CN1301300A - Pollen specific promoter - Google Patents
Pollen specific promoter Download PDFInfo
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- CN1301300A CN1301300A CN99805265A CN99805265A CN1301300A CN 1301300 A CN1301300 A CN 1301300A CN 99805265 A CN99805265 A CN 99805265A CN 99805265 A CN99805265 A CN 99805265A CN 1301300 A CN1301300 A CN 1301300A
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Abstract
A recombinant nucleic acid which comprises a promoter sequence of the ZmC5 gene in maize, or a variant or fragment thereof, which acts as a promoter in pollen. The DNA sequence of the promoter is depicted in figure 5. Expression cassettes and expression systems as well as transformation methods, transformed plants including the promoter sequence of the invention are also claimed. The nucleic acid may be used inter alia in the production of male sterile plants and/or hybrids as well as in the transformation of pollen.
Description
The present invention relates to the pollen specific promoter sequence, contain construct and the transgenic plant cells and the plant of this promotor, transform the method for pollen and the method for using this promotor controlling plant fertilizability.
For the required inherited character with two kind of plant imports a kind of plant, as kind or hybrid, traditional method is cross breeding.For the hybrid of acquired character unanimity reliably, must guarantee that mother plant can self-pollination.
By guaranteeing that the parent can accomplish this point for male sterile.It is known producing male sterile multiple technologies, also is that this area recommends to use.Wherein a kind of method is removed flower pesticide or the tassel of female mother plant with comprising artificially or mechanize.This plant has only the pollen by the male parent just to have fertilizability, therefore thereafter on behalf of hybrid.Yet, this method effort and not exclusively reliable, because some females may not be removed tassel, perhaps in some cases, plant also can grow second tassel after being removed tassel.In addition, in this system, the self-pollination of male parent's energy, the therefore essential male and female parent of physical sepn, thus make the results of hybrid become easy.This segmentation planting method is very suitable for corn, because its pollen is light and amount is big.Yet this method also is not suitable for the heavier kind of pollen and male and female floral organ is included in same kind in spending, as wheat, and paddy rice.
Prevent that the chemical process that pollen produces from also being known, United States Patent (USP) 4,801,326 have described and can be applicable to plant or the compound of soil to prevent that pollen from producing.Yet this technology is also required great effort, and is not very reliable.Must use enough compounds at interval with reasonable time and can not take place to guarantee pollination, this can pollute environment.In addition, these compounds are also very expensive.
Genetically engineered provides another kind of alternative method, takes this to produce in plant, keeps and/or recovers male sterile subsequently.
Some systems have been described, wherein inducible promoter can according to the externalizing compound use or validity starts or closes fertilizability.For example, WO 90/08830 has described by expressing and has destroyed the male sterile of the biosynthetic proteinic gene order cascade system inducing plant of pollen.WO 93/18171 has described use GST promotor inducible expression chalcone synthase (chs), and has recovered to have the fertilizability of the male sterile plants of sterile proterties by " knocking out " endogenous chs gene.
Among the WO 93/25695 (PGS) diverse ways has been described, this method relies on uses tapetum specific efficient promoter to express pollen lethal gene barnase in tapetal cell, this enzyme is vital to the generation of pollen, therefore can destroy pollen and produce.Can use recover the gene barstar recover fertilizability (Mariani etc., nature, Vol357:384-387).
It will be favourable directly controllably expressing in pollen pollen is produced influential gene.In case of necessity, fertilizability should be able to be resumed.
Drive expression promoter specifically in the tissue that the moiety of genetically engineered male sterility system is to use in pollen or pollen produces or growth is relied on.
A lot of the characteristic genes encoding pair cell wall metabolism of specific expressed or high expression level can active protein in the pollen of pollen and sprouting, for example, participates in the enzyme of pectin degrading with coding; Polygalacturonase (.. such as SM Brown, vegetable cell (1990) 2:263-274 .. such as SJ Tebbutt, molecular biology of plants.(1994) 25:283-299), pectate lyase (HJ Rogers etc., molecular biology of plants, 20:493-502 (1992), RAWing etc., molecular biology of plants, 14:17-28 (1989)) and pectin methyl esterase (PME) (JH Mu etc., molecular biology of plants, 25:539-544 (1994)) gene that gene has homology.
Other gene of high expression level in pollen comprises Codocyte skelemin (.. such as I.Lopez, Proc.Natl, Acad Sci USA 93:7415-7420 (1996) .. such as HJ Rogers, .. such as plant magazine 4:875-882 (1993) and CJ Staiger, plant magazine 4; 631-641 (1993)), the Vitamin C oxidase of inferring, the gene of Kunitz protein inhibitor and a lot of its functions can not be by other genes of inferring with the homology of known.Studied the temporal expression of these genes, found in sophisticated microsporocyte, the expression amount in late period that a lot of genes take place sporule is the highest.In some cases, illustrated the continuous expression in the pollen tube (AK Kononowicz etc., vegetable cell 4; 513-524 (1992)).These genes are called as " late gene ".The great majority in this stage are expressed from vegetative cell rather than sexual cell, and most of late genes may be transcribed by vegetative nucleus; But only having confirmed that " late period " gene is transcribed by vegetative nucleus obtains (D.Twell, plant magazine 2:887-892 (1992).Found that the different classes of gene of expressing in the flower pesticide has different expression programs, can detect expression first soon behind the four-strand stage, and expression amount straight line minimizing before pollen maturation.The main effect of these " in early days " genes may embody in the process of sporule differentiation and growth rather than pollen tube growth.In addition, United States Patent (USP) 5.086,169 (Mascarenhas) has been described isolated first pollen specific promoter from corn.
