Method for increasing betacyanin content of suaeda heteroptera cluster buds and application of method

Abstract

The invention belongs to the technical field of plant treatment, and particularly relates to a method for increasing the betacyanin content of suaeda heteroptera cluster buds and application thereof.In the process of replacing a culture medium after primary culture of the suaeda heteroptera cluster buds, NaCl is added into the culture medium or the pH of the culture medium is adjusted to 6.5-7.0, and the plant hormone concentration of a cluster bud induced proliferation culture medium is obtained; the influence of environmental conditions on betacyanin biosynthesis is determined.

Description

Technical Field

[0001] The invention belongs to the technical field of plant treatment, and particularly relates to a method for increasing the betalain content of Suaeda salsa buds and an application thereof. Background Art

[0002] Betalain is a highly water-soluble plant pigment with strong antioxidant capacity. Suaeda salsa can metabolize betalain to reduce plant damage in a comprehensive adverse environment. Due to its high antioxidant and coloring properties, betalain is in high market demand in many fields such as medicine, food, and chemical engineering. With increasing attention to food safety, natural plant-derived pigments are gradually replacing synthetic pigments in the industrial sector. However, betalain sources are mostly limited to plants such as beets and amaranth, and the plant growth cycle is relatively long. Plant tissue culture can reduce costs and time, while also allowing for the production of large quantities of betalain after processing. Therefore, this study used sterile seedlings of Suaeda salsa as the material to investigate the high-proliferation system induced by clustered shoots and explore the effects of environmental conditions on betalain biosynthesis. This study aims to provide theoretical and technical support for betalain production in Suaeda salsa and address the challenges of betalain biosynthesis. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides a method for increasing the betalain content of Suaeda salsa clumping shoots and its application, and uses Suaeda salsa as the main body for tissue culture to explore the effects of different environmental conditions on betalain biosynthesis.

[0004] The present invention is achieved by providing a method for increasing the betalain content of Suaeda salsa clumping buds. During the process of replacing the culture medium after the primary culture of Suaeda salsa clumping buds, NaCl is added to the culture medium or the pH of the culture medium is adjusted to 6.5-7.0.

[0005] Preferably, the culture medium is MS medium with 30 g / L sucrose, 6.0 g / L agar powder, 0.5 mg / L 6-BA and 0.2 mg / L NAA added thereto.

[0006] Preferably, the amount of NaCl added is 400 mM.

[0007] Preferably, the pH of the culture medium is adjusted by adding NaHCO 3 or NaOH to the culture medium. When NaHCO 3 is added, the pH of the culture medium is adjusted to 6.5; when NaOH is added, the pH of the culture medium is adjusted to 7.0.

[0008] Preferably, the culture temperature of the Suaeda winged buds is 25°C.

[0009] The present invention also provides an application of the method for increasing the betalain content in Suaeda salsa clumping sprouts in promoting the expression of betalain synthesis genes in Suaeda salsa clumping sprouts.

[0010] Preferably, the betalain synthesis genes in the clustered shoots of Suaeda salsa include Ss-CYP76C1, Ss-UGE1 and Ss-CYP76AD1, which have the nucleotide sequences shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0011] More preferably, when NaCl is added to the culture medium, the expression levels of Ss-CYP76C1 and Ss-UGE1 increase; when NaHCO3 is added to the culture medium to adjust the pH of the culture medium to 6.5, the expression level of Ss-CYP76AD1 increases.

[0012] Compared with the prior art, the advantages of the present invention are:

[0013] The present invention provides a method for increasing the betalain content in clustered buds of Suaeda salsa and its application. Using sterile seedlings of Suaeda salsa as materials, the plant hormone concentration of the proliferation culture medium for clustered bud induction is determined, and the influence of environmental conditions on betalain biosynthesis is determined. Specifically:

[0014] 1. It was found that the amount of betalain produced by adjusting the pH of the culture medium to 6.5 by adding NaHCO3 to the culture medium for the induction and proliferation of Suaeda salsa was 73.4423 mg·g -1 , the highest among all treatment groups.

