CN104725495B - Cotton GhWRKY51 transcription factors and its encoding gene and application - Google Patents
Cotton GhWRKY51 transcription factors and its encoding gene and application Download PDFInfo
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Classifications
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- 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
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Abstract
The present invention relates to cotton WRKY transcription factors and its applied technical field, specifically a kind of cottonGhWRKY51 transcription factors and its encoding gene and application.CottonGhWRKY51 transcription factors, be as（a）Or（b）Shown in albumen：（a）Protein shown in SEQ ID NO.2；（b）By protein shown in SEQ ID NO.2 by the replacement of one or more amino acid, missing or/and insertion it is derivative obtain still haveGhWRKY51 functional transcription factors or active protein variant.The present inventionGhWRKY51It shows as significantly raising in application JA, ABA, GA, is that expression variation is little using SA, is found when being handled using arid and NaClGhWRKY51Gene expression is significantly raised.The transgene tobacco of overexpression can generate high resistance.
Description
Technical field
The present invention relates to cotton WRKY transcription factors and its applied technical field, specifically a kind of cotton GhWRKY51 transcriptions
The factor and its encoding gene and application.
Background technology
Cotton is important industrial crops, and fiber is the important source material of textile industry in the world.Cotton or oil simultaneously
One of the important sources of material, feed and other raw materials of industry.However the yield and quality of cotton is often subject to insect pest, disease and each
The influence of kind adverse circumstance.In order to ensure that grain security, the cultivated area and planting site of cotton will change.Especially it is being unfavorable for
Planting the salt-soda soil of cereal crops and punja plant cotton becomes the direction that cotton is studied.In order to solve this problem must
It must reinforce Resistance Strain of Cotton against breeding research.By excavating a series of anti contravariance related genes, degeneration-resistant cotton is formulated using technique for gene engineering
Flower new material can accelerate to cultivate the adaptable and high new cotton variety process of yield.It is ground for what degeneration-resistant cotton variety was cultivated
Study carefully the cultivation that maximum difficult point is degeneration-resistant resource, however the method for conventional breeding is difficult to effectively obtain these degeneration-resistant resources.With
Population constantly increase, environmental pollution, unreasonable irrigation, excessive to apply the factors such as chemical fertilizer, the salinization of soil is more serious,
Agricultural production and increasingly sharpen using the contradiction between arable land.Therefore, improve the salt tolerant energy of the resistance especially cotton of cotton
Power has a very important significance for expanding the planting range of cotton, developing saline and alkaline soil.
Transcription factor (transcription factor) is to refer to that spy occurs with the cis-acting elements of eukaryotic gene
Opposite sex interaction and the DNA binding protein for having the effect of activating or inhibiting to transcription, transcription factor regulate and control between complicated albumen
Interaction network.Existing transcription factor includes mainly:MYB, WRKY, bZIP, AP2/EREBP, their wide participation plant droughts,
The regulation and control of the abiotic stresses such as salt tolerant.WRKY transcription factors are important turns that one kind of discovered in recent years is widely present in plant
Record factor superfamily.WRKY albumen is by the W-box of specific recognition downstream target gene promoter region, to participate in plant
Growth and development and degeneration-resistant reaction process.Numerous studies show WRKY transcription factors in the biotic reaction process of response plant
It plays a significant role.The research for participating in plant biological stress in relation to WRKY transcription factors in recent years is gradually reported, but about cotton
The research that flower WRKY participates in abiotic stress is few.
Other than playing particularly significant effect in terms of the signal transduction in stress resistance of plant, WRKY transcription factor families are also joined
With the physiology courses such as regulating growth of plants and metabolism, including Seed Development, seed germination and dormancy, fruit at
Ripe, roots development, the aging of blade, embry ogenesis etc..The few members of WRKY transcription factors have also assisted in plant senescence process
And it played an important role.
Invention content
The present invention is intended to provide a kind of cotton GhWRKY51 transcription factors, be such as (a) or (b) shown in albumen：
(a) protein shown in SEQ ID NO.2；
(b) protein shown in SEQ ID NO.2 is passed through into the replacement of one or more amino acid, missing or/and insertion
And what derivative obtained still has GhWRKY51 functional transcription factors or active protein variant.
It is such as SEQ ID the second object of the present invention is to provide a kind of cotton GhWRKY51 transcription factor encoding genes
Base sequence shown in NO.1；Or replacing for one or more bases is carried out on the basis of base sequence shown in SEQ ID NO.1
The base sequence variant for changing, lacking or/and being inserted into, and the encoded albumen of base sequence variant still has GhWRKY51 transcriptions
The function or activity of the factor.
