CN101967482B - Promoter BglosP536 and preparation method and application thereof - Google Patents
Promoter BglosP536 and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a promoter, in particular to a promoter of a monocotyledon such as paddy rice and a preparation method and application of the promoter. The promoter of the invention has a nucleotide sequence shown as SEQ ID NO:1 or is selected from the following variants which have promoter functions: 1) a nucleotide sequence hybridized with the nucleotide sequence shown as SEQ ID NO:1 under a highly strict condition; 2) a nucleotide sequence for performing substitution, deletion, addition and modification of one or more basic groups on the nucleotide sequence shown as SEQ ID NO:1; and 3) a nucleotide sequence having at least 90 percent of sequence identity with the nucleotide sequence shown as SEQ ID NO:1. The invention also relates to a preparation method of the promoter and the application of the promoter to the regulation and control of target gene expression and rice breeding in the monocotyledon.
Description
Technical field
The present invention relates to a kind of promotor, particularly a kind of monocotyledons is the 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 correct combination of template, 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 influences 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 is meant 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 constitutive promoter CsVMV efficiently is isolating 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.With these promotor replaced C aMV 35S promoter, can more effectively in monocotyledons, drive transcribing of foreign gene.Naomi etc. have cloned corresponding promotor respectively from the tryptophan synthetase beta subunit gene of Arabidopis thaliana and phytochrome gene, with its replaced C aMV 35S promoter, 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 ubiquitinpromoter), Actin promotor (Plant Actin promoter) and Adh-1 promotor (Maize alcohol dehydrogenase 1 promoter).
Factors such as Ub i promotor is low with its starting efficiency height, the degree that methylates, stabilization characteristics of genetics and gaining great popularity.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 corn gene group, 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 application more and more widely owing to the control effect of emphasizing to genetic expression in the transgenosis of monocotyledons good character.
Adh-1 promoter regulation ethanol dehydrogenase (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 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 be regulated and control the plant efficient rate and express the foreign gene with 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, efficient promotor such as potent promotor of some in the dicotyledons such as CsVMV 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 2469 bases, shown in SEQ ID NO:1:
AGAAAACTGGACTGCCTTGGAGAGATGAGTTCAAAGGTACAACATTCTAAGTTCTTCTGTTTATGAGAATAATGTTTACAATATGTTAAGATTCAGAATTGCCTTGTGAAACTCCTAAATATATTTTTACTGAACATGGAACTACAAGTGTTATATTGCACAGTAAAATACATTAAACAAATTTAATAATATACTTTTGTACTAATAAATATTTTTCATATTATATCAAGACTTTACTCGAGTTTTCTCGGTCCCATCATTGGTTCTCTGAAGCATGGTTCATCTCTATTTTTTTTAGCATCATAAGAAATCGGATAAGATTGAGCTCATATTTCACAAATGCGAATCAATTTTACTGTAAAATAGAACTCAAATGAACTGAGACTGGAGAGCAAGATAGAAGTGGCAACATATGGAAATTGAAAAGTCATTTTGGTATTTATCTGAAAGCTTTATTGAGCTTTGTGAATGGACCTTGCGTGAGCTCTGAAACCAGCTTACCACCGTTAGTTCCAGCTCGGTTTTCTTTTTTTCCCCTTAAAGGTTTCATGCGTATCGTGACAGATGTTACATAATGACAAATTCCCCAGCTGGAGCACCTTTATCCCTGCTGTTTGCATGAAATTAGCTTGTCTTGTAGTTCCCTCCAGCAAAAAGAAGTCTGAAACAAAACAACATTTCGAAAAAAAGGCATCCATGAGTTAGCATTTCTACAGTTGTCTATAGAGGGGAAGGCTGCACGACAAAGTTTCCAGGCTTGGAAACAACCTCTTATGTAAAATTTTTCGTATGTATCAGATGATTTGTTTGCGTTACGGCATCTCCACCTAACATCACCTTCATCATGCGCCTATGGTCTTTCTCTTGCCTGTTTTATACGTAAAATTGGAAACGACAGAAACTTTTGCCATCTTTATTAAAGGAAGGCAAATATGCAAATATAGGCATCAAGATCACAGTTAGTGGATTATCATCTTTGTAGGTTAACATGTCCTACCCCAGGGGAGCTTATACTCAAGTACTCCATGCATTTTCATGAAATGAGAAAAAACGATTTTTAAGAGAAATGTACTTTCTTGTATTTATGCCAAATGGCAAGGACTGAAAGGGAAAAACTAAGAAAGGGAACGTTACAGTAAGGCTCTGTGGGGACTGGGGACTTCAGAGAAACGTGAACCCTGCTTCCTTCCTCTGCATGAACATAACACCAGAGGTTTCCAGCCTTTCACACAGTTGTTGATGGCTTCACACAATTCATCTCTACCTCCTGACTCTTTATAAGGACCCCCAGCATCACCACAATTGCACAAGTACAGGCATTAGATCCACAAGAACACTTGGGCAGGCAAGCACCTCTTTGATCTTTAAGCCGTTGTTATGTTCTATTTCTGAGCATATGGTTTCTAGTTATATTCTTTTTCTTCATTCGTTTCATATCTTTGAAGTGTTGATGCAAATGCGGTGAACAACTATCAACTGTGTACTCTCCAAGTGAATGCGAATAATCATTTCCTGTGAGAATTGTGGGCTAGATAAACGAATGAAATGCTGTTTTATCTATGTCATGTGTGGAAATTTAGTTAATTTTCCGGTCTTTTTATGCATTGAGATGGGTATGCTGTTTTTTTAGTTGGGTCCCATCATCTTGAGAATTCTTTCAAATTTCCTTTTCTTTATCCTATATAAAGGATAGAGAAGGCGTATGCCTAGGTGCACCAACCCTGAAAGTTTTATTCTAATTGCGGGAATGGTTTGTAATTTTTGCTTGTTCAGGTTCTTTTTCGTGGCCTTTCTTTTTTTTCCCCTTATTTTGCTTAGTCTTTCACAGTCCAATTTTTGGGAAGTAGTATATCTTAGTTTGGTCCTAAGGCACCATGTTGTACTGCAGGAAAAAAAAGAGTAATTGTATTCTGTTTTTTCCTTGATTACTATATCCCTGTTTTAATTAATTTTGTGCCTTTGTTGTTTGATGTTGGAACTTCAATGCCCATAATTAGTCATTTGACTTGTTTTGGGTTTTGACGCTATCTTGAGTGCCATAGGAAACTGGTAGAATTTAGTAATAATTTTATATAGACTGAATGTTGAGCCCACCACAAATGGTTTCCTTCTGTACAAGTATTTAATAACTCAAGCACAGGAAACATCAGATCTCTAATCTAAAGGTTAACAATGGGCTCAAGCAGGAGCAGTAGTTCAGCTCTATCTGTATATTTAGAAGGGCTGGATCTACCTGTCCACCAGCTTTTAATTTTACCCTGGCAGCTGGATAACTTCTTGTCTGTTAATTTCATTTAGTGCTGTGTTATTTTCTTCTTGTTGTTCAGGATGGATGCTTTTGAATTTCTGGAATTTCGTATTTTGTTCTATCTCTTTATGAAATGACGTTATGGCACACTTTTTCTGCATATTCTTGATGAAAATAATTACCTAGTCATTTTTTTAGTTGCAGGTTTGTCTGGGACTTTG(SEQID NO：1)
