A method for increasing the content of proanthocyanidins in Spirodendrum

Abstract

The present invention belongs to the technical field of natural material synthesis, and specifically relates to a method for increasing the content of proanthocyanidins in Spirodela. In order to promote the application of Spirodela in ecological engineering, the present invention discloses a method for increasing the content of proanthocyanidins in Spirodela, wherein the proanthocyanidin content of Spirodela is increased by culturing Spirodela with a sugar-containing culture medium, culturing Spirodela with a hormone-containing culture medium, culturing Spirodela with light of different wavelengths, or culturing Spirodela with a nitrogen-deficient and / or phosphorus-deficient culture medium, and the proanthocyanidin content of Spirodela can also be increased by increasing the expression of genes such as 4CL1, F3'H1, F3'H3, F3'H4, ANS1, PAL1, F3'H2, ANR3 and F3H through genetic engineering means. The method of the present invention is helpful for developing and utilizing Spirodela to produce proanthocyanidins, turning Spirodela into treasure, and also provides a theoretical basis for the molecular mechanism of environmental factors regulating the biosynthesis of proanthocyanidins in Spirodela.

Description

Technical Field

[0001] The invention belongs to the technical field of natural material synthesis, and particularly relates to a method for increasing the content of Spirodela proanthocyanidin. Background Art

[0002] Spirodela polyrrhiza is a floating plant on the water surface of the Lemna family and the genus Lemna. It has the advantages of strong adaptability, fast reproduction, short growth cycle, salvage and recycling, high nutritional value and water purification. Spirodela polyrrhiza has a high flavonoid content and is a very promising flavonoid resource plant. The content of flavonoid compounds in plants will be affected by environmental factors. Among them, proanthocyanidin (PA) is an important flavonoid compound in plants. It is a defense method for plants to deal with biological and non-biological stresses (microbial pathogens, insects and ultraviolet rays, etc.). It can play a strong biological activity in the body, such as anti-oxidation, anti-bacterial and anti-inflammatory, anti-tumor, cardiovascular protection and protection of the central nervous system. It is also a natural antioxidant with high safety and strong biological activity. At present, there have been studies on the accumulation of flavonoids using duckweed, and many studies have proved that environmental factors will affect the accumulation of proanthocyanidins in plants, but in specific studies, it is necessary to consider the influence of factors such as different plant species, varieties and growth environment. However, there are no reports on the effects of environmental factors on the synthesis of proanthocyanidins in Spirodendrum and its mechanism. Spirodendrum is a rapidly reproducing aquatic plant that is widely used in ecological engineering. If its ability to produce proanthocyanidins can be developed and utilized, it will help to comprehensively utilize Spirodendrum for water purification in ecological engineering and turn waste into treasure.

[0003] Molecular sequencing technology has gone through the development of the first to the fourth generation, and the throughput and accuracy have been continuously improved. Now the most widely used is the second-generation sequencing technology. Transcriptome refers to all the transcription products of a specific cell or tissue in a specific developmental period. Transcriptomics has been a hot topic in molecular biology research in recent years, and transcriptome sequencing based on second-generation sequencing technology is its core technology. The second-generation transcriptome sequencing technology has many advantages, such as high throughput, automation, high resolution and high sensitivity of sequencing. This technology can not only study species with known genome sequence information and expand the gene database of the species, but also be used to study species with unknown genome sequence information, obtain genetic information of new species, and can be used for annotation of gene function, mining unknown genes and exploring unknown small RNAs. At present, the biosynthetic pathway of proanthocyanidins is mainly composed of phenylpropanoid, core flavonoid-anthocyanidin pathway and proanthocyanidin-specific pathway. The expression of genes of related enzymes in the proanthocyanidin biosynthetic pathway will directly affect the synthesis of proanthocyanidins in plants. Therefore, on the basis of studying the effects of environmental factors on the biosynthesis of proanthocyanidins in Spirodela, further exploring the enzyme genes related to the biosynthesis of proanthocyanidins based on the second-generation transcriptome sequencing data of Spirodela can provide a theoretical basis for the molecular mechanism of environmental factors regulating the biosynthesis of proanthocyanidins in Spirodela, and lay the foundation for subsequent research on the functions of key genes, which has important application prospects. Summary of the invention

[0004] The present invention utilizes iron salt catalysis-ultraviolet spectrophotometry to discover environmental factors that can affect the proanthocyanidin content of Spirodela, and simultaneously mines enzyme genes related to the biosynthesis of proanthocyanidins based on the second-generation transcriptome sequencing data of Spirodela, thereby proposing a method that can effectively increase the proanthocyanidin content of Spirodela.

