CN104726486B - A kind of breeding method for the sweet wormwood for absorbing benzene - Google Patents
A kind of breeding method for the sweet wormwood for absorbing benzene Download PDFInfo
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Abstract
The invention discloses a kind of breeding method for the sweet wormwood for absorbing benzene, this method is to import Rhd genes in sweet wormwood by plant expression vector, screens the sweet wormwood for the benzene that is absorbed, concretely comprises the following steps：Rhd genes are cloned from bacterial strain bacillus, build the plant expression vector of the gene containing Rhd, use is agriculture bacillus mediated, and Rhd genes are transferred into blue or green punt-pole and cultivate screening regeneration plant, obtains the transgenosis green grass or young crops punt-pole plant for absorbing benzene.In closed benzene fumatorium, common plant benzene is absorbed as 0.12mg/m2When .h, the transgenosis green grass or young crops punt-pole plant benzene that the present invention obtains absorbs highest 0.23mg/m2.h, it is nearly 2 times of nontransformed control green grass or young crops punt-pole content that its benzene, which absorbs,.The method of the present invention is significant for removing the benzene discharged in house fitting-up.
Description
Technical field
It the present invention relates to the use of genetic engineering and cultivate plant field, be specifically that a kind of cultivated using transgenic technology can inhale
The method for receiving the sweet wormwood of benzene.
Background technology
Home decoration is essential in people's daily life, and there is various industrial chemicals, chemical industry in ornament materials to close
Domestic finishing material is increasingly being used for into material, especially with ropy decoration and finishing material, indoor pollution is more
Seriously, the chemical contamination of the new house release after finishing turns into the problem of very serious, comes to people's health care belt potentially hazardous.It is indoor
Domestic environment pollution causes extensive concern and research, and one of four global environmental problems are exactly indoor environmental pollution.Room
Interior decoration pollution can cause bronchitis and lung cancer chronic lung disease breathing problem, and there are some researches show leukemia of children and household
The chemical substance discharged after finishing has certain correlation.So-called indoor environment refers to relative residing for people's Working Life activity
The interior space of closing, refer to the indoor activity place such as office and family's residential houses.The dirt that fitment process and completion discharge later
Dye material mainly has benzene, formaldehyde, ammonia and radon radiation etc..
Benzene has special fragranced, colourless, is volatile aromatic hydrocarbon ring class compound, including benzene, toluene, ethylbenzene, neighbour,
Between, paraxylene etc., priority pollutants are classified as by developed country.It is family because various construction materials largely use benzene
Occupy one of volatile materials main in finishing.Suction benzene can cause Chronic Benzene Poisoning for a long time, and its reason is central nervous system
System is suppressed.The benzene discharged in finishing, containing benzene and toluene and dimethylbenzene, it is mainly derived from some oil used in furniture
Paint, glue, solvent, coating, adhesive, wallpaper etc..Benzene is classified as one of carcinogen in house fitting-up by many experts.Connect for a long time
Tactile benzene can cause genetoxic to cause lame property, hematotoxicity, show as uncomfortable in chest, dizzy vomiting, nausea, it is drowsiness, trigger anaemia and in
Pivot nerve blurred vision, blood disease, there is skin hypersensitivity eczema, bronchitis, laryngeal edema and blood platelet decline etc.；Women
Anemia during pregnancy and vomiting, miscarriage, fetal congenital deformity defect etc..Chronic Benzene Poisoning can hinder marrow hemopoiesis function
Hinder, cause alpastic anemia, research shows that benzene of the leukaemia also with house fitting-up release has certain association.
