CN106636030B - Plant drought GAP-associated protein GAP EtSnRK2.2 and its encoding gene and application - Google Patents
Plant drought GAP-associated protein GAP EtSnRK2.2 and its encoding gene and application 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 present invention relates to genetic engineering fields, in particular it relates to plant drought GAP-associated protein GAP EtSnRK2.2 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 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, and in particular to plant drought GAP-associated protein GAP EtSnRK2.2 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..SnRK2 family gene is functionally shown
Otherness has 9 genes to be induced by mannitol or NaCl hyperosmotic stress in arabidopsis in SnRK family member, and 5 genes are by ABA
Induction, but not by induction of chilling stress.SnRK gene in rice, by protein phosphorylation analysis shows all members can be high
Stress activation is seeped, but only these three genes of OsSAPK8, OsSAPK9 and OsSAPK10 are by ABA inducing expression.In wheat,
First SnRK2 member is isolated PKABA1 from the wheat embryo cDNA library that ABA is handled, and the expression of PKABA1 removes
It is induced by ABA and drought stress.TaSnRK2.4 gene overexpression can enhance transgenic arabidopsis to arid, the side of body with high salt
Compel patience, while not will cause the growth dwarfism of plant.TaSnRK2.7 gene function analysis is shown, in glycometabolism, is reduced
It plays an important role in the physiological and biochemical procedures such as osmotic potential, the activity for enhancing Photosystem I I and promotion plant establishment；
The arabidopsis of TaSnRK2.8 gene overexpression has certain stress tolerance to arid, low temperature, with high salt, high temperature etc..
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 EtSnRK2.2.
Another object of the present invention is to provide the gene for encoding above-mentioned plant drought correlation egg EtSnRK2.2.
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 EtSnRK2.2.
Drought resistant correlative protein EtSnRK2.2 provided by the present invention derives from hair fringe couchgrass, and amino acid sequence is such as
Shown in SEQ ID NO.1.
Protein kinase of the invention is made of 343 amino acid residues, is SnRK albuminoid kinases.From SEQ ID NO.1
Amino terminal 10-30 amino acids residue be ATP binding domain, from the 115-130 amino acids residue of SEQ ID NO.1
For serine/threonine binding domain.
SEQ ID NO:1
In order to make albumen EtSnRK2.2 convenient for purifying, the egg that can be formed in the amino acid sequence shown in SEQ ID NO.1
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.1 sequence, transcription factor EtSnRK2.2 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.
EtSnRK2.2 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 EtSnRK2.2 gene belong to this hair
Bright protection scope.
The recombinant expression carrier of EtSnRK2.2 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 EtSnRK2.2, 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 EtSnRK2.2 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 EtSnRK2.2 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, hair fringe couchgrass, arabidopsis, rice, corn, cucumber, tomato, poplar, turfgrass, lucerne
Place etc..
The present invention for experimental material, is obtained with the stronger hair fringe couchgrass of drought resistance (Elytrigia trichophora L.)
Degeneration-resistant relevant EtSnRK2.2 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 specific embodiment the present invention will be further described.
Detailed description of the invention
Fig. 1 shows EtSnRK2.2 transgenic wheat Molecular Detection result.M:Trans2K Plus DNAmarker；7: yin
Property control；2-6 is different transgenic wheat strains.
Fig. 2 shows EtSnRK2.2 transgenic wheat drought canopy drought resisting qualification result, wherein the capital winter 18 is receptor control；446-
Isosorbide-5-Nitrae 63-2,660-1,471-3 are different transgenic lines.
Fig. 3 shows EtSnRK2.2 transgenic wheat field Physiological Indices of Drought Resistance measurement result, mainly determines 446-1,
463-2,660-1,471-3 transgenic line and the soluble sugar and chlorophyll fluorescence in receptor capital winter 18.
Fig. 4 shows that EtSnRK2.2 transgenic wheat difference strain phenotype compares, mainly investigated 421-1,437-8,
The data such as 471-2,582-1 transgenic line and the grain number per spike in receptor capital winter 18, plant height, spike length.
