CN105296443A - Plant drought-resistant salt-tolerant associated protein EeSAPK7 as well as encoding gene and application thereof - Google Patents
Plant drought-resistant salt-tolerant associated protein EeSAPK7 as well as encoding gene and application thereof Download PDFInfo
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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 invention relates to the field of genetic engineering, and in particular to plant drought-resistant salt-tolerant associated protein EeSAPK7 as well as an encoding gene and an application thereof. The amino acid sequence of the protein is as shown in SEQ ID NO.1, and the gene sequence of the protein is as shown in SEQ ID NO.2. The plant drought-resistant salt-tolerant associated protein and the encoding gene of the protein have very important theoretical and practical significance for improving and enhancing stress resistance of arabidopsis thaliana, increasing the yield, accelerating stress resistance molecular breeding process and effectively saving water resource.
Description
Technical field
The present invention relates to genetically engineered field, particularly, the present invention relates to a kind of plant drought, protein related to salt tolerance EeSAPK7 and encoding gene thereof and application.
Background technology
Wheat, as one of important food crop of China, occupies very important status in national economy.But, every year because the environment stress condition such as arid, saline and alkaline drastically influence the yield and quality of wheat, govern China's wheat grain security.Genetic engineering technique is utilized to further investigate the relation between plant and abiotic stress from molecular level, disclose plant to environment stress intracellular signaling and gene expression regulation molecule mechanism, clone anti contravariance related because cultivate the degeneration-resistant new germ plasm of crop to provide candidate's adversity gene resource.
Sucrose non-fermented related protein kinase enzyme family (SnRKs) plays an important role in many physiological processs of plant, such as hormone signal conduction, the growing of abiotic stress and plant.SnRK protein kinase belongs to serine/threonine protein kitase super families, due to the similarity of gene order and the difference of gene structure, is divided into three subfamilies respectively: SnRK1, SnRK2 and SnRK3.SnRK protein kinase three subfamilies have similar constructional feature, and N-end has the kinase domain of one section of energy and other protein-interactings, and structure is height change in three families.
SnRK2 family gene functionally shows certain otherness, and have 9 genes to be oozed by height in Arabidopis thaliana in SnRK family member and coerce (N.F,USP MANNITOL or NaCl) induction, 5 genes are induced by ABA, but all not by induction of chilling stress.In paddy rice, identify 10 SnRK2 protein kinase family genes, called after OsSAPK1 ~ OsSAPK10; By protein phosphorylation analysis, SnRK gene in paddy rice, shows that all members can be oozed by height and coerces activation, but only have these three genes of OsSAPK8, OsSAPK9 and OsSAPK10 by ABA abduction delivering.In wheat, first SnRK2 member is separated the PKABA1 obtained from the wheat embryo cDNA library of ABA process, and the expression of PKABA1 is except induced by ABA and drought stress.In addition, in wheat, multiple SnRK family member is also identified, as TaSnRK2.3, TaSnRK2.4, TaSnRK2.7 and TaSnRK2.8.TaSnRK2.3 gene overexpression can strengthen the stability of transgenic arabidopsis cytolemma and the stress tolerance to arid, low temperature; TaSnRK2.4 gene overexpression can strengthen the stress tolerance of transgenic arabidopsis to arid, high salt, can not cause the growth dwarfism of plant simultaneously.TaSnRK2.7 gene function analysis shows, and plays an important role in the physiological and biochemical procedures such as carbohydrate metabolism, reduction osmotic potential, the activity strengthening Photosystem I I and promotion plant establishment; The Arabidopis thaliana of TaSnRK2.8 gene overexpression all has certain stress tolerance to arid, low temperature, high salt, high temperature etc.
In sum, SnRK protein kinase, at the Response to stress of regulating plant, improves in the resistance of plant and plays vital effect, can produce huge pushing effect and economic benefit to breeding for stress tolerance and agriculture production.Therefore, the resistance utilize drought resisting, the wild plant E. elongata of salt tolerant is experiment material, clone, being separated degeneration-resistant relevant SnRK protein kinase gene improvement and improving crop has very important significance.
Summary of the invention
The object of this invention is to provide a kind of plant drought, protein related to salt tolerance EeSAPK7.
The gene that another object of the present invention is to provide the above-mentioned plant drought of coding, salt tolerant is correlated with egg EeSAPK7.
