CN101310882A - Method for repairing heavy metal and organic compound contaminated soil using transgenic plant - Google Patents
Method for repairing heavy metal and organic compound contaminated soil using transgenic plant Download PDFInfo
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- CN101310882A CN101310882A CNA2007101076883A CN200710107688A CN101310882A CN 101310882 A CN101310882 A CN 101310882A CN A2007101076883 A CNA2007101076883 A CN A2007101076883A CN 200710107688 A CN200710107688 A CN 200710107688A CN 101310882 A CN101310882 A CN 101310882A
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Abstract
The invention discloses a method for restoring soil compositely polluted by heavy metals and organics by using a transgenic plant and is technically characterized in that foreign genes which can strengthen the efficiency for restoring the organic pollution of the plant and foreign genes which can improve the tolerance and the accumulation to a plurality of metals of the plant are introduced to the plant so as to establish a transgenic plant which has a better restoring effect on the pollution by the heavy metals and the organics. The method of the invention has a better restoring effect on the polluted soil of the three types as follows: (1) the soil polluted by organic pollutants of halohydrocarbon compounds such as trichloroethylene, dibromethane, and the like; (2) the soil polluted by various heavy metals such as Cd, Cr, Cu, Mn, Zn and Pb; (3) the soil compositely polluted by the heavy metals and the organics. In addition, the method of the invention comprises the procedures of establishing the transgenic plant, the screening and cultivation of light-demanding plants and the restoring of polluted soil by the transgenic plant. The method of the invention has broad application prospect in the restoring of the soil compositely polluted by the heavy metals and the organics.
Description
One, technical field
The present invention relates to biotechnology and field of environment protection, particularly foreign gene is introduced anti-combined pollution plant and be applied to heavy metal and the method for organic compound contaminated soil reparation.
Two, background technology
Along with the intensivization development of urbanization, industrialization, motor vehicle exhaust emission and agricultural, environmental problem more and more is subjected to people and pays close attention to, and the soil pollution that heavy metal, organic matter cause also becomes the hot issue that environment, soil scientists are studied day by day.Soil not only directly influences crop yield and quality after being polluted by heavy metal and organic matter, also can influence health and safety by food chain.The strategy that utilizes green plants to clear the pollution off, promptly to have a cost low for phytoremediation technology, and environmental friendliness and characteristics such as recycling are a kind of very promising environmental pollution original position governance ways.Genetically modified plants can obtain better soil remediation effect owing to have better tolerance and accumulation.Use the genetically modified plants repairing polluted soil, become one of emphasis of various countries scientist research.
The genetically modified plants repairing heavy metal pollution
In genetically modified plants were repaired, the plant huge legendary turtle was closed the peptide technology and obtains more application.U.S. scientist Bennett etc. has compared the repairing effect of the Indian mustard of expression gamma glutamyl cysteine synthetase (ECS) or glutathione synthetase (GS) or ATP sulfurylase (APS) gene to soil respectively, the result shows, express the plant of gamma glutamyl cysteine synthetase (ECS) or glutathione synthetase (GS), the level that the plant huge legendary turtle is closed peptide and glutathione increases, and the tolerance and the accumulation of cadmium also improved.Express the plant of ATP sulfurylase, the content of its glutathione and mercaptan all increases.The heavy metal cumulant of ECS and GS plant cauline leaf is significantly higher than wild strain, and wherein cadmium is 1.5 times, and zinc is 1.5-2 times, and for chromium, copper, lead, the cumulant of ECS plant is 2.4-3 a times of wild strain.The heavy metal amount that all transfer-gen plants are removed from soil all is significantly higher than wild strain, and the reduction of cadmium, zinc is respectively 25% and 6% in the soil.In above-mentioned several transfer-gen plant, the putting up the best performance of ECS plant.
2006, U.S. scientist Li etc. expressed gamma glutamyl cysteine synthetase (ECS) gene in arabidopsis, made the content of glutathione in cauline leaf and root increase 2-5 doubly than wild strain, had effectively improved the tolerance of plant to arsenic, mercury.Professors Pomponi etc. close the peptide gene with the plant huge legendary turtle of arabidopsis and express in tobacco, have improved tolerance and the accumulation of plant to Cd.2007, American scholar Banuelos etc. changed cysteine lyase (cpSL) and cysteine methylferase (SMT) gene in the Indian mustard over to respectively and express, and repair the soil of selenium contamination with transfer-gen plant.As a result, the plant of the commentaries on classics cpSL gene of plantation in spring is increased to original 2 times to the cumulant of selenium, compares with the wild type plant, and selenium concentration has improved 1.8 times in the leaf.Selenium concentration has improved 1.7 times than wild type plant in the plant leaf of commentaries on classics SMT gene.The plant of the commentaries on classics SMT gene of autumn plantation is 1.6 times of wild plant to the cumulant of selenium, and the concentration of selenium is all higher in the stem, leaf.These results show that the transgenosis Indian mustard of cpSL, SMT significantly improves the accumulation capability of selenium.Researchers such as LeDuc express ATP sulfurylase and cysteine transmethylase respectively simultaneously in arabidopsis and India's mustard, make plant improve 4-9 doubly to the accumulation of selenium.2007, researchers such as the Banuelos of the U.S. carried out the field experiment of transgenosis Indian mustard, had expressed cysteine lyase or cysteine transmethylase in transfer-gen plant, and the result shows that the accumulation of selenium has improved 1.6-2 doubly.Gasic etc. close the peptide gene with the plant huge legendary turtle of arabidopsis and express in Indian mustard, have improved the tolerance of plant to cadmium.
