CN100436584C - Seed specificity promoter, core function fragment and application thereof - Google Patents

Abstract

The invention discloses the seed specificity promoter and application. The promoter is one of the nucleic acid sequences: 1 DNA sequence of SEQ ID No.:5; 2 nucleic acid sequences have 90% consanguinity with DNA sequence of SEQ ID No.:5, promotor gene transcription function and seed specificity; 3 nucleic acid sequences can cross with DNA sequence of SEQ ID No.:5 at rigorous condition. The invention clarify the expression regulate and control mechanism of MBL in betelnut plant, and provides the theoretical basis for MBL gene transferring to routine crop; the invention has unique dominance and wide prospect for expressing the polypeptide drug functional protein gene.

Description

Seed specific promoters and core function fragment and application

Technical field

The present invention relates to plant gene promoter and application thereof, particularly relate to a seed specific promoters and an application in the plant quality improvement thereof that derives from Semen Capparis.

Background technology

Different genes specifically expressing in different developmental phases and different tissues is the needs that plant itself grows.Due to different genes specifically expressing without any confusion in the organism, key are that special cis-acting elements and specific transcription factor have nothing in common with each other in its promotor.Studies show that, generally have the element of various control tissue specific expression in the tissue-specific promoter simultaneously, its expression specificity is by the common decision of kind, quantity and the relative position etc. of these elements.In the genetically engineered improvement of plant, on purpose use efficient tissue-specific promoter, it is non-specific in recipient plant to overcome constitutive promoter startup foreign gene, continue and efficiently express the waste that is caused, even the heterologous protein of great expression can be broken the original metabolic balance of plant, hinder the normal growth of plant to grow, and in needing the particular organization of great expression, cross low falling flat because of expression amount again sometimes, therefore, further investigate these tissue-specific promoters and not only help to illustrate phytomorph, grow, basic theory such as physiology and metabolism, and be with a wide range of applications, be emphasis and the difficult point in the plant genetic engineering research for a long time always.

Seed development comprises the coordination in succession of series of genes to be expressed, and reason is that the promotor of these genes is activated in succession, and the expression regulation mechanism of therefore studying these genes and promotor thereof has important theory and realistic meaning.At present, the more seed specific promoters of research mainly is endosperm specificity promoter and embryo in the dicotyledons and the cotyledon specificity promoter in the monocotyledons.

At present, the seed specific promoters of dicotyledons research mainly concentrates on the napin gene promoter of rape (Brassica) and 11S, the 7S protein gene promoter of beans.Napin albumen is the seed storage protein that extensively is present in the brassica plant, and 1.7S albumen is otherwise known as.Up to now, the napin protein gene of many plants, as napA, napB, BcNA1 and pGNA etc. obtain clone and order-checking (Xiong Xinghua etc. in succession, the clone and the sequential analysis of Semen Brassicae campestris specifically expressing napin gene promoter, biotechnology, 13 (3): 4-5,2003), analyze their regulating and controlling sequence and find nucleotide sequence and the closely related (Ellerstrom etc. of seed-specific expression that some is specific, Functional dissection of a napin gene promoter:identification of promoterelements required for embryo and endosperm-specific transcription, PlantMolecular Biology, 32:1019-1027,1996).Zhang etc. have been separated to the partial sequence nap300 (Zhang etc. of napinB promotor from the genomic dna of rape H165, Identification ofseed-specific promoter nap300and its comparison with 7S promoter, Pro.Nat.Acad.Sci, 12 (10): 737-741,2002), the promotor of it and 7S protein gene is carried out sequence alignment to be found, some sequences of high conservative have during evolution been comprised in the napinB promotor, as rich AT sequence, the napin-motif conserved sequence, RY tumor-necrosis factor glycoproteins and G-box etc., these cis-acting elements also are the binding site (Wu etc. of nucleoprotein, Analysis of 5 ' region of glutelin genes from wild rice species, Bot Bull Acad Sin, 37:41,1996), may play important regulation to seed-specific expression.

The happy grade of the blue or green lattice of wealth sound cloned soybean beta-conglycinin gene α subunit promoter fragment BCSP666, the promotor of it and α ' subunit gene is compared, though find the sequence homology very low (30-40%) between these promotors, but the quantity of contained promoter sequence element is last very similar with distance, the RY tumor-necrosis factor glycoproteins element that all has multiple copied, the AGCCCA sequential element, napin-motif sequential element and rich AT sequence (the blue or green lattice of wealth sound are happy etc., Scientia Agricultura Sinica, 38 (3): 454-461,2005).The promotor of proof α ' subunit genes such as Beachy has stronger seed-specific, and above-mentioned these promoter sequence elements and the closely related (Beachy etc. of seed specific sexual function, Accumulation and assembly of soy-bean β-conglycinin in seads oftransfomed petunia plants, The EMBO, 4 (12): 3047-3053,1985).Cahoon etc. utilize the promotor of α ' subunit gene successfully to express fatty acid dehydrogenase gene in somatic embryos of soybean, further confirm its seed specific activity and showed its application prospects (Cahoon etc., Production offatty acid componcnts of meadow foam oil in somatic soybean embryos, PlantPhysiol., 124:243-251,2000).

