CN101054411B - Corn calcium adjusting phosphatase B analogy albumen, coding gene and application thereof - Google Patents

Abstract

The present invention discloses a calcium-modulated phosphatase B analog protein derived from corn, and its coding gene and uses in promoting plant saline resistance. The protein is one of the following amino acid residue: SEQ ID NO:1 in list; a protein substituted, deleted or added with one to ten amino acid residue from SEQ ID NO:1 in list and promote saline resistance. The inventive protein and coding gene has significance to plant saline resistance research, promote plant saline resistance and related character improvement.

Description

Corn calcium adjusting phosphatase B analogy albumen and encoding gene thereof and application

Technical field

The present invention relates to vegetable-protein and encoding gene thereof and application, particularly relate to a calcium adjusting phosphatase B analogy albumen that derives from corn (calcineurin B-like protein, CBL) and encoding gene and its application in improving plant salt endurance.

Background technology

The soil salinization is the restriction plant growth and causes one of main abiotic stress of crop failure.The salt damage that the whole world has the sewage farming of 20% arable land and 50% to be subjected in various degree approximately threatens, and the trend that increases is year by year arranged.China's salt marsh land area is about 1.4 hundred million mu, accounts for the nearly 7% of total cultivated area, also has more than 200,000,000 mu in salt marsh wasteland in addition.Cultivating the salt tolerant crop kind by genetically engineered is the most effective, one of the approach efficiently of development and use salinate fields.But up to now, in the gene that separates and identified, the resistant gene of salt that can utilize for genetically engineered seldom, and some is from bacterium and fungi, its security is often controversial.Therefore, further from the Mechanism of Salt-tolerant of gene level research plant, separation and excavation resistant gene of salt are significant from plant.

Too much be salt stress again because of salinity in soil or the environment to the harm that plant produces.Salt stress comprises mainly to the harm of plant that ion murder by poisoning, osmotic stress, nutrition are coerced with oxygen and the aspect such as coerces:

(1) ion is poisoned: think that at present the main component of harm plant-growth is Na in the solonchak ⁺, Na ⁺Entering cell in a large number is the major cause that causes salt damage.Na ⁺Murder by poisoning be many-sided.At first, Na ⁺With Ca ²⁺Ionic radius very close, so Na ⁺Bonded Ca on the replaceable cell membrane system ²⁺, the stability of membrane structure and integrity are damaged, the permeability of film increases, and the film function is impaired.Its result can cause that soluble substance (comprises K in the cell ⁺Deng) exosmose, be the H that can make on the vacuole skin on the other hand ⁺The proton pump activity change causes the H of film both sides ⁺The proton concentration gradient descends even disappears, the vacuole alkalization, and cell micro-environment changes.Secondly, Na ⁺With K ⁺Similar chemical structure and physical property are arranged, the Na of a large amount of accumulation in the tenuigenin ⁺Meeting and K ⁺Form competition, the K of direct substitution enzyme active center ⁺Or combine, thereby cause the forfeiture of many enzymic activitys with the inhibitory area of some enzyme.Therefore, Na in the tenuigenin ⁺/ K ⁺Ratio may be decision Na ⁺Toxic key.

(2) osmotic stress: salt stress and osmotic stress have indivisible relation.Too much salt branch reduces soil water potential, causes the water uptake by plant roots difficulty, even moisture exosmoses in the body, causes the major reason of hazard of plant so osmotic stress is a salt stress.

(3) nutrition is coerced: plant exists competitive relation between the close element of some chemical structures and physical property in absorbing the process of mineral element.Many results of study prove, the Na of high density ⁺Can suppress plant to K ⁺And Ca ²⁺Deng absorption and accumulation that must element, thereby cause the shortage and the ion imbalance of these elements in the cell.K ⁺Be the essential macroelement of plant, all play an important role at aspects such as keeping cell growth and turgescence, adjusting stomatal movement and enzymic activity.Therefore keep K in the cell ⁺, Na ⁺Balance is the important mechanisms of plant salt tolerance.Ca ²⁺Be not only the essential composition of keeping plant cell structures, enzymic activity and many metabolic reactions, the more important thing is as the multiple signal pathway in the direct mediated cell of second messenger, thereby in the response of plant, bring into play important regulating effect environment stress and growth signal.Increase external source Ca ²⁺Concentration can alleviate salt stress the murder by poisoning of plant is confirmed by many experiments.

(4) oxygen is coerced: high salt can also be broken the balance of active oxygen in the vegetable cell, causes active oxygen excessively to accumulate.Active o content increases can cause peroxidation of film fat or film fat fat abstraction, thereby causes the integrity of film impaired, can cause damage to macromole such as protein and nucleic acid in addition.

Plant is called salt tolerance to the adaptive faculty of salt stress.The Mechanism of Salt-tolerant of research plant, separating resistant gene of salt and cultivating the salt tolerant high-yield variety by genetically engineered is the unremitting pursue of Chinese scholars for many years.By research to the model plant Arabidopis thaliana, the researchist from the molecular level the Mechanism of Salt-tolerant to plant certain understanding has been arranged, and cultivate the strain system that some salt tolerances obviously improve by transgenic technology.But up to now, the resistant gene of salt of being cloned into is very limited, wherein can improve plant salt endurance, have the resistant gene of salt of actual application value very few especially by genetic engineering means.Below be the result of study of at present relevant plant salt tolerance mechanism:

Keeping of ionic equilibrium: under hypersaline environment, keep intracellular ionic equilibrium, especially K ⁺, Na ⁺Balance is the important mechanisms of plant salt tolerance.Existing research data shows, plant need be regulated by several approach keeping of ionic equilibrium, comprise the absorption that limits salt, increase effluxing and separating of salinity of salt, and the control salinity carries out long-distance transportation from root system to overground part absorbing.

(1) restriction Na ⁺Interior stream: plasma membrane is to Na ⁺Impervioursness be the restriction Na ⁺Diffuse into the natural cover for defense of cell, but under hypersaline environment, a large amount of Na are arranged still ⁺Enter cell.About Na ⁺How entering vegetable cell does not still have final conclusion, may be by following approach: the one, and outer rectification type cationic channel, hypersaline environment and plasma membrane depolarize have increased the possibility of this channel opener, Na ⁺Flow into cell along huge electrochemical gradient.Therefore the open adjusting of this ionic channel of any minimizing can both limit Na+ and enters cell and strengthen salt tolerance.The 2nd, (voltage-insensitive monovalent cation channel, VIC), but its molecular basis is still indeterminate to the insensitive monovalent cation passage of voltage.The 3rd, by low close K ⁺System.Na ⁺, K ⁺The film transportation of striding vie each other, show that the two has similar absorption mechanism.K ⁺Absorption by high close K ⁺With low close K ⁺Two kinds of systems carry out.At low K ⁺During (10-30 μ M) environment, by the close K of height ⁺System absorbs, and is not subjected to Na ⁺Hinder.The close K of this height ⁺System may be exactly by K ⁺/ H ⁺Carrier HKTI forms altogether.And at high K ⁺During (more than the 300 μ M) environment, by low close K ⁺System absorbs, and show as low Na+, K+ selectivity this moment, and Na+ may enter cell by this approach.Therefore under salt stress, start high close K ⁺System, thereby absorption of K specifically ⁺And prevention Na ⁺Interior stream just becomes the important mechanisms of plant salt tolerance.

(2) Na ⁺Efflux and separating: by the Na on the plasma membrane ⁺/ H ⁺Antiporter is with Na ⁺Transporting cell may be that higher plant effluxes Na ⁺Main mechanism.Plasma membrane Na ⁺/ H ⁺Antiporter is encoded by multigene family, and the SOS1 that is separated to from Arabidopis thaliana promptly belongs to one of this family member.The SOS1 transgenation causes Arabidopis thaliana to sodium-chlor and lithium chloride hypersensitization, and SOS1mRNA expression amount in the soft tissue of tip of a root epidermic cell and root, stem, food value of leaf ectosome and synplasm intersection is very high, and the film transport experiment proves that further SOS1 albumen has Na ⁺Transport activity.These evidences show SOS1 albumen Na in arabidopsis cell ⁺Stride film and have critical function in effluxing.With Na too much in the cell ⁺Be stored in the vacuole is that plant reduces another important mechanisms that Na+ poisons.This process is by the Na on the vacuole skin ⁺/ H ⁺Finish to transporter, from plants such as Arabidopis thaliana, be separated to the encoding gene of this proteinoid in recent years.Na ⁺Vacuole in storage on the one hand with Na ⁺Separate with sensitive compositions such as enzyme in the tenuigenin and plasma membranes, simultaneously can also balance by the extracellular osmotic stress that high salt caused, make cell absorb moisture as much as possible.

