CN104099345A - Coding sequence and application of arabidopsis function unknown gene H38 - Google Patents
Coding sequence and application of arabidopsis function unknown gene H38 Download PDFInfo
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Abstract
The invention relates to a coding sequence of an arabidopsis function unknown gene H38. The coding sequence comprises a nucleotide sequence shown by SEQ ID NO.1 in a sequence table, or a nucleotide sequence having at least 70 percent of homology with the DNA sequence shown in the SEQ I D NO.1, or a polyribonucleotide of an amino acid sequence shown by SEQ ID NO.2 in a coding sequence table, or a sequence having at least 70 percent of homology with the amino acid sequence of the SEQ ID NO.2 by replacement, deletion or addition of the amino acid residue sequence of SEQ I D NO.2 through more than one amino acid residue. The invention further discloses the application of the coding sequence provided by the invention in adjusting plant heat resistance, so that plant heat resistance can be adjusted, a warranty is provided for improving plant stress resistance under a high temperature condition, so as to be convenient for breeding heat resisting breeds of crops and vegetables. Therefore, harms caused by high temperature to the yield and the quality of industrial crops can be slowed down.
Description
Technical field
The present invention relates to plant genetic engineering field, be specifically related to a kind of Arabidopis thaliana Unknown Function gene
h38encoding sequence and application.
Background technology
Heat evil, refers to that the temperature of working as plant growth environment exceedes its optimum growth temperature and causes cell impaired on a large scale, thereby causes metabolism disorder, finally causes the one of necrocytosis to coerce reaction.Heat evil between grow up develop of plant can directly cause the loss of crop yield, and has report in various crop in as paddy rice, tomato, Kidney bean, Wheat and Corn and Cotton.Because atmosphere greenhouse Gas Emission increases and surface ground coverage minimizing, global climate has the trend warming gradually.2007, IPCC(Intergovernmental Panel on Climate Change) the Fourth Assessment Report demonstration, on average rise 0.74 DEG C in the global surface temperature of 1906-2005, and predict future 20 years will warm with the speed that increases about 0.2 DEG C for every 10 years, to the end of this century (2090-2099) earth's surface medial temperature with respect to exceeding more than 1 DEG C between 1980-1999.And along with the rising of global climate, agriculture production and food safety will face severeer situation.Plant is actionless, and in its life cycle, often meets with hostile environment as the variation of temperature; Therefore, for the plant that survives must be evolved out complicated, meticulous defense mechanism regulates its biochemical and physiological adaptive faculty under adverse environmental factor by changing the expression of gene.
Thermal excited transcryption factor (Heat stress factors, Hsfs) by identification heat shock protein (heat stress proteins, Hsps) heat shock element of promoter region (heat stress elements, HSE) to activate its expression be the primary mechanism of plant to thermal response.In Arabidopis thaliana,
hsfsfamily is by 21 member compositions.Wherein,
hsfA1smain heat resistanceheat resistant regulatory factor,
hsfA2with
hsfA3most important to the heat impedance extending.These genes all hot response gene of each inherent regulation one class (comprise
hsps) expression.HsfB1 and HsfB2b are also that plant heat resistanceheat resistant is necessary.But because they lack activation domain, HsfBs is by suppressing category-A
hsfsgene (as
hsfA2with
hsfA7a) mediation indirectly regulate
hspsthe expression of gene.
