CN106367423B - NAC films combination transcription factor gene GmNTL7 and its application in soybean WILLIAMS-DARLING Ton 82 - Google Patents
NAC films combination transcription factor gene GmNTL7 and its application in soybean WILLIAMS-DARLING Ton 82 Download PDFInfo
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Abstract
The invention discloses the NAC films combination transcription factor gene GmNTL7 in a kind of soybean WILLIAMS-DARLING Ton 82 and the plant expression vectors containing the gene GmNTL7.The invention also discloses the applications that the gene GmNTL7 improves its salt stress, drought stress tolerance in Arabidopsis plant.Experiments have shown that, transfer-gen plant of the present invention has obtained significantly improving than the salt resistance ability of nontransgenic plants, this provides theoretical foundation and practical basis to improve crop drought resistance, salt tolerance, indicate gene of the present invention can be widely used for cultivating drought resisting, salt tolerant plant variety.
Description
Technical field
The invention belongs to technical field of biological genetic engineering more particularly to a kind of soybean (Glycine max (L.) Merr)
NAC films combination transcription factor gene --- GmNTL7 and its application in WILLIAMS-DARLING Ton 82 (Williams 82).
Background technology
Plant is exposed in natural environment, is often subject to the influence of various environment-stress, such as arid, extreme temperature
Degree, nutrient dificiency and pest and disease damage etc..These environment-stress not only constrain the growth and development of plant, while also resulting in grain work
The underproduction of object.Higher plant own growth and development and to the response of environmental change be by regulate and control target gene expression come reality
It is existing.And the interaction of transcription factor and gene cis-acting elements, it can be as the molecular switch of gene expression regulation.Exactly
The interaction of transcription factor and degeneration-resistant functional gene promoter region cis-acting elements has activated the table of anti contravariance related gene
It reaches, improves the synthesis resistance of plant.There is a kind of special transcription factor, is referred to as film combination because it contains one section of transmembrane region
Transcription factor (Membrane-bound transcription factors, MTFs).Film combination transcription factor is directly incorporated in
In intracellular membrane structure (such as cytoplasma membrane, endoplasmic reticulum, nuclear membrane etc.), in a dormant state, change when by external environment
After stimulation, film combination transcription factor is just discharged from film, is changed into state of activation, and function in transporte to cells core.
MTFs has been observed that in yeast, prokaryotes and animals and plants, and confirms that it can be by activating genes of interest
It expresses to exercise multiple functions.It is worth noting that, most plant membrane combination transcription factor is adjusting plant to the environment side of body
Compel to play an important role in response.NAC transcription factor family was the distinctive a kind of transcription factor family of plant, after 2006
Kim etc. has found NAC transcription factor family in arabidopsis for the first time, and there are MTFs types, and are named as NTM1 (NAC WITH
TRANSMEMBRANE MOTIF 1) since, at present in plant, the function of having 9 NAC film combination transcription factors is sent out
Pick and report.Such as table 1：
The function of 1 plant membrane NAC combination transcription factors of table
Since plant membrane combination transcription factor finds that so far, people have been achieved with the research of film combination transcription factor for the first time
Major progress is related to many aspects such as growth and development of plants, hormone signal response.It of particular concern is, film combines
Transcription factor all has highly important regulation and control to the adaptation of the adverse circumstances such as the disease resistance of plant, winter resistance, drought resistance and salt tolerance and makees
With.But breakthrough achievement in research is still lacked to the research of its signal transduction and regulatory mechanism at present, and is focused mostly in pattern
In plant Arabidopsis thaliana.Through retrieval, the report of soybean NAC film combination transcription factors and its correlation function yet there are no.
Invention content
For current technology situation, the object of the present invention is to provide a kind of soybean (Glycine max (L.) Merr) prestige
NAC films combination transcription factor gene --- GmNTL7 and its application in Lian Musi 82 (Williams 82).
