CN103045638A

CN103045638A - Plants having improved growth characteristics and method for making the same

Info

Publication number: CN103045638A
Application number: CN2012105354751A
Authority: CN
Inventors: V·弗兰卡德; C·鲁兹; A·I·桑兹莫林纳罗
Original assignee: CropDesign NV
Current assignee: CropDesign NV
Priority date: 2004-08-16
Filing date: 2005-08-16
Publication date: 2013-04-17
Also published as: CN101040050A; ZA200701304B

Abstract

The present invention concerns a method for improving the growth characteristics of plants by increasing activity in a plant of an RNA-binding protein or a homologue thereof, wherein said RNA-binding protein or homologue thereof is either: (i) a polypeptide having RNA-binding activity and comprising either 2 or 3 RNA 10 recognition motifs (RRMs) and a motif having at least 75% sequence identity to motif I: PIYEAAVVALPVVVKERLVRILRLGIATRYD and/or a motif having at least 50% sequence identity to motif II: RFDPFTGEPYKFDP; or (ii) an RBP1 polypeptide or homologue thereof having (a) RNA-binding activity; (b) two RRM domains, (c) the following two motifs: (i) KIFVGGL; and (ii) 15 RPRGFGF, allowing for up to three amino acid substitutions and any conservative change in the motifs; and (d) having at least 20% sequence identity to the amino acid represented by SEQ ID NO: 15. The invention also concerns to transgenic plants having introduced therein an RNA-binding protein-encoding nucleic acid or variant thereof, which plants have improved growth characteristics relative to corresponding wild type plants. The present invention also concerns constructs useful in the methods of the invention.

Description

Has plant of improvement growth characteristics and preparation method thereof

The application be submitted on August 16th, 2005, denomination of invention divides an application for the PCT application PCT/EP2005/054034's of " having plant of improvement growth characteristics and preparation method thereof ", it is on April 12nd, 2007 that described PCT application enters the date in China national stage, and application number is 200580034841.3.

Technical field

Relate generally to biology field of the present invention, and relate to the method for improving plant growth characteristic.More specifically, the present invention relates to come the improving plant growth characteristic by the activity of rna binding protein or its homologue in the increase plant, especially the method for productive rate.The invention still further relates to have increases active rna binding protein or the plant of its homologue, and these plants have the growth characteristics of improvement with respect to corresponding wild-type plant.Rna binding protein or its homologue useful in the inventive method have RNA in conjunction with activity, and comprise 2 or 3 RNA identification motifs (RRM), and comprise with motif I:PYEAAVVALPVVVKERLVRILRLGIATRYD and have the motif of at least 75% sequence identity and/or the motif that has at least 50% sequence identity with motif II:RFDPFTGEPYKFDP.Rna binding protein or its homologue useful in the inventive method also can be RBP1 polypeptide or its homologues with following feature: (a) RNA is in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF allow at the most three aminoacid replacement and arbitrarily conservative change in the motif; (d) amino acid according to the preference ordering that increases and SEQ IDNO:15 representative has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity.The present invention also provides useful in the methods of the invention construct.

Technical background

The continuous growth of world population and be used for the minimizing of tilled the land supplys of agricultural promotes farming research towards the future development of raising farm efficiency.The ordinary method of farm crop and gardening improvement is the plant that utilizes breeding technique to identify to have desired characteristic.Yet this breeding technique has several shortcomings, and namely these technology are normally labor-intensive, and produces the plant that usually contains the allogeneic heredity component, and these hereditary components are not always to produce the desirable proterties of transmitting from mother plant.Molecular biological progress has allowed the germplasm (germplasm) of human reconstruction animal and plant.Plant genetic engineering need to separate and operate genetic material (usually with DNA or RNA form) and subsequently with this genetic material introduced plant.This techniques enable is enough sent farm crop or the plant of economy, agricultural or Horticultural Characters with multiple improvement.A proterties with special economic interests is productive rate.Productive rate is generally defined as from the economic worth of farm crop can measure output.It can define according to quantity and/or quality.Productive rate directly depends on several factors, for example: the quantity of organ and size, plant structure (for example branch amount), seed production and more factor.Development of root, dietetic alimentation and stress tolerance also are the important factors that determines productive rate.Can increase by optimizing one of above-mentioned factor the productive rate of farm crop.

The ability of the multiple growth characteristics of improvement plant will as: the fields such as production, arboriculture (aboriculture), Horticulture and forestry that increase farm crop, plant breeding, ornamental plant have many application.The growth characteristics of improvement, such as productive rate, the algae that also can be used for using at bio-reactor produces (be used for the biotechnology production such as medicine, antibody or vaccine substance, or be used for the bio-transformation of organic waste) and other this class field.

Have now found that, compare with corresponding wild-type plant, the activity of rna binding protein or its homologue makes plant have the growth characteristics of improvement in the increase plant, described rna binding protein or its homologue have RNA in conjunction with active and comprise 2 or 3 RNA identification motifs (RRM), and comprise with motif I:PYEAAVVALPVVVKERLVRILRLGIATRYD and have the motif of at least 75% sequence identity and/or the motif that has at least 50% sequence identity with motif II:RFDPFTGEPYKFDP.Have now found that, compare with corresponding wild-type plant that the activity of RBP1 polypeptide or its homologue makes plant have the growth characteristics of improvement in the increase plant.The polypeptide that described RBP1 polypeptide or its homologue refer to have following feature: (a) RNA is in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF allow at the most three aminoacid replacement and arbitrarily conservative change in the motif; (d) amino acid according to the preference ordering that increases and SEQ ID NO:15 representative has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity.

Rna binding protein transcribe with the post-transcriptional level Enhancer elements in have vital role.Synthetic, the processing that extends horizontally to the RNA molecule of regulating and the institute in the turnover comprise premessenger RNA montage, polyadenylation, mRNA transhipment, translation and stability/decay in steps.Regulating mainly is directly to finish by rna binding protein, or the function of indirectly regulating other regulatory factor by rna binding protein is finished.The RNA-protein interaction is many-sided key link that cellular metabolism, cytodifferentiation and growth and pathogen infection copy.RNA identification motif or RRM usually are present in the multiple rna binding protein and relate to all and transcribe post-treatment, and wherein from one to four copy of RRM number of each protein changes.RRM is about 80 amino acid whose zones of containing several high conservative residues, it is the subunit order of two weak points that wherein some are gathered, RNP-1 (eight aggressiveness) and RNP-2 (six aggressiveness) (Birney etc., Nucleic Acids Research, 1993, Vol.21, No.25,5803-5816).

196 protein that contain RRM of Arabidopis thaliana (Arabidopsis) genome encoding, the example are RBP1 (Lorkovic etc., Nucleic Acids Research, 2002, Vol.30, No.3,623-635).They report that the RRM of AtRBP1 is the most similar to the RRM of metazoan Musashi protein.Except AtRBP1, Lorkovic etc. have also described three protein that have similarity with AtRBP1 and Musashi protein.Separating first the concurrent RBP1 that expresses in the tissue of division fast now from the RBP1 of Arabidopis thaliana (Arabidopsis thaliana) by (Plant Cell Physiol.41 (3): 282-288 (2000)) such as Suzuki is a kind of rna binding protein (shown in Suzuki etc. 2000), comprises two RRM.

Summary of the invention

According to one embodiment of the invention, the method of improving plant growth characteristic is provided, it comprises the activity that increases rna binding protein in the plant or its homologue, described rna binding protein or its homologue have RNA and identify motifs (RRM) in conjunction with active and 2 or 3 RNA, and comprise with motif I:PYEAAVVALPVVVKERLVRILRLGIATRYD and have the motif of at least 75% sequence identity and/or the motif that has at least 50% sequence identity with motif II:RFDPFTGEPYKFDP.

According to one embodiment of the invention, the method for improving plant growth characteristic is provided, it comprises the activity that increases RBP1 polypeptide in the plant or its homologue, and described RBP1 polypeptide or its homologue have following feature: (a) RNA is in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL; (ii) RPRGFGF allows at the most three aminoacid replacement and any conservative change in the motif; (d) amino acid according to the preference ordering that increases and SEQ ID NO:15 representative has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity.

Advantageously, carry out method of the present invention and make plant have the growth characteristics of multiple improvement, the productive rate, particularly seed productive rate that especially increase.

Term defined herein " productive rate of increase " means with respect to corresponding wild-type plant, increase in following arbitrary or many aspects: (i) one or more parts of plant, particularly the biomass (weight), the root biomass that increases or any other that increase of (can gather in the crops) part can be gathered in the crops the biomass that part increases on the ground; (ii) the seed productive rate that increases comprises the increase of seed biomass (seed weight), and it can be in every strain plant seed weight or take the increase of single seed as the basis; (iii) (full) seed amount that increases; (iv) seed size that increases, it also may affect the composition of seed; (v) the seed volume that increases, it also may affect the composition of seed; (vi) harvest index that increases, it is expressed as the productive rate that can gather in the crops part (such as seed) and the ratio of total biomass; The thousand seed weight (TKW) that (vii) increases, it is to calculate from the gross weight of full seed number.The TKW that increases may be derived from seed size and/or the seed weight of increase.

Take corn as example, the increase of productive rate can show as following one or more: the increase of per hectare or every acre of number of plant, the increase of every strain plant spike number, increase of line number, a row grain number, grain weight, thousand seed weight, fringe length/diameter etc.Take rice as example, the increase of productive rate can show as following one or more: the quantity of per hectare or every acre of plant, the panicle number of every strain plant, each paniculiform spikelet number, each paniculiform number of spending, the increase of the full rate of seed, increase of thousand seed weight etc.The increase of productive rate is the mutagenic structure of possibility also, and the result who perhaps can be used as the structure of change occurs.

According to preferred aspect, carry out method of the present invention and produce the plant with increase productive rate.Therefore, the invention provides the method that increases plant yield, the method comprises the activity that increases rna binding protein in the plant or its homologue, described rna binding protein or its homologue have RNA and identify motifs (RRM) in conjunction with active and 2 or 3 RNA, and comprise with motif I:PYEAAVVALPVVVKERLVRILRLGIATRYD and have the motif of at least 75% sequence identity and/or the motif that has at least 50% sequence identity with motif II:RFDPFTGEPYKFDP.According to another preferred aspect of the present invention, the method that increases plant yield is provided, described method comprises the activity that increases RBP1 polypeptide in the plant or its homologue, described RBP1 polypeptide or its homologue have following feature: (a) RNA is in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF allow at the most three aminoacid replacement and arbitrarily conservative change in the motif; (d) amino acid according to the preference ordering that increases and SEQ ID NO:15 representative has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity.

Because transgenic plant of the present invention have the productive rate of increase, for the growth velocity of their life cycle respective stage, these plants may present the growth velocity (in life cycle of their parts at least) of increase with respect to corresponding wild-type plant.The growth velocity that increases may be specific to one or more parts (comprising seed) of plant, perhaps may basically spread all over whole plant.Have the plant that increases growth velocity even may present early flowering.The increase of growth velocity may appear at one or more stages in plant life cycle, perhaps appears in the process in whole plant life cycle basically.At the commitment in plant life cycle, the growth of growth velocity may show as the vigor of enhancing.The increase of growth velocity can change the harvest cycle of plant, makes the plant can be than other possible situation more late sowing kind and/or faster results.If growth velocity fully increases, may allow to sow the more seed of kindred plant species (for example sow and gather in the crops rice plants, subsequently in sowing in vegetative period of a routine and gather in the crops more rice plants).Same, if growth velocity increases fully, may allow to sow the more seed of different plant species (for example sow and gather in the crops rice plants, subsequently, such as sowing and optional results soybean, potato or any plant that other is fit to).Also may be from the extra number of times of same rootstock harvester in the situation of some plants.The harvest cycle that changes plant may cause every acre year biomass yield increase (because increase of (for example in 1 year) any specified plant Growth and yield number of times).Compare with wild type counterparts, the increase of growth velocity also may allow more wide region cultivation transgenic plant because the regional limits of Planting Crops during usually by plantation when (early season) or results (season in evening) hostile environment condition determined.If the shortening harvest cycle can be avoided this class unfavourable condition.Can a plurality of parameters from the growth curve that growth experiment is drawn determine growth velocity, this class parameter can be: T-Mid (plant reaches 50% required time of its largest amount) and T-90 (plant reaches its largest amount 90% required time) etc.

