CN111286508A - Ginkgo biloba flavonoid 3`,5`-hydroxylase GbF3`5`H1 gene and its protein and application - Google Patents

Abstract

The invention discloses a ginkgo biloba flavonoid 3',5'-hydroxylase GbF3'5'H1 gene, a protein and an application thereof, belonging to the technical field of genetic engineering. The base sequence of the flavonoid 3',5'-hydroxylase GbF3'5'H1 gene is shown in SEQ ID NO.1, and the amino acid of the flavonoid 3',5'-hydroxylase GbF3'5'H1 The sequence is shown in SEQ ID NO.2. The gene sequence of the flavonoid 3', 5'-hydroxylase GbF3'5'H1 is derived from Ginkgo biloba. The invention clones the flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene from Ginkgo biloba, and systematically identifies its function, and finds that the gene can increase the content of plant flavonoid-related metabolites, And can participate in and promote the synthesis of flavonoid-related metabolites in transgenic poplar.

Description

Ginkgo biloba flavonoid 3′,5′-hydroxylase GbF3′5′H1 gene and its protein and application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to the 3', 5'-hydroxylase GbF3'5'H1 gene of ginkgo biloba flavonoids and its protein and application.

Background technique

The CYP450 protein family is the largest family of plant genes, and plant CYP450 genes account for about 1% of the total genes in the plant genome. Currently, CYP450 has received extensive attention due to its basic function in plants, which is involved in the metabolism of exogenous chemicals and endogenous biochemicals. In particular, CYP450 is involved in plant secondary metabolism, mainly involved in the synthesis of structural macromolecules, pigments and defense compounds; and the catabolism of various hormones or signaling molecules. CYP450 is a heme-dependent oxidase with mixed functions. Some important reactions catalyzed by CYP450 play important roles in the synthesis of phenylpropane and flavonoids in plants. For example, it can alter the content of anthocyanins downstream of the flavonoid pathway and thus affect the appearance of pigments.

Flavonoids are an important class of secondary metabolites in plants with diverse physiological functions and are considered to be an important part of chemical defense mechanisms. Its synthesis starts with phenylalanine and malonyl-coenzyme A, and gradually synthesizes aromatic compounds with diverse structures. It is further modified into different subclasses of flavonoids by different kinds of enzymes. Many genes involved in flavonoid synthesis have been cloned, and the biosynthetic pathway of flavonoids in Arabidopsis has been well studied. Compared with the single-copy gene of Arabidopsis, the multi-gene family of Ginkgo biloba controls various steps of the biosynthetic pathway of flavonoids, forming a more complex network. Relevant studies have shown that the biosynthesis of flavonoids is regulated by key enzyme genes in the pathway. Although some key enzymes in the Ginkgo flavonoid biosynthesis pathway (GbCHS, GbCHI, GbF3H, and GbFLS) are catalytically active in E. coli, it is unclear how these enzymes synthesize flavonoids in Ginkgo biloba due to the lack of related mutants.

Ginkgo biloba is an ancient relict plant, considered a "living fossil", and it is the only extant species of the Ginkgo family. Its origin can be traced back to more than 200 million years ago, and it is a unique species representative of plant phylogeny and has an important evolutionary position. Because ginkgo has good adaptability to the environment and climate, it has been cultivated all over the world. In addition, Ginkgo biloba is also an important medicinal tree species because its leaves are rich in flavonoids and terpenoids. It can be seen that Ginkgo biloba is a chemical factory for the production of secondary metabolites with important pharmacological activities. To date, Ginkgo biloba extract EGB761 is one of the most well-known products in Ginkgo biloba and has been widely used, including the prevention of Alzheimer's disease and cerebrovascular dysfunction. Although there are many difficulties in the supply of Ginkgo biloba and the chemical synthesis is far from being applied to industrial production, there is a huge development potential and economic value. However, since the regeneration system of Ginkgo biloba has not been established, it is currently impossible to breed Ginkgo biloba genotypes with high flavonoid production through genetic transformation.

