CN100398656C - Transgened-tobacco for degrading organic mercury pollution - Google Patents
Transgened-tobacco for degrading organic mercury pollution Download PDFInfo
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- CN100398656C CN100398656C CNB2003101084491A CN200310108449A CN100398656C CN 100398656 C CN100398656 C CN 100398656C CN B2003101084491 A CNB2003101084491 A CN B2003101084491A CN 200310108449 A CN200310108449 A CN 200310108449A CN 100398656 C CN100398656 C CN 100398656C
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Abstract
The present invention provides an organic mercury lyase merB gene suitable for being expressed in plants. The gene is obtained by reforming the sequence of merB genes for converting catalytic methyl mercury in ions into ion mercury. The present invention also relates to a transgenic plant with strong resistance to organic mercury, which is obtained by transferring reformed merB genes into plants. The transgenic plant can be used for degrading organic mercury pollutants in soil.
Description
Technical field
The invention belongs to phytology and prevention and cure of pollution field, specifically, the present invention relates to transformation, organic mercury is polluted the method for the transgenic plant (especially tobacco) that high resistance is arranged with the merB gene preparation of transforming organic mercury lyase merB gene.The invention still further relates to the method for polluting with described transgenic plant degraded organic mercury.
Background technology
Mercury is widely used in chemical industry, paper-making industry, and mining industry and national defense industry are the one of the chief elements of soil, water and air heavy metal contamination.Mercury in the environment has 3 kinds of forms of simple substance mercury, ionic mercury and organic mercury (as methyl mercury).Some microorganism can make the low inorganic mercury of toxicity be transformed into the high methyl mercury of toxicity, and the physiology toxicity of methyl mercury is thousands of times of simple substance mercury.It is very fast that fish absorb the speed of methyl mercury, causes biomagnification by food chain, and be difficult for discharging in vivo." minamata disease " of Japan is exactly because the mercury pollution fish and shellfish causes.
A kind of predominant bacteria is arranged in the mercury contaminated soil, have methyl mercury is changed into ionic mercury, ionic mercury changes simple substance mercury into, and it is discharged intravital ability.Someone successively isolates merA and merB gene from this bacterium, merA genes encoding Mercuryreductase wherein, and the catalysis ionic mercury changes the process of simple substance mercury into; The enzyme of merB genes encoding is called as organic mercury lyase (organomercurial lyase), and the catalysis methyl mercury changes ionic mercury into.Two gene combined action will be converted into the mercury of organic gasiform simple substance mercury.
Generally speaking, the detoxifcation of inorganic mercury compound can be realized by merA expression of gene product.Yet organomercurial detoxifcation needed for two steps transformed at least, and promptly organic mercury is degraded to inorganic mercury, and inorganic mercury is vaporizated into simple substance mercury.
The toxicity of simple substance mercury is little, and the mercury that is discharged in the atmosphere has been diluted to safe concentration.Utilize this principle, people attempt merA or merB gene transfered plant, adopt gene engineering method to create transgenic plant, and purpose is the form that exists that transforms mercury, repairs organic mercury or inorganic mercury Contaminated soil and water body.
Doctor Meagher leader's research group at first shifts merA and merB gene on the model plant Arabidopis thaliana, obtain the transgenic plant of anti-mercury and volatilization mercury.Yet, it is found that directly the transgenic plant that merA and merB obtained with bacterium still are difficult to satisfactory to organomercurial resistance.
Therefore, this area presses for exploitation has the transgenic plant of high resistance to organic mercury, so as can be effectively and the environment of degrading cheaply in organic mercury pollute.
Summary of the invention
Purpose of the present invention just provides a kind of preparation has the methods of the transgenic plant of very high resistance to organic mercury, and these transgenic plant organic mercury that is used to degrade is polluted.
In a first aspect of the present invention, provide the present invention that a kind of polypeptide with organic mercury degradation function also is provided, it is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the organic mercury degradation function by (a) polypeptides derived.
The present invention also provides a kind of polynucleotide of the aforementioned polypeptides of encoding.In preference, a kind of isolating polynucleotide are provided, it is the merB gene that is particularly suitable for expressing in vegetable cell through transforming, it comprises a nucleotide sequence, this nucleotide sequence coded polypeptide with aminoacid sequence shown in the SEQ ID NO:2, and the absolute difference of the degree of the G+C among the degree of the G+C in the described nucleotide sequence and the SEQ ID NO:1 is less than 5%.
In another preference, the difference of the degree of the G+C among the degree of the G+C in the described nucleotide sequence and the SEQ ID NO:1 is less than 2%.
