CN107760656A - Applications of the corn Stress Related Protein kinases SAPK10 in osmotic stress - Google Patents
Applications of the corn Stress Related Protein kinases SAPK10 in osmotic stress Download PDFInfo
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- CN107760656A CN107760656A CN201610707358.7A CN201610707358A CN107760656A CN 107760656 A CN107760656 A CN 107760656A CN 201610707358 A CN201610707358 A CN 201610707358A CN 107760656 A CN107760656 A CN 107760656A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- 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
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Abstract
The invention provides a kind of Stress Related Protein kinasesZmSAPK10Gene, the amino acid sequence that it is encoded is as shown in SEQ ID No.1, and the nucleotide sequence of the gene coded sequence is as shown in SEQ ID No.2.The present inventionZmSAPK10Gene participates in the resistance to osmotic stress process of plant by gene regulation, significant for cultivating the transgenic crop with certain environment stress resistance.
Description
Technical field
The present invention relates to genetic engineering field, specifically, is related to corn Stress Related Protein kinases ZmSAPK10 genes
And its application.
Background technology
Plant growth and the environment of development are complicated and changeable, arid, high temperature, extreme low temperature and disease in many changes
The adverse circumstances such as substance are an important factor for influenceing growth and development of plants.Plant forms a set of to survive and continue species
The regulatory mechanism of these adverse circumstances of effective perception and response.In numerous regulatory mechanisms, the phosphorylation of albumen and dephosphorization
Acidization plays vital effect in the Signaling transduction networks of cellular level.Wherein, the albumen for being responsible for phosphorylation swashs
Enzyme (protein kinase) and responsible dephosphorylized phosphoprotein phosphatase (protein phosphatase) are to be prevalent in
All organisms, and phosphorylation and the dephosphorylation reaction being catalyzed by them are almost related to all physiology and pathology mistake
The expression of journey, such as gene, the growing of cell, the transduction of signaling molecule and photosynthesis and transpiration.For albumen
The research of kinases is focused mostly in animal or yeast, and the research to plant protein kinase is started late, but is developed comparatively fast, wherein right
Higher plant Stress Related Protein kinases (stress-activated protein kinase, SAPK), also referred to as sucrose is non-
The research of glycolysis related protein kinase (sucrose non-fermenting-1-related protein kinase, SnRK)
Progressively warm up.
The SnRK protein kinases of higher plant with animal AMPK (AMP-activated protein kinase,
AMPK) and in yeast SNF1 (sucrose non-fermenting-1, SNF1) collectively constitutes SNF1 protein kinase superfamilies.
In addition, SnRK protein kinases family can be divided into tri- subfamilies of SnRK1, SnKR2 and SnKR3 again.Wherein, SnKR2 albumen is swashed
The sequential analysis of protein result of enzyme family shows that it contains conservative kinase domain in N, but C-terminal sequence variation is larger, conservative
Property is relatively low, thus it is speculated that relevant with the variation of its function.By to known genomic data（Including EST）Scanning analysis hair
Existing, SnRK2 is existed only in plant, and it is probably a kind of gene family specific to plant to illustrate them, in fact it could happen that in plant
Early stage evolve in.
Understanding of the people to SnRK2 just gradually increases intensification, wherein the research in mode in plant Arabidopsis thaliana is more deep.
