CN105349505B - A kind of plant drought, protein related to salt tolerance AsSnRK and its encoding gene and application - Google Patents
A kind of plant drought, protein related to salt tolerance AsSnRK and its encoding gene and application Download PDFInfo
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- CN105349505B CN105349505B CN201510890663.XA CN201510890663A CN105349505B CN 105349505 B CN105349505 B CN 105349505B CN 201510890663 A CN201510890663 A CN 201510890663A CN 105349505 B CN105349505 B CN 105349505B
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Abstract
The present invention relates to genetic engineering fields, in particular it relates to a kind of plant drought, protein related to salt tolerance AsSnRK 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
AsSnRK 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 such as arid, saline and alkaline condition is about 80,000,000,000 kilograms of the underproduction caused by China's wheat, the production of wheat is drastically influenced
Amount and quality, restrict China's wheat grain security.With the development of modern molecular biology, using technique for gene engineering from point
The relationship between plant and abiotic stress is furtherd investigate in sub- level, discloses plant to environment stress signal transduction and gene table
Up to regulatory molecule mechanism, theoretical basis is provided to cultivate the degeneration-resistant new germ plasm of crop.
In recent years, identify, illustrate by the structure of protein kinase and functional analysis it is various under the conditions of gene expression regulation
Mechanism get the attention.Sucrose non-fermented related protein kinase enzyme family (SnRKs) is in many physiology courses of plant
It plays an important role, such as hormone signal conduction, abiotic stress and growth and development of plant etc.[45-48].SnRK albumen swashs
Enzyme belongs to serine/threonine protein kitase super families, due to the similitude of gene order and the difference of gene structure, is divided
It is respectively for three subfamilies: SnRK1, SnRK2 and SnRK3.Three subfamilies of SnRK protein kinase have similar structure special
Point, the end N- have one section can with the kinase domain of other protein-interactings, and structure be in three families height become
Change.
First SnRK2 member is PKABA1, PKABA1 isolated from the wheat embryo cDNA library that ABA is handled
Expression except being induced by ABA and drought stress.SnRK2 family gene functionally shows certain otherness, arabidopsis
There are 9 genes to be induced in middle SnRK family member by hyperosmotic stress (mannitol or NaCl), 5 genes are induced by ABA, but not
By induction of chilling stress.Arabidopsis SnRK2.6 gene controls gas by participating in main metabolic processes and ABA approach
The aperture in hole.SnRK gene in rice, by protein phosphorylation analysis shows all members can be activated by hyperosmotic stress, still
Only these three genes of OsSAPK8, OsSAPK9 and OsSAPK10 are by ABA inducing expression.SnRK2 family gene is obtained in wheat to exist
Functionally also there is certain difference, the resistance that plant can be remarkably reinforced in TaSnRK2.4 gene is overexpressed in arabidopsis;
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 is overexpressed to arid, low temperature, with high salt
Etc. stress have certain patience.Therefore, it cloned using the oat of drought resisting, salt tolerant, separate degeneration-resistant correlation SnRK protein kinase gene
Improvement and the resistance for improving crop, can generate huge impetus and economic benefit to breeding for stress tolerance and agricultural production, have
Extremely important application prospect.
Summary of the invention
The object of the present invention is to provide a kind of plant droughts, protein related to salt tolerance AsSnRK.
Another object of the present invention is to provide the gene for encoding above-mentioned plant drought, salt tolerant correlation egg AsSnRK.
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 recombinant cells comprising said gene.
Another object of the present invention provides the application of above-mentioned plant drought, protein related to salt tolerance AsSnRK.
Drought resisting provided by the present invention, protein related to salt tolerance AsSnRK derive from oat, amino acid sequence such as SEQ ID
Shown in NO.1.
Protein kinase of the invention is made of 360 amino acid residues, is SnRK albuminoid kinases.From SEQ ID NO.1
Amino terminal 25-40 amino acids residue be ATP binding domain, from the 128-142 amino acids residue of SEQ ID NO.1
For serine/threonine binding domain.
SEQ ID NO.1
In order to make albumin A sSnRK 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 AsSnRK of the invention can be artificial synthesized,
Its encoding gene can be first synthesized, then carries out biological expression and obtains.
AsSnRK 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 AsSnRK gene belong to the present invention
Protection scope.
The recombinant expression carrier of AsSnRK 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.
