CN101037693A - New CkNHX gene and shearing decorative gene CkNHXn, method for cultivating inverse-resistant plant - Google Patents
New CkNHX gene and shearing decorative gene CkNHXn, method for cultivating inverse-resistant plant Download PDFInfo
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Abstract
The invention provides a novel CkNHX genen and excision adorn gene CkNHXn thereof. The gene stems from Caragana korshinskii Kom, its gene codes Na+/H+ antiporter gene. The cDNA obtained by clone constructs yeast expression carrier and plant expression carrier. The yeast mutant strain vertifies salt resistance thereof by complament experiment. Moreover, a transgenic plant is also obtained. By testing salt resistance of transformed plant, transformation of the gene can make the transgenic plant with salt resistance.
Description
Technical field
The present invention relates to a kind of Na
+/ H
+Counter transport PROTEIN C kNHX and modified protein CkNHXn.Particularly, the present invention relates to a kind of NHX class Na that derives from desert shrub plant caragana microphylla (Caragana korshinskii Kom)
+/ H
+Counter transport albumen, called after CkNHX, with and modified protein CKNHXn.The invention still further relates to this class of coding Na
+/ H
+The proteic gene of counter transport contains the carrier of this genoid and utilizes this genoid to cultivate the method for salt-tolerant plant.
Background technology
Because the variation of ecotope, add the salinization in arid, short of rain and soil, can cause the big area salinification that can till the land, have a strong impact on human existence (Wang W X, Vinocur B, AltmanA (2003) .Plant responses to drought, salinity and extremetemperatures:towards genetic engineering for stress tolerance.Planta, 218:1-14).How to improve the saltings, safeguard ecotope, suppress soil erosion, restore vegetation, cultivating the resistance of reverse plant is one of major objective of crop, the breeding of woods herbage.Especially traditional forest salt tolerant, drought resisting anti-adversity (being) genetic improvement approach mainly adopt methods such as cross-breeding, selection breeding, but because the forest tree propagation cycle is long, the genetic heterozygosity height, the hereditary mechanism of many proterties is not clear, makes that utilizing the conventional breeding means to be difficult to directive breeding goes out to have the forest new variety of salt-tolerance character.Genetic engineering technique is combined with the conventional breeding method, be expected cultivating the new seeds of genetically modified anti-salt, for long-range, the efficient and lasting breeding in China western part provides safeguard.
Na
+/ H
+The counter transport protein gene coding be a class ionic membrane translocator that is positioned on the vacuole skin, can pass through in the intracellular region territoryization sodium ion, keep ion running balance in the born of the same parents, greatly eliminate this class ion and coerce influence and destruction normal physiological metabolism of plant and cell tissue structure, improve resistance of reverse (the Apse M P of plant, Aharon G S, Snedden W A, Blumwald E (1999) .Salttolerance conferred by overexpression of a vacuolar Na
+/ H
+Antiporterin Arabidopsis.Science, 285:1256-1258).Such assignment of genes gene mapping is on the vegetation water vacuolar membrane, can strengthen salt tolerance (the Venema K of plant, Quintero F J, Pardo J M, and DonaireJ P (2002) The Aaradbidopsis Na+/H+exchanger AtNHX1 catalyzes lowaffinity Na+and K+transport in reconstituted liposomes.JBC 277 (4): 2413-2418).Do not retrieve at present gene C kNHX as yet from the coding NHX of shrub plant caragana microphylla.
The contriver separates, is cloned into coding NHX class Na from desert shrub plant caragana microphylla (Caragana korshinskii Kom)
+/ H
+The proteic gene of counter transport, called after CkNHX, coded albumen called after CkNHX.In addition, this albumen has been carried out shearing modification, made up than the active high CkNHXn albumen of CkNHX albumen salt tolerant.In addition, make up the expression vector that is applicable to single, double cotyledon plant respectively, transformed dicotyledonous model plant tobacco and woody model plant Cortex Populi Tomentosae.Transfer-gen plant has tangible salt tolerance.
Summary of the invention
An object of the present invention is to provide a kind of CkNHX gene with strong salt tolerance, this gene source is in caragana microphylla (Caragana korshinskii Kom), and its nucleotide sequence and aminoacid sequence are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively.Should be pointed out that NHX class Na of the present invention
+/ H
+Counter transport albumen carries out the amino acid whose replacement of one or several non-conservative region, insertion or lacks resulting functional analogue also reaching purpose of the present invention.Thereby the present invention also comprises with the aminoacid sequence shown in the SEQ ID NO:2 having at least 90% homology, especially has at least 95% homology, but has the functional analogue of CkNHX gene activity in the caragana microphylla simultaneously.
Another object of the present invention provides the gene of a kind of caragana microphylla CkNHX that encodes, at the gene shown in the SEQ ID NO:1 is the NHX genoid that derives from caragana microphylla, contain the conservative Na_H_exchanger structural domain sequence of NHX genoid, but be not limited to listed sequence.Because the coded albumen of this genoid is positioned on the leaf vacuolar membrane, is plant membrane albumen, difficult in the world at present this class purifying protein of acquisition, but can adopt complementary method to verify the protein function of this coded by said gene.The yeast function complementation experiment of this gene shows the salt tolerance that can improve yeast salt tolerant sensitive strain really that changes over to of this gene.
Another object of the present invention provides the shearing gene of a kind of caragana microphylla CkNHXn that encodes, described sequence is seen SEQ ID NO:3, the aminoacid sequence of its encoded protein is seen SEQ ID NO:4, and its yeast complementation experiment presents the salt tolerant activity strong than total length CkNHX albumen.
Persons of ordinary skill in the art may appreciate that gene of the present invention is carried out replacement, the insertion of one or several Nucleotide or lack resulting analogue with its identical function being also contained in equally in the scope of the present invention.
A further object of the present invention provides a kind of method of cultivating salt-tolerant plants, and this method comprises with gene C kNHX of the present invention or CkNHXn structure plant expression vector; Transform plant tissue with the expression vector that makes up; The plant transformed tissue cultivating is become plant.
Another purpose of the present invention provides a kind of expression vector that comprises gene C kNHX of the present invention or CkNHXn.Can use any expression vector that can guide foreign gene in plant, to express.These plant expression vectors include but not limited to, the double base agrobacterium vector, and pBIN19 for example, pBI121, pB221 contains the promotor of DRE, ARE element and the carrier that is used for the monocotyledons micropellet bombardment.Carrier also can comprise 3 ' untranslated zone.3 ' untranslated zone is meant a kind of part of gene, but it contain a kind of comprise the polyadenylic acid signal and any other effect mRNA precursor processing or the dna fragmentation of genetic expression.3 ' untranslated zone of CkNHX gene of the present invention can be used to express in plant.
Carrier of the present invention also can contain suitable promotor.Can use any strong promoter in the present invention.These promotors include but not limited to cauliflower mosaic virus (CaMV 35S) and Ubiqutin promotor.It can use separately or be used in combination with other plant promoter.
Expression vector of the present invention also can comprise enhanser when needed, no matter be translational enhancer or transcriptional enhancer.These enhanser zones include but not limited to ATG initiator codon and neighboring region.Initiator codon must with the reading frame of coding region in the same way, to guarantee the translation of whole sequence.The translation control signal can be multiple different source with initiator codon, can be natural or synthetic.Translation initiation region can be from the transcription initiation zone, or from structure gene.
