CN102746391A - Arsenic-resistance related protein PvArrp1, and coding gene and application thereof - Google Patents
Arsenic-resistance related protein PvArrp1, and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses arsenic-resistance related protein PvArrp1, and a coding gene and application thereof. The protein (PvArrp1 protein) provided in the invention is derived from Pteris vittata L. and is (a) protein composed of an amino acid sequence represented by sequence 1 in a sequence table, or (b) protein derived from sequence 1, wherein the protein is obtained through replacement and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence represented by sequence 1 in the sequence table and is related to resistance of plants to arsenic stress. When the coding gene of the protein provided by the invention is introduced into a plant, accumulation of arsenic in the plant can be substantially reduced, and resistance of the plant to arsenic stress is remarkably enhanced. According to the invention, effective approaches for breeding economic crops or crops with resistance to arsenic and low accumulation of arsenic are provided, and the invention is of a significant value to the field of agriculture.
Description
Technical field
The present invention relates to a kind of arsenic resistance-associated protein PvArrp1 and encoding sox and application.
Background technology
Arsenic is a kind of deleterious chemical element, and its common existence form is arsenate [Arsenate, As (V)] or arsenite [Arsenic, As (III)].
Arsenic contamination at home and abroad all is a very outstanding environmental problem, and China is one of the most serious country of arsenic contamination in the world.Human economic activity can directly cause comparatively serious arsenic contamination, comprise ore smelting, burning of coal, scoriaceously abandon, process hides refuse, DYE PRODUCTION, wood preservation, contain arsenic agricultural chemicals, chemical and biological weapons and Utilization of pesticides etc.Arsenic in the environment all has very big harm to human and plant-animal, can pass through respiratory tract, food or skin exposure and get into human body, and arsenic contamination has caused global a lot of place to poison relevant pandemic with arsenic at present.
Accumulation can accumulate arsenic in the plant of in containing arsenic soil, planting, and the mankind take in arsenic indirectly through the edible part (comprising root, stem, leaf, fruit, seed etc.) of edible these plants, thus harm people's health.In view of the extensive existence of arsenic in environment, and also do not have highly effective method original position to remove the arsenic in soil and the underground water at present, the accumulation that reduces arsenic in the plant materials through the biotechnology means is significant.
Summary of the invention
The purpose of this invention is to provide a kind of arsenic resistance-associated protein PvArrp1 and encoding sox and application.
Protein provided by the invention (PvArrp1 albumen) available from Herba pteridis vittatae (Pteris vittata L.), is (a) or (b) as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
The resistance of (b) aminoacid sequence shown in the sequence in the sequence table 1 being coerced arsenic through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with plant relevant by sequence 1 deutero-protein.
In order to make the protein in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (b) but in the protein synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Proteinic encoding sox in above-mentioned (b) can be through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The gene of encoding said proteins (PvArrp1 gene) also belongs to protection scope of the present invention.
Said gene is (1) or (2) or (3) or (4) described dna molecular as follows:
(1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 1st to 1137 Nucleotide;
(2) dna molecular shown in the sequence 2 in the sequence table;
(3) the dna sequence dna hybridization that under stringent condition, limits and the dna molecular of coded plant arsenic stress resistance GAP-associated protein GAP with (1) or (2);
(4) dna sequence dna that limits with (1) or (2) has the dna molecular of 90% above homology and plant arsenic stress resistance GAP-associated protein GAP at least.
Said stringent condition be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain said gene all belong to protection scope of the present invention.
Available existing expression vector establishment contains the recombinant expression vector of said gene.Said expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for micropellet bombardment.Said expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.Said polyadenylic acid signal can guide polyadenylic acid to join 3 ' end of mRNA precursor.When using said gene constructed recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can use separately or be used in combination with other promotor; In addition; When using gene constructed recombinant expression vector of the present invention; Also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc.; But must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of identifying and screening, can process used expression vector, can produce enzyme or the gene of luminophor, antibiotic marker thing or the anti-chemical reagent marker gene etc. of colour-change as adding to encode with resistance.Also can not add any selected marker, directly according to phenotypic screen.