Alcohol soluble protein gene promotor and glutenin gene promotor are the emphasis and the focuses of monocotyledons seed specific promoters research.Study more alcohol soluble protein gene and glutenin gene and comprise corn (Zea mays L.) 19KD, 22KD zein gene (Heideckerr etc., Structural analysis of plant genes, Annual.Rev.Plant physiol, 37:439-446,1986), wheat glutenin, a/ β-gliadin (Reeves andOkita, Analysis of a/ β-gliadin genes from dipliod and hexaploid wheats, Gene, 52:257-266,1987), γ-gliadin gene, barley B ₁-hordein gene (Forde etc., Nucleotide sequence of B, hordein gene and the identification of possibleupstream regulatory elements in endosperm storage protein genes from barley, wheat and maize, Nucleic Acids Res., 13:7327-7339,1985) and the 4a gene (Zhao Jun etc. of paddy rice, the rice paddy seed prolamine 4a expression of gene combined promoter of being connected is regulated and control, Chinese science C collects, 26 (2): 156-163,1996) etc.In the promotor of all glutenin genes, all contain GCN4 motif, AACA motif and GCAA motif, and their position is basic identical; In the promotor of alcohol soluble protein gene, except that containing said elements, also contain Prolamin-box (prolamine frame).The research of Wu etc. thinks that the GCN4 motif is the critical elements of decision endosperm specific expression, the combination of GCN4 motif, AACA motif and ACCT motif is competent (Wu etc. for the endosperm specific expression characteristic, Analysis of 5 ' region of glutelin genes from wild ricespecies, The Plant J., 23 (3): 415-421,2000).

Semen Capparis (Capparis masaikai L é vl.), have another name called horse gold capsule, Ma Jinnan, Tai Ji (Yunnan Chinese medicine another name), Root of Common Javatea (Xichou, Yunnan), purple betel nut or water betel nut (Baise of Guangxi), Capparaceae (Capparidaceae) Chinese lime belongs to (Capparis) plant, mainly be distributed in China torrid zone, subtropical zone, be the regional a kind of perennial evergreen woody climber of high height above sea level (800-1600m), the climbing shrub that originates in provinces and regions such as China Guangxi, southeastern Yunnan and South of Guizhou, to ten several meters, also is a kind of rare wild fruit tree in imminent danger up to several meters.Can be used as medicine after the seed peeling of Semen Capparis, be the important medicine source of " shangqing pill ".Kind of benevolence hardship, sweet, cold, have hasten parturition, contraception, clearing heat and detoxicating, control swelling and pain in the throat, evil pyogenic infections from tumour or sore, promoting production of body fluid and nourishing the lung, aid digestion, go the spot acute diseases such as cholera and sunstroke and multiple efficacies such as sober up.Its kind benevolence food also have prolonged sweet taste, be because of being rich in (Liu etc. due to the monellin mabinlin, Purification, complete aminoacid sequence and structural characterization of the heat-stable sweet protein, mabinlin II, Eur.J Biochem., 211:281-287,1993).

The seed of Semen Capparis is flat volution, and embryo is positioned at kind of benevolence central authorities, and plumular axis is the strip dish of hypertrophy around cotyledon, and plumular axis is reserve protein and greasy main place.Sweet ingredient in the Semen Capparis seed is mabinlin (MBL) albumen (claiming Ma Binling or mabinlin), it is the main storage protein of Semen Capparis seed, account for 4% (Hu Zhong etc. of kind of benevolence dry weight, the research I of mabinlin, Yunnan plant research, 5 (2): 207-212,1983), account for 1.4% (Liu etc. of full seed dry weight, Purification, complete amino acid sequence andstructural characterization of the heat-stable sweet protein, mabinlin II, Eur.J Biochem., 211:281-287,1993), this albumen is a kind of alkaline white protein (albumin) that has, and is present in the proteoplast of plumular axis cell, accounts for 90% of proteoplast total protein content, its sugariness is 375 times (comparatively basic with weight ratio) of sucrose approximately, and the sweet taste threshold value is about 0.1%.

Semen Capparis is one of 6 kinds of plants that produce sweet protein in the whole world, and is exclusive, the unique sweet plant protein resource plant of China.Mabinlin mabinlin has 4 kinds of isoproteins, be respectively MBL I-1, II, III, IV, the cDNA sequence of these protein genes is all cloned and is checked order, wherein the molecular weight of MBLII is 10.4KD, iso-electric point is 11.3, more stable under acidic conditions, and still can keep its sweet taste activity (Liu etc. at 100 ℃ of insulation 48h, Purification, complete amino acid sequence and structural characterizationof the heat-stable sweet protein, mabinlin II, Eur.J Biochem., 211:281-287,1993).Compare with other monellin, though the proteic sugariness of mabinlin is not very high (is about as the sugariness of monellin thaumatin sucrose 3000 times), but its acid resistance is good, the thermostability height, and be the highest (Faus etc. of thermostability in all monellins, Recent developments in the characterization andbiotechnological production of sweet-tasting proteins, Appl.Microbiol.Biotechnol., 53 (2): 145-51,2000), therefore promise to be most new food additive, even become the part substitute of sweeting agent sucrose, therefore further investigate significant to Semen Capparis and monellin mabinlin thereof.

As the mabinlin mabinlin that accounts for plumular axis proteoplast total protein content 90%, the promotor of its gene may be the strong promoter of seed-specific expression, and as a kind of white protein, the promotor of its gene may have and the different using value of known seed specific promoters (as the isogenic promotor of legA, napA, napB and Zein), so the clone of mabinlin gene (MBL) promotor has crucial meaning.

