CN106636032B - A kind of plant drought GAP-associated protein GAP EeSnRK2.7 and its encoding gene and application - Google Patents
A kind of plant drought GAP-associated protein GAP EeSnRK2.7 and its encoding gene and application Download PDFInfo
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Abstract
The present invention relates to genetic engineering fields, in particular it relates to a kind of plant drought GAP-associated protein GAP EeSnRK2.7 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.1.Drought resistant correlative protein and its encoding gene of the invention improves yield, accelerates degeneration-resistant molecular breeding process to improvement, enhancing Resistance of Wheat To Adversity, 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 GAP-associated protein GAP EeSnRK2.7
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, clones anti contravariance related because cultivating
The degeneration-resistant new germ plasm of crop provides candidate adversity gene resource.
Sucrose non-fermented related protein kinase enzyme family (SnRKs) plays important work in many physiology courses of plant
With.
SnRK2 family gene functionally shows certain otherness, there is 9 bases in arabidopsis in SnRK family member
Because being induced by hyperosmotic stress, 5 genes are induced by ABA, but not by induction of chilling stress.In rice, 10 SnRK2 are identified
Protein kinase family gene, is named as OsSAPK1~OsSAPK10；SnRK gene in rice passes through protein phosphorylation analytical table
Bright all members can be activated by hyperosmotic stress, but only these three genes of OsSAPK8, OsSAPK9 and OsSAPK10 are by ABA
Inducing expression.
Therefore, it clones, separate degeneration-resistant correlation SnRK protein kinase gene improvement and improve the resistance of crop with very
Important meaning.
Summary of the invention
The object of the present invention is to provide a kind of plant drought GAP-associated protein GAP EeSnRK2.7.
Another object of the present invention is to provide the gene for encoding above-mentioned plant drought correlation egg EeSnRK2.7.
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 GAP-associated protein GAP EeSnRK2.7.
Drought resistant correlative protein EeSnRK2.7 provided by the present invention derives from E. elongata, and amino acid sequence is such as
Shown in SEQ ID NO.2.
Protein kinase of the invention is made of 342 amino acid residues, is SnRK albuminoid kinases.From SEQ ID NO.2
Amino terminal 10-30 amino acids residue be ATP binding domain, from the 112-128 amino acids residue of SEQ ID NO.2
For serine/threonine binding domain.
SEQ ID NO.1
In order to make albumen EeSnRK2.7 convenient for purifying, the egg that can be formed in the amino acid sequence shown in SEQ ID NO.2
The amino terminal or carboxyl terminal of white 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-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Disclosed SEQ ID NO.2 sequence, transcription factor EeSnRK2.7 of the invention can be closed manually according to the present invention
At can also first synthesize its encoding gene, then carry out biological expression and obtain.
EeSnRK2.7 encoding gene according to the present invention has the cDNA sequence as shown in SEQ ID NO.1.
SEQ ID NO.2
Expression cassette, recombinant expression carrier, transgenic cell line and recombinant bacterium containing EeSnRK2.7 gene belong to this hair
Bright protection scope.
The recombinant expression carrier of EeSnRK2.7 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 EeSnRK2.7, any one can be added before its transcription initiation nucleotide
The enhanced promoter of kind 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 weight containing EeSnRK2.7 gene
Group 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 EeSnRK2.7 gene transfered plant cell can get to the enhancing of the abiotic stress tolerances such as arid and salt
Transgenic cell line and transgenic plant.The expression vector for carrying encoding gene can be by using Ti-plasmids, Ri plasmid, plant
The conventional biology methods such as viral vectors, 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 for experimental material, is obtained with the stronger E. elongata of drought resistance (Elytrigia trichophora L.)
Degeneration-resistant relevant EeSnRK2.7 albumen and its encoding gene have been arrived, and has been conducted into wheat, has significantly improved transgenic wheat
Drought resistance.Drought resistant correlative protein and its encoding gene of the invention improves yield, accelerates to resist to improvement, enhancing Resistance of Wheat To Adversity
Inverse molecular breeding process, and water resource is effectively saved with highly important theoretical and practical significance.With reference to the accompanying drawing and
The present invention will be further described for specific embodiment.
Detailed description of the invention
Fig. 1 shows the Molecular Detection of EeSnRK2.7 transgenic wheat, wherein 1-9: different transgenic positive plant；
10: negative control；11:H2O control；M:Marker；
Fig. 2 shows EeSnRK2.7 transgenic wheat jointing stage root system statistic analysis result, wherein ESK-1, -2 is not
Same transgenic line；The capital winter 18 is receptor control；
Fig. 3 shows that EeSnRK2.7 difference transgenic wheat strain phenotype compares.
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 EeSnRK2.7 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 (GIBCOBRL, CAT.NO.18374-058) and 3 ' RACE kits (GIBCOBRL,
CAT.NO.18373-019 the full length sequence 1029bp of EeSnRK2.7 gene) is obtained.
