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Zea mays zinc iron-regulated transporter ZmZIP4 and ZmZIP6 genes and applications thereof

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

Inactive 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 ZmZIP4 and ZmZIP6 genes and applications thereof
  • Zea mays zinc iron-regulated transporter ZmZIP4 and ZmZIP6 genes and applications thereof
  • Zea mays zinc iron-regulated transporter ZmZIP4 and ZmZIP6 genes and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Cloning of ZmZIP4 gene and ZmZIP6 gene of maize zinc and iron regulated transporters

[0075] 1. Processing of plant material

[0076] 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.

[0077] 2. Extraction of corn total RNA

[0078] Total corn RNA was extracted using Trizol extraction kit.

[0079] 3. Synthesis of cDNA

[0080] (1) To remove DNA,...

Embodiment 2

[0093] Example 2 Expression patterns of ZmZIP4 and ZmZIP6 in seedlings, embryos and endosperms

[0094] 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:

[0095] cDNA 2.0 μL, ExTaq 0.1 μL, 2×GCI buffer 10.0 μL, 10 mM dNTP mix 0.8 μL, upstream primer RTZmZIP4F / RTZmZIP6F (10 μM / μL) 1.0 μL, downstream primer RTZmZIP4R / RTZmZIP6R (10 μM / μL) 1.0 μL, ddH 2 O5.1 μL, 20.0 μL in total;

[0096] RTZmZIP4F5'-CCTTCTTCTCGCTCACCGCT-3'

[0097] RTZmZIP4R5'-AGCCTCGGGTTGCTGAAGT-3'

[0098] RTZmZIP6F5'-CATCGCACAGGCTGGTTTTG-3'

[0099]RTZmZIP6R5'-TTCCAGCCGAAAGTGAACCA-3'

[0100] 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.

[010...

Embodiment 3

[0107] Example 3 Bioinformatics analysis of ZmZIP4 and ZmZIP6

[0108] ZmZIP4 and ZmZIP6 genes are located on chromosome 4 and 8 of maize, respectively. ZmZIP4 consists of 3 exons and 2 introns, and ZmZIP6 consists of 2 exons and 1 intron. ZmZIP4 encodes 386 amino acids, and ZmZIP6 gene encodes 396 amino acids. According to the amino acid sequence comparison of maize ZIP proteins, ZmZIP4 protein contains 6 transmembrane domains, and ZmZIP6 protein contains 8 transmembrane domains. There is a histidine-rich variable region between the 4 transmembrane regions, which may be related to the binding and transport of metal ions. Phylogenetic tree analysis showed that ZmZIP4 and OsZIP4 were on the same branch, and ZmZIP6 and OsZIP6 were on the same branch ( Figure 5 ), OsZIP4 is a zinc transporter, so ZmZIP4 and ZmZIP6 may be zinc-iron transporters.

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Abstract

The invention discloses Zea mays zinc iron-regulated transporter ZmZIP4 and ZmZIP6 genes and applications thereof. The Zea mays zinc iron-regulated transporter ZmZIP4 and ZmZIP6 genes are separated from Zea mays, and the cDNA sequences of the genes are represented by SEQ ID No.1 and SEQ ID No.3 respectively. Subcellular localization shows that a zinc iron-regulated transporter encoded by the two genes is localized in the plasma membrane and endoplasmic reticulum of a cell. Quantitative real-time RT-PCR expression analysis shows that the ZmZIP4 plays a role in the embryo growth process, and the ZmZIP6 is related to embryo maturation. Yeast complementation experiments show that each of the ZmZIP4 and the ZmZIP6 has a zinc and iron transportation function. The separated ZmZIP4 and ZmZIP6 genes have important application prospects in the regulation of zinc and iron absorption, transportation and storage of plants, and promotion of the embryo maturation, and radicle and germ growth.

Description

technical field [0001] The present invention relates to metal ion transporter genes isolated from plants, in particular to regulatory transporter ZmZIP4 and ZmZIP6 genes isolated from corn and related to the absorption, transport or storage of zinc and iron, and the present invention further relates to regulatory transporter ZmZIP4 or ZmZIP6 The application of genes in regulating the ability of plants to absorb, transport or store zinc or iron, to promote the development or maturation of embryos, and to increase the zinc and iron content of 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 b...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00
Inventor 李素贞李宏博陈景堂黄亚群祝丽英
Owner HEBEI AGRICULTURAL UNIV.
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