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Rice dehydrin protein with antibacterial activity and application thereof

A technology of antibacterial activity, protein, applied in the field of genetic engineering

Inactive Publication Date: 2011-05-04
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there is no report that dehydrin protein can inhibit the growth of bacteria

Method used

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  • Rice dehydrin protein with antibacterial activity and application thereof
  • Rice dehydrin protein with antibacterial activity and application thereof
  • Rice dehydrin protein with antibacterial activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the acquisition of dehydrin clone and the growth curve of bacteria

[0028] According to the screening method of Cheng et al. (Cheng et al, 2009), the expression library from rice roots was used for screening. After induction with IPTG, the colonies that turned blue with trypan blue staining were selected. After two rounds of induced expression verification, 35 colonies were selected. The clones were terminally sequenced, and the sequencing results were analyzed by BLASTX. It was found that 7 clones encoded Dehydrin protein, 4 of which were genes encoding SK3-Dehydrin protein, and their numbers were RR46, RR51, RR142 and RR306, respectively.

[0029] Take the overnight bacteria of RR46 clone, inoculate 100 mL of LB liquid medium containing 100 μg / mL ampicillin at a ratio of 1%, shake culture at 37 ° C until the OD600 value is about 0.1, take out 50 mL and add IPTG (final concentration 1 mmol / L) to induce, and the remaining As an uninduced control, samples ...

Embodiment 2

[0030] Embodiment 2, the cloning of dehydrin gene and the expression of protein

[0031]Using the RR46 plasmid DNA as a template, the dehydrin gene was amplified and cloned into the pET28a expression vector (Zhai Congjie et al. 2009), and the cloned pET28a-Dehydrin plasmid was transformed into Escherichia coli BL21-DE3-pLysS strain, and picked Inoculate a single colony into 3 mL LB liquid medium containing 50 μg / mL kanamycin, and culture overnight at 37°C with shaking. Transfer 100 mL of LB liquid medium containing 50 μg / mL kanamycin according to 1% inoculum amount, culture at 37 °C until OD600 = 0.6-0.8, take out 50 mL and add IPTG to a final concentration of 1 mmol / L, induce for 3 hours, and culture the rest of the bacteria for the same time . Centrifuge, discard the supernatant, wash the bacteria 3 times, each time with 15mL sterile water. Resuspend in 5 mL PBS buffer, sonicate on ice (on / off=5S / 5S) for 10 min, 12000 r / min, centrifuge at 4°C for 10 min, and the supernatan...

Embodiment 3

[0032] Embodiment 3, the inhibition of dehydrin protein to bacterial growth

[0033] Use a file puncher to cut a circular piece of filter paper with a diameter of about 0.7 cm, dry it after sterilization, spread it on the surface of the freshly prepared medium containing the bacteria to be tested, add 20 μL of the liquid protein to be tested on the filter paper, and keep at 37 °C Cultivate overnight, observe the inhibition zone and take pictures the next day. The results show that the bacterial lysate has obvious antibacterial effect on Bacillus pumilus, Bacillus subtilis, Sarcina luteus and Staphylococcus aureus, but it has obvious antibacterial effect on Escherichia coli, Xanthomonas oryzae had no visible antibacterial effect ( figure 2 ), the purified Dehydrin protein had no antibacterial activity (data not attached).

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Abstract

The invention discloses rice dehydrin protein with antibacterial activity. The dehydrin protein contains one of the following amino acid residue sequences: (1) protein of SEQ ID NO:1 in a sequence table; (2) protein with antibacterial activity, which is obtained by replacing and / or deleting and / or adding amino acid residues of the SEQ ID NO:1 in the sequence table by one or several amino acid residues; and (3) segments with over 15 amino acids of the SEQ ID NO:1 in the sequence table. The in-vitro expressed rice dehydrin protein, the protein segments and in-vitro synthesized segments in the invention all have antibacterial activity for special Gram-positive bacteria. The dehydrin protein in plants, disclosed by the invention, is extracted for inhibiting the Gram-positive bacteria. Resistance to biological stress can be improved by transplanting encoding genes of the rice dehydrin protein provided by the invention into plants.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a rice dehydrin protein with antibacterial activity and its application. Background technique [0002] Antibiotics can inhibit or kill pathogenic microorganisms, and their application plays an important role in ensuring human health. However, due to the overuse of antibiotics, bacterial resistance has increased, leading to a decline in the efficacy of antibiotics. Antimicrobial peptides are small molecular polypeptides with antibacterial activity existing in organisms, and they are an important line of defense for organisms against the invasion of pathogenic microorganisms. Antimicrobial peptides can act on the cell membrane of pathogenic bacteria to depolarize the membrane or form "holes", causing the contents of the cell to flow out or peptide molecules to enter the cell and interact with the target, thereby killing the bacteria. Because antimicrobial peptides can act on mu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29A01N63/02A01N47/44A01P1/00C12N5/10
Inventor 刘国振成雄鹰翟从劼李莉云刘丽娟兰金苹
Owner HEBEI AGRICULTURAL UNIV.
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