NAC films combination transcription factor gene in soybean WILLIAMS-DARLING Ton 82 of the present invention, it is characterised in that：The gene
It is named as soybean WILLIAMS-DARLING Ton 82NAC film combination transcription factor gene GmNTL7, the nucleotide sequence such as SEQ of the gene cDNA
Shown in ID No.1.
The present invention also provides a kind of plants containing NAC films combination transcription factor gene in above-mentioned soybean WILLIAMS-DARLING Ton 82
Expression vector, it is characterised in that：The plant expression vector is named as expression vector pK2GW7::GmNTL7, the expression vector gram
Grand Region Nucleotide sequence is as shown in SEQ ID No.4.
NAC film combination transcription factor genes are improving plant drought resisting, salt tolerant in soybean WILLIAMS-DARLING Ton 82 of the present invention
In application.
Wherein：The plant is preferably soybean or arabidopsis.Further, the arabidopsis is Col-0 wild types, soybean
Kind is Shandong beans 11.
In the present invention, applicant's isolated NAC films combination transcription factor gene-from soybean Williams 82
GmNTL7, which, which is gone in model plant arabidopsis, proves its function, as a result obtained drought resisting, salt tolerance improve turn base
Because of Arabidopsis plant.
Specifically, soybean WILLIAMS-DARLING Ton 82NAC film combination transcription factor genes GmNTL7 is expressed in Arabidopsis plant
The illustration of GmNTL7 applications.
Applicant is cloned into NAC film combination transcription factor genes GmNTL7 in 82 plant of Williams first；Sharp mistake
Gateway systems are by GmNTL7 genes by, (see Fig. 1), then passing through the side of conversion on BP reaction formings to pDONR221 carriers
Method is largely cloned in bacillus coli DH 5 alpha, then carries out LR reactions and this gene GmNTL7 is connected to expression vector
On pK2GW7 (see Fig. 2), and expressed in bacillus coli DH 5 alpha；Then screening transgenic Escherichia coli, and conversion plasmid is extracted,
Finally conversion plasmid is transferred in agrobacterium strains GV3101.Conversion agrobacterium strains are transferred to model plant arabidopsis, are verified
Express the function of gene GmNTL7.
The beneficial effects of the invention are as follows：Using existing plant gene engineering technology, the present invention clones obtain for the first time
Williams82NAC film combination transcription factor genes are simultaneously expressed in arabidopsis, and render transgenic arabidopsis obtains non-turn
The ability of high-salt stress tolerance not available for gene arabidopsis.Fully demonstrate soybean WILLIAMS-DARLING Ton of the present invention
82NAC films combination transcription factor gene GmNTL7 is played an important role in raising plant in drought stress and salt stress, in advance
Show gene of the present invention can be widely used for cultivate drought resisting, salt tolerant plant variety.
Description of the drawings
Fig. 1 is entry vector pDONR221 collection of illustrative plates.
Fig. 2 is plant expression vector pK2GW7 collection of illustrative plates.
Fig. 3 is the CDS full-length clone electrophoretograms of 82 NAC transcription factor gene GmNTL7 of Williams,
Wherein：M is Marker, and swimming lane 1,2,3,4 is the CDS full-length clones of gene.
Fig. 4 is 35S::The arabidopsis pure lines screening of GmNTL7 transgenosis.
Fig. 5 is 82 NAC transcription factor gene GmNTL1 RT-PCR expression analysis in transgenic arabidopsis of Williams.
Fig. 6 is 35S::GmNTL7 transgenic Arabidopsis plants phenotypic analyses,
Wherein：A figures are salt-resistance analysis of the transfer-gen plant under the conditions of 0,100,150mmol/L NaCl, and B figures are to turn
Drought Resistance Analysis under the conditions of gene plant 0,200,250mmol/L mannitol.
Fig. 7 is the Subcellular Localization of the protein product of GmNTL7 gene expressions,
Wherein：ER：Endoplasmic reticulum；Empty vector：Specificity is positioned at the albumen of endoplasmic reticulum.