Carrying out method of the present invention makes plant have the growth velocity of increase.Therefore, the invention provides the method that increases plant growth rate, the method comprises the activity that increases rna binding protein in the plant or its homologue, described rna binding protein or its homologue have RNA and identify motifs (RRM) in conjunction with active and 2 or 3 RNA, and comprise with motif I:PYEAAVVALPVVVKERLVRILRLGIATRYD and have the motif of at least 75% sequence identity and/or the motif that has at least 50% sequence identity with motif II:RFDPFTGEPYKFDP.Another method that increases plant growth rate also is provided, and described method comprises the activity that increases RBP1 polypeptide in the plant or its homologue, and described RBP1 polypeptide or its homologue have following feature: (a) RNA is in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF allow at the most three aminoacid replacement and any conservative change in the motif; (d) amino acid according to the preference ordering that increases and SEQ ID NO:15 representative has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity.

Be in without under the stress conditions plant, or be exposed to respect to control plant and multiplely slightly coerce down, the increase of productive rate and/or growth velocity occurs in it.Usually plant is replied to be exposed to by more slowly growth and coerces.Under the severe water stress condition, plant even can stop growing fully.On the other hand, slightly coerce to be defined as in this article when plant and be exposed to this, do not cause the plant forfeiture that stops growing fully to restart any of energy for growth and coerce.Because the development of the farming method (irrigation, fertilising, pesticide treatments), the crop plants of cultivation usually can not run into severe water stress.Therefore, by the impaired feature of not expecting in the agricultural that is grown to serve as of slight stress-inducing.Slightly coerce is that the typical case that plant may contact coerces.These coerce may be the plant daily biology that is exposed to it and/or abiotic (environment) coerce.The coercing of typical abiotic or environment comprises by temperature stress, salt stress, water that the heat of abnormality or cold/freezing temperature produces coerces (arid or excessive water).Abiotic stress also can be caused by pharmaceutical chemicals.Biological coercing generally is that those that caused by pathogenic agent such as bacterium, virus, fungi and insect are coerced.

Can in any plant, advantageously modify above-mentioned growth characteristics.

The ancestors of whole plant, plant and the part of offspring and plant contained in term used herein " plant ", comprises seed, branch, stem, leaf, root, flower (comprising stem tuber) and tissue and organ, and wherein above-mentioned every kind comprises goal gene/nucleic acid.Suspension culture, callus, embryo, meristematic zone, gametophyte, sporophyte, pollen and sporule also contained in term " plant ", and wherein above-mentioned every kind equally also comprises goal gene/nucleic acid.

The plant that can be used for especially in the inventive method comprises the whole plants that belong to vegitabilia (Viridiplantae) superfamily, particularly unifacial leaf and dicotyledons comprise the feed or the feed leguminous plants that are selected from following species, ornamental plant, food crop, arbor or shrub: Acacia species (Acacia spp.), maple species (Acer spp.), Actinidia species (Actinidia spp.), Aesculus species (Aesculus spp.), New Zealand kauri (Agathis australis), Albizia amara, Alsophila tricolor, Andropogon species (Andropogon spp.), Arachis species (Arachis spp.), betel nut (Areca catechu), Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula (Betula spp.), Btassica (Brassica spp.), Bruguiera conjugata (Bruguiera gymnorrhiza), Burkea africana, palas (Butea frondosa), Cadaba farinosa, Zhu Ying Pittosporum species (Calliandra spp.), daye tea (Camellia sinensis), Canna generalis Bailey (Canna indica), Capsicum species (Capsicum spp.), Cassia species (Cassia spp.), apart from pea (Centroema pubescens), Chaenomeles species (Chaenomeles spp.), Chinese cassia tree (Cinnamomum cassia), fruitlet coffee (Coffea arabica), Colophospermum mopane, variation coronule flower (Coronillia varia), Cotoneaster serotina, hawthorn species (Crataegus spp.), Cucumis species (Cucumis spp.), Cupressus species (Cupressus spp.), Cyathea dealbata Quinces Quince (Cydonia oblonga), Japanese cypress (Cryptomeria japontca), Cymbopogon species (Cymbopogon spp.), Cynthea dealbata Quinces Quince (Cydonia oblonga), Dalbergia monetaria, Da Ye Rhizome of Fortune's Drynaria (Davallia divaricata), mountain horseleech species (Desmodium spp.), coarse freshwater mussel shellfish fern (Dicksonia squarosa), Diheteropogon amplectens, Dioclea spp, sickle Dolichos species (Dolichos spp.), Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp. Finger-millet (Eleusine coracana), Eragrostis species (Eragrestis spp.), Erythrina species (Erythrina spp.), eucalyptus species (Eucalyptus spp.), Euclea schimperi, Eulalia villosa, Fagopyrum species (Fagopyrum spp.), feijoa (Feijoa sellowiana), Fragaria species (Fragaria spp.), Moghania species (Flemingia spp), Freycinetia banksii, Geranium thunbergii, ginkgo (Ginkgo biloba), Glycine javanica, Gliricidia spp, upland cotton (Gossypium hirsutum), Grevillea species (Grevillea spp.), Guibourtia coleosperma, rock Astragalus species (Hedysarum spp.), Hemarthria compressa (Hemarthia altissima), turn round Huang Mao (Heteropogon contortus), barley (Hordeum vulgare), Hyparrhenia rufa, Herba Hyperici Erecti (Hypericum erectum), Hyperthelia dissoluta, spend front yard indigo plant (Indigo incarnata) in vain, Jris species (Iris spp.), Leptarrhena pyrolifolia, lespedeza species (Lespediza spp.), Lettuca spp., Leucaena leucocephala, Loudetia simplex, sieve beans (Lotonus bainesii) that pause, Lotus species (Lotus spp.), Macrotyloma axillare, Malus species (Malus spp.), Manihot esculenta, alfalfa (Medicago sativa), metasequoia (Metasequoia glyptostroboides), powder bajiao banana (Musa sapientum), Nicotiana species (Nicotianum spp.), donkey food Macroptilium species (Onobrychis spp.), Ornithopus spp., Oryza species (Oryza spp.), Peltophorum africanum, Pennisetum species (Pennisetum spp.), Persea gratissima, green winter Solanum species (Petunia spp.), Phaseolus species (Phaseolus spp.), betel nut bamboo (Phoenix canariensis), Phormium cookianum, Photinia species (Photinia spp.), white spruce (Picea glauca), Pinus species (Pinus spp.), pea (Pisum sativum), alpine totara (Podocarpus totara), Pogonarthria fleckii, Pogonarthria squarrosa, Populus species (Populus spp.), algarroba (Prosopis cineraria), Pseudotsuga menziesii (Mirbel) Franco (Pseudotsuga menziesii), Pterolobium stellatum, European pear (Pyrus communis), oak species (Quercus spp.), Rhaphiolepsis umbellata, delicious rod is spent palm fibre (Rhopalostylis sapida), Rhus natalensis, Europe gooseberry (Ribes grossularia), currant species (Ribes spp.), acacia (Robinia pseudoacacia), rose species (Rosa spp.), rubus species (Rubus spp.), Salix species (Salix spp.), Schyzachyrium sanguineum, parasol pine (Sciadopitys verticillata), sequoia sempervirens (Sequoia sempervirens), big tree (Sequoiadendron giganteum), dichromatism chinese sorghum (Sorghum bicolor), spinach species (Spinacia spp.), Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, tadehagi ohashi species (Tadehagi spp.), southern cypress (Taxodiumdistichum), Arabic Herba Themedae japonicae (Themeda triandra), Clover species (Trifolium spp.), Triticum species (Triticum spp.), tsuga heterophylla (Tsuga heterophylla), genus vaccinium species (Vaccinium spp.), Vetch species (Vicia spp.), grape (Vitis vinifera), the fertile gloomy flower (Watsonia pyramidata) of cone fringe, common calla (Zantedeschia aethiopica), corn (Zea mays), Amaranthus, arithoke, asparagus, cabbage, brassica oleracea var gemmifera, Caulis et Folium Brassicae capitatae, rape, Radix Dauci Sativae, Cauliflower, celery, the green wild cabbage of the garment or robe made of feathers, flax, kale; root of Szemao crotalaria; oil grain rape; gumbo; onion; potato; rice; soybean; strawberry; sugar beet; sugarcane; Sunflower Receptacle; tomato; pumpkin; tea tree and algae etc.According to the preferred embodiment of the invention, described plant is crop plants, such as soybean, Sunflower Receptacle, rape, clover, Semen Brassicae campestris, cotton, tomato, potato or tobacco.Further preferred plant is monocotyledons, such as sugarcane.Preferred plant is cereal, such as rice, corn, wheat, barley, grain, rye, Chinese sorghum or oat.

Can increase by the level that improves rna binding protein in the plant activity of rna binding protein or its homologue.Alternatively, when the rna binding protein level does not increase, perhaps even when the rna binding protein level reduces, also can increase its activity.When this situation appears at the polypeptide natural characteristics and changes, for example, have more active mutant forms by preparation than wild type peptide.Similarly, can increase by the level that improves RBP1 polypeptide protein in the plant activity of RBP1 polypeptide or its homologue.Alternatively, when the RBP1 level does not change, perhaps even when RBP1 polypeptide level reduces, also can increase its activity.When this situation appears at the polypeptide natural characteristics and changes, for example, have more active mutant by preparation than wild-type.

The term " rna binding protein or its homologue " of this paper definition refers to have RNA and identifies the polypeptide of motifs (RRM) in conjunction with active and 2 or 3 RNA, and comprises with motif I:PYEAAVVALPVVVKERLVRILRLGIATRYD and have the motif of at least 75%, 80%, 85%, 90% or 95% sequence identity and/or the motif that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity with motif II:RFDPFTGEPYKFDP.This term also refers to such aminoacid sequence, and described aminoacid sequence has at least 13%, 15%, 17%, 19%, 21%, 23%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity according to the aminoacid sequence of the preference ordering that increases and SEQ ID NO:2 representative.

Can use routine techniques well-known in the art easily to identify " rna binding protein or its homologue " in the above range of definition.For example, use technology well-known in the art can determine easily in external or body that RNA is in conjunction with activity.The example of external test comprises: use nucleic acid that North-Western and/or South-Western analyze in conjunction with measuring .Plant Cell Physiol.41 (3): 282-288 (2000) such as () Suzuki; Use the crosslinked RNA of UV in conjunction with mensuration; The electrophoretic mobility shift assay of rna binding protein (Smith, RNA-Protein Interactions-A Practical Approach 1998, University of Cambridge).The example of in vivoassay comprises TRAP (translation repression measuring method) (Paraskeva E, Atzberger A, Hentze MW:A translational repression assay procedure (TRAP) for RNA-protein interactions in vivo.PNAS 1998 Feb 3; 95 (3): 951-6.).

Can determine easily whether the amino acid of polypeptide and SEQ ID NO:2 representative has at least 13% identity by sequence alignment.The method of sequence alignment is well-known in the art, and these class methods comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.The comparison that the algorithm (J.Mol.Biol.48:443-453,1970) of GAP application Needleman and Wunsch is searched two complete sequences minimizes the maximization of coupling number and room number.The per-cent of BLAST algorithm sequence of calculation identity, and the similarity between two sequences carried out statistical study.The software of carrying out the BLAST analysis can obtain publicly from biotechnology infonation center.For example use VNTI AlignX multiple ratio to program, by compare (see for example shown in Figure 1 comparison) of search sequence (preferred protein sequence) with known rna binding protein sequence, can differentiate easily that the aminoacid sequence with SEQ ID NO:2 representative has rna binding protein or its homologue of at least 13% identity, described VNTI AlignX multiple ratio is to the clustal W algorithm (InforMax of program based on modification, Bethesda, MD, http://www.informaxinc.com), the band have vacant position open point penalty be 10 and the room extend to 0.05 default setting.

The person skilled in the art can also easily identify with motif I:PYEAAVVALPVVVKERLVRILRLGIATRYD to have the motif of at least 75%, 80%, 85%, 90% or 95% sequence identity and/or the motif that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% sequence identity with motif II:RFDPFTGEPYKFDP.This can be by comparing and searching for the homology zone and easily realize.

Following table 1 is presented at the motif I that finds among the sequence SEQ ID NO:2 and II and the sequence identity percentage ratio of corresponding motif in cognate rna is combined albumen.Useful rna binding protein may contain motif I or II in the method for the invention, or motif I and II.

Table 1: the motif that in rna binding protein and its homologue, finds.

The example that falls into the polypeptide of " rna binding protein or its homologue " definition comprises following sequence: SEQ ID NO:2 is from tobacco; SEQ ID NO:4 is the protein prediction from rice BAC clone (NCBI accession number AL731884); SEQ ID NO:6 is the rice protein prediction (fragment) from cDNA (NCBI accession number AK059444); SEQ ID NO:8 is the corn protein prediction (fragment) from cDNA (NCBI accession number AY105295); And SEQ ID NO:10 is the rice sequence (NCBI accession number BAC83046) of total length.

