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Exogenous gene expression system of Chlamydomonasreinhardtii and method for constructing and producing PHB transgenic algae

An exogenous gene and expression system technology, applied in the field of constructing and producing poly-β-hydroxybutyric acid transgenic algae, can solve the problems of inability to chemically synthesize plastics, difficulty in industrial production of PHB, infertility of transgenic plants, etc., and achieve fast reproduction speed. , the effect of easy separation and extraction, and easy fixed-point integration

Active Publication Date: 2006-07-26
SHENZHEN UNIV
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Problems solved by technology

[0011] Although the technology of bacterial fermentation to produce PHB is relatively mature, it is difficult to reduce the price due to the need for expensive fermentation substrates and energy consumption for bacterial culture, and it cannot compete with the current cheap chemically synthesized plastics.
The use of transgenic plants to produce PHB also has problems such as long growth cycle, large space occupation, and infertility of transgenic plants, which brings great difficulties to the realization of industrialized production of PHB.

Method used

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  • Exogenous gene expression system of Chlamydomonasreinhardtii and method for constructing and producing PHB transgenic algae
  • Exogenous gene expression system of Chlamydomonasreinhardtii and method for constructing and producing PHB transgenic algae
  • Exogenous gene expression system of Chlamydomonasreinhardtii and method for constructing and producing PHB transgenic algae

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Experimental program
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Embodiment 1

[0043] Embodiment 1: Selection and cultivation of transgenic recipient algae

[0044] The transgenic acceptor used in the present invention is Chlamydomonas reinhardtii cc-849 (Chlamydomonas reinhardtii cc-849) (purchased from the Chlamydomonas Genetic Center of Duke University, Durham, NC 27708USA) as the acceptor for transgenic manipulation, and the strain is cell wall defective type Chlamydomonas reinhardtii strains.

[0045] The culture medium used when Chlamydomonas reinhardtii is cultivated is TAP medium, and the composition of 1L medium is as follows: Tris 2.42g, 4 times of Beijerinck salts (containing 16g NH per liter 4 Cl, 2g CaCl 2 2H 2 O, 4gMgSO 4 ·7H 2 O) 25mL, 1M potassium phosphate buffer 1mL, trace element mixture (containing 11.4g H per liter 3 BO 3 , 22g ZnSO 4 ·7H 2 O, 5.06g MnCl 2 4H 2 O, 4.99 g 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 2 O, 50gNa 2 EDTA) 1mL, glacial acetic acid 1mL, H 2 O 975...

Embodiment 2

[0047] Example 2: Isolation of key enzyme genes for PHB biosynthesis and construction of expression vectors

[0048] (1) Cloning of the key enzyme gene of PHB biosynthesis: the chromosomal DNA of Alcaligenes eutropha H16 was extracted (see: Ye Liang, Li Cong et al., Science Bulletin, cloning, sequence analysis and analysis of 3-ketothiolyase gene phbA Functional Detection, 1999, 44(4):398-402), as a template for PCR amplification.

[0049] Primers were designed according to the published phbA gene sequence (GenBank accession number: J04987), PrA1: 5-CACCATGACTTACGTTGTC-3′; PrA2: 5GAAGAGCTCTTCCTTTATTT-3′. PCR program: Denaturation at 95°C for 1 minute, renaturation at 53°C for 1 minute, extension at 74°C for 90 seconds, 35 cycles. Experimental results A 1.2kb specific fragment was amplified, which was digested with SacI and EcoRV and then ligated into the SacI and EcoRV sites of pBluescript SK+ to obtain the pSKA-1 plasmid.

[0050] Primers were designed according to the phbB...

Embodiment 3

[0079] Example 3: Genetic transformation of Chlamydomonas reinhardtii

[0080] (1) Transformation of Chlamydomonas reinhardtii by "bead milling method"

[0081] Chlamydomonas reinhardtii CC-849 was cultured to the logarithmic phase in TAP medium, and the number of cells was about 1-2×10 6 Cells / mL, collected by centrifugation at room temperature (20-25°C), resuspended with TAP solution, and adjusted the cell concentration to 2×10 8 cells / mL. Pipette 300 μL of the suspension into a 5 mL test tube (containing sterilized quartz sand), cut the plasmid containing the exogenous gene into a line (including pH105A124, p105B124 and pH105C124), add 1 μg-2 μg to the test tube, CC- The 849 / quartz sand / exogenous DNA mixture was shaken rapidly for 15 seconds. Transfer the mixture to a 25mL centrifuge tube with a screw cap, add 10mL sterilized TAP culture solution, and incubate overnight at 25°C on a full-temperature shaking shaker at 60 rpm (the light condition is 90μE / m 2 / s). Collect...

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Abstract

The invention discloses an expression system and constructing PHB transgene algae method of rhine chlamydomonas exogenesis gene, which comprises the following parts: promotor, exogenesis-goal gene, screening badge and rhine chlamydomonas acceptor algae strain, wherein the method comprises the following: A, selecting and cultivating acceptor algae strain; B, cloning key enzyme gene by PHB biosynthesizing; C, expressing carrier of PHB biosynthesizing key enzyme rhine chlamydomonas; D, using PHB biosynthesizing key enzyme to lead in rhine chlamydomonas from fungus Bacillus alcaligenes with bead-milling method, gene-gun method or electrization; obtaining bivalence or transgene Chlamydomonas of producing PHB by screen selecting and molecule detecting. The poly-hydroxybutyric acid is compounded by acting in conjunction of PHB compounding key enzyme of phbA, phbB and phbC gene coding, which uses acetylcoenzyme A as substrate by photosynthesis. The method simplifies the operating, which reduces cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to a Chlamydomonas reinhardtii exogenous gene expression system, and also relates to a method for constructing and producing poly-β-hydroxybutyric acid (PHB) transgenic algae. Background technique [0002] With the continuous development of society, plastic products have become a necessity of human social life. Due to the slow photodegradation and biodegradation of chemically synthesized plastics in the natural environment, 14 The results of C isotope tracking and investigation of the degradation of plastics in soil show that the decomposition rate of plastics varies with environmental conditions (such as rainfall, air permeability, temperature, etc.), but overall, the decomposition rate is very slow, and it is generally believed that it takes 200 -400 years, the problem of "white pollution" caused by its plastic waste has become a growing environmental problem. The development of biodegrad...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N1/12C12P7/42
Inventor 胡章立王潮岗宋艳茹
Owner SHENZHEN UNIV
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