10 family members, wherein AtSnRK2.2, AtSnRK2.3 and AtSnRK2.6/OST1 (Open are shared in arabidopsis
Stomata1 the process closed by the stomata of ABA mediations) is participated in.And its two other family member, AtSnRK2.4 and
AtSnRK2.10 is then primarily involved in the plant growth of root and morphogenesis under salt stress.Equally, SnRK3（Also referred to as calcium adjusts phosphorus
Sour enzyme B albuminoids interaction protein kinase C IPK (calcineurin B-like protein interacting protein
kinase)）Gene family contains 26 family members altogether in arabidopsis, and wherein CIPK3 participates in the germination process of plant seed,
And CIPK24 then participates in resisting for plant pair salt stress.In addition, there are some researches show cipk24 mutant plants to salt stress more
Sensitivity, and it is overexpressed the CIPK24 then more preferable salt tolerances of render transgenic plant acquisition.Though it is interesting that cipk3 mutant plants
The sensitivity to ABA is so shown as, and causes seed germination rate to decline, but the transfer-gen plant for being overexpressed CIPK3 does not increase
Add the resistance to ABA, equally also without the germination rate of increase seed.These results of study show that the gene of a family is compiled
Although the protein sequence of code, with higher similarity, it is even entirely different that its function there may be redundancy, and to adverse circumstance
Responsive genes are also required to more careful research, so as to ensure its smooth application in genetic engineering improves breeding.
Therefore, the research at present for plant SnRK2 genes is exactly a field in the ascendant, some only researchs
As a result simply the desk study in this field, the research work of this respect are substantially carried out on model plant arabidopsis
's.For important crops（Such as rice, corn, wheat）This genoid research still in the early stage, more particularly to corn
The functional study of SAPK family genes is less.
The content of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of corn Stress Related Protein to swash
The application of enzyme ZmSAPK10 genes.
In order to realize the object of the invention, present invention firstly provides a kind of corn Stress Related Protein kinases ZmSAPK10 bases
Cause, the amino acid sequence that it is encoded is as shown in SEQ ID No.1.
Preferably, the nucleotide sequence of the coded sequence of the gene is as shown in SEQ ID No.2.
Present invention also offers the primer pair for expanding ZmSAPK10 gene coded sequences, the primer pair includes：
Forward primer F：5'- atggaccgggcggcgctcacgctgg -3'；
Reverse primer R：5'- tcaaatagcatacacgatttcccca -3'.
Present invention also offers the carrier containing forementioned gene.
Present invention also offers the engineering bacteria containing forementioned gene.
Present invention also offers application of the forementioned gene in Osmotic Stress Tolerance In Plant is improved.
Further, forementioned gene is transformed into plant by carrier or engineering bacteria, obtains genetically modified plants plant
Strain, so as to improve the resistance of Plant Osmotic Stress.
Specially：
1）Using corn cDNA as template, it is connected, is passed through with pGWC-T carriers using foregoing primer pair amplifies ZmSAPK10 gene orders
Sequencing is crossed to determine to be named as pGWC-ZmSAPK10 after obtained sequence is consistent with purpose fragment；
2）PGWC-ZmSAPK10 carriers and plant expression vector pEarleyGate100 are subjected to restructuring connection by LR enzymes, so
DH5 α competent cells are converted afterwards, obtain recombinant clone, after PCR is detected, send monoclonal to sequencing, correctly clone is sequenced
It is named as pEarleyGate100-ZmSAPK10；
3）The carrier of preparation is converted into Agrobacterium GV3101 bacterial strains；
4）Using Agrobacterium GV3101 bacterial strains conversion plant, obtain homozygous conversion positive seedling strain；
5）Transgenic line dibbling is cultivated on the MS culture mediums containing 325mM mannitol (mannitol), obtains resistance to infiltration
The transfer-gen plant of stress.
The beneficial effects of the present invention are：
Present invention firstly discovers that the albumen of ZmSAPK10 codings participates in the resistance to osmotic stress of plant by regulating and controlling downstream related gene
Process.Further such that ZmSAPK10 genes of the present invention by gene regulation come participate in Drought Stress Tolerance in Plants process turn into can
Can, it is significant for cultivating the transgenic crop with certain environment stress resistance.
Brief description of the drawings
Fig. 1 is plant expression vector schematic diagram in the embodiment of the present invention 1.
