CN103966254B - A kind of transcription factor that can be applicable to regulate plant trait - Google Patents

Abstract

The present invention relates to a kind of transcription factor that can be applicable to regulate plant trait, by changing the expression in plant of this transcription factor, if the significantly improvement plant type of plant, flowering time, the maturation time of results organ reality and size.The transcription factor of the present invention can be applicable to create the plant of ideal character.

Description

A kind of transcription factor that can be applicable to regulate plant trait

Technical field

The invention belongs to biotechnology and botany field；More particularly it relates to an can be applicable to The transcription factor of regulation plant trait.

Background technology

In the case of nature, spontaneous or Mutation induction the frequency of gene is low, and beneficial mutation is the most less；Further, certain The kind available germ plasm resource of plant is owing to being restricted by characteristics such as reproduction isolation, and is confined to very limited amount of In the range of, make the genetic improvement of plant be restricted to a great extent.In currently available technology, plant Genetic improvement is based on gene mutation and sexual hybridization.

Along with the development of molecular biology, people are to external source gene into cells and transformant after leading people's cell Regeneration, is explored and has been studied, established practicable genetic conversion system so that plant gene work Journey technology becomes better and approaching perfection day by day.People are applied to genetic modification of plants technique for gene engineering, solve conventional genetic Some difficult problems present in improvement, progressively develop into a kind of new genetic improvement means, change to plant genetic Good it is filled with new vitality.Developing rapidly of modern biotechnology, makes the research outstanding achievement of plant genetic engineering tire out Tired, bring new hope and dawn to the survival and development of the mankind.Although plant genetic engineering changes in heredity Application in good yet suffers from many problems, but its using value increasingly comes into one's own.Pass through plant gene Engineering, inserts exogenous genetic material, is incorporated in the genome of recipient plant so that it is plant offspring Stably heredity and expression in strain, so that recipient plant obtains new character.

Plant gene engineering technology is utilized to carry out genetic improvement, it is possible to break the reproduction isolation between species, make not It is possibly realized with heredity exchange between species, may utilize gene bank create condition for widening plant: plant gene Engineering provides the new technological means creating variation, substantially reduces the genetic modification of plants cycle: use simultaneously Gene in genetic engineering genetic improvement is studied the clearest mostly, and the plant purpose character of improvement is clear and definite, Selection approach is effective, makes initiation plant produce directed variation and be oriented and be selected to possibility: additionally by Some crucial character of improvement plant, make the Main Agronomic Characters of original kind (being) be carried to a great extent High.

The popularization of genetically modified crops will alleviate chief crop poor quality, low yield not before, pest and disease damage year by year Increase the weight of, shortage of water resources, soil degradation, salt-soda soil, Altoxicity and a large amount of ring using pesticide to cause The problems such as environment pollution, provide by force for Ensuring Food Safety, ecological safety and raising agricultural product international competitiveness Power technical support.

Along with deepening continuously of genetic engineering research, it is possible to be cloned into more new gene and transgenic technology Can be further improved, multiple genes be oriented operation and also will be possibly realized, this changes at conventional genetic It is difficult in good.But, in view of the multiformity of plant gene, this area be necessary further from All polygenes find for changing the relevant gene of plant trait, for create preferable plant plant type, Improve plant nutrient ingredient and fruit properties.

Summary of the invention

It is an object of the invention to provide a kind of transcription factor that can be applicable to regulate plant trait.

In a first aspect of the present invention, it is provided that a kind of method regulating plant trait, described method includes: adjust The expression of SlZFP2 polypeptide in joint plant.

In a preference, described method also includes subsequent step: from the expression of regulation SlZFP2 polypeptide After plant in select and compare character for check plant and obtain the plant of regulation.

In another preference, described plant is selected from: grass, plant of Solanaceae.

In another preference, described grass is selected from: Oryza sativa L., Semen Maydis, Semen Tritici aestivi, Fructus Hordei Vulgaris, swallow Wheat, rye (Secale cereale L.), Sorghum vulgare Pers..

In another preference, described plant of Solanaceae is selected from: Fructus Lycopersici esculenti, Rhizoma Solani tuber osi, Fructus Solani melongenae, Fructus Capsici, cigarette Grass, Fructus Lycii.

In another preference, described SlZFP2 polypeptide is selected from lower group: (a) such as SEQ ID NO:2 ammonia The polypeptide of base acid sequence；(b) by SEQ ID NO:2 aminoacid sequence through one or more (such as 1-20； Preferably 1-10；More preferably 1-5) amino acid residue replacement, lack or add and formed, and have The polypeptide derivative by (a) of (a) polypeptide function；Or the peptide sequence that (c) and (a) limits has more than 80% (preferably More than 85%, more preferably more than 90%, such as 95%, 98%, 99%) homology and there is (a) polypeptide function The polypeptide derivative by (a).

In another preference, described method includes: improve the expression of SlZFP2 polypeptide in plant；Thus: Promote flowering of plant time advance；Postpone fruit maturation time；Increase plant branching or the quantity of tiller； Increase carotenoid and/or content of lycopene in plant (preferably pulp class plant) fruit；Reduction is planted The height of thing plant；The weight and/or the minimizing plant seed quantity that reduce plant seed (include making plant not produce Non-hibernating eggs)；And/or promote that plant leaf blade diminishes.

In another preference, described method includes: proceeded to by the polynucleotide of coding SlZFP2 polypeptide Plant (such as cell, tissue, organ or seed), it is thus achieved that be transformed into described polynucleotide plant (as cell, Tissue, organ or seed).

In another preference, the sequence such as SEQ ID NO of the polynucleotide of described coding SlZFP2 polypeptide: 1, or the nucleotide sequence of the degeneracy of SEQ ID NO:1, or polypeptide and the SEQ ID NO:1 of coding The nucleotide variants that coded polypeptide function is identical.

In another preference, the method that the polynucleotide of coding SlZFP2 polypeptide proceed to plant is included:

(S1) Agrobacterium of expression vector is carried in offer, and described expression vector contains polynucleotide, its coding SlZFP2 albumen；

(S2) cell or tissue or the organ of plant are contacted with the Agrobacterium in step (S1), so that described Polynucleotide proceed to plant cell, tissue, organ or seed.

In another preference, described method also includes:

(S3) plant cell of polynucleotide, tissue, organ or seed described in having proceeded to are selected；With

(S4) by the plant cell in step (S3), tissue, organ or seed regeneration plant.

In another preference, described method includes:

(S1) Agrobacterium of expression vector is carried in offer, and described expression vector contains polynucleotide, its coding SlZFP2 polypeptide；

(S2) cell or tissue or the organ of plant are contacted with the Agrobacterium in step (S1), so that described Polynucleotide proceed to plant cell, tissue, organ or seed.

In another preference, described method includes: reduce the expression of SlZFP2 polypeptide in plant, thus:

Reduce plant branching or the quantity of tiller；And/or

Reduce carotenoid and/or content of lycopene in plant (preferably pulp class plant) fruit.

In another preference, in described reduction plant, the expression of SlZFP2 polypeptide includes:

By disturb described SlZFP2 polypeptide coded sequence express disturbing molecule proceed to plant cell, tissue, Organ or seed, thus lower the expression of SlZFP2 polypeptide in plant.

In another preference, in described reduction plant, the method for the expression of SlZFP2 polypeptide includes:

I () provides the Agrobacterium carrying the carrier that may interfere with gene expression, described carrier is selected from lower group:

A () contains SlZFP2 gene or the carrier of genetic fragment (antisense molecule) that opposite direction starts；

B () is containing the composition that can form specificity interference SlZFP2 gene expression (or transcribing) in plant The carrier of disturbing molecule；

(ii) cell of plant, tissue or organ are contacted with the Agrobacterium in step (i), so that described load Body proceeds to plant cell, tissue or organ.

It is preferred that described method also includes: (iii) selects and proceeded to the plant cell of described carrier, tissue Or organ；(iv) plant cell, tissue or the neomorph in step (iii) is become plant.

In another aspect of this invention, it is provided that a kind of SlZFP2 polypeptide or its encoding gene purposes, it is used for adjusting Joint plant trait.

In a preference, described SlZFP2 polypeptide or its encoding gene are used for: promote flowering of plant Time advance；Postpone fruit maturation time；Increase plant branching or the quantity of tiller；Increase plant (relatively Goodly for pulp class plant) carotenoid and/or content of lycopene in fruit；Reduce the height of plant Degree；Reduce the weight of plant seed and/or reduce plant seed quantity (including making plant not produce seed)；Promote Enter plant leaf blade to diminish；And/or the molecular marker as plant identification character.

In another preference, if after testing in plant tissue SlZFP2 expression of polypeptides higher than a particular value (such as the value of average SlZFP2 expression of wild type same plant), the most comparatively speaking, during described flowering of plant Between in advance；Fruit maturation time retardation；The quantity of branch or tiller increases；In fruit carotenoid and/ Or content of lycopene increases；The height reduction of plant；The weight of seed and/or quantity reduce or do not produce kind Son；Or plant leaf blade diminishes.If in plant tissue, SlZFP2 expression of polypeptides is less than this particular value after testing, The most comparatively speaking, the branch of described plant or the quantity of tiller reduce；Or in fruit carotenoid and/or kind Lycopene content reduces.