[0015] 2. Transcriptome analysis of clustered shoots treated with 400 mM NaCl and pH 6.5 (NaHCO3) revealed that the synthesis pathways of secondary metabolites and key enzymes (oxidoreductases and glycosyltransferases) were significantly upregulated, indicating that Ss-CYP76C1, Ss-UGE1 and Ss-CYP76AD1 were all related to betalain synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The growth state of cluster buds induced by cluster bud proliferation medium under different concentrations of plant growth hormone, among which experimental group 1 was MS+6-BA 1.0 mg·L -1 +NAA 0.5 mg·L -1 , Experimental group 2: MS+6-BA 0.5 mg·L -1 +NAA0.2 mg·L -1 , Experimental group 3: MS+6-BA 0.1 mg·L -1 +NAA 0.2 mg·L -1 ;

[0017] Figure 2 The proliferation times of clustered buds under different periods and different concentrations of plant growth hormone, where A is the induction period, B is the subculture period, and C is the second subculture period. In each period, experimental group 1 was MS+6-BA 1.0 mg·L -1 +NAA 0.5 mg·L -1 , Experimental group 2: MS+6-BA 0.5 mg·L -1 +NAA 0.2 mg·L -1 , Experimental group 3: MS+6-BA 0.1 mg·L -1 +NAA0.2 mg·L -1 ;

[0018] Figure 3 The growth status of Suaeda salsa buds treated with different concentrations of NaCl, where the top row from left to right are 0mM, 100mM, 200mM; the bottom row from left to right are 300mM, 400mM, 500mM;

[0019] Figure 4 The proliferation times of clustered buds of Suaeda salsa at different periods and different concentrations of NaCl treatment, where A is the induction period, B is the subculture period, and C is the second subculture period;

[0020] Figure 5 The growth state of clustered shoots was obtained by adjusting the culture medium with NaHCO3 at different pH values, where A was pH 6.0, B was pH 6.5, C was pH 7.0, and D was pH 7.5.

[0021] Figure 6 The proliferation times of clustered buds at different periods and in NaHCO3-adjusted medium at different pH values, where A is the induction period, B is the subculture period, and C is the second subculture period;

[0022] Figure 7 The growth state of clustered shoots was obtained by adjusting the culture medium with NaOH at different pH values, including pH 6.0 for A, pH 6.5 for B, pH 7.0 for C, and pH 7.5 for D.

[0023] Figure 8 The proliferation times of clustered buds at different periods and in NaOH-adjusted medium at different pH values, where A is the induction period, B is the subculture period, and C is the second subculture period;

[0024] Figure 9 The growth status of clustered buds under different temperature treatments, from left to right: 15℃, 20℃, 25℃, 30℃;

[0025] Figure 10The proliferation times of cluster buds under different periods and temperature treatments, where A is the induction period, B is the subculture period, and C is the second subculture period;

[0026] Figure 11 is the standard curve of betalain;

[0027] Figure 12 Synthesis of betalain OH from sprouts under different treatments - Inhibition ability;

[0028] Figure 13 The DPPH radical scavenging rate of betalain synthesized by axillary buds under different treatments;

[0029] Figure 14 are the expression levels of Ss-CYP76C1, Ss-CYP76AD1 and Ss-UGE1, where A is the expression level of Ss-CYP76C1 under NaHCO3 treatment; B is the expression level of Ss-CYP76AD1 under NaHCO3 treatment; C is the expression level of Ss-UGE1 under NaCl treatment; and D is the expression level of Ss-CYP76C1 under NaCl treatment. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] Example 1: Effects of different culture conditions on the proliferation of clustered shoots of Suaeda salsa.

[0032] Plant tissue culture is a biotechnology technique that utilizes the totipotency of plant cells to obtain tissues or cells by culturing explants in sterile culture media. Due to its short experimental cycles and low costs, it is often used in plant breeding and rapid propagation. Therefore, the present invention utilizes plant tissue culture to induce and propagate clustered shoots in Suaeda salsa.

[0033] 1.1: Effects of different plant hormone ratios on the proliferation of clustered shoots of Suaeda winged.