(3) Na ⁺Transportation in vivo and distribution: restriction Na ⁺Transport to overground part from root, and Na+ on the ground the properly distributed between each organ of portion and the tissue be the another important factor that constitutes salt tolerance.Control Na ⁺The step of long-distance transportation is Na ⁺Quick loading in the epiblem cell and in the unloading of xylem.Control root Na in the Arabidopis thaliana ⁺Fortune is SAS1 to a component of overground part, and its sudden change causes Na in the overground part ⁺Content increases several times and Na in the root ⁺Content is constant.Another is with Na ⁺Component in the xylem unloading is SOS1.The salinity of sos1 mutant overground part accumulation under the moderate salt concn is less than wild-type, and therefore its overground part infers that than the more salinity of wild-type accumulation SOS1 may promote Na under the moderate salt concn under high salt condition ⁺Transport to overground part from xylem, and SOS1 may can limit this transport process under high salt condition, thereby alleviate Na ⁺Harm to the overground part vegetative point.

Accent oozes the synthetic of material and salt stress associated protein: the interior synthetic accent of Salt Stress-induced plant materials oozes material and divides two classes: a class is the small molecules organic compound, comprise proline(Pro), trimethyl-glycine, N.F,USP MANNITOL, trehalose, sorbyl alcohol and polyamines etc., another kind of is that protein/accent oozes albumen (osmotin).Synthesize some in addition and separate toxenzyme, as glutathione s-transferase, soluble epoxide hydrolase, super-oxide enzyme, catalase and ascorbate peroxidase enzyme etc., and the albumen of many Unknown Function.The effect of these materials mainly is to regulate osmotic equilibrium, remove active oxygen or stop its pair cell structure and macromolecular damage.Environment stresses such as arid, high temperature and low temperature also can be induced the synthetic of these materials; this conforms to its mechanism of action; because these abiotic stresses can cause osmotic stress and active oxygen injury to plant, and the accumulation of these protective substance is one of important mechanisms of plant stress-resistance.

The salt stress ionic equilibrium signal transduction pathway-SOS approach of Arabidopis thaliana: the generation of plant salt endurance is by multiple signal pathway crosstalk, coefficient result.These signal pathways relate to ionic equilibrium, osmoregulation, detoxifcation reaction and all many-sides such as cell fission and growth regulating.At present in higher plant except the SOS approach of Arabidopis thaliana, understand few to the composition and the signal cascade reaction thereof of other approach.

The discovery of SOS approach starts from Arabidopis thaliana salt hypersensitization mutant, i.e. sos (salt overly sensitive) mutant choice.Since 1996, (Wu, S.J. such as the Zhu of Arizona, USA university, Ding, L., Zhu, J.K. (1996) SOS1, a genetic locus essential for salt tolerance and potassiumacquisit í on.Plant Cell 8:617-627; Liu, J., Ishitani, M., Halfter, U., Kim, C.S., Zhu, J.K. (2000) The Arabidopsis thaliana SOS2 gene encodes a proteinkinase that is required for salt tolerance.Proc.Natl.Acad.Sci.USA 97:3730-3734; Liu, J., Zhu, J.K. (1998) A calcium sensor homolog required forplant salt tolerance.Science 280:1943-1945; Shi, H., Kim, Y.S., Guo, Y., Stevenson, B., Zhu, J.K. (2003) The Arabidopsis SOS5 locus encodes a putativecell surface adhesion protein and is required for normal cell expansion.PlantCell 15:19-32). by research, take the lead in making a breakthrough aspect the higher plant salt tolerance mechanism to Arabidopis thaliana sos mutant.They obtain 5 groups of non-allelic genes mutant altogether, called after sos1-sos5, and be separated to 5 corresponding resistant gene of salt, i.e. SOS1-SOS5 by map based cloning.The sos mutant all shows the supersensitivity that sodium-chlor and lithium chloride are coerced.At present comparatively clear to the function of SOS1, SOS2 and SOS3, its coded product acts on same bars approach.A kind of plasma membrane Na of SOS1 genes encoding ⁺/ H ⁺The antiport body, its N end contains 12 hydrophobic membrane spaning domains, the C end is positioned at tenuigenin one side for long wetting ability afterbody, may with the Na in the competent cell matter ⁺Concentration is relevant, and it is the effector molecule of SOS approach.SOS1 gene transcription and proteic transport activity thereof are regulated by the part of SOS2 and SOS3 at least.SOS2 is called AtCIPK24 again, a kind of Ser/Thr protein kinase of encoding, its N end have one to yeast SNF1 kinases (sucrose-non-fermenting protein kinase) and the similar catalyst structure domain of Mammals AMP activated protein kinase (AMP-activated protein kinases), C end is regulatory region, contains the FISL motif of being made up of 21 amino-acid residues of a high conservative.The series deletion analysis shows that the FISL motif is the necessary zone of mediation SOS2 and SOS3 bonded.SOS3 is called CBL4 again, and its coded product is a kind of calcium ion-binding protein, with the B subunit (CNB) and the animal nerve calcium sensor (NCS) of yeast and animal calcineurin higher homology is arranged.Infer contain in its aminoacid sequence 3 with calcium ion in conjunction with relevant EF chiral structure territory and N end myristoylation characteristic sequence.Experimental results show that calcium ion combination and N end myristoylation all are essential for the function of SOS3 in salt tolerance.Based on above-mentioned result of study, (Halfter such as Zhu, U., Ishitani, M., Zhu, J.K. (2000) The Arabidopsis SOS2 protein kinasephysically interacts with and is activated by the calcium-binding protein SOS3.Proc.Natl.Acad.Sci.USA 97:3735-3740; Qiu, Q.S., Guo, Y., Dietrich, M.A., Schumaker, K.S., Zhu, J.K. (2002) Regulation of SOS1, a plasma membrane Na ⁺H ⁺Exchanger in Arabidopsis thaliana, by SOS2 and SOS3.Proc.Natl.Acad.Sci.USA 99:8436-8441; Zhu, J.K. (2002) Salt and drought stress signaltransduction in plants.Annu.Rev.Plant Biol.53,247-273.) the SOS model of Arabidopis thaliana salt stress signal transduction has been proposed: in the SOS approach, the SOS3 impression combines and activates its kinase activity with its target protein SOS2 then by the calcium signal that salt stress excited; Activatory SOS3-SOS2 complex body further activates SOS1 on the plasma membrane by phosphorylation, again by SOS1 with Na too much in the cytosol ⁺Outside the transporte to cells, thereby kept intracellular ionic equilibrium.This model is further verified by the reconstruction experiment of SOS approach in yeast.

Resistant gene of salt divides two classes, one class is a functional gene, the effector molecule of its product for directly in salt tolerance, working, as the various small molecules of encoding transfer the gene that oozes the material synthetic enzyme to conciliate the gene of toxenzyme class, and the encoding gene of various film transport proteins, aquaporin and some metabolic enzymeses relevant with salt stress also belongs to this type of.Another kind of is regulatory gene, and its product is positioned at the upstream of effector molecule in the salt signal transduction pathway, directly or indirectly the expression of functional gene or the activity of its product is regulated.The adjusting albumen of having identified comprises calcium ion induction albumen, protein kinase and transcription factor etc.Over nearly 20 years; both at home and abroad from various biologies; comprise and be separated to many salt-resistant related genes in bacterium, yeast, algae and the higher plant; but can improve transgenic plant salt tolerance person through functional verification and only account for a wherein small part; comprise that the coding accent oozes the functional gene of material synthetic enzyme, film transport protein and ionic pump, antioxidant and protectiveness enzyme, and the minority regulatory gene.Although these genes of bibliographical information can improve the salt tolerance of transgenic plant, most very limited for the improvement degree of salt tolerance, in agriculture production, still there is not practical value.Cause the reason of this present situation mainly to be the complicacy of plant salt endurance itself and the limitation of used resistant gene of salt.Plant salt endurance is the quantitative character by controlled by multiple genes, and the formation of salt tolerance is the result of a plurality of physiological processs comprehensive action on different levels such as organ, tissue and cell, may relate to the comprehensive regulation of some signal pathways.At present, goal gene used in the plant salt tolerance genetically engineered belongs to the salt tolerant functional gene mostly, transgenosis in recipient cell, be subjected to unavoidably relevant regulatory pathway influence and can not normal expression even take place reticent, adding majority is that single-gene transforms, therefore, the effect of improving plant salt endurance by this approach is very limited.The associated metabolic process of regulating and control recipient plant by conversion salt tolerant regulatory gene may be the effective way that overcomes this limitation.At present, the regulatory gene of having separated and can improve plant salt endurance by transgenosis checking seldom comprises AtGSK1 (glycogen synthase kinase) gene of OsCDPK7 (calcium ion deopendent protein kinase) gene, Arabidopis thaliana of encoding gene (DREB, Alfinl, MsPRP2 and Tsi1 etc.), zymic CaN (calcineurin) gene and HAL1 gene, the paddy rice of several transcription factors and RS (SR-like montage albumen) gene etc.Wherein some gene (as DREB and OsCDPK7 etc.) can also improve the resistance of transgenic plant to environment stresses such as low temperature and arids.

