CN106367423A - Membrane-bound NAC transcription factor gene GmNTL7 in soybean Williams 82 and application thereof - Google Patents
Membrane-bound NAC transcription factor gene GmNTL7 in soybean Williams 82 and application thereof Download PDFInfo
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Abstract
The invention discloses a membrane-bound NAC transcription factor gene GmNTL7 in soybean Williams 82 and a plant expression vector containing the gene GmNTL7. The invention also discloses application of the gene GmNTL7 in increasing the tolerance of arabidopsis thaliana plants to salt stress and drought stress. Shown by experimental evidence, the salt resistance of the transgenic plants disclosed by the invention is increased to a great extent as compared with non-transgenic plants, thereby providing a theoretical base and a practice foundation for increasing the drought resistance and salt resistance of crops, and foreshowing that the gene disclosed by the invention can be widely used in culturing drought resistant and salt resistant plant species.
Description
Technical field
The invention belongs to technical field of biological genetic engineering, more particularly, to a kind of Semen sojae atricolor (glycine max (l.) merr)
Nac film combination transcription factor gene gmntl7 and its application in WILLIAMS-DARLING Ton 82 (williams 82).
Background technology
Plant is exposed in the middle of natural environment, is often subject to the impact 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 promoter of plant, also result in grain simultaneously and make
The underproduction of thing.The growth of higher plant own growth and the response to environmental change are by regulating and controlling the expression of genes of interest Lai real
Existing.And the interaction of transcription factor and gene cis acting element, can be used as the molecular switch of gene expression regulation.Exactly
The interaction of transcription factor and degeneration-resistant functional gene promoter region cis acting element have activated the table of anti contravariance related gene
Reach, improve the comprehensive resistance of plant.There is the special transcription factor of a class, be 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, when being changed by external environment
After stimulation, film combination transcription factor just discharges from film, is changed into state of activation, and functions in transporte to cells core.
Mtfs all has been observed that in yeast, prokaryote and animals and plants, and confirms that it can pass through activating genes of interest
Express and to exercise several functions.It should be noted that most plant membrane combines transcription factor is adjusting plant to the environment side of body
Compel to play an important role in response.Nac transcription factor family is the distinctive class transcription factor family of plant, continues 2006
Kim etc. finds that nac transcription factor family has mtfs type first in arabidopsiss, and is named as ntm1 (nac with
Transmembrane motif 1) since, at present in plant, the function of existing 9 nac film combination transcription factor is sent out
Pick and report.As table 1:
Table 1 plant membrane nac combines the function of transcription factor
Since with reference to transcription factor, plant membrane finds that so far, people have been achieved with to the research of film combination transcription factor first
Major progress, is related to the many aspects such as growth and development of plants, hormone signal response.Of particular concern is, film combination
Transcription factor adapts to adverse circumstances such as the disease resistance of plant, winter resistance, drought resistance and salt tolerances to be respectively provided with highly important regulation and control to be made
With.But the research to its signal transduction and regulatory mechanism still lacks breakthrough achievement in research at present, and focuses mostly in pattern
In plant Arabidopsis thaliana.Through retrieval, yet there are no the report of Semen sojae atricolor nac film combination transcription factor and its correlation function.
Content of the invention
For current technology situation, it is an object of the invention to provide a kind of Semen sojae atricolor (glycine max (l.) merr) prestige
Nac film combination transcription factor gene gmntl7 and its application in Lian Musi 82 (williams 82).
Nac film combination transcription factor gene in Semen sojae atricolor WILLIAMS-DARLING Ton 82 of the present invention it is characterised in that: described gene
It is named as Semen sojae atricolor WILLIAMS-DARLING Ton 82nac film combination transcription factor gene gmntl7, the nucleotide sequence such as seq of this gene cdna
Shown in id no.1.
Present invention also offers a kind of plant containing nac film combination transcription factor gene in above-mentioned Semen sojae atricolor WILLIAMS-DARLING Ton 82
Expression vector it is characterised in that: described plant expression vector is named as expression vector pk2gw7::gmntl7, this expression vector gram
Grand Region Nucleotide sequence is as shown in seq id no.4.
