CN105296443B - A kind of plant drought, protein related to salt tolerance EeSAPK7 and its encoding gene and application - Google Patents
A kind of plant drought, protein related to salt tolerance EeSAPK7 and its encoding gene and application Download PDFInfo
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- CN105296443B CN105296443B CN201510890523.2A CN201510890523A CN105296443B CN 105296443 B CN105296443 B CN 105296443B CN 201510890523 A CN201510890523 A CN 201510890523A CN 105296443 B CN105296443 B CN 105296443B
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- 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.)
- C12N9/12—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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
Abstract
The present invention relates to genetic engineering fields, in particular it relates to a kind of plant drought GAP-associated protein GAP EeSAPK7 and its encoding gene and application.The amino acid sequence of the albumen is as shown in SEQ ID NO.1, and gene order is as shown in SEQ ID NO.2.Drought resisting, protein related to salt tolerance and its encoding gene of the invention improves yield, accelerates degeneration-resistant molecular breeding process to improvement, enhancing arabidopsis resistance, and effective water resource of saving has highly important theoretical and practical significance.
Description
Technical field
The present invention relates to genetic engineering fields, in particular it relates to a kind of plant drought, protein related to salt tolerance
EeSAPK7 and its encoding gene and application.
Background technique
The wheat cereal crops one of important as China, play a very important role in national economy.However, every
Year because the environment stresses condition such as arid, saline and alkaline drastically influences the yield and quality of wheat, restricts China's wheat grain security.
The relationship between plant and abiotic stress is furtherd investigate from molecular level using technique for gene engineering, discloses plant to adverse circumstance
Stress signal conduction and gene expression regulation molecule mechanism are cloned anti contravariance related because cultivating the degeneration-resistant new germ plasm of crop provides time
Select adversity gene resource.
Sucrose non-fermented related protein kinase enzyme family (SnRKs) plays important work in many physiology courses of plant
With, such as hormone signal conduction, abiotic stress and growth and development of plant etc..SnRK protein kinase belongs to serine/Soviet Union's ammonia
Pka acid super families are divided into three subfamily difference due to the similitude of gene order and the difference of gene structure
It is: SnRK1, SnRK2 and SnRK3.Three subfamilies of SnRK protein kinase have similar design feature, and there is one section of energy at the end N-
With the kinase domain of other protein-interactings, and structure is height change in three families.
SnRK2 family gene functionally shows certain otherness, there is 9 bases in arabidopsis in SnRK family member
Because being induced by hyperosmotic stress (mannitol or NaCl), 5 genes are induced by ABA, but not by induction of chilling stress.In rice,
10 SnRK2 protein kinase family genes are identified, OsSAPK1~OsSAPK10 is named as；SnRK gene in rice, passes through egg
White phosphorus acidification is analysis shows all members can be activated by hyperosmotic stress, but only OsSAPK8, OsSAPK9 and OsSAPK10
These three genes are by ABA inducing expression.In wheat, first SnRK2 member is the wheat embryo cDNA library handled from ABA
In isolated PKABA1, the expression of PKABA1 by ABA and drought stress except being induced.In addition, also being identified in wheat
Multiple SnRK family members, such as TaSnRK2.3, TaSnRK2.4, TaSnRK2.7 and TaSnRK2.8.TaSnRK2.3 gene crosses table
Danone enough enhances the stability of transgenic arabidopsis cell membrane and the stress tolerance to arid, low temperature；TaSnRK2.4 gene crosses table
Danone enough enhances transgenic arabidopsis to arid, stress tolerance with high salt, while not will cause the growth dwarfism of plant.
TaSnRK2.7 gene function analysis is shown, in glycometabolism, is reduced osmotic potential, enhances the activity of Photosystem I I and is promoted plant
It takes root for waiting and play an important role in physiological and biochemical procedures；The arabidopsis of TaSnRK2.8 gene overexpression to arid, low temperature, it is with high salt,
High temperature etc. has certain stress tolerance.
In conclusion SnRK protein kinase is improved and is played in the resistance of plant to pass in the Response to stress for adjusting plant
Important role can generate huge impetus and economic benefit to breeding for stress tolerance and agricultural production.Therefore, drought resisting, resistance to is utilized
The wild plant E. elongata of salt is experimental material, clones, separates degeneration-resistant correlation SnRK protein kinase gene improvement and improve
The resistance of crop has very important significance.
Summary of the invention
The object of the present invention is to provide a kind of plant droughts, protein related to salt tolerance EeSAPK7.
Another object of the present invention is to provide the gene for encoding above-mentioned plant drought, salt tolerant correlation egg EeSAPK7.
It is a further object of the present invention to provide the recombinant vectors comprising said gene.
It is a further object of the present invention to provide the transgenic cell lines comprising said gene.
