CN102115745B - Promoter BgIosP556, and preparation method and application thereof - Google Patents
Promoter BgIosP556, and preparation method and application thereof Download PDFInfo
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- CN102115745B CN102115745B CN 200910238991 CN200910238991A CN102115745B CN 102115745 B CN102115745 B CN 102115745B CN 200910238991 CN200910238991 CN 200910238991 CN 200910238991 A CN200910238991 A CN 200910238991A CN 102115745 B CN102115745 B CN 102115745B
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Abstract
The invention provides a promoter BgIosP556, and a preparation method and application thereof. The promoter provided by the invention has a nucleotide sequence disclosed as SEQ ID NO:1, or has a variant with promoter functions from: (1) a nucleotide sequence crossbred with the nucleotide sequence disclosed as SEQ ID NO:1 under highly strict conditions, (2) a nucleotide sequence formed by carrying out substitution, deletion and addition of one or a plurality of basic groups on the nucleotide sequence disclosed as SEQ ID NO:1, and (3) a nucleotide sequence which is at least 90% identical to the nucleotide sequence disclosed as SEQ ID NO:1. The invention also relates to a preparation method of the promoter, and application of the promoter in the control of the expression of a target gene in a monocotyledon.
Description
Technical field
The present invention relates to a kind of promotor, particularly a kind of monocotyledons promotor of paddy rice for example, and the Preparation method and use of described promotor.
Background technology
Promotor is an integral part of gene, is usually located at structure gene 5 ' end upstream, is the section of DNA sequence of RNA polymerase identification, combination and transcriptional start.Promotor can instruct holoenzyme (holoenzyme) with the template correct combination, the activation RNA polymerase, and promotor gene is transcribed, thus controlling gene is expressed the time of origin of (transcribing) and the degree of expression.In transgenic plant, promotor is one of important factor that affects transgene expression efficient, and selecting high efficiency promotor is the key of high-efficient expression foreign gene.
Transcriptional profile according to promotor can be divided into it 3 classes: constitutive promoter, tissue or organ specific promoters and inducible promoter.So-called constitutive promoter refers to that the genetic expression of different tissues organ and etap does not have notable difference, thereby is referred to as constitutive promoter under the constitutive promoter regulation and control.In the dicotyledons the constitutive promoter of normal use be cauliflower mosaic virus (CaMV) 35S promoter.Another kind of efficiently constitutive promoter CsVMV separates from cassava vein mosaic virus (cassava vein mosaic virus).
People pay much attention to the constitutive promoter from plant clone itself.For example Actin muscle (actin) and the isogenic promotor of ubiquitin (ubiquitin) are cloned.Replace the CaMV 35S promoter with these promotors, can more effectively in monocotyledons, drive transcribing of foreign gene.The Naomi philosophy has been cloned corresponding promotor from the tryptophan synthase subunit gene of Arabidopis thaliana and phytochrome gene, replace the CaMV 35S promoter with it, in transgene tobacco, also obtained good expression effect (Plant biotechnology, 2002,19 (1): 19-26).
Promotor common in the monocotyledons gene has: Ubi promotor (Plant ubiquitin promoter), Actin promotor (Plant Actin promoter) and Adh-1 promotor (Maize alcohol dehydrogenase1promoter).
The Ubi promotor gains great popularity with the factor such as its starting efficiency is high, methylation is low, stabilization characteristics of genetics.At present, from a lot of ubiquitin genes, separated and obtained promoter sequence, comprise Ubi-1 promotor, paddy rice ubiquitin RUBQ2 promotor, Arabidopis thaliana ubiquitin promoter, Sunflower Receptacle ubiquitin UbB1 promotor, tobacco ubiquitin Ubi.U4 promotor, potato ubiquitin Ubi7 promotor, tomato ubiquitin Ubi1-1 promotor in the Maize genome, barley ubiquitin Mub1 promotor.Corn ubiquitin Ubi-1 promotor has been widely used in the monocotyledonss such as corn, wheat, paddy rice, and paddy rice ubiquitin RUBQ2 promotor also has more application in paddy rice and sugarcane.
Actin promotor nineteen ninety is found in paddy rice first by McElroy of Cornell University etc., belongs to strong constitutive promoter.The Actin promotor acts in the unifacial leaf Gramineae significantly, but the gene regulating function in the plant that contiguous section belongs to is but very undesirable.Therefore, many correlative studys are sought the Actin promotor by other monocotyledonss, and success is found in banana, muskmelon, corn and Arabidopis thaliana successively.The Actin promotor has obtained using more and more widely in the transgenosis of monocotyledons good character owing to the control effect of emphasizing to genetic expression.
Adh-1 promoter regulation ADH (alcohol dehydrogenase) gene, most important to the expression of plant ethanol dehydrogenase under anaerobic environment.The Adh-1 promotor is to monocotyledons particularly cereal grass such as paddy rice, oat and barley, and small part dicotyledons such as tobacco, and the adjusting function of gene improves 10-50 doubly than CaMV (cauliflower mosaic virus CaMV) 35S promoter.The Adh-1 promotor is mainly used in monocotyledons, and is all very limited to the regulating effect that most dicotyledon genes are expressed.
Monocotyledons is angiospermous main monoid, and the Gramineae in the monocotyledons, Liliaceae, Palmae and Rhizoma Arisaematis etc. are very important agricultural crops.The potent promotor of monocotyledons gene can the regulating plant high-efficient expression has the foreign gene of specialized character, and is great to the molecular breeding Research Significance of good crop.
In potent promotor Related Research Domain, find and verified many monocotyledonous promotors.In addition, the efficient promotor such as the potent promotor of some in the dicotyledons such as CsVMV (Cassava Vein Mosaic Virus) promotor, tomato E8 promotor, Resveratrol synthase gene Vst1 promotor also has very strong gene regulating effect in monocotyledons.
Although above-mentioned known monocotyledonous promotor has been arranged, the inventor is by the further investigation to rice genome, a kind of new Monocotyledon promoter is provided, described promotor can be used in destination gene expression in the regulation and control monocotyledons, provides a kind of new instrument and selection for destination gene expression in the research monocotyledons simultaneously.
Summary of the invention
One aspect of the present invention provides the Monocotyledon promoter of nucleotide sequence shown in a kind of SEQ of having ID NO:1.In the present invention, the concrete base sequence length of described promotor is 2927 bases, shown in SEQ ID NO:1:
GTCAGCCCCTTCTCGCTCATTCTGCTCCTCCTCCGGCTGCCAAGTCTCATCCTTCTCTAGATCTTGGCAGCGGAAGCAAGCTAGCATCGGCGGCGCCGTCGGAGGCGGACCTCGCGCAGCTCTCGACGGTGAGGAGGTGCACGGGGAGGAGCGGCCTGCGGTAAGGTGAGCAAGGACCTGATCCATGGCGGCGATGGCGCACTGTGTTGTCCTAGTGCCTGTCCTCGTCATCGCCTCCTCCCTTGTTGCGGCGGCGTACGGGGCGGCCTTGCGTTCGCTCAGTGCCAACGAAGGCAAGGGAGAGGTCAACACCGACACCGCCGTCGACCTCATCACCACCAGCCGAACCTCAACACTGACGCAGGGTGGTCTCGCCAGAGCCGTGGCCATCGTTAGGTTGGTCTCACCTTTGTCTCCTTTTTTTTTAATCCCTGCGAAGTCCACCTTCGATGCCCCGATGCCACGATTGACGTTGCCGCGTCGCTGGTTGATTTCGTCGCTAAGATTTGGAGAAGGAGGCTGTGACGGCCGGAGGAGGAAAAGGAGAGGGGAGGGGCTGACGGGTAGGGTCCTCATTGATTTCCACACGGGCACACCACGATGACATGTCACGTAGGCAAGAGTGACAAATAGTTAAGAAATCATCTATCTCCAGTGGTAAATTATTAAGCGTAAATCTAAGAGGGGTATTTGGATAATTGCCAAATCTAAAAGTAGTAAATAGTTAAATTCCCCAGATTAGATTGGGGATGCGTGAAGTTCAGGCGAGCAAGGCATGTAAACACTGACGCAAATCGCAATCACGCAATACATCACTACAGCAGCAGCGGATCAAAGATCAACAACTCCATTTCCCAAAATTTAATCCATAATGAAAGACATACACTGATATACAAGAGCCTAAACATTTACTAATCATCAACAAAAGATAGCTAGGAATTAATTAAATAATACAGCTACGCCAACAGTGCGTACGTCAGCACCATTCACCCCACGCTTTTTTAGCGCAATGTGTAGCCACCCAGCGGCATTGTCAGAAAAAGTTGAGGGCTTTTTTGGTGCAGCCTTGGAGTGTTGGACTACAACAACTCAGTGCGCGAACGAAAACTTTTAGCGTGTTATATGGATATTAATTTAAAGTATTAAATGTAGACTAATAATAAAAATAAATTATAAATTTTGTCTGAAAACTACGTGACGAATTTATTAAGTCTAGTTAATCTGTTATTAGCAAATATTTACTGTAGCACCATATTGTTAAATCATAGAGCAGTTAGGTTTAAAATATCCGTATCGCAATCTACACGTAATCTGTGTAATTAGTTTTTTTATATATTTAATACTTCATGTACGGATGTGACAGGGTGAAAAATTATTGTTTAGAAACTAAACAGGCTCTAAATTATAAGCACCGCGATATTATCAGAGGTTTGATGATGAAGTCGATGGTCATCATCATAATCTCTCTCAACGTGCCTGCGAGTCTCATTGCCCCCCAACTCCCCCGCCCACGAAATGATTCCCCTGCTCGCCGGCGTATGTCGCAAGCAACACAGCGATCCAACAACGAGCCTCGCGCCCTCGCGGCTCCACCACCTGCGCACCGCCGCATCTGCGTTTTCTCCGTGCTCCGAACCCGACATCCTTCGCCACCGGGTCCGACCGGCCGGCCCGTACCCAAAAAGTCAATTTTCCCGGGCCCCACATGTCAGTGAGGTACCAGCCCATCCCCCGGTCCTAAACAACTTAACCACCGGTGAACAACTGAGCACCGATGCCCCACCTCGCAGAGATAATTATAAAGTATTAGGGCCTCTTTGAATCGCAGGATTGAGAAAACGTAGGAATAGGAAAAACGTAGGATTTTAATAGGAATGTAAGTTTAAAACAGAGGATTGCAAAACACAGGAAAAACATAGGAATGACCGTTTGATTGAACGGTGAGAGAGAGACTTAAAGGAAAGTTAGCAAGAGGTTGAAGCTCTTGCTAAGGGCCTCTTTGAATCGCAGGGTAGAAAAAAACGGAGAAATAAGAAAAACGCAGGATTCTGACAAGAATTGAAGTGTAAAACAGAGGATTGCAAAACACAGGAAAAACACATGAATGGTCGTTTGATTGGAGCGCAGGAAAAACGCAGGAATCGGATAAGAGAGATAGACTCAAAGGAAATTTTCCAAGAGGTTGGAGCTCTTGCTAAATTTCCTCCAAAATCCACATGCAATGTGTCA