Application of uPEP1 polypeptide in increasing content of total flavonoids in ginkgo biloba

Abstract

The invention discloses application of uPEP1 polypeptide in increasing the content of total flavonoids of ginkgo biloba, and relates to the technical field of biology, transcriptome analysis shows that the expression quantity of a GbCCR6 gene in family members is the highest. Further translator analysis finds that an upstream open reading frame (uORF) for coding 14 amino acids exists in a 5 'UT R region, a product of the uPEP1 polypeptide is named as uPEP1 polypeptide, and subsequent exogenous application test finds that the exogenous application of the uPEP1 polypeptide can improve the content of total flavonoids in ginkgo leaves, so that the content of total flavonoids in ginkgo leaves can be increased, and the content of total flavonoids in ginkgo leaves can be increased. The research result not only provides key technical support for improving the industrial value of the medicinal gingko and promoting sustainable development of environment-friendly agriculture and forestry industries, but also lays an important theoretical foundation for industrial development of high-added-value economic forests.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of uPEP1 polypeptide in increasing the content of total flavonoids in Ginkgo biloba. Background Art

[0002] Ginkgo biloba L. is a relict plant endemic to my country, possessing significant medicinal and edible value. Its leaves are rich in secondary metabolites such as flavonoids and terpene lactones, exhibiting multiple biological activities, including antioxidant, anti-inflammatory, and anti-aging properties. They possess both significant scientific research value and enormous industrial economic potential. However, the content of secondary metabolites in ginkgo leaves is regulated by both genetic background differences and environmental factors, necessitating a strategy combining cultivation optimization with molecular breeding to enhance the accumulation of target products. Due to the long breeding cycle for ginkgo varieties and the difficulty of gene editing, current approaches primarily focus on optimizing nutrient supply through multi-fertilizer ratios and on environmental interventions such as exogenous hormone induction to enhance the synthesis and accumulation of medicinally active substances.

[0003] As a class of short-chain molecules composed of amino acids, polypeptides play an important role in natural growth regulation in plant growth and development and stress response. For example, the plant polypeptide CLV3 promotes high plant yield by regulating the maintenance of the shoot apical meristem, while systemin promotes the accumulation of defensive compounds by activating the jasmonic acid signaling pathway, thereby enhancing the plant's stress resistance. In recent years, studies have found that upstream open reading frames (uORFs) and their encoded polypeptides (uORF-encoded peptides, uPEPs) play a key role in plant growth and defense regulation. Functional uPEPs can precisely regulate the expression of downstream genes at the translation level through cis- or trans-action mechanisms, providing a new regulatory tool for improving forest metabolic traits.

[0004] Cinnamoyl-CoA reductase (CCR) is a key enzyme in the phenylpropanoid metabolic pathway, affecting downstream metabolic balance by competitively consuming common precursors of phenylpropanoid metabolism. Studies have shown that targeted inhibition of CCR expression can redirect carbon flow from lignin synthesis to the flavonoid pathway, significantly increasing the accumulation of flavonoids in plants. Currently, functional research on CCR genes has mainly focused on the transcriptional level, while its regulatory network at the translational level has not been fully elucidated. Therefore, systematic analysis of the uORF element of the Ginkgo biloba GbCCR gene and the function of the uPEP it encodes is expected to reveal the molecular mechanism of translational regulation in metabolic diversion and provide new regulatory targets for the targeted optimization of flavonoid biosynthesis. Summary of the Invention

[0005] The present invention aims to provide the use of the uPEP1 polypeptide for increasing the total flavonoid content of Ginkgo biloba. This research aims to elucidate the molecular mechanism by which uPEP, upstream of the GbCCR gene, regulates flavonoid metabolism and, based on this, develop polypeptide preparations that specifically enhance flavonoid biosynthesis. This research achievement will not only provide key technical support for increasing the industrial value of medicinal Ginkgo biloba and promoting the sustainable development of environmentally friendly agriculture and forestry, but also lay an important theoretical foundation for the industrial development of high-value-added economic forests.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] The first object of the present invention is to provide the use of uPEP1 polypeptide in increasing the total flavonoid content of Ginkgo biloba. The uPEP1 polypeptide is encoded by the upstream open reading frame of the 5'UTR region of the GbCCR6 gene. The amino acid sequence of the uPEP1 polypeptide is shown in SEQ ID NO.2.

[0008] A further improvement is that the application is specifically: during the process of raising or cultivating ginkgo seedlings, uPEP1 polypeptide is applied exogenously to promote the increase of the total flavonoid content in ginkgo leaves.

[0009] A further improvement is that the increase in the total flavonoid content of Ginkgo biloba is specifically reflected in the increase in the content of broccoli scutellariae, a flavonol, a naringenin, a dihydroflavonoid, and epicatechin, a flavanol.

