CN103059115A - Plant salt-tolerance-related protein AtSDIP1, and coding gene and application thereof - Google Patents
Plant salt-tolerance-related protein AtSDIP1, and coding gene and application thereof Download PDFInfo
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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
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, at least 20% arable land and surpass 50% irrigate land in the impact that is subject in varying degrees salt damage is arranged in the world.Some arids or semiarid zone, because steam output is large, quantity of precipitation is little, causing the salinity in the soil (mainly is NaCl and NaCO
3) a large amount of accumulation.Some oceanfronts, the higher or inwelling of ground water table, also can cause upper soll layer to accumulate more salinity (mainly is 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 rapid decline of the irrigateed land area that causes of water resources shortage, fully the development and utilization saltings has become the important topic that concerns human survival and development.
Salt stress is one of main limiting factor of growth and development of plants.According to the tolerance difference of plant for salt stress, plant can be divided into halophytes and glycophyte two classes.Halophytes be can be lower than at osmotic potential-edatope of 3.3MPa in growth and finish the natural phant colony of the life history, otherwise then be glycophyte.
Most plants especially farm crop belongs to glycophyte, and is responsive to salt stress.When they were grown in the saltings, owing to be subjected to the salt stress effect, poor growth, came off at often blade flavescence, death, has a strong impact on photosynthesis, sometimes even whole strain plant withered death, thereby caused crop production reduction.Salt stress has seriously influenced global grain yield, therefore, discloses the mechanism of plant reply salt stress, and improves accordingly the saline-alkaline tolerance of plant, has 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 salinity is too high in the soil, and the osmotic potential of soil is reduced, and can cause the 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 that causes; The 3rd, when plant is subject to salt stress, can accumulate in a large number active oxygens in the cell, the active oxygen of high density then can cause the destruction of film fat, protein and nucleic acid.
The research plant is for the molecular mechanism of response and the tolerance of salt stress, seeks anti-salt-related gene and be focus in the last few years, for the farm crop improvement with cultivate salt tolerant crop and have great importance.
Summary of the invention
The purpose 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 (Arabidopsis thalianaecotype Columbia) of Colombia, called after AtSDIP1 albumen is following (a) or (b) or (c):
(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or more amino-acid residues (such as 1-25,1-20,1-15,1-10,1-5 or 1-3 amino-acid residue) and/or disappearance and/or interpolation and relevant with plant salt endurance protein of being derived by sequence 1;
(c) has 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 with the aminoacid sequence of sequence 1 and the relevant protein of being derived by sequence 1 with plant salt endurance.
In order to make the protein in (a) be convenient to purifying, N-terminal or C-terminal that can the protein that the aminoacid sequence shown in the sequence 1 forms in by sequence table 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) but or the protein synthetic (c), also can synthesize first its encoding gene, carry out again biological expression and obtain.Above-mentioned (b) or (c) in protein encoding gene can by will be in the dna sequence dna shown in the sequence in the sequence table 2 codon of one or several amino-acid residue of disappearance, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
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 such as sequence in the sequence table 2 from the dna molecular shown in 5 ' the terminal 85-744 position Nucleotide;
2) coding region such as sequence in the sequence table 2 from the dna molecular shown in 5 ' the terminal 85-747 position Nucleotide;
3) dna molecular shown in the sequence 2 in the sequence table;
4) under medium stringent condition with 1) or 2) or 3) the dna sequence dna hybridization that limits 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 that limits and the dna molecular of coding salt tolerance associated protein;
6) with 1) or 2) or 3) dna sequence dna that limits 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 also can be expression vector.Contain in the expression vector (recombinant expression vector) of 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 with described AtSDIP1 gene insertion vector pCAMBIA1300-221 obtains.
