Application of LmSCL15 in regulating chlorogenic acid and luteolin in Lonicera macgyffiensis

Abstract

The present application relates to the technical field of biological genes, and particularly relates to application of LmSCL15 of Lonicera macgyffi rea for regulating chlorogenic acid and luteolin. The nucleotide sequence of the LmSCL15 gene is shown as SEQ ID NO:1, and the amino acid sequence encoded by the LmSCL15 gene is shown as SEQ ID NO:2. The present application constructs an expression vector of the LmSCL15 gene, overexpresses the LmSCL15 gene in a wild type tomato, and finds that the chlorogenic acid and luteolin contents in the LmSCL15 overexpressed plant are obviously increased. The expression level of the LmSCL15 gene is verified by using a molecular biology detection technology, and the chlorogenic acid and luteolin content changes are measured by using an HPLC method. The results show that with the development process of the plant advancing, the expression level of the LmSCL15 gene, the chlorogenic acid and luteolin contents in the transgenic plant all show an upward trend. It is shown that the gene has the function of inducing the plant to increase the chlorogenic acid and luteolin contents. Through the present application, key gene resources and technical means can be provided for medicinal plant quality improvement and high active ingredient new variety cultivation.

Description

Technical Field

[0001] This invention relates to the field of biogenetics, and in particular to the application of LmSCL15 of Lonicera japonica in regulating chlorogenic acid and luteolin. Background Technology

[0002] Lonicera macranthoides Hand.-Mazz., belonging to the genus Lonicera in the family Caprifoliaceae, is the primary legal source plant for the medicinal herb *Lonicera macranthoides* listed in the *Pharmacopoeia of the People's Republic of China*. It is widely cultivated in southwestern my country and is a pillar crop of local economies in many regions. Traditionally used to treat carbuncles, boils, colds due to wind-heat, and fever caused by febrile diseases, this herb possesses clear heat-clearing, detoxifying, and wind-heat-dispersing effects. It has a large market demand in traditional Chinese medicine decoction pieces, medicinal extracts, and health and daily chemical products, demonstrating significant economic value. Therefore, the development of high-quality germplasm breeding and the enhancement of the accumulation of its intrinsic medicinal components are core and essential needs for the industry's growth.

[0003] Chlorogenic acid is the core phenolic acid active substance in *Lonicera japonica*, possessing multiple pharmacological effects including broad-spectrum antibacterial, anti-inflammatory, antipyretic, liver-protective, and antioxidant stress-resistance properties. It is a mandatory quality control indicator under the *Lonicera japonica* section of the current *Chinese Pharmacopoeia*, directly setting the minimum acceptable level for the medicinal material. Luteolin is a characteristic flavonoid active component of the plant, possessing pharmacological activities such as respiratory anti-inflammatory repair, antiviral activity, and free radical scavenging. Although not listed in the legally mandated testing standards for *Lonicera japonica* in the pharmacopoeia, it has long been used in scientific research and production as an important marker for evaluating the quality of *Lonicera japonica* and developing deep-processed products. These two types of substances together constitute the core material basis of the efficacy of *Lonicera japonica*, and their accumulation levels directly determine the commercial grade, clinical application value, and industrial development potential of the medicinal material.

[0004] Current research has largely clarified the biosynthetic pathways of chlorogenic acid and luteolin in Lonicera japonica, and identified a number of functional genes that regulate single pathways of phenolic acids and flavonoids respectively. However, key target genes that can simultaneously and synergistically regulate the two metabolic pathways and synchronously increase the accumulation of chlorogenic acid and luteolin are still very scarce, which limits the progress of targeted molecular breeding of new varieties with high effective components.

[0005] To address the shortcomings of existing technologies, this invention, for the first time, identified the LmSCL15 gene from *Lonicera japonica*. This gene belongs to the GRAS transcription factor family and exhibits significantly differential expression during flower development, consistent with the accumulation trends of chlorogenic acid and luteolin. By constructing an overexpression vector and transforming it into tomato, it was found that LmSCL15-overexpressing plants showed significantly increased levels of chlorogenic acid and luteolin, along with enhanced expression of key genes related to phenylpropanoid and flavonoid metabolism pathways. This suggests that the gene may promote the accumulation of active ingredients by regulating the secondary metabolic network. This discovery fills a gap in research on the involvement of GRAS transcription factors in the regulation of active ingredients in *Lonicera japonica*, providing new molecular targets and theoretical basis for the quality improvement of medicinal plants and the cultivation of new varieties with high active ingredients.

