CN102747099A - Application of rice gene OsbZIP46 in heat resistance and cold resistance regulation - Google Patents
Application of rice gene OsbZIP46 in heat resistance and cold resistance regulation Download PDFInfo
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Abstract
The invention which belongs to the technical field of the plant gene engineering concretely relates to an application of rice gene OsbZIP46 in the heat resistance and cold resistance regulation. The nucleotide sequence of the gene is represented by SEQ ID NO: 1 in a sequence table, and an amino acid sequence coding the gene is represented by SEQ ID NO: 2 in the sequence table. In the invention, a candidate gene screening method is adopted to clone to the gene OsbZIP46 controlling the heat resistance and the cold resistance of the rice, and the excess expression of the OsbZIP46 gene enhances the conduction capability to ABA signals, so the heat resistance and the cold resistance of transgenic rice are improved, and the function and the application approach of the gene are confirmed.
Description
Technical field
The present invention relates to paddy gene engineering field.Be specifically related to: (1) obtains a kind of rice Os bZIP46 gene that can improve high temperature and low temperature tolerance through separation, clone and functional verification, the application of (2) this gene in paddy rice heat impedance and cold resistance genetic improvement.The present invention adopts the candidate gene screening method; Be cloned into the gene OsbZIP46 of control paddy rice heat impedance and cold resistance; Overexpression OsbZIP46 gene has strengthened the transmissibility to the ABA signal, has improved transgenic paddy rice heat resistanceheat resistant and anti-cold ability, has confirmed the function and the application approach of this gene.
Background technology
Abiotic stress can cause the extensive underproduction of farm crop, the restriction agricultural development like arid, high salt, extreme temperature meeting serious harm growth and development of plant such as (heat, cold).The anti-adversity ability that improves farm crop has become the key issue of being badly in need of solution.Through transgenic technology farm crop are carried out the resistance genetic improvement, the crop varieties of cultivating resistance of reverse seems important all the more, becomes one of major objective of agricultural cience and farming techniques research.Seeking the gene that can really be used for the resistance genetic improvement in the plant is key wherein.
Plant can produce corresponding responsing reaction receiving when coercing, and the variation on a series of physiology taking place and growing reduces or eliminates and coerce the harm that brings to plant.This responsing reaction of plant is a complex process that relates to polygene, many signal pathways.Many genes be proved to be receive adverse circumstance induce and plant to play a role aspect the patience of adverse circumstance (Shinozaki etc., 1997).These genes and expression product thereof can be divided three classes: participate in the gene and the product of signal cascade amplification system and transcriptional control, like some kinases and transcription factor; Directly to protection microbial film and acting gene of protein and expression product thereof; Take in the protein relevant with water and ionic with transhipment.Wherein transcription factor plays important effect in the responsing reaction of plant to abiotic stress.They are constantly synthetic under stress-inducing, regulate and control the expression of a series of downstream genes, with signal transmission and amplification, cause the change of plant physiology and biochemistry aspect, finally make it produce corresponding resistance.Possibly participate in arid, high temperature and cryogenic replying (Yoh Sakuma etc., 2009) like the DREB2A transcription factor of Arabidopis thaliana.Through genetically engineered, part adverse circumstance response transcription factor has been successfully applied to the paddy rice anti contravariance genetic breeding, as the transgenic paddy rice that utilizes the transcription factor SNAC2 of paddy rice NAC family to cultivate has significantly improved anti-salt and anti-cold energy power .2008 such as () Hu; The CBF3 of Arabidopis thaliana is carried out the composition overexpression in paddy rice, significantly improved the drought-enduring salt resistance ability of paddy rice, also part has improved the resistance to cold of paddy rice, does not cause the variation (Oh etc., 2005) on other phenotypes of plant simultaneously.