[0034] Hormone concentration in plant culture media is one of the factors that influences the growth of tissue culture seedlings and forms the foundation of all culture systems. Axillary buds from sterile Suaeda salsa seedlings were explanted and used in MS medium to investigate the effects of the interaction between the plant growth hormones 6-BA and NAA on the growth of clustered shoots. The goal was to identify the optimal plant hormone concentration for clustered shoot induction and proliferation. Figure 1The growth status of the three groups of tissue culture seedlings can be observed. Among them, cluster buds were induced in experimental group 2 during the second subculture period and the growth status was good; cluster bud growth was not observed in experimental groups 1 and 3, and the growth status of the axillary buds in experimental group 3 appeared white.

[0035] based on Figure 2 Analysis of experimental data reveals that during the secondary subculture stage, the phytohormone combination used in Experimental Group 2 significantly enhanced the proliferation of clustered shoots, achieving a multiplication factor of 5.59±0.08, approximately twice that of the control groups (Experimental Groups 1 and 3). Further analysis of the longitudinal comparison data in Table 1 reveals that the proliferation advantage of Experimental Group 2 is clearly stage-dependent: at the three key stages of culture, namely the induction phase, the subculture phase, and the secondary subculture phase, the multiplication factor of this group was statistically significantly different from that of the other two groups (p<0.05). This consistent difference suggests that the hormone combination used in Experimental Group 2 provides important technical parameters for subsequent large-scale seedling production. This phenomenon may be related to the activation of growth and metabolic pathways by the phytohormone combination.

[0036] Table 1: Effects of the interaction between 6-BA and NAA on the proliferation of cluster shoots

[0037] Combine Figure 1 and Figure 2 The proliferation rate of experimental group 2 was as high as 5.0, which was about twice that of the other two experimental groups, and clusters of buds grew during the growth process. Therefore, the hormone concentration ratio of experimental group 2 will be used in different environmental treatments in the future. That is, 0.5 mg·L -1 6-BA and 0.2 mg·L -1 NAA induced and proliferated the buds, enabling them to synthesize betalain under different environmental conditions.

[0038] 1.2: Effects of different NaCl concentrations on the proliferation of clustered buds of Suaeda winged.

[0039] Depend on Figure 3, it can be seen that at 300 mM and 400 mM NaCl treatment concentrations, the plants grew well compared to the control group and had a purple-red appearance. Among them, when treated with 400 mM NaCl, the leaves and stems of the plants were pink, and the color depth was better than that of the other experimental groups; when treated with 300 mM NaCl, the leaves of the plants were pink and the other parts were green; when treated with 200 mM NaCl, the stems of the plants were pink, and when treated with 100 mM NaCl, the plants grew well, most of the plants were green, and a few parts were pink; when treated with 500 mM NaCl, the plants showed dehydration and albinism, and a small part of the tissue was pink.

[0040] From Table 2 and Figure 4 The control group's proliferation rates were significantly higher than those of the experimental group during the induction, subculture, and secondary subculture phases. In the experimental group, treatment with 400 mM NaCl resulted in higher proliferation rates during the induction, subculture, and secondary subculture phases compared to the other experimental groups. The proliferation rate during the secondary subculture phase was 5.21 ± 0.06, second only to the control group. It is speculated that different NaCl treatment conditions have a certain retarding effect on the growth and development of clustered shoots in Suaeda salsa.

[0041] Table 2 Effects of NaCl concentration on the proliferation of cluster buds

[0042]

[0043] That is, when treated with 400 mM NaCl, cluster bud growth could be observed after secondary subculture, and the proliferation multiple was 5.21±0.06, which was better than other experimental groups. The cluster buds of Suaeda winged saltwort appeared pink. It is speculated that under the treatment of 400 mM NaCl, the betalain content in the cluster buds of Suaeda winged saltwort was the highest.

[0044] 1.3: Effect of NaHCO3 on the proliferation rate of cluster buds.

[0045] Depend on Figure 5 It can be seen that when the pH of the culture medium is adjusted by NaHCO3 to 6.0 and 6.5, it can be obviously observed that the plant is in good growth condition. When the pH of the culture medium is adjusted to 6.0 by adding NaHCO3, the leaves of the plant are observed to be pink. When the pH of the culture medium is adjusted by NaHCO3 to 6.5, clustered buds of the plant are observed to grow, and the whole plant is purple-red. Not only the clustered bud growth state but also the color depth are better than the control group and other experimental groups. When the pH of the culture medium is adjusted by NaHCO3 to 7.0 and 7.5, the plant growth state is white and no longer grows.

