CN101063138A - Plant flower organ specificity promoter and its application - Google Patents
Plant flower organ specificity promoter and its application Download PDFInfo
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- CN101063138A CN101063138A CN 200710098728 CN200710098728A CN101063138A CN 101063138 A CN101063138 A CN 101063138A CN 200710098728 CN200710098728 CN 200710098728 CN 200710098728 A CN200710098728 A CN 200710098728A CN 101063138 A CN101063138 A CN 101063138A
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Abstract
The invention discloses a special promoter of plant flower organ and appliance, which is characterized by the following: (1) possessing nucleic acid sequence of sequence 1 in sequence table; or (2) possessing nucleic acid sequence complemented with the nucleic acid sequence; or (3) possessing nucleic acid sequence with 60% or above 60% homologous property with (1) or (2) nucleic acid sequence; or (4) possessing nucleic acid sequence crossed with the nucleic acid sequence of (1), (2) or (3) under strict crossing condition. This promoter possesses strong driving force and high specificity, which can foster new breed dissimilar with wild-type acceptor.
Description
Technical field
The present invention relates to a kind of plant promoter, particularly relate to a kind of plant flower organ specificity promoter and application thereof.
Background technology
Promotor is the most important factor in the gene expression regulation factor, and it determines substantially whether a gene is expressed, when expressed and where express.By the mode of action and function, promotor can be divided into constitutive promoter, specificity promoter and inducible promoter three major types [Wang Guanlin, Fang Hongjun, 2002, plant genetic engineering philosophy and technique (second edition), Beijing, science tech publishing house] substantially.This mode classification has reflected dissimilar promotors functional characteristics separately basically, but in some cases, one type promotor often has the characteristic of other type promotor concurrently.
Inducible promoter (inducible promoter) is meant that the gene of its control under the stimulation of some specific physics, chemistry and bio signal (being referred to as " elicitor " or " inducible factor "), can increase transcriptional level significantly.It is characterized by, the gene of the type promotor control is not expressed under the condition that does not have inducible factor to exist or is had only low-down expression (being also referred to as " background expression "), but in case be subjected to inducing of inducible factor, the expression of gene amount rapidly and increase considerably.Inducible promoter is usually classified according to its inducement signal and is named, for example: hormone induction promotor [Xu D etc., 1993, Plant Mol Biol, 22 (4): 573-588; TaylorJE etc., 1995, Plant J, 7 (1): 129-134], chemically inducible promoter (Williams S etc., 1992, Biol/Technol, 10:540-543; Summary is seen: Padidam M, 2003, Curr Opinion in PlantBiol, 6 169-177), photoinduction promoter [Sheen JY etc., 1987, Plant Mol Biol, 8 (3): 227-238; Matsuoka M etc., 1994, Plant J, 6 (3): 311-319], heat shock promoter [SchofflF etc., 1989, Mol Gen Genet, 217 (2-3): 246-53], wound-induced promotor [Farmer EE etc., 1992, Plant Cell, 4:129-134; Carrera E etc., 1998, Plant J, 15 (6): 765-771], fungal induction promotor (Fukuda Y etc., 1994, Plant Mol Biol, 24 (3): 485-493) with symbiotic bacterium evoked promoter (Miao GH etc., 1993, Plant Cell, 5:781-786) etc.How many inducible promoters often also has organ and/or tissue specificity, for example tobacco Whitfield's ointment evoked promoter PR-1a mainly drive gene in blade, express (Uknes S etc., 1993, Plant Cell, 5:159-169).
Constitutive promoter (constitutive promoter) is meant that under the type promotor control, the expression of structure gene is constant on certain level substantially, does not also have notable difference at the expression level of Different Organs and/or tissue.Be characterized in: be subjected to the expression of the structure gene of its control to have persistence, but do not have the space-time specificity; RNA is relative with the protein expression amount constant, be not subjected to inducing of extraneous factor, for example: corn Ubiqultin promotor, Actinl promotor (the Wang etc. of paddy rice, Molecular and Cellular Biology, 12 (8): 3399-3406 (1992)), United States Patent (USP) the 5641876th) and Cauliflower dish mosaic virus (CaMV35SRNA) (Odell etc., Nature, 313:810-812 (1985)) etc.
Organ or tissue's specificity promoter (organ-and/or tissue-specific promoter), the expression that is meant its regulatory gene often only occurs in a certain of plant materials or organ and/or tissue that some is specific, perhaps often only occurs in a certain of growth and development of plants or some specified phase.It is characterized in that being subjected to the genetic expression of its control or adjusting to have temporal and spatial, and often show the characteristic of growing adjusting.For example, root-specific promoter (Yamamoto YT etc., 1991, Plant Cell, 3:371-382), blade specific promoter [Taylor, WC, 2001, Plant Mol Biol, 46 (3): 325-333), fruit-specific promoter (Pear JR etc., 1989, Plant Mol Biol, 13:639-651), flower specific promoter (Van tunen etc., 1988, EMBO J., 7:1257), endosperm specificity promoter [Colot V etc., 1987, EMBO J, 6 (12): 3559-3564], cotton fiber specific promotor (Ma DP etc., 1997, Biochem Biophys Acta, 1344:111-114) with phloem specific promotor [Bostwick DE etc., 1994, Plant Mol Biol, 26:887-897] or the like.
Reproductive growth is the important stage of higher plant in the life history, and is subjected to biologist and agronomist's extensive concern as the formation and development of the vitals-floral organ of carrying out reproductive process always.The flower development process of plant can be divided into 4 stages (Koornneefm et al; Ann.Rev.Plant Physiol Plant MolBiol; 1998,49:345-370): floral induction, floral meristem form, the floral organ original hase produces and the floral organ maturation.Therefore, the growth course of floral organ is a high complexity, orderly Physiology and biochemistry and morphogenetic process.The expression of a large amount of specific genes is arranged in this course.