F3′5′H has a wide range of flavonoid substrate activities and participates in the biosynthetic pathway of flavonoid-related metabolites. At present, the F3′5′H gene has been well studied in the ornamental plant petunia. And F3'5'Hs have been isolated from a variety of plants, such as grape, snapdragon, Fuguiju, tomato, etc., but have not been studied in Ginkgo biloba.

SUMMARY OF THE INVENTION

In view of the problems in the prior art, the technical problem solved by the present invention is to separate and obtain the F3'5'H gene involved in the synthesis of flavonoid-related metabolites from Ginkgo biloba, and the present invention separates and obtains ginkgo biloba flavonoids 3', 5'- Hydroxylase GbF3'5'H1 gene, and provides the amino acid sequence of ginkgo flavonoid 3', 5'-hydroxylase GbF3'5'H1, and also provides ginkgo flavonoid 3', 5'-hydroxylase GbF3 Application of '5'H1 gene in increasing the content of plant flavonoid-related metabolites. The present invention clones the flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene from Ginkgo biloba, and finds that the gene can increase the content of plant flavonoid-related metabolites, and can participate in and promote flavonoids in transgenic poplars Synthesis of related metabolites.

In order to solve the above problems, the technical scheme adopted in the present invention is as follows:

The base sequence of Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene is shown in SEQ ID NO.1.

The amino acid sequence of Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 is shown in SEQ ID NO.2.

The expression vector containing the above-mentioned Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene.

The expression vector containing the Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene, the ccdB gene located downstream of the CaMV 35S promoter was cloned into the PBI121 vector using the Gateway technology to construct a Pro35S::GbF3 '5'H1 expression vector.

The application of the above-mentioned Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene in increasing the content of plant flavonoid-related metabolites. The method comprises the following steps: (1) constructing an expression vector containing the Ginkgo biloba flavonoid 3',5'-hydroxylase GbF3'5'H1 gene; (2) transforming the constructed expression vector into a plant or plant cell; ( 3) Cultivate and screen to obtain transgenic plants.

The application of the Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene in improving the content of plant flavonoid-related metabolites, using the Gateway technology to clone the ccdB gene located downstream of the CaMV 35S promoter into PBI121 On the vector, an expression vector containing Pro35S::GbF3'5'H1 was constructed.

For the application of the Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene in increasing the content of plant flavonoid-related metabolites, the constructed expression vector is transformed into plants by using Agrobacterium strain EHA105.

The application of the Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene in improving the content of flavonoid-related metabolites in a plant, the plant being poplar.

The application of the Ginkgo biloba flavonoid 3',5'-hydroxylase GbF3'5'H1 gene in improving the content of plant flavonoid-related metabolites, the flavonoid-related metabolites include catechol, phenylalanine, 4',5-dihydroxy-7-glucosoxyflavanone.

Beneficial effects: Compared with the existing technology, the advantages of the present invention include:

(1) The results of the present invention not only provide information for the characteristics and functions of the ginkgo GbF3'5'H1, but also help to better understand the synthesis and underlying molecular mechanism of flavonoids.

(2) The present invention cloned the gene of the flavonoid 3', 5'-hydroxylase GbF3'5'H1 from Ginkgo biloba, and systematically identified its function, and found that the gene can improve the metabolism of plant flavonoids It can participate in and promote the synthesis of flavonoid-related metabolites in transgenic poplars.

Description of drawings

Figure 1 shows the expression profile of GbF3'5'H1 gene in Ginkgo biloba detected by qRT-PCR, in which Figure 1A shows the expression pattern of GbF3'5'H1 in various tissues, and Figure 1B shows the GbF3'5'H1 gene in Ginkgo biloba at different times. The expression law diagram of ;

Figure 2 shows the results of GbF3'5'H1 transgenic plants and non-transgenic plants, wherein Figure 2A is the semi-quantitative PCR amplification of non-transgenic and transgenic poplars containing the target fragment, and Figure 2B is qRT-PCR detection 8 The relative expression levels of GbF3′5′H1 in each transgenic line and non-transgenic poplar, Figure 2C is the growth status of non-transgenic and transgenic poplar at 45 days;