Except huge legendary turtle was closed the peptide technology, other genetically modified plants also were used for the heavy metal pollution reparation.
The mammiferous abc transporter genes of heterogenous expression such as Japan scientist Yazaki K can improve simultaneously to organic and tolerance inorganic pollution in tobacco, thereby, be expected to be used for the phytoremediation of combined pollution.But it is stable inadequately that ABC expresses.Sasaki etc. express bacterium mercury transporter gene in arabidopsis, made up super mercury accumulation plant.Kim etc. express abc transporter AtPDR8 in arabidopsis, improved the tolerance to cadmium.2006, the sulfate transmission subbase that American scholar Lindblom etc. are affine with height obviously improved plant to f Cr, Cd, the tolerance of V and W because of expressing in India's mustard.Japan scientist Sasaki etc. expresses the mercury protein called membrane transporters of Thiobacillus in arabidopsis, made up super anti-mercury plant, and its mercury accumulation is 2 times of wild plant.2006, Nagata etc. expressed bacterium phosphokinase gene in tobacco, had improved mercury tolerance and the accumulation of plant.
Genetically modified plants are repairing organic polluted
Trichloro-ethylene (TCE) etc. is the bigger organic pollution of contaminant capacity, and is particularly more to phreatic pollution.P450 2E1 enzyme can the multiple pollutant of oxidation, comprise trichloro-ethylene, bromine ethene, chloroform, carbon tetrachloride and vinyl chloride etc., effective to multiple main soil pollutant, so P450 2E1 (CYB 2E1) is considered to strengthen the candidate gene of plant organic contamination remediation efficiency.
(16) such as U.S. scientist Doty change human cell's chromogene P450 2E1 over to tobacco, use its repairing polluted soil.P450 2E1 transgene tobacco reaches 640 times to the metabolic capability of TCE than blank strain raising.Transfer-gen plant has obviously increased the picked-up of bromine ethene and debromination.So P450 2E1 plant can be used for the phytoremediation that halide pollutes, effectively repair halide and pollute.2006, Britain scientist Rylott etc. expressed the Cytochrome P450 of degraded T4 (RDX) in arabidopsis, improved its tolerance to RDX, and in containing the RDX environment, the growth of cauline leaf and root all is better than wild plant.Greece scholar Karavangeli etc. make up transgene tobacco, express the glutathione s-transferase gene of corn, have improved the degradation capability to chloro-acetanilide herbicide.2006, professor Liao of Fudan University waits and has furtherd investigate the degraded of transgenic arabidopsis to catechol, bacterium chlorine catechol dioxygenase gene is expressed in arabidopsis, and transgenic arabidopsis shows the ability of obviously high removing catechol, can be as the phytoremediation of fragrant pollutant.
Express a plurality of genes simultaneously and can produce synergy
Inui H etc. express human P450 gene: CYP1A1 in potato, CYP2B6, and CYP2C9 and CYP2C19, the result shows, changes CYP1A1 simultaneously over to, CYP2B6, and the plant T1977 of CYP2C19 gene show the cross resistance to multiple herbicide; Express the plant 2C9-57R2 of CYP2C19 gene, sulfonylurea drugs is had strong tolerance; Express the plant 2C19-12R1 of CYP2C9 gene, multiple herbicide is had stronger cross tolerance power.The above results shows the polygenes synergy, has improved the repair ability of transgenic potato.Kawahigashi etc. adopt plasmid pIKBACH, with the gene C YP1A1 of human cell's cytochrome p 450, CYP2B6 and CYP2C19 express in paddy rice simultaneously, increased substantially the tolerance of paddy rice, can carry out phytoremediation the soil of organic contamination to herbicide atrazine and isopropyl methoxalamine.Wawrzynski etc. close the peptide synthetase gene with serine acetyltransferase gene, gamma-glutamyl cysteine enzyme gene and plant huge legendary turtle and express in plant simultaneously, make the accumulation of cadmium improve 8 times, and express the plant of term single gene, and the cumulant of cadmium improves less.This shows, expresses a plurality of genes simultaneously, has synergy.
