CN101270364A

CN101270364A - Method for improving plant absorption and tolerance methanal, plant expression vector and application

Info

Publication number: CN101270364A
Application number: CNA2008100583419A
Authority: CN
Inventors: 陈丽梅; 李昆志; 潘正波; 宋中邦; 赵艳
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2008-04-30
Filing date: 2008-04-30
Publication date: 2008-09-24

Abstract

The invention discloses a method for improving assimilation of formaldehyde and tolerance to formaldehyde of a plant, expression vectors and application of the plant, which belongs to the technical field of plant gene engineering. In the method, double genes containing DAS and DAK are fed into the chloroplasts of plant leaf cells, thus establishing a formaldehyde assimilation path, and a mechanism to assimilate formaldehyde with the photosynthesis of the plant so as to detoxicate formaldehyde in plant cells, create decorative plants with the specific function of decontaminating formaldehyde in indoor air and controlling the pollution of formaldehyde in indoor air, and maintain health of human bodies. The expression vectors of the plant are vector pK2-35S-PrbcS-*T-DAS and vector pH2-35S-PrbcS-*T-DAK. Compared with the control plant, the plant can thrust up in the environment polluted by formaldehyde, the quantity of formaldehyde assimilated is increased by 20 to 70 percent, and the speed to assimilate formaldehyde is improved by 16 to 27 percent.

Description

Improve method and the plant expression vector and the application of plant absorbing and tolerance methanal

Technical field

The invention belongs to plant genetic engineering field, be specifically related to a kind of method that improves plant absorbing and tolerance methanal, use the required plant expression vector of this method and construction process and their application.

Background technology

Formaldehyde (HCHO) is widely used in the industrial production, is the raw material of making resin, tackiness agent, paint, plastics, regenerated fiber, is the most widely used chemical raw material of adhesives industries.Along with the raising of expanding economy and living standards of the people, the building and ornament materials that the various raw materials that contain formaldehyde are made have entered into family.People move in the new home and will carry out upholstery and upgrade furniture, and this is the main source of room air formaldehyde pollution just, makes formaldehyde become a kind of ubiquitous atmospheric pollution gas.Long-term contact low dosage formaldehyde can cause chronic respiratory tract disease, cause snuff cancer, colorectal carcinoma, brain tumor, menoxenia, nuclear transgenation, in the dna single chain commissure and DNA and protein commissure and suppress dna damage reparation, pregnancy syndrome, cause neonatal chromosome disorder, leukemia, the comprehensive disease of so-called finishing (sick-house) that Here it is.With respect to benzene,toluene,xylene, free toluene diisocyanate (TDI), the volatile organic compounds objectionable impuritiess such as (VOC) in the paint, formaldehyde has latency period long (3-15), hides characteristics such as dark, that distribution is wide, volatile, it is difficult to administer, harm is big.Remove the normally used method of pollution formaldehyde at present and have three kinds: one is to use environment-friendly materials; The 2nd, the ventilation of windowing; The 3rd, remove pollution with plant or stain remover, use stain remover that the possibility of secondary pollution is arranged.Removing formaldehyde with the indoor cultivation plant pollutes, be the mode of the most natural, environmental protection, scientific research proof some plant really can absorb decomposing formaldehyde, but the very limited (Giese etc. of receptivity and speed, 1994, Plant Physiol.104:1301-1309).

Formaldehyde is a kind of very active compound, because energy and protein, nucleic acid and lipid produce nonspecific reaction (Feldman etc.; Prog Nucleic Acid Res Mol Biol; 1973,13:1-49), therefore very high toxicity concerning comprising plant, all biologies arranged all.But, also can produce the formaldehyde of lower concentration, so nearly all biological PARA FORMALDEHYDE PRILLS(91,95) all has detoxifcation mechanism separately in vivo by various demethylation reactions.Most of plants of occurring in nature belong to the photosynthetic autotrophs biology, mainly with CO ₂Be carbon source, most of plant is by the photosynthetic Kelvin approach fixation of C O that circulates ₂Formaldehyde also is the metabolic a kind of intermediate product of plant one carbon (C1) compound, but most of formaldehyde does not exist with unbound state in plant materials, but be attached to (Chen etc. on endogenous nucleophilic compound such as gsh or the tetrahydrofolic acid (THFA), 1997, Plant Physiol 1175:299-309).

Biological chemistry and the genetic formaldehyde dehydrogenase (FALDH) that studies have shown that dependence-Triptide are to be responsible for the metabolic main enzyme of formaldehyde in eukaryote, and this kind of enzyme is prevalent in the animal and plant tissue.FALDH in the ADH2 genes encoding Arabidopis thaliana has only a spot of expression in plant.A recently Hispanic research group obtains the transgenic arabidopsis of FALDH overexpression, their result of study shows that improving the enzymic activity of FALDH in transgenic plant can make them absorb the effectiveness raising 25% of external source formaldehyde, and by suppressing altogether or the activity of antisense expression reduction FALDH in transgenic plant, the speed that can make them absorb formaldehyde significantly descends, compare with wild-type, the detoxification ability of PARA FORMALDEHYDE PRILLS(91,95) also reduces greatly, this is the first research (Achkor etc., Plant physiology.132:2248-2255) that strengthens plant PARA FORMALDEHYDE PRILLS(91,95) detoxification with transgenic method.

Before life was born, formaldehyde was one of composition of primeval soup on the earth, and the relation of formaldehyde and biology has begun to continue existing tens years so far from its birth.In the meantime, rise in vivo for preventing concentration of formaldehyde, organism has been set up mechanism cleverly, makes it existence better in containing the environment of formaldehyde, and this machine-processed high development appears in the microorganism that can assimilate one-carbon compound.No matter be prokaryotic organism or eukaryote, everyly can utilize C1 compound (methane, methyl alcohol, formaldehyde, methane amide etc.) microorganism, in they metabolic initial stages, all produce formaldehyde, all have in their cell to become the formaldehyde that produces from various C1 compounds to be the assimilation approach of the moiety of cell and the oxidative pathway that obtains cell institute energy requirement from formaldehyde.In methylotrophy type microorganism, formaldehyde is a crucial intermediate product in the metabolic process of good several C1 compounds, and its participation dissimilation and assimilation process (Quayle etc., 1978,42:251-273).Ribulose list phosphoric acid approach (RuMP) is one of bacteria formaldehyde assimilation approach, among the RuMP fixedly the key enzyme of formaldehyde be 6-hexulose phosphate synthetic enzyme (HPS) and fructose-1, 6-diphosphate isomerase (PHI).Because ribulose-5-phosphate and fructose-1, 6-diphosphate all are Kelvin's round-robin intermediate products, we are expected at the HPS and the PHI that if can successfully express bacterium in the chloroplast(id) of plant, and formaldehyde just can assimilate by photosynthesis so.Our nearest result of study shows that overexpression is from HPS and the PHI of methylotrophic bacteria Mycobacterium gastri MB19 in the chloroplast(id) of Arabidopis thaliana and tobacco, can strengthen the resistance of transgenic plant PARA FORMALDEHYDE PRILLS(91,95) and absorption and with ability (the Chen et al. of oxidizing gases and liquid formaldehyde, Plant cell and Physilo., 2005).This just proves and can utilize plant gene engineering technology that the formaldehyde assimilation approach of microorganism is incorporated in the carbon dioxide fixation approach of plant.

The methylotrophy yeast has the efficient metabolic mechanism that utilizes one-carbon compound methyl alcohol, in the pathways metabolism of methyl alcohol, methyl alcohol at first is oxidized to formaldehyde by alcohol oxidase, formaldehyde is a critical intermediate product in the methyl alcohol metabolic process, it is on the tapping point of methyl alcohol assimilation and alienation approach, formaldehyde is a critical intermediate product in the methyl alcohol metabolic process, and it is on the tapping point of methyl alcohol assimilation and alienation approach.First reaction in constructed by dihydroxy acetone synthetase (DAS) the catalysis formaldehyde assimilation approach, formaldehyde in the DAS catalysis yeast and xylulose 5-P form otan and Glycerose 3-phosphoric acid.After methyl alcohol is oxidized to formaldehyde by alcohol oxidase enzyme, by it formaldehyde fixed (Yurimoto etc., 2005, The ChemicalRecord.5:367-375) to D-xylulose 5-phosphate molecule.Confirmed that by mutant and enzymatic property analysis the physiological action of DAS is the key enzyme of methyl alcohol assimilation, is positioned in the peroxysome.From methylotrophic yeast bacterium candiyeast (Candida boidinii), be cloned into the gene (DHAS1) of encoding D AS at present, further investigation result confirms that DAS mainly participates in the assimilation of formaldehyde in this yeast cell but not dissimilation or detoxification (Sakai etc., 1998, American Society for Microbiology.180:5885-5890).The DAS zymoprotein is a homodimer that contains diphosphothiamine, experience is transported in the peroxysome after Dimerized in tenuigenin, confirmed that now the N end of this enzyme contains the signal peptide sequence (Sakai etc. of peroxysome, 1998, American Society for Microbiology.180:5885-5890).