Another object of the present invention provides the application of above-mentioned plant drought, protein related to salt tolerance EeSAPK7.
Drought resisting provided by the present invention, protein related to salt tolerance EeSAPK7 derive from E. elongata, amino acid sequence
As shown in SEQ ID NO.1.
Protein kinase of the invention is made of 357 amino acid residues, is SnRK albuminoid kinases.From SEQ ID NO.1
Amino terminal 10-34 amino acids residue be ATP binding domain, from the 119-131 amino acids residue of SEQ ID NO.1
For serine/threonine binding domain.
SEQ ID NO.1
In order to make albumen EeSAPK7 convenient for purifying, the albumen that can be formed in the amino acid sequence shown in SEQ ID NO.1
The amino terminal or carboxyl terminal of matter connect upper label as shown in Table 1.
The sequence of 1 label of table
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tagII | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Disclosed SEQ ID NO.1 sequence according to the present invention, transcription factor EeSAPK7 of the invention can be artificial synthesized,
Its encoding gene can also be first synthesized, then carries out biological expression and obtains.
EeSAPK7 encoding gene according to the present invention has the cDNA sequence as shown in SEQ ID NO.2.
SEQ ID NO.2
Expression cassette, recombinant expression carrier, transgenic cell line and recombinant bacterium containing EeSAPK7 gene belong to the present invention
Protection scope.
The recombinant expression carrier of EeSAPK7 gene can be contained with existing plant expression vector construction.
The plant expression vector includes double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment.The plant
Object expression vector also may include 3 ' end untranslated regions of foreign gene, that is, include polyadenylation signals and any other participation
The DNA fragmentation of mRNA processing or gene expression.The bootable polyadenylic acid of polyadenylation signals is added to the 3 ' of mRNA precursor
End, such as Agrobacterium crown gall nodule induction (Ti) plasmid gene (such as kermes synzyme Nos gene), non-the turning over of the end of plant gene 3 ' transcription
It translates area and all has similar functions.
It, can be plus any one before its transcription initiation nucleotide when constructing recombinant plant expression vector using EeSAPK7
Enhanced promoter or constitutive promoter, such as the ubiquitin promoter of cauliflower mosaic virus (CaMV) 35S promoter, corn
(Ubiquitin), they can be used alone or are used in combination with other plant promoters；In addition, using gene structure of the invention
When building plant expression vector, enhancer also can be used, including translational enhancer or transcriptional enhancer, these enhancer regions can be with
It is ATG initiation codon or neighboring region initiation codon etc., but must be identical as the reading frame of coded sequence, it is entire to guarantee
The correct translation of sequence.The source of the translation control signal and initiation codon be it is extensive, can be it is natural, can also be with
It is synthesis.Translation initiation region can come from transcription initiation region or structural gene.
For the ease of transgenic plant cells or plant are identified and screened, plant expression vector used can be carried out
Processing, as be added the coding that can be expressed in plant can produce color change enzyme or luminophor gene (gus gene,
Luciferase genes etc.), resistant antibiotic marker (gentamicin marker, kanamycins marker etc.) or anti-
Chemical reagent marker gene (such as anti-herbicide gene).From the security consideration of genetically modified plants, any selectivity can be not added
Marker gene directly screens transformed plant with adverse circumstance.
It is a further object to provide a kind of methods for cultivating plant with adverse resistance.
The method provided by the present invention for cultivating plant with adverse resistance, is by any of the above-described kind of recombination containing EeSAPK7 gene
Expression vector imports in plant cell, obtains plant with adverse resistance.
The carrier that foreign gene can be guided to express in plant using any one, by SnRK egg provided by the present invention
White kinases EeSAPK7 gene transfered plant cell can get and turn to abiotic stress tolerance enhancings such as arid and salt
Gene cell system and transgenic plant.The expression vector for carrying encoding gene can be by using Ti-plasmids, Ri plasmid, phytopathy
The conventional biology methods such as poisonous carrier, directly delivered DNA, microinjection, conductance, mediated by agriculture bacillus convert plant cell or group
It knits, and the plant tissue of conversion is cultivated into plant.The plant host being converted is either monocotyledon, is also possible to double
Cotyledon plant, such as: arabidopsis, wheat, E. elongata, arabidopsis, rice, corn, cucumber, tomato, poplar, turfgrass, lucerne
Place etc..
The present invention with drought resisting, the stronger E. elongata of salt tolerance (Elytrigia elongate L.) be experimental material,
Degeneration-resistant relevant EeSAPK7 albumen and its encoding gene have been obtained, and has been conducted into arabidopsis, has significantly improved the anti-of plant
Drought, salt tolerance.Drought resisting, protein related to salt tolerance and its encoding gene of the invention is improved and is produced to improvement, enhancing arabidopsis resistance
It measures, accelerate degeneration-resistant molecular breeding process, and effectively save water resource there is highly important theoretical and practical significance.
