CN105001313B - Bermuda grass ' C299 ' dehydrin protein Dehydrin-S and its encoding gene and probe - Google Patents
Bermuda grass ' C299 ' dehydrin protein Dehydrin-S and its encoding gene and probe Download PDFInfo
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Abstract
The present invention relates to a kind of Bermuda grass ' C299 ' dehydrin protein Dehydrin S and its encoding gene and probe, the protein is the protein of following (a) or (b)：(a) protein being made of the amino acid sequence as shown in SEQ ID NO.4；(b) amino acid sequence shown in SEQ ID NO.4 passes through substitution, lacks or adds one or several amino acid and has the protein as derived from (a) of Bermuda grass ' C299 ' dehydrin protein activity.The present invention also provides the probes of a kind of nucleic acid sequence for encoding above-mentioned protein and the above-mentioned nucleic acid sequence of detection；The present invention is to regulate and control the environment stresses physiological responses such as Bermuda grass ' C299 ' drought resisting using technique for gene engineering, so as to achieve the purpose that improve turfgrass drought-resistant ability, provides theoretical foundation for molecular breeding, has very big application value.
Description
Technical field
A kind of important protected protein dehydrin protein in Bermuda grass ' C299 ' drought stress response process of the present invention
Dehydrin-S and its encoding gene and probe, and in particular to a kind of Bermuda grass ' C299 ' dehydrin protein Dehydrin-S and
Its encoding gene and probe.
Background technology
Bermuda grass (Cynodon dactylon) is very important warm season turf, is planted for grass family perennial herb
Object has flourishing rhizome and stolon, the speed of growth is fast, power of regeneration is strong, into fast, heat-resisting, the resistance to trample in level ground, quality it is very thin,
The advantages that color and luster is good is at home and abroad widely used in sports ground, esplanade, park and soil and slope protection lawn etc..Therefore it is season type
One of application value highest, most widely used grass seeds in turfgrass enjoy the title of warm season turf " main cultivated orages ", great society
Meeting, economy and the ecological value.The drought-resistant ability of Bermuda grass is very strong, year evapotranspiration less than 400mm, be only the agricultures such as corn, wheat
A quarter of crop yearly water consumption or so, therefore be the excellent material on the water-saving lawn of planting.In Bermuda grass, ' C299 ' belongs to
One of best kind of ornamental value, but its drought resistance is slightly weak compared with other kinds, therefore improves the drought tolerance of Bermuda grass ' C299 ',
It has very important significance to the application and ornamental value of warm season turf.
Dehydrins (dehydrin) belong to II families of LEA-, are that one kind in plant has high heat stability, hydrophilic
The LEA protein (late embryogensis abundant proteins) of property, can be in Embryos Development of Plant later stage and inverse
Great expression under border, is widely present in plant kingdom.It can be expressed in the case where plant is in the adverse environmental factors such as arid, in adverse circumstance following table
Power and the plant stress-resistance ability reached has close ties, and the expression quantity in resistant plant is higher than the plant to adverse circumstance sensitivity.
It has thus been shown that the drought resisting of it and plant has close relationship.We are according to the method for heterologous clone from Bermuda grass ' C299 '
Obtain similar dehydrin gene with clone, Q-PCR the results show that the gene in ' C299 ' with the increase of degree of drought and
Expression quantity increases, it was demonstrated that the gene plays an important role during ' C299 ' drought resisting.
The encoding gene of Dehydrins is cloned from various plants to be come, including：It is arabidopsis, rice, barley, Oak Tree, red
Hai Lan etc..But the clone for turfgrass plant Dehydrins, expression pattern and protein sequence are unclear.At present, do not have any
With Bermuda grass ' C299 ' dehydrin protein structure and its relevant document report of coding gene sequence.
Invention content
It is an object of the invention to fill up the clone of Bermuda grass ' C299 ' Dehydrin-S genes, expression pattern analysis and
The blank of Bermuda grass ' C299 ' Dehydrin-S albumen, the present invention also provides a kind of nucleic acid sequence for encoding above-mentioned protein with
And the probe of the detection nucleic acid sequence；The invention discloses Bermuda grass ' C299 ' Dehydrin-S albumen and its nucleic acid sequence exist
Expression pattern in Bermuda grass ' C299 ' stress during drought stress, for from now on using technique for gene engineering to Dehydrin-S genes
The space-time characterisation of expression is regulated and controled, so as to for improve the drought-resistant stress ability of Bermuda grass and breeding work provide theory according to
According to very big application value.
