High-yielding lutein transgenic chlorella and preparation thereof
A kind of chlorella and transgenic technology, applied in the field of genetic engineering, can solve the problems of limited access to lutein, increased labor and land costs, etc.
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Embodiment 1
[0051] Embodiment 1, the construction of recombinant plasmid pCAMBIA2301-idi
[0052] 1. Cloning of Escherichia coli idi gene and construction of related vectors
[0053] 1. Cloning of Escherichia coli idi gene
[0054] Using Escherichia coli DH5α as material, the genome of Escherichia coli DH5α was extracted by phenol-chloroform method;
[0055] The details are as follows: take 500uL of DH5α bacterial liquid, and centrifuge at 12000r for 5min to obtain bacterial cells. Add 650uL of lysate, shake for 2min, and put in a 65°C water bath until the lysate becomes clear. Add an equal volume of balanced phenol: chloroform: isoamyl alcohol (25:24:1), shake and mix, and centrifuge at 13000r for 15min. Aspirate the supernatant and gently transfer it to a new tube, then extract once with an equal volume of chloroform:isoamyl alcohol (24:1), and centrifuge at 13000r for 15min. Aspirate the supernatant and move it to a new tube, add 1 / 10 volume of NaAc, and 2 times the volume of absolut...
Embodiment 2
[0089] Embodiment 2, the acquisition of the trans idi Chlorella of high-yielding lutein
[0090] 1. Obtaining trans idi chlorella
[0091] The transformation of Chlorella was mediated by Agrobacterium as follows:
[0092] (1) Preparation of Chlorella protoplasts
[0093] Chlorella STIO02 was deposited in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on November 30, 2012, and the preservation number is CGMCC No. .6951, with the proposed taxonomic designation Chlorella vulgaris.
[0094] A single algal colony of Chlorella STIO02 (CGMCC 6951; also known as wild-type Chlorella) was picked and inoculated in FC medium, and cultured on a shaking table at 28°C until the algae grew out.
[0095] Transfer to 50mL fresh FC medium with a 1% inoculum amount, and culture on a shaker at 28°C until logarithmic phase.
[0096] Collect the algal cells...
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