More preferably, the difference of the degree of the G+C among the degree of the G+C in the described nucleotide sequence and the SEQ ID NO:1 is less than 1%.
In another preference, described nucleotide sequence has the nucleotide sequence shown in SEQ ID NO:1 or 3.
More preferably, 15 Nucleotide in initiator codon ATG upstream contain plant translation consensus sequence AGAACCAC.
In a second aspect of the present invention, a kind of carrier is provided, it contains the polynucleotide of the merB gene of transformation of the present invention.
In a third aspect of the present invention, a kind of host cell is provided, it contains the above-mentioned carrier of the present invention, perhaps is integrated with the polynucleotide of the merB gene of transformation of the present invention in the karyomit(e) of described host cell.
In another preference, described cell is a vegetable cell, especially tobacco, paddy rice, wheat, seeding corn and other crops.
In a fourth aspect of the present invention, a kind of method that changes the anti-organic mercury resistance of plant is provided, it comprises step:
(1) provides the Agrobacterium of carrying expression vector, described expression vector contains the nucleotide sequence of the merB that encodes, nucleotide sequence coded polypeptide with aminoacid sequence shown in the SEQ ID NO:2, and the absolute difference of the degree of the G+C among the degree of the G+C in the described nucleotide sequence and the SEQ ID NO:1 is less than 5%;
(2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (1), thereby make the nucleotide sequence of coding merB change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell or the tissue or the organ of nucleotide sequence that changes coding merB over to;
(4) vegetable cell in the step (3) or tissue or neomorph are become plant.
In another preference, described plant is tobacco, paddy rice, wheat or seeding corn and other crops.More preferably, described plant is a tobacco.
Aspect of the present invention five, the transgenic plant that obtain with aforesaid method of the present invention are provided.
In sixth aspect present invention, the purposes of transgenic plant of the present invention is provided, the organic mercury that they are used to degrade in the soil pollutes.
Description of drawings
Figure 1A and 1B have shown the nucleotide sequence and the amino acid sequence coded thereof of the merB gene (merBhe gene) that the present invention transforms.
Fig. 2 has shown the structural representation of merBhe expression vector pJR1::merBhe.
Fig. 3 has shown the Southern and the Northern results of hybridization of merBhe transgene tobacco.Wherein,
(a) Southern hybridization. the 1st, 3 swimming lane is respectively MerBhe-13 and two transgenic lines of MerBhe-14, and the 2nd swimming lane is a wild-type tobacco, and the 4th swimming lane is the probe of merBhe gene.
(b) Northern hybridization. the 1st, 3 swimming lane is respectively MerBhe-13 and two transgenic lines of MerBhe-14, and the 2nd swimming lane is a wild-type tobacco, and the 4th swimming lane is the probe of merBhe gene
Fig. 4 has shown that the merBhe transgene tobacco is at the germination and the growing state that contain on the 0.5 μ mol/L PMA substratum.Wherein, (a) wild-type tobacco, (b) transgenic line MerBhe-14.
Embodiment
The inventor is through discovering, the merA of bacterium or merB are directly imported plant, and can't to obtain that organic mercury is had the major cause of the plant of high resistance be that the G+C content of bacterial gene is too high, in plant materials, do not express or expression level very low.Suitably reduce the content of G+C, obtain the merB gene of transformation, the merB gene of transforming is imported tobacco, the result has obtained the transgenic plant of organomercurial resistance far above prior art.Finished the present invention on this basis.
As used herein, term " the merB gene of transformation " or " merB gene of the present invention " are used interchangeably, refer to such nucleotide sequence, this nucleotide sequence coded polypeptide with aminoacid sequence shown in the SEQ ID NO:2, and the absolute difference of the degree of the G+C among the degree of the G+C in the described nucleotide sequence and the SEQ ID NO:1 is less than 5%.Preferably, this absolute difference is less than 2%, more preferably less than 1%, best less than 0.5%.
As used herein, term " merBhe gene " refers to have the nucleic acid molecule of nucleotide sequence shown in the SEQ ID NO:1.Wherein the G+C degree is 51.8%.This term also can be included in 20 in upstream from start codon and terminator codon downstream with interior Nucleotide, the sequence shown in SEQ ID NO:3, and wherein the G+C degree is 48.6%.
In the present invention, term " merB albumen " or " merB polypeptide " are used interchangeably, all refer to have the merB aminoacid sequence albumen or the polypeptide of (SEQ ID NO:2).They comprise the merB that contains or do not contain initial methionine.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
The present invention also comprises fragment, derivative and the analogue of merB.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of natural merB of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound merge formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence of this polypeptide of purifying).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " merB polypeptide " refers to have the active SEQ ID of merB NO.2 polypeptide of sequence.This term also comprises having and the identical function of merB variant form (degraded organic mercury function), SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises active fragments and the reactive derivative of merB.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of merB DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-merB polypeptide to obtain.The present invention also provides other polypeptide, as comprises merB polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the fragment of merB polypeptide.Usually, this fragment have the merB peptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids.
