CN103725677B

CN103725677B - Tissue-specific promoter and purposes thereof

Info

Publication number: CN103725677B
Application number: CN201310581104.1A
Authority: CN
Inventors: 于彩虹
Original assignee: Beijing Dbn Biotech Co Ltd; Beijing Dabeinong Technology Group Co Ltd
Current assignee: Beijing Dabeinong Biotechnology Co Ltd
Priority date: 2013-11-18
Filing date: 2013-11-18
Publication date: 2016-06-08
Anticipated expiration: 2033-11-18
Also published as: CN103725677A

Abstract

The present invention relates to a kind of tissue-specific promoter and purposes thereof, does the nucleotide sequence of tissue-specific promoter comprise the sequence of following (a) or (b): (a) has SEQ simultaneously? ID? nucleotide sequence shown in the 920-932 position Nucleotide of NO:1,947-961 position Nucleotide, 1507-1519 position Nucleotide, 1963-1971 position Nucleotide and 2018-2028 position Nucleotide; The nucleotide sequence of b nucleotide sequence hybridization that () limits with (a) under strict conditions. Tissue-specific promoter of the present invention is separated the ABCG15 gene from rice variety 93-11 first; Simultaneously, it is possible to regulation and control object heterologous nucleotide sequence is specific expressed in plant anther tissue, and particularly tapetal tissue, lays a good foundation for formulating novel male sterile line.

Description

Tissue-specific promoter and purposes thereof

Technical field

The present invention relates to a kind of tissue-specific promoter and purposes thereof, particularly relate to promotor and the purposes thereof of the long-grained nonglutinous rice ABCG15 gene of a kind of anther tapetum tissue specific expression.

Background technology

The expression of allogeneic dna sequence in plant host depends on the promotor being operably connected having and working in plant host. The selection of promoter sequence will determine the time that allogeneic dna sequence is expressed in plant host and position. Therefore, when needs are expressed in plant is preferably organized, it may also be useful to tissue-specific promoter. On the contrary, when needs are expressed in whole vegetable cell, it may also be useful to constitutive promoter. Such as, by the heredity manipulation to Plant Genome, make it to contain the tissue-specific promoter being operatively connected with allos anti insect gene so that anti-insect material is expressed specifically in susceptible plant tissues, it is possible to the resistance realizing plants against insects invasion and attack increases. Heterologous nucleotide sequence preferential expression in targeted tissue decreases constitutive promoter and starts heterologous nucleotide sequence in whole vegetable cell and transcribe the plant resources consumption occurred.

Flower pesticide is the major portion that stamen produces pollen, and the wall of flower pesticide is made up of epidermal area, layer of fibers, middle layer and tapetum. Tapetum is the special cells layer around pollen sac, tool double-core or coenocytism, and cell includes more RNA and protein, and has the nutritive substance such as grease and carotenoid, has the effect that supply pollen granule grows required nutriment. In some cases, it is desirable to some important functional gene only specifically expressing in anther tissue. Such as, in hybrid seeding process, flower pesticide becomes, as the supplier of pollen, the key formulating novel male sterile line, it would therefore be desirable to have the anther tissue specific expressing promoter that the heterologous nucleotide sequence relevant to affecting anther and pollen development is operatively connected.

ABCG15 belongs to ABC(ATPBindingcassette) transfer protein G subfamily member (PaulJ.Verrieretal.TrendsinPlantScience2008.13(14): 151-159), report that the promotor of ABCG15 gene is separated from japonica rice as anther tapetum tissue specificity expression promoter and identifies there is not the correlative study deriving from long-grained nonglutinous rice ABCG15 gene promoter so far.

Summary of the invention

It is an object of the invention to provide a kind of tissue-specific promoter and purposes thereof, namely new separation, from the ABCG15 gene promoter of rice variety 93-11, enables object heterologous nucleotide sequence efficient specifically expressing in photosynthetic tissue.

For achieving the above object, the present invention provides a kind of tissue-specific promoter, and its nucleotide sequence comprises the sequence of following (a) or (b):

A () has the nucleotide sequence shown in the 920-932 position Nucleotide of SEQIDNO:1,947-961 position Nucleotide, 1507-1519 position Nucleotide, 1963-1971 position Nucleotide and 2018-2028 position Nucleotide simultaneously;

The nucleotide sequence of b nucleotide sequence hybridization that () limits with (a) under strict conditions.

Further, the nucleotide sequence of described tissue-specific promoter comprises the sequence of following (a) or (b):

A () has the nucleotide sequence shown in the Nucleotide of 900-2066 position of SEQIDNO:1;

Preferably, the nucleotide sequence of described tissue-specific promoter comprises the sequence of following (a) or (b):

A () has the nucleotide sequence shown in SEQIDNO:1;

Above-mentioned stringent condition can be at 6 �� SSC(Trisodium Citrate), 0.5%SDS(sodium lauryl sulphate) in solution, hybridize at 65 DEG C, then respectively wash film 1 time with 2 �� SSC, 0.1%SDS and 1 �� SSC, 0.1%SDS.

For achieving the above object, present invention also offers a kind of expression cassette comprising the described tissue-specific promoter being operably connected with object heterologous nucleotide sequence.

Further, described object heterologous nucleotide sequence coding target protein matter.

For achieving the above object, present invention also offers a kind of DNA construct comprising described expression cassette.

For achieving the above object, present invention also offers a kind of recombinant vectors comprising described DNA construct.

For achieving the above object, present invention also offers a kind of host cell comprising described recombinant vectors.

