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Method for obtaining desmodesmus NMX451 and genetic transformation method of desmodesmus NMX451

A genetic transformation method and technology of algae chains, which are applied in the field of screening and genetic transformation of oleaginous microalgae, can solve the problems that the biomass and oil production are difficult to achieve industrial production, and the oleaginous microalgae are difficult to adapt to open culture, etc. , to achieve the effect of reducing costs and preventing damage

Active Publication Date: 2014-02-12
INST OF AQUATIC LIFE ACAD SINICA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some microalgae, such as salina and spirulina, have been able to carry out factory culture and production, while most oil-producing microalgae are difficult to adapt to open culture, and some oil-producing microalgae can adapt to open culture. However, it is difficult to meet the needs of industrialized production, and it is necessary to further optimize the culture conditions, or properly carry out genetic modification to increase production, so as to reduce costs

Method used

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  • Method for obtaining desmodesmus NMX451 and genetic transformation method of desmodesmus NMX451
  • Method for obtaining desmodesmus NMX451 and genetic transformation method of desmodesmus NMX451
  • Method for obtaining desmodesmus NMX451 and genetic transformation method of desmodesmus NMX451

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Effect test

example 1

[0053] In water area 1m 2 In the runway pool, 100L of BG11 medium containing high-concentration sodium bicarbonate (12.6g / L) was injected, and inoculated with algae strain NMX451 ( Figure 5 ), the water surface light intensity is 80μE / m 2 ·s, during the cultivation period, the temperature is maintained between 25-27°C. Cultured to the optical density value (OD 750 ) was 1.8, the algae liquid was harvested by flocculation and centrifugation; the flocculation system was: pH 7, CuSO 4 The concentration of chitosan is 18.75μM, and the concentration of chitosan is 7.5μg / L; 20L of algae liquid is harvested the next day, and 20L of high-alkaline BG11 medium is added to it. The volume remained at about 100L; the harvest lasted for one month, and a total of 16 batches of algae liquid were collected; among the collected batches, the wet and dry weights of algal cells did not change significantly (Table 3). Add 4L of chloroform / methanol (1:1) for every 100g of dry algae powder to ex...

Embodiment 2

[0057] ① Inoculate the algae cells into a 250ml Erlenmeyer flask and culture it to the logarithmic phase (growth about 6 days). 2 s) Count the cells under a microscope, collect the algal cells by centrifugation at 6000rpm, wash twice with sterilized 375mM sorbitol, resuspend, and concentrate the cells to 5×10 9 pcs / ml, dispense into sterilized 1.5ml microcentrifuge tubes, 100μl per tube, put on ice for use; extract plasmid pHB4785 by alkaline lysis, purify with 13% PEG8000, dissolve in sterilized ddH 2 O, cut into a linear shape with PstI, after detecting the concentration on a Biophotometer spectrophotometer, mix it with algae cells at 5 μg per tube for later use.

[0058] ②Use Bio-Rad's electric pulse instrument for electrotransfer. The diameter of the electric shock cup is 1mm. The electrotransfer conditions are: voltage 1.2KV, resistance 500Ω, capacitance 50μF. Transfer each 100μL of cells after electric pulse to 10ml of fresh culture in advance. In the sterilized tube of...

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Abstract

The invention discloses a method for obtaining desmodesmus NMX451 and a genetic transformation method of the desmodesmus NMX451 and relates to screening of oil-producing microalgae and a genetic transformation technology thereof. Green alga separated from a wild environment is subjected to 18S rDNA (ribosomal deoxyribonucleic acid) sequence analysis and morphological identification and is determined to be a desmodesmus member, and a sequence of the green alga is SEQ NO.1. The genetic transformation method of the green alga comprises the following steps: (1) constructing a nuclear gene transformation plasmid with a resistance gene; (2) transforming the desmodesmus NMX451 by adopting an electrotransfer method; (3) detecting whether a transformant is positive or nor through PCR (polymerase chain reaction); (4) proving that an exogenous gene enters host DNA (deoxyribonucleic acid) through DNA blot hybridization. The desmodesmus NMX451 can tolerate high-concentration sodium bicarbonate, and oil content can be 27.63% of dry cell weight; algae cells of the desmodesmus NMX451 also contain high proportion of linolenic acid; by adopting the method for obtaining the desmodesmus NMX451, the exogenous gene can be introduced into the desmodesmus NMX451, so that the desmodesmus NMX451 can obtain resistance to zeocin and also can express a protein with an economic value.

Description

technical field [0001] The invention relates to the screening of oil-producing microalgae and its genetic transformation technology, in particular to a method for obtaining and genetic transformation of the algae NMX451. [0002] Specifically, the present invention collects water samples from the wild, obtains an oil-producing algae strain capable of open culture—Centronella NMX451 through separation and purification, and uses gene recombination to construct a strain with the expression For the plasmid of the resistance gene, the DNA is transferred into the cells of Zonaria cerevisiae by means of electric pulse, and the transformant is obtained through zeocin resistance screening, in which the plasmid with the resistance fragment or foreign gene is integrated into the host genome. Background technique [0003] Microalgae are a large class of lower plants, many of which are single-celled, can perform photosynthesis, fix carbon dioxide, release oxygen, and synthesize oil or po...

Claims

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Application Information

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IPC IPC(8): C12N1/12C12N15/79C12N1/13C12R1/89
Inventor 孔任秋徐旭东余桂兰高宏方仙桃
Owner INST OF AQUATIC LIFE ACAD SINICA
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