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: the cDNA clone of hair fringe couchgrass drought resisting correlation EtSnRK2.2 gene
The hair fringe couchgrass seedling of growth 30 days or so is carried out Osmotic treatment 5 hours, hair 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 1032bp of EtSnRK2.2 gene) is obtained.
The total serum IgE that hair fringe couchgrass seedling is extracted with Trizol, with superscript II (invitrogen) reverse transcription
Enzyme reverse transcription acquires cDNA.According to EtSnRK2.2 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 '-TTACAAAGGGCACACGAAATCC-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, which is connect with pGEM-T Easy (Promega), then converts Escherichia coli
DH5 α competent cell is contained according to the acillin resistance marker screening positive clone on pGEM-T Easy carrier
Recycle the recombinant plasmid of segment.Using T7 the and SP6 promoter sequence on the recombinant plasmid vector as primer pair, it carries out nucleotide
Sequencing, sequencing result show that the open reading frame (ORF) of the EtSnRK2.2 gene expanded is oneself of SEQ ID No.2
5 ' the deoxyribonucleotides of end the 1st to 1032, encoding amino acid sequence are the protein of SEQ ID No.1.Sequence will be contained
The recombinant vector of EtSnRK2.2 gene shown in SEQ ID No.2 is named as pTE-EtSnRK2.2.
EtSnRK2.2 gene is a new gene, is further carried out in hair fringe couchgrass genome with primer P1 and P2
Amplification, the genome sequence size of the gene is consistent with cDNA length scale as the result is shown, does not contain intron sequences.
Embodiment 2: with the drought resistance of EtSnRK2.2 genes amplification plant
1, the building of recombinant expression carrier
1) building of Ubi-EtSnRK2.2 recombinant expression carrier
The cDNA obtained using the total serum IgE reverse transcription of hair fringe couchgrass as template, with contain HindIII and BamHI connector sequence
The special primer of column carries out PCR amplification；Then HindIII and BamHI double digestion PCR product recycles, and digestion products forward direction is inserted
Enter between HindIII the and BamHI restriction enzyme site after the Ubi promoter of carrier pNT112, obtain recombinant vector Ubi::
EtSnRK2.2。
Primer sequence is as follows:
EtSnRK2.2[HindIII]5’-TCAAGCTTATGGATCGGTACGAGGTGGT-3’
EtSnRK2.2[BamHI]5’-GG GGATCCTTACAAAGGGCACACGAAATCC-3’
2, transgenic wheat acquisition and Function Identification
1) acquisition of transgenic wheat material
The recombinant expression carrier pUbi::EtSnRK2.2 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::EtSnRK2.2, 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 '-GGAGATTATGAACCACCGGT-3 ',
P4 (downstream primer): 5 '-TCTCCGGGATGGTTATTCGC-3 '.
PCR identification is carried out to Ubi::EtSnRK2.2 transgenic wheat, positive transgenic plant can get through PCR amplification
As a result 1000bp or so band obtains and turns 56 plants of Ubi::EtSnRK2.2 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 is shown.
2) EtSnRK2.2 transgenic wheat Identification of Drought
In the case that the time of infertility only pour turn green water, non-irrigated canopy drought resistance is carried out to the EtSnRK2.2 transgenic line of plantation
Screening.Drought resisting phenotypic evaluation in field is found, it is preferable that EtSnRK2.2 transgenic line holds green property, boot leaf still be able to normally into
Row photosynthesis, kernel grouting is sufficient, and mass of 1000 kernel increases significant；And compare hold green property poor, boot leaf and other position leaves of capital winter 18
Piece jaundice, early ageing are serious, cannot proceed normally photosynthesis (Fig. 2).Soluble sugar, chlorophyll fluorescence physiological index determining hair
Existing, Fluorometer, the soluble sugar content of EtSnRK2.2 transgenic line accumulate more (Fig. 3) than control.In addition,
From the species test of harvest data analysis found that the characters such as grain number per spike, root system, spike length of transgenic line are superior to compare, to make
It obtains EtSnRK2.2 transgenic line drought tolerance and significantly increases (Fig. 4).