When constructing recombinant plant expression vector using AsSnRK, it can increase plus any before its transcription initiation nucleotide
Strong type 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 the recombination table by any of the above-described kind containing AsSnRK gene
Up in vector introduction plant cell, plant with adverse resistance is obtained.
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 AsSnRK gene transfered plant cell can get and turn base to the enhancing of the abiotic stress tolerances such as arid and salt
Because of cell line and transgenic plant.The expression vector for carrying encoding gene can be by using Ti-plasmids, Ri plasmid, plant virus
The conventional biology methods such as carrier, directly delivered DNA, microinjection, conductance, mediated by agriculture bacillus convert plant cell or tissue,
And the plant tissue of conversion is cultivated into plant.The plant host being converted is either monocotyledon, is also possible to Shuangzi
Leaf plant, such as: arabidopsis, wheat, oat, arabidopsis, rice, corn, cucumber, tomato, poplar, turfgrass, lucerne place.
The present invention for experimental material, has obtained degeneration-resistant phase with drought resisting, the stronger oat of salt tolerance (Avena sativaL.)
The AsSnRK albumen and its encoding gene of pass, and it is conducted into arabidopsis, significantly improve drought resisting, the salt tolerance of plant.This hair
Bright drought resisting, protein related to salt tolerance and its encoding gene improves yield, accelerates degeneration-resistant point to improvement, enhancing arabidopsis resistance
Sub- breeding process, and water resource is effectively saved with 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 identification of the T1 for AsSnRK transgene tobacco strain.M:marker;1: negative control;2: water control;3~
11: different transgene tobacco strains.
Fig. 2 is the identification of transgene tobacco drought tolerance.CK is wild-type tobacco, and L1, L2, L3 are that 3 independent AsSnRK turn
Genetic tobacco strain.
Fig. 3 transgene tobacco Salt-Tolerance Identification.CK is wild-type tobacco, and L1, L2, L3 are that 3 independent AsSnRK turn base
Because of tobacco line.
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: oat drought resisting, salt tolerant correlation AsSnRK gene cDNA clone.
The oat seedlings of growth 30 days or so are carried out Osmotic treatment 5 hours, extract oat total serum IgE with Trizol.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 AsSnRK gene it is complete
Long sequence 1081bp.
The total serum IgE that oat seedlings are extracted with Trizol is inverted with superscript II (invitrogen) reverse transcriptase
Record acquires cDNA.According to AsSnRK coding sequence design primer P1 and P2.The cDNA obtained using reverse transcription as template,
PCR amplification is carried out with primer P1 and P2.The sequence of primer P1 and P2 are as follows:
P1:5 '-ATGGATCGGTACGAGGTGGTG-3 ',
P2:5 '-AATGGTTTGGAACGATCGGGGG-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 AsSnRK gene expanded is SEQ ID
No.2 from the deoxyribonucleotide of 5 ' end the 1st to 1081, encoding amino acid sequence is the protein of SEQ ID No.1.
The recombinant vector of AsSnRK gene shown in the ID of SEQ containing sequence No.2 is named as pTE-AsSnRK.
The sequence of AsSnRK gene is compared, does not find homologous protein gene in oat, it was demonstrated that AsSnRK gene
It is a new gene.
Embodiment 2: with the drought resisting of AsSnRK genes amplification plant, salt tolerance
1, the building of recombinant expression carrier
1) building of 35S::AsSnRK recombinant expression carrier
The cDNA obtained using the total serum IgE reverse transcription of oat is template, with specifically drawing containing SmaI and SpeI joint sequence
Object carries out PCR amplification;Then SmaI and SpeI double digestion PCR product recycles, by digestion products forward direction insertion carrier pBI121's
Between SmaI and SpeI restriction enzyme site after CaMV 35S promoter, recombinant vector p35S::AsSnRK is obtained.
Primer sequence is as follows:
AsSnRK[SmaI]5’-TCCCCCGGG ATGGATCGGTACGAGGTGGTG-3’
AsSnRK[SpeI]5’-GGACTAGT AATGGTTTGGAACGATCGGGGG-3’
2, transgene tobacco acquisition and Function Identification
1) acquisition of transgene tobacco
The recombinant expression carrier p35S::AsSnRK of above-mentioned building is converted into Agrobacterium tumefaciems EHA105 with freeze-thaw method respectively,
The Agrobacterium tumefaciems EHA105 transformation of tobacco for using p35S::AsSnRK again, is carried out with the MS culture medium of the kanamycins containing 100mg/L
Screening, obtains positive transgenic plant.The positive transgenic plant that screening obtains is cooked into further evaluation and screening, PCR institute with PCR
Pair of primers is P3 and P4.