[0046] From Table 3 and Figure 6It can be seen that when the pH of the solid culture medium was adjusted to 6.5 by NaHCO3, its proliferation multiples were significantly higher than those of the control group and other experimental groups during the induction period, subculture period, and secondary subculture period, and the proliferation multiples were 4.74±0.05 higher than that of the control group; when the pH of the solid culture medium was adjusted to 7.0 and 7.5 by NaHCO3, the proliferation multiples of the plant was 0 during the secondary subculture period, that is, the alkaline salt concentration in the plant was too high, and the intracellular pH was difficult to maintain a steady state, resulting in plant death.

[0047] Table 3 Effects of NaHCO3 on the proliferation of buds induced by different pH values of the culture medium

[0048]

[0049] Combined with Table 3, Figure 5 and Figure 6 It can be concluded that when the pH of the culture medium was adjusted to 6.5 by NaHCO3, the proliferation rate of the clustered shoots of Suaeda winged was 4.74±0.05, and the plant as a whole appeared purple-red, which was darker than the color of the 400 mM NaCl treatment. It is speculated that its content in the body was higher than that of the other experimental groups and the 400 mM NaCl treatment group.

[0050] 1.4: Effect of NaOH treatment on the proliferation rate of cluster buds.

[0051] Depend on Figure 7 It can be seen that when the pH of the culture medium was adjusted by NaOH to 6.0 and 6.5, it can be obviously observed that the plants were in good growth condition. When the pH of the culture medium was adjusted by NaOH to 6, it can be observed that the plants were in good growth condition and the leaves of the plants were pink; when the pH of the culture medium was adjusted by NaOH to 6.5, the plants were in good growth condition and the leaves were pink; when the pH of the culture medium was adjusted by NaOH to 7.0, the plants were in good growth condition and buds grew, and the leaves were purple-red; when the pH of the culture medium was adjusted by NaOH to 7.5, the growth condition of the plants was slightly albinic, and the whole plant was light pink.

[0052] From Table 4 and Figure 8 As can be seen from the results, when the pH of the solid culture medium was adjusted with NaOH, the control group experimented with pH = 6.0. The control group had significant proliferation times during the induction period, subculture period, and second subculture period, with a proliferation time of 5.59 ± 0.08. As the pH of the culture medium increased, at pH = 6.5, the proliferation time of Suaeda salsa decreased throughout the entire cycle, suggesting that its growth was inhibited by the chemical treatment. When the culture medium pH was treated at 7.0, the proliferation time of Suaeda salsa was second only to the control group, with a brief growth retardation during the induction period, but the proliferation time was around 5.46 ± 0.07 during the second subculture period. When the culture medium pH was 7.5, the proliferation time was 2.22 ± 0.16, suggesting that it is not suitable for the growth of Suaeda salsa.

[0053] Table 4 Effects of NaOH on the proliferation of buds induced by different pH values of the culture medium

[0054]

[0055] 1.5: Effects of different temperatures on the proliferation of clustered buds of Suaeda winged.

[0056] Depend on Figure 9 It can be seen that the plants grow slowly under the culture conditions of 15℃, 20℃ and 30℃; under the culture conditions of 25℃, the plants grow well and the leaves appear pink.

[0057] From Table 5 and Figure 10 As can be seen from the results, at an ambient temperature of 25°C, the proliferation rate of the buds of Suaeda salsa was significantly higher than that of the other experimental groups during the induction, subculture, and secondary subculture periods, with the proliferation rate ranging from 4 to 5. At ambient temperatures of 15°C, 20°C, and 30°C, the proliferation rate of the buds was all around 2 to 3. This indicates that at an ambient temperature of 25°C, the proliferation rate of the buds of Suaeda salsa was higher than that of the other groups.

[0058] Table 5 Effects of different temperature treatments on the proliferation of cluster buds

[0059]

[0060] Example 2: Effects of different culture conditions on the betalain content in the clustered shoots of Suaeda salsa.

