Application of polygonatum cyrtonema cyclase PcAS1 in promotion of hematoxylin A synthesis

Abstract

The invention discloses an application of polygonatum cyrtonema cyclase PcAS1 in promoting synthesis of hematoxylin A. The cyclase PcAS1 is derived from polygonatum cyrtonema, a high isoflavone compound hematoxylin A is synthesized in a plant body, and according to the application, a recombinant plant expression vector for expressing a PcAS1 gene is constructed and transformed into the plant to realize synthesis of the hematoxylin A. Based on multiple omics data such as whole genome, re-sequencing and transcriptome of polygonatum cyrtonema, key cyclase PcAS1 closely related to homoisoflavone biosynthesis is systematically mined. Through a tobacco transient expression system and a polygonatum cyrtonema hairy root transformation system, it is proved for the first time that cyclase PcAS1 can significantly promote synthesis of high isoflavone hematoxylon A, and biosynthesis of hematoxylon A is achieved in polygonatum cyrtonema for the first time.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology and relates to the application of a polysaccharide cyclase PcAS1 in promoting the synthesis of hematoxylin A. Background Technology

[0002] Polygonatum is a perennial herb belonging to the genus Polygonatum in the family Asparagaceae. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is one of my country's important medicinal and edible plant resources, widely distributed in provinces such as Zhejiang, Yunnan, Guizhou, Hunan, Jiangxi, and Anhui. Polygonatum cyrtonema Hua is one of the three source species of medicinal Polygonatum listed in the 2020 edition of the *Chinese Pharmacopoeia*. It possesses various pharmacological activities, including enhancing immunity, anti-oxidation, and regulating blood sugar. Its rhizomes are rich in flavonoids, polysaccharides, saponins, and other active ingredients, especially possessing high levels of isoflavones, giving it unique medicinal value.

[0003] High-isoflavones possess a unique structure, with a C6-C3-C1-C6 core, one carbon atom more than common flavonoids (C6-C3-C6), exhibiting carbon 9 specificity. Modern pharmacological studies have shown that high-isoflavones possess strong biological activities in anti-neuroinflammatory and glycemic regulation, making them an important direction for the development of new natural product drugs. Among them, high-isoflavones sappanone A can specifically bind to IMP2 to inhibit neuroinflammation. Its unique neuroprotective mechanism provides a novel target for developing next-generation safe and effective drugs for the treatment of neurodegenerative diseases, and related research results were selected as one of the top ten medical advances in China in 2017. However, high-isoflavones are extremely rare in nature, have low content, are difficult to extract, and their biosynthetic pathways or key enzymes remain unclear.

[0004] Current research generally considers 4,4'-dihydroxy-2'-methoxychalcone as the precursor for the synthesis of high isoflavones. However, 4,4'-dihydroxy-2'-methoxychalcone loses its nucleophilicity due to the 2'-methoxy substitution, suggesting that its cyclization mechanism differs from that of common flavonoids, which undergo nucleophilic cyclization at the 2'-hydroxyl group to form flavanones. However, since Dewick proposed the high isoflavone cyclization reaction 50 years ago, the enzyme responsible for the key cyclization step has remained undiscovered, becoming a technological bottleneck restricting the biosynthesis and industrial utilization of high isoflavones.

[0005] Cyclization reactions in plant secondary metabolic pathways mainly involve families of chalcone isomerases (CHI), terpenoid synthases (TPS), and cytochrome P450 (CYP450) enzymes. Current research and technologies lack clearly validated functional enzymes that catalyze the cyclization of high isoflavones or the generation of high isoflavones, and no complete pathway or transformation system for the synthesis of the high isoflavone hematoxylin A has been constructed in plant systems.

