Plant strong salt-resistant gene AtNHXS1 and its coding protein and application

A salt-tolerant gene and plant technology, applied in the field of plant genetic engineering, can solve unreported problems and achieve the effect of improving salt resistance

Inactive Publication Date: 2008-09-10
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of this technology to improve the performance of the Na+ / H+ antiporter gene and its protein has not been reported at home and abroad.

Method used

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  • Plant strong salt-resistant gene AtNHXS1 and its coding protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Arabidopsis Na + / H + Cloning of the antiporter gene

[0031] Arabidopsis thaliana total RNA was extracted from the young leaves of Arabidopsis thaliana with TRizol reagent, and the total RNA was used as a template for reverse transcription to synthesize the first strand of cDNA. Using the synthesized cDNA as a template and referring to the cDNA sequence of AtNHX1, the following two primers were designed (the 5' end contains SmaI and SalI restriction sites respectively):

[0032] AtR(5'-GC GTC GAC TCAAGCCTTACTAAGATCAGGAGG-3')

[0033] AtF(5'-TCC CCCGGG ATGTTGGA TTCTCTAGTG-3')

[0034] Arabidopsis Na + / H + The antiporter gene AtNHX1 was connected to the pGM-T vector and transformed into Escherichia coli DH5a, and the sequence of the cloned AtNHX1 was confirmed by sequencing.

Embodiment 2

[0035] Example 2. DNA shuffling of the AtNHX1 gene

[0036] 1) Preparation of starting materials Using pGM-T-AtNHX1 as a template, AtNHX1 was amplified by PCR with Taq DNA polymerase, purified and used as starting materials for DNA shuffling.

[0037] 2) Random enzyme digestion with DNase I Take 10 μg of purified AtNHX1 and add it to 50 μl enzyme digestion reaction system (10 mM Tris-HCl, pH7.4, 50 mM MnCl 2 ), react at 15°C for 10 min. Add DNase I0.15U, mix well, 15°C, 2min, 90°C, 10min. The product goes through 2.5% agarose gel electrophoresis, and the gel containing 100-200bp fragments is excised and recovered.

[0038] 3) PCR without primers Take 1 μg of recovered small fragments and add to 50 μl reaction system (10×Pfubuffer, 0.2 mM each dNTP, 0.6 U / μl Pfu polymerase). The PCR program was reaction conditions: pre-denaturation at 94°C for 60s, denaturation at 94°C for 30s, annealing at 50°C for 30s, extension at 72°C for 30s, a total of 40 cycles, and finally extension a...

Embodiment 3

[0041] Example 3. Construction of expression shuffling library and resistance to high salt Na + / H + Screening for antiporter genes

[0042] The recombinant product obtained in Example 2 was digested with Sal I and Sma I, purified and connected to the same digested yeast expression vector pYPGE15, and the constructed expression shuffling library was introduced into the yeast mutant strain W303-1BΔena1- 4::HIS3Δnhx1::TRP1, spread on the APG selection medium containing 100mM NaCl (without uracil), culture at 30°C for 2 days, and carry out high-salt Na + / H + High-throughput screening of antiporter genes. Finally, a normal-growing yeast mutant containing a new plant strong salt-tolerant gene AtNHXS1 was obtained.

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Abstract

The invention provides a novel plant strong salt tolerant gene AtNHXS1 acquired through DNA reshuffling technology, and the ionic transport activity of Na+/H+ antiporter protein coded by the gene is stronger than that of wild Na+/H+ antiporter protein ATNHX1. A nucleotide sequence of the gene as shown in SEQ ID NO:1 also comprises genes of the nucleotide sequence of sequence 1 in a sequence table with the homology between 70 and 100 percent, or a nucleotide sequence of an amino acid sequence of sequence 2 in a coded sequence table. The new Na+/H+ antiporter has a protein of the amino acid sequence of sequence 2 in the sequence table, protein of the amino acid sequence of the sequence 2 with the homology between 70 and 100 percent, or protein which replaces, deletes or adds one or a plurality of amino acids in the amino acid sequence of the sequence 2 with the same homology. The invention also provides the construction of recombinant vector, the transgenic plant and other methods so as to apply the gene and the protein, thereby cultivating a novel variety of transgenic plant with strong capacity of salt tolerance or other improved biologic characters.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to the use of DNA shuffling technology to obtain significantly improved plant Na + / H + The antiporter gene, the invention also relates to the use of the gene to breed transgenic salt-tolerant plants. Background technique [0002] Soil salinization is an important abiotic stress factor affecting crop production and ecological environment. At present, about 20% of the cultivated land and nearly 50% of the irrigated land in the world are seriously harmed by salinity (Flowler T J, Yeo A R. Breeding for salinity resistance in crop plants. Where next? Aust.J.Plant Physiol.1995 , 22: 875-884). There are about 500 million mu of saline-alkali land in my country, and its area has a tendency to increase continuously. Cultivating salt-tolerant plants is an effective way to promote the development and utilization of saline-alkali land, improve soil and ecological management. Theref...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/63C07K14/415A01H1/00
Inventor 夏涛徐凯洪平
Owner EAST CHINA NORMAL UNIV
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