CN100336904C - Pathogen from rice, chemically inducible promoter and their use - Google Patents
Pathogen from rice, chemically inducible promoter and their use Download PDFInfo
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Abstract
The present invention aims to provide a DNA sequence segment. The DNA sequence segment is separated from rice, is invaded by rice blast fungus, is induced by salicylic acid and jasmonic acid, and can be used as pathogen and a chemical induction type promoter. The DNA sequence segment is a transcription regulation region for rice hydroperoxl fatty acid hydroxylating an epoxide hydrolase gene (POG). The present invention is characterized in that the present invention has a promoter region nucleotide sequence from the first position to the 2789th position shown in SEQ ID NO. 1. The insertion, the replacement and/or the deletion of one or a plurality of nucleotide sequences are carried out on the nucleotide sequence, function analogs are obtained, and the purpose of the present invention is achieved. The present invention can be used as pathogen and chemical induction type promoters, molecular marker probes, and rice re-separation control sequence, or is used for the related control sequence of other transgenic plants for cultivating disease resistance plants.
Description
Invention field
The invention belongs to plant genetic engineering field, the invention provides a kind of can being induced by pathogen and salicylic acid (SA), jasmonic (JA) activates and starts the inducible genes promotor that the DNA that is associated with it transcribes, but the present invention relates to comprise the carrier of this inducible genes promotor or its variant of deriving, host and cell, this promotor provided by the invention can be used as pathogen-inducible promoter and is applied to drive and gives the genetic expression of plant host with pathogen resistance, or induces in the destination gene expression because of chemical substance treatment such as salicylic acid and jasmonics and to use.
Technical background
Plant can be subjected to the influence of hundreds and thousands of kinds of microorganisms in process of growth, with these microorganism long term processes in, the plant multiple defense mechanism of resisting the invasion and attack of potential pathogen on every side of evolving out gradually so that himself with the competition of multiple microorganism in win.Totally it seems, Passive Defence and epigamic two classes of initiatively defending that plant becomes second nature to the machine-processed grouping of resisting of pathogen, play a leading role (Agrios, Plant PathologyFourth edition 1999) resisted in the pathogenic bacteria in epigamic active defence plant.Follow the resistance expression to pathogenic bacteria, partial necrocytosis, i.e. anaphylaxis (hypersensitive response is called for short HR) usually take place on every side rapidly in host plant in the pathogen infection site.From the identification of R/Avr between host plant and the pathogenic bacteria,, wherein related to the process of signal identification, transduction and the cascade amplification of a series of complexity to the plant disease-resistant Expression of Related Genes.Plant is to the defence of pathogen and unsuccessful in some cases, cause plant susceptible, but the responsing reaction to the cause of disease intrusion also takes place in plant in this case, just the speed of responsing reaction is too slow or degree is too light, make plant have little time signal is passed to system of defense, and cause of disease is made effective opposing.
Studies show that lipoxygenase (LOX) approach and plant are closely related to the defense response of pathogen.Intravital linolenic acid of plant (Linolenic acid) and linolic acid polyunsaturated fatty acids such as (Linoleic acid) under aerobic conditions through lipoxygenase catalysis oxygenation, generate hydroperoxyl radical lipid acid (fatty acid hydroperoxide), again through the final meta-bolites that generates of the effect of a series of different enzymes with certain physiological function.Because the key enzyme of this pathways metabolism is a lipoxygenase, it catalytic reaction controlling the speed of response of this approach, be the rate-limiting step of this pathways metabolism, therefore be called as lipoxygenase pathway (Gardner, Hort.Science 30:197-205,1995).The defense response of lipoxygenase pathway and plant mainly shows following three reverse side: 1. involved in plant anaphylaxis, infected by pathogen plant is that LOX and related enzyme activity thereof increase and the consistent (Berkman of HR reaction process, Ingram, Physiol.Mol.Plant.Pathol., 45:229-246,1994; Barker, Orlandi, Aunu.Rev.Phytopathol., 33:299-325,1995; Shewfelt, Purvis, Hort.Science, 32:213-218,1995); 2. the signaling molecule of synthetic inducing plant defence genetic expression, can defend expression of gene by activated plant by LOX approach synthetic jasmonic compounds, make the resistance (Dixon of plant generation to pathogen, Lamb, Annu.Rev.Plant Physiol.Plant Mol.Biol., 41:339-3671990; Choi, Bostock, Proc.Natl.Acad.Sci.USA, 91:2329-2333,1994).3. participate in the synthetic of antimicrobial substance, in plant LOX approach, hydroperoxyl radical lipid acid produces in a large number as aldehydes, divinyl acetaldehyde class etc. by the catalytic branched metabolic pathway of hydroperoxide lyase has the intermediate product of higher anti-microbial activity or end product (FEBS Letters such as Stumpe, 507:372-376,2001; Itoh etc., J.Bio.Chem.276:3620-3627,2001).
Hydroperoxyl radical lipid acid hydroxylation cyclooxygenase (POG) is the terminal enzyme that participates in the LOX approach, hydroxylating (the J.Biol.Chem.252:199-204 such as Ishimaru that can catalysis depends on hydroperoxyl radical compound and hydrogen peroxide, 1977) and epoxidation reaction (Gardner etc., J.Biol.Chem.268:6971-6977,1993).POG has vital role in the metabolism of hydroperoxyl radical fatty acid cpds, its catalytic activity is found (Blee etc. in many kind of plant such as pea, soybean, paddy rice, corn and potato tuber, J.Biol.Chem., 265:12887-12894,1990).And existing report (Plant Cell Physiol.31:1117-1122 such as Ohta, 1990 and Plant Physiol.97:94-98,1991) show paddy rice active increasing sharply of energy induced strong POG when infected by rice blast, catalysis LOX product forms antimicrobial compoundss such as hydroxyl or epoxy hydroxy fatty acid, and causes the synthetic of oxidation of fat acids plant protecting chemical.
