CN105612171B - The method for adjusting the seed in plant and organ size - Google Patents

The method for adjusting the seed in plant and organ size Download PDF

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CN105612171B
CN105612171B CN201480045197.9A CN201480045197A CN105612171B CN 105612171 B CN105612171 B CN 105612171B CN 201480045197 A CN201480045197 A CN 201480045197A CN 105612171 B CN105612171 B CN 105612171B
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plant
polypeptide
expression
seq
eod1
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CN105612171A (en
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李云海
夏天
李娜
杰克·杜梅尼尔
迈克尔·贝文
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Institute of Genetics and Developmental Biology of CAS
Plant Bioscience Ltd
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Institute of Genetics and Developmental Biology of CAS
Plant Bioscience Ltd
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Abstract

The present invention relates to plant E3 ubiquitin ligases (referred to as DA2), and seed and organ size are controlled with DA1 synergistic effect.The method for increasing plant products is provided, this method includes that the expression or activity of DA2 are reduced in lacking DA1 expression or active plant.The method for additionally providing the plant with increased yield and generating this kind of plant.

Description

The method for adjusting the seed in plant and organ size
Technical field
The present invention relates to the sizes of the seed and organ that change plant, such as the method to improve plant products.
Background technique
The size of seed and organ is on the agronomy under heredity control and ecological important character (Alonso- Blanco,C.PNAS USA 96,4710-7(1999)；Song,X.J.Nat Genet 39,623-30(2007)；Weiss, J.Int J Dev Biol 49,513-25(2005)；Dinneny,J.R.Development 131,1101-10(2004)； Disch,S.Curr Biol 16,272-9(2006)；Science 289,85-8(2000)；Horiguchi,G.Plant J 43,68-78(2005)；Hu,Y Plant J 47,1-9(2006)；Hu,Y.Plant Cell 15,1951-61(2003)； Krizek,B.A.Dev Genet 25,224-36(1999)；Mizukami,Y.PNAS USA 97,942-7(2000)；Nath, U.Science 299,1404-7(2003)；Ohno,C.K.Development 131,1111-22(2004)；Szecsi, J.Embo J 25,3912-20(2006)；White,D.W.PNAS USA 103,13238-43(2006)；Horvath, B.M.Embo J 25,4909-20(2006)；Garcia,D.Plant Cell 17,52-60(2005).Seed and organ are most Whole size is constant in given species, and seed and organ size variation are very big between species, this show plant have with Coordinate and timely mode controls the regulatory mechanism of seed and organ growth.However, although the importance of seed and organ size, About the final organ and the molecule of seed size that control in plant and knowing little for genetic mechanism.
Usage quantity trait locuses (QTL), which have been located in plant, is included in tomato, soybean, corn and rice In the genetic regulation of seed size is studied.So far, in disclosed document, two kinds of genes authenticated (Song,X.J.Nat Genet 39,623-30(2007)；Fan,C.Theor.Appl.Genet.112,1164-1171 (2006)), both genes are potential two kinds of main QTL of rice granularity；But the molecular mechanism of these genes still needs It illustrates.In arabidopsis, to the seed weight influenced in germplasm (accession) hybridization between Ler and Cvi and/or A kind of locus of the ten of length is positioned { Alonso-Blanco, 1999 are same as above }, but not yet identifies corresponding gene.Most Nearly research has revealed that AP2 and ARF2 participates in control seed size.Unfortunately, ap2 and arf2 mutant has than open country The raw lower fertilizability of type (Schruff, M.C.Development 137,251-261 (2006)；Ohto,M.A.PNAS USA 102,3123-3128(2005)；Jofuku,K.D.PNAS USA 102,3117-3122(2005)).In addition, being planted using mutation The research of object authenticated worked by cell proliferation or expand influence several positive and negatives regulation of organ size because Son { Krizek, B.A.Dev Genet 25,224-36 (1999)；Mizukami,Y.Proc Natl Acad Sci U S A 97,942-7(2000)；Nath,U.Science 299,1404-7(2003)；Ohno,C.K.Development 131,1111- 22(2004)；Szecsi,J.Embo J 25,3912-20(2006)；White,D.W.PNAS USA 103,13238-43 (2006)；Horvath,B.M.Embo J25,4909-20(2006)；Garcia,D.Plant Cell 17,52-60(2005). Horiguchi,G.Plant J 43,68-78(2005)；Hu,Y Plant J 47,1-9(2006)Dinneny, J.R.Development 131,1101-10(2004))。
It is related to several factor known effect seed sizes of ubiquitin related activity.Growth limiting factor DA1 is ubiquitin receptor And including combining two ubiquitin of ubiquitin to interact motif (UIM) in vitro, and da1-1 mutant is by influencing ovule Parent integument and form big seed (Li et al. people, 2008).(it encodes E3 ubiquitinbond to an enhancer (EOD1) of da1-1 Enzyme BIG BROTHER (BB) (Disch et al., 2006；Li et al. people, 2008) seed size of the mutation collaboration enhancing da1-1 in Phenotype, to indicate DA1 and EOD1/BB synergistic effect to control seed size.In rice, the wide volume that 2 (GW2) are weighed with grain of grain The quantitative trait locus (QTL) of code E3 ubiquitin ligase is by limitation cell division come Control granularity (Song et al., 2007). GW2 homologue (the Ta-GW2 in wheat is authenticated；Bednarek et al. is 2012).It needs to be encoded by rice qSW5/GW5 Agnoprotein matter come limit the granularity in rice (Shomura et al., 2008；Weng et al., 2008).GW5 is in yeast two-hybrid Physically interact in measurement with poly- ubiquitin, thus show GW5 may participate in Ubiquitin-Proteasome Pathway (Weng et al., 2008).However, whether both unclear factors work in maternal tissue in rice and/or zygote tissue.
Other factors for identifying the final size of both control seed and organ will not only promote to the size control in plant The understanding of the mechanism of system, and can for example have in terms of improving crop yield and plant biomass for generating bio-fuel There is a large amount of practical application.
Summary of the invention
The present inventor authenticated plant E3 ubiquitin ligase (referred to as DA2), pass through the pearl of limitation development seed Cell Proliferation by regulates and controls the final size of seed and organ.It has been surprisingly found that DA2 and DA1 synergistic effect and independence Seed and organ size are controlled in EOD1.As target therefore it can be used for improving plant products using DA2 and DA1 and/or EOD1.
An aspect of of the present present invention provides a kind of method of yield for increasing plant, comprising:
The expression or activity of the intracellular DA2 polypeptide of the plant are reduced,
Wherein the plant lacks DA1 expression or activity.
Another aspect provides a kind of methods of yield for increasing plant, comprising:
The expression or activity of the intracellular DA2 polypeptide of the plant are reduced,
Wherein the plant lacks EOD1 expression or activity.
Another aspect provides a kind of methods of yield for increasing plant, comprising:
The expression or activity of the intracellular DA2 polypeptide of the plant are reduced,
Wherein the plant lacks DA1 and EOD1 expression or activity.
Another aspect provides a kind of methods of yield for increasing plant, comprising:
The expression or activity of the intracellular DA2 polypeptide of the plant are reduced or eliminated, and；
I) expression or activity of the intracellular DA1 polypeptide are reduced or eliminated,
Ii the expression or activity of the intracellular EOD1) are reduced or eliminated, and/or
Iii) in the dominant negative DA polypeptide of the cell inner expression.
Another aspect provides a kind of methods for generating and having the plant of increased yield, comprising:
The expression for lacking both DA1, EOD1 or DA1 and EOD1 or active plant cell are provided,
It will be eliminated by conversion or the expression of DA2 polypeptide or active heterologous nucleic acids is inhibited to be incorporated in the plant cell, And；
Plant described in cytothesis from one or more conversion.
Detailed description of the invention
Fig. 1 shows seed and organ size in da2-1 mutant.1A shows Col-0, da2-1 and 35S::DA2#1 kind The projected area of son.Seed is divided into three groups (>0.13,0.12-0.13 and<0.12mm2).Every group of value is expressed as being analyzed Total number seeds percentage.1B shows every silique number seeds of Col-0, da2-1 and 35S::DA2#1.Angle on stem Fruit (from fourth angle fruit to the tenth silique) is for measuring every silique number seeds.1C shows Col-0, da2-1 and 35S::DA2#1 Every plant seed weight.1D shows every plant seed number of Col-0, da2-1 and 35S::DA2#1.1E show Col-0, The height of da2-1 and 35S::DA2#1 plant.Numerical value (B-E) is provided as the average value ± SE relative to wild type numerical value, if It is set to 100%.Compared to wild type, * *, P < 0.01 and *, P < 0.05 (student t inspection).Ruler item: F, 1cm；G, 1mm
Fig. 2 shows 4 day age plant (F) of Col-0 (left side), da2-1 (centre) and 35S::DA2#1 (right side) and Col-0 (on), da2-1 (centre) and 35S::DA2#1 (under) flower (G).
Fig. 3 shows DA1 and DA2 synergistic effect to control seed size.3A shows Col-0, da1-1, da2-1 and da1-1 The dry seeds of da2-1.3B shows 10 day age seedling of Col-0, da2-1, da1-1 and da1-1 da2-1 (from left to right).3C shows The seed weight of Col-0, da1-1, da2-1 and da1-1 da2-1 out.3D shows Col-0, da1-ko1, da2-1 and da1-ko1 The seed weight of da2-1.Numerical value is provided as the average value ± SE relative to corresponding wild type numerical value, is arranged to 100%.It compares In wild type, * *, P < 0.01 and *, P < 0.05 (student t inspection).Ruler item: A, 0.1mm；B, 1m
Fig. 4 shows DA1 and DA2 synergistic effect to control seed size.Upper left illustrate 10 day age Col-0, da1-1, The cotyledon area of da2-1 and da1-1 da2-1 seedling.Upper right illustrates 10 day age Col-0, da1-ko1, da2-1 and da1-ko1 The cotyledon area of da2-1 seedling.Lower-left illustrates the fence in the cotyledon of Col-0, da1-1, da2-1 and da1-1da2-1 plumule The average area of cell.Bottom right illustrates Col-0, da1-1, da1-1 da2-1, da1-ko1 da2-1, da1-ko1 dar1-1 With the projected area of da1-ko1 dar1-1 da2-1 seed.Numerical value is as the average value ± SE relative to corresponding wild type numerical value It provides, is arranged to 100%.Compared to wild type, * *, P < 0.01 and *, P < 0.05 (student t inspection).Ruler item: A, 0.1mm；B, 1m
Fig. 5 shows the cell Proliferation in parent integument of the DA1 and DA2 synergistic effect to control development seed.(5A-5D) point The mature ovule of Col-0, da1-1, da2-1 and da1-1 da2-1 are not shown.The ovule of da2-1 mutation collaboration enhancing da1-1 is big It is small.
Fig. 6 shows (left figure) Col-0 × Col-0 (c/c) F1, da2-1 × da2-1 (d2/d2) F1, Col-0 × da2-1 (c/d2) projected area of F1 and da2-1 × Col-0 (d2/c) F1 seed and (middle graph) Col-0 × Col-0 (c/c) F1, da1-ko1 da2-1×da1-ko1 da2-1(dd/dd)F1、Col-0×da1-ko1 da2-1(c/dd)F1、da1-ko1 The projected area of da2-1 × Col-0 (dd/c) F1 seed.Right figure is shown with da1-ko1 da2-1 double-mutant pollen pair Da1-ko1/+da2-1/+ plant pollination, so as to cause the intracutaneous da1-ko1/+da2-1/+ (a) of da1-ko1/+da2-1/+ kind, The hair of da1-ko1/+da2-1da2-1 (b), da1-ko1/da1-ko1 da2-1/+ (c) and da1-ko1 da2-1 (d) plumule Projection seed area after educating.It measures come the da1-ko1/+da2- for da1-ko1 da2-1 double-mutant pollen fertilization of using by oneself The projected area of the single seed of 1/+ plant.These seeds are mutated further Genotyping for da1-ko1 and da2-1.Number It is uncorrelated to the variation of the size of these seeds (P > 0.05, student t are examined) according to display da1-ko1 and da2-1 mutation.Numerical value is made To provide relative to the average value ± SE of corresponding wild type numerical value, it is arranged to 100%.Compared to wild type, * *, P < 0.01 (is learned Raw t is examined).Ruler item: A-D, 0.5mm.
Fig. 7 shows the projected area of (left figure) Col-0, da1-1, da2-1 and da1-1 da2-1 maturation ovule；It is (intermediate Figure) cell in 6DAP and 8DAP in the external integument of Col-0, da1-1, da2-1 and da1-1 da2-1 seed number；With And (right figure) from the external integument length and cell number of single seed calculate in 6DAP and 8DAP Col-0, da1-1, da2-1 and The average length of cell in the external integument of da1-1 da2-1 seed.
Fig. 8 A shows DA2 gene structure.Indicate initiation codon (ATG) and terminator codon (TAA).It is closed frame instruction Coded sequence, open frame indicates 5 ' and 3 ' non-translational regions, and the line between frame indicates introne.T- in DA2 gene is shown DNA insertion point (da2-1).Fig. 8 B shows the RING structural domain that DA2 protein includes prediction.
Fig. 9 shows the E3 ubiquitin ligase activity of DA2.By MBP-DA2 and the DA2 of mutation (MBP-DA2C59S and MBP- DA2N91L) fusion protein is surveyed for E3 ubiquitin ligase activity in the presence of E1, E2 and His- ubiquitin (His-Ub) It is fixed.Respectively by detecting ubiquitination egg with anti-His antibody (anti-His) and anti-MBP antibody (anti-MBP) immunoblotting (IB) White matter.Lower arrow indicates MBP-DA2 protein, and upper arrow shows ubiquitination MBP-DA2 protein.
Figure 10 shows the projected area (above) of Col-0, da2-1, COM#6, COM#8 and COM#10 seed, wherein COM It is the da2-1 Coding Sequence Transformed with DA2 driven by the promoter of their own；Col-0, da2-1, COM#6, COM#8 and DA2 in the petal area (middle graph) of COM#10 plant and Col-0, da2-1, COM#6, COM#8 and COM#10 seedling The quantitative Real time RT-PCR analysis (following figure) of gene expression.Numerical value (D and E) is as the average value ± SE relative to da2-1 numerical value It provides, is arranged to 100%.Compared to da2-1 mutant, * *, P < 0.01 (student t inspection).
Figure 11 shows the expression pattern of DA2.11A shows the quantitative Real time RT-PCR analysis of DA2 gene expression.From root (R), Stem (S), leaf (L), seedling (Se) and inflorescence (In) separate total serum IgE.11B-11N is shown to be supervised by pDA2:GUS transgene expression The DA2 expression activity of survey.It observes four GUS expression systems, and all shows similar mode, although they are in the strong of dyeing Degree aspect is slightly different.4 day age seedling (11B), 10 day age seedling (11C), the inflorescence (11D) spent, the petal (11E- just developed 11G), in the stamen (11H and 11I) just developed, the carpel (11J-11L) just developed and the ovule just developed (11M and 11N) The active tissue chemical analysis of GUS.Ruler item: B-D, 1mm；E-N, 0.1mm.
Figure 12 shows DA1 and directly interacts in vitro with DA2.By GST-DA1, GST-DA1R358K, GST-DA1- UIM, GST-DA1-LIM, GST-DA1-LIM+C and GST-DA1-C are pulled down by the MBP-DA2 being fixed on amylose resin Come (PD) and is analyzed using anti-GST antibody by immunoblotting (IB).
Figure 13 shows the schematic diagram of the DA1 containing specific protein domains and its derivative.The DA1 protein of prediction Including two UIM motifs, single LIM domain and C-terminal region.
Figure 14 shows DA1 and interacts in vivo with DA2.Ben's tobacco (Nicotiana benthamiana) leaf is logical Cross the Agrobacterium tumefaciens (Agrobacterium that injection possesses 35S:Myc-DA1 and 35S:GFP-DA2 plasmid Tumefaciens) GV3101 cell is converted.By gross protein GFP-Trap-A immunoprecipitation, and respectively with anti-GFP Antibody and anti-Myc antibody detect immunoblotting.Myc-DA1 is detected in the GFP-DA2 compound of immunoprecipitation, to indicate There are physical correlations between DA1 and DA2 in plant.
Figure 15 shows the da2-1 mutation increased organ size of body display.15A shows Col-0, da2-1 and 35S:DA2#1 Petal length (PL), petal width (PW), petal area (PA), sepal area (SA), the carpel length (CL), long stamen of plant Length (LSL) and short stamen length (SSL).15B shows the 5th leaf area of Col-0, da2-1 and 35S::DA2#1 plant. 15C shows the weight of Col-0, da2-1 and 35S::DA2#1 flower.15D shows the maximum width region of Col-0 and da2-1 petal In paraxial epidermal cell size.15E shows the size of the palisade cell in the 5th leaf of Col-0 and da2-1.Open flower (stage 14) is used to measure the size (15A), the weight (C) of flower and the size (15D) of epidermal cell of petal.Numerical value (A-E) is made To provide relative to the average value ± SE of corresponding wild type numerical value, it is arranged to 100%.Compared to wild type, * *, P < 0.01 (is learned Raw t is examined).
