CN102911936B - Two paddy rice MAR (matrix attachment region) sequences and application thereof - Google Patents

Abstract

The invention provides two nucleotide sequences of MAR (matrix attachment region) derived from paddy rice, which are shown in SEQIDNO.1 and SEQIDNO.2. The MAR nucleotide sequences comprise core motifs of the MAR sequences, and the core motifs of the MAR sequences have the characteristics of AT richness, replication origins, curved DNA (deoxyribonucleic acid), kinky DNA, topoisomerase II recognition sites, ATC principle and MAR recognition. According to the invention, the nucleotide sequences derived from paddy rice are connected to two sides of an exogenous gene to construct an expression vector and transform the expression vector to a plant, thus greatly improving the expression level and stability of the exogenous gene, effectively reducing or eliminating transgene silencing phenomenon, being beneficial to promoting development and application of plant transgene technology and having notable economic benefit and social benefit.

Description

Article two, paddy rice MAR sequence and application thereof

Technical field

The present invention relates to plant genetic engineering field, more specifically, the present invention relates to MAR sequence and the application thereof in two paddy rice sources.

Background technology

The develop rapidly of plant transgenic technology becomes the effective way of problems such as solving global crisis in food, shortage of resources, ecological degeneration and labor force's minimizing, and the fields such as agricultural, medicine, environmental protection and the energy have been produced to far-reaching influence.In the evolution of plant transgenic technology, the phenomenon of transgene silencing (transgene silencing) is one of important factor affecting its development.The phenomenon that how to overcome transgene silencing has become one of focus of plant transgenic technology development.At present, researchist mainly transforms foreign gene by modification or adds the method for regulating and controlling sequence to overcome the phenomenon of transgene silencing.

MAR/SAR (Matrix/Scaffold attachment region or Matrix/Scaffold association region, nuclear matrix land/nuclear skeleton attachment region) be the section of DNA sequence of can specificity in eukaryotic gene group combining closely on nuclear matrix, in eukaryotic gene group, exist in a large number.Existing research shows, does not have sequence homology between MAR sequence, there is no conserved sequence, but contains a plurality of common core primitives.These core primitives comprise: be rich in AT, replication origin, curved DNA, kink DNA, be rich in TG, topoisomerase II recognition site, plant conservative primitive, ATC principle and MAR recognition feature etc.Generally speaking, MAR sequence is all rich in AT, and other primitives are not that each MAR sequence possesses.MAR sequence length fluctuation range is larger, and the shortest tens base pairs of ability the longest reach nearly 8000 base pairs, but most MAR sequence length is between 600 to 1200 base pairs.The specific binding of MAR sequence and nuclear matrix has high conservative on evolving, and MAR sequence and the nuclear matrix in different plant species source can be combined closely.Meanwhile, study and also show, MAR sequence is present on karyomit(e) in a large number, is generally all positioned at intergenic region, and most of MAR sequences and miniature inverted repeat transposable element coexist.

Feature due to MAR sequence itself, does not have conservative property and sequence homology, adds MAR sequence distribution range extremely extensive, One's name is legion, and most of MAR sequences do not have again stronger foreign gene adjusting function, therefore, it is quite difficult separated and cloning handy MAR sequence.Perfect along with the development of information biology and gene order-checking, some new MAR forecasting techniquess that have been born, for separation and the clone of MAR provides a kind of new approach.MarFinder wherein (MAR-Wiz) be by British scientist Dr. Singh exploitation the earliest for the technology of MAR sequence prediction, there is higher predicted reliability.

Since the separated purifying of MAR sequence, its feature and function just enjoy researchist's concern, cotransformation is carried out in the both sides that researchist is connected in reporter gene by MAR sequence, finds that MAR sequence can make its efficient, stably express in acceptor in the situation that not changing reporter gene copy number.At present, the mechanism of action of MAR sequence is still without final conclusion, but its adjusting function is really extensively admitted.Basic approval MAR sequence all has obvious regulating and controlling effect to the transformation efficiency of foreign gene, expression level and stability and genetic stability in plant transgenic technology, can effectively reduce or eliminate the generation of transgene silencing phenomenon.

In plant transgenic technology, all exogenous dna fragments that derive from nonhost source are all considered to unsafe, all may have the problem of biological safety.The use that the DNA fragmentation of selection Hosts is replaced foreign DNA as far as possible, has become the megatrend of current researchist in plant transgenic technology.In the research of transgenic paddy rice, the expression that the MAR sequence of selecting paddy rice to originate regulates and controls foreign gene is considered to a kind of safer mode.