Available existing plant expression vector construction contains the recombinant expression vector of described AtSDIP1 gene.Described plant expression vector comprises the 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 zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant plant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also enhanser be can use, translational enhancer or transcriptional enhancer comprised, but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of colour-change as adding the coding that in plant, to express.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 the purpose plant, obtains the transgenic plant that salt tolerance is higher than described purpose plant.Carry described gene expression vector can 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 by using, and the plant tissue that transforms is cultivated into plant.Described AtSDIP1 gene specifically can import in the described purpose plant by described recombinant expression vector.Described purpose plant is monocotyledons or dicotyledons.Described dicotyledons specifically can be Arabidopis thaliana, such as the environmental Arabidopis thaliana of 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 that salt tolerance is 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 the purpose plant, obtains the transgenic plant that salt tolerance is lower than described purpose plant.Described " suppressing the expression of gene described in the purpose plant " specifically can realize by import interference fragment in the purpose plant.Described interference fragment is comprised 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 shown in the sequence 3 of sequence table.Described interference fragment can import in the described purpose plant by interference carrier.Described interference carrier specifically can be the recombinant plasmid pCAMBIA1300-221-AtSDIP1-RNAi that the multiple clone site with described interference fragment insertion vector pCAMBIA1300-221 obtains.Carry described interference fragment expression vector can 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 by using, and the plant tissue that transforms is cultivated into plant.Described purpose plant is monocotyledons or dicotyledons.Described dicotyledons specifically can be Arabidopis thaliana, such as the environmental Arabidopis thaliana of Colombia.
Available existing plant expression vector construction contains the recombinant expression vector of described interference fragment.Described plant expression vector comprises the 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 zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant plant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be combined with other plant promoter; In addition, when making up plant expression vector, also enhanser be can use, translational enhancer or transcriptional enhancer comprised, but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of colour-change as adding the coding that in plant, to express.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
The present invention provides the foundation for the response of salt stress and the molecular mechanism of tolerance for the research plant, has great importance for farm crop improvement and cultivation salt tolerant crop.
Description of drawings
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 growth conditions of the plant of each strain.
Fig. 5 is that the 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 purpose of the present invention, these specific embodiments equally within the scope of the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.
" be correlated with "/" being operably connected " refer to the nucleotide sequence that two physics or function are relevant.For example, to such an extent as to if promotor or adjusting dna sequence dna and coding RNA or protein DNA sequence are operably connected or locate and regulate the expression level that dna sequence dna will affect coding or structural DNA sequence, claim so promotor or regulate dna sequence dna and coding RNA or protein DNA sequence " relevant ".
" mosaic gene " is recombinant nucleic acid sequence, promotor or regulate be operably connected coding mRNA or as the nucleotide sequence of protein expression of nucleotide sequence wherein, or with the coding mRNA or relevant as the nucleotide sequence of protein expression so that the adjusting 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 normally is operably connected of finding such as occurring in nature.
" encoding sequence ": refer to directly to specify the nucleotide sequence of the aminoacid sequence of its protein, it can be DNA, cDNA, nucleotide sequence synthetic or restructuring.The border of encoding sequence is determined by open reading frame usually.5 ' end of open reading frame is defined by initiator codon, and 3 ' end is defined by terminator codon.Initiator codon is generally ATG, perhaps also can be other initiator codon such as GTG or TTG.Terminator codon comprises for example TAA, TAG, TGA.
" cDNA " refers to the DNA with the RNA complementary element, strand or double-stranded dna molecular that it comes from the mRNA molecule reverse transcription that derives from eukaryotic maturation, process montage by ThermoScript II.So, do not contain any intron that originally may be present in the corresponding with it genomic dna sequence among the cDNA.
Corresponding to: in the context of the invention " corresponding to " mean when 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, when comparing 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 of reference 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 used " expression cassette " means to instruct and is fit to the nucleotide sequence that specific nucleotide sequence is expressed in the host cell, comprises the promotor that is operably connected with the purpose nucleotide sequence, the described purpose nucleotide sequence termination signal that is operably connected.Usually, it also comprises the required sequence of the correct translation of nucleotide sequence.The expression cassette that comprises the purpose nucleotide sequence can be chimeric, and meaning one of its composition at least is allos with respect to its one of other composition at least.Expression cassette also can be naturally occurring, but obtains to be used for the expression cassette of heterogenous expression with recombinant forms.Yet usually, expression cassette is allos with respect to the host, that is, the specific nucleic acid sequence of expression cassette is not natural to be appeared in the host cell, must be introduced into by transformation event the precursor of host cell or host cell.The expression of nucleotide sequence can be controlled by constitutive promoter or inducible promoter in the expression cassette, and wherein only when host cell is exposed to some specific outside stimuluss, described inducible promoter is initial transcribing.If the situation of multicellular organisms, such as plant, promotor also can be to particular organization, or organ or etap are special.
" gene " is the localized area that is positioned at genome, except aforementioned nucleic acid sequence encoding, comprises the nucleotide sequence that other mainly is modulability, and described modulability nucleotide sequence is responsible for the expression of encoding part, namely 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 and the not natural relevant nucleotide sequence of its host cell that is introduced into to comprise 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 when with reference to the polypeptide of nucleic acid sequence encoding the polypeptide of same acid sequence being arranged, this nucleotide sequence be " isocoding " with reference to nucleotide sequence.
