CN106834338A - The expression vector of arabidopsis gene REM16 and its in the regulation and control interim application of plant blossom - Google Patents

Abstract

The invention discloses arabidopsis geneREM16Expression vector and its application in Controlling the flowering period, the present invention proved by experimental analysis, arabidopsis geneREM16Function with florescence control, and obtain arabidopsis geneREM16Over-express vector and gene silencing vector, overexpressionREM16Gene causes and bloom in transgenic Arabidopsis plants related gene expression change and promote arabidopsis to do sth. in advance to bloom, and early floral formation is shown as compared with the flowering time of WT lines；SilenceREM16Gene causes and bloom in transgenic Arabidopsis plants related gene expression change and postpone arabidopsis and bloom, and late floral formation is shown as compared with WT lines.By analyze transgenic line bloom related gene expression so that it is determined thatREM16The function that gene regulation is bloomed.Technical scheme is significant for plant florescence control.

Description

Arabidopsis geneREM16Expression vector and its regulation and control plant blossom it is interim Using

Technical field

The invention belongs to technical field of molecular biology, and in particular to arabidopsis geneREM16Expression vector and its The interim application of regulation and control plant blossom.

Background technology

The depolymerization of microfilaments factor（ADF/cofilin）It is widely present in eukaryotic, to maintaining the morphosis of cell, thin Born of the same parents' transport, energy conversion, information transmission, cell division and differentiation etc. play very important effect.Early-stage Study shows that plan is southern The change of mustard depolymerization of microfilaments factors A DF1 expression quantity can influence the florescence of plant,ADF1The reduction of expression quantity causes delay of blooming. Wildtype Arabidopsis thaliana was about bloomed at 35 ± 1 days, there is 17 lotus throne leaves；AndADF1Gene silencing plant blossom postpones 2 compared with wild type Week, lotus throne number of sheets mesh increases to 26.Thereafter, people are deeply parsed by gene knockoutADFDividing in growth and development of plants Subfunction.Arabidopsisadf4Mutant has longer hypocotyl and epidermal cell.ArabidopsisADF9Mutant（adf9-1,adf9-2）The apical dominance enhancing of plant, the second branch number of its inflorescence is less than wild type, but the length of branch compares wild type It is long；Mutant flowering time is earlier than wild type under long-day growth conditions, while lotus throne number of sheets mesh is fewer than wild type, and short-day Under growth conditions no notable difference is compared with wild type.Additionally,ADF9The expression of conversion period related gene of blooming can be regulated and controled Amount.These researchs show that the direct involved in plant of ADF albumen grows and florescence control, but its regulation and control target gene, participate in Key regulator and molecular regulation mechanism etc. it is all unclear.

The content of the invention

The invention provides arabidopsis geneREM16Expression vector and its in the regulation and control interim application of plant blossom.This Invention screens from arabidopsis cDNA library that to have obtained more than ten ADF1 mutual with arabidopsis ADF1 as bait, by yeast two-hybrid Make protein molecular, obtain gene of the present inventionREM16(gene is numberedAT3G53310), and it has been experimentally confirmed it Regulate and control the function at arabidopsis florescence.

For achieving the above object, the present invention is achieved using following technical scheme：

The invention provides a kind of arabidopsis geneREM16Over-express vector pCambia1300-221-HA, the overexpression The nucleotide sequence of carrier pCambia1300-221-HA such as sequence table SEQ ID No:Shown in 2, the over-express vector contains Such as sequence table SEQ ID No:Gene shown in 1REM16, the over-express vector pCambia1300-221-HA is for regulating and controlling to intend Southern mustard advance flowering period.

Further：By geneREM16With the pCambia1300-221-HA carriers with XbaI and KpnI restriction enzyme sites The double digestion of KpnI and XbaI is carried out respectively, according still further to genetic fragment and carrier 4：1 ratio is entered under the catalysis of T4 ligases 16 DEG C of row overnight connects 20h and is obtained.

The invention provides a kind of arabidopsis geneREM16Gene silencing vector pB7GWIWG2 (II) .0, the gene The nucleotide sequence such as sequence table SEQ ID No of silent carrier pB7GWIWG2 (II) .0:Shown in 3, the gene silencing vector PB7GWIWG2 (II) .0 is used to regulate and control arabidopsis flowering delay.

Present invention also offers arabidopsis geneREM16Application in arabidopsis florescence is regulated and controled.

Further：Containing such as sequence table SEQ ID No in the arabidopsis of the Controlling the flowering period:Overexpression shown in 1 Carrier pCambia1300-221-HA or such as sequence table SEQ ID No:Gene silencing vector pB7GWIWG2 (II) .0 shown in 2.

Further：The arabidopsis geneREM16Function with florescence control, geneREM16Overexpression plant table It is now early floral formation, geneREM16Silence plant shows as late floral formation.

Further：The geneREM164-6 days more early than WT lines of overexpression plant blossom time, lotus throne leaf 2 fewer than wild type of number；And the geneREM162-3 days more late than WT lines of silence plant blossom time, lotus throne leaf Number and wild type do not have significant changes.

Further：The geneREM16It is related to Photoperiod pathway in overexpression plantGI、COAnd downstream of blooming is opened Flower integron and flower_meristem_identity geneFT、LFY、AP1Expression quantity be above WT lines, vernalization approachFLC's Expression quantity is less than WT lines, gibberellin pathwayRGA、RGL1Expression quantity and wild type ratio there is no notable difference.

Further：The geneREM16It is related to Photoperiod pathway in silence plantGI、COAnd downstream of blooming is bloomed Integron and flower_meristem_identity geneFT、LFY、AP1Expression quantity be below WT lines, vernalization approachFLCTable It is higher than WT lines, gibberellin pathway up to amountRGA、RGL1Expression quantity and wild type ratio there is no notable difference.

The advantages of the present invention：Using existing plant gene engineering technology, the present invention is infected by inflorescence The method of method obtains arabidopsis geneREM16Overexpression and gene silencing transgene carrier and its transgenic line, by grinding Study carefully overexpression and gene silencing transfer-gen plant phenotype, and by its with the same terms under the WT lines flowering time that grows and Lotus throne number of sheets mesh is compared.Proved by experimental analysis, arabidopsis geneREM16 (AT5G25520)With florescence control Function, overexpressionREM16Gene causes and bloom in transgenic Arabidopsis plants related gene expression change and promote arabidopsis to carry Prematurity, shows as early floral formation compared with the flowering time of WT lines；SilenceREM16Gene causes transgenic arabidopsis Related gene expression of being bloomed in plant changes and postpones arabidopsis and bloom, and late floral formation is shown as compared with WT lines.It is logical Cross analysis transgenic line bloom related gene expression so that it is determined thatREM16The function that gene regulation is bloomed.Skill of the invention Art scheme is significant for plant florescence control.

Brief description of the drawings

Fig. 1 is wild type and mutantREM16Gene expression amount is analyzed（**：P<0.01）.

Fig. 2 compares for wild type with mutant flowering time and lotus throne leaf, wherein：A () is bloomed for wild type with mutant Time compares；B () is wild type and mutant flowering time statistical analysis；C () is that wild type is united with mutant lotus throne number of sheets mesh Meter analysis (* *：P<0.01).

Fig. 3 is that wild type and mutant are bloomed related gene expression amount analysis (* *：P<0.01).

Specific embodiment

Technical scheme is further described in detail with specific embodiment below in conjunction with the accompanying drawings.

Reagent used in present invention experiment etc. is purchased from companies such as TAKARA, Promega, Sigma.

The LB culture medium prescriptions used in experiment：0.5% dusty yeast, 1%NaCl, 1% peptone.

Other agent prescriptions：See《Molecular cloning》The third edition.

Embodiment 1,REM16The clone of gene

1st, the plantation of arabidopsis

The surface sterilization of arabidopsis seed：50 arabidopsis seeds subject to sterilization are put into 1.5 ml centrifuge tubes, 1 is added The ethanol of ml 75%（TritonX-100 containing 0.03% volume ratio）Concussion sterilization 1 min, then with 70% ethanol shake sterilize 1 min（Twice）, seed is drawn onto on aseptic filter paper with suction nozzle finally and is dried up, then clicked and entered 1/2 MS with aseptic toothpick Take out to be placed in illumination box after 4 DEG C of d of vernalization 3 in culture medium, in refrigerator and cultivate.

Condition of culture is as follows：16 h illumination/8 h dark alternate illuminations, 22 DEG C of temperature, 100 μm of olm of light intensity^-2·s^-1.Then, in treating that illumination grows and Arabidopsis thaliana Seedlings moved on into soil in 7-10 days, cover preservative film and take off film in 3-7 days, after in culturing room Culture, the same illumination box of condition.

2nd, arabidopsis Total RNAs extraction

(1) arabidopsis material is put into the mortar of precooling, adds liquid nitrogen to grind to form uniform powder rapidly.Note ground To ensure that material is immersed in liquid nitrogen in journey；

Etc. (2) after liquid nitrogen volatilization, 100-200 mg plant powders are transferred in 1.5 ml centrifuge tubes immediately, it is then rapid plus Enter 1 ml Trizol extract solutions, vortex concussion makes sample be fully dissolved in extract solution, room temperature places 5 min；

(3) 4 DEG C, 12,000 rpm are centrifuged 11 min, and 0.8 ml supernatants are transferred in 1.5 new ml centrifuge tubes；Again plus Enter 0.2 ml chloroforms and acutely vibrate 15 sec of mixing, room temperature places 2-5 min until layering.

(4) 4 DEG C, 12,000 rpm are centrifuged 11 min, and 0.4 ml supernatants are transferred in 1.5 new ml centrifuge tubes；

0.4 ml isopropanols are added, 15 mixing solution, -20 DEG C of 30 mim of placement are spun upside down.

(5) 4 DEG C, 12,000 rpm are centrifuged 11 min, abandon supernatant, and precipitation is washed with the ethanol of 0.9 ml 75% twice, and 4 DEG C, 12,000 rpm are centrifuged 5 min；

(6) supernatant is abandoned, is uncapped and RNA about 2-5 min is dried in superclean bench, 40 μ l RNase-Free water are added, 60 Fully dissolving RNA 10 min in DEG C；

(7) the OD values and concentration of RNA sample are surveyed with ultraviolet specrophotometer, A260/A280 reaches 1.8-2.0 and is preferred；Agar The quality of sugared detected through gel electrophoresis.

3rd, the reverse transcription of RNA

Refer to the Prime Script of TaKaRa^TM RT reagent Kit with gDNA Eraser kits（RR047A）Say Bright book.

4th, PCR amplifications

With above-mentioned reverse transcription synthesis cDNA as template, PCR method amplification gene fragments.

(1) PCR primer is synthesized by Shanghai Sani's biotechnology Services Co., Ltd：

Primer REM16-For:5’-ATG GCT GAC GAT AGC GAAT-3’（SEQ ID No:4）；

Primer REM16-Rev:5’-TCA AGC ATC TCT ACG CAA GAC ATG-3’ （SEQ ID No:5）.

(2) PCR reaction systems

Component	Volume (μ l)/pipe
		10×PCR Buffer	2.5
dNTP Mixture	1
		Arabidopsis cDNA	3
Forward primer（10 μΜ）	1
		Reverse primer（10 μΜ）	1
rTaq	0.15
			18.5
Cumulative volume	25

PCR reaction conditions：94^oThe min of C predegenerations 5；94^oC is denatured 30 s, 62^oC anneal 30 s, 72^oC extends 50s, 27 Individual circulation；72^oC extends 7 min.

5th, PCR primer is reclaimed

1% agarose gel electrophoresis detection after PCR reactions（110 V 17 min）, after in gel imaging instrument observe result simultaneously Take pictures.Referring next to TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 kit explanations Book reclaims PCR primer, is recovered to arabidopsis geneREM16 (AP2/B3-like transcriptional factor family protein), the geneREM16 CDS sequences such as sequence table SEQ ID No:Shown in 1.

6th, connect

The REM16 genetic fragments for obtaining will be above reclaimed to be connected on cloning vector pMD18-T Vector by TA clones, 16^oC is overnight.Linked system is as follows：

Component	Volume (μ l)/pipe
		Solution I	5
pMD18-T Vector	1
		DNA recovery products	4
Cumulative volume	10

7th, the preparation and conversion of bacillus coli DH 5 alpha competent cell

The preparation of bacillus coli DH 5 alpha competent cell

(1) activation of DH5 α strains：The DH5 α strains of preservation are taken out from -80 DEG C of ultra low temperature freezers, training is inverted for 37 DEG C after line Support；

(2) picking single bacterium colony is inoculated in 10 mL LB（-）In fluid nutrient medium, 37 DEG C, the h of 200 rpm incubated overnights 12；

(3) by 1:100 take and are inoculated in 50 ml LB fluid nutrient mediums, 37 DEG C, 200 rpm cultivate to OD600 values be 0.4- 0.6；

(4) bacterium solution is transferred in 50 mL centrifuge tubes, 10 min is placed on ice, 4 DEG C, 2500 g are centrifuged 5 min, abandon supernatant；

(5) the 0.1M MgCL of 20mL precoolings are added₂Suspension thalline, 4 DEG C, 2500g centrifugation 5min abandon supernatant；

(6) the 0.1M CaCL of 50 mL precoolings are added₂Suspension thalline, the min of ice bath 20,4 DEG C, 2500 g are centrifuged 5 min, abandon Clearly；

(7) the 0.1M CaCL2 suspension thallines of 10 mL precoolings are added, 1 mL glycerine is added and is fully mixed, the packing of each EP pipe Saved backup in -80 DEG C of refrigerators with liquid nitrogen flash freezer after 100 μ L.

Transformed competence colibacillus cell

(1) competent cell is taken out from -80 DEG C of ultra low temperature freezers in thawed on ice, 10 μ L recombinant plasmids is added to impression In state cell, mixing, the min of ice bath 30 are played in suction；

(2) 42 DEG C of s of heat shock 90, the immediately min of ice bath 5；

(3) 900 μ L LB are added（-）Culture medium, 37 DEG C, 200 rpm, 1.5 h；

(4) 4000 g room temperatures are centrifuged 5 min, abandon 800 μ L of supernatant, and the resuspended thalline of remaining liq is coated with Amp resistances On LB solid mediums, flat board is absorbed in 37 DEG C of positive 30 min that place to liquid, be inverted flat board, 12 h are cultivated in 37 DEG C Overnight.

8th, the acquisition and identification of recombinant plasmid

Single bacterium colony is chosen after TA is cloned carries out bacterium solution PCR checkings, positive bacterium solution sample presentation to Shanghai Sani Bioisystech Co., Ltd DNA sequencing checking is carried out, correct bacterium solution will be sequenced and be extracted plasmid for subsequent experimental.

Embodiment 2,35S::REM16 CDS expression vector establishments

1st, the acquisition of genes of interest

Design of primers and the system and condition of PCR reactions：

（1）It is template so that correct recombinant plasmid is sequenced, KpnI-REM16-For and XbaI-REM16 Rev are expanded for primer PCR The REM16 full-length genes coding of 861 bp（CDS）Sequence, the primer sequence is as follows：

KpnI-REM16-For：5’-CGGGGTACCCCG ATG GCT GAC GAT AGC GAAT-3’ （SEQ ID No:6）；

XbaI-REM16- Rev：5’-GCTCTAGAGC TCA AGC ATC TCT ACG CAA GAC ATG-3’ （SEQ ID No:7）；

PCR reaction systems and condition are with embodiment 1.

2nd, the structure of expression vector

By the glue reclaim product of previous step and with XbaI and KpnI restriction enzyme sites pCambia1300-221-HA carriers difference The double digestion of KpnI and XbaI is carried out, according still further to 4:1 ratio is overnight connected under the catalysis of T4 ligases.Reaction system And condition is as follows：

Endonuclease reaction system and condition：

Component	Volume (μ L)/pipe
			1
	1
		10×M buffer	2
Glue reclaim product/carrier	8
			8
Cumulative volume	20

37 DEG C of 2.5 h of incubation, then respectively reclaim digestion products.

By T4 ligases by genes of interestREM16Fragment is with 16 DEG C of binary vector pCambia1300-221-HA overnight 20 h are connected, linked system is as follows：

Component	Volume (μ L)/pipe
		10×T4 DNA ligase Buffer	2.5
	4
		PCambia1300-221-HA digestion recovery products	1
T4 DNA ligase	1
			1.5
Cumulative volume	10

Escherichia coli are converted after the completion of connection, then choosing single bacterium colony and shaking bacterium and extract recombinant plasmid carries out double digestion and bacterium solution PCR is tested Card, extra large Sani Bioisystech Co., Ltd sequencing is served by the positive strain containing recombinant plasmid, and the sequencing result for obtaining passes through NCBI blast are analyzed, certain CDS sequences containing REM16 in positive plasmid.So far 35S has been obtained::REM16 CDS are expressed Carrier pCambia1300-221-HA（Sequence such as SEQ ID No:Shown in 2）.

Embodiment 3,REM16-RNAi carrier builds

1st, anchor tip is contained（AttB1, attB2）Purpose fragment acquisition

Sequencing is correctly included into purpose fragmentREM16The 18-T recombinant plasmids of gene dilute 1000 times, with the plasmid after dilution It is template, is primer with the upstream and downstream primer for adding attB joints according to above-mentioned clone's condition, clones purpose fragment.Gel is reclaimed Purpose fragment.

2nd, BP recombining reactions system and condition

Component	Volume (μ L)/pipe
		The attB-PCR recovery products of belt lacing	3
pDONR221 vector (150ng/µL)	2
		BP enzymes	2
TE Buffer, PH 8.0	to 10

25 DEG C of 20 h of culture, each reaction adds 1 μ l Proteinase K, 37 DEG C of min of water-bath 10.Conversion Escherichia coli sense By state cell, picking single bacterium colony, positive strain plasmid is extracted in bacterium solution PCR checkings.

3rd, LR recombining reactions system and condition：

Component	Volume (μ L)/pipe
		Entry clone (previous step BP reacts plasmid)	3
pB7GWIWG2(II).0 vector (150 ng/µL)	2
		LR enzymes	2
TE Buffer, PH 8.0	To 10

25 DEG C of 20 h of culture, each reaction adds 1 μ l Proteinase K, 37 DEG C of min of water-bath 10.Conversion Escherichia coli sense By state cell, picking single bacterium colony, positive strain plasmid, as arabidopsis gene are extracted in bacterium solution PCR checkingsREM16Gene sink Silent carrier pB7GWIWG2 (II) .0（Sequence such as SEQ ID No:Shown in 3）.

The conversion of embodiment 4, arabidopsis and the screening of transfer-gen plant

1st, the preparation of Agrobacterium competent cell

(1) the Agrobacterium GV3101 bacterium solutions oese deposited of being gone bail for from ultra low temperature freezer draws plate activation, obtains single bacterium colony；

(2) oese picking single bacterium colony is used, 5 ml liquid YEP are inoculated in（-）In culture medium, 28 DEG C, 200 rpm cultures 12 h；

(3) liquid culture is inoculated into new liquid YEP（-）In culture medium, concussion and cultivate to OD₆₀₀It is 0.4-0.6；

(4) by the h of bacterium solution ice bath 0.5,4 DEG C, 5000 rpm, 5 min；

(5) supernatant is abandoned, with the NaCl suspension thallines of the M precoolings of 10 mL 0.15,4 DEG C, 5000 rpm, 5 min；

(6) supernatant is abandoned, with the CaCl of the mM precoolings of 10 mL 20₂Resuspended thalline, is distributed into 200 μ l/ pipes in 1.5 mL centrifuge tubes In, with quick freeze in liquid nitrogen after being saved backup in ultra low temperature freezer.

2nd, the carrier conversion Agrobacterium GV3101 that will be built（Freeze-thaw method）

(1) Agrobacterium competent cell is taken out from -80 DEG C of ultra low temperature freezers slowly to melt on ice, add 10 μ l restructuring matter Grain, the h of ice bath 0.5 freezes 60 s in liquid nitrogen, after be placed in 37 DEG C of min of water-bath 5；

(2) 950 μ l liquid YEP are added（-）Culture medium, 28 DEG C, 200 rpm cultivate 3 h；

(3) 12000 rpm, 1 min collects thallines leave and take 100 μ l supernatant back dissolving thalline；

(4) bacterium solution is applied to LB（Containing antibiotic）On solid medium, first 28 DEG C just put culture 0.5 h, afterwards be inverted culture 2- 3 days；

(5) bacterium solution PCR reactions checking.

3rd, the conversion of arabidopsis

With reference to the agriculture bacillus mediated colored dip method of the transformation of Arabidopsis thaliana of Clough et al..Authenticated positive colony bacterium is taken in containing There are 50 mg/l rifampins（Rif）In the LB fluid nutrient mediums of corresponding resistant, 28 DEG C, 220 rpm concussion and cultivates to OD₆₀₀Extremely 1.0 - 1.8.6000 rpm are centrifuged 5 min collects thallines, and with containing 5 % sucrose, 0.04 % Silwet L-77 infect liquid and hang Thalline is floated, now OD₆₀₀0.8 or so.Arabidopsis floral is dipped into and infects 10-15 s in liquid.Then the arabidopsis that will have been infected Plant is put into overlay film moisturizing above in camera bellows, and long-day culture in culturing room is put in after the h of light culture 24.

4th, the screening and identification of genetically modified plants

The first generation seed that acquisition will be infected is named as T₁Generation, arabidopsis seed will be layered on after surface sterilization 1/2 MS+ On 30 mg/l hygromycin (Hyg) or 5 mg/l glufosinate-ammoniums (PPT) culture mediums, vernalization treatment 3-4 days in 4 DEG C of refrigerators are placed in, then 16 h illumination/8 h dark alternate illuminations culture 10 days in incubator, positive seedling can survive always and color is greener, cloudy Property seedling can gradually turn yellow lethal.The dark alternately light of 16 h illumination/8 h in culturing room will be finally put in positive transplantation of seedlings to soil According to culture to results T₂For seed（Individual plant is received）.The T2 of results is carried out into homozygote identification for seed paving resistance plate, if positive seedling It is 3 with negative seedling ratio:1 is homozygous lines, and the positive seedling of homozygosis is transplanted seedlings in soil again according to screening technique before Cultivated to the T for harvesting in culturing room₃For seed（Individual plant is received）, the screening and culturing in resistant panel, now seedling should be complete green Prove homozygote.The Arabidopsis thaliana Seedlings of 10 d are grown in the long-day, seedling extraction total serum IgE is taken and is obtained cDNA through reverse transcription, will CDNA dilute five times as template, by the analysis of fluorescent quantitative PCR technique, experimental result as shown in figure 1,REM16Overexpression It is related in plantREM16Gene expression amount is significantly higher than WT lines, andREM16In gene silencing plantREM16Gene table WT lines are significantly lower than up to amount.

Embodiment 5, transfer-gen plant phenotype and differenceREM16Expression quantity influence is bloomed the analysis of related gene

1st, flowering time and lotus throne number of sheets mesh statistical analysis

Choose under long-day conditions（16 h illumination/8 h are dark）Grow normal arabidopsis wild type（WT）Plant and mutation Each 40 plants of body plant, carries out flowering time and lotus throne number of sheets mesh statistical analysis.

Statistics as shown in Fig. 2 from map analysis, when growing to 5 weeks, wild type（WT）Arabidopsis plant has been completed From transformation from nutrient growth to reproductive growth, flowering, lotus throne number of sheets mesh is about 10；REM16Overexpression plant blossom 4-6 days more early than wild type of time, about 2 fewer than wild type of lotus throne number of sheets mesh；AndREM16Gene silencing plant blossom time ratio In 2-3 days wild type evenings, lotus throne number of sheets mesh and wild type do not have significant changes.Thereby determine that arabidopsis geneREM16 (AT5G25520)Function with florescence control, overexpression mutant shows as early floral formation, and gene silencing mutant shows as Late floral formation.

2nd, it is differentREM16The expression quantity influence of gene is bloomed the analysis of related gene expression

The Arabidopsis thaliana Seedlings of 10 d are grown in the long-day, seedling extraction total serum IgE is taken and is obtained cDNA through reverse transcription, cDNA is diluted five Times as template, by the analysis of fluorescent quantitative PCR technique, experimental result as shown in figure 3,REM16It is related in overexpression plant Photoperiod pathwayGI、COAnd bloom integron and the flower_meristem_identity gene in downstream of bloomingFT、LFY、AP1Expression quantity It is above WT lines, vernalization approachFLCExpression quantity be less than WT lines, gibberellin pathwayRGA、RGL1Expression quantity There is no notable difference with wild type ratio, this withREM16The phenotype of overexpression plant early blossoming is consistent.REM16Gene silencing plant In be related to Photoperiod pathwayGI、COAnd bloom integron and the flower_meristem_identity gene in downstream of bloomingFT、LFY、AP1's Expression quantity is below WT lines, vernalization approachFLCExpression quantity be higher than WT lines, gibberellin pathwayRGA、RGL1's Expression quantity and wild type ratio do not have notable difference, this withREM16The phenotype of gene silencing plant evening flower is consistent.

Above evidence showsREM16Participation has regulated and controled the expression of related gene of partly blooming, and causesREM16Overexpression is dashed forward Variant shows as early floral formation,REM16Gene silencing mutant shows as late floral formation.

The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to foregoing reality Example is applied to be described in detail the present invention, for the person of ordinary skill of the art, still can be to foregoing implementation Technical scheme described in example is modified, or carries out equivalent to which part technical characteristic；And these are changed or replace Change, do not make the spirit and scope of the essence disengaging claimed technical solution of the invention of appropriate technical solution.

SEQUENCE LISTING

<110>Qingdao Agricultural University

<120>The expression vector of arabidopsis gene REM16 and its in the regulation and control interim application of plant blossom

<130>

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 861

<212> DNA

<213>Artificial sequence

<400> 1

atggctgacg atagcgaatt gtatcctcgt ttcttcaaag tgttcctcgt tgaatcggct 60

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acagtgaagc tccaaggtct tggaggaaaa ctttggactg tgagcttgaa gaaaataagc 180

ggagctgcgt atcttactag aggatggcca aaattcgcag aggaacatga gctgaagaac 240

ggagagttca tgacatttgt gtatgatggt catcgtacct ttgaagtgag tgtctttgat 300

cgttggggta gcaaagaggt cagagctgaa atacaagcca taccactttc tgattctgat 360

tctgattctg ttgtggaaga cgaaaaagac tcaactgatg ttgttgaaga tgatgatgat 420

gaagatgaag atgaagatga agatgatgat ggtagctttg atgaagatga agaaatcagc 480

cagagtcttt atccgattga cgaagagact gcaaccgatg ctgcagtttt tgaagggaac 540

ttggacgtgg aagctttaac taatccacac tttccgacta cgctcaagaa caggatttat 600

gaactgttga tcccagccaa tgtggtgaag gataataacc ttgaatttgg ttcatccatc 660

aagtacatcg atggagaagg aaccttagta gggctaagag gaaaatgggc tgataagagg 720

gtttgcttca aaggatggga caggatttgc agaaggaaca ggctcaaaaa gcaccaagat 780

actgtcgaat gcgagcttct tcatgatgac cagaagatgg ttcattccat ccgagtccat 840

gtcttgcgta gagatgcttg a 861

<210> 2

<211> 10221

<212> DNA

<213>Artificial sequence

<400> 2

gaattcccga tctagtaaca tagatgacac cgcgcgcgat aatttatcct agtttgcgcg 60

ctatattttg ttttctatcg cgtattaaat gtataattgc gggactctaa tcataaaaac 120

ccatctcata aataacgtca tgcattacat gttaattatt acatgcttaa cgtaattcaa 180

cagaaattat atgataatca tcgcaagacc ggcaacagga ttcaatctta agaaacttta 240

ttgccaaatg tttgaacgat cggggaaatt cgagctcact agtggatcct cactaaagac 300

tagcataatc tggaacatca taaggataca tcaatgatgc gtagtcaggc acgtcgtatg 360

ggtacataag actagcataa tctggaacat cataaggata gcccccgggg tcgacggtac 420

cccgctcgac gcgcctcgag atcctctaga gtcccccgtg ttctctccaa atgaaatgaa 480

cttccttata tagaggaagg gtcttgcgaa ggatagtggg attgtgcgtc atcccttacg 540

tcagtggaga tatcacatca atccacttgc tttgaagacg tggttggaac gtcttctttt 600

tccacgatgc tcctcgtggg tgggggtcca tctttgggac cactgtcggc agaggcatct 660

tcaacgatgg cctttccttt atcgcaatga tggcatttgt aggagccacc ttccttttcc 720

actatcttca caataaagtg acagatagct gggcaatgga atccgaggag gtttccggat 780

attacccttt gttgaaaagt ctcaattgcc ctttggtctt ctgagactgt atctttgata 840

tttttggagt agacaagtgt gtcgtgctcc accatgttga cgaagatttt cttcttgtca 900

ttgagtcgta agagactctg tatgaactgt tcgccagtct ttacggcgag ttctgttagg 960

tcctctattt gaatctttga ctccatggcc tttgattcag tgggaactac ctttttagag 1020

actccaatct ctattacttg ccttggtttg tgaagcaagc cttgaatcgt ccatactgga 1080

atagtacttc tgatcttgag aaatatatct ttctctgtgt tcttgatgca gttagtcctg 1140

aatcttttga ctgcatcttt aaccttcttg ggaaggtatt tgatttcctg gagattattg 1200

ctcgggtaga tcgtcttgat gagacctgct gcgtaagcct ctctaaccat ctgtgggtta 1260

gcattctttc tgaaattgaa aaggctaatc tggggacctg caggcatgca agcttagctt 1320

ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 1380

tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga 1440

tcgcccttcc caacagttgc gcagcctgaa tggcgaatgc tagagcagct tgagcttgga 1500

tcagattgtc gtttcccgcc ttcagtttaa actatcagtg tttgacagga tatattggcg 1560

ggtaaaccta agagaaaaga gcgtttatta gaataacgga tatttaaaag ggcgtgaaaa 1620

ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac agggttcccc tcgggatcaa 1680

agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag 1740

ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg 1800

cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac 1860

tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc 1920

cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc 1980

tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc 2040

aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg 2100

gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc 2160

ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg 2220

accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc 2280

gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg 2340

gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg 2400

gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa 2460

gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc 2520

gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg 2580

acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg 2640

ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg 2700

gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc 2760

gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg 2820

cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc 2880

tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc 2940

cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg 3000

cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac 3060

gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc 3120

ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt 3180

gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg 3240

cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg 3300

cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga 3360

gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct 3420

tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa 3480

atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta 3540

agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac 3600

acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag 3660

atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag 3720

ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg 3780

cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg 3840

aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg 3900

aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg 3960

gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc 4020

aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc 4080

gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg 4140

gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc 4200

gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg 4260

tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg 4320

ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga 4380

accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg 4440

acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa 4500

cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg 4560

gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga 4620

gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga 4680

tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc 4740

ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca 4800

gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct 4860

gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg 4920

aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag 4980

catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa 5040

aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca 5100

ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca 5160

tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac 5220

ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg 5280

aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc 5340

gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac 5400

cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc 5460

tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 5520

gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 5580

ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat 5640

actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 5700

aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 5760

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 5820

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 5880

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 5940

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 6000

gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 6060

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 6120

atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 6180

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 6240

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 6300

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6360

ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6420

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6480

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 6540

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg cattctaggt 6600

actaaaacaa ttcatccagt aaaatataat attttatttt ctcccaatca ggcttgatcc 6660

ccagtaagtc aaaaaatagc tcgacatact gttcttcccc gatatcctcc ctgatcgacc 6720

ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc gcttctccca agatcaataa 6780

agccacttac tttgccatct ttcacaaaga tgttgctgtc tcccaggtcg ccgtgggaaa 6840

agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc atacagctcg cgcggatctt 6900

taaatggagt gtcttcttcc cagttttcgc aatccacatc ggccagatcg ttattcagta 6960

agtaatccaa ttcggctaag cggctgtcta agctattcgt atagggacaa tccgatatgt 7020

cgatggagtg aaagagcctg atgcactccg catacagctc gataatcttt tcagggcttt 7080

gttcatcttc atactcttcc gagcaaagga cgccatcggc ctcactcatg agcagattgc 7140

tccagccatc atgccgttca aagtgcagga cctttggaac aggcagcttt ccttccagcc 7200

atagcatcat gtccttttcc cgttccacat cataggtggt ccctttatac cggctgtccg 7260

tcatttttaa atataggttt tcattttctc ccaccagctt atatacctta gcaggagaca 7320

ttccttccgt atcttttacg cagcggtatt tttcgatcag ttttttcaat tccggtgata 7380

ttctcatttt agccatttat tatttccttc ctcttttcta cagtatttaa agatacccca 7440

agaagctaat tataacaaga cgaactccaa ttcactgttc cttgcattct aaaaccttaa 7500

ataccagaaa acagcttttt caaagttgtt ttcaaagttg gcgtataaca tagtatcgac 7560

ggagccgatt ttgaaaccgc ggtgatcaca ggcagcaacg ctctgtcatc gttacaatca 7620

acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt 7680

ccgaatagca tcggtaacat gagcaaagtc tgccgcctta caacggctct cccgctgacg 7740

ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg 7800

gggagctgtt ggctggctgg tggcaggata tattgtggtg taaacaaatt gacgcttaga 7860

caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga attaattcgg 7920

gggatctgga ttttagtact ggattttggt tttaggaatt agaaatttta ttgatagaag 7980

tattttacaa atacaaatac atactaaggg tttcttatat gctcaacaca tgagcgaaac 8040

cctataggaa ccctaattcc cttatctggg aactactcac acattattat ggagaaactc 8100

gagcttgtcg atcgacagat ccggtcggca tctactctat ttctttgccc tcggacgagt 8160

gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc 8220

cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc 8280

gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata 8340

gagttggtca agaccaatgc ggagcatata cgcccggagt cgtggcgatc ctgcaagctc 8400

cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca 8460

gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat 8520

gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac 8580

gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc 8640

gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc 8700

aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg 8760

gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatag cctccgcgac 8820

cggttgtaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc 8880

acggcgggag atgcaatagg tcaggctctc gctaaactcc ccaatgtcaa gcacttccgg 8940

aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc 9000

ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg 9060

agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag 9120

aaacttctcg acagacgtcg cggtgagttc aggctttttc atatctcatt gccccccggg 9180

atctgcgaaa gctcgagaga gatagatttg tagagagaga ctggtgattt cagcgtgtcc 9240

tctccaaatg aaatgaactt ccttatatag aggaaggtct tgcgaaggat agtgggattg 9300

tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg aagacgtggt 9360

tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact 9420

gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt 9480

gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc 9540

gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt ggtcttctga 9600

gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca tgttatcaca 9660

tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt 9720

gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga tagcctttcc 9780

tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc ttttgatgaa 9840

gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc tttgttgaaa 9900

agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg agtagacgag 9960

agtgtcgtgc tccaccatgt tggcaagctg ctctagccaa tacgcaaacc gcctctcccc 10020

gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc 10080

agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 10140

tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga 10200

aacagctatg accatgatta c 10221

<210> 3

<211> 12652

<212> DNA

<213>Artificial sequence

<400> 3

ctagagggcc cgacgtcgca tgcctgcagg tcactggatt ttggttttag gaattagaaa 60

ttttattgat agaagtattt tacaaataca aatacatact aagggtttct tatatgctca 120

acacatgagc gaaaccctat aagaacccta attcccttat ctgggaacta ctcacacatt 180

attctggaga aaaatagaga gagatagatt tgtagagaga gactggtgat ttttgcggac 240

tctagcatgg ccgcgggata tcacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa 300

tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta cataatactg 360

taaaacacaa catatccagt cactatggcg gccgcattag gcaccccagg ctttacactt 420

tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atccggctta ctaaaagcca 480

gataacagta tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt 540

atacccgaag tatgtcaaaa agaggtgtgc tatgaagcag cgtattacag tgacagttga 600

cagcgacagc tatcagttgc tcaaggcata tatgatgtca atatctccgg tctggtaagc 660

acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag 720

gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc tgacgagaac 780

agggactggt gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct 840

gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct 900

ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat 960

cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat 1020

cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct 1080

gatgttctgg ggaatataaa tgtcaggctc ccttatacac agccagtctg caggtcgacc 1140

atagtgactg gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct 1200

aatttaatat attgatattt atatcatttt acgtttctcg ttcagctttc ttgtacaaag 1260

tggtgatatc actagtgcgg ccgcctgcag gtcgaccata tggtcgacct gcaggcggcc 1320

gcactagtga tgctgttatg ttcagtgtca agctgacctg caaacacgtt aaatgctaag 1380

aagttagaat atatgagaca cgttaactgg tatatgaata agctgtaaat aaccgagtat 1440

aaactcatta actaatatca cctctagagt ataatataat caaattcgac aatttgactt 1500

tcaagagtag gctaatgtaa aatctttata tatttctaca atgttcaaag aaacagttgc 1560

atctaaaccc ctatggccat caaattcaat gaacgctaag ctgatccggc gagattttca 1620

ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc 1680

caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac 1740

cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag 1800

ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt 1860

atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt 1920

ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg 1980

cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc 2040

cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc 2100

agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc 2160

aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc 2220

gtctgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag 2280

tggcagggcg gggcgtaaac gcgtggatca gcttaatatg actctcaata aagtctcata 2340

ccaacaagtg ccaccttatt caaccatcaa gaaaaaagcc aaaatttatg ctactctaag 2400

gaaaacttca ctaaagaaga cgatttagag tgttttacca agaatttctg tcatcttact 2460

aaacaactaa agatcggtgt gatacaaaac ctaatctcat taaagtttat gctaaaataa 2520

gcataatttt acccactaag cgtgaccaga taaacataac tcagcacacc agagcatata 2580

tattggtggc tcaaatcata gaaacttaca gtgaagacac agaaagccgt aagaagaggc 2640

aagagtatga aaccttacct catcatttcc atgaggttgc ttctgatccc gcgggatatc 2700

accactttgt acaagaaagc tgaacgagaa acgtaaaatg atataaatat caatatatta 2760

aattagattt tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca 2820

ctatggtcga cctgcagact ggctgtgtat aagggagcct gacatttata ttccccagaa 2880

catcaggtta atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc 2940

cccgataacg gagaccggca cactggccat atcggtggtc atcatgcgcc agctttcatc 3000

cccgatatgc accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact 3060

ggccaggggg atcaccatcc gtcgcccggg cgtgtcaata atatcactct gtacatccac 3120

aaacagacga taacggctct ctcttttata ggtgtaaacc ttaaactgca tttcaccagt 3180

ccctgttctc gtcagcaaaa gagccgttca tttcaataaa ccgggcgacc tcagccatcc 3240

cttcctgatt ttccgctttc cagcgttcgg cacgcagacg acgggcttca ttctgcatgg 3300

ttgtgcttac cagaccggag atattgacat catatatgcc ttgagcaact gatagctgtc 3360

gctgtcaact gtcactgtaa tacgctgctt catagcacac ctctttttga catacttcgg 3420

gtatacatat cagtatatat tcttataccg caaaaatcag cgcgcaaata cgcatactgt 3480

tatctggctt ttagtaagcc ggatcctaac tcaaaatcca cacattatac gagccggaag 3540

cataaagtgt aaagcctggg gtgcctaatg cggccgccat agtgactgga tatgttgtgt 3600

tttacagtat tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat 3660

atcattttac gtttctcgtt cagctttttt gtacaaactt gtgatatcac tagtgcggcc 3720

gcctgcaggt cgactagaat agtaaattgt aatgttgttt gttgtttgtt ttgttgtggt 3780

aattgttgta aaaatacgga tcgtcctgca gtcctctcca aatgaaatga acttccttat 3840

atagaggaag ggtcttgcga aggatagtgg gattgtgcgt catcccttac gtcagtggag 3900

atatcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt ttccacgatg 3960

ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg cagaggcatc ttgaacgata 4020

gcctttcctt tatcgcaatg atggcatttg taggtgccac cttccttttc tactgtcctt 4080

ttgatgaagt gacagatagc tgggcaatgg aatccgagga ggtttcccga tattaccctt 4140

tgttgaaaag tctcaatagc cctttggtct tctgagactg tatctttgat attcttggag 4200

tagacgagag tgtcgtgctc caccatgttg acgaagattt tcttcttgtc attgagtcgt 4260

aaaagactct gtatgaactg ttcgccagtc ttcacggcga gttctgttag atcctcgatc 4320

tgaatttttg actccatggc ctttgattca gtaggaacta ctttcttaga gactccaatc 4380

tctattactt gccttggttt atgaagcaag ccttgaatcg tccatactgg aatagtactt 4440

ctgatcttga gaaatatatc tttctctgtg ttcttgatgc agttagtcct gaatcttttg 4500

actgcatctt taaccttctt gggaaggtat ttgatctcct ggagattatt actcgggtag 4560

atcgtcttga tgagacctgc cgcgtaggcc tctctaacca tctgtgggtc agcattcttt 4620

ctgaaattga agaggctaat cttctcatta tcggtggtga acatggtatc gtcaccttct 4680

ccgtcgaact ttcttcctag atcgtagaga tagagaaagt cgtccatggt gatctccggg 4740

gcaaaggaga tcagcttggc tctagtcgac catatgggag agctcaagct tagcttgagc 4800

ttggatcaga ttgtcgtttc ccgccttcag tttaaactat cagtgtttga caggatatat 4860

tggcgggtaa acctaagaga aaagagcgtt tattagaata acggatattt aaaagggcgt 4920

gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca accacagggt tcccctcggg 4980

atcaaagtac tttgatccaa cccctccgct gctatagtgc agtcggcttc tgacgttcag 5040

tgcagccgtc ttctgaaaac gacatgtcgc acaagtccta agttacgcga caggctgccg 5100

ccctgccctt ttcctggcgt tttcttgtcg cgtgttttag tcgcataaag tagaatactt 5160

gcgactagaa ccggagacat tacgccatga acaagagcgc cgccgctggc ctgctgggct 5220

atgcccgcgt cagcaccgac gaccaggact tgaccaacca acgggccgaa ctgcacgcgg 5280

ccggctgcac caagctgttt tccgagaaga tcaccggcac caggcgcgac cgcccggagc 5340

tggccaggat gcttgaccac ctacgccctg gcgacgttgt gacagtgacc aggctagacc 5400

gcctggcccg cagcacccgc gacctactgg acattgccga gcgcatccag gaggccggcg 5460

cgggcctgcg tagcctggca gagccgtggg ccgacaccac cacgccggcc ggccgcatgg 5520

tgttgaccgt gttcgccggc attgccgagt tcgagcgttc cctaatcatc gaccgcaccc 5580

ggagcgggcg cgaggccgcc aaggcccgag gcgtgaagtt tggcccccgc cctaccctca 5640

ccccggcaca gatcgcgcac gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag 5700

aggcggctgc actgcttggc gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg 5760

aggaagtgac gcccaccgag gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg 5820

aggccgacgc cctggcggcc gccgagaatg aacgccaaga ggaacaagca tgaaaccgca 5880

ccaggacggc caggacgaac cgtttttcat taccgaagag atcgaggcgg agatgatcgc 5940

ggccgggtac gtgttcgagc cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct 6000

ggccggtttg tctgatgcca agctggcggc ctggccggcc agcttggccg ctgaagaaac 6060

cgagcgccgc cgtctaaaaa ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt 6120

cgctgcgtat atgatgcgat gagtaaataa acaaatacgc aaggggaacg catgaaggtt 6180

atcgctgtac ttaaccagaa aggcgggtca ggcaagacga ccatcgcaac ccatctagcc 6240

cgcgccctgc aactcgccgg ggccgatgtt ctgttagtcg attccgatcc ccagggcagt 6300

gcccgcgatt gggcggccgt gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc 6360

ccgacgattg accgcgacgt gaaggccatc ggccggcgcg acttcgtagt gatcgacgga 6420

gcgccccagg cggcggactt ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt 6480

ccggtgcagc caagccctta cgacatatgg gccaccgccg acctggtgga gctggttaag 6540

cagcgcattg aggtcacgga tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc 6600

aaaggcacgc gcatcggcgg tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt 6660

cttgagtccc gtatcacgca gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc 6720

gttcttgaat cagaacccga gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa 6780

attaaatcaa aactcatttg agttaatgag gtaaagagaa aatgagcaaa agcacaaaca 6840

cgctaagtgc cggccgtccg agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg 6900

cagacacgcc agccatgaag cgggtcaact ttcagttgcc ggcggaggat cacaccaagc 6960

tgaagatgta cgcggtacgc caaggcaaga ccattaccga gctgctatct gaatacatcg 7020

cgcagctacc agagtaaatg agcaaatgaa taaatgagta gatgaatttt agcggctaaa 7080

ggaggcggca tggaaaatca agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt 7140

ggaggaacgg gcggttggcc aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc 7200

actggaaccc ccaagcccga ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca 7260

aatcggcgcg gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca 7320

gcggcaacgc atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg 7380

aatccgcaaa gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc 7440

caagggcgac gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga 7500

tagtcgcagc atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg 7560

cgaggtgatc cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg 7620

catggccagt gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc 7680

catgaaccga taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt 7740

tgcggacgta ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt 7800

agaaacctgc attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa 7860

gaacggccgc ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt 7920

aaagagcgaa accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg 7980

cgagatcaca gaaggcaaga acccggacgt gctgacggtt caccccgatt actttttgat 8040

cgatcccggc atcggccgtt ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga 8100

agccagatgg ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa 8160

gttctgtttc accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa 8220

ggaggaggcg gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg 8280

cgaagcatcc gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg 8340

ggaaaaaggt cgaaaaggtc tctttcctgt ggatagcacg tacattggga acccaaagcc 8400

gtacattggg aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc 8460

acacatgtaa gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt 8520

aaaacttatt aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac 8580

agccgaagag ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac gccccgccgc 8640

ttcgcgtcgg cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa 8700

tctaccaggg cgcggacaag ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg 8760

caccctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg 8820

agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 8880

cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag 8940

tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg 9000

gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc 9060

ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 9120

aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 9180

aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 9240

gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 9300

gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 9360

tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 9420

ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 9480

ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 9540

tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 9600

tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 9660

ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 9720

aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 9780

ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 9840

tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgcatga 9900

tatatctccc aatttgtgta gggcttatta tgcacgctta aaaataataa aagcagactt 9960

gacctgatag tttggctgtg agcaattatg tgcttagtgc atctaatcgc ttgagttaac 10020

gccggcgaag cggcgtcggc ttgaacgaat ttctagctag acattatttg ccgactacct 10080

tggtgatctc gcctttcacg tagtggacaa attcttccaa ctgatctgcg cgcgaggcca 10140

agcgatcttc ttcttgtcca agataagcct gtctagcttc aagtatgacg ggctgatact 10200

gggccggcag gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg attttgccgg 10260

ttactgcgct gtaccaaatg cgggacaacg taagcactac atttcgctca tcgccagccc 10320

agtcgggcgg cgagttccat agcgttaagg tttcatttag cgcctcaaat agatcctgtt 10380

caggaaccgg atcaaagagt tcctccgccg ctggacctac caaggcaacg ctatgttctc 10440

ttgcttttgt cagcaagata gccagatcaa tgtcgatcgt ggctggctcg aagatacctg 10500

caagaatgtc attgcgctgc cattctccaa attgcagttc gcgcttagct ggataacgcc 10560

acggaatgat gtcgtcgtgc acaacaatgg tgacttctac agcgcggaga atctcgctct 10620

ctccagggga agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat 10680

caagccttac ggtcaccgta accagcaaat caatatcact gtgtggcttc aggccgccat 10740

ccactgcgga gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga 10800

cgccaactac ctctgatagt tgagtcgata cttcggcgat caccgcttcc cccatgatgt 10860

ttaactttgt tttagggcga ctgccctgct gcgtaacatc gttgctgctc cataacatca 10920

aacatcgacc cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata gactgtaccc 10980

caaaaaaaca tgtcataaca agaagccatg aaaaccgcca ctgcgccgtt accaccgctg 11040

cgttcggtca aggttctgga ccagttgcgt gacggcagtt acgctacttg cattacagct 11100

tacgaaccga acgaggctta tgtccactgg gttcgtgccc gaattgatca caggcagcaa 11160

cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt gtttcaaacc 11220

cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag tctgccgcct 11280

tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat cgagtggtga 11340

ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga tatattgtgg 11400

tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt taatgtactg 11460

aattaacgcc gaattgaatt atcagcttgc atgccggtcg atctagtaac atagtagatg 11520

acaccgcgcg cgataattta tcctagtttg cgcgctatat tttgttttct atcgcgtatt 11580

aaatgtataa ttgcgggact ctaatcataa aaacccatct cataaataac gtcatgcatt 11640

acatgttaat tattacatgc ttaacgtaat tcaacagaaa ttatatgata atcatcgcaa 11700

gaccggcaac aggattcaat cttaagaaac tttattgcca aatgtttgaa cgatctgctt 11760

gactctaggg gtcatcagat ttcggtgacg ggcaggaccg gacggggcgg caccggcagg 11820

ctgaagtcca gctgccagaa acccacgtca tgccagttcc cgtgcttgaa gccggccgcc 11880

cgcagcatgc cgcggggggc atatccgagc gcctcgtgca tgcgcacgct cgggtcgttg 11940

ggcagcccga tgacagcgac cacgctcttg aagccctgtg cctccaggga cttcagcagg 12000

tgggtgtaga gcgtggagcc cagtcccgtc cgctggtggc ggggggagac gtacacggtc 12060

gactcggccg tccagtcgta ggcgttgcgt gccttccagg gacccgcgta ggcgatgccg 12120

gcgacctcgc cgtccacctc ggcgacgagc cagggatagc gctcccgcag acggacgagg 12180

tcgtccgtcc actcctgcgg ttcctgcggc tcggtacgga agttgaccgt gcttgtctcg 12240

atgtagtggt tgacgatggt gcagaccgcc ggcatgtccg cctcggtggc acggcggatg 12300

tcggccgggc gtcgttctgg gctcatggta gatcccctcg atcgagttga gagtgaatat 12360

gagactctaa ttggataccg aggggaattt atggaacgtc agtggagcat ttttgacaag 12420

aaatatttgc tagctgatag tgaccttagg cgacttttga acgcgcaata atggtttctg 12480

acgtatgtgc ttagctcatt aaactccaga aacccgcggc tcagtggctc cttcaacgtt 12540

gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc gtcatcggcg ggggtcataa 12600

cgtgactccc ttaattctca tgtatgataa ttcgagggta cccggggatc ct 12652

<210> 4

<211> 19

<212> DNA

<213>Artificial sequence

<400> 4

atggctgacg atagcgaat 19

<210> 5

<211> 24

<212> DNA

<213>Artificial sequence

<400> 5

tcaagcatct ctacgcaaga catg 24

<210> 6

<211> 31

<212> DNA

<213>Artificial sequence

<400> 6

cggggtaccc cgatggctga cgatagcgaa t 31

<210> 7

<211> 34

<212> DNA

<213>Artificial sequence

<400> 7

gctctagagc tcaagcatct ctacgcaaga catg 34

Claims (9)

1. a kind of arabidopsis geneREM16Over-express vector pCambia1300-221-HA, it is characterised in that：The overexpression The nucleotide sequence of carrier pCambia1300-221-HA such as sequence table SEQ ID No:Shown in 2, the over-express vector contains Such as sequence table SEQ ID No:Gene shown in 1REM16, the over-express vector pCambia1300-221-HA is for regulating and controlling to intend Southern mustard advance flowering period.

2. arabidopsis gene according to claim 1REM16Over-express vector pCambia1300-221-HA, its feature It is：By geneREM16KpnI is carried out respectively with the pCambia1300-221-HA carriers with XbaI and KpnI restriction enzyme sites With the double digestion of XbaI, according still further to genetic fragment and carrier 4：1 ratio carries out 16 DEG C under the catalysis of T4 ligases and overnight connects 20h is met to be obtained.

3. a kind of arabidopsis geneREM16Gene silencing vector pB7GWIWG2 (II) .0, it is characterised in that：The gene sinks The nucleotide sequence such as sequence table SEQ ID No of silent carrier pB7GWIWG2 (II) .0:Shown in 3, the gene silencing vector PB7GWIWG2 (II) .0 is used to regulate and control arabidopsis flowering delay.

4. arabidopsis geneREM16Application in arabidopsis florescence is regulated and controled.

5. arabidopsis gene according to claim 4REM16Application in arabidopsis florescence is regulated and controled, its feature exists In：Containing such as sequence table SEQ ID No in the arabidopsis of the Controlling the flowering period:Over-express vector shown in 1 PCambia1300-221-HA or such as sequence table SEQ ID No:Gene silencing vector pB7GWIWG2 (II) .0 shown in 2.

6. arabidopsis gene according to claim 4REM16Application in arabidopsis florescence is regulated and controled, its feature exists In：The arabidopsis geneREM16Function with florescence control, geneREM16Overexpression plant shows as early floral formation, base CauseREM16Silence plant shows as late floral formation.

7. arabidopsis gene according to claim 6REM16Application in arabidopsis florescence is regulated and controled, its feature exists In：The geneREM164-6 days more early than WT lines of overexpression plant blossom time, lotus throne number of sheets mesh fewer than wild type 2 Piece；And the geneREM162-3 days more late than WT lines of silence plant blossom time, lotus throne number of sheets mesh and wild type do not have Have significant change.

8. arabidopsis gene according to claim 4REM16Application in arabidopsis florescence is regulated and controled, its feature exists In：The geneREM16It is related to Photoperiod pathway in overexpression plantGI、COAnd bloom integron and the flower point in downstream of blooming Raw organization decided geneFT、LFY、AP1Expression quantity be above WT lines, vernalization approachFLCExpression quantity less than wild Type plant, gibberellin pathwayRGA、RGL1Expression quantity and wild type ratio there is no notable difference.

9. arabidopsis gene according to claim 4REM16Application in regulation and control arabidopsis is bloomed, it is characterised in that： The geneREM16It is related to Photoperiod pathway in silence plantGI、COAnd blooming for downstream of blooming and spends mitogenetic group at integron Knit decision geneFT、LFY、AP1Expression quantity be below WT lines, vernalization approachFLCExpression quantity higher than wild type plant Strain, gibberellin pathwayRGA、RGL1Expression quantity and wild type ratio there is no notable difference.