CN101979546B

CN101979546B - Promoter BgIosP574, preparation method and application thereof

Info

Publication number: CN101979546B
Application number: CN2009102389865A
Authority: CN
Inventors: 张耕耘; 朱江阳; 张厚宝; 邓倩; 刘小菊
Original assignee: BGI Shenzhen Co Ltd
Current assignee: Shenzhen Huada Gene Agriculture Holding Co ltd
Priority date: 2009-12-31
Filing date: 2009-12-31
Publication date: 2012-01-18
Anticipated expiration: 2029-12-31
Also published as: CN101979546A

Abstract

The invention relates to a promoter, in particular to the promoter of monocotyledons such as rice and the like, and a preparation method and application of the promoter. The promoter of the invention has a nucleotide sequence which is shown by sequence identifier number 1 (SEQ ID No. 1) or has a variant with promoter function selected from the following variants: 1) a nucleotide sequence which is hybridized with the nucleotide sequence shown by the SEQ ID No. 1 under highly strict conditions; 2) a nucleotide sequence which is formed by substituting, missing, adding and modifying one or more basic groups from the nucleotide sequence shown by the SEQ ID No. 1; and 3) a nucleotide sequence which has at least 90 percent of sequence identity with the nucleotide sequence shown by the SEQ ID No.1. The invention also relates to the preparation method of the promoter and application of the promoter to adjustment and control of target gene expression in the monocotyledons.

Description

A kind of promotor BgIosP574, Preparation Method And The Use

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 said promotor.

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 dehydrogenase1promoter).

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 McEl roy 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 ADH (alcohol dehydrogenase) gene, most important to the expression of plant ethanol dehydrogenase under anaerobic environment.The Adh-1 promotor is to monocotyledons particularly cereal grass such as paddy rice, oat and barley and few part dicotyledons such as tobacco, and the adjusting function of gene improves 10-50 doubly than CaMV (cauliflower mosaic virus CaMV) 35S promoter.The Adh-1 promotor is mainly used in monocotyledons, and is all very limited to the regulating effect that most dicotyledon genes are expressed.

Monocotyledons is angiospermous main monoid, and the Gramineae in the monocotyledons, Liliaceae, Palmae and Rhizoma Arisaematis etc. are very important agricultural crops.The 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 (Cassava Vein Mosaic Virus) 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 the regulation and control monocotyledons, for destination gene expression in the research monocotyledons a kind of new instrument and selection is provided 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 said promotor is 2886 bases, shown in SEQ ID NO:1:

TGCAAGCACCTGTTCCACGTCGGATGCATCGACATGTGGCTCCACTCCCACTCCACGTGTCCTCTCTGCAGGGCCAGCGTGGAGCCTCTTGGCAAGGAGACTCCTCTCAAGGATCAGGCACCTCCAGTGTAGCGCAACGGGTCGGACCATGTCATAGATTTCCGACGAGACTCTCTCCACGTAAAGCCGCAGCGACGTTGCATCCTGACATGTGGATCTGTAGGGGATGGGATCTATTTGTGAGTGACAACGTTTGGAGATAAGTTACGTAAGAGGGTCTTGATCTACGTGGATTTGAGCTCTCATTTGAATTACGAGAATTCGATACGGGTGGGTGTTAGGTTTTCTCCATAGATGTCGTATTGTTTGATGTGTAGAAATGGAAGTTTTCTTTGGGGGTAATTCATTACCTGAGTAATGTGTTTGTAAATATATCAACGATTTAGGTTTAAACAGAGTCTTGTATGCTGGTGAGCATAATGTCTTCAGTTCATATGGGAGGACCCGTTTTTTTTGCTCTACGGATCAAACAATTTCACATTTGTATGCTGGTGAAATTTTGGATTATACTTCAGCATTGAGAAGGCGTGAATCATCATGGTGTTTGGGGTGGAAATGGCAGGATGAGATGGCCGTCTGACGTCTCTGATGACTGATTGTTTCTTCAGAGAGGTGTGTAATAGAGTACCGAACTCCAAAGTCAGCGCACTTTATTTGTCCTTCTTATCTAATCTTTTATAGTTTAGTCACTAAACTTCAGTGGAAAATCATTCAAAGTCCCAGCAATTGAGTATATCATATAGTGTCGATGATCTCATCGTTTCACTTTGACACAGTAAATTTTGGGAATAGTTATATGTTGGCACTGGGTTGAGTATTTTGTCCAATATAATCAGTTGTTTTCAATCGTGTAGAAAAGAAAGGGACATTTGTATAATTTATTGAAAAGATGTATATACATGAGCATGCATGGTTAAAAAAACCCATCCGGATTAAGAAGGGGGTGGGCATTTGGTTTTCTTGGTTTATTAGAACTTTTGCTCAGTAATAATACAAACAGAAGAAGTTGAAAGGGAAATAGAAACCAAGCCGAATAAACGAAAGCAACCCGGTTTGGTTTTTTCTCTAGTTTTCCTACCAAACACTTGGTCATGCATGAATGTGCTACACCGATCCTCCACCCACCTCTCTCAGCATCTCATTGCATCATGCGATATAATACACATGTTTTGCTTTTGGACCCTAAGTTTAATCTTGTATTGGGCCTTGGGCCAAAGATAGAATAAAAATCCAAAAGATATAGAAGGTGGGAAGCATAGAAGTAATGGAGGTAAGCTGACATCTCGAATTTTTTGGTTTGTTTGATTTCTTGGGCAACAACCGAACAGAAACCCGAAGACCAATCTAGCCAAAAACAAAAGCCAAAGAGGGCACTGAAGACTGAAGAAATCGATACATCAGGTCAGTTCGGCTTGGTTTTGTTCAGTCTTTCAGTGCTCAGCGATTTTTCGTCCACTCCACTTTTGTTTTTTTATTAGAGTGAATATATTAATTCAAACGATTACACTACGCTTCGCCTGCACAAACCTAGGAGCCCTAGAGGATAATATGGAAGAATACACAGGTGTATACAAAGAAGAGAATGCAAAGGCATCATAACCTTGTTAGCTAAGTGGCTTAGAAAACTGGAAGCTGGTGCTTGGCTTTATCTTTTTTCTATAGAAAGACTACACATGCTATTGAACCATATGAACCACATAGTTTTCTCATAAACCGGACAATTAAAGAGTGTGTTCTTTGAGAGTGAGGATGAACTGTTGGTGAGTCTCTATGGAAATTAACTCATCTGGATCCAAACTTTGTCCCATCTCAAATCCACTTATTCCTCTTAGGATATAGAAATTTTCTTGAGACCGCACACATGACTTAGCGTAAATATAGCAAACTTCCTATTCTCTTATGAATTGAGCTATGACTACCTTCACTCTTTATAGTACAGTGAATATAAAGTAAGTCATATTCTATTTATACACTCATTTGTTGTGCCAAACTAGTAATCAATTAGAGTCATTGTCTTTATCTTTGCTCATCGAAGAACACATTATAATTCTCTTTATGGACAAATCATGGTAACATATTCAACTACCGAGGGCCTAACTCTCCTTGCACACTTAGAAAATATGATTAGTCTTAATAATCTATGTTGTCTTTAATCACCAAGACTCAAATAAAGGGTCTAGATGTGTATAACGAAGAGCTAATTGCAAACTAACCAACAAAAACCATATATTTTTTTTCTCTTGATGTTTACTTAATCTAACTAAAAGCGACTTTAGTAGTTTGGACAATTTGATGATCCATTAAAGTTTTTAGATATGACCACACCTCGTAGGTATACAAGAGTCGCAAATATTTATCAGCTAATAAAAAGTACCAAAAGTAAGATATGCTTGAAAACAACAGATAATGATGTAACTGGTCAAGCTTAAGTAAATGGATTAATACAGGTTACAAAGTTGTTGAAGAAACAAACAAGTCTATCAACTTTTTGATTGCTTGTAAAACTTTGTAAAGTACAAAAGAAGAAAGAGTATACACAAAAATGCTAGAAGCTAACAAGTTTGTTGTTGCAAAAGAACACTTAATGCATTTGGCTAGAAACTTTATGAGAAAATGAACACATTTGGCATGTACGAAACACGTCAAAAAGTATATGGTATGAATCATTTCAACAACATCCTTTGATGTAGTGACTTCTTTGGACCAAAAATCTTGCATTGTACACAATCCTTGTCCACAGGTTAACAAACTTCATAAGCACCTTGGATCCCAAAGCCACTTTTATTGGCTATAAAAACCACCACCTACGCACTTGCAATCACATCG(SEQ?ID?NO：1)

In the present invention, the promoter sequence shown in the SEQ ID NO:1 is called promotor BgIosP574, or abbreviates the P574 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 said 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 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.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 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 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 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, said replacement, 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.

Among the present invention, described promotor derives from monocotyledons, and is concrete, and said monocotyledons is a paddy rice, and for example said paddy rice is a Japan fine (Oryza sativa L.ssp.japonica cv.Nipponbare).

Another aspect of the present invention also relates to a kind of recombinant vectors that contains Monocotyledon promoter according to the invention.Said recombinant vectors can be through being inserted into cloning vector with above-mentioned promotor or expression vector obtains.

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 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, said recombinant vectors is the p8+P574 recombinant vectors.

Another aspect of the present invention also relates to the reconstitution cell of the said recombinant vectors that contains Monocotyledon promoter according to the invention.Said reconstitution cell can be converted into host cell through the said recombinant vectors that will contain Monocotyledon promoter according to the invention and obtain.

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 one embodiment of the invention, said reconstitution cell is reorganization agrobacterium tumefaciens (Agrobacteriumtumefaciens) EHA105-P574.

Another aspect of the present invention also relates to a kind of monocotyledons callus, and said callus transforms has promotor of the present invention.In one embodiment of the invention, said monocotyledons is a paddy rice.Said paddy rice includes but not limited to; 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 etc. (above-mentioned rice varieties all can available from emblem, Anhui good fortune ltd of merchant farmers') etc.In another embodiment of the present invention, said paddy rice is that Japan is fine.

Another aspect of the present invention also relates to a kind of method for preparing promotor according to the 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 the pcr amplification primer that is 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, said 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 said method comprises the step with the callus of Monocotyledon promoter transforming monocots according to the invention.In one embodiment of the invention, the trans-utilization of said monocotyledons callus contain the reconstitution cell of Monocotyledon promoter according to the invention.In one embodiment of the invention, utilized aforesaid reorganization Agrobacterium EHA105-P574 in the conversion process of said monocotyledons callus.In a specific embodiments of the present invention, said monocotyledonous callus is the rice callus tissue, and particularly, said 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 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.

