CN103421785A - Identification and utilization of low-temperature induced paddy rice promoter PC1 - Google Patents
Identification and utilization of low-temperature induced paddy rice promoter PC1 Download PDFInfo
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Abstract
The invention belongs to the technical field of paddy rice gene engineering, and particularly relates to application of a low-temperature induced paddy rice promoter PC1 which is obtained through separation, clone and function verification to the low-temperature resistant genetic improvement of paddy rice. The nucleotide sequence of the promoter is shown in SEQ ID NO: 1. Stress expression profile data analysis shows that the PC1 gene is subject to strong low-temperature inducible expression. The low-temperature induced paddy rice promoter PC1 is obtained through cloning by adopting the PCR method, a DX2181GFP-a-PC1 expression vector is built, enzyme activity reporting the gene protein GUS of transgenic plants under low-temperature stress is detected through transformation of paddy rice middle flower 11 and tobacco, and that the promoter is subject to strong low-temperature inducible expression is testified.
Description
Technical field
The invention belongs to the plant gene engineering technology field.Relate to isolation identification and the application of the special inducible promoter of a kind of plant adverse circumstance.By separating and identify the promotor of a paddy rice low inducible genes, can be applied to, in the genetically engineered of plant, particularly improve the purpose of plant frigostabile to reach genetic modification of plants.
Background technology
Paddy rice is most important food crop, and the whole world approximately has population more than half to using paddy rice as main food, and plantation is worldwide all arranged.It is a kind of optical-and thermal-sensitive crop that originates from Perenniporia martius.The envrionment conditions that the plantation of paddy rice requires is harsher.In high latitude, temperate zone and semi-tropical very large regional extent, low temperature is that the main environment of restriction rice yield is coerced, and particularly regional time in early spring at these, rice seedlings damages to plants caused by sudden drop in temperature especially responsive to chilling temperature, can cause rice seedlings poor growth, Huang Miao, suppress to tiller.In South Asia and South East Asia, the soil that surpasses 70,000,000 hectares meets with such low temperature stress often.And meet with low temperature at Rise's boot period, it is also the havoc to rice yield.Along with molecular biological development, transgenosis has become the effective means of research functional genomics, and just progressively obtains in recent years feasibility study and generally accept by transgenosis approach improvement stress resistance of plant.There is the lot of documents report can improve to a certain extent the resistance (Dubouzet etc. of plant by overexpression Stress response genes involved in plant, O OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt-and cold-responsive gene expression.Plant J33,751-763.2003; Wang etc., Overexpression of a rice OsDREB1F gene increases salt, drought, and low temperature tolerance in both Arabidopsis and rice.Plant molecular biology67,589-602.2008; Hu etc., Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice.Plant molecular biology67,169-181.2008; Ye etc., Identification and expression profiling analysis of TIFY family genes involved in stress and phytohormone responses in rice.Plant molecular biology71,291-305.2009.But the common use of these overexpressions in report is all constitutive promoter, although constitutive promoter overexpression effect is high, usually can be accompanied by inevitable negative effect, as plant-growth and growth are had a negative impact.Although the promotor (Yamaguchi-Shinozaki etc. that had bibliographical information to isolate from plant to be subject to Stress response, Characterization of the expression of a desiccation-responsive rd29gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants.Mol Gen Genet23:331-340,1993; Kasuga etc., A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought-and low-temperature stress tolerance in tobacco by gene transfer.Plant Cell Physiol45:346-350,2004; Xiao etc., Over-expression of a LEA gene in rice improves drought resistance under the field conditions.Theor Appl Genet115:35-46,2007), even but rare very strong adverse circumstance inducible promoter is applied to plant genetic engineering in important food crop paddy rice.The PC1 promotor is the promotor that expressed by the low temperature induced strong.
