Application of chlamydomonas reinhardtii RFC1 gene in regulation of cadmium tolerance of chlamydomonas reinhardtii

A technology for the resistance of Chlamydomonas reinhardtii, which is applied in the application field of Chlamydomonas reinhardtii RFC1 gene in regulating the cadmium tolerance of Chlamydomonas reinhardtii, can solve the problems of complex operation and high cost, and achieve simple methods, significant resistance to heavy metal cadmium, The effect of mature technology system

Inactive Publication Date: 2018-03-23
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In comparison, traditional physical and chemical methods often have the characteristics of high cost and complicat

Method used

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  • Application of chlamydomonas reinhardtii RFC1 gene in regulation of cadmium tolerance of chlamydomonas reinhardtii
  • Application of chlamydomonas reinhardtii RFC1 gene in regulation of cadmium tolerance of chlamydomonas reinhardtii
  • Application of chlamydomonas reinhardtii RFC1 gene in regulation of cadmium tolerance of chlamydomonas reinhardtii

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Screening of Chlamydomonas reinhardtii heavy metal cadmium-resistant mutant algal beads

[0035] (1) Preparation for inserting the target fragment

[0036] First extract the PJMG-aphⅧ plasmid (Zhangfeng Hu, Yinwen Liang, Wei He, Junmin Pan * , CiliaDisassembly with Two Distinct Phases of Regulation, Cell Reports, 2015, 10(11): 1803-1810; containing aphⅧ fragment, the sequence is shown in SEQ ID NO: 3), and then digested with EcoRI and cutsmartBuffer, the digested product Agarose gel electrophoresis, the target fragment is cut and recovered, such as figure 1 As shown, the band indicated by the arrow is the target fragment that needs to be recovered. Gel recovery mainly uses Sangon’s Gel Recovery Kit (article number: B518191-0100). According to the weight of the gel strip, add Buffer B2 3-6 times the weight of the gel strip, and use 20 μL of Elution Buffer to elute the DNA fragments. Around 10ng / μL.

[0037] (2) Preparation of Chlamydomonas Competent Cells ...

Embodiment 2

[0043] Example 2: Identification of Mutant Genes of Chlamydomonas reinhardtii Heavy Metal Cadmium-Resistant Mutant Algal Strain

[0044] (1) Extraction of mutant genomic DNA

[0045] a: Put the heavy metal cadmium-resistant mutant TC8 screened above into the liquid medium with a pipette tip. After the cells grow to the logarithmic phase, collect the cells into a 1.5mL EP tube, centrifuge at 14,000rpm at room temperature for 1min, and discard. The precipitate was frozen with liquid nitrogen and stored at -80°C for future use.

[0046] b: Take out the cells collected above with liquid nitrogen, add 700 μL of preheated CTAB lysate, and put them in a water bath at 65°C for about one hour, during which time they are inverted and mixed several times to fully lyse the cells.

[0047] c: After the cells are fully lysed, add 700 μL of a mixed solution of phenol: chloroform: isoamyl alcohol = 25:24:1 to the EP tube, shake the EP tube, mix well, and centrifuge at 14000 rpm for 10 min. ...

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Abstract

The invention discloses application of a chlamydomonas reinhardtii RFC1 gene in the regulation of cadmium tolerance of chlamydomonas reinhardtii and belongs to the fields of biotechnology and environment pollution control. A chlamydomonas reinhardtii mutant strain capable of resisting cadmium chloride obviously is acquired by randomly inserting an aphVIII segment (SEQ ID NO: 3) containing a paromomycin resistance gene into chlamydomonas, and further identification discovers that the cadmium resistance of the mutant strain is caused by inserting the aphVIII segment into the RFC1 gene (the CDS sequence is as shown in SEQ ID NO: 1). By reducing the expression of the chlamydomonas reinhardtii RFC1 gene, the RFC1 gene is deactivated or deleted so as to improve the cadmium tolerance of the chlamydomonas reinhardtii; the chlamydomonas reinhardtii strain with enhanced cadmium tolerance and acquired through a gene engineering method can be applied to the monitoring and treatment of a cadmium-polluted water area and provides a new direction for sewage treatment.

Description

technical field [0001] The invention relates to the fields of biotechnology and environmental pollution control, in particular to the application of the Chlamydomonas reinhardtii RFC1 gene in regulating the cadmium tolerance of the Chlamydomonas reinhardtii. Background technique [0002] With the continuous improvement of modern industrialization, environmental pollution has become a global problem. Due to the unreasonable development of some factories and unreasonable industrial development, a series of environmental pollution problems have been caused, among which the most serious pollution is water and the atmosphere. The pollution of heavy metal cadmium is one of the main industrial pollution. Both industrial waste gas and waste water and automobile exhaust contain a large amount of cadmium. The nature of heavy metal pollution in the water, soil, and atmosphere will continue to accumulate, and finally the ecological environment will be destroyed, and the survival and dev...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/405C12N1/13C02F3/32C12R1/89C02F101/20
CPCC02F3/322C02F2101/20C07K14/405
Inventor 田广梅李丽丽章伟雄田诚彭海李论刘艳玲王露胡长峰
Owner JIANGHAN UNIVERSITY
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