The contriver has isolated another only specific expressed promotor in the pollen tissue.This promotor gets self-separation from " late period " of corn pollen expressing gene ZmC5.
Therefore, according to a first aspect of the invention, provide the promoter sequence or its variant or its pulsating recombinant nucleic acid sequence that contain corn ZmC5 gene, described nucleotide sequence can be used as the promotor in the pollen.
Term used herein " segment " comprises one or more zones of the basic sequence that keeps promoter activity.When segment contains one or more zone, can directly they be linked together, perhaps they are separated with other base.
Term used herein " variant " refers to any replacement of above-mentioned nucleotide sequence being carried out one or more Nucleotide, and variation is modified, and replaces, and lacks or interpolation, still shows the sequence of pollen promoter expression.This term also comprises the sequence that can hybridize basically with above-mentioned nucleotide sequence.
Term used herein " ZmC5 gene " refers to the corn gene of the sequence of coding 563 amino acid longs described herein.The cDNA sequence of this sequence of encoding limits in EMBL Y13285.
Promoter sequence of the present invention is included among the clone NCIMB 40915 that was preserved in state-run industry and marine bacteria preservation center on January 26th, 1998.This promoter sequence is can be by the described Sal I segment that obtains hereinafter.Promoter region is positioned at the zone of being made up of the about 2kb sequence in corn ZmC5 genetic transcription initiation site upstream hereinafter shown in Figure 1.
According to the preferred embodiments of the invention, provide and contained promoter sequence or its variant or its pulsating recombinant nucleic acid sequence, described promoter sequence contains the part of dna sequence dna shown in Figure 5 at least or the promotor that contains at least with Fig. 5 coding has the part of the encoding sequence of similar substantially active promotor.
Term " substantially similar activity " comprises the dna sequence dna of and hybridization complementary with DNA of the present invention, the promotor that described sequence encoding is worked in pollen.Preferred described hybridization is carried out under low and highly tight condition, or carries out under marginal condition.Usually, low stringent condition is about 60 ℃-Yue 65 ℃ temperature, 3 x SSC, and the height stringent condition is about 65 ℃, 0.1x SSC.SSC is 0.15M NaCl, the damping fluid title of 0.015M trisodium citrate.The concentration of 3 x SSC is three times of SSC, and the rest may be inferred.
Can use pollen specific promoter of the present invention, can disturb pollen to produce or the gene of viability causes male sterile by driving, or in pollen granule specific expressed goal gene.
According to a second aspect of the invention, promoter sequence can with needs at pollen, especially the gene that late period, pollen was expressed in generating forms the part of expression cassette together.Described gene comprises pollen or pollen is generated influential gene.Described gene can be the gene that participates in the control male fertility, and coding kills the gene of insect toxins (but target is the insect of food with pollen then), maybe can strengthen or modify the gene of the nutritive value of pollen.In addition, the expression that can use promoter sequence to drive selective marker transforms to be used for pollen.Suitably the example of selectable marker gene comprises antibiotics resistance gene, and as kalamycin resistance gene, hygromycin gene and PAT resistant gene are to identify stable transformant according to kind of (as corn, paddy rice, wheat).
Term " expression cassette " and term " DNA construct ", " heterozygote " and " puting together body " is synonym, it comprises directly or indirectly and regulates promotor bonded effector, thereby forms box.The bonded example provides the proper spacing base indirectly, as promotor and target gene intermediary intron sequences.Dna sequence dna can further be positioned on the different carriers, therefore, needn't be positioned on the identical carrier.Term described in the present invention " fusion " comprises direct or indirect combination equally.Described construct also comprises plasmid and the phage that is suitable for transforming the purpose cell.
According to preferred embodiment, expression cassette of the present invention contains above-mentioned promoter sequence, this sequence that is in the appropriate location can be controlled the deleterious expression of gene of pollen development, as the barnase of encoding, adenine nucleotide transfer protein, the tubulin of sudden change, the gene of (desired among the WO 97/04116) T-urf or treahalose phosphate Phosphoric acid esterase (TPP).
For example, WO 93/25695 has described the purposes of the gene of Codocyte toxic protein barnase, and this gene is subjected to the control of tapetum specific efficient promoter.The expression of barnase in tapetal cell has destruction to these cells, and can destroy the generation of pollen.
Ribozyme is the RNA molecule of energy catalysis endonuclease scission reaction.Their can trans catalyzed reactions and can the different sequences of target, therefore, can substitute antisense nucleic acid and become the instrument that modulate gene is expressed.(1994) Mol Gen Genet 245 (465-470) such as Hasselhof and Gerlach ((1988) natural Vol 334:585-591) or Wegener have illustrated by ribozyme expression gene in plant and have produced trans-dominant mutation.In case of necessity, can use the expression of pollen specific promoter control ribozyme of the present invention so that they are specific expressed in pollen.
Baulcombe (1997) has described by using reproducible viral rna vector (Amplicons
TM) method of gene silencing in the render transgenic plant, described method also can be used as the instrument that knocks out endogenous gene expression.The advantage of this method is the target gene that produces dominant mutation (i.e. count enable under hybridism) and knock out all copies, also can knock out isotype.This is tangible advantage in as the wheat of hexaploid.Can drive the expression that knocks out the gene function copy by using inducible promoter then, thereby recover fertilizability.By pollen specific promoter is inserted Amplicon as assembly
TMCarrier, genetic expression will be carried out in pollen specifically.
