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Spartina anglica sodium hydrogen pump protein gene SaNHX and application thereof

A sodium-hydrogen pump, rice grass technology, applied in the application, genetic engineering, plant genetic improvement and other directions, to achieve huge economic value and application prospects, improve the effect of salt resistance

Inactive Publication Date: 2008-03-26
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on the gene of sodium hydrogen pump protein in halophytes belonging to the same family as rice has not been reported yet.
Before the publication of the present invention, there has not been any disclosure or report of the protein sequence of the gramineous halophyte Spartina hydrogen pump protein sequence and its nucleic acid sequence mentioned in this patent application

Method used

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  • Spartina anglica sodium hydrogen pump protein gene SaNHX and application thereof
  • Spartina anglica sodium hydrogen pump protein gene SaNHX and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Cloning of Spartina Grass SaNHX gene and its sequence information and homology analysis

[0024] 1. Tissue and RNA Isolation

[0025] Spartina comes from Luoyuan Bay, Fujian Province. Part of the tissue was taken, crushed with a mortar, added to a 50 mL tube filled with lysate, and fully shaken, then total RNA was extracted (TRIzol Reagents, Invitrogen, NY, USA).

[0026] 2. Full-length cDNA clone of SaNHX

[0027] According to the conserved amino acid sequence of rice sodium hydrogen pump protein, primers were designed, and using the principle of homologous gene cloning, the full-length cDNA clone was carried out by using the RACE method (Invitrogen kit), which was carried out in three stages:

[0028] (1) 3′-RACE

[0029] 2003SN3' (793bp) was obtained by PCR (AUAP+GSP3-3), recovered, cloned and sequenced. The sequencing results were BLASTed on NCBI, and its nucleic acid sequence and encoded protein were known to have high homology with the known model p...

Embodiment 2

[0039] Example 2: Southern hybridization analysis of Spartina SaNHX gene

[0040] Genomic DNA of Spartina was extracted according to the method of Paterson et al. (1993). Take 1 μg of genomic DNA and digest with EcoRI and HindIII respectively. Digested products were separated by 0.7% agarose gel electrophoresis (30-50V, O / N), and then the gel was treated on a shaker: 0.125N HCl soaking for 15min, denaturing solution (1.5M NaCl, 0.5N NaOH) soaking 30min, soak in neutralizing solution (1.5MNaCl, 0.5M Tris-HCl pH 7.2) for 30min. Finally the DNA was blotted to HybondN with 20×SSC + on nylon membrane.

[0041] Pre-hybridization and hybridization were carried out according to conventional methods (Jin Dongyan et al., 1996; Hybond N + mannual, Amersham).

[0042] Probe and its label: The probe used for hybridization is derived from a DNA fragment about 800 bp in the cDNA of SaNHX gene. Labeling was performed overnight with the Primer-a-Gene kit from Promega.

[0043] The membran...

Embodiment 3

[0046] Example 3: Northern hybridization analysis of Spartina SaNHX gene

[0047] After cultivating Spartina with Hoagland nutrient solution for 5 days, NaCl was added to a final concentration of 400mmol / L, and the stress time was 0, 1, 3, 6, 10 and 24 hours, respectively. The total RNA of the leaves was extracted for Northern analysis. The results are shown in Figure 3 . It can be seen from the figure that the expression of SaNHX was induced by salt stress, and reached a high level after 6 hours of stress, the expression level was still high after 10 hours of stress, and the expression level was high after 24 hours, but decreased. The expression level of the untreated control was also present, but lower. This result is basically consistent with the semi-quantitative RT-PCR results in time.

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Abstract

The present invention is cord grass sodium hydrogen pump protein gene SaNHX and its application. The sodium hydrogen pump protein gene SaNHX has the DNA sequence as shown in sequence 1 of the sequence list or the DNA sequence with over 90 % homology with the sequence 1 limited DNA sequence and coding protein with the same function. The gene SaNHX coded cord grass sodium hydrogen pump protein is protein with the amino acid residue sequence as shown in sequence 2 of the sequence list or derivative protein of the sequence 2 through the substitution, deletion or addition of one or several amino acid residues and with the same activity as the protein with the amino acid residue sequence in sequence 2. The gene of the present invention is significant in cultivating new variety of salt tolerant crop, especially salt tolerant rice.

Description

technical field [0001] The invention relates to the fields of molecular biology, enzymology, physiology, genetic engineering and the like. Specifically, the present invention relates to a sodium-hydrogen pump protein (Spartina anglica) expressed in Spartina anglica + / H + antiporter, SaNHX) and nucleic acid sequences and their applications. Background technique [0002] There are 1 billion hectares of saline-alkali soil in the world, accounting for 7% of the land area (Szaboles, 1994); 7.7 million hectares of arable land are affected by salinity, accounting for about 5% of the arable land area (Munns et al., 1999). Salt damage has a great impact on the yield of crops, resulting in a substantial decline in yield. [0003] When the salt content in the soil is too high, it will cause salt stress to plants, which will harm the normal growth and development of plants. It is mainly manifested in two aspects: first, the water shortage caused by the high solute concentration in ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N5/10
Inventor 兰涛段远霖吴为人周元昌
Owner FUJIAN AGRI & FORESTRY UNIV
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