CN106086038B - Artemisia apiacea WRKY transcription factor coding sequence, cloning method and application - Google Patents

Abstract

The invention discloses the cloning of the WRKY gene coding sequence of Artemisia annua and its application. Specifically, it includes the cloning of the gene AaWRKY2, the construction of a plant expression vector containing the gene, and the activation of the gene on the specific gene promoter of the artemisinin biosynthesis pathway. The nucleotide sequence of the above-mentioned Artemisia annua AaWRKY2 gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. The invention also discloses that the AaWRKY2 gene has the characteristic of being able to activate the expression of two gene promoters, ADS and DBR2, which are specific for the biosynthesis pathway of artemisinin. The AaWRKY2 gene in the present invention can be applied to quality improvement of Artemisia annua through overexpression, etc., and can increase the content of artemisinin in Artemisia annua.

Description

A kind of Qinghao WRKY transcription factor coding sequence and cloning method and application

technical field

The invention relates to the technical field of genetic engineering, in particular to AaWRKY2, a coding sequence of a WRKY class transcription factor of Artemisia annua and its application.

Background technique

Metabolism in plants is divided into primary metabolism and secondary metabolism. Primary metabolites (such as sugars, lipids and nucleic acids) exist in all plants and are necessary for plants to maintain cell life activities, while plant secondary metabolites refer to A large class of small-molecule organic compounds that are not necessary for plant growth and development in plants, the synthesis and distribution of which are specific to species, tissues and organs, and production and development. For example, artemisinin, a specific drug component for treating malaria, is synthesized and stored only in the secretory glandular hairs on the surface of the plant Artemisiaannua L.. In recent years, with the gradual deepening of the research on the components of Chinese herbal medicines, it has been found that the active components of many Chinese herbal medicines are plant secondary metabolites, such as tanshinone in Salvia miltiorrhiza and vinca alkaloids in periwinkle.

The content of most plant secondary metabolites in natural plants is extremely low, and the use of chemical synthesis methods is complex and costly, and the biosynthetic pathways of many plant secondary metabolites are unclear and cannot be realized. Chemical total synthesis. Therefore, researchers began to explore other ways to increase the content of secondary metabolites in plants. Considering that the synthetic pathways of plant secondary metabolites are complex, the number of genes involved in the reaction is large, and it is affected by various factors such as development and environment, it is sometimes difficult to modify a single gene in the pathway. Transcription factors can usually regulate the expression of multiple enzyme genes in the biosynthetic pathway of a certain plant secondary metabolite, thereby regulating the biosynthesis of the secondary metabolite.

At present, it has been reported in Artemisia annua that transcription factors can effectively regulate the expression of genes specific to the artemisinin synthesis pathway, thereby regulating the biosynthesis of artemisinin, such as AaORA1 transcription factor, AaWRKY2 transcription factor, AaMYC2 transcription factor and so on. Therefore, cloning transcription factors that can regulate the expression of genes specific to the biosynthetic pathway of artemisinin is of great significance for improving and increasing the content of artemisinin in Artemisia annua.

SUMMARY OF THE INVENTION

In view of the above-mentioned defects of the prior art, the present invention provides the following technical solutions to improve the content of artemisinin in Artemisia annua:

The present invention provides a coding sequence of a WRKY class transcription factor of Artemisia annua, the coding sequence is denoted as AaWRKY2, and the nucleotide sequence of AaWRKY2 is shown in SEQ ID NO.1.

The present invention also provides a coding sequence of a WRKY class transcription factor of Artemisia annua, and the amino acid sequence coded by AaWRKY2 is shown in SEQ ID NO.2.

The present invention also provides a polypeptide whose amino acid sequence is shown in SEQ ID NO.2.