On the one hand, the present invention provides the Bermuda grass ' C299 ' Dehydrins with drought stress response function and defencive function
Dehydrin-S albumen, the protein that the protein is made of the amino acid sequence as shown in SEQ ID NO.4；Or by
Amino acid sequence shown in SEQ ID NO.4 passes through substitution, lacks or adds one or several amino acid and with Bermuda grass
The protein of ' C299 ' Dehydrins Dehydrin-S protein specificities.The protein is in different drought coerces phase process in cell
In expression quantity there are larger differences.
Preferably, the protein for amino acid sequence shown in SEQ ID NO.4 by 1~50 amino acid missing,
It is inserted into and/or replaces or add sequence obtained from amino acid within 1~20 in C-terminal and/or N-terminal.
It is further preferred that the protein is 1~10 amino acid in amino acid sequence shown in SEQ ID NO.4 by property
The sequence that matter is similar or similar amino acid is replaced and formed.
On the other hand, the present invention provides a kind of nucleic acid sequences for encoding above-mentioned protein.
Preferably, the nucleic acid sequence is specially：(a) base sequence is as shown in SEQ ID NO.3 the 1st~495；Or
(b) there is the sequence of at least 70% homology with the nucleic acid shown in SEQ ID NO.3 the 1st~495；Or (c) can be with SEQ ID
The sequence that nucleic acid shown in NO.3 the 1st~495 is hybridized.
Preferably, the nucleic acid sequence is specially 1~90 in the nucleic acid sequence shown in SEQ ID NO.3 the 1st~495
Missing, insertion and/or the substitution of a nucleotide and 5 ' and/or 3 ' end addition 60 with inner nucleotide formed sequences.
In addition, the present invention also provides a kind of probe for detecting above-mentioned nucleic acid sequence, the probe is with above-mentioned nucleic acid
The nucleic acid molecules of 8~100 continuous nucleotides of sequence, the probe can be used in detection sample with the presence or absence of coding Bermuda grass
The nucleic acid molecules of ' C299 ' Dehydrin-S gene-correlations.
In the present invention, " DNA of separation ", " DNA of purifying " refer to that the DNA or segment are located under native state
It is separated in the sequence of its both sides, also refers to the DNA or segment and separated with the component with nucleic acid under native state, and
The protein with accompanying in cell separates.
In the present invention, term " Bermuda grass ' C299 ' Dehydrin-S albumen coded sequences ", which refers to coding, has Bermuda grass
The nucleotide sequence of the polypeptide of ' C299 ' Dehydrins Dehydrin-S protein actives, the as shown in SEQ ID NO.3 the 1st~495
Position nucleotide sequence and its degenerate sequence.The degenerate sequence refers to, positioned at the 1st~495 nucleosides shown in SEQ ID NO.3
In acid, there are one or multiple codons be encoded same amino acid degenerate codon replace after generation sequence.Due to
The degeneracy of codon, so with the 1st~495 nucleotide sequence homology shown in SEQ ID NO.3 down to about 70%
Degenerate sequence can also encode out the sequence shown in SEQ ID NO.4.The term further includes the nucleosides shown in SEQ ID NO.3
The nucleotide sequence of the homology of acid sequence at least 70%.
The term further includes the identical function that can encode natural Bermuda grass ' C299 ' Dehydrin-S albumen, SEQ ID
The variant form of sequence shown in NO.3.These variant forms include (but being not limited to)：Usually 1~90 nucleotide lacks
It loses, be inserted into and/or replace and be added to 60 at 5 ' and/or 3 ' ends with inner nucleotide.
In the present invention, term " Bermuda grass ' C299 ' Dehydrin-S albumen " refers to Bermuda grass ' C299 '
The polypeptide of sequence shown in the SEQ ID NO.4 of Dehydrin-S protein actives.The term, which further includes, to be had and natural Bermuda grass
' C299 ' Dehydrin-S albumen identical functions, SEQ ID NO.4 sequences variant form.These variant forms include (but
It is not limited to)：Usually the missing of 1~50 amino acid, insertion and/or substitution and C-terminal and/or N-terminal addition one
It is a or be amino acid within 20.For example, in the art, when being substituted with similar nature or similar amino acid, usually
The function of protein will not be changed.For another example, adding one or several amino acid in C-terminal and/or N-terminal will not generally also change
Become the function of protein.The term further includes the active fragment and reactive derivative of Bermuda grass ' C299 ' Dehydrin-S albumen.
