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Salicornia europaea SePSS protein, and coding gene and application thereof

A coding gene and coding technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as cell osmotic stress, ion balance disruption, poisoning, etc.

Active Publication Date: 2019-12-31
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the plasma membrane of the cell is depolarized, the ion channel on the membrane is opened and loses its specificity, which leads to changes in the ion concentration in the cell and destroys the ion balance inside the cell, thus causing osmotic stress and ion toxicity to the cell.

Method used

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  • Salicornia europaea SePSS protein, and coding gene and application thereof
  • Salicornia europaea SePSS protein, and coding gene and application thereof
  • Salicornia europaea SePSS protein, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1, discovery of Salicornia sePSS protein and its coding gene

[0077] Total RNA was extracted from aerial parts of Salicornia praecox and reverse transcribed into cDNA. After a large number of sequence analysis, expression analysis and functional verification, a DNA coding sequence of phosphatidylserine synthase was found from cDNA, as shown in sequence 2 of the sequence listing, and its encoded protein is shown in sequence 1 of the sequence listing .

[0078] The protein shown in Sequence 1 of the sequence listing is named SePSS protein, which consists of 425 amino acid residues, and has nine transmembrane domains without signal peptide. The gene encoding SePSS protein is named as SePSS gene, and its full-length CDS is 1728bp.

Embodiment 2

[0079] Embodiment 2, the expression analysis of SePSS gene

[0080] After 30 days of sowing Salicornia seeds, the seedlings with consistent growth were taken and cultured for different time (0, 3, 6, 12, 24, 72 or 168 hours), and the aerial parts were taken for gene expression detection.

[0081] Extract the total RNA from the aerial part, reverse transcribe it into cDNA, use the cDNA as a template, and use real-time quantitative PCR (Real-time quantitative PCR, qRT-PCR) to detect the expression of SePSS gene (use primers SePSS-qPCR-Forward and The primer pair composed of primer SePSS-qPCR-Reversed was used for detection), and the α-tubulin gene was used as an internal reference (the primer pair composed of primer α-tubulin-qPCR-Forward and primer α-tubulin-qPCR-Reversed was used for detection).

[0082] α-tubulin-qPCR-Forward: 5'-CGAAAGTTGGGGGCTCGAAG-3';

[0083] α-tubulin-qPCR-Reversed: 5'-CCCCGGAACCCAAAGACTTTG-3';

[0084]SePSS-qPCR-Forward: 5'-GCGATGATGCTCGGTTGTTC-3'; ...

Embodiment 3

[0087] Example 3, SePSS protein subcellular localization

[0088] 1, pCAMBIA1300-35S::SePSS-GFP carrier: Insert the double-stranded DNA molecule described in Sequence 2 of the sequence listing between the BamHI restriction sites of pCAMBIA1300-35S::GFP vector to obtain pCAMBIA1300-35S::SePSS- GFP vector (sequenced and verified).

[0089] 2. pCAMBIA2300-35S::HDEL-mCherry vector: use the double-stranded DNA molecule described in sequence 3 (HDEL-mCherry fragment) of the sequence listing to replace the fragment between the SalI and HindIII restriction sites of the pCAMBIA2300 vector to obtain pCAMBIA2300- 35S::HDEL-mCherry vector (verified by sequencing).

[0090] 3. Introduce the pCAMBIA1300-35S::SePSS-GFP vector into C58 Agrobacterium to obtain recombinant bacteria 35S::SePSS-GFP. The recombinant bacteria 35S::SePSS-GFP was inoculated in LB liquid medium containing 20μM AS, 10mM MES, 100μM Kan and 100μM Rif and shaken (28°C, 180rpm) to OD 600 = 1.8. After the culture system...

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Abstract

The invention discloses a salicornia europaea SePSS protein, and a coding gene and application thereof. The invention proves for the first time that the SePSS gene of salicornia europaea is induced and expressed by salt through experiments, and an expression level is highest when treated with 200 mM NaCl for 12 h and 800 mM NaCl for 24 h. The SePSS gene is used to complement an Arabidopsis thaliana atpss mutant, phenotypes such as PS content, cytoplasmic membrane polarity, chloroplast content, fresh weight, and root length of the complementary strain are all restored, and the restoration effect is the same as that of the AtPSS gene. Under 150 mM NaCl treatment conditions, a fresh weight of a SePSS-overexpression strain is significantly higher than that of wild-type Arabidopsis thaliana. The results show that SePSS has the PS synthesis function and is very important for maintaining polarity of cytoplasmic membrane, and overexpression of the SePSS gene can improve salt tolerance of Arabidopsis thaliana. The work provides a theoretical basis and genetic resource for genetic improvement of stress-resistant crops.

Description

technical field [0001] The invention relates to a salt hornwort SePSS protein and its coding gene and application. Background technique [0002] Soil salinization is a widespread problem worldwide. Saline-alkali land poses a serious threat to agricultural production and restricts economic development. People have taken many measures to improve the saline-alkali land, including the use of traditional agricultural methods such as water conservancy projects, but the effect is not significant. With the development of biological science and technology, the use of genetic engineering technology to improve crops and improve the salt tolerance of crops has become an effective way to use saline-alkali land. [0003] The plasma membrane is a barrier for the exchange of substances inside and outside the cell, and plays an important role in maintaining a stable intracellular environment, nutrient absorption, cell recognition, and signal transmission. The plasma membrane of the cell g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/12C12N15/54A01H5/00A01H6/20
CPCC12N9/1288C12N15/8273C12Y207/08008
Inventor 李银心郭杰台方吕素莲
Owner INST OF BOTANY CHINESE ACAD OF SCI
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