The present invention also provides a recombinant expression vector comprising an open reading frame sequence of the nucleotide sequence shown in SEQ ID NO.1, and the open reading frame sequence shown in SEQ ID NO.3. Its construction method includes the following steps:

Step 1. Design and amplify the open reading frame sequence of the AaWRKY2 gene according to the sequence of SEQ ID NO.1. The amplification primers are as follows:

Forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3';

Step 2. The PCR amplification product is recovered and purified, and then connected to the pENTR/D-TOPO vector;

Step 3. The pEarlygate104-AaWRKY2 plant expression vector containing the target gene was constructed by LR reaction between the pENTR/D-TOPO vector linked into the AaWRKY2 gene and the pEarlygate104 plant expression vector.

The present invention also provides a recombinant expression transformant comprising the open reading frame sequence of the nucleotide sequence shown in SEQ ID NO.1, and the open reading frame sequence shown in SEQ ID NO.3 Show.

Further, the host strain of the above recombinant expression transformant is Agrobacterium.

The invention also provides the application of the coding sequence AaWRKY2 of the above-mentioned WRKY transcription factor of Artemisia annua in increasing the content of artemisinin.

The present invention also provides the cloning method of the above-mentioned Artemisia annua WRKY transcription factor coding sequence AaWRKY2, the cloning method comprising the following steps:

Step 1. Extraction and purification of total RNA, using various general plant total RNA extraction kits to extract and purify to obtain total RNA of Artemisia annua leaves;

Step 2. Reverse the total RNA of Artemisia annua leaves into cDNA with reverse transcriptase;

Step 3. Using the above cDNA as a template, by designing gene-specific primers and amplifying by PCR, a PCR product is obtained, and the gene-specific primers are:

Forward primer P1: 5'-AAGATGGAATATGGAAGAGG-3'

Reverse primer P2: 5'-ATCAAACAAAGAGTCCACAGAC-3;

Step 4. The PCR product is recovered, purified and sequenced to obtain the nucleotide sequence shown in SEQ ID NO.1.

The present invention also provides a method for rapidly detecting the biological function of the amino acid sequence encoded by AaWRKY2, the method comprising the following steps:

Step 1. Design and amplify the open reading frame sequence of the AaWRKY2 gene according to the sequence of SEQ ID NO.1. The amplification primers are as follows:

Forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3';

Step 2. The PCR amplification product is recovered and purified, and then connected to the pENTR/D-TOPO vector;

Step 3. Construct the pEarlygate104-AaWRKY2 plant expression vector containing the gene of interest by LR reaction between the pENTR/D-TOPO vector linked into the AaWRKY2 gene and the pEarlygate104 plant expression vector;

Step 4. Connect the promoter ProADS of the ADS gene unique to the artemisinin synthesis pathway in Artemisia annua into the pGreenII0800-LUC vector to construct the plant dual-fluorescein detection report vector pGreenII0800-ProADS;

Step 5. Connect the promoter ProCYP71AV1 of the unique CYP71AV1 gene in the artemisinin synthesis pathway in Artemisia annua into the pGreenII0800-LUC vector to construct a plant dual-fluorescein detection reporter vector pGreenII0800-ProCYP71AV1;

Step 6. Connect the promoter ProDBR2 of the DBR2 gene unique to the artemisinin synthesis pathway in Artemisia annua into the pGreenII0800-LUC vector to construct a plant dual-fluorescein detection report vector pGreenII0800-ProDBR2;

Step 7. Connect the promoter ProALDH1 of the ALDH1 gene unique to the artemisinin synthesis pathway in Artemisia annua into the pGreenII0800-LUC vector to construct a plant dual-fluorescein detection report vector pGreenII0800-ProALDH1;

Step 8. Transform the pEarlygate104-AaWRKY2 plant expression vector and the plant dual fluorescein detection reporter vector pGreenII0800-ProADS, pGreenII0800-ProCYP71AV1, pGreenII0800-ProDBR2 and pGreenII0800-ProALDH1 into Agrobacterium respectively to obtain an Agrobacterium engineering strain containing the target vector;

Step 9. After mixing the Agrobacterium engineering strain containing the pEarlygate104-AaWRKY2 plant expression vector and the Agrobacterium engineering strain containing the plant dual fluorescein detection report vector, inject it into tobacco leaves that have grown for 5 weeks by injection infection;

Step 10. Take the tobacco leaves that have been cultured for 2 days after injection, freeze them with liquid nitrogen and grind them into powder;

Step 11: Detecting the fluorescence intensity with a Promega-Dual-Luciferase detection kit to determine the activation of the AaWRKY2 gene and the promoter of a gene specific to the artemisinin synthesis pathway.

