CN106350525A

CN106350525A - Rice grain gene DSS and encoded proteins and application thereof

Info

Publication number: CN106350525A
Application number: CN201610985544.7A
Authority: CN
Inventors: 万建民; 江玲; 张胜忠; 冯志明; 牟昌龄; 刘世家; 刘喜; 田云录; 赵志刚; 王益华; 刘裕强; 陈亮明
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2016-11-09
Filing date: 2016-11-09
Publication date: 2017-01-25
Anticipated expiration: 2036-11-09
Also published as: CN106350525B

Abstract

The invention discloses a rice grain gene DSS and encoded proteins and application thereof. The cloned gene (DSS) has a nucleotide sequence shown as SEQ ID NO:1 and an amino acid sequence shown as SEQ ID NO: 3. According to cloning and identification of the rice grain gene (DSS) and expression analysis of the gene, the functions of the gene are verified, mutant dss seeds can be enlarged due to over-expression of DSS and can restore to the sizes of wild seeds, and the sizes of the wild seeds can be narrowed to a certain degree by inhibiting expression of the gene. Therefore, the gene has certain effects in the aspect of yield and quality breeding of rice.

Description

A kind of rice grain shape gene dss and its coded protein and application

Technical field

The invention belongs to genetic engineering field is and in particular to a kind of gene dss of control rice grain shape, and it encodes egg White matter and application.

Background technology

Oryza sativa L. is important cereal crops, and it provides the Energy intaking demand of world population about 21%.In recent years, Oryza sativa L. The increase of per unit area yield encounters bottleneck, how to improve Oryza sativa L. in the case that Monitoring of Paddy Rice Plant Area is basically unchanged or declines year by year Total output, Ensuring Food Safety, is the significant challenge currently facing.The yield of Oryza sativa L. by number of productive ear, number of grain per ear and Grain waits factors composition again, and seed size is the direct determiner of rice grain weight.Seed size not only affects rice yield, It is an important quality trait.Large number of and widely distributed for the crowd of food with Oryza sativa L., people can be according to the hobby of oneself And social mores are selecting the type of edible rice.Such as, people's hobby seed of the U.S., southern china and most of Asian countries Elongated rice varieties, and the resident on the ground such as Korea S, Japan and north of China then prefers the short and round rice varieties of seed.Cause This illustrates the regulatory mechanism of seed size and is to improve rice yield and improve rice quality using desirable genes in breeding One Critical policies.

The grain type character of Oryza sativa L. includes that grain length, grain be wide, grain is thick and the mutual ratio between them.Typically now think grain The heredity long, grain is wide and grain is thick is all by controlled by multiple genes.Grain type related gene is divided by regulating cell or extends, impact grain husk The processes such as the grouting of the size of shell and seed, to determine size and the shape of seed, finally affect the yield of crop.Most of water Grain of rice type gene is to detect grain type qtls by building genetical population, then carries out finely positioning by backcross population and find, As gs3, gw2, gs5, gw6a etc..Other participates in the gene of regulation and control hormone-content and signal transduction, also assists in rice grain The regulation and control of type, such as d1, d2, brd2, arf2, gn1a etc..

Although the report of existing many grain type related genes at this stage, really use in produce reality and few, study carefully It is an extremely complex character that its reason is mainly rice grain shape, by polygenic regulation and control, and at this stage for its research master Still to concentrate on the clone of individual gene, the regulated and control network of their complexity is also known little about it.Therefore for rice grain shape On the one hand research needs the interaction and regulated and control network between our research clone genes, on the other hand then needs us gram Grand more type genes, to disclose the molecular mechanism of grain type regulation and control more fully hereinafter.

Content of the invention

It is an object of the invention to disclosing a kind of nucleotide sequence of rice grain shape related gene dss.Its nucleotide sequence As shown in seq id no.1, containing 7550bp.

Second object of the present invention also provides the protein sequence of described rice grain shape related gene dss coding, its amino Acid sequence as shown in seq id no.3, containing 473 aminoacid.

The encoding gene of described protein be preferably following 1) or 2) or 3) described in dna molecule:

1) the dna molecule shown in seq id no.1；

2) the dna molecule shown in seq id no.2；

3) under strict conditions with 1) or 2) the dna molecule of the dna sequence hybridization that limits and encoding said proteins；

4) with 1) or 2) or 3) the dna sequence that limits has more than 90% homology, and coded plant grain type associated protein Dna molecule.

Recombinant expression carrier containing gene described in any of the above falls within protection scope of the present invention.

Can use the recombinant expression carrier that existing plant expression vector construction contains described gene.

Described plant expression vector includes double base agrobacterium vector and can be used for carrier of plant micropellet bombardment etc..Described plant Thing expression vector also can comprise 3 ' end untranslated regions of exogenous gene, that is, comprise polyadenylation signals and any other participation Mrna processing or the dna fragment of gene expression.The bootable polyadenylic acid of described polyadenylation signals is added to the 3 ' of mrna precursor End, such as Agrobacterium crown gall nodule induction (ti) plasmid gene (as kermes synzyme no gene), plant gene are (as soybean storage egg White gene) untranslated region of 3 ' end transcriptions is respectively provided with similar functions.

During using described gene constructed recombinant plant expression vector, can be plus any one before its transcription initiation nucleotide Enhancement mode promoter or constitutive promoter, such as cauliflower mosaic viruses (camv) 35s promoter, the ubiquitin promoter of Semen Maydiss (ubiquitin), they be can be used alone or are used in combination with other plant promoters；Additionally, the gene using the present invention When building plant expression vector, it is also possible to use enhancer, including translational enhancer or transcriptional enhancer, these enhancer regions can To be atg start codon or neighboring region start codon etc., but must be identical with the reading frame of coded sequence, whole to ensure The correct translation of individual sequence.The source of described translation control signal and start codon is extensive, can be natural, also may be used To be synthesis.Translation initiation region can come from transcription initiation region or structural gene.

For the ease of being identified to transgenic plant cells or plant and screening, plant expression vector used can be carried out Processing, such as add the coding that can express in plant can produce the enzyme of color change or luminophor gene (gus gene, Luciferase genes etc.), there is the antibiotic marker thing (gentamycin label, kanamycin label etc.) of resistance or anti- Chemical reagent marker gene (as anti-herbicide gene) etc..From the security consideration of transgenic plant, any selectivity can be not added with Marker gene, directly screens transformed plant with adverse circumstance.

Described recombinant expression carrier can be restructuring over-express vector or restructuring interference carrier.

Restructuring over-express vector can be in the weight with restricted enzyme kpni and spei double digestion carrier pcambia1390 The recombiant plasmid that described gene (dss) obtains is inserted in group site.Pcambia1390 containing dss is named as pcambia1390-dss.

Restructuring interference carrier can be in the kpn i and sac i recombination site of lh-fad2-1390rnai carrier and mlu i The recombiant plasmid that the Partial Fragment inserting described gene (dss) respectively with bamh i recombination site obtains.By the lh- containing dss Fad2-1390rnai is named as lh-fad2-1390rnai-dss.