Invention also provides the analogue of merB or polypeptide.The difference of these analogues and natural merB polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology.
In the present invention, " merB conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala (A) | Val；Leu；Ile | Val |
| Arg (R) | Lys；Gln；Asn | Lys |
| Asn (N) | Gln；His；Lys；Arg | Gln |
| Asp (D) | Glu | Glu |
| Cys (C) | Ser | Ser |
| Gln (Q) | Asn | Asn |
| Glu (E) | Asp | Asp |
| Gly (G) | Pro；Ala | Ala |
| His (H) | Asn；Gln；Lys；Arg | Arg |
| Ile (I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu (L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys (K) | Arg；Gln；Asn | Arg |
| Met (M) | Leu；Phe；Ile | Leu |
| Phe (F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro (P) | Ala | Ala |
| Ser (S) | Thr | Thr |
| Thr (T) | Ser | Ser |
| Trp (W) | Tyr；Phe | Tyr |
| Tyr (Y) | Trp；Phe；Thr；Ser | Phe |
| Val (V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The nucleotide sequence that the present invention relates to comprises that also the absolute difference of degree of the degree of G+C and the G+C among the SEQ ID NO:1 is less than 5% nucleotide sequence.Wherein this difference can be calculated by following formula:
G+C per-cent-SEQ ID the NO:1 of difference=certain sequence or 3 G+C per-cent
The Nucleotide full length sequence of the merB of transformation of the present invention or its fragment can use the method for synthetic, pcr amplification method or recombination method to obtain usually.For example at first carrying out complete sequence according to the sequence of SEQ ID NO:1 synthesizes.Usually, can synthesize a plurality of small segments earlier, and then connect and to obtain the very long fragment of sequence.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with reorganization.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.Also available PCR method obtains the merB gene that the present invention transforms.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or merB encoding sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the merB polypeptide of reorganization.In general following steps are arranged:
(1). with the polynucleotide (or varient) of coding of the present invention merB polypeptide, or with the recombinant expression vector that contains these polynucleotide proper host cell that transforms or transduce;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, the merB polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus or other carriers.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains merB DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell such as yeast; Vegetable cell; Insect cell etc.Preferred host cell is a vegetable cell.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.Transform plant and also can use methods such as Agrobacterium-mediated Transformation or particle gun conversion, for example leaf dish method.Can use ordinary method regeneration plant for plant transformed cell, tissue or organ, thereby obtain the plant that anti-organic mercury resistance improves.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
On the other hand, the present invention also comprises merB DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment or chimeric antibody.Antibody of the present invention can be prepared by the known various technology of those skilled in that art.
Major advantage of the present invention is:
(a) the merB gene of Gai Zaoing is particularly suitable for expressing in vegetable cell.
(b) the anti-organic mercury resistance of the transgenic plant of Huo Deing is far above prior art.
Therefore, the merB of the present invention for a change anti-organic mercury resistance of plant provides new approach, thereby has great application prospect.Can import by encoding gene, change the anti-organic mercury resistance of existing good variety of crops, can obtain anti-organomercurial wheat, paddy rice or other variety of crops, thereby the organic mercury in the degraded environment pollutes merB.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
1 materials and methods
(i) vegetable material.Test plant is tobacco (Nicotiana tabacum).Adopt ordinary method sterilization tobacco seed,, and obtain aseptic seedling, get the acceptor material of the blade of 25～30d seedling age as genetic transformation at MS substratum upper seeding wheel.
(i i) bacterial classification, plasmid, toolenzyme.Bacterial classification comprises bacillus coli DH 5 alpha, Agrobacterium LBA4404 (United States Patent (USP) 5659121 and United States Patent (USP) 5808034), and testing used cloning vector is T-easy (Promega company product), pBluescript KS (+); Plant gene expression vector is pJR1 (United States Patent (USP) 5659121 and a United States Patent (USP) 5808034), and above-mentioned plasmid and bacterium all are that this area is commonly used, disclose in numerous patent documentations or can buy by the commercial channel.Restriction enzyme, modifying enzyme are available from Huamei Bio-Engrg Co..