The present invention identifies a rice starter PC1 who is subject to the low temperature induced strong, by gene adverse circumstance express spectra and promoter activity quantitative analysis, show, this promotor can be used for to the expression of resistance genes involved crops such as paddy rice, thereby effectively improve the degeneration-resistant border performance of plant.
Summary of the invention
The objective of the invention is the clone, identify a plant endogenous promotor that is subject to the low temperature stress abduction delivering, and utilize this promotor to build expression vector, by genetic transforming method, reach and control the purpose that target gene is expressed by low temperature induction specifically.
The present invention implements by the following technical programs:
At first be the promotor of separating the candidate gene that is subject to the expression of low temperature induced strong.Selected candidate gene is the paddy rice native gene, and grass institute is special, its concrete function also be not in the news (KOME annotation AK061293).This gene is spent seedling stage of 11 and is subject to strong rising abduction delivering (as Fig. 2) in low temperature stress in rice varieties.The promotor of this gene separated derives from rice varieties " Japan is fine ", called after PC1.The PC1 promotor is to have the sequence shown in base 1-1801 position in accompanying drawing 1, contain the ABA response element ABRELATERD1 (Simpson etc. relevant to adverse circumstance in 243-247, the 1429-1434 base site of this sequence, Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence.Plant J33:259-270,2003, Nakashima etc., Transcriptional regulation of ABI3-and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of rabidopsis.Plant Mol Biol.60:51-68, 2006), 242-248 in this sequence, 1484-1490, 1630-1636, 1747-1753 base site is contained to adverse circumstance and calcium and is regulated and controled relevant response element ABRERATCAL (Kaplan etc., Rapid Transcriptome Changes Induced by Cytosolic Ca2+Transients Reveal ABRE-Related Sequences as Ca2+-Responsive cis Elements in Arabidopsis.Plant Cell.18:2733-2748, 2006), 1303-1308 in this sequence, (the Dunn etc. with low temperature response associated responses element LTRE1HVBLT49 are contained in 1522-1527 base site, Identification of promoter elements in a low-temperature-responsive gene (blt4.9) from barley (Hordeumvulgare L.) Plant Mol Biol38:551-564, 1998), contain (the Baker etc. with low temperature response associated responses element LTRECOREATCOR15 in the 1432-1436 of this sequence base site, The 5 '-region ofArabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought-and ABA-regulated gene expression.Plant Mol Biol24:701-713, 1994, Jiang etc., Requirement of a CCGAC cis-acting element for cold induction of the BN115 gene from winter Brassica napus Plant Mol Biol30:679-684,1996, Busk etc., Regulation ofabscisic acid-induced transcription Plant Mol Biol37:425-435,1998, Kim etc., Light signalling mediated by phytochrome plays an important role in cold-induced gene expression through the C-repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana Plant J29:693-704,2002), contain the response element MYB2CONSENSUSAT (Abe etc. relevant to dehydration in 1610-1615 base site, Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling.Plant Cell15:63-78,2003), contain the response element MYCATERD1 (Simpson etc. relevant to dehydration in the 81-86 of this sequence base site, Two different novel cis-acting elements oferd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence.Plant J33:259-270,2003, Tran etc., Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress.Plant Cell16:2481-2498, 2004), 81-86 in this sequence, 242-247, 307-312, 357-362, 377-382, 667-672, 693-698, 1286-1291, 1600-1605, response element MYCCONSENSUSAT (the Oh etc. relevant to dehydration are contained in 1610-1615 base site, Arabidopsis CBF3/DREB1Aand ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth..Plant Physiology138:341-351, 2005, Chinnusamy etc., Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants.J Exp Bot.55:225-236,2004).
Promotor PC1 provided by the present invention contains in zone a plurality of cis-acting elements relevant to adverse circumstance (as shown in Figure 1), can play responsing reaction to adverse circumstance, dormin (ABA), low temperature, dehydration specifically.Spend 11 in the GUS expression vector rice transformation acceptor kind that the applicant utilizes the PC1 promotor to build, can when transfer-gen plant is subject to environment stress, induce consumingly the expression of reporter gene GUS.Effect of the present invention refers to embodiment.
