CN103966254A - Transcription factor capable of being used to adjust plant traits - Google Patents

Transcription factor capable of being used to adjust plant traits Download PDF

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CN103966254A
CN103966254A CN201310032971.XA CN201310032971A CN103966254A CN 103966254 A CN103966254 A CN 103966254A CN 201310032971 A CN201310032971 A CN 201310032971A CN 103966254 A CN103966254 A CN 103966254A
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plant
slzfp2
polypeptide
expression
fruit
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CN103966254B (en
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肖晗
翁林
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Center for Excellence in Molecular Plant Sciences of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Abstract

The invention relates to a transcription factor capable of being used to adjust plant traits. The plant shape, the blossom period, and the mature time and size of the harvest organs such as fruits are prominently improved through changing the expression of the transcription factor in plants. Thus the transcription factor can be used to create plants with ideal traits.

Description

A kind of transcription factor that can be applicable to regulating plant proterties
Technical field
The invention belongs to biotechnology and phytology field; More specifically, the present invention relates to a kind of transcription factor that can be applicable to regulating plant proterties.
Background technology
In nature situation, gene is spontaneous or to bring out the frequency of sudden change low, and useful sudden change still less; And the available germ plasm resource of certain kind of plant is owing to being subject to the restriction of the characteristics such as reproduction isolation, and be confined in very limited scope, the genetic improvement of plant is restricted to a great extent.In currently available technology, genetic modification of plants is taking transgenation and sexual hybridization as basis.
Along with molecular biological development, the regeneration of people's transformant to foreign gene transfered cell and after leading people's cell, explores and studies, and has set up practicable genetic conversion system, and plant gene engineering technology is become better and approaching perfection day by day.People are applied to genetic modification of plants genetic engineering technique, have solved some difficult problems that exist in conventional genetic improvement, progressively develop into a kind of new genetic improvement means, have injected new vitality to genetic modification of plants.The develop rapidly of modern biotechnology, achieves great success the research of plant genetic engineering, has brought new hope and dawn to the mankind's the survival and development.Although the application of plant genetic engineering in genetic improvement still exists many problems, its using value more and more comes into one's own.By plant gene engineering technology, exogenous genetic material is inserted, is incorporated in the genome of recipient plant, make its Inheritance and expression stably in offspring plant, thereby make recipient plant obtain new proterties.
Utilize plant gene engineering technology to carry out genetic improvement, can break the reproduction isolation between species, make heredity interchange between different plant species become possibility, can utilize gene pool to create condition for widening plant: the technique means that plant genetic engineering provides new creation to make a variation, greatly shortened the genetic modification of plants cycle: mostly study comparatively clearly for the gene of genetically engineered genetic improvement simultaneously, the plant object proterties of improvement is clear and definite, selection approach is effective, make to cause plant generation determinate variation and carry out orthoselection and become possibility: in addition by some crucial proterties of improvement plant, the Main Agronomic Characters of former kind (being) is improved to a great extent.
The popularization of genetically modified crops will alleviate staple crop poor quality, low yield not before, disease and pest increases the weight of year by year, shortage of water resources, Soil degradation, saltings, Altoxicity and use in a large number the problems such as the environmental pollution that agricultural chemicals causes, for Ensuring Food Safety, ecological safety and raising agricultural-food international competitiveness provide powerful technical support.
Along with deepening continuously of genetically engineered research, can be cloned into more new gene and transgenic technology can be further improved, and multiple genes is carried out to directional operation and also will become possibility, and this is difficult to realize in conventional genetic improvement.But in view of the diversity of plant gene, this area is necessary to find from all polygene further for changing the relevant gene of plant trait, for creating desirable plant plant type, improving plant nutrient ingredient and fruit properties.
Summary of the invention
The object of the present invention is to provide a kind of transcription factor that can be applicable to regulating plant proterties.
In a first aspect of the present invention, a kind of method of regulating plant proterties is provided, described method comprises: the expression of SlZFP2 polypeptide in regulating plant.
In a preference, described method also comprises subsequent step: from regulate the plant the expression of SlZFP2 polypeptide, select the plant of comparing control plant proterties and obtain adjusting.
In another preference, described plant is selected from: grass, plant of Solanaceae.
In another preference, described grass is selected from: paddy rice, corn, wheat, barley, oat, rye, Chinese sorghum.
In another preference, described plant of Solanaceae is selected from: tomato, potato, eggplant, capsicum, tobacco, matrimony vine.
In another preference, described SlZFP2 polypeptide is selected from lower group: (a) as the polypeptide of SEQ ID NO:2 aminoacid sequence; (b) by one or more SEQ ID NO:2 aminoacid sequence process (as individual in 1-20; Preferably 1-10; More preferably 1-5) replacement, disappearance or the interpolation of amino-acid residue form, and have (a) polypeptide function by (a) derivative polypeptide; Or the peptide sequence (c) and (a) limiting has more than 80% (preferably more than 85%, more preferably more than 90%, as 95%, 98%, 99%) homology and have (a) polypeptide function by (a) derivative polypeptide.
In another preference, described method comprises: the expression that improves SlZFP2 polypeptide in plant; Thereby: promote flowering of plant time advance; Postpone fruit maturation time; The quantity that increases plant branching or tiller; Increase carotenoid and/or content of lycopene in plant (being preferably pulp class plant) fruit; Reduce the height of plant plant; Reduce the weight of plant seed and/or reduce plant seed quantity (comprise and make plant not produce seed); And/or promote that plant leaf diminishes.
In another preference, described method comprises: the polynucleotide of coding SlZFP2 polypeptide are proceeded to plant (as cell, tissue, organ or seed), obtain the plant (as cell, tissue, organ or seed) that is transformed into described polynucleotide.
In another preference, the sequence of the polynucleotide of described coding SlZFP2 polypeptide is as SEQ ID NO:1, or the nucleotide sequence of the degeneracy of SEQ ID NO:1, or the polypeptide polypeptide function identical nucleotide diversity body coded with SEQ ID NO:1 of coding.
In another preference, the method that the polynucleotide of coding SlZFP2 polypeptide is proceeded to plant comprises:
(S1) provide the Agrobacterium of carrying expression vector, described expression vector contains polynucleotide, its coding SlZFP2 albumen;
(S2) cell or tissue of plant or organ are contacted with the Agrobacterium in step (S1), thereby make described polynucleotide proceed to vegetable cell, tissue, organ or seed.
In another preference, described method also comprises:
(S3) select the vegetable cell, tissue, organ or the seed that have proceeded to described polynucleotide; With
(S4) by the vegetable cell in step (S3), tissue, organ or seed regeneration plant.
In another preference, described method comprises:
(S1) provide the Agrobacterium of carrying expression vector, described expression vector contains polynucleotide, its coding SlZFP2 polypeptide;
(S2) cell or tissue of plant or organ are contacted with the Agrobacterium in step (S1), thereby make described polynucleotide proceed to vegetable cell, tissue, organ or seed.
In another preference, described method comprises: reduce the expression of SlZFP2 polypeptide in plant, thereby:
The quantity that reduces plant branching or tiller; And/or
Reduce carotenoid and/or content of lycopene in plant (being preferably pulp class plant) fruit.
In another preference, in described reduction plant, the expression of SlZFP2 polypeptide comprises:
The disturbing molecule that disturbs the encoding sequence of described SlZFP2 polypeptide to express is proceeded to vegetable cell, tissue, organ or seed, thereby lower the expression of SlZFP2 polypeptide in plant.
In another preference, in described reduction plant, the method for the expression of SlZFP2 polypeptide comprises:
(i) provide the Agrobacterium of carrying the carrier that can disturb genetic expression, described carrier is selected from lower group:
(a) the SlZFP2 gene that contains startup in the other direction or the carrier of gene fragment (antisense molecule);
(b) carrier of the disturbing molecule that contains the composition that can form specificity interference SlZFP2 genetic expression (or transcribing) in plant materials;
(ii) cell of plant, tissue or organ are contacted with the Agrobacterium in step (i), thereby make described carrier proceed to vegetable cell, tissue or organ.
Preferably, described method also comprises: (iii) select the vegetable cell, tissue or the organ that have proceeded to described carrier; (iv) vegetable cell, tissue or neomorph in step (iii) are become to plant.
