CN106632629A - Cotton WRKY transcription factor GarWRKY5 for regulating stress tolerance of plants and application - Google Patents
Cotton WRKY transcription factor GarWRKY5 for regulating stress tolerance of plants and application Download PDFInfo
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Abstract
The invention discloses a cotton WRKY transcription factor GarWRKY5 for regulating stress tolerance of plants and application. Gossypium wild species gossypium aridum WRKY gene GarWRKY5 has a sequence of SEQ ID No. 2 in a sequence table. Electronic cloning and an RT-PCR technology is utilized to separate a plant stress tolerance related gene GarWRKY5, functional identification is performed by converting arabidopsis thaliana, it is proved that the transcription factor can regulate salt tolerance, and by overexpression of the gene, the stress tolerance of the plants is obviously improved.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, is a kind of cotton transcription factor for just regulating and controlling plant stress tolerance
GarWRKY5。
Background technology
Plant can face various different and changeable poor environments in the growth and development process of its whole life cycle, often
(such as salt damage, arid, low temperature), and these abiotic stress factors can cause bad impact to growing for plant.Agriculture
Crop is important bio-energy source for the survival of mankind, and in abiotic stress, arid and high salt are used as two kinds of masters
The abiotic stress wanted has a strong impact on the yield and quality of crop.According to the incomplete statistics of the World Food Programme of the United Nations, full generation
The various salinized soil areas in boundary are nearly 1,000,000,000 hectares, each bioclimatic zone of world five continents are distributed widely in, especially in arid
And semiarid zone, wherein being distributed in nearly 100,000,000 hectares of China, another aspect China is vast in territory, seashore line length, possesses big
The beach saline-alkali ground of amount.Cotton is described as salt-soda soil pioneer crop, cotton anti-adversity ability is further excavated, by Cotton Production to edge
Seabeach applies and the transfer of inland salt-soda soil is very urgent, is the needs of grain security, while to effectively utilizes soil and surely
Field planting cotton production, the sustainable development tool for realizing agricultural production is of great significance.
Plant WRKY Transcription Factor is the peculiar new zinc finger-type transcription regulatory factor of newfound plant in recent years.WRKY leads to
The W boxes (T) crossed in specific bond target gene promoters (T) TGAC (C/T) regulating and controlling the transcriptional level of target gene, involved in plant
Response to abiotic stress, finally makes certain stress in plant body reach balance.WRKY transcription factors are distributed widely in plant
In, many WRKY transcription factors of substantial amounts of document report can by high-salt stress abduction delivering, but some can also be by high salt
Stress suppresses expression.The diversity of WKRY transcription factors role in the salt response of plant, additionally, many researchs are also indicated that, it is single
One WRKY transcription factors may participate between the multiple environment stress of many regulation and control and mediation unlike signal path as regulatory factor
Interaction.At present, WRKY transcription factor family members, and grinding in some model plants are all had found in various plants
Study carefully it is middle parsed the transcription factor in the function and regulated and control network, but cotton of plant response environment stress with resistance to inversely related
The fresh understatement road of research of WRKY transcription factors, therefore, excavation is studied and using WRKY transcription factors related to adverse circumstance in cotton,
The raising of inverse ability resistance to cotton has great importance.
The content of the invention
It is an object of the invention to provide it is a kind of regulation and control plant stress tolerance cotton WRKY transcription factors GarWRKY5 and its should
With.
The cotton WRKY transcription factor gene GarWRKY5 of regulation and control plant stress tolerance of the present invention, its cDNA nucleotides
Sequence is as shown in SEQ ID NO.2.
The invention also discloses the protein of the cotton GarWRKY5 gene codes of regulation and control plant stress tolerance, with sequence table
Amino acid sequence described in middle SEQ ID NO.3.
The invention provides the plant expression vector containing above-mentioned cotton WRKY transcription factors GarWRKY5.More specifically
It is, by GarWRKY5 gene clonings to pCAMBIA2301, to obtain the expression of pCAMBIA2301-CaMV35S-GarWRKY5 plants and carry
Body.
