CN103172719A - Stress-resistance-related SOS3-like calcium-ion-binding protein, and coding gene and application thereof - Google Patents
Stress-resistance-related SOS3-like calcium-ion-binding protein, and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a stress-resistance-related calcineurin-B-like protein, and a coding gene and an application thereof. The application provided by the invention is specifically the application of a protein composed of an amino acid sequence represented by sequence 1 in the sequence table in the following (a1) or (a2): (a1) regulating plant salt resistance; and (a2) breeding salt-resistant plant varieties. As a result of experiments, when the coding gene of the protein composed of the amino acid sequence represented by sequence 1 in the sequence table is expressed in an arabidopsis mutant sos3, the salt-sensitive phenotype of the arabidopsis mutant sos3 can be inhibited. When the gene is over expressed in an arabidopsis wild-type Columbia, plant salt resistance can be improved.
Description
Technical field
The invention belongs to biological technical field, relate to the similar calcium ion-binding protein of a kind of degeneration-resistant relevant SOS3 and encoding gene and application.
Background technology
The adverse circumstance such as the soil salinization, arid is the main environment limiting factor of plant-growth, growth.Plant is in order to tackle adverse circumstance, develop in the long-term evolution process and form a cover perception environment stress, conduction adverse circumstance signal, and the finely regulating that responds on molecule, cell and physiological level mechanism (Li Ruifen, Zhang Junwen, Wei Jianhua, Ma Rongcai, 2009.Functions and Mechanisms of the CBL-CIPK signaling system in plant response to abiotic stresses.Progress in Natural Science, 19(6): 667-676).Ca
2+The signal pathway that relies on comprises at the multiple bioprocess of plant play a significant role in the environment stress response (Kudla J., Batistic O.﹠amp; Hashimoto K., 2010.Calcium signals:the lead currency of plant information processing.The Plant Cell, 22:541-563).At deciphering Ca
2+Signal, reply that in the process of Adversity-stressed Plant, the calcium ion susceptor is important signal transmitter, Push And Release (the Luan S. of regulation and control stress response gene, Kudla J., Rodriguez-Concepcion M., Yalovsky S., Gruissem W., 2002.Calmodulins and calcineurin B-like proteins:calcium sensors for specific signal response coupling in plants.The Plant Cell, 14,389-400).According to the difference of function and structure, the calcium ion susceptor is divided into response type and dependency calcium ion susceptor.The former can be in conjunction with Ca
2+, have again kinase activity, as calcium ion deopendent protein kinase (CDPKs); And the latter can only be in conjunction with Ca
2+, and could transmit the calcium signal, calcium ion-binding protein as similar in SOS3 (SOS3-LIKE CALCIUM BINDING PROTEIN is called for short SCaBP) after doing mutually with special protein kinase.
SCaBP type calcium receptor is that a class exists only in plant and animal calcineurin B subunit (calcineurin B, CNB) and nervus centralis calcium receptor (neuronal calcium sensors, NCS) extremely similar small molecular protein, therefore be named as calcineurin B-like albumen, i.e. CBL or CBL calcium receptor.Because the SOS approach finds that early people also are called SCaBPs(Liu J with this susceptor, Zhu JK, 1998.A calcium sensor homolog required for plant salt tolerance.Science, 280:1943-1945; Kudla J, Xu Q, Harfter K, Gruissem W, and Luan S, 1999.Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals.Proc.Natl.Acad.Sci.USA., 96:4718-4723).All identify at present 10 SCaBPs in Arabidopis thaliana and paddy rice, form with 26 AtCIPKs, 30 OsCIPKs respectively and make mutually complex body, the expression of regulation and control downstream gene.So far, the regulation and control that some SCaBPs participate in Stress response have been identified.Participate in as SCaBP5/CBL1 and SCaBP7/CBL9 regulation and control (the Xu J that low K+ coerces, Li H-D, Chen L-Q, Wang Y, Liu L-L, He L, and Wu W-H, 2006.A protein kinase, interacting with two calcineurin B-like proteins regulates K
+Transporter AKT1in Arabidopsis.Cell125:1347-1360; Li L, Kim B-G, Cheong Y H, Pandey G K, and Luan S, 2006.A Ca
2+Signaling pathway regulates a K channel for low-K
+Response in Arabidopsis.Proc Natl Acad Sci USA, 103 (33): 12625-12630); SOS3/CBL4 and SCaBP8/CBL10 participate in the SOS regulatory pathway, both make mutually respectively salt tolerance (the Qiu Q-S of regulating plant root and overground part with AtCIPK24/SOS2 in the SOS approach, Guo Y, Dietrich M, Schumaker KS, Zhu J-K, 2002.Regulation of SOS1, a plasma membrane Na
+/ H
+Exchanger in Arabidopsis thaliana, by SOS2and SOS3.Proc Natl Acad Sci USA, 99:8436-8441; Uan RD, Lin HX, Mendoz I, Zhang YG, Cao WH, Yang YQ, Shang M, Chen SY, Pardo JM, and Guo Y, 2007.SCABP8/CBL10, a Putative Calcium Sensor, Interacts with the Protein Kinase SOS2to Protect Arabidopsis Shoots from Salt Stress.Plant Cell, 19:1415-1431); AtSCaBP4/CBL5 participates in the response of drought stress, but not clear concrete regulatory pathway (Cheong YH, Sung SJ, Kim B-G, Pandey GK, Cho J-S, Kim K-N﹠amp; Luan S, 2010.Constitutive overexpression of the calcium sensor CBL5confers osmotic or drought stress tolerance in Arabidopsis.Molecules and Cells29,159-165).The also involved in plant regulation and control of growing of part SCaBPs.As seen, SCaBP has diversified biological function as the calcium ion susceptor.Carried out in recent years part crop SCaBP/CBL-CIPK functional study (Mahajan S, Sopory S K, Tuteja N, 2006.Cloning and characterization of CBL-CIPK signalling components from a legume (Pisum sativum) .FEBS Journal, 273 (5): 907-925), and obtained certain progress.Because different plant species homology SCaBP gene may have difference in functionality or new function, the clone replys the SCaBP gene of various environment stresses, might resolve certain degeneration-resistant regulatory pathway.But the material major part of research is confined to model plant or glycophyte, non-Special germplasm at present.Some special habitats germplasm materials may have deciphering adverse circumstance Ca
2+The special pathway of signal excavates clone's special habitats plant signal component gene, to effectively utilizing genetic resources Crop Improvement resistance significant.