[0006] Therefore, we propose the application of LmSCL15 of Lonicera japonica in regulating chlorogenic acid and luteolin in this invention. Summary of the Invention

[0007] The purpose of this invention is to provide the application of LmSCL15 in regulating chlorogenic acid and luteolin in Lonicera japonica, which fills the research gap in the participation of GRAS transcription factors in regulating the active components of Lonicera japonica. The LmSCL15 gene can serve as a key molecular target to regulate chlorogenic acid and luteolin.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The LmSCL15 gene, and / or the LmSCL15 protein, are used in regulating chlorogenic acid content in plants, and / or in the preparation of drugs or reagents for regulating chlorogenic acid content in plants, wherein the nucleotide sequence of the LmSCL15 gene is shown in SEQ ID NO: 1.

[0010] The amino acid sequence of the LmSCL15 protein is shown in SEQ ID NO: 2.

[0011] The regulation is positive regulation, which increases the chlorogenic acid content in plants by promoting LmSCL15 gene expression and / or increasing the LmSCL15 protein content.

[0012] The LmSCL15 gene, and / or the LmSCL15 protein, in regulating luteolin content in plants, and / or the application of drugs or reagents for regulating luteolin content in plants.

[0013] The regulation is positive regulation, which increases the content of luteolin in plants by promoting the expression of the LmSCL15 gene and / or increasing the content of LmSCL15 protein.

[0014] The LmSCL15 gene, and / or any of the following applications of the LmSCL15 protein:

[0015] (1) To improve phenylpropane metabolism, and / or to prepare drugs or reagents that improve phenylpropane metabolism;

[0016] (2) Promote flavonoid metabolism, and / or, prepare drugs or reagents that promote flavonoid metabolism.

[0017] A method for increasing the content of chlorogenic acid and / or luteolin in plants by promoting the expression of the LmSCL15 gene in plants and / or increasing the content of LmSCL15 protein in plants, thereby increasing the content of chlorogenic acid and / or luteolin in plants.

[0018] The present invention has at least the following beneficial effects:

[0019] This invention reveals that the contents of chlorogenic acid and luteolin in plants expressing the LmSCL15 gene both show an increasing trend, indicating that this gene has the effect of inducing tomato plants to increase the contents of chlorogenic acid and luteolin. Through this invention, key gene resources and technical means can be provided for the quality improvement of medicinal plants and the breeding of new varieties with high active ingredients. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Phylogenetic tree construction of L.mSCL15 Lonicera grahami;

[0022] Figure 2 Dynamic changes in chlorogenic acid content at different developmental stages of Lonicera japonica;

[0023] Figure 3 A graph showing the changes in luteolin content in flowers of Lonicera lycopersicum at different developmental stages;

[0024] Figure 4 Relative expression levels of LmSCL15 in transgenic plants at different growth stages;

[0025] Figure 5 Electrophoresis image of RNA from Lonicera macrantha;

[0026] Figure 6 Construction diagram of LmSCL15 overexpression vector;

[0027] Figure 7 Image of LmSCL15 overexpression plants;

[0028] Figure 8 Graphs showing the chlorogenic acid content of different transgenic lines of WT and LmSCL15;

[0029] Figure 9 Graphs showing the detection of luteolin content in different transgenic lines of WT and LmSCL15;

[0030] Figure 10 Graph showing the expression patterns of key enzyme genes in the biosynthetic pathways of chlorogenic acid and luteolin in transgenic plants;

[0031] Figure 11. Analysis of the expression characteristics of some transcription factors in transgenic plants. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] The main objective of this invention is to provide the application of LmSCL15 from Lonicera japonica in regulating chlorogenic acid and luteolin, as detailed below:

[0034] Example 1: This example discloses the cloning and bioinformatics analysis of the LmSCL15 gene of Lonicera lyrata.

[0035] Using flower tissues from different developmental stages of Lonicera macranthoides as experimental material, total RNA was extracted ( Figure 5 The cDNA was obtained by reverse transcription. Specific primers were designed based on the transcriptome sequencing data to amplify LmSCL15 using cDNA as a template (see...). Figure 6 The complete open reading frame (ORF) sequence of gene A) was obtained. The PCR amplification product was recovered and purified after detection by 1% agarose gel electrophoresis, ligated into a cloning vector, and sequenced for verification.

[0036] Sequencing analysis revealed that the cloned LmSCL15 gene contains 1587 bp bases, and its nucleotide sequence is shown in SEQ ID NO.1. It is predicted to encode a protein consisting of 523 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2.