Said recombinant expression vector specifically can be said gene is imported the recombinant plasmid that the MCS of pSN1301 plasmid obtains.
The present invention also protects a kind of method of cultivating transgenic plant, is said gene is imported in the purpose plant, obtains the resistance that arsenic is coerced is higher than the transgenic plant of said purpose plant.Said gene specifically can import in the said purpose biology through said recombinant expression vector.Carry the recombinant expression vector of said gene can be through using conventional biological methods such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity led, agriculture bacillus mediated, particle gun transform the cell or tissue of said biology.
Said purpose plant can be monocotyledons or dicotyledons.Said dicotyledons can be Arabidopis thaliana, like the environmental Arabidopis thaliana of Colombia.
Said arsenic specifically can arsenate or the form of arsenite exist.Said arsenite specifically can be Sodium metaarsenite.Said arsenate specifically can be sodium arseniate.
The present invention also protects said albumen or the application of said gene in the resistance that the regulation and control plant is coerced arsenic.
With PvArrpl gene transfered plant provided by the invention; Can significantly reduce the accumulation of arsenic in the plant materials; And remarkable enhancement of plant resistance that arsenic is coerced, the present invention provides valid approach for cultivating anti-arsenic and low arsenic accumulation cash crop or farm crop, has great value for agriculture field.
Description of drawings
Fig. 1 is on plant the moves to new 1/2GM solid medium and cultivate the phenotype after 7 days.
Fig. 2 contains on the 1/2GM solid medium of 10 μ M Sodium metaarsenites (AsIII) and cultivates the phenotype after 7 days for plant moves to.
Fig. 3 contains the 1/2GM solid medium of 150 μ M sodium arseniates (AsV) and cultivates the phenotype after 7 days for plant moves to.
Fig. 4 coerces root elongation quantitative statistics down for plant at 10 μ M AsIII.
Fig. 5 is the statistics of plant fresh weight under 10 μ M AsIII coerce.
Fig. 6 coerces root elongation quantitative statistics down for plant at 300 μ M AsV.
Fig. 7 is the statistics of plant fresh weight under 300 μ M AsV coerce.
Fig. 8 is that plant is handled 7d back root part total arsenic content mensuration at 5 μ M AsIII.
Fig. 9 is that plant over-ground part total arsenic content after 5 μ M AsIII handle 7d is measured.
Figure 10 is that plant is handled 7d back root part total arsenic content mensuration at 10 μ M AsV.
Figure 11 is that plant over-ground part total arsenic content after 10 μ M AsV handle 7d is measured.
Figure 12 is the Subcellular Localization of PvArrpl-GFP fusion rotein at the Arabidopis thaliana root.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
Herba pteridis vittatae (Pteris vittata L.): the public can obtain from Chinese Academy of Sciences's plant research; Reference: Yang XX; Chen H; Dai XJ; Xu WZ, He ZY, Ma be of vacuolar compartmentalization of arsenic in the hyperaccumulator Pteris vittata.Chinese Science Bulletin 54 (22) M.2009.Evidence: 4229-4233..
The pSN1301 plasmid: the public can obtain from Chinese Academy of Sciences's plant research; Reference: Guo J; Dai X; Xu W, Ma be GSH1 and AsPCS1 simultaneously increases the tolerance and accumulation of cadmium and arsenic in Arabidopsis thaliana.Chemosphere 72 (7) M.2008.Overexpressing: 1020-1026..
Agrobacterium strains c58: the public can obtain from Chinese Academy of Sciences's plant research; Reference: Lv S; Wang H; Yu B; Meng W, Cao be of Arabidopsis HIS1-3 gene and construction of its expression vector.Journal of Hefei University of Technology.Natural Science 35 (1) S.2012.Cloning: 120-123..
The environmental Arabidopis thaliana (Col) of Colombia: available from Arabidopsis Biological Resource Center (ABRC).
The 1/2GM solid medium: on the basis of 1/2MS liquid nutrient medium, every liter is added 10g sucrose, 0.5gMES and 8g agar; PH5.9.
1/2GM solid sugar-free culture-medium: on the basis of 1/2MS liquid nutrient medium, every liter is added 0.5gMES and 8g agar; PH5.9.