Fig. 3 is a graph showing the measurement results of differential metabolites between transgenic plants and non-transgenic plants, wherein Fig. 3A is an expression graph of differential metabolites between transgenic and non-transgenic seedling groups, and Fig. 3B is 4',5-dihydroxy-7 - The results of the content of glucooxyflavanone in transgenic and non-transgenic seedlings, Figure 3C is the content of catechol, phenylalanine, piceatannol and resveratrol in transgenic and non-transgenic seedlings Result graph.

Detailed ways

In order to make the above objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention will be described in detail below with reference to specific embodiments.

Example 1 Cloning and identification of Ginkgo biloba flavonoid 3′,5′-hydroxylase GbF3′5′H1 gene

Ginkgo DNA was extracted using the Plant Genomic DNA Kit (cetyltrimethylammoniumbromide (CTAB)) (Zoman, Beijing, China).

RNAprep Pure Plant kit (TIANGEN, Beijing, China) was used to extract total RNA from Ginkgo biloba leaves, and specific primers for GbF3′5′H1 gene were designed based on Ginkgo biloba transcriptome data (NCBI Short Reads Archive (SRA) database under access number SRP 137637) . The full-length cDNA sequence of GbF3'5'H1 was cloned by rapid amplification of cDNA ends (RACE). Nested primers were designed to amplify full-length cDNA using the SMATer RACE 5'/3' Kit (Clontech, Japan). All primers were designed using Oligo 6.0 software (as shown in Table 1). Amplification by PCR, recovery and purification of gel cutting, and transfer into E. coli competent cells via pMD19-T vector. Colonies were detected by PCR, and positive colonies were selected for Sanger sequencing. The full length of GbF3'5'H1 was obtained by splicing 5' and 3'-RACE sequences, and the open reading frame (ORF) was predicted using NCBI ORFFinder. Afterwards, the GbF3′5′H1 ORF was PCR-amplified in the following program: 95°C for 3 min; 35 cycles of 95°C for 30s, 55°C for 40s, 72°C for 90s; and a final extension at 72°C for 10 min. The PCR product was detected by 1% agarose gel electrophoresis, and the target fragment was recovered by the Agarose Gel DNA purification kit (TaKaRa, Dalian, China), and then the target fragment was ligated to the pMD19-T vector and transformed into Escherichia coli TOP10. Take a single colony for culture. Screened positive clones were sent to Sanger sequencing.

Table 1 Primer sequence information

In order to isolate the full-length cDNA of GbF3'5'H gene, the predicted GbF3'5'H gene was detected by 5'RACE and 3'RACE, and then named GbF3'5'H1. The base sequence of Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene is shown in SEQ ID NO.1, the amino acid of Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 The sequence is shown in SEQ ID NO.2. The full-length cDNA sequence of the gene is 1959 bp, including an ORF of 1527 bp, flanked by 42 bp 5'-UTR region and 387 bp 3'-URT region, and the termination codon is TGA.

Example 2 Transcription and expression levels of GbF3'5'H1 gene

To examine the transcription and expression levels of the GbF3'5'H1 gene, quantitative PCR (qRT-PCR) analysis was performed. Total RNA was extracted with RNAprep Plant Kit (TIANGEN, Beijing, China), and then total RNA was transcribed with PrimeScript ^™ RTMaster Mix (TAKAEA, Dalian, China). The cDNA was diluted 5-fold as template. The GbF3'5'H1 primers and internal reference gene primers designed for qRT-PCR amplification are shown in Table 1. qRT-PCR analysis was performed on the ABI ViiA 7Real-time PCR platform using the FastStart Universal SYBR Green Master with ROX for RT-PCR Kit (Roche, Indianapolis, IN, USA). The 10 μL reaction system and PCR reaction program were as follows: 95°C for 2 min; 95°C for 15s and 95°C for 1 min under 40 cycles. Three biological replicates were prepared for each sample and the average relative expression was calculated. Relative expression levels were calculated using the 2- ^ΔΔCt method. Duncan's multiple test was performed using SPSS 22.0 software (SPSS Inc., Chicago, IL, USA), and p value < 0.05 was considered statistically significant.