Fig. 2 is the PCR detections of ZmSAPK10 transgenic line target gene in the embodiment of the present invention 3；
OE1-2：ZmSAPK10 difference transgenic line target gene amplifications；CK：Positive control (is overexpressed with ZmSAPK10 and carried
Body is pcr template)；WT：Negative control (using arabidopsis wild type Col0 DNA as pcr template)；gene：For ZmSAPK10 expansion
Open up fragment；Bar：For the extension fragment of Bar genes.
Fig. 3 is for transgenic arabidopsis in the embodiment of the present invention 4 with WT lines in the culture containing Different stress condition
Growing state compares on base.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
The structure of expression vector of the embodiment 1 containing ZmSAPK10 genes
1st, the sequence of target gene known to foundation（As shown in SEQ ID No.2）, primer is designed, is expanded from corn cDNA
ZmSAPK10 gene orders, and reclaim purpose fragment with Ago-Gel kit.
2nd, the fragment of recovery is connected with pGWC-T carriers, then converts DH5 α competent cells, obtain positive monoclonal,
Sequencing is sent, by sequence alignment, the monoclonal consistent with former sequence is obtained, shakes bacterium and extract the monoclonal plasmid, named
For pGWC-ZmSAPK10.
3rd, LR enzymes are added after pGWC-ZmSAPK10 and pEarleyGate100 plasmids are mixed, are obtained after being reacted by LR
Recombinant vector pEarleyGate100-ZmSAPK10（As shown in Figure 1）.DH5 α competent cells are converted afterwards, by the sun of acquisition
Property monoclonal send sequencing, by sequence alignment confirm it is errorless.
Conversion and screening of the embodiment 2 containing corn SAPK10 genes
1st, the ZmSAPK10 overexpression vectors built are transferred to Agrobacterium GV3101.
2nd, with being stained with colored dip-dye（floral dipping）Method arabidopsis thaliana transformation wild type.
3rd, then the T1 obtained after conversion is directly seeded into Nutrition Soil and grown, normal growth is about for seed vernalization 3 days
After two weeks, with 0.5 ‰ PPT（phosphinothricin）Spray T1 and screen transformed plant for arabidopsis.
4th, because plant overexpression vector used carries anti-PPT（phosphinothricin）Basta genes, it
Plant can be transferred to target gene during conversion, so spraying PPT（phosphinothricin）It is most of afterwards not turn
It is dead to change plant, and transformed plant remains able to normal growth.Transformed plant harvests T2 for seed respectively according to different strains.
5th, after harvesting each transgenic line T2 for seed, seed is carried out disinfection processing with 0.5% NaClO.Then dibbling
In containing 0.5 ‰ PPT（phosphinothricin）MS culture medium flat plates in (each transgenic line needs about 50 kinds
Son), using wild type as negative control.
6th, vernalization is placed in 22 DEG C of illumination boxs after three days and grown, and growth of seedling situation is observed after one week.Wild type exists
Containing 0.5 ‰ PPT（phosphinothricin）MS culture mediums in can not survive；T2 for heterozygote transgenic line due to
The separation of meeting producer and independent assortment during generation filial generation, therefore some is understood without PPT
（phosphinothricin）The seed of resistance occurs；Only T2 on behalf of homozygote transgenic line seed can all containing
PPT（phosphinothricin）Survived in the MS culture mediums of resistance.
Transfer-gen plant nucleic acid PCR analysis of the embodiment 3 containing corn SAPK10 genes
1st, using the STb gene of the positive plant of extraction as template, enter performing PCR with ZmSAPK10 gene primers and expand, positive plant energy
1089bp band is enough amplified, and WT lines can not amplify purpose band, as shown in Figure 2.
2nd, after harvesting each transgenic line T2 for seed, seed is carried out disinfection processing with 0.5% NaClO.Then dibbling
In containing 0.5 ‰ PPT（phosphinothricin）MS culture medium flat plates in (each transgenic line needs about 50 kinds
Son), using wild type as negative control.