In another aspect of this invention, it is provided that the purposes of a kind of material reducing SlZFP2 expression of polypeptides, use In: reduce plant branching or the quantity of tiller；And/or reduce in plant (preferably pulp class plant) fruit Carotenoid and/or content of lycopene.

In another preference, the material of described reduction SlZFP2 expression of polypeptides is to disturb described SlZFP2 The disturbing molecule of gene expression.

In another aspect of this invention, it is provided that a kind of disturbing molecule disturbing described SlZFP2 gene expression, It contains the structure shown in formula (I): Seq_Forward-X-Seq_ReverselyFormula (I),

In formula (I), Seq_ForwardFor SlZFP2 genetic fragment (334-723 in preferably SEQ ID NO:1 Nucleotide sequence shown in Wei), Seq_ReverselyFor with Seq_ForwardComplementary polynucleotide；

X is for being positioned at Seq_ForwardAnd Seq_ReverselyBetween intervening sequence, and described intervening sequence and Seq_ForwardWith Seq_ReverselyThe most complementary.

In another preference, the structure shown in formula (I), after proceeding to plant cell, forms two shown in formula (II) Level structure:

Formula (II),

In formula (II), Seq_Forward、Seq_ReverselyIt is as defined above with X and states, | | represent at Seq_ForwardAnd Seq_Reversely Between the relation being substantially complementary.

In another preference, described intervening sequence itself does not constitute complementary duplex structure.

In another aspect of this invention, it is provided that a kind of carrier, described carrier contains described disturbing molecule.

The other side of the present invention due to this disclosure, be to those skilled in the art aobvious and It is clear to.

Accompanying drawing explanation

Fig. 1, over-express vector pWL007 schematic diagram.

Fig. 2, RNAi carrier pWL009 schematic diagram.

Fig. 3, SlZFP2 allelic expression.Wherein A and E is the miscellaneous of SlZFP2 justice rna probe Knot fruit, without clear signal.B-D and F-H is the results of hybridization of SlZFP2 antisense RNA probes.Scale For 200um.

Fig. 4, overexpression and expression inhibiting are in the phenotype of plant type, fruit and seed.

A-C, respectively three weeks seedlings, 45 days seedling stem freehand section and the photo of 45 days little shoot roots.

D, pWL007 transformation plant 103 ripening of fruits photo, upper row is non-transgenic reference, under Row is 103 transfer-gen plant fruits.

E-F, respectively pWL009 transformed plant seedling and comparison.

G, RNAi plant inflorescence is beared fruit difficulty.

The M82 transfer-gen plant of H, pWL007 and pWL009.

J-H, four pWL007 transformation plant fruit maturation times (from pollination to natural law during fruit annesl) With hundred seed weights (unit is milligram), NonTrans is non-transgenic reference.

The mechanism that Fig. 5, gene regulation abscisic acid (ABA) synthesize.

A: three pWL007 transgenic LA1589 strains and the pore opening of non-transgenic reference thereof.Often Individual strain takes 3-5 individual plant 50-60 pore altogether and observes measurement on ultramicroscope, and data are strain gas The means standard deviation of hole size.

B: the statistics of germination speed.Three pWL007 transgenic LA1589 strains and non-turn base Because comparison seed germinates in absorbent paper, timing statistics 0-7 days during D0-D7, repeat for 3 times, each repetition 50 seeds.Data are average germination percentage ± standard deviation.

C: overexpression SlZFP2 is (by the M82 transgenic being derived from pWL007 in ABA mutant sit By hybridization transformation in sit mutant, sit mutant is from U.S.'s Tomato Germplasms center, TGRC). Picture is the rip cutting figure of mature fruit.

D: three pWL007 transgenic lines and the ABA content of non-transgenic reference thereof.Blade, Hua He The ABA content assaying method in three periods of ripe green fruit sees list of references Fu, X., et al., J Exp Bot, 2012.63(1):p.341-54。

E:ABA synzyme and transporter gene are in pWL007 transgenic and non-transgenic reference plant leaf Expression change.NOT, SIT, SlAO1, SlAO2 and FLC are the Chief enzyme of ABA synthesis Encoding gene, SlABCG40 is the Potential Vector gene of Fructus Lycopersici esculenti ABA transhipment.Quantitative RT PCR analysis material Material is for transgenic line 103 and to blade when impinging upon 45 days, and result shows the table relative to SleIF4a gene The amount of reaching means standard deviation.

F:ABA synthase gene SIT and FLC expression change in RNAi plant.RNA extracts (bloom from the RNAi plant (different independent transformation plant) of four LA1589 and the flower of LA1589 comparison The same day).

SlZFP2 Yu ABA synthase gene SlAO1 promoter is analyzed in G: co-immunoprecipitation analysis (ChIP) Combination.Material therefor is transfer-gen plant and comparison blade, ChIP and the quantitative PCR of 30 days seedling ages (qPCR) analysis method sees list of references Ito, S., et al., Proc.Natl.Acad.Sci.U.S.A., 2012.109(9):p.3582-3587。

H: gel stops the combination of (EMSA) experimental verification SlAO1 promoter and SlZFP2.Probe from The PCR primer of ChIP experiment (G figure), is carried out with GST-SlZFP2 (total length) fusion protein of bacterial expression External EMSA tests.EMSA method sees list of references Peng, H., et al., Cancer Res, 2002. 62(13):p.3773-81。

The mechanism of Fig. 6, SlZFP2 gene regulation plant blossom.The expression shown in C, D and F in figure All make reference with Fructus Lycopersici esculenti SleIF4a expression.

The LA1589 transgenic of A:pWL007 and the nontransgenic plants photo when 45 days.

The LA1589 transgenic of B: four pWL007 and the flowering time statistics of nontransgenic plants.Open The true leaf number before occurring with first inflorescence of taking time represents, utilizes student T-test (student ' s t-test) The relatively difference between transfer-gen plant and the nontransgenic plants being isolatable from same transgenic T0 generation.Sample Number N=15.

C: Fructus Lycopersici esculenti floral genes SFT in transfer-gen plant and the expression of non-transgenic reference all ages and classes blade Change.Blade for quantitative RT-PCR takes from 45 days plant, and nontransgenic plants has 16 to see leaf, And transfer-gen plant has 12 visible leaves.

D:SFT changes in the expression of four RNAi plant.Total serum IgE is taken from pWL009 and is turned LA1589 Flower tissue (blooming the same day) in the present age (T0).

The flowering time statistics of E:ABA mutant and WT lines thereof.ABA synthesis mutant not, Sit and flc is given by TGRC.Flowering time statistical method is with Fig. 4 B.

F:SFT expression change in ABA synthesis mutant and WT lines blade thereof.Blade takes same The blade (from the 5th leaf of stem apex down number, 45 days plant) of one developmental stage.The expression of Fructus Lycopersici esculenti sft does Negative control.

The combination of SlZFP2 Yu SFT promoter is analyzed in G: co-immunoprecipitation analysis (ChIP).Material therefor is Transfer-gen plant and comparison blade, ChIP and the quantitative PCR (qPCR) of 30 days seedling ages analyze method ginseng See reference document Ito, S., et al., Proc.Natl.Acad.Sci.U.S.A., 2012.109 (9): p. 3582-3587。

H: gel stops the combination of (EMSA) experimental verification SFT promoter and SlZFP2.Probe is from ChIP The PCR primer of experiment (G figure), carries out external with GST-SlZFP2 (total length) fusion protein of bacterial expression EMSA tests.EMSA method sees list of references Peng, H., et al., Cancer Res, 2002.62 (13): p.3773-81。

The Subcellular Localization of Fig. 7, SlZFP2.

Fig. 8, transgenic Fructus Lycopersici esculenti carotenoid content change.

A: by transcriptome analysis, finds that SlZFP2 mainly expresses at Fructus Lycopersici esculenti Post flowering；

B: utilize Semiquatitative RT-PCR assay to demonstrate SlZFP2 and express to be regulated and controled by fruit development；

C: carry out sequential analysis of protein by MEME software and show that SlZFP2 polypeptide contains a C₂H₂Zinc Finger domain (domain 1) and a domain (domain 2) that Transcription inhibition may be participated in；

The conservation of amino acids analysis of D-E: two domains.

Fig. 9, overexpression SlZFP2 change tomato plant plant type and fruit maturation (T1 generation).

A: four pWL007 convert the transgenic and non-turn that the transformed plant selfing of LA1589 separates The gene plant photo when about 45 days；

B:SlZFP2 transgenic expression analysis in pWL007 converts the transformed plant blade of LA1589；

C:HA-SlZFP2 protein expression in four pWL007 convert the transformed plant of LA1589 Level.Western blot is with in HA monoclonal antibody (Sigma) detection rotaring gene plant blade HA-SlZFP2 protein level；

D-G: four pWL007 convert the transformed plant of LA1589 T1 for time plant height, branch amount, The phenotype statistics of flowering time and fruit maturation.

Each genotype observes 5 strains altogether, and fruit maturation has added up at least 15 fruits of every strain, it is illustrated that each The character means standard deviation of genotype.