Promotor is the dna sequence dna that regulatory gene is transcribed, be usually located at institute's regulating and expressing gene the upstream, have the growth of responding to or environmental stimulus or change expression of gene in the tissue specificity mode.The regulating and controlling effect that utilization can be experienced the gene promoter of pathogenic bacterium inducing just can make the defence gene express selectively in plant materials, prevents and treats disease effectively.Reaching this purpose has two means available, the one, separate and utilize suitable being subjected to pathogen inductive plant gene promoter to come to link to each other to form mosaic gene conversion plant with the defence gene, once there was report will injure the inductive promotor rice transformation that links to each other with the potato proteinase inhibitor gene, transgenic paddy rice (the Duan etc. of anti-snout moth's larva have successfully been obtained, Nature Biotech.14:494-498,1996).Be exactly the trans-acting factor that separates the gene induced expression of regulation and control defence in addition, the expression with this factor of regulation and control comes the goal of regulation and control expression of gene then.Determine in the gene promoter region that to the important cis-acting elements of this gene induced expression be the basis of separating trans-acting factor of special mutual work with it, merge the conversion plant with the promoter region excalation and with reporter gene, can determine the cis-regulating element important abduction delivering by examining report expression of gene situation.At present this method of utilization has been found some relevant with the pathogen infection abduction delivering special cis-regulating element (Dixon etc., Annu.Rev.Phytopathol, 32:479-501,1994 from the defence gene promoter region; Grary etc., Proc.Natl.Acad.Sci.USA, 89:9230-9234 1992).
Paddy rice POG gene and transcriptional control region nucleotide sequence thereof yet there are no report so far, owing to known its can be expressed by the rice blast fungus induced strong, and infer that it has participated in disease-resistant signal conduction.Therefore its transcription regulatory region has just comprised the promoter element that energy rapid answer pathogen stimulates, use the POG gene promoter and give the gene of plant host and link to each other with pathogen resistance, change susceptible plant over to, just can under the situation that pathogenic agent is invaded, start the goal gene that is associated and in plant host, express, give the ability that plant is effectively resisted disease.
Studies show that in a large number simultaneously that Whitfield's ointment (SA) and jasmonic (JA) participate in many plant defense gene transcription regulation and control and HR reaction (Durner etc., TrendsPlant Sci., 2:266-274,1997 as the endogenous signal molecule of key; Penninckx etc., Plant Cell, 8:2309-2323,1996), by inference when plant and cause of disease are done mutually, at different pathogens, plant may take independently defence signal transduction pathway JA mediation and/or the SA mediation to regulate and control corresponding defense response Proc.Natl.Acad.Sci.USA.95:15107-1511 such as (, 1998) Thomma.But concrete mechanism is still unclear, and as the participant of the disease-resistant signal conduction of regulation and control, the POG gene promoter can also provide the chance of cis element, transcription factor and the stream signal composition of qualitative identification of cell to relating in the replying of SA and JA.Provide foundation for understanding the conduction of plant disease-resistant signal.
Summary of the invention
The analogue development body nucleotide sequence fragment that order of the present invention is to provide a kind of POG gene transcription control region nucleotide sequence or its to have identical function, be a kind of can by pathogen such as rice blast fungus infects and the stimulation of chemistry such as salicylic acid (SA), jasmonic (JA) are induced and activated and start the inducible promoter that the DNA that is associated with it transcribes.Can order about in pathogenic agent or chemical substance and induce the goal gene that is associated with it under the situation in plant host, to express, give the ability that plant is effectively resisted disease, worm.Specifically, be the startup of the abduction delivering that from paddy rice, is separated to, the control region dna fragmentation of transcribing, it is characterized in that it have with shown in the SEQ ID NO:1 the 1st to the 2789th promoter region nucleotide sequence, but also should be pointed out that nucleotide sequence shown in the SEQ ID NO:1 of the present invention is carried out insertion, the replacement of one or more nucleotide sequences and/or lack resulting functional analogue also reaching purpose of the present invention.Thereby the present invention also comprises with the nucleotide sequence shown in the SEQID NO:1 having at least 50% homology, preferred at least 90% homology, and have the analogue of identical function simultaneously.
Expression vector and its recombinant host cell that provides the analogue that contains above-mentioned inducible promoter sequence and have identical function to derive variant also is provided another object of the present invention.Recombinant host cell has recombinant microorganism and recombinant plant cell, and recombinant microorganism comprises recombinant bacteria or/and recombination yeast, and recombinant microorganism wherein has the reorganization Agrobacterium, the Agrobacterium EHA105 that especially recombinates, the preferred paddy rice recombinant plant of recombinant plant cell cell.According to inducible promoter provided by the invention, can make up the binary expression vector that comprises this promotor, its promotor downstream can chimeric user the interested gene that will express.These genes can be both to deposit in the paddy rice according to the source, also can be the effable heterologous genes (as nontoxic gene etc.) outside the paddy rice; It can be the gene (as defending gene etc.) that suppresses the pathogenic bacteria expansion.General experimenter all can choose these interested genes from relevant scientific and technical literature, database or handbook.The expression vector technology that makes up among the present invention is that those of ordinary skill in the art grasps, and can operate according to the associated viscera of documents such as (molecular cloning experiment guide, second edition, cold spring harbor laboratories 1989) such as Sambrook.As a same reason, transform the microorganism that microorganism cells can obtain recombinating, utilize reorganization Agrobacterium-mediated Transformation vegetable cell can obtain the recombinant plant cell with above-mentioned expression vector.This technology also is that the those skilled in the art in present technique field can realize.
The present invention also comprises and contains such recombinant plant cell at least or start from cytocerastic like this plant or plant part tissue.
