Zinc/iron-regulated transporter genes ZmZIPs from zea mays and application thereof
A transporter, zinc and iron technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem of few research reports
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Embodiment 1
[0077] Example 1 Cloning of ZmZIPs Gene of the Zinc and Iron Regulatory Transporter in Corn
[0078] 1. Treatment of plant materials
[0079] First soak the vermiculite with Hoagland nutrient solution, sow the corn inbred line X178 seeds in the seedling tray, cover it with a layer of dry vermiculite, cultivate in the greenhouse (16h light / 8h dark, 26℃), 12 days seedling When it grows to 2 leaves and one core, it is transferred to the standard Hoagland nutrient solution and grown for 6 days to 3 leaves and one core (the nutrient solution is changed every 3 days). The corn seedlings with three leaves and one core are in the standard nutrient solution and without zinc, iron, copper, manganese, After 0, 6, 12, 24, 48, 96 hours of treatment under high zinc and iron conditions, the shoots and roots of the seedlings were harvested, and then quickly frozen in liquid nitrogen and stored at -80°C for total RNA extraction.
[0080] 2. Extraction of corn total RNA
[0081] Trizol method was used...
Embodiment 2
[0094] Example 2 Expression pattern of ZmZIPs in seedling, embryo and endosperm
[0095] Dilute the reverse-transcribed cDNA in step 3 of Example 1 by 10 times as a template for the PCR reaction. The PCR reaction system is as follows:
[0096] cDNA2.0μL, ExTaq0.1μL, 2×GCI buffer 10.0μL, 10mMdNTPmix0.8μL, upstream primer RTZmZIP1F / RTZmZIP2F / RTZmZIP3F / RTZmZIP7F / RTZmZIP8F(10μM / μL) 1.0μL, downstream primer RZmZRZRZRZRZRZRZRZR / RTZMIP2R / RTZmZIP1 (10μM / μL)1.0μL, ddH 2 O5.1μL, total 20.0μL;
[0097] RTZmZIP1F5'-CCTCTCTGCGTTGGTTGCTCT-3'
[0098] RTZmZIP1R5'-TTGATGGTTGTTTTCTGGTCGT-3'
[0099] RTZmZIP2F5'-CCACAAATGGCACGAGGTCT-3'
[0100] RTZmZIP2R5'-CGAAGACGGAGTGGAAGCAAA-3'
[0101] RTZmZIP3F5'-GCCTCTTGTTGGTGCCCTTA-3'
[0102] RTZmZIP3R5'-TCAACAATGAACGCTGTAGTGCT-3'
[0103] RTZmZIP7F5'-ACTAGGTGGGTGCATTGCTCAG-3'
[0104] RTZmZIP7R5'-TGCCAGCAGATACCGAGTCAA-3'
[0105] RTZmZIP8F5'-CGTGTCATCGCTCAGGTTCTTG-3'
[0106] RTZmZIP8R5'-CCCTCGAACATTTGGTGGAAG-3'
[0107] PCR reaction conditions: 94°C pre-denaturation f...
Embodiment 3
[0113] Example 3 Bioinformatics analysis of ZmZIPs
[0114] ZmZIPs are composed of 367-483 amino acids and contain 6-9 transmembrane domains. There is a histidine-rich variable region between the third and fourth transmembrane regions, which may be related to the binding and transport of metal ions. Phylogenetic tree analysis shows that ZmZIP1 has a close evolutionary relationship with AtIAR1 and OsIAR1. In addition, ZmZIP3 and ZmZIP4 form a gene cluster with OsZIP3 and OsZIP4, ZmZIP2 is adjacent to OsZIP2, and the zinc transporter OsZIP1, AtZIP2 and AtZIP11 are on the same branch, ZmZIP5 and ZmZIP7 are on the same branch, and the relationship between ZmZIP8 and OsZIP6 and OsZIP6 is evolved. More recently, these results show that the five ZmZIPs isolated in the present invention may be zinc-iron transporters.
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