<110>Beijing City Agriculture and Forestry Institute
<120>plant drought GAP-associated protein GAP EtSnRK2.2 and its encoding gene and application
<160>2
<210> 1
<211> 343
<212> PRT
<213>couchgrass
<400> 1
MDRYEVVRDI GSDNFGVAKL VRDVRTKEHF AVKFIKRGRK IDEHVQREIM NHRSLKHPNI 60
IRFKEVVLTP THLAIIMEYA SGGELFQRIC NAGRFSEDEG RFFFQQLISG VSYCHSMQVC 120
HRDLKLENTL LDGSVAPRLK ICGFGYSKSS VLHSQPKSTV GTPAYIAPEV LSRREYDGKV 180
ADVWSCGVTL YVMLVGAYPF EDPDEPRNFR KTITRILSVQ YSVPDYVRVS MDCIHLLSRI 240
FVGNPQQRIT IPEIKNHPWF LKRLPVEMTD EYQRSMQLAD MNTPSQSLEE AMAIIHEARK 300
PGDSALGIAG QVARLGSMDL DDIDFDDIDD IDIENSGDFV CPL 343
<210> 2
<211> 1032
<212> DNA
<213>couchgrass
<400> 2
atggatcggt acgaggtggt gagggacatc gggtccgaca acttcggggt ggcgaagctg 60
gtgcgggacg tcaggaccaa ggagcacttc gccgtcaagt tcatcaagcg aggccgcaag 120
attgatgaac atgttcaaag ggagattatg aaccaccggt cactcaagca tccaaatatt 180
atccgattca aggaggtcgt gctaactccc acacatttgg caataattat ggaatatgcc 240
tctgggggcg agctatttca aaggatttgc aacgcaggga gatttagcga ggatgaggga 300
aggttcttct tccaacaatt gatttctgga gtgagctatt gtcactctat gcaagtatgt 360
catagagatt tgaaactaga gaatactctc ttggatggta gtgttgcacc tcgactcaag 420
atttgtggct tcggttactc caagtcttct gtcttgcatt ctcaaccgaa gtcaactgtt 480
ggcacaccgg catacatcgc cccggaggtc ctctctagaa gagagtatga tggaaaggtc 540
gctgatgttt ggtcttgtgg agtaacgctc tatgtgatgc ttgtcggggc atatcctttc 600
gaggaccctg atgagccaag gaacttccgc aaaacaatca ctaggatact cagtgtacag 660
tactctgttc cggactacgt tcgagtctcc atggattgca tacatctgct gtcccgcatt 720
ttcgttggaa atcctcagca gcgaataacc atcccggaga tcaagaacca tccatggttc 780
ctcaaacgcc tgcccgttga gatgaccgat gagtaccaaa ggagcatgca gttggcagac 840
atgaacacgc cgtcacagag cctggaagaa gccatggcga tcatccatga ggcacggaaa 900
ccgggtgata gcgccctagg gattgctggg caggttgccc gcctggggag catggatcta 960
gatgacattg atttcgacga tatcgacgac attgacattg agaacagcgg ggatttcgtg 1020
tgccctttgt aa 1032
Claims (6)
1. a kind of plant drought GAP-associated protein GAP EtSnRK2.2, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.1.
2. a kind of gene studies on plant drought-resistance EtSnRK2.2, which is characterized in that encode plant drought phase described in claim 1
Close albumen EtSnRK2.2.
3. gene studies on plant drought-resistance EtSnRK2.2 as claimed in claim 2, which is characterized in that its base sequence such as SEQ
Shown in ID NO.2.
4. the recombinant vector comprising gene studies on plant drought-resistance EtSnRK2.2 described in Claims 2 or 3.
5. the recombinant bacterial strain comprising gene studies on plant drought-resistance EtSnRK2.2 described in Claims 2 or 3.
6. application of the plant drought GAP-associated protein GAP EtSnRK2.2 described in claim 1 in terms of improving plant drought resistance, the plant
Object is that wheat or hair fringe are laid down wheat.
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| Abscisic acid-inducible protein kinase [Triticum urartu];LIng.H.Q等;《Genbank：EMS57069.1》;20150520;全文 * |
| 小麦SnRK2.2基因克隆、表达载体构建及转化;张照贵等;《山东农业科学》;20141231;第46卷(第11期);摘要，第3页右栏 * |
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