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Ammonium salt absorption-related protein and encoding gene and application thereof

A technology for encoding genes and proteins, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as low utilization rate

Active Publication Date: 2012-02-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In my country's maize production, the amount of nitrogen fertilizer application is too high, but the utilization rate is low is a common phenomenon (Ju et al., 2009, Proc Natl Acad Sci USA, 106: 3041-3046)

Method used

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  • Ammonium salt absorption-related protein and encoding gene and application thereof
  • Ammonium salt absorption-related protein and encoding gene and application thereof
  • Ammonium salt absorption-related protein and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the cloning of maize ammonium transporter gene ZmAMT1; 1

[0040] 1. Construction of maize cDNA library

[0041] Seedling root RNA of maize inbred line B73 (Schnable et al., Science 2009: 1112-1114; publicly available from China Agricultural University) was extracted to construct a cDNA library. The total RNA extraction steps, mRNA purification steps and cDNA library construction steps were all operated according to the methods provided in the instructions attached to the respective kits. The steps are briefly described as follows:

[0042] 1. Total RNA extraction

[0043] Take 200 mg of fresh corn root and grind it in liquid nitrogen; add 1 ml of Trizol extract provided by RNA extraction kit (invitrogen company, catalog number 15596-018), shake at room temperature for 5 minutes; then add 200 μl of chloroform, shake for 30 seconds, Centrifuge at 4°C, 12,000 rpm for 15 minutes; take the supernatant, add 0.5ml of isopropanol, let stand at room temperature...

Embodiment 2

[0134] Example 2, ZmAMT1; cloning of 1 gene, construction of yeast expression vector, transformation and yeast functional complementation

[0135] 1. Amplification of full-length ZmAMT1; 1 gene cDNA

[0136] According to the ZmAMT1;1 sequence obtained by yeast screening, a pair of primers P1-F and P1-R were designed with the following sequences:

[0137] P1-F: 5'-GAAAGATGTCGACGTGCGCGGCGGA-3';

[0138] P1-R: 5'-TTTTACACCTGGCTGCTGGTCGCCG-3'.

[0139] Among them, P1-F just contains the translation start codon ATG, and P1-R contains the translation stop codon TAA; the cDNA reverse-transcribed from the total RNA of the corn inbred line B73 root is used as a template, and the high-fidelity pfu enzyme (Promega Company, catalog No.: M7745) was amplified to obtain a ZmAMT1; 1 gene fragment (its nucleotide sequence is from the 78th to the 1574th nucleotide at the 5' end of Sequence 2) containing the complete open reading frame.

[0140] The reaction system is:

[0141] 2μg total RNA...

Embodiment 3

[0151] Example 3, Real-Time PCR analysis of ammonium-induced expression characteristics of maize ZmAMT1; 1 gene

[0152] Hydroponic corn inbred line B73 seedlings, with Hoagland nutrient solution (composition is K 2 SO 4 0.5mM, MgSO 4 .7H 2 O0.6mM, KH 2 PO4 0.1mM, CaCl 2 .2H 2 O 0.5mM, NH 4 NO 3 2mM,H 3 BO 3 1 μM, MnSO 4 .H 2 O 0.5 μM, ZnSO 4 .7H 2 O 0.5 μM, CuSO 4 .5H 2 O 0.2 μM, Na 2 MoO 4 .2H 2 O 0.07μM, NaFe-EDTA 0.1mM (pH 5.7) culture, and change the nutrient solution every 2 days. When growing to the three-leaf stage (20 days after germination), move into the Hoagland nutrient solution without ammonium nitrate and carry out the pretreatment of nitrogen deficiency for 4 days, and then transfer to the nutrient solution containing 4mM potassium nitrate (KNO 3 ) and 2mM (NH 4 ) 2 SO 4 The roots were harvested after 0, 1, 3, 6, 12, and 24 hours of cultivation. A part of the roots was used to determine the rate of ammonium absorption, and the other pa...

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Abstract

The invention discloses a protein ZmAMT1;1 and an encoding gene and application thereof. The protein is derived from corn and related to plant root ammonium salt absorption. The protein is the following protein: (a) protein consisting of amino acid sequences expressed by a sequence 1 in a sequence table; or (b) protein which is derived from the (a), is subjected to substitution and / or deletion and / or addition of one or more amino acid residues from the amino acid residue sequence of the sequence 1 in the sequence table and is related to the corn ammonium salt absorption. The expression of theencoding gene ZmAMT1;1 of the protein in arabidopsis can obviously enhance the ammonium salt absorption capacity of the arabidopsis. The invention provides more characteristic gene resources for researching high-efficiency ammonium salt absorption of crops, and plays important role in researching nutrient high-efficiency performance of genetic engineering improved plants.

Description

technical field [0001] The invention relates to a protein, its coding gene and application in the field of plant genetic engineering, in particular to a protein related to ammonium salt absorption, its coding gene and its application. Background technique [0002] Nitrogen is one of the essential mineral nutrients for plants, and is an essential component for the synthesis of proteins, nucleic acids and many biologically active substances. Nitrogen deficiency often results in stunted plant growth, chlorosis of mature leaves with concomitant accumulation of anthocyanins (Diaz et al., 2006, Plant Cell Physiology 47:74-83). The extensive use of nitrogen fertilizers in agricultural production has greatly increased crop yields. Currently, about 80 to 90 million tons of nitrogen fertilizer are applied globally every year, and it is predicted that it will increase to 240 million tons by 2050 (Tilman et al., 1999, Proc Natl Acad Sci USA96: 5995-6000). The increase in energy costs ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A01H5/00
Inventor 袁力行顾日良段凤莹张福锁
Owner CHINA AGRI UNIV