The polypeptide that refers to have following characteristics such as the term " RBP1 or its homologue " of definition here: (a) RNA is in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF allow at the most three aminoacid replacement and any conservative change in the motif; (d) amino acid according to the preference ordering that increases and SEQ ID NO:15 representative has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity.Conservative replacement table is (for example sees Creighton (1984) Proteins.W.H.Freeman and Company and see the following form 4) well-known in the art.

Can use the well-known routine techniques of those skilled in the art easily to identify to belong to " RBP1 polypeptide or its homologue " of above definition.For example, can determine easily as mentioned above that RNA is in conjunction with activity.

In addition, the RRM structural domain is well-known in the art and is comprised of about 80-90 amino acid; They have and comprise four chains arranging with α/β interlayer form and the structure of two spirals, sometimes RNA in conjunction with in have the 3rd spiral.The protein that contains the RRM structural domain has mode configuration.Can use SMART to identify RRM structural domain (a Simple Modular Architecture Research Tool:Identification of signaling domains, the .PNAS such as Schultz, 95,5857-5864 (1998)) (http://smart.embl-heidelberg.de/).See again Letunic etc., and Recent improvements to the SMART domain-based sequence annotation resource (Nucleic Acids Res.30 (1), 242-244).

The method that can use as mentioned above comparison determines easily that by sequence alignment whether polypeptide has at least 20% identity with the amino acid of SEQ ID NO:2 representative.

Because the RBP1 polypeptide comprises the zone of high conservative, those skilled in the art are by comparing the RBP1 sequence that can easily identify other with those of the conserved regions of arbitary inquiry sequence and known RBP1 sequence.The example of these conserved regions comprises following two motifs: (i) KIFVGGL and (ii) RPRGFGF allow at the most three aminoacid replacement and any conservative change in the motif.

The example of polypeptide that belongs to " RBP1 polypeptide or its homologue " definition comprises: all from At1g58470 (SEQ ID NO:15), At4g26650 (SEQ ID NO:17), At5g55550 (SEQ ID NO:19), At4g14300 (SEQ ID NO:21), At3g07810 (SEQ ID NO:23), At2g33410 (SEQ ID NO:25) and the At5g47620 (SEQ ID NO:27) of Arabidopis thaliana; NP_921939.1 (SEQ ID NO:29) from rice; AK067725 (SEQ ID NO:31) and AK070544 (SEQ ID NO:33) corresponding to the RBP1 polypeptide of rice mRNA coding; Partial protein prediction from EST (expressed sequence tag) from the CK210974 (SEQ ID NO:35) of wheat with from the CA124210 (SEQ IDNO:37) of sugarcane.

Although show relatively low sequence homology (approximately being low to moderate 25%), the full length protein that RPB1 protein and all have 2 RRM structural domains structurally is high conservative.Can easily find thus the rbp1 gene (see above example from rice, sugarcane and wheat, it is accredited as RBP1 protein here for the first time) in other plant species.Following table 2 is presented at the identity percentage ratio of some sequences that show in the comparison of Fig. 3.

Table 2:RBP1 protein sequence and SEQ ID NO:15 are based on the homology of comprehensive whole sequence alignment

It should be understood that, term RBP1 polypeptide or its homologue are not limited to the sequence by SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQID NO:33, SEQ ID NO:35 and SEQ ID NO:37 representative, but meeting any polypeptide of following standard, described standard is: (a) RNA is in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF allow at the most three aminoacid replacement and any conservative change in the motif; (d) amino acid according to the preference ordering that increases and SEQ IDNO:15 representative has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity, and described polypeptide is useful in carrying out method of the present invention.

Coding RNA can be any natural or synthetic nucleic acid in conjunction with the nucleic acid of albumen or its homologue.Rna binding protein or its homologue such as above this paper definition are by rna binding protein coding nucleic acid/genes encoding.Therefore the term " rna binding protein coding nucleic acid/gene " such as definition here is coding as the rna binding protein of above this paper definition or any nucleic acid/gene of its homologue.The example of rna binding protein coding nucleic acid comprises by those of arbitrary representative among SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7 and the SEQ ID NO:9.Rna binding protein coding nucleic acid/gene and functional variant thereof are applicable to implementing method of the present invention.The functional variant of rna binding protein coding nucleic acid/gene comprise rna binding protein coding nucleic acid/gene part and/or can with the nucleic acid of rna binding protein coding nucleic acid/gene recombination.Term with regard to functional variant " function " refers to such variant (i.e. part or hybridization sequences), its coding has RNA in conjunction with the polypeptide of activity and preferably and additionally has at least one RRM, preferred 2 or 3 RRM and more preferably have one of following at least motif: have the motif of at least 75% sequence identity and/or the motif that has at least 50% sequence identity with motif II:RFDPFTGEPYKFDP with motif I:PYEAAVVALPVVVKERLVRILRLGIATRYD.Term " function " also can refer to make plant have the growth characteristics of improvement such as the coding RNA of the above this paper definition nucleic acid in conjunction with albumen or its homologue when it is introduced in plant and expresses.

The nucleic acid of coding RBP1 polypeptide or its homologue can be any natural or synthetic nucleic acid.RBP1 polypeptide or its homologue such as above this paper definition are by rbp1 nucleic acid/genes encoding.Therefore the term " rbp1 nucleic acid/gene " such as definition here is coding as the RBP1 polypeptide of above this paper definition or any nucleic acid/gene of its homologue.The example of rbp1 nucleic acid comprises by those of arbitrary representative among SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and the SEQ ID NO:36.Rbp1 nucleic acid/gene and functional variant thereof are applicable to implementing method of the present invention.The functional variant of rbp1 nucleic acid/gene comprise rbp1 nucleic acid/gene part and/or can with the nucleic acid of rbp1 nucleic acid/gene recombination.Term with regard to functional variant " function " refers to such variant (i.e. part or hybridization sequences), the polypeptide of its coding has RNA in conjunction with activity and has at least one RRM structural domain, preferred 2 RRM structural domains and more preferably have following two motifs: (i) KIFVGGL and (ii) RPRGFGF allow at the most three aminoacid replacement and arbitrarily conservative change in the motif.Term " function " also can refer to encode such as the RBP1 polypeptide of above this paper definition or the nucleic acid of its homologue, makes plant have the growth characteristics of improvement when it is introduced in plant and expresses.

Term as defined herein partly refers to the protein-bonded dna fragmentation of coding RNA, be at least 180,300,500 or 700 according to the preference ordering length of nucleotides that increases, and the polypeptide of this part coding has RNA in conjunction with active and at least 1 RRM, among preferred two or three RRM and motif I or the II at least one, preferred two are had both at the same time.For example, can prepare part by the rna binding protein coding nucleic acid is carried out one or more disappearances.Can use this part maybe it can be blended in other coding (or non-coding) sequence with the form of separating and produce the protein that makes up some activity with (for example), one of them be that RNA is in conjunction with activity.When being blended in other encoding sequence, the polypeptide that the polypeptide that is produced by translation can partly be predicted greater than rna binding protein.Preferably, described funtion part is the part by the nucleic acid of arbitrary representative among SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and the SEQ ID NO:9.

Term with regard to rbp1 nucleic acid refers to that partly the dna fragmentation and the described fragment coding that comprise at least 80 Nucleotide have RNA in conjunction with the polypeptide of activity, and has at least one RRM structural domain, preferred two RRM structural domains and further preferably have following two motifs: (i) KIFVGGL and (ii) RPRGFGF.For example, can prepare part by rbp1 nucleic acid is carried out one or more disappearances.Can use this part maybe it can be blended in other coding (or non-coding) sequence with the form of separating and produce the protein that makes up various active with (for example), one of them be that RNA is in conjunction with activity.When being blended in other encoding sequence, can be greater than the rbp1 fragment polypeptide of prediction by the polypeptide that translation produces.Preferably, this funtion part is the part by the nucleic acid of arbitrary representative among SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and the SEQID NO:36.

The rna binding protein variant of another type is under the stringent condition that reduces, preferably under stringent condition can with the nucleic acid such as the nucleic acid/gene recombination of this paper rna binding protein defined previously coding, this hybridization sequences coding has RNA in conjunction with the polypeptide of activity, and has at least 1 RRM, preferred two or three RRM, and at least one of motif I or II, preferred two.According to the preference ordering that increases, the length of nucleotides of this hybridization sequences is at least 180,300,500 or 700.Preferably, this hybridization sequences can with the nucleic acid hybridization by arbitrary representative among SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and the SEQ ID NO:9.

Similarly, the variant rbp1 of another type is under the stringent condition that reduces, preferably under stringent condition can with the nucleic acid such as this paper rbp1 nucleic acid/gene recombination defined previously, this hybridization sequences coding has RNA in conjunction with the polypeptide of activity, and has at least 1 RRM, preferred two RRM, and further preferably have following two motifs: (i) KIFVGGL and (ii) RPRGFGF.This hybridization sequences preferred length is at least 80 Nucleotide.Preferably, this hybridization sequences can with the nucleic acid hybridization by arbitrary representative among SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34 and the SEQ ID NO:36.

Refer to wherein the process that the basic nucleotide sequence of homologous complementary is annealed each other such as the term " hybridization " of definition here.Crossover process can occur in solution fully, and namely complementary nucleic acid is all in solution.Crossover process also can be carried out in this case, and namely one of complementary nucleic acid is fixed on the matrix, on magnetic bead, sepharose 4B or any other resin.In addition, crossover process also can so be carried out, namely wherein one of complementary nucleic acid is fixed on solid support such as nitrocellulose or the nylon membrane, perhaps for example be fixed on the siliceous glass support (latter is referred to as nucleic acid array or microarray, or is referred to as nucleic acid chip) such as photolithograph.For hybridization is occured, usually make nucleic acid molecule thermally denature or chemical modification, so that two strands is unwind into two strands, and/or remove hairpin structure or other secondary structure in the single-chain nucleic acid.The severity of hybridization is subjected to the impact of conditions such as temperature, salt concn, ionic strength and hybridization buffer composition.Under the stringent condition that reduces, preferably under stringent condition, hybridize.The example of stringent condition shows in following table 3.Stringent condition is at least as strict as condition A-L; The stringent condition that reduces is at least as strict as condition M-R.

Table 3: the example of stringent condition

" hybrid length " is the expection length of hybrid nucleic acid.When the nucleic acid hybridization of known array, can and differentiate that by sequence alignment conservative region as herein described determines hybrid length.

In hybridization and lavation buffer solution, can (1 * SSPE be 0.15M NaCl, 10mM NaH with SSPE ₂PO ₄With 1.25mM EDTA, pH7.4) replacement SSC (1 * SSC is 0.15M NaCl and 15mM Trisodium Citrate); Washing was 15 minutes after hybridization was finished.Hybridization and washing can additionally comprise the fragmentation salmon sperm DNA, 0.5% trisodium phosphate of 5 * Denhardt ' s reagent, 0.5-1.0%SDS, 100 μ g/ml sex change and up to 50% methane amide. ^*Tb-Tr: for the heterozygote of expection length less than 50 base pairs, hybridization temperature should be than the melting temperature(Tm) T of heterozygote _mLow 5-10 ℃; Determine T according to following equation _mFor hybrid length less than 18 base pairs, Tm (℃)=2 * (A+T base number)+4 * (G+C base number).For hybrid length between 18 to 49 base pairs, Tm (℃)=81.5+16.6 * log ₁₀[Na ⁺]+0.41 * (%G+C)-(600/N), wherein N is the base number in the heterozygote, [Na ⁺] be the ([Na of 1 * SSC of Na ion concentration in the hybridization buffer ⁺]=0.165M).± the present invention is contained with peptide nucleic acid(PNA) (PNA) or the nucleic acid arbitrary or a plurality of DNA of replacement that modifies or RNA hybridization mating partner.

Rna binding protein coding nucleic acid or its variant can be derived from any natural or artificial source.Can be from microbe-derived such as bacterium, yeast or fungi, or from plant, algae or animal (comprising the people) source isolating nucleic acid/gene or its variant.Can modify this nucleic acid from its natural form composition and/or genome environment by meticulous manual operation.This nucleic acid preferred plant source, or from identical plant species (for example from will to exotic plant species wherein) or from different plant species.Described nucleic acid can from dicotyledonous species, preferably from Solanaceae (Solanaceae), more preferably separate from tobacco.More preferably, the rna binding protein coding nucleic acid that separates from tobacco is represented by SEQ ID NO:1, and rna binding protein aminoacid sequence such as SEQ ID NO:2 representative.