[0006] Therefore, utilizing naturally enriched plants with high isoflavone content to discover and verify key enzymes for synthesizing high isoflavones, especially cyclase genes, and developing related genetic transformation systems to construct plant bioreactors (Polygonatum sibiricum and tobacco), thereby establishing an efficient synthesis system for high isoflavone natural products such as hematoxylin A, has important theoretical significance and application value. Summary of the Invention

[0007] The purpose of this invention is to overcome the technical difficulties in the prior art, such as the unclear synthesis pathway of the high isoflavone hematoxylin A and the unknown key catalytic enzyme, and to provide an application of the Polygonatum polyantha cyclase PcAS1 in the synthesis of hematoxylin A. Through high-throughput resequencing, transcriptomics, and metabolomics techniques in Polygonatum polyantha var. ...

[0008] On the one hand, the present invention provides an application of Polygonatum polyanthracyclase PcAS1 in promoting the synthesis of hematoxylin A. The cyclase PcAS1 is derived from Polygonatum polyanthracyclase and is used in plants to synthesize high isoflavone hematoxylin A. The application is achieved by constructing a plant overexpression vector expressing the PcAS1 gene and transforming it into plants to realize the expression and synthesis of hematoxylin A.

[0009] According to the present invention, the nucleotide sequence of the above-mentioned PcAS1 gene is represented by SEQ ID NO: 1, or the amino acid sequence encoded by the PcAS1 gene is shown as SEQ ID NO: 2.

[0010] According to the present invention, the above expression vector is the pCAMBIA1380 vector, which contains the PcAS1 structural gene, the CaMV35S promoter, the GFP fluorescent reporter gene, and the NOS terminator.

[0011] According to the present invention, the above-mentioned synthetic route of hematoxylin A is to generate hematoxylin A using PcAS1.

[0012] According to the present invention, the above-mentioned plant is Nicotiana benthamiana or Polygonatum multiflorum.

[0013] On the other hand, the present invention provides a method for promoting the synthesis of hematoxylin A, the method comprising preparing a modified plant containing the Polygonatum polyanthracum PcAS1 gene, wherein the expression of the Polygonatum polyanthracum PcAS1 gene is increased in the modified plant compared with the wild-type plant; wherein the nucleotide sequence of the Polygonatum polyanthracum PcAS1 gene is as shown in SEQ ID NO: 1.

[0014] According to the present invention, the above-mentioned plant is Nicotiana benthamiana or Polygonatum multiflorum.

[0015] On the other hand, the present invention provides a method for constructing a plant expression system for expressing the cyclase PcAS1, comprising the following steps:

[0016] 1) Amplify the PcAS1 gene in Polygonatum multiflorum;

[0017] 2) The PcAS1 gene was constructed into the pCAMBIA1380 expression vector;

[0018] 3) Transform the pCAMBIA1380 expression vector into the Agrobacterium strain;

[0019] 4) Introduce Agrobacterium strains into plants via Agrobacterium-mediated transient or stable transformation;

[0020] 5) Detect GFP signal, cyclase PcAS1 expression, and hematoxylin A metabolites in plants.

[0021] According to the present invention, the above-mentioned plant is Nicotiana benthamiana or Polygonatum multiflorum.

[0022] This invention, based on multi-omics data including the whole genome, resequencing, and transcriptome of *Polygonatum multiflorum*, systematically identifies the key cyclase PcAS1 closely related to the biosynthesis of high isoflavones. Through a transient expression system in tobacco and a root transformation system of *Polygonatum multiflorum*, it is demonstrated for the first time that the cyclase PcAS1 can significantly promote the synthesis of the high isoflavone hematoxylin A. Furthermore, the biosynthesis of hematoxylin A in *Polygonatum multiflorum* is achieved for the first time, successfully creating *Polygonatum multiflorum* germplasm rich in hematoxylin A. This provides crucial technical support for the efficient synthesis, functional study, clinical application, and quality control of high isoflavone natural products. The main achievements of this invention are as follows:

[0023] (1) Based on multi-omics joint analysis, the candidate cyclase PcAS1 with high isoflavone content was systematically identified.