One-year-old Ginkgo biloba seedlings (leaf, stem, root, petiole) and 25-year-old Ginkgo biloba (pipe, bud) were used as materials to study the tissue expression of this gene in Ginkgo biloba. One-year-old Ginkgo biloba seedlings cultivated in the greenhouse of Nanjing Forestry University were used as experimental materials. At different leaf development stages, leaves were collected once a month from April to October to study the expression pattern of the gene. After collection, plant material was snap-frozen in liquid nitrogen and placed in an ultra-low temperature freezer at -80°C.

The expression analysis results are shown in Figure 3. The expression level of the gene in the stem is set to 1. In Figure 1A, R refers to the root, S refers to the stem, L refers to the leaf, and K refers to the seed kernel; B Refers to the bud, and P refers to the petiole; in Figure 1B, the expression level of the GbF3′5′H1 gene in June is set to 1, and the time expression pattern: 4, 5, 6, 7, 8, 9, and 10 represent April, respectively , May, June, July, August, September, October. The results showed that GbF3′5′H1 was accumulated in leaves (significantly higher than other sites), followed by petioles, with no expression in roots and nuclei (as shown in Figure 1A). From April to October, Ginkgo biloba development experienced different periods (as shown in Figure 1B). The results of GbF3′5′H1 gene expression showed that the mRNA expression level of Ginkgo biloba leaves was the highest in April (more than 24 times that in June, significantly higher than other periods), and the expression level in July was weak (only 0.3 expression level).

Example 3 Heterologous overexpression of GbF3'5'H1 in poplar

The ORF of the GbF3'5'H1 cDNA was amplified by PCR, and the ccdB gene located downstream of the CaMV 35S promoter was cloned into the PBI121 vector using Gateway technology (Invitrogen, CA, Carlsbad). The vector containing Pro35S::GbF3'5'H1 was introduced into Agrobacterium strain EHA105 for transformation. Using the stable and efficient genetic transformation system of Populus davidiana×Populus bolleana, the GbF3′5′H1 gene of Ginkgo biloba was transgenic into Populus sanguinis. The tissue culture seedlings of P. chinensis were grown under 16h light and 8h dark photoperiod and 25℃(day) and 18℃(night).

In order to study the function of GbF3'5'H1 in plants, multiple 35S::GbF3'5'H1 poplar transgenic lines were verified by PCR after kanamycin (Kan) resistance screening. Eight independent transgenic lines were randomly selected, and the expression levels of GbF3'5'H1 were detected by semi-quantitative PCR and qRT-PCR (see Table 1 for primers). The results showed that GbF3′5′H1 was successfully expressed in 8 transgenic lines, and the results of semi-quantitative PCR and qRT-PCR analysis were consistent (as shown in Figures 2A and 2B, WT is a non-transgenic poplar in the figure, L1-L10 are transgenic poplar lines). qRT-PCR analysis showed that the expression level of GbF3′5′H1 was the highest in transgenic line L4 (the relative expression level was 426 times that of non-transgenic seedlings), followed by transgenic line L1 and line L6. Eight transgenic lines The expression levels were higher than those of non-transgenic poplar. It can be seen from Figure 2C that both the transgenic and non-transgenic seedlings can grow healthily.