In the present invention, the promoter sequence shown in the SEQ ID NO:1 is called promotor BgIosP536, or abbreviates the P536 promotor as.
This promotor is a constitutive promoter, and the rice callus tissue that has this promotor and GUS is after GUS dyeing experiment, and described rice callus tissue becomes blueness.
Another aspect of the present invention relates to the promotor that has with nucleotide sequence complementary sequence 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) to the nucleotide sequence shown in the SEQ ID NO:1 carry out one or more bases replacement, disappearance, interpolation modified nucleotide sequences 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 a foundation in conjunction with the melting temperature(Tm) (Tm) of mixture or probe with nucleic acid for example.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 with the salt of hybridization or ionic strength conditions and/or one or the washing of repeatedly stringency be 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 stringencies such as * SSC=height.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 low relatively salt and/or high-temperature condition.Sambrook etc. (Sambrook, J. etc. (1989) molecular cloning, laboratory manual, Cold Spring HarborPress, Plainview N.Y.) provides the hybridization conditions that comprises medium stringency and high stringency.
For ease of explanation, the suitable moderate stringent condition that is used to detect polynucleotide of the present invention and other multi-nucleotide hybrid comprises: 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 following each washed twice 20 minutes.It will be appreciated by those skilled in the art that and easily to operate the hybridization stringency, as changing the 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 replacement, disappearance, interpolation modified nucleotide sequences of the nucleotide sequence shown in the SEQ ID NO:1 being carried out one or more bases, be meant respectively or simultaneously and hold at the 5 ' end and/or 3 ' of described nucleotide sequence, 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 of representing with continuous integral number are one by one respectively modified.
In the present invention, described replacement, disappearance, the interpolation modified nucleotide sequences that nucleotide sequence shown in the SEQ ID NO:1 is carried out as above-mentioned one or more bases has and the same or analogous promoter activity of nucleotide sequence shown in the SEQ IDNO:1.
Describe by a kind of polynucleotide, the nucleotide sequence that it had for example with the reference nucleotide sequence of SEQID NO:1 at least " identity " of tool 95% be meant: in per 100 Nucleotide of the reference nucleotide sequence of SEQ IDNO: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, wherein the Nucleotide of Cha Ruing 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 position 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, the algorithm that is used for determining sequence identity and sequence similarity percentage ratio is for example BLAST and BLAST 2.0 algorithms, and they are described in (1990) J.Mol.Biol.215:403-410 such as (1977) Nucl.Acid.Res.25:3389-3402 such as Altschul and Altschul respectively.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.The software of carrying out the BLAST analysis can be obtained by the public by state-run biotechnology information center (NCBI).
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 and the same substantially polynucleotide sequence of 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 particularly, described monocotyledons is a paddy rice, and for example described paddy rice is Japanese eyeball (Oryza sativa L.ssp.japonicacv.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, for example: pUC18, pUC19, pUC118, pUC119, pMD19-T, pMD20-T, pMD18-T SimpleVecter, pMD19-T Simple Vecter etc.
Be suitable for making up expression vector of the present invention and include but not limited to, for example: pBI121, p13W4, pGEM etc.
