ADTW6E gene for regulating and controlling color of peanut endopleura and application of ADTW6E gene

Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to an ADTW6E gene for regulating and controlling the color of peanut endopleura and application of the ADTW6E gene. The nucleotide sequence of the ADTW6E gene is as shown in SEQ ID NO.1, and the amino acid sequence of protein expressed by the ADTW6E gene is as shown in SEQ ID NO.2. Compared with a wild type, transgenic arabidopsis is turned into purple through over-expression of ADTW6E, and the anthocyanin content of plants is increased. Theoretical support can be provided for subsequent explanation of a regulation mechanism of plant pigment accumulation, and a foundation is laid for genetic improvement and new germplasm creation of peanuts.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, and more specifically, relates to the regulation of the inner seed coat color of peanuts. ADTW6E Genes and their applications. Background Technology

[0002] Arachis ( Arachis Originating in South America, peanuts are an important source of vegetable oil and protein, and can also be used as livestock feed. In addition, peanuts can be eaten fresh. Aside from their taste, the color of the peanut shell also affects the value of fresh peanuts. Because the inner shell of peanuts comes in a variety of colors, including black, red, white, and multicolored, these peanuts are also called colored peanuts.

[0003] Existing research has clearly established that the black and purple colors in colored peanuts are primarily due to the accumulation of anthocyanins. Anthocyanins play an important role in anti-oxidation and anti-aging processes; furthermore, they also play a significant role in maintaining human growth and development, enhancing immunity, and protecting the cardiovascular system. Therefore, identifying genes that regulate the color of the peanut inner seed coat is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a method for regulating the color of peanut inner seed coat. ADTW6E Genes and their applications to solve the aforementioned technical problems.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for regulating the color of peanut inner seed coat. ADTW6E Genes, the ones mentioned ADTW6E The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] This invention applies molecular biology techniques to clone peanuts. ADTW6E Genes are genetically transformed and overexpressed. ADTW6E Genes can cause plants to turn purple and increase their anthocyanin content. This invention can provide theoretical support for elucidating the regulatory mechanism of plant pigment accumulation and lay the foundation for peanut genetic improvement and new germplasm creation.

[0007] The present invention also provides the above. ADTW6E A gene-expressed protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant expression vector comprising the above-mentioned... ADTW6E Gene.

[0009] Furthermore, the aforementioned ADTW6EGene insertion into the pCAMBIE2300-OMC overexpression vector Kpn I Japanese Pst Obtained between I restriction endonuclease sites.

[0010] The recombinant engineered bacteria include the aforementioned recombinant expression vector.

[0011] Furthermore, the recombinant engineered bacteria is obtained by transferring the recombinant expression vector into Agrobacterium EHA105.

[0012] The present invention also provides the following ADTW6E The application of the gene, the protein, the recombinant expression vector, and the recombinant engineered bacteria in regulating plant color, wherein the regulation is to make the plant color turn purple.

[0013] Furthermore, turning plants purple is achieved by increasing the anthocyanin content in the plants.

[0014] Furthermore, the plants include peanuts and Arabidopsis thaliana.

[0015] The present invention has the following beneficial effects: This invention is the first discovery ADTW6E The application of genes in regulating color in Arabidopsis thaliana. By... ADTW6E Overexpression of the gene in Arabidopsis thaliana resulted in a purple color and increased anthocyanin content in the plant leaves. This invention provides theoretical support for elucidating the regulatory mechanisms of plant color changes and lays the foundation for peanut genetic improvement and new germplasm creation. Attached Figure Description

[0016] Picture 1 Electrophoresis diagram for PCR identification of ADTW6E overexpressing transgenic plants. 1 is the DNA marker; 2-23 are ADTW6E transgenic positive plants; 24 is the negative control.

[0017] Picture 2 To determine the phenotypes of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana overexpressing ADTW6E, A represents the color phenotype of the transgenic lines in MS medium; B represents the color phenotype of leaves of wild-type and transgenic plants under normal growth conditions; and C represents the color phenotype of pods of wild-type and transgenic plants under normal growth conditions.

[0018] Picture 3 The graph shows the anthocyanin content detection results, where A is the experimental result graph and B is the statistical analysis graph of the experimental results. Detailed Implementation

[0019] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0020] Example 1: ADTW6E Gene identification and cloning.