Wild barley (Hordeum brevisubulatum (Trin.) Link) is the good forage that a kind of perennial facultative salt of Gramineae Hordeum is given birth to, mainly be distributed in West Siberia, Mongolia and the provinces and regions such as China northeast, North China, Xinjiang, Tibet and Inner Mongol, it is the constructive species on salinization and Alkalization Meadow grassland, can well grow on the soil of saltiness 0.6-1.0%, its salt tolerance can match in excellence or beauty with Puccinellia tenuiflora etc., has important application and scientific research value.But forefathers only are confined to the research of the aspects such as form, dissection, physiology, and research is less aspect the clone of degeneration-resistant regulatory gene and functional analysis.
Summary of the invention
The purpose of this invention is to provide the similar calcium ion-binding protein of a kind of degeneration-resistant relevant SOS3 and encoding gene and application.
Application provided by the present invention is specially following A or B:
A: the protein (called after HbSCaBP) that is comprised of the aminoacid sequence shown in sequence in sequence table 1 is at following a1) or a2) in application:
A1) regulating plant salt resistance;
A2) seed selection salt-resistant plant kind.
B: the encoding gene (called after HbSCaBP) of the protein that is comprised of the aminoacid sequence shown in sequence in sequence table 1 is at following a1) or a2) in application:
A1) regulating plant salt resistance;
A2) seed selection salt-resistant plant kind.
The present invention also provides a kind of method of cultivating the salt tolerant transgenic plant.
The method of cultivation salt tolerant transgenic plant provided by the present invention specifically can comprise the steps:
A) import the encoding gene of the protein that is formed by the aminoacid sequence shown in sequence in sequence table 1 in the purpose plant, obtain expressing the transgenic plant of described encoding gene;
B) obtain comparing with described purpose plant the transgenic plant that salt tolerance improves from step a) gained transgenic plant.
In above-mentioned application or method, the encoding gene (being the HbSCaBP gene) of the described protein that is comprised of the aminoacid sequence shown in sequence in sequence table 1 is following 1) to 4) in arbitrary described DNA molecular:
1) encoding sequence be in sequence table sequence 2 from the DNA molecular shown in the 85th to 741 Nucleotide of 5 ' end;
2) DNA molecular shown in sequence 2 in sequence table;
3) under stringent condition with 1) or 2) aminoacid sequence shown in sequence 1 forms in the DNA molecule hybridize that limits and coding sequence table protein DNA molecule;
4) with 1) or 2) or 3) DNA molecular that limits has the protein DNA molecule of the composition of the aminoacid sequence shown in sequence 1 in 90% above homology and coding sequence table.
In aforesaid method, the encoding gene of the described protein that is comprised of the aminoacid sequence shown in sequence in the sequence table 1 specifically recombinant expression vector of the encoding gene by containing described protein imports in described purpose plant.
In above-mentioned application or method, described plant namely can be dicotyledons, also can be monocotyledons.
In one embodiment of the invention, described plant is dicotyledons, is specially Arabidopis thaliana, more is specially Arabidopis thaliana wild-type kind Columbia or Arabidopsis Mutants sos3.
Protein provided by the present invention is the similar calcium ion-binding protein of SOS3, the name be called HbSCaBP, derive from wild barley (Hordeum brevisubulatum (Trin.) Link), be following a) or b):
A) protein that is formed by the aminoacid sequence shown in sequence in sequence table 1;
B) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to the plant anti-salt protein that is derived by sequence 1;
In sequence table, sequence 1 is comprised of 218 amino-acid residues.
For the ease of the purifying of HbSCaBP albumen, label as shown in the table on N-terminal that can the protein that the amino acid residue sequence of sequence 1 forms in by sequence table or C-terminal connect.
Table: the sequence of label
| The label residue sequence |
| Poly-Arg5-6(is generally 5) RRRRR |
| Poly-His2-10(is generally 6) HHHHHH |
| FLAG8DYKDDDDK |
| Strep-tag?II8WSHPQFEK |
| c-myc10EQKLISEEDL |
But above-mentioned HbSCaBP albumen synthetic in a) also can first synthesize its encoding gene, then carries out biological expression and obtain.Above-mentioned b) the HbSCaBP albumen in can obtain by the following method: with the codon of one or several amino-acid residue of disappearance in the DNA sequence dna shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or connect the encoding sequence of label shown in table at its 5 ' end and/or 3 ' end and obtain encoding gene, then carry out biological expression and obtain HbSCaBP albumen.
The nucleic acid molecule of described HbSCaBP albumen of encoding also belongs to protection scope of the present invention.
Described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA, hnRNA or tRNA etc.
In one embodiment of the invention, described nucleic acid molecule is specially coding described HbSCaBP(wild barley SOS3 similar calcium ion-binding protein) gene (called after HbSCaBP) of albumen; Described HbSCaBP gene is following 1) to 4) in arbitrary described DNA molecular:
1) encoding sequence be in sequence table sequence 2 from the DNA molecular shown in the 85th to 741 Nucleotide of 5 ' end;
2) DNA molecular shown in sequence 2 in sequence table;
3) under stringent condition with 1) or 2) DNA molecule hybridize that limits and the DNA molecular of the described HbSCaBP albumen of encoding;
4) with 1) or 2) or 3) DNA molecular that limits has the DNA molecular of 90% above homology and the described HbSCaBP albumen of encoding;
Above-mentioned stringent condition can be with 6 * SSC, and the solution of 0.5%SDS is hybridized under 65oC, then uses 2 * SSC, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Wherein, sequence 2 is comprised of 1159 Nucleotide, and the 85th to 741 is its encoding sequence, the protein shown in sequence 1 in the code sequence list.
The recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain above-mentioned nucleic acid molecule also belong to protection scope of the present invention.
Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
Described recombinant expression vector can be used existing plant expression vector construction.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vector.Described plant expression vector also can comprise 3 ' of foreign gene and hold untranslated regional, namely comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any enhancement type, composing type, organizing specific type or inducible promoter before its transcription initiation Nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter Rd29A etc., they can use separately or be combined with other plant promoter; In addition, when using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhansers zone can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process recombinant expression vector used, as add the coding that to express in plant can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of colour-change.Also can not add any selected marker, directly with adverse circumstance screening transformed plant.