TTCCATACGAATTTCATAGGATTTGGAAAGCTTCAATCCTTTGAATCAAAGGCCCAAATAGGAAAATTTCCTATAGGATTTAAATCCTATGAAATTTCTACATAAATCCTTTGATTTAAAGGGGCCCTAAATTTCCTCCAAAATCTCTATAGGATTGTCCATTCCATATGAATTTCAAAGAATTGGATAGGATTTAATCCTTTGTTTTAAAGGGCTTTATAGGAAATTTTCCTATAGGATTGAAATACTCTAAAATTCCTGTGTTTTCCTCCAAATCTAAGGAGCCCTTAACCGGGGAAATCGCTACCTAATCTGGCCTAACCGAGGTAATTATCGAGTGGGATTAACCTTCGTTCGGGGCGGTGCTCGGGTCGGGCGTCCAGATCTCGGGCTCGCTGAGCTGTCGCCATCGACCCGGAGTCCCTCCCGCACCAGCTAACCTTTGCTTCCGTTGGAAGCGCCAGCCAGCGCTCGCCGTTGTCACCTGTCACACTGCGTTGCACGGATTATCGTTTTTAATTAATCGTCACCAGATTGTTTTTTTTCTTTCTTTTTGCAAATCTCGCTTCGCTTTTGGAGTGGGAGGAATTAATTGTGGAGTTGGGTAGATCGATCAGGGGCAGCGCGAGCGAGGCCCGGTTGGTAGCAGTCGGTGCGTGTTTGTGCGTGTGCGCGCTGCTTCTGCTTGCATCGTGGTG(SEQ ID NO：1)
In the present invention, the promoter sequence shown in the SEQ ID NO:1 is called promotor BgIosP556, or referred to as the P556 promotor.
This promotor is constitutive promoter, and behind the GUS Coloration experiment, described Rice Callus becomes blueness with the Rice Callus of this promotor and GUS.
Another aspect of the present invention relates to the promotor that has with the sequence of nucleotide sequence complementation shown in the SEQ ID NO:1.
Another aspect of the present invention, what also relate to Monocotyledon promoter shown in the SEQ ID NO:1 has a following variant of being selected from of promoter function:
1) under high stringent condition with the nucleotide sequence of the nucleotide sequence hybridization shown in the SEQ ID NO:1,
2) nucleotide sequence shown in the SEQ ID NO:1 is carried out the nucleotide sequence that the replacement, disappearance, interpolation of one or more bases are modified, and
3) has the nucleotide sequence of at least 90% sequence identity with the nucleotide sequence shown in the SEQ ID NO:1.
" stringency " degree of used condition was classified when typically, " hybridization conditions " was according to measurement hybridization.The stringency degree can be take nucleic acid for example in conjunction with the melting temperature(Tm) (Tm) of mixture or probe as foundation.For example, " maximum stringency " typically occurs in about Tm-5 ℃ (being lower than 5 ℃ of probe Tm); " high stringency " occurs in following about 5-10 ℃ of Tm; " medium stringency " occurs in following about 10-20 ℃ of probe Tm; " low stringency " occurs in following about 20-25 ℃ of Tm.As an alternative, perhaps further, hybridization conditions can take the salt of hybridization or ionic strength conditions and/or one or the washing of repeatedly stringency as foundation.For example, the extremely low stringency of 6 * SSC=; 3 * SSC=is low to moderate medium stringency; The medium stringency of 1 * SSC=; 0.5 the high stringency that waits of * SSC=.On function, can adopt maximum stringency condition to determine and the tight same or near tight same nucleotide sequence of hybridization probe; And adopt high stringency condition to determine to have an appointment 80% or the nucleotide sequence of multisequencing identity more with this probe.
For the application that requires highly selective, typically expectation adopts relatively restricted condition to form crossbred, for example, selects relatively low salt and/or high-temperature condition.Sambrook etc. (Sambrook, J. etc. (1989) molecular cloning, laboratory manual, Cold Spring Harbor Press, Plainview, N.Y.) provide the hybridization conditions that comprises medium stringency and high stringency.
For ease of explanation, comprise for detection of the suitable moderate stringent condition of polynucleotide of the present invention and other multi-nucleotide hybrid: with 5 * SSC, 0.5%SDS, the prewashing of 1.0mM EDTA (pH8.0) solution; Under 50-65 ℃, in 5 * SSC, hybridize and spend the night; Subsequently with contain 2 of 0.1%SDS *, 0.5 * and 0.2 * SSC 65 ℃ of lower each washed twice 20 minutes.It will be appreciated by those skilled in the art that easily to operate the hybridization stringency, as changing saltiness and/or the hybridization temperature of hybridization solution.For example, in another embodiment, the tight hybridization conditions of suitable height comprises above-mentioned condition, and difference is that hybridization temperature is elevated to for example 60-65 ℃ or 65-70 ℃.
In the present invention, described under high stringent condition with the nucleotide sequence of the nucleotide sequence hybridization shown in the SEQ ID NO:1, it has and the same or analogous promoter activity of nucleotide sequence shown in the SEQ ID NO:1.
In the present invention, described nucleotide sequence shown in the SEQ ID NO:1 is carried out the nucleotide sequence that the replacement, disappearance, interpolation of one or more bases are modified, refer to hold at the 5 ' end and/or 3 ' of described nucleotide sequence respectively or simultaneously, and/or sequence inside for example is no more than 2-45, perhaps be no more than 2-30, perhaps be no more than 3-20, perhaps be no more than 4-15, perhaps be no more than 5-10, the replacement, disappearance, the interpolation that perhaps are no more than 6-8 the base that represents with continuous integral number are one by one respectively modified.
In the present invention, describedly nucleotide sequence shown in the SEQ ID NO:1 is carried out the nucleotide sequence that replacement, disappearance, interpolation such as above-mentioned one or more bases modify have and the same or analogous promoter activity of nucleotide sequence shown in the SEQ ID NO:1.
Describe by a kind of polynucleotide, its nucleotide sequence that has for example with the reference nucleotide sequence of SEQ ID NO:1 at least " identity " of tool 95% refer to: in per 100 Nucleotide of the reference nucleotide sequence of SEQ ID NO:1, the nucleotide sequence of these polynucleotide is except containing the difference that reaches 5 Nucleotide, and the nucleotide sequence of these polynucleotide is identical with reference sequences.In other words, in order to obtain the identical polynucleotide of nucleotide sequence and reference nucleotide sequence at least 95%, nearly 5% Nucleotide can be deleted or by another nucleotide substitution in the reference sequences; Maybe some Nucleotide can be inserted in reference sequences, the Nucleotide that wherein inserts can reach reference sequences total nucleotide 5%; Or in some Nucleotide, the combination of have deletion, inserting and replacing, wherein said Nucleotide reach reference sequences total nucleotide 5%.These sudden changes of reference sequences can occur in 5 or 3 terminal positions of reference nucleotide sequence, or any place between these terminal positions, they or be dispersed in separately in the Nucleotide of reference sequences, or be present in the reference sequences with the group of one or more vicinities.
In the present invention, algorithm that be used for to determine sequence identity and sequence similarity percentage ratio is for example BLAST and BLAST 2.0 algorithms, and they are described in respectively (1990) J.Mol.Biol.215:403-410 such as (1977) Nucl.Acid.Res.25:3389-3402 such as Altschul and Altschul.For example adopt described in the document or default parameters, BLAST and BLAST 2.0 can be used for determining nucleotide sequence homology percentage ratio of the present invention.Carrying out software that BLAST analyzes can be obtained by the public by state-run biotechnology information center.