In Semen sojae atricolor WILLIAMS-DARLING Ton 82 of the present invention, nac film combination transcription factor gene is improving plant drought resisting, salt tolerant
In application.
Wherein: described plant is preferably Semen sojae atricolor or arabidopsiss.Further, described arabidopsiss are col-0 wild types, Semen sojae atricolor
Kind is Shandong bean 11.
In the present invention, applicant separates from Semen sojae atricolor williams 82 and obtains nac film combination transcription factor gene
Gmntl7, this gene is gone to proves its function in model plant arabidopsiss, result obtained drought resisting, salt tolerance improve turn base
Because of Arabidopsis plant.
Specifically, Semen sojae atricolor WILLIAMS-DARLING Ton 82nac film combination transcription factor gene gmntl7 expresses in Arabidopsis plant
The illustration of gmntl7 application.
Applicant is cloned into nac film combination transcription factor gene gmntl7 first in williams 82 plant；Sharp mistake
Gmntl7 gene is passed through bp reaction forming to (see Fig. 1) on pdonr221 carrier by gateway system, then passes through the side of conversion
Method is cloned in escherichia coli dh5 α in a large number, then carries out lr reaction and this gene gmntl7 is connected to expression vector
On pk2gw7 (see Fig. 2), and express in escherichia coli dh5 α；Then screening transgenic escherichia coli, and extract conversion plasmid,
Finally conversion plasmid is proceeded in agrobacterium strains gv3101.Conversion agrobacterium strains are proceeded to model plant arabidopsiss, checking
Have expressed the function of gene gmntl7.
The invention has the beneficial effects as follows: using existing plant gene engineering technology, the present invention clones first and obtains
Williams82nac film combination transcription factor gene is simultaneously expressed in arabidopsiss, and render transgenic arabidopsiss obtain non-turn
The ability of the high-salt stress toleration not available for gene arabidopsiss.Fully demonstrate Semen sojae atricolor WILLIAMS-DARLING Ton of the present invention
82nac film combination transcription factor gene gmntl7 has important effect in raising plant in drought stress and salt stress, in advance
Show that gene of the present invention can be widely used for cultivating drought resisting, the plant variety of salt tolerant.
Brief description
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 electrophoretogram of williams 82 nac transcription factor gene gmntl7,
Wherein: m is marker, swimming lane 1,2,3,4 is the cds full-length clone of gene.
Fig. 4 is the arabidopsiss pure lines screening of 35s::gmntl7 transgenic.
Fig. 5 is williams 82 nac transcription factor gene gmntl1 rt-pcr expression analysis in transgenic arabidopsis.
Fig. 6 is 35s::gmntl7 transgenic Arabidopsis plants phenotype analytical,
Wherein: a figure is salt-resistance analysis under the conditions of 0,100,150mmol/l nacl for the transfer-gen plant, and b figure is 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 expression,
Wherein: er: endoplasmic reticulum；Empty vector: specificity is positioned the albumen of endoplasmic reticulum.
Fig. 8 turns the pcr detection figure of Shandong bean 11 for williams 82 nac film combination transcription factor gene gmntl7,
Wherein: upper figure is the result that 35s-f/r primer carries out pcr amplification, figure below is carried out for 35s-f+gmntl7-r primer
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；
Upper in figure: swimming lane 1,2,3,4,5,6,7,8,9,10,11 is and turns gmntl7 transgenic positive plant；Lower in figure:
Swimming lane 1,2,4 is to turn gmntl7 transgenic positive plant.
Fig. 9 changes for expression amount under 35s::gmntl7 transgenic Arabidopsis plants abiotic stress,
Wherein: a figure be 200mmol/l nacl process 0,1, transgenic Arabidopsis plants root after 12h, stem, gmntl7 in leaf
Expression changes；B figure be 20%peg process 0,1, transgenic Arabidopsis plants root after 12h, stem, gmntl7 expression quantitative change in leaf
Change.