Identify in the world at present, separation and functional study the promotor of some flower organ specificities.Some flower organ specificity promoters drive foreign gene and express in a plurality of relatively independent unit of floral organ, for example, the zb8 promoters driven reporter gene GUS of PAL family expresses (Zhu Q et al in flower pesticide, bud, holder and the filigree of transgenic paddy rice, 1995, Plant Mol.Biol., 29:535-550), other flower organ specificity promoters then have the specificity of a certain specific unit of floral organ, for example, Rice Anther specificity promoter (Tsuchiya T etc., 1994, Plant Mol Biol, 20:1189-1193; Wu Xiaohuai etc., 2003, Science Bulletin, 48:2154-2161), tomato pollen specificity promoter LAT52 (Twell D etc., 1989, Mol GenGenet, 217:240-245), TomA108 (Xu XS and Chen RD, 2006, Physiol and Biochem is 25:231-240) with tobacco anther tapetum specific efficient promoter TA29 (Koltunow AM etc., 1990, PlantCell, 2:1201-1224) or the like.People's research finds that also a lot of genes in the metabolic gene of the volatile chemical of the cyanidin(e) pathways metabolism and the fragrance of a flower all have flower organ specificity.For example, petunia cinnamophenone (CHS) gene promoter have the specific expressed characteristic of very strong petal (Van tunen etc., 1988, EMBO J., 7:1257).
The research of flower organ specificity gene and promotor thereof, not only controlling gene and the network thereof of understanding floral organ differentiation, formation, g and D had important significance for theories, and to utilizing genetic engineering technique improvement plant to have important use value, particularly at the artificial male sterility gene engineering of crop (Mariani etc., 1990, Nature, 347:737-741; Xiao etc., 2004, Chinese invention patent, ZL00109108.5), genetically engineered (Lee etc. such as colored shape, pattern and florescence control of flowers, 2003, Chinese biological engineering magazine 23:42-46) has broad application prospects with the shortening the juvenile phase of fruit tree and by shifting to an earlier date genetically engineered aspects such as florescence adjustment fruit Time To Market.
Wild cabbage (Brassica oleracea L.) is that the Cruciferae rape belongs to important vegetable crop, in the main vegetable growing of world many countries conduct.In addition, the limited cross compatibility of other plant of Cruciferae such as it and rape is that very big contribution has been made in the breed improvement of rape etc.For example, swede type rape has been one of the major oil colza class that comprises many countries and regions of China.
The clone of wild cabbage floral organ genes involved and promotor thereof obtains tangible progress, has cloned genes involveds such as the not affine genes involved Thl1 of selfing, Thl2, ARC1 gene and male sterile.These specific genes, particularly its promotor have begun to be applied to genetic engineering breeding (Bhalla etc., 1998, Mol Breeding, 4 (12): 531-541).Simultaneously, utilize the work of other plant flower specificity promoter improvement wild cabbage also to obtain some progress, as, (Acta Agriculture Ahanghai such as Zhu Yuying, 2001,17 (1): 79-82) TA29 and Barnase gene fusion are imported wild cabbage, observing transfer-gen plant has the male sterile of staminody and sterile plant to occur.Along with to the raising of environmental protection consciousness and raising that Food Quality is required, people are also more and more stronger to utilizing plant-derived gene to improve the requirement of plant or crop.
Summary of the invention
The purpose of this invention is to provide a kind of plant flower organ specificity promoter and application thereof.
Plant flower organ specificity promoter provided by the present invention, its nucleotide sequence is:
(1), sequence 1 described nucleotide sequence in the sequence table; Or
(2), with (1) described nucleotide sequence complementary nucleotide sequence; Or
(3), the nucleotide sequence that has 60% or 60% above homology with (1) or (2) described nucleotide sequence; Or
(4), be listed in the nucleotide sequence that to hybridize under the rigorous hybridization conditions with (1), (2) or (3) described nucleotides sequence.
Above-mentioned rigorous hybridization conditions can be at 2 * SSC, in the solution of 0.1%SDS, hybridizes under 68 ℃ and washes film 2 times, each 5min; Perhaps at 0.5X SSC, in the solution of 0.1%SDS, under 68 ℃, hybridize and wash film 2 times, each 15min.
The expression cassette that contains above-mentioned flower specific promoter also belongs to protection scope of the present invention.
In described expression cassette, the inverted defined gene of the downstream syndeton gene of described flower organ specificity promoter, regulatory gene, structure gene, the inverted defined gene of regulatory gene or the little RNA that can disturb native gene to express are used for the expression of the little RNA of the inverted defined gene of drives structure gene, regulatory gene, structure gene, the inverted defined gene of regulatory gene, natural little RNA or synthetic.
The recombinant expression vector that contains above-mentioned flower organ specificity promoter also belongs to protection scope of the present invention, and described recombinant expression vector is to contain above-mentioned expression cassette and plasmid, virus or the constructed recombinant vectors of vehicle expression vector; Described recombinant expression vector is the reorganization plant expression vector; Described recombinant plant expression vector comprise above-mentioned expression cassette and described expression cassette can be passed on enter plant host cell, tissue or organ and offspring thereof and can or convenient at least described expression cassette be incorporated in host's the genome, it includes but not limited to binary vector, closes carrier altogether.
Above-mentioned recombinant expression vector can be by using conventional biological method transformed plant cells or tissue or organs such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, agriculture bacillus mediated or particle gun, obtain transgenic plant cells or tissue or organ and differentiation, regenerated whole plant and clone or its offspring thus.
The present invention also provides a kind of preparation method of above-mentioned flower organ specificity promoter, being that genomic dna with wild cabbage is a template, is that sequence 2 and nucleotide sequence are that a pair of primer of sequence 3 carries out pcr amplification and obtains the floral organ promotor in the sequence table in the sequence table with nucleotide sequence.