In another aspect of this invention, provide a kind of SlZFP2 polypeptide or its encoding gene purposes, for regulating plant proterties.
In a preference, described SlZFP2 polypeptide or its encoding gene are used for: promote flowering of plant time advance; Postpone fruit maturation time; The quantity that increases plant branching or tiller; Increase carotenoid and/or content of lycopene in plant (being preferably pulp class plant) fruit; Reduce the height of plant plant; Reduce the weight of plant seed and/or reduce plant seed quantity (comprise and make plant not produce seed); Promote that plant leaf diminishes; And/or as the molecule marker of plant identification proterties.
In another preference, if after testing in plant tissue SlZFP2 expression of polypeptides higher than a particular value (as the value of the average SlZFP2 expression amount of wild-type same plant), comparatively speaking, described flowering of plant time advance; Fruit maturation time retardation; Branch or the quantity of tillering increase; In fruit, carotenoid and/or content of lycopene increase; The height of plant reduces; The weight of seed and/or quantity reduce or do not produce seed; Or plant leaf diminishes.If in plant tissue, SlZFP2 expression of polypeptides is lower than this particular value after testing, comparatively speaking, the branch of described plant or the quantity of tillering reduce; Or carotenoid and/or content of lycopene reduction in fruit.
In another aspect of this invention, provide a kind of purposes of material of the SlZFP2 of reduction expression of polypeptides, for: the quantity that reduces plant branching or tiller; And/or reduce carotenoid and/or content of lycopene in plant (being preferably pulp class plant) fruit.
In another preference, the material of described reduction SlZFP2 expression of polypeptides is the disturbing molecule that disturbs described SlZFP2 genetic expression.
In another aspect of this invention, provide a kind of disturbing molecule that disturbs described SlZFP2 genetic expression, it contains the structure shown in formula (I): Seq forward-X-Seq oppositelyformula (I),
In formula (I), Seq forwardfor SlZFP2 gene fragment (being preferably the nucleotide sequence shown in 334-723 position in SEQ ID NO:1), Seq oppositelyfor with Seq forwardcomplementary polynucleotide;
X is for being positioned at Seq forwardand Seq oppositelybetween intervening sequence, and described intervening sequence and Seq forwardand Seq oppositelynot complementary.
In another preference, the structure shown in formula (I) is proceeding to after vegetable cell, forms the secondary structure shown in formula (II):
formula (II),
In formula (II), Seq forward, Seq oppositelywith the definition of X as above-mentioned, || be illustrated in Seq forwardand Seq oppositelybetween complementary relation substantially.
In another preference, itself does not form complementary duplex structure described intervening sequence.
In another aspect of this invention, provide a kind of carrier, described carrier contains described disturbing molecule.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Brief description of the drawings
Fig. 1, over-express vector pWL007 schematic diagram.
Fig. 2, RNAi carrier pWL009 schematic diagram.
Fig. 3, SlZFP2 allelic expression.Wherein A and E are the results of hybridization of SlZFP2 justice rna probe, without clear signal.B-D and F-H are the results of hybridization of SlZFP2 antisense RNA probes.Scale is 200um.
Fig. 4, overexpression and expression inhibiting are in the phenotype of plant type, fruit and seed.
A-C, be respectively the photo of three weeks seedlings, the free-hand section of 45 days seedling stems and 45 days little shoot roots.
D, pWL007 transformation plant 103 ripening of fruits photos, upper row is non-transgenic contrast, lower row is 103 transfer-gen plant fruits.
E-F, pWL009 transformed plant seedling and contrast respectively.
G, the RNAi plant inflorescence difficulty of bearing fruit.
The M82 transfer-gen plant of H, pWL007 and pWL009.
J-H, four pWL007 transformation plant fruit maturation time (number of days from pollination during to fruit annesl) and hundred seed weights (unit be milligram), NonTrans is that non-transgenic contrasts.
Fig. 5, the synthetic mechanism of gene regulating dormin (ABA).
A: the pore opening of three pWL007 transgenosis LA1589 strains and non-transgenic contrast thereof.Each strain is got common 50-60 the pore of 3-5 individual plant and on electron microscope, is observed measurement, and data are the means standard deviation of strain pore opening.
B: the statistics of seed germination speed.Three pWL007 transgenosis LA1589 strains and non-transgenic contrast seed thereof germinate on thieving paper, and timing statistics 0-7 days when D0-D7, repeats 50 seeds of each repetition for 3 times.Data are average germination percentage ± standard deviation.
C: overexpression SlZFP2 in ABA mutant sit (by hybridizing transformation to sit mutant, sit mutant is from U.S.'s Tomato Germplasms center, TGRC by the M82 transgenosis that is derived from pWL007).Picture is the rip cutting figure of mature fruit.
D: the ABA content of three pWL007 transgenic lines and non-transgenic contrast thereof.The ABA content assaying method in blade, flower and three periods of ripe green fruit is referring to reference Fu, X., et al., J Exp Bot, 2012.63 (1): p.341-54.
E:ABA synthetic enzyme and the transporter gene expression amount in pWL007 transgenosis and non-transgenic adjoining tree blade changes.NOT, SIT, SlAO1, SlAO2 and FLC are the synthetic main key enzyme encoding gene of ABA, and SlABCG40 is the Potential Vector gene of tomato ABA transhipment.Quantitative RT-PCR analysis of material be transgenic line 103 and when impinging upon 45 days blade, result is shown the expression amount means standard deviation with respect to SleIF4a gene.
F:ABA synthase gene SIT and the FLC expression in RNAi plant changes.RNA extracts the flower (blooming the same day) from the RNAi of four LA1589 plant (different independent transformed plants) and LA1589 contrast.
G: the combination of SlZFP2 and ABA synthase gene SlAO1 promotor is analyzed in co-immunoprecipitation analysis (ChIP).Material therefor is transfer-gen plant and the contrast blade thereof of 30 days seedling ages, ChIP and quantitative PCR (qPCR) analytical procedure is referring to reference 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 promotor and SlZFP2.Probe, from the PCR product of ChIP experiment (G figure), carries out external EMSA experiment with GST-SlZFP2 (total length) fusion rotein of bacterial expression.EMSA method is referring to reference Peng, H., et al., Cancer Res, 2002.62 (13): p.3773-81.
The mechanism of Fig. 6, SlZFP2 gene regulating plant blossom.Expression amount shown in C, D and F in figure is all done reference with tomato SleIF4a expression amount.
The LA1589 transgenosis of A:pWL007 and the non-transgenic plant photo in the time of 45 days.
B: the LA1589 transgenosis of four pWL007 and the flowering time of non-transgenic plant statistics.True leaf number before flowering time occurs with first inflorescence represents, utilizes relatively transfer-gen plant and separate the difference between the non-transgenic plant in same transgenosis T0 generation of student T-test (student ' s t-test).Sample number N=15.
C: tomato floral genes SFT changes in the expression of transfer-gen plant and non-transgenic contrast different ages blade.Blade for quantitative RT-PCR is taken from 45 days plant, and non-transgenic plant has 16 to see leaf, and transfer-gen plant has 12 visible leaves.
D:SFT changes in the expression of four RNAi plant.Total RNA takes from pWL009 and turns the flower tissue (blooming the same day) of the LA1589 present age (T0).
The flowering time statistics of E:ABA mutant and wild-type plant thereof.ABA synthesis mutant not, sit and flc are so kind as to give by TGRC.Flowering time statistical method is with Fig. 4 B.
The expression of F:SFT in ABA synthesis mutant and wild-type plant leaf thereof changes.Blade is got the blade (from down the 5th leaf of number of stem apex, 45 days plant) of same developmental stage.The expression amount of tomato sft does negative contrast.
G: the combination of SlZFP2 and SFT promotor is analyzed in co-immunoprecipitation analysis (ChIP).Material therefor is transfer-gen plant and the contrast blade thereof of 30 days seedling ages, ChIP and quantitative PCR (qPCR) analytical procedure is referring to reference 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 promotor and SlZFP2.Probe, from the PCR product of ChIP experiment (G figure), carries out external EMSA experiment with GST-SlZFP2 (total length) fusion rotein of bacterial expression.EMSA method is referring to reference 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 transcribing group analysis, find that SlZFP2 mainly expresses after tomato blooms;
B: utilize sxemiquantitative RT-PCR to verify that SlZFP2 expression is regulated and controled by fruit development;
C: carry out sequential analysis of protein by MEME software and show that SlZFP2 polypeptide contains a C 2h 2zinc finger domain (structural domain 1) and one may participate in transcribing the structural domain (structural domain 2) of inhibition;
D-E: the amino acid conservative Analysis of two structural domains.