The bZIP transcription factor contains alkaline leucine zipper motif, and the wide participation plant is replied abiotic stress, has identified many bZIP transcription factors relevant with adverse circumstance so far.Liao etc. think in 131 bZIP genes in the soybean, have more than 1/3rd with plant to reply drought, salt, cold at least a relevant (Liao et al., 2008) of coercing with four kinds of treatment condition of ABA.The bZIP transcription factor participates in depending on the stress response of ABA.ABA is a plant abiotic stress signal transduction path signal of interest molecule; ABA's is synthetic in abiotic stress and the Exogenous ABA ability inducing plant body; BZIP albumen also is activated thereupon and combines with ABA response element ABRE (ABA responsive element); Start the expression (Choi etc., 2000) of downstream gene.Identified the bZIP transcription factor that many participation adverse circumstances are replied in the Arabidopis thaliana.The method of usefulness yeast One-Hybrid such as Choi is isolated 4 kinds of adverse circumstance inductive bZIP albumen from Arabidopis thaliana, be called ABF1, ABF2/AREB1, ABF3/DPBF5 and ABF4/AREB2 respectively.Wherein the expression of ABF2/AREB1, ABF3/DPBF5 and ABF4/AREB2 receives inducing of ABA, arid and high salt, induces and the expression of ABF1 is caught a cold, and they have regulated the expression of a large amount of anti contravariance related genes simultaneously.Overexpression ABF2 has improved the susceptibility of plant to glucose, and has influenced the patience of plant to drought, salt and heat.ABF3 and ABF4 play an important role in Arabidopis thaliana ABA and the transmission of adverse circumstance signal, behind overexpression ABF3 and the ABF4, have improved the susceptibility of plant to ABA, have reduced transpiration rate, have improved plant drought resisting and saline-alkaline tolerance (Choi et al., 2000; Kang et al., 2002; Kim et al., 2004; Oh et al., 2005; Zhou et al., 2006).ABI5 is the relevant bZIP class transcription factor of studying in another Arabidopis thaliana of many and ABA signal conduction.ABI5 can be induced by arid, salt and ABA, and overexpression ABI5 ability render transgenic plant is improved its drought-resistant ability to the ABA hypersensitization.Identified also in the paddy rice that some have the bZIP transcription factor gene of degeneration-resistant effect, like OsABI5, OsbZIP23 (Zou etc., 2008; Xiang etc., 2008).
The OsbZIP46 gene that the present invention relates to is one of bZIP family member in the paddy rice, and its biological function is not reported as yet.The present invention is through candidate gene screening and evaluation; Verified OsbZIP46 gene participation paddy rice replying to ABA processing, high temperature and low temperature stress; Overexpression OsbZIP46 gene shows the conduction of enhancing to the ABA signal; Improved transgenic paddy rice heat resistanceheat resistant and anti-cold ability, so the present invention is for the today that takes place frequently at extreme weather conditions, cultivates extreme temperature tolerance enhanced paddy rice is had great importance.
Summary of the invention
The object of the invention relates to the application of a bZIP transcription factor family member OsbZIP46 gene in control paddy rice heat impedance and cold resistance improvement.Handle the back in the paddy rice adverse circumstance and rise in the transcription factor of expressing, one of them gene belongs to the bZIP transcription factor family, the applicant with this unnamed gene be OsbZIP46 (
ORyza
SAtiva
STress-induced
TRanscription factor 46).The present invention separates and uses a kind of dna fragmentation of the OsbZIP46 of comprising gene, and this fragment is given paddy rice heat impedance enhanced ability under hot conditions, under coldcondition, strengthens the ability of cold resistance.Wherein, the described nucleotide sequence that contains the OsbZIP46 gene is shown in sequence table SEQ NO:1, and sequence length is 975bp, and its corresponding amino acid sequence is shown in SEQ ID NO:2, and its aminoacid sequence is 324.
Carrying OsbZIP46 expression carrier of the present invention can be through using Ti-plasmids, plant viral vector, directly DNA conversion; Microinjection, conventional biotechnological means such as electroporation imports vegetable cell (Weissbach, 1998; Method for Plant Molecular Biology VIII; Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition)).
Can use to comprise that OsbZIP46 expression carrier of the present invention transforms host's (comprising the paddy rice various plants), cultivate heat resistanceheat resistant plant variety and anti-cold plant variety.
Gene of the present invention is expressed by high temperature induction; Therefore can with gene of the present invention be connected into suitable expression vector after any interested high temperature induction promotor combines; And the conversion plant host, but under hot conditions the abduction delivering gene, improve the plant heat impedance.
Below in conjunction with accompanying drawing and embodiment the present invention is further specified.
Description of drawings
Sequence table SEQ ID NO:1 is the nucleotide sequence that includes the OsbZIP46 gene coding region of separating clone of the present invention, and sequence length is 975bp, and its corresponding proteinic aminoacid sequence is shown in SEQ ID NO:2, and aminoacid sequence is 324.