Fig. 8 is the PCR detection figures that 82 NAC film combination transcription factor genes GmNTL7 of Williams turn Shandong beans 11,
Wherein：Upper figure is 35S-F/R primers progress PCR amplification as a result, figure below is the progress of 35S-F+GmNTL7-R primers
The result of PCR amplification；
Swimming lane M is Marker, and swimming lane positive control compares for positive plasmid；Swimming lane negative control is that negative plant pair is shone；
In upper figure：Swimming lane 1,2,3,4,5,6,7,8,9,10,11 is to turn GmNTL7 transgenic positive plant；In figure below：
Swimming lane 1,2,4 is to turn GmNTL7 transgenic positive plant.
Fig. 9 is 35S::Expression amount changes under GmNTL7 transgenic Arabidopsis plants abiotic stress,
Wherein：A figures are GmNTL7 in transgenic Arabidopsis plants root, stem, leaf after 200mmol/L NaCl processing 0,1,12h
Expression quantity changes；B figures are 20%PEG processing 0,1, GmNTL7 expression quantitative change in transgenic Arabidopsis plants root, stem, leaf after 12h
Change.
Specific implementation mode
The clone of embodiment 1, GmNTL7
The extraction of 82 total serum IgEs of 1.1Williams
(1) 82 vegetable materials of Williams are put into mortar, being ground into powder using liquid nitrogen (directly applies to
Following experiment or jelly is saved backup in -80 DEG C of ultra low temperature freezers)；
(2) etc. after liquid nitrogen volatilization, 100-200mg plant powders are transferred in 1.5ml centrifuge tubes immediately, then rapidly
1ml Trizol extracting solutions are added, the concussion that is vortexed makes sample be fully dissolved in extracting solution, is placed at room temperature for 5min；
(3) 4 DEG C, 12,000rpm, 10min is centrifuged, 0.9ml supernatants are transferred in new 1.5ml centrifuge tubes, then is added
Enter 0.2ml chloroforms and shake vigorously and mix well 15sec, is placed at room temperature for 2-5min；
(4) 4 DEG C, 12,000rpm, 10min is centrifuged, 0.4ml supernatants are transferred in new 1.5ml centrifuge tubes, then is added
Enter 0.4ml isopropanols, spins upside down 15 mixing solution, be placed at room temperature for 15mim；
(5) 4 DEG C, 12,000rpm, 10min is centrifuged, supernatant is abandoned, precipitation is washed twice with the ethyl alcohol of 1ml 75%, 4 DEG C, 8,
000rpm centrifuges 5min；
(6) supernatant is abandoned, uncap the upper dry RNA about 2-5min in superclean bench, and 40 μ l RNase-Free water are added,
RNA 10min are fully dissolved in 60 DEG C；
(7) the OD values and concentration of RNA sample, A are surveyed with ultraviolet specrophotometer260/A280Reach 1.7-2.0 to be preferred；Agar
The quality of sugared detected through gel electrophoresis.
The reverse transcription of 1.2 RNA
(1) following substance (40 μ l reaction systems) is sequentially added into centrifuge tube：
(2) gently after mixing, 65 DEG C of denaturation 5min are inserted on ice, ice bath at least 1min immediately；
(3) following substance is sequentially added into centrifuge tube
(4) gently after mixing, 42 DEG C of water bath with thermostatic control 1h, 65 DEG C of denaturation 10min, -20 DEG C save backup.
1.3 GmNTL7 gene clonings
GmNTL7 OX-F：5’-AA AAA GCA GGC TATGGGTGCCGTCGTCGA-3’
GmNTL7 OX-R：5’-AGAA AGCTGGTCAAGGAGAG ACACATCT-3’
(1) high fidelity enzyme Primer Star carry out following (the 50 μ l of reaction system of the first step amplification of Gateway systems
System)：
Amplification condition is as follows：
After reaction, reaction solution is detected in 0.8%TAE agarose gel electrophoresis.