Calcium ion plays keying action as intracellular important second messenger in the transmittance process of vegetable cell to various environment-stress and self growth signal.Environment stresses such as high salt, arid and low temperature all can trigger the free calcium ion concentration moment rising in the plant cytoplasm, thereby produce the calcium signal.It is generally acknowledged that the calcium signal that is triggered by the different adverse circumstance factors has specificity, and discerned and transmit, and then activate a series of signal cascade reaction in its downstream, finally cause cell response by corresponding calcium ion induction albumen.Therefore, calcium ion induction albumen is the member of upstream in the various calcium signal pathways, and the transmission of calcium signal and the cell response reaction of mediation thereof are had very crucial regulating effect.So far the calcium ion induction albumen of finding in higher plant mainly contains three classes, the first kind is calmodulin and calmodulin associated protein family, second class is calcium ion deopendent protein kinase (CDPKs) family, the 3rd class be find recently with albumen like the calcineurin category-B (calcineurin B-like proteins, CBLs) family.Respectively there are ten members in CBL family in Arabidopis thaliana and paddy rice, wherein the AtCBL1 of Arabidopis thaliana, 4,9 and the OsCBL2 of paddy rice separated and carried out Function Identification.AtCBL4/SOS3 is first separated and identify CBL gene, has important regulatory function in the salt stress ionic equilibrium signal pathway of Arabidopis thaliana, but does not see it can improve salt tolerance in transgenic plant report so far as yet.AtCBL1 participates in the response of Arabidopis thaliana to low temperature, arid and salt stress, its overexpression in Arabidopis thaliana can be improved salt tolerance and the drought tolerance of plant, but the frost resistance of transfer-gen plant obviously reduces, and this has influenced this gene to a certain extent and has been worth in the resistant gene of salt application in engineering.AtCBL9 participation Arabidopis thaliana is to the response of ABA (dormin) and play important regulatory role in ABA is synthetic.OsCBL2 participation paddy rice aleurone cell replys GA's (Plant hormones regulators,gibberellins/acid), and may be in aleurone cell's vacuolization of GA signal pathway mediation, i.e. functionating in the formation of aleuron vesicle.The CBL gene is peculiar by higher plant, has not yet to see the report of the CBL gene of isolating definite functions from other plant.

Summary of the invention

The purpose of this invention is to provide a calcium adjusting phosphatase B analogy albumen (calcineurin B-Like protein, CBL).

Calcium adjusting phosphatase B analogy albumen provided by the present invention, name is called ZmCBL4, derives from Zea corn (Zeamays L.), is one of following amino acid residue sequences:

1) the SEQID NO:1 in the sequence table;

2) with the amino acid residue sequence of SEQ ID NO:1 through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein that improves the plant salt tolerance sexual function.

SEQID NO:1 in the sequence table is made up of 211 amino-acid residues; wherein; be and the relevant EF chiral structure territory of calcium ion combination to be N end myristoylation feature motif from aminoterminal 44-57 position, 81-92 position, 118-129 position and 162-173 amino acids residue from aminoterminal 1-6 amino acids residue.

One to ten amino-acid residue of described replacement, disappearance or interpolation can be the amino-acid residue in the non-structural domain, and its change can not exert an influence to this proteic function.

The gene (ZmCBL4) of code book invention calcium adjusting phosphatase B analogy albumen, its cDNA is one of following nucleotide sequence:

1) dna sequence dna of SEQ ID NO:2 in the sequence table;

2) dna sequence dna of SEQ ID NO:1 in the code sequence tabulation;

3) dna sequence dna with SEQ ID NO:2 qualification has 90% above homology and has the nucleotide sequence that improves the plant salt tolerance sexual function;

4) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with SEQ ID NO:2 in the sequence table.

The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

SEQID NO:2 in the sequence table is by 636 based compositions, and its encoding sequence is that coding has the protein of the amino acid residue sequence of SEQ ID NO:1 in the sequence table from 5 ' end 1-633 bit base.

Its genomic gene is one of following nucleotide sequence:

1) dna sequence dna of SEQ ID NO:3 in the sequence table;

2) with sequence table in the dna sequence dna that limits of the SEQ ID NO:3 nucleotide sequence that has 90% above homology and have raising plant salt tolerance sexual function;

3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with SEQ ID NO:3 in the sequence table.

The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

SEQ ID NO:3 in the sequence table is by 1994 based compositions, from 5 ' end 1-82 bit base is the 1st exon of this genomic gene, from 5 ' end 83-199 bit base is the 1st intron of this genomic gene, from 5 ' end 200-282 bit base is the 2nd exon of this genomic gene, from 5 ' end 283-392 bit base is the 2nd intron of this genomic gene, from 5 ' end 393-452 bit base is the 3rd exon of this genomic gene, from 5 ' end 453-527 bit base is the 3rd intron of this genomic gene, from 5 ' end 528-636 bit base is the 4th exon of this genomic gene, from 5 ' end 637-833 bit base is the 4th intron of this genomic gene, from 5 ' end 834-886 bit base is the 5th exon of this genomic gene, from 5 ' end 887-1530 bit base is the 5th intron of this genomic gene, from 5 ' end 1531-1605 bit base is the 6th exon of this genomic gene, from 5 ' end 1606-1716 bit base is the 6th intron of this genomic gene, from 5 ' end 1717-1829 bit base is the 7th exon of this genomic gene, from 5 ' end 1830-1939 bit base is the 7th intron of this genomic gene, is the 8th exon of this genomic gene from 5 ' end 1940-1994 bit base.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification ZmCBL4.

Another object of the present invention provides a kind of method that improves plant salt endurance.

The method of raising plant salt endurance provided by the present invention, be to have 90% above homology with described corn calcium adjusting phosphatase B analogy albumen gene ZmCBL4 or with ZmCBL4 and dna sequence dna importing plant tissue, cell or the organ of the same protein of encoding, plant salt endurance obtains to improve.

In the method for above-mentioned raising plant salt endurance, described corn calcium adjusting phosphatase B analogy albumen gene ZmCBL4 both can be the cDNA sequence of ZmCBL4, also can be the genomic gene sequence of ZmCBL4; With the dna sequence dna that ZmCBL4 has 90% above homology and coding same protein, be the cDNA of ZmCBL4 or genomic gene sequence to be separated and/or modified and/or design with known method obtain.What it should be appreciated by those skilled in the art is; the minor alteration of Nucleotide identity may cause the reduction or the reinforcement of this gene usefulness in the specific gene sequence; and (for example in some application; antisense or suppress technology altogether) in, partial sequence plays a role equally effectively through regular meeting and full length sequence.The method that gene order changes or shortens, and the method for testing the validity of these genes that change all is well known to those skilled in the art.

Described corn calcium adjusting phosphatase B analogy albumen gene ZmCBL4 or its homologous sequence can import plant tissue, cell or organ by the plant expression vector that contains ZmCBL4 or its homologous sequence; The carrier that sets out that is used to make up described plant expression vector can be any one and can be used for the carrier etc. that agrobacterium tumefaciens or Agrobacterium rhizogenes transform the binary vector of plant or can be used for the plant micropellet bombardment, as pCAMBIA serial carrier, PER8, PX6, pBI serial carrier, pBin serial carrier or other plant expression vector of deriving, the described carrier that sets out also can be the carrier that can duplicate in prokaryotic organism, as pUC serial carrier or pBluescript serial carrier etc.

When using ZmCBL4 or its homologous sequence to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter; Described constructive expression's promotor can be cauliflower mosaic virus (CAMV) 35S promoter, corn Ubiquitin promotor or paddy rice actin1 promotor etc.; Described tissue specificity expression promoter can be the seed-specific expression promotor, flower specific expresses promotor or pollen specific is expressed promotor, as 2S1 promotor (GenBank number: NM_118848.2, GI:30687489) and NapinA (GenBank number: M64633.1, GI:349405) promotor; Described inducible promoter can be inductive promotors such as being subjected to ABA, ethene or chemistry; Above-mentioned promotor can be used separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene (gus gene of luminophor as adding the coding that in plant, to express, the GFP gene, luciferase genes etc.), antibiotic marker thing (neomycin phosphotransferase (NPTII) gene with resistance, hygromix phosphotransferase (Hygromycin phosphotransferase) gene, gentamicin marker or kantlex marker etc.) or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.Described host plant cell, tissue or the organ that contains neomycin phosphotransferase gene can be screened by kantlex or its substituted derivatives such as G418 etc., and the host plant cell, tissue or the organ that contain hygromycin phosphotransferase gene can be screened by Totomycin.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.After aforesaid method screens, also can adopt Southern, PCR or dot blot equimolecular detection means that transfer-gen plant is detected, whether transform goal gene to determine it.

Wherein, be the carrier that sets out with pCAMBIA3301, the plant expression vector that contains ZmCBL4 of structure is p3301-ZmCBL4.

The plant expression vector that carries corn calcium adjusting phosphatase B analogy albumen gene ZmCBL4 of the present invention or its homologous sequence can be by using protoplastis-chemical mediated method (Ca ²⁺, PEG), combination transformed plant cells, tissue or the organ of any or several method in the conventional biological methods such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, pollen tube, microinjection, electricity swash, particle gun, and plant transformed cell, tissue or organ cultivated into plant; Described tissue and organ can comprise fruit pod, callus, stem apex, blade and the seed etc. of host plant.

