CN108893479A - A method of sweet potato is improved to the adverse circumstance property resisted using IbMPK6 gene - Google Patents
A method of sweet potato is improved to the adverse circumstance property resisted using IbMPK6 gene Download PDFInfo
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- CN108893479A CN108893479A CN201810731705.9A CN201810731705A CN108893479A CN 108893479 A CN108893479 A CN 108893479A CN 201810731705 A CN201810731705 A CN 201810731705A CN 108893479 A CN108893479 A CN 108893479A
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- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- 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
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Abstract
It is specially a kind of to improve sweet potato to the method for the adverse circumstance property resisted using IbMPK6 gene the invention belongs to genetic engineering field；Relate generally to the functional verification and application of IbMPK6.The gene is overexpressed in sweet potato, the transgenic plant of acquisition significantly improves sweet potato resistance of reverse, and method is to obtain recombinant vector for after the cDNA homologous recombination of IbMPK6 to the CaMV35S strong promoter in over-express vector pMDC84.Agrobacterium_mediated method is recycled, the transgenic plant for being overexpressed the gene is obtained.It handles and tests by environment stress, be found in adjoining tree and compare, transgenic plant significantly improves the lower temperature resistance and salt tolerance of sweet potato.
Description
Technical field
The invention belongs to genetic engineering fields, and in particular to be overexpressed protein kinase in sweet potato main breed Xushu29
IbMPK6 gene.
Background technique
Abiotic stress is to influence plant growth and development, leads to the principal element of crop production reduction.Sweet potato is planted in China
Extensively, first place in the world is accounted in terms of cultivated area, total output.Sweet potato is because of its impoverishment tolerant, manageability, the high and abundant nutrition of yield
Value, be widely applied in people's current consumption, animal feed and industrial fuel, be after rice, wheat and corn etc. it
One of most important crops afterwards.But the abiotic environments factor such as low temperature, saline and alkaline limits the yield and Sweet Potato Industry of sweet potato
Development.
Mitogen-activated protein kinases (MAPK, Mitogen-activated Protein Kinase) are extensive
The signaling molecule being present in eucaryote, is made of MAPKKK, MAPKK and MAPK, by cascading successively phosphorylation reaction side
Formula participates in extracellular signal amplification and intracellular signal transmitting, participates in biology or abiotic Stress responses, hormone response, cell point
It splits, growth and development and procedural apoptosis etc..MAPK cascades complicated composition, and is interweaved, redundancy, while having pleiotropism, only
There is a small number of cascades to be elaborated.With high salt, low temperature stress is responded as MKK1/MKK2-MPK4/MPK6 is cascaded；MKK6-MPK4/
MPK11 grades be associated in regulating cell division on play important function；MEKKK1-MKK2-MPK4/MPK6 cascade signal participates in plant
Resist low temperature stress etc..20 MAPK are shared in arabidopsis, according to by the phosphorylation site of upstream MAPKK, MPK6 belongs to TEY base
A group in sequence.Arid, low temperature and high-salt stress all can induce the expression of AtMPK6.It is heterologous by genetic engineering means in recent years
Or homologous overexpression MPK6 kinases, improve plant abiotic stress tolerance.Such as in arabidopsis, AtMPK6 can induce SOS1 table
It reaches, maintains the ionic equilibrium of intraor extracellular, increase salt tolerance.In rice, OsMPK6 participates in the metabolic pathway of trehalose, improves
The expression quantity of OsTPP1 improves the cold resistance of rice.
Sweet potato kinases IbMPK6 responds a variety of abiotic stress, but its biological function is still not clear.The present invention is logical
It crosses transgenic approach and verifies its gene function, and improve sweet potato to the resistance of reverse of environment.
Summary of the invention：
The object of the present invention is to provide one kind by 35S strong promoter control under, in sweet potato main breed Xu potato 29
IbMPK6 gene is overexpressed to improve the cold resistance of sweet potato and the method for salt tolerance.
The technical scheme is that：A method of it is described using IbMPK6 gene raising sweet potato to the adverse circumstance property resisted
Method is by being overexpressed IbMPK6 gene to improve the property resisted of sweet potato plant pair adverse circumstance.
