Method for improving yield of chlamydomonas reinhardtii biological hydrogen production through soybean hemoglobin genes

A technology of hemoglobin and biological hydrogen production, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., can solve the problems of affecting hydrogen production efficiency, affecting Chlamydomonas hydrogen production efficiency, growth inhibition of Chlamydomonas, etc. question

Inactive Publication Date: 2010-09-29
SHANGHAI NORMAL UNIVERSITY
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Problems solved by technology

The current way to reduce the oxygen content in Chlamydomonas cells is to inhibit the activity of photosynthetic system II (PS II), thereby inhibiting the release of oxygen from photolyzed water. Hydrogen production efficiency; on the other hand, the growth of Chlamydomonas was severely inhibited under anoxic conditions, which ultimately affected the hydrogen production efficiency

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  • Method for improving yield of chlamydomonas reinhardtii biological hydrogen production through soybean hemoglobin genes
  • Method for improving yield of chlamydomonas reinhardtii biological hydrogen production through soybean hemoglobin genes
  • Method for improving yield of chlamydomonas reinhardtii biological hydrogen production through soybean hemoglobin genes

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Embodiment 1

[0068] Chlamydomonas reinhardtii and its cultivation:

[0069] Chlamydomonas reinhardtii is a representative species of photosynthetic hydrogen production by microalgae because of its fast growth rate, low culture cost, and high hydrogenase activity. In order to facilitate transgene manipulation, the present invention prefers Chlamydomonas reinhardtii species cc849 with a cell wall deficiency.

[0070]①Normal culture conditions: 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, the intensity of sunlight (100-200mol photonsm -2 the s -1 ), the liquid culture is at 50~100ml Tris-Acetate-Phosphate (abbreviated as TAP) medium, initial pH7.2, horizontal shaker rotating speed 100~130rpm, every 5~6 days 1% inoculation subculture; Solid TAP (plate ) medium contains 1.5% agar powder, and the preservation and purification of the algae species is to pick a single clone...

Embodiment 2

[0074] Cloning of leghemoglobin subunit lba gene:

[0075] By standard methods known to those skilled in the art (Sambrook et al., Molecular Cloning. New York: Cold Spring Harbor Laboratory Press. 1998) and the manufacturer's instructions for the reagents used (QIAGEN TM , Promega TM ) to extract the total RNA and synthesize cDNA of soybean, and to detect the concentration and purity of the RNA. The specific implementation method is: take 0.1 g of soybean mature root nodules, grind them with liquid nitrogen, extract the total RNA of soybeans according to the QIAGEN RNeasy Plant Mini Kit reagent manufacturer's instructions, and detect its concentration and purity by ultraviolet spectrophotometry. with DNase I Water bath at 37°C for 30 minutes to remove the mixed DNA in the RNA; then add 25mM EDTA and bathe in water at 65°C for 10 minutes to terminate the activity of DNase I. Single-stranded cDNA was synthesized according to Promega TM AMVRT protocol manufacturer’s instruc...

Embodiment 3

[0080] Construction of Chlamydomonas reinhardtii chloroplast expression vector:

[0081] The Chlamydomonas reinhardtii chloroplast expression vector cg40 was 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 Vaistij et al. (The Plant Journal, 2000, 21 (5): 469-482) for vector construction. The specific implementation method is: use restriction endonucleases SmalI and SacI to digest the soybean Iba gene fragment and the carrier cg40 obtained by the above-mentioned clone respectively, and the purified Iba gene fragment is connected with T4DNA ligase through the above two enzyme cutting sites. After the aadA gene in the plasmid cg40 forms the aadA-lba fusion gene, the Chlamydomonas chloroplast expression vector cg401-1-lba ( figure 1 ), transformed Escherichia coli DH5α, extracted the plasmid, and identified by restriction endonuclease SmaI and SacI double digestion, obtai...

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Abstract

The invention belongs to a biological hydrogen production technology, particularly to a method for improving the yield of chlamydomonas reinhardtii biological hydrogen production through soybean hemoglobin genes. Traditional chlamydomonas reinhardtii hydrogen production has the disadvantages that chlamydomonas reinhardtii hydrogenase is sensitive to oxygen and is easily inhabited by the oxygen to inactivate; and the hydrogen production effect of chlamydomonas is limited. The invention discloses application of soybean hemoglobin globulin subunit genes in chlamydomonas reinhardtii hydrogen production, the soybean hemoglobin globulin subunit genes lba are constructed in a chlamydomonas reinhardtii chloroplast expression vector, and the expression vector is transformed into chlamydomonas reinhardtii chloroplast, so that the lba genes are expressed in the chlamydomonas reinhardtii chloroplast. The decrease of the oxygen content in a closed culture system of the transformed chlamydomonas reinhardtii is markedly quicker than that of the chlamydomonas reinhardtii of the untransformed genes, the oxygen content can be kept at a lower level, and the hydrogen yield is markedly increased; the method is applicable for all microalgae for hydrogen production.

Description

technical field [0001] The invention belongs to biological hydrogen production technology, in particular to a method for improving the production of biological hydrogen production of Chlamydomonas reinhardtii by soybean hemoglobin gene. Background technique [0002] Energy shortage and environmental pollution have become two major problems facing the development of contemporary human society, and the development and utilization of clean energy is imminent. Hydrogen has a high energy density, only water is produced after combustion, and CO with greenhouse effect is not produced 2 And other toxic gases, no pollution to the environment, is an ideal clean energy with great development potential. The method for preparing hydrogen in the prior art is to prepare hydrogen through water electrolysis, but this method consumes electric energy, and actually exchanges electric energy for hydrogen energy. Biological hydrogen production is one of the important ways to solve the problem o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P3/00C12N15/29C12N15/63C12N1/13C12R1/89
Inventor 吴双秀阎光宇许丽丽黄瑞王荣荣王全喜
Owner SHANGHAI NORMAL UNIVERSITY
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