CN103074342A - Inducible promoter for pathogenic bacteria of rice - Google Patents
Inducible promoter for pathogenic bacteria of rice Download PDFInfo
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Abstract
The invention relates to the technical field of plant gene engineering and provides an inducible promoter for pathogenic bacteria of rice. The promoter can be activated by rice bacterial leaf blight bacterium xanthomonas PXO99 and PXO61, a rice baeterial leaf streak bacterium RS105 and a corn banded sclerotial blight bacterium YWK-196, and can also be activated by abscisic acid, gibberellic acid, salicylic acid and sodium chloride. Truncation of the promoter verifies that two sections from -513bp to -411bp and from -411bp to -309bp are critical areas induced by the pathogenic bacteria. Various plant expression vectors can be constructed with the adoption of the promoter, and the promoter can be used for improving plant quality and other beneficial production traits through a plant gene engineering technology.
Description
Technical field
The present invention relates to the plant gene engineering technology field, a rice pathogens evoked promoter is provided, can be used for improving disease resistance of plant and other useful production traitss by plant gene engineering technology.
Background technology
The plant disease that is caused by fungi, bacterium, virus and nematode is that crop production is brought tremendous influence.Plant has the different resistance mechanism of two covers: the one, and Passive Defence mechanism, refer to the morphological structure that some resistances are relevant, such as epidermis and hard cell walls (Brady JD, Fry S.Formation of di-isodityrosine and loss of isodityrosine in the cell walls of tomato cell-suspension cultures treated with fungal eli citors or H
2O
2.Plant Physiol 1997,115:87-92.).Another is Initiative Defense, and Initiative Defense is that resistant gene and pathogenic bacteria identify and mutual the work causes.The acceptor molecule of disease-resistant gene (R) the coding exciton of the exciton of pathogen nontoxic gene (avr) coding and host plant response interacts and produces disease resistance response.The plant disease-resistant defense response generally experiences signal identification, signal conduction, Defense gene expression three link.All types of disease-resistant mechanism all will rely on Defense gene expression in the Initiative Defense, and the validity of opposing depends on the space-time characterisation of genetic expression.Just because of the spatial and temporal expression characteristic of Analysis of Defence Genes Involved, its promotor often has some special cis-acting elements, and people can utilize Analysis of Defence Genes Involved, especially its cis element to provide favourable instrument for the genetically engineered improvement.Most disease-resistant genes are all encoded and are had the albumen (NBS-LRR) of nucleotide binding site and leucine repetition in the plant, (McHale L, Tan X, Koehl P, Michelmore RW.Plant NBS-LRR proteins:adaptable guards.Genome Biol 2006,7:212.).But the promotor research for this genoid is also fewer.
Plant gene promoter is important cis-acting elements, is the dna sequence dna that is positioned at structure gene 5 ˊ end upstream, is the center of transcriptional control.Since the strain transgenic plant were come out from nineteen eighty-three first, promotor was engineered study hotspot always, selects the promotor of suitable and effective expression to establish solid basis for engineered research.The organizing specific promotor can make the expression of foreign gene only occur in some specific organ or tissue position, and often shows the growth control characteristic; Inducible promoter can make foreign gene that some signal is produced response, only expresses under distinctive signal stimulates.The great advantage of these promotors is: it has overcome foreign gene that constitutive promoter starts and nonspecificly in recipient plant continues, efficiently expresses the waste that causes, and satisfies the demand of some needs foreign gene specifically expressing.Tissue specificity or abduction delivering promotor all are the Focal point and difficult points of plant genetic engineering research all the time.
At present, found some tissue specificities or abduction delivering promotor, and found the corresponding cis factor of existence on it, for promotor research is afterwards laid a good foundation.The Sar8.2b gene of tobacco is induced by Whitfield's ointment (SA), has found several cis acting factors in its promotor, comprises the as-1 factor, GT21 and Dof binding sequence.SA induces Sar8.2b genetic expression may be present in (Song F in the cis factor in-728 to-927bp and-197 to-351bp zone, Goodman RM.Cloning and identification of the promoter of the tobacco Sar8.2b gene, a gene involved in systemic acquired resistance.Gene 2002,290:115-124.).Maize sucrose synthetic enzyme I only expresses (Yang NS in phloem cell, Russell D.Maize sucrose synthase-promoter directs phloem cell specific expression of gus gene in transgenic tobacco plants.Proc Natl Acad Sci 1990,87:4144-4148.).The PsGNS2 promotor is specifically expressing (Buchner P in seed only, Rochat C, Wuilleme S, Boutin JP.Characterization of a tissue-specific anddevelopmentally regulated beta-1,3-glucanase gene in pea (Pisum sativum) .Plant Mol Biol 2002,49:171-186.).