More detailed technical scheme is as described below:
A kind of isolated paddy DNA molecule, its nucleotide sequence as shown in SEQ ID NO:1 and Fig. 1, the nucleotide sequence that wherein promotor PC1 comprises the 1-1801 bit base in the sequence shown in Fig. 1.
The DNA molecular of described a kind of separation, except basic promoter element (TATA-box), also comprise the combination of a plurality of Stress response cis-acting elements (ABRELATERD1, ABRERATCAL, LTRE1HVBLT49, LTRECOREATCOR15, MYB2CONSENSUSAT, MYCATERD1, MYCCONSENSUSAT) in its zone.
Obviously, the content that the present invention asks for protection comprises following feature:
The paddy rice expression vector of all or part of sequence construct of described PC1 promotor.
The all or part of sequence construct paddy rice of described PC1 promotor expression vector, the method for cultivating the improvement plant by genetic transformation.
The all or part of sequence construct paddy rice of described PC1 promotor expression vector, the adversity resistant plant material of cultivating by genetic transformation.The material of described adversity resistant plant refers to plant, seed or cell clone.
According to above technical scheme, obviously, other people beyond applicant or applicant can utilize PC1 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 that comprises paddy rice, wheat, corn etc., can certainly be to comprise some other important cash crop, for example corn, cotton, rape or tomato.
The accompanying drawing explanation
Sequence table SEQ ID NO:1 is the nucleotide sequence of the rice starter PC1 that clones of the present invention, and sequence length is 1801, and two of this sequence is designed with primer (that is: 1-18bp, 1784-1801bp section).
Fig. 1: the nucleotide sequence audio-visual picture that is the present invention's PC1 promotor of cloning.In figure: the underscore sequence is amplification PC1 promotor primer sequence used.What shadow zone showed is basic promoter element sequence.Italic boldface letter to add the wavy line display space be Stress response element core sequence.
Fig. 2: demonstration be the PC1 promotor Rice Seedlings kind " in spend 11 ", can be by abduction delivering consumingly when plant is subject to low temperature stress.5 time points are successively: before 0H, low temperature stress; 6H, low temperature (4 ℃, following low temperature all refers to this temperature) are coerced 6 hours; 12H, low temperature stress 12 hours; 18H, low temperature stress 18 hours; Under R24H, 28 ℃, recover 24 hours.
Fig. 3: be the plasmid figure of empty carrier DX2181GFP-a and the DX2181GFP-a-PC1 expression vector figure that the present invention builds.This DX2181GFP-a-PC1 carrier is containing hygromycin resistance screening-gene (HRG), and promotor PC1 is fused to gus gene 5 ' end non-translational region.The plasmid figure that the upper figure of Fig. 3 is empty carrier DX2181GFP-a, figure below of Fig. 3 is the DX2181GFP-a-PC1 expression vector figure that the present invention builds.
Fig. 4: demonstration be the GUS activity of gus gene under 4 ℃ of cold Stress treatments under the PC1 promotor is controlled.DX2181 DX2181GFP-a empty carrier spends 11 in transforming, in contrast family; PC1-12, PC1-15, PC1-17, PC1-22 and PC1-30 spend independently transgenic positive family of 11 5 of obtaining during DX2181GFP-a-PC1 transforms.3 time points are successively: before c0, low temperature stress; C6, low temperature stress 6 hours; C24, low temperature stress 24 hours.
Fig. 5: demonstration be the GUS activity of gus gene transformation of tobacco under 4 ℃ of cold Stress treatments under the PC1 promotor is controlled.DX2181-1 and DX2181-2 are the tobaccos that the DX2181GFP-a empty carrier transforms, family in contrast, and PC1-1, PC1-2 and PC1-3 are independently tobaccos of 3 strains that transform of DX2181GFP-a-PC1.2 time points are successively: before c0, low temperature stress; C24, low temperature stress 24 hours.