Rbp1 nucleic acid or its variant can be derived from any natural or artificial source.Can be from microbe-derived such as bacterium, yeast or fungi, or from plant, algae or animal (comprising the people) source isolating nucleic acid/gene or its variant.Can modify this nucleic acid from its natural form composition and/or genome environment by meticulous manual operation.This nucleic acid preferred plant source, or from identical plant species (for example from will to exotic plant species wherein) or from different plant species.Described nucleic acid can from dicotyledonous species, preferably from cruciate flower (Brassicaceae) section, more preferably separate from Arabidopis thaliana.More preferably, the rbp1 that separates from Arabidopis thaliana is represented by SEQ ID NO:14, and RBP1 aminoacid sequence such as SEQ ID NO:15 representative.

Can increase by introducing genetic modification (preferably at rna binding protein encoding gene seat) activity of rna binding protein or its homologue.Similarly, can increase by introducing genetic modification (preferably at the rbp1 locus) activity of RBP1 polypeptide or its homologue.Here the locus of definition means the genome district, and it comprises goal gene and upstream of coding region or downstream 10KB.

For example, can introduce genetic modification by following any (or multiple) method: TDNA activation, TILLING, site-directed mutagenesis, homologous recombination or by in plant, introducing and expressing coding RNA in conjunction with the nucleic acid of albumen or its homologue or by in plant, introducing and express the nucleic acid of coding RBP1 polypeptide or its homologue.Introduce after the genetic modification, succeeded by the activity of selecting the active of rna binding protein increase or selecting the RBP1 polypeptide to increase, the increase of described activity makes plant have the growth characteristics of improvement.

T-DNA activation tagging (Science (1992) 1350-1353 such as Hayashi) relates to the insertion of T-DNA, this T-DNA contains promotor (also can be translational enhancer or intron) usually, it is at genome district or gene coding region upstream or the downstream 10KB of goal gene, and makes in configuration promotor can instruct the expression of target gene.Usually natural promoter is destroyed to the regulation and control of expression of target gene, and gene is by the promotor control of new introducing.Promotor generally is contained among the T-DNA.For example, infect this T-DNA radom insertion Plant Genome by Agrobacterium (Agrobacterium) and cause near the gene overexpression that inserts the T-DNA.The transgenic plant that obtain are owing near the gene overexpression promotor of introducing shows the dominant phenotype.The promotor of introducing can be arbitrarily can be in desirable organism (be plant in this kind situation) instructs the promotor of genetic expression.For example, composing type, that organize preference, the cell type preference and promotor induction type all are applicable to T-DNA and activate.

Also can genetic modification be introduced rna binding protein encoding gene seat by TILLING (the local sudden change of the genome of targeted induction) technology.This is a kind of induced-mutation technique, for generation of and/or identify, and the last variant that separates the rna binding protein coding nucleic acid that can present the rna binding protein activity (or rbp1 coding nucleic acid) of mutagenesis.TILLING also allows to select to carry the plant of this kind mutation variants.These mutant even may to present higher rna binding protein than its natural form gene active.TILLING combines high-density mutagenesis and high-throughput screening method.The step that TILLING generally follows is: (a) EMS mutagenesis (Redei and Koncz, 1992; Feldmann etc., 1994; Lightner and Caspar, 1998); (b) DNA preparation and individual the merging; (c) pcr amplification in purpose zone; (d) sex change and annealing are to form heteroduplex; (e) DHPLC, the heteroduplex that wherein exists in the storehouse can detect extra peak at color atlas; (f) evaluation of mutated individual; (g) order-checking of sudden change PCR product.The method of TILLING is (McCallum NatBiotechnol.2000 Apr well-known in this area; 18 (4): 455-7, by Stemple summary, 2004 (TILLING-a high-throughput harvest for functional genomics.Nat Rev Genet.2004Feb; 5 (2): 145-50.)).

Site-directed mutagenesis can be used for producing the variant of rna binding protein coding nucleic acid or its part, described variant retentive activity, and namely RNA is in conjunction with activity.Can finish site-directed mutagenesis by some methods, (current protocols in molecular biology.Wiley edits the method for the modal PCR of being based on Http:// www.4ulr.com/products/currentprotocols/index.html).Site-directed mutagenesis can be used for producing the variant of rna binding protein coding nucleic acid or its part, described variant retentive activity, and namely RNA is in conjunction with activity.Similarly, site-directed mutagenesis can be used for producing the variant of RBP1 coding nucleic acid or its part, described variant retentive activity, and namely RNA is in conjunction with activity.Site-directed mutagenesis also can be used for producing the variant of RBP1 coding nucleic acid or its part, described variant retentive activity, and namely RNA is in conjunction with activity.

TDNA activation, TILLING and site-directed mutagenesis are to produce the example that new allelotrope and rna binding protein variant (this variant keeps the rna binding protein function) maybe can produce the technology of new allelotrope and rbp1 variant (this variant keeps the RBP1 function), so it is useful in the method for the invention.

The homologous recombination permission is introduced selected nucleic acid to the appointment selected location in the genome.Homologous recombination is the conventional standard technique of using in the bio-science, and it is used for low organism such as yeast or the mosses (such as physcomitrella) of waiting.The method of in plant, carrying out homologous recombination not only in model plant, be described (Offringa etc., Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium-mediated transformation.1990 EMBO is Oct J.1990; 9 (10): 3077-84), and at crop plants, as obtaining describing (Terada R in the rice, Urawa H, Inagaki Y, Tsugane K, Iida S.Efficient gene targeting by homologous recombination in rice.Nat Biotechnol.2002.lida and Terada:A tale of two integrations, transgene and T-DNA:gene targeting by homologous recombination in rice.Curr Opin Biotechnol.2004 Apr; 15 (2): 132-8).The nucleic acid of institute's target (it may be that the rna binding protein coding nucleic acid that above defines or its variant or its may be rbp1 nucleic acid or its variants that above defines) does not need targeted rna binding-protein gene seat or target rbp1 locus, but can be introduced into for example zone of high expression level.The nucleic acid of institute's target can be the allelotrope of improvement, and it is used for replacing native gene or additionally being incorporated into native gene.

According to the preferred embodiments of the invention, nucleic acid by introducing in plant and expression coding RNA Binding peptide or its homologue can be improved the growth characteristics of plant, described polypeptide or its homologue have RNA and identify motifs (RRM) in conjunction with active and 2 or 3 RNA, and it comprises with motif I:PYEAAVVALPVVVKERLVRILRLGIATRYD and has the motif of at least 75% sequence identity and/or the motif that has at least 50% sequence identity with motif II:RFDPFTGEPYKFDP.

The preferred method of introducing genetic modification (it does not need in the rna binding protein locus in this case) is to introduce in plant and express the coding RNA of as mentioned definition in conjunction with the nucleic acid of albumen or its homologue.

Another preferred embodiment according to the present invention can be by introducing and express the nucleic acid improving plant growth characteristic of coding RBP1 polypeptide or its homologue in plant.

A preferred method introducing genetic modification (it does not need in the rbp1 locus in this case) is to introduce and express the coding RBP1 polypeptide of as mentioned definition or the nucleic acid of its homologue in plant.As mentioned above RBP1 polypeptide or its homologue have: (a) RNA is in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF allow at the most three aminoacid replacement and arbitrarily conservative change in the motif; (d) amino acid according to the preference ordering that increases and SEQID NO:15 representative has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity.

" homologue " of protein contains peptide, oligopeptides, polypeptide, protein and enzyme, it has amino acid whose replacement, disappearance and/or insertion with respect to the unmodified protein matter of discussing, and have to its derived from similar biologic activity and the functionally active of unmodified protein matter.In order to produce such homologue, the amino acid of protein can be by other amino acid substitution with similar quality (such as similar hydrophobicity, wetting ability, antigenicity, form or break the tendency of αhelix or β laminated structure).Conservative replacement table is well-known in this area (for example seeing Creighton (1984) Proteins.W.H.Freeman and Company).Following table provides the example that conserved amino acid replaces.

Table 4: the example that conserved amino acid replaces

Residue	The conservative replacement	Residue	The conservative replacement
				Ala	Ser	Leu	Ile、Val
Arg	Lys	Lys	Arg、Gln
				Asn	Gln、His	Met	Leu、Ile
Asp	Glu	Phe	Met、Leu、Tyr
				Gln	Asn	Ser	Thr、Gly
Cys	Ser	Thr	Ser、Val
				Glu	Asp	Trp	Tyr
Gly	Pro	Tyr	Trp、Phe
				His	Asn、Gln	Val	Ile、Leu
Ile	Leu、Val

The homologue of two kinds of specific forms also contained in term " homologue ", and it comprises ortholog sequence and paralog sequence, and it is contained for the evolution concept of describing the gene ancestral relationship.Term " paralog " relates to the collateral line gene that the gene replication in species gene group inside produces.Term " ortholog " relates to because species form the homologous gene in the different organisms that produce.

For example can easily find for example ortholog thing in the monocotyledons species by carrying out so-called mutual (reciprocal) blast search.This can by use the sequence of studying (for example SEQID NO:1 or 2 or SEQ ID NO:14 or 15) realize by a blast for any sequence library (such as the public obtainable ncbi database that can find at http://www.ncbi.nlm.nih.gov).For example, if the ortholog thing in the searching rice should carry out in 28,469 full length cDNA clones for rice (Oryza sativa) Nipponbare that can obtain from NCBI the blast that studies sequence.When beginning from Nucleotide, BLASTn or the tBLASTX with standard default can be used, maybe when beginning from protein, BLASTP or the TBLASTN with standard default can be used.Blast result can filter.Then use the result or the full length sequence among the unfiltered result that filter to carry out reverse blast (secondary blast) for the sequence of research sequence source organism certainly.Then the result of blast relatively for the first time and for the second time.When described secondary blast result provides when having the highest similarity with rna binding protein coding nucleic acid or rna binding protein polypeptide, be and find the ortholog thing, for example, if one of organism is tobacco, find so the paralog thing.For RBP1, when described secondary blast result provides when having the highest similarity with rbp1 nucleic acid or RBP1 polypeptide, be and find the ortholog thing, for example, if one of organism is Arabidopis thaliana, find so the paralog thing.Can use Clustal W in the situation of extended familys, it is visual to help cluster succeeded by contiguous threaded tree.

Homologue may be the form of protein " replacement variant ", namely has at least a residue to be removed in aminoacid sequence, and inserts different residues in this position.Aminoacid replacement is the replacement of single residue normally, but determines it also may is that cluster replaces by the functional limitations factor of polypeptide; Insert usually at about 1 to 10 amino-acid residue order of magnitude.Preferably, aminoacid replacement comprises conservative aminoacid replacement.

Homologue also can be the form of protein " insertion variant ", namely introduces one or more amino-acid residues in the predetermined position of protein.Insertion can comprise the fusion of aminoterminal and/or carboxyl terminal, and the inner insertion of single or multiple amino acid whose sequence.The general order of magnitude will be less than the fusion of amino or carboxyl terminal in the insertion of the aminoacid sequence inside of about 1 to 10 residue.The example of amino or carboxyl terminal fused protein or peptide be included in the binding domains of the activating transcription factor of using in the yeast two-hybrid system or activation structure territory, bacteriophage coat protein matter, (Histidine) 6-label, glutathione S-transferase label, a-protein, maltose binding protein, Tetrahydrofolate dehydrogenase, Tag100 epi-position, c-myc epi-position,

Epi-position, lacZ, CMP (calmodulin binding peptide), HA epi-position, protein C epi-position and VSV epi-position.

The homologue of protein " disappearance variant " form is characterised in that removes one or more amino acid from protein.

Can use well-known peptide synthetic technology in this area, such as the solid phase method of peptide synthesis etc., or prepare the amino acid variant of protein by recombinant DNA processing ease ground.Dna sequence dna working method for generation of the replacement of protein, insertion or disappearance variant is well-known in this area.For example, producing the technology that replaces sudden change in the DNA predetermined position is that those skilled in that art are well-known, comprise M13 mutagenesis, T7-Gen vitro mutagenesis (USB, Cleveland, OH), QuickChange site-directed mutagenesis (Stratagene, San Diego, CA), site-directed mutagenesis or other site-directed mutagenesis method of PCR mediation.

Rna binding protein or its homologue can be that derivative or RBP1 polypeptide or its homologue can be derivatives." derivative " comprises peptide, oligopeptides, polypeptide, protein and enzyme, with the natural generation form of protein (for example SEQ ID NO:2 representative or in the RBP1 situation SEQ ID NO:15 representative) aminoacid sequence compares, it can comprise replacement, disappearance or the interpolation natural and amino-acid residue that non-natural produces." derivative " of protein contains peptide, oligopeptides, polypeptide, protein and enzyme, compares with the aminoacid sequence of the natural generation form of polypeptide, and it can comprise the amino-acid residue that natural that change, glycosylated, acylations or non-natural produces.The one or more non-aminoacid replacement that derivative can also comprise with respect to its aminoacid sequence that is derived from (for example is incorporated into reporter molecules or other part of aminoacid sequence covalently or non-covalently, such as the reporter molecules that is conducive to detect of combination with it), and the amino-acid residue that non-natural produces for the aminoacid sequence of natural generation protein.