[0024] Based on 177 natural germplasm resources of Polygonatum multiflorum, a high-quality whole-genome SNP variant library was constructed, and targeted metabolomics data of the corresponding materials were obtained. On this basis, genome-wide association analysis (GWAS) was used to precisely locate two major QTL sites highly associated with high isoflavone accumulation, located on chromosomes Chr04 and Chr05, respectively. Two potential metabolic gene clusters were identified within a 1Mb region upstream and downstream of the major SNP. Combining gene function annotation within the candidate region, biogenic biosynthetic pathway prediction, and cross-species homology comparison, the cyclic gene PcAS1 was finally screened as an important candidate gene for high isoflavone biosynthesis.

[0025] (2) Functional study of PcAS1 gene in transient tobacco conversion and biosynthesis of high isoflavones

[0026] The coding sequence of the PcAS1 gene was obtained by cloning, and a 35S-driven plant expression vector was constructed and transiently introduced into the model plant *Nicotiana benthamiana*. The expression efficiency of the vector in tobacco was verified by GFP fluorescence signal. Metabolic detection using UPLC-MS / MS showed that hematoxylin A was not detected in wild-type tobacco, but it was successfully detected in the PcAS1 gene overexpression strain, reaching a content of 102.40 ng / g fresh weight. This clearly demonstrates that the cyclase PcAS1 catalyzes the cyclization reaction of high isoflavones to generate hematoxylin A, and is a key catalytic enzyme missing in the high isoflavone synthesis pathway.

[0027] (3) Constructing PcAS1 gene overexpressing plants of Polygonatum multiflorum and constructing lines with high accumulation of hematoxylin A.

[0028] The PcAS1 gene of *Polygonatum multiflorum* was further introduced into the rooting system of *Polygonatum multiflorum*, resulting in PcAS1 gene overexpression lines. Fluorescence signal detection and qRT-PCR results confirmed that the expression level of the PcAS1 gene in *Polygonatum multiflorum* was 5.35 times that of the wild type. UPLC-MS / MS detection of the accumulation of high levels of isoflavone metabolites in the transformed hairy roots showed that hematoxylin A was not detected in the wild-type hairy roots, while the content of hematoxylin A in the PcAS1 gene overexpression lines reached 18.26 ng / ml, achieving the first in vivo synthesis of hematoxylin A in *Polygonatum multiflorum*.

[0029] This invention reveals for the first time the function of the polysaccharide cyclase PcAS1 in the biosynthesis of high-isoflavone hematoxylin A in Polysaccharide, breaking through a long-standing bottleneck in the research of this type of natural product synthesis. Using a stable genetic transformation system based on the root development of Polysaccharide, a Polysaccharide strain with high hematoxylin A accumulation was obtained. This provides core technical support and theoretical basis for the efficient heterologous synthesis of natural drugs, the genetic improvement of Polysaccharide germplasm, and its industrialization, and has broad application prospects in biomedicine and health products. Attached Figure Description

[0030] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0031] Figure 1 This invention provides a genome-wide association analysis and candidate gene clusters for Polygonatum multiflorum.

[0032] Figure 2 This invention aims to identify gene co-expression modules significantly associated with high isoflavone levels through co-expression network analysis of Polygonatum multiflorum.

[0033] Figure 3This study analyzes the correlation between the hub gene, a co-expression module of Polygonatum multiflorum, and HIF1 in this invention.

[0034] Figure 4 The comparative transcriptome analysis of Polygonatum polyantha for this invention further identified the PcAS1 gene in Polygonatum polyantha.

[0035] Figure 5 The image shows the fluorescence of the PcAS1 gene of Polygonatum multiflorum in tobacco, as presented in this invention.

[0036] Figure 6 This is a liquid chromatography diagram for in vivo functional verification of the Polygonatum multiflorum PcAS1 gene in tobacco according to the present invention; CK: injection of isoliquiritin substrate only; PcOMT: injection of PcOMT3+ substrate only; PcAS: PcOMT3+PcAS1+ substrate group.

[0037] Figure 7 This is a secondary mass spectrum of the PcOMT3+PcAS1+ substrate group of the present invention.