" separation " nucleic acid molecule or " separation " protein are that the artificially is separated with its natural surroundings and existed, and are not nucleic acid molecule or the protein of natural product therefore.The nucleic acid molecule or the protein that separate can exist with purified form, perhaps may reside in the non-natural environment, for example in recombinant host cell or the transgenic plant.
Natural: as to refer to the gene that in the genome of no transformed cells, exists.
Natural existence: term " natural existence " is used for describing can be at the object of occurring in nature discovery, and they are different from artificial object that produces.For example, can separate from natural source, protein or the nucleotide sequence of not having a mind to carry out existence in manually modified, the organism (comprising virus) in the 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 individual cells or the culturing cell form of separating, or as high organized unit as, plant tissue for example, the part of plant organ or whole plant.
The plant unit that " plant cell cultures " means the various etap as, protoplastis for example, cell cultures cell, the cell in the plant tissue, pollen, pollen tube, ovule, blastular, the culture of zygote and embryo.
" vegetable material " refers to leaf, stem, and root, the part of flower or flower, fruit, pollen, ovum, zygote, seed is transplanted a 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, such as root, and stem, leaf, bud or embryo.
Here used " plant tissue " means to be organized into one group of vegetable cell of structure and function unit.Comprise in the plant or any tissue of plant in the 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 independent 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, and it comprises the binding site of RNA polymerase, and the transcribing of initiate dna.Promoter region also can comprise other element as the genetic expression instrumentality.
" protoplastis " is the vegetable cell that does not have cell walls or the separation of parts of fine cell wall is only arranged.
" regulatory element " refers to participate in controlling the sequence that nucleotide sequence is expressed.Regulatory element comprises promotor and the termination signal of the purpose nucleotide sequence that is operably connected.Usually they also comprise the required sequence of the correct translation of nucleotide sequence.
" reorganization " nucleic acid is by Shuffling Method, as described herein the nucleic acid of any Shuffling Method generation.Produce reorganization nucleic acid by mode artificial and alternatively circulation (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 purpose nucleic acid; Can before or after any reconstitution steps, carry out this screening step.In some (but not all) reorganization embodiments, be desirably in and carry out many wheel restructuring before the screening 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 recombinating 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 one of following sequence comparison algorithm or range estimation to measure, have at least 60%, preferred 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 zone at least about 100 residues, most preferably, basic identical at least about the sequence in 150 residues.In particularly preferred embodiments, sequence is basic identical in the whole length in coding region.And essentially identical nucleic acid or protein sequence have essentially identical function.
In order to carry out sequence relatively, usually, a sequence compares with the detection sequence as canonical sequence.When utilizing sequence comparison algorithm, will detect and canonical sequence is input in the 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 will calculate and detect sequence with respect to the percentage sequence identity of canonical sequence.
For example, by Smith ﹠amp; Waterman, local homology's algorithm of Adv.Appl.Math.2:482 (1981) is by Needleman ﹠amp; Wunsch, the sequence analysis algorithm of .J.Mol.Biol.48:443 (1970) is by Pearson ﹠amp; Lipman, the similarity retrieval method of Proc.Nat ' l.Acad.Sci.USA85:2444 (1988), (GAP in the Wisconsin Genetics software package, BESTFIT, FASTA and TFASTA are implemented in computerize by these algorithms, Genetics Computer Group, 575Science Dr., Madison, WI) or by range estimation (usually referring to, Ausubel etc. hereinafter) can carry out comparing for the best of sequence relatively.
An examples of algorithms that is suitable for measuring percentage sequence identity and sequence similarity is the BLAST algorithm, and at Altschul etc. .J.Mol.Biol.215:403-410 has described this algorithm in (1990).Can obtain carrying out the software that BLAST analyzes by the public of NCBI (http://www.Ncbi.nlm.nih.gov/).This algorithm comprises: being tested and appraised out and searching in the sequence length is the short word of W and at first identify the high score sequence to (HSPs), described short word with database sequence in the word of equal length coupling or satisfy some on the occasion of the threshold values T that scores when comparing.T is called adjacent words score threshold (Altschul etc., 1990).These initial neighborhood word are hit the longer HSPs that goes to find to comprise them as the clue that begins to search.Then, these words hit along the extension far away as far as possible of the both direction of each sequence, no longer increase until score value is compared in accumulation.For nucleotide sequence, with parameter M (the award score value that mates in pairs residue; Always greater than zero) and N (the point penalty value of mispairing residue; Always 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 that obtains, because one or more negative score value residues comparison accumulation, the accumulation score value reaches or is lower than zero, or any of two sequences be when 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 relatively be default value.For aminoacid sequence, the BLASTP program is used word length value (W) 3, and expected value (E) 10 and BLOSUM62 score matrix (referring to, Henikoff ﹠amp; Henikoff, Proc.Natl.Acad.Sci.USA89:10915 (1989)) be default value.
