Method for adjusting content of active ingredients in motherwort plants

Abstract

The invention belongs to the technical field of motherwort planting, and particularly relates to a method for adjusting the content of active ingredients in motherwort plants, and indoleacetic acid is applied to motherwort. According to the invention, indoleacetic acid is applied to leonurus, so that the content of active ingredients in the leonurus, such as any one of rutin, leonurine, caffeic acid and ferulic acid, is increased.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation, especially the field of motherwort cultivation technology, and specifically relates to a method for regulating the content of active ingredients in motherwort plants. Background Technology

[0002] Motherwort, also known as Leonurus japonicus, is the fresh or dried aerial part of the plant Leonurus japonicus, belonging to the Lamiaceae family. With the decline of wild resources, the market currently relies mainly on cultivated resources. Motherwort cultivation is primarily concentrated in Asia, such as China, Japan, and South Korea. In China, the cultivation area is relatively large, mainly distributed in Henan, Hubei, Hunan, and Jiangxi provinces. Motherwort typically thrives in sunny, well-drained soil and under suitable climatic conditions. The yield of motherwort has consistently fallen short of market demand for high-quality medicinal herbs; therefore, improving both the yield and quality of motherwort through standardized cultivation practices is one of the urgent issues to be addressed in motherwort production.

[0003] Modern pharmacological studies have shown that motherwort contains a variety of active ingredients, and its medicinal value mainly stems from the synergistic effects of these active components, including alkaloids, flavonoids, and phenolic acids. Rutin, leonurine, caffeic acid, ferulic acid, and quercetin are considered its core active ingredients. However, current motherwort cultivation techniques and methods for regulating active ingredients still suffer from low and unstable levels of these ingredients. Traditional cultivation methods rely on the natural environment and do not target secondary metabolic pathways, leading to large fluctuations in the content of target components (such as leonurine and rutin). Summary of the Invention

[0004] The purpose of this invention is to provide a method for regulating the content of active ingredients in motherwort plants, in order to solve the problems of low cultivation efficiency and insufficient accumulation of active ingredients in motherwort in the prior art, and to provide a method for optimizing the content of active ingredients in motherwort by external application of IAA.

[0005] The present invention adopts the following technical solution:

[0006] A method for regulating the content of active ingredients in motherwort plants involves applying indoleacetic acid to motherwort.

[0007] As a preferred method, indoleacetic acid is applied during the leaf stage, early flowering stage, or full bloom stage of Leonurus japonicus.

[0008] Preferably, the concentration of indoleacetic acid is 30-50 mg / L.

[0009] Preferably, the concentration of indoleacetic acid is 40 mg / L.

[0010] Preferably, the active ingredient is any one or more of rutin, leonurine, caffeic acid, ferulic acid, and quercetin.

[0011] Preferably, salicylic acid and methyl jasmonate are applied simultaneously with indoleacetic acid.

[0012] Preferably, the concentration of the salicylic acid is 10-20 mg / L.

[0013] Preferably, the concentration of methyl jasmonate is 35-50 μM.

[0014] As a preferred option, indoleacetic acid and salicylic acid are applied simultaneously during the initial flowering stage of Leonurus japonicus. The active ingredients are rutin, leonurine, caffeic acid, ferulic acid and quercetin.

[0015] As a preferred method, spray the motherwort three times during the initial flowering stage, with each application spaced 5-7 days apart.

[0016] By implementing the above technical solution, the present invention has the following beneficial effects:

[0017] This invention increases the content of any one of the active ingredients in Leonurus japonicus, such as rutin, leonurine, caffeic acid, and ferulic acid, by applying indoleacetic acid to Leonurus japonicus.

[0018] This invention applies indoleacetic acid, salicylic acid, and methyl jasmonate to Leonurus japonicus during the initial flowering stage, which can simultaneously increase the content of multiple active ingredients, so as to meet the acquisition of multiple active ingredients at the same time. Attached Figure Description

[0019] Figure 1 Figures showing the growth of Leonurus japonicus at the leaf stage under different IAA treatments;

[0020] Figure 2 The effects of external application of IAA on the changes of various growth indicators of Leonurus japonicus at different growth stages were investigated, including (a) plant height, (b) stem diameter, (c) leaf length, (d) leaf width, (e) east-west crown width, (f) north-south crown width, and (g) number of tillers.

[0021] Figure 3 Table showing the determination of phenotypic indicators of Leonurus japonicus at different growth stages after external application of IAA;

[0022] Figure 4 Biomass of Leonurus japonicus at maturity after external application of IAA;

[0023] Figure 5 The results show the content of indicative components of Leonurus japonicus at different growth stages after external application of IAA.

[0024] Figure 6The effect of external application of IAA on the effective components of Leonurus japonicus at different stages;

[0025] Figure 7 Gene expression level analysis (a) Correlation analysis between sequencing samples (b) Principal component analysis plot between sequencing samples;

[0026] Figure 8 This is a distribution map of transcription factors for differentially expressed genes.

