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Soybean eIF5A gene subtypes and genetic engineering bacteria, and applications thereof

A technology of genetically engineered bacteria and soybeans, applied in the fields of molecular biology and genetic engineering, to achieve the effect of promoting growth and reproduction, strong growth and reproduction ability, and slow aging

Pending Publication Date: 2016-12-07
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are no reports on the cloning and functional research of soybean eIF5A gene at home and abroad

Method used

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  • Soybean eIF5A gene subtypes and genetic engineering bacteria, and applications thereof
  • Soybean eIF5A gene subtypes and genetic engineering bacteria, and applications thereof
  • Soybean eIF5A gene subtypes and genetic engineering bacteria, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] Example 1 Electronic cloning, determining the three subtype sequences of the soybean eIF5A gene and positioning:

[0149] First search the cDNA and amino acid sequences of known eIF5a genes in other species in the NCBI database (http: / / www.ncbi.nlm.nih.gov): Cassava (AF266464), Medicago sativa (AF416338), Solanumlycopersicum (AF296083), Hevea brasiliensis (AF516357), Euphorbia esula (AF225297), Senecio vernalis (AJ238624), Tamarix androssowii (AY587771), etc.

[0150] The above gene sequence is used as the seed sequence; 839,014 soybean EST sequences (http: / / www.ncbi.nlm.nih.gov / dbEST) and soybean genome sequence (http: / / www.phytozome.net) are used as the database File, use the local BLAST software for sequence homology comparison, the parameters are set as: E≤1e-10, Score≥100; use the splicing software CAP3 (http: / / pbil.univ-lyon1.fr / cap3.php) to align All matched EST sequences are clustered and spliced ​​to obtain different types of contig sequences, that is, differe...

Embodiment 2

[0155] Expression of embodiment 2GmeIF5A gene in yeast:

[0156] 1. Extraction of soybean total RNA

[0157] Extraction of soybean total RNA: Total RNA was extracted from the leaves of soybean Suinong No. 10 by Trizol method. Take an appropriate amount of sample and grind it into powder in liquid nitrogen, take 50-100 mg into a 115 mL centrifuge tube, add 1 mL of Trizol solution, let stand at room temperature for 5 min, add 200 μL of chloroform, shake vigorously for 15 s, then take the upper aqueous phase into another centrifuge tube Add 500μL of isopropanol, let it stand for 10min, and centrifuge at 12 000r / min 4℃ for 10min. Discard the supernatant, add 75% ethanol to wash, then dry at room temperature or in vacuum for 5-10 minutes, add 100 μL of ddH2O to dissolve, and measure the OD260 and OD280 values ​​of the RNA sample with a UV spectrophotometer. The OD260 / OD280 ratios of the two samples are 1.93 and 1.96, respectively. The ratio is close to 2.0, indicating that the RN...

Embodiment 3

[0270] Example 3: The final expression levels of GmeIF5A1, 2, and 3 genes in Pichia pastoris GS115 cells reached 11.3%, 10.5%, and 11.7% of the total soluble protein in the cells, equivalent to 1.5-1.7g / L.

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Abstract

The invention relates to soybean eIF5A gene subtypes and genetic engineering bacteria, and applications thereof; the soybean eIF5A gene subtypes GmeIF5A1, GmeIF5A2 and GmeIF5A3 have the sequences respectively shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The invention also provides the genetic engineering bacteria for producing a soybean eIF5A protein, the gene subtypes are expressed, and the soybean eIF5A protein is produced; an expression vector is a pPIC3.5K plasmid; the gene subtypes are located between an EcoR I enzyme restriction site and an Not I enzyme restriction site on the pPIC3.5K plasmid, recombinant plasmids are formed, and a host is pichia pastoris GS115. The genetic engineering bacteria for producing the soybean eIF5A protein can be applied for fermentation production of the soybean eIF5A protein. The gene subtypes can be applied for high yielding of the soybean eIF5A protein, and promotion of cell proliferation or plant resistance to environmental stress and the like.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to soybean eIF5A gene subtypes, recombinant genetic engineering bacteria and applications. Background technique [0002] Eukaryotic initiation factor 5A (eIF5A) plays a key role in the continuous proliferation of cells. eIF5A is the only protein known to contain a post-translational modification to form the amino acid derivative hypusine [Nε-(4-aminobutyl-2-hydroxybutyl) lysine]. [0003] Since the discovery of the eIF5A gene, it has been widely concerned, including eIF5A in animals, plants, and humans has been continuously cloned and analyzed. [0004] In terms of its function, eIF5A is based on the initial function of protein synthesis, and promotes the proliferation of cells or the aging and death of cells by promoting the transfer of some specific mRNAs and the expression of related specific genes, or the response and resistance to environmental stress....

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N1/19C12P21/02C12N15/82C12R1/84
Inventor 双宝李文滨韩英鹏
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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