In an embodiment of the present invention, in described recombinant expression vector, the promotor of the described HbSCaBP genetic transcription of startup is specially cauliflower mosaic virus 35s promotor (CaMV35S promotor).
More specifically, the described recombinant expression vector recombinant plasmid for obtaining after between the multiple clone site BamH I that described HbSCaBP gene (sequence 2 the 85th to 741) is inserted into the pGreen0029 carrier and EcoR I.
Described expression cassette is by the promotor that can start described HbSCaBP genetic expression, described HbSCaBP gene, and transcription termination sequence forms.
Described recombinant vectors, described expression cassette, described transgenic cell line or described recombinant bacterium are at following a1) or a2) in application also belong to protection scope of the present invention:
A1) regulating plant salt resistance;
A2) seed selection salt-resistant plant kind;
In above-mentioned application or method, described plant namely can be dicotyledons, also can be monocotyledons.
In one embodiment of the invention, described plant is dicotyledons, is specially Arabidopis thaliana, more is specially Arabidopis thaliana wild-type kind Columbia or Arabidopsis Mutants sos3.
In the present invention, the described regulating plant salt resistance above all a1) all is specially the raising salt resistance of plants; The method of the described seed selection salt-resistant plant kind above all a2) all specifically can comprise the step that the plant that described HbSCaBP expressing quantity is higher hybridizes as the parent.
The present invention's clone identification from the living wild barley of Special germplasm-salt goes out the HbSCaBP gene of a strong response salt stress.One of this genes encoding participates in the similar calcium ion-binding protein of SOS3 that the adverse circumstance signal transmits, and it mainly is positioned on cytoplasmic membrane.Experiment showed, that the HbSCaBP gene can suppress its quick salt phenotype after Arabidopsis Mutants sos3 crosses table, Columbia crosses the salt tolerance that expression can improve plant in the Arabidopis thaliana wild-type.As seen, HbSCaBP albumen is important regulatory factor in the salt tolerant regulator control system.
Description of drawings
Fig. 1 is the systematic evolution tree analysis of HbSCaBP and Arabidopis thaliana, paddy rice and Chinese sorghum SCaBP family.
Fig. 2 is the expression analysis of HbSCaBP gene under different biologies are coerced.Wherein, a is that wild barley root and stem are coerced 6h respectively under 350mM NaCl, 350mM N.F,USP MANNITOL and 10%PEG6000,4 ℃ of cold 12h that coerce, and the expression of the HbSCaBP gene under contrast (CK, undressed wild barley root and stem) condition; B is that wild barley root and stem are coerced 24h at 350mM NaCl, 350mM N.F,USP MANNITOL, 10%PEG6000 and 150 μ M ABA respectively, 4 ℃ of cold 48h that coerce, and the expression of HbSCaBP gene under contrast (CK, undressed wild barley root and stem) condition.
Fig. 3 is the PCR detected result that turns HbSCaBP gene Arabidopis thaliana.Wherein, a is T
2In generation, change the qualification result of the Arabidopsis Mutants sos3 of recombinant expression vector pGreen0029-HbSCaBP over to, and s1, s3, s5, s9, s15 and s17 represent 6 different transgenic lines; B is T
3In generation, change the qualification result of the Arabidopis thaliana wild-type Columbia of recombinant expression vector pGreen0029-HbSCaBP over to, and w3, w4, s5, w11, w16 and w18 represent 6 different transgenic lines.In a and b, " plasmid " is with the contrast of recombinant expression vector pGreen0029-HbSCaBP as template; " empty carrier " is with the contrast of carrier pGreen0029 as template; Swimming lane M is DNA molecular amount standard.
Fig. 4 is T
2For the germination test of the homozygous lines (s5, s9 and s17) that turns HbSCaBP gene Arabidopsis Mutants sos3 and the result of growth of seedling test.Wherein, a is transgenic line (s5, s9 and s17) and Arabidopsis Mutants sos3 in the statistics that contains the percentage of germination on NaCl different concns (0,100,125 and 150mM) substratum; B is transgenic line (s5, s9 and s17) and Arabidopsis Mutants sos3 in the statistics that contains the average single-strain fresh weight after coercing 2 weeks of growth on NaCl different concns (0,100,125 and 150mM) substratum; C is transgenic line (s5, s9 and s17), Arabidopsis Mutants sos3 and not genetically modified Arabidopis thaliana wild-type Columbia(WT) at the growth phenotype photo that contains after coercing 2 weeks of growth on NaCl different concns (0,100,125 and 150mM) substratum.
Fig. 5 is T
3For the germination test of the homozygous lines (W5, W11 and W16) that turns HbSCaBP gene Arabidopis thaliana wild-type Columbia and the result of growth of seedling test.Wherein, a is transgenic line (W5, W11 and W16) and not genetically modified Arabidopis thaliana wild-type Columbia(WT) in the statistics that contains the percentage of germination on NaCl different concns (0,100,125 and 150mM) substratum; B is transgenic line (W5, W11 and W16) and not genetically modified Arabidopis thaliana wild-type Columbia(WT) contain coerce 2 weeks of growth on NaCl different concns (0,100,125 and 150mM) substratum after the statisticses of average single-strain fresh weights; C is transgenic line (W5, W11 and W16) and not genetically modified Arabidopis thaliana wild-type Columbia(WT) at the growth phenotype photo that contains after coercing 2 weeks of growth on NaCl different concns (0,100,125 and 150mM) substratum.
Embodiment
The experimental technique that uses in following embodiment is ordinary method if no special instructions.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Arabidopis thaliana wild-type Columbia and Arabidopsis Mutants sos3:http: //the www.arabidopsis.org website.Wherein, Arabidopsis Mutants sos3 is numbered CS3864, and mutation type surface is that blade surface is smooth, without or few epidermal hair, quick salt is described in detail and is seen " Liu J P﹠amp; Zhu J K J, 1997.An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance.Proc.Natl.Acad.Sci.USA, 94:14960-14964 ".
The pGreen0029 carrier: http://www.pgreen.ac.uk/ orders on the net.
Agrobacterium Gv3101(contains assistant's carrier pSoup): available from Chinese plasmid vector strain cell pnca gene preservation center-Biovector Science Lab.
