CN101818168A - Application of AtNRT1.8 gene to strengthening resistance of crops to stress of heavy metals or salts - Google Patents
Application of AtNRT1.8 gene to strengthening resistance of crops to stress of heavy metals or salts Download PDFInfo
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Abstract
The invention relates to application of an AtNRT1.8 gene to strengthening the resistance of crops to the stress of heavy metals or salts, in particular to application of an AtNRT1.8 gene or encoded protein or polypeptide thereof to strengthening the resistance of plants to the stress of heavy metals, salts or draught. The invention also provides a genetic engineering method for strengthening the resistance of plants to the stress of heavy metals, salts or draught by utilizing the AtNRT1.8 gene, a vector and a host cell containing the AtNRT1.8 gene, a method for preparing transgenosis plants and the transgenosis plants. The method can strength the resistance of the plants to the stress of the heavy metals, the salts and the draught and has active effects for improving the yield and properties of crops and wide application potential.
Description
Technical field
The present invention relates to plant biological engineering and plant improvement genetically engineered field.Specifically, the present invention relates to the AtNRT1.8 gene and strengthening crop, and improve method and the transgenic plant of plant the resistance of heavy metal, salt or drought stress by changing described gene over to the purposes in the resistance of heavy metal, salt or drought stress.
Background technology
Heavy metal cadmium causes the major reason of murder by poisoning to be significantly to suppress important nitrogen assimilation process to plant, causes the plant materials nitrogen stress, the degraded of body internal protein, and therefore a direct result for crop is exactly the serious underproduction.In addition, heavy metal such as the accumulation in the rice, also can cause the decline of security quality at crop edible position, causes great safety hazard for the human or animal who eats these contaminated food.Wherein famous public hazard incident is exactly " itai-itai " incident that occurs in Japan last century, and reason causes bone soft frangible with regard to the rice that is the edible for a long time cadmium pollution of resident.
Since the reform and opening-up, the industry of China is developed rapidly, but environmental practice relatively lags behind, and caused comprising the serious environmental pollution of heavy metal.According to the incomplete statistics before 10 years, China is subjected to nearly 2,000 ten thousand hectares of the cultivated area of heavy metal contamination, accounts for 1/5 of total area under cultivation, is subjected to the grain of heavy metal contamination to reach 1,200 ten thousand tons, and since the grain drop in production that heavy metal contamination causes also on 1,000 ten thousand tons.The monitoring of State Environmental Protection Administration shows that heavy metal contamination has the trend (http://www.sepa.gov.cn/natu/yjsp/) of continuous deterioration in China.
Therefore, separate and illustrate the key gene of regulation and control plant heavy metal resistance, and cultivate the heavy metal resistance crop, will on producing, produce huge economic and social benefit by transgenic technology.
The another one serious problems that China's agricultural faces are owing to long-term irrigate the salinification that causes and coerce, how to improve crop to the resistance capacity of salt for improving output, the grain security that solves China also is significant.
AtNRT1.8 gene (AGI numbering: At4g21680) belong to the NRT1 gene family, be also referred to as the POT/PTR gene family, in the model plant Arabidopis thaliana, have 53 family members and (see the summary of Tsay YF, FEBSLetters, 2007,581:2290-2300), the low-affinity nitrate radical translocator of mainly encoding also finds to have the part member oligopeptides translocator of encoding.AtNRT1.8 gene order and structure thereof are as shown in Figure 1.
Yet, the research or the report that concern between any AtNRT1.8 of relating to gene and plant (especially crop) resistance to heavy metal, salt or drought stress are not arranged in this area as yet.
Summary of the invention
Clear and definite just AtNRT1.8 gene of purpose of the present invention and crop be to the relation between the resistance of heavy metal, salt or drought stress, provides a kind of new approach for strengthening crop to the resistance of heavy metal, salt or drought stress.Another object of the present invention provide have the enhanced heavy metal, preparation method and the transgenic plant of the transgenic plant of salt or drought stress resistance.
In a first aspect of the present invention, provide AtNRT1.8 gene or its encoded protein matter or polypeptide strengthening plant to the resistance of heavy metal, to the resistance of salt stress or to the purposes in the resistance of arid.
In a preference, described AtNRT1.8 gene is an Arabidopis thaliana AtNRT1.8 gene.
In another preference, described plant is dicotyledons or monocotyledons, preferred crop.
In another preference, described plant is selected from: grass, Malvaceae cotton, Cruciferae Brassica plants, feverfew, plant of Solanaceae, labiate or samphire, preferred Arabidopis thaliana, cotton, rape, paddy rice, corn, wheat, barley or Chinese sorghum, more preferably paddy rice, corn, wheat, barley, Arabidopis thaliana or cotton most preferably are paddy rice, corn, wheat, Arabidopis thaliana or cotton.
In an embodiment of the invention, the sequence of described protein or polypeptide is selected from:
(a) SEQ ID NO:3; Or
(b) in the aminoacid sequence that (a) limits, strengthen plant active to the resistance of heavy metal or salt stress by (a) deutero-protein or polypeptide through replacing, lack or adding one or several amino acid and have.
In yet another embodiment of the present invention, described AtNRT1.8 gene is the sequence of coding protein of the present invention or polypeptide.
In yet another embodiment of the present invention, described AtNRT1.8 gene is selected from:
(i) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5; Or
(ii) under stringent condition with the sequence hybridization that (ii) limits and have and strengthen the active molecule of plant to the resistance of heavy metal or salt stress.
In yet another embodiment of the present invention, described enhancing plant shows as the resistance of heavy metal: reduce the heavy metal content in the plant.
In a preference, described enhancing plant shows as the resistance of heavy metal: do not have heavy metal contamination, heavy metal contamination to reduce than the non-transgenic plant of the following plantation of the same terms.
In yet another embodiment of the present invention, described heavy metal, salt stress or arid comprise by coercing that following material or situation cause: cadmium, lead, mercury, arsenic, copper, chromium, antimony, tin, zinc, barium, bismuth, nickel, cobalt, manganese, iron, vanadium, sodium-chlor, sodium sulfate, yellow soda ash, sodium bicarbonate or lack of water.
In a preference, described heavy metal stress or salt stress are preferably: cadmium is coerced, lead is coerced, mercury is coerced, arsenic is coerced, sodium-chlor is coerced, sodium sulfate is coerced, and yellow soda ash is coerced, and sodium bicarbonate is coerced.
In a preference, described reduction plant heavy metal content is preferably the content that reduces cadmium, lead, mercury, arsenic.
In a second aspect of the present invention, a kind of carrier is provided, described carrier contains the AtNRT1.8 gene.
In a preference, contain Arabidopis thaliana AtNRT1.8 gene in the described carrier.
In a preference, the sequence of described AtNRT1.8 gene is selected from: (i) SEQ ID NO:1 (corresponding to the sequence among Fig. 1), SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5; Or
(ii) under stringent condition with the sequence hybridization that (i) limits and have and strengthen the active molecule of plant to the resistance of heavy metal or salt stress.
In a preference, described carrier is selected from: bacterial plasmid, phage, yeast plasmid, vegetable cell virus or mammalian cell virus, preferred pEGFP-1, pBI121, pCAMBIA1300, pCAMBIA1301, pCAMBIA2301 or pHB.
In a third aspect of the present invention, a kind of genetically engineered host cell is provided, described host cell contains carrier of the present invention.
In a preference, described host cell is selected from prokaryotic cell prokaryocyte, eukaryotic cell or higher eucaryotic cells such as low, preferred bacterium cell, yeast cell or vegetable cell, more preferably intestinal bacteria, streptomycete, Agrobacterium, yeast, Agrobacterium most preferably, described Agrobacterium includes but not limited to: EHA105, SOUP1301 or C58.
In a fourth aspect of the present invention, a kind of method for preparing transgenic plant is provided, described method comprises:
(1) with the construction transformed plant cells cell, tissue or the organ that contain the AtNRT1.8 gene;
(2) selection changes vegetable cell, tissue or the organ of AtNRT1.8 gene over to; With
(3) vegetable cell, tissue or neomorph in the step (2) are become plant,
Wherein, the transgenic plant of gained strengthen to some extent to the more unconverted plant of the resistance of heavy metal or salt stress.
In a preference, described construction is carrier or the host cell that contains the AtNRT1.8 gene.
In another preference, integrated the AtNRT1.8 gene on the karyomit(e) in vegetable cell, tissue or the organ of step (2).
In another preference, described method comprises following concrete steps:
(a) provide the carrier that contains the AtNRT1.8 gene;
(b) provide the host cell that carries the carrier in the step (a);
(c) vegetable cell or tissue are contacted with host cell in the step (b), thereby make AtNRT1.8 gene transferred plant cell, and be incorporated on the karyomit(e) of vegetable cell;
(d) selection changes vegetable cell, tissue or the organ of AtNRT1.8 gene over to; With
(e) vegetable cell, tissue or neomorph in the step (d) are become plant,
Wherein said transgenic plant strengthen to some extent to the more unconverted plant of the resistance of heavy metal or salt stress.
In another preference, described AtNRT1.8 gene is an Arabidopis thaliana AtNRT1.8 gene.
In another preference, described plant is selected from: grass, Malvaceae cotton, Cruciferae Brassica plants, feverfew, plant of Solanaceae, labiate or samphire, preferred Arabidopis thaliana, cotton, rape, paddy rice, wheat, barley, corn or Chinese sorghum, more preferably paddy rice, corn, wheat, barley, Arabidopis thaliana or cotton most preferably are paddy rice, corn, wheat, Arabidopis thaliana or cotton.
