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Application of chlamydomonas reinhardtii LRR (Leucine Rich Repeat) gene in regulating and controlling cadmium resistance of chlamydomonas reinhardtii

A reinhardtii and tolerance technology is applied in the application field of the Chlamydomonas reinhardtii LRR gene in regulating the cadmium tolerance of Chlamydomonas reinhardtii, which can solve the problems of unrelated research reports on the regulation of heavy metal cadmium metabolism, and achieve stable traits, The method is simple and the method is feasible

Inactive Publication Date: 2018-03-06
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that leucine-rich repeat sequences widely exist in plant disease resistance genes, but there is no related research report on the regulation of heavy metal cadmium metabolism

Method used

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  • Application of chlamydomonas reinhardtii LRR (Leucine Rich Repeat) gene in regulating and controlling cadmium resistance of chlamydomonas reinhardtii
  • Application of chlamydomonas reinhardtii LRR (Leucine Rich Repeat) gene in regulating and controlling cadmium resistance of chlamydomonas reinhardtii
  • Application of chlamydomonas reinhardtii LRR (Leucine Rich Repeat) gene in regulating and controlling cadmium resistance of chlamydomonas reinhardtii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Obtaining Chlamydomonas transformants with randomly inserted aphⅧ fragments by electroporation

[0036] (1) Using the previously constructed plasmid pJMG-aphⅧ (Zhangfeng Hu, Yinwen Liang, Wei He, Junmin Pan * , Cilia Disassembly with Two Distinct Phases of Regulation, CellReports, 2015, 10(11): 1803-1810), the plasmid contains the aphⅧ fragment encoding paromomycin resistance, and the pJMG-aphⅧ plasmid is digested overnight ( EcoRI digestion), and then the digested plasmid was subjected to DNA agarose gel electrophoresis to separate the aphⅧ fragment (such as figure 1 shown), the gel was recovered to obtain the aphⅧ fragment (sequence shown in SEQ ID NO: 3).

[0037] (2) The wild-type Chlamydomonas 21gr was transferred from the TAP solid medium to the air-blown bottle containing the TAP liquid medium, and cultured for 3 days under continuous light conditions to make the concentration reach 1.5×10 7 cells / mL. The cultured cells were transferred to the Erlen...

Embodiment 2

[0042] Example 2: Screening transformants to obtain cadmium chloride-resistant mutant algal strains

[0043] (1) Pick the Chlamydomonas transformants from the TAP solid medium containing paromomycin to the TAgP solid medium with a sterilized toothpick, and each transformant corresponds to a number. Then put it under the photoperiod and cultivate it for 3 days, and then pick it on the TAgP solid medium containing 0.6mM cadmium chloride, the numbers correspond to the numbers on the TAgP solid medium, and the wild type 21gr is used as a control. The screening concentration of 0.6mM cadmium chloride is the optimal screening concentration of heavy metal cadmium-resistant mutants obtained in the applicant's previous experiments. For details, see patent 201611022254.9.

[0044] (2) Put the 0.6mM cadmium chloride solid medium containing the transformants to cultivate under the photoperiod for 3-4 days, then observe and record the growth situation of the transformants on the 0.6mM cadm...

Embodiment 3

[0045] Example 3: Extracting the genome of the mutant

[0046] (1) Pick the mutant from the TAgP solid medium with a sterilized toothpick and culture it in the TAP liquid medium for 4 days.

[0047] (2) Collect the cells, centrifuge at 2500rpm for 3min, discard the supernatant, resuspend the cells with 4mL of liquid medium, transfer 1mL of the cells to a 1.5mL EP tube with a pipette gun, centrifuge at 14000rpm for 1min, discard the supernatant, and place the cells in Freeze in liquid nitrogen and store at -80°C for later use.

[0048] (3) Take out the frozen cells and add 650 μL of preheated CTAB lysate, pipette and mix the cells thoroughly, place them in a water bath at 65°C for 1 hour, and invert the EP tube every 10 minutes to fully lyse them.

[0049] (4) Add 650 μL of PCI (phenol: chloroform: isoamyl alcohol = 25:24:1). PCI should be shaken well before adding. After adding, invert the EP tube up and down to mix well, and centrifuge at 14,000 rpm for 10 min.

[0050] (5)...

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Abstract

The invention discloses application of a chlamydomonas reinhardtii LRR (Leucine Rich Repeat) gene in regulating and controlling cadmium resistance of chlamydomonas reinhardtii, and belongs to the technical field of biologics and the field of environmental pollution improvement. According to the application, an aphVIII fragment with a paromomycin resistance gene is randomly inserted into chlamydomonas, then a chlamydomonas reinhardtii mutant alga strain which is remarkable in cadmium chloride resistance is cultured, and further identification shows that the cadmium sensitivity of the mutant alga strain is caused since the aphVIII (SEQ ID NO:3) fragment is inserted into an LRR gene (a CDS (Cadmium Sulfide) sequence is shown in SEQ ID NO:1 in the specification). By reducing expression of thechlamydomonas reinhardtii LRR gene, deactivating the LRR gene or by deleting the LRR gene, the cadmium resistance of the chlamydomonas reinhardtii can be improved; a cadmium resistance improved chlamydomonas reinhardtii strain obtained by means of genetic engineering can be applied to monitoring and improvement of cadmium polluted water areas, cadmium polluted water environments can be treated with the cadmium resistance improved chlamydomonas reinhardtii strain, the operation is simple and feasible, and the cost is low.

Description

technical field [0001] The invention relates to the fields of biotechnology and environmental pollution control, and in particular to the application of the Chlamydomonas reinhardtii LRR gene in regulating the cadmium tolerance of the Chlamydomonas reinhardtii. Background technique [0002] With the development of heavy industry, environmental pollution is becoming more and more serious, especially heavy metal pollution. Heavy metals have a relative density greater than 5g / cm 3 Metals and heavy metals are difficult to be degraded in soil, atmosphere and water environment. At present, the main treatment methods are physical treatment, chemical treatment and biological treatment, among which the consumables for physical and chemical treatment are relatively expensive and the operation is cumbersome. Biological treatment is to enrich heavy metals through some microorganisms and algae, and then recover heavy metals by recycling microorganisms and algae. In summary, biological ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N1/13C07K14/405C12R1/89
Inventor 胡潇李丽丽彭海章伟雄田广梅刘艳玲高利芬唐宇云魏志新胡长峰
Owner JIANGHAN UNIVERSITY
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