Gamma-tocopherol tranferase, gene and use thereof
A technology of phenol methyl transferase, applied in the field of γ-tocopherol methyl transferase
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Embodiment 1
[0019] Example 1. Cloning of γ-tocopherol methyltransferase gene
[0020] 1) Extraction of total RNA from head cabbage
[0021] Take 2.5 g of terminal buds of 9-day-old seedlings of Brassica oleracea L.Var.Capitata L., grind them with liquid nitrogen, and quickly add 15 mL of RNA extraction buffer preheated at 65°C ( 100mmol / L Tris-HCl, pH8.0, 2% CTAB, 2% PVP4000, 25mmol / LEDTA, 2.0mol / L NaCl, 0.5g / L Spermidine and 2% β-mercaptoethanol), invert and mix well. Extract twice with an equal volume of chloroform and isoamyl alcohol mixed solution (V / V=24:1), take the last supernatant solution, add 1 / 4 volume of 10mol / L LiCl, place it at 4°C overnight, and the next day Centrifuge at 4°C and 10,000rpm to recover the RNA precipitate in the above solution, and then dissolve the precipitate in 500 μL of SSTE solution (10mmol / L Tris-HCl pH8.0, 1.0mol / L NaCl, 0.5% SDS, 1mmol / L EDTA pH8.0 ), extracted twice more with a mixed solution of chloroform and isoamyl alcohol, added twice the volum...
Embodiment 2
[0032] Example 2. Preparation of recombinant plant constitutive expression vector containing γ-tocopheryl methyltransferase structural gene, recombinant plant seed-specific expression vector and recombinant Agrobacterium
[0033] The plasmid pTMTL was digested with BamH I and Sal I, and small DNA fragments were recovered. Similarly, the plant constitutive expression vector pBin438 and the seed-specific expression vector p7S438 were double-digested to recover large fragments of the DNA vector. Take these large DNA vector fragments and connect them with the above-mentioned small DNA fragments respectively. After transforming competent Escherichia coli JM109 by electric shock, spread on LB solid medium containing Kan (kanamycin). Transformants were identified by PCR and double digestion with BamH I and Sal I, and JM109 cells containing recombinant plasmid pBin-TMTL or p7S-TMTL were picked.
[0034] Plasmids pBin-TMTL and p7S-TMTL were extracted by alkaline method, respectively ...
Embodiment 3
[0037] Example 3. Preparation of recombinant microorganism expression vector containing γ-tocopherol methyltransferase structural gene and recombinant microorganism, expression of γ-TMT, enzyme activity analysis of expressed γ-TMT and antibody preparation of γ-TMT
[0038] 1) Recombinant Escherichia coli expression vector containing γ-tocopherol methyltransferase structural gene and preparation of recombinant Escherichia coli and induced expression of γ-TMT
[0039] In order to enable the γ-tocopherol methyltransferase structural gene to be correctly expressed in Escherichia coli and the expressed protein to be active, we designed a An upstream primer RT5: 5'-CGGGATCCACCATGACAACGACGGCAAC-3', this primer contains a BamHI restriction site, when it is amplified by RT-PCR with RT3 primer, the obtained 930bp product encodes a 2 - A truncated protein of the chloroplast guide peptide. The RT-PCR amplification conditions were the same as in Example 1. The PCR amplification products wer...
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