In one embodiment of the invention, described recombinant vectors is the p8+P536 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, for example: agrobacterium tumefaciens cell LBA4404, EHA105, GV3101 etc.
In one embodiment of the invention, described reconstitution cell is reorganization agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105-P536.
Another aspect of the present invention also relates to a kind of monocotyledons callus, and described callus transforms promotor of the present invention.In one embodiment of the invention, described monocotyledons is a paddy rice.Described paddy rice includes but not limited to, for example: 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, accurate two excellent 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 (above-mentioned rice varieties all can available from emblem, Anhui good fortune company limited of merchant farmers') etc.In another embodiment of the present invention, described paddy rice is Japanese eyeball.
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 is right,
2) be template with the fine genomic dna of paddy rice Japan, use pcr amplification primer designed in the step 1) carrying out pcr amplification.
Those skilled in the art are known, and it is right to design corresponding pcr amplification primer 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 one embodiment of the invention, utilized aforesaid reorganization Agrobacterium EHA105-P536 in the conversion process of described monocotyledons callus.In a specific embodiments of the present invention, described monocotyledonous callus is the rice callus tissue, 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 that agrobacterium mediation converted, virus-mediated conversion, microinjection, particle bombardment, particle gun transform 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 coat the DNA that transforms) embryo callus or embryo's exploitation.In addition, the method for the transforming monocots that can also adopt is a protoplast transformation.After the gene transformation, the employing method in common is screened and regeneration is integrated with the unitary plant of expression.
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, for example: 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 a paddy rice, and described particularly 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 is fine for Japan, in spend 9, in spend 10, in 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 excellent 718, accurate two is excellent 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.
Promotor of the present invention can become a kind of new promotor as genetically modified instrument start-up of monocotyledons, especially paddy rice, low expressing gene conversion seedling screens in order to carry out, the breeding research of plant flower organ abortion equimolecular facilitates, thereby shortens the seed selection time of improved seeds greatly.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 to regulate and control the 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 BgIosP536.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 BgIosP536, wherein, swimming lane 1:1kb DNA Ladder Marker, swimming lane 2:PCR amplified production, swimming lane 3:100bp DNALadder Marker, the size of the band of the LadderMarker that the numeral 3000 on its right side and 2000 expressions are pointed, unit all is bp.
Fig. 2 is the pCAMBIA-1301 plasmid synoptic diagram that is used to make up the p8 plasmid.
Fig. 3 is multiple clone site and a GUS sequence synoptic diagram partly in the p8 plasmid synoptic diagram.
Fig. 4 is a p8 plasmid synoptic diagram.
Fig. 5 is the GUS coloration result of the rice callus tissue through transforming.Wherein, the rice callus tissue (left side) that is transformed by the reorganization agrobacterium tumefaciens p8+P536 that has promotor P536 sequence of the present invention presents blueness after GUS dyeing; Rice callus tissue (contrast, the right side) color after GUS dyeing that does not have the reorganization 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 to illustrate 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 (for example with reference to works such as J. Sa nurse 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 1P536 promoter fragment and the structure of pMD18-T+P536 recombinant vectors
Pcr amplification
Use plant genome DNA to extract test kit (TIANGEN novel plant genome DNA extracting reagent kit; catalog number (Cat.No.): DP320-02) extraction paddy rice Japan is fine (was preserved in Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center on December 18th, 2009; it is Chinese typical culture collection center (CCTCC); deposit number is CCTCC-P200910) genomic dna; according to the sequence of this promotor in paddy rice Japan eyeball gDNA; design one couple of PCR specificity amplification primer (upstream primer F1 at head and the tail respectively; add restriction enzyme site Kpn I and protection base; downstream primer R1 adds restriction enzyme site Sbf I and protection base).The fine gDNA of paddy rice Japan with said extracted is a template, and (TaKaRa, DRR100B) polysaccharase carries out pcr amplification to use high-fidelity Ex Taq.As shown in table 1.
Table 1 gene promoter amplification PCR system
The pcr amplification program is: 94 ℃ of pre-sex change 5min, 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:GGggtaccAGAAAACTGGACTGCCTTGGA (SEQ ID NO:2), wherein lowercase is represented Kpn I restriction enzyme site.
Downstream primer R1:TGcctgcaggCAAAGTCCCAGACAAACCTGC (SEQ ID NO:3), wherein lowercase is represented Sbf I restriction enzyme site.
Pcr amplification product separates through 1.0% agarose gel electrophoresis, obtains size and is the band of 2487bp (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+P536 recombinant vectors
Will as the above-mentioned pcr amplification product that obtains carry out T/A clone (the pMD18-T plasmid, TaKaRa, D103A), transformed into escherichia coli, picking positive colony order-checking (shown in SEQ ID NO:4), it is accurate prove.