[0005] To achieve the above object, the present invention is implemented by the following technical solutions:

[0006] The present invention provides a method for increasing the proanthocyanidin content of Spirodela, which uses at least one of the following methods to increase the proanthocyanidin content of Spirodela:

[0007] (1) Spirodela edulis is cultured in a sugar-containing medium at a culture temperature of 23°C ± 2°C, a light intensity of 4000 lx ± 500 lx, and a light / dark cycle of 16 h / 8 h, wherein the sugar includes sucrose and fructose; sucrose and fructose can promote the synthesis of Spirodela edulis proanthocyanidins within a certain concentration range, and sucrose has the best induction effect. The longer the time, the more proanthocyanidins accumulate;

[0008] (2) using a hormone-containing medium to culture Spirodela edulis at a temperature of 23°C ± 2°C, a light intensity of 4000 lx ± 500 lx, and a light / dark cycle of 16 h / 8 h, wherein the hormones include naphthaleneacetic acid (NAA) and methyl jasmonate (MeJA); a certain concentration of NAA and MeJA can promote the biosynthesis of proanthocyanidins;

[0009] (3) Spirodela was cultured in a medium containing 2% sucrose at a temperature of 23°C ± 2°C, with dark (dark-proof) or red light, a light intensity of 4000 lx ± 500 lx, and a light / dark cycle of 16 h / 8 h. Darkness can induce more proanthocyanidins than light conditions, and red light is more conducive to the accumulation of proanthocyanidins in Spirodela than blue light.

[0010] (4) Spirodendrum edulis was cultured in nitrogen-deficient and / or phosphorus-deficient medium at a temperature of 23°C ± 2°C, a light intensity of 4000 lx ± 500 lx, and a light / dark cycle of 16 h / 8 h. The concentrations of nitrogen and phosphorus had a significant effect on proanthocyanidins. As the concentration of the elements decreased, more proanthocyanidins accumulated.

[0011] (5) The expression level of at least one gene among 4CL1, F3'H1, F3'H3, F3'H4, ANS1, PAL1, F3'H2, ANR3 and F3H is increased by genetic engineering. Based on the second-generation transcriptome data of Spirodendrum edulis cultured with 1% and 3% sucrose, the present invention explores its differentially expressed genes at the molecular level, screens genes with p-adjust<0.05 and |log2FC|≥1 as significantly differentially expressed genes, and finds that the expression levels of enzyme genes related to proanthocyanidin synthesis are relatively high, among which there are 3 significantly upregulated genes, namely 4CL1, F3'H1 and ANS1, which may be the key genes promoting the production of proanthocyanidins. Among all the genes in Spirodendrum edulis, 27 are enzyme genes that are annotated to the proanthocyanidin synthesis metabolic pathway and expressed. At the same time, the present invention uses real-time fluorescence quantitative PCR technology to determine the expression of enzyme genes related to proanthocyanidin biosynthesis, and finds that sugars, light, hormones, nitrogen and phosphorus can regulate the gene expression of proanthocyanidin synthesis-related enzymes, so that the coordinated expression of these genes promotes or inhibits the synthesis of proanthocyanidins by Spirodendrum. The expression levels of the four genes 4CL1, F3'H1, F3'H3 and F3'H4 have the same rules as the proanthocyanidin content accumulated in Spirodendrum, and play a key role in the continuous synthesis of proanthocyanidins by Spirodendrum. Under light-proof conditions, the expression of PAL1, F3'H2, ANR3 and F3H increased significantly, and it is speculated that these four genes play an important role in the synthesis of proanthocyanidins by Spirodendrum under light-proof conditions.

[0012] In one embodiment, the mass content of sugar in the sugar-containing culture medium is 1% to 7%.

[0013] The proanthocyanidin content of Spirodela in sucrose increases with the increase of sugar concentration; the proanthocyanidin content of Spirodela in fructose increases with the increase of fructose concentration, but tends to be stable after reaching 5%.

[0014] In one embodiment, the wavelength of the red light is 660 nm.

[0015] In one embodiment, the hormone in the hormone-containing culture medium is naphthylacetic acid, and the content of naphthylacetic acid is less than 1 mg / L.

[0016] Low concentrations (especially 0.02 mg / L) of naphthaleneacetic acid (NAA) can promote the synthesis of proanthocyanidins, while high concentrations (1-2 mg / L) can inhibit the synthesis of proanthocyanidins.

[0017] In one embodiment, the hormone in the hormone-containing culture medium is methyl jasmonate, and the content of methyl jasmonate is 0.02-2 mg / L.

