CN110066808A

CN110066808A - Application of the GY3 gene in control spikelets per panicle of rice and single plant yield

Info

Publication number: CN110066808A
Application number: CN201910195096.4A
Authority: CN
Inventors: 邢永忠; 吴比; 毛东海
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-03-14
Filing date: 2019-03-14
Publication date: 2019-07-30
Anticipated expiration: 2039-03-14
Also published as: CN110066808B

Abstract

The invention belongs to plant genetic engineering fields.More particularly to application of the GY3 gene in control spikelets per panicle of rice and single plant yield.The gene is located at the 3rd chromosome long arm end of rice, controls spikelets per panicle of rice character function.The sequence of the gene coding region is as shown in SEQ ID NO:1, and protein sequence is as shown in SEQ ID NO:2.Its coded sequence of the allele of the gene is as shown in SEQ ID NO:3.Retrotransposon insetion sequence is as shown in SEQ ID NO:4 in GY3 gene promoter.Comparing sequencing proves, there are the insertions of the retrotransposon of a 3882bp in wide affine japonica rice 02428 and the special green promoter of long-grained nonglutinous rice, leads to the reduction of 02428 expression quantity, 02428 allelotype is caused to be increased production.The grains per panicle and yield for overexpressing plant are remarkably decreased.It matches with GY3 low expression production-increasing function in NIL-02428.

Description

Application of the GY3 gene in control spikelets per panicle of rice and single plant yield

Technical field

The present invention relates to field of plant genetic project technology.More particularly to GY3 gene control spikelets per panicle of rice and Application in single plant yield.The present invention is to positioned at the control spikelets per panicle of rice gene of the 3rd chromosome long arm end of rice one GY3 has carried out separation clone, functional verification and as the application in the improvement of rice key variety yield traits.

Background technique

Rice is the important cereal crops of the mankind, and as the growth of population and improvement of living standard and urbanization are led It causes cultivable area constantly to reduce, harsh demand, therefore, the genetic improvement one of rice yield is proposed to the growth of rice yield It is directly the important goal of breeding.Rice yield is mainly determined that the product of number of grain per ear, mass of 1000 kernel and tiller number is constituted by single plant yield Single plant yield, meanwhile, the length at heading stage determines the regional adaptability of kind, and suitable plant height is guaranteeing life high as far as possible It is resistant to lodging while object amount, to guarantee the stabilization of rice yield.

Numerous genes for influencing yield have been cloned in rice.Gn1a (Ashikari M etc., Cytokinin Oxidase regulates rice grain production, Science, 2005,309:741-745) it is Japanese clone An influence yield gene, encoding cytokinin oxidases OsCKX2 can promote the degradation of the basic element of cell division, be mutated The basic element of cell division obtains accumulation to increase yield afterwards.(Huang X etc., Natural variation the at the such as Huang DEP1locus enhances grain yield in rice, Nat Genet, 2009,41:494-497) clone one directly Vertical fringe gene DEP1, it encodes a unknown phosphotidylethanolabinding binding protein (PEBP), by the expression for regulating and controlling OsCKX2 It can promote cell division to influence activity and the division growth of cell, the gene mutation of separate living tissue, spike of rice is made to become close, branch stalk Number increases, every tassel seed number increases.(Zha X etc., Over-expression of the rice LRK1gene such as Zha Improves quantitative yield components, Plant Biotechnol J, 7:611-20) one richness of clone Repeating receptor-like kinase enzyme gene LRK1, LRK1 containing leucine is a plasmalemma protein, and the constitutive expression in young fringe, being overexpressed should Gene can increase grains per panicle.Ideotype gene OsSPL14 encode class Squamosa promoter binding protein and by The regulation of micro-RNA OsmiR156, in vegetative growth phase, OsSPL14 controls rice tillering；In reproductive stage, The high expression of OsSPL14 promotes fringe branch, research shows that a point mutation of OsSPL14 has upset OsmiR156 pairs The regulation of OsSPL14, after OsSPL14 mutation, so that rice tillering is reduced, grain number per spike and mass of 1000 kernel increase, while stalk becomes It is sturdy, lodging tolerance enhancing, and then improve yield (Jiao Y etc., Regulation of OsSPL14by OsmiR156defines ideal plant architecture in rice,Nat Genet,42:541-544).Other one A little genes have pleiotropism, such as Ghd7 (Weiya Xue etc., Natural variation in Ghd7is an important regulator of heading date and yield potential in rice,Nat Genet, 2008,40:761-767), Ghd8 (Wenhao Yan etc., A major QTL, Ghd8, plays pleiotropic roles in regulating grain productivity,plant height,and heading date in rice,Mol Plant, 2011,4:319-330), Ghd7.1 (Wenhao Yan etc., Natural variation in Ghd7.1plays an important role in grain yield and adaptation in rice,Cell Res,2013,23:969- Etc. 971) have the function of increasing yield and plant height, but they require to extend breeding time as cost.

Summary of the invention

It is an object of the invention to overcome the deficiencies of existing technologies, a control rice grain yield gene GY3 has been cloned (especially control spikelets per panicle of rice and single plant yield) and its application.This gene in the case where not extending heading stage, Increase production key variety significantly, there is preferable production application prospect.This unnamed gene of clone is GY3 gene by applicant (Grain yield 3)。

The present invention has cloned one using the method for map based cloning and has been located at the 3rd chromosome long arm end, can increase water simultaneously The gene of rice Secondary branch, grains per panicle and single plant yield can increase 10 Secondary branch under near isogenic lines background, About 50 grains per panicles and about more than 10 grams of single plant yields.Meanwhile being found by analysis, japonica rice carries the equipotential of volume increase Gene, and long-grained nonglutinous rice only partially carries volume increase allele, can improve long-grained nonglutinous rice yield using this yield gene.By japonica rice In excellent allele key variety " 93-11 " is imported by the method for continuous backcross, " bright extensive 63 " and " special blueness " all have Significant production-increasing function.Therefore GY3 gene has very strong application value.

It is described that technical scheme is as follows:

(1) it QTL scanning and the building of near isogenic lines: utilizes and (comes from Jiangsu Province's agricultural sciences with rice varieties " 02428 " Institute) and " special green " (TQ) (Guangdong Academy of Agricultural Sciences's Huang Yaoxiang academician's breeding and give) hybridization acquisition F₁, utilize SSR marker RM570 and RM7389 (primer sequence reference sitehttp://www.gramene.org/Design) the true hybrid of identification, selects very miscellaneous Kind is continuously returned 4 acquisition BC with 02428₄F₁, building process is referring to FIG. 1, plantation introgressive line BC₄F₂Segregating population, and carry out The QTL of Correlated Yield Characters is scanned, and detects the QTL of control a spikelets per panicle of rice and yield in the 3rd end of chromosome QGY3 (Bi Wu etc., Two quantitative trait loci for grain yield and plant height on chromosome 3are tightly linked in coupling phase in rice,Mol Breeding,2015, 35:156).In order to further verify and clone qGY3, BC is selected₄F₂Number is that M43 single plant and recurrent parent are returned in group And it is selfed and obtains BC₅F₂Segregating population is found that homozygous 02428 allelotype has and is increased by the genetic analysis to segregating population The effect of production can increase about 50 grains per panicles and about more than 10 grams of single plant yield, that is, complete GY3 near isogenic lines Building and Primary Location.

