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Dunaliella gene knockout vector and application thereof

A gene knockout, Dunaliella technology, applied in the use of vectors to introduce foreign genetic material, microorganism-based methods, microorganisms, etc., can solve the problems of difficult extraction, adverse health effects of chemical additives, and high costs

Inactive Publication Date: 2011-09-21
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Most of the lycopene on the market is a natural product extracted from tomato, but since the content of lycopene in tomato is only about 0.002%, the extraction is difficult, costly and low in purity, and a large number of tomatoes are grown for tomato red Vegetarian extraction needs to consume a lot of land resources
[0006] There are also some reports on the preparation of lycopene by microbial fermentation. U.S. patents US3097146, US3369974 and Chinese patent 200510090996.0 propose a method for preparing lycopene by fermentation of B. trispora. However, these methods require the addition of aminopyridine, etc. Chemical agents interfere with the biosynthetic pathway of molds to achieve the accumulation of lycopene, and the residues of chemical additives in the product will have adverse effects on human health
Chinese patent 200710122895 invented a method for accumulating lycopene by fermentation of bacteria (Streptomyces fissures), but due to the limitation of the supply of its synthetic precursor, the yield of lycopene produced according to this method is only 0.4% to 0.7% %Cell dry weight range, there is still no substantial breakthrough and improvement
[0009] Both lycopene and β-carotene are carotene substances, and they have similar chemical structures. The idea of ​​allowing Dunaliella to directly synthesize and accumulate lycopene like biosynthesis of β-carotene is very attractive, because so far So far, no organisms capable of synthesizing and accumulating lycopene with such high efficiency have been found in the world

Method used

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  • Dunaliella gene knockout vector and application thereof
  • Dunaliella gene knockout vector and application thereof
  • Dunaliella gene knockout vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1, Construction of Dunaliella lycopene-β-cyclase gene knockout vector

[0040] Step 1, Extraction of Dunaliella Genomic DNA

[0041] Centrifuge at 5000r / min to collect DsMG liquid medium (Mu Chunlin, Chen Xiwen, Hou Zhaoli. Isolation and species identification of β-carotene Dunaliella in industrial production. Salt Industry and Chemical Industry, 2009, 38(4): 25-29) For Dunaliella cells, add 750 μL preparation solution (containing 2.5mL extract solution, 2.5mL nuclear lysate solution, 1mL 5% (w / v) sodium N-lauroyl sarcosinate for every 100mg cells, mix well, weigh 0.024g Sodium bisulfite, dissolved, mixed. The extract was 350mmol / L sorbitol, 100mmol / LTris (pH7.5), 5mmol / L EDTA. The nuclear lysate was 200mmol / L Tris HCl (pH7.5), 50mmol / L EDTA, 2.0mol / LNaCl, 2% CTAB), incubate at 55°C for 2h (invert and mix once every 15min), aliquot, 750μL per centrifuge tube; add 750μL chloroform / isoamyl alcohol (24:1) to each tube , invert and mix well, centrifuge at room te...

Embodiment 2

[0053] Example 2, Electrotransformation of lycB gene knockout vector pDs-GKO-cat and screening and identification of transformants

[0054] a. Cultivate Dunaliella cells to the logarithmic growth phase (1.0×10 6 / mL), centrifuge at 4000r / min for 5min at 4°C, and collect the cells;

[0055] b. Operation on ice, electric shock buffer (composed of: 0.28mol / L NaCl, 5mmol / L KCl, 25mmol / L CaCl 2 , the Hepes of 20mmol / L, the mannitol of 200mmol / L, the sorbitol of 200mmol / L, the Tween-20 of 0.05%, the glycerol of 0.4mol / L) suspension cell, wash 2 times;

[0056] c. Resuspended to a cell density of 1.0×10 7 / mL; Take 90-100 μL of the resuspended algae liquid, add the final concentration of 10 ng / μL carrier and 20 ng / μL protamine DNA, mix well, and ice-bath for 10 min;

[0057] d. 6kV / cm electric shock, ice bath for 10min, add 1mL DsMG, dark culture for 12h;

[0058] e. Light: dark = 14:10 culture, and finally plated on a solid plate containing 100 μg / mL chloramphenicol (instruction...

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Abstract

The invention relates to Dunaliella gene engineering, in particular to a Dunaliella lycopene-beta-cyclase (LycB) gene knockout vector and application thereof. The LycB gene knockout vector comprises a selection marker gene and is connected to a 3' end homologous recombination region and a 5' homologous recombination region of the LycB gene. The LycB gene knockout vector is applied to culturing LycB gene knockout Dunaliella. The process of biologically synthesizing beta-carotene by using LycB gene knockout Dunaliella is stopped at the lycopene stage; and under the stress condition, high-content lycopene can be accumulated and the accumulation quantity is 3 to 4 percent of the dry weight of the Dunaliella.

Description

technical field [0001] The invention relates to the field of Dunaliella genetic engineering, in particular to a Dunaliella lycopene-β-cyclase (Lycopene β-cyclase, LycB) gene knockout vector and application thereof. Background technique [0002] Lycopene (Lycopene) is a fat-soluble natural pigment. Its molecule is a straight-chain hydrocarbon composed of 11 conjugated double bonds and 2 non-conjugated double bonds. It belongs to carotenoids. Mainly found in tomato, watermelon, red grapefruit and other plants. Studies in the past ten years have shown that lycopene has a strong antioxidant activity, and its ability to scavenge singlet oxygen is 100 times that of vitamin E, 2.2 times that of β-carotene, and the effect of scavenging hydroxyl free radicals is better than that of β-carotene. It is 32 times stronger than antioxidant, which is one of the strongest antioxidants found in nature. Studies have found that lycopene has superior physiological functions. It not only has an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12R1/89
Inventor 陈德富李伯平陈喜文牟春琳
Owner NANKAI UNIV
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