Microorganism energy Partial digestion benzene, benzene degradation bacteria mainly have pseudomonad, acinetobacter calcoaceticus, bacillus, Rhodococcus sp, promise
Cattell bacterium, Ralstonia bacterium, Bacillus alcaligenes etc..In the degradation process of benzene, bacterium oxidizing aromatic hydrocarbons compound first, initially
The oxygen in air is incorporated into substrate by the catalytic action of enzyme, ring hydroxylating dioxygenase (Ring hydroxylation
Dioxygenase, Rhd) rate-limiting step that initial action is benzene biodegradation process is catalyzed, benzene homologues dioxygenase is to degrade
Key enzyme in journey, dioxygenase are directly to add two oxygen atoms to form catechols on phenyl ring；Monooxygenase is then directly attacked
Phenyl ring or oxidized side chain, and then catechol is generated, then crack phenyl ring by catechol dioxygenase effect, generate aldehyde
Or acid, aldehyde or acid are again by tricarboxylic acid cycle oxidation Decomposition into CO2And water.Acetone can be generated after the aromatic rings fracture of benzene
Acid, butanedioic acid, acetic acid, fumaric acid and acetaldehyde, these materials can be utilized by microorganism, and energy and synthetic cell are provided for it
Structural molecule.(CO is resolved into after first ring crack solution of aromatic rings of benzene homologues2And pyruvic acid), second ring also can be by same
On attack hence into degradation process.
Interior decoration volatilization is discharged into the benzene-like compounds in air, is diffused into by stomata and hole skin in plant tissue,
Into in plant cell, reacted with the relevant enzyme in cytoplasm and organelle, benzene can be changed into other materials, finally
It is released in the form of carbon dioxide and water.Medicinal ingredient in sweet wormwood can be divided into volatility and non-volatile 2 parts.
Chemical composition point contains in sweet wormwood (Artemisia annual L.)：Flavones, sequiterpene, cumarin and volatile oil.It is non-volatile
Composition is mainly qinghaosu.Volatile ingredient is mainly volatile oil, including different punt-pole ketone, punt-pole ketone, levo-camphor, by olein, borneol,
The composition such as trip's alkene, sesquialter Chu alcohol, cloves alkene, caryophyllene oxide wherein cloves alkene, punt-pole ketone, different punt-pole ketone, camphor, borneol etc. are living
Property is higher, and sweet wormwood plant is tall and big, fragrant odour, is positioned over and newly repaiies in cell or newly-decorated house, can suppress or clearly
Eliminate the unusual smell, the sweet wormwood volatile ingredient such as chemical substance such as alkene, ketone, alcohols diffuses in atmosphere, can consume in air and partly be harmful to
Chemical substance benzene, reach the purpose of removing.
The content of the invention
For the release of benzene in existing house fitting-up, people train to removing the household plant demand of benzene in air pollution
The ring hydroxylating dioxygenase overexpression transgene abrotanum educated.The technical problems to be solved by the invention are in sweet wormwood genome
The gene of ring hydroxylating dioxygenase synthesis can be increased by importing, and strengthen the expression of its original gene, and it is new to obtain transgene abrotanum
Strain, to improve its ring hydroxylating dioxygenase synthesis capability, make that benzene in blade cell super-transformed can be sucked in its blade
Purpose, this transgene abrotanum strain is set to turn into the removing plant for removing the benzene discharged in house fitting-up.
Ring hydroxylating dioxygenase is the key enzyme that benzene is removed in organism, strengthens Rhd gene activities, can improve green grass or young crops
The activity of wormwood artemisia plant ring hydroxylating dioxygenase, it is of the invention by bacillus (Bacillus) ring hydroxylating dioxygenase gene
The conversion of Rhd gene clonings imports sweet wormwood, improves the ring hydroxylating dioxygenase synthesis in sweet wormwood, obtains epipodium hydroxylating and add dioxygen
The genetic engineering sweet wormwood kind of enzymatic activity, a kind of new method is provided to cultivate the sweet wormwood of ring hydroxylating dioxygenase high activity.
It is contemplated that the plant expression vector of structure Rhd genes, and recombined engineering sweet wormwood plant is obtained, cultivated using transgenic technology
The sweet wormwood of ring hydroxylating dioxygenase high activity lays the foundation.