The invention of embodiment 1, PvArrp1 albumen and encoding sox thereof
Herba pteridis vittatae (Pteris vittataL.) belongs to pteridophyte Pteridaceae Pteris.
The contriver from Herba pteridis vittatae from having found an albumen relevant, with its called after PvArrp1 albumen with the arsenic resistance.The proteic aminoacid sequence of PvArrp1 is shown in the sequence 1 of sequence table.The proteic unnamed gene of PvArrp1 of will encoding is the PvArrpl gene, and its ORFs is shown in the sequence 2 of sequence table.
Acquisition of embodiment 2, transgenic plant and evaluation
One, the structure of recombinant expression vector
1, extracts total RNA of Herba pteridis vittatae blade.
Total RNA reverse transcription of 2, extracting with step 1 is cDNA.
3, the cDNA that obtains with step 2 is a template, and the primer of forming with PvArrplS and PvArrp1A obtains pcr amplification product to carrying out pcr amplification.
PvArrp1S：5’-ATGGAGAACTCAAGCGCGGAGCGGA-3’；
PvArrp1A：5’-CTAAACAGAAGGCCCCTTCCTCTGA-3’。
Pcr amplification program: 98 ℃ of 10s, 55 ℃ of 30s, 72 ℃ of 70s, 35 circulations; 72 ℃ of 10min; 10 ℃ of preservations.
4, the pcr amplification product with step 3 is a template, and the primer that adopts PvArrplS1/PvArrp1A1 to form is right, obtains pcr amplification product.
PvArrp1S1：5’-
ggatccATGGAGAACTCAAGCGCGGAGCGGA-3’；
PvArrp1A1：5’-
ggtaccCTAAACAGAAGGCCCCTTCCTCTGA-3’。
The sequence of square frame mark is the sequence with pSN1301 plasmid generation homologous recombination; Be respectively to the homology arm of the partial sequence at the BamHI restriction enzyme site upper reaches of pSN1301 plasmid with to the homology arm of the partial sequence in the KpnI restriction enzyme site downstream of pSN1301 plasmid; So pcr amplification product can with pSN1301 plasmid generation homologous recombination, replace the BamHI of pSN1301 plasmid and the small segment between the KpnI restriction enzyme site with the sequence between BamHI and the KpnI restriction enzyme site
Pcr amplification Program Synchronization rapid 3.
5,, reclaim skeleton carrier (about 13kb) with restriction enzyme BamHI and KpnI double digestion pSN1301 plasmid.
6, the carrier framework of the pcr amplification product of step 4 and step 5 is recombinated be connected (adopting CloneEZ recombinant clone test kit and by specification to carry out), obtain recombinant plasmid pSN1301-PvArrp1.According to sequencing result, it is following that recombinant plasmid pSN1301-PvArrpl is carried out structrual description: the small segment between pSN1301 plasmid BamHI and the KpnI restriction enzyme site is replaced for the PvArrpl gene shown in the sequence 2 of sequence table.
7, the pcr amplification product with step 3 is a template, and the primer that adopts the PvArrplS2/PvArrp1A2 composition obtains pcr amplification product to carrying out pcr amplification.
PvArrp1S2：5’-
ctcgagATGGAGAACTCAAGCGCGGAGCGGA-3’；
PvArrp1A2：5’-
ggtaccAACAGAAGGCCCCTTCCTCTGAAAG-3’。
The sequence of square frame mark is the sequence with pGFP121 plasmid generation homologous recombination; Be respectively to the homology arm of the partial sequence at the XhoI restriction enzyme site upper reaches of pGFP121 plasmid with to the homology arm of the partial sequence in the KpnI restriction enzyme site downstream of pGFP121 plasmid; So pcr amplification product can with pGFP121 plasmid generation homologous recombination, replace the XhoI of pGFP121 plasmid and the small segment between the KpnI restriction enzyme site with the sequence between XhoI and the KpnI restriction enzyme site.
Pcr amplification Program Synchronization rapid 3.
8,, reclaim skeleton carrier (about 13kb) with restriction enzyme XhoI and KpnI double digestion pGFP121 plasmid (gus gene of pBI121 plasmid is replaced with the recombinant plasmid that the GFP gene obtains).