[0061] 2.1: Drawing of the standard curve of betalain.

[0062] Use 1 mg·mL -1 The control solution was prepared from the standard stock solution, and the absorbance was measured at a wavelength of 538 nm using an enzyme reader. Three replicates were performed each time and the average value was taken. The betaine concentration was used as the Y axis, and the OD 538 As the x-axis, make a standard curve. The standard curve formula is: Y=0.2208 X+0.01780,R 2 =0.9992. R 2 >0.999, good linear regression, see Figure 11 .

[0063] 2.2: Effect of different NaCl concentrations on the betalain content in the clustered shoots of Suaeda salsa.

[0064] Salt-alkali mudflats are generally located in coastal areas, so the soil contains high concentrations of sodium ions, which exerts a certain degree of environmental stress on the osmotic pressure of plant cells. Suaeda salsa mostly grows in salt-alkali mudflats and can utilize ions in the soil for secondary metabolism during its growth process, causing its aerial parts to appear purple-red. Research has found that Suaeda salsa biosynthesizes betalain under adverse conditions. Therefore, the present invention performs treatments with different salt concentrations to screen out salt concentration treatment conditions that have high proliferation rates and high betalain content.

[0065] Betacyanin synthesized in the buds induced by different salt treatments was extracted, and the absorbance was measured at a wavelength of 538 nm using a microplate reader. Three replicates were performed each time, and the average value was substituted into the betacyanin standard curve formula to calculate the betacyanin content, which is plotted in Table 6. According to Table 6, it can be concluded that under salt treatment conditions, the betacyanin content of Suaeda salsa at 400 mM NaCl concentration was higher than that of the other salt experimental groups, with a betacyanin content of 57.9077 mg·g -1 .

[0066] Table 6 Betacyanin content in NaCl-treated cluster sprouts

[0067]

[0068] 2.3: Effect of different NaHCO3 concentrations on the betalain content in the clustered shoots of Suaeda salsa.

[0069] Tidal flat soils contain numerous harmful salts. In addition to neutral salts, primarily NaCl, alkaline salts also contribute. Suaeda salsa can utilize its internal buffers to lower intracellular pH, maintaining its own homeostatic environment while synthesizing secondary metabolites. This results in the plant developing a purple-red coloration, indicating the biosynthesis of large amounts of betalain. Therefore, this experiment will utilize alkaline treatments with NaHCO₃ and NaOH to investigate their effects on betalain content under conditions of high bud proliferation.

[0070] Betacyanin synthesized in the buds induced by different NaHCO3 treatments was extracted, and the absorbance was measured at a wavelength of 538 nm using a microplate reader. Three replicates were performed each time, and the average value was substituted into the betacyanin standard curve formula to calculate the betacyanin content, which is plotted in Table 7. According to Table 7, it can be concluded that the amount of betacyanin synthesized by Suaeda salsa in the NaHCO3-adjusted pH 6.5 group was higher than that in the other NaHCO3 groups, with a betacyanin content of 73.4423 mg·g -1 .

[0071] Table 7 Betacyanin content synthesized by clustered sprouts treated with NaHCO3

[0072]

[0073] 2.4: Effect of different NaOH concentrations on the betalain content in the clustered shoots of Suaeda salsa.

[0074] Betacyanin synthesized in the buds induced by different NaOH treatments was extracted, and the absorbance was measured at a wavelength of 538 nm using a microplate reader. Three replicates were performed each time, and the average value was substituted into the betacyanin standard curve formula to calculate the betacyanin content, which is plotted in Table 8. According to Table 8, it can be concluded that the amount of betacyanin synthesized by Suaeda salsa in the NaOH-adjusted pH 7.0 group was higher than that in the other NaOH groups, with a betacyanin content of 27.3823 mg·g -1 .

[0075] Table 8 Betalain content of clustered sprouts treated with NaOH

[0076]

[0077] In summary, when NaOH was added to adjust the pH of the culture medium to 7.0, the proliferation times of the clustered shoots of Suaeda salsa reached 4-5, and the biological content of betalain was the highest.

[0078] 2.5: Effect of temperature treatment on the proliferation rate of cluster buds and betalain content.

