CN103421788B - Separation and utilization of stress response paddy rice promoter OsSN1P - Google Patents
Separation and utilization of stress response paddy rice promoter OsSN1P Download PDFInfo
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Abstract
The invention belongs to the technical field of paddy rice gene engineering and particularly relates to separation and utilization of a stress response paddy rice promoter OsSN1P. The promoter responding to various abiotic stresses, such as drought, high salt, high temperature, ABA and the like is obtained through separation, cloning and function verification, and the nucleotide sequence of the promoter is shown in SEQ ID NO: 1. The stress response paddy rice promoter OsSN1P can improve the specific expression of a gene related to stress tolerance in the abiotic stress environment, and can be used for genetically modifying the resistance ability of paddy rice to abiotic stress.
Description
Technical field
The invention belongs to field of plant genetic.Be specifically related to be separated, clone and obtain one by functional verification and can improve anti contravariance related gene specifically expressing in abiotic stress environment to the promotor that multiple abiotic stress (arid, high salt, high temperature, ABA etc.) is replied, be applied to the ability of genetic improvement paddy rice tolerance abiotic stress.The present invention adopts pcr amplification and merges the method for gus reporter gene genetic transformation, clone obtains controlling paddy rice abiotic stress response promotor SN1P, by Real-time PCR, transfer-gen plant GUS dyes and observes and GUS enzyme activity determination, prove that this promotor is by abiotic stress (arid, high temperature, oxidative stress) induction, can be applicable to the genetic improvement of paddy rice.
Background technology
Under nature or agricultural production conditions, the growth and development process of plant can be subject to the impact of various environmental factors.It is even dead that many abiotic stress conditions such as arid, high salt, extreme temperature usually can quickly make plant come to harm.Concerning agriculture production, these poor environments are the most direct, the most important factors affecting crop yield and quality, therefore become the bottleneck restricting many regional agricultural developments.Along with the development of modern molecular biology technique, the function of research anti contravariance related gene and regulatory mechanism, and utilize these genes to cultivate the crop varieties with excellent resistance, become one of important goal of agricultural cience and farming techniques research.
Because plant can not arbitrarily movement in its process of growth, therefore in long-term evolution and natural selection, gradually define a set of self-protective mechanism resisting poor environment.Plant experiences the expression by resistance relevant protein in signal transduction procedure regulation cell after extraneous adverse circumstance signal, so adjust self metabolism, physiological status or form to adapt to adverse environment, maintain basic survival activity.Plant senses after environment stress through a series of intracellular signaling and transcriptional control, start the expression of a large amount of and stress response genes involved in downstream, produce as LEA proteinoid, ooze tune albumen, antifreeze protein, channel protein etc. some make cell from the material of stress damage, and then plant is strengthened the tolerance of coercing (Valliyodan B, Nguyen H T.Understanding regulatory networks and engineering for enhanced droughttolerance in plants.Curr Opin Plant Biol, 2006, 9:189-195).In recent years, just progressively obtain feasibility study in recent years by transgenic approach improvement stress resistance of plant and generally accept.Much research has been had to confirm, in plant, overexpression Stress response genes involved can improve resistance (the .Overexpressing aNAM such as Hu of plant to a certain extent, ATAF, and CUC (NAC) transcription factorenhances drought resistance and salt tolerance in rice.Proc Natl Acad Sci U S A, 2006,103:12987-12992; The .A homolog of human ski-interacting protein in rice positively reg μ Lates cell viability and stress tolerance.Proc Natl Acad Sci such as Hou, 2009,106 (15): 6410-6415).But report in these overexpressions usually be all constitutive promoter; although constitutive promoter overexpression effect is high; but usually can along with inevitable negative effect; poison as produced plant or burden or the (Puranik etc. such as render transgenic plant is lethal; NAC proteins:regulation and role in stress tolerance.Trends Plant Sci; 2012,17 (6): 369-381).Although there has been bibliographical information to isolate promotor (Kasuga etc. by Stress response from plant, Acombination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought-andlow-temperature stress tolerance in tobacco by gene transfer.Plant Cell Physiol, 2004,45:346-350; Xiao etc., Over-expression of a LEA gene in rice improves drought resistance under the field conditions.Theor Appl Genet, 2007,115 (1): 35-46), also considerably less but adverse circumstance inducible promoter to be applied to the successful examples of important food crop paddy rice anti contravariance breed of variety.NAC (NAM, ATAF, and CUC) transcription factor is the distinctive class transcription factor of plant, find that there are 151 member (Nuruzzaman etc. in this family in paddy rice up to now, Genome-wide analysis of NAC transcription factor family in rice.Gene, 2010,465:30-44), many research report NAC transcription factor growing and playing vital effect in process in Stress response plant.Our research shows have quite a few member to be subject to abiotic stress abduction delivering (Fang etc. in NAC family, Systematic sequence analysis and identification of tissue-specific or stress-responsive genes of NACtranscription factor family in rice.Mol Genet Genomics, 2008,280 (6): 547-563).