(iii) to the improvement and design of merB sequence, artificial complete synthesis and vector construction.The merB gene of the transformation of the present invention's design is called as merBhe, and concrete sequence is shown in Fig. 1 and SEQ ID NO:3, and wherein coding region sequence is shown in SEQ ID NO:1.The sequence of design is carried out full gene with ordinary method synthesizes.(SEQ ID NO:3) is cloned in the T-easy carrier with synthetic merBhe sequence, and subclone is in pBluescriptKS (+) plasmid subsequently.The merBhe fragment is inserted in the pJR1 carrier, be built into merBhe expression carrier pJR1::merB.Adopt freeze-thaw method to change agrobacterium tumefaciens lba4404 over to.
(iv) genetic transformation.The conversion of tobacco is carried out with conventional leaf dish method, and method is as follows: the Agrobacterium of incubated overnight is centrifugal, and thalline 10 times of suspensions of MS liquid nutrient medium.Tobacco leaf is cut into the fritter of 0.5cm * 0.5cm, puts into bacterium liquid and soak 10min, blot the substratum that lies in MS+6-BA 3mg/L behind the unnecessary bacterium liquid and cultivate 2d in advance.Be transferred to then on differentiation and the screening culture medium (MS+6-BA 3mg/L+Km 50mg/L+ Pyocianil Cb 500mg/L), cultivate altogether down at 25 ℃., downcut bud and change on the root media (1/2MS+NAA 1mg/L+Cb 300mg/L) and take root when high to 2cm Deng bud is long.Treat to be transplanted to after root grows in the basin alms bowl, be cultured in the greenhouse and bloom.Each transfer-gen plant is labeled as a transgenic line, gathers in the crops seed and numbering respectively by each fruit.
(the v) kalamycin resistance of transgenic seed screening.The seed of gathering in the crops from transformed plant is sowed after surface sterilization at the 1/2MS solid medium that contains 45mg/L kantlex (Km) and is screened, and chooses green transplantation of seedlings behind the 21d in vermiculite and perlitic artificial soil, make its in the controlled environment chamber in growth.
(the vi) mensuration of organic mercury resistance.(PMA) represents organic mercury with Phenylmercuric Acetate.In the MS substratum, add PMA, be mixed with the substratum that different PMA concentration are handled, make its concentration of treatment be respectively 0,0.05,0.10,0.15,0.20,0.25,0.30,0.35 μ mol/L.Get the seed of transformed plant, containing on the MS solid medium of different PMA, cultivating indoor cultivation through sowing after the surface sterilization.The seed that can germinate and grow green true leaf is regarded as the seed of anti-PMA, and the seed that the back lobus cardiacus of can not sprouting or germinate bleaches is considered as not anti-PMA.Whether in addition, add Km and PMA simultaneously in substratum, it is synchronous to the resistance of Km and PMA to observe transfer-gen plant.
For confirm transfer-gen plant in seedling stage the resistance to PMA, in kind has the soil of tobacco transgenic seedling, add PMA, judge its resistance according to the growth of seedlings situation.
(vii) Southern and Northern hybridization.CTAB method with routine is extracted the total DNA of tobacco transformed plant, behind EcoRI digestion 3h, and electrophoresis on 0.7% sepharose.DNA is transferred on the nylon membrane (amersham).With XbaI/SalI double digestion pJR1::merBhe carrier, obtain with
32P-dCTP is the synthesising probing needle of marker, and at 42 ℃ of prehybridization 4h, the prehybridization damping fluid contains 25% methane amide, the salmon sperm DNA of 100 μ g/mL sex change, 10 μ g/mL yeast rnas, 5 * Denhardt ' s reagent, 50mmol/L sodium phosphate (pH 6.5), 5 * SSC and 0.2%SDS.In the prehybridization damping fluid, add probe, incubation 24h under same temperature through sex change.At room temperature use rinsing damping fluid (0.05mol/L NaH
2PO
4, 0.05mol/L Na
2HPO
4, 5 * SSC and 25% methane amide) and the rinsing Hybond membrane, continue rinsing with 2 * SSC/0.02%SDS down at 42 ℃ then.Adding intensifying screen after the filter membrane oven dry exposes to the X ray sheet.From the blade of transgene tobacco and wild-type tobacco, extract total RNA respectively, with
32P-dCTP is that the merBhe gene fragment of mark is that probe carries out Northern hybridization, and hybridization temperature is 58 ℃.
2 results
2.1merB gene order transformation and plant expression vector construction
Increased the content of A and T in the improved merBhe gene (Fig. 1), but do not changed aminoacid sequence, the aminoacid sequence shown in the SEQ ID NO:2 of still encoding.In addition, merBhe ATG code of sequence ATG upstream has also comprised plant translation consensus sequence AGAACCAC, makes it more to meet Eukaryotic sequence signature.