Paddy rice is one of most important food crop, and the fungal disease of paddy rice and Micobial Disease all can cause output to reduce and quality descends.The separation of paddy disease-resistant gene cloning and promotor can make us understand better the mutual work of host and pathogenic bacteria and lay the foundation for genetically engineered improves.Therefore how to utilize the separation of paddy disease-resistant gene cloning and promotor to obtain disease-resistant plant and become one of problem demanding prompt solution.
Summary of the invention
The present inventor is for the situation of above-mentioned prior art, a rice pathogens evoked promoter is provided, this promotor can be by bacterial blight of rice germ Xanthomonas campestris PXO99, PXO61, Xanthomonas campestris pv. Oryzicola RS105 and corn sheath blight fungus YWK-196 activate, simultaneously, also can be activated by dormin, gibberic acid, Whitfield's ointment, sodium-chlor processing.By this promotor is carried out brachymemma, determined-513bp to-411bp and-411 to-310bp is pathogeny evoked critical area.Can utilize promotor of the present invention to be built into various plant expression vectors, be used for improving plant quality and other useful production traitss by plant gene engineering technology.
The contriver at first provides a rice pathogens evoked promoter, contains in this promoter gene sequence just like the dna sequence dna shown in the sequence table SEQ ID NO.1.
This promotor derives from I in Rice RBB13(Oryza sativa), be one of following nucleotide sequence:
Dna sequence dna or partial dna sequence shown in the SEQ ID NO.1 in the sequence table.
SEQ ID NO.1 in the sequence table is by 2257 based compositions.Holding the 2198th bit base from 5 ˊ is transcription initiation site, be designated as+1.
Wherein the TATA frame is positioned at transcription initiation site upstream-30 to-25 bit bases, and CAAT box is positioned at-72 to-69 bit bases, these elements are basic promoter element in transcribing.
From the-1239 at 5 ˊ end ,-183 bit bases are yellow induced element ACGTATERD1; From the-1793 at 5 ˊ end ,-1750 ,-682 bit bases are embryo and endosperm specific expression element CANBNNAPA; Be dehydration response element CBFHV from 5 ˊ end, the-128 bit bases; From the-1474 at 5 ˊ end ,-1137 ,-837 ,-168 bit bases are heat shock protein gene expression element CCAATBOX1; Holding the-55 bit bases from 5 ˊ is dark response element CDAATCAB2; From the-2079 at 5 ˊ end ,-1773 ,-1243 ,-739 bit bases are cupric ion abduction delivering element CURECORECR; From the-1714 at 5 ˊ end ,-1276 ,-1005 ,-906 ,-725 ,-587 ,-380 ,-333 ,-230 bit bases are DBP gene expression element DOFCOREZM; From the-2069 at 5 ˊ end ,-1631 ,-1572 ,-1566 ,-1527 ,-220 bit bases are storage protein gene Expression element EBOXBNNAPA; From the-2119 at 5 ˊ end ,-1252 ,-1186 bit bases are enhancer element EECCRCAH1; Holding the-336 bit bases from 5 ˊ is nitrogen response element EMHVCHORD; From the-1805 at 5 ˊ end ,-1670 ,-1441 ,-1071 ,-698 ,-392 ,-284 ,-227 ,-212 bit bases are photoinduction element GATABOX; From the-2137 at 5 ˊ end ,-315 bit bases are pathogenic bacteria and salt ion abduction delivering element GT-1; Holding the-683 bit bases from 5 ˊ is ABA abduction delivering element PROXBBNNAPA; From the-1854 at 5 ˊ end ,-726 bit bases are GA induced element PYRIMIDINEBOXOSRAMY1A; From the-1517 at 5 ˊ end ,-950 bit bases are Induced by Salicylic Acid element WBOXATNPR1.The sequencing results of 2H16 promotor p2H16 shows that the 2H16 gene is subjected to the regulation and control of complicated factor in paddy rice resistance and stress-inducing process.
Verify by brachymemma simultaneously, determined pathogeny evoked relevant section be positioned at-513bp to-411bp and-411bp is to-309bp, after having determined above-mentioned pathogeny evoked critical area, can be easily with other disease-resistant related genes by the genetic engineering modified disease resistance of plant that improves.