Figure 16 shows DA1 and DA2 synergistic effect to control seed size.16D show Col-0, da1-ko1, da2-1 and The petal area of da1-ko1 da2-1 flower.16E shows the maximum of Col-0, da1-ko1, da2-1 and da1-ko1da2-1 petal The size of paraxial epidermal cell in width regions.16F shows the seed weight of Col-0, eod1-2, da2-1 and eod1-2da2-1 Amount.16G shows the petal area of Col-0, eod1-2, da2-1 and eod1-2da2-1.Open flower (stage 14) is for measuring The size (16D and 16G) of petal and the size (16E) of epidermal cell.It is worth (16D-G) as relative to corresponding wild type numerical value Average value ± SE is provided, and is arranged to 100%.Compared to wild type, * *, P < 0.01 and *, P < 0.05 (student t inspection).Ruler item: 0.1mm。
Figure 17 shows the overexpression limitation organ growth of DA2.17A shows Col-0,35S:DA2#2 and 35S:DA2#4's Petal area.17B shows the expression of the DA2 in Col-0,35S:DA2#2 and 35S:DA2#4 seedling.It is worth (A and B) conduct Average value ± SE relative to Col-0 value is provided, and is arranged to 100%.Compared to wild type, * *, P < 0.01 (student t inspection).
Figure 18 shows the overexpression limitation organ growth of DA2L.18A shows Col-0,35S:DA2L#1,35S:DA2L# 3,20 day age plant of 35S:DA2L#4,35S:DA2L#5 and 35S:DA2L#6.18B shows Col-0,35S:DA2L#1,35S: 30 day age plant of DA2L#3,35S:DA2L#4,35S:DA2L#5 and 35S:DA2L#6.18C show Col-0,35S:DA2L#1, The RT-PCR analysis of DA2L expression in 35S:DA2L#3,35S:DA2L#4,35S:DA2L#5 and 35S:DA2L#6 seedling.To from First chain cDNA of 2 week old seedling preparation carries out RT-PCR.By cDNA by reference to ACTIN2 standard.Ruler item: A, 1cm, B, 1cm
Figure 19 shows the overexpression limitation seed and organ growth of GW2.19A shows Col-0,35S:GW2#1,35S: 30 day age plant of GW2#2,35S:GW2#3,35S:GW2#6 and 35S:GW2L#7.19B shows Col-0,35S:GW2#1,35S: The projected area of GW2#2,35S:GW2#3,35S:GW2#6 and 35S:GW2L#7 seed.19C show Col-0,35S:GW2#1, The quantitative real-time RT-PCR of GW2 gene expression in 35S:GW2#2,35S:GW2#3,35S:GW2#6 and 35S:GW2L#7 seedling Analysis.Numerical value (B) is provided as the average value ± SE relative to Col-0 numerical value, is arranged to 100%.Compared to wild type, * *, P < 0.01 (student t inspection).Ruler item: A, 1cm
Specific embodiment
The present invention relates to pass through the expression for changing plant E3 ubiquitin ligase DA2 or active Binding change DA1 and/or EOD1 Expression or activity come change influence yield plant trait (such as seed and organ size) method.Preferably, in plant Change the expression or activity of DA2 and DA1.
The expression or activity of DA2 can be changed prior to, concurrently with, or after the expression or activity for changing DA1 and/or EOD1.Example Such as, in some embodiments, the expression or activity of DA2 polypeptide can be changed in one or more plant cells, the plant is thin Born of the same parents have had following one: the DA1 expression of change or activity, the EOD1 expression of change or activity or the DA1 and EOD1 that change Expression or activity.
Provided herein is for example increasing the method for the yield of plant by increasing organ or seed size, the method includes It provides and lacks DA1 and/or EOD1 expression or active plant, and reduce the DA2 in one or more cells of the plant Expression.In other embodiments, the expression or activity of the DA1 and/or EOD1 in one or more plant cells, institute can be reduced Plant cell is stated with reduced DA2 polypeptide expression or activity.
Other methods may include the expression of the DA2 in the one or more cells for reduce plant and reduce one or more The expression or activity of DA1, EOD1 or DA1 and both EOD1 in cell.
The method for generating the plant that there is increased yield relative to wild-type plant is also provided herein, comprising:
(a) the following terms is incorporated in plant cell by converting:
(i) the first heterologous nucleic acids, the nucleic acid reduce the expression of DA2 polypeptide,
(ii) the second heterologous nucleic acids, the nucleic acid reduce the expression of one of DA1 polypeptide and EOD1 polypeptide, and optionally,
(iii) third heterologous nucleic acids, the nucleic acid reduce the expression of the other of DA1 polypeptide and EOD1 polypeptide, and
(b) plant described in the cytothesis from one or more conversion.
Generate the other methods with the plant of increased yield can include:
It provides and lacks DA1 and/or EOD1 expression or activity, the active plant cell of preferably DA1,
The heterologous nucleic acids of the activity of reduction DA2 polypeptide or expression are incorporated in the plant cell by converting, and；
Plant described in cytothesis from the conversion.
After regeneration, it may be selected that there is reduced DA2 polypeptide active or expression and reduction relative to wild-type plant DA1 and/or EOD1 activity or expression plant.
The combination of reduced DA2 expression and reduced DA1 and/or EOD1 expression cooperates with the seed and/or device for increasing plant The size of official, to increase plant products.
One of plant or a variety of characters relevant to yield can be expressed by reduced DA2 or activity combines reduction DA1 and/or EOD1 expression or activity are improved.For example, the control or wild type that can not yet be reduced relative to the expression of DA2 polypeptide One or more of service life, organ size and the seed size of plant increase plant.
The expression of DA2, DA1 or EOD1 or activity can reduce at least in method described herein relative to wild-type plant 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98%.In some preferred implementations In example, expression or activity are reduced to zero or are substantially zero (i.e. expression or activity are eliminated).
The method of the present invention includes the expression or activity of the DA2 polypeptide in the one or more cells for changing plant.
DA2 polypeptide is the E3 ubiquitin ligase found in plant.The DA2 that expression or activity are lowered as described herein Polypeptide may include RING structural domain (Stone, S.L. et al. (2005)), it is therefore preferable to C5HC2, C5NC2 or C5TC2 RING knot Structure domain.Suitable RING structural domain can be made of the amino acid sequence of SEQ ID NO:1；
C(X)2C(X)11CC(X)4CX2CX7(H/N/T)X6CX2C。(SEQ ID NO:1)
For example, suitable RING structural domain can be made of the amino acid sequence of SEQ ID NO:2；
CPICFL(Y/F)YPSLNRS(K/R)CC(S/M/T/A)K(G/S)ICTECFL(Q/R)MK(P/N/S/V/T/N) (T/P)(H/N/T)(T/S)(A/T/C)(R/Q/K)PTQCP(F/Y)C
(SEQ ID NO:2)
In some embodiments, the H/N/T residue at the position 33 in the RING structural domain of SEQ ID NO:2 can be T Or N.
In some preferred embodiments, DA2 polypeptide may include RING structural domain, and the RING structural domain has in table 1 Shown in amino acid sequence (SEQ ID NO:3-19), such as arabidopsis DA2 (SEQ ID NO:11), arabidopsis DAL2 (SEQ ID NO:13) or rice GW2 (SEQ ID NO:7) or its variant.For example, RING structural domain can have the amino acid of the following terms Sequence: residue 59 to 101, the SEQ ID NO:21 (Rc_GI- of SEQ ID NO:20 (Pt_GI-224061326.pro) The residue 59 to 101 of residue 59 to 101, SEQ ID NO:22 (Vv_GI-147817790.pro) 255578534.pro), Residue 59 to 101, the SEQ ID NO:24 (At_GI- of SEQ ID NO:23 (Gm_GI-356549538.pro) Residue 61 to 103, the SEQ of residue 59 to 101, SEQ ID NO:25 (Ta_GI 408743661.pro) 18411948.pro) Residue 61 to 103, the SEQ ID NO:27 (Bd_GI-357140854.pro) of ID NO:26 (Hv_GI-164371454.pro) Residue 61 to 103, SEQ ID NO:28 (Os_GI-115445269.pro) residue 62 to 104, SEQ ID NO:29 (Sb_ GI-242064618.pro the residue 65 of residue 63 to 105, SEQ ID NO:30 (Zm_GI-220961719.pro)) to 107, the residue 61 to 103 of SEQ ID NO:31 (Ta_GI-408743658.pro), SEQ ID NO:32 (Bd_GI- Residue 62 to 104, the SEQ of residue 43 to 85, SEQ ID NO:33 (Os_GI-218197613.pro) 357125256.pro) The residue 62 to 104 or SEQ ID NO:35 (Sb_GI-242092026.pro) of ID NO:34 (Zm_GI-260935347.pro) Residue 62 to 104.
Other suitable RING domain sequences can be used standard sequence analysis technology as described herein (for example, simple Module tectonic cycle period tool (SMART)；EMBL Heidelberg, DE) identify.
DA2 polypeptide may also include the first apokoinou construction domain.First apokoinou construction domain can be located at the upstream of RING structural domain (i.e. In N-terminal side).The first suitable apokoinou construction domain can be made of the amino acid sequence of SEQ ID NO:36.
Q (Q/ is not present) GLY (P/M/N/V/Q/L/V/E) (H/S/N) (P/K/R) D (I/V) D (L/I/H/V/Q) (K/R) KL
(R/K)(R/K)LI(V/L)(E/D)(A/S/T)KLAPC
(SEQ ID NO:36)
In some preferred embodiments, DA2 polypeptide may include the first shared of DA2 amino acid sequence shown in table 2 Structural domain, such as the residue 20 to 45 of SEQ ID NO:20, the residue 20 to 45 of SEQ ID NO:21, SEQ ID NO:22's are residual Base 20 to 45, the residue 20 to 45 of SEQ ID NO:23, the residue 20 to 45 of SEQ ID NO:24, SEQ ID NO:25 residue 21 to 46, the residue 21 of the residue 21 to 46 of SEQ ID NO:26, the residue 21 to 46 of SEQ ID NO:27, SEQ ID NO:28 To the 46, residue 21 to 46 of SEQ ID NO:29, the residue 21 to 46 of SEQ ID NO:30, SEQ ID NO:31 residue 21 to 46, the residue 23 to 48 of the residue 4 to 29 of SEQ ID NO:32, the residue 23 to 48 of SEQ ID NO:33, SEQ ID NO:34 Or the residue 23 to 48 of SEQ ID NO:35.
DA2 polypeptide may also include the second apokoinou construction domain.Second apokoinou construction domain can be located at the downstream of RING structural domain (i.e. On C-terminal side).Second apokoinou construction domain can be made of the amino acid sequence of SEQ ID NO:37.
(N/S)YAVEYRG(V/G)K(T/S)KEE(K/R)(G/S)(V/T/I/F/L/M)EQ(L/I/V/F)EEQ(R/L/ K)VIEA(Q/K)(I/M)RMR(H/Q)(K/Q)(E/A)
(SEQ ID NO:37)。
In some preferred embodiments, DA2 polypeptide may include the second shared of DA2 amino acid sequence shown in table 2 Structural domain, such as the residue 106 to 141 of SEQ ID NO:20, the residue 106 to 141 of SEQ ID NO:21, SEQ ID NO:22 Residue 106 to 141, the residue 106 to 141 of SEQ ID NO:23, the residue 106 to 141 of SEQ ID NO:24, SEQ ID The residue 107 to 143 of NO:25, the residue 107 to 143 of SEQ ID NO:26, SEQ ID NO:27 residue 107 to 143, SEQ The residue 108 to 144 of ID NO:28, the residue 109 to 145 of SEQ ID NO:29, SEQ ID NO:30 residue 111 to 147, The residue 107 to 143 of SEQ ID NO:31, the residue 90 to 125 of SEQ ID NO:32, SEQ ID NO:33 residue 108 to 143, the residue 108 to 143 of the residue 108 to 143 of SEQ ID NO:34 or SEQ ID NO:35.
Standard sequence analysis skill as described herein can be used in other examples of the first and second suitable domain sequences Art is (for example, simple module tectonic cycle period tool (SMART)；EMBL Heidelberg, DE) identify.
In some preferred embodiments, the DA2 polypeptide that expression or activity are lowered as described herein may include SEQ Second shared knot of the RING structural domain of ID NO:2, the first apokoinou construction domain of SEQ ID NO:36 and SEQ ID NO:37 Structure domain.
For example, DA2 polypeptide may include RING domain sequence listed above, the first apokoinou construction domain sequence and second Any combination of apokoinou construction domain sequence.
Suitable DA2 polypeptide may include the amino acid sequence such as any of the SEQ ID NO 20 to 35 that lists in table 2 Or it can be one variant in these sequences.In some preferred embodiments, DA2 polypeptide may include SEQ ID NO:28 Or 33 (OsGW2) amino acid sequence, SEQ ID NO:24 (AtDA2), SEQ ID NO:25 or SEQ ID NO:31 (TaGW2) Or the variant that any of can be these sequences with E3 ubiquitin ligase activity.
For SEQ ID NO:20 to any of 35 or other with reference to DA2 sequence variant DA2 polypeptides may include with It is described with reference to DA2 sequence have at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, the amino acid sequence of at least 90%, at least 95% or at least 98% sequence identity.
DA2 polypeptide for the variant of SEQ ID NO:20 to any of 35 may also include the sequence with SEQ ID NO:2 The RING structural domain of column, the first apokoinou construction domain of sequence with SEQ ID NO:36 and the sequence with SEQ ID NO:37 Second apokoinou construction domain of column.The example of suitable sequence is as listed above out.In some preferred embodiments, DA2 polypeptide can RING structural domain, the first apokoinou construction domain and the second apokoinou construction domain including SEQ ID NO:20 to any of 35.
The nucleic acid of encoding D A2 polypeptide may include selected from by JN896622.1 GI:408743658 (TaGW2-A)；With The nucleotide sequence listed or can be in the data base entries of the group of JN896623.1 GI:408743660 (TaGW2-B) composition It is one variant in these sequences.
In some preferred embodiments, the nucleic acid of encoding D A2 polypeptide may include coding AtDA2, AtDAL2, OsGW2, The nucleotide sequence of TaGW2-A or TaGW2-B or the variant that any of can be these DA2 sequences, coding have DA2 Active polypeptide.
DA2 polypeptide and code nucleic acid, institute can be identified using routine sequence analysis technology in any target plant species Plant species specifically crop is stated, such as wheat, barley, corn, rice, soybean；And other agricultural plant.
DA2 expression or active reduction show the prominent of the activity or expression for cooperateing with enhancing to reduce DA1 herein in plant Become the effect to the Correlated Yield Characters in plant.In a preferred embodiment, method described herein may include reducing to lack Both DA2 expression in DA1 expression or active plant or the DA1 in reduction plant and DA2 expression.
DA1 polypeptide be the ubiquitin receptor found in plant and in Li et al. people (2008), Wang et al. (2012) and It is described in detail in WO2009/047525.The DA1 polypeptide that expression or activity are lowered as described herein may include LIM domain, protect The C-terminal structural domain and one or more UIM structural domain kept.
LIM domain includes two Zn zinc-finger motifs and can have amino acid sequence (SEQ ID NO:38)；
C(X)2 C(X)16-23 (H/C)(X)2/4 (C/H/E)(X)2 C(X)2 C(X)14-21 (C/H)(X)2/1/3 (C/H/D/E)X
Wherein X is any amino acid and Zn coordination residue underlines.
Zn coordination residue in LIM domain can be C, H, D or E, preferably C.
In some preferred embodiments, LIM domain may include CXXC, HXXCXXCXXC and HxxC motif, and wherein X is Any amino acid.For example, LIM domain may include amino acid sequence (SEQ ID NO:39)；
C(X)2 C(X)16-23 (H)(X)2 (C)(X)2 C(X)2 C(X)14-21 H(X)2 CX
Wherein X is any amino acid and Zn coordination residue underlines.
In some embodiments, LIM domain may include the amino acid sequence of AtDA1 LIM domain；
CAGCNMEIGHGRFLNCLNSLWHPECFRCYGCSQPISEYEFSTSGNYPFHKACY
(SEQ ID NO:40；Zn coordination residue underlines)
Other LIM domains include the LIM domain of DA1 amino acid sequence shown in table 3, such as SEQ ID NO:41 (Si_GI-514815267.pro) residue of residue 141 to 193, SEQ ID NO:42 (Bd_GI-357157184.pro) 123 to 175, the residue 155 to 207 of SEQ ID NO:43 (Br_DA1b.pro), SEQ ID NO:44 (Br_DA1a.pro) Residue 172 to 224, the residue 172 to 224 of SEQ ID NO:45 (At_GI-15221983.pro), SEQ ID NO:46 (Tc_ GI-508722773.pro the residue 117 of residue 117 to 169, SEQ ID NO:47 (Gm_GI-356564241.pro)) to 169, the residue 121 to 173 of SEQ ID NO:48 (Gm_GI-356552145.pro), SEQ ID NO:49 (Vv_GI- The residue 122 to 174 of residue 119 to 171, SEQ ID NO:50 (Vv_GI-359492104.pro) 302142429.pro), Residue 125 to 177, the SEQ ID NO:52 (Os_GI- of SEQ ID NO:51 (Sl_GI-460385048.pro) The residue 124 to 176 of residue 516 to 568, SEQ ID NO:53 (Os_GI-115466772.pro) 218197709.pro), Residue 150 to 202, the SEQ ID NO:55 (Bd_GI- of SEQ ID NO:54 (Bd_GI-357160893.pro) The residue 124 to 176 of residue 132 to 184, SEQ ID NO:56 (Sb_GI-242092232.pro) 357164660.pro), Residue 147 to 199, the SEQ ID NO:58 (At_GI- of SEQ ID NO:57 (Zm_GI-212275448.pro) The residue 162 to 214 of residue 190 to 242, SEQ ID NO:59 (At_GI-145360806.pro) 240256211.pro), Residue 1240 to 1291, the SEQ ID NO:61 (At_GI- of SEQ ID NO:60 (At_GI-22326876.pro) Residue 347 to 402, the SEQ of residue 80 to 122, SEQ ID NO:62 (At_GI-30698240.pro) 30698242.pro) Residue 286 to 341, the SEQ ID NO:64 (At_GI-334188680.pro) of ID NO:63 (At_GI-15240018.pro) Residue 202 to 252.
