CN102363782B

CN102363782B - Rice histone deacetylases gene HDT701 promoter and application thereof

Info

Publication number: CN102363782B
Application number: CN 201110327877
Authority: CN
Inventors: 段俊; 张伟; 赵金会; 刘夏
Original assignee: South China Botanical Garden of CAS
Current assignee: South China Botanical Garden of CAS
Priority date: 2011-10-25
Filing date: 2011-10-25
Publication date: 2013-06-19
Anticipated expiration: 2031-10-25
Also published as: WO2013060074A1; CN102363782A

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

A kind of paddy rice histone deacetylase gene HDT701 promotor and application thereof

Technical field:

The invention belongs to genetically engineered and rice breeding technology field; be specifically related to a kind of clone of paddy rice histone deacetylase gene HDT701 upstream of coding region promoter sequence-paddy rice histone deacetylase gene HDT701 promotor, and the application of this promotor in transgenic paddy rice.

Background technology:

Promotor refers to can be combined with RNA polymerase and form on DNA molecular the zone of transcription initiation complex, is usually located at the upstream of encoding gene.Have a large amount of cis acting original papers in promoter fragment, thereby transcription factor makes gene have the spatial and temporal expression specificity by be combined 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 commonly using in plant genetic engineering is divided into 3 classes 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, there is no Space-time speciality.In genetically engineered commonly used to have at present commonly used in plant expression vector be constitutive promoter, as 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 affects 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 like this gene overexpression that plant materials is caused 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 development of modern genetic engineering technology has obtained a lot of transgenic plant in recent years, but food-safety problem popularization and the development of transgenic crop have but been restricted.In transgenic paddy rice, foreign gene is expressed in endosperm and is caused foreign protein to accumulate therein, and being affects the restricted principal element of transgenic paddy rice development that some have fine quality.For this phenomenon, utilizing organizing specific type promotor to make foreign gene only express and not express to reduce food safety risk in the edible part of the mankind in vegetative organ 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 is to provide 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 SEQ ID NO.1 in sequence table;

(b) can hybridize and have with the DNA sequence dna of (a) nucleotide sequence of identical function under rigorous condition;

(c) with (a) or DNA sequence dna (b) have homology more than 90%, and also have the nucleotide sequence of promoter function.

Paddy rice histone deacetylase gene HDT701 promotor as 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 starts by the HDT701 promotor fusion expression vector that gus gene is expressed with multiple clone site place's formation that the paddy rice histone deacetylase gene HDT701 promotor as shown in SEQ ID NO.1 is inserted into carrier PCAMBIA1391Z, obtains transfer-gen plant by agrobacterium-mediated transformation rice transformation mature embryo callus.detect and GUS dyeing by the PCR to transgenic paddy rice, result shows that we are successful the HDT701 promotor is incorporated in paddy gene, and the gus gene of HDT701 promoters driven all has expression in the organ except endosperm at transgenic paddy rice, illustrate that thus the sequence as 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 expression in the organ except endosperm at transgenic paddy rice, namely just express in the part organ-tissue in transgenic paddy rice, this promotor specificity in a organized way is described thus, it is a tissue-specific promoter.

Therefore, second purpose of the present invention is to provide 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 is to provide a kind of Host Strains, it is characterized in that, contains above-mentioned expression vector.

Described Host Strains is preferably Agrobacterium EHA105.

The 4th purpose of the present invention is to provide the application that a kind of paddy rice histone deacetylase gene HDT701 promotor is expressed in paddy rice at the startup downstream target gene.

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 transgenic engineering; the expression product of the goal gene that makes is in certain organ or tissue's position accumulation; increase the local expression amount; also can avoid simultaneously the unnecessary waste of plant nutrition, therefore good application prospect 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 builds and to complete enzyme and 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, take the 1-8 swimming lane as 8 rotaring gene plant blades, the wild-type rice leaf is the 9th swimming lane, genetically modified 3,4,6, No. 7 paddy rice can 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, A is the HDT701 gene, 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, 1 is stem, and 2 is pulvinus, and 3 is callus, 4 is clever shell, 5 is blade, and 6 is young root, and 7 is clever shell and flower pesticide, 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 transfer-gen plant Different Organs is subject to coldly coercing rear gus protein relative reactivity and relatively scheming.