Except calculating percentage sequence identity, the BLAST algorithm also carries out the statistical analysis of similarity between two sequences (referring to, Karlin ﹠amp for example; Altschul, Proc.Nat'l.Acad.Sci.USA90:5873-5787 (1993)).It is minimum and probability (P (N)) that the similarity that the BLAST algorithm provides is measured, and it provides the accidental indication that the probability of coupling occurs between two Nucleotide or aminoacid sequence.For example, if detect nucleotide sequence and be less than approximately 0.1 with reference to the minimum of nucleotide sequence comparison and probability, more preferably less than about 0.01, most preferably be less than approximately 0.001, think that so to detect nucleotide sequence similar to canonical sequence.
Two essentially identical another indexs of nucleotide sequence are the mutually hybridization under stringent condition of two molecules.Phrase " specific hybrid " refers to that under stringent condition, molecule only is combined with specific nucleotide sequence, forms duplex or hybridization when this sequence is present among complex mixture (for example, total cell) DNA or the RNA." basic combination " refers to complementary hybridization between probe nucleic acid and target nucleic acid, and comprises less mispairing, can tolerate described mispairing by the severity that reduces hybridization medium, to realize the expectation detection of target nucleic acid sequence.
" stringent hybridization condition " and " strictly hybridizing rinsing condition " is sequence dependent in nucleic acid hybridization is tested such as Southern and Northern hybridization context, and is different under the varying environment parameter.Long sequence is at the 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 among the New York.Usually, for the particular sequence under restriction ionic strength and pH, high stringency hybridization and rinsing condition are chosen as and are lower than heat fusion joint (T
m) about 5 ℃.Typically, under " stringent condition ", probe will with the hybridization of its target subsequence, and not with other sequence hybridization.
Temperature when Tm is (under limiting ionic strength and pH condition) 50% target sequence with the probe hybridization of complete coupling.For specific probe, very strict condition is chosen as and equals T
mAt filter membrane the example of a stringent hybridization condition of hybridizing more than the complementary nucleic acid of 100 complementary residues being arranged in Southern or Northern trace 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 ℃, about 15 minutes of 0.15M NaCl.The example of strict rinsing condition is at 65 ℃, 0.2x SSC rinsing 15 minutes (referring to, Sambrook, hereinafter, the description of SSC damping fluid).Usually, before high severity rinsing, hang down the severity rinsing to remove the background probe signals.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 for example.For short probe (for example, about 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 about 0.01 to 1.0Na ionic concn (or other salt) usually, and typical temperature is about 30 ℃ at least.Also can obtain stringent condition by adding destabilizing agent such as methane amide.Usually, in specific cross was measured, the just irrelevant viewed value of probe of noise signal to noise ratio was high by 2 * and (or higher) show the detection of specific hybridization.If the protein of their codings of the nucleic acid of not hybridizing mutually under stringent condition is essentially identical, they are still essentially identical so.For example, when the maximum codon degeneracy creation nucleic acid that allows with genetic code copies, this situation will appear.
The below is the example that hybridization/rinsing condition arranges, described condition can be used for clone and 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, hybridize among the 1mM EDTA, at 50 ℃, 2X SSC, rinsing among the 0.1%SDS more is desirably in 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO
4, hybridize among the 1mM EDTA, at 50 ℃, 1X SSC, rinsing among the 0.1%SDS more is desirably in 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO
4, hybridize among the 1mM EDTA, at 50 ℃, 0.5X SSC, rinsing among the 0.1%SDS, preferably, and at 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO
4, hybridize among the 1mM EDTA, at 50 ℃, 0.1X SSC, rinsing among the 0.1%SDS, more preferably, and at 50 ℃, 7% sodium lauryl sulphate (SDS), 0.5M NaPO
4, hybridize among the 1mM EDTA, at 65 ℃, 0.1X SSC, rinsing among the 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 usually substantially identical with the second protein, and for example, wherein two protein is only owing to preservative replacement and difference.