Paddy rice (Oryza sativa) is as a kind of important food crop, and its degeneration-resistant improvement is the target that people make great efforts always.In today that natural climate condition is day by day severe, meaning and the urgency of cultivating degeneration-resistant new rice variety are apparent.The SN1 gene that the present invention relates to is one of paddy rice NAC family member, the promotor of SN1 gene is demonstrated by multiple abiotic stress abduction delivering by Real-Time Fluorescent Quantitative PCR Technique, shown by promoter activity quantitative analysis, gus reporter gene under SN1 gene promoter drives is expressed and is significantly risen under arid, high temperature and oxidative stress conditions, this promotor can be used for the abduction delivering of resistance genes involved in the crops such as paddy rice, thus effectively improve the performance of plant resisting abiotic adverse circumstance, for the degeneration-resistant New Crop Varieties of cultivation by significant.
Summary of the invention
Object of the present invention relates to the application of promotor in paddy rice anti contravariance improvement of a paddy rice NAC family gene SN1.SN1 gene is the gene of a coding NAC albumen.This gene pairs comprises arid, high temperature, low temperature, oxidative stress, injures and all has response at interior multiple abiotic stress.The present invention is separated and applies a kind of promoter fragment of SN1 gene, and this fragment gives the downstream goal gene ability that expression amount significantly rises under the oxidative stress conditions of arid, high temperature and MV simulation.Wherein, described SN1P promotor nucleotide sequence length is 2233bp, and its nucleotide sequence is as shown in sequence table SEQ NO:1.
The present invention implements by the following technical programs:
First be separated the promotor to the candidate gene that Stress response is expressed.Selected candidate gene is paddy rice native gene, belongs to NAC transcription factor family, called after SN1 (TIGR ID is LOC_OsO1g09550).This gene is all subject to induction and rises and express (as Fig. 2) under the Drought at seedling stage of rice varieties " in spend 11 ", high temperature, high-salt stress and ABA process.The promotor of this gene be separated derives from rice varieties " Japan is fine ", called after SN1P.SN1P promotor has the sequence as shown in base 1-2233 in Fig. 1; The ABA response element ABRELATERD1 (Simpson etc. relevant to adverse circumstance are contained at 52-56,1894-1898 base position of this sequence, Two different novelcis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stressand dark-induced senescence.Plant J, 2003,33:259-270; Nakashima etc., Transcriptional regulation of ABI3-and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings ofArabidopsis.Plant Mol Biol, 2006,60:51-68); The relevant anaerobism response element ANAERO2CONSENSUS (Mohanty etc. of adverse circumstance are contained at 317-334,2228-2233 base position, Detection and preliminary analysis of motifs in promoters ofanaerobically induced genes of different plant species.Ann Bot, 2005,96:669-681); Relevant high temperature response element CCAATBOX1 (the Rieping M of adverse circumstance is contained at 953-957,1408-1412 base position, Schoffl F, Synergistic effect of upstreamsequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genesin transgenic tobacco.Mol Gen Genet, 1992,231:226-232; Haralampidis etc., Combinatorial interaction of ciselements specifies the expression of the Arabidopsis AtHsp90-1gene.Plant Physiol, 2002,129:1138-1149; Wenkel etc., CONSTANS and the CCAAT Box Binding Complex Share a Functionally Important Domain andInteract to Regulate Flowering of Arabidopsis.Plant Cell, 2006,18:2971-2984); The relevant low temperature response element LTRE1HVBLT49 (Dunn etc. of adverse circumstance are contained at 1217-1222 base position, Identification of promoter elements in alow-temperature-responsive gene (blt4.9) from barley (Hordeum vulgare L.) .Plant Mol Biol, 1998,38:551-564).