Although not all thermal response gene is all subject to
hsfsadjusting, but so far find most of heat resistanceheat resistant regulatory factors all with
hsfsgene association.Early stage at heat stress, second messenger Ca in born of the same parents
2+/ CaM accepts thermal signal, and Hsf is also subject to phosphorylation and the dephosphorylized adjusting of Ser/Thr Phosphoric acid esterase PP7 of calmodulin-binding protein kinase (calmodulin-binding protein kinase) CBK3 in conjunction with the activity of HSE.Dehydration response element can activate in conjunction with albumen (dehydration responsive element binding protein) DREB2A and DREB2C
hsfA3promotor, regulates then
hspsexpression and the heat impedance of plant.Heat stress can be induced the accumulation in endoplasmic reticulum region of false folding and metaprotein.The transcription factor of endoplasmic reticulum location
bZIP28participate in the expression of the albumen Related to repair gene that regulates endoplasmic reticulum region, and then affect the heat impedance of plant.Hormone also participates in the adjusting of heat impedance, and wherein Whitfield's ointment (SA) partly depends in the process of heat stress reaction
hsf-Hspapproach.The sub-multiprotein bridging of the co-activation factor 1c (MBF1c) that transcribes of an animals and plants circle high conservative is the hinge of Genes For Plant Tolerance thermal conditioning net.In the process regulating in heat resistanceheat resistant
mBF1cbe positioned at the upstream of SA and ethene; Also control on the other hand
hsfB2a,
hsfB2bwith
dREB2athe expression of gene.And above-mentioned mention
dREB2a,
mBF1cwith
bZIP28the expression of gene all depends on
hsfA1s.
Although had been found that the gene that many heat resistanceheat resistants regulate, hot regulated and control network also discloses far away.The factor of one of them restriction is to have quite a few thermal response gene function completely unknown, as 30% front 100
hsfA1in mutant, lowering and expressing significant gene is Unknown Function gene.Exploring these Unknown Function genes correspondingly becomes a huge challenge of finely regulating net, also can bring unlimited opportunity to disclose hot regulatory mechanism multi-facetedly simultaneously.
Summary of the invention
Technical requirements
Arabidopis thaliana Unknown Function gene is provided
h38nucleotide sequence and aminoacid sequence and its application in plant heat resistance property, utilize
h38downward expression or overexpression change plant heat resistance property, give security for improving crop anti-adversity under hot conditions, for use in the heat resistanceheat resistant breed breeding of farm crop and vegetables, slow down thus the injury that high temperature causes yield of commercial crops and quality.
The present invention is achieved in that
A kind of Arabidopis thaliana Unknown Function gene
h38encoding sequence, it has as SEQ ID NO in sequence table: the nucleotide sequence as shown in the of 1, or with SEQ ID NO: the DNA sequence shown in 1 has the nucleotide sequence of 70% homology at least; Or there are the polynucleotide of the aminoacid sequence shown in SEQ ID NO:2 in code sequence list; Or replacement, disappearance or the interpolation through more than one amino-acid residue by the amino acid residue sequence of SEQ ID NO:2, there is the sequence that has 70% homology with the aminoacid sequence of SEQ ID NO:2 at least.
Arabidopis thaliana Unknown Function gene
h38encoding sequence be SEQ ID NO in sequence table: the protein shown in 2 belongs to the hot corresponding protein of small molecules.
Arabidopis thaliana Unknown Function gene H38 is positioned plastosome, can reduce thermal excited transcryption factor (
hsfA2,
hsfA3,
hsfA7adeng), heat shock protein (
hsp101,
hsp15.7-CI (r), Hsp18.5 (r),
hsp17.6-CI, Hsp17.8deng), GolS1 and other plastosome gene location (
atDjB1,
mtHSC70-1,
atMge2, SHOT1deng) in normal or heat stress expression after treatment.
h38in mutant plant
h38violent downward expressed, and weakens than the thermotolerance of wild-type plant, transforms raising by complementation
h38in mutant plant
h38content can recover
h38the heat impedance of mutant, explanation
h38there is the effect of regulating plant heat impedance.
Beneficial effect
The present invention discloses play-by-play Arabidopis thaliana Unknown Function gene both at home and abroad first
h38sequence, its expression amount is lowered the function that can reduce plant heat resistance property does not at present have relevant report.Experimental result of the present invention shows Arabidopsis Mutants
h38in
h38expression amount than the remarkable reduction of wild-type, therefore its thermotolerance also weakens.Meanwhile, by
h38middle complementary conversion
pMDC83-H38carrier can recover
h38thermo-responsive phenotype.Therefore the present invention proposes to utilize
h38downward expression or overexpression change plant heat resistance property, give security for improving crop anti-adversity under hot conditions, for use in the heat resistanceheat resistant breed breeding of farm crop and vegetables, slow down thus the injury that high temperature causes yield of commercial crops and quality.