Further, in the above-mentioned step 8, the host bacteria is the Agrobacterium GV3101 strain; in the above-mentioned step 9, the mixing ratio of the Agrobacterium engineering strain comprising the pEarlygate104-AaWRKY2 plant expression vector and the Agrobacterium engineering strain comprising the plant double fluorescein detection report carrier is: Mix at a 3:1 concentration.

In the present invention, various vectors known in the art can be used for cloning the AaWRKY2 gene, such as commercially available vectors, including plasmids, cosmids, and the like. In the present invention, the AaWRKY2 plant expression vector is constructed, and various vectors known in the art can also be used, such as the commercially available pCAMBIA series vectors; in the present invention, the promoter dual fluorescein reporter vector can also be constructed using known in the art. Various other vectors, such as commercially available vectors of Promega company; the Agrobacterium involved in the present invention is Agrobacterium tumefaciens strain GV3101, which can be publicly purchased from the market.

The present invention will be further described below with reference to the accompanying drawings to fully illustrate the purpose, technical features and technical effects of the present invention.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

Figure 1 shows that, in a preferred embodiment, transient transformation of the AaWRKY2 gene in tobacco significantly increases the expression activity of the two gene promoters, ADS and DBR2.

Detailed ways

The embodiments of the present invention are described in detail below: the present embodiment is implemented on the premise of the technical solution of the present invention, and provides a detailed implementation manner and a specific operation process, but the protection scope of the present invention is not limited to the following implementations example. The experimental method of unreceipted specific conditions in the following examples, usually according to normal conditions, molecular cloning such as Sambrook: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer's recommended conditions.

Example 1, Example 1 Cloning of AaWRKY2 gene of Artemisia annua

1. Artemisia annua is cultivated in an artificial climate chamber, and the growth conditions are photoperiod 18h/6h (light/dark), 25°C;

2. Extraction of total RNA from Artemisia annua leaves. Take about 100 mg of young and tender Artemisia annua leaf tissue material, place it in liquid nitrogen and fully grind it to a powdery state, and extract total leaf RNA according to the method of the plant total RNA extraction kit (Tiangen Biochemical, Beijing). 3 μL of the obtained plant total RNA was subjected to agarose gel electrophoresis to identify the quality of the total RNA, and then the concentration of the total RNA was measured on a NanoDrop (Thermo Fisher, USA) spectrophotometer.

3. Gene cloning. Using the extracted total RNA as the template (500ng), according to the method of the reverse transcription kit PrimeScript1st Strand cDNA Synthesis Kit (TaKaRa, Dalian), reverse transcription to produce the first-strand cDNA; through the designed specific primers, the cDNA is used as the template PCR amplification was carried out, and the specific primer sequences were as follows:

Forward primer P1: 5'-AAGATGGAATATGGAAGAGG-3'

Reverse primer P2: 5'-ATCAAACAAAGAGTCCACAGAC-3

The total volume of the PCR reaction was 50 μL, and the reaction system was: 5 μL 10×KOD buffer, 5 μL dNTPs, 4 μL MgSO4, 1 μL forward primer, 1 μL reverse primer, 1 μL cDNA template, 1 μL KOD enzyme, and ddH2O supplemented to 50 μL.

PCR amplification conditions, pre-denaturation at 95°C for 3 min, 35 cycles: 95°C, 30sec; 54°C, 30sec; 68°C, 100sec, and a final extension at 68°C for 5min.

After the PCR product was recovered and purified, the blunt end vector pLB (product of Tiangen Biochemical Co., Ltd.) was connected and sequenced to obtain the pLB-AaWRKY2 plasmid vector.

Through the above steps, the sequence of AaWRKY2 gene in Artemisia annua is obtained as shown in SEQ ID NO.1, and its protein coding sequence is deduced as shown in SEQ ID NO.2.