[0037] Bioinformatics analysis indicates that this gene belongs to the GRAS transcription factor family and contains typical GRAS conserved domains such as VHIID, PFYRE, and SAW. Phylogenetic analysis shows that LmSCL15 clusters with SCL15 proteins from species such as *Chrysanthemum indicum* (AgSCL15) and *Radish* (RsSCL15), indicating a close phylogenetic relationship. Figure 1 The fact that the SCL15 protein is distributed in a different branch than that of species such as Arabidopsis thaliana indicates that it has undergone sequence variation and functional differentiation during long-term evolution.

[0038] The nucleotide sequence of the LmSCL15 gene, SEQ ID NO.1, is as follows:

[0039]

[0040] The amino acid sequence of the protein encoded by the LmSCL15 gene, SEQ ID NO.2, is as follows:

[0041] MLMEEDDLENKPCMFHECSALQAAEKSFYDVLGQKYPPSPDSHPPFQSHPIFVKKTHNREDKSDDECVHQERSTNKLLARNYADESEDSDFVMYDKVLLCAVLNPGLHDECPFEEAALEEKKFQQNPQPK QPRPRSRKHKKKVVDLTSLLTQCAQAISISDIITTDGLLKQIREYSWPHGDGTERLAHYFANAIEARLAGTGTALYSAFVTRRIMAKDVLKAYQAYVGACPFHKMSNIFANKSIGRLARKATRLHVIDFGI LYGFQWPCLIQGLSLRPGGPPKLRITGIDFPQPGFRPAKRVEDTGRRLADYCKRFNVPFEYNAIAKKWESIRLEDLKIDRDGNEMLVVNSLYRLRNVTDEIVEAHSPRDVVLNLIKRINPDMFMHGVVNGS YSAPFFVTRFREALFHFSTLFDMFEATMAREDMQRMVHEQEVFARDAMNVIACEGGARIERPETYRQWQARNVRAGFRQVGLYRNIVKEVSRKVRARYHGDYLVDEDSNWMLQGWKGRVLYALSCWKPMQD

[0042] In this embodiment, the full-length sequence of the LmSCL15 gene of Lonicera grahami was cloned and obtained. Its gene family affiliation and conserved structural domains were identified through bioinformatics methods, providing a core target sequence for subsequent gene function verification.

[0043] Example 2: Analysis of metabolites and LmSCL15 expression levels at different flower development stages of Lonicera japonica.

[0044] Using three flower development stages of *Lonicera grayi*—the budding stage (FS1), the large white stage (FS2), and the golden flower stage (FS3)—as materials, ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) was employed to detect metabolites. (See...) Figure 2 )

[0045] The results showed that the chlorogenic acid content continuously increased with the flower development process, reaching a higher level at the golden flowering stage (FS3). This indicates that the chlorogenic acid content of Lonicera japonica shows a continuous upward trend at different flower development stages.

[0046] UPLC-MS / MS technology was used to detect and analyze luteolin in flowers at different developmental stages. Figure 3 )

[0047] The results showed that the content of luteolin was not significantly different between FS1 and FS2 stages, but increased significantly in FS3 stage, indicating that luteolin mainly accumulates rapidly in the later stages of flower development. This result suggests that luteolin accumulation varies among different flower development stages of *Lonicera japonica*.

[0048] Simultaneously, analysis of LmSCL15 expression levels at different flower development stages (FS1–FS3) based on transcriptome data revealed a significant differential expression pattern, with a marked upregulation at FS3, highly consistent with the accumulation trends of chlorogenic acid and luteolin. This result suggests that LmSCL15 may be involved in regulating flower development and the accumulation of medicinal active ingredients in *Lonicera japonica*. Figure 4 )

[0049] Therefore, we can determine the natural accumulation pattern of target active ingredients (chlorogenic acid and luteolin) during the development of Lonicera japonica, and verify that there is a positive correlation between the expression pattern of the LmSCL15 gene and the accumulation of metabolites, suggesting that it may be involved in regulating the accumulation of medicinal active ingredients.

[0050] Example 3: Construction of the LmSCL15 recombinant expression vector

[0051] Using the LmSCL15 gene obtained in Example 1 as a template, the complete coding sequence was amplified by PCR. Homologous recombination technology was then used to ligate the LmSCL15 gene into the intermediate vector 8GWN. Sequencing confirmed the correct sequence was then performed. Figure 6 B) was recombined into the plant overexpression vector CaMV35S-K303 using the GatewayLR Clonase II kit to construct the plant overexpression vector K303-LmSCL15.