[0018] 0.02-2 mg / L of methyl jasmonate (MeJA) can promote the biosynthesis of proanthocyanidins. The higher the concentration, the more obvious the promotion effect.

[0019] In one embodiment, the nitrogen content in the nitrogen-deficient medium is 37.45 mg / L. The phosphorus content in the phosphorus-deficient medium is 3.88 mg / L.

[0020] Nitrogen deficiency (37.45 mg / L) and phosphorus deficiency (3.88 mg / L) will promote the synthesis of proanthocyanidins by Spirodendrum affine, while nitrogen abundance (749 mg / L) and phosphorus abundance (77.5 mg / L) will inhibit its synthesis.

[0021] In one embodiment, the sugar-containing culture medium, hormone-containing culture medium, nitrogen-deficient and / or phosphorus-deficient culture medium are all prepared using B5 culture medium.

[0022] In one embodiment, the pH of the sugar-containing medium, hormone-containing medium, nitrogen-deficient and / or phosphorus-deficient medium is 5.5.

[0023] In one embodiment, the genetic engineering approach includes CRISPR / Cas9-mediated gene editing technology.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] Spirodela, as an aquatic plant that can reproduce rapidly, is widely used in ecological engineering. Proanthocyanidins are important flavonoid compounds in plants, and have the characteristics of high safety and strong biological activity. The present invention discloses a method for increasing the proanthocyanidin content of Spirodela, which increases the proanthocyanidin content of Spirodela by culturing Spirodela with a sugar-containing culture medium, culturing Spirodela with a hormone-containing culture medium, culturing Spirodela with light of different wavelengths, or culturing Spirodela with a nitrogen-deficient and / or phosphorus-deficient culture medium, and can also increase the proanthocyanidin content of Spirodela by increasing the expression of genes such as 4CL1, F3'H1, F3'H3, F3'H4, ANS1, PAL1, F3'H2, ANR3 and F3H through genetic engineering. The method of the present invention is helpful to develop and utilize Spirodela to produce proanthocyanidins, turning Spirodela into treasure, and also provides a theoretical basis for the molecular mechanism of environmental factors regulating the biosynthesis of proanthocyanidins in Spirodela, and also lays a foundation for further research on key gene functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effects of sugars on the proanthocyanidin content of Spirodendrum; (a) the effect of sucrose on the proanthocyanidin content, (b) the effect of fructose on the proanthocyanidin content, (c) the effect of rhamnose on the proanthocyanidin content;

[0027] Figure 2 The effect of light on the proanthocyanidin content of Spirodendrum truncatum;

[0028] Figure 3 The effects of hormones on the proanthocyanidin content of Spirodela; (a) the effect of NAA on the proanthocyanidin content, (b) the effect of 6-BA on the proanthocyanidin content, (c) the effect of MeJA on the proanthocyanidin content;

[0029] Figure 4 The effects of nitrogen and phosphorus concentrations on the proanthocyanidin content of Spirodendrum affine; (a) the effect of nitrogen on the proanthocyanidin content, (b) the effect of phosphorus on the proanthocyanidin content;

[0030] Figure 5 Schematic diagram of the proanthocyanidin biosynthesis pathway; Note: The triangle-marked ones indicate enzymes with upregulated gene expression and related to proanthocyanidin biosynthesis;

[0031] Figure 6The results of gel electrophoresis of total RNA; Note: in order from left to right. Lanes 1, 5, 9, 15, 16, 23, 30, 34: MARKER; Lanes 2-4, 6-8: phosphorus deficiency, normal, phosphorus abundance, nitrogen deficiency, normal, nitrogen abundance; Lanes 10-14: white light, dark, red light, blue light, red and blue 1:1; Lanes 17-22, 24-26: NAA (0, 0.02, 2 mg / L), MeJA (0, 0.02, 2 mg / L), 6-BA (0, 0.02, 2 mg / L); Lanes 27-29, 31-33, 35-37: sucrose (0, 1%, 7%), fructose (0, 1%, 7%), rhamnose (0, 1%, 7%).