Expression cassette containing gene described in any of the above (dss) and recombinant bacterium belong to protection scope of the present invention.

Expand described gene (dss) total length or the primer pair of arbitrary fragment falls within protection scope of the present invention, described Primer pair preferred primer1/primer2, primer3/primer4, primer5/primer6 and primer7/primer8；Its Middle primer1 sequence as shown in seq id no.4, primer2 sequence as shown in seq id no.5, primer3 sequence such as seq Shown in id no.6, primer4 sequence as shown in seq id no.7, primer5 sequence as shown in seq id no.8, primer6 Sequence as shown in seq id no.9, primer7 sequence as shown in seq id no.10, primer8 sequence such as seq id no.11 Shown.

The positioning primer (being shown in Table 1) being related to during this gene of map based cloning, except primer indel3-29, rm426, Outside rm168, rm1350, rm8277 and rm3225, remaining primer is that this experiment needs and the primer of designed, designed, and these voluntarily set The primer of meter falls within protection scope of the present invention.

Beneficial effect:

The rice grain shape related gene dss of the present invention can affect the size of rice grain.Under mutant background, overexpression This gene may result in rice grain shape to become big.Under wild type background, the expression of this locus gene is suppressed to may result in rice grain shape one Determine diminishing of degree.Described gene and mutant can apply to theoretical research and the genetic improvement that rice grain shape regulates and controls, from And so that the yield and quality of Oryza sativa L. is further improved.

Brief description

Fig. 1 is wild type wt (nanjing35) and mutant dss phenotype analytical.

The Oryza glutinosa comparison diagram of a wild type wt and mutant dss；

The brown rice comparison diagram of b wild type wt and mutant dss；

The average grain length of c wild type wt and mutant dss；

The average grain of d wild type wt and mutant dss is wide；

The average mass of 1000 kernel of e wild type wt and mutant dss；

The average single plant yield of f wild type wt and mutant dss；

The plant type figure in g wild type wt and mutant dss heading period；

H wild type wt and each internode of mutant dss stem and fringe comparison diagram；

The each internode of stem of i wild type wt and mutant dss and spike length degree statistics.

Fig. 2 is the finely positioning of dss.

Fig. 3 is over-express vector pcambia1390 plasmid map.

The interference carrier lh-fad2-1390rnai plasmid map that Fig. 4 transforms for Li Hui.

Fig. 5 is that the grain type of transgenic overexpression plant is observed.

A compares dss and the plant type figure in overexpression plant heading period；

B compares the relative expression quantity of dss gene in dss and overexpression plant；

C wild type wt, mutant dss and overexpression strain grain type and clever shell cross section；

D wild type wt, mutant dss and overexpression strain lemma cross section periphery prothenchyma (of wood) number.

The grain type that Fig. 6 disturbs plant for transgenic is observed.

A compares wt and transgenic disturbs the plant type figure after plant heading；

B compares wt and transgenic disturbs the relative expression quantity of dss gene in plant；

C wild type wt, mutant dss and transgenic interference plant grain type and clever shell cross section；

D wild type wt, mutant dss and transgenic interference plant lemma cross section periphery prothenchyma (of wood) number.

Specific embodiment

Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method, if no special instructions, is conventional method.Test material used in following embodiments, if no special instructions, is certainly Routine biochemistry reagent shop is commercially available.

Embodiment 1, the discovery of coding rice grain shape gene

First, the acquisition of rice grain shape mutant

Rice grain shape mutant dss (decreased seed size) is that japonica rice variety nanjing 35 passes through co⁶⁰Radiation After mutation, screening obtains.Through for many years, multipoint relaying, the phenotype of dss is stable.The method of investigation grain type: latter 30 days of heading, take master Fringe top 1/4 seed, measures using slide gauge is wide to the grain length and grain not having a seed, wild type and each 10 weights of mutant Multiple.Investigation mass of 1000 kernel method: wild type and mutant respectively take 1000 seeds, weigh weight, are respectively repeated 5 times.Investigation individual plant produces Amount method: latter 30 days of heading, wild type and all grain harvests of every plant of mutant are weighed, be respectively repeated 10 times.By Fig. 1 Shown, find that the grain length of dss, grain are wide and mass of 1000 kernel type significantly diminishes compared with wild type, single plant yield significantly reduces.In addition mutant The plant height of dss significantly reduces compared with wild type, and each internode all has and shortens in various degree.The tiller number of mutant dss dramatically increases.

2nd, the acquisition of rice grain shape related gene

It is maternal with rice variety 9311, mutant dss obtains f for paternal hybrid₁, the f after selfing afterwards₂Segregating population (8000 plants).In f₂Select phenotype and the extremely similar individual plant of mutant dss in colony, extract dna using its blade.Using covering Ssr the and indel labelling of rice genome carries out linkage analysises, and the preliminary gene finding control rice grain shape is present in labelling Between rm426 and indel3-29.Afterwards between rm426 and indel3-29, designed, designed ssr and indel labelling simultaneously combine The phenotype of individual plant reduces positioning interval further, reduces between a2 and indel3-3 the most at last between positioning area, and a9 is altogether Separation marking.According to the sequence prediction that Japan is fine, the physical distance of this section is about 140kb, finds this section by sequencing Lack 18kb (Fig. 2)

The method of above-mentioned ssr labeled analysis is as described below:

(1) extract total dna of above-mentioned selection individual plant as template, concrete grammar is as follows:

1. take 0.2 gram about of Oryza sativa L. young leaflet tablet, be placed in 2.0ml eppendorf pipe, in pipe, place a steel ball, The eppendorf pipe installing sample is freezed 5min in liquid nitrogen, is placed in pulverizing sample on 2000 type geno/grinder instruments 1min.

2. 660 μ l extracting solution (tris-hcl containing 100mm (ph 8.0), 20mm edta (ph 8.0), 1.4m are added The solution of nacl, 0.2g/ml ctab), whirlpool device is acutely vortexed and mixes, ice bath 30min.

3. 40 μ l 20%sds, 65 DEG C of temperature bath 10min, mixing of gently turning upside down every two minutes are added.

4. 100 μ l 5m nacl are added, gentle mixing.

5. add 100 μ l 10 × ctab, 65 DEG C of temperature bath 10min, be interrupted mixing of gently turning upside down.

6. add 900 μ l chloroforms, fully mix, 12000rpm is centrifuged 3min.

7. transfer supernatant, to 1.5ml eppendorf pipe, adds 600 μ l isopropanols, mixes, and 12000rpm is centrifuged 5min.

8. abandon supernatant, precipitate with 70% (volumn concentration) ethanol rinse once, room temperature airing.

9. add 100 μ l 1 × te (121 grams of tris are dissolved in 1 liter of water, adjust ph value to 8.0 solution obtaining with hydrochloric acid) molten Solution dna.

10. 2 μ l electrophoresis detection dna mass are taken, and with du800 spectrophotometric determination concentration (beckman instrument inc.u.s.a).

(2) dna of said extracted is diluted to about 20ng/ μ l, carries out pcr amplification as template；

Pcr reaction system (10 μ l): dna (20ng/ul) 1ul, forward primer (2pmol/ul) 1ul, downstream primer (2pmol/ul) 1ul, 10xbuffer (mgcl₂Free) 1ul, dntp (10mm) 0.2ul, mgcl₂(25mm) 0.6ul, rtaq (5u/ul) 0.1ul, ddh₂O5.1ul, common 10ul.

Pcr response procedures: 94.0 DEG C of degeneration 5min；94.0 DEG C of degeneration 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, altogether Circulation 35 times；72 DEG C of extension 7min；10 DEG C of preservations.Pcr reaction is carried out in mj research ptc-225 thermal cycler.

Above-mentioned primer development process is as follows:

(1) ssr marker development

The ssr labelling of public collection of illustrative plates is integrated with Rice Genome Sequence, is downloaded the bac/pac near mutational site Cloned sequence.With potential in ssrhunter (Li Qiang etc., heredity, 2005,27 (5): 808-810) or ssrit software search clone Ssr sequence (number of repetition >=6)；The sequence of these ssr and its neighbouring 400～500bp is existed by blast program in ncbi Line is compared with corresponding long-grained nonglutinous rice sequence, if both ssr numbers of repetition are variant, tentatively infers the pcr of this ssr primer There is polymorphism in product between Xian, round-grained rice；Recycle primer premier 5.0 software design ssr primer, and by Shanghai English fine horse Bioisystech Co., Ltd synthesizes.Paired for the ssr of designed, designed primer equal proportion is mixed, detects it in 9311 Hes Polymorphism between nanjing35, shows the molecular marker that polymorphic person is used as finely positioning dss.For positioning molecule a1, A2, a13, a15 and a19 labelling is shown in Table 1.

The pcr product detection of ssr labelling:

Amplified production is analyzed with 8% native polyacrylamide gel electrophoresises.With the dna ladder of 50bp as contrast ratio Compared with the molecular size range of amplified production, silver staining develops the color.

(2) indel marker development

Indel design of primers: 9311 and nanjing35 partial sector near dss position are sequenced, and Compare, find the snps existing between the two, use software design indel labelling based on these snps, use simultaneously The corresponding another primer of primer premier 5.0 software design, primer indel3-3 and indel3-4 for positioning is shown in Table 1.

The pcr reaction system of indel labeled analysis: dna (20ng/ul) 2ul, primer1 (10pmol/ul) 2ul, Primer2 (10pmol/ul) 2ul, 10xbuffer (mgcl₂Free) 2ul, dntp (10mm) 0.4ul, mgcl₂(25mm) 1.2ul, rtaq (5u/ul) 0.4ul, ddh₂O 10ul, cumulative volume 20ul.

Amplified reaction is carried out on ptc-200 (mj research inc.) pcr instrument: 94 DEG C of 3min；94 DEG C of 30sec, 55 DEG C (primer different, adjusted) 45sec, 72 DEG C of 2.5min, 35 circulations；72℃5min.

Pcr product purification reclaims, and carries out by test kit (Beijing tiangen company) step.Pcr product is digested overnight Afterwards, with being separated by electrophoresis in the agarose gel of 1-4%, observe under uviol lamp through eb dyeing and take pictures.Dcaps with 8% non- Degeneration page glue separates, silver staining.

Table 1 is used for the molecular marker of gene mapping

(3) acquisition of grain type gene

According to the site design primer of positioning, sequence is as described below:

Primer1:

5'—cgggagcggaagagatta—3'(seq id no.4)

Primer2:

5'—tcggtaaacctttctgaactt—3'(seq id no.5)

With primer1 and primer2 as primer, with the cdna of nanjing35 as template, carry out pcr amplification and obtain purpose Gene.This contains whole coding regions of this gene to the amplified production of primer.

Amplified reaction with kod enzymatic amplification (purchased from toyobo company), in ptc-200 (mj research inc.) pcr instrument On carry out: 94 DEG C of 2min；98 DEG C of 10sec, 60 DEG C of 30sec, 68 DEG C of 5min, 35 circulations；68℃20min.Pcr product is reclaimed It is connected to after purification (purchased from takara company) on carrier pmb18t carrier, conversion escherichia coli dh5 α competent cell (is purchased from Tiangen company), select positive colony, be sequenced.

Sequencing results show, the fragment that pcr reaction obtains comprises the nucleotide sequence shown in seq id no.2, compiles The protein (see seq id no.3) of 473 amino acid residue compositions of code, the albumen shown in seq id no.3 is named as dss.

Embodiment 2, the acquisition of transgenic plant and identification

First, restructuring over-express vector and interference carrier build

With the cdna of nanjing35 as template, carry out pcr amplification and obtain dss gene, pcr primer sequence is as follows:

Primer3 (sequence shown in underscore is kpn i recombination site):

5'—ttctgcactaggtaccatgcagcggcggcgggc—3'(seq id no.6)

Primer4 (sequence shown in underscore is spe i recombination site):

5'—ggactagtttaaacatcatatacgggc—3'(seq id no.7)

Above-mentioned primer is located at the coding region original position of gene shown in seq id no.2 and coding region terminator position, expands Volume increase thing contains the complete coding region of this gene, by pcr product recovery purifying.Usinghd cloning kit Recombination kit (takara company) is by pcr product cloning in carrier pcambia1390 (Fig. 3).

In-fusion recombining reaction system (10 μ l): pcr product 10-200ng, reclaims through kpn i and spe i double digestion Pcambia1390 carrier 50-200ng, 5 × in-fusion hd enzyme premix 2 μ l, deionized water to 10μl.Pipette tips piping and druming is placed in after 50 DEG C of reaction 15min of mixed system on ice after mixing, and takes 2 μ l reaction system heat shock methods to turn Change escherichia coli dh5 α competent cell (tiangen company).Cell will be totally converted be uniformly coated on card containing 100mg/l that is mould On the lb solid medium of element.37 DEG C of culture 12-16h, picked clones positive colony, it is sequenced.

The structure of interference carrier, equally with the cdna of nanjing35 as template, carries out pcr amplification and obtains dss gene, pcr Primer sequence is as follows:

Primer5 (sequence shown in underscore is kpn i recombination site):

5'—ttctgcactaggtaccaggcctggccgttgcttattgcgtttg—3'(seq id no.8)

Primer6 (sequence shown in underscore is sac i recombination site):

5'—ctgacgtaggggcgatagagctccggtatctaattgccgctgat—3'(seq id no.9)

Primer7 (sequence shown in underscore is bamh i recombination site):

5'—cggggatccgtcgactacgccgttgcttattgcgtttg—3'(seq id no.10)

Primer8 (sequence shown in underscore is mlu i recombination site):

5'—aggtggaagacgcgttaccggtatctaattgccgctgat—3'(seq id no.11)

The product of above-mentioned primer5/6 and primer7/8 amplification is respectively designated as rnai1 and rnai2, all contains this The partial coding region of gene, by pcr product recovery purifying.UsingHd cloning kit recombination kit (takara company) is by pcr product cloning in carrier lh-fad2-1390rnai (Fig. 4).