Another object of the present invention is to provide the recombinant vectors comprising said gene.
Another object of the present invention is to provide the transgenic cell line 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, and its aminoacid sequence is as shown in SEQIDNO.1.
Protein kinase of the present invention is made up of 357 amino-acid residues, is SnRK proteinoid kinases.Being ATP binding domain from the N-terminal 10-34 amino acids residue of SEQIDNO.1, is serine/threonine binding domain from the 119-131 amino acids residue of SEQIDNO.1.
SEQIDNO.1
In order to make albumen EeSAPK7 be convenient to purifying, label as shown in table 1 can be connected at the N-terminal of the protein be made up of the aminoacid sequence shown in SEQIDNO.1 or C-terminal.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tagII | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
SEQIDNO.1 sequence disclosed according to the present invention, transcription factor EeSAPK7 of the present invention can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.
EeSAPK7 encoding gene according to the present invention has cDNA sequence as shown in SEQIDNO.2.
SEQIDNO.2
Expression cassette containing EeSAPK7 gene, recombinant expression vector, transgenic cell line and recombinant bacterium all belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of EeSAPK7 gene.
Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor, as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene 3 ' hold the non-translational region of transcribing all to have similar functions.
When using EeSAPK7 to build recombinant plant expression vector, any one enhancement type promotor or constitutive promoter can be added before its transcription initiation Nucleotide, as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CaMV) 35S promoter, corn, they can be used alone or are combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.
For the ease of identifying transgenic plant cells or plant and screening, can process plant expression vector used, the coding can expressed in plant as added can produce enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, the antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance or the chemical resistance reagent marker gene (as anti-weedkiller gene) etc. of colour-change.From the security consideration of transgenic plant, any selected marker can not be added, directly with adverse circumstance screening transformed plant.
Another object of the present invention is to provide a kind of method of cultivating plant with adverse resistance.
The method of cultivation plant with adverse resistance provided by the present invention, is imported in vegetable cell by any one recombinant expression vector containing EeSAPK7 gene above-mentioned, obtains plant with adverse resistance.
The carrier utilizing any one can guide foreign gene to express in plant, by SnRK protein kinase EeSAPK7 gene transfered plant cell provided by the present invention, the transgenic cell line to abiotic stress tolerance enhancings such as Drought and salts and transfer-gen plant can be obtained.The plant tissue of conversion by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, conventional biology methods transformed plant cells or the tissue such as agriculture bacillus mediated, and is cultivated into plant by the expression vector carrying encoding gene.The plant host be converted both can be monocotyledons, also can be dicotyledons, as: Arabidopis thaliana, wheat, E. elongata, Arabidopis thaliana, paddy rice, corn, cucumber, tomato, willow, turfgrass, lucerne place etc.
The present invention with drought resisting, E. elongata (ElytrigiaelongateL.) that salt tolerance is stronger for experiment material, obtain degeneration-resistant relevant EeSAPK7 albumen and encoding gene thereof, and imported Arabidopis thaliana, significantly improve the drought resisting of plant, salt tolerance.Drought resisting of the present invention, protein related to salt tolerance and encoding gene thereof, to improvement, enhancing Arabidopis thaliana resistance, improve output, accelerate degeneration-resistant molecular breeding process, and effectively saving water resources have very important theoretical and practical significance.
Below in conjunction with drawings and the specific embodiments, the present invention will be further described.
Accompanying drawing explanation
Fig. 1 is that T1 detects for transgenic arabidopsis PCR.M:Trans2KPlusDNAmarker; 1: negative control; 2-8 is transgenic arabidopsis strain.
Fig. 2 is the long phenotypic evaluation of EeSAPK7 transgenic arabidopsis substratum root.CK is wildtype Arabidopsis thaliana; L1-L5 is different transgenic arabidopsis strains.
Fig. 3 is the qualification of EeSAPK7 transgenic arabidopsis drought tolerance.CK is wildtype Arabidopsis thaliana; L1, L3, L4, L5 are different transgenic arabidopsis strains.
Fig. 4 is EeSAPK7 transgenic arabidopsis sieve box Salt-Tolerance Identification.CK is wildtype Arabidopsis thaliana; L1, L3, L4, L5 are different transgenic arabidopsis strains.