A kind of method of cultivating disease-resistant plant that provides is provided, it is characterized in that the analogue development body that uses the aforesaid dna sequence dna of the present invention and have identical function suppresses with direct or indirect, destroy the gene of pathogenic bacteria expansion and existence, the enzyme gene of key in plant protecting chemical synthetic gene or its production process, and the heterologous gene that can effectively suppress the germ expansion that can express in paddy rice is (as nontoxic gene, sense-rna etc.) tabling, form the fusion dna sequence, and directly or with the carrier that contains this fusion sequence transform plant, obtain disease-resistant or anti-sick plant, expression vector of the present invention transforms plant and can transform by number of ways, and the preferred agrobacterium-mediated transformation of embodiments of the invention transforms plant.
The present invention also comprise a kind of in plant the method for marking protein, it is characterized in that the dna fragmentation of promotor of the present invention with the coding goal gene carried out being operatively connected to use as control region, specifically, wherein said promotor can be induced activation by Whitfield's ointment, jasmonic or its analogue, and starts destination gene expression.
For further understanding, do following explanation again to the implication of foregoing invention content and the various words that relate to:
The present invention adopts by the method for protein purification to gene clone.From rice blast fungus inductive rice cDNA library, be cloned into hydroperoxyl radical lipid acid hydroxylation cyclooxygenase (POG) gene, its sequence is shown in SEQID NO:2, with it is probe Screening of Rice genomic library, obtained to comprise the genomic fragment of this gene, therefrom clone gene 5 ' ending regulating sequence, this sequence has the base sequence shown in the SEQ ID:1, its promoter activity is preferably infected by rice blast fungus induces, also can be induced simultaneously by salicylic acid and jasmonic, the accumulation of the gene transcript of being regulated and control increases with the prolongation of the time of processing, by analyzing the part that the regulating and controlling effect of inferring this sequence is the conduction of paddy disease-resistant signal.
Relate to 5 ' ending regulating sequence in this specification sheets and mainly be meant 5 ' the end upstream sequence that begins from transcription initiation site (corresponding to the 2290th Nucleotide of SEQID:1), comprise the important cis-regulating element of controlling downstream gene expression corresponding to this section sequence.Comprise the sequence (the 2786th to 2788 Nucleotide of corresponding SEQ ID:1) simultaneously from transcription initiation site to downstream translation initiation site ATG.Be positioned such that according to the important promoter element of sequence signature with this sequence: transcription initiation site is orientated 1 as, TATA box (corresponding to the 2265th to 2270 Nucleotide of SEQ ID:1) is positioned at-25 to-20, and CAAT box (corresponding to the 2209th to 2212 Nucleotide of SEQ ID:1) is positioned at-79 to-76.
Among the present invention regulating and controlling sequence segmental " clone " be meant from the natural rice genome of nature isolated, one section promoter sequence that can directly separately utilize without any change.According to sequence of the present invention, can design a pair of primer of upstream and downstream, obtain once more with round pcr, also can utilize its label probe of design from the rice genome library, to screen acquisition, even can synthesize acquisition with classical enzyme process or chemical process, these approach all are that persons skilled in the art can be accomplished.
" variant of deriving " of the gene 5 ' ending regulating sequence among the present invention refers in SEQ ID NO:1 sequence on the basis of sequence between 1 to 2789 Nucleotide, adds or an isolating once more part, a few part or its combination through fragment deletion, sequence change, base.
The gene 5 ' ending regulating sequence among the present invention and the promoter activity of the variant of deriving thereof can such as β-glucuronidase (GUS), be confirmed with the expression of GUS by at the chimeric reporter gene in promotor downstream.A preferred embodiment is to utilize the sophisticated method of (EMBO J., 6:3901～3907,1987) foundation such as Jerfferson.According to embodiments of the invention, obtained forming by the cell of the above-mentioned chimeric DNA fragment that has mixed one or more copies in the genome basically, and one or more copy transfer can be given offspring's plant, this dna fragmentation can be expressed in host plant cell by special driving reporter gene by way of expectations.
The present invention also provides the roughly section at promotor cis element place, made up the variant of deriving that lacks step by step from its 5 ' end for this reason, and be fitted to before the gus gene of the binary vector pCAMBIA1301 (professor Jerfferson provides) that removes 35S, more than operation all is the basic skills that those skilled in the art use always, preferred scheme is the primer that the suitable convenient restriction enzyme site that connects is carried in design, with the round pcr acquisition of increasing from original series.
The preferred embodiments of the invention are can be operatively connected by this inducible promoter and goal gene, goal gene is placed under its control, expression system that can autotelic switch gene, this expression system can make plant the favourable time or opportunity (infecting) expressing gene as cause of disease, make plant effectively resist infecting of pathogen.
Another purposes of the present invention is that goal gene is connected with above-mentioned evoked promoter, using external source chemicals (as JA, SA) induces realization target protein matter at the intravital controlled expression of plant, in this respect, the application of SA or JA can be adopted any form, for example plant is carried out foliage spray or irrigation.
The method of using the dna fragmentation that comprises goal gene and be connected with above-mentioned evoked promoter to import paddy rice among the present invention is conspicuous in this area, also is that persons skilled in the art are accessible.The activity of variant of deriving above-mentioned promotor or its detects and utilizes this promotor to carry out molecule manipulation targetedly, all inevitably will carry out in botanical system, an embodiment preferred is to adopt agrobacterium mediation method (Hiei etc., Plant J., 6:271～282,1994; Hiei etc., Plant Mol.Biol., 35:205～218,1997) it is imported in the paddy rice; The experimenter also can select following method for transformation for use flexibly according to the skill level of oneself: (Horsch etc., Science, 234:496,1984 such as protoplastis fusion method, particle bombardment, electric shocking method, PEG method, pollen tube introductory technique; Barton, Cell32:1033,1983; Acta Phytophysiol.Sin such as Liu, 21:195～205; Science such as Horsch, 227:1229,1985)
Activated other of the promotor according to the present invention variant of deriving, after transgenosis is finished, during the tissue culture from kanamycin-resistant callus tissue to the back immigration physical environment (as the field) of taking root, transforming tissue or the individual activity that similar constitutive expression is arranged, this is because in the tissue culture medium (TCM) during stimulating activity composition or the group training due to its difference with natural plant (as similar damage), promptly loses substantially after recover in the field moving into.
