Gamma-tocopherol tranferase, gene and use thereof

A technology of phenol methyl transferase, applied in the field of γ-tocopherol methyl transferase

Inactive Publication Date: 2004-07-07
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no new gene of γ-TMT has been obtained from economic crops, and it is transformed into non-model plant economic crops to increase the content of α-tocopherol in crops and their products and change the content of α-tocopherol reports on the distribution of

Method used

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  • Gamma-tocopherol tranferase, gene and use thereof
  • Gamma-tocopherol tranferase, gene and use thereof
  • Gamma-tocopherol tranferase, gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

A whole-length cDNA sequence of gamma-tocopherol methyltransferase (gamma-TMT) cloned from the Brassica oleracea L.Var, capitata L. is disclosed. The gamma-TMT and its coding gene are also disclosed. The prokaryotic expression plasmid, plant constitution and specific expression plasmid in plant seed are configured. After said gamma-TMT structure gene is transferred to high-expression microbes, a biocatalytic method is used to methylate the gamma-tocopherol to obtain alpha-tocopherol and the transgenic plant with high content of alpha-tocopherol.

Description

technical field [0001] The invention belongs to the field of gamma-tocopherol methyltransferase (gamma-tocopherolmethyltransferase, gamma-TMT) genetic engineering. Specifically, it relates to a gamma-tocopherol methyltransferase from head cabbage (Brassica oleracea L.Var.capitata L.), a gamma-tocopherol methyltransferase gene and its structural gene and its recombinant plasmid ; Relating to its recombinant plasmid with constitutive or seed-specific expression in plants, to improve the content of α-tocopherol in plants and corresponding products and to change the distribution of α-tocopherol (especially to improve the concentration of α-tocopherol in seeds and oils) phenol content); at the same time, it also relates to a recombinant plasmid that specifically expresses γ-TMT in microorganisms, and uses a bioreactor to catalyze the production of α-tocopherol. Background technique [0002] Vitamin E (also known as tocopherol) is divided into two types: natural and synthetic. A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/00C12N1/19C12N1/21C12N9/10C12N15/54C12N15/63C12P17/06
Inventor 蔡文启欧阳青韩天富孙卉樊春涛张玉满吴存祥白羊年
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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