Summary of the invention

In view of above situation, in order to obtain the MAR(matrix attachment region in paddy rice source, MAR) sequence, reduce or elimination transgene silencing phenomenon obstruction and negative impact that to plant transgenic technology, development causes, the invention provides the MAR sequence in two paddy rice sources, and be applied in plant transgenic technology, improved expression level and the stability of foreign gene, effectively reduce or eliminated the generation of transgene silencing phenomenon.

The nucleotide sequence that the invention provides the MAR in two paddy rice sources, the nucleotide sequence of described MAR is as shown in SEQ ID NO.1 and SEQ ID NO.2.

The nucleotide sequence of described MAR comprises the core primitive of MAR sequence; The core primitive of described MAR sequence comprises and is rich in AT, replication origin, curved DNA, kink DNA, topoisomerase II recognition site, ATC principle and MAR recognition feature.

Another object of the present invention is the nucleotide sequence of the MAR in described paddy rice source to be applied in plant transgenic technology, and obtains the transfer-gen plant of stability and high efficiency expression alien gene.This application is the nucleotide sequence of the MAR in described paddy rice source to be connected into the both sides of foreign gene, construction of expression vector, and by expression vector transformed plant and obtain transgenic plant;

Described foreign gene is gus gene;

Described plant is monocotyledons;

Described plant is dicotyledons;

Described monocotyledons is any in paddy rice, corn, wheat, Chinese sorghum, oat, rye, barley, millet, sugar-cane and dogstail;

Described dicotyledons is any in soybean, pod, Semen Brassicae campestris, cotton, Sunflower Receptacle, tomato, potato, beet, alfalfa, cloves and peanut.

Remarkable advantage of the present invention:

The present invention has obtained the MAR sequence in two new paddy rice sources, and be applied in plant transgenic technology, can improve widely expression level and the stability of foreign gene, effectively reduce or eliminate the generation of transgene silencing phenomenon, help lend some impetus to development and the application of plant transgenic technology, there is significant economic benefit and social benefit.

Accompanying drawing explanation

Fig. 1 is the sequence of the synthetic multiple clone site short-movie section of artificial design and the restriction enzyme restriction enzyme site comprising thereof.

Fig. 2 is plant expression vector pCAMBIA1300-M1- gusplasmid map.

Fig. 3 is plant expression vector pCAMBIA1300-M2- gusplasmid map.

The positive contrast expression vector of Fig. 4 pCAMBIA1300- gusplasmid map.

Fig. 5 is the electrophoresis result that the PCR of hygromycin gene Hyg detects.The the 1st to the 3rd swimming lane is for turning pCAMBIA1300- gusthe plant of positive control transgenic paddy rice; The the 4th to the 6th swimming lane is for turning pCAMBIA1300-M1- gusthe positive plant of paddy rice; The the 7th and the 8th swimming lane is the negative control of non-transgenic paddy rice; The the 9th to the 11st swimming lane is for turning pCAMBIA1300-M2- gusthe positive plant of paddy rice; The 12nd swimming lane is ddH ₂the negative control of O; The 13rd swimming lane is DNA Marker.

Fig. 6 is the analysis alive of the GUS enzyme of transgenic paddy rice blade.Turning M1 paddy rice refers to and turns pCAMBIA1300-M1- gusthe transgenic paddy rice of plasmid; Turning M2 paddy rice refers to and turns pCAMBIA1300-M2- gusthe transgenic paddy rice of plasmid; Positive control refers to and turns pCAMBIA1300- gusthe transgenic paddy rice of plasmid; Negative control refers to non-transgenic paddy rice.

Embodiment

The MAR sequence in two paddy rice of the present invention source obtains as follows:

1) utilize the information of rice genome database in conjunction with the forecast analysis of MAR sequence prediction technology MAR-Wiz, to determine candidate's MAR sequence;

2), according to the MAR sequences Design synthetic primer of determining candidate, the method by pcr amplification clones candidate MAR sequence from oryza sativa genomic dna, and through sequence verification; Separation and clone candidate MAR sequence;

3) by candidate MAR sequence separated and that be cloned into paddy rice through sequential analysis, whether checking candidate MAR sequence meets the sequence signature of MAR;

4) by the candidate MAR sequence construct after checking in the both sides of plant transgene (reporter gene GUS), and by construction rice transformation, obtain transgenic rice plant;

5) by detecting the expression level of reporter gene GUS in transgenic paddy rice blade and not containing the comparison that contrasts transgenic paddy rice of MAR sequence, analyze and judge whether candidate MAR sequence has improved expression level and the stability of foreign gene, thereby determining whether candidate MAR sequence is needed paddy rice MAR sequence.