Brief description of the drawings
Fig. 1 is Arabidopis thaliana
h38homologous sequence comparison chart;
With Arabidopis thaliana
h38sequence is template, carries out BLAST comparison in Uniprot Protein Data Bank, threshold value E is set and is less than 10
-4, the representative homologous sequence that screening is obtained utilizes the comparison of ClustalX 1.8.3 software to obtain result;
Fig. 2 is plant expression vector schematic diagram;
Clone obtains
h38coding sequence and by its with
pMD18-Tempty carrier connects acquisition
pMD18-H38, after order-checking is correct, with Pme1 and Asc1 respectively enzyme cut
pMD18-H38with
pMDC83empty carrier, reclaims gene fragment and carrier segments, then connects, transforms, screens acquisition
pMDC83-H38positive colony;
Fig. 3 is screening
h38middle conversion
pMDC83-H38positive complementary materials;
The transgenic seed disinfecting is seeded in to 15 days (A) of 1/2MS+ Ticarcillin/Clavulanate Acid 300mg/L+Totomycin 25mg/L substratum growth; Employing PCR method detects
h38mutant (B), and complementary materials (C);
Fig. 4 is
h38carrier structure schematic diagram and the expression analysis of mutant and conversion complementary materials;
Extract 7 days greatly
h38mutant, wild-type and complementary materials
cO55the RNA of plant, reverse transcription is lived and is obtained cDNA, then adopts qRT-PCR to analyze
h38expression amount;
Fig. 5 is
h38comparison schematic diagram after mutant, wild-type and 45 DEG C of pyroprocessing of complementary materials;
By the planting seed disinfecting, in 1/2MS substratum growth 7 days, 45 DEG C of pyroprocessing 30min, transferred under 22 DEG C of normal growth conditions and grow 7 days, take a picture and add up mortality ratio, and mutant survival rate significantly declines, and complementary materials and wild-type are suitable;
Fig. 6 is
h38mutant and wild-type regulate other changes in gene expression schematic diagram under normal and 38 DEG C of pyroprocessing conditions;
7 is the largest
h38mutant and wild-type plant after 1 hour, are extracted corresponding RNA in normal condition and 38 DEG C of pyroprocessing, and change with the expression that qRT-PCR detects corresponding candidate gene.
h38can regulate and comprise
hsfwith
hspin the expression of interior many heat resistanceheat resistant genes involveds.
Embodiment
embodiments of the invention 1: Arabidopis thaliana Unknown Function gene
h38acquisition and homologous gene analysis.
In Tair database, retrieve Arabidopis thaliana
h38the sequence of gene, the both sides primer (forward primer: [5 '-ATGTCGTTGATTCCAAGCTTC-3 '] of design gene coded sequence; Reverse primer: [5 '-TCAGCCAGAGATCTGGATAG-3 ']), for cloning Arabidopis thaliana
h38sequence.And being connected to pMD18-T carrier, the correct clone of sequencing analysis is used for next step analysis.In Uniprot Protein Data Bank, H38 protein sequence is carried out to BLAST analysis, setting threshold is that E value is less than 10
-4, by ClustalX 1.8.3 software comparison for the sequence obtaining, concrete outcome is with reference to Fig. 1.
, extract Arabidopis thaliana total RNA.
(1) extraction of RNA is with reference to (TRIZOL
tMkit RNA extracts reagent specification sheets)
(2) get 100mg fresh plant tissue sample liquid nitrogen grinding,
(3) add 1ml TRIZOL, room temperature (20-25 DEG C, lower same) is placed 10min,
(4) add 200 μ l chloroforms, thermal agitation 30s, room temperature is placed 5min,
(5) 12000g, 10min, 4 DEG C, get 500 μ l supernatants to new pipe, add 1ml dehydrated alcohol, after mixing, place 20min for-20 DEG C,
(6) 12000g, 10min, 4 DEG C, remove supernatant, add 1ml 75% ethanol (dehydrated alcohol and DEPC H
2the volume ratio of O),
(7) 10000g, 5min, 4 DEG C, remove supernatant, dry air 15min,
(8) add 20-30 μ l RNase-free H
2o 50-60 DEG C dissolves 5min.
