CN105037514A

CN105037514A - Bermuda grass 'Tifway' dehydrin-L, encoding gene and probe thereof

Info

Publication number: CN105037514A
Application number: CN201510179177.7A
Authority: CN
Inventors: 周鹏; 安渊; 吕爱敏; 张荻; 李姣姣
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2015-04-15
Filing date: 2015-04-15
Publication date: 2015-11-11
Anticipated expiration: 2035-04-15
Also published as: CN105037514B

Abstract

The invention relates to bermuda grass 'Tifway' dehydrin-L, an encoding gene and a probe thereof. The dehydrin-L is a protein represented as (a) or (b), wherein (a) is a protein composed of the amino acid sequence represented as the Seq ID No.4 and (b) is a protein which is a derivative from the protein (a), is obtained through substitution, deficiency or addition of one or more amino acids and has the bermuda grass 'Tifway' dehydrin-L activity. The invention also provides a nucleic acid sequence for encoding the proteins and the probe for detecting the nucleic acid. In the invention, adversity stress physiological response, such as drought resistance and the like, of the bermuda grass 'Tifway' are regulated through genetic engineering technologies, thereby improving the drought resistance of turfgrass. The invention provides theoretical evidence for molecular breeding and has a huge application value.

Description

Bermuda grass ' Tifway ' dehydrin protein Dehydrin-L and encoding gene thereof and probe

Technical field

One important protected protein dehydrin protein Dehydrin-L in Bermuda grass of the present invention ' Tifway ' drought stress response process and encoding gene thereof and probe, be specifically related to a kind of Bermuda grass ' Tifway ' dehydrin protein Dehydrin-L and encoding gene thereof and probe.

Background technology

Bermuda grass (Cynodondactylon) is very important warm season turf, for Gramineae per nnial herb, there is flourishing rhizome and stolon, the advantages such as fast growth, regenerative power are strong, one-tenth fast, heat-resisting, the resistance to trample in level ground, quality is very thin, color and luster is good, are widely used in playground, esplanade, park and soil and slope protection lawn etc. at home and abroad.Therefore be one of grass seeds that in warm season turf, Turf characteristic is high, the most most widely used, enjoy the title of warm season turf " main cultivated orages ", have society, economy and the ecological value.The drought-resistant ability of Bermuda grass is very strong, year, evapotranspiration was lower than 400mm, only about 1/4th of the farm crop such as corn, wheat yearly water consumption, the Bermuda grass kind (as ' Tifway ') of some drought resisting still can well grow at below annual rainfall 250mm, is therefore the excellent material on planting water saving lawn.

Dehydrins (dehydrin) belongs to LEA-II family, that one in plant materials has high heat stability, hydrophilic lea protein (lateembryogensisabundantproteins), can under Embryos Development of Plant later stage and adverse circumstance great expression, be extensively present in vegitabilia.It is expressed under being in the adverse environmental factors such as arid plant, and its power expressed under adverse circumstance and plant stress-resistance ability have close ties, and the expression amount in resistant plant is higher than the plant to adverse circumstance sensitivity.Show thus, the drought resisting of it and plant has close relationship.

Bermuda grass ' Tifway ' is one of kind the most drought-enduring in Bermuda grass.By protein immunoblot and gene expression analysis, determine that Dehydrins and ' Tifway ' are drought-enduring closely related.We suppress subtractive library by setting up ' Tifway ' arid cDNA of 10 days, and therefrom obtain the est sequence of a new gene, it is high with the barley dehydrin protein DHN4 gene similarity of report, shows this genes encoding dehydrin protein.RT-PCR result proves, along with the increase of degree of drought, expression amount raises this gene, expression amount in " Tifway " is significantly higher than the expression amount in arid responsive Bermuda grass kind, this show this dehydrin gene and plays an important role in Bermuda grass drought resisting process.

The encoding gene of Dehydrins clones out from various plants, comprising: Arabidopis thaliana, paddy rice, barley, Oak Tree, Rhizophora stylosa etc.But the clone of turfgrass plant Dehydrins, expression pattern and protein sequence be it be unclear that.At present, any bibliographical information relevant to Bermuda grass dehydrin protein structure and coding gene sequence thereof is not had.