Otan is deleterious compound to yeast cell, dihydroxyacetone kinase in yeast (DAK) participates in the detoxification of otan, its catalytic reaction makes the otan phosphorylation, forms avirulent phosphodihydroxyacetone, can be yeast cell and utilizes.This enzyme is prevalent in the most organism, in conjunction with genetics and biochemical method, from yeast yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), be cloned into the gene (YML070W/DAK1 and YML053W/DAK2) of two encoding D AK, their coded protein is homodimer (Molin etc., 2003, The journal ofbiological chemistry.278:1415-1423), the protein positioning of the DAK genes encoding of from pichia pastoris phaff (Pichia pastoris), being cloned into (L ü ers etc. in tenuigenin, Yeast.1998, Jun 15,14 (8): 759-71).

Also do not change at present the plant of DAS or DAK gene, other genetic manipulations also of no use improve the report that indoor plant absorbs formaldehyde.X 5P and phosphodihydroxyacetone and Glycerose 3-phosphoric acid all are Kelvin's round-robin intermediate products, if can be in the chloroplast(id) of plant overexpression from saccharomycetic DAS and DAK, DAS just can utilize Kelvin's round-robin intermediate product X 5P to be the fixing acceptor of formaldehyde so, by its catalysis is the reaction of low thing with X 5P and formaldehyde, produce otan and Glycerose 3-phosphoric acid, make the otan phosphorylation of the toxic effect of pair cell again by the effect of DAK, form phosphodihydroxyacetone, phosphodihydroxyacetone and Glycerose 3-phosphoric acid can be used as Kelvin's round-robin intermediate product, reenter Kelvin's approach that circulates, so just can make up a formaldehyde assimilation approach, saccharomycetic formaldehyde assimilation approach is incorporated in the carbon dioxide fixation approach (Kelvin circulate approach) of plant with DAS and DAK.

Summary of the invention

The objective of the invention is to overcome natural phant and absorb decomposing formaldehyde ability and the very limited defective of speed, utilize genetic engineering technique in the chloroplast(id) of plant leaf cell overexpression from saccharomycetic DAS and DAK gene, thereby make up the formaldehyde assimilation approach of an effective absorption and metabolism formaldehyde, utilize photosynthesis of plants assimilation formaldehyde, to improve the ability of plant absorbing and tolerance methanal, creation has the special ornamental plant that purifies the indoor formaldehyde pollution, safeguards the health of human body.

For achieving the above object, the invention provides required special plant expression vector of a kind of this method of methods and applications that improves plant absorbing and tolerance methanal and their application.

Method provided by the invention is to utilize genetic engineering technique overexpression constructed by dihydroxy acetone synthetase (DAS) and dihydroxyacetone kinase (DAK) in the chloroplast(id) of plant leaf cell dual-gene, utilize a formaldehyde assimilation of the dual-gene structure of DAS and DAK approach (as Fig. 1), set up the mechanism of utilizing photosynthesis of plant assimilation formaldehyde, make the formaldehyde energy detoxification that enters in the vegetable cell, started the method that improves plant absorbing and tolerance methanal.

The invention provides the required a pair of raising plant absorbing of application the inventive method and the plant expression vector of tolerance methanal is pK2-35S-PrbcS-*T-DAS carrier and pH2-35S-PrbcS-*T-DAK carrier.Dedicated carrier pK2-35S-PrbcS-*T-DAS is the plant expression vector that contains constructed by dihydroxy acetone synthetase DAS gene; Dedicated carrier pH2-35S-PrbcS-*T-DAK is the plant expression vector that contains dihydroxyacetone kinase DAK gene.

The present invention also provides the inventive method and the application of a pair of plant expression vector of the present invention on the morning glory plant, and this application has obtained good effect.

The invention has the beneficial effects as follows, use method of the present invention and a pair of plant expression vector provided by the invention, transform the transgenic plant that plant obtained, than not changeing the dual-gene wild-type plant of DAS and DAK and only changeing the plant of single DAS gene and DAK gene, in the environment that has formaldehyde to pollute, not only growing way is good, and the amount of absorption formaldehyde has improved 20%～70%, the speed raising 16%～27% of absorption formaldehyde.

Technical scheme of the present invention is as follows:

One, improve the method for plant absorbing and tolerance methanal, this method may further comprise the steps:

(1), the recombinant vectors pK2-35S-PrbcS-*T-DAS of DAS gene and DAK gene and the structure of pH2-35S-PrbcS-*T-DAK

1, the structure of recombinant plant expression vector pK2-35S-PrbcS-*T-DAS

(1) amplification of the gene of candiyeast DAS

From GenBank, search the full-length gene order of candiyeast (Candida boidinii) DAS, the GenBank accession number of its DAS gene is AF086822, and the following a pair of primer of implementation sequence (the DAS gene is also at Sakai etc., 1998, American Society for Microbiology.180:5885-5890 report):

DAS5：caccgcATGcCTCTCGCAAAAGCTGCTTC

DAS3：ggatccTTATTGATCATGTTTTGGTTTTTC

5 ' end primer DAS5, end adds the caccgc characteristic sequence, and changes the G behind the ATG into c and form the SphI restriction enzyme site thus; 3 ' end primer DAS3, end adds the BamHI restriction enzyme site; Genomic dna with candiyeast (Candida boidinii) is a template amplification, obtains the full length DNA of DAS;

(2), the TA of DAS gene clone

Reclaim and purifying DAS full-length gene fragment, and be connected on the pMD18-T carrier, adopt alkaline lysis method of extracting plasmid DNA, cut detection by PCR detection and enzyme and obtain recombinant plasmid pMD-DAS;

(3), the structure of entry vector pENTR*-PrbcS-*T-DAS

(another of the inventor applied for a patent to cut pENTR*-PrbcS-*T-GFP with SphI and BamHI, application number is 200710066422.9) and pMD-DAS, reclaim carrier pENTR*-PrbcS-*T segment and DAS gene segment, connect PENTR*-PrbcS-*T and DAS gene segment with ligase enzyme, obtain entry vector pENTR*-PrbcS-*T-DAS.

(4), the structure of DAS gene plant expression vector pK2-35S-PrbcS-*T-DAS

LR by the Gateway technology reacts the PrbcS-*T-DAS subclone in plant expression vector pK2GW7 (the purpose carrier of Gateway, Billy's stone VIB/Gent company), and the expression of plants that obtains the DAS gene carries pK2-35S-PrbcS-*T-DAS.

Recombinant vectors pK2-35S-PrbcS-*T-DAS of the present invention, it contains promotor PrbcS and chloroplast(id) positioning sequence * T, is right after the DAS gene thereafter, and the GenBank accession number of DAS gene is: AF086822.

2, the structure of recombinant vectors pH2-35S-PrbcS-*T-DAK

(1), the amplification of the gene of pichia spp DAK

From GenBank, search the full-length gene order of pichia pastoris phaff (Pichia pastoris) DAK, the GenBank accession number of its DAK gene is AF019198, and the following a pair of primer of implementation sequence, (the DAK gene is also at L ü ers etc., Yeast.1998, Jun 15,14 (8): the 759-71 report):

DAK5：CACCGCatgCctagtaaacattgggattac

DAK3：CTCGAGctacaacttggtttcagatttg

5 ' end primer DAK5, end adds the caccgc characteristic sequence, and changes the t behind the ATG into C and form the SphI restriction enzyme site thus; 3 ' end primer DAK3, end adds the XhoI restriction enzyme site; Genomic dna with pichia pastoris phaff (Pichia pastoris) is a template amplification, obtains the full length DNA of DAK;

(2), the TOPO of DAK gene clone

Reclaim and purifying DAK gene fragment, to pENTR-TOPO carrier (available from invitrogen company), adopt alkaline lysis method of extracting plasmid DNA, detect and sequencing analysis acquisition recombinant plasmid pENTR-TOPO-DAK by PCR by TOPO clone technology subclone.

(3), the structure of entry vector pENTR*-PrbcS-*T-DAK

(another of the inventor applied for a patent to cut pENTR*-PrbcS-*T-GFP with SphI and XhoI, a kind of gateway cloning entry vector and construction process thereof and application, application number is 200710066422.9) and pENTR-TOPO-DAK, reclaim carrier pENTR*-PrbcS-*T segment and DAK gene segment, connect PENTR*-PrbcS-*T and DAK gene segment with ligase enzyme, obtain entry vector pENTR*-PrbcS-*T-DAK.

(4), the structure of DAK gene plant expression vector pH2-35S-PrbcS-*T-DAK

LR by the Gateway technology reacts the PrbcS-*T-DAK subclone in plant expression vector pH2GW7 (the purpose carrier of Gateway, Billy's stone VIB/Gent company), obtains the plant expression vector pK2-35S-PrbcS-*T-DAK of DAS gene.The GenBank accession number of DAK gene is AF019198.

In above-mentioned carrier, the upstream of DAS and DAK gene is added with photoinduction type promotor PrbcS and chloroplast(id) positioning sequence.Photoinduction type promotor (PrbcS) that the present invention uses and chloroplast(id) positioning sequence are promotor and the chloroplast(id) positioning sequences of the rbcS-3C that separates from the genome of tomato, be a dna segment (Sugita et al. by the 1.9kb of HindIII cutting generation, 1987, MGG, 209:247-256).