[0027] Figure 9 Volcano plot for differentially expressed genes;

[0028] Figure 10 Heatmap of differentially expressed genes in Leonurus japonicus;

[0029] Figure 11 For differential expression analysis of Wien genes;

[0030] Figure 12 KEGG enrichment analysis among different groups of Leonurus japonicus samples;

[0031] Figure 13 Analysis of gene expression patterns in the biosynthetic pathway of leonurine;

[0032] Figure 14 Expression pattern analysis of nine target differentially expressed genes. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0034] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

[0035] Example 1: Effects of external application of IAA on the growth and development of Leonurus japonicus at different growth stages

[0036] During its growth and development, Leonurus japonicus undergoes dynamic changes due to the transfer of matter and energy. Furthermore, environmental disturbances lead to variations in phenotypic traits and the accumulation of secondary metabolites at different stages and under different conditions. This chapter investigates the effects of different gradients of intracellular adsorption (IAA) on the growth and development of Leonurus japonicus at different growth stages by measuring agronomical indicators, antioxidant enzyme activity, MDA content, carotenoid content, chlorophyll content, and active ingredient content.

[0037] 1.1 Experimental Materials and Methods

[0038] 1.1.1 Experimental Materials

[0039] Test reagent: IAA, produced by Shanghai Yuanye Biotechnology Co., Ltd.

[0040] The plant material used in this embodiment consisted of 33 wild Leonurus japonicus seedlings, with seeds collected from Emeishan City, Sichuan Province, and cultivated in greenhouses after sowing. During the experiment, exogenous spraying of different concentrations of IAA was carried out at the leaf stage, early flowering stage, and before the full bloom stage. Leaf tissues of Leonurus japonicus were collected at the leaf stage, early flowering stage, and full bloom stage for subsequent experiments.

[0041] Table 1. Experimental Material Information

[0042]

[0043] 1.1.2 Experimental Design

[0044] The plant growth regulator used in this experiment was indoleacetic acid (IAA). Five different treatment levels were set up. After the motherwort seedlings had recovered from transplanting, exogenous IAA was applied to the motherwort plants at the leaf stage, before the initial flowering stage, and before the full bloom stage. Ten days after the exogenous IAA application, various indicators were measured. The agent was sprayed evenly on the plant surface using a sprayer until the leaves of the motherwort plant were dripping with water. The auxin concentration was set at 20 mg / L, 40 mg / L, 80 mg / L, and 160 mg / L. Each treatment group was sprayed until the leaf surface was dripping with water. A control group (CK) was sprayed with water. Ten days later, fresh leaves were collected for subsequent experimental measurements. The collected materials were wrapped in aluminum foil and flash-frozen in dry ice and stored in a refrigerator at -80°C.

[0045] 1.2 Experimental Methods

[0046] 1.2.1 Determination of agronomic traits

[0047] Following the phenological schedule of Leonurus japonicus (Table 2.3), three Leonurus japonicus plants were randomly selected. Exogenous IAA was sprayed 10 days before each stage. Various indicators of Leonurus japonicus under different treatments at different stages were measured and recorded on time. The measurement method was based on Jiang Huanian's scheme.

[0048] Table 2. Phenological Stages of Motherwort

[0049]

[0050] 1.2.2 Results

[0051] After measuring various phenotypic indicators of Leonurus japonicus at different time stages, differences were found among the sample groups. External application of 20 mg / L IAA at each time stage had the most significant promoting effect on the height growth of Leonurus japonicus plants. The growth of Leonurus japonicus at different IAA concentrations during the leaf stage is shown in [reference needed]. Figure 1At the leaf stage, early flowering stage, and full bloom stage, the plant height of IAA1 was 27.17 cm, 125.00 cm, and 136.10 cm, respectively, representing increases of 25.21%, 10.30%, and 11.01% compared to the control (CK) (P<0.05). The most significant increase in plant height occurred from the leaf stage to the early flowering stage, while the rate of increase gradually slowed from the early flowering stage to the full bloom stage. Figure 2 (a) Stem diameter is an important indicator of plant growth status. During the initial and full-blown flowering stages, the stem diameter of *Leonurus japonicus* showed a trend of first increasing and then decreasing with increasing IAA concentration. Furthermore, external application of different concentrations of IAA significantly promoted the increase in stem diameter. At the initial flowering stage, the stem diameter with IAA2 was the largest, at 10.86 mm, an increase of 22.43% compared to the control (CK). At the full-blown flowering stage, the stem diameter with IAA2 was the largest, at 11.61 mm, a significant increase of 16.92% compared to the CK (P<0.05). Figure 2 (b) External application of low-concentration IAA during the leaf stage and full bloom stage effectively increased the leaf width of Leonurus japonicus. IAA2 showed the most significant increase, increasing leaf width by 12.99% and 14.07% relative to the control (CK) at the leaf stage and full bloom stage, respectively (P<0.05). The leaf width of the control (CK) showed a trend of first increasing and then decreasing from the leaf stage to the full bloom stage. External application of low-concentration IAA during the leaf stage and full bloom stage effectively increased the leaf length of Leonurus japonicus. IAA2 showed the most significant increase, increasing leaf length by 3.52% and 12.12% relative to the control (CK) at the leaf stage and full bloom stage, respectively (P<0.05). Figure 2 (c)). At different stages, external application of 40 mg / L IAA significantly increased the leaf area of ​​Leonurus japonicus. The leaf area of ​​IAA2 at the leaf stage, early flowering stage, and full bloom stage was 70.85 cm². 2 75.06cm 2 88.22cm 2 Compared with the control (CK), the tillering rates increased by 17.09%, 6.82%, and 27.84% respectively (P<0.05). At the leaf stage, IAA2 had the highest number of tillers, while IAA3 had the highest number of tillers at both the initial flowering and full bloom stages. Figure 2 (g)). See the table for the determination of performance indicators of Leonurus japonicus at different growth stages. Figure 3 .

