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Method for screening chlamydomonas reinhardtii cycle adjustment and control defect mutants

A technology of rhinestones and mutants, applied in the field of microorganisms, can solve the problems of affecting proliferation and cycle disorder, and achieve the effect of short operation method, short time-consuming, easy to popularize and apply

Active Publication Date: 2020-05-01
XUZHOU NORMAL UNIVERSITY
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Problems solved by technology

[0003]Chlamydomonas cell cycle regulation involves the regulation of Chlamydomonas cilia deassembly, release of centrosomes, formation of spindles, and subsequent cell division. Defects in each link will cause cycle disturbance, thereby affecting its proliferation

Method used

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  • Method for screening chlamydomonas reinhardtii cycle adjustment and control defect mutants

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Embodiment 1

[0038] A. Prepare TAP liquid medium and TAP solid medium plate containing paro resistance, and set aside; B. Pick 21gr of Chlamydomonas reinhardtii in TAP liquid medium, under the temperature of 23±0.5℃, continuous light, 8000 Lx light intensity Under the conditions of air blowing culture for 3-4 days, cultivate Chlamydomonas cells to a concentration of 1-2×10 7 cells / mL, and then transferred to another TAP liquid medium so that the initial concentration was 1×10 6 cells / mL, cultured under continuous light for about 20 h, the final concentration can generally reach 5×10 6 C. Use the square wave electric shock method (BTX ECM830, set the parameters to 500 V, 4 ms, 6 bands, and the interval time is 100 ms), insert the anti-Paromyces vegetarian aphVIII Genes, which cause random site mutations, are spread on plates containing 10 μg / mLTAP + Paro, and cultured upside down for 7-10 days at a temperature of 23±0.5°C, a photoperiod (14 h during the day / 10 h at night), and a light int...

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Abstract

The invention discloses a method for screening chlamydomonas reinhardtii cycle adjustment and control defect mutants. The method comprises specific steps that after a chlamydomonas reinhardtii mutantlibrary is established, cell cycle adjustment and control defect mutants are directly screened, wherein primary screening is performed according to the size of the diameter of a monoclone, and in re-screening, the concentration of cells is detected through a microplate reader OD434, so that the ratio of a 24h light absorption value to a 48h light absorption value is calculated, and chlamydomonas cell cycle adjustment and control defect mutants are screened and identified reference to a wild type reproduction ratio. According to the method disclosed by the invention, changes of the quantity ofchlamydomonas reinhardtii cells are detected through the microplate reader, so that the chlamydomonas cell cycle adjustment and control defect mutants are screened and identified. The method is simpleand convenient to operate and high in efficiency, cell cycle adjustment and control defect mutants can be quickly and stably obtained, and a base is established for subsequent experiments.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a method for screening a cycle regulation defect mutant of Chlamydomonas reinhardtii. Background technique [0002] Chlamydomonas reinhardtii is a single-cell eukaryotic algae, characterized by rapid growth, short generation cycle, simple cell cycle regulation, easy to cultivate, can be cultured with TAP liquid medium, and can also grow monoclonal on a plate, etc. It was selected as a model organism and widely used to study photosynthesis, lipid metabolism, cilia movement, cell division and other processes. Previous studies have also proved that the important research value of Chlamydomonas is its simple way to obtain mutants. For example, in 1930, Moiwus was able to successfully isolate mutants of Chlamydomonas cells. Nowadays, it is possible to efficiently and quickly obtain a large number of mutants by means of electroporation, and use this as a basis to screen m...

Claims

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Application Information

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IPC IPC(8): C12N15/87C12N13/00C12N1/13C40B40/02C12R1/89
CPCC12N15/87C12N13/00C12N1/12C40B40/02Y02A40/80
Inventor 王亮温馨李春红王雯陆军郑元林
Owner XUZHOU NORMAL UNIVERSITY
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