Embodiment
1, PC1 isolation of promoter and evaluation
By rice varieties " in spend 11 ", (Chinese Academy of Agricultural Sciences's crop investigations provides, the kind of openly applying) low temperature induction gene expression spectrum analysis, found a gene that is subject to low temperature induced strong (low temperature stress later stage expression amount improves more than 100 times), its TIGR (http://rice.plantbiology.msu.edu/) ID is LOC03g19070, be named as PC1, full-length cDNA corresponding in KOME database (http://cdna01.dna.affrc.go.jp/cDNA/) is numbered AK061293.
Next step is exactly the promotor of separating this gene.Concrete steps are as follows: the scope that finds the genome sequence (NC_008394.4) of the japonica rice that this gene pairs answers " Japan is fine " and choose the transcription initiation site upstream 2.0Kb of this gene at NCBI (http://www.ncbi.nlm.nih.gov/) is carried out pcr amplification as candidate's promoter region.Design primer PC1-F (5 '-CAG
CTGCAGGCTGCTTGCCTTGACTGA-3 ') and PC1-R (5 '-CAG
GGATCCCGCTCTTGCTTTCGCCTA-3 '), and at primer 5 ' end add restriction enzyme site PstI and BamHI (underline and mean by italic, three bases before restriction enzyme site are the protection base).At first utilize primer PC1-F and PC1-R with " Japan is fine " genomic dna (CTAB method extracting, Zhang etc., genetic diversity and differentiation of indica anjaponica rice detected bv RFLP analysis, 1992, TheorAppl Genet, 83,495-499) for template, increased, reaction system is that 20uL GC buffer I system is (purchased from precious biotechnology (Dalian) company limited, be TaKaRa company), reaction conditions is: 94 ℃ of denaturation 5min; 94 ℃ of 30sec, 56 ℃ of 30sec, 72 ℃ of 2mim, 32 circulations; 72 ℃ are extended 7min.The PCR product is connected on pGEM-T Easy carrier, screening positive clone order-checking (ABI3730 sequenator, Applied Biosystem, order-checking completes at national plant gene center [Wuhan]), result confirms: institute's extension increasing sequence is the PC1 promoter sequence of expection, and it comprises a plurality of dehydrations, low temperature response and ABA Stress response cis-acting elements (as Fig. 1).
2, detect the abduction delivering of paddy rice native gene PC1
The rice varieties " in spend 11 " of take is material, plants in the earth of booth, and 3 leaves carry out 4 ℃ of low temperature stress during the phase.Get and coerce front c0 time point sample, coerce and within 6 hours, get c6 time point sample, coerce and within 12 hours, get c12 time point sample, coerce and within 18 hours, get c18 time point sample, coerce and within 24 hours, get c24 time point sample, coerce rear recovery and within 12 hours, get r12 time point sample.Total RNA adopts TRIZOL reagent (purchased from Invitrogen company) to extract (extracting method is according to above-mentioned TRIZOL reagent specification sheets), utilizes ThermoScript II SSIII (purchased from Invitrogen company) by the synthetic cDNA (method is according to Invitrogen company ThermoScript II reagent specification sheets) of its reverse transcription.The synthetic cDNA of the above-mentioned reverse transcription of take is template, with primer (5 '-GGAGTTCATCGCCAAATTCC-3 ' and 5 '-CGTTGTACTGCAGCCATGAGA-3 '), the PC1 gene is carried out to special pcr amplification (the long 68bp of amplified production).Use primer (5 '-TGGCATCTCTCAGCACATTCC-3 ' and 5 '-TGCACAATGGATGGGTCAGA-3 ') to do specific amplified (the long 76bp of amplified production) to paddy rice Actin1 gene simultaneously, using and carry out quantitative analysis as internal reference.Reaction conditions is: 95 ℃ of 10sec, 95 ℃ of 5sec, 60 ℃ of 34sec, 40 circulations.Carry out the fluoroscopic examination real-time quantitative analysis in reaction process.Result shows that this promotor is subject to strong rising abduction delivering in the seedling stage of rice varieties " in spend 11 " in low temperature stress, coerce 24 hours point rising multiples and be about 170 times (as Fig. 2).
