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Cold-adapted ribonuclease R and coding gene and application thereof

A technology of ribonuclease and coding genes, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as poor stability

Active Publication Date: 2020-10-27
HARBIN INST OF TECH AT WEIHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The ribonuclease R extracted from animals and plants is easily restricted by season, climate and region, and its stability is poor, so it is difficult to be directly applied to related fields such as industrial production

Method used

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  • Cold-adapted ribonuclease R and coding gene and application thereof
  • Cold-adapted ribonuclease R and coding gene and application thereof
  • Cold-adapted ribonuclease R and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Cloning and sequencing analysis of ribonuclease R gene.

[0019] Antarctic microbes Psychrobacter sp. ANT206 was activated in 2216E liquid medium, cultured to the middle and late logarithmic growth phase (about 4d), and the total gene DNA of the strain was extracted by combining CTAB method and phenol-chloroform extraction method. Using the extracted total DNA as a template, PCR was performed using degenerate primers.

[0020] Upstream primer: 5'- ATGANGGAATGATCRACGCCT -3'

[0021] Downstream primer: 5'- ANGTTTGGATCATTNACTCAT -3'

[0022] Amplification conditions were: denaturation at 94°C for 1 min, annealing at 54°C for 1 min, extension at 72°C for 90 s, and 30 cycles. Then, agarose gel electrophoresis was used to detect the band containing the target gene cold-adaptive ribonuclease R and sequenced. By analyzing the sequencing results, a gene with a complete reading frame sequence of 2 313 bp in full length was obtained. The nucleotide sequence is shown in SEQ ID ...

Embodiment 2

[0024] Expression and purification of ribonuclease R gene

[0025] According to the determined full-length sequence of cold-adapted ribonuclease R, primers containing restriction sites were redesigned.

[0026] Upstream primer: 5’-ACT GGATCC ATGTCAAACCAAGATC -3'

[0027] Downstream primer: 5’-TAC CTCGAG CGCTCTTTTTACTACT -3'

[0028] The lines are Bam Hi, xho I restriction site.

[0029] The cold-adapted ribonuclease R gene and pET-28a (+) double-digested gel recovery product were connected in proportion with T4 ligase to construct a recombinant expression vector. Transform recombinant expression vectors into competent cells E. coli In BL21, positive clone screening and enzyme digestion verification were carried out.

[0030] The recombinant strain obtained by screening was induced to express by IPTG. Inoculate the recombinant bacteria into LB medium and cultivate to OD at 32-40°C 600 0.6-0.8, IPTG was added to the medium to a final concentration of 0.5-1.5 mM, ...

Embodiment 3

[0032] Study on the Enzymatic Properties of Recombinant Cold Adaptive Ribonuclease R

[0033] (1) Determination of the optimum reaction temperature: Under the conditions of different temperatures (0 ~ 50 ℃) and pH 6.0, the enzymatic activity of cold-adapted ribonuclease R was determined, and the highest enzymatic activity was set as 100%, and the activity at other temperatures was The ratio of the enzyme activity to the highest enzyme activity is the relative enzyme activity of the enzyme solution to be tested at this temperature. The result is as figure 2 As shown, the results show that the optimum action temperature of the enzyme is between 28 ~ 32 ℃, and the enzyme activity is maintained at more than 85% of the highest enzyme activity at 20 ~ 35 ℃. When the temperature exceeds 35 ℃, with the temperature As the temperature increases, the enzyme activity gradually decreases, and when the temperature reaches above 50 °C, the enzyme activity is basically lost.

[0034] (2) T...

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Abstract

The invention discloses a cold-adapted ribonuclease R and a coding gene and application thereof. Firstly, a novel cold-adapted ribonuclease R coding gene is cloned from Psychrobactersp. in Antarctic sea ice microorganisms, and an amino-acid residue sequence of the cold-adapted ribonuclease R is shown in SEQ ID No. 2. Sequence analysis shows that a protein sequence encoded by the gene contains a total of 770 amino acid residues, belonging to the RNR superfamily, and the highest homology with the published protein sequences in the current database is 90.07%. The ribonuclease R gene has an expression way realized by using plasmid pET-28 (+) to construct a recombinant expression vector containing the ribonuclease R gene, transferring the recombinant expression vector into Escherichia coli hostcells and inducing the expression of the ribonuclease R gene. The optimal catalytic temperature of the expression product, ribonuclease R, of the invention is about 30 DEG C, and 50% or above of thehighest enzyme activity is maintained between 15 and 40 DEG C; and 85% or above of the highest enzyme activity is maintained with the presence of 0-3 M NaCl, and the expression product has broad application prospects. The expression product of the invention has outstanding cold adaptability, relatively good stability and certain salt resistance, opening up the potential application of the expression product in food, medicine and molecular biology related fields.

Description

technical field [0001] The invention belongs to the related fields of enzyme engineering, food and medicine, and in particular relates to a cold-adapted ribonuclease R and its encoding gene and application. Background technique [0002] Ribonuclease (Ribonuclease, RNase) is an important nucleolytic enzyme in organisms. Its function is to hydrolyze the phosphodiester bonds between nucleic acid residues to generate mononucleotides or oligonucleotides, which play a role in the process of RNA metabolism. play an important role. The RNase family has high application value in the fields of food, medicine and molecular biology research because of its special biological activity and pharmacological function. Ribonuclease R is a member of the ribonuclease family and mainly acts on the 3'-5' phosphodiester bond in nucleic acid. Ribonuclease R can participate in the control of the type and quantity distribution of RNA in cells, and can also modify mRNA and degrade different types of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/22C12N15/70C12N1/21C12R1/19
CPCC12N9/22C12N15/70
Inventor 侯艳华王亚彤王全富王一帆
Owner HARBIN INST OF TECH AT WEIHAI
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