[0038] Figure 8 The present invention relates to the high content of isoflavone hematoxylin A in tobacco.

[0039] Figure 9 The fluorescence image of the rhizome of Polygonatum sibiricum PcAS1 gene is shown in this invention.

[0040] Figure 10 The relative expression level of the PcAS1 gene in Polygonatum multiflorum is shown in this invention.

[0041] Figure 11 This is a liquid chromatography diagram for validating the function of the Polygonatum multiflorum PcAS1 gene in Polygonatum sibiricum of the present invention. CK: wild type; PcAS: PcAS1 bacterial culture treatment.

[0042] Figure 12 This is a secondary mass spectrum of the cyclase PcAS1 of the present invention.

[0043] Figure 13 The present invention relates to the high content of isoflavone hematoxylin A in Polygonatum sibiricum. Detailed Implementation

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] 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 specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0047] 1. Experimental Materials

[0048] 1.1 Experimental Materials

[0049] The Nicotiana benthamiana used in the experiment was cultured in the plant growth chamber of the Forest Medicinal Herbs Team, National Key Laboratory of Zhejiang A&F University (culture temperature: 25℃, light duration: 16h·d). -1 Polygonatum multiflorum was obtained from the orchard of Zhejiang Agricultural and Forestry University (cultivation conditions were natural).

[0050] 1.2 Chemical Reagents

[0051] The main reagents used in this experiment are shown in Table 1.1 below.

[0052] Table 1.1 Main reagents required for the experiment

[0053]

[0054]

[0055] 1.3 Instruments and Equipment

[0056] Lanyan constant temperature metal bath; AB104-N electronic analytical balance; PCR instrument; Thermo nanodrop 2000 spectrophotometer; Sigma benchtop high-speed refrigerated centrifuge; D-37520 high-speed centrifuge; DK-S24 electric thermostatic water bath; SpectraMax 190 microplate reader; DHG-9070HA precision forced convection drying oven; GENSYS10S UV-Vis ultraviolet spectrophotometer; #EAA06 cross-flow growth chamber; BCM-1300-A clean bench; GI100DWS high-temperature autoclave; ALPHA1-2LD Plus freeze dryer; plant in vivo transformation system, etc.

[0057] 1.4 Main Solution Formulation

[0058] 1.4.1LB medium

[0059]

[0060] Autoclave at 121°C for 20 minutes, then cool and store at 4°C. Liquid LB medium should be without agar.

[0061] 1.4.2 Preparation of antibiotic stock solution

[0062] (1) Ampicillin (100 mg / mL) -1 )

[0063] Ampicillin (Apm) 1000mg

[0064] ddH2O (autoclaved) 10mL

[0065] After dissolution, the solution was filtered through a 0.22μm microporous membrane for sterilization, then dispensed into sterilized 2mL centrifuge tubes and stored at -20℃.

[0066] (2) Kanamycin (100 mg / mL) -1 )

[0067] Kanamycin (Kan) 1000mg

[0068] ddH2O (autoclaved) 10mL

[0069] After dissolution, the solution was filtered through a 0.22μm microporous membrane for sterilization, then dispensed into sterilized 2mL centrifuge tubes and stored at -20℃.

[0070] (3) Rifampin (100 mg·mL) -1 )

[0071] Rifampin 1000mg

[0072] Dimethyl sulfoxide (DMSO) 10 mL

[0073] After dissolution, the solution was filtered through a 0.22μm microporous membrane for sterilization, then dispensed into sterilized 2mL centrifuge tubes and stored at -20℃.

[0074] 1.4.3 Preparation of Tobacco Transient Expression Injection Buffer

[0075]

[0076] MES and Na3PO4 solutions are prone to bacterial contamination; after contamination, the stock solution needs to be prepared anew. Acetyleugenone needs to be dissolved in N,N-dimethylformamide before aliquoting, and repeated freeze-thaw cycles should be avoided as much as possible. Injection buffer should be prepared fresh for each use. 1.4.4 Preparation of Resuspension of Polygonatum sibiricum Hairy Root Infection

[0077]

[0078] Adjust the pH to 5.7 using KOH. Acetyleugenone needs to be dissolved in N,N-dimethylformamide before aliquoting, avoiding repeated freeze-thaw cycles as much as possible. The resuspension should be prepared fresh before use.

