CN103059115B

CN103059115B - Plant salt-tolerance-related protein AtSDIP1, and coding gene and application thereof

Info

Publication number: CN103059115B
Application number: CN201310003774.5A
Authority: CN
Inventors: 谢旗; 张华伟; 崔凤
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2013-01-06
Filing date: 2013-01-06
Publication date: 2014-06-04
Anticipated expiration: 2033-01-06
Also published as: CN103059115A

Abstract

The invention discloses a plant salt-tolerance-related protein AtSDIP1, and a coding gene and an application thereof. The protein provided by the invention is the following (a), or (b), or (c): (a) a protein composed of an amino acid sequence represented by the sequence 1 in the sequence table; (b) a protein derived from the sequence 1 and is related to plant salt tolerance, wherein the amino acid sequence of the sequence is subjected to substitution and/or deletion and/or addition of one or more amino acid residues; (c) a protein derived from the sequence 1 and is relates to plant salt tolerance, wherein the protein has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 95%, at least 98%, or at least 99% homology with the sequence 1. The invention provides a basis for the researches of plant response and tolerability molecular mechanisms against salt stress, and has important significance to crop improvement and salt-tolerant crop cultivation.

Description

Plant salt tolerance associated protein AtSDIP1 and encoding gene thereof and application

Technical field

The present invention relates to a kind of plant salt tolerance associated protein AtSDIP1 and encoding gene and application.

Background technology

According to incompletely statistics, there is in the world at least 20% arable land and exceed 50% irrigate land in the impact that is subject in varying degrees salt damage.Some arids or semiarid zone, because steam output is large, quantity of precipitation is little, causing the salinity in soil (is mainly NaCl and NaCO ₃) accumulation in a large number.Some oceanfronts, the higher or inwelling of ground water table, also can cause upper soll layer to accumulate more salinity (is mainly NaCl and MgSO ₄).And irrational irrigation method and the long-term fresh water desalinization of soil by flooding or leaching have caused the secondary salinization in a large amount of farmlands.Along with the sharp increase of world population, the aggravation of s Urbanization In Developing Countries process, and the sharply decline of the irrigateed land area that causes of water resources shortage, fully development and utilization saltings has become the important topic that is related to human survival and development.

Salt stress is one of main limiting factor of growth and development of plants.The plant of tolerance difference according to to(for) salt stress, can be divided into plant halophytes and glycophyte two classes.Halophytes is in the edatope lower than-3.3MPa, to grow and to complete the natural phant colony of the life history at osmotic potential, otherwise is glycophyte.

Most plants especially farm crop belongs to glycophyte, to salt stress sensitivity.When they are grown in saltings, owing to being subject to salt stress effect, poor growth, often blade flavescence, death, comes off, and has a strong impact on photosynthesis, even whole strain plant withered death sometimes, thus cause crop production reduction.Serious impact of salt stress global grain yield, therefore, disclose the mechanism of plant reply salt stress, and improve accordingly the saline-alkaline tolerance of plant, become the important foundation that promotes agriculture production.

Salt stress is mainly manifested in the following aspects to the impact of plant-growth: the first, and in soil, salinity is too high, and the osmotic potential of soil is reduced, and can cause water uptake by plant roots difficulty, causes plant moisture to wane; The second, the ion that the accumulation of sodium ion, chlorion and vitriol causes is poisoned, and causes potassium, calcium, phosphorus, nitrogen intake deficiency, thus the Nutrient Stress causing; The 3rd, in the time that plant is subject to salt stress, in cell, can accumulate in a large number active oxygens, the active oxygen of high density can cause the destruction of film fat, protein and nucleic acid.

Research plant is for the molecular mechanism of response and the tolerance of salt stress, and finding anti-salt-related gene is focus in the last few years, has great importance for farm crop improvement and cultivation salt tolerant crop.

Summary of the invention

The object of this invention is to provide a kind of plant salt tolerance associated protein AtSDIP1 and encoding gene and application.

Protein provided by the invention, from the environmental Arabidopis thaliana of Colombia (Arabidopsis thalianaecotype Columbia), called after AtSDIP1 albumen is following (a) or (b) or (c):

(a) protein being formed by the aminoacid sequence shown in sequence in sequence table 1;

(b) by the one or more amino-acid residues of aminoacid sequence process of sequence 1 replacement and/or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to plant salt endurance of (as 1-25 is individual, 1-20 is individual, 1-15 is individual, 1-10 is individual, 1-5 is individual or 1-3 amino-acid residue);

(c) there is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% homology to the aminoacid sequence of sequence 1 and the relevant protein being derived by sequence 1 with plant salt endurance.

In order to make the protein in (a) be convenient to purifying, N-terminal that can the protein that the aminoacid sequence shown in sequence 1 forms in by sequence table or C-terminal connect label as shown in table 1.

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6(is generally 5)	RRRRR
Poly-His	2-10(is generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag?II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned (b) or (c) in protein can synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.Above-mentioned (b) or (c) in the encoding gene of protein can be by lacking the codon of one or several amino-acid residue 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 the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.

The gene (AtSDIP1 gene) of described AtSDIP1 albumen of encoding also belongs to protection scope of the present invention.

Described gene can be following 1) or 2) or 3) or 4) or 5) or 6) or 7) DNA molecular:

1) coding region is if sequence in sequence table 2 is from the DNA molecular as shown in 5 ' end 85-744 position Nucleotide;

2) coding region is if sequence in sequence table 2 is from the DNA molecular as shown in 5 ' end 85-747 position Nucleotide;

3) DNA molecular shown in sequence 2 in sequence table;

4) under medium stringent condition with 1) or 2) or 3) the DNA sequence dna hybridization limiting and the DNA molecular of coding salt tolerance associated protein;

5) under high stringent condition with 1) or 2) or 3) the DNA sequence dna hybridization limiting and the DNA molecular of coding salt tolerance associated protein;

6) with 1) or 2) or 3) DNA sequence dna limiting has the DNA molecular of at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% homology and coding salt tolerance associated protein;

7) with 1) to 6) in the DNA molecular of arbitrary described DNA molecular reverse complemental.

The present invention also protects the active fragments of described protein or the active fragments of described gene.