The pcr product 10-200ng of in-fusion recombining reaction system (10 μ l): rnai1, uses kpn i and sac for the first time I double digestion reclaims lh-fad2-1390rnai carrier 50-200ng, 5 × in-fusion hd enzyme premix 2 μ l, Deionized water to 10 μ l, pipette tips piping and druming is placed on ice by after 50 DEG C of reaction 15min of mixed system after mixing, and second Pcr product 10-200ng, the bamh i and mlu i double digestion of secondary use rnai2 reclaims the lh-fad2- being connected with rnai1 fragment 1390rnai carrier 50-200ng, 5 × in-fusion hd enzyme premix 2 μ l, deionized water to 10 μ l.Pipette tips piping and druming is placed in after 50 DEG C of reaction 15min of mixed system on ice after mixing, and takes 2 μ l reaction system heat shock method conversions big Enterobacteria dh5 α competent cell (tiangen company).It is uniformly coated on being totally converted cell containing 100mg/l kanamycin On lb solid medium.37 DEG C of culture 12-16h, picked clones positive colony, it is sequenced.

Sequencing result shows, has obtained the recombinant expression carrier containing dss gene shown in seq id no.2, will contain dss Pcambia1390 be named as pcambia1390-dss, the lh-fad2-1390rnai containing dss is named as lh-fad2- 1390rnai-dss.

2nd, the acquisition of recombinational agrobacterium

Pcambia1390-dss and lh-fad2-1390rnai-dss is converted respectively Agrobacterium eha105 bacterium with thermal shock method Strain (purchased from handsome company of the U.S.), obtains recombinant bacterial strain, extracts plasmid and carries out pcr and enzyme action identification.Pcr and enzyme action are just identified True recombinant bacterial strain is respectively designated as eh-pcambia1390-dss and eh-lh-fad2-1390rnai-dss.

3rd, the acquisition of transgenic plant

By eh-pcambia1390-dss rice transformation mutant dss, eh-lh-fad2-1390rnai-dss rice transformation Nanjing35, method particularly includes:

(1) 28 DEG C of culture eh-pcambia1390-dss and eh-lh-fad2-1390rnai-dss16 hour, collects bacterium Body, and be diluted in the n6 fluid medium containing 100 μm of ol/l (sigma company, c1416) to concentration be od₆₀₀≈ 0.5, obtains Obtain bacterium solution；

(2) by the mutant dss and nanjing 35 Mature Embryos of Rice embryo callus of culture to month and step (1) bacterium solution mixed infection 30min, filter paper proceed to after blotting bacterium solution co-cultivation culture medium (n6 solid co-cultivation medium, Sigma company) in, 24 DEG C co-culture 3 days；

(3) wound healing of step (2) is seeded in containing 100mg/l paromomycin (phyto technology Laboratories company) n6 solid screening culture medium on for the first time screen (16 days)；

(4) picking health wound healing proceeds to programmed screening in the n6 solid screening culture medium containing 100mg/l paromomycin, Every 15 days subcultures are once；

(5) picking health wound healing proceeds to and screens for the third time in the n6 solid screening culture medium containing 50mg/l paromomycin, Every 15 days subcultures are once；

(6) picking kanamycin-resistant callus tissue proceeds to differentiation on division culture medium；

Obtain the t of seedling differentiation₀For positive plant.With mutant dss and nanjing35 as negative control.

4th, the identification of transfer-gen plant

1st, pcr Molecular Identification

The t that step 3 is obtained₀Extract genome dna for positive plant, with genome dna as template, utilize The primer on primer primer3 and seq id no.2 near pcambia1390 upper seq id no.2 insertion point left margin Primer4 is expanded (primer3:5' as primer pairttctgcactaggtaccatgcagcggcggcgggc—3' (seq id no.6) and primer4:5'ggactagtTtaaacatcatatacgggc 3'(seq id no.7)), amplification Length 1446bp.Using the primer primer4 near seq id no.2 insertion point left margin on lh-fad2-1390rnai and Primer primer5 on seq id no.2 is expanded (primer5:5' as primer pairttctgcactaggtaccaggcctgGccgttgcttattgcgtttg 3'(seq id no.8) and primer6:5'ctgacgtaggggcgatagagctcCggtatctaattgccgctgat 3'(seq id no.9)), amplification length 483bp. Pcr reaction system: dna (20ng/ul) 2ul, primer5 (10pmol/ul) 2ul, primer6 (10pmol/ul) 2ul, 10xbuffer(mgcl₂Free) 2ul, dntp (10mm) 0.4ul, mgcl₂(25mm) 1.2ul, rtaq (5u/ul) 0.4ul, ddh₂O 10ul, cumulative volume 20ul.Amplified reaction is carried out on ptc-200 (mj research inc.) pcr instrument: 94 DEG C 3min；94 DEG C of 30sec, 55 DEG C of 45sec, 72 DEG C of 1min, 35 circulations；72℃5min.

Reclaim pcr product with test kit (Beijing tiangen company) purification.Pcr product is examined with 1% sepharose electrophoresis Survey.

2nd, phenotypic evaluation

Respectively by t₀In generation, turns eh-pcambia1390-dss and eh-lh-fad2-1390rnai-dss plant, nanjing35 It is planted in Agricultural University Of Nanjing's rice test station with mutant dss, cross-sectional slices observation and expression are carried out to small ear after heading Amount analysis, and investigate grain type.As shown in Figure 5, proceed to eh-pcambia1390-dss transfer-gen plant dss-oe1 and In dss-oe2, the expression of dss significantly improves, and the size of seed significantly increases compared with mutant dss, returns to the water of wild type Flat.As shown in Figure 6, the expression proceeding to dss in transfer-gen plant r4 and r7 of eh-lh-fad2-1390rnai-dss is notable Reduce, seed size significantly diminishes compared with wild type wt.Illustrate that dss can affect the grain type of seed really.

