CN105693836A - Cold-resistant gene of rubber trees, cold-resistant protein and application of cold-resistant gene and cold-resistant protein - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and relates to a cold-resistant key gene HbICE1 of rubber trees, a cold-resistant protein and application of the cold-resistant key gene and the cold-resistant protein to the aspect of improving the cold resistance of plants. The gene HbICE1 related to the cold resistance of the rubber trees is cloned, CDS sequences of the gene are shown as sequences SEQ ID NO.1, and protein sequences of the gene are shown as sequence tables SEQ ID NO.2. The cold-resistant gene, the cold-resistant protein and the application have the advantages that as shown by genetic transformation, the cold resistance of Arabidopsis thaliana can be improved by the aid of the gene, the gene can be applied to selectively breeding cold-resistant varieties of the rubber trees and genetically improving the rubber trees, and a novel gene resource can be provided for the rubber trees.

Description

A kind of rubber tree Cold resistant genes and albumen and application thereof

Technical field

The invention belongs to plant genetic engineering field, be specifically related to a kind of rubber tree Cold resistant genes HbICE1 and albumen and the application in improving rubber tree cold tolerance thereof。

Background technology

Para rubber tree (Heveabrasiliensis (Willd.exA.Juss.) Muell.) is the main source of natural rubber, is not only the most important specialty economies crop in Hainan Province, the strategic resources that Ye Shi China is important。Para rubber tree originates in Amazon forest, for perennial deciduous tree, it it is typical tropical rain forest tree, well-grown under the environment of high temperature, high humidity, quiet wind and fertile soil, but low temperature is very sensitive, can not resist cold (PriyadarshanPM.Contributionsofweathervariablesforspecifi cadaptationofrubbertree (HeveabrasiliensisMuell.-Arg) clones [J] .GenetMolBiol, 2003,26 (4): 435-440.)。At present, mainly produce glue country in the world and be respectively positioned in the climate zone of suitable rubbers tree planting, such as Malaysia, Thailand, Indonesia etc.；And Zhi Jiao district of China belongs to tropical monsson climate, it is mainly distributed on Hainan, Guangdong, Guangxi, Fujian, Yunnan, and frequently suffers from the invasion and attack of the natural disaster such as low temperature and typhoon。Cold damage is one of the Major Natural Disasters in Zhi Jiao district of China, not only affects Rubber Yield, and directly threatens existence (PriyadarshanPM, HoaTTT, HuasunH, the de of rubber treePS.Yieldingpotentialofrubber (Heveabrasiliensis) insub-optimalenvironments [J] .JournalofCropImprovement, 2005,14 (1-2): 221-247.)。As the cold wave at the beginning of 2008 makes trees large area withered, bring heavy economic losses to China's Rubber Industry。Although rubber tree has been successfully introduced China, but cold damage is still restriction and plants the bottleneck of glue industry development, and therefore parsing rubber tree resists the molecule mechanism of cold damage stress is one of significant problem being badly in need of solution in natural rubber production。