For achieving the above object, present invention also offers the anther tissue of a kind of plant containing described expression cassette in its cell.

Further, described plant is corn, Chinese sorghum, barley, oat, paddy rice or wheat.

For achieving the above object, present invention also offers a kind of method expressing object heterologous nucleotide sequence in plant, comprising: stable being integrated in vegetable cell of object heterologous nucleotide sequence being operably connected with described tissue-specific promoter.

Preferably, described object heterologous nucleotide sequence is expressed in anther tissue. More preferably, described anther tissue is tapetal tissue.

Preferably, the protein of described object heterologous nucleotide sequence coding-control plant fertility. The protein of described control plant fertility is the protein of control plants male sterility. The protein of described control plants male sterility is the protein affecting flower pesticide and/or pollen development.

For achieving the above object, present invention also offers a kind of described tissue-specific promoter for the purposes expressing object heterologous nucleotide sequence preferential in plant anther tissue.

Preferably, described plant is corn, Chinese sorghum, barley, oat, paddy rice or wheat.

In the present invention, term " promotor " refers to DNA regulatory region, usually starts the TATA box of RNA synthesis containing the applicable transcription initiation site that rna plymerase ii can be guided at specific encoding sequence. Promotor in addition containing other recognition sequence, can generally be positioned at upstream or the 5 ' end of TATA box, be called upstream promoter element, and they affect transcription initiation rate. Generally acknowledging ground, owing to promoter region nucleotide sequence of the present invention is own through determining, in the 5 ' non-translated region of the present invention's concrete promoter region upstream, separation andpreconcentration regulatory element belongs to prior art further. Therefore, promoter region of the present invention comprises upstream regulatory elements further, it gives any heterologous nucleotide sequence tissue specific expression being operably connected with promoter sequence of the present invention, it will be preferred that specific expressed in anther tissue, it is more preferable to ground is specific expressed in tapetal tissue.

In the present invention, term " gene " refers under suitable regulation and control region (such as plant expressible promoter region) controls any DNA fragmentation containing the region of DNA territory (" the region of DNA territory transcribed ") being transcribed into RNA molecule (such as mRNA) in cell. Therefore, the DNA fragmentation that gene may connect containing several operability, such as promotor, the 5 ' non-translation leader sequence, coding region and the 3 ' non-translation region containing polyadenylation site. Endogenous plant gene is the gene of natural discovery in plant species. Mosaic gene is any gene usually not found in plant species, or under its natural environment its promotor and the region of DNA territory partly or entirely transcribed or with this gene at least another regulates and controls the unrelated any gene in region.

The anther tissue that the feature of the promoter sequence of the present invention's separation is to provide object heterologous nucleotide sequence is specific expressed. Described anther tissue is that the part in cryptomere is expanded on filigree top, is the major portion that stamen produces pollen, and the wall of flower pesticide is made up of epidermal area, layer of fibers, middle layer and tapetum. Tapetum is the special cells layer around pollen sac, tool double-core or coenocytism, and cell includes more RNA and protein, and has the nutritive substance such as grease and carotenoid, has the effect that supply pollen granule grows required nutriment. In the present invention, term " anther tissue specificity " refers to as specific purpose, and the high degree of specificity of object heterologous nucleotide sequence in plant (such as rice plants (" Rice Anther tissue specificity ")) anther tissue cell is expressed. In other words, object heterologous nucleotide sequence is mainly expressed in the anther tissue of plant, and the DNA transcript level in the non-anther tissue of plant is that cannot detect or very low (every microgram total serum IgE is lower than about 0.2 pik).

Those skilled in the art can easily identify according to identical object and utilize promotor functionally of equal value. Have in essence to the nucleotide sequence shown in the 900-2066 position Nucleotide of the 920-932 position Nucleotide containing SEQIDNO:1 of the present invention, 947-961 position Nucleotide, 1507-1519 position Nucleotide, 1963-1971 position Nucleotide and the nucleotide sequence shown in the Nucleotide of 2018-2028 position or SEQIDNO:1 or the nucleotide sequence shown in SEQIDNO:1 or the DNA sequence dna with the similar promoter activity of the long-grained nonglutinous rice ABCG15 gene promoter of the nucleotide sequence of promoter activity part, be the function equivalent of these promotors. These function equivalence promotors can be hybridized with the long-grained nonglutinous rice ABCG15 gene promoter region containing above-mentioned nucleotide sequence under strict conditions.

In hybridization technique, oneself knows that all or part of of nucleotide sequence is used as probe, optionally hybridizes with other the corresponding nucleotide sequence existed in the cloned genomic dna fragment or cDNA fragment (such as genomic library or cDNA library) colony of selected organism. Hybridization probe can be genomic DNA fragment, cDNA fragment, RNA fragment or other oligonucleotide, it is possible to the group that can be detected as³²P or other any detectable marker substance markers. Such as, therefore, hybridization probe can be prepared by mark according to the synthetic oligonucleotide of sequence of the present invention. The method preparing hybridization probe and construction cDNA and genomic library be this area oneself know and be disclosed in the people such as Sambrook (1989) molecular cloning: the laboratory manual (second edition, ColdSpringHarborLaboratoryPress, Plainview, NewYork).

The hybridization of sequence can carry out under strict conditions. Described " stringent condition " refers to that probe will exceed and the condition of other sequence hybridization (as at least 2 times to background) to detecting degree with the hybridization of its target sequence. Stringent condition has sequence dependent, and different because of the difference of environment. By the severity of control hybridization and/or wash conditions, it is possible to the target sequence (homology detection) of qualification and probe 100% complementation. Selectively, it is possible to regulate stringent condition to allow some sequence mismatch to detect the similarity (allos detection) of relatively low degree. Usually, probe length is shorter than about 1000 Nucleotide, it is preferable that be shorter than 500 Nucleotide.