Summary of the invention

The object of the invention is to fill up the blank of the clone of Bermuda grass ' Tifway ' Dehydrin-L gene, expression pattern analysis and Bermuda grass ' Tifway ' Dehydrin-L albumen, present invention also offers a kind of above-mentioned nucleic acid sequences to proteins and detect the probe of described nucleotide sequence of encoding; The invention discloses Bermuda grass ' Tifway ' Dehydrin-L albumen and the expression pattern of nucleotide sequence in Bermuda grass ' Tifway ' stress during drought stress thereof, regulate and control for utilizing the space-time characterisation of genetic engineering technique to Dehydrin-L genetic expression from now on, thus be that the raising drought-resistant ability of coercing of Bermuda grass and breeding work provide theoretical foundation, there is very large using value.

On the one hand, the invention provides Bermuda grass ' Tifway ' the Dehydrins Dehydrin-L albumen with drought stress response function and defencive function, the protein that described protein is made up of the aminoacid sequence such as shown in SEQIDNO.4; Or by the aminoacid sequence shown in SEQIDNO.4 through replacing, lacking or add one or several amino acid and there is the protein of Bermuda grass ' Tifway ' Dehydrins Dehydrin-L protein specificity.There is larger difference in the expression amount that this protein is coerced in phase process in cell in different drought.

Preferably, described protein for aminoacid sequence shown in SEQIDNO.4 is through 1 ~ 50 amino acid whose disappearance, insertion and/or replacement, or within C-terminal and/or N-terminal add 1 ~ 20 amino acid and the sequence that obtains.

Further preferred, described protein for 1 ~ 10 amino acid in aminoacid sequence shown in SEQIDNO.4 replace by the similar or close amino acid of character and the sequence that formed.

On the other hand, the invention provides the above-mentioned nucleic acid sequences to proteins of a kind of coding.

Preferably, described nucleotide sequence is specially: (a) base sequence is as shown in SEQIDNO.3 1st ~ 543; Or the nucleic acid shown in (b) and SEQIDNO.3 1st ~ 543 has the sequence of the homology of at least 70%; Or (c) can carry out the sequence of hybridizing with the nucleic acid shown in SEQIDNO.3 1st ~ 543.

Preferably, described nucleotide sequence is specially the disappearance of 1 ~ 90 Nucleotide in the nucleotide sequence shown in SEQIDNO.3 1st ~ 543, insertion and/or replacement, and 5 ' and/or 3 ' sequence of being formed with inner nucleotide of end interpolation 60.

In addition, present invention also offers a kind of probe detecting above-mentioned nucleotide sequence, described probe is the nucleic acid molecule with above-mentioned nucleotide sequence 8 ~ 100 continuous nucleotides, and this probe can be used for detecting the nucleic acid molecule that whether there is coding Bermuda grass ' Tifway ' Dehydrin-L gene-correlation in sample.

In the present invention, " DNA of separation ", " DNA of purifying " refer to, the sequence that this DNA or fragment have been arranged in its both sides from native state is separated, also refer to that this DNA or fragment with under native state are separated with the component of nucleic acid, and separate with the protein accompanied in cell.

In the present invention, term " Bermuda grass ' Tifway ' Dehydrin-L albumen coded sequence " refers to that coding has the nucleotide sequence of the polypeptide of Bermuda grass ' Tifway ' Dehydrins Dehydrin-L protein-active, 1st ~ 543 nucleotide sequences as shown in SEQIDNO.3 and degenerate sequence thereof.This degenerate sequence refers to, is arranged in the Nucleotide of 1st ~ 543 shown in SEQIDNO.3, have one or more codon replace by the degenerate codon of same amino acid of encoding after produce sequence.Due to the degeneracy of codon, the sequence shown in SEQIDNO.4 so the degenerate sequence being low to moderate about 70% with the nucleotide sequence homology of 1st ~ 543 shown in SEQIDNO.3 also can be encoded out.This term also comprises the nucleotide sequence with the homology at least 70% of the nucleotide sequence shown in SEQIDNO.3.

This term also comprises the variant form of sequence shown in the identical function of natural Bermuda grass ' Tifway ' Dehydrin-L albumen of encoding, SEQIDNO.3.These variant forms comprise (but being not limited to): be generally the disappearance of 1 ~ 90 Nucleotide, insertion and/or replacement, and 5 ' and/or 3 ' end be added to 60 with inner nucleotide.

