CN102363782A - Rice histone deacetylases gene HDT701 promoter and application thereof - Google Patents
Rice histone deacetylases gene HDT701 promoter and application thereof Download PDFInfo
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Abstract
The invention discloses a rice histone deacetylases gene HDT701 promoter and an application thereof. The sequence of the promoter is one of the following nucleotide sequences: (a) the nucleotide sequence shown in the sequence table SEQ ID NO.1; (b) the nucleotide sequence which can be hybridized with a DNA sequence of 1) under rigorous conditions and has same function; (c) the nucleotide sequence which has more than 90% homology with the DNA sequence of a) or b) and also has the function of the promoter. According to the invention, the promoter of the HDT701 gene is cloned from the rice, wherein the cloned promoter is the rice histone deacetylases gene HDT701 promoter which can promote the expression of downstream gene in some organs of the rice, and the rice histone deacetylases gene HDT701 promoter is a tissue specificity promoter, so that the promoter can be used in the transgenic engineering, the expression products of the target gene can be accumulated at certain organ or tissue so as to increase local expression quantity, and simultaneously, unnecessary waste of plant nutrient can be reduced, consequently, the promoter has good application prospect in the transgenic engineering.
Description
Technical field:
The invention belongs to genetically engineered and rice breeding technology field; Be specifically related to a kind of clone-paddy rice histone deacetylase gene HDT701 promotor of paddy rice histone deacetylase gene HDT701 upstream of coding region promoter sequence, and the application of this promotor in transgenic paddy rice.
Background technology:
Promotor is meant the zone that can combine and form transcription initiation complex on the dna molecular with RNA polymerase, is usually located at the upper reaches of encoding sox.In promoter fragment, have a large amount of cis acting original papers, thereby transcription factor makes gene have the spatial and temporal expression specificity through combine transcribing of regulatory gene with the cis acting original paper.So the separation of promotor and functional analysis are the important contents of genetically engineered research.
The promotor of using always in the plant genetic engineering is divided into 3 types by its mode of action and function: composing type, organizing specific type and inducible promoter.Constitutive promoter in a organized way in all expression of promotor gene, have persistence, do not have the space-time specificity.In the genetically engineered commonly used to have at present commonly used in plant expression vector be constitutive promoter, like the Ubiquitin promotor of cauliflower mosaic virus CaMV 35S promoter, corn etc.But foreign gene continues in recipient plant, expression efficiently tends to cause that the form of plant changes, and influences plant and grows normally, even causes plant dead.
Organizing specific type promotor can make the expression product of goal gene in the position accumulation of certain organ or tissue, increases the local expression amount.The injury of having avoided gene overexpression that plant materials is caused like this also can be avoided the unnecessary waste of plant nutrition simultaneously.So the research of organizing specific type promotor receives much concern in recent years.
Comprise a lot of food crop along with the technological development of modern genetic engineering has obtained a lot of transgenic plant in recent years, but food-safety problem the popularization and the development of transgenic crop have but been restricted.Foreign gene is expressed in endosperm and is caused foreign protein to accumulate therein in the transgenic paddy rice, and being influences some transgenic paddy rices with fine quality to develop restricted principal element.To this phenomenon, utilizing organizing specific type promotor to make foreign gene only in vegetative organ, express and not express to reduce food safety risk in the human consumption part is an important channel that promotes that transgenic paddy rice and transgenic crop are promoted.
Summary of the invention:
First purpose of the present invention provides a kind of tissue-specific paddy rice histone deacetylase gene HDT701 promotor that has.
Paddy rice histone deacetylase gene HDT701 promotor of the present invention is characterized in that its sequence is one of following nucleotide sequences:
(a) nucleotide sequence shown in the SEQ ID NO.1 in the sequence table;
(b) under rigorous condition, can and have the nucleotide sequence of identical function with the dna sequence dna of (a) hybridization;
(c) with (a) or dna sequence dna (b) have the homology more than 90%, and also have the nucleotide sequence of promoter function.
