 Induction results

[0071] According to Table 1, Agrobacterium rhizogenes B has strong infection ability on the stem segments of Amas sterile seedlings. When the OD of the bacterial solution is 0.6, the infection time is 5 min, the co-culture time is 2 d, and the bacteria are removed, the hairy roots can be induced after 10 d. The induction time is short, the induction efficiency is high, the anthocyanin-producing hairy roots can be obtained in a short time, and the proliferation of experimental materials is realized. 600

[0072] 7.2 Screening and overexpression of anthocyanin induction factors.

[0073] The bHLH and MYBA1 genes were used as target genes to construct vectors (based on pCambia1301 plasmid), and the constructed vectors were respectively transferred into the optimal Agrobacterium rhizogenes B and infected plant explants to realize the induction of target genes on the anthocyanin-producing hairy roots of Amas stem segments. The method of the application can obtain hairy roots in a short time, the bacteria are removed after 2 d of co-culture, and the hairy roots can be obtained after 10 d. The induction efficiency is high (100 explants can obtain hairy roots).

[0074] The induction results of the bHLH and MYBA1 genes on the hairy roots of Amas stem segments are shown in Figure 1. The results show that after the MYBA1 gene vector is constructed, transferred into the optimal Agrobacterium rhizogenes B and infected plant explants, the red hairy roots of the MYBA1 gene are successfully induced, and can produce anthocyanin. After the bHLH gene vector is constructed, transferred into the Agrobacterium rhizogenes B and infected plant explants, the hairy roots are successfully induced, but cannot biosynthesize anthocyanin.

[0075] Comparative Example 1

[0076] ​Species comparison.

[0077] The hairy roots were induced from Nicotiana tabacum var Xanthinc sterile seedlings as a control, and the experimental method was the same as that of Amas hairy root induction.

[0078] The experimental results are shown in Figure 2.

[0079] As shown in Figure 2, Agrobacterium rhizogenes D has strong infection ability on Ntx leaves, and can induce hairy roots when the OD 600 is 0.6 for 5 min, and the co-culture is 2 d. However, the induction time of hairy roots is as long as 40 d, and other strains cannot induce hairy roots in 40 d.

[0080] Comparative Example 2

[0081] Species comparison.

[0082] The hairy roots were induced from Nicotiana tabacum var Xanthinc sterile seedlings as a control, and the experimental method was the same as that of Amas hairy root induction.

[0083] The experimental results are shown in Figure 3. As shown in Figure 3, strains A and E have strong hairy root induction effect on Arabidopsis, while the other strains cannot induce Arabidopsis root. When the OD 600 of the bacterial solution is 0.6 for 5 min, and the co-culture is 2 d, both Arabidopsis leaves and petioles can induce hairy roots, and GUS staining is blue, proving that Agrobacterium carrying GUS gene is transferred into plant tissue, but the whole hairy root induction time is as long as 40 d.

[0084] Comparative Example 3

[0085] Tissue comparison.

[0086] The hairy roots were induced from Nicotiana tabacum var Xanthinc sterile seedlings as a control, and the experimental method was the same as that of Amas hairy root induction.

[0087] The experimental results are shown in Tables 2 and 3. Figure 4 is a diagram of the experimental results of some tissue type comparison.

[0088] Table 2 Stem segment experimental results

[0089] Table 3 Leaf experimental results

[0090] As shown in Tables 2 and 3, Agrobacterium rhizogenes B has strong infection ability on Amas sterile seedling stems, and can induce hairy roots when the OD 600The Amas stem segments were infected with Agrobacterium rhizogenes B at OD

[0091] Comparative Example 4

[0092] Strain comparison.

[0093] The hairy roots were induced from the Amas aseptic seedlings of different strains, and the experimental method was the same as that of the Amas hairy root induction.

[0094] The experimental results are shown in Table 4.

[0095] Table 4 Experimental results

[0096] Figure 5 is a comparison of the induction efficiency of strains. The Amas stem segments were used as explants for hairy root induction, and the induction of some of the explants is shown in the figure.