9, the carrier framework of the pcr amplification product of step 7 and step 8 is recombinated be connected, obtain recombinant plasmid pGFP121-PvArrp1.According to sequencing result, it is following that recombinant plasmid pGFP121-PvArrpl is carried out structrual description: the small segment between pGFP121 plasmid XhoI and the KpnI restriction enzyme site is replaced sequence 2 for sequence table from the PvArrpl gene shown in 5 ' the terminal 1-1137 position Nucleotide.
Two, the acquisition of transgenic plant
1, recombinant plasmid pSN1301-PvArrpl is imported agrobacterium strains c58, obtain the Agrobacterium of recombinating.
2, through flower infusion method (Clough; S.J.; And Bent; A.F. (1998) .Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J 16, and the Agrobacterium of 735-743.) will recombinating imports the environmental Arabidopis thaliana of Colombia, obtains T1 for seed.
3, T1 is transplanted to resistant plant in the soil for seed results back screening resistant plant in 1/2GM solid sugar-free culture-medium (containing the 20mg/L Totomycin), and results T2 cultivates for seed and with it and is plant (T2 is for plant).
Get respectively T1 for plant and T2 for plant, carry out GUS dyeing earlier and identify.The method that GUS dyeing is identified: when treating that plant grows to 6-8 sheet true leaf, the clip blade carries out GUS dyeing, presents the blue corresponding plant of blade and carries out Molecular Identification.The method of Molecular Identification: total RNA and the reverse transcription of carrying plant leaf are cDNA; Primer with PvArrp1S and PvArrp1A form is identified (target sequence is the band of about 1.1kb) to the primer of forming to the cDNA from each sample being carried out PCR, it is transfer-gen plant that PCR is accredited as the male plant.For plant, if its T2 is accredited as the positive for the equal PCR of plant, then this plant is the transfer-gen plant that isozygotys for a certain T1, and this plant and offspring thereof are 1 transgenic line that isozygotys.
T2 produces T3 for seed for the transfer-gen plant selfing.
The T3 of two transfer-gen plant strain systems of picked at random (L9 strain system and L11 strain system) carries out the evaluation of step 4 for seed.
Three, change the acquisition of empty carrier adjoining tree
Replace recombinant plasmid pSN1301-PvArrp1 to carry out the operation of step 2 with the pSN1301 plasmid, obtain changeing the empty carrier adjoining tree.
Four, each Performance Testing of transgenic plant and adjoining tree
The T3 of L9 strain system is respectively as follows identified (normal illumination cultivate be illumination 16 hour, dark 8 hour) for seed, the T3 that changes the empty carrier adjoining tree for the environmental Arabidopis thaliana of seed and Colombia for the T3 of seed, L11 strain system:
1, phenotypic evaluation
First group: point after the seed disinfection is sowed on the 1/2GM solid medium, keeps flat lucifuge and place 4 ℃ of 2d, move to 22 ℃ of normal illuminations cultivation 3d in the incubator then, seedling is moved on the new 1/2GM solid medium cultivate 7d then.
Second group: point after the seed disinfection is sowed on the 1/2GM solid medium; Keep flat lucifuge and place 4 ℃ of 2d; Move to 22 ℃ of normal illuminations cultivation 3d in the incubator then, seedling is moved on the 1/2GM solid medium that contains 10 μ M Sodium metaarsenites (AsIII) cultivate 7d then.
The 3rd group: point after the seed disinfection is sowed on the 1/2GM solid medium, keeps flat lucifuge and place 4 ℃ of 2d, move to 22 ℃ of normal illuminations cultivation 3d in the incubator then, seedling is moved on the 1/2GM solid medium that contains 150 μ M sodium arseniates (AsV) cultivate 7d then.