Multiple cis-acting elements relevant to adverse circumstance (as shown in Figure 1) is contained in promotor SN1P region provided by the present invention, can play responsing reaction specifically to the adverse circumstances such as high temperature, low temperature, Oxdative stress and ABA.GUS expression vector (as shown in Figure 5) rice transformation acceptor kind that applicant utilizes SN1P promotor to build " in spend No. 11 ", can the expression (as shown in Figure 7 and Figure 8) of induced reporter gene GUS when transgenic line is subject to environment stress.Effect of the present invention refers to embodiment.
Detailed technical scheme is as described below:
A kind of isolated paddy DNA molecule, its nucleotide sequence as shown in Fig. 1 and sequence table SEQ ID NO:1, the nucleotide sequence shown in 1-2233 bit base during wherein promotor SN1P comprises as shown in Fig. 1 and sequence table SEQ ID NO:1 sequence.
The DNA molecular of above-mentioned separation, in its region except basic promoter element (as TATA-box), also comprise the combination of multiple Stress response cis-acting elements (as ABRELATERD1, ANAERO2CONSENSUS, CCAATBOX1 and LTRE1HVBLT49).
The described all or part of sequence of SN1P promotor may be used for the paddy rice expression vector built.
The described all or part of sequence of SN1P promotor can be applicable to build paddy rice expression vector, and cultivate improvement plant by the method for genetic transformation, preferred plant is paddy rice.
Utilize above-mentioned SN1P promotor all or part of sequence construct paddy rice expression vector, by the method for genetic transformation may be used for cultivate adversity resistant plant material, described adversity resistant plant refer to plant, seed or cell clonal.
According to above technical scheme, other people beyond applicant or applicant can utilize SN1P promotor provided by the present invention to build the expression vector conversion of plant of anti contravariance related gene to improve the resistance of plant.Recipient plant can be the cereal crop comprising paddy rice, wheat, corn etc., can certainly be comprise some other important cash crop, such as corn, cotton, rape or tomato etc.
Below in conjunction with drawings and Examples, the present invention will be further described.
Accompanying drawing explanation
Sequence table SEQ ID NO:1 is the nucleotide sequence of the rice starter SN1P that the present invention clones, and sequence length is 2233bp.
Fig. 1: SN1P promoter sequence (basic promoter element structure).Basic promoter element sequence dash area shows, and Stress response ABRELATERD1, ANAERO2CONSENSUS, CCAATBOX1, LTRE1HVBLT49 element core sequence is by italic and underline and represent.
The expression of Fig. 2: SN1 gene under multiple adverse circumstance.Each processing sample is: arid (drought) processes 0h, 3h, 6h, 12h; High salt (salt) processes 0h, 3h, 6h, 12h; High temperature (heat) processes 0min, 1h, 3h, 6h; Low temperature (cold) processes 0h, 1h, 6h, 24h; Flooding stress (flood) processes 0h, 6h, 12h, 24h; Injury (wound) processes 0h, 1h, 6h, 12h; Oxidative stress (H
2o
2) process 0h, 1h, 3h, 12h; ABA (100 μMs) processes 0h, 0.5h, 3h, 6h.
The histoorgan expression pattern (in figure, histoorgan is followed successively by from left to right: callus, root, stem apex, leaf, leaf sheath, 1cm fringe, 5cm fringe, 10cm fringe, gynoecium, stamen and embryo) of Fig. 3: SN1P promotor.
The plasmid map of Fig. 4: DX2181G carrier.
The collection of illustrative plates of Fig. 5: the expression vector DX2181G-SN1P that the present invention builds.This carrier contains hygromycin resistance screening-gene (hygromycin), and promotor SN1P is fused to gus gene 5 ' and holds non-translational region.
Fig. 6: SN1P be SN1P promotor of the present invention drive under this promotor transgenic paddy rice normal growing conditions the expression of downstream gus reporter gene (in figure, stained tissue Organ naming is followed successively by from left to right from top to bottom: differentiation initial stage callus, differentiation seedling, blade tip, blade, pulvinus, auricle and the tip of a leaf, leaf sheath, leaf sheath profile, root, clever shell, rataria, seed).