With reference to the accompanying drawing and specific embodiment the present invention will be further described.
Detailed description of the invention
Fig. 1 is that T1 is detected for transgenic arabidopsis PCR.M:Trans2K Plus DNAmarker；1: negative control；2-8
For transgenic arabidopsis strain.
Fig. 2 is EeSAPK7 transgenic arabidopsis culture medium root long phenotypic evaluation.CK is wildtype Arabidopsis thaliana；L1-L5 is not
With transgenic arabidopsis strain.
Fig. 3 is the identification of EeSAPK7 transgenic arabidopsis drought tolerance.CK is wildtype Arabidopsis thaliana；L1, L3, L4, L5 are difference
Transgenic arabidopsis strain.
Fig. 4 is that EeSAPK7 transgenic arabidopsis sieves box Salt-Tolerance Identification.CK is wildtype Arabidopsis thaliana；L1, L3, L4, L5 are
Different transgenic arabidopsis strains.
Specific embodiment
Do not make the experimental methods of molecular biology illustrated in following embodiment, referring to " Molecular Cloning:A Laboratory guide "
Listed specific method carries out in one book of (third edition) J. Pehanorm Brooker, or carries out according to kit and product description.
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.
Embodiment 1: E. elongata drought resisting, salt tolerant correlation EeSAPK7 gene cDNA clone.
The E. elongata seedling of growth 30 days or so is carried out Osmotic treatment 5 hours, long fringe is extracted with Trizol and lays down wheat
Careless total serum IgE.Using 5 ' RACE kits (5 ' RACE System for Rapid Amplification of cDNA Ends
Kit) (GIBCOBRL, CAT.NO.18374-058) and 3 ' RACE kits (3 ' RACE System for Rapid
Amplification of cDNA Ends Kit) (GIBCOBRL, CAT.NO.18373-019) obtain EeSAPK7 gene it is complete
Long sequence 1074bp.
The total serum IgE that E. elongata seedling is extracted with Trizol, with superscript II (invitrogen) reverse transcription
Enzyme reverse transcription acquires cDNA.According to EeSAPK7 coding sequence design primer P1 and P2.The cDNA obtained with reverse transcription
For template, PCR amplification is carried out with primer P1 and P2.The sequence of primer P1 and P2 are as follows:
P1:5 '-ATGGAGAGGTACGAGCTGCT-3 ',
P2:5 '-TCAGCTGATGTGGAACTCACCGCT-3 '.
0.8% agarose gel electrophoresis detection is carried out to PCR product, obtains the band that molecular weight is about 1kb or so, and it is pre-
Phase result is consistent.The segment is recycled with Ago-Gel QIAquick Gel Extraction Kit (TIANGEN).By the recycling segment and pGEM-T
Easy (Promega) connection turns connection product referring to the method (Proc Natl Acad Sci, 69:2110) of Cohen etc.
Change bacillus coli DH 5 alpha competent cell, according to positive gram of the screening of acillin resistance marker on pGEM-T Easy carrier
It is grand, obtain the recombinant plasmid containing recycling segment.Using T7 the and SP6 promoter sequence on the recombinant plasmid vector as primer pair its
Nucleotide sequencing is carried out, sequencing result shows that the open reading frame (ORF) of the EeSAPK7 gene expanded is SEQ ID
No.2 from the deoxyribonucleotide of 5 ' end the 1st to 1074, encoding amino acid sequence is the protein of SEQ ID No.1.
The recombinant vector of EeSAPK7 gene shown in the ID of SEQ containing sequence No.2 is named as pTE-EeSAPK7.
The sequence of EeSAPK7 gene is compared, does not find homologous protein gene in E. elongata, it was demonstrated that
EeSAPK7 gene is a new gene.
It is further expanded in E. elongata genome with primer P1 and P2, as the result is shown the genome of the gene
Sequence size is consistent with cDNA length scale, does not contain intron sequences.
Embodiment 2: with the drought resisting of EeSAPK7 genes amplification plant, salt tolerance
1, the building of recombinant expression carrier
1) building of 35S-EeSAPK7 recombinant expression carrier
The cDNA obtained using the total serum IgE reverse transcription of E. elongata is template, with containing SmaI and SpeI joint sequence
Special primer carries out PCR amplification；Then SmaI and SpeI double digestion PCR product recycles, and digestion products forward direction is inserted into carrier
Between SmaI and SpeI restriction enzyme site after the CaMV 35S promoter of pBI121, recombinant vector p35S::EeSAPK7 is obtained.
