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Salt-tolerant sinorhizobium meliloti NSM18 and special gene cluster thereof

A Sinorhizobium, gene cluster technology, applied in genetic engineering, plant genetic improvement, application, etc., to achieve the effect of excellent nodulation nitrogen fixation ability

Inactive Publication Date: 2012-07-25
CHINA AGRI UNIV
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  • 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

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  • 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 amplification conditions of PCR are:

[0063]

[0064] Reaction conditions

[0065] 94°C for 5 minutes

[0066] 94°C for 30s, 56°C for 30s, 72°C for 30s, 30 cycles

[0067] 72℃10min

[0068] A DNA fragment with a size of about 497 bp was obtained by PCR amplification, and the amplified product was recovered and purified by electrophoresis, and then connected to the pGEM-T easy vector. The plasmid was extracted by a...

Embodiment 2

[0099] Embodiment 2, acquisition and detection of recombinant Escherichia coli

[0100]One, the acquisition of the complete sequence of ectABC gene used in the present invention

[0101] 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:

[0102] NectupE 5'-GCGAATTCGGCGCAGATCACAGGAATACAA-3' (EcoRI site)

[0103] NectdnX 5'-GCTCTAGAGCTCTCCCTCAGCCAGGG-3'(XbaI site)

[0104] The PCR amplification conditions are as follows:

[0105] 5×Phusion GC Buffer (contains Mg 2+ ) 10μl

[0106] dNTP (2.5mmol / L each) 4μl

[0107] Upstream primer (NectupE) 1 μl (0.5 μmoL / L

[0108] Downstream primer (NectdnX) 1μl (0.5μmoL / L)

[0109] 1μl (1~10ng) of genomic DNA of Halophagous Netherensis DSM 20541

[0110] Phusion DNA Polymerase 0.5μl (2U)

[0111] wxya 2 O 32.5 μl

[0112] Total volume 50μl

[0113] The PCR amplification conditions are:

[0114] D...

Embodiment 3

[0138] Embodiment 3, the acquisition of recombinant rhizobia and its detection

[0139] 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.

[0140] 1. Obtaining recombinant rhizobia

[0141] 1. Construction of recombinant expression vector

[0142] 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 ...

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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

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Application Information

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Patent Type & Authority Patents(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