In the present invention, term " Bermuda grass ' Tifway ' Dehydrin-L albumen " refer to have Bermuda grass ' Tifway ' Dehydrin-L protein-active SEQIDNO.4 shown in the polypeptide of sequence.This term also comprise have with natural Bermuda grass ' Tifway ' Dehydrin-L albumen identical function, the variant form of SEQIDNO.4 sequence.These variant forms comprise (but being not limited to): be generally 1 ~ 50 amino acid whose disappearance, insertion and/or replacement, and add one or amino acid within being 20 at C-terminal and/or N-terminal.Such as, in the art, when replacing with similar nature or similar amino acid, the function of protein can not usually be changed.Again such as, add at C-terminal and/or N-terminal the function that or several amino acid also can not change protein usually.This term also comprises active fragments and the reactive derivative of Bermuda grass ' Tifway ' Dehydrin-L albumen.

The variant form of Bermuda grass of the present invention ' Tifway ' Dehydrin-L albumen comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutants, under high or low high stringency conditions can to the albumen coded by the DNA of the relevant DNA hybridization of Bermuda grass ' Tifway ' Dehydrin-L and the polypeptide utilizing the antiserum(antisera) of Bermuda grass ' Tifway ' Dehydrin-L albumen to obtain or albumen.

In the present invention, " Bermuda grass ' Tifway ' Dehydrin-L conservative variation polypeptide " refers to compared with the aminoacid sequence shown in SEQIDNO.4, have 10 amino acid at the most replace by the similar or close amino acid of character and form polypeptide.These conservative variation's polypeptide preferably carry out replacing according to table 1 and produce.

Table 1

Initial residue	Representational replacement	Preferred replacement
			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 also comprises the analogue of Bermuda grass ' Tifway ' Dehydrin-L albumen or polypeptide.The difference of these analogues and Bermuda grass ' Tifway ' Dehydrin-L related polypeptide can be the difference on aminoacid sequence, also can be the difference do not affected on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant that is natural or induction.Induce variation body can be obtained by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also by site-directed mutagenesis or the biological technology of other known moleculars.Analogue also comprises the analogue with the residue (as D-amino acid) being different from natural L-amino acids, and has the analogue of amino acid (as β, gamma-amino acid) that is that non-natural exists or synthesis.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide enumerated.

(usually the not changing primary structure) form of modification comprises: the chemically derived form of the polypeptide that body is interior or external is as acetylize or carboxylated.Modify and also comprise glycosylation, as carried out glycosylation modified and polypeptide that is that produce in those in the synthesis of polypeptide and processing or further procedure of processing.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) by being exposed to by polypeptide and completing.Modified forms also comprises the sequence with phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Also comprise and modified thus improve its anti-proteolysis performance or optimize the polypeptide of solubility property.

In the present invention, the expression pattern of methods analyst Bermuda grass ' Tifway ' the Dehydrin-L gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of the mRNA transcript namely analyzing Bermuda grass ' Tifway ' Dehydrin-L gene in cell.

The present invention detects in sample the detection method that whether there is Bermuda grass ' Tifway ' Dehydrin-L related nucleotide sequences, and comprise and hybridizing with above-mentioned probe and sample, then whether detection probes there occurs combination.This sample is the product after pcr amplification, and wherein pcr amplification primer corresponds to Bermuda grass ' Tifway ' Dehydrin-L related nucleosides coding sequences, and can be positioned at both sides or the centre of this encoding sequence.Primer length is generally 15 ~ 50 Nucleotide.

In addition, according to Bermuda grass of the present invention ' Tifway ' Dehydrin-L nucleotide sequence and aminoacid sequence, can on the homology basis of nucleic acid homology or marking protein, screening Bermuda grass ' Tifway ' Dehydrin-L associated homologous gene or homologous protein.

In order to obtain the dot matrix with Bermuda grass ' Tifway ' Dehydrin-L genes involved, can screen Bermuda grass ' Tifway ' cDNA library with DNA probe, these probes are under low high stringency conditions, use ³²what P was correlated with to Bermuda grass ' Tifway ' Dehydrin-L all or part ofly does radioactivity mark and obtains.The cDNA library being suitable for screening is the library from Bermuda grass ' Tifway '.The method built from the cDNA library of interested cell or tissue is that biology field is well-known.In addition, many such cDNA libraries also can buy, such as, purchased from Clontech, Stratagene, PaloAlto, Cal..This screening method can identify the nucleotide sequence of the gene family relevant to Bermuda grass ' Tifway ' Dehydrin-L.