Fig. 7: in kanamycin-resistant callus tissue SN1P promotor control under the gus gene oxidative stress process of simulating at arid, high temperature, waterlogging and methyl viologen (MV) under expression activity, show GUS coloration result in figure.
Fig. 8: the GUS enzyme under the oxidative stress process that in kanamycin-resistant callus tissue, SN1P promoters driven gus reporter gene is simulated at arid, high temperature, waterlogging and methyl viologen (MV) is lived quantitative analysis results.
Fig. 9: be middle interstitial granules pEGEM-T Vectorde collection of illustrative plates of the present invention.
Figure 10: be the plasmid map inserting SN1P gene fragment (right side see Figure 10) in middle interstitial granules pEGEM-T Vectorde.
Embodiment
Following embodiment defines the present invention, and describes the present invention in separation SN1P promotor, and clone includes the DNA fragmentation of SN1P promotor, and the method for checking SN1P promoter function.According to following description and these embodiments, those skilled in the art can determine essential characteristic of the present invention, and when not departing from spirit and scope of the invention, various change and amendment can be made to the present invention, being suitable for different purposes and condition to make it.
1, the separation andpreconcentration of SN1P promotor
(Institute of Crop Science, Chinese Academy of Agricultural Science is derived from by rice varieties " in spend No. 11 ", the open rice varieties promoted) adverse circumstance expression pattern analysis, find a gene that multiple adverse environmental factor is replied, its TIGR (http://rice.plantbiology.msu.edu/) ID is LOC_Os01g09550, be named as SN1, corresponding pac clone number is P0710E05.
Next step is exactly the promotor being separated this gene.Concrete steps are as follows: the genome sequence (P0710E05) of the japonica rice " Japan is fine " finding this gene pairs to answer at NCBI (http://www.ncbi.nlm.nih.gov/) the scope choosing the transcription initiation site upstream 2.5kb of this gene alternatively promoter region carry out pcr amplification.Design primer SN1P-F (5 '-AA
cTGGAAACCGTGAGAC-3 ') and SN1P-R (5 '-AA
gCTGCTTTCACAAGTTTA-3 '), and hold interpolation restriction enzyme site SalT (underline by italic and represent, two bases before restriction enzyme site are protection base) respectively at primer 5 '.First genomic dna (be shown in: Zhang etc. by CTAB method extracting oryza sativa genomic dna document with " Japan is fine " to utilize primer SN1P-F and SN1P-R, genetic diversity and differentiation ofindica an japonica rice detected by RFLP analysis.Theor Appl Genet, 1992,83:495-499) for template increases, reaction system is 20 μ LLAbufferII systems (purchased from TaKaRa company), and reaction conditions is: 94 DEG C of denaturation 4min; 94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 2min 30sec, 32 circulations; 72 DEG C extend 7min.The PCR primer that amplification obtains is connected into pGEM-T (purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, the i.e. U.S. Promega agency of corporation) on carrier, screening positive clone is cut and (the ABI3730 sequenator that checks order by enzyme, Applied Biosystem, order-checking completes in National Botanical gene center [Wuhan]), result confirms: institute's extension increasing sequence is the SN1P promoter sequence of expection, it comprises multiple high temperature, low temperature, anaerobism, ABA Stress response cis-acting elements (as Fig. 1), the recombinant plasmid vector of acquisition is named as pGEM-SN1P.