(2) 2500 plants of near isogenic lines the determination of the finely positioning of GY3 and candidate gene: has been planted within 2014 in Wuhan Background F₂Big group carries out the finely positioning of gene, by the assignment of genes gene mapping in 1 about 60kb of common indicium RM7389 and InDel Tag ID Section (Fig. 1).By carrying out function prediction to the section gene, mono- coding for cytokinin of LOC_Os03g64070 is found Kinase gene LOG-like 5 (its CDS sequence as shown in SEQ ID NO:1, the exactly GY3 base named later of the present invention Cause), and sequencing is compared to LOG-like 5, compared with " special green ", there is the reversion of a 3882bp in " 02428 " promoter Stand insertion, and have the insertion of a 9bp segment at 5 ' UTR region 33bp of the upstream ATG, it is inserted into/lacks for retrotransposon It loses design primer combination and identifies all recombination single plants, it is found that it is isolated with phenotype.5 coding for cytokinin core of LOG-like Glycosides 5 '-monophosphate phosphoric acid nucleoside hydrolase, can promote the synthesis of the basic element of cell division.Conventional Real Time is utilized simultaneously (Kenneth J.Livak etc., Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2^-ΔΔC _TMethod.Methods, 2001,25 (4): 402-408) method it is closer The expression of LOG-like 5 in isogenic line children's fringe, discovery " special green " genotype have higher expression quantity.Therefore, the present invention will It is determined that being candidate gene.

(3) functional verification of GY3 gene: amplify LOG-like's 5 from rice " special green " using the method for PCR CDS, and be connected on pU1301 carrier, candidate gene is transferred in " 02428 " using agrobcterium-mediated transformation. And phenotype investigation is carried out in T0 generation, compared to transgene negative plant, the transgenic plant for overexpressing LOG-like 5 is average every It is 56 that fringe, which can increase floret bears, and single plant yield increases by 11 grams, therefore assert that the candidate gene LOG-like 5 of GY3 has and increase Add the function of grains per panicle and single plant yield.

(4) GY3 gene increases production rice backbone parent significantly: being sequenced by the comparison to rice key variety, finds Xian It is all special green allelotype that GY3 is most of in rice key variety, so, by being returned in main Indica Rice Restorer Lines " 93- 11 ", " japonica rice superior allelic is imported in bright extensive 63 " and " special green ", the backbone parent single plant yield after improvement significantly increases Add.

Beneficial effects of the present invention:

The rice GY3 gene that the present invention clones can dramatically increase the every fringe grain husk flower of rice in the case where not influencing heading stage Several and yield, can be by the application that changes its expression quantity to realize High-yield Rice Breeding.

Detailed description of the invention

Fig. 1: general technical route map of the invention.Description of symbols: rice varieties " 02428 " hybridize production with " special green " Raw F₁Afterwards, it is recurrent parent with rice varieties 02428, obtains BC by being continuously returned₄F₁, selfed seed is collected, BC is planted₄F₂Point The body that peels off carries out QTL scanning.The QTL of grains per panicle and yield is controlled to one in the 3rd chromosome long arm end point detection QGY3 selects M43 single plant and continues to be returned and be selfed with 02428 to obtain BC₅F₂Group, for the genetic analysis of GY3 and fine fixed Position.

Fig. 2: important recombination single plant in finely positioning of the present invention.Description of symbols: what is indicated above black line is to be used for The label title of identification recombination single plant genotype, the genotype for recombinating single plant are generally side genotype homozygosis other side genotype There is separation in the offspring of recombination single plant in heterozygosis, heterozygous genotypes.So plantation recombinates individual plant selfing offspring BC respectively₅F₃Family System, to all single plants Marker Identification genotype in each family, and according to homozygous special in the more same family of genotype results Green genotype and homozygous 02428 genotype single plant whether there is the significant difference of yield Relevant phenotype, to infer the gene of GY3 Type.As shown in Figure 2, Secondary branch (secondary branch) Defined daily doses (grain number is listed respectively Per panicle) and yield (yield) species test data, TQ^Mean±SDGenotype homozygosis is represented in each family as TQ phenotype The mean+SD of value, 02428^Mean±SDGenotype homozygosis is represented in each family as the average value of 02428 phenotypic number ± standard deviation.From Fig. 2 it can be found that there are the significant differences of phenotype between the 2nd, 3,4,5 and 9 family different genotype, therefore Determine that GY3 is located in the region of RM7389 to ID1.

Fig. 3: the comparison of near isogenic lines (NIL) stem two kinds of genotype of fringe in the present invention.Description of symbols: closely equal bases Because of the phenotype of owner's stem fringe, near isogenic lines NIL-02428 has more Secondary branch compared near isogenic lines NIL-TQ And number of grain per ear.

Fig. 4: being the stem fringe phenotype of near isogenic lines and high yield allele importing key variety that the present invention constructs. Description of symbols: A schemes in Fig. 4, the B figure in Fig. 4 and the C figure in Fig. 4 are respectively " special blueness " (TQ) and " TQ^GY3", " bright extensive 63 " (MH63) and " MH63^GY3" and " 93-11 " and " 93-11^GY3" in phenotype, in which: A1, B1, C1 are whole plant type figure, A2, B2, C2 and A3, B3, C3 are maturity period fringe type figure, and Cong Tuzhong is it can be found that the importing of GY3 makes spike of rice obviously become larger, Secondary branch Become more, can play the role of increasing these kind fringe sizes.

Fig. 5: used over-express vector constructs schematic diagram in the present invention.Description of symbols: the present invention is expanded using PCR Increase the CDS of GY3 out, with KpnI single endonuclease digestion, more grams for illustrating black arrow instruction in PU1301 expression vector are connected to one-step method On grand site.

Specific embodiment

To the explanation of sequence table:

SEQ ID NO:1 is the nucleotide sequence of the code area (CDS) for the GY3 gene that the present invention separates.

SEQ ID NO:2 is the protein sequence of GY3 gene coding.

SEQ ID NO:3 is the allele (GY3 of GY3 gene^TQ) code area (CDS) nucleotide sequence.

SEQ ID NO:4 is the allele (GY3 of GY3 gene^TQ) coding protein sequence.

SEQ ID NO:5 is retrotransposon nucleotide sequence of the rice " 02428 " in GY3 upstream region of gene.

It is scanned simultaneously in the 3rd end of chromosome to control grains per panicle and single plant from the introgressive line of rice varieties " special green " The QTL of yield selects the M43 single plant and 02428 that SSR marker RM16217 is TQ genotype and continues backcrossing purifying genetic background, subtracts Interference of the difference of few genetic background to the assignment of genes gene mapping utilizes BC to construct near isogenic lines NIL (GY3)₅F₂Separate group Body carries out genetic analysis and QTL effect detection.The BC of 2500 plants of screening₅F₂Recombinate single plant in group, and after carrying out to recombination single plant Generation test, thus finely positioning GY3, finally by the GY3 assignment of genes gene mapping in the area of the region about 60kb from SSR marker RM7389 to ID1 Section.Include a mitogen-activated enzyme gene LOG-like 5 in this section, the comparison of LOG-like 5 is sequenced, discovery There is the insertion/deletion of 9bp between parent in 5 ' UTR of the upstream ATG, and genotype identification is carried out to recombination single plant and finds this 9bp Missing isolated completely with phenotype, this gene is set to candidate gene.The green allelotype of the spy of this gene is connected to " 02428 " is converted on pU1301 overexpression vector, is tailed off in T0 for multiple transgenic positive single plant grains per panicles, single plant yield Reduced phenotype, it is thus identified that LOG-like5 is exactly GY3 gene.