<110>Agricultural University Of Nanjing

<120>a kind of rice grain shape gene dss and its coded protein and application

<160> 21

<210> 1

<211> 7550

<212> dna

<213>Oryza Oryza sativa L. (oryza sativa var. nipponbare)

<220>

<223>gene order of seed grain type related gene dss

<400> 1

aaagaaaagg gctagagtct aagaggggag agagagagag agagagagag gaaggaaccc 60

ttcgtcctcg tcgcctcact tttgtctccc actccttcgt cggcggctcg gctcggcgca 120

ggcgcagcgg ggagaagacc aaggaggagg gagaggcgga cgcgggagcg gaagagatta 180

atttccagtg gggtttgggg cgcggtgagg aggtgaggga tgcagcggcg gcgggcgcag 240

acgtgggcgg gggtggggaa gacggcgcag gcggcggcgg cgcacgcggc gctcttctgc 300

ttcacgctcc tcctcgcgct caaagtcgac ggccgcacag cctactcctg gtggtaacgc 360

tccctgctct aaaccctagc tagcaccctc ccccgtcctc cgccgccacg ggcctccgct 420

tcgatctggc ttagtggttc ccgcgagatt gcgagttggg ggtggaatta gtggctggcg 480

tagtggtttt tttggggggg tttccatcgg atgcagatgc aggctgaggg gggaatcgga 540

ctagtccttt gcgtgcggtt gagagtcgtc gcctgcttgt ggtggtctgg tgaactaatt 600

ttctccacca gaacgattca gctctttcgt gtccttgtgt ttggacattg atgagttgat 660

gtaggcattt tgctcacgca gagggtgtgc ttcggttagt ctttagctga tgctgggcca 720

cagttcctgg ttctactgaa ggctgcactt tgagagctcg ttcttatttg gttatgtatt 780

tgtttgagta acccagcgct cttgttccta taatgatggt agcagattca gaagacattg 840

ttgactgctt tgtctgtagc agtgaaggta ttaataccga atgctaattc tgctatttcc 900

atcatgcagg attatattca tccctctatg gctatttcat ggcattgttg cccgtggaag 960

gttttcaatg ccagcccctt cgcttcctca tggccgtcat gtaagaattg gtatcaattt 1020

ctctagttag tatgcacgtg cttaccccac attttgatct gatgggtttc tatttgtatg 1080

tagtgggctc cttgccattc aattgttgca gcgccgttgc ttattgcgtt tgagctgctg 1140

ctttgcatat atctcgaaag tttgagaggt aggttcttag gttatctaga tgagagaagc 1200

tttggcgacc ataaacgaat ttcctggagg ctcttatacc ccttctctct gtttgttctt 1260

ccgcagttaa aagtaagccg actgttgatt tgaagattgt attccttcct cttctggcct 1320

ttgaagtgat tattcttgct gacaatttca ggtaatgaat ctgaacaatt attttgcatg 1380

cctttaatac tgtatcagta aatgcacaaa cgcactacca attggcacga ttgtatgtgc 1440

cttactgttt tatctgctta tgtattctgc aagtcttaag tgctgaatgt aactagtttt 1500

tttttccaag attgtttttc tgactaagca gctagcactt ataaactgtg ccatgaactc 1560

gtaatatgtc ctaacctatt tttggttacg tctgggattg tagtaagacg acctgaccta 1620

ttgtcaaata tggtatgcac cttcaagagc agctaacgcg ttttagatta taaatttctt 1680

gcaaccaaaa gaattcaatt atctggtgtt ctgttgttct caatggccgg ttcttggtgt 1740

ctgtcttttt ctccactgga aagaatactg aaattactca actgcctctt tgtttatttt 1800

tgcacactgc ttaggctgtg ttcgccagtc cacgttccca accggaacag tacgcgcgga 1860

aaacggagcg gtccattagc gcgtaattaa ttaagtatta gctatttttt tttcaaaaat 1920

agattaattt gattttttaa gcaacttttg tatagaaact ttttgcaaaa aacacaccgt 1980

ttaacagttt gaaaagtgtg cgcgtggaaa acgagggaga ggggttggaa aaaggggtgc 2040

cgaacacagc cttagtcagg ttcaatgata tctttctgtt ggttgcaaaa taaattaatc 2100

atgatcatta tactcatatg gtttctgttg gttgcaaaat aaattaataa ttatcattat 2160

acttgacgac ttggtatgta gagggtagca ctaagtgctt gtttatcatt gcttgtttga 2220

tagcaggttg aattatctca attacaaata tactcaagtc tactgttgat attattctta 2280

gttttgttat tccgtacaac attttttttg ctacataata ataaatggtg gcattctatc 2340

caaaagttac aaatggtgtt tttaaaaggt aaatttcgca atactgaact accatttgca 2400

aaactatcgc aaaagacaca tgtttattca caattttgga gaactacact ttttagttgc 2460

aaaatgtgca gcaaaactac actcctatca gagaaacagg tctgataggt tgggccctct 2520

catcagtcat cagtatttca tccgggttgt ttctgtttct gatgcatgtg ttggttaaaa 2580

agaaacagcc tacacatgca acactgacta gtggacctag cctgtcaggc tcattggcat 2640

tttcagtaat ttagttttgt gaaacttttc acaaccaaag gtattcctcc gaaatcgtcg 2700

caaaagtgtg tttgcgattg ttttagcgat ggcttttgtc tggtttaatg aaattgaatc 2760

attgttaaaa tttagcttgt cacacaatca gtactgttgt tggtatgcaa cgcataaatt 2820

ggatgcaatt cataaagata tgtgctgacc aaatcatgaa ccttaatttg gccaaaaata 2880

tctagagata ttagttgtta atacaatagt agcgctaaag ttaatttgga tgttaacctt 2940

tatttttttt ccttgcattg gtttgactat tcagaatgtg tagagcttta atgccaggag 3000

atgaagaaag tatgagcgat gaagctattt gggagacact tcctgtgagt ataagtacta 3060

gtaactagtg gttcttttta agaaattatc tccattagct tcaatttgag cttaactttt 3120

atttactata agtaaccata cttctaccaa atcatcttat aaaaatatat tagaaacttt 3180

taaatacgat gataactaaa ttagagagct gaatatcatg caacaaaatg cattattttt 3240

cagtccattt ttagtggaat actggattgg aataatttta ttgataagaa taaatcagct 3300

gcaaggatga aagtagaatc tcatgaactt gtcaattgac gatgctgtat tttcacatta 3360

tggagcctat atgttggact tgtaccagct taatatgggt tgatgattta ttaagtttaa 3420

caaagcaaaa catatacagt gaacaaatat tcactacata atgaagtaaa ttctgttcaa 3480

agtttaagag aattaaacct tgatcagggc ttgtgtttat agaattatgg tttgatcatt 3540

tcattctatc tgatactcta tttgtcacag cagaatgtac cgatttacat tgtaatattt 3600

cacaccgatg taaagtctat gaatgaataa cattgtgtgt ggctctttta taagttatct 3660

atgatatcat atttcttatc ttgtgctgac tgccgtgttc tagaatagtg tggactatac 3720