Below in conjunction with specific embodiment, further set forth the present invention, but the invention is not restricted to this.The present invention's experiment material used is commercially available purchase product if no special instructions.(experiment material of following examples be Japanese fine paddy rice ( oryza sativa L. japonica cv.))

embodiment 1:the screening of candidate MAR sequence

According to the content of AT-Richness, cance high-expression gene, choose be rich in AT-Richness and cance high-expression gene karyomit(e) for screening candidate MAR sequence.At BGI(Beijing Genomics Institute, BGI) check chromosomal gene mapping in rice genome database, the functional gene of selected genes compact district (bunch) DNA sequence dna of compartment is as sequence to be analyzed, length is generally 3 ~ 5kb.Selection AT content is positioned at the sequence of " 65%≤AT%≤80% ", then by MAR-Wiz on-line analysis (network address: http://genomecluster.secs.oakland.edu/) carry out multiple sieve.

MAR-Wiz not only contains common MAR sequence signature primitive, as ORI Pattern, TG-Richness Pattern, Curved DNA Pattern, Kinked DNA Pattern, Topo II Pattern, AT-Richness Pattern and ATC Rule etc., also comprised and adapted to this research from " the Plant MAR Consensus " of the needs of rice genome screening MAR sequence.

While using MAR-Wiz to analyze sequence to be measured, first at MAR Finder Home Page, submit sequence to be measured, select as required this research of MAR Rules(to choose all primitives and rule in Core MAR Rules and New MAR Rules), use default parameters or reset correlation parameter (as Sliding Window Parameters and Peak Detection Parameter, this study general is selected default parameters).Submit after sequence, MAR-Wiz is by the detailed analysis result output of sequence to be measured, according to MAR-Wiz analytical results, select the sequence of " Minimum Potential >=1.0000 possess (Core Region) and whole MAR Motifs between core area " as candidate MAR sequence.As said in literature review, TG-Richness is only that some MAR sequences are all.Meanwhile, in multiple sieve series row, only there is minority sequence to contain TG-Richness.The multiple sieve series row that therefore, also can " not containing TG-Richness, but contain between core area or possess higher MAR Potential " are included candidate MAR sequence in.The signature analysis of candidate MAR sequence is as shown in table 1.

Core primitive kind and the number thereof of contained MAR sequence in table 1 candidate MAR sequence

embodiment 2:the clone of candidate MAR sequence

The extraction of 2.1 rice leaf genomic dnas

Extract plant genome DNA, adopt CTAB method, process is as follows:

Get the rice leaf of about 0.1g and clay into power in liquid nitrogen, appropriate powdered material is transferred in 1.5mlEP pipe.1.5 * CTAB the damping fluid that adds 600 μ l preheatings (95 ℃), then 65 ℃ of temperature are bathed 45min.Take out EP pipe, add 600 μ l chloroforms after being cooled to room temperature: primary isoamyl alcohol (24:1) mixes, and then the centrifugal 10min of 10000rpm, draws supernatant liquor in another new EP pipe after taking out.Add the Virahol of 2/3 volume to mix, ice bath to DNA filament occurs.Centrifugation DNA, outwells supernatant liquor a little.Add 600 μ l75% washing with alcohol, place 30min.Outwell supernatant liquor, under room temperature, dry up DNA.Add 100 μ l ddH ₂o dissolving DNA.Electrophoresis detection plant genomic DNA content.

2.2 pcr amplification candidate MAR sequences

According to candidate's two MAR sequences, two pairs of primers are synthesized in design respectively:

（SEQ ID NO.3）：M1F 5'-GCGCAGCTGATTCCTCCCTCTACCTATGTTTG-3'

(SEQ ID NO.4): M1R 5'-GCGCAGCTGCATCCCTGTAAAAGGAAGAGTAG-3' and

（SEQ ID NO.5）M2F 5'-GCGCAGCTGTCAATACACAAGTCCGAGTCGC-3'

(SEQ ID NO.6) M2R 5'-GCGCAGCTGACCTCCTCCAAAACCTAATCAC-3' be used for increasing candidate MAR sequence M1 and M2.

PCR reaction system is as follows:

Constituent concentration volume

Genomic templates DNA 50 ng/ μ l 1 ul

dNTP 2.5 mM 4 ul

10 times of 5 ul of Taq enzyme buffer liquid

Primer 10 μ M 5 ul

Taq enzyme 2.5 U

Add ddH2O to cumulative volume 50 ul.

PCR response procedures is respectively:

M1:94 ℃, 5 minutes; 94 ℃, 1 minute, 58 ℃, 1 minute, 72 ℃, 1 minute, 29 circulations; 72 ℃ are extended 10 minutes.

