CN102146406B - Promoter BgIosP535, and preparation method and use thereof - Google Patents

Abstract

The invention relates to a promoter BgIosP535, and a preparation method and use thereof. The promoter has a nucleotide sequence which is selected from any of the following groups and has a prompter function: a, a nucleotide sequence represented by SEQ ID No.1; b, a nucleotide sequence complementary to the SEQ ID No.1; c, a nucleotide sequence which can be crossed with the nucleotide sequence a or the nucleotide sequence b under a highly strict condition; d, a nucleotide sequence obtained by modifying the nucleotide sequence a or the nucleotide sequence b by substituting, losing and increasing one or more bases; and e, a nucleotide sequence having an identity of at least 90 percent with the nucleotide sequence a or the nucleotide sequence b. The invention also relates to the use of the promoter in the control over the expression of a target gene in monocotyledons or dicotyledons and the use of the promoter in seed breeding.

Description

A kind of promotor BgIosP535

Technical field

The present invention relates to the gene regulating field.Particularly, the present invention relates to a kind of promotor, specifically is the promotor BgIosP535 of paddy rice, and the preparation method of said promotor.The invention still further relates to the purposes of said promotor destination gene expression in regulation and control monocotyledons or dicotyledons.

Background technology

Promotor is an integral part of gene, is usually located at structure gene 5 ' the end upper reaches, is the section of DNA sequence of RNA polymerase identification, combination and transcriptional start.Promotor can instruct holoenzyme (holoenzyme) correctly to combine with 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 3 types with it: 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.The constitutive promoter that the most often uses in the dicotyledons is 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; Good expression effect (Plant biotechnology, 2002,19 (1): 19-26) in transgene tobacco, have also been obtained.

Promotor common in the monocotyledons gene has: Ubi promotor (Plant ubiquitin promoter), Actin promotor (Plant Actin promoter) and Adh-1 promotor (Maize alcohol dehydrogenase 1promoter).

The Ubi promotor gains great popularity with factor such as its starting efficiency is high, the degree that methylates 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, yam 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 through 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 emphasical control effect to genetic expression in the transgenic 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 few 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 strong effect 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 the strong promotor Related Research Domain of imitating, find and verified many monocotyledonous promotors.In addition, efficient promoters such as strong effect promotor of some in the dicotyledons such as CsVMV promotor, tomato E8 promotor, resveratrol synthase gene Vst1 promotor also have very strong gene regulating effect in monocotyledons.

Although above-mentioned known monocotyledonous promotor has been arranged; The inventor is through the further investigation to rice genome; A kind of new Monocotyledon promoter is provided; Said promotor can be used in destination gene expression in regulation and control monocotyledons and the dicotyledons, for destination gene expression in research monocotyledons and the dicotyledons a kind of new instrument and selection is provided simultaneously.

Summary of the invention

In first aspect, the invention provides a promoter gene BgIosP535 with promoter activity.The nucleotide sequence of said promotor is shown in SEQ ID NO:1.

CGTGTGTGAGGTGTCCGAAGTACTAAGAGCTGCAAAGTCAGTTTAACTGCTTCTGTACTAGCGGCAAATTTCCAATTTGCCAATTCGATTGTGTTTGTCCAGAATTGGTTGCTAACAATTTCCGGTGACTCTACCGGTCAGAAACAATTTACTGGTAATTCTCTGAAGTTTCTGAAGTCTGAACCATCTCTTTTCGGACGGGGACACTTTCTGTTCAGGAAAGCTCTGTCTCTTTTTTCCTGGCTTCAGGACATTTACATCAACAAAAACAACTTGATCCACAATCTCGATTTTTACCGAGTTTTCGCATCCCCTCCTGAATTGAATGGTTCTAGAACAAAATAATGTGATCCAATGAAGAATAGATGCGCCAGCAATATCTTAGTGCATCCACAATGATTATCTATAAGCTCTCTACAAGAGATCTATGTCAGTATATTTTCTTATTTGGAAGAGATTAAATGAAGAGAGAGAACAAAACTATCTACTAACCTGGAGATAGTCTATAGAGAAAAACGAGGCAATGAATTAGAGAGCTATAGATACCCATGTAGACATACTATTGAGATGGTTTACTATTAATCTAGTTTATTGCTGAGATATACATGTTTTATAGAGAGCACCTTACTTTACCATTGCGGGTGCTCTTATGCCTACCCTTTATTAAGACAGGCATGAACAGAAGGCCAGCCAATAGAAATCTGAGTTGCCAGTTGCCACACAGATGTTGGCAAGCTCATACAGGTGGTTCTCAGCCTCTGATTGTTGCCAAACCCCTGGTTCAGAATTCAGAGTTCATCAGAGCCTCAGAAATGGTAAACCTTCGGAGTTCAGATGGACCTCAACAAAACAATACTAGGTTGTTCTGTCGCCAAATTGTTCTGTAAATTCTACACATACTTGTCGTAAATTTGCGTGCTGATTGACCATGATGCTGCAGCAAATCTGCAAATGCAAGTGTAACCAAAGAGCGTATAGGTTGTTCTGTACTTTTCTGATAAGAAATCGCATCTGAAAGGAGAAGGAAATAATTAAAAAAAAAAACAGTTTGGCTGTTTGTTTGTCCTCACCTAGAGAGACGCACCGAGGACCACAAAATCCCATGCAACCAAAGAATCATTCGCCACAAAGTATTCGCTCCTCCACCTCGCACCCTCGAACAGCGCGCACGATGCTAACCCGAACACAAAAAAAAAAAATCACAAAAGTACAACACCTACCATGACATGTGGGTCCCACTACACGTGGGGCCCCACGGATCAGTCGCACGGCTCACGTACAGGCCGGTACAGCGCCCCCGCGCGGGCCCCAGCGCATAGACAGGTGGGGCCCACGGCTTCCCTCACGGGACAAACAAACACGCCACAGGAGTCGCCATTTCACGTGCG(SEQ?ID?NO：1)。

In one embodiment, the present invention relates to contain and be selected from following any one group and have the promotor of the nucleotide sequence of promoter function:

Nucleotide sequence shown in a, the SEQ ID NO:1;

B, with SEQ ID NO:1 complementary nucleotide sequence;

C, under high stringent condition can with the nucleotide sequence of the nucleotide sequence hybridization of above-mentioned a or b;

D, nucleotide sequence shown in above-mentioned a or the b carried out displacement, disappearance, the interpolation modified nucleotide sequences of one or more bases; And

E, the nucleotide sequence that has at least 90% identity with nucleotide sequence shown in above-mentioned a or the b.

In second aspect, the invention provides a kind of nucleic acid construct, the gene order that said nucleic acid construct comprises promotor of the present invention and can be operatively connected with promotor, wherein said promotor is identical or different with said gene order source.

In the third aspect, the invention provides a kind of carrier, said carrier comprises promotor of the present invention or nucleic acid construct.In one embodiment, said plasmid is pMD18-T plasmid or p8 plasmid.In one embodiment, said promotor or nucleic acid construct sequence are between KpnI restriction enzyme site and SbfI restriction enzyme site.