In addition, after conversion being had corn calcium adjusting phosphatase B analogy albumen gene ZmCBL4 of the present invention or carrying out succeeding transfer culture, can therefrom further filter out the transfer-gen plant that genotype is isozygotied with the transfer-gen plant of dna sequence dna that ZmCBL4 has 90% above homology and a coding same protein.

Method of the present invention all is suitable for dicotyledons and monocotyledons, therefore, describedly both dicotyledonss such as Arabidopis thaliana, rape, peanut, cotton, soybean, Sunflower Receptacle, palm tree, olive, castor-oil plant, potato or tobacco can be derived from, also monocotyledonss such as corn, paddy rice, wheat, barley, oat, rye, jowar, millet or turfgrass can be derived from by plant transformed cell, tissue or organ.

The invention provides a calcium adjusting phosphatase B analogy albumen ZmCBL4 and an encoding gene thereof that derives from corn.Corn salt tolerant regulatory gene ZmCBL4 provided by the invention belongs to higher plant CBL gene family member, and its proteins encoded has important regulatory function in the calcium ion signal transduction pathway of plant to the salt stress response, and the salt tolerance of plant is strengthened.Transgenic experiments result shows, its overexpression in Arabidopis thaliana sos3 mutant can be recovered the salt tolerant phenotype of mutant fully, and overexpression can obviously improve the salt tolerance of transgenic line in seed germination phase and growth of seedling phase in wild-type.The Mechanism of Salt-tolerant of inferring the ZmCBL4 transfer-gen plant is: this gene can be experienced the calcium signal that is triggered by salt stress, and the ionic equilibrium signal transduction pathway of startup plant, be the SOS approach, thereby make cell keep ionic equilibrium under hypersaline environment, plant salt endurance is enhanced.Albumen of the present invention and encoding gene thereof are for the plant salt tolerance Study on Mechanism, and improve the salt tolerance of plant and the improvement of correlated character has important theory and practical significance, to in the resistant gene of salt engineering improvement of plant, play a significant role, have a extensive future.

Below in conjunction with specific embodiment the present invention is described in further details.

Description of drawings

Figure 1A is the agarose gel electrophoretogram of ZmCBL4 full length gene cDNA pcr amplification product

Figure 1B is the agarose gel electrophoretogram of ZmCBL4 gene genomic dna pcr amplification product

Fig. 2 is that the Southern blot of ZmCBL4 copy number in the corn gene group analyzes collection of illustrative plates

Fig. 3 A is that the ZmCBL4 gene is in the expression performance analysis result in the corn seedling blade under the different stress conditions

Fig. 3 B is ZmCBL4 gene expression performance analysis result in maize seedling roots under different stress conditions

Fig. 4 is the specific expressed level of ZmCBL4 in the corn different tissues

Fig. 5 cuts the evaluation electrophoretogram for the enzyme of the plant expression vector p3301-ZmCBL4 of structure

Fig. 6 is the PCR evaluation electrophoretogram of transgenic arabidopsis T1 for goal gene ZmCBL4 in the positive plant

Fig. 7 A is that ZmCBL4 analyzes collection of illustrative plates at ZmCBL4 transgenic arabidopsis sos3 mutant T3 for the Northernblot in the homozygous lines

Fig. 7 B is that ZmCBL4 analyzes collection of illustrative plates at ZmCBL4 transgenosis wild-type Arabidopis thaliana T3 for the Northern blot in the homozygous lines

Fig. 8 A is that the curved root of seedling is tested the ZmCBL4 transgenic arabidopsis sos3 mutant T3 of detection for the salt tolerance qualification result of homozygous lines on additional different concns sodium-chlor of MS or lithium chloride substratum

Fig. 8 B is that seed germination and growth of seedling are tested the ZmCBL4 transgenic arabidopsis sos3 mutant T3 of detection for the salt tolerance qualification result of homozygous lines on additional different concns sodium-chlor of MS or lithium chloride substratum

Fig. 9 ties up to salt tolerance qualification result on additional finite concentration sodium-chlor of MS or the lithium chloride substratum for ZmCBL4 transgenosis wild-type Arabidopis thaliana T3 for strain

Figure 10 A is that ZmCBL4 transgenosis wild-type Arabidopis thaliana T3 ties up to the seed germination rate statistics on the MS substratum that adds different concns sodium-chlor for strain

Figure 10 B is that ZmCBL4 transgenosis wild-type Arabidopis thaliana T3 ties up to the seed germination rate statistics on the MS substratum that adds the different concns lithium chloride for strain

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3 ^RdEdition, 2001, NY, Cold SpringHarbor).The primer and dna sequence dna are given birth to worker's biotechnology company limited by Shanghai and are synthesized.

The acquisition of embodiment 1, corn calcium adjusting phosphatase B analogy albumen gene ZmCBL4 and gene organization's structure thereof and genome copy number are analyzed

One, the amplification of the acquisition of the full length cDNA sequence of corn calcium adjusting phosphatase B analogy albumen gene ZmCBL4 and full-length cDNA and genomic dna

1, the acquisition of corn calcium adjusting phosphatase B analogy albumen gene ZmCBL4 cDNA sequence

With the amino acid residue sequence of the calcium adjusting phosphatase B analogy albumen (CBL) of Arabidopis thaliana at NCBI; in the genome database such as MaizeGDB and TIGR the various sequences of corn are carried out TBLASTN and BLASTP search; and the sequence that obtains spliced assembling; the result obtains 9 contigs altogether; the i.e. CBL encoding sequence of the corn of 9 suppositions; then the CBL amino acid residue sequence of these 9 suppositions and the amino acid residue sequence of 10 CBL in the Arabidopis thaliana are carried out sequence alignment and cluster analysis; found that one with the encoding sequence of AtSOS3/AtCBL4 (GenBank number: AF192886) the highest gene order of homology; this sequence has the nucleotide sequence of SEQ ID NO:4 in the sequence table; by 1417bp based composition; its encoding sequence has the nucleotide sequence of SEQ ID NO:2 in the sequence table; by 636 based compositions; the amino acid residue sequence of SEQ ID NO:1 in the code sequence tabulation; SEQID NO:1 in the sequence table is made up of 211 amino-acid residues; with the consistence of AtSOS3/AtCBL4 be 57%; similarity is 72%; The sequencing results shows has 4 (to be respectively among the SEQ ID NO:1 from aminoterminal 44-57 position in conjunction with relevant EF chiral structure territories with calcium ion in the amino acid residue sequence of this genes encoding; the 81-92 position; 118-129 position and 162-173 amino acids residue) and N end myristoylation feature motif (among the SEQ ID NO:1 from aminoterminal 1-6 amino acids residue); with this unnamed gene is ZmCBL4, its proteins encoded called after ZmCBL4.

2, the amplification of corn calcium adjusting phosphatase B analogy albumen gene ZmCBL4 cDNA sequence

According to a pair of primer of sequences Design (SN1 and AB1) at ZmCBL4 gene complete encoder block two ends, primer sequence is as follows:

SN1 (upstream primer): 5 '-GATCCATGGGCTGCGCGACGTCCAA-3 ';

AB1 (downstream primer): 5 '-GTGGGTCACCATACGCAGATGTACGCAAAC-3 '.

Total RNA of corn drought 21 (being provided by the crop investigations of Shanxi Province Academy of Agricultural Sciences) self-mating system seedling is provided with hot phenol method, with the M-MLV ThermoScript II of Promega company and with reference to synthetic its cDNA of specification sheets reverse transcription and as template, under the guiding of primer SN1 and AB1, with the total length eDNA of conventional PCR method amplification ZmCBL4.After reaction finishes; pcr amplification product is carried out 1.4% agarose gel electrophoresis to be detected; (swimming lane M is detected result: 100bp Marker shown in Figure 1A; swimming lane 1 and 2 is: pcr amplification product); obtained the dna fragmentation of 674bp through amplification; conform to expected results; reclaim and this fragment of purifying; connect among the carrier pGEM-T Easy (Promega company); to connect product transformed into escherichia coli (E.coli) DH5 α competent cell (three rich companies) again; screening positive clone; the upgrading grain; obtain containing the segmental recombinant plasmid of purpose; called after pGEM-ZmCBL4; it is checked order; pGEM-ZmCBL4 recombinant plasmid to 3 independent PCR has carried out sequencing altogether; sequencing result shows the full length cDNA sequence that has obtained the correct ZmCBL4 of sequence; nucleotide sequence with SEQ ID NO:2 in the sequence table; by 636 based compositions; the amino acid residue sequence of SEQ ID NO:1 in the code sequence tabulation; wherein, from 5 ' end 132-171 position; the 243-276 position; 354-387 position and 486-519 bit base coding and the relevant EF chiral structure territory of calcium ion combination are from 5 ' end 1-18 bit base coding N end myristoylation feature motif.