2, the expression level of the endogenous SN1 gene of paddy rice is detected
Applicant selects japonica rice variety " in spend No. 11 " as the material of expression pattern analysis.Selection normal growth to 4 leaf phase seedling carries out various adverse circumstance and dormin (ABA) process.Osmotic treatment makes its seasoning, samples after 0h, 3h, 6h, 12h; High-salt stress is solution seedling root being immersed in 200mM NaCl, samples after 0h, 3h, 6h, 12h; High temperature stress seedling is put into 42 DEG C of phytotrons, samples after 0h, 1h, 3h, 6h; Low temperature stress seedling is put into 4 DEG C of phytotrons, samples after 0h, 1h, 6h, 24h; Flooding stress is placed in by seedling to fill in the four sides light transmission container of water, samples after 0h, 6h, 12h, 24h; Injury process carries out physical abuse with tweezers to seedling, samples after 0h, 1h, 6h, 12h; Oxidative stress is that seedling root is immersed in 1%H
2o
2in solution, sample after 0h, 1h, 3h, 12h; Dormin (ABA) process is behind the uniform spray water rice plants surface of dormin (ABA) with 100 μMs and is affixed by seedling root, samples after 0h, 0.5h, 3h, 6h.Tissue expression spectrum analysis still for material, gets the tissue sample (callus, root, stem apex, leaf, leaf sheath, 1cm fringe, 5cm fringe, 10cm fringe, gynoecium, stamen, embryo) of each developmental stage with japonica rice variety " in spend 11 ".The extraction of total serum IgE adopts TRIZOL reagent (purchased from Invitrogen company) to extract, extracting method is according to above-mentioned TRIZOL reagent specification sheets), utilize ThermoScript II SSIII (purchased from Invitrogen company) by its reverse transcription synthesis cDNA (method is according to Invitrogen company ThermoScript II reagent specification sheets), reaction conditions is: 65 DEG C of 5min, 50 DEG C of 120min, 70 DEG C of 10min.The cDNA synthesized with above-mentioned reverse transcription is template, carries out special pcr amplification with primer (SN1F:5 '-GCACGTCTTCGAGCACAAAA-3 ' and SN1R:5 '-TGGCTGGTAAGGCCATTCTG-3 ') to SN1 gene.Use primer UF (5 '-AACCAGCTGAGGCCCAAGA-3 ') and UR (5 '-ACGATTGATTTAACCAGTCCATGA-3 ') to carry out specific amplification to paddy rice Ubiquitin gene, to carry out quantitative analysis as internal reference simultaneously.Reaction conditions is: 95 DEG C of 5min; 95 DEG C of 10sec, 60 DEG C of 5sec, 72 DEG C of 34sec, 45 circulations.Fluoroscopic examination real-time quantitative analysis is carried out in reaction process.Result shows, SN1 promotor (its sequence is as shown in SEQ NO:1) is induced SN1 gene to rise and expressed (Fig. 2) under arid, high salt, high temperature, oxidative stress and ABA process.Distribution expression pattern analyzing and testing finds that SN1 all has expression, expression amount higher (Fig. 3) in root, leaf and stamen in each histoorgan.
3, the structure of SN1P promoters driven gus reporter gene carrier and genetic transformation
In order to analyze the function of SN1P promotor, applicant constructs SN1P and drives the expression vector of gus reporter gene and be transformed in japonica rice variety and spend in No. 11, from the function of this gene promoter of GUS activity research of transgenic line.
SN1P drives the expression vector establishment method of gus reporter gene as follows: first carry out SalI single endonuclease digestion to the pGEM-SN1P plasmid obtained in embodiment 1, reclaims exogenous sequences.Simultaneously, the enzyme that uses the same method cuts GUS expression vector DX2181G, and (Fig. 4 is shown in by DX2181G vector plasmid collection of illustrative plates, DX2181G reconstructs by the Genetic Transformation in Higher Plants carrier pCAMBIA1301 basis commonly used in the world, Agrobacterium-mediated genetic transformation carrier containing gus reporter gene), enzyme cuts complete, with chloroform: primary isoamyl alcohol (volume ratio 24: 1) extracting, purifying digestion products.Ligation is done, thereafter transformation of E. coli Top10 (this intestinal bacteria Top10 bacterial strain is purchased from Invitrogen company) with the DX2181G carrier that the endonuclease bamhi and enzyme that comprise SN1P promotor are cut.Cut screening positive clone by enzyme and verify exogenous sequences direction of insertion, (the SN1P promoter sequence on carrier is exactly the nucleotide sequence shown in SEQ ID NO:1 the recombinant plasmid vector of acquisition to be named as SN1P-DX2181G, sequence length is 2233bp, and the recombinant plasmid vector SN1P-DX2181G Vector map that the present invention builds is shown in Fig. 5).
