CN110128516A - Barley moisture-proof controlling gene HvERF2.11, albumen and its application in breeding - Google Patents
Barley moisture-proof controlling gene HvERF2.11, albumen and its application in breeding Download PDFInfo
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Abstract
The invention discloses barley moisture-proof controlling gene HvERF2.11, albumen and its applications in breeding.Barley moisture-proof geneHvERF2.11CDS sequence as shown in SEQ ID NO:1.Clone obtains moisture-proof gene to the present invention from moisture-proof barley for the first timeHvERF2.11, and prove that the gene is related with barley moisture-proof, it, will using the method for genetic engineeringHvERF2.11Gene overexpression is into arabidopsis, and compared with the control group, transgenic plant moisture-proof ability significantly increases, explanationHvERF2.11The overexpression of gene improves the moisture-proof of plant.In the present inventionHvERF2.11The technologies such as the clone of gene and conversion, transgenosis are that the molecule mechanism for studying barley moisture-proof and Breeding Application are laid a good foundation, and have wide Breeding Application prospect and certain economic value.
Description
Technical field
The invention belongs to gene engineering technology field more particularly to a barley moisture-proof controlling genesHvERF2.11And its
Breeding Application.
Background technique
Wet injury (waterlogging) refers to anaerobic environment caused by soil moisture is excessive, to crop normal growth and development institute
The harm of generation.Wet injury has generation all over the world, especially more serious in states such as North America, Australia, China, India.
With the deterioration of global ecological environment, extreme weather occur frequency be continuously increased, drought and waterlogging occur frequently, wet injury at
For one of the main abiotic stress factor for influencing production estimation.According to statistics, worldwide, about 16% arable land is equal
By different degrees of wet flood harm, wet injury often leads to crop failure about 20%-30%, and serious wet injury even results in total crop failure.
China is to be influenced more serious one of country by wet flood in the world, and about 30% cultivated area is by different degrees of every year
Flood influences, and in increased trend year by year.Only in 2013, China because flood cause farmland disaster area up to 1.19 ×
107hm2, cause 300,000,000,000 yuan or more of direct economic loss.
Plant forms multiple gene coordinate expression regulation and control models during long-term evolution, to adapt to environment stress.Closely
Nian Lai has turned to the mirror to transcription factor to the research of plant responding environment stress from the clone of individual feature gene and Function Identification
The research of fixed and adjusting function.One transcription factor relevant to environment stress can regulate and control a series of correlation function bases in downstream simultaneously
The expression of cause changes.Therefore, the effect that transcription factor improves crop anti-adversity may be more preferable than individual feature gene.Rice,
The moisture-proof controlling gene cloned in the plants such as arabidopsis, Kiwi berry belongs to transcription factor ERF-VIIs subtribe gene, big at present
As the moisture-proof controlling gene of transcription factor, there is not been reported in wheat.
Summary of the invention
The object of the present invention is to provide a barley moisture-proof genesHvERF2.11And its Breeding Application, it clones and constructsHvERF2.11Expression vector, will using transgenic technologyHvERF2.11Channel genes arabidopsis, to transgenic arabidopsis
Carry out wet injury Stress treatment, confirmationHvERF2.11With moisture-proof activity, genetic resources and reason are provided for barley moisture-proof breed improvement
By foundation.
By bioinformatic analysis, we identify 54 in barley and belong to ERF gene family；Systematic evolution tree point
Analysis discoveryHvERF2.11With Hypoxia Stress related gene in arabidopsisHRE1、HRE2、RAP2.12AndRAP2.2Evolutionary relationship
Recently.It is analyzed under barley Different Organs, different condition by qRT-PCR technologyHvERF2.11Gene expression dose has been cloned resistance to
In wet barley TF58HvERF2.11Gene, and willHvERF2.11Gene is transferred to arabidopsis, demonstrates the function of the gene, to
It lays the foundation for barley variety patience molecular marker assisted selection breeding.
