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Halobacillus sodium/hydrogen antiporter gene sdmlT and identification

A technology of antiporter and bacillus, applied in the field of Bacillus halophilus sodium/hydrogen antiporter gene sdmlT and identification, can solve the problems of unknown specific function, no guiding direction, lack of guiding significance, etc., and achieve enrichment of microbial salt tolerance Alkali gene resources, effect of improving salt-alkali tolerance

Active Publication Date: 2018-12-21
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0009] The exact function of SdmlT remains unknown; the functional characterization of SdmlT and its responsibility for the transport of 2-hydroxycarboxylic acids and Na + , Li + The functional analysis of isocations lacks guiding significance for further revealing the molecular mechanism of HCT family proteins transporting 2-hydroxycarboxylic acid and the molecular mechanism of sodium (lithium) / hydrogen antiporter salt resistance, and the existing technology has used the gene sdmlT as There is no guiding direction for the construction of genetically engineered strains with high salt-alkali tolerance

Method used

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  • Halobacillus sodium/hydrogen antiporter gene sdmlT and identification
  • Halobacillus sodium/hydrogen antiporter gene sdmlT and identification
  • Halobacillus sodium/hydrogen antiporter gene sdmlT and identification

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Experimental program
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Embodiment 1

[0080] Embodiment 1. Online biological software analyzes the transmembrane region and hydrophobicity of SdmlT protein, such as figure 1 shown;

[0081] The present invention utilizes online prediction websites http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / and http: / / web.expasy.org / protscale / to conduct bioinformatics analysis on the protein sequence encoded by the sdmlT gene, It was found that the protein SdmlT encoded by this gene has 11 transmembrane regions (as shown in Table 1), and the protein exhibits high hydrophobicity.

[0082] Table 1 The putative transmembrane region of SdmlT protein

[0083]

[0084] figure 2 It is the alignment of the amino acid sequences of SdmlT and homologues provided in the examples of the present invention;

[0085] Ht_pCitS, putative CitS from Halobacillus trueperi (Accession No. CDQ28507); Hs_pCitS, putative CitS from Halobacillus sp. BAB-2008 (Accession No. ZP_20420905); Bsp_pCitS, putative CitS from Bacillus sp. putative CitS from Bac...

Embodiment 2

[0086] The construction process of embodiment 2pET19-sdmlT prokaryotic expression vector; as image 3 shown;

[0087] According to the sdmlT gene sequence, first use Primer5 software to design SEQID NO.3: sdmlT-F; SEQIDNO.4: sdmlT-R specific primers (as shown in Table 2), and introduce Nde I at the 5' and 3' ends of the gene respectively , BamH I two restriction sites, and then carry out the amplification of sdmlT gene (as shown in Table 3), the length of the expected amplified intermediate sequence fragment is about 1327bp.

[0088] Table 2 Hz_sdmlT gene expression vector construction primer sequence

[0089]

[0090] Table 3 PCR amplification reaction

[0091]

[0092] Add "A" to the end of the above PCR product and connect it into the pEASY-T3 vector, then transform it into Trans1-T1 competent cells, and perform blue-white screening to select subclones. The pEASY-T3-sdmlT connected with the sdmlT gene was treated with Nde I and BamH I two endonucleases, and the exp...

Embodiment 3

[0099] Identification of embodiment 3 sdmlT gene physiological function:

[0100] In order to identify the physiological function of the sdmlT gene, the present invention uses KNabc / pET19 and DH5α / pET19 as negative controls, and transfers the bacterial solutions of the subclones KNabc / pET19-sdmlT and DH5α / pET19-sdmlT to 1% inoculum containing different salts concentration( Figure 7 &8) in fresh LBK medium for 24h.

[0101] 1. Salt-alkali tolerance test of KNabc / pET19-sdmlT

[0102] The salt-alkali tolerance test of SdmlT in Escherichia coli (E.coli) KNabc showed that the gene sdmlT can provide Escherichia coli (E.coli) KNabc resistance to 0.4M NaCl, 5mM LiCl and alkaline pH (Figure 7), suggesting that the gene sdmlT is saline-alkali tolerant and may have Na + / H + Antiporter activity.

[0103] 2. Salt-alkali tolerance test of DH5α / pET19-sdmlT:

[0104] The salt-alkali tolerance test of SdmlT in wild-type Escherichia coli (E.coli) DH5α showed that the gene sdmlT could in...

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Abstract

The invention belongs to the technical fields of molecular biology and genetic engineering, and discloses a novel halobacillus sodium / hydrogen antiporter gene sdmlT and identification. The novel halobacillus sodium / hydrogen antiporter gene sdmlT is a complete open reading frame ORF, and the nucleotide sequence of the ORF is shown as SEQ ID NO.1. The identification method comprises the following steps: a segment containing a salt-and-alkali-tolerant gene is first obtained; a prokaryotic expression vector containing the sdmlT gene is constructed; and the physiological function of the gene and the function of a protein coded by the gene are identified. The sdmlT gene which is obtained from halobacillus andaensis by the invention can remarkably increase the salt-and-alkali-tolerant capabilityof a salt-sensitive mutant strain KNabc of Escherichia coli; the sdmlT gene proposed by the invention enriches microbial salt-and-alkali-tolerant gene resources, can be used in the construction of highly effectively salt-and-alkali-tolerant engineering bacteria, stress-resistant crops or the like, and particularly has significant value in plants tolerant to salt or alkali stress.

Description

technical field [0001] The invention belongs to the technical fields of biology and genetic engineering, and in particular relates to a Bacillus halophilus sodium / hydrogen antiporter gene sdmlT and its identification. Background technique [0002] Stress factors such as drought, salinity, low temperature and other stress factors in the environment have an important impact on the growth and development of plants, and in severe cases, they will cause large-scale production reduction of crops. Among them, salinity is considered to be one of the most harmful abiotic stress factors. The high saline environment not only has a toxic effect on the bacterial cells themselves, but also the high concentration of osmotic pressure and ionic pressure seriously affects the normal physiological functions of the bacteria. At the same time, the bacteria that have important economic value in agricultural production and can be used as microbial fertilizers have very limited salt-alkali toleran...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/32C12N15/70C12N15/90C12N1/21C05F11/08C12N15/82A01H5/00C12R1/19
CPCC05F11/08C07K14/245C07K14/32C12N15/70C12N15/8273C12N15/902
Inventor 姜巨全孟琳王艳红徐桐张正来刘贺男张伟
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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