[0079] 2. Experimental Methods

[0080] 2.1 Multi-omics mining of the PcAS1 gene in Polygonatum multiflorum

[0081] First, combining resequencing data from 177 Polygonatum polyanthemum accessions and high isoflavone HIF1 content data, Emmax software was used to perform genome-wide association analysis (P-value < 1e-6) to identify leader SNPs and screen candidate genes in upstream and downstream 1Mb intervals. A batch of candidate genes were then identified through GWAS. Second, combining transcriptomic and metabolomic data (6 time periods and 7 tissues), a weighted gene co-expression network was established to screen modules significantly associated with high isoflavone content. Further significant association modules were selected, and correlation analysis was performed between genes in these modules and HIF1 compound content to screen candidate genes (r > 0.90 and P-value < 0.001). The transcriptional expression profiles of H32_23 (low HIF) and high-quality material H26_13 (high HIF) were compared to screen genes with significant changes (P-value ≤ 0.001 and Fold Change > 10). Finally, the high isoflavone candidate gene PcAS1 was determined using the above methods.

[0082] 2.2 Transient transformation of the Polygonatum multiflorum PcAS1 gene into tobacco

[0083] (1) Construction of PcAS1 gene vector. The PcAS1 gene of Polygonatum cyrtonema was amplified by PCR and homologously recombined into the pCAMBIA1380 vector to construct the single-gene plant overexpression vector pCAMBIA1380-PcAS1. The single-gene plant expression vector was transformed into GV3101 competent cells, plated on Kan and Rif double-antibiotic plates and grown at 28°C for two days. After Agrobacterium colonies grew, single colonies were selected and transferred to 1 mL of double-antibiotic LB liquid medium and cultured at 28°C and 200 rpm. The bacterial culture was then transferred to 50 mL of double-antibiotic LB liquid medium and cultured overnight until the OD600 was about 0.6-0.8.

[0084] (2) Prepare injection buffer for transient expression of tobacco.

[0085] (3) Centrifuge the above bacterial culture at 3000 rpm for 10 min, discard the supernatant, add injection buffer to resuspend the precipitate, and continue to centrifuge at 3000 rpm for 10 min. Discard the supernatant and repeat twice.

[0086] (4) The precipitate was resuspended in the injection buffer, and the final Agrobacterium tumefaciens bacterial solution had an OD600 between 0.3 and 0.5.

[0087] (5) Use a disposable 1mL syringe to co-inject the bacterial solution containing pCAMBIA1380-PcOMT3 and the substrate, and to co-inject the mixed bacterial solution of pCAMBIA1380-PcAS1+pCAMBIA1380-PcOMT3 and the substrate, and to inject the substrate only.

[0088] (6) Incubate the tobacco in the dark for 2-3 days after injection.

[0089] 2.3 Metabolome of transient tobacco conversion

[0090] 2.3.1 Sample Extraction

[0091] (1) Cut off tobacco leaves that showed significant gene expression in qRT-PCR results, wrap them in aluminum foil and freeze them in liquid nitrogen, and store them at -80℃.

[0092] (2) The sample was subjected to vacuum freeze-drying and then ground with a grinder until it was powder.

[0093] (3) Dissolve 50 mg of sample powder in 1 mL of 70% methanol internal standard extract.

[0094] (4) Add 500 μL of petroleum ether, vortex for 5 min, let stand to separate into layers, and centrifuge at 12000 rpm for 10 min at 4℃.

[0095] (5) After centrifugation, all the supernatant was filtered through a 0.22 μm PTFE membrane and stored at -20℃ for LC-MS / MS analysis.