Summary of the invention

The purpose of this invention is to provide a promotor with mabinlin gene of organ of multiplication, particularly seed-specific.

Organ of multiplication specificity promoter provided by the present invention, name is called MBL-P695, derives from Chinese lime platymiscium Semen Capparis (Capparis masaikai L é vl.), and its core function fragment is one of following nucleotide sequence:

1) dna sequence dna of SEQ ID NO:2 in the sequence table;

2) dna sequence dna of SEQ ID NO:3 in the sequence table;

3) dna sequence dna of SEQ ID NO:4 in the sequence table;

4) with sequence table in the nucleotide sequence of the dna sequence dna hybridization that limits of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

SEQ ID NO:2 in the sequence table is by 300 based compositions; SEQ ID NO:3 in the sequence table is by 424 based compositions; SEQ ID NO:4 in the sequence table is by 510 based compositions.

Described seed specific promoters MBL-P695 with above-mentioned core function fragment is one of following nucleotide sequence:

1) dna sequence dna of SEQ ID NO:5;

2) with sequence table in the dna sequence dna that limits of SEQ ID NO:5 have 90% above homology and have the nucleotide sequence of promotor gene functional transcription and seed-specific;

3) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with SEQ ID NO:5 in the sequence table.

The rigorous condition of above-mentioned height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, wash film under 65 ℃ of conditions.

SEQ ID NO:5 in the sequence table is by 695 based compositions.

The expression vector, transgenic cell line and the host bacterium that contain promotor of the present invention and core function fragment thereof all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention in amplification MBL-P695 and the core function fragment thereof.

The invention provides seed specific promoters MBL-P695 and core function fragment thereof, this promotor is to adopt the promotor of the kind benevolence monellin mabinlin gene that the GenomeWalking method clones from Chinese lime is born in the year of horse betel nut (Capparis masaikai L é vl.).Arabidopis thaliana transgenosis evidence MBL-P695 and core function fragment thereof can be given gus reporter gene high level expression in plant plant propagation organ (particularly seed), have stronger tissue specificity.One aspect of the present invention helps deeply to illustrate the expression regulation mechanism of mbl gene in Semen Capparis, and be the conventional crop of mbl gene genetic transformation, established theoretical basis as soybean, corn, paddy rice, coffee and various gourd, fruit and vegetables etc., to in the plant quality-improving of (comprising unifacial leaf and dicotyledons, especially spermatophyte), play a significant role; On the other hand, because MBL is a kind of white protein, and many functional proteins (as enzyme) and physiologically active ingredient etc. all are white proteins, so the promotor MBL-P695 of mbl gene of the present invention and core function fragment thereof also demonstrate unique advantage and potential application prospect to polypeptide drugs class functional protein expression of gene.

Below in conjunction with specific embodiment the present invention is described in further details.

Description of drawings

Figure 1A is the agarose gel electrophoresis detected result of first round nest-type PRC amplified production

Figure 1B second takes turns the agarose gel electrophoresis detected result of nest-type PRC amplified production

Fig. 2 is the The sequencing results that contains the dna fragmentation of promotor MBL-P695 sequence

Fig. 3 is the physical map of plant expression vector pVKH-35S-GUS-pA

Fig. 4 is the double digestion qualification result of the plant expression vector of the agarose gel electrophoresis detected result of the MBL-P779 series deletion fragment of pcr amplification and contrast LegA-P and the MBL-P779 series deletion fragment that merges gus gene, LegA-P

Fig. 5 A is the GUS histochemical stain result of MBL-Pa, MBL-Pb, MBL-Pc and MBL-Pd transgenic arabidopsis root, stem, leaf

Fig. 5 B is the GUS histochemical stain result of MBL-Pa, MBL-Pb, MBL-Pc and MBL-Pd transgenic arabidopsis flower

Fig. 6 is the GUS histochemical stain result of MBL-Pa, MBL-Pb, MBL-Pc and MBL-Pd transgenic arabidopsis seed

Fig. 7 for the MBL-Pb transgenic arabidopsis bloom the back the 6th, 10,12,14,16,18,20 day seed GUS original position histochemical stain result

Fig. 8 is the GUS original position histochemical stain result of MBL-Pb transgenic arabidopsis seed different sites

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, the primer sequence is given birth to worker's biotechnology company limited by Shanghai and is synthesized, used Semen Capparis (Capparis masaikai L é vl.) is picked up from the hillside thicket about the Jingxi County domestic height above sea level 1500m in old state of Baise of Guangxi area, and the locals is the water betel nut.

Embodiment 1, contain the clone of the dna fragmentation of Semen Capparis mbl gene promotor MBL-P695

One, the clone who contains the dna fragmentation of Semen Capparis mbl gene promotor MBL-P695 sequence

Universal GenomeWalker test kit with Clontech Laboratory Inc company is cloned the dna fragmentation that contains Semen Capparis mbl gene promotor (called after MBL-P695) sequence, and detailed process is as follows:

1, design of primers

According to the special nested primers of Semen Capparis (Capparis masaikai L é vl.) the cDNA sequence (GI:1817545) of mbl gene and 2 mbl genes of the requirement of mentioned reagent box specification sheets design, primer sequence is as follows:

pM1：5’-acctcgataacggtggtctggatgga-3’

pM2：5’-ggtctggatggaggcgttcgctagga-3’，

Again pM1 and pM2 are formed 2 couples of primer pM1/AP1 and pM2/AP2 with joint primer AP1, the AP2 that test kit carries respectively.