Typically, stringent condition is lower than about 1.5MNa ion pH7.0 to 8.3 time salt concn, typically about 0.01 to 1.0MNa ionic concn (or other salt), temperature at least about 30 DEG C of short probe (such as 10 to 50 Nucleotide), at least about 60 DEG C of long probe (as more than 50 Nucleotide). Also stringent condition can be obtained by adding destabilizing agent such as methane amide. Low stringency condition, such as, is included in 30-35% methane amide, 1MNaCl, 1%SDS(sodium laurylsulfonate) buffered soln in 37 DEG C of hybridization, 1 �� to 2 �� SSC(20 �� SSC=3.0MNaCl/0.3M trisodium citrate) in 50-55 DEG C of washing. Moderate stringency, such as, is included in the buffered soln of 40-45% methane amide, 1.0MNaCl, 1%SDS 37 DEG C of hybridization, in 0.5 �� 55-60 DEG C of washing to 1 �� SSC. Height stringent condition, such as, is included in the buffered soln of 50% methane amide, 1MNaCl, 1%SDS 37 DEG C of hybridization, 60-65 DEG C of washing in 0.1 �� SSC. Non-necessary ground, lavation buffer solution can contain the SDS of about 0.1% to 1%. Hybridization time generally less than about 24 hours, about 4 to 12 hours usually.

The function of washing after special hybridization typically, key factor is ionic strength and the temperature of final washing soln. For DNA-DNA crossbred, T_mCan from Meinkoth and Wahl(1984) equation estimation of Anal.Biochem.138:267-284: T_m=81.5 DEG C of+16.6(logM)+0.41(%GC)-0.61(%form)-500/L; Wherein M is the volumetric molar concentration of univalent cation, and %GC is guanylic acid and the cytidylic acid(CMP) per-cent in DNA, and %form is the per-cent of methane amide in hybridization solution, and L is the length of crossbred in base pair. T_mIt is 50% complementary target sequence and match the temperature (under the ionic strength and pH of regulation) of probe hybridization completely. The mispairing of every 1% needs T_mReduce about 1 DEG C; Therefore, T_mHybridization and/or wash conditions can be conditioned with the sequence hybridization with required identity. Such as, if the sequence sought has >=the identity of 90%, T_m10 DEG C can be reduced. Generally, the stringent condition of selection is the thermal melting point (T lower than particular sequence_m) about 5 DEG C, and it is complementary under the ionic strength and pH of regulation. But, height stringent condition can be applied lower than thermal melting point (T_m) hybridization of 1,2,3 or 4 DEG C and/or washing; Moderate stringency can be applied lower than thermal melting point (T_m) hybridization of 6,7,8,9 or 10 DEG C and/or washing; Low stringent condition can be applied lower than thermal melting point (T_m) hybridization of 11,12,13,14,15 or 20 DEG C and/or washing. Apply this equation, hybridization and cleaning composition and required T_m, the condition that those of ordinary skill in the art can understand hybridization and/or washing soln changes with the change of stringency. If required mispairing degree makes T_mLower than 45 DEG C (aqueous solution) or 32 DEG C (formamide soln), it is preferable that increase SSC concentration so that higher temperature can be used. The guide of nucleic acid hybridization sees Tijssen(1993) biological chemistry and Molecular Biology Lab's technology-with nucleic acid probe hybridization, I part, the 2nd chapter (Elsevier, NewYork); (1995) molecular biology modernism the 2nd chapter (GreenePublishingandWiley-Interscience, NewYork) is edited with people such as Ausubel. See the people such as Sambrook (1989) molecular cloning: laboratory manual (second edition, ColdSpringHarborLaboratoryPress, Plainview, NewYork).

Therefore, there is promoter activity and comprise in the present invention with the separation sequence of promoter sequence of the present invention or the hybridization of its fragment under strict conditions. These sequences and sequence of the present invention be 40%-50% homology at least approximately, about 60%, 65% or 70% homology, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology. Namely the scope of sequence iden is distributed at least approximately 40%-50%, about 60%, 65% or 70% homology, even at least about sequence iden of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or bigger.

Other function equivalence promotor contains such nucleotide sequence, the 920-932 position Nucleotide containing SEQIDNO:1 can be used, 947-961 position Nucleotide, 1507-1519 position Nucleotide, nucleotide sequence shown in 1963-1971 position Nucleotide and 2018-2028 position Nucleotide, or the nucleotide sequence shown in the Nucleotide of 900-2066 position of SEQIDNO:1, or at least about 25 of the nucleotide sequence shown in SEQIDNO:1, preferred at least about 50, the nucleotide sequence that particularly Oligonucleolide primers of at least about 100 continuous nucleotides increases in polymerase chain reaction.

Can also building Artificial promoters, it comprises the internal sequence of the anther tissue specificity of the decision promotor in 5 ' regulation and control region of the nucleotide sequence shown in SEQIDNO:1. Described Artificial promoters may comprise " core promoter " or " TATA box region " of another promotor can expressed in plant, such as the CaMV35S " TATA box region " described in WO93/19188. The suitability of the promoter region containing described Artificial promoters can be identified in the detection of appropriate tissue, the suitably expression of etap by the suitable fusion of they and object heterologous nucleotide sequence and object heterologous nucleotide sequence.