In addition, invention of the present invention also provides the recombinant vectors that contains p2H16, and called after 1381GFP::p2H16.
In sum, the present inventor provides a rice pathogens evoked promoter p2H16 in the world first, the Transgenic Rice proof finds that transfer-gen plant is subject to ABA, SA, GA, Xanthomonas campestris PXO99, PXO61, xanthomonas oryzae pv. oryzicola RS105 and corn sheath blight fungus YWK-196 induce rear GFP activity that remarkable rising is all arranged, and illustrate that p2H16 contains the corresponding responsing reaction factor.The expression characteristic of p2H16 in model plant shows that it is the relevant promotor of a cause of disease.The functional study of p2H16 can be the expression regulation mechanism and the concrete function that disclose 2H16 and lays the first stone, and also can be applicable in the genetically engineered improvement of Genetic Engineering for Disease Resistance in Rice and resistance.
Description of drawings
Fig. 1. the flower plant is subject to Xanthomonas campestris PXO99, PXO61 in the promotor rice transformation of the present invention, bacterial stripe germ RS105 induces as a result gray-scale map of rear GFP Fluirescence observation;
H wherein
2O is contrast, found that inoculation carries out Fluirescence observation behind the 24h, can observe very strong fluorescence, illustrates that this promotor is subject to inducing of above-mentioned pathogenetic bacteria;
Fig. 2. the flower plant is subject to corn sheath blight fungus YWK-196 and induces as a result gray-scale map of rear GFP Fluirescence observation in the promotor rice transformation of the present invention;
Do not meet bacterium CK and be contrast, found that inoculation carries out Fluirescence observation behind the 24h, can observe very strong fluorescence, illustrate that this promotor is subject to inducing of above-mentioned pathogenic fungi;
Fig. 3. the flower plant is subject to NaCl in the promotor rice transformation of the present invention, and SA, GA, ABA induce as a result gray-scale map of rear GFP Fluirescence observation;
Found that and carry out Fluirescence observation after processing 36h, NaCl, SA has very strong fluorescence after processing, and GA, after ABA processes fluorescence intensity a little less than, NaCl is described, SA, GA, ABA all can induce this promoter expression, just induce intensity different;
Fig. 4. the flower plant is subject to bacterial leaf spot pathogenic bacteria PXO99 and induces as a result gray-scale map of rear GFP Fluirescence observation in the different brachymemma promotor rice transformations of the present invention;
Found that inoculation is carried out Fluirescence observation behind the 24h, clipping-720bp is to-this section of 309bp, and fluorescence intensity obviously weakens, and illustrates that this section exists and pathogeny evoked relevant element;
Fig. 5. different brachymemma promotors of the present invention are GFP Fluirescence observation gray-scale map as a result after being subject to corn sheath blight fungus YWK-196 behind the transient expression on this life cigarette and inducing;
Found that inoculation is carried out Fluirescence observation behind the 24h, clipping-513bp to-411bp and-411bp is to-these two sections of 309bp, fluorescence intensity has obviously and weakens, and illustrates with pathogeny evoked relevant element to be present in this two sections.
Fig. 6 is the colored synoptic diagram of Fig. 1;
Fig. 7 is the colored synoptic diagram of Fig. 2;
Fig. 8 is the colored synoptic diagram of Fig. 3;
Fig. 9 is the colored synoptic diagram of Fig. 4;
Figure 10 is the colored synoptic diagram of Fig. 5.
Embodiment
Further definition the present invention in following examples, according to above description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and in the situation that does not depart from spirit and scope of the invention, can make various changes and modification to the present invention, so that its applicable various uses and condition.Except special indicating, the state of the art that is of the present invention;
Clone and the sequential analysis of embodiment 1 paddy rice 2H16 gene promoter p2H16
According to the fine 2H16 gene of paddy rice Japan (the GENBANK number of logging in is: AK058338) upstream 2300bp primers (5 ˊ-CAGACGGCTACTGTCCATCA-3 ˊ, its sequence is shown in SEQ ID NO.2; 5 ˊ-GCGAGAGCAGGAGGAGAGAC-3 ˊ, its sequence is shown in SEQ ID NO.3) be used for obtaining the promotor of gene.