Figure 10, overexpression SlZFP2 change tomato plant plant type and fruit maturation (T2 generation).

A: three SlZFP2 (pWL007) process LAN strain fruit maturation changes, DPA:days post anthesis (natural law after pollination)；

B: four SlZFP2 (pWL007) process LAN strains plant height when 45 days；

C: four SlZFP2 (pWL007) strain changes of number of branches when 45 days；

D and E: four transformation plant fruit sizes and the statistics of number seeds.In A "-" and "+" show respectively Do not contain and containing SlZFP2 transgenic fragment；

B-E:NonTrans is non-transgenic reference；

Data are from 5 strains/strain, every strain 15-20 fruit of statistics.In figure, all transgenic are homozygous strain System, all plant are LA1589 background.

Figure 11, overexpression SlZFP2 change tomato plant leaf blade size and lateral bud growth.

Figure 12, the SlZFP2 overexpressing plants (401,402 etc. represent process LAN strain) of Fructus Lycopersici esculenti M82 Phenotype.

A:SlZFP2 (pWL007) converts the transfer-gen plant of M82.401 and 402 plant of bottom and Branch photo was from 3 months plant；

B:pWL007 turns the gene expression analysis of M82 plant.The total serum IgE extracted from blade is used for semidefinite Amount RT-PCR analyzes；

C: five pWL007 turn the sectional view of M82 fruits/plant；

D: three pWL007 turn M82 plant (Lycoperdon polymorphum Vitt cylindricality) and non-transgenic reference (black surround white background cylindricality) Fruit weight compare.Nontransgenic plants is for being isolatable from transgenic T0 for self progeny.

Figure 13, SlZFP2 overexpressing plants without the HA label phenotype such as (502,503).Each little The strain numbering that digitized representation below figure is different, WT and LA1589 is non-transgenic reference.Offspring's property When shape is investigated, comparison used is the nontransgenic plants (NonTrans) separated from same transgenic T0 generation, Each strain analyzes transgenic and each 3 strains of nontransgenic plants.TrSlZFP2/eIF4a6 and EndoSlZFP2/eIF4a6 represents the transcript and eIFA4a6 transcript imported with endogenous SlZFP2 respectively Ratio, reflects the transcriptional level of this gene.The P value of display significance level is that T tests (Student ' s t-test) Calculate.

Grafting experiments between Figure 14, SlZFP2 transfer-gen plant and nontransgenic plants, 103N and 103 Represent non-transgenic and transfer-gen plant respectively.Grafting is to carry out, above horizontal line between plant at about 3 weeks For scion, lower section is stock.

Figure 15, RNAi interference carrier pWL009 has been directed respectively into Fructus Lycopersici esculenti LA1589 and M82.In figure 206, 208,209 and 210 strains be pWL009 convert LA1589 RNAi transformed plant, 501,509, 510 and 512 convert the RNAi transformed plant of M82 for pWL009.Each numbering represent one independent Transformation plant.

Figure 16, overexpression SlZFP2 affect Stoma of Leaves and the seed germination sensitivity to ABA.

The pore of LA1589 plant (102,103, the 105) blade of A: three SlZFP2 transgenic compares；

B: different ABA process the blade impact on stomatal movement；

The C:ABA impact on three SlZFP2 transgenic LA1589 germinations.

Figure 17, in Fructus Lycopersici esculenti LA1589 overexpression SlZFP2 gene pairs floral genes SFT spending, send out Educate the expression impact of fruit (after pollination 5 and 10 days) and ripe green fruit.The black cylindricality of the most special mark in figure Figure is the transfer-gen plant that SlZFP2 turns LA1589, white bar diagram non-transgenic reference.Dpa, awards Natural law (day post anthessis) after powder.LA0534 and LA0535 is not and sit, flc mutant respectively Wild type control (NIL).

Figure 18, SlZFP2 overexpression in Oryza sativa L. causes plant tillering number to increase.Wherein, CK is not Spending 11 in Oryza sativa L. containing SlZFP2 transgenic, NO1-NO11 is for turning SlZFP2 trans-genetic hybrid rice.

Figure 19, the impact of ABA content, material during in Fructus Lycopersici esculenti LA1589, overexpression SlZFP2 is on blade Material turns the homozygous transgenic plant of LA1589 for pWL007, during sampling nontransgenic plants have 17 visible Leaf, transfer-gen plant has 12 visible leaves, is after planting 45 days plant.

Figure 20, by each ABA synthase gene of qRT-PCR quantitative analysis at the SlZFP2 of LA1589 Expression in rotaring gene plant blade.

Figure 21, ABA synthesize, transport and respond related gene at several RNAi (pWL009) transformation plant The expression of flower, WT is non-transgenic LA1589 comparison.Material therefor and qRT-PCR analyze and figure 5F is the same, is the experiment of same batch.

Detailed description of the invention

The present inventor, through in-depth study, finds by changing SlZFP2 expression in plant, can With the plant type (side shoot number, plant height) of notable improvement plant if, flowering time, the maturation of results organ reality Time and size.Therefore the method for the present invention can be applicable to create desirable plants plant type, obtain high hycopene, The new variety of plant of the nutrient substance such as high beta-carotene, has on the genetic improvement of plant products and quality Good application prospect.

As used herein, the plant (or crop) of the present invention is the plant of the conversion operation being appropriate to gene, as Various crops, flower plant or forestry plant etc..Described plant can be such as (being not limited to): dicotyledonous Plant, monocotyledon or gymnosperm.More specifically, described plant includes, but is not limited to: Fructus Lycopersici esculenti, Capsicum annuum L., American Avocado Tree, Cortex cinnamomi japonici (Ramulus Cinnamomi), Camphora, Nicotiana tabacum L., Semen Tritici aestivi, Fructus Hordei Vulgaris, rye (Secale cereale L.), Oryza sativa L., Semen Maydis, Sorghum vulgare Pers., Radix Betae, Fructus Mali pumilae, pears, Lee, Fructus Persicae, Fructus Pruni, Fructus Pruni pseudocerasi, Fructus Fragariae Ananssae, rasp berry, blackberry, bean, Seem Lablab Album, Semen Pisi sativi, Semen sojae atricolor, Brassica campestris L, Mustard, Semen Papaveris, olive, Helianthi, Cortex cocois radicis, castor oil plant, cacao bean, Semen arachidis hypogaeae, calabash, Fructus Cucumidis sativi, west Melon, Cotton Gossypii, Caulis et Folium Lini, Fructus Cannabis, Corchorus olitorius L., mandarin orange, Fructus Citri Limoniae, grapefruit, Herba Spinaciae, piemarker lettuce, Germinatus Phragmitis, cabbage, Chinese cabbage, Plantula Brassicae chinensis, Radix Dauci Sativae, Bulbus Allii Cepae, Rhizoma Solani tuber osi, nut, coffee, Fructus Solani melongenae, Caulis Sacchari sinensis, Folium Camelliae sinensis, Fructus Piperis, Vine, oyster fiber crops grass, Fructus Musae, natural rubber tree and ornamental plant etc..

As a kind of optimal way, described " plant " includes but not limited to: grass, Solanaceae are planted Thing etc..Such as, described " plant " includes but not limited to: Oryza sativa L., Semen Tritici aestivi, Semen Maydis, Fructus Lycopersici esculenti, Ma Ling Potato, Fructus Solani melongenae, Fructus Capsici, Nicotiana tabacum L. etc..

As used herein, selection suitably " check plant " is the customary part of experimental design, " check plant " Corresponding wild-type plant or the corresponding plant without genes of interest can be included.Check plant is usually identical plant Species or even identical with plant to be assessed kind.Check plant can also be to lose transgenic because of separation to plant The individuality of thing.Check plant refers not only to full plants as used herein, also refers to plant part, including seed and kind Subdivision.

Present invention additionally comprises the fragment of SlZFP2 polypeptide, derivant and analog.As used herein, term " sheet Section ", " derivant " refer to be kept substantially the biology that the SlZFP2 polypeptide of the present invention is identical with " analog " Learn function or the polypeptide of activity.It is one or more that the polypeptide fragment of the present invention, derivant or the like can be that (i) has The polypeptide that conservative or non-conservative amino acid residue (preferably conservative amino acid) is replaced, and such replacement Amino acid residue can may not be and encoded by genetic code, or (ii) is residual at one or more aminoacid Base has the polypeptide of substituted radical, or (iii) mature polypeptide (such as extends the polypeptide half-life with another compound Compound, such as Polyethylene Glycol) merge the polypeptide that formed, or (iv) additional aminoacid sequence is fused to this polypeptide Sequence and the polypeptide that formed are (such as targeting sequencing or secretion sequence or the sequence being used for this polypeptide of purification or proteinogen sequence Row, or fusion protein).The skilled skill in this area is belonged to according to these fragments of definition, derivant and analog herein Scope known to art personnel.It is preferred that SlZFP2 polypeptide of the present invention includes a kind of fusion protein, it is The fusion protein that HA label is connected and composed with SlZFP2；This fusion protein has stable in plant The effect of SlZFP2 polypeptide function.