[0096] 2.3.2 Chromatographic and Mass Spectrometry Acquisition Conditions

[0097] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) (ExionLC). TM AD (https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (Applied Biosystems 6500QTRAP, https: / / sciex.com.cn / ).

[0098] Liquid phase conditions mainly include:

[0099] 1) Column: Agilent SB-C18 1.8μm, 2.1mm × 100mm;

[0100] 2) Mobile phase: Phase A is ultrapure water (with 0.1% formic acid added), and Phase B is acetonitrile (with 0.1% formic acid added);

[0101] 3) Elution gradient:

[0102] 0.00–9.00 min: The proportion of phase B increases linearly from 5% to 95%;

[0103] 9.00-10.00 min: Phase B ratio is 95%;

[0104] 10.00-11.00 min: Phase B proportion decreases to 5%;

[0105] 11.00-14.00 min: Phase B ratio is 5%;

[0106] 4) Flow rate 0.35 mL / min; column temperature 40℃; injection volume 2 μL.

[0107] Mass spectrometry conditions mainly include:

[0108] Electrospray ionization (ESI) temperature was 500 °C; ion spray voltage (IS) was 5500 V (positive ion mode) / -4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and collision-induced ionization parameters were set to high. QQQ scans were performed using MRM mode with the collision gas (nitrogen) set to medium. DP and CE for each MRM ion pair were optimized through further declustering potential (DP) and collision energy (CE). A specific set of MRM ion pairs was monitored at each epoch based on the metabolites eluted within each epoch.

[0109] 2.4 Transgenic Polygonatum multiflorum PcAS1 gene Polygonatum

[0110] Select a bacterial solution with a correct bacterial test result for infection. Wash the rhizomes of *Polygonatum sibiricum*. Make incisions on the surface of the young rhizomes with a blade, focusing on the tender white areas. Simultaneously, use a needle to create small wounds on the surface of the rhizomes. Cut a small number of fibrous roots, retaining most of them. Then soak in an infection solution prepared with *Agrobacterium rhizogenes* K599 using a vacuum device at a pressure of 8 kPa for 120 seconds, repeating the process three times. Sow the treated *Polygonatum sibiricum* in a mixture of high-humidity vermiculite, and pour the infection solution back into the container. Induction culture of hairy roots is performed under the following conditions: temperature 26℃ / 22℃ (day / night), light 16h / dark 8h, light intensity 50 μmol·m⁻¹. -2 ·s -1 During growth induction, the plant was irrigated weekly with a root-promoting nutrient solution (1 / 4 Hoagland's solution). Fluorescence was observed in the fibrous roots of explants after two months under a microscope.

[0111] 2.5 qRT-PCR analysis of transgenic Polygonatum sibiricum

[0112] 2.5.1 Extraction of RNA from Polygonatum sibiricum

[0113] Total RNA was extracted from Polygonatum cyrtonema using the TaKaRa MiniBEST Plant RNA Extraction Kit, the instructions of which can be found on the TaKaRa website (https: / / www.takarabiomed.com.cn / ). After loading an appropriate amount of total RNA sample from Polygonatum cyrtonema, electrophoresis was performed at 110V for approximately 15 minutes to check RNA integrity. The electrophoresis buffer needed to be replaced frequently, and the operation should be as rapid as possible to minimize RNA degradation. RNA concentration and A230 / A260 and A260 / A280 ratios are parameters for judging the quality of the extracted RNA, obtained by measuring 1 μL of total RNA sample using a spectrophotometer.

[0114] 2.5.2 Synthesis of the first strand of cDNA

[0115] RNA was extracted from Polygonatum multiflorum and processed according to PrimeScript. TM The RT reagent kit with gDNA Eraser (Perfect Real Time) was used to obtain cDNA. Genomic DNA removal was performed according to the specifications in Table 2.1. The mixture was gently mixed and incubated at 42°C for 2 min. After the reaction, the sample was placed on ice. Reverse transcription was then performed according to the specifications in Table 2.2. The mixture was gently mixed and incubated at 37°C for 15 min, followed by incubation at 85°C for 5 seconds. After the reaction, the sample was stored at -20°C.