[0097] The experimental results show that Agrobacterium rhizogenes B has strong infection ability on the explants of Amas aseptic seedlings. When the OD 600 The hairy roots were induced after 10 days of infection at OD

[0098] Comparative Example 5

[0099] Comparison of biomass of engineering materials

[0100] The Amas aseptic seedlings were used as materials for hairy root induction, and an equal amount was inoculated into MS liquid medium and MS solid plate culture, respectively, and sampled for biomass determination after 18 days of growth (according to the growth cycle of the measured hairy roots, the biomass was the largest at 18 days and began to decline later, so 18 days was selected for biomass determination).

[0101] The experimental results of liquid and solid Amas hairy root growth are shown in Figure 6. The experimental results show that the biomass of Amas hairy roots cultured in liquid and solid media for 18 days was 8.3577 g, which was higher than the biomass of 0.8931 g in MS solid plate, and the liquid culture was 9.3 times that of solid culture, resulting in a higher anthocyanin content of 10.2 mg / g.FW in liquid culture than 0.092 mg / g.FW in solid culture.

[0102] Although the above embodiments have been described in detail, it should be understood that the above embodiments are only some embodiments of the present application, but not all embodiments of the present application, and other embodiments can be obtained by those skilled in the art without creativity on the basis of the above embodiments, and these embodiments all belong to the protection scope of the present application.

Claims

1. A method for inducing anthocyanin and its glycoside derivatives-producing Antirrhinum majus hairy roots, characterized by, The method comprises the following steps: The method comprises the following steps: The method comprises the following steps:

2. The induction method of claim 1, wherein, The inducing gene comprises a gene encoding a MYB transcription factor or a mutant thereof.

3. The induction method of claim 1, wherein, The Agrobacterium rhizogenes comprises Agrobacterium rhizogenes MSU440.

4. The induction method of claim 1, wherein, The Antirrhinum majus explants comprise Antirrhinum majus stem segments.

5. The induction method of claim 4, wherein, The gene encoding a MYB transcription factor comprises a MYBA1 gene.

6. The induction method of claim 1, wherein, The nucleotide sequence of the MYBA1 gene is shown in SEQ ID NO.

1.

7. The induction method of claim 6, wherein, The co-culture uses a co-culture medium containing acetosyringone; and the bacteria-free culture uses a bacteria-free culture medium containing carbenicillin and timentin.

8. The induction method of claim 1, wherein, In the co-culture medium, the concentration of acetosyringone is 100 μmol / L; in the bacteria-free culture medium, the mass concentration of timentin is 100-300 mg / L, and the mass concentration of carbenicillin is 100-300 mg / L.

9. The induction method of claim 1, wherein, The infection time is 3-8 min.

10. The induction method of claim 9, wherein, The method for constructing the Agrobacterium rhizogenes containing the inducing gene comprises the following steps: constructing the inducing gene into a vector to obtain a recombinant vector, and transforming the recombinant vector into the Agrobacterium rhizogenes to obtain the Agrobacterium rhizogenes containing the inducing gene. The vector comprises pCambia1301 or pCambia1301-GUS.

12. A method for the production of anthocyanins and their glycoside derivatives by Antirrhinum majus hairy roots induced by the induction method according to any one of claims 1 to 10, comprising the following steps:

11. The application of the Antirrhinum majus hairy roots induced by the inducing method of any one of claims 1-10 in the production of anthocyanins and glycoside derivatives thereof.

13. The method of claim 12, wherein, The Antirrhinum majus hairy roots are subjected to liquid culture to obtain a product containing anthocyanins and glycoside derivatives thereof.

14. A method for screening genes inducing anthocyanins and their glycoside derivatives production in Antirrhinum majus hairy roots based on the induction method according to any one of claims 1 to 10, characterized in that, The temperature of the liquid culture is 25-29℃; and the rotation speed of the liquid culture is 90-120 rpm. The method comprises the following steps: The method comprises the following steps: The method comprises the following steps: The method comprises the following steps: The Agrobacterium rhizogenes containing different genes to be screened is used to infect Antirrhinum majus explants, co-culture is performed, bacteria-free culture is performed, and genes capable of producing Antirrhinum majus hairy roots of anthocyanins and glycoside derivatives thereof are screened as genes for inducing Antirrhinum majus hairy roots to produce anthocyanins and glycoside derivatives thereof.

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