The bioactive fragment of any SlZFP2 polypeptide can be applied in the present invention.Here, The bioactive fragment of SlZFP2 polypeptide is meant that referring to as is a peptide species, and it still can keep total length All or part of function of SlZFP2 polypeptide.Under normal circumstances, described bioactive fragment at least keeps The activity of the total length SlZFP2 polypeptide of 50%.Under still more preferential conditions, described active fragment can keep complete The activity of 60%, 70%, 80%, 90%, 95%, 99% or the 100% of long SlZFP2 polypeptide.

In the present invention, term " SlZFP2 polypeptide " refers to the SEQ ID NO with SlZFP2 polypeptide active: The polypeptide of 2 sequences.This term also includes having and SlZFP2 polypeptide identical function, SEQ ID NO:2 The variant form of sequence.These variant forms include (but being not limited to): several (usually 1-50, relatively Good ground 1-30, more preferably 1-20, most preferably 1-10, the most more preferably such as 1-8,1-5) amino The disappearance of acid, insert and/or replace, and C-terminal and/or N-terminal add one or several (usually Within 20, within preferably 10, within being more preferably 5) aminoacid.Such as, in this area In, when replacing with similar nature or similar aminoacid, generally will not change the function of protein.Again Such as, one is added or several aminoacid generally also will not change protein at C-terminal and/or N-terminal Function.This term also includes active fragment and the reactive derivative of SlZFP2 polypeptide.

The variant form of polypeptide includes: homologous sequence, conservative variant, allelic variant, natural mutation Body, induced mutants, under the conditions of high or low stringency can with SlZFP2 polypeptid DNA hybridization DNA Coded albumen and utilize the polypeptide or albumen that the antiserum of anti-SlZFP2 polypeptide obtains.The present invention Additionally provide other polypeptide, as comprised the fusion protein of SlZFP2 polypeptide or its fragment.

High (such as same with the sequence shown in SEQ ID NO:2 of any with described SlZFP2 peptides homologous Source property is 70% or higher；Preferably, homology is 80% or higher；It is furthermore preferred that homology is 90% Or higher, such as homology 95%, 98% or 99%) and the albumen with SlZFP2 polypeptide identical function It is also included in the present invention.

Invention also provides for the analog of SlZFP2 polypeptide or polypeptide.These analog and natural SlZFP2 polypeptide Difference can be the difference on aminoacid sequence, it is also possible to be not affect the difference on the modified forms of sequence Different, or have both at the same time.These polypeptide include the natural or genetic variant of induction.Induction variant is permissible Obtained by various technology, as produced random mutagenesis by radiating or be exposed to mutagenic agent, also by fixed Point mutagenesis or the biological technology of other known moleculars.Analog also includes having and is different from natural L-amino The analog of residue (such as D-aminoacid) of acid, and there is non-naturally-occurring or synthesis aminoacid (as β, gamma-amino acid) analog.Should be understood that the polypeptide of the present invention is not limited to the above-mentioned representativeness enumerated Polypeptide.

Although should be understood that the SlZFP2 polypeptide of the present invention is preferably obtained from plant of Solanaceae (such as Fructus Lycopersici esculenti), but available from Other plant with Fructus Lycopersici esculenti SlZFP2 polypeptide very high homology (such as have more than 80%, such as 85%, 90%, 95%, Even 98% sequence thereto) other polypeptide also within the scope of the present invention contemplates.Aligned sequences homogeny Method and kit for be also well known in the art, such as BLAST.

The invention still further relates to code book invention SlZFP2 polypeptide or the polynucleotide sequence of its conservative variation's polypeptide Row.Described polynucleotide can be DNA form or rna form.DNA form includes cDNA, base Because of group DNA or the DNA of synthetic.DNA can be strand or double-strand.DNA can be Coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be with the volume shown in SEQ ID NO:1 Region sequence is identical or the variant of degeneracy for code.As used herein, " variant of degeneracy " is in the present invention In refer to encode the protein with SEQ ID NO:2, but with the coding region sequence shown in SEQ ID NO:1 Arrange differentiated nucleotide sequence.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 include: the code sequence of an encoding mature polypeptide Row；The coded sequence of mature polypeptide and various additional coding sequence；The coded sequence of mature polypeptide (with optionally Additional coding sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " can be the polynucleotide including coding said polypeptide, it is possible to To be the polynucleotide also including additional code and/or non-coding sequence.

The invention still further relates to the variant of above-mentioned polynucleotide, its coding has identical aminoacid sequence with the present invention The polypeptide of row or the fragment of polypeptide, sum analogous to general Dedekind sum.The variant of these polynucleotide can be natural The variant that raw allelic variant or non-natural occur.These nucleotide variants include replace variant, Deletion variants and insertion variant.As known in the art, allelic variant is replacing of polynucleotide Changing form, it is probably the replacement of one or more nucleotide, lacks or insert, but will not be from substantially changing Become the function of the polypeptide of its coding.

The invention still further relates to have at least 50%, the most extremely between above-mentioned sequence hybridization and two sequences Few 70%, the polynucleotide of more preferably at least 80% homogeny.The present invention be more particularly directed under strict conditions with The interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " refers to: (1) Hybridization under relatively low ionic strength and higher temperature and eluting, such as 0.2 × SSC, 0.1%SDS, 60 DEG C； Or added with denaturant during (2) hybridization, such as 50% (v/v) Methanamide, 0.1% calf serum/0.1%Ficoll, 42 DEG C Deng；Or (3) only homogeny between two sequences, at least more than 90%, just sends out when more preferably more than 95% Raw hybridization.Further, the polypeptide of interfertile polynucleotide encoding and the mature polypeptide shown in SEQ ID NO:2 There are identical biological function and activity.

The invention still further relates to and the nucleic acid fragment of above-mentioned sequence hybridization.As used herein, " nucleic acid fragment " Length at least containing 15 nucleotide, preferably at least 30 nucleotide, more preferably at least 50 nucleoside More than acid, preferably at least 100 nucleotide.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid To determine and/or to separate the polynucleotide of coding SlZFP2 polypeptide.

The nucleotide full length sequence of the SlZFP2 gene of the present invention or its fragment generally can use PCR TRAP, The method of recombination method or synthetic obtains.For PCR TRAP, can be according to disclosed in this invention relevant Nucleotide sequence, especially open reading frame sequence design primer, and with commercially available cDNA storehouse or by this CDNA storehouse prepared by conventional method known to the skilled person, as template, expands and obtains relevant sequence.

The present invention also relates to comprise the carrier of described polynucleotide, and with described carrier or SlZFP2 The host cell that gene order produces through genetic engineering.

In the present invention, SlZFP2 polypeptide polynucleotide sequence can be plugged in recombinant expression carrier, as long as can be Replicating in host and stable, any plasmid and carrier can be used.One key character of expression vector is Usually contain origin of replication, promoter, marker gene and translation and control element.

Comprise above-mentioned suitable DNA sequence and suitable promoter or control the carrier of sequence, Ke Yiyong In converting suitable host cell, allow it to marking protein.Host cell can be prokaryotic cell, Such as bacterial cell；Or the eukaryotic cell such as low, such as yeast cells；Or higher eucaryotic cells, as plant is thin Born of the same parents.Representative example has: escherichia coli, streptomyces, Agrobacterium；Fungal cell's such as yeast；Plant is thin Born of the same parents etc..

When described polynucleotide are expressed in higher eucaryotic cells, if inserting enhancer sequence in the carrier Time will make to transcribe to be strengthened.Enhancer is the cis-acting factors of DNA, generally about has 10 to 300 Individual base pair, acts on promoter transcribing with enhancing gene.How persons skilled in the art are aware that Select suitable carrier, promoter, enhancer and host cell.

Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.Turn Change plant and can use the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as spraying, leaf disk method, Oryza sativa L. children Embryo conversion method etc..Plant cell, tissue or organ for converting can use conventional method regeneration plant, Thus the plant that acquired character changes.

In a particular embodiment of the present invention, overexpression SlZFP2 polypeptide in plant of Solanaceae Fructus Lycopersici esculenti, can To dramatically speed up the plant blossom time, when during to bloom, the lobe numbers of plant is for index, overexpressing plants Early 3-5 sheet leaf is bloomed, and fruit maturation is postponed 5-7 days, and plant bud dormancy weakens, branch increases, and plant is slightly Having dwarfing, ABA content in plant leaf, flower and fruit reduces, and lycopene in fruit and β-recklessly Radix Raphani cellulose content increases, and Grain Dormancy decreases.But suppress this transcription factor endogenous table in Fructus Lycopersici esculenti Reaching, plant does not has significant change with WT lines in the trophophase, but side shoot reduces, and bears fruit and is suppressed, Easily shedding.

Vivo immunization is co-precipitated it is demonstrated experimentally that SlZFP2 polypeptide can be combined in multiple ABA synthase gene With in the promoter of floral genes SFT, external EMSA experiment also confirms its specificity combined.One is It is a regulation and control ABA synzyme base that row experiment demonstrates this transcription factor from the many aspects such as hereditary, biochemical Because of the newcomer expressed, directly participate in regulation and control flowering of plant approach.