In the present invention, the nucleotide sequence that nucleotide sequence shown in described and the SEQ ID NO:1 has at least 90% sequence identity comprises the polynucleotide sequence substantially same with the disclosed sequence of SEQ ID NO:1, for example when adopting methods described herein (for example adopting the BLAST of canonical parameter to analyze), compare with polynucleotide sequence of the present invention and to contain at least 90% sequence identity, preferred at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or those sequences of higher sequence identity.
In the present invention, the nucleotide sequence of the nucleotide sequence shown in described and the SEQ ID NO:1 with sequence identity of at least 90% has and the same or analogous promoter activity of nucleotide sequence shown in the SEQ ID NO:1.
Among the present invention, described promotor derives from monocotyledons, and is concrete, and described monocotyledons is paddy rice, and for example described paddy rice is Japan fine (Oryza sativa L.ssp.japonica cv.Nipponbare).
Another aspect of the present invention also relates to a kind of recombinant vectors that contains Monocotyledon promoter of the present invention.Described recombinant vectors can be by being inserted into cloning vector with above-mentioned promotor or expression vector obtains.
The cloning vector that is suitable for making up recombinant vectors of the present invention includes but not limited to, such as: pUC18, pUC19, pUC118, pUC119, pMD19-T, pMD20-T, pMD18-T Simple Vecter, pMD19-T Simple Vecter etc.
Be suitable for making up expression vector of the present invention and include but not limited to, such as: pBI121, p13W4, pGEM etc.
In one embodiment of the invention, described recombinant vectors is the p8+P556 recombinant vectors.
Another aspect of the present invention also relates to the reconstitution cell of the described recombinant vectors that contains Monocotyledon promoter of the present invention.Described reconstitution cell can be converted into host cell by the described recombinant vectors that will contain Monocotyledon promoter of the present invention and obtain.
The host cell that is suitable for making up reconstitution cell of the present invention includes but not limited to, such as: agrobacterium tumefaciens cell LBA4404, EHA105, GV3101 etc.
In one embodiment of the invention, described reconstitution cell is restructuring agrobacterium tumefaciens (Agrobacteriumtumefaciens) EHA105-P556.
Another aspect of the present invention also relates to a kind of monocotyledons callus, and described Transformation of Callus has promotor of the present invention.In one embodiment of the invention, described monocotyledons is paddy rice.Described paddy rice includes but not limited to, Japan is fine, in spend 9, in spend 10, in spend 11, the Taibei 309, No. 8, Danjiang River, cloud rice No. 2, Shanyou 63, Shan excellent 608, Feng You 22, Guizhou Province excellent 88, II excellent 416, II excellent 107, II excellent 128, II excellent 718, Zhunliangyou 527, No. 1, river farming, assorted 0152, Anhui rice 88, Anhui rice 90, Anhui rice 92, Anhui rice 94, Anhui rice 96, Anhui rice 185, Anhui rice 187, Anhui rice 189, Anhui rice 191, Anhui rice 193, Anhui rice 195, Anhui rice 197, Anhui rice 199, Anhui rice 201, Anhui rice 203, Anhui rice 205, Anhui rice 207, and Tianjin former 101 etc. (above-mentioned rice varieties all can available from emblem, Anhui good fortune company limited of merchant farmers').In another embodiment of the present invention, described paddy rice is that Japan is fine.
Another aspect of the present invention also relates to a kind of method for preparing promotor of the present invention, comprises the steps:
1) according to the nucleotide sequence shown in the SEQ ID NO:1, design pcr amplification primer pair,
2) take the fine genomic dna of paddy rice Japan as template, use step 1) in designed pcr amplification primer to carrying out pcr amplification.
Those skilled in the art are known, can design corresponding pcr amplification primer pair according to the base complementrity principle according to purpose nucleotide sequence to be amplified.In one embodiment of the invention, described pcr amplification primer is to shown in SEQ ID NO:2 and SEQ ID NO:3.
Another aspect of the present invention also relates to a kind of method of regulating and control destination gene expression in the monocotyledons, and described method comprises the step with the callus of Monocotyledon promoter transforming monocots of the present invention.In one embodiment of the invention, the trans-utilization of described monocotyledons callus contain the reconstitution cell of Monocotyledon promoter of the present invention.In a specific embodiments of the present invention, described monocotyledonous callus is Rice Callus, and particularly, described paddy rice is that Japan is fine.
In the present invention, can adopt the plant gene transformation technology that goal gene is inserted in the Plant Genome, comprise agrobacterium mediation converted, virus-mediated conversion, microinjection, particle bombardment, via Particle Bombardment Transformation and electroporation etc.This area is known, and agriculture bacillus mediated gene transformation often is used to the gene transformation of monocotyledons and dicotyledons, but other transformation technology also can be used for monocotyledonous gene transformation of the present invention.Certainly, the another kind of method that is suitable for transforming monocots of the present invention is particle bombardment (micro-gold or tungsten particle attached bag are covered the DNA of conversion) embryo callus or embryo's exploitation.The method of the transforming monocots that can also adopt in addition, is protoplast transformation.After the gene transformation, adopt general method to screen and regenerate and be integrated with the plant of expressing the unit.
Among the present invention, can utilize the described monocotyledons of described Monocotyledon promoter regulation and control destination gene expression to include but not limited to, such as: paddy rice, wheat, corn, millet, sugarcane, Chinese sorghum, barley etc.
Another aspect of the present invention also relates to Monocotyledon promoter of the present invention is regulated and control destination gene expression in monocotyledons application.In one embodiment of the invention, utilizing the goal gene of promoter regulation of the present invention is GUS.In one embodiment of the invention, described monocotyledons is paddy rice, and concrete described paddy rice is that Japan is fine.
For realizing the purpose of above-mentioned regulation and control destination gene expression, promotor of the present invention can be used with the form of single copy and/or multiple copied, also can with promotor coupling well known in the prior art.
Another aspect of the present invention relates to the purposes of promotor of the present invention in rice breeding.Described paddy rice spends 9 for Japan is fine, middle, middlely spend 10, middlely spend 11, the Taibei 309, No. 8, Danjiang River, cloud rice No. 2, Shanyou 63, Shan are excellent 608, Feng You 22, the Guizhou Province is excellent 88, II is excellent 416, II is excellent 107, II is excellent 128, II is excellent 718, Zhunliangyou 527, No. 1, river farming, assorted 0152, Anhui rice 88, Anhui rice 90, Anhui rice 92, Anhui rice 94, Anhui rice 96, Anhui rice 185, Anhui rice 187, Anhui rice 189, Anhui rice 191, Anhui rice 193, Anhui rice 195, Anhui rice 197, Anhui rice 199, Anhui rice 201, Anhui rice 203, Anhui rice 205, Anhui rice 207, and Tianjin former 101.In one embodiment of the invention, described paddy rice is that Japan is fine.
It is sub as the instrument start-up of monocotyledons, especially Transgenic Rice that promotor of the present invention can become a kind of new promotor, low expressing gene transformation seedlings screens in order to carry out, the breeding research of plant flower organ abortion equimolecular facilitates, thereby shortens greatly the seed selection time of improved seeds.Promotor of the present invention can be widely used in monocotyledonss such as cultivating paddy rice, wheat, corn, millet, sugarcane, Chinese sorghum, barley.
The beneficial effect of the invention
In order to seek new Monocotyledon promoter, be used for regulation and control monocotyledons destination gene expression, provide new instrument and selection for the genetic expression of research monocotyledons simultaneously, the inventor obtains by information biology research, and adopts biological experiment to verify the function of described promotor BgIosP556.Particularly, described promotor can be regulated and control the gus gene expression in paddy rice.
Description of drawings
Fig. 1 is the pcr amplification detected result of promotor BgIosP556, wherein, swimming lane 1:200bp DNA LadderMarker, swimming lane 2:PCR amplified production, swimming lane 3:1kb DNA Ladder Marker, the size of the band of the Ladder Marker that the numeral 2000 on its right side and 3000 expressions are pointed, unit all is bp.
Fig. 2 is for the pCAMBIA-1301 plasmid synoptic diagram that makes up the p8 plasmid.
Fig. 3 is the synoptic diagram of multiple clone site and GUS Sequence in the p8 plasmid synoptic diagram.
Fig. 4 is p8 plasmid synoptic diagram.
Fig. 5 is the GUS coloration result of the Rice Callus through transforming.Wherein, the Rice Callus (left side) that is transformed by the restructuring agrobacterium tumefaciens p8+P556 with promotor P556 sequence of the present invention presents blueness after GUS dyeing; Rice Callus (contrast, the right side) color after GUS dyeing without the restructuring agrobacterium tumefaciens p8 plasmid of promoter sequence of the present invention does not change.
Embodiment
Below in conjunction with embodiment embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person among the embodiment, according to the described technology of the document in this area or condition (such as works such as reference J. Pehanorm Brookers, " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
The pcr amplification of embodiment 1P556 promoter fragment and the structure of pMD18-T+P556 recombinant vectors
Pcr amplification
Use plant genome DNA to extract test kit (TIANGEN plant genes group DNA extraction test kit; catalog number (Cat.No.): DP320-02) extract the fine seed of paddy rice Japan and (be preserved in Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center on December 18th, 2009; it is Chinese Typical Representative culture collection center (CCTCC); deposit number is CCTCCNo:P200910) genomic dna; according to the sequence of this promotor in the fine gDNA of paddy rice Japan; design one couple of PCR specificity amplification primer (upstream primer F1 at head and the tail respectively; add restriction enzyme site EcoR I and protection base; downstream primer R1 adds restriction enzyme site Sbf I and protection base).Take the fine gDNA of the paddy rice of said extracted Japan as template, high-fidelity Ex Taq
TM(TaKaRa, DRR100B) polysaccharase carries out pcr amplification.As shown in table 1.