Bermuda grass of the present invention ' Tifway ' Dehydrin-L associated nucleotide full length sequence or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.

After obtaining relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.

In addition, also by chemosynthesis, sudden change is introduced in protein sequence of the present invention.

Except producing with recombination method, the fragment also available solid phase technique of albumen of the present invention, is produced (people such as Stewart, (1969) Solid phase peptide synthssis, WHFreemanCo., SanFrancisco by direct peptide synthesis; MerrifieldJ. (1963) J.AmChem.Soc85:2149-2154).Synthetic protein can carry out by hand or automatically in vitro.Such as, can by 431A type peptide synthesizer (FosterCity, CA) of AppliedBiosystems from dynamic synthetic peptide.Each fragment of chemosynthesis albumen of the present invention can be distinguished, then chemically connected the molecule producing total length.

Utilize Bermuda grass of the present invention ' Tifway ' Dehydrin-L albumen, by various conventional screening assays, can filter out and relevant to Bermuda grass ' Tifway ' Dehydrin-L albumen interactional material occur, or inhibitor and antagonist etc.

Bermuda grass ' Tifway ' is as turfgrasses, and application is very extensive, and its market requirement is also very large.The important response protein of the present invention first in cloned dog root of the tooth ' Tifway ' stress during drought stress and the encoding sequence of protective protein Dehydrin-L; and adopt the expression pattern of the methods analyst Dehydrin-L gene of fluorescence real-time quantitative PCR; for the spatial and temporal expression utilizing genetic engineering technique to regulate and control Dehydrin-L gene from now on; thus provide theoretical foundation for improving turf grass drought resistance, breeding of new variety aspect, there is very large using value.

Accompanying drawing explanation

By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:

Fig. 1 is Homology search (GAP) result of Bermuda grass of the present invention ' Tifway ' Dehydrin-L gene and the nucleotide sequence without the hidden son grass of awns (Cleistogenessongorica) dehydrin gene mRNA;

Fig. 2 is Homology search (FASTA) result of the aminoacid sequence of Bermuda grass of the present invention ' Tifway ' Dehydrin-L albumen and E. elongata (Lophopyrumelongatum) dehydrin albumen, wherein, identical amino acid marks with amino acid monocase between two sequences.

Fig. 3 is the expression amount change of Bermuda grass ' Tifway ' Dehydrin-L gene in stress during drought stress;

Fig. 4 verifies with Bermuda grass ' Tifway ' Dehydrin positive monoclonal bacterial plaque PCR;

Fig. 5 be wild-type with Bermuda grass ' Tifway ' Dehydrin-L transgenic arabidopsis in the relative percentage of water loss of the plant of different drought stress time;

Fig. 6 is wild-type and Dehydrin-L transgenic Arabidopsis plants drought stress Phenotypic Observation;

The plant relative conductivity value that Fig. 7 is wild-type and Dehydrin-L transgenic arabidopsis when drought stress 4 days;

The plant Dehydrin Quantitative analysis of gene expression that Fig. 8 is wild-type and Dehydrin-L transgenic arabidopsis when drought stress 4 days.

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.These embodiments are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, such as Sambrook equimolecular clone: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.

embodiment 1, Bermuda grass ' Tifway ' Dehydrin-L gene clone

1. the acquisition of vegetable material

Get Bermuda grass ' Tifway ' leaf tissue, for extracting RNA;

The extracting of 2.RNA

By " RNApreppure plant total RNA extraction reagent box " extracted total RNA (Trizol:Invitrogen), by the integrity of denaturing formaldehyde gel electrophoresis qualification RNA, then in upper purity and the concentration measuring RNA of spectrophotometer (ThermoScientificNANODROP1000Spectrophotometer);

3. the full-length clone of gene

According to setting up the est sequence and protein function annotation result that suppress the separation of subtractive library (SSH) to obtain in Bermuda grass ' Tifway ' stress during drought stress, obtain Bermuda grass ' Tifway ' Dehydrin-L gene core fragment.Adopt RACE method (SMARTer ^tMrACEcDNAAmplificationKit:Clonetech) carry out cDNA full-length clone, a point three phases carries out:

(1) RT-PCR obtains gene intermediate segment

The RNA of extraction is carried out reverse transcription (PrimeScript II 1stStrandcDNASynthesisKit: precious biotechnology (Dalian) company limited), with the first chain cDNA for template, primer Dehydrin-LF (SEQIDNO.1) and Dehydrin-LR (SEQIDNO.2) is utilized to carry out PCR, amplification obtains 203bp fragment, reclaim and be connected on pMD18-TSimplevector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, at ABI377 sequenator (Perkin-Elmer, USA) check order on, sequencing result is by carrying out the BLAST existing database of (http://blast.ncbi.nlm.nih.gov/) comparison (GenBank) in NCBI website, know its nucleotide sequence and proteins encoded and known to the hidden son grass of awns, E. elongata, orchardgrass (Bermudagrass), the homology of the Dehydrin gene of ragimillet is very high, tentatively think that it is a Dehydrin gene,

(2)3′RACE

Two take turns the amplification that nest-type PRC completes 3 ' end sequence.

The first round: UPM+3 '-GSP1 (5 '-GCGAACAGTCCGTGATAACTGTCTGTCA-3 ')

Second takes turns: NUP+3 '-GSP2 (5 '-TCGTGTAACATGATAAGATGGTCAGCCA-3 ')

UPM and NUP provides for test kit.3 ' RACE obtains the 3 ' end sequence (228bp) of Bermuda grass ' Tifway ' Dehydrin-L, reclaim, be connected on pMD18-TSimplevector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, at ABI377 sequenator (Perkin-Elmer, USA) check order on, sequencing result is by carrying out the BLAST existing database of (http://blast.ncbi.nlm.nih.gov/) comparison (GenBank) in NCBI website, know that its nucleotide sequence and proteins encoded and the known homology without awns hidden son grass and orchardgrass Dehydrin gene are very high,

(3)5′RACE

With 5 ' RACEreadycDNA for template, take turns by two the amplification that nest-type PRC completes 5 ' end sequence,

The first round: UPM+5 '-GSP1 (5 '-ACCATCTTATCATGTTACACGAACGTCG-3 ')

Second takes turns: NUP+5 '-GSP2 (5 '-GAACGTCGTGACAGACAGTTATCACGGA-3 ')

UPM and NUP provides for test kit.5 ' RACE obtains the 5 ' end sequence (705bp) of Bermuda grass ' Tifway ' Dehydrin-L gene, reclaim after connecting and check order with the method above, the sequencing result of the sequence obtained by above-mentioned 3 kinds of methods is spliced, to splice sequence submits to BLAST to analyze, result proves that the Dehydrin gene newly obtained from Bermuda grass ' Tifway ' is the gene that a dehydrin protein is relevant really, the ORFFinding (http://www.ncbi.nlm.nih.gov/gorf) of sequencing result in conjunction with NCBI is predicted, initiator codon and the terminator codon of Bermuda grass ' Tifway ' Dehydrin-L gene are found, according to the sequence obtained, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F (5 '-ATGGAGCACCAGGGACAGTACGGC-3 '), ORF-R (5 '-TTAGTGCTGGCCGGGGAGCTTCTC-3 '), be that template carries out PCR with Bermuda grass ' Tifway ' cDNA, amplification obtains the complete encoding sequence (SEQIDNO.3) of 543bp Bermuda grass ' Tifway ' Dehydrin-L albumen.

embodiment 2, the sequence information of Bermuda grass ' Tifway ' Dehydrin-L gene and homology analysis

Bermuda grass of the present invention ' Tifway ' Dehydrin-L full length gene opening code-reading frame sequence is 543bp, and detailed sequence is shown in sequence shown in SEQIDNO.3.Derive the aminoacid sequence of Bermuda grass ' Tifway ' Dehydrin-L albumen according to opening code-reading frame sequence, totally 180 amino-acid residues, molecular weight is 18.2kDa, and iso-electric point (pI) is 8.78, and detailed sequence is shown in sequence shown in SEQIDNO.4;

The opening code-reading frame sequence of Bermuda grass ' Tifway ' Dehydrin-L and the aminoacid sequence blast program of proteins encoded thereof are carried out Nucleotide and protein homology search in Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundantGenBankCDStranslations+PDB+SwissProt+Superd ate+PIR database, found that it and the homogeny on nucleotide level without the careless Dehydrin gene of the hidden son of awns (accession number: FJ972827.1) with 78%, (Query: the coding gene sequence of Bermuda grass ' Tifway ' Dehydrin-L as shown in Figure 1, Sbjct: the mRNA sequence without the careless Dehydrin of the hidden son of awns), on amino acid levels, it and E. elongata Dehydrin gene (accession number: AAC05922.1) also have the consistence of 68% and the similarity of 66%, as shown in Figure 2 (the aminoacid sequence of Query: Bermuda grass ' Tifway ' Dehydrin-L albumen, the aminoacid sequence of Sbjct: E. elongata Dehydrin albumen).As can be seen here, all there is higher homology in the Dehydrin gene of Bermuda grass ' Tifway ' Dehydrin-L gene and other known species from nucleic acid or protein level.