[0052] During peak flowering, the entire Leonurus japonicus plant was dug up and its dry and wet biomass was measured. See the results below. Figure 4The biomass of Leonurus japonicus plants initially increased and then decreased with increasing IAA concentration. IAA1 had the highest biomass, with aboveground wet weight, underground wet weight, and underground dry weight of 116.97g, 33.90g, 36.57g, and 10.67g, respectively, representing increases of 2.67%, 5.05%, and 16.36% compared to the control (CK). External application of low-concentration IAA can increase the biomass of Leonurus japonicus.

[0053] Example 2: Effect of external application of IAA on the content of active ingredients in Leonurus japonicus at different growth stages

[0054] 2.1 The experimental materials are the same as in Example 1.

[0055] 2.2 Chromatographic conditions

[0056] Referring to and improving Tan Yajie's

[104] method for determining the active components of Leonurus japonicus by ultra-high performance liquid chromatography (UPLC), the leaf parts of Leonurus japonicus under different treatments and at different stages were determined. The UPLC chromatographic conditions were as follows: Waters ACQUITYUPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) was used, with 0.1% formic acid aqueous solution (A) and 100% acetonitrile solution (B) as the mobile phase, gradient elution, flow rate of 0.3 mL / min, column temperature of 30℃, injection volume of 1.0 μL, and detection wavelength of 300 nm. (See Table 3)

[0057] Table 3 Elution gradient of mobile phase

[0058] Time (minutes) 0.1% Formic A (%) 100% Acetonitrile B (%) 0 90 10 2 70 30 4 50 50 7 20 80 8.5 90 10 10 90 10

[0059] 2.3 Preparation of the reference standard solution

[0060] Weigh 4.08 mg of rutin, 3.97 mg of leonurine, 2.99 mg of caffeic acid, 3.55 mg of ferulic acid, and 2.28 mg of quercetin, dissolve them in 2 mL of 70% ethanol, and dilute to 10 mL. Prepare reference standard solutions with mass concentrations of 0.408, 0.397, 0.299, 0.355, and 0.228 mg / mL for rutin, caffeic acid, ferulic acid, and leonurine, respectively. Filter the solutions through a 0.22 μm filter membrane and transfer them to sample vials for later use.

[0061] 2.4 Preparation of Sample Solution

[0062] Weigh 0.05 g of Leonurus japonicus leaf powder, add 2.0 mL of 70% ethanol to dissolve it, and extract by ultrasonication for 2 h. Centrifuge at 12,000 r / min for 20 min using a high-speed refrigerated centrifuge. Filter the supernatant of Leonurus japonicus through a 0.22 μm filter membrane and place it in a sample vial to obtain the test solution. Inject 1.0 μL of the test solution according to the chromatographic conditions in Table 3 for determination. Each sample should be determined in triplicate.

[0063] 2.5 Results

[0064] The results of the determination of the active ingredient content of motherwort at different growth stages are as follows ( Figure 5 ).

[0065] From the leaf stage to the full bloom stage, the rutin content in Leonurus japonicus showed a trend of first increasing and then decreasing, reaching its maximum at the initial flowering stage. At the initial flowering stage, external application of IAA significantly increased the rutin content in Leonurus japonicus, with IAA2 showing the highest rutin content at 24.766 mg / g, a significant increase of 13.84% compared to the control (CK).

[0066] The content of leonurine varied considerably at different stages, with the highest content observed during the peak flowering period. External application of IAA can effectively increase the leonurine content in motherwort. The highest leonurine content was 6.36 mg / g in IAA2, which was 61.42% higher than that in the control group.