Extract I in Rice RBB13 genomic dna, as template, carry out pcr amplification, response procedures is as follows: 94 ℃ of 5min of predeformation, 94 ℃ of 40s of sex change, the 54 ℃ of 40s that anneal extend 72 ℃ of 2min, react 35 circulations, 72 ℃ of 7min of rear extension, after the end, with DNA recovery test kit recovery and the purifying amplified fragments of Kang Wei company, then the dna fragmentation with purifying is connected to (Promega company) among the carrier pGEM-T, incubated at room 1h, Transformed E .coli DH5 ɑ competent cell is selected positive colony upgrading grain, and order-checking is finished by the large gene of China.The result through behind the pcr amplification, obtains the dna fragmentation that length is 2257bp take I in Rice RBB13 genomic dna as template, has the dna sequence dna of sequence table SEQ ID NO.1.
Compare with the promoter sequence of the 2H16 that has cloned, there is the consecutive miss of 73bp in the dna fragmentation of this 2257bp as a result, with this promotor called after p2H16.And with the above-mentioned recombinant vectors called after pGEM-T::p2H16 that contains p2H16.First base of 2H16cDNA sequence is defined as transcription initiation site, the 2198th bit base that namely SEQ ID NO.1 holds from 5 ˊ in the sequence table.
Use PLACE(Higo K, Ugawa Y, Iwamoto M, Korenaga T.Plant cis-acting regulatory DNA-elements (PLACE) .Nucl Acids Res1999,27:297-300.) software carries out sequential analysis to the promotor nucleotide sequence (the SEQ ID NO.1 in the sequence table) of paddy rice 2H16.With above-mentioned software the cis-acting elements among the 2H16 gene promoter p2H16 is searched for, predicted, contain as a result the homologous sequence of numerous and known Eukaryotic cis element in this promotor.Wherein, first base of 2H16cDNA sequence is defined as transcription initiation site (be designated as+1), it is the 2198th bit base that SEQ ID NO.1 holds from 5 ˊ in the sequence table, wherein the TATA frame is positioned at transcription initiation site upstream-30 to-25 bit bases, and CAAT box is positioned at-72 to-69 bit bases, these elements are basic promoter element in transcribing.From the-1239 at 5 ˊ end,-183 bit bases are yellow induced element ACGTATERD1 (Simpson SD, Nakashima K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K.Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence.Plant J2003,33:259-270.); From the-1793 at 5 ˊ end,-1750,-682 bit bases are embryo and endosperm specific expression element CANBNNAPA (EllerstromM, Stalberg K, Ezcurra I, Rask L.Functional dissection of a napin gene promoter:identification of promoter elements required for embryo and endosperm-specific transcription.Plant Mol Biol 1996,32:1019-1027.); Be dehydration response element CBFHV (Svensson JT from 5 ˊ end, the-128 bit bases, Crosatti C, Campoli C, Bassi R, Stanca AM, Close TJ, Cattivelli L.Transcriptome analysis of cold acclimation in barley albina and xantha mutants.Plant Physiol 2006,141:257-270.); From the-1474 at 5 ˊ end,-1137,-837,-168 bit bases are heat shock protein gene expression element CCAATBOX1 (Rieping M, Schoffl F.Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobacco.Mol Gen Genet 1992,231:226-232.); Holding the-55 bit bases from 5 ˊ is dark response element CDAATCAB2 (Maxwell BB, Andersson CR, Poole DS, Kay SA, Chory J.HY5, circadian clock-assosiated 1, and a cis-lelement, DET1 dark response element, mediate DET1 regulation of chlorophyll a/b-binding protein 2 expression.Plant Physiol 2003,133:1565-1577.); From the-2079 at 5 ˊ end,-1773,-1243,-739 bit bases are cupric ion abduction delivering element CURECORECR (Quinn JM, Merchant S.Two copper-responsive elements associated with the chlamydomonasCyc6 gene function as targets for transcriptional activators.Plant Cell 1995,7:623-628.); From the-1714 at 5 ˊ end,-1276,-1005,-906 ,-725 ,-587,-380,-333 ,-230 bit bases be DBP gene expression element DOFCOREZM (Yanagisawa S.Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize.Plant J 2000,21:281-288.); From the-2069 at 5 ˊ end,-1631,-1572,-1566,-1527,-220 bit bases are storage protein gene Expression element EBOXBNNAPA (Stalberg K, Ellerstom M, Ezcurra I, Ablov S, Rask L.Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds.Planta 1996,199:515-519.); From the-2119 at 5 ˊ end ,-1252 ,-1186 bit bases are enhancer element EECCRCAH1 (Yoshioka S, Taniguchi F, Miura K, Inoue T, Yamano T, Fukuzawa H.The novel Myb transcription factor LCR1 regulates the CO