P3 (upstream primer): 5 '-ATGCCTCTGGCGGTGAGCTA-3 ',
P4 (downstream primer): 5 '-TGCGCTGTTCAGGATTTCCA-3 '.
PCR identification is carried out to 35S::AsSnRK transgene tobacco, positive transgenic plant can get 750bp through PCR amplification
As a result left and right band obtains and turns 25 plants of 35S::AsSnRK tobacco (Fig. 1).
PBI121 empty carrier is imported into tobacco simultaneously, method is same as above, and as control, obtain 11 strains turns empty carrier cigarette
Grass (the transgene tobacco T that screening obtains2Representative is shown).
2) transgene tobacco drought tolerance is identified
Further to detect transgenic tobacco plant to the patience of drought stress, first to T2 for transgene tobacco 5%
It is cultivated on PEG culture medium.It is found by culture in 30 days, control growth is obviously suppressed, and blade turns to be yellow substantially;And turn base
Because plant is preferable than compareing, blade is greener, shows drought-enduring phenotype.Further control and transgenic line are tied up in flowerpot and controlled
It water process 45 days, after rehydration after the 10th day, is taken a picture to the phenotype of transgenic line it has been observed that L1, L2, L3 transgenic line
System substantially can restoration ecosystem, survival rate respectively reaches 75%, 60%, 45%, and wild-type tobacco can hardly survive, survival
Rate is 0%, illustrates that the overexpression of AsSnRK gene can enhance the drought resistance (Fig. 2) of transgenic tobacco plant.
3) transgene tobacco Salt-Tolerance Identification
For identify transgenic plant salt tolerance, to the similar wild-type tobacco plants of growing way and the cigarette for turning AsSnRK gene
Careless plant carries out Salt Tolerance Analysis.T2 is cultivated on 250mMNaCl culture medium for transgene tobacco first.By 20 days
Culture discovery, control growth are obviously suppressed, and blade turns to be yellow death substantially;And L1, L2, L3 rotaring gene plant blade are greener,
Can normal growth show the phenotype of salt tolerant.Salt-Tolerance Identification is further carried out in flowerpot.It pours every other week
It 1 liter of 250mMNaCl solution, is periodically observed and is taken a picture.Analysis shows, sent out after salt treatment the 25th day by tobacco phenotypes
Existing, the degradation of WT lines leaf chlorophyll is serious, holds green property and is substantially reduced;And L1, L2 transgenic leaf chlorophyll content compared with
It is preferable to hold green property for height.Survival rate statistics display, the survival rate of L1, L2 transgenic line respectively reach 45%, 34%, and wild
The survival rate of type is only 5% or so;In addition, carrying out measuring chlorophyll content discovery, L1, L2 transgenic line to the above salt-resistance strain
The chlorophyll content of system is higher than wild-type tobacco, illustrates that the overexpression of AsSnRK gene can effectively enhance and improve transgenosis
The salt tolerance (Fig. 3) of tobacco.
PBLAST is carried out with wheat SnRK2 family gene known to ncbi database to AsSnRK gene to compare, albumen
Homology range is from 28% to 95%, and there are notable differences for gene structure and amino acid composition, therefore AsSnRK gene has newly
Newness belongs to new discovery protein kinase in oat.
Claims (1)
1. plant drought, salt-resistant related geneAsSnRKApplication in terms of improving plant drought, salt tolerance, geneAsSnRK's
Base sequence is as shown in SEQ ID NO. 2.
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CN109666659B (en) * | 2018-11-07 | 2020-09-04 | 北京市农林科学院 | Plant drought-resistant and salt-tolerant protein AsCIPK14 as well as encoding gene and application thereof |
CN111593064B (en) * | 2019-02-01 | 2021-08-31 | 中国科学院植物研究所 | Method for improving salt tolerance of rice by inhibiting OsSDM gene expression |
CN114716522B (en) * | 2020-12-22 | 2023-07-07 | 中国农业大学 | Application of KIN10 protein and related biological materials thereof in saline-alkali tolerance of plants |
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