(2) the purifying recycling (Tiangeng kit) of clone gene segment
1) gel with target fragment will be cut to be put into 1.5ml centrifuge tubes and claim gel weight, the molten of 3 times of volumes is added
Glue, 60 DEG C of colloidal sol 10min are constantly overturn during colloidal sol；
2) it after gel melts completely, is all drawn in recovery column, places a moment；
3) room temperature, 12000rpm centrifuge 30Sec, abandon solution；
4) rinsing liquid of 700 μ l is added in column, 12000rpm centrifuges 1min, abandons rinsing liquid；
5) rinsing liquid of 500 μ l is added in column, 12000rpm centrifuges 1min, abandons rinsing liquid；
6) void column, 12000rpm centrifuge 2min；
7) recovery column, which is uncapped, dries 1-2min, is put into new clean 1.5ml centrifuge tubes, and the 40 μ l that 60 DEG C of preheatings are added go out
Bacterium water or EB buffer solutions place 2min；
8) 12000rpm centrifuges 1min, and acquired solution is to recycle segment.
(3) high fidelity enzyme Primer Star carry out the second step amplification of Gateway systems.
Attb primer sequences：
attb-F：5’-G GGG ACA AGT TTG TAC AAA AAA GCA GGC T-3’
attb-R：5’-GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3’
Reaction system is following (50 μ l systems)：
Amplification condition is as follows：
After reaction, reaction solution is detected in 0.8%TAE agarose gel electrophoresis, sees Fig. 3.
The BP of 1.4 clone gene segments reacts
It is as follows that BP reacts (Gateway systems) system：
25 DEG C react 8 hours-overnight.
The preparation (sterile working) of 1.5 E. coli competents
(1) DH5 α strains are taken to be inoculated in 20ml LB liquid mediums, 37 DEG C of shaking table cultures are stayed overnight；
(2) 1 is pressed:100 are inoculated in 50ml LB liquid mediums, 37 DEG C, and 200rpm is cultivated 1 hour, until OD600Value is
0.4-0.6；
(3) bacterium solution is put on ice for 30min；
(4) 4 DEG C, 4200rpm, 10min is centrifuged, abandons supernatant, the CaCl of the 0.1M of 10ml precoolings is added2Suspension thalline；
(5) bacterium solution is put on ice for 10min；
(6) 4 DEG C, 4200rpm, 10min is centrifuged, abandons supernatant, the CaCl of the 0.1M of 2ml precoolings is added2Suspension thalline, packing
In 1.5ml centrifuge tubes, current or addition 30% glycerine of final volume saves backup for -80 DEG C after liquid nitrogen flash freezer.
The plasmid conversion (sterile working) of 1.6 Escherichia coli
(1) 1-5 μ l Plasmid DNA or connection product are added in the competent cell of 50 μ l, flick centrifuge tube mixing, ice
Bathe 30min；
(2) 42 DEG C, tepidarium heat shock 90sec, ice bath 2-3min immediately；
(3) 1ml LB culture mediums, 37 DEG C of culture 40-50min are added；
(4) room temperature, 4000rpm centrifuge 3min, collect thalline；
(5) bacterium is coated on the culture plate containing corresponding antibiotic, 37 DEG C of inversion overnight incubations.
1.7 colibacillus PCRs are verified
Reaction system is following (20 μ l systems)：
Amplification condition is as follows：
After reaction, reaction solution is detected in 0.8%TAE agarose gel electrophoresis.
1.8 DNA sequencing
The positive single bacterium colony that picking contains recombinant plasmid is shaken overnight with the liquid LB containing Kan (25mg/L), is then served
Hai Boya Bioisystech Co., Ltd is sequenced, and obtains sequencing result：Gene cDNA sequence is as shown in sequence table SEQ ID No.1.
By sequence analysis, the nucleotide homology 100% of above-mentioned cDNA sequence and soybean Willms82, three times experiments have shown that
What is obtained is exactly Willms82 NAC film combination transcription factor genes, is named as GmNTL1, the cDNA's of the gene GmNTL1
Nucleotide sequence is as shown in SEQ ID No.1.
