CN107164347A - Control Culm of Rice rugosity, tiller number, grain number per spike, mass of 1000 kernel and the ideotype gene NPT1 of yield and its application - Google Patents

Abstract

It can increase Culm of Rice rugosity, per fringe grain number per spike, mass of 1000 kernel and the ideotype gene NPT1 of yield and its application the invention discloses a kind of.NPT1 gene functions and its expression quantity height are with Culm of Rice rugosity, per fringe grain number per spike, grain weight and yield are closely related, one excellent allelic variation type npt1 of the gene, the gene expression amount is caused to be remarkably decreased, Culm of Rice thickening, every fringe grain number per spike is caused to increase again with grain, so as to increase yield.Further study show that NPT1 encodes the deubiquitinating enzymes of a similar mankind OTUB1 albumen, it is by regulating and controlling OsSPL14 protein stabilities, so as to realize the regulation and control to paddy rice strain (fringe) type.Npt1 is polymerize with excellent economical character controlling gene dep1 1, rice yield can be further improved.NPT1 is as the new gene for controlling important Agronomic Traits in Rice, and its clone is that rice molecular pyramiding breeding and yield improvement provide certain theory and technology and supported.

Description

Control the preferable strain of Culm of Rice rugosity, tiller number, grain number per spike, mass of 1000 kernel and yield Type gene NPT1 and its application

Technical field

The invention belongs to plant biotechnology field.In particular it relates to a kind of increase Culm of Rice rugosity, every fringe Grain number per spike, the gene of mass of 1000 kernel and yield and its application.

Background technology

Paddy rice is one of important cereal crops, has supported the population in the whole world about 1/2nd.Eighties of last century sixties, The popularization of half breeding wheat for semidwarfness causes whole world rice yield to be significantly improved.Since nearly 30 years, although rice heterosis Using and its rearing new variety achieve larger progress in terms of rice yield lifting, but the increase rate of yield is still not Demand of the growing population to grain can be met.It is expected that the year two thousand fifty size of population is up to 8,900,000,000, it means that the year two thousand fifty grain Food production needs raising 50%.In face of population sharp increase, environment constantly deteriorates, the severe shape of the continuous reduction of arable area Gesture, how to effectively improve rice yield turns into a very important task in agricultural production.

The main number of productive ear in unit area of rice yield, the factors composition such as per fringe grain number per spike and mass of 1000 kernel.Tiller Number determines number of productive ear, and tiller is that influence paddy rice spike number is how many and and then influence one of Main Agronomic Characters of its yield；One Secondary branch stalk, Secondary branch and setting percentage determine every fringe grain number per spike；Seed size (length and width, thickness) determines mass of 1000 kernel.Meanwhile, Heading stage, lodging tolerance, disease-resistant resistance to inverse characteristic also contributes to rice yield.Rice panicle type refers to size and the growth of Rice Panicle Posture, is that the important component grain number per spike of plant type of rice and fringe develop closely related, little Hua numbers determine every fringe grain number per spike；Thousand Grain weight is closely related with Grain filling in grain size and grain maturation, and these economical characters are all close with plant yield Correlation, and these Main Agronomic Characters such as plant height, tiller number, tillering angle, fringe portion form, grain type etc. are all typical complexity Economical character, is controlled by multiple quantitative trait locus (Quantitative Trait Locus, QTLs) and envirment factor.

In rice breeding, ideotype, which is moulded, has played the genetic improvement of rice varieties huge effect.Traditional Plant Height of Rice is higher, and stalk is weak, low to nitrogenous fertilizer tolerance, easily lodging, yields poorly；Semi-dwarf gene sd1 introducing, makes plant height Significantly reduce, stalk thickening, tiller number increases, resistance to fertilizer is improved with lodging tolerance, therefore yield is significantly improved.In order to enter One step improves rice yield, eighties of last century International Rice eighties institute (International Rice Research Institute, IRRI) propose ideotype (New Plant Type) breeding plan, that is, it is bred as few tiller, big fringe, stalk thick Strong ideotype paddy rice.At the same time, China's breeding man proposes the theory of Japonica Rice for Super High Yield, it is indicated that erect head breeding Be after adapting to the another important breeding Morphological evolution selection of Super-high-yielding after half breeding wheat for semidwarfness, and be bred as with " distant round-grained rice 5 " and " Shennong-265 " etc. is a collection of high-yield rice new varieties of representative, considerably improves the unit yield of China japonica rice.Early stage, this The gene DEP1 for controlling the Erect Panicle character has been cloned in laboratory from high yield japonica rice kind " Shennong-265 ", it is found that its is excellent Allele dep1-1 can increase every fringe grain number per spike and improve yield, and served in " super hybridization rice " breeding of China to Close important effect.

The content of the invention

In order to further parse the molecular regulation mechanism of rice high yield plant type, we utilize having that International Rice is cultivated The parental rice IR66167-27-5-1-6 of ideotype feature, is separated and cloned with map-based cloning by QTL positioning Control the ideotype and the key gene NPT1 of yield.On this basis, science of heredity, molecular biology, life are fully utilized The multiple means such as thing chemistry, have extensively studied the biological function of NPT1 genes and its molecular basis of adjusting and controlling rice yield, are High-yield Rice Breeding provides corresponding theoretical foundation, also provides important gene resource for rice molecular design and context.

Culm of Rice rugosity, tiller number, the preferable strain per fringe grain number per spike, mass of 1000 kernel and yield are controlled the present invention relates to a kind of Type gene NPT1 and its application.In particular it relates to which NPT1 is improving crops (for example, paddy rice, wheat, barley, jade Rice, sorghum etc.) tiller number, per fringe grain number per spike, mass of 1000 kernel, yield, the New function in terms of stalk rugosity and resistance to lodging ability and Using.More particularly it relates to NPT1 and its homologous gene from the agriculture such as paddy rice, wheat, barley, corn, sorghum Crop.

The present inventor utilizes a few tiller from International Rice Research Institute, big fringe and the sturdy parental rice of stalk IR66167-27-5-1-6 and China japonica rice variety spring river 06 hybridize constructed recombinant inbred lines, find one of strain It is that RIL52 shows ideotype feature, including the increase of stalk rugosity, tillering number is reduced, and Grain number per spike and yield are notable The characters such as increase.Hybridize constructed F to RIL52 and rice variety Zhejiang spoke 802₂The field that colony has carried out completely random is real Test, tiller number has been investigated respectively, per fringe grain number per spike and stalk rugosity this 3 characters, with reference to the phenotypic number and molecule mark of each character Count evidence, and qtl analysis finds that have one on No. 8 chromosome long arms controls stalk rugosity, grain number per spike and tiller number simultaneously Main effect QTL, is named as qNPT1.The BC built followed by RIL52 and Zhejiang spoke 802₂F₂And BC₂F₃Colony, by qNPT1 essences Fine positioning is in No. eight chromosome long arm end 4.1Kbp of paddy rice physical extent, and the section only has candidate's base of a prediction Cause.On this basis, by IR66167-27-5-1-6 and in spend 11 hybridization, and spend 11 backgrounds in being constructed to objective trait (ZH11) NIL, it is found that ZH11-npt1 stalks are more sturdy, tiller number is slightly reduced, and plant height is basically unchanged, but per fringe Grain number per spike and mass of 1000 kernel are all significantly increased, and single plant yield is dramatically increased.

Paddy rice NPT1 encodes a deubiquitinating enzymes (OsOTUB1), is homologous genes of the mankind OTUB1 in paddy rice, because The gene is also referred to as OsOTUB1 genes by this, and the two terms abbreviation in the present invention is used interchangeably.Research finds paddy rice OsOTUB1(SEQ ID NO：15, the i.e. albumen of paddy rice NTP1 gene codes, also referred to as NPT1 albumen in the present invention, the two Term can also used interchangeably) there is the activity of catalysis K48 and K63 ubiquitin chain depolymerization.The present inventor is to OsOTUB1 albumen Subcellular Localization is studied, it is found that OsOTUB1-GFP fusion proteins are positioned in cytoplasm and nucleus.In addition, building The pOsOTUB1 of OsOTUB1 its own promoters driving：：GUS plant expression vectors, are obtained by agriculture bacillus mediated method Transfer-gen plant.Organ and tissue to transfer-gen plant different development stage carry out GUS dyeing and find that the gene is in paddy rice Whole developmental stage has expression, the significantly high expression especially in fibrovascular system and separate living tissue.

By transgene method, OsOTUB1 gene functions are demonstrated.It was found that the gene moderately express or lose by reduction Function mutation can cause tiller number to reduce, but can increase stalk rugosity, per fringe grain number per spike, mass of 1000 kernel, and then dramatically increase production Amount.On the contrary, the overexpression gene can then produce uneven class sizes phenotype and trigger cell death.Pass through yeast two-hybrid screening interaction Albumen finds that OsOTUB1 albumen can interact with ubiquitin binding enzyme OsUBC13, and suppresses its function.Further study show that OsOTUB1 and plant type of rice and the positive regulatory factor OsSPL14 interactions between protein of yield, and by influenceing OsSPL14 protein stabilities Come controlling plant type of rice and yield.

Meanwhile, it is same that the present inventor is cloned into OsOTUB1 respectively from wheat, barley, corn, sorghum, mouse and the mankind The cDNA sequence of source gene, and demonstrating them by transgenic research has the control Culm of Rice similar with OsOTUB1 thick Degree, tiller number, grain number per spike, the biological function of mass of 1000 kernel and yield.

Excellent allele npt1 is imported in high yield japonica rice kind force fortune round-grained rice 7, production statistical is surveyed in multiple years field Analysis finds that npt1 and dep1-1 polymerizations can realize that rice yield is further lifted on the basis of main breed high yield.

It is an object of the invention to provide the reason of a controllable stalk rugosity, tiller number, grain number per spike, mass of 1000 kernel and yield Think plant type gene NPT1, the new gene resource being worth with important breeding utilization is provided for rice high yield Molecular design breeding.

Therefore, the present invention provides following：

In a first aspect, the present invention provides a kind of Ideal Rice Plant Type gene NPT1 and its allele npt1, wherein Npt1 can increase the stalk rugosity of paddy rice, per fringe grain number per spike, mass of 1000 kernel and single plant yield.The nucleosides that both genes are related to Acid sequence includes：

(1)SEQ ID NOs：Nucleotide sequence shown in any one in 1-4；

(2) hybridize with the complementary series of the nucleotide sequence of (1) in medium stringency condition, under preferably high stringent hybridization condition Nucleotide sequence；

(3) have at least 70% with the nucleotide sequence of (1), preferably at least 80%, more preferably at least 90%, especially extremely The nucleotide sequence of few 95% or 98% or 99% homogeneity；

(4) with the protein of the nucleotide sequence coded same amino acid sequence of (1) but because of the degeneracy of genetic code The different nucleotide sequence in sequence；

(5) nucleotide sequence of one of following amino acid sequence is encoded：SEQ ID NO：Amino acid sequence shown in 15, or Person, due to one or more (such as 1-25,1-20,1-15,1-10,1-5,1-3) amino acid residues replacement, Missing and/or insertion and with SEQ ID NO：The different amino acid sequence of amino acid sequence shown in 13, or, with SEQ ID NO：Amino acid sequence shown in 15 has at least 70%, preferably at least 80%, more preferably at least 90%, especially at least 95% Or 98% homogeneity amino acid sequence；

(6) active fragment of any one nucleotide sequence in (1)-(6)；

(7) the complementary nucleotide sequence of any one nucleotide sequence in (1)-(6).

In second aspect, the present invention provides the promoter sequence of the gene and its allele described in first aspect, and it is such as SEQ ID NO：Shown in 5 or 6.

Table 1.SEQ ID NOs：1-6 sequence names and its source

SEQ ID NO：	Title	Source
			1	NPT1 cDNA sequences	Zhejiang spoke 802
2	Npt1 cDNA sequences	IR66167-27-5-1-6
			3	NPT1 gDNA sequences	Zhejiang spoke 802
4	Npt1 gDNA sequences	IR66167-27-5-1-6
			5	NPT1 promoter sequences	Zhejiang spoke 802
6	Npt1 promoter sequences	IR66167-27-5-1-6

In the third aspect, the present invention provides the homologous gene of the paddy rice NPT1 genes of first aspect, and the homologous gene comes Uralensis Fisch, barley, corn, sorghum, soybean, rape, cotton or tomato are come from, it encodes SEQ ID NOs：In 16-23 Amino acid sequence shown in any one.

Specifically, the homologous gene includes SEQ ID NOs：Nucleotide sequence shown in any of 7-14.

The nucleotide sequence of the homologous gene of the paddy rice NPT1 genes of table 2.

SEQ ID NO：	Title	Source
			7	TuNPT1 cDNA sequences	Uralensis Fisch
8	HvNPT1 cDNA sequences	Barley
			9	ZmNPT1 cDNA sequences	Corn
10	SbNPT1 cDNA sequences	Sorghum
			11	GmNPT1 cDNA sequences	Soybean
12	BnNPT1 cDNA sequences	Rape
			13	GhNPT1 cDNA sequences	Cotton
14	SlNPT1 cDNA sequences	Tomato

In fourth aspect, the present invention provides a kind of polypeptide of separation (also referred to as albumen), and it is included from such as the following group amino acid sequence The amino acid sequence selected in row：

(1)SEQ ID NOs：Amino acid sequence shown in any one in 15-23,

(2) due to one or more (such as 1-25,1-20,1-15,1-10,1-5,1-3) amino acid The replacement of residue, missing and/or insertion and with SEQ ID NO：The different ammonia of amino acid sequence shown in any one in 15-23 Base acid sequence,

(3) with SEQ ID NOs：Amino acid sequence shown in any one in 15-23 has at least 70%, preferably at least 80%th, the amino acid sequence of more preferably at least 90%, especially at least 95% or 98% or 99% homogeneity,

(4) active fragment of (1) or (2) or (3) described amino acid sequence,

(5) the present invention relates to polynucleotide molecule coding amino acid sequence.

Wherein, OsNPT1 encodes homologous proteins of the OTUB1 in paddy rice, therefore is named as OsOTUB1 to its encoding proteins, The amino acid sequence of protein sequence and its homologous protein is by SEQ ID NOs：Shown in 15-23, referring specifically to table 3 below.

The title and its source of table 3.NPT1 protein sequences and its misfolded proteins

At the 5th aspect, the present invention provides a kind of recombinant precursor, and it contains the present invention first to described in the third aspect NPT1 or allele npt1 related polynucleotides.Carrier used in wherein described construct is cloning vector or for table Up to the expression vector of the polynucleotides.

At the 6th aspect, the present invention provides a kind of recombinant host cell, and it contains the restructuring described in fifth aspect present invention Construct, or it is integrated with its genome the present invention first to the NPT1 described in the third aspect or allele npt1 multinuclears Thuja acid.The host cell can be selected from plant cell or microbial cell, such as Bacillus coli cells, agrobatcerium cell, Preferred plant cell, most preferably rice cell.The cell can be separation, in vitro, culture or be plant one Part.

At the 7th aspect, the present invention provides the polynucleotides or polypeptide of first to fourth aspect or the restructuring structure of the 5th aspect Build body or the 6th aspect recombinant host cell improvement rice plants character in purposes.

The invention further relates to improve the method for rice plants character, this method includes preparing containing first to fourth aspect The rice plant of the construct of polynucleotides or the 5th aspect, for example, the restructuring that methods described can be included in terms of the 6th is planted Thing cytothesis transfer-gen plant, or obtain transfer-gen plant with the recombinant microorganism cell transfecting rice plant of the 6th aspect. The character includes but is not limited to：Stalk rugosity, Grain number per spike and yield etc..

The present invention also provides a kind of method for the paddy rice for cultivating output increased, and this method includes：With the restructuring of the 7th aspect Host cell infection rice plant, which obtains the decline of NPT1 gene expression doses or amino acid sequence change, to be caused under protein function The transgenic rice plant of drop, reduces NPT1 gene expression doses particular by RNAi technology or passes through CRISPR/Cas9 And initiative NPT1 gene expression doses decline or NPT1 albumen lose or miopragia transgenic paddy rice.

In eighth aspect, the present invention provides the purposes of the npt1 genes, and it is used to increase crop stem rugosity, every fringe Grain number per spike, mass of 1000 kernel, and then improve the yield of crop.

At the 9th aspect, the present invention relates to a kind of method for the paddy rice for cultivating output increased.This method includes：From the 6th hair The bright restructuring paddy rice host cell regeneration of transgenic rice plant comprising npt1 allele, or by reducing NPT1 genes Expression, or change NPT1 protein biological functions, or by Tilling technologies, that is, be exactly directional induction gene Group mutating technology obtain NPT1 gene expression doses decline or NPT1 albumen lose or miopragia paddy rice or will bag The rice plant of the allele containing npt1 hybridizes with another plant, preferably go out tiller number slightly reduce, stalk thickening, per fringe grain number per spike Increase with mass of 1000 kernel, and then the increased plant of yield.

Invention technician is it should be understood that the research based on the present inventor is found, i.e. reduce the expression of NPT1 genes Amount, or make the miopragia of its NPT1 albumen expressed, it can act as increase rice tillering number, stalk rugosity, per fringe fringe The effect of grain number, mass of 1000 kernel and yield, can reduce the expression quantity of NPT1 genes, or make its table by selecting appropriate technology The miopragia of the NPT1 albumen reached, and then rice breeding is carried out on this basis.

