High-yielding lutein transgenic chlorella and preparation thereof

A kind of chlorella and transgenic technology, applied in the field of genetic engineering, can solve the problems of limited access to lutein, increased labor and land costs, etc.

Inactive Publication Date: 2013-04-17
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, lutein is mainly derived from marigolds. However, due to the increase in labor and land costs, obtaining lutein from marigolds is subject to certain restrictions

Method used

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  • High-yielding lutein transgenic chlorella and preparation thereof
  • High-yielding lutein transgenic chlorella and preparation thereof
  • High-yielding lutein transgenic chlorella and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the construction of recombinant plasmid pCAMBIA2301-idi

[0052] 1. Cloning of Escherichia coli idi gene and construction of related vectors

[0053] 1. Cloning of Escherichia coli idi gene

[0054] Using Escherichia coli DH5α as material, the genome of Escherichia coli DH5α was extracted by phenol-chloroform method;

[0055] The details are as follows: take 500uL of DH5α bacterial liquid, and centrifuge at 12000r for 5min to obtain bacterial cells. Add 650uL of lysate, shake for 2min, and put in a 65°C water bath until the lysate becomes clear. Add an equal volume of balanced phenol: chloroform: isoamyl alcohol (25:24:1), shake and mix, and centrifuge at 13000r for 15min. Aspirate the supernatant and gently transfer it to a new tube, then extract once with an equal volume of chloroform:isoamyl alcohol (24:1), and centrifuge at 13000r for 15min. Aspirate the supernatant and move it to a new tube, add 1 / 10 volume of NaAc, and 2 times the volume of absolut...

Embodiment 2

[0089] Embodiment 2, the acquisition of the trans idi Chlorella of high-yielding lutein

[0090] 1. Obtaining trans idi chlorella

[0091] The transformation of Chlorella was mediated by Agrobacterium as follows:

[0092] (1) Preparation of Chlorella protoplasts

[0093] Chlorella STIO02 was deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​November 30, 2012, and the preservation number is CGMCC No. .6951, with the proposed taxonomic designation Chlorella vulgaris.

[0094] A single algal colony of Chlorella STIO02 (CGMCC 6951; also known as wild-type Chlorella) was picked and inoculated in FC medium, and cultured on a shaking table at 28°C until the algae grew out.

[0095] Transfer to 50mL fresh FC medium with a 1% inoculum amount, and culture on a shaker at 28°C until logarithmic phase.

[0096] Collect the algal cells...

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Abstract

The invention discloses a preparation method of a high-yielding lutein transgenic chlorella. The preparation method aims to transfer an IPP (Isopentenyldiphosphate) isomerase encoding gene to an objective chlorella to obtain a transgenic chlorella, wherein the lutein yield of the transgenic chlorella is higher than that of the objective chlorella; and the amino acid sequence of the IPP isomerase is the sequence 2 in the sequence table. Tests show that, the method provided by the invention, can transfer an IPP (Isopentenyldiphosphate) isomerase encoding gene expression box to a wild-type chlorella to obtain the transgenic chlorella of which lutein content is increased by 30.95 percent maximumly compared with the wild-type chlorella and the lutein yield is increased by 36.77 percent maximumly compared with the wild-type chlorella; and the transgenic chlorella is the high-yielding lutein transgenic chlorella.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a high-yielding lutein transgenic chlorella and a preparation method thereof. Background technique [0002] Lutein is an oxygen-containing carotenoid, which plays a very important role in living organisms. Lutein has antioxidant activity that protects against diseases such as cancer, cardiovascular disease, and age-related macular degeneration. In addition, as a natural pigment, lutein can be used as a pigment additive in food, medicine and health care products. At present, lutein is mainly derived from marigolds. However, due to the increase of labor and land costs, obtaining lutein from marigolds is limited. Chlorella is rich in lutein, and chlorella has the advantages of fast growth, heterotrophic culture, easy large-scale cultivation and algae improvement. The use of chlorella to synthesize lutein has attracted more and more attention. . [0003] At present, the methods a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N1/13C12N15/63C12N9/90C12N1/21C07C403/24C12R1/89C12R1/01
Inventor 林祥志马瑞娟荣辉林汝榕程汝滨王昭凯杨善军马勇陈水波柯秀蓉李惠丽
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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