Base sequence shown in SEQ ID NO.1 is hybrid with it and encodes still having of obtaining under strict conditions
GhWRKY51 functional transcription factors or active DNA molecular also belong to protection scope of the present invention.Shown in the SEQ ID NO.1
Base sequence be by the libraries salt resistance SSH obtain est sequence after, cloned from cotton leaf using RACE technologies.
The third object of the present invention is to provide cotton GhWRKY51 transcription factor encoding genes and is improving plant to the adverse circumstance side of body
Compel the application in resistance.
SEQ ID NO.1 are a kind of cotton transcription factor encoding genes cloned from cotton leaf, are named as
GhWRKY51, open reading frame 546bp encode the protein being made of 181 amino acid, as shown in SEQ ID NO.2.This turn
The transcriptional expression that the factor starts specific gene is recorded, the change of plant physiology and biochemistry is caused, to protect the growth and development of plant, is increased
Add plant to environment stress resistance.
In order to make albumen shown in above-mentioned (a) convenient for purifying, can be formed in the amino acid sequence shown in SEQ ID NO.2
The upper label as shown in Table 1 of amino terminal or carboxyl terminal connection of protein.
The sequence of 1 label of table
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Albumen shown in above-mentioned (b) can be artificial synthesized, also can first synthesize its encoding gene, then carries out biological expression and obtain.
The encoding gene of albumen shown in above-mentioned (b) can be by will lack one or several amino in base sequence shown in SEQ ID NO.1
The codon of sour residue, and/or the missense mutation of one or several base-pairs is carried out, and/or connected at its 5 ' end and/or 3 ' ends
The coded sequence of label shown in table 1 obtains.
Recombinant vector (cloning vector or plant expression vector) or recombinant bacterium or transgenosis containing above-mentioned encoding gene are thin
Born of the same parents system also belongs to protection scope of the present invention.
Further, the present invention is inserted into cotton GhWRKY51 transcription factor encoding genes in the multiple cloning sites of pPZP111 and obtains
The recombinant vector arrived.
It is a further object of the present invention to provide a kind of methods of genetically modified plants that cultivating resistance to environment stress, specially will be upper
It states recombinant vector to imported into plant, cultivates screening and obtain the genetically modified plants improved to environment stress resistance.
Above-mentioned plant is dicotyledon and monocotyledon, preferably tobacco, cotton and corn.Above-mentioned environment stress includes
It is with high salt or arid.
The present invention designs specific primer according to the relevant est sequence of cotton salt stress, using 3 ', 5 '-RACE technologies, from
The gene is cloned in cotton leaf, is named as GhWRKY51.Find that the gene is composing type table in cotton using qPCR analyses
It reaches, the expression quantity in spending is higher, and expression quantity is relatively low in flower bud.GhWRKY51 shows as showing in application JA, ABA, GA
Write up-regulation, be that expression variation is little using SA, find GhWRKY51 gene expressions significantly when being handled using arid and NaCl on
It adjusts.The transgene tobacco of overexpression can generate high resistance.Therefore it is trained using the degeneration-resistant albumen and gene of the present invention
Adversity resistant plant is educated, will be held out broad prospects in the applications such as the expansion of plant environment adaptability and planting range.
Description of the drawings
Fig. 1 is that cotton GhWRKY51 analyzes comparison diagram with other plant WRKY gene phylogenetic tree.
Fig. 2 is specific expressed comparison diagrams of the GhWRKY51 in cotton different tissues organ.
Fig. 3 is expression analysis schematic diagram of the cotton GhWRKY51 genes under SA processing.
Fig. 4 is expression analysis schematic diagram of the cotton GhWRKY51 genes under JA processing.
Fig. 5 is expression analysis schematic diagram of the cotton GhWRKY51 genes under ABA processing.
Fig. 6 is cotton GhWRKY51 genes in GA3Expression analysis schematic diagram under processing.
Fig. 7 is expression analysis schematic diagram of the cotton GhWRKY51 genes under Osmotic treatment.
Fig. 8 is expression analysis schematic diagram of the cotton GhWRKY51 genes under salt treatment.
Fig. 9 is the building process schematic diagram of recombinant cloning vector pBWRKY51.
Figure 10 is the PCR testing results of transgenic tobacco plant.
Figure 11 is the RT-PCR testing results of transgenic tobacco plant.
Specific implementation mode
The present invention is further explained in the light of specific embodiments, but the present invention is not limited to following embodiments.