The carrier, expression cassette, transgenic cell line or the recombinant bacterium that contain described AtSDIP1 gene all belong to protection scope of the present invention.Described carrier can be that cloning vector can be also expression vector.In the expression vector (recombinant expression vector) that contains described AtSDIP1 gene, the upstream of described AtSDIP1 gene has promotor, and the downstream of described AtSDIP1 gene has terminator.Described recombinant expression vector specifically can be the recombinant plasmid pCAMBIA1300-221-AtSDIP1 that the multiple clone site of described AtSDIP1 gene insertion vector pCAMBIA1300-221 is obtained.

The recombinant expression vector that available existing plant expression vector construction contains described AtSDIP1 gene.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, 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.While using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can be used alone or are combined with other plant promoter; In addition, while using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, 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 region or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene of luminophor, have antibiotic marker thing or the anti-chemical reagent marker gene etc. of resistance.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

The present invention also protects a kind of method of cultivating transgenic plant, is described AtSDIP1 gene is imported in object plant, obtains the transgenic plant of salt tolerance higher than described object plant.Carry described gene expression vector can by using, Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated, and the plant tissue of conversion is cultivated into plant.Described AtSDIP1 gene specifically can import in described object plant by described recombinant expression vector.Described object plant is monocotyledons or dicotyledons.Described dicotyledons specifically can be Arabidopis thaliana, Arabidopis thaliana as environmental in Colombia.

The present invention also protects a kind of method of cultivating salt-tolerant plant, is transfer-gen plant and the plant hybridization that sets out that above arbitrary described method is obtained, and obtains the filial generation of salt tolerance higher than the described plant that sets out.

The present invention also protects a kind of method of cultivating transgenic plant, is the expression that suppresses gene (AtSDIP1 gene) described in object plant, obtains the transgenic plant of salt tolerance lower than described object plant.Described " suppressing the expression of gene described in object plant " specifically can realize by import interference fragment in object plant.Described interference fragment is made up of described gene (AtSDIP1 gene), the reverse complementary sequence of described gene (AtSDIP1 gene) and intervening sequence between the two.Described interference fragment specifically can be as shown in the sequence of sequence table 3.Described interference fragment can import in described object plant by interference carrier.Described interference carrier specifically can be the recombinant plasmid pCAMBIA1300-221-AtSDIP1-RNAi that the multiple clone site of described interference fragment insertion vector pCAMBIA1300-221 is obtained.Carry described interference fragment expression vector can by using, Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated, and the plant tissue of conversion is cultivated into plant.Described object plant is monocotyledons or dicotyledons.Described dicotyledons specifically can be Arabidopis thaliana, Arabidopis thaliana as environmental in Colombia.

The recombinant expression vector that available existing plant expression vector construction contains described interference fragment.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, 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.While using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can be used alone or are combined with other plant promoter; In addition, while building plant expression vector, also can use enhanser, comprise translational enhancer or transcriptional enhancer, 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 region or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene of luminophor, have antibiotic marker thing or the anti-chemical reagent marker gene etc. of resistance.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

The present invention is that research plant provides the foundation for the response of salt stress and the molecular mechanism of tolerance, has great importance for farm crop improvement and cultivation salt tolerant crop.

Accompanying drawing explanation

Fig. 1 is the structural representation of recombinant plasmid pCAMBIA1300-221-AtSDIP1.

Fig. 2 is the structural representation of recombinant plasmid pCAMBIA1300-221-AtSDIP1-RNAi.

Fig. 3 is the relative expression quantity of AtSDIP1 gene in each strain.

Fig. 4 is the photo of the growth conditions of the plant of each strain.

Fig. 5 is that experimental group plant is according to the grouping standard of growth conditions.

Fig. 6 is that each strain is according to the grouping situation of growth conditions.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.It will be understood by those skilled in the art that and can use other specific embodiments to realize object of the present invention, these specific embodiments equally within the scope of the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.

" be correlated with "/" being operably connected " refer to two physics or the relevant nucleotide sequence of function.For example, if promotor or regulate DNA sequence dna and coding RNA or protein DNA sequence to be operably connected or locate to such an extent as to regulates the DNA sequence dna will impact coding or the expression level of structural DNA sequence, claims promotor or adjusting DNA sequence dna and coding RNA or protein DNA sequence " relevant " so.

" mosaic gene " is recombinant nucleic acid sequence, wherein promotor or regulate be operably connected coding mRNA or as the nucleotide sequence of protein expression of nucleotide sequence, or to coding mRNA or relevant as the nucleotide sequence of protein expression, make to regulate nucleotide sequence can regulate transcribing or expressing of associated nucleic acid sequences.The adjusting nucleotide sequence of mosaic gene is not the associated nucleic acid sequences that is normally operably connected of being found as occurring in nature.

" encoding sequence ": refer to the nucleotide sequence of the aminoacid sequence of directly specifying its protein, it can be DNA, cDNA, nucleotide sequence synthetic or restructuring.The border of encoding sequence is determined by open reading frame conventionally.5 ' end of open reading frame is defined by initiator codon, and 3 ' end is defined by terminator codon.Initiator codon is generally ATG, or can be also that other initiator codon is as GTG or TTG.Terminator codon comprises for example TAA, TAG, TGA.

" cDNA " refers to the DNA with RNA complementary element, and it is strand or the double-stranded DNA molecular from deriving from eukaryotic maturation, come through the mRNA molecule reverse transcription of montage by ThermoScript II.So, in cDNA, do not contain any intron that originally may be present in corresponding with it genomic dna sequence.

Corresponding to: in the context of the invention " corresponding to " mean in the time that the nucleic acid coding sequence of different SDIP1 genes or protein or aminoacid sequence are compared mutually, " corresponding to " nucleic acid or the amino acid of some counting position is to compare with these positions, but need not to be with respect to specific SDIP1 nucleic acid or the amino acid in these precise figures positions of nucleic acid coding sequence or aminoacid sequence separately.Equally, while comparison when the coding of specific SDIP1 or aminoacid sequence and with reference to the coding of SDIP1 or aminoacid sequence, " corresponding to " be to compare with these positions with reference to SDIP1 sequence with reference to this specific SDIP1 sequence amplifying nucleic acid or the amino acid of some counting position of SDIP1 sequence, but need not to be at this specific SDIP1 protein nucleic acid or amino acid in these precise figures positions of nucleic acid coding sequence or aminoacid sequence separately.