Promoter sequence of the present invention and fragment thereof, when being assembled into DNA structure and make promoter sequence be operably connected with object heterologous nucleotide sequence, manipulate any plant for heredity. What described " being operably connected " referred between the promoter sequence of the present invention with the 2nd sequence functional is connected, and wherein promoter sequence starts and regulates transcribing of the DNA sequence dna corresponding to the 2nd sequence. Generally, it is operably connected and refers to that the nucleotide sequence being connected is continuous print, be adjacent to if desired in conjunction with two protein-coding regions, and in same reading frame. In this way it would be possible, promotor nucleotide sequence form with object heterologous nucleotide sequence described mosaic gene in expression cassette together with provide, with in object plant express. This kind of expression cassette provides a large amount of restriction site to insert object heterologous nucleotide sequence, and described object heterologous nucleotide sequence will be subject to comprising the transcriptional regulatory of the regulatory region of promoter sequence of the present invention. Expression cassette can contain at least one additional gene that cotransformation enters organism in addition. Selectively, additional gene can be provided on multiple expression cassette.

Expression cassette can contain selectable marker gene in addition. Generally, expression cassette will comprise the selected marker for selecting transformant. Described selected marker is for selecting the cell or tissue transformed. Described selected marker includes but not limited to, the gene (such as coding neomycin phosphotransferase II(NPT) of coding antibiotics resistance), the gene of phosphomannose isomerase (PMI) and the gene of hygromix phosphotransferase (HPT), and the gene of conferring herbicide resistance is such as Glufosinate ammonium, bromoxynil, imidazolone type and 2,4-dichlorphenoxyacetic acid (2,4-D).

Expression cassette comprises along 5 '-3 ' promoter sequence of the present invention of transcribing of direction, Translation initiator, object heterologous nucleotide sequence and transcribing and translation termination district of working in plant. Object heterologous nucleotide sequence can be natural or to plant host external source or allos. Selectively, object heterologous nucleotide sequence can be native sequences or selectivity synthesis sequence. " external source " refers to there is not described importing transcription initiation region in the natural phant importing transcription initiation region. Such as, mosaic gene comprises the promoter sequence of the present invention, the promoter sequence of the present invention operationally encoding sequence with the promoter sequence being different from the present invention be connected.

Terminate the promoter sequence that district can derive from the present invention, it is possible to derive from the object heterologous nucleotide sequence being operatively connected, maybe can derive from other source. Traditional termination district can obtain from the Ti-plasmids of soil Agrobacterium, as carnitine synthetic enzyme and rouge alkali synthetase (NOS) terminate district.

In prepared by expression cassette, it is possible to manipulate different DNA fragmentations to provide the DNA sequence dna in suitable direction, and provide suitable reading frame in applicable. So, can apply and accept son or connexon in conjunction with DNA fragmentation, maybe can carry out other manipulation with provide convenience restriction site, remove unnecessary DNA, remove restriction site etc. Object for this reason, it is possible to relate to vitro mutagenesis, primer reparation, restriction, annealing, replace, as changed and conversion.

Under appropriate circumstances, object heterologous nucleotide sequence can be optimized to be increased in the expression amount transformed in plant. Namely the preferred codon synthetic gene of available plant is to improve expression.

Well known in the art, other sequence modification can improve the gene expression dose in cell host. These include but not limited to, remove the tumor-necrosis factor glycoproteins of the poly-adenosine signal of coding vacation, exon: intron splice site signal, transposon, and other fully symbolizes the sequence that may be unfavorable for genetic expression. The G-C content of sequence can be adjusted to the mean level (ML) specifying host cell, quotes the gene expression dose that in host cell, oneself knows and calculates. May, modification sequence is to avoid the hair clip formula mRNA secondary structure of prediction.

In expression cassette or recombinant vectors, expression cassette can contain 5 ' leader sequence in addition. Described leader sequence can play a part to improve transcriptional efficiency. Described leader sequence is known in the art, and includes but not limited to, picornavirus leader sequence, such as EMCV leader sequence (encephalomyocarditis 5 ' non-coding region); Potato virus group leader sequence, such as tobacco etch virus (TEV) leader sequence, corn short and small mosaic virus (MDMV) leader sequence and human immunoglobulin heavy chain's associated proteins (BiP); From alfalfa mosaic virus coating protein mRNA(AMVRNA4) non-translation leader sequence; Tobacco mosaic virus (TMV) (TMV) leader sequence; With corn yellows spot virus (MCMV) leader sequence. Other oneself element of improvement transcriptional efficiency of knowing, such as intron etc. can also be used.

The promoter sequence of the present invention can be used to start transcribing of at least part of complementary antisense construct of messenger RNA(mRNA) (mRNA) with object heterologous nucleotide sequence. Build antisense base sequences to hybridize with corresponding mRNA. As long as antisense sequences grows to and can hybridize with corresponding mRNA and disturb it to express, so that it may carry out the modification of antisense sequences. Have 70% in this way, it is possible to use with corresponding antisense sequences, it is preferable that 80%, it is more preferable to the antisense construct of 85% sequence iden. In addition, a part for antisense base sequences can be used to destroy the expression of target gene. Generally, the sequence of at least 50 Nucleotide, 100 Nucleotide, 200 Nucleotide or more Nucleotide can be used.