Following case study on implementation further defines the present invention, and describes separation clone and the functional verification of GY3 gene.According to Description below and these embodiments, those skilled in the art can determine essential characteristic of the invention, and without departing from this In the case where spirit and range, various changes and modifications can be made to the present invention, so that it is applicable in various uses and item Part.

The positioning of embodiment 1:GY3 and effect analysis

(1) discovery of GY3: " special using rice varieties " 02428 " (Jiangsu Province Agriculture Science Institute give) and rice varieties It is green " (Guangdong Academy of Agricultural Sciences's Huang Yaoxiang academician's breeding and give) hybridize, acquisition cenospecies F₁, and with " 02428 " work For recurrent parent, 4 acquisition BC of continuous backcross₄F₁And be selfed, plant BC₄F₂Family (each family plants 24 plants), is collected each The phenotype of the characters such as family yield and grains per panicle, in conjunction with " 02428 " and " TQ " recombinant inbred lines (RILs) genetic map structure Building SSR marker used, (document is shown in: the hair East Sea i.e. Mao, Donghai etc., Epistasis and complementary gene action adequately account for the genetic bases of transgressive segregation Of kilo-grain weight in rice.Euphytica, 2011,180:261-271), identify genotype, utilize Dan Yin It whether there is the chain relevant QTL of grains per panicle and yield between plain variance analysis two kinds of genotype of each SSR marker. Finally the SSR marker RM148 (www.gramene.org) in the 3rd chromosome long arm end nearby identifies one while controlling The QTL qGY3 of grains per panicle and single plant yield.

(2) building and Effect Evaluation of GY3 near isogenic lines: spikelets per panicle of rice is influenced in order to further parse this The gene of single plant yield, the present invention have planted the BC of 140 single plants₄F₂Group identifies the gene of each single plant with SSR marker Type constructs local section genetic linkage maps using MapMaker, then utilizes and use 2.5 software of WinQTL cartographer Selecting composite interval mapping method to carry out QTL scanning, (document is shown in: Zeng ZB.Precision mapping of 5quantitative trait loci.Genetics, 1994,136:1457-1468), in BC₄F₂GY3 is located in the 3rd dye Between colour solid end SSR marker RM148 and RM16211, and according to 140 single plant progeny test BC₄F₃The phenotype of family, can be with GY3 is located between RM16211 and RM7389 (document is shown in: Bi Wu etc., Two quantitative trait loci for grain yield and plant height on chromosome 3are tightly linked in coupling phase in rice,Mol Breeding,2015,35:156).According to BC₄F₂And BC₄F₃Family positioning result is inconsistent, pushes away Disconnected may be since background is impure to influence positioning result, meanwhile, applicant selects RM16217 homozygosis according to genotype and is The single plant " M43 " of " TQ is special green ", continues backcrossing once with " 02428 " for " M43 " and is selfed, obtain BC₅F₂Group is used for GY3 The evaluation of effect and the finely positioning of gene.In BC₅F₂30 plants of homozygosis 02428 (NIL-02428) He Teqing are selected in group respectively (NIL-TQ) genotype single plant (fringe type is shown in Fig. 3) investigates grains per panicle, Defined daily doses, Primary branch, Secondary branch, thousand Grain weight and single plant yield, as a result such as table 1.

1 near isogenic lines yield traits of table compare

As it can be seen from table 1 GY3 is that the single plant of 02428 allelotype has increase by 50 under near isogenic lines background More grains per panicles and Defined daily doses increase by 15 or more Secondary branch and about 15 grams of single plant yield.

(3) finely positioning of GY3: in 2500 plants of BC₅F₂Inside group, marked using the self-designed InDel of applicant ID8 and RM16217 identifies each single plant genotype.The single plant for selecting the recombination of two sides marker genetype carries out progeny test, identification Progeny test F₃The genotype of each single plant in family, and detect F₃It whether there is between two kinds of homozygous genotype single plants of family The significant difference of phenotype, to infer the genotype of GY3 in recombination single plant.By increasing molecule mark between ID8 and RM16217 Remember and identifies that the genotype of recombination single plant can be located in GY3 the area of the 60kb of RM7389 to ID1 in conjunction with the genotype of GY3 In domain.The sequence such as table 2 of present invention InDel label newly developed.

The sequence of 2 GY3 finely positioning primer of table

5 (the accession number of gene LOG-like annotated in finely positioning region comprising one as basic element of cell division kinase LOC_Os03g64070) 9 genes such as (http://rapdblegacy.dna.affrc.go.jp/).LOG(Takashi K Deng Direct control of shoot meristem activity by a cytokinin-activating Enzyme, 2007,445:652-655) it is that a basic element of cell division activation enzyme gene, mutation spike of rice become smaller in rice, it is serious prominent Become the missing for leading to floral organ.In arabidopsis (Kuroha T etc., Functional analyses of LONELY GUY cytokinin-activating enzymes reveal the importance of the direct activation Pathway in Arabidopsis, 2009,21:3152-3169) in have similar function.To the overall length of LOG-like 5 CDS and promoter are compared sequencing, find the retrotransposon Gypsy-like LTR- for having 3882bp in 02428 promoter Retrotransposon from rice (long terminal repeat) insertion, while 02428 apart from translation initiation position There is the missing of a GACCAGCAG at point ATG upstream 33bp.The difference of 3 SNP is identified in 02428 and special green code area, but The change of albumen coded sequence is not caused.5 expression quantity of LOG-like is detected and is found, compared to NIL-02428, NIL-TQ table It is higher up to measuring.So inferring that the insertion of retrotransposon may cause the reduction of expression quantity, to increase grain number per spike and improve yield.

Using the insertion/deletion of retrotransposon, devise 3 primer CL1F, CL1R and CL1-2F (primer sequence is shown in Table 3) Identify that whether there is or not the insertions of retrotransposon in rice.With primer combination if CL1F+CL1R and CL1-2F+CL1R is to parent 02428 PCR amplification is carried out with TQ, can identify the insertion that whether there is retrotransposon in parent, parent 02428 can smoothly amplify CL1-2F+CL1R and CL1F+CL1R cannot be amplified, be the type for thering is retrotransposon to be inserted into, and special blueness can amplify CL1F+ CL1R and CL1-2F+CL1R cannot be amplified, be the type of no retrotransposon insertion.Applicant is to 533 parts of rice germplasms simultaneously (State Key Laboratory of Crop Genetic Improvent is collected, http://ricevarmap.ncpgr.cn/ for group V2/) DNA carry out PCR amplification, essentially all of japonica rice be all have retrotransposon insertion (CL1-2F+CL1R can be expanded and CL1F+ CL1R cannot be expanded), and long-grained nonglutinous rice only part kind have retrotransposon insertion (CL1F+CL1R can expand and CL1-2F+CL1R not Can expand), meanwhile, applicant also identifies more than 100 parts of wild rice, and a part has retrotransposon insertion, and a part does not have then Have.Expand the primer sequence such as table 3.

