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Detecting method for construction of genetic transformation system of phaeodactylum tricomutum

A technology of genetic transformation system and detection method, which is applied in the rapid detection field of microalgae detection and Phaeodactylum tricornutum genetic transformation system construction.

Inactive Publication Date: 2012-03-21
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the establishment of the transformation system of Phaeodactylum tricornutum needs to extract genomic DNA and detect it by PCR or Southern blotting. These two methods are cumbersome to operate and not easy to master

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] (1) GFP gene recombinant plasmid construction, extraction of recombinant plasmid for Phaeodactylum tricornutum transformation;

[0015] (2) Gene gun transformation, resuspend the algal cells harvested by centrifugation with fresh medium, take about 5 × 10 7 One algae cell was spread on the center of a solid plate with a f / 2 diameter of about 2-2.5 cm 1 hour before the bombardment, and 10 μL of microparticle bullets (coated with 1.0 μg of plasmid DNA) were placed in the center of the DNA slide, and dried with sterile air; The plate was placed on the second layer of the gene gun for bombardment; after the bombarded algal cells were cultured under light for 24-48 hours, the algal cells were washed with 500 μL of fresh medium and reapplied in a medium containing 100 μg·mL -1 On the solid medium of bleomycin (Zeocin) grow single algal colony;

[0016] (3) Observation with a fluorescence microscope, picking a single algal drop containing 100 μg·mL -1 After 7 days of culture...

Embodiment 2

[0020] (1) GFP gene recombinant plasmid construction, extraction of recombinant plasmid for Phaeodactylum tricornutum transformation;

[0021] (2) Gene gun transformation, resuspend the algal cells harvested by centrifugation with fresh medium, take about 5 × 10 7 One algae cell was spread on the center of a solid plate with a f / 2 diameter of about 2-2.5 cm 1 hour before the bombardment, and 10 μL of microparticle bullets (coated with 1.0 μg of plasmid DNA) were placed in the center of the DNA slide, and dried with sterile air; The plate was placed on the second layer of the gene gun for bombardment; after the bombarded algal cells were cultured under light for 36 hours, the algal cells were washed with 500 μL of fresh medium and reapplied in a medium containing 100 μg·mL -1 On the solid medium of bleomycin (Zeocin) grow single algal colony;

[0022] (3) Observation with a fluorescence microscope, picking a single algal drop containing 100 μg·mL -1 After 7 days of culture in...

Embodiment 3

[0026] (1) GFP gene recombinant plasmid construction, extraction of recombinant plasmid for Phaeodactylum tricornutum transformation;

[0027] (2) Gene gun transformation, resuspend the algal cells harvested by centrifugation with fresh medium, take about 5 × 10 7 One algae cell was spread on the center of a solid plate with a f / 2 diameter of about 2-2.5 cm 1 hour before the bombardment, and 10 μL of microparticle bullets (coated with 1.0 μg of plasmid DNA) were placed in the center of the DNA slide, and dried with sterile air; The plate was placed on the second layer of the gene gun for bombardment; after the bombarded algal cells were cultured under light for 48 hours, the algal cells were washed with 500 μL of fresh medium and reapplied in a medium containing 100 μg·mL -1 On the solid medium of bleomycin (Zeocin) grow single algal colony;

[0028] (3) Observation with a fluorescence microscope, picking a single algal drop containing 100 μg·mL -1 After 7 days of culture in...

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Abstract

The invention provides a detecting method for construction of a genetic transformation system of phaeodactylum tricomutum. The detecting method is characterized by comprising the following steps of: (1) construction of GFP gene recombinant plasmid, and extraction of the recombinant plasmid for carrying out transformation on the phaeodactylum tricomutum; (2) transformation by a gene gun; and (3) observation of a fluorescence microscope. The detecting method has the advantages of being simple, fast, easy to implement, economic and applicable.

Description

technical field [0001] The invention relates to a detection method for microalgae, in particular to a rapid detection method for constructing a genetic transformation system of Phaeodactylum tricornutum, belonging to the technical field of microalgae molecular biology. technical background [0002] The establishment of a genetic transformation system is the basis for realizing the expression of foreign genes in the host, and it is also the most direct platform for studying gene functions and regulating gene expression. Among algae, the transformation system of Chlamydomonas reinhardtii is quite mature, and it is currently the only biological material that has established three sets of genetic transformation of nucleus, chloroplast and mitochondria. The functions of many endogenous genes have been directly verified through its transformation system. Eukaryotic microalgae such as Volvox, Chlorella, and Salina have also been successfully transformed. Due to the siliceous shell...

Claims

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Application Information

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IPC IPC(8): C12Q1/02
Inventor 朱葆华陈常杰潘克厚刘冬梅杨官品
Owner OCEAN UNIV OF CHINA
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