MS is dull and stereotyped: contain 4.3g/L MS powder, 20g/L sucrose, 6g/L agar, pH5.8.Each concentration is final concentration.Wherein the MS powder is Sigma company product, and its catalog number is M5519.
Acquisition and the evaluation of embodiment 1, the gene HbSCaBP relevant to plant salt endurance
One, the acquisition of the gene HbSCaBP relevant to plant salt endurance
1, the wild barley material is cultivated
Saline Forage-wild barley (Hordeum brevisubulatum (Trin.) Link) picks up from salinification grassland, the Inner Mongol.At ambient temperature, after the wild barley seed is soaked 12h with deionized water, put into 4 ℃ of refrigerator overnight.Deionized water rinsing 3 times with sterilization is put in the culture dish of moistening gauze 25 ℃ of rudiments.After treating its most of germination in 4～5 days, forward in the vial that fills the 1/2Hoagland nutrient solution of sterilization (with lighttight paper bag firmly), at temperature 22-23 ℃, intensity of illumination 1000-3000 μ mol m
-2s
-1, grow under the condition of illumination 12h/ days, dark 12h/ days, wait growing to two leaves wholeheartedly the time, adding final concentration in the 1/2Hoagland nutrient solution is that the NaCl of 430mmo/L coerces, and coerces after 30min standby.
Wherein, Hoaglan nutrient solution formula: 0.51g/L KNO
3, 0.82g/L Ca (NO
3)
2, 0.49g/L MgSO
47H
20,0.136g/L KH
2PO
4Add again lmL Fe EDTA solution (compound method: dissolve respectively 7.45g Na
2EDTA, 5.57g FeSO
47H
20 in 200mL distilled water, heating.Constantly stir Na
2EDTA solution and FeS0
4Solution mixes, and is settled to 1L); Then add lmL A-Z solution (formula: 2.80mg/L H
3BO
3, 0.08mg/LCuS0
45H
2O, 0.22mg/L ZnSO
47H
2O, 81mg/L MgCl
26H
2O, 0.09mg/L HMoO4H
2O).Above each concentration is the final concentration of respective components in corresponding solution.The 1/2Hoagland nutrient solution is the solution of rear gained that the concentration of each component in above-mentioned Hoaglan nutrient solution is reduced by half.
2, HbSCaBP gene clone
The acquisition of (1) 5 ' terminal sequence
The root of wild barley seedling of coercing lower processing 30min at 430mmo/L NaCl in the step 1 is as experiment material, simultaneously take the root of untreated wild barley seedling as control material.Experiment material and control material are wrapped with aluminum tissue paper respectively indicated, drop into immediately in liquid nitrogen fixingly ,-80 ℃ save backup.According to Trizol(Invitrogen, USA) the total RNA that processes wild barley root and control material through salt is extracted in test kit explanation, residual DNA in the total RNA of DNase I digestion, phenol/chloroform/primary isoamyl alcohol mixed solution (volume ratio 25:24:1) extracted total RNA, spectrophotometric determination OD
260And OD
280, according to OD
260Value is calculated the output of RNA; According to OD
260/ OD
280The quality of value judgement RNA.According to Superscript III(Invitrogen) the test kit explanation, getting the total RNA reverse transcription of 200ng is cDNA.According to document (Rivandi J., Miyazaki J., Hrmova M., Pallotta M., Tester M.﹠amp; Collins N.C., 2011.ASOS3homologue maps to HvNax4, a barley locus controlling an environmentally sensitive Na
+Exclusion trait.Journal of Experimental Botany, the barley gene HvNax4(Genbank:HM175878 of report 62:1201-1216)) design homologous primer Hb-F1 and Hb-R1, carry out pcr amplification take above-mentioned gained cDNA as template.PCR program: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 1min, 30 circulations; 72 ℃ are extended 7min, 10 ℃ of preservations.To pGEM-T carrier (Promega, USA), mono-clonal is that Beijing Bo Maide biotech company checks order with the PCR product cloning.5 ' the terminal sequence that obtains is by the exactness of online NCBI blast analysis confirmation sequence.
Hb-F1:5 '-GTAGAGTGCGGAGCTTATTCTCTCGCC-3 ' (the 262-288 position of Genbank:HM175878);
Hb-R1:5 '-AGGTGCAGCTCACGCACTTCTGAACG-3 ' (reverse complementary sequence of the 1980-2005 position of Genbank:HM175878).
The acquisition of (2) 3 ' terminal sequences
The rear discovery of comparing of the 5 ' terminal sequence that above-mentioned pcr amplification is obtained and barley template sequence (Genbank:HM175878), this sequence and barley template sequence (Genbank:HM175878) are variant in a plurality of sites of 3 ' non-coding region (3 ' UTR).In order to obtain 3 ' correct UTR sequence, designed the primer Hb-3RACE-GSP1 of amplification 3 ' terminal sequence at terminator codon TAA precontract 200bp place, coding region.According to the operation instructions of BD SMARTTM RACE cDNAAmplification test kit (Clontech, Cat.No.634914, USA), the cDNA in the above-mentioned steps carries out the nest-type PRC amplification as template.PCR program: 94 ℃ of denaturation 2min; 94 ℃ of sex change 30sec, 63 ℃ of annealing 30sec, 72 ℃ are extended 1min, 30 circulations; 72 ℃ are extended 7min, 10 ℃ of preservations.To pGEM-T carrier (Promega, USA), mono-clonal obtains 3 ' terminal sequence in the order-checking of Beijing Bo Maide biotech company with the PCR product cloning.
Hb-3RACE-GSP1:5 '-CGACCTGTGCCTCTCCGATAGCGCCG-3 ' (the 1204-1229 position of Genbank:HM175878, the 510-535 position of sequence 2)
(3) acquisition of HbSCaBP full length gene sequence
5 ' the terminal sequence that step (1) is obtained and 3 ' terminal sequence of step (2) gained splice, and obtain the full length cDNA sequence of this gene.The result demonstration, the cDNA total length of splicing gained is 1159bp, as shown in sequence in sequence table 2, wherein the 636th Nucleotide is a.In the amplification procedure of many experiments, the present inventor must be also that in sequence table, the 636th Nucleotide of sequence 2 is the situation of g.5 ' non-translational region 84bp of sequence 2,3 ' non-translational region 418bp, initiator codon is ATG, terminator codon is TAA, the 85th to 741 of open reading frame 657bp(sequence 2), be HbSCaBP with this unnamed gene.Protein (HbSCaBP albumen) in described HbSCaBP gene coded sequence table shown in sequence 1.