LIM domain sequence can be used standard sequence analysis technology (for example, simple module tectonic cycle period tool (SMART)； EMBL Heidelberg, DE) identify.
In addition to LIM domain, DA1 albumen may also include carboxy-terminal end region, the carboxy-terminal end region have with it is following Every sequence at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, the amino acid sequence of at least 95% or at least 98% amino acid identities: the residue 198 to 504 of SEQ ID NO:41, The residue 180 to 487 of SEQ ID NO:42, the residue 212 to 514 of SEQ ID NO:43, SEQ ID NO:44 residue 229 to 532, the residue of the residue 229 to 532 of SEQ ID NO:45, the residue 174 to 478 of SEQ ID NO:46, SEQ ID NO:47 174 to 474, the residue 178 to 478 of SEQ ID NO:48, the residue 176 to 462 of SEQ ID NO:49, SEQ ID NO:50 Residue 179 to 482, the residue 182 to 486 of SEQ ID NO:51, the residue 573 to 878 of SEQ ID NO:52, SEQ ID NO: 53 residue 181 to 486, the residue 207 to 512 of SEQ ID NO:54, the residue 189 to 491 of SEQ ID NO:55, SEQ ID The residue 181 to 486 of NO:56, the residue 204 to 508 of SEQ ID NO:57, SEQ ID NO:58 residue 247 to 553, SEQ The residue 219 to 528 of ID NO:59, the residue 1296 to 1613 of SEQ ID NO:60, SEQ ID NO:61 residue 128 to 450, the residue of the residue 404 to 702 of SEQ ID NO:62, the residue 343 to 644 of SEQ ID NO:63, SEQ ID NO:64 256 to 587.
The carboxy-terminal end region of DA1 albumen may include metallopeptidase motif HEMMH (SEQ ID NO:65).
Carboxy-terminal end region may also include the EK (X) between LIM domain and HEMMH motif8R(X)4SEEQ(SEQ ID NO:66) or EK (X)8R(X)4SEQ (SEQ ID NO:67) motif.
Other than LIM domain and conservative carboxy-terminal end region, DA1 albumen may include UIM1 structural domain and UIM2 knot Structure domain.UIM1 and UIM2 structural domain can be located between the N-terminal and LIM domain of DA1 albumen.
UIM1 structural domain can be made of the sequence of SEQ ID NO:68 and UIM2 structural domain can be by SEQ ID NO:69's Sequence composition.
p---pLpbAl pb.Sbp-.pp p(SEQ ID NO:68)
p---pLpbAl pb.Sbp-spp p(SEQ ID NO:69)
Wherein；
P is polar amino acid residues, for example, C, D, E, H, K, N, Q, R, S or T；
B is big amino acid residue, for example, E, F, H, I, K, L, M, Q, R, W or Y；
S is p1 amino acid residue, for example, A, C, D, G, N, P, S, T or V；
L is aliphatic amino acid residue, such as I, L or V；
Be be not present or any amino acid, and
It is any amino acid.
Standard sequence analysis technology (example as described herein can be used in other examples of UIM1 and UIM2 domain sequence Such as, simple module tectonic cycle period tool (SMART)；EMBL Heidelberg, DE) identify.
In some preferred embodiments, DA1 polypeptide may include；
The LIM domain of SEQ ID NO:39,
C-terminal region, the residue 229 to 532 or SEQ NO 41 to 44 of the C-terminal region and SEQ ID NO:45 or The equivalent area of any one in 46 to 64 has at least 20% sequence identity, such as listed above and including EK (X)8R(X)4SEEQ or EK (X)8R(X)4SEQ motif and HEMMH motif,
The UIM structural domain of SEQ ID NO:66, and
The UIM structural domain of SEQ ID NO:67.
DA1 albumen may include plant DA1 albumen shown in table 3 amino acid sequence (SEQ ID NO:41 to 64) or can To be one homologue or variant in these sequences, the homologue or variant have DA1 activity.For example, DA1 polypeptide can Including amino acid sequence shown in table 3 (SEQ ID NO:41 to 64) or it can be one variant in these sequences, institute Variant is stated with DA1 activity.
For example, DA1 polypeptide may include AtDA1, AtDAR1, AtDAR2, AtDAR3, AtDAR4, AtDAR5, AtDAR6, AtDAR7、BrDA1a、BrDA1b、BrDAR1、BrDAR2、BrDAR3-7、BrDAL1、BrDAL2、BrDAL3、OsDA1、 OsDAR2、OsDAL3、OsDAL5、PpDAL1、PpDAL2、PpDAL3、PpDAL4、PpDAL5、PpDAL6、PpDAL7、PpDAL8、 The amino acid sequence of SmDAL1, SmDAL2 or ZmDA1, preferably AtDA1, AtDAR1, BrDA1a, BrDA1b, OsDA1 or One homologue or variant in the amino acid sequence of ZmDA1 or these sequences.
In some preferred embodiments, DA1 polypeptide may include AtDA1 (AT1G19270；NP_173361.1GI: 15221983) amino acid sequence or the variant that can be this sequence, the variant have DA1 activity.
Standard sequence analysis tool can be used to reflect for other DA1 protein sequences including the above-mentioned characteristic features listed Not.Technical staff can easily identify the nucleic acid sequence of the encoding D A1 albumen in any target plant species.
DA1 albumen in target plant species can have for the variant for the DA1 albumen reference amino acid sequence listed herein Amino acid sequence.
For with reference to DA1 sequence variant DA1 polypeptide, such as any of SEQ ID NO 41 to 64, it may include with institute State reference sequences at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, The amino acid sequence of at least 90%, at least 95% or at least 98% sequence identity.
The specific amino acid sequence variation occurred in plant species can exist with the reference sequences difference listed herein In 1 amino acid, 2,3,4,5-10,10-20,20-30,30-50 or the insertion more than 50 amino acid, addition, substitution or lack It loses.
In some embodiments, the DA1 polypeptide for the variant of the AtDA1 sequence of SEQ ID NO:45 may include with sequence The UIM1 structural domain of QENEDIDRAIALSLLEENQE (SEQ ID NO:70) and have sequence D EDEQIARALQESMVVGNSP The UIM2 structural domain of (SEQ ID NO:71).
DA1 polypeptide for the variant of the AtDA1 sequence of SEQ ID NO:45 may include having following sequence of LIM structure Domain:
ICAGCNMEIGHGRFLNCLNSLWHPECFRCYGCSQPISEYEFSTSGNYPFHKAC
(SEQ ID NO:72)
The nucleic acid of encoding D A1 polypeptide may include selected from by NM_101785.3GI:42562170 (AtDA1)；NM_ 001057237.1GI:115454202(OsDA1)；The database of the group of BT085014.1GI:238008663 (ZmDA1) composition The nucleotide sequence listed in entry can be one variant in these sequences, the variant encoding active DA1 polypeptide.
In some preferred embodiments, the nucleic acid of encoding D A1 polypeptide may include AtDA1 (NM_101785.3GI: 42562170), the core of ZmDA1 (BT085014.1GI:238008663), OsDA1 (NM_001057237.1GI:115454202) Nucleotide sequence or the variant that any of can be these sequences, coding retain the active polypeptide of DA1.
DA1 polypeptide and code nucleic acid, the plant object can be identified using routine sequence analysis technology in plant species Kind specifically crop, such as wheat, barley, corn, rice and other agricultural plant.
In some preferred embodiments, the DA1 activity in one or more cells of plant can be by one Or dominant negative DA1 polypeptide is expressed in multiple cells to reduce (see, for example, Li et al. people (2008)；WO2009/047525； Wang et al. is 2012).The plant for expressing dominant negative DA1 polypeptide can have da1-1 phenotype.
The dominant negative allele of DA1 polypeptide may include that there is mutation (such as to replace or lack at conservative R residue Lose) DA1 polypeptide, the R residue is located at the position of the position 358 of arabidopsis DA1 amino acid sequence, corn DA1 amino acid sequence It sets at the equivalent position in 333 or another kind DA1 amino acid sequence.For example, the dominant negative allele of DA1 polypeptide may include Guarantor at the position of the position 333 of the position 358 or corn DA1 amino acid sequence that are equivalent to arabidopsis DA1 amino acid sequence Keep the mutation of R residue.In a preferred embodiment, conservative R residue can be replaced by K.
The position 358 of the SEQ ID NO:45 for being equivalent to arabidopsis DA1 in DA1 amino acid sequence or SEQ ID Conservative R residue at the position of the position 333 of the corn DA1 of NO:57 is located in DA1 amino acid sequence and corresponds to SEQ ID At the position of the R358 of the R333 and SEQ ID NO:45 of NO:57, i.e., it is in other motifs and structural domain relative to DA1 albumen Same position in.Conservative R residue is located between the LIM domain in C-terminal region and HEMMH peptase motif, and in DA1 It is completely conservative under same sequence background in albumen.Conservative R residue may include the EK (X) in C-terminal region8R(X)4SEEQ (SEQ ID NO:66) or EK (X)8R(X)4In SEQ (SEQ ID NO:67) motif.
Conservative R residue can compare these conservative C-terminal regions by using standard sequence analysis and comparison tool And identifies and use arrow logo in the sequence of table 3.
The nucleic acid of the dominant negative allele of encoding D A albumen can be generated by any convenient technology.For example, can Site directed mutation is used on the nucleic acid of encoding D A1 polypeptide to change in the R333 with the R358 of arabidopsis DA1 or corn DA1 Conservative R residue at equivalent position, such as change into K.Reagent and kit for being mutated in vitro are commercially available.
In some embodiments, the nucleic acid for encoding dominant negative DA1 polypeptide as described herein can operationally connect It is connected to heterologous regulatory sequence, such as promoter, such as composing type, induction type, tissue specificity or development-specific promoter.Coding The nucleic acid of dominant negative DA1 polypeptide may include in one or more carriers.For example, the dominant negative etc. of encoding D A1 albumen The nucleic acid of the mutation of position gene can also be cloned into expression vector and be expressed in plant cell as described below to change and plant Object phenotype.
In other embodiments, in the endogenous DA1 nucleic acid that mutation can be introduced in plant, so that by mutant DA1 The DA1 polypeptide of nucleic acid encode has dominant negative activity.
The nucleic acid of the DA1 polypeptide of encoding dominant negative feminine gender can its initial separation from identical plant species or mutation in table It reaches or is expressed in different plant species or mutation (i.e. heterologous plant).
What DA2 was expressed in plant reduces or eliminates in the expression or active mutation pair for also showing enhancing reduction EOD1 herein The effect of Correlated Yield Characters in plant.
Method described herein may include the DA2 expression reduced lack in EOD1 expression or active plant or activity or drop The expression or activity of DA2 and both EOD1 in low plant.In a preferred embodiment, plant can also lack DA1 activity or institute Stating method in addition may include the DA1 expression reduced or eliminated in the plant.
EOD1 polypeptide is the E3 ubiquitin ligase found in plant and in Disch et al. (2006), Li et al. people (2008) and in WO2009/047525 it is described in detail.
The EOD1 polypeptide that expression or activity are lowered as described herein may include EOD structural domain.Suitable EOD structural domain It can be made of the amino acid sequence of SEQ ID NO:73；
(E/K)RCVICQ(L/M)(K/R/G/T/E)Y(K/R)(R/I)(G/K)(D/N/E)(R/Q/K/L)Q(I/M/V) (K/N/T/A)L(L/P)C(K/S)H(V/A)YH(S/T/G/A)(E/Q/D/S/G)C(I/G/T/V)(S/T)(K/R)WL(G/T/ S)INK(V/I/A/K)CP(V/I)C(SEQ ID NO:73)
In some preferred embodiments, EOD1 polypeptide may include the EOD structure with the amino acid sequence of the following terms Domain: residue 195 to 237, the SEQ ID NO:75 (Sb_GI- of SEQ ID NO:74 (Zm_GI-223973923.pro) The residue 195 to 237 of residue 195 to 237, SEQ ID NO:76 (Zm_GI-226496789.pro) 242042045.pro), Residue 218 to 260, the SEQ ID NO:78 (Os_GI- of SEQ ID NO:77 (Os_GI-222624282.pro) The residue 197 to 239 of residue 196 to 238, SEQ ID NO:79 (Bd_GI-357113826.pro) 115451045.pro), Residue 193 to 235, the SEQ ID NO:81 (Rc_GI- of SEQ ID NO:80 (Sl_GI-460410949.pro) The residue 150 to 192 of residue 187 to 229, SEQ ID NO:82 (Pt_GI-224059640.pro) 255582236.pro), Residue 194 to 236, the SEQ ID NO:84 (Gm_GI- of SEQ ID NO:83 (Gm_GI-356548935.pro) The residue 194 to 236 of residue 194 to 236, SEQ ID NO:85 (Vv_GI-359487286.pro) 356544176.pro), Residue 189 to 231, the SEQ ID NO:87 (Pp_GI- of SEQ ID NO:86 (Tc_GI-508704801.pro) The residue 190 to 232 of residue 192 to 234, SEQ ID NO:88 (Cr_GI-482561003.pro) 462414664.pro), The residue 195 to 237 of SEQ ID NO:89 (At_GI-22331928.pro) or the residue 195 to 237 of SEQ ID NO:90 (Sl_GI-460370551.pro), as shown in table 4.
Other suitable EOD domain sequences can be used standard sequence analysis technology as described herein (for example, simple mould Block tectonic cycle period tool (SMART)；EMBL Heidelberg, DE) identify.
The EOD1 polypeptide that expression or activity are lowered as described herein may include SEQ ID NO as listed in Table 4 Any of 74 to 90 amino acid sequence.In some preferred embodiments, EOD1 polypeptide may include SEQ ID NO:89 (AtEOD1) or the amino acid sequence of SEQ ID NO:77 or 78 (OsEOD1) or the variant that can be this sequence it is general, to retain E3 Element connection enzymatic activity.
May include for SEQ ID NO:74 to any of 90 or other EOD1 polypeptides for referring to the variant of EOD1 sequence With it is described with reference to EOD1 sequence have at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, The amino acid sequence of at least 80%, at least 90%, at least 95% or at least 98% sequence identity.
It may also include for the EOD polypeptide of the variant of SEQ ID NO:74 to any of 90 with SEQ ID NO:73's The EOD structural domain of sequence.The example of suitable sequence is as listed above out.
The nucleic acid for encoding EOD1 polypeptide may include the core listed in the data base entries selected from the group being made of the following terms Nucleotide sequence: XM_002299911.1GI:224059639 (PtEOD1)；XM_002531864.1GI:255582235 (RcEOD1)；XM_002279758.2GI:359487285(VvEOD1)；XM_003542806.1GI:356548934 (GmEOD1a)；XM_003540482.1GI:356544175(GmEOD1b)；XM_002468372.1GI:242042044 (SbEOD1)；NM_001147247.1GI:226496788(ZmEOD1)；Or NP_001030922.1GI:79316205 (AtEOD1；At3g63530), or it can be one variant in these sequences.
In some preferred embodiments, the nucleic acid of coding EOD1 polypeptide may include the nucleosides for encoding AtEOD1 or OsEOD1 Acid sequence or the variant that any of can be these sequences, the variant coding have the active polypeptide of EOD1.
Expression can be identified using routine sequence analysis technology in any target plant species or activity is as described herein The EOD1 polypeptide and code nucleic acid being lowered, the plant species specifically crop, such as wheat, barley, corn, rice, big Beans；And other agricultural plant.
DA2 mutation in plant is also showing the combination of collaboration enhancing DA1 and EOD1 mutation to the yield in plant herein The effect of correlated traits.
Method described herein is not limited to specific plant species, and DA2, DA1 and/or EOD1 expression or activity can It is reduced in any target plant species, as described herein.
DA1, DA2 or EOD1 polypeptide in target plant species can have correspondence DA1, DA2 or EOD1 to list herein The amino acid sequence of the variant of reference amino acid sequence.DA1, DA2 or EOD1 for the variant for the reference sequences listed herein is more Peptide may include with the reference sequences have at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, the amino acid sequence of at least 80%, at least 90%, at least 95% or at least 98% sequence identity.
The specific amino acid sequence variation occurred in plant species can exist with the reference sequences difference listed herein In 1 amino acid, 2,3,4,5-10,10-20,20-30,30-50 or the insertion more than 50 amino acid, addition, substitution or lack It loses.
DA1, DA2 or EOD1 nucleic acid in target plant species can have correspondence DA1, DA2 or EOD1 to list herein The nucleotide sequence of the variant of reference nucleotide sequence.For example, Variant nucleotide sequences can be the reference DA1 listed herein, The homologue or allele of DA2 or EOD1 sequence, and can be with the difference with reference to DA1, DA2 or EOD1 nucleotide sequence Place be one or more of nucleic acid (such as 2,3,4,5-10,10-20,20-30,30-50 or more than 50) nucleotide Addition, one or more of insertion, deletion or substitution, add so as to cause one or more amino acid in the polypeptide of coding Add, insertion, deletion or substitution.Certainly, the variation on the amino acid sequence of coding without influence including being carried out on nucleic acid.DA1, DA2 or EOD1 code nucleic acid may include the sequence for having at least 20% or at least 30% sequence identity with reference nucleic acid sequence, Preferably at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95% or At least 98%.Sequence identity is in above description.