In a preference, the described method for preparing transgenic plant, also comprise transgenic plant and non-transgenic plant or the hybridization of other transgenic plant that to obtain with aforementioned any method of the present invention, thereby obtain to comprise the filial generation of AtNRT1.8 gene, described filial generation strengthens to some extent to the more unconverted plant of the resistance of heavy metal or salt stress.
In a preference, the plant of hybridization usefulness belongs to same section or not equal plant in classification, be preferably same section plant.Described plant optimization is grass, Malvaceae cotton, Cruciferae Brassica plants, feverfew, plant of Solanaceae, labiate or samphire, preferred Arabidopis thaliana, cotton, rape, paddy rice, wheat, barley, corn or Chinese sorghum, more preferably paddy rice, corn, wheat, barley, Arabidopis thaliana or cotton most preferably are paddy rice, corn, wheat, Arabidopis thaliana or cotton.
In another preference, described filial generation has stable inherited character.
In a fifth aspect of the present invention, the purposes of the transgenic plant that make with the method for the invention is provided, and described transgenic plant are used for producing: the agricultural-food, heavy metal contamination that do not have heavy metal contamination are coerced crop or are drought-resistantly coerced crop than agricultural-food, anti-heavy metal stress crop, the salt tolerant of the non-transgenic plant reduction of the following plantation of the same terms.
In a preference, described heavy metal, salt or drought stress comprise by coercing that following material or situation cause: cadmium, lead, mercury, arsenic, copper, chromium, antimony, tin, zinc, barium, bismuth, nickel, cobalt, manganese, iron, vanadium, sodium-chlor, sodium sulfate, yellow soda ash, sodium bicarbonate or lack of water.
In another preference, described heavy metal stress or salt stress are preferably: cadmium is coerced, lead is coerced, mercury is coerced, arsenic is coerced, sodium-chlor is coerced, sodium sulfate is coerced, yellow soda ash is coerced or sodium bicarbonate is coerced.
In another preference, described crop is Arabidopis thaliana, cotton, rape, paddy rice, wheat, barley, corn or Chinese sorghum, more preferably paddy rice, corn, wheat, barley, Arabidopis thaliana or cotton.
In a sixth aspect of the present invention, a kind of transgenic plant are provided, it comprises the AtNRT1.8 gene.
In a preference, described transgenic plant strengthen to some extent to the resistance of heavy metal, salt or drought stress.
In another preference, the sequence of described AtNRT1.8 gene is selected from coding and is selected from down the protein of group or the sequence of polypeptide: (a) SEQ ID NO:3; Or (b) in the aminoacid sequence that (a) limits, strengthen plant active to the resistance of heavy metal or salt stress by (a) deutero-protein or polypeptide through replacing, lack or adding one or several amino acid and have.
In a preference, described AtNRT1.8 gene is selected from: (i) SEQ ID NO:1 (corresponding to the sequence among Fig. 1), SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5; Or (ii) under stringent condition with the sequence hybridization that (i) limits and have and strengthen the active molecule of plant to the resistance of heavy metal or salt stress.
In another preference, described plant is dicotyledons or monocotyledons, preferred crop.
In another preference, described plant is selected from: grass, Malvaceae cotton, Cruciferae Brassica plants, feverfew, plant of Solanaceae, labiate or samphire, preferred Arabidopis thaliana, cotton, rape, paddy rice, wheat, barley, corn or Chinese sorghum, more preferably paddy rice, corn, wheat, barley, Arabidopis thaliana or cotton most preferably are paddy rice, corn, wheat, Arabidopis thaliana or cotton.
It should be understood that others of the present invention because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1: AtNRT1.8 gene order and structure thereof.Wherein, the capitalization part is the exon of gene, and small letter partly is the intron between the exon.
Fig. 2: the expression of AtNRT1.8 gene in adverse circumstance (stresses).Wherein, Fig. 2 A: root is carried out AtNRT1.8 expression level after different time is handled with different concns Cd; Fig. 2 B, C and D are for coercing processing after 6 hours, the expression of AtNRT1.8 with various.Wherein, the expression of " S " among Fig. 2 B stem; " L " represents leaf; " R " root.The acid-treated sample of " JA " expression jasmine among Fig. 2 C; " SA " represents Whitfield's ointment; " Na " represents NaCl; The sample that " Cu " expression is handled with copper; The sample that " Zn " expression is handled with zinc; " Ctl " represents contrast." ACC " expression 1-amino-cyclopropane yl carboxylic acid among Fig. 2 D, it is the precursor that ethene generates, handling plant and handle plant with ethene ETH with ACC has similar effects; " R " represents root, and " L " represents leaf.
Fig. 3: the electric Physiological Analysis result of xenopus leavis oocytes injection research AtNRT1.8 gene in vitro transport activity.Wherein, " noinj " expression is without the sample of injection.
Fig. 4: expression and the location of AtNRT1.8 gene in Arabidopis thaliana.Wherein: Fig. 4 A-4E is the acquisition of directly taking pictures or cut into slices by GUS dyeing; Fig. 4 F-4M transforms onion epidermis cell and use agrobacterium mediation method arabidopsis thaliana transformation wild-type WS, the acquisition of taking pictures then under Laser Scanning Confocal Microscope by particle gun.
Fig. 5: regulation and control (the 50uM Cd+50mM NO of AtNRT1.8 gene pairs plant Cd resistance
3 -).
Fig. 6: regulation and control (the 50mM NaCl+50mM NO of AtNRT1.8 gene pairs plant NaCl resistance
3 -).
Fig. 7: Cd is lotus throne leaf and the accumulation of fruit in the pod in AtNRT1.8 gene mutation body (nrt1.8, OE-1) and corresponding wild-type (WS, Col 0).
Fig. 8: the reallocation of AtNRT1.8 gene regulating nitrate under the heavy metal cadmium stress conditions.
Fig. 9: the regulation and control of AtNRT1.8 gene pairs plant drought.
Embodiment
The inventor has carried out long-term and deep research to structure, location and the function etc. of AtNRT1.8 gene, location and functional analysis, mutation type surface research etc. by the analysis of adverse circumstance factor abduction delivering, dna encoding the protein or polypeptide, proved between heavy metal, salt or the arid resistance of AtNRT1.8 gene and plant to exist confidential relation, the high expression level of this gene can strengthen the resistance of plant to heavy metal, salt or drought stress.On this basis, the inventor has finished the present invention.
Particularly, the contriver's studies show that AtNRT1.8 genes encoding NO
3 -(main nitrogenous fertilizer form) translocator, it is expressed adverse circumstances such as the hard to bear heavy metal of energy, salt stress, arid and induces, and transfer-gen plant obviously improves the resistance of heavy metal, salt stress, arid.Therefore this gene has bigger application potential the raising crop aspect environment stress abilities such as heavy metal, salt.
AtNRT1.8 gene and encoded protein matter or polypeptide
In the present invention, term " AtNRT1.8 albumen or polypeptide " or " protein of AtNRT1.8 genes encoding or polypeptide " refer to protein or the polypeptide by AtNRT1.8 genes encoding of the present invention, also comprise in this definition having heavy metal or the above-mentioned protein of salt stress resistance or the variant form of polypeptide that improves plant.The protein of described AtNRT1.8 genes encoding or the sequence of polypeptide can be selected from: (a) SEQ ID NO:3; Or (b) in the aminoacid sequence that (a) limits, strengthen plant active to the resistance of heavy metal or salt stress by (a) deutero-protein or polypeptide through replacing, lack or adding one or several amino acid and have.
Protein of the present invention or polypeptide can be the products of natural purifying, or the product of chemosynthesis, or use recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).AtNRT1.8 albumen or polypeptide are preferably by Arabidopis thaliana AtNRT1.8 gene or its homologous gene or family gene coding among the present invention.
The variant form of protein of the present invention or polypeptide comprises (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, for example 1,2,3,4,5,6,7,8,9 or 10) amino acid whose disappearance, insertion and/or replacement, and at C-terminal and/or N-terminal interpolation one or several (being generally in 20, preferably is in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change the function of protein or polypeptide usually with the close or similar amino acid of performance.Again such as, add the function that or several amino acid also can not change protein or polypeptide usually at C-terminal and/or N-terminal, AtNRT1.8 protein for example of the present invention or polypeptide can comprise or not comprise initial methionine residues and still have the activity that strengthens plant heavy metal or salt stress resistance.
Can adopt radiation or be exposed to mutagenic compound and get off to produce random mutagenesis, also can obtain protein or polypeptide in above-mentioned (b) by site-directed mutagenesis method or other known Protocols in Molecular Biology.Can utilize the encoding sequence of code for said proteins or polypeptide to make up transgenic plant, and the proterties of observing these transgenic plant whether to some extent improvement screen and differentiate gained protein or polypeptide.
The host used according to the recombinant production scheme, protein of the present invention or polypeptide can be glycosylated, maybe can be nonglycosylated.This term also comprises proteic active fragments of AtNRT1.8 and reactive derivative.