In order to use the plant expression vector of Gateway technique construction DAS and DAK gene, the present invention DAS and DAK gene alias carrier pENTR*-PrbcS-*T-GFP (another patent of the inventor: a kind of gateway cloning entry vector and construction process thereof and application, application number: the GFP gene 200710066422.9), the entry vector pENTR*-PrbcS-*T-DAS and the pENTR*-PrbcS-*T-DAK of generation DAS and DAK gene.

(2), the detection of genetic transformation of Agrobacterium and transformant

The competent cell of preparation Agrobacterium, the electricity consumption impulse method changes above-mentioned plant expression vector pK2-35S-PrbcS-*T-DAS that builds and pH2-35S-PrbcS-*T-DAK in the Agrobacterium over to, is being added with screening transformant bacterium colony on the flat board of spectinomycin.With the lysate of Agrobacterium bacterium colony template as the PCR reaction, make PCR with the upstream and downstream Auele Specific Primer of DAS and DAK gene and detect the transformant bacterium colony, be used to transform plant through the transformant bacterium colony of bacterium colony PCR affirmation.

(3), with the Agrobacterium-mediated Transformation plant that contains DAS and DAK gene plant expression vector

The picking single colony inoculation of Agrobacterium that carries plasmid pK2-35S-PrbcS-*T-DAS and pH2-35S-PrbcS-*T-DAK is cultivated in the LB liquid nutrient medium respectively, and centrifugal collection thalline suspends with the MS liquid nutrient medium again.Plant tissue with the agroinfection that suspends breaks up easily obtains seedling by tissue culture then, utilizes antibiotic-screening to obtain transgenic plant again.

(4), DAS and DAK gene in transgenic plant the insertion situation and the detection of transcriptional level

In order to confirm to contain DAS gene and DAK gene really, the transgenic plant that screen are done further to identify with PCR method with the transgenic plant that antibiotic-screening obtains.At first extracting the genomic dna of transgenic plant, is template then with the plant genome DNA, makes PCR with the upstream and downstream Auele Specific Primer of DAS gene and DAK gene and detects, and the transfer-gen plant of confirming through PCR is used for the RT-PCR analysis.

In order to investigate DAS gene and DAK gene transcription situation in the transfer-gen plant that contains DAS gene and DAK gene, from transgenic plant, extract total RNA, reversing is used for RT-PCR behind the green one-tenth cDNA and analyzes, and detects DAS gene and the transcriptional level of DAK gene in transgenic plant.With cDNA is template, makes PCR with DAS and DAK gene upstream and downstream Auele Specific Primer,, be used for the detection of the resistance and the uptake rate of plant PARA FORMALDEHYDE PRILLS(91,95) through the transfer-gen plant of RT-PCR affirmation.

(5), the resistance of plant PARA FORMALDEHYDE PRILLS(91,95) detects

With the transgenic plant seedling and not the wild-type seedling of transgenic plant move into and to be added with on the MS solid medium of formaldehyde, under continuous light, cultivate for some time after the making plant phenotype change.If it is better than wild-type growing way to change the plant of DAS and DAK, illustrate that the formaldehyde of introducing assimilates approach and can bring into play predictive role in plant, the ability render transgenic plant of enhancing plant formaldehyde assimilation strengthens for the resistance of liquid formaldehyde.

In order to verify the resistance of transgenic plant to gas formaldehyde, the transgenic plant seedling moved in the sealed vessel that the MS substratum is arranged cultivate, the centrifuge tube of one 500 μ l is placed in the back of taking root in container, add a certain amount of 37% formaldehyde solution in centrifuge tube.Continuous light is observed the upgrowth situation of transgenic plant after cultivation for some time, and measure gas formaldehyde concentration in the container with the formaldehyde determination instrument, if the upgrowth situation of transgenic plant obviously is better than the wild-type plant, illustrate that transgenic plant strengthen the resistance of gas formaldehyde.

(6), the detection of transgenic plant PARA FORMALDEHYDE PRILLS(91,95) uptake rate

Formaldehyde can with Nash reagent (Ammonium Acetate: 15%, Glacial acetic acid: 0.3%, methyl ethyl diketone: 0.2%) chemical reaction takes place and produce coloured material, the maximum absorption wavelength of this material is 410nm, the typical curve that can prepare formaldehyde in view of the above, can calculate the content of formaldehyde in the reaction solution according to the typical curve of HCHO, utilize this method can measure the uptake rate of plant PARA FORMALDEHYDE PRILLS(91,95).Get a certain amount of vegetable material and put into little triangular flask, after adding formaldehyde solution is handled certain hour, take out a certain amount of treatment solution, add water to 1ml, add 1ml Nash reagent again and after 30 minutes, measure OD 30 ℃ of insulations ₄₁₀, calculate concentration of formaldehyde remaining in the treatment solution according to typical curve.The formaldehyde that treatment solution is remaining is few more, illustrates that the uptake rate of plant PARA FORMALDEHYDE PRILLS(91,95) is big more.

The Agrobacterium of the genetic transformation of the Agrobacterium described in (two) step is C58Cl (pPMP90) in the aforesaid method; The usefulness described in (three) step contains the Agrobacterium-mediated Transformation plant of DAS and DAK gene plant expression vector in the aforesaid method, and its plant is the plant of suitable indoor cultivation.

Used method, reagent, instrument is molecular biology experiment and the breadboard ordinary method of genetically engineered, reagent and instrument if no special instructions in the aforesaid method.

Two, improve a pair of plant expression vector of plant absorbing and tolerance methanal, this a pair of plant expression vector contains pK2-35S-PrbcS-*T-DAS carrier and pH2-35S-PrbcS-*T-DAK carrier

The DAS gene that contains candiyeast in the described pK2-35S-PrbcS-*T-DAS carrier, the GenBank accession number of its DAS gene is AF086822; The DAK gene that contains pichia spp in the described pH2-35S-PrbcS-*T-DAK carrier, the GenBank accession number of its DAK gene is AF019198; The DAS gene of described candiyeast and the DAK upstream region of gene of pichia spp all have the promotor PrbcS and the chloroplast(id) positioning sequence * T of Rubisco small subunit.

The initial vector of above-mentioned pK2-35S-PrbcS-*T-DAS plant expression vector is pK2GW7, and the initial vector of pH2-35S-PrbcS-*T-DAK plant expression vector is pH2GW7.

The construction process of pK2-35S-PrbcS-*T-DAS plant expression vector and pH2-35S-PrbcS-*T-DAK plant expression vector is as follows:

1, the structure of recombinant plant expression vector pK2-35S-PrbcS-*T-DAS

(1) amplification of the gene of candiyeast DAS

From GenBank, search the full-length gene order of candiyeast (Candida boidinii) DAS, and the following a pair of primer of implementation sequence (the DAS gene is also at Sakai etc., 1998, American Society for Microbiology.180:5885-5890 report):

DAS5：caccgcATGcCTCTCGCAAAAGCTGCTTC

DAS3：ggatccTTATTGATCATGTTTTGGTTTTTC

(2), the TA of DAS gene clone

(3), the structure of entry vector pENTR*-PrbcS-*T-DAS

(4), the structure of DAS gene plant expression vector pK2-35S-PrbcS-*T-DAS

Recombinant vectors pK2-35S-PrbcS-*T-DAS of the present invention, it contains promotor PrbcS and chloroplast(id) positioning sequence * T, is right after the DAS gene thereafter, and the GenBank accession number of DAS gene is AF086822.

2, the structure of recombinant vectors pH2-35S-PrbcS-*T-DAK

(1), the amplification of the gene of pichia spp DAK

From GenBank, search the full-length gene order of pichia pastoris phaff (Pichia pastoris) DAK, and the following a pair of primer of implementation sequence (the DAK gene is also at L ü ers etc., Yeast.1998, and Jun 15,14 (8): the 759-71 report):

DAK5：CACCGCatgCctagtaaacattgggattac

DAK3：CTCGAGctacaacttggtttcagatttg

(2), the TOPO of DAK gene clone

(3), the structure of entry vector pENTR*-PrbcS-*T-DAK

(4), the structure of DAK gene plant expression vector pH2-35S-PrbcS-*T-DAK

LR by the Gateway technology reacts the PrbcS-*T-DAK subclone in plant expression vector pH2GW7 (the purpose carrier of Gateway, Billy's stone VIB/Gent company), obtains the plant expression vector pK2-35S-PrbcS-*T-DAK of DAS gene.The GenBank accession number of DAK gene is: AF019198.

Three, the application of the plant expression vector of the inventive method or a pair of raising plant absorbing of the present invention and tolerance methanal is the application on morning glory plant (Petunia hyrida).