Sequence similarity and identity are typically referenced to algorithm GAP (Wisconsin Package, Accelerys, San Diego USA) Lai Dingyi.GAP compares two complete sequences using Needleman and Wunsch algorithm, and the algorithm makes The number matched maximizes and minimizes the number in vacancy.In general, using default parameter, wherein gap creation penalty=12 Hes Gap extension penalties=4.It the use of GAP may be preferred, but other algorithms can also be used, such as (it is used BLAST The method of Altschul et al. (1990) J.Mol.Biol.215:405-410), FASTA (its use Pearson and Lipman (1988) method of PNAS USA 85:2444-2448) or Smith-Waterman algorithm (Smith and Waterman (1981) J.Mol Biol.147:195-197) or TBLASTN program for being same as above of Altschul et al. (1990), generally use default ginseng Number.Specifically, psi-Blast algorithm (Nucl.Acids Res. (1997) 253389-3402) can be used.
Sequence, which compares, to be carried out in the overall length of correlated series as described herein.
Suitable variant amino acids and nucleotide sequence can be used standard sequence analysis technology in any target plant species Middle identification.
For DA1, DA2 or EOD1 nucleotide sequence of the variant for reference DA1, DA2 or EOD1 nucleic acid sequence listed herein Can selectively it hybridize with the nucleic acid sequence or its complement under strict conditions.
Stringent condition includes for example, being the identical sequence of about 80%-90% for hybridization, in 0.25M at 42 DEG C Na2HPO4In (pH 7.2), 6.5%SDS, 10% dextran sulfate hybridized overnight and at 55 DEG C in 0.1X SSC, 0.1%SDS In finally wash.For being detected as greater than about 90% identical sequence, suitable condition is included at 65 DEG C in 0.25M Na2HPO4, pH 7.2,6.5%SDS, in 10% dextran sulfate hybridized overnight and at 60 DEG C in 0.1X SSC, 0.1%SDS In finally wash.
In plant nucleic acid preparation may particularly suitable alternative solution be 5x SSPE (final 0.9M NaCl, 0.05M sodium phosphate, 0.005M EDTA pH 7.7), 5X step on the solution of Ha Teshi solution, 0.5%SDS, at 50 DEG C or 65 DEG C Overnight.As needed, washing can carry out or at 50 DEG C -60 DEG C in 0.2x SSC/0.1%SDS in 1x SSC/ at 65 DEG C It is carried out in 0.1%SDS.
Nucleic acid as described herein can be wholly or partially synthetic.Especially, they can be recombination, be not The nucleic acid sequence (discontinuous extend) found together in nature has been connected or artificial combination in another way.Alternatively, it May directly be synthesized, such as use automatic synthesizer.
The expression of DA2 nucleic acid and DA1 and/or EOD1 nucleic acid can by any convenient technology plant one or more It is reduced or eliminated in cell.
It is in this field for reducing the expression or active method of DA2 polypeptide and DA1 and/or EOD1 polypeptide in plant It is well known and be described more fully hereinafter in.In some embodiments, the expression of active DA2, DA1 and/or EOD1 polypeptide can By will be reduced in the nucleic acid sequence being mutated in introduced plant cell, preferably eliminate, it is described in the nucleic acid sequence encoding more The expression of peptide or the regulation nucleic acid sequence.The mutation can destroy the expression or function of DA2, DA1 and/or EOD1 polypeptide.It is suitable The mutation of conjunction includes knocking out and striking low mutation.In some embodiments, mutation can produce the dominant negative allele of DA1.So It afterwards can be from the cytothesis plant of the mutation.Nucleic acid can be mutated by being inserted into or lacking one or more nucleotide.For The technology of mutation, inactivation or knockout target gene is well known in the art (see, for example, In Vitro Mutagenesis Protocols；Methods in Molecular Biology (second edition) Ed Jeff Braman；Sambrook J et al. 2012.Molecular Cloning:A Laboratory Manual (the 4th edition) CSH Press；Current Protocols in Molecular Biology；Ed Ausubel et al. (2013) Wiley).In some embodiments, it can be compiled by genome Mutation is introduced in target EOD1, DA2 or DA1 gene by the technology of collecting, such as RNA guides nucleic acid zymotechnic such as CRISPR, zinc finger core Sour enzyme (ZFN) and transcriptional activators sample effector nuclease (TALEN) (Urnov, F.D. et al. Nature reviews.Genetics 11,636-646(2010)；Joung, J.K. et al. Nature reviews.Molecular cell biology 14,49-55(2013)；Gasiunas, G. et al. PNAS USA 109, E2579-2586 (2012)；Cong, L. etc. People Science 339,819-823 (2013)).
It reduces expression or active series jump may include one or more nucleotide relative to wild-type nucleotide sequences Missing, insertion or substitution, gene magnification or methylation such as supermethylation increase or decrease.One or more of mutation It can be in the coding or noncoding region of nucleic acid sequence.Mutation in the code area of the gene of encoder element can prevent overall length living Property protein translation, i.e. truncated mutant, or allow overall length but the translation of inactive or impaired functional protein, i.e., missense is prominent Become.Mutation or epigenetic variation such as methylation in the noncoding region of the gene of encoder element, such as in controlling element can be prevented The only transcription of the gene.Nucleic acid including one or more series jumps can be compiled for example by the activity of the change of controlling element There is code active variant polypeptide or codified reduce or elimination to have seldom or without expression wild type more in the cell Peptide.Nucleic acid including one or more series jumps can relative to unmutated sequence have one, two, three, four or More mutation.
For example, can by corresponding to SEQ ID NO:89 position 44 position at introduce mutation as missing, insertion or Replace, for example, A to T replaces, to reduce, preferably eliminate the activity of EOD1.SEQ ID NO is equivalent in EOD1 polypeptide sequence: Standard sequence analysis and comparison tool can be used to identify for the position of 89 position 44, as shown in table 4.
Standard sequence analysis technology can be used, such as by planting compared with the reference sequences listed herein in any target Identify DA2, DA1 and EOD1 coded sequence in object species.
The mutation of expression suitable for elimination activity DA2, DA1 and/or EOD1 polypeptide will be aobvious and easy for technical staff See.
In some preferred embodiments, it can will reduce or eliminate DA2 expression or active mutation is introduced to plant cell In, the plant cell expresses dominant negative DA1 polypeptide and optionally includes the heterologous core for i) encoding EOD1 repressor nucleic acid Acid or ii) reduce EOD1 expression or active mutation.
It in some embodiments, can be by being reduced in the plant cell in the cell inner expression heterologous nucleic acids of plant The expression of DA1, DA2 and/or EOD1 polypeptide, heterologous nucleic acids coding or Transcription inhibition daughter nucleus acid, for example, repressor RNA or RNAi molecule.Repressor RNA inhibits the expression of its target polypeptide (i.e. DA1, DA2 or EOD1) in plant cell.
Nucleic acid as described herein can be wholly or partially synthetic.Specifically, they can be recombination, it is The nucleic acid sequence (discontinuous extend) not found together in nature has been connected or artificial combination in another way.Alternatively, They may directly have been synthesized, such as use automatic synthesizer.
Nucleic acid can of course be double-strand or single-stranded, cDNA or genomic DNA or RNA.Depending on design, nucleic acid be can be Wholly or partially synthetic.In general, technical staff will be appreciated that when nucleic acid includes RNA, referring to for the sequence to shown in should be solved It is interpreted as referring to that wherein U is replaced by T to RNA equivalent.
" heterologous " instruction is the base for intervening the nucleotide that will be discussed by people using genetically engineered or recombination method Gene/sequence sequence that cause/sequence or regulation are discussed is introduced in the cell of plant or its ancestors.To plant cell For heterologous nucleotide sequence can be it is non-naturally occurring in the cell of the type, mutation or species (that is, exogenous Or it is external) or can be in the subcellular of the cell or genomic context non-naturally occurring sequence or can be with It is the sequence that non-natural regulates and controls in the cell, be connected to non-natural controlling element with can be operated.
It is well known in the art for inhibiting the expression of the target polypeptide in plant cell.Suitable repressor nucleic acid can be phase For DA1, DA2 and/or EOD1 gene with target DA1, DA2 and/or EOD1 gene of antisense or ariyoshi orientation or both insertion All or part of copy, so as to realize the target gene expression reduction.See, for example, van der Krol et al., (1990)The Plant Cell 2,291-299；Napoli et al., (1990) The Plant Cell 2,279-289； Zhang et al., (1992) The Plant Cell 4,1575-1588 and US-A-5,231,020.The other improvements of the method It can be at WO95/34668 (Biosource)；Angell&Baulcombe(1997)The EMBO Journal 16,12:3675- 3684；And it is found in Voinnet&Baulcombe (1997) page Nature389: the 553.
In some embodiments, the ariyoshi that repressor nucleic acid can be the expression of DA1, DA2 and/or EOD1 polypeptide inhibits Son.
Suitable ariyoshi repressor nucleic acid can be double-stranded RNA (Fire A. et al. Nature, Vol 391, (1998)). The silencing that dsRNA is mediated is gene specific and commonly referred to as RNA interference (RNAi).RNAi is two step process.It is first First, dsRNA is cracked in the cell to generate the about 21-23nt length with the end 5' phosphate and the short jag of 3' (about 2nt) Short interfering rna (siRNA).SiRNA targeting is for destroying specific corresponding mRNA sequence (Zamore P.D.Nature Structural Biology,8,9,746-750,(2001)。
SiRNA (sometimes referred to as Microrna), which passes through to combine complementation RNA and trigger mRNA, to be eliminated (RNAi) or prevents mRNA It translates into protein and carrys out down-regulation of gene expression.SiRNA can be obtained by processing compared with long dsrna, and worked as and sent out in nature Usually there is exogenous origin now.Small-RNA interfering (miRNA) is that the endogenous obtained by processing short hair clip encodes Small non-coding RNA.SiRNA and miRNA can inhibit the translation of the target sequence for carrying partial complementarity and the mRNA without RNA cracking, and And degradation carries the mRNA of the sequence of complete complementary.
Therefore, the present invention provides the RNAi sequence based on DA1, DA2 and/or EOD1 nucleic acid sequence for inhibiting DA1, DA2 And/or the purposes of the expression of EOD1 polypeptide.For example, RNAi sequence can correspond to reference DA2, DA1 or EOD1 core listed herein The segment of nucleotide sequence can be its variant.
SiRNA molecule is usually double-strand, and in order to optimize the effective of the downward of the function of the target gene of RNA mediation Property, preferably the length of selection siRNA molecule and sequence are to ensure correctly to identify the siRNA (RISC by RISC compound Compound mediates the identification by the siRNA of mRNA target) and so that the siRNA is as short as being enough to reduce host and answer It answers.
MiRNA ligand is usually region that is single-stranded and having the partial complementarity for making the ligand be capable of forming hair clip. MiRNA is from DNA transcription but the untranslated RNA sequence at protein.The DNA sequence dna ratio miRNA long of coding miRNA.This DNA Sequence includes miRNA sequence and approximate reversed complement.When this DNA sequence dna is transcribed into single strand RNA molecule, miRNA sequence Column and its base pairing of reverse mutual complement are to form partially double stranded RNA section.The design of microRNA seqeunce in John et al., PLoS Biology, 11 (2) discuss on 1862-1879,2004.
Usually, it is intended that the RNA molecule of the effect of simulation siRNA or miRNA has the ribonucleotide between 10 and 40 (or its synthetic analogues), ribonucleotide more preferably between 17 and 30, it is 19 and 25 more preferable between ribose core Thuja acid and the most preferably ribonucleotide between 21 and 23.In some embodiments of the invention using double-strand siRNA, The molecule can have the symmetrical 3' jag of such as one or two (ribose) nucleotide, usual dTdT 3' jag UU.Based on disclosure provided herein, technical staff can easily design suitable siRNA and miRNA sequence, such as using Resource such as siRNA finder (Ambion).SiRNA and miRNA sequence can be synthetically produced and exogenous add to cause gene It lowers or is generated using expression system (such as carrier).In a preferred embodiment, siRNA is synthesized syntheticly.
It can be processed in cell compared with long dsrna to generate siRNA (see, for example, Myers (2003) Nature Biotechnology 21:324-328).Longer dsRNA molecule can have the symmetrical of such as one or two (ribose) nucleotide 3' or 5' jag, or can have flat end.Longer dsRNA molecule can be 25 nucleotide or longer.Preferably, longer The length of dsRNA molecule is the nucleotide between 25 and 30.It is highly preferred that the length of longer dsRNA molecule is 25 and 27 Between nucleotide.Most preferably, the length of longer dsRNA molecule is 27 nucleotide.Carrier pDECAP can be used to express Length is the dsRNA (Shinagawa et al., Genes and Dev., 17,1340-5,2003) of 30 nucleotide or more.
Another alternative solution is expression short hairpin RNA molecule (shRNA) in cell.ShRNA is more steady than synthesis siRNA It is fixed.ShRNA is made of the short inverted repeats by small ring sequence separates.One inverted repeats and gene target are mutual It mends.In cell, shRNA is processed by siRNA, the siRNA degradation target gene mRNA and inhibition expression by DICER.In In preferred embodiment, by endogenously generating shRNA (in the cell) from carrier transcription.It can be by rna plymerase iii Promoter such as people H1 or 7SK promoter or the lower carrier with coding shRNA sequence of rna plymerase ii promoter control transfect cell Come in the intracellular generation shRNA.Alternatively, can be by synthesizing shRNA (in vitro) exogenously from carrier transcription.Then may be used ShRNA is directly introduced into the cell.Preferably, shRNA molecule includes the partial sequence of DA1, DA2 and/or EOD1.Example Such as, the length of shRNA sequence is between 40 and 100 bases, and more preferably length is between 40 and 70 bases.The stem of hair clip Length preferably between 19 and 30 base-pairs.The stem may include that G-U is matched so that hairpin structure is stablized.
It can be preferably incorporated in the intracorporal nucleic acid sequence of load by transcription and to recombinate prepare siRNA molecule, longer dsRNA Molecule or miRNA molecule.Preferably, siRNA molecule, longer dsRNA molecule or miRNA molecule include the reference listed herein The partial sequence of DA2, DA1 or EOD1 nucleotide sequence or its variant.
In other embodiments, repressor nucleic acid can be the Antisense Suppression of the expression of DA1, DA2 and/or EOD1 polypeptide Son.Come in down-regulation of gene expression using antisense sequences, nucleotide sequence is placed in the control of the promoter in " inverted orientation " Under, so that transcription generates RNA, the RNA is complementary with the normal mRNA transcribed from " ariyoshi " chain of target gene.See, for example, Rothstein et al., 1987；Smith et al., (1988) Nature 334,724-726；Zhang et al., (1992) The Plant Cell 4,1575-1588；English et al., (1996) The Plant Cell 8,179-188.Antisense technology is also It summarizes in Bourque, (1995), Plant Science 105,125-149 and Flavell (1994) PNAS USA 91, In 3490-3496.
Antisense Suppression daughter nucleus acid may include from for reference DA2, DA1 or EOD1 nucleotide sequence listed herein or its change The antisense sequences of at least ten nucleotide of the nucleotide sequence of the segment of body.
It may be preferred that the expression for lowering target sequence sequence with that there are sufficient sequences in the target sequence is same One property, but the complete complementarity of sequence or similitude are not required.One or more nucleotide in used sequence It can be different from target gene.Therefore, the sequence used in the downward of gene expression according to the present invention can be selected from obtainable The variant of those wild-type sequence (such as gene) or this sequence.
The sequence need not include open reading frame or it is specified will be interpretable RNA.It is corresponded to it may be preferred that existing Enough homologys of antisense and ariyoshi RNA molecule are to hybridize.There may be the downwards of gene expression, even if in used sequence In the case where there is about 5%, 10%, 15% or 20% or more mispairing between target gene.Effectively, homology should be enough to use In the downward that gene expression occurs.
Repressor RNA molecule may include the sense or antisense chain of the nucleic acid sequence of encoding D A2, DA1 and/or EOD1 polypeptide 10-40 nucleotide.
Repressor nucleic acid can be operably coupled to allogeneic promoter, such as tissue specificity or inducible promoter. For example, integument and seed specific promoters can be used for two or more in specifically lower levelling development ovule and seed DA1, DA2 and/or EOD1 nucleic acid are to increase final seed size.
In some preferred embodiments, DA2 repressor nucleic acid can with encoding dominant negative feminine gender DA1 polypeptide and optionally It is expressed in the plant cell of the nucleic acid of ground EOD1 repressor nucleic acid.
The nucleic acid for encoding repressor nucleic acid and/or dominant negative DA1 polypeptide may include in one or more carriers.
The nucleic acid for encoding repressor nucleic acid as described herein and/or dominant negative DA1 polypeptide can operationally connect It is connected to heterologous regulatory sequence, such as promoter, such as composing type as described above, induction type, tissue specificity or development-specific Promoter.