2-responsive gene Cah1, encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii.Plant Cell 2004,16:1466-1477.); Holding the-336 bit bases from 5 ˊ is nitrogen response element EMHVCHORD (Muller M, Knudsen S.The nitrogen response of a barley C-hordein promoter is controlled by positive and negative regulation of the GCN4 and endosperm box.Plant J 1993,4:343-355.); From the-1805 at 5 ˊ end ,-1670 ,-1441,-1071,-698 ,-392 ,-284,-227,-212 bit bases are photoinduction element GATABOX (Gilmartin PM, Sarokin L, Memelink J, Chua NH.Molecular light switches for plant genes.Plant Cell 1990,2:369-378.); From the-2137 at 5 ˊ end,-315 bit bases are pathogenic bacteria and salt ion abduction delivering element GT-1 (Park HC, Kim ML, Kang YH, Jeon JM, Yoo JH, Kim MC, Park CY, Jeong JC, Moon BC, Lee JH, Yoon HW, Lee SH, Chung WS, Lim CO, Lee SY, Hong JC, Cho MJ.Pathogen-and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1box that interactswith a GT-1-like transcription factor.Plant Physiol 2004,135:2150-2161.); Holding the-683 bit bases from 5 ˊ is ABA abduction delivering element PROXBBNNAPA (Ezcurra I, Ellerstrom M, Wycliffe P, Stalberg K, Rask L.Interaction between composite elements in the napA promoter:both the B-box ABA-responsive complex and the RY/G complex are necessary for seed-specific expression.Plant Mol Biol 1999,40:699-709.); From the-1854 at 5 ˊ end,-726 bit bases are GA induced element PYRIMIDINEBOXOSRAMY1A (Mena M, Cejudo FJ, Isabel-Lamoneda I, Carbonero P.A role for the DOF transcription factor BPBF in the regulation of gibberellin-responsive genes in barley aleurone.Plant Physiol 2002,130:111-119.); From the-1517 at 5 ˊ end,-950 bit bases are Induced by Salicylic Acid element WBOXATNPR1 (Chen W, Provart NJ, Glazebrook J, Katagiri F, Chang HS, Eulgem T, Mauch F, Luan S, Zou G, Whitham SA, Budworth PR, Tao Y.Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses.Plant Cell 2002,14:559-574.).The sequencing results of 2H16 promotor p2H16 shows that the 2H16 gene is subjected to the regulation and control of complicated factor in paddy rice resistance and stress-inducing process.
Controlling element analysis in the table 12H16 promoter sequence
1. N represents A, C, G or T; R represents A or G
Spend 11 in the recombinant vectors of embodiment 2 structure 2H16 promoter sequences and GFP gene and the rice transformation
Take I in Rice RBB13 DNA as template, use forward primer, its sequence is (5 ˊ-ATG shown in SEQ ID NO.4
GTCGACCAGACGGCTACTGTCCATCA-3 ˊ, band underscore base is restriction enzyme SalI recognition site) and reverse primer, its sequence is (5 ˊ-ATG shown in SEQ ID NO.5
CTGCAG.GCGAGAGCAGGAGGAGAGAC-3 ˊ, band underscore base is restriction enzyme PstI recognition site) the amplification promoter fragment, the PCR response procedures is as follows: 94 ℃ of 5min of denaturation, 94 ℃ of 40s of sex change, the 62 ℃ of 40s that anneal extend 72 ℃ of 2min, react 35 circulations, 72 ℃ of 7min of rear extension obtain the fragment of sequence SEQ ID NO.1-2197bp-+60bp zone, and the recognition site of restriction enzyme SalI and PstI on adding respectively at the sequence two ends.After reaction finishes, the PCR product is carried out 1.0% agarose gel electrophoresis detect, reclaim and purifying purpose fragment the resulting PCR product of sequence verification, the PCR product carries out enzyme with SalI and PstI to be cut, and is connected with plant expression vector 1381GFP through the same enzyme double digestion.The upstream that the promoter fragment of above acquisition is connected to the GFP gene obtains recombinant vectors, and called after 1381GFP::p2H16 transforms Agrobacterium EHA105 with electrization with recombinant vectors.