Figure 10 is that transfer-gen plant is subject to variously coercing rear gus protein relative reactivity and relatively scheming.

Embodiment:

Following examples are to further illustrate 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, to carry out according to the molecular cloning experiment guide or according to the experiment condition that manufacturers advises as extraction of plasmid etc., as: DNA reclaims the DNA gel recovery test kit that adopts Dongsheng biotech firm, operates by its specification sheets.

The promotor of embodiment 1:OSHDT701 gene: the clone of HDT701 promotor

1, Japanese fine as experiment material take the paddy rice japonica rice variety, extract genomic dna as template by the CTAB method.

2, the sequence of known paddy gene OSHDT701 (Genebank accession number be AK072845) upper according to NCBI, get initiator codon front 2KB design primer, carries out pcr amplification take the fine genomic dna of japonica rice variety Japan as template.

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 denaturation 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), then be cloned in carrier pMD18-T, spends the night 16 ℃ of connections.

Connect product and transform competent escherichia coli cell JM109 with the heat shock method, the mono-clonal that grows is carried out bacterium colony PCR to be identified, choose the positive colony that has changed the pMD18-T that is connected with the purpose fragment over to and serve the order-checking of extra large Ying Jun Bioisystech Co., Ltd, obtain the sequence of amplified fragments, its sequence is as 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 in embodiment 1 is correct.The plasmid that extracts and carrier PCAMBIA1391Z (its structure 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.There is a SpeI restriction enzyme site at 700bp place in the HDT701P fragment, there is no the SpeI restriction enzyme site on PCAMBIA1391Z, identifies 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 by 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) obtain transfer-gen plant with spending in the plant fusion expression vector PCAMBIA1391Z-HDT701P Introduced into Rice japonica rice variety that builds in the callus of 11 mature embryos.

Agriculture bacillus mediated genetic transformation step is as follows:

1, callus induction

Ripe rice paddy seed shells, and is 70% Ethanol Treatment 1min with volume fraction, and massfraction is 1% mercuric chloride solution sterilization 15min; Aqua sterilisa cleans seed 3～5 times, front 3-5min several times, last 10min.The seed that disinfects is inoculated on inducing culture cultivated for 2 weeks in 26 ℃ of darkrooms.

2, callus subculture

Inoculate the scultellum that will expand after two weeks and scale off from seed, change on new inducing culture, select the granular little callus of beige after 2 weeks, be put in dark lower the cultivation for 2 weeks on subculture medium.

3, preculture

Select a certain size particulate state callus, be put on inducing culture dark lower preculture 3 days, 26 ℃ of temperature.

4, Agrobacterium activation

The Agrobacterium EHA105 that contains carrier PCAMBIA1391Z-HDT701P that builds containing of embodiment 2 is being rule with the YM solid medium (the YM substratum belongs to substratum of the prior art) of 50mg/L kantlex and 25mg/L Vetstrep is upper, and 28 ℃ of inversions were cultivated 48 hours; Choose single colony inoculation and cultivate 24-48h on 28 ℃ of shaking tables in containing identical antibiotic 5ml YM liquid nutrient medium, get that in the identical antibiotic YM liquid nutrient medium that 1ml is inoculated in 30ml, shaking culture makes OD600=0.6-0.8, 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 preculture callus of 3 days is put into above-mentioned agrobacterium suspension soak 15min, shake every several minutes the centre, blots unnecessary bacterium liquid with sterilization filter paper after the taking-up callus; Then be seeded in the dark 3d of cultivation on common substratum, 22 ℃ of temperature.

6, the cleaning of callus and screening

Callus with the aqua sterilisa that is added with the 500mg/L cephamycin after to common cultivation is cleaned 4-5 time, before each 4-5min several times, then last 10min transfers to the aseptic filter paper suck dry moisture, callus is linked into 26 ℃ of dark cultivations on screening culture medium, and every two all subcultures once.Twice rear kanamycin-resistant callus tissue that the president makes new advances on old callus of subculture.

7, break up and take root

The kanamycin-resistant callus tissue that grows to a certain size is transferred to the dark 7d of cultivation on the pre-differentiation 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 grow seedling in 26 ℃ of illumination boxs.The seedling that grows is changed on the screening root media take root.