Recombinant plasmid vector SN1P-DX2181G is transferred in rice varieties " in spend 11 " by agriculture bacillus mediated rice transformation method (its concrete step of converting is as described below), through preculture, infect, callus that Dual culture, screening have damp mould rope 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 the method (Hiei etc. of people's reports such as Hiei, Efficient transformation of rice, Oryza sativa L., mediated by Agrobacterium and sequenceanalysis of the boundaries of the T-DNA.Plant J, 1994,6:271-282) on basis improvement carry out.
The concrete genetic transformation step of the present embodiment is as follows:
(1) electricity transforms: by the destination carrier SN1P-DX2181G (plasmid map is shown in Fig. 5) of final promoters driven gus reporter gene, use 1800v voltage, electricity is transformed into Agrobacterium EHA105 bacterial strain, be coated onto on the conventional LA substratum selected with corresponding resistance, filter out positive colony, for following conversion callus.
(2) callus induction: shelled by the rice paddy seed of maturation " in spend 11 ", then uses the ethanolic soln process 1 minute of 70%, 0.15% mercury chloride (HgCl successively
2) solution seed-coat sterilizes 15 minutes; Seed is cleaned 4-5 time again with aqua sterilisa, sterile seed is placed on (composition sees below) on inducing culture and cultivates acquisition callus, gained callus is transferred to inducing culture (composition sees below) and is placed in dark place's cultivation 4 weeks, culture temperature 25 ± 1 DEG C.
(3) callus subculture: select glassy yellow, consolidation and the embryo callus subculture of relatively dry, is put in the upper dark lower cultivation of callus subculture medium (composition sees below) 2 weeks, culture temperature 25 ± 1 DEG C.
(4) preculture: select consolidation and the embryo callus subculture of relatively dry, is put in the upper dark lower cultivation of pre-culture medium (composition sees below) 2 weeks, culture temperature 25 ± 1 DEG C.
(5) Agrobacterium is cultivated: on the LA substratum selected with corresponding resistance, (composition sees below) preculture Agrobacterium EHA105 (derives from Australian CAMBIA laboratory commercial strains, carry promoter vector SN1P-DX2181G of the present invention, Vector map is shown in Fig. 5) two days, culture temperature 28 DEG C; Described Agrobacterium is transferred to suspension medium (composition sees below) inner, 2-3 hour cultivated by 28 DEG C of shaking tables.
(6) Agrobacterium is infected: pre-incubated callus be transferred in the good bottle of sterilizing; Regulate the suspension of Agrobacterium to OD
6000.8-1.0; Callus is soaked 30 minutes in agrobacterium suspension; Transfer callus blots to the good filter paper of sterilizing; Then the upper cultivation of Dual culture base (composition sees below) 3 days are placed on, culture temperature 19-20 DEG C.
(7) callus washing and selection are cultivated: aqua sterilisa washing callus is to cannot see Agrobacterium; To be immersed in the aqua sterilisa containing 400ppm Pyocianil (CN) 30 minutes; Transfer callus blots to the good filter paper of sterilizing; Shift callus again to select 2-3 time to Selective agar medium (composition sees below) is upper, within each 2 weeks, (the Pyocianil concentration of screening is for the first time 400ppm, second time and later Pyocianil concentration are 250ppm, and Totomycin concentration is 250ppm).
(8) break up: kanamycin-resistant callus tissue is transferred to the upper dark place of pre-division culture medium (composition sees below) and cultivates 5-7 week; Shift the callus of pre-differentiation culture to (composition sees below) on division culture medium, cultivate under illumination (4000lx), culture temperature 26 DEG C.
(9) take root: cut the root that differentiation phase produces; Then transfer them in root media and cultivate 2-3 week under illumination (4000lx), culture temperature 26 DEG C.
(10) transplant: wash the remaining medium on root off, the seedling with good root system is proceeded to greenhouse, kept moisture moistening at initial several days simultaneously.
Nutrient media components and formula thereof: (1) reagent and solution abbreviation: the abbreviation of the plant hormone in the present invention used by substratum is expressed as follows: 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 (CaseinEnzymatic Hydrolysate, caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); N6max (a large amount of ingredient solution of N6); N6mix (N6 trace ingredients solution); MSmax (a large amount of ingredient solution of MS); MSmix (MS trace ingredients solution).(2) main solution formula:
1) preparation of N6 substratum macroelement mother liquor [10 times of concentrated solutions (10X)]:
Dissolve one by one, be then settled to 1000ml under room temperature.