3, the rice transformation of PC1 promoters driven reporter gene
Embodiment of the present invention important step is exactly build the gus gene expression vector of PC1 promotor and be transformed in rice varieties " in spend 11 ", detects quantitatively the abduction delivering activity of the low temperature stress of PC1 promotor.Concrete operations are as follows:
At first the PCR product of the PC1 promotor of separation in implementation step 1 is connected into to pGEM-T Easy carrier (purchased from Promega company), transforms bacillus coli DH 5 alpha (purchased from Promega company) and also obtain positive colony.Cut from pGEM-T Easy positive colony recovery PC1 and be connected to again GUS expression vector DX2181GFP-a (seeing the upper figure of Fig. 3) by PstI and BamHI enzyme, after enzyme is cut the checking positive colony, import to rice varieties " in spend in 11 " by agriculture bacillus mediated rice transformation system, through preculture, infect, cultivate altogether, callus that screening has hygromycin resistance, break up, take root, practice transplantation of seedlings, obtain transfer-gen plant.Agriculture bacillus mediated paddy rice (japonica rice subspecies) genetic conversion system is mainly applied the method for the people such as Hiei report (referring to Efficient transformation of rice, Oryza sativa L., mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, 1994, Plant Journal6:271-282) also improve on its basis optimization.To not connect the DX2181GFP-a empty carrier rice transformation kind " in spend in 11 " of external source fragment in contrast simultaneously.Key step and reagent are as follows:
(1) reagent and solution abbreviation
In the present invention, the abbreviation of substratum plant hormone used 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 (Casein Enzymatic Hydrolysate, caseinhydrolysate); HN (Hygromycin B, Totomycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); 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 concentrated solutions (10X)]:
Dissolve one by one, then under room temperature, be settled to 1000ml.
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 the 800ml distilled water and be heated to 70 ℃, adding b diammonium disodium edta (Na
2EDTA2H
2O) 3.73 grams keep 2 hours after fully dissolving in 70 ℃ of water-baths, are settled to 1000ml, and 4 ℃ save backup.
4) VITAMIN stock solution (100X) preparation
Add water and be settled to 1000ml, 4 ℃ 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, the preparation of 4-D stock solution (1mg/ml):
8) preparation of 6-BA stock solution (1mg/ml):
Weigh 6-BA100mg, dissolve 5 minutes with 1ml1N potassium hydroxide, be settled to 100ml, room temperature preservation after then adding 10ml distilled water dissolve complete.
9) preparation of naphthylacetic acid (NAA) stock solution (1mg/ml):
Weigh NAA100mg, dissolve 5 minutes with 1ml1N potassium hydroxide, be settled to 100ml after then adding 10ml distilled water dissolve complete, 4 ℃ save backup.
10) preparation of indolylacetic acid (IAA) stock solution (1mg/ml):
Weigh IAA100mg, dissolve 5 minutes with 1ml1N potassium hydroxide, be settled to 100ml, 4 ℃ of standby 300ml distilled water and ferric sulfate (FeSO of adding of preservations after then adding 10ml distilled water dissolve complete in a large triangular flask
47H
2O) 2.78g.In another large triangular flask, add 300ml distilled water to use.
11) preparation of glucose stock solution (0.5g/ml):
Weigh glucose 125g, then with distilled water, dissolve and be settled to 250ml, after sterilizing, 4 ℃ save backup.
