Method for detecting cadmium pollution through variation of genetic expression of brassica plant

A technology of gene expression and Brassica genus, applied in the field of molecular biology detection of environmental pollution, achieves the effects of strong sensitivity and specificity, improved identification efficiency, and high practical value

Inactive Publication Date: 2013-02-06
CENT SOUTH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The object of the present invention is to overcome the deficiency of traditional cadmium pollution detection method, provide a kind of fast, specificity good, the method for the high sensitivity detection cadmium pollution

Method used

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  • Method for detecting cadmium pollution through variation of genetic expression of brassica plant
  • Method for detecting cadmium pollution through variation of genetic expression of brassica plant
  • Method for detecting cadmium pollution through variation of genetic expression of brassica plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Identification of up-regulated and down-regulated genes specifically expressed in Brassica juncea under cadmium induction.

[0033] Select plump and clean mustard seeds, soak them in 75% ethanol for 1 min, then sterilize them in 0.1% mercuric chloride for 10 min, wash them with sterile water for 3 times, soak them in sterile water for 5 hours, and blot the excess water with filter paper , inoculated on 1 / 2 MS solid medium with pH 6.0, containing 5g / L sucrose and 0.7% agar. Cadmium (CdCl 2 ) concentration level (as Cd 2+ 0mg / kg, 0.5mg / kg, 1.0mg / kg and 1.5mg / kg, respectively, grown in a greenhouse under sterile conditions for 15 days, the day-to-night ratio was 14 / 10h, and the day-to-night temperature was 22 / 16°C. The light intensity is 200 μmol m –2 the s –1 . Rapeseed root in solid medium was taken for experiment.

[0034]Collect cadmium-treated and control mustard roots, extract total RNA, hybridize and analyze with 29K Arabidopsis expression chips, and perform ...

Embodiment 2

[0050] 1) Take paddy soil from the surrounding farmland of Hunan University of Science and Technology in Xiangtan, Hunan. The content of Pb is 55.4 mg / kg, the content of Zn is 157.8 mg / kg, the content of Cd is 0.17 mg / kg, and the pH value is 6.5.

[0051] 2) Take a flower pot with a diameter of 20cm and a height of 20cm. The following cultures were placed in the flower pots: paddy soil, referred to as control; paddy soil and 0.50 mg / kg Cd, referred to as treatment 1; paddy soil and 1.00 mg / kg Cd, referred to as treatment 2; paddy soil and 1.50 mg / kg Cd, referred to as treatment 2; Referred to as treatment 3. The volume of the culture is about 4 / 5 of the volume of the flower pot. There is a watertight plate under the flower pot. After the pot is installed, water 200ml per pot and place it in the natural environment of Hunan University of Science and Technology for 1 month.

[0052] 3) In September, sow the mustard seeds in the above-mentioned flower pots, place them in the na...

Embodiment 3

[0060] 1) Collect mustard greens grown in cadmium-contaminated soil and non-polluted soil around a tailing mine in Hunan. Taking non-polluted soil as a control, the cadmium content was determined by atomic absorption spectrometry to be 0.21mg / kg, which is in line with the national soil environmental quality secondary standard (GB15168-1995). The content of cadmium in cadmium-contaminated soil was determined to be 2.23mg / kg by atomic absorption spectrometry. The roots of mustard were collected for extraction of total RNA. The extracted total RNA was reverse transcribed.

[0061] 2) Taking the expression of Actin gene as a reference, the sequence of forward primer ACTF and ACTF is: 5'-GACATTCAACCTCTTGTTTGC-3', and the sequence of reverse primer ACTR is: 5'-CTGCTCGTAGTCAAGAGCAATG-3'; detection by semi-quantitative PCR The expression of 6 all up-regulated genes and 6 all down-regulated genes specifically expressed under the induction of mustard cadmium, the specific operation is...

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Abstract

The invention discloses a method for detecting cadmium pollution through variation of genetic expression of a brassica plant. The method comprises steps as follows: extracting total RNA (Ribose Nucleic Acid) from a cadmium-processed brassicajuncea root and a compared brassicajuncea root; hybridizing with a 29K arabidopsis chip, so as to obtain a cadmium induced up-regulated gene and a cadmium induced down-regulated gene under specific expression; designing primers in conserved regions of genes; carrying out a semiquantitative PCR (Polymerase Chain Reaction) method to sieve and determine six up-regulated genes and six down-regulated genes with the specific expression of brassicajuncea when cadmium concentration is more than 1.00mg / kg based on a standard of increasing or reducing 2.00 timesof expression quantity; utilizing variation of gene expression in the root of the brassica plant in primer detection environments of 12 genes; if luminance of a product of the brassica plant in the environment to be detected and amplified by the primers of 6 up-regulated genes is increased over 2.00 times that of contrast, and the luminance of the product of the brassica plant in the environment to be detected and amplified by the primers of 6 down-regulated genes is decreased over 2.00 times that of the contrast, determining that concentration of cadmium in the environment to be detected is more than 1.00mg / kg, thus determining that whether the brassica plant in the environment to be detected is subjected to the cadmium pollution. The detection method is high in sensitivity and high in specificity, and can be used for quickly detecting whether content of the cadmium in the environment of the brassica plant is suitable for agricultural production.

Description

technical field [0001] The invention relates to the technical field of detecting environmental pollution by molecular biology methods, in particular to a method for detecting cadmium pollution by using the expression changes of genes specifically induced by cadmium mustard. Background technique [0002] The detection of cadmium in the traditional environment is usually detected by atomic absorption spectrometer. This method requires expensive instruments, a good experimental platform and high costs, which is not conducive to popularization among grassroots environmental detection units and users. Therefore, a fast, simple and convenient method is invented. Inexpensive detection methods for detecting cadmium contamination are necessary. [0003] Polymerase chain reaction (PCR) is currently the most popular technique for amplifying DNA fragments in vitro. It is fast, sensitive and specific. If this technique can be used to detect cadmium-related gene , will have important si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 严明理冯涛向言词刘丽莉柴立元李夕兵
Owner CENT SOUTH UNIV
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