The PCR system of table 1 gene promoter amplification
The pcr amplification program is: 94 ℃ of denaturation 5mi n, and then with 94 ℃ of sex change 45s, 55 ℃ of annealing 50s, 72 ℃ are extended 90s, carry out 35 reaction cycle, and last 72 ℃ are extended 7min.
Wherein, upstream primer F1:GgaattcGTCAGCCCCTTCTCGCTCA (SEQ ID NO:2), wherein lowercase represents EcoR I restriction enzyme site.
Downstream primer R1:TGcctgcaggCACCACGATGCAAGCAGAAGC (SEQ ID NO:3), wherein lowercase represents Sbf I restriction enzyme site.
Pcr amplification product separates through 1.0% agarose gel electrophoresis, obtains size and is the band of 2944bp (Fig. 1), uses TIANGEN sepharose DNA to reclaim test kit (catalog number (Cat.No.): DP209-03) carry out purifying and reclaim.
The structure of pMD18-T+P556 recombinant vectors
Pcr amplification product obtained above is carried out T/A clone (pMD18-T plasmid, TaKaRa, D103A), transform intestinal bacteria, picking positive colony order-checking (shown in SEQ ID NO:4) proves accurately.
Wherein, T/A clone's condition of contact is as follows:
T/A linked system: 10 μ l
pMD18-T 1μl
2*solution I 5μl
Pcr amplification product (recovery Insert Fragment) 10ng～20ng, fixed according to its concentration
DdH
2O polishing to 10 μ l
(the new sesame in Ningbo SDC-6) the middle connection more than the 8h, obtains the pMD18-T+P556 recombinant vectors at the energy-conserving intelligent thermostatic bath in 16 ℃.To transform as follows intestinal bacteria through the product after the above-mentioned connection:
(Dong Yang of Kunming Institute of Zoology, Chinese Academy of Sciences provides to take out the competent cell 100 μ l DH5 α that prepare according to Calcium Chloride Method shown in " molecular cloning experiment guide " (third edition, Science Press) from Ultralow Temperature Freezer; Perhaps can be from for example: Shanghai be given birth to the worker and is buied), after melting on ice, add the as above connection product of gained of 10 μ l, it is the pMD18-T+P556 recombinant vectors, stir evenly gently ice bath 30min, 42 ℃ of heat shock 60s, ice bath 5min, the SOC substratum (concrete prescription sees " molecular cloning experiment guide ", the third edition, Science Press for details) that adds 4 ℃ of precoolings of 600 μ l, 37 ℃ of 220rpm recovery 45min, the centrifugal 30s of 8000rpm removes supernatant, leaves and takes 150 μ l, with the mixture after the remaining resuspended precipitation of 150 μ l supernatants, blow gently evenly, granulated glass sphere coating LB (kantlex) is dull and stereotyped, and (concrete prescription sees " molecular cloning experiment guide ", the third edition for details, Science Press), be inverted cultivation 16h～24h for 37 ℃.Acquisition contains the recombination bacillus coli of pMD18-T+P556 cloning vector, called after DH5 α-P556.The large Gene science limited-liability company of Shenzhen China checks order to the P556 in the pMD18-T+P556 cloning vector, and the result is as follows:
G
GAATTC 
TTCTGCTCCTCCTCCGGCTGCCAAGTCTCATCCTTCTCTAGATCTTGGCAGCGGAAGCAAGCTAGCATCGGCGGCGCCGTCGGAGGCGGACCTCGCGCAGCTCTCGACGGTGAGGAGGTGCACGGGGAGGAGCGGCCTGCGGTAAGGTGAGCAAGGACCTGATCCATGGCGGCGATGGCGCACTGTGTTGTCCTAGTGCCTGTCCTCGTCATCGCCTCCTCCCTTGTTGCGGCGGCGTACGGGGCGGCCTTGCGTTCGCTCAGTGCCAACGAAGGCAAGGGAGAGGTCAACACCGACACCGCCGTCGACCTCATCACCACCAGCCGAACCTCAACACTGACGCAGGGTGGTCTCGCCAGAGCCGTGGCCATCGTTAGGTTGGTCTCACCTTTGTCTCCTTTTTTTTTAATCCCTGCGAAGTCCACCTTCGATGCCCCGATGCCACGATTGACGTTGCCGCGTCGCTGGTTGATTTCGTCGCTAAGATTTGGAGAAGGAGGCTGTGACGGCCGGAGGAGGAAAAGGAGAGGGGAGGGGCTGACGGGTAGGGTCCTCATTGATTTCCACACGGGCACACCACGATGACATGTCACGTAGGCAAGAGTGACAAATAGTTAAGAAATCATCTATCTCCAGTGGTAAATTATTAAGCGTAAATCTAAGAGGGGTATTTGGATAATTGCCAAATCTAAAAGTAGTAAATAGTTAAATTCCCCAGATTAGATTGGGGATGCGTGAAGTTCAGGCGAGCAAGGCATGTAAACACTGACGCAAATCGCAATCACGCAATACATCACTACAGCAGCAGCGGATCAAAGATCAACAACTCCATTTCCCAAAATTTAATCCATAATGAAAGACATACACTGATATACAAGAGCCTAAACATTTACTAATCATCAACAAAAGATAGCTAGGAATTAATTAAATAATACAGCTACGCCAACAGTGCGTACGTCAGCACCATTCACCCCACGCTTTTTTAGCGCAATGTGTAGCCACCCAGCGGCATTGTCAGAAAAAGTTGAGGGCTTTTTTGGTGCAGCCTTGGAGTGTTGGACTACAACAACTCAGTGCGCGAACGAAAACTTTTAGCGTGTTATATGGATATTAATTTAAAGTATTAAATGTAGACTAATAATAAAAATAAATTATAAATTTTGTCTGAAAACTACGTGACGAATTTATTAAGTCTAGTTAATCTGTTATTAGCAAATATTTACTGTAGCACCATATTGTTAAATCATAGAGCAGTTAGGTTTAAAATATCCGTATCGCAATCTACACGTAATCTGTGTAATTAGTTTTTTTATATATTTAATACTTCATGTACGGATGTGACAGGGTGAAAAATTATTGTTTAGAAACTAAACAGGCTCTAAATTATAAGCACCGCGATATTATCAGAGGTTTGATGATGAAGTCGATGGTCATCATCATAATCTCTCTCAACGTGCCTGCGAGTCTCATTGCCCCCCAACTCCCCCGCCCACGAAATGATTCCCCTGCTCGCCGGCGTATGTCGCAAGCAACACAGCGATCCAACAACGAGCCTCGCGCCCTCGCGGCTCCACCACCTGCGCACCGCCGCATCTGCGTTTTCTCCGTGCTCCGAACCCGACATCCTTCGCCACCGGGTCCGACCGGCCGGCCCGTACCCAAA AAGTCAATTTTCCCGGGCCCCACATGTCAGTGAGGTACCAGCCCATCCCCCGGTCCTAAACAACTTAACCACCGGTGAACAACTGAGCACCGATGCCCCACCTCGCAGAGATAATTATAAAGTATTAGGGCCTCTTTGAATCGCAGGATTGAGAAAACGTAGGAATAGGAAAAACGTAGGATTTTAATAGGAATGTAAGTTTAAAACAGAGGATTGCAAAACACAGGAAAAACATAGGAATGACCGTTTGATTGAACGGTGAGAGAGAGACTTAAAGGAAAGTTAGCAAGAGGTTGAAGCTCTTGCTAAGGGCCTCTTTGAATCGCAGGGTAGAAAAAAACGGAGAAATAAGAAAAACGCAGGATTCTGACAAGAATTGAAGTGTAAAACAGAGGATTGCAAAACACAGGAAAAACACATGAATGGTCGTTTGATTGGAGCGCAGGAAAAACGCAGGAATCGGATAAGAGAGATAGACTCAAAGGAAATTTTCCAAGAGGTTGGAGCTCTTGCTAAATTTCCTCCAAAATCCACATGCAATGTGTCATTCCATACGAATTTCATAGGATTTGGAAAGCTTCAATCCTTTGAATCAAAGGCCCAAATAGGAAAATTTCCTATAGGATTTAAATCCTATGAAATTTCTACATAAATCCTTTGATTTAAAGGGGCCCTAAATTTCCTCCAAAATCTCTATAGGATTGTCCATTCCATATGAATTTCAAAGAATTGGATAGGATTTAATCCTTTGTTTTAAAGGGCTTTATAGGAAATTTTCCTATAGGATTGAAATACTCTAAAATTCCTGTGTTTTCCTCCAAATCTAAGGAGCCCTTAACCGGGGAAATCGCTACCTAATCTGGCCTAACCGAGGTAATTATCGAGTGGGATTAACCTTCGTTCGGGGCGGTGCTCGGGTCGGGCGTCCAGATCTCGGGCTCGCTGAGCTGTCGCCATCGACCCGGAGTCCCTCCCGCACCAGCTAACCTTTGCTTCCGTTGGAAGCGCCAGCCAGCGCTCGCCGTTGTCACCTGTCACACTGCGTTGCACGGATTATCGTTTTTAATTAATCGTCACCAGATTGTTTTTTTTCTTTCTTTTTGCAAATCTCGCTTCGCTTTTGGAGTGGGAGGAATTAATTGTGGAGTTGGGTAGATCGATCAGGGGCAGCGCGAGCGAGGCCCGGTTGGTAGCAGTCGGTGCGTGTTTGTGCGTGTGCGCGCT
CCTGCAGGCA(SEQ ID NO：4)
Above sequence in, with underscore be restriction enzyme site (EcoRI and Sbf I), with square frame is primer sequence.