Rna binding protein or its homologue can be by the alternative splicing variant codings of rna binding protein nucleic acid/gene.RBP1 polypeptide or its homologue can be by the alternative splicing variant codings of rbp1 nucleic acid/gene.The intron of selecting contained wherein in term used herein " alternative splicing variant " and/or exon is cut, replace or the variant of the nucleotide sequence that adds.Such variant has kept the biological activity of protein, and this can be by the selectively functional fragment realization of retaining protein.Such splice variant can be natural or artificial.The method that produces this splice variant is well-known in the art.Preferred splice variant is the splice variant of the nucleic acid of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7 and SEQ ID NO:9 representative.The polypeptide of preferred splice variant coding has kept RNA in conjunction with active and have at least one RRM, at least one among preferred 2 or 3 RRM and motif I or the II, and preferably both have concurrently.The splice variant of preferred RBP1 is the splice variant by the nucleic acid of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27, SEQID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 and SEQ IDNO:37 representative.The polypeptide of how preferred splice variant coding has kept RNA in conjunction with active and have preferred two RRM structural domains and further preferred following two motifs: (i) KIFVGGL and (ii) RPRGFGF, the change that allows three aminoacid replacement at the most in the motif and guard arbitrarily.

Homologue can also be by the allele variant coding of coding RNA in conjunction with the nucleic acid of albumen or its homologue, the allele variant of the nucleic acid of arbitrary representative among preferred SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and the SEQ ID NO:9.Further preferably have RNA in conjunction with active and have at least one RRM by the polypeptide of allele variant coding, preferred 2 or 3 RRM and motif I or II genuine at least one, preferably both have concurrently.Homologue can also be by the allele variant coding of the nucleic acid of coding RBP1 polypeptide or its homologue, preferably by the allele variant of the nucleic acid of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQID NO:23, SEQ ID NO:25 or SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:37 representative.Further preferably has RNA in conjunction with active and preferred 2 RRM structural domains and following two motifs by the polypeptide of allele variant coding: (i) KIFVGGL and (ii) RPRGFGF, the change that allows three aminoacid replacement at the most in the motif and guard arbitrarily.The natural existence of allele variant, and these natural allelic purposes are contained in the method for the present invention.Allele variant comprises single nucleotide polymorphism (SNP), and small-sized insertion/deletion (INDEL).The size of INDEL is usually less than 100bp.SNP and INDEL form one group of maximum sequence variants in the naturally occurring polymorphism strain of most of organisms.

According to a preferred aspect of the present invention, the expression of rna binding protein coding nucleic acid or its variant increases or increases.According to a preferred aspect of the present invention, the expression of rbp1 nucleic acid or its variant increases or increases.The gene that to improve or increase or the method for gene product expression have sufficient record in this area, it comprises, for example by the use of crossing expression, transcriptional enhancer or translational enhancer of suitable promoters driven.The appropriate location (generally being the upstream) that the nucleic acid that is used as the separation of promotor or enhancer element can be introduced non-allos form polynucleotide, thereby the expression of rise rna binding protein coding nucleic acid or its variant.For example, can and/or replace by sudden change, disappearance, body changes endogenesis promoter interiorly and (sees Kmiec, U.S. Patent number 5,565,350; Zarling etc., PCT/US93/03868), perhaps with the promotor of separating in the suitable direction of gene of the present invention with in apart from the introduced plant cell, thereby the expression of controlling gene.

If wish expression of polypeptides, usually to comprise polyadenylation region at the 3 '-end in peptide coding zone.Polyadenylation region can be derived from natural gene, multiple other plant gene or T-DNA.For example, 3 ' of adding end sequence can be derived from kermes magnetic synthase or octopine synthase gene or alternatively from other plant gene or suboptimum selection of land from any other eukaryotic gene.

Also can in 5 ' non-translational region of part encoding sequence or encoding sequence, add intron sequences, be increased in the ripe courier's who accumulates in the kytoplasm quantity.Show, but the intron of the montage that comprises in the transcription unit of plant and animal expression construct all can increase genetic expression up to 1000 times at mRNA and protein level simultaneously, Buchman and Berg, Mol.Cell biol.8:4395-4405 (1988); Callis etc., Genes Dev.1:1183-1200 (1987).Usually when this intron be placed on transcription unit 5 ' terminal near the time, its effect that improves genetic expression reaches maximum.Corn intron A dh1-S introne 1,2 and the use of 6, Bronze-1 intron be well known in the art.Usually see The Maize Handbook, the 116th chapter, Freeling and Walbot edit, Springer, N.Y. (1994).

The present invention also provides genetic constructs and carrier, to promote introducing and/or the expression for the nucleotide sequence of the inventive method.

Therefore, the gene construct that provides comprises:

(i) rna binding protein coding nucleic acid or its variant;

(ii) one or more control sequences that can drive the nucleotide sequences expression of (i); With optional

(iii) transcription termination sequence.

The gene construct that also provides comprises:

(i) rbp1 nucleic acid or its variant;

(iii) transcription termination sequence.

Can use the well-known recombinant DNA technology of those skilled in the art to make up the construct that is used for the inventive method.Gene construct can be inserted in (commercially available) carrier, described carrier is suitable for transforming the cells goal gene that enters plant and be suitable for transforming.

Use comprises the carrier conversion of plant of aim sequence (being rna binding protein coding nucleic acid or its variant or rbp1 nucleic acid or its variant).Aim sequence effectively is connected in one or more control sequences (being connected at least promotor).In this article all tradable uses of term " controlling element ", " control sequence " and " promotor ", and general reference can affect the regulation and control nucleotide sequence that its catenation sequence is expressed in context.Above-mentioned term is contained and is derived from typical eukaryotic gene group gene and (comprises the TATA box that has or do not have CCAAT box sequence, it is essential for accurate transcription initiation) transcription regulating nucleotide sequence, and other controlling element (being upstream activating sequence, enhanser and silencer), it is by replying growth stimulation and/or outside stimulus or changing genetic expression in tissue-specific mode.This term has also comprised the transcription regulating nucleotide sequence of classical prokaryotic gene, and it can comprise-35 box sequences and/or-10 box transcription regulating nucleotide sequences in the case.Synthetic fusion molecule or derivative also contained in term " controlling element ", and it gives, activates or improve the expression of cell, tissue or organ amplifying nucleic acid molecule.Term used herein " effectively connects " and refers to the functional connection between promoter sequence and goal gene, thereby makes transcribing of the promoter sequence initial goal gene of energy.

Advantageously, can use the expression of the promoters driven nucleotide sequence of any type.Promotor can be inducible promoter, namely replys the stimulation of growth, chemistry, environment or physics, has the transcription initiation of inducing or increase.The example of inducible promoter is stress induced promoter, namely is exposed to the promotor of various abiotic stress condition activation of lower time when plant.In addition or alternative, described promotor can be to organize the promotor of preference, namely can organize at some, such as initial promotor of transcribing preferentially in the tissues such as leaf, root, seed.

Preferably, rna binding protein coding nucleic acid or its variant effectively are connected in the promotor of seed preference.The promotor of seed preference is preference, but need not to drive in seed tissue the promotor of expressing single-mindedly.Preferred seed tissue is endosperm.Preferred promotor is prolamine (prolamin) promotor, for example from the prolamine promotor (SEQ ID NO:11) of rice.Should be clear and definite be the rna binding protein coding nucleic acid that application of the present invention is not limited to SEQ ID NO:1 representative, the expression of rna binding protein coding nucleic acid when also being not limited to by the prolamine promoters driven.

Preferably, rbp1 nucleic acid or its variant effectively be connected in can be in branch the promotor of preference ground express nucleic acid.Preferably, can in branch, the promotor of preference ground express nucleic acid have the express spectra similar to β expansion protein promoter, for example shown in Figure 5.Most preferably, can be in branch the promotor of preference ground express nucleic acid from the β expansion protein promoter (SEQ ID NO:38) of rice.Should be clear and definite be the rbp1 nucleic acid that application of the present invention is not limited to SEQ ID NO:14 representative, also be not limited to the expression of rbp1 nucleic acid when being driven by β expansion protein promoter.

Choose wantonly, can also in the construct of introduced plant, use one or more terminator sequences.Control sequence contained in term " terminator ", and this dna sequence dna is positioned at transcription unit's end, 3 ' processing of transmission of signal initiation primary transcript and polyadenylation and the termination of transcribing.Other controlling element can comprise the enhanser of transcribing and translating.Those skilled in the art will know and be suitable for carrying out terminator of the present invention and enhancer sequence.

Genetic constructs of the present invention also is included in the starting point of keeping in the particular cell types and/or copying required replication sequence.Example be when needs with genetic constructs as additive type genetic elements (such as plasmid or a glutinous grain molecule) when in bacterial cell, keeping.Preferred replication orgin includes, but is not limited to f1-ori and colE1.

Genetic constructs can randomly comprise selectable marker gene.As used herein, term " selectable marker gene " comprises any gene of giving cell phenotype, and the expression of this gene in cell is conducive to identify and/or the cell of choice for use nucleic acid construct transfection of the present invention or conversion.Suitable mark can be selected from the mark with microbiotic or Herbicid resistant, and it is introduced new metabolic characteristic or allows visual selection.The example of selectable marker gene comprises the gene (for example npt II of phosphorylation Liu Suanyan NEOMYCIN SULPHATE and kantlex, or the hpt of phosphorylation Totomycin) of giving antibiotics resistance, and (for example bar provides the resistance to Basta to the gene of conferring herbicide resistance; AroA or gox provide the resistance to glyphosate), or the gene (using seminose as the manA of sole carbon source as allowing plant) of metabolic characteristic is provided.Visual mark cause form color (β-glucuronidase for example, GUS), luminous (for example luciferase) or fluorescence (green fluorescent protein GFP and derivative thereof).

The present invention is also contained can be by the plant of the inventive method acquisition.Therefore the present invention provides and can by the plant of the inventive method acquisition, introduce rna binding protein coding nucleic acid or its variant or rbp1 nucleic acid or its variant in this plant.

The present invention also is provided for producing the method for transgenic plant with improvement growth characteristics, its be included in introduce in the plant and expressed rna in conjunction with encoding histone nucleic acid or its variant.

More specifically, the invention provides the method for generation of the transgenic plant with improvement growth characteristics, the method comprises:

(i) in plant or vegetable cell, introduce rna binding protein coding nucleic acid or its variant; With

(ii) culturing plants cell under the condition of Promoting plant growth and growth.

The present invention also is provided for producing the method for the transgenic plant with improvement growth characteristics, and it is included in introduces and express rbp1 nucleic acid or its variant in the plant.

(iii) in plant or vegetable cell, introduce rbp1 nucleic acid or its variant; With

(iv) culturing plants cell under the condition of Promoting plant growth and growth.

Can be with the direct introduced plant cell of nucleic acid or plant itself (comprising any other parts of introducing tissue, organ or plant).According to preferred feature of the present invention, preferably by transforming the nucleic acid introduced plant.

Here indication term " conversion " is contained exogenous polynucleotide is shifted into host cell, and does not consider the method for transfer.Can use genetic constructs of the present invention to transform by organ occurs or the embryogenetic plant tissue that carries out clonal expansion that can continue and from its whole plant that regenerates.Concrete tissue is selected and will and be changed by the clonal expansion system decision of available and the concrete species of conversion that are suitable for most.Exemplary target tissue comprises the meristematic tissue (for example apical meristem, axillalry bud and root meristematic tissue) of leaf dish, pollen, embryo, cotyledon, hypocotyl, megagamete, callus, existence, and the meristematic tissue of inducing (for example cotyledon meristematic tissue and hypocotyl meristematic tissue).Can be with polynucleotide instantaneous or stably introduce host cell, and can, for example keep nonconformable state as plasmid.Alternatively, it can be integrated into host genome.The transformed plant cells that obtains can be then used in the plant that the mode that well known to a person skilled in the art is regenerated and transformed.

