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Pseudomonas Na+/H+ antiporter protein gene and its cloning process

A technology of antiporter and Pseudomonas, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve the problems of long experiment period, time-consuming and expensive

Inactive Publication Date: 2007-04-25
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Method 1 needs to construct the genome library of the organism in advance, then chemically synthesize radioactive or non-radioactive DNA probes, and then perform a large number of Southern hybridizations, which is expensive, time-consuming, and the experimental cycle is long;

Method used

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  • Pseudomonas Na+/H+ antiporter protein gene and its cloning process
  • Pseudomonas Na+/H+ antiporter protein gene and its cloning process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0255] Example 1: Cloning of Na from extremely salt-tolerant Pseudomonas + / H + Antiporter gene (nha A), its steps are:

[0256] 1. Culture Pseudomonas in a medium containing 0.5mol / L NaCl at a temperature of 28°C and shake at a speed of 100rpm until the middle and late stages of exponential growth;

[0257] 2. The total DNA of Pseudomonas was extracted by conventional alkaline denaturation method, and the purity was detected by ultraviolet spectroscopy, and its OD 260 / OD 280 The ratio should be ≥ 1.8.

[0258] 3. According to the Na of several organisms + / H + The sequence design primers of the antiporter gene, wherein a pair of primers that can obtain PCR amplification products more stably are as follows:

[0259] 5' end primer: 5'A CCCGGG ATGATTATGGCCAACAGC 3'

[0260] 3' end primer: 5'T GGATCC TCAAACTGATGGACGCAA 3'

[0261] In order to facilitate the construction of prokaryotic gene expression vectors, Sma I and BamHI restriction sites (underlined) were added t...

Embodiment 2

[0313] Similar to Example 1, the difference is that Pseudomonas was inoculated in a medium containing 0.7 mol / L NaCl, kept at a constant temperature of 24°C, and cultured with shaking at a speed of 150 rpm until the middle and late stages of exponential growth, about 20 hours. The total DNA of the bacteria was extracted by conventional alkaline denaturation method, and the purity was detected by ultraviolet spectroscopy, and its OD 260 / OD 280 The ratio should be ≥ 1.8.

[0314] According to Na according to several organisms + / H + The sequence design primers of the antiporter gene, wherein a pair of primers that can obtain PCR amplification products more stably are as follows:

[0315] 5' end primer: 5'A CCCGGG ATGATTATGGCCAACAGC 3'

[0316] 3' end primer: 5'T GGATCC TCAAACTGATGGACGCAA 3'

[0317] In order to facilitate the construction of prokaryotic gene expression vectors, Sma I and BamHI restriction sites (underlined) were added to the 5' end primer and the 3' end...

Embodiment 3

[0362] Similar to Example 1, the difference is that Pseudomonas is inoculated in a medium containing 0.4 mol / L of NaCl at a temperature of 30° C., and cultured with shaking at a speed of 110 rpm until the middle and late stages of exponential growth; conventional alkali denaturation method extracts pseudomonas The total DNA of Monascus, the purity was detected by ultraviolet spectroscopy, and its OD 260 / OD 280 The ratio should be ≥ 1.8.

[0363] According to Na according to several organisms + / H + The sequence design primers of the antiporter gene, wherein a pair of primers that can obtain PCR amplification products more stably are as follows:

[0364] 5' end primer: 5'A CCCGGG ATGATTATGGCCAACAGC 3'

[0365] 3' end primer: 5'T GGATCC TCAAACTGATGGACGCAA 3'

[0366] In order to facilitate the construction of prokaryotic gene expression vectors, Sma I and BamHI restriction sites (underlined) were added to the 5' end primer and the 3' end primer respectively.

[0367] A...

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Abstract

This invention is fake unicell bacteria Na+ / H+ antiport albumen gene and its clone method, it relates to one kind of gene clone. One kind of fake unicell bacteria Na+ / H+ antiport albumen structure gene and one kind of quickly and economic clone method are provided, gene length is 1089bp, coding 362 amino acids. Procedures are that extra anti-salt fake unicell bacteria is inoculated and cultivated, whole DNA is extracted, primer is designed, PCR reaction and amplified product is gel imaged system sweep record, then the amplified about 1.1kb tripe is cut down. One gland piao lin deoxyribonucleotide (A) is added to end of PCR product, above clear liquid is abandoned, and 20muL weight distilled water is added to dissolve DNA. Carrier is connected with DNA to form single colony. White colony is selected and recombination particle is extracted by caustic cracking solution. Goal gene is identificated and anti-salt level of transformant is detected, clone gene of transformant is sequenced and weight plasmid of transformant is identificated, then colone gene is estimated.

Description

technical field [0001] The present invention relates to a kind of gene cloning, relate in particular to a kind of Pseudomonas Na + / H + Antiporter gene (nha A) cloning. Background technique [0002] At present, the conventional method of cloning a known gene from prokaryotes is to search for biological information to obtain the sequence of other biological genes, and then follow two technical routes to clone the gene: [0003] 1. Synthesize the oligonucleotide DNA probe of the gene, label the molecular probe with a radioactive isotope or a non-radioactive chemical substance (such as a fluorescent substance), and carry out Southern hybridization of the probe with the previously constructed biological genome library , fish out the DNA fragment containing the target gene, and sequence the fragment; construct an expression recombinant plasmid containing the fragment, transform the recipient cell, and detect the new protein and its new biological function obtained by the transf...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/10C12Q1/68
Inventor 刘广发陈启伟
Owner XIAMEN UNIV
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