Sequencing result shows that the P556 promoter sequence is correct in the pMD18-T+P556 cloning vector of acquisition.
The structure of embodiment 2p8+P556 recombinant vectors
(catalog number (Cat.No.): operational manual DP103-03), the conversion that makes up from embodiment 1 have the cloning vector pMD18-T+P556 that extracts bacillus coli DH 5 alpha-P556 of promotor P556 with P556 promoter sequence of the present invention according to the little extraction reagent kit of the common plasmid of TIANGEN; Carry out enzyme with corresponding restriction enzyme EcoR I (NEB) and Sbf I (NEB) behind the purifying and cut, reclaim corresponding promotor Insert Fragment, and connect with the carrier large fragment that the p8 plasmid reclaims after with identical digestion with restriction enzyme respectively.
Gained is connected product p8+P556 recombinant vectors to be transformed according to " molecular cloning the experiment guide " (third edition, the competent cell DH5 α of the preparation of Calcium Chloride Method Science Press), be inverted for 37 ℃ and cultivate 16～24h, after son to be transformed grew bacterium colony, the picking mono-clonal carried out the PCR detection and enzyme is cut evaluation.
The p8 plasmid construction
Employed p8 plasmid is that (Dong Yang of Kunming Institute of Zoology, Chinese Academy of Sciences provides by the pCAMBIA-1301 plasmid among the present invention; Perhaps can buy from for example auspicious Gene Tech. Company Limited of Shanghai state, the primary source of the pCAMBIA-1301 plasmid of the said firm is The CAMBIA Bios (biological open source) Licensee, Australia) transform in the following manner and make up, be described as follows:
With Kpn I/Nco I (NEB) double digestion plasmid pCAMBIA-1301 (referring to Fig. 2), reclaim large fragment.According to synthetic following sequence: the GGTACCAAGCTTACTAGTCCTGCAGGTCTAGAGGATCCGTCGACCATGG (SEQ ID NO:5) (restriction enzyme site that comprises is Kpn I/HindIII/Spe I/Sbf I/Pst I/Xba I/BamH I/Sal I/Nco I) of the restriction endonuclease sites that adopts, with reclaiming behind the Kpn I/Nco I double digestion, (Dong Yang of Kunming Institute of Zoology, Chinese Academy of Sciences provides to be connected the rear t of conversion op10 cell with the above-mentioned large fragment that reclaims; Perhaps can be from for example: Suo Laibao Science and Technology Ltd. in Beijing buys).Screen transformant with primer GCTTCCGGCTCGTATGTTGT (SEQ ID NO:6)/GAGTCGTCGGTTCTGTAAC (SEQ ID NO:7), by the PCR detection method, be the transformant (referring to Fig. 4) that the transformant of 350bp is the p8 plasmid that contains multiple clone site that needs make up and GUS sequence with amplified fragments.
Length 2353 bases of the multiple clone site in the described p8 plasmid and GUS sequence, shown in SEQ ID NO:8 (referring to Fig. 3):
GTTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTAT
GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTAC
GAGCTC 
AAGCTT 
C G 
GGATCC 
C 
(SEQ IDNO：8)
As above constructed p8 plasmid among the present invention shown in the sequence, EcoR I/Sac I/KpnI/Hind III/Spe I/Sbf I/Pst I/Xba I/BamH I/Sal I/Nco I restriction enzyme site in its multiple clone site is respectively with adding frame and underscore represents, the used primer GCTTCCGGCTCGTATGTTGT/GAGTCGTCGGTTCTGTA AC (being SEQ ID NO:6 and 7) of screening transformant represents with double underline, the GUS sequence represents that with italic its intron sequences adds shading with italic respectively and illustrates.
The structure of recombinant vectors p8+P556
According to restriction enzyme EcoR I (NEB) and Sbf I (NEB) operation instructions, according to resulting cloning vector pMD18-T+P556 in the following condition Processing Example 1, and the p8 plasmid that makes up as mentioned above.
Wherein, the enzyme tangent condition of cloning vector pMD18-T+P556 and p8 plasmid is as follows:
Enzyme is cut system: 50 μ l
Sterilized water 34.8 μ l
10*buffer H 5μl
EcoR I 0.1μl(10U)
Sbf I 0.1μl(10U)
Cloning vector pMD1g-T+P556 or p8 plasmid 10 μ l (＜1000ng)
Use TIANGEN sepharose DNA to reclaim test kit (catalog number (Cat.No.): DP209-03) reclaim respectively the p8 plasmid of cutting through enzyme, and promotor P556 Insert Fragment, according to T4 ligase enzyme (TaKaRa, D2011A) operation instructions, connect according to following condition:
Linked system: 10 μ l
10*T4buffer 1μl
The promotor P556 Insert Fragment 10-20ng that reclaims, fixed according to its concentration
Sterilized water polishing to 9.5 μ l
T4ligase(TaKaRa，D2011A) 0.5μl
T4buffer melts on ice, the about 20ng of p8 plasmid vector add-on after enzyme is cut, and the P556 fragment among the present invention adds 10-20ng.(the new sesame in Ningbo is SDC-6) more than the middle connection 8h at the energy-conserving intelligent thermostatic bath in 16 ℃.
The competent cell DH5 that 100 μ l Calcium Chloride Method are made takes out from Ultralow Temperature Freezer, after melting, adds the connection product above the 10 μ l on ice, stir evenly gently ice bath 30min, 42 ℃ of heat shock 60s, ice bath 5min adds the SOC of 4 ℃ of precoolings of 600 μ l, 37 ℃ of lower 220rpm recovery 45min, the centrifugal 30s of 8000rpm, remove supernatant, leave and take 150 μ l, blow gently even, granulated glass sphere coating LB (kan) is inverted for 37 ℃ and cultivates 16～24h.Obtain recombinant vectors p8+P556.
Detect as primer carries out PCR to gained recombinant vectors p8+P556 take F1 (SEQ ID NO:2) and R1 (SEQ ID NO:3) respectively, to contain required promotor P556 among the conclusive evidence gained recombinant vectors p8+P556.Cut screening by EcoR I/SbfI enzyme and contain recombinant vectors p8+P556 transformant.
The preparation of embodiment 3 restructuring agrobacterium tumefaciens EHA105-P556 cells
With p8+P556 recombinant vectors and the p8 plasmid in contrast of method structure transform respectively according to " molecular cloning the experiment guide " (third edition as described in Example 2, the agrobacterium tumefaciens EHA105 Agrobacterium tumefaciens EHA105 of the method for calcium chloride Science Press) preparation (was preserved in Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center on December 24th, 2009, it is Chinese Typical Representative culture collection center (CCTCC), deposit number is CCTCC No:M 209315) competent cell, concrete grammar is as follows:
Agrobacterium tumefaciens competent cell EHA105 is taken out in Ultralow Temperature Freezer, as for thawing on ice.After the thawing, the p8+P556 recombinant vectors and p8 plasmid and the p8 empty carrier in contrast that add respectively 5 μ l, mixing gently, ice bath 10min, put into the freezing 5min of liquid nitrogen, 37 ℃ of 5min that thaw, the LB liquid nutrient medium that adds 800 μ l normal temperature, 28 ℃ of 160rpm recovery 3h, the centrifugal 30s of 8000rpm sucks supernatant, staying 200 μ l blows even, coat and be added with (50mg/l Kan, 10mg/l Rif specifically fill a prescription referring to table 4) on the two anti-YM culture medium flat plates of kan-rif (kantlex-Rifampin).Be inverted for 28 ℃ and cultivated 2-3 days.
Carry out PCR detects and cuts the screening transformant by EcoRI/Sbf I enzyme take F1 (SEQ ID NO:2) and R1 (SEQ ID NO:3) as primer.
What pcr amplification went out that about 2944bp band and enzyme cut out about 2937bp band is the restructuring agrobacterium tumefaciens of recombinant vectors p8+P556.
Among the present invention, according to the restructuring Agrobacterium with recombinant vectors p8+P556 that obtains such as above-mentioned method, called after restructuring agrobacterium tumefaciens EHA105-P556.
According to the method for the invention, the contrast restructuring Agrobacterium with the p8 plasmid that obtains, called after restructuring agrobacterium tumefaciens EHA105-p8.
The inducing and transforming of embodiment 4 Rice Callus
Inducing paddy rice callus in accordance with the following steps, and transform described callus with restructuring agrobacterium tumefaciens EHA105-P556 and restructuring agrobacterium tumefaciens EHA105-p8 respectively.
