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Construction method and applications of carrier with chlamydomonas endogenous gene knocked out and exogenous gene expressed

An endogenous gene and exogenous gene technology, which is applied to the construction and application of vectors for knocking out endogenous genes and expressing exogenous genes in Chlamydomonas, can solve the problems of not being widely used and low efficiency.

Active Publication Date: 2017-07-25
INST OF AQUATIC LIFE ACAD SINICA +1
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  • Abstract
  • Description
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Problems solved by technology

As with other model organisms, both forward and reverse genetics techniques are widely used in Chlamydomonas research, and the recent CRISPR / Cas9 gene editing technology has not been widely used due to its low efficiency

Method used

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  • Construction method and applications of carrier with chlamydomonas endogenous gene knocked out and exogenous gene expressed
  • Construction method and applications of carrier with chlamydomonas endogenous gene knocked out and exogenous gene expressed
  • Construction method and applications of carrier with chlamydomonas endogenous gene knocked out and exogenous gene expressed

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Embodiment Construction

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0021] Comparison of the conversion efficiency of two electrotransfer vectors

[0022] Reagent preparation:

[0023] 20% starch: weigh 4g of starch in a 50ml centrifuge tube, wash once with absolute ethanol, and centrifuge at 1000rpm / min for 2min. Pour off the supernatant, wash twice with ultrapure water, and centrifuge. Resuspend in 70% ethanol and make up to 20ml. Leave at room temperature.

[0024] Starch working solution: mix the above 20% starch upsid...

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Abstract

The invention provides a construction method and applications of a carrier with a chlamydomonas endogenous gene knocked out and an exogenous gene expressed. The carrier comprises a hygromycin resistance maker gene, wherein a splicing donor sequence (TCCATTTGCAG / GATGTTCGA) from the RubisCO gene, three termination codons in tandem duplication, and one transcription terminator are arranged at each of two ends of the maker gene. For the carrier, a 1.9kb fragment generated by Spel I and Cla I double digestion is adopted for carrying out electric shock for transforming chlamydomonas reinhardtii cells, an insertion carrier becomes one part of mRNA even if the carrier is inserted into the chlamydomonas gene intron, and genetic transcription stops at the three termination codons in tandem duplication and the transcription terminator. Compared with the 2.6kb AphVIII insertion carrier without SIS, the newly constructed carrier has the improved mutant transcription termination efficiency.

Description

technical field [0001] The invention relates to a carrier construction method and application for knocking out Chlamydomonas endogenous genes and expressing exogenous genes. Background technique [0002] Chlamydomonas reinhardtii is a model organism for studying plant photosynthesis, flagella cilia, and stress response. It is called "biological yeast" and a "biological factory" for producing antibodies and other drugs. Like other model organisms, both forward and reverse genetics techniques are widely used in Chlamydomonas research, and the recent CRISPR / Cas9 gene editing technology has not been widely used due to its low efficiency. Therefore, constructing a large-scale Chlamydomonas mutant library provides a method to obtain specific gene mutants of Chlamydomonas. [0003] A certain exogenous fragment is electrotransformed into Chlamydomonas cells, and the exogenous fragment will be randomly inserted into the Chlamydomonas genome, and mutants inserted into the target gene...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/66C12N1/13C12R1/89
CPCC12N15/66C12N15/79
Inventor 黄开耀邓璇王宇蕾
Owner INST OF AQUATIC LIFE ACAD SINICA
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