Two, the phylogenetic analysis of HbSCaBP
The similar calcium ion-binding protein of SOS3 (SCaBP) the family member number of logging in the Genbank of NCBI inquiry Arabidopis thaliana, paddy rice and Chinese sorghum.Wherein, 10 Arabidopis thaliana SCaBPs family members' Genbank:AtSCaBP1, AAC26009; AtSCaBP2, AAG28400; AtSCaBP3, AAG10059; AtSCaBP4, AAG28401; AtSCaBP5, AAC26008; AtSCaBP6, AAC26010; AtSCaBP7, AAL10301; AtSCaBP8, AAO72364; AtSCaBP9, AAL10300; SOS3, AAG28402.10 paddy rice SCaBPs family members' Genbank:OsSCaBP01, ABA54176; OsSCaBP02, NP_001067190; OsSCaBP03, NP_001050704; OsSCaBP04, NP_001056148; OsSCaBP05, NP_001043483; OsSCaBP06, NP_001066223; OsSCaBP07, Q3HRP0; OsSCaBP08, Q3HRN9; OsSCaBP09, ABA54184; OsSCaBP10, Q3HRN7.8 Chinese sorghum SCaBPs family members' Genbank:SbSCaBP01, FJ901259; SbSCaBP02, FJ901264; SbSCaBP03, FJ901265; SbSCaBP04, FJ901261; SbSCaBP05, FJ901263; SbSCaBP06, FJ901262; SbSCaBP07, FJ901260; SbSCaBP08, FJ901266.Adopt ClustalX software that the aminoacid sequence of all derivations is compared, in conjunction with ortho position phase connection (Neighbor-joining) constructing system evolutionary tree, and adopt the Bootstraping method to evolutionary tree test (bootstrap value〉1000).
Result shows, compares with systematic evolution tree the analysis showed that the homology the highest (61%) of SOS3 in HbSCaBP and Arabidopis thaliana SCaBP family with Arabidopis thaliana, paddy rice and Chinese sorghum SCaBP family; In paddy rice SCaBP family, be up to 82% with the homology of OsSCaBP4/OsCBL4; In Chinese sorghum SCaBP family, the homology with SbSCaBP4/SbCBL4, SbSCaBP5/SbCBL5 and SbSCaBP8/SbCBL8 is 63%, 68% and 62% respectively.From the molecular evolutionary trees analysis, HbSCaBP and SOS3, OsSCaBP4 and SbSCaBP4 belong to same subtribe, and they belong to the ortholog body on evolving.As seen in the sequence table described in step 1, the protein shown in sequence 1 (HbSCaBP albumen) is the similar calcium ion-binding protein of SOS3.
The wild barley seedling being cultured to two leaves wholeheartedly the time, carrying out respectively following arbitrary processing: 350mM NaCl, 350mM N.F,USP MANNITOL, 10%(massfraction) PEG6000 or 150 μ M ABA process 6h or 24h; Process 12h or 48h for 4 ℃.After processing, get respectively root and stem, extract total RNA and DNA digestion, carry out reverse transcription according to Superscript III test kit (Invitrogen) and obtain cDNA.Simultaneously with undressed wild barley seedling root and stem in contrast.
Adopt online software Primer3(http: //frodo.wi.mit.edu/) according to HbSCaBP full length gene cDNA(sequence 2) design special primer Hb-F and Hb-R; Determine that by trial test 18S rRNA is the reference gene of stably express, with reference to primer (forward primer: 5 '-ACGGCTACCACATCCAAGGA-3 ' as middle in Publication about Document " Michael W Christiansen; Preben B Holm and Per L Gregersen.Characterization of barley (Hordeum vulgare L.) NAC transcription factors suggests conserved functions compared to both monocots and dicots.BMC Research Notes2011,4:302 "; Reverse primer: 5 '-CAGGATTGGGTAATTTGCGC-3 ') synthetic reference gene primer.First increase by regular-PCR, and at 2%(w/v) specificity of checking primer on sepharose.Then adopt SYBR Premix Ex Taq
TM(Perfect Real Time, Takara, Ca#DRR041A) is at Step One Plus
TMQuantitative real time PCR Instrument carries out Real-time PCR reaction.Pcr amplification program: 95 ℃ of denaturation 30s; 95 ℃ of sex change 5s, 60 ℃ of annealing 30s, 40 circulations.Reaction system (20 μ L): 10 μ L SYBR Premix Ex Taq
TMMaster, 0.4 μ L ROX Dye, 2 μ L cDNA, each 10 μ M of upstream and downstream primer, distilled water complements to 20 μ L.At MicroAmp Fast Optical96-Well Reaction Plate(ABI, Ca#4346906) plate and mulch film MicroAmp
TMMove in the PCR instrument after Optical Adhesive Film (ABI, Ca#4311971).Adopt 2
-Δ Δ CtMethod (reference: " Livak﹠amp; Schmittgen, 2001.Analysis of relative gene expression data using real-time quantitative PCR and the2
-Δ Δ CtMethod.Methods25,402-408. ") carry out the gene relative expression and analyze.
Hb-F:5 '-CACTGCCCTATCTCCAGGAC-3 ' (the 653-672 position of sequence 2);
Hb-R:5 '-GTGGCAGGCATGGTTCTTAT-3 ' (reverse complementary sequence of the 738-757 position of sequence 2).
Result is being coerced early stage (6-12h) as shown in Figure 2, and 350mM N.F,USP MANNITOL, 10%PEG6000 and 4 ℃ of cold expression amounts of coercing HbSCaBP gene in rear wild barley root, stem are compared with undressed contrast (CK), and difference is all not obvious; Only 350mM NaCl coerces down, and the expression amount in HbSCaBP gene Barley Roots out of office significantly strengthens, and is approximately contrast 18 times of (CK) expression amount, and that the expression amount in stem changes is not obvious (in Fig. 2 a).This shows that HbSCaBP genetic expression has space-time characteristics, coerces early stage its and only responds salt stress various, illustrates that this gene is relevant with salt tolerance.Coercing the later stage (24-48h), 350mM NaCl, 350mM N.F,USP MANNITOL, 10%PEG6000 and after 150 μ M ABA coerce 24h in wild barley root, stem the HbSCaBP gene expression amount compare with undressed contrast (CK), difference is all not remarkable; Only after 4 ℃ of cold 48h of coercing, the expression amount of HbSCaBP gene barley root out of office increases to some extent, approximately reaches 2.4 times of contrast (CK) expression amount, but the expression amount in cauline leaf still not significantly (b in Fig. 2) compared with the control.These expression analysis further illustrate, and wild barley HbSCaBP gene pairs permeates, cold-peace ABA stress response is not obvious; Strong in the early stage response of salt stress, and not obvious in the later stage, show that this gene might participate in the signal conduction of early stage salt stress or the regulation and control of adaptibility gene.
