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Method for breeding high-yield photosynthetic-hydrogen-production chlamydomonas reinhardtii through cell nucleus insertion mutagenesis

A technology of hydrogen rheindulae and rheindala, which is applied in the field of nuclei insertion mutagenesis and breeding of high-yield photosynthetic hydrogen-producing Chlamydomonas reinhardtii, which can solve the problems that cannot solve the problem of the hydrogen production rate of Chlamydomonas reinhardtii and limit the application and development of biological photosynthetic hydrogen production technology. And other issues

Inactive Publication Date: 2010-10-20
SHANGHAI NORMAL UNIVERSITY
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Problems solved by technology

Hydrogenase is sensitive to oxygen and leads to low hydrogen production rate of Chlamydomonas reinhardtii, which is an important reason that limits the industrialization of this technology
The prior art has already conducted relatively in-depth research on the hydrogen-producing metabolism process and culture conditions of Chlamydomonas reinhardtii, but the hydrogen-producing metabolic process of Chlamydomonas reinhardtii is related to photosynthetic electron transfer, non-photosynthetic electron transfer, starch metabolism, respiratory metabolism, and fermentation metabolism. It is closely related to multiple metabolic processes such as photosynthesis, and its hydrogen production is largely affected by these metabolisms, so it has not been able to solve the technical problem of low hydrogen production rate of Chlamydomonas reinhardtii
[0003] The low hydrogen production rate of the existing Chlamydomonas reinhardtii limits the application and development of biological photosynthetic hydrogen production technology

Method used

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  • Method for breeding high-yield photosynthetic-hydrogen-production chlamydomonas reinhardtii through cell nucleus insertion mutagenesis
  • Method for breeding high-yield photosynthetic-hydrogen-production chlamydomonas reinhardtii through cell nucleus insertion mutagenesis
  • Method for breeding high-yield photosynthetic-hydrogen-production chlamydomonas reinhardtii through cell nucleus insertion mutagenesis

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Experimental program
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Effect test

Embodiment 1

[0080] Cultivation of Chlamydomonas reinhardtii:

[0081] Chlamydomonas reinhardtii has fast growth rate, low culture cost and high hydrogenase activity, so it is a representative species of microalgae for photosynthetic hydrogen production. In order to facilitate transgene manipulation, the present invention prefers Chlamydomonas reinhardtii species cc849 with a cell wall deficiency.

[0082] ①Normal culture conditions of Chlamydomonas reinhardtii: According to "The Chlamydomonas Sourcebook: a comprehensive guide to biology and laboratory use. New York: Academic Press. 1989" edited by Harris, preferably at 25±1°C, sunlight intensity (100-200 μmol photons m -2 the s -1 ), liquid culture is in 50~100ml Tris-Acetate-Phosphate (TAP) culture medium, initial pH7.2, horizontal shaker speed 100~130rpm, every 5~6 days 1% inoculation subculture; Solid TAP plate culture medium Containing 1.5% agar powder, the preservation and purification of the algal species is to pick a single clone...

Embodiment 2

[0086] Preparation of Chlamydomonas reinhardtii nuclear transformation vector and preparation of glass beads:

[0087] 1. Extraction and purification of plasmid pSP124S: described by standard methods known to those skilled in the art (Sambrook et al., Molecular Cloning. New York: Cold Spring Harbor Laboratory Press. 1998) and Lumbreras et al. Methods (The Plant Journal, 1998, 14:441-448) and TIANGEN plasmid extraction kit (TIANprep Mini kit) were used to extract and purify the vector pSP124S. Specifically, take 1-5 mL of overnight cultured E. coli liquid containing the pSP124S plasmid, centrifuge at 12,000 rpm for 1 min at 4°C to collect bacterial cells, add 250 μl of P1 (with RNase A added in advance) and 250 μl of P2, and gently flip up and down for 6- 8 to fully lyse the cells; then add 350 μl of P3, immediately turn it gently up and down for 6-8 times to fully mix the cells, centrifuge at 12,000 rpm for 10 min at 4°C, transfer the supernatant to the adsorption column CB3, ...

Embodiment 3

[0093] Nuclear transformation of Chlamydomonas reinhardtii and screening of transformants:

[0094] ① Transformation of the nucleus of Chlamydomonas reinhardtii:

[0095] Transform the nucleus of Chlamydomonas reinhardtii by the glass bead transformation method of Kindle (Proc.Natl Acad.Sci.US A.1990, 87:1228 1232), take 100ml and grow to the middle and late logarithmic phase (about 4~5×10 6 ), Chlamydomonas reinhardtii with healthy and active cells, collected algal cells by centrifugation at 4000rpm for 5min at 25°C, washed three times with fresh TAP, and resuspended the algal cells with fresh TAP to make the cell concentration 2×10 8 cells / ml. Pipette 300μl of Chlamydomonas suspension into a 15ml test tube, which contains 0.3g of sterilized glass beads, and add 5μg of the purified DNA of the linearized plasmid pSP124S into the test tube, and the mixture is shaken at the maximum on the WH-816 vortex shaker Intensity, 45-degree oblique vortex oscillation for 15-30 seconds. ...

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Abstract

The invention relates to a biological hydrogen production technology, in particular to a method for breeding high-yield photosynthetic-hydrogen-production chlamydomonas reinhardtii through cell nucleus insertion mutagenesis. The traditional chlamydomonas reinhardtii has low hydrogen production rate and limits the application and the development of a biological and photosynthetic hydrogen production technology. The method for obtaining a high-hydrogen-production mutant strain through the random insertion mutagenesis of chlamydomonas reinhardtii cell nuclei comprises the following steps: cultivation of the chlamydomonas reinhardtii; preparation of a chlamydomonas reinhardtii cell nucleus transformation vector and a glass bead; chlamydomonas reinhardtii cell nucleus transformation and transformant screening; analysis of the hydrogen yield and the oxygen consumption of chlamydomonas reinhardtii transformant hydrogen-production cultivation; analysis of the integration and the expression thereof of ble genes in a chlamydomonas reinhardtii mutant strain; and analysis of the influence of recombinant leghemoglobin hemH-1ba on the growth of the chlamydomonas reinhardtii. The invention has the advantages that the biological hydrogen production yield of the obtained high-hydrogen-production chlamydomonas reinhardtii is 7 times of the original yield; the method provides a good experimental material and a good basic condition for deeply researching the hydrogen-production metabolic mechanism of the chlamydomonas reinhardtii.

Description

technical field [0001] The invention relates to biological hydrogen production technology, in particular to a method for breeding high-yield photosynthetic hydrogen production Chlamydomonas reinhardtii by cell nucleus insertion mutagenesis. Background technique [0002] Biophotosynthetic hydrogen production is one of the important ways for the sustainable production of clean energy in the future. Many microgreen algae and cyanobacteria, including Chlamydomonas reinhardtii, can induce hydrogenase (H 2 ase) gene expression, hydrogenase is transported to the chloroplast, and the H produced in photosynthesis + and e - Synthetic H 2 , released outside the cell, is an ideal model for the sustainable production of hydrogen using solar energy. Chlamydomonas reinhardtii grows fast, has low cultivation cost and high hydrogenase activity, and is a microalgae photosynthetic hydrogen-producing alga species with great development potential. The low hydrogen production rate of Chlamydo...

Claims

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Application Information

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IPC IPC(8): C12N1/13C12P3/00C12Q1/68G01N21/31C12R1/89
Inventor 吴双秀王荣荣许丽丽黄瑞王全喜
Owner SHANGHAI NORMAL UNIVERSITY
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