Among the present invention, the said monocotyledons of said Monocotyledon promoter regulation and control destination gene expression capable of using includes 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 according to the invention is regulated and control destination gene expression in monocotyledons application.In one embodiment of the invention, utilizing the goal gene of promoter regulation according to the invention is GUS.In one embodiment of the invention, said monocotyledons is a paddy rice, and concrete said paddy rice is that Japan is fine.

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.

Another aspect of the present invention relates to the purposes of promotor according to the invention in rice breeding.Said 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 etc.; In one embodiment of the invention, said 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; For the genetic expression of research monocotyledons new instrument and selection is provided simultaneously; The inventor obtains through information biology research, and adopts biological experiment to verify the function of said promotor BgIosP574.Particularly, said 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 BgIosP574; Wherein, Swimming lane 1:1kb DNA LadderMarker, swimming lane 2:PCR amplified production, swimming lane 3:100bp DNA Ladder Marker; The size of the band of the Ladder Marker that the numeral 2000 in its left side and 3000 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 MCS 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 through the rice callus tissue that transforms.Wherein, the rice callus tissue (left side) that is transformed by the reorganization agrobacterium tumefaciens p8+P574 that has promotor P574 sequence according to the 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

To combine 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 1P574 promoter fragment and the structure of pMD18-T+P574 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; Be Chinese typical culture collection center (CCTCC), deposit number is CCTCC-P200910) genomic dna, according to the sequence of this promotor in the fine gDNA of paddy rice Japan; Design one couple of PCR specificity amplification primer (upstream primer F1 at head and the tail respectively; Add restriction enzyme site 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, high-fidelity Ex Taq ^TM(TaKaRa, DRR100B) polysaccharase carries out pcr amplification.As shown in table 1.

Table 1 gene promoter amplification PCR system

The pcr amplification program is: 94 ℃ of preparatory 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:GGggtaccTGCAAGCACCTGTTCCACGTC (SEQ ID NO:2), wherein lowercase is represented Kpn I restriction enzyme site.

Downstream primer R1:TGcctgcaggCGATGTGATTGCAAGTGCGTA (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 2904bp (Fig. 1), uses TIANGEN sepharose DNA to reclaim test kit (catalog number (Cat.No.): DP209-03) carry out purifying and recovering.

The structure of pMD18-T+P574 recombinant vectors

With 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 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 polishing to 10 μ l

(the new sesame in Ningbo SDC-6) the middle connection more than the 8h, obtains the pMD18-T+P574 recombinant vectors at the energy-conserving intelligent thermostatic bath in 16 ℃.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 be from for example: Shanghai be given birth to the worker and is buied), after melting, add 10 μ l as above the connection product of gained, i.e. pMD18-T+P574 recombinant vectors on ice; Stir ice bath 30min, 42 ℃ of heat shock 60s, ice bath 5min gently; The SOC substratum (concrete prescription sees " molecular cloning experiment guide ", the third edition, Science Press for details) that adds 4 ℃ of precoolings of 600 μ l, 37 ℃ of 220rpm recovery 45min; The centrifugal 30s of 8000rpm removes supernatant, leaves and takes 150 μ l; With the mixture 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 cultivate 16h～24h.Acquisition contains the recombination bacillus coli of pMD18-T+P574 cloning vector, called after DH5 α-P574.The big Gene science limited-liability company of Shenzhen China checks order to the P574 in the pMD18-T+P574 cloning vector, and the result is following:

GG GGTACC

GGATGCATCGACATGTGGCTCCACTCCCACTCCACGTGTCCTCTCTGCAGGGCCAGCGTGGAGCCTCTTGGCAAGGAGACTCCTCTCAAGGATCAGGCACCTCCAGTGTAGCGCAACGGGTCGGACCATGTCATAGATTTCCGACGAGACTCTCTCCACGTAAAGCCGCAGCGACGTTGCATCCTGACATGTGGATCTGTAGGGGATGGGATCTATTTGTGAGTGACAACGTTTGGAGATAAGTTACGTAAGAGGGTCTTGATCTACGTGGATTTGAGCTCTCATTTGAATTACGAGAATTCGATACGGGTGGGTGTTAGGTTTTCTCCATAGATGTCGTATTGTTTGATGTGTAGAAATGGAAGTTTTCTTTGGGGGTAATTCATTACCTGAGTAATGTGTTTGTAAATATATCAACGATTTAGGTTTAAACAGAGTCTTGTATGCTGGTGAGCATAATGTCTTCAGTTCATATGGGAGGACCCGTTTTTTTTGCTCTACGGATCAAACAATTTCACATTTGTATGCTGGTGAAATTTTGGATTATACTTCAGCATTGAGAAGGCGTGAATCATCATGGTGTTTGGGGTGGAAATGGCAGGATGAGATGGCCGTCTGACGTCTCTGATGACTGATTGTTTCTTCAGAGAGGTGTGTAATAGAGTACCGAACTCCAAAGTCAGCGCACTTTATTTGTCCTTCTTATCTAATCTTTTATAGTTTAGTCACTAAACTTCAGTGGAAAATCATTCAAAGTCCCAGCAATTGAGTATATCATATAGTGTCGATGATCTCATCGTTTCACTTTGACACAGTAAATTTTGGGAATAGTTATATGTTGGCACTGGGTTGAGTATTTTGTCCAATATAATCAGTTGTTTTCAATCGTGTAGAAAAGAAAGGGACATTTGTATAATTTATTGAAAAGATGTATATACATGAGCATGCATGGTTAAAAAAACCCATCCGGATTAAGAAGGGGGTGGGCATTTGGTTTTCTTGGTTTATTAGAACTTTTGCTCAGTAATAATACAAACAGAAGAAGTTGAAAGGGAAATAGAAACCAAGCCGAATAAACGAAAGCAACCCGGTTTGGTTTTTTCTCTAGTTTTCCTACCAAACACTTGGTCATGCATGAATGTGCTACACCGATCCTCCACCCACCTCTCTCAGCATCTCATTGCATCATGCGATATAATACACATGTTTTGCTTTTGGACCCTAAGTTTAATCTTGTATTGGGCCTTGGGCCAAAGATAGAATAAAAATCCAAAAGATATAGAAGGTGGGAAGCATAGAAGTAATGGAGGTAAGCTGACATCTCGAATTTTTTGGTTTGTTTGATTTCTTGGGCAACAACCGAACAGAAACCCGAAGACCAATCTAGCCAAAAACAAAAGCCAAAGAGGGCACTGAAGACTGAAGAAATCGATACATCAGGTCAGTTCGGCTTGGTTTTGTTCAGTCTTTCAGTGCTCAGCGATTTTTCGTCCACTCCACTTTTGTTTTTTTATTAGAGTGAATATATTAATTCAAACGATTACACTACGCTTCGCCTGCACAAACCTAGGAGCCCTAGAGGATAATATGGAAGAATACACAGGTGTATACAAAGAAGAGAATGCAAAGGCATCATAACCTTGTTAGCTAAGTGGCTTAGAAAACTGGAAGCTGGTGCTTGGCTTTATCTTTTTTCTATAGAAAGACTACACATGCTATTGAACCATATGAACCACATAGTTTTCTCATAAACCGGACAATTAAAGAGTGTGTTCTTTGAGAGTGAGGATGAACTGTTGGTGAGTCTCTATGGAAATTAACTCATCTGGATCCAAACTTTGTCCCATCTCAAATCCACTTATTCCTCTTAGGATATAGAAATTTTCTTGAGACCGCACACATGACTTAGCGTAAATATAGCAAACTTCCTATTCTCTTATGAATTGAGCTATGACTACCTTCACTCTTTATAGTACAGTGAATATAAAGTAAGTCATATTCTATTTATACACTCATTTGTTGTGCCAAACTAGTAATCAATTAGAGTCATTGTCTTTATCTTTGCTCATCGAAGAACACATTATAATTCTCTTTATGGACAAATCATGGTAACATATTCAACTACCGAGGGCCTAACTCTCCTTGCACACTTAGAAAATATGATTAGTCTTAATAATCTATGTTGTCTTTAATCACCAAGACTCAAATAAAGGGTCTAGATGTGTATAACGAAGAGCTAATTGCAAACTAACCAACAAAAACCATATATTTTTTTTCTCTTGATGTTTACTTAATCTAACTAAAAGCGACTTTAGTAGTTTGGACAATTTGATGATCCATTAAAGTTTTTAGATATGACCACACCTCGTAGGTATACAAGAGTCGCAAATATTTATCAGCTAATAAAAAGTACCAAAAGTAAGATATGCTTGAAAACAACAGATAATGATGTAACTGGTCAAGCTTAAGTAAATGGATTAATACAGGTTACAAAGTTGTTGAAGAAACAAACAAGTCTATCAACTTTTTGATTGCTTGTAAAACTTTGTAAAGTACAAAAGAAGAAAGAGTATACACAAAAATGCTAGAAGCTAACAAGTTTGTTGTTGCAAAAGAACACTTAATGCATTTGGCTAGAAACTTTATGAGAAAATGAACACATTTGGCATGTACGAAACACGTCAAAAAGTATATGGTATGAATCATTTCAACAACATCCTTTGATGTAGTGACTTCTTTGGACCAAAAATCTTGCATTGTACACAATCCTTGTCCACAGGTTAACAAACTTCATAAGCACCTTGGATCCCAAAGCCACTTTTATTGGCTATAAAAACCACCACC

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 P574 promoter sequence is correct in the pMD18-T+P574 cloning vector of acquisition.

The structure of embodiment 2p8+P574 recombinant vectors

(catalog number (Cat.No.): operational manual DP103-03), the conversion that makes up from embodiment 1 have bacillus coli DH 5 alpha-P574 of promotor P574 and extract the cloning vector pMD18-T+P574 that has P574 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 Kpn I (NEB) and Sbf I (NEB) 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+P574 recombinant vectors to be transformed according to " molecular cloning the experiment guide " (third edition; The competent cell DH5 α of the preparation of Calcium Chloride Method Science Press); Be inverted for 37 ℃ and cultivate 16～24h; After son to be transformed grew bacterium colony, the picking mono-clonal carried out the PCR detection and enzyme is cut evaluation.