The nucleotide sequence of the IbMPK6 gene is as shown in sequence table SEQ ID No.1.
In one embodiment of the invention, the overexpression is that upstream and downstream primer is expanded IbMPK6 gene order.
The upstream and downstream primer is IbMPK6F and IbMPK6R, nucleotide sequence such as sequence table SEQ ID No.2 and SEQ
Shown in ID No.3.
A kind of in the present invention to improve sweet potato to the method for the adverse circumstance property resisted using IbMPK6 gene, it includes following steps
Step 1：Construct recombinant vector CaMV35S-IbMPK6.
Step 2：Transfer vector plasmid is transferred in Agrobacterium GV3101 bacterial strain, kanamycins is added in YEP culture medium,
Through plate screening, single colonie is obtained, single colonie genome is extracted and carries out PCR verifying, obtain the agriculture bar for being overexpressed IbMPK6 gene
Bacterium recombinant bacterial strain.
Step 3：Sweet potato callus is infected using agrobacterium-mediated transformation, hygromycin is added in MS culture medium and carries out selective training
It supports, until callus grows up to seedling.
Step 4：Seedling leaf DNA is extracted, according to carrier GFP gene design primer SEQ ID No.4 and SEQ ID No.5
Identify transgenic plant；And design qRT-PCR primer SEQ ID No.6 and SEQ ID No.7, screening transgenic strain.
Step 5：Gene IbMPK6 is verified in the abiotic stress tolerance for improving sweet potato.
The Agrobacterium single colonie of overexpression IbMPK6 gene in the present invention is seeded in liquid YEP medium, and 30 DEG C overnight
Culture.It takes 100 μ l to be incubated overnight liquid to be transferred in the fresh YEP culture medium containing 100 μM of acetosyringones, 30 DEG C of culture 4-
6 hours, culture solution OD600Reach 0.8-1.0, thalline were collected by centrifugation.
What described in the present invention filtered out cross, and table plant places carries out expanding in the greenhouse numerous, and intensity of illumination is 150 μm of ol
m-2s-1, humidity 85%, light application time 16h/8h (light dark), 25 DEG C of temperature.
The present invention also provides a kind of application of bMPK6 gene in sweet potato plant.
Beneficial effects of the present invention：
The present invention identifies the function of IbMPK6 gene, and significantly improves sweet potato plant pair by being overexpressed IbMPK6 gene
The property resisted of low temperature and salt stress.
Detailed description of the invention
Fig. 1：Recombinant vector CaMV35S-IbMPK6 schematic diagram
Fig. 2：PCR identifies sweet potato transgenic plant
Fig. 3：QRT-PCR screens sweet potato transgenic plant
Fig. 4：IbMPK6 is overexpressed transgenic plant and improves sweet potato abiotic stress tolerance
Specific embodiment
The present invention is described in further detail below by embodiment.
Embodiment 1
The building of recombinant vector CaMV35S-IbMPK6
Using the cDNA of Xu's potato 29 as template, using published upstream and downstream primer PCR amplification IbMPK6 gene, it is connected to T-
On blunt carrier, and converted into bacillus coli DH 5 alpha competent cell.Kanamycins is added in LB culture medium, passes through
Plate screening obtains single colonie, extracts single colonie genome and carries out PCR verifying, shakes bacterium by the single colonie of verifying and extracts plasmid
It is sequenced.The gateway vector construction technology for recycling Invitrogen company to provide, by the cDNA homologous recombination of IbMPK6
To after the CaMV35S strong promoter of over-express vector pMDC84, recombinant vector CaMV35S-IbMPK6 is obtained.