The extraction of 1.9 e. coli plasmid dnas
(1) picking monoclonal is inoculated in the LB liquid medium that 10ml contains corresponding antibiotic, 37 DEG C of culture 8h；
(2) room temperature, 12,000rpm, 1min is centrifuged, thalline is collected；
(3) supernatant is abandoned, the solution I of 100 μ l low temperature precooling is added, vibrates suspension thalline；
(4) add the solution II of 200 μ l Fresh, fast fast overturning mixing, ice bath 5min；
(5) solution III of 150 μ l low temperature precooling is added after solution clarification, gently ice bath 5min after mixing；
(6) 4 DEG C, 12000rpm, 10min is centrifuged, is drawn in supernatant to new 1.5ml centrifuge tubes；
(7) isometric phenol/chloroform/isoamyl alcohol (25/24/1) is added, vibrates mixing；
(8) room temperature, 12,000rpm, 10min is centrifuged, transfer upper strata aqueous phase is added in another new 1.5ml centrifuge tubes
Isometric chloroform: isoamyl alcohol (24: 1), then extract primary, oscillation mixing；
(9) room temperature, 12,000rpm, 10min is centrifuged, transfer upper strata aqueous phase is added in another new 1.5ml centrifuge tubes
Isometric isopropanol, mixing simultaneously place 30min in -20 DEG C；
(10) room temperature, 12,000rpm, 10min is centrifuged, supernatant is abandoned and retains precipitation.
(11) it washs precipitation twice with 75% ethyl alcohol, is dried in vacuo 5min, is dissolved in 40 μ l aqua sterilisas, put to -20 DEG C and preserve
It is spare.
The LR of 1.10 clone gene segments reacts
It is as follows that LR reacts (Gateway systems) system：
25 DEG C are reacted 8 hours.
The preparation of 1.11 E. coli competents and plasmid conversion (with 1.5,1.6)
1.12 colibacillus PCRs verification (with 1.7)
The extraction of 1.13 e. coli plasmid dnas (with 1.9)
Verification is transferred to the positive pK2GW7 of pK2GW7 carriers::The cloning region nucleotide sequence such as SEQ of GmNTL7 plasmids
Shown in ID No.1.
The preparation (sterile working) of 1.14 Agrobacterium competence
(1) Agrobacterium GV3101 strains are taken to be inoculated in 10ml YEP fluid nutrient mediums, 28 DEG C of shaking table cultures are stayed overnight；
(2) 1 is pressed：50 are inoculated in 50ml YEP fluid nutrient mediums, 28 DEG C of shaken cultivations 3-4 hours, until OD600Value is
0.4-0.6；
(3) 4 DEG C, 4,200rpm, 10min is centrifuged, thalline is collected；
(4) supernatant is abandoned, the NaCl suspension thallines of the 0.15mol/L of 10ml precoolings are added；
(5) step 3 is repeated；
(6) supernatant is abandoned, the CaCl of the 20mmol/L of 2ml precoolings is added2Suspension thalline is sub-packed in 1.5ml centrifuge tubes, existing
With or be added final volume 7%DMSO saved backup for -80 DEG C after liquid nitrogen flash freezer.
1.15 the plasmid conversion (sterile working) of Agrobacterium
(1) 10 μ l Plasmid DNA are added in the competent cell of 50 μ l, flick centrifuge tube mixing, ice bath 30min；
(2) liquid nitrogen flash freezer 1min；Then 37 DEG C of water-bath 5min, immediately ice bath 2-3min；
(3) 1ml YEP culture mediums, 28 DEG C of culture 2-4h are added；
(4) room temperature, 4,000rpm, 3min is centrifuged, thalline is collected；
(5) bacterium is coated on the YEP culture plates containing corresponding antibiotic, 28 DEG C are inverted culture 48h.
The PCR verifications of 1.16 conversion Agrobacteriums
Reaction system is following (20 μ l systems)：
Amplification condition is as follows：
After reaction, reaction solution is detected in 0.8%TAE agarose gel electrophoresis.