The p8 plasmid construction

Employed p8 plasmid is that (Dong Yang of Kunming Institute of Zoology, Chinese Academy of Sciences provides by the pCAMBIA-1301 plasmid among the present invention; Perhaps can buy from for example auspicious Gene Tech. Company Limited of Shanghai state; The primary source of the pCAMBIA-1301 plasmid of the said firm is The CAMBIA Bios (biological open source) Licensee; Australia) transform and make up according to following mode, 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/HindIII/Spe I/Sbf I/Pst I/Xba I/BamH I/Sal I/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 (SEQ ID NO:7); Through 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 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:8 (referring to Fig. 3):

GTTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTAT GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTAC GAGCTC

AAGCTT C

G

GGAT CC

C

(SEQ?IDNO：8)

As above constructed p8 plasmid among the present invention shown in the sequence; EcoR I/Sac I/KpnI/Hind III/Spe I/Sbf I/Pst I/Xba I/BamH I/Sal I/Nco I restriction enzyme site in its MCS is respectively with adding frame and underscore is represented; The used primer GCTTCCGGCTCGTATGTTGT/GAGTCGTCGGTTCTGTA AC (being SEQ ID NO:6 and 7) 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+P574

According to restriction enzyme Kpn I (NEB) and Sbf I (NEB) operation instructions, handle resulting cloning vector pMD18-T+P574 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+P574 and p8 plasmid is following:

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+P574 or p8 plasmid 10 μ l (＜1000ng)

Use TIANGEN sepharose DNA receive test kit (catalog number (Cat.No.): DP209-03) reclaim the p8 plasmid of cutting through enzyme respectively, and promotor P574 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

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

The promotor P574 that reclaims inserts fragment 10-20ng, and is fixed according to its concentration

Sterilized water polishing to 9.5 μ l

T4ligase(TaKaRa，D2011A) 0.5μl

T4buffer melts on ice, the about 20ng of p8 plasmid vector add-on after enzyme is cut, and the P574 fragment among the present invention adds 10-20ng.(the new sesame in Ningbo is SDC-6) more than the middle connection 8h at the energy-conserving intelligent thermostatic bath in 16 ℃.

The competent cell DH5 that 100 μ l Calcium Chloride Method are made takes out from Ultralow Temperature Freezer, after melting, adds the connection product above the 10 μ l on ice, stirs gently; Ice bath 30min, 42 ℃ of heat shock 60s, ice bath 5min adds the SOC of 4 ℃ of precoolings of 600 μ l; 37 ℃ of following 220rpm recovery 45min, the centrifugal 30s of 8000rpm removes supernatant, leaves and takes 150 μ l; Blow evenly gently, granulated glass sphere coating LB (kan) is inverted for 37 ℃ and cultivates 16～24h.Obtain recombinant vectors p8+P574.

Be that primer carries out the PCR detection to gained recombinant vectors p8+P574 with F1 (SEQ ID NO:2) and R1 (SEQ ID NO:3) respectively, to contain required promotor P574 among the conclusive evidence gained recombinant vectors p8+P574.Cut screening through Kpn I/SbfI enzyme and contain recombinant vectors p8+P574 transformant.

The preparation of embodiment 3 reorganization agrobacterium tumefaciens EHA105-P574 cells

To transform respectively according to " molecular cloning the experiment guide " (third edition like the p8+P574 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 (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 following:

EHA105 takes out in Ultralow Temperature Freezer with the agrobacterium tumefaciens competent cell, as for thawing on ice.After the thawing, add p8+P574 recombinant vectors and the p8 plasmid of 5 μ l respectively and, mixing gently, ice bath 10min as the p8 empty carrier of contrast; Put into the freezing 5min of liquid nitrogen, 37 ℃ of 5min that thaw add the LB liquid nutrient medium of 800 μ l normal temperature, 28 ℃ of 160rpm recovery 3h; The centrifugal 30s of 8000rpm inhales and removes supernatant, stays 200 μ l and blows even; Coat and be added with (50mg/l Kan, 10mg/l Rif specifically fill a prescription referring to table 4) on the two anti-YM culture medium flat plates of kan-rif (kantlex-Rifampin).Be inverted for 28 ℃ and cultivated 2-3 days.

With F1 (SEQ ID NO:2) and R1 (SEQ ID NO:3) is that primer carries out the PCR detection and cuts the screening transformant through Kpn I/Sbf I enzyme.

What pcr amplification went out that about 2904bp band and enzyme cut out about 2896bp band is the reorganization agrobacterium tumefaciens of recombinant vectors p8+P574.

Among the present invention, according to the reorganization Agrobacterium that has recombinant vectors p8+P574 that obtains like above-mentioned method, called after reorganization agrobacterium tumefaciens EHA105-P574.

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 said callus with reorganization agrobacterium tumefaciens EHA105-P574 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 Youxiaolin sterilization 30min of available chlorine 1.5%, 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 2), seal with sealing film; 29.5 ℃ illumination cultivation 3-4 week;

2) choose the callus (yellow-white, drying, diameter 1-3mm) of active growth, 29.5 ℃ of illumination cultivation are 3 days on new N6D substratum;

3) the constructed single bacterium colony of reorganization agrobacterium tumefaciens (reorganization agrobacterium tumefaciens EHA105-P574 or reorganization agrobacterium tumefaciens EHA105-p8) of picking such as embodiment 3 respectively; In adding microbiotic (50mg/l Kan; YM substratum 10mg/lRif) (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 said reorganization agrobacterium tumefaciens cell (reorganization agrobacterium tumefaciens EHA105-P574 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 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 petridish, 28 ℃ of dark 48-60h that cultivate;

6) infected callus is placed the 50ml sterile tube, shake cleaning, become clarification until supernatant with aqua sterilisa; Callus is soaked in the sterilized water that contains 500mg/l Pyocianil (Cb) 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 illumination cultivation 3-4 weeks.