M2:94 ℃, 5 minutes; 94 ℃, 1 minute, 57 ℃, 1 minute, 72 ℃, 2 minutes, 29 circulations; 72 ℃ are extended 10 minutes.

The pcr amplification product of M1 and M2 reclaims respectively and is connected into T carrier pMD19-T(TaKaRa company, Dalian) in, through sequence verification and sequence alignment, clone MAR sequence meets experiment expection.

embodiment 3:the structure of plant expression vector

The acquisition of 3.1 candidate MAR intermediate carrier pMD19T-M1 and pMD19T-M2

After the pcr amplification product of M1 and M2 is reclaimed through sepharose, TA clone's method is connected in T carrier pMD19-T, obtains respectively intermediate carrier pMD19T-M1 and pMD19T-M2.

The acquisition of 3.2 GUS expression cassette CaMV35S:GUS:Tnos

Restriction enzyme hindiII and ecorI double digestion plasmid pCAMBIA1300-AGS obtains GUS fragment, after processing and fill end through Klenow klenow fragment, be inserted into by kpni is mono-to be cut and passes through in the plasmid pSPROK that T4 polysaccharase fills processing, obtains plasmid pSPGUSA.

3.3 synthetic of multiple clone site short-movie section and the acquisition of intermediate carrier pMD19-Ts-MCS

In order to facilitate the structure of carrier, the multiple clone site (design as shown in Figure 1) of one section of short-movie section has been synthesized in artificial design, and synthetic multiple clone site is inserted into T carrier pMD19T-Simple(TaKaRa company, Dalian) in obtain plasmid pMD19-Ts-MCS.

3.4 plant expression vector pCAMBIA1300-M1- gusand pCAMBIA1300-M2- gusacquisition

Restriction enzyme pvuiI single endonuclease digestion plasmid pMD19T-M1 obtains MAR sequence M1 fragment, and the mode with inverted repeat of being taken up in order of priority is inserted into plasmid pMD19-Ts-MCS's stui site and ecorV site obtains plasmid pMD19Ts-MCS-M1.Use again restriction enzyme avriI digested plasmid pMD19Ts-MCS-M1 obtains the M1 fragment of two inverted repeats and fills end with Klenow, is inserted into hindiII is mono-to be cut and Klenow fills in the plasmid pCAMBIA1300 of end and obtains plasmid pCAMBIA1300-M1.With hindiII and ecorI double digestion plasmid pSPGUSA Klenow fill end and obtain GUS expression cassette fragment, are inserted into plasmid pCAMBIA1300-M1's hpai site, builds and obtains plant expression vector pCAMBIA1300-M1- gus, plasmid map as shown in Figure 2.

By same structure flow process, obtain plant expression vector pCAMBIA1300-M2- gus, plasmid map as shown in Figure 3.

3.5 positive control expression vector pCAMBIA1300- gusacquisition

Restriction enzyme hindiII and ecorI double digestion plasmid pSPGUSA obtains GUS expression cassette fragment, is inserted in the plasmid pCAMBIA1300 of same double digestion, build obtain plant expression vector pCAMBIA1300- gus, plasmid map as shown in Figure 4.

embodiment 4:the acquisition of transgenic paddy rice

4.1 NB culture medium prescriptions (minimum medium)

N6 is a large amount of: saltpetre KNO ₃2830 mg.L ^-1, sulfate of ammoniac (NH ₄) ₂sO ₄463 mg.L ^-1, potassium primary phosphate KH ₂pO ₄400 mg.L ^-1, magnesium sulfate MgSO ₄7H ₂o 185 mg.L ^-1, calcium chloride CaCl22H2O 166 mg.L ^-1;

B5 trace: boric acid H ₃bO ₄3 mg.L ^-1, Liu Suan Manganese MnSO ₄h ₂o 7.58 mg.L ^-1, zinc sulfate ZnSO ₄7H ₂o 2 mg.L ^-1, potassiumiodide KI 0.75 mg.L ^-1, Sodium orthomolybdate Na2MoO ₄2H ₂o 0.25 mg.L ^-1, copper sulfate CuSO ₄5H ₂o 0.025 mg.L ^-1, cobalt chloride CoCl ₂6H ₂o 0.025 mg.L ^-1;

Molysite: ferrous sulfate FeSO ₄7H ₂o 27.8 mg.L ^-1, disodium ethylene diamine tetraacetate Na ₂eDTA 37.3 mg.L ^-1;

Inositol: inositol Myo-inositol 100 mg.L ^-1;

Organic composition: vitamin ThiamineHCl 10 mg.L ^-1, pyridoxine hydrochloride PyridoxineHCl 1 mg.L ^-1, nicotinic acid Niacin 1 mg.L ^-1, caseinhydrolysate Casamino acids 300 mg.L ^-1, glutamine Glutamine 250 mg.L ^-1, proline(Pro) Proline 500 mg.L ^-1, glycine Glycine 2 mg.L ^-1, sucrose Sucrose 30000 mg.L ^-1, agar Phytagel 2400 mg.L ^-1

PH value 5.8-5.9.