Fig. 9, overexpression SlZFP2 change tomato plant plant type and fruit maturation (T1 generation).
A: transgenosis and the photo of non-transgenic plant in the time of 45 days left and right that the transformed plant selfing of four pWL007 conversion LA1589 is separated;
The expression analysis of B:SlZFP2 transgenosis in the transformed plant blade of pWL007 conversion LA1589;
The protein expression level of C:HA-SlZFP2 in the transformed plant of four pWL007 conversion LA1589.Western blot detects the HA-SlZFP2 protein level in rotaring gene plant blade with HA monoclonal antibody (Sigma);
D-G: the transformed plant that four pWL007 transform LA1589 T1 for time the phenotype statistics of plant height, branch amount, flowering time and fruit maturation.
Each genotype has been observed 5 strains altogether, and fruit maturation has been added up at least 15 fruits of every strain, illustrates each genotypic proterties means standard deviation.
Figure 10, overexpression SlZFP2 change tomato plant plant type and fruit maturation (T2 generation).
A: three SlZFP2 (pWL007) cross expression strain fruit maturation and change, DPA:days post anthesis (number of days after pollination);
B: four SlZFP2 (pWL007) cross and express the plant height of strain in the time of 45 days;
C: the variation of four SlZFP2 (pWL007) strain branch number in the time of 45 days;
D and E: the statistics of four transformation plant fruit sizes and number seeds.In A, "-" and "+" shows respectively not containing and contains SlZFP2 transgenic fragment;
B-E:NonTrans is non-transgenic contrast;
Data are from 5 strains/strain, and 15-20 fruit added up in every strain.In figure, all transgenosiss are homozygous lines, and all plant are LA1589 background.
Figure 11, overexpression SlZFP2 change tomato plant blade size and lateral bud growth.
The SlZFP2 overexpression plant of Figure 12, tomato M82 (401,402 etc. represented expression strain) phenotype.
A:SlZFP2 (pWL007) transforms the transfer-gen plant of M82.401 and 402 plant of below and branch photo were from 3 months plant;
B:pWL007 turns the gene expression analysis of M82 plant.The total RNA extracting from blade is for semi-quantitative RT-PCR analysis;
C: five pWL007 turn the sectional view of M82 fruits/plant;
D: three pWL007 turn M82 plant (grey cylindricality) and contrast with non-transgenic the fruit weight comparison of (black surround white background cylindricality).Non-transgenic plant is for separating from transgenosis T0 for self progeny.
Figure 13, not containing the SlZFP2 overexpression plant of HA label (502,503 etc.) phenotype.The different strain numbering of digitized representation of each little figure below, WT and LA1589 are non-transgenic contrasts.When characters of progenies is investigated, contrast used is the non-transgenic plant (NonTrans) of separating from same transgenosis T0 generation, and each strain has been analyzed transgenosis and each 3 strains of non-transgenic plant.TrSlZFP2/eIF4a6 and endoSlZFP2/eIF4a6 represent respectively to import and the transcript of endogenous SlZFP2 and the ratio of eIFA4a6 transcript, reflect the transcriptional level of this gene.Show that the P value of significance level is T test (Student ' s t-test) calculating.
Grafting experiment between Figure 14, SlZFP2 transfer-gen plant and non-transgenic plant, 103N and 103 represents respectively non-transgenic and transfer-gen plant.Grafting is to carry out between the plant of left and right at 3 weeks, and horizontal line top is scion, and below is stock.
Figure 15, RNAi interference carrier pWL009 have imported respectively tomato LA1589 and M82.In figure, 206,208,209 and 210 strains are the RNAi transformed plant that pWL009 transforms LA1589,501,509, the 510 and 512 RNAi transformed plants for pWL009 conversion M82.Each numbering represents an independently transformation plant.
Figure 16, overexpression SlZFP2 affect Stoma of Leaves and the susceptibility of seed germination to ABA.
A: the pore comparison of three genetically modified LA1589 plant of SlZFP2 (102,103,105) blade;
B: different ABA process the impact of blade on stomatal movement;
The impact of C:ABA on three SlZFP2 transgenosis LA1589 seed germinations.
Figure 17, in tomato LA1589, overexpression SlZFP2 gene pairs floral genes SFT is spending, is growing the expression impact of fruit (pollination after 5 and 10 days) and ripe green fruit.In figure, the black column diagram of special mark is not the transfer-gen plant that SlZFP2 turns LA1589, the contrast of white cylindricality diagram non-transgenic.Dpa, number of days (day post anthessis) after pollination.LA0534 and LA0535 are respectively not and sit, the wild-type contrast (near isogenic line) of flc mutant.
Figure 18, SlZFP2 overexpression in paddy rice causes plant tillering number to increase.Wherein, CK is not for spending 11, NO1-NO11 for turning SlZFP2 trans-genetic hybrid rice containing in the genetically modified paddy rice of SlZFP2.
Figure 19, in tomato LA1589 overexpression SlZFP2 on blade in the impact of ABA content, material is the transfer-gen plant that isozygotys that pWL007 turns LA1589, when sampling, non-transgenic plant has 17 visible leaves, and transfer-gen plant has 12 visible leaves, is after planting 45 days plant.
Figure 20, by the expression of each ABA synthase gene of qRT-PCR quantitative analysis in the SlZFP2 of LA1589 rotaring gene plant blade.
The expression level that Figure 21, ABA synthesize, transport and response genes involved is spent at several RNAi (pWL009) transformation plant, WT is non-transgenic LA1589 contrast.Material therefor is the same with Fig. 5 F with qRT-PCR analysis, is same batch of experiment.
Embodiment
The inventor, through deep research, finds by changing the expression of SlZFP2 in plant materials, can significantly improve plant plant type (side shoot number, plant height) if, flowering time, real maturation time and the size of results organ.Therefore the new variety of plant that method of the present invention can be applicable to create desirable plants plant type, obtains the nutritive substances such as high hycopene, high β-carotene has a good application prospect on the genetic improvement of plant biomass and quality.
As used herein, plant of the present invention (or crop) is the plant that is applicable to the conversion operation of carrying out gene, as various farm crop, flower plant or forestry plant etc.Described plant is such as being (being not limited to): dicotyledons, monocotyledons or gymnosperm.More specifically, described plant includes, but is not limited to: tomato, green pepper, avocado, cassia bark, camphor, tobacco leaf, wheat, barley, rye, paddy rice, corn, jowar, beet, apple, pears, Lee, peach, apricot, cherry, strawberry, rasp berry, blackberry, blueberry, beans, French beans, pea, soybean, rape, mustard, opium poppy, olea, Sunflower Receptacle, coconut, Viscotrol C plant, cocoa beans, peanut, cucurbit, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, natsudaidai, spinach, piemarker lettuce, asparagus, cabbage, Chinese cabbage, Plantula Brassicae chinensis, Radix Dauci Sativae, onion, potato, nut, coffee, eggplant, sugarcane, tealeaves, pepper, grapevine, oyster fiber crops grass, banana, natural rubber tree and ornamental plant etc.
As a kind of optimal way, described " plant " includes but not limited to: grass, plant of Solanaceae etc.Such as, described " plant " includes but not limited to: paddy rice, wheat, corn, tomato, potato, eggplant, capsicum, tobacco etc.
As used herein, selecting suitable " control plant " is the customary part of experimental design, and " control plant " can comprise corresponding wild-type plant or the corresponding plant without goal gene.Control plant is generally identical plant species or or even the kind identical with plant to be assessed.Control plant can be also the individuality of losing transgenic plant because of separation.Control plant not only refers to complete plant as used herein, also refers to plant part, comprises seed and plants subdivision.