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 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-P574 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-P574 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 is as shown in Figure 5; After the rice callus tissue (Fig. 5 left side) of the Agrobacterium tumefaciens mediated conversion of reorganization of the p8+P574 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. 5 is right) of Agrobacterium tumefaciens mediated conversion.The result shows that P574 promotor of the present invention is expressed gus gene has regulating and controlling effect.

Employed relevant culture medium prescription explanation is 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 Pottasium Hydroxide, seal the back and sterilize by ordinary method.

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, zero(ppm) water is settled to 1L, and 4 ℃ of preservations are subsequent use.

N ₆Micro mother liquor (1000X): 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 (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 zero(ppm) water, 70 ℃ of temperature are bathed 2h, cool off back 4 ℃ of preservations and are no more than 1 month.

N ₆VITAMINs stock solution (1000X): 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 3YM liquid nutrient medium (containing 50mg/L Kan, 10mg/L Rif):

Conventional sterilization adds after being cooled to room temperature

Table 4YM solid medium (containing 50mg/L Kan, 10mg/L Rif):

Table 5AAM substratum

Add 1N Pottasium Hydroxide and regulate pH value to 5.2, conventional sterilization.

AAM macro (10X): 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 (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), zero(ppm) water is settled to 1L, and 4 ℃ of preservations are subsequent use.

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

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 ₆VITAMINs stock solution (1000X): all see table 2.

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 accompanying claims and any equivalent thereof.

SEQUENCE?LISTING

< 110>Shenzhen Huada Genetic Technology Co., Ltd

< 120>a kind of promotor BgIosP574, Preparation Method And The Use

<130>P2009-1-0023

<160>8

<170>PatentIn?version?3.5

<210>1

<211>2886

<212>DNA

< 213>paddy rice Japan fine (Oryza sativa L.ssp.japonica cv.)