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 EeSnRK2.7 coding sequence design primer P1 and P2.It is obtained with reverse transcription
CDNA is template, carries out PCR amplification with primer P1 and P2.The sequence of primer P1 and P2 are as follows:
P1:5 '-ATGGATCGGTACGAGGTGGT-3 ',
P2:5 '-TCACAACGGGCACACGAAG-3 '.
0.8% agarose gel electrophoresis detection is carried out to PCR product, obtains the band that molecular weight is about 1kb or so.Use fine jade
Sepharose QIAquick Gel Extraction Kit recycles the segment.The recycling segment is connect with pGEM-T Easy (Promega), referring to Cohen
Deng method (Proc Natl Acad Sci, 69:2110), by connection product convert bacillus coli DH 5 alpha competent cell, root
According to the acillin resistance marker screening positive clone on pGEM-T Easy carrier, the recombination matter containing recycling segment is obtained
Grain.Using T7 the and SP6 promoter sequence on the recombinant plasmid vector as primer pair, it carries out nucleotide sequencing, sequencing result
The open reading frame (ORF) for showing the EeSnRK2.7 gene expanded is SEQ ID No.1 from 5 ' end the 1st to 1029
Deoxyribonucleotide, encoding amino acid sequence are the protein of SEQ ID No.2.It will contain shown in sequence SEQ ID No.1
The recombinant vector of EeSnRK2.7 gene is named as pTE-EeSnRK2.7.
The sequence of EeSnRK2.7 gene is compared, does not find homologous protein gene in E. elongata, it was demonstrated that
EeSnRK2.7 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 resistance of EeSnRK2.7 genes amplification plant
1, the building of recombinant expression carrier
1) building of Ubi-EeSnRK2.7 recombinant expression carrier
The cDNA obtained using the total serum IgE reverse transcription of E. elongata is template, with containing KpnI and BamHI joint sequence
Special primer carries out PCR amplification；Then KpnI and BamHI double digestion PCR product recycles, and digestion products forward direction is inserted into carrier
Between KpnI and BamHI restriction enzyme site after the Ubi promoter of pNT112, recombinant vector Ubi::EeSnRK2.7 is obtained.
Primer sequence is as follows:
EeSnRK2.7[KpnI]5’-TCAGGTACCATGGATCGGTACGAGGTGGT-3’
EeSnRK2.7[BamHI]5’-GGGGATCC TCACAACGGGCACACGAAG-3’
2, transgenic wheat acquisition and Function Identification
1) acquisition of transgenic wheat material
The recombinant expression carrier pUbi::EeSnRK2.7 of above-mentioned building is converted into Agrobacterium tumefaciems with freeze-thaw method respectively
C58C1, then with the Agrobacterium tumefaciems C58C1 transformed wheat of pUbi::EeSnRK2.7, cultivated with the MS of the Basta containing 200mg/L
Base is screened, and positive transgenic plant is obtained.The positive transgenic plant that screening obtains is cooked into further identification sieve with PCR
It selects, pair of primers used in PCR is P3 and P4.
P3 (upstream primer): 5 '-GGATCCGGGAACTTCGGGGT-3 ',
P4 (downstream primer): 5 '-TCTCATTGTCAATGTCGTCG-3 '.
PCR identification is carried out to Ubi::EeSnRK2.7 transgenic wheat, positive transgenic plant can get through PCR amplification
As a result 800bp or so band obtains and turns 35 plants of Ubi::EeSnRK2.7 wheat (Fig. 1).
PNT112 empty carrier is imported into wheat simultaneously, method is same as above, as control, obtain 10 strains to turn empty carrier small
Wheat screens the transgenic wheat T of acquisition2Representative shows,.
2) EeSnRK2.7 transgenic wheat Identification of Drought
The excellent EeSnRK2.7 transgenic line based material of the economical character selected is carried out one in Beijing Shunyi, Fangshan
Year two o'clock cell production identification.By to jointing stage water, nonirrigated farmland cell material carry out aerial part fresh weight, under ground portion root system
Statistical analysis, the results showed that, the difference EeSnRK2.7 transgenic line based material such as ESK-1, ESK-2 is total fresh under the conditions of water ground
Weight, total root length are significantly higher than the control capital winter 18；Total fresh weight, total root length under the conditions of nonirrigated farmland, which are also significantly greater than, to be compareed
The capital winter 18 shows stronger drought resistance (Fig. 2).
In the case that the time of infertility only pour turn green water, to Fangshan, Beijing plantation EeSnRK2.7 transgenic line carry out field
Between screening of drought resistance.The discovery of drought resisting phenotypic evaluation, EeSnRK2.7 transgenic line hold that green property is preferable, and boot leaf still is able to normally
Photosynthesis is carried out, kernel grouting is sufficient, and mass of 1000 kernel increases significant；And compare hold green property poor, boot leaf and other positions of capital winter 18
Yellow leaf, early ageing are serious, cannot proceed normally photosynthesis (Fig. 3).In addition, from the species test of harvest data analysis found that turning
The characters such as plumpness, the mass of 1000 kernel of the seed of gene strain are superior to compare, so that EeSnRK2.7 transgenic line drought resisting
Property significantly increases (Fig. 3).