[0021] A type of substance that can change the color of plants to purple. ADTW6E The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0022] SEQ ID NO.1: ATGGAGGGATCCATAGGCCTAAGAAAAGGTGCATGGGCTAAGGTGGAAGATGACCTTCTAAGAGCTTGTGTTGAACAATATGGAGAAGGAAAGTGGCACCTAGTTCCTTCTAGAGCGGGGTTGAACAGATGCCGCAAAAGCTGTAGATTGCGATGGTTGAATTATCTGAAACCAAATATAAAGCGAGGAGAGTTCGCTGAAGATGAAGTTGATCTCATGATTAGAATGCATAGACTTTTGGGAAACAGATGGTCCTTAATTGCTGGAAGACTTCCGGGAAGAACGCCAAACGATGTGAAGAATTACTGGAACACCTATGTTCGAAGGAATAATAAGCACACTTCATCCTCATCAACACCATCAGTACCCTCTCCTTCATCAGTGGTGACGACAGTGAAACGTCATGACCATATTAATCATCAGGTAATAAAACCTCATCCCCGAACTTTCTCGAAAGCATCGCCATGGTTATTATTAAAGAAAACATCTGCAAGTGGTGATCATAATAATAGCAAGCAAAGGGATGGGGCCAAAGCAGAAGAAGAGTGCACCGATAATAATAATAATGGTTCTGGTGGTGGATGTGATGATGGGGACACGTGTCGGGACAAGAACGGTGGCAAAGGGGATGAAGAGAAGTTGAACGACGGATGCTTACTCTCAGTCTCAGGTGCTGAGGAAGAAGAAGAAGAAGAAGAAGACTGGAAGCTGCTTTTACCTGACTTCAATTGGGATGCTCATAATGATGATCACTTCTTGAAGGACGCTTCTGATGCTGACCAGGATATGCTTATTAATGTCCATGGTCAAACTTGGAGTGATATCCTTCTTGATATCAATTTGTGGGATCCACAATAA。

[0023] ADTW6E The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.2.

[0024] SEQ ID NO.2: MEGSIGLRKGAWAKVEDDLLRACVEQYGEGKWHLVPSRAGLNRCRKSCRLRWLNYLKPNIKRGEFAEDEVDLMIRMHRLLGNRWSLIAGRLPGRTPNDVKNYWNTYVRRNNKHTSSSSTPSVPSPSSVVTTVKRHDHINHQ VIKPHPRTFSKASPWLLLKKTSASGDHNNSKQRDGAKAEEECTDNNNNGSGGGCDDGDTCRDKNGGKGDEEKLNDGCLLSVSGAEEEEEEEDWKLLLPDFNWDAHNDDHFLKDASDADQDMLINVHGQTWSDILLDINLWDPQ.

[0025] This invention uses the cultivated peanut variety Tifrunner as the experimental material, which was provided by the laboratory of the College of Grassland Science, Qingdao Agricultural University. It has been published in the literature "Holbrook, CC, Culbreath AK Registration of 'Tifrunner' peanut. Journal of Plant Registrations, 2007, 1(2): 124."

[0026] 1. Primer design Designing amplifications using Primer 5.0 software ADTW6E Specific primers for the target fragment and the addition of homologous arms, respectively ADTW6E -F and ADTW6E -R, ADTW6E The nucleotide sequence of -F is shown in SEQ ID NO.3. ADTW6E The nucleotide sequence of -R is shown in SEQ ID NO.4.

[0027] SEQ ID NO. 3: atttggagaggacagggtaccATGGAGGGATCCATAGGCCTA.

[0028] SEQ ID NO. 4: agagccctggcatgcctgcagTTATTGTGGATCCCACAAATTGATA.

[0029] 2. RNA extraction Total RNA was extracted from the experimental materials using the FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics-rich). The entire procedure was performed according to the kit's instructions. The extracted total RNA was then used as a template for reverse transcription to obtain cDNA. The FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics-rich) was purchased from Novizan.

[0030] The reverse transcription procedure is as follows: The first step is to denature the RNA template. Add 1 μL of RNA and 7 μL of ddH2O to an RNase-free centrifuge tube, heat at 65°C for 5 min, quickly cool on ice, and let stand on ice for 2 min.

[0031] The second step is to remove genomic DNA. Add 2 μL of 5×gDNA wiper mix, mix thoroughly by pipetting, and react at 42°C for 2 min.

[0032] The third step involves preparing the first-strand cDNA synthesis reaction solution by adding 2 μL of 10×RT Mix, 2 μL of HiScript III Enzyme Mix, and 1 μL of Oligo(dT). 20 After adding VN and 5 μL of ddH2O, gently mix with a pipette. Finally, incubate at 50°C for 45 min in an RT-PCR instrument to obtain cDNA.