Paddy rice histone deacetylase gene HDT701 promotor shown in SEQ ID NO.1 of the present invention, its cis acting original paper is analyzed as shown in table 1:
Table 1: paddy rice histone deacetylase gene HDT701 promotor cis acting original paper is analyzed
The present invention is inserted into the fusion expression vector of the MCS place formation of carrier PCAMBIA1391Z by the expression of HDT701 promotor startup gus gene with the paddy rice histone deacetylase gene HDT701 promotor shown in SEQ ID NO.1, obtains transfer-gen plant through agrobacterium-mediated transformation rice transformation mature embryo callus.Detect and GUS dyeing through PCR transgenic paddy rice; The result shows our successful being incorporated into the HDT701 promotor in the paddy gene; And HDT701 promoter-driven GUS gene all has expression in the organ of transgenic paddy rice except that endosperm; Explain that thus the sequence shown in SEQ ID NO.1 of the present invention is a promotor; Called after paddy rice histone deacetylase gene HDT701 promotor, this promotor starts gus gene all has expression in the organ of transgenic paddy rice except that endosperm, promptly in transgenic paddy rice, just in the part organ-tissue, express; This promotor specificity in a organized way is described thus, is a tissue-specific promoter.
Therefore, second purpose of the present invention provides a kind of expression vector, it is characterized in that, contains paddy rice histone deacetylase gene HDT701 promotor.
The 3rd purpose of the present invention provides a kind of host bacterium, it is characterized in that, contains above-mentioned expression vector.
Described host bacterium is preferably Agrobacterium EHA105.
The 4th purpose of the present invention provides a kind of paddy rice histone deacetylase gene HDT701 promotor and starting the application that the downstream goal gene is expressed in paddy rice.
The present invention clones the promotor of HDT701 gene from paddy rice: paddy rice histone deacetylase gene HDT701 promotor; This promotor can start in the part organ of downstream gene in paddy rice to be expressed; It is a tissue-specific promoter; Therefore can be applied in the transgenic engineering, the expression product of the goal gene that makes increases the local expression amount in certain organ or tissue's position accumulation; Also can avoid simultaneously the unnecessary waste of plant nutrition, therefore good prospects for application is arranged in transgenic engineering.
Description of drawings:
Fig. 1 is that the PCR product gel of HDT701 promotor reclaims detection figure, and M is the 2KB molecular weight marker, and 1 is purpose product band.
Fig. 2 is the structural representation of binary vector PCAMBIA1391Z.
Fig. 3 is that expression vector PCAMBIA1391Z-HDT701P structure completion enzyme is cut detection figure, and M is the 2KB molecular weight marker, and 1 is HindIII/speI enzyme slitting band, and 2 is HindIII/PstI enzyme slitting band.
Fig. 4 is that PCR detects positive transgenic rice plant figure; M is the 2KB molecular weight marker; With the 1-8 swimming lane is 8 rotaring gene plant blades; The wild-type rice leaf is the 9th swimming lane, and genetically modified 3,4,6, No. 7 paddy rice can both obtain the GUS purpose band of 399bp, wild-type do not obtain the purpose band as negative control.
Fig. 5 is the semi-quantitative expressed analysis demonstration figure of HDT701 gene in root, the leaf in generative phase, stem and the clever shell in paddy rice young root, spire, generative phase, and A is the HDT701 gene, and B is Actin gene (accession number is AK100267); 1 is seedling; 2 is stem, and 3 is the leaf in generative phase, and 4 is clever shell; 5 is the root in generative phase, and 6 is young root.
Fig. 6 is the coloration result figure that the HDT701 promotor merges GUS dyeing transgenic line, and 1 is stem, and 2 is pulvinus; 3 is callus, and 4 is clever shell, and 5 is blade; 6 is young root, and 7 is clever shell and flower pesticide, and 8 is the root in generative phase; 9 for sprouting the seedling of different number of days, and 10 is the clever shell of mature seed, and 11 is the endosperm of the seed of sprouting.
Fig. 7 is transgenic paddy rice young root and spire semithin section figure as a result, and 1 is young root rip cutting figure, and 2 is the young root sectional view, and 3 is the spire sectional view.
Fig. 8 is that transfer-gen plant different times, different tissues organ gus protein relative reactivity are relatively schemed.