The variant form of Bermuda grass ' C299 ' the Dehydrin-S albumen of the present invention includes：Homologous sequence, conservative variation
Body, allelic variant, natural mutation, induced mutants, can be with Bermuda grass ' C299 ' under high or low high stringency conditions
The encoded albumen of DNA of Dehydrin-S correlation DNA hybridizations and utilize Bermuda grass ' C299 ' Dehydrin-S albumen
More peptide or proteins that antiserum obtains.
In the present invention, " Bermuda grass ' C299 ' Dehydrin-S conservative variation's polypeptides " refer to shown in SEQ ID NO.4
Amino acid sequence compare, there are at most 10 amino acid to be replaced by amino acid with similar or analogous properties and form polypeptide.This
A little conservative variation's polypeptides are replaced preferably based on table 1 and are generated.
Table 1
| Initial residue | Representative substitution | Preferred substitution |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Invention further includes the analog of Bermuda grass ' C299 ' Dehydrin-S albumen or polypeptide.These analogs and Bermuda grass
The difference of ' C299 ' Dehydrin-S related polypeptides can be the difference on amino acid sequence or not influence repairing for sequence
It adorns formal difference or haves both at the same time.These polypeptides include natural or induction genetic variant.Induce variant can be with
Obtained by various technologies, such as generate random mutagenesis by radiating or being exposed to mutagens, can also by site-directed mutagenesis or
The technology of other known molecular biology.Analog is further included with residue (such as D- amino different from natural L-amino acids
Acid) analog and with it is non-naturally occurring or synthesis amino acid (such as β, gamma-amino acid) analog.Ying Li
Solution, polypeptide of the invention are not limited to the above-mentioned representative polypeptide enumerated.
Modification (not changing primary structure usually) form includes：The chemical derivative form of in vivo or in vitro polypeptide such as acetyl
Change or carboxylated.Modification further includes glycosylation, is carried out in the synthesis and processing of polypeptide or in further processing step such as those
Glycosylation modified and generation polypeptide.This modification can carry out glycosylated enzyme (such as mammal by the way that polypeptide is exposed to
Glycosylase or deglycosylation enzyme) and complete.Modified forms are further included with phosphorylated amino acid residue (such as phosphoric acid junket ammonia
Acid, phosphoserine, phosphothreonine) sequence.It further includes and is modified to improve its anti-proteolytic properties or optimization
The polypeptide of solubility property.
In the present invention, method analysis Bermuda grass ' C299 ' the Dehydrin-S genes of real-time fluorescence quantitative PCR can be used to produce
The expression pattern of object, that is, analyze Bermuda grass ' C299 ' Dehydrin-S genes presence or absence of the mRNA transcripts in cell and
Quantity.
It whether there is the detection side of Bermuda grass ' C299 ' Dehydrin-S related nucleotide sequences in present invention detection sample
Method, including being hybridized with above-mentioned probe with sample, then whether detection probe has occurred combination.The sample is PCR amplification
Product afterwards, wherein PCR amplification primer correspond to Bermuda grass ' C299 ' Dehydrin-S related nucleosides coding sequences, and can position
In the both sides of the coded sequence or centre.Primer length is generally 15~50 nucleotide.
In addition, Bermuda grass ' C299 ' Dehydrin-S nucleotide sequences according to the present invention and amino acid sequence, Ke Yi
Nucleic acid homology or express protein homology basis on, screen Bermuda grass ' C299 ' Dehydrin-S associated homologous gene or
Homologous protein.
In order to obtain with the dot matrix of Bermuda grass ' C299 ' Dehydrin-S related genes, dog tooth can be screened with DNA probe
Root ' C299 ' cDNA library, these probes are under low high stringency conditions, are used32P is relevant to Bermuda grass ' C299 ' Dehydrin-S
All or part is done obtained by radioactivity label.The cDNA library for being suitable for screening is the library from Bermuda grass ' C299 '.
The method for building the cDNA library from interested cell or tissue is that biology field is well-known.In addition,
Many such cDNA libraries can also be bought, such as purchased from Clontech, Stratagene, Palo Alto, Cal..This
Kind screening technique can identify and the nucleotide sequence of the relevant gene families of Bermuda grass ' C299 ' Dehydrin-S.