Various sequence provided by the invention can be used as molecular mark probe, can also can be used for the relevant regulating and controlling sequence of other plant as separate this sequence again in paddy rice, and these all are contents involved in the present invention.
Be convenient to further understand the present invention by the following drawings explanation
Description of drawings
The abduction delivering dynamic N orthern of accompanying drawing 1. paddy rice POG genes analyzes, among the figure from left to right each swimming lane be respectively the accumulation of transcribing of handling POG gene mRNA after 0,8,16,24,36,60 and 72 hour, every kind of processing is last sample contrast with 17SrDNA.Wherein A is clear water simulation inoculation contrast, and B is inoculation rice blast fungus affinity microspecies 007, and C is the non-affinity microspecies 131 of inoculation rice blast fungus, and D is that SA handles, and E is that JA handles, and F is that dormin (ABA) is handled, and G is a physical abuse, and H is uviolizing.
Accompanying drawing 2.POG gene promoter 5 ' end is the active column synoptic diagram of GUS abduction delivering of the transgenic paddy rice blade of each variant driven GUS genetic expression of disappearance in various degree, if change the negative contrast of transgenic paddy rice (WP) of no promoter-driven GUS reporter gene over to, establish the positive contrast of transgenic paddy rice (35S) that changes over to CaMV35S promoter-driven GUS gene.Illustrate: the various from left to right SA of being treated to, JA, rice blast fungus microspecies 131, rice blast fungus microspecies 007, physical abuse and water contrast.GUS activity unit be pmol 4-MU/mg protein/minute.
Specific embodiments
For a better understanding of the present invention, illustrate further by the following examples, but be not limitation of the present invention.
Embodiment 1 rice blast fungus infects the clone of induced gene POG promotor
1. the purifying of paddy rice hydroperoxyl radical lipid acid hydroxylation cyclooxygenase (POG)
Prepare rice varieties and like to know the rising sun (Oryza sativa cv Aichiasahi), when treating that the 4-5 leaf is extracted out, rice leaf with non-affinity Pyricularia oryzae inoculation is an experiment material, by selectivity extracting membranin, the ammonium sulfate precipitation impurity elimination of 20% saturation ratio and 45% saturation ratio ammonium sulfate precipitation, DEAE-Toyopearl anionresin, DEAE-Sepharose anionresin, CM-Toyopearl (pH5.1) cationic exchange, steps such as CM-Toyopearl (pH4.5) cationic exchange and ultrafiltration and concentration, purifying rice blast fungus infect inducibility POG, and obtained electrophoretically pure zymoprotein.Adopt the method for the specific Lysyl endopeptidase of Methionin enzymolysis, the-terminal amino acid sequence of POG is measured, obtain partial amino-acid series.
2.POG the clone of gene
The rice leaf of non-affinity Pyricularia oryzae inoculation, get the rice leaf behind the 48h, guanidinium isothiocyanate (GT) method extracts total RNA, with poly A fast m RNA purification kit (Stratagene, USA) purified mRNA is that template makes up paddy disease-resistant cDNA phage library with ZAP Express cDNA Gigapack II Gold cloning test kit with this mRNA.
According to the proteic aminoacid sequence design of the part POG that obtains degenerated primer, specific amplified goes out the fragment of a 105bp from constructed paddy disease-resistant library, is the disease-resistant library of probe Screening of Rice with this fragment, takes turns screening through second and third, separation and purification candidate positive colony is again through confirming.Selection comprises the segmental phage of bigger insertion and carries out external cutting, discharges to contain to insert segmental cyclisation phagemid, carries out sequencing.The bed board of relevant phage library screening, change concrete operations steps such as film, fixing, hybridization cutting and undertaken by cDNA Gigapack II Gold cloning test kit specification sheets.Paddy rice POG whole gene cDNA sequence is shown in SEQ ID:2.
3.POG the clone of gene promoter
With paddy rice POG gene cDNA fragment is that probe Screening of Rice genomic library (is provided by professor Liu Yaoguang of Agricultural University Of South China, structure and screening method are seen Gene such as Liu, 282:247-255,2002) acquisition comprises the oryza sativa genomic dna fragment of POG, be probe carry out Southren to positive colony hybridizes with 5 ' end of POG gene cDNA, and subclone has the fragment of POG5 ' side sequence, comprises this segmental plasmid and name and be POGH, measure sequence, software is inferred the promoter element in the sequence.Relevant Southren changes film, hybridization, concrete operations such as subclone are according to (molecular cloning experiment guides such as Sambrook, 1989) etc. the associated viscera of document is carried out, 1st to 2789th the nucleotide sequence of 5 ' control region sequence of paddy rice POG gene shown in SEQ ID:1, wherein the 1st to the 2290th Nucleotide is promoter region, and the 2291st to the 2789th Nucleotide is 5 ' non-translational region (5 ' UTR).