Description of drawings

Fig. 1 makes up the principle of formaldehyde assimilation approach with DAS and DAK

In the chloroplast(id) of plant, express constructed by dihydroxy acetone synthetase (DAS) and dihydroxyacetone kinase (DAK) from the methylotrophic yeast bacterium, DAS can utilize Kelvin's round-robin intermediate product X 5P (Xu5P) to be the fixing acceptor of formaldehyde, by its catalysis is the reaction of low thing with X 5P and formaldehyde (HCHO), produce otan (DHA) and 3-phoshoglyceric acid (3-PGA), effect by DAK makes the DHA phosphorylation again, form phosphodihydroxyacetone (DHAP), DHAP and 3-PGA can be used as Kelvin's round-robin intermediate product, reenter Kelvin's approach that circulates.

The detection of Fig. 2 yeast genomic dna

1: the pichia spp genome; 2: the candiyeast genome; 3:D2000 DNA marker.

The TA clone strategy of Fig. 3 DAS gene

The amplification of Fig. 4 DAS gene and TA clone

(A) be masterplate amplification DAS gene with the candiyeast gene DNA.M:D2000DNA marker; The pcr amplification product of 1-4:DAS.(B) electrophoresis detection of recombinant plasmid pMD18-DAS.1: over against shining (molecular weight is the plasmid DNA of 4.8kb); 2: recombinant plasmid pMD18-DAS.(C) PCR of recombinant plasmid pMD18-DAS check.M: λ-DNA/Hind III DNA marker; 1～5: with pMD18-DAS is the PCR product of masterplate with DAS5 and DAS3 primer amplification.(D) enzyme of recombinant plasmid pMD18-DAS is cut detection.1: the product of cutting pMD18-DAS with SphI and BamHI enzyme; 2: λ-DNA/Hind III DNA marker.

The construction strategy of Fig. 5 entry vector pENTR*-PrbcS-*T-DAS

The detection of Fig. 6 entry vector pENTR*-PrbcS-*T-DAS

(A) electrophoresis detection of pENTR*-PrbcS-*T-DAS plasmid.1: over against shining (molecular weight is the plasmid DNA of 6.1kb); 2: negative contrast (molecular weight is the plasmid DNA of 4.0kb); The 3:pENTR*-PrbcS-*T-DAS plasmid DNA.(B) enzyme of pENTR*-PrbcS-*T-DAS is cut detection.M: λ-DNA/Hind III DNA marker; The EcoRV enzyme of 1-2:pENTR*-PrbcS-*T-DAS plasmid is cut product; (C) PCR of pENTR*-PrbcS-*T-DAS detects (primer is DAS5 and DAS3).1: positive control (is the product of masterplate amplification with the candiyeast genome); 2-4: with pENTR*-PrbcS-*T-DAS is the product of masterplate amplification; M: λ-DNA/Hind III DNA marker.

The construction strategy of Fig. 7 DAS plant expression vector

The detection of Fig. 8 DAS plant expression vector

(A) electrophoresis detection of pK2-35S-PrbcS-*T-DAS plasmid.1: plasmid pK2GW7; 2: plasmid pENTR*-PrbcS-*T-DAS; 3-8: recombinant plasmid pK2-35S-PrbcS-*T-DAS.(B) PCR of pK2-35S-PrbcS-*T-DAS detects.1-3: with the pK2-35S-PrbcS-*T-DAS plasmid is masterplate amplification PCR products (primer is PrbcS5 and DAS3), M:500bp DNA marker.

The construction strategy of Fig. 9 pENTR-TOPO-DAK carrier

The detection of Figure 10 pENTR-TOPO-DAK carrier

(A) be masterplate amplification DAK gene with the pichia spp gene DNA.M:D2000 DNA marker; The pcr amplification product of 1-2:DAK.(B) electrophoresis detection of recombinant plasmid pENTR-TOPO-DAK.1: over against shining (molecular weight is the plasmid DNA of 4.5kb); 2:pENTR-TOPO; 3-4: recombinant plasmid pENTR-TOPO-DAK.(C) enzyme of recombinant plasmid pMD 18-DAS is cut detection.1: the product of cutting pENTR-TOPO-DAK with SphI and XhoI enzyme; 2:D2000DNA marker.(D) PCR of recombinant plasmid pENTR-TOPO-DAK check.1～3: with pENTR-TOPO-DAK is the PCR product of masterplate with DAK5 and DAK3 primer amplification; M: λ-DNA/Hind III DNA marker.

The construction strategy of Figure 11 pENTR*-PrbcS-*T-DAK carrier

The detection of Figure 12 pENTR*-PrbcS-*T-DAK carrier

(A) electrophoresis detection of pENTR*-PrbcS-*T-DAK plasmid.1: over against shining (molecular weight is the plasmid DNA of 6.0kb); 2: negative contrast (molecular weight is the plasmid DNA of 4.0kb); The 3:pENTR*-PrbcS-*T-DAK plasmid DNA.(B) enzyme of pENTR*-PrbcS-*T-DAK is cut detection.M: λ-DNA/Hind III DNA marker; The EcoRV enzyme of 1-4:pENTR*-PrbcS-*T-DAK plasmid is cut product.(C) PCR of pENTR*-PrbcS-*T-DAK detects (primer is DAK5 and DAK3).1: positive control (is the product of masterplate amplification with the pichia spp genome); 2-4: with pENTR*-PrbcS-*T-DAK is the product of masterplate amplification; M: λ-DNA/Hind III DNA marker.

The construction strategy of Figure 13 DAK plant expression vector

The detection of Figure 14 DAK plant expression vector

(A) electrophoresis detection of pH2-35S-PrbcS-*T-DAK plasmid.1: plasmid pKHGW7; 2: plasmid pENTR*-PrbcS-*T-DAK; 3-10: recombinant plasmid pH2-35S-PrbcS-*T-DAK.(B) PCR of pH2-35S-PrbcS-*T-DAK detects.1-2: with the pH2-35S-PrbcS-*T-DAK plasmid is masterplate amplification PCR products (primer is PrbcS5 and DAK3); M:500bp DNA marker.

The PCR of Figure 15 Agrobacterium bacterium colony detects

1: the Agrobacterium of unconverted plasmid; The PCR of the Agrobacterium-mediated Transformation daughter colony of 2～3:pH2-35S-PrbcS-*T-DAK (primer is PrbcS5 and DAK3) amplified production; The PCR of the Agrobacterium-mediated Transformation daughter colony of 4～6:pK2-35S-PrbcS-*T-DAS (primer is PrbcS5 and DAS3); M:500bp DNA Marker.

The genetic transformation procedures of Figure 16 plant

(A) the leaf dish of agroinfection is not having cultivation altogether on the MS1 substratum of antibiotin.(B) the leaf dish after common the cultivation is in the formation that is added with induced bud on the antibiotic MS4 division culture medium.(C) at the bud that forms on the MS4 division culture medium formation of inducing root on the biorefractive root media is being arranged.

The PCR that Figure 17 changes DAS and DAK gene morning glory detects

(A) PCR that changes the DAS morning glory detects.1-3: the plant that changes DAS; 4: negative contrast (not genetically modified wild-type); 5: λ-DNA/Hind III DNA marker; 6: over against shining (is the PCR product of template amplification with pK2-35S-PrbcS-*T-DAS).(B) PCR that changes DAK gene morning glory detects: 1-2: the plant that changes DAK; 3: negative contrast (not genetically modified wild-type), 4: over against shining (is the PCR product of template amplification with pH2-35S-PrbcS-*T-DAK); 5:DNA marker.(C) PCR that changes DAS and DAK gene morning glory detects.1:DNA marker; 2-6: the amplified production that changes DAS and DAK plant genome PCR; 7: negative contrast (wild-type); 8: over against shining (pMD18-DAS plasmid): 9: over against shining (pENTR-TOPO-DAK plasmid).

The RT-PCR that Figure 18 changes DAS and DAK gene morning glory detects

(A) detection of DAS gene transcription level.1: over against shining (is the PCR product of template amplification with plasmid pMD18-DAS); 2: negative contrast (wild-type); 3: the plant that changes DAS; 4: the plant that changes DAK; 5: the plant that changes DAS and DAK; 6: λ-DNA/Hind III DNA marker.(B) detection of DAK gene transcription level.1: λ-DNA/Hind III DNAmarker; 2: negative contrast (wild-type); 3: the plant that changes DAK and DAK; 4: the plant that changes DAS; 5: the plant that changes DAK; 6: over against shining (is the PCR product of template amplification with the pENTR-TOPO-DAK plasmid).

Figure 19 transgenosis morning glory is to the detection of liquid formaldehyde resistance capacity

(A) wild-type; (B) change the DAK plant; (C) change the DAS plant; (D) change DAS and DAK plant.

Figure 20 transgenosis morning glory is to the detection of gas formaldehyde resistance capacity

(A) change the DAS plant; (B) change DAS and DAK plant; (C) change the DAK plant; (the concentration of formaldehyde in the D culture vessel.

Figure 21 transgenosis morning glory absorbs the mensuration of liquid formaldehyde speed

(A) change the mensuration that DAS gene morning glory plant absorbs formaldehyde speed.WT: wild-type; S3, S9, S12, S7, S5: the morning glory plant that changes the DAS gene; (B) change the mensuration that DAS and DAK gene morning glory plant absorb formaldehyde speed.WT: wild-type; Ks1, ks3, ks4, ks7, ks8, ks10, ks15, ks16, ks23: the morning glory plant that changes DAS and DAK gene.