In model plant arabidopsis, signal pathway and the key gene of plant reply low temperature stress have been identified very abundant all, the conduction of its signal and molecular action pattern are as follows: ICE1 (INDUCEROFCBFEXPRESSION) is the activating transcription factor of CBF gene, at low ambient temperatures, it can specifically bind on the MYC cis acting element of CBF promoter, activate the expression (ChinnusamyV of CBF, OhtaM, KanrarS, LeeBH, HongX, AgarwalM, ZhuJK.ICE1:aregulatorofcold-inducedtranscriptomeandfreez ingtoleranceinArabidopsis [J] .GenesDev, 2003, 17 (8): 1043-1054.)。CBF activating transcription factor is able to the class transcription factor in conjunction with CRT/DRE core parts, and CRT/DRE element (C-repeat/Dehydrationresponsiveelement) is with the CCGAC class cis acting element being core sequence。CBF gene can be expressed rapidly under low temperature induction, and it is attached on CRT/DRE element, thus starting the expression of the downstream functional genes such as COR, promote plant cold resistance (BakerSS, WilhelmKS, ThomashowMF.The5 '-regionofArabidopsisthalianacor15ahascis-actingelementsth atconfercold-, drought-andABA-regulatedgeneexpression [J] .PlantMolBiol, 1994,24 (5): 701-713；StockingerEJ, GilmourSJ, ThomashowMF.ArabidopsisthalianaCBF1encodesanAP2domain-co ntainingtranscriptionalactivatorthatbindstotheC-repeat/D RE, acis-actingDNAregulatoryelementthatstimulatestranscripti oninresponsetolowtemperatureandwaterdeficit [J] .PNAS.1997,94 (3): 1035-1040.)。Arabidopsis comprises 4 CBF transcription factor, respectively CBF1, CBF2, CBF3 and CBF4, overexpression CBF1 or CBF3 in arabidopsis, the cold tolerance of transfer-gen plant to be substantially better than wild arabidopsis (GilmourSJ, SeboltAM, SalazarMP, EverardJD, ThomashowMF.OverexpressionoftheArabidopsisCBF3transcript ionalactivatormimicsmultiplebiochemicalchangesassociated withcoldacclimation [J] .PlantPhysiol, 2000, 124 (4): 1854-1865.)。Being found that 2 ICE genes in arabidopsis at present, respectively ICE1 and 1CE2, ICE1 encode the bHLH type activating transcription factor of a similar MYC, and the overexpressing plants tolerance to cold of ICE1 is significantly stronger than wildtype Arabidopsis thaliana。After ICE2 gene is proceeded to arabidopsis, transgenic arabidopsis can be survived and normal growth under the low temperature environment of-20 DEG C, and the expression of CBF1 can be remarkably reinforced (FursovaOV in transgenic line, PogorelkoGV, TarasovVA.IdentificationofICE2, ageneinvolvedincoldacclimationwhichdeterminesfreezingtol eranceinArabidopsisthaliana [J] .Gene, 2009,429 (1): 98-103.)。Otherwise, cold coercing is shown as hypersensitization by mutant ice1。The result of transcriptome analysis shows: in ice1 mutant, the expression of the transcription factor of the gene of the cold stress regulatory of 40% and 46% cold stress regulatory is all lowered。And, the cold acclimation protein participating in Ca2+ oscillations, receptor protein kinase and lipid signal in ice1 mutant has been also affected by impact (LeeBH, HendersonDA, ZhuJK.TheArabidopsiscold-responsivetranscriptomeanditsre gulationbyICE1 [J] .PlantCell, 2005,17 (11): 3155-3175.)。Further, the result that the transcript profile chip data of the cold response of mutant and transgenic arabidopsis carries out bioinformatic analysis shows, ICE1 is a vital activating transcription factor in the signal pathway of low temperature stress, the transcriptional level control network of ICE-CBF transcriptional cascade reaction plays a significant role (Benedictetal., 2006)。And, R2R3 type myb transcription factor MYB15 can be combined with each other with ICE1, and it is attached to CBF promoter region Myb recognition site, expression (the CatherineB of negative regulation CBF, MattG, JohanT, NormanH, VaughanH.Consensusbydemocracy.Usingmeta-analysesofmicroa rrayandgenomicdatatomodelthecoldacclimationsignalingpath wayinArabidopsis [J] .PlantPhysiol, 2006,141 (4): 1219-1232.)。In addition, SMUO (smallubiquitin-relatedmodifier) E3 ligase SIZ1 and ubiquitin degradation E3 ligase HOS1 can modify ICE1 and then regulation and control ICE-CBF signal path (DongCH from level after translation, AgarwalM, ZhangY, XieQ, ZhuJK.Thenegativeregulatorofplantcoldresponses, HOS1, isaRINGE3ligasethatmediatestheubiquitinationanddegradati onofICE1 [J] .PNAS, 2006,103 (21): 8281-8286.)。Visible, ICE1 is activating transcription factor important in low temperature stress signal transmission path, is the key factor connecting cold signal and downstream response gene。

But, the research for Para rubber tree low temperature stress signal pathway is also in the stage at the early-stage。So far, above-mentioned signal path includes HbICE1 and is not yet cloned in interior most gene or identifies, this becomes the bottleneck in the cold-resistant study mechanism of rubber。

Summary of the invention

In view of the deficiencies in the prior art, it is an object of the invention to clone a kind of rubber tree Cold resistant genes HbICE1 and albumen, the plant tolerance to low temperature can being effectively improved after this gene overexpression, thus being applied to the cultivation of cold-resistant variety。

