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Rice copper transporter gene OsCtr5 and application thereof in copper polluted soil and water restoration

A rice and gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as the inability to meet the needs of copper pollution control

Inactive Publication Date: 2014-03-19
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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  • Rice copper transporter gene OsCtr5 and application thereof in copper polluted soil and water restoration
  • Rice copper transporter gene OsCtr5 and application thereof in copper polluted soil and water restoration
  • Rice copper transporter gene OsCtr5 and application thereof in copper polluted soil and water restoration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Sequence and structural analysis of the OsCtr5 gene

[0021] 1. Prediction of OsCtr5 gene structure

[0022] 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 gene AtCOPT1 sequence is highly homologous to a section (81886 to 82341) of a 125650bp sequence (GenBank registration number: AC109595.2) from rice variety Nipponbare located on rice chromosome 5. In the rice genome database TIGR (http: / / rice.plantbiology.msu.edu / ), this rice sequence homologous to the OsCtr1 gene is annotated as a gene, and its number is LOC_Os05g35050. The applicant of the present invention named this gene OsCtr5 (Oryza sativa c topper tr ansporter 5).

[0023] 2. Isolation and cloning of OsCtr5 gene from rice variety Zhonghua 11

[0024]Zhonghua 1...

Embodiment 2

[0030] Example 2: Expression profile analysis of OsCtr5 gene after copper treatment

[0031] In order to verify whether the OsCtr5 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 OsCtr5 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

[0034] Example 3: Analysis of subcellular localization of OsCtr5 gene-encoded product

[0035] 1. Construction of genetic transformation vector

[0036] 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.

[0037] Using the full-length OsCtr5 gene clone as a template, PCR technology was used to amplify the coding segment DNA of OsCtr5 gene. PCR primers were OsCtr55F (5'CGG GGTACC ATGGACATGGGCGGC-3') (the underline represents the KpnI restriction endonuclease digestion site) and OsCtr56R (5'-GCT GGATCC CAGCACACGGGGTCG-3') (the underline represents the BamHI restriction endonuclease digestion site) ( figure 2 ). The PCR product was connected to the T-A cloning carrier pGEM-T (Promega, USA), and the connected carrier was electrotran...

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Abstract

The invention relates to the field of plant genetic engineering technology, in particular to the isolated cloning and function verification of a DNA fragment including a rice copper transporter gene OsCtr5. The OsCtr5 gene encodes a copper transporter protein and can strengthen the capability of the rice for absorbing and accumulating the copper. The fragment and the exogenous regulatory sequence thereof are directly transported in the rice, and the capability of the genetically modified rice of the over-expressed OsCtr5 for absorbing and accumulating the copper is markedly strengthened. The rice material of the over-expressed OsCtr5 is planted to reduce the copper content in the soil and the surrounding water body and restore the copper pollution of 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 OsCtr5. OsCtr5 gene is an important copper transport gene in rice. After overexpressing the OsCtr5 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 OsCtr5 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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IPC IPC(8): C12N15/29C12N15/82
Inventor 王石平袁猛
Owner HUAZHONG AGRI UNIV