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Transgenic regulation and control miRNA (micro ribonucleic acid) gene algae capable of realizing continuous photosynthetic hydrogen production and creation method of gene algae

A construction method and gene technology, which are applied in the field of trans-regulated miRNA gene algae and their construction, can solve the problems of no industrial application prospect, decreased growth and hydrogen release capacity, difficulty in separating green algae from culture medium, etc.

Inactive Publication Date: 2014-02-12
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the difficulty in separating the green algae from the medium, the growth and hydrogen release capacity of the algae will be greatly reduced after the algae are immobilized, so this method of hydrogen production has no industrial application prospects.

Method used

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  • Transgenic regulation and control miRNA (micro ribonucleic acid) gene algae capable of realizing continuous photosynthetic hydrogen production and creation method of gene algae
  • Transgenic regulation and control miRNA (micro ribonucleic acid) gene algae capable of realizing continuous photosynthetic hydrogen production and creation method of gene algae
  • Transgenic regulation and control miRNA (micro ribonucleic acid) gene algae capable of realizing continuous photosynthetic hydrogen production and creation method of gene algae

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Selection and cultivation of embodiment 1 transgenic recipient algae strain

[0062] The recipients of the transgenic operation selected in the present invention are cell wall-deficient Chlamydomonas reinhardtii (Chlamydomonas reinhardtii, cc-849, purchased from the Chlamydomonas Genetic Center of Duck University, USA) and cytochrome b-deficient Chlamydomonas reinhardtii (cc-2654, purchased from Chlamydomonas Genetics Center, Duck University, USA).

[0063] The culture medium used when the above-mentioned Chlamydomonas reinhardtii was cultivated was TAP medium, and the components of the TAP medium were as follows: Tris2.42g, 4× Beijerinck salts (containing 16g NH per liter 4 Cl, 2g CaCl 2 .2H 2 O,4g MgSO 4 .7H 2 O) 25mL, 1M potassium phosphate buffer 1mL, Trace trace element mixture (containing 11.4g H per liter 3 BO 3 ,5.6g MnCl 2 .4H 2 O,22g ZnSO 4 .7H 2 O, 4.99g FeSO 4 .7H 2 O, 1.61g CoCl 2 .6H 2 O, 1.57g CuSO 4 .5H 2 O,1.1g(NH 4 ) 6 Mo 7 o 24 .4H...

Embodiment 2

[0065] Example 2 Screening or designing regulation miRNAs

[0066] (1) Screening of intracellular miRNAs

[0067] After Chlamydomonas reinhardtii was cultured under sulfur deficiency stress, its hydrogen production increased, the expression of its miRNAs was detected, the miRNAs whose expression changed were counted, and a small RNA library was established (BMC Genomics, 2012, 13:108), and It was found that the expression of Chlamydomonas miRNAs in the established small RNA library changed significantly (more than 2 times), and the expression of most miRNAs was up-regulated, and some predicted target genes were screened from the established small RNA library. They are miRNAs related to photosystem II and whose expression levels are significantly upregulated, such as miRNA909.1, miR1150.3, miR1166.1, miR1158. Figure 9 For the selected miRNAs and their predicted target genes. Here, it is preferable to select miR1150.3 and miR1166.1 for the next step to construct the framework...

Embodiment 3

[0071] Embodiment 3 regulates the construction and synthesis of miRNAs expression backbone

[0072] When miRNAs in green algae are expressed in cells, they first transcribe precursors containing a backbone structure. Therefore, when overexpressing intracellular miRNAs, it is also necessary to select an expression backbone for the designed "regulatory miRNA". In this example, the natural cre-MIR1162MI0006223 with high expression abundance in Chlamydomonas reinhardtii was selected as the backbone precursor of the miRNAs to be screened. The mature sequences of intracellular regulatory miRNAs screened from the small RNA library were replaced with the mature sequences of miRNAs in cre-MIR1162MI0006223 to obtain a complete framework for regulating the expression of miRNAs.

[0073] In this example, miR1150.3 and miR1166.1 were screened from the library for research, and other miRNAs in the library could be constructed and synthesized according to this method. The mature sequences o...

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Abstract

The invention discloses transgenic regulation and control miRNA (micro ribonucleic acid) gene algae capable of realizing continuous photosynthetic hydrogen production. Regulation and control miRNA is screened from a green algae miRNAs library or is designed for a green algae functional gene affecting photosynthetic efficiency, transgenic algae capable of inducing the expression of the regulation and control miRNA through such manners as light intensity or temperature and the like is created, and the activity of a green algae photosynthetic system II is indirectly regulated and controlled by means of inhibition of the miRNAs on a target gene to control the hypoxia environment in algae cells so as to enable the green algae cells to continuously produce hydrogen; a culture medium does not need to be alternately changed in the hydrogen production process of the transgenic regulation and control miRNA gene algae created by the method, and thus the defect that the culture medium needs to be alternately changed to realize the continuous hydrogen production of the existing green algae to cause great reduction on the growth and hydrogen production ability of the culture medium is overcome, and the transgenic regulation and control miRNA gene algae has an industrial application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a sustainable photosynthetic hydrogen production transregulated miRNA gene algae and a construction method thereof. Background technique [0002] In the 1940s, Gafforn and his collaborators first discovered that Scenedesmus uses its reversible hydrogenase, which can absorb hydrogen to fix carbon dioxide under anaerobic conditions and release hydrogen under light conditions [Gaffron H, Rubin J. Fermentative and photochemical production of hydrogen in algae. J. Gen. Physiol., 1942(26): 219-240.]. Subsequently, photosynthetic hydrogen desorption was found in many green algae (such as Chlamydomonas reinhardtii, Chlorella, etc.). Because these green algae can obtain hydrogen energy from the most abundant resources in nature (light and water), and the hydrogenase activity in green algae is more than 100 times that of cyanobacteria and photosynthetic bacteria, the International Energy Agency...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N1/13C12R1/89
Inventor 胡章立李辉庄晓珊张立舒龙飞
Owner SHENZHEN UNIV
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