Prokaryotic expression vector of rice metallothionein gene OsMT-1-2a and application thereof

A metallothionein, prokaryotic expression technology, applied in the use of vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve the problems of unclear ROS scavenging ability, unclear scavenging ability, etc., and achieve strong scavenging of superoxide ions and The effect of hydroxyl radical capacity

Inactive Publication Date: 2014-04-23
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] There are 11 metallothionein genes in the rice genome, except for OsMT2b and OsMT-1-4b, Whether the other 9 OsMT genes have ROS scavenging ability is not clear. We found in the previous research that in rice ROS-a

Method used

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  • Prokaryotic expression vector of rice metallothionein gene OsMT-1-2a and application thereof
  • Prokaryotic expression vector of rice metallothionein gene OsMT-1-2a and application thereof
  • Prokaryotic expression vector of rice metallothionein gene OsMT-1-2a and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Prokaryotic expression vector Pet-32A- OsMT-1-2a build

[0038] (1) Primer design: according to the rice in the NCBI database OsMT-1-2a Gene (accession number: D15602) sequence and prokaryotic expression vector Pet-32A multiple cloning restriction site, design a pair of specific primers:

[0039] OsMT-1-2a -5': GAATTCatgtcgtgctgcggaggaaactg

[0040] OsMT-1-2a -3': CTCGAGcttgccgcagttgcaggggttgc

[0041] Primer OsMT-1-2a -5' contains restriction endonuclease EcoR I recognition site, OsMT-1-2a- 3' Contains a restriction enzyme xho I recognition site.

[0042] (2) Extraction of total RNA

[0043] After the rice flower was separated from the plant, it was quickly put into liquid nitrogen and ground to powder, and the ground material was transferred to an eppendorf tube, and 1 mL of Trizol was added, oscillated and mixed, and 0.2 mL of chloroform was added, oscillated and mixed, 12000rpm, 4 Centrifuge at ℃ for 10 min, carefully absorb the su...

Embodiment 2

[0056] Embodiment 2: prokaryotic expression vector Pet-32A- OsMT-1-2a Prokaryotic expression of

[0057] The recombinant plasmid Pet-32A- OsMT-1-2a Transformed into E. coli BL21 (DE3) Competent cells were spread on LB (Amp-resistant) solid plates, and the recombinant colonies of Pet-32A-OsMT-1-2a were picked and placed in LB (Amp-resistant) liquid medium, 37°C, Cultivate overnight on a shaker at 200 rpm, inoculate on the same LB medium at a ratio of 1:100, culture with shaking at 37°C until OD600 is 0.6, add IPTG with a final concentration of 1 mmol / L to induce at 37°C for 6 hours, 6000 rpm The cells were collected by centrifugation for 10 min at 1 / min, and the supernatant and inclusion bodies were separated after sonication. According to SDS-PAGE electrophoresis analysis, compared with the empty vector Pet-32A, there was an obvious protein band with a molecular weight of about 32 kD in the protein of the Pet-32A-OsMT-1-2a recombinant cell, which was consistent with the p...

Embodiment 3

[0058] Embodiment 3: the purification of protein

[0059]Since the prokaryotic expression vector Pet-32A contains a His-tag in the C segment, and the C-terminus of the expressed protein contains 6 histidines, Ni-NTA can be used for purification. Add 50 ml of lysate (50 mmol / L NaH2PO4 and 300 mol / L NaCl, 8 mol / L Urea, 20 mmol / L imidazole, pH8.0) to 50 ml of bacteria prepared in LB medium, stir at room temperature for 30 min, and then Centrifuge at 12000 r / min for 30 minutes. Take the supernatant, pass through the Ni-NTA resin column, and wash twice with 10 ml of washing solution (50 mmol / L NaH2PO4 and 300 mmol / L NaCl, 8 mol / Lurea, 20 mmol / L imidazole, pH8.0), Then it was eluted with eluent (50 mmol / L NaH2PO4 and 300 mmol / L NaCl, 250 mmol / L imidazole, 8 mol / L Urea, pH8.0). SDS-PAGE electrophoresis analysis found that there was a clear band at about 32 kD in the Pet-32A-OsMT-1-2a strain, and a clear band at 23 kD in the supernatant of the protein expressed by the Pet-32A stra...

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Abstract

The invention discloses a prokaryotic expression vector of the rice metallothionein gene OsMT-1-2a and an application thereof. The vector contains the rice OsMT-1-2a protein gene, a T7 promoter and a terminator, a ribosome bind site (RBS) and a His label; and the upstream part of the OsMT-1-2a gene is the promoter, and the downstream part of the T7 promoter has an operation sub-sequence which can be induced by IPTG. The obtained in-vitro expression protein Pet-32A-OsMT-1-2a has relatively strong capacity of removing superoxide ions and hydroxyl radicals, and a feasible way is provided for the function and effect of clearing reactive oxygen of the OsMT-1-2a; and the activity level of the OsMT-1-2a protein in plant can be improved through the strategy of genetic engineering so as to improve the stress resistance of the plant.

Description

technical field [0001] The present invention relates to rice metallothionein gene OsMT-1-2a The prokaryotic expression vector and application thereof belong to the field of genetic engineering. Background technique [0002] The growth and development of plants requires suitable external conditions. When the temperature, humidity, moisture, and salt concentration change sharply, or when air pollution, ultraviolet radiation, pesticides, and pathogens act on plants, a large amount of active oxygen will be produced in the plant. (Reactive Oxygen Species, ROS), leading to oxidative stress response. [0003] The protective mechanisms for effectively scavenging reactive oxygen species in plants are divided into enzymatic and non-enzymatic systems. Enzymatic systems include superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), gluten Glutathione reductase (GR), glutathione peroxidase (...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/82
Inventor 胡丽芳贺浩华刘世强朱昌兰边建民彭小松贺晓鹏傅军如陈小荣欧阳林娟
Owner JIANGXI AGRICULTURAL UNIVERSITY
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