[0067] From the leaf stage to the full bloom stage, the ferulic acid content in Leonurus japonicus showed a trend of first decreasing and then increasing. During the leaf stage, the ferulic acid content was significantly higher than in other growth stages. During the leaf stage, external application of IAA significantly increased the ferulic acid content. With increasing IAA concentration, the ferulic acid content showed a trend of first increasing and then decreasing, with IAA2 showing the highest ferulic acid content at 2.67 mg / g, a significant increase of 136.56% compared to the control (CK).

[0068] From the leaf stage to the full bloom stage, the caffeic acid content in motherwort shows a trend of first decreasing and then increasing. During the leaf stage, the caffeic acid content in motherwort is significantly higher than in other growth stages.

[0069] During the leaf stage, external application of IAA can effectively increase the accumulation of caffeic acid in Leonurus japonicus. Among them, IAA2 has the highest caffeic acid content of 1.39 mg / g, which is significantly higher than the ferulic acid content of CK by 63.53%.

[0070] External application of IAA had no significant effect on the quercetin content in Leonurus japonicus. During the full bloom stage, the quercetin content was low, below the detection limit.

[0071] Depend on Figure 6It can be seen that the same active ingredient has significant differences under different concentrations of IAA at different times. As the plant grows and develops, the cumulative content of each substance shows a trend of first increasing and then decreasing. The content is highest at the initial flowering stage. Since the harvesting period of motherwort is specified in the pharmacopoeia as the initial flowering stage, the initial flowering stage is the focus of observation. At the initial flowering stage, as the concentration of externally applied IAA increases, the total content of each substance shows a trend of first increasing and then decreasing. Among them, the total content of IAA2 group (40 mg / L) is the highest.

[0072] Example 3: Effects of IAA on Leonurus japonicus at different stages based on comparative transcriptomics analysis

[0073] Exogenous plant growth regulators directly or indirectly affect plant growth. Currently, transcriptomics technology has been successfully applied to various plant responses to different plant growth regulators. This study used *Leonurus japonicus* leaves treated with 40 mg / L IAA and a control group at three different stages (leaf stage, early flowering stage, and full bloom stage) as research materials. RNA-seq data analysis was performed to analyze the expression profiles of key enzymes in the leonurine biosynthesis pathway under different treatments. A co-expression network was constructed from pathway genes and transcripts in *Leonurus japonicus* leaves at different stages and under different treatments to further identify candidate genes involved in regulating leonurine synthesis.

[0074] 3.1 Experimental Materials and Methods

[0075] 3.1.1 Experimental Materials

[0076] Metabolomics analysis was performed on Leonurus japonicus from three different periods: those without IAA and those with 40 mg / L IAA. Leaves from the leaf stage, early flowering stage, and full bloom stage were collected at each harvest, mixed, bagged, and sealed. Six groups were sampled, with three replicates per group, for metabolomics analysis. The sample grouping is shown in Table 4 below:

[0077] Table 4 Sample Grouping

[0078]

[0079] 3.1.2 Experimental Apparatus

[0080] The experimental instruments used in this embodiment are listed in Table 5.

[0081] Table 5 Experimental Instruments

[0082]

[0083] 3.2 Experimental Methods

[0084] 3.2.1 Sample Extraction

[0085] After grinding the Leonurus japonicus samples stored at -80℃ with liquid nitrogen, RNA was extracted from 18 Leonurus japonicus samples using an RNA extraction kit. The extracted RNA was quantified and quality tested using a Qubit 2.0 Fluorometer and an Agilent 2100 Bioanalyzer. The qualified RNA solutions were stored at -80℃ for later use.

[0086] 3.2.2 Construction of transcriptome cDNA library and sequencing

[0087] After the motherwort samples passed quality inspection, the cDNA library construction, quality control, and sequencing were completed by Wuhan Benabio Biotechnology Co., Ltd. using the Llumina NovaSeq high-throughput sequencing platform.

[0088] 3.2.3 Data quality control and sequence alignment to the reference genome: HISAT2 v2.0.5 was used to align clean reads to the reference genome for subsequent analysis.

[0089] 3.2.4 Gene Expression Analysis FPKM is generally regarded as a standard for gene expression values ​​in RNA-seq technology. By calculating the FPKM value of each sample in all genes, the distribution of gene expression levels in different samples is displayed through box plots, showing the differences in gene expression levels between individuals at different times and under different treatments, and the correlation coefficients between different sample groups are calculated to draw the corresponding heatmaps.

[0090] 3.2.5 Differentially expressed gene screening and analysis: DESeq2 was used for analysis. Genes with P<0.01 and fold change (FC)≥1.5 were selected as differentially expressed genes for enrichment. GO and KEGG pathway annotation and enrichment analysis were used to conduct in-depth research.