In order to realize the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is a kind of training for the sweet wormwood for absorbing benzene
Method is educated, is comprised the following steps：
1) genetic fragment of clone strain bacillus ring hydroxylating dioxygenase, Rhd genetic fragments are designated as, and will
Rhd genetic fragments are built into recombinant plasmid pCB-Rhd；
2) recombinant plasmid pCB-Rhd channel genes sweet wormwoods, sweet wormwood of the acquisition comprising Rhd genes are planted using transgenic technology
Strain；PCB-Rhd is imported by Agrobacterium competent cell EHA105 using electric shocking method, then using agrobacterium-mediated transformation by the agriculture
Bacillus competent cell EHA105 imports sweet wormwood, forms the sweet wormwood plant for including Rhd genes.
Specifically, the DNA sequence dna of the Rhd genes is SEQ ID NO.1 in the present invention.
By the above-mentioned transformation carried out to sweet wormwood genome, the ability that sweet wormwood absorbs benzene can be increased substantially, is made
Remove the plant of the benzene discharged in house fitting-up.
It is same kind that the present invention, which is used for as the sweet wormwood of Rhd genetic recipient parents,.Rhd genes are imported in sweet wormwood can be with
The ability that sweet wormwood absorbs benzene is improved, in closed benzene fumatorium, common plant benzene is absorbed as 0.12mg/m2When .h, of the invention
It is up to 0.23mg/m that the transgenosis green grass or young crops punt-pole plant benzene arrived, which absorbs,2.h, it is nearly the 2 of nontransformed control green grass or young crops punt-pole activity that its benzene, which absorbs,
Times.The method of the present invention is significant (such as Fig. 5) for removing the benzene discharged in house fitting-up.
Brief description of the drawings
Fig. 1 is to convert Escherichia coli Ecoli DH5 α competent cells with pET28a-Rhd, after recon single bacterium colony culture,
Again after extracting plasmid enzyme restriction identification and sequencing, then it is transformed into expressive host bacterium BL2l (DE3) and carries out protein expression, it is different
IPTG concentration (isopropyl-β-D-thiogalactoside) stimulates the PCR amplification figures of recombinant protein progress induced expression；
M indicates Marker in figure, and 1 is control Vector, and 2,3,4,5,6 be that (wherein concentration is respectively different IPTG concentration
0.01mmol/L, 0.05mmol/L, 1mmol/L, 1.5mmol/L, 2.0mmol/L, 2.5mmol/L) induced expression protein
SDS-PAGE。
Fig. 2 plant expression vector structure charts；
Be pCAMBIA2301-Rhd vector construction schematic diagrames in figure, before target gene plus zymoprotein promoter or
Person's enzyme promoters adds terminator behind target gene.
Fig. 3 is the PCR specific amplification result schematic diagrams of candidate's transfer-gen plant；
M indicates Marker in figure, and V is only transfects Vector, and 1,2,3,4,5,6 swimming lanes are different candidate's positive plants, i.e.,
Transfect the positive plant of Rhd genes.
Fig. 4 is the real-time quantitative PCR testing result of candidate's transfer-gen plant；
Wherein, ordinate represents transcriptional level, and Vector represents adjoining tree, and Rhd represents candidate's transfer-gen plant.
Fig. 5 is number of the positive transgenic plant with adjoining tree blade to the absorbability of benzene in closed benzene fumatorium
Datagram according to statistics, in figure, Vector represents control, and Rhd represents candidate's transfer-gen plant.
Arabic numerals in Fig. 4 and Fig. 5 after " Rhd- " represent the numbering of different transfer-gen plants.
Embodiment
The invention will be further described with reference to the accompanying drawings and examples, but should not be construed the above-mentioned theme of the present invention
Scope is only limitted to following embodiments.Without departing from the idea case in the present invention described above, known according to ordinary skill
Knowledge and customary means, make various replacements and change, all should include within the scope of the present invention.
Material：Using conventional wild sweet wormwood, derive from Xiushan, Chongqing county within 2008, Chongqing medicines higher junior college with
5 generations of general planting.