3, the amplification of corn calcium adjusting phosphatase B analogy albumen gene ZmCBL4 genomic dna

Extract the genomic dna of corn drought 21 self-mating system seedling with the CTAB method, under the guiding of primer SN1 and AB1, with the genomic dna of conventional PCR method amplification ZmCBL4.After reaction finishes, pcr amplification product is carried out 1.0% agarose gel electrophoresis to be detected, (swimming lane M is detected result: 100bp Marker shown in Figure 1B, swimming lane 1 and 2 is: pcr amplification product), obtained the dna fragmentation of 2032bp through amplification, conform to expected results, reclaim and this fragment of purifying, connect among the carrier pGEM-T Easy, will connect product transformed into escherichia coli (E.coli) DH5 α competent cell again, screening positive clone, the upgrading grain, obtain containing the segmental recombinant plasmid of purpose, called after pGEM-ZmCBL4Z checks order to it, sequencing result shows the genomic dna that has obtained the correct ZmCBL4 of sequence, nucleotide sequence with SEQ ID NO:3 in the sequence table, by 1994 based compositions, the amino acid residue sequence of SEQ IDNO:1 in the code sequence tabulation.

Two, ZmCBL4 gene organization structure and genome copy number are analyzed

1, gene organization's structural analysis

Step 1 is carried out the sequence alignment analysis through the cDNA sequence and the genomic dna sequence of the ZmCBL4 gene of order-checking acquisition, the result shows that the genomic dna of this gene has 7 introns and 8 exons, wherein, from 5 ' end 1-82 bit base is the 1st exon of this genomic gene, from 5 ' end 83-199 bit base is the 1st intron of this genomic gene, from 5 ' end 200-282 bit base is the 2nd exon of this genomic gene, from 5 ' end 283-392 bit base is the 2nd intron of this genomic gene, from 5 ' end 393-452 bit base is the 3rd exon of this genomic gene, from 5 ' end 453-527 bit base is the 3rd intron of this genomic gene, from 5 ' end 528-636 bit base is the 4th exon of this genomic gene, from 5 ' end 637-833 bit base is the 4th intron of this genomic gene, from 5 ' end 834-886 bit base is the 5th exon of this genomic gene, from 5 ' end 887-1530 bit base is the 5th intron of this genomic gene, from 5 ' end 1531-1605 bit base is the 6th exon of this genomic gene, from 5 ' end 1606-1716 bit base is the 6th intron of this genomic gene, from 5 ' end 1717-1829 bit base is the 7th exon of this genomic gene, from 5 ' end 1830-1939 bit base is the 7th intron of this genomic gene, is the 8th exon of this genomic gene from 5 ' end 1940-1994 bit base.

2, carry out the analysis of genome copy number with the Southern hybrid method

With the Southern hybrid method ZmCBL4 gene is carried out the copy number analysis, method is: the genomic dna of getting 30 μ g corn inbred lines drought, 21 seedling, carry out single endonuclease digestion digestion with restriction enzyme EcoRI, EcoRV, HindIII, BamHI, Kpn I and Sac I respectively, enzyme is cut product carry out the separation of 0.8% agarose gel electrophoresis, shift liquid with alkali then and (see Sambrook, et al., " Molecular Cloning:A Laboratory Manual ", 3 ^RdEdition, 2001, NY, Cold Spring Harbor) be transferred to the Southern blotting membrane, the full-length cDNA with the ZmCBL4 gene is that probe is hybridized (Prime-a-Gene Labelling System test kit and reference reagent box specification sheets with Promega company prepare probe) again; Prehybridization and hybridization solution are Church damping fluid (seeing Sambrook, et al., " Molecular Cloning:A Laboratory Manual ", 3rd edition, 2001, NY, Cold Spring Harbor); After hybridization finishes, use 2 * SSC+0.5%SDS, 1 * SSC+0.5%SDS, 0.5 * SSC+0.5%SDS and 0.1 * SSC+0.1%SDS film washing liquid under 65 ℃, respectively to wash film once successively, each 15min, sop up the moisture on film surface then with thieving paper, wrap the back with preservative film and press the phosphorus plate, carry out image scanning after three days, the result shows that the ZmCBL4 gene has only a copy in the corn gene group as shown in Figure 2.

The abduction delivering of embodiment 2, ZmCBL4 gene and tissue specific expression analysis

One, the expression performance analysis of ZmCBL4 gene under different stress conditions

At first analyze the expression of ZmCBL4 gene under different stress conditions (250mM NaCl, 20mM LiCl, 100 μ M ABA, 20%PEG solution) with conventional Northern blot method, the result does not all detect hybridization signal, show that this expression of gene abundance is very low, therefore use real-time fluorescence quantitative PCR (Real-time quantitativePCR instead, RT-qPCR) technology is analyzed the expression of ZmCBL4 gene under different stress conditions, and concrete grammar may further comprise the steps:

1, coerces processing

Cultivate corn drought 21 self-mating system seedling to tri-leaf period with Sha Peifa, careful whole strain seedling and the clean root sand of taking out from flowerpot, (prescription is referring to Chen Jianxun with the Hogland nutritive medium, Wang Xiaofeng. plant physiology experiment instructs (second edition), press of South China Science ﹠ Engineering University, in February, 2006) under aeration condition, adapt to cultivation three days, change 250mM NaCl then respectively into, 20mM LiCl, 100 μ M ABA and 20%PEG solution (with the preparation of Hogland nutritive medium) are coerced processing, learn from else's experience to coerce and handle 0,2,6, seedling after 12 and 24 hours, root system and blade separately and are rapidly dropped into liquid nitrogen, be transferred in-76 ℃ of refrigerators and preserve.

2, RT-qPCR detects

Coerce the root system of milpa of processing and total RNA of blade through difference in the extraction step 1 respectively with hot phenol method, and digest with RQI DNase (Promega company), pollute to remove genomic dna, be synthetic its cDNA of template reverse transcription with total RNA then, again with 10 times of templates of synthetic cDNA dilution as RT-qPCR.Then, ((GenBank number: X15704) as internal standard gene, the applied sample amount to different sample RNA/cDNA templates when data analysis carries out homogenization to the gene of α-tubulin) with the corn alpha-tubulin.According to real-time fluorescence quantitative PCR primer design principle (Applied Biosystems), Auele Specific Primer with Primer Express 2.0 software design goal gene ZmCBL4 and internal standard gene α-tubulin, the genomic dna that may exist in the template eDNA pollutes amplification is exerted an influence, sense primer is located at respectively in the different exons with antisense primer, and primer sequence is as follows: the primer of testing goal gene ZmCBL4 expression level:

RTS3 (upstream primer): 5 '-TCAGTGTGTTCCACCCTAAAGCA-3 '

RTA3 (downstream primer): 5 '-ATCAAGCAGCGCCAAGACCAT-3 ',

At primer to RTS3﹠amp; RTA3 guiding is contemplated to 128bp with cDNA as the amplified production length of template down, is contemplated to 963bp with genomic dna as the amplified production length of template.

Detect the primer of internal standard gene α-tubulin expression level:

TS6 (upstream primer): 5 '-GAGCATGGCATTCAGGCTGACG-3 '

TA6 (downstream primer): 5 '-TCAACAAAAACAGCACGGGGCA-3 ',

At primer to TS6﹠amp; TA6 guiding is contemplated to 128bp with cDNA as the amplified production length of template down, is contemplated to 987bp with genomic dna as the amplified production length of template.

With above-mentioned cDNA through 10 times of dilutions as template, respectively at primer to RTS3﹠amp; RTA3 and TS6﹠amp; Carrying out RT-qPCR under the guiding of TA6 detects, the PCR reaction system is: 2 * SYBR GreenIPCR Master Mix (ABI company), 12.5 μ L, RTS3 (or TS6) (10 μ M) 0.5 μ L, RTA3 (or TA6) (10 μ M) 0.5 μ L, cDNA template (dilution in 1: 10) 1 μ L is with sterilization ddH ₂O postreaction system to 25 μ L.The PCR reaction conditions is: 50 ℃ of 2min of elder generation; 95 ℃ of 10min then; 94 ℃ of 30s again, 60 ℃ of 30s, 72 ℃ of 18s, totally 40 circulations; Last 95 ℃ of 15s, 60 ℃ of 15s, 95 ℃ of 15s, 1 circulation.After reaction finishes, pcr amplification product is carried out 2.0% (w/v) agarose gel electrophoresis to be detected, the result is 128bp to the length of the amplified production of goal gene ZmCBL4 and internal standard gene α-tubulin, and is consistent with expected results, and illustrating does not have genomic dna to pollute.With untreated samples (0h) is with reference to sample, according to 2 ^{-Δ Δ Ct}Formula (Livak, K.J., Schmittgen, T.D. (2001) Analysis of relativegene expression data using real-time quantitative PCR and the 2 ^{-Δ Δ CT}Method.Methods 25:402-408) it is dynamic to calculate the expression that ZmCBL4 coerces under the treatment condition in difference.The result shows, coerces down at 250mM NaCl, and ZmCBL4 expression amount in blade is reduced rapidly, and expression amount slowly increases in root system; Coerce down at 20mM LiCl, the ZmCBL4 changes of expression level is dynamically similar to sodium-chlor in the root system, then is to increase sharply earlier in blade, is reduced to the basal expression level then again; 100 μ M ABA handle the expression of ZmCBL4 are raised rapidly, especially in root system on modulation factor of amplitude modulation more obvious, the last modulation factor of amplitude modulation during this external entire treatment in root system and the blade all shows the wave variation; 20%PEG coerces the expression level of ZmCBL4 is obviously reduced, and changes more obvious in blade.The concrete expression of ZmCBL4 dynamically and the relative changing value of different time points is seen Fig. 3 A (expression of ZmCBL4 in blade is dynamic) and Fig. 3 B (expression of ZmCBL4 in blade is dynamic).Above-mentioned test-results shows, ZmCBL4 has certain basis to express in the corn seedling phase, high salt, height ooze or exogenous aba treatment can make its expression pattern change, show that this gene participates in corn seedling to the replying of these environment stresses, and might in different calcium signal pathways, bring into play regulating effect.

