CN110452917A - The application of bryony VyGOLS gene and its coding albumen in drought stress - Google Patents
The application of bryony VyGOLS gene and its coding albumen in drought stress Download PDFInfo
- Publication number
- CN110452917A CN110452917A CN201910872930.9A CN201910872930A CN110452917A CN 110452917 A CN110452917 A CN 110452917A CN 201910872930 A CN201910872930 A CN 201910872930A CN 110452917 A CN110452917 A CN 110452917A
- Authority
- CN
- China
- Prior art keywords
- gene
- vygols
- plant
- bryony
- drought
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01123—Inositol 3-alpha-galactosyltransferase (2.4.1.123), i.e. galactinol-synthase
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to bryoniesVyGOLSThe application of gene and its coding albumen in drought stress, belongs to field of plant genetic project technology.The transgenic technology that strong promoter (cauliflower mosaic virus 35 S promoter) driving principle is utilized in the present invention, by Yanshan Mountain grapeVyGOLSThe overexpression carrier of gene is transferred in arabidopsis, to obtain transgenic Arabidopsis plants；It is demonstrated experimentally that the Arabidopsis plant relative to conversion empty carrier, overexpressionVyGOLSGene leads to the expression of the accumulation of degeneration-resistant related substances and gene related to drought tolerance in transgenic arabidopsis, the enhancing of transgenic plant drought resistance.Therefore, bryonyVyGOLSGene and its recombinant expression carrier can be used in plant drought breeding for quality.
Description
Technical field
The present invention relates to bryoniesVyGOLSThe application of gene and its coding albumen in drought stress, belongs to plant gene
Field of engineering technology.
Background technique
Grape is many kinds of, has important edible value and economic value.Arid (Drought) develops grape growth
Process and yield and quality have serious influence, have become one of the principal element for restricting grape growth and improving fruit quality,
The especially frequent appearance of the variation of global climate in recent years and south China arid, makes grape industry by very big threat.
Under the overall background for worldwide all suffering from water shortage problem, drought resisting grape resource, research grape anti-drought gene are excavated to mentioning
High grape drought resistance cultivates drought resisting new varieties and water-saving culture etc. all with important scientific value and meaning.
The drought resistance (Drought resistance) of grape is the quantitative character by controlled by multiple genes, to adverse circumstances such as arids
Response and the participation for meeting the needs of multiple genes, and the regulation by number of ways.In mild drought stress, osmotic adjustment is
Plant adapts to the main path of water deficit, on the one hand plant improves the transcription water of synthesis osmotic adjustment relative enzyme gene
It is flat, promote the expression of gene, increases the accumulation of expression product；The enhancing of another aspect plant is anti-oxidant, detoxifies relative enzyme gene (such as
SOD, CAT, GST etc.) expression.When coercive intensity exceeds the ability of osmotic adjustment, the dehydration of LEA albumen and carbohydrate
Protective substances accumulation, protects large biological molecule and biofilm system from damage.The drought-resistant ability of grape depends on itself as a result,
Institutional framework and physiological property, be the difference of molecular level after all.With the announcement of grape whole genome sequence, perhaps
Gene mostly relevant to drought stress and its regulatory factor have been separated and have been cloned, and are transformed into grape or model plant
And its function is analyzed and verified.
GOLS is the key that gossypose synthesis rate-limiting enzyme.Currently, having identified 7 in arabidopsisGOLSGene and
Three possibleGOLSGene,AtGOLS1It is arid, with high salt and high temperature stress inducing expression；AtGOLS2By arid and the side of body with high salt
Compel inducing expression；AndAtGOLS3Gene is only by low temperature stress inducing expression.The overexpression in arabidopsisAtGOLS2Gene makes
Galactinol and gossypose are largely accumulated in Transgenic plant leaf, and significantly improve plant drought-resistant ability and Strong oxdiative
The tolerance of agent-methyl viologen (MV).It overexpresses in riceAtGOLS2, render transgenic rice drought-resistance ability significantly improves, together
When under drought condition transgenic paddy rice strain yield be significantly higher than control.By the galactinol synthase in a salt mustard
(TsGOLS2) import in arabidopsis, improve the salt tolerant of transgenic arabidopsis and ability (Selvaraj, the M. of resistance to osmotic stress
G., et al., (2017). Overexpression of an Arabidopsis thaliana galactinol
synthase gene improves drought tolerance in transgenic rice and increased
Grain yield in the field. Plant biotechnology journal, 15 (11), 1465-1477.).With
Qiang Guang, high temperature handles simultaneously or hydrogen peroxide treatment Arabidopsis plant can makeAtGOLS1WithAtGOLS2Gene mRNA expression amount
Rise.ArabidopsisAtGOLS1The mutant atgols1 of gene can't detect galactinol by high temperature stress rear blade
With cotton seed sugar accumulation, this result shows thatAtGOLS1The function of gene may be responsible for synthesizing inositol gala under heat-shock stress
Glucosides.In tobacco overexpress clover galactinol synthase gene (MfGOLS1) improve transgene tobacco it is cold-resistant and
Resistance to penetrating power, while RFOs content significantly improves in transgenic plant body.
Although having had in some species in the prior artGOLSThe report of gene, but in bryonyGOLSBase
Because not yet someone reports that people are for bryonyGOLSThe concrete function of gene also lacks understanding.
Summary of the invention
The object of the present invention is to provide a kind of bryoniesVyGOLSGene can increase degeneration-resistant correlation in transgenic plant
The accumulation of substance and the expression of gene related to drought tolerance promote the enhancing of transgenic plant drought resistance.