, synthetic cDNA the first chain of reverse transcription
The reverse transcription of cDNA the first chain adopts RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas), and operation is carried out with reference to used kit explanation.
, clone's H38 gene
Carry out PCR amplification taking cDNA as template, obtained an Arabidopis thaliana Unknown Function gene
h38, its base sequence is SEQ ID NO: the nucleotide sequence shown in 1.Its corresponding protein sequence is SEQ ID NO: the aminoacid sequence shown in 2.
embodiments of the invention 2:arabidopis thaliana Unknown Function gene
h38application in regulating plant thermotolerance.
,
h38the structure of expression vector and
h38the complementation of plant transforms
PCR amplification is obtained
h38gene is connected to
pMD18-Tcarrier (Takara company), be transformed in competent cell Trans5 α (Transgene company), utilize penbritin screening, use M13F [5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 '] and M13R [5 '-GAGCGGATAACAATTTCACACAGG-3 '] the positive clone of insertion of carrier primer amplification qualification simultaneously, adopt plasmid extraction kit (the raw work in Shanghai) to extract
pMD18-H38and
pMDC83empty carrier plasmid, utilize Pme1 and Asc1 double digestion, reclaim object fragment, with T4 ligase enzyme connection (Fermentas), and be transformed in competent cell Trans5 α, utilize kantlex screening, and carry out PCR qualification through carrier primer (35S starts word aligning primer) with gene primer (gene downstream primer), idiographic flow schematic diagram is with reference to Fig. 2
Bacterium colony PCR authentication method
(1) select 10-15 single bacterium colony in 0.02M NaOH solution, 94 DEG C of sex change 10min are for subsequent use,
(2) configuration PCR reaction mixture (Shanghai raw work 2 × Taq PCR reaction kit):
2×Taq PCR Mixture 10μl
Forward primer (M13 F) 1 μ l
Reverse primer (M13 R) 1 μ l
Bacterium liquid template 2 μ l after denaturation
dd H
2O 6μl
(3) carry out PCR reaction, time and the temperature of reaction are as follows:
94℃ 3min
94℃ 30s，
58℃ 45s
72℃ 30s，30cycles
72℃ 5min
(4) get 8-10 μ l PCR product and carry out electrophoresis detection, pick out positive colony.
The conversion process of Arabidopis thaliana:
Reagent preparation
Inducing culture (200ml): take 10g sucrose (mass ratio is 5%) and be fully dissolved in 200ml distilled water, added the raw work in 20 μ l SilwetL(Shanghai before transforming).
Step of converting
(1) from resistance plate, the single bacterium colony of the Agrobacterium that contains target plasmid of picking activation activates 24 h to the substratum of 5ml LB+gentamicin 25mg/L+kantlex 50 mg/L, and 28 DEG C, 220 rpm.
(2) according to the ratio of 1:50, the bacterium liquid of activation is inoculated into the culture medium culturing 8-12h of 200ml LB+gentamicin 25mg/L+kantlex 50 mg/L, 28 DEG C, 220 rpm.
(3) the centrifugal 15min of room temperature 5000rpm.Abandon supernatant, Agrobacterium precipitation is suspended in the inducing culture of fresh configuration.
(4) colored and angle fruit by removing
h38mutant plant is directly dipped to agrobacterium suspension 30s.
(5) lucifuge overnight incubation, is then normally cultured to seed maturity.
,
h38screening and the checking of complementary transgenic arabidopsis
the screening of transformed the seed
(1) the new Arabidopis thaliana transgenosis T0 seed of receiving is placed under the sun and shines 1-2 days, is distributed into the every pipe of 200mg.
(2), with 75% ethanol disinfection 1min, then use 50% 84 thimerosal (clean auntie, commercially available) sterilization 3 min.