At the tenth aspect, the present invention has found OsOTUB1 albumen (that is, paddy rice by yeast two-hybrid screening interaction albumen NPT1 albumen) it can be interacted with ubiquitin binding enzyme OsUBC13, and suppress its function.The present inventor has found OsUBC13 mistakes first Amount expression transfer-gen plant can increase yield, and this shows that rice breeding can be carried out using OsUBC13 genes.Therefore, this hair Bright to be related to a kind of method that every fringe grain number per spike and the increased paddy rice of yield are cultivated by overexpression OsUBC13, this method includes Transgenic rice plant is obtained with the recombinant host cell transfection rice plant of the recombinant precursor containing OsUBC13, is utilized In the paddy rice ubiquitin binding enzyme OsUBC13 and NPT1 albumen of OsUBC13 codings effect, resulting transgenic rice plant NPT1 activity is affected, therefore increase is per fringe grain number per spike and yield.Wherein described cell is microbial cell, the microorganism Cell is preferably Escherichia coli or agrobatcerium cell.Its action principle is OsUBC13 coding paddy rice ubiquitin binding enzyme OsUBC13, The albumen directly with NPT1 interactions between protein, enzymatic activity is suppressed by NPT1 albumen, therefore overexpression OsUBC13 transgenosis water The similar NPT1 protein actives of rice decline paddy rice, by increasing capacitance it is possible to increase paddy rice grain number per spike and yield.Wherein, the egg of OsUBC13 gene codes Bai Xulie such as SEQ ID NO：Shown in 36, in a specific embodiment, the nucleotide sequence such as SEQ of OsUBC13 genes ID NO：Shown in 35.

Tenth on the one hand, by CRISPR/cas9 gene editings technology knock out NPT1 or homologous gene TuNPT1, The increased crop of yield produced by HvNPT1, ZmNPT1, SbNPT1, GmNPT1, BnNPT1, GhNPT1, SlNPT1.It is described above Crop is preferably crops, is limited such as paddy rice, wheat, barley, corn, sorghum, soybean, rape, cotton, tomato, but not only In these crops.

12nd aspect, a kind of molecular marker assisted selection pyramiding breeding method for cultivating high-yield rice kind, this method Hybridize including the use of the parental rice comprising excellent allele npt1 with another parental rice comprising dep1-1 genes, rear Go out strain or kind containing npt1 and dep1 gene pyramidings according to molecular selection in generation.Wherein dep1-1 gene codes SEQ ID NO：Amino acid sequence (i.e. DEP1 albumen) shown in 34, in a specific embodiment, dep1-1 genes Nucleotides sequence such as SEQ ID NO：Shown in 33.

13rd aspect, the present inventor are further study show that OsOTUB1 and plant type of rice and the positive regulatory factor of yield OsSPL14 interactions between protein, and by influenceing OsSPL14 protein stabilities come controlling plant type of rice and yield.Therefore, the present invention is carried , should for a kind of by strengthening the method that plant type of rice modulin OsSPL14 cultivates every fringe grain number per spike and the increased paddy rice of yield Method is included with recombinant precursor and recombinant host cell containing NPT1 genes, the acquisition transgenic paddy rice plant of rice transformation plant Strain, is derived from the enhanced transfer-gen plant of OsSPL14 functions, wherein the cell is microbial cell, the microorganism is thin Born of the same parents are preferably Escherichia coli or agrobatcerium cell.Its action principle is NPT1 and OsSPL14 albumen direct interactions, science of heredity Prove that OsSPL14 is located at NPT1 downstreams, its function is suppressed by NPT1.

Wherein rice Os SPL14 protein sequences such as SEQ ID NO：Shown in 38, rice Os SPL14cDNA sequences such as SEQ ID NO：Shown in 37.

Brief description

Fig. 1 control Ideal Rice Plant Type strains qNPT1 QTL positioning.(a) spring river 06 and International Rice institute ideotype water Rice IR66167-27-5-1-6 hybridizes and obtains RIL by single seed descent, and wherein RIL52 shows ideotype Feature, engineer's scale, 20cm；(b) fringe portion phenotype compares, engineer's scale, 5cm；(c) tiller number statistical analysis；(d) grain number per spike statistical Analysis；(e) rugosity statistical analysis in the middle part of a section stalk is fallen；(f) Culm of Rice rugosity, grain number per spike, tiller number main effect QTL point are controlled Analysis.

Fig. 2 separate and cloned qNPT1 using map based cloning method.(a) by qNPT1 genes finely positioning in 4.1kb area In, its candidate gene encodes a deubiquitinating enzymes, is homologous gene of mankind's OTUB1 genes in paddy rice；(b) screens area Between haplotype analysis.

Fig. 3 .OsOTUB1 functional verifications.(a) plant type of rice compares, engineer's scale, 20cm；(b) CRISPR/cas9 methods are created Paddy rice osotub1-C1 mutant processed, green portion is sgRNA target sequences, and RED sector is PAM sequences；(c) heading stage compares Analysis；(d) plant height comparative analysis；(e) a section diameter statistical analysis is fallen；(f) tiller number comparative analysis；(g) spike length degree compares point Analysis；(h) per the stalk number comparative analysis of fringe Their First Branch；(i) per two grades of branch stalk number comparative analysis of fringe；(j) compare per fringe grain number per spike Analysis；(k) mass of 1000 kernel comparative analysis；(l) single plant yield statistical analysis.

Fig. 4 real-time quantitative PCRs detect expression of the OsOTUB1 genes in paddy rice different tissues.R：The tip of a root；C：Stem Stalk；LB：Blade；LS：Leaf sheath；SAM：Shoot apical meristem；YP0.2: 0.2 centimetre of young fringe；YP6：06 centimetre of fringe；YP12：12 lis Meter Sui.

Fig. 5 pOsOTUB1 under ZH11 backgrounds：：GUS transfer-gen plant GUS coloration results.(a) OsOTUB1 is in plumule With root expression, engineer's scale, 1cm；(b) the crosscutting display OsOTUB1 in root maturation zone is expressed in pericycle and bast, engineer's scale, 200μm；(c) OsOTUB1 is in the significantly high expression of tip of a root quiescent center, engineer's scale, 200 μm；(d) the crosscutting display of a section stalk is fallen OsOTUB1 is expressed in stalk vascular bundle, engineer's scale, 100 μm；(e) the young fringe dyeing of different development stage, found in young fringe period OsOTUB1 expression is most strong, and fringe development later stage is gradually reduced, engineer's scale, 1cm；(f) OsOTUB1 is expressed in developmental glume, Engineer's scale, 1mm.

Fig. 6 .OsOTUB1-GFP fusion proteins are positioned in cytoplasm and nucleus.(a) OsOTUB1-GFP fusion proteins Positioned in the cell of tip of a root elongation zone, engineer's scale, 20 μm；(b) OsOTUB1-GFP fusion proteins are primary in seedling leaf sheath dissociation in 10 days Plastid is positioned, engineer's scale, 10 μm.

Fig. 7 .OsOTUB1 overexpression transfer-gen plant phenotypic analyses.(a) grouting later stage plant type compares.OsOTUB1 is excessive Express transgenic plant, which shows different degrees of dwarfing, tiller reduction, blade, uneven class sizes isophenous；Engineer's scale, 20cm； (b) fringe portion form compares.OsOTUB1 overexpression transgenic lines show different degrees of fringe and diminished, and branch stalk is reduced；Ratio Chi, 5cm；(c) leaf morphology comparative analysis.OsOTUB1 overexpressions transgenic line OE-8# and OE-13# is in heading early stage leaf There are uneven class sizes in piece；Engineer's scale, 2cm；(d) Trypan Blue finds OsOTUB1 overexpression transgenic lines OE-8# and OE- 13# occurs in that different degrees of apoptosis；(e) OsOTUB1 gene expression amounts are analyzed in overexpression strain；(f) OsOTUB1 overexpression transfer-gen plant plant heights show different degrees of reduction；(g) OsOTUB1 overexpressions transfer-gen plant Tiller number shows different degrees of reduction；(h) OsOTUB1 overexpressions transfer-gen plant is shown in various degree per fringe grain number per spike Reduce.

Fig. 8 rice Os OTUB1 albumen has K48 and K63 ubiquitin chain Disaggregating activities.

Fig. 9 .OsOTUB1 and OsUBC13 interactions controlling plant type of rice and yield.(a) yeast two-hybrid checking OsOTUB1 with OsUBC13 interactions between protein；(b) Pull-down experimental analyses OsOTUB1 and OsUBC13 interactions between protein；(c) BiFC experimental analyses OsOTUB1 and OsUBC13 interactions between protein, engineer's scale, 10 μm；(d) under ZH11 backgrounds, the plant type of OsUBC13 transfer-gen plants Compare, engineer's scale, 20cm；(e) a section stalk rugosity of falling compares, engineer's scale, 0.5mm；(f) fringe type compares, engineer's scale, 5cm； (g) grain type compares, engineer's scale, 2mm；(h) OsUBC13 gene expression doses are analyzed；(i) tiller number statistical analysis；(j) per fringe fringe Grain number statistical analysis；(k) mass of 1000 kernel statistical analysis；(l) a section rugosity statistical analysis is fallen.

The SBP domains of Figure 10 .OsSPL14 albumen participate in OsOTUB1 interactions between protein.(a) yeast two-hybrid is verified OsOTUB1ΔN^1-80With OsSPL14 Δs N2^1-100Interaction；(b) schematic diagram of OsSPL14 Protein Deletion Mutants；(c)BiFC The different structure territory of experimental analysis OsSPL14 albumen and OsOTUB1 interactions between protein, engineer's scale, 10 μm.

Figure 11 .OsOTUB-OsSPL14 molecular modules controlling plant type of rice.(a) BiFC experiment prove OsOTUB1 with OsSPL14 SBP domains interaction；(b) Co-IP experiments prove OsOTUB1 and OsSPL14 interactions between protein；(c) in NIL- Under npt1 backgrounds, reduction OsSPL14 expression can cause the character such as the increase of its tiller number and grain number per spike reduction, and similar ZH11 is wild Type phenotype；And ZH11-OsSPL14^WFPMaterial phenotype is similar to NIL-npt1, engineer's scale, 20cm；(d) in NIL-npt1 backgrounds Under, OsSPL14 gene expressions are reduced, every fringe grain number per spike can be caused to reduce, and ZH11-OsSPL14^WFPMaterial phenotype is similar to NIL-npt1, engineer's scale, 5cm；(e) OsSPL14 gene expression doses are analyzed；(f) tiller number statistical analysis；(g) per fringe fringe grain Number statistical analysis；(h) a section stalk rugosity statistical analysis is fallen.

The amino acid alignment analysis of Figure 12 .OTUB1 and its homologous gene.

The energy complementation ZH11-npt1 mutation of Figure 13 overexpressions corn, barley, mouse and mankind OTUB1 homologous genes Phenotype.(a) grouting later stage plant type compares, and is overexpressed the tiller increase of transfer-gen plant, and stalk attenuates, engineer's scale, 20cm；(b) Fringe portion form compares, and the branch stalk for being overexpressed transfer-gen plant is reduced, and is reduced per fringe grain number per spike, engineer's scale, 5cm；(c)OsOTUB1 Gene expression dose is analyzed；(d) tiller number statistical analysis；(e) per fringe grain number per spike statistical analysis；(f) section stalk middle part is fallen directly Footpath statistical analysis.

The excellent allele npt1 of Figure 14 and dep1-1 pyramiding breedings lifting rice yield.(a) be in the milk later stage plant type ratio Compared with engineer's scale, 20cm；(b) fringe portion form compares, engineer's scale, 5cm；(c) grain type compares, engineer's scale, 2mm；(d) a section stem is fallen Stalk rugosity compares, engineer's scale, 0.5mm；(e) number of vascular bundles statistical analysis；(f) heading stage statistical analysis；(g) plant height statistical Analysis；(h) tiller number statistical analysis mass of 1000 kernel statistical analysis；(i) per fringe grain number per spike statistical analysis；(j) mass of 1000 kernel statistical analysis； (k) output statistics is analyzed.

Figure 15 reduction OsOTUB1 (i.e. NPT1) gene expression genetically modified plants yield increases.(a) be in the milk later stage plant type ratio Compared with engineer's scale, 20cm；(b) fringe portion form compares, engineer's scale, 5cm；(c) OsOTUB1 gene expression doses are analyzed；(d) per fringe Grain number per spike statistical analysis；(e) a section stalk rugosity statistical analysis is fallen；(f) single plant yield statistical analysis.

Figure 16 show OsOTUB1 sgRNA boxes sequence (SEQ ID NO：24)：U6a-sgRNA box monocotyledons.Its The sequence that middle lowercase is shown is target sequence, and the sequence that decline runic underscore is shown is sgRNA coded sequences.

Embodiment

The in-depth study by extensive, the present invention, which has cloned one, can change Culm of Rice rugosity, tiller number, per fringe The ideotype gene NPT1 of grain number per spike and yield, the gene is located at No. eight chromosome long arm of paddy rice；The gene expression amount drops The mutation of low or afunction can cause tiller number to reduce, and stalk thickening increases per fringe grain number per spike, mass of 1000 kernel and yield.

Plant Transformation

In a particularly preferred embodiment, at least one control grain of the invention is expressed in higher organisms such as plant The albumen of wide and grain weight.The nucleotide sequence for the gene that the control grain of the present invention is wide and grain is heavy can be inserted into expression cassette, so Afterwards preferably, by the expression cassette stable integration in the Plant Genome.In another preferred embodiment, by the control The nucleotide sequence of wide and grain weight the gene of granulation is included in the virus of non-pathogenic self-replacation.The plant converted according to the present invention Thing can be monocotyledon or dicotyledon, including but not limited to corn, wheat, barley, rye, sweet potato, beans, pea, Witloof, lettuce, wild cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, winter squash, south Melon, hemp, zucchini, apple, pears , Wen Quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, rasp berry, blackberry, blueberry, spinach Trailing plants, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, beet, sunflower, oil seed rape, clover, cigarette Grass, carrot, cotton, clover, rice, potato, eggplant, cucumber, Arabidopsis and xylophyta such as coniferous tree and deciduous tree.It is special You Xuanshi not paddy rice, wheat, barley, corn, oat or rye.

Once by desired nucleotide sequence conversion enter specified plant species in, can have been bred in the species it or It is transferred into other kinds of same species with traditional breeding method, particularly including in commercial variety.

Preferably, the nucleotide sequence of the present invention is expressed in genetically modified plants, phase is thus caused in genetically modified plants Answer the biosynthesis of the wide albumen of grain.By this way, the genetically modified plants with Ameliorative character can be produced.In order to be planted in transgenosis Nucleotide sequence of the present invention is expressed in thing, nucleotide sequence of the present invention may need to modify and optimize.All organisms have spy Fixed codon usage bias, this is known in the art, can keep nucleotide sequence coded ammonia of the present invention Base changes its codon to meet plant-preference while sour.Moreover, from having at least about 35%, preferably more than about 45%, more Preferably more than 50%, the most preferably greater than about coded sequence of 60%GC contents can be best implemented with high-caliber table in plant Reach.Although preferred gene order can fully be expressed in monocotyledon and dicot plant species, it can modify Sequence to adapt to the specific codons preference and G/C content preference of monocotyledon or dicotyledon because these preferences by It is proved to be different (Murray etc., Nucl.Acids Res.17：477-498(1989)).In addition, nucleotide sequence can be screened To find the presence for the unconventional splice site for causing message truncation.Utilize Patent Application Publication EP 0 385 962 (Monsanto), the method described in EP 0 359 472 (Lubrizol) and WO 93/07278 (Ciba-Geigy), uses ability Site-directed mutagenesis technology known to domain, PCR and synthetic gene build the institute for carrying out needing to carry out in these nucleotide sequences There is change, those change as described above.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that the following example is only used for further illustrating The present invention, and it is not limited to the spirit and scope of the present invention.

It should be noted that it should be appreciated by those skilled in the art that reagent, enzyme used in following embodiments etc. are except spy Do not mentionlet alone outside bright, be the reagent or enzyme of the pure rank of analysis commercially available from Reagent Company.

Embodiment 1：Ideal Rice Plant Type gene NPT1 clone

The present inventor is by Chinese Japonica Rice kind spring river 06 and International Rice institute ideotype strain IR66167-27-5-1-6 Hybridization harvest F₁, and RIL is obtained by single seed descent；Wherein RIL52 strains have the increase of stalk rugosity, tiller number Mesh is slightly reduced, the phenotype (Fig. 1 a-e) for the ideotype feature that grain number per spike is dramatically increased.By RIL52 and rice variety Zhejiang spoke 802 Hybridization obtains F₁, F₂The blade that 196 individual plants are randomly selected in offspring extracts DNA, while a section middle part stalk is thick for correspondence investigation The characters such as degree, tiller number and every fringe grain number per spike.Polymorphism analysis is carried out using SSR primer pairs RIL52 and Zhejiang spoke 802, selection has Polymorphic primer, respectively to 196 F₂Individual plant enters performing PCR amplification, according to electrophoresis result and mapping software Mapmaker Version 3.0 requires that every individual plant for RIL52 banding patterns occur is labeled as B, and the individual plant for the banding pattern of Zhejiang spoke 802 occur is labeled as A, The individual plant for occurring RIL52 and the banding pattern of Zhejiang spoke 802 simultaneously is labeled as H.Utilize Win QTL Cartographer V2.5 and recombination region Between the method mapped carry out QTL positioning and Effect Estimation, as a result find there is a control simultaneously on No. 8 chromosome long arm Culm of Rice rugosity (Culm Diameter), tiller number (Tiller Number) and per fringe grain number per spike (Grain Number) Main effect QTL, is named as qNPT1 (Fig. 1 f).