Primer sequence is as follows:
EeSAPK7[SmaI]5’-TCCCCCGGGGATGGAGAGGTACGAGCTGCT-3’
EeSAPK7[SpeI]5’-GGACTAGTTCAGCTGATGTGGAACTCACCGCT-3’
2, transgenic arabidopsis acquisition and Function Identification
1) acquisition of transgenic arabidopsis
The recombinant expression carrier p35S::EeSAPK7 of above-mentioned building is converted into Agrobacterium tumefaciems with freeze-thaw method respectively
EHA105, then with the Agrobacterium tumefaciems EHA105 arabidopsis thaliana transformation of p35S::EeSAPK7, with the MS of the kanamycins containing 100mg/L
Culture medium is screened, and positive transgenic plant is obtained.The positive transgenic plant that screening obtains is cooked into further identification with PCR
It screens, pair of primers used in PCR is P3 and P4.
P3 (upstream primer): 5 '-GGTCACGCCGACGCACCTGG-3 ',
P4 (downstream primer): 5 '-TGCAGTCTTGGGATACGTGG-3 '.
PCR identification is carried out to 35S::EeSAPK7 transgenic arabidopsis, positive transgenic plant can get through PCR amplification
As a result 500bp or so band obtains and turns 34 plants of 35S::EeSAPK7 arabidopsis (Fig. 1).
PBI121 empty carrier is imported into arabidopsis simultaneously, method is same as above, and as control, obtain 15 strains turns empty carrier
Arabidopsis (transgenic arabidopsis that screening obtains is represented with T3 to be shown).
2) transgenic arabidopsis root long statisticallys analyze under ABA, PEG stress
In order to verify transgenic arabidopsis and wild type to ABA, PEG stress tolerance, 50 μM are added on MS culture medium
ABA or 5%PEG carries out the analysis of root system phenotypic evaluation.On the MS culture medium of 50 μM of ABA, the root long of L1, L2 and L5 with it is wild
Type arabidopsis root long is essentially identical, and L3, L4 strain are slightly more longer than wildtype Arabidopsis thaliana root system；In the MS culture medium of 5%PEG
On, L4, L5 transgenic line are considerably longer than wildtype Arabidopsis thaliana, and lateral root number is more, and blade is also bigger than wild type；L1, L2 and L3
The root long of transgenic line is more slightly longer than wildtype Arabidopsis thaliana, illustrates that L4, L5 for turning EeSAPK7 gene are drought-enduring on culture medium
Effect is more apparent (Fig. 2).
3) transgenic arabidopsis drought tolerance is identified
To detect transgenic Arabidopsis plants drought tolerance, lower to drought stress 20 days, turn EeSAPK7 within the 10th day after rehydration
Gene phenotype carries out photograph observation (Fig. 3).At Osmotic treatment the 20th day, wildtype Arabidopsis thaliana and transgenic arabidopsis all withered
It is listless, there is the phenomenon that mortality.Find that wildtype Arabidopsis thaliana is all dead, transgenic arabidopsis L4, L5 within the 10th day after rehydration
Plant part has restored normal condition, and survival rate is 35.7%, 30% respectively, and survival rate is significantly greater than wildtype Arabidopsis thaliana, table
The bright EeSAPK7 gene that turns significantly improves the drought resistance of arabidopsis.
4) transgenic arabidopsis Salt-Tolerance Identification
The identification of salt stress patience further has been carried out to transgenic Arabidopsis plants.The consistent transgenosis of upgrowth situation is intended
Southern mustard and wild type pour 250mM salt water simultaneously, handle the 20th day salt stress, turn EeSAPK7 gene phenotype within the 5th day after rehydration
Carry out photograph observation.Phenotypic evaluation is the results show that the phenomenon that mortality, but L4 and L5 transgenic line occurs in wildtype Arabidopsis thaliana
Normal condition has been restored by system, and the survival rate of L4 is that the survival rate of 15.2%, L5 is 65% (Fig. 4).And the quasi- south of L1 and L3 transgenosis
Mustard strain and wildtype Arabidopsis thaliana are all dead, show that the overexpression of EeSAPK7 gene improves the resistance to of L4, L5 transgenic line
Salt.
Claims (1)
1. plant drought, salt-resistant related geneEeSAPK7Application in terms of improving plant drought, salt tolerance, geneEeSAPK7
Base sequence as shown in SEQ ID NO. 2.
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| CN108530524A (en) * | 2018-04-18 | 2018-09-14 | 山东省果树研究所 | The application of birch-leaf pear Pb4RMYB genes and its coding albumen in improving plant salt endurance |
| CN109666681B (en) * | 2018-11-07 | 2020-09-04 | 北京市农林科学院 | Plant drought-resistant and salt-tolerant protein EeCIPK26 as well as coding gene and application thereof |
| CN109777790B (en) * | 2018-11-08 | 2020-09-01 | 北京市农林科学院 | Plant drought-resistant and salt-tolerant associated protein EeSAPK4, and coding gene and application thereof |
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