The invention also discloses applications of the gene GarWRKY5 in salt-tolerant plant is cultivated.Specifically by GarWRKY5
Gene is proceeded in purpose plant by plant expression vector.The plant is preferably model plant arabidopsis.
Beneficial effects of the present invention：Using existing plant gene engineering technology, using electronic cloning and RT-PCR technology,
Separate and the identification cotton gene sequence information of resistance to inversely related, and gene is proceeded to by arabidopsis, Jing by Agrobacterium tumefaciems flower-dipping method
Crossing resistance to inverse phenotypic evaluation proves that the resistance to inverse power of transfer-gen plant is remarkably reinforced.
Description of the drawings
The amplification of Fig. 1 GarWRKY5 full length gene cDNA sequences.
The structure of Fig. 2 plant expression vector pCAMBIA2301-CaMV35S-GarWRKY5
(A) Escherichia coli pCAMBIA2301-CaMV35S-GarWRKY 5PCR detections electrophoresis result
(B) the digestion identification of pCAMBIA2301-CaMV35S-GarWRKY5.
The PCR identifications of Fig. 3 transfer-gen plants
PCR amplification vector pCAMBIA2301-CaMV35S-GarWRKY5 resistance arabidopsis genes of interest.
The PCR identifications of Fig. 4 transfer-gen plants and the RT-PCR identifications of pure lines plant
Turn identification of the pCAMBIA2301-CaMV35S-GarWRKY5 genes arabidopsis pure lines in DNA and cDNA levels.
The comparison of Fig. 5 germination period salt tolerances
On the MS culture mediums of 0mM and 150mM NaCl, the comparison of wild type and transgenic line germination period salt tolerance with
Statistical analysis.
The comparison of Fig. 6 Seedling Salt-tolerances.
This figure represents the growing state in wild type and transgenic line seedling stage under the conditions of 0mM, 100mM and 150mM NaCl.
The comparison of Fig. 7 germination period drought tolerances
Transgenic arabidopsis and wild type be in MS and on the MS culture mediums containing 20%PEG6000, the comparison of sprouting situation.
The comparison of Fig. 8 Seedling Stage drought tolerances
Transgenic arabidopsis and wild type be in MS and on the MS culture mediums containing 20%PEG6000, the comparison of root growth situation
The comparison of Fig. 9 seedling stage drought tolerances
Wild type and transgenic line are processed respectively with the clear water and solution containing 20%PEG6000mM of same volume, are observed
The growing state in its seedling stage.
Specific embodiment
The acquisition of embodiment 1, GarWRKY5 genes
The extraction of 1.1 RNA
Extract RNA
(1) the fresh cotton tissues of 0.5g are taken, 0.1g crosslinked polyethylenes arsenic pyrrolidone (PVPP) is added, in liquid nitrogen 3 fully
Powder is ground to, agar end is proceeded to rapidly in 10ml centrifuge tubes, plus 5ml CTAB extracts and 500 μ L0.1M pH8.0
Tris-HCl, 65 DEG C of water-bath 20min, midway upset is mixed；
(2) equal-volume chloroform is added fully to mix, ice bath stands 10min；
(3) 4 DEG C, 10000rpm centrifugation 20min.It is sub-packed in 4 1.5ml centrifuge tubes；
(4) suct clear, the 8M LiCl for adding 1/3 volume are mixed, -70 DEG C of 30min or -20 DEG C are overnight；
(5) 4 DEG C, 10000rpm centrifugation 20min.Supernatant is abandoned, after 70% ethanol is washed twice, is dried up and is precipitated and dissolved in 30 μ
LDEPC water；
(6) DNase and 25U RNase Inhabitors of the 10U without RNase activity, 10 × buffer digestion 30min are added
Afterwards plus equal-volume chloroform, extracting is once；
(7) supernatant is transferred in new pipe, plus 1/10 volume 3M pH 5.2NaAc and isopyknic isopropanol or 2.5 times
The absolute ethyl alcohol of volume, -20 DEG C stand overnight or -70 DEG C of ice bath 3h；
(8) 4 DEG C, 10000rpm centrifugation 20min abandon supernatant, and 70% ethanol is dissolved in 30 μ L DEPC water after washing twice.