Embodiment

Further specify essentiality content of the present invention below in conjunction with accompanying drawing with embodiments of the invention, but do not limit the present invention with this.

Used reagent and instrument are among the following embodiment of the present invention:

Reagent is mainly molecular biology experiment reagent, and all the other reagent are homemade analytical pure.

Instrument is molecular biology and genetically engineered laboratory common instrument.

Method therefor is ordinary method if no special instructions in the embodiment of the invention.

The structure of embodiment 1, DAS genetically modified plants expression vector pK2-35S-PrbcS-*T-DAS

(1) preparation and the detection of candiyeast (Candida boidinii) genomic dna

The used candiyeast (Candida boidinii) of the present invention is available from Chinese industrial microbial strains preservation center, and the CTAB method is adopted in the preparation of candidiasis genomic dna.Get 1.5mL bacterium liquid in 4 ℃ of centrifugal 2min of 4000rpm, abandon most supernatant liquor, collect thalline.Add 400 μ l, 2 * CTAB (NaCl 1.4M, CTAB 2% for Tris-HCl pH 7.5100mM, EDTA 20mM) homogenate, 65 ℃ of insulation 20min add behind the 500 μ l chloroform mixings the centrifugal 10min of 13000rpm under room temperature.Shift supernatant, add the 10%CTAB of 50 μ l, add 650 μ l Virahols and placed room temperature 1 hour, 4 ℃ of centrifugal 25min. of 12000rpm, precipitation is washed one time with 500 μ l, 75% alcohol, and vacuum-drying adds the TE dissolving that 20 μ l contain RNase, and 37 ℃ are incubated one hour.Get 2 μ l genomic dnas and carry out electrophoresis detection with 1% sepharose, result (Fig. 2) illustrates that the genomic dna quality of extracting meets the requirements.

(2), the amplification of DAS gene and TA clone

The amplification of DAS gene and TA clone's strategy as shown in Figure 3, (the GenBank accession number of DAS gene is AF086822 to the first-selected full-length gene order of searching DAS from GenBank, the DAS gene is also at Sakai etc., 1998, American Societyfor Microbiology.180:5885-5890 report), and design a pair of primer, sequence is as follows:

DAS5：caccgcATGcCTCTCGCAAAAGCTGCTTC

DAS3：ggatccTTATTGATCATGTTTTGGTTTTTC

5 ' end primer DAS5 end adds the caccgc characteristic sequence, and changes the G behind the ATG into c and form the SphI restriction enzyme site thus; 3 ' end primer DAS3 end adds the BamHI restriction enzyme site.

Make PCR with DAS gene upstream and downstream Auele Specific Primer DAS5 and DAS3, in the PCR reaction mixture, add the candiyeast genomic dna of 50ng as template, the Auele Specific Primer DAS5 and DAS3, the 4 μ ldNTP (2.5mM) that add 75ng simultaneously, Extaq (the 5U/ μ l) polysaccharase of 10 * Extaq of 5 μ l reaction Buffer and 0.25 μ l (Japan is precious biological), adding distilled water, to make the reaction final volume be 50 μ l.In 94 ℃ of heating 2 minutes, then according to 94 ℃, 30 seconds, 55 ℃, 30 seconds, 72 ℃, 2 minutes program was carried out 30 round-robin reactions, carried out PCR reaction amplification in 10 minutes program of 72 ℃ of prolongation reactions at last and obtained the DAS gene on the PCR instrument.After reaction is finished, separate the pcr amplification product (Fig. 4 A) of DAS by agarose gel electrophoresis.Reclaim and purifying DAS full-length gene (2.1kb), use the TA clone test kit of precious biological (TaKaRa) to be connected on pMD18-T (the precious biotech firm in the Dalian) carrier then, experimental implementation is undertaken by the specification sheets of test kit, after spending the night, reaction uses reaction mixture transformed into escherichia coli competence DH5 α (available from sky root biochemical technology company), adopt alkaline lysis method of extracting plasmid DNA, through 1% agarose gel electrophoresis (Fig. 4 B), choose the big or small recombinant plasmid pMD18-DAS that conforms to theoretical value and do further PCR detection, make PCR with DAS gene upstream and downstream Auele Specific Primer DAS5 and DAS3, the recombinant plasmid of subclone success all can amplify the DAS gene DNA fragment (Fig. 4 C) about 2.1kb.Multiple clone site according to positive recombinant plasmid pMD-DAS carrier two ends, with SphI and BamHI double digestion recombinant plasmid, detect enzyme through 1% agarose gel electrophoresis and cut product, the recombinant plasmid pMD-DAS of successful connection produces 2 bands, article one, be the DAS gene DNA insertion fragment (Fig. 4 D) about 2.0kb, another is the carrier segment of 2.7kb, proves that through sequential analysis the insertion segment in this carrier is the full-length gene of DAS.Reaffirm be the plasmid of successful connection after, transformed into escherichia coli DH5 α chooses single bacterium colony and carries out liquid culture again, with test kit plasmid purification pMD18-DAS.

(3), the structure of the ABC of cloning vector pENTR*-PrbcS-*T-DAS of Gateway

The construction strategy of pENTR*-PrbcS-*T-DAS as shown in Figure 5, (another of the inventor applied for a patent to cut the plasmid vector pENTR*-PrbcS-*T-GFP of purifying with SphI (Fermentas) and BamHI (Fermentas), application number is 200710066422.9) and pMD18-DAS, separate the carrier that has cut and insert fragment by agarose gel electrophoresis, recovery pENTR*-PrbcS-*T-GFP is cut the carrier segment pENTR*-PrbcS-*T (4.0kb) of back generation and the dna segment (2.1kb) of the DAS gene that pMD18-DAS is cut generation from gel, uses the ligase enzyme test kit connection pENTR*-PrbcS-*T of precious biological (TaKaRa) and the dna segment of DAS gene to produce entry vector pENTR*-PrbcS-*T-DAS then.Transform high-level efficiency (10 with the ligation mixture ⁸) competent escherichia coli cell (DH5 α, it root biochemical technology), be applied on the flat board that is added with kantlex (Km, 50 μ g/ml) transforming good intestinal bacteria, in 37 ℃ of incubated overnight, screening Km resistance recon bacterium colony, extract plasmid (Fig. 6 A) from Km resistance recon bacterium colony, carry out enzyme with EcoRV (Fermentas) and cut detection, the plasmid of successful connection produces two bands on agarose gel electrophoresis figure, one that molecular weight is little is 2.1kb, and another is 4.0kb (Fig. 6 B).Select the plasmid vector pENTR*-PrbcS-*T-DAS of successful connection, carry out PCR with DAS gene upstream and downstream Auele Specific Primer and detect (Fig. 6 C), reaffirm be the plasmid of successful connection after, again transformed into escherichia coli DH5 α, choose single bacterium colony and carry out liquid culture, with test kit plasmid purification pENTR*-PrbcS-*T-DAS.

(4), the structure of DAS gene plant expression vector

The construction strategy of DAS gene plant expression vector as shown in Figure 7, the LR reaction by the Gateway technology the PrbcS-*T-DAS subclone in plant expression vector pK2GW7 (the purpose carrier of Gateway is available from Belgian VIB/Gent company).Concrete way is: with the purpose carrier pK2GW7 of plasmid extraction test kit purifying Gateway, in the LR of Gateway reaction system, add pENTR*-PrbcS-*T-DAS and each 150ng of pK2GW7,1 μ l LR Clonase II Enzyme Mix (Invitrogen), be mixed and spend the night, PrbcS-*T-DAS is incorporated into the plant expression carrier plasmid pK2-35S-PrbcS-*T-DAS of acquisition DAS among the pK2GW7 by the effect of intergrase in 25 ℃ of reactions.Transform high-level efficiency (10 with reaction mixture ⁸) competent escherichia coli cell (DH5 α, it root biochemical technology), be applied to and be added with spectinomycin (Spe transforming good intestinal bacteria, 50 μ g/ml) on the flat board, in 37 ℃ of incubated overnight, screening Spe resistance recon bacterium colony, from Spe resistance recon bacterium colony, extract plasmid (Fig. 8 A), downstream Auele Specific Primer DAS3 with Auele Specific Primer PrbcS5 that is arranged in the PrbcS zone and DAS carries out the PCR detection, the plasmid of successful integration all can amplify the band (Fig. 8 B) of a 2.4kb, be confirmed to be the plasmid of successful connection after, transformed into escherichia coli DH5 α again, choose single bacterium colony and carry out liquid culture, use the test kit plasmid purification.