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 (reclaim and 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+P536 recombinant vectors at the energy-conserving intelligent thermostatic bath in 16 ℃.Will be through the transformed into escherichia coli as follows of 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+P536 recombinant vectors, stir evenly ice bath 30min, 42 ℃ of heat shock 60s gently, 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, mixture with the remaining resuspended post precipitation of 150 μ l supernatants, blow evenly gently, 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+P536 cloning vector, called after DH5 α-P536.The big Gene science limited-liability company of Shenzhen China checks order to the P536 in the pMD18-T+P536 cloning vector, and the result is as follows:
GG
GGTACC 
GAGATGAGTTCAAAGGTACAACATTCTAAGTTCTTCTGTTTATGAGAATAATGTTTACAATATGTTAAGATTCAGAATTGCCTTGTGAAACTCCTAAATATATTTTTACTGAACATGGAACTACAAGTGTTATATTGCACAGTAAAATACATTAAACAAATTTAATAATATACTTTTGTACTAATAAATATTTTTCATATTATATCAAGACTTTACTCGAGTTTTCTCGGTCCCATCATTGGTTCTCTGAAGCATGGTTCATCTCTATTTTTTTTAGCATCATAAGAAATCGGATAAGATTGAGCTCATATTTCACAAATGCGAATCAATTTTACTGTAAAATAGAACTCAAATGAACTGAGACTGGAGAGCAAGATAGAAGTGGCAACATATGGAAATTGAAAAGTCATTTTGGTATTTATCTGAAAGCTTTATTGAGCTTTGTGAATGGACCTTGCGTGAGCTCTGAAACCAGCTTACCACCGTTAGTTCCAGCTCGGTTTTCTTTTTTTCCCCTTAAAGGTTTCATGCGTATCGTGACAGATGTTACATAATGACAAATTCCCCAGCTGGAGCACCTTTATCCCTGCTGTTTGCATGAAATTAGCTTGTCTTGTAGTTCCCTCCAGCAAAAAGAAGTCTGAAACAAAACAACATTTCGAAAAAAAGGCATCCATGAGTTAGCATTTCTACAGTTGTCTATAGAGGGGAAGGCTGCACGACAAAGTTTCCAGGCTTGGAAACAACCTCTTATGTAAAATTTTTCGTATGTATCAGATGATTTGTTTGCGTTACGGCATCTCCACCTAACATCACCTTCATCATGCGCCTATGGTCTTTCTCTTGCCTGTTTTATACGTAAAATTGGAAACGACAGAAACTTTTGCCATCTTTATTAAAGGAAGGCAAATATGCAAATATAGGCATCAAGATCACAGTTAGTGGATTATCATCTTTGTAGGTTAACATGTCCTACCCCAGGGGAGCTTATACTCAAGTACTCCATGCATTTTCATGAAATGAGAAAAAACGATTTTTAAGAGAAATGTACTTTCTTGTATTTATGCCAAATGGCAAGGACTGAAAGGGAAAAACTAAGAAAGGGAACGTTACAGTAAGGCTCTGTGGGGACTGGGGACTTCAGAGAAACGTGAACCCTGCTTCCTTCCTCTGCATGAACATAACACCAGAGGTTTCCAGCCTTTCACACAGTTGTTGATGGCTTCACACAATTCATCTCTACCTCCTGACTCTTTATAAGGACCCCCAGCATCACCACAATTGCACAAGTACAGGCATTAGATCCACAAGAACACTTGGGCAGGCAAGCACCTCTTTGATCTTTAAGCCGTTGTTATGTTCTATTTCTGAGCATATGGTTTCTAGTTATATTCTTTTTCTTCATTCGTTTCATATCTTTGAAGTGTTGATGCAAATGCGGTGAACAACTATCAACTGTGTACTCTCCAAGTGAATGCGAATAATCATTTCCTGTGAGAATTGTGGGCTAGATAAACGAATGAAATGCTGTTTTATCTATGTCATGTGTGGAAATTTAGTTAATTTTCCGGTCTTTTTATGCATTGAGATGGGTATGCTGTTTTTTTAGTTGGGTCCCATCATCTTGAGAATTCTTTCAAATTTCCTTTTCTTTATCCTATATAAAGGATAGAGAAGGCGTATGCCTAGGTGCACCAACCCTGAAAGTTTTATTCTAATTGCGGGAATGGTTTGTAATTTTTGCTTGTTCAGGTTCTTTTTCGTGGCCTTTCTTTTTTTTCCCCTTATTTTGCTTAGTCTTTCACAGTCCAATTTTTGGGAAGTAGTATATCTTAGTTTGGTCCTAAGGCACCATGTTGTACTGCAGGAAAAAAAAGAGTAATTGTATTCTGTTTTTTCCTTGATTACTATATCCCTGTTTTAATTAATTTTGTGCCTTTGTTGTTTGATGTTGGAACTTCAATGCCCATAATTAGTCATTTGACTTGTTTTGGGTTTTGACGCTATCTTGAGTGCCATAGGAAACTGGTAGAATTTAGTAATAATTTTATATAGACTGAATGTTGAGCCCACCACAAATGGTTTCCTTCTGTACAAGTATTTAATAACTCAAGCACAGGAAACATCAGATCTCTAATCTAAAGGTTAACAATGGGCTCAAGCAGGAGCAGTAGTTCAGCTCTATCTGTATATTTAGAAGGGCTGGATCTACCTGTCCACCAGCTTTTAATTTTACCCTGGCAGCTGGATAACTTCTTGTCTGTTAATTTCATTTAGTGCTGTGTTATTTTCTTCTTGTTGTTCAGGATGGATGCTTTTGAATTTCTGGAATTTCGTATTTTGTTCTATCTCTTTATGAAATGACGTTATGGCACACTTTTTCTGCATATTCTTGATGAAAATAATTACCTAGTCATTTTTTTAGTT
CCTGCAGGCA(SEQ ID NO：4)
In the top sequence, what be with underscore is restriction enzyme site, and what be with square frame is primer sequence.