[0032] Figure 7 The effect of sucrose on genes related to proanthocyanidin synthesis;

[0033] Figure 8 The effect of fructose on genes related to proanthocyanidin synthesis;

[0034] Fig. 9 The effect of rhamnose on genes related to proanthocyanidin synthesis;

[0035] Fig.10 The effect of light on genes related to proanthocyanidin synthesis;

[0036] Fig.11 The effect of NAA on genes related to proanthocyanidin synthesis;

[0037] Fig.12 The effect of 6-BA on genes related to proanthocyanidin synthesis;

[0038] Fig.13 The effect of MeJA on genes related to proanthocyanidin synthesis;

[0039] Fig.14 The effect of nitrogen on genes related to proanthocyanidin synthesis;

[0040] Fig.15 The effect of phosphorus on genes related to proanthocyanidin synthesis. DETAILED DESCRIPTION

[0041] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0042] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0043] Example 1 Determination of the proanthocyanidin content of Spirodendrum based on the DB12 / T 885-2019 proanthocyanidin determination standard

[0044] (1) Preparation of culture medium: ① Preparation of sugar-containing culture medium: Sucrose, fructose and rhamnose were used. 0 g, 0.5 g, 1.5 g, 2.5 g and 3.5 g of sugar were weighed and added to 50 mL / bottle of B5 culture medium to prepare 0% (control group), 1%, 3%, 5% and 7% (mass percentage) sugar-containing B5 culture medium. The pH was adjusted to 5.5 and sterilized at 120°C for 15 min. ② Preparation of hormone-containing culture medium: normal B5 culture medium was divided into 50 mL / bottle, and 2% sucrose was added, pH was adjusted to 5.5, and sterilized at 120℃ for 15 min. Three 1 mg / mL hormone storage solutions (sterile) of MeJA, NAA and 6-BA were prepared. In the clean bench, 0 μL, 1 μL, 10 μL, 50 μL, and 100 μL of hormone storage solutions were respectively added to the sterilized B5 culture medium to prepare hormone culture medium with concentrations of 0 mg / L (control group), 0.02 mg / L, 0.2 mg / L, 1 mg / L, and 2 mg / L. ③ Preparation of culture medium for illumination: 2% sucrose was added to B5 culture medium, 50 mL per bottle, pH was adjusted to 5.5, and sterilized at 120℃ for 15 min. It was divided into five groups: white light (control group), dark, red light (LED lamp beads: 660 nm), blue light (LED lamp beads: 445 nm), and red and blue light 1:1 mixture. ④ Configuration of culture medium with different concentrations of nitrogen and phosphorus: The contents of nitrogen and phosphorus in normal B5 culture medium are 374.5mg / L and 38.75mg / L respectively. The content of single element in the deficiency culture medium is reduced by 10 times on the basis of the normal culture medium, and the content of element in the abundance culture medium is increased by 2 times on the basis of the normal culture medium. The modified components in B5 culture medium are listed in Table 1. The normal B5 culture medium is used as the control group, and 2% sucrose is added to both, the pH is adjusted to 5.5, and sterilized at 120℃ for 15min.

[0045] (2) Spirodela culture: Spirodela was placed in 50 mL of B5 medium containing 1% (mass concentration) sucrose for pre-culture until maturity, with a light / dark cycle of 16h / 8h, a culture temperature of 23℃±2℃, and a light intensity of 4000lx±500lx. Subsequently, 0.5 g of Spirodela was transferred to 50 mL / bottle of culture medium (treated with different environmental factors) using sterile tweezers, with a culture temperature of 23℃±2℃, and with a light intensity of 4000lx±500lx and a light / dark cycle of 16h / 8h, except for the 24h dark treatment under dark conditions, and the culture was carried out for 7d and 14d, respectively. Six bottles of 50 mL culture medium were set up for each group under different environmental factor treatments, with three biological replicates for 7d and 14d, respectively.

[0046] (3) Determination of proanthocyanidin content: The above-mentioned Spirodendrum duckweed materials cultured for 7 days and 14 days were taken to extract and determine the proanthocyanidin content of Spirodendrum duckweed using the DB12 / T885-2019 proanthocyanidin determination standard, and a standard curve y=0.00396x was prepared for the relationship between proanthocyanidin concentration and absorbance. The results are as follows: Figure 1 to Figure 4 .