Example 4 Differential metabolites between transgenic and non-transgenic plants

In order to determine whether overexpression of GbF3′5′H1 affects the synthesis of flavonoid-related metabolites in transgenic plants, non-targeted metabolic detection and analysis of differential metabolites in the leaves of transgenic poplar and non-transgenic seedlings in Example 3 were used, and metabolism was determined. relative concentration of the expressed substance. Untargeted metabolome profiling employs the following methods:

1. Sample processing includes the following steps:

1) Precisely weigh 60 mg of each plant sample, put it into a 1.5 mL centrifuge tube, and add 40 μL of internal standard (L-2-chloro-phenylalanine, 0.3 mg/mL, prepared in methanol);

2) Add two small steel balls and 360 μL of cold methanol in turn, and place them in a -20°C refrigerator for 2 minutes;

3) put into the grinder and grind (60Hz, 2min);

4) ultrasonic extraction in ice water bath for 30min;

5) Add 200 μL of chloroform, vortex in a vortex machine (60 Hz, 2 min), add 400 μL of water, and vortex in a vortex machine (60 Hz, 2 min);

6) ultrasonic extraction in ice water bath for 30min;

7) -20℃ for 30min;

8) Centrifuge at low temperature for 10min (13000rpm, 4°C), take 300μL of supernatant and put it into a glass derivatization bottle;

9) The quality control sample (QC) is prepared by mixing equal volumes of the extracts of all samples, and the volume of each QC is the same as that of the sample;

10) Dry the sample with a centrifugal concentrator desiccator.

11) 80 μL of methoxyamine hydrochloride pyridine solution (15 mg/mL) was added to the glass derivatization vial, and after vortexing for 2 min, the oximation reaction was carried out in a shaking incubator at 37° C. for 90 min.

12) After taking out the sample, add 80 μL of BSTFA (containing 1% TMCS) derivatization reagent and 20 μL of n-hexane, and add 11 internal standards (C8/C9/C10/C12/C14/C16, 0.8 mg/mL; C18/ C20/C22/C26/C28, 0.4 mg/mL, all prepared in chloroform) 10 μL, vortexed for 2 min, and reacted at 70° C. for 60 min.

13) After taking out the sample, place it at room temperature for 30 minutes and perform GC-MS metabolomics analysis.

2. Gas chromatography-mass spectrometry analysis conditions

Chromatographic conditions: DB-5MS capillary column (30m×0.25mm×0.25μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas is high-purity helium (purity not less than 99.999%), flow rate 1.0mL/min, feed The temperature of the sample port was 260°C. The injection volume was 1 μL, the split injection ratio was 4:1, and the solvent delay was 5 min.

Program temperature: the initial temperature of the column oven is 60 °C, and the temperature is programmed to 125 °C at 8 °C/min, 5 °C/min to 210 °C; 10 °C/min to 270 °C, 20 °C/min to heat up to 305 °C Hold for 5min.

Mass spectrometry conditions: electron bombardment ion source (EI), ion source temperature 230°C, quadrupole temperature 150°C, electron energy 70eV. Scanning mode is full scan mode (SCAN), mass scanning range: m/z 50-500.

3. Differential metabolite screening

A combination of multidimensional analysis and single-dimensional analysis was used to screen differential metabolites between groups. In the OPLS-DA analysis, the variable weight value can be used to measure the impact strength and explanatory power of the expression pattern of each metabolite on the classification and discrimination of each group of samples, and to mine the differential metabolites with biological significance. Generally, metabolites with VIP>1 are considered as are differential metabolites. The t test (student's t test) was used to further verify the significance of the metabolite differences between groups. The screening criteria were that the VIP value of the first principal component of the OPLS-DA model was >1, and the p value was <0.05. Among them, the fold change is the ratio of the average content of metabolites in the two groups.