First group of plant moves on the new 1/2GM solid medium and the photo of cultivating after 7 days is seen Fig. 1.Second group of plant moves on the 1/2GM solid medium that contains 10 μ M Sodium metaarsenites (AsIII) and the photo of cultivating after 7 days is seen Fig. 2.The 3rd group of plant moves to the 1/2GM solid medium that contains 150 μ M sodium arseniates (AsV) and the photo cultivated after 7 days is seen Fig. 3.During each group was handled, the growing state that changes the empty carrier adjoining tree was all consistent with the growing state of Colombia ecotype Arabidopis thaliana.The result shows that the growing state of transfer-gen plant in the environment that contains AsIII or AsV significantly is superior to the environmental Arabidopis thaliana of Colombia.Heterogenous expression PvArrp1 gene does not influence the Arabidopis thaliana growth under the normal growth condition, but under As II I and AsV treatment condition, can significantly strengthen the arsenic resistance of Arabidopis thaliana, alleviates arsenic and handles the inhibition to the Arabidopis thaliana growth.
2, the evaluation of root elongation and fresh weight
First group of (AsIII 0 μ M; AsV 0 μ M): point after the seed disinfection is sowed on the 1/2GM solid medium; Keep flat lucifuge and place 4 ℃ of 2d; Move in the incubator 22 ℃ of normal illuminations then and cultivate 3d, measure long (L1) continued of root of seedling and cultivate 7d, measure the root long (L2) of seedling once more and weigh.
Second group (AsIII10 μ M): point after the seed disinfection is sowed on the 1/2GM solid medium; Keep flat lucifuge and place 4 ℃ of 2d; Move to 22 ℃ of normal illuminations cultivation 3d in the incubator then; Measure behind the root long (L1) of seedling seedling moved on the 1/2GM solid medium that contains 10 μ M Sodium metaarsenites (AsIII) and cultivate 7d, measure the root long (L2) of seedling once more and weigh.
The 3rd group (AsV 300 μ M): point after the seed disinfection is sowed on the 1/2GM solid medium; Keep flat lucifuge and place 4 ℃ of 2d; Move to 22 ℃ of normal illuminations cultivation 3d in the incubator then; Measure behind the root long (L1) of seedling seedling moved on the 1/2GM solid medium that contains 300 μ M sodium arseniates (AsV) and cultivate 7d, measure the root long (L2) of seedling once more and weigh.
Root elongation (mm)=L2-L1; Be meant the increasing amount of main root length in the cultivation of 7d afterwards of transplanting seedlings.
Each strain is that every group of experiment got 6 seeds and carried out above-mentioned steps, carries out revision test three times, results averaged.
The root elongation of first group of plant and second group of plant is seen Fig. 4, and fresh weight is seen Fig. 5.The root elongation of first group of plant and the 3rd group of plant is seen Fig. 6, and fresh weight is seen Fig. 7.During each group was handled, the root growth amount of changeing the empty carrier adjoining tree and fresh weight were all consistent with Colombia ecotype Arabidopis thaliana.Compare with Colombia environmental Arabidopis thaliana, under 10 μ M AsIII conditions, cultivated 7 days, the root elongation increase of L9 and L11 is above 10 times; Fresh weight has increased about 1 times; Under 300 μ MAsV conditions, the root elongation of L9 and L11 increases above 2 times, and fresh weight has increased about 1 times.The result shows that the growing state of transfer-gen plant in the environment that contains AsIII or AsV significantly is superior to the environmental Arabidopis thaliana of Colombia.
3, the evaluation of plant arsenic content
First group: point after the seed disinfection is sowed on the 1/2GM solid medium; Keep flat lucifuge and place 4 ℃ of 2d; Move to 22 ℃ of normal illuminations cultivation 3d in the incubator then, seedling is moved on the 1/2GM solid medium that contains 5 μ M Sodium metaarsenites (AsIII) cultivate 7d then.
Second group: point after the seed disinfection is sowed on the 1/2GM solid medium, keeps flat lucifuge and place 4 ℃ of 2d, move to 22 ℃ of normal illuminations cultivation 3d in the incubator then, seedling is moved on the 1/2GM solid medium that contains 10 μ M sodium arseniates (AsV) cultivate 7d then.
Each strain system gets 50 seeds and carries out above-mentioned steps, carries out revision test three times, results averaged.