Cytotoxicity or destructive gene need be expressed the recovery gene to regain fertilizability as the instrument that destroys the pollen generation.Suitable construct also contains expression cassette, wherein contain the nucleotide sequence that can overcome described detrimental influence, as recover gene (as barnase is barstar, or TPP being TPS) or coding to the sequence of deleterious gene for construct that justice or antisense are arranged.
The another kind of method that the control deleterious gene is expressed is to use the operator gene sequence.Can by WO90/08830 is described will be as lac, tet, the operator gene sequence of 434 grades is inserted promoter region.The repressor molecule can combine and stop downstream gene (as to the deleterious gene of pollen development) to transcribe (Wilde etc. with these operator gene sequences then.(1992)EMBO?J.11,1251)。In addition, can be by (Lehming etc.(1987) EMBO.6,3145-3153) described preparation binding ability enhanced operator gene sequence to some extent is as Lac I Δ His mutant.This mutant the 17th amino acids changes Histidine into by tyrosine, thereby can strict control express.Unite use with the inducible expression of repressor and can express the inactivation gene of closing.
Take this, can in expression system, mix expression cassette with fertilizability by controlling plant mentioned above.
Term " expression system " refers to said system can be at suitable biology, and tissue is expressed in cell or the substratum.Described system can contain one or more expression cassettes, also can contain to guarantee to regulate other component that promotor increases expression of target gene by using.
According to a third aspect of the invention we, provide expression system, it contains:
(a) specific expressed first promoter sequence in pollen;
(b) first gene can destroy the biological generation of pollen when expressing under its control at described pollen specific promoter;
(c) second promoter sequence, it is to the existence or the shortage reaction to some extent of exogenous chemical inducer; With
(d) second gene, its coding can suppress described first expression of gene, maybe can suppress the proteinic element of described first genes encoding, and can operate the control that links to each other and be subjected to this promoter sequence with described second promoter sequence.
Said elements (a), (b), (c) and (d) can by one or two independently carrier provide, but preferably be contained in the identical carrier to guarantee altogether-to separate.Suitable interaction so that take place in available their conversions or cotransformation vegetable cell between the element.
Second promoter sequence and second gene provide chemical switch for first gene.When second promoter sequence reacts to some extent to the existence of exogenous chemical inducer, use chemical inducer can make second gene begin to play a role to pollen or plant, take this to contend with the effect of first gene.If second promoter sequence only has activity when lacking chemical inducer, the shortage of chemical inducer can play effect similar to the above so.
Element (c) and (d) be suitable for taking the form of expression cassette, contain the nucleotide sequence that can overcome described detrimental influence in this expression cassette, as recover gene (as barnase is barstar, or TPP is TPS) or coding for the sequence of deleterious gene for construct that justice or antisense are arranged, or be the gene of repressor molecule of encoding when used operator gene sequence and inducible promoter operationally interconnect.
Expression system of the present invention also can contain selective marker, as herbicide resistance gene or antibiotics resistance gene to identify a stable transformant according to kind of (as corn, paddy rice, wheat).The existence of herbicide resistance gene also can be selected the male sterile filial generation in the segregating population.
Transform plant with this expression system and can cause producing male sterile plants, the method that produces this kind of plant constitutes a fourth aspect of the present invention.
The expression system of this embodiment according to the present invention (generation of the great-hearted pollen of gene pairs wherein is harmful) can be used for producing heterozygote, and still, when male-sterile line can be prepared to homozygote, this expression system was particularly useful.When using " late period " promotor, during as above-mentioned ZmC5 promotor, because gene product was expressed in the late period of pollen development, the sterility of main transformant and pollen that heterozygote produces was separated by 1: 1, promptly 50% pollen can be educated, but therefore self-pollination produces the seed of non--heterozygosis.
For the sterile plants that obtains isozygotying, must use suitable compound, as the AlcA/R switch is safener for ethanol or for the GST switch, inducible promoter is played a role recover expression of gene to drive, take this deactivation deleterious gene, allow to carry out self-pollination according to following modes.It is heterozygous genes type MSRcs--gamete MSRcs that MS=dominance male sterile Rcs=induction type recovers ... self-pollination is carried out after the chemical induction
MSRcs ...MSRcs MSMSRcsRcs ...MSRcs... MSRcs--- ......
All pollen in homozygote generation (MSMSRcsRcs) all are sterile, and 50% pollen in heterozygote generation (MSRcs--) is sterile, invalid (...) all pollen of filial generation all can educate.Use vital dye, can identify the plant that produces 100% pollen sterile as the dye pollen of these strains of DAP I.Perhaps, can repeat to induce and self-pollination, and analyze the separation of filial generation sterility these isolating colonies.Obviously, all filial generations that obtain by the strain self-pollination of isozygotying self be isozygoty with male sterile, promptly they will produce 100% sterile pollen, and the offspring who is produced by the self-pollination of heterozygosis strain will continue to separate sterility.Perhaps, if cause male sterile gene to be connected with the gene of conferring herbicide resistance, can use the herbicide spray offspring in the seedling stage, herbicide tolerant will be separated with the sterility gene.Can identify and select the male sterile parent to be used to produce heterozygote by this method.