Bacterial strain：Bacterial strain bacillus (Bacillus) is that this laboratory is isolated from certain Furniture Factory's runoff water,
(competent cell prepares and takes CaCl Escherichia coli Ecoli DH5 α competence2Method), Agrobacterium EHA105
Plasmid：PMD18-T carriers are purchased from TaKaRa biotech companies, prokaryotic expression carrier pET-28a (+), and plasmid carries
Body pCAMBIA2301.
Enzyme and main agents:Various restriction enzymes such as EcoR I and BamH I, BamHI and HindIlI, Taq DNA
Polymerase, IPTG, dNTP etc. are purchased from TaKaRa biotech companies, and primer synthesis, DNA sequencing are by Shanghai Sheng Gong technology companys.
PCR primer is synthesized by Sangon biotech companies, and random primer labelling kit is purchased from magnificent biological skill engineering company.
Protocols in Molecular Biology
What is be not specifically noted in the following example is carried out according to conventional laboratory conditions, as molecular cloning is taken
Laboratory manual (the New York that SAMBROOK.T etc. writes:Cold Spring Harbor Laboratory PreSS,1989)
Described in condition, or take manufacturer provide kit requirement condition carry out.
Embodiment 1
1.1Rhd gene cloning
Degenerate primer：
Sense primer DNA sequence dna is SEQ ID NO.2：
5′-TGCATATATTAGAGGCACCTCCCTCCGT-3′。
Anti-sense primer DNA sequence dna is SEQ ID NO.3：
5′-TACACTACACGACTCCCAACAGGAACCTC-3′。
PCR primer designs according to ring hydroxylating dioxygenase conserved amino acid sequence:
Sense primer DNA sequence dna is SEQ ID NO.4：
5′-ATTAC TCAGCTACTTGCTTGCCTGCCGTTAC-3′。
Anti-sense primer DNA sequence dna is SEQ ID NO.5：
5′-TACGTGCTAGCT TGCTTCACATTCAGTCACCGTCAG-3′。
Bacillus extraction of plasmid DNA extracts bacillus plasmid according to the method on the specification of kit.PCR
Reaction system：Template DNA 1 μ L, SEQ ID NO.2 and SEQ ID each 1 μ L of NO.3, Taq enzyme 0.2 μ L, 10 × PCR buffer
2 μ L, MgCl21 0.4 μ L of μ L, dNTP, use ddH20 supplies the μ L of cumulative volume 20.PCR reaction conditions：95 DEG C of 4min of pre-degeneration；Pre-expansion
Increase 92 DEG C of 1min, 58 DEG C of 1min, 72 DEG C of 3min, 6 circulations；93 DEG C of 1min, 52 DEG C of 1min, 72 DEG C of 2min, 35 circulations, 72 DEG C
Extend 10min.Agarose gel electrophoresis detects pcr amplification product, and specific fragment is reclaimed using DNA gel QIAquick Gel Extraction Kit, gram
Grand connection plasmid vector pMDl8-T, the positive recombinant plasmid of acquisition is sequenced, identification confirms standby.
Separated and recovered by agarose gel electrophoresis and purify pcr amplification product Rhd gene DNAs (1.8kb) fragment, then
It is subcloned into and their TA cloned plasmids pMD18-T-Rhd is obtained on pMD18-T carriers, confirms through PCR and digestion identification, then
It is measured sequence.After sequence analysis is correct, Rhd gene segments are cut out from pMD18-T-Rhd with NdeI and XhoI, Ya Ke
Between grand insertion prokaryotic expression carrier pET28a EcoR I and BamH I site, prokaryotic expression carrier pET28a-Rhd is obtained.
Escherichia coli Ecoli DH5 α competent cells are converted with pET28a-Rhd, picking recon single bacterium colony, are seeded to containing 50mg/L
In the LB fluid nutrient mediums of kanamycins, nutrient solution dilution is seeded to kanamycins containing 50mg/L for 100 times by 37 DEG C of overnight incubation
LB fluid nutrient mediums in, 37 DEG C of shaking table cultures of 250r/min, cultivate to OD600For 0.6-1.0 constantly, IPTG induction weights are added
The expression of histone, 30 DEG C of 150r/min shaking table cultures, using empty carrier pET-28a (+) recombinant bacterium as negative control, passes through
Set different IPTG concentration (isopropyl-β-D-thiogalactoside) to carry out induced expression to recombinant protein, carry out SDS-PAGE
Whether electrophoresis detection recombinant protein expresses.After extracting plasmid enzyme restriction identification and sequencing simultaneously, then it is transformed into expressive host bacterium BL2l
(DE3) protein expression (such as Fig. 1) is carried out in.After preliminary protein expression confirms, by recombinant plasmid with restriction enzyme BamHI and
HindIlI carries out digestion, reclaims about 1.4kb small fragments, and this fragment is connected to the plasmid of identical digestion with T4DNA polymerases
On carrier pCAMBIA2301, purpose is exactly in purpose base before target gene plus zymoprotein promoter or enzyme promoters
Because adding terminator (such as Fig. 2) below, construction step is as described in embodiment 1.2-1.4：(or other equivalent methods).
1.2 build intermediate carrier pMD18-T-p35s-gfp*gus-nos
PMD18-T and pCAMBIA2301 are used as basic building element, is built in pMD18-T-p35s-gfp*gus-nos
Between carrier.Specific implementation step is as follows, and pair of primers is designed by the base sequence of p35s-gfp*gus-nos on pCAMBIA2301
Sense primer NDA sequences are 5 '-CTCAGAACAGAAGCCATCAAAAGC-3 ' (SEQ ID NO.8)；Anti-sense primer NDA sequences
For 5 '-CGTCTTCGTGAGAGACATCCTGT-3 ' (SEQ ID NO.9), and introduce respectively on the primer of upstream and downstream restricted
Restriction enzyme site, in order to the structure of expression vector.Using pCAMBIA2301 plasmids as masterplate, PCR amplification gfp*gus fusions
The expression cassette of gene, pMD18-T carriers are then attached to, inverted screening, the sequencing of picking monoclonal, which compares, to be confirmed.
1.3 structure intermediate carriers
PMD18-T-p35s-Rhd-nos structure.Based on described pMD18-T-p35s-gfp*gus-nos,
With the gfp*gus fusions of Rhd genes replacement thereon in recombinant plasmid vector pMD18-T-Rhd.Specific implementation step:With
Spe I and BstE II double digestion pMD18-T-Rhd and pMD18-T-p35s-gfp*gus-nos, recovery Rhd genetic fragments and
PMD18-T-p35s-gfp*gus-nos large fragments, Rhd genetic fragments are connected with large fragment respectively, are converted picking monoclonal, are carried
Plasmid is taken to do PCR detections and digestion verification, confirmation is errorless, and it is SEQ ID NO.4 and SEQ ID NO.5 to identify primer.
1.4 structure plant binary expression vector pCAMBIA2301-Sps
Based on described pMD18-T-p35s-Rhd-nos, the expression cassette containing Rhd fusions is placed in plant
In binary expression vector pCAMBIA2301, plant binary expression vector pCAMBIA2301-Rhd is built into.Specific implementation operation:
With Sma I and the double digestion pCAMBIA2301 of pst I, large fragment is reclaimed, with Swa I and the double digestion pMD18-T-p35s- of Pst I
Rhd-nos, reclaim Rhd expression cassette.Rhd expression cassettes are connected with pCAMBIA2301 large fragments, picking monoclonal is converted, carries
Plasmid is taken to do PCR detections and digestion verification.