The expression of Fig. 1 .OsbZIP46 gene under multiple adverse circumstance and HORMONE TREATMENT.Each handles sample: arid (drought) is handled 0d, 3d, 5d, rehydration 12h; High salt (salt) is handled 0h, 3h, 6h, 12h; Low temperature (cold) is handled 0h, 3h, 6h, 12h; High temperature (heat) is handled 0h, 2h, 6h, 12h; Oxidative stress (H
2O
2) processing 0h, 3h, 6h, 12h.HORMONE TREATMENT: jasmonic (JA) is handled 0h, 1h, 3h, 6h; Dormin (ABA), indolylacetic acid (IAA), Plant hormones regulators,gibberellins (GA), phytokinin (KT), it all is 0h that brassinolide (BR) is handled, 3h, 6h, 12h.
The expression amount of Fig. 2 .OsbZIP46 overexpression vector synoptic diagram and overexpression plant detects, and CK is the wild-type contrast.
The germination test phenotype of the overexpression material of Fig. 3 .OsbZIP46 under ABA handles, ox2, ox13, ox15 are the overexpression family, and ck9, ck15 are the negative family of separating of transgenic, and ZH11 is a wild-type.
The percentage of germination statistics of the overexpression material of Fig. 4 .OsbZIP46 under ABA handles, ox2, ox13, ox15 are the overexpression family, and ck9, ck15 are the negative family of separating of transgenic, and ZH11 is a wild-type.
The overexpression material of Fig. 5 .OsbZIP46 the seedling phase to ABA sensitivity test phenotype, OsbZIP46U-15-OX, OsbZIP46U-2-OX are the overexpression family, OsbZIP46U-15-NCK, OsbZIP46U-2-NCK are the negative family of the transgenic separated.
The overexpression plant of Fig. 6 .OsbZIP46 is plant height and the long statistics of root to the ABA sensitivity test in the seedling phase, and 15-OX, 2-OX are the overexpression family, and 15-NCK, 2-NCK are the negative family of separating of transgenic.
Phenotype under Fig. 7 .OsbZIP46 overexpression plant high temperature stress, OX is the overexpression family, CK is the wild-type contrast.
Survival rate statistics behind Fig. 8 .OsbZIP46 overexpression plant high temperature stress, OsbZIP46U-2, OsbZIP46U-9, OsbZIP46U-15 are the overexpression family, ZH11 is the wild-type contrast.
Phenotype under Fig. 9 .OsbZIP46 overexpression plant low temperature stress, OX is the overexpression family, CK is the wild-type contrast.
Survival rate statistics behind Figure 10 .OsbZIP46 overexpression plant high temperature stress, OsbZIP46U-2, OsbZIP46U-9, OsbZIP46U-15 are the overexpression family, ZH11 is the wild-type contrast.
Embodiment
Following examples have defined the present invention, and have described the present invention at the dna fragmentation that separates, the clone includes OsbZIP46 gene complete coding section, and the method for checking OsbZIP46 gene function.All or part of implementation step according to following description; Those skilled in the art can confirm essential characteristic of the present invention; And under the situation that does not depart from spirit and scope of the invention, can make various changes and modification to the present invention, so that it is suitable for different purposes and conditions.
1, detecting the endogenous OsbZIP46 gene of paddy rice receives adverse circumstance to induce level
For tentatively whether judging the OsbZIP46 gene with degeneration-resistant relevant, whether the present invention has at first detected paddy rice native gene OsbZIP46 and induced by adverse circumstance.The applicant selects the material of japonica rice variety " in spend 11 " (being called for short ZH11, from Institute of Crop Science, Chinese Academy of Agricultural Science) as expression pattern analysis for use.Grow to 4 the leaf phase seedling carry out the processing of various adverse circumstances and hormone.It is not water to let its seasoning that arid is handled, 0d, and 3d, 5d, rehydration was taken a sample after 12 hours; High-salt stress is the solution that the seedling root is immersed in 200mM NaCl, 0h, and 3h, 6h takes a sample behind the 12h; Low temperature stress is that seedling is put into 4 ℃ of phytotrons, 0h, and 3h, 6h takes a sample behind the 12h.High temperature stress is that seedling is put into 42 ℃ of phytotrons, 0h, and 2h, 6h takes a sample behind the 12h.Oxidative stress is that the seedling root is immersed in 1%H
2O