Fig. 9 is that the gus protein relative reactivity was relatively schemed after the transfer-gen plant Different Organs received cold coercing.
Figure 10 is that the gus protein relative reactivities were relatively schemed after transfer-gen plant received various coercing.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; Like the extraction of plasmid etc. is to carry out according to the molecular cloning experiment guide or the experiment condition of advising according to manufacturers; As: DNA reclaims the dna gel that adopts Dong Sheng biotech firm and reclaims test kit, operates by its specification sheets.
The promotor of embodiment 1:OSHDT701 gene: the clone of HDT701 promotor
1, fine with paddy rice japonica rice variety Japan is experiment material, extracts genomic dna as template through the CTAB method.
2, going up the sequence of known paddy gene OSHDT701 (the Genebank accession number is AK072845) according to NCBI, get initiator codon front 2KB design primer, is that template is carried out pcr amplification with the fine genomic dna of japonica rice variety Japan.
Upstream primer: F:5 '-TGAAGCTTTAAGGCGAATAAGCGAAAC-3 '
Downstream primer: R:5 '-CTCTGCAGGAAGAACCCTAGAAAAGAAA-3 '
The PCR reaction is undertaken by the 50ul system, and reaction parameter is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 58.9 ℃ of annealing 45s, 72 ℃ are extended 2min, circulate 35 times; Extend 5min after 72 ℃.Pcr amplification product reclaims purifying after 1.0% agarose gel electrophoresis separates (Fig. 1), be cloned into then among the carrier pMD18-T, spends the night 16 ℃ of connections.
Connect product with heat shock method transformed into escherichia coli competent cell JM109; The mono-clonal that grows is carried out bacterium colony PCR to be identified; Choose and changed the positive colony that is connected with the segmental pMD18-T of purpose over to and serve the order-checking of extra large Ying Jun Bioisystech Co., Ltd, obtain the sequence of amplified fragments, its sequence is shown in SEQ ID NO.1; Clip size is 1844bp, with this sequence called after HDT701.
The structure of embodiment 2:HDT701-GUS fusion expression vector
The positive colony large quantity extracting plasmid that order-checking among the embodiment 1 is correct.Plasmid that extracts and carrier PCAMBIA1391Z (its structure is as shown in Figure 2) carry out enzyme with HindIII and PstI simultaneously and cut; Reclaim purpose fragment, connection; Can obtain with HDT701 as promotor, its downstream gene is the expression vector PCAMBIA1391Z-HDT701P of GUS.At the segmental 700bp of HDT701P place a SpeI restriction enzyme site is arranged, on PCAMBIA1391Z, do not have the SpeI restriction enzyme site, identify that positive plasmid can obtain the small segment (Fig. 3) of 0.7KB so cut fusion vector with HindIII and SpeI enzyme.Positive plasmid changes Agrobacterium EHA105 over to through freeze-thaw method, and bacterium colony PCR identifies that the positive colony that changes expression vector PCAMBIA1391Z-HDT701P over to is used for rice conversion.
Embodiment 3: agrobacterium tumefaciens rice transformation mature embryo callus
Adopt the genetic transforming method (Hiei etc. of Agrobacterium EHA105 mediation; Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA; 1994, Plant Journal 6:271-282) with obtaining transfer-gen plant in the callus of spending 11 mature embryos in the plant fusion expression vector PCAMBIA1391Z-HDT701P importing paddy rice japonica rice variety that builds.
Agriculture bacillus mediated genetic transformation step is following:
1, callus induction
Sophisticated rice paddy seed shells, and the use volume(tric)fraction is 70% Ethanol Treatment 1min, and massfraction is 1% mercuric chloride solution sterilization 15min; Aqua sterilisa cleans seed 3～5 times, preceding 3-5min several times, last 10min.The seed that disinfects is inoculated on the inducing culture in 26 ℃ of darkrooms cultivated for 2 weeks.
2, callus subculture
Inoculate the scultellum that will expand after two weeks and scale off, change on the new inducing culture, select the granular little callus of beige after 2 weeks, be put in dark 2 weeks of cultivation down on the subculture medium from seed.