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 coded albumen of the sequence of AtNRT1.8 albumen coded sequence hybridization and polypeptide or the albumen that utilizes the proteic antiserum(antisera) of anti-AtNRT1.8 to obtain.The present invention also can use other polypeptide, as comprises AtNRT1.8 albumen or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the proteic soluble fragments of AtNRT1.8.Usually, this fragment have the AtNRT1.8 protein sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
As used herein, term " AtNRT1.8 gene " or " plant AtNRT1.8 gene " are used interchangeably, all be meant a kind of sequence of encode AtNRT1.8 albumen of the present invention or polypeptide, itself and Arabidopis thaliana AtNRT1.8 gene order (referring to SEQ ID NO:1) can the height homology or under stringent condition with the molecule of described gene order hybridization or with above-mentioned numberator height homologous family gene molecule, described expression of gene has some improvement to heavy metal or the salt stress resistance tool of plant.
Disclose AtNRT1.8 and homogenic sequence thereof in the prior art, included but not limited to: AGI numbering: At4g21680, see Tsay YF summary (FEBS Letters, 2007,581:2290-2300); GenBank Accession:NM_118288, GenBank Accession:AK118142.These genes known in the art include in the present invention.
AtNRT1.8 gene of the present invention can be selected from: (i) SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:4 and SEQ ID NO:5 (its correspond respectively to MATDB AGI code:At4g21680 (
Http:// mips.gsf.de/proj/plant/jsf/athal/index.jsp), At4g21680 coding region, GenBank Accession:NM_118288 and GenBank Accession:AK 118142); Or (ii) under stringent condition with sequence hybridization that (i) limits and active molecule with improvement crop seed proterties.
As used herein, term " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only the homogeny between two sequences at least 50%, preferred more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, be more preferably 95% and just hybridize when above.For example, described sequence can be the complementary sequence of sequence defined in (a).
AtNRT1.8 gene nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
Should understand, AtNRT1.8 gene of the present invention is preferably available from Arabidopis thaliana, available from (as having more than 50% of other plant with Arabidopis thaliana AtNRT1.8 gene height homology, preferred more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more preferably more than 85% as 85%, 90%, 95% even 98% sequence homogeny) other gene also within the equivalency range that the present invention preferably considers.The Method and kit for of aligned sequences homogeny also is that this area is known, as BLAST.
Plant and to the resistance of heavy metal, salt or drought stress
As used herein, described " plant " has no particular limits, and includes, but is not limited to: grass, Malvaceae cotton, Cruciferae Brassica plants, feverfew, plant of Solanaceae, labiate or samphire etc.Preferred described plant is a crop.
As used herein, term " crop " is meant the plant of economically valuable in grain, cotton, wet goods agricultural and industry, and its economic worth may be embodied on the useful positions such as the seed, fruit, root, stem, leaf of this plant.Crop includes but not limited to: dicotyledons or monocotyledons.Preferred monocotyledons is a grass, more preferably paddy rice, wheat, barley, corn, Chinese sorghum etc.Preferred dicotyledons includes but not limited to: Malvaceae cotton, Cruciferae Brassica plants etc., more preferably cotton, rape etc.
As used herein, term " heavy metal stress " is meant: when referring to that plant grows in soil that contains the high density heavy metal or water body, it grows and is suppressed, even dead phenomenon.Cause the heavy metal of heavy metal stress to include, but is not limited to: cadmium, lead, mercury, arsenic, copper, chromium, antimony, tin, zinc, barium, bismuth, nickel, cobalt, manganese, iron or vanadium.AtNRT1.8 gene of the present invention or its encoded protein matter or polypeptide can strengthen the resistance of plant to heavy metal, and the raising of this resistance can show as and compare without the control plant that described gene, protein or polypeptide are handled: the heavy metal content on (preferably top) reduces, does not have heavy metal contamination, grows uninfluenced or the degree of susceptibility reduction in the presence of heavy metal in the described plant.
As used herein, term " salt stress " is meant: when referring to that plant grows in soil that contains the high density salinity or water body, it grows and is suppressed, even dead phenomenon.Causing salt to belong to the salt of coercing includes, but is not limited to: sodium-chlor, sodium sulfate, yellow soda ash or sodium bicarbonate.AtNRT1.8 gene of the present invention or its encoded protein matter or polypeptide can strengthen the resistance of plant to salt stress, and the raising of this resistance can show as and compare without the control plant that described gene, protein or polypeptide are handled: described plant is growing uninfluenced or degree of susceptibility reduction or can survive under higher salt concn in the presence of the high density salinity.
As used herein, term " drought stress " is meant: when plant grew in the soil of lack of water or other drought environment, it grew and is suppressed, even dead phenomenon.AtNRT1.8 gene of the present invention or its encoded protein matter or polypeptide can strengthen the resistance of plant to drought stress, and the raising of this resistance can show as and compare without the control plant that described gene, protein or polypeptide are handled: described plant under the lack of water condition grow uninfluenced or degree of susceptibility reduces or can survive under more arid condition.
Carrier, host and transgenic plant
The invention still further relates to the carrier that comprises the AtNRT1.8 gene, and the host cell that produces through genetically engineered with this carrier, and the transgenic plant that obtain high expression level AtNRT1.8 by transgenosis.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize encoding sequence of the present invention to can be used to express or produce the AtNRT1.8 albumen of reorganization.In general following steps are arranged:
(1), or transforms or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide with the proteic polynucleotide of coding AtNRT1.8 of the present invention (or varient);
(2) host cell of in suitable medium, cultivating; With
(3) separation from substratum or cell, protein purification or polypeptide.
Among the present invention, term " carrier " is used interchangeably with " recombinant expression vector ", refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus or other carrier.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 AtNRT1.8 encoding sequence 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.Preferred pEGFP-1, pBI121, pCAMBIA1300, pCAMBIA1301, pCAMBIA2301 or the pHB of using among the present invention.
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 or polypeptide.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 etc.In the present invention, preferably adopt Agrobacterium as host 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.
Term among the present invention " transgenic plant ", " transformant " or " transforming plant " are used interchangeably, and all refer to the cell, organ, tissue or the plant that change AtNRT1.8 gene of the present invention over to and stablize high expression level AtNRT1.8 albumen or polypeptide that obtain by conventional transgenic method.
Transform plant and 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 disease 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.
Major advantage of the present invention
Major advantage of the present invention is:
(1) provide the new purposes of AtNRT1.8 gene and encoded protein matter or polypeptide, it can be used for effectively improving the resistance of plant to heavy metal, salt or drought stress;
(2) transgenic plant are provided, for grain, cotton, wet goods production and processing provide good raw material and product with improvement heavy metal, salt or drought stress resistance;
(3) provide the new way of improving plant heavy metal, salt or drought stress resistance, thereby had great application prospect.
Embodiment
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 (for example can be according to normal condition with reference to " molecular cloning experiment guide " as people such as Sambrook, English name is " Molecular Cloning:A Laboratory Manual ", the third edition, 2001, Cold Spring Harbor Laboratory press) or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The regulating and controlling effect of embodiment 1. heavy metals and other multiple adverse circumstance (Stress) factor pair AtNRT1.8 gene
The contriver finds that by the gene chip analysis AtNRT1.8 is subjected to cadmium to induce the strongest (data are not shown) in 53 POT gene family members.The sequence of used AtNRT1.8 gene as shown in Figure 1 in the present embodiment.
Induced whether shown in gene chip by cadmium in order to prove conclusively this gene, and further disclose it and induced by other adverse circumstance factor, carried out Northern cross experiment (test method: the Northern hybridization in the classical molecular biology of plants, reference " molecular cloning experiment guide " 532-552 page or leaf).
Above-mentioned test-results shows: the AtNRT1.8 gene is subjected to the abduction delivering of cadmium to have the time and concentration relies on effect (seeing Fig. 2 A), and the secondary effect osmotic stress and the oxidative stress that not coerced by cadmium are regulated and control (shown in Fig. 2 B, PEG is a kind of high permeate agent, can cause osmotic stress to plant, AtNRT1.8 gene high expression plant does not show the function of impermeabilisation).Except cadmium, the AtNRT1.8 gene also is subjected to the abduction delivering (Fig. 2 C) of heavy metal copper (Cu) and zinc (Zn).
In addition, the AtNRT1.8 gene is subjected to adverse circumstance signaling molecule ethene (it is the synthetic precursor substance of ethene for ACC, 1-amino-cyclopropane-1-carboxylic acid; Ethene is the signaling molecule of multiple adverse circumstance, under a lot of adverse circumstances, and plant ethene meeting high expression level) the induced strong expression, induced by JA (jasmonic).
The internally-oriented low-affinity nitrate transport protein of embodiment 2.AtNRT1.8 genes encoding (Nitrate
Transporter)
The AtNRT1.8 gene belongs to the PTR gene family, also is called NRT1 family or POT family simultaneously.Because this family gene has been found the nitrate transport protein of both having encoded, the oligopeptides translocator of also encoding, therefore in order to detect the transhipment substrate of AtNRT1.8 gene coded protein, the contriver has studied AtNRT1.8 gene in vitro transport activity by the mode of xenopus leavis oocytes injection.
Test method: with the UTR sequence subclone at the encoding sequence of AtNRT1.8 gene and two ends to the pGEMHE carrier, become cRNA by in-vitro transcription, be expelled to xenopus leavis oocytes (available from Capital Normal University), carry out electric Physiological Analysis (voltage clamp is available from Dagan company, and model is TEV-200A VOLTAGECLAMP).Test-results as shown in Figure 3.