Bermuda grass ' C299 ' the Dehydrin-S associated nucleotides full length sequence or its segment of the present invention can usually use PCR
Amplification, recombination method or artificial synthesized method obtain.It, can be according to related nucleosides disclosed in this invention for PCR amplification method
Acid sequence, especially open reading frame sequence design primer, and with commercially available cDNA libraries or by known to those skilled in the art
Conventional method prepared by cDNA libraries as template, amplification and related sequence.When sequence is longer, it is often necessary to carry out two
Secondary or multiple PCR amplification, the segment for then again amplifying each time are stitched together by proper order.
After related sequence is obtained, it is possible to obtain related sequence in large quantity with recombination method.This is typically by it
Carrier is cloned into, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
It is introduced into protein sequence of the present invention in addition, can will be also mutated by chemical synthesis.
Other than being generated with recombination method, solid phase technique also can be used in the segment of albumen of the present invention, by direct synthetic peptide and
Produced (Stewart et al., (1969) Solid phase peptide synthssis, WH Freeman Co., San Francisco；
Merrifield J.(1963)J.Am Chem.Soc 85:2149-2154).In vitro synthetic protein can by hand or from
It is dynamic to carry out.It for example, can be with the 431A types peptide synthesizer (Foster City, CA) of Applied Biosystems from dynamic circuit connector
Into peptide.Each segment of chemical synthesis albumen of the present invention can be distinguished, then chemically connected point to generate overall length
Son.
Using Bermuda grass ' C299 ' the Dehydrin-S albumen of the present invention, by various conventional screening assays, can filter out
The substance to interact related to Bermuda grass ' C299 ' Dehydrin-S albumen or inhibitor and antagonist etc..Bermuda grass
' C299 ' as turfgrasses, application is extremely wide, and the market demand is also very big.The present invention clones Bermuda grass ' C299 ' for the first time
The coded sequence of important response protein and protective protein Dehydrin-S in stress during drought stress, and it is fixed in real time using fluorescence
The expression pattern of the method analysis Dehydrin-S genes of PCR is measured, to regulate and control Dehydrin-S using technique for gene engineering from now on
The spatial and temporal expression of gene so as to which to improve turf grass drought resistance, breeding of new variety aspect provides theoretical foundation, has very big
Application value.
Description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is Bermuda grass ' C299 ' the Dehydrin-S genes of the present invention and the hidden sub- grass (Cleistogenes of no awns
Songorica) Homology search (GAP) result of the nucleotide sequence of dehydrin gene mRNAs；
Fig. 2 is Bermuda grass ' C299 ' the Dehydrin-S albumen of the present invention and E. elongata (Lophopyrum
Elongatum) Homology search (FASTA) of the amino acid sequence of dehydrin albumen is as a result, wherein, identical amino acid is two
It is marked between a sequence with amino acid monocase；
Fig. 3 is expression quantity variation of Bermuda grass ' C299 ' the Dehydrin-S genes in stress during drought stress.
Fig. 4 is to be verified with Bermuda grass ' C299 ' Dehydrin positive monoclonal bacterial plaques PCR；
Fig. 5 is the plant of wild type and Bermuda grass ' C299 ' Dehydrin-S transgenic arabidopsis in different drought stress time
The opposite percentage of water loss of strain；
Fig. 6 is wild type and Dehydrin-S transgenic Arabidopsis plants drought stress Phenotypic Observations；
The plant relative conductivity value that Fig. 7 is wild type and Dehydrin-S transgenic arabidopsis in drought stress 4 days；
The plant Dehydrin genes that Fig. 8 is wild type and Dehydrin-S transgenic arabidopsis in drought stress 4 days
Express quantitative analysis.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail.Following embodiment will be helpful to the skill of this field
Art personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that this field
Those of ordinary skill for, without departing from the inventive concept of the premise, can also make it is several deformation and change
Into.
These belong to protection scope of the present invention.Such as Sambrook equimoleculars clone：Laboratory manual (New
York:
Cold Spring Harbor Laboratory Press, 1989) condition described in or according to manufacturer
Institute
It is recommended that condition.
