Oryza sativa copper transporter gene OsCtr1 and application thereof in remediation of copper contaminated soil and water body

A gene and rice technology, which is applied in the field of isolation, cloning, functional verification and application of the rice copper transporter gene OsCtr1, can solve the problems such as the inability to meet the needs of copper pollution control.

Inactive Publication Date: 2011-01-12
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these plant species are far from meeting the needs of different soil copper pollution control in different environments.

Method used

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  • Oryza sativa copper transporter gene OsCtr1 and application thereof in remediation of copper contaminated soil and water body
  • Oryza sativa copper transporter gene OsCtr1 and application thereof in remediation of copper contaminated soil and water body
  • Oryza sativa copper transporter gene OsCtr1 and application thereof in remediation of copper contaminated soil and water body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Sequence and structural analysis of the OsCtr1 gene

[0022] 1. Prediction of OsCtr1 gene structure

[0023] The applicant of the present invention uses a section of cDNA sequence (Kampfenkel et al., 1995) of the copper transporter gene AtCOPT1 of Arabidopsis thaliana, uses BLAST method (Altschul etc., 1997) to search nucleotide database GenBank (http: / / www.ncbi. nlm.nih.gov), found that the cDNA sequence was highly homologous to a section (positions 104141 to 104626) of a 166421bp long sequence (GenBank accession number: AP003106.1) located on rice chromosome 1 from the rice variety Nipponbare. In the rice genome database TIGR (http: / / rice.plantbiology.msu.edu / ), this rice sequence homologous to the AtCOPT1 gene is annotated as a gene, and its number is LOC_Os01g56420. The applicant of the present invention named this gene OsCtr1 (Oryza sativa copper transporter 1).

[0024] 2. Isolation and cloning of OsCtr1 gene from rice variety Zhonghua 11

[0025]Zho...

Embodiment 2

[0031] Example 2: Expression profile analysis of OsCtr1 gene after copper treatment

[0032] In order to verify whether the OsCtr1 gene is involved in the regulation of copper uptake and transport in rice, the applicant of the present invention firstly adopted quantitative reverse transcription-PCR (quantitative reverse transcription-PCR, qRT-PCR) technology (Qiu et al., 2007) to analyze the OsCtr1 gene's effect on copper. Treatment response. The applicant of the present invention used hydroponics to grow rice (Ishimaru et al., 2006). The hydroponic culture medium adopts the standard formula (Ishimaru et al., 2006). The content of copper salt (copper sulfate) in the standard hydroponic culture solution is 0.2 μM. In the copper deficiency stress treatment experiment, no copper salt was added to the culture medium; in the copper excess stress treatment experiment, the copper salt content in the culture medium was 50 μM. The rice variety Zhonghua 11 was hydrocultured to the 4-...

Embodiment 3

[0035] Example 3: Analysis of subcellular localization of OsCtr1 gene-encoded product

[0036] 1. Construction of genetic transformation vector

[0037] The genetic transformation vector mediated by Agrobacterium used in the present invention is pU1391 ( Figure 4 ). pU1391 is a commonly used rice genetic transformation vector (Cai et al., 2008). It carries the maize ubiquitin gene promoter (P Ubi ) and green fluorescence protein (GFP) tags.

[0038] Using the full-length OsCtr1 gene clone as a template, PCR technology was used to amplify the coding segment DNA of the OsCtr1 gene. PCR primers are OsCtr11F (see Example 1) and OsCtr12R (5'-CGG GGATCC CAGCAGGCCGGGTCG-3') (the underline represents the BamHI restriction endonuclease digestion site) ( figure 2 ). The PCR product is connected with the T-A cloning carrier pGEM-T (Promega Company of the United States), and the connected carrier is electrotransformed (the electrotransformer is a product of eppendorf Company, an...

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Abstract

The invention relates to the technical field of plant gene engineering, in particular to isolation, cloning and function verification of DNA fragments containing Oryza sativa copper transporter gene OsCtr1. The OsCtr1 encodes a copper transporter protein, and can enhance the copper absorption and accumulation capability of Oryza sativa. The segment and the extraneous adjustment sequence thereof are directly transported into the Oryza sativa, thereby obviously enhancing the copper absorption and accumulation capability of the transgenic Oryza sativa with excessive OsCtr1 expression. The Oryza sativa with excessive OsCtr1 expression can be planted to reduce the content of copper in soil and surrounding water body and remedy the copper pollution in the soil and the water body.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. It specifically relates to the isolation and cloning, functional verification and application of a rice copper transporter gene OsCtr1. OsCtr1 gene is an important copper transport gene in rice. After overexpressing the OsCtr1 gene, the copper content in the aboveground and underground parts of rice plants increased significantly. It can be used to remediate copper pollution in soil and water by planting rice materials overexpressing OsCtr1 to reduce the copper content in soil and surrounding water. Background technique [0002] As an important raw material, copper has a very wide range of uses in many fields such as industry, agriculture, and transportation. With the development of the economy, the demand for copper continues to increase. In the process of copper mining, smelting and use, it is inevitable to cause the discharge and residue of copper-containing waste, result...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82
Inventor 王石平袁猛
Owner HUAZHONG AGRI UNIV
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