1) the fine seed of paddy rice Japan is shelled, 70% ethanol surface sterilization 30s, then with the clorox sterilization 30min of available chlorine 1.5%, during acutely shake, clean 5 times with aqua sterilisa after the sterilization; Seed after the sterilization is placed on the N6D substratum (specifically filling a prescription referring to table 2), seal with sealed membrane; 29.5 ℃ illumination cultivation 3-4 week;
2) choose the callus (yellow-white, drying, diameter 1-3mm) of active growth, 29.5 ℃ of illumination cultivation are 3 days on new N6D substratum;
3) the constructed single bacterium colony of restructuring agrobacterium tumefaciens (restructuring agrobacterium tumefaciens EHA105-P556 or restructuring agrobacterium tumefaciens EHA105-p8) of picking such as embodiment 3 respectively, in adding microbiotic (50mg/l Kan, upper streak culture 3 days of YM substratum 10mg/lRif) (specifically filling a prescription referring to table 3, table 4), 28 ℃ of culture temperature; The above-mentioned restructuring agrobacterium tumefaciens of scraping places the AS (Acetosyringone that has added 30 μ l 100mM respectively, Syringylethanone) in the 30ml AAM substratum (specifically filling a prescription referring to table 5), gentle resuspended described restructuring agrobacterium tumefaciens cell (restructuring agrobacterium tumefaciens EHA105-P556 or restructuring agrobacterium tumefaciens EHA105-p8);
4) callus with succeeding transfer culture places the sterilization culture dish; To pour in the culture dish such as the restructuring agrobacterium tumefaciens suspension of step 3 preparation, callus will be immersed wherein 15min;
5) outwell restructuring agrobacterium tumefaciens suspension, callus is sopped up unnecessary liquid with sterilization thieving paper; On N6-AS substratum (prescription is referring to table 6), put a sterilization filter paper, add the AAM substratum of 1ml such as the above-mentioned AS of containing, callus is transferred on the filter paper; The sealing culture dish, 28 ℃ of dark 48-60h that cultivate;
6) infected callus is placed the 50ml sterile tube, shake cleaning with aqua sterilisa, until supernatant liquor becomes clarification; Callus is soaked in the sterilized water that contains 500mg/l Pyocianil (Cb) to kill the restructuring agrobacterium tumefaciens; Remove moisture unnecessary on the callus with sterilization thieving paper, then transfer them on the N6-AS substratum that contains 1mg/l hygromycin B (HmB) and 50mg/l Carb; Seal culture dish with sealed membrane, 29.5 ℃ of illumination cultivation 3-4 weeks.
The expression of GUS in embodiment 5 Rice Callus
For detecting the expression through goal gene GUS in the Rice Callus of embodiment 4 described conversions, according to Chen S Y etc. at Journal of Integrative Plant Biology, 2008,50 (6): the described method of 742-751, dye to the Rice Callus that transforms with restructuring agrobacterium tumefaciens EHA105-P556 or restructuring Agrobacterium EHA105-p8 respectively.
The prescription of GUS staining fluid (1ml): 610 μ l 0.2M Na
2HPO
4Solution (pH=7.0); 390 μ l 0.2M NaH
2PO
4Solution and 10 μ l 0.1M X-gluc.
The Rice Callus that transforms with restructuring agrobacterium tumefaciens EHA105-P556 or restructuring agrobacterium tumefaciens EHA105-p8 respectively is immersed in the GUS staining fluid, 37 ℃ of insulations are blue to occurring, Taking Pictures recording, the result as shown in Figure 5, present blueness after the Rice Callus of the restructuring Agrobacterium-Mediated Transformation of the p8+P556 recombinant vectors through containing promotor (Fig. 5 is left) is dyed, Rice Callus (in contrast, Fig. 5 is right) color after GUS dyeing of the p8 plasmid restructuring Agrobacterium-Mediated Transformation through not containing promotor does not change.The result shows that P556 promotor of the present invention is expressed gus gene has regulating and controlling effect.
Employed relevant culture medium prescription is described as follows in the embodiment of the invention:
Below in the relevant substratum alleged " conventional sterilization " refer to the sterilization of following condition: 121 ℃ of lower vapor sterilizations 20 minutes.
Table 2N6D substratum
Regulate pH value to 5.8 with 1N potassium hydroxide, according to a conventional method sterilization after the sealing.
N
6Macro mother liquor (20X): saltpetre 56.60g, potassium primary phosphate 8.00g, ammonium sulfate 9.26g, sal epsom 3.70g, calcium chloride 3.32g, distilled water is settled to 1L, and 4 ℃ save backup.
N
6Micro mother liquor (1000X): potassiumiodide 0.80g, boric acid 1.60g, manganous sulfate 3.33g, zinc sulfate 1.50g, distilled water is settled to 1L, and 4 ℃ save backup.
Molysite (Fe
2EDTA) stock solution (100X): with 3.73g b diammonium disodium edta (Na
2EDTA2H
2O) and 2.78g FeSO
4.7H
2O dissolves respectively, mixes and usefulness.Be settled to 1000ml with distilled water, 70 ℃ of temperature are bathed 2h, cool off rear 4 ℃ of preservations and are no more than 1 month.
N
6VITAMIN stock solution (1000X): vitamins B
10.10g, vitamins B
60.05g, nicotinic acid 0.05g, glycine 0.20g, adding distil water is settled to 100ml, filtration sterilization, 4 ℃ of preservations were no more than for 1 week.
Table 3YM liquid nutrient medium (containing 50mg/L Kan, 10mg/L Rif):
Conventional sterilization adds after being cooled to room temperature
Table 4YM solid medium (containing 50mg/L Kan, 10mg/L Rif):
Table 5AAM substratum
Add 1N potassium hydroxide and regulate pH value to 5.2, conventional sterilization.
AAM macro (10X): 2.5g magnesium sulfate heptahydrate (MgSO
47H
2O), 1.5g Calcium dichloride dihydrate (CaCl
22H
2O), 1.33g sodium dihydrogen phosphate dihydrate (NaH
2PO
4.2H
2O), distilled water is settled to 1L, and 4 ℃ save backup.
The single water manganous sulfate of AAM micro (100X): 0.7g (MnSO
4H
2O), 0.2g Zinc Sulphate Heptahydrate (ZnSO
47H
2O), 0.075g potassiumiodide (KI), 0.3g boric acid (H
3BO
3), 25mg Sodium Molybdate Dihydrate (Na
2MoO
4.2H
2O), 2.5mg cupric sulfate pentahydrate (CuSO
4.5H
2O), 2.5mg CoCL2 6H2O (CoCl
2.6H
2O), distilled water is settled to 1L, and 4 ℃ save backup.
AAM organic (1000X): 0.75g glycine (Glycine), 0.1g nicotinic acid (Nicotinic acid), 0.1g VB
6(Pyridoxine), 1g VB
1(Thiamine), distilled water is settled to 100ml, and 4 ℃ save backup.
Molysite (Fe
2EDTA) stock solution (100X): see Table 2.
Table 6N6-AS is substratum altogether
Transfer pH to 5.2.
N
6Macro mother liquor (20X), N
6Micro mother liquor (1000X), molysite (Fe
2EDTA) stock solution (100X), N
6VITAMIN stock solution (1000X): all see Table 2.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
SEQUENCE LISTING
＜110〉Shenzhen Huada Genetic Technology Co., Ltd
＜120〉a kind of promotor BgIosP556, Preparation Method And The Use
<130>P2009-1-0014
<160>8
<170>PatentIn version 3.5
<210>1
<211>2927
<212>DNA
＜213〉paddy rice Japan fine (Oryza sativa L.ssp.japonica cv.)