embodiment 3, Bermuda grass ' Tifway ' Dehydrin-L gene is at the different expression of drought stress different steps

1. the acquisition of material: the blade of Bermuda grass ' Tifway ' drought stress different steps (0 day, 5 days, 10 days, 15 days) material is sampled.Drop at once in liquid nitrogen after sample is wrapped with aluminium platinum paper respectively, then proceed to stored for future use in-80 DEG C of Ultralow Temperature Freezers;

The extraction of 2.RNA: utilize RNApreppure plant Total RNAs extraction (Trizol:Invitrogen); Extract the total serum IgE in Bermuda grass ' Tifway ' different sample tissue;

The determination of the integrity of 3.RNA, purity, concentration: with plain agar sugar gel electrophoresis (gum concentration 1.2; %0.5 × TBE electrophoretic buffer; 150v, 15min) detect integrity, in electrophoretic band, maximum rRNA brightness should be 1.5 ~ 2.0 times of Article 2 rRNA brightness, otherwise represents the degraded of rRNA sample; Purity good RNA, A ₂₆₀/ A ₂₈₀and A ₂₆₀/ A ₂₃₀be about about 2.0, calculate rna content by spectrophotometric determination OD value;

The acquisition of 4.cDNA: with the total serum IgE of 500ng for template, according to precious biotech firm TaKaRaPrimeScript ^tMit is for subsequent use that RTreagentKitPerfectRealTime test kit operation instructions carries out reverse transcription acquisition cDNA;

5. design Auele Specific Primer to carry out real-time fluorescence quantitative PCR analyzing gene at each organ and the expression amount in tissue, according to the Bermuda grass obtained ' Tifway ' Dehydrin-L gene order, primer-design software is utilized to be designed for the Auele Specific Primer that in Real-timePCR, Dehydrin-L gene quantification is analyzed, primer qDHNF (5 '-CGGAGGAAGAAGGGAATC-3 '), primer qDHNR (5 '-AGTTCCGGAGCCGGTCA-3 '), reference gene is rrna 18S gene, primer is 18S-F (5 '-GTGACGGGTGACGGAGAATT-3 '), 18S-R (5 '-GACACTAATGCGCCCGGTAT-3 '),

6. make the typical curve of goal gene and reference gene: with EASYDilution (test kit provides), standard substance cDNA solution is carried out gradient dilution, then respectively with dilution after cDNA solution for template, carry out Real-timePCR amplification with the Auele Specific Primer of goal gene and reference gene, reaction terminates rear drafting solubility curve and typical curve; Analyze solubility curve, judge whether the solubility curve of goal gene and reference gene obtains simple spike, to judge to use this primer can obtain single pcr amplification product; By the appropriate dilutions multiple of typical curve determination template cDNA;

7. the Real time PCR of goal gene in testing sample: with the cDNA Article 1 chain synthesized for template, quantitative fluorescence analysis is carried out respectively by the primer amplified of goal gene and internal reference gene, Real-timePCR reaction is carried out on BIO-RADChromo4 real-time quantitative instrument, reaction system is 20 μ L, reaction adopts three-step approach, 94 DEG C of sex change 20s, then 40 circulations: 94 DEG C of 15s; 58 DEG C of 15s; 72 DEG C of 25s; Whether, after each amplification completes, all do solubility curve, be special generation to check amplified production;

8. adopt 2 ^{-△ △ Ct}method makes relative quantitative assay, and result shows Bermuda grass ' Tifway ' increasing the weight of along with drought stress degree, and its Dehydrin-L expression level significantly rises (Fig. 3).The 5th day of drought stress, the 10th day, when the 15th day, the expression level of this gene was respectively 110.9,136.8 and 313.4 times of adjoining tree, illustrates that this gene and drought stress respond significant correlation, has obvious spatio-temporal difference.

embodiment 4, Bermuda grass ' Tifway ' Dehydrin-L gene transformation model plant Arabidopis thaliana