Two, detect the tissue specific expression situation of ZmCBL4 gene

With with step 1 in identical RT-qPCR method detect the specific expressed situation of ZmCBL4 gene in different tissues and organ, (1 is the young root in tri-leaf period to the result as shown in Figure 4; 2 is the spire in tri-leaf period; 3 is the matured root of tasseling stage; 4 is the ripe stem of tasseling stage; 5 is the climax leaves of tasseling stage; 6 is the female fringe of tasseling stage; 7 is the tassel of tasseling stage; 8 is the filigree that has just spued), show that there is significant difference in the expression abundance of ZmCBL4 gene in different tissues or organ, wherein minimum to express abundance in climax leaves, stem and the tassel of tasseling stage, and the highest in female fringe.If as the reference sample, the relative expression's multiple change in other tissue or organ is between 1.05-10.98 with the climax leaves of tasseling stage.In addition, ZmCBL4 expression of gene abundance is lower than spire in the young root in tri-leaf period, and be in the climax leaves nearly 3 times in the matured root of tasseling stage, this tissue specific expression pattern hint ZmCBL4 gene may participate in the adjusting to some signal pathway in the development process of corn.

The acquisition of embodiment 3, ZmCBL4 transgenic arabidopsis and salt tolerance thereof are identified

One, the acquisition of ZmCBL4 transgenic arabidopsis

1, the structure of ZmCBL4 plant expression vector

NcoI and BstEII restriction enzyme site according to primer SN1 and the design of AB1 two ends, carry out double digestion with restriction enzyme NcoI and BstEII to containing ZmCBL4 full-length cDNA fragment carrier pGEM-ZmCBL4, reclaim the also ZmCBL4 cDNA fragment of purifying 660bp, with its plant expression vector pCAMBIA3301 (p3301 with the same enzyme double digestion of warp, Australia CAMBIA company) carrier connects, make ZmCBL4 under the driving of CaMV35S promotor, and then will connect product transformed into escherichia coli (E.coli) DH5 α competent cell, screening positive clone, the upgrading grain carries out single endonuclease digestion and NcoI﹠amp with BamH I successively; The BstEII double digestion is identified, enzyme is cut product carry out the detection of 1.0% agarose gel electrophoresis, and (swimming lane 1 is a BamHI single endonuclease digestion product to the result, and swimming lane 2 is Nco I﹠amp as shown in Figure 5; BstE II double digestion product, swimming lane M is 1kb Marker), can obtain the dna fragmentation of 9073bp and 844bp through BamH I single endonuclease digestion, through Nco I﹠amp; The BstEII double digestion can obtain the dna fragmentation of about 9257bp and 660bp, conforms to expected results, shows the plant expression vector of the ZmCBL4 that has obtained insertion sequence and correct position, called after p3301-ZmCBL4.

2, arabidopsis thaliana transformation

Get the about 1 μ g of plasmid p3301-ZmCBL4 that step 1 obtains, it is transformed Agrobacterium GV3101 competent cell (three rich companies), (contain kantlex Kan 100 μ g/mL at 28 ℃, YEB solid medium, Rifampin Rif 125 μ g/mL) go up to cultivate two days, select then three mono-clonals at primer to SNI﹠amp; Be bacterium liquid PCR under the guiding of ABI and identify that the result all amplifies the target stripe of 674bp, show that goal gene ZmCBL4 has transformed among the Agrobacterium GV3101.To identify that good bacterium liquid is inoculated in is added with in kantlex Kan 100 μ g/mL and the antibiotic liquid YEB of the Rifampin Rif 125 μ g/mL substratum again, 28 ℃ of following shaking culture to OD600 be 0.5-0.8,4 ℃, the centrifugal 15min collection of 5000rpm bacterium.Bacterial sediment is suspended with the infiltration damping fluid that 1 * MS macroelement (Murashige and Skoog, 1962) adds 5% sucrose, subsequently with being stained with colored method arabidopsis thaliana transformation sos3 mutant plant (Liu andZhu, 1998) and wild-type plant.(primer is to SNI﹠amp in conjunction with PCR method with 0.5 ‰ ppt (weedicide) for transformation generation; ABI) screen and identify, with the not negative contrast of transfer-gen plant, wherein, transgenosis T1 for the PCR qualification result of plant as shown in Figure 6 (swimming lane M:1kb Marker, T1 is for plant for swimming lane 1-10:10 strain transgenosis, amplifies the 674bp target stripe; Swimming lane 11: transgenosis negative control not), show the cDNA that all is integrated with ZmCBL4 in the genome of transfer-gen plant.

3, the Northern blot of ZmCBL4 transgenic arabidopsis detects

Detect the expression of ZmCBL4 in transgenic arabidopsis with Northern blot method, may further comprise the steps:

1) extracts total RNA of transgenic arabidopsis with hot phenol method;

2) preparation sex change glue: take by weighing the 0.72g agarose, add 43.2mL DEPC treating water, the microwave oven fusing, to be cooled to about 60 ℃, add 10.8mL formaldehyde and 6mL 10 * MOPS, add a small amount of EB again, fall glue behind the mixing;

3) preparation of sample: the total RNA of 20 μ g is mended to 9 μ l with the DEPC treating water, add 10 * MOPS, 4 μ l successively, methane amide 20 μ l, formaldehyde 7 μ l, mixing, 65 ℃ of water-bath 10min, ice bath 5min, centrifugal, solution is collected into the pipe end;

4) electrophoresis: every duplicate samples adds 4 μ l, 10 * Loading buffer (sample-loading buffer) and goes up sample, is electrophoretic buffer with 1 * MOPS, earlier with 30V voltage electrophoresis, treat that sample leaves the point sample hole after, strengthen voltage to 50V, electrophoresis 5-6h;

5) change film: after electrophoresis finishes, use DEPC treating water rinsing gel 3 times, each 15min; Cut the unnecessary glue of gel limit, and cut a little angle to show direction; One disk is added alkali shifts liquid, frame lastblock sheet glass, on put 4 layers of thieving paper of being wider than gel, two is dipped in the liquid, between filter paper bubble can not be arranged; Gel point sample hole is placed on the filter paper down, drain bubble therebetween, put the water proof bar around the blob of viscose well; All be laid on the glue a block length is wide, repave four filter paper with the identical size of film, drain bubble therebetween than the nylon membrane of the big 1mm of gel (shifted in the liquid and evenly soaked into Amersham company) at alkali; Addend layer paper handkerchief pressed a sheet glass and 750g weight on it, inhale seal 5-6h; Film is taken off, mark in pencil, (0.3M NaCl, 0.03M citric acid pH7.0) are put to filter paper after the middle rinsing, and super clean bench dries up at 2 * SSC; Film is sandwiched between filter paper and the two blocks of glass, and in 80 ℃ of baking 0.5-1h, with the preservative film parcel, 4 ℃ of preservations are standby;

6) mark of probe, hybridize and wash membrane method with the Southern blot hybridization among the embodiment 1.

The result obtains Arabidopis thaliana sos3 mutant ZmCBL4 transgenosis T3 altogether for 30 of homozygous lines, wild-type ZmCBL4 transgenosis T3 is for 20 of homozygous lines, get wherein 6 Arabidopis thaliana sos3 mutant ZmCBL4 transgenosis T3 respectively for homozygous lines (SS1, SS13, SS23, SS24, SS28, SS29) and 6 wild-type ZmCBL4 transgenosis T3 for homozygous lines (SW8, SW9, SW16, SW17, SW18, SW19) carrying out Northern blot with aforesaid method analyzes, be contrast with Arabidopis thaliana sos3 mutant (sos3) and the wild-type Arabidopis thaliana (WT) that transforms the p3301 empty carrier respectively, the detected result of Arabidopis thaliana sos3 mutant ZmCBL4 transgenic line is (sos3:sos3 mutant contrast shown in Fig. 7 A; SS1-SS29:6 transgenic line; RRNA is the amount ginseng of applied sample amount), the detected result of wild-type ZmCBL4 transgenic line is (WT: wild-type contrast shown in Fig. 7 B; SW8-SW19:6 transgenic line; RRNA is the amount ginseng of applied sample amount), above-mentioned transgenic line all can detect ZmCBL4 than strongly expressed, prove to have obtained positive transgenic line of Arabidopis thaliana sos3 mutant ZmCBL4 and wild-type ZmCBL4 positive transgenic line.