[0010] A further improvement is that the exogenously applied concentration of the uPEP1 polypeptide is 50 uM and the number of exogenous applications is 2-3 times.

[0011] The second object of the present invention is to provide a polypeptide preparation comprising a solvent and an artificially synthesized uPEP1 polypeptide, wherein the uPEP1 polypeptide is encoded by the upstream open reading frame of the 5'UTR region of the GbCCR6 gene, and the amino acid sequence of the uPE P1 polypeptide is shown in SEQ ID NO.2.

[0012] A further improvement is that the concentration of the uPEP1 polypeptide in the polypeptide preparation is 50 uM.

[0013] A further improvement is that the solvent is phosphate buffer.

[0014] The second object of the present invention is to provide a use of any of the above-mentioned polypeptide preparations in increasing the total flavonoid content of Ginkgo biloba.

[0015] A further improvement is that the application is specifically: during the process of raising or cultivating ginkgo seedlings, the polypeptide preparation is sprayed onto the leaves.

[0016] A further improvement is that the spraying times are 2-3 times and the spraying amount is 15 mL / plant.

[0017] The present invention provides the following beneficial effects:

[0018] Transcriptome analysis of the present invention revealed that the GbCCR6 gene has the highest expression level among family members. Further translationome analysis revealed the presence of an upstream open reading frame (uORF) encoding 14 amino acids in its 5'UTR region, the product of which was named uPEP1 polypeptide. Subsequent exogenous application experiments revealed that exogenous application of uPEP1 polypeptide can increase the total flavonoid content of ginkgo leaves. This research achievement will not only provide key technical support for enhancing the industrial value of medicinal ginkgo and promoting the sustainable development of environmentally friendly agriculture and forestry industries, but also lay an important theoretical foundation for the industrial development of high-value-added economic forests. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 1 shows the sequencing and analysis of the Ginkgo ribosomal imprint. (a) shows the experimental design: based on the Ginkgo genome sequenced plants, root, stem, leaf, and fruit tissues were collected for translation and peptide group sequencing analysis; (b) shows the length distribution of ribosomal protected fragments; (c) and (d) show the genome-wide identification of open reading frames and their encoded peptides.

[0020] Figure 2 Analysis of the Ginkgo CCR gene family. (a) shows the chromosomal location and environmental response expression profile of GbCCR (*P < 0.05, **P < 0.05; JS: Jiangsu, YN: Yunnan, XJ: Xinjiang); (b) shows the gene structure characteristics; (c) shows the phylogenetic relationship;

[0021] Figure 3 The exogenous application of uPEP1 peptide at different concentrations and different treatment times increases the total flavonoid content of Ginkgo biloba. In the figure, (a) shows the structure of the GbCCR6 gene; (b)-(d) show the total flavonoid content test results of samples collected after 1, 2, and 3 exogenous application treatments, respectively.

[0022] Figure 4 Multi-omics analysis of Ginkgo uPEP1 exogenously treated samples. (a)-(d) Comparative analysis of the contents of the flavonoid compound pinocembrin, the flavonol chrysoeriol, the dihydroflavonoid naringenin, and the flavanol epicatechin (-)-epicatechin; (e) and (f) are expression analyses of GbCCR6 at the transcriptional and protein levels, respectively. CK is the control group; uPEP1 is the treated group. DETAILED DESCRIPTION

[0023] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0024] 1. Materials and Reagents

[0025] Unless otherwise specified, the following methods can be carried out according to conventional methods. The materials and reagents used can be obtained through commercial channels.

[0026] 2. Methods

[0027] To reveal the translation potential of the non-coding regions of the Ginkgo biloba genome, translational and peptide sequencing was performed on root, stem, leaf, and fruit tissue samples of Ginkgo biloba. Figure 1 (a)). A total of 112 million clean ribosomal footprint reads were generated. After rigorous quality control, more than two-thirds of the reads were successfully aligned to the reference genome, of which approximately 78% of the ribosomal footprints were located in known coding regions ( Figure 1 (b)). Integrating the results of peptide sequencing analysis, a large number of active translation events were found in the "non-coding" regions, including: 1656 uORFs and 42 uPEPs were identified in the 5'UTR segment, 739 intronic ORFs (iORFs) and 364 their encoded peptides were identified in the intron region, and 302 downstream ORFs (dORFs) and 24 their encoded peptides were detected in the 3'UTR segment ( Figure 1 (c) Figure 1 (d)). These findings lay the foundation for further exploration of the potential biological functions and regulatory mechanisms of "non-coding" sequences.