Here " expression cassette " used means to instruct and is applicable to the nucleotide sequence that in host cell, specific nucleotide sequence is expressed, and comprises the promotor being operably connected with object nucleotide sequence, the described object nucleotide sequence termination signal that is operably connected.Conventionally, it also comprises the required sequence of the correct translation of nucleotide sequence.The expression cassette that comprises object nucleotide sequence can be chimeric, and meaning one of its composition is at least allos with respect to its one of other composition at least.Expression cassette can be also naturally occurring, but obtains the expression cassette for heterogenous expression with recombinant forms.But conventionally, expression cassette is allos with respect to host, that is, the specific nucleic acid sequence of expression cassette is not natural to be appeared in host cell, must be introduced into by transformation event the precursor of host cell or host cell.In expression cassette, the expression of nucleotide sequence can be subject to constitutive promoter or inducible promoter control, and wherein only in the time that host cell is exposed to some specific outside stimuluss, described inducible promoter is initial transcribing.If the situation of multicellular organisms, as plant, promotor can be also to particular organization, or organ or the etap special.

" gene " is the localized area that is positioned at genome, except aforementioned nucleic acid sequence encoding, comprises the nucleotide sequence that other is mainly modulability, and described modulability nucleotide sequence is responsible for the expression of encoding part, transcribes and translate control.Gene also can comprise other 5 ' and 3 ' non-translated sequence and terminator sequence.The element that further can exist is, for example intron.

" allos " nucleotide sequence is the not natural relevant nucleotide sequence of host cell being introduced into it, comprises the natural multiple copied that has nucleotide sequence that non-natural exists.

" homologous recombination " is the mutual exchange of the intermolecular nucleic acid fragment of homologous nucleic acid.

When nucleic acid sequence encoding is when having the polypeptide of same acid sequence with reference to the polypeptide of nucleic acid sequence encoding, this nucleotide sequence be " isocoding " with reference to nucleotide sequence.

" separation " nucleic acid molecule or " separation " protein are that artificially separates and exists with its natural surroundings, are not therefore nucleic acid molecule or the protein of natural product.The nucleic acid molecule or the protein that separate can exist with purified form, or may reside in non-natural environment, for example, in recombinant host cell or transgenic plant.

Natural: refer to the gene that exists in the genome of no transformed cells.

Natural existence: term " natural existence " is for describing the object that can find at occurring in nature, and they are different from the artificial object producing.For example, can separate from natural source, protein or the nucleotide sequence of not having a mind to carry out existence in manually modified, organism (comprising virus) in laboratory are " natural existence ".

" nucleic acid molecule " or " nucleotide sequence " is can be from the list of any source separation or the linear fragment of double-stranded DNA or RNA.In the context of the invention, preferably, nucleic acid molecule is DNA fragmentation." nucleic acid molecule " also claims polynucleotide molecule.

" plant " is any plant, particularly spermatophyte in any etap.

" vegetable cell " is structure and the physiology unit of plant, comprises protoplastis and cell walls.Vegetable cell can be separate individual cells or culturing cell form, or as high organized unit as, for example plant tissue, a part for plant organ or whole plant.

The plant unit that " plant cell cultures " means the various etap as, for example protoplastis, cell cultures cell, the cell in plant tissue, pollen, pollen tube, ovule, blastular, the culture of zygote and embryo.

" vegetable material " refers to leaf, stem, root, the part of flower or flower, fruit, pollen, ovum, zygote, seed, cutting, cell or tissue culture, or any other parts or the product of plant.

" plant organ " is clearly and the obviously part of structurizing and differentiation of plant, as root, and stem, leaf, bud or embryo.

Here " plant tissue " used means to be organized into one group of vegetable cell of structure and function unit.Comprise any tissue of plant in plant or in culture.This term includes but not limited to whole plant, plant organ, plant seed, tissue culture and any vegetable cell group that is organized into structure and/or functional unit.The combined utilization of any particular type plant tissue that this term and above maybe this definition of enumerating comprise or separately application are not meaned and are got rid of any other type plant tissue.

" promotor " is the DNA sequence dna of coding region upstream untranslated, the binding site that it comprises RNA polymerase, and the transcribing of initiate dna.Promoter region also can comprise other element as genetic expression instrumentality.

" protoplastis " is the vegetable cell that there is no cell walls or only have the separation of parts of fine cell wall.

" regulatory element " refers to participate in controlling the sequence that nucleotide sequence is expressed.Promotor and termination signal that regulatory element comprises the object nucleotide sequence that is operably connected.Conventionally they also comprise the required sequence of the correct translation of nucleotide sequence.

" reorganization " nucleic acid is by Shuffling Method, the nucleic acid that any Shuffling Method produces as described herein.Produce reorganization nucleic acid by mode artificial and circulation alternatively (physically or in fact) the two or more nucleic acid (or character string) of recombinating.Usually, in Shuffling Method, utilize a step or multistep screening step to identify object nucleic acid; Can before or after any reconstitution steps, carry out this screening step.In some (but not all) reorganization embodiments, be desirably in before screening and carry out many wheel restructuring to increase the diversity in storehouse to be screened.Alternatively, can be cycled to repeat all processes of restructuring and screening.Based on context, reorganization can refer to all processes of restructuring and screening, or alternately, can only refer to the restructuring part of all processes.

Basic identical: the phrase " basic identical " in two nucleic acid or protein sequence context refers to when comparing and comparing to obtain maximum at once, as utilize below one of sequence comparison algorithm or range estimation to measure, have at least 60%, preferably 80%, more preferably 85%, more preferably 90%, even more preferably two or more sequences or the subsequence of 95% and most preferably at least 99% Nucleotide or amino-acid residue identity.Preferably, basic identity is present in the sequence area at least about 50 residue length, more preferably on the region at least about 100 residues, most preferably, basic identical at least about the sequence in 150 residues.In particularly preferred embodiments, in the whole length in coding region, sequence is basic identical.And essentially identical nucleic acid or protein sequence have essentially identical function.

In order to carry out sequence comparison, conventionally, sequence as canonical sequence and with detect sequence comparison.In the time utilizing sequence comparison algorithm, detection and canonical sequence are input in computer, if necessary specify the coordinate of subsequence, and the parameter of specified sequence algorithm routine.Then,, according to selected program parameter, sequence comparison algorithm detects the percentage sequence identity of sequence with respect to canonical sequence by calculating.

