Novel plant strong-salt resistance gene NHXFS1, encoding protein and use thereof

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

Inactive Publication Date: 2009-04-22
EAST CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the application of this technology to improve the performance of the N

Method used

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  • Novel plant strong-salt resistance gene NHXFS1, encoding protein and use thereof
  • Novel plant strong-salt resistance gene NHXFS1, encoding protein and use thereof
  • Novel plant strong-salt resistance gene NHXFS1, encoding protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1. Arabidopsis, rice, chrysanthemum Na + / H + Cloning of the antiporter gene

[0039] Total RNA was extracted from the young leaves of Arabidopsis thaliana, rice and chrysanthemum with TRizol reagent, and reverse transcription was performed using the total RNA as a template to synthesize the first strand of cDNA. Using the synthesized cDNA as a template and referring to the cDNA sequences of AtNHX1, OsNHX1, and DmNHX1, the following primers were designed (the 5' end contains SmaI and SalI restriction sites respectively):

[0040] AtR(5'-GC GTC GAC TCAAGCCTTACTAAGATCAGGAGG-3')

[0041] AtF(5'-TCC CCCGGG ATGTTGGA TTCTCTAGTG-3')

[0042] OsR(5'-ACGC GTC GAC TCATCTTCCTCCATGGC-3')

[0043] OsF(5'-TCC CCCGGG ATGGGGATGGAGGTGGCG-3')

[0044] DmR (5'-GC GTC GAC TTAGTTTCTTTCTTTCATCTTC-3')

[0045] DmF(5'-TC CCCCGG GATGGTGTTCGATTC-3')

[0046] Arabidopsis Na + / H + Antiporter gene AtNHX1, rice Na + / H + Antiporter genes OsNHX1 and chrysanthemum Na...

Embodiment 2

[0047] Example 2. DNA shuffling of AtNHX1, OsNHX1, DmNHX1 genes

[0048] 1) Preparation of starting materials Using pGM-T-AtNHX1, pGM-T-OsNHX1, and pGM-T-DmNHX1 as templates, use PfuDNA polymerase to PCR amplify the three genes, and use them as starting materials for DNA shuffling after purification .

[0049] 2) Random enzyme digestion with DNase I Determine the concentration of purified DNA by ultraviolet absorption method, mix AtNHX1, OsNHX1, and DmNHX1 with a content of 9ug, so that the ratio of the three gene substances is 1:1:1. Take 40ul of mixed AtNHX1, OsNHX1, DmNHX1 and add it to 50μl enzyme digestion reaction system (10mM Tris-HCl, pH7.5, 50mM MnCl 2 ), react at 15°C for 10 min. Add DNase I 0.15U, mix well, 15°C, 1min16s, 90°C, 10min. The product is electrophoresed through 2.5% agarose gel, and the gel containing 100-200bp fragments is excised and recovered.

[0050] 3) PCR without primers Take 1 μg of recovered small fragments and add to 50 μl reaction system (1...

Embodiment 3

[0053] Example 3. Construction of expression shuffling library and resistance to high salt Na + / H + Screening of antiporter gene NHXFS1

[0054] 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 W303-1B△ by the lithium acetate method ena1-4::HIS3 △nhx1::TRP1, spread on the APG selection medium without uracil, culture at 30°C for 2 days, and select Na + / H + The yeast strain of the antiporter gene was further screened by increasing the NaCl concentration, and finally a normal growth yeast mutant was obtained on the selection medium with a concentration of 200mM NaCl, which contained a new plant Strong salt tolerance gene NHXFS1.

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Abstract

The invention relates to the field of plant genetic engineering and provides a novel plant strong salt-tolerant gene NHXS1 obtained by a restructuring technology. A nucleotide sequence of the plant strong salt-tolerant gene NHXS1 is SEQ ID NO: 1, or a DNA sequence which has 70 to 100 percent homology with the SEQ ID NO: 1 nucleotide sequence or a DNA sequence for coding a protein sequence of SEQ ID NO: 2. Na + / H + antiporter NHXS1 colded by the gene has stronger ion transport activity than the wild-type Na + / H + antiporter AtNHX1. The invention also provides methods for construction of recombinant vectors and transgenic plants to apply the genes and proteins, and can culture a novel breed of transgenic plant with stronger salt-tolerant property or other improved biological characteristics.

Description

technical field [0001] The present invention relates to the field of plant genetic engineering, in particular to the use of DNA family 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 mana...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/63C12N5/10C07K14/415A01H1/00
CPCC12N15/8273C07K14/705
Inventor 夏涛张辉徐凯
Owner EAST CHINA NORMAL UNIVERSITY
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