Therefore, the invention provides described SlZFP2 polypeptide or the purposes of its encoding gene, be used for regulating The character of plant；Or for the regulation useful material of plant trait, (that is: described material is by adjusting for screening The expression of joint SlZFP2 polypeptide regulates plant trait).As a kind of optimal way, described SlZFP2 Polypeptide can be used for: promotes flowering of plant time advance；Postpone fruit maturation time；Increase plant branching Or the quantity of tiller；Increase carotenoid and/or tomato red in plant (preferably pulp class plant) fruit Cellulose content；Reduce the height of plant；Reduce the weight of plant seed and/or reduce plant seed quantity (bag Include and make plant not produce seed)；And/or promote that plant leaf blade diminishes.

Agonist or antagonist of the invention still further relates to SlZFP2 and application thereof.Agonist due to SlZFP2 Or the expression of antagonist scalable SlZFP2 and/or regulation SlZFP2 activity etc., therefore, described The agonist of SlZFP2 or antagonist also by the impact of SlZFP2 is regulated plant trait, thus can reach Purpose to improvement plant.

Any improve SlZFP2 polypeptide activity, the stability improving SlZFP2 polypeptide, promote SlZFP2 The transcription and translation of the expression of polypeptide, prolongation SlZFP2 polypeptide effective acting time or promotion SlZFP2 Material is used equally to the present invention, as can be used for regulating plant trait, particularly promotes the flowering of plant time to carry Before；Postpone fruit maturation time；Increase plant branching or the quantity of tiller；Increase plant (preferably Pulp class plant) carotenoid and/or content of lycopene in fruit；Reduce the height of plant；Fall The weight of low plant seed and/or minimizing plant seed quantity (including making plant not produce seed)；And/or promote Plant leaf blade diminishes.Any reduce SlZFP2 polypeptide activity, the stability reducing SlZFP2 polypeptide, The expression of suppression SlZFP2 polypeptide, minimizing SlZFP2 polypeptide effective acting time or reduction SlZFP2's The material of transcription and translation is used equally to the present invention, as lower adjustment, antagonist or the inhibitor of SlZFP2 (that is: lowering the material of SlZFP2 expression of polypeptides), the antibody of SlZFP2 polypeptide as described in anti-, interference is described The disturbing molecule (as formed the disturbing molecule of microRNA) that the encoding gene of SlZFP2 polypeptide is expressed.Institute Lower adjustment, antagonist or the inhibitor stated can be used for regulating plant trait, particularly reduces plant branching or divides The quantity of tiller；And/or reduce carotenoid and/or tomato red in plant (preferably pulp class plant) fruit Cellulose content.After knowing target sequence, the method for the disturbing molecule that preparation interference specific gene is expressed is ability Known to the personnel of territory.

The invention still further relates to a kind of method improveing plant, the method includes regulating SlZFP2 in described plant The expression of polypeptide.

On the one hand, the invention provides a kind of method regulating plant trait, described method includes: make institute State plant overexpression SlZFP2 polypeptide, thus: promote flowering of plant time advance；Postpone fruit Maturation time；Increase plant branching or the quantity of tiller；Increase plant (preferably pulp class plant) fruit Middle carotenoid and/or content of lycopene；Reduce the height of plant；Reduce the weight of plant seed And/or reduce plant seed quantity (including making plant not produce seed)；And/or promote that plant leaf blade diminishes.

On the other hand, present invention also offers a kind of method regulating plant trait, described method includes:: Reduce the expression (including making SlZFP2 polypeptide not express or low expression) of SlZFP2 polypeptide in described plant, from And: reduce plant branching or the quantity of tiller；And/or reduce in plant (preferably pulp class plant) fruit Carotenoid and/or content of lycopene.

After the purposes knowing described SlZFP2 polypeptide, can use well known in the art multiple Method regulates the expression of described SlZFP2 polypeptide.Will than such as by approach known to those skilled in the art The ceneme (such as expression vector or virus etc.) carrying SlZFP2 gene is delivered on target spot, and is allowed to table Reach the SlZFP2 polypeptide of activity.

In addition it is also possible to use multiple method well known in the art to reduce the expression of SlZFP2 polypeptide Or it is allowed to loss of expression, such as by ceneme (the such as expression vector or disease of carrying antisense SlZFP2 gene Poison etc.) it is delivered on target spot so that cell or plant tissue are not expressed or reduce expression SlZFP2 polypeptide.

As one embodiment of the present invention, by the gene of coding SlZFP2 polypeptide by conventional method It is cloned in suitable carrier, imports to the described recombinant vector with exogenous gene to express described In the plant cell of SlZFP2 polypeptide, described plant cell is made to express SlZFP2 polypeptide.Can be by by institute State Plant cell regeneration and become plant, it is thus achieved that the plant of overexpression SlZFP2 polypeptide.

Preferably, it is provided that a kind of method preparing transgenic plant, including:

(1) encoding gene of the SlZFP2 polypeptide of external source is proceeded to plant cell, tissue, organ or tissue, Obtain the plant cell of encoding gene, tissue, organ or the seed being transformed into SlZFP2 polypeptide；With

(2) plant cell of the encoding gene having proceeded to external source SlZFP2 polypeptide that step (1) obtained, group Knit, organ or seed regeneration plant.

As the preferred example of one, described method includes step:

(s1) Agrobacterium of expression vector is carried in offer, and described expression vector contains the volume of SlZFP2 polypeptide Code gene；

(s2) plant cell, tissue, organ are contacted with the Agrobacterium in step (s1), so that SlZFP2 The encoding gene of polypeptide proceeds to plant cell, and is incorporated on the chromosome of plant cell；

(s3) plant cell of the encoding gene proceeding to SlZFP2 polypeptide, tissue, organ or seed are selected； And

(s4) by the plant cell in step (s3), tissue, organ or seed regeneration plant.

Other method increasing SlZFP2 gene or the expression of its homologous genes is well known in the art.Such as, SlZFP2 gene or its homogenic expression can be strengthened by driving with strong promoter.Or pass through Enhancer (such as Oryza sativa L. waxy gene First Intron, Actin gene First Intron etc.) strengthens this The expression of SlZFP2 gene.The strong promoter being applicable to the inventive method includes but not limited to: 35S promoter, Oryza sativa L., the Ubi promoter etc. of Semen Maydis.

Preferably, additionally provide and a kind of reduce the method for the expression of SlZFP2 polypeptide in plant, described side Method includes:

(1) disturbing molecule of interference SlZFP2 gene expression is proceeded to plant cell, tissue, organ or seed, Obtain and be transformed into the plant cell of described disturbing molecule, tissue, organ or seed；With

(2) plant cell of described disturbing molecule, tissue, organ or seed have been proceeded to by what step (1) obtained Regeneration plant.

As the preferred example of one, described method includes step:

I () provides the Agrobacterium carrying the carrier that may interfere with gene expression, described carrier is selected from lower group:

The encoding gene of a SlZFP2 polypeptide that () starts containing opposite direction or the load of genetic fragment (antisense molecule) Body；

(b) containing can be formed in plant specificity interference SlZFP2 polypeptide encoding gene express (or turn Record) the carrier of disturbing molecule of composition；

(ii) cell of plant, tissue or organ are contacted with the Agrobacterium in step (i), so that described load Body proceeds to plant cell, tissue or organ.

It is preferred that described method also includes:

(iii) select and proceeded to the plant cell of described carrier, tissue or organ；With

(iv) plant cell, tissue or the neomorph in step (iii) is become plant.

Nucleotide sequence based on SlZFP2 gene, can be designed that import can be formed after plant special Property interference SlZFP2 gene expression the polynucleotide of molecule.Specificity to be considered and interference during design Efficiency.The preparation method of disturbing molecule is had no particular limits by the present invention, includes but not limited to: chemistry Synthetic method, in vitro transcription method etc..Should be understood that those skilled in the art are knowing SlZFP2 gene and planting After the dependency of physical property shape, described disturbing molecule can be prepared with various approach, thus be used for regulating Plant trait.Described disturbing molecule can be transported in plant by transgenic technology, or also can use Multiple technologies known in the art are transported in plant.

Particularly preferred mode as the present invention, it is provided that the disturbing molecule of a kind of excellent effect, described Disturbing molecule can the expression of specific interference SlZFP2 gene；And empirical tests, it has good doing Disturb the effect of SlZFP2 gene expression.Described disturbing molecule is containing 334-723 in SEQ ID NO:1 The molecule of the nucleotide sequence shown in Wei.Can be formed after it is transferred in plant and there is SEQ ID NO: The disturbing molecule of nucleotide sequence shown in 1.

Present invention also offers a kind of disturbing molecule, described disturbing molecule contains following structure: Seq_ForwardFor SlZFP2 genetic fragment (nucleotide sequence shown in 334-723 position in preferably SEQ ID NO:1), Seq_ReverselyFor with Seq_ForwardComplementary polynucleotide；X is for being positioned at Seq_ForwardAnd Seq_ReverselyBetween intervening sequence, And described intervening sequence and Seq_ForwardAnd Seq_ReverselyThe most complementary.

Described disturbing molecule, after importing in plant, can be folded into stable loop-stem structure, loop-stem structure Stem both sides comprise the two sequences being substantially complementary.That is, form secondary structure as follows:

Wherein, ‖ represents at Seq_ForwardAnd Seq_ReverselyBetween the relation being substantially complementary.Described stem ring knot Structure can be acted on by the various materials in plant, process or shear further, and forms double-strand RNA(dsRNA)。

Generally, described disturbing molecule is positioned on expression vector.