By spending 11 in the agrobacterium mediation converted paddy rice, the young plant that differentiates transplanted to soil grow.Further verify the detection transfer-gen plant with PCR method.Results transfer-gen plant seed.The transfer-gen plant planting seed also is used for promoter function and the Characteristics in soil.Described method for transformation all adopts prior art.
Embodiment 3 detects the p2H16 transfer-gen plant to the induced reaction of pathogenic bacteria, salt and plant hormone
Be taken at the greenhouse and grow the T1 in about 3 weeks for spending 11 transgenosis seedling to do various processing in the paddy rice.Use respectively bacterial blight of rice pathogenic bacteria Xanthomonas campestris PXO99, PXO61, xanthomonas oryzae pv. oryzicola RS105, corn sheath blight fungus YWK-196, this three Plant Hormone of NaCl and Whitfield's ointment, gibberic acid and dormin is processed rice seedling, and arranges that to be untreated be contrast.Detect the power of different transgenic line different treatment front and back GFP activity.Wherein bacterial leaf spot pathogenic bacteria and slice germ on the PSA substratum 28 ℃ cultivated 2 days, resuspended with sterilized water, adopt injection inoculation, got plant leaf analysis behind the inoculation pathogenic bacteria in 1 day; The corn sheath blight fungus is having on the PDB substratum of toothpick 25 ℃ to cultivate 2 days, and the inoculation of toothpick embedding inlay technique was got plant leaf analysis after 1 day; The treatment process of NaCl is: be that the NaCl of 100mM directly waters in the soil that is filled in paddy growth with concentration, process and get blade analysis after 36 hours; The treatment process of hormone is: be respectively the ABA of 25 μ M with concentration, the SA of the GA of 50 μ M and 200 μ M directly sprays rice seedling.All HORMONE TREATMENT times are 36 hours.
Through the GFP analytical resultss of the transgenic plant of above-mentioned different treatment as shown in the figure, wherein inoculate transfer-gen plant GFP behind PXO99, PXO61, the RS105 active with inoculation H
2O has compared obvious enhancing (Fig. 1); The transfer-gen plant GFP activity of inoculation behind the YWK-196 with do not meet bacterium CK and compared obvious enhancing (Fig. 2); Transfer-gen plant GFP activity after NaCl, SA process has been compared obvious enhancing with being untreated, GA, and the GFP fluorescence intensity of the transfer-gen plant after ABA processes is weak (Fig. 3).
Spend 11 in the 2H16 promoter sequence of the different brachymemmas of embodiment 4 structures and the recombinant vectors of GFP gene and the rice transformation
Take I in Rice RBB13 DNA as template, use respectively forward primer, its sequence such as SEQ ID NO.6,7,8, (5 ˊ-TATCCACACTGTAATTTTTGTGAT-3 ˊ shown in 9,5 ˊ-ATAATTTGTAAATCATGTACACGTA-3 ˊ, 5 ˊ-AGAGGACATCAGAAACACGC-3 ˊ, 5 ˊ-CTACCCTACTATTTATATCAGAACA-3 ˊ) and reverse primer, its sequence is (5 ˊ-GACCACCGTACGTAGGCTTA-3 ˊ) amplification promoter fragment shown in SEQ ID NO.10, the PCR response procedures is as follows: 94 ℃ of 5min of denaturation, 94 ℃ of 40s of sex change, the 50 ℃ of 40s that anneal extend 72 ℃ of 2min, react 35 circulations, 72 ℃ of 7min of rear extension obtain sequence SEQ ID NO.11,12,13, fragment-1742bp-+60bp of 14 ,-1259bp-+60bp ,-720bp-+60bp and-309bp-+60bp zone.After reaction finishes, the PCR product is carried out 1.0% agarose gel electrophoresis detect, reclaim and purifying purpose fragment, the resulting PCR product of sequence verification, the PCR product is connected with the plant expression vector pCXGFP-P that cuts through the XcmI enzyme.The upstream that the promotor truncated segment of above acquisition is connected to the GFP gene obtains recombinant vectors, with electrization recombinant vectors is transformed Agrobacterium EHA105.
By spending 11 in the agrobacterium mediation converted paddy rice, the young plant that differentiates transplanted to soil grow.Verify further that with PCR method detecting transfer-gen plant also is used for promoter function and the Characteristics.Described method for transformation all adopts prior art.