Embodiment 1The clone of Bermuda grass ' C299 ' Dehydrin-S genes
1. the acquisition of vegetable material
Bermuda grass ' C299 ' leaf tissue is taken, for extracting RNA；
The extracting of 2.RNA
With " RNA prep pure plant total RNA extraction reagents box " extracted total RNA (Trizol：Invitrogen), first is used
The integrality of aldehyde denaturation gel electrophoresis identification RNA, then in spectrophotometer (Thermo Scientific NANODROP 1000
Spectrophotometer the purity and concentration of RNA is measured on)；
3. the full-length clone of gene
According to the isolated EST sequences that inhibition subtractive library (SSH) is established in Bermuda grass ' C299 ' stress during drought stress
Row and protein function annotation are as a result, obtain Bermuda grass ' C299 ' Dehydrin-S gene core segments.Using RACE methods
(SMARTerTMRACE cDNA Amplification Kit:Clonetech) carry out cDNA full-length clones, divide three phases into
Row：
(1) RT-PCR obtains gene intermediate segment
The RNA of extraction is subjected to reverse transcription (II 1st Strand cDNA Synthesis Kit of Prime Script:It is precious
Bioengineering (Dalian) Co., Ltd), using the first chain cDNA as template, utilize primer Dehydrin-S F (SEQ ID NO.1)
PCR is carried out with Dehydrin-S R (SEQ ID NO.2), amplification obtains 203bp segments, recycles and be connected to pMD18-T
On Simple vector carriers, by the use of RV-M and M13-47 as universal primer, using terminate object fluorescent marker (Big-Dye,
Perkin-Elmer, USA) method, be sequenced on ABI377 sequenators (Perkin-Elmer, USA), sequencing result
By carrying out BLAST (http in NCBI websites://blast.ncbi.nlm.nih.gov/) compare existing database
(GenBank), know its nucleic acid sequence and coding albumen grass hidden with known no awns, E. elongata, orchardgrass
(Bermudagrass), the homology of the Dehydrin genes of ragimillet is very high, it was initially believed that it is a Dehydrin base
Cause；
(2)3′RACE
Two wheel nest-type PRCs complete the amplification of 3 ' end sequences.
The first round：UPM+3’-GSP1(5′-GCGAACAGTCCGTGATAACTGTCTGTCA-3′)
Second wheel：NUP+3’-GSP2(5′-TCGTGTAACATGATAAGATGGTCAGCCA-3′)
UPM and NUP are provided for kit.3 ' RACE obtain the 3 ' end sequences of Bermuda grass ' C299 ' Dehydrin-S
(217bp), recycling, is connected on pMD18-T Simple vector carriers, by the use of RV-M and M13-47 as universal primer, adopts
With terminate object fluorescent marker (Big-Dye, Perkin-Elmer, USA) method, ABI377 sequenators (Perkin-Elmer,
USA it is sequenced on), sequencing result in NCBI websites by carrying out BLAST (http://
Blast.ncbi.nlm.nih.gov/) compare existing database (GenBank), know its nucleic acid sequence and coding albumen with
That knows is very high with the homology of orchardgrass Dehydrin genes without the hidden son grass of awns；
(3)5′RACE
Using 5 ' RACE ready cDNA as template, the amplification of 5 ' end sequences is completed by two wheel nest-type PRCs,
The first round：UPM+5’-GSP1(5′-ACCATCTTATCATGTTACACGAACGTCG-3′)
Second wheel：NUP+5’-GSP2(5′-GAACGTCGTGACAGACAGTTATCACGGA-3′)
UPM and NUP are provided for kit.5 ' RACE obtain 5 ' end sequences of Bermuda grass ' C299 ' Dehydrin-S genes
(643bp) is sequenced, the survey of sequence that will be obtained by above-mentioned 3 kinds of methods after recycling connection with method as above
Sequence result is spliced, and splicing sequence is submitted BLAST analyses, as a result proves what is newly obtained from Bermuda grass ' C299 '
Dehydrin genes are a relevant gene of dehydrin protein really, by the ORF Finding of sequencing result combination NCBI
(http://www.ncbi.nlm.nih.gov/gorf) prediction, it was found that Bermuda grass ' C299 ' Dehydrin-S genes rise
Beginning codon and terminator codon according to the sequence of acquisition, design specificity at initiation codon and terminator codon respectively
Primer ORF-F (5 '-ATGGAGCACCAGGGACAGTACGGC-3 '), ORF-R (5 '-TTAGTGCTGGCCGGGGAGCTTCTC-
3 ') PCR, is carried out by template of Bermuda grass ' C299 ' cDNA, amplification obtains 495bp Bermuda grass ' C299 ' Dehydrin-S albumen
Complete encoding sequence (SEQ ID NO.3).