Embodiment 2
Agrobacterium-mediated transformation infects sweet potato callus
The Agrobacterium single colonie that picking is overexpressed IbMPK6 gene is seeded in liquid YEP medium, and 30 DEG C are incubated overnight.It takes
100 μ l are incubated overnight liquid and are transferred in the fresh YEP culture medium containing 100 μM of acetosyringones, 30 DEG C culture 4-6 hours,
Culture solution OD600Reach 0.8-1.0, thalline were collected by centrifugation.Thallus is resuspended in fresh containing 100 μM of acetosyringones
In YEP culture medium, it is adjusted to OD600To 0.8.Sweet potato callus is added in bacterium solution, after ambient temperature with gentle shakes 30min.Callus is moved
On to the MS solid medium containing 100 μM of acetosyringones after dark culture 2-3d, the bacterium solution on callus surface is washed away with clear water, it will
Callus moves on the MS solid medium containing hygromycin, cultivates under light, during which constantly replaces culture medium, until callus grow up to it is small
Seedling.
Embodiment 3
Gene IbMPK6 is verified in the abiotic stress tolerance for improving sweet potato
The overexpression IbMPK6 transgenic line filtered out and adjoining tree (Xu's potato 29) place carry out expanding in the greenhouse it is numerous,
Intensity of illumination is 150 μm of ol m-2s-1, humidity 85%, light application time 16h/8h (light dark), 20-25 DEG C of temperature.After cultivating 3 weeks
It chooses the consistent sweet potato's seedlings of growing way and carries out abiotic stress processing, and experiment is repeated 3 times.Low temperature stress：Experimental material is moved
Enter and handle 48h in 4 DEG C of artificial incubator, recovery is placed at room temperature for for 24 hours；High-salt stress：Use the culture containing 250mM NaCl
Liquid pours plant 20d, restores normal and pours 10d.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Sequence table
<110>Xuzhou Agriculture Science Inst., Xuhuai area, Jiangsu (Xuzhou Sweetpotato Center)
<120>A method of sweet potato is improved to the adverse circumstance property resisted using IbMPK6 gene
<130> 2018.4.4
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1173
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atggacgctg gttcggctca gccggcggac acggtgatgt cggaggctgc gccacctcag 60
cctccagtgc ccgggatcga caacattccg gcgacactca gtcacggcgg cagattcatc 120
caatacaaca tcttcggcaa cgtctttgag gtcacagcca agtacaagcc tcccatcatg 180
cctatcggca aaggcgccta tggaatcgtc tgttctgctt tgaattcgga aacgaatgag 240
cacgtagcga taaaaaaaat agcgaatgct tttgataaca aaatcgatgc caagaggact 300
ctgcgtgaga tcaagttgct tcgccacatg gatcacgaaa atattgttgc aattagagat 360
ataataccac ctcctcagag agcgtcattc aatgacgttt atattgcata tgagcttatg 420
gacactgatc ttcatcaaat tattcgctct aatcagtcat tatcagagga gcactgtcag 480
tatttcttgt atcagattct ccgtgggttg aaatacatac attctgcaaa tgttttgcac 540
agggacttga aacccagcaa tctactattg aatgccaatt gtgatctgaa aatatgtgat 600
tttggattag ctcgtgtgac atctgaaaca gattttatga ctgaatatgt tgtgactaga 660
tggtaccggc cacctgagct attattgaac tcttctgact acactgcagc cattgatgta 720
tggtcagtgg gttgtatttt catggaattg atggatcgga aacccctatt ccctggtaga 780
gatcatgtcc accagctacg tctgcttatg gagctaatag gtacaccttc ggaggctgaa 840
atggagtttt tgaatgagaa tgcgaaaaaa tatatccggc agcttccatt atatcgtcgc 900
cagtcattta ctgaaaggtt tccacatgcg caccctggtg ctattgatct tgttgagaaa 960
atgttgacat ttgatcctag gcgaagaatt accgttgaag gtgcacttgc gcatccttac 1020
ctaacatcac tccatgatat cagtgacgag ccaagttgca cgactccctt taactttgat 1080
tttgagcagc atgcattgag tgaggagcag atgaaggagc tgatttaccg agaggctgtt 1140
gctttcaacc ccgagtttga gctgcaaatg tga 1173
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atggacgctg gttcggctca 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
catttgcagc tcaaactcgg 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gagaggacca tcttgttcaa 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
catccatgcc atgtgtaatc 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gcggcagatt catccaatac 20
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
tatcgctacg tgctcattcg 20
Claims (8)
1. a kind of improve sweet potato to the method for the adverse circumstance property resisted using IbMPK6 gene, it is characterised in that：The method passed through
IbMPK6 gene is expressed to improve the property resisted of sweet potato plant pair adverse circumstance.