Present invention additionally comprises the plant utilizing any one method aforementioned to obtain, described plant includes: proceed to SlZFP2 gene or its homogenic transgenic plant；Or SlZFP2 expression of polypeptides amount (includes low table Reach or do not express) plant etc. that reduces.

Any suitable conventional means can be used, implement described including reagent, temperature, pressure condition etc. Method.

Moreover, it relates to utilize SlZFP2 polypeptide or its encoding gene to plant as a kind of gene transformation The tracking labelling of strain offspring.The invention still further relates to utilize SlZFP2 polypeptide or its encoding gene to divide as one Sub-labelling, by the expression of SlZFP2 polypeptide, the character of plant identification in detection plant.Also can profit With the plant characteristic of SlZFP2 gene-correlation as the cue mark of hybrid true during hybrid seeding.

Additionally, the method that the present invention also provides for screening the potential material of scalable plant trait.Knowing After the purposes of the SlZFP2 polypeptide stated, multiple method well known in the art can be used to screen regulation plant The potential material of character.In a kind of optimal way of the present invention, it is provided that a kind of screening regulates plant trait The method of potential material, described method includes: candidate substances and the system expressing SlZFP2 polypeptide are connect Touch, detect the candidate substances impact on SlZFP2 polypeptide；If described candidate substances can improve or reduce SlZFP2 The expression of polypeptide, or promote or the activity of suppression SlZFP2 polypeptide, then show that it is to can be used for regulating plant Character.The material that these Preliminary screening go out may make up a screening storehouse, in order to people may finally therefrom sieve Selecting can be for the regulation useful material of plant trait.

The growth cycle of crop is decision crop yield, plants adaptive particularly important factor, opening of crop Take time and controlled, by when blooming of genetic improvement crop by multiple environmental factors and inherent genetic factor Between, agricultural production has important economic worth.The transcription factor SlZFP2 effect that present invention discover that In flowering time control gene conservative in each species, therefore utilizing when blooming of genetic improvement crop It is likely to be of universality between, can apply to Different Crop.Transcription factor involved in the present invention is utilized also may be used To be effectively increased or to reduce the branch amount of plant, it is applied to the Plant-type Breeding of crop, it is thus achieved that ideotype.

Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are only used for The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example Method, generally writes according to normal condition such as J. Pehanorm Brooker etc., Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.Unless additionally said Bright, otherwise percentage ratio and number are calculated by weight.

Embodiment 1, the separation of gene, vector construction and transgenic method

Gene segregation and vector construction

SlZFP2 Gene segregation is from tomato breeding lines LA1589 (Solanum pimpinefolium).Utilize Trizol The total serum IgE of extraction synthesizes cDNA through reverse transcriptase, first uses primer XP0034: 5 '-ATGTTCCAGATTACGCTCTCGAGATGAGTTATGAACCAAACACGG-3 ' and XP0036:CGGGATCCAACATAATCGCCCTTGAGTAGA amplifies SlZFP2 code sequence Row, are cloned into pMD18-T carrier (purchased from Takara company), the most again with XP0035: GCCACCATGGTTTACCCATACGATGTTCCAGATTACGCTCTCGAG and XP0036 expands target fragment from the above-mentioned plasmid errorless through order-checking, and PCR primer is connected to PMD18-T carrier.Primer XP0035 sequence label Han HA, is used for building HA-SlZFP2 fusion protein. Order-checking determine errorless after, utilize restricted enzyme BamHI to cut out HA-SlZFP2 fragment, and be connected to Same with on the Agrobacterium binary vector pHX20 of BamHI enzyme action, pHX20 is adapted from pZH01 (pZH01 It is adapted from pCAMBIA1300, refers to document Xiao, H., et al., Functional analysis of the rice AP3homologue OsMADS16by RNA interference.Plant Mol Biol, 2003.52 (5): p. 957-66).PHX20 is by reclaiming containing carrier framework after restricted enzyme SalI digestion pZH01 Large fragment, carries out carrier with T4DNA ligase and forms from connecting, and this carrier eliminates cauliflower mosaic virus GUS bonding pad between CaMV35S promoter and Agrobacterium tumefaciens opaline synzyme NOS terminator. Obtaining the process LAN i.e. p35S:HA-SlZFP2 of plasmid pWL007, wherein HA-SlZFP2 is connected to composing type Promoter CaMV 35S downstream, the upstream of NOS terminator.Plasmid imports to Agrobacterium by heat shock method GV3101 bacterial strain, for Plant Transformation.

Over-express vector pWL007 schematic diagram such as Fig. 1, wherein, p35S, cauliflower mosaic virus CaMV35S Promoter；HA, is derived from influenza virus hemagglutinin surface antigen (Human influenza hemagglutinin) Protein tag, aminoacid sequence is YPYDVPDYA, and sequence is synthetic；Nos-t, Agrobacterium tumefaciems Rouge alkali synthetase NOS terminator.In addition to HA-SlZFP2, other DNA sequence is from plasmid backbone pHX20.PHX20 contains hygromycin gene Hyg that 35S drives^R(the choosing when transgenic screens Select labelling) and bacterial selectable markers kalamycin resistance gene Kan^R。

The structure of RNAi interference carrier pWL009 is (purchased from intending south with pFGC5941RNAi binary vector Mustard resource center ABRC) it is skeleton, by SlZFP2 sequence fragment (334-723 position in SEQ ID NO:1) The AscI/SwaI site (forward connection) that is connected on pFGC5941 in forward and reverse mode respectively and BamHI/XbaI site (Opposite direction connection), this plasmid information and detailed cloning process see document Kerschen, A., et al., Effectiveness of RNA interference intransgenicplants.FEBS Lett, 2004. The SlZFP2 fragment of 566 (1-3): p.223-8, this 401bp is to utilize primer XP0028: GCTCTAGAGGCGCGCCGAGTTGAGTTCAACCCTACG and XP0029: CGCGGATCCATTTAAATAATCGCCCTTGAGTAGAATC passes through PCR from LA1589 CDNA expands, and underlined sequences is the artificial restriction enzyme enzyme recognition site added, for carrier Build.Plasmid imports to Agrobacterium GV3101 bacterial strain by heat shock method, for Plant Transformation.

RNAi carrier pWL009 schematic diagram is as shown in Figure 2.PWL009 is based on RNAi interference carrier PFGC5941 builds.PFGC5941 contains selectable plant marker Bar gene and antibacterial selects mark Note Kan^RGene.CHSA Intron, petunia chalcone synthase A gene (chalcone synthase A) Intron.OCS3 ', 3 ' ploy A districts of octopine synthase gene (OCS).

RT-PCR primer such as table 1.

Table 1, RT-PCR primer

Plant Transformation

Utilize the Agrobacterium-medialed transformation method of cotyledon, respectively pWL007 and pWL009 is imported to Fructus Lycopersici esculenti Strain LA1589 (the nearly source wild species of cultivated tomato, available from U.S.'s Tomato Germplasms center TGRC) and M82 (processing type Fructus Lycopersici esculenti, available from Israel Hebrew University ofJerusalem).PWL007 also utilizes Agrobacterium-mediated transformation proceeds to spend in rice varieties 11.Fructus Lycopersici esculenti Agrobacterium-mediated Transformation sees McCormick, S., Transformation of tomato with Agrobacterium tumefaciens.In Plant Tissue Culture Manual, Lindsey, K., ed. (Kluwer Academic Publishers, Dordrecht, The Netherlands, 1991) .B6:p.1-9 method is carried out, and rice conversion method uses Hiei, Y., et al., Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and Sequence analysis of the boundaries of the T-DNA.Plant J, 1994.6 (2): p.271-82 side Method.PWL007 transfer-gen plant is screened by HYG, and pWL009 utilizes glyphosate (PPT) to screen.

Embodiment 2, SlZFP2 allelic expression and Subcellular Localization

1, allelic expression

Utilize the DNA sequence of SlZFP2, Fructus Lycopersici esculenti Wild related germplasm LA1589 plant is carried out in situ hybridization Detection SlZFP2 expression in different tissues.Method sees reference document Coen, E.S., et al., Cell, 1990. 63(6):p.1311-22。

Result such as Fig. 3.Show that SlZFP2 is mainly at spire (A), lateral bud (B), flower primordium (D), flower pesticide and ovule (F), seed (G and H) is expressed.

2, the Subcellular Localization of SlZFP2

Fluorescence protein gene YFP is to pass through from Clontech company, the structure of control vector p35S:YFP PCR amplification YFP gene is cloned on pHX20, p35S:SlZFP2-YFP fusion expression vector be by The complete encoding sequence of SlZFP2 is connected to YFP on p35S:YFP through NotI enzyme action after utilizing PCR amplification The corresponding site of Sequences upstream, forms fusion protein.P35S:SlZFP2-YFP and p35S:YFP is directed respectively into agriculture Bacillus strain GV3101.