2In the solution, 0h, 3h, 6h takes a sample behind the 12h.HORMONE TREATMENT is with the dormin of 100 μ M (ABA), brassinolide (BR), indolylacetic acid (IAA), phytokinin (KT); Uniformly behind the spray water rice plants surface and be added to the seedling root, jasmonic (JA) is pressed time point 0h, 1h respectively for Plant hormones regulators,gibberellins (GA), jasmonic (JA); 3h, the 6h sampling, other presses time point 0h; 3h, 6h, 12h sampling.The extraction of total RNA adopts TRIZOL reagent (available from Invitrogen company) to extract; Process for extracting is according to TRIZOL reagent specification sheets; Utilize ThermoScript II SSIII (available from Invitrogen company) with the synthetic cDNA (method is according to Invitrogen company ThermoScript II reagent specification sheets) of its reverse transcription; Reaction conditions is: 65 ℃ of 5min, 50 ℃ of 60min, 70 ℃ of 10min.With above-mentioned reverse transcription synthetic cDNA is template, with primer (OsbZIP46RealT-1025F:5 '-CTTGGACCAAAGGCAAAGAGA-3 ' and OsbZIP46RealT-1097R:5 '-ACCGGAGACTCTGGCTTCAG-3 ') the OsbZIP46 gene is carried out special pcr amplification.Use primer (actin76F:5 '-TGGCATCTCTCAGCACATTCC-3 ' and actin76R:5 '-TGCACAAT GGATGGGTCAGA-3 ') to paddy rice Actin1 gene (accession number: X16280) do specific amplified (the long 76bp of amplified production), simultaneously to carry out quantitative analysis as internal reference.Reaction conditions is: 95 ℃ of 10sec; 95 ℃ of 5sec, 60 ℃ of 34sec, 45 circulations.Carry out the fluoroscopic examination real-time quantitative analysis in the reaction process.The result shows, OsbZIP46 gene (SEQ NO:1) is in arid, high salt, and high temperature, inducing behind the oxidative stress rises expresses, and the OsbZIP46 gene receives ABA, and IAA induces to rise and expresses (Fig. 1).
2, the structure and the genetic transformation of OsbZIP46 gene overexpression carrier
The structure of overexpression carrier:
In order to study the degeneration-resistant function of OsbZIP46 gene, the applicant is with its overexpression in paddy rice, and the function of this gene is studied in expectation from the phenotype of transfer-gen plant.The overexpression carrier construction method is following:
At first through searching: LOC_Os06g10880 at the note of rice genome note website TIGR (http://rice.plantbiology.msu.edu/) OsbZIP46 gene number; With the note of KOME (http://cdna01.dna.affrc.go.jp/cDNA/) OsbZIP46 gene number: AK103188; Prediction OsbZIP46 is that a bZIP family gene (see shown in the SEQ ID NO:1 by the complete nucleotide sequence of this gene; Its coding region length of nucleotides is 975bp, and the nucleotide sequence corresponding amino acid sequence is 324).In crop genetic improvement National Key Laboratory of Hua Zhong Agriculture University bright extensive equilibration in 63 time of infertility cDNA library (Chu etc., 2003), retrieve a cDNA clone who contains OsbZIP46 gene coding region 5 ' partial sequence (clone number: BI103-O13) (the external disclosed network address in this library: http://redb.ncpgr.cn/modules/redbtools/).Picking should the clone from the library, and the extracting plasmid utilizes universal primer SP6 (5 '-ATTTAGGTGACACTATA-3 '), T7 (5 '-TAATACGACTCACTATAGGG-3 ') to carry out sequence verification from two ends, and examining order is accomplished on the ABI7500 instrument.Then with the KOME DB in the full-length cDNA of OsbZIP46 gene carry out sequence relatively, confirm that clone BI103-O13 comprises the complete ORFs (ORF, open reading frame) of OsbZIP46 gene.Clone BI103-O13 plasmid reclaims exogenous segment with KpnI and BamHI double digestion; Simultaneously; The enzyme that uses the same method is cut the genetic transformation carrier pCAMBIA1301U that carries the Ubiquitin promotor, and (pCAMBIA1301U reconstructs on the plant genetic conversion carrier pCAMBIA1301 basis of using always in the world; Carry the agriculture bacillus mediated genetic transformation carrier of corn ubiquitin promotor) with composing type and overexpression characteristic; Enzyme cuts complete, uses chloroform: primary isoamyl alcohol (volume ratio is 24: 1) extracting, purifying enzyme is cut product.Do ligation with comprising the endonuclease bamhi of OsbZIP46 gene and pCAMBIA1301U carrier that enzyme is cut, thereafter transformed into escherichia coli DH10 β (this intestinal bacteria DH10 β bacterial strain is available from Promega company).Cut screening positive clone through enzyme, the recombinant plasmid vector of acquisition is named as OsbZIP46-1301U (Fig. 2 A).