" synthesize " nucleotide sequence that refers to comprise non-existent constitutional features in the 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 the dna molecular stable integration and enter in the purpose organism genome.
" conversion/genetically modified/restructuring " refer to introduce the host organisms of heterologous nucleic acids molecule, such as bacterium or plant.Nucleic acid molecule can stably be integrated into host genome or nucleic acid molecule also can be used as the extrachromosomal molecule existence.This extrachromosomal molecule can be self-replicating.The cell that transforms, 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 not contain the wild-type organisms of 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, the cDNA sequence, the genomic dna sequence that comprises exon and intron, the DNA of chemosynthesis and RNA sequence, and justice and corresponding antisense strand are arranged.
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, the cold spring port, New York; Ausubel etc., 1989, molecular biology current techniques, GreenePublishing Assoc iates ﹠amp; 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,
The PCR strategy, Academic Press, Inc., SanDiego; And Erlich (volume), 1992, round pcr, Oxford University Press, New York) finish.
Plant Transformation:
In embodiment particularly preferably, in higher organism style such as plant, express at least a albumen of giving the salt tolerance of the present invention.Nucleotide sequence of the present invention can be inserted in the expression cassette, then preferably, the 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 the Agrobacterium.The plant that transforms according to the present invention can be monocotyledons or dicotyledons, includes 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 such as softwood tree and deciduous trees.Particularly preferably be paddy rice, wheat, barley, corn, oat, rye, sugarcane, beet, soybean, potato.Preferably, being used for plant of the present invention is rape section plant.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 also can be other plant of rape section.
In case the nucleotide sequence conversion with expectation enters in the specified plant species, can in these species, breed it or with traditional breeding method its be shifted other kind that enters same 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 changes simultaneously of the present invention to meet plant-preference.And, from having at least about 35%, preferably more than about 45%, more preferably more than 50%, most preferably can realize best high-caliber expression in the plant more than the encoding sequence of about 60%GC content.Although can in monocotyledons and dicotyledons species, express fully preferred gene order, but can special codon preference and the GC content preference of modification sequence to adapt to 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 is blocked with searching.Utilize publication application EP 0 385 962 (Monsanto), method described in EP 0 359 472 (Lubrizol) and the WO93/07278 (Ciba-Geigy), with site-directed induced-mutation technique well known in the art, PCR and synthetic gene make up the institute that carries out needing to carry out and change in these nucleotide sequences, such as above-mentioned those changes.
In one embodiment of the present invention, can prepare synthetic gene according to disclosed method in the United States Patent (USP) 5,625,136 that is incorporated herein by reference document here.In the method, utilized the preferred codon of corn, i.e. that amino acid whose single cipher in the most normal coding corn.The preferred codon of the corn of specific amino acids can derive from, for example the known sequence of corn.At Murray etc., instructed the corn codon of 28 genes of maize plant to use among the Nucleic Acids Research17:477-498 (1989), the disclosure of incorporating this piece document here into is reference.
Nucleotide sequence can be optimized by this way so that the expression in any plant.Generally acknowledge all of gene order or any part can be optimized or synthetic.That is, 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 consensus sequence (NAR15:6643-6653 (1987)) that plant is fit to, and Clonetech has proposed the sub-consensus sequence of further translation initiation (1993/1994 catalogue, 210 pages).These consensus sequences are fit to use with nucleotide sequence of the present invention.To comprising described nucleotide sequence, until and comprise ATG (keeping simultaneously second amino acid not modified) or alternately until and comprise in the construction of the GTC (have and modify second amino acid whose possibility of transgenosis) behind the ATG and introduce this sequence.
Can with 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 in plant, expressing, comprise composing type, induction type, sequential is regulated, 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 the target species.Therefore, can use at leaf, stem or cane, fringe, inflorescence (for example, spike, panicle, cob etc.), root, and/or express nucleotide sequence of the present invention in the seedling.Although having proved the many promotors that derive from dicotyledons is operational in monocotyledons, vice versa, but ideally, select the dicotyledons promotor to be used for the expression of dicotyledons, monocotyledonous promotor is used for the expression of monocotyledons.Yet, the origin of selected promotor without limits, the expression that drives nucleotide sequence in the expectation cell is just enough as long as promotor works.