[0052] The specific primer sequences used in the construction process are as follows:

[0053] name Primer sequence (5'-3') K303-LmSCL15F ATACTTCCAACTAGTGCGGCATGAAAGTACCCTTTACTTCACCAC K303-LmSCL15R ATGGTCATCCCGGGACCTGCAGGTTATCCTTGAGACATTAACACCTCCAGGCTGACGTGGCA K303-F GGCTTGTCCCGCGTCATCGGCGGG

[0054] The recombinant expression vector was then transformed into E. coli DH5α for amplification, and the results were verified by kanamycin resistance screening and colony PCR. Figure 6 (C) The plasmid was extracted and transformed into Agrobacterium tumefaciens GV3101 to obtain the engineered strain.

[0055] This embodiment mainly constructs a plant overexpression vector carrying the LmSCL15 gene and an engineered Agrobacterium strain to prepare the necessary molecular tools for carrying out plant genetic transformation operations.

[0056] Example 4: Construction and molecular identification of LmSCL15 transgenic plants.

[0057] Using the Micro-Tom tomato variety as the recipient material, genetic transformation was carried out via Agrobacterium-mediated transformation. The specific process included:

[0058] (1) Pre-culture of explants

[0059] Take young, sterile tomato leaves, cut them into pieces about 0.5 cm² in size, and place them in MB medium containing 2,4-D and KT. Pre-culture them in the dark for 1–2 days.

[0060] (2) Agrobacterium infection

[0061] Agrobacterium GV3101 carrying the K303-SCL15 expression vector was cultured in LB liquid medium at 28°C with shaking until the OD600 was approximately 0.4. After centrifugation to collect the bacterial cells, they were resuspended in an infection solution containing acetylsyringone (AS). Tomato leaf explants were then immersed for 8 min with gentle shaking.

[0062] (3) Co-cultivation

[0063] The infected explants were placed in a co-culture medium and incubated in the dark at 25°C for 2 days.

[0064] (4) Resistance screening and bud induction

[0065] The co-cultured explants were transferred to a selection medium containing kanamycin and cephalosporin, and cultured for 3–4 weeks at 25±2℃ under 12 h light conditions to induce resistant shoots. Figure 7 A)

[0066] (5) Rooting culture

[0067] The resistant shoots were transferred to 1 / 2 MB medium containing IAA for rooting culture. Figure 7 (B)

[0068] (6) Transplanting

[0069] After the plants have rooted, they are transplanted into nutrient soil for cultivation to obtain LmSCL15 gene-transgenic tomato plants. Figure 7 D in the middle

[0070] DNA was then extracted from young leaves of transgenic and wild-type tomatoes and identified using PCR.

[0071] The PCR reaction system is as follows:

[0072] reagents Dosage ddH2O 19 μl F primer (10 μM) 2 μl R primer (10 μM) 2 μl cDNA template 2 μl PrimeSTAR® Max DNA Polymerase 25 μl

[0073] The PCR reaction procedure is as follows:

[0074] Amplification conditions: 98°C for 2 min; 98°C for 10 s, 55~60°C for 20 s, for a total of 25~35 cycles; 72°C at 2K / min; 72°C for 5 min; store at 4°C.

[0075] PCR results showed that all transgenic plants amplified specific bands of the same size as the target gene. Further qRT-PCR analysis of LmSCL15 gene expression levels revealed that LmSCL15 expression was significantly higher in transgenic plants than in wild-type plants. Figure 7 (C)

[0076] Example 5: Analysis of the regulatory effect of LmSCL15 on the accumulation of chlorogenic acid and luteolin.

[0077] Leaves from transgenic tomato plants and wild-type (WT) plants at different developmental stages were collected, and the changes in chlorogenic acid content were determined by HPLC and UPLC-MS / MS methods, respectively. Figure 8 ) and changes in the content of luteolin () Figure 9 ).

[0078] The results showed that as the plant developed, the chlorogenic acid content in the LmSCL15 transgenic plants generally increased continuously, and was significantly higher than that in the wild-type plants. These results indicate that LmSCL15 can significantly promote the accumulation of chlorogenic acid in plants and enhance the activity of the phenylpropanone metabolic pathway.

[0079] Meanwhile, the luteolin content in LmSCL15-overexpressing plants was significantly higher than that in wild-type plants. This indicates that LmSCL15 can promote the activity of flavonoid metabolic pathways and increase the biosynthesis level of luteolin.