[0079] Betacyanin synthesized in the buds induced by different temperature treatments was extracted, and the absorbance was measured at a wavelength of 538 nm using a microplate reader. Three replicates were performed each time, and the average value was substituted into the betacyanin standard curve formula to calculate the betacyanin content, which is plotted in Table 9. According to Table 9, it can be concluded that the amount of betacyanin synthesized by Suaeda salsa cultured at an ambient temperature of 25°C was higher than that of the other temperature treatment groups, with a betacyanin content of 15.7880 mg·g -1 .

[0080] Table 9 Betacyanin content of clustered sprouts during temperature treatment

[0081]

[0082] 2.6: Antioxidant activity of betalain synthesized by Suaeda salsa sprouts under different environmental treatments.

[0083] Under different treatment conditions, the synthesis of betalain may be related to the removal of reactive oxygen species (ROS). ROS activates related pathways and induces the upregulation of key enzymes in betalain synthesis, thereby reducing the damage caused by adverse environments to plants. - Free radical inhibition ability and DPPH free radical inhibition ability test, antioxidant trend as follows Figure 12 and Figure 13 Show. Figure 12 Yes OH- The free radical inhibition ability and antioxidant activity of the treated group were significantly higher than those of the control group. The betalain OH synthesized under NaHCO3 treatment - The free radical inhibition ability was significantly higher than that of other experimental groups and 1‰ VC, indicating that under this condition, the cluster buds were in an adverse growth state. In order to adapt to the growth environment, they reduced the damage of free radicals in the body to the plants by metabolizing and synthesizing betacyanin.

[0084] Figure 13 It can be seen that the DPPH scavenging ability of the control group was less than 50%. Among the treatment groups, the scavenging abilities of 400 mM salt treatment and NaHCO3 treatment were higher than those of the control group and NaOH test group, and the scavenging ability under NaHCO3 treatment conditions was significantly as high as about 60%, indicating that DPPH-related free radicals have been expressed in plants under normal and adverse conditions.

[0085] In summary, under the treatment of NaCl, NaHCO3 and NaOH, Suaeda salsa was in an adverse growth process. It was speculated that the relevant metabolic pathways were activated and a large amount of betacyanin was synthesized to achieve the purpose of scavenging free radicals in the body. Among them, Suaeda salsa had the strongest adverse reaction in the NaHCO3 environment, with OH - Free radicals are the main scavenging targets.

[0086] Example 3: Screening of genes related to betalain synthesis in Suaeda salsa.

[0087] Suaeda salsa exhibits distinct phenotypes under normal growth and after treatment with 400 mM NaCl and pH 6.5 (NaHCO₃). In the treated groups, particularly those treated with pH 6.5 (NaHCO₃), betalains accumulated significantly in the shoots, demonstrating antioxidant activity, suggesting activation of betalain-related metabolic pathways. Transcriptomic analysis revealed significant upregulation of related metabolic pathways (secondary metabolite synthesis) and key enzymes (oxidoreductases and glycosyltransferases) in GO and KEGG enriched pathways, suggesting that the high accumulation of betalains in Suaeda salsa may be linked to the elevated expression of genes supporting photosynthetic carbon fixation and secondary metabolite synthesis. Table 10 shows upregulated genes related to glycosyltransferases and oxidoreductases in the transcriptomes of NaCl vs. CK and NaHCO₃ vs. CK.

[0088] Table 10 Upregulated genes of key enzymes in betalain-related metabolic pathways

[0089]

[0090] 3.1: Verification of candidate genes for betalain biosynthesis in Suaeda salsa.

[0091] 3.1.1: Extraction of RNA and reverse cDNA.

[0092] RNA was extracted using the EASYspin Universal Plant RNA Rapid Extraction Kit (Adlai, RN52). The RNA concentration was determined and diluted using a reverse transcription kit (Darling Biotechnology, DLR102) to synthesize cDNA as template DNA for RT-qPCR testing.

[0093] Table 11 cDNA reaction system

[0094]

[0095] 3.1.2: Primer design.