The promoter sequence of the present invention also can be used for starting justice direction nucleotide sequence transcribe the expression suppressing endogenous gene in plant. The method that the nucleotides sequence in application justice direction is listed in plant inhibition of gene expression be this area oneself know. The method is usually directed to transform plant with the DNA structure containing promotor, and promotor may be operably coupled to the nucleotide sequence that a few part is equivalent to endogenous gene transcription, and drives it to express in plant. Typically, the sequence of described nucleotide sequence and endogenous gene transcription has essence Shangdi sequence iden, it is preferable that exceed about 65% sequence iden, it is more preferable to ground exceedes about 85% sequence iden, it is most preferred that ground exceedes about 95% sequence iden.

The promoter sequence of the present invention is used to the tissue specific expression of object heterologous nucleotide sequence. The sequence that " heterologous nucleotide sequence " exists with referring to non-natural together with promoter sequence. Although described nucleotide sequence and promoter sequence are allos, but may be homology or natural or allos or external source to plant host. The heterologous nucleotide sequence being operationally connected with the promotor of the present invention can encode target protein matter. The example of this kind of heterologous nucleotide sequence includes but not limited to, coding gives the nucleotide sequence of following resistant polypeptides: abiotic stress is such as arid, temperature, salinity, ozone and weedicide, or biology stress attack such as pathogenic agent, comprise insect, virus, bacterium, fungi and threadworms, and prevent the disease that these organisms are adjoint; The protein of all right coding-control plant fertility, it is preferable that the protein of control male plant fertility.

A lot of for gene crucial male fertile, comprise and stop spore development (AbortedMicrospores, i.e. AMS) gene, NEF1 gene, AtGPAT1 gene, dde2-2 suddenlys change (its defect demonstrating allene oxide synthase gene), flp1(facelesspollen-1) gene, the dead 1(MaleMeiocyteDeath1 of male parent cell) gene, tapetum specificity zinc finger gene TAZ1, tapetum factor of determination 1(TapetumDeterminant1) gene, MS1 gene, DPW(MS2) gene, MS3 gene, MS5 gene, MS7 gene, MS8 gene, MS9 gene, MS10 gene, MS11 gene, MS12 gene, MS13 gene, MS14 gene, MS17 gene, MS20 gene, MS22 gene, MS23 gene, MS24 gene, MS25 gene, MS26 gene, MS27 gene, MS28 gene, MS29 gene, MS30 gene, MS31 gene, MS32 gene, MS33 gene, MS34 gene, MS36 gene, MS37 gene, MS38 gene, MS43 gene, MS45 gene, MS48 gene, MS50 gene.

In addition, the nucleotide sequence affecting male fertility expressed in plant male tissue in the present invention also can encoding carbohydrate degraded or modifying enzyme, amylase, debranching factor and polygalacturonase, such as ��-amylase gene, plant hormone, rolB, cytotoxin, diphtheria toxin, DAM methylase, avidin or protokaryon regulator control system can be selected from. Citing, the RNase-T1 of aspergillus oryzae (Aspergillusoryzae) or the RNase(of bacillus amyloliquefaciens (Bacillusamyloliquefaciens) is named as " barnase ") breaking of tapetal cell of induced expression in tapetum, cause male sterile. The rolB genes encoding of agriculture bacillus (Arobacteriumrhizogenes) is by discharging free indoles from indolol-��-glucoside thus disturb the enzyme of growth hormone metabolism. There are some researches show, in tobacco, the pollen sac of rolB gene is specific expressed creates the withered plant of pollen sac, and wherein, the generation of pollen greatly reduces, and therefore rolB gene is the example that can be used for controlling the gene that pollen produces.

Transformation Protocol and scheme that nucleotide sequence imports plant are different according to the directed plant that transforms or plant cell type, i.e. monocotyledons or dicotyledons. Appropriate methodology nucleotide sequence being imported vegetable cell and insert in Plant Genome subsequently includes but not limited to, Agrobacterium-medialed transformation, trace launch bombardment, the direct DNA that DNA takes in protoplastis, electroporation or silicon whisker mediation imports.

The cell transformed can grow into plant in a conventional manner. These plants are cultivated, and pollinate with identical conversion strain or different conversion strains, the identified phenotype feature needed for crossbred expression obtained. Can cultivate two generations or many generations with ensure to keep with stablizing with heredity needed for the expression of phenotype feature, then results can ensure to obtain the seed of required phenotype feature representation.

The present invention provides a kind of tissue-specific promoter and purposes thereof, has the following advantages:

1, it is separated first. Tissue-specific promoter of the present invention is separated the ABCG15 gene from rice variety 93-11 first.

2, tissue specific expression. It is specific expressed in plant anther tissue that tissue-specific promoter of the present invention can regulate and control object heterologous nucleotide sequence, particularly tapetal tissue, lays a good foundation for formulating novel male sterile line.

Below by drawings and Examples, the technical scheme of the present invention is described in further detail.

Accompanying drawing explanation

The recombinant cloning vector pT-prOsATS1 that Fig. 1 is tissue-specific promoter of the present invention and purposes thereof builds schema;

The recombinant expression vector DBN-prOsATS1 that Fig. 2 is tissue-specific promoter of the present invention and purposes thereof builds schema;

Fig. 3 is the morphological observation figure of the mutant plants Osats1 of tissue-specific promoter of the present invention and purposes thereof;

Fig. 4 is the pollen development figure of the mutant plants Osats1 of tissue-specific promoter of the present invention and purposes thereof;

Fig. 5 is the pollen development figure of the transgenic rice plant-ATS1 of tissue-specific promoter of the present invention and purposes thereof.

Embodiment

The technical scheme of tissue-specific promoter of the present invention and purposes thereof is described further below by specific embodiment.