In case identify to finish, can use this sterile strain of isozygotying, by being produced the F1 heterozygote by not modified inbreeding male parent strain cross-pollination.Particular content sees below:
Female parent male parent
MSMSRcsRcs * * * * * * gamete MSRcs * * *
MSRcs MSRcs *** MSRcs*** MSRcs***F1 *** MSRcs*** MSRcs***
Therefore, all F1 seeds all are hybrids, and its sterility is a heterozygosis, this means that the pollen of each plant 50% can be educated.In the crop as corn, this is enough great-hearted pollen, can guarantee to pollinate completely in the monoblock field because of the amount of each male flower pollen that fringe produces is considerable.For wheat, this also is enough effective pollen, does not have production loss in the time of guaranteeing self-pollination, thereby this is to have reduced the loss that wind causes because flower is still closed during pollination.In the kind of collecting the plant nutrition part, the reduction of pollen viability is not the factor that will consider.
Another advantage of the cenospecies producer is: if the peasant wants to keep the plantation after the F2 seed is provided with, will cause production loss because of heterotic forfeiture.Particular content sees below: F1 MSRcs*** gamete MSRcs (debility) * * *
MSRcs does not have * * * MSRcsF2
* * does not have * * * * * *
By using inducible promoter to activate, these methods also can be replied the sterility in the heterozygote production process for example.
According to a fifth aspect of the invention, provide the method for controlling plant fertilizability, described method comprises with above-mentioned expression system and transforms described plant, and when fertilizability is replied, activation-inducing type promotor.
Suitable inducible promoter comprises the promotor by using outside chemical irritant such as herbicide-safener to control.The example of inducible promoter comprises for example two-component system, as the alcA/aIcR gene switching promoter systems of describing in our the open International Application No. WO 93/21334, the GST promotor of describing among the moulting hormone on off system of describing in our the open International Application No. WO 96/37609 or disclosed International Patent Application WO 90/08826 and the WO 93/031294 (above-mentioned patent application is all listed this paper in as a reference).This paper is called this promoter systems " switch activated son ".The switch compound of uniting use with switch activated son is that agricultural goes up acceptable compound, thereby makes this system particularly useful to method of the present invention.
If pollen specific promoter of the present invention is used to obtain male infertility,, can not reply fertilizability fully by this method because pollen is monoploid.This means to activate in the pollen that recovers to produce after the gene only has 50% can educate.But the expression of using promotor of the present invention be the highly tissue-specific fact therewith conclusion run counter to.
This characteristic makes uses promotor of the present invention particularly useful in some very special application.For example, when needs transformed pollen, it was very desirable using the promotor of pollen specific.An example of known this application is MAGE LITER (male kind is to transform), and it is described in (vegetable cell report 14 (1995) 273-278) such as Stoger.In this method, transform pollen by microparticle bombardment.Use pollen specific promoter to drive for example selectable marker gene.Promotor is a pollen specific, and this fact has been given several advantages.At first, mark is not only expressed in the rest part plant at pollen, so remaining plant tissue does not contain unwanted mark.In addition, have only and have selective marker in the pollen and make to transform once more and need not to have different selective markers and become possibility that it is more or less freely promptly to make gene pile up by ordinary method.Pollen transforms why important reasons is a pollen need be stood the sporophyte etap, promptly produces monoploid and amphiploid plant.This means and a step to obtain homozygosity.Perhaps, can use the pollen of conversion to pollinate in wild-type plant, thereby obtain carrying the genetically modified seed of importing, this also is than traditional path for transformation method more fast.
Can expression system of the present invention be imported plant or vegetable cell by any operable method, described method comprises that the agrobacterium tumefaciens by containing the Ti-plasmids of recombinating infects, electroporation, micro-injection vegetable cell and protoplastis, microparticle bombardment, bacterium bombardment, especially " fiber " or " (phage) whisker " method, transform with pollen tube, used concrete grammar depends on the specified plant kind that is transformed.Under proper condition transformant is regenerated as complete plant then, wherein new nuclear substance is stabilized and mixes genome.The unifacial leaf and the dicotyledons that can obtain transforming with this method.Can be with reference to the document of describing currently known methods in detail.This method has constituted another aspect of the present invention.
Method of the present invention can be used for producing multiple heterozygosis plant, as wheat, and paddy rice, corn, cotton, Sunflower Receptacle, sugar beet and lettuce, coleseed and tomato.
The plant that contains the vegetable cell of above-mentioned gene expression in plants system and contain these cells constitutes another aspect of the present invention jointly.
According to a ninth aspect of the invention, provide reproducible viral DNA carrier (Amplicon
TM), it contains above-mentioned recombinant nucleic acid.
According to the tenth aspect of the invention, provide the method that transforms pollen cell with above-mentioned expression system.
Specifically describe the present invention by embodiment below with reference to accompanying drawings.
Fig. 1 illustrates the sequence of ZmC5 cDNA and arranges, and this cDNA has the 2.4kb segment in its corresponding gene 5 ' zone.Transcription initiation site represents with *, underscore be the translation initiation site of inferring.Sa=SalⅠ,S=SmaⅠ,Sp=SphⅠ,H=HindⅢ,X=XhoⅠ,P=PstⅠ；
Fig. 2 has shown the Southern trace of corn (inbred lines A188) genomic dna.Each swimming lane contains the genomic dna of 15 micrograms through digestion, and the digestive ferment of swimming lane 1 is an Eco R I, and swimming lane 2 is the Hind III, and swimming lane 3 is the BamH I.With the Southern trace and through radiolabeled ZmC5 cDNA probe hybridization.
Fig. 3 demonstrates the 18S RNA of the multiple total RNA of corn tissue through the gel of ethidium bromide staining and the Northern trace of the identical gel of surveying with ZmC5 cDNA probe.(A) the total RNA with the multiple corn tissue of 10 micrograms goes up the sample gel; (B) separate from stem with 10 micrograms, the small ear of different developmental phases, total RNA of pollen and sprouting pollen goes up the sample gel.