For example, by Smith & Waterman, local homology's algorithm of Adv.Appl.Math.2:482 (1981), by Needleman & Wunsch, .J.Mol.Biol.48:443 the sequence analysis algorithm of (1970), by Pearson & Lipman, the similarity retrieval method of Proc.Nat ' l.Acad.Sci.USA85:2444 (1988), (GAP in Wisconsin Genetics software package is implemented in computerize by these algorithms, BESTFIT, FASTA and TFASTA, Genetics Computer Group, 575Science Dr., Madison, WI) or by range estimation (conventionally referring to, Ausubel etc., below) can carry out the best comparison for sequence relatively.

An examples of algorithms that is suitable for measuring percentage sequence identity and sequence similarity is BLAST algorithm, and at Altschul etc. .J.Mol.Biol.215:403-410 has described this algorithm in (1990).Can obtain carrying out the software of BLAST analysis by the public of NCBI (http://www.Ncbi.nlm.nih.gov/).This algorithm comprises: be tested and appraised out and search the short word that in sequence, length is W and first identify high score sequence to (HSPs), described short word mates or meets some on the occasion of the threshold values T that scores in the time comparing with the word of equal length in database sequence.T is called adjacent words score threshold (Altschul etc., 1990).These initial neighborhood word are hit the longer HSPs that goes discovery to comprise them as the clue that starts to search.Then, these words hit the extension far away as far as possible of the both direction along each sequence, until accumulation comparison score value no longer increases.For nucleotide sequence, (mate in pairs the award score value of residue with parameter M; Always be greater than zero) and N (the point penalty value of mispairing residue; Always be less than zero) calculating accumulation score value.For aminoacid sequence, with the matrix computations accumulation score value of scoring.When accumulation comparison score value falls quantity X after rise from the maximum value obtaining, due to one or more negative score value residues comparison accumulation, accumulation score value reaches or lower than zero, or any of two sequences be while reaching home, and the word of each direction hits to extend and stops.The parameter W of BLAST algorithm, T and X have determined susceptibility and the speed of comparison.BLASTN program (for nucleotide sequence) is used word length value (W) 11, expected value (E) 10, cutoff value 100, M=5, N=-4 and two chains be relatively default value.For aminoacid sequence, BLASTP program is used word length value (W) 3, expected value (E) 10 and BLOSUM62 score matrix (referring to, Henikoff & Henikoff, Proc.Natl.Acad.Sci.USA89:10915 (1989)) be default value.

Except calculating percentage sequence identity, BLAST algorithm also carry out similarity between two sequences statistical analysis (referring to, for example Karlin & Altschul, Proc.Nat'l.Acad.Sci.USA90:5873-5787 (1993)).It is minimum and probability (P (N)) that the similarity that BLAST algorithm provides is measured, and it provides the accidental indication that occurs the probability mating between two Nucleotide or aminoacid sequence.For example, if detect nucleotide sequence and be less than approximately 0.1 with reference to minimum and the probability of nucleotide sequence comparison, more preferably less than approximately 0.01, be most preferably less than approximately 0.001, think that so detection nucleotide sequence is similar to canonical sequence.

Two essentially identical another indexs of nucleotide sequence are the hybridization mutually under stringent condition of two molecules.Phrase " specific hybrid " refers to that under stringent condition, molecule is only combined with specific nucleotide sequence, forms duplex or hybridization in the time that this sequence is for example present in, in complex mixture (, total cell) DNA or RNA." basic combination " refers to complementary hybridization between probe nucleic acid and target nucleic acid, and comprises less mispairing, can tolerate described mispairing by reducing the severity of hybridization medium, to realize the expectation detection of target nucleic acid sequence.

In nucleic acid hybridization is tested as Southern and Northern hybridization context, " stringent hybridization condition " and " strictly hybridizing rinsing condition " is sequence dependent, and is different under varying environment parameter.Longer sequence is at comparatively high temps specific hybrid.At Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part i the 2nd chapter " Overview of principles of hybridization and the strategyof nucleic acidprobe assays " Elsevier, can find a large amount of guides of nucleic acid hybridization in New York.Usually, for the particular sequence limiting under ionic strength and pH, high stringency hybridization and rinsing condition are chosen as lower than heat fusion joint (T _m) approximately 5 ℃.Typically, under " stringent condition ", probe will with the hybridization of its target subsequence, and not with other sequence hybridization.

Tm be (limiting under ionic strength and pH condition) 50% target sequence with mate completely probe hybridization time temperature.For specific probe, very strict condition is chosen as and equals T _m.In Southern or Northern trace, on filter membrane, having the example of a stringent hybridization condition of hybridizing more than the complementary nucleic acid of 100 complementary residues is at 42 ℃, has 50% methane amide of 1mg heparin, spends the night and carries out this hybridization.The example of high severity rinsing condition is 72 ℃, 0.15M NaCl approximately 15 minutes.The example of strict rinsing condition is at 65 ℃, 0.2x SSC rinsing 15 minutes (referring to, Sambrook, below, the description of SSC damping fluid).Conventionally, before high severity rinsing, carry out low severity rinsing to remove background probe signals.For for example, more than for the duplex of 100 Nucleotide, the example of middle severity rinsing is 45 ℃, 1x SSC rinsing 15 minutes.For for example, more than for the duplex of 100 Nucleotide, the example of low severity rinsing is 40 ℃, 4-6x SSC rinsing 15 minutes.For short probe (for example, approximately 10 to 50 Nucleotide), stringent condition is usually included in the salt concn of about 1.0M Na of being less than of pH7.0 to 8.3 ion, and approximately 0.01 to 1.0Na ionic concn (or other salt) conventionally, and typical temperature is at least approximately 30 ℃.Also can obtain stringent condition by adding destabilizing agent as methane amide.Usually, in specific cross is measured, noise signal to noise ratio just the irrelevant viewed value of probe high by 2 × (or higher) show the detection of specific hybridization.If the protein of their codings of the nucleic acid of hybridization is not essentially identical mutually under stringent condition, they are still essentially identical so.For example, in the time that the maximum codon degeneracy allowing with genetic code is created nucleic acid copy, just there will be this situation.

The example that hybridization/rinsing condition arranges below, described condition can be for clone with the present invention with reference to the essentially identical homologous nucleotide sequence of nucleotide sequence: with reference to nucleotide sequence with reference to nucleotide sequence preferably at 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, in 1mM EDTA, hybridize, at 50 ℃, 2X SSC, rinsing in 0.1%SDS, is more desirably in 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, in 1mM EDTA, hybridize, at 50 ℃, 1X SSC, rinsing in 0.1%SDS, is more desirably in 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, in 1mM EDTA, hybridize, at 50 ℃, 0.5X SSC, rinsing in 0.1%SDS, preferably, and at 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, in 1mM EDTA, hybridize, at 50 ℃, 0.1X SSC, rinsing in 0.1%SDS, more preferably, and at 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄, in 1mM EDTA, hybridize, at 65 ℃, 0.1X SSC, rinsing in 0.1%SDS.

