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Formaldehyde dehydrogenase gene CcFALDH and use thereof

A technology of formaldehyde dehydrogenase and gene, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem that formaldehyde dehydrogenase gene has not been reported yet

Inactive Publication Date: 2014-10-22
HARBIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Xu et al. used the dynamic box simulation method to study that the Chlorophytum potted plant system has a long-term purification effect on formaldehyde, and the purification rate is greater during the day than at night, but the change of light has no significant effect on the purification rate.
Glutathione-dependent formaldehyde dehydrogenase plays an important role in plant formaldehyde metabolism. Although Arabidopsis, pea, corn, rice and pothos have been reported, the formaldehyde dehydrogenase gene and related applications in Chlorophytum have not been seen so far. to report

Method used

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  • Formaldehyde dehydrogenase gene CcFALDH and use thereof
  • Formaldehyde dehydrogenase gene CcFALDH and use thereof
  • Formaldehyde dehydrogenase gene CcFALDH and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, the cloning of Chlorophytum formaldehyde dehydrogenase gene

[0030] From the NCBI database (http: / / www.ncbi.nlm.nih.gov / ), search and save the formaldehyde dehydrogenase sequence of higher plants, using the known Arabidopsis, pea, corn, rice and pothos in the database (NM_123761.3, Y11029, AK099733.1, AB618795) formaldehyde dehydrogenase sequences were compared. Degenerate primers were designed according to the conserved amino acid sequences. In order to facilitate the construction of recombinant plant expression vectors, the 5' ends of the upstream and downstream primers were respectively introduced BamH I and Sac I restriction enzyme sites, primer sequences are as follows:

[0031] FALDH-F: CGGAATTCATGGCKACHSAGGSHCAGG

[0032] FALDH-R: CGAGCTCTYASAYYTGCRTVKCMAG

[0033] RNA extraction: RNAprep pure Plant Kit (TianGen) was used to extract RNA from leaves of Chlorophytum comosum treated with 5mM formaldehyde for 1 hour, and reverse-transcribed into cDNA...

Embodiment 2

[0039] Embodiment 2, the construction of plant expression vector pBI-35S-FALDH

[0040] Restriction sites BamH I and Sac I were respectively introduced into the primers during gene cloning. The cloning vector pMDCcFALDH plasmid containing the CcFALDH gene was double digested with BamH I and Sac I. For the enzyme digestion system, refer to the manual of restriction endonuclease double digestion reaction of Takara Company, and react at 37°C for 12 hours. The target fragment of CcFALDH with cohesive ends was recovered.

[0041] The plasmid DNA of the binary plant expression vector pBI121 was digested with BamH I and Sac I, reacted at 37°C for 12 hours, and the large fragment, namely the pBI121 vector backbone, was recovered.

[0042] Ligate the fragment of interest to the parent vector. The ligation product was transformed into TOP10 competent cells. Single colonies were picked for PCR identification. Plasmids were extracted from colonies positive for PCR, and BamH I and Sac...

Embodiment 3

[0043] Embodiment 3, detection of CcFALDH gene transformed plant and transgenic plant

[0044] The successfully constructed plant expression vector pBI-35S-FALDH was introduced into Agrobacterium EHA105 by electroporation, and colony PCR identification was carried out, and a product of about 1200bp was obtained. Extract the PCR-positive colony plasmid, carry out enzyme digestion, and obtain a fragment of about 1200bp, which proves that the transformation vector has been transferred into Agrobacterium ( figure 2 -f).

[0045] Inoculate single Agrobacterium colonies identified by colony PCR into 5ml YEB liquid medium containing 100 μg / ml KM, culture overnight at 28°C with shaking at 200 rpm, and transfer them into 50ml YEB liquid medium the next day , at 28°C, shake culture at 200 rpm to OD60, when the value is 0.4-0.6, collect the bacteria, suspend with MS liquid medium, and set aside.

[0046] Transfer the recombinant overexpression vector pBI-35S-FALDH obtained above into ...

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Abstract

The invention relates to the field of plant gene engineering and especially relates to a use of a bracketplant formaldehyde dehydrogenase gene and a plant expression vector in formaldehyde purifying plant cultivation. Through separation and function identification of the bracketplant gene FADLH, use of the gene CcFADLH in formaldehyde absorption and metabolism of plants is ensured. The invention discloses a formaldehyde dehydrogenase gene nucleotide sequence and a protein amino acid sequence coded by the formaldehyde dehydrogenase gene nucleotide sequence. The gene CcFADLH has full length of 1140bp and can code a protein comprising 379 amino acid residues. An experiment proves that the gene can improve a formaldehyde absorption rate of gloxinia. The gene has a wide application prospect and a large economic value in improvement of an indoor formaldehyde purification capability of ornamental plants based on gene engineering.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and plant genetic engineering, and specifically relates to a formaldehyde dehydrogenase gene expressed in Chlorophytum Chlorophytum and capable of improving the ability of plants to absorb formaldehyde—CcFALDH gene, a recombinant plant expression vector containing the gene, and a recombinant plant expression vector containing the gene. The transgenic plant transformant of the above recombinant plant expression vector and the application of CcFALDH gene in improving the ability of plants to absorb and purify formaldehyde. Background technique [0002] Formaldehyde is an essential chemical raw material in industrial manufacturing. Formaldehyde gas emitted from formaldehyde products has become a common decoration indoor air pollutant. As a common indoor environmental pollutant, the harm of formaldehyde to the human body is mainly reflected in stimulating toxicity, nervous system toxicity, ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/82A01H5/00
Inventor 郭长虹宋丽莉尹琳微
Owner HARBIN NORMAL UNIVERSITY
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