Specific embodiment
Embodiment 1, the clone of gmntl7
The extraction of the total rna of 1.1williams 82
(1) williams 82 vegetable material is put in mortar, be ground into powder using liquid nitrogen and (directly apply to
Following experiment or freeze saves backup in -80 DEG C of ultra cold storage freezers)；
Etc. (2), after liquid nitrogen volatilization, immediately 100-200mg plant powder is transferred in 1.5ml centrifuge tube, then rapidly
Add 1ml trizol extracting solution, vortex concussion makes sample be fully dissolved in extracting solution, room temperature places 5min；
(3) 4 DEG C, 12,000rpm, it is centrifuged 10min, 0.9ml supernatant is transferred in new 1.5ml centrifuge tube, then plus
Enter 0.2ml chloroform and acutely vibrate mixing 15sec, room temperature places 2-5min；
(4) 4 DEG C, 12,000rpm, it is centrifuged 10min, 0.4ml supernatant is transferred in new 1.5ml centrifuge tube, then plus
Enter 0.4ml isopropanol, spin upside down 15 mixing solution, room temperature places 15mim；
(5) 4 DEG C, 12,000rpm, it is centrifuged 10min, abandons supernatant, precipitate twice with the washing with alcohol of 1ml 75%, 4 DEG C, 8,
000rpm, is centrifuged 5min；
(6) abandon supernatant, uncap and rna about 2-5min is dried in the superclean bench, add 40 μ l rnase-free water,
Fully dissolving rna 10min in 60 DEG C；
(7) od value and the concentration of rna sample, a is surveyed with ultraviolet spectrophotometer260/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) sequentially add following material (40 μ l reaction system) in centrifuge tube:
(2) after gently mixing, 65 DEG C of degeneration 5min, it is inserted on ice immediately, ice bath at least 1min；
(3) sequentially add following material in centrifuge tube
(4) after gently mixing, 42 DEG C of water bath with thermostatic control 1h, 65 DEG C of degeneration 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 carries out following (the 50 μ l of reaction system of the first step amplification of gateway system
System):
Amplification condition is as follows:
After reaction terminates, reactant liquor detects in 0.8%tae agarose gel electrophoresiies.
(2) purification of clone gene fragment reclaims (Tiangeng test kit)
1) gel cutting with purpose fragment is put into 1.5ml centrifuge tube and claim gel weight, add the molten of 3 times of volumes
Glue, 60 DEG C of colloidal sol 10min, constantly overturn during colloidal sol；
2) after gel melts completely, all it is drawn in recovery column, place a moment；
3) room temperature, 12000rpm, it is centrifuged 30sec, abandon solution；
4) add the rinsing liquid of 700 μ l, 12000rpm in post, be centrifuged 1min, abandon rinsing liquid；
5) add the rinsing liquid of 500 μ l, 12000rpm in post, be centrifuged 1min, abandon rinsing liquid；
6) void column, 12000rpm, it is centrifuged 2min；
7) recovery column is uncapped and is dried 1-2min, puts into new clean 1.5ml centrifuge tube, adds 40 μ l of 60 DEG C of preheatings to go out
Bacterium water or eb buffer, place 2min；
8) 12000rpm centrifugation 1min, resulting solution as reclaims fragment.
(3) high-fidelity enzyme primer star carries out the second step amplification of gateway system.
Attb primer sequence:
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 system):
Amplification condition is as follows:
After reaction terminates, reactant liquor detects in 0.8%tae agarose gel electrophoresiies, sees Fig. 3.
The bp reaction of 1.4 clone gene fragments
Bp reaction (gateway system) system is as follows:
25 DEG C react 8 hours-overnight.
The preparation (sterile working) of 1.5 E. coli competent
(1) dh5 α strain is taken to be inoculated in 20ml lb fluid medium, 37 DEG C of shaking table cultures are overnight；
(2) it is inoculated in 50ml lb fluid medium by 1:100,37 DEG C, 200rpm cultivates 1 hour, to od600It is worth and be
0.4-0.6；
(3) bacterium solution is put on ice for 30min；
(4) 4 DEG C, 4200rpm, it is centrifuged 10min, abandons supernatant, add the cacl of the 0.1m of 10ml pre-cooling2Suspension thalline；
(5) bacterium solution is put on ice for 10min；
(6) 4 DEG C, 4200rpm, it is centrifuged 10min, abandons supernatant, add the cacl of the 0.1m of 2ml pre-cooling2Suspension thalline, subpackage
In 1.5ml centrifuge tube, now with or add final volume 30% glycerol save backup for -80 DEG C after liquid nitrogen flash freezer.