In fourth aspect, the invention provides a kind of reconstitution cell that comprises promotor of the present invention, nucleic acid construct or carrier.In one embodiment, said reconstitution cell is recombinant Bacillus coli cells or reorganization agrobatcerium cell.In another embodiment, said bacterial strain is agrobacterium tumefaciens EHA105-P535.In one embodiment, the invention provides a kind of plant callus, plant explants or plant that comprises promotor of the present invention, nucleic acid construct, carrier or reconstitution cell.In one embodiment, said plant callus is the rice callus tissue.In one embodiment, said plant is monocotyledons or dicotyledons, preferred Oryza or Nicotiana, more preferably paddy rice or tobacco, the most preferably fine or tobacco NC89 of paddy rice Japan.

In aspect the 5th, the invention provides the method for the promoter sequence shown in a kind of SEQ of preparation ID NO:1, comprising: with the fine genomic dna of paddy rice Japan is template, uses a pair of amplimer to increase.In one embodiment, said amplimer designs to head and the tail respectively according to the sequence of SEQ ID NO:1 in the fine gDNA of paddy rice Japan.In another embodiment, said amplimer is SEQ ID NO:2 and SEQ ID NO:3.In one embodiment, said amplimer 5 ' end also connects one section restriction enzyme site and/or protection base.For example, said amplimer is SEQ ID NO:4 and SEQ ID NO:5, and wherein 5 ' of SEQ ID NO:4 and SEQID NO:5 end contains KpnI restriction enzyme site and SbfI restriction enzyme site respectively.

In aspect the 6th, the invention provides the purposes of the sequence shown in the SEQ ID NO:1 as promotor.In one embodiment, said purposes is the purposes of destination gene expression in the regulation and control plant, and said goal gene is GUS preferably.In another embodiment, said plant is monocotyledons or dicotyledons, preferred Oryza or Nicotiana, more preferably paddy rice or tobacco, the most preferably fine or tobacco NC89 of paddy rice Japan.

In aspect the 7th; The invention provides a kind of regulate gene expression method; Said method comprises step: promotor of the present invention, nucleic acid construct, carrier or reconstitution cell are converted into plant, preferably are converted into plant callus, make said promotor be positioned at 5 ' end of said gene.In one embodiment, said plant is monocotyledons or dicotyledons, preferred Oryza or Nicotiana, more preferably paddy rice or tobacco, the most preferably fine or tobacco NC89 of paddy rice Japan.

For realizing the purpose of above-mentioned regulate 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.

In eight aspect; The invention provides a kind of method for preparing transgenic plant; Be included under the condition of effective generation plant and cultivate reconstitution cell of the present invention, plant callus, plant explants or plant, said plant is monocotyledons or dicotyledons, preferred Oryza or Nicotiana; More preferably paddy rice or tobacco, the most preferably fine or tobacco NC89 of paddy rice Japan.

In aspect the 9th; The invention provides the purposes that promotor of the present invention, nucleic acid construct, carrier, reconstitution cell, plant callus, plant explants or plant are used to regulate and control plant destination gene expression and breeding; Said plant is monocotyledons or dicotyledons; Preferred Oryza or Nicotiana, more preferably paddy rice or tobacco, the most preferably fine or tobacco NC89 of paddy rice Japan.

Preservation information

Culture title: tobacco NC89 seed.

Preservation date: on November 12nd, 2010.

Depositary institution: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center, promptly Chinese typical culture collection center (CCTCC).

Deposit number: CCTCC NO:P201017.

Description of drawings

Fig. 1 illustrates the collection of illustrative plates of plasmid pCAMBIA-1301.

Fig. 2 illustrates the collection of illustrative plates of the p8 plasmid that contains MCS that needs make up and GUS sequence.

Fig. 3 illustrates rice callus and organizes the painted photo of gus.Organize with the rice callus of the Agrobacterium tumefaciens mediated conversion of reorganization of the p8+P535 recombinant vectors that contains promotor and to present blueness (right side) after dyed.As contrast, through the p8 plasmid that does not the contain promotor rice callus tissue color after GUS dyeing of Agrobacterium tumefaciens mediated conversion do not change (left side) of recombinating.

Fig. 4 illustrates the painted photo of root gus of paddy rice transgenic seedling.Present blueness (right side) with the root of the paddy rice seedling of the Agrobacterium tumefaciens mediated conversion of reorganization of the p8+P535 recombinant vectors that contains promotor after dyed.As contrast, through the p8 plasmid that does not the contain promotor root color after GUS dyeing of paddy rice seedling of Agrobacterium tumefaciens mediated conversion do not change (left side) of recombinating.

Fig. 5 illustrates paddy rice transgenic seedling leaf gus dyeing photo.Present blueness (right side) with the leaf of the paddy rice seedling of the Agrobacterium tumefaciens mediated conversion of reorganization of the p8+P535 recombinant vectors that contains promotor after dyed.As contrast, through the p8 plasmid that does not the contain promotor leaf color after GUS dyeing of paddy rice seedling of Agrobacterium tumefaciens mediated conversion do not change (left side) of recombinating.

Fig. 6 illustrates the painted Photomicrograph of leaf gus (opticmicroscope, * 3) of transgene tobacco.Present blueness (right side) after the leaf of the tobacco that transforms with reorganization agrobacterium tumefaciens EHA105-P535 is dyed.The leaf of contrast tobacco color after GUS dyeing do not change (left side).

Fig. 7 illustrates the painted Photomicrograph of transgene tobacco root gus (opticmicroscope, * 3).Present blueness (right side) with the root of the tobacco of reorganization agrobacterium tumefaciens EHA105-P535 mediated transformation after dyed.The root of contrast tobacco color after GUS dyeing do not change (left side).

Embodiment

In first aspect, the invention provides a promoter gene BgIosP535 with promoter activity.In one embodiment, the present invention relates to contain and be selected from following any one group and have the promotor of the nucleotide sequence of promoter function:

Nucleotide sequence shown in a, the SEQIDNO:1;

B, with SEQIDNO:1 complementary nucleotide sequence;

C, under high stringent condition can with the nucleotide sequence of the nucleotide sequence hybridization of above-mentioned a or b;

D, nucleotide sequence shown in above-mentioned a or the b carried out displacement, disappearance, the interpolation modified nucleotide sequences of one or more bases;

E, the nucleotide sequence that has at least 90% identity with nucleotide sequence shown in above-mentioned a or the b.

In the present invention, described promotor derives from monocotyledons.Particularly, said monocotyledons is a paddy rice, and for example said paddy rice is a Japan fine (Oryza sativaL.ssp.japonica cv.Nipponbare).Promotor of the present invention can the expression of regulatory gene in plant, particularly monocotyledons.In one embodiment of the invention, said monocotyledons is an Oryza.In another embodiment of the present invention, said Oryza is a paddy rice.In another embodiment of the present invention, said is that paddy rice Japan is fine.

The all right controlling gene of promotor of the present invention is in dicotyledons, and said dicotyledons has for example tobacco, soybean, yam, broad bean, radish, peanut etc.Tobacco is typical genetically engineered model plant.Select tobacco to carry out transgenic research in an embodiment, to verify the effect of promotor of the present invention in dicotyledons.Experimental result shows that this promotor can work in transgene tobacco.