<400>1
gtcagcccct tctcgctcat tctgctcctc ctccggctgc caagtctcat ccttctctag 60
atcttggcag cggaagcaag ctagcatcgg cggcgccgtc ggaggcggac ctcgcgcagc 120
tctcgacggt gaggaggtgc acggggagga gcggcctgcg gtaaggtgag caaggacctg 180
atccatggcg gcgatggcgc actgtgttgt cctagtgcct gtcctcgtca tcgcctcctc 240
ccttgttgcg gcggcgtacg gggcggcctt gcgttcgctc agtgccaacg aaggcaaggg 300
agaggtcaac accgacaccg ccgtcgacct catcaccacc agccgaacct caacactgac 360
gcagggtggt ctcgccagag ccgtggccat cgttaggttg gtctcacctt tgtctccttt 420
ttttttaatc cctgcgaagt ccaccttcga tgccccgatg ccacgattga cgttgccgcg 480
tcgctggttg atttcgtcgc taagatttgg agaaggaggc tgtgacggcc ggaggaggaa 540
aaggagaggg gaggggctga cgggtagggt cctcattgat ttccacacgg gcacaccacg 600
atgacatgtc acgtaggcaa gagtgacaaa tagttaagaa atcatctatc tccagtggta 660
aattattaag cgtaaatcta agaggggtat ttggataatt gccaaatcta aaagtagtaa 720
atagttaaat tccccagatt agattgggga tgcgtgaagt tcaggcgagc aaggcatgta 780
aacactgacg caaatcgcaa tcacgcaata catcactaca gcagcagcgg atcaaagatc 840
aacaactcca tttcccaaaa tttaatccat aatgaaagac atacactgat atacaagagc 900
ctaaacattt actaatcatc aacaaaagat agctaggaat taattaaata atacagctac 960
gccaacagtg cgtacgtcag caccattcac cccacgcttt tttagcgcaa tgtgtagcca 1020
cccagcggca ttgtcagaaa aagttgaggg cttttttggt gcagccttgg agtgttggac 1080
tacaacaact cagtgcgcga acgaaaactt ttagcgtgtt atatggatat taatttaaag 1140
tattaaatgt agactaataa taaaaataaa ttataaattt tgtctgaaaa ctacgtgacg 1200
aatttattaa gtctagttaa tctgttatta gcaaatattt actgtagcac catattgtta 1260
aatcatagag cagttaggtt taaaatatcc gtatcgcaat ctacacgtaa tctgtgtaat 1320
tagttttttt atatatttaa tacttcatgt acggatgtga cagggtgaaa aattattgtt 1380
tagaaactaa acaggctcta aattataagc accgcgatat tatcagaggt ttgatgatga 1440
agtcgatggt catcatcata atctctctca acgtgcctgc gagtctcatt gccccccaac 1500
tcccccgccc acgaaatgat tcccctgctc gccggcgtat gtcgcaagca acacagcgat 1560
ccaacaacga gcctcgcgcc ctcgcggctc caccacctgc gcaccgccgc atctgcgttt 1620
tctccgtgct ccgaacccga catccttcgc caccgggtcc gaccggccgg cccgtaccca 1680
aaaagtcaat tttcccgggc cccacatgtc agtgaggtac cagcccatcc cccggtccta 1740
aacaacttaa ccaccggtga acaactgagc accgatgccc cacctcgcag agataattat 1800
aaagtattag ggcctctttg aatcgcagga ttgagaaaac gtaggaatag gaaaaacgta 1860
ggattttaat aggaatgtaa gtttaaaaca gaggattgca aaacacagga aaaacatagg 1920
aatgaccgtt tgattgaacg gtgagagaga gacttaaagg aaagttagca agaggttgaa 1980
gctcttgcta agggcctctt tgaatcgcag ggtagaaaaa aacggagaaa taagaaaaac 2040
gcaggattct gacaagaatt gaagtgtaaa acagaggatt gcaaaacaca ggaaaaacac 2100
atgaatggtc gtttgattgg agcgcaggaa aaacgcagga atcggataag agagatagac 2160
tcaaaggaaa ttttccaaga ggttggagct cttgctaaat ttcctccaaa atccacatgc 2220
aatgtgtcat tccatacgaa tttcatagga tttggaaagc ttcaatcctt tgaatcaaag 2280
gcccaaatag gaaaatttcc tataggattt aaatcctatg aaatttctac ataaatcctt 2340
tgatttaaag gggccctaaa tttcctccaa aatctctata ggattgtcca ttccatatga 2400
atttcaaaga attggatagg atttaatcct ttgttttaaa gggctttata ggaaattttc 2460
ctataggatt gaaatactct aaaattcctg tgttttcctc caaatctaag gagcccttaa 2520
ccggggaaat cgctacctaa tctggcctaa ccgaggtaat tatcgagtgg gattaacctt 2580
cgttcggggc ggtgctcggg tcgggcgtcc agatctcggg ctcgctgagc tgtcgccatc 2640
gacccggagt ccctcccgca ccagctaacc tttgcttccg ttggaagcgc cagccagcgc 2700
tcgccgttgt cacctgtcac actgcgttgc acggattatc gtttttaatt aatcgtcacc 2760
agattgtttt ttttctttct ttttgcaaat ctcgcttcgc ttttggagtg ggaggaatta 2820
attgtggagt tgggtagatc gatcaggggc agcgcgagcg aggcccggtt ggtagcagtc 2880
ggtgcgtgtt tgtgcgtgtg cgcgctgctt ctgcttgcat cgtggtg 2927
<210>2
<211>26
<212>DNA
＜213〉artificial sequence
<400>2
ggaattcgtc agccccttct cgctca 26
<210>3
<211>31
<212>DNA
＜213〉artificial sequence
<400>3
tgcctgcagg caccacgatg caagcagaag c 31
<210>4
<211>2944
<212>DNA
＜213〉artificial sequence
<400>4
ggaattcgtc agccccttct cgctcattct gctcctcctc cggctgccaa gtctcatcct 60
tctctagatc ttggcagcgg aagcaagcta gcatcggcgg cgccgtcgga ggcggacctc 120
gcgcagctct cgacggtgag gaggtgcacg gggaggagcg gcctgcggta aggtgagcaa 180
ggacctgatc catggcggcg atggcgcact gtgttgtcct agtgcctgtc ctcgtcatcg 240
cctcctccct tgttgcggcg gcgtacgggg cggccttgcg ttcgctcagt gccaacgaag 300
gcaagggaga ggtcaacacc gacaccgccg tcgacctcat caccaccagc cgaacctcaa 360
cactgacgca gggtggtctc gccagagccg tggccatcgt taggttggtc tcacctttgt 420
ctcctttttt tttaatccct gcgaagtcca ccttcgatgc cccgatgcca cgattgacgt 480
tgccgcgtcg ctggttgatt tcgtcgctaa gatttggaga aggaggctgt gacggccgga 540
ggaggaaaag gagaggggag gggctgacgg gtagggtcct cattgatttc cacacgggca 600
caccacgatg acatgtcacg taggcaagag tgacaaatag ttaagaaatc atctatctcc 660
agtggtaaat tattaagcgt aaatctaaga ggggtatttg gataattgcc aaatctaaaa 720
gtagtaaata gttaaattcc ccagattaga ttggggatgc gtgaagttca ggcgagcaag 780
gcatgtaaac actgacgcaa atcgcaatca cgcaatacat cactacagca gcagcggatc 840
aaagatcaac aactccattt cccaaaattt aatccataat gaaagacata cactgatata 900
caagagccta aacatttact aatcatcaac aaaagatagc taggaattaa ttaaataata 960
cagctacgcc aacagtgcgt acgtcagcac cattcacccc acgctttttt agcgcaatgt 1020
gtagccaccc agcggcattg tcagaaaaag ttgagggctt ttttggtgca gccttggagt 1080
gttggactac aacaactcag tgcgcgaacg aaaactttta gcgtgttata tggatattaa 1140
tttaaagtat taaatgtaga ctaataataa aaataaatta taaattttgt ctgaaaacta 1200
cgtgacgaat ttattaagtc tagttaatct gttattagca aatatttact gtagcaccat 1260
attgttaaat catagagcag ttaggtttaa aatatccgta tcgcaatcta cacgtaatct 1320
gtgtaattag tttttttata tatttaatac ttcatgtacg gatgtgacag ggtgaaaaat 1380
tattgtttag aaactaaaca ggctctaaat tataagcacc gcgatattat cagaggtttg 1440
atgatgaagt cgatggtcat catcataatc tctctcaacg tgcctgcgag tctcattgcc 1500
ccccaactcc cccgcccacg aaatgattcc cctgctcgcc ggcgtatgtc gcaagcaaca 1560
cagcgatcca acaacgagcc tcgcgccctc gcggctccac cacctgcgca ccgccgcatc 1620
tgcgttttct ccgtgctccg aacccgacat ccttcgccac cgggtccgac cggccggccc 1680
gtacccaaaa agtcaatttt cccgggcccc acatgtcagt gaggtaccag cccatccccc 1740
ggtcctaaac aacttaacca ccggtgaaca actgagcacc gatgccccac ctcgcagaga 1800
taattataaa gtattagggc ctctttgaat cgcaggattg agaaaacgta ggaataggaa 1860
aaacgtagga ttttaatagg aatgtaagtt taaaacagag gattgcaaaa cacaggaaaa 1920
acataggaat gaccgtttga ttgaacggtg agagagagac ttaaaggaaa gttagcaaga 1980
ggttgaagct cttgctaagg gcctctttga atcgcagggt agaaaaaaac ggagaaataa 2040
gaaaaacgca ggattctgac aagaattgaa gtgtaaaaca gaggattgca aaacacagga 2100
aaaacacatg aatggtcgtt tgattggagc gcaggaaaaa cgcaggaatc ggataagaga 2160
gatagactca aaggaaattt tccaagaggt tggagctctt gctaaatttc ctccaaaatc 2220
cacatgcaat gtgtcattcc atacgaattt cataggattt ggaaagcttc aatcctttga 2280
atcaaaggcc caaataggaa aatttcctat aggatttaaa tcctatgaaa tttctacata 2340
aatcctttga tttaaagggg ccctaaattt cctccaaaat ctctatagga ttgtccattc 2400
catatgaatt tcaaagaatt ggataggatt taatcctttg ttttaaaggg ctttatagga 2460
aattttccta taggattgaa atactctaaa attcctgtgt tttcctccaa atctaaggag 2520
cccttaaccg gggaaatcgc tacctaatct ggcctaaccg aggtaattat cgagtgggat 2580
taaccttcgt tcggggcggt gctcgggtcg ggcgtccaga tctcgggctc gctgagctgt 2640
cgccatcgac ccggagtccc tcccgcacca gctaaccttt gcttccgttg gaagcgccag 2700
ccagcgctcg ccgttgtcac ctgtcacact gcgttgcacg gattatcgtt tttaattaat 2760
cgtcaccaga ttgttttttt tctttctttt tgcaaatctc gcttcgcttt tggagtggga 2820
ggaattaatt gtggagttgg gtagatcgat caggggcagc gcgagcgagg cccggttggt 2880
agcagtcggt gcgtgtttgt gcgtgtgcgc gctgcttctg cttgcatcgt ggtgcctgca 2940
ggca 2944
<210>5
<211>49
<212>DNA
＜213〉artificial sequence
<400>5
ggtaccaagc ttactagtcc tgcaggtcta gaggatccgt cgaccatgg 49
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<400>6
gcttccggct cgtatgttgt 20
<210>7
<211>19
<212>DNA
＜213〉artificial sequence
<400>7
gagtcgtcgg ttctgtaac 19
<210>8
<211>2353
<212>DNA
＜213〉artificial sequence
<400>8
gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 60
taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 120
aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 180
atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 240
tacgaattcg agctcggtac caagcttact agtcctgcag gtctagagga tccgtcgacc 300
atggtagatc tgagggtaaa tttctagttt ttctccttca ttttcttggt taggaccctt 360