Continue to be returned Zhejiang spoke 802 after RIL52 and Zhejiang spoke 802 are hybridized, obtain BC₁F₂Colony；Choose similar RIL52 plant The individual plant of phenotype 389.Clear and definite 93 individual plants of phenotype are further therefrom chosen, DNA is extracted and carries out coarse positioning.Using in target Region filters out 8 InDel marks, and target gene is positioned between No. 8 chromosome long arms P6049 and P351.Utilize residue 296 individual plants carry out finely positioning, and target gene is positioned between P5528 and P3914.By the BC of similar RIL52 phenotypes₁F₂ Individual plant continues to be returned with ZF802, obtains the BC of objective trait₂F₂5326, sample is separated, is continued with the development of screens interval 6 In-Del label screenings exchange individual plant, and the 4.1Kbp that most target gene is positioned between P139 and P143 at last is interval interior, its In include 1 predicted gene.One deubiquitinating enzymes of the gene code, are homologous genes of the mankind OTUB1 in paddy rice, by it It is named as OsOTUB1 (Fig. 2 a).RAP-DB(http：//rapdb.dna.affrc.go.jp/) predict that OsOTUB1 has two Transcript, wherein OsOTUB1.1 have 8 extrons, 7 intrones, code area 825bp；OsOTUB1.2 has aobvious outside 5 Son, 4 intrones, code area 597bp.Haplotype analysis are carried out to screens interval, find to derive from IR66167-27-5-1-6 The peculiar 4 SNP differences of section and single base insertion difference, one of SNP is located at promoter (Fig. 2 b).

QNPT1 identify with separate used in molecular labeling be mark based on PCR, including SSR marker and voluntarily set Count InDel marks.SSR marker is all from the microsatellite marker linkage map that McCouch etc. (2001,2002) is delivered；STS is marked With ssr analysis instrument (http：//www.gramene.org/gramene/searches/ssrtool) analysis cloned sequence sieve The reproducible SSR target sequences of microsatellite are selected, then primer is carried out to these target sequences with Primer5 analysis softwares and are set Meter.75 pairs are have selected from these primers to be uniformly distributed on chromosome, and the good mark of polymorphism is used for heredity between parent Background is screened, and part Primer and sequence are shown in Table 4.PCR programs slightly change progress according to Panaud etc. (1996) method, Specially every μ l amplification reaction systems of pipe 20, including：0.15 μM of SSR primers, 200 μM of dNTPs, 1 × PCR reaction buffers (50mM KCl, 10mM Tris-HCl pH 8.3,1.5mM MgCl₂, 0.01% gelatin), 50~100ng template DNAs, 1U Taq enzyme；Response procedures are：94 DEG C of DNA denaturation 5min, circulation (94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 1min) 36 times, 72 DEG C again Extend 5min.The PCR primer amplified carries out electrophoresis with 6% polyacrylamide denaturant gel, and voltage is adjusted to 300V, electric at room temperature Swimming about 3 hours.After electrophoresis is finished, silver staining record banding pattern or gel imaging.

Using the middle DNA for spending 11 (ZH11) as template, 2.5Kbp OsOTUB1.1 and OsOTUB1.2 promoter are expanded respectively Area, insertion pCAMBIA-2300 carriers (referring to Hajdukiewicz et al., 1994) constitute and carry paddy rice its own promoter Carrier pCAMBIA-OsOTUB1.1-2300 and pCAMBIA-OsOTUB1.2-2300.Meanwhile, with ZH11 young fringe material CDNA is template, expands OsOTUB1.1 and OsOTUB1.2 coding region sequence, respectively insertion vector pCAMBIA- OsOTUB1.1-2300 and pCAMBIA-OsOTUB1.2-2300, constitutes plant expression vector pOsOTUB1.1：：OsOTUB1.1 And pOsOTUB1.2：：OsOTUB1.2.ZH11-npt1 is converted by agriculture bacillus mediated method, and obtains transfer-gen plant.Grind Study carefully discovery pOsOTUB1.1：：OsOTUB1.1 transgenic positive plant Primary branch and Secondary branch are reduced, and fringe is elongated, grain It is elongated, significantly reduced per tassel seed number, stalk attenuates, and tiller increases, the phenotype of similar ZH11 wild types；And pOsOTUB1.2：： The similar ZH11-npt1 of OsOTUB1.2 transgenic positive plant phenotypes.Result above shows that OsOTUB1.1 transcripts have function, ZH11-npt1 phenotype can be recovered.

Embodiment 2：The middle structure for spending 11 background NILs

The present inventor will spend 11 in RIL52 continuous backcrosses, choose stalk thickening, tiller reduction, the increased plant of grain number per spike, And detection is tracked to target zone using InDel molecular labelings P135；Meanwhile, the section beyond objective trait is carried on the back Scape is scanned, and final obtain is used for follow-up grind with the middle NIL ZH11-npt1 materials for spending 11 genetic backgrounds to be sufficiently close to Study carefully.Grouting later stage ZH11-npt1 shows sturdy stalk, available tillering reduction and per the increased phenotype of fringe grain number per spike.Enter one Walk statistical analysis to find, compared to ZH11 wild types, ZH11-npt1 internode stalk rugosity is dramatically increased, available tillering is reduced greatly About 2, spike length slightly shorten (length 18-20cm), increase 50-70, single plant yield increase about 10% (Fig. 3) per fringe grain number per spike.

Embodiment 3：NPT1 functional verification

Root (root), young tender stem stalk (culm), the blade of a pair of NIL seedling of 11 backgrounds are spent in extracting respectively (leaf blade), leaf sheath (leaf sheath), apical meristem (SAM), the young fringes (young panicle) of 0.2cm, 6cm The total serum IgE of fringe and 12cm fringes, reverse transcription is obtained after cDNA, quantitative PCR detection OsOTUB1 different tissues and device in a pair of materials Expression in official, hair OsOTUB1 has the expression of higher level, the especially expression quantity in young fringe in the whole developmental stage of paddy rice It is high.Compared to ZH11, expression of the OsOTUB1 in ZH11-npt1 different tissues and organ presents a certain degree of downward (Fig. 4).

With plasmid pB1121 (Chen et al., 2003) for template amplification GUS sequences, GUS fragments and carrier framework are reclaimed And connect, constitute carrier pCAMBIA：：GUS-2300；2.5kb OsOTUB1.1 promoter sequences are expanded, digestion is reclaimed and inserted Enter pCAMBIA：：GUS-2300, is built into pOsOTUB1：：GUS.ZH11 is transferred to by agriculture bacillus mediated method, to turning The organ of gene plant different development stage and tissue carry out GUS dyeing and found, the gene paddy rice whole developmental stage all There is expression, especially significantly expressed in fibrovascular system.Seedling dyeing to growth one week, it was observed that OsOTUB1 is in plumule With root expression significantly (Fig. 5 a)；Have in the quiescent center (quiescent center, QC) and root cap of Root apical meristem Express (Fig. 5 b)；The pericycle expression (Fig. 5 c) in root；There is expression (Fig. 5 d) in stalk vascular bundle；The different times developed in fringe There is expression, gradually reduced to later stage experssion, is concentrated mainly in glume (Fig. 5 e, f).

Using pCAMBIA-2300 as skeleton carrier, then OsOTUB1.1 promoter insertion vectors sequentially insert Remove the OsOTUB1.1 coded sequences and GFP coded sequences of terminator codon, construct its own promoter driving OsOTUB1.1-GFP fusion protein plant expression vectors pOTUB1：OsOTUB1.1-GFP simultaneously converts ZH11.To transgenic seedlings Tip of a root direct tablet compressing is observed, it is found that OsOTUB1.1-GFP fusion proteins are positioned at cytoplasm and (Fig. 6 a) in nucleus.Further Transgenic positive plant shoots leaf sheath protoplast is digested, also demonstrate that OsOTUB1.1-GFP fusion proteins are positioned at cytoplasm With (Fig. 6 b) in nucleus.

Using pCAMBIA-2300 as skeleton carrier, pActin is constructed：：OsOTUB1.1 Overexpression vectors, and pass through agriculture The method of bacillus mediation is transferred in ZH11, real-time quantitative PCR detection OsOTUB1.1 gene expression amounts, obtains scale Up to transgenic line OE-8 and OE-13.Show that fringe is small, downgrade two superelevation expression strains grouting later stages, blade has obvious Uneven class sizes phenotype, further to occur the blade of uneven class sizes carry out Trypan Blue, find part cell occur in that cell is dead Die.Statistical analysis finds that OsOTUB1.1 superelevation expression causes Primary branch, Secondary branch and significantly subtracting per fringe grain number per spike Few, plant height reduction shows dosage effect (Fig. 7).

The ubiquitination of target protein and deubiquitination regulation and control play cell and vital movement important function.Paddy rice OsOTUB1 encodes a deubiquitinating enzymes, take part in the depolymerization of ubiquitin chain.By OsOTUB1.1, it is public that OsOTUB1.2 is building up to GE Take charge of on prokaryotic expression carrier pGEX-4T-1, be transferred in Rosetta (DE3) bacterial strain induction expression protein and purify, buy Boston The OTUB1 albumen of Biochem companies and two kinds of substrate ubiquitin chain Tetra-ub.By GST-OsOTUB1.1, GST-OsOTUB1.2 It is incubated 1 hour jointly in 37 DEG C with two kinds of substrate ubiquitin chain Tetra-ub respectively with OTUB1, carries out external deubiquitination experiment. Western Blot detections find that Tetra-ub-K48 and Tetra-ub-K63 can be cracked by OsOTUB1.1, and can not be by OsOTUB1.2 is cracked, and this shows that the OsOTUB1.1 for possessing entire catalytic avtive spot can not only be catalyzed the solution of 48 ubiquitin chains It is poly-, and 63 ubiquitin chain depolymerization can be catalyzed.Further catalysis of the analysis OsOTUB1.1 to K48 and K63 ubiquitin chains is lived Property, find the two, OsOTUB1.1 depolymerization ability to K48 ubiquitin chain different with the depolymerization ability of K63 ubiquitin chains to K48 It is stronger, substantially by the whole depolymerization of substrate, form Ub1；And the depolymerization ability to K63 ubiquitin chains is weaker, formed Ub3, Ub2 and Ub1 (Fig. 8).

Embodiment 4：NPT1/OsOTUB1 interaction albumen OsUBC13 is identified and functional verification

OsOTUB1.1 is built on pGBKT7 (Clontech companies), using yeast-two hybrid technique from Rice Panicle Interaction albumen is screened in cDNA library.It is tentatively flat in-Ade/-His/-Leu/-Trp auxotrophs using the method for cotransformation Positive colony 200 is obtained on plate, positive colony 40 is obtained after blue hickie screening.Extract plasmid and convert Escherichia coli again, Confirm that OsUBC13 (Os01g48280), OsSPL14 (Os08g39890) interact with OsOTUB1.1 respectively.

OsUBC13 encodes a ubiquitin binding enzyme, its homologous protein UBC13 in animal directly with OTUB1 interactions, and Suppressed by OTUB1.By OsOTUB1.1 insertion pGEX-4T-1 prokaryotic expression carriers (being purchased from GE), pGEX-4T- is built into OsOTUB1.1；OsUBC13 insertion pET-28a prokaryotic expression carriers (being purchased from Novagen), are built into pET-28a-OsUBC13. It is transferred to respectively in coli strain Rosetta with reference protein GST empty carriers (be purchased from GE) simultaneously, induced expression and purifying is obtained Obtain GST, GST-OsOTUB1.1 and His-OsUBC13 albumen.By GST, GST-OsOTUB1.1 respectively with Sepharose 4B 4 DEG C be incubated 1 hour after, add His-OsUBC13 albumen.4 DEG C be incubated 2 hours after Western Blot detect, GST pull- Down experiments prove that GST-OsOTUB1.1 and His-OsUBC13 interacts.OsOTUB1.1 is connected to pSY-735-35S- CYFP-HA (Bracha-Drori, K.et al, 2004) carrier, expresses YFP^C-OsOTUB1.1；OsUBC13 is connected to pSY- 736-35S-nYFP-EE (Bracha-Drori, K.et al, 2004) carrier, expresses YFP^N-OsUBC13.7 are dissociated using enzyme process Its leaf sheath of rice seedling first, obtains protoplast.The method cotransformation protoplast mediated by PEG, as a result shows YFP^C- OsOTUB1.1 and YFP^N- OsUBC13 occurs to interact (Fig. 9 a-c) in nucleus and cytoplasm.

Using pCAMBIA-2300 as skeleton carrier, p35S is built respectively：OsUBC13 and p35S：：OsUBC13-RNAi plants Expression vector converts ZH11, finds overexpression OsUBC13 plant plant height without significant change, tiller is slightly reduced, stalk thickening, one Secondary branch stalk and the increase of Secondary branch number, grain number per spike increase, phenotype are similar to NIL-npt1.To falling, internode stalk middle part is horizontal Cut, find stalk thickening, thicken, the increase of vascular bundle number.And OsUBC13 expression is reduced by RNAi, it is found that transgenosis is planted Strain plant height is without significant change, but tiller reduction, stalk attenuate, Primary branch and Secondary branch quantity are reduced, subtracted per fringe grain number per spike Few, phenotype is similar to the phenotype of OsOTUB1.1 overexpressing plants.To crosscutting in the middle part of the first internode under its fringe, it is found that stalk becomes Carefully, vascular bundle number is reduced.Result above shows that OsOTUB1 has the function (Fig. 9 d-i) of suppressing OsUBC13.

Embodiment 5：NPT1/OsOTUB1 interaction albumen OsSPL14 is identified and functional verification

OsSPL14 encodes a transcription factor, and it is regulated and controled on rna level by miR156.OsSPL14 transcriptional levels Increase with protein level so that rice tillering is reduced, grain number per spike and mass of 1000 kernel increase, while stalk becomes sturdy, energy resistant to lodging Power strengthens, and then improves yield.In order to further confirm that OsOTUB1 and OsSPL14 interaction and interaction domain, build Expression YFP^N-OsSPL14 total lengths and different segment deletion transient expression vectors, respectively with expression YFP^C- OsOTUB1.1 table Up to the instantaneous cotransformation rice leaf sheath protoplast of carrier.Observation finds OsOTUB1.1 and OsSPL14 SBP- domain phase interactions With (Figure 10, Figure 11 a).This shows that the SBP domains of paddy rice SPL transcription factors are that OsOTUB1.1 and OsSPL14 interacts Key structure domain.Co-immunoprecipitation (Co-immunoprecipitation, Co-IP) is study interaction of in vivo proteins one Plant experimental method.Extract ZH11 backgrounds pActin：：OTUB1.1-GFP transfer-gen plants total protein is incubated for 4 DEG C with anti-GFP antibody 1h is educated, 40 μ L Protein G sepharose 4Bs are then added after 4 DEG C of common incubation 1h.Supernatant is outwelled to rinse three times and addition 2 × SDS sample-loading buffers, boiling water bath carries out sds polyacrylamide electrophoresis after 5 minutes.Western Blot detections are found OsOTUB1 interacts (Figure 11 b) in vivo with OsSPL14 albumen.

Using pCAMBIA-2300 as skeleton carrier, pActin is built：：OsSPL14-RNAi plant expression vectors are converted ZH11-npt1, observation transfer-gen plant finds that reduction OsSPL14 expressions cause plant tillering to dramatically increase, and fringe portion is once Branch stalk and Secondary branch number are reduced, and are reduced per fringe grain number per spike, partly similar to ZH11 phenotypes.By excellent allele OsSPL14^WFPZH11 backgrounds are hybridized to, ZH11-OsSPL14 is found^WFPTiller is reduced, fringe portion Primary branch and Secondary branch number Increase, Grain number per spike increase, similar to ZH11-npt1 phenotypes.These researchs show that OsSPL14 is in OsOTUB1 downstreams, the two The morphogenesis (Figure 11 c-h) of common regulation plant type of rice (fringe type).

Embodiment 6：The clone of NPT1 homologous genes and functional analysis

The lane database provided at NCBI websites (www.ncbi.nih.nlm.gov), passes through Basic logical Alignment search tool (BLAST) compare, obtain wheat, barley, corn, sorghum, arabidopsis, soybean, mouse and The homologous cDNA sequence of people, respectively Uralensis Fisch TuOTUB1 (SEQ ID NO：7), barley HvOTUB1 (SEQ ID NO：8), corn ZmOTUB1 (SEQ ID NO：9), sorghum SbOTUB1 (SEQ ID NO：10), arabidopsis AtOTUB1 (SEQ ID NO：11), soybean GmOTUB1 (SEQ ID NO：12), mouse MmOTUB1 (SEQ ID NO：13) with mankind HsOTUB1 (SEQ ID NO：14), the homology for the albumen that they are encoded is respectively：TuOTUB1(SEQ ID NO：16) with rice Os OTUB1 (SEQ ID NO：4) similitude is 86.43%；HvOTUB1(SEQ ID NO：17) similitude with rice Os OTUB1 is 86.79%； ZmOTUB1(SEQ ID NO：19) similitude with rice Os OTUB1 is 69.38%；Sb OTUB1(SEQ ID NO：18) it is same Rice Os OTUB1 similitudes are 68.10%；AtOTUB1(SEQ ID NO：20) similitude with rice Os OTUB1 is 59.80%；GmOTUB1(SEQ ID NO：21) similitude with rice Os OTUB1 is 61.32%；MmOTUB1(SEQ ID NO：22) similitude with rice Os OTUB1 is 38.70%；HsOTUB1(SEQ ID NO：23) with the similar of rice Os OTUB1 Property be 39.12% (Figure 12).

OsOTUB1 homologous gene is separated from corn, barley, mouse and the mankind respectively by homologous clone method ZmOTUB1, HvOTUB1, MmOTUB1 and HsOTUB1, construct pOsOTUB1 respectively：TuOTUB1、pOsOTUB1：HvOTUB1、 pOsOTUB1：MmOTUB1 and pActin：HsDEP1 carriers, convert ZH11-npt1 rice plants；Transgenic rice plant phenotype Comparative analysis shows that overexpression corn, barley, mouse and mankind OTUB1 homologous genes can the plants of complementation ZH11-npt1 paddy rice The mutant phenotype of strain, this shows that these homologous gene functions have conservative (Figure 13).