Two nucleotide sequences or another substantially identical index of protein are protein immunological cross-reaction or the specific combination of protein and second nucleic acid encoding of the first nucleic acid encoding.Therefore, protein is conventionally substantially identical with the second protein, and for example, wherein two protein are only different due to preservative replacement.

" synthesize " nucleotide sequence that refers to comprise non-existent constitutional features in native sequences.For example, claim that similar artificial sequence dicotyledonous and/or monocotyledons gene G+C content and normal codon distribution synthesizes more nearly.

" conversion " is the process of introducing heterologous nucleic acids in host cell or organism, and especially, " conversion " means DNA molecular stable integration and enter in object organism genome.

" conversion/genetically modified/restructuring " refer to introduce the host organisms of heterologous nucleic acids molecule, as bacterium or plant.Nucleic acid molecule can stably be integrated into host genome or nucleic acid molecule also can be used as extrachromosomal molecule existence.This extrachromosomal molecule can be self-replicating.The cell transforming, tissue, or plant is interpreted as the final product that not only comprises conversion process, also comprises its transgenosis filial generation." non-transformed ", " not genetically modified ", or " nonrecombinant " host refers to the wild-type organisms that does not contain heterologous nucleic acids molecule, for example bacterium or plant.

Term used herein " polynucleotide ", " polynucleotide molecule ", " polynucleotide sequence ", " encoding sequence ", " open reading frame (ORF) " etc. comprise strand or double-stranded DNA and RNA molecule, can comprise one or more protokaryon sequences, cDNA sequence, the genomic dna sequence that comprises exon and intron, the DNA of chemosynthesis and RNA sequence, and have justice and corresponding antisense strand.

The method of producing and operate polynucleotide molecule disclosed herein and oligonucleotide molecules is well known by persons skilled in the art, and can according to the recombinant technology of having described (referring to Maniatis etc., 1989, molecular cloning, laboratory manual, press of cold spring harbor laboratory, cold spring port, New York; Ausubel etc., 1989, molecular biology current techniques, GreenePublishing Assoc iates & Wiley Intersc ience, NY; Sambrook etc., 1989, molecular cloning, laboratory manual, the 2nd edition, press of cold spring harbor laboratory, cold spring port, New York; Innis etc. (volume), 1995, pCR strategy, Academic Press, Inc., SanDiego; And Erlich (volume), 1992, round pcr, Oxford University Press, New York) complete.

Plant Transformation:

In embodiment particularly preferably, express at least one albumen of giving salt tolerance of the present invention in higher organism style in as plant.Nucleotide sequence of the present invention can be inserted in expression cassette, then preferably, expression cassette stable integration is in described Plant Genome.In another preferred implementation, nucleotide sequence is included in the virus of non-pathogenic self-replacation.In another preferred implementation, nucleotide sequence is included in Agrobacterium.The plant transforming according to the present invention can be monocotyledons or dicotyledons, include but not limited to corn, wheat, barley, rye, sweet potato, beans, pea, witloof, lettuce, wild cabbage, Cauliflower, asparagus broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, winter squash, pumpkin, hemp, zucchini, apple, pears, Wen Quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, rasp berry, blackberry, blueberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, Chinese sorghum, sugarcane, beet, Sunflower Receptacle, oil seed rape, trifolium, tobacco, Radix Dauci Sativae, cotton, clover, rice, potato, eggplant, cucumber, Arabidopsis and xylophyta are as softwood tree and deciduous trees.Particularly preferably be paddy rice, wheat, barley, corn, oat, rye, sugarcane, beet, soybean, potato.Preferably, be rape section plant for plant of the present invention.Preferably, described plant is brassica plant, comprises black mustard (Brassica nigra), colea (Brassicanapus), wild cabbage (Brassica oleraceae), turnip (Brassica rapa), brassicacarinata (Brassicacarinata), leaf mustard (Brassica juncea).Described plant can be also other plant of rape section.

Once the nucleotide sequence of expectation be transformed and is entered in specified plant species, can breed it or its is shifted to other kind that enters same species with traditional breeding method in these species, in commercial variety.

Preferably, in transgenic plant, express nucleotide sequence of the present invention, in transgenic plant, cause thus the biosynthesizing of the albumen of giving accordingly plant salt endurance.By this way, can produce the transgenic plant with Ameliorative character.In order to express nucleotide sequence of the present invention in transgenic plant, nucleotide sequence of the present invention may need to modify and optimize.All organisms have specific codon usage bias, and this is known in the art, can keep nucleotide sequence coded amino acid whose its codon that simultaneously changes of the present invention to meet plant-preference.And, from having at least about 35%, preferably more than approximately 45%, more preferably more than 50%, most preferably can realize best high-caliber expression in plant more than the encoding sequence of about 60%GC content.Although can express fully preferred gene order in monocotyledons and dicotyledons species, but can modification sequence to adapt to special codon preference and the GC content preference of monocotyledons or dicotyledons, because these preferences have been proved to be different (Murray etc., Nucl.Acids Res.17:477-498 (1989)).In addition, can screen nucleotide sequence and cause existing of unconventional splice site that information blocks to find.Utilize publication application EP 0 385 962 (Monsanto), method described in EP 0 359 472 (Lubrizol) and WO93/07278 (Ciba-Geigy), with site-directed induced-mutation technique well known in the art, PCR and synthetic gene build and carry out needing in these nucleotide sequences the institute carrying out to change, as above-mentioned those changes.

In one embodiment of the present invention, can prepare synthetic gene according to being incorporated herein by reference disclosed method in the United States Patent (USP) 5,625,136 of document here.In the method, utilize the preferred codon of corn, that amino acid whose single cipher in the corn of the most often encoding.The preferred codon of corn of specific amino acids can derive from, the known sequence of for example corn.At Murray etc., in Nucleic Acids Research17:477-498 (1989), instruct the corn codon of 28 genes of maize plant to use, the disclosure that is incorporated to this piece of document is here reference.