1.17 GmNTL7 protein subcellulars position
(1) blade (being typically 5-7 pieces true leaf) being fully deployed is chosen on the 3-4 weeks plant never to bloom.
(2) middle part of blade is cut into the small item of 0.5-1mm with new single-edge blade by (adhesive tape tear also can), ensures that section does not have
The extruding of tissue.If situation is good, every gram of fresh weight can generate 107 protoplasts, and (40-60ml enzyme solutions digest 100-150 piece leaves
Son).For routine experiment, 5-10ml enzyme solutions, which digest 10-20 blade, can generate 0.5-1*10-6 protoplast, can be used for
25-100 sample (1-2*10-4 protoplast of each sample)
(3) it is transferred to blade item is rapid and soft in the enzyme solution got ready (5-10ml enzyme solutions add 10-25 blade), use
Blunt-ended forceps make leaf both sides section all enter enzyme solution.
(4) leaf item is vacuumized into 30min in the dark.
(5) (40rpm, 60-90min) continues the static enzymolysis of room temperature in the dark at least 3 hours.Enzyme solution is in green after jog
Color illustrates that protoplast disintegrates down.
(6) microexamination protoplast is used, normal arabidopsis mesophyll protoplast should about 30-50 μm.
(7) enzyme/protoplast is diluted in equal volume with W5 solution.
(8) excessive moisture is cleaned and sucked to the ethyl alcohol on 75 μm of nylon wires with water, be used in combination W5 to soak, then filter above-mentioned
Mixture.
(9) supernatant is transferred to 30ml round bottom centrifuge tubes, 100g (or<1000rpm) centrifuge 1-2min.It is eliminated as much as
Clearly, it and softly rotates and protoplast is resuspended.
(10) blood counting chamber is used to be counted to protoplast under 100 power microscopes, in the ratio W5 of 2*10-5ml-1
Protoplast is resuspended.30min is placed on ice.
(11) after 15min, protoplast will start to be deposited in tube bottom.W5 is sucked as far as possible with pipettor, and it is heavy not encounter
It forms sediment.Protoplast is resuspended in 2*10-5ml-1 ratios MMG, and is placed on room temperature.
(12) 10 μ l DNA (plasmid 10-20 μ g for being equivalent to 5-10kb) are added in 2ml centrifuge tubes.
(13) 100 μ l protoplasts (2*10-4) are added, it is soft to mix.
(14) 110ml PEG solution is added, tapping centrifuge tube mixes well and (can turn 6-10 sample every time).
(15) it is placed at room temperature for 5-10min.
(16) with 440 μ l W5 dilutions, soft mixing is to terminate conversion.
(17) 100g centrifuges 2min, removes supernatant.
(18) it uses 1ml W5 that protoplast is resuspended and is placed in 6 porocyte culture plates.
(19) incubated at room temperature certain time.
(20) protoplast is resuspended, 100g centrifuges 2min and collects.
(21) removal supernatant directly observes living cells, and the albumen production of GmNTL7 expression is observed under laser confocal microscope
The positioning of object in the cell.
Embodiment 2, the function that 82 NAC film combination transcription factor genes GmNTL7 of Williams are expressed in arabidopsis are tested
Card
2.1 flower infestation method arabidopsis thaliana transformations
(1) when arabidopsis (Col-0 wild types) grows to bolting 1cm, top is cut to the generation to induce side to give birth to inflorescence；
(2) in conversion the previous day, the Agrobacterium GV3101 containing expression vector plasmid that 1ml was activated is taken to be added to containing corresponding
In the 40ml YEP culture mediums of antibiotic and 50 μ g/ml rifampins, 28 DEG C of shake cultures to OD600About 1.0-1.2；
(3) room temperature, 4,200rpm, 10min is centrifuged, thalline is collected, with dip dyeing liquid for shell (5% sucrose, 0.05%Silwet L-
77) thalline is resuspended, makes OD600About 0.8；
(4) Agrobacterium is dripped on inflorescence with pipettor and is disseminated, after all inflorescences are all infected, arabidopsis is put
Enter and vacuumizes 1min in vacuum desiccator；
(5) inflorescence is covered with freshness protection package, being protected from light culture as 20-22 DEG C cuts off top exposing inflorescence for one day, is further cultured for one
Freshness protection package, culture to seed maturity are thrown off after it.