The method of the complementary sequence of a nucleotide sequence of the known acquisition of those skilled in the art.And; In order to realize the regulation and control of promotor to gene; Can the complementary sequence of said gene be connected to 5 ' formation nucleic acid construct of the complementary sequence of said promotor; Obtain the complementary sequence of said nucleic acid construct then, thereby can utilize the complementary sequence of said nucleic acid construct to realize the regulation and control of promotor a gene.

Utilizing the evaluation and the examination of Nucleotide hybridization carrying out Nucleotide is well known to a person skilled in the art." stringency " degree of used condition was classified when typically, " hybridization conditions " was according to measurement hybridization.It is foundation that the stringency degree can combine the melting temperature(Tm) (Tm) of mixture or probe with for example nucleic acid.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.Alternatively, perhaps further, hybridization conditions can with salt or the ionic strength conditions of hybridization and/or one or repeatedly stringency washing 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.Say from function, can adopt maximum stringency condition to confirm and the tight same or near tight same nucleotide sequence of hybridization probe; And adopt high stringency condition to confirm 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 Harbor Press, 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 * with 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, said under high stringent condition with the nucleotide sequence of 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; Said displacement, 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 at the 5 ' end and/or the 3 ' end of said nucleotide sequence, and/or sequence inside for example is no more than 2-45, perhaps is 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 displacement, 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, said displacement, disappearance, the interpolation modified nucleotide sequences that nucleotide sequence shown in the SEQ ID NO:1 is carried out like above-mentioned one or more bases has and the same or analogous promoter activity of nucleotide sequence shown in the SEQ ID NO:1.

Describe through a kind of polynucleotide; The nucleotide sequence that it had for example with the reference nucleotide sequence of SEQ ID NO:1 at least " identity " of tool 95% be meant: 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 by deletion or by another nucleotide substitution in the reference sequences; Maybe can some Nucleotide be inserted in reference sequences, the Nucleotide that wherein inserts can reach reference sequences total nucleotide 5%; Or in some Nucleotide, have deletion, insert and the combination of replacement, 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; The algorithm that is used for confirming 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 definite nucleotide sequence homology percentage ratio of the present invention.Carrying out software that BLAST analyzes can be obtained by the public through state-run biotechnology information center.

In the present invention; The nucleotide sequence that nucleotide sequence shown in said and the SEQ ID NO:1 has at least 90% sequence identity comprises and the same basically 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 said 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.

In second aspect, the invention provides a kind of nucleic acid construct, the gene order that said nucleic acid construct comprises promotor of the present invention and can be operatively connected with promotor, wherein said promotor is identical or different with said gene order source.

In this article, said nucleic acid construct can comprise the promoter sequence shown in the SEQ ID NO:1 or the nucleotide sequence of its complementary sequence.Said nucleic acid construct can prepare through the promoter sequence shown in the SEQ ID NO:1 or its complementary sequence are operably connected with other required sequences.Perhaps, said nucleic acid construct can be through directly synthetic preparation.

In the third aspect, the invention provides a kind of carrier, said carrier comprises promotor of the present invention or nucleic acid construct.In one embodiment, said plasmid is pMD18-T plasmid or p8 plasmid.In one embodiment, said promotor or nucleic acid construct sequence are between KpnI restriction enzyme site and SbfI restriction enzyme site.The method that makes up plasmid is known in this area.For example, pMD18-T plasmid and p8 plasmid have been made up in an embodiment of the present invention.

The cloning vector that is suitable for making up recombinant vectors according to the invention includes but not limited to, for example: pUC18, pUC19, pUC118, pUC119, pMD19-T, pMD20-T, pMD18-T Simple Vecter, pMD19-T SimpleVecter 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 fourth aspect, the invention provides a kind of reconstitution cell that comprises promotor of the present invention, nucleic acid construct or carrier.In one embodiment, said reconstitution cell is recombinant Bacillus coli cells or reorganization agrobatcerium cell.The method that plasmid is imported bacterium is as known in the art.For example, said reconstitution cell can be converted into host cell through the said recombinant vectors that will contain promotor of the present invention and obtains.In an embodiment of the present invention, plasmid is imported into agrobacterium tumefaciens (Agrobacterium tumefaciens), specifically is agrobacterium tumefaciens EHA105-P535.

The host cell that is suitable for making up reconstitution cell according to the invention includes but not limited to, for example: agrobacterium tumefaciens cell LBA4404, EHA105, GV3101 etc.

In aspect the 5th, the invention provides the method for the promoter sequence shown in a kind of SEQ of preparation ID NO:1, comprising: with the fine genomic dna of paddy rice Japan is template, uses a pair of amplimer to increase.

Can use that known method prepares the promotor shown in the SEQ ID NO:1 among the present invention, for example the direct de novo synthesis of chemical synthesis or from genome the said promoter sequence of amplification.In one embodiment, said amplimer designs to head and the tail respectively according to the sequence of SEQ ID NO:1 in the fine gDNA of paddy rice Japan.It is right that those skilled in the art can design corresponding pcr amplification primer according to the base complementrity principle according to purpose nucleotide sequence to be amplified.For example, primer of the present invention can be CGTGTGTGAGGTGTCCGAAGTA (SEQIDNO:2) and CGCACGTGAAATGGCGACTCC (SEQ IDNO:3).In one embodiment, said amplimer 5 ' end also connects one section restriction enzyme site and/or protection base.For example, the amplimer that uses in an embodiment is SEQ ID NO:4 and SEQ ID NO:5, and wherein 5 ' of SEQ ID NO:4 and SEQ ID NO:5 end contains KpnI restriction enzyme site and SbfI restriction enzyme site respectively.Connecting one section restriction enzyme site and/or protect base at amplimer 5 ' end is method commonly used in the gene amplification, and those skilled in the art can realize through conventional method.

In aspect the 6th, the invention provides the purposes of the sequence shown in the SEQ ID NO:1 as promotor.In one embodiment, said purposes is the purposes of destination gene expression in the regulation and control plant, and said goal gene is GUS preferably.In another embodiment, said plant is monocotyledons or dicotyledons, preferred Oryza or Nicotiana, more preferably paddy rice or tobacco, the most preferably fine or tobacco NC89 of paddy rice Japan.

In aspect the 7th; The invention provides a kind of regulate gene expression method; Said method comprises step: promotor of the present invention, nucleic acid construct, carrier or reconstitution cell are converted into plant, preferably are converted into plant callus, make said promotor be positioned at 5 ' end of said gene.In one embodiment, said plant is monocotyledons or dicotyledons, preferred Oryza or Nicotiana, more preferably paddy rice or tobacco, the most preferably fine or tobacco NC89 of paddy rice Japan.

Nucleotide sequence, structure nucleic acid construct, plasmid or bacterium are imported plant can use method commonly used in this area.For example, 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 according to the 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.The method of the transforming monocots that can also adopt in addition, 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.

For realizing the purpose of above-mentioned regulation and control destination gene expression, promotor according to the 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.