Fig. 4 has shown
(A) physical map of ZmC5::uidA construct and catenation sequence.By *,
With ◆ the A residue of expression corresponds respectively to the ZmC5 transcription initiation site, removes T to the A sudden change in Hind III site (in order to be easy to the clone) and 3 ' end of ZmC5 promoter region.Underscore be the uidA translation initiation site.(B) demonstrate the active group of the GUS fractional analysis that indigo plant is dyed isolating transgenosis ZmC5::uidA tobacco pollen.(C) derive from ZmC5 promoter activity in the transgene tobacco tissue of two transgenic strain GC5-2 (Bai Zhu) and GC5-7 (gray columns).The data description of each strain non-transgenic contrast is the mean value of standardized twice independent experiment.The stage of sprouting is as described below: Bud-1 is equivalent to the bud (sporule of reduction division/four-strand stage) of 5-8mm, the bud of Bud-2:10-12mm (mononuclear microspore), the bud of Bud-3:13-15mm (After microspore mitosis), Bud-4:(17-22mm) in early days to double-core gametophyte in mid-term, Bud-5:(27-45mm) double-core gametophyte in mid-term to late period (Tebbutt etc., document is the same).
Fig. 5 has shown the dna sequence dna of coding corn ZmC5 promoter sequence.The A of underscore is the transcription initiation site of inferring, and the ATG of black matrix and underscore is a translation initiation site.
Fig. 6 has shown the expression cassette that contains C5-barnase/barstar-no.
Embodiment 1ZmC5 cDNA and genomic clone
According to standard method (.. such as FM Ausubel, the molecular biology fresh approach, Wiley, New York (1990)), use at cDNA probe screening corn (inbred lines A188) pollen of pollen and stem poly (A)-RNA preparation and sprout pollen (HJ Rogers etc., plant magazine 4 (1993) 875-882) the cDNA library.Pick out 1101 clones altogether, all clones all demonstrate with through radiolabeled pollen cDNA rather than through the hybridization of radiolabeled stem DNA.The random choose colony is measured 5 ' terminal sequence, with sequence and up-to-date database comparison.Clone and a known pectin methyl esterase with 800bp inset has significant sequence identity.Screen pollen cDNA library once more with this cDNA inset, the ZmC5 clone (ZmC5c) of evaluation and order-checking total length.
Use PCR segment screening corn (inbred lines B73) genomic library (8 * 10
6Individual plaque), described segment is corresponding to the 5 ' 270bp of the cDNA clone ZmC5c through causing (Ausubel etc., document is the same) mark at random.A positive colony ZmC5g is carried out plaque purification.Subclone is compared with ZmC5c (preservation is NCIMB40915) to the pulsating sequence of ZmC5g 2.5kb Sal I of pUC19, and the result shows that two sequences are overlapping, and they are identical, and what the clone represented is identical gene.Eclipsed is described and is shown in Fig. 1 between related gene group clone's 2.5kb segment and the cDNA.
Use and encoding sequence (data not shown) Nucleotide 1 to 21 (5 '-ACCTAGGAGAGCCTTTGCCAT-3 ') and 56 to 82 (5 '-AGCGGGTGACGGTGGCGACCACACCGA-3 ') two Oligonucleolide primers mappings of complementary transcription initiation site.The transcription initiation site of product clearly is positioned at infers the only A Nucleotide (Fig. 1) at 15 base places of ATG upstream.Found that other pollen specific gene has 5 ' long non-translated sequence .. such as (, molecular biology of plants (1994) 25:283-299) SJ Tebbutt, as if therefore, this ZmC5g zone is short unusually.As calculated, the free energy of this 5 ' short UTL is 0.9kJ/mol (M.Zuker, an Enzymology method.(1989) 180:262-288), make it can not form any stable secondary structure.
Identify the open reading frame of 1692bp, the ATG initiator codon (Fig. 1) of inferring and consensus sequence .. such as (, EMBO J6:(1987) HA Lutcke 43-48) meet preferably.The TATAA primitive of inferring (CP Joshi, nucleic acids research, 15:6648-6653 (1987)) is from-32, yet, do not find findable discernible CAAAT primitive in a lot of other plant genes at 60bp place, transcription initiation site (Fig. 1) upstream.ZmC5c lacks clear discernible AATAAA polyadenylation site signal, this no wonder: 30% plant gene lacks discernible AATAAA primitive .. such as (, vegetable cell 2 (1990) 1261-1272) BD Mogen.In ZmC5c, one section sequence that polyA adds 5 the A residues in 16bp place, upstream, site can be used as polyadenylation signal, but has found the AATAAA primitive and the poly (A) of other pollen specific transcript
+Distance between the interpolation site is greater than mean distance (Tebbutt etc., document is the same).
The ZmC5 aminoacid sequence that embodiment 2 infers
The ZmC5 aminoacid sequence of inferring (563 amino acid) is compared with EMBL and GenBank database, disclose high-level homology with plant (30.9%-41.4%) and microorganism PME (18.6%-20.8%).The arrangement of aminoacid sequence demonstrates the conservative property that plant and microorganism sequence mainly are confined to c-terminal of protein, conserved regions comprise 4 may be the catalytic domain or the avtive spot of enzyme the zone (D.Albani etc., molecular biology of plants (1991) 16:501-513, .. such as 0 Marcovic, protein science 1 1288-1292 (1992)).Vitro mutagenesis aspergillus niger PME .. such as (, biotechnology communication 18:621-626 (1996)) B Duwe demonstrates the avtive spot that I district histidine residues conservative in ZmC5 may be positioned at enzyme, and in aspergillus niger, enzymic activity needs this histidine residues.Yet in several PME of plant and originated from fungus, this Histidine is replaced by other amino-acid residue, and this shows that the not all PME of this histidine residues is necessary.When comparing plant PME, Bp19 gene (D.Albani etc. with respect to the expression of B.napus " in early days " pollen, document is the same), the PPE gene that ZmC5 and P.inflata " late period " pollen is expressed .. such as (, molecular biology of plants 25:539-544 (1994)) JH Mu relation is closer.
