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Reed gamma-glutamyl cysteine synthetase gene PcGCS and its application

A technology of cysteine ​​and glutamyl, applied in the direction of enzymes, the use of carriers to introduce foreign genetic material, biochemical equipment and methods, etc., to achieve the effect of improving the ability to enrich heavy metals

Inactive Publication Date: 2007-11-07
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after searching, it has not been reported that the gamma-glutamylcysteine ​​synthetase gene of reed is transferred into bentgrass to improve its anti-heavy metal performance

Method used

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  • Reed gamma-glutamyl cysteine synthetase gene PcGCS and its application
  • Reed gamma-glutamyl cysteine synthetase gene PcGCS and its application
  • Reed gamma-glutamyl cysteine synthetase gene PcGCS and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The cloning of embodiment 1, PcGCS

[0021] 1.1 Extraction of total RNA from reed

[0022] (1) Put the reed material into a pre-cooled mortar, add liquid nitrogen and quickly grind it into a uniform powder. Pay attention to ensure that the material is immersed in liquid nitrogen during the grinding process.

[0023] (2) After the liquid nitrogen volatilizes, quickly transfer the material to a pre-cooled centrifuge tube, add 1ml TRIZOL solution for every 50-100mg of tissue material, mix well, place at room temperature for 5min, centrifuge at 12000r / min, 2-8℃ for 10min To precipitate.

[0024] (3) Add 0.2ml of chloroform to every 1ml of TRIZOL solution, oscillate for 15sec to mix well, leave at room temperature for 5min, centrifuge at 12000r / min, 2-8°C for 15min.

[0025] (4) Take the supernatant, add 0.5ml of isopropanol to every 1ml of TRIZOL, mix well, and place at room temperature for 10-20min.

[0026] (5) Centrifuge at 12000r / min for 10min at 2-8°C, discard the ...

Embodiment 2

[0111] Embodiment 2, the construction of plant expression vector

[0112] 2.1 Obtaining the target gene

[0113] Primer design and PCR reaction system and conditions:

[0114] (1) Design the following pair of PCR amplification primers, and introduce the two enzyme cutting sites SacI and XbalI contained in the plant expression vector pROK2 (purchased from Bao Biological Engineering Co., Ltd.).

[0115] P11: 5'-CGAGCTCGA TGG AAG AAA ACC ATG TTA TCG-3'

[0116] SacI

[0117] P12: 5'-GCTCTAGAGCTCA GTA TAA CAA GTG CTC G-3'

[0118] wxya

[0119] (2) PCR reaction system:

[0120] 10×PCR buffer 2μl

[0121] MgCl 2 (25mmol / L) 1.5μl

[0122] dNTP (each 250μmol / L) 0.2μl

[0123] P11 (4μmol / L) 1μl

[0124] P12 (4μmol / L) 1μl

[0125] Recombinant plasmid pGEM-GCS 1μl

[0126] rTaq (TaKaRa) 0.2μl

[0127] Sterile water 13.1μl

[0128] (3) PCR reaction conditions are

[0129] 94°C for 5min; 94°C for 30sec, 60°C for 30sec, 72°C for 2min, 35 cycles; finally 72°C for 7min. The PCR...

Embodiment 3

[0159] Example 3. Functional verification of reed γ-glutamyl-cysteine ​​synthetase gene PcGCS

[0160] 3.1 Construction of yeast expression vector

[0161] 3.1.1 Obtaining the target gene

[0162] (1) Primer design: A pair of PCR amplification primers (P9, P10) were designed to introduce two restriction sites BamHI and NotI contained in the yeast expression vector pDR195 (purchased from Bao Biological Engineering Co., Ltd.).

[0163] P9: 5’CGG GAT CCA TGG AAG AAA ACC ATG TTA TCG 3’

[0164] BamHI

[0165] P10: 5'ATT TGC GGC CGC TCA GTA TAA CAA GTG CTC G 3'

[0166] NotI

[0167] (2) PCR reaction system:

[0168] 10×PCR buffer 2μl

[0169] MgCl 2 (25mmol / L) 1.5μl

[0170] dNTP (each 250μmol / L) 0.2μl

[0171] P9 (4μmol / L) 1μl

[0172] P10 (4μmol / L) 1μl

[0173] Recombinant plasmid pGEM-GCS 1μl

[0174] rTaq (TaKaRa) 0.2μl

[0175] Sterile water 13.1μl

[0176] (3) PCR reaction conditions are

[0177] 94°C for 5min; 94°C for 30sec, 60°C for 30sec,...

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Abstract

The present invention discloses one kind of reed gamma-gluamyl-cysteine synthetase gene PcGCS and its plant expression vector. The present invention discloses also the application of gene PcGCS in culturing heavy metal resisting plant, especially Agrostis stolonifera. Experiment shows that the transgenic plant of the present invention has greatly raised heavy metal enriching capacity.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a reed-enriched heavy metal-related gene—γ-glutamylcysteine ​​synthetase gene PcGCS and its application. Background technique [0002] With the development of industry and the intensification of human production activities, heavy metal pollution has become a global concern. At present, there are tens of millions of acres of heavy metal-contaminated soil in our country, which has caused great harm to human health. Therefore, the effective removal of heavy metal pollution has become a very urgent task. [0003] Using transgenic plant technology to transfer new traits into high-biomass plants, in order to develop an efficient transgenic phytoremediation system, is a technology with broad application prospects for soil remediation of heavy metal pollution. [0004] Genetic engineering has made some relevant research progress in the enrichment of heavy met...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/82
Inventor 向凤宁赵翠珠夏光敏于延冲乔孟
Owner SHANDONG UNIV
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