Attention: organic composition can not autoclaving, must use filter suction filtration ,-20 ℃ of preservations after packing

The cultivation of 4.2 callus

The tender seed of children after water intaking rice children tassel blossom 12-15 d, peels off clever shell, in 75% ethanol sterilizing 0.5-1 min, then receives solution sterilization 15-30 min with 10-30% hypochlorous acid, and in super clean bench, aseptic water washing 3-4 time, is placed on aseptic filter paper and dries.With tweezers and dissecting needle, take out rataria, be placed in inducing culture (minimum medium+2,4-D, 2 mg L ^-1; PH 5.8-5.9) upper, 27 ° of C secretly cultivate.After 10-15 d, excise the bud scale of rataria etc., the cell that only leaves end expands body, transfers in new inducing culture (minimum medium+2,4-D, 2 mg L ^-1; PH 5.8-5.9), in, every 20 d succeeding transfer culture 1 time on inducing culture, obtains afterwards the embryo callus rapidly of growing 3 times and makes Agrobacterium-mediated Transformation thereafter.

4.3 agrobacterium mediation converted:

With reference to the specification sheets of BIO-RAD company electric exciter, plant expression vector p1300-LF-AGCSGS is transformed in agrobacterium tumefaciens lba4404 by electrization.

Get eugonic embryo callus in sterilizing culture dish, add respective concentration (OD ₆₀₀value 1-2) after agrobacterium liquid (agrobacterium tumefaciens lba4404 after above-mentioned carrier transforms) soaks 3-5 min, callus is taken out and dried, then transfer in the common substratum that is covered with an aseptic filter paper (minimum medium+2,4-D, 2 mg L ^-1+ AS 100 μ M; PH 5.2) upper, 28 ℃ of lucifuges are cultivated 2-3 d altogether.After cultivating altogether, callus is transferred in sterile petri dish, in super clean bench, rinsed with sterile water is 3 times, then with containing Pyocianil 250 mg L ^-1aseptic washing 1 time to kill except Agrobacterium, with aseptic filter paper, blot after taking out callus, transfer in containing Pyocianil 250 mg L ^-1with hygromycin 50 mg L ^-1or PPT 15 mg L ^-1screening culture medium (minimum medium+2,4-D, 2 mg L ^-1+ Pyocianil 250 mg L ^-1+ hygromycin 50 mg L ^-1(or PPT 15 mg L ^-1, pH 5.8-5.9) and upper 28 ℃ of lucifuges cultivation screening resistant calli.The callus infecting is cultivated after 20-30 d in screening culture medium, and the resistant calli that edge is grown is transferred and in new screening culture medium, continued screening 1-2 time.

The resistant calli obtaining in screening culture medium screening is transferred in division culture medium (minimum medium+KT 10 mg L ^-1+ NAA 0.4 mg L ^-1; PH 5.8-5.9) upper, 28 ℃ of dark 7-10 d that cultivate, then move to (14 h/d) under illumination and cultivate, and grow gradually subsequently green point, are finally divided into seedling.After seedling breaks up completely, transferring, (1/2 MS inorganic salt (referring to that inorganic salt content reduces by half)+MS organic composition (refers to the organic composition in NB culture medium prescription (minimum medium), comprises vitamin ThiamineHCl 10 mg.L in root media ^-1, pyridoxine hydrochloride PyridoxineHCl 1 mg.L ^-1, nicotinic acid Niacin 1 mg.L ^-1, caseinhydrolysate Casamino acids 300 mg.L ^-1, glutamine Glutamine 250 mg.L ^-1, proline(Pro) Proline 500 mg.L ^-1, glycine Glycine 2 mg.L ^-1, sucrose Sucrose 30000 mg.L ^-1, agar Phytagel 2400 mg.L ^-1)+hygromycin 30 mg L ^-1(or PPT 10 mg L ^-1); PH 5.8-5.9) 28 ℃, illumination 14 h/d cultivate 14-21 d and take root and screen.The resistance seedling obtaining after taking root washes away after root substratum, moves in 1/10 MS suspension culture 3-5 d and can grow new root, then transplants in greenhouse or paddy field, solarium.

The PCR of 4.4 transgenic paddy rices detects

Adopt CTAB method to extract plant genome DNA, process is as described in 2.1 joints of [embodiment 2].