Sequencing result shows that the P536 promoter sequence is correct in the pMD18-T+P536 cloning vector of acquisition.
The structure of embodiment 2 carriers-p8+P536 recombinant vectors
(catalog number (Cat.No.): operational manual DP103-03), the conversion that makes up from embodiment 1 have bacillus coli DH 5 alpha-P536 of promotor P536 and extract the cloning vector pMD18-T+P536 that has P536 promoter sequence of the present invention according to the little extraction reagent kit of the common plasmid of TIANGEN; Behind the purifying with corresponding restriction enzyme Kpn I (NEB, B0101S) and Sbf I (NEB, R0140T) carry out enzyme and cut, reclaim corresponding promotor and insert fragment, and connect with the big fragment of carrier that the p8 plasmid reclaims after with identical digestion with restriction enzyme respectively.
Gained is connected product p8+P536 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 CAMBIABios (biological open source) Licensee, Australia) transform in the following manner and make up, specify as follows:
With Kpn I/Nco I (NEB) double digestion plasmid pCAMBIA-1301 (referring to Fig. 2), reclaim big fragment.According to synthetic following sequence: the GGTACCAAGCTTACTAGTCCTGCAGGTCTAGAGGATCCGTCGACCATGG (SEQ ID NO:5) (restriction enzyme site that comprises is Kpn I/Hind III/Spe I/Sbf I/PstI/Xba I/BamH I/Sa II/Nco I) of the restriction endonuclease sites that is adopted, with reclaiming behind the Kpn I/Nco I double digestion, be connected back conversion top10 cell (Dong Yang of Kunming Institute of Zoology, Chinese Academy of Sciences provides with the above-mentioned big 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 (SEQID NO:7), by the PCR detection method, the transformant that has amplified fragments and be 350bp is the transformant (referring to Fig. 4) of the p8 plasmid that contains multiple clone site that needs make up and GUS sequence.
Length 2353 bases of multiple clone site in the described p8 plasmid and GUS sequence, shown in SEQID NO:8 (referring to Fig. 3):
GTTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTAT
GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTAC
GAGCTC 
AAGCTT 
GGAT CC 
(SEQ ID NO：8)
As above constructed p8 plasmid among the present invention shown in the sequence, EcoRI/Sac I/Kpn I in its multiple clone site/Hind III/Spe I/Sbf I/Pst I/Xba I/BamH I/Sa II/Nco I restriction enzyme site is respectively with adding frame and underscore is represented, the used primer GCTTCCGGCTCGTATGTTGT/GAGTCGTCGGTTCTGTAAC (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+P536
According to restriction enzyme Kpn I and Sbf I (NEB) operation instructions, according to resulting cloning vector pMD18-T+P536 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+P536 and p8 plasmid is as follows:
Enzyme is cut system: 50 μ l
Sterilized water 34.8 μ l
10*buffer H 5μl
Kpn I 0.1μl(10U)
Sbf I 0.1μl(10U)
Cloning vector pMD18-T+P536 or p8 plasmid 10 μ l (＜1000ng)
Use TIANGEN sepharose DNA reclaim test kit (catalog number (Cat.No.): DP209-03) reclaim the p8 plasmid of cutting through enzyme respectively, and promotor P536 inserts fragment, according to the T4 ligase enzyme (TaKaRa, D2011A) operation instructions connect according to following condition:
Linked system: 10 μ l
10*T4buffer 1μl
The promotor P536 that reclaims inserts fragment 10～20ng, determines 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 P536 fragment among the present invention adds 10ng.(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 α 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 ice bath 30min, 42 ℃ of heat shock 60s gently, ice bath 5min adds the SOC of 4 ℃ of precoolings of 600 μ l, 37 degree 220rpm recovery 45min, 8000rpm is from 30s, remove supernatant, leave and take 150 μ l, blow even gently, granulated glass sphere coating LB (kan) is inverted for 37 ℃ and cultivates 16～24h.Obtain recombinant vectors p8+P536.
Be that primer carries out the PCR detection to gained recombinant vectors p8+P536 with F1 (being SEQ ID NO:2) and R1 (being SEQ ID NO:3) respectively, to contain required promotor P536 among the conclusive evidence gained recombinant vectors p8+P536.Cut screening by enzyme and contain recombinant vectors p8+P536 transformant.
The preparation of embodiment 3 reorganization agrobacterium tumefaciens EHA105-P536 cells
To transform respectively according to " molecular cloning the experiment guide " (third edition as p8+P536 recombinant vectors and the p8 plasmid in contrast that method as described in the embodiment 2 makes up, the 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 culture collection center (CCTCC), deposit number is CCTCC M 209315) competent cell, concrete grammar is as follows:
EHA105 takes out in Ultralow Temperature Freezer with the agrobacterium tumefaciens competent cell, as for thawing on ice.After the thawing, add p8+P536 recombinant vectors and p8 plasmid and p8 empty carrier in contrast, the mixing gently of 5 μ l respectively, ice bath 10min puts 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 inhales and removes supernatant, staying 200 μ l blows even, coat and be added with (50mg/lKan, 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.
With F1 (being SEQ ID NO:2) and R1 (being SEQ ID NO:3) is that primer carries out the PCR detection and cuts the screening transformant by EcoR I/Pst I enzyme.
What pcr amplification went out that about 2487bp band and enzyme cut out about 2476bp band is the reorganization agrobacterium tumefaciens of recombinant vectors p8+P536.
Among the present invention, according to the reorganization Agrobacterium that has recombinant vectors p8+P536 that obtains as above-mentioned method, called after reorganization agrobacterium tumefaciens EHA105-P536.
According to the method for the invention, the contrast reorganization Agrobacterium that has the p8 plasmid that obtains, called after reorganization agrobacterium tumefaciens EHA105-p8.
The inducing and transforming of embodiment 4 rice callus tissues
According to following steps inducing paddy rice callus, and transform described callus with reorganization agrobacterium tumefaciens EHA105-P536 and reorganization 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, the sterilization back is cleaned 5 times with aqua sterilisa; Seed after the sterilization is placed on the N6D substratum (specifically filling a prescription referring to table 2), seal with sealing film; 29.5 3～4 weeks of ℃ illumination cultivation;
2) choose active growth callus (yellow-white, drying, diameter 1～3mm), 29.5 ℃ of illumination cultivation are 3 days on new N6D substratum;
3) the constructed single bacterium colony of reorganization agrobacterium tumefaciens (reorganization agrobacterium tumefaciens EHA105-P536 or reorganization agrobacterium tumefaciens EHA105-p8) of picking such as embodiment 3 respectively, in adding microbiotic (50mg/l Kan, 10mg/l Rif) YM substratum (specifically filling a prescription referring to table 3, table 4) was gone up streak culture 3 days, 28 ℃ of culture temperature; Scrape respectively and get above-mentioned reorganization agrobacterium tumefaciens and place the AS (Acetosyringone that has added 30 μ l 100mM, Syringylethanone) in the 30ml AAM substratum (specifically filling a prescription) referring to table 5, gentle resuspended described reorganization agrobacterium tumefaciens cell (reorganization agrobacterium tumefaciens EHA105-P536 or reorganization agrobacterium tumefaciens EHA105-p8);
4) callus with succeeding transfer culture places the sterilization culture dish; To pour in the culture dish as the reorganization agrobacterium tumefaciens suspension of step 3 preparation, callus will be immersed wherein 15min;
5) outwell reorganization agrobacterium tumefaciens suspension, callus is sopped up excess 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, become clarification until supernatant liquor with aqua sterilisa; Callus is soaked in the sterilized water that contains 500mg/l Pyocianil (Carb) to kill the reorganization agrobacterium tumefaciens; Remove redundant moisture on the callus with sterilization thieving paper, then it is transferred on the N6-AS substratum that contains 1mg/l hygromycin B (HmB) and 50mg/l Carb; With sealing the film phonograph seal culture dish, 29.5 ℃ of 3～4 weeks of illumination cultivation.
The expression of GUS in the embodiment 5 rice callus tissues
For detecting expression through goal gene GUS in the rice callus tissue of embodiment 4 described conversions, according to Chen SY etc. at Journal of Integrative Plant Biology, 2008,50 (6): the described method of 742-751, dye to the rice callus tissue that transforms with reorganization agrobacterium tumefaciens EHA105-P536 or reorganization 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 tissue that transforms with reorganization agrobacterium tumefaciens EHA105-P536 or reorganization 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 tissue of the Agrobacterium tumefaciens mediated conversion of reorganization of the p8+P536 recombinant vectors through containing promotor (Fig. 5 left side) is dyed, do not change through recombinate rice callus tissue (Fig. 5 right side in contrast) color after GUS dyeing of Agrobacterium tumefaciens mediated conversion of the p8 plasmid that does not contain promotor.The result shows that P536 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 " be meant the sterilization of following condition: 121 ℃ of following vapor sterilizations 20 minutes.
Table 2N6D substratum
Regulate pH value to 5.8 with 1N potassium hydroxide, sterilization according to a conventional method after 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 ℃ of preservations are standby.
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 ℃ of preservations are standby.
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 back 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 preservations were no more than for 1 week.
Table 3YM liquid nutrient medium (containing 50mg/L Kan, 10mg/L Rif):
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 ℃ of preservations are standby.
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 (CoCl
2.6H
2O), distilled water is settled to 1L, and 4 ℃ of preservations are standby.
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 ℃ of preservations are standby.
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 table
＜110〉Shenzhen Huada Genetic Technology Co., Ltd
＜120〉a kind of promotor BgIosP536, Preparation Method And The Use
<130>IDC090179
<160>8
<170>PatentIn version 3.2
<210>1
<211>2469
<212>DNA
＜213〉paddy rice Japan fine (Oryza sativa L.ssp.japonica cv.)
<400>1
agaaaactgg actgccttgg agagatgagt tcaaaggtac aacattctaa gttcttctgt 60
ttatgagaat aatgtttaca atatgttaag attcagaatt gccttgtgaa actcctaaat 120
atatttttac tgaacatgga actacaagtg ttatattgca cagtaaaata cattaaacaa 180
atttaataat atacttttgt actaataaat atttttcata ttatatcaag actttactcg 240
agttttctcg gtcccatcat tggttctctg aagcatggtt catctctatt ttttttagca 300
tcataagaaa tcggataaga ttgagctcat atttcacaaa tgcgaatcaa ttttactgta 360
aaatagaact caaatgaact gagactggag agcaagatag aagtggcaac atatggaaat 420
tgaaaagtca ttttggtatt tatctgaaag ctttattgag ctttgtgaat ggaccttgcg 480
tgagctctga aaccagctta ccaccgttag ttccagctcg gttttctttt tttcccctta 540
aaggtttcat gcgtatcgtg acagatgtta cataatgaca aattccccag ctggagcacc 600
tttatccctg ctgtttgcat gaaattagct tgtcttgtag ttccctccag caaaaagaag 660
tctgaaacaa aacaacattt cgaaaaaaag gcatccatga gttagcattt ctacagttgt 720
ctatagaggg gaaggctgca cgacaaagtt tccaggcttg gaaacaacct cttatgtaaa 780
atttttcgta tgtatcagat gatttgtttg cgttacggca tctccaccta acatcacctt 840
catcatgcgc ctatggtctt tctcttgcct gttttatacg taaaattgga aacgacagaa 900
acttttgcca tctttattaa aggaaggcaa atatgcaaat ataggcatca agatcacagt 960
tagtggatta tcatctttgt aggttaacat gtcctacccc aggggagctt atactcaagt 1020
actccatgca ttttcatgaa atgagaaaaa acgattttta agagaaatgt actttcttgt 1080
atttatgcca aatggcaagg actgaaaggg aaaaactaag aaagggaacg ttacagtaag 1140
gctctgtggg gactggggac ttcagagaaa cgtgaaccct gcttccttcc tctgcatgaa 1200
cataacacca gaggtttcca gcctttcaca cagttgttga tggcttcaca caattcatct 1260
ctacctcctg actctttata aggaccccca gcatcaccac aattgcacaa gtacaggcat 1320
tagatccaca agaacacttg ggcaggcaag cacctctttg atctttaagc cgttgttatg 1380
ttctatttct gagcatatgg tttctagtta tattcttttt cttcattcgt ttcatatctt 1440
tgaagtgttg atgcaaatgc ggtgaacaac tatcaactgt gtactctcca agtgaatgcg 1500
aataatcatt tcctgtgaga attgtgggct agataaacga atgaaatgct gttttatcta 1560
tgtcatgtgt ggaaatttag ttaattttcc ggtcttttta tgcattgaga tgggtatgct 1620
gtttttttag ttgggtccca tcatcttgag aattctttca aatttccttt tctttatcct 1680
atataaagga tagagaaggc gtatgcctag gtgcaccaac cctgaaagtt ttattctaat 1740
tgcgggaatg gtttgtaatt tttgcttgtt caggttcttt ttcgtggcct ttcttttttt 1800
tccccttatt ttgcttagtc tttcacagtc caatttttgg gaagtagtat atcttagttt 1860
ggtcctaagg caccatgttg tactgcagga aaaaaaagag taattgtatt ctgttttttc 1920
cttgattact atatccctgt tttaattaat tttgtgcctt tgttgtttga tgttggaact 1980
tcaatgccca taattagtca tttgacttgt tttgggtttt gacgctatct tgagtgccat 2040
aggaaactgg tagaatttag taataatttt atatagactg aatgttgagc ccaccacaaa 2100
tggtttcctt ctgtacaagt atttaataac tcaagcacag gaaacatcag atctctaatc 2160
taaaggttaa caatgggctc aagcaggagc agtagttcag ctctatctgt atatttagaa 2220
gggctggatc tacctgtcca ccagctttta attttaccct ggcagctgga taacttcttg 2280
tctgttaatt tcatttagtg ctgtgttatt ttcttcttgt tgttcaggat ggatgctttt 2340
gaatttctgg aatttcgtat tttgttctat ctctttatga aatgacgtta tggcacactt 2400
tttctgcata ttcttgatga aaataattac ctagtcattt ttttagttgc aggtttgtct 2460
gggactttg 2469
<210>2
<211>29
<212>DNA
＜213〉artificial sequence
<400>2
ggggtaccag aaaactggac tgccttgga 29
<210>3
<211>31
<212>DNA
＜213〉artificial sequence
<400>3
tgcctgcagg caaagtccca gacaaacctg c 31
<210>4
<211>2487
<212>DNA
＜213〉artificial sequence
<400>4
ggggtaccag aaaactggac tgccttggag agatgagttc aaaggtacaa cattctaagt 60
tcttctgttt atgagaataa tgtttacaat atgttaagat tcagaattgc cttgtgaaac 120
tcctaaatat atttttactg aacatggaac tacaagtgtt atattgcaca gtaaaataca 180
ttaaacaaat ttaataatat acttttgtac taataaatat ttttcatatt atatcaagac 240
tttactcgag ttttctcggt cccatcattg gttctctgaa gcatggttca tctctatttt 300
ttttagcatc ataagaaatc ggataagatt gagctcatat ttcacaaatg cgaatcaatt 360
ttactgtaaa atagaactca aatgaactga gactggagag caagatagaa gtggcaacat 420
atggaaattg aaaagtcatt ttggtattta tctgaaagct ttattgagct ttgtgaatgg 480
accttgcgtg agctctgaaa ccagcttacc accgttagtt ccagctcggt tttctttttt 540
tccccttaaa ggtttcatgc gtatcgtgac agatgttaca taatgacaaa ttccccagct 600
ggagcacctt tatccctgct gtttgcatga aattagcttg tcttgtagtt ccctccagca 660
aaaagaagtc tgaaacaaaa caacatttcg aaaaaaaggc atccatgagt tagcatttct 720
acagttgtct atagagggga aggctgcacg acaaagtttc caggcttgga aacaacctct 780
tatgtaaaat ttttcgtatg tatcagatga tttgtttgcg ttacggcatc tccacctaac 840
atcaccttca tcatgcgcct atggtctttc tcttgcctgt tttatacgta aaattggaaa 900
cgacagaaac ttttgccatc tttattaaag gaaggcaaat atgcaaatat aggcatcaag 960
atcacagtta gtggattatc atctttgtag gttaacatgt cctaccccag gggagcttat 1020
actcaagtac tccatgcatt ttcatgaaat gagaaaaaac gatttttaag agaaatgtac 1080
tttcttgtat ttatgccaaa tggcaaggac tgaaagggaa aaactaagaa agggaacgtt 1140
acagtaaggc tctgtgggga ctggggactt cagagaaacg tgaaccctgc ttccttcctc 1200
tgcatgaaca taacaccaga ggtttccagc ctttcacaca gttgttgatg gcttcacaca 1260
attcatctct acctcctgac tctttataag gacccccagc atcaccacaa ttgcacaagt 1320
acaggcatta gatccacaag aacacttggg caggcaagca cctctttgat ctttaagccg 1380
ttgttatgtt ctatttctga gcatatggtt tctagttata ttctttttct tcattcgttt 1440
catatctttg aagtgttgat gcaaatgcgg tgaacaacta tcaactgtgt actctccaag 1500
tgaatgcgaa taatcatttc ctgtgagaat tgtgggctag ataaacgaat gaaatgctgt 1560
tttatctatg tcatgtgtgg aaatttagtt aattttccgg tctttttatg cattgagatg 1620
ggtatgctgt ttttttagtt gggtcccatc atcttgagaa ttctttcaaa tttccttttc 1680
tttatcctat ataaaggata gagaaggcgt atgcctaggt gcaccaaccc tgaaagtttt 1740
attctaattg cgggaatggt ttgtaatttt tgcttgttca ggttcttttt cgtggccttt 1800
cttttttttc cccttatttt gcttagtctt tcacagtcca atttttggga agtagtatat 1860
cttagtttgg tcctaaggca ccatgttgta ctgcaggaaa aaaaagagta attgtattct 1920
gttttttcct tgattactat atccctgttt taattaattt tgtgcctttg ttgtttgatg 1980
ttggaacttc aatgcccata attagtcatt tgacttgttt tgggttttga cgctatcttg 2040
agtgccatag gaaactggta gaatttagta ataattttat atagactgaa tgttgagccc 2100
accacaaatg gtttccttct gtacaagtat ttaataactc aagcacagga aacatcagat 2160
ctctaatcta aaggttaaca atgggctcaa gcaggagcag tagttcagct ctatctgtat 2220
atttagaagg gctggatcta cctgtccacc agcttttaat tttaccctgg cagctggata 2280
acttcttgtc tgttaatttc atttagtgct gtgttatttt cttcttgttg ttcaggatgg 2340
atgcttttga atttctggaa tttcgtattt tgttctatct ctttatgaaa tgacgttatg 2400
gcacactttt tctgcatatt cttgatgaaa ataattacct agtcattttt ttagttgcag 2460
gtttgtctgg gactttgcct gcaggca 2487
<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
ggaagcaacg 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. one kind by nucleotide sequence shown in the SEQ ID NO:1 or the promotor formed with its complementary nucleotide sequence.
2. a recombinant vectors is characterized in that, described recombinant vectors contains the described promotor of claim 1.
3. the recombinant vectors of claim 2, wherein said recombinant vectors is the p8+P536 recombinant vectors.
4. reconstitution cell that contains claim 2 or 3 described recombinant vectorss.
5. the described reconstitution cell of claim 4, it is reorganization Agrobacterium EHA105-P536.
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 is right;
2) be template with the fine genomic dna of paddy rice Japan, use pcr amplification primer designed in the step 1) carrying out pcr amplification.
7. the method for claim 6, wherein said 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 the described promotor transforming monocots of claim 1, wherein said monocotyledons is a paddy rice, and described monocotyledons callus is the rice callus tissue.
9. the method for claim 8, wherein said paddy rice are that Japan is fine.
10. method according to claim 8 has been utilized the described reorganization of claim 5 Agrobacterium EHA105-P536 in the conversion process of wherein said rice callus tissue.
11. the purposes of the described promotor of claim 1 destination gene expression in the regulation and control monocotyledons, wherein said goal gene is GUS, and described monocotyledons is a paddy rice.
12. the purposes of claim 11, wherein said paddy rice are that Japan is fine.
13. the purposes of the described promotor of claim 1 in rice breeding.
14. the purposes of claim 13, wherein said 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, accurate two excellent 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. the purposes of claim 14, wherein said paddy rice are that Japan is fine.
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| CN2009102495760A CN101967482B (en) | 2009-12-30 | 2009-12-30 | Promoter BglosP536 and preparation method and application thereof |
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| CN101967482B true CN101967482B (en) | 2011-12-28 |
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