For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, comprise adding the alternative mark of plant.Spendable selected marker comprises the enzyme to antibiotics resistance, and microbiotic comprises gentamicin, Totomycin, kantlex etc.Equally, can use the enzyme that produces the compound of discerning by colour-change (for example GUS) or luminous (for example luciferase), or anti-chemical reagent (for example removing the agent of withering).In addition, also can any selection markers.
Expression vector of the present invention can be by using Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, importing such as electroporation, laser beam perforation vegetable cell.
Can use method plant transformed host of the present invention to comprise monocotyledons and dicotyledons, for example: willow, mesquite, graminous pasture and crop (wheat, corn, paddy rice), clover, soybean, rape, cotton, peanut etc.
Description of drawings
Fig. 1 .CkNHX gene cDNA full length sequence, SEQ ID NO:1.
The aminoacid sequence of Fig. 2 .CkNHX coded by said gene, SEQ ID NO:2.
The coded membranin of Fig. 3 .CkNHX stride the topological diagram that film and phosphorylation site distribute, be phosphorylation site shown in the circle wherein, the italic numeral is the amino acid sites of phosphorylation, all the other are the possible film place amino acid sites of striding; CYT is expressed as the kytoplasm interface; VAC represents leaf bubble inner face.
Fig. 4 .CkNHXn gene ORF nucleotide sequence, SEQ ID NO:3.
The aminoacid sequence of Fig. 5 .CkNHXn coded by said gene, SEQ ID NO:4.
Fig. 6 .CkNHX gene 5 ' UTR nucleotide sequence, SE9 ID NO:12.
Fig. 7 .CkNHX gene 3 ' UTR nucleotide sequence, SEQ ID NO:13.
The design of graphics of the Yeast expression carrier pYES2-NHX of Fig. 8 .CkNHX.
The design of graphics of the Yeast expression carrier pYES2-NHXn of Fig. 9 .CkNHXn.
Figure 10. change the yeast salt tolerant complementation test of CkNHX and CkNHXn gene.
Figure 11 .CkNHX plant expression vector pCkNHX design of graphics.
Figure 12. the tobacco plant of Agrobacterium transfection CkNHX, transgene tobacco screens used antibiotic concentration, Kan 600mg/L, Cb 500mg/L.
The PCR of Figure 13 .CkNHX transgene tobacco identifies.
The RT-PCR of Figure 14 .CkNHX transgene tobacco identifies.
Figure 15. the salt tolerance experiment of tobacco plant, (A): the salt tolerant test of Nicotiana gossei; (B): the NaCl resistance test of changeing the tobacco 300mM of CkNHX.
Embodiment
Describe the present invention in detail below by embodiment.Those of ordinary skill in the art should be appreciated that following embodiment is not the purpose that is used to limit, and substantial protection domain of the present invention is limited by accompanying Claim.
Agents useful for same is as follows among the embodiment:
High frequency high fidelity Pyrobest Taq, Premix Ex Taq, AMV, RNA enzyme inhibitors, various restriction enzyme, One Step RNA PCR test kit, pMD18-T Vector are available from precious (TaKaRa) company of giving birth in Dalian; The DNA purifying reclaims test kit available from ancient cooking vessel state biotech company; T4 dna ligase and RNase A are available from MBI company; ABA, DNase (RNase free) are available from Sigmag company.
The clone of embodiment 1, caragana microphylla CkNHX gene
Get 2-3 month caragana microphylla seedling (forming) of growth for material, spend the night and induce with 100uM ABA (Sigma company) available from eremophyte garden, Turfan, Chinese Academy of Sciences Xinjiang caragana microphylla seed germination.Get blade, after cleaning, put into the homogenizer after DEPC handles, sterilizes, add TRIzol (Invitrogen) grinding, homogenate, room temperature was placed 5 minutes, added chloroform extracting twice, got supernatant, with the total RNA of isopropanol precipitating, after the dry air, be dissolved in an amount of DEPC water.Primer with band polyT carries out reverse transcription acquisition cDNA.
The primer sequence of polyT is as follows:
Oligo?dT-Primer：5’-CGAGCGGCCGCCCGGGCAGGTTTTTTTTTTTTTTTT-3’(SEQ?IDNO：5)
The RT reaction conditions is as follows:
DEPC water 85uL
RNase inhibitor (40U/uL) 5uL
Oligo?dT-Primer 10uL
RNA 10uL
110uL altogether
Bathed 10 minutes in 65 ℃ of temperature, immediately in quenching, adding then on ice
MgCl
2 40uL
10XRNA?Buffer 20uL
dNTP?mixture 20uL
AMV 10uL
90uL altogether
Reaction volume is 200uL altogether
Reaction conditions is 1.30 ℃, 10ain; 2.42 ℃, 1h; 3.99 ℃, 5min; 4.5 ℃, 5min.
Get the following 9 pairs of primers of an amount of above-mentioned reverse transcription product (three forward primer and three reverse primer combinations) and carry out pcr amplification (designed primer is the nest-type PRC amplimer, and the amplified fragments that obtains has overlapping mutually).
Forward primer:
L1 5’-GCAGGGTTTCAGGTGAAAAAGAAGCA-3’(SEQ?ID?NO：6)
L2 5’-GAAAAATGGAAGTTTGTTAGTGATAGTCC-3’(SEQ?ID?NO：7)
L3 5’-CACCATGTCGGGGCACACTCAAC-3’(SEQ?ID?NO：8)
Reverse primer:
R1 5’-GTACAATGATGATGATGATGGGTCAGAG-3’(SEQ?ID?NO：9)
R2 5’-AGATGGCTTATGGTACACGGCCTTCTCG-3’(SEQ?ID?NO：10)
R3 5’-CTTCTCAACGCCATTGATGACCATTG-3’(SEQ?ID?NO：11)
The PCR condition
1.?95℃ 1min
2.?94℃ 30s
3.?60℃ 40s
4. 2 29 circulations of 72 ℃ of 2min
5.?72℃ 10min
6. 4 ℃ of termination reactions
(L1 is about 1.5Kb respectively with the fragment that R1, R2, R3 combination are increased respectively with the PCR product; L2 is about 0.8Kb respectively with the fragment that R1, R2, R3 combination are increased respectively; L3 is about 0.5Kb respectively with the fragment of R1, R2, R3 combination amplification respectively) be connected respectively to and be used for order-checking on the pUCm-T carrier (MBI company), after adopting public DNAMAN software known in this field to carry out sequence assembly above-mentioned 9 sequencing sequences, compare with known NHX genoid (AF515632) in the ncbi database on the World Wide Web, what prove conclusively that above-mentioned PCR is cloned into is NHX genoid fragment.
Be that the following primer of stencil design carries out 5 ', 3 '-RACE amplification respectively with this section from above-mentioned reverse transcription reaction fragment through the sequence of splicing again, obtain gene 5 ' and 3 ' terminal sequence (SEQ ID NO:12 and SEQ ID NO:13).The concrete grammar operation is referring to the BDSMART of BD Biosciences Clontech
TMRACE cDNA Amplification Kit (Cat.No.634914).