The product cloning that this is complete synthesis obtains the merBhe gene identical with forecasting sequence through order-checking on the T-easy carrier, wherein the coding region is 639bp.Because the content of G+C obviously reduces (reducing to 48.6% by 55.3%) in the synthetic merBhe sequence, therefore, the coding region of the merBhe gene that is obtained meets Eukaryotic sequence signature more.
Cut the pBluescript carrier with the XbaI/SalI enzyme, obtain the encoding sequence of merBhe gene, be inserted in the pJR1 binary vector, be built into pJR1::merBhe plant gene expression vector (Fig. 2) with same restriction enzyme site.With freeze-thaw method this carrier is transformed among the Agrobacterium LBA4404.
2.2 tobacco transforms and the screening of anti-organic mercury
Adopt leaf dish method to transform, the adventitious bud induction culture base is MS+6-BA 3mg/L+Km 50mg/L+Cb 500mg/L, root media is that 1/2MS+NAA 1mg/L+Cb 300mg/L is on the Km screening culture medium, tobacco leaf begins to expand behind the switching 14d, thicken, leaf margin begins to produce gauffer, and the callus of adularescent generates.The differentiation of visible bud behind the 22d.After this, a part of regeneration bud bleaches and eliminates.Cultivate the green regenerating bud to the 2cm after, carry out root culture, root growth stalwartness about 1 month is transplanted in the basin alms bowl in greenhouse.This experiment obtains 120 strain regrowths, transplant survival 86 strains.Each transfer-gen plant is received separately kind, numbering.
Extracting total DNA respectively from the blade of transfer-gen plant, is that probe is hybridized with the dna fragmentation of merBhe gene, the result in 4 DNA samples, have the DNA sample of 3 transfer-gen plants to show hybridization signal (Fig. 3 a).Wherein the merBhe gene is that single copy inserts in two transfer-gen plants of MerBhe-13 and MerBhe-14.From the blade of above-mentioned 2 transgenic lines and wild-type tobacco, extract total RNA respectively, with
32The merBhe gene fragment of P-dCTP mark is that probe carries out Northern hybridization, and the result hybrid belt (Fig. 3 b) all occurred in MerBhe-13 and two transgenic lines of MerBhe-14.Illustrating that the merBhe gene is fastened in this strain has obtained expression.
Table 1 is with the screening situation of different mercury concentration screening T1 for the transgene tobacco seed.
The table 1T1 for the transgene tobacco seed at the seedling rate that contains on the screening culture medium of PMA
A)
A) measure at 35d after planting
Table 1 is the result show, the contrast seed promptly can not be emerged when the concentration of PMA is 0.5 μ mol/L, and the germination of transgenic seed is fine.But some seeds germinated had been died afterwards.Therefore those seedling that grow green true leaf are just calculated seedling.Seedling rate reduces with the concentration increase of PMA, seedling rate was 13% when wherein the MerBhe-13 strain tied up to 2.5 μ mol/L, 3.0 still have 5% seedling rate during μ mol/L, can not emerge when seedling rate was 6%, 3.0 μ mol/L when the MerBhe-14 strain tied up to 2.5 μ mol/L.
Because plant expression vector contains plant selectable marker gene NPTII (neomycin phosphotransferase gene), utilize the substratum screening seed that contains Km, can determine gene transformation rate and transfer-gen plant.Whether synchronous for understanding anti-Km of transfer-gen plant and anti-mercury, the inventor is with containing Km, mercury, 3 kinds of substratum screenings of Km+ mercury transgenic seed.The screening concentration of mercury adopts the lethal concentration of tobacco, changes merBhe gene seed and screens with PMA 0.5 μ mol/L; The screening concentration of Km is 50mg/L.In the kind sub-group of MerBhe-13 strain system, the ratio of anti-Km and anti-mercury is higher, almost accounts for 2/3 (table 2) of the seed of handling; The MerBhe-14 strain is in the colony, and the ratio of anti-Km and anti-mercury is lower, is less than 1/3 of the seed of handling.Contain at the same time on the substratum of Km and PMA, the ratio of two anti-seedling is near the ratio of monoclonal antibody in the MerBhe-13 strain system, and the two anti-ratio of MerBhe-14 strain system is starkly lower than monoclonal antibody, and the not anti-PMA of plant of some anti-Km is described, and the not anti-Km of the plant of some anti-mercury.