ttctcttttt atttttttga gctttgatct ttctttaaac tgatctattt tttaattgat 420
tggttatggt gtaaatatta catagcttta actgataatc tgattacttt atttcgtgtg 480
tctatgatga tgatgatagt tacagaaccg acgactcgtc cgtcctgtag aaaccccaac 540
ccgtgaaatc aaaaaactcg acggcctgtg ggcattcagt ctggatcgcg aaaactgtgg 600
aattgatcag cgttggtggg aaagcgcgtt acaagaaagc cgggcaattg ctgtgccagg 660
cagttttaac gatcagttcg ccgatgcaga tattcgtaat tatgcgggca acgtctggta 720
tcagcgcgaa gtctttatac cgaaaggttg ggcaggccag cgtatcgtgc tgcgtttcga 780
tgcggtcact cattacggca aagtgtgggt caataatcag gaagtgatgg agcatcaggg 840
cggctatacg ccatttgaag ccgatgtcac gccgtatgtt attgccggga aaagtgtacg 900
tatcaccgtt tgtgtgaaca acgaactgaa ctggcagact atcccgccgg gaatggtgat 960
taccgacgaa aacggcaaga aaaagcagtc ttacttccat gatttcttta actatgccgg 1020
aatccatcgc agcgtaatgc tctacaccac gccgaacacc tgggtggacg atatcaccgt 1080
ggtgacgcat gtcgcgcaag actgtaacca cgcgtctgtt gactggcagg tggtggccaa 1140
tggtgatgtc agcgttgaac tgcgtgatgc ggatcaacag gtggttgcaa ctggacaagg 1200
cactagcggg actttgcaag tggtgaatcc gcacctctgg caaccgggtg aaggttatct 1260
ctatgaactc gaagtcacag ccaaaagcca gacagagtct gatatctacc cgcttcgcgt 1320
cggcatccgg tcagtggcag tgaagggcca acagttcctg attaaccaca aaccgttcta 1380
ctttactggc tttggtcgtc atgaagatgc ggacttacgt ggcaaaggat tcgataacgt 1440
gctgatggtg cacgaccacg cattaatgga ctggattggg gccaactcct accgtacctc 1500
gcattaccct tacgctgaag agatgctcga ctgggcagat gaacatggca tcgtggtgat 1560
tgatgaaact gctgctgtcg gctttcagct gtctttaggc attggtttcg aagcgggcaa 1620
caagccgaaa gaactgtaca gcgaagaggc agtcaacggg gaaactcagc aagcgcactt 1680
acaggcgatt aaagagctga tagcgcgtga caaaaaccac ccaagcgtgg tgatgtggag 1740
tattgccaac gaaccggata cccgtccgca aggtgcacgg gaatatttcg cgccactggc 1800
ggaageaacg cgtaaactcg acccgacgcg tccgatcacc tgcgtcaatg taatgttctg 1860
cgacgctcac accgatacca tcagcgatct ctttgatgtg ctgtgcctga accgttatta 1920
cggatggtat gtccaaagcg gcgatttgga aacggcagag aaggtactgg aaaaagaact 1980
tctggcctgg caggagaaac tgcatcagcc gattatcatc accgaatacg gcgtggatac 2040
gttagccggg ctgcactcaa tgtacaccga catgtggagt gaagagtatc agtgtgcatg 2100
gctggatatg tatcaccgcg tctttgatcg cgtcagcgcc gtcgtcggtg aacaggtatg 2160
gaatttcgcc gattttgcga cctcgcaagg catattgcgc gttggcggta acaagaaagg 2220
gatcttcact cgcgaccgca aaccgaagtc ggcggctttt ctgctgcaaa aacgctggac 2280
tggcatgaac ttcggtgaaa aaccgcagca gggaggcaaa caagctagcc accaccacca 2340
ccaccacgtg tga 2353
Claims (15)
1. promotor, its nucleotide sequence or are the complementary sequence of nucleotide sequence shown in the SEQ ID NO:1 shown in SEQ ID NO:1.
2. a recombinant vectors is characterized in that, described recombinant vectors contains promotor claimed in claim 1.
3. recombinant vectors according to claim 2, it is characterized in that, described recombinant vectors is the p8+P556 recombinant vectors, described p8 plasmid is the large fragment that obtains after the pCAMBIA-1301 plasmid is cut with Kpn I and Nco I enzyme, is connected resulting plasmid with Kpn I with the fragment that obtains after Nco I enzyme is connected with SEQ ID NO:5; The sequence of described P556 is shown in the SEQ ID NO:1.
4. a reconstitution cell that contains claim 2 or 3 described recombinant vectorss is characterized in that, described reconstitution cell is for getting rid of the reconstitution cell outside the vegetable cell.
5. reconstitution cell claimed in claim 4, it is to contain the Agrobacterium EHA105 reconstitution cell that right requires the carrier of 1 promotor or claim 2 or 3.
6. a method for preparing the promotor described in the claim 1 comprises the steps:
1) according to the nucleotide sequence shown in the SEQ ID NO:1, design pcr amplification primer pair;
2) take the fine genomic dna of paddy rice Japan as template, use step 1) in designed pcr amplification primer to carrying out pcr amplification.
7. method according to claim 6 is characterized in that, described pcr amplification primer is to being SEQ ID NO:2 and SEQ ID NO:3.
8. method of regulating and control genetic expression in the monocotyledons, described method comprises the step with the callus of promotor transforming monocots claimed in claim 1, and wherein, described monocotyledons is paddy rice, and described callus is Rice Callus.
9. the method for genetic expression in the regulation and control monocotyledons according to claim 8 is characterized in that described paddy rice is that Japan is fine.
10. according to claim 8 or 9 described methods, utilized restructuring Agrobacterium EHA105-P556 claimed in claim 5 in the conversion process of wherein said monocotyledons callus.
11. the purposes of promotor claimed in claim 1 destination gene expression in the regulation and control monocotyledons, wherein said goal gene is GUS, and described monocotyledons is paddy rice.
12. the purposes of promotor according to claim 11 destination gene expression in the regulation and control monocotyledons is characterized in that described paddy rice is that Japan is fine.
13. the purposes of promotor claimed in claim 1 in rice breeding.
14. purposes according to claim 13, it is characterized in that, described paddy rice is that Japan is fine, in spend 9, in spend 10, in spend 11, the Taibei 309, No. 8, Danjiang River, cloud rice No. 2, Shanyou 63, Shan excellent 608, Feng You 22, Guizhou Province excellent 88, II excellent 416, II excellent 107, II excellent 128, II excellent 718, Zhunliangyou 527, No. 1, river farming, assorted 0152, Anhui rice 88, Anhui rice 90, Anhui rice 92, Anhui rice 94, Anhui rice 96, Anhui rice 185, Anhui rice 187, Anhui rice 189, Anhui rice 191, Anhui rice 193, Anhui rice 195, Anhui rice 197, Anhui rice 199, Anhui rice 201, Anhui rice 203, Anhui rice 205, Anhui rice 207, and Tianjin former 101.
15. purposes according to claim 14 is characterized in that, described paddy rice is that Japan is fine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910238991 CN102115745B (en) | 2009-12-31 | 2009-12-31 | Promoter BgIosP556, and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910238991 CN102115745B (en) | 2009-12-31 | 2009-12-31 | Promoter BgIosP556, and preparation method and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN102115745A CN102115745A (en) | 2011-07-06 |
| CN102115745B true CN102115745B (en) | 2013-04-24 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 200910238991 Expired - Fee Related CN102115745B (en) | 2009-12-31 | 2009-12-31 | Promoter BgIosP556, and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103627707A (en) * | 2013-10-28 | 2014-03-12 | 华大基因杨凌创新研究院有限公司 | Promoter OsP006 as well as preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1063506A (en) * | 1990-05-18 | 1992-08-12 | 卢布里绍尔遗传学股份有限公司 | Be used for recombinant promoter in monocotyledons genetic expression |
| CN1494594A (en) * | 2001-03-27 | 2004-05-05 | 独立行政法人农业生物资源研究所 | Vegetative growth-specific promoter and genetically modified plants obtained thereby |
| WO2007075925A3 (en) * | 2005-12-23 | 2007-12-06 | Arcadia Biosciences Inc | Promoter sequence obtained from rice and methods of use |
-
2009
- 2009-12-31 CN CN 200910238991 patent/CN102115745B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1063506A (en) * | 1990-05-18 | 1992-08-12 | 卢布里绍尔遗传学股份有限公司 | Be used for recombinant promoter in monocotyledons genetic expression |
| CN1494594A (en) * | 2001-03-27 | 2004-05-05 | 独立行政法人农业生物资源研究所 | Vegetative growth-specific promoter and genetically modified plants obtained thereby |
| WO2007075925A3 (en) * | 2005-12-23 | 2007-12-06 | Arcadia Biosciences Inc | Promoter sequence obtained from rice and methods of use |
Non-Patent Citations (3)
| Title |
|---|
| Sasaki T et al."登录号：AP004002.3".《GENBANK》.2008, |
| Sasaki T et al."登录号:AP004002.3".《GENBANK》.2008, * |
| 柏亚男 等.水稻基因启动子OsBTF3p的克隆和启动活性分析.《中国农业科学》.2009,第42卷(第3期), * |
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| CN102115745A (en) | 2011-07-06 |
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