The surface sterilization of 2.2 arabidopsis seeds
Appropriate arabidopsis neutron subject to sterilization is put into 1.5ml centrifuge tubes, the ethyl alcohol that 1ml 75% is added (contains
The TritonX-100 of 0.03% volume ratio) concussion disinfection 1min, then 1min (twice) is sterilized with 70% ethyl alcohol concussion, finally
Seed is drawn onto on aseptic filter paper with suction nozzle and is dried up, is then clicked and entered in culture medium with sterile toothpick.
The screening of 2.3 transfer-gen plants
Surface sterilization is carried out for seed to the T0 of harvest, is then spread evenly across afterwards on 1/2MS tablets and (contains corresponding antibiosis
Plain Baste).Phjytotron growth is moved into after vernalization treatment 3d.It sprouts about 10 days, the bottle-green plant of cotyledon is planted for transgenosis
Strain, and cotyledon is sent out light green or even the plant of yellow is nontransgenic plants.Transfer-gen plant is transferred to growth in soil until harvest
T1 is obtained for transgenic seed, for plant single plant sowing, every plant of seed collected continues to screen T1, is 3 by offspring's segregation ratio:
Harvest T2 is grown to after the positive plant transplanting of 1 (positive: negative) for transgenic seed, after single plant sowing, every plant of kind collected
Son can obtain pure lines T2 for transgenic seed through screening, see Fig. 4.
The extraction of 2.4 plant RNAs
(1) vegetable material is put into mortar, using liquid nitrogen be ground into powder (directly apply to following experiment or
Person is frozen to be saved backup in -80 DEG C of ultra low temperature freezers)；
(2) etc. after liquid nitrogen volatilization, 100-200mg plant powders are transferred in 1.5ml centrifuge tubes immediately, then rapidly
1ml Trizol extracting solutions are added, the concussion that is vortexed makes sample be fully dissolved in extracting solution, is placed at room temperature for 5min；
(3) 4 DEG C, 12,000rpm, 10min is centrifuged, 0.9ml supernatants are transferred in new 1.5ml centrifuge tubes, then is added
Enter 0.2ml chloroforms and shake vigorously and mix well 15sec, is placed at room temperature for 2-5min；
(4) 4 DEG C, 12,000rpm, 10min is centrifuged, 0.4ml supernatants are transferred in new 1.5ml centrifuge tubes, then is added
Enter 0.4ml isopropanols, spins upside down 15 mixing solution, be placed at room temperature for 15mim；
(5) 4 DEG C, 12,000rpm, 10min is centrifuged, supernatant is abandoned, precipitation is washed twice with the ethyl alcohol of 1ml 75%, 4 DEG C, 8,
000rpm centrifuges 5min；
(6) supernatant is abandoned, uncap the upper dry RNA about 2-5min in superclean bench, and 40 μ l RNase-Free water are added,
RNA 10min are fully dissolved in 60 DEG C；
(7) the OD values and concentration of RNA sample, A are surveyed with ultraviolet specrophotometer260/A280Reach 1.7-2.0 to be preferred；Agar
The quality of sugared detected through gel electrophoresis.
The reverse transcription of 2.5 RNA
(1) following substance (40 μ l reaction systems) is sequentially added into centrifuge tube：
(2) gently after mixing, 65 DEG C of denaturation 5min are inserted on ice, ice bath at least 1min immediately；
(3) following substance is sequentially added into centrifuge tube
(4) gently after mixing, 42 DEG C of water bath with thermostatic control 1h, 65 DEG C of denaturation 10min, -20 DEG C save backup.
The RealTime-PCR of 2.6 transfer-gen plants is detected
PCR method screening transgenic positive plants：By the cDNA of resistant plant GmNTL7 gene RT-PCR primers
Carry out PCR detections.