Embodiment 2The sequence information and homology analysis of Bermuda grass ' C299 ' Dehydrin-S genes
Bermuda grass ' C299 ' the Dehydrin-S full length gene opening code-reading frames sequence of the present invention is 495bp, detailed sequence
See sequence shown in SEQ ID NO.3.The ammonia of Bermuda grass ' C299 ' Dehydrin-S albumen is derived according to opening code-reading frame sequence
Base acid sequence, totally 164 amino acid residues, molecular weight 16.7kDa, isoelectric point (pI) are 8.81, and detailed sequence is shown in SEQ ID
Sequence shown in NO.4；
The opening code-reading frame sequence of Bermuda grass ' C299 ' Dehydrin-S and its amino acid sequence for encoding albumen are used
BLAST programs are in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS
Nucleotide and protein homology search are carried out in translations+PDB+SwissProt+Superdate+PIR databases,
As a result, it has been found that the hidden sub- grass Dehydrin gene (accession number of it and no awns：FJ972827.1) with 82% on nucleotide level
The phase same sex, (Query as shown in Figure 1：The coding gene sequence of Bermuda grass ' C299 ' Dehydrin-S；Sbjct：Without the hidden son grass of awns
The mRNA sequence of Dehydrin)；On amino acid levels, it is with E. elongata Dehydrin gene (accession number：
AAC05922.1) also there are 64% consistency and 64% similitude, (Query as shown in Figure 2：Bermuda grass ' C299 '
The amino acid sequence of Dehydrin-S albumen；Sbjct：The amino acid sequence of E. elongata Dehydrin albumen).Thus may be used
See, the Dehydrin genes of Bermuda grass ' C299 ' Dehydrin-S genes and other known species are from nucleic acid or albumen
All there is higher homology in level.
Embodiment 3Bermuda grass ' C299 ' Dehydrin-S genes are in the different expression of drought stress different phase
1. the acquisition of material：To the blade of Bermuda grass ' C299 ' drought stress different phase (0 day, 5 days, 10 days) material into
Row sampling.It is put into liquid nitrogen at once after sample is wrapped respectively with aluminium platinum paper, is then transferred to storage in -80 DEG C of ultra low temperature freezers and treats
With；
The extraction of 2.RNA：Utilize RNA prep pure plant Total RNAs extractions (Trizol：Invitrogen)；Extract dog
Total serum IgE in root of the tooth ' C299 ' difference sample tissues；
The integrality of 3.RNA, purity, concentration determine：With plain agar sugar gel electrophoresis (gum concentration 1.2%； 0.5×
TBE electrophoretic buffers；150v, 15min) detection integrality, in electrophoretic band maximum rRNA brightness it is bright to should be Article 2 rRNA
Otherwise 1.5~2.0 times of degree represent the degradation of rRNA samples；Purity preferable RNA, A260/A280And A260/A230About 2.0
Left and right, with spectrophotometric determination OD values and calculates rna content；
The acquisition of 4.cDNA：Using the total serum IgE of 500ng as template, according to precious biotech firm TaKaRa PrimeScriptTM
It is spare that RT reagent Kit Perfect Real Time kits operating instruction carries out reverse transcription acquisition cDNA；
5. design specific primer analyzes gene in each organ and the expression in tissue to carry out real-time fluorescence quantitative PCR
Amount, according to Bermuda grass ' C299 ' the Dehydrin-S gene orders obtained, is designed for using primer-design software
The specific primer that Dehydrin-S gene quantifications are analyzed in Real-time PCR, primer qDHN F (5 '-
CGGAGGAAGAAGGGAATC-3 '), primer qDHN R (5 '-TCTCCTTGATCTTGTCCAT-3 '), reference gene is ribosomes
18S genes, primer be 18S-F (5 '-GTGACGGGTGACGGAGAATT-3 '), 18S-R (5 '-
GACACTAATGCGCCCGGTAT-3′)；
6. make the standard curve of target gene and reference gene：With EASY Dilution (kit offer) by standard
Product cDNA solution carries out gradient dilution, then respectively using the cDNA solution after dilution as template, with target gene and reference gene
Specific primer carry out Real-time PCR amplifications, draw solubility curve and standard curve after reaction；Analysis dissolving is bent
Can line, judges whether the solubility curve of target gene and reference gene obtains simple spike, to judge obtain list using the primer
One pcr amplification product；The appropriate dilutions multiple of template cDNA is determined by standard curve；
7. the Real time PCR of target gene in sample to be tested：Using first chain of cDNA of synthesis as template, point
Quantitative fluorescence analysis is not carried out with the primer amplified of target gene and internal reference gene, Real-time PCR reactions exist
It is carried out on 4 real-time quantitative instrument of BIO-RAD Chromo, reaction system is 20 μ L, and reaction is connect using three-step approach, 94 DEG C of denaturation 20s
40 cycles：94℃15s；58℃15s；72℃25s；Every time after the completion of amplification, solubility curve is done, to examine amplified production
Whether it is specifically to generate；
8. using 2-△△CtMethod makees relative quantitative assay, the results showed that Bermuda grass ' C299 ' adds with drought stress degree
Weight, Dehydrin-S expressions significantly rise (Fig. 3).The 5th day of drought stress, at the 10th day, the expression water of the gene
Flat is respectively 20.7 and 24.2 times of adjoining tree, it is significantly correlated to illustrate that the gene is responded with drought stress, when having apparent
Empty otherness.