Embodiment 7：Excellent allele npt1 and dep1-1 pyramiding breeding application

DEP1 is the main effect QTL of a control paddy rice Erect Panicle and yield traits, its excellent allele dep1-1 energy Paddy rice stem apex meristematic activity is improved, increase improves the yield (invention authorized referring to the applicant per fringe grain number per spike Patent application, the unnamed gene was at that time in this application dep1, later in order to distinguish by Application No. 201110029759.9 The Different Variation type of same DEP1 genes, original dep1 is renamed as dep1-1).In order to evaluate dep1-1 and Npt1 assembly effects, npt1 is imported carry dep1-1 (SEQ ID NO by the method for hybridization：33) high yield japonica rice kind is military Round-grained rice 7 (WYJ7-dep1-1) is transported, by molecular marker assisted selection and continuous backcross 6 times, WYJ7-NPT1-dep1-1 is obtained With a pair of NILs of WYJ7-npt1-dep1-1.Multiple years field survey production analysis find, excellent allele npt1 and Dep1-1 polymerizations do not influence rice ear sprouting period and plant height, and tiller number slightly has reduction, but can substantially increase stalk thickening, per fringe fringe grain Number and mass of 1000 kernel, and then realize that yield increases (Figure 14).The molecular labeling being related in selection is P135 and Pd1, sequence See table 5.

Embodiment 8：NPT1 and its homologous gene are knocked out using CRISPR/cas9 systems

Compared by Basic logical alignment search tool (BLAST), obtain wheat, barley, jade Rice, sorghum, soybean, rape, cotton and the homologous cDNA sequence of tomato.It is conservative with homologous genes encoding protein amino acid sequence Region is target sequence, designs target site.Sequence is as follows：

Paddy rice：5′-tcagctggaaagtgttctgcagg

Wheat：5′-ttaaggcgaacacgaggagatgg

Barley：5′-ttaagacggacacgaggagatgg

Corn：5′-aagctttatgttttcctacctgg

Sorghum：5′-aagctttatgttctcctacttgg

Soybean：5′-attcgtcgtactcgaggagatgg

Rape：5′-gttgcaatcaggcgaacaagagg

Cotton：5′-gcagccattagaagaacgcgagg

Tomato：5′-gctgccattagaagaacacgtgg

Target sequence is connected into sgRNA expression cassettes (sequence SEQ ID NO by design primer：24-32), monocotyledon side digestion Side connection insertion pYLCRISPR/Cas9P_ubiPYLCRISPR/Cas9P is even inserted in-MH, dicotyledon side trimming_35S- DH, side Method has obtained Transgenic Rice positive plant at present referring in particular to document (Ma et al., 2015).Extract transgenic paddy rice The DNA of different strains, the gDNA sequences containing target site are expanded by PCR, are reclaimed PCR primer and are simultaneously sequenced, it was found that not similar shape The target site of formula knocks out strain,.Wherein osotub1-C1 mutant gDNA lacks 7 bases, causes frameshift mutation, encoding proteins Amino acid sequence is terminated in advance.Compared to ZH11, osotub1-C1 mutant shows similar NIL-npt1 phenotypes (Fig. 3).

Embodiment 9：Reduce the increase of NPT1 gene expression genetically modified plants yield

5 ' end the 300bp of PCR amplifications NPT1 genes (i.e. OsOTUB1 genes) code area cDNA sequence, constructs and possesses The RNAi interference carriers pActin of palindrome：：RNAi-OsOTUB1, and be transferred to by agriculture bacillus mediated method OsOTUB1 gene expressions show different degrees of downward and show ZH11-npt1 classes in ZH11, transgenic positive plant As phenotype.Compare two materials of ZH11 and RNAi-OsOTUB1, it is found that RNAi-OsOTUB1 transgenic line fringes shorten, stalk Thickening, Primary branch and the increase of Secondary branch number, per the increase of fringe grain number per spike, yield increase (Figure 15).

Part primer and sequence used in table 4.QTL analyses and map based cloning

Part primer and sequence used in the vector construction of table 5.

It should be understood that although with reference to its exemplary embodiment, particularly shown and description is carried out to the present invention, It should be understood by those skilled in the art that without departing substantially from spirit of the invention as defined in appended claims Under conditions of scope, the change of various forms and details can be carried out wherein, any of various embodiments can be carried out Combination.

Bibliography：

Kiewicz P, Svab Z, and Maliga P. (1994) .The small, versatilepPZP family ofAgrobacterium binary vectors for plant transformation.Plant Molecular Biology 25.989-994.

Chen P-Y, Wang C-K, Soong S-C, and To K.-Y (2003) .Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from Transgenic plants.Molecular Breeding 11,287-293.

Ma X, Zhang Q, Zhu Q, et al. (2015) .A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.Mol Plant 8,1274-1284.

Bracha-Drori K, Shichrur K, Katz A, et al. (2004) .Detection of protein- protein interactions in plants using bimolecular fluorescence complementation.Plant J；40：419-427.

Sequence table

<110>Inst. of Genetics and Development Biology, CAS

<120>Control the ideotype gene NPT1 of Culm of Rice rugosity, tiller number, grain number per spike, mass of 1000 kernel and yield and its answer With