PCR detects Totomycin (hyg) resistant gene in regeneration plant, according to the synthetic pair of primers of the sequence-specific of Totomycin (hyg) resistant gene, as follows:

(SEQ ID NO.7) upstream primer is: 5 '-TACACAGCCATCGGTCCAGA-3 '

(SEQ ID NO.8) downstream primer is: 5'-TAGGAGGGCGTGGATATGTC-3'

PCR reaction system is as described in 2.2 joints of [embodiment 2].

PCR response procedures: 94 ℃, 5 minutes; 94 ℃, 1 minute, 56 ℃, 1 minute, 72 ℃, 1 minute, 35 circulations; 72 ℃ are extended 10 minutes.

The result that transgenosis PCR detects is shown in Figure 5, and the sample that can amplify 840bp band is transgenic paddy rice positive plant, and result shows that sample 1-6 and 9-11 are transgenic positive plant.

embodiment 5:the GUS enzyme of transgenic paddy rice blade is lived and is analyzed

The extraction of 5.1 rice leaf gus proteins

Blade is chosen young leaflet tablet near blade tip position, and about 5-10mg puts into 2ml centrifuge tube, and every pipe adds 600 μ l GUS extraction buffer(50mM NaHPO ₄(phosphoric acid buffer), pH7.0,10 mM 2-mercaptoethanol, 10mM Na ₂eDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100).Then every pipe adds a diameter 6mm stainless shot, on the sample milling machine TissueLyser of QIAGEN company with the frequency vibrations grind away of per minute 20 times 5 minutes.Then on refrigerated centrifuge 4 ℃ 13000 revs/min centrifugal 10 minutes, supernatant is proceeded in 96 hole microwell plates, note recording the corresponding hole number of sample.Silica-gel plate sealing, be stored in 4 ℃ standby, within 2 days, detected as early as possible.

The mensuration of 5.2 total proteins

The quantitative assay of total protein adopts classical Bradford method (1976), and concrete operations are with reference to the Bradford quantification of protein test kit (catalog number (Cat.No.): specification sheets PA102) carries out of biological (Beijing) company of sky root.Xylene Brilliant Cyanine G G-250 dye liquor is oneself preparation, and BSA standard substance adopt mentioned reagent box to provide, and test adopts 96 hole microwell plates to measure.Reaction system adopts 285 μ l Xylene Brilliant Cyanine G G-250 dye liquor+15 μ l protein solutions, in standard curve making, increase the concentration range of standard substance BSA, standard substance concentration is chosen respectively 0,1/15,2/15,3/15,4/15,5/15,6/15,7/15,8/15,9/15 mg/ml and is measured.With different BSA standard substance concentration and corresponding OD ₅₉₅value is made scatter diagram in Excel table, and obtains both dependency equation and degree of variation R ²value.If the concentration of testing sample exceeds the scope of this typical curve, need suitably after dilution, to redeterminate, final concentration value need to convert and go back according to concrete extent of dilution.

The making of 5.3 GUS enzymes fluorometric assay typical curve alive

GUS enzyme activity determination mainly carries out with reference to the service manual of DyNA Quant 200 Fluorometer of Pharmacia Biotech company.Take 4-MU sodium salt (molecular weight 198.20) 19.8mg and be dissolved in 100ml deionized water, obtain 4-MU stock solution A.Get 10 μ l 4-MU stock solution A and join in 10ml deionized water, obtain 4-MU stock solution B.The 4-MU stock solution B that adds 0,25,50,100,150,200,300,400,500,600,700,800,900,1000 μ l according to every pipe, then with the Carbonate stop buffer of 0.2M, supply 1000 μ l respectively, every pipe is got reading on 200 μ l deblooming detectors and is measured.With the fluorescence reading that different 4-MU concentration is corresponding different, in Excel table, make scatter diagram, and obtain both dependency equation and degree of variation R ²value.

The enzymic catalytic reaction of 5.4 gus protein enzymes

Get 96 hole microwell plates and be placed in precooling on ice, then every hole adds rapidly the GUS assay buffer of 50 μ l precoolings, then adds fast the testing sample protein solution of every hole 50 μ l, and shrouding paper seals.Put into fast 37 ℃ of incubators and be incubated 45 minutes, then every hole adds rapidly the Carbonate stop buffer(0.2M Na of 900 μ l 0.2M ₂cO ₃) termination reaction.