In order to achieve the above-mentioned object of the invention, the technical scheme adopted by the invention is as follows: a kind of barley moisture-proof regulates and controls related egg
White, the albumen is for following (a) or (b):
(a) protein that the amino acid sequence shown in SEQ ID NO:2 forms；Particular sequence is MCGGAILAGFIPPSAAA
AAAKAAATAKKKQQQRSVTADSLWTGLRKKADEEDFEADFRDFERDSSEEEDDEVEEVPPPPAPATAGFAFAAAAE
VALRAPARRDAAVQHDGPAAKQVKRVRKNQYRGIRQRPWGKWAAEIRDPSKGVRVWLGTYDTAEEAARAYDAEARK
IRGKKAKVNFPEDAPTVQKSTLKPTAAKSAKLAPPPKACEDQPFNHLSRGDNDLFAMFAFSDKKVPAKPTDSVDSL
LPVKHLAPTEAFGMNMLSDQSSNSFGSTDFGWDDEAMTPDYTSVFVPSAAAMPAYGEPAYLQGGAPKRMRNNFGVA
VLPQGNGAQDIPAFDNEVKYSLPYVESSSDGSMDNLLLNGAMQDGASSGDLWSLDELFMAAGGY；
(b) by the amino acid sequence of SEQ ID NO:2 by one or several amino acid residues substitution and/or missing and/or
Addition and the protein as derived from SEQ ID NO:2 relevant to the regulation of barley moisture-proof.
The present invention also provides the genes of coding foregoing proteins.The gene is DNA any in following (a1)-(a3)
Molecule；
(a1) DNA molecular shown in SEQ ID NO:1；
(a2) hybridize under strict conditions with (a1) DNA sequence dna limited and the DNA of encoding barley moisture-proof regulation GAP-associated protein GAP divides
Son；
(a3) at least have 70% with (a1) DNA sequence dna limited, at least have 75%, at least with 80%, at least with 85%, extremely
Less with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% or at least have it is 99% homologous
Property and encoding barley moisture-proof regulation GAP-associated protein GAP DNA molecular.
The present invention also provides the expression cassette containing forementioned gene, recombinant vector, recombinant microorganism or transgenic cell lines.
It is a further object to provide the applications of (b1) or (b2) or (b3):
(b1) albumen, or, the gene, or, containing the expression cassette of the gene, recombinant vector, recombinant microorganism or turning
Gene cell system, the application in anti-barley wet injury stress；
(b2) albumen, or, the gene, or, containing the expression cassette of the gene, recombinant vector, recombinant microorganism or turning
Gene cell system, the application in cultivation barley, arabidopsis moisture-proof new varieties；
(b3) albumen, or, the gene, or, containing the expression cassette of the gene, recombinant vector, recombinant microorganism or turning
Gene cell system, the application in anti-arabidopsis wet injury stress.
The present invention also provides a kind of methods for cultivating plant moisture-proof kind, and the gene is transferred to purpose plant or institute
Expression cassette, the recombinant vector stated convert purpose plant, obtain genetically modified plants；The wet injury stress resistance of the genetically modified plants is high
In the purpose plant.The purpose plant is barley, arabidopsis.The resistance to wetting phase of barley described in the Expressed in Transgenic Plant
Close albumen.
HvERF2.11DNA segment described in gene is as shown in sequence table SEQ ID NO.1, or substantially corresponds to SEQ ID
DNA sequence or its function shown in NO.1 are equivalent to the Partial Fragment of sequence shown in SEQ ID NO.1.To the gene into
The analysis of row sequence, showsHvERF2.11Gene coding region overall length is 1158 bp, encodes 385 amino acid, and molecular weight is
41.38kDa, isoelectric point 5.14 contain conservative AP2 structural domain.Overexpressing sequence shown in sequence table SEQ ID NO.1 can be with
Enhance the plants such as arabidopsis and barley to the patience of wet injury.
Said gene can be applied to the moisture-proof ability of improvement barley, and concrete operations are as follows:
(1) it the acquisition of gene: by bioinformatic analysis, obtains in barleyHvERF2.11The full-length gene order of gene, mentions
Moisture-proof strain TF58 root system total serum IgE is taken, is arrived using RT-PCR amplificationHvERF2.11Gene order, amplified production is connected to
On pGEM-Teasy carrier, had through sequencingHvERF2.11The clone of gene.
(2) plant expression vector construction and genetic transformation: barley is constructed using the Gateway technology of Invitrogen companyHvERF2.11Gene overexpression vector.It, will using BP Clonase enzymeHvERF2.11Gene and pDONR221 carrier
Connection constructs entry vector, then utilizes LR Clonase enzyme, entry vector is connect with pB2GW7 carrier, constructs
ContainHvERF2.11The overexpression vector of gene.It will be contained by frozen-thawed methodHvERF2.11The overexpression vector of gene is led
Enter in Agrobacterium Gv3101.It, will using Agrobacterium-mediated genetic transformation methodHvERF2.11Coded sequence imports table in arabidopsis
It reaches.Positive transgenic Arabidopsis plant is screened by antibiotic-screening, RT-PCR etc..