The genetic transformation step:
Through agriculture bacillus mediated rice genetic method for transformation (its concrete steps are described below) above-mentioned overexpression vector OsbZIP46-1301U is transferred in the rice varieties " in spend 11 "; Through the callus of cultivating in advance, infecting, cultivating altogether, screening having hygromycin resistance, break up, take root, practice seedling, transplanting, obtain transfer-gen plant.Above-mentioned agriculture bacillus mediated paddy rice (in spend 11) genetic transforming method (system) is at people's reported method such as Hiei (Hiei etc.; Efficient transformation of rice; Oryza sativa L., mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J; 6:271-282,1994) improve on the basis and carry out.The concrete genetic transformation step of present embodiment is following:
(1) electricity transforms: use 1800v voltage, final overexpression destination carrier OsbZIP46-1301U electricity is transformed into Agrobacterium EHA105 bacterial strain, be coated onto on the LA substratum that has corresponding resistance selection, filter out positive colony, be used for following conversion callus.
(2) callus induction:, used 70% Ethanol Treatment then successively 1 minute with spending 11 to shell in the sophisticated rice paddy seed; 0.15% mercury chloride (HgCl
2) surface sterilization 15 minutes; Wash seed 4-5 time with sterilization; Should be placed on (composition is seen after) on the inducing culture by sterile seed; Place dark place to cultivate 4 weeks, 25 ± 1 ℃ of temperature postvaccinal inducing culture.
(3) callus subculture: select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in subculture medium (composition is seen after) and go up dark 2 weeks, 25 ± 1 ℃ of the temperature of cultivating down.
(4) cultivate in advance: select the embryo callus subculture of consolidation and relatively dry, be put in pre-culture medium (composition is seen after) and go up dark 2 weeks, 25 ± 1 ℃ of the temperature of cultivating down.
(5) Agrobacterium is cultivated: (composition is seen after) cultivated Agrobacterium EHA105 (derive from CAMBIA, commercial bacterial strain carries overexpression vector OsbZIP46-1301U of the present invention) two days, 28 ℃ of culture temperature in advance on the LA substratum that has corresponding resistance selection; Described Agrobacterium is transferred to suspension culture base (composition is seen after), cultivated 2-3 hour on 28 ℃ of shaking tables.
(6) Agrobacterium is infected: pre-incubated callus is transferred in the good bottle of sterilization; The suspension-s of regulating Agrobacterium is to OD
6000.8-1.0; Callus was soaked in agrobacterium suspension 30 minutes; Shift on the good filter paper of callus to sterilization and blot; Be placed on common substratum (composition is seen after) then and go up cultivation 3 days, culture temperature 19-20 ℃.
(7) callus washing and selection are cultivated: aqua sterilisa washing callus is to cannot see Agrobacterium; Be immersed in the aqua sterilisa that contains 400ppm Pyocianil (CN) 30 minutes; Shift on the good filter paper of callus to sterilization and blot; Shift callus and select 2-3 time, each 2 weeks (screening Pyocianil concentration for the first time is 400ppm, and be 250ppm later the second time, Totomycin concentration 250ppm) to selecting substratum (composition is seen after) to go up.
(8) differentiation: kanamycin-resistant callus tissue is transferred to presorting substratum (composition is seen after) goes up dark place cultivation 5-7 days; The callus of presorting of transfer cultivation (composition is seen after) to division culture medium, illumination (3500lux) is cultivated down, 26 ℃ of temperature.
(9) take root: cut the root that differentiation phase produces; Then it is transferred to and cultivates 2-3 week, 26 ℃ of temperature in the root media under the illumination.
(10) transplant: wash the residual substratum on the root off, the seedling that will have good root system changes the greenhouse over to, divides moistening at initial several Tian Bao water holding simultaneously.
Nutrient media components and prescription thereof:
(1) reagent and solution abbreviation
The abbreviation of the used plant hormone of substratum is represented as follows among the present invention: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin, Pyocianil); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indolylacetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4 dichlorophenoxyacetic acid); AS (Acetosringone, Syringylethanone); CH (Casein Enzymatic Hydrolysate, caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (Dimethyl Sulfoxide, DMSO 99.8MIN.); N6max (a large amount of composition solution of N6); N6mix (N6 trace ingredients solution); MSmax (a large amount of composition solution of MS); MSmix (MS trace ingredients solution).