2) preparation of N6 substratum trace element mother liquor [100 times of concentrated solutions (100X)]
Dissolve under room temperature and be settled to 1000ml.
3) molysite (Fe
2eDTA) preparation of stock solution (100X)
Prepare 800ml distilled water and be heated to 70 DEG C, adding b diammonium disodium edta (Na
2eDTA2H
2o) 3.73 grams, keep 2 hours in 70 DEG C of water-baths after fully dissolving, be settled to 1000ml, 4 DEG C save backup.
4) vitamins stock liquid (100X) preparation
Add water and be settled to 1000ml, 4 DEG C save backup.
5) preparation of MS substratum macroelement mother liquor (10X)
Dissolve under room temperature and be settled to 1000ml.
6) preparation of MS substratum trace element mother liquor (100X)
Dissolve under room temperature and be settled to 1000ml.
7) 2,4-D stock solutions, 6-BA stock solution, naphthylacetic acid (NAA) stock solution, indolylacetic acid (IAA) stock solution: 1 is mg/ml.
8) glucose storage liquid: 0.5g/ml.
9) preparation of AS stock solution: weigh AS0.392g, DMSO10ml.
(3) for the culture medium prescription of rice transformation
1) callus inducing medium
Adding distil water, to 900ml, 1N potassium hydroxide adjust ph to 5.9, boils and is settled to 1000ml, is dispensed into 50ml triangular flask (25ml/ bottle), sealing sterilizing.
2) subculture medium
Adding distil water, to 900ml, 1N potassium hydroxide adjust ph to 5.9, boils and is settled to 1000ml, is dispensed into 50ml triangular flask (25ml/ bottle), sealing sterilizing.
3) pre-culture medium
Adding distil water, to 250ml, 1N potassium hydroxide adjust ph to 5.6, seals sterilizing.Use front heating for dissolving substratum and add 5ml glucose storage liquid and 250 μ l AS stock solutions, (25ml/ ware) in culture dish is poured in packing into.
4) Dual culture base
Adding distil water, to 250ml, 1N potassium hydroxide adjust ph to 5.6, seals sterilizing.Use front heating for dissolving substratum and add 5ml glucose storage liquid and 250 μ l AS stock solutions, (the every ware of 25ml/) in culture dish is poured in packing into.
5) suspension medium
Adding distil water is to 100ml, and adjust ph, to 5.4, is dispensed in the triangular flask of two 100ml, sealing sterilizing.1ml glucose storage liquid and 100 μ l AS stock solutions are added before using.
6) Selective agar medium
Adding distil water is to 250ml, and adjust ph, to 6.0, seals sterilizing.Dissolve substratum before using, add 250 μ l HN and 400ppmCN, (25ml/ ware) in culture dish is poured in packing into.
7) pre-division culture medium
Adding distil water, to 250ml, 1N potassium hydroxide adjust ph to 5.9, seals sterilizing.Dissolve substratum before using, add 250 μ lHN and 200ppm CN, (25ml/ ware) in culture dish is poured in packing into.
8) division culture medium
Adding distil water is to 900ml, 1N potassium hydroxide adjust ph to 6.0.Boil and be settled to 1000ml, being dispensed into 50ml triangular flask (50ml/ bottle), sealing sterilizing.
9) root media
Adding distil water is to 900ml, 1N potassium hydroxide adjust ph to 5.8.Boil and be settled to 1000ml, being dispensed into and taking root (25ml/ pipe) in pipe, sealing sterilizing.
4, SN1P promoter expression Pattern recognition
SN1P promotor (see SEQ ID NO:1) is controlled the conversion carrier SN1P-DX2181G that gus reporter gene constructs and transform " in spend 11 ", at different developmental phases tissue site, GUS dyeing (100ml GUS prescription of its dyeing liquor: 0.1M Na has been carried out to transfer-gen plant
2hPO
4[pH7.0] 40ml, 0.5M EDTA2ml, Triton X-1001ml, K4 [Fe (CN)
6] 85mg, K3 [Fe (CN)
6] 66mg, X-Gluc100mg, paraxin 10mg, methyl alcohol 20ml, distilled water 37ml).By GUS tissue staining (dyeing process: transfer-gen plant different tissues organ staining fluid is dyeed, spend the night in 37 DEG C of thermostat containers, observe after adding destainer process after discarding staining fluid, take pictures), confirmation gus reporter gene is respectively organized at callus, blade, the tip of a leaf, auricle, stem, root, clever shell and gynoecium etc. under normal growing conditions all higher expression (see Fig. 6).