The plant of cultivating after 7 days is taken off from substratum, and each plant carries out following operation respectively: respectively over-ground part and underground part (root) are put into baking oven, 80 ℃ of baking 6h, title dry weight after giving a baby a bath on the third day after its birth time with ultrapure water.The material of claiming put into to disappear boil pipe and add mixing acid [nitric acid: the vitriol oil: perchloric acid=4:1:0.5 (volume ratio); It is pure to be top grade] 1ml; Room temperature is placed cold disappearing and is boiled and spend the night (add acid back room temperature leave standstill be called cold disappearing boil), and 250 ℃ disappear and boil 4h then, are settled to 20ml with zero(ppm) water; (Thermo Electron corporation USA) measures arsenic content with inductive coupling plasma emission spectrograph iCAP6300 behind the filter paper filtering.
The root total arsenic content of first group of plant is seen Fig. 8, and the over-ground part total arsenic content is seen Fig. 9.The root total arsenic content of second group of plant is seen Figure 10, and the over-ground part total arsenic content is seen Figure 11.During each group was handled, the root total arsenic content that changes the empty carrier adjoining tree and over-ground part total arsenic content were all consistent with Colombia ecotype Arabidopis thaliana.Compare with Colombia environmental Arabidopis thaliana; Under 5 μ M AsIII conditions, cultivated 7 days; L9 and L11 root total arsenic content have reduced by 92% and 91% respectively, and the over-ground part total arsenic content has reduced by 63% and 68% respectively, under 10 μ M AsV conditions, cultivate 7 days; L9 and L11 root total arsenic content have reduced by 62% and 61% respectively, and the over-ground part total arsenic content has reduced by 18% and 24% respectively.The result shows that the total arsenic content of transfer-gen plant in the environment that contains AsIII or AsV significantly is lower than the environmental Arabidopis thaliana of Colombia.
Five, the proteic location of PvArrp1
Replace recombinant plasmid pSN1301-PvArrp1 to carry out the operation of step 2 with recombinant plasmid pGFP121-PvArrp1, obtain transgenic plant.The two-photon fluorescence observed result of the shoot root portion of transgenic plant is seen Figure 12, and the result shows that GFP fluorescence is positioned on the cytolemma.
Claims (9)
1. protein is (a) or (b) as follows:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
The resistance of (b) aminoacid sequence shown in the sequence in the sequence table 1 being coerced arsenic through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with plant relevant by sequence 1 deutero-protein.
2. coding claim 1 said proteic gene.
3. gene as claimed in claim 2 is characterized in that: said gene is (1) or (2) or (3) or (4) described dna molecular as follows:
(1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 1st to 1137 Nucleotide;
(2) dna molecular shown in the sequence 2 in the sequence table;
(3) the dna sequence dna hybridization that under stringent condition, limits and the dna molecular of coded plant arsenic stress resistance GAP-associated protein GAP with (1) or (2);
(4) dna sequence dna that limits with (1) or (2) has the dna molecular of 90% above homology and plant arsenic stress resistance GAP-associated protein GAP at least.
4. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 said genes.
5. recombinant expression vector as claimed in claim 4 is characterized in that: said recombinant expression vector is that said gene is imported the recombinant plasmid that the MCS of pSN1301 plasmid obtains.
6. a method of cultivating transgenic plant is that claim 2 or 3 said genes are imported in the purpose plant, obtains the resistance that arsenic is coerced is higher than the transgenic plant of said purpose plant.
7. method as claimed in claim 6 is characterized in that: claim 2 or 3 said genes import in the said purpose plant through the said recombinant vectors of claim 5.
8. like claim 6 or 7 described methods, it is characterized in that: said purpose plant is dicotyledons or monocotyledons; Said dicotyledons is specially Arabidopis thaliana.
9. said albumen of claim 1, or claim 2 or 3 said genes, the application in the resistance that the regulation and control plant is coerced arsenic.
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| CN106916213A (en) * | 2015-12-25 | 2017-07-04 | 中国农业大学 | A kind of albumin A sT and its encoding gene and the application in plant stress tolerance |
| CN109295016A (en) * | 2017-07-24 | 2019-02-01 | 清华大学 | Protein relevant to enduring high-concentration methanol |
| CN109295016B (en) * | 2017-07-24 | 2021-11-02 | 清华大学 | Proteins associated with tolerance to high concentrations of methanol |
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