Obtain final product cotton RNA.
The synthesis of 1.2 cDNA
System:
The synthesis of the chains of cDNA first
1.3 cDNA clone
According to this laboratory nonirrigated farmland cotton transcript profile sequencing (Xu et al., 2013) (De novo transcriptome
sequencing and comparative analysis of differentially expressed genes in
Gossypiumaridum under salt stress) 6 est sequences obtaining, sequence assembly obtains an a length of 1238pb length
Sequence, its nucleotide sequence is as shown in SEQ ID NO.1.With software ORF Finder (http://
Www.ncbi.nlm.nih.gov/gorf/gorf.html) complete ORF is predicted.ORF sequences both sides design upstream primer 5 '-
AGCAAACATGTCTTGGAAC-3 ' (SEQ ID NO.4) and-CGGCCTTTCAAAACTGA -3 ' (the SEQ ID of downstream primer 5 '
NO.5), RT-PCR is carried out with nonirrigated farmland cotton cDNA (being synthesized by step 1.2), is obtained comprising whole encoder block
GarWRKY5cDNA is cloned, and total length 921bp (SEQ ID NO.2) is (Fig. 1).The fragment is reclaimed, PTG19-T- carriers (north is cloned into
Jing Quanshijin Bioisystech Co., Ltd) in identification, sequencing.Reference literature cai et al., 2014 (Genome-wide
analysis of the WRKY transcription factor gene family in Gossypiumraimondii
And the expression of orthologs in cultivated tetraploid cotton), we will separate
To nonirrigated farmland cotton WRKY gene be named as GarWRKY5, its coding amino acid sequence as shown in SEQ ID NO.3.
1.3.1 PCR reaction systems (50 μ L)
1.3.2 PCR amplification programs：94℃3min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 3min, 34 circulations；72℃
10min；4 DEG C of insulations.
C, amplification obtain the ORF sequences of the gene, reclaim amplified production, and are cloned into PTG19-T carriers, positive gram of screening
Grand, sequencing is completed by the handsome company in Shanghai.
The expression analysis of 1.4 genes
1.4.1 PCR reactions
1. with cDNA as template, primer is
GarWRKY5-RT-F：5‘-GCCTTGTCATTTCATGGTGGA-3’(SEQ ID NO.6)
GarWRKY5-RT-R：5‘-GGGTTGTCGTTGCCTTGC-3’(SEQ ID NO.7)
2. PCR reaction systems：
The structure of embodiment 2, plant expression vector
The structure of 2.1 pCAMBIA2301-CaMV35S-GarWRKY5 plant expression vectors
Plant expression vector pCAMBIA2301-CaMV35S plasmids (Feng Juan etc., 2013；Gossypium wild species nonirrigated farmland cotton protein
The clone of kinase gene GarCIPK8 and functional analysis).From XbaI and Knp I respectively to pCAMBIA2301-CaMV35S and mesh
Genetic fragment GarWRKY5 carry out digestion, carrier large fragment and genes of interest fragment are reclaimed, with converting after the connection of T4 ligases
Escherichia coli Trans1-T1 competent cells (are purchased from Beijing Quanshijin Biotechnology Co., Ltd), obtain final product after identification recon
To the plant expression vector with genes of interest.
The digestion of pCAMBIA2301-CaMV35S plasmids and genes of interest fragment
Plasmid double digestion system is as follows：
In 30 DEG C of digestions, reaction time >=5h.Ago-Gel carries out electrophoresis detection to double digestion product after double digestion.
GarWRKY5 fragment double digestion systems：
Reclaim pCAMBIA2301-CaMV35S carriers large fragment and genes of interest fragment.
2.1.1 the connection of the pCAMBIA2301 large fragments that gene is obtained with digestion
Coupled reaction system：
2.1.2 Escherichia coli are converted
Connection product converts competent escherichia coli cell, 37 DEG C of vibrations in the LB fluid nutrient mediums containing Amp100mg/L
Culture.