Recombinant plasmid has genetic fragment Rhd, and the fragment is located at CaMV 35S promoters downstream, and enzyme is carried out to recombinant plasmid
Identification is cut, the recombinant plasmid by identification is named into pCB-Rhd., feminine gender is used as using empty carrier pET-28a (+) recombinant plasmid
Control, the identification of PCR augmentation detections, it is SEQ ID NO.4 and SEQ ID NO.5 (such as Fig. 3) to identify primer
The Rhd channel genes sweet wormwood genomes that embodiment 2 contains recombinant plasmid pCB-Rhd
1) electric shocking method is taken to convert Agrobacterium competent cell the recombinant plasmid pCB-Rhd plasmids of above-mentioned preparation
EHA105:PCB-Rhd is imported into Agrobacterium competent cell EHA105, obtains EHA105/p Rhd.Implementation steps are according to English
Process plate 5《Molecular biology mustard laboratory manual》(Science Press .2002), using what is carried on Biorad companies electroporation apparatus
Agrobaterium (Agrobacterium) electric shock programs.The Agrobacterium of the plant expression vector of importing converts for sweet wormwood.
2) conversion and regeneration of sweet wormwood：The conversion of Agrobacterium is carried out first, prepares Agrobacterium competence and conversion, will be turned
The Agrobacterium bacterium solution of change is coated on containing kanamycins 60Lg/mL, rifampin 30Lg/mL, streptomysin 30Lg/mL solid YEP
On culture medium flat plate, 28 DEG C are cultivated 18~24h, the conversion of the fresh single bacterium colony of inoculated and cultured and non-transformed Agrobacterium, are extracted respectively
DNA, and implement PCR amplifications by template of the DNA of extraction, identified with PCR method.Primer is SEQ ID NO.2
With SEQ ID NO.3, in the control (CK) of setting in addition to target DNA fragment is not inserted, other compositions are consistent with processing
.PCR amplification program:96 DEG C of 5min, 92 DEG C of 45s, 65 DEG C of 45s, 72 DEG C of 45s, 35 circulations, 72 DEG C of 5min. agarose gel electrophoresis
After observe
3) conversion of sweet wormwood explant：Conversion is carried out to sweet wormwood using leaf disk method and chooses young piece from sweet wormwood aseptic seedling top,
0.6cm × 0.6cm vanelets are cut into, soak 8min using the Agrobacterium bacterium solution of conversion, net more bacterium solution is blotted with aseptic filter paper,
Inoculation adds the MS culture mediums containing 2mg/L 6-BA again, in (25 ± 1) DEG C 6~7d of culture, during which keeps in dark.Then turn
Resistance screening is carried out on to the MS culture mediums with 480mg/L carbenicillins (Cb) and 12mg/L hygromycin.It is further cultured for 3-4
In week, select both ends and grow small callus hypocotyl, gradually grow green resistant budses and the non-resistance bud of white.About 1cm is grown to Deng tender shoots
It is long, the root media with identical antibiotic is gone to, is taken root after waiting 2 weeks and grows up to complete sweet wormwood plantlet.Plantlet passes through
Hardening, transplanting, growth, growth course simultaneously produce seed.PCR method is taken to identify transgene abrotanum, extraction regeneration sweet wormwood DNA is
Template, pcr amplification reaction condition are as follows：96 DEG C of pre-degenerations 5min, 92 DEG C of denaturation 1min, 58 DEG C of annealing 45S, 72 DEG C of extension 3min
15S, totally 34 circulations, last 72 DEG C of extensions 10min.By agarose gel electrophoresis, transgene abrotanum amplified with containing Rhd
The recombinant plasmid PCR identical bands of gene, and empty carrier pET-28a (+) is contaminated without corresponding amplified band (such as Fig. 3).
Expression of the embodiment 3 using Real-time PCR Analysis Rhd genes in positive sweet wormwood plant
Extract PCR test positive bioengineering sweet wormwood plant total serum IgEs, reverse transcription cDNA.Design reference gene β-
Actin primer, demarcate internal reference.
Sense primer SEQ ID NO.6:5′-GCCACCGATCCAGACACTGTACTTCC-3′.