5, SN1P promotor adverse circumstance induced activity qualification
The abduction delivering that embodiment of the present invention are exactly the quantitative and qualitative analysis gus gene that detects SN1P promoters driven in SN1P-DX2181G transgenic line under adverse circumstance comprises the oxidative stress that arid, high temperature, waterlogging and methyl viologen (MV) simulate is active, thus verifies the function of this gene promoter.Concrete steps are as follows:
The callus of the SN1P-DX2181G vector paddy rice " in spend No. 11 " adopting the present invention to build, third time screening stage get kanamycin-resistant callus tissue sample carry out normal condition respectively under and the mensuration of GUS staining analysis under stress conditions and GUS activity.Wherein arid (drought) is coerced is that kanamycin-resistant callus tissue is placed in seasoning on clean filter paper; It is kanamycin-resistant callus tissue is placed in 42 DEG C of thermostat containers that high temperature (heat) is coerced; The oxidative stress that methyl viologen (MV) is simulated is immersed in the MV solution of 2 μMs by kanamycin-resistant callus tissue, samples after coercing 3h.
GUS dyeing process is identical with embodiment 4.GUS active level analytical procedure is as follows: sample liquid nitrogen grind away, by GUS extract (50mM Na
2hPO
4, pH7.0,10mM β-mercaptoethanol, 10mM Na
2eDTA, 0.1%Sarkosyl, 0.1%Triton-100) extracting total protein, takes out a certain amount of albumen and carries out the analysis of GUS active level from sample.The GUS being obtained raw sample by analysis of fluorescence result is more alive than enzyme.Concrete steps are as follows: by Bradford method (see A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem, 1976,72:248-254) record the total protein concentration of sample, the total protein of the equal in quality of certain volume is measured again, with gross protein extract, 0.4ml GUS Extraction buffer (the 50mM Na of identical amount according to concentration pipettor
2hPO
4[pH7.0], 10mM β-mercaptoethanol, 10mMNa
2-EDTA, 0.1%sarkosyl, 1%Triton X-100), the 10 miniature ketone-glucuronide (MUG) of μ l40mM substrate 4-methyl (4-methylumbelifferyl-β-D-glucuronide) within 37 DEG C of water-bath 1h after, add 1.6ml reaction terminating liquid (0.2MNa
2cO
3); Each reaction arranges three repetitions.Each sample is measured at exciting light 365nm, the fluorescent value at utilizing emitted light 455nm place, simultaneously using the fluorescent value of 50nM MU as reference with Tecan grating type microplate reader infinite M200.And then calculated the ratio enzyme work of gus protein in each sample by above data, i.e. the amount (unit pmol MU/min/ μ g protein) of 1 μ g total protein per minute reaction substrate MU.
GUS coloration result shows, the expression of the gus reporter gene under SN1P promoters driven can be detected, but expression amount is in lower level under normal condition in kanamycin-resistant callus tissue; And after the oxidative stress that arid, high temperature and methyl viologen (MV) are simulated, the expression amount of gus reporter gene significantly rises (see Fig. 7).The result of gus protein active level analysis shows, SN1P promotor controls the induced strong that lower gus protein activity is subject to the oxidative stress of arid, high temperature and methyl viologen simulation, in transgenic resistance callus the activity of gus protein after the oxidative stress that arid, high temperature and methyl viologen are simulated be coerce respectively before about 11 times, 8 times and 12 times (as Fig. 8).This result confirms that SN1P promotor is a multiple abiotic stress inducible promoter, and the stress inducibility that may be used for target gene is expressed.
Sequence table
Claims (1)
1. the paddy rice Stress response type promotor OsSN1P of a kind of separation as shown in sequence table SEQ ID NO:1 at paddy gene to the application in the specifically expressing under the oxidative stress of arid, high temperature, waterlogging and methyl viologen simulation.
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