The structure of embodiment 2, DAK gene recombined vector pH2-35S-PrbcS-*T-DAK

(1) preparation and the detection of pichia pastoris phaff (Pichia pastoris) genomic dna

Pichia pastoris phaff (Pichia pastoris), available from Chinese industrial microbial strains preservation center, the CTAB method is adopted in the preparation of pichia pastoris phaff bacterium genomic dna.Get 1.5mL bacterium liquid in 4 ℃ of centrifugal 2min of 4000rpm, abandon most supernatant liquor, collect thalline.Add 400 μ l, 2 * CTAB (NaCl 1.4M, CTAB 2% for Tris-HCl pH 7.5100mM, EDTA 20mM) homogenate, 65 ℃ of insulation 20min add behind the 500 μ l chloroform mixings the centrifugal 10min of 13000rpm under room temperature.Shift supernatant, add the 10%CTAB of 50 μ l, add 650 μ l Virahols and placed room temperature 1 hour, 4 ℃ of centrifugal 25min. of 12000rpm, precipitation is washed once with 500 μ l, 75% alcohol, and vacuum-drying adds the TE dissolving that 20 μ l contain RNase, and 37 ℃ are incubated one hour.Get 2 μ l genomic dnas and carry out electrophoresis detection with 1% sepharose, result (Fig. 2) illustrates that the genomic dna quality of extracting meets the requirements.

(2) amplification of DAK gene DNA and TOPO clone

The amplification of DAK gene DNA and TOPO clone's strategy as shown in Figure 9, (the GenBank accession number of DAK gene is AF019198 to the first-selected full-length gene order of searching DAK from GenBank, the DAK gene is also at L ü ers etc., Yeast.1998, Jun 15,14 (8): the 759-71 report), and design a pair of primer, sequence is as follows:

DAK5：CACCGCatgCctagtaaacattgggattac

DAK3：CTCGAGctacaacttggtttcagatttg

5 ' end primer DAK5 end adds the CACCGC characteristic sequence, and changes the t behind the ATG into C and form the SphI restriction enzyme site thus; 3 ' end primer DAK3 end adds the XhoI restriction enzyme site.

Make PCR with DAK gene upstream and downstream Auele Specific Primer DAK5 and DAK3, in the PCR reaction mixture, add the pichia spp genomic dna of 50ng as template, the Auele Specific Primer DAK5 and DAK3, the 4 μ ldNTP (2.5mM) that add 75ng simultaneously, PrimeSTARHS (the 5U/ μ l) archaeal dna polymerase of 5 * PrimeSTAR of 10 μ l reaction Buffer and 0.5 μ l (Japan is precious biological), adding distilled water, to make the reaction final volume be 50 μ l.In 94 ℃ of heating 2 minutes, then according to 94 ℃, 45 seconds, 55 ℃, 30 seconds, 72 ℃, 2 minutes program was carried out 30 round-robin reactions, carried out PCR reaction amplification in 10 minutes program of 72 ℃ of prolongation reactions at last and obtained the DAK gene on the PCR instrument.After reaction is finished, separate the pcr amplification product (Figure 10 A) of DAK by agarose gel electrophoresis.Recovery and purifying DAK full-length gene fragment, the product of DAK is flat terminal dna fragmentation, therefore uses the pENTR of Invitrogen company ^TMDirectional

The Cloning test kit, the flat terminal dna fragmentation that is recovered to is subcloned in the pENTR/D-TOPO carrier, experimental implementation is undertaken by the specification sheets of test kit, after spending the night, room temperature reaction uses reaction mixture transformed into escherichia coli competence DH5 α (available from sky root biochemical technology company), adopt alkaline lysis method of extracting plasmid DNA, through 1% agarose gel electrophoresis (Figure 10 B), choosing the recombinant plasmid pENTR-TOPO-DAK that conforms to theoretical value of size carries out enzyme with SphI and XhoI (Fermentas) and cuts detection, the plasmid of successful connection produces two bands on agarose gel electrophoresis figure, one that molecular weight is little is the DAK gene insertion segment of 1.8kb, and another is the carrier segment (Figure 10 C) of 2.6kb.Choose the correct plasmid of connection and do further PCR detection, make PCR with DAK gene upstream and downstream Auele Specific Primer DAK5 and DAK3, the recombinant plasmid of subclone success all can amplify the dna fragmentation (Figure 10 D) about 1.8kb, proves the full-length gene of DAK gene through sequential analysis.

(3), the structure of the ABC of cloning vector pENTR*-PrbcS-*T-DAK of Gateway

The construction strategy of pENTR*-PrbcS-*T-DAK as shown in figure 11, cut the plasmid vector pENTR*-PrbcS-*T-GFP and the pENTR-TOPO-DAK of purifying with SphI (Fermentas) and BamHI (Fermentas), separate the carrier that has cut and insert fragment by agarose gel electrophoresis, recovery pENTR*-PrbcS-*T-GFP is cut the carrier segment pENTR*-PrbcS-*T (4.0kb) of back generation and the dna segment (1.8kb) of the DAK gene that pENTR-TOPO-DAK is cut generation from gel, uses the ligase enzyme test kit connection pENTR*-PrbcS-*T of precious biological (TaKaRa) and the dna segment of DAK gene to produce entry vector pENTR*-PrbcS-*T-DAK then.Transform high-level efficiency (10 with the ligation mixture ⁸) competent escherichia coli cell (DH5 α, it root biochemical technology), be applied on the flat board that is added with kantlex (Km, 50 μ g/ml) transforming good intestinal bacteria, in 37 ℃ of incubated overnight, screening Km resistance recon bacterium colony, extract plasmid (Figure 12 A) from Km resistance recon bacterium colony, carry out enzyme with EcoRV (Fermentas) and cut detection, the plasmid of successful connection produces two bands on agarose gel electrophoresis figure, one that molecular weight is little is 0.75kb, and another is 5.0kb (Figure 12 B).Select the plasmid vector pENTR*-PrbcS-*T-DAK of successful connection, carry out PCR with DAK gene upstream and downstream Auele Specific Primer and detect (Figure 12 C), reaffirm be the plasmid of successful connection after, again transformed into escherichia coli DH5 α, choose single bacterium colony and carry out liquid culture, with test kit plasmid purification pENTR*-PrbcS-*T-DAK.

(4), the structure of DAK gene plant expression vector

The construction strategy of DAK gene plant expression vector as shown in figure 13, the LR reaction by the Gateway technology the PrbcS-*T-DAK subclone in plant expression vector pH2GW7 (the purpose carrier of Gateway is available from Belgian VIB/Gent company).Concrete way is: with the purpose carrier pH2GW7 of plasmid extraction test kit purifying Gateway, in the LR of Gateway reaction system, add pENTR*-PrbcS-*T-DAK and each 150ng of pH2GW7,1 μ l LR Clonase II Enzyme Mix (Invitrogen), be mixed and spend the night, PrbcS-*T-DAK is incorporated into the plant expression carrier plasmid pH2-35S-PrbcS-*T-DAK of acquisition DAK among the pH2GW7 by the effect of intergrase in 25 ℃ of reactions.Transform high-level efficiency (10 with reaction mixture ⁸) competent escherichia coli cell (DH5 α, it root biochemical technology), be applied to and be added with spectinomycin (Spe transforming good intestinal bacteria, 50 μ g/ml) on the flat board, in 37 ℃ of incubated overnight, screening Spe resistance recon bacterium colony, from Spe resistance recon bacterium colony, extract plasmid (Figure 14 A), carrying out PCR with DAS upstream and downstream Auele Specific Primer DAS5 and DAS3 detects, the plasmid of successful integration all can amplify the band (Figure 14 B) of a 2.1kb, be confirmed to be the plasmid of successful connection after, transformed into escherichia coli DH5 α again, choose single bacterium colony and carry out liquid culture, use the test kit plasmid purification.

The plant expression vector that embodiment 3, usefulness contain DAS and DAK gene transforms Agrobacterium

The competent cell of preparation Agrobacterium, the electricity consumption impulse method changes above-mentioned plant expression vector pK2-35S-PrbcS-*T-DAS that builds and pH2-35S-PrbcS-*T-DAK in the Agrobacterium (C58Cl (pPMP90)) over to, screens transformant being added with on the flat board of spectinomycin.The plasmid that takes a morsel adds in the Agrobacterium competent cell, gently mixing; The electricity that mixture is joined precooling transforms in the cup, at the bottom of knocking a glass body gently mixed liquid being dropped down onto glass; Electricity is transformed cup place electric conversion instrument (BIO-RAD) chute, with the electric shock cup of 1mm and 200 ohm, the parameter of 2.5kV/0.2cm shocks by electricity, take out electricity after the electric shock immediately and transform cup, add the 0.5mlSOC substratum rapidly, mixing is transferred in the centrifuge tube of 1.5ml; 28 ℃, the 200rpm shaking table is cultivated 3-5h; Under the room temperature, the centrifugal 1min of 7500rpm abandons most of supernatant, keeps 100 μ l with cell suspension; Agrobacterium has been coated on the LB solid medium of spectinomycin (Spe, 50 μ g/ml), cultivated for 28 ℃ and obtained single bacterium colony in 2 days; At first put into 20 μ l ddH with toothpick picking Agrobacterium bacterium colony ₂Among the O, handle for 98 ℃ and take out the template of 10 μ l Agrobacterium lysates after 15 minutes as the PCR reaction.Making PCR with the downstream Auele Specific Primer of the Auele Specific Primer PrbcS5 that is arranged in the PrbcS zone and DAS and DAK gene detects, transform successful bacterium colony and all can amplify the DAS of 2.3kb and the DAK gene band (Figure 15) of 2.1kb, the transformant bacterium colony of confirming through bacterium colony PCR is used to transform plant.

