Buckwheat Na+/H+ antiporter FtNHX and coding gene and application thereof

An antiporter, buckwheat technology, applied in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2010-09-08
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Another object of the present invention is to

Method used

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  • Buckwheat Na+/H+ antiporter FtNHX and coding gene and application thereof
  • Buckwheat Na+/H+ antiporter FtNHX and coding gene and application thereof
  • Buckwheat Na+/H+ antiporter FtNHX and coding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Buckwheat Na + / H + Acquisition of genes encoding antiporters

[0036] 1.1 Design of degenerate primers

[0037] By comparing the cloned Na in organisms + / H + The amino acid sequence of the antiporter, looking for the conserved region, and designing a pair of degenerate primers based on the sequence of the conserved region:

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

[0039] dpNHXR: 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 buckwheat total RNA

[0041] RNA was extracted from buckwheat (F. tataricum Gaertn) using RNAiso Reagent Kit (TAKARA Code: D312). The concentration and quality of RNA were determined by UV spectrophotometry, and the integrity of RNA was detected by electrophoresis on agarose denaturing gel containing formaldehyde.

[0042] 1.3 Buckwheat Na + / H + Amplification of antiporter-specific small fragments

[0043] The first-strand cDNA of buckwheat wa...

Embodiment 2

[0050] Embodiment 2 Buckwheat Na + / H + Analysis of the Complete Sequence of the Antiporter Gene FtNHX

[0051] The sequence is 2052bp in full length, has the DNA sequence of SEQ ID No.1, and 288-1949bp is its complete coding region. The encoded protein contains 553 amino acid residues and has the amino acid residue sequence of SEQ ID No.2.

[0052] The cDNA includes an open reading frame (Open Reading Frame, ORF) of 1662bp, a 5' non-translated region (Non Translated Region, NTR) of 287 bases, a 3' untranslated region of 75 bases and a 28-base polyA tail. The gene was named FtNHX, and the encoded protein was named FtNHX.

[0053] The amino acid sequence of FtNHX is similar to other cloned Na + / H + Antiporters have high homology. Prediction of FtNHX with all Na + / H + The common structural features of antiporters include a hydrophobic N-terminal, 8 transmembrane regions and a C-terminal hydrophilic region with regulatory functions, among which LFFIYLLPPI is highly cons...

Embodiment 3

[0056] Example 3 Construction of expression vector FtNHX-pYPGE15

[0057] 3.1 According to the isolated buckwheat Na + / H + For the nucleotide sequence of the antiporter gene FtNHX, Xbal and Sal I enzyme cleavage site sequences and several protective bases are added to the two ends of the designed primer to facilitate subsequent connection with the carrier:

[0058] FTNHX-F: 5'-GC TCTAGA ATGTCGACCCTCATCGATCTT-3'

[0059] FTNHX-R: 5'-GC GTC GAC ATGACTTCACTGAACATGATG-3'

[0060] The cDNA synthesized by the 5'-RACE reaction was used as a template for PCR reaction.

[0061] 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 L...

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Abstract

The invention relates to the field of plant gene engineering, providing a buckwheat Na+/H+ antiporter FtNHX. The buckwheat Na+/H+ antiporter FtNHX is provided with SEQ ID No:2 amino acid residue sequence in a sequence table, or the buckwheat Na+/H+ antiporter FtNHX is obtained by the substitution, deletion or adding of one or multiple amino acid residues by SEQ ID No: 2 in the sequence table, has the same activity with the SEQ ID No:2 amino acid residue sequence and is derived by SEQ ID No:2. The gene for coding the protein is preferably selected from a nucleotide sequence comprising the 288th-1949th bit of SEQ ID No:1, or the gene is a nucleotide sequence which has more than 90% of homology with a DNA sequence specified by the SEQ ID No:1 and codes same functional protein. The invention also provides methods for recombinant vector construction, transgenic plant and the like to apply the above gene and protein, and can cultivate new improved gene plant species with stronger salt tolerance or other biological characters.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and relates to an important ion transporter and its coding gene and application, specifically a Na + / H + Antiporters and their coding genes and applications, especially involving buckwheat Na + / H + Antiporters and their coding genes and applications. Background technique [0002] Salt damage is an important limiting factor of crop yield. Understanding the salt-resistant mechanism of plants, cloning salt-resistant genes and transferring them to salt-sensitive crops, and cultivating new salt-resistant transgenic varieties are of great significance to China, which has more than 500 million mu of salt wasteland. [0003] In order to resist salt stress, plants have formed a series of salt-tolerant mechanisms, mainly through the accumulation of small molecules of non-toxic organic solutes and ions in the cytoplasm, and the compartmentalization in the vacuole to achieve osmotic balance. In...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A01H5/00
Inventor 李翠黑倩徐凯张辉夏涛
Owner EAST CHINA NORMAL UNIV
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