Table 2 T1 is for the reaction of merBhe transgene tobacco to Km and PMA
A)
A) measure at 35d after planting.Km+ shows anti-kantlex, and Hg+ shows anti-organic mercury
Theoretically, in the transfer-gen plant body, the integration of T-DNA imports merBhe and NPTII gene simultaneously.When Km resistance and mercury resistance were inconsistent, best explanation was, the merBhe of some transfer-gen plant or NPTII gene do not express or expression level very low.
2.3 the form of transfer-gen plant performance
Fig. 4 shows that on the substratum that contains 0.5 μ mol/L PMA, the tobacco seed of wild-type does not germinate, and the seed germination situation of transgenic line MerBhe-13 and MerBhe-14 is normal substantially, and can normal growth.When PMA concentration was brought up to 1.0 μ mol/L, though transgenic seed can germinate and grow, the stretching, extension of hypocotyl and blade began to occur unusual phenomenon, showed some hypocotyl cripetura and inclination, and blade diminishes and twists.Along with the rising of PMA concentration, the intensity of anomaly of growth of seedling increases.
Through the MerBhe-13 of PMA preliminary screening seedling, transplant in basin alms bowl soil, with the nutritive medium pouring that contains PMA.The result shows that the dead threshold concentration of wild-type tobacco PMA is 0.1 μ mol/L, and under this concentration, seedling is cultivated the 5d rear blade and begins jaundice and dead gradually.Different therewith, the MerBhe-13 seedling is healthy growth under the condition of 0.1 μ mol/LPMA.Along with the increase of PMA concentration, growth begins to be subjected to inhibition in various degree, shows the minimizing of the number of blade, the reduction of plant height, and the flavescence of leaf look, root system diminishes.Wherein during 2.0 μ mol/L, the bottom Ye Huanghua death of plant, but upper leaf still is a yellow-green colour, young leaves is a light green, can keep growth.When PMA concentration is 2.5 μ mol/L, all leaf yellows, but if change over to when yellow in the substratum that does not contain PMA, after 1 week, it is green that young leaves changes, and shows that injury can reverse.3.0 the injury of μ mol/L occurs early, and is also irreversible.Therefore, 2.5 μ mol/L PMA are deadly threshold concentrations of MerBhe-13 transgenic line, compare not genetically modified tobacco seedling, and anti-mercury has improved 25 times.
3 discuss
Phytoremediation technology (phytoremediation) is a kind of green ecological technology that development in recent years is got up, wherein utilizing heavy metal super-enriched plant (hyperaccumulator) to repair is the hot fields of current environment biological study, and it is economic, effective method for governing pollution.At present existing Cd, Co, Cr, Cu, Mn, Ni, Pb, the report that super enriching plants such as Zn and As are found, but plant is difficult for enrichment mercury comparatively speaking, does not also have the report of mercury super enriching plant at present.Therefore, utilize the natural super enriching plant of mercury to come absorbing enriched, thereby the possibility of getting rid of mercury from environment is very little.The present invention is by modification and the transfer of merBhe, create transgenic plant, the result obtains the transgenic lines to the high anti-effect of organic mercury performance, and these transgenic plant can be used to absorb and transforms the organic mercury in the contaminate environment, are the effective ways of mercury contaminated soil improvement.
In the transgenic plant body, merA expression of gene product has the detoxification that absorbs and volatilize ionic mercury, this point has obtained confirmation on transgenic arabidopsis, yellow poplar and tobacco, meanwhile, the merB gene is an inorganic mercury with organomercurial transformation on transgenic arabidopsis.Yet the merB transgenic plant can't make the inorganic mercury volatilization.Make plant finish of the volatilization of the mercury of organic to gaseous mercury, two kinds of selections can be arranged: the one, in this plant, import two genes of merA and merB simultaneously, the 2nd, with merA transfer-gen plant and the hybridization of merB transfer-gen plant, its offspring's genome has this two genes simultaneously.As this hybridization of father and mother, the filial generation that is obtained can be converted into organic mercury gasiform simple substance mercury with the same of expection to people such as Bizily with merA and merB transgenic arabidopsis.Up to the present, most of mercury toxicide researchs concentrate on the transgenic arabidopsis.Because model plant Arabidopis thaliana individuality is very little, can not be used for large-scale phytoremediation.Adopt the method processing soil of phytoremediation and the mercury pollution in the water body, must adopt the bigger plant of biomass.In this respect, tobacco is a kind of more satisfactory plant.