2, the nest-type PRC amplification contains the dna fragmentation of Semen Capparis mbl gene promotor MBL-P695 sequence

1) structure in GenomeWalker library

Extract the genomic dna of Semen Capparis blade with ordinary method, and with Dra I, EcoR V, Pvu II and four kinds of flat terminal restriction endonucleases of Stu I the Semen Capparis blade genomic dna that extracts is carried out enzyme with reference to mentioned reagent box specification sheets and cut digestion, jointing obtains four kinds of GenomeWalker libraries.

2) nest-type PRC amplification

The GenomeWalker library that makes up with step 1) is a template, carries out 2 respectively under the guiding of pM1/AP1 and two pairs of primers of pM2/AP2 and takes turns nest-type PRC, and first round PCR reaction conditions is: 94 ℃ of 2s of elder generation, 72 ℃ of 3min, 7 circulations; 94 ℃ of 2s then, 67 ℃ of 3min, 37 circulations; Last 67 ℃ of 4min: second takes turns the PCR reaction conditions is: 94 ℃ of 2s of elder generation, 70 ℃ of 3min, 5 circulations; 94 ℃ of 2s then, 65 ℃ of 3min, 30 circulations; Last 65 ℃ of 4min.Every take turns reaction and finish after, all amplified production being carried out 1% agarose gel electrophoresis detects, the agarose gel electrophoresis detected result of first round nest-type PRC amplified production is (swimming lane M:DNA MarkerDL2000+15000 shown in Figure 1A, swimming lane S: with Stu I GenomeWalker library is the pcr amplification product of template, swimming lane P: with PvuII GenomeWalker library is the pcr amplification product of template, swimming lane E: with EcoRV Genome Walker library is the pcr amplification product of template, swimming lane D: with Dra I GenomeWalker library is the pcr amplification product of template), second takes turns agarose gel electrophoresis detected result (swimming lane M:DNAMarker DL2000+15000 shown in Figure 1B of nest-type PRC amplified production, swimming lane P: with PvuII GenomeWalker library is the pcr amplification product of template, swimming lane E: with EcoRV GenomeWalker library is the pcr amplification product of template, swimming lane D: with Dra IGenomeWalker library is the pcr amplification product of template), four bands on recovery and the purifying Dra I library swimming lane and the band on the swimming lane of PvuII library, to reclaim dna fragmentation and be connected to carrier pMD18-T available from (Takara company), Transformed E .coli DH5 α competent cell, select positive colony upgrading grain, order-checking, the result is respectively 337bp by the wherein length of 3 dna fragmentations that increases in the Dra I library, 672bp and 885bp, these 3 dna fragmentations all are the special purpose fragments of mbl gene upstream, and preceding 2 fragments all are comprised in the 3rd the longest fragment, the length that long segment is removed behind the joint primer is 851bp, the sequence of the long 72bp of its 3 ' end and the homology of mbl gene cDNA5 ' terminal sequence reach 100%, show and successfully be cloned into the sequence that contains mbl gene 5 ' upstream promotor, it is SEQ ID № in the sequence table: 1 nucleotide sequence, by 779 based compositions, with this fragment name MBL-P779.With Biology WorkBench 3.2 softwares this sequence is carried out the restriction enzyme site analysis, found that to have 7 Dra I restriction enzyme sites in this fragment, this is the reason that is gone out multi-ribbon by Dra I amplified library.

Two, contain the sequential analysis of the dna fragmentation of Semen Capparis mbl gene promotor MBL-P695 sequence

Blastn program with the NCBI website compares to the dna fragmentation MBL-P779 that contains Semen Capparis mbl gene promotor MBL-P695 sequence that step 1 obtains, the result does not find sequence homology information, show the promotor MBL-P695 of isolating Semen Capparis mbl gene be a new promotor.Its conserved sequence element is predicted and analyzed with online software NNPP (http://www.fruitfly.org/seq_tools/promoter.html) and PLACE (http://www.dna.affrc.go.jp/PLACE/signalscan.html), the result as shown in Figure 2, from 5 ' end 220-270 bit base is the core promoter area I, from 5 ' end 677-727 bit base is core promoter area I I, discovery removes in this promoter sequence and contains transcription initiation site (from 5 ' end the 260th and the 717th bit base), TATA-box is (from 5 ' end 230-238 bit base, from 5 ' end 310-318 bit base, from 5 ' end 685-691 bit base), and ACGT-box is (from 5 ' end 544-552 bit base, from 5 ' end 677-684 bit base) outside the basic promoter element, also contain RY-repeat (from 5 ' end 663-668 bit base), napin-motif (from 5 ' end 625-631 bit base), E-box is (from 5 ' end 36-41 bit base, from 5 ' end 652-657 bit base), I-box is (from 5 ' end 170-175 bit base, from 5 ' end 544-548 bit base, from 5 ' end 568-572 bit base) and the CAAT tumor-necrosis factor glycoproteins (hold the 275-278 bit base from 5 ', from 5 ' end 297-300 bit base, from 5 ' end 361-364 bit base, from 5 ' end 455-458 bit base) etc. the relevant conserved sequence element of a large amount of seed specific expressions, the hint mbl gene may be subjected to the regulation and control in seed development stage, and its promotor is the promotor of a seed specific expression.