[0028] For Ginkgo biloba, genome annotation identified 11 CCR gene family members distributed on 6 chromosomes. Transcriptome analysis based on Ginkgo clones from Jiangsu, Yunnan, and Xinjiang showed that GbCCR6 was the most expressed gene, and multiple GbCCR genes showed environment-specific expression patterns ( Figure 2 (a)). Comparative analysis showed that the CCR gene family is highly conserved in Ginkgo biloba, and its functional module structure is consistent ( Figure 2 (b)). Phylogenetic analysis further revealed sequence homology between GbCCR and other species, indicating its functional conservation during plant evolution ( Figure 2 (c)).

[0029] 2.1. Obtaining uPEP1 polypeptide

[0030] Transcriptome analysis showed that the GbCCR6 gene (NGDC, Gene ID: evm.TU.chr2.284) was the most highly expressed gene in the family. Further translational analysis revealed the presence of an upstream open reading frame (uORF) encoding 14 amino acids in its 5'UTR region, the sequence of which is shown in SEQ ID NO.1. The product encoded by the uORF was named uPEP1 polypeptide, and its amino acid composition is MALRFSGDGLHKFF (SEQ ID NO.2) ( Figure 3 (a)).

[0031] 2.2 Artificial Synthesis of uPEP1 Peptide

[0032] (1) Select the resin for synthesis based on the sequence of the polypeptide and determine the amount of resin required based on the number of polypeptides to be synthesized.

[0033] (2) Place the resin in a peptide receiving bottle or reaction column, soak it in 5-8 mL / g of DMF for about 10 minutes, then drain it, add 5-8 mL / g of 20% hexahydropyridine (piperidine) DMF solution, place it on a shaker at room temperature or stir it with nitrogen, shake it for about 5 minutes, drain it again, add 5-8 mL / g of 20% hexahydropyridine DMF solution, place it on a shaker at room temperature or stir it with nitrogen, shake it for 15 minutes, drain it again, wash the resin with DMF 5-9 times, and drain it.

[0034] (3) Add the calculated amount of Fmoc (9-fluorenylmethoxycarbonyl [can be used as an amino protecting group]) protected amino acid, condensing agent, and HOBt to the peptide receiving bottle, and then add 5-10 mL / g of DMF and alkaline reagent. Then, place it on a shaker or nitrogen agitation at room temperature, shake for 20-60 minutes, and then drain (wash the resin with DMF 3 times, anhydrous methanol 3 times, and dichloromethane 3 times, and drain). Or wash it with DMF 6 times and then drain it.

[0035] Place approximately 10-30 resin pellets in a test tube, add 1 mL of Kaiser reagent, and heat in a water bath at 100°C for 3-5 minutes. If the resin is colorless, the reaction is complete; if it is blue or lavender, the reaction is incomplete. Add the Fmoc-protected amino acid, condensing agent, and HOBt to the peptide collection bottle, then add 5-10 mL / g of resin and the base reagent. Repeat the reaction until the Kaiser test color is colorless or nearly colorless.

[0036] (4) Add 20% hexahydropyridine (piperidine) DMF solution 5-10 mL / g, seal well, place on a shaker at room temperature, shake for about 5 minutes, then drain, react again, place on a shaker at room temperature or stir with nitrogen, shake for about 15 minutes, then drain, wash the resin three times with DMF, wash three times with anhydrous methanol, wash three times with dichloromethane, or wash 5-9 times with DMF and drain.

[0037] (5) Synthesize the next amino acid according to the sequence, and repeat the operation process of step 3 and step 4.

[0038] (6) After synthesizing the polypeptide according to the polypeptide sequence, wash the resin with ether 3-9 times, then drain and dry.

[0039] (7) Prepare the cleavage reagent. Prepare the cleavage reagent according to the sequence. The cleavage reagent mainly includes trifluoroacetic acid, water, phenol (or p-cresol), dithiol, triethylsilane, anisole, etc. The commonly used ones are: trifluoroacetic acid: water: p-cresol: dithiol 92.5:2.5:2.5:2.5 (volume ratio) trifluoroacetic acid: water: p-cresol: dithiol: anisole 82.5:5:5:5:2.5 (volume ratio), etc. Then add the polypeptide resin to the cleavage reagent solution, 6-15mL / g, seal it, shake it at room temperature for 1-4h, filter it, wash it with trifluoroacetic acid, add anhydrous ether to the filtrate, and precipitate it. Wash the solid with ether several times and then dry it. The crude polypeptide is obtained and the polypeptide is purified according to the purity requirements.