The present invention also comprises fragment, derivative and the analogue of SlZFP2 polypeptide.As used herein, term " fragment ", " derivative " refer to and substantially keep biological function or the active polypeptide that SlZFP2 polypeptide of the present invention is identical with " analogue ".Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferably conservative amino acid residue), and the amino-acid residue of such replacement can not be also to be encoded by genetic code, or (ii) in one or more amino-acid residues, there is the polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as extending the compound of polypeptide transformation period, for example polyoxyethylene glycol) merge the polypeptide that forms, or (iv) additional aminoacid sequence be fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for sequence or the proteinogen sequence of this polypeptide of purifying, or fusion rotein).Belong to the known scope of those skilled in the art according to these fragments of definition, derivative and analogue herein.Preferably, SlZFP2 polypeptide of the present invention comprises a kind of fusion rotein, and it is the fusion rotein that HA label and SlZFP2 are connected and composed; This fusion rotein has the effect of stablizing SlZFP2 polypeptide function in plant materials.
The bioactive fragment of any SlZFP2 polypeptide can be applied in the present invention.Here, the implication of the bioactive fragment of SlZFP2 polypeptide refers to as a peptide species, and it still can keep all or part of function of the SlZFP2 polypeptide of total length.Under normal circumstances, described bioactive fragment at least keeps the activity of 50% total length SlZFP2 polypeptide.Under preferred condition, described active fragments can keep 60%, 70%, 80%, 90%, 95%, 99% or 100% activity of total length SlZFP2 polypeptide.
In the present invention, term " SlZFP2 polypeptide " refers to the polypeptide of the SEQ ID NO:2 sequence with SlZFP2 polypeptide active.This term also comprises having and variant form SlZFP2 polypeptide identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best, also better for 1-8,1-5) amino acid whose disappearance, insertion and/or replacement, and add one or several (being generally in 20, is preferably in 10, is more preferably in 5) amino acid at C-terminal and/or N-terminal.For example, in the art, while replacement with the close or similar amino acid of performance, conventionally can not change the function of protein.Again such as, the function of adding or several amino acid and conventionally also can not change protein at C-terminal and/or N-terminal.This term also comprises active fragments and the reactive derivative of SlZFP2 polypeptide.
The variant form of polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, albumen that can be coded with the DNA of SlZFP2 polypeptid DNA hybridization under high or low stringency condition and polypeptide or the albumen that utilizes the antiserum(antisera) of anti-SlZFP2 polypeptide to obtain.The present invention also provides other polypeptide, as the fusion rotein that comprises SlZFP2 polypeptide or its fragment.
Any and described SlZFP2 peptides homologous high (such as with the homology of the sequence shown in SEQ ID NO:2 be 70% or higher; Preferably, homology is 80% or higher; Preferred, homology is 90% or higher, as homology 95%, 98% or 99%) and the albumen with SlZFP2 polypeptide identical function be also included within the present invention.
Invention also provides the analogue of SlZFP2 polypeptide or polypeptide.The difference of these analogues and natural SlZFP2 polypeptide can be the difference on aminoacid sequence, can be also the difference not affecting on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant natural or induction.Induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has the analogue of non-natural amino acid (as β, gamma-amino acid) that exist or synthetic.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide exemplifying.
Should understand, although SlZFP2 polypeptide of the present invention is preferably available from plant of Solanaceae (as tomato), but within the scope of also considering in the present invention available from other plant and other polypeptide tomato SlZFP2 polypeptide height homology (as have more than 80%, as 85%, 90%, 95%, even 98% sequence homogeny).The Method and kit for of aligned sequences homogeny is also that this area is known, for example BLAST.
The invention still further relates to the polynucleotide sequence of code book invention SlZFP2 polypeptide or its conservative property variation polypeptide.Described polynucleotide can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " refers to that coding has the protein of SEQ ID NO:2 in the present invention, but with the differentiated nucleotide sequence of coding region sequence shown in SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence (with optional additional code sequence) and the non-coding sequence of mature polypeptide.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise coding said polypeptide, can be also the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or the fragment of polypeptide, analogue and derivative with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing in fact the function of polypeptide of its coding.
The invention still further relates between above-mentioned sequence hybridization and two sequences and have at least 50%, preferably at least 70%, the more preferably polynucleotide of at least 80% homogeny.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " refers to: (1) at the hybridization compared with under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, and 0.1%SDS, 60 DEG C; Or (2) hybridization time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.; Or (3) only at the homogeny between two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in SEQ ID NO:2.
The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment ", containing 15 Nucleotide, is at least better at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the polynucleotide of coding SlZFP2 polypeptide.
The Nucleotide full length sequence of SlZFP2 gene of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.
The present invention also relates to the carrier that comprises described polynucleotide, and the host cell producing through genetically engineered by described carrier or SlZFP2 gene order.
In the present invention, SlZFP2 polypeptide polynucleotide sequence can be inserted in recombinant expression vector, as long as can copy in host and stablize, any plasmid and carrier can be used.A key character of expression vector is conventionally to contain replication orgin, promotor, marker gene and translation controlling elements.
Comprise above-mentioned suitable DNA sequence dna and the suitable carrier of promotor or control sequence, can be for transforming suitable host cell, with can marking protein.Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell is as yeast; Vegetable cell etc.
When described polynucleotide are expressed in higher eucaryotic cells, be enhanced if will make to transcribe insert enhancer sequence in carrier time.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs, act on promotor transcribing with enhancing gene conventionally.Persons skilled in the art are all known the suitable carrier of How to choose, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.Conversion of plant can use the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as spraying, Ye Panfa, Rice Young Embryo conversion method etc.Can use ordinary method regeneration plant for the vegetable cell, tissue or the organ that transform, thus the plant that acquired character changes.
In specific embodiments of the invention, overexpression SlZFP2 polypeptide in plant of Solanaceae tomato, can significantly accelerate the plant blossom time, when the lobe numbers of plant is as index when blooming, overexpression plant early 3-5 sheet leaf blooms, fruit maturation is postponed 5-7 days, plant bud dormancy weakens, branch increases, and plant is slightly downgraded, and the ABA content in plant leaf, flower and fruit reduces, Lyeopene in fruit and content beta-carotene increase, and Grain Dormancy decreases.But suppress the endogenous expression of this transcription factor in tomato, plant and wild-type plant do not have considerable change in the vegetative phase, but side shoot minimizing is beared fruit and is suppressed, easily shedding.
In body, co-immunoprecipitation experiment showed, that SlZFP2 polypeptide can be combined in the promotor of multiple ABA synthase genes and floral genes SFT, and the specificity of its combination has also been confirmed in external EMSA experiment.Series of experiments has proved that from the many aspects such as hereditary, biochemical this transcription factor is a newcomer that regulation and control ABA synthase gene is expressed, and participates in the regulating plant approach of blooming directly.
Therefore, the invention provides described SlZFP2 polypeptide or the purposes of its encoding gene, for the proterties of regulating plant; Or for screening the material useful for regulating plant proterties (that is: described material carrys out regulating plant proterties by the expression that regulates SlZFP2 polypeptide).As a kind of optimal way, described SlZFP2 polypeptide can be used for: promote flowering of plant time advance; Postpone fruit maturation time; The quantity that increases plant branching or tiller; Increase carotenoid and/or content of lycopene in plant (being preferably pulp class plant) fruit; Reduce the height of plant plant; Reduce the weight of plant seed and/or reduce plant seed quantity (comprise and make plant not produce seed); And/or promote that plant leaf diminishes.
The invention still further relates to the agonist of SlZFP2 or antagonist and uses thereof.Due to the agonist of SlZFP2 or the expression of the adjustable SlZFP2 of antagonist and/or regulate the activity etc. of SlZFP2, therefore, the agonist of described SlZFP2 or antagonist also can be by the impact of SlZFP2 is carried out to regulating plant proterties, thereby reach the object of improvement plant.