[0091] 3.2.6 Pathway Gene Expression Pattern Analysis: Gene and protein sequences of key enzymes in the biosynthesis of leonurine were downloaded from the NCBI (National Center for Biotechnology Information) database. The sequences were then analyzed using the Blast Compare Two Segments [Sets] function in TBtools software. <big file>The function uses downloaded protein sequences of key enzymes as an index to establish a local protein database of key enzymes in the biosynthesis of leonurine. Using all protein sequences from *Leonurus japonicus* as target sequences, a Blastomy search is performed with an E-value of 1e-5. Sequences with ≥80% consistency in the output results are then selected for use as genes in the key pathway of leonurine synthesis. Related heatmaps are generated using the chiplot website (https: / / www.chiplot.online / dubble_matrices_correlation).

[0092] 3.2.7 WGCNA was used to perform WGCNA analysis on the expression levels of genes involved in the leonurine synthesis pathway and all genes in 18 samples. First, hierarchical clustering was performed using the pheatmap program. Then, an appropriate soft threshold was determined, TOM values ​​were calculated to construct a scale-free network and divide it into modules. Finally, association analysis was performed between pathway genes and each module.

[0093] 3.2.8 qRT-PCR Validation Nine differentially expressed genes from Leonurus japonicus were randomly selected for qRT-PCR validation to verify the accuracy of the transcriptome data. First, the extracted total RNA was reverse transcribed. Then, primers were designed using genes related to enzymes in the leonurine and flavonoid biosynthesis pathways as templates, and TUB was used as an internal reference gene for detection. The primer sequences are shown in Table 6.

[0094] Using cDNA as a template, PCR was followed by electrophoretic separation and visualization. qRT-PCR was performed using the QuantStudio 6Flex Real-time PCR system, and the relative expression levels of the genes were calculated using the Ct value method (2-ΔΔCt).

[0095] Table 6 qRT-PCR Primer Sequences

[0096]

[0097] 4.3 Results and Analysis 4.3.1 RNA Extraction and Detection Total RNA was extracted from *Leonurus japonicus* at the leaf stage, early flowering stage, and full flowering stage after external application of 40 mg / L IAA. This RNA was used to construct a transcriptome database for *Leonurus japonicus*. The results showed that the OD260 / 280 ratios of all samples were between 2.16 and 2.19, and the OD260 / 280 ratios were between 2.19 and 2.28. The RNA quality was satisfactory and could be used for subsequent analysis (Table 7).

[0098] Table 7. Results of Total RNA Quality Inspection of Leonurus japonicus

[0099]

[0100] 3.3.2 Data Quality Summary The clean reads data obtained after processing the original data are summarized, and the results are shown in Table 8.

[0101] Table 8. Transcriptome sequencing output statistics

[0102]

[0103] 4.3.3 Reference Genome Alignment Clean reads were aligned with the *Leonurus japonicus* reference genome. The alignment results showed that groups CK2_1, CK2_2, and CK2_3 had low alignment rates, presumably due to contamination during collection. We increased their sequencing depth to ensure accuracy. For the remaining groups, over 83% of reads were completely aligned to the reference genome, with alignment rates for multiple alignment positions below 1.95% (Table 9). These results further demonstrate the good sequencing results and the acceptable quality of clean reads, allowing for further data analysis.

[0104] Table 9. Statistics of Reference Sequence Alignment Results

[0105]

[0106] 3.3.4 Gene Expression Level Analysis

[0107] Correlation heatmaps were plotted using the FPKM values ​​of 18 Leonurus japonicus samples. The minimum correlation coefficient was 0.98, and the maximum inter-group correlation coefficient was 0.972. Figure 7 Principal component analysis (PCA) can be used to evaluate the differences and reproducibility between biological replicates of different sample groups. The three growth stages of Leonurus japonicus can be clearly distinguished. There are also significant differences in gene expression levels between the IAA group (exogenously treated with IAA) and the water control group (CK).

[0108] 3.3.5 Screening for differentially expressed genes

[0109] To investigate the effects of external IAA application on Leonurus japonicus at different growth stages, the differentially expressed genes (DEGs) of leaf parts in the IAA and CK groups at different growth stages were statistically analyzed. A total of 9 differentially expressed genes were obtained across the 9 differentially expressed groups. The changes in the number of differentially expressed genes among the six groups are shown in Table 10 below. CK2_CK1: 7,093 differentially expressed genes (3,673 upregulated, 3,420 downregulated); CK3_CK1: 7,686 differentially expressed genes (3,804 upregulated, 3,882 downregulated); CK3_CK2: 9,852 differentially expressed genes (4,965 upregulated, 4,887 downregulated); IAA1_CK1: 388 differentially expressed genes (248 upregulated, 140 downregulated); IAA2_CK2: 4,889 differentially expressed genes (2,670 upregulated, 2,219 downregulated). IAA3_CK3 contains 1,556 differentially expressed genes (576 upregulated and 980 downregulated).