Following all equal references of genetic manipulation method《Molecular cloning》(Sambrook J et al., 1989) described progress；Have
Close the application note that kit uses the purchased kit of reference；Restriction enzyme used and other toolenzymes are purchased from NEB public affairs
Department.
Cotton material is degeneration-resistant cotton variety Shanxi cotton 19, is carried by Cotton Research Institute, Shanxi Academy of Agricultural Sciences variety source room
For.Tobacco bred is SR1, is provided by Microbe Inst., Chinese Academy of Sciences Wu Jiahe researcher, bacillus coli DH 5 alpha, 5 '-Full
RACE Kit, 3 '-Full RACE Kit and SYBR Premix Ex Taq are purchased from TaKaRa companies, pGEM-T Easy
Vector System are purchased from Promega companies, Revert Aid H Minus First Strand cDNA Synthesis
Kit is purchased from Fermentas companies, and Gen Clean pillar Ago-Gel DNA QIAquick Gel Extraction Kits, TRIzol kits are purchased from upper
Extra large JaRa bioengineering Co., Ltd.
The clone of 1 cotton GhWRKY51 transcription factor cDNA sequences of embodiment and expression analysis
1. the extraction of total serum IgE and the amplification of cotton GhWRKY51 gene EST target fragments
19 seed of cotton variety Shanxi cotton is impregnated with sterile water, is placed in 25 DEG C of illumination box and handles 48h, the photoperiod
Dark for 12h illumination 12h, kind is in vermiculite and Nutrition Soil (1 after waiting seeds to show money or valuables one carries unintentionally:1) it is positioned in greenhouse in the compost mixed
Emergence, 27 DEG C of temperature, natural lighting.Win the root of cotton seedling, stem, leaf and cotton full-bloom stage respectively in cotton development stage
Flower bud, flower and bell sample use TRIzol kits extraction cotton RNA.
With reference to cotton salt stress est sequence, design specific primer carries out RT-PCR amplifications, observe in cotton leaf whether
There is the expression of GhWRKY51 genes.PCR amplification is the result shows that the gene has certain expression, explanation can be straight in blade
It connects and clones the gene from blade.It is solidifying from agarose in order to further confirm the associated clip that expanded product is the gene
Recycling PCR product, which is connected in carrier T, on glue is sequenced, the result shows that the est sequence one of obtained sequence and institute's reference
It causes, which is subjected to Blast comparisons on NCBI, it is very high with the homology of arabidopsis, confirm that this sequence is cotton
The gene order of GhWRKY51.
2. cotton GhWRKY51 gene clonings
(1) synthesis of the first chains of cDNA
The first chains of cDNA synthesis for expression analysis uses Revert Aid H Minus First Strand cDNA
Synthesis Kit are carried out.
The synthesis of the first chains of cDNA of gene cloning respectively refers to 5 '-Full RACE Kit of TaKaRa, 3 '-Full
RACE Kit User Manual are carried out.
(2) design and synthesis of primer
25 '-RACE antisense primers are separately designed according to one section of sequence of GhWRKY51 genes in cotton est database：
GhWRKY51-R1, GhWRKY51-R2 and 23 '-RACE sense primers GhWRKY51-F1, GhWRKY51-F2.Design of primers knot
Fruit is as follows：
GhWRKY51-F1：5’-GTTGGGACTGGGACATCGTATTGC-3’
GhWRKY51-F2：5’-CCCCAATCCAAGGAATTACTAT-3’
GhWRKY51-R1：5’-TGGGCTCTCATGATTATGAACACCT-3’
GhWRKY51-R2：5’-TCCTCTTCTTCACATTGCATCCTCC-3’
All primers of this experiment are synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.
(3) RACE methods clone gene
Target fragment is carried out according to 5 '-Full RACE Kit of TaKaRa, 3 '-Full RACE Kit User Manual
The amplification of 5 ' and 3 ' ends.
5 '-RACE and 3 '-RACE products, 2% agarose gel electrophoresis are detected, in the UV lamp cut target fragment
Glue recycles, and is connected to pGEM-T4On carrier, E. coli competent DH5 α are converted, carry out blue hickie screening, picking hickie, bacterium
After falling PCR detections, by positive colony sequencing.Sequencing is completed by Shanghai Sheng Gong bioengineering Co., Ltd.
