Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Salt-tolerant sinorhizobium meliloti NSM18 and special gene cluster thereof

A technology of gene clusters and genes, applied in the direction of genetic engineering, plant gene improvement, application, etc., to achieve the effect of excellent nodulation and nitrogen fixation ability

Inactive Publication Date: 2010-11-03
CHINA AGRI UNIV
View PDF0 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the transfer and expression between different species of microorganisms has not been reported

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Salt-tolerant sinorhizobium meliloti NSM18 and special gene cluster thereof
  • Salt-tolerant sinorhizobium meliloti NSM18 and special gene cluster thereof
  • Salt-tolerant sinorhizobium meliloti NSM18 and special gene cluster thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1, Obtaining the complete sequence of the ectoine synthetic gene

[0058] 1. Partial fragments of ectB gene

[0059] In the present invention, the ectoine synthesis gene was isolated from N. stelienckii DSM 20541 (purchased from German Culture Collection of Microorganisms). Gene isolation is achieved through degenerate primer amplification to obtain partially conserved fragments. The primer sequences are shown in the table below:

[0060] ectB-up 5'-GCNGGNGCNYTNAAYTAYGGNCAYAA-3'

[0061] ectB-dn 5'-ARNCCNCCYTCNCCYTGNACNGTYTC-3'

[0062] The PCR amplification conditions are:

[0063] Component volume

[0064] 2×GC rich buffer I 25μl

[0065] dNTP Mixture (2.5mmoL / L each) 4μl

[0066] ectB-up (20mmoL / L) 1μl

[0067] ectB-dn (20mmoL / L) 1μl

[0068] Genomic DNA 1 μl (1-10

[0069] ng)

[0070] TaKaRa LA Taq 0.25μl

[0071] wxya 2 O 50μl

[0072] Reaction conditions

[0073] 94℃5min

[0074] 94°C for 30s, 56°C for 3...

Embodiment 2

[0114] Embodiment 2, acquisition and detection of recombinant Escherichia coli

[0115] One, the acquisition of the complete sequence of ectABC gene used in the present invention

[0116] A pair of primers were designed using primer premier 5.0 to amplify the entire sequence of ectABC gene including promoter and terminator sequences. The primer sequences are as follows:

[0117] NectupE 5'-GC GAATTC GGCGCAGATCACAGGAATACAA-3' (EcoRI site)

[0118] NectdnX 5'-GC TCTAGA GCTCTCCCTCAGCCAGGG-3'(XbaI site)

[0119] The PCR amplification conditions are as follows:

[0120] 5×Phusion GC Buffer (contains Mg 2+ ) 10μl

[0121] dNTP (2.5mmoL / L each) 4μl

[0122] Upstream primer (NectupE) 1 μl (0.5 μmoL / L

[0123] Downstream primer (NectdnX) 1μl (0.5μmoL / L)

[0124] 1μl (1~10ng) of genomic DNA of Halophagous Netherensis DSM 20541

[0125] Phusion DNA Polymerase 0.5μl (2U)

[0126] wxya 2 O 32.5 μl

[0127] Total volume 50μl

[0128] The PCR amplification conditions are:

...

Embodiment 3

[0153] Embodiment 3, the acquisition of recombinant rhizobia and its detection

[0154] Sinorhizobium meliloti is an important agricultural microorganism, which can form nodules with alfalfa and other forages to fix nitrogen and increase the yield of forages. Its symbiotic system has important application prospects in agriculture and animal husbandry. However, this symbiotic system is often inhibited by salt stress. Based on the high expression of the ectoine synthesis gene in Escherichia coli, it can be used as a salt-tolerant gene resource in important agricultural strains.

[0155] 1. Obtaining recombinant rhizobia

[0156] 1. Construction of recombinant expression vector

[0157] The recombinant plasmid pGEM-Nect was digested with EcoRI and HindIII, and the digested fragment was recovered by gel, and connected to pLAFR3 (preserved by China Agricultural University. The non-patent literature that recorded this vector is Sun Dong, Tang Lili, Wang Qianqian, etc., 2006, High ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses salt-tolerant sinorhizobium meliloti NSM18 and a special gene cluster thereof. The gene cluster is expressed as a sequence 1 in a sequence table. The gene cluster is transferred to sinorhizobium meliloti to construct an engineering strain NSM18. Salt-tolerant experiments show that the engineering strain can tolerate the salt concentration conditions of 1.4mol / L NaCl, 1.5mol / L KCl and 0.6mol / L LiCl; and the engineering strain can be grown at the temperature of 42 DEG C, while a contrast cannot be grown, and the engineering strain shows high-temperature resistance. Moreover, apart from the advantages, the engineering strain NSM18 still keeps good nodulation and nitrogen fixation capabilities.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a salt-tolerant Sinorhizobium meliloti NSM18 and its special gene cluster. Background technique [0002] Drought and soil salinization are a major problem in agriculture, especially in arid or semi-arid climates. It is estimated that about 40% of the arable land in the world is oversalted, and the area of ​​intensive agriculture affected by salinization may reach 400-950 million hectares. What's more serious is that due to the impact of the El Niño climate, the global temperature rises, the sea level rises, coupled with the influence of industrial pollution, agricultural irrigation, improper fertilization and other human factors, the area of ​​secondary salinized soil is still growing at an annual rate of 3 % speed scaling. Using bioengineering technology to improve the stress resistance of crops and agricultural microorganisms is one of the economical and effective methods...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/63C12N5/10C12N1/00C12N15/74C12N1/21C12P17/12C12R1/01
Inventor 杨苏声张博王磊冯德芹刘文彦
Owner CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com