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Alfalfa Na+/H+ reverse transport protein gene and its clone and use

A technology of antiporter and alfalfa, applied in the field of molecular biology and biology, can solve problems such as less obvious effects

Inactive Publication Date: 2006-07-19
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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  • Alfalfa Na+/H+ reverse transport protein gene and its clone and use
  • Alfalfa Na+/H+ reverse transport protein gene and its clone and use
  • Alfalfa Na+/H+ reverse transport protein gene and its clone and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0074] Embodiment 1: Alfalfa Na + / H + Cloning method of antiporter gene

[0075] (1) Extraction of RNA: Total RNA was extracted by CTAB method or RNA kit.

[0076] (2) Synthesis of the first strand of DNA: take 1 μg of total RNA, add 4 μl of 5× reaction buffer, 2 μl of 10 mM deoxyribonucleic acid (dNTP), 0.5 μl of ribonuclease inhibitor (40-200u / μl), primer oligodT (1 μg / μl) 1 μl, reverse transcriptase (10u / μl) 2 μl, react at 42°C for 60 minutes, then place at 85°C for 10 minutes to terminate the reaction.

[0077] (3) PCR reaction: polymerase chain reaction (PCR) reagents and conditions are:

[0078] First mix the following reagents together:

[0079] 10×Reaction Buffer 5μl

[0080] Deoxynucleotide mixture (dNTP) 4μl

[0081] Forward primer (5μM) 4μl

[0082] Reverse primer (5μM) 4μl

[0083] Template cDNA 4μl

[0084] Taq DNA polymerase 0.5μl

[0085] Total volume 50μl

[0086] The PCR reaction conditions are: 94°C for 3 minutes; then enter the following cycle: 9...

Embodiment approach 2

[0092] Embodiment 2: Alfalfa Na + / H + The sequence of the antiporter gene (MsNHX1) is shown in the "Summary of the Invention" section.

Embodiment approach 3

[0093] Embodiment 3: Construction of expression vector

[0094] (1) According to the separated Na + / H + Nucleotide sequence of antiporter gene, design primers:

[0095] Forward primer: 5′-TATTCTAGACGAGGTGGCGACCGGCATGG-3′

[0096] Reverse primer: 5′-GACGAGCTCCTTAACTACGGTCTTCTGC-3′

[0097] The polymerase chain reaction was performed using the cDNA reverse-transcribed from the root total RNA as a template.

[0098] (2) Take 2 μl of the PCR product and connect it to the pGEM-T easy vector, and the operation steps are carried out according to the instructions of pGEM-T easy and pGEM-T easy Vector system produced by Promega Company. Then Escherichia coli DH5α strain was transformed and grown overnight on LB plates coated with 5-bromo-4-chloro-3-indole-β-D-galactoside and X-gal containing ampicillin (100 μg / ml). Pick white colonies and culture them overnight in LB liquid medium. Then the plasmid DNA was extracted by alkaline method and sequenced.

[0099] (3) Cut the gene fr...

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Abstract

The present invention relates to cloning, recombination, salt endurance function analysis and applications of Na+ / H+ reverse transport protein MsNHX genes in alfalfa, and belongs to molecular biology and biological technology field. The total RNA is extracted from roots of alfalfa, then 1 microgramme total RNA is reversely transcripted into cDNA. According to conservative amino acid sequences in Na+ / H+ reverse transport protein in other plants, a pair of degenerated primers are designed to execute a routine polyase chain reaction (PCR), wherein the PCR product is connected to a pGEM-T carrier to converse DH5 alpha cells and execute sequence determination. Then, 3' and 5' tail ends rapid amplifications are executed to obtain cDNA with full-length. Right expression carriers are further constructed for conversion of arabidopsis thaliana. The transgenic arabidopsis thaliana possesses higher salt resistant capability. Hence the transgenic double-seminal leaf crops such as heart trefoil will improve its salt resistant capability, yield and quality, then the planted area of dicotyledon at alkaline land can be expanded to generate huge economic benefit and social benefit.

Description

(1) Technical field: [0001] The present invention relates to Na in alfalfa + / H + The cloning and recombination of the antiporter MsNHX1 gene and the analysis and application of salt tolerance function belong to the field of molecular biology and biotechnology. (two) background technology: [0002] Salt stress can cause ion osmotic stress, generate 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, improving the salt tolerance of crops has attracted more and more attention. Under high-salt conditions, plants can alleviate the toxicity caused by salt stress by producing stress proteins and soluble osmoregulatory substances. Early salt-tolerant genetic engineering mainly focused on scavenging free radicals and increasing osmotic adjustment substances. The salt-tolerant ability of transgenic plants was improved, but the effect was not obvious. Recently, Na...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07H21/04C12P19/34C12N15/82C12N15/10A01H1/00
Inventor 张宪省安宝燕李加瑞李祥
Owner SHANDONG AGRICULTURAL UNIVERSITY
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