[0110] Table 10 Screening of differentially expressed genes

[0111]

[0112] 3.3.6 Transcription factor analysis of differentially expressed genes

[0113] Using eggNOG (eggNOG-mapper(embl.de)) annotation, differentially expressed transcription factors from Leonurus japonicus were predicted and screened. A total of 1,012 differentially expressed transcription factor genes were identified from the differential metabolites, distributed in different transcription factor families. Figure 8 The top five transcription factor families with the most differentially expressed genes are AP2, MYB, bHLH, C2H2, and NAC, with 80, 67, 64, 48, and 45 differentially expressed genes, respectively.

[0114] 3.3.6.1 Volcano Plot of Differentially Expressed Genes

[0115] Volcano plots were generated for the DEGs in each group of Leonurus japonicus for comparison. The results are as follows: Figure 9 As shown, the distribution of differentially expressed gene levels is relatively concentrated, with most ranging between 0 and 10, while the expression levels of some differentially expressed genes vary considerably.

[0116] 3.3.6.2 Cluster analysis of differentially expressed genes

[0117] After unifying all the DEGs grouped by difference comparisons, hierarchical cluster analysis was performed, and a cluster heatmap was plotted. The results are as follows: Figure 10 As shown, the clustering results can be roughly divided into three expression patterns. The first type of DEGs is highly expressed during the leaf stage and lowly expressed during the initial flowering and full bloom stages; the second type of DEGs is highly expressed during the initial flowering stage and lowly expressed during the leaf stage and full bloom stages; the third type of DEGs is highly expressed during the full bloom stage and lowly expressed during the leaf stage and initial flowering stage.

[0118] 3.3.6.3 Wien analysis of differentially expressed genes

[0119] Wien analysis was used to screen for unique or common DEGs between the IAA group and the control CK group, and for unique or common DEGs between the CK group at three different time points. The overlap of DEGs between different comparison groups was demonstrated. The results are as follows: Figure 11 As shown, at the leaf stage, there were 129 DEGs specific to IAA1_CK1; at the initial flowering stage, there were 4,294 DEGs specific to IAA2_CK2; and at the full bloom stage, there were 1,110 DEGs specific to IAA3_CK3. In addition, 42 DEGs were specifically expressed in IAA_CK at all three stages.

[0120] There are 1,371 common differentially expressed genes in the CK2_CK1, CK3_CK1, and CK3_CK2 groups, 1,179 CK2_CK1-specific DEGs, 1,304 CK3_CK1-specific DEGs, and 2,013 CK3_CK2-specific DEGs.

[0121] 3.3.7 GO Functional Annotation and Enrichment Analysis of Differentially Expressed Genes

[0122] To investigate the differences in growth and development between Leonurus japonicus treated with exogenous IAA and Leonurus japonicus treated with water as a control, this study performed GO functional annotation and enrichment analysis on the DEGs of Leonurus japonicus samples under different treatments at the same growth and development stage.

[0123] 3.3.7.1 GO Functional Annotation of Differentially Expressed Genes

[0124] At the leaf stage, the DEGS in IAA1_CK1 contained 28 secondary GO functional categories, including 10 biological processes (BP), 8 cell components (CC), and 10 molecular functions (MF). At the initial flowering stage, the DEGS in IAA2_CK2 and at full bloom, the DEGS in IAA3_CK3 both contained 30 secondary GO functional categories, including 10 BP, 10 CC, and 10 MF. The DEGS in CK2_CK1 and CK3_CK2 both contained 30 secondary GO functional categories, each including 10 BP, CC, and MF.

[0125] 3.3.8 Functional enrichment and KEGG enrichment analysis

[0126] To investigate the effects of external application of IAA on Leonurus japonicus at different growth stages, this section compares and analyzes the DEGs of the IAA group and the CK sample group at the same growth stage, as well as the DEGs of the CK sample group at different growth stages.

[0127] 87, 952, and 301 DEGs were obtained from IAA1_CK1, IAA2_CK2, and IAA3_CK3, respectively. Comparative analysis of DEGs from IAA_CK at the same growth stage revealed that the DEGs obtained from IAA1_CK1 were mainly enriched in the biosynthesis of keratinized amber and wax, flavonoid synthesis, linoleic acid metabolism, secondary metabolite synthesis, and phenylpropane biosynthesis. The enrichment of keratinized amber and wax biosynthesis pathways was most significant, with the phenylpropane biosynthesis pathway showing the highest number of enriched DEGs.

[0128] IAA2_CK2 was significantly enriched in glycolysis signaling pathways, starch and sucrose metabolism, plant secondary metabolite pathways, biosynthesis of cutin amber and wax, and galactose metabolism. Among these, the enrichment in the cutin pentose phosphate pathway was the most significant, while the starch and sucrose metabolism pathways showed the highest number of DEGs enriched.