(3) with sterile water wash seed 3-4 time, be then distributed in 1/2MS+Ticarcillin/Clavulanate Acid 300mg/L+Totomycin 25mg/L substratum with 0.1% agarose uniform spreading, 4 DEG C of vernalization 3 days.
(4) in the controlled environment chamber in cultivate 15 days, artificial culture chamber condition: temperature: 22 DEG C, humidity: 80%, intensity of illumination: 80-200 μ mol/m
2/ s, the photoperiod: 16h illumination, 8h dark culturing (Fig. 3 A).
qualification
Be used for the extraction of the transformed plant DNA of PCR
(1) take the fresh leaf tissue of about 100mg and put into the 1.5ml centrifuge tube of high-temperature sterilization.
(2) add 400 μ l extraction buffers (200mM Tris-HCl pH7.5,250mM NaCl, 25mM EDTA, 0.5% SDS), room temperature is ground rapidly.
(3) centrifuge tube that turns upside down is gently counted 5-10 time, leaves standstill 10min.
(4) 12000rpm, 10min, room temperature.Get supernatant, add 800 μ l dehydrated alcohols ,-20 DEG C of precipitation 1-2 h.
(5) 12000rpm, 15min, room temperature, removes supernatant.
(6) add sterilized water 100 μ l dissolvings and slightly put forward DNA precipitation, getting 2 μ l is template, carries out PCR.
program
The preparation of PCR reaction mixture is with plasmid bacterium colony PCR qualification,
h38mutant qualification employing upstream primer [5 '-CCCGACTAACAAAACAAATTTTTAC-3 '] and downstream primer [5 '-ATACATGAGCACTGGACCGAC-3 '] and carrier primer [5 '-ATTTTGCCGATTTCGGAAC-3 '] be combined into performing PCR detection with downstream primer.Complementary materials is combined into performing PCR with plant expression vector middle and upper reaches 35S promoter sequence and goal gene downstream primer [5 '-GGCGCGCCAGCCGTCTTCGTCGCG-3 '] and detects, preparation PCR reaction mixture (Shanghai raw work 2 × Taq PCR reaction kit):
2×Taq PCR Mixture 10μl
Forward primer 1 μ l
Reverse primer 1 μ l
DNA 2μl
dd H
2O 6μl
The time of reaction and temperature are done as follows:
94℃ 3min，
94℃ 30s，
58℃ 45s
72℃ 30s，30cycles
72℃ 5min
Detected result demonstration,
h38homozygote mutant adds downstream primer amplification without band with upstream primer, carrier primer adds downstream primer band, heterozygote middle and upper reaches primer adds downstream primer and carrier primer and adds downstream primer amplification and have band, add downstream primer amplification at wild-type middle and upper reaches primer and have band, carrier primer adds downstream primer without band, and result is shown as (Fig. 3 B).
The all transformed plants of complementary materials all can amplify the big or small electrophoretic band of expection, and negative control does not have, and shows to have contained foreign gene DNA fragmentation in transgenic arabidopsis genome, and result is shown as (Fig. 3 C).
,
h38the expression checking of mutant and complementary transgenic arabidopsis
(1) extraction of RNA is with reference to (TRIZOL
tMkit RNA extracts reagent specification sheets), specific experiment and operating process are with part 1 1.1.
(2) the employing RevertAid H Minus First Strand cDNASynthesis Kit (Fermentas) of reverse transcription cDNA the first chain, operation is carried out with reference to used kit explanation.
(3) that qRT-PCR detects employing is SYBR Green Realtime PCR Master Mix plus(TOYUBO, QPK-212) test kit, operation reference reagent box specification sheets completes, detection primer forward primer [5 '-GAAGATGGAGGAAACGGTAGAG-3 '], reverse primer [5 '-GGCTAGACGTCCATGATTGAA-3 '].
(4) qRT-PCR response procedures is as follows:
94℃ 2min
94℃ 15s，
58℃ 15s
72℃ 30s，read plate，45cycles
72℃ 5min
h38t-DNA insertion mutation body (Fig. 4 A),
h38gene is not expressed (Fig. 4 C).Complementary materials is utilized conversion carrier
pMDC-H38(Fig. 4 B),
h38content significantly increases (Fig. 4 C).