(3) transgenic plant moisture-proof is analyzed: 35 days transgenosis of normal growth and wildtype Arabidopsis thaliana strain are carried out
Submerging treatment.After submerging treatment 2 weeks, the phenotypic difference of observation wet injury stress Transfer-gen plant and WT lines, to demonstrate,prove
It is brightHvERF2.11The ability of gene pairs improvement arabidopsis moisture-proof.
The present invention provides a kind of new method to the resistance of wet injury for enhancing plant, is improved and is made by genetic engineering means
The moisture-proof of object not only overcomes the traditional breeding method period short, and it is easy to operate, be easy to get moisture resistant material.The present invention is for the first time
Clone has obtained barley moisture-proof controlling geneHvERF2.11, and the gene is transferred to by arabidopsis by the method for mediated by agriculture bacillus
In, through over moisture damage identification and analysis, it was demonstrated that transgenic plant is significantly improved compared with the moisture-proof ability of WT lines, it was demonstrated that the present invention
Gene be with a wide range of applications in terms of improving barley moisture-proof.
Detailed description of the invention
Fig. 1 barleyHvERF2.11Gene order RT-PCR amplified production,
Marker:DL2000DNA Marker (Dalian precious biology), by 2000bp, 1000bp, 750bp, 500bp, 250bp with
And six DNA segment compositions of 100bp；
Fig. 2 barleyHvERF2.11Differential expression of the gene in barley Different Organs and arabidopsis,
A: after wet injury stress, barleyHvERF2.11Differential expression of the gene in TF58 Different Organs, L: blade;AR: no
Determine root;SR: seminal root;NR: root section;S: tassel.B:HvERF2.11Gene is in different transgenic arabidopsis strains and open country
Differential expression in raw type strain；
The moisture-proof phenotypic difference of transgenic arabidopsis strain and WT strain under Fig. 3 submerging treatment 2 weeks and collating condition,
(A) plant strain growth phenotype；(B) plant height；(C) cauline leaf fresh weight；(D) cauline leaf dry weight；(E) root length；(F) survival rate；
The variation of Fig. 4 difference waterflooding period transgenic line and WT strain Physiological Index in Leaves,
A-E distinguishes SOD, CAT, POD, ADH enzymatic activity and proline content, and data indicate mean+SD, and test 3 is secondary
Object repeats.*With**Respectively represent the difference between transgenic line and wild type reach it is significant and it is extremely significant it is horizontal (*P <
0.05; * *P< 0.01)；
Fig. 5 difference wet injury coerces the differential expression of period transgenic arabidopsis and wild type moisture-proof related gene,
A-F is respectively representedAtSOD1Gene,AtCAT1Gene,AtPOD1Gene,AtADH1Gene,AtPDC1AndAtACO1Gene, arabidopsisAtActinThe reference gene that gene is expressed as different genes.Numerical value is duplicate average by 3
Value ± standard deviation indicates.*With**It respectively represents the difference between transgenic line and wild type and reaches significant and extremely significant level.
Specific embodiment
Invention is further described in detail combined with specific embodiments below.