Embodiment 5 detects the p2H16 transfer-gen plant of different brachymemmas to the induced reaction of pathogenic bacteria
Be taken at the greenhouse and grow the T0 in about 3 weeks for spending 11 transgenosis seedling to do various processing in the paddy rice.With bacterial blight of rice pathogenic bacteria Xanthomonas campestris PXO99 Inoculated Rice seedling.Detect the power of the rear GFP activity of different brachymemma promotor transgenic line inoculations.Wherein bacterial leaf spot pathogenic bacteria on the PSA substratum 28 ℃ cultivated 2 days, resuspended with sterilized water, adopt injection inoculation, got plant leaf analysis behind the inoculation pathogenic bacteria in 1 day.
Through the GFP analytical resultss of the transgenic plant of above-mentioned inoculation as shown in the figure, behind the inoculation PXO9924h,-1742bp-+60bp,-1259bp-+60bp and-blade fluorescence intensity and the total length promotor-2197bp-+60bp of 720bp-+60bp is basically identical, and-fluorescence intensity of 309bp-+60bp compares obviously with total length and weakens (as shown in Figure 4), illustrate with pathogeny evoked relevant element to be present in-720bp is to-this zone of 309bp.
Embodiment 6 structure-720bp to the recombinant vectors of the different brachymemma promoter sequences of-309bp and GFP gene and on this life cigarette transient expression
For the further accurately pathogeny evoked relevant section in location, p-720bp has carried out again brachymemma to-this section of 309bp.Take I in Rice RBB13DNA as template, use respectively forward primer, its sequence such as SEQ ID NO.15,16, (5 ˊ-CTACTTACAAAACTAATTATATAAAA-3 ˊ shown in 17,5 ˊ-AGGCTAATCATGGATTAATTAGG-3 ˊ, 5 ˊ-GTGTCCAAATATCCGATGTGATA-3 ˊ) and reverse primer, its sequence is (5 ˊ-GACCACCGTACGTAGGCTTA-3 ˊ) amplification promoter fragment shown in SEQ ID NO.10, and the PCR response procedures is as follows: 94 ℃ of 5min of denaturation, 94 ℃ of 40s of sex change, 50 ℃ of 40s anneal, extend 72 ℃ of 1min, react 35 circulations, 72 ℃ of 7min of rear extension, obtain sequence SEQ ID NO.18, fragment-614bp-+60bp of 19,20 ,-513bp-+60bp and-411bp-+60bp zone.After reaction finishes, the PCR product is carried out 1.0% agarose gel electrophoresis detect, reclaim and purifying purpose fragment, the resulting PCR product of sequence verification, the PCR product is connected with the plant expression vector pCXGFP-P that cuts through the XcmI enzyme.The upstream that the promotor truncated segment of above acquisition is connected to the GFP gene obtains recombinant vectors, with electrization recombinant vectors is transformed Agrobacterium GV3101.
With cultured Agrobacterium GV3101 and p19K 10mM MgCl
2Resuspended, transfer OD
600=1.0, this life of injection Tobacco Leaves was used for transient expression behind the injection 3-5d and detects after equal-volume mixed.
Embodiment 7 detection-720bp are to the different brachymemma promotors of-309bp induced reaction to pathogenic bacteria behind transient expression on this life cigarette
This life Tobacco Leaves behind the injection Agrobacterium 5d is cut, be tiled in the inoculation dish, cultured corn sheath blight fungus YWK-196 is taken off circular bacterium piece with punch tool, tip upside down on the tobacco leaf, get the vaccination blade behind 25 ℃ of processing of moisturizing 24h and analyze.
Through the tobacco leaf GFP of above-mentioned inoculation analytical results as shown in the figure, behind the inoculation YWK-19624h,-614bp-+60bp and-the blade fluorescence intensity of 513bp-+60bp with-720bp-+60bp is basically identical, and-fluorescence intensity of 411bp-+60bp and-513bp-+60bp and-309bp-+60bp has obviously and weakens (Fig. 5), illustrate-513bp to-411bp and-411bp extremely-these two sections of 309bp are with pathogeny evoked relevant.
More than these presentation of results 2H16 promotor be subject to pathogenic bacteria, NaCl, ABA, GA, SA and induce, there is corresponding response element on it, it is the relevant promotor of a cause of disease, simultaneously by brachymemma checking, determined pathogeny evoked relevant section be positioned at-513bp to-411bp and-411bp is to-309bp.After having determined above-mentioned pathogeny evoked critical area, can be easily with other disease-resistant related genes by the genetic engineering modified disease resistance of plant that improves.