The tobacco plant of wild-type is owing to lack external source merB gene, can't transform PMA effectively, therefore PMA is shown the susceptibility of height, why transgene tobacco shows resistance to PMA, be because it absorbs PMA, and under the effect of merB gene expression product, the PMA (organic mercury) of organic be converted into the mercury of ionic state.Consequently the intravital ionic mercury concentration of plant increases, and plant itself also becomes the super enrichment carrier of mercury ion.In experiment of the present invention, when PMA concentration was 0.5 μ mol/L, emerging and growing of merB transgene tobacco was normal basically, and wild-type tobacco can not germinate, and illustrates that transgene tobacco shows resistance completely to PMA under this concentration; Along with the rising of PMA concentration,, be subjected to increasing inhibition though transfer-gen plant can be grown.This is because the cumulative ionic mercury is on the increase in the transfer-gen plant body, and the hazard rating of plant tissue is strengthened gradually.Because of plant absorbs PMA the concentration of phenyl ring is increased simultaneously.The intravital phenyl ring of plant also might work the mischief to growth above after a certain amount of.But in this type of experiment, the harm of phenyl ring is not tangible.Human methyl mercury and PMA screening merB transgenic arabidopsis such as Bizily, the screening effect that found that both is consistent.
The merBhe transgene tobacco up to 3.0 μ mol/L, can further be converted into the simple substance mercury of low toxicity by merBhe mercury ion that gene action produces to the resistance of PMA, and this process depends on the effect of merA gene.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉transgene tobacco of degraded organic mercury pollution
<130>036598
<160>3
<170>PatentIn version 3.1
<210>1
<211>639
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(639)
＜223〉merB encoding sequence
<400>1
atgaaacttg ctccatatat tttagaatta ttaacttcag ttaatagaac taatggtact 60
gcagatctat tagtacctct acttagagaa ctagctaaag gtagaccagt ttcacgaacg 120
acacttgcct ggattctcga ctggcccgct gagcgagtgg ccgccgtact cgaacaggcc 180
accagtaccg aatatgacaa agatgtgaac atcatcggct acggcctcac cttgcgcgag 240
acttcgtatg tctttgaaat tgacgaccgc cgtctgtatg cctggtgcgc gctggacacc 300
ttgatatttc cggcgctgat cggacgtaca gctcgcgtct catcacattg cgctgcaacc 360
ggagcaccgg tttcactcac ggtttcaccc agcgagatac aggctgtcga acctgccgtc 420
atggcggtgt ccttggtatt gccgcaggaa gcagccgacg ttcgtcagtc cttctgttgc 480
catgtacatt tctttgcatc tgtcccgacg gcggaagact gggcctcaaa gcatcaagga 540
ttggaaggat tagcaattgt aagtgttcat gaagctttag gtttaggtca agaatttaat 600
cgacatctat tacaaacaat gtcatctaga acaccttga 639
<210>2
<211>212
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉organic mercury lyase
<400>2
Met Lys Leu Ala Pro Tyr Ile Leu Glu Leu Leu Thr Ser Val Asn Arg
1 5 10 15
Thr Asn Gly Thr Ala Asp Leu Leu Val Pro Leu Leu Arg Glu Leu Ala
20 25 30
Lys Gly Arg Pro Val Ser Arg Thr Thr Leu Ala Trp Ile Leu Asp Trp
35 40 45
Pro Ala Glu Arg Val Ala Ala Val Leu Glu Gln Ala Thr Ser Thr Glu
50 55 60
Tyr Asp Lys Asp Val Asn Ile Ile Gly Tyr Gly Leu Thr Leu Arg Glu
65 70 75 80
Thr Ser Tyr Val Phe Glu Ile Asp Asp Arg Arg Leu Tyr Ala Trp Cys
85 90 95
Ala Leu Asp Thr Leu Ile Phe Pro Ala Leu Ile Gly Arg Thr Ala Arg
100 105 110
Val Ser Ser His Cys Ala Ala Thr Gly Ala Pro Val Ser Leu Thr Val
115 120 125
Ser Pro Ser Glu Ile Gln Ala Val Glu Pro Ala Val Met Ala Val Ser
130 135 140
Leu Val Leu Pro Gln Glu Ala Ala Asp Val Arg Gln Ser Phe Cys Cys
145 150 155 160
His Val His Phe Phe Ala Ser Val Pro Thr Ala Glu Asp Trp Ala Ser
165 170 175
Lys His Gln Gly Leu Glu Gly Leu Ala Ile Val Ser Val His Glu Ala
180 185 190
Leu Gly Leu Gly Gln Glu Phe Asn Arg His Leu Leu Gln Thr Met Ser
195 200 205
Ser Arg Thr Pro
210
<210>3
<211>658
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉contain the merBhe of upstream and downstream partial sequence
<400>3
ctctagaacc acaatgaaac ttgctccata tattttagaa ttattaactt cagttaatag 60
aactaatggt actgcagatc tattagtacc tctacttaga gaactagcta aaggtagacc 120
agtttcacga acgacacttg cctggattct cgactggccc gctgagcgag tggccgccgt 180
actcgaacag gccaccagta ccgaatatga caaagatgtg aacatcatcg gctacggcct 240
caccttgcgc gagacttcgt atgtctttga aattgacgac cgccgtctgt atgcctggtg 300