<400>1

tgcaagcacc?tgttccacgt?cggatgcatc?gacatgtggc?tccactccca?ctccacgtgt 60

cctctctgca?gggccagcgt?ggagcctctt?ggcaaggaga?ctcctctcaa?ggatcaggca 120

cctccagtgt?agcgcaacgg?gtcggaccat?gtcatagatt?tccgacgaga?ctctctccac 180

gtaaagccgc?agcgacgttg?catcctgaca?tgtggatctg?taggggatgg?gatctatttg 240

tgagtgacaa?cgtttggaga?taagttacgt?aagagggtct?tgatctacgt?ggatttgagc 300

tctcatttga?attacgagaa?ttcgatacgg?gtgggtgtta?ggttttctcc?atagatgtcg 360

tattgtttga?tgtgtagaaa?tggaagtttt?ctttgggggt?aattcattac?ctgagtaatg 420

tgtttgtaaa?tatatcaacg?atttaggttt?aaacagagtc?ttgtatgctg?gtgagcataa 480

tgtcttcagt?tcatatggga?ggacccgttt?tttttgctct?acggatcaaa?caatttcaca 540

tttgtatgct?ggtgaaattt?tggattatac?ttcagcattg?agaaggcgtg?aatcatcatg 600

gtgtttgggg?tggaaatggc?aggatgagat?ggccgtctga?cgtctctgat?gactgattgt 660

ttcttcagag?aggtgtgtaa?tagagtaccg?aactccaaag?tcagcgcact?ttatttgtcc 720

ttcttatcta?atcttttata?gtttagtcac?taaacttcag?tggaaaatca?ttcaaagtcc 780

cagcaattga?gtatatcata?tagtgtcgat?gatctcatcg?tttcactttg?acacagtaaa 840

ttttgggaat?agttatatgt?tggcactggg?ttgagtattt?tgtccaatat?aatcagttgt 900

tttcaatcgt?gtagaaaaga?aagggacatt?tgtataattt?attgaaaaga?tgtatataca 960

tgagcatgca?tggttaaaaa?aacccatccg?gattaagaag?ggggtgggca?tttggttttc 1020

ttggtttatt?agaacttttg?ctcagtaata?atacaaacag?aagaagttga?aagggaaata 1080

gaaaccaagc?cgaataaacg?aaagcaaccc?ggtttggttt?tttctctagt?tttcctacca 1140

aacacttggt?catgcatgaa?tgtgctacac?cgatcctcca?cccacctctc?tcagcatctc 1200

attgcatcat?gcgatataat?acacatgttt?tgcttttgga?ccctaagttt?aatcttgtat 1260

tgggccttgg?gccaaagata?gaataaaaat?ccaaaagata?tagaaggtgg?gaagcataga 1320

agtaatggag?gtaagctgac?atctcgaatt?ttttggtttg?tttgatttct?tgggcaacaa 1380

ccgaacagaa?acccgaagac?caatctagcc?aaaaacaaaa?gccaaagagg?gcactgaaga 1440

ctgaagaaat?cgatacatca?ggtcagttcg?gcttggtttt?gttcagtctt?tcagtgctca 1500

gcgatttttc?gtccactcca?cttttgtttt?tttattagag?tgaatatatt?aattcaaacg 1560

attacactac?gcttcgcctg?cacaaaccta?ggagccctag?aggataatat?ggaagaatac 1620

acaggtgtat?acaaagaaga?gaatgcaaag?gcatcataac?cttgttagct?aagtggctta 1680

gaaaactgga?agctggtgct?tggctttatc?ttttttctat?agaaagacta?cacatgctat 1740

tgaaccatat?gaaccacata?gttttctcat?aaaccggaca?attaaagagt?gtgttctttg 1800

agagtgagga?tgaactgttg?gtgagtctct?atggaaatta?actcatctgg?atccaaactt 1860

tgtcccatct?caaatccact?tattcctctt?aggatataga?aattttcttg?agaccgcaca 1920

catgacttag?cgtaaatata?gcaaacttcc?tattctctta?tgaattgagc?tatgactacc 1980

ttcactcttt?atagtacagt?gaatataaag?taagtcatat?tctatttata?cactcatttg 2040

ttgtgccaaa?ctagtaatca?attagagtca?ttgtctttat?ctttgctcat?cgaagaacac 2100

attataattc?tctttatgga?caaatcatgg?taacatattc?aactaccgag?ggcctaactc 2160

tccttgcaca?cttagaaaat?atgattagtc?ttaataatct?atgttgtctt?taatcaccaa 2220

gactcaaata?aagggtctag?atgtgtataa?cgaagagcta?attgcaaact?aaccaacaaa 2280

aaccatatat?tttttttctc?ttgatgttta?cttaatctaa?ctaaaagcga?ctttagtagt 2340

ttggacaatt?tgatgatcca?ttaaagtttt?tagatatgac?cacacctcgt?aggtatacaa 2400

gagtcgcaaa?tatttatcag?ctaataaaaa?gtaccaaaag?taagatatgc?ttgaaaacaa 2460

cagataatga?tgtaactggt?caagcttaag?taaatggatt?aatacaggtt?acaaagttgt 2520

tgaagaaaca?aacaagtcta?tcaacttttt?gattgcttgt?aaaactttgt?aaagtacaaa 2580

agaagaaaga?gtatacacaa?aaatgctaga?agctaacaag?tttgttgttg?caaaagaaca 2640

cttaatgcat?ttggctagaa?actttatgag?aaaatgaaca?catttggcat?gtacgaaaca 2700

cgtcaaaaag?tatatggtat?gaatcatttc?aacaacatcc?tttgatgtag?tgacttcttt 2760

ggaccaaaaa?tcttgcattg?tacacaatcc?ttgtccacag?gttaacaaac?ttcataagca 2820

ccttggatcc?caaagccact?tttattggct?ataaaaacca?ccacctacgc?acttgcaatc 2880

acatcg 2886

<210>2

<211>29

<212>DNA

< 213>artificial sequence

<400>2

ggggtacctg?caagcacctg?ttccacgtc 29

<210>3

<211>31

<212>DNA

< 213>artificial sequence

<400>3

tgcctgcagg?cgatgtgatt?gcaagtgcgt?a 31

<210>4

<211>2904

<212>DNA

< 213>artificial sequence

<400>4

ggggtacctg?caagcacctg?ttccacgtcg?gatgcatcga?catgtggctc?cactcccact 60

ccacgtgtcc?tctctgcagg?gccagcgtgg?agcctcttgg?caaggagact?cctctcaagg 120

atcaggcacc?tccagtgtag?cgcaacgggt?cggaccatgt?catagatttc?cgacgagact 180

ctctccacgt?aaagccgcag?cgacgttgca?tcctgacatg?tggatctgta?ggggatggga 240

tctatttgtg?agtgacaacg?tttggagata?agttacgtaa?gagggtcttg?atctacgtgg 300

atttgagctc?tcatttgaat?tacgagaatt?cgatacgggt?gggtgttagg?ttttctccat 360

agatgtcgta?ttgtttgatg?tgtagaaatg?gaagttttct?ttgggggtaa?ttcattacct 420

gagtaatgtg?tttgtaaata?tatcaacgat?ttaggtttaa?acagagtctt?gtatgctggt 480

gagcataatg?tcttcagttc?atatgggagg?acccgttttt?tttgctctac?ggatcaaaca 540

atttcacatt?tgtatgctgg?tgaaattttg?gattatactt?cagcattgag?aaggcgtgaa 600

tcatcatggt?gtttggggtg?gaaatggcag?gatgagatgg?ccgtctgacg?tctctgatga 660

ctgattgttt?cttcagagag?gtgtgtaata?gagtaccgaa?ctccaaagtc?agcgcacttt 720

atttgtcctt?cttatctaat?cttttatagt?ttagtcacta?aacttcagtg?gaaaatcatt 780

caaagtccca?gcaattgagt?atatcatata?gtgtcgatga?tctcatcgtt?tcactttgac 840

acagtaaatt?ttgggaatag?ttatatgttg?gcactgggtt?gagtattttg?tccaatataa 900

tcagttgttt?tcaatcgtgt?agaaaagaaa?gggacatttg?tataatttat?tgaaaagatg 960

tatatacatg?agcatgcatg?gttaaaaaaa?cccatccgga?ttaagaaggg?ggtgggcatt 1020

tggttttctt?ggtttattag?aacttttgct?cagtaataat?acaaacagaa?gaagttgaaa 1080

gggaaataga?aaccaagccg?aataaacgaa?agcaacccgg?tttggttttt?tctctagttt 1140

tcctaccaaa?cacttggtca?tgcatgaatg?tgctacaccg?atcctccacc?cacctctctc 1200

agcatctcat?tgcatcatgc?gatataatac?acatgttttg?cttttggacc?ctaagtttaa 1260

tcttgtattg?ggccttgggc?caaagataga?ataaaaatcc?aaaagatata?gaaggtggga 1320

agcatagaag?taatggaggt?aagctgacat?ctcgaatttt?ttggtttgtt?tgatttcttg 1380

ggcaacaacc?gaacagaaac?ccgaagacca?atctagccaa?aaacaaaagc?caaagagggc 1440

actgaagact?gaagaaatcg?atacatcagg?tcagttcggc?ttggttttgt?tcagtctttc 1500

agtgctcagc?gatttttcgt?ccactccact?tttgtttttt?tattagagtg?aatatattaa 1560

ttcaaacgat?tacactacgc?ttcgcctgca?caaacctagg?agccctagag?gataatatgg 1620

aagaatacac?aggtgtatac?aaagaagaga?atgcaaaggc?atcataacct?tgttagctaa 1680

gtggcttaga?aaactggaag?ctggtgcttg?gctttatctt?ttttctatag?aaagactaca 1740

catgctattg?aaccatatga?accacatagt?tttctcataa?accggacaat?taaagagtgt 1800

gttctttgag?agtgaggatg?aactgttggt?gagtctctat?ggaaattaac?tcatctggat 1860

ccaaactttg?tcccatctca?aatccactta?ttcctcttag?gatatagaaa?ttttcttgag 1920

accgcacaca?tgacttagcg?taaatatagc?aaacttccta?ttctcttatg?aattgagcta 1980

tgactacctt?cactctttat?agtacagtga?atataaagta?agtcatattc?tatttataca 2040

ctcatttgtt?gtgccaaact?agtaatcaat?tagagtcatt?gtctttatct?ttgctcatcg 2100

aagaacacat?tataattctc?tttatggaca?aatcatggta?acatattcaa?ctaccgaggg 2160

cctaactctc?cttgcacact?tagaaaatat?gattagtctt?aataatctat?gttgtcttta 2220

atcaccaaga?ctcaaataaa?gggtctagat?gtgtataacg?aagagctaat?tgcaaactaa 2280

ccaacaaaaa?ccatatattt?tttttctctt?gatgtttact?taatctaact?aaaagcgact 2340

ttagtagttt?ggacaatttg?atgatccatt?aaagttttta?gatatgacca?cacctcgtag 2400

gtatacaaga?gtcgcaaata?tttatcagct?aataaaaagt?accaaaagta?agatatgctt 2460

gaaaacaaca?gataatgatg?taactggtca?agcttaagta?aatggattaa?tacaggttac 2520

aaagttgttg?aagaaacaaa?caagtctatc?aactttttga?ttgcttgtaa?aactttgtaa 2580

agtacaaaag?aagaaagagt?atacacaaaa?atgctagaag?ctaacaagtt?tgttgttgca 2640

aaagaacact?taatgcattt?ggctagaaac?tttatgagaa?aatgaacaca?tttggcatgt 2700

acgaaacacg?tcaaaaagta?tatggtatga?atcatttcaa?caacatcctt?tgatgtagtg 2760

acttctttgg?accaaaaatc?ttgcattgta?cacaatcctt?gtccacaggt?taacaaactt 2820

cataagcacc?ttggatccca?aagccacttt?tattggctat?aaaaaccacc?acctacgcac 2880

ttgcaatcac?atcgcctgca?ggca 2904

<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

1. promotor, its nucleotide sequence or are the complementary sequence of nucleotide sequence shown in the SEQ ID NO:1 shown in SEQ ID NO:1.

2. a recombinant vectors is characterized in that, said recombinant vectors contains the described promotor of claim 1.

3. recombinant vectors according to claim 2 is characterized in that, said recombinant vectors is the p8+P574 recombinant vectors.

4. reconstitution cell that contains claim 2 or 3 said recombinant vectorss.

5. the described reconstitution cell of claim 4, it is reorganization Agrobacterium EHA105-P574.

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;

7. method according to claim 6 is characterized in that, 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, said method comprises the step with the callus of the described promotor transforming monocots of claim 1, and wherein, said monocotyledons is a paddy rice, and said callus is the rice callus tissue.

9. the method for genetic expression is characterized in that in the regulation and control monocotyledons according to claim 8, and said paddy rice is that Japan is fine.

10. according to Claim 8 or 9 described methods, utilized the described reorganization of claim 5 Agrobacterium EHA105-P574 in the conversion process of wherein said monocotyledons callus.

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 said monocotyledons is a paddy rice.

12. the purposes of promotor according to claim 11 destination gene expression in the regulation and control monocotyledons is characterized in that said paddy rice is that Japan is fine.

13. the purposes of the described promotor of claim 1 in rice breeding.

14. purposes according to claim 13; It is characterized in that; Said 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.

15. the purposes according to claim 14 is characterized in that, said paddy rice is that Japan is fine.