12) preparation of AS stock solution:
Weigh AS0.392g, DMSO10ml, divide and be filled in the 1.5ml centrifuge tube, and 4 ℃ save backup.
13) 1N potassium hydroxide stock solution
Weigh potassium hydroxide 5.6g, and dissolve and be settled to 100ml with distilled water, room temperature preservation is standby.
(3) for the culture medium prescription of rice transformation
1) inducing culture
Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides and installs to 50ml triangular flask (25ml/ bottle), the sealing sterilizing.
2) subculture medium
Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides and installs to 50ml triangular flask (25ml/ bottle), the sealing sterilizing.
3) pre-culture medium
Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, the sealing sterilizing.
Use front heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (25ml/ ware) in culture dish poured in packing into.
4) be total to substratum
Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, the sealing sterilizing.
Use front heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (the every ware of 25ml/) in culture dish poured in packing into.
5) suspension medium
Adding distil water, to 100ml, is regulated pH value to 5.4, divides and installs in the triangular flask of two 100ml, the sealing sterilizing.
Add 1ml glucose stock solution and 100 μ l AS stock solutions before use.
6) select substratum
Adding distil water, to 250ml, is regulated pH value to 6.0, the sealing sterilizing.
Dissolve substratum before using, add 250 μ l HN and 400ppm CN, (25ml/ ware) in culture dish poured in packing into.
7) pre-division culture medium
Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.9, the sealing sterilizing.
Dissolve substratum before using, add 250 μ l HN and 200ppm CN, (25ml/ ware) in culture dish poured in packing into.
8) division culture medium
Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 6.0.
Boil and be settled to 1000ml, dividing and install to 50ml triangular flask (50ml/ bottle), the sealing sterilizing.
9) root media
Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.8.
Boil and be settled to 1000ml, dividing and install to (25ml/ pipe) in the pipe of taking root, the sealing sterilizing.
(4) agriculture bacillus mediated genetic transformation step
3.1 callus of induce
(1) ripe rice paddy seed is shelled, then use successively 70% Ethanol Treatment 1 minute, 0.15% mercury chloride (HgCl
2) seed-coat sterilization 15 minutes;
(2) wash seed 4-5 time with sterilizing;
(3) seed is placed on inducing culture;
(4) postvaccinal substratum is placed in to dark place and cultivates 4 weeks, 25 ± 1 ℃ of temperature.
3.2 callus subculture
Select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in dark lower the cultivation 2 weeks on subculture medium, 25 ± 1 ℃ of temperature.
3.3 preculture
Select the embryo callus subculture of consolidation and relatively dry, be put in dark lower the cultivation 2 weeks on pre-culture medium, 25 ± 1 ℃ of temperature.
3.4 Agrobacterium is cultivated
(1) preculture Agrobacterium EHA105 (deriving from CAMBIA, commercial bacterial strain) two days on the LA substratum of selecting with corresponding resistance, 28 ℃ of temperature;
(2) Agrobacterium is transferred in suspension medium, cultivates 2-3 hour on 28 ℃ of shaking tables.
3.5 Agrobacterium is infected
(1) pre-incubated callus is transferred in the bottle that sterilizing is good;
(2) regulate the suspension of Agrobacterium to OD
6000.8-1.0;
(3) callus is soaked 30 minutes in agrobacterium suspension;
(4) shift callus blots to the good filter paper of sterilizing; Then be placed on common substratum and cultivate 2 days, temperature 19-20 ℃.
3.6 callus washing and selection are cultivated
(1) aqua sterilisa washing callus is to cannot see Agrobacterium;
(2) be immersed in containing in the aqua sterilisa of 400ppm Pyocianil (CN) 30 minutes;
(3) shift callus blots to the good filter paper of sterilizing;
(4) shift callus and select 2-3 time on substratum to selecting, each 2 weeks.(hygromycin selection concentration is 400ppm for the first time, is 250ppm for the second time later)
3.7 differentiation
Kanamycin-resistant callus tissue is transferred to dark place on pre-division culture medium and cultivates 5-7 week;
Shift the callus of pre-differentiation culture to division culture medium, cultivate under illumination, 26 ℃ of temperature.