2. a kind of method using IbMPK6 gene raising sweet potato to the adverse circumstance property resisted according to claim 1, feature
It is：The nucleotide sequence of the IbMPK6 gene is as shown in sequence table SEQ ID No.1.
3. a kind of method using IbMPK6 gene raising sweet potato to the adverse circumstance property resisted according to claim 2, feature
It is：Clone the primer SEQ ID No.2 and SEQ ID No.3 of gene described in claim 2.
4. a kind of method using IbMPK6 gene raising sweet potato to the adverse circumstance property resisted according to claim 3, feature
It is：The overexpression is that upstream and downstream primer is expanded IbMPK6 gene order.
5. a kind of method using IbMPK6 gene raising sweet potato to the adverse circumstance property resisted according to claim 1, feature
It is, comprises the following steps：
Step 1：Construct recombinant vector CaMV35S-IbMPK6；
Step 2：Transfer vector plasmid is transferred in Agrobacterium GV3101 bacterial strain, kanamycins is added in YEP culture medium, through flat
Screen choosing, obtains single colonie, extracts single colonie genome and carries out PCR verifying, obtains the Agrobacterium weight for being overexpressed IbMPK6 gene
Group bacterial strain；
Step 3：Sweet potato callus is infected using agrobacterium-mediated transformation, hygromycin is added in MS culture medium and carries out selective culture,
Until callus grows up to seedling；
Step 4：Seedling leaf DNA is extracted, according to GFP6 gene design primer SEQ ID No.4 and SEQ ID No.5 on carrier；
Identify transgenic plant；And design qRT-PCR primer SEQ ID No.6 and SEQ ID No.7, screening transgenic strain；
Step 5：Gene IbMPK6 is verified in the abiotic stress tolerance for improving sweet potato.
6. a kind of method using IbMPK6 gene raising sweet potato to the adverse circumstance property resisted according to claim 5, feature
It is, the Agrobacterium single colonie that picking is overexpressed IbMPK6 gene is seeded in liquid YEP medium, and 30 DEG C are incubated overnight；Take 100
μ l is incubated overnight liquid and is transferred in the fresh YEP culture medium containing 100 μM of acetosyringones, 30 DEG C culture 4-6 hours, culture
Liquid OD600Reach 0.8-1.0, thalline were collected by centrifugation.
7. a kind of method using IbMPK6 gene raising sweet potato to the adverse circumstance property resisted according to claim 5, feature
It is, the overexpression plant that filters out, which is placed, to carry out expanding in the greenhouse numerous, and intensity of illumination is 150 μm of ol m-2s-1, humidity 85%,
Light application time 16h/8h (light dark), 25 DEG C of temperature.
8. a kind of IbMPK6 gene utilizes application of the step in sweet potato plant described in claim 5.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810731705.9A CN108893479A (en) | 2018-07-05 | 2018-07-05 | A method of sweet potato is improved to the adverse circumstance property resisted using IbMPK6 gene |
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| CN201810731705.9A CN108893479A (en) | 2018-07-05 | 2018-07-05 | A method of sweet potato is improved to the adverse circumstance property resisted using IbMPK6 gene |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725367A (en) * | 2021-03-01 | 2021-04-30 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato sucrose invertase gene IbINV and application thereof |
-
2018
- 2018-07-05 CN CN201810731705.9A patent/CN108893479A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| HO SOO KIM等: "Molecular characterization of biotic and abiotic stress-responsive MAP kinase genes, IbMPK3 and IbMPK6, in sweetpotato", 《PLANT PHYSIOLOGY AND BIOCHEMISTRY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725367A (en) * | 2021-03-01 | 2021-04-30 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato sucrose invertase gene IbINV and application thereof |
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Application publication date: 20181127 |