(1) conversion carrier is built

Respectively from initiator codon and terminator codon design Auele Specific Primer Dehydrin_OFR-S (5 '-AA gGATCCaTGGAGCACCAGGGACAGTA-3 '), Dehydrin_ORF-A (5 '-A aCTAGTgTGCTGGCCGGGGAGCTT-3 ') and introduce BamHI and SpeI restriction enzyme site respectively in full length gene sequence both sides, be that template carries out PCR with Bermuda grass ' Tifway ' cDNA.Reclaim PCR primer and be connected on pMD18-TSimplevector carrier, picking mono-clonal bacterial plaque carries out PCR checking.There are 2 members (Dehydrin-L, Dehydrin-S) in Bermuda grass ' Tifway ' Dehydrin gene family, the comparatively long segment of about 550bp is Dehydrin-L (Fig. 4, swimming lane 1-4), extracts positive colony bacterium liquid plasmid.Object fragment of plasmid and PHB binary transformation vector are carried out BamHI and SpeI double digestion, reclaim enzyme cut after PHB carrier and Dehydrin-L fragment, Dehydrin-L and PHB carrier is connected the structure conversion carrier that spends the night by application T4 ligase enzyme in 16 DEG C of water-baths, and by vector Agrobacterium GV3101.

(2) Dehydrin-L arabidopsis thaliana transformation

1. shake Agrobacterium in advance: choose positive monoclonal and contain in the YEP liquid nutrient medium of 50mg/LKan, 50mg/L gentamicin, 25mg/LRif to 25ml, 28 DEG C, 200rpm shakes bacterium 24h;

2. spread cultivation Agrobacterium: spread cultivation to containing in 400mLKan resistance YEP substratum by the Agrobacterium bacterium liquid shaken in advance with 1:100,28 DEG C, 200rpm, cultivates 13-16h, be cultured to absorbancy OD ₆₀₀reach between 1.5-2.0 and receive bacterium, receiving bacterium condition is 23 DEG C, 5000rpm, 8min;

3. transformed plant: (needing transforming the day before yesterday or transforming the little Hua cutting off angle fruits all on plant the same day and bloomed and show money or valuables one carries unintentionally) prepares the 1/2MS solution of 500mL containing 5% sucrose, with a small amount of MS solution, the Agrobacterium of collecting precipitation is hanged, shake up, SilwetL-77 and the 10 μ L6-BA (mother liquor is 1mg/mL) of 0.04% (v/v) are added in remaining sucrose solution, stir evenly, before conversion, the two is mixed, base of the plant and inflorescence are immersed in 50s in bacterium liquid, taking-up drains bacterium liquid, put into disposable plastic bag, sealing, moisturizing.After all plant transformation, black box on cover, lucifuge cultivates 24h.Take out plant afterwards, erect plants is placed, waters Aquaponic, ensure that plant moisture is sufficient.

(3) screening of transgenic positive strain

Plant after conversion is sowing after angle fruit all maturation, in the desiccation culture ware being lined with filter paper, room temperature is placed one week, make seed all dry, use 50 object stainless steel sift filter seed afterwards, except chamfer fruit, collect transgenosis T0 for seed and be seeded in cave dish in, carry out Resistance of Seedling screening with 0.05% (v/v) glyphosate, obtain T1 for transfer-gen plant, continue screening until obtain T3 for homozygote transfer-gen plant.

embodiment 5,arabidopis thaliana Dehydrin-L transfer-gen plant drought stress Physiologic Studies

(1) Arabidopis thaliana Dehydrin-L transfer-gen plant percentage of water loss detects

Shear Arabidopis thaliana wild-type and Dehydrin-L rotaring gene plant blade 0.5g, be positioned in aluminium box, take leaf weight in different steps (0-12h), calculate different plant loses water rate.Pass in time, Dehydrin-L transfer-gen plant percentage of water loss is starkly lower than adjoining tree and is about 5-15% (Fig. 5), illustrates that Dehydrin-L gene has better water tariff collection effect to plant under in vitro drought condition.