Two, the salt tolerance of transgenic line is identified

1, the ZmCBL4 gene is expressed in Arabidopis thaliana sos3 mutant and can be recovered its salt tolerance fully

Select to detect ZmCBL4 through Northern blot in the step 1 and express 3 stronger sos3 mutant T3, with curved root test of seedling and seed germination test it is carried out salt tolerance respectively and identify for ZmCBL4 transgenosis homozygous lines (SS1, SS13 and SS23).The curved root test method of seedling is: the seedling that will sprout 5d age on the MS substratum is transferred to MS (MS solid medium: add 20g sucrose in 1 liter of MS minimum medium, 10g agar, pH 5.7), MS adds on three kinds of different solid mediums that 100-175mM (100,125,150 or 175mM) NaCl and MS add 10-20mM (10,12,15 or 20mM) LiCl, the tip of a root is vertically placed growth up, and 12d takes pictures; The seed germination test method is: with the seed of transfer-gen plant respectively dibbling add on three kinds of different solid mediums that 125-175mM (125,150 or 175mM) NaCl and MS add 12-20mM (12,15,17 or 20mM) LiCl in MS, MS, sprout 8d (MS) down at 22 ℃, take pictures behind 14d (MS that contains 125-175mM NaCl) and the 12d (MS that contains 12-20mMLiCl).Arabidopis thaliana sos3 mutant (sos3) and wild-type Arabidopis thaliana (WT) with conversion p3301 empty carrier are contrast.

The curved root test-results of seedling is (is example with the SS1 strain) shown in Fig. 8 A, the seed germination test-results is (is example with SS1 and SS13 strain) shown in Fig. 8 B, coerce down at 100-175mM sodium-chlor, the salt tolerance of ZmCBL4 transgenic line is all obviously compared according to the sos3 mutant is strong does not have difference with wild-type; Coerce down at the 10-20mM lithium chloride, the salt tolerance of 3 transgenic lines is not only obviously strong than sos3 mutant, and obviously surpasses the wild-type contrast, proves that ZmCBL4 gene of the present invention can improve the salt tolerance of plant.

2, the ZmCBL4 gene is expressed in the Arabidopis thaliana wild-type and can obviously be improved its salt tolerance:

Select to detect ZmCBL4 through Northern blot in the step 1 and express 4 stronger Arabidopis thaliana wild-type T3 for homozygous lines (SW16, SW17, SW18 and SW19), it is identified at seed germination and the salt tolerance of growth of seedling phase, is contrast with not genetically modified wild-type Arabidopis thaliana (WT).Authentication method is: according to ordinary method with seed disinfection, be inoculated in MS, MS additional 125,150 or 175mM NaCl respectively, and on the solid plate substratum of MS additional 12,15,17 or 20mM LiCl, 4 ℃ of vernalization 4d, move to (22 ℃ of normal growth conditions then, 16h illumination/d) cultivate down, level or vertical placement, observed and recorded seed germination and growth of seedlings situation.

Wherein, add 150mM NaCl at MS behind the sprouting 10d down at 22 ℃, and the growth of seedling situation of SW16, SW17 on the solid plate substratum of the additional 17mM LiCl of MS as shown in Figure 9, on common MS substratum, transgenic line and wild-type do not have difference to impinging upon on seed germination and the growth of seedling situation, but coerce down at different concns sodium-chlor or lithium chloride, the seed germination of transgenic line and growth of seedling situation thereof all obviously are better than the wild-type contrast, and this difference is coerced particularly outstanding down at lithium chloride.To sprout (expanding into standard with green cotyledon) 5d on MS+150mM NaCl substratum is example, shown in Figure 10 A, four transgenic lines, the germination rate that is SW16, SW17, SW18 and SW19 is respectively 76.10%, 76.37%, 73.74% and 77.42%, and wild-type WT is 56.76%, differs about 20%; Shown in Figure 10 B, after sprouting 5d on the MS+15mM LiCl substratum, the seed germination rate of above-mentioned four transgenic lines is all more than 70%, and wild-type WT has only 11.24%.At 125mM or 175mM NaCl, and 12mM or 20mM LiCl coerce down, and this difference still exists.When sodium chloride concentration reached 150mM, the wild-type growth of seedling was slow, yellow even withered gradually, and transgenic line seedling look normal, still can keep the speed of growth faster; When sodium chloride concentration reached 175mM, wild-type was difficult to grow true leaf and withered gradually, and the transgenic line seedling keeps green substantially, still can keep certain growth.Coerce down at 15mM LiCl, the albefaction death gradually of most of wild-type seedling, and the transgenic line seedling all grows fine; When lithium chloride concentration increased to 17mM, though the part wild type seeds can be sprouted, most cotyledon colors were turned white, can not continued growth and wither, and transgenic line seedling look normal, and growing way is still better; When lithium chloride concentration during up to 20mM, the seed of wild-type almost can not be sprouted, and the seed major part of transgenic line still can be sprouted, and has the green seedling more than 30% at least.

Above-mentioned experimental result shows, ZmCBL4 is the homologous gene of AtSOS3/AtCBL4, involved in plant is to the signal transduction process of salt stress response, it is not only normal wild type plant salt tolerance and forms a necessary key gene, and its overexpression can be significantly improved the salt tolerance of seed germination phase and growth of seedling phase, so this gene can be used for the genetically engineered improvement of plant salt endurance.

Sequence table

<160>4

<210>1

<211>211

<212>PRT

＜213〉Zea corn (Zea mays L.)

<400>1

Met?Gly?Cys?Ala?Thr?Ser?Lys?Gln?Phe?Ser?Arg?Ser?Ala?Pro?Ala?His

1 5 10 15

Ala?Asp?Pro?Ala?Val?Leu?Ala?Thr?Gln?Thr?Ser?Phe?Thr?Met?Asn?Glu

20 25 30

Val?Glu?Ala?Leu?Tyr?Glu?Leu?Tyr?Lys?Lys?Leu?Ser?Cys?Ser?Ile?Val

35 40 45

Lys?Asp?Gly?Leu?Ile?His?Lys?Glu?Glu?Phe?Gln?Leu?Ala?Leu?Phe?Arg

50 55 60

Asn?Ser?Arg?Arg?Ala?Asn?Leu?Phe?Ala?Asp?Arg?Val?Phe?Asp?Leu?Phe

65 70 75 80

Asp?Leu?Lys?Arg?Asn?Gly?Val?Ile?Asp?Phe?Glu?Glu?Phe?Val?Arg?Ser

85 90 95

Leu?Ser?Val?Phe?His?Pro?Lys?Ala?Asp?Thr?Ser?Glu?Lys?Thr?Ala?Phe

100 105 110

Ala?Phe?Lys?Leu?Tyr?Asp?Leu?Arg?Gly?Thr?Gly?Tyr?Ile?Glu?Lys?Glu

115 120 125

Glu?Leu?Arg?Glu?Met?Val?Leu?Ala?Leu?Leu?Asp?Glu?Ser?Asp?Leu?Cys

130 135 140

Leu?Ser?Asp?Ser?Thr?Val?Glu?Thr?Ile?Val?Asp?Asn?Thr?Phe?Ser?Gln

145 150 155 160

Ala?Asp?Ser?Asn?Gly?Asp?Gly?Arg?Ile?Asp?Pro?Glu?Glu?Trp?Glu?Glu

165 170 175

Phe?Val?Lys?Arg?Asn?Pro?Ala?Thr?Leu?Arg?Asn?Met?Thr?Leu?Pro?Tyr

180 185 190

Leu?Gln?Asp?Ile?Thr?Met?Ser?Phe?Pro?Ser?Phe?Ile?Met?Arg?Ser?Glu

195 200 205

Ala?Ser?Asp

210

<210>2

<211>636

<212>DNA

＜213〉Zea corn (Zea mays L.)

<400>2

atgggctgcg?cgacgtccaa?gcagttcagc?aggagcgcgc?cggcgcacgc?ggatccggcc 60

gtgctggcga?cccagacctc?attcacgatg?aacgaggtgg?aggcgctgta?cgagctgtac 120

aagaagctga?gctgctccat?cgtcaaagac?gggctcatcc?acaaggagga?gttccagctc 180

gccttgttca?ggaacagcag?gagagcgaac?ctctttgcag?acagggtctt?cgatctgttc 240

gatctcaagc?ggaacggagt?catcgatttc?gaggagttcg?tgcggtcgct?cagcgtgttc 300

caccctaaag?cagatacgtc?ggagaagacg?gcgttcgctt?tcaagctgta?tgatctgagg 360

gggacaggct?acatcgagaa?agaagagctg?agagagatgg?tcttggcgct?gcttgatgag 420

tccgacctct?gcctttcaga?cagcaccgtc?gagacgatcg?tcgataacac?gttcagccaa 480

gcggactcga?acggagatgg?caggatagat?cctgaagaat?gggaggagtt?cgtgaagagg 540

aacccggcaa?cgttaaggaa?catgactctc?ccctatctgc?aggacatcac?catgtcattt 600

ccgagcttca?taatgcgttc?agaagccagt?gactga 636

<210>3

<211>1994

<212>DNA

＜213〉Zea corn (Zea mays L.)