(2) main solution formula
1) preparation of N6 substratum macroelement mother liquor [10 times of liquid concentrators (10X)]:
Dissolving is settled to 1000ml under the room temperature then one by one.
2) preparation of N6 substratum trace element mother liquor [100 times of liquid concentrators (100X)]
Dissolving and be settled to 1000ml under the room temperature.
3) molysite (Fe
2EDTA) preparation of stock solution (100X)
Prepare the 800ml distilled water and be heated to 70 ℃, add b diammonium disodium edta (Na
2EDTA2H
2O) 3.73 grams, fully the dissolving back kept 2 hours in 70 ℃ of water-baths, was settled to 1000ml, and 4 ℃ of preservations are subsequent use.
4) VITAMINs stock solution (100X) preparation
Add water and be settled to 1000ml, 4 ℃ of preservations are subsequent use.
5) preparation of MS substratum macroelement mother liquor (10X)
Dissolving and be settled to 1000ml under the room temperature.
6) preparation of MS substratum trace element mother liquor (100X)
Dissolving and be settled to 1000ml under the room temperature.
7) 2, the preparation of 4-D stock solution (1mg/ml):
Take by weighing 2,4-D 100mg with 1ml1N Pottasium Hydroxide dissolving 5 minutes, adds the 10ml dissolved in distilled water then and is settled to 100ml after fully, under room temperature, preserves.
8) preparation of 6-BA stock solution (1mg/ml):
Take by weighing 6-BA 100mg,, add the 10ml dissolved in distilled water then and be settled to 100ml, room temperature preservation after fully with 1ml 1N Pottasium Hydroxide dissolving 5 minutes.
9) preparation of naphthylacetic acid (NAA) stock solution (1mg/ml):
Take by weighing NAA 100mg, with 1ml1N Pottasium Hydroxide dissolving 5 minutes, add the 10ml dissolved in distilled water then and be settled to 100ml after fully, 4 ℃ of preservations are subsequent use.
10) preparation of indolylacetic acid (IAA) stock solution (1mg/ml):
Take by weighing IAA 100mg,, add the 10ml dissolved in distilled water then and be settled to 100ml after fully, preserve to be equipped with for 4 ℃ and in a big triangular flask, add 300ml zero(ppm) water and ferric sulfate (FeSO with 1ml1N Pottasium Hydroxide dissolving 5 minutes
47H
2O) 2.78g.In another big triangular flask, adding 300ml zero(ppm) water uses.
11) preparation of glucose stock solution (0.5g/ml):
Take by weighing glucose 125g, be settled to 250ml with dissolved in distilled water then, the back 4 ℃ of preservations of sterilizing are subsequent use.
12) preparation of AS stock solution:
Take by weighing AS 0.392g, DMSO 10ml divides to be filled in the 1.5ml centrifuge tube, and 4 ℃ of preservations are subsequent use.
13) 1N Pottasium Hydroxide stock solution
Take by weighing Pottasium Hydroxide 5.6g, and be settled to 100ml with dissolved in distilled water, room temperature preservation is subsequent use.
(3) be used for the culture medium prescription that rice genetic transforms
1) inducing culture
Adding distil water is to 900ml, and 1N Pottasium Hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides to install to 50ml triangular flask (25ml/ bottle), seals sterilization.
2) subculture medium
Adding distil water is to 900ml, and 1N Pottasium Hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides to install to 50ml triangular flask (25ml/ bottle), seals sterilization.
3) pre-culture medium
Adding distil water is to 250ml, and 1N Pottasium Hydroxide is regulated pH value to 5.6, seals sterilization.Use preceding heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (25ml/ ware) in the petridish poured in packing into.
4) be total to substratum
Adding distil water is to 250ml, and 1N Pottasium Hydroxide is regulated pH value to 5.6, seals sterilization.Use preceding heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (the every ware of 25ml/) in the petridish poured in packing into.
5) suspension culture base
Adding distil water is regulated pH value to 5.4 to 100ml, divides to install in the triangular flask of two 100ml, seals sterilization.Add 1ml glucose stock solution and 100 μ l AS stock solutions before using.
6) select substratum
Adding distil water is regulated pH value to 6.0 to 250ml, seals sterilization.The dissolving substratum adds 250 μ l HN and 400ppm CN before using, and (25ml/ ware) in the petridish poured in packing into.