[0096] Gene IDs were obtained using transcriptome data. The cds sequences of Ss-CYP76C1, Ss-CYP76AD1, and Ss-UGE1 were obtained from the NCBI website (http: / / www.ncbi.nlm.nih.gov / ) and primers were designed (Table 12).

[0097] Table 12 Real-time fluorescence quantitative PCR primer sequences

[0098]

[0099] 3.1.3: Verification of candidate genes related to betalain synthesis.

[0100] In order to verify the reliability of the transcriptome data and verify the response of Suaeda salsa to the betalain secondary metabolic pathway under NaCl and NaHCO3 treatment, the related genes Ss-CYP76C1, Ss-CYP76AD1, and Ss-UGE1 were upregulated.

[0101] RNA was extracted from the treatment and control groups and reverse transcribed to obtain cDNA. RT-qPCR was performed to verify the Ss-CYP76C1, Ss-CYP76AD1, and Ss-UGE1 genes (Table 13). The -△△Ct formula was used to analyze the significant differences by t-test using SPSS software. P < 0.05 was considered as , P < 0.01 and P < 0.001 for Mark the significance.

[0102] Table 13 RT-qPCR reaction procedure

[0103]

[0104] The results are as follows Figure 14As shown, the expression levels of Ss-CYP76C1 and Ss-UGE1 genes increased under 400 mM NaCl treatment compared with the control group. Ss-UGE1 is a key enzyme gene in the betalain biosynthesis pathway. Its significant expression suggests that the UDP-glycosyltransferase gene is induced by 400 mM NaCl treatment. Therefore, it is speculated that the increase in betalain content under 400 mM NaCl treatment is related to the increased expression of Ss-UGE1. Ss-CYP76C1 is a key enzyme gene in the betalain biosynthesis pathway. The significant expression of Ss-CYP76C1 compared with the control group suggests that the monooxygenase gene expression of betalain biosynthesis genes is increased under 400 mM NaCl and pH 6.5 (NaHCO₃) treatment, promoting the increase in betalain content in Suaeda salsa.

[0105] Under pH 6.5 (NaHCO3) treatment, the gene expression level of Ss-CYP76AD1 was significantly higher than that of the control group, indicating that under pH 6.5 (NaHCO3) treatment, tyrosinase synthesized with betalain was induced to express, which promoted the increase of betalain content in the clustered shoots of Suaeda salsa.

Claims

1. A method for increasing the betalain content of Suaeda salsa buds, characterized in that: During the process of replacing the culture medium after the primary culture of Suaeda salsa buds, NaCl is added to the culture medium or the pH of the culture medium is adjusted to 6.5-7.

0.

2. The method for increasing the betalain content of Suaeda salsa clump sprouts according to claim 1, wherein: The culture medium is prepared by adding 30 g / L sucrose, 6.0 g / L agar powder, 0.5 mg / L 6-BA and 0.2 mg / L NAA to the MS culture medium.

3. The method for improving the betalain content of Suaeda salsa buds according to claim 1, wherein: The amount of NaCl added is 400 mM.

4. The method for increasing the betalain content of Suaeda salsa buds according to claim 1, wherein: The pH of the culture medium was adjusted by adding NaHCO3 or NaOH to the culture medium. When NaHCO3 was added, the pH of the culture medium was adjusted to 6.5; when NaOH was added, the pH of the culture medium was adjusted to 7.

0.

5. The method for increasing the betalain content of Suaeda salsa buds according to claim 1, wherein: The culture temperature of the clustered buds of Suaeda winged is 25℃.

6. Use of the method for increasing the betalain content in the clustered shoots of Suaeda salsa according to any one of claims 1 to 5 in promoting the expression of betalain synthesis genes in the clustered shoots of Suaeda salsa.

7. The use according to claim 6, characterized in that The betalain synthesis genes in the clustered buds of Suaeda salsa include Ss-CYP76C1, Ss-UGE1 and Ss-CYP76AD1, which have the nucleotide sequences shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively.

8. The use according to claim 7, characterized in that When NaCl was added to the culture medium, the expression levels of Ss-CYP76C1 and Ss-UGE1 increased; when NaHCO3 was added to the culture medium to adjust the pH of the culture medium to 6.5, the expression level of Ss-CYP76AD1 increased.

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