Preferred constitutive promoter comprises CaMV35S and 19S promotor (Fraley etc., the U.S. Patent number 5,352,605 that on October 4th, 1994 announced).Preferred promotor derives from any of several actin genes of expressing in most cell types in addition.Can easily modify (Mol.Gen.Genet.231:150-160 (1991)) the described promoter expression cassettes such as McElroy to be used for the expression of ThLEA1 gene, this expression casette is particularly suitable for using in the monocotyledons host.
Another preferred constitutive promoter derives from ubiquitin, and it is the known another kind of gene product that accumulates 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 the transgenic plant.Develop the corn ubiquitin promoter in the transgenosis monocotyledons system, and announced the carrier that discloses its sequence among the EP0342926 and be used for the monocotyledons conversion in patent.Ubiquitin promoter is adapted at transgenic plant, the particularly expression of new vertical compact panicle gene in the monocotyledons.
Except the selection that is fit to promotor, the structure of AtSDIP1 protein expression can be connected to the transcription terminator that is fit in heterologous nucleotide sequence downstream in the plant.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 that works in plant can be with in the present invention.
Also can introduce other sequence in the expression cassette of the present invention.These sequences comprise proves the sequence that strengthens expression, such as intron sequences (for example deriving from Adhl and bronzel) and virus leader sequence (for example deriving from TMV, MCMV and AMV).
Might be preferably the expression of nucleotide sequence of the present invention be targeted to different cellular localizations in the 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 enzyme that transgenosis is coded has adopted technology well known in the art.Usually, operate coding derives from the DNA of 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 makes up 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 purposes is well known in the art.
The a large amount of conversion carriers that can be used for Plant Transformation are that the Plant Transformation those skilled in the art are known, and nucleic acid molecule of the present invention can be united use with any this carrier.The selection of carrier will depend on for the preferred transformation technology and the target plant species that transform.For some target species, can be preferably different microbiotic or herbicide selective mark.Usually the selected marker that is used in the conversion comprises nptII gene (the Messing ﹠amp that gives kantlex and associated antibiotic resistance; Vierra., 1982.Gene19:259-268; 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 of the hph gene to the microbiotic hygromycin resistance; And give dhfr gene (Bourouis etc. to the methatrexate resistance Diggelmann, MolCell Biol4:2929-2931),, J.2 1983.EMBO (7): 1099-1104), give EPSPS gene (the U.S. patent No.: 4,940 to the glyphosate resistance, 935 and 5,188,642), and the mannose-6-phosphate isomerase gene (U.S. Patent number 5 of metabolism seminose ability is provided, 767,378 and 5,994,629).Yet the selection of selected marker is not vital to the present invention.
Another preferred embodiment in, nucleotide sequence of the present invention directly is transformed in the plastom.The major advantage of plastid transformation is that plastid does not need the essence modification just can express bacterial gene usually, and plastid can be expressed a plurality of open reading frame under single promotor control.At United States Patent (USP) 5,451, in 513,5,545,817 and 5,545,818, among the PCT application number WO95/16783 and McBride etc., (1994) Proc.Nati.Acad.Sci.USA91 has at large described the plastid transformation technology among the 7301-7305.The basic fundamental that chloroplast(id) transforms comprises that the zone that for example utilizes biological bombardment or protoplast transformation (for example conversion of calcium chloride or PEG mediation) will be arranged in clone's plastid DNA of goal gene and selective marker flank introduces suitable target tissue together.1 to the 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 is providing point mutation to the chloroplast(id) 16S rRNA of spectinomycin and/or streptomycin resistance and rps12 gene as the selective marker (Svab that transforms, 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 per approximately 100 target blades bombardment 1 time has produced stable homogeneity transformant.The cloning site that exists between these marks can be used for producing for the plastid targeting vector that imports foreign gene (Staub, J.M., and Maliga, P. (1993) EMBO J.12,601-606).By using dominant selectable marker, remarkable increase (the Svab that 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 transformation frequency, Z. and Maliga, 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 that is used for plastid transformation is known in the art, and comprises within the scope of the present invention.Usually, need about 15 to 20 cell division cycle to reach the homogeneity state after the conversion.Expression level gene inserted the advantage that the plastid in all several thousand the annular plastom copies that exist in each vegetable cell expresses to have utilized copy number to be much higher than the nuclear expression gene by homologous recombination, so that can easily surpass 10% of total solvable plant protein.In a preferred embodiment, nucleotide sequence of the present invention is inserted in the plastid targeting vector, and conversion enters in the plant host plastom of expectation.Obtained to belong to for the plastom that contains nucleotide sequence of the present invention the plant of homogeneity, preferably, this plant has the ability of expressing nucleotide sequence high-levelly.