The acquisition of the first embodiment, Osats1 mutant plants and morphological observation

Mutant plants Osats1(Sichuan Agricultural University collaborative project obtains) it is the spontaneous mutation material identifying discovery in indica hybrid seed selection process, this material does not have significant difference (Fig. 3) in plant vegetative growth stage and wild-type, but flower pesticide is suppressed in early days in growth, white is presented at flower development later stage flower pesticide, thin and hollow, finally cannot produce pollen, show as male sterile completely (Fig. 4).

The acquisition of the 2nd embodiment, anther-specific expression promotor prOsATS1 and analysis

Find that Osats1 mutant plants is by ABCG15 gene (Loc_Os06g40550 by Fine Mapping; As shown in SEQ ID NO:2) the middle afunction mutant deleted 12bp and cause, the sequence of mutant plants Osats1 is as shown in SEQ ID NO:3.

Analyzing and find, the ABCG15 upstream region of gene 2066bp being positioned at Fine Mapping has RNA polymerase recognition site, as shown in SEQ ID NO:1, and called after prOsATS1. This promotor is expressed specifically in flower pesticide, particularly tapetal tissue.

By NSITE-PL on-line prediction find prOsATS1(SEQIDNO:1) on comprise 5 core controlling elements (http://linux1.softberry.com/berry.phtml topic=nsitep&group=programs&subgroup=promoter), specifying information is in table 1:

The core controlling element caluclate table of table 1:prOsATS1

Numbering	Position	Controlling element	Controlling element binding factor
				1	920-932bp	WRKY11BS2	WRKY11
2	947-961bp	E2F BS	E2F
				3	1507-1519bp	CytRE	unknown nuclear factor
4	1963-1971bp	Sph-core	VP1(VIVAPAROUS1)
				5	2018-2028bp	GA-5	BPC1

It can thus be seen that the core regulating and controlling sequence of prOsATS1 is the 900-2066 position of sequence 1, this section of sequence is likely on prOsATS1 to can be used for the most short data records recovering Osats1 mutant pollen fertility.

The structure of the 3rd embodiment, recombinant expression vector and recombinant expression vector transformation Agrobacterium

1, the recombinant cloning vector containing prOsATS1 promoter sequence is built

PrOsATS1 promoter sequence is connected into cloning vector pGEM-T(Promega, Madison, USA, CAT:A3600) on, operation steps is undertaken by Promega company product pGEM-T carrier specification sheets, wherein, obtaining recombinant cloning vector pT-prOsATS1, it builds flow process, and (Amp represents ampicillin resistance gene as shown in Figure 1; F1 represents the replication orgin of phage f1; LacZ is LacZ initiator codon; SP6 is SP6RNA polymerase promoter; T7 is t7 rna polymerase promotor; PrOsATS1 is prOsATS1 promoter sequence (SEQIDNO:1); MCS is multiple clone site).

Then by recombinant cloning vector pT-prOsATS1 heat shock method transformation of E. coli T1 competent cell (Transgen, Beijing, China, CAT:CD501), its hot shock condition is: 50 �� l intestinal bacteria T1 competent cells, 10 �� l plasmid DNA (recombinant cloning vector pT-prOsATS1), 42 DEG C of water-baths 30 seconds; 37 DEG C of shaking culture 1 hour (under 100rpm rotating speed shaking table shake), scribble IPTG(isopropylthio-��-D-galactoside on surface) and the bromo-4-of X-gal(5-chloro-3-indoles-��-D-galactoside) LB flat board (the Tryptones 10g/L of penbritin (100 mg/litre), yeast extract 5g/L, NaCl10g/L, agar 15g/L, adjusts pH to 7.5 with NaOH) upper grow overnight. Choose extracting waste bacterium colony, LB liquid nutrient medium (Tryptones 10g/L, yeast extract 5g/L, NaCl10g/L, penbritin 100mg/L, with NaOH adjust pH to 7.5) under temperature 37 DEG C of conditions overnight incubation. Its plasmid of alkalinity extraction: by bacterium liquid centrifugal 1min under 12000rpm rotating speed, remove supernatant liquor, the precipitation thalline solution I (25mMTris-HCl, 10mMEDTA(ethylenediamine tetraacetic acid (EDTA)) of 100 �� l ice precoolings, 50mM glucose, pH8.0) suspend; Add the solution II (0.2MNaOH, 1%SDS(sodium lauryl sulphate) that 150 �� l newly prepare), pipe is put upside down 4 times, mixing, puts 3-5min on ice; Add the ice-cold solution III of 150 �� l (4M Potassium ethanoate, 2M acetic acid), fully mixed even immediately, place 5-10min on ice; Centrifugal 5min in temperature 4 DEG C, rotating speed 12000rpm when, adds 2 times of volume dehydrated alcohols in supernatant liquor, and mixed even rear room temperature places 5min; Centrifugal 5min in temperature 4 DEG C, rotating speed 12000rpm when, abandons supernatant liquor, precipitation concentration (V/V) be 70% washing with alcohol after dry; Add 30 �� l containing RNase(20 �� g/ml) TE(10mMTris-HCl, 1mMEDTA, PH8.0) dissolution precipitation; Water-bath 30min at temperature 37 DEG C, digestion RNA; Save backup in temperature-20 DEG C.

The plasmid extracted is after NotI and SacII enzyme cuts qualification, positive colony is carried out sequence verification, result shows that the described prOsATS1 promoter sequence of insertion in recombinant cloning vector pT-prOsATS1 is the nucleotide sequence shown in SEQ ID NO:1, and namely prOsATS1 promoter sequence correctly inserts.