Example 2. Construction of a plant expression vector comprising the AaWRKY2 gene

1. Construction of the intermediate vector pENTR/D-TOPO-AaWRKY2.

Design and amplify the open reading frame sequence of AaWRKY2 gene according to the sequence information of SEQ ID NO.1, and the amplification primers are as follows:

Forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3'

The pENTR/D-TOPO vector was purchased from Invitrogen Company, which is the entry vector of the company's Gateway cloning technology. According to the requirements of the product manual, four bases of CACC were added before the ATG base of the forward primer. Using the pLB-AaWRKY2 plasmid as the template, PCR amplification was carried out with the blunt-end high-fidelity enzyme KOD. The PCR product was recovered and purified, and then connected to the pENTR/D-TOPO vector by Gateway cloning technology. The specific method was according to Invitrogen's pENTR/D-TOPO. cloning kit instructions.

2. Construction of a plant expression vector containing the target gene. According to Invitrogen's LR ClonaseIIEnzyme kit, the intermediate vector pENTR/D-TOPO-AaWRKY2 and pEarlygate104 plant expression vector were subjected to LR reaction. The reaction system was prepared according to the kit instructions, placed in a metal bath at 25°C for 3 hours, and transformed into E. coli DH5α receptors The positive clone was verified by PCR, and finally the pEarlygate104-AaWRKY2 plant expression vector containing the target gene was obtained.

Example 3. Construction of artemisinin synthesis pathway-specific gene promoter dual-fluorescein reporter vector

1. Amplify the promoter of the specific gene of artemisinin synthesis pathway by PCR. According to the sequence information of the ADS gene promoter of Artemisia annua (GenBank: DQ448297.1) in the NCBI database, specific primers for ADS gene promoter amplification were designed. The forward and reverse specific primers contain Kpn I and Pst I restriction sites respectively. The primer sequences are as follows :

ProADS F 5’-ggtaccACCGGGGACCTCTAGAGATC-3’,

ProADS R 5'-ctgcagGATTTTACAAACTTTGAA-3'.

Similarly, according to the sequence information of the promoter of Artemisia annua CYP71AV1 gene (GenBank: FJ870128.1) in the NCBI database, CYP71AV1 promoter-specific primers containing Kpn I and Pst I restriction sites were designed. The primer sequences are as follows:

ProCYP F 5’-ggtaccATGGGTCAATTTCGGGTTG-3’,

ProCYP R 5'-ctgcagTGCTTTTAGTATACTCTTC-3';

According to the sequence information of the DBR2 gene promoter of Artemisia annua (GenBank: KC118524.1) in the NCBI database, DBR2 promoter-specific primers containing Kpn I and Pst I restriction sites were designed. The primer sequences are as follows:

ProDBR2 F 5’-ggtaccAAGATGAGATAGGGAACTAAC-3’,

ProDBR2 R 5'-ctgcagTATTGAGTTTGATGTTGACC-3';

According to the sequence information of the ALDH1 gene promoter of Artemisia annua (GenBank: KC118522.1) in the NCBI database, ALDH1 promoter-specific primers containing Kpn I and Pst I restriction sites were designed. The primer sequences are as follows:

ProALDH1 F 5’-ggtaccATGAACCATTAGAAGGGAAGG-3’,

ProALDH1 R 5'-ctgcagCTTTGTTTTTTATGAAA-3';

Using the genomic DNA of Artemisia annua as a template, the above four promoter fragments were amplified by PCR and recovered and purified.

2. The promoter PCR product was ligated into a dual fluorescein reporter vector. The above PCR product was double digested with Kpn I and Pst I, and the digested fragments were recovered. The four recovered fragments were connected to the pGreenII0800-LUC vector fragment recovered after double digested with Kpn I and Pst I to construct a plant. Dual fluorescein detection reporter vectors pGreenII0800-ProADS, pGreenII0800-ProCYP71AV1, pGreenII0800-ProDBR2 and pGreenII0800-ProALDH1.