The result shows: AtNRT1.8 gene coded protein unitransport NO
3 -, and be an internally-oriented low-affinity translocator.Term " internally-oriented low-affinity translocator " is meant: the albumen of this genes encoding can be transported NO in the export-oriented tenuigenin of tenuigenin
3 -, and only to the NO of high density
3 -Vigor is arranged, in low concentration of NO
3 -Turn-over capacity is very poor under the condition.
Expression and the location of embodiment 3.AtNRT1.8 gene in Arabidopis thaliana
Determine the tissue expression feature and the Subcellular Localization situation of gene, have important suggesting effect for the mechanism of action of clear and definite gene.For this reason, the contriver has further studied expression and the location situation of AtNRT1.8 gene in Arabidopis thaliana.
With reference to Dawar Hussain etc., The Plant Cell " vegetable cell ", the 16th volume, the method that 1327-1339 put down in writing, the promoter region and the reporter gene GUS of gene are merged, and perhaps the encoder block of gene and reporter gene GFP merge, and are used for arabidopsis thaliana transformation or onion epidermis cell.In situ hybridization is respectively with forward sequence and the reverse sequence of AtNRT1.8 and organize the mRNA hybridization of this life.Test-results as shown in Figure 4.
The result shows: the fractional analysis of GUS group shows that the AtNRT1.8 expression of gene is mainly expressed in the vascular system of plant, and is positioned at center pillar, and in situ hybridization leads to the same conclusion (Fig. 4 A-E), has confirmed the center pillar specifically expressing pattern of AtNRT1.8.Further section analysis revealed: this expression of gene is positioned at xylem parenchyma cell (Fig. 4 B-C), points out this gene may participate in NO
3 -Long-distance transhipment.Further utilize GFP and AtNRT1.8 gene Fusion albumen positioning analysis to show to be onion cell transient expression or all AtNRT1.8 is positioned cytoplasmic membrane (Fig. 4 F-M), in conjunction with the internally-oriented NO of this genes encoding at the intravital stably express of Arabidopis thaliana
3 -The fact of translocator infers that AtNRT1.8 may play from xylem unloading NO
3 -Effect.
Embodiment 4.AtNRT1.8 gene transgenic plant is to the resistance of heavy metal stress, salt stress
For clear and definite AtNRT1.8 in the intravital concrete physiological action of plant, the contriver has studied the gene knockout mutant and the phenotype of overexpression mutant under heavy metal and salt stress adverse environmental factor of this gene respectively.
The method of the resistant phenotype observation of mutant and the developmental state of statistics root system is as follows: with WS and nrt1.8, and Col 0 and OE-1 are (wherein, the nrt1.8 mutant is the mutant of AtNRT1.8 afunction, WS is a wild-type, Col 0 is another kind of wild-type commonly used, after OE-1 is the sterilization of AtNRT1.8 overexpression mutant (35S::POT1/Col 0) seed, be seeded at the 1/4PNS substratum (according to the formulated in the following document: Richard N.Arteca and Jeannette M.Arteca, A novel method for growing Arabidopsis thaliana plants hydroponically, the novel method of water planting plantation Arabidopis thaliana, PHYSIOLOGIA PLANTARUM 108:188-193.2000) on, 4 ℃ of refrigerator vernalization 2 days, place then in the phytotron (16h/8h photoperiod, 22 ℃) and cultivated 5 days.
The seedling that the picking growth conditions is comparatively close is transferred to the heavy metal and the 50mM NO that contain 50uM Cd, Pb, Hg or As respectively
3 -4 kinds of 1/4PNS substratum on, each strain system chooses 8 seedlings, and good their positions on substratum at this moment of mark.Plant after the transfer after 10 days, is observed their growing state in continued growth on the flat board, and adds up the increased value of their main root length in 10 days, and Taking Pictures recording.
Phenotype during salt stress is handled changes research and adopts above-mentioned similar experimental technique to carry out.Different with aforesaid method is: coerce the used substratum of test for containing 50mM NO
3 -With 50mM NaCl or Na
2CO
3The 1/4PNS substratum.
The result shows: the nrt1.8 mutant of AtNRT1.8 afunction is coerced responsive (Fig. 5) more with respect to wild-type WS to cadmium, and correspondingly be, overexpression mutant (35S::POT1/Col 0) obviously improves with respect to the resistance of wild-type contrast (Col 0) for cadmium, and the long increased value of corresponding root is seen Fig. 5.The test of adopting Pb, Hg or As heavy metal to carry out has also obtained similar result.The above results shows: AtNRT1.8 gene regulating plant is to the resistance capacity of heavy metal, and the overexpression of this gene can improve the resistance of plant to heavy metal.
Simultaneously, also observe similar phenotype in salt stress is handled, the long increased value of corresponding root is seen (Fig. 6).This shows that AtNRT1.8 may be on broad sense more plays regulating effect to plant for the resistance of multiple adverse circumstance.
Under embodiment 5. heavy metal stress, the influence that AtNRT1.8 distributes to the plant heavy metal
The result of study of previous embodiment shows that AtNRT1.8 effect gene plant is to the resistance of heavy metal, and whether the contriver has further studied the AtNRT1.8 gene also can influence heavy metal in the intravital distribution of plant, and this is extremely important to the using value that improves this gene.
Adopted inductively coupled plasma mass spectrometry (ICP-MS) to measure the content of Cd in the mutant, concrete grammar is as follows:
1) AtNRT1.8 gene mutation body (nrt1.8, OE-1) and corresponding wild-type (WS, Col 0) are transferred on the 1/4PNS liquid nutrient medium in growth on the 1/2MS flat board after 5 days, cultivated for 4 weeks;
2) they are transferred on the 1/4PNS liquid nutrient medium that contains 20uM Cd handled 3 days, prepare to draw materials;
3) material earlier uses ultrapure washing once, 5 minutes, uses the CaCl of 25mM then
2(the pH value is about 5.0) cleaning material 2 times each 4 minutes, re-uses ultrapure washing once, 5 minutes at last;
4) material that will get places 80 ℃ baking oven, oven dry 24h; After treating finish-drying, take by weighing dry weight, each mutant and wild-type all repeat to weigh 3 times;
5) material after weighing is placed the ICP measuring tube, the record dry weight adds other 70% concentrated nitric acid of extra-pure grade 1ml then in pipe, spend the night and clear up;
6) second day, in 95 ℃ of water-baths, disappear and boil about 30 minutes, until can't see the visible precipitation;
7) add the ultrapure water of 13ml in the ICP measuring tube, make the overall solution volume after thoroughly clearing up reach 14ml, promptly dilute 14 times.In add ultrapure water and be diluted to 14ml, promptly dilute 14 times, use ICP-MS (to measure the amount of contained Cd as ELAN DRC-e available from perkinelmer company model then.
The measurement result of ICP-MS shows: AtNRT1.8 influences plant Cd really and coerces down Cd in the intravital distribution of plant, and in WS and nrt1.8, in lotus throne leaf and fruit pod, the content of Cd all is lower than nrt1.8 among the WS.And in crossing expression strain system, this influence of AtNRT1.8 is more obvious, is among the OE-1 crossing the expression strain, its on the ground the content in part fruit pod and the lotus throne leaf all significantly be lower than contrast Col 0 (Fig. 7).In crossing expression strain system, other also observes similar ICP phenotype.
Adopt and use the same method, the test of carrying out with Pb, Hg or As heavy metal has also obtained similar result.
This function of AtNRT1.8 gene is very helpful to the food safety that improves farm crop.
Embodiment 6.AtNRT1.8 gene pairs NO
3
-
The regulation and control of under the heavy metal cadmium stress conditions, reallocating
How to regulate and control the resistance capacity of plant to adverse circumstance for understanding AtNRT1.8, the contriver has analyzed the interior NO of plant materials under the cadmium stress conditions
3 -In the intravital reallocation situation of plant.
Adopt the NO in the HPLC method measurement plant tissue
3 -Content, concrete grammar is as follows:
(1) after AtNRT1.8 mutant nrt1.8 and OE-1 and corresponding wild-type WS, Col 0 material use the 1/4PNS liquid nutrient medium or contain the 1/4PNS liquid nutrient medium processing 3d of 20 μ M Cd, divide over-ground part and underground part to draw materials;
(2) every part of material uses ultrapure water to clean each 2 minutes 4 times;
(3) use thieving paper to blot and remain in structural moisture content, on balance, weigh then, write down the fresh weight value of each sample, place the eppendorf pipe of 1.5ml;
(4) according to the 30ul/mg ratio, toward wherein adding ultrapure water;
(5) use boiling water to boil 20 minutes, place the liquid nitrogen quick-frozen then ,-80 ℃ of refrigerator overnight;
(6) the centrifugal material of 20800g is 5 minutes; Collect supernatant to clean eppendorf pipe;
(7) the membrane filtration supernatant of use 0.45um then with 20 times of each diluted samples, uses HPLC (Agilent, 1200series, chromatographic column is PARTISIL 10SAX (strong anion exchange) column (Whatman, Clifton, NJ), measure sample;
The condition determination of HPLC is: moving phase: 45mM-50mM KH
2PO
4-H
3PO
4(pH2.9-3.0); Flow velocity: 1mL/min; Column temperature: 30 ℃; Detect wavelength: 210nm; Sensing range: 50pmol-200pmol (5 μ L-500 μ L); Appearance time: about 8min; The time that is provided with of every pin is 13 minutes.