2.1.3 the identification of recon
1. PCR identifies (Fig. 2A)
2. picking single bacterium colony is inoculated in 37 DEG C of shaken cultivations in LB fluid nutrient mediums of the 1.5mL containing kanamycins, uses
GarWRKY22 gene-specific primer GarWRKY5F-XbaI：5’-GCTCTAGAATGGAGAAGAAGAACGAGAA-3’(SEQ
ID NO.8) and GarWRKY5R-KpnI:5'-GGGGTACCCTACTGATCTTCTTCCTTTGC-3'(SEQ ID NO.9) carry out
PCR is expanded, and agarose gel electrophoresis is detected whether containing expected fragment.PCR response procedures are as follows：：94℃5min；94℃
30sec, 55 DEG C of 45sec, 72 DEG C of 1min30sec, 36 circulations；72℃10min；4 DEG C of insulations.Digestion identification (Fig. 2 B) of plasmid
Plasmid is extracted with alkaline denaturation, choosing XbaI and the enzymes of Knp I carries out digestion, and ibid, Ago-Gel is electric for digestion system
Swimming has detected whether expected fragment.
3. pCAMBIA2301-CaMV35S-GarWRKY5 is extracted and preserved
The positive single bacterium colony that 1. and 2. picking is identified is inoculated in 37 DEG C of mistakes in LB fluid nutrient mediums of the 5mL containing card Na penicillin
Night shaken cultivation, with plasmid extraction kit (AxyPrep Plasmid Miniprep Kit, the limited public affairs of healthy and free from worry life science
Department) extract plasmid pCAMBIA2301-CaMV35S-GarWRKY5 and by plasmid be stored in -20 DEG C it is standby.
The competent preparation of embodiment 3, Agrobacterium and conversion
The competent preparations of 3.1 Agrobacterium EHA105
(1) picking EHA105 single bacterium colonies, in being inoculated in 5ml LB fluid nutrient mediums, 28 DEG C, 200rpm concussion and cultivates are overnight
It is 0.4 to OD600 values；
(2) with 1：100 are inoculated in 400-500ml LB culture mediums (in 1L triangular flasks), and it is 0.6- to shake bacterium to OD600
0.8, ice bath 10min；
(3) bacterium solution is collected in the 50ml centrifuge tubes of precooling, 4 DEG C, 5000rpm is centrifuged 5min；
(4) supernatant is abandoned, precipitation sterilized water fully suspends, 4 DEG C, 5000rpm, 5min is centrifuged；Repeat this process 3 times,.
(5) in washed thalline plus 1ml (according to thalline it is how many depending on) contain 10% sterile glycerol resuspension cell.
(6) be distributed into 50 μ L often to manage, liquid nitrogen flash freezer, be placed in -80 DEG C it is standby.
3.2 electric shocking methods convert Agrobacterium competent cell EHA105
1. Agrobacterium competent cell is taken out in freeze thawing on ice.
2. add 2 μ l pCAMBIA2301-CaMV35S-GarWRKY5 DNAs in 50 μ l competent cells, use pipette tips
It is gently mixed mixing.
3. take out cell and proceed to (electric shock -20 DEG C of precoolings of cup) in electric shock cup with the mixture of plasmid, blot electric shock cup surface
Water, will electric shock cup be put between the electrode of electric conversion instrument, shock by electricity 4-5s under 2400V high pressure.
4. electric shock cup is taken out, 1ml LB fluid nutrient mediums is rapidly added without antibiotic), mix and shift mixed liquor and arrive
In 1.5ml centrifuge tubes, 28 DEG C, 200rpm shaken cultivations 3h.
5. take 100ul bacterium solutions to coat containing on Rif (50mg/L) and Kan (100mg/L) LB flat boards, 28 DEG C are inverted culture 2-
3 days.
Note：
1. cell should be slowly added in electric shock cup with the mixture of plasmid along cell wall, it is to avoid produce bubble.
2. electric shock cup outside is dried before shocking by electricity.
3. coated plate bacterium solution amount can be adjusted according to bacterial concentration.
3.3 thalline PCR are identified
Thalline PCR method and program are ibid (with step 2.1.4).