[0129] IAA3_CK3 was significantly enriched in diterpenoid biosynthesis, secondary metabolite biosynthesis, α-linolenic acid metabolism, cyanoamino acid metabolism, and carotenoid biosynthesis, with the highest number of DEGs enriched in secondary metabolite biosynthesis.

[0130] 1479, 1744, and 2224 DEGs were obtained from CK2_CK1, CK3_CK1, and CK3_CK2, respectively. Comparative analysis of DEGs from IAA_CK at the same growth stage revealed that CK2_CK1 was significantly enriched in the pentose phosphate pathway, photosynthesis-antennae protein pathway, photosynthesis, starch and sucrose metabolism, and galactose metabolism pathway. The pentose phosphate pathway showed the most significant enrichment, while the starch and sucrose metabolism pathways showed the highest number of enriched DEGs.

[0131] CK3_CK1 was enriched in ribosomes, α-linolenic acid metabolism, glycerophospholipid metabolism, linolenic acid metabolism, and ribosome biosynthesis. Among these, the enrichment in the ribosome pathway was the most significant, and the number of DEGs enriched was the largest.

[0132] CK3_CK2 was enriched in the ribosome, pentose phosphate pathway, lysine biosynthesis, photosynthesis, and pentose phosphate pathway. Among these, the enrichment in the ribosome pathway was the most significant, and the number of DEGs enriched was the largest.

[0133] Depend on Figure 12 It was found that IAA1_CK1, IAA2_CK2, and IAA3_CK3 were all enriched in the pathways of secondary metabolite synthesis, phenylpropane biosynthesis, and linoleic acid metabolism. Notably, the phenylpropane biosynthesis pathway is a precursor pathway for leonurine synthesis. A total of 14 genes were enriched, including PAL1 (evm.model.CM061053.1.1265) and PAL2 (evm.model.CM061053.1.1266), genes upstream of leonurine biosynthesis. During the full bloom period, compared with no IAA application, the expression levels of PAL1 and PAL2 increased by 3.66-fold and 3.59-fold, respectively, which is consistent with the trend of leonurine content changes in the non-target metabolite group during the full bloom period.

[0134] 3.3.9 Pathway Gene Expression Pattern Analysis To investigate the differences in the growth and development of Leonurus japonicus, this study analyzed the expression patterns of the leonurine synthesis pathway in the leaves of Leonurus japonicus under different treatments at different stages. The differential gene expression patterns of the leonurine synthesis pathway are as follows: Figure 13 As shown, a total of 33 DEGs were annotated that participate in the regulation of the leonurine synthesis pathway in Leonurus japonicus. After clustering, the DEGs in the Leonurus japonicus synthesis pathway were roughly divided into three categories: one category was highly expressed during the leaf stage and lowly expressed during the initial flowering and full flowering stages; another category was highly expressed during the initial flowering stage and lowly expressed during the leaf and full flowering stages; and the third category was highly expressed during the full flowering stage.

[0135] To further investigate the biosynthesis of leonurine, 40 genes related to leonurine biosynthesis were identified based on transcriptome data. Analysis of gene expression levels revealed that NAGK was present in leonurus japonicus treated with exogenous IAA.

[0136] The expression levels of genes PAL1 (evm.model.CM061057.1.2827), NAGPR (evm.model.CM061057.1.145), F5H (evm.model.CM061057.1.3800), and AO (evm.model.CM061056.1.1085) were higher than those in the control group at all stages. Enrichment results revealed 14 genes enriched, including PAL1 (evm.model.CM061053.1.1265) and PAL2 (evm.model.CM061053.1.1266), genes involved in the upstream pathway of leonurine biosynthesis. At full bloom, compared with no IAA application, the expression levels of PAL1 and PAL2 increased by 3.66-fold and 3.59-fold, respectively, consistent with the leonurine content change trend in the non-target metabolite group during full bloom. Furthermore, C4H in Leonurus japonicus...

[0137] The variation trends of (evm.model.CM061055.1.2523) and 4-hydroxycinnamic acid (pC oumaric Acid) are consistent; the variation trends of C3H (evm.model.CM061057.1.1016) and caffeic acid (C affeic acid) are consistent; and the variation trends of SCPL (evm.model.CM061057.1.817) and leonurine are consistent.

[0138] 3.3.10 qRT-PCR validation of transcriptome data

[0139] To verify the reliability of the transcriptome results, this study selected nine genes with significant differences in expression within the leonurine and flavonoid pathways, combining DRGs and FPKM values. Using a method that comprehensively considers DEG function and FC values, nine genes with significant differential expression were randomly selected in the phenylpropane pathway and leonurine synthesis for qPCR validation to verify the accuracy and reliability of the transcriptome sequencing results (see details). Figure 14 The validation results showed that the expression profiles of the selected DEGs were basically consistent with the transcriptome sequencing results, exhibiting the same upregulation or downregulation trend.