The present invention also provides bryony VyGOLS albumen, which can promote to accumulate degeneration-resistant correlation in transgenic plant
Substance causes transgenic plant drought resistance to enhance.
The present invention also provides include bryonyVyGOLSThe recombinant expression carrier of gene, the carrier carry bryonyVyGOLSGene, therefore can overexpressVyGOLSGene, and then enhance plant drought resistance.
The present invention also provides above-mentioned to include bryonyVyGOLSThe preparation method of the recombinant expression carrier of gene, can
The carrier is made.
The present invention also provides above-mentioned bryoniesVyGOLSGene and recombinant expression carrier are in plant variety breeding
Using drought resistant plant variety can be obtained.
To achieve the goals above, the technical scheme adopted by the invention is that:
BryonyVyGOLSGene, the amino acid sequence of coding is as shown in SEQ ID NO.2.
The transgenic technology of strong promoter (cauliflower mosaic virus 35 S promoter) driving principle is utilized in the present invention, it willVyGOLSThe overexpression carrier of gene is transferred in arabidopsis, to obtain transgenic Arabidopsis plants；It is demonstrated experimentally that relative to
Convert the Arabidopsis plant of empty carrier, overexpressionVyGOLSGene leads to the accumulation of degeneration-resistant related substances in transgenic arabidopsis
With the expression of gene related to drought tolerance, the enhancing of transgenic plant drought resistance.
BryonyVyGOLSGene, nucleotide sequence is as shown in the position 186-1196 in SEQ ID NO.1.
Above-mentioned nucleotides sequence is classified as naturally occurring sequence in bryony, and it is excellent can also to carry out codon according to the sequence
Change, obtained optimization also has same effect.
Bryony VyGOLS albumen, amino acid sequence is as shown in SEQ ID NO.2.
Bryony VyGOLS albumen is the albumen for containing 336 amino acid, which can promote in transgenic plant
Accumulating degeneration-resistant related substances causes transgenic plant drought resistance to enhance.
Recombinant expression carrier, the recombinant expression carrier include bryonyVyGOLSGene, the bryonyVyGOLSBase
The nucleotide sequence of cause is as shown in the position 186-1196 in SEQ ID NO.1.
Recombinant expression carrier in the present invention is plant Overexpression vector, and target gene can be overexpressed in plant.
The preparation method of recombinant expression carrier, comprising: according to the sequence as shown in the position 186-1196 in SEQ ID NO.1
Design primer clones the bryonyVyGOLSGene, then by the bryonyVyGOLSGene is connected to pCAMBIA2300
On plant expression vector to get.
By bryony in the present inventionVyGOLSGene open reading frame is connected to plant Overexpression vector pCAMBIA2300
On, it is capable of forming recombinant expression carrier pCAMBIA2300-VyGOLS.
Above-mentioned bryonyVyGOLSApplication of the gene in plant variety breeding；Specifically, being educated in plant drought kind
Application in kind；More specifically, the application in arabidopsis drought-resistant variety breeding.Above-mentioned recombinant expression carrier is in plant
Application in breeding for quality；Specifically, the application in plant drought breeding for quality；More specifically, in arabidopsis drought resisting product
Application in kind breeding.
The present invention is cloned and isolated from the grape of the Yanshan Mountain and is completely compiled with gene related to drought tolerance by plant gene engineering technology
The DNA fragmentation of code section, and the function of the gene is demonstrated, degeneration-resistant correlative in transgenic arabidopsis after discovery overexpression
The accumulation of matter and the expression of gene related to drought tolerance, the enhancing of transgenic plant drought resistance.Therefore, bryonyVyGOLSGene and its again
Group expression vector can be used in plant drought breeding for quality.
Detailed description of the invention
Fig. 1 is bryony in the present inventionVyGOLSGene expression characteristics analysis chart；
Fig. 2 is bryony in the present inventionVyGOLSThe qualification figure of gene overexpression carrier；
Fig. 3 is transfer of the present inventionVyGOLSThe PCR qualification figure of gene Arabidopsis plant；
Fig. 4 is transfer of the present inventionVyGOLSThe Identification of Drought figure of gene Arabidopsis plant；
Fig. 5 is transfer of the present inventionVyGOLSThe Physiological Characteristics Analysis figure of gene Arabidopsis plant；
Fig. 6 is the expression analysis figure of transgenic Arabidopsis plant medium drought resistant related gene of the present invention.
Specific embodiment
The present invention is described in further detail combined with specific embodiments below.It is each to implement in addition to specified otherwise
Equipment used in example and test example and reagent are commercially available.
BryonyVyGOLSThe embodiment 1 of gene
Bryony in the present embodimentVyGOLSGene, nucleotide sequence is as shown in the position 186-1196 in SEQ ID NO.1.
The embodiment 1 of bryony VyGOLS albumen
Bryony VyGOLS albumen in the present embodiment, amino acid sequence is as shown in SEQ ID NO.2.
The embodiment 1 of recombinant expression carrier
Recombinant expression carrier includes bryony in the present embodimentVyGOLSGene, the bryonyVyGOLSThe nucleotides sequence of gene
Column are as shown in the position 186-1196 in SEQ ID NO.1.
The embodiment 1 of the preparation method of recombinant expression carrier
The preparation method of recombinant expression carrier in the present embodiment, comprising: according to as shown in the position 186-1196 in SEQ ID NO.1
Primers, clone the bryonyVyGOLSGene, then by the bryonyVyGOLSGene is connected to
On pCAMBIA2300 plant expression vector to get.
BryonyVyGOLSThe embodiment 1 of application of the gene in plant variety breeding
Bryony in the present embodimentVyGOLSGene can increase the accumulation and drought resisting phase of degeneration-resistant related substances in transgenic plant
The expression of correlation gene promotes the enhancing of transgenic plant drought resistance, therefore can be used for the application in plant drought breeding for quality, has
The breeding that can be used for arabidopsis drought-resistant variety of body.
The embodiment 1 of application of the recombinant expression carrier in plant variety breeding
Recombinant expression carrier includes bryony in the present embodimentVyGOLSGene, therefore can be transformed into plant, with
To the plant variety of drought resisting.
1 grape of test exampleVyGOLSThe expression characterization of gene is analyzed
After 16 d of Yanshan Mountain Tissue culture the seedling of grape squamous subculture, robust growth is selected to show consistent seedling for the processing of various adverse circumstances.
Osmotic treatment: grape seedling is extracted from culture medium, and being placed on filter paper and being exposed to room temperature is (32 ± 1) DEG C, phase
It is 55% to humidity, handles under conditions of the photoperiod is 10 h of illumination 14 h/ dark, is sampled in 0,2,6,12,24 h.
Low-temperature treatment: tissue-cultured seedling is placed in temperature is (4 ± 1) DEG C, relative humidity 75%, photoperiod are that 14 h/ of illumination is black
It cultivates under conditions of dark 10 h, is sampled in 0,2,6,12,24 h.
Salt stress: 20 mL, 100 mmolL is added in triangular flask-1NaCl solution, temperature be (25 ± 1) DEG C,
It cultivates under conditions of relative humidity is 75%, the photoperiod is 10 h of illumination 14 h/ dark, is sampled in 0,2,6,12,24 h.
The control that isometric distilled water is handled as salt stress is added in triangular flask.The tissue-cultured seedling conduct normally cultivated
The control of arid and low-temperature treatment.
In the Yanshan Mountain grape of 8 ~ 10 a of grown in field, grape fruit is taken in veraison, taking root system in full-bloom stage, (first is newborn
Lateral root), stem (stem section of the 4th ~ 5 leaf under new expansion leaf), leaf (the 4th ~ 5 under new expansion leaf), inflorescence and tendril (newborn branch
The 1st branch) etc. samples.Grape leave total serum IgE is extracted with plus plant total RNA extraction reagent box (Tiangeng).Common reverse transcription
Use PrimeScriptII1st Strand cDNA Synthesis Kit(TaKaRa) synthesis the first chain of cDNA.
Specific steps are as follows: being added in PCR pipe: mers(50 μM of Random 6) 1 μ L, dNTP Mixture
2 μ g, RNase free dH of (10 mM each) 1 μ L, Total RNA2O polishing mixes well, brief centrifugation to 10 μ L
Make solution to PCR pipe bottom.65 DEG C of 5 min of reaction in PCR instrument, on ice chilling.
According toVyGOLSGene order designs real-time fluorescence quantitative PCR primer,
Forward primer sequence is qRT-VyGOLS-F:5'-GGGGACTATGTGAAAGGGGTT-3'(such as SEQ ID NO.3 institute
Show)；
Reverse primer sequences are qRT-VyGOLS-R:5'-GGATTTGGTTCTCAGGAGGG-3'(as shown in SEQ ID NO.4).
WithVyGAPDHGene is internal reference,
Forward primer sequence is qRT-VyGAPDH-F:5'-CCCTTGTCCTCCCAACTCT-3'(as shown in SEQ ID NO.5)；
Reverse primer sequences are qRT-VyGAPDH-R:5'-CCTTCTCAGCACTGTCCCT-3'(as shown in SEQ ID NO.6).
Real-time fluorescence quantitative PCR is according to TaKaRa SYBR Premix Ex Taq II (Perfect Real
Time) illustrate in Bio-Rad IQ5 Real-Time PCR Detection System(Bio-Rad Laboratories,
Herc μ Les, CA) on carry out.The reaction system of 25 μ L: the reverse transcription template of 1 μ L；Forward and reverse each 1 μ L of primer； 12.5 μ
2 × SYBR Premix Ex Taq (2 ×) of L；The nuclease-free water of 9 μ L.Response procedures are as follows: 95 DEG C,
30 s；40 cycles of 95℃ for 5 s；57℃ for 30 s；72℃ for 30 s.As a result 2 are used-ΔΔC(t)Method into
Row analysis.
As a result as shown in Figure 1, the results showed thatVyGOLSGene is mainly expressed in root system, secondly in leaf expression quantity compared with
Height, the expression quantity in stem, flower, fruit, tendril are lower；The 2h after low temperature, arid, high salt treatment,VyGOLSTranscript quickly accumulates
It is tired, in the main road 6h peak value, then gradually decrease.
2 bryony of test exampleVyGOLSThe building of gene overexpression carrier
To study grapeVyGOLSThe function of gene will includeVyGOLSThe ORF piece of total 1011bp including gene coding region
Section is correctly inserted on plant Overexpression vector pCAMBIA2300- GFP.
It is cloned into according to early periodVyGOLSGene ORF sequence, design can expandVyGOLSThe upstream and downstream of gene ORF is drawn
ObjectVyGOLS- ORF-F andVyGOLS-ORF-R；According to the restriction enzyme site on pCAMBIA2300-GFP carrier, in primerVyGOLS5 ' the ends of-ORF-F add restriction enzyme siteXbaI, particular sequence are as follows:
VyGOLS-ORF-XbaI-F:5 '-GGGTCTAGAATGGCCCCAGGAGTGCCCGCAGA-3 ' is (such as SEQ ID NO.7 institute
Show)；
In primerVyGOLS5 ' the ends of-ORF-R add restriction enzyme siteKpnI, particular sequence are as follows:
VyGOLS-ORF-KpnI-R:5 '-GGGGGTACCTCAAGCAGCAGAGGGTGCGGGAA-3 ' is (such as SEQ ID NO.8 institute
Show).
With pMD18-T-VyGOLSPlasmid is template, is usedVyGOLS-ORF-XbaI-F withVyGOLS-ORF-KpnI-R is carried out
Amplification is connected to pMD19-T cloning vector after recycling purpose band, converts TOP10 competent cell, in the LB training of additional Amp
It supports and carries out blue hickie screening on base, detected respectively by bacterium solution PCR and plasmid enzyme restriction, pMD19-T-VyGOLSPositive colony send public affairs
Department's sequencing.WithXbaI、KpnI double digestion recombinant cloning vector pMD19-T-VyGOLSWith plant expression vector pCAMBIA2300-
GFP recycles linearized vector and target fragment, connects and convert TOP10, and through Kan antibiotic-screening, picking monoclonal shakes bacterium,
Plasmid enzyme restriction detection is proposed after bacterium solution detection.Testing result is as shown in Fig. 2, M:DNA molecular mass standard；Swimming lane 1:VyGOLSGene
The double digestion of Overexpression vector is identified；Swimming lane 2:VyGOLSGene overexpression carrier；The result shows that being constructed in this test example
Success plant expression vector pCAMBIA2300-VyGOLS.It is transformed into Agrobacterium, for transfecting plant.
3 grape of test exampleVyGOLSOverexpression of the gene in arabidopsis
By containing recombinant plant expression vector Agrobacterium scribing line culture LB plate (Gent containing 60 mg/L, 100 mg/L's
Kan it crosses on), 24 h is cultivated under the conditions of being placed in 28 DEG C；Picking monoclonal is (additional corresponding anti-in 10 mL LB liquid mediums
Raw element) in, 24 h are cultivated under the conditions of 28 DEG C；5 mL bacterium solutions are taken to be transferred in the fresh LB liquid medium of 50 mL, at 28 DEG C
Under the conditions of continue to cultivate, until bacterium solution OD600 reaches 0.6 or so；It is transferred in centrifugal bottle or centrifuge tube, under room temperature, revolving speed
10 min are centrifuged for 4000 rpm, remove supernatant collection thallus；Be resuspended in permeabilization buffer (0.5 × MS, 5 % sucrose,
0.03% Silwet L-77(GE Health)), adjust OD600 to 0.8；Fruit pod existing in arabidopsis floral is removed, inflorescence
Be completely immersed in penetrating fluid 10-30 s(or with pipettor directly will infiltration drop on inflorescence), remove immediately arabidopsis leaf or
Penetrating fluid in stalk, plant is lain in pallet, is covered pallet with plastic film, film is removed after 24 h, in greenhouse
Continue to cultivate；To improve transformation efficiency, infected again after 7 days with same method；Arabidopsis plant by conversion carries out just
Often management carries out sowing when fruit pod existing white.
To what is obtained above by kanamycins preliminary screeningVyGOLSTransgenic plant and conversion empty carrier plant, into one
Step is identified in DNA level, extracts total DNA using improved SDS Trace bio-element method.Respectively with above extractedVyGOLSTransgenic plant and the DNA for converting empty carrier plant are template, upstream primer are designed on 35S promoter, with gene
Special downstream primer forms primer pair, carries out PCR detection.Primer is as follows:
Detection primer-F:5 '-GAAGATGCCTCTGCCGACAGTG-3 ' (as shown in SEQ ID NO.9)；
Detection primer-R:5 '-AGTACTCCGTCGGCTACTGCCA-3 ' (as shown in SEQ ID NO.10).Reaction system (25 μ
L) are as follows: 10 × buffer, 2.5 μ L；d NTPs 0.5 µL；0.3 μ L of Taq enzyme；ddH2O 16.2 µL；Primer F 1.5
µL；Primer R 1.5 µL；DNA 2.5 µL.Response procedures are as follows: 94 DEG C of 5 min of initial denaturation；35 circulations, 94 DEG C of denaturation
30 S, 58 DEG C of annealing 30 S, 72 DEG C of 1 min of extension；72 DEG C of extension 10min, 4 DEG C of preservations.PCR product is solidifying in 1% agarose
Electrophoresis detection is carried out on glue.
Testing result is as shown in figure 3, M:DNA molecular mass standard；Swimming lane 1: with ddH2O is that template carries out PCR；Swimming lane 2:
Arabidopsis DNA to convert empty carrier carries out PCR as template；Swimming lane 3: using pCAMBIA2300-VyGOLS Plasmid DNA as template
Carry out PCR；Swimming lane 4-6: the transgenic arabidopsis DNA to convert VyGOLS gene carries out PCR as template.It can from figure
Out, this test example, which has successfully been obtained, turnsVyGOLSGene Arabidopsis plant, is respectively designated as OE#1, OE#2, OE#3.
The Identification of Drought of 4 transgenic Arabidopsis plants of test example
VyGOLSAfter the arabidopsis (EV expression) of transgenic plant and conversion empty carrier is grown 7 days on MS culture medium, transfer
Into nutritive cube, normal its seedling for growing into stalwartness of 20 angels of watering.Then stop watering to Arabidopsis thaliana Seedlings and be done
Drought processing, until the 7th day part Arabidopsis plant blade occur apparent dehydration wither here symptom.All plant are carried out later
Rehydration observed plant growth condition after 48 hours.
The phenotype of Arabidopsis plant is by photographing to record behind Osmotic treatment front and back and rehydration, as a result as shown in figure 4, from figure
In 4 it can be seen that compared with converting empty carrier arabidopsis, turnVyGOLSThe drought resisting of gene Arabidopsis plant OE#1, OE#2, OE#3
Ability is remarkably reinforced.
5 transgenic Arabidopsis plants Analysis of The Physiological And Biochemical Properties of test example
The measurement of percentage of water loss:VyGOLSTransgenic plant takes about 0.2 g with after conversion empty carrier normal plants 3 weeks respectively
Lotus throne leaf carry out percentage of water loss measurement.The lotus throne leaf taken is placed on dry filter paper, it is primary every 10 min measurement
The fresh weight (FW) of blade, percentage of water loss measurement terminates when surveying to 50 min.By the fluid loss measured each time and survey for the first time
The ratio of fixed fresh weight is as percentage of water loss.
The measurement of Electrolytic leakage (conductivity): blade is fitted into centrifuge tube, is settled to 10 with super deionized water
ML measures the electric conductivity value of solution, is denoted as and boils preceding C1 after vibrating 1 hour at room temperature.Then solution is placed in boiling water together with blade
After boiling 10 min, etc. temperature be cooled to room temperature measurement electric conductivity value, be denoted as C2.By the ratio (C1/C2) of C1 and C2 as opposite
Electrolyte leakage value.
Testing result is as shown in figure 5, the expression quantity of VyGOLS gene detects in (A) transgenic Arabidopsis plants；(B) turn
Survival rate statistics of the VyGOLS gene Arabidopsis plant after Osmotic treatment 18d；(C) it is opposite to turn VyGOLS gene Arabidopsis leaf
Percentage of water loss；(D) turn relative conductivity of the VyGOLS gene Arabidopsis plant after Osmotic treatment 18d.It can be seen from the figure that turning
VyGOLS gene expression amount is higher in gene Arabidopsis plant, and survival rate is significantly improved relative to the plant of conversion empty carrier；Phase
For converting empty carrier plant, turning percentage of water loss and conductivity in VyGOLS gene Arabidopsis plant is significantly reduced.
6 transgenic arabidopsis gene related to drought tolerance expression analysis of test example
Transgenic arabidopsis blade total serum IgE after extracting Osmotic treatment with plus plant total RNA extraction reagent box.Common reverse transcription
Use PrimeScriptII1st Strand cDNA Synthesis Kit(TaKaRa) synthesis the first chain of cDNA.Concrete operations step
It is rapid as follows: to be added in PCR pipe: mers(50 μM of Random 6) 1 μ L, dNTP Mixture(10 mM each) 1 μ L,
Total RNA 2 μ g, RNase free dH2O polishing to 10 μ L, mixes well, brief centrifugation makes solution to PCR pipe bottom.
65 DEG C of 5 min of reaction in PCR instrument, on ice chilling.Using arabidopsis AtActin as reference gene, detection transgenic arabidopsis is planted
Strain medium drought resistant related geneAtCOR15A、AtERD15、AtRD29A、AtP5CS1The expression of gene.The designed following institute of primer
Show:
QRT-AtActin-F:5 '-CGGTGGTTCTATCTTGGCATC-3 ' (as shown in SEQ ID NO.11)；
QRT-AtActin-R:5 '-GTCTTTCGCTTCAATAACCCTA-3 ' (as shown in SEQ ID NO.12)；
qRT-AtCOR15A- F:5 '-CAGCGGAGCCAAGCAGAGCAG-3 ' (as shown in SEQ ID NO.13)；
qRT-AtCOR15A- R:5 '-CATCGAGGATGTTGCCGTCACC-3 ' (as shown in SEQ ID NO.14)；
qRT-AtERD15-F:5 '-CCAGCGAAATGGGGAAACCA-3 ' (as shown in SEQ ID NO.15)；
qRT-AtERD15-R:5 '-ACAAAGGTACAGTGGTGGC-3 ' (as shown in SEQ ID NO.16)；
qRT-AtRD29A-F:5 '-GTTACTGATCCCACCAAAGAAGA-3 ' (as shown in SEQ ID NO.17)；
qRT-AtRD29A-R:5 '-GGAGACTCATCAGTCACTTCCA-3 ' (as shown in SEQ ID NO.18)；
qRT-AtP5CS1- F:5 '-CGACGGAGACAATGGAATTGT-3 ' (as shown in SEQ ID NO.19)；
qRT-AtP5CS1- R:5 '-GATCAGAAATGTGTAGGTAGC-3 ' (as shown in SEQ ID NO.20).
Real-time fluorescence quantitative PCR is according to TaKaRa SYBR Premix Ex Taq II(Perfect Real
Time) illustrate in Bio-Rad IQ5 Real-Time PCR Detection System(Bio-Rad Laboratories,
Herc μ Les, CA) on carry out.The reaction system of 25 μ L: the reverse transcription template of 1 μ L；Forward and reverse each 1 μ L of primer； 12.5 μ
2 × SYBR Premix Ex Taq (2 ×) of L；The nuclease-free water of 9 μ L.Response procedures are as follows: 95 DEG C,
30 s；40 cycles of 95℃ for 5 s；57℃ for 30 s；72℃ for 30 s.As a result 2 are used-ΔΔC(t)Method into
Row analysis.
Testing result is as shown in fig. 6, from fig. 6 it can be seen that under drought condition, with conversion empty carrier arabidopsis phase
Than transgenic Arabidopsis plants medium drought resistant related gene expression amount is significantly raised, shows the bryony in the present inventionVyGOLSBase
Cause can increase the expression of the accumulation of degeneration-resistant related substances and gene related to drought tolerance in transgenic plant, promote transgenic plant
Drought resistance enhancing.
<110>University Of Science and Technology Of He'nan
<120>bryonyVyGOLSThe application of gene and its coding albumen in drought stress
<160> 20
<170> SIPOSequenceListing 1.0
<211> 1011
<212> DNA
<213>bryony
<221> VyGOLSGene
<400> 1
gcgaaaccgc cccctttttg ttggtacccg ggaaaccggc cattacggcc ggggaggaac 60
aaaggcaaag tagggtggca tccacagtgt tgctggttta ctttcccaac cctcctcacc 120
accaactctc tccttaacat ttttcttgca ccaacttgaa atctcacccc aaataaacca 180
caacaatggc cccaggagtg cccgcagatg tgtttacagc cggcggaaag gtttccaccc 240
tcaacgcagg ctactcaaag ggggcctacg tcacattttt agctggaaac ggggactatg 300
tgaaaggggt tgttgggttg gctaagggtt tgcgcaaggt gaagagcgcg taccctcttg 360
tggttgcaat gttgccggat gtgcctgagg agcaccgtga gatcttaaag tctcagggct 420
gcataattcg tgaaattgag cccatctacc ctcctgagaa ccaaatccag tttgcaatgg 480
catactacgt catcaactat tccaaactcc gtatttggaa tttcgaggaa tacagcaaga 540
tggtgtattt ggatgctgat atccaagttt acgacaacat agaccacctt atggacgccc 600
cggacggcta cttttacgcg gtaatggact gcttctgtga gaagacatgg agtcacactc 660
cccagtactc cgtcggctac tgccagcagt gcccggacaa ggtgacttgg cccgctgaga 720
tgggttcacc tccacctttg tacttcaacg ctgggatgtt cgtctttgag cctagccgtt 780
tgacttatga aagccttctc catactctac ggatcactcc tccgaccgcc tttgccgagc 840
aagatttctt gaacatgttc ttccaacaca tgtacaagcc catccctctc gtatacaact 900
tggttctagc aatgctgtgg cgccacccgg agaacgttga gctcgaccag gtcaaggtgg 960
tgcactactg tgctgctgga tcaaagccat ggagatacac tgggaaagaa gcaaacatgg 1020
agagagagga catcaagatg ttggtagcca aatggtggga catttacaat gataagtctc 1080
tggatttcaa ggctgaggac agtgttccag agggagaagg attctctagg ccatcgatca 1140
tggcttccat gcctgagcct gcaatctcct atattcccgc accctctgct gcttgaagat 1200
tacaaatctt taggagagag tgtattgaag ctcagggtgt gatctatctc tttttctatt 1260
taataccttt tccaaaggct acttggt 1287
<211> 336
<212> PRT
<213>bryony
<221>VyGOLS albumen
<400> 2
MET Ala Pro Gly Val Pro Ala Asp Val Phe Thr Ala Gly Gly Lys
1 5 10 15
Val Ser Thr Leu Asn Ala Gly Tyr Ser Lys Gly Ala Tyr Val Thr
20 25 30
Phe Leu Ala Gly Asn Gly Asp Tyr Val Lys Gly Val Val Gly Leu
35 40 45
Ala Lys Gly Leu Arg Lys Val Lys Ser Ala Tyr Pro Leu Val Val
50 55 60
Ala MET Leu Pro Asp Val Pro Glu Glu His Arg Glu Ile Leu Lys
65 70 75
Ser Gln Gly Cys Ile Ile Arg Glu Ile Glu Pro Ile Tyr Pro Pro
80 85 90
Glu Asn Gln Ile Gln Phe Ala MET Ala Tyr Tyr Val Ile Asn Tyr
95 100 105
Ser Lys Leu Arg Ile Trp Asn Phe Glu Glu Tyr Ser Lys MET Val
110 115 120
Tyr Leu Asp Ala Asp Ile Gln Val Tyr Asp Asn Ile Asp His Leu
125 130 135
MET Asp Ala Pro Asp Gly Tyr Phe Tyr Ala Val MET Asp Cys Phe
140 145 150
Cys Glu Lys Thr Trp Ser His Thr Pro Gln Tyr Ser Val Gly Tyr
155 160 165
Cys Gln Gln Cys Pro Asp Lys Val Thr Trp Pro Ala Glu MET Gly
170 175 180
Ser Pro Pro Pro Leu Tyr Phe Asn Ala Gly MET Phe Val Phe Glu
185 190 195
Pro Ser Arg Leu Thr Tyr Glu Ser Leu Leu His Thr Leu Arg Ile
200 205 210
Thr Pro Pro Thr Ala Phe Ala Glu Gln Asp Phe Leu Asn MET Phe
215 220 225
Phe Gln His MET Tyr Lys Pro Ile Pro Leu Val Tyr Asn Leu Val
230 235 240
Leu Ala MET Leu Trp Arg His Pro Glu Asn Val Glu Leu Asp Gln
245 250 255
Val Lys Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro Trp Arg
260 265 270
Tyr Thr Gly Lys Glu Ala Asn MET Glu Arg Glu Asp Ile Lys MET
275 280 285
Leu Val Ala Lys Trp Trp Asp Ile Tyr Asn Asp Lys Ser Leu Asp
290 295 300
Phe Lys Ala Glu Asp Ser Val Pro Glu Gly Glu Gly Phe Ser Arg
305 310 315
Pro Ser Ile MET Ala Ser MET Pro Glu Pro Ala Ile Ser Tyr Ile
320 325 330
Pro Ala Pro Ser Ala Ala
335 336
<211> 21
<212> DNA
<213>artificial sequence
<221> qRT-VyGOLS-F
<400> 3
ggggactatg tgaaaggggt t 21
<211> 20
<212> DNA
<213>artificial sequence
<221> qRT-VyGOLS-R
<400> 4
ggatttggtt ctcaggaggg 20
<211> 19
<212> DNA
<213>artificial sequence
<221> qRT-VyGAPDH-F
<400> 5
cccttgtcct cccaactct 19
<211> 19
<212> DNA
<213>artificial sequence
<221> qRT-VyGAPDH-R
<400> 6
ccttctcagc actgtccct 19
<211> 32
<212> DNA
<213>artificial sequence
<221> VyGOLS-ORF-XbaI-F
<400> 7
gggtctagaa tggccccagg agtgcccgca ga 32
<211> 32
<212> DNA
<213>artificial sequence
<221> VyGOLS-ORF-KpnI-R
<400> 8
gggggtacct caagcagcag agggtgcggg aa 32
<211> 22
<212> DNA
<213>artificial sequence
<221>detection primer-F
<400> 9
gaagatgcct ctgccgacag tg 22
<211> 22
<212> DNA
<213>artificial sequence
<221>detection primer-R
<400> 10
agtactccgt cggctactgc ca 22
<211> 21
<212> DNA
<213>artificial sequence
<221> qRT-AtActin-F
<400> 11
cggtggttct atcttggcat c 21
<211> 22
<212> DNA
<213>artificial sequence
<221> qRT-AtActin-R
<400> 12
gtctttcgct tcaataaccc ta 22
<211> 21
<212> DNA
<213>artificial sequence
<221> qRT-AtCOR15A-F
<400> 13
cagcggagcc aagcagagca g 21
<211> 22
<212> DNA
<213>artificial sequence
<221> qRT-AtCOR15A-R
<400> 14
catcgaggat gttgccgtca cc 22
<211> 20
<212> DNA
<213>artificial sequence
<221> qRT-AtERD15-F
<400> 15
ccagcgaaat ggggaaacca 20
<211> 19
<212> DNA
<213>artificial sequence
<221> qRT-AtERD15-R
<400> 16
acaaaggtac agtggtggc 19
<211> 23
<212> DNA
<213>artificial sequence
<221> qRT-AtRD29A-F
<400> 17
gttactgatc ccaccaaaga aga 23
<211> 22
<212> DNA
<213>artificial sequence
<221> qRT-AtRD29A-R
<400> 18
ggagactcat cagtcacttc ca 22
<211> 21
<212> DNA
<213>artificial sequence
<221> qRT-AtP5CS1-F
<400> 19
cgacggagac aatggaattg t 21
<211> 21
<212> DNA
<213>artificial sequence
<221> qRT-AtP5CS1-R
<400> 20
gatcagaaat gtgtaggtag c 21
Claims (8)
1. bryonyVyGOLSGene, it is characterised in that: the amino acid sequence that it is encoded is as shown in SEQ ID NO.2.
2. bryony according to claim 1VyGOLSGene, it is characterised in that: its nucleotide sequence such as SEQ ID
Shown in the position 186-1196 in NO.1.
3. bryony VyGOLS albumen, it is characterised in that: its amino acid sequence is as shown in SEQ ID NO.2.
4. recombinant expression carrier, it is characterised in that: the recombinant expression carrier includes bryonyVyGOLSGene, the bryonyVyGOLSThe nucleotide sequence of gene is as shown in the position 186-1196 in SEQ ID NO.1.
5. the preparation method of recombinant expression carrier as claimed in claim 4, it is characterised in that: include: according to such as SEQ ID
Primers shown in the position 186-1196 in NO.1, clone the bryonyVyGOLSGene, then by the wild Portugal
GrapeVyGOLSGene be connected on pCAMBIA2300 plant expression vector to get.
6. bryony as described in claim 1VyGOLSGene or recombinant expression carrier as claimed in claim 4 are in plant
Application in breeding for quality.
7. bryony according to claim 6VyGOLSThe application of gene or recombinant expression carrier, it is characterised in that: In
Application in plant drought breeding for quality.
8. bryony according to claim 7VyGOLSThe application of gene or recombinant expression carrier, it is characterised in that: In
Application in arabidopsis drought-resistant variety breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910872930.9A CN110452917B (en) | 2019-09-16 | 2019-09-16 | Wild grape VyGOLS gene and application of encoding protein thereof in drought stress |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910872930.9A CN110452917B (en) | 2019-09-16 | 2019-09-16 | Wild grape VyGOLS gene and application of encoding protein thereof in drought stress |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110452917A true CN110452917A (en) | 2019-11-15 |
| CN110452917B CN110452917B (en) | 2022-08-02 |
Family
ID=68492109
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910872930.9A Active CN110452917B (en) | 2019-09-16 | 2019-09-16 | Wild grape VyGOLS gene and application of encoding protein thereof in drought stress |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110452917B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444738A (en) * | 2021-06-22 | 2021-09-28 | 郑州大学 | Application of cotton GhGOLS2 gene in controlling cotton seed germination |
| CN115094084A (en) * | 2022-06-06 | 2022-09-23 | 福建省农业科学院水稻研究所 | Application of rice OsRS gene in breeding of high-seed activity rice |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1259996A (en) * | 1997-04-28 | 2000-07-12 | 味之素株式会社 | Raffinose synthetase gene, process for producing, same, and transformed plant |
| CN107586809A (en) * | 2017-10-09 | 2018-01-16 | 中国科学院天津工业生物技术研究所 | A kind of method of living things catalysis synthesis soyabean oligosaccharides |
| CN110079537A (en) * | 2019-05-27 | 2019-08-02 | 河南科技大学 | Vitis mitogen response regulation factor Ⅴ vRR gene and its coding albumen and application |
-
2019
- 2019-09-16 CN CN201910872930.9A patent/CN110452917B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1259996A (en) * | 1997-04-28 | 2000-07-12 | 味之素株式会社 | Raffinose synthetase gene, process for producing, same, and transformed plant |
| CN107586809A (en) * | 2017-10-09 | 2018-01-16 | 中国科学院天津工业生物技术研究所 | A kind of method of living things catalysis synthesis soyabean oligosaccharides |
| CN110079537A (en) * | 2019-05-27 | 2019-08-02 | 河南科技大学 | Vitis mitogen response regulation factor Ⅴ vRR gene and its coding albumen and application |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: ""PREDICTED: Vitis vinifera galactinol synthase 1 (LOC1 00260266), mRNA"，Accession Number：XM_002265911.3", 《GENBANK》 * |
| TAO ZHOU ET AL.: ""Molecular Cloning and Characterization of GhGolS1, a Novel Gene Encoding Galactinol Synthase from Cotton (Gossypium hirsutum)"", 《PLANT MOL BIOL REP》 * |
| 冯睿杰 等: ""葡萄肌醇半乳糖苷合成酶基因VvGolS2-4的克隆及表达特性分析"", 《华北农学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444738A (en) * | 2021-06-22 | 2021-09-28 | 郑州大学 | Application of cotton GhGOLS2 gene in controlling cotton seed germination |
| CN113444738B (en) * | 2021-06-22 | 2022-07-05 | 郑州大学 | Application of cotton GhGOLS2 gene in controlling cotton seed germination |
| CN115094084A (en) * | 2022-06-06 | 2022-09-23 | 福建省农业科学院水稻研究所 | Application of rice OsRS gene in breeding of high-seed activity rice |
| CN115094084B (en) * | 2022-06-06 | 2023-10-24 | 福建省农业科学院水稻研究所 | Application of rice OsRS gene in breeding high-seed-activity rice |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110452917B (en) | 2022-08-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110845590B (en) | Wild grape VyPPR gene and application of encoding protein thereof in drought stress | |
| CN105254726B (en) | ERF class transcription factor relevant to plant stress-resistance and its encoding gene and application | |
| CN106661583A (en) | Plants and methods to improve agronomic characteristics under abioticstress conditions | |
| CN102766618A (en) | Rice OsICL protein and coding gene thereof, and application of the two | |
| CN109879944B (en) | EAR1 protein related to plant drought resistance and coding gene and application thereof | |
| CN102181456B (en) | Cotton flowering hormone GhFT and vector, construct, cell and polypeptide thereof | |
| CN110452917A (en) | The application of bryony VyGOLS gene and its coding albumen in drought stress | |
| CN101880674B (en) | Date tree aquaporin gene and application in improvement on plant drought resistance and salt resistance | |
| CN110129291A (en) | Barley moisture-proof controlling gene HvACO1, albumen and its application in breeding | |
| CN106749580B (en) | Plant salt tolerance GAP-associated protein GAP TaPUB15-D and its encoding gene and application | |
| CN102477435A (en) | Method for improving plant drought resistance using Poncirus trifoliata transcription factor gene PtrABF | |
| CN109628475B (en) | Application of brassinolide synthetic gene PaCYP724B1 in regulation and control of plant branches | |
| CN112391405B (en) | Application of tea tree hexokinase CsHXK3 gene in regulation of plant growth and development and enhancement of cold resistance | |
| CN105175522B (en) | Crowtoe AP2/ERF transcription factors and its encoding gene and application | |
| CN111996197B (en) | Salt-tolerant gene and protein of pyrus betulaefolia, recombinant vector and application | |
| CN109207487B (en) | Rape stain-resistant gene BnalPP1, and preparation method and application thereof | |
| CN114214333A (en) | Gene for regulating and controlling development of plant leaf epidermal hair and thickness of secondary wall and application thereof | |
| CN102586264B (en) | Method for improving plant yield | |
| CN111423500A (en) | SiMYB56 protein and application of encoding gene thereof in regulation and control of plant drought resistance | |
| CN114941003B (en) | Wild grape vyMPBQ gene, protein coded by same and application thereof | |
| CN109810984A (en) | A kind of relevant Sesame SiGolS6 of drought resisting and its application | |
| CN114875044B (en) | Wild grape VyVTE gene, protein coded by same and application thereof | |
| CN114606244B (en) | Astragalus sinicus AGL18 gene and application thereof | |
| CN113604475B (en) | Application of cotton GH_D03G1517 gene in promotion of drought resistance and salt tolerance | |
| CN102757970B (en) | Eucalyptus PGEF12 gene as well as plant expression vector thereof, host cell thereof and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |