CN104805084A - Rice leaf intersection angle epigenetic regulation site and application thereof - Google Patents

Abstract

The invention belongs to the field of plant molecular biology, and concretely relates to a rice leaf intersection angle epigenetic regulation site. The epigenetic regulation site controls the rice leaf intersection angle by changing the expression of flanking gene.

Description

The commitment site of rice leaf intersection angle and application thereof

Technical field

The invention belongs to field of plant molecular biology, be specifically related to a kind of commitment site of rice leaf intersection angle, described commitment site controls the Leaf angle of paddy rice by the expression changing other adjacent gene.

Background technology

Paddy rice, as important food crop, is also monocotyledonous model plant simultaneously.Ideal Rice Plant Type is morphological structure and the basic substance of SOYBEAN IN HIGH-YIELD BREEDING.Leaf angle is the important component part of plant type of rice, refers to the angle between blade and stem stalk.The size of Leaf angle particularly sword-like leave Leaf angle regulates the photosynthetic turnout of paddy rice to a great extent, and then affects rice yield.Rice leaf angle is one of important factor of SOYBEAN IN HIGH-YIELD BREEDING ideotype, is subject to the regulation and control of brassinolide (BR).Endogenous BR concentration directly control rice leaf intersection angle size, and BR disappearance or susceptibility reduce rice leaf all can be caused upright, illustrates that BR can the plant type of adjusting and controlling rice effectively.There are some researches show, reduce BR signal and rice leaf can be made upright, the rational close planting being conducive to paddy rice finally improves output (Sakamoto etc., 2006).At present, utilize forward and reverse-genetics approach to identify the member of many paddy rice BR pathways metabolisms, people have been had paddy rice BR metabolic process and is more in depth familiar with.Mori in 2002 etc. identify the relevant mutant brdl (brassinosteriod-dependent1) of BR route of synthesis in paddy rice.Brdl mutant shows as typical BR and lacks symptom: stem stalk is seriously downgraded, and bending blade is stiff, and in deformity, fringe shape and grain etc. are less than normal, and can recover mutation type surface after external source applies BL.This is the mutant that the BR synthesis determined first in paddy rice is correlated with.The BR6ox homology called after OsBR6ox of this gene and tomato and Arabidopis thaliana, this enzyme belongs to Cytochrome P450 family.Subsequently, Hong etc. identify BR shortage property Dwarf Mutants brd2, and this mutant also shows obvious BR shortage property symptom: upright blade, plant is downgraded.Brd2 gene is the homologous sequence of Arabidopis thaliana DIM1/DWF1 gene, and major catalytic BR biosynthesizing early stage 24-methylene radical sterol (24-methylenecholesterol, 24-MC) is to the conversion of Brassicasterin (campesterol, CR).OsLIC encodes a conservative CCCH type zinc finger protein, and the inhibitors as BR signal regulates its synthesis to affect the molecule mechanism (Wang etc., 2008a) of Leaf angle.In paddy rice, encode the BU1 gene of HLH albumen as the response factor of BR, Plant Leaf angle after its process LAN, can will be made to become large (Tanaka etc., 2009).Above BR synthesis mutant analytic explanation, BR has vital role in the Leaf angle regulation and control of paddy rice.

Study discovery in addition, GAST gene family member OsGSR1 can do mutually with the enzyme DIM/DWF1 transformed to Brassicasterin (campesterol) with catalysis 24-methylene radical sterol (24-methylenecholesterol) in BR biosynthesizing.Illustrate that OsGSR1 regulates and controls BR biosynthesizing by directly acting on BR synthetic enzyme DIM/DWF1, and then regulate rice leaf intersection angle (Wang etc., 2009).There is being similar to plant and lack the phenotype that BR causes in OsGSR1RNAi plant, comprises that primary root shortens, upright blade and fertility reduce.The endogenous BR content of OsGSR1RNAi plant reduces, simultaneously its Dwarfing phenotypes can be executed outward BL recover.In view of BR is to the regulating effect of rice leaf intersection angle, the synthesis or the signal susceptibility related gene that change BR can as the effective means of improvement plant type of rice., BR molecular mechanism is deeply resolved meanwhile, make the breeding molecular element that these genes are expected to become new, improve the rice plant type of water to increase rice yield by genetic manipulation BR approach.

MITEs (mininature inverted-repeats transposable elements) is that a class transposon important in plant all exists (Feng et al., 2002 in many eukaryotes and prokaryotic gene group; Feschotte et al., 2002).MITEs fragment less (usual 80-500bp), and in genome, have a lot of copies, great majority are all present in the non-coding region (intron near gene, 5 '-UTRs, 3 '-UTRs), expression (Oki et al., 2008 of neighbouring gene may be regulated and controled; Piriyapongsaand Jordan, 2008).Different from Arabidopis thaliana, in paddy rice, MITEs is the transposon (Jiang et al., 2004) that copy number is the highest.MITEs transcribes the RNA that rear formation has loop-stem structure, can produce siRNA and miRNA two micromolecular RNA, and it is the intermediate (Piriyapongsa and Jordan, 2008) of siRNA to miRNA evolution transition process.SiRNA441, siRNA446 of being produced by MITE in paddy rice may as positive regulatory factors when paddy rice response ABA signal pathway and arid, salt, the abiotic stress such as cold.They play a role (Yan et al., 2011) mainly through degraded target gene F-box gene mRNA.Genetic analysis shows, in plant 24-nt siRNA effect path, key protein comprises DCL3, RDR2 and AG04, they, by participating in the generation of siRNA and the formation of RISC, come the DNA of controlling gene group distinguished sequence and histone methylated modification, thus carry out silence to target gene.There are two DCL3 genes in paddy rice, OsDCL3a is in paddy rice each position expression amount higher (Kapoor et al., 2008) in chip data display.From evolutionary analysis, OsDCL3a and AtDCL3a homology is higher, and it is responsible for the generation of 24nt lmiRNA and the expression (Wu et al., 2010) by carrying out DNA methylation modification regulation and control target gene to its target gene site.Meanwhile, OsDCL3a is also responsible for the 24-ntsiRNA (Song et al., 2012) that processing produces tumor-necrosis factor glycoproteins region in paddy rice.We find in field observation, and the transgenic line (3a-3 and 3a-1) knocking out rice Os DCL3a all shows Leaf angle and becomes large, the anormogenesis phenotype of dwarfing.Compared with Arabidopis thaliana, containing more abundant transposon or tumor-necrosis factor glycoproteins in rice genome, and these sequences exist near most paddy gene.The 24-nt siRNA deriving from transposon or tumor-necrosis factor glycoproteins can express to regulate and control it by carrying out DNA or histone methylated apparent modification to the adjacent gene in side, finally regulates the whole etap of paddy rice subtly.

Paddy rice is one of most important food crop of China, is related to the basic guarantee of China's grain security and people's lives.Plant type of rice breeding is one of focal issue of modern breeding scholar concern always.Along with the widespread use of the means such as molecular biology, functional genomics and high-flux sequence, commitment rice leaf intersection angle related gene, and then the molecular mechanism research affecting plant type of rice deepens continuously, important clue and foundation can be provided for the Main Agronomic Characters of molecular improvement crop.

This research provides the commitment site of rice leaf intersection angle first, which not only provides a kind of method of plant type of rice breeding, and provides a practical scheme for solving modern molecular design and context problem, has great importance.

Summary of the invention

The present invention contains 2 BR anabolism and 1 rice leaf intersection angle related gene, other adjacent MITEs produces 24-ntsiRNA can the expression of these genes of commitment, for plant type of rice breeding provides important clue and foundation, can be used as the new tool of Screening of Rice ideotype resource.

Rice leaf intersection angle commitment site provided by the present invention, comprise the adjacent MITEs site, side of 2 BR anabolism and 1 rice leaf intersection angle related gene, the nucleotide sequence in adjacent MITEs site, described side is respectively as shown in SEQ ID NO:1, SEQIDNO:2 and SEQ IDNO:3.