The material of transcribing and translating of the activity of any SlZFP2 of raising polypeptide, the stability that improves SlZFP2 polypeptide, the expression that promotes SlZFP2 polypeptide, prolongation SlZFP2 polypeptide effective acting time or promotion SlZFP2 all can be used for the present invention, as can be used for regulating plant proterties, particularly promote flowering of plant time advance; Postpone fruit maturation time; The quantity that increases plant branching or tiller; Increase carotenoid and/or content of lycopene in plant (being preferably pulp class plant) fruit; Reduce the height of plant plant; Reduce the weight of plant seed and/or reduce plant seed quantity (comprise and make plant not produce seed); And/or promote that plant leaf diminishes.The material of transcribing and translating of the activity of any SlZFP2 of reduction polypeptide, the stability that reduces SlZFP2 polypeptide, the expression that suppresses SlZFP2 polypeptide, minimizing SlZFP2 polypeptide effective acting time or reduction SlZFP2 all can be used for the present invention, as lower adjustment, antagonist or the inhibitor (that is: lowering the material of SlZFP2 expression of polypeptides) of SlZFP2, as the antibody of anti-described SlZFP2 polypeptide, disturb the disturbing molecule (as formed the disturbing molecule of microRNA) of the encoding gene expression of described SlZFP2 polypeptide.Described lower adjustment, antagonist or inhibitor can be used for regulating plant proterties, the quantity that particularly reduces plant branching or tiller; And/or reduce carotenoid and/or content of lycopene in plant (being preferably pulp class plant) fruit.Obtaining after cicada target sequence, it is well known in the art that the method for the disturbing molecule of specific gene expression is disturbed in preparation.
The invention still further relates to a kind of method that improves plant, the method comprises the expression that regulates SlZFP2 polypeptide in described plant.
On the one hand, the invention provides a kind of method of regulating plant proterties, described method comprises: make described plant overexpression SlZFP2 polypeptide, thereby: promote flowering of plant time advance; Postpone fruit maturation time; The quantity that increases plant branching or tiller; Increase carotenoid and/or content of lycopene in plant (being preferably pulp class plant) fruit; Reduce the height of plant plant; Reduce the weight of plant seed and/or reduce plant seed quantity (comprise and make plant not produce seed); And/or promote that plant leaf diminishes.
On the other hand, the present invention also provides a kind of method of regulating plant proterties, described method comprises:: reduce the expression (comprise SlZFP2 polypeptide is not expressed or low expression) of SlZFP2 polypeptide in described plant, thereby: the quantity that reduces plant branching or tiller; And/or reduce carotenoid and/or content of lycopene in plant (being preferably pulp class plant) fruit.
After the purposes of the SlZFP2 polypeptide described in cicada, can adopt several different methods well known in the art to regulate the expression of described SlZFP2 polypeptide.Such as can the ceneme (such as expression vector or virus etc.) that carry SlZFP2 gene being delivered on target spot by the known approach of those skilled in the art, and make it the SlZFP2 polypeptide of expression activity.
In addition, also can adopt several different methods well known in the art reduce the expression of SlZFP2 polypeptide or make it loss of expression, such as the ceneme of carrying antisense SlZFP2 gene (such as expression vector or virus etc.) is delivered on target spot, make cell or plant tissue not express or reduce and express SlZFP2 polypeptide.
As one embodiment of the present invention, the gene of coding SlZFP2 polypeptide is cloned in suitable carrier by conventional method, the described recombinant vectors with foreign gene is imported in the vegetable cell that can express described SlZFP2 polypeptide, make described vegetable cell express SlZFP2 polypeptide.Can, by by described vegetable cell regeneration plant, obtain the plant of overexpression SlZFP2 polypeptide.
Preferably, provide a kind of method of preparing transgenic plant, having comprised:
(1) encoding gene of the SlZFP2 polypeptide of external source is proceeded to vegetable cell, tissue, organ or tissue, obtain vegetable cell, tissue, organ or the seed of the encoding gene that is transformed into SlZFP2 polypeptide; With
(2) vegetable cell, tissue, organ or the seed regeneration plant plant of the encoding gene that has proceeded to external source SlZFP2 polypeptide step (1) being obtained.
As the preferred example of one, described method comprises step:
(s1) provide the Agrobacterium of carrying expression vector, the encoding gene that described expression vector contains SlZFP2 polypeptide;
(s2) vegetable cell, tissue, organ are contacted with the Agrobacterium in step (s1), thereby make the encoding gene of SlZFP2 polypeptide proceed to vegetable cell, and be incorporated on the karyomit(e) of vegetable cell;
(s3) select vegetable cell, tissue, organ or the seed of the encoding gene that proceeds to SlZFP2 polypeptide; And
(s4) by the vegetable cell in step (s3), tissue, organ or seed regeneration plant.
Other method that increases SlZFP2 gene or the expression of its homologous gene is that this area is known.For example, thus can be by driving and strengthen SlZFP2 gene or its homogenic expression with strong promoter.Or strengthen the expression of this SlZFP2 gene by enhanser (as paddy rice waxy gene First Intron, Actin gene First Intron etc.).The strong promoter that is applicable to the inventive method includes but not limited to: 35S promoter, the Ubi promotor of paddy rice, corn etc.
Preferably, also provide a kind of method that reduces the expression of SlZFP2 polypeptide in plant, described method comprises:
(1) disturbing molecule that disturbs SlZFP2 genetic expression is proceeded to vegetable cell, tissue, organ or seed, obtain the vegetable cell, tissue, organ or the seed that are transformed into described disturbing molecule; With
(2) vegetable cell that has proceeded to described disturbing molecule, tissue, organ or the seed regeneration plant that step (1) are obtained.
As the preferred example of one, described method comprises step:
(i) provide the Agrobacterium of carrying the carrier that can disturb genetic expression, described carrier is selected from lower group:
(a) contain the encoding gene of SlZFP2 polypeptide or the carrier of gene fragment (antisense molecule) that start in the other direction;
(b) carrier of the disturbing molecule of the composition that contains the encoding gene expression (or transcribing) that can form specificity interference SlZFP2 polypeptide in plant materials;
(ii) cell of plant, tissue or organ are contacted with the Agrobacterium in step (i), thereby make described carrier proceed to vegetable cell, tissue or organ.
Preferably, described method also comprises:
(iii) select the vegetable cell, tissue or the organ that have proceeded to described carrier; With
(iv) vegetable cell, tissue or neomorph in step (iii) are become to plant.
Based on the nucleotide sequence of SlZFP2 gene, can design the polynucleotide that can form the molecule of specificity interference SlZFP2 genetic expression after plant materials importing.When design, to consider the efficiency of specificity and interference.The present invention has no particular limits the preparation method of disturbing molecule, includes but not limited to: chemical synthesis, in-vitro transcription method etc.Should be understood that those skilled in the art are after obtaining the dependency of cicada SlZFP2 gene and plant trait, can prepare described disturbing molecule with various approach, thereby for regulating plant proterties.Described disturbing molecule can be transported in plant materials by transgenic technology, or also can adopt multiple technologies known in the art to be transported in plant materials.
As particularly preferred mode of the present invention, a kind of disturbing molecule of effect excellence is provided, described disturbing molecule can be specific the expression of interference SlZFP2 gene; And empirical tests, it has the effect of good interference SlZFP2 genetic expression.Described disturbing molecule is the molecule that contains the nucleotide sequence shown in 334-723 position in SEQ ID NO:1.After in it is transferred to plant materials, can form the disturbing molecule with nucleotide sequence shown in SEQ ID NO:1.
The present invention also provides a kind of disturbing molecule, and described disturbing molecule contains following structure: Seq forwardfor SlZFP2 gene fragment (being preferably the nucleotide sequence shown in 334-723 position in SEQ ID NO:1), Seq oppositelyfor with Seq forwardcomplementary polynucleotide; X is for being positioned at Seq forwardand Seq oppositelybetween intervening sequence, and described intervening sequence and Seq forwardand Seq oppositelynot complementary.
Described disturbing molecule, after importing in plant materials, can be folded into stable loop-stem structure, and the stem both sides of loop-stem structure comprise substantially complementary two sequences.Also, form secondary structure as follows:
Wherein, ‖ is illustrated in Seq forwardand Seq oppositelybetween complementary relation substantially.Described loop-stem structure can further be acted on, process or shear by the various materials in plant materials, and forms double-stranded RNA (dsRNA).
Conventionally, described disturbing molecule is positioned on expression vector.
The present invention also comprises the plant that utilizes aforementioned any method to obtain, and described plant comprises: proceeded to SlZFP2 gene or its homogenic transgenic plant; Or the plant that SlZFP2 expression of polypeptides amount (comprise low expression or do not express) reduces etc.
Can adopt any suitable conventional means, comprise that reagent, temperature, pressure condition etc. implement described method.