[0116] Table 2.1 Genomic DNA Removal Reaction System

[0117]

[0118] Table 2.2 Reverse Transcription Reaction System

[0119]

[0120] 2.5.3 qRT-PCR analysis

[0121] qRT-PCR reactions use a rapid real-time PCR kit Fast SYBR qPCR was performed using SuperMi. Primer sequences are shown in Table 2.3 and were synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The qRT-PCR reaction system is shown in Table 2.4, and the reaction procedure is shown in Table 2.5.

[0122] Table 2.3 qRT-PCR primer sequences

[0123]

[0124] Table 2.4 qRT-PCR reaction system

[0125]

[0126] Table 2.5 qRT-PCR reaction procedure

[0127]

[0128] 2.6 Metabolome of transient conversion of Polygonatum odoratum

[0129] 2.6.1 Sample Extraction

[0130] Same as above 2.3.1.

[0131] 2.6.2 Chromatography-Mass Spectrometry Acquisition Conditions

[0132] Same as above 2.3.2.

[0133] 3 Experimental Results

[0134] 3.1 Multi-omics mining of the PcAS1 gene in Polygonatum multiflorum

[0135] Combining resequencing data from 177 Polygonatum polyanthemum accessions and high isoflavone HIF1 content data, genome-wide association analysis (P-value < 1e-6) was performed using Emmax software to identify leader SNPs and screen candidate genes within upstream and downstream 1Mb intervals. Figure 1 ); Combining transcriptomic and metabolomic data (6 time periods and 7 tissues), a weighted gene co-expression network was established to screen modules significantly associated with high isoflavone content. Figure 2 Further, correlation analysis between gene expression and HIF1 levels was conducted. Figure 3 Candidate genes were screened (r>0.90 and P-value<0.001), and the transcriptional expression profiles of H32_23 (low HIF) and the high-quality material H26_13 (high HIF) were compared. Figure 4 Genes with significant changes (P-value ≤ 0.001 and Fold Change > 10) were screened, and the PcAS1 gene of Polygonatum polyanthae on chromosome 4 was finally identified as an important candidate gene for high isoflavone cyclization.

[0136] 3.2 Transient transformation of the Polygonatum multiflorum PcAS1 gene into tobacco

[0137] Based on a fluorescent reporter gene (such as GFP) linked to the target gene, fluorescence phenotype screening of transgenic tobacco leaves was performed using fluorescence microscopy. The results showed that the experimental group exhibited stronger fluorescence than the control group. Figure 5Using UPLC-MS / MS, no chromatographic peak of hematoxylin A was detected in the characteristic ion chromatogram of the control group, and a secondary mass spectrum could not be obtained. No obvious peak of hematoxylin A was found in the characteristic ion chromatogram of the PcOMT3+ substrate, and a secondary mass spectrum could not be obtained. Combined with quantitative results, the content of hematoxylin A in this group was only 0.10 ng / ml, making accurate quantitative analysis impossible. The characteristic ion chromatogram of the PcOMT3+PcAS1+ substrate group showed a peak at 10.01 min. Figure 6 The secondary mass spectrum of the high-isoflavone hematoxylin A was extracted, with a mass-to-charge ratio (m / z) of 283.08, confirming the presence of hematoxylin A. Figure 7 The concentration of hematoxylin A was 10.24 ng / ml, and the content was 102.40 ng / g wetweight. Figure 8 The above experiments show that wild-type tobacco does not possess the enzyme catalyzing the synthesis of isoflavones. When PcOMT3 is introduced, a large amount of isoflavone precursors are synthesized in tobacco, while very low amounts of isoflavones are produced, indicating that PcOMT3 may be the key enzyme determining isoflavone production. Overexpression of PcAS1 in Polygonatum in tobacco increases the content of isoflavones synthesized more than 100 times, indicating that this enzyme may have undergone novel functionalization, exhibiting strong catalytic activity and determining the yield of isoflavones.