The fluorometric assay that 5.5 GUS enzymes are lived

Substantially the fluorescence measurement instrument FLUOROSCAN ASCENT FL specification sheets with reference to Thermo Scientific company operates.The spectral filter that the spectral filter that filter set selective exitation wavelength is 355nm and emission wavelength are 460nm, scale-up factor is 5/1, scan mode is endpoint determination, using do not add any sample blank well as blank negative control.Adopt the microwell plate of 96 hole enzyme plates to measure, every hole application of sample 200 μ l.If the maximum value that the fluorescent value of sample is greater than or is near the mark in curve, this sample needs suitably after dilution, to resurvey.

The analysis of 5.6 GUS enzyme live datas

The unit definition of GUS enzyme activity is the mole number of the 4-MU with fluorescence activity that produces of the GUS zymoprotein catalysis of unit mass in the unit time, is specially the picomole number (being pmol/mg/min) of the 4-MU that every milligram of zymoprotein catalysis of per minute produces.GUS enzyme live data is all converted into the enzyme activity unit of standard, according to different enzyme activity units, judges gusthe expression level of gene.

Turn expression vector pCAMBIA1300-M1- gus, pCAMBIA1300-M2- gusturn pCAMBIA1300-with positive control gustransgenic paddy rice and the GUS enzyme of the single-strain blade of the negative control non-transgenic paddy rice measurement result of living as shown in Figure 6.The GUS enzyme loose joint of negative control is bordering on zero level, negligible.The GUS enzyme of positive control is lived mostly in 300-1400(unit: pmol/mg/min), and average about 660(unit: pmol/mg/min).Turn pCAMBIA1300-M1- gusthe GUS enzyme of transgenic paddy rice live mostly in 2000-5000(unit: pmol/mg/min), average about 3300(unit: pmol/mg/min).Turn pCAMBIA1300-M2- gusthe GUS enzyme of transgenic paddy rice live mostly in 9000-13000(unit: pmol/mg/min), average about 12400(unit: pmol/mg/min).No matter presentation of results, put down or the GUS enzyme of colony average alive from the individual plant GUS enzyme running water of transgenic paddy rice, and the existence of MAR sequence has all effectively improved the expression level of foreign gene.In addition, from the difference between individual plant, obviously little than the intragroup difference of positive control with the transgenic paddy rice of MAR sequence, show that the existence of MAR sequence has improved the expression stability of foreign gene.

Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make according to the present invention various changes and distortion, and these changes and distortion all should belong to the scope of claims of the present invention.

<110> Fujian Province Agriculture Science Academy, Institute of Biotechnology

Two paddy rice MAR sequences of <120> and application thereof

<160> 8

<210> 1

<211> 978

<212> DNA

The fine paddy rice of <213> Japan ( oryza sativa L. japonica cv.)

<400> 1

attcctccct ctacctatgt ttgcaaaggt agcttttaaa tttggtacag cttttttcaa 60

gctaattttg gattttaact ctttttaata ataattttag gtcatcttct tttacttttc 120

ttcataacaa tataaaagtt gagtaacaaa aaacaatgaa gaaagcccga aaatctaaaa 180

tcacaaattg tagcatcaca gtggactacg ccctccatct aaaaatatat aagtattttt 240

ttacggaaat taagggaaaa tgtgaaatta aatgagaaaa gtttgtgata agttaagaca 300

tgagaaagta aaaatagaga aattaaatga gagatgattg tgattgaagt agctagaaat 360

tgatatgttt tgagataatt tttttagtta gaagatgcta ttattagggt gcattctaaa 420

gtgcttttga acggatgatt agccatgtgt aaaacgagaa gcgtgataag tatatgatta 480

attgggtatt agttattaaa atcttgaaaa acatatattt tttatttttt taaagcaact 540

cgttatatag gaagttttta gaagaaacgt aacgtttagc aattttaaat gtgtgctaac 600

aaaaattgag gaagtagcca aactaaaatg gagttttgaa cgtgttgagt ctaatttaca 660

taatctcaat gatttagttt aaacatgatc acttttactc aatgcagaat ttatttatct 720

gctaacttga ggatgaagcc attgaagccg gtcactttga tttacttctt gttctttctt 780

ttctcatctt ctcgtaattc ttctcactgt aggagaagta tagggcgttg gcttttctgt 840

caataaaaca aaagaaaatt atatagatca cctcactaat ctcttcccgc atcagttgat 900

cggacgactt aggttctagg gttttagaat tgagagagtt ggggcgtccc ccctcctact 960

cttcctttta cagggatg 978

<210> 2

<211> 1777

<212> DNA

The fine paddy rice of <213> Japan ( oryza sativa L. japonica cv.)