[0040] 2.3. Exogenous application of uPEP1 polypeptide

[0041] Artificially synthesized uPEP1 polypeptide (purity ≥ 95%) was used, and gradient concentration treatment solutions of 0, 12.5, 25, and 50 uM were prepared in 10 mM phosphate buffer. Twelve ginkgo seedlings were selected from each group for foliar spraying (about 15 mL / plant). After 2 hours of light-proof and moisturizing, they were routinely cultured. Leaves were collected and treated repeatedly every 48 hours for a total of 3 times. After the leaves were collected, they were quickly frozen in liquid nitrogen and stored at -80°C.

[0042] The results showed that ( Figure 3 (b) Figure 3 (c) and Figure 3 (d) Treatment with 50uM uPEP1 peptide for 2 or 3 times significantly increased the total flavonoid content in Ginkgo biloba leaves, which increased by 39.14% and 32.04% respectively compared with the control group (CK, 0uM uPEP1).

[0043] 2.4 Multi-omics integrated analysis

[0044] The samples of the control group (CK, 0uM uPEP1) and the C50 treatment group (50uM uPEP1) sampled after the third treatment in step 2.3 were selected and commissioned to Shanghai Zhongke New Life Biotechnology Co., Ltd. for metabolomic, transcriptomic and proteomic sequencing analysis.

[0045] For plant metabolome analysis, after a series of experimental processes including sample preparation, QC preparation, LC-MS / MS mass spectrometry analysis, and data analysis, the results showed that the content of various flavonoids was significantly increased after 50uM uPEP1 treatment ( Figure 4 (a) Figure 4 (b) Figure 4 (c) and Figure 4 (d)). For example, the levels of pinocembrin, a flavanone, chrysoeriol, naringenin, and (-)-epicatechin in Ginkgo biloba leaves increased 6-22 times after treatment compared to the control.

[0046] In addition, T7 transcriptome library sequencing analysis showed that GbCCR6 expression was not affected by the treatment at the transcriptional level. Proteomic sequencing analysis, through a series of experimental processes including protein extraction, peptide hydrolysis, liquid chromatography-tandem mass spectrometry (LC-MS / MS) DIA data acquisition, database retrieval, qualitative and quantitative result analysis, and bioinformatics analysis, showed that the protein expression of the GbCCR6 gene was reduced by 82.93% ( Figure 4 (e) Figure 4 (f)). Furthermore, the results showed that exogenous treatment with uPEP1 polypeptide inhibited the translation expression of the GbCCR6 gene, thereby directional regulating the carbon flow from lignin synthesis to the flavonoid pathway, significantly improving the synthesis and accumulation of flavonoids in Ginkgo leaves.

[0047] 3. Conclusion

[0048] The research of the present invention found that exogenous application of uPEP1 polypeptide can increase the total flavonoid content of ginkgo leaves. This research result will not only provide key technical support for enhancing the industrial value of medicinal ginkgo and promoting the sustainable development of environmentally friendly agricultural and forestry industries, but also lay an important theoretical foundation for the industrial development of high-value-added economic forests.

[0049] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. The application of uPEP1 polypeptide in increasing the total flavonoid content of Ginkgo biloba is characterized in that: The uPEP1 polypeptide is encoded by the upstream open reading frame of the 5'UTR region of the GbCCR6 gene. The amino acid sequence of the uPEP1 polypeptide is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The application is specifically: during the process of raising or cultivating ginkgo seedlings, uPEP1 polypeptide is applied exogenously to promote the increase of the total flavonoid content in ginkgo leaves.

3. The use according to claim 1, characterized in that The improvement of the total flavonoid content of Ginkgo biloba is specifically reflected in the increase of the content of picrotoxin of the flavanone class, chrysanthemum flavonoid class, naringenin of the dihydroflavonoid class, and epicatechin of the flavanol class.

4. The use according to claim 2, characterized in that The exogenous application concentration of the uPEP1 polypeptide is 50 uM, and the number of exogenous applications is 2-3 times.

5. A polypeptide preparation, characterized in that The invention comprises a solvent and an artificially synthesized uPEP1 polypeptide, wherein the uPEP1 polypeptide is encoded by an upstream developed reading frame of the 5'UTR region of the GbCCR6 gene, and the amino acid sequence of the uPEP1 polypeptide is shown in SEQ ID NO.

2.

6. A polypeptide preparation according to claim 5, characterized in that: The concentration of the uPEP1 polypeptide in the polypeptide preparation is 50 uM.

7. A polypeptide preparation according to claim 5, characterized in that: The solvent is phosphate buffer.

8. Use of the polypeptide preparation according to any one of claims 5 to 7 for increasing the total flavonoid content of Ginkgo biloba.

9. The use according to claim 8, characterized in that The application is specifically as follows: during the process of raising or cultivating ginkgo seedlings, the polypeptide preparation is sprayed onto leaves.

10. The use according to claim 9, characterized in that The spraying times were 2-3 times, and the spraying amount was 15 mL / plant.

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