Using 5 '-RACE and 3 '-RACE technologies, GhWRKY51 gene cDNAs are obtained respectively from cotton leaf total serum IgE
The sequence at 5 ' and 3 ' ends.Gel extraction, product are connected in carrier T after RACE technologies amplification acquisition purpose band, select the positive
As a result cloning and sequencing shows that 5 '-RACE product lengths are 450bp, 3 '-RACE product lengths are 250bp.By the two segments into
Row splicing obtain GhWRKY51 full length genes be 700bp, including open reading frame 546bp, encode 181 amino acid, molecule
Amount is 44.8KDa, isoelectric point pI=5.21 (as shown in SEQ ID NO.1 and SEQ ID NO.2).
3.GhWRKY51 gene order systematic evolution trees are built
The amino acid sequence of the WRKY51 of different plants is chosen in NCBI, chooses different types of WRKY in the database
Albumen builds the chadogram in relation to WRKY albumen using MEGA4 programs.It has chosen different plants and includes inhomogeneous WRKY
Albumen carries out the structure of systematic evolution tree with cotton GhWRKY51, by Fig. 1 it will be seen that GhWRKY51 and other plants
WRKY51 albumen has higher affiliation, belongs to I class WRKY, has height with cocoa (Theobroma cacao) WRKY51
The affiliation of degree.This chadogram also shows WRKY and constantly evolves and make a variation in plant situation, illustrates WRKY in plant
It is middle to execute important physiology and biochemical function.
4. expression analysis of cotton GhWRKY51 genes under the conditions of different tissues and different disposal
(1) acquisition and processing setting of sample
Salt treatment is tested：19 seed of cotton variety Shanxi cotton is planted in the greenhouse, is chosen the consistent cotyledon period seedling of growing way and is used
To do different disposal.Salt stress pours cotton seedling with 300mmol/LNaCl.The cotton seedling handled with clear water is as a contrast, each to locate
Manage 3 repetitions.0,2,4,8 and 12h time points take plant leaf to be put into quick-frozen in liquid nitrogen and then be stored in -80 DEG C of ice after treatment
It is for use in case.
Osmotic treatment：Above-mentioned similar cotton seedling, drought stress stops watering Continuous Drought and coerces 12 days, until blade goes out
Now wilt.The plant normally watered is as a contrast.0,2,4,6,8,10 and 12 day time point took plant leaf to be put into after treatment
It is quick-frozen and then be stored in for use in -80 DEG C of beneficial refrigerators in liquid nitrogen.
HORMONE TREATMENT：Above-mentioned similar cotton seedling carries out the processing of hormon in the greenhouse, and the method for processing is according to report
Road carries out.The concentration for spraying hormone JA, SA, ABA, GA is respectively 100mmol/L, 2mmol/L, 50mmol/L, 1 μm of ol/L.Place
Different time points take plant leaf to be put into quick-frozen in liquid nitrogen and then be stored in -80 DEG C of refrigerators for use after reason.
(2) Quantitative Real-time RT-PCR methods
Using Quantitative Real-time RT-PCR (qRT-PCR) method, detection GhWRKY51 Levant Cotton Root,
The expression of the different periods after expression and its hormone induction and adverse circumstance induction in stem, leaf, bud, flower, bell
The pair of primers of GhWRKY51 amplifications is GhWRKY51-F：5’-GTTGGGACTGGGACATCGTA-3’；GhWRKY51-R：5’-
CTGTTTTTGACCGACTTTTTCC-3’.With cotton ubiquitin gene (accession number：AY189972 it) is used as internal standard gene, is expanded
Increasing pair of primers is Ubi-F：5’-AAGACCTACACCAAGCCCAAG-3’；Ubi-R：5’-ACACTCCGCATTAGGACACTC-
3’.Ubiquitin is in charge of amplification with target gene with machine.
Reaction system used in qRT-PCR is 25 μ l, and wherein template is 0.5 μ l cDNA after quantitative dilution.Amplification condition
For：95 DEG C, 1min；95 DEG C, 5s；59 DEG C, 30s；72 DEG C, 30s；78 DEG C, 82 DEG C, 85 DEG C of each read plates are primary, and 40 cycles are arranged,
Draw solubility curve.Using ubiquitin genes as internal reference, biological experiment is repeated 3 times.GhWRKY51 is acquired with Ct methods are compared
Relative expression quantity of the gene in root, stem, leaf, bud, flower, bell, wherein calculation formula Y=10△Ct/3× 100% (△ Ct are interior
Mark the difference of gene C t and target gene Ct, i.e. △ Ct=CtGhubi–CtGhWRKY51)；With 2 softwares of Opticon Monitor
The Ct values of sample are calculated, and use 2-△△CtComputational methods acquire the relative expression quantity of sample under different disposal.