2, the recombinant expression vector containing prOsATS1 promotor is built

With restriction enzyme SacI and KpnI respectively enzyme cut recombinant cloning vector pT-prOsATS1 and expression vector DBNBC-01(carrier framework: pCAMBIA2301(CAMBIA mechanism can provide)), the prOsATS1 promoter sequence fragment cut is inserted between SacI and the KpnI site of expression vector DBNBC-01, conventional enzymatic cleavage methods carrier construction is utilized to be well-known to those skilled in the art, being built into recombinant expression vector DBN-prOsATS1, it builds flow process (Kan: kanamycin gene as shown in Figure 2; RB: right margin; PrOsATS1: long-grained nonglutinous rice ABCG15 gene promoter (SEQIDNO:1); GOsATS1: long-grained nonglutinous rice ABCG15 gene (SEQIDNO:2); Nos: the terminator (SEQIDNO:4) of rouge alkali synthetase gene; Ubi: corn Ubiquitin(ubiquitin) gene promoter (SEQIDNO:5); PMI: Phophomannose isomerase gene (SEQIDNO:6); LB: left side circle).

By recombinant expression vector DBN-prOsATS1 heat shock method transformation of E. coli T1 competent cell, its hot shock condition is: 50 �� l intestinal bacteria T1 competent cells, 10 �� l plasmid DNA (recombinant expression vector DBN-prOsATS1), 42 DEG C of water-baths 30 seconds; 37 DEG C of shaking culture 1 hour (under 100rpm rotating speed shaking table shake); Then at LB solid plate (the Tryptones 10g/L containing 50mg/L kantlex (Kanamycin), yeast extract 5g/L, NaCl10g/L, agar 15g/L, adjust pH to 7.5 with NaOH) upper under temperature 37 DEG C of conditions cultivation 12 hours, choose extracting waste bacterium colony, at LB liquid nutrient medium (Tryptones 10g/L, yeast extract 5g/L, NaCl10g/L, kantlex 50mg/L, with NaOH adjust pH to 7.5) under temperature 37 DEG C of conditions overnight incubation. Its plasmid of alkalinity extraction. Identify after the plasmid restriction enzyme SacI of extraction and KpnI enzyme are cut, and positive colony is carried out order-checking qualification, result shows that the nucleotides sequence of recombinant expression vector DBN-prOsATS1 between SacI and KpnI site is classified as nucleotide sequence shown in SEQ ID NO:1, i.e. prOsATS1 promoter sequence.

3, recombinant expression vector transformation Agrobacterium

Oneself is transformed into Agrobacterium LBA4404 (Invitrgen through building correct recombinant expression vector DBN-prOsATS1 liquid nitrogen method, Chicago, USA, CAT:18313-015) in, its conversion condition is: 100 �� L Agrobacterium LBA4404s, 3 �� L plasmid DNA (recombinant expression vector); It is placed in liquid nitrogen 10 minutes, 37 DEG C of warm water bath 10 minutes; It is inoculated in LB test tube in temperature 28 DEG C, rotating speed are 200rpm when to cultivate 2 hours by the Agrobacterium LBA4404 after transforming, be applied to the Rifampin containing 50mg/L (Rifampicin) and 100mg/L kantlex (Kanamycin) LB flat board on until grow positive monoclonal, choose and get Colony Culture and extract its plasmid, carrying out digestion verification with restriction enzyme A hdI and XbaI, result shows that recombinant expression vector DBN-prOsATS1 structure is entirely true.

The acquisition of the 4th embodiment, transgenic rice plant and analysis

1, transgenic rice plant is obtained

The agriculture bacillus infestation method conveniently adopted, the long-grained nonglutinous rice mutant Osats1(Sichuan Agricultural University collaborative project with the recessive allelotrope ABCG15 that isozygotys of sterile culture is obtained) callus and the 3rd embodiment in agriculture bacillus Dual culture described in 3, so that the T-DNA(in the recombinant expression vector DBN-prOsATS1 of in the 3rd embodiment 2 structures is comprised prOsATS1 promoter sequence, OsATS1 gene, the promoter sequence of corn Ubiquitin gene, PMI gene and Nos terminator sequence) it is transferred in rice chromosome group, obtain transgenic rice plant-ATS1.