1.6 colibacillary plasmids convert (sterile working)
(1) 1-5 μ l plasmid dna or connection product are added in the competent cell of 50 μ l, flick centrifuge tube and mix, ice
Bath 30min；
(2) 42 DEG C, tepidarium heat shock 90sec, ice bath 2-3min immediately；
(3) 1ml lb culture medium, 37 DEG C of culture 40-50min are added；
(4) room temperature, 4000rpm, it is centrifuged 3min, collects thalline；
(5) bacterium is coated on the culture plate containing corresponding antibiotic, 37 DEG C of inversion overnight incubation.
1.7 escherichia coli pcr checkings
Reaction system is following (20 μ l system):
Amplification condition is as follows:
After reaction terminates, reactant liquor detects in 0.8%tae agarose gel electrophoresiies.
1.8 dna sequencings
The positive single bacterium colony that picking contains recombiant plasmid is shaken overnight with the liquid lb containing kan (25mg/l), then serves
Hai Boya Bioisystech Co., Ltd is sequenced, and obtains sequencing result: gene cdna sequence is as shown in sequence table seq id no.1.
Through sequence analysis, above-mentioned cdna sequence and the nucleotide homology 100% of Semen sojae atricolor willms82, test proof three times
Obtain is exactly willms82 nac film combination transcription factor gene, is named as gmntl1, the cdna's of described gene gmntl1
Nucleotide sequence is as shown in seq id no.1.
The extraction of 1.9 escherichia coli plasmid dna
(1) picking monoclonal is inoculated in the lb fluid medium that 10ml contains corresponding antibiotic, 37 DEG C of culture 8h；
(2) room temperature, 12,000rpm, it is centrifuged 1min, collects thalline；
(3) abandon supernatant, add the solutions i of 100 μ l low temperature pre-coolings, vibrate suspension thalline；
(4) add freshly prepared solutions i i of 200 μ l, fast upset soon mixes, ice bath 5min；
(5) solutions i ii of 150 μ l low temperature pre-coolings, ice bath 5min after gently mixing are added after solution clarification；
(6) 4 DEG C, 12000rpm, it is centrifuged 10min, draw supernatant to new 1.5ml centrifuge tube；
(7) add isopyknic phenol/chloroform/isoamyl alcohol (25/24/1), vibration mixes；
(8) room temperature, 12,000rpm, it is centrifuged 10min, transfer upper strata aqueous phase, in another new 1.5ml centrifuge tube, adds
Isopyknic chloroform: isoamyl alcohol (24: 1), then extract once, vibration mixes；
(9) room temperature, 12,000rpm, it is centrifuged 10min, transfer upper strata aqueous phase, in another new 1.5ml centrifuge tube, adds
Isopyknic isopropanol, mixes and places 30min in -20 DEG C；
(10) room temperature, 12,000rpm, it is centrifuged 10min, abandon supernatant and retain precipitation.
(11) precipitated twice with 75% washing with alcohol, be vacuum dried 5min, be dissolved in 40 μ l aquesterilisa, put and preserve to -20 DEG C
Standby.
The lr reaction of 1.10 clone gene fragments
Lr reaction (gateway system) system is as follows:
25 DEG C are reacted 8 hours.
The preparation of 1.11 E. coli competent and plasmid conversion (with 1.5,1.6)
1.12 escherichia coli pcr checkings (with 1.7)
The extraction (with 1.9) of 1.13 escherichia coli plasmid dna
Checking proceeds to the cloning region nucleotide sequence such as seq of the positive pk2gw7::gmntl7 plasmid of pk2gw7 carrier
Shown in id no.1.
The competent preparation of 1.14 Agrobacteriums (sterile working)
(1) Agrobacterium gv3101 strain is taken to be inoculated in 10ml yep fluid medium, 28 DEG C of shaking table cultures are overnight；
(2) it is inoculated in 50ml yep fluid medium by 1:50,28 DEG C of shaken cultivation 3-4 hours, to od600It is worth and be
0.4-0.6；
(3) 4 DEG C, 4,200rpm, it is centrifuged 10min, collects thalline；
(4) abandon supernatant, add the nacl suspension thalline of the 0.15mol/l of 10ml pre-cooling；
(5) repeat step 3；
(6) abandon supernatant, add the cacl of the 20mmol/l of 2ml pre-cooling2Suspension thalline, is sub-packed in 1.5ml centrifuge tube, existing
With or add final volume 7%dmso save backup for -80 DEG C after liquid nitrogen flash freezer.