The nucleic acid for encoding repressor nucleic acid as described herein and/or dominant negative DA1 polypeptide may include in nucleic acid structure It builds on body or carrier.The construct or carrier are preferably adapted for conversion table into plant cell and/or in plant cell It reaches.Carrier is especially in double-strand or single-stranded linear or annular form any plasmid, clay, bacteriophage or Agrobacterium binary Carrier, it is self can shifting or moveable that the carrier can be or can not be, and the carrier can be by being integrated into There is (for example, plasmid that autonomous duplication has replication orgin) to convert protokaryon or eukaryon in cellular genome or chromosome other places Host, specifically plant host.
It specifically include shuttle vector, shuttle vector means natively or passes through design can be in two different organisms The DNA medium of middle duplication, the DNA medium can be selected from actinomyces and relative species, (such as the high plant of bacterium and eukaryon Object, mammal, yeast or fungi) cell.
Construct or carrier as described above including nucleic acid need not be including promoter or other regulating and controlling sequences, especially such as Carrier described in fruit, which is not used in, to be introduced into nucleic acid in cell to be used to recombinate into genome.
Construct and carrier may also include by assign may be selected phenotype (such as to the resistance of antibiotic) gene form can Select genetic marker, the antibiotic such as kanamycins, hygromycin, glufosinate (phosphinotricin), chlorsulfuron, first Aminopterin, gentamicin, spectinomycin, imidazolone type, glyphosate and d- amino acid.
Those skilled in the art can for example in microorganism or plant cell carrier construction and designed for recombination base Because of the scheme of expression.It may be selected or construct the suitable carrier for containing regulating and controlling sequence appropriate, the regulating and controlling sequence includes promoter Sequence terminates sub-piece, polyadenylation sequence, enhancer sequence, marker gene and other sequences appropriate.For into one Details is walked, see, for example, Molecular Cloning:a Laboratory Manual: the 3 edition, Sambrook et al., 2001, Cold Spring Harbor Laboratory Press and Protocols in Molecular Biology, second Version, Ausubel et al. edit John Wiley&Sons, 1992.Previously used has extensively successfully specific journey on plant Sequence and carrier are by Bevan, Nucl.Acids Res. (1984) 12,8711-8721) and Guerineau and Mullineaux, (1993)Plant transformation and expression vectors.In:Plant Molecular Biology Labfax (Croy RRD is edited) Oxford, BIOS Scientific Publishers, the 121-148 pages description.
When selected gene construct is introduced cell, it is necessary to consider well-known to those having ordinary skill in the art certain examine Consider.The nucleic acid being inserted into should include that will assemble in the construct of the Effective Regulation element of driving transcription.There must be available Method construct being transported in cell.Once in cell membrane, endogenous will occur or will not be integrated into for construct In chromosomal material.Finally, target cell type is preferably so that cell can be reproduced into full plants.
It is desirable for the construct of the expression of the nucleic acid of enhancing coding repressor nucleic acid or dominant negative DA1 polypeptide and turns Change method.The single copy of gene, which is integrated into the genome of plant cell, may be beneficial to make gene silencing effect minimum Change.Equally, control integration complexity can be in this regard it is beneficial.In this regard it is particularly interesting that using according to example Plant cell is converted such as the minimum gene expression construct of european patent number EP1407000B1, the patent is for this purpose It is hereby incorporated herein by.
Technology well-known to those having ordinary skill in the art can be used for for nucleic acid construct and carrier being introduced in plant cell with Generate the genetically modified plants with characteristic as described herein.
Agrobacterium conversion is that a kind of method of conversion plant species is widely used in by those skilled in the art.It generates Stable fertile genetically modified plants be now it is conventional in the art (see, for example, Toriyama, et al. (1988) Bio/ Technology 6,1072-1074；Zhang, et al. (1988) Plant Cell Rep.7,379-384；Zhang, et al. (1988)Theor Appl Genet 76,835-840；Shimamoto, et al. (1989) Nature 338,274-276； Datta, et al. (1990) Bio/Technology 8,736-740；Christou, et al. (1991) Bio/Technology 9, 957-962；Peng, et al. (1991) International Rice Research Institute, Manila, Philippines 563-574；Cao, et al. (1992) Plant Cell Rep.11,585-591；Li, et al. (1993) Plant Cell Rep.12,250-255；Rathore, et al. (1993) Plant Molecular Biology 21,871- 884；Fromm, et al. (1990) Bio/Technology 8,833-839；Gordon-Kamm, et al. (1990) Plant Cell 2,603-618；D'Halluin, et al. (1992) Plant Cell 4,1495-1505；Walters, et al. (1992) Plant Molecular Biology 18,189-200；Koziel, et al. (1993) Biotechnology 11,194-200；Vasil, I.K.(1994)Plant Molecular Biology 25,925-937；Weeks, et al. (1993) Plant Physiology 102,1077-1084；Somers, et al. (1992) Bio/Technology 10,1589-1594；WO92/14828； Nilsson, O. et al. (1992) Transgenic Research 1,209-220).
Other methods, such as particle or partickle bombardment (US 5100792, EP-A-444882, EP-A-434616), electroporation (EP 290395, WO 8706614), microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. (1987) Plant Tissue and Cell Culture, Academic Press), direct DNA absorb (DE 4005152, WO 9012096, US 4684611), liposome-mediated DNA intake (such as Freeman et al. Plant Cell Physiol.29:1353 (1984)) or vortex methods (such as Kindle, PNAS U.S.A.87:1228 (1990d)) in soil It in the case that Bacillus conversion is inefficient or invalid, such as in some gymnosperm species may be preferred.It is planted for converting The physical method of object cell is summarized in Oard, 1991, Biotech.Adv.9:1-11.
Alternatively, the combination of different technologies can be used for enhancing the efficiency of conversion process, such as coated with Agrobacterium micro- Partickle bombardment (EP-A-486234) or microparticle bombardment then co-culture (EP-A- with Agrobacterium to induce wound 486233)。
After conversion, can be for example from individual cells, callus or leaf dish aftergrowth, such as standard in the art 's.Substantially any plant can all regenerate completely from the cell, tissue and organ of plant.Available technology summary in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, I, II and III volumes, Laboratory Procedures and Their Applications, Academic Press, 1984 and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, in 1989.
The specific choice of transformation technology will be converted the efficiency of certain plants species by it and practice the warp of people of the invention It tests and determines with preference with specific selection method.It will be clear to the skilled person that by the transformation system of nucleic acid into plant cells The specific choice of system is not limitation required in this invention or of the invention, nor the selection of the technology for plant regeneration.
After conversion, can identify and/or select to express with reduceds DA2 expression and reduced DA1 and/or EOD1 or Active plant cell.It can be from the plant cell aftergrowth.
Plant as described above that also lack both DA1, EOD1 or DA1 and EOD1 with reduced DA2 activity or expression Object can sexual or vegetative propagation or growth to generate filial generation or offspring.Can sexual or vegetative propagation or growth from one or more thin The filial generation of the regenerated plant of born of the same parents or offspring.Plant or its filial generation or offspring can hybridize with other plant or with itself.
DA1, DA2 and/or EOD1 amino acid or nucleic acid sequence can be used as molecular marker come grow it is sexual or asexual The table of one or more DA1, DA2 and/or EOD1 polypeptides out as listed above in plant is measured before, during or after breeding It reaches or active.A kind of method can include:
The group of plant is provided,
The amount of the expression of DA1, DA2 and/or EOD1 polypeptide in one of described group or various plants is measured, and
Identifying in the group has reduced DA1, DA2 and/or EOD1 polypeptide relative to other members of the group One or more plants of expression.
The plant population can be generated as described above.
In some embodiments, a kind of method can include:
Make the first and second plant hybridizations to generate the group of progeny plants；
The expression of one of described progeny plants in the group or a variety of DA1, DA2 and EOD1 polypeptides is measured, with And
Identify the expression of DA1, DA2 and/or EOD1 polypeptide described in the group progeny plants reduced relative to control.
One or both of first plant and second plant can be generated as described above.
The expression of DA2 and DA1 and/or EOD1 polypeptide is reduced relative to control (for example, other members of the group) Progeny plants can show relative to the increased seed of the control and/or organ size and can have higher plant products.
In some embodiments, DA1 and EOD1 amino acid or nucleic acid sequence can be used as molecular marker to measure in plant One or more DA1 and/or EOD1 polypeptides expression or activity, so as to identify lack DA1 and/or EOD1 plant or plant Cell, the expression of DA2 polypeptide or activity can be lowered as described above in the plant or plant cell.A kind of method can wrap It includes:
The group of plant is provided,
The amount of the expression of the DA1 and/or EOD1 polypeptide in one of described group or various plants is measured, and
Identifying in the group has reduced DA1 and/or EOD1 polypeptide expression relative to other members of the group One or more plants.
DA2 expression or activity can be reduced in the plant identified using method as described above.
Can by DA1 in the following one or more cells for measuring the plant come differential plant or progeny plants i), The amount of DA2 and/or EOD1 polypeptide, ii) the measurement plant one or more cells in DA1, DA2 and/or EOD1 mRNA Amount or iii) nucleic acid of DA1, DA2 and/or EOD1 polypeptide in the one or more cells for encoding the plant is surveyed Sequence and the presence for identifying one or more mutation.
The plant identified further can be bred or be hybridized, for example, with have reduced DA1, DA2 and/or EOD1 table The other plant reached hybridizes or from hybridization to generate self-mating system.DA1, DA2 and/or EOD1 in the group of progeny plants can be measured The expression of polypeptide or activity and identification have reduced DA1, DA2 and/or EOD1 expression or active one or more offsprings Plant.
In some embodiments, the amount of the expression of DA1, DA2 and/or EOD1 can be measured under protein level.A kind of side Method can include:
The group of plant is provided,
The amount of DA1, DA2 and/or EOD1 polypeptide in one or more plants of the group is measured, and described in identification There are in group relative to other members of the group one or more plants of reduced DA1, DA2 and/or EOD1 polypeptide amount Object.
Advantageously, immunological technique such as Western blotting can be used, using combination DA1, DA2 or EOD1 polypeptide and display and plant The less combination of other antigens in object or the antibody without combination.For example, DA1, DA2 and/or EOD1 polypeptide in plant cell Amount can measure in the following manner: make include the plant cell sample be directed to DA1, DA2 or EOD1 polypeptide Antibody or other specific binding members are in contact, and measure the combination of DA1, DA2 or EOD1 polypeptide and the sample. The amount for DA1, DA2 or EOD1 polypeptide that the amount instruction of the combination of specific binding members is expressed in cell.
It can be in one or more cells of plant, preferably in aerial part or tissue from plant, in tender shoots Vascular system and nascent and secondary meristem cell in measure the amount of DA1, DA2 and/or EOD1 polypeptide.
In other embodiments, the expression of DA1, DA2 or EOD1 polypeptide can be measured under nucleic acid level.For example, can measure The amount of the nucleic acid of encoding D A1, DA2 or EOD1 polypeptide.Generating has a kind of method of the plant of increased Correlated Yield Characters can Include:
The group of plant is provided,
Measure in the cell of one or more plants of the group nucleic acid of encoding D A1, DA2 or EOD1 polypeptide for example The level or amount of mRNA, and
Identifying in the group has reduced DA1, DA2 or EOD1 code nucleic acid relative to other members of the group Amount one or more plants.
The level or amount of code nucleic acid in plant cell can for example pass through the coding core of the transcription in the detection cell The amount of acid measures.Standard technique such as RNA trace or RT-PCR can be used to carry out for this.
Alternatively, can the expression of determination influences DA1, DA2 or EOD1 polypeptide or the presence of active sequence variations.Generation has Another method of the plant of increased growth and/or biomass can include:
The group of plant is provided,
Measure the nucleic acid of encoding D A1, DA2 and/or EOD1 polypeptide in the cell in one or more plants of the group One of or a variety of sequence variations (for example, polymorphism, mutation or supermethylation region) presence,
Wherein one or more sequence variations reduce DA1, DA2 and/or EOD1 polypeptide of the coding expression or Activity, and
Identify has the expression for reducing DA1, DA2 and/or EOD1 relative to other members of the group in the group Or one or more plants of active one or more sequence variations.
DA1, DA2 and/or EOD1 polypeptide and code nucleic acid are being described in more detail above.
The presence of one or more sequence variations can be by detecting variant nucleic in one or more plant cells in nucleic acid The presence of sequence is measured by detecting by the presence of the variant polypeptide of the nucleic acid sequence encoding.Preferred variable nucleic acid sequence Different detection technique includes ARMSTMAllele specific amplification, OLA, ALEXTM, COPS, Taqman, molecular beacon, RFLP with And the PCR and FRET technology based on restriction site.
For measure the nucleic acid of encoding D A1, DA2 or EOD1 polypeptide in plant cell amount or encoding D A1, DA2 or Present or absent many suitable methods of sequence variations in the nucleic acid of EOD1 polypeptide are obtainable (ginsengs in this field See for example (see, for example, Molecular Cloning:a Laboratory Manual: the 3 edition, Sambrook&Russell (2001)Cold Spring Harbor Laboratory Press NY；Current Protocols in Molecular Biology, Ausubel et al. edit John Wiley&Sons (1992)；DNA Cloning,The Practical Approach Series (1995), series editor D.Rickwood and B.D.Hames, IRL Press, Oxford, UK and PCR Protocols:A Guide to Methods and Applications (Innis, et al. 1990.Academic Press,San Diego,Calif.)).Many current methods for detection sequence variation by Nollau et al., Clin.Chem.43,1114-1120,1997；And in standard textbook such as " Laboratory Protocols for Mutation Detection ", U.Landegren are edited, Oxford University Press, and 1996 and " PCR ", second edition Newton&Graham, BIOS Scientific Publishers Limited is summarized in 1997.
Preferred polypeptide sequence mutation includes immunoassays, immunoassays be it is well-known to those having ordinary skill in the art, Such as A Practical Guide to ELISA, D M Kemeny, Pergamon Press 1991；Principles and Practice of Immunoassay, second edition, C P Price&D J Newman, 1997, by America & Canada The Stockton Press and Macmillan Reference of Britain is published.
In some embodiments, nucleic acid or its amplification region can be sequenced with identify or measure polymorphism therein or The presence of mutation.Can by by the known array of sequence obtained and DA1, DA2 or EOD1 (for example, as in sequence database It is listed) it is compared to identify polymorphism or mutation.Alternatively, can be by sequence obtained and the corresponding core from control cell The sequence of acid is compared.Specifically, the one or more polymorphisms for causing the reduction of function but not exclusively eliminating can be measured Or the presence of mutation.Can be used in series of standards technology any is sequenced.The sequencing of amplified production can be related to for example And with isopropanol precipitating, resuspension and using TaqFS+Dye terminator sequencing kit (such as from GE Healthcare UK Ltd UK) sequencing.Extension products can carry out electrophoresis in 377 DNA sequencer of ABI and use Sequence Navigator soft Part analyzes data.
It can be for right relative to increased or enhancing Correlated Yield Characters (such as increased seed or organ size) are compareed It is identified as being tested with the progeny plants that reduced DA1, DA2 and/or EOD1 are expressed.
The progeny plants identified further can be bred or be hybridized, such as (be returned with first or second plant hybridization Hand over) or hybridize certainly to generate self-mating system.
Can relative to comparison needle to seed size, organ size and/or plant products come to the progeny plants identified into Row test.
The plant generated as described herein can lack DA2 expression or activity and can also lack DA1 expression or activity, EOD1 Expression or activity or both DA1 and EOD1 expression or activity.
Inhibition daughter nucleus can be encoded by the mutation of one or more nucleotide in plant code sequence and/or by expression The heterologous nucleic acids of acid reduce or eliminate the expression or activity of DA2, DA1 and EOD1 in plant.In some preferred embodiments In, the activity of the DA1 in plant can be reduced or eliminated by the heterologous nucleic acids of the DA1 polypeptide of expression encoding dominant negative feminine gender.
Therefore plant may include heterologous nucleic acids, the heterologous nucleic acids encode repressor nucleic acid such as siRNA or shRNA, described Repressor nucleic acid reduces the expression of one or more of DA1, DA2 and EOD1 or the DA1 polypeptide of encoding dominant negative feminine gender.
It can be in plant as described herein using mutation, any combination of repressor nucleic acid.For example, plant may include i) Heterologous nucleic acids and the fgs encoder for reducing the repressor nucleic acid of mutation, the coding reduction EOD1 expression of DA2 activity or expression are aobvious Property negative DA1 polypeptide nucleic acid heterologous nucleic acids；Ii) coding reduces the heterologous nucleic acids of the repressor nucleic acid of DA2 expression, reduces The heterologous nucleic acids of the nucleic acid of the mutation and dominant negative DA1 polypeptide of fgs encoder of EOD1 expression；Iii) coding reduces EOD1 With the heterologous nucleic acids of the nucleic acid of the heterologous nucleic acids and dominant negative DA1 polypeptide of fgs encoder of the repressor nucleic acid of DA2 expression；Or Iv the heterologous nucleic acids of the nucleic acid of the mutation and dominant negative DA1 polypeptide of fgs encoder of EOD1 and DA2 activity or expression) are reduced.
In other embodiments, plant may include i) reducing DA2 activity or the mutation expressed, coding to reduce DA1 and express The heterologous nucleic acids of repressor nucleic acid；Ii) coding reduces the heterologous nucleic acids of the repressor nucleic acid of DA2 expression, reduces the prominent of DA1 expression Become；Iii) coding reduces the heterologous nucleic acids of the repressor nucleic acid of DA1 and DA2 expression；Iv) reduce DA1 and DA2 activity or expression Mutation；Or v) reduce the heterologous nucleic acids and volume of the repressor nucleic acid of mutation or the coding reduction DA2 expression of DA2 activity or expression The heterologous nucleic acids of the nucleic acid of the DA1 polypeptide of code encoding dominant negative feminine gender.