Embodiment 1HvERF2.11The clone of gene
Design barleyHvERF2.11The special primer P of gene1Forward primer: 5'-CCAGTCAGAGATGGTCAAGG-3'(SEQ
ID NO.3) and P2Reverse primer: 5'-CATCCATCGTCTTGCTGAG-3'(SEQ ID NO.4), it is extracted using CTAB method resistance to
Wet stock TF58 root system total serum IgE, reverse transcription synthesize cDNA and utilize primer P using reverse transcription cDNA as template1And P2Amplify as
The CDS sequence of barley moisture-proof gene, gene shown in SEQ ID NO:1HvERF2.11CDS sequence 1158bp (figure
1)。
Specific step is as follows:
(1) CTAB (cetyl trimethylammonium bromide) Extraction buffer [2% (W/V) CTAB, NaCl is added into centrifuge tube
1 .4mol/L, EDTA (ethylenediamine tetra-acetic acid) 20mmol/L, TrisHCl 100mmol/L, 2% (W/V) PVP] and 10% β-
Mercaptoethanol preheats in water-bath；
(2) grind barley root system liquid nitrogen is cooling, be added in extracting solution, mix, 65 DEG C water-bath 10 minutes；
(3) isometric chloroform is added: isoamyl alcohol (volume ratio 24:1) mixed liquor is mixed by inversion, and stands 10min, and 4 DEG C
12000g is centrifuged 10min；
(4) supernatant is taken, is repeated step (3)；
(5) supernatant is taken, the LiCl of final concentration of 2mol/L, ice bath 10-12 hours, 11000rpm, 4 DEG C centrifugation 15min is added,
Supernatant is abandoned, cleans precipitating twice with 75% ethyl alcohol, is dissolved in stand-by in suitable DEPC (pyrocarbonic acid diethyl ester) processing water；
(6) it is template that root system total serum IgE is extracted from barley strain TF58, (is purchased from Thermo Fisher using reverse transcriptase
Scientific company) by its reverse transcription synthesize first chain of cDNA, reaction condition are as follows: 65 DEG C of 5min, 42 DEG C of 50min, 70 DEG C
10min；
(7) barley ethylene transcription factor gene is amplified from the cDNA that RNA reverse transcription obtains using above-mentioned primer P1 and P2HvERF2.11CDS sequence；
Reaction condition: 94 DEG C of initial denaturation 4min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min, 33 circulations；72 DEG C of extensions
10min.The PCR product that amplification obtains is connected into pMD18-T carrier (purchased from precious bioengineering Dalian Co., Ltd), converts large intestine
Bacillus competent cell, screening positive clone are simultaneously sequenced, and obtain required full-length gene.It extracts and carries from positive colonyHvERF2.11The plasmid of gene C DS sequence.
Embodiment 2HvERF2.11The building of gene overexpression carrier and genetic transformation
In order to preferably analyze geneHvERF2.11Biological function, further willHvERF2.11Gene is in arabidopsis
It realizes and is overexpressed, construct barley using the Gateway technology of Invitrogen companyHvERF2.11Gene overexpression vector.Benefit
It, will with BP Clonase enzymeHvERF2.11Gene is connect with pDONR221 carrier, is constructed entry vector, is then utilized
Entry vector is connect by LR Clonase enzyme with pB2GW7 carrier, and building containsHvERF2.11The overexpression of gene
Carrier.It will be contained by frozen-thawed methodHvERF2.11The overexpression vector of gene imports in Agrobacterium Gv3101.Utilize agriculture bar
The genetic transformation that bacterium mediates, willHvERF2.11Coded sequence imports in arabidopsis.It is sieved by antibiotic-screening and RT-PCR etc.
Choosing is positive to be turnedHvERF2.11Gene plant.
It is as follows that this tests practical genetic transforming method:
(1) culture of Agrobacterium
Firstly, in solid LB media (10g/L peptone, 5g/L yeast extract, 10g/L chlorine with corresponding resistance selection
Change sodium, Kan 100mg/L, agar 1.5g/L) on preculture carryHvERF2.11The Agrobacterium of gene 48 hours, culture temperature
28 DEG C of degree；Picking preculture Agrobacterium single colonie, be inoculated in the selection of corresponding resistance LB liquid medium (10g/L peptone,
5g/L yeast extract, 10g/L sodium chloride, Kan 100mg/L) in, it is stayed overnight in 28 DEG C of 200rpm shaking table cultures, until bacterial concentration
OD600 value is about the .0 of 0 .8~1.
(2) inflorescence method infects arabidopsis
Using the inflorescence infestation method arabidopsis thaliana transformation of improvement.Picking containsHvERF2.11The Agrobacterium single colonie of gene plasmid turns
Enter in 100mL LB liquid medium, is placed in 200r/min in 28 DEG C of shaking tables and is incubated overnight, reach Agrobacterium bacterial concentration
For OD600 in 0 .8~1 .0,4000r/min is centrifuged 10min, after abandoning supernatant, contains sucrose 5g, SilwetL-77 with 100mL
The LB liquid medium of 50 μ L suspension Agrobacterium bacterium solution again.It, will by the back-off of arabidopsis flowerpot in the beaker containing bacteria suspension
The inflorescence of arabidopsis is immersed in 10~20s in bacterium solution, and after the lower culture for 24 hours of dark, it is strong to be placed in 25 DEG C of temperature, humidity 60%, illumination
It is cultivated in 3000~4000lx of degree and the illumination box of 16h/8h photoperiod, waters and cultivate plant every other day, received to Post flowering
Obtain seed.After disinfection 5min in the arabidopsis seed of harvest 8%NaClO alcoholic solution (95% alcohol), selecting
It is screened on culture medium (MS+50mg/L kanamycins+7g/L agar+30g/L sucrose).