Result such as Fig. 7.Diagram contains the Agrobacterium of the two carrier respectively and infects Ben Shi Nicotiana tabacum L. N.benthamiana Blade expression after 3 days, confocal laser scanning microscope is positioned at cell to SlZFP2-YFP signal In core, illustrate that SlZFP2 encodes a nuclear locating sequence.Detailed agroinfection and fluorescence-detecting step See document Tsai, C.W., et al., J Virol, 2005.79 (9): p.5304-14.

Embodiment 3, overexpression and expression inhibiting are in the phenotype of Fructus Lycopersici esculenti plant type, fruit and seed

1, the character mutation analysis of Tomato Root System, fruit

As embodiment 1 prepares the Fructus Lycopersici esculenti of transgenic, observe SlZFP2 overexpression and suppress expression in plant type, really The real phenotype with seed.Such as Fig. 4, except H is from addition to M82 transgenic, and remaining is all from LA1589 in figure Transgenic line.OE represents the plant of the overexpression SlZFP2 being transformed by plasmid pWL007, RNAi The SlZFP2 expression inhibiting plant come for pWL009.#102,103,104 and 105 are respectively four independences Convert the plant in source, after isozygotying, be referred to as strain.Each strain has added up the maturation of 15-20 fruit in 5 strains Seed weight in change and 10 fruits, data are 5 strain mean+SD.Result shows, overexpression SlZFP2 causes root system to reduce, and plant branch increases, and stem stalk attenuates, and fruit maturation is postponed, and seed lightens；And Then affect plant by the expression of RNAi interference SlZFP2 to bear fruit.

2, transgenic Fructus Lycopersici esculenti (converting LA1589) carotenoid content change

By transcriptome analysis, find that SlZFP2 mainly expresses (Fig. 8 A) at Fructus Lycopersici esculenti Post flowering, further profit Demonstrate SlZFP2 with Semiquatitative RT-PCR assay to express by fruit development regulation and control (Fig. 8 B).Pass through MEME Software carries out sequential analysis of protein and shows that SlZFP2 contains a C₂H₂Zinc finger domain (domain in Fig. 8 C 1) and one may participate in Transcription inhibition domain (domain 2 in Fig. 8 C), Fig. 8 D and E show this two The conservation of amino acids of individual domain.Root in Fig. 8 B figure, cotyledon, lower ovule take from 7-10 days LA1589 Seedling, before blooming, the fruit in petal, flower and each period takes from the plant of about 2 months.In Fig. 8 C figure The first two letter of each species ZFP2 protein name represent the first letter of this species latin name.MEME Analysis sees reference document Bailey, T.L., et al., Nucl.Acids Res., 2006.34 (suppl2): p. W369-W373。

The carotenoid content of SlZFP2 Transgenic tomato fruit changes such as table 2.

Table 2

The mensuration of carotene metabolite sees list of references Fu, X., et al., J Exp Bot, 2012.63 (1): p.341-54.Mature Green and Turning represents fruit and is in ripe green fruit phase and colour-change period.103N It is pWL007 overexpression and the non-transgenic reference of pWL009RNAi transgenic line respectively with 208N (NonTrans), each of which separates from same transgenic T0 plant selfing, as compareing them and turning Gene plant is compared has same genetic background, because experiencing identical tissue culture procedures.

Result shows: overexpression SlZFP2 increases carotenoid and content of lycopene, and suppresses SlZFP2 expresses and then reduces carotenoid and content of lycopene, and illustrating can be by regulation and control SlZFP2's Expression improves the carotenoid content of material such as the lycopene of fruit.

3, T1 is for Transgenic Tomato Plants plant type (converting LA1589) and fruit maturation

Observe pWL007 and convert the T1 that the transformed plant (102,103,104,105) of LA1589 is separated For transgenic and nontransgenic plants (NonTrans)；To SlZFP2 transgenic at pWL007 transformed plant blade In expression be analyzed；SlZFP2 protein level in detection rotaring gene plant blade；Observe pWL007 Convert the transformed plant of LA1589 T1 for time the phenotype of plant height, branch amount, flowering time and fruit maturation Statistics.

Result such as Fig. 9, illustrates that the Fructus Lycopersici esculenti LA1589 transfer-gen plant of overexpression SlZFP2 has and substantially divides The increase of branch number, plant becomes short, and flowering time shifts to an earlier date, and fruit maturation is substantially postponed, and there is agent Graded effect, the transfer-gen plant effect i.e. isozygotied becomes apparent from.

4, T2 is for Transgenic Tomato Plants plant type (converting LA1589) and fruit maturation

Observe pWL007 and convert transformed plant (SlZFP2 (pWL007)) the process LAN T2 of LA1589 for strain System (102,103,104,105) fruit maturation change, plant height, the change of number of branches, fruit size and Number seeds.Result such as Figure 10, illustrates compared with non-transgenic reference (NonTrans), overexpression SlZFP2 LA1589 transgenic second filial generation fruits/plant maturation postpone 5-10 days, plant height substantially reduces and branch Number increases, and the fruit weight of some strain decreases.

5, in LA1589, overexpression SlZFP2 changes tomato plant leaf blade size and lateral bud growth

Observe blade and the lateral bud of the transformed plant of pWL007 conversion LA1589, found that overexpression SlZFP2 causes blade to diminish, lateral bud dormant trait reduces, such as Figure 11.

6, the SlZFP2 overexpressing plants phenotype of Fructus Lycopersici esculenti M82

Observe the phenotype that pWL007 converts the transfer-gen plant of M82, carry out Semiquatitative RT-PCR assay gene table Reach analysis (B), by the crosscutting observation cross section of fruits/plant, transfer-gen plant (Lycoperdon polymorphum Vitt cylindricality) and non-transgenic The fruit weight of comparison (black surround white background cylindricality) compares.Result such as Figure 12, mistake in cultivated tomato kind M82 Scale reaches SlZFP2 and causes plant branch substantially to increase (A), and affects the formation of seed in fruit, produces nothing Seed or the few fruit (C-D) of seed.

7, the SlZFP2 overexpressing plants phenotype without HA label

Build the over-express vector p35:SlZFP2 without HA label, by SlZFP2 full length cDNA sequence (restriction endonuclease BamHI/SalI is utilized to connect between the 35S promoter and the Nos terminator that are cloned on pHX20 To between promoter and terminator).Plasmid p35S:SlZFP2 passes through agriculture bacillus mediated importing Fructus Lycopersici esculenti LA1589.

Result such as Figure 13, illustrates that in Fructus Lycopersici esculenti LA1589, the overexpression transfer-gen plant without HA label is also Can substantially increase plant number of branches and make plant Blooming.

8, the Grafting experiments of SlZFP2 transfer-gen plant

Grafting experiments result such as Figure 14 between Fructus Lycopersici esculenti SlZFP2 overexpressing plants and nontransgenic plants, Show that the number of branches that SlZFP2 overexpression causes increases mainly by affecting aerial parts, i.e. lateral bud Formation, and there is no obvious relation with under ground portion, illustrate that SlZFP2 overexpression can reduce lateral bud Dormancy, promote lateral bud growth.After 103 are grafted onto 103, scion growth recovery is very slow, so while Still there is the phenotype that 103 blades are less, but number of branches is not so good as without grafting and is grafted onto non-transgenic stock Plant.

9, suppression SlZFP2 expresses tomato growth and the impact of fruit development

RNAi interference carrier pWL009 has been directed respectively into Fructus Lycopersici esculenti LA1589 and M82.Result such as Figure 15, Branch amount and flowering time are added up in about 45 days plant, and each strain compares and is isolatable from same transgenic 3 transgenic of T0 plant and 3 nontransgenic plants.Germination percentage added up 7 days in the going out of each material Bud number, average germination rate is to identify at PCR to determine that genotype (whether containing transgenic) seed afterwards is averagely sent out Natural law needed for bud.Containing transgenic (pWL009) with without turning base in each strain simultaneously when comparing the 6th day The chitting piece number of cause.Because RNAi plant bears fruit difficulty, number seeds is little, and each strain is only analyzed 15-20 grain.Result illustrates, by SlZFP2 in RNAi method suppression Fructus Lycopersici esculenti LA1589 and M82 Gene expression, reduces plant number of branches and postpones the sprouting speed of seed, but to flowering time fruit size Not impact.

10, the expression in different tissues of SlZFP2 Accelerate bloom gene SFT

Overexpression SlZFP2 gene in Fructus Lycopersici esculenti LA1589, observes floral genes SFT and is spending, growing (fruit reaches final size, will enter the maturation period for fruit (after pollination 5 and 10 days) and ripe green fruit Green fruit) in expression.Result such as Figure 17, the reduction of overexpression SlZFP2 gene is combined with SFT SPGB gene expression, SPGB is (each at Fructus Lycopersici esculenti ABA synthesis mutant not, sitflc and mutant sft of blooming Mutant is all obtained from U.S.'s Tomato Germplasms center TGRC) in express also below wild type, SlZFP2 is described The expression of regulation and control SFT and SPGB is by ABA route of synthesis.