In addition, the invention still further relates to and utilize SlZFP2 polypeptide or its encoding gene tracking mark as a kind of gene transformation plant offspring.The invention still further relates to and utilize SlZFP2 polypeptide or its encoding gene as a kind of molecule marker, by detecting the expression of SlZFP2 polypeptide in plant, the proterties of plant identification.Also can utilize the plant characteristic of SlZFP2 gene-correlation as the cue mark of true hybrid in hybrid seeding process.
In addition, the present invention also provides the method for the potential material of the adjustable plant trait of screening.After the purposes of the SlZFP2 polypeptide described in cicada, can adopt several different methods well known in the art to screen the potential material of regulating plant proterties.In a kind of optimal way of the present invention, a kind of method of the potential material that screens regulating plant proterties is provided, described method comprises: candidate substances is contacted with the system of expressing SlZFP2 polypeptide, detect the impact of candidate substances on SlZFP2 polypeptide; If described candidate substances can improve or reduce the expression of SlZFP2 polypeptide, or the activity of promotion or inhibition SlZFP2 polypeptide, show that it is to can be used for regulating plant proterties.The material that these preliminary screening go out can form a screening storehouse so that people finally can therefrom filter out can be for the useful material of regulating plant proterties.
The growth cycle of crop is decision crop yield, plants adaptive very important factor, the flowering time of crop is controlled by multiple environmental factors and inherent gene, by the flowering time of genetic improvement crop, in agriculture production, there is important economic worth.The transcription factor SlZFP2 that the present invention finds acts on flowering time control gene conservative in each species, therefore on the flowering time that utilizes genetic improvement crop, may have ubiquity, can be applied to Different Crop.Utilize transcription factor involved in the present invention can also effectively increase or reduce the branch amount of plant, be applied to the Plant-type Breeding of crop, obtain ideotype.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, writes molecular cloning experiment guide, the third edition, Science Press, the condition described in 2002, or the condition of advising according to manufacturer conventionally as J. Pehanorm Brooker etc. according to normal condition.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Separation, vector construction and the transgenic method of embodiment 1, gene
Gene isolation and vector construction
SlZFP2 gene isolation is from tomato breeding lines LA1589 (Solanum pimpinefolium).Utilize total RNA that Trizol extracts to synthesize cDNA through reversed transcriptive enzyme, first use primer XP0034:5 '-ATGTTCCAGATTACGCTCTCGAGATGAGTTATGAACCAAACACGG-3 ' and XP0036:CGGGATCCAACATAATCGCCCTTGAGTAGA to amplify SlZFP2 encoding sequence, be cloned into pMD18-T carrier (purchased from Takara company), and then with XP0035:GCCACCATGGTTTACCCATACGATGTTCCAGATTACGCTCTCGAG and XP0036 from the above-mentioned target fragment that increases through the errorless plasmid that checks order, and PCR product is connected to pMD18-T carrier.Primer XP0035 is containing HA sequence label, for building HA-SlZFP2 fusion rotein.Order-checking determine errorless after, utilize restriction enzyme BamHI to cut out HA-SlZFP2 fragment, and be connected on the same Agrobacterium binary vector pHX20 cutting with BamHI enzyme, from pZH01, (pZH01 transforms from pCAMBIA1300 in pHX20 transformation, refer to document Xiao, H., et al., Functional analysis of the riceAP3homologue OsMADS16by RNA interference.Plant Mol Biol, 2003.52 (5): p.957-66).PHX20 is by reclaiming the large fragment that contains carrier framework after restriction enzyme SalI digestion pZH01, carry out carrier with T4DNA ligase enzyme and form from connecting, this carrier has been removed the GUS joining region between cauliflower mosaic virus CaMV35S promotor and agrobacterium tumefaciens rouge alkali synthetase NOS terminator.Obtaining expression plasmid pWL007 is p35S:HA-SlZFP2, and wherein HA-SlZFP2 is connected to constitutive promoter CaMV35S downstream, the upstream of NOS terminator.Plasmid imports to Agrobacterium GV3101 bacterial strain by heat shock method, for Plant Transformation.
Over-express vector pWL007 schematic diagram is as Fig. 1, wherein, and p35S, cauliflower mosaic virus CaMV35S promotor; HA, is derived from the albumen label of influenza virus hemagglutinin surface antigen (Human influenza hemagglutinin), and aminoacid sequence is YPYDVPDYA, and sequence is synthetic; Nos-t, agrobacterium tumefaciens rouge alkali synthetase NOS terminator.Except HA-SlZFP2, other DNA sequence dna is from plasmid skeleton pHX20.PHX20 contains the hygromycin gene Hyg that 35S drives r(selective marker while screening for transgenosis) and bacterium selective marker kalamycin resistance gene Kan r.
The structure of RNAi interference carrier pWL009 is taking pFGC5941RNAi binary vector (purchased from the ABRC of Arabidopis thaliana resource center) as skeleton, SlZFP2 sequence fragment (334-723 position in SEQ ID NO:1) is connected to AscI/SwaI site (forward connection) and BamHI/XbaI site (Opposite direction connection) on pFGC5941 in forward and reverse mode respectively, this plasmid information and detailed clone's step are referring to document Kerschen, A., et al., Effectiveness of RNA interference intransgenicplants.FEBS Lett, 2004.566 (1-3): p.223-8, the SlZFP2 fragment of this 401bp is to utilize primer XP0028:GCTCTAGAGGCGCGCCGAGTTGAGTTCAACCCTACG and XP0029:CGCGGATCCATTTAAATAATCGCCCTTGAGTAGAATC to come from LA1589cDNA amplification by PCR, underscore sequence is the artificial restriction enzyme enzyme recognition site adding, for vector construction.Plasmid imports to Agrobacterium GV3101 bacterial strain by heat shock method, for Plant Transformation.
RNAi carrier pWL009 schematic diagram as shown in Figure 2.PWL009 builds based on RNAi interference carrier pFGC5941.PFGC5941 contains foliage filter screening mark Bar gene and bacterium selective marker Kan rgene.CHSA Intron, petunia chalkane synthetase A gene (chalcone synthase A) intron.OCS3 ', 3 ' ploy A district of octopine synthase gene (OCS).
RT-PCR primer is as table 1.
Table 1, RT-PCR primer
Plant Transformation
Utilize the agriculture bacillus mediated method for transformation of cotyledon, respectively pWL007 and pWL009 are imported to tomato breeding lines LA1589 (the nearly source wild species of cultivation tomato, available from U.S.'s Tomato Germplasms center TGRC) and M82 (processed-type tomato, available from Israel Hebrew University ofJerusalem).PWL007 also utilizes agrobacterium-mediated transformation to proceed in rice varieties and spends 11.Tomato Agrobacterium-mediated Transformation is referring to McCormick, S., Transformation of tomato with Agrobacterium tumefaciens.In Plant Tissue CultureManual, Lindsey, K., ed. (Kluwer Academic Publishers, Dordrecht, TheNetherlands, 1991) .B6:p.1-9 method is carried out, rice conversion method adopts Hiei, Y., et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium andsequence analysis of the boundaries of the T-DNA.Plant J, 1994.6 (2): p.271-82 method.PWL007 transfer-gen plant screens by hygromycin B, and pWL009 utilizes glyphosate (PPT) screening.
Embodiment 2, SlZFP2 allelic expression and Subcellular Localization
1, allelic expression
Utilize the DNA sequence dna of SlZFP2, the nearly edge wild species of tomato LA1589 plant is carried out in situ hybridization and detect the expression of SlZFP2 in different tissues.The method document Coen that sees reference, E.S., et al., Cell, 1990.63 (6): p.1311-22.
Result is as Fig. 3.Show that SlZFP2 mainly expresses at spire (A), lateral bud (B), flower primordium (D), flower pesticide and ovule (F), seed (G and H).
2, the Subcellular Localization of SlZFP2
Fluorescence protein gene YFP is from Clontech company, the structure of control vector p35S:YFP be by pcr amplification by YFP gene clone to pHX20, p35S:SlZFP2-YFP fusion expression vector is to cut and be connected to corresponding site, the upper YFP sequence upstream of p35S:YFP through NotI enzyme after the complete encoding sequence of SlZFP2 is utilized to pcr amplification, forms fusion rotein.P35S:SlZFP2-YFP and p35S:YFP import respectively agrobacterium strains GV3101.