<130> IB178051

<160> 38

<170> PatentIn version 3.1

<210> 1

<211> 825

<212> DNA

<213>NPT1 cDNA from Zhejiang spoke 802

<400> 1

atgggcgggg actactacca ctcgtgctgc ggcgaccccg accccgacct ccgcgcgccc 60

gaggggccca agctgccgta cgtcggggac aaggaacctc tctccacttt agccgctgag 120

tttcagtctg gcagccccat tttacaggag aaaataaagt tgcttggtga acagtatgat 180

gctttaagaa ggacacgagg agatggaaac tgcttttatc gaagctttat gttttcctac 240

ttggaacata tcctagagac acaagacaaa gctgaggttg agcgcattct aaaaaaaatt 300

gagcagtgca agaagactct tgcagatctt ggatacattg agttcacctt tgaagatttc 360

ttctctatat tcattgatca gctggaaagt gttctgcagg gacatgaatc ctccataggg 420

gccgaagagc ttctagaaag aaccagggat cagatggttt ctgattatgt tgtcatgttc 480

tttaggtttg tcacctctgg tgaaatccaa aggagggctg agttcttcga accattcatc 540

tctggcttga caaattcgac tgtggttcag ttctgcaagg cttccgtgga gccgatgggc 600

gaggaaagtg accatgtcca cataattgcc ctatcagatg cgttgggtgt gccaatccgt 660

gtgatgtacc tagacagaag ctcatgtgat gctggaaata taagtgtgaa ccaccatgat 720

ttcagccctg aggccaattc atcggacggt gctgctgctg ctgagaaacc ttacattact 780

ttgctctacc gtcctggtca ctacgacatt ctctacccga agtga 825

<210> 2

<211> 825

<212> DNA

<213>Npt1 cDNA from IR66167-27-5-1-6

<400> 2

atgggcgggg actactacca ctcgtgctgc ggcgaccccg accccgacct ccgcgcgccc 60

gaggggccca agctgccgta cgtcggggac aaggaacctc tctccacttt agccgctgag 120

tttcagtctg gcagccccat tttacaggag aaaataaagt tgcttggtga acagtatgat 180

gctttaagaa ggacacgagg agatggaaac tgcttttatc gaagctttat gttttcctac 240

ttggaacata tcctagagac acaagacaaa gctgaggttg agcgcattct aaaaaaaatt 300

gagcagtgca agaagactct tgcagatctt ggatacattg agttcacctt tgaagatttc 360

ttctctatat tcattgatca gctggaaagt gttctgcagg gacatgaatc ctccataggg 420

gccgaagagc ttctagaaag aaccagggat cagatggttt ctgattatgt tgtcatgttc 480

tttaggtttg tcacctctgg tgaaatccaa aggagggccg agttcttcga accattcatc 540

tctggcttga caaattcgac tgtggttcag ttctgcaagg cttccgtgga gccgatgggc 600

gaggaaagtg accatgtcca cataattgcc ctatcagatg cgttgggtgt gccaatccgt 660

gtgatgtacc tagacagaag ctcatgtgat gctggaaata taagtgtgaa ccaccatgat 720

ttcagccctg aggccaattc atcggacggt gctgctgctg ctgagaaacc ttacattact 780

ttgctctacc gtcctggtca ctacgacatt ctctacccga agtga 825

<210> 3

<211> 3825

<212> DNA

<213>Npt1 gDNA sequences from Zhejiang spoke 802

<400> 3

atgggcgggg actactacca ctcgtgctgc ggcgaccccg accccgacct ccgcgcgccc 60

gaggggccca agctgccgta cgtcggggac aaggtgagat gttgacgcct ctctctcttt 120

ctctgtctct ctcgctcgct ttgactcatc tgcgctttga ctcatctgcg gtcgatagat 180

ttgttcatgt ggtagaaatg ggtctgaatc gtggtaagac gcccagtgtt gccatgccag 240

tatccgctag ttgtgccagc aggtgaggcg atagatcagt cctgttagtc tagttggatg 300

ctgattgttg gtcatcatta ctgttgtatt ggtgctccat ttctatgtga attgacattt 360

taaggcgtct atacaagcag tggggactag aatttggata acaagtaaca atttcccctt 420

attgcgtcat cttaaaggat aagaggagtt atcagatgct ggattttctc ctttattttt 480

agtcgtggtg cctggaataa tggagattgg ctgaacaagt tcatatcagt tgtgtccatt 540

ttcatcctct ggatggtcag tccttaagta ttgtcaggcg ctattgttgt gagttgtaac 600

tgttgtactt gattttttag cttcgttggt gaactgatgt ttggaggttc atgcagaagt 660

cagaaccatg gggaattaga atttggatga acagacagca attacctttc gtgttctctt 720

ctaggaaaag aagggtttat ctgttatcat ttgctggatg tgctccttac ttaatattta 780

tgcatggaat aatagagatg gccaaacaag tttgctccat agcgtattat gattctagga 840

taaaagtggt gtcatccttt aatcgtcaca cctaggacaa taaatgtgaa ggacgaactt 900

gttgatgcag aataatagcc tatgctagtc aattcagcac agaaactgag gttaaatgtg 960

tcccaaaagt ttcagttaag gttcacacta ggatttacac gaacagaaca aatctgcaaa 1020

tatagtccat atgagaaggt ggagagctac atacacacaa tttcatatga aatggaaaat 1080

gatgttggca ctaaacttgg atttaggtca agtagttaga tatttgatgc ctcaaattca 1140

ttttgttctg ttaattgcaa gcaccttttc acaatggagg acactaatgc attgctatga 1200

ttatctctgt gtttgtacag ttattttagg ctatcgtatg actttcttct gctttcattt 1260

gtgttcatta ttgatatctt ttgaaccttg caggaacctc tctccacttt agccgctgag 1320

tttcagtctg gcagccccat tttacaggag aaaataaagg tccatatgga tttggcagtt 1380

ataatatgta atagacatat tttggttgat ctatcgtatc aatggatggt gcttcaattt 1440

gttcttataa tttcttgctt ggtctgcagt tgcttggtga acagtatgat gctttaagaa 1500

ggacacgagg agatggaaac tgcttttatc gaagctttat gttttcctac ttggtattat 1560

ttttggtctg tttccataca aactttgact attttataag ctgatgatct tatcatttgc 1620

ttctaggaac atatcctaga gacacaagac aaagctgagg ttgagcgcat tctaaaaaaa 1680

attgagcagt gcaagaagac tcttgcagat cttggataca ttgagttcac ctttgaagat 1740

ttcttctctg taagtcttta ttgttacttt gtgtggtcct ccttacttat cctgttcaat 1800

tgctgttttg caacttatgc cagatgtatt ccctctgaat agtatgaaga tctgtccgat 1860

tattttcatg tatgcttgtt tgcatttcct ttttagatgt tcctggaata atttttgtat 1920

gagctagtta taatgagagc ttgtgcattt tcctgtcatg caacaaatta aatactagtg 1980

tctaatcctt gtgcattgtt aataactttg aaaatgatta gccttgaaga ttggtccatt 2040

atatatatgt tcacttgttt cttagttagg atcactcacc agtcaccctt ctgaagttca 2100

taatgtatca cttataagta agctagcaaa acaaaatttg gactgtttgt agccacccag 2160

aacccaaata gatggatttc acattatttt ctactggctt tgggagttat ttgatcgatg 2220

ctagtacaac gttgaaattt gggtagttga gatgcgtttt tcacaaagga ctcctttatt 2280

ggtgcttgat ctacaactgg tgttttactt ttttacaaaa aaatgtaatc tccttgcagt 2340

gcactcaaat tattgcaacc tccttcctta tgttcccacc ctcattattt tcagatattc 2400

attgatcagc tggaaagtgt tctgcaggga catgaatcct ccatagggta aatatcctag 2460

agttatattt gtatccttaa tgcatatgac caataatcat gtattaacaa caaagcaatt 2520

tttgtaattg tttataaagt atggcatgtc catcataaat gttttccttc tgtagtgaat 2580

ctattttgtt ttcctgtatc cttagggccg aagagcttct agaaagaacc agggatcaga 2640

tggtttctga ttatggtttg tacatccaga tatgtgtagt atgcctttct ctgctttgct 2700

ctcattattt aactatgtct tctttagttg tcatgttctt taggtttgtc acctctggtg 2760

aaatccaaag gagggctgag ttcttcgaac cattcatctc tggcttgaca aattcgactg 2820

tggttcaggt tagctccata cttccatttt atgagggttt gtacagtcgt tggggaggta 2880

ttatgaggta caatacctcc aggtaccggg agttaccaca cataagcata aaatgtgtgg 2940

tgcctctcca aggaccacaa gaaatctctt cattatattt gtaatgcaca gagcagagag 3000

tacagacaaa tagacctgca ctctgcattt tcattaagta tttagatgtg agattattct 3060

atgttttatc tctcttgtta gtattttttg ctctgtttta taatggaagt tcattttctt 3120

gggaactgtc attcacaaaa caatgagtta tcgtaccctg ccatttagta gggaatttgg 3180

tggtaaaaaa ccattaactt tttcttcaat tttgtgcctt ctgcacaagg tgggataggg 3240

catatattgt ggaacaaaag agtgcacaat gactaattat ttagtatgca tcacactgga 3300

gtatgatata ctagtggaaa ggttatggca aaataccatg atagtagctt gatagattag 3360

caggtccgta agtatttttc caatgataat gttttattca ttaaactgta gcaggataaa 3420

atctacttat gcaccttttt ttcatgagta gcaaacaatg cattctctgg tttgaaaaac 3480

ttgttcaagt tgcagtgtgt tattccaatc cgtgtttgtg tgacaagcaa ttgctggagt 3540

tactgatcct gagttcaatt caatttgcag ttctgcaagg cttccgtgga gccgatgggc 3600

gaggaaagtg accatgtcca cataattgcc ctatcagatg cgttgggtgt gccaatccgt 3660

gtgatgtacc tagacagaag ctcatgtgat gctggaaata taagtgtgaa ccaccatgat 3720

ttcagccctg aggccaattc atcggacggt gctgctgctg ctgagaaacc ttacattact 3780

ttgctctacc gtcctggtca ctacgacatt ctctacccga agtga 3825

<210> 4

<211> 3824

<212> DNA

<213>Npt1 gDNA sequences from IR66167-27-5-1-6

<400> 4

atgggcgggg actactacca ctcgtgctgc ggcgaccccg accccgacct ccgcgcgccc 60

gaggggccca agctgccgta cgtcggggac aaggtgagat gttgacgcct ctctctctct 120

ctgtctctct cgctcgcttt gactcatctg cgctttgact catctgcggt cgatagattt 180

gttcatgtgg tagaaatggg tctgaatcgt ggtaagacgc ccagtgttgc catgccagta 240

tccgctagtt gtgccagcag gtgaggcgat agatcagtcc tgttagtcta gttggatgct 300

gattgttggt catcattact gttgtattgg tgctccattt ctatgtgaat tgacatttta 360

aggcgtctat acaagcagtg gggactagaa tttggataac aagtaacaat ttccccttat 420

tgcgtcatct taaaggataa gaggagttat cagatgctgg attttctcct ttatttttag 480

tcgtggtgcc tggaataatg gagattggct gaacaagttc atatcagttg tgtccatttt 540

catcctctgg atggtcagtc cttaagtatt gtcaggcgct attgttgtga gttgtaactg 600

ttgtacttga ttttttagct tcgttggtga actgatgttt ggaggttcat gcagaagtca 660

gaaccatggg gaattagaat ttggatgaac agacagcaat tacctttcgt gttctcttct 720

aggaaaagaa gggtttatct gttatcattt gctggatgtg ctccttactt aatatttatg 780

catggaataa tagagatggc caaacaagtt tgctccatag cgtattatga ttctaggata 840

aaagtggtgt catcctttaa tcgtcacacc taggacaata aatgtgaagg acgaacttgt 900

tgatgcagaa taatagccta tgctagtcaa ttcagcacag aaactgaggt taaatgtgtc 960

ccaaaagttt cagttaaggt tcacactagg atttacacga acagaacaaa tctgcaaata 1020

tagtccatat gagaaggtgg agagctacat acacacaatt tcatatgaaa tggaaaatga 1080

tgttggcact aaacttggat ttaggtcaag tagttagata tttgatgcct caaatttatt 1140

ttgttctgtt aattgcaagc accttttcac aatggaggac actaatgcat tgctatgatt 1200

atctctgtgt ttgtacagtt attttaggct atcgtatgac tttcttctgc tttcatttgt 1260

gttcattatt gatatctttt gaaccttgca ggaacctctc tccactttag ccgctgagtt 1320

tcagtctggc agccccattt tacaggagaa aataaaggtc catatggatt tggcagttat 1380

aatatgtaat agacatattt tggttgatct atcgtatcaa tggatggtgc ttcaatttgt 1440

tcttataatt tcttgcttgg tctccagttg cttggtgaac agtatgatgc tttaagaagg 1500

acacgaggag atggaaactg cttttatcga agctttatgt tttcctactt ggtattattt 1560

ttggtctgtt tccatacaaa ctttgactat tttataagct gatgatctta tcatttgctt 1620

ctaggaacat atcctagaga cacaagacaa agctgaggtt gagcgcattc taaaaaaaat 1680

tgagcagtgc aagaagactc ttgcagatct tggatacatt gagttcacct ttgaagattt 1740

cttctctgta agtctttatt gttactttgt gtggtcctcc ttacttatcc tgttcaattt 1800

ctgttttgca acttatgcca gatgtattcc ctctgaatag tatgaagatc tgtccgatta 1860

ttttcatgta tgcttgtttg catttccttt ttagatgttc ctggaataat ttttgtatga 1920

gctagttata atgagagctt gtgcattttc ctgtcatgca acaaattaaa tactagtgtc 1980

taatccttgt gcattgttaa taactttgaa aatgattagc cttgaagatt ggtccattat 2040

atatatgttc acttgtttct tagttaggat cactcaccag tcacccttct gaagttcata 2100

atgtatcact tataagtaag ctagcaaaac aaaatttgga ctgtttgtag ccacccagaa 2160

cccaaataga tggatttcac attattttct actggctttg ggagttattt gatcgatgct 2220

agtacaacgt tgaaattttg ggtagttgag atgcattttt cacaaaggac tcctttattg 2280

gtgcttgatc tacaactggt gttttacttt tttacaaaaa aatgtaatct ccttgcagtg 2340

cactcaaatt attgcaacct ccttccttat gttcccaccc tcattatttt cagatattca 2400

ttgatcagct ggaaagtgtt ctgcagggac atgaatcctc catagggtaa atatcctaga 2460

gttatatttg tatccttaat gcatatgacc aataatcatg tattaacaac aaagcatttt 2520

ttgtaattgt ttataaagta tggcatgtcc atcataaatg ttttccttct gtagtgaatc 2580

tattttgttt tcctgtatcc ttagggccga agagcttcta gaaagaacca gggatcagat 2640

ggtttctgat tatggtttgt acatccagat atgtgtagta tgcctttctc tgctttgctc 2700

tcattattta actatgtctt ctttagttgt catgttcttt aggtttgtca cctctggtga 2760

aatccaaagg agggccgagt tcttcgaacc attcatctct ggcttgacaa attcgactgt 2820

ggttcaggtt agctccatac ttccattgta tgagggtttg tacagttgtt ggggaggtat 2880

tatgaggtac aatacctcca ggtaccggga gttaccacac ataagcataa aatgtgtggt 2940

gcctctccaa ggaccacaag aaatctcttc attatatttg taatgcacag agcagagagt 3000

acagacaaat agacctgcac tctgcatttt cattaagtat ttagatgtga gattattcta 3060

tgttttatct ctcttgttag tattttttgc tctgttttat aatggaagtt cattttcttg 3120

ggaactgtca ttcacaaaac aatgagttat cgtaccctgc catttagtag ggaatttggt 3180

ggtaaaaaac cattaacttt ttcttcaatt ttgtgccttc tgcacaaggt gggatagggc 3240

atatattgtg gaacaaaaga gtgcacaatg actaattatt tagtatgcat cacactggag 3300

tatgatatac tagtggaaag gttatggcaa aataccatga tagtagcttg atagattagc 3360

aggtccgtaa gtatttttcc aatgataatg ttttattcat taaactgtag caggataaaa 3420

tctacttatg cacctttttt tcatgagtag caaacaatgc attctctggt ttgaaaaact 3480

tgttcaagtt gcagtgtgtt attccaatcc gtgtttgtgt gacaagcaat tgctggagtt 3540

actgatcctg agttcaattc aatttgcagt tctgcaaggc ttccgtggag ccgatgggcg 3600

aggaaagtga ccatgtccac ataattgccc tatcagatgc gttgggtgtg ccaatccgtg 3660

tgatgtacct agacagaagc tcatgtgatg ctggaaatat aagtgtgaac caccatgatt 3720

tcagccctga ggccaattca tcggacggtg ctgctgctgc tgagaaacct tacattactt 3780

tgctctaccg tcctggtcac tacgacattc tctacccgaa gtga 3824

<210> 5

<211> 2462

<212> DNA

<213>The kb promoter sequences of NPT1 2.5 from Zhejiang spoke 802

<400> 5

gagttgaagt tgttgctgct gtcataagta ctatctgcta aatgggcaca ctcctagcat 60

tattagaact gagaaatatc ccaagcaatg aaagcgacaa aaaagtaccc gtttgaagac 120

atgattgaca tggtcacatc aaacaccgga catcaacatc taaatgtaca taacaaggcc 180

aaaataattt tcgatgctgg ttggtgctac caagtcccac gtatgatact taagaatcaa 240

tcatgaatat tacaaatcaa gtcaaactac gttatgtatt gaactcttat aattactgca 300

acatatcaca ctggaatttc ctatggtaat tcctcgccag ccttatccta cccatccctt 360

gcagtatatt aagagcatca acaacaaaca tgattcaaga caacttttat taacactgaa 420

caacataaat tgggaacaaa acaaaccact tggaggcatg attaggataa tcggtattaa 480

agaactggac atcacaattc acaactagat gttgaaataa tacctgtctc ttctttggct 540

catggcaggt gtcagtgaaa tatactgatg ctccaagaga gctggaagca ccgtttccac 600

gtaatcaaaa tgtccttttc gtttgctgca atcaacctta aagggctctt ttgatgctat 660

ctcttcaggc atgtccttta caacttccac ataacctctg gtttgctcaa tgaagtaatc 720

aacatcgaaa acgtctgcaa atccactgac agagaatacc aataagtgat gaactacctt 780

ttgaacagaa ataaactgca taactacaag tagcacagtc gttcatcttg tagagtgatt 840

ctcataccta gattcattcc agtaggcagc aacctcaaac ttgggcagaa ccattgttgc 900

gttgagaagg cgcgcaaccg caattccatc acatagctga agaaacattc ggaagaataa 960

ttacaaccag gagtaacata ataacatagc cagttgaaat cacattcgcc ttgcaatgtg 1020

aaaattttca taaataatct gaaaatttag ttatgccact atatatcatg caacctgcct 1080

ccacgacatt ttaatcatgg agtagaagat aaaacatatg atccccctca ttgaccctac 1140

tatcttacta cttgtgcatg gccgaacgat ctaacagcga aatccagaaa gccaacactc 1200

atttgatccc actaacaacg gaagagagaa acgctagccg agatcgctta acgtacatcg 1260

cgtcgcagct ggttgagccc gccgtagcag tcgatccgga tgtacccatt cctcctcgac 1320

ggagctgcag aagaagagga ggttcaaaac cgcaatcacc accacagtct caagcagaga 1380

tgtccactac ccggatcctt aaacccaaac cacaaatcac ggcgaggtct cacccggcat 1440

tgccgcccgc caccacccgc acgaccgcca ctccgccacc cgccgctgcg cccatatgac 1500

ccgcgacccc gacgccgacg gcgactcctc cctaaagacc aaaagcgagt aagcgagatc 1560

cgtaagcttc tggaacaatc tcgagcatca gctgcaagag gtgaggctgg gccgcgtacc 1620

tggaggtggg aagagtgaag aagaaaggcg gagaggaggg tggagagagg aggaagtaga 1680

gcgcgggggc gaggaagatg accggtagga ggatgcggac gcggctgcgc gcccaccacg 1740

ccgccggcga cgccgacgac gacatcgcct cgccgcgaga agcactggat ctgatcggcc 1800

gccgcctcca cgccggagtg gagagcgtat ataagctcgt cagaatgtgg gcccgtggct 1860

atgtgggccc accatgtcat cgacgcttat caagatcgag cggtggcgtg aggaaaccgg 1920

taggggtggg ggggctaacc aatcggaaac gcgtaataac tcacccgcgg ttcactttct 1980

ccttatgaca cgtgggccca tctcttcctg gacccacctg tcagttaccc ttacggcctc 2040

cactctgagg atctaaacgt aaaaacgaat ttatcggagg gcttatccgc gaggggaaaa 2100

aaacgcgcac ttatttctcg ccttcgccga gatctcggaa gagaagaaca cgcaccgcgg 2160

ggagagggga gagaagcgga aagctccacc gaatcgaagc ccccacacac gcgaagctgg 2220

cgcgggaggc ggccgacgcg agcgcccgga agcgcaaggc ggcggacggc ggcggggagg 2280