Test-results is as shown in Figure 7: under the situation that does not have cadmium to coerce, and the NO between nrt1.8 and the wild-type contrast WS
3 -No matter content still is in the root, all not have marked difference in leaf; And under the cadmium stress conditions, the two is the NO at position on the ground
3 -Content all descends to some extent, but does not have significant difference each other, in the root at underground position, and the NO in the wild-type
3 -Apparently higher than mutant nrt1.8, this mainly is owing to NO in the wild-type root
3 -Due to content rises.
In conjunction with the test-results of front, can infer: because under the cadmium stress conditions, the AtNRT1.8 gene by abduction delivering, makes the NO of higher concentration in the xylem vessel in root
3 -Discharge, in the root cells around getting back to, thereby effectively alleviate because cadmium is coerced the root NO that causes
3 -Density loss.
Embodiment 7.AtNRT1.8 gene transgenic plant is to the resistance of arid
Get the mature seed of Ws, nrt1.8, Col 0, OE-1 (AtNRT1.8 overexpression mutant (35S::POT1/Col 0)), OE-18 (another strain system of AtNRT1.8 overexpression mutant), vernalization 3 days, cast to the dull and stereotyped cultivation of 1/2MS 6 days, move to trinity mixture fill, vermiculite again, water respectively equivalent water, contain 6mM NH
4NO
3Nutrient solution.18 days afterwards with NH
4NO
3The plant of handling is divided two portions, and a part is watered NH
4NO
3(concentration is 6mM), another part change waters (NH
4)
2SO
4(concentration is 6mM).Choose the excised leaf weighing after 10 days, calculate fluid loss.Every kind of plant is got 6 in the identical substantially blade of growing way, and 0,30min, 60min, 120min, 180min weighing fresh weight, 3 parallel.Fluid loss (%)=(W-Wi)/Wi*100%.The fluid loss of preliminary experiment proof Ws is lower than nrt1.8, and promptly Ws is than more drought resisting of nrt1.8.The fluid loss of OE-18, OE-1 is lower than Col 0, and promptly OE-18, OE-1 are than Col 0 drought resisting.
Get the mature seed of Ws, nrt1.8, Col 0, OE-1, five kinds of plant of OE-18, vernalization 3 days casts to the dull and stereotyped cultivation of 1/2MS 6 days, moves to trinity mixture fill again, and every group is provided with 6 parallel sample.Carry out arid after 21 days at normal irrigation and handle, and whether the observation plant there is the drought resisting phenotype.Observe phenotype and find OE-1, OE-18 than Col 0 drought resisting, and OE-1 drought-resistant ability stronger (referring to Fig. 9).
Embodiment 8.AtNRT1.8 transgenic paddy rice is to the resistance of heavy metal stress, salt stress and arid
The AtNRT1.8 subclone in the paddy rice expression plasmid, is carried out the paddy rice transgenosis, thereby obtain the resistant rice transfer-gen plant.Transfer-gen plant is carried out Physiological Analysis (method is with embodiment 4 and embodiment 7), and whether the plant that changes AtNRT1.8 with observation over to can improve the resistance of paddy rice to heavy metal, salt or drought stress.
Agrobacterium tumefaciens rice transformation mature embryo schedule of operation
One, transformed bacteria liquid is prepared:
(1) first day morning: from the bacterial classification of-80 ℃ of preservations picking a little in 5mlYEP (Rif+Kan) liquid nutrient medium, 28 ℃ of cultivations, 100rpm.
(2) the next morning: from the YEP nutrient solution that contains Agrobacterium, draw 1～2ml, change 25～50mlAB (Rif+Kan50) liquid nutrient medium over to, 28 ℃, 100rpm, after 4 hours to the OD600=0.5.
Two, cultivate altogether
Second day afternoon: survey the OD600 value, the centrifugal 5000rpm of bacterium liquid, 15min, bacterial sediment are suspended in AAM (AS100) and to bacterium liquid OD600=0.4～0.6 bacterium liquid are poured in the triangular flask that rice callus is housed, and make callus soak wherein 20 minutes, and rock frequently; Blot bacterium liquid with aseptic paper, the callus that had soaked is transferred on the NBD that contains 2.5%Phytagel (AS100) substratum that is lined with one deck aseptic filter paper cultivated altogether 2～3 days.Each culture dish adds the abundant moistening aseptic filter paper of 1mlAAM (AS100) nutrient solution again.
Three, screening
Callus is blotted (changing filter paper one time) with aseptic filter paper, go to and screen kanamycin-resistant callus tissue (about 45 days, the centre is changed screening culture medium one time) (the dark cultivation) on the screening culture medium.
Screen used substratum: screen for the first time: NBD+Cef700ul+Hyg25mg/L; Programmed screening: NBD+Cef500ul+Hyg50mg/L.
Four, pre-differentiation:
The rice callus of screening growth is gone to pre-differentiation substratum, and ((pH=5.7 does not have 2,4-D)+Car250+BAP2mg/L+NAA1mg/L+ABA5mg/L), cultivates 10 days to contain the NB of 0.45%Phytagel.
Five, differentiation
To go to division culture medium (1/2MS (pH=5.7～the 5.8)+sucrose 20g/L that contains 0.45%Phytagel) through the rice callus of pre-differentiation and go up seedling differentiation (illumination, 15-30 days).
Six, take root:
Green seedling gone on the root media (1/2MS (pH=5.7～the 5.8)+sucrose 20g/L that contains 0.45%Phytagel) take root.
The rice callus tissue culture
(1) peels off kind of a skin;
(2) 75% alcohol disinfectings 1～2 minute;
(3) aseptic water washing is 2～3 times;
(4) 20～30 minutes (sterilized waters: clorox=3: 1～4: 1) of clorox sterilization
(5) aseptic water washing is 4～5 times;
(6) aseptic filter paper blots;
(7) insert the NBD substratum;
Cut callus after (8) 7～10 days and change same medium over to, in 1～2 all follow-up generations, once can transform.
The result who transfer-gen plant is carried out Physiological Analysis shows: the rice plant that changes AtNRT1.8 over to is for heavy metal stress (especially cadmium, lead, mercury or arsenic), salt stress (especially NaCl or Na
2CO
3) and the arid resistance all increase.
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〉the AtNRT1.8 gene strengthens the application of farm crop to the resistance of heavy metal or salt stress