3.8 take root
(1) cut the root that differentiation phase produces;
(2) then transfer them in root media, cultivate 2-3 week, 26 ℃ of temperature under illumination.
3.9 transplant
Wash the residual substratum on root off, the seedling that will have good root system proceeds to greenhouse, at initial several days, keeps moisture moistening simultaneously.
4, the evaluation of promotor PC1 low temperature induction expression activity
The applicant adopts DX2181GFP-a-PC1 carrier (as Fig. 3) rice transformation " in spend in 11 " of structure, obtains 13 strains of transgenic positive plant; The DX2181GFP-a empty carrier rice transformation " in spend in 11 " that does not connect the external source fragment obtains 8 strains of transgenic positive plant.Choose 5 and turn the positive family of promotor and 1 and turn empty carrier positive control family and T2 is carried out to Totomycin (HN) resistance screening for seed germinate, the young plant kind of germination is grown to 4 leaf phases in the earth of little red bucket and do 4 ℃ of low temperature stress.Wherein the low temperature stress method as described in example 2 above, is got and is coerced the sample that front c0, x coerce 6 hours c6, coerce tri-time points of 24 hours c24.What every duplicate samples was got is the compound sample (being no less than 10 strains) of this family.
Sample liquid nitrogen grind away, by GUS extract (50mM Na
2HPO4, pH7.0,10mM β-mercaptoethanol, 10mMNa
2EDTA, 0.1%Sarkosyl, 0.1%Triton-100) the extracting total protein, from sample, a certain amount of albumen of taking-up and is carried out the analysis of GUS active level.The GUS that obtains raw sample by the analysis of fluorescence result is more alive than enzyme.Concrete steps are as follows: by the Bradford method (referring to A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.1976, Anal Biochem, 72:248-254) record the total protein concentration of sample, measure again the total protein of the equal in quality of certain volume with pipettor according to concentration, with the gross protein extract of same amount+0.4mlGUS Extraction buffer+10 μ l40mM substrate MUG (4-methylumbelifferyl-β-D-glucuronide) after 37 ℃ of water-bath certain hours (in 1h), add 1.6ml reaction terminating liquid (0.2M Na
2CO
3), each reaction arranges three technology and repeats.Measure each sample at exciting light 365nm with Tecan grating type microplate reader infinite M200, the fluorescent value at utilizing emitted light 455nm place is usingd the fluorescent value of 50nM MU as reference simultaneously.And then live by the ratio enzyme that above data calculate gus protein in each sample, i.e. the amount of 1ug total protein per minute reaction substrate MU (the pmol MU/min/ug protein of unit).
Measure and find by the gus protein active level, the gus protein activity of PC1 promotor under controlling is subject to the induced strong of low temperature stress, in six independent transgenosis familys gus protein at low temperature stress approximately 3 to 11 times (as the Fig. 4) before being to coerce of the activity in the time of 24 hours.