Embodiment 3 estimates the ZmC5 number gene
With survey corn (inbred lines A188) genome Southern trace through radiolabeled total length ZmC5 cDNA inset, described trace contains 15 micrograms through the BamH I, the DNA of EcoR I or the digestion of Hind III.At least there are two similar genes in two of each trace swimming lane strong hybrid belt hint corn gene groups among Fig. 2.Other several bands that demonstrate than weak signal hint that this gene family also contains several more incoherent members.
The space of embodiment 4ZmC5 and temporal expression
Survey with ZmC5c cDNA and to contain 10 micrograms and derive from the Northern trace of total RNA of 8 corn tissues to measure the expression of gene program.Only detect the transcript (Fig. 3 (A)) that is about 2.0kb in the pollen of pollen and sprouting, as if this shows that in the detection limit of this technology this expression of gene is confined to this two tissues.At leaf, root, stem, cob does not detect signal in endosperm or the embryo.In the small ear growth course, also detect the expression program.Fig. 3 (B) demonstrates to contain and derives from 0.25,0.5 and the 1.0cm small ear, mature pollen and sprout the Northern trace of total RNA of pollen.The applied sample amount that shows RNA in the swimming lane at each gel through the gel of ethidium bromide staining equates.
With the carmine dyeing of acto-flower pesticide small ear is divided into several stages, finds that flower pesticide contains the cell that is in following phases: premeiotic sporpogenous cell (0.25cm), I prophase mid-term (0.5cm), mature pollen (1.0cm).Yet some between the continuous stages are overlapping to be inevitably because between two Xiao Hua in the identical small ear etap different.Northern analysis revealed ZmC5 expresses the pollen be confined to sophisticated pollen that splits and sprouting, does not detect expression in comprising any other corn tissue of small ear, and described small ear contains and is in sporule stages of cell early takes place.
Embodiment 5 expresses ZmC5 promotor/GUS construct in transgenic plant
Transcribe fusions (Fig. 4 A) between preparation ZmC5g5 ' zone and the reporter gene beta-Glucuronidase, and use this fusions by Agrobacterium-mediated Transformation method transformation of tobacco.By following preparation construct: use two Sph I sites to remove-61 to+403 3 ' end, one of them Sph I site is positioned at the 2.5kb Sal I segment that contains ATG 5 ' 2kb sequence, and one is positioned at polylinker (Fig. 1).Directly the PCR segment with the digestion of Sph I replaces, described PCR segment comprises-61 to+1 zone, be positioned at other restriction site (BamH I, Hind III, Sal I) of 3 ' end and remove the Hind III site mutation (Fig. 1 and 4A) that is positioned at transcription initiation site.Use 3 ' Sal I site of primary 5 ' Sal I site and importing to excise the ZmC5 promoter region in the Sma I site that is cloned into pGUS then.Then ZmC5 promotor-UID-A is transcribed fusions and be transferred to pBin 19 carriers (Fig. 4 A), and be used for the Nicotianatabacun var Samsun roundleaf sheet conversion of Agrobacterium-mediation.Select transformant on kantlex, former generation transgenic plant that regenerate are cultivated the generation for T2 with 2 positive plant of transgene expression.
Pollen granule in the flower pesticide that the collection transgenic plant are split is pressed J.A.Jefferson (molecular biology of plants report (1987) 5:387-405) its GUS activity of described dyeing.Two plant are the positive (Fig. 4 B) that demonstrates about 50% blue dyeing flower powder.In the non-transgenic contrast, do not detect blueness.In order to study the number of integration site,, offspring's kalamycin resistance is marked the plant selfing of two transgenic strains.Among the offspring of transgenic plant GC5-2,303 is kalamycin resistance, and 8 average proportions is 38: 1 to the kantlex sensitivity, shows to have two integration sites at least.The offspring's of transgenic plant GC5-7 kalamycin resistance: the average proportions of kantlex sensitivity is 3.8: 1, show and have only single integration site (the expection ratio of an integration site is 3: 1, the expection ratio of two integration sites is 15: 1, and the expection ratio of three integration sites is 63: 1).
From the multiple tissue of the flower pesticide that comprises 5 etap, prepare extract, analyze fir GUS by fluorometry and express (Jefferson, document is the same).Fig. 4 (C) has shown the GUS activity of two transgenic plant.Except that the developmental and sophisticated flower pesticide that splits, in other tissue, only detect very low-level expression.In tobacco, the stage that bud is grown and the length relevant (Tebutt etc., document is the same) of bud, but this depends on growth conditions.Therefore, Bud-1 is equivalent to the sporule of reduction division/four-strand stage, Bud-2: mononuclear microspore, Bud-3: After microspore mitosis, Bud-4: in early days to double-core gametophyte in mid-term, Bud-5: mid-term to late period the double-core gametophyte.By sporule stage of DAP I dyeing (data not shown) assessment show the expression sequential of ZmC5 promotor in the tobacco and its expression (according to the Northern data) in corn meet better (Fig. 3; Fig. 4 C).Therefore, in the natural surroundings of corn and transgene tobacco, the ZmC5 promotor worked in the late period of pollen development, non-activity in fact before After microspore mitosis.Some differences of expressing of existence between two transgenic strains, GC5-2 has the expression of higher level in the great majority tissue that is tried.The difference that often has expression level in the transgenosis colony, this is because due to the insertion site of transgenosis 9SLA (Hobbs etc., molecular biology of plants 21:17-26 (1993)).