The primer of retrotransposon insertion/deletion identification positioning in 3 GY3 promoter of table

The transgenosis functional verification of embodiment 2:GY3

(1) convert the acquisition of segment: using primer shown in table 4, amplification produces the DNA fragmentation of GY3, institute from parent special blueness It is as follows with program and PCR reaction condition: 1. 94 DEG C 4 minutes, 2. 94 DEG C 30 seconds, 3. 58 DEG C 30 seconds, 4. 72 DEG C 30 seconds 1 minute, 5. From step 2. -4. recycle 35 times, 6. 72 DEG C 15 minutes, 7. 4 DEG C of preservations.PU1301 is crop genetic improvement where the applicant The plant expression vector of National Key Laboratory's transformation, by being inserted into a corn in pCAMBIA1301 multiple cloning sites Ubiquitin promoter and NOS (synthase polyadenylation signal) terminator (Fig. 5).To utilize Ubiquitin promoter driving foreign gene is overexpressed (Qiu D etc., OsWRKY13mediates rice disease resistance by regulating defense-related genes in salicylate-and jasmonate- dependent signaling,Molecular plant-microbe interactions:MPMI,2007,20:492- 499).PU1301 carrier KpnI single endonuclease digestion tries PCR product and carrier digestion products using the recycling of Fermentas company Agent box recycles (method is shown in specification, article No. #K0513), and do one-step method connection (method referring to Gibson Dg etc., Enzymatic assembly of DNA molecules up to several hundred kilobases,Nature Methods,2009,6:343-345)。

The overexpression transgene carrier of 4 GY3 of table constructs the primer

(2) using connection product electrotransformation e. coli DH10B, (electroporation is eppendorf electroporator 2510, voltage parameter 1800v, application method are shown in that specification, Escherichia coli DH10B bacterial strain are purchased from promega company), connection Product is applied on the LA plate of the kanamycins containing 30mg/L, and picking single bacterium falls within kanamycins of the 1mL containing 30mg/L after 16h In LB culture medium, cultivate 12h after extract plasmid, using KpnI digestion detection positive colony (operating method is shown in J. Pehanorm Brooker, Not Ritchie, T Manny A Disi write EF, and Huang Peitang, Wang Jiaxi etc. is translated, Molecular Cloning:A Laboratory guide (third edition), Science Press, 2002 editions).Positive colony is sequenced, confirms exogenous sequences without mutation.

(3) 02428 is converted: by the carrier electrotransformation built in step (2) to Agrobacterium (A.tumefaciens) EHA105 (is purchased from the Australia laboratory CAMBIA), with reference to the electrotransformation method of connection product in step (2).Genetic transformation Method (Hiei etc., Efficient transformation of rice (Oryza of 02428 method with reference to Hiei et al. report sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries Of the T-DNA.Plant J, 1994,6:271-282) it carries out.

(4) transgenic plant in T0 generation is obtained.Transgenosis single plant using GUS primer (table 4) identify, can amplify be Transgenic positive single plant, cannot expand is transgene negative single plant.

4 transgenosis single plant positive identification primer of table

(5) it is investigated by the yield Relevant phenotype for being overexpressed plant to T0 generation, finds the every of transgenic positive plant Fringe floret bears reduce by 50 or so, and Defined daily doses reduce by 40 or so, and Secondary branch reduces by 10 or so, lead to single plant yield Yield strain more negative than 02428 reduces about 26 grams or so, so that demonstrating GY3 gene reduces rice single plant by improving expression quantity Yield, this is high with GY3 expression quantity in NIL-TQ, and the result that single plant yield reduces is consistent.So far, GY3 gene is by success gram Grand, it reduces rice Secondary branch by the raising of expression quantity, reduces grains per panicle and Defined daily doses, and reduce rice Single plant yield.

5 transgenosis single plant yield correlated traits of table is investigated

Embodiment 3: with 02428^GY3Allele improves backbone parent

By being combined using the primer of CL1F+CL1R to rice varieties 93-11 (Lixiahe region in Jiangsu's research of agricultural science Institute's breeding and give), bright extensive 63 (Fujian Sanming Institute of Agricural Science thanks to Huaan academician's breeding and give) He Teqing it is (wide Eastern Shanxi Academy of Agricultural Sciences Huang Yaoxiang academician breeding and give) carry out genotype identification, discovery all without retrotransposon be inserted into.? In self-mating system (NIL), for NIL-TQ, NIL-02428 has the function of increasing yield.In order to verify japonica rice equipotential base Because whether the GY3 of type equally has the function of volume increase under long-grained nonglutinous rice background, the present invention selects 93-11, special green and bright extensive 63 conduct Receptor parent obtains F after hybridizing respectively using 02428 as donor parents₁, and be continuously returned 4 times with receptor parent, and use Primer combination CL1F+CL1R and CL1-2F+CL1R selects GY3 gene, obtains 93-11, bright extensive 63 respectively and spy is green GY3 is the BC of 02428 genotype under background₄F₁, selfing acquisition BC₄F₂, plant BC₄F₂And the correlated traits for influencing yield is investigated (Fig. 4).Yield traits investigate and all have good effect of increasing production.

The key step of genetic transformation of the invention, culture medium and its method of preparation are as described below:

(1) abbreviation of plant hormone used in culture medium is expressed as follows in reagent and the solution abbreviation present invention: 6-BA (6- BenzylaminoPurine, 6-benzyladenine)；CN (Carbenicillin, carbenicillin)；KT (Kinetin, excitement Element)；NAA (Napthalene acetic acid, methyl α-naphthyl acetate)；IAA (Indole-3-acetic acid, heteroauxin)；2, 4-D (2,4-Dichlorophenoxyacetic acid, 2,4- dichlorphenoxyacetic acids)；AS (Acetosringone, acetyl fourth Ketone musk)；CH (Casein Enzymatic Hydrolysate, caseinhydrolysate)；HN (Hygromycin B, hygromycin)； DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide)；N6max (N6 a great number of elements ingredient solution)；N6mix (the micro member of N6 Plain ingredient solution)；MSmax (MS a great number of elements ingredient solution)；MSmix (MS Trace Elements solution)

(2) main solution formula

1) N6 culture medium a great number of elements mother liquor (being prepared according to 10 times of concentrates (10X)):

Mentioned reagent is dissolved one by one, is then settled to 1000 milliliters with distilled water at room temperature.

2) N6 culture medium microelement mother liquor (is prepared according to 100 times of concentrates (100X)

Mentioned reagent is dissolved at room temperature and is settled to 1000 milliliters with distilled water.

3) molysite (Fe2EDTA) storage liquid (being prepared according to 100X concentrate)

3.73 grams of b diammonium disodium edtas (Na2EDTA2H2O) and 2.78 grams of FeSO47H2O are dissolved respectively, are mixed Merging is settled to 1000 milliliters with distilled water, until 70 DEG C warm bath 2 hours, 4 DEG C save backup.