[0033] 3. Gene cloning Using the obtained cDNA as a template, primers were used... ADTW6E -F and ADTW6E PCR amplification was performed using -R to obtain the target fragment of 1098 bp.

[0034] The PCR reaction system consisted of 1 μL of cDNA template, 5 μL of 2×Phanta Flash Master Mix (DyePlus), and 0.5 μL of... ADTW6E -F and 0.5 μL ADTW6E -R, and finally make up to 10μL with ddH2O.

[0035] PCR reaction procedure: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 10 seconds, 55℃ annealing for 5 seconds, 72℃ extension for 4 seconds / kb, 34 cycles; 72℃ final extension for 5 minutes. Products were subjected to agarose gel electrophoresis.

[0036] 4. Gel recovery and sequencing The PCR amplification products were recovered from the gel and sequenced. The sequencing sequence is shown in SEQ ID NO.1.

[0037] After comparison ADTW6E The coding region sequences of the genes are identical, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0038] Example 2: Recombinant plant overexpression vector pCAMBIE2300-OMC- ADTW6E The construction.

[0039] 1. Double enzyme digestion of the overexpression vector use Kpn I and PST The pCAMBIE2300-OMC expression vector was double-digested with restriction endonucleases, and the DNA was recovered. Specific steps were performed according to the NEB instruction manual and the DNA purification kit instructions. The purified digested products were stored at -20°C.

[0040] The product purification kit, FastPure® Gel DNA Extraction Mini Kit, was purchased from Novizan.

[0041] 2. Homologous recombination to connect the target gene and the overexpression vector The target gene fragment was ligated to the double-digested overexpression vector using a homologous recombination kit. For specific steps, please refer to the instructions for the homologous recombination kit.

[0042] Among them, the homologous recombination kit: ClonExpress® Ultra One Step Cloning Kit; the homologous recombination kit: Novizan, Nanjing, China.

[0043] PCR reaction system: Take 2 μL of target fragment, 3 μL of linear vector and 5 μL of 2×ClonExpress Mix; PCR reaction procedure: ligation at 50℃ for 30 min, followed immediately by 4℃.

[0044] 3. Transformation of Escherichia coli with recombinant plasmids The ligation product was transferred into E. coli competent cells DH5α. For specific steps, please refer to the E. coli competent cells DH5α instruction manual.

[0045] Among them, Escherichia coli competent cells DH5α were provided by Shanghai Weidi Biotechnology Co., Ltd.

[0046] After transformation, the *E. coli* culture was spread onto LB agar containing 50 mg / L kanamycin and incubated upside down at 37°C for 36 h. Single colonies were picked using a sterile pipette tip and identified by colony PCR using p2300-F / Tc1M-R primers, followed by sequencing. The p2300-F primer sequence is shown in SEQ ID NO. 5, and the Tc1M-R primer sequence is shown in SEQ ID NO. 6.

[0047] SEQ ID NO. 5: TATTGTGAAGATAGTGGAAAAGGAA.

[0048] SEQ ID NO. 6: GGTCAGCATCAGAAGCGTCC.

[0049] Activate, preserve, and extract plasmids from the correctly sequenced monoclonal bacterial culture. Refer to the plasmid extraction instructions for specific steps.

[0050] Among them, the FastPure Plasmid Mini Kit plasmid extraction kit is from Novizan, Nanjing, China.

[0051] LB medium: LB broth, model HB0128, Soo-Ao, Qingdao, China.

[0052] 4. Transformation of Agrobacterium with recombinant plasmids Will ADTW6E The overexpression vector plasmid was transformed into Agrobacterium tumefaciens competent cells EHA105, following the instructions for Agrobacterium tumefaciens competent cells. The transformed Agrobacterium culture was plated on LB agar containing 50 mg / L kanamycin and 50 mg / L rifampin, and incubated at 28°C for 36 h. Single colonies were picked using a sterile pipette tip and identified by colony-linked PCR using p2300-F / Tc1M-R primers. The correctly identified single colonies were activated and preserved to obtain cells containing pCAMBIE2300-OMC- ADTW6E Agrobacterium with recombinant vector. Agrobacterium competent cells EHA105: purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0053] Example 3: Verification of transgenic function ADTW6E Screening and phenotypic analysis of transgenic Arabidopsis plants.