3, cultivate in advance
Select the sized granular callus, be put on the inducing culture and cultivated 3 days 26 ℃ of temperature under the dark in advance.
4, Agrobacterium activation
The Agrobacterium EHA105 that contains carrier PCAMBIA1391Z-HDT701P that builds containing of embodiment 2 is gone up line at the YM solid medium that has 50mg/L kantlex and 25mg/L Vetstrep (the YM substratum belongs to substratum of the prior art), be inverted for 28 ℃ and cultivated 48 hours; Choose single colony inoculation and in containing identical antibiotic 5ml YM liquid nutrient medium, cultivate 24-48h on 28 ℃ of shaking tables; Get that shaking culture makes OD600=0.6-0.8 in the identical antibiotic YM liquid nutrient medium that 1ml is inoculated in 30ml; Centrifugal collection thalline; With the resuspended thalline of AAM liquid nutrient medium that is added with 200umol/L Syringylethanone (As), be used for contaminating.
5, Agrobacterium is infected
The callus of cultivating 3 days is in advance put into above-mentioned agrobacterium suspension soak 15min, middle every separated several minutes shakes, blots unnecessary bacterium liquid with sterilization filter paper after the taking-up callus; Be seeded in the dark 3d of cultivation on the common substratum then, 22 ℃ of temperature.
6, the cleaning of callus and screening
With the aqua sterilisa that is added with the 500mg/L cephamycin callus after cultivating is altogether cleaned 4-5 time; Before each several times 4-5min, last 10min transfers to the aseptic filter paper suck dry moisture then; Callus is linked into 26 ℃ of dark cultivations on the screening culture medium, and per two all subcultures once.Twice back kanamycin-resistant callus tissue that the president makes new advances on old callus of subculture.
7, break up and take root
Long kanamycin-resistant callus tissue to a certain size is transferred to the dark 7d of cultivation on the presorting screening culture medium that is added with ABA, transfers to again after dry 3 days on the differentiation screening culture medium on three layers of aseptic filter paper, cultivate until growing seedling in 26 ℃ of illumination boxs.Changing the seedling that grows over to screening takes root on the root media.
8, transplant
Seedling after will taking root is taken one week of room temperature condition lower refining seedling, plants in the flowerpot soil normal management then.
Embodiment 4: the evaluation of transfer-gen plant
Treat that transfer-gen plant is long after a certain size, clip leaf CTAB method is extracted genomic dna, and designs primer it is carried out the PCR detection, and primer is:
F：5’-GGAGTGAAGAGTATCAGTGTGC-3’
R：5’-GATAATCATCGCAAGACCG-3’。
The PCR reaction parameter is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 90s, circulate 30 times; Prolong 10min after 72 ℃.Pcr amplification product separates through 1.0% agarose gel electrophoresis.Expection PCR product sheet segment length is 399bp.The result is as shown in Figure 4; M is a molecular weight marker; With the 1-8 swimming lane is 8 rotaring gene plant blades; The wild-type rice leaf is the 9th swimming lane, and genetically modified 3,4,6, No. 7 paddy rice can both obtain the GUS purpose band (positive plant) of 399bp, wild-type do not obtain the purpose band as negative control.Obtain to change over to the positive transgenic rice plant of PCAMBIA1391Z-HDT701P carrier thus.
Embodiment 5: the expression of semi-quantitative analysis goal gene HDT701 gene in root, the leaf in generative phase, stem and the clever shell in paddy rice young root, spire, generative phase.
The root of fetching water respectively rice young root, spire, generative phase, the leaf in generative phase, stem and clever shell carry out the extraction of RNA, carry out semi-quantitative expressed analysis then:
The process for extracting of RNA and semi-quantitative expressed analytical procedure are following:
Used primer sequence:
F：5’-TGTGAGCCTGAAGATGAACG-3’，
R：5’-GAGAGGGTTCCAATGACTAGC-3’
1, the extraction of RNA: (Trizol method)
(1) gets the various organ-tissues of 100mg, add liquid nitrogen and be ground into powder.
(2) add 1ml Trizol, room temperature leaves standstill, and melts fully until sample, continues to be ground to lysate with pestle again and is transparence.
