White clover Na+/H- antiport protein gene TrNHX1, encoding protein and use thereof

A technology of antiporter and white clover, applied to the Na+/H+ antiporter of white clover and its coding gene and application field

Inactive Publication Date: 2008-12-17
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the invention is to provide white clover Na + / H + antiporter gene TrNHX1

Method used

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  • White clover Na+/H- antiport protein gene TrNHX1, encoding protein and use thereof
  • White clover Na+/H- antiport protein gene TrNHX1, encoding protein and use thereof
  • White clover Na+/H- antiport protein gene TrNHX1, encoding protein and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, white clover Na + / H + Acquisition of genes encoding antiporters

[0036] 1.1 Design of degenerate primers:

[0037] By comparing the cloned Na in organisms + / H + Antiporter amino acid sequence, find the conserved region, and design a pair of degenerate primers based on the conserved region sequence:

[0038] jbNHX1: 5'-CC(A / T)CC(G / C)AT(C / T)AT(A / C)TTCAATGCA GG(C / G / T)TTTCA-3'

[0039] jbNHX1: 5'-(T / A / C)ACAACACC(C / T)TC(A / G / T)CC(A / G)AA(G / T)AC(A / C)AGACTGTA-3'

[0040] 1.2 Extraction of total RNA from white clover

[0041] Trizol kit (Invitrogen) was used to extract RNA from white clover (Trifolium repens L.) seedlings (sowed in common soil of vermiculite:perlite=3:1, grown at room temperature), extracted with chloroform, Precipitate with isopropanol, wash with 75% ethanol, dissolve in 50 μL DEPC water after natural drying, put in water bath at 55°C for 10 minutes, and then store at -20°C. The concentration and quality of RNA were determined by UV spec...

Embodiment 2

[0049] Embodiment 2, white clover Na + / H + Antiporter gene TrNHX1 and white clover Na + / H + Antiporter full sequence analysis

[0050] The full length of the sequence is 2394bp, which has the DNA sequence of SEQ ID No.1, 451-2076bp is its complete coding region, and the encoded protein contains 541 amino acid residues, and has the amino acid residue sequence of SEQID No.2.

[0051] The cDNA includes an open reading frame (Open Reading Frame, ORF) of 1626 bp, a 5' non-translated region (Non Translated Region, NTR) of 450 bases, a 3' untranslated region of 289 bases and a 29-base polyA tail. The gene was named TrNHX1.

[0052] White Clover Na + / H + The amino acid sequence of the antiporter TrNHX1 and other cloned Na + / H + Antiporters have high homology. In order to analyze different Na + / H + For the genetic relationship between the antiporters, the inventors conducted a phylogenetic analysis and analyzed the amino acid sequence with Phylip software. Find some r...

Embodiment 3

[0065] Embodiment 3, construction of expression vector TrNHX1-pYPGE15

[0066] 3.1 According to the isolated white clover Na + / H + For the nucleotide sequence of the antiporter gene TrNHX1, SmaI and SalI restriction site sequences and several protective bases are added at both ends of the designed primers to facilitate subsequent connection with the carrier:

[0067] TrNHX1-F: 5'-TCC CCCGGG ATGGCTATTGAAATT-3'

[0068] TrNHX1-R: 5'-ACGC GTC GAC TTAACGCCATAAATTACC-3'

[0069] The cDNA synthesized by the 5'-RACE reaction was used as a template for PCR reaction.

[0070] 3.2 The product after the PCR reaction was subjected to agarose gel electrophoresis and recovered with the Nucleotrap Gel Extraction Kit (Clontech). Take 5 μL of the recovered product and connect it to the pGM-T vector. The operation steps were carried out according to the instructions of the TIANGEN product pGM-T cloning kit. . Then Escherichia coli DH5α strain was transformed and grown overnight on LB ...

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Abstract

The invention discloses a Trifolium repens.L Na<+> / H<+> antiporter protein gene TrNHX1 coming from the Trifolium repens.L, and the coded protein and the application thereof. The gene is one of the following nucleotide sequences: (1) SEQ ID No: 1 in a sequence table; (2) the polynucleotide of an SEQ ID No: 2 protein sequence in a coded sequence table; (3) a DNA sequence of functional protein with the same code, which has the homology by at least 70 percent with the limited DNA sequence of the SEQ ID No: 1 in the sequence table. The results of the functional complementation assay of TrNHX1 with salt-sensitive single and double mutants of yeast prove that the gene can recover the salt resistance to a certain extent. The gene of the invention can be applied to the transformation of the Trifolium repens.L, leguminous plants and other plants as well as the culture of new salt-resistant varieties of the plants or other new varieties of transgenic plants, the biological characters of which are improved.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, specifically a Na + / H + Antiporters and their coding genes and applications, especially involving Na + / H + Antiporters and their coding genes and applications. Background technique [0002] In nature, there are obvious differences in the adaptation of plants to salt. There are salt-sensitive sweet soil plants and salt-tolerant halophytes. Although some halophytes require Na for growth + , but too much Na in the soil + remains one of the major limiting factors for plant growth in the world's vast salt wastelands. The harm of salt to plants includes two aspects: first, the water deficit caused by the high solute concentration in the soil; second, the ion poisoning caused by excessive salt ions absorbed by plants while absorbing water, especially Na + poison. Although halophytes can adapt to high-salt environments, the enzymes in their cytoplasm cannot adapt to high Na + level, wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/63C12N15/82C12N5/10A01H1/00A01H5/00
Inventor 唐睿夏涛徐凯李翠
Owner EAST CHINA NORMAL UNIV
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