Experimental results show that, flower specific promoter Bofs of the present invention can start reporter gene GUS specifically expressing in petal, flower pesticide, column cap and the pollen of the floral organ of transgene tobacco, and all do not detect expression at vegetative organ, and representative has thereafter showed identical characteristics; Illustrate that promotor Bofs provided by the present invention not only has strong driven nature, but also have good flower organ specificity, and this strong driven nature and specificity can stably pass to the offspring.Therefore, separation provided by the present invention is a flower organ specificity promoter stable, that motivating force is strong and specificity is high from the promotor Bofs of wild cabbage (Brassica oleracea L).
Plant flower organ specificity promoter of the present invention can be expressed in different plants or crop and genetic stability, described plant or crop are meant polycarpeae, according to different plant classification methods, described plant or crop can include but not limited to angiosperm and gymnosperm, monocotyledons and dicotyledons, herbaceous plant, vine and xylophyta, yearly plant and perennial plant, aquatic and terrestrial plant and sexual and asexually propagated plant, wherein according to aquatic and classification terrestrial plant, described plant or crop are preferably terrestrial plant.
With promotor of the present invention downstream be connected native gene inverted defined gene, can disturb little RNA that native gene expresses (comprise natural with little RNA synthetic) or foreign gene, the construction expression box also connects with different expression vector, be transformed in the plant, utilize it to spend different in nature expression activity, the described inverted defined gene of specific expressed its control or guidance, foreign gene or little RNA in floral organ.In transfer-gen plant, make its native gene inverted defined gene, can disturb during little RNA that native gene expresses (comprise natural with little RNA synthetic) or expression of exogenous gene be confined to spend, got rid of of the expression of these genes at other position of plant materials.
Flower organ specificity promoter of the present invention, not only the molecule mechanism scheduling theory research for the differentiation of floral organ, formation, g and D provides important molecular element, also be the plant genetic engineering breeding, particularly genetic engineering breedings such as flower plant flower shape, pattern, fragrance and florescence control provide crucial molecular element.Utilize this promoters driven native gene inverted defined gene, can disturb little RNA that native gene expresses or foreign gene to transform the cell or tissue and progeny cell thereof of plant after, can cultivate colored shape, pattern, the fragrance of a flower and fertility etc. be different from the acceptor wild-type flowers crop new variety, cultivate the male sterile crop varieties and cultivate the garden plants etc. of eliminating or reducing " willow catkins flying in the air " significantly.
Description of drawings
Fig. 1 is flower organ specificity promoter Bofs clone's electrophorogram.
Fig. 2 is the dna sequence dna of flower organ specificity promoter Bofs.Among the figure, the underscore person is a primer sequence, and italic person is known the site by the restriction enzyme that adds, and 5 ' end is Hind III site, and 3 ' end is Bam HI site.
Fig. 3 drives the structure schema of the plant expression vector pRDBofsG of gus gene (being the Bofs::GUS fusion gene) for flower organ specificity promoter Bofs.
Fig. 4 is that the enzyme that contains the plant expression vector pRDBofsG of flower organ specificity promoter Bofs driving gus gene (being the Bofs::GUS fusion gene) is cut evaluation figure.
Fig. 5 is for changeing the PCR evaluation figure of pRDBofsG tobacco T0 for plant.
Fig. 6 is for changeing pRDBofsG tobacco T0 for the plant and the GUS dyeing photo of transgenosis adjoining tree petal not.
Fig. 7 is for changeing pRDBofsG tobacco T0 for the plant and the GUS dyeing photo of transgenosis adjoining tree column cap not.
Fig. 8 is for changeing pRDBofsG tobacco T0 for the plant and the GUS dyeing photo of transgenosis adjoining tree flower pesticide not.
Fig. 9 is for changeing pRDBofsG tobacco T0 for the plant and the GUS dyeing photo of transgenosis adjoining tree pollen not.
Embodiment
Promotor nucleotide sequence described in the present invention can be that wherein one or more Nucleotide replace, the nucleotide sequence of disappearance, insertion or inversion, promptly artificial mutant or " natural " mutant of isolating nucleotide sequence, it keeps its promoter function; It can also be the fusion sequence of the conservative regulating and controlling sequence (" motif " or " box ") of described nucleotide sequence and other promoter sequence or promoter region." promotor " described in the present invention recorded initiation site (+1) upstream and had the guide RNA polysaccharase and start the function of transcribing on the tram, but is not limited near the part this zone; In addition, it also contain being used to of being associated with protein except that RNA polymerase regulate another of expression must the zone." promoter region " described in the present invention is defined as and contains the above zone of the promotor of definition." promoter activity " described in the present invention but be meant when be connected to the downstream of promotor with the phraseology of certain gene, and import among the host, this host shows when having the ability of producing this gene product in the host or outside the host and function, this promotor have start active.Usually, it is the downstream that the easy proteinic gene (reporter gene) qualitative or detection by quantitative of coding is connected to this promotor, this gene is imported in host, and detect expressed protein, can determine the active of specific promotor or not have the effectiveness of this promotor or this promotor.Structure gene described in the present invention is meant one section nucleotide sequence of can encode certain protein or other active substance function, comprises RNA or dna sequence dna.Described regulatory gene is meant one section nucleotide sequence that certain RNA of its coding or protein or other active substance can be regulated or regulate and control the expression of other structure gene, comprises RNA or dna sequence dna.Described just gene comprises above-mentioned structure gene and regulatory gene.Described inverted defined gene is meant RNA complementary RNA or the dna sequence dna with said structure gene or regulatory gene coding.Described native gene is meant the gene from host self, comprises RNA or dna sequence dna.Described foreign gene is meant any one section nucleotide sequence, and has coding certain protein or other active substance function, comprises natural and RNA synthetic or dna sequence dna, and this sequence does not combine in normal circumstances with anther specific promoter.Described little RNA is meant that separation is from RNA sequence fragment organism or synthetic, its length is generally 20-26 deoxynucleotide or can be sheared into the RNA fragment of 20-26 deoxynucleotide after importing the dormitory cell, and itself is nontoxic or toxicity is extremely low to organism.
Any plant vector that expression vector described in the present invention is meant is well known in the prior art, can express in plant, for example pBin19, pBI121 (U.S. ClonTech company product) and pCAMBIA series (Australian CAMBIA center product) etc.
That conversion described in the present invention is meant is well known in the prior art, can be with any methods for plant transformation of exogenous gene transfered plant cell or plant tissue, as agrobacterium-mediated transformation and particle gun etc.
Host cell described in the present invention or host tissue or host's organ and progeny cell thereof be meant all vegetable cells or plant tissue or plant organ or by these cells, tissue or organ by tissue differentiation or asexual embryo regeneration and the whole plant (comprising seed) of fully-developed.
Term " nucleotide sequence " or " nucleotide sequence " refer to contain the sequence of naturally occurring Nucleotide or nucleoside monomers.This sequence also comprises the monomer of the non-natural existence with identity function or sequence modification or that replace of its part.
Term " flower organ specificity promoter " is meant that the gene of expressing is not or have the floral organ of the plant of basal expression to express and the startup word of not expressing at other organs of plant materials under promotor control.
Term " sequence with 60% or 60% above homology " is meant with the sequence in (1) or (2) compares same or analogous those nucleotide sequences of the nucleotide sequence that has more than 60% or 60% or nucleotide sequence, these sequences play a role in essentially identical mode and can drive the anther-specific expression of its downstream gene, they may be because modification or the sudden change on the local structure comprises artificial mutation and unartificial sudden change with the difference of (1) or (2) middle sequence.
Term " sequence of hybridization " is meant can be under rigorous hybridization conditions and the nucleotide sequence of the sequence hybridization of (1), (2), (3) or (4)." rigorous hybridization conditions " is meant well known by persons skilled in the art, perhaps can be at molecular biology or genetically engineered experiment guide, and " Molecular Cloning " (3 for example
RdEd) (Sambrook etc., Cold Spring Harbor Laboratory, New York, 2001) find in the general scheme in (hereinafter to be referred as " " molecular cloning " third edition "), be specially at 2 * SSC, in the solution of 0.1%SDS, under 68 ℃, hybridize and wash film 2 times.Each 5min.0.5X SSC in the solution of 0.1%SDS, is hybridized under 68 ℃ and is washed film 2 times.Each 15min.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
T0 representative is shown seed that transfer-gen plant and the clone thereof that is obtained by callus, T1 represent that T0 produces for selfing and by plant and clone that it grew up to.
The clone of embodiment 1, wild cabbage (Brassica oleracea L) floral organ specific promoter Bofs with separate
1, the extraction of the total DNA of wild cabbage (Brassica oleracea L)
Get head cabbage varieties " black leaf butch haircut " (available from Vegetable ﹠ Flower Inst., Chinese Academy of Agriculture Science) leaflet tablet 1-2g of greenhouse pot culture, extract total DNA with CTAB method (" molecular cloning " third edition).Get 1-5ul DNA sample, measure its concentration and purity with ultraviolet spectrophotometer, the agarose gel electrophoresis with 0.8% detects DNA purity and integrity.With the DNA that extracts in-20 ℃ of preservations.
2, the recovery of pcr amplification and amplified fragments
Design and synthesize following two primers (primer 1 and primer 2) amplification wild cabbage floral organ specific promoter, later clone and structure held the recognition sequence site (the underscore sequence in the following primer sequence) that has added restriction enzyme HindIII and Bam HI respectively at 5 ' of two primers for convenience
Primer 1:5 '-
AAGCTTAGCAGCACGAATGAAGTTC-3 ' (
Hind III)
Primer 2: 5 '-
GGATCCTTGTAGTGAGAAAACTCGGGGAA-3 ' (
Bam HI)
The wild cabbage genomic dna that extracts with step 1 is a template, carries out the PCR reaction:
Reaction system is:
Template DNA: 800ng;
10X Exbuffer： 2ul；
DNTP mixture (2.5mM): 2ul;
ExTaq DNA Polymerase(5U/u1)：0.5ul；
Primer 1 (10uM): 2ul;
Primer 2 (10uM): 2ul;
Aseptic double distilled water (sddH2O): complement to 20ul.
PCR response procedures: earlier pre-94 ℃ of 5min of sex change; 94 ℃ of 30sec again, 50 ℃ of 30sec, 72 ℃ of 1min 30sec, 35 circulations; Last 72 ℃ of 10min.
After the PCR reaction is finished, get the 15ul amplified production through 1.0% agarose gel electrophoresis, ultraviolet lamp downcuts the specific fragment that is positioned at about 1200bp with disposable blade down fast, reclaiming test kit with dna fragmentation reclaims and purifying (Easy-NA Gel Extraction Kit, Germany Omeg-Bio/TEK product), be dissolved in 50ulddH
2Among the O ,-20 ℃ of preservations are standby.
3, reclaim segmental subclone and order-checking
The fragment that step 2 is reclaimed is connected to plasmid vector pUCm-T (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's product), resulting recombinant plasmid is transformed into coli strain DH5 α competent cell by " freeze-thaw method " (" molecular cloning " third edition), containing on the LB solid medium of penbritin 100mg/L in 37 ℃ of incubated overnight, the white colony of growing on the picking flat board inserts in the LB liquid nutrient medium that contains penbritin 100mg/L in 37 ℃ of incubated overnight.When bacterial concentration reaches OD
600It is 0.6 o'clock, centrifugal collection thalline, extract plasmid by a small amount of alkaline lysis (" molecular cloning " third edition), behind Restriction Enzyme HindIII and BamHI double digestion, use 1.0% agarose gel electrophoresis, should be under the ultraviolet lamp visible molecular size of correct recombinant vectors approximately be respectively 2800bp (carrier band) and 1200bp (object tape) two bands (Fig. 1, among the figure, swimming lane 1 is DNA sized molecules standard I---Marker I; Swimming lane 2 is the HindIII+BamHI cleavage map of the cloning vector of the above-mentioned pcr amplified fragment of insertion).To cut by above-mentioned enzyme and identify that contain DH5 α that 1200bp inserts segmental plasmid (pUBofs) or contain this plasmid delivers order-checking (the learned biotechnology in former Shanghai Services Co., Ltd of commercial order-checking company, now incorporated Introgen company into), will show correct recombinant vectors called after pUBofs through order-checking.Sequencing result shows that the fragment that above-mentioned pcr amplification obtains is a wild cabbage floral organ specific promoter, sequence as shown in Figure 2, the nucleotide sequence that wild cabbage floral organ specific promoter has sequence 1 in the sequence table will have the wild cabbage floral organ specific promoter called after Bofs of the nucleotide sequence of sequence 1 in the sequence table.Among Fig. 2, the underscore person is used primer sequence (above-mentioned primer 1 and primer 2), and italic is the restriction enzyme enzyme recognition site (HindIII and BamHI) that adds.
The structure flow process that the floral organ specific promoter drives GUS fusion gene (Bofs::GUS) plant expression vector pRDBofsG as shown in Figure 3, concrete grammar is as follows:
With pUBofs behind restriction enzyme HindIII and BamHI double digestion, Bofs fragment (approximately 1200bp), reclaim test kit (Easy-NAGel Extraction Kit with freeze-thaw method (" molecular cloning " third edition) or dna fragmentation, Germany Omeg-Bio/TEK product) reclaims and purifying floral organ specific promoter Bofs fragment, link to each other with the big fragment (being approximately 13.6kb) of the plant expression vector pRD410 (Canadian PBI product) that passes through HindIII and BamHI double digestion, obtain recombinant plasmid.Resultant recombinant plasmid is transformed into coli strain DH5a with " freeze-thaw method " (" molecular cloning " third edition).The DH5 α that transforms is containing on the LB solid medium of kantlex 50mg/L in 37 ℃ of incubated overnight, and single bacterium colony of growing on the picking flat board inserts in the LB liquid nutrient medium that contains kantlex 50mg/L and cultivates in 37 ℃ of shaken overnight.When bacterial concentration reaches OD600 value 0.5-0.6, centrifugal collection thalline, by above-mentioned a small amount of alkaline lysis method of extracting plasmid, behind restriction enzyme Hindll and BamHI double digestion, at 1.0% agarose gel electrophoresis, visible molecular size is about 13.5kb (pRD410 carrier band) and 1200bp (Bofs) two bands respectively under ultraviolet lamp, and carried out sequence verification, enzyme has been cut and checked order and show and contain floral organ specific promoter Bofs fragment and the pRD410 enzyme is cut the big segmental recombinant expression vector called after pRDBofsG that obtains.Gus gene needs the floral organ specific promoter to drive expression among the pRDBofsG.
The evaluation of embodiment 3, commentaries on classics Bofs fusion gene (Bofs::GUS) tobacco
1, the evaluation of conversion of Agrobacterium and transformant
Use CaCl
2The competent cell of method (" molecular cloning " third edition) preparation agrobacterium tumefaciens bacterial strain LBA4404 (U.S. LifeTechnology company product).Utilize plant expression vector pRDBofsG that freeze-thaw method (" molecular cloning " third edition) will contain Bofs fusion gene (Bofs::GUS) to change the competent cell of the LBA4404 of preparation over to.With the LBA4404 cell inoculation that transforms to the YEB solid medium that contains Streptomycin sulphate (Str) 100mg/L and kantlex (Kan) 50mg/L, place 28 ℃ in the dark to cultivate 48-72h, single bacterium colony on the picking flat board, access contains the YEB liquid nutrient medium of Streptomycin sulphate 100mg/L and kantlex 50mg/L, cultivates 28 ℃ of shaken overnight.When the concentration of culture reaches OD
600During value 0.4-0.6, bacterium liquid (1.5-2ml) takes a morsel, by above-mentioned a small amount of alkaline lysis method of extracting plasmid, identify with restriction enzyme HindIII and BamHI double digestion, at 1.0% agarose gel electrophoresis, under ultraviolet lamp visible molecular size approximately be respectively 13.6kb (carrier band) and 1200bp (Bofs) two bands (Fig. 4, among Fig. 4, swimming lane 1 is the Hind III+Bam HI double digestion collection of illustrative plates of pRDBofsG; Swimming lane 2 is two λ DNA sized molecules standard---λ DNA/HindIII+EcoRI that cut), and order-checking is identified.The result shows that plant expression vector pRDBofsG has successfully changed Agrobacterium LBA4404 over to.Enzyme cut and check order and identify and show the Agrobacterium LBA4404 clone called after pRDBofsG/LBA4404 that contains plant expression vector pRDBofsG.
2, change the acquisition of Bofs fusion gene (changeing pRDBofsG) tobacco
1) preparation of Agrobacterium
Picking carries the single bacterium colony of Agrobacterium LBA4404 (pRDBofsG/LBA4404) of plant expression vector pRDBofsG, be inoculated in 5ml and contain in the YEB liquid nutrient medium of Streptomycin sulphate (Str) 100mg/L and kantlex (Kan) 50mg/L, 28 ℃ of shaking culture incubated overnight (approximately 12h) are with activation.Get the activatory agrobacterium liquid, join in the YEB liquid nutrient medium that contains Str 100mg/L and Kan 50mg/L, continue to be cultured to OD in 1: 100 ratio
600Value is 0.4-0.6; The centrifugal 5min of 5000rpm collects thalline; Wash thalline once with 1/2 MS0 liquid nutrient medium (MS inorganic salt+B5 VITAMIN+inositol 0.1g/L+ sucrose 30g/L, pH 5.80), and it is diluted in the 1/2 MS liquid nutrient medium of centrifugal preceding 3 times of volumes of bacterium liquid, prepare to infect usefulness.
2) regeneration of conversion of tobacco and transformed plant
The conversion of tobacco according to leaf dish method (Horsch RB etc., 1985, Science 227:1229-1231) carries out.Choose tobacco (kind " NC89 ", seed is available from the Chinese Academy of Agricultural Sciences) aseptic seedling of about 30 days seedling ages, downcut fresh and tender dark green blade, produce leaf dish explant with the punch tool of diameter 9mm; Freshly prepd explant is dropped in off-the-shelf Agrobacterium (LBA4404/pRDBofsG) the bacterium liquid, infect 15min; Take out the leaf dish, absorb the Agrobacterium bacterium liquid of leaf panel surface remnants with autoclaved thieving paper, placing solid regenerated substratum (MS inorganic salt+B5 VITAMIN+inositol 0.1g/L+BA 2.0mg/L+IAA 0.5mg/L+ sucrose 30g/L+ agar powder 0.7%, pH 5.8) to go up the dark place cultivated 2 days altogether; The leaf dish that to cultivate altogether then forwards the solid regenerated substratum (1500-2500Lx under light that contains Pyocianil (Carb) 500mg/L and Kan 50mg/L to, the dark 8h of light 16h) carries out screening and culturing, change a subculture every 2-3 week, and reduce Carb to 200mg/L gradually.When treating Kan resistant buds length to 1-1.5cm, the solid root media (MS inorganic salt+B5 VITAMIN+inositol 0.1g/L+IAA 0.5mg/L+ sucrose 30g/L+ agar powder 0.7%, pH 5.80) that contains Carb 200mg/L and Kan 50mg/L is changed in its cutting-out gone up root induction.The commentaries on classics pRDBofsG tobacco plant of the complete kalamycin resistance of 58 strains that obtains is used for Molecular Identification.
3) Molecular Identification of commentaries on classics pRDBofsG tobacco
At first to step 2) the Kan resistance that obtains changes the pRDBofsG tobacco plant and carries out PCR and identify.Extracting according to above-mentioned CTAB method changes the pRDBofsG tobacco (Kan resistant strain) and the blade genomic dna of transgene tobacco contrast strain not, and the Auele Specific Primer at usefulness flower organ specificity promoter Bofs two ends is to (primer 1:5 '-AAG CTT AGC AGC ACG AAT GAA GTT C-3 '; Primer 2: 5 '-GGA TCC TTG TAG5 '-AAG CTT AGC AGC ACG AAT GAA GTT C-3 '; Primer 2: 5 '-GGA TCC TTG TAGTGA GAA AAC TCG GGG AA-3 ') carry out pcr amplification, commentaries on classics pRDBofsG tobacco is all increased and obtains the big or small object tape of 1200bp that is, unconverted contrast tobacco does not then amplify this target fragment, and (part pcr amplification result as shown in Figure 5 in the corresponding position, among Fig. 5, swimming lane 1-7 is that different commentaries on classics pRDBofsG tobacco (Kan resistant strain) T0 is for plant; Swimming lane NC89 is not transfer-gen plant contrast (negative control); Swimming lane Bofs is vector plasmid contrast (positive control); Swimming lane M is a DNA sized molecules standard (marker)).This result shows that tentatively target gene (Bofs::GUS) has been incorporated in the tobacco gene group.
The histological chemistry that embodiment 4, commentaries on classics pRDBofsG tobacco gus gene are expressed is detected
And have simultaneously commentaries on classics pRDBofsG tobacco T0 that kalamycin resistance and PCR be positive for strain system (strain is 2,6,7,9,11,12,14) and not genetically modified tobacco NC89 adjoining tree (CK) test-tube plantlet in one week of triangular flask split shed hardening, move into the flowerpot that common flower nutrition soil is housed then, in the greenhouse, cultivate, bagging is preserved moisture and is removed bag after a week, carries out Routine Management.
Change the histological chemistry that pRDBofsG tobacco gus gene expresses and detect according to Jefferson RA[1987 PlantMol Biol Rep, 5 (4): 387-405] method carry out.At the different development stage of floral organ (according to document (Cao Kehao, China Agricultural University's master thesis, the clone of the sequence of rice Os g6B promotor and functional analysis and intestinal bacteria argE gene and to the conversion of tobacco, 2003) method divides development of floral organs period), to change pRDBofsG tobacco and contrast thereof (tobacco bred " NC89 ", seed is available from the Chinese Academy of Agricultural Sciences) fresh root, stem, leaf, calyx, petal, filigree, ovary, column cap and flower pesticide incubation 24-48h in X-GLuc solution dyes, then with painted vegetable material respectively with the thorough rinsing of 70% and 100% ethanol decolouring and fixing, at the microscopically observing samples and take pictures.
The painted result of floral organ GUS is as shown in table 1, the result shows, each organ of not genetically modified tobacco NC89 adjoining tree (CK) all can not dye, and in the positive T0 of 7 PCR of the commentaries on classics pRDBofsG tobacco that is detecting for strain is, the flower pesticide of all strain systems can both be colored (Fig. 8 to some extent, A is not genetically modified tobacco NC89 adjoining tree among Fig. 8, and B is for changeing pRDBofsG tobacco T0 plant).5 strain systems (removing 2 and 12) are colored (A is not genetically modified tobacco NC89 adjoining tree among Fig. 6 and Fig. 7 for Fig. 6, Fig. 7, and B is changes pRDBofsG tobacco T0 plant) to some extent in petal, column cap.In strain is pollen being colored in various degree in 6 and 11 (Fig. 9, A is not genetically modified tobacco NC89 adjoining tree among Fig. 9, B is for changeing pRDBofsG tobacco T0 plant).Filigree, ovary etc. all do not dye blueness in all strain systems.These results demonstrate the painted flower organ specificity of GUS, show that promptly promotor Bofs has flower organ specificity and drives active.Fig. 6 is not transgenosis contrast (A) and the petal GUS dyeing of changeing pRDBofsG tobacco (B); Fig. 7 is contrast (A) and the column cap GUS dyeing of changeing pRDBofsG tobacco (B).Fig. 8 is not genetically modified contrast (A) and the flower pesticide GUS dyeing of changeing pRDBofsG tobacco (B).Fig. 9 is not genetically modified contrast (A) and the pollen GUS dyeing of changeing pRDBofsG tobacco (B).Vegetative organ of commentaries on classics pRDBofsG tobacco such as root, stem, leaf are not seen the GUS activity.
Table 1, change the pRDBofsG tobacco T0 GUS dyeing in generation
| Strain system | Development of floral organs period | Flower pesticide | Pollen | Column cap | Petal |
| 2 | V | - | - | - | - |
| IV | - | - | - | - | |
| III | - | - | - | - | |
| II | ++ | - | - | - | |
| 6 | V | +++ | +++ | +++ | +++ |
| IV | - | - | - | +++ | |
| III | + | - | + | +++ | |
| 7 | V | + | - | - | - |
| III | ++ | - | - | ++ | |
| 9 | V | - | - | - | +++ |
| III | +++ | - | +++ | +++ | |
| II | + | - | + | ++++ | |
| 11 | V | - | ++ | + | +++ |
| IV | + | - | + | ++ | |
| III | - | - | - | - | |
| II | - | - | - | - | |
| 12 | V | ++ | - | - | - |
| IV | ++ | - | - | - | |
| III | ++ | - | - | - | |
| II | ++ | - | - | - | |
| I | - | - | - | - | |
| 14 | V | - | - | ++ | +++ |
| IV | ++ | - | ++ | +++ | |
| III | - | - | ++++ | +++ | |
| Contrast | - | - | - | - | - |
Annotate: "+" expression GUS stained positive in the table; " +++" represent that dyeing is the strongest; "-" expression GUS dyeing is negative,
Embodiment 5: change pRDBofsG tobacco T1 and express for GUS
Is to be inoculated into the seed germination substratum (1/2MS) that contains and do not contain Kan after 6,11 and 14 seeds of being tied (T1 generation) and not genetically modified contrast seed (NC89) surface sterilization thereof with changeing pRDBofsG tobacco T0 for strain, the dark cultivation 7 days is placed under the light in 25 ± 1 ℃ of cultivations.On the substratum that does not contain Kan, change pRDBofsG tobacco and not genetically modified contrast seed and can both germinate (percentage of germination is more than 95%), and seedling is green.On the substratum that contains 100mg/L Kan, though the part that has of not genetically modified contrast seed also can be germinateed all albefaction and death of seedling; But the seed seedling that changes the pRDBofsG tobacco has nearly 70% to be green, and growth is normal.Cultivated about 30 days, with to change pRDBofsG tobacco T0 be 6,11 and 14 T1 for strain for Kan resistance seedling each at random choosing wherein a strain carry out test tube and cultivate, obtain getting its vegetative organ behind the seedlings of vigorous growth and carry out GUS dyeing.The T1 seedling is long to be got the leaf dish when 2-3 sheet true leaf is arranged and carries out external anti-Kan and test, maturation is grown in 3 strains of selecting and remain at random of each strain system, in the growth course, get different organs and organize the method for having stated according to preamble to carry out GUS dyeing, to determine to change stability and the specificity that Bofs::GUS reaches in the T1 representative in the pRDBofsG tobacco.The result is as shown in table 2.
Table 2, commentaries on classics pRDBofsG tobacco T1 dye for the GUS of Kan resistance seedling
| Strain system | Root | Stem | Leaf | Flower pesticide | Pollen | Filigree | Ovary | Column cap | Petal | Sepal |
| 6 | - | - | - | +++ | + | - | - | ++ | ++ | - |
| 11 | - | - | - | +++ | + | - | - | +++ | ++ | - |
| 14 | - | - | - | +++ | ++ | - | - | ++ | ++ | - |
| Contrast | - | - | - | - | - | - | - | - | - | - |
Annotate: "+" expression GUS stained positive in the table; " +++" represent that dyeing is the strongest; "-" expression GUS dyeing is negative,
As can be seen from Table 2, in changeing the pRDBofsG tobacco, can stably pass to the offspring by Bofs driven GUS gene, and gus gene is not expressed at the offspring plant test-tube plantlet of all detections and the vegetative organ of greenhouse seedling, only expresses in floral organ specifically.
The above embodiments result shows and proves conclusively, wild cabbage provided by the present invention (Brassica oleracea L) promotor Bofs not only has strong driven nature, but also have good flower organ specificity, and this strong driven nature and specificity can stably pass to the offspring.Therefore, separation provided by the present invention is a flower organ specificity promoter stable, that motivating force is strong and specificity is high from the promotor Bofs of wild cabbage (Brassicaoleracea L).
Sequence table
<160>1
<210>1
<211>1244
<212>DNA
＜213〉the Cruciferae rape belongs to wild cabbage (Brassicia.oleracea L.)