A kind of rubber tree Cold resistant genes HbICE1 of offer and albumen application in the cold-resistant breed of variety of rubber tree are provided。

In order to realize the above-mentioned purpose of the present invention, inventor utilizes the rubber transcript profile sequencing data that its seminar's early stage completes, the full length cDNA sequence of rubber tree low temperature stress signal pathway key gene HbICE1 it is successfully obtained by electronic cloning and RACE technology, and it being cloned into HbICE1 by pcr amplification and order-checking merit, sequence is 1410bp。Owing to the ICE gene of rubber tree is cloned first, so being HbICE1 by this unnamed gene。After this gene is carried out transgenic checking, it has been found that this gene of overexpression can significantly improve the cold tolerance of arabidopsis。And by fluorescent quantitative PCR technique, have detected the change that this gene tackles the expression of Different stress in arabidopsis。Utilize the protein subcellular positioning scenarios of confocal laser scanning microscope HbICE1, it has been found that this protein localization is in nucleus。

In consideration of it, first aspect of the present invention provides rubber tree cold-resistant factor HbICE1 albumen, its aminoacid sequence is shown in SEQ ID NO:2。

The second aspect of the invention provides the polynucleotide sequence encoding a kind of rubber tree cold-resistant factor HbICE1 albumen。Preferably, this sequence is shown in SEQ ID NO:1, or the polynucleotide sequence shown in SEQIDNo.1 adds through nucleotide, deletes, replaces, the conservative mutation modified and the conservative variant that obtains。

The third aspect of the invention provides the expression vector comprising above-mentioned polynucleotide sequence；Wherein preferred expression vector is pMD18-T carrier, pCAMBIA1300 carrier or PEGAD carrier。

The fourth aspect of the invention provides the cell comprising above-mentioned expression vector；Wherein preferred cell is bacillus coli DH 5 alpha or Agrobacterium GV3101。

The present invention is found by experimental study, and in rubber tree, the above-mentioned Cold resistant genes HbICE1 of overexpression, can be effectively improved the plants such as the arabidopsis tolerance to low temperature；Therefore, the fifth aspect of the invention provides above-mentioned aminoacid sequence SEQIDNO:2 or nucleotide sequence SEQIDNO:1 application in plant cold resistance breed of variety。

Compared with prior art, Cold resistant genes HbICE1 in the rubber tree that the present invention relates to, it is up to 85% with Homologous gene sequences similarity degree in other identified at present species, belongs to a brand-new gene。This gene can be effectively improved the cold tolerance of arabidopsis after overexpression；And expressed protein localization is in nucleus, is regulated and controled by low temperature stress, it is especially suitable for the cultivation of the cold-resistant kind of rubber tree。

Accompanying drawing explanation

Fig. 1 is rubber tree ICE1 and other species ICE evolution of amino acid tree analysis chart；

Fig. 2 is that pEGAD-HbICE1 recombinant expression carrier builds flow chart；

Fig. 3 is that pEGAD-HbICE1 recombinant expression carrier converts Agrobacterium bacterium colony PCR qualification electrophoretogram；

Fig. 4 is HbICE1 process LAN plant genome identification electrophoretogram；

Fig. 5 is the HbICE1 expression analysis chart of HbICE1 process LAN plant；

Fig. 6 is that overexpression rubber tree ICE1 gene can improve arabidopsis cold tolerance；

Fig. 7 is the HbICE1 laser confocal microscope detection figure being positioned in nucleus；

Fig. 8 is HbICE1 gene relative expression quantity in rubber tree different tissues；

Fig. 9 is HbICE1 expression pattern under low temperature stress processes。

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is made more detailed description。According to description below and these embodiments, those skilled in the art may determine that the basic feature of the present invention。And without departing from the spirit and scope of the invention, it is possible to the present invention is made various change and amendment, so that its applicable various uses and condition。Such as, the polynucleotide sequence of this gene add through nucleotide, delete, replace, the conservative mutation modified and the conservative variant that obtains falls within protection scope of the present invention。