[0047] from Figure 1 It can be seen that, in the case of 7 days of cultivation, the PA content of Spirodela in sucrose increased with the increase of sugar concentration; the PA content of Spirodela in fructose increased with the increase of concentration, but tended to be stable after reaching 5%; the PA content of Spirodela in rhamnose increased slightly, but the trend was not obvious. On the 14th day, the PA accumulated by Spirodela in sucrose and fructose was more than that at 7 days, and the accumulation trend was consistent. After treating Spirodela with rhamnose for 14 days, the PA content dropped significantly, and tended to be stable at 5%. This shows that culturing Spirodela with different concentrations of sugar and different types of sugar will have different effects on the PA content. Figure 2 It can be seen that after culturing Spirodela in different light conditions for 7 days, the order of PA content produced is darkness > red light > blue light > red and blue mixed light ≈ white light, indicating that light avoidance treatment is conducive to Spirodela accumulation of more PA; the PA content in Spirodela after monochromatic light treatment is between white light and light avoidance, and red light is more conducive to Spirodela accumulation of PA than blue light, and the results of red-blue 1:1 mixed light and white light treatment are consistent. After 14 days of cultivation, the PA accumulated by Spirodela under different light conditions increased, and the accumulation of PA content was: darkness > red light > red and blue mixed light ≈ white light > blue light. Red and blue mixed light induced Spirodela to produce more PA in the last 7 days, while the PA content in blue light became less than that in white light. This shows that darkness can induce more PA than light conditions, and different light conditions will have different effects on the PA content. Figure 3 It can be seen that after 7 days of cultivation, there was no significant difference in the induction of PA by hormones. On the 14th day of cultivation, there was a significant difference in the accumulation of PA. Compared with the blank group, 6-BA had an inhibitory effect on the production of PA, and the higher the concentration, the more obvious the inhibitory effect. Low concentrations of NAA significantly promoted the synthesis of PA, while the PA content decreased at high concentrations. Even when the NAA concentration was greater than 1 mg / L, the PA in Spirodendrum edulis was less than that in the blank group. The PA of Spirodendrum edulis in MeJA increased with the increase of hormone concentrations and tended to be stable at 2 mg / L. This shows that culturing Spirodendrum edulis with different concentrations of hormones and different types of hormones will have different effects on the PA content. Figure 4It can be seen that in the case of 7 days of cultivation, whether it is lack of elements or abundance, the accumulation of PA is not affected much; on the 14th day of cultivation, the nitrogen and phosphorus concentrations have a significant effect on PA. As the element concentration decreases, the more PA accumulates, especially in the nitrogen-deficient group (37.45 mg / L), the PA content is twice that of the nitrogen-rich group (749 mg / L). This shows that with the increase of cultivation time, the nitrogen and phosphorus concentrations will have different effects on the PA content of Spirodendrum affine. Nitrogen deficiency and phosphorus deficiency (3.88 mg / L) will promote the synthesis of PA in Spirodendrum affine, while nitrogen and phosphorus abundance (77.5 mg / L) will inhibit its synthesis.

[0048] Table 1 Partial composition of B5 medium (the concentration of components not listed is the same)

[0049]

[0050]

[0051] Note: For the normal B5 culture medium formula, please refer to Baidu Library.

[0052] Example 2 Analysis of genes of enzymes related to proanthocyanidin biosynthesis using second-generation transcriptome sequencing

[0053] Based on the second-generation transcriptome data of Spirodendrum edulis cultured with 1% and 3% sucrose for 12 days (commissioned by Meiji Biotechnology), the differentially expressed genes were explored at the molecular level, and the expression levels of the genes were quantitatively analyzed using the expression quantification software RSEM. The quantitative index was TPM. Genes with p-adjust < 0.05 and |log2FC| ≥ 1 were screened in the transcriptome data as significantly differentially expressed genes. Enzyme genes related to proanthocyanidin synthesis were found, and compared with GO, KEGG and other databases using BLASTX software to obtain their functional annotation information. The expression of enzyme genes related to proanthocyanidin biosynthesis was found and analyzed in the phenylpropanoid pathway, core flavonoid-anthocyanidin pathway and proanthocyanidin-specific pathway. As shown in Table 2 and Figure 5As shown in the figure, the expression levels are relatively high, and there are 3 significantly up-regulated genes, including 1 differentially expressed gene (4CL1) in the phenylpropanoid anabolism pathway and 2 genes (ANS1, F3'H1) in the flavonoid anabolism pathway. These 3 genes may be key genes for promoting the production of proanthocyanidins. Among all the genes in Spirodendrum truncatum, 27 enzyme genes are annotated to the proanthocyanidin anabolism pathway and expressed, of which 12 genes are annotated to the phenylpropanoid pathway, 3 are annotated to the enzyme PAL, and 3 are annotated to the enzyme C4H. Although there is no significant difference in the expression of these genes, the expression levels are relatively high and all are up-regulated. There are also 6 genes annotated to the enzyme 4CL, among which the differentially expressed 4CL1 plays an important role in the synthesis of 4-coumaroyl-CoA, the reactant of chalcone synthase. The three enzymes PAL, C4H, and 4CL play a key role in the synthesis of proanthocyanidin precursors. In addition, 15 genes were annotated to the flavonoid synthesis pathway, and 2 genes were annotated to the enzymes CHS and CHI, respectively. Although these 2 genes were not differentially expressed, their expression levels were particularly high. As upstream genes entering the core flavonoid-anthocyanidin pathway, their high expression helps to accumulate more flavonoid secondary metabolites. 5 genes are related to the synthesis of flavonoid hydroxylases, among which F3'H1 is an upregulated differentially expressed gene. The expression levels of F3H and F3'H2 are relatively high, which plays a key role in catalyzing the synthesis of dihydroflavanone alcohol, the substrate of DFR enzyme. 2 highly expressed genes are related to the synthesis of DFR, which is the key enzyme that catalyzes the production of colorless anthocyanidins from dihydroflavanone alcohol. 2 genes are related to the synthesis of ANS enzymes, and ANS1 is a differentially expressed gene, while ANS2 is not differentially expressed but has a high expression level, which helps to convert colorless anthocyanidins into colored anthocyanidins. The remaining four genes were involved in the specific pathway of proanthocyanidin synthesis and were related to the synthesis of LAR and ANR enzymes. Although there was no significant difference in expression, the expression levels were significantly higher than those of other flavonoid metabolic pathways, which was beneficial to the accumulation of proanthocyanidins.

