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Couchgrass sodium ion reverse transport protein gene and its cloning method and use

A technology of antiporter protein and sodium ions, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as ineffective effects

Inactive Publication Date: 2005-08-17
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Early salt-tolerant genetic engineering mainly focused on scavenging free radicals and increasing osmotic adjustment substances. The salt-tolerant ability of transgenic plants has improved, but the effect is not obvious

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0087] Implementation mode 1: Elytrigia Na + Cloning method of antiporter gene

[0088] 1. Extraction of total RNA: use one-step method or RNA kit to extract total RNA

[0089] 2. Synthesis of the first strand of cDNA: Take 2 micrograms of total RNA, add 4 microliters of 5× reaction buffer, 10mM deoxyribonucleic acid (dNTP) 2 microliters, ribonuclease inhibitor (40-200u / microliter) 0.5 microliters, primer oligodT (1μg / μl) 1μl, reverse transcriptase (10u / μl) 2μl, react at 42°C for 60 minutes, stop the reaction at 85°C, and dilute to 200μl.

[0090] 3. PCR reaction: Polymerase chain reaction (PCR) reagents and conditions are:

[0091] First mix the following reagents together:

[0092] 10× reaction buffer 5μl

[0093] Deoxynucleotide Mix (dNTP) 4μl

[0094] Forward primer (5μM) 4μl

[0095] Reverse primer (5μM) 4μl

[0096] Template cDNA 4μl

[0097] TaqDNA polymerase 0.5μl

[0098] Total volume 50μl

[0099]The PCR reaction conditions are: 94°C for 3 minutes, then enter the follow...

Embodiment approach 2

[0105] Implementation mode 2: Elytrigia Na + The antiporter gene ENHX1 has the following sequence:

[0106] (1) SEQ ID NO 1 information

[0107] (a) Sequence characteristics

[0108] * Length: 1955 base pairs

[0109] * Type: Nucleic Acid

[0110] * Chain type: Double chain

[0111] * Topological structure: linear

[0112] (b) Molecular type: cDNA

[0113] (c) Assumption: No

[0114] (d) Antonym: No

[0115] (e) Original source: Elytrigia

[0116] (f) Sequence description: SEQ IN NO.1

[0117] 1 ATCCGCCGAG GTGGCGACCG GCATGGGGCT CGATTTGGGA GCCATCGCTC TCAAGTACAC-60

[0118] 61 GGGGCTGGCG GTGTCGGACC ACGGCTCCAT CGTCGCCATC AACATCTTCA TCGCGCTGCT-120

[0119] 121 CTGCGGCTGC ATTGTCTTCG GCCACCTGCT CGAGGGGAAC CGCTGGGTCA ATGAGTCCAC-180

[0120] 181 CACCGCGCTT GTCCTGGGGC TCATCACCGG TGGTGTGATT CTGATCTGCT CCAAAGGGGT-240

[0121] 241 GAATTCGCGC ATCCTTATCT TCAGCGAGGA TATTTTCTTC ATCTACTTGC TCCCGCCCAT-300

[0122] 301 CATTTTTAAC GCCGGGTTTC AAGTAAAGAA AAAGCAATTC TTCCGCAACT TTGCGACAAT-360

[0...

Embodiment approach 3

[0168] Embodiment 3: Construction of expression vector

[0169] 1. According to the separated Na + Nucleotide sequence of antiporter gene, design primers:

[0170] Forward primer: 5'-TATTCTAGACGAGGTGGCGACCGGCATGG-3'

[0171] Reverse primer: 5'-GACGAGCTCCTTAACTACGGTCTTCTGC-3'

[0172] Perform polymerase chain reaction using the cDNA reverse-transcribed from the total RNA of the root as a template.

[0173] 2. Take 2μl PCR and connect with pGEM-T vector. The operation steps are carried out according to the instructions of Promega product pGEM-T and pGEM-T easyVector system. Then transform Escherichia coli DH5α strain, and grow overnight on an LB plate containing ampicillin (100 μg / ml) coated with 5-bromo-4-chloro-3-indole-β-D-galactoside and X-gal. . Pick white colonies and culture them in LB liquid medium overnight. Plasmid DNA was extracted by alkaline method and sequenced.

[0174] 3. Cut the gene from the pGEM-T vector with two restriction enzymes XabI and SacI, and ligate it wi...

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Abstract

The present invention belongs to the cloning, recombination and salt endurance function analysis of couchgrass sodium ion reverse transport protein ENHX1 gene, and belongs to the field of molecular biology and biological technology. General RNA is first extracted from couchgrass root and then inverse transcripted into cDNA. A pair of facultative primer is designed based on the conservative amino acid sequence of other plant's sodium ion reverse transport protein CycD2 for conventional PCR, land the PCR product is connected to pGEM-The vector to convert DH5 alpha cell and determine sequence. Then, whole length cDNA is obtained via fast amplification in 3' and 5' ends. Further, positive-sense expression vector is constituted to convert pseudomustard.

Description

(1) Technical field: [0001] The present invention relates to Na in Elytrigia + The cloning and recombination of the antiporter ENHX1 gene and the analysis and application of salt tolerance functions belong to the fields of molecular biology and biotechnology. (2) Background technology: [0002] Salt stress can cause ion osmotic stress, produce active oxygen, destroy the osmotic potential of plants and the balance of ion distribution, resulting in plant damage, especially affecting the yield of crops. Therefore, increasing the salt tolerance of crops has attracted more and more attention. Under high salinity, plants can reduce the toxicity caused by salt stress by producing stress proteins and soluble osmotic adjustment substances. Early salt-tolerant genetic engineering mainly focused on scavenging free radicals and increasing osmotic adjustment substances. The salt-tolerant ability of transgenic plants has been improved, but the effect is not obvious. Recently, Na located on the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H1/00C07H21/04C12N15/10C12N15/29C12P19/34
Inventor 张宪省乔卫华李兴国李全梓
Owner SHANDONG AGRICULTURAL UNIVERSITY
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