One, turn the acquisition of HbSCaBP gene Arabidopis thaliana plant
1, the structure of recombinant expression vector pGreen0029-HbSCaBP
HbSCaBP gene (sequence 2) the design primer that 1 amplification obtains according to above-described embodiment adds BamH I and EcoR I restriction enzyme site, and primer sequence is as follows:
HbSCaBP-BamHI-F:5 '-T
GGATCCATGGGCTGCGTGTCGTCGTC-3 ' (underscore is partly the recognition sequence of BamH I, and sequence thereafter is the 85-104 position of sequence 2)
HbSCaBP-EcoRI-R:5 '-T
GAATTCTTATTTGCTGATTCCGCTGTAATCGTCG-3 ' (underscore is partly the recognition sequence of EcoR I, and sequence thereafter is the reverse complementary sequence of the 714-741 position of sequence 2)
HbSCaBP gene shown in sequence 2 is as template in the sequence table of synthetic, carry out pcr amplification with primer HbSCaBP-BamHI-F and HbSCaBP-EcoRI-R, with restriction enzyme BamH I and EcoR I, the PCR product is carried out double digestion, reclaim enzyme and cut product, be connected with the pGreen0029 carrier (containing CaMV35S promotor and Nos terminator) through same double digestion, obtain recombinant plasmid.Recombinant plasmid is changed in intestinal bacteria, resistance screening, the picking positive colony carries out liquid culture with positive colony, extracts the positive colony plasmid and carries out sequence verification.To show through order-checking the recombinant plasmid called after pGreen0029-HbSCaBP of DNA fragmentation shown in the 85-741 position of having inserted sequence 2 in the sequence table between the multiple clone site BamH of pGreen0029 carrier I and EcoR I.In recombinant expression vector pGreen0029-HbSCaBP, the promotor that starts HbSCaBP genetic expression is the CaMV35S promotor.
2, turn the acquisition of HbSCaBP gene Arabidopis thaliana plant
The recombinant expression vector pGreen0029-HbSCaBP of step 1 gained and empty carrier pGreen0029 are shocked by electricity respectively be transformed into Agrobacterium Gv3101(and contain assistant's carrier pSoup).The Agrobacterium called after Gv3101/pGreen0029-HbSCaBP of recombinant expression vector pGreen0029-HbSCaBP will be changed over to; The Agrobacterium called after Gv3101/pGreen0029 of pGreen0029 empty carrier will be changed over to.
(the former is the quality percentage composition to preparation transformed bacteria liquid for 5% sucrose, 0.05%Silwet L-77, and the latter is volume percent; OD
600=0.8～1.2).Dip in colored method (reference " Clough S.J.﹠amp according to what Clough and Bent proposed; Bent A.F., 1998.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.The Plant Journal16,735-743 "), respectively Arabidopis thaliana wild-type Columbia and Arabidopsis Mutants sos3 are contaminated conversion with the transformed bacteria liquid of restructuring Agrobacterium Gv3101/pGreen0029-HbSCaBP and Gv3101/pGreen0029.Results T
0For seed.Screen transformant containing on the MS flat board of 50mg/L Kan, obtain T
1For the resistance seedling, with T
1For resistance seedling self propagated.On the one hand, the screening offspring separates than the T that is the Arabidopsis Mutants sos3 that changes recombinant expression vector pGreen0029-HbSCaBP over to of 3:1
1For strain, from its self progeny (T
2Generation) screening next stage offspring (T in
3Generation) be all the strain of Kan resistance, obtain the T that turns HbSCaBP gene Arabidopsis Mutants sos3 that isozygotys
2For the resistance seedling.On the other hand, results T
1In generation, change the self progeny (T of the Arabidopis thaliana wild-type Columbia strain of recombinant expression vector pGreen0029-HbSCaBP over to
2Generation), with T
2By self propagated, screening the offspring separate than being the T of 3:1 for the resistance seedling
2For strain, from its self progeny (T
3Generation) screening next stage offspring (T in
4Generation) be all the strain of Kan resistance, obtain the T that turns HbSCaBP gene Arabidopis thaliana wild-type Columbia that isozygotys
3For the resistance seedling.
Further to above T
2Generation or T
3The HbSCaBP gene Arabidopis thaliana seedling that turns in generation carries out PCR and detects, and take the genomic dna that turns HbSCaBP gene Arabidopis thaliana seedling as template, the primer that PCR detects use is primer HbSCaBP-BamHI-F and the HbSCaBP-EcoRI-R in step 1.The Arabidopis thaliana wild-type Columbia and the Arabidopsis Mutants sos3 plant that change empty carrier over to are set simultaneously contrast as two empty carriers, not genetically modified Arabidopis thaliana wild-type Columbia and Arabidopsis Mutants sos3 plant are set contrast as two non-transgenics.
The PCR detected result changes the Arabidopis thaliana wild-type Columbia and the different T that change the Arabidopsis Mutants sos3 of recombinant expression vector pGreen0029-HbSCaBP over to of recombinant expression vector pGreen0029-HbSCaBP over to as shown in Figure 3
2Or T
3All increase for strain and obtained the fragment that length is about 660bp.Detect and carry out PCR with two empty carrier contrasts of same primer pair and two non-transgenic contrasts, all do not obtain above-mentioned amplified fragments.
Obtain altogether 11 T that independently isozygoty
2In generation, turn HbSCaBP gene Arabidopsis Mutants sos3 strain, and 12 T that independently isozygoty
3In generation, turn HbSCaBP gene Arabidopis thaliana wild-type Columbia strain.Therefrom select healthy growth, 3 more T of grain weight
2In generation, turn HbSCaBP gene Arabidopsis Mutants sos3 homozygous lines s5, s9 and s17, and 3 T
3For turning HbSCaBP gene Arabidopis thaliana wild-type Columbia homozygous lines W5, W11 and the W16 salt resistance identification and analysis for following step 2.