Embodiment 4, usefulness contain the Agrobacterium-mediated Transformation plant of DAS and DAK gene plant expression vector

With the experimental arrangement of Agrobacterium-mediated Transformation plant as shown in figure 16, at first the picking single colony inoculation of Agrobacterium that carries the pK2-35S-PrbcS-*T-DAS plasmid (contains Spe, 100 μ g/ml) in the LB of 50ml substratum, and 180rpm cultivates 24h, treats bacterium liquid OD for 28 ℃ ₆₀₀To about 1.0, centrifugal 10min (3000rpm), precipitation thalline.Suspend centrifugal 10min (3000rpm), precipitation thalline again with the MS liquid nutrient medium about 10ml.Repeat above operation 2～3 times.The MS liquid nutrient medium resuspension that adds certain volume at last makes the OD of thalline ₆₀₀Value is 0.5.The present invention chooses easy plant transformed morning glory and does conversion test.The aseptic seedling of preparation petunia (Petunia hyrida), the blade of aseptic seedling is cut into pieces the leaf dish, in the Agrobacterium bacterium liquid for preparing, contaminate 15-20min, after blotting with aseptic thieving paper, be tiled in the last dark of callus inducing medium MS1 (MS+NAA0.21 μ g/ml+BAP 0.02 μ g/ml) and cultivate 2 days (Figure 16 A) altogether, explant is transferred to induce (Figure 16 B) that carries out bud on the bud inducing culture MS4 (MS+NAA 0.53 μ g/ml+BAP 0.5 μ g/ml) that contains kantlex (50 μ g/ml), and about 15 days subcultures once.After having treated that blastogenesis becomes, change induce (Figure 16 C) that carries out root on the MS substratum that contains kantlex (50 μ g/ml) over to, obtain to change over to the plant of DAS gene.Preparation contains the agrobacterium suspension of the expression vector pK2-35S-PrbcS-*T-DAK of DAK then, infect the plant leaf dish that changes the DAS gene according to above-mentioned Transformation Program, to cultivate altogether 2 days on the infected leaf dish immigration MS1 substratum, transfer to again on the bud inducing culture MS4 of Totomycin (25 μ g/ml) and carry out inducing of bud, after having treated that blastogenesis becomes, change on the MS substratum that contains Totomycin (25 μ g/ml) and carry out inducing of root, obtain to change over to the plant of DAS and DAK gene at last.

Embodiment 5, DAS and DAK gene in transgenic plant the insertion situation and the detection of transcriptional level

In order to confirm to contain DAS and DAK gene really, the transfer-gen plant that screens is done further to identify with PCR method from the transfer-gen plant that the floral leaf dish of leading a cow that transforms produces.At first adopt the CTAB method to extract Plant Genome: to take by weighing and place the 1.5ml centrifuge tube about plant leaf 100mg, add liquid nitrogen and be ground to Powdered with special pestle; Add 900 μ l and be preheating to 2 * CTAB damping fluid (NaCl 1.4M, CTAB 2% for Tris-HCl pH 7.5 100mM, EDTA 20mM) of 65 ℃, 65 ℃ of degree heating in water bath take out cooling after 20 minutes; Add 500 μ l chloroforms-primary isoamyl alcohol mixed solution (24: 1) and shake up, 4 ℃ of centrifugal 10min (7500rpm) back is shifted supernatant and is managed to 1.5ml EP; Add 500 μ l chloroforms-primary isoamyl alcohol mixed solution (24: 1) once more and shake up 4 ℃ of centrifugal 10min (7500rpm); Take out supernatant and place new EP pipe, add 1/10 volume 3M pH5.2 sodium-acetate and equal-volume Virahol, shake up back 4 ℃ of centrifugal 20min (12000rpm); Abandon supernatant, after twice of 75% ethanol cleaning, drying is with the dissolving of TE damping fluid and the degradation of rna that contain RNase.With the plant genome DNA is template, making PCR with DAS and DAK gene upstream and downstream Auele Specific Primer detects, the plant that successfully changes the DAS gene over to all can amplify the DAS band (Figure 17 A and 17C) of 2 1kb, and the plant that successfully changes the DAK gene over to all can amplify the DAK band (Figure 17 B and 17C) of 1.8kb.The transfer-gen plant of confirming through PCR is used for the RT-PCR analysis.

In order to investigate DAS and DAK gene transcription situation in containing the transgenosis morning glory strain system of DAS and DAK gene; from transgenic plant, extract total RNA; reversing is used for RT-PCR behind the green one-tenth cDNA and analyzes, and detects DAS and the transcriptional level of DAK gene in transgenic plant.Adopt TRIzoL Reagent (Invitrogen) to extract RNA, get the about 0.1g of young leaf of plant, the TRIzoL extracting solution that adds 1ml grinds in mortar, room temperature moves into centrifuge tube after leaving standstill 5min, add the 0.2ml chloroform again, the vibration mixing, centrifugal 15min (12000rpm) shifts supernatant liquor to new pipe, add the 0.5ml Virahol, the mixing room temperature is placed 10min, and 4 ℃ of centrifugal 10min (12000rpm) abandon supernatant, precipitation is cleaned with 75% ethanol 1ml, 4 ℃ of centrifugal 5min (7500rpm) abandon ethanol vacuum-drying and precipitate or dry naturally, with 20 μ l diethylpyrocarbonate (DEPC) treating water dissolving RNA.Use RevertAid ^TMM-MuLV Reverse Transcriptase Kit (Fermentas) carries out the synthetic of cDNA, get the about 0.1 μ g-5 μ g of the total RNA of plant, oligo (dT) 50ng, 10mM dNTP mix 1 μ l complements to 10 μ l with the DEPC treating water, behind the mixing, of short duration centrifugal it is collected in managed at the end, places 65 ℃ of heating 5min, ice bath 10min, add reaction mixture 9 μ l (5 * reaction buffer, 4 μ l, 25mM MgCl ₂4 μ l, 0.1M DTT 2 μ l, RNA enzyme inhibitors 1 μ l), with the said mixture mixing, of short duration centrifugal it is collected in managed at the end, and 25 ℃ of insulation 2min add 1 μ l RevertAid ^TMM-MuLV ReverseTranscriptase, with the said mixture mixing, of short duration centrifugal it is collected in managed at the end, 25 ℃ of insulation 20min, 42 ℃ of insulation 70min synthesize cDNA then.With cDNA is template, make PCR with DAS and DAK gene upstream and downstream Auele Specific Primer, the plant that successfully changes DAS over to all can amplify the DAS band (Figure 18 A) of 2.1kb, the plant that successfully changes the DAK gene over to all can amplify the DAK band (Figure 18 B) of 1.8kb, proves that DAS and DAK gene can successfully transcribe in transgenic plant.Be used for the analysis of formaldehyde resistance and uptake rate through the transfer-gen plant of RT-PCR affirmation.

The resistance of embodiment 6, commentaries on classics DAS and DAK gene plant PARA FORMALDEHYDE PRILLS(91,95) detects

In the transgenosis morning glory, whether bring into play predictive role in order to detect DAS and DAK; with the transgenic plant seedling and not the wild-type seedling of transgenic plant move into and to be added with on the MS solid medium of formaldehyde (concentration is 8mM); cultivate at 25 ℃ of continuous lights, the making plant phenotype changes (Figure 19) after 32 days.The plant (Figure 19 D) that presentation of results changes DAS and DAK simultaneously over to is better than wild-type (Figure 19 A) growing way, only change DAS (Figure 19 C) or DAK (Figure 19 B) growing way of plant is similar to the growing way of wild-type, illustrate that the formaldehyde of introducing assimilates approach and can bring into play predictive role in plant, strengthen the assimilative capacity of plant PARA FORMALDEHYDE PRILLS(91,95), the render transgenic plant strengthens for the resistance of liquid formaldehyde.

In order to verify the resistance of transgenic plant to gas formaldehyde, the transgenic plant seedling moved in the sealed vessel that the MS substratum is arranged cultivate, in container, place 500 μ l centrifuge tubes of an opening after taking root in about 15 days, and in centrifuge tube, add the formaldehyde solution of a certain amount of (30 μ l) 37%.25 ℃ of continuous lights were cultivated after 49 days, observed the upgrowth situation (Figure 20) of transgenic plant, and with the gas formaldehyde concentration in the formaldehyde determination instrument mensuration container.The plant (Figure 20 B) that presentation of results changes DAS and DAK simultaneously over to is better than the growing way of only changeing DAS (Figure 20 A) or DAK (Figure 20 C) plant, the concentration ratio of residual formaldehyde only changes few 30% (Figure 20 D) of plant of DAS or DAK in the culture vessel that changes DAS and DAK plant over to, proof changes DAS and the DAK gene plant is accelerated the uptake rate of gas formaldehyde, and resistance strengthens.

