CN102604963B - Separation, cloning and application of Poncirus trifoliata EARLYFLOWERING 5 (PtELF5) gene - Google Patents

Abstract

The invention relates to separation, cloning and application of Poncirus trifoliata EARLYFLOWERING 5 (PtELF5) gene, in particular to the separation of the PtELF5 gene from Poncirus trifoliata and the cloning of the PtELF5 gene. The nucleotide sequence of the PtELF5 gene is SEQ ID No: 1 as shown in the sequence table, and the corresponding amino acid sequence is SEQ ID No: 2 as shown in the sequence table, comprises a 1623bp open reading frame and 540 encoded amino acids and has the isoelectric point of 9.62 and the predicted molecular weight of 57.2kD. After the PtELF5 gene is introduced intoarabidopsis to verify the function, the obtained transgenic arabidopsis plant flowers early obviously, and the vegetative growth period of the transgenic arabidopsis plant is shortened obviously. It proves that the PtELF5 gene functions as the positive regulatory factor in Poncirus trifoliata flowering regulating process, and the PtELF5 gene can be over-expressed to culture the novel variety of Poncirus trifoliata with a short juvenile phase.

Description

Separating clone and the application of oranges and tangerines early blossoming gene PtELF5

Technical field

The present invention relates to the plant gene engineering technology field.Be specifically related to separating clone, functional verification and application thereof that oranges and tangerines become to spend genes involved PtELF5.The PtELF5 gene is the positive regulatory factor that oranges and tangerines become a key in the flower process, overexpression PtELF5 significantly shortening the juvenile phase the model plant Arabidopis thaliana the virgin phase, cause early blossoming.So the positive regulatory factor that this gene may be oranges and tangerines blooms impels the oranges and tangerines early flowering, enter the Adulthood then can lay the foundation for accelerating the oranges and tangerines breeding.

Background technology

Fruit tree mostly is greatly the perennial woody plant, will experience the very long virgin phase from planting seed to blossoming and bearing fruit first.The virgin phase short such as peach, Lee, 3-4 is bloomed, long such as oranges and tangerines, walnut, 8-12 is bloomed, longer such as ginkgo, Chinese torreya etc., the virgin phase reaches ten several years even many decades.Because body volume is large, Tong Qichang, genetic background are complicated, has greatly hindered the perennial woody plant, particularly genetics research, breeding process and the new variety take results floral organ, fruit and seed as main fruit tree and commodity trees are promoted.Therefore, disclose the virgin phase growth of orchard fruit and Flowering mechanism and have important theory and practice meaning.

Oranges and tangerines are the large fruit of the first in the world, are the large international trade agricultural-food in the third place in the world that is only second to wheat and maize.Because the virgin phase of oranges and tangerines is longer, is generally 6-8 and does not wait.The long virgin phase makes the genetic breeding cycle of oranges and tangerines extremely very long, adds that nucellus embryo disturbs, the factor affecting such as sexual organ abortion and the upper height of heredity heterozygosis so that the research of the conventional breeding of oranges and tangerines and genetic mechanism difficult, make slow progress.Therefore disclose the molecule mechanism of oranges and tangerines Flowering mechanism, not only help to accelerate the oranges and tangerines breeding process, change oranges and tangerines and bear fruit period and fruit ripening stage, and have an important theory significance, for the development of Genetic Mechanisms research, breeding practice and production tech potential far reaching significance is arranged also.

We are under the early stage grant of national natural science foundation, and having excavated has the real trifoliate orange of the early blossoming variation of important value-early (promise that genetic resources disclosure of origin registry form is provided and provides this resource proves for Pang, et al.2006) to the virgin phase research of xylophyta.Real trifoliate orange (Precocious trifoliate orange early, Poncirus trifoliata L.Raf.) is the strain variation of finding in the Yichang, namely have then 20% to bloom in sowing, Second Year can all be bloomed, and have the characteristic (Zhang, the et al.2008 that downgrade and several times a year bloom; Zhang, et al.2009; Zhang, et al.2009).Early real trifoliate orange only needs just can enter generative growth phase through of short duration vegetative growth phase, so be considered to study preferably the material of xylophyta Flowering determination as a natural trifoliate orange class early blossoming mutant.Subtract hybridization (2D-SSH) by real trifoliate orange early and common trifoliate orange are suppressed difference in the two dimension of virgin phase and Adulthood respectively early stage, screen 532 ESTss relevant with phasic development and become the flower genes involved with tens of, such as PtTFL1, PtFLC, PtFT, PtBAM and PtF-box etc.Wherein the PtELF5 gene is a new gene that is separated in the real trifoliate orange morning, the function between them is opposite (Noh et al because existing certain homology to name still at encoding sequence with the EARLYFLOWERING 5 (ELF5) of Arabidopis thaliana for it, 2004), it is the repressor gene of blooming in Arabidopis thaliana, is a cone production gene in oranges and tangerines.Find by in situ hybridization, obviously raise at bud differentiation conversion stage expression amount.In the process that becomes flower to change, can suppress the to bloom expression of repressor gene PtFLC of this gene.In addition, this gene of overexpression can significantly shorten the virgin phase of plant, Blooming in the model plant Arabidopis thaliana.A lot of floral genes that separate all are the homologous genes of Arabidopis thaliana in the past, and excavate less to the own peculiar gene of xylophyta, although and this gene has certain homology with Arabidopis thaliana ELF5, but homology is lower, and functions reversed, we infer that this gene is the distinctive early blossoming gene of xylophyta, may have more widely aspect the virgin phase the shortening xylophyta to use.

Summary of the invention

The object of the invention is to separate the gene of a virgin phase growth of control oranges and tangerines, this gene does not all have homologous sequence in model plant and nearly source species, only there is certain similarity in the EARLYFLOWERING 5 (ELF5) with Arabidopis thaliana, the applicant is with its called after PtELF5, but ELF5 is one and checks floral genes in Arabidopis thaliana, and PtELF5 is into the flower promotion factor in the present invention, and this gene can shorten virgin stage phase of plant greatly.Can cultivate greatly shortening and economic worth and the higher Citrus Cultivars of quality of a kind of virgin phase so use this gene.

The present invention is achieved through the following technical solutions:

The present invention is from Fructus Aurantii (Precocious trifoliate orange, Poncirus trifoliata L.Raf.) separates the new gene PtELF5 that obtains the virgin phase growth of a regulating plant in, the nucleotide sequence of this gene is (wherein 137-1759 is the coding region of this gene) shown in sequence table SEQ NO:1, its coding aminoacid sequence shown in sequence table SEQ ID NO:2, its 540 amino acid of encoding.

Described gene PtELF5 grows relevant with the virgin phase of oranges and tangerines.The rear model plant that directly changes over to is combined in the complete translation district (Coding sequence) of this gene with 35S promoter, transfer-gen plant is compared with the wild-type contrast and has been presented extreme early blossoming, and do not have the floral organ unusual phenomenon, lay a good foundation for further utilizing this gene; And find that by in situ hybridization this gene has presented very high expression in the process that becomes flower to change, illustrate with become to spend in close relations.Can suppress the to bloom expression of repressor PtFLC of this gene is found in real-time quantitative research, then causes the plant early blossoming.

The present invention provides a kind of new method for shortening the virgin phase of xylophyta.The method is by transgenic technology overexpression PtELF5, suppresses to bloom and suppresses expression and then the shortening the juvenile phase of sub-PtFLC.Adopting this class transgenic technology to create short child phase plant is that traditional breeding technology institute is inaccessiable.

In the embodiments of the invention part, the applicant has set forth separation, functional verification and the application process of PtELF5 gene and the characteristics of this gene.For the oranges and tangerines breeding lays the foundation.More detailed technological invention details is provided by following examples, but is not the protection domain that limits this invention.

Description of drawings

Sequence table SEQ ID NO:1 is the nucleotide sequence of the PtELF5 gene of separating clone of the present invention, and sequence length is 2271bp, and wherein 137-1759 is the coding region.

Sequence table SEQ ID NO:2 is the aminoacid sequence of PtELF5 genes encoding.

Fig. 1 is techniqueflow chart of the present invention.

Virgin phase of the early real trifoliate orange of Fig. 2 (A) and Adulthood total RNA agarose electrophoresis detected result, M:DNA molecular weight standard DL20001: total RNA of virgin phase material; 2: total RNA of Adulthood material.(B) the mRNA electrophoresis detection result that separates with the Adulthood of virgin phase of real trifoliate orange early, M:DNA molecular weight standard DL2000; 1: the mRNA of virgin phase material; 2: the mRNA of Adulthood material.(C) the joint joint efficiency is analyzed, M:DNA molecular weight standard DL2000; 2,4:Tester-1 and Tester-2 increase with the inner special primer of Actin; 1,3:Tester-1 and Tester-2 use respectively the inner special primer of Actin and joint primer amplification.(D) 24 clones for detection of the Insert Fragment size that randomly draw.(E) PCR detects the poor efficient that subtracts.

The screening of the reverse northern of Fig. 3 subtracted library, arrow represents positive colony.(A) film of the reverse probe hybridization of expression.(B) film of expression forward probe hybridization.

Fig. 4 (A) PtELF5 and PtFLC are at the relative quantification in the real trifoliate orange early of virgin phase.(B) PtELF5 and PtFLC are at the Adulthood relative quantification in the real trifoliate orange early.(C) PtELF5 and PtFLC are at the Adulthood relative quantification in the real trifoliate orange different tissues early.Wherein the spring tip is the meristematic tissue on top, and fruit is for spending rear 30 days.The expression of all genes Actin homogenization, all experiments are carried out 4 times and are repeated.

Fig. 5 detects PtELF5 (a, b, c, d) and PtFLC (e, f, g, h) in the expression of different etap of real trifoliate orange apical meristem early by in situ hybridization.(a, e) is the meristematic tissue on virgin phase top.(b, f) is the Adulthood nutrition apical meristem in period.(c, g) is the flower differentiation apical meristem in period.(d, h) is for using up full differentiation period.(i) be the results of hybridization with just PtELF5.Scale is 100 microns.

Fig. 6 is that the plant overexpression vector 35S::PtELF5 of one of them embodiment of the present invention makes up flow process.

The phenotype analytical of Fig. 7 PtELF5 in Colombia's type Arabidopis thaliana.A: be grown in wild-type Arabidopis thaliana under the long day condition; B: be grown in the transgenic arabidopsis under the long day condition.The transfer-gen plant (d) of comparing with wild-type (c) does not in addition present the unusual of floral organ, illustrates that this gene only is the bloom controlling gene.

Embodiment

Embodiment 1: utilize the poor relevant regulatory gene of hybridization separation that subtracts of inhibition

1.1 vegetable material and RNA separate

Mutant " early real trifoliate orange " (Zhang Jinzhi etc. with Fructus Aurantii (Poncirus trifoliata L.Raf.), Identification ofearly-flower-related ESTs in an early-flowering mutant of trifoliate orange (Poncirus trifoliata) by suppression subtractive hybridization and macroarray analysis.Tree Physiology 200828:1449-1457) be parent material, getting the spring tip of real trifoliate orange different developmental phases morning and the meristematic tissue on virgin phase different times top is material, Adulthood and virgin phase " early real trifoliate orange " all is planted in country of Hua Zhong Agriculture University oranges and tangerines breeding central nursery, morning Adulthood, real trifoliate orange was the tree of 3-5, and repeatedly bloomed.Because the spring tip is that early real trifoliate orange becomes the main position of flower, thus the Adulthood material take the terminal bud of the spring tip and five buds subsequently as material, respectively the natural time 1,3,5,7,9, draw materials November.Early real trifoliate orange seed is being transplanted after the successful vernalization under the condition of self-sow, water with nutritive medium during this time, guarantee that its upgrowth situation is good, the material of virgin phase respectively 3,6,9, get apical meristem December, Adulthood and virgin phase are got respectively three trees when drawing materials, liquid nitrogen flash freezer-80 ℃ preservation after mixing.

Morning of different times, real trifoliate orange tissue was according to the method isolation of RNA (Zhang, et al.2008) of Zhang Jinzhi etc.Utilize Clontech PCR-Select ^TMCDNA Subtractive Kit (Clontech, USA) carries out that inhibition is poor to subtract hybridization, makes up respectively the library that positive contrast subtracts.In the forward library, the virgin phase, the Adulthood obtained the gene relevant with early blossoming like this as detecting son (Tester) in the forward subtracted library as driveing son (Driver); In reverse library, the virgin phase, the Adulthood, concrete grammar was as follows as driveing son as detecting son:

1.2 the first chain cDNA is synthetic

1) for Tester, each is mixed in Driver in the 0.2ml centrifuge tube in the following order;

mRNA(4μg) 2-4μl

cDNA synthesis Primer(10μmol/L) 1μl

Add water without the RNA enzyme to cumulative volume 5 μ l such as needs;

2) 70 ℃ of 2min in the PCR instrument place renaturation 2min on ice; Of short duration centrifugal;

3) every pipe adds following component:

Table 1: the composition of the first chain reverse transcription (test kit provides)

4) light shaking and briefly centrifugal, in the PCR instrument, 42 ℃ 1.5 hours;

5) place on ice, stop synthetic article one chain, and enter immediately next program.

1.3 the second chain cDNA is synthetic

1. add following composition in the synthetic reaction tubes of the first chain cDNA:

Table 2: the composition (test kit provides) that the second chain is synthetic

2. mixing is also briefly centrifugal, and 16 ℃ of temperature were bathed 2 hours, added 2 μ l T4DNA Polymerase mixings;

3.16 ℃ 30min adds 4 μ l, 20 * EDTA/Glycogen Mix termination reaction;

4. add 100 μ l phenol: chloroform: primary isoamyl alcohol (volume ratio: 25: 24: 1), fully concussion, the centrifugal 10min of 14000g/min;

5. shift supernatant to and newly manage, add 100 μ l chloroforms: primary isoamyl alcohol (volume ratio: 24: 1) is to water;

6. in the supernatant that separates, add 40 μ l 4mol/L ammonium acetates and 300 μ l, 95% ethanol, the mixing that fully vibrates, the centrifugal 20min of room temperature 14000g/min; Abandon supernatant, add the ethanol of 500 μ l 80%;

7. beat mixing, centrifugal 10 minutes of room temperature 14000g/min with careful suction of pipettor;

8. carefully abandon supernatant liquor, dry air 10 minutes with the water dissolution of 50 μ l, is got 5 μ l in another pipe, is stored in-20 ℃.

1.4RsaI enzyme cut

1) add following ingredients in a centrifuge tube:

Table 3: the composition of endonuclease reaction (test kit provides)

2) concussion mixing and briefly centrifugal, 37 ℃ of enzymes were cut 6 hours;

3) get 5 μ l enzymes and cut the product analysis enzyme and cut efficient, add 2.5 μ l 20x EDTA/Glycogen Mix termination reactions;

4) add 50 μ l phenol: chloroform: primary isoamyl alcohol (volume ratio is 25: 24: 1), fully shake mixing;

5) the centrifugal 10min of 14000g/min shifts the new 0.5ml centrifuge tube of supernatant liquor to;

6) add isopyknic chloroform: primary isoamyl alcohol (volume ratio is 24: 1), fully shake mixing, the centrifugal 10min of 14000g/min shifts supernatant in a new 0.5ml pipe;

7) add 25 μ l 4mol/L ammonium acetates and 187.5 μ l, 95% ethanol, fully shake complete mixing;

8) the centrifugal 20min of 14000g/min adds 200 μ l, 80% ethanol;

9) the centrifugal 10min of 14000g/min, air-dry 5-10min on the super clean bench is dissolved in 5.5 μ l sterilized waters ,-20 ℃ of preservations;

10) the 5 μ l enzymes that will reserve are cut the double-stranded cDNA that product and previous step retain and are utilized 1.2% agarose to carry out electrophoresis detection.

1.5 the connection of joint

1) cuts from μ l enzyme of upper steps 5.5 and get 1 μ l the cDNA and add 5 μ l sterilized waters;

2) mixed solution (Master Mix) of preparing jointing is put into a 0.5ml centrifuge tube and is added following component:

Table 4: the composition of ligation (test kit provides)

3) system that connects is got enzyme after the dilution and is cut Tester cDNA and be divided into two pipes, adds different joints and forms two linked systems:

Table 5: the composition of ligation (test kit provides)

4) in a new pipe, mix 2 μ l Teste1 and 2 μ l Tester 2;

5) with above respectively manage brief centrifugal mixing after, and bathe in 16 ℃ of temperature and to spend the night;

6) add 1 μ l EDTA/Glycogen Mix termination reaction;

7) 72 ℃ of 5min deactivation T4 ligase enzymes are of short duration centrifugal;

8) get the not poor cDNA of subtracting and be diluted in 1ml H ₂Among the O, be used for the pcr amplification of back, preserve sample in-20 ℃.

1.6 the detection of joint efficiency

1) Tester-1 after the ligation and Tester-2 are as template, respectively take 3 of housekeeping gene Actin ' (ActinS:5 '-CCGACCGTATGAGCAAGGAAA-3), 5 ' end (ActinA:5 '-TTCCTGTGGACAATGGATGGA-3 ') and PCR primerl (5 '-CTAATACGACTCACTATAGGGC-3 ') carry out pcr amplification as primer, relatively the amplification efficiency of the corresponding product of different primers is judged joint efficiency.

2) dilution connects product (Tester 1 and Tester 2), respectively gets 1 μ l and joins respectively in the sterilized water of 100 μ l, mixes following component in 4 centrifuge tubes:

The main primer composition of table 6 detection system

3) prepare mixed reaction solution (Master Mix), be sequentially added into following composition:

The main primer composition (test kit provides) of table 7 detection system

4) fully shake mixing;

5) respectively get in the pipe in 2 steps of 22 μ l Master Mix to the, fully shake mixing;

6) temperature is bathed 75 ℃ of 5min of hybrid reaction system, begins immediately thermal cycling (can not take out from the PCR instrument):

94℃ 30s

7) every system is got 5 μ l and is detected in 2.0% agarose/EB glue.

1.7 for the first time hybridization

1) poor subtract hybridize before, first 4 * Hybridization buffer room temperature is placed 30min, if there is precipitation, heat at 37 ℃ and to come dissolution precipitation in 10 minutes.Each library is carried out respectively forward and oppositely poorly subtracted hybridization, and the forward alternative is as follows, and is reverse then be forward detection sample and the exchange of driveing sample;

Table 8 is hybridized the major ingredient (test kit provides) of system for the first time

2) cover sample with a mineral oil, and of short duration centrifugal, make mineral oil cover the surface of sample fully;

3) after sample places 98 ℃ of 1.5min;

4) hybridize 8h (note to surpass 12h scarcely, after the time, enter next stage immediately) in 68 ℃ of PCR instrument.

1.8 for the second time hybridization

Attention: the sample with hybridization does not shift out the PCR instrument.Two samples after the hybridization will be mixed to together for the first time, add immediately fresh sex change driver cDNA.

1) following component is packed into a sterile tube:

Table 9 is hybridized the major ingredient (test kit provides) of system for the second time

2) get 1 μ l said mixture to a 0.5ml pipe;

3) 98 ℃ of temperature are bathed 1.5min;

4) carry out following program to reach the purpose that hybridization sample 1,2 and fresh driver are mixed simultaneously;

5) take out the driver of just sex change from the PCR instrument, can mix simultaneously driver and hybridization sample 1 and 2 by following steps, can guarantee that like this driver of two hybridization samples and new sex change mix simultaneously;

A) liquid-transfering gun is transferred to 15 μ l;

B) gently the rifle head is touched mineral oil and the interface of hybridizing sample 2;

C) will hybridize sample 2 all sucks in the rifle head;

D) from pipe, take out rifle head and suck a small amount of air;

E) repeating step b-d sucks new driver cDNA in the rifle head;

F) sample that mixes in the rifle head is all changed over to hybridization sample 1 place centrifuge tube;

G) repeatedly inhale up and down and beat abundant mixing;

6) the brief centrifugal sample that makes all is sunken to the pipe end, and 68 ℃ are spent the night;

7) add 200 μ l lysates (test kit provides) to reaction tubes, repeatedly inhale and beat mixing;

8) 68 ℃ of 7min are stored in-20 ℃ of refrigerators.

Subdue effect 1.9 utilize Actin to detect

1. get respectively each the 3 μ l of inhibition PCR product and add 27 μ l distilled waters and do 10 times of dilutions second time that do not carry out behind subtractive hybridization and the subtractive hybridization, the product after the dilution is as template, with Actin primer checking Using Suppression Subtractive Hybridization effect respectively;

2. from above every pipe, get 1 μ l and add respectively the 0.5ml centrifuge tube;

3. base fluid (Master Mix) is reacted in preparation in the following order:

Table 10 detects the main PCR composition of subduing effect reaction

4. abundant mixing, each pipe in 2 steps adds reaction base fluid 24 μ l;

5. add a dropstone wax oil, the simple centrifugal liquid that makes was gathered in the pipe end;

6.PCR amplification, loop parameter is:

94℃ 30s

7. after finishing 18 circulations, cancellation subtracts and does not subdue each 5 μ l of PCR product and places new centrifuge tube respectively, gives over to follow-up checking.Remain 20 μ lPCR products and again put back to the PCR instrument and proceed circulation by aforementioned condition, equally in 23,28 and 33 circulations, cancellation subtracts and does not subdue each 5 μ l of product and gives over to electrophoresis respectively;

8. get respectively PCR product 5 μ l electrophoresis observation product formational situation on 1XTBE damping fluid 2.0% agarose gel of above-mentioned different cycle indexes.

1.10 twice PCR amplification

1) preparation of template: the hybridization sample and the not poor Tester of subtracting of 1 μ l that get 1 μ l dilution are put in the 0.2ml centrifuge tube;

2) the according to the form below order adds following reagent preparation PCR Master Mix in new centrifuge tube:

The major ingredient of for the first time PCR reaction of table 11

3) quick centrifugal mixing adds 24 μ l MasterMix in each sample;

4) add a mineral oil in each centrifuge tube, 75 ℃ of temperature are bathed the 5min extension sub;

5) begin immediately PCR:

94℃ 5min

72℃ 10min

6) each sample is respectively got 8 μ l for analyzing PCR result;

7) getting for the first time, PCR product 3 μ l are diluted in the 27 μ l sterilized waters;

8) diluent of getting 1 μ l step 7 is put in the good centrifuge tube of mark;

9) according to the form below sequentially adds for the second time PCR Master Mix of following ingredients preparation in centrifuge tube:

The major ingredient of for the second time PCR reaction of table 12

10) fully mixing is also centrifugal fast, adds 24 μ lMaster Mix in the centrifuge tube of step 10;

11) PCR that brings into operation:

94℃ 25s

72℃ 10min

12) get 8 μ l and be used for analyzing PCR result;

13) be stored in-20 ℃ for subsequent use;

1.11PCR the retrieve and purification of product

Amplified production is put into 2.0ml centrifuge tube with careful cutting-out of fragment with clean blade through behind the agarose gel electrophoresis under ultraviolet lamp, operation is reclaimed the test kit specification sheets by Shanghai biotechnology company limited gel and carried out:

(1) add Binding Buffer in the ratio of 500 μ l/100mg sepharoses, place 55 ℃ of water-baths, add thermosol, every 2min mixing once, until glue thoroughly melts;

The Binding Buffer that (2) will contain the purpose fragment is transferred in the UNIQ-10 post that is placed in the 2ml collection tube, and room temperature is placed 2-3min, the centrifugal 0.5min of 12000g/min;

(3) take off the UNIQ-10 post, outwell the waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, add the Washbuffer of 500 μ l, the centrifugal 0.5min of room temperature 12000g/min;

(4) repeating step is 3 times;

(5) take off the UNIQ-10 post, again the UNIQ-10 post is put into collection tube, the centrifugal 2-3min of room temperature 12000g/min;

(6) the UNIQ-10 post is put into the aseptic centrifuge tube of a 1.5ml, added the sterilized water of 25 μ l in pillar film central authorities, 5min are placed in 55 ℃ of water-baths;

(7) the centrifugal 2-3min of room temperature 12000g/min, the liquid of collecting in the 1.5ml centrifuge tube is the dna fragmentation of recovery.

1.12 the preparation of competent cell

(1) picking DH5 α intestinal bacteria original seed bacterium, streak culture on the LB agar plate with transfering loop, fresh single bacterium colony that picking white is full is inoculated in the 100ml LB liquid nutrient medium, and 37 ℃ of constant-temperature tables are cultured to OD ₆₀₀Reach 0.4-0.5;

(2) will shake the aseptic 50ml centrifuge tube that muddy bacterium liquid is transferred to a precooling, ice bath 10min, 4 ℃ of centrifugal 10min of 4500g/min outwell supernatant to reclaim precipitation;

(3) the 0.1mol/L CaCl of adding 10ml precooling ₂The suspension bacterial precipitation, ice bath 30min, 4 ℃ of centrifugal 10min of 4500g/min outwell supernatant and again reclaim bacterial precipitation;

(4) add 3ml 0.1mol/LCaCl ₂Solution Eddy diffusion bacterial precipitation;

(5) glycerol adding is to final concentration 15%-20%, and mixing is distributed into every pipe 100 μ l, and is frozen in-80 ℃.

1.13 the clone of purpose product

Purpose product and 16 ℃ of connections of pGMT carrier (Promega, the U.S.) of purifying are spent the night.The ligation system is: 10 * Ligation Buffer1.0 μ l, purified product 5.0 μ l, T4DNA ligase 1.0 μ l, pGMT carrier 1 μ l, ddH ₂O 2 μ l totally are 10 μ l.

(1) on the LB flat board that contains 50 μ g/ml penbritins, smear 10 μ l IPTG (beta-galactosidase enzymess, 100mg/ml) (5-bromo-4-chloro-3-indoles-β-D-galactoside 20mg/ml), is secretly putting about 1h on the super clean bench with 40 μ lX-Gal;

(2) take out colibacillary competence, place freeze thawing on ice, add 10 μ l and connect product, inhale gently with liquid-transfering gun and beat mixing, place 30min on ice;

(3) heat shock: with centrifuge tube at 42 ℃ of lower water-bath 90s;

(4) iced: as fast centrifuge tube to be transferred to ice bath, 2-3min;

(5) recovery: every pipe adds 450 μ l liquid LB substratum, turn at 37 ℃ of shaking tables 250/min shakes 1h;

(6) spread ware: get 100 μ l bacterium liquid and evenly coat on the LB flat board that has prepared;

(7) cultivate: 37 ℃ of lower inversions are cultivated about 16h, namely can be observed blue white alternate bacterium colony.White colony is the transformant that contains the external source Insert Fragment, and blueness is that carrier is from the transformant that connects;

(8) evaluation of positive colony: select white single bacterium colony with sterilizing toothpick, put into the triangular flask that fills 5ml liquid LB substratum (50 μ g/ml penbritin), overnight incubation (12-16h) on 37 ℃ of shaking tables.

1.14 the structure of subtracted library

1) in each hole of 384 hole microbial culture plates, adds 50 μ l LB liquid nutrient mediums (containing penbritin 50 μ g/ml), in single colony inoculation 384 well culture plates of picking white;

2) cultivate 12h for 37 ℃, visible substratum becomes muddy, in each culture hole, adds the glycerine of sterilization, to final concentration 15%;

3) culture plate is placed under 4 ℃ leave standstill 2d, behind the complete mixing of glycerine ,-80 ℃ of preservations.

1.15 the preparation of hybridization probe

The specification sheets of the DIG High Primer DNA Labeling and Detection Starter Kit I of Stones in Accordance with German Roche company, the quality of getting about 3 μ g preferably cDNA fragment is carried out mark, altogether 2 probes of mark: the poor underproduction thing of forward and reverse poor underproduction physical prospecting pin.In order to eliminate the joint consensus sequence to the impact of hybridization signal, remove joint sequence with the excision of RsaI enzyme respectively before forward and the reverse poor underproduction thing probe mark.

1.16 the amplification of Insert Fragment and some film

Take the bacterium liquid of 1 μ l overnight incubation as template, (test kit provides Nested PCR primer 2R, 10 μ mol/L) and each 0.5 μ l of Nested PCR primer1 (test kit provides, 10 μ mol/L) primer, 2.5 μ l, 10 * PCR reaction buffer (+Mg ²⁺), 0.5 μ l dNTP, 0.2 μ l Tap (worker is given birth in Shanghai) adds water polishing 25 μ l, and cycling condition is: 94 ℃ of 5min; 94 ℃ of 35s, 68 ℃ of 35s, 72 ℃ of 1.5min; 35 circulations, 72 ℃ of 10min, 25 ℃ of preservations.Increase complete after, PCR product electrophoresis detection on 1.2%EB glue of getting 8 μ l reject to be inserted too little fragment (less than 200bp) and biobelt product.Get product and the abundant mixing of the freshly prepared 0.6mol/L NaOH of 5 μ l of 5 μ l bacterium colony PCR, get 2 copies of mixture corresponding points nylon membrane (Nylon membranes of 1.0 μ l, positively charged, Grmany), after the some film is complete nylon membrane is placed 0.5mol/L Tris-HCl and the 5min of pH7.5, then use the distilled water rinsing, toast 40min in 120 ℃ of baking ovens.

1.17 hybridization and colour developing

Hybridization is carried out according to the specification sheets of the DIG of Roche Holding Ag test kit, and concentration and probe concentration final in the hybridization solution is about 35ng/ml, and 42 ℃ of hybridization are spent the night.Wash film twice with 25 ℃ of 2 * Saline-Sodium Citrate (SSC)+0.1% sodium lauryl sulphate (SDS), then each 5min uses 65 ℃ of 0.5 * SSC (0.1%SDS) elutriants to wash film twice, each 15min.After the immune response, color reaction adopts the NBT (tetrazolium pale blue) of this test kit/BCIP (5-bromo-4-chloro-3-indolylphosphate salt) system colour developing, developing time is 1-16h, it is complete with TE (pH 8.0) color development stopping to develop the color, and utilizes gel imaging system to scan the colour developing result under visible light.

1.18 choosing of differential cloning

Two hybridization signals that repeat film of contrast in the forward subtracted library, only choosing has signal or obviously is better than the oppositely poor clone's (strength of signal is more than 1.5 times) who subtracts probe with the poor probe hybridization signal that subtracts of forward with the poor probe that subtracts of forward; In reverse library, in contrast, select the clone and utilize T7 or the unidirectional order-checking of SP6 primer to give birth to worker's biotechnology company limited in Shanghai.

1.19 sequence homology retrieval

Utilize VecScreen (http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html) to remove carrier and joint sequence obtains the EST fragment.In the GenBank storehouse, carry out sequence homology relatively by the Blast research tool first, analyze the aminoacid sequence that these EST and those genes have homology and match; Then utilizing search oranges and tangerines database to carry out sequence alignment (http://harvest.ucr.edu/); The judging criterion of gene function is with reference to the standard of the draft model plants such as paddy rice, Arabidopis thaliana.

The result of present embodiment presents: adopt the CTAB method to separate early real trifoliate orange total RNA in different periods of growing, clear through the band of electrophoresis detection 28S and 18SRNA, ratio between the two is between 1.8-2.0, and this illustrates that total RNA keeps complete, does not degrade.Simultaneously the point sample hole is clean, illustrate do not have polyphenol, the pollution (Fig. 2 A) of polysaccharide and protein.Utilize the detection of ultraviolet spectrophotometer to show: the A260/280 value is about 1.9, and the A260/230 value illustrates that the integrity of extracting RNA is better about 2.0, and purity is higher, is fit to the separation of next step mRNA.The purifying of mRNA adopts Oligotex mRNA separating kit in this experiment, and the mRNA electrophoresis detection of separation illustrates the mRNA that is separated to more complete (Fig. 2 B), for next step test is had laid a good foundation between 0.5-4kb.The concentration of adjusting mRNA with spectrophotometer is carried out next step two strands to about the 0.5mg/ μ l and is synthesized.

Synthetic and the enzyme of double-stranded cDNA is cut the synthetic quality of effect cDNA and RsaI enzyme and is cut that effect directly affects efficient that joint connects and final difference subtracts efficient.Get respectively the mRNA of 4 μ g Adulthoods and virgin phase material, adopt PCR-Select ^TMCDNA Subtraction Kit test kit (Clontech, USA) provides the synthetic cDNA of cDNA synthetic agent double-stranded, and parent material adopts mRNA to avoid in the process of synthetic cDNA because the pollution that the existence of DNA may cause.Can find out that from experimental result the synthetic double-stranded cDNA size of real trifoliate orange morning Tong Qihe Adulthood all is distributed between 0.5kb and the 4kb, substantially meet in size plant transcription characteristics originally, also the size with mRNA is more consistent, and double-stranded synthesize very successful is described.

The joint joint efficiency is the poor key link that subtracts efficient of directly impact, and joint efficiency will more than 25%, can descend otherwise difference subtracts efficient at least greatly.This experiment shows the detected result of Tester-1 and Tester-2 joint joint efficiency, the inner special primer of Actin and joint primer extension product and the inner a pair of special primer amplified production brightness of Actin are more or less the same, this explanation joint efficiency is at least in 70%-80% (gel imaging system swept brilliance area ratio), connect respond wellly, can guarantee the preferably poor efficient (Fig. 2 C) that subtracts.In addition, the poor success or not that subtracts hybridization finally will subtract efficient by difference and embody, and it is higher that difference subtracts efficient, and in subtracted library, the gene abundance of institute's enrichment specifically expressing is also higher.Present embodiment is to subtract with the not poor abundance that subtracts the constitutive expression gene by difference in the method detection PCR product second time of PCR to judge the poor efficient that subtracts, and the primer is the inner a pair of special primer of Actin.The amplification of grads PCR shows that in not poor underproduction thing, the Actin fragment occurs at 25 circulation times, and the Actin fragment just occurs after 35 circulations in poor underproduction thing, contrast 10 circulations in evening.As seen the abundance of constitutive expression gene reduces greatly in poor underproduction thing, proves that difference subtracts hybridization and reached comparatively satisfied effect (Fig. 2 E).

After the two-wheeled pcr amplification is complete, gets respectively the first round and second of 8 μ l and take turns PCR product electrophoresis detection.Detected result shows that the amplification of two-wheeled selective PCR is normal, and this has also reflected the high-level efficiency that joint connects from the side, if the joint joint efficiency is too low, can have a strong impact on the expanding effect of two-wheeled PCR.The fragment length of two-wheeled pcr amplification is distributed between the 0.3-1kb, is uniform Smear shape, and mean size conforms to the desired expectation size of test kit about about 500bp.Behind positive contrast subtracts second time PCR product purification, be connected with carrier and be transformed among the intestinal bacteria DH-5 α, each spreads 3 20cm culture dish, by blue hickie screening, forward and oppositely the library respectively obtain about 5000 clones.Picking separates 2304 of good, clear full white clones and places 384 hollow plates that 50 μ lLB (containing Amp) are housed, seal with sealed membrane, 37 ℃ of incubator overnight incubation, obtaining at last the forward storage capacity is that two differences that 2304 clones and reverse storage capacity are 1920 clones subtract the hybridization library.Every plate is randomly drawed 24 clones, and by detecting with the quality of T7 and SP6 carrier primer PCR amplification Insert Fragment to subtracted library, the result of electrophoresis detection shows all can amplify Insert Fragment.The size distribution of Insert Fragment between 0.2-0.7kb, minority clone's Insert Fragment more than 1kb, average Insert Fragment size (Fig. 2 D) about 0.5kb, only a few clone's PCR product is two bands, may be that picking white clone is wrong.

2304 Insert Fragments that obtain by pcr amplification from the forward library carry out reverse northern hybridization, there are notable difference in the fragment of amplification and the hybridization signal of 2 probes, most fragments with not poor subtract material prepare the probe hybridization signal a little less than, poor to subtract the probe hybridization signal stronger with forward, screen by contrasting 2 copy film hybridization signals, obtain altogether 467 clones that obvious differential expression is arranged, account for selected clone's 17.8%.Figure .2 is numbered two films of 10 to subtract and oppositely the poor probe hybridization that subtracts is by the result of reverse northern dot hybridization screening with forward is poor respectively, and arrow is depicted as the differential expression clone.The reverse storehouse of same method obtains 463 clones that notable difference is arranged, and accounts for selected clone's 24%.

Embodiment 2:PtELF5 gene cloning and sequential analysis

In the sequence that embodiment 1 separates, a fragment that the frequency of occurrences is very high is arranged, present certain similarity with the ELF5 of Arabidopis thaliana, according to this gene fragment, designed the inner special primer P1:5 ' of one pair of genes GSP PtELF5 primer (5 '-ATGACAGAGCCATCCCCAGAGCCTAAT-3 ') and 3 ' GSP PtELF5 prime r (5 '-GAAGGGTGAGACTCCAGTCATGTTTAG-3 '), the length of amplification gene is respectively 300bp.Strategy (the Tian that the full-length gene clone adopts the terminal rapid amplifying of library and cDNA to be combined, et al.2003): take the library phage DNA as template, with carrier primer λ TripIEx25 ' LD-Insert screening primer (5 '-CTCCGAGATCTGGACGAGCT-3 ') and 3 ' GSP Primer amplification cDNA5 ' end, with 5 ' GSPprimer and carrier primer 3 ' λ TripIEx2 sequencing primer (5 '-TAATACGACTCACTATAGGG-3 ') pairing amplification cDNA 3 ' end, then utilize the method for information biology to be stitched together.Concrete experimental technique is as follows: pcr template 1 μ l (template is the cDNA amplification library of 1000 times of dilutions), inner each the 1 μ l (10 μ mol/L) of special primer of carrier primer and gene, 0.25 μ l high-fidelity PyrobestDNA polymerase (precious biotechnology Dalian company limited), 2.5 μ l 10 * Pyrobest DNA polymerase buffer, 0.5 μ l dNTPs (10mmol/L) adds ddH ₂O to 25 μ l.PCR loop parameter: 94 ℃ of abundant sex change 5min; 94 ℃ of sex change 30s, 64 ℃ of annealing 30s, 72 ℃ are extended 1.5min, 35 circulations; Last 72 ℃ are extended 10min.Amplified production detects at 1.2%Agarose/EB gel.Reclaim respectively 5 ' with 3 ' end pcr amplified fragment, get 1000 times of diluents of 1 μ l as the pcr amplification template, verify that with the further nested amplification of 3 ' GSP primer and 5 ' GSP primer PCR system and working procedure are the same.After conclusive evidence 5 ' with 3 ' end PCR reclaims fragment and can amplify the big or small fragment of expectation, it is cloned into pMD18-T (Takara, Japan) carrier.Bacterium liquid send the Wuhan Company order-checking of the large genome company of China.Prediction and the BLAST analytical procedure (Altschul etc., 1997) of utilizing GENSCAN (http://genes.mit.edu/GENSCAN.html) to carry out gene structure are carried out the gene homology analysis.

The cDNA sequence of PtELF5 total length is 1800bp, and prediction has comprised the ORF of 1623bp.The full length DNA sequence of this gene also obtains by chromosome walking simultaneously, show by the sequential analysis to DNA and cDNA: this gene exists 10 exons and 9 introns in reading frame, the aminoacid sequence that sequential analysis is derived has comprised 540 amino acid, has predicted the molecular weight of a 57.2KD.(K:43,7.96% that in these amino acid, have comprised 75 alkalescence; R:26,4.81%; H:6,1.11%) and 52 acidic amino acid (D:22,4.07%; E:30,5.56%), and predicted one 9.62 iso-electric point.Utilize the database (http://www.expacy.org) of protein characteristic to identify two module (position of these coded amino acids is 2-9 and 123-423) and WW domain (7-92) that are rich in Methionin.Presented 60% homology (NP_201070) by the protein of Blastx homology search prediction and the aporepressor ELF5 of blooming of Arabidopis thaliana, the NpwBP albumen with the people has also presented very high homology simultaneously.

Embodiment three: PtELF5 and the PtFLC real-time quantitative analysis in real trifoliate orange early

For further checking PtFLC and PtELF5 gene are in the early expression of real trifoliate orange different tissues, different times, this research detects with Real time round pcr.The extraction of material collection and RNA is according to embodiment 1.Detect the primer through the PrimerExpress software design.The RNA that extracts processes 15min through 37 ℃ of DNaseI and pollutes to remove DNA.RNA after the processing synthesizes the first chain cDNA with the reverse transcription test kit reverse transcription of MBI company.Adopt the ABI7500 real-time PCR to carry out Real time pcr amplification.With gene to be detected (PtELF5:PtELF5S 5-ATCACCCTACGCTGAATCCT-3, PtELF5A 5-GGTACGGGCAAAGCAACATC-3; PtFLC:PtFLCS5-AAGCGGCGTAGCGGATTGAT-3, PtFLCA 5-TGCCTTAGGTGCTCCTGAAG-3) and the special primer (ActinS, ActinA) of reference gene with

GREEN Master Mix (Applied Biosystems, USA) mixes, and then joins in the reaction tubes that contains template, and reaction system is 10 μ l.Response procedures is: 50 ℃ of 2min, 95 ℃ of 1min, (95 ℃ of 15s, 60 ℃ of 1min) 40 circulations.The data that produce are analyzed in Excel after Sequence Detector Version 1.3.1 software (Applied Biosystems, USA) transforms.

The result of present embodiment shows that PtFLC and PtELF5 are higher at the expression amount of real virgin phase of trifoliate orange of whole morning, and when plant enters December, PtFLC and PtELF5 express and begin to strengthen, and illustrates that they are early serving as extremely important role in the real trifoliate orange early blossoming process.Yet PtFLC early has higher expression amount in the spring tip in real trifoliate orange January in the Adulthood, rising along with temperature, expression amount reduces gradually, after autumn, along with the reduction of temperature, expression amount gos up again to some extent, to November, expression amount has been raised to a higher level, and the result shows the in close relations of the expression amount of PtFLC and temperature.In the expression investigation of Adulthood, PtELF5 has higher expression amount in March and May, and be the bud differentiation period of early real trifoliate orange and bloom period in this two period, and higher its expression amount and the seasonality of real trifoliate orange early of having hinted of expression amount in this bloomed closely related in two period.In Arabidopis thaliana, FLC is suppressed by ELF5, and the expression that ELF5 suppresses FLC causes that then the FLC expression amount descends.And see that the expression amount of PtFLC decreases in March and May early in the real trifoliate orange, early exist in the real trifoliate orange and the similar bloom controlling mode of Arabidopis thaliana vernalization regulation and control model (figure .4).

Embodiment four: the space expression analysis in PtFLC and the PtELF5 apical meristem

The method that in situ hybridization is reported in earlier stage with reference to this laboratory (Li et al, 2010).The preparation of PtFLC and PtELF5 specific probe: utilize primer (PtELF5S, PtELF5A; PtFLCS, PtFLCA) amplify specific fragment, then reclaim specific fragment and be connected on the pGEM-T carrier, for in-vitro transcription used.Before transcribing, determine the closure of specific fragment on the pGEM-T carrier through order-checking first, be positive-sense strand and antisense strand so which kind of enzyme transcribes out when determining in-vitro transcription.After PtFLC and PtELF5 specific fragment will be housed carrier carry out linearization for enzyme restriction, linearizing endonuclease is ApaI and BstXI.Concrete experimental implementation is according to DIGNorthern StarterKit test kit specification sheets (Roche, Germany).

The result of present embodiment presents in the apical meristem of the vegetative bud of virgin phase apical meristem and the spring Adulthood tip, PtFLC has presented higher expression amount in whole apical meristem, these positions are the positions of in the future carrying out bud differentiation, have also presented higher expression at the fringe region of leaf primordium and spire; In the bud differentiation stage, PtFLC expresses stronger in apical meristem and leaf primordium; In the flowering phase, PtFLC has higher expression amount in stamen, but does not detect the expression of this gene at apical meristem.By the expression analysis of real-time quantitative, PtFLC has higher expression at leaf, in spending, and expression amount is lower in root and fruit.

The expression of PtELF5 in virgin phase and spring Adulthood tip apical meristem also investigated (figure .5) by in situ hybridization.As can be seen from the results, in virgin phase apical meristem, PtELF5 expression amount in tender tissue is higher, such as positions such as the tender blade of children, axillalry buds, does not still detect the expression of PtELF5 at apical meristem; In the vegetative bud of Adulthood, in whole apical meristem and axillalry bud, can both detect it and express, do not find the expression that it is special, just higher a little at mitotically active position expression amount.Spending differential period, the expression of PtELF5 can both detect at whole flower primordium, and expression amount is higher.In the flowering phase, it is very high to detect in the expression of this gene and the stamen expression amount in all floral organs, has similar expression pattern to PtFLC.May two genes at this moment the phase be the relation of vying each other, the space real-time quantitative detects with PtFLC and has similar expression pattern, have higher expression amount at leaf, in spending, and expression amount is lower in root and fruit.

Embodiment five: PtELF5 is the ectopic expression analysis in Arabidopis thaliana

(buy from the CAMBIA company of Australia according to the pCAMBIA1301 carrier, the coding region sequence of multiple clone site Fig. 6) and PtELF5 gene, as follows with Primer Premier 5.0 software designs go out to increase upstream and downstream its sequence of PCR primer of the whole coding region of PtELF5 gene according to the principle of general design primer:

Forward primer：5’-GCCATGG CGTTTCGTGGTTGCAGTTCG-3’

Reverse primer：5’-GGGTAACC CTGGCTCTTCATTACCTTGT-3’

Carry out pcr amplification take PtELF5 gene cloning as template.The annealing temperature of pcr amplification is 58 ℃.Comprise 100ng cDNA in the reaction system of 25 μ l, 1 * damping fluid, 2.5mM MgCl ₂, 0.25mM dNTP, (aforementioned damping fluid and Taq polysaccharase Lithuania) add the above-mentioned primer of 0.5 μ M available from Fermentas company to 0.5U Taq polysaccharase.PCR reaction is finished by following program on ABI 9700 (Applied Biosystem) amplification instrument: 94 ℃, and 5 minutes, 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 50 seconds, 72 ℃ were extended 35 circulations 90 seconds; 72 ℃ were extended 15 minutes after circulation was finished.Produce a single PCR band product, behind 1% agarose gel electrophoresis, use

Dna gel reclaims test kit (available from Omega company, the U.S.) and reclaims special band, and extraction step is consulted and used explanation.The double digestion system: the reaction cumulative volume is 20 μ l, wherein contains the purified product 10 μ l of PCR, 10 * G damping fluid (available from MBI company), 2 μ l, each 1 μ l of NcoI and BstEII, distilled water 6 μ l.37 ℃ of enzymes cut spend the night after purifying reclaim.The double digestion system of pCAMBIA1301 carrier: the reaction cumulative volume is 20 μ l, wherein contain the pCAMBIA1301 carrier DNA 8 μ l that obtain through plasmid extraction, 10 * G damping fluid (available from MBI company), 2 μ l, each 1 μ l of NcoI and BstEII adds distilled water 8 μ l.Cutting the rear purifying that spends the night in 37 ℃ of enzymes reclaims.The mol ratio of inserting PtELF5 gene and carrier pCAMBIA1301 in the ligation system is 3: 1, the reaction cumulative volume is 10 μ l, wherein contain 10 * Buffer, 1 μ l, T4DNA ligase enzyme 1 μ l, the double digestion of PtELF5 gene reclaims product 4 μ l, the double digestion of pCAMBIA1301 carrier reclaims product 2 μ l, distilled water 2 μ l.16 ℃ of reactions 14-16 hour.Connect product and transform coli strain DH5 α, screening positive clone in containing the LB solid plate of 50mg/L kantlex, the extracting plasmid carries out that enzyme is cut and PCR identifies, order-checking determines not have the reading frame sudden change, acquisition contains the recombinant clone that inserts the purpose fragment, with its called after pCAMBIA1301-PtELF5 recombinant vectors, use freeze-thaw method (with reference to the Pehanorm Brooker, Huang Peitang translates, " molecular cloning experiment guide " third edition, Science Press, 2002) recombinant vectors pCAMBIA1301-PtELF5 is imported among the Agrobacterium EHA105.The method (Mei et al, 2010) that the Arabidopis thaliana cultivated ginseng is reported in earlier stage according to this laboratory.Transformation of Arabidopsis thaliana adopts inflorescence dip method (Clough and Bent 1998).Arabidopis thaliana is the annual herb plant, and the wild-type Arabidopis thaliana probably needs 25d from being seeded into to bloom.By time, stem leaf number and the lotus throne number of sheets that the statistics Arabidopis thaliana is bloomed, verify the functional analysis with the genes involved of blooming.The positive seedling of PtELF5 presents early blossoming (Fig. 7, table .13) in various degree under the condition of long day.And PtELF5 checks in Arabidopis thaliana and blooms, self constitutive expression has presented late flower, presented early blossoming behind the insertion mutation, can infer that by both different results PtELF5 has presented the control methods different from Arabidopis thaliana in real trifoliate orange early, possible this gene differentiation occurred in the process of long-term evolution.

Table 13 35S:PtELF5 T ₂Flowering time analysis for transfer-gen plant

^aPlant-growth is (16 hours illumination/8 hour dark) under the condition of long day of 22 degree in temperature; ^bThe calculating of fate is from being seeded into inflorescence length to about the 1cm; ^cThe number of sheets is the total number of sheets in the time of the long 1cm of arriving of inflorescence.* there were significant differences with wild-type (P＜0.05) in representative.

The main reference document

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Claims (3)

1. the oranges and tangerines early blossoming gene PtELF5 of a separating clone, its nucleotide sequence is shown in sequence table SEQ ID NO:1.

2. oranges and tangerines early blossoming gene PtELF5 claimed in claim 1, the aminoacid sequence of its coding is shown in sequence table SEQ ID NO:2.

3. claim 1 or 2 described oranges and tangerines early blossoming gene PtELF5 are shortening the virgin interim application of Arabidopis thaliana.

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