, Arabidopis thaliana heat shock experiment
Isozygoty
h38mutant,
h38complementary transgenosis T3 is seeded in 1/2MS solid medium for plant and wild-type (WT) simultaneously, and 22 DEG C, 16h/8h (light/dark) growth is after one week, and 45 DEG C of heat shock 30min, observe plant existing state after one week.Experimental result shows,
h38mutant declines with respect to wild-type Arabidopis thaliana surviving rate after pyroprocessing, and thermotolerance obviously reduces (Fig. 5), and complementary materials can make
h38survival rate return to wild-type level.
, gene expression analysis
RNA extracts, and the program of reverse transcription and qRT-PCR is all with 2.3, the primer that fluorescent quantitation (qRT-PCR) uses.
Data analysis adopts Excel 2010, and graphics software adopts Photoshop CS6 to process.
Result: comprise
hsf(
hsfA2,
hsfA3,
hsfA7adeng) with
hsp(
hsp101,
hsp15.7-CI,
hsp18.5-CI,
hsp17.6-CI,
hsp17.8-CIdeng) and
golS1expression at interior multiple heat stress genes involveds is all suppressed, plastosome location
atDjB1,
mtHSC70-1,
atMge2with
sHOT1all suppressed (Fig. 6) of expression.
SEQUENCE LISTING
Sequence table
<110> Guizhou Province Grass Industry Research Institute
<120> Arabidopis thaliana Unknown Function gene
h38encoding sequence and application
<130> nm:
<160> 40
<170> PatentIn version
<210> 1
<211> 264
<212> DNA
<213> Arabidopsis (Arabidopis thaliana)
<400>
ATGCG ACGTT TTCCT GCTCT TATAG AGAAG TTAAA ACCGA TCTTC ACGGT 50
CTCAG GATCA AACGG TAAAG TGGTA AGAAC CATTG TACCG AAGAA ACCTG 100
TGAAC GAGAA CATTA GTGAA AGCGA AACAA TGAAG AAGAT GGAGG AAACG 150
GTAGA GCCTA TGGTT GCCTT AAGTA GACCA CCACC ATTTT CACCA TTTGT 200
CGGTC CAGTG CTCAT GTATT CCCTG CTTCA ATCAT GGACG TCTAG CCGCG 250
ACGAA GACGG CTAG 264
<210> SEQ ID NO. 2
<211> 1
<212> 87
<213> Arabidopsis (Arabidopis thaliana)
<400>
MRRFPALIEK LKPIFTVSGS NGKVVRTIVP KKPVNENISE SETMKKMEET 50
VEPMVALSRP PPFSPFVGPV LMYSLLQSWT SSRDEDG- 87
<210> 3
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
h38the sequence of gene, designs the forward primer of gene coded sequence for cloning Arabidopis thaliana with Primer Premier 5.0
h38sequence.
<400> 3
ATGTC GTTGA TTCCA AGCTT C 21
<210> 4
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
h38the sequence of gene, designs the reverse primer of gene coded sequence for cloning Arabidopis thaliana H38 sequence with Primer Premier 5.0.
<400> 4
TCAGC CAGAG ATCTG GATAG 20
<210> 5
<211> 25
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
h38the sequence of mutant upstream and the each 1000bp in downstream, the forward primer of then using Primer Premier 5.0 software design on position upstreams for detection of
h38whether mutant isozygotys.
<400> 5
CCCGA CTAAC AAAAC AAATT TTTAC 25
<210> 6
<211> 2
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
h38the sequence of mutant upstream and the each 1000bp in downstream, the reverse primer of then using Primer Premier 5.0 software design on position downstreams for detection of
h38whether mutant isozygotys.
<400> 6
ATACA TGAGC ACTGG ACCGA C 21
<210> 7
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> provides and detects universal support primer with reference to Arabidopis thaliana SALK mutant website, for detection of
h38whether mutant isozygotys.