(3) add the 0.2ml chloroform, acutely shake 30s, room temperature 5min.
(4) 12000rmp, 4 ℃ centrifugal, 15min.
(5) suct the colourless water of layer, move in another EP pipe (about 0.5ml).
(6) add the equal-volume Virahol ,-20 ℃, 30min.
(7) 12000rmp, 4 ℃ centrifugal, 10min.The visible micro-RNA deposition in the pipe bottom
(8) abandon supernatant, centrifuge tube is inverted on the paper handkerchief suck dry moisture;
(9) add 1ml 75% ethanol, abandon supernatant (cleaning twice), dry air DNA;
(10) with the water 50 μ l dissolving that contains DNAaseI, put one hour for 37 ℃, add again like the DHPC water of 550ul repeating step 3-11 then;
(11) ,-20 ℃, get 1 μ l and add 99 μ l DEPC water survey OD260/OD280 with 40 μ l DHPC water dissolution;
(12) calculating concentration and purity ,-70 ℃ of preservations.
2, cDNA first chain is synthetic
The following solution of preparation in the PCR of RNase free pipe:
Total?RNA 1μg
DEPC handles H
2O supplies 15.5 μ l
70 ℃ of water-bath 10min put on ice;
Add following reagent again:
Above-mentioned mixed appearance is in 37 ℃ of insulation 1h, and 72 ℃ of 10min enzyme that goes out is lived.The reverse transcription product dilution is used for the pcr amplification template for 5 times.
3, PCR reaction
Amplification system is:
The RT-PCR response procedures is: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30sec, Tm value renaturation 1min, 72 ℃ are extended 1min, 35 circulations; 72 ℃ are extended 10min.
The PCR product carries out electrophoresis; Electrophoretic band obtains testing gene and internal control gene band peak area integrated value separately through the scanning of density scan appearance; Calculate and respectively organize both ratio of sample, the result is as shown in Figure 5, and goal gene HDT701 all has expression in obtaining tissue.
Embodiment 6: to the GUS staining analysis of positive transfer-gen plant
The seed of getting leaf, stem, root, clever shell and the sprouting different number of days of the positive transgenic rice plant that changes the PCAMBIA1391Z-HDT701P carrier over to of embodiment 4 respectively immerses in the GUS dye liquor, and 37 ℃ are spent the night, the observation of taking pictures after the absolute ethyl alcohol decolouring.As shown in Figure 6, gus gene all has expression in the organ of paddy rice except that endosperm.Explanation thus; The HDT701 fragment of sequence is a promotor shown in SEQ ID NO.1; Called after paddy rice histone deacetylase gene HDT701 promotor, this promotor starts gus gene all has expression in the organ of transgenic paddy rice except that endosperm, and promptly this promotor only starts downstream gene and in the part organ-tissue, expresses in transgenic paddy rice; Explaining that thus this promotor has tissue specificity, is a tissue-specific promoter.
GUS dye liquor: 50mmol/L sodium phosphate buffer (pH7.0), 5mmol/L K
4Fe (CN)
6, 5mmol/L K
3Fe (CN)
6, 10mmol/L EDTA, 0.1%TritonX-100,1.0mg/mL X-Gluc.
Embodiment 7: the mensuration of positive transfer-gen plant being carried out Different Organs gus protein relative reactivity
Root, stem, leaf, the leaf sheath of getting the positive transgenic paddy rice that the embodiment 4 of vegetative growth phase and reproductive stage obtains respectively carry out the gus protein relative reactivity and measure.
Gus protein relative reactivity land surveying method is following:
(1) get the 0.1g material in the 1.5ml centrifuge tube, add liquid nitrogen, grinding powder adds the 600ul zyme extract.
(2) 13000rpm, 4 ℃ of centrifugal 10min get supernatant.
(3) get the supernatant of 50ul, add that to make final volume in the sterilization deionized water of 50ul be 100ul, measure proteic content with the Xylene Brilliant Cyanine G method.
(4) get the supernatant that 50ul contains GUS and join 450ul in the detection liquid (2mmol/L MUG solution) of 37 ℃ of preheatings.Rapid fully mixing, and take out 50ul at once and join 1950ul reaction terminating liquid (0.2mol/L Na
2CO
3), with 0 point of this pipe as enzymatic reaction.