The abduction delivering characteristic of embodiment 2 POG genes
To like knowing that the rising sun (Oryza sativa cv Aichiasahi) serves as for trying the water rice varieties.Being that rice blast fungus (Magnaporthe grisea) the 131# physiological strain of non-affinity and the rice blast fungus 007# physiological strain of affinity are inoculum to this kind.Rice blast fungus spore preparation method is with (Can.J.Biol., 66:730-735,1988) such as Peng.When rice seedling the 5th leaf nearly launched fully, (106/ml) spray inoculation paddy rice, illumination cultivation again after 100% relative humidity, 28 ℃ of lucifuges are cultivated 36h was contrast with the inoculation of 0.02% Tween-20 SIMULATION OF SPRAY then with the rice blast fungus spore suspension.After inoculation, take a sample clip the 5th leaf central part during sampling when 0h, 8h, 16h, 24h, 36h, 48h, 60h, 72h respectively.Other physics, chemical treatment are as follows: JA handles: the rice leaf that is sprayed on 100 μ M JA is sealed in the inoculation tank then; Mechanical wounding: after clamping the rice leaf with flint paper, tangible scar occurs along vein direction scraping blade face to blade face; Uviolizing: will put in order the strain rice leaf up, and under ultraviolet lamp, shine 15min; SA and ABA handle: be respectively that 10mM SA or 600 μ M ABA are sprayed on rice leaf with concentration.Mechanical wounding, uviolizing, SA and dormin (ABA) are suitably preserved moisture after handling.0h, 8h, 16h, 24h, 36h, 48h, 60h, 72h take a sample respectively after the above-mentioned processing, and be standby in-80 ℃ of preservations after the liquid nitrogen quick freezing.The GT method is extracted total RNA, is that probe carries out Northern hybridization with the POG gene cDNA.
The result shows, the inoculation rice blast fungus after 16 hours the POG gene transcript begin that accumulation is arranged, the accumulation of time lengthening gene transcript also increases thereupon, but the accumulation of inoculating gene transcript in the material of non-affinity microspecies will be higher than the material of inoculating the affinity microspecies same period, show that the POG gene is induced by rice blast fungus to infect to induce, inducing does not have little specific specificity, but but this genetic expression of non-affinity microspecies induced strong infers that this gene participates in the defense response that paddy rice is infected rice blast fungus.SA and JA also can induce the POG gene, and uviolizing needs just to observe after 72 hours the accumulation of gene transcript, and ABA, physical abuse almost can not induce POG genetic expression (accompanying drawing 1).
5 ' the end gradual change of embodiment 3 POG promotors lacks the acquisition of the variant of deriving
POG gene 5 end upstream regulation and control fragment are put into database and are retrieved, and use software analysis, infer the cis element position that it is possible, then according to these positions, method with Reversetranscriptase_polymerasechainreaction has obtained 6 POG promotors 5 ' end gradual change deletion fragment, be respectively called after GPF (corresponding to the 163rd to 2789 Nucleotide of SEQID.1), G200 (corresponding to the 807th to 2789 Nucleotide of SEQ ID:1), G150 (corresponding to the 1286th to 2789 Nucleotide of SEQ ID.1), G100 (corresponding to the 1635th to 2789 Nucleotide of SEQ ID.1), G75 (corresponding to the 2038th to 2789 Nucleotide of SEQ ID.1), G42 (corresponding to the 2363rd to 2789 Nucleotide of SEQ ID.1).Wherein GPF carries out pcr amplification by Oligonucleolide primers 5 ' tcttggcgtc gaggagc 3 ' of synthetic and 5 ' tcgacatcag gcgccgacga g 3 ' to plasmid POGH and obtains, G200 is obtained by NcoI+SpeI double digestion POGH plasmid, G150 carries out pcr amplification by the Oligonucleolide primers SEQ5 ' tcttggcgtc gaggagc 3 ' of synthetic and 5 ' cataagtcat ctttagtcat acc 3 ' to plasmid POGH and obtains, G100 is obtained by NcoI+BamHI double digestion POGH plasmid, G75 is obtained by NcoI+ScaI double digestion POGH plasmid, and G42 carries out pcr amplification by Oligonucleolide primers 5 ' tcttggcgtc gaggagc 3 ' of synthetic and 5 ' ttagcctgct attgtacctg c3 ' to plasmid POGH and obtains.
The binary vector structure chimeric of embodiment 45 end upstream regulation and control fragment and a series of variants of deriving thereof with gus gene
In above-mentioned promotor 5 ' the end gradual change deletion fragment, by being cloned in the T-easy carrier that PCR obtains, respectively binary vector pCAMBIA1301 is carried out double digestion with identical restriction enzyme, the fragment SalI+NcoI on the selection T-easy of restriction enzyme with the variant of deriving on being cloned into T-easy.Product behind two groups of double digestions reclaims with reclaiming test kit (Beijing ancient cooking vessel state) respectively: (1) binary vector pCAMBIA1301 is the remainder that cuts original 35S, and (2) T-easy carrier is that to reclaim what downcut be tape starting and its variant part of deriving.Last (1) (2) two portions that will reclaim respectively by a certain percentage mix and connect with the T4 ligase enzyme, the variant fragment of deriving that obtains with the double digestion mode described in the embodiment 2, directly with through the pCAMBIA1301 of identical double digestion carrier link to each other, connect product transformed into escherichia coli DH5 α bacterial strain, and cut the affirmation of identifying and check order through enzyme.
The fusion gene rice transformation that embodiment 5 POG promoters driven GUS express
1. binary vector transforms agrobacterium tumefaciens
Respectively above-mentioned binary vector plasmid DNA is imported agrobacterium tumefaciens (EHA105) competent cell with freeze-thaw method, cultivate for 28 ℃ and form single bacterium colony.The single colony inoculation of the Agrobacterium that picking transforms is in the YM liquid nutrient medium that contains 50 μ g/ml Kanamycin, and 28 ℃, 220rpm shakes training 16hr, directly is PCR with bacterium liquid and identifies.
2. Agrobacterium tumefaciens mediated rice conversion
Prepare rice varieties and like to know and place rising sun immature seed on the solid inducing culture (NB), secretly cultivate evoked callus through extruding the paddy rice rataria after the surface sterilization.Peel callus after about 5-7 days, change on the freshly prepared subculture medium (NB), succeeding transfer culture is about 5 days under the same conditions; Perhaps prepare the paddy rice mature embryo callus, select succeeding transfer culture 5-7 days, the yellowish callus of color and luster cultivates altogether.Select state preferably callus cultivated altogether 15-20 minute with an amount of agrobacterium suspension (OD 0.3-0.5), change solid over to altogether on the substratum, 26 ℃ dark culturing 2-3 days.The callus that is total to after cultivating is placed on the screening culture medium that contains 50mg/lHygromycin, and 26 ℃ of dark cultivations 14 days forward to and continue on the freshly prepared screening culture medium to screen 14 days to growing resistant calli.From the resistant calli that after the two-wheeled screening, grows, the resistant calli of selecting the milk yellow densification goes on the division culture medium that contains 50mg/LHygromycin, and dark earlier the cultivation 3 days goes to then under the 15h/d illumination condition and cultivate, general through about 15-25 days, there is green point to occur.Further differentiate seedling after 30-40 days.When the bud of resistant calli differentiation grows to about 2cm, seedling is moved on on the root media, cultivate about two weeks, can in the greenhouse, transplant and bury.