[0054] Table 2 Expression of enzyme genes related to proanthocyanidin synthesis pathway

[0055]

[0056] Example 3 Determination of the expression level of proanthocyanidin biosynthesis-related enzyme genes

[0057] 1. Total RNA extraction and quality testing

[0058] (1) Extraction of total RNA from Spirodela ovale cultured under different environmental factors: Total RNA was extracted using TaKaRa MiniBEST Plant RNA Extraction Kit according to the instructions.

[0059] (2) RNA quality testing: ① Determine the integrity of the extracted total RNA by agarose gel electrophoresis. Figure 6 As shown in the figure, there are two clear bands under each sample, namely 18S and 28S, indicating that the extracted total RNA has good integrity. There are small bands other than 28S and 18S, which may be due to some degradation of RNA during the electrophoresis process, but it does not affect the final result. ② The concentration and purity of the extracted total RNA were detected using the ultra-micro nucleic acid protein analyzer nano-600. A260 / A280 were slightly greater than 2.0, indicating that the total RNA may be slightly degraded. A260 / A230 was between 1.7 and 2.1, and the value of A260 / A230 was less than 1.8, indicating that salt ions may remain. In general, the quality of these total RNAs meets the requirements and can be used for subsequent experiments.

[0060] 2. Analysis of the expression levels of genes related to the synthesis of proanthocyanidins in Spirodendrum under different environmental factors

[0061] (1) cDNA synthesis: Takara Prime ScriptTM RTreagent Kit with gDNAEraser (Perfect Real Time) was used to synthesize cDNA from the extracted total RNA according to the instructions.

[0062] (2) Primer design: First, based on the results of the second-generation transcriptome sequencing, Actin (Spipo0G0103500.v2), a commonly used internal reference gene in plants and with relatively high expression and stability in the transcriptome data, was selected as the internal reference gene. Oligo7 was used to design primers for the gene sequences in Table 2 (Table 3). The main parameters for primer design were: length 17-25 bp, annealing temperature (Tm) between 45-65°C, PCR product length between 80-200 bp, and the appearance of primer secondary structures such as hairpin structures, dimers, and mismatches was avoided as much as possible. The designed primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0063] (3) Determination of the optimal annealing temperature of primers and preparation of standard curve: Real-time fluorescence quantitative PCR was performed using the TB Green Premix Ex TaqII kit to explore the optimal annealing temperature of primers. The program was set as follows: first, pre-denaturation was performed at 95°C for 30 seconds, and one cycle was performed; then, reaction was performed at 95°C for 5 seconds, and then extension amplification was performed within the selected temperature range for 30 seconds, and the cycle was repeated 40 times. The melting curve was increased from 65°C to 95°C at a rate of 0.5°C every 5 seconds. The results of the optimal annealing temperature are shown in Table 3. The reverse transcribed cDNA was diluted in a 10-fold dilution gradient to obtain a total of 5 concentrations. Each concentration was technically repeated 3 times, and a standard curve of the primer was drawn, and the annealing temperature was changed to the optimal annealing temperature. According to the standard curve, the amplification efficiency of the internal reference gene and all genes was between 90% and 110%, and the determination coefficient R 2 All of them were greater than 0.990, and the selected primers could be used for subsequent verification experiments.

