CN106916829A - A kind of rubber tree bloom controlling gene HbFT1 and its clone with application - Google Patents

Abstract

The invention belongs to gene engineering technology field, and in particular to a kind of rubber tree blooms controlling gene HbFT1, and its clone and application are further disclosed.The bloom cDNA of controlling gene HbFT1 of rubber tree of the present invention is included such as SEQ ID No：Nucleotide sequence shown in 1.The present invention obtains rubber tree in Para rubber tree and blooms the full-length cDNA of controlling gene HbFT1 first, and is analyzed by rubber tree HbFT1 Tissue-specific expressions.

Description

A kind of rubber tree bloom controlling gene HbFT1 and its clone with application

Technical field

The invention belongs to gene engineering technology field, and in particular to a kind of rubber tree blooms controlling gene HbFT1, one is gone forward side by side Step discloses its clone and application.

Background technology

Higher plant life cycle mainly includes that seed is sprouted, nutrition organs grows, blooms, being fertilized, embryonic development and kind The processes such as son formation.Wherein, the process of blooming is related to from nutrient growth to the conversion of reproductive growth, the generation of floral organ, Yi Ji Generation, transmission and interaction of the lower internal various signals of envirment factor effect etc., occupy Central places in plant life cycle Position, and it is closely related with crop yield and quality.

Flower induction is the process that plant is changed from nutrient growth to reproductive growth, it by it is endogenous development signal and environment because The strict control of son, flower induction is formed by a kind of complicated signal pathway " network ", is an extremely complex process.According to In recent years to the research of the adjustment signal approach of blooming of the several modes plant such as arabidopsis, toad's-mouth, corn, especially to intending south The research of mustard, proposes the 4 main biological pathway regulation and control grown and bloom of influence plant, including from master regulation on the way Footpath

(Autonomous pathway), vernalization approach (Vernalization), GA regulatory pathways (Gibberellic ) and Photoperiod approach (Photoperiod) acid.Wherein autonomous regulatory pathway and GA regulatory pathways are that plant is mainly inherent Control factors, and vernalization approach and Photoperiod approach are then extraneous main Control factors.

FT (FLOWERING LOCUS T) gene is the key gene in florescence control approach.It is located at flower development approach In Rendezvous Point, the signal from the difference flower development approach such as Photoperiod pathway, vernalization approach and autonomous pathway is integrated, in plant Played an important role in flower development.FT albumen is the chief component of florigen, in different genetic approach of blooming, on Trip key gene regulation and control FT genes, make FT albumen move to shoot apical meristem (SAM) from bast, with band alkalescence leucine Zipper protein (basic region-leucine zipper, b-ZIP) transcription factor FD (FLOWERING LOCUS D) is in stem Point promotes flowering transition by the interaction of protein, and by transcriptional activation floral meristem characteristic gene AP1 (APETALA1) flower development process is started.

The FT genes of arabidopsis belong to FT/TFL1 gene families, are also called phosphotidylethanolabinding binding protein (phosphatidyletha nolamine-binding protein, PEBP) gene family, because its albumen easily with phosphatidyl second Hydramine is combined and gained the name.Arabidopsis FT genes are subject to Photoperiod, and flowering of plant is postponed under ft mutant long-day environment (Kobayashi Y,Kaya H,Goto K,et al.1999.A pair of related genes with antagonistic roles in mediating flowering signals.Science 286,1960-1962.)。FT Homologous gene PtFT1 transgenic poplars can show flowering phenotype in very short time, while short-day can also be resisted Under the conditions of induce growth stop and bud dormancy phenomenon generation (Bohlenius H, Huang T, Charbonnel- Campaa L,et al.2006.CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees.Science 312,1040-43.).Willow FT2 is also proved to be had The effect of Accelerate bloom, while poplar adjusted and controlled seasonality is bloomed by the regulation and control of photoperiod function (Hsu CY, Liu Y, Luthe DS et al.2006.Poplar FT2shortens the juvenile phase and promotes seasonal flowering.The Plant Cell 18,1846-1861.).Tomato FT homologous genes SFT regulation and control are multiple Different growth courses, such as transformation of blooming, the maturation of blade, elongation of stem etc. (Shalit A, Rozman A, Goldshmidt A,et al.2009.The flowering hormone florigen functions as a general systemic regulator of growth and termination.Proc.Nati.Acad.Sci.USA.106,8392- 8397.).PdFT1 and PdFT2 with different expression patterns because speculating have not in eastern cottonwood (Populus deltoides) Same biological function (Igasaki T, Watanabe Y, Nishiguchi M, et al.2008.The FLOWERING LOCUS T/TERMINAL FLOWER 1family in Lombardy poplar.Plant and Cell Physiology 49, 291-300.).In pear tree, speculate that PcFT1 has the function of Accelerate bloom according to expression analysis, and PcFT2 then may regulation and control The aging of the blade of pear tree and the dormancy of bud.Overexpression PcFT2 genes can cause the generation of tobacco early blossoming phenotype in tobacco, Grafting research shows that PcFT2 albumen has mobility.But the pear tree for turning PcFT2 genes can not but show early blossoming phenotype, and Be the leaf abscission and bud for postponing to cause by short-day and low temperature dormancy (Freiman A, Golobovitch S, Yablovitz Z,et al.2015.Expression of flowering locus T2transgene from Pyrus communis L.delays dormancy and leaf senescence in Malus×domestica Borkh,and causes early flowering in tobacco.Plant Science 241,164-176.).BvFT1 is exercised in beet Bloom suppression function, its expression is suppressed by vernalization treatment, and BvFT2 is then cone production person (Pin PA, Benlloch R,Bonnet D,et al.2010.An antagonistic pair of FT homologs mediates the control of flowering time in sugar beet.Science 330,1397-1400.).It can be seen that, FT not only exists Played an important role during flowering transition, but also participate in some processes of growth regulation.

Natural rubber is always the important strategic materials and reserved resources of China, at present in China, rubber tree new varieties Seed selection is still based on traditional artificial pollination breeding.Because rubber tree is slow-growing, juvenile phase from postembryonal development is to blooming The 5-8 years are needed, and some proterties only can just be showed in the manhood, and this hinders entering for Rubber Tree Breeding by serious Journey.At present, to rubber tree, especially the Flowering mechanism of Para rubber tree has not been reported with Cloning of Genes Related and functional study. Rubber tree is become civilized controlling gene clone and its biological function research and exploration, specify it and bloomed in rubber tree and changed Effect in journey, is that the cultivation for accelerating rubber tree new product using molecular breeding technology lays the foundation.

The content of the invention

Therefore, the technical problems to be solved by the invention are to provide a kind of rubber tree to bloom controlling gene HbFT1, go forward side by side One step discloses its clone and application.

Controlling gene HbFT1, the gene in order to solve the above technical problems, a kind of rubber tree of the present invention blooms The cDNA of HbFT1 includes such as SEQ ID No：Nucleotide sequence shown in 1.

Described rubber tree blooms the cDNA nucleotide sequences such as SEQ ID of controlling gene HbFT1, the gene HbFT1 No：Shown in 1.

Bloomed the invention also discloses a kind of described rubber tree the albumen of controlling gene HbFT1 codings, including such as SEQ ID No：Amino acid sequence shown in 2.

The albumen of controlling gene HbFT1 codings, its amino acid sequence such as SEQ ID specifically, described rubber tree blooms No：Shown in 2.

Bloomed controlling gene HbFT1, the genomic DNA bag of the gene HbFT1 the invention also discloses a kind of rubber tree Include such as SEQ ID No：Nucleotide sequence shown in 3.

Described rubber tree blooms the genomic DNA nucleotide sequence such as SEQ of controlling gene HbFT1, the gene HbFT1 ID No：Shown in 3.

The invention also discloses a kind of recombinant plant expression load of controlling gene HbFT1 of being bloomed containing described rubber tree Body, the carrier is recombinant plasmid pXCS-HbFT1.

Carrier in the recombinant plant expression vector is pMD19-T carriers.

Bloomed the invention also discloses the rubber tree described in a kind of clone the method for controlling gene HbFT1, including following step Suddenly：

(1) template is mixed into the blade of Para rubber tree and inflorescence cDNA, designs following primer and enter performing PCR amplification, obtained Obtain HbTF1 gene open reading frames；

HbFT1OF：GGAATTCATGCCTAGTGACAGAG；

HbFT1OR：CCCCCCGGGGCATCTTCTTCC；

(2) based on HbTF1 gene open reading frames, design causes the UTR and 3 ' of synthesis 5 ' UTR as follows；

HbFT1-5UF：GTAAAAGCTGGATACCTCAGGCTCC；

HbFT1-5UR：CACCAACATGAACCCTAGGTTGGTT；

HbFT1-GSP3^1st：GTGAGCGACATCCCTGGAACA；

HbFT1-GSP3^2nd：CGAAGACAGACAATCAACCCACC；

(3) according to the acquisition of 5 ' UTR and 3 ' UTR, following special primer is designed, carries out the expansion of target gene HbFT1 total lengths Increase；

HbFT1-F：GTAAAAGCTGGATACCTCAGGCTCCAAAC；

HbFT1-R：ATGGGGTCAAATGTGGTAGAAAAAT.

The invention also discloses described rubber tree bloom controlling gene HbFT1 for prepare promotion rubber tree Blooming Accelerator application.

The present invention obtains rubber tree in Para rubber tree and blooms the full-length cDNA of controlling gene HbFT1 first, and passes through Rubber tree HbFT1 Tissue-specific expressions are analyzed, and HbFT1 genes are expressed in stabilization vane, the male flower bloomed and female flower, but Expression quantity is all than relatively low, and its expression quantity is raised with the increase at age in the stabilization vane of tree not of the same age, in the main florescence 3-5 months Expression quantity is extremely low, but is peaked the phase to 9-10 month tables.

The present invention shows that the transfer-gen plant of HbFT1 genes shows excessively by arabidopsis wild type genetic transformation Early blossoming phenotype, and with the formation of terminal inflorescence.Meanwhile, gene overexpression in ft-10 mutant arabidopsis, ft-10 dashes forward Becoming phenotype all can effectively be reverted to wild type phenotype, in addition some bloom it is also more early than WT lines.These results are equal Show that HbFT1 homologous genes there are very big potentiality to exercise the function of FT homologous genes.Therefore the gene can be used as Para rubber tree Highly useful regulation and control candidate gene of blooming, can be realized to rubber tree by gain-of-function mutation or afunction mutation research The Effective Regulation bloomed.

Brief description of the drawings

In order that present disclosure is more likely to be clearly understood, below according to specific embodiment of the invention and combine Accompanying drawing, the present invention is further detailed explanation, wherein,

Fig. 1 for HbFT1 genes tissue specific expression analysis (10 age tree roots, stem, bronze leaf, change colour leaf, pale green leaf, Stabilization vane, bud, the inflorescence of the first stage of development, the male flower bloomed, the female flower bloomed, pericarp and seed)；

Fig. 2 is HbFT1 genes in two expression patterns of different age of tree rubber tree Various Seasonals；

Fig. 3 is influence of the different photoperiods to HbFT1 gene expressions；

Fig. 4 is influence of the different temperatures to HbFT1 gene expressions；

Fig. 5 is the functional verification of rubber tree HbFT1 genetic transformation wildtype Arabidopsis thalianas；

Fig. 6 is that HbFT1 gene pairs ft-10 mutation type surfaces recover confirmatory experiment.

Specific embodiment

Method described in following embodiments, unless otherwise instructed, is conventional method.

The Para rubber tree of embodiment 1 is bloomed the acquisition of controlling gene HbFT1

Arabidopsis, the cDNA sequence of the FT genes of castor-oil plant for being logged in NCBI are analyzed, by searching for the rubber tree set up Blade transcript profile database, the piece segment information obtained by way of homologous comparison, is then spliced, then predict open reading And design DNA sequence dna and cDNA sequence that special primer acquisition includes entire open reading frame.Specific method is as follows：

(1) acquisition of HbTF1 gene opens reading frame

Gene-specific primer design is as follows：

HbFT1OF：GGAATTCATGCCTAGTGACAGAG；

HbFT1OR：CCCCCCGGGGCATCTTCTTCC.

7-33-97 (Rubber Institute, Chinese Academy of Agricultural Science's cultivation) blades and inflorescence are ground with Para rubber tree heat CDNA is mixed into template (being obtained with random primed reverse transcription), with HbFT1OF and HbFT1OR as primer, its final concentration of 0.4 μ Mol/L, enters performing PCR amplification in 20 μ l reaction systems.

PCR amplification programs are：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, 35 Individual circulation, 72 DEG C extend 10min eventually.Amplified production is connected with pMD19-T carriers, in conversion Escherichia coli EHA105 and is surveyed Sequence.Finally, the entire open reading frame of HbFT1 is obtained.

(2) 5 ' and 3 ' end UTR

Carry out building RACE cDNA libraries according to SMARTTMcDNA Library Construction Kit specifications (kit is purchased from Clontech companies), 5 ' UTR are obtained by 5 ' RACE, and adapter-primer used has 3, respectively：

QT：

CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTT；

Q1：GAGGACTCGAGCTCAAGC；With

Q0：CCAGTGAGCAGAGTGACG.

(Dieffenbacher C W, moral Vickers strangles the yellow trainings of G R.PCR technology experiment guides to 5 ' RACE operating processes reference literatures Hall translates Beijing:Science Press, 1998:268-277).CDNA sequence according to HbFT1 designs 2 primers and is respectively：

HbFT1-5UF：GTAAAAGCTGGATACCTCAGGCTCC；

HbFT1-5UR：CACCAACATGAACCCTAGGTTGGTT.

Specifically amplification program is：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, 35 Individual circulation, 72 DEG C extend 10min eventually.

3 ' the UTR of HbFT1 are obtained by 3 ' RACE：3 adapter-primers one that adapter-primer used and 5 ' RACE are used Sample, while 3 ' RACE operating processes are also that (Dieffenbacher C W, moral Vickers strangles G R.PCR technology experiment guides to reference literature Huang Peitang translates Beijing:Science Press, 1998:268-277).First with QT primers as reverse transcription primer, reverse transcription program is pressed Book is carried out as directed for reverse transcription.Afterwards respectively with the GSP3 of following HbFT1 genes^1stWith Q0 for primer carries out 3 groups of first round respectively PCR, afterwards respectively using 50 times of dilution PCR primers as the masterplate of the second wheel PCR.When carrying out the second wheel PCR, then respectively with this 2 The following GSP3 of individual gene^2ndBeing combined as primer pair with Q1 carries out the second wheel PCR, and product digs glue and obtains by electrophoresis；

HbFT1-GSP3^1st：GTGAGCGACATCCCTGGAACA；

HbFT1-GSP3^2nd：CGAAGACAGACAATCAACCCACC；

Amplification program is：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 are followed Ring, 72 DEG C extend 10min eventually.5 ' and 3 ' end RACE products are respectively 122bp and 113bp, and product is carried out after cutting glue purification Cloning and sequencing.

(3) clone of HbFT1 full length genes

According to the acquisition of 5 ' and 3 ' UTR, it is designed to expand the special primer of total length again：

HbFT1-F：GTAAAAGCTGGATACCTCAGGCTCCAAAC；

HbFT1-R：ATGGGGTCAAATGTGGTAGAAAAAT.

Masterplate is mixed into rubber tree blade and inflorescence cDNA, the amplification of target gene HbFT1 total lengths is carried out.

Amplification program is：95 DEG C of predegenerations 3min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 are followed Ring, 72 DEG C extend 10min eventually.The PCR primer of acquisition is connected and is sequenced with pMD19-T, and sequencing shows the piece of above-mentioned acquisition Rubber tree needed for section is the present invention blooms controlling gene, is designated as HbFT1, and the fragment has SEQ ID No in sequence table：1 Shown nucleotide sequence, its total length is 763 nucleotides, is 528 ORFs of nucleotides comprising a length 5 '-UTR of (ORF, from 123-650, the 5 ＇ ends nucleotide sequence of sequence 1), 122 nucleotides are (from 5 ' ends of sequence 1 the 1-122 nucleotide sequence) and 113 3 '-UTR of nucleotides (holding 651-763 nucleotide sequence from the 5 ' of sequence 1), One length of the gene code is 118 amino acid (see SEQ ID No in sequence table：2).

7-33-97 leaves genomic DNAs are ground as template with Para rubber tree heat simultaneously, enters performing PCR amplification (program ibid institute State), sequencing result shows to obtain the genomic dna sequence of rubber tree floral genes, and the sequence has SEQ ID in sequence table No：3 nucleotide sequence, totally 2692 nucleotides contains three length and is respectively the interior of 144,1181 and 604 nucleotides Containing son (holding 323-466,529-1709 and 1751-2354 nucleotide sequence from the 5 ' of sequence 3).

The rubber tree HbFT1 Tissue-specific expressions of embodiment 2 are analyzed

Each nutritive issue and organ of multiplication (bag of the 10 age trees of the rubber tree 7-33-97 of March collection are chosen in this research Include root, stem, bronze leaf, discoloration leaf, pale green leaf, discoloration leaf, stabilization vane, bud, the inflorescence of the first stage of development, the male flower bloomed, The female flower bloomed) and August part collection pericarp and seed, the RNA that them are extracted respectively is standby.

After entering DnaseI digestion to above-mentioned obtained RNA reverse transcription was carried out according to reverse transcription reagent box.By fluorescence Quantitative technique carries out the specific expressed analysis of target gene tissue.

Quantifying primer is：

HbFT1-QF：TACACATCAACGGTACACACCAGTC；

HbFT1-QR：CCAAAACATCCCCTATCACACG.

Result as shown in Figure 1 shows that HbFT1 genes only have in stabilization vane, the male flower bloomed and in female flower low amounts to express.

Embodiment 3HbFT1 genes are in two expression patterns of different age of tree rubber tree Various Seasonals

Stabilization vane with 2 age trees and 10 age trees is research object, the capturing material since March, and every month gathers once, Untill November.Quantitative fluorescence analysis are carried out to HbFT1 genes afterwards, quantitative primer is with foregoing tissue specific expression point Analysis primer.

Result as shown in Figure 2 shows that HbFT1 genes show similar expression mould in two different age of tree rubber trees Formula, 3 between June, HbFT1 gene expression amounts are all very low always, since July, in two age of tree stabilization vanes HbFT1 expression quantity starts to gradually rise, wherein the HbFT1 expression quantity in 10 age tree stabilization vanes is in September part highest, and 2 age trees are steady The HbFT1 expression quantity determined in leaf then shows highest in October.Then, HbFT1 expression quantity is gradually reduced directly in two age of tree trees To November, now, HbFT1 gene expression amounts turn again to minimum.What is more important, HbFT1 genes are in 10 age trees Expression quantity nearly all than 2 age trees in it is high.

Influence of the different photoperiods of embodiment 4 to HbFT1 gene expressions

40 plants of the same 3 months big seedlings of growing way are divided into two groups, every group 20 plants.One of which culture is at 28 DEG C Long-day environment (16h illumination/8h is dark)；Another set is then cultivated in 28 DEG C of short-day environment (8h illumination/16h is dark). After being cultivated 30 days under two kinds of environment, (24h) each two hour is gathered once two groups of blades of plant within one day respectively, altogether Collection 12 times.Quantitative fluorescence analysis are ibid.

Result as shown in Figure 3 shows, expression quantity of the HbFT1 genes under short-day environment is apparently higher than under long-day environment Expression quantity.Under short-day environment, HbFT1 genes all show metastable table at 12 points in morning in the evening from 8 points of morning to Up to level (in addition to occurring an expression peak when the 12 noon), after 12 points of morning, its expression gradually rises, in morning Upper 6 o'clock shows highest expression, then starts to decrease up to 8 points of morning again.Under long-day environment, HbFT1 bases Although because expression is very low but also shows some small expression wave phenomenons.

Influence of the different temperatures of embodiment 5 to HbFT1 gene expressions

40 plants of 3 months big seedlings are divided into two groups, every group 20 plants.First group of plant is planted in an incubator, The inside sets 28 DEG C, 32 DEG C and 36 DEG C three temperature periods；Another set is then cultivated in another incubator, the inside difference 28 DEG C, 24 DEG C, 20 DEG C, 16 DEG C and 8 DEG C five temperature periods of setting.Each constant temperature 3 days, is then transferred to next temperature successively Degree.In order to avoid photoperiod and biological clock may be unified in and sample on time 10 points of every morning to the influence of HbFT1 expression.

Result as shown in Figure 4 shows that, at 28 DEG C, HbFT1 gene expression amounts reach highest, when temperature is higher than or low When 28 DEG C, its expression quantity can be suppressed, and thus illustrate, 28 DEG C be rubber tree HbFT1 gene expressions the induction being best suitable for Temperature.

The functional verification of the rubber tree HbFT1 genetic transformation wildtype Arabidopsis thalianas of embodiment 6

Connected with HbFT1 genes using pXCS plant expression vectors (being preserved by Rubber Institute, Chinese Academy of Agricultural Science) Connect structure arabidopsis thaliana transformation wild type conversion carrier.Specific embodiment is as follows：

Separately design the primer of the target gene for building plant expression vector：It is used for building plant expression vector Primer be the special primer of gene described in embodiment 1, wherein the positive primer 5 ' end of gene is respectively added with EcoRI digestions position Point, anti-primer 5 ' is held respectively added with XmaI restriction enzyme sites.The PCR primer of acquisition is connected and is sequenced with pMD19-T carriers.Most Afterwards, the correct plasmid of sequencing is extracted, double digestion pXCS and the correct plasmid of sequencing is distinguished with EcoRI and XmaI, connected by T4DNA Connect enzyme and build the plant expression vector of HbFT1 target genes, and be named as pXCS-HbFT1.

For infecting for wildtype Arabidopsis thaliana, (Clough S J and Bent A are F.1998.Floral for reference literature dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J 16,735-743.) T0 that obtains for seed, first soaks it with running water, and It is positioned over vernalization 3 days in 4 DEG C of environment darkrooms.Sowed afterwards on the vermiculite soaked through 1/2M culture mediums in 22 DEG C of long-day Ambient growth (16h/8h), when seedling germinates, is sprayed with the herbicide that concentration is 10mg/L, is planted to screen resistance Strain.After 10 days, normal seedling will be grown and be transplanted to Nutrition Soil (Nutrition Soil：Vermiculite=2：1) sowing is grown and waited in.It is positive The detection of plant is by southern methods, the method reference literature (Blanc G, Baptiste C, Oliver G, Martin F,Montoro P.2006.Efficient Agrobacterium tumefaciens-mediated transformation of embryogenic calli and regeneration of Hevea brasiliensis Müll Arg.plants.Plant Cell Rep 2006,24,724-733)。

Gene T3 is cultivated with wild type in equivalent environment respectively for transfer-gen plant, and is compared they is bloomed Phenotype compares, and as a result sees Fig. 5 and table 1.

The 35S of table 1::The wild-type transgenic arabidopsis and wild type control of HbFT1 are bloomed and are compared

* 0.01 is represented<P<0.05；* represents P<0.01.

Result shows, all 35S::The wild-type transgenic strain of HbFT1/wt can show different degrees of early blossoming Phenotype, WT lines are bloomed for 25 days or so, and transgenic line is tied up to 17-19 days and bloomed, and flowering time and wheel seat number of sheets mesh are true Fullsized wild type will be lacked.It is similar to wildtype Arabidopsis thaliana phenotype in addition to blade is smaller and plant is shorter, all show Go out normal inflorescence phenotype.

Embodiment 7HbFT1 gene pairs ft-10 mutation type surfaces recover confirmatory experiment

In the HbFT1 target gene plant expression vector arabidopsis thaliana transformation ft-10 mutant that will be built, reference literature (Clough, S.J.and Bent, A.F. (1998) Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J.16:735- 743.).The T0 of acquisition first soaks it with running water, and be positioned over vernalization 3 days in 4 DEG C of environment darkrooms for seed.Sow afterwards In 22 DEG C of long-day ambient growths (16h/8h) on the vermiculite soaked through 1/2M culture mediums, when seedling germinates, with dense Spend for the herbicide of 10mg/L is sprayed, to screen resistant plant.After 10 days, normal seedling will be grown and be transplanted to nutrition Native (Nutrition Soil：Vermiculite=2：1) sowing is grown and waited in.

Ft-10 mutant transfer-gen plant is cultivated with wild type, ft-10 mutant plants in equivalent environment respectively, and They and wild type, the flowering phenotype of ft-10 mutant plants are respectively compared, Fig. 6 and table 2 below is as a result seen.

The 35S of table 2::The ft-10 mutant transgenic arabidopsis and ft-10 mutant and wild type of HbFT1 are bloomed ratio Compared with

* 0.01 is represented<P<0.05；* represents P<0.01.

Result shows that ft-10 mutant plants flowering time is 42 days or so, and ft-10 mutant transfer-gen plant is all The different degrees of early blossoming phenotype occurred in that than ft-10 mutant earlier, and the 6 plants of 35S for choosing::HbFT1/ft-10 transgenosis Strain even shows the flowering phenotype also more early than wildtype Arabidopsis thaliana and grows leaf on less wheel seat leaf and stem.

Obviously, above-described embodiment is only intended to clearly illustrate example, and not to the restriction of implementation method.It is right For those of ordinary skill in the art, can also make on the basis of the above description other multi-forms change or Change.There is no need and unable to be exhaustive to all of implementation method.And the obvious change thus extended out or Among changing still in the protection domain of the invention.

Sequence table

<110>Rubber Institute, Chinese Academy of Agricultural Science

<120>A kind of rubber tree bloom controlling gene HbFT1 and its clone with application

<160> 3

<170> PatentIn version 3.5

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<211> 763

<212> cDNA

<213>Hevea rubber

<400> 1

GTAAAAGCTG GATACCTCAG GCTCCAAACA ATATATATAT ACATGCCTGT ATGTCATTGA 60

ATTCTACACA TCAACGGTAC ACACCAGTCC ACTACTAGCT AGTGCTTCTC ATAGTTAGAA 120

AAATGCCTAG TGACAGAGAT CCTCTTGCTA TTGGGCGTGT GATAGGGGAT GTTTTGGACC 180

CTTTCGCAAG GTCTATCTCC CTCCAGGTCA CCTACAATAG CAGAGATCAT GTCACCAATG 240

GTTGTGAGCT CAAACCCTCT CAAATTGTCA ACCAACCTAG GGTTCATGTT GGTGGTGATG 300

ATTTAAGGAC CTTCTATACT TTGGTTATGG TGGACCCTGA CGCACCTAGC CCCAGTGACC 360

CAAATCTCAG AGAATACTTG CATTGGCTGG TGACTGATAT TCCAGCCACT ACAGGGACAA 420

GCTTTGGGCA AGAGGTTGTG TGCTATGAGA GCCCAGCACC ATCGGTGGGG ATTCATCGGT 480

TTGTTTTCAT ATTGTTTCGG CAACTGGGGA GGCAGACCGT GTATGCGCCT GGTTGGCGTC 540

AGAATTTCAA CACTAGAGAC TTTGCTGAGC TTTACAATCT TGGATCGCCG GTGGCCGCTG 600

TTTACTTTAA CTGCCAGAGG CAGAGTGGCA CCGGGGGAAG AAGATGCTAA CTCATCAAGT 660

AATAATAATG GGGTCCCTTT CTCCAAGGTT AATGAGTACT GAACTGTATT TCCAAACTAT 720

CTCAATAATA TTTTTCTACC ACATTTGACC TCATAAAAGA AAT 763

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<211> 175

<212> PRT

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Met Pro Ser Asp Arg Asp Pro Leu Ala Ile Gly Arg Val Ile Gly Asp

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Val Leu Asp Pro Phe Ala Arg Ser Ile Ser Leu Gln Val Thr Tyr Asn

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Ser Arg Asp His Val Thr Asn Gly Cys Glu Leu Lys Pro Ser Gln Ile

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Val Asn Gln Pro Arg Val His Val Gly Gly Asp Asp Leu Arg Thr Phe

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Tyr Tyr Leu Val Met Val Asp Pro Asp Ala Pro Ser Pro Ser Asp Pro

65 70 75 80

Asn Leu Arg Glu Tyr Leu His Trp Leu Val Thr Asp Ile Pro Ala Thr

85 90 95

Thr Gly Thr Ser Phe Gly Gln Glu Val Val Cys Tyr Glu Ser Pro Ala

100 105 110

Pro Ser Val Gly Ile His Arg Phe Val Phe Ile Leu Phe Arg Gln Leu

115 120 125

Gly Arg Gln Thr Val Tyr Ala Pro Gly Trp Arg Gln Asn Phe Asn Thr

130 135 140

Arg Asp Phe Ala Glu Leu Tyr Asn Leu Gly Ser Pro Val Ala Ala Val

145 150 155 160

Tyr Phe Asn Cys Gln Arg Gln Ser Gly Thr Gly Gly Arg Arg Cys

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GTAAAAGCTG GATACCTCAG GCTCCAAACA ATATATATAT ACATGCCTGT ATGTCATTGA 60

ATTCTACACA TCAACGGTAC ACACCAGTCC ACTACTAGCT AGTGCTTCTC ATAGTTAGAA 120

ATTCTACACA TCAACGGTAC ACACCAGTCC ACTACTAGCT AGTGCTTCTC ATAGTTAGAA 180

CTTTCGCAAG GTCTATCTCC CTCCAGGTCA CCTACAATAG CAGAGATCAT GTCACCAATG 240

GTTGTGAGCT CAAACCCTCT CAAATTGTCA ACCAACCTAG GGTTCATGTT GGTGGTGATG 300

ATTTAAGGAC CTTCTATACT TTGGTAACTT CTTCTTCTTC TTCTTCTTCT TCTTCTTCTT 360

CTTTCATAAT AGAATCTTAT TATGCACATT TGGTTTCTGT ATATATCCTA TGGTTTTTTA 420

CGTGTGAGAG ATAGATATAT GGTTGAGATT CTATTCGCTC GACGCAGGTT ATGGTGGACC 480

CTGACGCACC TAGCCCCAGT GACCCAAATC TCAGAGAATA CTTGCATTGG TACCTTCTTG 540

ATTCTCTCTC CTTCTCTCTG GCCTCCACTA TTCACACACA ACTACACGTA TATGCACGTT 600

TGAGTTTTGG TGCTCTAGGC TTAATTAATA TATGATATCT AATATATATG TATTCATTCA 660

TTTTCATCTT TCTGAAACAA ACTTCAACAA TATAATCCAT GCACAAAGCC TCCCTCTTCA 720

ACTAACCGGG CATGGGTGAA TTGTCAATAG CTTGGAGAGT TAATGGTAGT TGGCGACTTA 780

CACTCGTGGA AATCATCAAG CCCCATATGA AAGCTTACAA AGCTATCTAT AGAAAGCGAG 840

ATGTGGTAGT GCCACCATAT AATATAACAT CAAAAATTTA TTTTATATGT GAATTTTATA 900

TTTTATCTTG AATTCATTGT GAATTGAATC TAAATAAGAA TTTTCCTGCT TATCCTCATT 960

CCATACTTGC TTCTAGAAAT AGATACACAC CTGTCTTTCT TCTACTCCTC TGTGCAACTT 1020

TTTTTTTTTC AGTAATTAAT TGATTGGCCC CGAATTTTTC AAGACTGACT TAAAAGAATA 1080

AGGTTTTATT TAATATCTCT AGAGCCTATG CTCTTTTATA TGCATGGAAT AATTATAAAC 1140

ATATGAGTGC ATGGTATACT AAATTTTAGT ACACAAGAAA TATCTTATAA ATTTCTAAAG 1200

AATGAATATA TGCATATGGA ATTAGGACGA TAAAGAACTT AAAAAATTAA ATATTGAGAT 1260

TTTAAAAACA AAAAATACTT TTTAGATTAA AAATATTTTA TTATTTTAAT ATTTTTTTTA 1320

TTATTCAATC ATGAAAGTTT ACTACCATGT GGAATCAATG AAATGAATAG GACTTGCTTG 1380

TCTTAACCTC AAGTGACAAT TATGTATCAC TTATAGAATG ATCATGTCTA ATTAAATTAA 1440

TAATTCAACA ATCGAGTGCT TCATTACTTC ACAATGTAAA TGACGCAAAA AAATGCCTTC 1500

TCGCCATCCT AGTCTATGGG CTGCCTGCAT AAGACATCAC ATCAAAAGGA ATCGATGATC 1560

GAGGTCCATC ATCTTTTCGA TGAAGTAGGG GCTTAAATAC AAATTCATTG TTATTGAATA 1620

TTGTGTGCGA TAATTAAGAG GAATTAATAC AACTCAAGGC TAGCTGATTA TGTGAACTAA 1680

CAGACTGTTT CTTTTATGAA ATTTTTGTAG GCTGGTGACT GATATTCCAG CCACTACAGG 1740

GACAAGCTTT GGTGAGTAGA TTTAATTACC TTAAAATTTT TTATATATCC TTCATTTTGA 1800

TGATCATATG TATGTTTCAA AAGTTTGTCA TTAATTTTAC ATCATTATTT TCTCCAACTT 1860

AATTCAGCAA ATTAATAATA TAAGTATTAG CAACACTTAT TTGAAGAAGG TCGAGAGGAA 1920

CTATATAATT AATACAGATA TAATAAAGGT TACTCCACCT ATATATATGC ACGGAAAGAG 1980

AGATTCATTT ATCTTTTTTT TTTTTTTTTT TTTCTCTCTC GAATCTCTTA ATTTTCTTGT 2040

GGTAGATATC GTGCGAGGCC ATCAGTATCA ATCTTATTCC ACCTGATGCA TCTACACAGA 2100

ATATATAGTG TGTGGGGTTG GGAGACAACA ATATGATGAT AAAAGCATAA GAAATCTTAT 2160

CAACATATGT GAAATTGAGA AGATTCTCTC TTTCCTTGAT CTAGAAGAAG ATACTTAAAA 2220

ATAAAAAAGC AAAACCATAT ACTTGAATCT TGTATATATA ATTTATGTAC TCGGTTGGTG 2260

ACTTGGTGTA TAAGTGCAAT GATAATGATG GTGTTATACA TTTTGTAATG ATGATGGTGG 2340

TGTTATACAT TGTAGGGCAA GAGGTTGTGT GCTATGAGAG CCCAGCACCA TCGGTGGGGA 2400

TTCATCGGTT TGTTTTCATA TTGTTTCGGC AACTGGGGAG GCAGACCGTG TATGCGCCTG 2460

GTTGGCGTCA GAATTTCAAC ACTAGAGACT TTGCTGAGCT TTACAATCTT GGATCGCCGG 2520

TGGCCGCTGT TTACTTTAAC TGCCAGAGGC AGAGTGGCAC CGGGGGAAGA AGATGCTAAC 2580

TCATCAAGTA ATAATAATGG GGTCCCTTTC TCCAAGGTTA ATGAGTACTG AACTGTATTT 2640

CCAAACTATC TCAATAATAT TTTTCTACCA CATTTGACCC CATAAAAGAA AT 2692

Claims (10)

1. a kind of rubber tree blooms controlling gene HbFT1, it is characterised in that the cDNA of the gene HbFT1 includes such as SEQ ID No：Nucleotide sequence shown in 1.

2. rubber tree according to claim 1 blooms controlling gene HbFT1, it is characterised in that the gene HbFT1's CDNA nucleotide sequences such as SEQ ID No：Shown in 1.

3. the rubber tree described in a kind of claim 1 or 2 bloom controlling gene HbFT1 coding albumen, it is characterised in that including Such as SEQ ID No：Amino acid sequence shown in 2.

4. rubber tree according to claim 3 bloom controlling gene HbFT1 coding albumen, it is characterised in that its amino Acid sequence such as SEQ ID No：Shown in 2.

5. a kind of rubber tree blooms controlling gene HbFT1, it is characterised in that the genomic DNA of the gene HbFT1 is included such as SEQ ID No：Nucleotide sequence shown in 3.

6. rubber tree according to claim 5 blooms controlling gene HbFT1, it is characterised in that the base of the gene HbFT1 Because of group DNA nucleotide sequence such as SEQ ID No：Shown in 3.

7. a kind of rubber tree containing described in claim 1 or 2 blooms the recombinant plant expression vector of controlling gene HbFT1, its It is characterised by, the carrier is recombinant plasmid pXCS-HbFT1.

8. recombinant plant expression vector according to claim 7, it is characterised in that in the recombinant plant expression vector Carrier is pMD19-T carriers.

9. a kind of rubber tree cloned described in claim 1 or 2 or 5 or 6 blooms the method for controlling gene HbFT1, and its feature exists In comprising the following steps：

(1) template is mixed into the leaves genomic DNA of Para rubber tree, or the cDNA of blade and inflorescence respectively, design is such as Lower primer enters performing PCR amplification, obtains HbTF1 gene open reading frames；

HbFT1OF：GGAATTCATGCCTAGTGACAGAG；

HbFT1OR：CCCCCCGGGGCATCTTCTTCC；

(2) based on HbTF1 gene open reading frames, design causes the UTR and 3 ' of synthesis 5 ' UTR as follows；

HbFT1-5UF：GTAAAAGCTGGATACCTCAGGCTCC；

HbFT1-5UR：CACCAACATGAACCCTAGGTTGGTT；

HbFT1-GSP3^1st：GTGAGCGACATCCCTGGAACA；

HbFT1-GSP3^2nd：CGAAGACAGACAATCAACCCACC；

(3) according to the acquisition of 5 ' UTR and 3 ' UTR, following special primer is designed, carries out the amplification of target gene HbFT1 total lengths；

HbFT1-F：GTAAAAGCTGGATACCTCAGGCTCCAAAC；

HbFT1-R：ATGGGGTCAAATGTGGTAGAAAAAT.

10. the rubber tree described in claim 1 or 2 or 5 or 6 bloom controlling gene HbFT1 for prepare promotion rubber tree shift to an earlier date The application of the accelerator bloomed.