Embodiment 7, transgenosis morning glory are to the detection of liquid formaldehyde uptake rate

Formaldehyde can with Nash reagent (Ammonium Acetate: 15%, Glacial acetic acid: 0.3%, methyl ethyl diketone: 0.2%) chemical reaction takes place and produce coloured material, the maximum absorption wavelength of this material is 410nm, the typical curve that therefore can prepare formaldehyde, can calculate the content of formaldehyde in the reaction solution according to the typical curve of HCHO, utilize this method can measure the uptake rate of plant PARA FORMALDEHYDE PRILLS(91,95).Get 0.3g left and right sides vegetable material and put into little triangular flask, add 10～20ml formaldehyde (4mM) solution-treated after 24 hours, take out 20～100 μ l treatment solutions, add water to 1ml, add 1ml Nash reagent again and after 30 minutes, measure OD 30 ℃ of insulations ₄₁₀, calculate the remaining concentration of formaldehyde of treatment solution (Figure 21) according to typical curve.The concentration of residual formaldehyde is the highest in the presentation of results wild-type morning glory treatment solution; only change the remaining concentration of formaldehyde of the morning glory blade treatment solution of DAS a little less than wild-type (Figure 21 A); but difference of them is not too obvious; and commentaries on classics DAS and DAK morning glory blade treatment solution residual formaldehyde concentration are significantly less than wild-type plant (Figure 21 B); compare with control plant; the amount of changeing the plant absorbing formaldehyde of DAS and DAK has improved 20%～70%; the speed that absorbs formaldehyde improves 16%～27%; the speed of as seen changeing DAS and DAK plant absorption formaldehyde generally is higher than the wild-type plant, proves and has only DAS and DAK to be present in the ability that could improve plant absorbing formaldehyde in the transgenic plant simultaneously.

Claims

1, improve the method for plant absorbing and tolerance methanal, this method may further comprise the steps:

(1), the structure of the recombinant vectors pK2-35S-PrbcS-*T-DAS of DAS

1) amplification of the gene of candiyeast DAS

Search the full-length gene order of candiyeast DAS from GenBank, its GenBank accession number is AF086822, and the following a pair of primer of implementation sequence:

DAS5：caccgcATGcCTCTCGCAAAAGCTGCTTC

DAS3：ggatccTTATTGATCATGTTTTGGTTTTTC

5 ' end primer DAS5, end adds the caccgc characteristic sequence, and changes the G behind the ATG into c and form the SphI restriction enzyme site thus; 3 ' end primer DAS3, end adds the BamHI restriction enzyme site; Genomic dna with candiyeast is a template amplification, obtains the full length DNA of DAS;

2), the TA of DAS gene clone

3), the structure of entry vector pENTR*-PrbcS-*T-DAS

With SphI and BamHI cutting pENTR*-PrbcS-*T-GFP and pMD-DAS, reclaim carrier pENTR*-PrbcS-*T segment and DAS gene segment, connect PENTR*-PrbcS-*T and DAS gene segment with the T4DNA ligase enzyme, obtain entry vector pENTR*-PrbcS-*T-DAS;

4), the structure of DAS gene plant expression vector pK2-35S-PrbcS-*T-DAS

LR by the Gateway technology reacts the PrbcS-*T-DAS subclone in plant expression vector pK2GW7, and the expression of plants that obtains the DAS gene carries pK2-35S-PrbcS-*T-DAS, and the GenBank accession number of its DAS gene is AF086822

Recombinant vectors pK2-35S-PrbcS-*T-DAS, DAS upstream region of gene are added with photoinduction type promotor PrbcS and chloroplast(id) positioning sequence * T, are right after the DAS gene thereafter;

(2), the structure of DAK gene recombined vector pH2-35S-PrbcS-*T-DAK

1), the amplification of the gene of pichia spp DAK

Search the full-length gene order of pichia pastoris phaff DAK from GenBank, its GenBank accession number is AF019198, and the following a pair of primer of implementation sequence:

DAK5：CACCGCatgCctagtaaacattgggattac

DAK3：CTCGAGctacaacttggtttcagatttg

5 ' end primer DAK5, end adds the caccgc characteristic sequence, and changes the t behind the ATG into C and form the SphI restriction enzyme site thus; 3 ' end primer DAK3, end adds the XhoI restriction enzyme site; Genomic dna with pichia pastoris phaff is a template amplification, obtains the full length DNA of DAK;

2), the TOPO of DAK gene clone

Reclaim and purifying DAK gene fragment, to the pENTR-TOPO carrier, adopt alkaline lysis method of extracting plasmid DNA, detect and sequencing analysis acquisition recombinant plasmid pENTR-TOPO-DAK by PCR by TOPO clone technology subclone;

3), the structure of entry vector pENTR*-PrbcS-*T-DAK

With SphI and XhoI cutting pENTR*-PrbcS-*T-GFP and pENTR-TOPO-DAK, reclaim carrier pENTR*-PrbcS-*T segment and DAK gene segment, connect PENTR*-PrbcS-*T and DAK gene segment with the T4DNA ligase enzyme, obtain entry vector pENTR*-PrbcS-*T-DAK;

4), the structure of DAK gene plant expression vector pH2-35S-PrbcS-*T-DAK

LR by the Gateway technology reacts the PrbcS-*T-DAK subclone in plant expression vector pH2GW7, obtains the plant expression vector pK2-35S-PrbcS-*T-DAK of DAS gene, and the GenBank accession number of its DAK gene is AF019198; The upstream of DAK gene is added with photoinduction type promotor PrbcS and chloroplast(id) positioning sequence * T among the recombinant vectors pK2-35S-PrbcS-*T-DAK;

(3), the detection of genetic transformation of Agrobacterium and transformant

The competent cell of preparation Agrobacterium, the electricity consumption impulse method changes above-mentioned plant expression vector pK2-35S-PrbcS-*T-DAS that builds and pH2-35S-PrbcS-*T-DAK in the Agrobacterium over to, is being added with screening transformant bacterium colony on the flat board of spectinomycin; With the lysate of Agrobacterium bacterium colony template as the PCR reaction, make PCR with the upstream and downstream Auele Specific Primer of DAS and DAK gene and detect the transformant bacterium colony, be used to transform plant through the transformant bacterium colony of bacterium colony PCR affirmation;

(4), with the Agrobacterium-mediated Transformation plant that contains DAS and DAK gene plant expression vector

The picking single colony inoculation of Agrobacterium that carries plasmid pK2-35S-PrbcS-*T-DAS and pH2-35S-PrbcS-*T-DAK is cultivated in liquid LB substratum respectively, and centrifugal collection thalline suspends with the MS liquid nutrient medium again.Plant tissue with the agroinfection that suspends breaks up easily obtains seedling by tissue culture then, utilizes antibiotic-screening to obtain transgenic plant again;

(5), DAS and DAK gene in transgenic plant the insertion situation and the detection of transcriptional level

With PCR method the transgenic plant that screen are done further to identify; At first adopting the CTAB method to extract the genomic dna of transgenic plant, is template then with the plant genome DNA, makes PCR with the upstream and downstream Auele Specific Primer of DAS gene and DAK gene and detects, and the transfer-gen plant of confirming through PCR is used for the RT-PCR analysis;

From transgenic plant, extract total RNA, be used for RT-PCR behind the green one-tenth cDNA that reverses and analyze, detect DAS gene and the transcriptional level of DAK gene in transgenic plant; Adopting TRIzoL Reagent (Invitrogen) to extract RNA, is template with cDNA, makes PCR with DAS and DAK gene upstream and downstream Auele Specific Primer, is used for the detection of the resistance and the uptake rate of plant PARA FORMALDEHYDE PRILLS(91,95) through the transfer-gen plant of RT-PCR affirmation.

2, the Agrobacterium of the genetic transformation of the Agrobacterium described in described (3) step of method of claim 1, it is characterized in that: described Agrobacterium is C58Cl (pPMP90).

3, the usefulness described in described (4) step of method of claim 1 contains the Agrobacterium-mediated Transformation plant of DAS and DAK gene plant expression vector, and it is characterized in that: described plant is the plant of suitable indoor cultivation.

4, improve a pair of plant expression vector of plant absorbing and tolerance methanal, it is characterized in that: this a pair of plant expression vector contains pK2-35S-PrbcS-*T-DAS carrier and pH2-35S-PrbcS-*T-DAK carrier; The DAS gene that contains candiyeast in the described pK2-35S-PrbcS-*T-DAS carrier; The DAK gene that contains pichia spp in the described pH2-35S-PrbcS-*T-DAK carrier; The DAS gene of described candiyeast and the DAK upstream region of gene of pichia spp all have the promotor PrbcS and the chloroplast(id) positioning sequence * T of Rubisco small subunit.

5, the described plant expression vector of claim 4 is characterized in that: the initial vector of described pK2-35S-PrbcS-*T-DAS plant expression vector is pK2GW7, and the initial vector of described pH2-35S-PrbcS-*T-DAK plant expression vector is pH2GW7.

6, the described plant expression vector of claim 4 is characterized in that: the GenBank accession number of the DAS gene of described candiyeast is AF086822; The DAK gene GenBank accession number of described pichia spp is AF019198.

7, the application of described method of claim 1 or the described a pair of plant expression vector of claim 4 is characterized in that: the application on the morning glory plant.