Embodiment
Do not make the experimental methods of molecular biology illustrated in following examples, all carry out with reference to concrete grammar listed in " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book, or carry out according to test kit and product description.
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.
Embodiment 1: E. elongata drought resisting, salt tolerant be correlated with EeSAPK7 gene cDNA clone.
Osmotic treatment is carried out 5 hours to the E. elongata seedling of growth about 30 days, extracts E. elongata total serum IgE with Trizol.Application 5 ' RACE test kit (5 ' RACESystemforRapidAmplificationofcDNAEndsKit) (GIBCOBRL, CAT.NO.18374-058) and 3 ' RACE test kit (3 ' RACESystemforRapidAmplificationofcDNAEndsKit) (GIBCOBRL, CAT.NO.18373-019) obtain the full length sequence 1074bp of EeSAPK7 gene.
Extract the total serum IgE of E. elongata seedling with Trizol, acquire cDNA with the reverse transcription of superscriptII (invitrogen) ThermoScript II.According to EeSAPK7 coding sequence design primer P1 and P2.The cDNA obtained with reverse transcription is template, carries out pcr amplification with primer P1 and P2.The sequence of primer P1 and P2 is as follows:
P1：5’-ATGGAGAGGTACGAGCTGCT-3’，
P2：5’-TCAGCTGATGTGGAACTCACCGCT-3’。
0.8% agarose gel electrophoresis detection is carried out to PCR primer, obtains the band that molecular weight is about about 1kb, conform to expected results.Reclaim test kit (TIANGEN) with sepharose and reclaim this fragment.This recovery fragment is connected with pGEM-TEasy (Promega), with reference to the method (ProcNatlAcadSci of Cohen etc., 69:2110), product conversion bacillus coli DH 5 alpha competent cell will be connected, according to the acillin resistance marker screening positive clone on pGEM-TEasy carrier, obtain the recombinant plasmid containing reclaiming fragment.With T7 and the SP6 promoter sequence on this recombinant plasmid vector, for primer pair, it carries out nucleotide sequencing, sequencing result show that the open reading frame (ORF) of the EeSAPK7 gene increased is SEQIDNo.2 from 5 ' end the 1 to 1074 deoxyribonucleotide, encoding amino acid sequence is the protein of SEQIDNo.1.By the recombinant vectors called after pTE-EeSAPK7 containing EeSAPK7 gene shown in sequence SEQIDNo.2.
The sequence of EeSAPK7 gene is compared, in E. elongata, does not find homologous protein gene, prove that EeSAPK7 gene is a new gene.
Increase in E. elongata genome with primer P1 and P2 further, the genome sequence size that result shows this gene is consistent with cDNA length scale, not containing intron sequences.
Embodiment 2: by drought resisting, the salt tolerance of EeSAPK7 genes amplification plant
1, the structure of recombinant expression vector
1) structure of 35S-EeSAPK7 recombinant expression vector
The cDNA obtained with the total serum IgE reverse transcription of E. elongata, for template, carries out pcr amplification with the special primer containing SmaI and SpeI joint sequence; Then SmaI and SpeI double digestion PCR primer reclaims, and between SmaI and the SpeI restriction enzyme site after the CaMV35S promotor of digestion products forward insertion vector pBI121, obtains recombinant vectors p35S::EeSAPK7.
Primer sequence is as follows:
EeSAPK7[SmaI]5’-TC
CCCCGGGGATGGAGAGGTACGAGCTGCT-3’
EeSAPK7[SpeI]5’-GG
ACTAGTTCAGCTGATGTGGAACTCACCGCT-3’
2, transgenic arabidopsis obtains and Function Identification
1) acquisition of transgenic arabidopsis
The recombinant expression vector p35S::EeSAPK7 of above-mentioned structure is used freeze-thaw method transform Agrobacterium tumefaciens EHA105 respectively, use the agrobacterium tumefaciens EHA105 arabidopsis thaliana transformation of p35S::EeSAPK7 again, screen with the MS substratum containing 100mg/L kantlex, obtain positive transgenic plant.Do further evaluation and screening by screening the positive transgenic plant PCR that obtains, PCR pair of primers used is P3 and P4.
P3 (upstream primer): 5 '-GGTCACGCCGACGCACCTGG-3 ',
P4 (downstream primer): 5 '-TGCAGTCTTGGGATACGTGG-3 '.