4) vitamins stock liquid (being prepared according to 100X concentrate)

Distilled water is added to be settled to 1000 milliliters, 4 DEG C save backup.

5) MS culture medium a great number of elements mother liquor (MSmax mother liquor) (being prepared according to 10X concentrate)

Mentioned reagent is dissolved at room temperature, and is settled to 1000 milliliters with distilled water.

6) MS culture medium microelement mother liquor (MSmin mother liquor) (being prepared according to 100X concentrate)

7) 2,4-D stores the preparation of liquid (1 mg/ml):

100 milligrams of 2,4-D are weighed, is dissolved 5 minutes with 1 milliliter of 1N potassium hydroxide, then plus 10 milliliters of distilled water have dissolved 100 milliliters are settled to after complete, is saved at room temperature.

8) 6-BA stores the preparation of liquid (1 mg/ml):

100 milligrams of 6-BA are weighed, is dissolved 5 minutes with 1 milliliter of 1N potassium hydroxide, then plus 10 milliliters of distilled water have dissolved 100 milliliters are settled to after complete, room temperature preservation.

9) preparation of methyl α-naphthyl acetate (NAA) storage liquid (1 mg/ml):

100 milligrams of NAA are weighed, is dissolved 5 minutes with 1 milliliter of 1N potassium hydroxide, then plus 10 milliliters of distilled water dissolutions are complete After be settled to 100 milliliters, 4 DEG C save backup.

10) preparation of heteroauxin (IAA) storage liquid (1 mg/ml):

100 milligrams of IAA are weighed, is dissolved 5 minutes with 1 milliliter of 1N potassium hydroxide, then plus 10 milliliters of distilled water dissolutions are complete After be settled to 100 milliliters, 4 DEG C save backup.

11) preparation of glucose storage liquid (0.5 grams per milliliter):

125 grams of glucose are weighed, then 250 milliliters is settled to distilled water dissolution, is saved backup for 4 DEG C after sterilizing.

12) AS stores the preparation of liquid:

0.392 gram of AS is weighed, 10 milliliters of DMSO dissolutions are added, dispense to 1.5 milliliters of centrifuge tubes, 4 DEG C save backup.

13) 1N potassium hydroxide storage liquid weighs 5.6 grams of potassium hydroxide, is settled to 100 milliliters with distilled water dissolution, room temperature is protected It deposits spare.

(3) it is used for the culture medium prescription of rice transformation

1) induced medium

Add distilled water to 900 milliliters, 1N potassium hydroxide adjusts pH value to 5.9, boils and is settled to 1000 milliliters, is dispensed into 50 milliliters of triangular flasks (25 milliliter/bottle), sterilize according to a conventional method after sealing (such as sterilize 25 minutes at 121 DEG C, following cultures Base sterilizing methods are identical as the sterilizing methods of basal culture medium).

2) subculture medium

Add distilled water to 900 milliliters, 1N potassium hydroxide adjusts pH value to 5.9, boils and is settled to 1000 milliliters, is dispensed into 50 milliliters of triangular flasks (25 milliliter/bottle), sealing, sterilize according to the above method.

3) pre-culture medium

Add distilled water to 250 milliliters, 1N potassium hydroxide adjusts pH value to 5.6, and sealing sterilizes according to the above method.

Using preceding heating for dissolving culture medium and 5 milliliters of glucose storage liquid and 250 microlitres of AS storage liquid are added, packing is poured into In culture dish (25 milliliters/ware).

4) base is co-cultured

Using preceding heating for dissolving culture medium and 5 milliliters of glucose storage liquid and 250 microlitres of AS storage liquid are added, packing is poured into In culture dish (25 milliliters/every ware).

5) suspension medium

Add distilled water to 100 milliliters, adjusts pH value to 5.4, be dispensed into two 100 milliliters of triangular flask, seal, by upper State method sterilizing.

1 milliliter of sterile dextrose storage liquid and 100 microlitres of AS storage liquid is added using preceding.

6) Selective agar medium

Add distilled water to 250 milliliters, adjusts pH value to 6.0, sealing sterilizes according to the above method.

Using preceding dissolution culture medium, 250 microlitres of HN (50 mg/ml) and 400 microlitres of CN (250 mg/ml) are added (25 milliliters/ware) are poured into culture dish in packing.(note: first time Selective agar medium carbenicillin concentration is 400 mg/litres, the Secondary and later Selective agar medium carbenicillin concentration is 250 mg/litres).

7) pre- differential medium

Add distilled water to 250 milliliters, 1N potassium hydroxide adjusts pH value to 5.9, and sealing sterilizes according to the above method.

Using preceding dissolution culture medium, 250 microlitres of HN (50 mg/ml), 250 microlitres of CN (250 mg/ml), packing is fallen Enter in culture dish (25 milliliters/ware).

8) differential medium

Add distilled water to 900 milliliters, 1N potassium hydroxide adjusts pH value to 6.0.

It boils and is settled to 1000 milliliters with distilled water, be dispensed into 50 milliliters of triangular flasks (50 milliliter/bottle), seal, by upper State method sterilizing.

9) root media

Add distilled water to 900 milliliters, adjusts pH value to 5.8 with 1N potassium hydroxide.

It boils and is settled to 1000 milliliters with distilled water, be dispensed into pipe of taking root (25 milliliters/pipe), sealing, by above-mentioned side Method sterilizing.

(4) Agrobacterium-mediated genetic transformation step

Callus induction

By 02428 mature rice paddy seed decladding, then successively with alcohol treatment 1 minute of 70%, 0.15% mercury chloride (HgCl2) the surface of the seed sterilizes 15 minutes；

With sterilizing washing seed 4-5 times；

Seed is put on the induction medium；

Culture medium after inoculation is placed at dark and is cultivated 4 weeks, 25 ± 1 DEG C of temperature.

3.2 callus subcultures select the embryo callus subculture of glassy yellow, consolidation and relatively dry, are put on subculture medium under dark Culture 2 weeks, 25 ± 1 DEG C of temperature.

3.3 preculture

The embryo callus subculture for selecting consolidation and relatively dry is put on pre-culture medium dark lower culture 2 weeks, 25 ± 1 DEG C of temperature.

The culture of 3.4 Agrobacteriums

1) in the LA culture medium with corresponding resistance selection, (preparation of LA culture medium is referring to J. Pehanorm Brooker etc., molecule Cloning experimentation guide, the third edition, golden winter wild goose etc. (translating), Science Press, 2002, Beijing) on preculture Agrobacterium EHA105 (should Bacterial strain discloses the agrobacterium strains used from CAMBIA company) two days, 28 DEG C of temperature；

Agrobacterium is transferred in suspension medium, is cultivated 2-3 hours on 28 DEG C of shaking tables.

3.5 Agrobacteriums are infected

1) callus of preculture is transferred in the bottle for bacterium of having gone out；

The suspension of Agrobacterium is adjusted to OD600 0.8-1.0；

Callus is impregnated 30 minutes in agrobacterium suspension；

It is blotted in transfer callus to the filter paper to have sterilized；It is then placed within to co-culture and be cultivated 3 days, 19-20 DEG C of temperature on base.