(3) organizing specific expression is analyzed
The total serum IgE of the organs such as root, stem, leaf, flower bud, flower and the young bell of cotton is extracted, and reverse transcription utilizes the cDNA at cDNA
Quantitative qRT-PCR analyses are carried out as template, the results showed that GhWRKY51 genes have expression (as schemed in these histoorgans
Shown in 2), illustrate that this gene is a constitutive expression gene, while finding that its expression quantity in spending is higher, secondly respectively
For leaf, stem, bell, root, the expression quantity in flower bud is minimum, and implying the function of the gene, there may be tissue specificities.
(4) the lower GhWRKY51 expression analysis of hormon processing
Utilize SA, JA, ABA and GA3Cotton is handled, respective treated RNA is extracted and reverse transcription is carried out at cDNA
QPCR is analyzed, the results showed that GhWRKY51 is in SA, JA, ABA and GA3Up-regulated expression is shown as under processing whithin a period of time
(as shown in Fig. 3, Fig. 4, Fig. 5 and Fig. 6).In JA, ABA and GA3GhWRKY51 expression shows as first being ramping up after processing
Then start to fall after rise, but the upper modulation of GhWRKY51 gene expressions is not so good as other three hormone signal molecules after SA processing.It says
Clear the gene can all there is responses to these hormones, but the pattern of response is not completely the same.
(5) expression analysis of arid and GhWRKY51 under salt treatment
There is wilting phenomenon after being stopped watering 12 days in Growth of Potted Cotton, we pick morning and wilt the blade and just restored
The plant leaf often to water carries out qPCR analyses, the results showed that GhWRKY51 genes are significantly induced, and expression quantity is control
3.3 times (as shown in Figure 7).Equally, cotton is handled with 300mM NaCl, sampling progress qPCR analyses, knot in different time
Fruit shows that GhWRKY51 gene expressions significantly improve in the cotton leaf of salt treatment, and especially after treatment 8 hours, transcriptional level was most
Height is 3.5 times (as shown in Figure 8) for compareing water process.GhWRKY51 gene pairs arid and salt are inverse as can be seen from the above results
Border, which has, significantly to be responded, and it is important to show that the gene may have the function of in the adaptation of these adverse circumstances.
Embodiment 2, the acquisition and functional analysis for turning GhWRKY51 genetic tobacco plant
1, the structure of the recombinant expression carrier pPZP-GhWRKY51 containing fusion GhWRKY51
(1) structure of binary expression vector pPZP111：
Binary expression vector provides for Institute of Microorganism, Academia Sinica Wu Jiahe researcher, containing by the double enhancers of band
CaMV35S promoters (DE35S), the Ω segments of TMV-RNAcDNA, the BamH I-Sal I segments from pBR322 and Nos
The exogenous gene expression frame of transcription terminator composition.
(2) structure of the recombinant expression carrier containing GhWRKY51 genes
In above-mentioned GhWRKY51 gene clonings to carrier T, BamH is added at 5 ' ends when designing clone gene primer
I restriction enzyme sites add Sal I restriction enzyme sites at 3 ' ends, expand the CDS sequences of the gene, be then attached in carrier T, should
Carrier is named as pBWRKY51, with restriction enzyme BamH I and Sal I digestion recombinant cloning vector pBWRKY51 and binary
Expression vector pPZP111, with T4DNA ligases by the CDS sequences of the GhWRKY51 cut be inserted into pPZP111 BamH I and
Recombinant plant expression vector pPZP-WRKY51, building process (RB as shown in Figure 9 are built between Sal I sites：Right margin；
NPT II：Neomycinsulphate gene；DE-35SP：The CaMV35S promoters of double enhancers；Nos：Terminator；LB：Left margin).
After recombinant expression carrier pPZP-WRKY51 conversion bacillus coli DH 5 alpha, select positive colony, carry out BamH I and
Sal I digestions and sequencing identification, the results showed that nucleotide sequences of the pPZP-WRKY51 between BamH I and Sal I sites be
From 1 nucleotide to 546 nucleotide in SEQ ID NO.1 in sequence table, i.e. the CDS sequences of gene GhWRKY51.
(3) recombinant expression carrier converts Agrobacterium tumefaciens
To having been built up correct recombinant plant expression vector pPZP-WRKY51 Agrobacterium tumefaciens are transformed into electric shocking method
It in LBA4404, is screened through kanamycins, selects positive colony, carry out PCR and sequence verification, the results showed that recombinant plant is expressed
Carrier pPZP-WRKY51 has been transferred to root agrobacterium.