<110>Beijing City Agriculture and Forestry Institute
<120>a kind of plant drought GAP-associated protein GAP EeSnRK2.7 and its encoding gene and application
<160>2
<210> 1
<211> 1029
<212> DNA
<213>E. elongata
<400> 1
atggatcggt acgaggtggt gagagacatc ggatccggga acttcggggt ggcgaagctg 60
gtgcgggacg tcaggaccaa ggagcacttc gccgtcaagt tcatcgagcg aggccacaag 120
attgatgaac atgttcaaag ggagattatg aaccaccggt cactcaagca tccaaatatt 180
attcgactca aggaggtcgt gctaactcct acacatttgg caatagttat ggagtatgcc 240
tctggcggtg agctatttga agggatttgc aatgcaggga gatttagcga ggatgaggga 300
aggtacttct tccgacaatt gatttctgga gtgagctatt gtcactctat gcaagtatgt 360
catagagatt tgaaactaga gaatactctc ttggatggta gtgtcgcacc tcgactcaag 420
atttgtgact ttggttactc caagtcttct gtcttgcact ctcaaccgaa gtcaactgtg 480
ggcacgccgg catacatcgc cccggaggtc ctctctaaaa gagagtatga tggaaaggtc 540
gccgatgttt ggtcttgtgg agtaaccctc tatgtgatgc ttgttggggc atatcctttc 600
gaggaccctg atgagccaag gaacttccgc aaaacgatca ctaggatact cagtgtacag 660
tactccgttc cggactacgt tcgagtctcg atggattgca cacatctgct gtcccgcatt 720
tttgttggaa atcctcagca gcgaataacc atcccagaga tcaagaacca tccatggttc 780
ctcaagagat tgcccgttga gatgaccgat gagtaccaaa gaagcatgca gttggcagac 840
atgaacacgc cgtcacggag tctggaagaa gccacggcga tcatccagga ggcgcagaaa 900
cctggcgata acgccctagg gattgctggg caggttgcct gcctggggag catggatcta 960
gacgacattg atttcgatat cgacgacatt gacaatgaga acagcgggga cttcgtgtgc 1020
ccgttgtga 1029
<210> 2
<211> 342
<212> PRT
<213>E. elongata
<400> 2
MDRYEVVRDI GSGNFGVAKL VRDVRTKEHF AVKFIERGHK IDEHVQREIM NHRSLKHPNI 60
IRLKEVVLTP THLAIVMEYA SGGELFEGIC NAGRFSEDEG RYFFRQLISG VSYCHSMQVC 120
HRDLKLENTL LDGSVAPRLK ICDFGYSKSS VLHSQPKSTV GTPAYIAPEV LSKREYDGKV 180
ADVWSCGVTL YVMLVGAYPF EDPDEPRNFR KTITRILSVQ YSVPDYVRVS MDCTHLLSRI 240
FVGNPQQRIT IPEIKNHPWF LKRLPVEMTD EYQRSMQLAD MNTPSRSLEE ATAIIQEAQK 300
PGDNALGIAG QVACLGSMDL DDIDFDIDDI DNENSGDFVC PL 342
Claims (7)
1. a kind of plant drought GAP-associated protein GAP EeSnRK2.7, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.2.
2. a kind of gene studies on plant drought-resistance EeSnRK2.7, which is characterized in that encode plant drought phase described in claim 1
Close albumen EeSnRK2.7.
3. gene studies on plant drought-resistance EeSnRK2.7 as claimed in claim 2, which is characterized in that its base sequence such as SEQ
Shown in ID NO.1.
4. the recombinant vector comprising gene studies on plant drought-resistance EeSnRK2.7 described in Claims 2 or 3.
5. the recombinant bacterial strain comprising gene studies on plant drought-resistance EeSnRK2.7 described in Claims 2 or 3.
6. application of the plant drought GAP-associated protein GAP EeSnRK2.7 described in claim 1 in terms of improving plant drought, wherein institute
Stating plant is E. elongata or wheat.
7. application of the gene studies on plant drought-resistance EeSnRK2.7 described in Claims 2 or 3 in terms of improving plant drought, wherein
The plant is E. elongata or wheat.
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| 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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Non-Patent Citations (2)
| Title |
|---|
| Characterization of a common wheat (Triticum aestivum L.) TaSnRK2.7 gene involved in abiotic stress responses;Hongying Zhang等;《J Exp Bot.》;20101028;第62卷(第3期);第977页左栏第2段，第981页右栏第2段，图7A * |
| Triticum aestivum serine-threonine protein kinase （W55a) mRNA;Xu,Z.S.等;《Genbank：DQ343300.1》;20060912;全文 * |
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