[0054] 1. Preparation of Arabidopsis thaliana plants In a clean bench, 200 Arabidopsis thaliana seeds were placed in a 1.5 mL centrifuge tube, 1 mL of ddH2O was added, and the tube was shaken before centrifugation in a small centrifuge. Then, 1 mL of 75% ethanol was added for sterilization for 30 seconds, followed by a second wash with ddH2O. Next, 10% NaClO was added for sterilization for 6 minutes, during which the centrifuge tube was shaken by hand. After washing three times with ddH2O, the tube was soaked in 1 mL of ddH2O for 45 minutes to allow for swelling. Using a 1 mL sterile pipette tip, the Arabidopsis thaliana seeds were evenly spread on 1 / 2 MS solid medium. After germination at 4°C for 3 days, the medium was transferred to a 24°C, 16h light / 8h darkness incubator. Once three true leaves had emerged, the seeds were transplanted into soil. The Arabidopsis thaliana continued to grow in the soil, with normal watering management, until bolting, at which point it was ready for genetic transformation.

[0055] 2. Preparation of infecting bacterial solution Select those containing pCAMBIE2300-OMC- ADTW6E Agrobacterium cells containing the recombinant vector were cultured overnight in LB liquid culture containing 50 mg / L kanamycin and 50 mg / L rifampin on a constant temperature shaker at 28°C and 200 rpm until OD reached. 600 The concentration was 0.8. Take 200 mL of the shaken bacterial culture and place it in a centrifuge tube. Centrifuge at 24°C and 8000 rpm for 10 min to collect the bacterial cells. Discard the supernatant after centrifugation and add a pre-prepared 5% sucrose solution. Mix well and resuspend to OD. 600 The bacterial suspension was prepared with a pH of approximately 1.0. Then, 0.03% (w / w) of surfactant sliwet-77 was added to obtain the inoculum for later use.

[0056] The 5% sucrose solution was prepared using a 1 / 2 MS solution. The 1 / 2 MS solution was prepared by adding 2.22g of MS powder and 8g of sucrose to a beaker, then adding ultrapure water to bring the volume to 1L. The solution was stirred with a magnetic stirrer until fully dissolved, and the pH was adjusted to 5.85. The surfactant sliwet-77 was purchased from Solarbio, model S9430.

[0057] 3. Infecting Arabidopsis thaliana using the flower-dipping method The Arabidopsis inflorescences were immersed in the infection solution for 60 seconds, immediately removed, and cultured in the dark for 24 hours, after which they were transferred to normal conditions for continued growth. After 7 days, the Arabidopsis inflorescences were smeared with the infection solution using cotton swabs for secondary infection.

[0058] After infection, Arabidopsis thaliana is managed normally until the seeds mature, and then the mature seeds of transgenic Arabidopsis thaliana are harvested.

[0059] In Arabidopsis inflorescence, the inflorescence refers to the unopened flower buds after removing the formed pods and open flowers.

[0060] The normal management method / conditions for Arabidopsis thaliana are as follows: Arabidopsis plants are cultured at 24℃ under 16h light / 8h darkness conditions, with water supplemented using Hoagland nutrient solution during this period. The Hoagland nutrient solution formula is shown in Table 1.

[0061] Table 1: Hoagland Nutrient Solution Formula 4. Identification of transgenic plants After sterilizing mature transgenic Arabidopsis seeds, they were evenly spread on 1 / 2 MS solid medium containing 50 μg / mL Kan (kanamycin). The specific steps were the same as those described in the Arabidopsis cultivation method above. Once the Arabidopsis seedlings had grown to 6 leaves, healthy leaves were harvested, and DNA was extracted using the CTAB method. Wild-type Arabidopsis plants were used as a negative control. p2300-F / Tc1M-R Primers were used to identify positive strains by PCR amplification and agarose gel electrophoresis.

[0062] The PCR amplification conditions were as follows: Arabidopsis DNA was used as a template, and RT-PCR identification was performed using the 2×Phanta Flash Master Mix (DyePlus) kit (Novizan, Nanjing, China).

[0063] PCR reaction system: Take 1 μL of DNA template, 5 μL of 2×Phanta Flash Master Mix (Dye Plus), 0.5 μL of each primer, and finally add ddH2O to make up to 10 μL.

[0064] PCR reaction procedure: Step 1: Pre-denaturation at 98℃ for 30 seconds; Step 2: Denaturation at 98℃ for 10 seconds, annealing at 55℃ for 5 seconds, extension at 72℃ for 5 seconds / kb, for 34 cycles; Final extension at 72℃ for 5 minutes.

[0065] The results of the identification are as follows Picture 1 As shown, the transgenic line contains a 1060bp fragment, while the wild-type line does not, indicating that... ADTW6E The overexpression vector has been successfully introduced into the Arabidopsis thaliana genome.