Result is as Fig. 7.Diagram infects the expression of Ben Shi tobacco N.benthamiana blade after 3 days containing the Agrobacterium of these two carriers respectively, and confocal laser scanning microscope is positioned at nucleus to SlZFP2-YFP signal, and a nuclear locating sequence of SlZFP2 coding is described.Detailed agroinfection and fluoroscopic examination step be referring to 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 tomato plant type, fruit and seed
1, the phenotype mutation analysis of Tomato Root System, fruit
As embodiment 1 prepares genetically modified tomato, observe SlZFP2 overexpression and suppress to express the phenotype at plant type, fruit and seed.As Fig. 4, in figure, except H is from M82 transgenosis, all the other are all from LA1589 transgenic line.OE represents the plant of the overexpression SlZFP2 being transformed by plasmid pWL007, and RNAi is the SlZFP2 expression inhibiting plant that pWL009 comes.#102,103,104 and 105 are respectively four independent plant that transform source, after isozygotying, are called strain.Each strain added up 15-20 fruit in 5 strains maturation change and 10 fruits in seed weight, 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; Disturbing the expression of SlZFP2 to affect plant by RNAi bears fruit.
2, transgenic Fructus Lycopersici esculenti (transforming LA1589) carotenoid content changes
By transcribing group analysis, find that SlZFP2 mainly expresses (Fig. 8 A) after tomato blooms, further utilize sxemiquantitative RT-PCR to verify that SlZFP2 expression is subject to fruit development regulation and control (Fig. 8 B).Carry out sequential analysis of protein by MEME software and show that SlZFP2 contains a C 2h 2zinc finger domain (structural domain 1 in Fig. 8 C) and one may participate in transcribing the structural domain (structural domain 2 in Fig. 8 C) of inhibition, and Fig. 8 D and E show the amino acid conservative property of these two structural domains.Root in Fig. 8 B figure, cotyledon, lower ovule are taken from 7-10 days LA1589 seedling, and before blooming, the fruit in petal, flower and each period is taken from the plant about 2 months.The first two letter of the each species ZFP2 protein name in Fig. 8 C figure represents the first letter of this species latin name.MEME analyzes the document Bailey that sees reference, T.L., et al., Nucl.Acids Res., 2006.34 (suppl2): p.W369-W373.
The carotenoid content of SlZFP2 Transgenic tomato fruit changes as table 2.
Table 2
The mensuration of carotene metabolite is referring to reference Fu, X., et al., J Exp Bot, 2012.63 (1): p.341-54.Mature Green and Turning represent that fruit is in the green fruit of maturation phase and Veraison.103N and 208N are respectively the non-transgenic contrasts (NonTrans) of pWL007 overexpression and pWL009RNAi transgenic line, they are respectively since same transgenosis T0 plant selfing separates, they have same genetic background compared with transfer-gen plant in contrast, because experience identical tissue culture procedures.
Result shows: overexpression SlZFP2 increases carotenoid and content of lycopene, suppress SlZFP2 and express and reduce carotenoid and content of lycopene, explanation can improve by the expression amount of regulation and control SlZFP2 the carotenoid substances content such as Lyeopene of fruit.
3, T1 is for Transgenic Tomato Plants plant type (transforming LA1589) and fruit maturation
The T1 that the transformed plant (102,103,104,105) of observation pWL007 conversion LA1589 is separated is for transgenosis and non-transgenic plant (NonTrans); Expression to SlZFP2 transgenosis in pWL007 transformed plant blade is analyzed; Detect the SlZFP2 protein level in rotaring gene plant blade; Observe transformed plant that pWL007 transforms LA1589 T1 for time the phenotype statistics of plant height, branch amount, flowering time and fruit maturation.
Result, as Fig. 9, illustrates that the tomato LA1589 transfer-gen plant of overexpression SlZFP2 has obvious branch amount object increase, and it is short that plant becomes, flowering time in advance, and fruit maturation obviously postpones, and have dosage effect, the transfer-gen plant effect of isozygotying is more obvious.
4, T2 is for Transgenic Tomato Plants plant type (transforming LA1589) and fruit maturation
The transformed plant (SlZFP2 (pWL007)) of observation pWL007 conversion LA1589 is crossed expression T2 and is changed for strain (102,103,104,105) fruit maturation, plant height, branch amount object changes, fruit size and number seeds.Result is as Figure 10, illustrate that contrast (NonTrans) compares with non-transgenic, the ripe 5-10 days that postpones of LA1589 transgenosis s-generation fruits/plant of overexpression SlZFP2, plant height obviously reduces and branch amount increases, and the fruit weight of some strain decreases.
5, in LA1589, overexpression SlZFP2 changes tomato plant blade size and lateral bud growth
Observe blade and the lateral bud of transformed plant that pWL007 transforms LA1589, found that overexpression SlZFP2 causes that blade diminishes, lateral bud dormancy reduces, as Figure 11.
6, the SlZFP2 overexpression plant phenotype of tomato M82
Observe the phenotype of the transfer-gen plant of pWL007 conversion M82, carry out sxemiquantitative RT-PCR gene expression analysis (B), the crosscut of fruits/plant is observed to square section, and transfer-gen plant (grey cylindricality) contrasts the fruit weight comparison of (black surround white background cylindricality) with non-transgenic.Result is as Figure 12, and in cultivation tomato variety M82, overexpression SlZFP2 causes plant branch obviously to increase (A), and affects the formation of seed in fruit, produces without seed or the few fruit (C-D) of seed.
7, do not contain the SlZFP2 overexpression plant phenotype of HA label
Build the not over-express vector p35:SlZFP2 containing HA label, be cloned between the 35S promoter and Nos terminator on pHX20 and (utilize restriction endonuclease BamHI/SalI to be connected between promotor and terminator) by SlZFP2 full length cDNA sequence.Plasmid p35S:SlZFP2 is by agriculture bacillus mediated importing tomato LA1589.
Result, as Figure 13, illustrates that in tomato LA1589, overexpression also can obviously not increase plant branch number and make plant Blooming containing the transfer-gen plant of HA label.
8, the grafting of SlZFP2 transfer-gen plant experiment
Grafting experimental result between tomato SlZFP2 overexpression plant and non-transgenic plant is as Figure 14, show that the branch number increase that SlZFP2 overexpression causes is mainly by affecting over-ground part, be the formation of lateral bud, and there is no obvious relation with underground part, illustrate that SlZFP2 overexpression can weaken the dormancy of lateral bud, promote the growth of lateral bud.After 103 graftings to 103, scion growth recovery is very slow, so although still have the phenotype that 103 blades are less, branch number is not as the plant to non-transgenic stock without grafting and grafting.
9, suppress SlZFP2 and express the impact on tomato growth and fruit development
RNAi interference carrier pWL009 has imported respectively tomato LA1589 and M82.Result is as Figure 15, and branch amount and flowering time are added up in 45 days left and right plant, and each strain has compared 3 transgenosiss and 3 the non-transgenic plant of separation from same transgenosis T0 plant.Percentage of germination has been added up the number that sprouts of each material in 7 days, and average germination rate is the seed required number of days that on average germinates after genotype (whether containing transgenosis) is determined in PCR qualification.While having compared the 6th day, in each strain, contain transgenosis (pWL009) simultaneously and do not contain genetically modified chitting piece number.Because of the RNAi plant difficulty of bearing fruit, number seeds is little, and each strain has only been analyzed 15-20 grain.Presentation of results, suppresses SlZFP2 genetic expression in tomato LA1589 and M82 by RNAi method, reduces plant branch number and postpones the sprouting speed of seed, but flowering time fruit size is not affected.
10, the expression in different tissues of SlZFP2 Accelerate bloom gene SFT
Overexpression SlZFP2 gene in tomato LA1589, observe floral genes SFT and spending, growing expression in fruit (latter 5 and 10 days of pollination) and ripe green fruit (fruit reaches final size, will enter the green fruit in stage of maturity).Result is as Figure 17, overexpression SlZFP2 gene reduces the SPGB genetic expression of being combined with SFT, SPGB expresses also lower than wild-type in tomato ABA synthesis mutant not, sitflc and the mutant sft of blooming (each mutant is all available from U.S.'s Tomato Germplasms center TGRC), illustrates that the expression of SlZFP2 regulation and control SFT and SPGB is by ABA route of synthesis.
