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Method for enhancing content of vitamin E in plants by gene cotransformation of gamma-tmt and hpt

A vitamin and co-transformation technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of low activity and high cost, and achieve the effect of improving the nutritional value of plants

Inactive Publication Date: 2010-06-16
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the cost is high, and the racemization of α-tocopherol produced is different from that of natural α-tocopherol, and the activity is also lower than that of natural α-tocopherol

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Cloning of Arabidopsis γ-tmt and hpt genes

[0025] 1. Extraction of Arabidopsis Genomic Total RNA

[0026] Take a small amount of young leaves of Arabidopsis thaliana (ecotype Colombian), freeze them quickly with liquid nitrogen, grind them quickly with a mortar, add them to a 1.5mL Eppendorf tube containing 1mL TRIzol Reagents (TRIzol Reagents, GIBCO BRL, USA), and fully After oscillating, place at room temperature for 5 minutes, add 200 μL of chloroform, shake vigorously for 15 see, place at room temperature for 2-3 minutes, then centrifuge at 4°C and 12,000 g for 15 minutes; pipette the supernatant (about 600 μL) into a clean 1.5 mL Eppendorf tube, Add an equal volume of isopropanol, mix by inverting, place at room temperature for 10 minutes, centrifuge at 4°C, 12,000g for 10 minutes; discard the supernatant, add 1mL 75% ethanol to wash, shake, and centrifuge at 4°C, 7,500g for 5 minutes; After drying at room temperature for 15-20 minutes, dissolve in an appropriat...

Embodiment 2

[0031] Construction of plant binary expression vector containing γ-tmt and hpt genes

[0032] 1. Intermediate vector pCAMBIA1304 + build

[0033] Select pBI121 and pCAMBIA1304 as the basic elements to construct the binary plant expression vector pCAMBIA1304 + . Specifically, pBI121 and pCAMBIA1304 were digested with HindIII and EcoRI; the pBI121 expression cassette was recovered, and the large fragment of pCAMBIA1304 was recovered; the two recovered products were connected, transformed into DH5α, screened on a kanamycin LB plate to obtain a single clone, and then extracted the plasmid , enzyme digestion verification (HindIII and EcoRI double enzyme digestion); that is, construct pCAMBIA1304 + .

[0034] 2 Construction of intermediate vectors pMD18T-γ-tmt and pMD18T-hpt

[0035] Firstly, primers were synthesized according to the restriction sites, restriction sites XbaI and SacI were introduced upstream and downstream of γ-tmt, and restriction sites BglII and BstEII were i...

Embodiment 3

[0042] Transformation of Arabidopsis thaliana with γ-tmt and hpt genes mediated by Agrobacterium tumefaciens to obtain transgenic plants

[0043] 1. Obtaining the engineering bacteria of Agrobacterium tumefaciens containing the binary plant expression vector of γ-tmt and hpt genes

[0044] The plant binary expression vector containing γ-tmt and hpt genes in Example 2 was transformed into Agrobacterium tumefaciens (such as GV3101, which was purchased from the University of Nottingham, UK used in this example), and verified by PCR. The results show that the plant binary expression vector containing γ-tmt and hpt genes has been successfully constructed into the strain of Agrobacterium tumefaciens.

[0045] 2. Agrobacterium tumefaciens mediates transformation of Arabidopsis thaliana with γ-tmt and hpt genes

[0046] 2.1 Planting of Arabidopsis thaliana

[0047] 2.1.1 Arabidopsis sterile culture

[0048] ①. Aseptic culture of Arabidopsis thaliana: wash the seeds in 15% sodium hy...

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Abstract

The invention belongs to the technical field of biology, in particular relates to a method for enhancing the content of vitamin E in plants by the cotransformation of double key enzyme genes of gamma-tmt and hpt. The method comprises the following steps of: cloning the genes of the gamma-tmt and the hpt from the plants, constructing a plant expression vector containing the DNA molecule, mediating by agrobacterium tumefaciens, introducing the genes of the gamma-tmt and the hpt into the plants simultaneously, regenerating plants, detecting the integration condition of the extraneous target genes of the gamma-tmt and the hpt by PCR, measuring the content of the vitamin E in the plants by a high-efficiency liquid chromatography-ultraviolet light detector and sieving to obtain a transgenic plant with the enhanced content of the vitamin E. The content of the vitamin E in the leaves of the obtained transgenic plant is obviously enhanced, and the highest content reaches 4 times of that of non-conversion check plants. The invention has an important meaning for enhancing the nutritive value of the plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for increasing the content of vitamin E (tocopherol), in particular to a method for co-transforming double key enzyme genes γ-tmt and hpt to increase the content of vitamin E in plants. Background technique [0002] Vitamin E is a lipid vitamin, and its natural products are divided into eight types according to the structure, namely α, β, γ, δ-tocopherol (tocopherol) and α, β, γ, δ-tocotrienol (tocotrienol), of which α-tocopherol has the highest biological activity and is considered to be the main active ingredient of vitamin E. [0003] Since the biological function of vitamin E was discovered in 1922 (Evans and Bishop, 1922), long-term medical research has shown that vitamin E is not only related to the reproductive system, but also to the central nervous system, digestive system, cardiovascular system and muscular system. Normal metabolism is closely related (Traber and Si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/82A01H1/00C12Q1/68G01N30/02
Inventor 孙小芬李殷唐克轩
Owner FUDAN UNIV
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