[0138] 3.3 Transformation of Polygonatum multiflorum PcAS1 gene into Polygonatum sibiricum

[0139] First, based on the fluorescent reporter gene (such as GFP) linked to the target gene, fluorescence microscopy was used to perform preliminary screening of the fluorescent phenotype of transgenic Polygonatum rhizomes. It was found that Polygonatum multiflorum PcAS1 showed obvious fluorescence in the hairy roots of Polygonatum multiflorum. Figure 9 Secondly, qRT-PCR experiments revealed that the relative expression level of the PcAS1 gene in the control group was 0.86, while the relative expression level in the experimental group was 5.35. Compared with the wild type, the expression level in the hairy roots overexpressed with Polygonatum multiflorum was increased by more than 5 times. Figure 10 Finally, using UPLC-MS / MS, the control group did not show a significant hematoxylin A peak in the qualitative analysis, making it impossible to obtain a secondary mass spectrum or accurately quantify the sample. The characteristic ion chromatogram of the PcAS1 group showed a significant hematoxylin A peak, with an elution time of 10.01 min. Figure 11 The secondary mass spectrum of the isoflavone hematoxylin A was extracted, with a mass-to-charge ratio (m / z) of 283.2, confirming the presence of hematoxylin A. Figure 12 Hematoxylin A (18.26 ng / ml) Figure 13The above experiments showed that wild-type Polygonatum multiflorum contains almost no hematoxylin A; however, when the PcAS1 gene was introduced into Polygonatum multiflorum, high levels of the isoflavone hematoxylin A were synthesized. Through overexpression of the PcAS1 gene, the biosynthetic pathway of high isoflavone hematoxylin A was successfully reconstructed in Polygonatum multiflorum. This pathway is absent in wild-type plants (hematoxylin A was not detected), achieving the species-specific synthesis of the target compound.

[0140] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a polysaccharide cyclase PcAS1 in promoting the synthesis of hematoxylin A, characterized in that, The cyclase PcAS1 is derived from Polygonatum polyanthae and synthesizes the high-isoflavone hematoxylin A in the plant. The application is achieved by constructing a PcAS1 gene overexpression vector and transforming the overexpression vector into the plant to realize the synthesis of hematoxylin A.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the PcAS1 gene is shown in SEQ ID NO: 1, or the amino acid sequence encoded by the PcAS1 gene is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that, The expression vector is the pCAMBIA1380 vector, which contains the PcAS1 structural gene, the CaMV35S promoter, the GFP fluorescent reporter gene, and the NOS terminator.

4. The application according to claim 1, characterized in that, The synthesis of hematoxylin A is catalyzed by the cyclase PcAS1 to generate hematoxylin A.

5. The application according to claim 1, characterized in that, The plant in question is either Nicotiana benthamiana or Polygonatum multiflorum.

6. A method for promoting the synthesis of hematoxylin A, the method comprising preparing a modified plant containing the PcAS1 gene of Polygonatum multiflorum, characterized in that, Compared to the wild-type plant, the expression of the Polygonatum multiflorum PcAS1 gene is increased in the modified plant; wherein the nucleotide sequence of the Polygonatum multiflorum PcAS1 gene is represented as SEQ ID NO:

1.

7. The method according to claim 6, characterized in that, The plant in question is either Nicotiana benthamiana or Polygonatum multiflorum.

8. A method for constructing a plant expression system for expressing the cyclase PcAS1, characterized in that, Includes the following steps: 1) Amplify the PcAS1 gene in Polygonatum multiflorum; 2) The PcAS1 gene was constructed into the pCAMBIA1380 expression vector; 3) Transform the expression vector into the Agrobacterium strain; 4) Introduce the Agrobacterium strain into plants via Agrobacterium-mediated transient or stable transformation; 5) Detect GFP signal, cyclase PcAS1 expression, and hematoxylin A metabolites in plants.

9. The method according to claim 8, characterized in that, The plant in question is either Nicotiana benthamiana or Polygonatum multiflorum.

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