Wherein, SEQ ID NO:1 is the Ditto-like MITE class transposon be made up of 47Sbp DNA sequence dna, be positioned at-the 888bp of OsGSR1LOC_Os06g15620 upstream region of gene to-411bp, this site can produce abundant 24-nt siRNA, by mediating apparent expression of modifying regulation and control side BR anabolism genes involved OsGSR1; SEQ ID NO:2 in sequence table is the Tourist-like MITE class transposon be made up of 308bp DNA sequence dna, be positioned at OsBR6ox LOC_Os03g40540 upstream region of gene-457bp to-150bp, this site can produce abundant 24-nt siRNA, by mediating apparent expression of modifying regulation and control side BR anabolism genes involved OsBR6ox; SEQ ID NO:3 in sequence table is Ditto-like MITE class transposon (2 the temperate zone japonica rice: Japan is fine be made up of 250bp DNA sequence dna, IRGC418), be positioned at LOC_Os08g19420 upstream region of gene-1177bp to-928bp, this site can produce abundant 24-nt siRNA, by mediating apparent expression of modifying regulation and control side rice leaf intersection angle related gene LOC_Os08g19420; SEQ IDNO:4 in sequence table is Ditto-like MITE class transposon (2 tropic Japonica: the IRGC17757 be made up of 156bp DNA sequence dna, IRGC328), be positioned at LOC_Os08g19420 upstream region of gene-1177bp to-928bp, this site sequence there are differences between warm tropic Japonica sequence, by the differential expression of regulation and control flanking genes LOC_Os08g19420, and then the difference of warm tropic Japonica Leaf angle can be caused.

Present invention also offers a kind of method detecting the commitment site of rice leaf intersection angle, described method comprises first detecting in rice material whether contain the complete transposon sequence as shown in SEQ ID NO:1, SEQ ID NO:2 and/or SEQ ID NO:3, and whether the expression of the apparent modification and other adjacent gene of then analyzing this site changes.From the commitment site of the rice leaf intersection angle described in the present invention, can fix a point to edit apparent modification on rice genome by CRISPR/Cas9 technology, and then the expression of adjusting and controlling rice leaf intersection angle related gene, the molecular designing of plant type of finally realizing ideal.

Present invention also offers a kind of molecule marker detecting rice leaf intersection angle, namely described molecule marker detects endogenous SEQ ID NO:1, SEQ ID NO:2 and/or SEQ ID NO:3 in different rice pest insects and whether there is natural variation, whether these natural variations of further analysis produce the change of apparent modification, thus judge whether this rice varieties meets the demand of Ideotype Breeding.Because this detection just can detect in the rice seedling phase, thus select the rice plant with excellent plant type, greatly can shorten the time of paddy rice traditional breeding method, save a large amount of man power and materials.

Present invention also offers the functional gene LOC_Os08g19420 of a new adjusting and controlling rice Leaf angle size, the genomic dna sequence of described gene is as shown in SEQ ID NO:5, the nucleotide sequence of its coding region is as shown in SEQ ID NO:6, and the aminoacid sequence of described genes encoding is as shown in SEQ ID NO:7.Process LAN functional gene provided by the present invention in paddy rice, can become large by the sword-like leave angle of transfer-gen plant; Lower the expression of described functional gene, then can reduce the Leaf angle of paddy rice, thus increase photosynthetic efficiency and the biological yield of rice plant.

Present invention also offers a kind of method of adjusting and controlling rice Leaf angle, described method is included in the functional gene LOC_Os08g19420 provided by the present invention of process LAN in paddy rice, makes the sword-like leave angle of described transfer-gen plant become large; Or lower the expression of described functional gene, the Leaf angle of described transfer-gen plant is diminished, thus increases the biological yield of rice plant.

Described in the present invention " expression of down function gene " includes but not limited to following methods, as the nucleotide sequence mutation caused by the method for physics or chemistry, chemical process comprises the mutagenesis caused with mutagenic compound process such as EMS, described sudden change can also be point mutation, also can be DNA disappearance or insertion mutation, can also be produced by RNAi gene silencing means, or the site-directed mutagenesis technique such as TALLEN, CRISPR/Cas9 obtain the plant after sudden change.

The present invention also comprises the construct containing SEQ ID NO:6 nucleotide sequence, and described construct comprises usually said carrier or expression cassette, can be used for the adjusting and controlling rice Leaf angle gene that process LAN is new.The promotor that promotor in described construct can be natural promoter or be substituted, its by drive connect nucleotides sequence and be listed in expression in plant.Promotor in construct can be constitutive promoter, includes but not limited to composing type viral promotors, such as cauliflower mosaic virus (CaMV) 19S and 35S promoter, or radix scrophulariae mosaic virus 35 S promoter, or ubiquitin promoter.

Also can comprise other component in above-mentioned construct, this depends primarily on object and the purposes of vector construction, such as, can comprise selectable marker gene, target or regulating and controlling sequence, critical sequences or homing sequence, intron etc. further.3 ' the end at desired heterologous nucleotide sequence is also included in plant by expression cassette has transcribing and translation termination of function.Terminator can be the terminator of gene provided by the present invention, also can be the terminator from external source.More specifically, above-mentioned terminator can be nopaline synthase or octopine synthase termination area.

In the process preparing expression cassette, can be operated multiple DNA fragmentation, be in proper orientation to provide, or be in the DNA sequence dna in correct reading frame.For reaching this object, adapter or joint can be used, DNA fragmentation is linked up, or comprise other operation further, with the restriction enzyme site etc. of providing convenience.

Further, in construct provided by the present invention, also selectable marker gene can be comprised, for selecting the cell or tissue through transforming.Described selectable marker gene comprises gives antibiotics resistance or the gene to Herbicid resistant.Suitable selectable marker gene includes but not limited to: chloramphenicol resistance gene, hygromycin gene, streptomycin resistance gene, miramycin resistant gene, sulfamido resistant gene, Glyphosate resistance gene, careless fourth phosphine resistant gene.Described selectable marker gene can also be the genes such as red fluorescent gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescence protein gene, anthocyanin p1.

Expression cassette provided by the present invention or carrier can be inserted into plasmid, clay, yeast artificial chromosome, bacterial artificial chromosome or other be applicable to being transformed in any carrier in host cell.Preferred host cell is bacterial cell, such as, in particular for cloning or storing polynucleotide or the bacterial cell for transformed plant cells, intestinal bacteria, Agrobacterium tumefaciems and Agrobacterium rhizogenes.When host cell is vegetable cell, expression cassette or carrier can be inserted in the genome of the vegetable cell be converted.Insertion can be location or random insertion.Preferably, be inserted through such as homologous recombination to realize.In addition, expression cassette or carrier can remain on outside karyomit(e).Expression cassette of the present invention or carrier can be present in the core of vegetable cell, chloroplast(id), plastosome and/or plastid.Preferably, expression cassette of the present invention or carrier are inserted in the chromosomal DNA of plant nucleolus.

Accompanying drawing explanation

Fig. 1 is MITE-siRNAs commitment BR anabolism genes involved OsGSR1 and OsBR6ox.(A, G) is the gene structure display of OsGSR1 and OsBR6ox respectively: black surround represents exon, white edge represents intron, grey frame table shows UTR, yellow frame table shows MITE transposon; The black arrow of transcriptional orientation represents, and black line indicates tiny RNA probe, sulphite order-checking region and ChIP-qPCR detection site from the top down respectively.(B, H) OsGSR1 and OsBR6ox gene region tiny RNA group and transcript profile signal, wild-type (blueness), mutant 3a-3 (green) and 3a-1 (redness); (C-D, I-J) tiny RNA hybridization verification OsGSR1 and OsBR6ox gene region SP8 and SP9 tiny RNA, real-time PCR detects OsGSR1 and OsBR6ox gene in wild-type, the transcriptional level in mutant 3a-3 and 3a-1; (E, K) sulphite sequencing analysis wild-type, the DNA methylation level in BSP7 and the BSP8 region in mutant 3a-3 and 3a-1.(F, L) ChIP-qPCR analyzes wild-type, and the H3k9me2 in the R8-R10 region in mutant 3a-3 and 3a-1 modifies level.

Fig. 2 is MITE-siRNAs commitment rice leaf intersection angle related gene LOC_Os08g19420.(A) be the gene structure display of LOC_Os08g19420: black surround represents exon, white edge represents intron, grey frame table shows UTR, yellow frame table shows MITE transposon; The black arrow of transcriptional orientation represents, and black line indicates tiny RNA probe, sulphite order-checking region and ChIP-qPCR detection site from the top down respectively; ; (B) LOC_Os08g19420 gene region tiny RNA group and transcript profile signal, wild-type (blueness), mutant 3a-3 (green) and 3a-1 (redness); (C-D) tiny RNA hybridization verification LOC_Os08g19420 gene region SP10 and SP11 tiny RNA, real-time PCR detects LOC_Os08g19420 gene in wild-type, the transcriptional level in mutant 3a-3 and 3a-1; (E) sulphite sequencing analysis wild-type, the DNA methylation level in BSP9 and the BSP10 region in mutant 3a-3 and 3a-1.(F) ChIP-qPCR analyzes wild-type, and the H3k9me2 in the R11-R15 region in mutant 3a-3 and 3a-1 modifies level.

Fig. 3 is process LAN LOC_Os08g19420 transfer-gen plant phenotype, the expression of LOC_Os08g19420 gene in the different transgenic line of (A) Northern hybridization check; (B-C) observe and add up the Leaf angle phenotype of process LAN LOC_Os08g19420 strain.

Fig. 4 is LOC_Os08g19420 commitment Mutation in warm tropic Japonica, (A) sequence alignment of LOC_Os08g19420 upstream MITE in 4 different japonica rice, yellow frame represents MITE transposon, blue arrow represents that MITE target site forward repeats, and red block represents MITE terminal repeat; (B) real-time PCR detects the expression of LOC_Os08g19420 in 4 different japonica rice.

Embodiment

According to specific embodiment, the present invention will be described below.It should be noted that, these embodiments are only used to the present invention is described, and can not be interpreted as limitation of the present invention by any way.In addition, unless stated otherwise, method involved is in the following embodiments ordinary method, can carry out with reference to " Molecular Cloning: A Laboratory guide " third edition or related products.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.

Table 1 synthesizes the primer of tiny RNA probe

The real-time PCR primer of table 2 gene expression detection situation

Table 3 detects the Bisulfite Sequencing primer of specific gene DNA methylation modification level

Table 4 detects the ChIP-qPCR primer of specific gene H3K9me2 modification level

The MITE sequential analysis that embodiment 1, rice leaf intersection angle related gene are contiguous

Utilize paddy rice tumor-necrosis factor glycoproteins database TIGR_Oryza_Repeats.v3.3 (ftp: //ftp.plantbiology.msu.edu), analyze the tumor-necrosis factor glycoproteins feature in 2 BR anabolism and 1 rice leaf intersection angle related gene district and upstream and downstream 2kb region thereof.Result shows, and all there is MITE class transposon (Figure 1A, 1G in the promoter region of OsGSR1, OsBR6ox and LOC_Os08g19420 gene; Fig. 2 A).

The contiguous MITE site of embodiment 2, rice leaf intersection angle related gene produces the analysis of 24-nt siRNA

Extract wild-type, mutant 3a-3 and 3a-1 total serum IgE, get 10 μ g total serum IgE PEGS000 separation and concentration tiny RNA respectively.Then according to Illumina company tiny RNA banking process, Illumina Hiseq2000 carries out smallRNA-seq order-checking.After obtaining small RNA-seq data, use Bowtie-0.12.7 by tiny RNA sequence alignment on rice genome (release7, http:// rice.Dlantbiology.msu.edu/).RP25M (reads Der twenty fivemillion) is utilized to be normalized correction to small RNA-seq data.Calculate 24-nt siRNA abundance using 500-nt as unit interval, compare the 24-nt siRNA bunch as differential expression that wild-type changes more than 3 times.

On the other hand, small RNA is utilized to hybridize the 24-nt siRNA bunch verifying differential expression in smal RNA-seq.Extract wild-type, mutant 3a-3 and 3a-1 total serum IgE, get 15-20 μ g total serum IgE 8M LiCI enrichment microRNA respectively.After preparation 15%7M Urea sex change glue carries out electrophoresis, semidrying transferring film is on Hybond-NX film.Then, after EDC is cross-linked, hybridize with the label probe of preparation.Use 4 groups of gene-specific primer SP8-SP11 (table 1) amplification tiny RNA probe template respectively, the tiny RNA probe that its in-vitro transcription becomes a-32p-UTP to mark by t7 rna polymerase.

In conjunction with microRNA sequencing data and small RNA hybrid experiment, analyze rice leaf intersection angle related gene further.Find that the MITE site of these 3 gene promoter areas can produce a large amount of 24-nt siRNA in wild-type, and in mutant, TE site 24-nt siRNA greatly reduces (Figure 1B-C, 1H-I; Fig. 2 B-C).

Mutant 3a-3 and 3a-1 described in the present embodiment refers to the mutating strain series having knocked out OsDCL3a gene in paddy rice.

The expression analysis of embodiment 3, rice leaf intersection angle related gene

Get 10 μ tg wild-types respectively, mutant 3a-3 and 3a-1 total serum IgE, then according to the special RNA-Seq banking process of Illumina company chain (Directional mRNA-Seq library prep v1.5), Illumina GAII carries out transcript profile order-checking.After obtaining RNA-seq data, use TopHat-1.3.0 by sequence alignment on rice genome (release7, http:// fiee.plantbiology msu.edu/).The sequence of the coupling multiple position of genome is dropped, and only carries out subsequent analysis to the sequence matching genome unique positions.With RPKM (reads per kilobase of exon model per million mappedreads), correction is normalized to RNA-seq data, and the transcriptional level (Mortazaviet al., 2008) of exon is weighed with RPKM > 3.Then WIG formatted file (the Zhang et al. of corresponding sequencing data is generated with MACS software, 2008), the expression (Nicol et al., 2009) of specific region is checked by Integrated Genome Browser (IGB).Between wild-type and mutant, differential expression genes adds up by DEGseq program, namely changes > 1.5 times and P value < 0.01 (Wang et al., 2010).Meanwhile, utilize real-time PCR to verify the gene of differential expression in RNA-seq.Extract wild-type, mutant 3a-3 and 3a-1 total serum IgE, M-MLV reverse transcription becomes eDNA, carries out real-timePCR (table 2) respectively with 4 pairs of gene-specific primers.

In conjunction with RNA-seq data and real-time PCR, analyze 2 BR anabolism and 1 rice leaf intersection angle related gene further.Result shows, and compared with wild-type, these 3 genes expression amount in mutant significantly raises (Figure 1B, 1D, 1H, 1J; Fig. 2 B, 2D).

Embodiment 4, MITE produce the detection of the apparent modification of 24-nt siRNA mediation

Adopt CTAB method to extract wild-type, mutant 3a-3 and 3a-1 genomic dna, Bisulfite Sequencing detects the MITE regional DNA producing 24-nt siRNA and to methylate modification situation.First Bisulfite Sequencing primer BSP7-BSP10 (table 3) of Methyl Primer Express software design 4 groups of specific regions is used.According to EZ DNAMethylation-GoldTM kit method process oryza sativa genomic dna, carry out BS-PCR and reclaim PCR primer and cloning and sequencing.Web-based Kismeth software on-line analysis (http://katahdin.mssm.edu/kismeth/revpage.pl) (Gruntman et al. is utilized after obtaining sequencing result,, and remove sibling clones by manual alignment 2008).In addition, after utilizing H3K9me2 antibody to carry out co-immunoprecipitation, carry out ChIP-qPCR (table 4) with 8 couples of Auele Specific Primer R8-R15 respectively.The internal reference verified using Anti-H3 as ChIP-qPCR, the negative contrast (Yin et al., 2008) that Os04g35560 modifies as H3K9me2 in paddy rice.

As can be seen from Fig. 1 E, 1K, Fig. 2 E, in wild-type and mutant, the MITE regional DNA methylation level producing 24-nt siRNA does not have significant difference.And to 3 contiguous rice leaf intersection angle related gene region H3K9me2 level detection displays, in mutant, the H3K9me2 level of surveyed area compares wild-type reduction (Fig. 1 F, 1L significantly; Fig. 2 F).Illustrate thus, the 24-nt siRNA that MITE produces, by changing the histone methylated modification of other adjacent gene and then have impact on the expression of contiguous gene on transcriptional level.