For agriculture bacillus mediated rice conversion, briefly, rice paddy seed is seeded in inducing culture (N6 salt 3.1g/L, N6 vitamin b6 usp, casein food grade 300mg/L, maltose 30g/L, 2, 4-dichlorphenoxyacetic acid (2, 4-D) 2mg/L, plant gel 3g/L, pH5.8) on, callus (step 1: callus of induce step) is induced from paddy rice mature embryo, afterwards, preferred callus, callus is contacted with agrobacterium suspension, at least one cell (step 2: infect step) that the construct of described mediated plant fertility can be passed on callus by its middle peasant bacillus. in this step, callus preferably immerses agrobacterium suspension (OD₆₆₀=0.3, infect substratum (N6 salt 3.1g/L, N6 vitamin b6 usp, casein food grade 300mg/L, sucrose 30g/L, glucose 10g/L, Syringylethanone (AS) 40mg/L, 2,4 dichlorophenoxyacetic acid (2,4-D) 2mg/L, pH5.4)) in start inoculation. Callus and agriculture bacillus Dual culture one period (3 days) (step 3: Dual culture step). Preferably, callus after infecting step at solid medium (N6 salt 3.1g/L, N6 vitamin b6 usp, casein food grade 300mg/L, sucrose 30g/L, glucose 10g/L, Syringylethanone (AS) 40mg/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 2mg/L, plant gel 3g/L, pH5.8) upper cultivate. After this Dual culture stage, there is " recovery " step. In " recovery " step, recovery media (N6 salt 3.1g/L, N6 vitamin b6 usp, casein food grade 300mg/L, sucrose 30g/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 2mg/L, plant gel 3g/L, pH5.8) at least exist in a kind of oneself know suppress agriculture bacillus growth microbiotic (cephamycin), do not add the selective agent (step 4: recovering step) of vegetable transformant. Preferably, callus having microbiotic but do not have on the solid medium of selective agent cultivate, taking eliminate agriculture bacillus and as infected cell provide decubation. Then, the callus of inoculation is containing cultivating the transformed calli (step 5: select step) that also growth selection on the substratum of selective agent (seminose). Preferably, callus is having the screening solid medium of selective agent (N6 salt 3.1g/L, N6 vitamin b6 usp, casein food grade 300mg/L, sucrose 5g/L, seminose 12.5g/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 2mg/L, plant gel 3g/L, pH5.8) upper cultivation, causes the cell selective growth transformed. Then, callus regeneration becomes plant (step 6: regeneration step), it may be preferred that at the callus containing growth on the substratum of selective agent in the upper cultivation of solid medium (N6 division culture medium and MS root media) with aftergrowth.

The resistant calli that screening obtains transfers to described N6 division culture medium (N6 salt 3.1g/L, N6 vitamin b6 usp, casein food grade 300mg/L, sucrose 20g/L, seminose 5g/L, 6-benzyl aminoadenine 2mg/L, naa 1mg/L, plant gel 3g/L, pH5.8), on, differentiation at 25 DEG C, is cultivated. Differentiation little Miao out transfers on described MS root media (MS salt 2.15g/L, MS vitamin b6 usp, casein food grade 300mg/L, sucrose 15g/L, plant gel 3g/L, pH5.8), is cultured to about 10cm height at 25 DEG C, moves to hot-house culture to solid. In greenhouse, every day cultivates at 28 DEG C.

2, transgenic rice plant is analyzed

Plant transgenic rice plant-ATS1 and wild rice plant respectively. Experimental result shows: compared with wild rice plant, transgenic rice plant-ATS1 not only plant vegetative growth there is no significant difference, and drive OsATS1 protein expression with the use of prOsATS1, anther development is normal, and can 100% recovery its deletion mutant pollen fertility, it is possible to normally solid (Fig. 5).

In sum, tissue-specific promoter of the present invention is separated the ABCG15 gene from rice variety 93-11 first; Simultaneously, it is possible to regulation and control object heterologous nucleotide sequence is specific expressed in plant anther tissue, and particularly tapetal tissue, lays a good foundation for formulating novel male sterile line.

It should be noted last that, above embodiment is only in order to illustrate the technical scheme of the present invention and unrestricted, although with reference to better embodiment to invention has been detailed explanation, it will be understood by those within the art that, the technical scheme of the present invention can be modified or equivalent replacement, and not depart from the spirit and scope of technical solution of the present invention.

Claims

1. a tissue-specific promoter, it is characterised in that, its nucleotide sequence is selected from the sequence of following (a) or (b):

Nucleotide sequence shown in (a) SEQIDNO:1;

The nucleotide sequence of b nucleotide sequence complementation that () and (a) limit.

2. one kind comprises the expression cassette of tissue-specific promoter described in the claim 1 being operably connected with object heterologous nucleotide sequence.

3. expression cassette according to claim 2, it is characterised in that, described object heterologous nucleotide sequence coding target protein matter.

4. one kind comprises the DNA construct of expression cassette described in Claims 2 or 3.

5. one kind comprises the recombinant vectors of DNA construct described in claim 4.

6. containing an anther tissue for the plant of expression cassette described in Claims 2 or 3 in its cell, described plant is corn or paddy rice.

7. in plant, express the method for object heterologous nucleotide sequence for one kind, it is characterized in that, comprising: be integrated in vegetable cell by stable for the object heterologous nucleotide sequence being operably connected with tissue-specific promoter described in claim 1, described plant is corn or paddy rice.

8. in plant, express the method for object heterologous nucleotide sequence according to claim 7, it is characterised in that, described object heterologous nucleotide sequence is expressed in anther tissue.

9. in plant, express the method for object heterologous nucleotide sequence according to claim 8, it is characterised in that, described anther tissue is tapetal tissue.

10. in plant, express the method for object heterologous nucleotide sequence according to claim 7, it is characterised in that, described object heterologous nucleotide sequence coding target protein matter.

11. express the method for object heterologous nucleotide sequence according to claim 10 in plant, it is characterised in that, the protein of described object heterologous nucleotide sequence coding-control plant fertility.

12. according to the method expressing object heterologous nucleotide sequence described in claim 11 in plant, it is characterised in that, the protein of described control plant fertility is the protein of control male plant fertility.

13. according to the method expressing object heterologous nucleotide sequence described in claim 12 in plant, it is characterised in that, the protein of described control male plant fertility is the protein affecting flower pesticide and/or pollen development.

Tissue-specific promoter described in 14. 1 kinds of claims 1 is used for preferentially expressing the purposes of object heterologous nucleotide sequence in plant anther tissue, and described plant is corn or paddy rice.

15. be used for preferentially expressing in plant anther tissue the purposes of object heterologous nucleotide sequence according to tissue-specific promoter described in claim 14, it is characterised in that, described object heterologous nucleotide sequence coding target protein matter.