Plasmid conversion (sterile working) of 1.15 Agrobacteriums
(1) 10 μ l plasmid dnas are added in the competent cell of 50 μ l, flick centrifuge tube and mix, ice bath 30min；
(2) liquid nitrogen flash freezer 1min；Then 37 DEG C of water-bath 5min, ice bath 2-3min immediately；
(3) 1ml yep culture medium, 28 DEG C of culture 2-4h are added；
(4) room temperature, 4,000rpm, it is centrifuged 3min, collects thalline；
(5) bacterium is coated on the yep culture plate containing corresponding antibiotic, be inverted culture 48h for 28 DEG C.
The pcr checking of 1.16 conversion Agrobacteriums
Reaction system is following (20 μ l system):
Amplification condition is as follows:
After reaction terminates, reactant liquor detects in 0.8%tae agarose gel electrophoresiies.
1.17 gmntl7 protein subcellular positioning
(1) choose fully deployed blade (typically 5-7 piece true leaf) on the 3-4 week plant never blooming.
(2) middle part of blade is cut into the little bar of 0.5-1mm it is ensured that tangent plane does not have with new single-edge blade by (adhesive tape tear also can)
The extruding of tissue.If situation is good, every gram of fresh weight can produce 107 protoplasts, and (40-60ml enzyme liquid digests 100-150 piece leaf
Son).For normal experiment, 5-10ml enzyme liquid digests 10-20 blade and can produce 0.5-1*10-6 protoplast, can be used for
25-100 sample (each sample with 1-2*10-4 protoplast)
(3) rapid and soft for blade bar is transferred to (5-10ml enzyme liquid adds 10-25 blade) in the enzyme liquid got ready, use
Blunt-ended forceps makes leaf bar both sides tangent plane all enter enzyme liquid.
(4) by leaf bar evacuation 30min in the dark.
(5) (40rpm, 60-90min) continues the static enzymolysis of room temperature in the dark at least 3 hours.After jog, enzyme liquid is in green
Color, illustrates that protoplast disintegrates down.
(6) use microscopy protoplast, normal arabidopsiss mesophyll protoplast should about 30-50 μm.
(7) w5 solution is used to dilute enzyme/protoplast equal-volume.
(8) with water, the ethanol on 75 μm of nylon wires is cleaned and sucked excessive moisture, and soaked with w5, then filter above-mentioned
Mixture.
(9) supernatant is proceeded to 30ml round bottom centrifuge tube, 100g (or < 1000rpm) centrifugation 1-2min.It is eliminated as much as
Clearly, and softly rotate resuspended protoplast.
(10) blood counting chamber is used under 100 power microscopes, protoplast to be counted, in the ratio w5 of 2*10-5ml-1
Resuspended protoplast.Place 30min on ice.
(11), after 15min, protoplast will start to be deposited in ttom of pipe.Suck w5 with pipettor as far as possible, it is heavy to encounter
Form sediment.In the 2*10-5ml-1 ratio resuspended protoplast of mmg, and it is placed on room temperature.
(12) 10 μ l dna (being equivalent to plasmid 10-20 μ g of 5-10kb) are added in 2ml centrifuge tube.
(13) 100 μ l protoplast (2*10-4) are added, soft mixing.
(14) add 110ml peg solution, rap centrifuge tube and fully mix (6-10 sample can be turned every time).
(15) room temperature places 5-10min.
(16) diluted with 440 μ l w5, soft mixing is to terminate converting.
(17) 100g centrifugation 2min, removes supernatant.
(18) 6 porocyte culture plates are inserted with the resuspended protoplast of 1ml w5.
(19) incubated at room temperature certain time.
(20) resuspended protoplast, 100g centrifugation 2min collects.
(21) remove supernatant and directly observe living cells, the albumen observing gmntl7 expression under laser confocal microscope produces
Thing positioning in the cell.