actgttcttt tcatcttata tgacacggct tctatttgct atcagcactt ttgggttgca 3780

atttctatgg tgtttcttat agctgctaca accttcacac ttttgaagct gtctggtaaa 3840

gtttcctgga tctccttttt gttttttgac cttcattatg gcatgtccta tttgttatgt 3900

gttttcaagc actgtagact gtagagttaa aaagtttgct tccttgcaca tgactaatat 3960

tgtttgtttg tctgctaagc ctctatagtt ggtaggatat ggctttctca aaagtcgctt 4020

cattgtttac tagagtggtt gtcacaatga cattcttcta agagtgatta ggtagttgca 4080

aatgagacaa tagttactaa aacaagatta gattgtcaat taaccataaa atctggaaac 4140

attacattca gcagcttggt tttgaaagcc aggcagtttt ctactatttg gaaatggctg 4200

gtgatttcac ctgaactggc cgggtttcta tttttggcag tttaaagcat aaattcgtgc 4260

aagttaaaac tatctttagt ataagcaata caatgttgga tagagagcaa aaagatattc 4320

ctagggttcc cgtgatgtga agacccacca gctgctttcc cattcacatg catatatgca 4380

acatttttcc atggtttctc actctaaaga gtgtaatctt ccaattccca acacaaaatc 4440

gaagtcagct tctccacact gaatcaaact ccttaatgca tttcatgtgg tcgattttct 4500

ttgacgatac tttcaatttg gtgatctatt acagttcttt tttttttggt atacgcaaaa 4560

gacttgtgta gcattaagga gtttgaatgt tacatcccct gcctagctcc atatagctgg 4620

gcaactacct aatgagtagt acaagattaa ttctcgtgat acaattgtgc gacctatgtg 4680

ccagagcgat gttagaccac agattgagcc atctttgctc ctgcaacgat ccagagtctt 4740

gcctctgcgg tgattgctgc aatcaccgca tccagttcct actttcccaa ttgaaaatcc 4800

ggtccttcaa gattacccaa gcgaccagga tccatcaaag ttctgagttg ttccagttac 4860

taggggctgc tttgtctgcc tcctccatga gcttttaagt gctacttttt aaatcaaatc 4920

atttattagt tcgatgtgat aagaacaaca tgttcagcaa actatcttag attgtacaat 4980

attcaggttt ttatttctct tcggcctaat tttcctctac tctgaaatgt tttgtgatat 5040

tattattgca tgacaggtga tgttggtgct ttgggatggt gggatttgtt tataaattat 5100

gggtgagact agtctcaata gcaattttct ttattaacga gatctgttat aatataatcc 5160

agcaccttct tttttgtaca gaatcgcgga gtgttttgca tttcttgttt gtactagatg 5220

gtttaatccc atgattcata aatctcctaa tcctggggag gctagctcat catcagcggc 5280

aattagatac cgtgattggg agagtggtct tctcctccca tcactagaag atcatgaaca 5340

agagaggctc tgtggtcttc ctgacatagg aggtcacgta atgaaaatac cactggtgat 5400

tttccaagtt ttgctttgta tgcgcttgga ggtacgtgtc atttatatat ttctattggg 5460

ttacatatgg ttgataaact ggtagatgca cttgtagaca gacattggat ggggattggg 5520

gagcttccag gaattgtttt ttaattatgt catgtaacag aacacagtaa cactatttgg 5580

aaaaaatgca aaacaagaac tttgtccatt ttctgagttc gtctaggggg tcaacgcttg 5640

ttagtggctt tttatcatga gctggatcaa taataatctt gaaaacatca tttgcttttg 5700

ttttttcagg gtacgcctcc tagtgctcag tatattccga tatttgcact gttctcccca 5760

ctgtttattt tacaaggcgc tggtgtcctt ttctctctag caagattgtt ggagaaggtt 5820

gttctactat tacgaaatgg accagttagt cctaattacc ttacaatctc atcaaaagtc 5880

cgtgattgct ttgcttttct tcatcgtggt tcaaggtaat atttgatagc tattatgagc 5940

tacttctcta tatgtttgtt ttcttgttgg cttatcttac tttgcatcac acaggcttct 6000

tggttggtgg tctattgatg aaggcagcaa agaagagcaa gcccggttat tctatactga 6060

atctactggg tacatgatag ttgacttcag cctgttcata ttgttaattt agatcctatt 6120

aagctggtca agttgtttca tttcctctat gtttacagtt ctttttgcgc atccacatcc 6180

actttctata ctgatttcct gcctggttgc cttttggttt taaggtacaa cacattttgt 6240

ggctatccac ctgaggtagt caggaaaatg cctaagaggg atcttgcaga agaggttaca 6300

ttctctcttt tcattttatt attgtttacc ttattaatgt tatgtgcact ctattttatc 6360

ataatataac tattttccta cttattatct ttcaggtatg gaggctccaa gcagctttgg 6420

gagagcaatc agaaattacc aaatgtacca agcaggaatt tgaaaggctt caaaatgtac 6480

catctccttg tgacttgtga agtttcatca ttttacatta tataaattgg tgcaatacat 6540

cctatagaca tgattgagtc cattaacttg aggacatgcc atttaggtcg ctcagcttac 6600

acaataagat cacatatgtc tgagtcgttc atgtttaagg agacttatgt gatatagcct 6660

tgaaactttt agcaaactac aattttaggt accgagaaat attgaattat caagtttgtg 6720

ggttcaagtg ggacatccat acaactctaa gaaactcatt tcattttaac cttttctgtt 6780

gttttattta agaacctaag tcactacagc tctatggcac taactgaaac ttccagagag 6840

gcagagagcg ctgatgatga tctgttggtt gtctgaccgg ctctttttcc tttgttgact 6900

aagtacttcc ttttccattt caggagaagg ttctttgtag gatttgctac gagggggaga 6960

tatgcatggt cttacttcct tgccggcaca gaacattatg caagtatgtt tccagtcact 7020

tgttaagcca ctttggatgc tcttacatgt tgatttggaa ctgacagttt tgttgatggt 7080

tctgtgatag gacttgttct gataagtgca agaaatgtcc aatctgccgt gtgcccattg 7140

aagaacgcat gcccgtatat gatgtttaaa cttcgctaac tcagatgaac gttacaaatt 7200

tgtacatgtt ggttgtgcaa tgtcgcgcca tgtagtctca atcacaactt taagctgatt 7260

gaggtttgca caagttcaga aaggtttacc gaatatggag aaaatataaa gcatatcatg 7320

tctaaccaaa agcatgaaaa ggtagttgat gatcattttg ccggttacaa ttatgtactg 7380

taagtatgtc atcggtggtt ttaacttttt tttttggtga tcgatagatg ctccagttag 7440

attgtgtagc atcttctcaa gtttatgcat tgtctgaatg taaataagaa tattgtcttg 7500

tttgagtgtt gtagtgctct ttggttgaga agagtagaaa agaaaaatgt 7550

<210> 2

<211> 1422

<212> dna

<213>Oryza Oryza sativa L. (oryza sativa var. nipponbare)