The DA1 polypeptide of encoding dominant negative feminine gender and/or the heterologous nucleic acids of repressor nucleic acid can be in identical or different expression On carrier and it can be incorporated in plant cell by routine techniques.
The example of the suitable plant used for any aspect according to invention as described herein includes monocotyledon With dicotyledonous high-grade plant, such as agricultural plant or crop, it is such as selected from the plant for the group being made of the following terms: Asian puccoon (Lithospermum erythrorhizon), Taxus, tobacco, cucurbit, carrot, Vegetables in Brassica, melon, capsicum, Portugal Grape tree, lettuce, strawberry, Brassica genus oily seed, beet, wheat, barley, corn, rice, soybean, pea, jowar, sunflower, Tomato, potato, pepper, chrysanthemum, carnation, linseed, hemp and rye.The plant generated as described above can sexual or nothing Sexual reproduction or growth are to generate filial generation or offspring.Can sexual or vegetative propagation or growth from the plants of one or more cytothesises Filial generation or offspring.Plant or its filial generation or offspring can hybridize with other plant or with itself.
Another aspect of the present invention provides genetically modified plants, and the genetically modified plants have into the cell in one or more DA2 polypeptide expression or activity reduce or elimination, wherein the plant lacks the expression of both DA1, EOD1 or DA1 and EOD1 Or activity.
The plant may include exogenous nucleic acid, the exogenous nucleic acid reduce or eliminate one of DA2, DA1 and EOD1 or The expression or activity of more persons.In some embodiments, genetically modified plants can express the active dominant negative DA1 for reducing DA1 Polypeptide.
In some embodiments, the plant can have reduction or elimination DA1, DA2 and EOD1 to express or can have DA2 and EOD1 reduce or elimination are expressed and can be expressed dominant negative DA1.
Except through the plant that method described herein generates, the present invention covers the plant, seed, selfing or hybridization Generation or offspring any clone and these any one of any part or brood body can be used for such as cutting and seed Breeding or breeding, it is sexual or asexual.Present invention also contemplates that plant, the plant is sexual or vegetative propagation the son of this plant Generation, clone or offspring or the plant, filial generation, clone or offspring any part or brood body.
Suitable plant can be generated by the above method.
The plant can relative to control wild-type plant (expression of i.e. wherein DA2 and optionally DA1 and/or EOD1 or The identical plant that activity not yet reduces) there is increased yield.For example, per unit area seed (such as grain) or other plant The quality of product can increase relative to check plant.
For example, one of plant or a variety of Correlated Yield Characters can be improved.Correlated Yield Characters may include service life, organ Size and seed size.
Correlated Yield Characters can improve relative to check plant, increase or enhance the Correlated Yield Characters in plant, in institute State the expression of the nucleic acid of encoding D A2 polypeptide in check plant do not eliminate or reduce again (i.e. wherein DA2 and optionally DA1 and/or The identical plant that the expression of EOD1 not yet reduces or eliminates).
Plant according to the present invention can be the plant of the not pure breeding in one or more characteristics.Botanical variety can be excluded, Botanical variety is registered in particular according to planting person's right.
DA1 shown herein in vivo with DA2 Physical interaction.Destroy or interfere the compound of the interaction can Suitable for increasing seed or organ size and improving plant products.
Identify the method for increasing the compound of plant products can include:
Effect of the measurement test compound to the combination of DA2 polypeptide and DA1 polypeptide,
In conjunction with the instruction compound that reduces or eliminates be applicable to increase plant products.
DA1 and DA2 polypeptide is being described in more detail above.
It is that DA1 and DA2 polypeptide can be separation or can recombinate in plant cell or endogenously express.
The compound for reducing or eliminating DA1/DA2 combination is applicable to processing plant to increase yield.
The specific of each that "and/or" should be considered as in two kinds of specific characteristics or component when using herein discloses Or without another one.For example, " A and/or B " should be considered as the specific disclosure of each of (i) A, (ii) B and (iii) A and B, It is individually listed just as each herein.
Unless context dictates otherwise, otherwise the description and definition of feature listed above are not limited to any tool of the invention In terms of body or embodiment and it is equally applicable to described all aspect and embodiment.
Other aspects of the present invention and embodiment provide wherein term " includes " by term " by ... form " substitute with The above that the aspect and embodiment and wherein term " includes " are substituted by term " substantially by ... form " Aspect and embodiment.
The All Files referred in the present specification are incorporated herein in its entirety by reference for all purposes.
The content of all data base entries referred in the present specification for all purposes it is whole by reference simultaneously Enter herein.This includes the version in the submission date of the application current any sequence.
Experiment
1. method
1.1 vegetable materials and growth conditions
Arabidopsis Columbia ecotype (Col-0) is used wild type system.All mutant are in Col-0 background Under.Da2-1 (SALK_150003) is obtained from arabidopsis inventory center NASC and ABRC preservation.It is true by PCR and sequencing Recognize T-DNA insertion.By seed 100% isopropanol surface sterilizing 1 minute and with 10% (v/v) household bleach surface sterilizing 10 minutes, at least three times with sterile water washing, stratification 3 days in the dark at 4 DEG C, on GM culture medium with 0.9% agar and The dispersion of 1% glucose, and then grown at 22 DEG C.By plant under long-day conditions (16 small time/8 hour dark) It is grown at 22 DEG C.
1.2 constructs and conversion
PDA2:DA2 construct is prepared by using the Gateway system of based on PCR.Using primer DA2proGW-F and DA2proGW-R expands the 1960bp promoter sequence of DA2.Then PCR product is cloned into pCR8/GW/TOPO TA clone In carrier (Invitrogen).Amplification DA2 CDS and then AscI that PCR product is cloned into Gateway carrier pMDC110 With the site KpnI to obtain DA2CDS-pMDC110 plasmid.Then it is reacted by LR and DA2 promoter is subcloned to DA2CDS- PMDC110 is to generate pDA2:DA2 construct.Plasmid pDA2:DA2 da2-1 is introduced to using Agrobacterium tumefaciens GV3101 to dash forward Transformant is selected in variant plant and on the culture medium containing hygromycin (30 μ g/ml).
35S:DA2 construct is prepared using the Gateway system of based on PCR.PCR product is subcloned using TOPO enzyme Into pCR8/GW/TOPO TA cloning vector (Invitrogen).Then DA2 gene is subcloned to containing 35S promoter In Gateway binary vector pMDC32 (Curtis and Grossniklaus, 2003).Using Agrobacterium tumefaciens GV3101 by matter Grain 35S:DA2 is introduced in Col-0 plant and selects transformant on the culture medium containing hygromycin (30 μ g/ml).
It expands the 1960bp promoter sequence of DA2 and PCR product is cloned into pGEM-T carrier using T4DNA ligase (Promaga) it in and is sequenced.Then DA2 promoter is inserted into the site SacI and NcoI of binary vector pGreen-GUS (Curtis and Grossniklaus, 2003) converts plasmid pDA2:GUS to generate.Using Agrobacterium tumefaciens GV3101 by matter Grain pDA2:GUS is introduced in Col-0 plant and selects transformant on the culture medium of (50 μ g/ml) containing kanamycin.Make 35S:GW2 construct is prepared with the Gateway system of based on PCR.PCR product is subcloned to pCR8/GW/ using TOPO enzyme In TOPO TA cloning vector (Invitrogen).Then GW2 gene is subcloned to the Gateway binary containing 35S promoter In carrier pMDC32 (Curtis and Grossniklaus, 2003).Plasmid 35S:GW2 is drawn using Agrobacterium tumefaciens GV3101 Enter into Col-0 plant and selects transformant on the culture medium containing hygromycin (30 μ g/ml).
1.3 morphology and cell analysis
It is weighed in 500 batch by using electronic analytical balance (METTLER MOLEDO AL104CHINA) maturation Dry seeds measure Average seed weights.The weight of five sample batch is measured for each seed seed lot.Use Lycra CCD (DFC420) it is taken pictures at Leica microscope (LEICA S8APO) to seed and is measured by using Image J software Seed size.The area measurement in petal (stage 14), leaf and cotyledon is carried out to generate digital picture by scanning organ, and Then by using Image J software reference area, length and width.It is big from DIC image measurement leaf, petal and embryo cell It is small.The biomass accumulation in flower (stage 14) is measured by weighing organ.
1.4 GUS dyeing
By sample (pDA2:GUS) in 1mM X-gluc, 100mM Na3PO4 buffer, respective 3mM K3Fe (CN) 6/ K4Fe (CN) 6,10mM EDTA and 0.1%Nodidet-P40 solution in dye, and be incubated at room temperature 6 hours.In After GUS dyeing, chlorophyll is removed using 70% ethyl alcohol.
1.5 RNA separation, RT-PCR and quantitative Real time RT-PCR analysis
Using RNeasy Plant Mini kit (TIANGEN, China) from arabidopsis root, stem, leaf, seedling and flower Sequence extracts total serum IgE.As described (Li et al. people, 2006) carries out reverse transcription (RT)-PCR.Use primer ACTIN2-F and ACTIN2- CDNA sample is based on actin transcript amount and is standardized by R.It is used with lightcycler 480engine (Roche) Lightcycler 480SYBR Green Master (Roche) carries out quantitative Real time RT-PCR analysis.ACTIN7 mRNA is used as Internal contrast, and the relative quantity of mRNA is calculated using threshold cycle method is compared.
The measurement of 1.6 E3 ubiquitin ligase activities
The coded sequence of DA2 is cloned into the site BamH I and PstI of pMAL-C2 carrier to generate construct MBP- DA2.By the DA2 (DA2C59S for generating mutation according to the instruction manual of more site directed mutation kits (Stratagene) And DA2N91L).
From the MBP-DA2's for expressing MBP-DA2 and mutation with the e. coli bl21 preparation of 0.4mM IPTG induction 2 hours Bacterial Lysates.By bacterium TGH dissolve buffer (50mM HEPES [pH 7.5], 150mM NaCl, 1.5mM MgCl2, 1mM EGTA, 1%Triton X-100,10% glycerol and protease inhibitor cocktail [Roche]) in dissolve and carry out Ultrasonic treatment.By lysate by centrifugal clarification and with amylose resin (New England Biolabs) together at 4 DEG C It is lower to be incubated for 30 minutes.Bead is washed by column buffer (20mM Tris pH7.4,200mM NaCl, 1mM EDTA), and It is balanced by reaction buffer (50mM Tris pH7.4,20mM DTT, 5mM MgCl2,2mM ATP).By 110ng E1 (Boston Biochem), 170ng E2 (Boston Biochem), 1 μ g His- ubiquitin (Sigma-Aldrich) and 2 μ g DA2-MBP or the DA2-MBP fusion protein of mutation are incubated for 2 hours at 30 DEG C in 20 μ l reaction buffers.
By being printed with the antibody for His (Abmart) and for the antibody mediated immunity of MBP (New England Biolabs) Mark detects poly-ubiquitin protein matter.
1.7 external protein-protein interactions
The coded sequence of DA1, da1-1 and DA1 derivative containing specific protein domains is cloned into pGEX- To generate GST-DA1, GST-DA1R358K, GST-DA1-UIM and GST- in BamH I and the Not I site of 4T-1 carrier DA1-LIM+C construct, and be cloned into the site EcoRI and XhoI of pGEX-4T-1 carrier with generate GST-DA1-LIM and GST-DA1-C construct.
For test protein-protein interaction, the bacterium that will contain about the MBP-DA2 fusion protein of 15 μ g is molten It solves product and contains GST-DA1, GST-DA1R358K, GST-DA1-UIM, GST-DA1-LIM, GST-DA1- of about 30 μ g The lysate of LIM+C or GST-DA1-C fusion protein merges.By 20 μ l amylose resin (New England Biolabs it) is added in each combined solution, continues to shake 1 hour at 4 DEG C.Bead is washed repeatedly with TGH buffer, And isolated protein is separated on 10%SDS- polyacrylamide gel and respectively with anti-GST antibody (Abmart) and Anti- MBP antibody (Abmart) is detected by western blot analysis.
1.8 co-immunoprecipitation
The coded sequence of DA1 and DA1-C is cloned into the site KpnI and BamHI of pCAMBIA1300-221-Myc carrier In with generate convert plasmid 35S::Myc-DA1 and 35S::Myc-DA1-C.PCR product is subcloned to pCR8/ using TOPO enzyme In GW/TOPO TA cloning vector (Invitrogen).Then DA2 gene is subcloned to containing 35S promoter and GFP gene Gateway binary vector pMDC43 in (Curtis and Grossniklaus, 2003).Using TOPO enzyme by sub- gram of PCR product It is grand into pCR8/GW/TOPO TA cloning vector (Invitrogen).Then PEX10 gene is subcloned to containing 35S and is started In the Gateway binary vector pH7FWG2 of son and GFP gene.
Ben's Tobacco Leaf is possessed to the Agrobacterium tumefaciens of 35S:Myc-DA1 and 35S:GFP-DA2 plasmid by injection GV3101 cell is converted, (Voinnet et al., 2003) as described previously.With Extraction buffer (50mM Tris/HCl (pH 7.5), 150mM NaCl, 20% glycerol, 2%Triton X-100,1mM EDTA, the mixing of 1 × adequate proteins enzyme inhibitor Object (Roche) and MG13220ug/ml) extract gross protein and to be incubated for 1 at 4 DEG C with GFP-Trap-A (Chromotek) small When.By bead washing buffer (50mM Tris/HCl (pH 7.5), 150mM NaCl, 0.1%Triton X-100 and 1 × adequate proteins Protease Inhibitor Cocktail (Roche)) washing 3 times.By immunoprecipitate in 10%SDS- polyacrylamide gel It is middle to separate and examined respectively with anti-GFP antibody (Beyotime) and anti-Myc antibody (Abmart) by western blot analysis It surveys.
1.9 accession number
The arabidopsis gene group project gene seat identifier for the arabidopsis gene being mentioned above is as follows: At1g19270 (NP_ 173361.1GI:15221983)(DA1)、At4g36860(NP_195404.6GI:240256211)(DAR1)、At1g78420 (NP_001185425.1GI:334183988) (DA2), At1g17145 (NP_564016.1GI:18394446) (DA2L) and At3g63530(NP_001030922.1GI:79316205)(EOD1/BB)。
2. result
2.1 da2-1 mutant generate big seed
In order to further appreciate that ubiquitin mediate seed size control mechanism, we have collected several microarrays study In the T-DNA obtained that discloses of the ubiquitin ligase genes of some predictions that is expressed in arabidopsis ovule and/or seed insert Enter and is and its seed growth phenotype is studied.From this screening, we be authenticated with the seed size changed Several T-DNA insertion mutation bodies.Discovery sequence with reference to big seed size mutant is specified by one in these mutant For da2-1 (DA means " big " in Chinese).By da2-1 generate seed it is bigger than wild type seeds and heavier (Figure 1A, 3C and 3D).In da2-1 the seed production of the number seeds of every silique and every plant those of be slightly higher than in wild type (Figure 1B and 1C).In contrast, compared to the total number of every plant seed in wild type, the total number of every plant seed is not shown in da2-1 It writes and increases (Fig. 1 D).Da2-1 plant is higher than WT lines (Fig. 1 E) in the maturity period.In addition, compared to WT lines, da2- 1 mutant plants form biggish flower and leaf and increased biomass (Fig. 2；Figure 15).Da2-1 mutant petal and leaf it is big Small increase is not as caused by biggish cell (Figure 15), thus instruction be petal and leaf cell number it is higher.
2.2 DA2 and DA1 act synergistically to control seed size, but controls seed size independently of EOD1
Da2-1 mutant shows weak but similar with da1-1 seed size phenotype (Li et al. people, 2008), refers to provide Show that DA1 and DA2 can be worked with common pathway.In order to test the genetic interaction between DA1 and DA2, we are made Da1-1 da2-1 double-mutant and determine its seed size.Although da2-1 mutant have it is more slightly greater than wild type and Heavier seed (Figure 1A, 3C and 3D), but da2-1 mutation collaboration enhancing da1-1 Seed size and weight phenotype (Fig. 3 A and 3C), genetic interaction is cooperateed between DA1 and DA2 in terms of seed size to disclose.The variation of seed size is reflected in In the size of plumule and gained seedling (Fig. 3 B).Further measure the cotyledon area of 10 day age seedling.Also it observes and passes through The collaboration that da2-1 is mutated the cotyledon size of da1-1 enhances (Fig. 3 B and 4).The mutant protein tool encoded by da1-1 allele There is the cathode active (Li et al. people, 2008) for DA1 and DA1 GAP-associated protein GAP (DAR1) (most closely related family member).
Double da1-ko1 dar1-1T-DNA insertion mutation body surfaces reveal da1-1 phenotype, and da1-ko1 and dar1-1 is mono- prominent Variant does not show apparent seed size phenotype (Li et al. people, 2008).Because da1-1 and da2-1 synergistic effect is to increase seed Expected da1-ko1 can be cooperateed with the phenotype of enhancing da2-1 by size.In order to test this point, da1-ko1 da2- has been made in we 1 double-mutant.As shown in fig.3d, the Seed size and weight phenotype of da2-1 is mutated collaboration enhancing also by da1-ko1.Into The one step surveying cotyledon area of 10 day age seedling.Da1-ko1 mutation collaboration enhances the cotyledon size phenotype (right side Fig. 4 of da2-1 On).Similarly, it was further observed that the petal size (Figure 16 D) of collaboration enhancing da2-1 is mutated by da1-ko1.These results are into one Step proves the synergistic effect destroyed while both DA1 and DA2.