The washing of 3.6 callus and selection culture

1) sterilizing water washing callus is to invisible Agrobacterium；

It is immersed in the aqua sterilisa of the carbenicillin containing 400 milligrams/L (CN) 30 minutes；

It is blotted in transfer callus to the filter paper to have sterilized；

Shift selection culture 2-3 times, every time 2 weeks in callus to Selective agar medium.

3.7 differentiation

1) kanamycin-resistant callus tissue is transferred on pre- differential medium and is cultivated 5-7 days at dark；

In the callus to differential medium of the pre- differentiation culture of transfer, cultivated under illumination, 26 DEG C of temperature.

3.8 taking root

1) root generated when differentiation is cut；

It is then transferred in root media under illumination and cultivates 2-3 weeks, 26 DEG C of temperature.

3.9 transplanting

The remaining medium on root is washed off, the seedling with good root system is transferred to crop field isolation environment, field management is same Common crop field.

Illustrate: this application involves rice material in, in the title of material plus " " number, with the material that " " number is not added Title is the same material.

Sequence table

<110>Hua Zhong Agriculture University

<120>application of the GY3 gene in control spikelets per panicle of rice and single plant yield

<141> 2019-03-04

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 741

<212> DNA

<213>rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(741)

<220>

<221> CDS

<222> (1)..(741)

<400> 1

atg atg atg gag aat agc agg gag cag cag ccg gag tcg tcg ccg gcg 48

Met Met Met Glu Asn Ser Arg Glu Gln Gln Pro Glu Ser Ser Pro Ala

1 5 10 15

aac aac aat agc aag aag aag aag aag aag aag acg gcg tcg cgg ttc 96

Asn Asn Asn Ser Lys Lys Lys Lys Lys Lys Lys Thr Ala Ser Arg Phe

20 25 30

cgg cgt gtg tgc gtg ttc tgc ggc agc agc ccc ggg aag aag gcg tcg 144

Arg Arg Val Cys Val Phe Cys Gly Ser Ser Pro Gly Lys Lys Ala Ser

35 40 45

tac cag gtg gcc gcc gtg cag ctg ggg cag cag ctg gtg gag cgc ggc 192

Tyr Gln Val Ala Ala Val Gln Leu Gly Gln Gln Leu Val Glu Arg Gly

50 55 60

atc gac ctg gtg tac ggc ggc ggc agc gtt ggg ctg atg ggg ctg gtg 240

Ile Asp Leu Val Tyr Gly Gly Gly Ser Val Gly Leu Met Gly Leu Val

65 70 75 80

tcc cgc gcc gtc cac gga ggc ggc ggg cac gtg gtg ggc gtg gtg ccc 288

Ser Arg Ala Val His Gly Gly Gly Gly His Val Val Gly Val Val Pro

85 90 95

aat ggc gtg ctg cca cgc gag ctg atc ggc gag acg ctg ggg gag gtg 336

Asn Gly Val Leu Pro Arg Glu Leu Ile Gly Glu Thr Leu Gly Glu Val

100 105 110

agg gcg gtg gga agc atg cac cag cgg aag gcg gag atg gcg cgg gag 384

Arg Ala Val Gly Ser Met His Gln Arg Lys Ala Glu Met Ala Arg Glu

115 120 125

tcg gac gcc ttc atc gcc ctc ccc ggc ggc tac ggc acg ctg gag gag 432

Ser Asp Ala Phe Ile Ala Leu Pro Gly Gly Tyr Gly Thr Leu Glu Glu

130 135 140

ctc ctc gag gtc atc acc tgg gct cag ctc cgc atc cac cac aag ccc 480

Leu Leu Glu Val Ile Thr Trp Ala Gln Leu Arg Ile His His Lys Pro

145 150 155 160

gtc ggc ctc ctc aac gtc gac ggc tac tac gac tcc ctg ctc gcc ttc 528

Val Gly Leu Leu Asn Val Asp Gly Tyr Tyr Asp Ser Leu Leu Ala Phe

165 170 175

atc gac aag gcc gtc cac gaa ggc ttc gtc tcg ccg ccc gcc cgc cgc 576

Ile Asp Lys Ala Val His Glu Gly Phe Val Ser Pro Pro Ala Arg Arg

180 185 190

atc atc gtc gcc gca ccc acc gcc tcc gac ctg ctc tgc aag ctc gag 624

Ile Ile Val Ala Ala Pro Thr Ala Ser Asp Leu Leu Cys Lys Leu Glu

195 200 205

gaa tac gtg ccg ccg ccg cac gac gcc acc gcc ctg aag ctc acc tgg 672

Glu Tyr Val Pro Pro Pro His Asp Ala Thr Ala Leu Lys Leu Thr Trp

210 215 220

gag atg tcc acc gta tcg gag cag cac gcc gga agc atc tac tcc ccc 720

Glu Met Ser Thr Val Ser Glu Gln His Ala Gly Ser Ile Tyr Ser Pro

225 230 235 240

aag ccc gac atg gca cgc tag 741

Lys Pro Asp Met Ala Arg

245

<210> 2

<211> 246

<212> PRT

<213>rice (Oryza sativa)

<400> 2

Met Met Met Glu Asn Ser Arg Glu Gln Gln Pro Glu Ser Ser Pro Ala

1 5 10 15

Asn Asn Asn Ser Lys Lys Lys Lys Lys Lys Lys Thr Ala Ser Arg Phe

20 25 30

Arg Arg Val Cys Val Phe Cys Gly Ser Ser Pro Gly Lys Lys Ala Ser

35 40 45

Tyr Gln Val Ala Ala Val Gln Leu Gly Gln Gln Leu Val Glu Arg Gly

50 55 60

Ile Asp Leu Val Tyr Gly Gly Gly Ser Val Gly Leu Met Gly Leu Val

65 70 75 80

Ser Arg Ala Val His Gly Gly Gly Gly His Val Val Gly Val Val Pro

85 90 95

Asn Gly Val Leu Pro Arg Glu Leu Ile Gly Glu Thr Leu Gly Glu Val

100 105 110

Arg Ala Val Gly Ser Met His Gln Arg Lys Ala Glu Met Ala Arg Glu

115 120 125

Ser Asp Ala Phe Ile Ala Leu Pro Gly Gly Tyr Gly Thr Leu Glu Glu

130 135 140

Leu Leu Glu Val Ile Thr Trp Ala Gln Leu Arg Ile His His Lys Pro

145 150 155 160

Val Gly Leu Leu Asn Val Asp Gly Tyr Tyr Asp Ser Leu Leu Ala Phe

165 170 175

Ile Asp Lys Ala Val His Glu Gly Phe Val Ser Pro Pro Ala Arg Arg

180 185 190

Ile Ile Val Ala Ala Pro Thr Ala Ser Asp Leu Leu Cys Lys Leu Glu

195 200 205

Glu Tyr Val Pro Pro Pro His Asp Ala Thr Ala Leu Lys Leu Thr Trp

210 215 220

Glu Met Ser Thr Val Ser Glu Gln His Ala Gly Ser Ile Tyr Ser Pro

225 230 235 240

Lys Pro Asp Met Ala Arg

245

<210> 3

<211> 741

<212> DNA

<213>rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(741)