<220>

<223>the cds sequence of seed grain type related gene dss

<400> 2

atgcagcggc ggcgggcgca gacgtgggcg ggggtgggga agacggcgca ggcggcggcg 60

gcgcacgcgg cgctcttctg cttcacgctc ctcctcgcgc tcaaagtcga cggccgcaca 120

gcctactcct ggtggattat attcatccct ctatggctat ttcatggcat tgttgcccgt 180

ggaaggtttt caatgccagc cccttcgctt cctcatggcc gtcattgggc tccttgccat 240

tcaattgttg cagcgccgtt gcttattgcg tttgagctgc tgctttgcat atatctcgaa 300

agtttgagag ttaaaagtaa gccgactgtt gatttgaaga ttgtattcct tcctcttctg 360

gcctttgaag tgattattct tgctgacaat ttcagaatgt gtagagcttt aatgccagga 420

gatgaagaaa gtatgagcga tgaagctatt tgggagacac ttcctcactt ttgggttgca 480

atttctatgg tgtttcttat agctgctaca accttcacac ttttgaagct gtctggtgat 540

gttggtgctt tgggatggtg ggatttgttt ataaattatg gaatcgcgga gtgttttgca 600

tttcttgttt gtactagatg gtttaatccc atgattcata aatctcctaa tcctggggag 660

gctagctcat catcagcggc aattagatac cgtgattggg agagtggtct tctcctccca 720

tcactagaag atcatgaaca agagaggctc tgtggtcttc ctgacatagg aggtcacgta 780

atgaaaatac cactggtgat tttccaagtt ttgctttgta tgcgcttgga gggtacgcct 840

cctagtgctc agtatattcc gatatttgca ctgttctccc cactgtttat tttacaaggc 900

gctggtgtcc ttttctctct agcaagattg ttggagaagg ttgttctact attacgaaat 960

ggaccagtta gtcctaatta ccttacaatc tcatcaaaag tccgtgattg ctttgctttt 1020

cttcatcgtg gttcaaggct tcttggttgg tggtctattg atgaaggcag caaagaagag 1080

caagcccggt tattctatac tgaatctact gggtacaaca cattttgtgg ctatccacct 1140

gaggtagtca ggaaaatgcc taagagggat cttgcagaag aggtatggag gctccaagca 1200

gctttgggag agcaatcaga aattaccaaa tgtaccaagc aggaatttga aaggcttcaa 1260

aatgagaagg ttctttgtag gatttgctac gagggggaga tatgcatggt cttacttcct 1320

tgccggcaca gaacattatg caagacttgt tctgataagt gcaagaaatg tccaatctgc 1380

cgtgtgccca ttgaagaacg catgcccgta tatgatgttt aa 1422

<210> 3

<211> 473

<212> prt

<213>Oryza Oryza sativa L. (oryza sativa var. nipponbare)