The preservation of 3.4 Agrobacteriums containing GarWRKY5 genes
The positive colony of the identification of picking 3.3 is inoculated into 28 DEG C in 5ml LB (Kan of the Rif and 100mg/L of 50mg/L),
200rpm shaken cultivations 1-2 days are until OD600=0.6-1.0.Then the 50% of sterilizing is added to the 1.5ml centrifuge tubes of sterilizing
The μ l of the glycerine 300 and μ l of bacterium solution 700, mix, and are stored in standby in -80 DEG C of refrigerator.
Embodiment 4, arabidopsis thaliana transformation and transgenosis functional verification
4.1 flower-dipping methods infect arabidopsis
4.1.1 the configuration (now with the current) of conversion fluid
1/2MSSucrose:μ l/100ml (the PH of 5%silwette-77 5:5.8)
4.1.2 conversion
1. the arabidopsis that about 20-30 titbit is grown in basin is selected, mature Fruit pod is cut, is poured within 2-3 days before conversion
Water.
2. with oese by 3.4 preserve the Agrobacterium inoculation containing GarWRKY5 genes to 28 DEG C in 1.5ml centrifuge tubes,
200rpm shaken cultivations 1-2 days.
3. in taking 5ul to 50ml LB (Kan of the Rif and 100mg/L of 50mg/L) culture medium, 28 DEG C, 200rpm vibration trainings
Support until OD600=0.6-1.0
4.700g centrifugation 5 minutes
5. supernatant is abandoned, and collects thalline, the MS conversion fluid resuspension thalline with 100ml are standby
6. plant to be transformed is inverted in into 45s in MS conversion fluids (bud will be totally immersed in conversion fluid).
7. bagging, as under regular culture conditions after half-light training sample 24h.
8. convert again once after one week, step is ibid.
The identification of 4.2 transgenic positive plant
4.2.1 sowing
1. will wait to sow in EB pipes of the son loaded on 2ml
2.70% ethanol (v/v) sterilization 1-3min, aseptic washing is once
3.15% NaClO (v/v) sterilization 5min, 9000rpm are centrifuged 2 minutes
4. sterile water wash 3-5 time
5. appropriate sterilized water is added
6.4 DEG C of light cultures 2-3 days
4.2.2 the identification of positive plant
1. the T that will be harvested0L containing 100mg is sowed at for seed–1On the 1/2MS culture mediums of kanamycins, observe after 2 weeks, turn
Gene plant grows true leaf and in green, and phenotype is normal, and nontransgenic plants growth rests on cotyledon period, and in Huang.
2. true leaf will be grown and greeny Arabidopsis plant will be transplanted in soil property matrix, and be placed in incubator and normally cultivate
Several weeks
3. gathering blade carries out PCR checkings (Fig. 4) of genomic level and transcript profile level, PCR amplification transfer-gen plants
Genomic DNA be template, primer gene-specific primer GarWRKY2F-XbaI (SEQ ID NO.8), GarWRKY5R-
KpnI (SEQ ID NO.9), PCR reaction systems and reaction condition are ibid.
The Resistance Identification of 4.3 transformation of Arabidopsis thaliana systems
4.3.1 the identification of transgenic line salt tolerance
4.3.1.1 the identification of transgenic line germination period salt tolerance
T3 generations pure lines arabidopsis transgenic seed and wild type seeds are chosen, seed tiles respectively to 0mM L after sterilizing- 1NaCl、150mM L-1On the 1/2MS sowing culture mediums of NaCl, light culture 2-3d；Then 18/22 DEG C is placed, 16hr illumination/8hr
4d. such as Fig. 5 is cultivated under conditions of dark, in 0mM L-1On the culture medium of NaCl, the sprouting of transgenic line and wild type is without bright
Significant difference different (such as Fig. 5 A), the sprouting situation of transfer-gen plant, transfer-gen plant on the MS culture mediums of the NaCl of L containing 150mmol
Sprouting situation be substantially better than wild type, statistics result also further demonstrates that transgenic progeny shows necessarily in germination period
Salt tolerance (such as Fig. 5 B).