The evaluation of embodiment 65 end upstream regulation and control fragment and a series of variant promoter activities of deriving thereof
Rice blast fungus inoculation: so that rice varieties is liked to know that the rising sun is that rice blast fungus (Magnaporthegrisea) the 131# physiological strain of non-affinity and the rice blast fungus 007# physiological strain of affinity are inoculum.(106/ml) spray inoculation paddy rice, illumination cultivation again after 100% relative humidity, 28 ℃ of lucifuges are cultivated 36h is contrast with 0.02% Tween-20 SIMULATION OF SPRAY inoculation (CK) then with the rice blast fungus spore suspension.48h sampling after inoculation, chemical treatment is as follows: JA handles: the rice leaf that is sprayed on 100 μ M MeJA is sealed in the inoculation tank then; SA handles: concentration is sprayed on rice leaf for 10mM SA.Above-mentioned processing is sampling respectively behind the 48h that suitably preserves moisture, clip the 5th leaf central part during sampling.The negative contrast of transgenic paddy rice (WP) that changes no promoter-driven GUS reporter gene over to is all established in above-mentioned processing, establishes transgenic paddy rice (35S) the positive contrast of commentaries on classics with the gus gene of CaMV35S promotor startup.
1. tissue staining methods are adopted in the active detection of GUS: the transgenic line through above-mentioned processing that will choose carries out the sterilized water surface cleaning, blot the back with aseptic thieving paper and immerse GUS staining fluid (1mM X-Gluc, 100mM phosphoric acid buffer PH7.5,10mM EDTA, the 5mM Tripotassium iron hexacyanide, 5mM yellow prussiate of potash), placed after bleeding 5 minutes with shaking table on 37 ℃, the 200rpm reaction is spent the night, and the rinsing of decolouring in 95% ethanol then is until colour developing.2. transgenic line 0.1 gram through above-mentioned processing that will choose grinds in liquid nitrogen fully, add 200 μ l extracting solution (50mM phosphoric acid buffer PH7.5, the 10mM mercaptoethanol, 0.1% Triton X-100,1mM EDTA), mix, centrifugal 10 minutes of 8000rpm, get 20 μ l supernatants and add 180 μ l with extracting the 2mM MUG reaction solution that damping fluid is prepared, 37 ℃ are incubated 1 hour, add 20 μ l 0.2M Na2CO3 solution termination reactions.The reaction solution of getting after 200 μ l stop is measured fluorescence under excitation wavelength 365nm and emission wavelength 455nm.The fluorescence of a series of diluting solns by assaying reaction product 4-MU is set up typical curve, and the protein content in conjunction with each sample calculates enzymic activity, and unit is 4-MU pmol/mg/ protein/min.The result shows, changes the POG promotor over to and respectively lacks transgenic paddy rice that structure drives gus gene can both detect gus gene under rice blast fungus and chemical induction expression, and its induced character is consistent with the POG gene.Comparing rice blast fungus and chemical substance with positive control induces down each structure of POG promotor all to show than the much higher activity of CaMV35S promotor commonly used in the plant expression system.Each structure is compared, and G200 has the highest promoter activity, and the activity of G100 is lower, and has all kept higher promoter activity than its long slightly or short slightly G150 and G75.Therefore can infer at the 1635th to 2038 Nucleotide of SEQ ID:1 to have the cis element that suppresses promoter activity, the shortest structure G42 has still kept very high reactivity, shows the core parts (accompanying drawing 2) that wherein comprised promotor.
Sequence table
＜110〉China Agricultural University
＜120〉a kind of cause of disease and chemical inducible promoter and purposes from paddy rice
＜130〉the good 2003-2 of Peng You
<160>2
<170>PatentIn?version?3.1
<210>1
<211>2789
<212>DNA
<213>Oryza?sativa?cv?Aichiasahi
<400>1
ataaactaaa?actaaacttc?caatgtgtac?gtatctgcgg?caggattctg?ctgcttgtcc 60
cccattaaat?tgtaatggtt?ccatcttctg?cttcgccata?tgatggtgct?tgctactact 120
caccggcgac?gacacgccta?cggccgccac?cgacttctgc?tgctcgacat?caggcgccga 180
cgagcctccg?cctactcgcg?ccgcggaagg?cgacgcaaca?gacctcgccg?acgacattct 240
gccctttcca?ccggagagtt?tctgctccat?caaaaacctg?cagaacaaaa?tcttcttcct 300
cacaaatccg?aaagataatt?gtggtcctca?ttttctgtcc?tccatcaccg?tcaacttaaa 360
tagagtatca?atacagcaac?ctccacacta?actactctcc?tggtcgctcg?gtcgtgcatg 420
cgtgcgggta?acaaaccatg?catgcagaac?attcaaaatg?actcaccatg?cagaacatag 480