The tissue chemical analysis of the acquisition of embodiment 2, Semen Capparis mbl gene promotor MBL-P695 and core function fragment thereof and Semen Capparis mbl gene promotor MBL-P695

One, the structure of the plant expression vector of the MBL-P779 series deletion fragment of fusion gus gene, LegA-P

Four pairs of primers of design carry out the series disappearance with the dna fragmentation MBL-P779 that contains Semen Capparis mbl gene promotor MBL-P695 that the method with PCR obtains embodiment 1 earlier, and add restriction enzyme Sac I and BamH I recognition site respectively at the two ends of deletion fragment, primer sequence is as follows:

The primer of deletion fragment MBL-Pa is used to increase:

Pa1:5 '-ctg GagctcAtcaaaatgctaaccgcct-3 ' (band underscore base is a restriction enzyme Sac I recognition site)

Pm:5 '-cat GgatccGggtgagtgtgtgttcttgt-3 ' (band underscore base is a restriction enzyme BamH I recognition site);

The primer of deletion fragment MBL-Pb is used to increase:

Pb1:5 '-ctg GagctcTaaaagaatctacaaaac-3 ' (band underscore base is a restriction enzyme Sac I recognition site)

Pm:5 '-cat GgatccGggtgagtgtgtgttcttgt-3 ' (band underscore base is a restriction enzyme BamH I recognition site);

The primer of deletion fragment MBL-Pc is used to increase:

Pc1:5 '-ctg GagctcTtatacaggtatagactcaat-3 ' (band underscore base is a restriction enzyme Sac I recognition site)

Pm:5 '-cat GgatccGggtgagtgtgtgttcttgt-3 ' (band underscore base is a restriction enzyme BamH I recognition site);

The primer of deletion fragment MBL-Pd is used to increase:

Pd1:5 '-ct GgagctcTttagtcatgtatgggacac-3 ' (band underscore base is a restriction enzyme Sac I recognition site)

Pm:5 '-cat GgatccGggtgagtgtgtgttcttgt-3 ' (band underscore base is a restriction enzyme BamH I recognition site).

The dna fragmentation MBL-P779 that contains Semen Capparis mbl gene promotor MBL-P695 that obtains with embodiment 1 is a template, under the guiding of above-mentioned 4 pairs of primers, carry out pcr amplification respectively, after reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, detected result is (swimming lane M:DNA Marker DL2000+15000 as shown in Figure 4, swimming lane a:MBL-Pa, swimming lane b:MBL-Pb, swimming lane c:MBL-Pc, swimming lane d:MBL-Pd), the result carries out pcr amplification with above-mentioned 4 pairs of primers and has obtained length respectively and be about 300bp, 424bp, the dna fragmentation of 510bp and 695bp, reclaim these 4 dna fragmentations and will reclaim fragment and be connected to respectively among the carrier pMD18-T, Transformed E .coli DH5 α competent cell, select positive colony upgrading grain, order-checking, sequencing result shows the serial deletion fragment that contains Semen Capparis mbl gene promotor MBL-P695DNA fragment MBL-P779 that above-mentioned 4 dna fragmentations obtain for embodiment 1 really, and the sequence two ends are added with restriction enzyme Sac I and BamH I recognition site respectively, with these 4 deletion fragments difference called after MBL-Pa (300bp), MBL-Pb (424bp), MBL-Pc (510bp) and MBL-Pd (695bp).With restriction enzyme Sac I and BamH I above-mentioned 4 deletion fragments are carried out double digestion then, again with 4 endonuclease bamhis respectively with through the plant expression vector pVKH-35S-GUS-pA of same enzyme double digestion (Hyg ^r13.29kb, physical map is seen Fig. 3) connect, make endonuclease bamhi replace the CaMV35S promotor of gus gene upstream among the pVKH-35S-GUS-pA, earlier recombinant vectors is carried out PCR and identify (detecting primer is the primer of each MBL-P779 deletion fragment of above-mentioned pcr amplification), the result has obtained size through pcr amplification and has been respectively 300bp, 424bp, the dna fragmentation of 510bp and 695bp, conform to expected results, with restriction enzyme Sac I and BamH I recombinant vectors being carried out double digestion again identifies, enzyme is cut qualification result (swimming lane M:DNA Marker DL2000+15000 as shown in Figure 4, the Sac I of swimming lane A:pVKH-35S-GUS-Pa and BamH I double digestion product, the Sac I of swimming lane B:pVKH-35S-GUS-Pb and BamH I double digestion product, the Sac I of swimming lane C:pVKH-35S-GUS-Pc and BamH I double digestion product, the Sac I of swimming lane D:pVKH-35S-GUS-Pd and BamH I double digestion product), PCR and Sac I all conform to expected results with BamH I double digestion qualification result, show obtained insertion sequence and position all correct contain the MBL-P779 series deletion fragment MBL-Pa (300bp) that merges gus gene respectively, MBL-Pb (424bp), the plant expression vector of MBL-Pc (510bp) and MBL-Pd (695bp), called after pVKH-35S-GUS-Pa successively, pVKH-35S-GUS-Pb, pVKH-35S-GUS-Pc and pVKH-35S-GUS-Pd.Four kind of plant expression vectors are checked order, and sequencing result shows that MBL-Pa has the nucleotide sequence of SEQ ID NO:2 in the sequence table, by 300 based compositions; MBL-Pb has the nucleotide sequence of SEQ ID NO:3 in the sequence table, by 424 based compositions; MBL-Pc has the nucleotide sequence of SEQ ID NO:4 in the sequence table, by 510 based compositions; MBL-Pd has the nucleotide sequence of SEQ ID NO:5 in the sequence table, by 695 based compositions.

