CN105017394B - Bermuda grass ' Tifway ' dehydrin protein Dehydrin-S and its encoding gene and probe - Google Patents
Bermuda grass ' Tifway ' dehydrin protein Dehydrin-S and its encoding gene and probe Download PDFInfo
- Publication number
- CN105017394B CN105017394B CN201510179091.4A CN201510179091A CN105017394B CN 105017394 B CN105017394 B CN 105017394B CN 201510179091 A CN201510179091 A CN 201510179091A CN 105017394 B CN105017394 B CN 105017394B
- Authority
- CN
- China
- Prior art keywords
- dehydrin
- tifway
- protein
- bermuda grass
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of Bermuda grass ' Tifway ' 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 ' Tifway ' 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 ' Tifway ' 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 ' Tifway ' 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 ' Tifway ' 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 Turf characteristic 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, the Bermuda grass kinds (as ' Tifway ') of some drought resistings annual rainfall 250mm with
Under remain to well grow, therefore be the excellent material on the water-saving lawn of planting.
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.
Bermuda grass ' Tifway ' is one of kind the most drought-enduring in Bermuda grass.Pass through protein immunoblot and gene expression
Analysis, determines that Dehydrins are drought-enduring closely related with ' Tifway '.We are inhibited by establishing ' Tifway ' arid cDNA of 10 days
Subtractive library therefrom obtains the est sequence of a new gene, it with report barley dehydrin protein DHN4 gene similitudes
It is high, show the gene code dehydrin protein.RT-PCR the results show that the gene with the increase of degree of drought expression quantity
Raising, the expression quantity in " Tifway " is significantly higher than the expression quantity in arid sensitive Bermuda grass kind, de- this show this
Water plain gene plays an important role during Bermuda grass 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 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 ' Tifway ' Dehydrin-S genes, expression pattern analysis with
And the blank of Bermuda grass ' Tifway ' Dehydrin-S albumen, the present invention also provides a kind of nucleic acid sequences for encoding above-mentioned protein
Arrange and detect the probe of the nucleic acid sequence；The invention discloses Bermuda grass ' Tifway ' Dehydrin-S albumen and its nucleic acid
Expression pattern of the sequence in Bermuda grass ' Tifway ' stress during drought stress, to utilize technique for gene engineering pair from now on
The space-time characterisation of Dehydrin-S gene expressions is regulated and controled, so as to improve the drought-resistant stress ability of Bermuda grass and breeding work
Theoretical foundation is provided, there is very big application value.
On the one hand, the present invention provides the Bermuda grass ' Tifway ' dehydrations with drought stress response function and defencive function
Plain 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 ' Tifway ' Dehydrins Dehydrin-S protein specificities.The protein is in different drought coerces phase process thin
There are larger differences for expression quantity in born of the same parents.
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 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 ' Tifway ' 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 ' Tifway ' Dehydrin-S albumen coded sequences ", which refers to coding, has Bermuda grass
The nucleotide sequence of the polypeptide of ' Tifway ' Dehydrins Dehydrin-S protein actives, as shown in SEQ ID NO.3 the 1st~
495 nucleotide sequences and its degenerate sequence.The degenerate sequence refers to, positioned at the 1st~495 core shown in SEQ ID NO.3
In thuja acid, there are one or multiple codons be encoded same amino acid degenerate codon replace after generation sequence.By
In 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 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 ' Tifway ' 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 ' Tifway ' Dehydrin-S albumen " refers to Bermuda grass ' Tifway '
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
' Tifway ' Dehydrin-S albumen identical functions, SEQ ID NO.4 sequences variant form.These variant forms include
(but being not limited to)：Usually the missing of 1~50 amino acid, insertion and/or substitution and add in C-terminal and/or N-terminal
Add one or for amino acid within 20.For example, in the art, when being substituted with similar nature or similar amino acid,
The function of protein is not usually changed.For another example, one or several amino acid are added generally also not in C-terminal and/or N-terminal
The function of protein can be changed.The term further includes the active fragment of Bermuda grass ' Tifway ' Dehydrin-S albumen and activity is spread out
Biology.
The variant form of Bermuda grass ' Tifway ' the Dehydrin-S albumen of the present invention includes：Homologous sequence, conservative become
Allosome, allelic variant, natural mutation, induced mutants, can be with Bermuda grass ' Tifway ' under high or low high stringency conditions
The encoded albumen of DNA of Dehydrin-S correlation DNA hybridizations and utilize Bermuda grass ' Tifway ' Dehydrin-S albumen
More peptide or proteins that antiserum obtains.