8, transplant

Seedling after taking root is taken one week of room temperature condition lower refining seedling, then plants in flowerpot soil normal management.

Embodiment 4: the evaluation of transfer-gen plant

After transfer-gen plant grew to a certain size, clip leaf CTAB method was extracted genomic dna, and it carries out the PCR detection to design primer pair, and primer is:

F：5’-GGAGTGAAGAGTATCAGTGTGC-3’

R：5’-GATAATCATCGCAAGACCG-3’。

The PCR reaction parameter is: 94 ℃ of denaturation 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.Result as shown in Figure 4, M is molecular weight marker, take the 1-8 swimming lane as 8 rotaring gene plant blades, the wild-type rice leaf is the 9th swimming lane, genetically modified 3,4,6, No. 7 paddy rice can 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 thus the positive transgenic rice plant of PCAMBIA1391Z-HDT701P carrier.

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, then carry out semi-quantitative expressed analysis:

The extracting method of RNA and semi-quantitative expressed analytical procedure are as follows:

Primer sequence used:

F：5’-TGTGAGCCTGAAGATGAACG-3’，

R：5’-GAGAGGGTTCCAATGACTAGC-3’

1, the extraction of RNA: (Trizol method)

(1) get the various organ-tissues of 100mg, add liquid nitrogen and be ground into powder.

(2) add 1ml Trizol, room temperature is standing, until sample melts fully, then continues to be ground to lysate with pestle 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 into (approximately 0.5ml) in another EP pipe.

(6) add the equal-volume Virahol ,-20 ℃, 30min.

(7) 12000rmp, 4 ℃ centrifugal, 10min.In the visible micro-RNA precipitation in pipe bottom

(8) abandon supernatant, centrifuge tube is inverted on paper handkerchief suck dry moisture;

(9) add 1ml 75% ethanol, abandon supernatant (cleaning twice), dry air DNA;

(10) with the water 50 μ l dissolvings that contain DNAaseI, put one hour for 37 ℃, then add as the DHPC water of 550ul repeating step 3-11 then;

(11) with 40 μ l DHPC water dissolution ,-20 ℃, get 1 μ l and add 99 μ l DEPC water to survey OD260/OD280;

(12) calculating concentration and purity ,-70 ℃ of preservations.

2, cDNA the first chain is synthetic

The following solution of preparation in the PCR of RNase free pipe:

Total RNA 1μg

DEPC processes H ₂O supplies 15.5 μ l

70 ℃ of water-bath 10min put on ice;

Add again following reagent:

Above-mentioned mixed sample is in 37 ℃ of insulation 1h, and 72 ℃ of 10min enzyme that goes out is lived.5 times of reverse transcription product dilutions are used for the pcr amplification template.

3, PCR reaction

Amplification system is:

The RT-PCR response procedures is: 94 ℃ of denaturation 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 reference gene band peak area integrated value separately through the scanning of density scan instrument, calculate and respectively to organize both ratio of sample, result as shown in Figure 5, goal gene HDT701 all has expression in obtained tissue.

Embodiment 6: to the GUS staining analysis of positive transfer-gen plant

Get respectively leaf, stem, root, the clever shell of the positive transgenic rice plant that changes the PCAMBIA1391Z-HDT701P carrier over to of embodiment 4 and sprout in the seed immersion GUS dye liquor of different number of days, 37 ℃ are spent the night, the observation of taking pictures after the dehydrated alcohol decolouring.As shown in Figure 6, gus gene all has expression in the organ except endosperm in paddy rice.Explanation thus; the HDT701 fragment of sequence is a promotor as shown in SEQ ID NO.1; called after paddy rice histone deacetylase gene HDT701 promotor; this promotor starts gus gene all expression in the organ except endosperm at transgenic paddy rice; namely this promotor only starts downstream gene and expresses in the part organ-tissue in transgenic paddy rice; illustrating 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 respectively the positive transgenic paddy rice that the embodiment 4 of vegetative growth phase and reproductive stage obtains carry out the gus protein relative reactivity and measure.

Gus protein relative reactivity land surveying method is as follows:

(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 the content of albumen 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 at once 50ul and join 1950ul reaction terminating liquid (0.2mol/L Na ₂CO ₃), with 0 point of this pipe as enzymatic reaction.

