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
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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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