CN102604963A - 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 into arabidopsis 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 possibly be oranges and tangerines blooms impels the oranges and tangerines early flowering, the entering Adulthood can lay the foundation for quickening the oranges and tangerines breeding then.

Background technology

Fruit tree is the perennial woody plant mostly, will experience the very long virgin phase from planting seed to blossoming and bearing fruit first.The virgin phase short like peach, Lee, 3-4 is bloomed, long like oranges and tangerines, walnut, 8-12 is bloomed, longer like ginkgo, Chinese torreya etc., the virgin phase reaches ten several years even many decades.Because body volume is big, Tong Qichang, genetic background are complicated, has greatly hindered the perennial woody plant, is the master's fruit tree and genetics research, breeding process and the new variety popularization of commodity trees with results floral organ, fruit and seed particularly.Therefore, the virgin phase of announcement orchard fruit grows and becomes flower mechanism to have important theory and practice significance.

Oranges and tangerines are the big fruit of the first in the world, are the big international trade agricultural-food in the third place in the world that is only second to wheat and corn.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, sexual organ abortion and heredity goes up factor affecting such as height heterozygosis, makes that the research of conventional breeding and genetic mechanism of oranges and tangerines is difficult, makes slow progress.Therefore disclose the molecule mechanism that oranges and tangerines become flower mechanism; Not only help to quicken the oranges and tangerines breeding process, change oranges and tangerines and bear fruit period and fruit ripening stage; And have an important significance for theories, for the development of hereditary mechanism research, breeding practice and production tech the potential far reaching significance is arranged also.

We have excavated early blossoming real trifoliate orange variation-morning (promise that provides the genetic resources source to disclose registry form and this resource of granting proves for Pang, et al.2006) that the virgin phase research of xylophyta is had important value under the early stage grant of national natural science foundation.Real trifoliate orange (Precocious trifoliate orange early; Poncirus trifoliata L.Raf.) is the strain variation of finding in the Yichang; Promptly have 20% to bloom then in sowing; Can all bloom in 1 year, and have characteristic (Zhang, the et al.2008 that downgrades and several times a year bloom; Zhang, et al.2009; Zhang, et al.2009).Early real trifoliate orange only needs just can get into generative growth phase through of short duration vegetative growth phase as a natural trifoliate orange class early blossoming two mutants, so be considered to study preferably the material that xylophyta blooms and determines.Early stage is through subtracting hybridization (2D-SSH) in that the two dimension inhibition of virgin phase and Adulthood is poor respectively to real trifoliate orange early and common trifoliate orange; Screen 532 ESTss relevant and become the flower genes involved with tens of, like PtTFL1, PtFLC, PtFT, PtBAM and PtF-box etc. with phasic development.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 of exist certain homology to name still at the EARLYFLOWERING 5 of encoding sequence and Arabidopis thaliana (ELF5) for it; 2004); It is the repressor gene of blooming in Arabidopis thaliana, is the promotion gene of blooming in oranges and tangerines.Find through in situ hybridization, obviously raise at bud differentiation conversion stage expression amount.In becoming the flower transforming process, 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 in the model plant Arabidopis thaliana, blooms in advance.A lot of in the past isolating floral genes all are the homologous genes of Arabidopis thaliana; And excavate less to the own peculiar gene of xylophyta; Though and this gene has certain homology with Arabidopis thaliana ELF5, homology is lower, and functions reversed; We infer that this gene is the distinctive early blossoming gene of xylophyta, possibly have more widely aspect the virgin phase the shortening xylophyta to use.

Summary of the invention

The objective 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, and the applicant is its called after PtELF5, but ELF5 is one and checks floral genes in Arabidopis thaliana; And PtELF5 becomes flower to promote the factor in the present invention, and this gene can shorten virgin stage phase of plant greatly.Can cultivate shortening and economic worth and the higher oranges and tangerines kind of quality greatly of a kind of virgin phase so use this gene.

The present invention realizes through following technical scheme:

The present invention is from Fructus Aurantii (Precocious trifoliate orange; Poncirus trifoliata L.Raf.) separates the new gene PtELF5 that obtains a virgin phase growth of regulation and control 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.With the complete translation area (Coding sequence) of this gene with directly change model plant over to after 35S promoter combines; 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 through in situ hybridization this gene has presented very high expression in becoming the flower transforming process, explain with become to spend in close relations.Real-time quantitative is discovered can suppress the to bloom expression of repressor PtFLC of this gene, causes the plant early blossoming then.

The present invention provides a kind of new method for shortening the virgin phase of xylophyta.This method is through transgenic technology overexpression PtELF5, suppresses to bloom and suppresses expression and then the shortening the juvenile phase of sub-PtFLC.Adopting this type 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 a 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) virgin phase and the Adulthood isolating mRNA electrophoresis detection result of early real trifoliate orange, 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 are with the inner special primer amplification of Actin; 1,3:Tester-1 and Tester-2 use inner special primer of Actin and joint primer amplification respectively.(D) 24 clones that are used to detect the insertion clip size that randomly draw.(E) PCR detects the poor efficient that subtracts.

The screening of the reverse northern of Fig. 3 subtracted library, arrow is represented 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 vertical meristematic tissue, and fruit is for spending back 30 days.All expression of gene are used the Actin homogenization, and all experiments are carried out 4 times and repeated.

Fig. 5 detects PtELF5 (a, b, c, d) and PtFLC (e, f, g, the h) expression in different etap of real trifoliate orange apical meristem morning through in situ hybridization.(a, e) is virgin vertical meristematic tissue of phase.(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 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 demonstrate the unusual of floral organ, explains that this gene only is the bloom controlling gene.

Embodiment

Embodiment 1: utilize the inhibition difference to subtract hybridization and separate relevant regulatory gene

1.1 vegetable material and RNA separate

Two mutants " real trifoliate orange early " (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 early, the spring tip of real trifoliate orange different developmental phases is a material with the vertical meristematic tissue of virgin phase different times; Adulthood and virgin phase " real trifoliate orange early " 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 the main position that early real trifoliate orange becomes flower, so the Adulthood material is a material with the terminal bud of the spring tip and five buds subsequently, 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, the Adulthood is got three trees respectively with the virgin phase when drawing materials, mix back liquid nitrogen flash freezer-80 ℃ preservation.

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 ^TM(Clontech USA) carries out the inhibition difference and subtracts hybridization cDNA Subtractive Kit, makes up the library that positive contrast subtracts respectively.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 following as driveing son as detecting son:

1.2 the first chain cDNA is synthetic

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

mRNA(4μg) 2-4μl

cDNA?synthesis?Primer(10μmol/L) 1μl

Water to the TV 5 μ l that add no RNA enzyme like needs;

2) 70 ℃ of 2min in the PCR appearance 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 appearance, 42 ℃ 1.5 hours;

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

1.3 the second chain cDNA is synthetic

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

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

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 isolating supernatant, 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, 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 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 cut product and the last double-stranded cDNA that retains of step utilizes 1.2% agarose to carry out electrophoresis detection.

1.5 the connection of joint

1) cuts from μ l enzyme of last 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) be template with Tester-1 after the ligation and Tester-2; Respectively with 3 of housekeeping gene Actin ' (ActinS:5 '-CCGACCGTATGAGCAAGGAAA-3), 5 ' end (ActinA:5 '-TTCCTGTGGACAATGGATGGA-3 ') and PCR primerl (5 '-CTAATACGACTCACTATAGGGC-3 ') they are that primer carries out pcr amplification; The amplification efficiency of the corresponding product of more 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, in 4 centrifuge tubes, mixes following component:

The main primer composition of table 6 detection architecture

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

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

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 thermal cycling (can not from the PCR appearance, take out) immediately:

94℃ 30s

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

1.7 hybridization for the first time

1) difference subtract hybridize before, earlier 4 * Hybridization buffer room temperature is placed 30min, if deposition, heat at 37 ℃ and to come dissolution precipitation in 10 minutes.Each library is carried out forward and the reverse poor hybridization that subtracts respectively, and the forward alternative is following, and is reverse then be the 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 MO, and of short duration centrifugal, make MO 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, get into next stage immediately) in 68 ℃ of PCR appearance.

1.8 hybridization for the second time

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

1) with the following component sterile tube of packing into:

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 and make hybridization appearance 1,2 and fresh driver while blended purpose to reach;

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

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

B) gently the rifle head is touched MO and the interface of hybridizing appearance 2;

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

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

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

F) all change blended sample in the rifle head over to hybridization appearance 1 place centrifuge tube;

G) inhale repeatedly 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, inhale repeatedly and beat mixing;

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

Subdue effect 1.9 utilize Actin to detect

1. getting 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 respectively, is template with the product after the dilution respectively, verifies inhibition subtractive hybridization effect with the Actin primer;

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

3. by following order preparation reaction base fluid (Master Mix):

Table 10 detects the main PCR composition of subduing effect reaction

4. abundant mixing, base fluid 24 μ l are reacted in each the pipe adding in 2 steps;

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 accomplishing 18 circulations, cancellation subtracts and does not subdue each 5 μ l of PCR product and place new centrifuge tube respectively, gives over to follow-up checking.Remain 20 μ lPCR products and put back to the PCR appearance again 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 give over to electrophoresis respectively;

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

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 table 11 PCR reaction for the first time

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

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

5) begin PCR immediately:

94℃ 5min

72℃ 10min

6) each sample is respectively got 8 μ l and is used to analyze 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 adds following ingredients preparation PCR Master Mix for the second time in proper order in centrifuge tube:

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

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 to analyze PCR result;

13) be stored in-20 ℃ subsequent use;

1.11PCR the recovery of product and purifying

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 uv lamp, operation is reclaimed the test kit specification sheets by Shanghai biotechnology ltd gel and carried out:

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

(2) will contain the segmental Binding Buffer of purpose and be transferred in the UNIQ-10 post that is placed in the 2ml collection tube, 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, once more 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 is streak culture on the LB agar plate with transfering loop, and fresh single bacterium colony that picking white is full is inoculated in the 100ml LB liquid nutrient medium, and 37 ℃ of constant temperature shaking 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 deposition;

(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 reclaim bacterial precipitation once more;

(4) add 3ml 0.1mol/LCaCl ₂The solution bacterial precipitation that suspends again;

(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, total system is 10 μ l.

(1) on the LB flat board that contains 50 μ g/ml penbritins, smear 10 μ l IPTG (beta-galactosidase enzymes, 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 following 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, shakes 1h at 37 ℃ of shaking table 250 commentaries on classics/min;

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

(7) cultivate: be inverted down for 37 ℃ and cultivate about 16h, promptly can be observed blue white alternate bacterium colony.White colony inserts segmental transformant for containing external source, and blueness is that carrier is from the transformant that connects;

(8) evaluation of positive colony: select white single bacterium colony with the sterilization toothpick, put into the triangular flask that fills 5ml liquid LB substratum (50 μ g/ml penbritin), overnight cultures (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 placed under 4 ℃ leave standstill 2d, treat the complete mixing of glycerine after ,-80 ℃ of preservations.

1.15 the preparation of hybridization probe

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

1.16 insert segmental amplification and put film

Bacterium liquid with 1 μ l overnight cultures is template, each 0.5 μ l of Nested PCR primer 2R (test kit provides, 10 μ mol/L) and 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.After amplification finished, PCR product electrophoresis detection on 1.2%EB glue of getting 8 μ l was rejected and is 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), places 0.5mol/L Tris-HCl and the 5min of pH7.5 with nylon membrane after the some film finishes; Use the distilled water rinsing then, 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 for 25 ℃ with 2 * Saline-Sodium Citrate (SSC)+0.1% sodium lauryl sulphate (SDS), each 5min uses 0.5 * SSC (0.1%SDS) elutriant to wash film twice for 65 ℃ then, each 15min.After the immunoreation; Coupling reaction adopts NBT (tetrazolium pale blue)/BCIP (5-bromo-4-chloro-3-indolylphosphate salt) the system colour developing of this test kit; Developing time is 1-16h, and colour developing finishes with TE (pH 8.0) color development stopping, utilizes gel imaging system under visible light, to scan the colour developing result.

1.18 choosing of differential cloning

The hybridization signal of two repetition films of contrast in the forward subtracted library is only chosen and to be subtracted probe with the forward difference and signal is arranged or subtract the probe hybridization signal with the forward difference and obviously be better than the reverse poor clone's (strength of signal is more than 1.5 times) who subtracts probe; 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 ltd 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 earlier, analyze the aminoacid sequence that these EST and those genes have homology and match through the Blast research tool; Utilizing search oranges and tangerines DB to carry out sequence alignment (http://harvest.ucr.edu/) then; The judgement criteria of gene function is with reference to the standard of draft model plants such as paddy rice, Arabidopis thaliana.

The result of present embodiment demonstrates: 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 explains that total RNA is kept perfectly, and does not degrade.Simultaneously the point sample hole is clean, explain do not have polyphenol, polysaccharide and proteinic pollution (Fig. 2 A).Utilize the detection of ultraviolet spectrophotometer to show: the A260/280 value is about 1.9, and the A260/230 value explains 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 isolating mRNA electrophoresis detection is explained 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.Carry out next step two strands to about the 0.5mg/ μ l synthesizes with the concentration of spectrophotometer adjustment mRNA.

Synthetic and the enzyme of double-stranded cDNA is cut effect cDNA synthetic quality and RsaI enzyme and is cut that effect directly influences efficient that joint connects and final difference subtracts efficient.Get the mRNA of 4 μ g Adulthoods and virgin phase material respectively, adopt PCR-Select ^TM(Clontech USA) provides the synthetic cDNA of cDNA synthetic agent double-stranded to cDNA Subtraction Kit test kit, and parent material adopts mRNA to avoid in the process of synthetic cDNA because the pollution that the existence of DNA possibly 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, meet plant transcription characteristics originally in size basically, also the size with mRNA is more consistent, explains that double-stranded synthetic is very successful.

The joint joint efficiency is the key link that directly influence difference subtracts efficient, and joint efficiency will can descend otherwise difference subtracts efficient more than 25% at least greatly.This experiment shows the detected result of Tester-1 and Tester-2 joint joint efficiency; 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 poor preferably efficient (Fig. 2 C) that subtracts.In addition, the poor success or not that subtracts hybridization finally will subtract efficient through difference and embody, and it is high more that difference subtracts efficient, and in subtracted library, the gene abundance of institute's enrichment specifically expressing is also high more.Present embodiment is to subtract with the not poor abundance that subtracts the constitutive expression gene through difference in the method detection PCR product second time of PCR to judge that difference subtracts efficient, and the primer is a pair of special primer in Actin inside.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 difference underproduction thing, compare according to 10 circulations in evening.It is thus clear that the abundance of constitutive expression gene reduces greatly in difference underproduction thing, prove that difference subtracts hybridization and reached comparatively satisfied effect (Fig. 2 E).

After the two-wheeled pcr amplification finishes, get the first round and second of 8 μ l respectively and take turns PCR product electrophoresis detection.Detected result shows that two-wheeled selectivity pcr amplification 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 petridish, and through blue hickie screening, forward respectively obtains about 5000 clones with reverse library.Picking separates 2304 of good, clear full white clones and places 384 hollow plates that 50 μ lLB (containing Amp) are housed; Seal with sealing film; 37 ℃ of incubator overnight cultures, obtaining the forward storage capacity at last 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, through insert fragment with the amplification of T7 and SP6 carrier primer PCR the quality of subtracted library is detected, and the result of electrophoresis detection shows all can amplify the insertion fragment.Insert segmental size distribution between 0.2-0.7kb, minority clone's insertion fragment is on average inserted clip size (Fig. 2 D) about 0.5kb more than 1kb, and only a few clone's PCR product is two bands, possibly be that picking white clone is wrong.

2304 that from the forward library, obtain through pcr amplification are inserted fragment and 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 prepn probe hybridization signal a little less than, to subtract the probe hybridization signal stronger with the forward difference, screens through contrasting 2 copy film hybridization signals; Obtain 467 clones that obvious differential expression is arranged altogether, account for selected clone's 17.8%.Figure .2 be numbered two films of 10 respectively with the forward difference subtract with the reverse poor probe hybridization that subtracts through reverse northern dot hybridization results of screening, 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%.

The clone and the sequential analysis of embodiment 2:PtELF5 gene

In embodiment 1 isolating sequence; 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 a pair of gene GSP PtELF5 primer (5 '-ATGACAGAGCCATCCCCAGAGCCTAAT-3 ') and 3 ' GSP PtELF5 prime r (5 '-GAAGGGTGAGACTCCAGTCATGTTTAG-3 '), the length of amplification gene is respectively 300bp.The full-length gene clone adopts library and the terminal rapid amplifying bonded of cDNA strategy (Tian; Et al.2003): with the library phage DNA is 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, utilize the method for information biology to be stitched together then.Concrete experimental technique is following: 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 ltd); 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 on 1.2%Agarose/EB gel.Reclaim 5 respectively ' 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 big genome company of China.Utilize GENSCAN (http://genes.mit.edu/GENSCAN.html) to carry out the prediction of gene structure and the homology analysis that BLAST analytical procedure (Altschul etc., 1997) is carried out gene.

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 through chromosome walking simultaneously; Sequential analysis through to DNA and cDNA shows: this gene exists 10 exons and 9 introns in reading frame; The sequential analysis deduced amino acid 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 DB (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) through 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 expression of real trifoliate orange different tissues, different times early, 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 handles 15min through 37 ℃ of DNaseI and pollutes to remove DNA.RNA after the processing is with the synthetic first chain cDNA of the reverse transcription test kit reverse transcription of MBI company.Adopt ABI7500 real-time quantitative PCR appearance 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 of internal control gene; ActinA) with

GREEN Master Mix (Applied Biosystems; USA) mix; Join then in the reaction tubes that contains template, reaction system is 10 μ l.Response procedures is: 50 ℃ of 2min, 95 ℃ of 1min, (95 ℃ of 15s, 60 ℃ of 1min) 40 circulations.(Applied Biosystems USA) transforms the back and in Excel, analyzes the data that produced through Sequence Detector Version 1.3.1 software.

The result of present embodiment shows, all than higher, when plant gets into December, PtFLC expresses with PtELF5 and begins enhancing at the expression amount of real virgin phase of trifoliate orange of whole morning for PtFLC and PtELF5, explains that they are serving as extremely important role in morning in the real trifoliate orange early blossoming process.Yet PtFLC early has higher expression in the spring tip in real trifoliate orange January in the Adulthood, and along with the rising of 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 expression amount and temperature in close relations of PtFLC.In the expression investigation of Adulthood; PtELF5 has higher expression in March and May; And this two periods be real trifoliate orange early bud differentiation period with bloom period, 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 in the real trifoliate orange early, 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

In situ hybridization is with reference to this laboratory reported method (Li et al, 2010) in early stage.The preparation of PtFLC and PtELF5 specific probe: utilize primer (PtELF5S, PtELF5A; PtFLCS PtFLCA) amplifies specific fragment, reclaim specific fragment then and be connected on the pGEM-T carrier, for in-vitro transcription used.Before transcribing, earlier confirm the closure of specific fragment on the pGEM-T carrier through order-checking, be positive-sense strand and antisense strand so that which kind of enzyme transcribes out when confirming 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 demonstrates in the apical meristem of the vegetative bud of the virgin phase apical meristem and the spring Adulthood tip; PtFLC has presented higher expression 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 in stamen, but does not detect this expression of gene at apical meristem.Through 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) through in situ hybridization.Can find out that from the result in virgin phase apical meristem, PtELF5 expression amount in tender tissue is higher,, still not detect the expression of PtELF5 at apical meristem such as positions such as the tender blade of children, axillalry buds; 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 expression amount is higher a little at mitotically active position.Spending differential period, PtELF5 is expressed in whole flower primordium and can both detects, and expression amount is higher.In the flowering phase, it is very high in all floral organs, can both to detect in this expression of gene and the stamen expression amount, has similar expression pattern with PtFLC.Maybe 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 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 according to the pCAMBIA1301 carrier from Australian CAMBIA company; The coding region sequence of MCS 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’

Clone's with the PtELF5 gene is that template is carried out pcr amplification.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 is reflected on ABI 9700 (Applied Biosystem) the amplification appearance and accomplishes by following program: 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 accomplished.Produce a single PCR band product; Behind 1% agarose gel electrophoresis; Reclaim test kit (available from Omega company with dna gel; The U.S.) reclaim special band, extraction step is consulted and used explanation.The double digestion system: the reaction TV 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.Cut the back purifying and recovering of spending the night at 37 ℃ of enzymes.The double digestion system of pCAMBIA1301 carrier: the reaction TV is 20 μ l, wherein contains through plasmid and extracts the pCAMBIA1301 carrier DNA 8 μ l that obtain, and 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.Cut the back purifying and recovering of spending the night in 37 ℃ of enzymes.The mol ratio of inserting PtELF5 gene and carrier pCAMBIA1301 in the ligation system is 3: 1; The reaction TV 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 transformed into escherichia coli bacterial strain DH5 α, screening positive clone in containing the LB solid plate of 50mg/L kantlex, extracting plasmid carry out that enzyme is cut and PCR identifies; Order-checking confirms not have the reading frame sudden change; Acquisition contains inserts the segmental recombinant clone of purpose, with its called after pCAMBIA1301-PtELF5 recombinant vectors, uses freeze-thaw method (with reference to Sa nurse Brooker; Huang Peitang translates; " molecular cloning experiment guide " third edition, Science Press, 2002) recombinant vectors pCAMBIA1301-PtELF5 is imported among the Agrobacterium EHA105.The Arabidopis thaliana cultivated ginseng is according to this laboratory reported method (Mei et al, 2010) in early stage.Arabidopis thaliana transforms and 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.Through time, stem leaf number and the lotus throne number of sheets that the statistics Arabidopis thaliana is bloomed, verify functional analysis with the genes involved of blooming.The positive seedling of PtELF5 demonstrates 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 after inserting sudden change; Can infer that through both different results PtELF5 has demonstrated the control methods different with 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 when inflorescence length arrives 1cm.* there were significant differences with wild-type (P＜0.05) in representative.

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Claims (4)

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. the described oranges and tangerines early blossoming of claim 1 gene PtELF5, its amino acid sequence coded is shown in sequence table SEQ ID NO:2.

3. claim 1 or 2 described genes are shortening the virgin interim application of plant.

4. the application of claim 3 wherein comprises and shortens the virgin interim application of oranges and tangerines.

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