7) presorting substratum
Adding distil water is to 250ml, and 1N Pottasium Hydroxide is regulated pH value to 5.9, seals sterilization.The dissolving substratum adds 250 μ lHN and 200ppm CN before using, and (25ml/ ware) in the petridish poured in packing into.
8) division culture medium
Adding distil water is to 900ml, and 1N Pottasium Hydroxide is regulated pH value to 6.0.Boil and be settled to 1000ml, divide to install to 50ml triangular flask (50ml/ bottle), seal sterilization.
9) root media
Adding distil water is to 900ml, and 1N Pottasium Hydroxide is regulated pH value to 5.8.Boil and be settled to 1000ml, divide installing in the pipe of taking root (25ml/ pipe), seal sterilization.
3, the expression amount of OsbZIP46 overexpression plant is identified
The present invention adopts the Northern hybridizing method that OsbZIP46 expression of gene in the transgenic rice plant of above-mentioned the 2nd step acquisition is detected.Total RNA of extraction blade (Trizol reagent Invitrogen), with the total RNA of 15 μ g electrophoretic separation in 1.2% agarose denaturant gel, goes to nylon membrane, through UV-crosslinked fixing, with
32The target gene probe hybridization of P mark, is hybridized, is washed film and development etc. and undertaken by the relevant experimental implementation method in " molecular cloning " (Science Press, 1999) mark of probe.The expression amount detected result shows that OsbZIP46 expression of gene amount significantly improves (Fig. 2 B) with respect to wild-type in the only about half of transfer-gen plant.
4, the ABA susceptibility of OsbZIP46 overexpression plant is identified
The transgenic family is the sterilization of shelling of the seed of just overexpression family, transgenic negative control family and wild-type to dormin (ABA) germination sensitivity Detection; Directly germinate containing on the MS culture medium flat plate of ABA concentration gradient, in culturing room's lucifuge growth and add up its percentage of germination.The result shows; On the MS substratum that contains 3 μ M and 6 μ MABA; The percentage of germination of overexpression family is starkly lower than contrast (negative family of transgenic and wild-type); On the MS substratum of no ABA or 1 μ MABA, both do not have significant difference by percentage of germination, and the susceptibility (Fig. 3 and Fig. 4) to ABA is described when overexpression OsbZIP46 gene causes strengthening transfer-gen plant and germinates.
Transgenic family ABA seedling growth sensitivity Detection is the sterilization of shelling of the seed with overexpression transgenic family and transgenic negative control family; Germinate containing on the MS substratum of 100mg/L Totomycin, selecting the good and consistent seed of growing way of germinateing transfer to contain 3 μ mol/LABA the MS substratum in culturing room's lucifuge growth.After one week of growth, observe phenotype and root length and the plant height of measuring plant respectively.The result shows the overexpression family in the ABA disposition, and root length and plant height are obviously compared according to lacking, and its growth receives the inhibition of ABA stronger, explains that the OsbZIP46 gene causes transfer-gen plant susceptibility to ABA when seedling growth to strengthen (Fig. 5 and Fig. 6).
Can find out that from the experimental result in above two stages the OsbZIP46 overexpression can strengthen the signal transmissibility of transfer-gen plant to ABA.
5, the high temperature stress phenotypic evaluation of OsbZIP46 overexpression plant
In order to verify the heat impedance of OsbZIP46 overexpression plant; The contriver has carried out the high temperature stress phenotypic evaluation to the overexpression plant; After three overexpression familys (OX) and wild-type family (in spend 11, or be called ZH11) germinateed, select consistent plant kind in catridge; Every barrel of kind transfer-gen plant and each 25 strain of wild-type contrast are put under the normal condition and are grown.The plant of normal 4 leaf phases of growth is moved to 42 ℃ of growth chambers carry out high temperature stress and handled 2 days, return to normal condition then and grow a week, take pictures and investigate the survival rate of plant, 3 repetitions are established in test.The result shows that in the high temperature stress treating processes, the blade of wild-type contrast curls very soon, and chlorosis is wilted, and the overexpression plant does not show intensive variation like this.Through high temperature stress and after the for some time of recovering to grow, the blade of wild-type (contrast) basically all withers, no greenery growth, and overexpression plant part blade still can recover, and can recover the new greenery of growing very soon.Further the statistics survival rate finds that wild-type contrast survival rate is lower than 40%, and the survival rate of overexpression plant is significantly higher than contrast (more than 60%).Compare with wild-type contrast, the overexpression plant shows stronger resistance to high temperature stress, and the heat impedance (Fig. 7 and Fig. 8) that has improved plant behind the OsbZIP46 overexpression is described.