Embodiment 4, Bermuda grass ' C299 ' Dehydrin-S genetic transformation model plant arabidopsis
(1) conversion carrier is built
Designed at initiation codon and terminator codon respectively specific primer Dehydrin_OFR-S (5 '-
AAGGATCCATGGAGCACCAGGGACAGTA-3 '), Dehydrin_ORF-A (5 '-
AACTAGTGTGCTGGCCGGGGAGCTT-3 ') and introduce in full length gene sequence both sides Bam HI and Spe I digestions position respectively
Point carries out PCR by template of Bermuda grass ' C299 ' cDNA.Recycling PCR product is simultaneously connected to pMD18-T Simple vector loads
On body, picking monoclonal bacterial plaque carries out PCR verifications.There are 2 members for Bermuda grass ' C299 ' Dehydrin gene families
(Dehydrin-L, Dehydrin-S), the relatively long segment of 500bp or so is Dehydrin-S (Fig. 4, swimming lane 4-6), and extraction is positive
Property clone bacterium solution plasmid.Target fragment plasmid and PHB binary transformation vectors are subjected to BamHI and Spe I double digestions, recycle enzyme
PHB carriers after cutting and Dehydrin-S segments, using T4 ligases in 16 DEG C of water-baths by Dehydrin-S and PHB carriers
Connection builds conversion carrier overnight, and carrier is converted Agrobacterium GV3101.
(2) Dehydrin-S arabidopsis thaliana transformations
1. shake Agrobacterium in advance：Positive monoclonal is chosen to 25ml Kan containing 50mg/L, 50mg/L gentamicins, 25mg/L
In the YEP fluid nutrient mediums of Rif, 28 DEG C, 200rpm shakes bacterium for 24 hours；
2. spread cultivation Agrobacterium：By the Agrobacterium bacterium solution shaken in advance with 1:100 spread cultivation to the resistance YEP culture mediums of Kan containing 400mL
In, 28 DEG C, 200rpm, 13-16h is cultivated, is cultivated to absorbance OD600Reach and bacterium is received between 1.5-2.0, it is 23 DEG C to receive bacterium condition,
5000rpm, 8min；
3. transformed plant：(need to cut off on the day of conversion the previous day or conversion silique all on plant and it is in full bloom with
And the little Hua to show money or valuables one carries unintentionally) the 1/2MS solution that 500mL contains 5% sucrose is prepared, the Agrobacterium of collection is precipitated with a small amount of MS solution and is hanged
It rises, shakes up, the Silwet L-77 and 10 μ L 6-BA (mother liquor 1mg/ of 0.04% (v/v) are added in into remaining sucrose solution
ML), stir evenly, by the two mixing before conversion, base of the plant and inflorescence are immersed in 50s in bacterium solution, taking-up drains bacterium solution, is put into
In disposable plastic bag, sealing, moisturizing.After by all plant transformations, flight data recorder on cover is protected from light culture for 24 hours.It takes out later
Plant places erect plants, pours Aquaponic, ensures that plant moisture is sufficient.