[0066] The specific propagation steps are as follows: In a clean bench, place 200 Arabidopsis thaliana seeds into a 1.5 mL centrifuge tube, add 1 mL of ddH2O, shake, and centrifuge. Then, add 1 mL of 75% ethanol for 30 seconds to sterilize, and wash again with ddH2O. Add 10% NaClO for 6 minutes to sterilize, shaking the centrifuge tube by hand during this time. After washing three times with ddH2O, add 1 mL of ddH2O to soak and allow to swell for 45 minutes. Using a 1 mL sterile pipette tip, pipette the Arabidopsis thaliana seeds evenly onto 1 / 2 MS solid medium. After germination at 4℃ for 3 days, transfer the culture dish to a 24℃, 16h light / 8h darkness incubator. Once three true leaves have emerged, transplant into soil. The Arabidopsis thaliana will continue to grow in the soil, with normal watering management, until the seeds mature. Harvest the transgenic Arabidopsis thaliana seeds.

[0067] 5. Color phenotype of transgenic Arabidopsis thaliana Wild-type and positive-positive transgenic Arabidopsis seeds were surface-sterilized with a 10% sodium hypochlorite solution for 10 min, then placed in 1 / 2 MS medium and cultured at 4°C for 3 days to break seed dormancy. Germination was then carried out in an incubator with alternating light and dark conditions for 16 h / 8 h. Color changes in the transgenic Arabidopsis were recorded during the culture period. After one week of normal growth, the seedlings were transplanted to a greenhouse for soil cultivation. Leaf color changes in both wild-type and transgenic Arabidopsis were then recorded.

[0068] The results are as follows Picture 2 As shown in Figure A, under MS culture conditions, the transgenic Arabidopsis roots turned purple. Picture 2 As shown in B and C, under soil cultivation conditions, the leaves and pods of transgenic Arabidopsis thaliana can turn purple, while the wild type shows no color change.

[0069] 6. Determination of anthocyanin content Collect wild type and ADTW6E The anthocyanin content of the genetically modified rosette leaves was extracted using a plant proanthocyanidin content detection kit (Beijing Box Biotechnology Co., Ltd.). For example... Picture 3 As shown, ADTW6E The anthocyanin content of the transgenic rosette leaves was significantly higher than that of the wild type.

[0070] The above results indicate that ADTW6E It participates in regulating the transformation of Arabidopsis thaliana into a purple phenotype and can be used to regulate color changes in other plants.

[0071] In production practice, the above-mentioned genes can be transformed into peanut explant cells, and then the transformed callus tissue can be cultured into plants. Through transgenic methods, plant expression vectors can be used to transform explant cells to cultivate peanuts with purple inner seed coats.

[0072] In production practice, the above-mentioned genes can also be used to enhance the efficiency and accuracy of breeding goals through molecular marker-assisted selection breeding methods. For example, molecular markers can be used to associate target genes with the purple trait of peanuts, and the presence of the target gene can be detected to achieve the purpose of selecting the target trait. This invention has discovered... ADTW6E The function and specific applications of genes in regulating plant color changes provide new genetic resources for flowering breeding and also contribute to further analysis. ADTW6E This lays the foundation for understanding the molecular mechanisms that regulate plant color changes.

[0073] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

Claims

1. A method for regulating the color of peanut inner seed coat ADTW6E Genes, characterized by, The ADTW6E The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The claim 1 ADTW6E Proteins expressed by genes are characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, It comprises the contents of claim 1 ADTW6E Gene.

4. The recombinant expression vector according to claim 3, characterized in that, It is the aforementioned ADTW6E Gene insertion into the pCAMBIE2300-OMC overexpression vector Kpn I and Pst Obtained between I restriction endonuclease sites.

5. Recombinant engineered bacteria comprising the recombinant expression vector of claim 3.

6. The recombinant engineered bacteria according to claim 5, characterized in that, The recombinant expression vector was obtained by transforming it into Agrobacterium EHA105.

7. The claim 1 ADTW6E The application of the gene, the protein of claim 2, the recombinant expression vector of any one of claims 3-4, or the recombinant engineered bacteria of any one of claims 5-6 in regulating plant color, characterized in that, The regulation described is to make the plant turn purple.

8. The application according to claim 7, characterized in that, The purple color of plants is achieved by increasing the anthocyanin content in the plants.

9. The application according to claim 8, characterized in that, The plants mentioned include peanuts and Arabidopsis thaliana.

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