<130>090797
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atg?gat?caa?aaa?gtt?aga?cag?ttt?gag?gtt?tgc?act?caa?gac?ggt?agc 48
Met?Asp?Gln?Lys?Val?Arg?Gln?Phe?Glu?Val?Cys?Thr?Gln?Asp?Gly?Ser
1 5 10 15
gtt?gat?cgt?cac?ggc?aat?cca?gct?atc?cga?gct?aat?acc?ggc?aaa?tgg 96
Val?Asp?Arg?His?Gly?Asn?Pro?Ala?Ile?Arg?Ala?Asn?Thr?Gly?Lys?Trp
20 25 30
ctc?act?gct?att?ctc?att?cta?gtg?aat?caa?gga?cta?gct?acg?ctt?gcg 144
Leu?Thr?Ala?Ile?Leu?Ile?Leu?Val?Asn?Gln?Gly?Leu?Ala?Thr?Leu?Ala
35 40 45
ttc?ttc?ggt?gta?gga?gtg?aat?ttg?gtt?ctg?ttt?ctg?act?cga?gtg?atg 192
Phe?Phe?Gly?Val?Gly?Val?Asn?Leu?Val?Leu?Phe?Leu?Thr?Arg?Val?Met
50 55 60
gga?caa?gac?aat?gca?gaa?gcg?gct?aat?aat?gtt?agt?aaa?tgg?aca?gga 240
Gly?Gln?Asp?Asn?Ala?Glu?Ala?Ala?Asn?Asn?Val?Ser?Lys?Trp?Thr?Gly
65 70 75 80
act?gtc?tat?atc?ttc?tct?ttg?ctt?ggt?gct?ttc?ctc?agt?gac?tct?tat 288
Thr?Val?Tyr?Ile?Phe?Ser?Leu?Leu?Gly?Ala?Phe?Leu?Ser?Asp?Ser?Tyr
85 90 95
tgg?gga?cgt?tac?aag?act?tgt?gct?atc?ttt?caa?gca?agt?ttc?gtt?gca 336
Trp?Gly?Arg?Tyr?Lys?Thr?Cys?Ala?Ile?Phe?Gln?Ala?Ser?Phe?Val?Ala
100 105 110
ggg?ttg?atg?atg?tta?tct?tta?tct?act?ggt?gcg?tta?ttg?ctt?gaa?cca 384
Gly?Leu?Met?Met?Leu?Ser?Leu?Ser?Thr?Gly?Ala?Leu?Leu?Leu?Glu?Pro
115 120 125
agt?ggt?tgt?gga?gtt?gaa?gat?tcg?ccg?tgt?aag?cct?cat?tcg?acg?ttt 432
Ser?Gly?Cys?Gly?Val?Glu?Asp?Ser?Pro?Cys?Lys?Pro?His?Ser?Thr?Phe
130 135 140
aag?acg?gtt?ctg?ttt?tat?ctg?tcg?gtg?tat?cta?atc?gcg?tta?ggg?tat 480
Lys?Thr?Val?Leu?Phe?Tyr?Leu?Ser?Val?Tyr?Leu?Ile?Ala?Leu?Gly?Tyr
145 150 155 160
ggt?ggt?tat?cag?ccg?aac?ata?gct?act?ttt?gga?gct?gat?cag?ttt?gat 528
Gly?Gly?Tyr?Gln?Pro?Asn?Ile?Ala?Thr?Phe?Gly?Ala?Asp?Gln?Phe?Asp
165 170 175
gcg?gag?gat?tcc?gtt?gaa?gga?cac?tcg?aaa?atc?gcg?ttt?ttc?agt?tac 576
Ala?Glu?Asp?Ser?Val?Glu?Gly?His?Ser?Lys?Ile?Ala?Phe?Phe?Ser?Tyr
180 185 190
ttt?tac?ttg?gct?ctg?aat?ctt?gga?tcg?ctc?ttc?tca?aat?act?gtc?ttg 624
Phe?Tyr?Leu?Ala?Leu?Asn?Leu?Gly?Ser?Leu?Phe?Ser?Asn?Thr?Val?Leu
195 200 205
ggt?tac?ttt?gag?gat?caa?ggg?gaa?tgg?ccg?ctt?gga?ttt?tgg?gcg?tct 672
Gly?Tyr?Phe?Glu?Asp?Gln?Gly?Glu?Trp?Pro?Leu?Gly?Phe?Trp?Ala?Ser
210 215 220
gct?ggc?tct?gct?ttt?gcg?ggg?tta?gtg?ctt?ttc?ttg?att?ggc?acg?cca 720
Ala?Gly?Ser?Ala?Phe?Ala?Gly?Leu?Val?Leu?Phe?Leu?Ile?Gly?Thr?Pro
225 230 235 240
aag?tac?cga?cac?ttt?acg?cct?aga?gag?agt?cct?tgg?tct?aga?ttc?tgc 768
Lys?Tyr?Arg?His?Phe?Thr?Pro?Arg?Glu?Ser?Pro?Trp?Ser?Arg?Phe?Cys
245 250 255
caa?gtg?ttg?gtt?gct?gca?aca?aga?aag?gct?aag?att?gat?gtg?cac?cat 816
Gln?Val?Leu?Val?Ala?Ala?Thr?Arg?Lys?Ala?Lys?Ile?Asp?Val?His?His
260 265 270
gaa?gag?ttg?aat?ctc?tat?gat?tct?gag?act?caa?tac?act?gga?gat?aag 864
Glu?Glu?Leu?Asn?Leu?Tyr?Asp?Ser?Glu?Thr?Gln?Tyr?Thr?Gly?Asp?Lys
275 280 285
aag?att?ctt?cat?acc?aaa?ggc?ttc?aga?ttc?ttg?gat?aga?gct?gct?att 912
Lys?Ile?Leu?His?Thr?Lys?Gly?Phe?Arg?Phe?Leu?Asp?Arg?Ala?Ala?Ile
290 295 300
gtt?aca?cct?gat?gat?gag?gct?gag?aaa?gta?gag?agc?gga?tcg?aaa?tac 960
Val?Thr?Pro?Asp?Asp?Glu?Ala?Glu?Lys?Val?Glu?Ser?Gly?Ser?Lys?Tyr
305 310 315 320
gat?cca?tgg?agg?ctc?tgc?tcg?gtg?act?caa?gtc?gaa?gaa?gtg?aaa?tgt 1008
Asp?Pro?Trp?Arg?Leu?Cys?Ser?Val?Thr?Gln?Val?Glu?Glu?Val?Lys?Cys
325 330 335
gta?tta?aga?ctc?tta?cca?atc?tgg?ctc?tgc?acc?atc?ctc?tac?tct?gtg 1056
Val?Leu?Arg?Leu?Leu?Pro?Ile?Trp?Leu?Cys?Thr?Ile?Leu?Tyr?Ser?Val
340 345 350
gtt?ttc?acc?caa?atg?gct?tca?ctg?ttc?gtt?gtg?caa?gga?gca?gcg?atg 1104
Val?Phe?Thr?Gln?Met?Ala?Ser?Leu?Phe?Val?Val?Gln?Gly?Ala?Ala?Met
355 360 365
aag?aca?aac?atc?aaa?aac?ttc?cgg?att?cca?gct?tca?agc?atg?tct?agt 1152
Lys?Thr?Asn?Ile?Lys?Asn?Phe?Arg?Ile?Pro?Ala?Ser?Ser?Met?Ser?Ser
370 375 380
ttc?gac?att?ctc?agt?gtc?gcc?ttc?ttc?atc?ttc?gca?tac?agg?cgg?ttt 1200
Phe?Asp?Ile?Leu?Ser?Val?Ala?Phe?Phe?Ile?Phe?Ala?Tyr?Arg?Arg?Phe
385 390 395 400
ctt?gat?cca?ctc?ttt?gca?aga?ctt?aac?aaa?aca?gaa?cgc?aac?aaa?ggt 1248
Leu?Asp?Pro?Leu?Phe?Ala?Arg?Leu?Asn?Lys?Thr?Glu?Arg?Asn?Lys?Gly
405 410 415
ctc?act?gag?ctt?cag?agg?atg?ggg?att?ggg?ctt?gtg?att?gcg?ata?atg 1296
Leu?Thr?Glu?Leu?Gln?Arg?Met?GlyIle?Gly?Leu?Val?Ile?Ala?Ile?Met
420 425 430
gcg?atg?att?tcc?gca?gga?atc?gta?gag?ata?cac?aga?ctg?aaa?aat?aag 1344
Ala?Met?Ile?Ser?Ala?Gly?Ile?Val?Glu?Ile?His?Arg?Leu?Lys?Asn?Lys
435 440 445
gaa?ccg?gaa?agt?gct?act?tcg?atc?tca?agc?tcc?agc?act?ttg?agc?att 1392
Glu?Pro?Glu?Ser?Ala?Thr?Ser?Ile?Ser?Ser?Ser?Ser?Thr?Leu?Ser?Ile
450 455 460
ttt?tgg?caa?gta?cct?cag?tac?atg?ttg?ata?ggt?gca?tca?gaa?gtg?ttc 1440
Phe?Trp?Gln?Val?Pro?Gln?Tyr?Met?Leu?Ile?Gly?Ala?Ser?Glu?Val?Phe
465 470 475 480
atg?tac?gtt?ggt?caa?ctc?gag?ttc?ttc?aac?agc?caa?gca?cca?acc?ggg 1488
Met?Tyr?Val?Gly?Gln?Leu?Glu?Phe?Phe?Asn?Ser?Gln?Ala?Pro?Thr?Gly
485 490 495
cta?aag?agc?ttt?gca?agc?gcg?cta?tgt?atg?gct?tca?ata?tct?ctt?ggg 1536
Leu?Lys?Ser?Phe?Ala?Ser?Ala?Leu?Cys?Met?Ala?Ser?Ile?Ser?Leu?Gly
500 505 510
aac?tat?gta?agc?agt?ttg?tta?gtt?tcc?att?gtc?atg?aag?atc?tct?aca 1584
Asn?Tyr?Val?Ser?Ser?Leu?Leu?Val?Ser?Ile?Val?Met?Lys?Ile?Ser?Thr
515 520 525
aca?gat?gat?gtg?cat?ggc?tgg?att?cct?gaa?aat?ctc?aac?aaa?gga?cac 1632
Thr?Asp?Asp?Val?His?Gly?Trp?Ile?Pro?Glu?Asn?Leu?Asn?Lys?Gly?His
530 535 540
ttg?gag?aga?ttc?tac?ttc?ctt?tta?gct?ggt?cta?acc?gct?gct?gat?ttc 1680
Leu?Glu?Arg?Phe?Tyr?Phe?Leu?Leu?Ala?Gly?Leu?Thr?Ala?Ala?Asp?Phe
545 550 555 560
gtg?gtt?tac?ttg?att?tgt?gcc?aaa?tgg?tac?aag?tat?att?aaa?tct?gaa 1728
Val?Val?Tyr?Leu?Ile?Cys?Ala?Lys?Trp?Tyr?Lys?Tyr?Ile?Lys?Ser?Glu
565 570 575
gca?agt?ttc?tct?gag?tcc?gta?act?gaa?gag?gag?gaa?gtc?tga 1770
Ala?Ser?Phe?Ser?Glu?Ser?Val?Thr?Glu?Glu?Glu?Glu?Val
580 585
<210>3
<211>589
<212>PRT
＜213〉artificial sequence
<400>3
Met?Asp?Gln?Lys?Val?Arg?Gln?Phe?Glu?Val?Cys?Thr?Gln?Asp?Gly?Ser
1 5 10 15
Val?Asp?Arg?His?Gly?Asn?Pro?Ala?Ile?Arg?Ala?Asn?Thr?Gly?Lys?Trp
20 25 30
Leu?Thr?Ala?Ile?Leu?Ile?Leu?Val?Asn?Gln?Gly?Leu?Ala?Thr?Leu?Ala
35 40 45
Phe?Phe?Gly?Val?Gly?Val?Asn?Leu?Val?Leu?Phe?Leu?Thr?Arg?Val?Met
50 55 60
Gly?Gln?Asp?Asn?Ala?Glu?Ala?Ala?Asn?Asn?Val?Ser?Lys?Trp?Thr?Gly
65 70 75 80
Thr?Val?Tyr?Ile?Phe?Ser?Leu?Leu?Gly?Ala?Phe?Leu?Ser?Asp?Ser?Tyr
85 90 95
Trp?Gly?Arg?Tyr?Lys?Thr?Cys?Ala?Ile?Phe?Gln?Ala?Ser?Phe?Val?Ala
100 105 110
Gly?Leu?Met?Met?Leu?Ser?Leu?Ser?Thr?Gly?Ala?Leu?Leu?Leu?Glu?Pro
115 120 125