In the present invention, " Bermuda grass ' Tifway ' Dehydrin-S conservative variation's polypeptides " refer to and SEQ ID NO.4 institutes
The amino acid sequence shown is compared, and has at most 10 amino acid to be replaced by amino acid with similar or analogous properties and form polypeptide.
These 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 ' Tifway ' Dehydrin-S albumen or polypeptide.These analogs and dog tooth
The difference of root ' Tifway ' Dehydrin-S related polypeptides can be the difference on amino acid sequence or not influence sequence
Modified forms on difference or have both at the same time.These polypeptides include natural or induction genetic variant.Induce variant
It can be obtained by various technologies, such as generate random mutagenesis by radiating or being exposed to mutagens, can also pass through direct mutagenesis
The technology of method or other known molecular biology.Analog is further included with residue (such as D- ammonia different from natural L-amino acids
Base 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 ' Tifway ' the Dehydrin-S genes of real-time fluorescence quantitative PCR can be used
The expression pattern of product, that is, analyze presence of the mRNA transcripts in cell of Bermuda grass ' Tifway ' Dehydrin-S genes with
No and quantity.
It whether there is the detection of Bermuda grass ' Tifway ' 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 that PCR expands
Product after increasing, wherein PCR amplification primer correspond to Bermuda grass ' Tifway ' Dehydrin-S related nucleosides coding sequences, and
The both sides or centre of the coded sequence can be located at.Primer length is generally 15~50 nucleotide.
It, can be in addition, Bermuda grass ' Tifway ' Dehydrin-S nucleotide sequences according to the present invention and amino acid sequence
On the homology basis of nucleic acid homology or expression protein, Bermuda grass ' Tifway ' Dehydrin-S associated homologous bases are screened
Cause or homologous protein.
In order to obtain with the dot matrix of Bermuda grass ' Tifway ' Dehydrin-S related genes, dog can be screened with DNA probe
Root of the tooth ' Tifway ' cDNA library, these probes are under low high stringency conditions, with 32P to Bermuda grass ' Tifway ' Dehydrin-S
Relevant all or part is done obtained by radioactivity label.The cDNA library for being suitable for screening is from Bermuda grass ' Tifway '
Library.The method for building the cDNA library from interested cell or tissue is that biology field is well-known
's.In addition, many such cDNA libraries can also be bought, such as purchased from Clontech, Stratagene, Palo Alto,
Cal..This screening technique can identify and the nucleotides sequence of the relevant gene families of Bermuda grass ' Tifway ' Dehydrin-S
Row.
Bermuda grass ' Tifway ' the Dehydrin-S associated nucleotides full length sequence or its segment of the present invention can usually be used
PCR amplification method, recombination method or artificial synthesized method obtain.It, can be according to related core disclosed in this invention for PCR amplification method
Nucleotide sequence, especially open reading frame sequence design primer, and with commercially available cDNA libraries or by those skilled in the art
CDNA libraries prepared by the conventional method known expand as template and obtain related sequence.When sequence is longer, it is often necessary to carry out
Twice 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 ' Tifway ' the Dehydrin-S albumen of the present invention, by various conventional screening assays, can screen
Go out the substance to interact related to Bermuda grass ' Tifway ' Dehydrin-S albumen or inhibitor and antagonist etc..
Bermuda grass ' Tifway ' is as turfgrasses, and application is extremely wide, and the market demand is also very big.The present invention is for the first time
Clone the code sequence of the important response protein and protective protein Dehydrin-S in Bermuda grass ' Tifway ' stress during drought stress
Row, and the expression pattern of the method analysis Dehydrin-S genes using fluorescence real-time quantitative PCR, to utilize genetic engineering from now on
The spatial and temporal expression of technical regulation Dehydrin-S genes, 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 ' Tifway ' 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 ' Tifway ' 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 ' Tifway ' the Dehydrin-S genes in stress during drought stress
Fig. 4 is to be verified with Bermuda grass ' Tifway ' Dehydrin positive monoclonal bacterial plaques PCR；
Fig. 5 is for wild type with Bermuda grass ' Tifway ' Dehydrin-S transgenic arabidopsis in different drought stress time
Plant is with respect to percentage of water loss；
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 further explained.These embodiments are merely to illustrate the present invention and do not have to
In limiting the scope of the invention.Test method without specific conditions in the following example, usually according to normal condition, such as
Sambrook equimoleculars are cloned：Laboratory manual (New York:Cold Spring Harbor Laboratory Press,
1989) condition described in or according to the normal condition proposed by manufacturer.