[0064] Table 3 Information on genes and primer sequences for fluorescence quantitative PCR

[0065]

[0066]

[0067] (4) Real-time fluorescence quantitative PCR analysis of gene expression under different environmental factors: The selected genes were amplified by real-time fluorescence quantitative PCR in the Spirodela cDNA template treated with different environmental factors, and the PCR results were analyzed using Bio-Rad CFX Manager software. -ΔΔCt The relative expression of the target gene was calculated by Figure 7 to Figure 15As shown. ① Sugar, light, hormones, nitrogen and phosphorus can regulate the gene expression of related enzymes in the PA biosynthesis pathway, so that the coordinated expression of these genes promotes or inhibits the synthesis of PA by Spirodela. Since there may be a negative feedback mechanism in the system, after Spirodela accumulates a large amount of PA, the expression of some genes will decrease to reduce the synthesis rate of PA to maintain the homeostasis of the system. The expression levels of the four genes 4CL1, F3'H1, F3'H3 and F3'H4 have the same pattern as the PA content accumulated in Spirodela, which plays a key role in the continuous synthesis of PA by Spirodela. ② In the medium containing 1% fructose or sucrose, the genes ANR1, F3H, F3'H1, F3'H3, F3'H4, ANS1, ANS2, CHS, CHI, DFR1, DFR2, LAR, PAL2, PAL3, 4CL1, 4CL4, 4CL6, C4H1, C4H2 and C4H3 of Spirodendrum rapae in the medium containing 1% fructose or sucrose were significantly upregulated compared with those in the medium containing 0%, which was consistent with the change trend of the actual measured PA content, indicating that these genes responded more actively to low concentrations of fructose and sucrose and played a more significant role. Rhamnose downregulated most genes of PA synthesis-related enzymes (ANR1, ANR2, ANR3, F3H, F3'H2, ANS1, ANS2, CHS, DFR1, LAR, PAL1, 4CL4, C4H2 and C4H3), and the higher the concentration of rhamnose, the more obvious the downregulation of expression. Rhamnose mainly inhibits the expression of these genes, thereby inhibiting the synthesis of PA. ③The existence pattern of ANS1, ANS2, LAR, ANR1, ANR2, ANR3, DFR1, DFR2, F3H, F3'H1, F3'H2, F3'H3, PAL3 and C4H1 genes is as follows: red light > red and blue mixed light > blue light, which is consistent with the actual PA content measurement pattern. Under light-avoiding conditions, the four genes PAL1, F3'H2, ANR3 and F3H were significantly upregulated, indicating that light-avoiding mainly promotes the upregulation of these four genes and increases the PA content of Spirodendrum affine. PAL1, F3'H2, ANR3 and F3H may play an important role in the synthesis of PA in Spirodendrum affine under light-avoiding conditions. ④Higher concentrations of NAA significantly inhibited the expression of genes such as PAL1, 4CL4, 4CL6, C4H3, ANR1, ANR2, ANR3, ANS1, ANS2, DFR1, LAR, CHI, CHS, F3H and F3'H2, thereby reducing the PA content of Spirodela. The expression of genes decreased significantly with the increase of 6-BA concentration, indicating that higher concentrations of 6-BA inhibited the expression of PA synthesis-related enzyme genes, thereby reducing the PA content of Spirodela. The four genes 4CL4, C4H1, F3'H4 and F3'H3 were highly expressed in high concentrations of MeJA, which played a key role in the synthesis of PA in high concentrations of MeJA.⑤PAL1, PAL2, 4CL2, 4CL4, 4CL6, C4H3, CHS, DFR2, F3'H1, F3'H3, F3'H4, ANR1 and ANS1 were induced to be highly expressed under nitrogen deficiency conditions, and 4CL2, 4CL4, 4CL6, C4H3, CHS, DFR1, DFR2, F3'H1, F3'H3, F3'H4, ANS1 and ANS2 were induced to be highly expressed under phosphorus deficiency conditions. It is mainly because the coordinated expression of these genes promoted the synthesis of more PA by Spirodendrum affine in nitrogen and phosphorus deficiency.