Nucleotide sequence can be optimized by this way so that the expression in any plant.All or any part of generally acknowledged gene order can be optimized or be synthetic., also can utilize sequence synthetic or that part is optimized.

Effectively initial for what translate, the sequence that may need to modify contiguous initial methionine.For example, by comprise known in plant effectively sequence can modify them.Joshi has proposed the applicable consensus sequence (NAR15:6643-6653 (1987)) of plant, and Clonetech has proposed the sub-consensus sequence of further translation initiation (1993/1994 catalogue, 210 pages).These consensus sequences are applicable to using together with nucleotide sequence of the present invention.To comprising described nucleotide sequence, until and comprise ATG (keeping second amino acid not modified) or alternately until and introduce this sequence in the construction that comprises GTC after ATG (have and modify second amino acid whose possibility of transgenosis) simultaneously.

Can using as its native sequences or as the new AtSDIP1 gene of the present invention who optimizes as mentioned above composition sequence operationally with the various promotors of expressing in plant, comprise composing type, induction type, sequential regulates, grow and regulate, Chemical Regulation, tissue preferably merges to prepare recombinant DNA molecules, i.e. mosaic gene mutually with tissue-specific promoter.The selection of promotor will be along with expression time and space requirement and is changed, and depends on target species.Therefore, can use at leaf, stem or cane, fringe, inflorescence (for example, spike, panicle, cob etc.), root, and/or in seedling, express nucleotide sequence of the present invention.Although it is operational having proved to derive from many promotors of dicotyledons in monocotyledons, vice versa, but ideally, select the expression of dicotyledons promotor for dicotyledons, monocotyledonous promotor is for the expression of monocotyledons.But, do not limit the origin of selected promotor, as long as working to drive, promotor expects that the expression of nucleotide sequence in cell is just enough.

Preferred constitutive promoter comprises CaMV35S and 19S promotor (Fraley etc., the U.S. Patent number 5,352,605 that on October 4th, 1994 announces).Preferred promotor derives from any of several actin genes of expressing in most cell types in addition.Can easily modify the expression of the described promoter expression cassettes such as McElroy (Mol.Gen.Genet.231:150-160 (1991)) for ThLEA1 gene, this expression casette is particularly suitable for using in monocotyledons host.

Another preferred constitutive promoter derives from ubiquitin, and it is the known another kind of gene product accumulating in many cell types.From several species, such as Sunflower Receptacle (Binet etc., 1991.Plant Science79:87-94), corn (Christensen etc., 1989.Plant Molec.Biol.12:619-632) and Arabidopis thaliana (the 1993.Plant Molec.Biol.21:895-906 such as Norris) cloned ubiquitin promoter, can be used in transgenic plant.Develop the corn ubiquitin promoter in transgenosis monocotyledons system, and announced in patent the carrier that discloses its sequence and transformed for monocotyledons in EP0342926.Ubiquitin promoter is adapted at transgenic plant, the particularly expression of new vertical compact panicle gene in monocotyledons.

Except being applicable to the selection of promotor, in plant, the structure of AtSDIP1 protein expression can be connected to the applicable transcription terminator in heterologous nucleotide sequence downstream.Can obtain several this terminators, and they are (for example derive from the tml of CaMV, derive from the E9 of rbcS) known in the art.Any known available terminator working in plant can be with in the present invention.

Also can be to introducing other sequence in expression cassette of the present invention.These sequences comprise to be proved to strengthen the sequence of expressing, for example, for example, as intron sequences (deriving from Adhl and bronzel) and virus leader sequence (deriving from TMV, MCMV and AMV).

Likely preferably the expression of nucleotide sequence of the present invention is targeted to different cellular localizations in plant.In some cases, possible expected location is in cytosol, and in other cases, possible preferred orientation is in some subcellular organelles.The Subcellular Localization of the coded enzyme of transgenosis has adopted technology well known in the art.Conventionally, operate coding derives from the DNA of the target peptide of the gene product of known targeted cells device, makes it to be fused to the upstream of nucleotide sequence.Become known for the many this target sequence of chloroplast(id), and known their functions in allos builds that proved.The expression of nucleotide sequence of the present invention also can be targeted in the endoplasmic reticulum or vacuole of host cell.The technology that realizes above-mentioned these objects is well known in the art.

The a large amount of conversion carriers that can be used for Plant Transformation are that Plant Transformation those skilled in the art are known, and nucleic acid molecule of the present invention can be combined use with any this carrier.The selection of carrier is by the preferred transformation technology and the target plant species that depend on for transforming.For some target species, microbiotic that can be preferably different or herbicide selective mark.Conventionally the selected marker being used in conversion comprises the nptII gene (Messing & Vierra., the 1982.Gene19:259-268 that give kantlex and associated antibiotic resistance, with Bevan etc., 1983.Nature304:184-187), give the bar gene to weedicide phosphinothricin resistance, (White etc., 1990.Nucl.Acids Res18:1062, with Spencer etc., 1990.Theor.Appl.Genet79:625-631), give (the Blochinger & Diggelmann of the hph gene to microbiotic hygromycin resistance, MolCell Biol4:2929-2931), with the dhfr gene (Bourouis etc. that give methatrexate resistance, 1983.EMBO is (7) J.2: 1099-1104), give EPSPS gene (the U.S. patent No.: 4 to glyphosate resistance, 940, 935 and 5, 188, 642), with the mannose-6-phosphate isomerase gene (U.S. Patent number 5 that metabolism seminose ability is provided, 767, 378 and 5, 994, 629).But the selection of selected marker is not vital to the present invention.