cgcagtgatc?cgaattttca?aaaaaaaaag?aatattcaag?tttctgcatc?actggtttaa 540
tggatctttt?ctcacaatcc?gttagtgagt?tgaagcccga?aaccctgatg?tgagttggac 600
aggtgtccga?tggttacaat?aggagtagct?actcctggga?gtagaatata?ggcttcatat 660
atatatatat?atatatatat?atatagtaaa?tttgtttggt?gctccatggt?gcccgggcac 720
cattgttgaa?attgagtata?tacccaattc?aaatgagtat?gaaacttttt?gcaattactt 780
aggtattaac?attaactact?ttactaacta?gttcaaagca?tgtttgtact?gaatttgaac 840
ttgtttttgt?tagattttga?ttctaggtat?atagcatcca?tacatataat?tagttaggta 900
tgtaatcatg?gattgtgaaa?taaagttaaa?tttgagttgt?tttgttgata?ggtatagata 960
tgaatcgaat?tattataact?aggtatatac?tcacaatttg?aaaattttga?aaaatttgag 1020
tgattttgta?cattagatac?catggtgccc?catatgagtg?tcctatatag?taaatttgtc 1080
ctatatatat?atatatatat?atgtttgttt?tgtgttgaaa?atatttctaa?ttttttctat 1140
aaacttgttt?ggacttaaaa?aaaatattga?ctagaaaaaa?attcaaaaca?acgtaaagaa 1200
aggaagtaga?atttagaagt?tgccctattt?tggccatgtt?tctttccata?agtcttaggg 1260
ctaaaaatta?gttctagggc?taaaacataa?gtcatcttta?gtcataccct?gtttgtttgg 1320
agggactaaa?agggactaaa?acctcattaa?atgacctatc?ttttagcact?tcatccaaca 1380
cctacatgct?tcatccattc?aatagtaggt?gcgctatagg?cctttagtct?aaattagcac 1440
ctcccctaga?gatttatgga?cctaggtaat?tttagcctct?tttagtccct?cctgtttagt 1500
attttagggg?ctaaaataga?ctaaagtgaa?gggacttatg?attagcccct?ccaaacaaac 1560
aacccgaagg?gagtactgtt?acatagtgat?tgacacatga?actgttgaca?gatggaactg 1620
tcagtacacg?ttggatccat?gctttagacg?tctcgacaat?aactttggat?ttgggatata 1680
ctagggaaac?aacgagggaa?tacaatcttc?gaattctagc?cttcgaccaa?gtcacccgcg 1740
cgcatgtacc?ttggacgacg?atgttgtcca?cttgccgtgc?gaacgtgtgg?aggtaaagcg 1800
aaaaaagact?aggtaaaacc?atgcaagcag?cacacgcgtg?cttatttact?gtttagttac 1860
atggacggat?gcaccaaata?atttcagcta?gacagcaatc?attccacacg?ttctcctctc 1920
ctgctccagc?tccccatccc?agatgaggtg?gcaacaacac?tgcttaaaca?cgccaccatc 1980
tcggccgccg?tccggccggc?cgtcttcgcg?agtgacgcag?ctcccatcag?cagcaacgag 2040
ctctgctagc?tttcgtctca?gcgctccgtc?ctgcggtctt?ttgggtctcg?aatcgcctca 2100
tgtggacccc?acaatcacgt?tggcaaatag?cagctgccct?ggtgctgctt?ctcacgtttt 2160
agagcaagtt?taatagtggc?cactgctagc?tccaattcat?caatagccaa?taaaataatt 2220
ggcttactat?actattaata?tatggtctca?tctgtcatac?atatattatg?tcttagagtc 2280
cgcgctgcag?ctggctacag?atctatagcc?tgctgctctt?ctctctcatc?ttttatctca 2340
ttaaaaatat?gtttacaggc?tattagcctg?ctattgtacc?tgctcttaga?gagagtgtgt 2400
cgtgggtgac?tattcagtct?cccntctcat?ttctctgcca?cctcatcact?tttgccatcg 2460
tggacacact?atcaccaggc?tccagctaat?ttttttcaat?tgtacgtgct?cttagtcgac 2520
gtacgtccaa?tttctttctc?caagaaagaa?cgccgaacaa?gcgccgacga?aaaacggttc 2580
ctcgttattt?ctctccaacg?cgcgcgcacg?ggctatttct?acccccgccg?ccgagtagtt 2640
aggcgtccaa?agtttcggga?gttcatcgtg?aacggtgatc?gatccaaaga?acaacaactc 2700
ggagcaaagc?tagctaacga?agttagttcc?aaacaaagct?agcttatcgc?gattagctag 2760
ctagaagaga?gttagctagg?cgccatgga 2789
<400>2
gttcatcgtg?aacggtgatc?gatccaaaga?acaacaactc?ggagcaaagc?tagctaacga 60
agttagttcc?aaacaaagct?agcttatcgc?gattagctag?ctagaagaga?gttagctagg 120
cgccatggag?ctaggcgtgc?cactgccacg?acggcccgtg?cccggtagct?acggcgtgcc 180
gttcgtctcg?gcggtgcgcg?accgcctcga?tttctactac?ttgcaggggc?aggacaagta 240
cttcgagtcg?cgcgccgaga?ggtacggctc?caccgtcgtc?cgcatcaacg?tcccgcctgg 300
cccattcatg?gcgcgcgacc?cccgcgtggt?ggcgctcctc?gacgccaaga?gcttccccgt 360
cctcttcgac?gtcgccaagg?tcgagaagcg?ggacgtgttc?accggcacgt?tcatgccgtc 420
cacctccctc?accggcggct?accgcgtctg?cgcctacctc?gacccgtccg?agcccaacca 480
cgccaagatc?aagcagctgc?tcctctccct?cctggtctct?cgcaaggacg?ccttcgtccc 540
ggtcttccgc?tccaatttcg?gcgcgctcct?cgacaccgtc?gagtcgcagc?tcgcgagcgg 600
cggcggcaag?tccgacttca?ccgccctcaa?cgatgccacc?tccttcgagt?tcatcggcga 660
ggcgtacttc?ggcgtgcgtc?cctccgcgtc?gagctccctc?ggcaccggcg?ggccgaccaa 720
ggccgccctg?tggctcctat?ggcagctcgc?cccgctcacc?acgctcggcc?tgcccatgat 780
catcgaggat?ccgctcctcc?acacgctgcc?gctgccaccc?ttcctcatca?gctccgacta 840
caaggcgctg?tacgcgtact?tcgccgccgc?ggcgtcgcag?gcgctcgacg?ccgccgaggg 900
ccttggcctg?tcgcgggagg?aggcctgcca?caacctgctg?ttcgcgacgg?tgttcaacag 960
ctacggcggc?ttcaagctgc?tgctcccgca?gatcctgtcg?cgcgtcgcgc?aggccggcga 1020
gaagctccac?gagaggctcg?ccgcggagat?acggagcgcg?gtggccgacg?ccggcggcaa 1080
cgtgacgctg?gccgctctgg?agaagatgga?gctgaccagg?tcggtggtgt?gggaggcgct 1140
gcggctggac?ccgccggtca?ggttccagta?cgggcgcgcc?aaggccgacc?tggagatcga 1200
gagccacgac?gcgtcgttcg?cgatcaagaa?gggggagatg?ctgttcggct?accagccgtg 1260
cgccaccagg?gacccgcggg?tgttcggcgc?cacggcgagg?gagttcgtcg?gcgaccggtt 1320
cgtcggcgag?gaggggagga?agctgctgca?atacgtgtac?tggtcgaatg?ggcgagagac 1380
ggagaaccct?agcgttgaca?acaagcagtg?ccccggcaag?aacctggtgg?tgctcgtcgg 1440
aaggctgttg?ctcgtcgagc?tcttcctccg?gtacgacacc?ttcaccgccg?aggccggcaa 1500
aaaggtggtc?atcaccgggg?tcaccaaagc?ttcaacctcc?gccgtcaatc?gtactgctta 1560