<220>

<223>aminoacid sequence of seed grain type related gene dss

<400> 3

met gln arg arg arg ala gln thr trp ala gly val gly lys thr

1 5 10 15

ala gln ala ala ala ala his ala ala leu phe cys phe thr leu

20 25 30

leu leu ala leu lys val asp gly arg thr ala tyr ser trp trp

35 40 45

ile ile phe ile pro leu trp leu phe his gly ile val ala arg

50 55 60

gly arg phe ser met pro ala pro ser leu pro his gly arg his

65 70 75

trp ala pro cys his ser ile val ala ala pro leu leu ile ala

80 85 90

phe glu leu leu leu cys ile tyr leu glu ser leu arg val lys

95 100 105

ser lys pro thr val asp leu lys ile val phe leu pro leu leu

110 115 120

ala phe glu val ile ile leu ala asp asn phe arg met cys arg

125 130 135

ala leu met pro gly asp glu glu ser met ser asp glu ala ile

140 145 150

trp glu thr leu pro his phe trp val ala ile ser met val phe

155 160 165

leu ile ala ala thr thr phe thr leu leu lys leu ser gly asp

170 175 180

val gly ala leu gly trp trp asp leu phe ile asn tyr gly ile

185 190 195

ala glu cys phe ala phe leu val cys thr arg trp phe asn pro

200 205 210

met ile his lys ser pro asn pro gly glu ala ser ser ser ser

215 220 225

ala ala ile arg tyr arg asp trp glu ser gly leu leu leu pro

230 235 240

ser leu glu asp his glu gln glu arg leu cys gly leu pro asp

245 250 255

ile gly gly his val met lys ile pro leu val ile phe gln val

260 265 270

leu leu cys met arg leu glu gly thr pro pro ser ala gln tyr

275 280 285

ile pro ile phe ala leu phe ser pro leu phe ile leu gln gly

290 295 300

ala gly val leu phe ser leu ala arg leu leu glu lys val val

305 310 315

leu leu leu arg asn gly pro val ser pro asn tyr leu thr ile

320 325 330

ser ser lys val arg asp cys phe ala phe leu his arg gly ser

335 340 345

arg leu leu gly trp trp ser ile asp glu gly ser lys glu glu

350 355 360

gln ala arg leu phe tyr thr glu ser thr gly tyr asn thr phe

365 370 375

cys gly tyr pro pro glu val val arg lys met pro lys arg asp

380 385 390

leu ala glu glu val trp arg leu gln ala ala leu gly glu gln

395 400 405

ser glu ile thr lys cys thr lys gln glu phe glu arg leu gln

410 415 420

asn glu lys val leu cys arg ile cys tyr glu gly glu ile cys

425 430 435

met val leu leu pro cys arg his arg thr leu cys lys thr cys

440 445 450

ser asp lys cys lys lys cys pro ile cys arg val pro ile glu

455 460 465

glu arg met pro val tyr asp val

470 473

<210> 4

<211> 18

<212> dna

<213>artificial sequence

<220>

<223> primer1

<400> 4

cgggagcgga agagatta 18

<210> 5

<211> 21

<212> dna

<213>artificial sequence

<220>

<223> primer2

<400> 5

tcggtaaacc tttctgaact t 21

<210> 6

<211> 33

<212> dna

<213>artificial sequence

<220>

<223> primer3

<400> 6

ttctgcacta ggtaccatgc agcggcggcg ggc 33

<210> 7

<211> 27

<212> dna

<213>artificial sequence

<220>

<223> primer4

<400> 7

ggactagttt aaacatcata tacgggc 27

<210> 8

<211> 43

<212> dna

<213>artificial sequence

<220>

<223> primer5

<400> 8

ttctgcacta ggtaccaggc ctggccgttg cttattgcgt ttg 43

<210> 9

<211> 44

<212> dna

<213>artificial sequence

<220>

<223> primer6

<400> 9

ctgacgtagg ggcgatagag ctccggtatc taattgccgc tgat 44

<210> 10

<211> 38

<212> dna

<213>artificial sequence

<220>

<223> primer7

<400> 10

cggggatccg tcgactacgc cgttgcttat tgcgtttg 38

<210> 11

<211> 39

<212> dna

<213>artificial sequence

<220>

<223> primer8

<400> 11

aggtggaaga cgcgttaccg gtatctaatt gccgctgat 39

<210> 12

<211> 21

<212> dna

<213>artificial sequence

<220>

<223>a13 forward primer

<400> 12

aggcgattcc catttgcttg c 21

<210> 13

<211> 22

<212> dna

<213>artificial sequence

<220>

<223>a13 downstream primer

<400> 13

agcagcgagg agggagaaga gg 22

<210> 14

<211> 25

<212> dna

<213>artificial sequence

<220>

<223>indel3-4 forward primer

<400> 14

cattctaaat gtgaccgtta tgtcc 25

<210> 15

<211> 20

<212> dna

<213>artificial sequence

<220>

<223>indel3-4 downstream primer

<400> 15

gtaccgggtc gctttgttct 20

<210> 16

<211> 20

<212> dna

<213>artificial sequence

<220>

<223>indel3-3 forward primer

<400> 16

gtggggaaca ggtttgaccg 20

<210> 17

<211> 19

<212> dna

<213>artificial sequence

<220>

<223>indel3-3 downstream primer

<400> 17

tgcagcgttt tcgcatcgt 19

<210> 18

<211> 22

<212> dna

<213>artificial sequence

<220>

<223>a15 forward primer

<400> 18

acggctccac gagaacatct gg 22

<210> 19

<211> 21

<212> dna

<213>artificial sequence

<220>

<223>a15 downstream primer

<400> 19

ctgctgcgga attgagcttg g 21

<210> 20

<211> 22

<212> dna

<213>artificial sequence

<220>

<223>a1 forward primer

<400> 20

caggatcgga caggatcaca gg 22

<210> 21

<211> 22

<212> dna

<213>artificial sequence

<220>

<223>a1 downstream primer

<400> 21

gctcctggcg cagctataga cc 22

<210> 22

<211> 23

<212> dna

<213>artificial sequence

<220>

<223>a2 forward primer

<400> 22

tccacgtgtt atcctctctt tgc 23

<210> 23

<211> 22

<212> dna

<213>artificial sequence

<220>

<223>a2 downstream primer

<400> 23

ccagattcct gcgttgtaca gg 22

<210> 24

<211> 23

<212> dna

<213>artificial sequence

<220>

<223>a19 forward primer

<400> 24

aagtgaggcg acgaggacga agg 23

<210> 25

<211> 23

<212> dna

<213>artificial sequence

<220>

<223>a19 downstream primer

<400> 25

aaacgcaacg cacagaagga agg 23

<210> 26

<211> 21

<212> dna

<213>artificial sequence

<220>

<223>indel3-9 forward primer

<400> 26

tgtcatcgtt gcatgtttgt t 21

<210> 27

<211> 21

<212> dna

<213>artificial sequence

<220>

<223>indel3-9 downstream primer

<400> 27

cagcagttct cgcatagtcc t 21

<210> 28

<211> 20

<212> dna

<213>artificial sequence

<220>

<223>rm426 forward primer

<400> 28

atgagatgag ttcaaggccc 20

<210> 29

<211> 20

<212> dna

<213>artificial sequence

<220>

<223>rm426 downstream primer

<400> 29

aactctgtac ctccatcgcc 20

<210> 30

<211> 21

<212> dna

<213>artificial sequence

<220>

<223>rm168 forward primer

<400> 30

tgctgcttgc ctgcttcctt t 21

<210> 31

<211> 20

<212> dna

<213>artificial sequence

<220>

<223>rm168 downstream primer

<400> 31

gaaacgaatc aatccacggc 20

<210> 32

<211> 23

<212> dna

<213>artificial sequence

<220>

<223>rm1350 forward primer

<400> 32

aggaacaccc aagagagtca tgc 23

<210> 33

<211> 22

<212> dna

<213>artificial sequence

<220>

<223>rm1350 downstream primer

<400> 33

gcaagaaagc tctgctccat gc 22

<210> 34

<211> 25

<212> dna

<213>artificial sequence

<220>

<223>rm8277 forward primer

<400> 34

cagcagagac tatagacact caagc 25

<210> 35

<211> 24

<212> dna

<213>artificial sequence

<220>

<223>rm8277 downstream primer

<400> 35

tgcctagcta ctctaggtga aacc 24

<210> 36

<211> 23

<212> dna

<213>artificial sequence

<220>

<223>rm3225 forward primer

<400> 36

gatagaggat tgggtgcgtg tgc 23

<210> 37

<211> 23

<212> dna

<213>artificial sequence

<220>

<223>rm3225 downstream primer

<400> 37

tacgccaacc aattccaaac acc 23

Sequence table

1

Claims

1. a kind of rice grain shape related gene dss, its nucleotide sequence is as shown in seq id no.1.

2. described in claim 1 rice grain shape related gene dss coding protein it is characterised in that aminoacid sequence such as Shown in seq id no.3.

3. protein according to claim 2 it is characterised in that: the encoding gene of described protein is following 1) or 2) or 3) dna molecule or 4):

1) the dna molecule shown in seq id no.1；

2) the dna molecule shown in seq id no.2；

4) with 1) or 2) or 3) the dna sequence that limits has more than 90% homology, and encode the dna molecule of rice grain shape albumen.

4. the recombinant expression carrier containing gene described in claim 1, expression cassette or recombinant bacterium.

5. recombinant expression carrier as claimed in claim 4, expression cassette or recombinant bacterium it is characterised in that: described recombinant expressed load Body is that gene described in insertion claim 1 between the kpn i and spe i double enzyme site of pcambia1390 carrier obtains The over-express vector of dss gene, or described recombinant expression carrier is kpn i and sac in lh-fad2-1390rnai carrier I recombination site and mlu i and bamh i recombination site insert the weight that the Partial Fragment of gene described in claim 1 obtains respectively Group interference expression vector.

6. the total length of gene described in amplification claim 1 and its primer pair of any fragment are it is characterised in that be selected from primer1/ In primer2, primer3/primer4, primer5/primer6 or primer7/primer8 any pair；primer1 Sequence as shown in seq id no.4, primer2 sequence as shown in seq id no.5, primer3 sequence such as seq id no.6 institute Show, primer4 sequence as shown in seq id no.7, primer5 sequence as shown in seq id no.8, primer6 sequence such as seq Shown in id no.9, as shown in seq id no.10, primer8 sequence is as shown in seq id no.11 for primer7 sequence.

7. the positioning primer being related to during gene described in map based cloning claim 1 is it is characterised in that be selected from following any Pair of primers:

A13 forward primer: seq id no.12, downstream primer: seq id no.13；

Indel3-4 forward primer: seq id no.14, downstream primer: seq id no.15；

Indel3-3 forward primer: seq id no.16, downstream primer: seq id no.17；

A15 forward primer: seq id no.18, downstream primer: seq id no.19；

A1 forward primer: seq id no.20, downstream primer: seq id no.21；

A2 forward primer: seq id no.22, downstream primer: seq id no.23；

A19 forward primer: seq id no.24, downstream primer: seq id no.25.

8. gene described in claim 1, protein described in claim 2, recombinant expression carrier, expression cassette described in claim 4 Or at least one application in plant breeding in recombinant bacterium.

9. a kind of change rice grain shape method, be by gene described in claim 1 in little particle mutant dss overexpression, Obtain the transgenic paddy rice that a type increases；Or the table of gene described in claim 1 in suppression conventional rice kind nanjing35 Reach, obtain the transgenic paddy rice that a type diminishes.

10. method according to claim 9 it is characterised in that: gene described in claim 1 pass through claim 4 or 5 institute State recombinant expression carrier to be directed respectively in little particle mutant dss or conventional rice nanjing35.