2, acquisition and the Molecular Detection of the tobacco plant of GhWRKY51 genes are transferred to
(1) it is transferred to the acquisition of the tobacco plant of GhWRKY51 genes
According to (Horch RB.A simple and general method for transferring genes into
plants.Science,1985,227:1229~1231) leaf disk method described in, by the tobacco SR1 (Nicotiana of sterile culture
Tabacum cv.SR1) blade is with the Agrobacterium tumefaciens co-cultivation containing pPZP-WRKY51 carriers, the weight that will be built above
T-DNA (including nptII genes and adversity gene GhWRKY51) in group plant expression vector pPZP-WRKY51 is transferred to tobacco
In genome, the tobacco plant of anti-kanamycins nptII genes and GhWRKY51 genes is obtained.
(2) it is transferred to the PCR amplification detection verification of GhWRKY51 genetic tobacco plant
Using the tobacco plant for being transferred to GhWRKY51 genes as material, (Paterson AH, Curt LB, Wendel JF.A is pressed
rapid method for extrac-tion of cotton(Gossypium spp.)genomic DNA suitable
for RFLP and PCR analysis.Plant Mol Bil Rep,1993,11:112-117) the method extracts it
DNA.The PCR special primers of design amplification GhWRKY51 Gene Partial segments, sequence are：
P1：5′-5’-GTTGGGACTGGGACATCGTATTGC-3’
P2：5 '-TGGGCTCTCATGATTATGAACACCT-3 ',
By Shanghai, Sheng Gong bioengineering Co., Ltd synthesizes, and the amplified production of this pair of primers should be the piece of 524bp or so
Section.
PCR amplification condition is：95 DEG C of pre-degeneration 3min；94 DEG C of denaturation 50sec, 58 DEG C of annealing 50sec, 72 DEG C extend
50sec, 32 cycles；72 DEG C of extension 5min, 16 DEG C of preservations.Simultaneously not to be transferred to WT lines and the clone of any gene
Carrier pBWRKY51 is as positive negative control.
With agarose gel electrophoresis detection PCR as a result, PCR results such as Figure 10 institutes of the regeneration plant of the anti-kanamycins in part
Show that (swimming lane M is 1kb DNA Marker；CK+ is the result for expanding cloning vector pBWRKY51；CK- is non-transgenic tobacco SR1
Result；1-14 is the result for being transferred to GhWRKY51 genetic tobaccos).These PCR are the result shows that gene has been integrated into and detected
In the genome of tobacco plant.
(3) GhWRKY51 transcriptional expressions are analyzed in transgene tobacco
According to total serum IgE in method described above extraction transgene tobacco and control tobacco leaf, and carry out reverse transcription acquisition
First chain cDNA.Then PCR amplification is carried out using methods described above.With agarose gel electrophoresis detect RT-PCR as a result,
(1 is the result for expanding cloning vector pPZP-WRKY51 to part PCR results as shown in figure 11；CK- is non-transgenic tobacco SR1's
As a result；2-5 is the result for being transferred to GhWRKY51 genetic tobacco strains YS1, YS2, YS3, YS4).These PCR the result shows that
GhWRKY51 genes may have been integrated into the genome of detected tobacco plant.
4, the salt resistance experiment of transgene tobacco
(1) transgenic tobacco plant and comparison of adjoining tree seedling under the conditions of salt treatment
Select the different transformation plant YS1, YS2 of T1 transgene tobaccos 5, YS3, YS4 and YS5 compare nontransgenic plants
Seed is seeded in after sterile-processed containing 100gL-1Positive-selecting is carried out on the 1/2MS culture mediums of Kan, growth is after a week
Positive transgenic seedling is transferred to containing 200mmolL-1Continued growth 12d on the 1/2MS culture mediums of NaCl observes transgenosis
The growing state of tobacco and wild-type tobacco.It is control with unconverted tobacco WT, each strain handles 100 seeds, picking 60
The positive seedling of strain carries out seedling percent under salt stress and detects.3 repetitions of each processing setting.Count tobacco germination percentage, plant
Root long, the dry weight of plant and fresh weight.
2 transgenic tobacco plant of table is with adjoining tree seedling in salt (200mmolL-1) comparative analysis under treatment conditions
It can see from table 2, in 200mmolL-1Under the conditions of salt treatment, turn the tobacco plants of GhWRKY51 genes with
Control is compared, and turns the tobacco plants of GhWRKY51 genes compared with the control, and root long increases 3.0 times, germination percentage increases 20%,
Reach 90% or more, plant weights and fresh weight increase 2 times or more.These results explanation specifically expressing under the conditions of salt treatment
GhWRKY51 genes have the function of remarkably promoting tobacco germination and grow that overexpression GhWRKY51 genes have enhancing tobacco
The function of salt tolerant.