Simultaneously, with from the promoter L egA-P of leguminA (legA) gene of the seed specific expression of pea (Pisum sativum) (Genbank number: GI:20777) as positive control, the cloning process of this promotor is: extract the pea seedlings genomic dna with the CTAB method, be template with the pea seedlings genomic dna that extracts then, at primer A1 (5 '-gt GgatccTttagaattatttttttag-3 ', band underscore base is a restriction enzyme BamH I recognition site) and A2 (5 '-ta TtcgaaAttctcttacaaccaacta-3 ', band underscore base is a restriction enzyme Hind III recognition site) guiding under carry out pcr amplification, obtaining length is the promoter L egA-P sequence of the legA gene of 1245bp, and restriction enzyme BamH I and Hind III recognition site on adding respectively at the sequence two ends.With restriction enzyme BamH I and Hind III the PCR product being carried out enzyme cuts, and be connected through the cloning vector pBluescript of same enzyme double digestion II (available from Takara company), to connect product Transformed E .coli DH5 α competent cell, select positive colony upgrading grain, order-checking, sequencing result shows that the homology of the promoter L egA-P sequence of insertion sequence and legA gene is 100%, and this is contained the recombinant clone plasmid called after pBS-legA of LegA-P.With the pBS-legA plasmid is template, at primer P1:5 '-cat GgatccGaatgttggttgtgatgcgg-3 ' (band underscore base is a restriction enzyme BamH I recognition site) and primer P2:5 '-ctg GagctcThe LegA-P of pcr amplification legA promotor under the guiding of ttggcgtctcattgattgac-3 ' (band underscore base is a restriction enzyme SacI recognition site), the recognition site of restriction enzyme Sac I and BamH I on adding respectively at the two ends of amplified fragments simultaneously, after reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, detected result is (swimming lane M:DNA Marker DL2000+15000 as shown in Figure 4, swimming lane e:LegA-P), obtain the dna fragmentation that length is about 1245bp through pcr amplification, with restriction enzyme Sac I and BamH I the LegA-P fragment of amplification is carried out double digestion again, again endonuclease bamhi is connected with plant expression vector pVKH-35S-GUS-pA through the same enzyme double digestion, earlier with primer P1 and P2 this recombinant vectors being carried out PCR identifies, with restriction enzyme Sac I and BamH I recombinant vectors being carried out double digestion again identifies, qualification result is (swimming lane M:DNA Marker DL2000+15000 as shown in Figure 4, the Sac I of swimming lane E:pVKH-35S-GUS-LegA-P and BamH I double digestion product), PCR and Sac I all conform to expected results with BamH I double digestion qualification result, show and obtained all correct plant expression vector that contains the LegA-P that merges gus gene of insertion sequence and position, called after pVKH-35S-GUS-LegA-P.

Two, transform the tissue chemical analysis of the Arabidopis thaliana of the plant expression vector that the serial deletion fragment of the MBL-P779 that merges gus gene, LegA-P are arranged respectively

1, the acquisition of transgenic arabidopsis

Under the mediation of Agrobacterium LBA4404, infiltrate conversion method with vacuum 5 plant expression vectors that step 1 makes up are transformed the ecotypic Arabidopis thaliana of Columbia (Arabidopsis thaliana) respectively with the carrier pVKH-35S-GUS-pA with 35S promoter (as the positive control of constitutive expression), screen positive transgenic arabidopsis plant.

2, MBL-P779 series deletion fragment drives the tissue specificity analysis of gus gene expression

T to step 1 acquisition ₂Root for transgenic arabidopsis, stem, leaf and flower detect with the tissue specificity of GUS original position histological chemistry activation analysis method to MBL-P779 and serial deletion fragment driving gus gene expression thereof, wherein, root, the expression of gus gene (a:pVKH-35S-GUS-Pa transformed plant shown in Fig. 5 A in stem and the leaf, the b:pVKH-35S-GUS-Pb transformed plant, the c:pVKH-35S-GUS-Pc transformed plant, the d:pVKH-35S-GUS-Pd transformed plant, the e:pVKH-35S-GUS-LegA-P transformed plant, the o:pVKH-35S-GUS-pA transformed plant, ck: negative control), spend expression (a:pVKH-35S-GUS-Pa transformed plant shown in Fig. 5 B of gus gene, the b:pVKH-35S-GUS-Pb transformed plant, the c:pVKH-35S-GUS-Pc transformed plant, the d:pVKH-35S-GUS-Pd transformed plant, the e:pVKH-35S-GUS-LegA-P transformed plant, the o:pVKH-35S-GUS-pA transformed plant, ck: negative control), each deletion fragment MBL-Pa of mbl gene promotor MBL-P779, MBL-Pb, MBL-Pc and MBL-Pd can not drive the root of gus gene at transgenic arabidopsis, stem, express (the figure a-figure d among Fig. 5 A) in the leaf, but MBL-Pd can drive gus gene in spending than strongly expressed (the figure d among Fig. 5 B), and MBL-Pa, the colored expression activity of MBL-Pb and MBL-Pc is very weak or not obvious.