Two, the salt resistance that turns HbSCaBP gene Arabidopis thaliana plant is identified
1, experimental technique
T with the step 1 gained
3In generation, turn HbSCaBP gene Arabidopis thaliana wild-type Columbia homozygous lines (W5, W11 and W16) and T
2The homozygous lines (s5, s9 and s17) that generation turns HbSCaBP gene Arabidopsis Mutants sos3 is experiment material, carries out the salt resistance that following germination test and growth of seedling analysis of experiments turn HbSCaBP gene Arabidopis thaliana plant.The Arabidopis thaliana wild-type Columbia and the Arabidopsis Mutants sos3 plant that change empty carrier over to are set simultaneously contrast (being denoted as respectively CK1 and CK2) as two empty carriers, not genetically modified Arabidopis thaliana wild-type Columbia and Arabidopsis Mutants sos3 plant are set contrast (being denoted as respectively WT and sos3) as two non-transgenics.
(1) germination test
Every strain is chosen 60 seeds at least, and disinfection is layered on the NaCl(0,100,125, the 150mM that contain respectively different concns) the MS flat board on, 4 ℃ of depositing treatment 2 days, at temperature 22-23 ℃, intensity of illumination 1000-3000 μ molm
-2s
-1, under the condition of light application time 16h/ days, growth is 2 days, and vertical the placement grown 7 days, recorded each strain percentage of germination.Experiment repeats 3 times, results averaged.
(2) growth of seedling test
To be transferred to NaCl(0,100,125, the 150mM that contains different concns the seedling that germinateed on the MS flat board 5 days) the MS flat board on, vertical-growth is observed upgrowth situation after 2 weeks, the fresh weight of 30 strain seedling is recorded in every processing at least, is used for calculating average single-strain fresh weight.Experiment repeats 3 times, results averaged.
2, result
(1) the HbSCaBP gene can suppress quick salt phenotype at Arabidopsis Mutants sos3 overexpression
T
2The result that generation turns the germination test of homozygous lines (s5, s9 and s17) of HbSCaBP gene Arabidopsis Mutants sos3 and growth of seedling test as shown in Figure 4.
A. germination test
Do not containing on the MS flat board of NaCl, the percentage of germination of not genetically modified Arabidopsis Mutants sos3 and transgenic line s5, s9 and s17 is 100%, there is no difference; Containing on the MS flat board of 100mM NaCl, the average germination percentage of transgenic line s5, s9 and s17 is respectively 85%, 90% and 88%, and the percentage of germination of not genetically modified Arabidopsis Mutants sos3 drops to 20%, transgenic line and parent contrast percentage of germination and are utmost point significant difference (p＜0.01), show that Arabidopsis Mutants sos3 has obvious quick salt phenotype; On the substratum that contains 125mM NaCl, not genetically modified Arabidopsis Mutants sos3 percentage of germination near 9%, and 3 transgenic lines percentage of germination reach approximately 58-69%, transgenic line and parent contrast percentage of germination and are utmost point significant difference (p＜0.01), and showing that the HbSCaBP gene is crossed expression at Arabidopsis Mutants sos3 can complementary its quick salt phenotype; Arabidopsis Mutants sos3 germinate hardly (percentage of germination is lower than 5%) on the substratum that contains 150mM NaCl, and 3 transgenic lines are still germinateed (37-45%), further show, the HbSCaBP gene is crossed expression at Arabidopsis Mutants sos3 can complementary its quick salt (in Fig. 4 a).Experimental result and the not genetically modified Arabidopsis Mutants sos3 of Arabidopsis Mutants sos3 plant (CK2) that changes empty carrier over to is basically identical, no difference of science of statistics.
B. growth of seedling test
Do not containing on the MS flat board of NaCl not genetically modified Arabidopsis Mutants sos3 and transgenic line s5, s9 and s17, and not genetically modified Arabidopis thaliana wild-type Columbia(WT) growth phenotype and single-strain fresh weight there is no difference; Increase along with salt concn, 3 transgenic line s5, s9 and s17, and not genetically modified Arabidopis thaliana wild-type Columbia(WT) growth of seedling is slack-off, individual plant seedling fresh weight descends, but the measurement result difference under three different salt concn is not remarkable, and not genetically modified Arabidopsis Mutants sos3 is containing all rear brownings of growth 2 on the MS flat board of 100mM NaCl, stop growing, its single-strain fresh weight and not genetically modified Arabidopis thaliana wild-type Columbia(WT) be utmost point significant difference (p＜0.01); Not genetically modified Arabidopsis Mutants sos3 just began albefaction afterwards at 5 days on the substratum that contains 125-150mM NaCl, not regrowth, and transgenic line s5, s9 and s17 and not genetically modified Arabidopis thaliana wild-type Columbia(WT) major part still is green, and the fresh weight of transgenic line s5, s9 and s17 is significantly greater than the fresh weight (b and c in Fig. 4) of not genetically modified Arabidopsis Mutants sos3.Experimental result and the not genetically modified Arabidopsis Mutants sos3 of Arabidopsis Mutants sos3 plant (CK2) that changes empty carrier over to is basically identical, no difference of science of statistics.
The result of above germination test and growth of seedling test fully shows, the quick salt phenotype that the HbSCaBP gene can complementary Arabidopsis Mutants sos3.
(2) the HbSCaBP gene can improve salt resistance at Arabidopis thaliana wild-type Columbia overexpression
T
3The result that generation turns the germination test of homozygous lines (W5, W11 and W16) of HbSCaBP gene Arabidopis thaliana wild-type Columbia and growth of seedling test as shown in Figure 5.
