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A tamarisk plasma membrane na + /h + antiporter gene and its application

A tamarisk quality and gene technology, applied in tamarix plasma membrane Na+/H+ antiporter gene and its application field, can solve problems that have not been seen, achieve the effect of improving salt tolerance and good application prospects

Active Publication Date: 2022-01-25
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current studies on halophyte plasma membrane Na + / H + The researches on antiporter gene in salt-tolerance function regulation and plant genetic engineering improvement mainly focus on some euhalophytes. There are few studies on this gene in halophytes such as tamarisk. The effect of this gene on improving soybean , cotton, Arabidopsis and other crops or plants, and the physiological effects of yeast salt tolerance, there is no report in this area after searching

Method used

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  • A tamarisk plasma membrane na <sup>+</sup> /h <sup>+</sup> antiporter gene and its application
  • A tamarisk plasma membrane na <sup>+</sup> /h <sup>+</sup> antiporter gene and its application
  • A tamarisk plasma membrane na <sup>+</sup> /h <sup>+</sup> antiporter gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Tamarix plasma membrane Na + / H + Cloning of antiporter gene

[0038] The RNA of Tamarix multibranches (from the Turpan Desert Botanical Garden, Chinese Academy of Sciences, Xinjiang) was extracted and used as a template to synthesize the first strand of cDNA. + / H + According to the alignment results of the antiporter gene sequences, the primers for the middle fragment were designed, and the primer sequences are as follows:

[0039] MS-F: 5'-ATGAATGATGGGACNGCNAT-3';

[0040] MS-R: 5'-ANNGCACTTTTCCTGCCANA-3';

[0041] N: A / G / C / T.

[0042] According to the RACE System for Rapid Amplification of cDNA Ends of Invitrogen Company, the Primer Premier5 software was used to design primers. According to the results of RACE, the CDS region of the gene was determined, and the full-length primer sequence was designed as follows:

[0043] F: 5'-ATGGCAGCGGTGTCCGAATT-3';

[0044] R: 5'-TCAAGAAGCATGACGGAAAGATAGC-3'.

[0045] Cloning was carried out with PrimeSTAR ...

Embodiment 2

[0047] Tamarix plasma membrane Na + / H + The antiporter gene was transformed into Arabidopsis WT and mutant plants, the main process is as follows:

[0048] 1. Construction of Arabidopsis overexpression vector

[0049] The Tamarix plasma membrane Na that cloned out in embodiment 1 + / H +The antiporter full-length gene was used as a template, and PrimeSTARGXL DNA Polymerase was used for cloning. The system was as follows (50 μL): 10 μL 5×PrimeSTAR GXL Buffer, 4 μL dNTP Mixture (2.5 mM each), 1 μL 1300-F, 1 μL 1300-R, 1 μL PrimeSTAR GXL DNA Polymerase, 1 μL template plasmid, ddH 2 O supplemented to 50 μL. in,

[0050] 1300-F:

[0051] 5'-GTACCCGGGGATCCTCTAGAATGGCAGCGGTGTCCGAATT-3';

[0052] 1300-R:

[0053] 5'-ATGCCTGCAGGTCGACTCTAGATCAAGAAGCATGACGGAAAGATAGC-3';

[0054] (2) PCR reaction program: 98°C for 3min; 98°C for 10s, 60°C for 15s, 68°C for 3min40s, 30 cycles; 68°C for 10min; 4°C for heat preservation

[0055] (3) Extraction of vector plasmid

[0056] The plas...

Embodiment 3

[0085]Using the overexpression transformation method mediated by Agrobacterium rhizogenes K599, the plasma membrane Na + / H + The transformation of antiporter gene, the main process is as follows:

[0086] 1. Transformation of Agrobacterium rhizogenes

[0087] (1) Take the competent Agrobacterium out of the -80°C refrigerator, put it on ice to melt, add the recombinant plasmid in Example 2, flick the tube wall to mix well, and put it on ice for 30 minutes. (2) Place the competent state in liquid nitrogen for rapid cooling for 90 seconds, quickly transfer it to a 37°C water bath for 60 seconds, and then transfer it to ice for 5 minutes. (3) Add 900 μL of LB liquid medium to the competent cells, and culture at 200 rpm in a constant temperature shaker at 28° C. for 2-4 hours. (4) Take 200 μL of the bacterial liquid and spread it on the LB solid medium containing Cannabidiol, and incubate at 28°C for 16 hours. (5) Pick a single colony and carry out the colony or bacterial liq...

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Abstract

The invention discloses a tamarisk plasma membrane Na + / H + The antiporter gene and its application belong to the technical field of genetic engineering. The Tamarix plasma membrane Na + / H + The nucleotide sequence of the antiporter gene is shown in SEQ ID No.1, and the amino acid sequence of the expressed protein is shown in SEQ ID No.2. The present invention clones Tamarix plasma membrane Na for the first time from Tamarix + / H + antiporter gene, through transformation of Arabidopsis, soybean, cotton and yeast, overexpressed Tamarix plasma membrane Na + / H + Arabidopsis thaliana, soybean, cotton and yeast with antiporter gene have lower salt tolerance than untransformed or transformed soybean, cotton or Arabidopsis plasma membrane Na + / H + antiporter gene was higher, indicating that Tamarix plasma membrane Na + / H + The antiporter gene has an outstanding effect in the application of improving the salt tolerance of plants or yeast, and has important application value in the field of improving the salt tolerance breeding of plants or strains.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a tamarisk plasma membrane Na + / H + Antiporter gene and its application. Background technique [0002] Soil salinization and desertification have become one of the main abiotic adversity factors that lead to the decrease of available arable land in the world and affect the growth and development of crops, and limit the improvement of yield and quality. They have become one of the worldwide problems that limit modern agricultural production and sustainable development. one. According to incomplete statistics, about 7% of the world's total land area is saline soil, and about 22% of irrigated land is being affected by increasing salt damage (Bhatnagar-Mathur et al., 2008). High concentrations of salt in the growth environment can cause plant cell osmotic and ion stress, nutrient imbalance and oxidative damage, and affect metabolic processes, resulting in cell ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H6/20A01H6/54A01H6/60
CPCC07K14/415C12N15/8273
Inventor 於丙军刘咏梅程聪车本宁刘询安靖
Owner NANJING AGRICULTURAL UNIVERSITY
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