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3-mercaptopyruvate sulfurtransferase and application thereof

A technology of mercaptopyruvate sulfur transferase, applied in the field of molecular biology

Active Publication Date: 2018-09-04
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] MST is an important enzyme in the process of cyanide detoxification in plants. This enzyme can transfer the sulfane sulfur atom on the substrate sodium mercaptopyruvate to cyanion to generate non-toxic thiocyanate and pyruvate. It has been reported that this protein interacts with H in plants 2 S generate about

Method used

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  • 3-mercaptopyruvate sulfurtransferase and application thereof
  • 3-mercaptopyruvate sulfurtransferase and application thereof
  • 3-mercaptopyruvate sulfurtransferase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1. Arabidopsis AtMST Gene cloning.

[0024] Take 4-week-old rosette leaves, extract total RNA (TRIzol Extraction Kit, TaKaRa Company), reverse transcribe into cDNA (Degenome Reverse Transcription Kit, abm Company), use the cDNA as a template, and use AtMST Specific primers for PCR, cloned AtMST Gene. Such as figure 1 It shows that after high-fidelity PCR using Arabidopsis cDNA as a template, a fragment with a size of 1140 bp is obtained as the target fragment; the negative control is ddH 2 O is the template, no bands were obtained; the fourth swimming lane is the recombinant pET28a- AtMST The fragments obtained from the bacterial liquid PCR detection of the construct indicated that the recombinant construct was constructed successfully.

Embodiment 2

[0025] Embodiment 2. T7:: AtMST Transformation of E. coli with recombinant plasmid, comparison experiment between E. coli induced by IPTG and E. coli without IPTG induction (method of preparing target protein and blank control experiment):

[0026] Preparation of experimental materials: extract total RNA from leaves of Arabidopsis thaliana, reverse transcribe into cDNA, clone AtMST gene (At1g79230), construct T7:: AtMST Recombinants were transformed into Escherichia coli BL21 (DE3) competent cells, and positive bacteria were obtained by kanamycin screening. A single colony was picked, identified by PCR, and successfully transformed Escherichia coli was obtained for experimentation.

[0027] The above two Escherichia coli were divided into two groups. One group was added with IPTG (final concentration: 0.05-0.5 mM), and the other group was added with an equal amount of double-distilled water, and cultured with shaking at 25-37°C for 12-20 h. Centrifuge at 10,000 rpm at 4°C ...

Embodiment 3

[0029] Example 3: H 2 Determination experiment of S yield.

[0030] Mix the reaction system according to the following formula: add 150 μmol·L -1 Sodium mercaptopyruvate, 100 μg protein, and the rest supplemented with PBS buffer solution with pH=7, the mixture was added to a 40 mL Erlenmeyer flask with a cover; the cap of a 1.5 mL centrifuge tube was cut off, and 500 μL of 1% zinc acetate solution; put the centrifuge tube into the Erlenmeyer flask and cover the Erlenmeyer flask cap. 37°C, shaking at 115 rpm, after fully reacting for 15 min, add 100 μL of 20 mM DPD and 30 mM FeCl respectively 3 The solution terminated the reaction, stood in the dark for 15 min, and measured the OD 670 . H was calculated from the standard curve 2 The yield of S.

[0031] image 3 The results showed that when induced by IPTG, AtMST The mercaptopyruvate sulfur transferase produced by gene expression can use sodium mercaptopyruvate as a substrate to catalyze H 2 S production, thereby incre...

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Abstract

The invention discloses a 3-mercaptopyruvate sulfurtransferase (AtMST) and an application thereof. The AtMST gene is cloned with Arabidopsis thaliana cDNA as a template to construct a recombinant pET28a-AtMST, the recombinant pET28a-AtMST is transferred into Escherichia coli BL21 (DE3), and IPTG induction expression and Ni column purification are performed, so it is proved that an MST protein adopts sodium mercaptopyruvate as a substrate and has an H2S generation catalysis activity. A transgenic plant having high resistance to biological stress and abiotic stress is established through a transgene technology in practical production by combining the wide and important functions of an H2S signal in a plant body.

Description

technical field [0001] The invention relates to a plant mercaptopyruvate sulfur transferase and an application thereof, belonging to the field of molecular biology. Background technique [0002] h 2 S is the third gas signaling molecule after nitric oxide (NO) and carbon monoxide (CO). In plants, H 2 S signal participates in various physiological processes of plants, such as promoting seed germination, root morphogenesis, enhancing leaf photosynthesis, regulating stomatal movement, and delaying plant senescence; H 2 S also participates in the process of plant resistance to various biotic and abiotic stresses, such as water stress, temperature stress, and heavy metal stress, by regulating gene expression and synergizing with plant hormones. [0003] In plants, H 2 S is mainly produced through enzymatic degradation. The currently identified catalytic plant endogenous H 2 The enzymes produced by S are shown in the table below. They all use cysteine ​​(Cys) as a substrate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P11/00
CPCC12N9/13C12N15/70C12P11/00C12Y208/01002
Inventor 裴雁曦解梦洁张丽萍贺烽
Owner SHANXI UNIV
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