Anti-sense primer SEQ ID NO.7:5′-ATTCAGGTGATGGTGTGAGCCACAC-3′.
Target gene Rhd target gene detection primer (such as SEQ ID NO of primer 3,4:4th, shown in 5).Amplification condition:94
DEG C pre-degeneration 4min；92 DEG C of denaturation 30S, 60 DEG C (reference genes) or 66 DEG C (target gene is adjustable) annealing 30S, 38 circulations,
72 DEG C of extension 10min, draw curve.As Fig. 4 can be seen that, target gene Rhd gene tables in positive organisms engineering sweet wormwood plant body
It is above compareing sweet wormwood plant up to amount, illustration purpose gene Rhd is not only incorporated into the genome of positive organisms engineering sweet wormwood plant
It is interior, and obtain effective expression (such as Fig. 4) in positive organisms engineering sweet wormwood plant vivo transcription level.
The sweet wormwood of embodiment 4 determines to benzene absorbability.
Take in confined reaction indoor measurement.Empty vector control sweet wormwood plant is first put into, the solution of 10 μ L benzene is sprayed
Day with fog note people is in confined reaction room, and opening built-in fans allows benzole soln to evaporate into mist completely, using Agilent
6890N gas chromatographs (Agilent companies of the U.S.) benzene homologues dedicated analysis gas chromatograph, benzene concentration one is measured every 3h
It is secondary, totally 24 hours.Benzene initial concentration is 12.5-32.8mg/m3.Then transgene abrotanum plant is put into the people confined reaction room
In, method is the same, and experiment is repeated 3 times, and places 3 basins sweet wormwood plant of the same race every time in the confined reaction room.Using Li-3000A
Leaf area instrument measures leaf area.It is unit for the ease of comparing the absorbability of sweet wormwood plant pair high concentration benzene by result conversion
The blade of chronomere's area absorbs the amount of benzene, and unit is mg/ (m2H) (such as Fig. 5).
Claims (2)
1. a kind of breeding method for the sweet wormwood for absorbing benzene, it is characterised in that comprise the following steps：
1) genetic fragment of clone strain bacillus ring hydroxylating dioxygenase, is designated as Rhd genetic fragments, and by Rhd bases
Because fragment is built into recombinant plasmid pCB-Rhd；
The DNA sequence dna of the Rhd genes is SEQ ID NO.1；
2) recombinant plasmid pCB-Rhd channel genes sweet wormwoods, acquisition are included into the sweet wormwood plant of Rhd genes using transgenic technology.
A kind of 2. breeding method for the sweet wormwood for absorbing benzene according to claim 1, it is characterised in that：Used in the step 2)
PCB-Rhd is imported Agrobacterium competent cell EHA105 by electric shocking method, then using agrobacterium-mediated transformation by the Agrobacterium sense
Sweet wormwood is imported by state cell EHA105, forms the sweet wormwood plant for including Rhd genes.
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| CN103421730B (en) * | 2012-12-21 | 2015-07-15 | 山东省科学院生物研究所 | Sphingobacterium multivorum capable of efficiently degrading multiring aromatics, and construction method thereof |
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| CN103421730B (en) * | 2012-12-21 | 2015-07-15 | 山东省科学院生物研究所 | Sphingobacterium multivorum capable of efficiently degrading multiring aromatics, and construction method thereof |
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| bph genes of the thermophilic PCB degrader, Bacillus sp.JF8:characterization of the divergent ring-hydroxylating dioxygenase and hydrolase genes upstream of the Mn-dependent BphC;Gouri Mukejee-Dhar,et al;《Microbiology》;20051201;摘要、第4146页左栏 * |
| PAHs降解基因及降解酶研究进展;曹晓星等;《生态学杂志》;20071231;第26卷(第6期);全文 * |
| 三叶草根系分泌物对多环芳香烃微生物降解及加氧酶的影响;王悦等;《应用生态学报》;20141130;第25卷(第11期);全文 * |
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