<400> 7
ATTTT GCCGA TTTCG GAAC 19
<210> 8
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> obtains PBI121 carrier sequence from ncbi database retrieval, therefrom choose 35S promoter district, then use the forward primer 35S of Primer Premier 5.0 software designs near promotor 3 ' end, detect complementary positive transformed plant for pcr amplification.
<400> 8
GGCGC GCCAG CCGTC TTCGT CGCG 24
<210> 9
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
h38the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
h38changes in gene expression.
<400> 9
GAAGA TGGAG GAAAC GGTAG AG 22
<210> 10
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
h38the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
h38changes in gene expression.
<400> 10
GGCTA GACGT CCATG ATTGA A 21
<210> 11
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
golS1the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
golS1changes in gene expression.
<400> 11
TCTCG TCCTT GCGAT GTTAT G 21
<210> 12
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
golS1the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
golS1changes in gene expression.
<400> 12
CGCTT CTTTC CCTGT GTATC T 21
<210> 13
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsp15.7-CI (r)the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsp15.7-CI (r)changes in gene expression.
<400> 13
CGGTG GTGGA AGTGA GTTT 19
<210> 14
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsp15.7-CI (r)the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsp15.7-CI (r)changes in gene expression.
<400> 14
AGAAC ACCAT TCTCC ACGTA AG 22
<210> 15
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsp18.5-CI (r)the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsp18.5-CI (r)changes in gene expression.
<400> 15
TCTGT ACTGG CGACA ACAAG 20
<210> 16
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsp18.5-CI (r)the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsp18.5-CI (r)changes in gene expression.
<400> 16
CTCAA CGAAC ACGAC AAGAA AC 22
<210> 17
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
atDjB1the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
atDjB1changes in gene expression.
<400> 17
GGCAA AGGAC TACCG AAGCA 20
<210> 18
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
atDjB1the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
atDjB1changes in gene expression.
<400> 18
GCACG CTGAC GTTCA TTCA 19
<210> 19
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
mtHSC70-1the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
mtHSC70-1changes in gene expression.
<400> 19
GTCGA GGACA TCAAG GCTAA GG 22
<210> 20
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
mtHSC70-1the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
mtHSC70-1changes in gene expression.
<400> 20
TTCCA TCGGA ACCAG ATGAT C 21
<210> 21
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
atMge2the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
atMge2changes in gene expression.
<400> 21
GGAGT GGAGA TGACT GAGAA AC 22
<210> 22
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
atMge2the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
atMge2changes in gene expression.
<400> 22
GCATC AGGGA CTTGG AACA 19
<210> 23
<211> 23
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
sHOT1the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
sHOT1changes in gene expression.
<400> 23
GGCCT TCATA GCTCG TCTAT ATG 23
<210> 24
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
sHOT1the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
sHOT1changes in gene expression.
<400> 24
CAACT GCTGC AAGAT CCAAA G 21
<210> 25
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsfA2the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsfA2changes in gene expression.
<400> 25
GGAAG CAGCG TTGGA TGTGA 20
<210> 26
<211> 23
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsfA2the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsfA2changes in gene expression.
<400> 26
TAGAT CTTGG CTGTC CCAAT CCA 23
<210> 27
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsfA3the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsfA3changes in gene expression.
<400> 27
GTTGA TGACC CGACT CTTGA C 21
<210> 28
<211> 23
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsfA3the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsfA3changes in gene expression.
<400> 28
GAGGA TCCCA AACTA CGAAG CTA 23
<210> 29
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsfA7athe sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsfA7achanges in gene expression.
<400> 29
TGCAT TCTTT CTCCA CGATT CTCC 24
<210> 30
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsfA7athe sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsfA7achanges in gene expression.
<400> 30
CAAAT TCCCA TCTCT CTGCT TCTA 24
<210> 31
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsp17.8the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsp17.8changes in gene expression.
<400> 31
TCCGA GTCAC CAGCT TTGCT CA 22
<210> 32
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsp17.8the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsp17.8changes in gene expression.
