Application of isocitrate dehydrogenase in improvement of formaldehyde absorption and metabolism capability of plants

A technology of isocitrate dehydrogenase and formaldehyde absorption, applied in the field of molecular biology and genetic engineering, to achieve the effects of facilitating planting, improving formaldehyde absorption efficiency, and easy storage

Active Publication Date: 2021-10-15
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, there are no reports about the role of Arabidopsis isocitrate dehydrogenase gene AtIDH3 in improving the process of plant absorption and metabolism of formaldehyde and the regulation of Arabidopsis isocitrate dehydrogenase by 14-3-3c protein

Method used

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  • Application of isocitrate dehydrogenase in improvement of formaldehyde absorption and metabolism capability of plants
  • Application of isocitrate dehydrogenase in improvement of formaldehyde absorption and metabolism capability of plants
  • Application of isocitrate dehydrogenase in improvement of formaldehyde absorption and metabolism capability of plants

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Construction of prokaryotic expression vector of AtIDH3 gene and its protein expression and purification

[0040] TRIzoL Reagent (Invitrogen) was used to extract the total RNA of Arabidopsis leaves, and the operation was carried out according to the instructions; 25 μL RNA was reverse-transcribed into cDNA with M-MLV Reverse Transcriptase (Fermentas Company) reverse transcription kit; 1 μL cDNA was used as a template, Perform PCR to amplify the target fragment. The CDS sequence (At4G35650) of Arabidopsis thaliana IDH3 was found in NCBI, and its double restriction sites (BamHI and SalⅠ) and gene-specific primers (F-ATGGCGAGAAGATCCGTTTC; R-CTCAAGAGCTGCTATGACAGCA) were designed by DNAman software. Use designed specific primers to carry out PCR amplification, gel recovery AtIDH3 gene fragment ( figure 1 A), store at -20°C for later use. Gel recovered AtIDH3 gene and pMD18-T for TA cloning to obtain pMD18-AtIDH3 vector, heat shock transformed DH5α, smeared in am...

Embodiment 2

[0043] Embodiment 2: Determination of isocitrate dehydrogenase enzyme activity

[0044] The protein concentration was determined using the BCA method protein quantification kit produced by Shanghai Jierui Bioengineering Co., Ltd., with bovine serum albumin (Bovine serum albumin, BSA) as the standard protein, and operating according to the Bradford method according to the kit operation manual. The enzyme activity was measured by spectrophotometry, and the enzyme activity assay system of IDH was 20mmol / L Tris-HCl, 5mmol / L MgCl 2 , 5mmol / L DL-isocitric acid trisodium or 0.5mmol / L DL-isocitric acid trisodium, 2mmol / L NAD + or 0.5mmol / L NADP + . At 25° C., a UV spectrophotometer was used to detect the change of light absorption value at 340 nm during the reaction.

[0045] (1) Optimum temperature and thermal stability of isocitrate dehydrogenase AtIDH3

[0046] In the above reaction system with other conditions unchanged, at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, in a water ...

Embodiment 3

[0056] Example 3: Preparation and titer detection of AtIDH3 protein rabbit antibody

[0057] 1 mL of the purified recombinant protein (containing approximately 5 mg of purified AtIDH3 protein) was emulsified with 1 mL of Freund's adjuvant at a ratio of 1:1. First add Freund's adjuvant into the mortar and emulsify several times; until a qualified water-in-oil agent is prepared, rabbits can be immunized. When the rabbit was immunized for the first time, it was emulsified with an equal volume of complete Freund's adjuvant, and the whole body of the rabbit was immunized several times in a small amount, and a small amount of antigen was injected at each point. Inject the antigen as evenly as possible into each immunization point through various methods such as intramuscular, subcutaneous or intradermal injection. Two weeks after the first immunization, the AtIDH3 antigen was emulsified with an equal volume of incomplete Freund's adjuvant to boost the rabbit. Weekly booster immuni...

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Abstract

The invention discloses an application of an arabidopsis thaliana isocitrate dehydrogenase gene AtIDH3 in improvement of formaldehyde absorption and metabolism capability of a plant. The nucleotide sequence of the arabidopsis thaliana isocitrate dehydrogenase gene AtIDH3 is as shown in SEQ ID NO:1. The AtIDH3 gene is recombined into a plant expression vector, wild tobacco is transformed, and AtIDH3 transgenic tobacco is obtained through screening; experimental results show that under the same concentration stress and the same time, the formaldehyde absorption effect of the overexpressed tobacco is better than that of the wild tobacco; under the stress of liquid formaldehyde, the expression quantity of AtIDH3 transcriptional level of wild tobacco and 14-3-3c overexpressed tobacco is obviously increased. Yeast double hybrid, Pull-down and Co-IP experiments prove that the 14-3-3c protein interacts with the AtIDH3, and the result shows that the over-expressed transgenic tobacco has higher formaldehyde absorption capacity than wild tobacco.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering, and in particular relates to the use of Arabidopsis isocitrate dehydrogenase gene AtIDH3 in improving plant formaldehyde absorption and metabolism. Background technique [0002] Formaldehyde is one of the pollutants that people are most often exposed to in daily life, so the treatment of formaldehyde pollution has always been widely concerned. Formaldehyde can react non-specifically with various macromolecules in organisms, resulting in the inactivation of macromolecules. Therefore, formaldehyde has multiple toxic effects on organisms and is associated with various human diseases. Formaldehyde enters the human body through the respiratory system and reaches the whole body through the blood circulation, causing various abnormal functions of the body. Any animal drinking water polluted by formaldehyde for a long time will also cause different degrees of harm to t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/82A01H5/00A01H6/82
CPCC12N9/0006C12N15/8243C12Y101/01041
Inventor 李昆志李方慧赵星年洪娟
Owner KUNMING UNIV OF SCI & TECH
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