The conversion of plant species is a kind of quite conventional technology at present.Advantageously, can use arbitrary in several method for transformation that goal gene is introduced suitable ancester cell.Method for transformation comprises with the chemical of liposome, electroporation, the picked-up of enhancing dissociative DNA, directly bombards, transforms and microprojection (microprojection) with virus or pollen to plant injection DNA, particle gun.Method can be selected from for protoplastis calcium/(1882, Nature 296,72-74 for Krens, F.A. etc. for the polyoxyethylene glycol method; Negrutiu I. etc., June 1987, Plant Mol.Biol.8,363-373), electroporation (the Shillito R.D. etc. of protoplastis, 1985 Bio/Technol 3,1099-1102), microinjection (Crossway A. etc., 1986, the Mol.Gen Genet 202 of vegetable material, 179-185), coated particle bombardment (the Klein T.M. etc. of DNA or RNA, 1987, Nature 327,70), (nonconformable) virus infection etc.The preferred protein-bonded transgenosis rice of conversion generation expressed rna of using any rice method for transformation of knowing to mediate via Agrobacterium, the method that described rice method for transformation is for example described in the how Publication about Document in office: disclosed European patent application EP 1198985A1, Aldemita and Hodges (Planta, 199,612-617,1996); Chan etc. (Plant Mol.Biol.22 (3) 491-506,1993), Hiei etc. (Plant is (2) 271-282 J.6,1994), its disclosure is introduced into as a reference as the full content of its statement.Transform as for corn, preferred method is as at (Nat.Biotechnol.1996 such as Ishida, 14 (6): 745-50) or (the Plant Physiol.2002 such as Frame, 129 (1): 13-22) described, its disclosure is introduced into as a reference as the full content of its statement.

Usually after transforming, select the vegetable cell or the groups of cells that there are one or more marks, described mark is by the expressive gene of plant coding that moves with the goal gene corotation, and the material regeneration with transforming that continues becomes whole plant.

After DNA transfer and the regeneration, can assess the conversion of plant of supposition, for example use existence, copy number and/or the genome constitutions of Southern analysis purposes gene.Alternative or extra, available Northern and/or Western analyze the expression level of the new DNA of introducing of monitoring, and these two kinds of technology all are that those skilled in the art are well-known.

The conversion of plant that produces can be bred in several ways, such as the breeding technique with clonal propagation or classics.For example, the first-generation (or T1) but the s-generation (or T2) transformant that the plant selfing that transforms obtains isozygotying, further by classical breeding technique breeding T2 plant.

The inverting biological body that produces can have various ways.For example, they can be the mosaics of transformant and no transformed cells; Clone's transformant (for example through transforming all cells that contains expression cassette); Transform and the graft of unconverted tissue (for example in plant, the rhizome of the conversion of grafting to the unconverted scion).

The present invention obviously extends to any vegetable cell or the plant that is produced by methods described herein, and the part of all plants and its vegetative propagule.The present invention contains also that the former generation that is produced by any aforesaid method transforms or the offspring of cell, tissue, organ or the whole plant of transfection, and unique requirement of described offspring is that the parent who produces with the inventive method presents same genotype and/or phenotypic characteristic.The present invention also comprises the host cell that contains separative rna binding protein nucleic acid or its variant.The preferred host cell of the present invention is vegetable cell.The present invention also extends to the part that plant can be gathered in the crops, such as, but not limited to seed, leaf, fruit, flower, stem culture, rhizome, stem tuber and bulb.

The purposes of rna binding protein nucleic acid or its variant and the purposes of rna binding protein or its homologue are also contained in the present invention.

A kind of such purposes relates to the growth characteristics that improve plant, particularly improves productive rate, especially the seed productive rate.The seed productive rate can comprise following one or more: (full) seed number of increase, the seed weight of increase, harvest index of increase etc.

Can use rna binding protein coding nucleic acid or its variant in the procedure of breeding, perhaps rna binding protein or its homologue are wherein identified the dna marker that can be connected in hereditarily rna binding protein encoding gene or its variant.Can use rna binding protein coding nucleic acid or its variant, perhaps rna binding protein or its homologue define molecule marker.Then this DNA or protein labeling can be used for the procedure of breeding, have the plant of the growth characteristics of change with selection.For example, rna binding protein encoding gene or its variant can be the nucleic acid by any one representative among SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7 and the SEQ ID NO:9.

The allele variant of rna binding protein encoding gene/nucleic acid also can be used for the auxiliary procedure of breeding of mark.Such procedure of breeding needs to use sometimes, and for example EMS mutagenesis is introduced allele variant by the mutagenic treatment of plant; Alternative, this program can begin with the allele variant of collecting what is called " natural " origin that is not intended to generation.Then identify allele variant by for example PCR.Be to select step in order to select the better allele variant of described sequence subsequently, described allele variant produces the growth characteristics of improvement in plant.The growth behavior that generally contains the different allele variant plants of studying to some extent sequence by monitoring is selected, for example arbitrary different allele variants among SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7 and the SEQ ID NO:9.Can in greenhouse or field, monitor growth behavior.How optional step comprises, will contain through evaluation plant and another plant hybridization of better allele variant.For example, can make the combination that produces in this way the purpose phenotypic characteristic.

Rna binding protein coding nucleic acid or its variant can also be as probes, be used for it is carried out the mapping of heredity and physics for the gene of a gene part, and the chain proterties mark of those genes of being linked to each other of conduct.These information can be used in plant breeding, to obtain having the strain of desired phenotype.This class of rna binding protein coding nucleic acid or its variant is used the one section nucleotide sequence that only needs at least 15 Nucleotide long.Rna binding protein coding nucleic acid or its variant also can be used as restriction fragment length polymorphism (RFLP) mark.Available rna binding protein coding nucleic acid or its variant are surveyed the Southern trace (Maniatis) of the plant genome DNA of restriction digest.Use subsequently computer program such as MapMaker (Lander etc., 1987) that the banding pattern that produces is carried out genetic analysis, to make up genetic map.In addition, can use nucleic acid to survey the Southern trace in the genomic dna that the restriction enzyme that contains one group of individuality is processed, described one group of individuality is the parent of the clear and definite genetic cross of representative and one group of individuality of filial generation.The separation of dna polymorphism is recorded and is used for calculating formerly with the genetic map rna binding protein coding nucleic acid of this colony's acquisition or the position (Botstein etc. (1980) Am.J.Hum.Genet.32:314-331) of its variant.

Derive generation and the purposes of probe of the plant gene that uses in genetic mapping is described among Bematzky and Tanksley (1986) the Plant Mol.Biol.Reporter 4:37-41.Described in a lot of publications with aforesaid method or its flexible form specific cDNA clone was carried out genetic mapping.For example, can use F2 hybrid Population, backcross population, panmictic population, the homogenic system of close relative and the individual mapping of other group.These methods are that those skilled in the art are well-known.

Nucleic acid probe also can be used for physical mapping and (namely settle sequence at physical map; See Hoheisel etc.: Non-mammalian Genomic Analysis:A Practical Guide, Academic press 1996, the 319-346 pages or leaves, and the reference of wherein quoting).

In another embodiment, nucleic acid probe can be used for direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet.7:149-154).Although the method inclination of at present FISH mapping uses large clone (several to a hundreds of KB; See (1995) the Genome Res.5:13-20 such as Laan), but the raising of susceptibility allows to use short probe in the FISH mapping.

Can use described nucleic acid to carry out for heredity and the multiple method based on nucleic acid amplification of physical mapping.Example comprises the polymorphism (CAPS of allele specific amplification (Kazazian (1989) J.Lab.Clin.Med11:95-96), pcr amplified fragment; Sheffield etc. (1993) Genomics16:325-332), allele-specific connects (Landegren etc. (1988) Science241:1077-1080), Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), Radiation hybrid mapping (Walter etc. (1997) Nat.Genet.7:22-28) and Happy mapping (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For implementing these methods, use the nucleotide sequence design and produce the primer pair that is used for amplified reaction or primer extension reaction.The design of this class primer is that those skilled in that art are well-known.Use the method for the genetic mapping of PCR-based, may need to identify the difference of crossing over corresponding to dna sequence dna between the parent of current nucleotide sequence zone mapping.Yet this is usually dispensable to drawing method.

Also find rna binding protein coding nucleic acid or its variant, perhaps rna binding protein or its homologue are as the purposes of growth regulator.Because it is useful that these molecules have been presented in the improving plant growth characteristic, they also are useful growth regulators, such as weedicide or growth stimulator.Therefore, the invention provides the composition as growth regulator, it comprises rna binding protein coding nucleic acid/gene or its variant or rna binding protein or its homologue and suitable carrier, thinner or vehicle.

The purposes of rbp1 nucleic acid or its variant and the purposes of RBP1 polypeptide or its homologue are also contained in the present invention.

Can use rbp1 nucleic acid or its variant in the procedure of breeding, perhaps RBP1 polypeptide or its homologue are wherein identified the dna marker that can be connected in hereditarily rbp1 gene or its variant.Can use rbp1 nucleic acid or its variant, perhaps RBP1 or its homologue limit molecule marker.Then this DNA or protein labeling can be used for the procedure of breeding, have the plant of the growth characteristics of change with selection.For example, rbp1 gene or its variant can be the nucleic acid by arbitrary representative among SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and the SEQ ID NO:36.

The allele variant of rbp1 also can be used for the auxiliary procedure of breeding of mark.Such procedure of breeding needs to use sometimes, and for example EMS mutagenesis is introduced allele variant by the mutagenic treatment of plant; Alternative, this program can begin with the allele variant of collecting what is called " natural " origin that is not intended to generation.Then identify allele variant by for example PCR.Be to select step in order to select the better allele variant of described sequence subsequently, described allele variant produces the growth characteristics of improvement in plant.The growth behavior that generally contains the different allele variant plants of studying to some extent sequence by monitoring is selected, for example the arbitrary different allele variants among SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and the SEQ ID NO:36.Can in greenhouse or field, monitor growth behavior.How optional step comprises, and will contain through evaluation plant and another plant hybridization of better allele variant.For example, can make the combination that produces in this way the purpose phenotypic characteristic.

Rbp1 nucleic acid or its variant can also be as probes, be used for it is carried out the mapping of heredity and physics for the gene of a gene part, and the chain proterties mark of those genes of being linked to each other of conduct.These information can be used in plant breeding, to obtain having the strain of desired phenotype.This class of rbp1 nucleic acid or its variant is used the one section nucleotide sequence that only needs at least 15 Nucleotide long.Rbp1 nucleic acid or its variant also can be used as restriction fragment length polymorphism (RFLP) mark.Available rbp1 nucleic acid or its variant are surveyed the Southern trace (Maniatis) of the plant genome DNA of restriction digest.Use subsequently computer program such as MapMaker (Lander etc., 1987) that the banding pattern that produces is carried out genetic analysis, to make up genetic map.In addition, can use nucleic acid to survey the Southern trace in the genomic dna that the restriction enzyme that contains one group of individuality is processed, described one group of individuality is the parent of the clear and definite genetic cross of representative and one group of individuality of filial generation.The separation of record dna polymorphism also is used for calculating formerly with the genetic map rbp1 nucleic acid of this colony's acquisition or the position (Botstein etc. (1980) Am.J.Hum.Genet.32:314-331) of its variant.

Derive generation and the purposes of probe of the plant gene that uses in genetic mapping is described among Bematzky and Tanksley (1986) the Plant Mol.Biol.Reporter 4:37-41.Described in a lot of publications with aforesaid method or its flexible form specific cDNA clone was carried out genetic mapping.For example, can use F2 hybrid Population, backcross population, panmictic population, the homogenic system of close relative and the mapping of other group of individuals.These class methods are that those skilled in the art are well-known.

The multiple method based on nucleic acid amplification that is used for heredity and physical mapping can use described nucleic acid to carry out.Example comprises the polymorphism (CAPS of allele specific amplification (Kazazian (1989) J.Lab.Clin.Med11:95-96), pcr amplified fragment; Sheffield etc. (1993) Genomics16:325-332), allele-specific connects (Landegren etc. (1988) Science241:1077-1080), Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), Radiation hybrid mapping (Walter etc. (1997) Nat.Genet.7:22-28) and Happy mapping (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For implementing these methods, use the nucleotide sequence design and produce the primer pair that is used for amplified reaction or primer extension reaction.The design of this class primer is that those skilled in that art are well-known.Use the method for the genetic mapping of PCR-based, may need to identify the difference of crossing over corresponding to dna sequence dna between the parent of current nucleotide sequence zone mapping.Yet this is usually dispensable to drawing method.

Also find rbp1 nucleic acid or its variant, perhaps RBP1 polypeptide or its homologue are as the purposes of growth regulator.Because it is useful that these molecules have been presented in the improving plant growth characteristic, they also are useful growth regulators, such as weedicide or growth stimulator.Therefore, the invention provides the composition as growth regulator, it comprises rbp1 or its variant or RBP1 polypeptide or its homologue and suitable carrier, thinner or vehicle.