Another preferred embodiment in, nucleotide sequence of the present invention is directly transformed in plastom.The major advantage of plastid transformation is that plastid does not need essence modification just can express bacterial gene conventionally, and plastid can be expressed the multiple open reading frame under single promotor control.At United States Patent (USP) 5,451, in 513,5,545,817 and 5,545,818, in PCT application number WO95/16783 and McBride etc., (1994) Proc.Nati.Acad.Sci.USA91, has at large described plastid transformation technology in 7301-7305.The basic fundamental that chloroplast(id) transforms for example comprises utilizes biological bombardment or protoplast transformation (conversion of for example calcium chloride or PEG mediation) to introduce applicable target tissue by being arranged in together with the region of clone's plastid DNA of goal gene and selective marker flank.1 to 1.5kb flank region, is called targeting sequencing, can promote and the homologous recombination of plastom, therefore allows displacement or the modification of plastom(e) specific region.At first; can utilize and provide the point mutation of chloroplast(id) 16S rRNA to spectinomycin and/or streptomycin resistance and rps12 gene as the selective marker (Svab transforming; Z.; Hajdukiewicz; P.; and Maliga, P. (1990) Proc.Nati.Acad.Sci.USA87,8526-8530; Staub, J.M., and Maliga, P. (1992) Plant Cell4,39-45).This frequency with approximately every 100 target blades bombardment 1 time has produced stable homogeneity transformant.The cloning site existing between these marks can be used for produce for import foreign gene plastid targeting vector (Staub, J.M., and Maliga, P. (1993) EMBO J.12,601-606).By using dominant selectable marker, the recessive rRNA of bacterium aadA gene substitution of coding spectinomycin detoxifying enzymes (spectinomycin-cletoxifying enzyme) aminoglycoside-3 '-adenylyl transferase or r-protein antibiotics resistance gene can obtain the remarkable increase (Svab of transformation frequency, and Maliga Z., P. (1993) Proc.Natl.Acad.Sci.USA90,913-917).In the past, this mark was successfully used to high frequency and transformed green alga Chlamydomonas reinhardtii plastom (Goldschmidt-Clermont, M. (1991) Nucl.Acids Res.19:4083-4089).Other selective marker for plastid transformation is known in the art, and comprises within the scope of the present invention.Conventionally, after conversion, need approximately 15 to 20 cell division cycle to reach homogeneity state.The plastid expression of gene being inserted in all several thousand the annular plastom copies that exist in each vegetable cell by homologous recombination has utilized copy number to be much higher than the advantage of nuclear expression gene, makes expression level can easily exceed 10% of total solvable plant protein.In a preferred embodiment, nucleotide sequence of the present invention is inserted in plastid targeting vector, and conversion enters in the plant host plastom of expectation.Obtained the plant that belongs to homogeneity for containing the plastom of nucleotide sequence of the present invention, preferably, this plant has the ability of expressing nucleotide sequence high-levelly.

The composition of MS substratum is in table 2.

Table 2MS medium component

Carrier pCAMBIA1300-221(plant binary expression vector): reference: Yiyue Zhang, ChengweiYang, Yin Li, Nuoyan Zheng, Hao Chen, Qingzhen Zhao, Ting Gao, Huishan Guo andQi Xie (2007) SDIR1 Is a RING Finger E3 Ligase That Positively RegulatesStress-Responsive Abscisic Acid Signaling in Arabidopsis.Plant Cell.19 (6): 1912 – 1929.

Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 strain: reference: Yiyue Zhang, Chengwei Yang, Yin Li, Nuoyan Zheng, Hao Chen, Qingzhen Zhao, Ting Gao, HuishanGuo and Qi Xie (2007) SDIR1 Is a RING Finger E3 Ligase That Positively RegulatesStress-Responsive Abscisic Acid Signaling in Arabidopsis.Plant Cell.19 (6): 1912 – 1929.

The environmental Arabidopis thaliana of Colombia (Arabidopsis thaliana ecotype Columbia): purchased from ABRC(ArabidopsisBiological Resource Center).

Embodiment 1, AtSDIP1 gene overexpression plant and AtSDIP1 gene inhibition are expressed the acquisition of plant

One, the structure of over-express vector

Small segment by the sequence of sequence table 2 between SmaI and the XbaI enzyme cutting site of the double chain DNA molecule replacement vector pCAMBIA1300-221 shown in 5 ' end 85-747 position Nucleotide, obtaining recombinant plasmid pCAMBIA1300-221-AtSDIP1(is over-express vector).The structural representation of recombinant plasmid pCAMBIA1300-221-AtSDIP1 is shown in Fig. 1, and AtSDIP1 gene is regulated and controled by 35S double-promoter.

Two, suppress the structure of expression vector

By the double chain DNA molecule shown in the sequence of sequence table 3 (in sequence 3, the reverse complemental fragment that is AtSDIP1 gene from the 1st to 663 Nucleotide of 5 ' end, the 664th to 853 Nucleotide are stuffer fragment, the 854th to 1516 Nucleotide are AtSDIP1 gene) small segment between BglII and the XbaI enzyme cutting site of replacement vector pCAMBIA1300-221, obtain recombinant plasmid pCAMBIA1300-221-AtSDIP1-RNAi(and suppress expression vector).The structural representation of recombinant plasmid pCAMBIA1300-221-AtSDIP1-RNAi is shown in Fig. 2.

Three, the acquisition of AtSDIP1 gene overexpression plant

1, over-express vector is imported in Agrobacterium tumefaciens EHA105 strain, obtain the edaphic bacillus of recombinating.

2, restructuring edaphic bacillus step 1 being obtained is by plant vacuum infiltration method (Bent AF and Clough SJ (1998) Agrobacterium germ-line transformation:transformation of Arabidopsis withouttissue culture.In Plant Molecular Biology Manual, 2nd ed, S.B.Gelvin and R.A.Schilperoot, eds (Dordrecht, The Netherlands:Kluwer Academic Publishers): 1-14) transform the environmental Arabidopis thaliana of Colombia, results seed (T ₀the seed in generation, the plant that this seed grows up to is T ₁for plant).

3, the seed of step 2 being gathered in the crops is placed in containing on the 1/2MS culture medium flat plate of 20 μ g/mL Totomycin, flat board is first placed to 2-4 days under 4 ℃ of dark conditions, then the tissue culture room (illumination 16 hours every days) that moves into 24 ℃ is cultivated 1 week, and seed (T is cultivated and gathers in the crops in the greenhouse that then moves into 24 ℃ ₁the seed in generation, the plant that this seed grows up to is T ₂for plant).

4, the seed of step 3 being gathered in the crops is placed in containing on the 1/2MS culture medium flat plate of 50 μ g/mL kantlex and carries out resistance screening, and gathers in the crops the T that single transfer-gen plant copying obtains ₂for the seed of resistant plant, (plant that this seed grows up to is T ₃for plant).If a certain T ₁the T obtaining for plant ₂in plant, the quantity of resistant plant and non-resistance plant ratio is about 3:1, and this T is described ₁it is the transfer-gen plant of single copy for plant.

5, the seed of step 4 being gathered in the crops is placed in containing on the 1/2MS culture medium flat plate of 50 μ g/mL kantlex and carries out resistance screening.If a certain T ₂the T obtaining for plant ₃be resistant plant for plant, this T is described ₂be the transfer-gen plant isozygotying for plant, this T ₂be the expression strain excessively of isozygotying for plant and offspring thereof.