Embodiment 3HvERF2.11Gene transgenic T3 is detected for seedling RT-PCR
In order to verify transgenic arabidopsis T3 for strain moisture-proof ability change whether be transferred toHvERF2.11Gene is related,
Using RT-PCR method in partial transgenic Arabidopsis plantHvERF2.11Gene expression is detected, and as a result sees Fig. 2,
Known to transgenic arabidopsis T3 for strain moisture-proof ability change be transferred toHvERF2.11The overexpression of gene is related.
Specific step is as follows:
It is extracted from transgenic arabidopsis T3 3 strains of generation using TRIZOL reagent (purchased from precious bioengineering Dalian Co., Ltd)
The total serum IgE (extracting method is operated referring to TRIZOL reagent specification) of plant, (is purchased from Thermo Fisher using reverse transcriptase
Scientific company) by its reverse transcription synthesize first chain of cDNA, reaction condition be 65 DEG C of 5min, 42 DEG C of 50min, 70 DEG C
10min.Detection first is carried out to the cDNA that reverse transcription obtains with the reference gene Actin of report and concentration adjusts, carries out PCR inspection
It surveys, it is ensured that reference gene can expand in control wildtype Arabidopsis thaliana and transgenic Arabidopsis plants.Then, according toHvERF2.11The sequence of gene carries out RT-PCR detection, reaction condition are as follows: 94 DEG C of initial denaturation 4min using primer P1 and P2；
94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1 .5min, 33 circulations；72 DEG C of extension 10min.The Ago-Gel electricity of amplified production
Swimming is detected in 3 transgenic lines the result shows that (Fig. 2)HvERF2.11The expression of gene.
4 turns of embodimentHvERF2.11The identification of Arabidopsis plant moisture-proof
In order to further analyze barley moisture-proof geneHvERF2.11Biological function, which is realized in arabidopsis
Overexpression, and the quasi- phenotypic characteristic from transgenic plant moisture-proof verifies geneHvERF2.11In wet injury stress regulatory
On biological function.Steps are as follows for specific experiment: what in early period, screening was obtainedHvERF2.11What genetic transformation arabidopsis obtained
T3 is for the seed in positive transgenic strain, selecting each 3 strains at random, and arabidopsis wild type seeds (WT) is used in control, 1/
Culture is middle in the controlled environment chamber to cultivate 35 days by seedling replanting into the basin alms bowl equipped with Nutrition Soil to 4 leaf phases on 2MS culture medium.
Transgenic line and wild type are put into water tank, submerging treatment is carried out, after waterflooding 2 weeks, observes transgenic line and wild type
The difference of material moisture-proof, and take the expression analysis of the material progress moisture-proof related gene of different Submergence time sections and correlation raw
The measurement for managing index, after waterflooding, measures the characters such as height of seedling, plant fresh weight, dry weight, the root length of different materials.As a result
Show that the plant height of wild type has dropped 49.09%, and 3 transgenic lines have dropped 30.54%, 27.41%, 32.48% respectively；
Wild type chlorophyll content has dropped 61.56%, and 3 transgenic lines have dropped 29.52%, 35.26%, 31.37% respectively；It is wild
Raw type cauline leaf fresh weight has dropped 65.8%, and 3 transgenic lines have dropped 50%, 19.67%, 17.86% respectively；Wild type cauline leaf is dry
51.01% is had dropped again, and 3 transgenic lines have dropped 46.25%, 40.5%, 16.1% respectively；Wild type root long has dropped
74.87%, 3 transgenic lines have dropped 59.69%, 41.47%, 41.88% respectively；After wet injury processing, wildtype Arabidopsis thaliana
Survival rate is only 27.61%, and the survival rate of transgenic arabidopsis is up to 78% or more (Fig. 3), shows to be overexpressedHvERF2.11Base
Because clearly enhancing the moisture-proof of arabidopsis.