4.3.1.2 the identification of seedling resistance of reverse test
Transgenosis is sheerly into arabidopsis seed and wild type seeds (WT) are together sowed in soil property matrix.After 15-20 days,
Respectively with the clear water of same volume, the NaCl solution of 150mM and 200mM is poured, and the upgrowth situation of plant, Zhi Daobiao are observed at any time
Reveal Resistant Difference.It is every time parallel to do 3 repetitions 9 plants in each Pen Portland, such as Fig. 6, under salt stress environment, transfer-gen plant
Salt tolerance apparently higher than wild type, illustrate GarWRKY5 regulation and control plant salt tolerance in terms of have certain facilitation.
4.3.2 the identification of transgenic line drought tolerance
4.3.2.1 the identification of transgenic line germination period drought tolerance
T3 generation pure lines arabidopsis transgenic seeds and wild type seeds are chosen, seed is laid in respectively 0 He after sterilizing
On the MS culture mediums of 20%PEG6000, light culture 2-3d；Then 18/22 DEG C is placed, is trained under conditions of 16hr illumination/8hr is dark
Foster 4d. such as Fig. 7, on the culture medium of MS, the sprouting situation no significant difference of transgenic line and wild type, containing 20%
The sprouting situation of transfer-gen plant is substantially better than wild type on the MS culture mediums of PEG6000, it was demonstrated that transgenic progeny is in germination period
Show certain drought tolerance.
4.3.2.2 the identification of transgenic line seedling drought tolerance test
T3 is chosen for arabidopsis transgenic seed and wild type seeds, using the wild type (as control) and T3 of sterilization for pure
Fit seed tiles respectively on MS sowing culture mediums, light culture 2-3d；Then 18/22 DEG C is placed, 16hr illumination/8hr is dark
Under conditions of cultivate
As a length of 1cm of plant root, choose the basically identical WT lines of root growing way and transgenic positive plant planted,
It is divided into two groups, is transferred to respectively on the MS solid mediums containing 0 and 20%PEG6000,8/22 DEG C, 16hr illumination/8hr dark
Under the conditions of cultivate, choose 3 familys of transfer-gen plant process, each process at least 10 young plants.After continuing to cultivate 10 days, observation
Transgenic arabidopsis and the root growth situation of wildtype Arabidopsis thaliana seedling.In whole growth course, MS flat boards are disposed vertically.As a result
Such as Fig. 8, the root length of transgenic and wild type is without significant difference on MS culture mediums, and is containing the MS solids of 20%PEG6000
On culture medium, there were significant differences with wild type for the root length of transgenic plant parts, shows obvious drought tolerance.
4.3.2.2 the identification of transgenic line seedling stage drought tolerance test
Transgenosis is sheerly into arabidopsis seed and wild type seeds (WT) are together sowed in soil property matrix.After 15-20 days,
Poured with the clear water and 20%PEG6000 solution of same volume respectively, the upgrowth situation of plant is observed at any time, until showing to resist
Sex differernce.It is every time parallel to do 3 repetitions 9 plants in each Pen Portland, such as Fig. 9, under Drought stress simulation environment, transfer-gen plant
Drought tolerance apparently higher than wild type, illustrate that GarWRKY5 has certain promotion at the resistance to inverse aspect of regulation and control plant.
Claims (6)
1. it is a kind of regulation and control plant stress tolerance cotton transcription factor GarWRKY5, it is characterised in that its sequence such as SEQ ID
Shown in No.2.
2. the protein that a kind of cotton transcription factor GarWRKY5 of the regulation and control plant stress tolerance by described in claim 1 is encoded,
It is characterized in that its amino acid sequence is as shown in SEQ ID No.3.
3. the expression vector of cotton transcription factor GarWRKY5 containing the regulation and control plant salt endurance described in claim 1.
4. expression vector according to claim 3, it is characterised in that be pCAMBIA2301-CaMV35S-GarWRKY5.
5. described in claim 1 regulation and control plant stress tolerance cotton transcription factor GarWRKY5 cultivate salt-tolerant plant in should
With.
6. application according to claim 5, it is characterised in that be to proceed to GarWRKY5 genes by plant expression vector
In purpose plant.
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Application publication date: 20170510 |