The composition of MS substratum sees 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 (Arabidopsis thaliana ecotype Columbia) of Colombia: available from ABRC(ArabidopsisBiological Resource Center).
One, the structure of over-express vector
From the SmaI of the double chain DNA molecule replacement vector pCAMBIA1300-221 shown in 5 ' the terminal 85-747 position Nucleotide and the small segment between the XbaI enzyme cutting site, obtaining recombinant plasmid pCAMBIA1300-221-AtSDIP1(is over-express vector with the sequence 2 of sequence table).The structural representation of recombinant plasmid pCAMBIA1300-221-AtSDIP1 is seen Fig. 1, and the AtSDIP1 gene is regulated and control by the 35S double-promoter.
Two, the structure that suppresses expression vector
With the double chain DNA molecule shown in the sequence 3 of sequence table (in the sequence 3, from 5 ' terminal the 1st to 663 reverse complemental fragment that Nucleotide is the AtSDIP1 gene, the the 664th to 853 Nucleotide is stuffer fragment, the the 854th to 1516 Nucleotide is the AtSDIP1 gene) BglII of replacement vector pCAMBIA1300-221 and the small segment between the XbaI enzyme cutting site, obtain recombinant plasmid pCAMBIA1300-221-AtSDIP1-RNAi(and namely suppress expression vector).The structural representation of recombinant plasmid pCAMBIA1300-221-AtSDIP1-RNAi is seen Fig. 2.
Three, the acquisition of AtSDIP1 gene overexpression plant
1, over-express vector is imported in the Agrobacterium tumefaciens EHA105 strain, obtain the edaphic bacillus of recombinating.
2, the restructuring edaphic bacillus that step 1 is obtained passes through 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 are T
1For plant).
3, the seed with step 2 results places on the 1/2MS culture medium flat plate that contains 20 μ g/mL Totomycin, flat board was placed 2-4 days under 4 ℃ of dark conditions first, then the tissue culture room (illumination 16 hours every days) that moves into 24 ℃ cultivated for 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 are T
2For plant).
4, the seed of step 3 being gathered in the crops places on the 1/2MS culture medium flat plate that contains 50 μ g/mL kantlex and carries out resistance screening, and gathers in the crops the T that single transfer-gen plant that copies obtains
2(plant that this seed grows up to is T for the seed of resistant plant
3For plant).If a certain T
1The T that obtains for plant
2The quantity of resistant plant and non-resistance plant ratio is about 3:1 in the plant, and this T is described
1Be the transfer-gen plant of single copy for plant.
5, the seed of step 4 being gathered in the crops places on the 1/2MS culture medium flat plate that contains 50 μ g/mL kantlex and carries out resistance screening.If a certain T
2The T that obtains for plant
3Be resistant plant for plant, this T is described
2Be the transfer-gen plant that isozygotys for plant, this T
2For plant and offspring thereof be isozygoty cross the expression strain.
Four, the 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 the 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 plant 2 age in week seedling, get at random the T that two AtSDIP1 gene inhibition are expressed strains (SDIP1-RNAi# 1 strain and SDIP1-RNAi# 2 strain)
3For plant 2 the week age seedling, get environmental Arabidopis thaliana (WT) plant of Colombia 2 age in week seedling, extracting total RNA and reverse transcription is cDNA, take cDNA as template the expression amount of AtSDIP1 gene in the plant of each strain being carried out PCR identifies, adopt the Actin2 gene as reference gene, as 1, calculate the relative expression quantity of AtSDIP1 gene in other each strain with the expression amount of the expression amount of AtSDIP1 gene in the environmental Arabidopis thaliana of Colombia.
Primer sequence for the identification of the AtSDIP1 gene is as follows:
SDIP1-qPCR-FW：5’-CGCAGGATTTTCTTGGTGAC-3’；
SDIP1-qPCR-Rev：5’-ACTGAAGCTGGGAGAGAGAC-3’。
Primer sequence for the identification of the Actin2 gene is as follows:
Actin2-qPCR-FW:5’-GGTAACATTGTGCTCAGTGGTGG-3’；
Actin2-qPCR-Rev:5’-AACGACCTTAATCTTCATGCTGC-3’。
Carry out repeated experiments three times, average, the results are shown in Figure 3.Cross the expression amount of expressing AtSDIP1 gene in the strain strain and significantly increase, the expression amount that suppresses AtSDIP1 gene in the expression strain significantly reduces.