<400>3

atgggctgcg?cgacgtccaa?gcagttcagc?aggagcgcgc?cggcgcacgc?ggatccggcc 60

gtgctggcga?cccagacctc?atgtgagcat?ctcacctccc?tccctccccc?tcacctccgg 120

caacgacagc?gccaattgcg?tgacgctgac?ccacgtctgc?tgcggctgcg?gctgttgtgt 180

tgcttgtgtg?gcatggcagt?cacgatgaac?gaggtggagg?cgctgtacga?gctgtacaag 240

aagctgagct?gctccatcgt?caaagacggg?ctcatccaca?aggtggggat?cgaccgacga 300

cccatctcgg?tacattccct?ggtttctgat?acttagatcg?cgccgctaac?gggttggagt 360

ctgtgctgtg?ttggtttggt?ctggtcggcc?aggaggagtt?ccagctcgcc?ttgttcagga 420

acagcaggag?agcgaacctc?tttgcagaca?gggtaagcat?ctcgtgctca?ttttcgccct 480

tacaggacac?aggaggagga?aactcagttc?gtgtttcgcc?gtcacaggtc?ttcgatctgt 540

tcgatctcaa?gcggaacgga?gtcatcgatt?tcgaggagtt?cgtgcggtcg?ctcagtgtgt 600

tccaccctaa?agcagatacg?tcggagaaga?cggcgtgtac?gaactgcata?cctcaatgtt 660

cattcatcat?cttcttcttc?tcgtgcatta?ggccatgttt?gcaacgaggg?attttttctc 720

tcgctaaaaa?tcctgagctt?ctcgcgtgaa?attcctgtac?ggttcggaat?ggccacaccg 780

agaaaggacg?gagatgacat?gtttgatttg?gacttttctc?atcctttatg?cagtcgcttt 840

caagctgtat?gatctgaggg?ggacaggcta?catcgagaaa?gaagaggtga?tccttcatgc 900

gttctcttgt?atattttcca?aagaaaaaaa?aacaaccaga?gcgcatgcca?tttaatgact 960

ggaaaccata?gtgtctgtgt?gtttttttag?ataatggacc?aaaatccggc?tttcgcctct 1020

ttcgaagata?aggttaagtt?cacacaactt?gaaacaaata?tccaacagct?cacaaaaata 1080

tgacaaaact?gtaagcctaa?aagagttaat?agcttcccta?tttctcaccg?actcaaactc 1140

acttaaaaat?taatttatta?caacccaatc?ctgtttgaaa?acctccaacc?ctgataatca 1200

aaaaactgca?tgaccaccga?ctctagatga?cgataagctt?ttcttaaaag?atctgctctt 1260

tttcactttt?ctgtagttgt?gcccgtagtc?tgagctaatg?agtacctttg?actagaacct 1320

gcaaataagt?caacatacgt?aatttattaa?atattaagtc?atttatactt?ttccaaaaag 1380

ctcagcacag?tgttgagaca?catgtaataa?tcagattctt?agtatttgga?tccatcccgt 1440

caaccaactc?ccaaccataa?tgcttgtgta?cactgcactt?gttcgattct?aatataaaac 1500

agtaactttg?ttcttgagaa?gcagctgaga?gagatggtct?tggcgctgct?tgatgagtcc 1560

gacctctgcc?tttcagacag?caccgtcgag?acgatcgtcg?ataacgtaac?tgatacactg 1620

ctactctccg?ctagtcatca?ggtgctagtt?tgcaaagatc?ccgtctccgt?tctaactcgt 1680

atcatatcgt?gctgctctgt?tcgcgttgga?gtgcagacgt?tcagccaagc?ggactcgaac 1740

ggagatggca?ggatagatcc?tgaagaatgg?gaggagttcg?tgaagaggaa?cccggcaacg 1800

ttaaggaaca?tgactctccc?ctatctgcag?tgcgtagcat?agcgtagcgc?atgtcctttt 1860

ctttcctttc?ctttccaaat?gatctttcct?tcaaaaaaaa?aagttctgac?tacttttgaa 1920

attactgttg?attgttcagg?gacatcacca?tgtcatttcc?gagcttcata?atgcgttcag 1980

aagccagtga?ctga 1994

<210>4

<211>1417

<212>DNA

＜213〉Zea corn (Zea mays L.)

<400>4

gcacgaggga?cgtggcgcga?gagggagagc?ggagaggggg?gctcaccaaa?ctgcaaagtc 60

tgtctcctga?ctcctcgccg?gcgtgccgga?tctcctgtct?ctctcgcggt?cggcggccgc 120

atagcctcgt?tcactcgcac?gacacagggc?aggggggcga?tgggctgcgc?gacgtccaag 180

cagttcagca?ggagcgcgcc?ggcgcacgcg?gatccggccg?tgctggcgac?ccagacctca 240

ttcacggtga?acgaggtgga?ggcgctgtac?gagctgtaca?agaagctgag?ttgctccatc 300

gtcaaagacg?ggctcatcca?caaggaggag?ttccagctcg?ccttgttcag?gaacagcagg 360

agagcgaacc?tctttgcaga?cagggtcttc?gatctgttcg?atctcaagcg?gaacggagtc 420

atcgatttcg?aggagttcgt?gcggtcgctc?agtgtgttcc?accctaaagc?agatacgtcg 480

gagaagacgg?cgttcgcttt?caagctgtat?gatctgaggg?ggacaggcta?catcgagaaa 540

gaagagctga?gagagatggt?cttggcgctg?cttgatgagt?ccgacctctg?cctttcagac 600

agcaccgtcg?agacgatcgt?cgataacacg?ttcagccaag?cggactcgaa?cggagatggc 660

aggatagatc?ctgaagaatg?ggaggagttc?gtgaagaaga?acccggcaac?gttaaggaac 720

atgactctcc?cctatctgca?ggacatcacc?atgtcatttc?cgagcttcat?aatgcgttca 780

gaagccagtg?actgaaatgt?ttgcgtacat?ctgcgtataa?ggaagcaccg?ttccatcagc 840

tctgctgttt?gttggagcct?acagcttaat?ccatggcacg?aggcaccgga?gaaagttcag 900

aatgtagcag?caacggccag?cagagtgcat?gagggttgct?gccatagctg?caggctgcgg 960

ccaaacaaga?tgatgaccaa?agattcatct?agatgtcagc?ttcagggttc?agatggtata 1020

ccaggcaacg?attttctttc?tcggcatcta?aacgccgtgg?atgttgcgtt?taaaaaattg 1080

ttgcagagga?tcaacaagtc?tgttcatcga?agcagtgcct?gagtttagaa?tcataagcta 1140

actgtctatt?gctgagatgc?tttcgtctag?atcttgcgtc?tagttgtaac?tgcaaggaaa 1200

gaataacttg?tggagagttg?gatttgttga?tgaaccttag?tagttcttga?cttggttagt 1260

tgttttcaca?tttcttcgca?gtgccagtgc?gtgcacttgt?aacttgcaaa?ttcttctcaa 1320

tccaccccta?atcgaagggt?ggatccagtt?ccgcatgtaa?tgaggcaata?tcatcaatag 1380

tttatgcttc?gtttatcgca?aaaaaaaaaa?aaaaaaa 1417

Claims (7)

1. from the calcium adjusting phosphatase B analogy albumen of corn, its amino acid residue sequence is shown in the SEQ IDNO:1 in the sequence table.

2. the cDNA gene of coding claim 1 described corn calcium adjusting phosphatase B analogy albumen, its nucleotide sequence is shown in the SEQ ID NO:2 in the sequence table.

3. the genomic gene of coding claim 1 described corn calcium adjusting phosphatase B analogy albumen, its nucleotide sequence is shown in the SEQ ID NO:3 in the sequence table.

4. the expression vector that contains described cDNA gene of claim 2 or the described genomic gene of claim 3.

5. the host bacterium that contains described cDNA gene of claim 2 or the described genomic gene of claim 3.

6. a method that improves plant salt endurance is cDNA gene transfered plant tissue, cell or the organ with the described corn calcium adjusting phosphatase B analogy albumen of claim 2, and plant salt endurance obtains to improve;

Described plant comprises monocotyledons and dicotyledons.

7. method according to claim 6 is characterized in that: the cDNA gene of the described corn calcium adjusting phosphatase B analogy albumen of claim 2 imports plant tissue, cell or organ by the plant expression vector that contains this gene; The carrier that sets out that is used to make up described plant expression vector is p3301, PER8, PX6, pBI serial carrier, pBin serial carrier, pCAMBIA serial carrier, pUC serial carrier or pBluescript serial carrier.

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