1, the clone of HbICE1 gene and analysis

Utilize the rubber transcript profile sequencing data that this seminar early stage completes, obtained the full-length cDNA of rubber tree low temperature stress signal pathway key gene HbICE1 by electronic cloning and RACE technology first。According to splicing sequential design pair of primers。Special primer is P1:5 '-CGGAATCCATGCTTGATACCGACTGGTA-3 ', P2 primer is 5 '-AACTGCAGCATCATTCCATGAAAGCCAG-3 ', and amplification obtains 3 ' terminal sequences of HbICE1 gene。Carry out pcr amplification with the cDNA that Para rubber tree latex RNA reverse transcription becomes for template, adopt the primer of above-mentioned design to utilize high-fidelity enzymatic amplification HbICE1 sequence。

Amplification method is: first extracting latex of panama rubber tree RNA, its detailed process is as follows: by material requested DEPC water soaking overnight, and secondary Nikkei high temperature sterilize post-drying gives standby completely；The latex of fresh collection is loaded in 50mL centrifuge tube, add 10mL and shift to an earlier date 2 × RNA Extraction buffer of pre-cooling, it is placed on and acutely shakes mixing 1min on ice, add 20mL water-saturated phenol (25) chloroform (24) isoamyl alcohol (1) mixed liquor, acutely concussion mixes 5min then ice bath 10min on ice。4 DEG C, 14000rpm is centrifuged 10min；Supernatant is transferred in new centrifuge tube, adds isopyknic water-saturated phenol (25) chloroform (24) isoamyl alcohol (1) mixed liquor, shake 5min, ice bath 5min on ice, 4 DEG C, 14000rpm, 10min；Collect supernatant, add isopyknic chloroform (24) isoamyl alcohol (1) mixed liquor, shake 5min, ice bath 5min on ice, 4 DEG C, 14000rpm, 10min；Collect supernatant, add 1/3 volume 8mol/LLiCl solution, shake 5min on ice, in the ratio of often pipe 1.0mL, mixed liquor is dispensed in 1.5mL centrifuge tube ,-20 DEG C of precipitates overnight。4 DEG C, 14000rpm is centrifuged 30min；Collect precipitation, be sequentially added into 750 μ LDEPC water and 250 μ L8mol/LLiCl solution wherein, with liquid-transfering gun piping and druming mixing, be again placed in-20 DEG C of precipitates overnight。4 DEG C, 14000rpm is centrifuged 30min；Collect precipitation, be sequentially added into 300 μ LDEPC water and 30 μ L3mol/LNaAc solution (pH5.2) and 750 μ L dehydrated alcohol, after liquid-transfering gun piping and druming mixing, be placed in-20 DEG C of precipitation a few hours or overnight。4 DEG C, 14000rpm is centrifuged 30min；Collecting precipitation, shift to an earlier date 75% absolute ethanol washing 3 times of pre-cooling with 500 μ L, 4 DEG C, 14000rpm is centrifuged 10min；Abandoning supernatant, extremely dried completely by bottom-up for centrifuge tube slant setting, finally with 20 μ LDEPC water by resolution of precipitate, take 2 μ L sample and carry out electrophoresis detection in the superclean bench ventilated, all the other are placed in-80 DEG C and save backup。Then with Reverse Transcription box (purchased from TOYOBO company, Japan) it is total cDNA by mRNA reverse transcription, detailed process is referring to description, again as template above forward and reverse primer coded sequence utilizing high-fidelity enzyme PrimeSTARTM (purchased from Takara company, Japan) to amplify HbICE1 from rubber tree leaf cDNA。PCR response procedures: 98 DEG C of degeneration 10s, 55 DEG C of annealing 15s, 72 DEG C extend 90s, 30 circulations。Product is after 1% agarose gel electrophoresis detection is for single band, test kit is reclaimed (purchased from Shanghai Sheng Gong company with glue, China, detailed process is referring to description) reclaim target stripe, it is cloned into pMD18-T carrier (purchased from Takara company, Japan), take 10 μ L connection products and carry out thermal shock conversion bacillus coli DH 5 alpha competence (with reference to works such as J. Pehanorm Brookers, Huang Peitang etc. translate, Molecular Cloning: A Laboratory guide (third edition), Science Press, 2002 editions), coat on the newly configured LB solid plate containing ampicillin/bromo-4-of isopropyl-β-D-thiogalactoside/5-chloro-3-indyl-B-D-galactose sweet (Amp/IPTG/X-gal), 37 DEG C of overnight incubation, select white macula some, in the LB liquid medium containing Amp50mg/L, in 37 DEG C of 200r/min shaken cultivation overnight to muddy, detect whether bacterium solution is positive with primer PCR, enzyme action detection sequence verification sequence (see sequence table SEQ IDNO.1 Suo Shi), examining order is completed by Shanghai Sheng Gong company。

