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Zea mays zinc iron-regulated transporter ZmITR1 gene and its applications

A transporter, zinc-iron technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of few research reports on ZIP family genes

Active Publication Date: 2014-12-17
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it is known that many transporters are involved in the zinc and iron ion balance network system in plants. Among them, the ZIP (Zinc-regulated transporters, Iron-regulated transporter-like proteins, ZIP) gene family can absorb divalent metal ions such as zinc and iron, Transportation and storage play an important role. In Arabidopsis, rice, barley, and soybean, some studies on ZIP family genes have been reported, but the specific mechanism of action of ZIP family genes in plants has not yet been fully understood. There are few research reports on ZIP family genes

Method used

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  • Zea mays zinc iron-regulated transporter ZmITR1 gene and its applications
  • Zea mays zinc iron-regulated transporter ZmITR1 gene and its applications
  • Zea mays zinc iron-regulated transporter ZmITR1 gene and its applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Cloning of ZmITR1 gene of maize zinc and iron regulated transporter

[0061] 1. Processing of plant material

[0062]First soak the vermiculite with Hoagland nutrient solution, sow the seeds of the corn inbred line X178 on the seedling tray, cover the top with a layer of dry vermiculite, and cultivate them in a greenhouse (16h light / 8h dark, 26°C). When it grows to 2 leaves and one heart, it is moved into the standard Hoagland nutrient solution and grows for 6 days to 3 leaves and one heart (the nutrient solution is changed every 3 days). After treatment under high zinc and iron conditions for 0, 6, 12, 24, 48, and 96 hours, the shoots and roots of the seedlings were harvested, respectively, and stored at -80°C for total RNA extraction after quick freezing in liquid nitrogen.

[0063] 2. Extraction of corn total RNA

[0064] The total RNA of maize was extracted by Trizol method.

[0065] 3. Synthesis of cDNA

[0066] (1) To remove DNA, prepare the reaction...

Embodiment 2

[0076] Example 2 Expression patterns of ZmITR1 in seedlings, embryos and endosperms

[0077] The reverse transcribed cDNA in Step 3 of Example 1 was diluted 10 times as a template for the PCR reaction, and the PCR reaction system was as follows:

[0078] cDNA 2.0 μL, ExTaq 0.1 μL, 2×GCI buffer 10.0 μL, 10 mM dNTP mix 0.8 μL, upstream primer RTZmITR1F (10 μM / μL) 1.0 μL, downstream primer RTZmITR1R (10 μM / μL) 1.0 μL, ddH 2 O5.1 μL, 20.0 μL in total;

[0079] RTZmITR1F5'-CACCACCTTCGTCGCCAT-3'

[0080] RTZmITR1R5'-TGTTGCCACCCTTCCTCC-3'

[0081] PCR reaction conditions: pre-denaturation at 94°C for 4 minutes; 30 cycles, each cycle of denaturation at 94°C for 45 seconds, annealing at 60°C for 1 minute, extension at 72°C for 1 minute; finally extending at 72°C for 10 minutes, cooling to 16°C, taking out the PCR product and putting it in Store at 4°C.

[0082] Detection of the expression level of the target gene: the reverse-transcribed cDNA in Step 3 of Example 1 was diluted 20 t...

Embodiment 3

[0090] Example 3 Bioinformatics analysis of ZmITR1

[0091] Bioinformatics analysis showed that ZmITR1, a zinc-iron-regulated transporter, was localized on the first chromosome of maize. ZmITR1, a zinc-iron-regulated transporter, consisted of 381 amino acids and contained 8 transmembrane domains. There is a histidine-rich variable region between them, which may be related to the binding and transport of metal ions. Phylogenetic tree analysis showed that ZmITR1 was closely related to AtIRT1, AtIRT2, OsIRT1, and OsIRT2 ( Figure 5 ), speculating that ZmITR1 may be an iron transporter.

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Abstract

The invention discloses a Zea mays zinc iron-regulated transporter ZmITR1 gene and its applications. The Zea mays zinc iron-regulated transporter ZmITR1 gene is separated from Zea mays, and the cDNA sequence of the gene is represented by SEQ ID No.1. Subcellular localization shows that the zinc iron-regulated transporter is localized in the plasma membrane and endoplasmic reticulum of a cell, has zinc and iron absorption, transportation and storage effects in plant cells, and particles in detoxification of zinc and iron ions. Quantitative real-time RT-PCR expression analysis shows expression the expression of the ZmITR1 in zinc or iron deficient root and overground parts rises, the expression in the later stage of embryo growth obviously rises, and the ZmITR1 plays an important role in the regulation of the embryo growth. Yeast complementation experiments show that the ZmITR1 gene has a zinc and iron complementation ability. The separated ZmITR1 gene has important application prospects in the regulation of zinc and iron absorption, transportation and storage of plants, promotion of the embryo growth and increase of the content of zinc and iron in grains of food crops.

Description

technical field [0001] The present invention relates to metal ion transporter genes isolated from plants, in particular to the regulatory transporter ZmITR1 gene isolated from corn and related to the absorption, transport or storage of zinc and iron. The present invention further relates to the regulatory transporter ZmITR1 gene in regulating The application in the ability of plants to absorb, transport or store zinc or iron, detoxify excessive zinc and iron to plants, and increase the content of zinc and iron in grain crop seeds belongs to the field of isolation and application of plant metal ion-regulated transporter genes. Background technique [0002] Zinc and iron are essential trace elements for organisms and play an important role in the growth and development of plants (Wintz H, Fox T, Wu YY, et al. Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal novel transporters involved in metal homeostasis . The Journal of biological chemistry 2003, 27...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415A01H5/00C12N15/82C12N15/29
Inventor 陈景堂李素贞李宏博祝丽英黄亚群
Owner HEBEI AGRICULTURAL UNIV.
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