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Cloning and applications of molecular chaperone protein OsATX1 capable of affecting copper transport and distribution in rice

A copper transport, rice technology, applied in the field of plant genetic engineering, can solve the problem of unclear understanding of the molecular mechanism of copper ions

Inactive Publication Date: 2019-01-01
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the molecular mechanism by which rice absorbs and distributes copper ions is not well understood.

Method used

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  • Cloning and applications of molecular chaperone protein OsATX1 capable of affecting copper transport and distribution in rice
  • Cloning and applications of molecular chaperone protein OsATX1 capable of affecting copper transport and distribution in rice
  • Cloning and applications of molecular chaperone protein OsATX1 capable of affecting copper transport and distribution in rice

Examples

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Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Sequence and structural analysis of the OsATX1 gene

[0026] Prediction of OsATX1 gene structure:

[0027] The applicant of the present invention utilizes a section of cDNA sequence of the copper ion chaperone protein gene AtATX1 of Arabidopsis, searches nucleotide database GenBank ( http: / / www.ncbi.nlm.nih.gov ), and found that the cDNA sequence was identical to a 126112bp sequence from the rice variety Nipponbare (GenBank registration number: AP003875.3 ) in a segment (16242 to 18197) is highly homologous. In the rice genome database TIGR ( http: / / rice.plantbiology.msu.edu / ), the rice sequence homologous to the AtATX1 gene was annotated as a gene, and its number was LOC_Os08g10480. The applicant named this gene OsATX1 gene (Antioxidantprotein1). The full-length genome sequence of OsATX1 is 1956bp, encoding 81 amino acids. The gene consists of 3 exons and 2 introns (see figure 2 , its nucleotide Xu sequence and coding region are shown in SEQ ID...

Embodiment 2

[0035] Embodiment 2: the expression analysis of OsATX1 gene after copper stress treatment

[0036] In order to verify whether the OsATX1 gene is involved in the regulation of copper uptake and transport in rice, the present invention firstly uses quantitative PCR (Quantitative real-time PCR, qRT-PCR) technology to analyze the response of the OsATX1 gene to copper stress treatment. The invention adopts the method of nutrient solution culture to plant rice. The nutrient solution culture medium adopts the standard formula. The copper salt (copper sulfate) content in the standard nutrient solution culture solution is 0.12 μmol. In the copper deficiency stress treatment experiment, a copper ion chelating agent, disodium phenanthrene disulfonate (BCS), was added to the culture medium, and the treatment concentration of the disodium phenanthrene disulfonate was 2 μmol. In the copper excess stress treatment experiment, the treatment concentration of copper in the culture solution wa...

Embodiment 3

[0038] Example 3: Analysis of subcellular localization of OsATX1 gene-encoded product

[0039] 1. Construction of OsATX1 subcellular localization transient expression vector

[0040] The genetic transformation vector mediated by Agrobacterium used in the present invention is pM999 (see structure Figure 4 ). It carries the CaMV35S promoter and a green fluorescent protein (GFP) tag. Using the full-length OsATX1 gene clone as a template, PCR technology was used to amplify the coding segment DNA of the OsATX1 gene. PCR primers were OsATX1-scF (5′-CG GAATTC ATGGCTGAGACTGTTGTGCTC-3′) (the underline represents the EcoR1 restriction endonuclease digestion site) and OsATX1-scF (5′-GG GGTACC TTAAGATGAAGCAGCAGTAGCTTC-3') (the underline represents the KpnI restriction endonuclease digestion site). The PCR product was connected to the T-A cloning vector pGEM-T (purchased from Promega, USA), and the connected carrier was electrotransformed (the electrotransformer is a product of epp...

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Abstract

The invention belongs to the technical field of plant genetic engineering, specifically relates to cloning and applications of a molecular chaperone protein OsATX1 capable of affecting copper transport and distribution in rice, and provides isolated cloning and functional verification of the DNA fragment of a rice copper transporter gene OsATX1. According to the present invention, the OsATX1 geneencodes a copper ion chaperone protein gene, the sequence of the protein encoded by the gene fragment is represented by EQ ID NO:3, and after the fragment and the exogenous regulatory sequence are directly transformed into rice, the transgene overexpressing the OsATX1 can significantly enhance the transport of copper from root to shoot and the transfer to young tissues and grains in rice; and it is expected to ensure the nutritional quality of rice by increasing the copper concentration in rice grains.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. It specifically relates to the cloning and application of a molecular chaperone protein OsATX1 that affects copper transport and distribution in rice. The invention includes the isolation, cloning, functional verification and application of the rice copper ion chaperone OsATX1. OsATX1 is an important copper ion chaperone protein in rice. Overexpression of OsATX1 gene leads to a significant increase in copper content in rice grains. Therefore, it is expected to increase the copper content in rice by planting rice materials overexpressing OsATX1, thereby ensuring the nutritional quality of crops. Background technique [0002] Copper (Cu) is one of the trace elements necessary for the growth and development of organisms, and plays an important role in the growth and development of organisms. Copper deficiency can endanger human health and cause immune deficiency (Collins and Kle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415A01H5/00A01H6/46
CPCC07K14/415C12N15/8243
Inventor 练兴明张园园
Owner HUAZHONG AGRI UNIV