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 that two AtSDIP1 gene inhibition are expressed strain (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 surface sterilization with 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 the 1/2MS culture medium flat plate that contains 100mMNaCl, flat board was placed 3 days under 4 ℃ of dark conditions, then the tissue culture room (illumination 16 hours every days) that moves into 24 ℃ was cultivated 14 days, took pictures and observed the growth conditions of plant.
Control group: aseptic seed is suspended in the agarose solution of 0.15g/100mL and bed board to the 1/2MS culture medium flat plate, flat board was placed 3 days under 4 ℃ of dark conditions, then the tissue culture room (illumination 16 hours every days) that moves into 24 ℃ was cultivated 14 days, took pictures and observed the growth conditions of plant.
Photo is seen Fig. 4.In control group was processed, the growth conditions of the plant of each strain did not have significant difference.In experimental group is processed, cross the growth conditions of expressing the strain plant and significantly be better than the environmental Arabidopis thaliana of Colombia, the plant that the growth conditions of the environmental Arabidopis thaliana of Colombia significantly is better than suppressing to express strain.
Carry out repeated experiments three times, each strain is got 100 seeds in each repeated experiments, and each 50 of experimental group and control groups according to the growth conditions of plant, divide into groups the plant of experimental group, and the grouping standard is as follows: the 1. plant of normal growth, see Fig. 5 A; 2. can only grow the plant of two true leaves, see Fig. 5 B; 3. cotyledon can turn green, but does not have the plant of true leaf, sees Fig. 5 C; 4. cotyledon can not turn green plant, sees Fig. 5 D.
After experimental group was processed, each strain was seen Fig. 6 according to the grouping situation of growth conditions.Cross the growth conditions of expressing the strain plant and significantly be better than the environmental Arabidopis thaliana of Colombia, the plant that the growth conditions of the environmental Arabidopis thaliana of Colombia significantly is better than suppressing to express strain.
Above result shows, crosses and expresses the salt tolerance that the AtSDIP1 gene can increase plant, suppresses to express the AtSDIP1 gene and can reduce the salt tolerance of plant.
Claims (10)
1. protein is following (a) or (b) or (c):
(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or more amino-acid residues and/or disappearance and/or interpolation and relevant with plant salt endurance protein of being derived by sequence 1;
(c) has 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 with the aminoacid sequence of sequence 1 and the relevant protein of being derived by sequence 1 with plant salt endurance.
2. the gene of coding claim 1 described protein.
3. gene as claimed in claim 2, it is characterized in that: described gene is following 1) or 2) or 3) or 4) or 5) or 6) or 7) dna molecular:
1) coding region such as sequence in the sequence table 2 from the dna molecular shown in 5 ' the terminal 85-744 position Nucleotide;
2) coding region such as sequence in the sequence table 2 from the dna molecular shown in 5 ' the terminal 85-747 position Nucleotide;
3) dna molecular shown in the sequence 2 in the sequence table;
4) under medium stringent condition with 1) or 2) or 3) the dna sequence dna hybridization that limits 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 that limits and the dna molecular of coding salt tolerance associated protein;
6) with 1) or 2) or 3) dna sequence dna that limits 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.
4. the active fragments of the described protein of claim 1, or, the active fragments of claim 2 or 3 described genes.
5. the carrier, expression cassette, transgenic cell line or the recombinant bacterium that contain claim 2 or 3 described genes.
6. a method of cultivating transgenic plant is that claim 2 or 3 described genes are imported in the purpose plant, obtains the transgenic plant that salt tolerance is higher than described purpose plant.
7. method as claimed in claim 6, it is characterized in that: described purpose plant is monocotyledons or dicotyledons.
8. a method of cultivating salt-tolerant plant is with claim 6 or the transfer-gen plant of 7 described methods acquisitions and the plant hybridization that sets out, and obtains the filial generation that salt tolerance is higher than the described plant that sets out.
9. method of cultivating transgenic plant is the expression that suppress claim 2 in the purpose plant or 3 described genes, obtains the transgenic plant that salt tolerance is lower than described purpose plant.
10. method as claimed in claim 9, it is characterized in that: described purpose plant is monocotyledons or dicotyledons.
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| SWARBRECK,D.ET AL.: "NCBI：NM_124490.4", 《NCBI》 * |
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