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Bacillus brevis formaldehyde dehydrogenase gene and plant expression vector and application thereof

A plant expression vector and formaldehyde dehydrogenase technology, applied in the field of genetic engineering, can solve the problems of low expression level and low ability to metabolize formaldehyde, etc.

Inactive Publication Date: 2013-08-28
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] For above-mentioned deficiency, the object of the present invention is to provide a kind of good stability, can improve the brevibacillus formaldehyde dehydrogenase gene of plant absorption, metabolism and tolerance formaldehyde ability simultaneously faldh Sequence and isolate and clone the primer sequence of the gene, and use the gene to transform plants to improve the absorption, metabolism and tolerance of formaldehyde, and overcome the shortcomings of the plant's own low expression level of key enzymes in formaldehyde metabolism and low ability to absorb and metabolize formaldehyde

Method used

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  • Bacillus brevis formaldehyde dehydrogenase gene and plant expression vector and application thereof
  • Bacillus brevis formaldehyde dehydrogenase gene and plant expression vector and application thereof
  • Bacillus brevis formaldehyde dehydrogenase gene and plant expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: faldh gene cloning

[0063] faldh Gene cloning strategies such as figure 1 shown. Find similar microbial species from GenBank (including Escherichia coli K-12, Paracoccus denitrificans , Photobacterium damselae subsp. Piscicida, Pichia pastoris , Rhodobacter sphaeroides 2.4.1) For the amino acid sequence of the formaldehyde dehydrogenase gene, design a pair of degenerate primers based on the conserved amino acid sequence, the sequence is as follows:

[0064] fld 3: GGNCAYGARCCNATGGGNATNGTNGARGA

[0065] fld 4: TCCATNCCNACRCARTCNATNACNACRTC

[0066] Genomic DNA was extracted by the following method: Pick a well-grown single colony and inoculate it in LB liquid medium for shaking culture at 30°C overnight; transfer 1% of the inoculum to 100ml of fresh LB liquid medium, and shake it for culture at 37°C 4h (OD 600 =2.0), 6000rpm / min centrifuge to collect bacteria; add SI solution (0.3M Sucrose, 25 uM Tris-HCL (pH8.0) 25mM EDTA) of 1 / 10 th...

Embodiment 2

[0070] Example 2: Construction of entry vector pENTR-2B- faldh

[0071] The entry vector pENTR-2B- faldh builds like Figure 4 shown. use Bam HI and xho I double digestion pMD18-T- faldh and pENTR-2B*, the cut vector and insert fragments were separated by agarose gel electrophoresis, and pMD18-T- faldh produced after being cut faldh The gene fragment (1.2kb) and the gateway clone (Gateway) entry vector pENTR-2B* were cut to generate the vector fragment pENTRT-2B*, and then the ligase kit of TaKaRa was used to connect pENTRT-2B* and faldh The DNA fragment of the gene produces the entry vector pENTR-2B- faldh ( Figure 4 ). Conversion of high efficiencies (10 8 ) Escherichia coli competent cells (DH5α, purchased from Tiangen Biochemical Technology Co., Ltd.), spread the transformed Escherichia coli on a plate added with kanamycin (Km, 50 μg / ml), at 37 o C culture overnight, screen the Km-resistant recombinant colony, extract the plasmid from the Km-resistant rec...

Embodiment 3

[0072] Embodiment 3: Plant expression vector pK 2 -35S- faldh build

[0073] Plant expression vector pK 2 -35S- faldh A build strategy such as Figure 6 shown. Through the LR response of Gateway technology, the faldh Subcloning into the plant expression vector pK 2 GW 7 (Gateway's destination carrier, Belgium VIB / Gent company). The specific method is: use the plasmid extraction kit to purify the Gateway's target vector pK 2 GW 7 , add pENTR-2B- to Gateway's LR reaction system faldh and pK 2 GW 7 150ng each, 1μl LR Clonase II Enzyme Mix (Invitrogen), mix well at 25 o C reaction overnight, through the action of integrase faldh Integrate into pK 2 GW7 obtained in faldh The plant expression vector plasmid pK2-35S- faldh , see the attached Figure 9 . Conversion of high efficiency (10 8 ) Escherichia coli competent cell DH5α (purchased from Tiangen Biochemical Technology Co., Ltd.), spread the transformed Escherichia coli on a plate added with spectinomyc...

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Abstract

The invention discloses a bacillus brevis formaldehyde dehydrogenase gene faldh, and a construction method and application of a plant expression vector of the gene, and provides a nucleotide sequence of the bacillus brevis gene faldh which can improve the endurance capability of formaldehyde and an amino acid sequence of protein encoded by the bacillus brevis gene. The gene is used for constructing a plant expression vector pK2GW7-35S-faldh, and the vector is transferred into a plant through agrobacterium tumefaciens in a mediate mode, so that the formaldehyde absorption, metabolization and endurance capability of the plant is improved, and the defects of relatively low expression levels, low formaldehyde absorption and metabolization capability of formaldehyde metabolism key enzyme of the plant are overcome; experiments prove that the formaldehyde (in liquid) absorption speed of a transgenic plant containing the formaldehyde dehydrogenase gene faldh is higher than that of a wild typeplant; glutathione-dependent formaldehyde dehydrogenase (FALDH) protein expressed by the transgenic plant has formaldehyde oxidation capability and high stability and can exert effects in the plant all the time; and the gas formaldehyde metabolization capability of the FALDH protein is greatly improved through excessive expression of the FALDH protein in the transgenic plant.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the formaldehyde dehydrogenase gene of brevis bacillus and the plant expression vector pK of the formaldehyde dehydrogenase gene thereof 2 -35S- faldh And the application of the gene in the transgenic plant with strong ability of improving plant absorption and tolerance to formaldehyde. Background technique [0002] Formaldehyde is a colorless gas with a strong pungent odor, easily soluble in water, alcohol and ether. It has a very strong reaction ability and can produce non-specific reactions with proteins, nucleic acids and lipids (Feldman et al., Prog Nucleic Acid Res Mol Biol, 1973, 13:1-49), and is a very active compound, so it is effective for all All organisms are highly toxic. Formaldehyde is widely used in industrial production as a raw material for the manufacture of resins, adhesives, paints, plastics, and man-made fibers, and is the most widely used c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/84A01H5/00C12R1/08
Inventor 年洪娟陈丽梅孟庆超程琴
Owner KUNMING UNIV OF SCI & TECH