<400>1
aagcttagca gcacgaatga agttcgtcaa gtttttaatt aggcttcgct tcttgtgatt 60
catcgaaaat ttatatcatt tcatacgttc attcttgttt tcatgtgact ttcctcttct 120
ctaccgtgag tctcatcaat ttcgtagatc gctatgttaa cgatccacgt atcatatata 180
caccttcttt ctatagccgt acgtatacca cacattacct catcccactt cctaacttat 240
ataattttac tactcagatc acaagtgtac gtatatcatg aagtcatttc ttctccttgt 300
cctactcctc tctttctttg tcggctctat cttcgctagt aggaattttc cgacgcaccc 360
ctatccaagt atgtatgctc ttcaattctc tctcactctc cttaatttta cccacctctt 420
tcactatctt caacgtcttt taacttgttc aattatgttc gtgtgggtgg gcaggtcata 480
atcatcatca tgtcggaatg atgggtagga aaatgaagcg tcagaggagg ccggacacgg 540
tgcaggtggc agggtctagg ctgccggact gctcacacgc gtgtggctca tgctccccat 600
gccgtcttgt gatggttagc ttcgtgtgtg catcgctaga ggaggctgag acttgtccca 660
tggcttataa gtgcatgtgc aagaacaaat cctaccccgt cccatgatga attagcctct 720
ctcacactta actctgtgca ttcagacgtt ttgtttcttt ccttttgctt cttcggataa 780
atgaccatgt gtatgtataa aatgcatctt ttcctttttt taattctgtt tgtctttttg 840
atatcttaaa cacagtttta cgaaacaaga ataagattag ttgagccact caaaagcgtg 900
gtcgactaaa ttgaaacaga aagccacaca actcattggg ctcttgttta tggcccatga 960
caccgcattt cagactgcaa caaccaaagt tgtagaaaga ataatattta aagggcacgt 1020
acatacgttg ttggcttcca ccaaactttg gaggctctct aataattagc acactccatt 1080
ctatgcattt gttacacacc ttctattttc aaccatttca tctcaccttt tttaaatgtt 1140
tccacagtta gctcagtaaa ttcactatat acagacatac accttccctc cacaagacca 1200
aacaaccaca ctaccttccc cgagttttct cactacaagg atcc 1244
<210>2
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
aagcttagca gcacgaatga agttc 25
<210>3
<211>29
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
ggatccttgt agtgagaaaa ctcggggaa 29
Claims (11)
1, a kind of plant flower organ specificity promoter, it is characterized in that: its nucleotide sequence is:
(1), sequence 1 described nucleotide sequence in the sequence table; Or
(2), with (1) described nucleotide sequence complementary nucleotide sequence; Or
(3), the nucleotide sequence that has 60% or 60% above homology with (1) or (2) described nucleotide sequence; Or
(4), be listed in the nucleotide sequence that to hybridize under the rigorous hybridization conditions with (1), (2) or (3) described nucleotides sequence.
2, the expression cassette that contains the described promotor of claim 1.
3, expression cassette according to claim 2 is characterized in that: the downstream of described promotor connects downstream syndeton gene, regulatory gene, the inverted defined gene of structure gene, the inverted defined gene of regulatory gene or the little RNA that can disturb native gene to express of described flower organ specificity promoter.
4, the recombinant expression vector that contains the described promotor of claim 1.
5, the recombinant expression vector that contains claim 2 or 3 described expression cassettes.
6, recombinant expression vector according to claim 5 is characterized in that: described recombinant expression vector is by claim 2 or 3 described expression cassettes and plasmid, virus or the constructed recombinant vectors of vehicle expression vector.
7, recombinant expression vector according to claim 6 is characterized in that: described recombinant expression vector is binary vector or closes carrier altogether.
8, transgenic plant cells or tissue or the organ that obtains by the arbitrary described recombinant expression vector transformed plant cells of claim 4-7 or plant tissue or plant organ.
9, the described flower organ specificity promoter of claim 1 is being cultivated the shape of not suiting, pattern, the fragrance of a flower and/or long shelf-life flower plant or crop varieties and/or strain, cultivate willow catkins flying in the air garden plants or crop varieties and/or strain that " willow catkins flying in the air " are eliminated or reduced significantly, cultivate enhancing of pollination fertility or the plant that weakens or crop varieties and/or strain or cultivate and to prevent to pollute the transgenic plant of its wild species and sibling species or the application in crop varieties and/or the strain by the pollen drift.
10, application according to claim 9 is characterized in that: described plant is a polycarpeae; Be preferably terrestrial plant.
11, the preparation method of the described anther specific promoter of a kind of claim 1, being that genomic dna with wild cabbage is a template, is that sequence 2 and nucleotide sequence are that a pair of primer of sequence 3 carries out pcr amplification and obtains promotor in the sequence table in the sequence table with nucleotide sequence.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101182523B (en) * | 2007-11-22 | 2010-11-24 | 中国农业大学 | Plants flower pesticide specificity promoter and uses thereof |
| CN105316333A (en) * | 2014-07-18 | 2016-02-10 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Identification and application of plant anther specific expression promoter pTaASG005 |
| CN109097364A (en) * | 2018-09-03 | 2018-12-28 | 深圳广三系农业科技有限公司 | A kind of identification and application of plant endosperm specificity expression promoter pOsEnS100 |
| CN110468136A (en) * | 2019-09-04 | 2019-11-19 | 中国农业大学 | A kind of flower specific promoter and its application |
| CN113416735A (en) * | 2021-03-17 | 2021-09-21 | 云南中烟工业有限责任公司 | Tobacco germ cell specific high expression gene and application thereof |
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2007
- 2007-04-25 CN CN200710098728A patent/CN100587071C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101182523B (en) * | 2007-11-22 | 2010-11-24 | 中国农业大学 | Plants flower pesticide specificity promoter and uses thereof |
| CN105316333A (en) * | 2014-07-18 | 2016-02-10 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Identification and application of plant anther specific expression promoter pTaASG005 |
| CN105316333B (en) * | 2014-07-18 | 2019-02-26 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | The identification and application of plant anther specific expression promoter pTaASG005 |
| CN109097364A (en) * | 2018-09-03 | 2018-12-28 | 深圳广三系农业科技有限公司 | A kind of identification and application of plant endosperm specificity expression promoter pOsEnS100 |
| CN110468136A (en) * | 2019-09-04 | 2019-11-19 | 中国农业大学 | A kind of flower specific promoter and its application |
| CN110468136B (en) * | 2019-09-04 | 2022-11-01 | 中国农业大学 | Flower-specific promoter and application thereof |
| CN113416735A (en) * | 2021-03-17 | 2021-09-21 | 云南中烟工业有限责任公司 | Tobacco germ cell specific high expression gene and application thereof |
| CN113416735B (en) * | 2021-03-17 | 2023-01-31 | 云南中烟工业有限责任公司 | Tobacco germ cell specific high expression gene and application thereof |
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