Japanese lawngrass Na<+>/H<+> antiporter gene ZjNHX1, encoding protein and application thereof

An anti-transporter, Zoysia technology, applied to the Na+/H+ anti-transporter of Japanese Zoysia and its encoding gene and application fields to achieve the effects of increasing yield and improving ecological environment

Inactive Publication Date: 2009-02-04
EAST CHINA NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the invention is to provide Japanese Zoysia Na + / H + Antiporter gene ZjNHX1

Method used

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  • Japanese lawngrass Na&lt;+&gt;/H&lt;+&gt; antiporter gene ZjNHX1, encoding protein and application thereof
  • Japanese lawngrass Na&lt;+&gt;/H&lt;+&gt; antiporter gene ZjNHX1, encoding protein and application thereof
  • Japanese lawngrass Na&lt;+&gt;/H&lt;+&gt; antiporter gene ZjNHX1, encoding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, Japanese Zoysia Na + / H + Cloning of the antiporter gene

[0032] 1. Extraction of total RNA

[0033] Zoysia japonica seedlings were taken, frozen in liquid nitrogen, ground with a mortar, and total RNA was extracted with TRIzol (Invitrogen) reagent.

[0034] 2. Japanese Zoysia Na + / H + Isolation of Antiporter cDNA Fragments

[0035] (1) Synthesis of the first strand of cDNA

[0036] The first strand of cDNA was synthesized using TaKaRa RNA PCR Kit (AMV) Ver.3.0. The reaction system of reverse transcription polymerase chain reaction (RT-PCR) is 10 μL, containing 1 μL RNA template, 1 μL dNTPs (each 10 mM), 2 μL MgCl 2 (25mM), 0.25μL RNase Inhibitor (40U / μL), 1μL Oligo dT-Adaptor Primer (0.25pmol / L), 1μL 10×RT Buffer, 0.5μL Reverse Transcriptase (5U / μL), 3.75μL RNase Free H 2 O. The RT-PCR reaction program was: 42°C for 30 minutes, 99°C for 5 minutes, and 5°C for 5 minutes. The first strand of cDNA was obtained as a template for degenerate PCR. ...

Embodiment 2

[0050] Example 2 ZjNHX1 sequence analysis

[0051] Zoysia japonica + / H + The full-length cDNA of the Antiporter gene includes a 1623bp open reading frame (OpenReading Frame, ORF), a 408-base 5'untranslated region (Non Translated Region, NTR), a 363-base 3'untranslated region and a 27-base base poly-A tail. This cDNA encodes a polypeptide of 540 amino acids, see sequence 2 in the sequence listing.

[0052] Using Clustal for different Na + / H + Amino acid sequence comparison analysis of antiporter, the amino acid sequence of ZjNHX1 and other cloned Na + / H + Antiporters have high homology (such as figure 1 shown), figure 1 The parts in black are consistent amino acids. ZjNHX1 86 LFFIYLLPPI 95 is highly conservative, which is Na + / H + Competitive inhibitor of the amlopyrazine-binding site of antiporters. The GenBank accession numbers of ZjNHX1, DmNHX1, AtNHX1, TrNHX1, OsNHX1, PaNHX1 and AlNHX1 are: ABY19311, ABN71591, AAF21755, ABV00895, BAA83337, BAD95562.1, AAV8...

Embodiment 3

[0054] Example 3 Construction of Yeast Expression Vector

[0055] 1. Design primers according to the nucleotide sequence of the isolated Zoysia japonica antiporter gene ZjNHX1:

[0056] PF2: 5'-CGGAATTCATGGGCCCCGGCGTGGTG-3'

[0057] PR2: 5'-ACGCGTCGACTCACCGTCCTCCATG-3'

[0058] Use 5'-RACE-Ready cDNA as a template for PCR reaction.

[0059] 2. The obtained PCR product was recovered and purified and connected to pGM-T vector, transformed into Escherichia coli, screened with blue and white spots, picked white clones, and sequenced for identification.

[0060] 3. Cut the gene from the pGM-T vector with restriction endonucleases EcorI and SalI, and connect it with the pYPGE15 vector that has been treated with the same restriction enzymes. The ligation product was transformed into Escherichia coli and screened on LB plates containing ampicillin. Pick a single colony and culture overnight in LB liquid medium. The plasmid DNA was extracted, identified by enzyme digestion and PCR...

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Abstract

The present invention discloses a Na / H antiporter gene ZjNHX1 coming from zoysia japonica, an encoding protein and an application thereof. The gene is one of the following nucleotide sequences: (1) SEQ ID No:1 in the sequence table; (2) SEQ ID No:2 protein in the coding sequence table, and a nucleotide sequence 70 percent homologous to the 409th position to 2031th position of SEQ ID No:1 of the sequence table; (3) a nucleotide sequence capable of being hybridized with the 409th position to 2031th position of SEQ ID No:1 sequence in the sequence table under the medium stringency condition. The result of a functional complementation experiment conducted between the ZjNHX1 and the single and the double mutant of salt-sensitive yeast shows that the gene can reinstate the anti-salt capability of the salt-sensitive yeast to a certain degree. The present invention is applicable to the cultivation of novel salt-tolerant plant varieties or other novel genetically modified plant varieties with improved biological characters.

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 in Zoysia japonica + / H + Antiporters and their coding genes and applications. Background technique [0002] Drought and salinity are important limiting factors that endanger plant growth and development, and are also a global problem currently restricting agricultural production. 20% of the world's arable land is threatened by salt damage, and 43% of the arable land is in arid and semi-arid areas. It is particularly important to study the effects of adversity stress on plants and the responses of plants, and to find and use various methods to improve plant stress resistance. The use of genetic engineering technology to breed new varieties of turfgrass resistant to salt and drought can be used in urban and rural beautification, environmental protection, etc., which has huge ecological...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N5/10A01H1/00A01H5/00
Inventor 杜艳华夏涛徐凯
Owner EAST CHINA NORMAL UNIV
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