United States Patent (USP) 20060150279A1, with rhamnosyltransferase gene rh1A or rh1B, or 2 genes are expressed in the tobacco simultaneously, have increased the tolerance of transfer-gen plant to pollutants such as copper.United States Patent (USP) 20060130173A1 changes P type ATPase ZntA gene over to arabidopsis, has improved the tolerance of plant to heavy metal.United States Patent (USP) 20060095982A1 expresses the plant huge legendary turtle and closes peptide in plant, improved the repair ability of plant to contaminated soil.Chinese patent CN1594583 expresses iaaM gene and ACC deaminase gene in petunia or tobacco, improved genetically modified plants to rich long-pending characteristic of the wide spectrum of different heavy metals such as copper, lead, cadmium, cobalt and resistance.
Combined pollution is repaired
Many areas exist heavy metal and organic combined pollution, and combined pollution brings more and seriously influences, and the task of therefore repairing combined pollution is arduous more.Present genetically modified plants, just changed corresponding gene at heavy metal pollution or organic matter pollution, therefore, can only be used for independent repairing heavy metal pollution or organic matter pollutes, and can not repair combined pollution, therefore, can repairing heavy metal pollution and the corresponding gene of organic contamination in a plant, express simultaneously, make it that tolerance and cumulant to heavy metal and organic pollution are provided simultaneously significantly, become the strong instrument of repairing combined contamination soil.
Three, summary of the invention
The present invention is based on above background, in the plant of anti-combined pollution, introduce the foreign gene that strengthens plant organic contamination remediation efficiency and can improve the foreign gene of plant multiple metal tolerance and accumulation, obtaining a kind ofly all has the genetically modified plants of better repairing effect to heavy metal and organic matter, for heavy metal and organic compound contaminated biological restoration provide a valid approach.The object of the present invention is to provide a kind of method of utilizing genetically modified plants remediating heavy metal and organic compound contaminated soil
Purpose of the present invention can realize as follows:
1. foreign gene is introduced anti-combined pollution plant
(1) structure contains the foreign gene that can strengthen plant organic contamination remediation efficiency and can improve the binary expression vector of plant to the foreign gene of multiple metal tolerance and accumulation
(2) utilize electric method for transformation that constructed plant binary expression vector is transformed Agrobacterium
(3) change foreign gene over to anti-combined pollution plant by agrobacterium-mediated transformation
2, the screening of transfer-gen plant
Select to break up on the culture medium and cultivate and the root induction cultivation applying antibiotic, filter out the resistance regeneration plant.
3, the check and analysis of transfer-gen plant
(1) PCR of transfer-gen plant detects
(2) enzyme of transfer-gen plant is cut and Southern hybridization detection
(3) the Western blotting detects the expression of exogenous gene product
4, the geobiont repairing test is carried out in the potted plant and field planting of transfer-gen plant test.
Plant transfer-gen plant there being halides to pollute or have on the soil of heavy metal, regularly the content to halides in the plant and heavy metal carries out check and analysis, simultaneously the content of beary metal in the soil is also carried out sample analysis, test is carried out at flowerpot and big Tanaka respectively.
The invention has the advantages that:
(1) can strengthen plant organic contamination remediation efficiency foreign gene and can improve foreign gene in plant the coordinate expression of plant to multiple metal tolerance and accumulation, can further improve plant to heavy metal and organic tolerance and accumulation ability.
(2) genetically modified plants of the present invention can be used for the reparation of organic material contaminated soils such as halogenated hydrocarbon compound such as trichloro-ethylene, Bromofume
(3) genetically modified plants of the present invention can be used for the reparation of various heavy contaminated soils such as Cd, Cr, Cu, Mn, Zn and Pb;
(4) genetically modified plants of the present invention can be used for the reparation of the combined contamination soil that above-mentioned heavy metal and organic matter cause.
Below in conjunction with specific embodiment the present invention is further described.
The specific embodiment
The structure of embodiment 1 plant binary expression vector and conversion Agrobacterium
To derive from people's Cytochrome P450 2 E1 genes and derive from colibacillary gamma glutamyl cysteine synthetase (after γ-ECS) gene is cut with restriction enzyme BamH1 and Sac1 enzyme, be connected to plant binary conversion carrier pCAMBIA2301G, the recombinant expression carrier called after pCAMBIA2301G-p450-ECS of acquisition.
Utilize electroporation that recombinant expression carrier pCAMBIA2301G-p450-ECS is transformed agrobacterium tumefaciens bacterial strain LBA4404.
Embodiment 2 Plant Transformation
Adopt agriculture bacillus mediated leaf disc transformation method, recombinant expression carrier pCAMBIA2301G-p450-ECS is changed in the alfalfa blade.