Example 4. Tobacco transient transformation detection gene and promoter activation

1. The acquisition of Agrobacterium engineering strains, the pEarlygate104 empty vector, pEarlygate104-AaWRKY2 expression vector and 4 promoter dual fluorescence detection report vectors were transformed into Agrobacterium tumefaciens GV3101 strain by freeze-thaw method to obtain agricultural products containing the empty vector. Bacillus engineering strains, Agrobacterium engineering strains containing AaWRKY2 gene and 4 Agrobacterium engineering strains containing promoters.

2. Expanded culture and treatment of Agrobacterium engineering strains. The above-mentioned 6 Agrobacterium engineering strains were expanded and cultured (5mL) in the LB medium containing 50mg/L rifampicin+20mg/L gentamicin+50mg/L kanamycin three antibiotics, 28 ℃, 220 rpm and incubate overnight. The next day, the concentration of the bacterial solution was measured, and the culture was stopped when the concentration of the Agrobacterium bacterial solution reached an OD600 value of about 2OD to 2.5OD. Centrifuge to collect Agrobacterium, and then resuspend the bacteria with 10 mM MgCl ₂ solution, and adjust the OD value of the resuspended bacteria to 0.6. Acetosyringone was added to the resuspended bacterial solution at a concentration of 200 mM. The above resuspended Agrobacterium solution was allowed to stand for 3 hours.

3. Instantaneous transformation of tobacco by injection infection. The above-mentioned statically treated Agrobacterium engineering strains containing empty vectors and 4 Agrobacterium engineering strains containing promoters were respectively mixed at a concentration ratio of 3:1 as a control group; Agrobacterium engineering strains and 4 Agrobacterium engineering strains containing promoters were also mixed at a concentration ratio of 3:1 as an experimental group. Through a 1 ml syringe, the mixed Agrobacterium was injected into tobacco leaves that had been growing for about 5 weeks, and then cultured in the dark for 1 day and then transferred to the light.

4. Detection of Dual-Luciferase. Using a circular hole punch with a diameter of 1.0 cm, take tobacco leaves that have been cultured for 2 days after injection, freeze them with liquid nitrogen and grind them into powder; use Promega Dual-Luciferase detection kit to detect the fluorescence intensity, and operate according to the instructions of Promega company. .

In the present invention, the AaWRKY2 gene can significantly activate the expression of two important structural gene promoters, ADS and DBR2 in the artemisinin biosynthesis pathway. Compared with the control group, AaWRKY2 can significantly activate and increase the expression activity of the ADS promoter to the control group. About 2.2 times, it can increase the expression activity of DBR2 promoter to about 2.5 times that of the control group, but it has no significant activation ability for the two promoters of CYP71AV1 and ALDH1. The present invention provides strong experimental evidence for further utilizing the gene to be overexpressed in Artemisia annua to increase the content of artemisinin in Artemisia annua.

The preferred embodiments of the present invention have been described in detail above. It should be understood that many modifications and changes can be made according to the concept of the present invention by those skilled in the art without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the prior art according to the concept of the present invention shall fall within the protection scope determined by the claims.

Claims (8)

1. A sweet wormwood WRKY transcription factor is characterized by being named as AaWRKY2, and the nucleotide sequence of AaWRKY2 is shown as SEQ ID No. 1.

2. The Artemisia annua WRKY transcription factor as claimed in claim 1, wherein the amino acid sequence of AaWRKY2 is shown as SEQ ID NO. 2.

3. A recombinant expression vector is characterized in that the recombinant expression vector comprises an open reading frame sequence of a nucleotide sequence shown as SEQ ID NO.1, and the open reading frame sequence is shown as SEQ ID NO. 3.

4. The use of the Artemisia annua WRKY transcription factor as claimed in claim 1 or 2 for increasing artemisinin content.