5, the low temperature stress phenotypic evaluation of OsbZIP46 overexpression plant
In order to verify the cold resistance of OsbZIP46 overexpression plant; The contriver has also carried out the low temperature stress phenotypic evaluation to the overexpression plant; After three overexpression familys (OX) and wild-type family (ZH11) germination; Select consistent plant kind in catridge, every barrel of kind transfer-gen plant and respectively 25 strains of wild-type contrast are put under the normal condition and are grown.The plant of normal 4 leaf phases of growth is moved to 4 degrees centigrade of freezers carry out low temperature stress and handle a week, return to normal condition then and grow a week, take pictures and investigate the survival rate of plant, 3 repetitions are established in test.The result shows that through low temperature stress and after the for some time of recovering to grow, the survival rate of overexpression plant is significantly higher than wild-type contrast (survival rate of overexpression plant is more than 50%, and the survival rate of wild-type plant is lower than 30%).Compare with wild-type contrast, the overexpression plant shows stronger resistance to low temperature stress, and the cold resistance (Fig. 9 and Figure 10) that has improved plant behind the OsbZIP46 overexpression is described.
Claims (2)
1. the application of rice Os bZIP46 gene in regulating and control high temperature resistant and low temperature genetic improvement, the nucleotide sequence that it is characterized in that this gene is shown in sequence table SEQ ID NO:1.
2. the application of rice Os bZIP46 gene in regulating and control high temperature resistant and low temperature genetic improvement, the amino acid sequence coded that it is characterized in that this gene is shown in sequence table SEQ ID NO:2.
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| CN103898078A (en) * | 2012-12-26 | 2014-07-02 | 中国科学院遗传与发育生物学研究所 | Paddy rice heat-resistant gene TOG1 and application thereof |
| CN106868017A (en) * | 2017-03-02 | 2017-06-20 | 湖南农业大学 | A kind of adjusting and controlling rice seedling stage heat resistance gene TBZ1 and application |
| CN109112140A (en) * | 2018-03-24 | 2019-01-01 | 华中农业大学 | Application of the OsSN28 gene in control rice high temperature resistant |
| CN111087456A (en) * | 2019-12-13 | 2020-05-01 | 浙江省农业科学院 | Transcription factor OsbZIP58 capable of generating variable shearing at high temperature and application thereof in regulation and control of high temperature resistance of rice |
| CN113004381A (en) * | 2019-12-20 | 2021-06-22 | 中国农业大学 | Application of ZmbZIP68 protein and coding gene thereof in regulating and controlling low-temperature stress tolerance of corn |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898078A (en) * | 2012-12-26 | 2014-07-02 | 中国科学院遗传与发育生物学研究所 | Paddy rice heat-resistant gene TOG1 and application thereof |
| CN103898078B (en) * | 2012-12-26 | 2016-04-20 | 中国科学院遗传与发育生物学研究所 | The heat-resisting gene TOG1 of paddy rice and application thereof |
| CN106868017A (en) * | 2017-03-02 | 2017-06-20 | 湖南农业大学 | A kind of adjusting and controlling rice seedling stage heat resistance gene TBZ1 and application |
| CN109112140A (en) * | 2018-03-24 | 2019-01-01 | 华中农业大学 | Application of the OsSN28 gene in control rice high temperature resistant |
| CN111087456A (en) * | 2019-12-13 | 2020-05-01 | 浙江省农业科学院 | Transcription factor OsbZIP58 capable of generating variable shearing at high temperature and application thereof in regulation and control of high temperature resistance of rice |
| CN111087456B (en) * | 2019-12-13 | 2021-11-19 | 浙江省农业科学院 | Transcription factor OsbZIP58 capable of generating variable shearing at high temperature and application thereof in regulation and control of high temperature resistance of rice |
| CN113004381A (en) * | 2019-12-20 | 2021-06-22 | 中国农业大学 | Application of ZmbZIP68 protein and coding gene thereof in regulating and controlling low-temperature stress tolerance of corn |
| CN113004381B (en) * | 2019-12-20 | 2022-07-15 | 中国农业大学 | Application of ZmbZIP68 protein and coding gene thereof in regulating and controlling low-temperature stress tolerance of corn |
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