Untargeted GC-MS analysis revealed 45 differential metabolites between the non-transgenic and transgenic groups (as shown in Figure 3A). The 45 differential metabolites are mainly divided into 9 categories, these differential metabolites may be caused by the overexpression of GbF3′5′H1 gene. The contents of 17 differential metabolites in transgenic poplars were higher than those in non-transgenic seedlings. These differentially up-regulated metabolites may be caused by the overexpression of GbF3′5′H1 gene in poplars, making the contents of these 17 metabolites significantly increased At the same time, the increase in the content of these metabolites may have a certain impact on the increase in the content of related metabolites. In addition, five metabolites (4′,5-dihydroxy-7-glucosoxyflavanone, catechol, phenylalanine, piceatannol and resveratrol) and flavonoid-related metabolites were found in transgenic seedlings. The metabolites showed differential expression, which indicated that GbF3′5′H1 might affect or interact with the contents of different types of flavonoids or other metabolites by affecting the contents of these five metabolites. The contents of three flavonoid-related differential metabolites were significantly up-regulated. Flavonoids belong to phenols in plant secondary metabolites, and 4′, 5-dihydroxy-7-glucosyloxyflavanone (4′, 5-dihydroxy-7-glucosyloxyflavanone) in transgenic seedlings is significantly higher than that in non-transgenic plants The expression level in the seedlings is about 3 times that of the non-transgenic seedlings (Fig. 3B), which is a certain promoting effect of the increase of the flavonoid content. The overexpression of the GbF3′5′H1 gene can produce a large amount of 4 ',5-dihydroxy-7-glucosoxyflavanone. The metabolite contents of catechol and phenylalanine were higher in transgenic plants than in wild-type plants (Fig. 3C). Among them, the increase of phenylalanine content in transgenic plants is beneficial to the biosynthesis of downstream flavonoids and other related metabolites. This is because the biosynthesis of flavonoids starts from the phenylalanine synthesis pathway, and its synthesis starts from phenylalanine and malonyl-CoA, and gradually synthesizes aromatic compounds with diverse structures. Therefore, increased phenylalanine content in transgenic plants favors the biosynthesis of downstream flavonoids and their related metabolites.

sequence listing

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<120> Ginkgo biloba flavonoid 3',5'-hydroxylase GbF3'5'H1 gene and its protein and application

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Claims (10)

1. Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene, the base sequence of which is shown in SEQ ID NO.1. 2. Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1, the amino acid sequence of which is shown in SEQ ID NO.2. 3. An expression vector containing the Ginkgo biloba flavonoid 3',5'-hydroxylase GbF3'5'H1 gene of claim 1. 4. The expression vector containing the Ginkgo flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene according to claim 3, wherein the ccdB located downstream of the CaMV 35S promoter is converted by Gateway technology The gene was cloned into PBI121 vector, and an expression vector containing Pro35S::GbF3'5'H1 was constructed. 5. The application of the Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene of claim 1 in increasing the content of plant flavonoid-related metabolites. 6. The application of Ginkgo biloba flavonoid 3' according to claim 5, 5'-hydroxylase GbF3'5'H1 gene in improving the content of plant flavonoid-related metabolites, is characterized in that, comprises the following steps: (1 ) constructing an expression vector containing the Ginkgo biloba flavonoid 3',5'-hydroxylase GbF3'5'H1 gene; (2) transforming the constructed expression vector into a plant or plant cell; (3) cultivating and screening to obtain a transgene plant. 7. The application of Ginkgo biloba flavonoid 3', 5'-hydroxylase GbF3'5'H1 gene according to claim 6 in improving the content of plant flavonoid-related metabolites, characterized in that, utilizing Gateway technology to locate CaMV The ccdB gene downstream of the 35S promoter was cloned into the PBI121 vector to construct an expression vector containing Pro35S::GbF3'5'H1. 8. The application of Ginkgo biloba flavonoid 3',5'-hydroxylase GbF3'5'H1 gene according to claim 6 in improving the content of plant flavonoid-related metabolites, characterized in that, using Agrobacterium strain EHA105 to The constructed expression vector is transformed into plants. 9. The application of Ginkgo biloba flavonoid 3',5'-hydroxylase GbF3'5'H1 gene according to claim 5 or 6 in improving the content of plant flavonoid-related metabolites, wherein the plant is Poplar. 10. The application of the Ginkgo biloba flavonoid 3',5'-hydroxylase GbF3'5'H1 gene according to claim 5 or 6 in increasing the content of plant flavonoid-related metabolites, wherein the flavonoid Relevant metabolites include catechol, phenylalanine, 4',5-dihydroxy-7-glucosoxyflavanone.

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