(2) resistance of the transgene tobacco to salt treatment
By the tobacco plant for being transferred to GhWRKY51, be not transferred to the wild-type tobacco plants Seed sterilization of any gene after, use
Sterile water impregnates, and is planted on tablet, is placed in 25 DEG C of illumination box, and the photoperiod is 12h illumination/12h dark, waits seedlings long
To transplanting when 1cm height in vermiculite and Nutrition Soil (1:1) in the compost mixed, Potted orchard is positioned in greenhouse, 25 DEG C of temperature,
Natural lighting.Seedling is grown 3 weeks or so, carries out salt treatment experiment.Just start to be handled 7 days with the NaCl of 100mM, then with 150mM's
NaCl is handled 7 days, is finally handled 2 weeks with 200mmol/LNaCl again, is then used the tap water desalinization of soil by flooding or leaching.
The results are shown in Table 3：The tobacco plant for turning GhWRKY51 genes of step 1 acquisition is other than YS7, remaining 6
(YSI to YS6) strain shows high anti-salt property, in ever-increasing salinity, until 28 days overwhelming majority
Plant remains able to restore normal growth, and non-transgenic tobacco plants (CKI) and empty vector control (CK2) were in salt treatment 28 days
It is i.e. all dead afterwards, it thus proves, the tobacco plant (in addition to YS7) for turning GhWRKY51 genes of acquisition has higher salt resistance energy
Power has higher saline-alkaline tolerance, this has very high application value to the research of breeding for salt resistance.
The salt resistance comparing result of 3 transgene tobacco of table
5, genetic stability detections of the fusion GhWRKY51 being transferred in transfer-gen plant
To understand the genetic stability of target gene, the pcr gene of the selfing generation plant of three plant is expanded and blocked
That chloramphenicol resistance is detected.
The seed (T1) for taking a transgenic tobacco plant selfing generation, according to the operating method of aseptic seedling, by the kind after sterilizing
Son is transferred on the MS plating mediums containing 200mg/ml kanamycins, is set culture in illumination box, is waited for that seedling grows one
White is quickly become to the plant of not anti-kanamycins after true leaf, and the seedling of anti-kanamycins still keeps green, counts each strain
The green seedling number and white seedling number of system, count the segregation ratio of kalamycin resistance.PCR amplification purpose is carried out to progeny plant (T1) simultaneously
Gene, method as described above, statistics resistance first filial generation separation situation.Experiment sets 3 repetitions.
Experimental result is as shown in table 4.The result shows that：Take the nptII genes (card in plant chromosome group to by expression vector
That mycin resistant gene) hereditary by the progress of single-gene law of segregation in genetically modified plants filial generation, transgenosis is chosen from filial generation
Homozygous line, and also show that 3 with the amplification of the target fragment of the GhWRKY51 genes of nptII close linkages:1 separation characteristic,
It is preliminary to show that the fusion GhWRKY51 of synthesis stablize heredity in transfer-gen plant offspring.
The separation situation of table 4 turns of GhWRKY51 genetic tobacco progeny of plants target gene and anti-kanamycins
Note：Kan in tableR、KanSAnti- kanamycins and kanamycins sensitivity response are indicated respectively.