5 '-RACE primer
5’-CGCCCAGTTCCAGTGGCCCAATATCCA-3’(SEQ?ID?NO：14)
3 '-RACE primer
5’-CTTGGAAGAGCAGCTTTTGTTTTCCCC-3’(SEQ?ID?NO：15)
On the basis of gene 5 ' and 3 ' sequence, design following Auele Specific Primer and adopt high-fidelity Pyrobest Taq to carry out pcr amplification acquisition gene ORF total length.
Forward primer (contain BamH I restriction enzyme site, illustrate) with underscore
5’-GCC
GGATCCCGTCTTTCTCCTCTGTCTC-3’(SEQ?ID?NO：16)
Reverse primer (contain Sac I restriction enzyme site, illustrate) with underscore
5’-CGCG
GAGCTCAATGATGATGATGATGGGTCAGAG-3’(SEQ?ID?NO：17)
The PCR reaction conditions is as follows:
Above-mentioned reverse transcription cDNA template plasmid 1 μ L
Each 1 μ L of forward and reverse primer
dNTP 2μL
ddH
2O 13.8μL
Pyrobest?Taq 0.2μL
10×buffer 2μL
Total： 20μL
The PCR program is:
a)95℃?2min
b)94℃?30s
c)55℃?1min
d)72℃?3min 2 29cycles
e)72℃?10min
f)4℃?pause
The CkNHX gene cds full length sequence that is obtained is 2547bp (SEQ ID NO:1), contain the 1618bp open reading frame, the polypeptide (SEQ ID NO:2) that 539 amino acid of encoding are formed, its 5 ' end is 652bp, 3 ' end is 262bp, and it is cloned among the pMD18-T Vector.
The structure of embodiment 2, CkNHXn gene
Reference report (Yamaguchi T, Apse M P, Shi H Z, and Blumwald E (2003) .Topological analysis of a plant vacuolar Na+/H+antiporter revealsa lumihal C terminus that regulates antiporter cation selectivity.PNAS.100 (21): 12510-12515), add that terminator codon forms the CkNHXn of C end brachymemma (peptide section place to go after being about to the 435th) (referring to Fig. 3) after utilizing following primer with the 435th amino acids.The primer is:
Forward primer is (containing Sac I site)
5’-CGCGCC
GAGCTCATGGCTTTTGAAATTGTCT-3’(SEQ?ID?NO：18)
Reverse primer is (containing Xba I site)
5’-CCGCGC
TCTAGACTAAGTCATCAAACCAAACACC-3’(SEQ?ID?NO：19)
Adopting high frequency high fidelity Pyrobest Taq, is that template is carried out pcr amplification with above-mentioned reverse transcription product, and method is the same.Obtained to be about 1.3kb size segmental CkNHXn gene (SEQ ID NO:3), the aminoacid sequence of its proteins encoded is shown in SEQ ID NO:4.This gene is connected into pMD18-T Vector, adopts Sac I and Xba I to carry out double digestion after the sequence verification, be connected into, be built into pYES-NHXn Yeast expression carrier (referring to Fig. 9) with the two pYES carriers of cutting (Invortrogene) of same enzyme.
1. the structure of Yeast expression carrier
Molecular biology method (work such as F. Ao Sibai, translate, fine works molecular biology experiment guide by Yan Ziying, Wang Hailin routinely.Science Press, 2001), introduce Sac I and Xba I site with round pcr respectively at CkNHX coding region and CkNHXn coding region two ends, adopt Sac I and Xba I double digestion, be connected into then with the two pYES2.0 (Invortrogene) that cut of same enzyme, be built into two Yeast expression carriers of pYES-NHX and pYES-NHXn.It makes up collection of illustrative plates shown in Fig. 8,9, through enzyme cut, PCR identifies and order-checking guarantee to insert fragment errorless after, change salt sensitive yeast mutant ANT3 over to and (see reference for details: Quintero FJ, Blattb MR, and Pardo JM (2000) .Functional conservationbetween yeast and plant endosomal Na
+/ H
+Antiporters.FEBS Lett 471,224-228).Concrete operations are with reference to Invortrogene pYES2.0protocol, Cat No.V825-20.
2. yeast salt tolerant function complementation experiment
1) unloaded pYES2.0, pYES-NHX, pYES-NHXn are changed over to yeast mutants ANT3 (Jose doctor M.Pardo present respectively, referring to document Quintero FJ, Blattb MR, andPardo JM (2000) .Functional conservation between yeast and plantendosomal Na
+/ H
+Antiporters.FEBS Lett 471,224-228), and in basic SC substratum (the detailed prescription with reference to Invortrogene pYES2.0protocol, CatNo.V825-20) 30 ℃ of overnight incubation in, be diluted to 0.3OD, induce in the SC substratum (prescription with reference to Invortrogene pYES2.0protocol, Cat No.V825-20) inducing culture 4-6hr in detail containing 2% semi-lactosi and 1% raffinose.
2) the bacterium liquid after will inducing carry out 10 times of serial gradient dilutions (1 *, 10 *, 100 *, 1000 * and 5000 *), (prescription is with reference to Invortrogene pYES2.0protocol in detail in the selectivity SC of 70mM NaCl plate culture medium for point 4 μ l, Cat No.V825-20) on, cultivated 4-5 days for 30 ℃, observe and take a picture.The results are shown in Figure 10.
In a single day because the salt sensitive yeast mutant ANT3 that obtains of institute is to salt sensitivity (promptly not salt tolerant), and in this yeast strain, change resistant gene of salt over to, can strengthen the salt resistance ability of this bacterial strain.In Figure 10, first behavior contains the growing state of yeast mutants ANT3 on 70mM NaCl substratum of empty carrier pYES2.0.As seen when bacterium liquid diluted 10 times, 100 times, it still can be grown; But when diluting 1000 times and 5000 times, then can not grow.And the bacterial strain that changes pYES-NHX and pYES-NHXn over to still can be grown when 1000 times and 5000 times of dilutions, and the pYES-NHXn bacterial strain of the third line to compare upgrowth situation better with pYES-NHX when 1000 times of dilutions.This yeast complementation experiment shows that changing over to of CkNHX and two gene orders of CkNHXn can strengthen the tolerance of this salt sensitive yeast mutant ANT3 to salt.The expressed albumen of these two gene fragments all has salt resistance ability, and expression excalation sequence C kNHXn has the salt tolerance stronger than CkNHX.
The structure of CkNHX expression vector molecular biology method routinely carries out that (F. Ao Sibai etc. work, Yan Ziying, Wang Hailin are translated, fine works molecular biology experiment guide.Science Press, 2001).The CkNHX gene that obtains with amplification among the embodiment 1 is a template, this gene ORF of pcr amplification (adopting pyrobestTaq (TaKaRa company)).
The primer is as follows:
Forward primer (containing BamH I site):
5’-CGCC
GGATCCATGGCTTTTGAAATTGTCTCAA-3’(SEQ?ID?NO：20)
Reverse primer (containing Sac I site):
5’-GTCA
GAGCTCTCAACGCCATTGATGACCATT-3’(SEQ?ID?NO：21)
PCR reaction system and condition are the same.