3rd, vernalization is placed in 22 DEG C of illumination boxs after three days and grown, and growth of seedling situation is observed after one week.Wild type exists
Containing 0.5 ‰ PPT（phosphinothricin）MS culture mediums in can not survive.
4th, T2 for heterozygote transgenic line due to during filial generation is produced can producer separation and independent assortment,
Therefore some is understood without PPT（phosphinothricin）The seed of resistance occurs；Only T2 is on behalf of homozygote transgenic line
The seed of system can be all containing PPT（phosphinothricin）MS culture mediums in survive.
Transfer-gen plant of the embodiment 4 containing SAPK10 genes is compared with WT lines resistance
By the dibbling of ZmSAPK10 transgenic lines on the MS flat boards containing 325mM mannitol, 4 DEG C of vernalization 3 days, 22 DEG C are placed in,
In the dark incubator in 16 small time/8 hour, photographic analysis after 14 days（As shown in Figure 3）.As can be seen from the results, containing
There is the wild type cotyledon development under mannitol Stress treatment slow and seedling greening-rate reduces, and the growing way of transgenic line is then obvious excellent
In WT lines.In addition both in normal MS culture mediums phenotype without significant difference.
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 made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. application of the gene of amino acid sequence shown in coding SEQ ID No.1 in improvement plant resists osmotic stress.
2. ZmSAPK10 genes according to claim 1, it is characterised in that the nucleotide sequence of the gene such as SEQ ID
Shown in No.2.
3. application according to claim 1 or 2, it is characterised in that be transformed into the gene by carrier or engineering bacteria
In plant, through screening, the genetically modified plants plant for improveing osmotic stress resistance is obtained.
4. application according to claim 3, it is characterised in that specifically comprise the following steps：
1）Using corn cDNA as template, it is connected using the primer pair amplifies ZmSAPK10 gene orders, and with pGWC-T carriers,
PGWC-ZmSAPK10 is named as after sequencing determines that obtained sequence is consistent with purpose fragment；
The primer pair includes：
Forward primer F：5'- atggaccgggcggcgctcacgctgg -3'；
Reverse primer R：5'- tcaaatagcatacacgatttcccca -3'；
2）PGWC-ZmSAPK10 carriers and plant expression vector pEarleyGate100 are subjected to restructuring connection by LR enzymes, so
DH5 α competent cells are converted afterwards, obtain recombinant clone, after PCR is detected, send monoclonal to sequencing, correctly clone is sequenced
It is named as pEarleyGate100-ZmSAPK10；
3）The carrier of preparation is converted into Agrobacterium GV3101 bacterial strains；
4）Using Agrobacterium GV3101 bacterial strains conversion plant, obtain homozygous conversion positive seedling strain；
5）Transgenic line is cultivated on MS culture mediums and MS culture mediums containing osmotic stress material mannitol respectively, obtained
The transfer-gen plant of resistance to osmotic stress.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610707358.7A CN107760656A (en) | 2016-08-23 | 2016-08-23 | Applications of the corn Stress Related Protein kinases SAPK10 in osmotic stress |
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| CN201610707358.7A CN107760656A (en) | 2016-08-23 | 2016-08-23 | Applications of the corn Stress Related Protein kinases SAPK10 in osmotic stress |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923213A (en) * | 2018-09-19 | 2020-03-27 | 南京农业大学 | Tea tree protein kinase gene CsCIPK sequence and application thereof |
| CN112575010A (en) * | 2020-12-14 | 2021-03-30 | 云南农业大学 | Reference gene for fluorescence quantification of different tissues of Chinese yam as well as primer and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923213A (en) * | 2018-09-19 | 2020-03-27 | 南京农业大学 | Tea tree protein kinase gene CsCIPK sequence and application thereof |
| CN112575010A (en) * | 2020-12-14 | 2021-03-30 | 云南农业大学 | Reference gene for fluorescence quantification of different tissues of Chinese yam as well as primer and application thereof |
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