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Sodium/hydrogen antiporter gene sdmlt of Bacillus halophilus and its 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: 2020-04-24
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Sodium/hydrogen antiporter gene sdmlt of Bacillus halophilus and its identification
  • Sodium/hydrogen antiporter gene sdmlt of Bacillus halophilus and its identification
  • Sodium/hydrogen antiporter gene sdmlt of Bacillus halophilus and its identification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0081] The present invention uses online prediction websites http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / and http: / / web.expasy.org / protscale / to perform 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 Putative transmembrane region of SdmlT protein

[0083]

[0084] figure 2 It is the comparison of the amino acid sequence of SdmlT and the homologue provided in the embodiment of the present invention;

[0085] Ht_pCitS,putative CitS from Halobacillus trueperi(AccessionNo.CDQ28507); Hs_pCitS,putative CitS from Halobacillus sp.BAB-2008(AccessionNo. ZP_20420905); Bsp_pCitS,putative CitS from Bacillus sp.SG-1(_01858 No.ZP); putative CitS from Bacillus halod...

Embodiment 2

[0086] Example 2 Construction process of pET19-sdmlT prokaryotic expression vector; 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. , BamH I two restriction sites, and then the sdmlT gene amplification (as shown in Table 3), the expected length of the amplified middle sequence fragment is about 1327bp.

[0088] Table 2 Sequences of primers constructed by Hz_sdmlT gene expression vector

[0089]

[0090] Table 3 PCR amplification reaction

[0091]

[0092] Add "A" to the end of the above PCR product and connect it to pEASY-T3 vector, then transfer it to Trans1-T1 competent cells, and perform blue-white spot screening to select subclones. Then treat the pEASY-T3-sdmlT with sdmlT gene with Nde I and BamH I, and do the same treatment on the expression vector pET19b at the same time, and recover and connect ...

Embodiment 3

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

[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 the subclones KNabc / pET19-sdmlT and DH5α / pET19-sdmlT are transferred to contain different salts at 1% inoculum. concentration( Figure 7 &8) Cultivate 24h in fresh LBK medium.

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

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

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

[0104] The salt-tolerance test of SdmlT in wild-type E.coli DH5α shows that the gene sdmlT can increase the NaCl resistance of E.coli DH5α from 0.8M to 1.2M, and it also improves E.coli ( ...

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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 field of biology and genetic engineering, and particularly relates to a sodium / hydrogen antiporter gene sdmlT and identification of Bacillus halophila. Background technique [0002] Stress factors such as drought, salinity, and low temperature in the environment have an important impact on the growth and development of plants, and in severe cases will cause a large-scale reduction in crop production. Among them, saline-alkali is considered to be one of the most harmful non-biological threat factors. The high-saline environment not only has a toxic effect on the bacterial cells itself, but the high concentration of osmotic pressure and ion pressure also seriously affect the normal physiological functions of bacteria. At the same time, bacteria that have important economic value in agricultural production and can be used as microbial fertilizers have limited salt-tolerance capabilities, such as rhizobia, nitrogen-fixing...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C07K14/32C12N15/70C12N15/90C12N1/21C05F11/08C12N15/82C12R1/19
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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