Embodiment 2, in arabidopsiss express williams 82 nac film combination transcription factor gene gmntl7 function test
Card
2.1 flower infestation method arabidopsis thaliana transformations
(1), when arabidopsiss (col-0 wild type) grow to bolting 1cm, top is cut to induce the generation of the raw inflorescence in side；
(2) in conversion the previous day, the Agrobacterium gv3101 containing expression vector plasmid that 1ml activated is taken to be added to containing corresponding
In the 40ml yep culture medium of antibiotic and 50 μ g/ml rifampicin, 28 DEG C of concussion and cultivates to od600It is about 1.0-1.2；
(3) room temperature, 4,200rpm, it is centrifuged 10min, collects thalline, with dip-dyeing solution (5% sucrose, 0.05%silwet l-
77) resuspended thalline, makes od600It is about 0.8；
(4) with pipettor, Agrobacterium is dripped to and contaminated on inflorescence, after all inflorescences are all infected, arabidopsiss are put
Enter evacuation 1min in vacuum desiccator；
(5) cover inflorescence with freshness protection package, cut off top within one day as 20-22 DEG C of lucifuge culture and expose inflorescence, be further cultured for one
Throw off freshness protection package after it, cultivate to seed maturity.
The surface sterilization of 2.2 arabidopsiss seeds
Arabidopsiss neutron subject to sterilization in right amount is put in 1.5ml centrifuge tube, adds the ethanol of 1ml 75% (to contain
The tritonx-100 of 0.03% volume ratio) concussion sterilization 1min, then shake sterilization 1min (twice) with 70% ethanol, finally
With suction nozzle, seed is drawn onto on aseptic filter paper and dries up, then clicked and entered in culture medium with aseptic toothpick.
The screening of 2.3 transfer-gen plants
For seed, surface sterilization is carried out to the t0 harvesting, is then spread evenly across afterwards on 1/2ms flat board (containing corresponding antibiosis
Plain baste).Phjytotron growth is moved into after vernalization treatment 3d.Sprout about 10 days, the bottle-green plant of cotyledon is planted for transgenic
Strain, and cotyledon is sent out light green or even the plant of yellow is nontransgenic plants.Transfer-gen plant is proceeded to growth in soil until harvesting
Obtain t1 for transgenic seed, t1 continues screening for plant individual plant sowing, the seed that every plant is collected, and offspring's segregation ratio is 3:
The positive plant of 1 (positive: negative) grows to results t2 for transgenic seed after transplanting, after individual plant sowing, every plant of kind collected
Son can obtain being sheerly t2 for transgenic seed through screening, sees Fig. 4.
The extraction of 2.4 plant rna
(1) vegetable material is put in mortar, using liquid nitrogen be ground into powder (directly apply to following experiment or
Person freeze save backup in -80 DEG C of ultra cold storage freezers)；
Etc. (2), after liquid nitrogen volatilization, immediately 100-200mg plant powder is transferred in 1.5ml centrifuge tube, then rapidly
Add 1ml trizol extracting solution, vortex concussion makes sample be fully dissolved in extracting solution, room temperature places 5min；
(3) 4 DEG C, 12,000rpm, it is centrifuged 10min, 0.9ml supernatant is transferred in new 1.5ml centrifuge tube, then plus
Enter 0.2ml chloroform and acutely vibrate mixing 15sec, room temperature places 2-5min；
(4) 4 DEG C, 12,000rpm, it is centrifuged 10min, 0.4ml supernatant is transferred in new 1.5ml centrifuge tube, then plus
Enter 0.4ml isopropanol, spin upside down 15 mixing solution, room temperature places 15mim；
(5) 4 DEG C, 12,000rpm, it is centrifuged 10min, abandons supernatant, precipitate twice with the washing with alcohol of 1ml 75%, 4 DEG C, 8,
000rpm, is centrifuged 5min；
(6) abandon supernatant, uncap and rna about 2-5min is dried in the superclean bench, add 40 μ l rnase-free water,
Fully dissolving rna 10min in 60 DEG C；
(7) od value and the concentration of rna sample, a is surveyed with ultraviolet spectrophotometer260/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) sequentially add following material (40 μ l reaction system) in centrifuge tube:
(2) after gently mixing, 65 DEG C of degeneration 5min, it is inserted on ice immediately, ice bath at least 1min；
(3) sequentially add following material in centrifuge tube
(4) after gently mixing, 42 DEG C of water bath with thermostatic control 1h, 65 DEG C of degeneration 10min, -20 DEG C save backup.