GmNTL7 gene RT-PCR primer sequences：
GmNTL7 RT-F：5’-AGCATGTGAATGGTTATCCT-3’
GmNTL7 RT-R：5’-GAGAAAGAATCAACAAAGCCT-3’
The reaction that general T aq enzymes carry out RealTime-PCR amplifications is following (15 μ l systems)：
Amplification condition is as follows：
Note：Internal standard gene selects GmTUB.
As a result see Fig. 5.
The lower GmNTL7 of 2.7 NaCl, PEG processing is in the intraorganic expression analysis of different tissues
(1) Col-0 plant are handled into 0h in 200mmol/L NaCl, takes its root, stem, leaf after 1h, 12h respectively, extracts RNA
RNA is reversed to cDNA (method is with 2.5), carries out RealTime-PCR detections (method is with 2.6) by (method is with 2.4) respectively.
(2) Col-0 plant are handled into 0h in 20%PEG, takes its root, stem, leaf after 1h, 12h respectively, (method is same by extraction RNA
2.4), RNA is reversed to cDNA (method is with 2.5) respectively, carry out RealTime-PCR detections (method is with 2.6).
2.8 mannitol and NaCl handle arabidopsis
(1) surface sterilization of arabidopsis seed (with 2.2).
(2) mannitol and NaCl processing
The Col-0 for bacterium of having gone out, GmNTL7-1, Gm NTL7-2 seed are spread evenly across respectively on 1/2MS tablets and (contain phase
The antibiotic Baste answered).Vernalization treatment moves into phjytotron growth after 3 days.About 4d is sprouted, root long to 1cm or so will be intended
Southern mustard seedling move to addition various concentration mannitol (250,300,350,400mmol/L) or NaCl (50,100,150,
On 1/2MS culture medium flat plates 200mmol/L).It waits for that seedling is grown to 11d, observes seedling root long upgrowth situation.
As a result see Fig. 6.
Claims (1)
1. soybean WILLIAMS-DARLING Ton 82NAC films combination transcription factor gene GmNTL7 is in improving soybean or arabidopsis drought resisting, salt tolerant
Application, wherein the cDNA nucleotide sequences of the gene GmNTL7 are as shown in SEQ ID No.1.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660554A (en) * | 2012-04-27 | 2012-09-12 | 山东大学 | Soybean holy bean 9# NAC transcription factor gene GmST1 and application thereof |
| CN102676541A (en) * | 2012-04-27 | 2012-09-19 | 山东大学 | NAC transcription factor gene GmST2 of soybean holy bean No.9 and application of NAC transcription factor gene GmST2 |
| CN103911392A (en) * | 2007-12-28 | 2014-07-09 | 瑞典树木科技公司 | Woody Plants Having Improved Growth Characteristics And Method For Making The Same Using Transcription Factors |
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| US8692064B2 (en) * | 2009-10-02 | 2014-04-08 | The Curators Of The University Of Missouri | Quantitative trait loci associated with soybean cyst nematode resistance and methods of their use |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911392A (en) * | 2007-12-28 | 2014-07-09 | 瑞典树木科技公司 | Woody Plants Having Improved Growth Characteristics And Method For Making The Same Using Transcription Factors |
| CN102660554A (en) * | 2012-04-27 | 2012-09-12 | 山东大学 | Soybean holy bean 9# NAC transcription factor gene GmST1 and application thereof |
| CN102676541A (en) * | 2012-04-27 | 2012-09-19 | 山东大学 | NAC transcription factor gene GmST2 of soybean holy bean No.9 and application of NAC transcription factor gene GmST2 |
Non-Patent Citations (2)
| Title |
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| Glycine max NAC transcription factor(SNAC38)mRNA,complete cds;Chung,E,et al;《Genbank:KF657305.1》;20131211 * |
| 大豆耐盐相关QTLs 鉴定和功能基因研究进展;王楠等;《遗传》;20160817;第38卷(第11期);992-1003 * |
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