(5) take out the reaction solution of 50ul then respectively respectively at 5min, 10min, 20min and 30min, change in the reaction terminating liquid of 1950ul.
(6) with spectrophotofluorometer under excitation wavelength 365nm, emission wavelength 455nm, measure the fluorescent value of different time points, calculate the amount of the MU that reaction generates;
(7) GUS is active in the calculation sample, the MU/ protein content (unit: pmolMU.mg-1.min-1) that the GUS activity=the unit time internal reaction generates.
GUS zyme extract: 0.1M phosphoric acid buffer (PH7.0) 50ml, 10%SDS 1ml, 0.5M EDTA (PH8.0) 2ml, methyl alcohol 20ml, Triton X-100 100ul, beta-mercaptoethanol 100ul, ddH
2O up to 100ml.
GUS detects liquid (MUG solution): MUG (C
16H
16O
94-methyl umbellate form ketone-beta-glucuronidase) molecular weight: 352.3,50mg MUG is dissolved in the GUS zyme extract of 72ml, be mixed with the concentration of 2mmol/L.
Reaction terminating liquid (0.2mol/L Na
2CO
3): the Na of preparation 500ml 0.2mol/L
2CO
3Solution: Na
2CO
310.6gH
2O up to 500ml
4-MU solution preparation: 4-MU (4-methyl umbellate form ketone) molecular weight: 176.2, claim that 0.14096g 4-MU solid is dissolved in 40ml reaction terminating liquid (0.2M Na
2CO
3) in, be mixed with the 4-MU solution of 20mM.And then be diluted to 1mM concentration, and be stored in-20 degree, wait until preparation MU typical curve.
Bradford solution preparation: take by weighing 100mg Xylene Brilliant Cyanine G medicine, be dissolved in 100ml 85% phosphoric acid and 50ml 95% alcohol mixeding liquid.After the dyestuff dissolving fully, complement to 1000ml with cold water, filter paper filtering keeps in Dark Place.
The result is as shown in Figure 8, Fig. 8 be presented in the Different Organs gus protein relative reactivity all be vegetative growth phase greater than reproductive stage, and vegetative growth phase the expression amount in root the strongest, be that expression amount in the stem is the strongest at reproductive stage.
Embodiment 8: the gus protein relative reactivity of transgenic paddy rice is measured under the different stress conditions
The transgenic paddy rice of the vegetative growth phase that changes the PCAMBIA1391Z-HDT701P carrier over to that embodiment 4 is obtained; Handle the gus protein relative reactivity of measuring root, stem, leaf, leaf sheath after 12 hours for 4 ℃; Concrete determination step is referring to embodiment 7; The result is as shown in Figure 9, and each organ gus protein relative reactivity after subzero treatment all descends, and the degree that just descends is variant.
Also measured the relative reactivity (young root is got 0.05g, and spire is got 0.05g) of the gus protein in two leaves, the one heart stage transgenic paddy rice under different stress conditions in addition, wherein coldly coerced that to handle be that transfer-gen plant is handled sampling in 12 hours in 4 ℃, measured; Pyroprocessing is transfer-gen plant to be handled in 40 ℃ took a sample in 12 hours, measures; Drought stress is that transfer-gen plant was taken a sample with 20% PEG processing in 12 hours, measures; Dark processing is transfer-gen plant to be positioned over handle sampling in 12 hours in the dark, measures, and concrete determination step is referring to embodiment 7, and each handles repetition 3 times.The result is shown in figure 10, and the gus protein relative reactivity can rise after the pyroprocessing, and the relative reactivity of subzero treatment, arid processing and reason back, dark place gus protein can descend.
Embodiment 9: semithin section:
1, the preparation of solution
A liquid: contain the Paraformaldehyde 96 of 2.5% LUTARALDEHYDE and 2% phosphoric acid buffer (0.1mol/L, pH7.2).