[0068] In summary, the present invention explored the effects of sugars, light, hormones, nitrogen and phosphorus on the PA content of Spirodela, used the second-generation transcriptome sequencing technology to analyze and screen the genes of enzymes related to PA synthesis in Spirodela, and used real-time fluorescence quantitative PCR to analyze the expression levels of genes of enzymes related to PA synthesis in Spirodela under different environmental factors. The main conclusions are as follows:

[0069] (1) Environmental factors can have different effects on the content of proanthocyanidins in Spirodela. Sugars: Sucrose and fructose can promote the synthesis of PA in Spirodela within a certain concentration. Sucrose has the best induction effect. The longer the time, the more PA accumulates. Rhamnose inhibits the synthesis of PA in Spirodela. The longer the time and the higher the concentration, the more obvious the inhibitory effect. Light: Darkness can induce more PA than light conditions. Red light is more conducive to the accumulation of PA in Spirodela than blue light. Hormones: 6-benzylaminopurine (6-BA) at a concentration of 0.02-2 mg / L can inhibit the production of PA, and the higher the concentration, the more obvious the inhibitory effect. Low concentration (0.02 mg / L) of naphthaleneacetic acid (NAA) can promote the synthesis of PA, but at high concentration (1-2 mg / L), it inhibits the synthesis of PA. Methyl jasmonate (MeJA) at 0.02-2 mg / L can promote the synthesis of PA. The higher the concentration, the more obvious the promotion effect. Nitrogen and phosphorus: Nitrogen deficiency (37.45 mg / L) and phosphorus deficiency (3.88 mg / L) will promote the synthesis of PA by Spirodendrum affine, while nitrogen abundance (749 mg / L) and phosphorus abundance (77.5 mg / L) will inhibit its synthesis.

[0070] (2) Based on the second-generation transcriptome data of Spirodendrum edulis cultured with 1% and 3% sucrose, genes with p-adjust < 0.05 and |log2FC| ≥ 1 were screened as significantly differentially expressed genes. It was found that the expression levels of enzyme genes related to proanthocyanidin synthesis were all high, among which 3 genes were significantly upregulated, namely 4CL1, F3'H1 and ANS1. These 3 genes may be key genes for promoting proanthocyanidin production. Among all the genes in Spirodendrum edulis, 27 enzyme genes were annotated to the PA synthesis metabolic pathway and expressed.

[0071] (3) Carbohydrates, light, hormones, nitrogen and phosphorus can regulate the gene expression of enzymes related to proanthocyanidin synthesis, so that the coordinated expression of these genes promotes or inhibits the synthesis of PA by Spirodela. The expression levels of the four genes 4CL1, F3'H1, F3'H3 and F3'H4 follow the same pattern as the PA content accumulated in Spirodela, and play a key role in the continuous synthesis of PA by Spirodela. Under light-proof conditions, the expression of PAL1, F3'H2, ANR3 and F3H increased significantly, suggesting that these four genes play an important role in the synthesis of PA by Spirodela under light-proof conditions.

[0072] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A method for increasing the content of proanthocyanidins in Spirodendrum truncatum, It is characterized in that At least one of the following methods is used to increase the proanthocyanidin content of Spirodendrum affine: (1) Using a sugar-containing B5 medium to culture Spirodela fasciata, the culture temperature is 23°C ± 2°C, the light intensity is 4000 lx ± 500 lx, and the light / dark cycle is 16 h / 8 h. The sugar-containing B5 medium is B5 medium + sugar, and the sugar is sucrose or fructose; the mass content of sugar in the sugar-containing medium is 1% to 7%; (2) Using a hormone-containing B5 medium to culture Spirodendrum truncatum, the culture temperature is 23°C ± 2°C, the light intensity is 4000 lx ± 500 lx, and the light / dark cycle is 16 h / 8 h. The hormone-containing B5 medium is B5 medium + hormone + 2% sucrose. The hormone is naphthylacetic acid or methyl jasmonate. The content of naphthylacetic acid is less than 1 mg / L, and the content of methyl jasmonate is 0.02-2 mg / L. (3) Spirodela was cultured in B5 medium supplemented with 2% sucrose at a temperature of 23°C ± 2°C, in darkness or red light, with a light intensity of 4000 lx ± 500 lx and a light / dark cycle of 16 h / 8 h. (4) Spirodendrum affine was cultured in nitrogen-deficient or phosphorus-deficient B5 medium at a temperature of 23°C±2°C, a light intensity of 4000 lx±500 lx, and a light / dark cycle of 16 h / 8 h. In the nitrogen-deficient medium, the nitrogen content was 37.45 mg / L; in the phosphorus-deficient medium, the phosphorus content was 3.88 mg / L.

2. A method for increasing the proanthocyanidin content of Spirodendrum according to claim 1, It is characterized in that The wavelength of the red light is 660 nm.

3. A method for increasing the proanthocyanidin content of Spirodendrum according to claim 1, It is characterized in that The pH of the sugar-containing culture medium, hormone-containing culture medium, nitrogen-deficient culture medium or phosphorus-deficient culture medium is 5.5.

Images