Embodiment 1, Bermuda grass ' Tifway ' Dehydrin-S genes clone
1. the acquisition of vegetable material
Bermuda grass ' Tifway ' 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 NANODROP1000
Spectrophotometer the purity and concentration of RNA is measured on)；
3. the full-length clone of gene
According to the isolated EST that inhibition subtractive library (SSH) is established in Bermuda grass ' Tifway ' stress during drought stress
Sequence and protein function annotation are as a result, obtain Bermuda grass ' Tifway ' 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 leads to
It crosses and carries 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 gene；
(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 ' Tifway ' Dehydrin-S
(228bp), 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/)
Existing database (GenBank) is compared, knows its nucleic acid sequence and coding albumen grass hidden with known no awns and orchardgrass
The homology of Dehydrin genes is very high；
(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 ' Tifway ' Dehydrin-S genes
It arranges (641bp), is sequenced after recycling connection with method as above, by the sequence obtained by above-mentioned 3 kinds of methods
Sequencing result is spliced, and splicing sequence is submitted BLAST analyses, as a result proves what is newly obtained from Bermuda grass ' Tifway '
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 ' Tifway ' 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 ' Tifway ' cDNA, amplification obtains 495bp Bermuda grass ' Tifway ' Dehydrin-S eggs
White complete encoding sequence (SEQ ID NO.3).
Embodiment 2, Bermuda grass ' Tifway ' Dehydrin-S genes sequence information and homology analysis
Bermuda grass ' Tifway ' the Dehydrin-S full length gene opening code-reading frames sequence of the present invention is 495bp, detailed sequence
Row are shown in sequence shown in SEQ ID NO.3.Bermuda grass ' Tifway ' Dehydrin-S albumen is derived according to opening code-reading frame sequence
Amino acid sequence, totally 164 amino acid residues, molecular weight 16.7kDa, isoelectric point (pI) is 8.81, and detailed sequence is shown in SEQ
Sequence shown in ID NO.4；
The opening code-reading frame sequence of Bermuda grass ' Tifway ' Dehydrin-S and its amino acid sequence for encoding albumen are used
Blast program is 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 83% on nucleotide level
The phase same sex, (Query as shown in Figure 1：The coding gene sequence of Bermuda grass ' Tifway ' Dehydrin-S；Sbjct：Without the hidden son of awns
The mRNA sequence of careless Dehydrin)；On amino acid levels, it is with E. elongata Dehydrin gene (accession number：
AAC05922.1) also there are 68% consistency and 68% similitude, (Query as shown in Figure 2：Bermuda grass ' Tifway '
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 ' Tifway ' Dehydrin-S genes and other known species are from nucleic acid or albumen
All there is higher homology in level.
Embodiment 3, Bermuda grass ' Tifway ' Dehydrin-S genes drought stress different phase different expression
1. the acquisition of material：To Bermuda grass ' Tifway ' drought stress different phase (0 day, 5 days, 10 days, 15 days) material
Blade be sampled.It is put into liquid nitrogen at once after sample is wrapped respectively with aluminium platinum paper, is then transferred to -80 DEG C of ultra low temperature freezers
Middle stored for future use；
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 ' Tifway ' 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 should be Article 2 rRNA brightness
1.5~2.0 times, otherwise represent rRNA samples degradation；Purity preferable RNA, A260/A280And A260/A230About 2.0 is left
The right side 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 ' Tifway ' 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 are 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 ' Tifway ' is with drought stress degree
It aggravates, Dehydrin-S expressions significantly rise (Fig. 3).The 5th day of drought stress, at the 10th day, the 15th day, the gene
Expression be respectively 4.83,272.2 and 717 times of adjoining tree, illustrate that the gene and drought stress response are significantly correlated,
With apparent spatio-temporal difference.