Embodiment 6 drives expression GUS by the AlcA inducible promoter in pollen
Plant conversion carrier is imported tobacco and tomato plant, and described carrier contains the AlcA promotor that drives the GUS expression and drives the 35S CaMV promotor that AlcR expresses.Before immersion liquid is induced as root with ethanol and afterwards, the GUS of institute in a organized way expresses.
After inducing, the GUS of tomato flower pesticide and pollen dyeing demonstrates tangible GUS and expresses.Wish that the pollen that derives from other kind also has identical result.
Embodiment 7 preparation C5-barnase box-dominance gametophyte male sterile boxes
By being inserted through the Sal I site of digestion, oligonucleotide joint MKLINK4 (5 '-TCGATTCGGCGGCCGCCG AA-3 ') use Not I recognition site to replace unique Sal I site among the pBluescriptSK+ (Stratagene).To carry the barnase coding region, be thereafter the corresponding segment place that the 0.9kbBamH I-Hind III segment of the barstar coding region of bacterium-promotor-driving is inserted modified pBluescript.Nos terminator on Hind III-Not I segment is inserted the corresponding segment of gained carrier.Then according to manufacturer's explanation, use the QuickChange system of Stratagene, and use oligonucleotide DAM-3A (5 '-GGTCGACTCTAGAGGAACCCCGGGTACCAAGC-3 ') and DAM-3S (5 '-GCTTGGTACCCGGGGTTCCTCTAGAGTCGACC-3 ') to remove unnecessary BamH I site.With BamH I complete digestion gained plasmid (being called pSK-BBN), with shrimp alkaline phosphotase dephosphorylation (37 ℃, 1 hour).Be connected with the 1.9kb BamH I segment of C5 5 ' flank region, digest to check existing and direction of inset respectively with BamH I and Pst I then.The gained plasmid is called as pSK-C5-BBN (Fig. 6).Then whole expression cassette is transferred among the binary plant conversion carrier pVB6 with EcoR I-pulsating form of Not I.By freeze-thaw method construct is imported agrobacterium tumefaciens, use standard technique that DNA is imported tobacco.
Embodiment 8 analyzes sterile transgenic plant
The situation of growing on kantlex during by tissue culture is selected former generation transformant, further confirms it by pcr analysis.In the greenhouse that plant culturing is extremely ripe, pollen is collected in the back of splitting from flower pesticide, uses vital staining to determine that pollen is that can educate or sterile.Expect that 50% pollen is sterile, these plants and wild-type plant are backcrossed (removing after the flower pesticide) or make its self-pollination, produce the offspring of 50% pollen sterility.
Other change of the present invention be will be apparent to those skilled in the art, and do not depart from the scope of the present invention.
Claims (27)
1. recombinant nucleic acid sequence, described nucleotide sequence contain promoter sequence or its variant or its segment of corn ZmC5 gene and can be used as promotor in the pollen.
2. the recombinant nucleic acid sequence of claim 1, it contains by the about 2kb sequence in corn ZmC5 genetic transcription initiation site upstream hereinafter shown in Figure 1.
3. claim 1 or 2 recombinant nucleic acid sequence, it contains promoter sequence or its variant or its segment, and described promoter sequence contains the part of dna sequence dna shown in Figure 5 at least or contains at least with the described promotor of Fig. 5 and has the part of the encoding sequence of similar substantially active promotor.
4. expression cassette, it contains in the claim 1 to 3 each nucleotide sequence, and the expression cassette that wherein is in the appropriate location can be controlled the gene that need express in pollen, and the product of described genes encoding produces pollen, kill insect toxins, or the nutritive value of enhancing or modification pollen is influential.
5. the expression cassette of claim 4, wherein said gene contains the deleterious gene of pollen development.
6. the expression cassette of claim 5, wherein said gene contains the coding barnase, adenine nucleotide transfer protein, the tubulin of sudden change, the gene of T-urf or treahalose phosphate Phosphoric acid esterase (TPP) or ribozyme.
7. the expression cassette of claim 4, wherein said gene contains selectable marker gene.
8. the expression cassette of claim 7, wherein selectable marker gene contains antibiotics resistance gene.
9. expression system, it contains in the claim 4 to 8 each expression cassette.
10. the expression system of claim 9, it contains the deleterious gene of pollen, also contain expression cassette, described expression cassette contains and can combined thing inductive promotor can operate second nucleotide sequence that links to each other, and described nucleic acid sequence encoding can overcome the peptide or the protein of described deleterious gene influence.
11. the expression system of claim 10, wherein said nucleotide sequence contains the recovery gene.
12. the expression system of claim 11, wherein said recovery gene is barstar or TPS.
13. the expression system of claim 10, wherein said nucleic acid sequence encoding for described deleterious gene for construct that justice or antisense are arranged to suppress its expression.
14. the expression system of claim 10, the wherein said second nucleic acid sequence encoding lac, tet, 434, the repressor of lac-His, it with each nucleotide sequence or Amplicon in the claim 1 to 3
TMCan operate first expression of gene that continuous operator gene sequence interacts and need express to stop in pollen.
15. each expression system of claim 10 to 14, wherein inducible promoter is AlcA/R, GST or can be by moulting hormone inductive promotor.