Ser?Gly?Cys?Gly?Val?Glu?Asp?Ser?Pro?Cys?Lys?Pro?His?Ser?Thr?Phe
130 135 140
Lys?Thr?Val?Leu?Phe?Tyr?Leu?Ser?Val?Tyr?Leu?Ile?Ala?Leu?Gly?Tyr
145 150 155 160
Gly?Gly?Tyr?Gln?Pro?Asn?Ile?Ala?Thr?Phe?Gly?Ala?Asp?Gln?Phe?Asp
165 170 175
Ala?Glu?Asp?Ser?Val?Glu?Gly?His?Ser?Lys?Ile?Ala?Phe?Phe?Ser?Tyr
180 185 190
Phe?Tyr?Leu?Ala?Leu?Asn?Leu?Gly?Ser?Leu?Phe?Ser?Asn?Thr?Val?Leu
195 200 205
Gly?Tyr?Phe?Glu?Asp?Gln?Gly?Glu?Trp?Pro?Leu?Gly?Phe?Trp?Ala?Ser
210 215 220
Ala?Gly?Ser?Ala?Phe?Ala?Gly?Leu?Val?Leu?Phe?Leu?Ile?Gly?Thr?Pro
225 230 235 240
Lys?Tyr?Arg?His?Phe?Thr?Pro?Arg?Glu?Ser?Pro?Trp?Ser?Arg?Phe?Cys
245 250 255
Gln?Val?Leu?Val?Ala?Ala?Thr?Arg?Lys?Ala?Lys?Ile?Asp?Val?His?His
260 265 270
Glu?Glu?Leu?Asn?Leu?Tyr?Asp?Ser?Glu?Thr?Gln?Tyr?Thr?Gly?Asp?Lys
275 280 285
Lys?Ile?Leu?His?Thr?Lys?Gly?Phe?Arg?Phe?Leu?Asp?Arg?Ala?Ala?Ile
290 295 300
Val?Thr?Pro?Asp?Asp?Glu?Ala?Glu?Lys?Val?Glu?Ser?Gly?Ser?Lys?Tyr
305 310 315 320
Asp?Pro?Trp?Arg?Leu?Cys?Ser?Val?Thr?Gln?Val?Glu?Glu?Val?Lys?Cys
325 330 335
Val?Leu?Arg?Leu?Leu?Pro?Ile?Trp?Leu?Cys?Thr?Ile?Leu?Tyr?Ser?Val
340 345 350
Val?Phe?Thr?Gln?Met?Ala?Ser?Leu?Phe?Val?Val?Gln?Gly?Ala?Ala?Met
355 360 365
Lys?Thr?Asn?Ile?Lys?Asn?Phe?Arg?Ile?Pro?Ala?Ser?Ser?Met?Ser?Ser
370 375 380
Phe?Asp?Ile?Leu?Ser?Val?Ala?Phe?Phe?Ile?Phe?Ala?Tyr?Arg?Arg?Phe
385 390 395 400
Leu?Asp?Pro?Leu?Phe?Ala?Arg?Leu?Asn?Lys?Thr?Glu?Arg?Asn?Lys?Gly
405 410 415
Leu?Thr?Glu?Leu?Gln?Arg?Met?Gly?Ile?Gly?Leu?Val?Ile?Ala?Ile?Met
420 425 430
Ala?Met?Ile?Ser?Ala?Gly?Ile?Val?Glu?Ile?His?Arg?Leu?Lys?Asn?Lys
435 440 445
Glu?Pro?Glu?Ser?Ala?Thr?Ser?Ile?Ser?Ser?Ser?Ser?Thr?Leu?Ser?Ile
450 455 460
Phe?Trp?Gln?Val?Pro?Gln?Tyr?Met?Leu?Ile?Gly?Ala?Ser?Glu?Val?Phe
465 470 475 480
Met?Tyr?Val?Gly?Gln?Leu?Glu?Phe?Phe?Asn?Ser?Gln?Ala?Pro?Thr?Gly
485 490 495
Leu?Lys?Ser?Phe?Ala?Ser?Ala?Leu?Cys?Met?Ala?Ser?Ile?Ser?Leu?Gly
500 505 510
Asn?Tyr?Val?Ser?Ser?Leu?Leu?Val?Ser?Ile?Val?Met?Lys?Ile?Ser?Thr
515 520 525
Thr?Asp?Asp?Val?His?Gly?Trp?Ile?Pro?Glu?Asn?Leu?Asn?Lys?Gly?His
530 535 540
Leu?Glu?Arg?Phe?Tyr?Phe?Leu?Leu?Ala?Gly?Leu?Thr?Ala?Ala?Asp?Phe
545 550 555 560
Val?Val?Tyr?Leu?Ile?Cys?Ala?Lys?Trp?Tyr?Lys?Tyr?Ile?Lys?Ser?Glu
565 570 575
Ala?Ser?Phe?Ser?Glu?Ser?Val?Thr?Glu?Glu?Glu?Glu?Val
580 585
<210>4
<211>2146
<212>DNA
＜213〉artificial sequence
<400>4
actctcaata?tcactattaa?atatctttct?cttttcactt?cttcactttc?ctcgattcct 60
gagttttcga?tccttttgtt?ctctaaccac?cggtttcctc?tttaaccggt?ctctggataa 120
tcatcataat?cattaggatc?atcaagataa?ggaatctttt?tgagtatatc?tctacaccaa 180
atcgaatcta?tggatcaaaa?agttagacag?tttgaggttt?gcactcaaga?cggtagcgtt 240
gatcgtcacg?gcaatccagc?tatccgagct?aataccggca?aatggctcac?tgctattctc 300
attctagtga?atcaaggact?agctacgctt?gcgttcttcg?gtgtaggagt?gaatttggtt 360
ctgtttctga?ctcgagtgat?gggacaagac?aatgcagaag?cggctaataa?tgttagtaaa 420
tggacaggaa?ctgtctatat?cttctctttg?cttggtgctt?tcctcagtga?ctcttattgg 480
ggacgttaca?agacttgtgc?tatctttcaa?gcaagtttcg?ttgcagggtt?gatgatgtta 540
tctttatcta?ctggtgcgtt?attgcttgaa?ccaagtggtt?gtggagttga?agattcgccg 600
tgtaagcctc?attcgacgtt?taagacggtt?ctgttttatc?tgtcggtgta?tctaatcgcg 660
ttagggtatg?gtggttatca?gccgaacata?gctacttttg?gagctgatca?gtttgatgcg 720
gaggattccg?ttgaaggaca?ctcgaaaatc?gcgtttttca?gttactttta?cttggctctg 780
aatcttggat?cgctcttctc?aaatactgtc?ttgggttact?ttgaggatca?aggggaatgg 840
ccgcttggat?tttgggcgtc?tgctggctct?gcttttgcgg?ggttagtgct?tttcttgatt 900
ggcacgccaa?agtaccgaca?ctttacgcct?agagagagtc?cttggtctag?attctgccaa 960
gtgttggttg?ctgcaacaag?aaaggctaag?attgatgtgc?accatgaaga?gttgaatctc 1020
tatgattctg?agactcaata?cactggagat?aagaagattc?ttcataccaa?aggcttcaga 1080
ttcttggata?gagctgctat?tgttacacct?gatgatgagg?ctgagaaagt?agagagcgga 1140
tcgaaatacg?atccatggag?gctctgctcg?gtgactcaag?tcgaagaagt?gaaatgtgta 1200
ttaagactct?taccaatctg?gctctgcacc?atcctctact?ctgtggtttt?cacccaaatg 1260
gcttcactgt?tcgttgtgca?aggagcagcg?atgaagacaa?acatcaaaaa?cttccggatt 1320
ccagcttcaa?gcatgtctag?tttcgacatt?ctcagtgtcg?ccttcttcat?cttcgcatac 1380
aggcggtttc?ttgatccact?ctttgcaaga?cttaacaaaa?cagaacgcaa?caaaggtctc 1440
actgagcttc?agaggatggg?gattgggctt?gtgattgcga?taatggcgat?gatttccgca 1500
ggaatcgtag?agatacacag?actgaaaaat?aaggaaccgg?aaagtgctac?ttcgatctca 1560
agctccagca?ctttgagcat?tttttggcaa?gtacctcagt?acatgttgat?aggtgcatca 1620
gaagtgttca?tgtacgttgg?tcaactcgag?ttcttcaaca?gccaagcacc?aaccgggcta 1680
aagagctttg?caagcgcgct?atgtatggct?tcaatatctc?ttgggaacta?tgtaagcagt 1740
ttgttagttt?ccattgtcat?gaagatctct?acaacagatg?atgtgcatgg?ctggattcct 1800
gaaaatctca?acaaaggaca?cttggagaga?ttctacttcc?ttttagctgg?tctaaccgct 1860
gctgatttcg?tggtttactt?gatttgtgcc?aaatggtaca?agtatattaa?atctgaagca 1920
agtttctctg?agtccgtaac?tgaagaggag?gaagtctgag?tcaggagcta?gagagttttg 1980
tgttttattt?taatgctgtg?aagcttttta?cagtaatttg?tttaaagctg?agcttgttct 2040
gtgatataaa?ctggtcttat?tgtgttttta?tttttgggtt?atactcttgt?gcaaagttta 2100
gttgatgttt?ctcaataaag?gataagttga?ttccacagtt?ccaaca 2146
<210>5
<211>2146
<212>DNA
＜213〉artificial sequence
<400>5
tactctcaat?atcactatta?aatatctttc?tcttttcact?tcttcacttt?cctcgattcc 60
tgagttttcg?atccttttgt?tctctaacca?ccggtttcct?ctttaaccgg?tctctggata 120
atcatcataa?tcattaggat?catcaagata?aggaatcttt?ttgagtatat?ctctacacca 180
aatcgaatct?atggatcaaa?aagttagaca?gtttgaggtt?tgcactcaag?acggtagcgt 240
tgatcgtcac?ggcaatccag?ctatccgagc?taataccggc?aaatggctca?ctgctattct 300
cattctagtg?aatcaaggac?tagctacgct?tgcgttcttc?ggtgtaggag?tgaatttggt 360
tctgtttctg?actcgagtga?tgggacaaga?caatgcagaa?gcggctaata?atgttagtaa 420
atggacagga?actgtctata?tcttctcttt?gcttggtgct?ttcctcagtg?actcttattg 480
gggacgttac?aagacttgtg?ctatctttca?agcaagtttc?gttgcagggt?tgatgatgtt 540
atctttatct?actggtgcgt?tattgcttga?accaagtggt?tgtggagttg?aagattcgcc 600
gtgtaagcct?cattcgacgt?ttaagacggt?tctgttttat?ctgtcggtgt?atctaatcgc 660
gttagggtat?ggtggttatc?agccgaacat?agctactttt?ggagctgatc?agtttgatgc 720
ggaggattcc?gttgaaggac?actcgaaaat?cgcgtttttc?agttactttt?acttggctct 780
gaatcttgga?tcgctcttct?caaatactgt?cttgggttac?tttgaggatc?aaggggaatg 840
gccgcttgga?ttttgggcgt?ctgctggctc?tgcttttgcg?gggttagtgc?ttttcttgat 900
tggcacgcca?aagtaccgac?actttacgcc?tagagagagt?ccttggtcta?gattctgcca 960
agtgttggtt?gctgcaacaa?gaaaggctaa?gattgatgtg?caccatgaag?agttgaatct 1020
ctatgattct?gagactcaat?acactggaga?taagaagatt?cttcatacca?aaggcttcag 1080