A. germination test
Do not containing on the MS flat board of NaCl not genetically modified Arabidopis thaliana wild-type Columbia(WT) be 100% with the percentage of germination of 3 transgenic line W5, W11 and W16, there is no difference, show that 3 transgenic lines are all normal aspect germination; Containing on the MS flat board of 100mM NaCl not genetically modified Arabidopis thaliana wild-type Columbia(WT) and the percentage of germination of 3 transgenic line W5, W11 and W16 all reduce, but wild-type (WT) is not obvious with the difference of 3 transgenic line percentage of germination; When NaCl concentration increases to 125mM, not genetically modified Arabidopis thaliana wild-type Columbia(WT) amplitude that reduces of percentage of germination is greater than 3 transgenic lines, and 3 transgenic line W5, W11 and W16 percentage of germination all are significantly higher than wild-type (WT) percentage of germination (p＜0.01); Particularly containing on the MS flat board of 150mMNaCl not genetically modified Arabidopis thaliana wild-type Columbia(WT) substantially can not germinate, and 3 transgenic lines still can germinate, percentage of germination is about in 11-16%(Fig. 5 a).Experimental result and the not genetically modified Arabidopis thaliana wild-type Columbia of Arabidopis thaliana wild-type Columbia plant (CK1) that changes empty carrier over to is basically identical, no difference of science of statistics.
B. growth of seedling test
Do not containing on the MS flat board of NaCl not genetically modified Arabidopis thaliana wild-type Columbia(WT) all there is no difference with growth phenotype and the single-strain fresh weight of 3 transgenic line W5, W11 and W16; Along with the increase of salt concn, 3 transgenic line W5, W11 and W16, and not genetically modified Arabidopis thaliana wild-type Columbia(WT) growth of seedling is slack-off, and single-strain fresh weight descends, but the Amplitude Ratio wild-type (WT) that transgenic line descends is little; Not genetically modified Arabidopis thaliana wild-type Columbia(WT on the MS of 150mMNaCl flat board) plant begins albefaction after coercing for 2 weeks, and the growth velocity of 3 transgenic line W5, W11 and W16 seedling is significantly higher than wild-type, and the average single-strain fresh weight that is mainly manifested in transgenic line is significantly greater than the average single-strain fresh weight (b and c in Fig. 5) of wild-type (WT).Experimental result and the not genetically modified Arabidopis thaliana wild-type Columbia of Arabidopis thaliana wild-type Columbia plant (CK1) that changes empty carrier over to is basically identical, no difference of science of statistics.
The result of above germination test and growth of seedling test fully shows, the HbSCaBP gene can improve the salt resistance of Arabidopis thaliana wild-type Columbia.
In a word, express no matter the HbSCaBP gene is crossed at the quick salt mutant of Arabidopis thaliana (Arabidopsis Mutants sos3) or in Arabidopis thaliana wild-type Columbia, all can improve the salt tolerance of plant.
Claims (10)
1. the protein that is comprised of the aminoacid sequence shown in sequence in sequence table 1 is at following a1) or a2) in application:
A1) regulating plant salt resistance;
A2) seed selection salt-resistant plant kind.
2. the encoding gene of the protein that is comprised of the aminoacid sequence shown in sequence in sequence table 1 is at following a1) or a2) in application:
A1) regulating plant salt resistance;
A2) seed selection salt-resistant plant kind.
3. cultivate the method for salt tolerant transgenic plant, comprise the steps:
A) import the encoding gene of the protein that is formed by the aminoacid sequence shown in sequence in sequence table 1 in the purpose plant, obtain expressing the transgenic plant of described encoding gene;
B) obtain comparing with described purpose plant the transgenic plant that salt tolerance improves from step a) gained transgenic plant.
4. application according to claim 1 and 2, or method claimed in claim 3 is characterized in that: the encoding gene of the described protein that is comprised of the aminoacid sequence shown in sequence in sequence table 1 is following 1) to 4) in arbitrary described DNA molecular:
1) encoding sequence be in sequence table sequence 2 from the DNA molecular shown in the 85th to 741 Nucleotide of 5 ' end;
2) DNA molecular shown in sequence 2 in sequence table;
3) under stringent condition with 1) or 2) aminoacid sequence shown in sequence 1 forms in the DNA molecule hybridize that limits and coding sequence table protein DNA molecule;
4) with 1) or 2) or 3) DNA molecular that limits has the protein DNA molecule of the composition of the aminoacid sequence shown in sequence 1 in 90% above homology and coding sequence table.
5. according to claim 3 or 4 described methods is characterized in that: the encoding gene of the described protein that is comprised of the aminoacid sequence shown in sequence in sequence table 1 is that the recombinant expression vector by the encoding gene that contains described protein imports in described purpose plant.
6. arbitrary described application or method according to claim 1-5, it is characterized in that: described plant is dicotyledons or monocotyledons.
7. following arbitrary biomaterial:
(I) protein, be following a) or b):
A) protein that is formed by the aminoacid sequence shown in sequence in sequence table 1;
B) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to the plant anti-salt protein that is derived by sequence 1;
(II) nucleic acid molecule of protein described in coding (I); Described nucleic acid molecule is following 1) to 4) in arbitrary described DNA molecular:
1) encoding sequence be in sequence table sequence 2 from the DNA molecular shown in the 85th to 741 Nucleotide of 5 ' end;
2) DNA molecular shown in sequence 2 in sequence table;
3) under stringent condition with 1) or 2) protein DNA molecule described in the DNA molecule hybridize that limits and coding claim 1;
4) with 1) or 2) or 3) DNA molecular that limits has 90% above homology and the protein DNA molecule described in claim 1 of encoding;
(II) recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain nucleic acid molecule described in (II).
8. biomaterial according to claim 7, it is characterized in that: described recombinant vectors is recombinant expression vector or recombinant cloning vector.
9. method according to claim 5, or biomaterial claimed in claim 8 is characterized in that: the promotor that in described recombinant expression vector, the encoding gene of the described protein of startup is transcribed is cauliflower mosaic virus 35s promotor.
10. the recombinant vectors described in claim 7, described expression cassette, described transgenic cell line or described recombinant bacterium are at following a1) or a2) in application:
A1) regulating plant salt resistance;
A2) seed selection salt-resistant plant kind;
Described plant is specially dicotyledons or monocotyledons.
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| CN114369604A (en) * | 2022-02-17 | 2022-04-19 | 中国农业大学 | Corn salt-resistant QTL gene and application thereof |
| CN116083459A (en) * | 2023-01-19 | 2023-05-09 | 云南中烟工业有限责任公司 | Tobacco calcium ion receptor related gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106831969A (en) * | 2017-04-11 | 2017-06-13 | 北京市农林科学院 | Plant salt tolerance GAP-associated protein GAP and its application |
| CN114369604A (en) * | 2022-02-17 | 2022-04-19 | 中国农业大学 | Corn salt-resistant QTL gene and application thereof |
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