3, MBL-P779 series deletion fragment starts active strength analysis

T to step 1 acquisition ₂Generation (and/or T ₃Generation also can) seed of transgenic arabidopsis starts active intensity with GUS original position histological chemistry activation analysis method to the serial deletion fragment of MBL-P779 and detects, the expression of gus gene is (a:pVKH-35S-GUS-Pa transformed plant as shown in Figure 6, the b:pVKH-35S-GUS-Pb transformed plant, the c:pVKH-35S-GUS-Pc transformed plant, the d:pVKH-35S-GUS-Pd transformed plant, the e:pVKH-35S-GUS-LegA-P transformed plant, the o:pVKH-35S-GUS-pA transformed plant, ck: negative control), MBL-Pb, the colour developing of MBL-Pc and MBL-Pd transfer-gen plant seed is all dark than the seed of MBL-Pa transfer-gen plant, and MBL-Pb, MBL-Pc and the interseminal colour developing depth of MBL-Pd transfer-gen plant difference are not remarkable, the shade of MBL-Pa transfer-gen plant seed and legA-P transfer-gen plant seed is approaching, the ability and the seed specific promoters legA-P that show MBL-Pa driving GUS expression are suitable substantially, and MBL-Pb, MBL-Pc, the driving force of MBL-Pd is strong than MBL-Pa all.The above results explanation MBL-Pd is the promotor MBL-P695 of Semen Capparis mbl gene, be one and have the organ of multiplication specificity, the strong promoter of seed-specific particularly, MBL-Pa, MBL-Pb and MBL-Pc are the essential sequence of promotor MBL-P695, i.e. core function fragment.

4, mbl gene promotor MBL-P695 drives the time series analysis of genetic expression

MBL-P695 core function fragment MBL-Pb T in step 1 acquisition ₂Bloom the back the 6th for transgenic arabidopsis, 10,12,14,16,18, its seed carried out the histochemical stain of GUS original position in 20 days, to analyze the sequential that promotor MBL-P779 drives genetic expression, the GUS coloration result of different time as shown in Figure 7, beginning in the 12nd day after blooming, promotor MBL-P695 begins to drive gus gene in seed expresses, reach climax (blueness is the darkest) to the 16th day expression level approximately, the GUS of seed colour developing afterwards maintains higher level always, show that promotor MBL-P695 drives genetic expression and is subjected to the regulation and control of etap, promotor MBL-P695 mainly drives gus gene in the middle and later periods of seed maturity and expresses, the accumulation of expression time and Arabidopis thaliana seed self storage protein mabinlin is synchronous, thereby proves further that also mbl gene promotor MBL-P695 is a seed specific promoters.

5, the expression and localization of mbl gene promotor MBL-P695 in seed

MBL-P695 core function fragment MBL-Pb T in step 1 acquisition ₂Different sites for the seed of transgenic arabidopsis carries out the histochemical stain of GUS original position, so that promotor MBL-P695 expressive site of promotor gene in seed is positioned, the result as shown in Figure 8, promotor MBL-P695 mainly drives gus gene and expresses in the embryo of transgenic arabidopsis seed, gus gene is not expressed in kind of skin, and wherein the expression activity in radicle, plumular axis is better than cotyledon; What echo mutually therewith is, the plumular axis of Semen Capparis seed embryo is grown and is expanded, cotyledon is less, plumular axis is the main storage place (Hu Zhong etc. of storage protein mabinlin, the research II of mabinlin, Yunnan plant research, 7 (2): 187-195, therefore 1985), in the plumular axis of above-mentioned transgenic arabidopsis seed, observe the strongly expressed of gus gene.

Sequence table

<160>5

<210>1

<211>779

<212>DNA

＜213〉the Chinese lime betel nut (Capparis masaikai L é vl.) of being born in the year of horse

<400>1

ttggcatttt?taaattttta?ttttatgttt?ttatacaaat?gtttttcaaa?aagtaaatat 60

aaaattcact?tttttagtca?tgtatgggac?acaaatttta?aattcatttt?cgaccctctt 120

caaaggtttt?cttatttaaa?atttttttaa?aaaaaattat?agacatatag?ataaggacct 180

attttttatt?ttattttttt?atctaaatta?agatccaact?tgatccacat?ataaaataaa 240

cccgatagaa?aatttattta?tacaggtata?gactcaattc?ataccaagcc?aaaaaccaat 300

ccgcaccatt?atataaataa?attaaagtta?gtggttacaa?ctttaaaaga?atctacaaaa 360

caattttatt?ttaatatata?tagaatttaa?aatattaaat?cgaagttatt?tttttacaaa 420

aaaaaaccta?gtaaattatg?aaattatttt?tgtgcaatta?tgcaaaaatc?aaaatgctaa 480

ccgcctaatt?tataagtata?ccttacaaaa?ggttatagat?ctccctctct?ctttaaattt 540

ttcgataacg?ttaaaacccg?caacttcgat?aatgtctcct?gctcaacacg?caacatattg 600

cgtgtattat?tatttgatca?ttgttacaca?taatcatctc?atctattctt?acacgtgatc 660

gccatgcaaa?gtcctcaacg?ttcgtataaa?tatacccacc?aaataatccc?ctcgtcacct 720

cactcatcac?ccaacaaaac?cctaataaca?agaacacaca?ctcacccaaa?accttagca 779

<210>2

<211>300

<212>DNA

＜213〉the Chinese lime betel nut (Capparis masaikai L é vl.) of being born in the year of horse