<220>

<221> CDS

<222> (1)..(741)

<400> 3

atg atg atg gag aat agc agg gag cag cag ccg gag tcg tcg ccg gcg 48

Met Met Met Glu Asn Ser Arg Glu Gln Gln Pro Glu Ser Ser Pro Ala

1 5 10 15

aac aac aat agc aag aag aag aag aag aag aag acg gcg tcg cgg ttc 96

Asn Asn Asn Ser Lys Lys Lys Lys Lys Lys Lys Thr Ala Ser Arg Phe

20 25 30

cgg cgt gtg tgc gtg ttc tgc ggc agc agc ccc ggg aag aag gcg tcg 144

Arg Arg Val Cys Val Phe Cys Gly Ser Ser Pro Gly Lys Lys Ala Ser

35 40 45

tac cag gtg gcc gcc gtg cag ctg ggg cag cag ctg gtg gag cgc ggc 192

Tyr Gln Val Ala Ala Val Gln Leu Gly Gln Gln Leu Val Glu Arg Gly

50 55 60

atc gac ctg gtg tac ggc ggc ggc agc gtt ggg ctg atg ggg ctg gtg 240

Ile Asp Leu Val Tyr Gly Gly Gly Ser Val Gly Leu Met Gly Leu Val

65 70 75 80

tcc cgc gcc gtc cac gga ggc ggc ggg cac gtg gtg ggc gtg gtg ccc 288

Ser Arg Ala Val His Gly Gly Gly Gly His Val Val Gly Val Val Pro

85 90 95

aat ggc gtg ctg cca cgc gag ctg atc ggc gag acg ctg ggg gag gtg 336

Asn Gly Val Leu Pro Arg Glu Leu Ile Gly Glu Thr Leu Gly Glu Val

100 105 110

agg gcg gtg gga agc atg cac cag cgg aag gcg gag atg gcg cgg gag 384

Arg Ala Val Gly Ser Met His Gln Arg Lys Ala Glu Met Ala Arg Glu

115 120 125

tcg gat gcc ttc atc gcc ctc ccc ggc ggc tac ggc acg ctg gag gag 432

Ser Asp Ala Phe Ile Ala Leu Pro Gly Gly Tyr Gly Thr Leu Glu Glu

130 135 140

ctc ctg gag gtc atc acc tgg gct cag ctc cgc atc cac cac aag ccc 480

Leu Leu Glu Val Ile Thr Trp Ala Gln Leu Arg Ile His His Lys Pro

145 150 155 160

gtc ggc ctc ctc aac gtc gac ggc tac tac gac tcc ctg ctc gcc ttc 528

Val Gly Leu Leu Asn Val Asp Gly Tyr Tyr Asp Ser Leu Leu Ala Phe

165 170 175

atc gac aag gcc gtc cac gaa ggc ttc gtc tcg ccg ccc gcc cgc cgc 576

Ile Asp Lys Ala Val His Glu Gly Phe Val Ser Pro Pro Ala Arg Arg

180 185 190

atc atc gtc gcc gcg ccc acc gcc tcc gac ctg ctc tgc aag ctc gag 624

Ile Ile Val Ala Ala Pro Thr Ala Ser Asp Leu Leu Cys Lys Leu Glu

195 200 205

gaa tac gtg ccg ccg ccg cac gac gcc acc gcc ctg aag ctc acc tgg 672

Glu Tyr Val Pro Pro Pro His Asp Ala Thr Ala Leu Lys Leu Thr Trp

210 215 220

gag atg tcc acc gta tcg gag cag cac gcc gga agc atc tac tcc ccc 720

Glu Met Ser Thr Val Ser Glu Gln His Ala Gly Ser Ile Tyr Ser Pro

225 230 235 240

aag ccc gac atg gca cgc tag 741

Lys Pro Asp Met Ala Arg

245

<210> 4

<211> 246

<212> PRT

<213>rice (Oryza sativa)

<400> 4

Met Met Met Glu Asn Ser Arg Glu Gln Gln Pro Glu Ser Ser Pro Ala

1 5 10 15

Asn Asn Asn Ser Lys Lys Lys Lys Lys Lys Lys Thr Ala Ser Arg Phe

20 25 30

Arg Arg Val Cys Val Phe Cys Gly Ser Ser Pro Gly Lys Lys Ala Ser

35 40 45

Tyr Gln Val Ala Ala Val Gln Leu Gly Gln Gln Leu Val Glu Arg Gly

50 55 60

Ile Asp Leu Val Tyr Gly Gly Gly Ser Val Gly Leu Met Gly Leu Val

65 70 75 80

Ser Arg Ala Val His Gly Gly Gly Gly His Val Val Gly Val Val Pro

85 90 95

Asn Gly Val Leu Pro Arg Glu Leu Ile Gly Glu Thr Leu Gly Glu Val

100 105 110

Arg Ala Val Gly Ser Met His Gln Arg Lys Ala Glu Met Ala Arg Glu

115 120 125

Ser Asp Ala Phe Ile Ala Leu Pro Gly Gly Tyr Gly Thr Leu Glu Glu

130 135 140

Leu Leu Glu Val Ile Thr Trp Ala Gln Leu Arg Ile His His Lys Pro

145 150 155 160

Val Gly Leu Leu Asn Val Asp Gly Tyr Tyr Asp Ser Leu Leu Ala Phe

165 170 175

Ile Asp Lys Ala Val His Glu Gly Phe Val Ser Pro Pro Ala Arg Arg

180 185 190

Ile Ile Val Ala Ala Pro Thr Ala Ser Asp Leu Leu Cys Lys Leu Glu

195 200 205

Glu Tyr Val Pro Pro Pro His Asp Ala Thr Ala Leu Lys Leu Thr Trp

210 215 220

Glu Met Ser Thr Val Ser Glu Gln His Ala Gly Ser Ile Tyr Ser Pro

225 230 235 240

Lys Pro Asp Met Ala Arg

245

<210> 5

<211> 3882

<212> DNA

<213>rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(3882)