Sequencing result shows, the cDNA total length of the HbICE1 cloned is 1410bp (see SEQ ID NO:1 Suo Shi), encodes 469 aminoacid (shown in SEQIDNO:2)。Protparam online tool is utilized to analyze the physicochemical property of HbICE1 protein, it was predicted that to analyze result and show: the molecular mass of HbICE1 albumen is about 51kDa, and theoretical isoelectric point, IP (pI) is 5.83, altogether includes 7114 atoms, and molecular formula is C₂₂₂₅H₃₅₂₈N₆₄₆O₆₉₅S₂₀, the instability index of HbICE1 albumen is 58.06, it was shown that HbICE1 albumen is unstable。Utilizing the hydrophilic and hydrophobic of online tool ProtScale analyzing proteins, result display HbICE1 is hydrophilic protein simultaneously。Utilizing TMHMM online tool (http://www.cbs.dtu.dk/services/TMHMM/) that Para rubber tree HbICE1 albumen carries out cross-film simultaneously and distinguish analysis, result shows that this albumen does not have trans-membrane region, and its also non-transmembrane protein is described。Finding through Multiple Sequence Alignment analysis, the ICE1 albuminoid of HbICE1 and other species has certain similarity, and HbICE1 has bHLH region, typical structure territory and the C-terminal conservative region of ICE1 albuminoid。Aminoacid sequence according to the HbICE1 gene code obtained after amplification order-checking, on NCBI, carry out Homology search after translating into albumen, obtain the protein sequence of 8 different plant species, the protein sequence of different from these for HbICE1 plants is carried out multiple ratio pair, constructing system cladogram, sees Fig. 1。All in all, the evolutionary relationship of HbICE1 and other species reaches unanimity, and the species the highest with rubber ICE1 similar sequences are closer with its sibship for leprosy and Semen Ricini, therefore can determine that the gene that we obtain is exactly the ICE1 gene of Para rubber tree from the angle of bioinformatic analysis。

2, overexpression vector construction

Owing to rubber tree is high megaphanerophyte, its genome sequence is unknown, and rubber tree genetic transfoumation also imperfection, it is thus achieved that the probability of rubber mutant is little, give clone and identify rubber-based because of function bring very big difficulty。In order to overcome the restriction of objective condition, and then the biological function of the rubber tree HbICE1 gene cloned is verified from genetic level, we construct pEGAD-HbICE1 plant expression vector, and are transformed in arabidopsis, intend analyzing the cold tolerance of overexpressing plants。

With the correct 19T-HbICE1 that checks order for template, pcr amplification is carried out with the primer of ECOR1 restriction enzyme site with design, utilize glue to reclaim test kit to be undertaken pcr amplification product reclaiming (purchased from Shanghai Sheng Gong company, China), utilize ECOR1 enzyme action to reclaim product and obtain the purpose fragment of HbICE1 gene, enzyme action PEGAD carrier simultaneously, and genes of interest fragment is mixed with the ratio of 0.8:1 with the PEGAD carrier large fragment of ECOR1 enzyme action, add T₄DNA ligase (purchased from Takara company) 1U, 1 × reaction buffer, sterilized water is supplemented to 10 μ L systems, 16 DEG C link 10 hours, take 10 μ L connection products and carry out thermal shock conversion bacillus coli DH 5 alpha competent cell, coat screening positive clone enzyme action detection on that (Km) 50mg/L resistance plate of newly configured card。Through PCR Testing and appraisal positive colony extracting plasmid。Last enzyme action test strip size is consistent with the length of former PCR primer, transformed bacteria does puncture tube and delivers to the raw work in Shanghai and check order。The flow process building pEGAD-HbICE1 plant expression vector is shown in Fig. 2。

3, the qualification of genetic transformation and transfer-gen plant

Heat shock method is adopted to forward in Agrobacterium GV3101 competent cell the plant expression vector pEGAD-HbICE1 built, with containing rifampicin (Rif) 100mg/L, the YEB solid resistance plate screening blocking that (Km) 50mg/L converts positive speckle, several positive speckles of picking carry out bacterium colony PCR detection, the band of about 1400bp is demonstrated through the detection of bacterium colony PCR rear electrophoresis, in the same size with HbICE1, see Fig. 3。The bacterial strain confirming as the positive preserves for follow-up genetic transformation。Flower-dipping method is utilized to be transformed in wildtype Arabidopsis thaliana by the Agrobacterium GV3101 containing purpose plasmid PEGAD-HbICE1。Concrete grammar is as follows: the Agrobacterium GV3101 inoculation containing purpose plasmid to 100ml contained in the dual anti-LB liquid medium of card sodium mycin and gentamycin, cultivates two days with the rotating speed of 250rpm in the shaking table of 28 DEG C。With the centrifugal 10min of the rotating speed of 5000rpm, collect thalline, then thalline is resuspended to 100ml5% sucrose and 0.05% surfactant solution in, Agrobacterium is fully resuspended。Time the plant to be transformed comparison that the flowers are in blossom is luxuriant, by the of short duration immersion about 15 to 30 seconds in Agrobacterium-mediated Transformation solution of the flower of arabidopsis。By transformed plant with, on lid lid, transformed plant being kept in Dark Place 1 day, then raise lid and normally cultivate。In order to improve transformation efficiency, it is possible to again plant is carried out Agrobacterium after converting one week and infect conversion。When seedling grows into two weeks, herbicide screening is utilized to go out to have the positive plant of basta resistance。Then utilize genomic kit (hundred Tykes, China) to extract the DNA of the positive Seedling of primary dcreening operation, utilize HbICE1 specific primer to carry out PCR and detect whether recombiant plasmid PEGAD-HbICE1 is successfully transferred to arabidopsis from DNA level。PCR rear electrophoresis testing result shows that the stripe size of band and the genes of interest amplified is consistent, sees Fig. 4。Tentatively prove that engineering bacteria successfully proceeds to Arabidopsis plant from DNA level。Further, whether we utilize the RNA that RNA test kit (sky root, China) extracts transfer-gen plant to carry out RT-PCR and detect the HbICE1 of rubber tree and express in arabidopsis, result show HbICE1 can in arabidopsis great expression, see Fig. 5。The positive transgenic plant identified is gone down to posterity, and obtains homozygote。

4, the qualification of transgenic line cold tolerance

In order to verify HbICE1 gene function in cold-resistant, transgenic line 9#, 11#, 17# and comparison WT (acceptor material used by transgenic) are planted in 1/2MS (sigma by inventor simultaneously, the U.S.) in culture medium, 4 DEG C of lucifuges process 3 days。Then being placed in illumination box and cultivate 14 days, condition of culture is as follows: temperature is 22 ± 1 DEG C, and relative humidity controls between 50%-60%, and intensity of illumination is 100 μm of olm-2s-1, and the photoperiod is long-day 16L/8D。Then by seedling 0 DEG C of cold adaptation 2 days ,-8 DEG C process 4h, are then placed in by the seedling processed under normal condition and cultivate 7 days, observe phenotype the rate of isozygotying of each plant of statistics of taking pictures。Test result indicate that: after freeze injury processes, in arabidopsis, the rate of isozygotying of the transgenic line of overexpression HbICE1 gene is apparently higher than compareing wild type (Fig. 6), prove that the gene HbICE1 that we clone in rubber tree has cold-resistant function really, be a functional gene that can have very much potentiality in the application of the cold-resistant breed breeding of rubber tree。

5, HbICE1 subcellular fraction learns the research of location

Owing to ICE1 is a transcription factor, and ExPASy analyzes and shows that this albumen of HbICE1 is likely to be positioned at nucleus, in order to study the Subcellular Localization of this albumen of HbICE1, explain the function of HbICE1 further, we construct fusion vector pCAMBIA1300-eGFP-HbICE1, primer sequence used in structure refers to table 1, and building process is similar to above-mentioned fusion vector pEGAD-HbICE1。

Table 1 builds pCAMBIA1300-eGFP-HbICE1 Vector PCR amplification gene specific primer

Electric shocking method is utilized to be transformed in Agrobacterium LBA4404 the fusion vector pCAMBIA1300-eGFP-HbICE1 that success builds。Being inoculated in the 5mL YEP fluid medium containing kanamycin, gentamycin and rifampicin by the Agrobacterium list bacterium colony LBA4404 containing purpose plasmid, 30 DEG C, 200rpm is overnight；Take 30 μ L culture fluid and go in the 30mL YEP fluid medium containing identical resistance, 30 DEG C, 200rpm to OD₆₀₀=1.5；5000rpm15min, abandons supernatant；Add 30mL re-suspension liquid resuspended, 5000rpm5min, abandons supernatant；Add re-suspension liquid and make OD₆₀₀=1.0, room temperature stands 2-3h。Draw bacterium solution with syringe, select the health tobacco blade that upgrowth situation is good, avoid vein from leaf back and bacterium solution is injected into Nicotiana tabacum L. from pore, index blade, cultivate 3-5d and faint yellow do position observation。Injection is had the leaf tissue film-making of genes of interest, utilizes laser confocal microscope to detect its positioning scenarios。The excitation wavelength of green fluorescence GFP and transmitting wavelength are 488nm and 508nm respectively。Observation indicate that: after control vector (pCAMBIA1300-eGFP) proceeds to Nicotiana tabacum L., detection green fluorescence can be full of whole cell, and in the tobacco leaf converting fusion vector pCAMBIA1300-HbICE1, green fluorescence overlaps (Fig. 7) with the fluorescent spots of nucleus specific dye DAPI, showing that HbICE1 is positioned nucleus, hint HbICE1 is a transcription factor。

6, the expression pattern analysis of HbICE1

In order to study HbICE1 expression in each tissue of rubber tree, we extract the RNA of six different tissues such as latex of panama rubber tree, blade, stem apex, bark, gynoecium and stamen, and reverse transcription becomes cDNA, carrying out fluorescence quantitative PCR detection with the cDNA in different tissues for template, concrete operation step is with reference to the RealTimePCR test kit of Takara company and ABI7500RealTimePCR instrument。The primer of the PCR of fluorescent quantitation refers to table 2。

Table 2HbICE1 gene specific primer

Using RH8 as internal reference, the result of quantitative fluorescent PCR shows, HbICE1 has expression in each tissue of rubber tree, and the expression in bark is the most notable, sees Fig. 8。In order to explain HbICE1 effect in rubber tree cold damage stress further, rubber tree is placed under 4 DEG C of low temperature and processes different time, detect the change of its expression by applicant, and result shows that HbICE1 is subject to induction of chilling stress, and reaches the highest after 3h, sees Fig. 9。Result above hint rubber tree HbICE1 gene plays an important role in the process of the cold-resistant signal transmission of rubber tree。

In sum, the gene HbICE1 that the present invention clones in rubber tree has cold-resistant function really, is a functional gene that can have very much potentiality in the application of the cold-resistant breed breeding of rubber tree。

Claims (10)

1. a rubber tree cold-resistant factor HbICE1 albumen, its aminoacid sequence is shown in SEQ ID NO:2。

2. encode the polynucleotide sequence of rubber tree cold-resistant factor HbICE1 albumen。

3. polynucleotide sequence according to claim 2, it is characterized in that: this sequence is shown in SEQ ID NO:1, or the polynucleotide sequence shown in SEQIDNo.1 adds through nucleotide, deletes, replaces, the conservative mutation modified and the conservative variant that obtains。

4. comprise the expression vector of polynucleotide sequence described in Claims 2 or 3。

5. expression vector according to claim 4, it is characterised in that: it is pMD18-T carrier, pCAMBIA1300 carrier or PEGAD carrier。

6. comprise the cell of expression vector described in claim 4 or 5。

7. cell according to claim 6, it is characterised in that: it is bacillus coli DH 5 alpha or Agrobacterium GV3101。

8. the application in plant cold resistance breed of variety of the aminoacid sequence SEQIDNO:2 described in claim 1。

9. the application in plant cold resistance breed of variety of the polynucleotide sequence SEQIDNO:1 described in Claims 2 or 3。

10. application according to claim 8 or claim 9, it is characterised in that: described plant is rubber tree, arabidopsis。