cgcgctggac accttgatat ttccggcgct gatcggacgt acagctcgcg tctcatcaca 360
ttgcgctgca accggagcac cggtttcact cacggtttca cccagcgaga tacaggctgt 420
cgaacctgcc gtcatggcgg tgtccttggt attgccgcag gaagcagccg acgttcgtca 480
gtccttctgt tgccatgtac atttctttgc atctgtcccg acggcggaag actgggcctc 540
aaagcatcaa ggattggaag gattagcaat tgtaagtgtt catgaagctt taggtttagg 600
tcaagaattt aatcgacatc tattacaaac aatgtcatct agaacacctt gaagcttc 658
Claims (10)
1. isolating polynucleotide, it is characterized in that, the polypeptide of aminoacid sequence shown in the nucleotide sequence coded SEQID NO:2 of described polynucleotide, and the absolute difference of the degree of the G+C among the degree of the G+C in the described nucleotide sequence and the SEQ ID NO:1 is less than 2%; Or described nucleotides sequence is classified SEQ ID NO:3 as.
2. polynucleotide as claimed in claim 1 is characterized in that, the difference of the degree of the G+C among the degree of the G+C in the described nucleotide sequence and the SEQ ID NO:1 is less than 0.5%.
3. polynucleotide as claimed in claim 1 is characterized in that, the difference of the degree of the G+C among the degree of the G+C in the described nucleotide sequence and the SEQ ID NO:1 is less than 1%.
4. polynucleotide as claimed in claim 1 is characterized in that described nucleotide sequence has the nucleotide sequence shown in SEQID NO:1 or 3.
5. a carrier is characterized in that, it contains the described polynucleotide of claim 1.
6. a host cell is characterized in that, it contains the described carrier of claim 5, perhaps is integrated with the described polynucleotide of claim 1 in the karyomit(e) of described host cell.
7. cell as claimed in claim 6 is characterized in that described cell is a vegetable cell.
8. method that changes the anti-organic mercury resistance of plant is characterized in that it comprises step:
(1) provides the Agrobacterium of carrying expression vector, described expression vector contains the nucleotide sequence of the merB that encodes, the polypeptide of aminoacid sequence shown in the nucleotide sequence coded SEQ ID NO:2, and the absolute difference of the degree of the G+C among the degree of the G+C in the described nucleotide sequence and the SEQ ID NO:1 is less than 2%, or described nucleotides sequence is classified SEQ ID NO:3 as;
(2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (1), thereby make the nucleotide sequence of coding merB change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell or the tissue or the organ of nucleotide sequence that changes coding merB over to;
(4) vegetable cell in the step (3) or tissue or neomorph are become plant.
9. method as claimed in claim 8 is characterized in that described plant is a tobacco.
10. the purposes of an anti-organic mercury resistance plant that obtains with the described method of claim 8 is characterized in that, the organic mercury that it is used to degrade in the soil pollutes.
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| CN100398656C true CN100398656C (en) | 2008-07-02 |
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| CN102604901B (en) * | 2012-03-22 | 2013-06-12 | 中山大学 | Heavy-metal mercury-resistance related protein DbsMerA and encoding genes and application thereof |
| CN103525849B (en) * | 2013-10-24 | 2016-01-20 | 舒海燕 | A kind of recombinant plasmid for removing trade effluent mercury pollution, construction process, recombinant bacterial strain and application |
| CN113873875A (en) * | 2019-01-15 | 2021-12-31 | 华盛顿大学 | Degradation of indoor air pollutants by genetically modified plants |
| CN113943677B (en) * | 2021-11-03 | 2023-04-28 | 华中农业大学 | Helcatylanicum strain for degrading methyl mercury and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5965796A (en) * | 1995-04-21 | 1999-10-12 | University Of Georgia Research Foundation Inc. | Metal resistance sequences and transgenic plants |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5965796A (en) * | 1995-04-21 | 1999-10-12 | University Of Georgia Research Foundation Inc. | Metal resistance sequences and transgenic plants |
Non-Patent Citations (1)
| Title |
|---|
| merB基因的序列修饰及转基因烟草对有机汞的高抗作用. 田吉林，沈瑞娟，何玉科.科学通报，第47卷第23期. 2002 * |
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