attcttggat?agagctgcta?ttgttacacc?tgatgatgag?gctgagaaag?tagagagcgg 1140
atcgaaatac?gatccatgga?ggctctgctc?ggtgactcaa?gtcgaagaag?tgaaatgtgt 1200
attaagactc?ttaccaatct?ggctctgcac?catcctctac?tctgtggttt?tcacccaaat 1260
ggcttcactg?ttcgttgtgc?aaggagcagc?gatgaagaca?aacatcaaaa?acttccggat 1320
tccagcttca?agcatgtcta?gtttcgacat?tctcaatgtc?gccttcttca?tcttcgcata 1380
caggcggttt?cttgatccac?tctttgcaag?acttaacaaa?acagaacgca?acaaaggtct 1440
cactgagctt?cagaggatgg?ggattgggct?tgtgattgcg?ataatggcga?tgatttccgc 1500
aggaatcgta?gagatacaca?gactgaaaaa?taaggaaccg?gaaagtgcta?cttcgatctc 1560
aagctccagc?actttgagca?ttttttggca?agtacctcag?tacatgttga?taggtgcatc 1620
agaagtgttc?atgtacgttg?gtcaactcga?gttcttcaac?agccaagcac?caaccgggct 1680
aaagagcttt?gcaagcgcgc?tatgtatggc?ttcaatatct?cttgggaact?atgtaagcag 1740
tttgttagtt?tccattgtca?tgaagatctc?tacaacagat?gatgtgcatg?gctggattcc 1800
tgaaaatctc?aacaaaggac?acttggagag?attctacttc?cttttagctg?gtctaaccgc 1860
tgctgatttc?gtggtttact?tgatttgtgc?caaatggtac?aagtatatta?aatctgaagc 1920
aagtttctct?gagtccgtaa?ctgaagagga?ggaagtctga?gtcaggagct?agagagtttt 1980
gtgttttatt?ttaatgctgt?gaagcttttt?acagtaattt?gtttaaagct?gagcttgttc 2040
tgtgatataa?actggtctta?ttgtgttttt?atttttgggt?tatactcttg?tgcaaagttt 2100
agttgatgtt?tctcaataaa?ggataagttg?attccacagt?tccaac 2146
Claims (10)
1.AtNRT1.8 gene or its encoded protein matter or polypeptide are strengthening plant to the resistance of heavy metal, to the resistance of salt stress or to the purposes in the resistance of arid.
2. purposes as claimed in claim 1 is characterized in that, the sequence of described protein or polypeptide is selected from:
(a) SEQ ID NO:3; Or
(b) in the aminoacid sequence that (a) limits, strengthen plant active to the resistance of heavy metal or salt stress by (a) deutero-protein or polypeptide through replacing, lack or adding one or several amino acid and have.
3. purposes as claimed in claim 2 is characterized in that, described AtNRT1.8 gene is the sequence of coding described protein of claim 2 or polypeptide.
4. purposes as claimed in claim 3 is characterized in that, described AtNRT1.8 gene is selected from:
(i) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5; Or
(ii) under stringent condition with the sequence hybridization that (ii) limits and have and strengthen the active molecule of plant to the resistance of heavy metal or salt stress.
5. purposes as claimed in claim 1 is characterized in that, described enhancing plant shows as the resistance of heavy metal: reduce the heavy metal content in the plant.
6. purposes as claimed in claim 1, it is characterized in that described heavy metal, salt stress or arid comprise by coercing that following material or situation cause: cadmium, lead, mercury, arsenic, copper, chromium, antimony, tin, zinc, barium, bismuth, nickel, cobalt, manganese, iron, vanadium, sodium-chlor, sodium sulfate, yellow soda ash, sodium bicarbonate or lack of water.
7. carrier, described carrier contains the AtNRT1.8 gene.
8. genetically engineered host cell, described host cell contains the described carrier of claim 7.
9. method for preparing transgenic plant, described method comprises:
(1) with the construction transformed plant cells cell, tissue or the organ that contain the AtNRT1.8 gene;
(2) selection changes vegetable cell, tissue or the organ of AtNRT1.8 gene over to; With
(3) vegetable cell, tissue or neomorph in the step (2) are become plant,
Wherein, the transgenic plant of gained strengthen to some extent to the more unconverted plant of the resistance of heavy metal or salt stress.
10. the purposes of the transgenic plant that make with the described method of claim 9, described transgenic plant are used for producing: the agricultural-food, heavy metal contamination that do not have heavy metal contamination are coerced crop or are drought-resistantly coerced crop than agricultural-food, anti-heavy metal stress crop, the salt tolerant of the non-transgenic plant reduction of the following plantation of the same terms.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100466509A CN101818168B (en) | 2009-02-26 | 2009-02-26 | Application of AtNRT1.8 gene to strengthening resistance of crops to stress of heavy metals or salts |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100466509A CN101818168B (en) | 2009-02-26 | 2009-02-26 | Application of AtNRT1.8 gene to strengthening resistance of crops to stress of heavy metals or salts |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101818168A true CN101818168A (en) | 2010-09-01 |
| CN101818168B CN101818168B (en) | 2012-07-04 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100466509A Expired - Fee Related CN101818168B (en) | 2009-02-26 | 2009-02-26 | Application of AtNRT1.8 gene to strengthening resistance of crops to stress of heavy metals or salts |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101818168B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255146A (en) * | 2013-02-06 | 2013-08-21 | 中国农业科学院作物科学研究所 | Arabidopsis thaliana nitrate transporter gene NRT2.1, encoding protein and applications thereof |
| CN105385696A (en) * | 2015-11-17 | 2016-03-09 | 江苏省农业科学院 | Tea tree NRT1 gene, protein and gene expression method |
| CN110885839A (en) * | 2019-12-31 | 2020-03-17 | 河南农业大学 | Corn arsenic stress resistance gene ZmASR1, and primer, coding product, linkage SNP and application thereof |
-
2009
- 2009-02-26 CN CN2009100466509A patent/CN101818168B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103255146A (en) * | 2013-02-06 | 2013-08-21 | 中国农业科学院作物科学研究所 | Arabidopsis thaliana nitrate transporter gene NRT2.1, encoding protein and applications thereof |
| CN105385696A (en) * | 2015-11-17 | 2016-03-09 | 江苏省农业科学院 | Tea tree NRT1 gene, protein and gene expression method |
| CN110885839A (en) * | 2019-12-31 | 2020-03-17 | 河南农业大学 | Corn arsenic stress resistance gene ZmASR1, and primer, coding product, linkage SNP and application thereof |
| CN110885839B (en) * | 2019-12-31 | 2022-06-28 | 河南农业大学 | Corn arsenic stress resistance gene ZmASR1, and primer, coding product, linkage SNP and application thereof |
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