Four, AtSDIP1 gene inhibition is expressed the acquisition of plant

Replace over-express vector with pressing down expression vector, other same step 3, strain is expressed in the inhibition that obtains isozygotying.

Five, turn the acquisition of empty carrier plant

Replace over-express vector with carrier pCAMBIA1300-221, other same step 3, what obtain isozygotying turns empty carrier strain.

Five, Molecular Identification

Get at random the T of two AtSDIP1 gene overexpression strains (SDIP1-OX#1 strain and SDIP1-OX#2 strain) ₃for the seedling in 2 week age of plant, get at random the T of two AtSDIP1 gene inhibition expression strains (SDIP1-RNAi#1 strain and SDIP1-RNAi#2 strain) ₃for the seedling in 2 week age of plant, get the seedling in 2 week age of environmental Arabidopis thaliana (WT) plant of Colombia, extracting total RNA reverse transcription is cDNA, take cDNA as template, the expression amount of AtSDIP1 gene in the plant of each strain is carried out to PCR evaluation, adopt Actin2 gene as reference gene, using the expression amount of the expression amount of AtSDIP1 gene in the environmental Arabidopis thaliana of Colombia as 1, calculate the relative expression quantity of AtSDIP1 gene in other each strain.

Primer sequence for the identification of AtSDIP1 gene is as follows:

SDIP1-qPCR-FW：5’-CGCAGGATTTTCTTGGTGAC-3’；

SDIP1-qPCR-Rev：5’-ACTGAAGCTGGGAGAGAGAC-3’。

Primer sequence for the identification of Actin2 gene is as follows:

Actin2-qPCR-FW:5’-GGTAACATTGTGCTCAGTGGTGG-3’；

Actin2-qPCR-Rev:5’-AACGACCTTAATCTTCATGCTGC-3’。

Carry out repeating for three times experiment, average, the results are shown in Figure 3.Cross the expression amount of expressing AtSDIP1 gene in strain strain and significantly increase, the expression amount that suppresses to express AtSDIP1 gene in strain significantly reduces.

Embodiment 2, AtSDIP1 gene overexpression plant and AtSDIP1 gene inhibition are expressed the Salt-Tolerance Identification of plant

Get at random the T of two AtSDIP1 gene overexpression strains (SDIP1-OX#1 strain and SDIP1-OX#2 strain) ₃for seed, get at random the T of two AtSDIP1 gene inhibition expression strains (SDIP1-RNA#1 strain and SDIP1-RNAi#2 strain) ₃for seed, get the seed of environmental Arabidopis thaliana (WT) plant of Colombia, get the T of empty carrier plant ₃for seed, carry out respectively following Salt-Tolerance Identification:

1, seed is carried out to surface sterilization by 10% SYNTHETIC OPTICAL WHITNER, then with sterilized water washing 3 times.

2, packet transaction

Experimental group: aseptic seed is suspended in the agarose solution of 0.15g/100mL and bed board to containing on the 1/2MS culture medium flat plate of 100mMNaCl, flat board is placed 3 days under 4 ℃ of dark conditions, then the tissue culture room (illumination 16 hours every days) that moves into 24 ℃ is cultivated 14 days, takes pictures and observes the growth conditions of plant.

Control group: aseptic seed is suspended in the agarose solution of 0.15g/100mL and bed board to 1/2MS culture medium flat plate, flat board is placed 3 days under 4 ℃ of dark conditions, then the tissue culture room (illumination 16 hours every days) that moves into 24 ℃ is cultivated 14 days, takes pictures and observes the growth conditions of plant.

Fig. 4 is shown in by photo.In control group is processed, the growth conditions of the plant of each strain does not have significant difference.In experimental group is processed, cross the growth conditions of expressing strain plant and be significantly better than the environmental Arabidopis thaliana of Colombia, the plant that the growth conditions of the environmental Arabidopis thaliana of Colombia is significantly better than suppressing to express strain.

Carry out repeating for three times experiment, repeat each strain in experiment at every turn and get 100 seeds, each 50 of experimental group and control group, according to the growth conditions of plant, divide into groups the plant of experimental group, and grouping standard is as follows: the 1. plant of normal growth, sees Fig. 5 A; The plant that 2. can only grow two true leaves, sees Fig. 5 B; 3. cotyledon can turn green, but there is no the plant of true leaf, sees Fig. 5 C; 4. cotyledon can not turn green plant, sees Fig. 5 D.

After experimental group is processed, each strain is shown in Fig. 6 according to the grouping situation of growth conditions.Cross the growth conditions of expressing strain plant and be significantly better than the environmental Arabidopis thaliana of Colombia, the plant that the growth conditions of the environmental Arabidopis thaliana of Colombia is significantly better than suppressing to express strain.

Above result shows, crosses to express AtSDIP1 gene and can increase the salt tolerance of plant, suppresses to express AtSDIP1 gene and can reduce the salt tolerance of plant.

Claims

1. cultivating a method for transgenic plant, is that the gene of the protein of the composition of the aminoacid sequence shown in sequence 1 in code sequence list is imported in object plant, obtains the transgenic plant of salt tolerance higher than described object plant; Described object plant is Arabidopis thaliana.

2. method according to claim 1, is characterized in that: described gene is following 1) or 2) or 3) DNA molecular: 1) coding region is if sequence in sequence table 2 is from the DNA molecular as shown in 5 ' end 85-744 position Nucleotide;

3) DNA molecular shown in sequence 2 in sequence table.

3. cultivating a method for salt-tolerant plant, is the transfer-gen plant that method described in claim 1 is obtained and the plant hybridization that sets out, and obtains the filial generation of salt tolerance higher than the described plant that sets out; The described plant that sets out is Arabidopis thaliana.

4. cultivating a method for transgenic plant, is to suppress the expression of the gene of the protein of the composition of the aminoacid sequence shown in sequence 1 in code sequence list in object plant, obtains the transgenic plant of salt tolerance lower than described object plant; Described object plant is Arabidopis thaliana.

5. method according to claim 3, is characterized in that: described gene is following 1) or 2) or 3) DNA molecular: 1) coding region is if sequence in sequence table 2 is from the DNA molecular as shown in 5 ' end 85-744 position Nucleotide;

3) DNA molecular shown in sequence 2 in sequence table.