With the Agrobacterium medium centrifugal that has the pCAMBIA2301G-p450-ECS plasmid of incubated overnight, thalline is suspended among the NaCL of 10ml0.15mol/L, and centrifugal collection thalline is suspended in the CaCL of 1ml 20mmol/L again
2Solution suspends, the alfalfa blade is cut into the fritter of 0.5X0.5cm, put into the bacterium liquid that contains the pCAMBIA2301G-p450-ECS plasmid and soak 20min, blot unnecessary bacterium liquid with filter paper, be placed on and cultivated altogether on the MS culture medium 3 days, and then forward on the screening and culturing base (MS+0.5mg/L6-BA+0.5mg/L 2,4-D+20mg/L Kanamycin+300mg/L Cefixime) that contains kanamycins and carry out Preliminary screening.Treat that bud grows to about 1cm, the leaf dish that sprouts is transferred on the root media (MS+70mg/LKanamycin+300mg/L Cefixime) induced the young shoot culture of rootage.After treating the root system stalwartness, transformed plant is transplanted in the greenhouse cultivates, obtain alfalfa and transform seedling.
Embodiment 3 transgenosis alfalfas are to the reparation experiment of cadmium, lead-TCE combined contamination soil
At first with heavy metal cadmium, plumbous with its inorganic salt solution (CdCl
25H2O, PbCl
2, analyze pure) form evenly be manured into soil, cadmium, lead and TCE are provided with high and low two kinds of concentration of treatment respectively, and (wherein cadmium is: 0.5,1.5mgkg
-1, lead: 100,300mgkg
-1, TCE is 10,100mgkg
-1), adopt L2
3Orthogonal design, and establish following contrast: the contaminated soil of contamination-free soil (2) the plantation wild type alfalfa of (1) plantation transgenic alfalfa, each processing is provided with 3 repetitions.
Test takes following steps that TCE is mixed with test soil:
1. each concentration is handled and is all used the needed TCE of proper amount of acetone solubility test earlier, joins respectively then in the soil of the required soil total amount 1/10 of experimental design, fully stirs.
2. step joins respectively in the soil of the required soil total amount 1/2 of experimental design after 1. the acetone volatilization is done in the soil again, fully stirs, and places 2d.
3. 2. add the whole soil of residue in the soil to step, place 15d, mix every day 1 time, divide at last to install in the plastic tub that has the chassis, every basin dress 2Kg soil, placed for 1 week after, broadcast into 20 of transgenosis alfalfa seeds, 6d final singling after germinateing, every basin 8-10 strain, daily management is kept adequate moisture with deionized water
Content of beary metal in the plant: plant sample is behind dry ashing, and with the HCl dissolving, the flame atomic absorption spectrophotometer method is measured.
Heavy metal in soil available state content: with the lixiviate of DTPA (diethylenetriaminepeacidcetic acidcetic) extractant, the flame atomic absorption spectrophotometer method is measured.
TCE content in the plant: after the plant sample pulverizing, put in the liquid nitrogen and smash to pieces, adopt the H2SO4-NaCl method to carry out extracting, the GC method is surveyed content.
TCE content in the soil: the pedotheque of collection adopts the microwave auxiliary extraction method to extract TCE, the TCE concentration determined by ultraviolet spectrophotometry in the extract after air-dry.
Cadmium content (mgkg in the clover in the potted plant experiment of table 1
-1)
Lead content (mgkg in the clover in the potted plant experiment of table 2
-1)
TCE content (mgkg in the clover in the potted plant experiment of table 3
-1)
Claims (4)
1. method of utilizing genetically modified plants remediating heavy metal and organic compound contaminated soil, it is characterized in that to strengthen the foreign gene of plant organic contamination remediation efficiency and can improving the foreign gene introduced plant of plant to multiple metal tolerance and accumulation, but obtain the genetically modified plants of remediating heavy metal and organic compound contaminated soil, in contaminated soil, plant genetically modified plants, repair contaminated soil.
2. according to the described a kind of method of utilizing genetically modified plants remediating heavy metal and organic compound contaminated soil of claim 1, it is characterized in that: the foreign gene that can strengthen plant organic contamination remediation efficiency is one of following gene: derive from mammiferous metallothionein (MT) gene, derive from the class MT gene of higher plant or derive from mammiferous Cytochrome P450 2E1 gene.
3. according to the described a kind of method of utilizing genetically modified plants remediating heavy metal and organic compound contaminated soil of claim 1, it is characterized in that: can improve plant is one of following gene to the foreign gene of multiple metal tolerance and accumulation: ACC deaminase gene, gamma-glutamyl cysteine synthetase gene (γ-ECS) or CAX-2 gene.
4. according to the described a kind of method of utilizing genetically modified plants remediating heavy metal and organic compound contaminated soil of claim 1, it is characterized in that: described plant is a kind of in the following plant: white poplar, tobacco, cauliflower, clover, Indian mustard.
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