Control and wild type under wet injury treatment conditions and transgenic arabidopsis activities of antioxidant enzymes (SOD, CAT, POD), second
The variation of alcohol dehydrogenase (ADH) activity and proline content as shown in figure 4, under normal growing conditions, transgenic line with it is wild
Type is in terms of physiological-biochemical level without notable difference；Wet injury is handled 3 days, and antioxidase and alcohol dehydrogenase in wild type and turn
Obviously increased in gene strain, wet injury is handled 6 days, increase up to maximum value, transgenic line increasing degree be substantially higher in
Non-transgenic system；After wet injury is handled 9 days, antioxidase and alcohol dehydrogenase activity begin to decline (Fig. 4 A, B, C, D).It is wet
Evil causes the proline content of wild type and transgenic line to obviously increase, but the increasing degree of wild type will be significantly greater than and turn
Gene strain (Fig. 4 E).Show that wet injury stress is lower to be overexpressedHvERF2.11Antioxidant Enzyme Systems are clear in genes amplification arabidopsis
Except the efficiency of harmful substance, the resistance that arabidopsis coerces wet injury is improved.
For identificationHvERF2.11The regulating effect of gene pairs wet injury stress response related gene, using qRT-PCR to turning base
Because gene relevant to activities of antioxidant enzymes, glycolysis and ethylene synthase in arabidopsis and wild type (AtSOD1,AtCAT1,AtPOD1,AtADH1,AtPDC11,AtACO1) carry out gene expression analysis.As shown in figure 5, under normal growing conditions,AtPOD1WithAtACO1Differential expression of the gene between wild type and transgenic arabidopsis reaches the level of signifiance, remaining gene does not have
Apparent differential expression；After wet injury stress, the expression of 6 genes is aobvious in wildtype Arabidopsis thaliana and transgenic arabidopsis
Up-regulated expression is write, and the ascensional range in transgenic arabidopsis is bigger；Wet injury is handled 3 days,AtSOD1、AtPOD1WithAtACO1
Gene expression dose reaches maximum value, and processing starts to gradually decrease (Fig. 5 A, C, F) after 6 days and 9 days；Wet injury is handled 3 days,AtCAT1、AtADH1WithAtPDC1The gene expression dose rate of climb is slower, and after wet injury is handled 6 days, gene expression dose reaches
Highest, wet injury are handled 9 days, and expression starts to reduce (Fig. 5 B, D, E).ShowHvERF2.11The controllable antioxygen of gene
Change enzyme, fermentation, gene relevant to wet injury stress in ethylene synthase approach, enhances the resistance that arabidopsis coerces wet injury.
Basic principles and main features and advantages of the present invention of the invention have been shown and described above.The skill of the industry
Art personnel it should be appreciated that the present invention is not limited to the above embodiments, the above embodiments and description only describe
The principle of the present invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these
Changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and
Its equivalent thereof.
Sequence table
<110>Yangzhou University
<120>barley moisture-proof controlling gene HvERF2.11, albumen and its application in breeding
<130> xhx2019041602
<141> 2019-04-16
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1158
<212> DNA
<213> Hordeum vulgare L.
<400> 1
atgtgcggcg gagccatcct cgcgggattc atcccgccgt cggcggccgc ggcggcggcc 60
aaggcagcgg cgacggccaa gaagaagcag cagcagcgca gcgtgacggc agactcgctc 120
tggacgggcc tgcggaaaaa ggcggacgag gaggacttcg aggccgactt ccgcgacttc 180
gagcgggact ccagcgagga ggaggacgac gaggtcgagg aggtcccccc tccgccggcg 240
ccggcgacgg ccgggttcgc cttcgccgcc gcggccgagg tcgcgctcag ggcccctgcc 300
cgccgagatg ctgctgttca acatgatgga cctgctgcca aacaagtaaa gcgcgttcgg 360
aagaatcagt acagagggat ccgccagcgt ccctggggga aatgggcagc tgaaatccgt 420
gaccctagca agggtgtccg ggtttggctc gggacatacg acactgctga ggaggcagcc 480
agggcatatg atgctgaggc ccgcaagatc cgtggcaaga aagccaaggt caattttcct 540
gaggatgctc cgactgttca gaagtctacc ctgaagccaa ctgctgctaa atcagcaaag 600
ctagctccac ctccgaaggc ctgcgaggat cagcctttca atcatctgag cagaggagac 660
aatgatttgt tcgcgatgtt tgccttcagt gacaagaagg ttcctgcaaa gccaactgac 720
agtgtggatt cccttcttcc agtgaaacac cttgccccca ccgaggcatt cggaatgaac 780
atgctctctg accagagcag caattcattt ggctccactg actttgggtg ggacgacgag 840
gccatgaccc cggactacac gtccgtcttc gtaccgagtg ctgctgccat gccggcgtac 900
ggcgagcccg cttacctgca aggcggagct ccaaagagaa tgaggaacaa ctttggcgta 960
gctgtgctgc ctcagggaaa tggtgcacaa gacatccctg cttttgacaa tgaggtgaag 1020
tactcgttgc cctacgttga gagcagctcg gacggatcta tggacaacct tttgctgaat 1080
ggtgcgatgc aggatggggc aagcagtggg gatctctgga gcctcgatga gctgttcatg 1140
gcagctggtg gttattga 1158
<210> 2
<211> 385
<212> PRT
<213> Hordeum vulgare L.
