Assimilation nitrate reductase gene and assimilation nitrite reductase gene both extracted from bacillus megaterium as well as applications of assimilation nitrate reductase gene and assimilation nitrite reductase gene

A technology of reducing enzymes and catalytic subunits, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as nitrate removal

Inactive Publication Date: 2014-04-16
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the above-mentioned deficiencies in the prior art, the present invention proposes a Bacillus megaterium assimilating nitrate reductase and nitrite reductase genes and their application, which can solve the problem of removing nitrate from the soil in facility greenhouses

Method used

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  • Assimilation nitrate reductase gene and assimilation nitrite reductase gene both extracted from bacillus megaterium as well as applications of assimilation nitrate reductase gene and assimilation nitrite reductase gene
  • Assimilation nitrate reductase gene and assimilation nitrite reductase gene both extracted from bacillus megaterium as well as applications of assimilation nitrate reductase gene and assimilation nitrite reductase gene
  • Assimilation nitrate reductase gene and assimilation nitrite reductase gene both extracted from bacillus megaterium as well as applications of assimilation nitrate reductase gene and assimilation nitrite reductase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Obtained sequences of assimilative nitrate reductase and nitrite reductase from strain NCT-2

[0036] 1.1 Source of the strain: Bacillus megaterium NCT-2 was isolated from a greenhouse in Chongming Island, Shanghai, which had been cultivated for 12-15 years. The preservation number is CGMCC NO.4698. It is No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, Chinese Institute of Microbiology; the preservation date is March 21, 2011.

[0037] 1.2 The strain was inoculated in the strain culture medium, placed at 30°C, 150 rpm, and shaken for 24 hours on a shaker.

[0038] Culture medium: KNO 3 10g, KH 2 PO 4 0.5g, KCl1g, MgSO 4 ·7H 2 O0.5g, CaCl 21mg, FeSO 4 ·7H 2 O10mg, glucose 15g, dilute to 1L with deionized water, adjust pH to 7.0 with NaOH, and autoclave.

[0039] 1.3 Genomic DNA extraction: Using the CTAB method, the genomic DNA is extracted and tested for quality. The requirements include the following three aspects: 1) Sample purity: A260...

Embodiment 2

[0046] Changes in gene expression of assimilative nitrate reductase and nitrite reductase in strain NCT-2 cultured at different nitrate concentrations

[0047] 2.1 Strain NCT-2 was cultured at different nitrate concentrations

[0048] 1) Different amounts of KNO were added to the inorganic salt medium 3 , so that the concentrations were: 10, 30 and 50 mM, and each concentration was set to 3 replicates.

[0049] 2) Negative control with (NH 4 ) 2 SO 4 As a nitrogen source, different amounts of (NH 4 ) 2 SO 4 The concentrations are respectively: 10, 30 and 50 mM, and each concentration has 3 replicates.

[0050] 3) Mother solution culture: with 10mM (NH 4 ) 2 SO 4 Nitrogen source inorganic salt culture strains. A 200mL culture medium in a Erlenmeyer flask was added to the incubator for 24 hours, and the culture conditions were 30°C and 180rpm. The negative control is the same.

[0051] 4) Add 95mL of culture medium and 5mL of bacteria solution to each Erlenmeyer fla...

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Abstract

The invention discloses an assimilation nitrate reductase gene and an assimilation nitrite reductase gene both extracted from bacillus megaterium as well as applications of the assimilation nitrate reductase gene and the assimilation nitrite reductase gene, and relates to the field of the biotechnology. The assimilation nitrate reductase gene is catalytic subunit nasC (Seq ID No.1) which codes assimilation nitrate reductase Nas; the assimilation nitrite reductase gene is large subunit nasD (Seq ID No.2) which codes assimilation nitrite reductase NiR. According to the invention, total RNA is extracted from the bacillus megaterium NCT-2, then cDNA synthesis is conducted to obtain a cDNA template, and function representation is realized through fluorogenic quantitative PCR. The assimilation nitrate reductase gene and the nitrite reductase gene can be further applied to research on removal of overmuch nitrate from soil.

Description

technical field [0001] The invention relates to a gene and application in the field of biotechnology, in particular to a Bacillus megaterium assimilating nitrate reductase and nitrite reductase gene and application. Background technique [0002] Since greenhouses and greenhouses are highly intensive facility cultivation formed under the premise of artificially changing the traditional open field, they have the characteristics of perennial high temperature, high humidity, no precipitation, high fertilization, high output, and super-intensive use. Compared with open soil, its special ecological environment and unreasonable water and fertilizer management lead to secondary salinization of soil. The accumulation of nitrate is not only one of the main characteristics of secondary salinization of protected cultivation soil, but also the leading factor causing physiological obstacles of protected cultivation crops, resulting in stunted growth of vegetable crops, reduced yield and l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12Q1/68B09C1/10C12R1/11
CPCC12N9/0044
Inventor 周培支月娥时唯伟卫星初少华冯海玮
Owner SHANGHAI JIAO TONG UNIV
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