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Identification method and application of coding protein Pm_MF

An identification method, a protein-encoding technology, which is applied in applications, biochemical equipment and methods, and microbial determination/inspection, etc., can solve problems such as difficulty in functional identification, lack of theoretical support and technical support, and no clear and suitable selection of genes. To achieve the effect of enriching microbial salt-alkali tolerance gene resources and improving salt-alkali tolerance

Pending Publication Date: 2018-07-27
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] There is no high-quality gene selection for the selection of genes in the development of saline-alkali-tolerant transgenic crops, and there is no clear and suitable selection for the selection of saline-alkali-tolerant microorganisms in the development of microbial fertilizers for saline-alkali soils, or the introduced genes; for specific functions that have not been annotated The gene Pm_mf lacks functional analysis and identification technology, especially it is responsible for the transport of various antibiotic drugs and cation Na + , Li + 、K + Difficulties in functional identification of
[0008] The existing research technology lacks sufficient theoretical and technical support for further understanding the relationship between the molecular mechanism of MF (major facilitator) family proteins transporting various antibiotic drugs and the molecular mechanism of sodium (lithium) / hydrogen antiporter salt-alkali tolerance. As a result, the application of bifunctional proteins cannot be cut into the root of the mechanism. Solving this problem will greatly improve the application value of the protein, and clarify how to make full use of the bifunctional advantages of the protein's salt-alkali resistance and multi-drug resistance to achieve bifunctionality. Working together in the research and development of microbial fertilizers and the introduction of effective avoidance of new environmental problems, it provides a new direction for fundamentally controlling the hazards of saline-alkali soil

Method used

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  • Identification method and application of coding protein Pm_MF
  • Identification method and application of coding protein Pm_MF
  • Identification method and application of coding protein Pm_MF

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Bioinformatics analysis of Pm_mf gene and protein Pm_MF, such as figure 1 shown.

[0128] Through the screening technology combining gene library and gene function complementation, a gene fragment related to salt-alkali tolerance was obtained from Zoonococcus littoralis, and the gene fragment related to salt-alkali tolerance contained a complete open reading frame ORF with a length of 1272bp , the nucleotide sequence of ORF is shown in SEQ ID NO.1; sequence analysis shows that it shows the highest homology with a putative MF (majorfacilitator) family transporter, which is 98%, so it is speculated that this gene also encodes A protein of the MF (major facilitator) family, so the gene was named Pm_mf.

[0129] The present invention uses online prediction websites http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / and http: / / web.expasy.org / protscale / to carry out bioinformatics analysis on the protein sequence encoded by Pm_mf gene, It was found that the protein Pm_MF encoded by...

Embodiment 2

[0132] The phylogenetic tree of Pm_MF and the identified Na+ / H+ antiporters and the homology comparison with homologues,

[0133] According to the predicted MF (major facilitator) family location, the present invention establishes a phylogenetic evolutionary tree of family homologues and identified Na+ / H+ antiporters, and the phylogenetic tree shows that the MF (majorfacilitator) family proteins form an independent branch, with a threshold of 99 %, but far from the identified Na+ / H+ antiporter or other protein phylogenetic relationship with Na+ / H+ antiporter activity, so it is considered that Pm_mf encodes a new Na+ / H+ antiporter. In the present invention, the homology comparison of homologs is carried out at the same time, and the results show a high degree of homology, and highly conserved amino acid residues are obtained, wherein the negatively charged amino acid residues include D67, D127, E187, D223, E341; Positive amino acid residues include R71; polar amino acid residue...

Embodiment 3

[0135] The construction process of pET-Pm_mf prokaryotic expression vector; as figure 2 shown;

[0136] According to the Pm_mf gene sequence, first use Primer5 software to design SEQID NO.3: Pm_mf-F; SEQID NO.4: Pm_mf-R specific primers (as shown in Table 1), and introduce Nde I at the 5' and 3' ends of the gene respectively , BamH I two enzyme cutting sites, then carry out the amplification of Pm_mf (as shown in table 2), expect to amplify the intermediate sequence fragment length about 1272bp (as shown in Table 2) figure 2 -A shown).

[0137] Table 1 Pm_mf gene expression vector construction primer sequence

[0138]

[0139] Table 2 PCR amplification reaction

[0140]

[0141] 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. Recombinant plasmid pEASY-Pm_mf was treated with Nde I and BamH I two kinds of endonucleases, and the ...

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Abstract

The invention belongs to the technical field of molecular biology and genetic engineering, and discloses an identification method and application of coding protein Pm_MF. The amino acid sequence of the protein Pm_MF is SEQ ID NO.2; the protein gene Pm_mf is a 1272bp complete open reading frame ORF; the nucleotide sequence of the ORF is shown as SEQ ID NO.1. The identification method comprises thefollowing steps of screening out a fragment containing a saline-alkaline tolerance gene, building a prokaryotic expression vector containing the Pm_mf gene, and the like. The protein provided by the invention can be used for building the efficient saline-alkaline tolerance engineering bacteria or stress resistance crops and the like; various medicine resistance protein functions can be used for gene engineering carrier screening marking; particularly, great values are realized by the duplex functions of saline-alkaline tolerance capability and the medicine resistance screening marking in aspects of saline-alkaline stress resistance gene engineering strains and plants.

Description

technical field [0001] The invention belongs to the technical fields of biology and genetic engineering, and in particular relates to an identification method and application of an encoding protein Pm_MF. The protein gene Pm_mf belongs to the new MF (majorfacilitator) family of Zoonococcus littoralis. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] It is understood that there are currently about 500 million mu of saline-alkali land in my country, and more than 100 million people in arid and semi-arid areas are under the threat of salinization of cultivated land. The saline-alkali land in Northeast China is to the west of the black soil area, and the black soil area is also threatened by salinity. The salinization of cultivated land in Xinjiang has seriously threatened the survival of local people and has become the most prominent livelihood issue. Therefore, the development and management of salin...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12Q1/686C12N1/21G01N33/68C12Q1/04C05G3/04C05G3/00C05G3/80
CPCC05G3/00C05G3/80C12N15/70C12Q1/04C12Q1/686G01N33/68G01N2333/245C12Q2525/131
Inventor 姜巨全韩波张瑞张正来徐桐孟琳邹俏郑秀桃
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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