(5) then take out respectively respectively the reaction solution of 50ul at 5min, 10min, 20min and 30min, change in the reaction terminating liquid of 1950ul.

(6) use spectrophotofluorometer under excitation wavelength 365nm, emission wavelength 455nm, measure the fluorescent value of different time points, calculate the amount of the MU of reaction generation;

(7) in calculation sample, GUS is active, 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 0.14096g 4-MU solid to be 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 100mg Xylene Brilliant Cyanine G medicine, be dissolved in 100ml 85% phosphoric acid and 50ml 95% alcohol mixeding liquid.After the dyestuff dissolve complete, complement to 1000ml with cold water, filter paper filtering keeps in Dark Place.

Result as shown in Figure 8, Fig. 8 be presented in Different Organs the gus protein relative reactivity be all vegetative growth phase greater than reproductive stage, and the expression amount in root is the strongest in vegetative growth phase, is that expression amount in stem is the strongest at reproductive stage.

Embodiment 8: under the Different stress condition, the gus protein relative reactivity of transgenic paddy rice is measured

The transgenic paddy rice of the vegetative growth phase that changes the PCAMBIA1391Z-HDT701P carrier over to that embodiment 4 is obtained, process 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, result as shown in Figure 9, each organ gus protein relative reactivity after subzero treatment all descends, and the degree that just descends is variant.

Also measured in addition the relative reactivity (young root is got 0.05g, and spire is got 0.05g) of the gus protein in two leaf one heart stage transgenic paddy rices under the Different stress condition, wherein cold Stress treatment is transfer-gen plant to be processed in 4 ℃ took a sample in 12 hours, measures; Pyroprocessing is transfer-gen plant to be processed 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 process sampling in 12 hours in dark, measures, and concrete determination step is referring to embodiment 7, and each is processed and repeats 3 times.Result as shown in figure 10, after pyroprocessing, the gus protein relative reactivity can rise, and subzero treatment, arid is processed and the dark place reason after the relative reactivity of gus protein can descend.

Embodiment 9: semithin section:

1, the preparation of solution

A liquid: the phosphoric acid buffer (0.1mol/L, pH7.2) that contains the paraformaldehyde of 2.5% glutaraldehyde and 2%.

B liquid: the phosphoric acid buffer (0.1mol/L, pH7.2) that contains 1% osmic acid

C liquid: phosphoric acid buffer (0.1mol/L, pH7.2)

2, fixing and embedding

(1) fixing: the suitable size that will shear dyes and the root and the leaf that took off the rice seedling of look drops into rapidly in A liquid, and 4 ℃ are fixedly spent the night.

(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 fraction is that the pre-cooled ethanol of 30%, 50%, 70%, 80%, 90%, 100% series dewaters step by step, every grade of 15min, 100% ethanol 2 times, each 30min.

(6) propylene oxide transition, the embedding of Epon812 ordinary method.Embedded material can carry out semithin section, is cut into the 2um slab, and Carl Zeiss microscopic examination is taken pictures.

Result as shown in Figure 7, Fig. 7 shows, GUS mainly concentrates on and expresses around fascicular 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 PLACE.Its result is as shown in table 1.

What more than enumerate at last, is only 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 from content derivation disclosed by the invention or all distortion of associating, all should think protection scope of the present invention.

Attached: as to 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 Mature Embryos of Rice 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) pre-division culture medium

Used time adds Totomycin and the 500mg/L cephamycin (breaking up in advance screening culture medium) of 50mg/L.

6) division culture medium

Used time adds Totomycin and the 500mg/L cephamycin (differentiation screening culture medium) of 50mg/L.

7) root media

Used time adds Totomycin and the 500mg/L cephamycin (screening root media) of 50mg/L.

Claims

1. a paddy rice histone deacetylase gene HDT701 promotor, is characterized in that, its sequence is:

Nucleotide sequence shown in sequence table SEQ ID NO.1.

2. an expression vector, is characterized in that, contains paddy rice histone deacetylase gene HDT701 promotor claimed in claim 1.

3. a Host Strains, is characterized in that, contains expression vector claimed in claim 2.

4. Host Strains according to claim 3, is characterized in that, described Host Strains is Agrobacterium EHA105.

5. the paddy rice histone deacetylase gene HDT701 promotor claimed in claim 1 application that starting downstream target gene and express in other rice organ except endosperm.