The method according to this invention is had the plant of improvement growth characteristics as previously mentioned.These favourable growth characteristics can also make up other economically favourable proterties, such as the proterties of further raising output, to the tolerance of various abiotic stress, change the proterties of various structural attitudes and/or biochemistry and/or physiologic character.

Accompanying drawing is described.

With reference to following accompanying drawing the present invention is described, wherein:

Fig. 1 shows the protein-bonded CLUSTAL multiple ratio of plant RNA pair.Motif I and II are added frame (BAC83046 lacks M2), and the RRM structural domain underlines.

Fig. 2 shows binary vector, and it is used for the tobacco rna binding protein under the control of rice expression prolamine promotor.

Fig. 3 shows the multiple ratio pair of plant RBP1 polypeptide.Gene pool protein or its coding nucleic acid have been shown.At refers to Arabidopis thaliana, and Os refers to rice.

Fig. 4 shows binary vector, and it is used for the Arabidopis thaliana RBP1 (confidential reference items CDS0078) under rice expression β expansion protein promoter (confidential reference items PRO0061) control.

Fig. 5 shows that the GUS that is driven by β expansion protein promoter expresses photo." C plant " photo is the GUS dyeing photo when about 5cm size is grown to by rice plant." B plant " photo is the GUS dyeing photo when about 10cm size is grown to by rice plant.Also can use the promotor with similar express spectra to be used for method of the present invention.

Fig. 6 has described the example of implementing useful sequence in the method according to this invention in detail.From SEQ ID NO:14, refer to for the At that provides number the MIP accession number ( Http:// mips.gsf.de/); Other identifier refers to the gene pool accession number.Capitalization represents encoding sequence, and lowercase refers to non-translational region (comprising 5 ' leader sequence, 3 ' non-translational region and intron).The chromosome position of gene is with the coordinate representation of ORF in contig number and the contig.

Embodiment

Describe the present invention with reference to the following example, described embodiment only is intended to explanation.

DNA operation: unless otherwise indicated, recombinant DNA technology is according to being described in (Sambrook etc. (2001) molecular cloning: laboratory manual, the third edition, cold spring harbor laboratory publishes, CSH, New York) or be described in Ausubel etc. (1994), Current Protocols in Molecular Biology, the first roll of Current Protocols and volume Two) standard method carry out.Standard material and the method for plant molecular operation are described in Plant Molecular Biology Labfase (1993) by R.D.D.Croy, are published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).

Embodiment 1: gene clone-tobacco rna binding protein encoding gene

The protein-bonded gene of coding RNA identifies from tobacco BY2 cell as expressed sequence tag at first and separates as partial sequence that described CDNA-AFLP experiment is used from the cDNA of synchronized tobacco BY2 cell culture (Nicotiana tabacum L.cv.Bright Yellow-2) preparation and carried out in the CDNA-AFLP experiment.Based on this CDNA-AFLP experiment, the BY2 label of identification of cell periodic adjustment also selects to be used for further clone.Use Expressed sequence tags screening tobacco cDNA library and separate complete sequence cDNA.

The synchronization of BY2 cell

Following by use aphid dwell rhzomorph with cell block at S phase synchronization morning tobacco BY2 (Nicotiana tabacum L.cv.Bright Yellow-2) culturing cell suspension.Keep as described the culturing cell suspension (Int.Rev.Cytol.132 such as Nagata, 1-30,1992) of tobacco Bright Yellow-2.Use 10 times of dilutions of fresh culture to be used for synchronization the static culture in 7 day age, described fresh culture has added aphid dwell rhzomorph (Sigma-Aldrich, St.Louis, MO; 5mg/l), a kind of archaeal dna polymerase α suppresses medicine.After 24 hours, make for several times cell releasing retardance and reenter cell cycle progression with the fresh culture washed cell.

RNA extracts and cDNA synthesizes

Use the total RNA of LiCI precipitation (Sambrook etc., 2001) and use Oligotex post (Qiagen, Hilden, Germany) from total RNA of 500 μ g, to extract poly (A according to manufacturer's explanation ⁺) RNA.Use biotinylated oligo-dT ₂₅Primer (Genset, Paris, France) and Superscript II (Life Technologies, Gaithersburg, MD) are by the poly (A of reverse transcription from 1 μ g ⁺) RNA begins synthetic the first chain cDNA.Use intestinal bacteria (Escherichia coli) ligase enzymes (Life Technologies), dna polymerase i (USB, Cleveland, OH) and RNA enzyme H (USB) to replace by chain and carry out the synthetic of the second chain.

CDNA-AFLP analyzes

The double-stranded cDNA of 500ng carries out aflp analysis (Vos etc., Nucleic Acids Res.23 (21) 4407-4414,1995 as described; Bachem etc., Plant are (5) 745-53 J.9, and 1996) and modification is arranged.The restriction enzyme of using is BstYI and MseI (Biolabs) and digested in other step at two minutes.After one of described enzyme of use carried out the restrictive diges-tion first time, 3 ' end fragment was collected on the Dyna pearl (Dynal, Oslo, Norway) by its biotinylated tail, and other fragment is by flush away.Use after second enzymic digestion, collect the restricted fragment of release and it is used as template in follow-up AFLP step.There are not the MseI primer of selective kernel thuja acid and the BstYI combination of primers that contains T or C at 3 ' least significant end to be used for pre-amplification.The PCR condition is (Vos etc., 1995) as described.The amplification mixture that obtains is diluted 600 times, get 5 μ l and be used for using P ³³The selective amplification of the BstYI primer of-mark and Amplitaq-Gold polymerase (Roche Diagnostics, Brussels, Belgium).In 5% polyacrylamide gel, use the system (Biorad) of Sequigel to separate amplified production.Dry gel exposure and scan at phosphoImager (Amersham Pharmacia Biotech, Little Chalfont, UK) on Kodak Biomax film.

The sign of AFLP fragment

To from gel, separate corresponding to the band of differential expression transcript (wherein containing the transcript corresponding to SEQ ID NO 1), and under the condition identical with selective amplification, the DNA of wash-out is used for again amplification.By to use polymerase chain reaction product direct Sequencing that selectivity BstYI primer increases again or after cloned sequence among the pGEM-T easy (Promega, Madison, WI) direct Sequencing, or obtain sequence information by the individual clone of order-checking.By Nucleotide and the protein sequence that exists in BLAST sequence alignment (Altschul etc., Nucleic Acids Res.25 (17) 3389-34021997) sequence that relatively obtains and the database that can openly obtain.When feasible, the EST that use is long or the cDNA sequence replacing sequence label of separation find the probability of remarkable homology with increase.Then clone from the physics cDNA of commercial tobacco cDNA library amplification corresponding to SEQ ID NO 1 according to following steps.

Gene clone

Use poly (A ⁺) the average inset of separation is the cDNA library of 1400bp from the not synchronized BY2 tobacco cell of active division.These library insets are cloned among the carrier pCMVSPORT6.0 (Life Technologies) that contains attB gateway box.From then on select 46000 clones to list in the 384 hole microtiter plates in the library, and follow duplicate place on NF.By using the radiolabeled tag library of the hundreds of kind clone that screening is arranged as probe (wherein containing the BY2 label corresponding to SEQ ID NO1).Separate positive colony (wherein contain with corresponding to the BY2 tag reactant of SEQ IDNO 1 clone), compare to its order-checking and with sequence label.If use label to hybridize unsuccessfully, by the full-length cDNA of following pcr amplification selection corresponding to label.Use primer3 program (http://www-genome.wi.mit.edu/genome_software/other/primer3.html) tag design Auele Specific Primer and be used for amplification cDNA inset partly with the universal support combination of primers.In pcr amplification, use the dna library of cloning from 50000,100000,150000 and 300000 cDNA as template.Separate amplified production from sepharose, compare to its clone, order-checking and with label.

Then, will be cloned in the suitable plant expression vector via LR Gateway reaction from the full-length cDNA corresponding to SEQ ID NO 1 of pCMVsport6.0 carrier library.

LR gateway reaction is cloned in CDS0701 in the plant expression vector

Then pCMV Sport 6.0p2461 is used for specify with the Gateway that is suitable for the rice conversion LR reaction of carrier.This carrier contains within the T-DNA border as the plant selectable marker of functional element and Gateway box, and described Gateway box is used for carrying out recombinating in the LR body with the aim sequence that is cloned in the donor carrier.The upstream of this Gateway box is the paddy Prolamin promoter, and it is used for the seed-specific expression of gene.

After the reconstitution steps, the expression vector (seeing Fig. 2) that produces transformed to enter agrobacterium strains LBA4404 and then transform enter rice plant.

Embodiment 2: rice transforms

Ripe dry seeds shelling with Japanese cultivated rice Nipponbare (NB).By in 70% ethanol, hatching one minute, then at 0.2%HgCl ₂In 30 minutes, the usefulness that continues distillation washing 6 times carried out disinfection in each 15 minutes.Then aseptic seed is sprouted on the substratum that contains 2,4-D (callus inducing medium).After around hatching in the dark, downcut the callus that embryogenetic scultellum derives and in identical substratum, breed.After two weeks, breed in other words callus by the other 2 all propagation of succeeding transfer culture in same medium.Before common cultivation 3 days, upload culture embryo generation callus fragment (active in order to promote cell fission) at fresh culture.The agrobacterium strains LBA4404 that will contain double base T-DNA carrier is used for cultivating altogether.Agrobacterium is inoculated in to contain in the suitable antibiotic AB substratum and at 28 ℃ and cultivated 3 days.Then collect bacterium and be suspended in liquid altogether in the culture medium to about 1 optical density(OD) (OD600).Then suspension is transferred to culture dish and callus was dipped in suspension 15 minutes.Subsequently callus is stained with dry doubling at filter paper and is transferred to altogether substratum and hatching 3 days in 25 ℃ in the dark of solid.In the presence of the selection material of suitable concn, the callus of cultivating is altogether containing 2, in the substratum of 4-D around 28 ℃ of dark growths.During this period, grow the resistant calli island (resistant callus islands) of Fast Growth.This substance transfer is hatched under illumination to regeneration culture medium, demonstrate embryo's generation potentiality and grow branch in ensuing four to five weeks.Branch is downcut and hatched for 2 to 3 weeks at the substratum that contains growth hormone from callus, with it from this media transfer to soil.The branch of hardening (shoot) is grown in the greenhouse under the condition in high humidity and short daytime.Then after five months, gather in the crops seed in transplanting three.The method is to provide transformant (Aldemita and Hodges, Planta, 199 612-617,1996 of individual gene seat above 50 ratio; Chan etc., Plant Mol.Biol.22 (3) 491-506,1993, Hiei etc., Plant J., 6 (2) 271-282,1994).

Embodiment 3: assessment and result

15 to 20 independently T0 rice transformants have approximately been produced.In former generation,, transformant transferred to greenhouse growth and results T1 seed by tissue culture room.5 events are kept, and wherein separation in 3: 1 of transgenosis existence/shortage occur the T1 offspring.By the expression of monitoring visable indicia, in each event, select about 10 T1 seedling that contain transgenosis (heterozygote and homozygote), and about 10 T1 seedling that lack transgenosis (invalid zygote) of similar number.4 T1 events were further assessed according to the appraisal procedure identical with T1 generation in T2 generation, but each event that described quilt is further assessed has more individuality.

Statistical study: F-check

Use double factor ANOVA (analysis of variance) as the statistical model of plant phenotype characteristic total evaluation.In all plants by all events of gene transformation of the present invention, all measuring parameters are carried out the F check.Carry out F and check to check the validity of gene in all transformation events, and verify the group effect to gene, also be called whole genetic effect.Be 5% probability level of F check with the threshold setting of real whole genetic effect significance.Significance F test value refers to genetic effect, and it means the difference that the existence of being not only gene or position cause phenotype.

3.1 the measurement of seed correlation parameter

Ripe former generation panicle gathered in the crops, packing, slug shape code, then in 37 ℃ of baking ovens dry three days.Then beat panicle and to all seed collections and counting.With blowing device full shell is separated with ghost.Abandon ghost, and again count remaining part.Full shell is weighed at analytical balance.Remaining full hull number is defined as the full seed number after separating step.Measure total seed productive rate by weighing from the whole full shell of plant results.Harvest index among the present invention is defined as total seed productive rate and ground area (mm ²) ratio multiply by the factor 10 ⁶

Following as a result table shows the p value to the F check of T1 and T2 assessment.The difference that also shows percentage ratio between transgenosis and the corresponding invalid zygote.For example, for the total seed weight in T1 generation, 3 in 4 strains are total seed weight positive (namely compare with the seed weight of corresponding invalid zygophyte, being shown as total seed weight increases (greater than 32%)).2 strains in 4 strains like this show that total seed weight significantly increases, and the p value in the F check is 0.061.