[0140] 3.4 Discussion

[0141] To gain a more detailed understanding of the expression and differential expression of related genes in Leonurus japonicus after treatment with the plant growth regulator IAA, this study selected three key stages in the growth and development of Leonurus japonicus (leaf stage, initial flowering stage, and full bloom stage) for RNA-Seq sequencing, obtaining a total of 126.93 Gb of data for subsequent analysis. Using Padj ≤ 0.001 as the screening criterion, pairwise comparisons were performed in each comparison group to identify 31,464 differentially expressed genes.

[0142] GO and KEGG enrichment analyses revealed similarities in the main enriched genes among the comparative groups at different time points. The IAA1_CK1, IAA2_CK2, and IAA3_CK3 comparative groups all showed enrichment in secondary metabolite synthesis, phenylpropane biosynthesis, and linoleic acid metabolism pathways, suggesting an overlap in the effects of exogenous IAA on Leonurus japonicus. The phenylpropane biosynthesis pathway is a precursor pathway for leonurine biosynthesis. To further investigate leonurine biosynthesis, 40 genes related to leonurine biosynthesis were identified based on transcriptome data. Analysis of gene expression levels revealed that the expression levels of NAGK (evm.model.CM061057.1.2827), NAGPR (evm.model.CM061057.1.145), F5H (evm.model.CM061057.1.3800), and AO (evm.model.CM061056.1.1085) in Leonurus japonicus treated with exogenous IAA were higher than those in the control group at all stages. Enrichment results showed that a total of 14 genes were enriched, including PAL (evm.model.CM061053.1.1265, evm.model.CM061053.1.1266), a gene in the upstream pathway of leonurus alkaloid biosynthesis. During the peak flowering period, compared with no IAA application, the expression levels of PAL (evm.model.CM061053.1.1265 and evm.model.CM061053.1.1266) increased by 3.66-fold and 3.59-fold, respectively. Furthermore, the expression trends of C4H (evm.model.CM061055.1.2523) and p-coumaric acid were consistent in Leonurus japonicus, as were those of C3H (evm.model.CM061057.1.1016) and caffeic acid, and SCPL (evm.model.CM061057.1.817) and leonurine. Exogenous IAA plays a regulatory role in the expression of pathway genes, further suggesting that exogenous IAA has a significant impact on the synthesis of secondary metabolites in Leonurus japonicus. After motherwort leaves were treated with exogenous IAA, some genes responded to maintain the plant's growth and development at a normal level. These results will contribute to the study of the response mechanism of motherwort to IAA treatment and the discovery of new genes related to its adaptation, in order to understand the mechanism by which IAA affects plant growth and development.

[0143] It is speculated that the candidate genes NAGK (evm.model.CM061057.1.2827), NAGPR (evm.model.CM061057.1.145), F5H (evm.model.CM061057.1.3800), AO (evm.model.CM061056.1.1085), and PAL (evm.model.CM061053.1.1265, evm.model.CM061053.1.1266) LjbHLH37 may play important roles in the leonurine synthesis pathway of Leonurus japonicus. Their specific functions require further verification. This study provides a preliminary reference for elucidating the mechanism by which exogenous IAA affects plant growth and development.< / big>

Claims

1. A method for regulating the content of active ingredients in Leonurus japonicus plants, characterized in that, Indoleacetic acid was applied to Leonurus japonicus.

2. The method for regulating the content of active ingredients in Leonurus japonicus plants according to claim 1, characterized in that, Apply indoleacetic acid during the leaf stage, early flowering stage, or full bloom stage of Leonurus japonicus.

3. The method for regulating the content of active ingredients in Leonurus japonicus plants according to claim 1, characterized in that, The concentration of indoleacetic acid is 30-50 mg / L.

4. The method for regulating the content of active ingredients in Leonurus japonicus plants according to claim 3, characterized in that, The concentration of indoleacetic acid is 40 mg / L.

5. The method for regulating the content of active ingredients in Leonurus japonicus plants according to claim 1, characterized in that, During the initial flowering stage of Leonurus japonicus, indoleacetic acid is applied, wherein the active ingredient is any one or more of rutin, leonurine, caffeic acid, ferulic acid, and quercetin.

6. The method for regulating the content of active ingredients in Leonurus japonicus plants according to claim 1, characterized in that, Indoleacetic acid is applied during the leaf stage and / or full bloom stage of Leonurus japonicus, wherein the active ingredient is any one or more of Leonurus alkaloid, stachydrine, and trigonelline.

7. The method for regulating the content of active ingredients in Leonurus japonicus plants according to claim 1, characterized in that, The content of active ingredients in Leonurus japonicus plants can be regulated by one or more of the genes NAGK, NAGPR, F5H, AO, PAL1, PAL2, C4H, SCPL, and LjbHLH37.

8. The method for regulating the content of active ingredients in Leonurus japonicus plants according to claim 1, characterized in that, The amount of indoleacetic acid to be sprayed is determined by the amount of water droplets falling from the leaves.

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