(3) screening of transgenic positive strain
Plant after conversion sowing after silique is all ripe, one is placed at room temperature in the desiccation culture ware for being lined with filter paper
Week make seed all dry, sieve filter seed with the stainless steel of 50 mesh later, remove silique, collect transgenosis T0 for seed simultaneously
It is seeded in hole tray, Resistance of Seedling screening is carried out with 0.05% (v/v) glyphosate, obtain T1 for transfer-gen plant, it is lasting to screen
Until T3 is obtained for homozygote transfer-gen plant.
Embodiment 5, arabidopsis Dehydrin-S transfer-gen plant drought stress Physiologic Studies
(1) arabidopsis Dehydrin-S transfer-gen plants percentage of water loss detects
Arabidopsis wild type and Dehydrin-S rotaring gene plant blade 0.5g are sheared, is positioned in aluminium box, in not same order
Section (0-12h) weighs leaf weight, calculates different plant percentages of water loss.Over time, Dehydrin-S transfer-gen plants dehydration
Rate is significantly lower than adjoining tree about 5-12% (Fig. 5), illustrates that Dehydrin-S genes have plant under in vitro drought condition
Better water tariff collection effect.
(2) arabidopsis Dehydrin-S transfer-gen plants drought stress Phenotypic Observation
Arabidopsis wild type and Dehydrin-S transfer-gen plants are planted in 22 DEG C of temperature, 6000 lux of light intensity, light
In the dark growth cabinet in 16 hours periods illumination/8 hour, it is carried out at the same time drought stress processing and carries out phenotype paired observation.
During drought stress 4 days, wild type has significant difference with Dehydrin-S transgenic Arabidopsis plants phenotype；Wild-type plant
Blade dries up wilting, and scape lodges；Dehydrin-S rotaring gene plant blade wilting degree is smaller, and scape is upright, plant
Growing state is also superior to wildtype Arabidopsis thaliana (Fig. 6).It is more preferable to illustrate that Dehydrin-S genetically modified plants have under drought condition
Drought resistance.
(3) arabidopsis Dehydrin-S transfer-gen plants conductivity detects
Arabidopsis wild type and the Dehydrin-S transfer-gen plants drought stress blade 0.2g of 4 days are sheared, is collected in
In 50ml centrifuge tubes, 20ml deionized waters are added in, is positioned on shaking table for 24 hours, measures conductivity initial value；It is put into high-pressure sterilizing pot
In (121 DEG C) handle 15 minutes, measure conductivity end value.Sample relative conductivity value is calculated than final value with initial value.It is dry
Wild-type plant relative conductivity value of the drought stress after 4 days is the relative conductivity of 87.7%, Dehydrin-S transfer-gen plants
Be worth is 68.4%, hence it is evident that less than WT lines (Fig. 7).Illustrate that Dehydrin-S genetically modified plants are thin under drought stress conditions
Born of the same parents' extent of injury is less than WT lines.
(3) wild type and transgenic Arabidopsis plants Dehydrin-S gene expression differences under drought stress conditions
Arabidopsis wild type and the Dehydrin-S transfer-gen plants drought stress blade 0.2g of 4 days are sheared, is pressedEmbodiment 3
Middle method extraction RNA, it prepares cDNA and carries out Real-time PCR Analysis.Dehydrin-S genes are determined in Real-time PCR
The specific primer of amount analysis for qDHN F (5 '-CGGAGGAAGAAGGGAATC-3 '), primer qDHN R (5 '-
TCTCCTTGATCTTGTCCAT-3 '), reference gene for intend south actin2 genes, primer be act-F (5 '-
CTTGCACCAAGCAGCATGAA-3 '), act-R (5 '-CCGATCCAGACACTGTACTTCCTT-3 ').Using 2-△△CtMethod is made
Relative quantitative assay, the results showed that the expression quantity of Dehydrin-S is higher in transgenic arabidopsis of the drought stress after 4 days, is interior
Join gene actin2 19.5 times are 1675 times (Fig. 8) of wild-type plant.Show that Dehydrin-S is not planted in wild type
It is expressed in strain.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (3)
1. a kind of protein that amino acid sequence by as shown in SEQ ID NO.4 forms.
2. a kind of nucleic acid for encoding protein described in claim 1.
3. nucleic acid as claimed in claim 2, it is characterized in that, the nucleic acid sequence is specially：
Base sequence is as shown in SEQ ID NO.3 the 1st~495.
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