B liquid: contain 1% osmic acid phosphoric acid buffer (0.1mol/L, pH7.2)
C liquid: phosphoric acid buffer (0.1mol/L, pH7.2)
2, fixing and embedding
(1) fixing: the suitable size dyeing that will shear and root and the leaf that took off the rice seedling of look drop in the A liquid 4 ℃ of fixed overnight rapidly.
(2) washing: with 4 ℃ of washings of C liquid six times, each 20min.
(3) fixing again: as in solution B, to fix 16 hours for 4 ℃.
(4) washing: with 4 ℃ of washings of C liquid 6 times, each 20min.
(5) volume(tric)fraction is that 30%, 50%, 70%, 80%, 90%, 100% serial pre-cooled ethanol dewaters step by step, every grade of 15min, 100% ethanol 2 times, each 30min.
(6) propylene oxide transition, the embedding of Epon812 ordinary method.The material that embedding is good can carry out semithin section, is cut into the 2um slab, and Carl Zeiss microscopic examination is taken pictures.
The result is as shown in Figure 7, and Fig. 7 shows that GUS mainly concentrates on fascicular expression on every side in young root and leaf, infers that perhaps the HDT701 promotor participates in the transportation of nutritive substance.
Embodiment 10: in order better to understand the function of HDT701 promotor, the HDT701 promotor is placed on has done the analysis of cis acting original paper on the PLACE.Its result is as shown in table 1.
What more than enumerate at last, only is several specific embodiments of the present invention.Obviously the invention is not restricted to above embodiment, many distortion can also be arranged.Those of ordinary skill in the art can derive all distortion of perhaps associating from content disclosed by the invention, all should think protection scope of the present invention.
Attach: relate to the substratum of using in the transgenic paddy rice process
1) induce, subculture medium:
2) AAM substratum
3) be total to substratum
The paddy rice mature embryo callus altogether substratum: N6 a large amount of+MS-Fe salt+B5 trace+B5 is organic+2,4-D2.0mg/L+CH 300mg/L+proline 500mg/L+ inositol 100mg/L+AS 100 μ M+ sucrose 30g/L+phytagel 3.0mg/L pH 5.2.
4) screening culture medium
The Totomycin of inducing culture+50mg/L+500mg/L cephamycin.
5) presorting substratum
Time spent adds Totomycin and the 500mg/L cephamycin (presorting screening culture medium) of 50mg/L.
6) division culture medium
Time spent adds Totomycin and the 500mg/L cephamycin (differentiation screening culture medium) of 50mg/L.
7) root media
Time spent adds Totomycin and the 500mg/L cephamycin (screening root media) of 50mg/L.
Claims (5)
1. paddy rice histone deacetylase gene HDT701 promotor is characterized in that its sequence is one of following nucleotide sequences:
(a) nucleotide sequence shown in the SEQ ID NO.1 in the sequence table;
(b) under rigorous condition, can and have the nucleotide sequence of identical function with the dna sequence dna of (a) hybridization;
(c) with (a) or dna sequence dna (b) have the homology more than 90%, and also have the nucleotide sequence of promoter function.
2. an expression vector is characterized in that, contains the described paddy rice histone deacetylase of claim 1 gene HDT701 promotor.
3. a host bacterium is characterized in that, contains the described expression vector of claim 2.
4. host bacterium according to claim 3 is characterized in that, described host bacterium is Agrobacterium EHA105.
5. the described paddy rice histone deacetylase of claim 1 gene HDT701 promotor is starting the application that the downstream goal gene is expressed in paddy rice.