The realtime-pcr detection of 2.6 transfer-gen plants
Pcr method screening transgenic positive plant: will have the cdna gmntl7 gene rt-pcr primer of the plant of resistance
Carry out pcr detection.
Gmntl7 gene rt-pcr primer sequence:
Gmntl7 rt-f:5 '-agcatgtgaatggttatcct-3 '
Gmntl7 rt-r:5 '-gagaaagaatcaacaaagcct-3 '
The reaction that common taq enzyme carries out realtime-pcr amplification is following (15 μ l system):
Amplification condition is as follows:
Note: internal standard gene selects gmtub.
Result is shown in Fig. 5.
2.7 nacl, peg process lower gmntl7 in the intraorganic expression analysis of different tissues
(1) col-0 plant is processed 0h in 200mmol/l nacl, after 1h, 12h, take its root, stem, leaf respectively, extract rna
(method same 2.4), rna is reversed to respectively cdna (method same 2.5), carries out realtime-pcr detection (method same 2.6).
(2) col-0 plant is processed 0h in 20%peg, after 1h, 12h, take its root, stem, leaf respectively, (method is same to extract rna
2.4), rna is reversed to respectively cdna (method same 2.5), carries out realtime-pcr detection (method same 2.6).
2.8 Mannitol and nacl process arabidopsiss
(1) surface sterilization (with 2.2) of arabidopsiss seed.
(2) Mannitol and nacl process
Col-0, gmntl7-1, gm ntl7-2 seed of bacterium of having gone out is spread evenly across on 1/2ms flat board respectively (containing phase
The antibiotic baste answering).Vernalization treatment moves into phjytotron growth after 3 days.Sprouting about 4d, root length to 1cm about, will intend
Southern mustard seedling move to add variable concentrations Mannitol (250,300,350,400mmol/l) or nacl (50,100,150,
On 1/2ms culture medium flat plate 200mmol/l).Treat that seedling length, to 11d, observes the long upgrowth situation of seedling root.
Result is shown in Fig. 6.
Claims (4)
1. in a kind of Semen sojae atricolor WILLIAMS-DARLING Ton 82 nac film combination transcription factor gene it is characterised in that: described unnamed gene be Semen sojae atricolor
The nucleotide sequence such as seq id no.1 institute of WILLIAMS-DARLING Ton 82nac film combination transcription factor gene gmntl7, this gene cdna
Show.
2. a kind of plant expression containing nac film combination transcription factor gene in Semen sojae atricolor WILLIAMS-DARLING Ton 82 described in claim 1 carries
Body it is characterised in that: described plant expression vector is named as expression vector pk2gw7::gmntl7, this expression vector cloning region
Nucleotide sequence is as shown in seq id no.4.
3. in Semen sojae atricolor WILLIAMS-DARLING Ton 82 described in claim 1 nac film combination transcription factor gene improving plant drought resisting, in salt tolerant
Application.
4. as claimed in claim 4 application it is characterised in that: described plant is Semen sojae atricolor or arabidopsiss.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110083234A1 (en) * | 2009-10-02 | 2011-04-07 | Nguyen Henry T | Quantitative Trait Loci Associated With Soybean Cyst Nematode Resistance And Methods Of Their Use |
| 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 |
-
2016
- 2016-09-30 CN CN201610867930.6A patent/CN106367423B/en active Active
Patent Citations (4)
| 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 |
| US20110083234A1 (en) * | 2009-10-02 | 2011-04-07 | Nguyen Henry T | Quantitative Trait Loci Associated With Soybean Cyst Nematode Resistance And Methods Of Their Use |
| 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 |
|---|
| CHUNG,E,ET AL: "Glycine max NAC transcription factor(SNAC38)mRNA,complete cds", 《GENBANK:KF657305.1》 * |
| 王楠等: "大豆耐盐相关QTLs 鉴定和功能基因研究进展", 《遗传》 * |
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