Carry out PCR qualification to 35S::EeSAPK7 transgenic arabidopsis, positive transgenic plant can obtain about 500bp band through pcr amplification, and result obtains and turns 35S::EeSAPK7 Arabidopis thaliana 34 strain (Fig. 1).
PBI121 empty carrier is imported Arabidopis thaliana, method is the same simultaneously, and in contrast, what obtain 15 strains turns empty carrier Arabidopis thaliana (the transgenic arabidopsis T3 representative that screening obtains is shown).
2) the long statistical study of lower transgenic arabidopsis root is coerced at ABA, PEG
In order to verify that transgenic arabidopsis and wild-type are to ABA, PEG stress tolerance, MS substratum adding 50 μMs of ABA or 5%PEG and carries out the analysis of root system phenotypic evaluation.On the MS substratum of 50 μMs of ABA, L1, L2 are long long substantially identical with wildtype Arabidopsis thaliana root with the root of L5, and L3, L4 strain is slightly more longer than wildtype Arabidopsis thaliana root system; On the MS substratum of 5%PEG, L4, L5 transgenic line is obviously longer than wildtype Arabidopsis thaliana, and side radical is more, and blade is also large than wild-type; The root length of L1, L2 and L3 transgenic line is more slightly longer than wildtype Arabidopsis thaliana, and L4, L5 drought-enduring effect comparatively obvious (Fig. 2) on substratum turning EeSAPK7 gene is described.
3) transgenic arabidopsis drought tolerance qualification
For detecting transgenic Arabidopsis plants drought tolerance, to drought stress lower 20 days, after rehydration, the EeSAPK7 gene phenotype that turns of the 10th day carried out photographs observation (Fig. 3).When Osmotic treatment the 20th day, wildtype Arabidopsis thaliana and transgenic arabidopsis were all wilted, and occur the phenomenon of mortality.Within after rehydration the 10th day, find, wildtype Arabidopsis thaliana is all dead, and transgenic arabidopsis L4, L5 plant part has recovered standard state, survival rate is 35.7% respectively, 30%, survival rate is obviously greater than wildtype Arabidopsis thaliana, shows to turn the drought resistance that EeSAPK7 gene significantly improves Arabidopis thaliana.
4) transgenic arabidopsis Salt-Tolerance Identification
Further the qualification of salt stress patience is carried out to transgenic Arabidopsis plants.Transgenic arabidopsis consistent for upgrowth situation and wild-type are watered 250mM salt solution simultaneously, and to salt stress process the 20th day, after rehydration, the EeSAPK7 gene phenotype that turns of the 5th day carried out photograph observation.Phenotypic evaluation result shows, and the phenomenon of mortality appears in wildtype Arabidopsis thaliana, but L4 and L5 transgenic line has recovered standard state, and the survival rate of L4 is the survival rate of 15.2%, L5 is 65% (Fig. 4).And L1 and L3 transgenic arabidopsis strain and wildtype Arabidopsis thaliana are all dead, show that the process LAN of EeSAPK7 gene improves the salt tolerance of L4, L5 transgenic line.
Claims (8)
1. plant drought, a protein related to salt tolerance EeSAPK7, is characterized in that, its aminoacid sequence is as shown in SEQIDNO.1.
2. plant drought, a salt-resistant related gene EeSAPK7, is characterized in that, encode plant drought protein related to salt tolerance EeSAPK7 according to claim 1.
3. plant drought, salt-resistant related gene EeSAPK7 as claimed in claim 2, it is characterized in that, its base sequence is as SEQIDNO.2.
4. comprise the recombinant vectors of plant drought described in Claims 2 or 3, salt-resistant related gene EeSAPK7.
5. comprise the recombinant bacterial strain of plant drought described in Claims 2 or 3, salt-resistant related gene EeSAPK7.
6. the application of plant drought, protein related to salt tolerance EeSAPK7 described in claim 1.
7. the application of plant drought, salt-resistant related gene EeSAPK7 described in Claims 2 or 3.
8. the application being applied in raising plant drought, salt tolerance aspect of plant drought, protein related to salt tolerance EeSAPK7 described in claim 1.
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| CN109777790A (en) * | 2018-11-08 | 2019-05-21 | 北京市农林科学院 | Plant drought, protein related to salt tolerance EeSAPK4 and its encoding gene and application |
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