The mechanism research of embodiment 4, SlZFP2 performance regulating and controlling effect
1, the synthetic mechanism of gene regulating dormin (ABA)
Analyze SlZFP2 overexpression and suppress the plant of expressing, the mechanism of research SlZFP2 performance regulating and controlling effect.As Fig. 5, show that overexpression SlZFP2 causes Stoma of Leaves to become large, seed germination speed improves, reduce the ABA content in the tissues such as flower and fruit, the expression of ABA synthase gene in overexpression plant leaf reduces, and disturb the expression of SlZFP2 by RNAi, reduce the expression of ABA synthase gene SIT and FLC in colored tissue, synthetic by conjunction with negative regulation ABA in the isogenic promotor of ABA synthase gene SlAO1 of SlZFP2.
Analyze SlZFP2 overexpression and suppress the plant of expressing, the mechanism of research SlZFP2 gene regulating plant blossom.Result is as Fig. 6.Show that overexpression SlZFP2 causes tomato to bloom in advance, SlZFP2 promotes that plant blossom is the expression that increases SFT by being directly combined in SFT promoter region.
2, the impact of overexpression SlZFP2 on blade ABA content
The relatively pore of SlZFP2 transgenosis LA1589 tomato plant blade, analyzes ABA processing and (sprouts 20 days left and right blades, after taking off, face up, be placed on containing 5mM KCl, 50 μ M CaCl 2, in 10mMMES-Tris (pH6.15) nutrient solution, under illumination condition, cultivate in the controlled environment chamber 2 hours, pore is opened, then blade is put into containing different concns (0,1.0,2.5,5.0 μ M) in the nutrient solution of ABA, continue to place 2 hours, then overlay film, electron microscopy observation, measures pore opening, the impact of the ABA of observation different concns on transfer-gen plant stomatal closure) impact of blade on stomatal movement, and the impact of ABA on three SlZFP2 transgenosis LA1589 plant seed germinations.Result is as Figure 16, show that transgenosis Stoma of Leaves is than non-transgenic plant leaf pore large (A), and to ABA more insensitive (B), the seed of gathering in the crops on overexpression plant in the time germinateing to the susceptibility of ABA also decrease (C).
3, the impact of overexpression SlZFP2 on blade ABA content
Measure the impact of overexpression SlZFP2 on blade ABA content, the measuring method of ABA is shown in that Fig. 5 illustrates.Result is as Figure 19, and in tomato LA1589, overexpression SlZFP2 reduces the ABA content in blade, the ABA in especially old blade.Material is the transfer-gen plant that isozygotys that pWL007 turns LA1589, and when sampling, non-transgenic plant has 17 visible leaves, and transfer-gen plant has 12 visible leaves, is after planting 45 days plant.
4, SlZFP2 participates in the expression level of regulation and control ABA synthase gene
Expression by each ABA synthase gene of qRT-PCR quantitative analysis in the SlZFP2 of LA1589 rotaring gene plant blade, result is as Figure 20.Materials and methods is consistent with Fig. 5 E, is same batch of experimental result, Fig. 5 E only show portion gene therein a period (genetically modified L6 and not genetically modified L10) data.Result shows, in the SlZFP2 overexpression plant of tomato LA1589, ABA synthase gene NOT, SIT, SlAO1, SlAO2 and the FLC expression in different development stage blade shows decline in various degree, correspondingly be that the expression of ABA transport agent SlABCG40 gene is also suppressed.
5, suppress that the expression in tomato of SlZFP2 gene is synthetic on ABA, the expression impact of transhipment and responsive genes
The expression level that research ABA synthesizes, transports and response genes involved is spent at several RNAi (pWL009) transformation plant.Result is as Figure 21, the SlZFP2 suppressing in tomato LA1589 by RNAi method expresses, expression impact on ABA synthase gene NOT and SlAO1 is not obvious, the expression of major effect SIT and FLC, the expression of ABA transport vehicle SlABCG40 is also affected not obvious, but ABA responsive genes Le16 and Le25 obviously rise.
Embodiment 5, SlZFP2 are for the impact of the proterties of paddy rice
To in plasmid pWL007 Introduced into Rice kind, spend 11, rice conversion is referring to reference Hiei, Y., et al., Plant J, 1994.6 (2): p.271-82.
Transfer-gen plant T0 for the tillering number statistics in flowering period as shown in figure 18.10 independent transformation plants all show as tiller number in transgenosis the present age to be increased.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (14)

1. a method for regulating plant proterties, is characterized in that, described method comprises: the expression of SlZFP2 polypeptide in regulating plant.
2. the method for claim 1, is characterized in that, described method also comprises subsequent step: from regulate the plant the expression of SlZFP2 polypeptide, select the plant of comparing control plant proterties and obtain adjusting.
3. the method for claim 1, is characterized in that, described plant is selected from: grass, plant of Solanaceae.
4. the method for claim 1, is characterized in that, described SlZFP2 polypeptide is selected from lower group:
(a) as the polypeptide of SEQ ID NO:2 aminoacid sequence;
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have (a) polypeptide function by (a) derivative polypeptide; Or
(c) with (a) limit peptide sequence have 80% above homology and have (a) polypeptide function by (a) derivative polypeptide.
5. the method for claim 1, is characterized in that, described method comprises: the expression that improves SlZFP2 polypeptide in plant; Thereby:
Promote flowering of plant time advance;
Postpone fruit maturation time;
The quantity that increases plant branching or tiller;
Increase carotenoid and/or content of lycopene in fruit;
Reduce the height of plant plant;
Reduce the weight of plant seed and/or reduce plant seed quantity; And/or
Promote that plant leaf diminishes.
6. method as claimed in claim 5, is characterized in that, comprising: the polynucleotide of coding SlZFP2 polypeptide are proceeded to plant, obtain the plant that is transformed into described polynucleotide.
7. method as claimed in claim 6, is characterized in that, comprising:
(S1) provide the Agrobacterium of carrying expression vector, described expression vector contains polynucleotide, its coding SlZFP2 polypeptide;
(S2) cell or tissue of plant or organ are contacted with the Agrobacterium in step (S1), thereby make described polynucleotide proceed to vegetable cell, tissue, organ or seed.
8. the method for claim 1, is characterized in that, described method comprises: reduce the expression of SlZFP2 polypeptide in plant, thereby:
The quantity that reduces plant branching or tiller; And/or
Reduce carotenoid and/or content of lycopene in fruit.
9. method as claimed in claim 8, it is characterized in that, in described reduction plant, the expression of SlZFP2 polypeptide comprises: the disturbing molecule that disturbs the encoding sequence of described SlZFP2 polypeptide to express is proceeded to vegetable cell, tissue, organ or seed, thereby lower the expression of SlZFP2 polypeptide in plant.
10. SlZFP2 polypeptide or its encoding gene purposes, for regulating plant proterties.
11. purposes as claimed in claim 10, is characterized in that, described SlZFP2 polypeptide or its encoding gene are used for:
Promote flowering of plant time advance;
Postpone fruit maturation time;
The quantity that increases plant branching or tiller;
Increase carotenoid and/or content of lycopene in fruit;
Reduce the height of plant plant;
Reduce the weight of plant seed and/or reduce plant seed quantity;
Promote that plant leaf diminishes; And/or
As the molecule marker of plant identification proterties.
12. 1 kinds are reduced the purposes of the material of SlZFP2 expression of polypeptides, for:
The quantity that reduces plant branching or tiller; And/or
Reduce carotenoid and/or content of lycopene in fruit.
13. 1 kinds are disturbed the disturbing molecule of described SlZFP2 genetic expression, and it contains the structure shown in formula (I):
Seq forward-X-Seq oppositelyformula (I),
In formula (I),
Seq forwardfor SlZFP2 gene fragment, Seq oppositelyfor with Seq forwardcomplementary polynucleotide;
X is for being positioned at Seq forwardand Seq oppositelybetween intervening sequence, and described intervening sequence and Seq forwardand Seq oppositelynot complementary.
14. 1 kinds of carriers, is characterized in that, described carrier contains the disturbing molecule described in claim 13.
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