[0080] Therefore, we can see that as the plant development progresses, the contents of chlorogenic acid and luteolin in the transgenic plants generally increase, and are significantly higher than those in the wild type. Further analysis revealed that LmSCL15 can promote the continuous accumulation of secondary metabolites related to the phenylpropanoid metabolic pathway and enhance the plant's ability to synthesize bioactive components. These results indicate that LmSCL15 plays an important positive regulatory role in the biosynthesis of chlorogenic acid and luteolin, and can be applied to the cultivation of new plant varieties with high bioactive components.

[0081] Example 6: Molecular mechanism analysis of LmSCL15-regulated related genes and transcription factors

[0082] Based on transcriptome sequencing data from wild-type and LmSCL15 transgenic plants, differentially expressed enzyme genes related to chlorogenic acid and luteolin synthesis in the phenylpropane pathway were screened. RNA was extracted from LmSCL15 transgenic plants and wild-type plants for transcriptome sequencing analysis.

[0083] Analysis results:

[0084] Overexpression of LmSCL15 significantly upregulated the expression of key structural genes in the phenylpropanone metabolic pathway, including PAL, C4H, 4CL, HCT, and HQT; simultaneously, the expression of genes related to the flavonoid synthesis pathway, such as CHS, CHI, and FNS, was enhanced. Furthermore, the expression of various transcription factors, including MYB, bHLH, WRKY, Homeobox, and members of the bZIP family, was significantly altered. Figure 10 , Figure 11 ).

[0085] The above results indicate that LmSCL15 can promote the accumulation of chlorogenic acid and luteolin by regulating the synergistic effects of structural genes and transcription factors related to phenylpropane metabolism and flavonoid metabolism.

[0086] Consequently, as the plant development progressed, the contents of chlorogenic acid and luteolin in the transgenic plants showed an overall increasing trend, and both were significantly higher than those in the wild type. Further analysis revealed that LmSCL15 could promote the continuous accumulation of secondary metabolites related to the phenylpropanoid metabolic pathway and enhance the plant's ability to synthesize bioactive components. These results indicate that LmSCL15 plays an important positive regulatory role in the biosynthesis of chlorogenic acid and luteolin, and can be applied to the breeding of new plant varieties with high bioactive components.

[0087] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. The application of the LmSCL15 gene and / or LmSCL15 protein in regulating chlorogenic acid content in plants, characterized in that, The nucleotide sequence of the LmSCL15 gene is shown in SEQ ID NO: 1; The amino acid sequence of the LmSCL15 protein is shown in SEQ ID NO: 2; The regulation is positive regulation, which increases the chlorogenic acid content in plants by promoting LmSCL15 gene expression and / or increasing the LmSCL15 protein content. The plant in question is a tomato.

2. The application of the LmSCL15 gene and / or LmSCL15 protein in the preparation of reagents for regulating chlorogenic acid content in plants, characterized in that, The nucleotide sequence of the LmSCL15 gene is shown in SEQ ID NO: 1; The amino acid sequence of the LmSCL15 protein is shown in SEQ ID NO: 2; The regulation is positive regulation, which increases the chlorogenic acid content in plants by promoting LmSCL15 gene expression and / or increasing the LmSCL15 protein content. The plant in question is a tomato.

3. The application of the LmSCL15 gene and / or LmSCL15 protein in regulating luteolin content in plants, characterized in that... The nucleotide sequence of the LmSCL15 gene is shown in SEQ ID NO: 1; The amino acid sequence of the LmSCL15 protein is shown in SEQ ID NO: 2; The regulation is positive regulation, which increases the content of luteolin in plants by promoting the expression of the LmSCL15 gene and / or increasing the content of LmSCL15 protein. The plant in question is a tomato.

4. The application of the LmSCL15 gene and / or LmSCL15 protein in the preparation of reagents for regulating luteolin content in plants, characterized in that, The nucleotide sequence of the LmSCL15 gene is shown in SEQ ID NO: 1; The amino acid sequence of the LmSCL15 protein is shown in SEQ ID NO: 2; The regulation is positive regulation, which increases the content of luteolin in plants by promoting the expression of the LmSCL15 gene and / or increasing the content of LmSCL15 protein. The plant in question is a tomato.

5. A method for increasing the content of chlorogenic acid and / or luteolin in plants, characterized in that, Increase the content of chlorogenic acid and / or luteolin in plants by promoting the expression of the LmSCL15 gene and / or increasing the content of LmSCL15 protein in plants; The nucleotide sequence of the LmSCL15 gene is shown in SEQ ID NO: 1; The amino acid sequence of the LmSCL15 protein is shown in SEQ ID NO: 2; The plant in question is a tomato.

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