gcgacgccgc ggcgacggtc ccggaggagg cggtgatggg ggaggcggcg gcggcagccg 2340

cggcccccga gccggtcgtc gaggggggag gagggggggg ggaggggttg aatcctaacc 2400

ctagcggcgg tgggggagga ggtggtggag ggtgctcgga ctccgtgtcg gtcgagctct 2460

cg 2462

<210> 6

<211> 2462

<212> DNA

<213>The kb promoter sequences of npt1 2.5 from IR66167-27-5-1-6

<400> 6

gagttgaagt tgttgctgct gtcataagta ctatctgcta aatgggcaca ctcctagcat 60

tattagaact gagaaatatc ccaagcaatg aaagcgacaa aaaagtaccc gtttgaagac 120

atgattgaca tggtcacatc aaacaccgga catcaacatc taaatgtaca taacaaggcc 180

aaaataattt tcgatgctgg ttggtgctac caagtcccac gtatgatact taagaatcaa 240

tcatgaatat tacaaatcaa gtcaaactac gttatgtatt gaactcttat aattactgca 300

acatatcaca ctggaatttc ctatggtaat tcctcgccag ccttatccta cccatccctt 360

gcagtatatt aagagcatca acaacaaaca tgattcaaga caacttttat taacactgaa 420

caacataaat tgggaacaaa acaaaccact tggaggcatg attaggataa tcggtattaa 480

agaactggac atcacaattc acaactagat gttgaaataa tacctgtctc ttctttggct 540

catggcaggt gtcagtgaaa tatactgatg ctccaagaga gctggaagca ccgtttccac 600

gtaatcaaaa tgtccttttc gtttgctgca atcaacctta aagggctctt ttgatgctat 660

ctcttcaggc atgtccttca caacttccac ataacctctg gtttgctcaa tgaagtaatc 720

aacatcgaaa acgtctgcaa atccactgac agagaatacc aataagtgat gaactacctt 780

ttgaacagaa ataaactgca taactacaag tagcacagtc gttcatcttg tagagtgatt 840

ctcataccta gattcattcc agtaggcagc aacctcaaac ttgggcagaa ccattgttgc 900

gttgagaagg cgcgcaaccg caattccatc acatagctga agaaacattc ggaagaataa 960

ttacaaccag gagtaacata ataacatagc cagttgaaat cacattcgcc ttgcaatgtg 1020

aaaattttca taaataatct gaaaatttag ttatgccact atatatcatg caacctgcct 1080

ccacgacatt ttaatcatgg agtagaagat aaaacatatg atccccctca ttgaccctac 1140

tatcttacta cttgtgcatg gccgaacgat ctaacagcga aatccagaaa gccaacactc 1200

atttgatccc actaacaacg gaagagagaa acgctagccg agatcgctta acgtacatcg 1260

cgtcgcagct ggttgagccc gccgtagcag tcgatccgga tgtacccatt cctcctcgac 1320

ggagctgcag aagaagagga ggttcaaaac cgcaatcacc accacagtct caagcagaga 1380

tgtccactac ccggatcctt aaacccaaac cacaaatcac ggcgaggtct cacccggcat 1440

tgccgcccgc caccacccgc acgaccgcca ctccgccacc cgccgctgcg cccatatgac 1500

ccgcgacccc gacgccgacg gcgactcctc cctaaagacc aaaagcgagt aagcgagatc 1560

cgtaagcttc tggaacaatc tcgagcatca gctgcaagag gtgaggctgg gccgcgtacc 1620

tggaggtggg aagagtgaag aagaaaggcg gagaggaggg tggagagagg aggaagtaga 1680

gcgcgggggc gaggaagatg accggtagga ggatgcggac gcggctgcgc gcccaccacg 1740

ccgccggcga cgccgacgac gacatcgcct cgccgcgaga agcactggat ctgatcggcc 1800

gccgcctcca cgccggagtg gagagcatat ataagctcgt cagaatgtgg gcccgtggct 1860

atgtgggccc accatgtcat cgacgcttat caagatcgag cggtggcgtg aggaaaccgg 1920

taggggtggg ggggctaacc aatcggaaac gcgtaataac tcacccgcgg ttcactttct 1980

ccttatgaca cgtgggccca tctcttcctg gacccacctg tcagttaccc ttacggcctc 2040

cactctgagg atctaaacgt aaaaacgaat ttatcggagg gcttatccgc gaggggaaaa 2100

aaacgcgcac ttatttctcg ccttcgccga gatctcggaa gagaagaaca cgcaccgcgg 2160

ggagagggga gagaagcgga aagctccacc gaatcgaagc ccccacacac gcgaagctgg 2220

cgcgggaggc ggccgacgcg agcgcccgga agcgcaaggc ggcggacggc ggcggggagg 2280

gcgacgccgc ggcgacggtc ccggaggagg cggtgatggg ggaggcggcg gcggcagccg 2340

cggcccccga gccggtcgtc gaggggggag gagggggggg ggaggggttg aatcctaacc 2400

ctagcggcgg tgggggagga ggtggtggag ggtgctcgga ctccgtgtcg gtcgagctct 2460

cg 2462

<210> 7

<211> 840

<212> DNA

<213>Uralensis Fisch TuNPT1 cDNA sequences

<400> 7

atgggcgggg actactacca cgcctgctgc ggcgacccgg accccgacca caagcccgag 60

ggaccccagg tgccgtacat cggtaacaag gaacctctct ccgccttagc agcagagttc 120

cagtctggca gccccatttt acaggagaaa ataaagttgc ttggtgaaca atatgatgct 180

ttaaggcgaa cacgaggaga tggaaactgc ttttatcgaa gctttatgtt ctcctacctg 240

gaacatatcc tagagacaca agatagagct gaggttgagc gcatcctaaa aaacattgaa 300

cagtgcaaga tgacactttc aggtcttgga tacattgaat tcacttttga agacttcttc 360

tctatgttca ttgaggagct gcaaaatgtt ctgcagggac acgaaacttc tattgggcct 420

gaagaacttc tagaaagaac cagggatcaa acgacttctg attatgttgt catgttcttt 480

aggtttgtta cctctggtga aattcaaagg agggctgagt tctttgaacc atttatttct 540

ggcttgacaa attcgaccgt ggctcagttt tgcaagtctt ctgtggagcc aatgggcgag 600

gaaagcgacc atgtgcacat tattgctctg tcagatgcgt taggggtgcc aatccgcgtg 660

atgtacctag accgaagctc ctgtgacaca ggcaatctaa gtgtgaacca ccatgatttc 720

attcctgcag ccaattcctc tgaaggtgat gctgcaatgg gattaaatcc ggctgaggag 780

aaaccttaca ttactctgct ctaccggcct ggtcactatg atattctcta cccaaagtga 840

<210> 8

<211> 840

<212> DNA

<213>Barley HvNPT1 cDNA sequences

<400> 8

atgggcgggg actactacca cgcctgctgc ggcgaccccg accccgaccc caagcccgag 60

ggaccccagg tgccgtacat cggtaacaag gaacctctct ccgccttagc agcagagttc 120

cagtctggca gccccatttt acaggagaaa ataaagttgc ttggtgaaca atatgatgct 180

ttaagacgga cacgaggaga tggaaactgc ttttatcgaa gctttatgtt ctcctacctg 240

gaacatatcc ttgagacaca agacagagct gaggttgagc gcatcctaaa aaacattgaa 300

caatgcaaga agacactttc aggtcttgga tacattgagt tcacttttga ggacttcttc 360

tctatgttca ttgaggagct gcaaaatgtt ctgcagggac acggaacttc tattgggcct 420

gaagaacttc tagaaagaac cagggatcag acgacttctg attatgttgt catgttcttt 480

agatttgtta cctctggtga aattcaaagg agggctgagt tctttgaacc atttatttct 540

ggcttgacaa attcgaccgt ggttcagttt tgcaagtctt ctgtggagcc aatgggcgag 600

gaaagtgacc atgtgcacat tattgctctg tcagatgcgt taggggtgcc aatccgcgtg 660

atgtacctag accgaagctc ttgtgacaca ggcaatctaa gtgtgaacca ccatgatttc 720

atccctgcag ccaattcctc tgaaggtgat gctgcaatgg gattaaatcc tgctgatgag 780

aaaccttaca ttactctgct ctaccggcct ggtcactatg acattctcta cccgaagtga 840

<210> 9

<211> 891

<212> DNA

<213>Corn ZmNPT1 cDNA sequences

<400> 9

atgggcgacg ttccacaggc gccgcacgct gcgggaggtg gagaagagtg ggcggggccg 60

gaccctaacc ctagcccgag cctcggcggc tgctcggacc ccgtgtcggt ggagctctcc 120

atgggcgggg actactaccg cgcctgctgc ggcgagcccg atcccgacat ccccgagggg 180

cccaagctgc cgtgcgttgg ggacaaggaa cctctctcct ctttagcagc tgagtttcag 240

tctggcagcc ccattttaca agagaaaatt aagttgcttg gcgagcaata tggtgcttta 300

agacgtacac gtggagatgg aaactgcttt tatcgaagct ttatgttttc ctacctggaa 360

cacatcctag agacacaaga caaagctgag gctgatcgca tcatggtaaa aattgaggaa 420

tgcaagaaaa cactcctctc tcttggatat attgagttca cttttgagga cttcttttcg 480

atattcattg aactgctgga aagtgttctg cagggacatg aaactcctat agggtttgtc 540

acttctggtg aaattcaaag gaggtctgac ttctttgaac cgttcatatc tggcttgaca 600

aattcaaccg tggttcagtt ctgcaaggct tctgtggaac ctatgggtga ggaaagtgac 660

catgtgcaca taattgccct atcagatgca ctaggcgtac caatccgtgt tatgtaccta 720

gaccgaagct cgtgtgacac tggcaacctg agcgtgaatc accacgattt catcccgtcg 780

gccaatgatt cggagggtga tgcggccacg acacctgctc ctgccacaga gaaaccgtac 840

atcactttgc tctaccgtcc tggccactac gatattctct acccaaagtg a 891

<210> 10

<211> 909

<212> DNA

<213>Sorghum SbNPT1 cDNA sequences

<400> 10

atgggcgacg tgccccaggc gccgcacgcc gcggaaggag gaggaggagg actggaggag 60

ggggcggtgc ccgaccctaa ccctagcccg agcctgagcc tcggcggctg ctcggacccc 120

gtgtcgctgg agctctccat gggcggggac tactaccgcg cctgctgcgg cgaccccgac 180

cccgacatcc ccgaggggcc caagctgccg tgcgttgggg aaaaggaacc tctctcctct 240

ttagcagccg agtttcagtc tggcagcccc attttacaag agaaaattaa gttgcttggc 300

gaacaatatg gtgctttaag acggacacgt ggagatggaa actgctttta tcgaagcttt 360

atgttctcct acttggaaca catcctagag acacaagaca aagctgaggc tgatcgcatc 420

atggtaaaaa ttgaggattg caagaagacg ctcctgtctc ttggatatat tgagttcact 480

tttgaggatt tctttgcgat attcattgat atgctggaaa gtgttctgca gggacatgaa 540

actcctatag ggtttgtcac ttctggtgaa attcaaagga ggtctgactt ctttgaacca 600

ttcatatctg gcttgacaaa ttcaactgtg gttcagttct gcaaggcttc tgtggaacct 660

atgggtgagg aaagtgacca tgttcacata attgccctat cggatgcact aggtgtacct 720

atccgtgtta tgtacctaga ccgaagctcg tgtgatactg gcaatctgag tgtgaatcac 780

catgatttca tcccttcgtc caatgcttct gagggtgatg ctgcgatgac atctactcct 840

gacgctgaga aaccttacat cactttgctc taccgtcctg gtcactatga tattctctac 900

ccaaagtga 909

<210> 11

<211> 906

<212> DNA

<213>Soybean GmNPT1 cDNA sequences

<400> 11

atgcagagta aagaagctgt tgtggaagat ggggaaataa agagtgtgac tgctgtaggg 60

tctgaaattg atgggtggac caattttggg gacgatgaca taatgcagca gcagtataca 120

attcagtctg aagaggctaa gaaagttcca tctgtgggcg acaaggaacc actgagtagc 180

ttagctgctg aatataaatc aggcagtcct atcttgctgg agaaaataaa ggtgcttgat 240

gagcaatacg ctgccattcg tcgtactcga ggagatggaa actgcttctt tcgaagcttt 300

atgttttcat atcttgagca tgttatgaaa tgtcaagacc aagcagaagt tgatcgtatc 360

caagccaatg ttgaaaaaag tagaaaagca ctgcagacct tgggttatgc agacttgact 420

tttgaagatt tttttgcgtt attccttgag cagctggaat ctgttattca agggaaagag 480

acttccataa gtcatgaaga gcttgttctt agaagccgag atcagtcagt atctgattat 540

gtcgttatgt tcttcagatt tgttacctct gccgcaatac aaaagcgcac agaatttttt 600

gaaccattca tactaggctt aactaataca acggtcgagc agttttgcaa atcatctgtt 660

gaaccaatgg gtgaagagag cgaccatgtg cacattactg ccctttcaga tgcattgggc 720

attccagtcc gtgttgtgta ccttgaccgc agctcaagtg atactggtgg tgtcagtgta 780

aatcatcatg atttcatgcc agtggctggt gatctcccaa atgctagttg cagctctgaa 840

aagaacattc ctttcatcac actactatat cgtcctggtc actatgacat cctctatcca 900

aaatga 906

<210> 12

<211> 903

<212> DNA

<213>Rape BnNPT1 cDNA sequences

<400> 12

atgcagaatc agaatgatac ggtgaaggat gatgcggagc tcgctgcttc catctcggct 60

gaacaatggg gatgctgttc agtggaggaa ccatcttttc aagatgatga agctgctaaa 120

gttccttatg ttggtgataa ggagcctatg tctagtttag ctgcagagta ccaagcaggg 180

agccccattt tgcttgagaa gataaaggta ctggacagtc aatatgttgc aatcaggcga 240

acaagaggag atggaaactg cttcttccga agttttatgt tctcttacct tgagcatatt 300

ttggaatcac aagatggtgc tgaagttgac cgtatcaagc tcaatgttga aaaatgtaga 360

aagaatctgc agaacttagg ctacacagat ttcacatttg aggacttctt tgcgttgttc 420

cttgagcaac tagatgacat cctccaagga ggcgaagagt ctataagcta tgatgagctg 480

gttaacagaa gtagagatca gtctgtttcc gactacattg tgatgttctt caggtttgtt 540

actgctggtg aaataaaaac gcgtgctgag ttcttcgagc cttttataac aggattatct 600

aataccacag tggatcagtt ttgcaagaca tcagttgaac cgatggggga agagagtgac 660

catattcaca taacagcttt gtcggacgcg cttggtgttg caatccgggt tgtgtatctt 720

gaccgtagct catgtgatac tggaggtggt gtcactgtga accatcacga ctttgttccc 780

gttggcagtg gcactaatga gaaagaagaa gcttcttctg ctgctccctt tataacattg 840

ctctatcgtc caggccatta cgatatcctc taccccaagg tattggagaa tgtggaaaaa 900

tga 903

<210> 13

<211> 912

<212> DNA

<213>Cotton GhNPT1 cDNA sequences

<400> 13

atgcaaaatc aggatggagc ggtagctgat agggaaaagg aatctgctgt atccattccc 60

atttctgaag ttgatgactg ggcaaatttc gcagatgatg atatcatgca gcaacaatct 120

gccattcatg ctgaggaggc caagaaaatt ccatttgttg gtgacaagga accactctct 180

atgttggcag ctgaatatga atcaggaagt cctatattgc tagaaaaaat aaaggtgctt 240

gatcagcaat atgcagccat tagaagaacg cgaggagatg gaaactgctt tttccggagt 300

tttatgtttt cataccttga acatattttg gaatctcaag accgtgctga ggttgatcgt 360

atcaaaggca atgttgaaga atgtagaaag ccacttcagc gcttagccca cacagatttt 420

acatttgagg actttttttc gttattcctt gagcagctgg aatgcgttct tcaaggaaat 480

gaagattcca taagtcagga tgaattaata ctaaggagtc gagatcagtc aatttccgac 540

tatgttgtaa tgttcttcag gtttgttacc tctggtgaaa tacgaaagcg atcagagttt 600

ttcgaaccct ttattttggg attaacaaat gcaacagtgg aacagttttg caagtcatcc 660

gtagagccaa tgggtgaaga gagtgatcat gtgcacatta ctgccctttc agacgctctg 720

ggcgtgccaa ttcgtgttgt gtaccttgat cggagctctt gtgacattgg tggtgttagt 780

gtaaaccatc atgattttct tcctacttcg ggcgataagt caaatgctaa aggtggtagc 840

actgtccctg tcaagccttt tattaccttg ctatatcgtc caggccacta tgacattctc 900

tatccaaagt ga 912

<210> 14

<211> 906

<212> DNA

<213>Tomato SlNPT1 cDNA sequences

<400> 14

atgcaggatc aggatgtgga tgtggatgtg gctgatgcag caaaagaaac gtttacatct 60

tctgaaactg atgactggaa aaaatacaag gatgatgata ttatgcaaca gcactctgcc 120

atacaagctg aacaagctgt aaaagttcca tttcttggtg ataaggaacc tttgtcctca 180

ttagaagctg aataccatct gggaaattca attgtgcttg agaaaataaa ggtgctgagt 240

gaacagtatg ctgccattag aagaacacgt ggagatggga actgcttttt ccgcagtttc 300

atgtttggtt accttgagca cattctggaa tcacaagatc acaatgaagt tcaacacatt 360

aaatctaata ttgaggaatg caagaagaca cttcaaagtt tgggctatgc agaattcacc 420

tttgaagact tttttgcatt attcctcgag caactcgata gtgttcttca aggtagcgaa 480

gattccataa gtcatgatga actcctgtgc agaagtcgtg atccatcaat ttctgactat 540

gttgttatgt tcttcagatt tgtgacatca ggtgaaataa gaaagcggtc cgagtttttc 600

gaaccattta ttctaggact aacaaatacc tcagtggagc agttttgcaa gtcagcagtg 660

gaacccatgg gtgaagagag tgatcatgtg caaattacag ccctatcaga tgcattgggt 720

gtaccgatcc gtgttgtata tcttgaccga agctcatgtg agaacaacag catcaatgta 780

aatcaccatg actttattcc tactagcagg gaggtcggga atagtgatgt ttccaagacc 840

acaaatcgtc catctatcac cctgctgtat cgcccagggc attatgacat tctgtacccc 900

aagtga 906

<210> 15

<211> 274

<212> PRT

<213>Rice Os OTUB1 albumen

<400> 15

Met Gly Gly Asp Tyr Tyr His Ser Cys Cys Gly Asp Pro Asp Pro Asp

1 5 10 15

Leu Arg Ala Pro Glu Gly Pro Lys Leu Pro Tyr Val Gly Asp Lys Glu

20 25 30

Pro Leu Ser Thr Leu Ala Ala Glu Phe Gln Ser Gly Ser Pro Ile Leu

35 40 45

Gln Glu Lys Ile Lys Leu Leu Gly Glu Gln Tyr Asp Ala Leu Arg Arg

50 55 60

Thr Arg Gly Asp Gly Asn Cys Phe Tyr Arg Ser Phe Met Phe Ser Tyr

65 70 75 80

Leu Glu His Ile Leu Glu Thr Gln Asp Lys Ala Glu Val Glu Arg Ile

85 90 95

Leu Lys Lys Ile Glu Gln Cys Lys Lys Thr Leu Ala Asp Leu Gly Tyr

100 105 110

Ile Glu Phe Thr Phe Glu Asp Phe Phe Ser Ile Phe Ile Asp Gln Leu

115 120 125

Glu Ser Val Leu Gln Gly His Glu Ser Ser Ile Gly Ala Glu Glu Leu

130 135 140

Leu Glu Arg Thr Arg Asp Gln Met Val Ser Asp Tyr Val Val Met Phe

145 150 155 160

Phe Arg Phe Val Thr Ser Gly Glu Ile Gln Arg Arg Ala Glu Phe Phe

165 170 175

Glu Pro Phe Ile Ser Gly Leu Thr Asn Ser Thr Val Val Gln Phe Cys

180 185 190

Lys Ala Ser Val Glu Pro Met Gly Glu Glu Ser Asp His Val His Ile

195 200 205

Ile Ala Leu Ser Asp Ala Leu Gly Val Pro Ile Arg Val Met Tyr Leu

210 215 220

Asp Arg Ser Ser Cys Asp Ala Gly Asn Ile Ser Val Asn His His Asp

225 230 235 240

Phe Ser Pro Glu Ala Asn Ser Ser Asp Gly Ala Ala Ala Ala Glu Lys

245 250 255

Pro Tyr Ile Thr Leu Leu Tyr Arg Pro Gly His Tyr Asp Ile Leu Tyr

260 265 270

Pro Lys

<210> 16

<211> 279

<212> PRT

<213>Uralensis Fisch TuOTUB1 albumen

<400> 16

Met Gly Gly Asp Tyr Tyr His Ala Cys Cys Gly Asp Pro Asp Pro Asp

1 5 10 15

His Lys Pro Glu Gly Pro Gln Val Pro Tyr Ile Gly Asn Lys Glu Pro

20 25 30

Leu Ser Ala Leu Ala Ala Glu Phe Gln Ser Gly Ser Pro Ile Leu Gln

35 40 45

Glu Lys Ile Lys Leu Leu Gly Glu Gln Tyr Asp Ala Leu Arg Arg Thr

50 55 60

Arg Gly Asp Gly Asn Cys Phe Tyr Arg Ser Phe Met Phe Ser Tyr Leu

65 70 75 80

Glu His Ile Leu Glu Thr Gln Asp Arg Ala Glu Val Glu Arg Ile Leu

85 90 95

Lys Asn Ile Glu Gln Cys Lys Met Thr Leu Ser Gly Leu Gly Tyr Ile

100 105 110