Claims (4)
1. rice pathogens evoked promoter, it is characterized in that: its gene order is shown in SEQ ID NO.1.
2. promotor according to claim 1, it is characterized in that: this promotor can be activated by pathogenic bacteria, perhaps can be activated when processing with dormin, gibberic acid, Whitfield's ointment, sodium-chlor.
3. promotor according to claim 1 is characterized in that: in the sequence-513bp to-411bp and-411bp to-309bp is pathogeny evoked critical area.
4. the application of promotor as claimed in claim 1 in plant quality improvement and plant resistance to environment stress improvement.
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| CN103540596A (en) * | 2013-10-10 | 2014-01-29 | 山东农业大学 | Corn pathogen induced promoter |
| CN103740723A (en) * | 2014-01-15 | 2014-04-23 | 海南大学 | Promoter induced by rice bacterial blight germ and bacterial streak germ and application |
| CN105567695A (en) * | 2016-03-08 | 2016-05-11 | 安徽省农业科学院水稻研究所 | Rice non-endosperm expression promoter SAFES3 and application thereof |
| CN105861506A (en) * | 2016-05-31 | 2016-08-17 | 安徽省农业科学院水稻研究所 | Rice endosperm non-expression promoter SAFES2 and separation method and application thereof |
| CN106148346A (en) * | 2016-07-15 | 2016-11-23 | 安徽省农业科学院水稻研究所 | A kind of isolated endosperm does not express promoter SAFES6 and application thereof |
| CN107475210A (en) * | 2017-09-07 | 2017-12-15 | 四川农业大学 | A kind of Bacterial Blight Resistance in Rice related gene OsABA2 and its application |
| CN107699566A (en) * | 2017-11-24 | 2018-02-16 | 江苏省农业科学院 | A kind of rice starter P71Z4 and its application by cause of disease induction |
| CN111264543A (en) * | 2020-01-09 | 2020-06-12 | 山东农业大学 | Purine base plant immunity inducing agent and application thereof |
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| CN103540596A (en) * | 2013-10-10 | 2014-01-29 | 山东农业大学 | Corn pathogen induced promoter |
| CN103740723A (en) * | 2014-01-15 | 2014-04-23 | 海南大学 | Promoter induced by rice bacterial blight germ and bacterial streak germ and application |
| CN103740723B (en) * | 2014-01-15 | 2015-05-20 | 海南大学 | Promoter induced by rice bacterial blight germ and bacterial streak germ and application |
| CN105567695B (en) * | 2016-03-08 | 2019-08-13 | 安徽省农业科学院水稻研究所 | A kind of rice non-endosperm expression promoter SAFES3 and its application |
| CN105567695A (en) * | 2016-03-08 | 2016-05-11 | 安徽省农业科学院水稻研究所 | Rice non-endosperm expression promoter SAFES3 and application thereof |
| CN105861506A (en) * | 2016-05-31 | 2016-08-17 | 安徽省农业科学院水稻研究所 | Rice endosperm non-expression promoter SAFES2 and separation method and application thereof |
| CN105861506B (en) * | 2016-05-31 | 2019-05-10 | 安徽省农业科学院水稻研究所 | A kind of paddy endosperm does not express promoter SAFES2 and its separation method and application |
| CN106148346A (en) * | 2016-07-15 | 2016-11-23 | 安徽省农业科学院水稻研究所 | A kind of isolated endosperm does not express promoter SAFES6 and application thereof |
| CN106148346B (en) * | 2016-07-15 | 2019-10-08 | 安徽省农业科学院水稻研究所 | A kind of endosperm isolated does not express promoter SAFES6 and its application |
| CN107475210A (en) * | 2017-09-07 | 2017-12-15 | 四川农业大学 | A kind of Bacterial Blight Resistance in Rice related gene OsABA2 and its application |
| CN107699566A (en) * | 2017-11-24 | 2018-02-16 | 江苏省农业科学院 | A kind of rice starter P71Z4 and its application by cause of disease induction |
| CN111264543A (en) * | 2020-01-09 | 2020-06-12 | 山东农业大学 | Purine base plant immunity inducing agent and application thereof |
| CN111264543B (en) * | 2020-01-09 | 2021-05-04 | 山东农业大学 | Purine base plant immunity inducing agent and application thereof |
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