Further measure the size of embryo cell and petal epidermal cell.Compared to the cell measured in its parental department The cell size of size, da1-1 da2-1 and da1-ko1 da2-1 double-mutant does not increase the (lower-left Fig. 4；Figure 16 E), to mention Proliferation process is limited for instruction DA1 and DA2 synergistic effect.
Da1-1 da2-1 double-mutant has the seed (Fig. 3 C, 3D and 4) bigger than da1-ko1 da2-1 double-mutant, This has phenotype more stronger than da1-ko1 consistent (Li et al. people, 2008) with the da1-1 allele that we are previously reported.da1-1 The size of seed is similar to the size of da1-ko1 dar1-1 double-mutant seed, because da1-1 allele, which has, is directed to DA1 With the cathode active (bottom right Fig. 4) (Li et al. people, 2008) of DAR1.Therefore, by the big of expected da1-1 da2-1 double-mutant seed The small size that may look like da1-ko1 dar1-1 da2-1 Trimutant seed.Then da1-ko1 has been made in we Dar1-1 da2-1 Trimutant and its seed size is studied.As shown in Figure 4, da1-ko1 dar1-1 The comparable size of da2-1 Trimutant seed must go up the size of da1-1 da2-1 double-mutant seed, but be greater than da1-ko1 The size of da2-1 double-mutant seed.Therefore, these genetic analyses further support da1-1 allele to DA1 and DAR1 two Person has negative effect (Li et al. people, 2008).
We previously authenticated the enhancer (EOD1) of da1-1, and the enhancer is and BIG BROTHER (BB) equipotential (Disch et al., 2006；Li et al. people, 2008).Eod1 mutation collaboration enhancing da1-1 seed size phenotype (Li et al. people, 2008).Similarly, the Seed size and weight phenotype of da2-1 by da1-1 and da1-ko1 collaboration enhancing (Fig. 3 A, 3C and 3D).Therefore whether inquiry DA2 and EOD1 can be worked with common pathway.In order to which the heredity measured between DA2 and EOD1 is closed System, we analyze eod1-2da2-1 double-mutant.Heredity compared to its parental department, between eod1-2 and da2-1 Interaction is (Figure 16) being substantially added for both seed weight and petal size, thus provide instruction DA2 with EOD1 separately works to influence seed and organ growth.
Act on to influence seed size to 2.3 DA2 parents
In view of the size of seed is by parent and/or zygote tissue effect, inquire DA2 be parent work or zygote It works on ground.In order to test this point, We conducted the reciprocal cross experiments between wild type and da2-1.As shown in Figure 6, Only effect of the da2-1 to seed size is observed when it is homozygosis that maternal plant is for da2-1 mutation.Regardless of pollen donor Genotype, the seed that is generated by parent wild-type plant is consistently greater than by the seed that parent da2-1 plant generates.This result Instruction da2-1 can act on to parent to influence seed size.Previously we worked to control kind with being proved DA1 also parent Sub- size (Li et al. people 2008).Since da1-ko1 is mutated the seed size phenotype (Fig. 3 D) of collaboration enhancing da2-1, we are into one Step has carried out the experiment of the reciprocal cross between wild type and da1-ko1 da2-1 double-mutant.Similarly, only in da1-ko1 da2- 1 observes effect (Fig. 6) of the da1-ko1 da2-1 to seed size when serving as maternal plant.
Da1-ko1/+ is caused to da1-ko1/+da2-1/+ plant pollination with da1-ko1 da2-1 double-mutant pollen The intracutaneous da1-ko1 da2-1 of da2-1/+ kind, da1-ko1/da1-ko1 da2-1/+, da1-ko1/+da2-1da2-1 and The development of da1-ko1/+da2-1/+ plumule.It further measures come da1-ko1 da2-1 double-mutant pollen fertilization of using by oneself The size of the single seed of da1-ko1/+da2-1/+ plant and to da1-ko1 and da2-1 mutation carry out Genotyping.As a result Show that da1-ko1 and da2-1 is mutated (Fig. 6) uncorrelated to the variation of the size of these seeds.In short, these analysis instructions DA1 Will not influence seed size with the plumule of DA2 and the genotype of endosperm, and need in the sporophytic tissues of maternal plant DA1 and DA2 is to control seed growth.
2.4 DA2 and DA1 act synergistically to influence the cell Proliferation in parent integument
Reciprocal cross works to determine seed size (Fig. 6) (Li et al. people, 2008) with showing DA1 and DA2 parent.Around embryo The integument of pearl is maternal tissue and forms kind of a skin after fertilization, can physically limit seed growth.Several studies have shown that The integument size of ovule determine seed size (Schruff et al., 2006；Adamski et al., 2009).Therefore inquiry DA1 and Whether DA2 is acted on by parent integument to control seed size.In order to test this point, we have studied 2 days after emasculation When the mature ovule from wild type, da1-1, da2-1 and da1-1 da2-1.Previous discovery (the Li et al. people, 2008) with us Unanimously, the size (Fig. 5 and 7) for being sized significantly greater than wild type ovule of da1-1 ovule.
Da2-1 ovule is also greater than wild type ovule (Fig. 5 and 7).The ovule size table of da2-1 mutation collaboration enhancing da1-1 Type, this is consistent with its cooperative interaction in terms of seed size.
We have studied wild types, the positive development seed in da1-1, da2-1 and da1-1 da2-1 in 6DAP and 8DAP External integument cell number.In wild type seeds, in 6DAP, the number of external integument cell is similar to the external integument in 8DAP The number (Fig. 7 middle graph) of cell, to indicate that the cell in the external integument of wild type seeds stops dividing completely in 6DAP. Similarly, the cell in the external integument of da1-1, da2-1 and da1-1 da2-1 seed stops cell Proliferation in 6DAP completely. Compared to the number of the external integument cell in wild type seeds, the number of the external integument cell in da1-1 and da2-1 seed is significant Increase (Fig. 7).The external integument cell number of da2-1 mutation collaboration enhancing da1-1.We further study after pollination 6 days With the external integument cell length of wild type at 8 days, da1-1, da2-1 and da1-1 da2-1 seed.Da1-1, da2-1 and da1-1 Cell in da2-1 external integument is considerably shorter than the cell (Fig. 7 right figure) in wild type external integument, increases to provide cell in integument Grow the instruction of the compensation mechanism between cell amplification.Therefore, these are the result shows that DA2 and DA1 acts synergistically to limit parent Cell Proliferation in integument.
2.5 DA2 encoding function E3 ubiquitin ligases
It authenticated da2-1 mutation (Fig. 8 A) in the case that T-DNA is inserted into the 7th exon of Gene A t1g78420. Using T-DNA specificity and flank primer and sequencing PCR product by PCR further confirms T-DNA insertion point. The full length mRNA of At1g78420 fails to detect in da2-1 mutant by Semiquatitative RT-PCR assay.We are in da2-1 plant With in 62 kinds of transgenic plants of separation their own promoter control under express At1g78420CDS.Almost all of turn Gene line shows the complementation (Figure 10) of da2-1 phenotype, to indicate that At1g78420 is DA2 gene.
In order to further characterize DA2 function, the specifically acquired phenotype of function, in WT lines and 77 kinds of separation The code area of DA2 is expressed in transgenic plant under the control of CaMV 35S promoter.The overexpression of DA2 causes seed big The reduction (Figure 1A, 1C and 1D) of the number seeds of the seed production and every plant of small, every plant.In addition, compared to wild type, The most of transgenic plants for over-expressing DA2 have compared with little Hua and leaf, compared with silicle, the plant height of reduction and reduction Biomass (Fig. 1 E, 2 and 15).These results further support effect of the DA2 in limitation seed and organ growth.
Predict the protein of 402 amino acid of the RING structural domain (59-101) that DA2 gene coding is predicted containing one (Fig. 8 B；Table 1).In order to study whether DA2 has E3 ubiquitin ligase activity, we are in expression in escherichia coli DA2, by it MBP-DA2 albumen is purified as the fusion protein with maltose-binding protein (MBP), and from soluble fraction.In E1 ubiquitin activating In the presence of enzyme, E2 conjugated enzyme, His- ubiquitin and MBP-DA2, poly- ubiquitin is observed by Western blotting using anti-His antibody Change signal (Fig. 9, from the 5th swimming lane of the left side).Anti- MBP engram analysis also indicates that MBP-DA2 by ubiquitination (Fig. 9, from the left side the 5th Swimming lane).However, in the absence of any one of E1, E2, His- ubiquitin or MBP-DA2, be not detected poly- ubiquitination (Fig. 9, From first to fourth swimming lane of the left side), to prove that DA2 is functional E3 ubiquitin ligase.RING motif is for RING zinc finger egg It is required (Xie et al., 2002) for white E3 ubiquitin ligase activity.Therefore, we have detected complete RING zinc finger knot Whether structure domain is needed for DA2 E3 connection enzymatic activity.List is generated by the way that cysteine -59 is sported serine (C59S) Amino acid substitution allele, because predicting that this mutation destroys RING structural domain (table 1 and 2).External ubiquitination measurement instruction E3 Connection enzymatic activity is eliminated (Fig. 9, from the 6th swimming lane of the left side) in the C59S mutant of DA2, to indicate complete RING structure Domain is needed for DA2 E3 ubiquitin ligase activity.We are further in 69 kinds of transgenosis of wild type Col-0 plant and separation DA2 C59S (35S:DA2C59S) has been over-expressed in plant.The seed size of transgenic plant is comparable to WT lines Seed size, but transgenic plant have high DA2 C59S expression, thus indicate DA2 C59S mutation influence DA2 exist Function in terms of seed growth.
Three kinds of RING type RING-H2, RING-Hca and RING-HCb are described in arabidopsis and five kinds are repaired RING type RING-C2, RING-v, RING-D, RING-S/T and RING-G (Stone et al., 2005) of decorations.It proposes in water The RING structural domain (C5HC2) (Song et al., 2007) of the new type found in rice GW2.Although the RING structure of the prediction of DA2 The spacing of cysteine in domain is similar to the spacing of the cysteine in the RING structural domain (C5HC2) of rice GW2, but DA2 RING structural domain lack the conservative histidine residues (Asn-91) (table 1 and 2) that are substituted by asparagine residue.Also in Shuangzi This amino acid substitution (table 1) is observed in the RING structural domain of the prediction of DA2 homologue in leaf plant such as soybean and rape.Cause This inquires whether this asparagine residue (Asn-91) is crucial for its E3 ubiquitin ligase activity.By by Asn- 91 sport leucine (N91L) to generate monamino acid and replace allele.External ubiquitination measurement shows that the N91L of DA2 is prominent Variant has E3 connection enzymatic activity (Fig. 9, from the 7th swimming lane of the left side), to show that Asn-91 may not be DA2E3 connection enzyme activity Needed for property.These are the result shows that the RING structural domain of DA2 can be the variant of the RING structural domain found in GW2.We Further DA2 N91L (35S:DA2N91L) has been over-expressed in WT lines and 26 kinds of transgenic plants of separation.Turn The seed of gene plant is less than wild type seeds, to show that DA2N91L can limit seed growth.
The homologue of 2.6 arabidopsis DA2
With the DA2 outside RING structural domain share the protein of significant homology arabidopsis and crop include rape, it is big (table 2) is found in beans, rice, corn and barley.The protein and DA2 of one of arabidopsis prediction share extensive amino Sour similitude and it is named as DA2 sample albumen (DA2L；At1g17145).As 35S:DA2 plant, DA2L overexpression System show compared with plantlet and organ (Figure 18), thus provide instruction DA2 have the function of with DA2L it is similar.Other plant species In analogous protein show with 39.2% -84.5% amino acid sequence identity (table 2) of DA2.It is same in cabbage type rape Source object and DA2 have highest amino acid sequence identity (84.5%) (table 2).Rice GW2 and arabidopsis DA2 has 43.1% ammonia Base acid sequence identity (table 2).Since the grain that the overexpression of GW2 reduces in rice is wide (Song et al., 2007), therefore inquire Whether DA2 exercises similar function with GW2 in terms of seed size control.Therefore GW2 has been over-expressed in WT lines. As 35S:DA2 and 35S:DA2L transgenosis system, WT lines are compared in the arabidopsis transgenic plant generation for over-expressing GW2 Smaller seed and organ, to indicate the conservative function of arabidopsis DA2 and rice GW2 in terms of seed and organ growth control Energy.
2.7 DA2 show similar expression pattern with DA1
In order to measure the expression pattern of DA2, the RNA from root, stem, leaf, seedling and inflorescence is passed through into quantitatively real-time RT- PCR is analyzed.DA2 mRNA (Figure 11 A) is detected in all plant organs tested.Start using containing DA2 Son: the active histochemistry's measurement of the GUS of the transgenic plant of GUS (pDA2:GUS) fusion has studied the tissue specificity of DA2 Expression pattern.Detect that GUS is active (Figure 11 B and 11C) in root, cotyledon, leaf and inflorescence.It is detected in phyllopodium and root Relatively high GUS is active (Figure 11 B and 11C).In spending, observed in young flower organ relatively more stronger than in presbyopic organ DA2 expresses (Figure 11 D-11L).Similarly, detected in compared with rataria pearl higher GUS activity in the old ovule of comparison (Figure 11 M and 11N).This shows that DA2 expression is regulated and controled over time and space.
2.8 DA1 and DA2 interacts in vitro and in vivo
Our genetic analysis shows DA1 and DA2 synergistic effect to limit seed and organ growth.Therefore external phase is used Interaction/involve whether Experimental Evaluation DA1 interacts with E3 ubiquitin ligase DA2.DA1 is expressed as gst fusion protein, and DA2 is expressed as MBP fusion protein.As shown in Figure 12 (from the first and second swimming lane of the left side), GST-DA1 is bound to MBP- DA2, and GST-DA1 is not associated with to negative control (MBP).This result instruction DA1 and DA2 physically interacts in vitro.
DA1 includes two ubiquitin interactions motif (UIM), single LIM domain and highly conserved C-terminal region (Figure 13) (Li et al. people, 2008).Further which structural domain of inquiry DA1 is needed for the interaction between DA1 and DA2. It will be in a series of DA1 derivatives containing specific protein structural domain of expression in escherichia coli: UIM structural domain there are two containing only DA1-UIM, only with LIM domain DA1-LIM, the DA1-LIM+C only containing LIM domain and C-terminal region and Only the DA1-C with C-terminal region is expressed as gst fusion protein (Figure 13).
DA2 is expressed as MBP fusion protein and is used to involve in experiment.As shown in Figure 12, GST-DA1-LIM+C and GST-DA1-C and MBP-DA2 interacts, but GST-DA1-UIM and GST-DA1-LIM are not associated with to MBP-DA2.This result The conservative C-terminal region and DA2 for indicating DA1 interact.
There is the mutation in C-terminal region in view of the mutant protein encoded by da1-1 allele (DA1R358K) Whether (Figure 13) (Li et al. people, 2008), inquiry DA1R358K mutation influence the interaction with DA2.In leading with MBP-DA2 GST-DA1R358K fusion protein is used in experiment out, we show that the mutation in DA1R358K does not influence between DA1 and DA2 It interacts (Figure 12, from left side third swimming lane).
In order to further study in plant possible correlation between DA1 and DA2, detected using co-immunoprecipitation analysis It interacts in vivo.35S:Myc-DA1 and 35S:GFP-DA2 are instantaneously co-expressed in Ben's Tobacco Leaf.In Ben's Tobacco Leaf In instantaneously co-express 35S:GFP and 35S:Myc-DA1 as negative control.By gross protein separate and with GFP-Trap-A agar Sugared bead is incubated with immunoprecipitation GFP-DA2 or GFP.Respectively with anti-GFP and anti-Myc antibody test sediment.Such as Figure 14 Shown in, Myc-DA1 is detected in the GFP-DA2 compound of immunoprecipitation, but is not detected in negative control (GFP), To which instruction is there are physical correlations between DA1 and DA2 in plant.Because involve measurement in DA1 C-terminal region with DA2 interacts (Figure 12), so further whether the C-terminal of inquiry DA1 in plant interacts with DA2.It is immunized coprecipitated It forms sediment analysis shows that detect the C-terminal region (Myc-DA1-C) of DA1 in GFP-DA2 compound, but not in negative control Detected in (PEX10-GFP, a kind of RING type E3 ubiquitin ligase) (Platta et al., 2009；Kaur et al., 2013).Cause This, the C-terminal region of these results instruction DA1 is needed for interacting in vitro and in vivo with DA2.
Seed size in higher plant is the key determinant of evolutionary adaptability, and the still weight in Crop Domestication Want economical character (Gomez, 2004；Orsi and Tanksley, 2009).
It acts on having authenticated parent to control several factors of seed size, such as ARF2/MNT, AP2, KLU/ CYP78A5, EOD3/CYP78A6 and DA1.However, heredity and molecular mechanism of these factors in seed size control are almost Totally unknown.It is big to control seed that we had previously demonstrated ubiquitin receptor DA1 and E3 ubiquitin ligase EOD1/BB synergistic effect Small (Li et al. people, 2008).