Embodiment 4, Bermuda grass ' Tifway ' 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 ') Bam HI and Spe I restriction enzyme sites and in full length gene sequence both sides are introduced respectively, using Bermuda grass ' Tifway ' cDNA as mould
Plate carries out PCR.Recycling PCR product is simultaneously connected on pMD18-T Simple vector carriers, and picking monoclonal bacterial plaque carries out
PCR is verified.Bermuda grass ' Tifway ' Dehydrin gene families there are 2 members (Dehydrin-L, Dehydrin-S),
The relatively long segment of 500bp or so is Dehydrin-S (Fig. 4, swimming lane 5-7), extracts positive colony bacterium solution plasmid.By target fragment
Plasmid carries out BamHI and Spe I double digestions with PHB binary transformation vectors, recycles the PHB carriers after digestion and Dehydrin-S pieces
Dehydrin-S, using T4 ligases in 16 DEG C of water-baths with PHB carriers connect and builds conversion carrier overnight by section, and by carrier
Convert 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 Rif
YEP fluid nutrient mediums in, 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 5-18% (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 better than wildtype Arabidopsis thaliana (Fig. 6).It is better 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 70.0%, 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 gene quantifications in Real-time PCR
The specific primer of analysis be 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 11.72 times are 1711 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510179091.4A CN105017394B (en) | 2015-04-15 | 2015-04-15 | Bermuda grass ' Tifway ' dehydrin protein Dehydrin-S and its encoding gene and probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510179091.4A CN105017394B (en) | 2015-04-15 | 2015-04-15 | Bermuda grass ' Tifway ' dehydrin protein Dehydrin-S and its encoding gene and probe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105017394A CN105017394A (en) | 2015-11-04 |
| CN105017394B true CN105017394B (en) | 2018-07-03 |
Family
ID=54407738
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510179091.4A Active CN105017394B (en) | 2015-04-15 | 2015-04-15 | Bermuda grass ' Tifway ' dehydrin protein Dehydrin-S and its encoding gene and probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105017394B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106432451B (en) * | 2016-11-25 | 2019-09-06 | 上海交通大学 | One kind can be used as the protectant albumen of enzyme activity |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101297039A (en) * | 2005-08-03 | 2008-10-29 | 斯瓦米那坦研究基金会 | Dehydrin gene from AVICENNIA MARINA responsible for conferring salt tolerance in plants |
-
2015
- 2015-04-15 CN CN201510179091.4A patent/CN105017394B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101297039A (en) * | 2005-08-03 | 2008-10-29 | 斯瓦米那坦研究基金会 | Dehydrin gene from AVICENNIA MARINA responsible for conferring salt tolerance in plants |
Non-Patent Citations (1)
| Title |
|---|
| 草坪草抗旱生理及相关基因的分析;胡龙兴;《中国博士学位论文全文数据库农业科技辑》;20101015(第10期);D048-26 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105017394A (en) | 2015-11-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102099476A (en) | Compositions and methods for improving plants | |
| KR20120101542A (en) | Gene dro1 controlling deep-rooted characteristics of plant and utilization of same | |
| CN105037514B (en) | Bermuda grass ' Tifway ' dehydrin protein Dehydrin-L and its encoding gene and probe | |
| CN108603198A (en) | Manipulate the composition and method of development of plants | |
| CN105837670B (en) | Afriocan agapanthus auxin response factor ApARF2 and its encoding gene and probe | |
| CN105440114B (en) | Eggplant cryptochrome gene SmCRY1 and application thereof | |
| CN105440116B (en) | Eggplant cryptochrome gene SmCRY2 and application thereof | |
| CN104745560B (en) | Eggplant chalcone synthase SmCHS1 albumen and its encoding gene | |
| CN108728450A (en) | A gene C dERF1 significantly induced by low temperature and its application in Bermuda grass | |
| CN105017394B (en) | Bermuda grass ' Tifway ' dehydrin protein Dehydrin-S and its encoding gene and probe | |
| CN105001313B (en) | Bermuda grass ' C299 ' dehydrin protein Dehydrin-S and its encoding gene and probe | |
| CN105037515B (en) | Bermuda grass ' C299 ' dehydrin protein Dehydrin-L and its encoding gene and probe | |
| CN103483437A (en) | Protein of key gene ApCO for photoperiod and flowering pathway of agapanthus praecox ssp.orientalis as well as coding gene and probe of gene | |
| CN104745561B (en) | Eggplant enzyme, namely chalcone isomerase SmCHI albumen and its encoding gene | |
| CN104961815B (en) | Afriocan agapanthus auxin signal transcription modulin Aux/IAA1 and its encoding gene and probe | |
| CN105085642B (en) | Afriocan agapanthus auxin signal transcription modulin Aux/IAA2 and its encoding gene and probe | |
| CN104961814B (en) | Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 and its encoding gene and probe | |
| CN102978194A (en) | Tulip chalcone isomerase TfCHI protein and coding gene thereof and probe | |
| CN103342741B (en) | Agapanthus praecox gibberellin receptor APGID1b protein, and encoding gene and probe thereof | |
| CN106946986B (en) | Afriocan agapanthus Y2SK2 type dehydrin protein and its encoding gene and probe | |
| CN106967724B (en) | Afriocan agapanthus SK3Type dehydrin protein and its encoding gene and probe | |
| CN107619436B (en) | Stress-resistant protein and coding gene thereof | |
| CN102533788B (en) | EADT1 gene capable of improving drought resistance of rice in growth period, coding sequence and application | |
| CN106811453B (en) | African agapanthus cathepsin B, coding gene and probe thereof, and application of African agapanthus cathepsin B | |
| CN103288942A (en) | African agapanthus flowering gene ApFT protein and coding gene and probe thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |