CN110387382A - Gene A RR5 is improving the application in plant drought ability - Google Patents
Gene A RR5 is improving the application in plant drought ability Download PDFInfo
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- CN110387382A CN110387382A CN201810333714.2A CN201810333714A CN110387382A CN 110387382 A CN110387382 A CN 110387382A CN 201810333714 A CN201810333714 A CN 201810333714A CN 110387382 A CN110387382 A CN 110387382A
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- 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
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Abstract
The present invention provides Gene A RR5 and is improving the application in plant drought ability, is overexpressed ARR5 gene respectively in arabidopsis and corn, can enhance the drought-resistant ability of genetically modified plants.ARR5 gene provided by the invention provides valuable genetic resources to cultivate drought-resistant plant new varieties, while having established theoretical basis with the molecule mechanism for resisting adverse environment for the mechanism of research plant response drought stress.
Description
Technical field
The present invention relates to genetic engineering and molecular biology fields, specifically, being related to Gene A RR5 is improving Genes For Plant Tolerance
Application in non-irrigated ability.
Background technique
Fixed growing plants is unavoidably by the influence of various environment stresses in nature, increasingly with water resource
Scarcity, drought stress not only influences growth and development, the geographical distribution of plant as one of main external environment factor, also tight
Ghost image rings the yield of crop.Therefore, the physiological acoustic signals and molecule mechanism of plant responding drought stress are studied, regulation is found and plants
The key gene of object response arid, the crop varieties for cultivating drought resisting provide valuable theories integration, to improve the production of crops
Amount.
Arabidopsis (Arabidopsis thaliana) belongs to crucifer, small in view of its genome, and growth is very fast
Etc. advantages, as model plant be widely used in Plant genetics, cell biology, molecular biology etc. research in.Research is quasi-
Various mechanism in southern mustard can provide newest Research Thinking and theoretical direction for the research of many crops.Generally pass through
The method that Agrobacterium infects inflorescence, related gene is gone in wildtype Arabidopsis thaliana, obtains the overexpression or clpp gene of the gene
Except strain, the detection of plant stress tolerance experiment is then carried out, function of the gene in adverse circumstance is studied, to obtain adverse circumstance
The genetic resources of response.
Corn (Zea mays) belongs to grass family Zea plant, is important cereal crops and forage crop, and
The highest crops of whole world total output.The adversity gene of arabidopsis can be imported the jade for needing to improve using transgenic technology
In rice inhereditary material, and make to represent the anti-adversity ability for revealing and stablizing heredity thereafter.It can be infected at present by Agrobacterium, by table
Maize calli is imported up to plasmid, regenerates resistant transgenic plant.Low-copy transgenic line is selected to carry out inverse
Border Phenotypic examination, obtaining has efficiently degeneration-resistant transgenic corns, provides excellent variety resource for agricultural production.
Summary of the invention
The object of the present invention is to provide Gene A RR5 to improve the application in plant drought ability.
In order to achieve the object of the present invention, Gene A RR5 provided by the invention is improving the application in plant drought ability,
In, the nucleotide sequence of the Gene A RR5 are as follows:
I) nucleotide sequence shown in SEQ ID NO:1；Or
Ii) nucleotide sequence shown in SEQ ID NO:1 be substituted, lack and/or increase one or more nucleotide and
Nucleotide sequence with the same function；Or
Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and nucleotide sequence with the same function,
The stringent condition be in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, it is miscellaneous at 65 DEG C
It hands over, and washes film with the solution；Or
Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and nucleotide with the same function
Sequence.
Arabidopsis ARR5 gene is to hold the 135th to the 1763rd from 5 ' by 2448 base compositions, the reading frame of the gene
Bit base.The gene reading frame is made of 5 exons and 4 intrones, and 5 exons are located at: reading frame the 1st is arrived
151st bit base, the 255th to the 335th bit base, the 430th to the 508th bit base, the 636th to the 707th bit base, the
1458 to the 1633rd bit base, remaining is intron sequences.ARR5 gene source is in Columbia ecotype arabidopsis, quasi-
Number in southern mustard genome database is At3g48100.
As arabidopsis ARR5 gene coded protein amino acid sequence as shown in SEQ ID NO:2.It should be understood that this field skill
Art personnel can disclosed amino acid sequence according to the present invention, do not influence its it is active under the premise of, replace, missing and/or increase
One or several amino acid obtain the mutant nucleotide sequence of the albumen.
It should be understood that those skilled in the art can in view of the degeneracy of codon and the preferences of different plant species codon
The codon for being suitble to particular species expression is used with as needed.
The present invention also provides the cloning vectors of ARR5 gene order or its segment containing plant drought or all kinds of expression to carry
Body, the host cell containing the carrier, conversion plant cell and transgenosis containing the gene order or its specific fragment
Plant.
The expression vector for carrying the target gene ARR5 can be by using Ti-plasmids, plant viral vector, direct DNA
The standard biologics technical method such as conversion, microinjection, electroporation imports (Weissbach, 1998, Method in plant cell
Plant Molecular Biology VIII, Academy Press, New York, the 411-463 pages；Geiserson and
Corey, 1998, Plant Molecular Biology, 2nd Edition)。
In aforementioned applications, the plant includes monocotyledon and dicotyledon, such as corn and arabidopsis etc..It will plant
Object drought resisting ARR5 gene is overexpressed in plant, including the use of any load that foreign gene can be guided to be overexpressed in plant
Body carries out.
The application specifically includes:
1) making plant includes ARR5 gene；Or
2) plant is made to be overexpressed the albumen of ARR5 gene coding.
The present invention also provides application of the Gene A RR5 in plant breeding, the purpose of the breeding is to improve plant drought energy
Power.
The present invention also provides the methods of the transgenic arabidopsis of building drought resisting: extracting arabidopsis total serum IgE, reverse transcription obtains
CDNA, using cDNA as template, F and R are primer, expand the CDS sequence of ARR5 gene, and amplified production is building up to plant expression and is carried
On body (such as pCM1307), Agrobacterium then is converted with the recombinant expression carrier obtained, then infects quasi- south with the Agrobacterium of conversion
Mustard (preferably wildtype Arabidopsis thaliana) inflorescence screens positive transgenic plant, obtains the transgenic arabidopsis of drought resisting.
In the present invention, the nucleotide sequence of the primers F and R are as shown in SEQ ID NO:3-4.
The present invention also provides the methods of the transgenic corns of building drought resisting: extracting arabidopsis total serum IgE, reverse transcription obtains
CDNA, using cDNA as template, F and R are primer, expand the CDS sequence of ARR5 gene, and amplified production is building up to plant expression and is carried
On body (such as pCUN), Agrobacterium then is converted with the recombinant expression carrier obtained, then corn is infected with the Agrobacterium of conversion and is cured
Injured tissue screens positive transgenic plant, obtains the transgenic corns of drought resisting.
The carrier pCUN is adapted from plasmid pCAMBIA1300, is that hygromycin gene is connected into pCAMBIA1300
It obtains.
In preceding method, the Agrobacterium is preferably GV3101.
After ARR5 gene overexpression of the invention, transgenic arabidopsis and corn show as drought resisting phenotype.In order to just
In being identified genetically modified plants and being screened, used carrier can be processed, plant alternative label is such as added
Or resistant antibiotic marker etc..
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
By the Gene A RR5 to response regulation in coded plant, (full name is ARABIDOPSIS RESPONSE to the present invention
REGULATOR 5) research, discovery be overexpressed the gene transgenic plant compared with WT lines have obvious drought resisting table
Type.ARR5 gene provided by the invention provides valuable genetic resources to cultivate drought-resistant plant new varieties, answers for research plant
Answer the mechanism of adverse circumstance signal and the molecule mechanism based theoretical of tolerance adverse environment.
Detailed description of the invention
Fig. 1 is the ARR5 albumen excess that Gene A RR5 is overexpressed arabidopsis strain OE-7 and OE-10 in the embodiment of the present invention 2
Expression figure.
Fig. 2 is that Gene A RR5 is overexpressed before arabidopsis strain OE-7 and OE-10 Osmotic treatment and does in the embodiment of the present invention 3
Plant strain growth situation photo after drought processing rehydration.
Fig. 3 is that Gene A RR5 is overexpressed at arabidopsis strain OE-7 and OE-10 percentage of water loss and arid in the embodiment of the present invention 3
Survival rate of plant statistical chart after reason rehydration.
Fig. 4 be the embodiment of the present invention 4 in Gene A RR5 be overexpressed corn strain Osmotic treatment rehydration after plant phenotype (A) and
The relative expression quantity (B) of Gene A RR5.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
PBSK carrier is common cloning vector in following embodiment, commercially available；PCM1307 carrier is by China Agricultural University
Biological institute Guo Yan professor laboratory give；Arabidopsis kind is Columbia ecotype；Agrobacterium GV3101 bacterial strain is common
Cloning vector.
Main agents in following embodiment are as follows: various restriction enzymes, Taq archaeal dna polymerase, T4 ligase,
Pyrobest Taq enzyme, KOD are purchased from biotech firms such as NEB, Toyobo；DNTPs is purchased from Genestar company；Plasmid is small to mention reagent
Box and Ago-Gel QIAquick Gel Extraction Kit are purchased from Shanghai JaRa bio-engineering corporation；MS culture medium, agar powder, agarose, ammonia benzyl
The antibiotic such as penicillin (Amp), kanamycins (Kan), gentamicin sulphate (Gen), rifampin (Rif) and Glucose,
BSA, nitrocellulose filter, LB Medium etc. are purchased from companies such as Sigma, Bio-Rad；It is various other used in embodiment
Chemical reagent is import or domestic analytical reagents.
Primer used in embodiment is synthesized by six directions Hua Da company, and carries out correlative measurement sequence.
The building of 1 ARR5 gene overexpression carrier of embodiment
For the molecular mechanism for understanding plant responding arid, from arabidopsis (Arabidopsis thaliana) genome gram
Grand ARR5 gene.It is analyzed according to coding region sequence, design primer F and R, the code area of the gene is amplified to come, is connected to
On over-express vector pCM1307 with 35S promoter.
The primer is (SEQ ID NO:3-4):
Upstream primer F:5 '-GTCGACATGGCTGAGGTTTTGCGTCCCGAGA-3 '
Downstream primer R:5 '-GGTACCTCAGATCTTTGCGCGTTTTAGCTGC-3 '
ARR5 gene is connected on the carrier pCM1307 with 35S promoter method particularly includes: be with cDNA first
Template is amplified ARR5 PCR product to be connected with pBSK carrier, connection product is named as using upstream and downstream primer
ARR5-pBSK；ARR5 is connected into pCM1307 carrier after correct ARR5-pBSK digestion is sequenced using SalI and KpnI, is connected
Product is named as 35S:HA/Flag-ARR5.
After the resulting plasmid enzyme restriction of previous step, electrophoresis detection, specific method are carried out are as follows: use SalI and KpnI digestion
35S:HA/Flag-ARR5, with 1% Ago-Gel, 120V, after 50mA electrophoresis, UVP Gel Documentation gel point
Analysis system scanning imagery.
The building of 2 ARR5 gene overexpression plant of embodiment
PCM1307 carrier described in embodiment 1 containing ARR5 gene is transformed into Agrobacterium GV3101 bacterial strain, then is transferred to
In wild-type Arabidopsis plants, arabidopsis transgenic seedlings are obtained.Method particularly includes: it will be containing the Agrobacterium inoculation for having purpose carrier
In tri- resistant to liquids culture solution of 100mL LB (Kan 50 μ g/mL, Rif 50 50 μ g/mL of μ g/mL, Gen), 28 DEG C of shaken cultivations
Overnight, to OD600Value is 1.0-2.0, and with 5000g, room temperature is centrifuged 15min, collects thallus；With 200mL conversion fluid (1/2MS, 5%
Sucrose, 40 μ L Silwet L-77) suspension thalline；Arabidopsis floral is immersed in 1min in the conversion fluid of Agrobacterium, puts on guarantor
Fresh bag moisturizing, which is placed in dark place, keeps its temperature lower, plant is taken out from freshness protection package within second day, is put back on illumination cultivation frame
Normal growth is to sowing.
PCM1307 carrier institute band screening resistant gene is hygromycin, with hygromycin resistance to arabidopsis transgenic seedlings into
Row screening, the T of acquisition1In generation, there is the positive seedling of hygromycin element resistance to carry out single plant sowing, then to T2It is anti-that hygromycin is carried out for seed
Property test, select that 3/4 is resistant and the strain of remaining 1/4 not no resistance, illustrate to carry target gene in the strain
Over-express vector by singly copy in the form of be inserted into.Plant in these strains with hygromycin resistance is removed, then carries out single plant
Sowing, then carry out hygromycin resistance screening illustrates that the transgenic line is homozygote if do not separated, which can be with
For breeding, Drought Stress processing experiment.
Isolated overexpression strain OE-7 and OE-10 in the present embodiment.Institute is detected using protein immunoblotting method
The protein expression of ARR5 in strain OE-7 and OE-10 must be overexpressed.
1) plant total protein is extracted
The 14d seedling grown on culture dish is wrapped with masking foil, is frozen rapidly spare in liquid nitrogen.With liquid nitrogen by plant
Material is clayed into power shape, and protein extract buffer is added, and in 4 DEG C after the concussion that is vortexed, 13000g is centrifuged 15min.
2) protein content in protein immunoblotting method detection OE-7 and OE-10 strain
The wild type of isoconcentration, the vegetable protein of OE-7 and OE-10 arabidopsis strain are taken, it is slow that 5 × SDS albumen loading is added
Fliud flushing, 100 DEG C are boiled 5min, after 90V voltage runs glue 15min, are changed to 120V voltage and are run glue 1h, 200mA transferring film 2h.Finally use
Protein content in Anti-HA antibody test OE-7 and OE-10 strain.Actin is as internal reference.
The gene expression that gained is overexpressed ARR5 in strain OE-7 and OE-10 is detected using semiquantitive PCR.
1) plant total serum IgE is extracted, and reverse transcription obtains cDNA.
2) ARR5 is expanded with primers F and R, expands 28 circulations.EF1 α is internal reference, expands 18 circulations.Arr5 mutant is made
For control.Wherein arr5 (CS25267) mutant is purchased from ABRC (Arabidopsis Biological Resources
Center) Arabidopsis Mutants library.
Testing result is shown in Fig. 1, it can be seen that the gene of ARR5 and protein content have in OE-7 and OE-10 transgenic line
Higher expression.
The method that ARR5 gene is overexpressed in other plants can be carried out with reference to the present embodiment.
Embodiment 3 is overexpressed the drought-resistant ability detection of ARR5 gene plant
First by ARR5 gene overexpression strain OE-7 and OE-10 obtained in wildtype Arabidopsis thaliana seedling and embodiment 2
And wild type grows 7d on culture dish, is then transferred to respectively in soil after cultivating 10d under normal condition, Osmotic treatment 7-
Phenotype is observed after 10d.Detect respectively simultaneously the ARR5 gene overexpression strain OE-7 for cultivating 10d in soil under normal condition and
The percentage of water loss of OE-10 and wild type.
(Fig. 2, Fig. 3) as the result is shown, OE-7 and OE-10 show the slow drought resisting phenotype of dehydration.This illustrates that ARR5 crosses table
Drought-resistance ability up to plant is significantly improved.It can be seen that the sub- ARR5 of arabidopsis response regulation can be mentioned significantly after being overexpressed
Tolerance of the high plant to arid.
The drought-resistant ability detection of ARR5 genetically modified plants is overexpressed in 4 corn of embodiment
The building process for being overexpressed ARR5 transgenic corns is as follows:
1) arabidopsis total serum IgE is extracted, reverse transcription obtains cDNA, and using cDNA as template, F and R are primer, expands ARR5 gene
CDS sequence, amplified production is building up on the expression vector pCUN after SpeI digestion with Infusion method, the recombination of acquisition
Expression vector is named as pCUN-ARR5；
2) Agrobacterium GV3101 is converted with pCUN-ARR5, then infects maize calli using the Agrobacterium of conversion, obtains
Obtain the transgenic corns seedling of drought resisting.
Wild-type corn seed and ARR5 gene overexpression corn strain OE-315 and the OE-316 kind of acquisition are planted in soil
In earth, and take pictures after carrying out Osmotic treatment 20 days.
(Fig. 4) as the result is shown, OE-315 and OE-316 show the phenotype of drought resisting.This illustrates the transgenosis that ARR5 is overexpressed
The drought-resistance ability of corn is significantly improved.It can be seen that the sub- ARR5 of arabidopsis response regulation can be significantly improved after being overexpressed
Tolerance of the plant to arid.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>China Agricultural University
<120>Gene A RR5 is improving the application in plant drought ability
<130> KHP181111831.5
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cttcttcatt agcatcaccg aaacttcttc atgttcttgc tgttgatgat agtatggttg 240
atcggaagtt catcgagcgg ttactcagag tctcatcttg taaaggtaac tgttgtgttt 300
gtgtttatca aaaaaaagtg ttgtgtttgt ttatcttgtg taatctgttt aagtacaaga 360
atctgacatt agttttgttc tgtctcagtt actgttgtcg atagtgcgac aagagcttta 420
caatatcttg gattagatgg agagaataat agttcggttg gatttgaggt aataaatctg 480
atcattgtgt ttaatatttt tgagagtgag aatccattca aatttggtgt taaaagattt 540
ttggtttgaa attatatttt aggatctgaa gattaatttg ataatgacgg attactctat 600
gcctgggatg actggatatg aactactgaa gaagatcaaa gtaagttttg ttgatgattc 660
aattgttttc acaaaattaa aatgaagttc tgatttgacg aaaaaaaatt taaaaaaaaa 720
tgaagttttg atctaatctt gtttatggat tttgaacttt ttaacaggaa tcatcagctt 780
tcagagaaat accagttgtg attatgtctt cagagaacat cttgcctcgt atcgataggt 840
atattctact ataataatct taactatgat ccttaattct tgtttaggtc aagattatgt 900
aggaccaaaa tttaccaaat atagtagatt ttgtgcattt attacttcag gagatgggga 960
tgattagatt tagagggtat tgtttgttta ggaaactttt taaagtaatc acactaattt 1020
aggatttgtg ataaagttac cgttaaagga tatgtattga ctaaattaaa ttgttgtata 1080
atcacaagat tttgttatga tagaaaaggt tattatatga ttacttttta actatcatgg 1140
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ggtcctaaaa tgatttttaa aatggatctt aggaataata aaatttatct tgtggccata 1260
aatttagaaa agaataatcc atcataagtt aagcaagatc ttctcttgag atttgattct 1320
atataagccg gtgacatgtt aggccctcca aaactttata atatatgttt taattgtttg 1380
ggttgcataa tcttaggtag ggtcatttat aaaatcccaa tcctcgtgat agacacctca 1440
catgcctttg cttattacta aggaaccaaa ccctctttac cagattaagt ttaatgatta 1500
tcatatatga gctggccatt ttctgtatcc aaagttataa actttatgga attagatcac 1560
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tgaagcctgt gaaattagct gatgtgaaga gattaagaga ttctttaatg aaagctgagg 1680
aaagagcttt taagaatatt atgcacaaga gagagcttga agctaatgat atctactcgc 1740
agctaaaacg cgcaaagatc tgagtttatc agattccacg tacgatatgt tctttaaaag 1800
ctcaaagatt cacacacatg catgtcctga ttctttcggc ttacaatttt tgagacatta 1860
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gatagaaatg taaattgtag acttggattc attagagagg agaagagata catttttttt 1980
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cattacacga agacaagcta atcttttgtc taattaaaat aaagctgtct acatgaatcg 2160
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gtagaaattt ccccaagttg ggttttatta agaccaagaa aattctagtc tcctagactt 2280
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Cys Lys Val Thr Val Val Asp Ser Ala Thr Arg Ala Leu Gln Tyr Leu
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Gly Leu Asp Gly Glu Asn Asn Ser Ser Val Gly Phe Glu Asp Leu Lys
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Ile Asn Leu Ile Met Thr Asp Tyr Ser Met Pro Gly Met Thr Gly Tyr
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Claims (10)
1. Gene A RR5 is improving the application in plant drought ability, which is characterized in that the nucleotide sequence of the Gene A RR5
Are as follows:
I) nucleotide sequence shown in SEQ ID NO:1；Or
Ii) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases one or more nucleotide and has
The nucleotide sequence of identical function；Or
Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and nucleotide sequence with the same function, it is described
Stringent condition be in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, hybridize at 65 DEG C, and
Film is washed with the solution；Or
Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and nucleotides sequence with the same function
Column.
2. application according to claim 1, which is characterized in that the plant includes monocotyledon and dicotyledon.
3. application according to claim 2, which is characterized in that the plant includes arabidopsis and corn.
4. application according to claim 1-3, which is characterized in that the application includes:
1) making plant includes ARR5 gene；Or
2) plant is made to be overexpressed the albumen of ARR5 gene coding.
5. application of the Gene A RR5 in plant breeding, which is characterized in that the purpose of the breeding is to improve plant drought ability,
Wherein, the definition of Gene A RR5 is the same as described in claim 1.
6. the method for constructing the transgenic arabidopsis of drought resisting, which is characterized in that arabidopsis total serum IgE is extracted, reverse transcription obtains cDNA,
Using cDNA as template, F and R are primer, expand the CDS sequence of ARR5 gene, amplified production is building up on plant expression vector,
Then Agrobacterium is converted with the recombinant expression carrier obtained, then infects arabidopsis floral with the Agrobacterium of conversion, screening is positive
Transgenic plant obtains the transgenic arabidopsis of drought resisting；
Wherein, the nucleotide sequence of the primers F and R are as shown in SEQ ID NO:3-4.
7. according to the method described in claim 6, it is characterized in that, the plant expression vector is pCM1307.
8. the method for constructing the transgenic corns of drought resisting, which is characterized in that arabidopsis total serum IgE is extracted, reverse transcription obtains cDNA, with
CDNA is template, and F and R are primer, expands the CDS sequence of ARR5 gene, amplified production is building up on plant expression vector, so
Agrobacterium is converted with the recombinant expression carrier obtained afterwards, then infects maize calli with the Agrobacterium of conversion, screening is positive
Transgenic plant obtains the transgenic corns of drought resisting；
Wherein, the nucleotide sequence of the primers F and R are as shown in SEQ ID NO:3-4.
9. according to the method described in claim 8, it is characterized in that, the plant expression vector is pCUN；Carrier pCUN transformation
It is to be connected into what hygromycin gene obtained in pCAMBIA1300 from plasmid pCAMBIA1300.
10. the method according to claim 6, which is characterized in that the Agrobacterium is GV3101.
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