Embodiment 5, process LAN LOC_Os08g19420 plant phenotype

From rice cDNA, with primer CX8996, (its nucleotide sequence is as shown in SEQ ID NO:44, containing XbaI enzyme cutting site) and CX8997 (its nucleotide sequence is as shown in SEQ ID NO:45, containing BglII restriction enzyme site) CDS of the LOC_Os08g19420 gene that increases, the nucleotide sequence of described CDS is as shown in SEQ ID NO:6.Then cut the CDS of LOC_Os08g19420 gene with XbaI and BglII enzyme, cut paddy rice over-express vector pCAMBIA2302-Ubiqutin-FLAG with SpeI and BamHI enzyme simultaneously, connect after respectively digestion products being reclaimed.Select the clone that sequencing result is correct, by Agrobacterium genetic transformation wild-type, Japan is fine.After screening positive transgenic strain, its phenotype is observed in plantation land for growing field crops.

As shown in Figure 3A, we identify 4 LOC_Os08g19420 process LAN strains, and have carried out Phenotypic Observation to it.Result shows these 4 process LAN strains compared with wild-type, and the sword-like leave angle shown in various degree becomes large abnormal phenotype (Fig. 3 B-C).Visible, LOC_Os08g19420 gene upregulation expresses the phenotype that may contribute to mutant Leaf angle and become large, have impact on plant type of rice.

Embodiment 6, warm tropic Japonica commitment Mutation are analyzed

Paddy rice temperature tropic Japonica based on sequence of resurveying: 2 temperate zone japonica rice (Japan is fine, IRGC418); 2 tropic Japonica (IRGC17757, IRGC328) genome sequences, the compare of analysis commitment site of rice leaf intersection angle related gene LOC_Os08g19420 (Ditto-like MITE transposon).The 250bp DNA sequence dna of temperate zone japonica rice has typical MITE transposon feature, namely comprise TAA target site direct repetitive sequence and red block instruction 14bp inverted terminal repeat sequence (Fig. 4 A) of blue arrow instruction, described temperate zone japonica rice 250bp has the nucleotide sequence of typical MITE transposon feature as shown in SEQ ID NO:3.Different from temperate zone japonica rice, tropic Japonica sequence then has excalation and base is replaced, and finally form incomplete Ditto-like MITE transposon (Fig. 4 A) by 156bp DNA sequence dna, its nucleotide sequence is as shown in SEQ ID NO:4.The natural variation that have detected Ditto-like MITE transposon further, on the impact of flanking genes, finds that incomplete Ditto-like MITE transposon may cause the up-regulated expression of flanking genes LOC_Os08g19420 (Fig. 4 B).The above results shows, the natural variation in rice leaf intersection angle commitment site well can select site for plant type of rice breeding provides, and has good application prospect.

Claims (10)

1. a DNA sequence dna, has the function of regulating plant Leaf angle, it is characterized in that described DNA sequence dna has the nucleotide sequence as shown in SEQID NO:5 or 6.

2. DNA sequence dna according to claim 1, is characterized in that the aminoacid sequence of described DNA sequence encoding is as shown in SEQIDNO:7.

3. an expression cassette, is characterized in that described expression cassette comprises DNA sequence dna according to claim 1.

4. an expression vector, is characterized in that described expression vector comprises expression cassette according to claim 3.

5. an engineering bacteria, is characterized in that described engineering bacteria contains expression vector according to claim 4.

6. the application of DNA sequence dna in regulating plant Leaf angle size, comprises the expression of raising or lowering associated dna sequence, thus affects the size of leaf angle, it is characterized in that the nucleotide sequence of described associated dna sequence is as shown in SEQ ID NO:5 or 6.

7. application according to claim 6, the expression of wherein said downward associated dna sequence refers to that the mode by suddenling change regulates and controls the expression of SEQ ID NO:5 or 6 in plant.

8. application according to claim 7, wherein said sudden change comprises the nucleotide sequence mutation caused by the method for physics or chemistry, chemical process comprises the mutagenesis caused with mutagenic compound process such as EMS, described sudden change can also be point mutation, also can be DNA disappearance or insertion mutation, can also be produced by RNAi gene silencing means, or the site-directed mutagenesis technique such as TALLEN, CRISPR/Cas9.

9. a transposon, has the function of regulating plant Leaf angle, it is characterized in that the nucleotide sequence of described transposon is as shown in SEQ ID NO:1,2,3 or 4.

10. the application of transposon according to claim 9 in the regulation and control of leaf angle.