Glu Phe Thr Phe Glu Asp Phe Phe Ser Met Phe Ile Glu Glu Leu Gln

115 120 125

Asn Val Leu Gln Gly His Glu Thr Ser Ile Gly Pro Glu Glu Leu Leu

130 135 140

Glu Arg Thr Arg Asp Gln Thr Thr Ser Asp Tyr Val Val Met Phe Phe

145 150 155 160

Arg Phe Val Thr Ser Gly Glu Ile Gln Arg Arg Ala Glu Phe Phe Glu

165 170 175

Pro Phe Ile Ser Gly Leu Thr Asn Ser Thr Val Ala Gln Phe Cys Lys

180 185 190

Ser Ser Val Glu Pro Met Gly Glu Glu Ser Asp His Val His Ile Ile

195 200 205

Ala Leu Ser Asp Ala Leu Gly Val Pro Ile Arg Val Met Tyr Leu Asp

210 215 220

Arg Ser Ser Cys Asp Thr Gly Asn Leu Ser Val Asn His His Asp Phe

225 230 235 240

Ile Pro Ala Ala Asn Ser Ser Glu Gly Asp Ala Ala Met Gly Leu Asn

245 250 255

Pro Ala Glu Glu Lys Pro Tyr Ile Thr Leu Leu Tyr Arg Pro Gly His

260 265 270

Tyr Asp Ile Leu Tyr Pro Lys

275

<210> 17

<211> 279

<212> PRT

<213>Barley HvOTUB1 albumen

<400> 17

Met Gly Gly Asp Tyr Tyr His Ala Cys Cys Gly Asp Pro Asp Pro Asp

1 5 10 15

Pro Lys Pro Glu Gly Pro Gln Val Pro Tyr Ile Gly Asn Lys Glu Pro

20 25 30

Leu Ser Ala Leu Ala Ala Glu Phe Gln Ser Gly Ser Pro Ile Leu Gln

35 40 45

Glu Lys Ile Lys Leu Leu Gly Glu Gln Tyr Asp Ala Leu Arg Arg Thr

50 55 60

Arg Gly Asp Gly Asn Cys Phe Tyr Arg Ser Phe Met Phe Ser Tyr Leu

65 70 75 80

Glu His Ile Leu Glu Thr Gln Asp Arg Ala Glu Val Glu Arg Ile Leu

85 90 95

Lys Asn Ile Glu Gln Cys Lys Lys Thr Leu Ser Gly Leu Gly Tyr Ile

100 105 110

Glu Phe Thr Phe Glu Asp Phe Phe Ser Met Phe Ile Glu Glu Leu Gln

115 120 125

Asn Val Leu Gln Gly His Gly Thr Ser Ile Gly Pro Glu Glu Leu Leu

130 135 140

Glu Arg Thr Arg Asp Gln Thr Thr Ser Asp Tyr Val Val Met Phe Phe

145 150 155 160

Arg Phe Val Thr Ser Gly Glu Ile Gln Arg Arg Ala Glu Phe Phe Glu

165 170 175

Pro Phe Ile Ser Gly Leu Thr Asn Ser Thr Val Val Gln Phe Cys Lys

180 185 190

Ser Ser Val Glu Pro Met Gly Glu Glu Ser Asp His Val His Ile Ile

195 200 205

Ala Leu Ser Asp Ala Leu Gly Val Pro Ile Arg Val Met Tyr Leu Asp

210 215 220

Arg Ser Ser Cys Asp Thr Gly Asn Leu Ser Val Asn His His Asp Phe

225 230 235 240

Ile Pro Ala Ala Asn Ser Ser Glu Gly Asp Ala Ala Met Gly Leu Asn

245 250 255

Pro Ala Asp Glu Lys Pro Tyr Ile Thr Leu Leu Tyr Arg Pro Gly His

260 265 270

Tyr Asp Ile Leu Tyr Pro Lys

275

<210> 18

<211> 296

<212> PRT

<213>Corn ZmOTUB1 albumen

<400> 18

Met Gly Asp Val Pro Gln Ala Pro His Ala Ala Gly Gly Gly Glu Glu

1 5 10 15

Trp Ala Gly Pro Asp Pro Asn Pro Ser Pro Ser Leu Gly Gly Cys Ser

20 25 30

Asp Pro Val Ser Val Glu Leu Ser Met Gly Gly Asp Tyr Tyr Arg Ala

35 40 45

Cys Cys Gly Glu Pro Asp Pro Asp Ile Pro Glu Gly Pro Lys Leu Pro

50 55 60

Cys Val Gly Asp Lys Glu Pro Leu Ser Ser Leu Ala Ala Glu Phe Gln

65 70 75 80

Ser Gly Ser Pro Ile Leu Gln Glu Lys Ile Lys Leu Leu Gly Glu Gln

85 90 95

Tyr Gly Ala Leu Arg Arg Thr Arg Gly Asp Gly Asn Cys Phe Tyr Arg

100 105 110

Ser Phe Met Phe Ser Tyr Leu Glu His Ile Leu Glu Thr Gln Asp Lys

115 120 125

Ala Glu Ala Asp Arg Ile Met Val Lys Ile Glu Glu Cys Lys Lys Thr

130 135 140

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

145 150 155 160

Ile Phe Ile Glu Leu Leu Glu Ser Val Leu Gln Gly His Glu Thr Pro

165 170 175

Ile Gly Phe Val Thr Ser Gly Glu Ile Gln Arg Arg Ser Asp Phe Phe

180 185 190

Glu Pro Phe Ile Ser Gly Leu Thr Asn Ser Thr Val Val Gln Phe Cys

195 200 205

Lys Ala Ser Val Glu Pro Met Gly Glu Glu Ser Asp His Val His Ile

210 215 220

Ile Ala Leu Ser Asp Ala Leu Gly Val Pro Ile Arg Val Met Tyr Leu

225 230 235 240

Asp Arg Ser Ser Cys Asp Thr Gly Asn Leu Ser Val Asn His His Asp

245 250 255

Phe Ile Pro Ser Ala Asn Asp Ser Glu Gly Asp Ala Ala Thr Thr Pro

260 265 270

Ala Pro Ala Thr Glu Lys Pro Tyr Ile Thr Leu Leu Tyr Arg Pro Gly

275 280 285

His Tyr Asp Ile Leu Tyr Pro Lys

290 295

<210> 19

<211> 302

<212> PRT

<213>Sorghum SbOTUB1 albumen

<400> 19

Met Gly Asp Val Pro Gln Ala Pro His Ala Ala Glu Gly Gly Gly Gly

1 5 10 15

Gly Leu Glu Glu Gly Ala Val Pro Asp Pro Asn Pro Ser Pro Ser Leu

20 25 30

Ser Leu Gly Gly Cys Ser Asp Pro Val Ser Leu Glu Leu Ser Met Gly

35 40 45

Gly Asp Tyr Tyr Arg Ala Cys Cys Gly Asp Pro Asp Pro Asp Ile Pro

50 55 60

Glu Gly Pro Lys Leu Pro Cys Val Gly Glu Lys Glu Pro Leu Ser Ser

65 70 75 80

Leu Ala Ala Glu Phe Gln Ser Gly Ser Pro Ile Leu Gln Glu Lys Ile

85 90 95

Lys Leu Leu Gly Glu Gln Tyr Gly Ala Leu Arg Arg Thr Arg Gly Asp

100 105 110

Gly Asn Cys Phe Tyr Arg Ser Phe Met Phe Ser Tyr Leu Glu His Ile

115 120 125

Leu Glu Thr Gln Asp Lys Ala Glu Ala Asp Arg Ile Met Val Lys Ile

130 135 140

Glu Asp Cys Lys Lys Thr Leu Leu Ser Leu Gly Tyr Ile Glu Phe Thr

145 150 155 160

Phe Glu Asp Phe Phe Ala Ile Phe Ile Asp Met Leu Glu Ser Val Leu

165 170 175

Gln Gly His Glu Thr Pro Ile Gly Phe Val Thr Ser Gly Glu Ile Gln

180 185 190

Arg Arg Ser Asp Phe Phe Glu Pro Phe Ile Ser Gly Leu Thr Asn Ser

195 200 205

Thr Val Val Gln Phe Cys Lys Ala Ser Val Glu Pro Met Gly Glu Glu

210 215 220

Ser Asp His Val His Ile Ile Ala Leu Ser Asp Ala Leu Gly Val Pro

225 230 235 240

Ile Arg Val Met Tyr Leu Asp Arg Ser Ser Cys Asp Thr Gly Asn Leu

245 250 255

Ser Val Asn His His Asp Phe Ile Pro Ser Ser Asn Ala Ser Glu Gly

260 265 270

Asp Ala Ala Met Thr Ser Thr Pro Asp Ala Glu Lys Pro Tyr Ile Thr

275 280 285

Leu Leu Tyr Arg Pro Gly His Tyr Asp Ile Leu Tyr Pro Lys

290 295 300

<210> 20

<211> 301

<212> PRT

<213>Soybean GmOTUB1 albumen

<400> 20

Met Gln Ser Lys Glu Ala Val Val Glu Asp Gly Glu Ile Lys Ser Val

1 5 10 15

Thr Ala Val Gly Ser Glu Ile Asp Gly Trp Thr Asn Phe Gly Asp Asp

20 25 30

Asp Ile Met Gln Gln Gln Tyr Thr Ile Gln Ser Glu Glu Ala Lys Lys

35 40 45

Val Pro Ser Val Gly Asp Lys Glu Pro Leu Ser Ser Leu Ala Ala Glu

50 55 60

Tyr Lys Ser Gly Ser Pro Ile Leu Leu Glu Lys Ile Lys Val Leu Asp

65 70 75 80

Glu Gln Tyr Ala Ala Ile Arg Arg Thr Arg Gly Asp Gly Asn Cys Phe

85 90 95

Phe Arg Ser Phe Met Phe Ser Tyr Leu Glu His Val Met Lys Cys Gln

100 105 110

Asp Gln Ala Glu Val Asp Arg Ile Gln Ala Asn Val Glu Lys Ser Arg

115 120 125

Lys Ala Leu Gln Thr Leu Gly Tyr Ala Asp Leu Thr Phe Glu Asp Phe

130 135 140

Phe Ala Leu Phe Leu Glu Gln Leu Glu Ser Val Ile Gln Gly Lys Glu

145 150 155 160

Thr Ser Ile Ser His Glu Glu Leu Val Leu Arg Ser Arg Asp Gln Ser

165 170 175

Val Ser Asp Tyr Val Val Met Phe Phe Arg Phe Val Thr Ser Ala Ala

180 185 190

Ile Gln Lys Arg Thr Glu Phe Phe Glu Pro Phe Ile Leu Gly Leu Thr

195 200 205

Asn Thr Thr Val Glu Gln Phe Cys Lys Ser Ser Val Glu Pro Met Gly

210 215 220

Glu Glu Ser Asp His Val His Ile Thr Ala Leu Ser Asp Ala Leu Gly

225 230 235 240

Ile Pro Val Arg Val Val Tyr Leu Asp Arg Ser Ser Ser Asp Thr Gly

245 250 255

Gly Val Ser Val Asn His His Asp Phe Met Pro Val Ala Gly Asp Leu

260 265 270

Pro Asn Ala Ser Cys Ser Ser Glu Lys Asn Ile Pro Phe Ile Thr Leu

275 280 285

Leu Tyr Arg Pro Gly His Tyr Asp Ile Leu Tyr Pro Lys

290 295 300

<210> 21

<211> 300

<212> PRT

<213>Rape BnOTUB1 albumen

<400> 21

Met Gln Asn Gln Asn Asp Thr Val Lys Asp Asp Ala Glu Leu Ala Ala

1 5 10 15

Ser Ile Ser Ala Glu Gln Trp Gly Cys Cys Ser Val Glu Glu Pro Ser

20 25 30

Phe Gln Asp Asp Glu Ala Ala Lys Val Pro Tyr Val Gly Asp Lys Glu

35 40 45

Pro Met Ser Ser Leu Ala Ala Glu Tyr Gln Ala Gly Ser Pro Ile Leu

50 55 60

Leu Glu Lys Ile Lys Val Leu Asp Ser Gln Tyr Val Ala Ile Arg Arg

65 70 75 80

Thr Arg Gly Asp Gly Asn Cys Phe Phe Arg Ser Phe Met Phe Ser Tyr

85 90 95

Leu Glu His Ile Leu Glu Ser Gln Asp Gly Ala Glu Val Asp Arg Ile

100 105 110

Lys Leu Asn Val Glu Lys Cys Arg Lys Asn Leu Gln Asn Leu Gly Tyr

115 120 125

Thr Asp Phe Thr Phe Glu Asp Phe Phe Ala Leu Phe Leu Glu Gln Leu

130 135 140

Asp Asp Ile Leu Gln Gly Gly Glu Glu Ser Ile Ser Tyr Asp Glu Leu

145 150 155 160

Val Asn Arg Ser Arg Asp Gln Ser Val Ser Asp Tyr Ile Val Met Phe

165 170 175

Phe Arg Phe Val Thr Ala Gly Glu Ile Lys Thr Arg Ala Glu Phe Phe

180 185 190

Glu Pro Phe Ile Thr Gly Leu Ser Asn Thr Thr Val Asp Gln Phe Cys

195 200 205

Lys Thr Ser Val Glu Pro Met Gly Glu Glu Ser Asp His Ile His Ile

210 215 220

Thr Ala Leu Ser Asp Ala Leu Gly Val Ala Ile Arg Val Val Tyr Leu

225 230 235 240

Asp Arg Ser Ser Cys Asp Thr Gly Gly Gly Val Thr Val Asn His His

245 250 255

Asp Phe Val Pro Val Gly Ser Gly Thr Asn Glu Lys Glu Glu Ala Ser

260 265 270

Ser Ala Ala Pro Phe Ile Thr Leu Leu Tyr Arg Pro Gly His Tyr Asp

275 280 285

Ile Leu Tyr Pro Lys Val Leu Glu Asn Val Glu Lys

290 295 300

<210> 22

<211> 303

<212> PRT

<213>Cotton GhOTUB1 albumen

<400> 22

Met Gln Asn Gln Asp Gly Ala Val Ala Asp Arg Glu Lys Glu Ser Ala

1 5 10 15

Val Ser Ile Pro Ile Ser Glu Val Asp Asp Trp Ala Asn Phe Ala Asp

20 25 30

Asp Asp Ile Met Gln Gln Gln Ser Ala Ile His Ala Glu Glu Ala Lys

35 40 45

Lys Ile Pro Phe Val Gly Asp Lys Glu Pro Leu Ser Met Leu Ala Ala

50 55 60

Glu Tyr Glu Ser Gly Ser Pro Ile Leu Leu Glu Lys Ile Lys Val Leu

65 70 75 80

Asp Gln Gln Tyr Ala Ala Ile Arg Arg Thr Arg Gly Asp Gly Asn Cys

85 90 95

Phe Phe Arg Ser Phe Met Phe Ser Tyr Leu Glu His Ile Leu Glu Ser

100 105 110

Gln Asp Arg Ala Glu Val Asp Arg Ile Lys Gly Asn Val Glu Glu Cys

115 120 125

Arg Lys Pro Leu Gln Arg Leu Ala His Thr Asp Phe Thr Phe Glu Asp

130 135 140

Phe Phe Ser Leu Phe Leu Glu Gln Leu Glu Cys Val Leu Gln Gly Asn

145 150 155 160

Glu Asp Ser Ile Ser Gln Asp Glu Leu Ile Leu Arg Ser Arg Asp Gln

165 170 175

Ser Ile Ser Asp Tyr Val Val Met Phe Phe Arg Phe Val Thr Ser Gly

180 185 190

Glu Ile Arg Lys Arg Ser Glu Phe Phe Glu Pro Phe Ile Leu Gly Leu

195 200 205

Thr Asn Ala Thr Val Glu Gln Phe Cys Lys Ser Ser Val Glu Pro Met

210 215 220

Gly Glu Glu Ser Asp His Val His Ile Thr Ala Leu Ser Asp Ala Leu

225 230 235 240

Gly Val Pro Ile Arg Val Val Tyr Leu Asp Arg Ser Ser Cys Asp Ile

245 250 255

Gly Gly Val Ser Val Asn His His Asp Phe Leu Pro Thr Ser Gly Asp

260 265 270

Lys Ser Asn Ala Lys Gly Gly Ser Thr Val Pro Val Lys Pro Phe Ile

275 280 285

Thr Leu Leu Tyr Arg Pro Gly His Tyr Asp Ile Leu Tyr Pro Lys

290 295 300

<210> 23

<211> 301

<212> PRT

<213>Tomato SlOTUB1 albumen

<400> 23

Met Gln Asp Gln Asp Val Asp Val Asp Val Ala Asp Ala Ala Lys Glu

1 5 10 15

Thr Phe Thr Ser Ser Glu Thr Asp Asp Trp Lys Lys Tyr Lys Asp Asp

20 25 30

Asp Ile Met Gln Gln His Ser Ala Ile Gln Ala Glu Gln Ala Val Lys

35 40 45

Val Pro Phe Leu Gly Asp Lys Glu Pro Leu Ser Ser Leu Glu Ala Glu

50 55 60

Tyr His Leu Gly Asn Ser Ile Val Leu Glu Lys Ile Lys Val Leu Ser

65 70 75 80

Glu Gln Tyr Ala Ala Ile Arg Arg Thr Arg Gly Asp Gly Asn Cys Phe

85 90 95

Phe Arg Ser Phe Met Phe Gly Tyr Leu Glu His Ile Leu Glu Ser Gln

100 105 110

Asp His Asn Glu Val Gln His Ile Lys Ser Asn Ile Glu Glu Cys Lys

115 120 125

Lys Thr Leu Gln Ser Leu Gly Tyr Ala Glu Phe Thr Phe Glu Asp Phe

130 135 140

Phe Ala Leu Phe Leu Glu Gln Leu Asp Ser Val Leu Gln Gly Ser Glu

145 150 155 160

Asp Ser Ile Ser His Asp Glu Leu Leu Cys Arg Ser Arg Asp Pro Ser

165 170 175

Ile Ser Asp Tyr Val Val Met Phe Phe Arg Phe Val Thr Ser Gly Glu

180 185 190

Ile Arg Lys Arg Ser Glu Phe Phe Glu Pro Phe Ile Leu Gly Leu Thr

195 200 205

Asn Thr Ser Val Glu Gln Phe Cys Lys Ser Ala Val Glu Pro Met Gly

210 215 220

Glu Glu Ser Asp His Val Gln Ile Thr Ala Leu Ser Asp Ala Leu Gly

225 230 235 240

Val Pro Ile Arg Val Val Tyr Leu Asp Arg Ser Ser Cys Glu Asn Asn

245 250 255

Ser Ile Asn Val Asn His His Asp Phe Ile Pro Thr Ser Arg Glu Val

260 265 270

Gly Asn Ser Asp Val Ser Lys Thr Thr Asn Arg Pro Ser Ile Thr Leu

275 280 285

Leu Tyr Arg Pro Gly His Tyr Asp Ile Leu Tyr Pro Lys

290 295 300

<210> 24

<211> 578

<212> DNA

<213>OsOTUB1 sgRNA expression cassette sequences

<400> 24

tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt ttaccatccg 60

aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc ccgtaaaaag 120

cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca ggctatcgag 180

atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg tcaggcgaaa 240

tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag ttggccggat 300

aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag cacttcgatt 360

cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc gcttagctag 420

agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccgtcag ctggaaagtg 480

ttctgcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 540

aaaagtggca ccgagtcggt gctttttttc aagagctt 578

<210> 25

<211> 578

<212> DNA

<213>TuOTUB1 sgRNA expression cassette sequences

<400> 25

tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt ttaccatccg 60

aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc ccgtaaaaag 120

cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca ggctatcgag 180

atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg tcaggcgaaa 240

tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag ttggccggat 300

aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag cacttcgatt 360

cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc gcttagctag 420

agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccgttaa ggcgaacacg 480

aggagagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 540

aaaagtggca ccgagtcggt gctttttttc aagagctt 578

<210> 26

<211> 578

<212> DNA

<213>HvOTUB1 sgRNA expression cassette sequences

<400> 26

tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt ttaccatccg 60

aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc ccgtaaaaag 120

cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca ggctatcgag 180

atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg tcaggcgaaa 240

tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag ttggccggat 300

aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag cacttcgatt 360

cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc gcttagctag 420

agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccgttaa gacggacacg 480

aggagagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 540

aaaagtggca ccgagtcggt gctttttttc aagagctt 578

<210> 27

<211> 578

<212> DNA

<213>ZmOTUB1 sgRNA expression cassette sequences

<400> 27

tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt ttaccatccg 60

aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc ccgtaaaaag 120

cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca ggctatcgag 180

atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg tcaggcgaaa 240

tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag ttggccggat 300

aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag cacttcgatt 360

cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc gcttagctag 420

agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccgaagc tttatgtttt 480

cctaccgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 540

aaaagtggca ccgagtcggt gctttttttc aagagctt 578

<210> 28

<211> 578

<212> DNA

<213>SbOTUB1 sgRNA expression cassette sequences

<400> 28

tggaatcggc agcaaaggat tttttcctgt agttttccca caaccatttt ttaccatccg 60

aatgatagga taggaaaaat atccaagtga acagtattcc tataaaattc ccgtaaaaag 120

cctgcaatcc gaatgagccc tgaagtctga actagccggt cacctgtaca ggctatcgag 180

atgccataca agagacggta gtaggaacta ggaagacgat ggttgattcg tcaggcgaaa 240

tcgtcgtcct gcagtcgcat ctatgggcct ggacggaata ggggaaaaag ttggccggat 300

aggagggaaa ggcccaggtg cttacgtgcg aggtaggcct gggctctcag cacttcgatt 360

cgttggcacc ggggtaggat gcaatagaga gcaacgttta gtaccacctc gcttagctag 420

agcaaactgg actgccttat atgcgcgggt gctggcttgg ctgccgaagc tttatgttct 480

cctactgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 540

aaaagtggca ccgagtcggt gctttttttc aagagctt 578

<210> 29

<211> 455

<212> DNA

<213>GmOTUB1 sgRNA expression cassette sequences

<400> 29

tggaatcggc agcaaaggat ttactttaaa ttttttctta tgcagcctgt gatggataac 60

tgaatcaaac aaatggcgtc tgggtttaag aagatctgtt ttggctatgt tggacgaaac 120

aagtgaactt ttaggatcaa cttcagttta tatatggagc ttatatcgag caataagata 180

agtgggcttt ttatgtaatt taatgggcta tcgtccatag attcactaat acccatgccc 240

agtacccatg tatgcgtttc atataagctc ctaatttctc ccacatcgct caaatctaaa 300

caaatcttgt tgtatatata acactgaggg agcaacattg gtcaattcgt cgtactcgag 360

gagagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 420

aagtggcacc gagtcggtgc tttttttcaa gagct 455

<210> 30

<211> 455

<212> DNA

<213>BnOTUB1 sgRNA expression cassette sequences

<400> 30

tggaatcggc agcaaaggat ttactttaaa ttttttctta tgcagcctgt gatggataac 60

tgaatcaaac aaatggcgtc tgggtttaag aagatctgtt ttggctatgt tggacgaaac 120

aagtgaactt ttaggatcaa cttcagttta tatatggagc ttatatcgag caataagata 180

agtgggcttt ttatgtaatt taatgggcta tcgtccatag attcactaat acccatgccc 240

agtacccatg tatgcgtttc atataagctc ctaatttctc ccacatcgct caaatctaaa 300

caaatcttgt tgtatatata acactgaggg agcaacattg gtcagttgca atcaggcgaa 360

caaggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 420

aagtggcacc gagtcggtgc tttttttcaa gagct 455

<210> 31

<211> 455

<212> DNA

<213>GhOTUB1 sgRNA expression cassette sequences

<400> 31

tggaatcggc agcaaaggat ttactttaaa ttttttctta tgcagcctgt gatggataac 60

tgaatcaaac aaatggcgtc tgggtttaag aagatctgtt ttggctatgt tggacgaaac 120

aagtgaactt ttaggatcaa cttcagttta tatatggagc ttatatcgag caataagata 180

agtgggcttt ttatgtaatt taatgggcta tcgtccatag attcactaat acccatgccc 240

agtacccatg tatgcgtttc atataagctc ctaatttctc ccacatcgct caaatctaaa 300

caaatcttgt tgtatatata acactgaggg agcaacattg gtcagcagcc attagaagaa 360

cgcggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 420

aagtggcacc gagtcggtgc tttttttcaa gagct 455

<210> 32

<211> 455

<212> DNA

<213>SlOTUB1 sgRNA expression cassette sequences

<400> 32

tggaatcggc agcaaaggat ttactttaaa ttttttctta tgcagcctgt gatggataac 60

tgaatcaaac aaatggcgtc tgggtttaag aagatctgtt ttggctatgt tggacgaaac 120

aagtgaactt ttaggatcaa cttcagttta tatatggagc ttatatcgag caataagata 180

agtgggcttt ttatgtaatt taatgggcta tcgtccatag attcactaat acccatgccc 240

agtacccatg tatgcgtttc atataagctc ctaatttctc ccacatcgct caaatctaaa 300

caaatcttgt tgtatatata acactgaggg agcaacattg gtcagctgcc attagaagaa 360

cacggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 420

aagtggcacc gagtcggtgc tttttttcaa gagct 455

<210> 33

<211> 825

<212> DNA

<213>Paddy rice dep1-1 cDNA sequence

<400> 33

atgggggagg aggcggtggt gatggaggcg ccgaggccca agtcgccgcc gaggtacccg 60

gacctgtgcg gccggcggcg gatgcagctg gaggtgcaga tcctgagccg cgagatcacg 120

ttcctcaagg atgagcttca cttccttgaa ggagctcagc ccgtttctcg ttctggatgc 180

attaaagaga taaatgagtt tgttggtaca aaacatgacc cactaatacc aacaaagaga 240

aggaggcaca gatcttgccg tctttttcgg tggatcggat caaaattgtg tatctgcatt 300

tcatgtcttt gctactgttg caagtgctca cccaagtgca aaagaccaag gtgcctcaat 360

tgttcttgca gctcatgctg cgacgagcca tgctgtaagc caaactgcag tgcgtgctgc 420

gctgggtcat gctgtagtcc agactgctgc tcatgctgta aacctaactg cagttgctgc 480

aagacccctt cttgctgcaa accgaactgc tcgtgctcct gtccaagctg cagctcatgc 540

tgcgatacat cgtgctgcaa accgagctgc acctgcttca acatctag 588

<210> 34

<211> 195

<212> PRT

<213>The amino acid sequence of paddy rice DEP1 albumen

<400> 34

Met Gly Glu Glu Ala Val Val Met Glu Ala Pro Arg Pro Lys Ser Pro

1 5 10 15

Pro Arg Tyr Pro Asp Leu Cys Gly Arg Arg Arg Met Gln Leu Glu Val

20 25 30

Gln Ile Leu Ser Arg Glu Ile Thr Phe Leu Lys Asp Glu Leu His Phe

35 40 45

Leu Glu Gly Ala Gln Pro Val Ser Arg Ser Gly Cys Ile Lys Glu Ile

50 55 60

Asn Glu Phe Val Gly Thr Lys His Asp Pro Leu Ile Pro Thr Lys Arg

65 70 75 80

Arg Arg His Arg Ser Cys Arg Leu Phe Arg Trp Ile Gly Ser Lys Leu

85 90 95

Cys Ile Cys Ile Ser Cys Leu Cys Tyr Cys Cys Lys Cys Ser Pro Lys

100 105 110

Cys Lys Arg Pro Arg Cys Leu Asn Cys Ser Cys Ser Ser Cys Cys Asp

115 120 125

Glu Pro Cys Cys Lys Pro Asn Cys Ser Ala Cys Cys Ala Gly Ser Cys

130 135 140

Cys Ser Pro Asp Cys Cys Ser Cys Cys Lys Pro Asn Cys Ser Cys Cys

145 150 155 160

Lys Thr Pro Ser Cys Cys Lys Pro Asn Cys Ser Cys Ser Cys Pro Ser

165 170 175

Cys Ser Ser Cys Cys Asp Thr Ser Cys Cys Lys Pro Ser Cys Thr Cys

180 185 190

Phe Asn Ile

195

<210> 35

<211> 462

<212> DNA

<213>Rice Os UBC13 gene orders

<400> 35

atggccaaca gcaacctccc ccggcgaatc atcaaggaga cgcagcgact cctcagcgag 60

ccagcgccgg gaatcagcgc gtctccgtcg gaggagaaca tgcgctactt caacgtcatg 120

atccttggcc cggcacagtc cccctatgaa ggtggagttt ttaagcttga actcttttta 180

cctgaggaat atcctatggc tgctccaaag gttaggttcc tgaccaaaat ataccacccc 240

aacattgaca agcttggtag gatatgcctt gacattctca aggacaaatg gagcccagcc 300

cttcagattc ggacagttct tttgagtatc caggcactcc taagtgcacc aaaccctgat 360

gatcctctct ctgataacat tgcaaagcac tggaaagcca atgaagcaga agctgttgaa 420

acagcaaagg agtggactcg cctgtatgcc agcggtgcat aa 462

<210> 36

<211> 153

<212> PRT

<213>The protein sequence of rice Os UBC13 gene codes

<400> 36

Met Ala Asn Ser Asn Leu Pro Arg Arg Ile Ile Lys Glu Thr Gln Arg

1 5 10 15

Leu Leu Ser Glu Pro Ala Pro Gly Ile Ser Ala Ser Pro Ser Glu Glu

20 25 30

Asn Met Arg Tyr Phe Asn Val Met Ile Leu Gly Pro Ala Gln Ser Pro

35 40 45

Tyr Glu Gly Gly Val Phe Lys Leu Glu Leu Phe Leu Pro Glu Glu Tyr

50 55 60

Pro Met Ala Ala Pro Lys Val Arg Phe Leu Thr Lys Ile Tyr His Pro

65 70 75 80

Asn Ile Asp Lys Leu Gly Arg Ile Cys Leu Asp Ile Leu Lys Asp Lys

85 90 95

Trp Ser Pro Ala Leu Gln Ile Arg Thr Val Leu Leu Ser Ile Gln Ala

100 105 110

Leu Leu Ser Ala Pro Asn Pro Asp Asp Pro Leu Ser Asp Asn Ile Ala

115 120 125

Lys His Trp Lys Ala Asn Glu Ala Glu Ala Val Glu Thr Ala Lys Glu

130 135 140

Trp Thr Arg Leu Tyr Ala Ser Gly Ala

145 150

<210> 37

<211> 1254

<212> DNA

<213>Rice Os SPL14 cDNA

<400> 37

atggagatgg ccagtggagg aggcgccgcc gccgccgccg gcggcggagt aggcggcagc 60

ggcggcggtg gtggtggagg ggacgagcac cgccagctgc acggtctcaa gttcggcaag 120

aagatctact tcgaggacgc cgccgcggca gcaggcggcg gcggcactgg cagtggcagt 180

ggcagcgcga gcgccgcgcc gccgtcctcg tcttccaagg cggcgggtgg tggacgcggc 240

ggagggggca agaacaaggg gaagggcgtg gccgcggcgg cgccaccgcc gccgccgccg 300

ccgccgcggt gccaggtgga ggggtgcggc gcggatctga gcgggatcaa gaactactac 360

tgccgccaca aggtgtgctt catgcattcc aaggctcccc gcgtcgtcgt cgccggcctc 420

gagcagcgct tctgccagca gtgcagcagg ttccacctgc tgcctgaatt tgaccaagga 480

aaacgcagct gccgcagacg ccttgcaggt cataatgagc gccggaggag gccgcaaacc 540

cctttggcat cacgctacgg tcgactagct gcatctgttg gtgagcatcg caggttcaga 600

agctttacgt tggatttctc ctacccaagg gttccaagca gcgtaaggaa tgcatggcca 660

gcaattcaac caggcgatcg gatctccggt ggtatccagt ggcacaggaa cgtagctcct 720

catggtcact ctagtgcagt ggcgggatat ggtgccaaca catacagcgg ccaaggtagc 780

tcttcttcag ggccaccggt gttcgctggc ccaaatctcc ctccaggtgg atgtctcgca 840

ggggtcggtg ccgccaccga ctcgagctgt gctctctctc ttctgtcaac ccagccatgg 900

gatactacta cccacagtgc cgctgccagc cacaaccagg ctgcagccat gtccactacc 960

accagctttg atggcaatcc tgtggcaccc tccgccatgg cgggtagcta catggcacca 1020

agcccctgga caggttctcg gggccatgag ggtggtggtc ggagcgtggc gcaccagcta 1080

ccacatgaag tctcacttga tgaggtgcac cctggtccta gccatcatgc ccacttctcc 1140

ggtgagcttg agcttgctct gcaggggaac ggtccagccc cagcaccacg catcgatcct 1200

gggtccggca gcaccttcga ccaaaccagc aacacgatgg attggtctct gtag 1254

<210> 38

<211> 417

<212> PRT

<213>The albumen of rice Os SPL14 codings

<400> 38

Met Glu Met Ala Ser Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly

1 5 10 15

Val Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Asp Glu His Arg Gln

20 25 30

Leu His Gly Leu Lys Phe Gly Lys Lys Ile Tyr Phe Glu Asp Ala Ala

35 40 45

Ala Ala Ala Gly Gly Gly Gly Thr Gly Ser Gly Ser Gly Ser Ala Ser

50 55 60

Ala Ala Pro Pro Ser Ser Ser Ser Lys Ala Ala Gly Gly Gly Arg Gly

65 70 75 80

Gly Gly Gly Lys Asn Lys Gly Lys Gly Val Ala Ala Ala Ala Pro Pro

85 90 95

Pro Pro Pro Pro Pro Pro Arg Cys Gln Val Glu Gly Cys Gly Ala Asp

100 105 110

Leu Ser Gly Ile Lys Asn Tyr Tyr Cys Arg His Lys Val Cys Phe Met

115 120 125

His Ser Lys Ala Pro Arg Val Val Val Ala Gly Leu Glu Gln Arg Phe

130 135 140

Cys Gln Gln Cys Ser Arg Phe His Leu Leu Pro Glu Phe Asp Gln Gly

145 150 155 160

Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn Glu Arg Arg Arg

165 170 175

Arg Pro Gln Thr Pro Leu Ala Ser Arg Tyr Gly Arg Leu Ala Ala Ser

180 185 190

Val Gly Glu His Arg Arg Phe Arg Ser Phe Thr Leu Asp Phe Ser Tyr

195 200 205

Pro Arg Val Pro Ser Ser Val Arg Asn Ala Trp Pro Ala Ile Gln Pro

210 215 220

Gly Asp Arg Ile Ser Gly Gly Ile Gln Trp His Arg Asn Val Ala Pro

225 230 235 240

His Gly His Ser Ser Ala Val Ala Gly Tyr Gly Ala Asn Thr Tyr Ser

245 250 255

Gly Gln Gly Ser Ser Ser Ser Gly Pro Pro Val Phe Ala Gly Pro Asn

260 265 270

Leu Pro Pro Gly Gly Cys Leu Ala Gly Val Gly Ala Ala Thr Asp Ser

275 280 285

Ser Cys Ala Leu Ser Leu Leu Ser Thr Gln Pro Trp Asp Thr Thr Thr

290 295 300

His Ser Ala Ala Ala Ser His Asn Gln Ala Ala Ala Met Ser Thr Thr

305 310 315 320

Thr Ser Phe Asp Gly Asn Pro Val Ala Pro Ser Ala Met Ala Gly Ser

325 330 335

Tyr Met Ala Pro Ser Pro Trp Thr Gly Ser Arg Gly His Glu Gly Gly

340 345 350

Gly Arg Ser Val Ala His Gln Leu Pro His Glu Val Ser Leu Asp Glu

355 360 365

Val His Pro Gly Pro Ser His His Ala His Phe Ser Gly Glu Leu Glu

370 375 380

Leu Ala Leu Gln Gly Asn Gly Pro Ala Pro Ala Pro Arg Ile Asp Pro

385 390 395 400

Gly Ser Gly Ser Thr Phe Asp Gln Thr Ser Asn Thr Met Asp Trp Ser

405 410 415

Leu

6

Claims (14)

1. control rice tillering number, stalk rugosity, the ideotype base per fringe grain number per spike, mass of 1000 kernel and yield of a kind of separation Cause, it encodes SEQ ID NO：Amino acid sequence shown in 15, is named as NPT1.

2. the gene described in claim 1, it includes SEQ ID NOs：Nucleotide sequence shown in 1 or 3.

3. the allele of the gene of claim 1, it includes SEQ ID NOs：Nucleotide sequence shown in 2 or 4, is ordered Entitled npt1.

4. the promoter sequence of the gene of claim 1 or its allele, it is such as SEQ ID NO：Shown in 5 or 6.

5. the homologous gene of the gene of claim 1, it encodes SEQ ID NOs：Amino acid sequence shown in any of 16-23 Row.

6. the homologous gene described in claim 5, it includes SEQ ID NOs：Nucleotide sequence shown in any of 7-14.

7. a kind of albumen of separation, its amino acid sequence such as SEQ ID NOs：Shown in any of 15-23.

8. a kind of recombinant precursor, it is characterised in that include the sequence any one of claim 1-6.

9. a kind of recombinant host cell, it is characterised in that include the sequence any one of claim 1-6；Or include right It is required that the recombinant precursor described in 8, wherein the cell is microbial cell, wherein the microbial cell is preferably large intestine bar Bacterium or agrobatcerium cell.

10. a kind of method for the paddy rice for cultivating output increased, this method includes：With the allele npt1 comprising claim 3 Recombinant host cell transfection rice plant obtain NPT1 gene expression doses decline or amino acid sequence change cause albumen The transgenic rice plant of function reduction, or by the rice plant containing the allele npt1 described in claim 3 and separately One rice plant hybridizes, wherein another rice plant is preferably Grain number per spike increase, the increased plant of yield.

11. a kind of method that every fringe grain number per spike and the increased paddy rice of yield are cultivated by overexpression OsUBC13, this method includes With recombinant precursor and recombinant host cell containing OsUBC13, rice transformation plant obtains transgenic rice plant, resulting Transgenic rice plant in NPT1 activity be affected, thus increase per fringe grain number per spike and yield, wherein the cell is micro- Biological cell, the microbial cell is preferably Escherichia coli or agrobatcerium cell.

12. a kind of pyramiding breeding method of the molecular marker assisted selection for the rice varieties for cultivating high yield, this method includes making With the parental rice comprising the allele npt1 described in claim 3 and another paddy rice for carrying excellent allele dep1-1 Parents, go out to have polymerize excellent allele npt1 and dep1-1 new lines or product in offspring according to molecular selection Kind.

13. a kind of crop that NPT1 or its homologous gene cultivation output increased are knocked out by CRISPR/cas9 gene editings technology Method, methods described include using NPT1 or its homologous gene as target spot, pass through CRISPR/cas9 gene editings technology knock out target Point gene, cultivates and obtains the crop of output increased, wherein the crop be selected from paddy rice, wheat, barley, corn, sorghum, soybean, Rape, cotton or tomato.

14. a kind of function by strengthening plant type of rice modulin OsSPL14 is cultivated per fringe grain number per spike and the increased water of yield The method of rice, this method is included with recombinant precursor and recombinant host cell containing NPT1, and rice transformation plant, which obtains, turns base Because of rice plant, the enhanced transfer-gen plant of OsSPL14 functions is derived from, wherein the cell is microbial cell, it is described Microbial cell is preferably Escherichia coli or agrobatcerium cell.