In this research, we, which authenticated, participates in control kind as the arabidopsis DA2 of another RING E3 ubiquitin ligase Sub- size.Genetic analysis shows DA2 and DA1 co-action to control final seed size, but independently of E3 ubiquitinbond Enzyme EOD1 controls final seed size.We further disclose DA1 and DA2 and physically interact.Our result is fixed The final seed size of justice control arabidopsis is related to the system based on ubiquitin of DA1, DA2 and EOD1.
Act on to control seed size to 2.9 DA2 parents
Da2-1 afunction mutant forms big seed and organ, and the plant for over-expressing DA2 generates small seed With organ (Figure 1A), thus indicate DA2 be seed and organ size control the negative factor.Unexpectedly, arabidopsis DA2 is nearest It is proposed as the positive regulatory factor of organ growth, although how to control seed about DA2 and organ growth is known nothing (Van Daele et al., 2012).In this research, we have enough evidences to prove that DA2 serves as seed and organ growth control The negative factor of system.Da2-1 afunction mutant forms big seed and organ (Fig. 1 to 4).Da2-1 mutation collaboration enhancing The seed and organ size phenotype (Fig. 1 to 4) of da1-1 and da1-ko1 supports this point.Da2-1 mutation also enhances eod1-2 Seed and organ size phenotype, so that further instruction da2-1 mutation promotes seed and organ growth.Da2-1 mutant exists The big ovule with more many cells, and the ovule size phenotype (Fig. 6) of da2-1 mutation collaboration enhancing da1-1 are formed in integument.
In addition, most of transgenic plants of overexpression DA2 and DA2L are (Fig. 2 smaller than WT lines；Scheme S9).This The corresponding expression of organ growth phenotype of a little transgenic plants is related (figure S4 and S9).Therefore, our data understand Ground proves that DA2 serves as the negative regulatory factor of seed and organ size.Several Arabidopsis Mutants with big organ are also formed greatly Seed (Krizek, 1999；Mizukami and Fischer, 2000；Schruff et al., 2006；Li et al. people, 2008；Adamski Et al., 2009), to show that the possibility between organ size and seed growth contacts.In contrast, several with big organ Other mutant show normal size seed (Hu et al., 2003；White, 2006；Xu and Li, 2011), thus indicator Official and seed size be not always positively related.These are the result shows that there is shared and different approach to control it for seed and organ Corresponding size.
Reciprocal cross experiment acts on to influence seed growth with showing DA2 parent, and the plumule of DA2 and endosperm genotype are not It will affect seed size (Fig. 6).Integument around ovule is maternal tissue and forms kind of a skin after fertilization.Parent integument is big Known variation (the Schruff for facilitating seed size of small change those of (such as observed in arf2, da1-1 and klu ovule) Et al., 2006；Li et al. people, 2008；Adamski et al., 2009).Mature da2-1 ovule is also greater than mature wild type ovule (Fig. 5 With 7).The integument size of da2-1 mutation also collaboration enhancing da1-1 ovule.It therefore, is control from total theme that these researchs show Parent integument size is one of the key mechanism for determining final seed size.It is of the same mind with this, it separates so far Seed size control plant maternal factor (such as KLU, ARF2 and DA1) influence integument size (Schruff et al., 2006；Li Et al., 2008；Adamski et al., 2009).
Integument or the size for planting skin are to expand by cell Proliferation and cell the processes of the two coordinations to determine.It is mature Cell number in the integument of ovule sets the growth potential that skin is planted after fertilization.For example, the generation of arf2 mutant has The big ovule of more many cells, so that big seed (Schruff et al. 2006) is generated, and klu mutant has less cell Small ovule, to generate seedlet (Adamski et al., 2009).Our result indicate that the integument of da1-1 and da2-1 seed There are more many cells than the integument of wild type seeds, and da1-1 and da2-1 synergistic effect is to promote the cell in integument to increase It grows.We have also observed that the cell in the external integument of da1-1, da2-1 and da1-1 da2-1 seed is than thin in wild type integument Born of the same parents are shorter, to show the possible compensation mechanism in parent integument between cell Proliferation and cell elongation.Therefore, it is possible to fill When the parent integument or kind skin of the physical constraint of seed growth can set the upper limit of final seed size.
The hereditary frame of the 2.10 seed size controls mediated for ubiquitin
DA2 coding has the RING knot of a prediction of any one of plant RING structural domain different from the previously described The protein in structure domain.The RING structural domain of DA2 and the RING structural domain of rice GW2 (C5HC2) share highest homology, but it is lacked A kind of weary conservative metal ligand amino acid (histidine residues) (Song et al., 2007) substituted by asparagine residue.Still The RING structural domain for being so possible to DA2 can be the variant of the RING structural domain found in GW2.Many RING type structural domains exist Be found in E3 ubiquitin ligase, ubiquitination substrate often target they to subsequent proteasome degradation (Smalle and Vierstra, 2004).It is function that we, which test the E3 activity for recombinating DA2 in external ubiquitinbond enzymatic determination and demonstrate DA2, Can property E3 ubiquitin ligase, thus show DA2 can target the positive regulatory factor of cell Proliferation with for by 26S proteasome into The degradation of row ubiquitin dependence.The protein of homology is shared in arabidopsis and other plant object with the DA2 outside RING structural domain It is found in kind.In arabidopsis, DA2 sample albumen (DA2L) and DA2 share extensive amino acid similarity.It is planted with 35S:DA2 Strain is the same, and DA2L overexpression system shows compared with plantlet (Figure 18), to indicate that DA2 can exercise similar function with DA2L. The homologue of DA2 is RING type (C5HC2) Protein G W2 (Song et al., 2007) in rice, known to serve as the negative of seed size Regulatory factor.However, heredity and molecular mechanism of the GW2 in seed size control are largely unknown in rice 's.
We previously authenticated DA1, and there is one kind ubiquitin to combine active ubiquitin receptor, the negative regulation as seed size The factor (Li et al. people, 2008).Modifier screens a kind of enhancer (EOD1) (Li et al. people, 2008) that authenticated da1-1, described Enhancer is and E3 ubiquitin ligase BB equipotential (Disch et al., 2006).The analysis of double eod1-2da1-1 mutant discloses Genetic interaction (Li et al. people, 2008) is cooperateed between DA1 and EOD1, to show that they can be by adjusting shared target Activity controls seed growth.Although the genetic interaction between da1-1 and eod1-2 also cooperates with enhancing seed and organ big It is small, but genetic analysis shows DA2 independently of EOD1 effect to influence seed growth, is used for show that DA2 and EOD1 can be targeted The different growth stimulants of degradation, wherein regulating and controlling jointly via DA1.Therefore, our discovery is established through three kinds of ubiquitin phases 3-protein d A1, DA2 and EOD1 of pass are used to control the frame of seed and organ size.In addition, it is observed that GW2's is excessive Seed and organ growth in expression limitation arabidopsis, to provide the finger of the possible conservative function in arabidopsis and rice Show.The combination for studying the rice homologue of GW2 and DA1 and EOD1 in rice may be very interesting to the effect of grain size.
Possible molecular mechanism of 2.11 DA1 and DA2 in seed size control
Our result proves that E3 ubiquitin ligase DA2 and ubiquitin receptor DA1 interact (Figure 12-in vitro and in vivo 14).However, it is unlikely be DA2 targeting DA1 with for proteasome degrade because the T-DNA of DA1 gene be inserted into dash forward The seed size phenotype (Fig. 3 and 4) of variant (da1-ko1) collaboration enhancing da2-1.However, many other types of ubiquitin modification Regulation protein (Schnell and the Hicke, 2003) in a manner of proteasome dependent/non-dependent.For example, single ubiquitination is involved in always In the activation of signal conductive protein, endocytosis and histone modification (Schnell and Hicke, 2003).In animal, ubiquitin Single ubiquitination of receptor eps15 depend on eps15 and E3 ligase Nedd4 family between interaction (Woelk et al., 2006).In contrast, it is also reported that the E3 dependent/non-dependent list ubiquitination (Hoeller et al., 2007) of ubiquitin receptor.It considers DA1 and DA2 interacts, and whether we test DA2 being capable of ubiquitination or single ubiquitination DA1.Exist in E1, E2 and ubiquitin Under, DA2-His has E3 ubiquitin ligase activity.However, in the presence of E1, E2, ubiquitin and DA2-His (E3), ours The DA1-HA of ubiquitination is not detected under reaction condition.Ubiquitin receptor can be via the bottom of the poly- ubiquitination of structural domain UIM and E3 Object interaction, and promote it to degrade (Verma etc., 2004) by proteasome.We had previously demonstrated the UIM knot of DA1 Structure domain can combine ubiquitin (Li et al. people, 2008).
In short, DA1 may participate in mediation in the case where DA1 is interacted by its C-terminal area and DA2 (Figure 12 and 14) Pass through the ubiquitination substrate of proteasome degradation DA2.A kind of mechanism can be related to the interaction of DA1 and DA2, facilitate DA1 Specifically identify the ubiquitination substrate of DA2.DA1 then can pass through the poly-ubiquitin chain of its UIM structural domain combination ubiquitination substrate And mediate the degradation of the poly- ubiquitination substrate.The important goal that seed production is crop breeding in worldwide is improved, And the size of seed is the important component of total seed production.We authenticated important regulating and controlling of the DA2 as seed size The factor influences seed size with DA1 co-action.
DA1 also influences seed growth with EOD1 synergistic effect.Dominant negative da1-1 is mutated the excessive of (Zmda1-1) Expression it is reported that increase the seed quality (Wang etc., 2012) of cereal, this demonstrate combine DA1 from different seed crops, The effect of DA2 and EOD1 in these crops to be engineered the prospect of big seed size.
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The comparison (SEQ ID NO:3-19) of table 1:DA2 RING structural domain.
The comparison (SEQ ID NO:20-35) of table 2:DA2 polypeptide
* identical residue is indicated
: indicate conservative residue
Semi-conservative residue is indicated
RING structural domain and the first and second apokoinou construction domains add frame.
The comparison (SEQ ID NO:41-64) of table 3:DA1 albumen
The comparison (SEQ ID NO:74-90) of table 4:EOD1 albumen

Claims (34)

1. a kind of quality, the method for the size of seed and organ of the per unit area seed for increasing plant, which comprises
The expression or activity of the intracellular DA2 polypeptide of the plant are reduced,
Wherein, the DA2 polypeptide includes the RING structural domain of SEQ ID NO:2, and wherein the plant has reduced DA1 Or reduced DA1 and EOD1 expression or activity.
2. according to the method described in claim 1, wherein the plant expresses dominant negative DA1 polypeptide.
3. according to claim 1 or method as claimed in claim 2, wherein the expression of the DA2 polypeptide or activity are in the plant It is eliminated in the cell of object.
4. a kind of quality, the method for the size of seed and organ of the per unit area seed for increasing plant, which comprises
The expression or activity of DA2 polypeptide are reduced, and；
The expression of the intracellular DA1 polypeptide of the plant or the expression or activity of activity or DA1 polypeptide and EOD polypeptide are reduced, Wherein, the DA2 polypeptide includes the RING structural domain of SEQ ID NO:2.
5. according to the method described in claim 4, wherein by that will be mutated in the nucleotide sequence for being introduced into the plant cell simultaneously And plant described in the cytothesis from mutation reduces the expression or activity of the EOD1 polypeptide, the nucleotide sequence coded institute It states EOD1 polypeptide or regulates and controls its expression.
6. according to the method described in claim 4, wherein by heterologous nucleic acids are incorporated in the plant cell reduce it is described The expression or activity of EOD1 polypeptide, the heterologous nucleic acids expression reduce the repressor nucleic acid of the expression of the EOD1 polypeptide.
7. according to the method described in claim 6, wherein by that will be mutated in the nucleotide sequence for being introduced into the plant cell simultaneously And plant described in the cytothesis from mutation reduces the expression or activity of the DA1 polypeptide, the nucleotide sequence coded institute It states DA1 polypeptide or regulates and controls its expression.
8. according to the method described in claim 6, wherein by heterologous nucleic acids are incorporated in the plant cell reduce it is described The expression or activity of DA1 polypeptide, the heterologous nucleic acids expression reduce the repressor nucleic acid of the expression of the DA1 polypeptide.
9. according to the method described in claim 6, wherein passing through the dominant negative DA1 polypeptide of cell inner expression in the plant To reduce the expression or activity of the DA1 polypeptide.
10. the method according to any one of claim 4 to 9, wherein by the way that the core for introducing the plant cell will be mutated Plant described in cytothesis in nucleotide sequence and from mutation reduces the expression or activity of the DA2 polypeptide, the nucleosides DA2 polypeptide described in sequences code regulates and controls its expression.
11. the method according to any one of claim 4 to 9, wherein by the way that heterologous nucleic acids are incorporated to the plant cell In reduce the expression or activity of the DA2 polypeptide, the heterologous nucleic acids expression reduces the repressor of the expression of the DA2 polypeptide Nucleic acid.
12. the method according to any one of claim 4 to 9, wherein the expression of the DA2 polypeptide or activity are in the plant It is eliminated in the cell of object.
13. a kind of method for the plant for generating the quality, the size of seed and organ with increased per unit area seed, institute The method of stating includes:
The expression for lacking both DA1 or DA1 and EOD1 or active plant cell are provided,
The expression for reducing DA2 polypeptide or active heterologous nucleic acids are incorporated to, the DA2 polypeptide includes the RING knot of SEQ ID NO:2 Structure domain；Or the expression for reducing DA2 polypeptide or active mutation are introduced into the plant cell, the DA2 polypeptide includes SEQ The RING structural domain of ID NO:2, and；
Plant described in cytothesis from one or more conversion.
14. according to the method for claim 13, wherein the plant cell includes the nucleotide sequence of the plant cell In mutation, the nucleotide sequence coded EOD1 polypeptide or regulate and control its expression；Or another heterologous nucleic acids, the heterologous core Acid expression reduces the repressor nucleic acid of the expression of the EOD1 polypeptide in the plant cell.
15. according to claim 13 or method of claim 14, wherein the plant cell lack DA1 polypeptide expression or Activity.
16. according to the method for claim 15, wherein the plant cell includes the nucleotide sequence of the plant cell In mutation, the nucleotide sequence coded DA1 polypeptide or regulate and control its expression；Another heterologous nucleic acids, the heterologous nucleic acids Expression reduces the repressor nucleic acid of the expression of the DA1 polypeptide in the plant cell；Or another heterologous nucleic acids, it is described heterologous Cell inner expression dominant negative DA1 polypeptide of the nucleic acid in the plant.
17. according to the method for claim 13, wherein the heterologous nucleic acids expression inhibiting daughter nucleus acid is to the plant cell In, the repressor nucleic acid reduces the expression of the DA2 polypeptide.
18. according to the method for claim 13, wherein the heterologous nucleic acids eliminate the DA2 polypeptide in the cell of the plant Expression or activity.
19. according to claim 1, method described in any one of 4 or 13, wherein the DA polypeptide of encoding dominant negative feminine gender and/or suppression The nucleic acid of system nucleic acid is operably coupled to allogeneic promoter.
20. according to the method for claim 19, wherein the promoter is tissue-specific promoter.
21. according to the method for claim 20, wherein the promoter is inducible promoter.
22. according to claim 1, method described in any one of 4 or 13, wherein heterologous nucleic acids are included in one or more loads In body.
23. according to claim 1 or method as claimed in claim 4, the method includes sexual or vegetative propagation or growth tools There are DA2 expression or filial generation or the offspring of the active plant of reduction.
24. according to claim 1, method described in any one of 4 or 13, wherein the DA2 polypeptide includes SEQ ID NO:36 The first apokoinou construction domain.
25. according to claim 1, method described in any one of 4 or 13, wherein the DA2 polypeptide includes SEQ ID NO:37 The second apokoinou construction domain.
26. according to claim 1, method described in any one of 4 or 13, wherein the DA2 polypeptide includes according to SEQ ID The amino acid sequence of NO:20 to any one of 35.
27. according to claim 1, method described in any one of 4 or 13, wherein the DA1 polypeptide includes SEQ ID NO:38 Or 39 LIM domain.
28. according to the method for claim 27, wherein the DA1 polypeptide includes C-terminal region, the C-terminal region packet Include the residue 229 to 532 of SEQ ID NO:45.
29. according to the method for claim 27, wherein the DA1 albumen includes according to appointing in SEQ ID NO:41 to 64 The sequence of one.
30. according to the method for claim 28, wherein the DA1 albumen includes according to appointing in SEQ ID NO:41 to 64 The sequence of one.
31. according to claim 1, method described in any one of 4 or 13, wherein to be included in the DA1 more by dominant negative DA1 The R to K at the position of the position 358 of the DA1 polypeptide of SEQ ID NO:45 is equivalent in the amino acid sequence of peptide to replace.
32. according to claim 1, method described in any one of 4 or 13, wherein the EOD1 polypeptide includes according to SEQ ID The sequence of NO:74 to any one of 90.
33. according to claim 1, method described in any one of 4 or 13, wherein the plant is higher plant.
34. according to the method for claim 33, wherein the plant is that the agricultural selected from the group being made of the following terms is planted Object: Asian puccoon, Taxus, tobacco, cucurbit, carrot, Vegetables in Brassica, melon, capsicum, vine, lettuce, strawberry, Brassica genus Oily seed, beet, wheat, barley, corn, rice, soybean, pea, jowar, sunflower, tomato, potato, pepper, chrysanthemum Flower, carnation, linseed, hemp and rye.
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