In eight aspect; The invention provides a kind of method for preparing transgenic plant; Be included under the condition of effective generation plant and cultivate reconstitution cell of the present invention, plant callus, plant explants or plant, said plant is monocotyledons or dicotyledons, preferred Oryza or Nicotiana; More preferably paddy rice or tobacco, the most preferably fine or tobacco NC89 of paddy rice Japan.

In aspect the 9th, the invention provides the purposes that promotor of the present invention, nucleic acid construct, carrier, reconstitution cell, plant callus or plant are used to regulate and control plant destination gene expression and breeding.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.For example, can use gene recombination method commonly used in this area, promotor of the present invention is imported the target gene 5 ' end of hoping in the Plant Genome that expression amount increases, realize that the expression of target gene increases.

In the present invention, term " monocotyledons " particularly, can be grass, more specifically, can include but not limited to for oryza plant paddy rice for example, for example includes but not limited to paddy rice, wheat, corn, millet, sugarcane, Chinese sorghum, barley etc.

In the present invention; Term " 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 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 (above-mentioned rice varieties all can available from emblem, Anhui good fortune ltd of merchant farmers') etc.

In the present invention, term " dicotyledons ", particularly; Can be plant of Solanaceae; More specifically, can be Nicotiana plant (tobacco), include but not limited to K326, K346, K394, NC82, NC89, G140, G28, G80, middle cigarette 90, Coker176, CV87 or the like

Embodiment

To combine specific embodiment that embodiment of the present invention are described in detail below, but it will be understood to those of skill in the art that the following example only is used to explain the present invention, and should not be regarded 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 through the conventional products of commercial acquisition.

The pcr amplification of embodiment 1:P535 promoter fragment and the structure of pMD18-T+P535 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) extract paddy rice Japan fine (be 200910238992.0 at application number, denomination of invention is preservation in the invention application of " a kind of promotor BgIosP587, Preparation Method And The Use "; And it is open on September 22nd, 2010; Deposit number is CCTCC P200910) genomic dna; According to the sequence of this promotor in the fine gDNA of paddy rice Japan, (upstream primer F1 adds restriction enzyme site KpnI and protection base to design the one couple of PCR specificity amplification primer at head and the tail respectively; Downstream primer R1 adds restriction enzyme site SbfI 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 preparatory sex change 5 minutes, and then with 94 ℃ of sex change 45 seconds, 55 ℃ of annealing 50 seconds, 72 ℃ were extended 90 seconds, carried out 35 reaction cycle, and last 72 ℃ were extended 7 minutes.

Wherein, upstream primer F1:GG GGTACCGTGTGTGAGGTGTCCGAAGTA, wherein underscore is represented the KpnI restriction enzyme site, (SEQ ID NO:4).

Downstream primer R1:TG CCTGCAGGCGCACGTGAAATGGCGACTCC, wherein underscore is represented the SbfI restriction enzyme site, (SEQ ID NO:5).

Pcr amplification product separates through 1.0% agarose gel electrophoresis, obtains size and is the band about 1380bp, and use TIANGEN sepharose DNA reclaims test kit (catalog number (Cat.No.): DP209-03) carry out purifying and recovering.

The structure of pMD18-T+P535 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, the order-checking of picking positive colony, with sequence shown in the SEQ ID NO:1 relatively, prove that it is accurately.

Wherein, T/A clone's condition of contact is following:

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 complements to 10 μ l

Under 16 ℃, (the new sesame in Ningbo SDC-6) the middle connection more than 8 hours, obtains the pMD18-T+P535 recombinant vectors at the energy-conserving intelligent thermostatic bath.To pass through product after the above-mentioned connection according to following method transformed into escherichia coli:

(Dong Yang of Kunming Institute of Zoology, Chinese Academy of Sciences provides from Ultralow Temperature Freezer, 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); Perhaps can give birth to the worker available from for example Shanghai), after melting, add 10 μ l as above the connection product of gained, i.e. pMD18-T+P535 recombinant vectors on ice; Stir ice bath 30 minutes, 42 ℃ of heat shocks 60 seconds, ice bath 5 minutes gently; Add 600 μ l the SOC of 4 ℃ of following precoolings substratum (concrete prescription sees " molecular cloning experiment guide ", the third edition, Science Press for details), under 37 ℃, recovered 45 minutes in 220rpm; Centrifugal 30 seconds of 8000rpm removes supernatant, leaves and takes 150 μ l; With the mixture of 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 " for details; The third edition, Science Press), be inverted for 37 ℃ and cultivated 16 hours～24 hours.Acquisition contains the recombination bacillus coli of pMD18-T+P535 cloning vector, called after DH5 α-P535.Shenzhen Huada Genetic Technology Co., Ltd checks order to the P535 in the pMD18-T+P535 cloning vector.

Sequencing result shows that the P535 promoter sequence is identical with SEQ IDNO:1 correct in the pMD18-T+P535 cloning vector of acquisition.

Embodiment 2: the structure of carrier-p8+P535 recombinant vectors

(catalog number (Cat.No.): operational manual DP103-03), the conversion that makes up from embodiment 1 have bacillus coli DH 5 alpha-P535 of promotor P535 and extract the cloning vector pMD18-T+P535 that has P535 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 KpnI and SbfI behind the purifying 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+P535 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 cultivation 16～24 hours down at 37 ℃; 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 CAMBIA Bios (biological open source) Licensee; Australia) transform and make up according to following mode, specify as follows:

With Kpn I/Nco I (NEB) double digestion plasmid pCAMBIA-1301 (referring to Fig. 1), reclaim big fragment.According to synthetic following sequence: the GGTACCAAGCTTACTAGTCCTGCAGGTCTAGAGGATCCGTCGACCATGG (SEQ IDNO:6) (restriction enzyme site that comprises is Kpn I/HindIII/Spe I/Sbf I/Pst I/Xba I/BamH I/SalI/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) available from for example Beijing Suo Laibao Science and Technology Ltd..Screen transformant with primer GCTTCCGGCTCGTATGTTGT (SEQ ID NO:7)/GAGTCGTCGGTTCTGTAAC (SEQ ID NO:8); Through the PCR detection method, the transformant that has amplified fragments and be 350bp is the transformant (referring to Fig. 2) of the p8 plasmid that contains MCS that needs make up and GUS sequence.

Length 2353 bases of MCS in the said p8 plasmid and GUS sequence, shown in SEQ ID NO:9:

GTTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTAT

GTGGAATTGTGAGCGGATAACAATTTCA

GTCCGTCCTGTAGAAACCCCAACCCGTGAAATCAAAAAACTCGACGGCCTGTGGGCATTCAGTCTGGATCGCGAAAACTGTGGAATTGATCAGCGTTGGTGGGAAAGCGCGTTACAAGAAAGCCGGGCAATTGCTGTGCCAGGCAGTTTTAACGATCAGTTCGCCGATGCAGATATTCGTAATTATGCGGGCAACGTCTGGTATCAGCGCGAAGTCTTTATACCGAAAGGTTGGGCAGGCCAGCGTATCGTGCTGCGTTTCGATGCGGTCACTCATTACGGCAAAGTGTGGGTCAATAATCAGGAAGTGATGGAGCATCAGGGCGGCTATACGCCATTTGAAGCCGATGTCACGCCGTATGTTATTGCCGGGAAAAGTGTACGTATCACCGTTTGTGTGAACAACGAACTGAACTGGCAGACTATCCCGCCGGGAATGGTGATTACCGACGAAAACGGCAAGAAAAAGCAGTCTTACTTCCATGATTTCTTTAACTATGCCGGAATCCATCGCAGCGTAATGCTCTACACCACGCCGAACACCTGGGTGGACGATATCACCGTGGTGACGCATGTCGCGCAAGACTGTAACCACGCGTCTGTTGACTGGCAGGTGGTGGCCAATGGTGATGTCAGCGTTGAACTGCGTGATGCGGATCAACAGGTGGTTGCAACTGGACAAGGCACTAGCGGGACTTTGCAAGTGGTGAATCCGCACCTCTGGCAACCGGGTGAAGGTTATCTCTATGAACTCGAAGTCACAGCCAAAAGCCAGACAGAGTCTGATATCTACCCGCTTCGCGTCGGCATCCGGTCAGTGGCAGTGAAGGGCCAACAGTTCCTGATTAACCACAAACCGTTCTACTTTACTGGCTTTGGTCGTCATGAAGATGCGGACTTACGTGGCAAAGGATTCGATAACGTGCTGATGGTGCACGACCACGCATTAATGGACTGGATTGGGGCCAACTCCTACCGTACCTCGCATTACCCTTACGCTGAAGAGATGCTCGACTGGGCAGATGAACATGGCATCGTGGTGATTGATGAAACTGCTGCTGTCGGCTTTCAGCTGTCTTTAGGCATTGGTTTCGAAGCGGGCAACAAGCCGAAAGAACTGTACAGCGAAGAGGCAGTCAACGGGGAAACTCAGCAAGCGCACTTACAGGCGATTAAAGAGCTCGATAGCGCGTGACAAAAACCACCCAAGCGTGGTGATGTGGAGTATTGCCAACGAACCGGATACCCGTCCGCAAGGTGCACGGGAATATTTCGCGCCACTGGCGGAAGCAACGCGTAAACTCGACCCGACGCGTCCGATCACCTGCGTCAATGTAATGTTCTGCGACGCTCACACCGATACCATCAGCGATCTCTTTGATGTGCTGTGCCTGAACCGTTATTACGGATGGTATGTCCAAAGCGGCGATTTGGAAACGGCAGAGAAGGTACTGGAAAAAGAACTTCTGGCCTGGCAGGAGAAACTGCATCAGCCGATTATCATCACCGAATACGGCGTGGATACGTTAGCCGGGCTGCACTCAATGTACACCGACATGTGGAGTGAAGAGTATCAGTGTGCATGGCTGGATATGTATCACCGCGTCTTTGATCGCGTCAGCGCCGTCGTCGGTGAACAGGTATGGAATTTCGCCGATTTTGCGACCTCGCAAGGCATATTGCGCGTTGGCGGTAACAAGAAAGGGATCTTCACTCGCGACCGCAAACCGAAGTCGGCGGCTTTTCTGCTGCAAAAACGCTGGACTGGCATGAACTTCGGTGAAAAACCGCAGCAGGGAGGCAAACAAGCTAGCCACCACCACCACCACCACGTGTGA(SEQ?ID?NO：9)。

As above constructed p8 plasmid among the present invention shown in the sequence; EcoR I/Sac I/Kpn I/HindIII/Spe I/SbfI/Pst I/Xba I/BamH I/Sal I/Nco I restriction enzyme site in its MCS is respectively with adding frame and underscore is represented; Used primer GCTTCCGGCTCGTATGTTGT (SEQ ID the NO:7)/GAGTCGTCGGTTCTGTAAC (SEQID NO:8) of screening transformant representes with double underline; The GUS sequence representes that with italic its intron sequences adds shading with italic respectively and illustrates.

The structure of recombinant vectors p8+P535

According to restriction enzyme KpnI and SbfI operation instructions, handle resulting cloning vector pMD18-T+P535 among the embodiment 1 according to following condition, and the p8 plasmid that makes up as stated.

Wherein, the enzyme tangent condition of cloning vector pMD18-T+P535 and p8 plasmid is following:

Enzyme is cut system: 50 μ l

Sterile distilled water 34.8 μ l

10×buffer?H 5μl

KpnI 0.1μl(10U)

SbfI 0.1μl(10U)

Cloning vector pMD18-T+P535 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 P535 inserts fragment, according to the T4 ligase enzyme (TaKaRa, D2011A) operation instructions connect according to following condition:

Linked system: 10 μ l

10 * T4 damping fluid, 1 μ l

P8 plasmid 1 μ l (20ng) after enzyme is cut

The promotor P535 that reclaims inserts fragment 10～20ng, confirms according to its concentration

Sterile distilled water complements to 9.5 μ l

T4 ligase enzyme (TaKaRa, D2011A) 0.5 μ l

The T4 damping fluid melts on ice, the about 20ng of p8 plasmid vector add-on after enzyme is cut, and the P535 fragment among the present invention adds 10ng.(the new sesame in Ningbo, SDC-6) middle connection is more than 8 hours 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, stirs gently; Ice bath 30 minutes, 42 ℃ of heat shocks 60 seconds, ice bath 5 minutes adds the SOC of 4 ℃ of precoolings of 600 μ l; Recovered 45 minutes with 220rpm at 37 ℃, centrifugal 30 seconds of 8000rpm removes supernatant, leaves and takes 150 μ l; Blow evenly gently, granulated glass sphere coating LB (kan) is inverted for 37 ℃ and cultivated 16～24 hours.Obtain recombinant vectors p8+P535.

Be that primer carries out the PCR detection to gained recombinant vectors p8+P535 with F1 (being SEQ ID NO:4) and R1 (being SEQ ID NO:5) respectively, to contain required promotor P535 among the conclusive evidence gained recombinant vectors p8+P535.Cut screening through KpnI and SbfI enzyme and contain recombinant vectors p8+P535 transformant.

The preparation of embodiment 3 reorganization agrobacterium tumefaciens EHA105-P535 cells

To transform respectively according to " molecular cloning the experiment guide " (third edition like the p8+P535 recombinant vectors of embodiment 2 said methods structures with as the p8 plasmid that contrasts; The agrobacterium tumefaciens EHA105 of the method for calcium chloride Science Press) preparation (be 200910238992.0 at application number, denomination of invention is preservation in the invention application of " a kind of promotor BgIosP587, Preparation Method And The Use "; And it is open on September 22nd, 2010; Deposit number is CCTCC M 209315) competent cell, concrete grammar is following:

EHA105 takes out in Ultralow Temperature Freezer with the agrobacterium tumefaciens competent cell, places on ice and thaws.After the thawing, add the p8+P535 recombinant vectors of 5 μ l and the p8 empty carrier of p8 plasmid and conduct contrast, mixing gently respectively; Ice bath 10 minutes was put into liquid nitrogen freezing 5 minutes, and 37 ℃ thawed 5 minutes; The LB liquid nutrient medium that adds 800 μ l normal temperature rocks recovery 3 hours with 160rpm under 28 ℃, left 30 seconds with 8000rpm; Supernatant is removed in suction; It is even to stay the 200l pressure-vaccum, coats to be added with kantlex and Rifampin (50mg/l Kan (specifically fills a prescription referring to table 2) on YM solid medium flat board 10mg/lRif).Be inverted for 28 ℃ and cultivated 2-3 days.

With F1 (being SEQ ID NO:4) and R1 (being SEQ ID NO:5) is that primer carries out the PCR detection and cuts the screening transformant through Kpn I/SbfI enzyme.

What pcr amplification went out that about 1380bp left and right sides band and enzyme cut out about 1380bp left and right sides band is the reorganization agrobacterium tumefaciens of recombinant vectors p8+P535.

Among the present invention, according to the reorganization Agrobacterium that has recombinant vectors p8+P535 that obtains like above-mentioned method, called after reorganization agrobacterium tumefaciens EHA105-P535.

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.

Embodiment 4: the inducing and transforming of rice callus tissue

According to following steps inducing paddy rice callus, and transform said callus with reorganization agrobacterium tumefaciens EHA105-P535 and reorganization agrobacterium tumefaciens EHA105-p8 respectively.

1) the fine seed of paddy rice Japan is shelled, 70% ethanol surface sterilization 30 seconds, then with the Youxiaolin sterilization of available chlorine 1.5% 30 minutes, during acutely shake, the sterilization back is with aqua sterilisa cleaning 5 times; Seed after the sterilization is placed on the N6D substratum (specifically filling a prescription referring to table 3), 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-P535 or reorganization agrobacterium tumefaciens EHA105-p8) of picking such as embodiment 3 respectively; In adding kantlex and Rifampin (50mg/lKan; 10mg/l Rif) on the YM solid medium 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 30mlAAM substratum (specifically filling a prescription) referring to table 4, gentle resuspended said reorganization agrobacterium tumefaciens cell (reorganization agrobacterium tumefaciens EHA105-P535 or reorganization agrobacterium tumefaciens EHA105-p8);

4) callus with succeeding transfer culture places the sterilization petridish; To pour in the petridish like the reorganization agrobacterium tumefaciens suspension of step 3 preparation, callus will be immersed wherein 15 minutes;

5) outwell reorganization agrobacterium tumefaciens suspension, callus is sopped up excess liquid with sterilization thieving paper; On N6-AS substratum (specifically filling a prescription), 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 referring to table 5; The sealing petridish, 28 ℃ of dark cultivations 48～60 hours;

6) infected callus is placed the 50ml sterile tube, shake cleaning, become clarification until supernatant with aqua sterilisa; Callus is soaked in the sterile distilled 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 HYG (HmB) and 50mg/l Carb; With sealing the film phonograph seal petridish, 29.5 ℃ of 3～4 weeks of illumination cultivation.

Embodiment 5: the expression of the GUS in the rice callus tissue

For detecting expression through goal gene GUS in the rice callus tissue of embodiment 4 said 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 tissue that transforms with reorganization agrobacterium tumefaciens EHA105-P535 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 0.2MNaH ₂PO ₄Solution and 10 μ l 0.1M X-gluc.

The rice callus tissue that transforms with reorganization agrobacterium tumefaciens EHA105-P535 or reorganization agrobacterium tumefaciens EHA105-p8 respectively is immersed in the GUS staining fluid; 37 ℃ of insulations are blue to occurring; Take pictures; The result is as shown in Figure 3; After the rice callus tissue (Fig. 3 is right) of the Agrobacterium tumefaciens mediated conversion of reorganization of the p8+P535 recombinant vectors that contains promotor is dyed, present blueness, recombinating through the p8 plasmid that does not contain promotor, color does not change after GUS dyes for the rice callus tissue (as contrast, Fig. 3 left side) of Agrobacterium tumefaciens mediated conversion.The result shows that P535 promotor of the present invention is expressed gus gene has regulating and controlling effect.

Embodiment 6: the expression of GUS in the transgenic paddy rice seedling

The callus that obtains among the embodiment 4 is transferred to MS-R division culture medium (concrete prescription is seen table 6) the differentiation seedling that contains 50mg/l HYG (HmB); With sealing the film phonograph seal petridish, 29.5 ℃ of illumination cultivation 3-4 weeks; Treat to transfer to when seedling grows to 3-4cm 1/2MS root media (the specifically filling a prescription) screening of taking root that contains 50mg/l HYG (HmB) referring to table 7.

The GUS dyeing course of transgenic paddy rice seedling is with the dyeing course of callus among the embodiment 5.The result is like Fig. 4, shown in 5; After root, the leaf (Fig. 4,5 right sides) of the paddy rice seedling of the Agrobacterium tumefaciens mediated conversion of reorganization of the p8+P535 recombinant vectors that contains promotor are dyed, present blueness; Do not change through recombinate root, leaf (as contrast, Fig. 4, the 5 left sides) color after GUS dyeing of paddy rice seedling of Agrobacterium tumefaciens mediated conversion of the p8 plasmid that does not contain promotor.The result shows that P535 promotor of the present invention is expressed gus gene has regulating and controlling effect.

Embodiment 7: GUS expresses in the transgene tobacco seedling

1. the tobacco aseptic seedling obtains:

● tobacco NC89 seed (was preserved in Chinese typical culture collection center (CCTCC) on November 12nd, 2010; Deposit number is CCTCC NO:P201017, and the depositary institution address is a China. Wuhan. and Wuhan University, postcode 430072) soak: with tobacco seed pack into (＜50/pipe) in the centrifuge tube of 1.5ml; Add the 1ml sterile distilled water; Beat several times with the pipettor suction, behind the replacing sterile distilled water, soaked at normal temperatures 24 hours;

● tobacco seed sterilization:, add alcohol-pickled 30 seconds of 1ml 75%, with pipettor sucking-off alcohol with the water of pipettor sucking-off immersion seed; Add 1ml10%H ₂O ₂Soak after 10 minutes, with pipettor sucking-off H ₂O ₂

● washing: clean five times with sterile distilled water, add the 1ml sterile distilled water at every turn and shook 1 minute;

● inoculation: aseptic filter paper blots the moisture of seed-coat; Being inoculated in MS solid medium (concrete prescription is seen table 8) with suction nozzle or aseptic toothpick again goes up and sprouts; About 10 of every ware; Place 28 ℃ of illumination cultivation chambers (16 little time/8 hour dark) to cultivate a week, intensity of illumination is 2000lx (all illumination cultivation materials of this experiment is all cultivated under this intensity of illumination);

● switching: after waiting to grow seedling, change fresh MS solid medium over to, every bottle of (Ф 6cm, 30ml substratum/bottle) 3 strain tobacco seedlings, acquisition tobacco aseptic seedling is cultivated about 5 weeks in 28 ℃ of illumination cultivation chambers (16 little time/8 hour dark).

2. the subculture of tobacco aseptic seedling is numerous with expansion

● cut off the blade and the root of tobacco aseptic seedling, cane is cut into the stem section (2cm-3cm) that has axillalry bud, then its morphology lower end vertically is inserted into fresh MS solid medium (axillalry bud can not insert in the substratum);

● stem explants that has axillalry bud of every bottle graft kind, place 28 ℃ of illumination cultivation chambers to cultivate about 5 weeks, use as material to be transformed.

3. the preparation of bacterium liquid before infecting:

● the single bacterium colony of the reorganization agrobacterium tumefaciens EHA105-P535 of picking embodiment 3 gained; Be inoculated into 10ml YM liquid nutrient medium and (contain Kan 50mg/L; Rif30mg/L) (specifically fill a prescription), under 28 ℃,, treat that bacterium liquid is muddy with the 250rpm shaken overnight referring to table 9; When also deposition not occurring, this bacterium liquid is put 4 ℃ of preservations;

● the bacterium liquid 20 μ l that go bail for and be stored in 4 ℃, (contain Kan 50mg/L, Rif30mg/L) in the 50ml centrifuge tube 28 ℃, the 250rpm shaken overnight treats that bacterium liquid is muddy, when also deposition occurring, stops to cultivate to be inoculated in 10ml YM liquid nutrient medium;

● get above-mentioned bacterium liquid 3ml and join 50ml YM liquid nutrient medium (specifically filling a prescription referring to table 9) in triangular flask 28 ℃, OD was cultivated in the concussion of 250rpm shaking table about 2 hours ₆₀₀During=0.5 left and right sides, use as infecting bacterium liquid.

4. infect:

● the bigger blade of clip on the tobacco aseptic seedling of cultivating for 5 weeks is kept in the petridish that the YM liquid nutrient medium is housed;

● the tapping and plugging machine with diameter 6mm breaks into leaf disc with tobacco leaf, is kept in another petridish that YM liquid nutrient medium is housed;

● the tobacco leaf disk transferred to be equipped with in the petridish that infects bacterium liquid;

● wave and culture ware gently, guarantee that Agrobacterium touches the leaf plate edge, soaked 10 minutes;

● the tobacco leaf disc that will infect is transferred to from agrobacterium suspension on the exsiccant aseptic filter paper, blots bacterium liquid till tobacco leaf disc does not drip bacterium liquid;

● tobacco leaf disc is inoculated on the RMOP solid medium (concrete prescription see table 10), the blade face up, about 10 of every ware;

● be inverted petridish, 28 ℃ of dark cultivations 3 days.

5. screening:

● the leaf disc of step 4 is transferred on the RMOP-TCH substratum (10mg/L Hyg) (concrete prescription see table 11), 10 in every ware, the blade face up, 28 ℃ of 2 weeks of illumination cultivation;

● per 2 all subcultures once, the bud of growing thickly appears in about 4 weeks.

6. take root:

● when regeneration bud grows to about 1-2cm, downcut bud and be inoculated into MST-TCH substratum (10mg/L Hyg) (concrete prescription is seen table 12), every bottle 1 strain tobacco seedling;

● 2 weeks were cultivated in 28 ℃ of illumination cultivation chambers;

Remove the little leaf of seedling base portion, transfer to fresh MST-TCH substratum (10mg/L Hyg), every bottle 1 strain tobacco seedling cultivated for 2 weeks.Get blade and root afterwards respectively and carry out GUS dyeing experiment, the same paddy rice of the prescription of GUS staining fluid and method.

The prescription of GUS staining fluid (1ml): 610 μ l 0.2MNa ₂HPO ₄Solution (pH=7.0); 390 μ l 0.2MNaH ₂PO ₄Solution and 10 μ l 0.1MX-gluc.

● root, the leaf of root, leaf and the contrast tobacco (not changing the empty carrier of goal gene over to) of transgene tobacco are immersed in respectively in the GUS staining fluid, and 37 ℃ of insulations are blue to occurring, (Fig. 6 and 7).The result is like Fig. 6, shown in 7, presents blueness with root, the leaf (Fig. 6,7 right sides) of the tobacco of reorganization agrobacterium tumefaciens EHA105-P535 mediated transformation after dyed, and the root of contrast tobacco, leaf (as contrast, Fig. 6,7 left sides) color after GUS dyeing does not change.

● employed relevant substratum is following in the embodiment of the invention:

With following the substratum of distilled water preparation, wherein alleged " the conventional sterilization " is meant the sterilization of following condition: 121 ℃ of following vapor sterilizations 20 minutes; With 1N Pottasium Hydroxide or 1N salt acid for adjusting pH value.

Table 2-YM solid medium (containing 50mg/L Kan, 10mg/L Rif) (every L):

Table 3-N6D substratum (every L):

N ₆Macro mother liquor (20 *): saltpetre 56.60g, potassium primary phosphate 8.00g, ammonium sulfate 9.26g, sal epsom 3.70g, calcium chloride 3.32g, zero(ppm) water is settled to 1L, and 4 ℃ of preservations are subsequent use;

N ₆Micro mother liquor (1000 *): potassiumiodide 0.80g, boric acid 1.60g, manganous sulfate 3.33g, zinc sulfate 1.50g, zero(ppm) water is settled to 1L, and 4 ℃ of preservations are subsequent use;

Molysite (Fe ₂EDTA) stock solution (100 *): 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 zero(ppm) water, 70 ℃ of temperature were bathed 2 hours, cooled off back 4 ℃ of preservations and were no more than 1 month;

N ₆VITAMINs stock solution (1000 *): vitamins B ₁0.10g, vitamins B ₆0.05g, nicotinic acid 0.05g, glycocoll 0.20g, adding distil water is settled to 100ml, filtration sterilization, 4 ℃ of preservations were no more than for 1 week.

Table 4-AAM substratum (every L):

AAM macro (10 *): 2.5g MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 (MgSO ₄7H ₂O), 1.5g Calcium dichloride dihydrate (CaCl ₂2H ₂O), 1.33g sodium dihydrogen phosphate dihydrate (NaH ₂PO ₄.2H ₂O), zero(ppm) water is settled to 1L, and 4 ℃ of preservations are subsequent use;

The single water manganous sulfate of AAM micro (100 *): 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), zero(ppm) water is settled to 1L, and 4 ℃ of preservations are subsequent use;

AAM organic (1000 *): 0.75g glycocoll (Glycine), 0.1g nicotinic acid (Nicotinic acid), 0.1gVB ₆(Pyridoxine), 1g VB ₁(Thiamine), zero(ppm) water is settled to 100ml, and 4 ℃ of preservations are subsequent use;

Molysite (Fe ₂EDTA) stock solution (100 *) is seen table 3.

Table 5-N6-AS substratum (every L):

N ₆Macro mother liquor (20 *), N ₆Micro mother liquor (1000 *), molysite (Fe ₂EDTA) stock solution (100 *) and N ₆VITAMINs stock solution (1000 *) is seen table 3.

Table 6-MS-R division culture medium (every L):

MS macro (20 *): an ammonium nitrate 33.0g, saltpetre 38.0g, potassium primary phosphate 3.4g, sal epsom 7.4g, calcium chloride 8.8g dissolves one by one, is settled to 1L with zero(ppm) water under the room temperature then, 4 ℃ of preservations;

MS micro (1000 *): manganous sulfate 16.90g, zinc sulfate 8.60g, boric acid 6.20g, potassiumiodide 0.83g, Sodium orthomolybdate 0.25g, copper sulfate 0.025g, NSC 51149 0.025g, mentioned reagent is at room temperature dissolved and is settled to 1L with zero(ppm) water, 4 ℃ of preservations;

MS VITAMINs stock solution (1000 *): vitamins B ₁0.010g, vitamins B ₆0.050g, nicotinic acid 0.050g, glycocoll 0.200g, adding distil water is settled to 100ml, filtration sterilization, 4 ℃ of preservations were no more than for 1 week;

Molysite (Fe ₂EDTA) stock solution (100 *) is seen table 3.

Table 7-1/2MS root media (every L):

MS macro (20 *) sees table 6;

MS micro (1000 *) and MS VITAMINs stock solution (1000 *) are seen table 6;

Molysite (Fe ₂EDTA) stock solution (100 *) is seen table 3.

Table 8-MS solid medium (every L):

Myo-inositol (500 *): the 5g inositol is dissolved in H ₂Behind the O, be settled to 100ml, 4 ℃ of preservations;

MS macro (20 *), MS micro (1000 *) and MS VITAMINs stock solution (1000 *) are seen table 6;

Molysite (Fe ₂EDTA) stock solution (100 *) is seen table 3;

MS organic (1000 *): VITMAIN B1,0.01g, Y factor, 0.05g, nicotinic acid B1,0.05g, glycocoll, 0.2g, adding distil water is settled to 100ml, filtration sterilization, 4 ℃ of preservations were no more than for 1 week.

Table 9-YM liquid nutrient medium (every L):

YM liquid nutrient medium (containing 50mg/L Kan, 10mg/L Rif): in refrigerative YM liquid nutrient medium, add 1ml kantlex (Kan) (50mg/ml), 0.2ml Rifampin (Rif) (50mg/ml).

Table 10-RMOP solid medium (every L):

MS Macro (20 *) and MS Micro (1000 *) see table 6;

Fe ₂EDTA stock solution (100 *) is seen table 3;

Myo-inositol (500 *) sees table 8.

Table 11-RMOP-TCH substratum (10mg/L Hyg) (every L)

MS Macro (20 *) and MS Micro (1000 *) see table 6;

Fe ₂EDTA stock solution (100 *) is seen table 3;

Myo-inositol (500 *) sees table 8.

Table 12-MST-TCH substratum (10mg/LHyg) (every L):

MS Macro (20 *) and MS Micro (1000 *) see table 6;

Fe ₂EDTA stock solution (100 *) is seen table 3;

Myo-inositol (500 *) sees table 8.

Although 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 appended claims and any equivalent thereof.

Claims (27)

1. promotor, said promotor are selected from following any one group and have the nucleotide sequence of promoter function:

Nucleotide sequence shown in a, the SEQ ID NO:1;

B, with SEQ ID NO:1 complementary nucleotide sequence.

2. a nucleic acid construct comprises the described promotor of claim 1, and the gene order that can be operatively connected with promotor, and wherein said promotor is identical or different with said gene order source.

3. carrier contains the nucleic acid construct of the promotor or the claim 2 of claim 1.

4. the carrier of claim 3, wherein said carrier are the pMD18-T plasmid or the p8 plasmid of nucleic acid construct that contains promotor or the claim 2 of claim 1.

5. a reconstitution cell contains the promotor of claim 1, the nucleic acid construct of claim 2 or the carrier of claim 3 or 4, and said reconstitution cell is recombinant Bacillus coli cells or reorganization agrobatcerium cell.

6. the reconstitution cell of claim 5, wherein said reconstitution cell is the reorganization agrobatcerium cell.

7. one group of primer is right, and said primer obtains the described promotor of claim 1 to being used for amplification, and it is characterized in that: two right primers of said primer contain the sequence shown in SEQ ID NO:2 and the SEQ ID NO:3 respectively.

8. the primer of claim 7 is right, and two right primers of wherein said primer also are connected with restriction enzyme site and/or protection base respectively at 5 ' end.

9. claim 7 or 8 primer are right, and two right primers of wherein said primer are respectively the sequence shown in SEQ ID NO:4 and the SEQ ID NO:5.

10. the method for preparing the promotor shown in the SEQ ID NO:1; Comprise: with the fine genomic dna of paddy rice Japan is template; Use a pair of amplimer to increase, said amplimer designs to head and the tail respectively according to the sequence of SEQ ID NO:1 in the fine genomic dna of paddy rice Japan.

11. the method for the promotor shown in the preparation SEQ ID NO:1 of claim 10, wherein used amplimer is that each described primer is right among the claim 7-9.

12. the method for a regulate gene expression, said method comprises step:

The carrier of the nucleic acid construct of the promotor of claim 1, claim 2, claim 3 or 4 or the reconstitution cell of claim 6 are imported plant, make said nucleotide sequence be positioned at 5 ' end of said gene.

13. the method for the regulate gene expression of claim 12, wherein said plant are plant callus or plant explants.

14. the method for the regulate gene expression of claim 12 or 13, wherein said plant are monocotyledons or dicotyledons.

15. the method for the regulate gene expression of claim 14, wherein said monocotyledons is an Oryza, and said dicotyledons is a Nicotiana.

16. the method for the regulate gene expression of claim 15, wherein said Oryza is a paddy rice, and said Nicotiana is a tobacco.

17. the method for the regulate gene expression of claim 16, wherein said paddy rice are that paddy rice Japan is fine, said tobacco is tobacco NC89.

18. a method for preparing transgenic plant is included in plant callus, plant explants or the plant of reconstitution cell of carrier or the claim 6 of the nucleic acid construct of cultivating a kind of promotor that contains claim 1, claim 2 under the condition of effective generation plant, claim 3 or 4.

19. the method for preparing transgenic plant of claim 18, wherein said plant are monocotyledons or dicotyledons.

20. the method for preparing transgenic plant of claim 19, wherein said monocotyledons is an Oryza, and said dicotyledons is a Nicotiana.

21. the method for preparing transgenic plant of claim 20, wherein said Oryza is a paddy rice, and said Nicotiana is a tobacco.

22. the method for preparing transgenic plant of claim 21, wherein said paddy rice are that paddy rice Japan is fine, said tobacco is tobacco NC89.

23. the carrier of the promotor of claim 1, the nucleic acid construct of claim 2, claim 3 or 4, the reconstitution cell of claim 6 or a kind of contains the purposes that plant callus, plant explants or the plant of reconstitution cell of nucleic acid construct, claim 3 or 4 the carrier or the claim 6 of promotor, the claim 2 of claim 1 is used to regulate and control plant destination gene expression and plant breeding.

24. the purposes of claim 23, wherein said plant are monocotyledons or dicotyledons.

25. the purposes of claim 24, wherein said monocotyledons is an Oryza, and said dicotyledons is a Nicotiana.

26. the purposes of claim 25, wherein said Oryza is a paddy rice, and said Nicotiana is a tobacco.

27. the purposes of claim 26, wherein said paddy rice are that paddy rice Japan is fine, said tobacco is tobacco NC89.

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