5, the evaluation of the tobacco instantaneous conversion of PC1 promoters driven reporter gene and promotor PC1 low temperature induction expression activity
The applicant adopts DX2181GFP-a-PC1 carrier (as Fig. 3) transformation of tobacco of structure.Method for transformation is as follows: picking transforms the Agrobacterium that expression plasmid is arranged and is cloned in 1ml and contains in corresponding antibiotic LB, and 28 degree shaking table 250rpm cultivate 24 hours, in the LB that contains kantlex (Kanamycin, 50 μ g/ml1/1000 volume ratios add in LB) in 5ml, add 100 μ l0.5M MES, 2 μ l100mM AS, inoculate 50 μ l Agrobacterium bacterium liquid, and 28 degree shaking table 250rpm are cultured to OD
600=1.0 (approximately 12-18 hour), 4000rpm normal temperature is collected thalline in centrifugal 10 minutes, uses 10mM MgCl
2resuspended to OD
600=1.0, with every milliliter of bacterium liquid, add the ratio of 2 μ l to add 100mMAS, standing more than 3 hours, get to be in and grow vigorous period (about one month, do not bloom) this uncured tobacco (Nicotiana benthamiana), suck bacterium liquid with syringe, remove syringe needle, prop up face of blade with finger, bacterium liquid is permeated into from the reverse side of blade, if injection has some setbacks, manufacture a small wound on available syringe needle blades, then bacterium liquid is injected from wound, place 24-48 hour under normal condition, the time of the expression in order to ensure expression vector in the tobacco body and prolongation protein expression, the preparation that uses the same method contains tomato bushy stunt virus gene p19 (Olivier Voinnety, Susana Rivas, Pere Mestre and David Baulcombe.An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.The Plant Journal, 2003, 33:949-956.) Agrobacterium of overexpression vector, with inject again tobacco after the Agrobacterium equivalent volumes of goal gene is mixed, independently tobacco is in contrast in 2 strains that the protein expression time of this method transforms the DX2181GFP-a empty carrier after for injection in 3-7 days, the DX2181GFP-a-PC1 carrier transforms the tobacco that 3 strains are independently grown.As coercing front 0 moment (C0), then plant is put into to 4 ℃ of subzero treatment while transforming latter the 36th hour, get the blade sample (C24) of processing 24 hours.Sample protein method for extracting and GUS measuring method are as described in example 4 above.
Measure and find by the gus protein active level, the gus protein activity of PC1 promotor under controlling is subject to the induced strong of low temperature stress, the activity of gus protein when low temperature stress 24h approximately 4 to 10 times (as Fig. 5) before being to coerce in 3 independent transgenic tobacco plants.
Appendix:
GUS Extraction buffer (cumulative volume 1L uses the distilled water constant volume): pH=7.0
0.2M Na
2HPO
4 152.5mL；
0.2M NaH
2PO
4 97.5mL；
14.3M β-ME 700uL；
Na
2-EDTA·2H
2O 3.72g；
Triton-X 100 1mL。
Claims (2)
1. one kind by the promotor PC1 of the special abduction delivering of low temperature, and its nucleotide sequence is as shown in SEQ ID NO:1.
2. the application of promotor PC1 claimed in claim 1 in controlling gene is expressed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104611337A (en) * | 2015-02-21 | 2015-05-13 | 吉林农业大学 | Low-temperature inducible promoter PCPI and application thereof |
| CN111763672A (en) * | 2020-06-30 | 2020-10-13 | 安徽省农业科学院水稻研究所 | Rice low-temperature inducible expression promoter Poscold10 and application thereof |
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| US6479734B2 (en) * | 1998-06-10 | 2002-11-12 | Kyushu University | DNA fragment responsive to low temperatures and a plant transformed with the DNA fragment |
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| US6479734B2 (en) * | 1998-06-10 | 2002-11-12 | Kyushu University | DNA fragment responsive to low temperatures and a plant transformed with the DNA fragment |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611337A (en) * | 2015-02-21 | 2015-05-13 | 吉林农业大学 | Low-temperature inducible promoter PCPI and application thereof |
| CN104611337B (en) * | 2015-02-21 | 2018-02-13 | 吉林农业大学 | Cold-inducible promoter PCP1 and application |
| CN111763672A (en) * | 2020-06-30 | 2020-10-13 | 安徽省农业科学院水稻研究所 | Rice low-temperature inducible expression promoter Poscold10 and application thereof |
| CN111763672B (en) * | 2020-06-30 | 2022-03-25 | 安徽省农业科学院水稻研究所 | Rice low-temperature inducible expression promoter Poscold10 and application thereof |
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