16. each expression system of claim 9 to 15, it further contains selective marker.
17. each expression system of claim 9 to 16, wherein the gene that need express in pollen links to each other with herbicide resistance gene.
18. expression system, it contains
(a) specific expressed first promoter sequence in pollen;
(b) first gene can destroy the biological generation of pollen when expressing under the control of its described pollen specific promoter;
(c) second promoter sequence, it is to the existence or the shortage reaction to some extent of exogenous chemical inducer; With
(d) second gene, its coding can suppress described first expression of gene, maybe can suppress the described first proteinic element, and can operate the control that links to each other and be subjected to this promoter sequence with described second promoter sequence.
19. produce the method for plant, described method comprises with each expression system transformed plant cells of claim 9 to 18.
20. vegetable cell, it contains each expression system of claim 9 to 18.
21. contain the plant of the cell of claim 20.
22. comprising with each expression system of claim 10 to 18, the male sterile method of inducing plant, described method transform plant.
23. the method for controlling plant fertilizability, described method comprise that the expression system with claim 11 or claim 18 transforms described plant, when fertilizability need be resumed, and activation-inducing type promotor.
24. the method for claim 23 is wherein by using compound to come activation-inducing type promotor to plant.
25. reproducible viral rna vector (Amplicon
TM), it contains each recombinant nucleic acid of claim 1 to 3.
26. transform the method for pollen, described method comprises that the expression system with claim 9 or claim 18 transforms pollen cell.
27. with reference to arbitrary accompanying drawing, aforesaid basically recombinant nucleic acid, expression cassette, expression system or method.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9803661.9 | 1998-02-20 | ||
| GB9803660.1 | 1998-02-20 | ||
| GBGB9803660.1A GB9803660D0 (en) | 1998-02-20 | 1998-02-20 | Expression system |
| GBGB9803661.9A GB9803661D0 (en) | 1998-02-20 | 1998-02-20 | Pollen specific promoter |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1301300A true CN1301300A (en) | 2001-06-27 |
Family
ID=26313163
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN99805265A Pending CN1301300A (en) | 1998-02-20 | 1999-01-22 | Pollen specific promoter |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP1054970A1 (en) |
| JP (1) | JP2002504335A (en) |
| KR (1) | KR20010041129A (en) |
| CN (1) | CN1301300A (en) |
| AU (1) | AU751438B2 (en) |
| BR (1) | BR9907997A (en) |
| CA (1) | CA2319079A1 (en) |
| DZ (1) | DZ2725A1 (en) |
| HU (1) | HUP0100787A3 (en) |
| IL (1) | IL137971A0 (en) |
| WO (1) | WO1999042587A1 (en) |
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- 1999-01-22 WO PCT/GB1999/000232 patent/WO1999042587A1/en not_active Application Discontinuation
- 1999-01-22 BR BR9907997-6A patent/BR9907997A/en not_active IP Right Cessation
- 1999-01-22 EP EP99902660A patent/EP1054970A1/en not_active Withdrawn
- 1999-01-22 AU AU22876/99A patent/AU751438B2/en not_active Ceased
- 1999-01-22 JP JP2000532527A patent/JP2002504335A/en active Pending
- 1999-01-22 CN CN99805265A patent/CN1301300A/en active Pending
- 1999-01-22 KR KR1020007009173A patent/KR20010041129A/en not_active Application Discontinuation
- 1999-01-22 CA CA002319079A patent/CA2319079A1/en not_active Abandoned
- 1999-01-22 HU HU0100787A patent/HUP0100787A3/en not_active Application Discontinuation
- 1999-01-22 IL IL13797199A patent/IL137971A0/en unknown
- 1999-02-17 DZ DZ990026A patent/DZ2725A1/en active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101240280B (en) * | 2002-07-31 | 2011-08-10 | 拜尔生物科学股份有限公司 | Corn root preferential promoters and uses thereof |
| CN102010864A (en) * | 2010-12-14 | 2011-04-13 | 安徽农业大学 | Maize pollen tissue specific promoter and expression vector thereof |
| CN102010864B (en) * | 2010-12-14 | 2012-10-17 | 安徽农业大学 | Maize pollen tissue specific promoter and expression vector thereof |
| CN104837334A (en) * | 2013-05-23 | 2015-08-12 | 深圳市作物分子设计育种研究院 | Establishment of maintainer plant line and sterile line and use thereof |
| CN109593778A (en) * | 2018-11-08 | 2019-04-09 | 浙江大学 | A kind of plant artificial intelligence male sterile line and application |
| WO2020211747A1 (en) * | 2019-04-17 | 2020-10-22 | 湖南杂交水稻研究中心 | Seed sorting method for fixing plant heterosis |
| CN111139257A (en) * | 2020-01-15 | 2020-05-12 | 南京农业大学 | Pear PMEI protein in-vitro expression method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| IL137971A0 (en) | 2001-10-31 |
| CA2319079A1 (en) | 1999-08-26 |
| EP1054970A1 (en) | 2000-11-29 |
| HUP0100787A2 (en) | 2002-04-29 |
| WO1999042587A1 (en) | 1999-08-26 |
| KR20010041129A (en) | 2001-05-15 |
| AU751438B2 (en) | 2002-08-15 |
| AU2287699A (en) | 1999-09-06 |
| JP2002504335A (en) | 2002-02-12 |
| HUP0100787A3 (en) | 2003-04-28 |
| DZ2725A1 (en) | 2003-09-01 |
| BR9907997A (en) | 2000-10-24 |
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