Show the 5:T1 result in generation

T1	Show the product coefficient that increases	Difference	Show the product coefficient that significantly increases	The p value of F check
					Total seed weight	In 43	＞32％	In 42	＜0.061
Harvest index	In 42	＞32％	In 42	＜0.09

Show the 6:T2 result in generation

T2	Show the product coefficient that increases	Difference	Show the product coefficient that significantly increases	The p value of F check
					Total seed weight	In 41	＞30％	In 41	＜0.064

Harvest index

In 41

＞40％

In 41

＜0.001

The gene clone of embodiment 4:AtRBP1

Use Arabidopsis thaliana Seedlings cDNA library (Invitrogen, Paisley, UK) to pass through pcr amplification Arabidopis thaliana AtRBP1 (CDS0078) as template.After the RNA reverse transcription that seedling is extracted, cDNA is cloned pCMV Sport 6.0.The average inset size in library is 1.5kb, and original clone's number is 1.59 * 10 ⁷Cfu.Original titre is confirmed as 9.6 * 10 ⁵Cfu/ml is 6 * 10 after for the first time amplification ¹¹Cfu/ml.After the plasmid extraction, the 200ng template is used for 50 μ l PCR mixtures.Comprise primer prm00405 (justice 5 ' ggggacaagtttgtacaaaaaagcaggcttcacaatggattatgatcggtacaagt tat3 ') and prm00406 (reverse complemental: 5 ' ggggaccactttgtacaagaaagctgggtttaaaagagtccaaagaatttcact 3 ') be used to pcr amplification for the AttB site of Gateway restructuring.Under standard conditions, use Hifi Taq archaeal dna polymerase to carry out PCR.The PCR fragment of 1209bp is amplified and also Application standard method purifying.Then carry out the first step of Gateway operation, the BP reaction, generation " entering the clone " p0073 according to the Gateway term during this period recombinates in PCR fragment and the pDONR201 plasmid body.Plasmid pDONR201 conduct The part of technology is available from Invitrogen.

Embodiment 5:AtRBP1 vector construction

Then will enter clone p0073 is used for reacting with the LR of p03069 (being used for the appointment carrier that rice transforms).This carrier contains as functional element within the T-DNA border: plant selectable marker; Visable indicia expression cassette and Gateway box, described Gateway box are used for carrying out restructuring in the LR body with the aim sequence that is cloned in " entering the clone ".The β expansion protein promoter of expressing in branch is positioned at the upstream of this Gateway box.

After the LR reconstitution steps, the expression vector (Fig. 4) that produces transformed to enter agrobacterium strains LBA4404 and then transform enter rice plant.Make rice plant's growth of conversion and follow and check in the parameter described in the embodiment 6.

The assessment of embodiment 6:AtRBP1 and result

15 to 20 independently T0 rice transformants have approximately been produced.In former generation,, transformant transferred to greenhouse growth and results T1 seed by tissue culture room.5 events are kept, and wherein separation in 3: 1 of transgenosis existence/shortage occur the T1 offspring.By the expression of monitoring visable indicia, in each event, select about 10 T1 seedling that contain transgenosis (heterozygote and homozygote), and about 10 T1 seedling that lack transgenosis (invalid zygote) of similar number.4 T1 events were further assessed according to the appraisal procedure identical with T1 generation in T2 generation, but each event that described quilt is further assessed has more individuality.No longer continue for a neutral strain in the first round.In T2 assessment, what relatively contain genetically modified 15 T2 seedling and similar number lacks genetically modified plant (invalid zygote).

Statistical study: F-check

Use double factor ANOVA (analysis of variance) as the statistical model of plant phenotype characteristic total evaluation.In all plants by all events of gene transformation of the present invention, all measuring parameters are carried out the F check.Carry out F and check to check the validity of gene in all transformation events, and verify the whole structure of gene, also be called whole genetic effect.Be 5% probability level of F check with the threshold setting of real whole genetic effect significance.Significance F test value refers to genetic effect, and it means the difference that the existence of being not only gene or position cause phenotype.

6.1 the measurement of seed correlation parameter

Ripe former generation panicle gathered in the crops, packing, slug shape code, then in 37 ℃ of baking ovens dry three days.Then beat panicle and to all seed collections and counting.With blowing device full shell is separated with ghost.Abandon ghost, and again count remaining part.Full shell is weighed at analytical balance.This operation produces seed correlation parameter group described below.

Following as a result table demonstration is from the p value of the F check that T1 assessment, T2 are assessed and the combination p value of checking from the F to T1 and T2 assessment.Combinatory analysis can be thought same event is carried out two experiments.This can and increase the confidence level of conclusion for detection of the consistence of two experiment effects.The method of using is the mixed mode method, and it considers multilevel data structure (i.e. experiment-event-segregant).Probability Detection by relatively card side's distribution obtains the P value.Each table give per generation transgenosis and corresponding invalid zygote between % difference.

6.1.1 ground area

Sum of all pixels by ground plant part after the calculating eliminating background is determined plant area on the ground.This value is to put at one time the mean value that obtains from different perspectives picture, and is converted into the physical surface value that represents with square millimeter by calibration.Experiment shows that the over-ground part plant area of this method measurement is relevant with the biomass that plant shoot divides.The results are shown in the following table 7 of T1 and T2 assessment.As shown in the table, from being significance to the p value (p value is 0.0011) of the F check of T2 assessment and the data (having the p value is 0.0287) of combination, it illustrates that the construct that exists has the remarkable positive to the ground areas of transgenic plant and affects in plant.

Table 7: ground area

6.1.2 the total seed production of every strain plant

Measure total seed production by weighing from the whole full shell of plant results.As shown in table 8 below, the p value of checking from the F to T1 and T2 combined evaluation is significance (the p value is 0.0287), and the existence of its explanation construct in plant has the significance impact to total seed weight of transgenic plant.

Table 8

6.1.3 seed is total

As shown in table 9 below, the p value of checking from the F to T1 and T2 combination (and T2 is independent) assessment is significance (the p value is 0.0006), and the existence of its explanation construct in plant has the significance impact to the seed sum of transgenic plant.

Table 9

Embodiment 7: the GUS that is driven by β expansion protein promoter expresses

Use " BP recombining reaction " that β is expanded the pDONR201 that protein promoter is cloned in the GatewayTM system and enter plasmid (Life Technologies).Identity and the base pair of the inset of determining the clone by checking order form, and additionally detect the plasmid that produces via restrictive diges-tion.

For promotor being cloned in before the reporter gene, then each being entered the clone and be used for " LR recombining reaction " (GatewayTM) with specifying carrier.Design effectively is connected in intestinal bacteria β glucuronidase (GUS) gene with this appointment carrier, and described connection is carried out via the replacement of the restructuring of the Gateway before gus gene box.Then the Application standard transformation technology transforms the conversion that enters agrobacterium strains LBA4044 and continue with the report carrier (comprising the promotor that effectively is connected in GUS) that produces and enters rice plant.

Produce transgenosis rice plant from the cell that transforms.Plant-growth is carried out under normal operation.

Use 90% ice-cold acetone to cover plant or plant part to be tested, hatched 30 minutes at 4 ℃.[15.76g Trizma HCl (Sigma T3253)+2.922g NaCl is in 1 liter of distilled water to use the Tris damping fluid, use NaOH to be adjusted to pH 7.0] wash 3 times, after each 5 minutes, this material is covered with Tris/ ferricyanate/X-Gluc solution [9.8ml Tris damping fluid+0.2ml ferricyanate mother liquor (0.33g potassium ferricyanate (Sigma P3667) is in 10ml Tris damping fluid)+0.2ml X-Gluc mother liquor (26.1mg X-Gluc (Europa Bioproducts ML 113A) is in 500 μ l DMSO)].Vacuum filtration 15 to 30 minutes.Hatch at 37 ℃ that plant or plant part reach 16 hours until blue the development as seen.With Tris damping fluid washing sample 3 times 5 minutes.Ethanol series with 50%, 70% and 90% is extracted chlorophyll (each is 30 minutes).

Claims

1. the method for improving plant growth characteristic, it comprises the activity that increases rna binding protein in the plant or its homologue, and wherein said rna binding protein or its homologue are RBP1 polypeptide or its homologue, and it has (a) RNA in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF, the change that allows three aminoacid replacement at the most in the motif and guard arbitrarily; (d) has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity according to the preference ordering that increases and the amino acid of SEQ ID NO:15 representative; With

Randomly select to have the plant of improvement growth characteristics.

2. according to claim 1 the method for improving plant growth characteristic, its be included in the plant introduce and expressed rna in conjunction with encoding histone nucleic acid or its functional variant, the rna binding protein of wherein said coding or its homologue are RBP1 polypeptide or its homologue, and it has (a) RNA in conjunction with active (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF, the change that allows three aminoacid replacement at the most in the motif and guard arbitrarily; (d) has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity according to the preference ordering that increases and the amino acid of SEQ ID NO:15 representative.

3. according to claim 2 method, wherein said variant be the rna binding protein coding nucleic acid part or can with the sequence of rna binding protein coding nucleic acid hybridization.

4. according to claim 2 or 3 method, wherein said rna binding protein coding nucleic acid or its functional variant are crossed in plant and are expressed.

5. each method in 4 according to claim 2, wherein said rna binding protein coding nucleic acid or its functional variant are plant origins, preferably from dicotyledons, further preferably from Cruciferae, more preferably nucleic acid is from Arabidopis thaliana.

6. each method in 5 according to claim 2, wherein said rbp1 nucleic acid or its functional variant effectively are connected in and can express the promotor of described nucleic acid in preference ground in branch.

7. according to claim 6 method, wherein said promotor has the express spectra similar to β expansion protein promoter.

8. each method in 7 according to claim 1, the plant growth characteristics of wherein said improvement is the productive rate that increases with respect to corresponding wild-type plant, comprises the phytomass of increase and/or the seed productive rate of increase.

9. according to claim 8 method, the seed productive rate of wherein said increase is selected from the following seed biomass of appointing one or more (i) to increase; (ii) (full) seed number that increases; (iii) seed size that increases; (iv) the seed volume that increases; (v) harvest index that increases; The thousand seed weight (TKW) that (vi) increases.

10. each method in 9 according to claim 1, the plant growth characteristics of wherein said improvement is the growth velocity that increases.

11. can be according to claim 1 each method obtains in 10 plant.

12. construct comprises:

(i) rbp1 coding nucleic acid or its variant, the RBP1 of wherein said coding has: (a) RNA is in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF, the change that allows three aminoacid replacement at the most in the motif and guard arbitrarily; (d) has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity according to the preference ordering that increases and the amino acid of SEQ ID NO:15 representative;

(ii) can drive one or more control sequences that the nucleotide sequence of (i) is expressed; With optional

(iii) transcription termination sequence.

13. construct according to claim 12, wherein said promotor can drive the expression in branch.

14. construct according to claim 13, wherein said promotor has similar express spectra to β expansion protein promoter.

15. use according to claim 12 the plant that each construct transforms in 14.

16. produce the method for the transgenic plant with improvement growth characteristics, the method comprises:

(i) introduce rbp 1 coding nucleic acid or its variant in plant, the RBP1 of wherein said coding has: (a) RNA is in conjunction with activity; (b) two RRM structural domains; (c) following two motifs: (i) KIFVGGL and (ii) RPRGFGF, the change that allows three aminoacid replacement at the most in the motif and guard arbitrarily; (d) has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence identity according to the preference ordering that increases and the amino acid of SEQ ID NO:15 representative; With

17. have by the transgenic plant of rbp1 nucleic acid or its variant being introduced the improvement growth characteristics that described plant produces.

18. according to claim 11,15 or 17 transgenic plant, wherein said plant is monocotyledons, and such as sugarcane, perhaps wherein said plant is cereal, such as rice, corn, wheat, barley, grain, rye, oat or Chinese sorghum.

19. according to claim 11, the part gathered in the crops of each plant in 15,17 or 18.

20. in the claim 4 in defined rna binding protein coding nucleic acid/gene or the claim 5 defined its variant or rna binding protein or its homologue particularly improve the especially purposes in the seed productive rate of productive rate in the improving plant growth characteristic.

21.rbp1 or defined RBP1 polypeptide or its homologue particularly improve the especially purposes in the seed productive rate of productive rate in the improving plant growth characteristic in its variant or the claim 2.

22. according to claim 20 or 21 purposes, wherein said seed productive rate comprises following one or more: (full) seed number of increase, the seed weight of increase, the harvest index of increase and the TKW of increase.