Priority Applications (2)
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| CN 201110327877 CN102363782B (en) | 2011-10-25 | 2011-10-25 | Rice histone deacetylases gene HDT701 promoter and application thereof |
| PCT/CN2011/084543 WO2013060074A1 (en) | 2011-10-25 | 2011-12-23 | Promoter of rice histone deacetylase gene hdt701 and use thereof |
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| CN 201110327877 CN102363782B (en) | 2011-10-25 | 2011-10-25 | Rice histone deacetylases gene HDT701 promoter and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774826A (en) * | 2015-03-20 | 2015-07-15 | 中国科学院华南植物园 | Histone deacetylase, encoding gene and applications thereof |
| CN104805065A (en) * | 2015-05-05 | 2015-07-29 | 中国科学院华南植物园 | Paddy rice histone deacetylase as well as encoding gene and application |
| CN104988140A (en) * | 2015-05-20 | 2015-10-21 | 中国科学院华南植物园 | Promoter from rice and application thereof |
| CN111041030A (en) * | 2019-12-31 | 2020-04-21 | 海南波莲水稻基因科技有限公司 | Promoter PCHF4 specifically expressed in rice pollen and application thereof |
| CN112409466A (en) * | 2019-08-21 | 2021-02-26 | 中国科学院微生物研究所 | Application of protein HDA703 in regulation and control of rice yield |
| CN112553224A (en) * | 2020-10-30 | 2021-03-26 | 广东省农业科学院农业生物基因研究中心 | Application of histone deacetylase gene OsHDT701 in prolonging life of plant seeds |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104975022B (en) * | 2014-04-08 | 2019-05-21 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | The identification and application of plant anther specific expression promoter pTaASG036 |
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| US20070118921A1 (en) * | 1999-07-20 | 2007-05-24 | Boukharov Andrey A | Genomic plant sequences and uses thereof |
| CN101792761A (en) * | 2009-10-23 | 2010-08-04 | 武汉大学 | Induction-enhanced tissue specific promoter and application thereof |
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| PL1687435T3 (en) * | 2003-11-17 | 2012-02-29 | Bayer Cropscience Nv | Insect resistance using inhibition of gene expression |
| CN102154363A (en) * | 2010-12-27 | 2011-08-17 | 浙江师范大学 | Recombinant vectors containing specific promoters of rice endosperm and use and plant thereof |
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- 2011-10-25 CN CN 201110327877 patent/CN102363782B/en not_active Expired - Fee Related
- 2011-12-23 WO PCT/CN2011/084543 patent/WO2013060074A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070118921A1 (en) * | 1999-07-20 | 2007-05-24 | Boukharov Andrey A | Genomic plant sequences and uses thereof |
| CN101792761A (en) * | 2009-10-23 | 2010-08-04 | 武汉大学 | Induction-enhanced tissue specific promoter and application thereof |
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| BOUKHAROV，A.A， ET AL.: "GX494091.1", 《GENBANK》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774826A (en) * | 2015-03-20 | 2015-07-15 | 中国科学院华南植物园 | Histone deacetylase, encoding gene and applications thereof |
| CN104774826B (en) * | 2015-03-20 | 2017-12-12 | 中国科学院华南植物园 | A kind of histone deacetylase and its encoding gene and application |
| CN104805065A (en) * | 2015-05-05 | 2015-07-29 | 中国科学院华南植物园 | Paddy rice histone deacetylase as well as encoding gene and application |
| CN104805065B (en) * | 2015-05-05 | 2017-10-27 | 中国科学院华南植物园 | A kind of paddy rice histone deacetylase and its encoding gene and application |
| CN104988140A (en) * | 2015-05-20 | 2015-10-21 | 中国科学院华南植物园 | Promoter from rice and application thereof |
| CN112409466A (en) * | 2019-08-21 | 2021-02-26 | 中国科学院微生物研究所 | Application of protein HDA703 in regulation and control of rice yield |
| CN111041030A (en) * | 2019-12-31 | 2020-04-21 | 海南波莲水稻基因科技有限公司 | Promoter PCHF4 specifically expressed in rice pollen and application thereof |
| CN111041030B (en) * | 2019-12-31 | 2021-09-10 | 海南波莲水稻基因科技有限公司 | Promoter PCHF4 specifically expressed in rice pollen and application thereof |
| CN112553224A (en) * | 2020-10-30 | 2021-03-26 | 广东省农业科学院农业生物基因研究中心 | Application of histone deacetylase gene OsHDT701 in prolonging life of plant seeds |
| CN112553224B (en) * | 2020-10-30 | 2022-04-08 | 广东省农业科学院农业生物基因研究中心 | Application of histone deacetylase gene OsHDT701 in prolonging life of plant seeds |
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| Publication number | Publication date |
|---|---|
| WO2013060074A1 (en) | 2013-05-02 |
| CN102363782B (en) | 2013-06-19 |
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