agccggccat?cacttcaggc?gtgggtcgtc?gatcatccac?tcaaccagct?ccgtcctacg 1620
catatgcaat?aagaaatact?cgtacctgaa?ttcttgcatg?gctaaacact?actaataagt 1680
gtatgtattc?tgtttatttg?ttcgattcca?tatttgtaat?tttgttgtta?ttgtttgctg 1740
ttgaattacg?attgtgtatt?ttccccaaaa?aaaaaaaaaa?aaaaaaaaaa 1790
Claims (14)
1. transcription regulatory region dna sequencing fragment that derives from paddy rice, it is a kind of nucleotide sequence fragment of paddy rice hydroperoxyl radical fat hydroxylation cyclooxygenase gene transcription control region, and its nucleotide sequence or its complementary nucleotide sequence are the nucleotide sequence of the 1st to the 2789th shown in the SEQ ID NO:1.
2. expression vector that contains the dna sequencing fragment of claim 1.
3. according to the expression vector of claim 2, after it is imported plant again, can start transcribing of genes involved, express, described genes involved is meant isolating disease-resistant gene in the plant, the defence gene, crucial enzyme in the perhaps defence process or proteic gene, the plant protecting chemical synthetic gene or the key enzyme genoid that perhaps have direct or indirect germicidal action, perhaps from cause of disease isolating nontoxic gene or with the sense-rna of the parasitic closely-related gene of cause of disease, or the gene of isolating active substance with disease resistance or the gene of synthetic their enzymes in animal or the microorganism.
4. the reorganization agrobacterium tumefaciens that contains the expression vector of claim 2.
5. method of cultivating disease-resistant plant, this method comprises that the expression vector that utilizes claim 2 carries out vegetable cell and transforms, and the dna sequencing fragment of claim 1 is incorporated in the karyomit(e) of vegetable cell, again the plant transformed cell culture is become plant.
6. according to the method for claim 5, wherein said vegetable cell method for transformation is Agrobacterium tumefaciens mediated by recombinating, protoplastis fusion, particle gun, electricity swash, PEG mediation or pollen tube introduction method carry out.
The dna sequencing fragment of claim 1 as molecular mark probe in the application that from paddy rice, separates again in the regulating and controlling sequence.
8. the functional analogue of the dna sequencing fragment of claim 1, described functional analogue be selected from following any: GPF, corresponding to the 163rd to 2789 Nucleotide of SEQ ID NO:1; G200 is corresponding to the 807th to 2789 Nucleotide of SEQ ID NO:1; G150 is corresponding to the 1286th to 2789 Nucleotide of SEQ ID NO:1; G75 is corresponding to the 2038th to 2789 Nucleotide of SEQ IDNO:1; G42 is corresponding to the 2363rd to 2789 Nucleotide of SEQ ID NO:1.
9. expression vector that contains the functional analogue of claim 8.
10. according to the expression vector of claim 9, after it is imported plant again, can start transcribing of genes involved, express, described genes involved is meant isolating disease-resistant gene in the plant, the defence gene, crucial enzyme in the perhaps defence process or proteic gene, the plant protecting chemical synthetic gene or the key enzyme genoid that perhaps have direct or indirect germicidal action, perhaps from cause of disease isolating nontoxic gene or with the sense-rna of the parasitic closely-related gene of cause of disease, or the gene of isolating active substance with disease resistance or the gene of synthetic their enzymes in animal or the microorganism.
11. contain the reorganization agrobacterium tumefaciens of the expression vector of claim 9.
12. a method of cultivating disease-resistant plant, this method comprise that the expression vector that utilizes claim 9 carries out vegetable cell and transforms, and the functional analogue of claim 8 is incorporated in the karyomit(e) of vegetable cell, again the plant transformed cell culture are become plant.
13. the method for claim 12, wherein said vegetable cell method for transformation is Agrobacterium tumefaciens mediated by recombinating, protoplastis fusion, particle gun, electricity swash, PEG mediation or pollen tube introduction method carry out.
14. the functional analogue of claim 8 as molecular mark probe in the application that from paddy rice, separates again in the regulating and controlling sequence.
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| 稻瘟病病原物诱导启动子的构建及表达 吕华飞 等，科学通报，第44卷第20期 1999 * |
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