<110>Cotton Research Institute, Shanxi Academy of Agricultural Sciences
<120>CottonGhWRKY51 transcription factors and its encoding gene and application
<160>2
<210>1
<211>546
<212>DNA
<213>Cotton
<221>CDS
<222>（1）…（546）
<400>1
ATGAGCTTCT GTCCTACTGA CCACTCAAAC CCTAACCCTA ATTCTATCTT 50
CTTCCCTGAA AATCCCGATC CGATGGCCGA CTTCGAGCTC TCTGATTACC 100
TTATGCTTGA TGCCGGTGTC TTCGAGGATG ATACATCGGA GAAGGGCATG 150
GGTGTCGCGA ATGAAAGTGC AACACCAAAA CATAGTAACA TAAGATGCAA 200
AAGTGGAGAG AAGAAAAGCA AGTTGGGACT GGGACATCGT ATTGCATTTA 250
GAATGAAATC AGAGATAGAA GCCATGGATG ATGGATATAA ATGGAGGAAA 300
TATGGGAAAA AGTCGGTCAA AAACAGCCCC AATCCAAGGA ATTACTATAA 350
ATGTGTAAGT GGAGGATGCA ATGTGAAGAA GAGGATAGAA AGGGACAGAG 400
ATGATAAAAG TTATGTGATA ACTACCTATG AAGGTGTTCA TAATCATGAG 450
AGCCCATACA CGTCTTACTG TAATCAAATG CCTCTTATGG CTCCTAATGC 500
TTGCATTTCC CAACCTTCTC CTTTCTCCTC TTCTTTTTCT ACATGA 546
<210>2
<211>181
<212>PRT
<213>Cotton
<222>（1）…（181）
<400>2
MET Ser Phe Cys Pro Thr Asp His Ser Asn Pro Asn Pro Asn Ser Ile Phe Phe Pro Glu 20
Asn Pro Asp Pro MET Ala Asp Phe Glu Leu Ser Asp Tyr Leu MET Leu Asp Ala Gly Val 40
Phe Glu Asp Asp Thr Ser Glu Lys Gly MET Gly Val Ala Asn Glu Ser Ala Thr Pro Lys 60
His Ser Asn Ile Arg Cys Lys Ser Gly Glu Lys Lys Ser Lys Leu Gly Leu Gly His Arg 80
Ile Ala Phe Arg MET Lys Ser Glu Ile Glu Ala MET Asp Asp Gly Tyr Lys Trp Arg Lys 100
Tyr Gly Lys Lys Ser Val Lys Asn Ser Pro Asn Pro Arg Asn Tyr Tyr Lys Cys Val Ser 120
Gly Gly Cys Asn Val Lys Lys Arg Ile Glu Arg Asp Arg Asp Asp Lys Ser Tyr Val Ile 140
Thr Thr Tyr Glu Gly Val His Asn His Glu Ser Pro Tyr Thr Ser Tyr Cys Asn Gln MET 160
Pro Leu MET Ala Pro Asn Ala Cys Ile Ser Gln Pro Ser Pro Phe Ser Ser Ser Phe Ser 180
Thr* 181
Claims (8)
1. cottonGhWRKY51 transcription factors, which is characterized in that be the protein as shown in SEQ ID NO.2.
2. cottonGhWRKY51 transcription factor encoding genes, which is characterized in that be the base sequence as shown in SEQ ID NO.1.
3. encoding gene described in claim 2 is improving plant to the application in environment stress resistance, the environment stress includes
It is with high salt or arid.
4. the recombinant vector containing encoding gene described in claim 2.
5. recombinant vector according to claim 4, which is characterized in that the recombinant vector is in the polyclonal of pPZP111
The recombinant vector that the encoding gene described in claim 2 obtains is inserted into site.
6. a kind of method of genetically modified plants that cultivating resistance to environment stress, which is characterized in that the recombination for obtaining claim 5 carries
Body is imported into plant, is cultivated screening and is obtained the genetically modified plants improved to environment stress resistance, the environment stress includes height
Salt or arid.
7. the method for the genetically modified plants according to claim 6 for cultivating resistance to environment stress, which is characterized in that the plant
Object is dicotyledon and monocotyledon.
8. the method for the genetically modified plants according to claim 7 for cultivating resistance to environment stress, which is characterized in that the plant
Object is tobacco, cotton and corn.
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| CN106632629A (en) * | 2016-10-18 | 2017-05-10 | 江苏省农业科学院 | Cotton WRKY transcription factor GarWRKY5 for regulating stress tolerance of plants and application |
| CN110872342B (en) * | 2018-08-14 | 2022-02-11 | 中国农业科学院棉花研究所 | Plant senescence-associated protein GhWRKY91, and coding gene and application thereof |
| CN112322628B (en) * | 2020-09-27 | 2022-11-15 | 湖北大学 | Transcription factor GhWRKY1-like gene for regulating and controlling verticillium wilt and drought resistance of cotton and application thereof |
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| CN104327173A (en) * | 2014-10-22 | 2015-02-04 | 江苏省农业科学院 | Cotton WRKY transcription factor GarWRKY22 for regulating salt tolerance of plants and application thereof |
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| CN104327173A (en) * | 2014-10-22 | 2015-02-04 | 江苏省农业科学院 | Cotton WRKY transcription factor GarWRKY22 for regulating salt tolerance of plants and application thereof |
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| Title |
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| 棉花转录因子GhWRKY4基因的克隆及特征分析;李光雷等;《棉花学报》;20130515;第25卷(第3期);第205页至206页第2段，第1.1-1.3节 * |
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