<400> 2
Met Cys Gly Gly Ala Ile Leu Ala Gly Phe Ile Pro Pro Ser Ala Ala
1 5 10 15
Ala Ala Ala Ala Lys Ala Ala Ala Thr Ala Lys Lys Lys Gln Gln Gln
20 25 30
Arg Ser Val Thr Ala Asp Ser Leu Trp Thr Gly Leu Arg Lys Lys Ala
35 40 45
Asp Glu Glu Asp Phe Glu Ala Asp Phe Arg Asp Phe Glu Arg Asp Ser
50 55 60
Ser Glu Glu Glu Asp Asp Glu Val Glu Glu Val Pro Pro Pro Pro Ala
65 70 75 80
Pro Ala Thr Ala Gly Phe Ala Phe Ala Ala Ala Ala Glu Val Ala Leu
85 90 95
Arg Ala Pro Ala Arg Arg Asp Ala Ala Val Gln His Asp Gly Pro Ala
100 105 110
Ala Lys Gln Val Lys Arg Val Arg Lys Asn Gln Tyr Arg Gly Ile Arg
115 120 125
Gln Arg Pro Trp Gly Lys Trp Ala Ala Glu Ile Arg Asp Pro Ser Lys
130 135 140
Gly Val Arg Val Trp Leu Gly Thr Tyr Asp Thr Ala Glu Glu Ala Ala
145 150 155 160
Arg Ala Tyr Asp Ala Glu Ala Arg Lys Ile Arg Gly Lys Lys Ala Lys
165 170 175
Val Asn Phe Pro Glu Asp Ala Pro Thr Val Gln Lys Ser Thr Leu Lys
180 185 190
Pro Thr Ala Ala Lys Ser Ala Lys Leu Ala Pro Pro Pro Lys Ala Cys
195 200 205
Glu Asp Gln Pro Phe Asn His Leu Ser Arg Gly Asp Asn Asp Leu Phe
210 215 220
Ala Met Phe Ala Phe Ser Asp Lys Lys Val Pro Ala Lys Pro Thr Asp
225 230 235 240
Ser Val Asp Ser Leu Leu Pro Val Lys His Leu Ala Pro Thr Glu Ala
245 250 255
Phe Gly Met Asn Met Leu Ser Asp Gln Ser Ser Asn Ser Phe Gly Ser
260 265 270
Thr Asp Phe Gly Trp Asp Asp Glu Ala Met Thr Pro Asp Tyr Thr Ser
275 280 285
Val Phe Val Pro Ser Ala Ala Ala Met Pro Ala Tyr Gly Glu Pro Ala
290 295 300
Tyr Leu Gln Gly Gly Ala Pro Lys Arg Met Arg Asn Asn Phe Gly Val
305 310 315 320
Ala Val Leu Pro Gln Gly Asn Gly Ala Gln Asp Ile Pro Ala Phe Asp
325 330 335
Asn Glu Val Lys Tyr Ser Leu Pro Tyr Val Glu Ser Ser Ser Asp Gly
340 345 350
Ser Met Asp Asn Leu Leu Leu Asn Gly Ala Met Gln Asp Gly Ala Ser
355 360 365
Ser Gly Asp Leu Trp Ser Leu Asp Glu Leu Phe Met Ala Ala Gly Gly
370 375 380
Tyr
385
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ccagtcagag atggtcaagg 20
<210> 4
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
catccatcgt cttgctgag 19
Claims (8)
1. a kind of barley moisture-proof regulates and controls GAP-associated protein GAP, which is characterized in that the albumen is for following (a) or (b):
(a) protein that the amino acid sequence shown in SEQ ID NO:2 forms；
(b) by the amino acid sequence of SEQ ID NO:2 by one or several amino acid residues substitution and/or missing and/or
Addition and the protein as derived from SEQ ID NO:2 relevant to the regulation of barley moisture-proof.