Obtain about 1.6Kb PCR product fragment, reclaim.Adopt BamH I and Sac I double digestion, be inserted into pBI121 binary expression vector (Chen PY, Wang CK, Soong SC, To KY (2003) Complete sequence of the binary vector pBI121 and its application incloning T-DNA insertion from transgenic plants.Molecular Breeding11:287-293) CaMV 35S promoter back in, obtain a binary expression vector, its collection of illustrative plates as shown in figure 11, after order-checking identifies that the insertion fragment is errorless, transform Agrobacterium LBA4404 (Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vetorstrategy based on separation of vir-and T-region of the Agrobacteriumtumefaciens Ti-plasmid.Nature 303:179-180), upgrading grain again, cut through enzyme, after PCR confirms, prove and successfully be transformed in the Agrobacterium, this expression vector can be directly used in plant, as tobacco, Cortex Populi Tomentosae, the conversion of plants such as graminous pasture.
The tobacco of embodiment 5, CkNHX gene transforms
1. the preparation of vegetable material
Do not containing the aseptic seedling that (Liu Minzhu, flowers tissue culture and batch production production, Geology Publishing House, in June, 2006,19-20 page or leaf) cultivates tobacco (W38) (plant institute of the Chinese Academy of Sciences) on the MS substratum of hormone, culture temperature is 25-28 ℃.
2. the cultivation of Agrobacterium
The Agrobacterium that will contain the purpose expression vector is inoculated into 20ml (30mg/L Str, 50mg/L Kan) YEP liquid nutrient medium, and (prescription is with reference to Invortrogene pYES2.0 protocol in detail, CatNo.V825-20) in, 28 ℃, 220rpm shakes bacterium 16-18h, makes Agrobacterium be cultured to logarithmic phase OD
600Value 0.6-0.8.
Get bacterium liquid 1-2mL and change 40mL over to and do not contain among the antibiotic YEP, 28 ℃, 220rpm shakes bacterium and spends the night.
3. transform
Pour bacterium liquid in the sterile petri dish (40mL/per), the blade of just drawing good wound, after putting into bacterium liquid and infecting 10-20min, with rinsed with sterile water once, the bacterium liquid on the blade is blotted slightly with sterilization filter paper, change (Liu Minzhu on the MS substratum of added with antibiotic not over to, flowers tissue culture and batch production production, the Geology Publishing House, in June, 2006,19-20 page or leaf).The blade inoculation of contaminating keeps flat and twists with the fingers substratum slightly on the MS substratum, cultivates altogether 2-4 days under 28 ℃ of dark culture condition, until seeing the Agrobacterium bacterial plaque.
4. select to cultivate:
The conversion blade that to cultivate altogether immediately, can see the Agrobacterium bacterial plaque is put into the screening division culture medium that contains microbiotic (Cb500mg/L, Kan 500mg/L), screening and culturing 3-4 week.After waiting to sprout, change in the root media that contains microbiotic (Cb 500mg/L, Kan 500mg/L).Obtain transfer-gen plant such as Figure 12.
Division culture medium is formed:
Contain 6-BA 2mg/L, NAA 0.1mg/L, Pyocianil 500mg/L, the MS substratum of kantlex 300mg/L.
Root media is formed:
Contain NAA 0.1mg/L, Pyocianil 500mg/L, the MS substratum of kantlex 300mg/L.
The tobacco transformant of embodiment 6, commentaries on classics CkNHX gene is identified
1) PCR that changes CkNHX tobacco plant genomic dna detects
(work such as F. Ao Sibai, Yan Ziying, Wang Hailin are translated, fine works molecular biology experiment guide to adopt the CTAB method.Science Press, 2001) extract the total DNA of plant, adopt the described primer of front vector construction (SEQ ID NO:20 and SEQ ID NO:21) to carry out PCR and detect result such as Figure 13.As seen has obvious pcr amplification band at the about 1.6kb of DNAMarker place, the wherein negative contrast of the 8th swimming lane (wild W38 tobacco gene group DNA); The positive contrast of the 5th swimming lane (is template with plasmid pCkNHX); The 6th swimming lane is negative contrast (is template with water); The 7th swimming lane is that (from top to bottom the DNA size is 19325,2000,1600,1000,750,500,250 to DNA MarkerDGL2000,100bp); 1-4 and 9-12 swimming lane are the PCR product of tobacco transformed plant genomic dna.This shows that the tobacco transformant that is obtained has contained this gene fragment.
2) tobacco changes CkNHX seedling RT-PCR detection
The RT-PCR that the detected positive transformed plant in front is carried out CkNHX detects, and CkNHX transcribes situation in the investigation transformed plant.According to routine techniques, adopt Trizol reagent to extract the RNA of transformed plant, adopt the one-step RT-PCR test kit of TakaRa company to carry out the RT-PCR detection, it the results are shown in Figure 14.The 1st swimming lane is that (from top to bottom the DNA size is 19325,2000,1600,1000,750,500,250 to DNA MarkerDGL2000,100bp); The positive tobacco transformed plant of 2-11 RT-PCR product; The negative contrast of the 12nd swimming lane (wild W38 tobacco), wherein the Actin primer with tobacco carries out transcriptional expression detection (being about 1Kb), and the CkNHX transcribed strand is in about 1.6Kb position.
This figure shows transcribe (the about 0.9kb) that tobacco Actin is all arranged in the positive tobacco conversion strain that is detected, and this CkNHX transcribed strand of visible 1.6Kb place in the transformant 2,5,6,7,8,9,10.This shows that CkNHX has obtained to transcribe in these transformants.
Used tobacco Actin primer sequence is as follows, and all operations is undertaken by one-stepRT-PCR test kit (DRR024A) operation instructions of TakaRa company.
Forward primer:
5’-GCTGGATTTGCTGGTGATGAT-3’(SEQ?ID?NO：22)
Reverse primer:
5’-CGATC?TGCAATGCCAGGAA-3’(SEQ?ID?NO：23)
The salt tolerance of embodiment 7, CkNHX genetic tobacco transformant is identified
With the tobacco plant of PCR and RT-PCR checking, on the substratum (MS) that has added different concns (100,200,300,400mM NaCl) NaCl, carry out the salt tolerant test.Carry out the salt tolerant test with the wild plant of same budding contrast 100,200,300, on the substratum of 400mM NaCl, to observe salt tolerance (Figure 15 A) in 42 days by a definite date.The result shows that wild plant in 42 days incubation times, can tolerate 200mM NaCl; In case and salt concn is when surpassing 300mM to 400mM, plant can't take root and begin yellow, so that death.
Figure 15 B is the salt tolerant experiment of the CkNHX transformed plant verified of front.Even as seen add the NaCl of 300mM, but change over to CkNHX still normal development of tobacco, take root growth.This explanation changes the CkNHX gene over to tobacco, can improve the salt tolerance of tobacco receptor.
The transfer-gen plant that can tolerate the NaCl of 300mM by genetically modified means acquisition is not arranged in the prior art at present as yet.