<400>2

atcaaaatgc?taaccgccta?atttataagt?ataccttaca?aaaggttata?gatctccctc 60

tctctttaaa?tttttcgata?acgttaaaac?ccgcaacttc?gataatgtct?cctgctcaac 120

acgcaacata?ttgcgtgtat?tattatttga?tcattgttac?acataatcat?ctcatctatt 180

cttacacgtg?atcgccatgc?aaagtcctca?acgttcgtat?aaatataccc?accaaataat 240

cccctcgtca?cctcactcat?cacccaacaa?aaccctaata?acaagaacac?acactcaccc 300

<210>3

<211>424

<212>DNA

＜213〉the Chinese lime betel nut (Capparis masaikai L é vl.) of being born in the year of horse

<400>3

taaaagaatc?tacaaaacaa?ttttatttta?atatatatag?aatttaaaat?attaaatcga 60

agttattttt?ttacaaaaaa?aaacctagta?aattatgaaa?ttatttttgt?gcaattatgc 120

aaaaatcaaa?atgctaaccg?cctaatttat?aagtatacct?tacaaaaggt?tatagatctc 180

cctctctctt?taaatttttc?gataacgtta?aaacccgcaa?cttcgataat?gtctcctgct 240

caacacgcaa?catattgcgt?gtattattat?ttgatcattg?ttacacataa?tcatctcatc 300

tattcttaca?cgtgatcgcc?atgcaaagtc?ctcaacgttc?gtataaatat?acccaccaaa 360

taatcccctc?gtcacctcac?tcatcaccca?acaaaaccct?aataacaaga?acacacactc 420

accc 424

<210>4

<211>510

<212>DNA

＜213〉the Chinese lime betel nut (Capparis masaikai L é vl.) of being born in the year of horse

<400>4

ttatacaggt?atagactcaa?ttcataccaa?gccaaaaacc?aatccgcacc?attatataaa 60

taaattaaag?ttagtggtta?caactttaaa?agaatctaca?aaacaatttt?attttaatat 120

atatagaatt?taaaatatta?aatcgaagtt?atttttttac?aaaaaaaaac?ctagtaaatt 180

atgaaattat?ttttgtgcaa?ttatgcaaaa?atcaaaatgc?taaccgccta?atttataagt 240

ataccttaca?aaaggttata?gatctccctc?tctctttaaa?tttttcgata?acgttaaaac 300

ccgcaacttc?gataatgtct?cctgctcaac?acgcaacata?ttgcgtgtat?tattatttga 360

tcattgttac?acataatcat?ctcatctatt?cttacacgtg?atcgccatgc?aaagtcctca 420

acgttcgtat?aaatataccc?accaaataat?cccctcgtca?cctcactcat?cacccaacaa 480

aaccctaata?acaagaacac?acactcaccc 510

<210>5

<211>695

<212>DNA

＜213〉the Chinese lime betel nut (Capparis masaikai L é vl.) of being born in the year of horse

<400>5

tttagtcatg?tatgggacac?aaattttaaa?ttcattttcg?accctcttca?aaggttttct 60

tatttaaaat?ttttttaaaa?aaaattatag?acatatagat?aaggacctat?tttttatttt 120

atttttttat?ctaaattaag?atccaacttg?atccacatat?aaaataaacc?cgatagaaaa 180

tttatttata?caggtataga?ctcaattcat?accaagccaa?aaaccaatcc?gcaccattat 240

ataaataaat?taaagttagt?ggttacaact?ttaaaagaat?ctacaaaaca?attttatttt 300

aatatatata?gaatttaaaa?tattaaatcg?aagttatttt?tttacaaaaa?aaaacctagt 360

aaattatgaa?attatttttg?tgcaattatg?caaaaatcaa?aatgctaacc?gcctaattta 420

taagtatacc?ttacaaaagg?ttatagatct?ccctctctct?ttaaattttt?cgataacgtt 480

aaaacccgca?acttcgataa?tgtctcctgc?tcaacacgca?acatattgcg?tgtattatta 540

tttgatcatt?gttacacata?atcatctcat?ctattcttac?acgtgatcgc?catgcaaagt 600

cctcaacgtt?cgtataaata?tacccaccaa?ataatcccct?cgtcacctca?ctcatcaccc 660

aacaaaaccc?taataacaag?aacacacact?caccc 695

Claims (12)

1, the core function fragment of seed specific promoters is one of following nucleotide sequence:

1) the SEQID NO:2 in the sequence table;

2) the SEQ ID NO:3 in the sequence table;

3) the SEQID NO:4 in the sequence table.

2, the expression vector that contains the described core function fragment of claim 1.

3, the transgenic cell line that contains the described core function fragment of claim 1.

4, the host bacterium that contains the described core function fragment of claim 1.

5, the application of the core function fragment of the described seed specific promoters of claim 1 in the plant quality improvement.

6, the seed specific promoters that contains the described core function fragment of claim 1.

7, promotor according to claim 6 is characterized in that: the base sequence of described promotor is shown in SEQ ID NO:5 in the sequence table.

8, the expression vector that contains claim 6 or 7 described promotors.

9, the transgenic cell line that contains claim 6 or 7 described promotors.

10, the host bacterium that contains claim 6 or 7 described promotors.

11, claim 6 or the 7 described seed specific promoters application in the plant quality improvement.

12, application according to claim 11 is characterized in that: described plant comprises monocotyledons and dicotyledons.

Images