5. The method for cloning the Artemisia annua WRKY transcription factor as claimed in claim 1, wherein the cloning method comprises the following steps:

step 1, extracting and purifying total RNA, namely extracting and purifying by adopting various general plant total RNA extraction kits to obtain total RNA of artemisia apiacea leaves;

step 2, reverse transcriptase is used for inverting the total RNA of the artemisia apiacea leaves into cDNA;

and 3, using the cDNA as a template, designing a gene specific primer, and amplifying by adopting a PCR method to obtain a PCR product, wherein the gene specific primer is as follows:

forward primer P1: 5'-AAGATGGAATATGGAAGAGG-3'

Reverse primer P2: 5' -ATCAAACAAAGAGTCCACAGAC-3;

and 4, recovering, purifying and sequencing the PCR product to obtain the nucleotide sequence shown as SEQ ID NO. 1.

6. The method of constructing a recombinant expression vector according to claim 3, comprising the steps of:

step 1, designing and amplifying AaWRKY2 gene open reading frame sequence according to SEQ ID NO.1 sequence, wherein amplification primers are as follows:

forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3', respectively;

step 2, recovering and purifying the PCR amplification product and connecting the PCR amplification product with a pENTR/D-TOPO vector;

and 3, constructing a pEarlygate104-AaWRKY2 plant expression vector containing the target gene by using the pENTR/D-TOPO vector connected with the AaWRKY2 gene and a pEarlygate104 plant expression vector in an LR reaction mode.

7. A method for rapidly detecting the biological function of the AaWRKY2 transcription factor of claim 2, comprising the steps of:

step 1, designing and amplifying AaWRKY2 gene open reading frame sequence according to SEQ ID NO.1 sequence, wherein amplification primers are as follows:

forward primer P3: 5'-CACCATGGAAGAGGTTGAAGCTG-3'

Reverse primer P4: 5'-AGCGATTGTGGATTGCACAGG-3', respectively;

step 2, recovering and purifying the PCR amplification product and connecting the PCR amplification product with a pENTR/D-TOPO vector;

step 3, constructing a pEarlygate104-AaWRKY2 plant expression vector containing a target gene by connecting the pENTR/D-TOPO vector connected with the AaWRKY2 gene with a pEarlygate104 plant expression vector in an LR reaction mode;

step 4, connecting a promoter ProADS of a special ADS gene in an artemisinin synthesis approach in artemisia apiacea into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII 0800-ProADS;

step 5, connecting a promoter ProCYP71AV1 of a specific CYP71AV1 gene in an artemisinin synthesis way in the sweet wormwood herb into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII0800-ProCYP71AV 1;

step 6, connecting a promoter ProDBR2 of a specific DBR2 gene in an artemisinin synthesis way in sweet wormwood herb into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII0800-ProDBR 2;

step 7, connecting a promoter ProALDH1 of a specific ALDH1 gene in an artemisinin synthesis way in sweet wormwood herb into a pGreenII0800-LUC vector to construct a plant double-fluorescein detection report vector pGreenII0800-ProALDH 1;

step 8, respectively transforming the pEarlygate104-AaWRKY2 plant expression vector and the plant double-fluorescein detection report vector pGreenII0800-ProADS, pGreenII0800-ProCYP71AV1, pGreenII0800-ProDBR2 and pGreenII0800-ProALDH1 into agrobacterium to obtain agrobacterium engineering strains containing target vectors;

step 9, mixing the agrobacterium engineering strain containing the pEarlygate104-AaWRKY2 plant expression vector and the agrobacterium engineering strain containing the plant double-fluorescein detection report vector, and injecting the mixture into tobacco leaves growing for 5 weeks in an injection infection mode;

step 10, taking the tobacco leaves which are cultured for 2 days after injection, quickly freezing the tobacco leaves by using liquid nitrogen, and grinding the tobacco leaves into powder;

and step 11, detecting the fluorescence intensity by adopting a Promega-Dual-Luciferase detection kit, and determining the activation effect of the AaWRKY2 gene and the specific gene promoter of the artemisinin synthesis pathway.

8. The method according to claim 7, wherein in step 8, the Agrobacterium is the GV3101 strain; in the step 9, the mixing ratio of the agrobacterium engineering strain containing the pEarlygate104-AaWRKY2 plant expression vector to the agrobacterium engineering strain containing the plant double-fluorescein detection report vector is 3:1, mixing.

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