2. encoding the gene of albumen described in claim 1.
3. gene according to claim 2, it is characterised in that: the gene is any in following (a1)-(a3)
DNA molecular；
(a1) DNA molecular shown in SEQ ID NO:1；
(a2) hybridize and the DNA molecular of encoding barley moisture-proof modulin with (a1) DNA sequence dna limited under strict conditions；
(a3) at least have 70% with (a1) DNA sequence dna limited, at least have 75%, at least with 80%, at least with 85%, extremely
Less with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% or at least have it is 99% homologous
The DNA molecular of property and encoding barley moisture-proof modulin.
4. expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing gene described in Claims 2 or 3.
5.(b1) or the application of (b2) or (b3):
(b1) albumen described in claim 1, or, gene described in Claims 2 or 3, or, containing gene described in Claims 2 or 3
Expression cassette, recombinant vector, recombinant microorganism or transgenic cell line, anti-barley wet injury stress in application；
(b2) albumen described in claim 1, or, gene described in Claims 2 or 3, or, containing gene described in Claims 2 or 3
Expression cassette, recombinant vector, recombinant microorganism or transgenic cell line, cultivating barley, answering in arabidopsis moisture-proof new varieties
With；
(b3) albumen described in claim 1, or, gene described in Claims 2 or 3, or, containing gene described in Claims 2 or 3
Expression cassette, recombinant vector, recombinant microorganism or transgenic cell line, anti-arabidopsis wet injury stress in application.
6. a kind of method for cultivating plant moisture-proof kind, which is characterized in that gene described in claim 2 or 3 is transferred to purpose
Plant or expression cassette as claimed in claim 4, recombinant vector convert purpose plant, obtain genetically modified plants；The transgenosis is planted
The wet injury stress resistance of object is higher than the purpose plant.
7. the method according to claim 6 for cultivating plant moisture-proof kind, which is characterized in that the purpose plant is big
Wheat, arabidopsis.
8. a kind of method for cultivating plant moisture-proof kind according to claim 6, which is characterized in that it is characterized in that, institute
The Expressed in Transgenic Plant stated barley moisture-proof described in claim 1 regulates and controls GAP-associated protein GAP.
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| CN114107333A (en) * | 2021-10-27 | 2022-03-01 | 上海市农业科学院 | Application of barley receptor kinase HvSERK1 in root hair growth |
| CN114262696A (en) * | 2021-11-16 | 2022-04-01 | 北京市农林科学院 | Plant flowering regulating related protein TaSOD and coding gene and application thereof |
| CN114874302A (en) * | 2022-05-27 | 2022-08-09 | 盐城师范学院 | Barley moisture-resistant gene HvMADS1, protein and application thereof in moisture stress resistance |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107333A (en) * | 2021-10-27 | 2022-03-01 | 上海市农业科学院 | Application of barley receptor kinase HvSERK1 in root hair growth |
| CN114262696A (en) * | 2021-11-16 | 2022-04-01 | 北京市农林科学院 | Plant flowering regulating related protein TaSOD and coding gene and application thereof |
| CN114262696B (en) * | 2021-11-16 | 2023-08-04 | 北京市农林科学院 | Plant flowering-regulating related protein TaSOD, and coding gene and application thereof |
| CN114014922A (en) * | 2022-01-05 | 2022-02-08 | 北京市农林科学院 | Protein for regulating and controlling plant salt tolerance, coding gene and application thereof |
| CN114874302A (en) * | 2022-05-27 | 2022-08-09 | 盐城师范学院 | Barley moisture-resistant gene HvMADS1, protein and application thereof in moisture stress resistance |
| CN114874302B (en) * | 2022-05-27 | 2023-08-01 | 盐城师范学院 | Barley moisture-resistant gene HvMADS1, protein and application thereof in moisture damage stress resistance |
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