<400> 5

tgtcacgccc cgaactagta ccgaccggaa ctagcccgtg acgctccaaa ttaacctgtt 60

aatcgatacc agtcccagga aacagtgctg gtatcgcagg aagacggatt atcacagcaa 120

cagaggtctc tttattatag agtagaggta cagtcatgtt gggctgcgga cagatcccga 180

gctcacaact gcattacaaa agggggaaac ggaagccagg acttggacca aacaacacag 240

gcgcgacttg ggaactaggc cgaaacccta aaactcatcg aagccggctt gctcctggaa 300

gaactcctca tcagcaggat ccgcttcatc ttcttcagca actgggggga ttatttatat 360

agagcaaggg tgagtacggg ggtactcagc aagccatggg aaataagtgt ttaatgcagg 420

cttcaaggaa ggctgttgtt tttgcagttg attttatttg aactcttttc taaaaacaac 480

taagtgagtg cttctcaaac gacacggatg agacagtgcg tctcgtccgg tcggagtatg 540

tgtaatgtat cagtctttag aattgattca aggttggcac ccggccaaca gcttttcaaa 600

cggccacccg ggccaacaac tttcaaacgg ccacccgggc ctagctgatc ccatcagctg 660

cagatttttc aatcatcgaa cccctttcca caacagcaat ttcacaagca gtagtcaaac 720

aaaactacgc taggaatcac ctcacatccg cccatgaccg tgggcacggc tattcgaaca 780

gcttgttaac ctctgcagag ggggtacact ttacccacac gacattacta acccggatct 840

cccagcccat ggggatccgc cacgtcggga gacctccaag ctttcatgac aaggcatttc 900

caaagccgac acaggtttac catatgccga cgagaggggt cccagaccaa caactggtta 960

ggtcccagac catactgtgc caggaagccc aggggtcctc cccgacacca ccccggcgaa 1020

tccacatgtc tctcggcatc aaggctcccc tgataagcta gttactcagc caggggtgtc 1080

ccattccacc catgtggtcg tacttgtctt atgttcggat gaaattccaa ggaaacggtc 1140

cttaggtgca agagcgggaa accgtacacc cggtacgttc cccggtccgc ggttttggaa 1200

attcatttag ttcgcaagca ccgacccagg tgtcgggttt tccaagtctt ttgtaaaacc 1260

caagttttac ccaagaagtt tttcagaatt ttaagtttga aggcgtccgt cgatactcgc 1320

acagggtgca cgaatatcga gacgcaacta gatggttaca aggggacacg atataacaat 1380

tatcaaagga aggatcaaat gcaacaaatt aggtaggtcc gccaatctgc cttgcagacg 1440

ggacaacaga ttaagtgcga tcctatcaat acataatatt tttcaagcaa cataattaaa 1500

tttcaattat aggctcaaga tgttcaaagg tggcttgcct tgctcgagat ctagagcttg 1560

atcctcgaaa tcctcgcact gcgggtcttc gggctccgaa actacacgca aaacgggaca 1620

actcaacaaa cggcgaaaat aaagccctat taatgacctc taagcgtgcc attagataga 1680

tctcgagatt tgaggaattt tggaagttga acggagtcaa acggatttac ggttgggaag 1740

atattgaatt tctaatatta ttggattttt ggtctaaagg aaaaatgatt taattaaatc 1800

ctttttggaa aagaaaagaa aagaaggagg gggaaaatta gacttccctc gggcggcgag 1860

ggcgcggccc gagagaaagg aggcggctcg gccgagctaa ctgggccggc cggcccgaaa 1920

ggcggcccaa agcgcgcgcg ggggagggga gggagggggc gccggtggac cgggctcacc 1980

tcgcgtggtc ccgagtggga cccgcttgtc agcggctcgg ctcaccgtgc ggagggagca 2040

cgcggcgcac gcgcgtgggc gggggaggga tgcggtgcac gcgcgcggtt cgcggtggaa 2100

cggatgcggc gggcccacgc gcagcctcac ggctcgcggt ggaacgcgcg cacggggagg 2160

ggctgaccgg ggtcaattcg acccggcctt ggccgagctt gcgccgacgt ggcgcctacg 2220

tggctgccac gcgggccggc gggaggtaga cgacgacgcc ggccggaacg aacggcggac 2280

gacagcggcg agcggcggag cgaaccacgg cgatataggc gaaagcgagc acaccgggtg 2340

gttgcacgag acgaggggag acgagccaac ggctcggatt cgccggaggg agcttgacgg 2400

cggcgaatcg cggcggcggc aaccggcggc gggaggaggg gaaaacggcg acgaggtcac 2460

aaggggccga ttcctggcgg cgagagcatc tacgcggcta cgggaatccg atgctagcgt 2520

tggattagga ggaaacgcat cgagggaggc cggcgacgag aggcgcttcc gagctcgggc 2580

agcgacagcg gcgagcacat ggcgagcggc ggcaacggtc ggggcggcgc cggctagcta 2640

cggggaggct actcgtgcta ctacccgagt ctaaggggag gagatggagc gaggagggag 2700

aacggaggga tgcactaccg tgcgggcggg gcgcagtgac gagaggccga cggcgcggga 2760

gtaatctctc cggcctcggg ccggggaaga ggaaggagag ggcgcgcccg gagtcccctt 2820

ccgcgtcctt gctcgtgccg gctcctccga cacgcgcaac gacgaaaggc gacggcgaac 2880

agagcacggg aggcggcggc aatggcgtgg aggcgaacgg gagaggaaag gaaaggggga 2940

agcttaggtt tataggggcg gcaatgtcag tttgggagag gggaaccgac attggacggt 3000

caagccgacg agctggcgct ccggctagcg gccaaaacgg cgacaggagg tgacgccggc 3060

gagagggaaa aaggaagaaa aggggggaga gaaagggggc ttgtcccttg cctcttcggg 3120

aaaaggaggg gggagcgggg cgacgcggca gagggaggag ggactctgcc tccgcccctt 3180

ggaggctagc gcgcggagtg gcggggccga ggcgatgatg acggcggggc ggtgtggagc 3240

ggagcggcga cacgggcgac aggcgcgagc aggcgctgac gacggcggcg accaggcggt 3300

cggccactcg gcactcgcgc gcggcttgca acgggaggcg cggcggatgg agtggcgcgg 3360

ctcgggcacg cgcgctggct gcggggccga gcggctgcag gcgtggcagg gcagcggggc 3420

cgggcggcgc agacagcgca gacagcgcag gcatgatgcg agcgacgacc acgcgggcgc 3480

gcgcgcgcga acgacgcgcg gggagcggca gcgagggagc gaagaggagg gctcggctcg 3540

gcgcgggcgg ctcacgcacg cgcgggcaga gcggagggag cggaggaggg gcgttcggcg 3600

cgagcggctc acgcgcacgc gcgcgcggcg cgcgggcgga gcggagcgga gggggagaga 3660

gagagagaga gagagagata gccggggagg gagggggaga tgggccgaga gagattcggc 3720

ccatcgaacc cagggggagg caaaatagac ttttgcggag gaatttgaga tctgagatgg 3780

cacggacact agacaacaag caaagaaaca aatttcgcaa ttagggtttt taagagataa 3840

ttttcccgct aggcgccacg acggaacggg cgctacagct tt 3882

Claims

1.GY3 gene is in the colored application in single plant yield of the every fringe grain husk of control rice, it is characterised in that the core of the gene coding region Nucleotide sequence is as shown in SEQ ID NO:1.

2.GY3 gene is in the colored application in single plant yield of the every fringe grain husk of control rice, it is characterised in that the albumen of gene coding Matter sequence is as shown in SEQ ID NO:2.

The allele of 3.GY3 gene is in the colored application in single plant yield of the every fringe grain husk of control rice, it is characterised in that the gene Code area nucleotide sequence as shown in SEQ ID NO:3.

The allele of 4.GY3 gene is in the colored application in single plant yield of the every fringe grain husk of control rice, it is characterised in that the gene Coding protein sequence as shown in SEQ ID NO:4.

5. GY3 gene as described in claim 1 exists in the colored application in single plant yield of the every fringe grain husk of control rice, feature In there is the retrotransposon nucleotide sequence of one section of missing in the upstream of GY3 gene, and the sequence is as shown in SEQ ID NO:5.