Embodiment 4, SlZFP2 play the study mechanism of regulating and controlling effect

1, the mechanism that gene regulation abscisic acid (ABA) synthesizes

Analyzing SlZFP2 overexpression and the plant of suppression expression, research SlZFP2 plays the machine of regulating and controlling effect Reason.Such as Fig. 5, showing that overexpression SlZFP2 causes Stoma of Leaves to become big, seed germination speed improves, and subtracts ABA content in the tissues such as few flower and fruit, ABA synthase gene is in overexpressing plants blade Express and reduce, and by the expression of RNAi interference SlZFP2, then reduce ABA synzyme in flower tissue The expression of gene SIT and FLC, by combining, ABA synthase gene SlAO1 is isogenic to be opened SlZFP2 The synthesis of negative regulation ABA on mover.

Analyze SlZFP2 overexpression and the plant of suppression expression, study SlZFP2 gene regulation plant blossom Mechanism.Result such as Fig. 6.Showing that overexpression SlZFP2 causes Fructus Lycopersici esculenti to bloom in advance, SlZFP2 promotes Plant blossom is the expression increasing SFT by being directly incorporated in SFT promoter region.

2, the overexpression SlZFP2 impact on blade ABA content

The relatively pore of SlZFP2 transgenic LA1589 tomato plant blade, analyzes ABA process and (sprouts About 20 days blades, face up after taking off, and are placed on containing 5mM KCl, 50 μMs of CaCl₂、10mM In MES-Tris (pH6.15) culture fluid, cultivate 2 hours under illumination condition in the controlled environment chamber, make pore open Open, then blade is put in the culture fluid containing variable concentrations (0,1.0,2.5,5.0 μM) ABA, continue Place 2 hours, then overlay film, electron microscopy observation, measure pore opening, observe variable concentrations ABA pair The impact that transfer-gen plant pore is closed) blade impact on stomatal movement, and ABA is to three SlZFP2 The impact of transgenic LA1589 plant seed germination.Result such as Figure 16, shows that transgenic leaf pore ratio is non- Rotaring gene plant blade pore is big (A), and more insensitive to ABA (B), the kind of results on overexpressing plants Son also decreases (C) when germinateing to the sensitivity of ABA.

3, the overexpression SlZFP2 impact on blade ABA content

Measuring the overexpression SlZFP2 impact on blade ABA content, the assay method of ABA is shown in Fig. 5 Explanation.Result such as Figure 19, the ABA during overexpression SlZFP2 reduces blade in Fructus Lycopersici esculenti LA1589 contain Amount, the ABA in the oldest blade.Material is the homozygous transgenic plant that pWL007 turns LA1589, During sampling, nontransgenic plants has 17 visible leaves, and transfer-gen plant has 12 visible leaves, is after planting 45 days plant.

4, SlZFP2 participates in the expression of regulation and control ABA synthase gene

Base is turned at the SlZFP2 of LA1589 by each ABA synthase gene of qRT-PCR quantitative analysis Because of the expression in plant leaf, result such as Figure 20.Material is consistent with Fig. 5 E with method, for a collection of Secondary experimental result, Fig. 5 E only shows (the L6 of transgenic and not genetically modified in portion gene period wherein L10) data.Result shows, in the SlZFP2 overexpressing plants of Fructus Lycopersici esculenti LA1589, and ABA synzyme Gene NOT, SIT, SlAO1, SlAO2 are different with FLC expression performance in different development stage blade The decline of degree, correspondingly be that the expression of ABA transport agent SlABCG40 gene is also suppressed.

5, suppression SlZFP2 gene expressing ABA synthesis, transhipment and the table of responsive genes in Fructus Lycopersici esculenti Reach impact

Research ABA synthesizes, transports and respond related gene at several RNAi (pWL009) transformation plant flower Expression.Result such as Figure 21, by the SlZFP2 table in RNAi method suppression Fructus Lycopersici esculenti LA1589 Reaching, the expression on ABA synthase gene NOT and SlAO1 affects inconspicuous, mainly affects SIT and FLC Expression, the expression on ABA transport vehicle SlABCG40 also affects inconspicuous, but ABA responsive genes Le16 and Le25 the most substantially rises.

Embodiment 5, SlZFP2 are for the impact of the character of Oryza sativa L.

To spend 11 in plasmid pWL007 Introduced into Rice kind, rice conversion sees list of references Hiei, Y., Et al., Plant J, 1994.6 (2): p.271-82.

Transfer-gen plant T0 for florescence tillering number statistical result as shown in figure 18.10 independent transformation Strain all shows as tiller number increase at transgenic the present age.

The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document It is individually recited as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, The present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within this Shen equally Please appended claims limited range.

Claims (13)

1. the method regulating plant trait, it is characterised in that described method includes: in regulation plant The expression of SlZFP2 polypeptide, described SlZFP2 polypeptide is the polypeptide of aminoacid sequence such as SEQ ID NO:2；

Described plant is selected from: plant of Solanaceae or Oryza sativa L.；

Described plant trait is: in the quantity of flowering of plant time, plant branching or tiller, fruit Carotenoid and/or the content of lycopene, the height of plant, the weight of plant seed and/or minimizing Plant seed quantity, or plant leaf blade size.

2. the method for claim 1, it is characterised in that described method also includes subsequent step: from Plant after the expression of regulation SlZFP2 polypeptide is selected character for comparing check plant and obtains regulation Plant.

3. the method for claim 1, it is characterised in that described plant of Solanaceae is selected from: Fructus Lycopersici esculenti, Rhizoma Solani tuber osi, Fructus Solani melongenae, Fructus Capsici, Nicotiana tabacum L., Fructus Lycii.

4. the method for claim 1, it is characterised in that described method includes: improve in plant The expression of SlZFP2 polypeptide；Thus:

Promote flowering of plant time advance；

Postpone fruit maturation time；

Increase plant branching or the quantity of tiller；

Increase carotenoid and/or content of lycopene in fruit；

Reduce the height of plant；

Reduce the weight of plant seed and/or reduce plant seed quantity；And/or

Promote that plant leaf blade diminishes.

5. method as claimed in claim 4, it is characterised in that including: by coding SlZFP2 polypeptide Polynucleotide proceed to plant, it is thus achieved that be transformed into the plant of described polynucleotide.

6. method as claimed in claim 5, it is characterised in that including:

(S1) Agrobacterium of expression vector is carried in offer, and described expression vector contains polynucleotide, its coding SlZFP2 polypeptide；

(S2) cell or tissue or the organ of plant are contacted with the Agrobacterium in step (S1), so that described Polynucleotide proceed to plant cell, tissue, organ or seed.

7. the method for claim 1, it is characterised in that described method includes: reduce in plant The expression of SlZFP2 polypeptide, thus:

Reduce plant branching or the quantity of tiller；And/or

Reduce carotenoid and/or content of lycopene in fruit.

8. method as claimed in claim 7, it is characterised in that SlZFP2 polypeptide in described reduction plant Expression include: to proceed to plant thin for the disturbing molecule expressed by the coded sequence disturbing described SlZFP2 polypeptide Born of the same parents, tissue, organ or seed, thus lower the expression of SlZFP2 polypeptide in plant.

9. SlZFP2 polypeptide or its encoding gene purposes, be used for regulating plant trait；Described SlZFP2 Polypeptide is the polypeptide of aminoacid sequence such as SEQ ID NO:2；

Described plant is selected from: plant of Solanaceae or Oryza sativa L.；

Described plant trait is: in the quantity of flowering of plant time, plant branching or tiller, fruit Carotenoid and/or the content of lycopene, the height of plant, the weight of plant seed and/or subtract Few plant seed quantity, or plant leaf blade size.

10. purposes as claimed in claim 9, it is characterised in that described SlZFP2 polypeptide or its coding Gene is used for:

Promote flowering of plant time advance；

Postpone fruit maturation time；

Increase plant branching or the quantity of tiller；

Increase carotenoid and/or content of lycopene in fruit；

Reduce the height of plant；

Reduce the weight of plant seed and/or reduce plant seed quantity；

Promote that plant leaf blade diminishes；And/or

Molecular marker as plant identification character.

The purposes of 11. 1 kinds of materials reducing SlZFP2 expression of polypeptides, is used for:

Reduce plant branching or the quantity of tiller；And/or

Reduce carotenoid and/or content of lycopene in fruit；

Described SlZFP2 polypeptide is the polypeptide of aminoacid sequence such as SEQ ID NO:2；

Described plant is selected from: plant of Solanaceae or Oryza sativa L.；

Described plant trait is: in the quantity of flowering of plant time, plant branching or tiller, fruit Carotenoid and/or the content of lycopene, the height of plant, the weight of plant seed and/or subtract Few plant seed quantity, or plant leaf blade size.

12. 1 kinds of disturbing molecules disturbing SlZFP2 gene expression, it contains the structure shown in formula (I):

Seq_Forward-X-Seq_ReverselyFormula (I),

In formula (I),

Seq_ForwardFor SlZFP2 genetic fragment, Seq_ReverselyFor with Seq_ForwardComplementary polynucleotide；

X is for being positioned at Seq_ForwardAnd Seq_ReverselyBetween intervening sequence, and described intervening sequence and Seq_ForwardWith Seq_ReverselyThe most complementary；The nucleotide sequence such as SEQ ID NO:1 of described SlZFP2 gene.

13. 1 kinds of carriers, it is characterised in that described carrier contains the disturbing molecule described in claim 12.