(2) Arabidopis thaliana Dehydrin-L transfer-gen plant drought stress Phenotypic Observation

Arabidopis thaliana wild-type and Dehydrin-L transfer-gen plant are planted in the growth cabinet of temperature 22 DEG C, light intensity 6000 lux, photoperiod 16 h light/8 h dark, carry out drought stress process simultaneously and carry out phenotype paired observation.During drought stress 4 days, wild-type and Dehydrin-L transgenic Arabidopsis plants phenotype have significant difference; Wild-type plant blade is withered wilts, and lodging appears in scape; Dehydrin-L rotaring gene plant blade wilting degree is less, and scape is upright, and plant strain growth situation is also obviously greater than wildtype Arabidopsis thaliana (Fig. 6).Illustrate that Dehydrin-L transgenic plant have better drought resistance under drought condition.

(3) Arabidopis thaliana Dehydrin-L transfer-gen plant specific conductivity detects

Shear Arabidopis thaliana wild-type and the Dehydrin-L transfer-gen plant drought stress blade 0.2g of 4 days, be collected in 50ml centrifuge tube, add 20ml deionized water, be positioned over 24h on shaking table, measure specific conductivity initial value; Put into high-pressure sterilizing pot (121 DEG C) process 15 minutes, measure specific conductivity end value.Sample relative conductivity value is calculated than final value with initial value.The wild-type plant relative conductivity value of drought stress after 4 days is the relative conductivity value of 87.7%, Dehydrin-L transfer-gen plant is 73.2%, is starkly lower than WT lines (Fig. 7).Illustrate Dehydrin-L transgenic plant under drought stress conditions cell extent of injury lower than WT lines.

(3) wild-type and transgenic Arabidopsis plants Dehydrin-L gene expression difference under drought stress conditions

Shear Arabidopis thaliana wild-type and the Dehydrin-L transfer-gen plant drought stress blade 0.2g of 4 days, press embodiment 3middle method is extracted RNA, preparation cDNA and is carried out Real-time PCR Analysis.In Real-timePCR, the Auele Specific Primer of Dehydrin-L gene quantification analysis is qDHNF (5 '-CGGAGGAAGAAGGGAATC-3 '), primer qDHNR (5 '-TCTCCTTGATCTTGTCCAT-3 '), reference gene is for intending southern actin2 gene, primer is act-F (5 '-CTTGCACCAAGCAGCATGAA-3 '), act-R (5 '-CCGATCCAGACACTGTACTTCCTT-3 ').Adopt 2 ^{-△ △ Ct}method makes relative quantitative assay, and result shows that the expression amount of Dehydrin-L in the transgenic arabidopsis of drought stress after 4 days is higher, is 1.52 times of reference gene actin2, is wild-type plant 16484 times (Fig. 8).Show that Dehydrin-L does not express in WT lines.

Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.

Sequence table

Claims

1. the protein of one kind following (a) or (b):

A protein that () is made up of the aminoacid sequence such as shown in SEQIDNO.4;

B the aminoacid sequence shown in () SEQIDNO.4 passes through replacement, lacks or adds one or several amino acid and has the protein derivative by (a) of Dehydrins enzymic activity.

2. protein as claimed in claim 1, it is characterized in that, described protein for aminoacid sequence shown in SEQIDNO.4 is through 1 ~ 50 amino acid whose disappearance, insertion and/or replacement, or within C-terminal and/or N-terminal add 1 ~ 20 amino acid and the sequence that obtains.

3. protein as claimed in claim 2, is characterized in that, described protein for 1 ~ 10 amino acid in aminoacid sequence shown in SEQIDNO.4 replace by the similar or close amino acid of character and the sequence that formed.

4. nucleic acid sequences to proteins described in a coding claim 1.

5. nucleotide sequence as claimed in claim 4, it is characterized in that, described nucleotide sequence is specially:

A () base sequence is as shown in SEQIDNO.3 1st ~ 543;

Or the nucleic acid shown in (b) and SEQIDNO.3 1st ~ 543 has the sequence of the homology of at least 70%;

Or (c) can carry out the sequence of hybridizing with the nucleic acid shown in SEQIDNO.3 1st ~ 543.

6. nucleotide sequence as claimed in claim 4, it is characterized in that, described nucleotide sequence is specially the disappearance of 1 ~ 90 Nucleotide in the nucleotide sequence shown in SEQIDNO.3 1st ~ 543, insertion and/or replacement, or 5 ' and/or 3 ' sequence of being formed with inner nucleotide of end interpolation 60.

7. for detecting a probe for nucleotide sequence as claimed in claim 4, it is characterized in that, described probe is the nucleic acid molecule including described nucleotide sequence 8 ~ 100 continuous nucleotides.