The IB056039-sequence table
SEQUENCE?LISTING
＜110〉Inst. of Genetics and Development Biology, CAS
Forestry Institue, Chinese Inst.of Forestry Sciences
<120>IB056039
＜130〉new CkNHX gene and shear modifying factor CkNHXn, and the method for cultivating plant with adverse resistance
<160>23
<170>PatentIn?version?3.1
<210>1
<211>2547
<212>DNA
＜213〉caragana microphylla (Caragana korshinskii Kom)
<400>1
cccgtctttc?tcctctgtct?ctttctttct?gtccacacca?cacggctctc?cctctctctc 60
tecctctctc?tctctctcta?aacctttgtc?ttccattttc?tctatatttc?cagagctttc 120
gttgccctct?ctttctctca?ctgtgactca?aattaataaa?aaaaatatct?cttaattttc 180
tttccttttc?gatttctcgt?tccttttcca?gctacctccc?ttgagcttca?cgtttatttt 240
aattctatta?atccctgctc?tgttggttca?ttctctattt?tcgtcttcgt?ttcgagcttg 300
atccctgcat?aattgttttg?gtggaacaat?tgcagagagc?tgaaaagtgt?tccaatttaa 360
cgggaattcg?aacagggggt?tggttcttag?ctttctctgt?tcgaattctt?tggacgttgg 420
gttccttgtg?tttttttaaa?tactcagaaa?aatcacagac?tcttcttcgc?aatgctgcta 480
tcaagctttc?tgagattctt?ttgttgacga?gatctgaagg?ttcttaaaat?ggatagctcg 540
gaaacataaa?tttggatcat?ttatactgga?ctttgaactt?ggcaactaag?caataatctg 600
aatctgtgtt?taaatctgct?tttgcaatta?tttattgtgg?agggtggacg?agatggcttt 660
tgaaattgtc?tcaaaattgc?aaaatctatc?cacttctgac?catgcctccg?tagtctccat 720
gaacttgttt?gtggcacttc?tatgtgcttg?tattgtagtt?ggccatcttc?ttgaggagaa 780
tcggtggatg?aatgagtcca?tcactgccct?tttgattggt?ctttgcactg?gcataatcat 840
tttgctgttt?agtggtggta?aaagctcgca?tattcttgtt?ttcagtgaag?atcttttctt 900
tatatacctt?ctgccgccta?taatattcaa?tgctgggttt?caggtgaaaa?agaagcagtt 960
ttttgtcaac?ttcatcacta?tcgtgtcgtt?tggggctatt?ggtacattag?tatcgtgtgt 1020
catcataact?ttgggtgtta?tgtatgcttt?taagaggatg?gatattgggc?cactggaact 1080
gggcgattat?ctagcaattg?gagcaatatt?tgccgcgaca?gattctgttt?gcacgttgca 1140
ggtgctaaat?caggatgaga?cacctttgct?gtacagtctt?gtatttgggg?agggtgttgt 1200
The IB056039-sequence table
gaacgatgct?acatcagtgg?tgcttttcaa?tgcaatccaa?agctttgacc?tcaaccgact 1260
taactcttta?attgctttgc?actttttggg?caatttcttg?tatttgttta?ttgcaagcac 1320
actgcttgga?gttttgacag?gtctgttcag?tgcttacgtt?attaaaaagc?tgtacattgg 1380
caggcactct?acagatcgtg?aggttgctct?tatgatgcta?atggcatacc?tctcctacat 1440
gctggctgaa?ttaagctatc?tgagtggcat?tctcactgta?ttcttttgtg?ggattgtcat 1500
gtctcattat?acttggcata?atgtgactga?gagttcaaga?atcactacca?agcatgcttt 1560
tgctaccttg?tcgtttgttg?ctgagatatt?tatcttcctt?tatgttggta?tggatgccct 1620
ggacattgaa?aaatggaagt?ttgttagtga?tagtcctgga?acatctgtag?cagcaagttc 1680
agtattgttg?ggtctagtcc?ttcttggaag?agcagctttt?gttttcccct?tatccttctt 1740
atccaacttg?accaaaaaat?caccgtatga?gaaaatctcc?ttcagacagc?aagtgatcat 1800
ttggtgggct?ggtcttatga?gaggtgctgt?ttcaatggca?ctcgcttata?atcagttcac 1860
catgtcgggg?cacactcaac?tgcgatgcaa?tgcaatcatg?atcactagca?ccatcactgt 1920
tgtgcttttc?agcacagtgg?tgtttggttt?gatgactaag?ccactcataa?ggcttttact 1980
acctcatcct?aaagcgacaa?acagcatgac?aaccacagaa?tcatctactc?caaaatcagc 2040
cattgtccca?cttctaggaa?gtgcccaaga?ttctgaagcc?gatcttgatg?gccatgagat 2100
tcatcgtccg?agcagtattc?gtgccttact?atccactcca?acgcacactg?ttcatcgatt 2160
atggcgaagg?tttgatgatt?cattcatgcg?tcctgttttt?ggtggcaggg?gttttgttcc 2220
tgtggaacct?ggctcaccaa?gtgaacgcaa?tggtcatcaa?tggcgttgag?aagaaagcca 2280
tgaaatatgt?aatgtgtgtt?gtaaactacg?tatgattgta?tgatttgtga?agatcaagca 2340
atgtatgtat?aataagaatc?acgtaatgaa?attttgttta?gttttttgtg?tagctagtac 2400
gagaaggccg?tgtaccataa?gccatctctg?cctcttctgt?aaactacaat?atttattaga 2460
ttctctgacc?catcatcatc?atcattgtac?taaaaatttg?attagctgaa?gctaaatatt 2520
ttcctttcca?caaaaaaaaa?aaaaaaa 2547
<210>2
<211>538
<212>PRT
＜213〉caragana microphylla (Caragana korshinskii Kom)
<400>2
Met?Ala?Phe?Glu?Ile?Val?Ser?Lys?Leu?Gln?Asn?Leu?Ser?Thr?Ser?Asp
1 5 10 15
His?Ala?Ser?Val?Val?Ser?Met?Asn?Leu?Phe?Val?Ala?Leu?Leu?Cys?Ala
20 25 30
Cys?Ile?Val?Val?Gly?His?Leu?Leu?Glu?Glu?Asn?Arg?Trp?Met?Asn?Glu
35 40 45
Ser?Ile?Thr?Ala?Leu?Leu?Ile?Gly?Leu?Cys?Thr?Gly?Ile?Ile?Ile?Leu
50 55 60
The IB056039-sequence table
Leu?Phe?Ser?Gly?Gly?Lys?Ser?Ser?His?Ile?Leu?Val?Phe?Ser?Glu?Asp
65 70 75 80
Leu?Phe?Phe?Ile?Tyr?Leu?Leu?Pro?Pro?Ile?Ile?Phe?Asn?Ala?Gly?Phe
85 90 95
Gln?Val?Lys?Lys?Lys?Gln?Phe?Phe?Val?Asn?Phe?Ile?Thr?Ile?Val?Ser
100 105 110
Phe?Gly?Ala?Ile?Gly?Thr?Leu?Val?Ser?Cys?Val?Ile?Ile?Thr?Leu?Gly
115 120 125
Val?Met?Tyr?Ala?Phe?Lys?Arg?Met?Asp?Ile?Gly?Pro?Leu?Glu?Leu?Gly
130 135 140
Asp?Tyr?Leu?Ala?Ile?Gly?Ala?Ile?Phe?Ala?Ala?Thr?Asp?Ser?Val?Cys
145 150 155 160
Thr?Leu?Gln?Val?Leu?Asn?Gln?Asp?Glu?Thr?Pro?Leu?Leu?Tyr?Ser?Leu
165 170 175
Val?Phe?Gly?Glu?Gly?Val?Val?Asn?Asp?Ala?Thr?Ser?Val?Val?Leu?Phe
180 185 190
Asn?Ala?Ile?Gln?Ser?Phe?Asp?Leu?Asn?Arg?Leu?Asn?Ser?Leu?Ile?Ala
195 200 205
Leu?His?Phe?Leu?Gly?Asn?Phe?Leu?Tyr?Leu?Phe?Ile?Ala?Ser?Thr?Leu
210 215 220
Leu?Gly?Val?Leu?Thr?Gly?Leu?Phe?Ser?Ala?Tyr?Val?Ile?Lys?Lys?Leu
225 230 235 240
Tyr?Ile?Gly?Arg?His?Ser?Thr?Asp?Arg?Glu?Val?Ala?Leu?Met?Met?Leu
245 250 255
Met?Ala?Tyr?Leu?Ser?Tyr?Met?Leu?Ala?Glu?Leu?Ser?Tyr?Leu?Ser?Gly
260 265 270
Ile?Leu?Thr?Val?Phe?Phe?Cys?Gly?Ile?Val?Met?Ser?His?Tyr?Thr?Trp
275 280 285
His?Asn?Val?Thr?Glu?Ser?Ser?Arg?Ile?Thr?Thr?Lys?His?Ala?Phe?Ala
290 295 300
Thr?Leu?Ser?Phe?Val?Ala?Glu?Ile?Phe?Ile?Phe?Leu?Tyr?Val?Gly?Met
305 310 315 320
Asp?Ala?Leu?Asp?Ile?Glu?Lys?Trp?Lys?Phe?Val?Ser?Asp?Ser?Pro?Gly
325 330 335
Thr?Ser?Val?Ala?Ala?Ser?Ser?Val?Leu?Leu?Gly?Leu?Val?Leu?Leu?Gly
340 345 350
Arg?Ala?Ala?Phe?Val?Phe?Pro?Leu?Ser?Phe?Leu?Ser?Asn?Leu?Thr?Lys
355 360 365
The IB056039-sequence table
Lys?Ser?Pro?Tyr?Glu?Lys?Ile?Ser?Phe?Arg?Gln?Gln?Val?Ile?Ile?Trp
370 375 380
Trp?Ala?Gly?Leu?Met?Arg?Gly?Ala?Val?Ser?Met?Ala?Leu?Ala?Tyr?Asn
385 390 395 400
Gln?Phe?Thr?Met?Set?Gly?His?Thr?Gln?Leu?Arg?Cys?Asn?Ala?Ile?Met
405 410 415
Ile?Thr?Ser?Thr?Ile?Thr?Val?Val?Leu?Phe?Ser?Thr?Val?Val?Phe?Gly
420 425 430
Leu?Met?Thr?Lys?Pro?Leu?Ile?Arg?Leu?Leu?Leu?Pro?His?Pro?Lys?Ala
435 440 445
Thr?Asn?Ser?Met?Thr?Thr?Thr?Glu?Ser?Ser?Thr?Pro?Lys?Ser?Ala?Ile
450 455 460
Val?Pro?Leu?Leu?Gly?Ser?Ala?Gln?Asp?Ser?Glu?Ala?Asp?Leu?Asp?Gly
465 470 475 480
His?Glu?Ile?His?Arg?Pro?Ser?Ser?Ile?Arg?Ala?Leu?Leu?Ser?Thr?Pro
485 490 495
Thr?His?Thr?Val?His?Arg?Leu?Trp?Arg?Arg?Phe?Asp?Asp?Ser?Phe?Met
500 505 510
Arg?Pro?Val?Phe?Gly?Gly?Arg?Gly?Phe?Val?Pro?Val?Glu?Pro?Gly?Ser
515 520 525
Pro?Ser?Glu?Arg?Asn?Gly?His?Gln?Trp?Arg
530 535
<210>3
<211>1305
<212>DNA
＜213〉caragana microphylla (Caragana korshinskii Kom)
<400>3
atggcttttg?aaattgtctc?aaaattgcaa?aatctatcca?cttctgacca?tgcctccgta 60
gtctccatga?acttgtttgt?ggcacttcta?tgtgcttgta?ttgtagttgg?ccatcttctt 120
gaggagaatc?ggtggatgaa?tgagtccatc?actgcccttt?tgattggtct?ttgcactggc 180
ataatcattt?tgctgtttag?tggtggtaaa?agctcgcata?ttcttgtttt?cagtgaagat 240
cttttcttta?tataccttct?gCcgcctata?atattcaatg?cagggtttca?ggtgaaaaag 300
aagcagtttt?ttgtcaactt?catcactatc?gtgtcgtttg?gggctattgg?tacattagta 360
tcgtgtgtca?tcataacttt?gggtgttatg?tatgctttta?agaggatgga?tattgggcca 420
ctggaactgg?gcgattatct?agcaattgga?gcaatatttg?ccgcgacaga?ttctgtttgc 480
acgttgcagg?tgctaaatca?ggatgagaca?cctttgctgt?acagtcttgt?atttggggag 540
ggtgttgtga?acgatgctac?atcagtggtg?cttttcaatg?caatccaaag?ctttgacctc 600
aaccgactta?actctttaat?tgctttgcac?tttttgggca?atttcttgta?tttgtttatt 660
The IB056039-sequence table
gcaagcacac?tgcttggagt?tttgacaggt?ctgttcagtg?cttacgttat?taaaaagctg 720
tacattggca?ggcactctac?agatcgtgag?gttgctctta?tgatgctaat?ggcatacctc 780
tcctacatgc?tggctgaatt?aagctatctg?agtggcattc?tcactgtatt?cttttgtggg 840
attgtcatgt?ctcattatac?ttggcataat?gtgactgaga?gttcaagaat?cactaccaag 900
catgcttttg?ctaccttgtc?gtttgttgct?gagatattta?tcttccttta?tgttggtatg 960
gatgccctgg?acattgaaaa?atggaagttt?gttagtgata?gtcctggaac?atctgtagca 1020
gcaagttcag?tattgttggg?tctagtcctt?cttggaagag?cagcttttgt?tttcccctta 1080
tccttcttat?ccaacttgac?caaaaaatca?ccgtatgaga?aaatctcctt?cagacagcaa 1140
gtgatcattt?ggtgggctgg?tcttatgaga?ggtgctgttt?caatggcact?cgcttataat 1200
cagttcacca?tgtcggggca?cactcaactg?cgatgcaatg?caatcatgat?cactagcacc 1260
atcactgttg?tgcttttcag?cacagtggtg?tttggtttga?tgtag 1305
<210>4
<211>434
<212>PRT
＜213〉caragana microphylla (Caragana korshinskii Kom)
<400>4
Met?Ala?Phe?Glu?Ile?Val?Ser?Lys?Leu?Gln?Asn?Leu?Ser?Thr?Ser?Asp
1 5 10 15
His?Ala?Ser?Val?Val?Ser?Met?Asn?Leu?Phe?Val?Ala?Leu?Leu?Cys?Ala
20 25 30
Cys?Ile?Val?Val?Gly?His?Leu?Leu?Glu?Glu?Asn?Arg?Trp?Met?Asn?Glu
35 40 45
Ser?Ile?Thr?Ala?Leu?Leu?Ile?Gly?Leu?Cys?Thr?Gly?Ile?Ile?Ile?Leu
50 55 60
Leu?Phe?Ser?Gly?Gly?Lys?Ser?Ser?His?Ile?Leu?Val?Phe?Ser?Glu?Asp
65 70 75 80
Leu?Phe?Phe?Ile?Tyr?Leu?Leu?Pro?Pro?Ile?Ile?Phe?Asn?Ala?Gly?Phe
85 90 95
Gln?Val?Lys?Lys?Lys?Gln?Phe?Phe?Val?Asn?Phe?Ile?Thr?Ile?Val?Ser
100 105 110
Phe?Gly?Ala?Ile?Gly?Thr?Leu?Val?Ser?Cys?Val?Ile?Ile?Thr?Leu?Gly
115 120 125
Val?Met?Tyr?Ala?Phe?Lys?Arg?Met?Asp?Ile?Gly?Pro?Leu?Glu?Leu?Gly
130 135 140
Asp?Tyr?Leu?Ala?Ile?Gly?Ala?Ile?Phe?Ala?Ala?Thr?Asp?Ser?Val?Cys
145 150 155 160
The IB056039-sequence table
Thr?Leu?Gln?Val?Leu?Asn?Gln?Asp?Glu?Thr?Pro?Leu?Leu?Tyr?Ser?Leu
165 170 175
Val?Phe?Gly?Glu?Gly?Val?Val?Asn?Asp?Ala?Thr?Ser?Val?Val?Leu?Phe
180 185 190
Asn?Ala?Ile?Gln?Ser?Phe?Asp?Leu?Asn?Arg?Leu?Asn?Ser?Leu?Ile?Ala
195 200 205
Leu?His?Phe?Leu?Gly?Asn?Phe?Leu?Tyr?Leu?Phe?Ile?Ala?Ser?Thr?Leu
210 215 220
Leu?Gly?Val?Leu?Thr?Gly?Leu?Phe?Ser?Ala?Tyr?Val?Ile?Lys?Lys?Leu
225 230 235 240
Tyr?Ile?Gly?Arg?His?Ser?Thr?Asp?Arg?Glu?Val?Ala?Leu?Met?Met?Leu
245 250 255
Met?Ala?Tyr?Leu?Ser?Tyr?Met?Leu?Ala?Glu?Leu?Ser?Tyr?Leu?Ser?Gly
260 265 270
Ile?Leu?Thr?Val?Phe?Phe?Cys?Gly?Ile?Val?Met?Ser?His?Tyr?Thr?Trp
275 280 285
His?Asn?Val?Thr?Glu?Ser?Ser?Arg?Ile?Thr?Thr?Lys?His?Ala?Phe?Ala
290 295 300
Thr?Leu?Ser?Phe?Val?Ala?Glu?Ile?Phe?Ile?Phe?Leu?Tyr?Val?Gly?Met
305 310 315 320
Asp?Ala?Leu?Asp?Ile?Glu?Lys?Trp?Lys?Phe?Val?Ser?Asp?Ser?Pro?Gly
325 330 335
Thr?Ser?Val?Ala?Ala?Ser?Ser?Val?Leu?Leu?Gly?Leu?Val?Leu?Leu?Gly
340 345 350
Arg?Ala?Ala?Phe?Val?Phe?Pro?Leu?Ser?Phe?Leu?Ser?Asn?Leu?Thr?Lys
355 360 365
Lys?Ser?Pro?Tyr?Glu?Lys?Ile?Ser?Phe?Arg?Gln?Gln?Val?Ile?Ile?Trp
370 375 380
Trp?Ala?Gly?Leu?Met?Arg?Gly?Ala?Val?Ser?Met?Ala?Leu?Ala?Tyr?Asn
385 390 395 400
Gln?Phe?Thr?Met?Ser?Gly?His?Thr?Gln?Leu?Arg?Cys?Asn?Ala?Ile?Met
405 410 415
Ile?Thr?Ser?Thr?Ile?Thr?Val?Val?Leu?Phe?Ser?Thr?Val?Val?Phe?Gly
420 425 430
Leu?Met
<210>5
<211>36
The IB056039-sequence table
<212>DNA
＜213〉artificial sequence
<400>5
cgagcggccg?cccgggcagg?tttttttttt?tttttt 36
<210>6
<211>26
<212>DNA
＜213〉artificial sequence
<400>6
gcagggtttc?aggtgaaaaa?gaagca 26
<210>7
<211>29
<212>DNA
＜213〉artificial sequence
<400>7
gaaaaatgga?agtttgt?tag?tgatagtcc 29
<210>8
<211>23
<212>DNA
＜213〉artificial sequence
<400>8
caccatgtcg?gggcacactc?aac 23
<210>9
<211>28
<212>DNA
＜213〉artificial sequence
<400>9
gtacaatgat?gatgatgatg?ggtcagag 28
<210>10
<211>28
<212>DNA
＜213〉artificial sequence
The IB056039-sequence table
<400>10
agatggctta?tggtacacgg?ccttctcg 28
<210>11
<211>26
<212>DNA
＜213〉artificial sequence
<400>11
cttctcaacg?ccattgatga?ccattg 26
<210>12
<211>652
<212>DNA
＜213〉artificial sequence
<400>12
cccgtctttc?tcctctgtct?ctttctttct?gtccacacca?cacggctctc?cctctctctc 60
tccctctctc?tctctctcta?aacctttgtc?ttccattttc?tctatatttc?cagagctttc 120
gttgccctct?ctttctctca?ctgtgactca?aattaataaa?aaaaatatct?cttaattttc 180
tttccttttc?gatttctcgt?tccttttcca?gctacctccc?ttgagcttca?cgtttatttt 240
aattctatta?atccctgctc?tgttggttca?ttctctattt?tcgtcttcgt?ttcgagcttg 300
atccctgcat?aattgttttg?gtggaacaat?tgcagagagc?tgaaaagtgt?tccaatttaa 360
cgggaattcg?aacagggggt?tggttcttag?ctttctctgt?tcgaattctt?tggacgttgg 420
gttccttgtg?tttttttaaa?tactcagaaa?aatcacagac?tcttcttcgc?aatgctgcta 480
tcaagctttc?tgagattctt?ttgttgacga?gatctgaagg?ttcttaaaat?ggatagctcg 540
gaaacataaa?tttggatcat?ttatactgga?ctttgaactt?ggcaactaag?caataatctg 600
aatctgtgtt?taaatctgct?tttgcaatta?tttattgtgg?agggtggacg?ag 652
<210>13
<211>278
<212>DNA
＜213〉artificial sequence
<400>13
gaagaaagcc?atgaaatatg?taatgtgtgt?tgtaaactac?gtatgattgt?atgatttgtg 60
aagatcaagc?aatgtatgta?taataagaat?cacgtaatga?aattttgttt?agttttttgt 120
gtagctagta?cgagaaggcc?gtgtaccata?agccatctct?gcctcttctg?taaactacaa 180
tatttattag?attctctgac?ccatcatcat?catcattgta?ctaaaaattt?gattagctga 240
agctaaatat?tttcctttcc?acaaaaaaaa?aaaaaaaa 278
<210>14
The IB056039-sequence table
<211>27
<212>DNA
＜213〉artificial sequence
<400>14
cgcccagttc?cagtggccca?atatcca 27
<210>15
<211>27
<212>DNA
＜213〉artificial sequence
<400>15
cttggaagag?cagcttttgt?tttcccc 27
<210>16
<211>28
<212>DNA
＜213〉artificial sequence
<400>16
gccggatccc?gtctttctcc?tctgtctc 28
<210>17
<211>34
<212>DNA
＜213〉artificial sequence
<400>17
cgcggagctc?aatgatgatg?atgatgggtc?agag 34
<210>18
<211>31
<212>DNA
＜213〉artificial sequence
<400>18
cgcgccgagc?tcatggcttt?tgaaattgtc?t 31
<210>19
<211>34
<212>DNA
＜213〉artificial sequence
The IB056039-sequence table
<400>19
ccgcgctcta?gactaagtca?tcaaaccaaa?cacc 34
<210>20
<211>32
<212>DNA
＜213〉artificial sequence
<400>20
cgccggatcc?atggcttttg?aaattgtctc?aa 32
<210>21
<211>31
<212>DNA
＜213〉artificial sequence
<400>21
gtcagagctc?tcaacgccat?tgatgaccat?t 31
<210>22
<211>21
<212>DNA
＜213〉artificial sequence
<400>22
gctggatttg?ctggtgatga?t 21
<210>23
<211>19
<212>DNA
＜213〉artificial sequence
<400>23
cgatctgcaa?tgccaggaa 19
Claims (14)
1. NHX class Na+/H+ counter transport albumen that derives from shrub plant caragana microphylla (Caragana korshinskii Kom), its aminoacid sequence is by shown in SEQ ID NO:2 or 4 or by carrying out the amino acid whose replacement of one or several non-conservative region, insertion or lack shown in the resulting sequence on the sequence of SEQ ID NO:2 or 4.
2. the proteic gene of counter transport of the claim 1 of encoding.
3. the gene of claim 2, its nucleotide sequence be by shown in SEQ ID NO:1 or 3, or by replacement, the insertion of on the sequence of SEQ ID NO:1 or 3, carrying out one or several Nucleotide or lack shown in the resulting sequence with identical function.
4. expression vector, it comprises the gene of claim 2 or 3.
5. the expression vector of claim 4, it is double base agrobacterium vector or plant expression vector.
6. the expression vector of claim 5, wherein said plant expression vector contains the promotor of DRE and/or ABRE element.
7. host cell, it comprises the described expression vector of claim 4.
8. the host cell of claim 7, it is Bacillus coli cells, yeast cell or agrobatcerium cell.
9. claim 2 or 3 gene obtain application in the transgenic plant transforming plant.
10. the application of claim 9, wherein said plant is selected from monocotyledons or dicotyledons.
11. the application of claim 10, wherein said monocotyledons is selected from the group of being made up of wheat, corn, paddy rice, graminous pasture, and described dicotyledons is selected from the group of being made up of willow, soybean, cotton and rape.
12. the application of the gene of claim 2 or 3 in coerce in raising plant stress-resistance border.
13. the application of claim 12, wherein said environment stress are salt stress.
14. a method of cultivating the plant of coercing in degeneration-resistant border comprises the plant expression vector that makes up the gene that contains right requirement 2 or 3; Plant expression vector transformed plant cells with described structure; With the plant transformed cell culture is become transfer-gen plant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100114934A CN100471953C (en) | 2006-03-15 | 2006-03-15 | New CkNHX gene and shearing decorative gene CkNHXn, method for cultivating inverse-resistant plant |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100114934A CN100471953C (en) | 2006-03-15 | 2006-03-15 | New CkNHX gene and shearing decorative gene CkNHXn, method for cultivating inverse-resistant plant |
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| Publication Number | Publication Date |
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| CN101037693A true CN101037693A (en) | 2007-09-19 |
| CN100471953C CN100471953C (en) | 2009-03-25 |
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| CNB2006100114934A Expired - Fee Related CN100471953C (en) | 2006-03-15 | 2006-03-15 | New CkNHX gene and shearing decorative gene CkNHXn, method for cultivating inverse-resistant plant |
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| CN (1) | CN100471953C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443591A (en) * | 2010-09-30 | 2012-05-09 | 中国科学院遗传与发育生物学研究所 | Application of CkGST gene |
| CN101701037B (en) * | 2009-11-05 | 2012-09-05 | 复旦大学 | Transporter protein for regulating and controlling salt resistance and drought resistance of plant and encoding gene and application thereof |
| CN103254297A (en) * | 2012-02-16 | 2013-08-21 | 中国科学院微生物研究所 | Stress resistance related protein IMSAL1 deriving from saline soda lake microorganisms, its coding gene and application |
| CN105713911A (en) * | 2016-02-29 | 2016-06-29 | 南京农业大学 | Screening method for artemisia japonica plasma membrane Na+/H+ reverse transport protein AjSOS1 salt tolerance key amino acid site |
| CN111088260A (en) * | 2020-01-16 | 2020-05-01 | 南京农业大学 | Radish salt-tolerant gene RsNHX1 and application thereof |
| CN112322629A (en) * | 2020-10-13 | 2021-02-05 | 河南农业大学 | Application of gene GhNHX4A in aspect of salt tolerance of plants |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421812B (en) * | 2013-05-03 | 2014-12-10 | 华中农业大学 | Method for cultivating high-temperature-resistant arabidopsis through gene KcNHX1 of karelinia caspia |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1238374C (en) * | 2003-04-09 | 2006-01-25 | 中国科学院植物研究所 | Na+/H+ retrograde transport protein of New Zedland spinach, its coding gene and application |
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2006
- 2006-03-15 CN CNB2006100114934A patent/CN100471953C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701037B (en) * | 2009-11-05 | 2012-09-05 | 复旦大学 | Transporter protein for regulating and controlling salt resistance and drought resistance of plant and encoding gene and application thereof |
| CN102443591A (en) * | 2010-09-30 | 2012-05-09 | 中国科学院遗传与发育生物学研究所 | Application of CkGST gene |
| CN102443591B (en) * | 2010-09-30 | 2013-09-04 | 中国科学院遗传与发育生物学研究所 | Application of CkGST gene |
| CN103254297A (en) * | 2012-02-16 | 2013-08-21 | 中国科学院微生物研究所 | Stress resistance related protein IMSAL1 deriving from saline soda lake microorganisms, its coding gene and application |
| CN103254297B (en) * | 2012-02-16 | 2015-01-07 | 中国科学院微生物研究所 | Stress resistance related protein IMSAL1 deriving from saline soda lake microorganisms, its coding gene and application |
| CN105713911A (en) * | 2016-02-29 | 2016-06-29 | 南京农业大学 | Screening method for artemisia japonica plasma membrane Na+/H+ reverse transport protein AjSOS1 salt tolerance key amino acid site |
| CN111088260A (en) * | 2020-01-16 | 2020-05-01 | 南京农业大学 | Radish salt-tolerant gene RsNHX1 and application thereof |
| CN112322629A (en) * | 2020-10-13 | 2021-02-05 | 河南农业大学 | Application of gene GhNHX4A in aspect of salt tolerance of plants |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100471953C (en) | 2009-03-25 |
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