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Strawberry glutathione-transferase FaGST gene of, express protein thereof and application thereof

A technology of glutathione and transferase, applied in the direction of transferase, application, genetic engineering, etc., can solve the problem of unclear process of anthocyanin

Active Publication Date: 2019-01-11
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the process by which anthocyanins are transported from the cytoplasm to the vacuole remains unclear

Method used

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  • Strawberry glutathione-transferase FaGST gene of, express protein thereof and application thereof
  • Strawberry glutathione-transferase FaGST gene of, express protein thereof and application thereof
  • Strawberry glutathione-transferase FaGST gene of, express protein thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Using the strawberry GST suspected gene sequence published by the strawberry genome (gene registration name: XM_004294173.2) as a template, the upstream primer GST1-F and the downstream primer GST1-R were designed using DNAMAN, and the cDNA of the "Sweet Charlie" strawberry fruit during the color conversion period was obtained. As a template, a PCR reaction was performed to clone the FaGST1 gene of "Sweet Charlie" strawberry. The gene sequence is 657 bp in length, and its base sequence is shown in SEQ ID NO. 1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO. 2. . The Fa GST1 gene of "Sweet Charlie" strawberry was transformed into pMD-18T vector to obtain pMD-18T-FaGST1 plasmid, and the plasmid was extracted for use.

[0020] Among them, the primer sequence is:

[0021] GST1-F: 5'-AATGGCGGATGAGGTTGTC-3',

[0022] GST1-R: 5'-CCACTTGGAATAAGAAACTG-3'.

[0023] The main conversion process is as follows:

[0024] (1) The ligation product is transformed in...

Embodiment 2

[0033] Using GST1-PHF and GST1-PHR as amplification primers, and using the pMD-18T-FaG ST1 plasmid obtained in Example 1 as a template, a PCR reaction was performed to recover the target gene product. Connect the target gene to the PHellsgate vector. After ligation at 25°C for 12 hours, 1μL reaction termination enzyme was added. The main process is as follows:

[0034] Heat shock method transforms DH5α Escherichia coli competent. Use pHellsgate2 interference vector for recombination and construction. At the 5'end of FaGST1, select fragments of about 300-500bp to design primers, and add attB site universal primers before specific primers as target fragment amplification primers. Strawberry cDNA was used as a template to amplify the target fragment. After the PCR product was recovered, it was recombined to the pHellsgate2 vector by BP reaction to construct the target vector. Select the correct monoclonal strain after colony PCR reaction, extract the plasmid and carry out the dou...

Embodiment 3

[0046] The single colony identified in Example 2 was selected for strawberry fruit infection. The octoploid fruits of the big green fruit period were selected as the test materials, and the injection method was used for fruit infection. The specific methods are as follows:

[0047] 1) Pick out the positive clone containing PHellsgate-FaGST1 plasmid and inoculate it into 15mL LB medium (containing 50mg L -1 Kana and 50mg L -1 Rifampicin), 28℃, 200rpm shake to OD 600 ≈2.0.

[0048] 2) Take 20mL of fresh LB liquid medium (containing 50mg L -1 Kana and 50mg L -1 Rifampicin), insert 1mL of Agrobacterium from step 1), shake to OD at 28°C, 200rpm 600 ≈0.6.

[0049] 3) Collect the bacterial solution and centrifuge at 6000 rpm for 3 min at room temperature.

[0050] 4) Resuspend the bacterial solution with the infection solution and centrifuge at 6000 rpm for 3 min at room temperature. Infection solution configuration: each configuration concentration is 1.0mol L -1 MES, 1.0mol L -1 MgCl 2 An...

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Abstract

The invention discloses a strawberry glutathione-transferase FaGST gene, an express protein thereof and an application thereof, wherein the base sequence of the strawberry glutathione transferase FaGST gene is shown as SEQ ID NO. 1. The strawberry glutathione-transferase FaGST gene is cloned, and the FaGST1 is found to be related to the accumulation of anthocyanin in strawberry fruit through an infection, and after the gene is silenced, the expression amount is reduced, and the expression amount of main anthocyanin synthesis genes such as CHS1, F3H1, ANS1, UGT1 and the like are inhibited. Therefore, the FaGST gene will be widely used in anthocyanin synthesis.

Description

Technical field [0001] The invention belongs to the technical field of plant cultivation, and specifically relates to a strawberry glutathione transferase FaGST gene and its expression protein and application. technical background [0002] Strawberry (Fragaria×ananassa Duch.) is a perennial herbaceous plant belonging to the genus Fragaria of the Rosaceae family. The fruit is bright in color, rich in aroma, sweet and sour, and has high nutritional and health value. It has the reputation of "Queen of Fruits". Strawberry fruits have been shown to have strong antioxidant activity, which is related to the polyphenol compounds contained in the fruit, especially anthocyanins. At the same time, anthocyanins are the main pigments that make up the color of strawberry fruits and are essential for the appearance of fruit. Anthocyanins also have important biological functions, such as being able to protect plants against ultraviolet rays, low temperature, and pests and diseases. Therefore, i...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/82A01H5/08A01H6/74
CPCC12N9/1088C12N15/825C12Y205/01018
Inventor 谢兴斌薛婷婷方从兵赵静王申冯欢石梦云宋佳丽
Owner ANHUI AGRICULTURAL UNIVERSITY
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