<400> 2

tcaatacaca agtccgagtc gcgaatagcc ttgtagttg atttgacatt acaagtttata 60

gtttatatgt tactccctct ctctttttat attactaga aaaatactcg tgcgttgcaaa 120

ggatgaagtc tattttaatc ttattattgt tatatggtt tagttaagat gaaattcacta 180

tgggagtacg cttggatata tattttttta gaaaatcat gagttgtagt taggagaccga 240

tcgtctcaag ttagcatgtg agtttttttt taaacagat ttcttatatg attcattctgt 300

attactaaaa gtgaacgatc ttaaaaaccg actcaaata cggatatgta tttccaaaagc 360

aaaacgaact taaaaaccga ctcatacacg gatgacgta ccaaaatacc agcaaaaacat 420

cttcaatttt tataatagta gagataaaga tagagaaga gatatagatt aaaaccaaaac 480

ataaaattaa aaccgactca aacacggatg acgaaccac ggaaacatct tcaatttttat 540

aaccgactca aatacggatg acggaccgcg gaaacatct ttaattttta taatagtagag 600

atgcccgttc attgcaacgg gtgaagtcta ttttaatct tattattaat atatggtttag 660

ttaagatgaa attcactgtg tgagttcgct tggatatat atatttttag aaaatcatgag 720

ctgcagttag gagttcgatc gtcttaagtt agcatgcga gtttttttaa acagatttctt 780

atatgattcc ttctgtatta acaaaagcaa acgaactta aaaaccgact catacacggat 840

gacgtaccaa aataccggca aaaatatctt caattttta taatagtaga gataagacata 900

agtcattttg attttttttt ctaatcaact ttgtgaagc aacatctaaa acaccaaatta 960

gtttcattaa atttaacatt gtatatattt tgataatat atttttttgt tgaaaaatgct 1020

actatatttt tctataaact taatcaaact tatcaaact taaagaattt tgactagaaaa 1080

aagtcaaaac aatttataat ataaaacgga gggagtata ttacaaaaca attcacaatgt 1140

caaaatattt tacagccact cttgttgctt ccaaggaat gaggatatat ggaaacatatc 1200

ttatgcataa atatcacgaa aattgatata aagaaatgg acatgtactc ctaaaagtatt 1260

acacatatat gcaaagtctc atattcaaat tctttatat tttagccgta gcaaaaaaaaa 1320

gtgaaaaatc tgataatttt aagagtaaaa gtgtgttac ggttaaaata taaagaatttg 1380

aagataaaat tttacgcata ggtgtagtac ttttaaatt tacgtgtcta atttttttaaa 1440

aaatttttat ggtatttgtt agttggtgtg tacagaaat tttgtttgta taggatatttt 1500

tccaagtaat ttttttccgt ttgttttagt tttttccct gttattttaa taagataaccg 1560

atcgattatt ggactttgct ttaaacaaaa attgcgtgg gctggtgacc acgtggtttgt 1620

acaaaaacaa taacggaaac acggagggat ctgcctaac tagtatccat aaattcaataa 1680

ctgaacgtgt gctcagagac attttcggat gatcctagg acttgctggc tccaaattaga 1740

ccaatgacgt cgttcgtgat taggttttgg aggaggt 1777

<210> 3

<211> 32

<212> DNA

<213> artificial sequence

<400> 3

gcgcagctga ttcctccctc tacctatgtt tg 32

<210> 4

<211> 32

<212> DNA

<213> artificial sequence

<400> 4

gcgcagctgc atccctgtaa aaggaagagt ag 32

<210> 5

<211> 31

<212> DNA

<213> artificial sequence

<400> 5

gcgcagctgt caatacacaa gtccgagtcg c 31

<210> 6

<211> 31

<212> DNA

<213> artificial sequence

<400> 6

gcgcagctga cctcctccaa aacctaatca c 31

<210> 7

<211> 20

<212> DNA

<213> artificial sequence

<400> 7

tacacagcca tcggtccaga 20

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<400> 8

taggagggcg tggatatgtc 20

Claims (4)

1. the nucleotide sequence of the MAR in a paddy rice source, is characterized in that: the nucleotide sequence of described MAR is as shown in SEQ ID NO.1.

2. the nucleotide sequence of the MAR in a paddy rice source, is characterized in that: the nucleotide sequence of described MAR is as shown in SEQ ID NO.2.

3. the application of the nucleotide sequence of the MAR in paddy rice as claimed in claim 1 or 2 source, it is characterized in that: this application is the nucleotide sequence of the MAR in described paddy rice source to be connected into the both sides of foreign gene, construction of expression vector, and by expression vector transformed plant and obtain transgenic plant; Described plant is paddy rice.

4. the application of the nucleotide sequence of the MAR in paddy rice according to claim 3 source, is characterized in that: described foreign gene is gus gene.