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Application of Chlamydomonas reinhardtii g3280.t2 gene for regulating and controlling cadmium tolerance of Chlamydomonas reinhardtii

A reinhardtii coat and tolerance technology is applied in the application field of the Chlamydomonas reinhardtii g3280.t2 gene in regulating the cadmium tolerance of Chlamydomonas reinhardtii, and can solve the problems of reducing urinary cadmium excretion, irritation, toxicity of heavy metal cadmium and the like, Achieving high efficiency, simple method and remarkable sensitivity

Inactive Publication Date: 2018-05-08
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The heavy metal cadmium is toxic and not an essential element for the human body. Cadmium in the human body enters and accumulates in the body through food, water, and air, so it will bring hidden dangers to the health of the human body, animals and plants
Cadmium can irritate the respiratory tract. Long-term exposure can cause anosmia, macular spots or yellow circles on the gums. Cadmium compounds are not easily absorbed by the intestines, but can be absorbed by the body through breathing and accumulate in the liver or kidneys, causing harm, especially for Kidney damage is the most obvious, and it can also lead to osteoporosis and softening. Long-term inhalation of cadmium can cause chronic poisoning, causing kidney damage. The main manifestation is a large amount of low molecular weight protein in the urine. The recycling function of the renal tubules is reduced, and the excretion of urinary cadmium is increased

Method used

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  • Application of Chlamydomonas reinhardtii g3280.t2 gene for regulating and controlling cadmium tolerance of Chlamydomonas reinhardtii
  • Application of Chlamydomonas reinhardtii g3280.t2 gene for regulating and controlling cadmium tolerance of Chlamydomonas reinhardtii
  • Application of Chlamydomonas reinhardtii g3280.t2 gene for regulating and controlling cadmium tolerance of Chlamydomonas reinhardtii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 The acquisition of Chlamydomonas insertion mutants

[0034] (1) Pick 21gr of wild-type Chlamydomonas reinhardtii algae strains in good growth state on the TAP solid medium to a blowing bottle equipped with TAP liquid medium, and cultivate under continuous light conditions to a concentration of 1 × 10 7 / mL and transferred to a 250mL sterile shaker flask, diluted with liquid TAP to a cell concentration of 1×10 6 / mL, cultured under continuous light on a shaker for 24 hours, to a final concentration of 4×10 6 / mL.

[0035] (2) Digest pJMG-aphⅧ plasmid with EcoRI-HF enzyme (Zhangfeng Hu, Yinwen Liang, Wei He, Junmin Pan * , Cilia Disassembly with Two Distinct Phases of Regulation, CellReports, 2015, 10(11):1803-1810), to obtain the aphⅧ fragment (sequence shown in SEQ ID NO: 3), which contains the Paromomyces resistance gene. The aphVIII fragment was then recovered by agarose gel electrophoresis. Such as figure 1As shown, M is DL5000 DNA Marker, 1 is the sam...

Embodiment 2

[0039] Example 2 Acquisition of Cadmium Chloride Sensitive Mutant Algae Strains

[0040] (1) According to the applicant's previous research results, 0.4mM cadmium chloride is the optimal concentration for screening sensitive mutant algal strains (see patent 201611022254.9 for details). When the transformants on the TAgP solid medium were cultured for about 3 days under the photoperiod, apply one-to-one corresponding numbers on the TAgP solid medium with 0.4mM cadmium chloride, culture under the photoperiod, and observe the state of the transformants. If the transformants On the 0.4mM cadmium chloride medium, it can not grow normally but the wild type can grow normally, so it is preliminarily considered that the mutant is a cadmium chloride sensitive mutant strain.

[0041] (2) Repeat the screening several times, inoculate nearly the same amount of wild-type algal strain 21gr and the initially identified cadmium chloride-sensitive mutant algal strain on the TAgP medium of 0.4mM...

Embodiment 3

[0042] Example 3 Acquisition of Insertion Mutation Gene and Mutation Site of Sensitive Mutant Algae Strain KY1

[0043] (1) Pick the mutant algae strain KY1 in good condition on the TAP solid medium into a gas blowing bottle filled with TAP liquid medium, and culture it with ventilation until the cell concentration is 4×10 6 / mL, the cells were collected, quick-frozen in liquid nitrogen, and stored at -80°C for later use.

[0044] (2) Genomic DNA of collected KY1 algae strains was extracted by CTAB method.

[0045] (3) Design specific primers F-Z2 and F-Z8 according to the nucleic acid sequence of the insert fragment aphⅧ, and design primer Q0 and the specific recognition sequence composed of Q0 and AluI, PstI, SacII, TaqI restriction endonucleases according to the characteristics of Chlamydomonas genome Degenerate primers DegAluI, DegPstI, DegSacII, DegTaqI. Using the extracted genomic DNA of the KY1 algae strain as a template, RESDA-PCR was performed to obtain four PCR pro...

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Abstract

The invention discloses application of Chlamydomonas reinhardtii g3280.t2 gene for regulating and controlling cadmium tolerance of Chlamydomonas reinhardtii, and belongs to the fields of biotechnologyand environmental pollution control. A Chlamydomonas reinhardtii mutation strain obviously sensitive to cadmium is obtained by inserting the aphVIII fragment (SEQ ID NO:3) containing a paromomycin resistance gene into chlamydomonas at random, and further identification discovers that the cadmium sensitivity of the mutation strain is caused by the fact that the aphVIII fragment is inserted into the g3280.t2 gene (CDS sequence is shown as SEQ ID NO: 1). By reducing the expression of the Chlamydomonas reinhardtii g3280.t2 gene, the g3280.t2 gene is inactivated or the cadmium tolerance of Chlamydomonas reinhardtii can be reduced by losing the gene; and the cadmium sensitive Chlamydomonas reinhardtiis train obtained through gene engineering means can be applied into water environments for monitoring and detection of cadmium pollution through biological means, and then a novel biological material is provided for sewage detection.

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 g3280.t2 gene in regulating the cadmium tolerance of the Chlamydomonas reinhardtii. Background technique [0002] Since the 21st century, with the development of science and technology, cadmium has been widely used in daily life: standard batteries, smoke bombs, phosphors, pigments, alloy manufacturing, etc. Cadmium compounds have also been widely used in the manufacture of plastic stabilizers, TV picture tube phosphors, etc. The discharge of a large amount of industrial waste water and waste makes cadmium ions enter the soil, air, and even food. The heavy metal cadmium is toxic and not an essential element for the human body. Cadmium enters and accumulates in the human body through food, water, and air, so it will bring hidden dangers to the health of the human body, animals and plants. Cadmium c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N1/13C07K14/405C12Q1/02C02F3/32C12R1/89C02F101/20
CPCC02F3/322C02F2101/20C07K14/405C12Q1/02G01N2333/405
Inventor 李丽丽刘艳玲章伟雄康研周俊飞胡潇田广梅魏志新王露方治伟
Owner JIANGHAN UNIVERSITY
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