Method for improving chlamydomonas reinhardtii hydrogen production amount of leghemoglobin ferrous chelate enzyme gene

A ferrochelatase and hemoglobin technology, applied in unicellular algae, introduction of foreign genetic material using a carrier, recombinant DNA technology, etc. question

Inactive Publication Date: 2010-07-14
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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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) and then inhibit the release of oxygen from photolyzed water. However, this method also inhibits the electron transfer efficiency of the photosynthetic chain and affects the production of Chlamydomonas Hydrogen efficiency, on the other hand, the growth of Chlamydomonas is also severely inhibited under anoxic conditions, which ultimately affects hydrogen production efficiency

Method used

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  • Method for improving chlamydomonas reinhardtii hydrogen production amount of leghemoglobin ferrous chelate enzyme gene
  • Method for improving chlamydomonas reinhardtii hydrogen production amount of leghemoglobin ferrous chelate enzyme gene
  • Method for improving chlamydomonas reinhardtii hydrogen production amount of leghemoglobin ferrous chelate enzyme gene

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Embodiment 1

[0078] Chlamydomonas reinhardtii and Bradyrhizobium soybean and their culture:

[0079] 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.

[0080] 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, the optimal condition is 25±1°C, and the intensity of sunlight (100-200 μmolphotons m -2 the s -1 ); liquid culture is at 50~100ml Tris-Acetate-Phosphate (TAP) medium, initial pH7.2, horizontal shaking table rotating speed 100~130rpm, every 5~6 days 1% inoculation subculture; Solid plate culture medium ( TAP) contains 1.5% agar powder. The preservation...

Embodiment 2

[0085] Cloning of leghemoglobin hemH gene and lba gene:

[0086] 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 kit used (QIAGEN TM , Promega TM ) to extract the total DNA of the bradyrhizobium soybean and the total RNA of the soybean, and then detect the concentration and purity of the total soybean RNA, and reverse transcribe it into cDNA. Specifically: when the OD of the bradyrhizobium solution 600 When the temperature is 0.6-0.8, take 10ml of bacterial solution, centrifuge at 10000rpm at 4°C for 15min, collect the bacterial cells, lyse the bacteria with 400μl TE, add 40μl of 10% SDS and Proteinase K (20mg / ml), then bathe in water at 56°C for 45min, add 400μl of phenol, 4 Centrifuge at 10,000 rpm for 10 min at ℃, extract the supernatant twice with phenol / chloroform / isoamyl alcohol (25:24:1) and once with chloroform, precipitate the t...

Embodiment 3

[0095] Construction of Chlamydomonas reinhardtii chloroplast expression vector:

[0096] 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. Respectively use restriction endonucleases SmalI and SacI to digest the soybean lba gene fragment obtained by the above clone and the vector cg40, and connect the purified lba gene fragment to the plasmid cg40 with T4 DNA ligase through the above two enzyme cutting sites After the aadA gene, the aadA-lba fusion gene is formed to obtain the vector cg401-1-lba, and then the hemH gene is connected to the plasmid cg401-1-lba after the aadA gene and before the lba gene by using the SacII restriction site with T4 DNA ligase , form aadA-hemH-lba fusion protein, and construct Chlamydomonas ch...

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Abstract

The invention relates to a biological hydrogen production technology, in particular to a method for improving the chlamydomonas reinhardtii hydrogen production amount of a leghemoglobin ferrous chelate enzyme gene. The traditional chlamydomonas reinhardtii hydrogen production method has the defects that chlamydomonas reinhardtii hydrogen enzymes are sensitive to oxygen and easily restricted by the oxygen to lose activity, and the chlamydomonas reinhardtii hydrogen production effect is limited. The invention discloses an application of a leghemoglobin ferrous chelate enzyme gene and a globulin subunit gene to hydrogen production by constructing the leghemoglobin ferrous chelate enzyme gene hemH and the globulin subunit gene lba in a chlamydomonas reinhardtii chloroplast expression vector, transferring the expression vector in the chlamydomonas reinhardtii chloroplast and expressing the hemH-lba gene in the chlamydomonas reinhardtii chloroplast. The invention has the advantages that the content of oxygen in a closed culture system of the transformed chlamydomonas reinhardtii is reduced obviously faster than that of the chlamydomonas reinhardtii of an un-transformed gene, the oxygen content is kept at a lower level, and the hydrogen production amount is obviously increased.

Description

technical field [0001] The invention relates to biological hydrogen production technology, in particular to a method for increasing the yield of hydrogen production by Chlamydomonas reinhardtii with leghemoglobin ferrochelatase gene. Background technique [0002] Hydrogen production by photosynthetic biology 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 intracellular hydrogenase (H 2 ase) gene expression, the H produced in photosynthesis + and e - Synthetic H 2 , released to the outside of the cell, biohydrogen production is an ideal model for hydrogen production 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. However, hydrogenase is extremely sensitive to oxygen and is easil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N1/13C12P3/00
Inventor 吴双秀黄瑞许丽丽王荣荣王全喜
Owner SHANGHAI NORMAL UNIVERSITY
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