<400> 32
TGCTT CGCCT GTTGT TGCCG A 21
<210> 33
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsp101the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsp101changes in gene expression.
<400> 33
AGGCT TGTGC GAATG TGAGA GTCC 24
<210> 34
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsp101the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsp101changes in gene expression.
<400> 34
GGCTG AAGCT TGTCT CTCAG GTCA 24
<210> 35
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsp17.6C-CIthe sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsp17.6C-CIchanges in gene expression.
<400> 35
TCGAT CCGTT CTCGC TGGAT GTTT 24
<210> 36
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
hsp17.6C-CIthe sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, detects for the method for fluorescent quantitation
hsp17.6C-CIchanges in gene expression.
<400> 36
CTTTA GCGTT TGTGA ACGCT GCCA 24
<210> 37
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
actin7the sequence of gene, then uses the forward primer of Primer Premier 5.0 software design genes, during for fluorescent quantitation as the difference of template concentrations between reference gene balance sample.
<400> 37
GATAT TCAGC CACTT GTCTG TGAC 24
<210> 38
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> is with retrieving Arabidopis thaliana in Tair database
actin7the sequence of gene, then uses the reverse primer of Primer Premier 5.0 software design genes, during for fluorescent quantitation as the difference of template concentrations between reference gene balance sample.
<400> 38
CATGT TCGAT TGGAT ACTTC AGAG 24
<210> 39
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> obtains pMD18-T carrier sequence from ncbi database retrieval, therefrom choose the sequence near T joint both sides, then use Primer Premier 5.0 software design forward primer M13F, detect the positive colony clone that transforms for pcr amplification.
<400> 39
CGCCA GGGTT TTCCC AGTCA CGAC 24
<210> 40
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> obtains pMD18-T carrier sequence from ncbi database retrieval, therefrom choose the sequence near T joint both sides, then use Primer Premier 5.0 software design forward primer M13R, detect the positive colony clone that transforms for pcr amplification.
<400> 40
GAGCG GATAA CAATT TCACA CAGG 24
Claims (2)
1. an Arabidopis thaliana Unknown Function gene
h38encoding sequence, it is characterized in that: it has as SEQ ID NO in sequence table: the nucleotide sequence as shown in the of 1, or with SEQ ID NO: the DNA sequence shown in 1 has the nucleotide sequence of 70% homology at least; Or there are the polynucleotide of the aminoacid sequence shown in SEQ ID NO:2 in code sequence list; Or replacement, disappearance or the interpolation through more than one amino-acid residue by the amino acid residue sequence of SEQ ID NO:2, there is the sequence that has 70% homology with the aminoacid sequence of SEQ ID NO:2 at least.
2. an Arabidopis thaliana Unknown Function gene as claimed in claim 1
h38the application of encoding sequence in regulating plant thermotolerance, it is characterized in that: Arabidopis thaliana Unknown Function gene
h38encoding sequence can improve plant heat resistance property.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109161552A (en) * | 2018-09-28 | 2019-01-08 | 浙江师范大学 | Increase the gene and application thereof of arabidopsis seed production |
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Non-Patent Citations (3)
| Title |
|---|
| AY086720.1: "Arabidopsis thaliana clone 270232 mRNA, complete sequence", 《GENBANK》, 27 June 2006 (2006-06-27) * |
| ERRU YU等: "Identification of Heat Responsive Genes in Brassica napus Siliques at the Seed-Filling Stage through Transcriptional Profiling", 《PLOS ONE》, vol. 9, no. 7, 11 July 2014 (2014-07-11), pages 101914 * |
| NM_118479.2: "Arabidopsis thaliana uncharacterized protein mRNA, complete cds", 《GENBANK》, 12 February 2014 (2014-02-12) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109161552A (en) * | 2018-09-28 | 2019-01-08 | 浙江师范大学 | Increase the gene and application thereof of arabidopsis seed production |
| CN109161552B (en) * | 2018-09-28 | 2021-05-25 | 浙江师范大学 | Gene for increasing seed yield of arabidopsis thaliana and use thereof |
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