CN101775071A - Protein for transferring saccharose and application of protein-coding gene thereof - Google Patents

Abstract

The invention discloses a protein for transferring saccharose and the application of a protein-coding gene thereof. The protein has one of the following sequences of amino acid residue: 1) amino acid residue sequence of SEQ ID No.1 in a sequence table; and 2) protein which is substituted and/or lost and/or added by one or a plurality of amino acid residue(s) with the SEQ ID No.1 amino acid residue sequence in the sequence table, has the function for transferring the saccharose, and is derived from the SEQ ID No.1. The yeast expressing system shows that the protein for transferring the saccharose has high saccharose appetency, has the typical characteristic of a saccharose/proton cotransporter, and is a key protein for deciding the supply of the saccharose and the yield of the rubber from a latex vessel. The gene of the protein for transferring the saccharose can be transformed into microorganisms or plants to improve the use efficiency of the microorganisms or the plants to the saccharose and the yield of the microorganisms or the plants. The gene of the protein for transferring the saccharose is transformed into saccharomyces cerevisiae to improve the absorption efficiency to the saccharose and the wine-making efficiency.

Description

The application of a kind of sucrose transporter and encoding gene thereof

Technical field

The present invention relates to a kind of sucrose transporter and encoding gene thereof and application.

Background technology

Natural rubber (cis _ 1,4_ polyisoprene, rubber hydrocarbon) is a kind of important basic industry raw material, and its comprehensive physicochemical property is better than utilizing the synthetic rubber of oil production.At present, the natural rubber more than 99% is from a tropical tree species _ Para rubber tree.Rubber is at a kind of eggcase cell, i.e. synthetic in the tenuigenin of latex dust, and the tenuigenin of latex dust also claims latex.The latex dust cell periodically breaks up generation by form layers, and the latex dust of each differentiation forms a line, in bark, be scattered in from the inside to surface a plurality of latex dusts row that are parallel to each other (d ' Auzac, J., Jacob, J.L.,

J.C., Cl é ment, A., Gallois, R., Crestin, H., Lacote, R., Pujade-Renaud, V.and Gohet, E.. (1997) Theregulation of cis-polyisoprene production (natural rubber) from Hevea brasiliensis.In:Pandalai S.G. (ed.) .Recent research developments in plant physiology.Vol.1.Trivandrum:Research Singpost, pp.273-332; Hao B.Z.and Wu J.L. (2000) Laticiferdifferentiation in Hevea brasiliensis:induction by exogenous jasmonic acid and linolenicacid.Ann.Bot.85,37-43).After the latex dust maturation, the adjacent cell wall of same row latex dust cell merges, and forms a network structure of UNICOM each other, but row are obstructed each other with row.This structure is beneficial to adopts glue, and the bark during rubber tapping cuts through glue cutter simple, and many latex dusts row are just cut off, and once rubber tapping just can be gathered in the crops a large amount of latex, and wherein 30～50%, rubber hydrocarbon.

Still do not know at present the biological significance that the rubber tree latex dust is real, but latex dust has been served more than 100 year (Ko for improving human lives's quality as " the green rubber plant " of a reusable edible, J.H., Chow, K.S.and Han, K.H. (2003) Transcriptome analysis reveals novel features of the molecular events occurringin the laticifers of Hevea brasiliensis (para rubber tree) .Plant Mol.Biol.53,479-492).The rubber biosynthesizing is the main metabolic activity of latex dust cell, rubber accounts for the (Jacob more than 90% of latex dust tenuigenin (latex) dry-matter, J.L.and Prevot, J.C. (1992) Metabolism of the laticiferous system and itsbiochemical regulation.In:Natural Rubber:Biology, Cultivation and Technology, SethurajMR, Mathew NM (eds), Amsterdam:Elsevier, pp116-136).Rubber is synthetic to be to be raw material with sucrose, generate first isoprenoid class material _ isopentenyl pyrophosphate (IPP) through more than 20 enzymatic reactions, this material is the biosynthetic direct substrate of macromolecule rubber hydrocarbon and elementary cell (Sando, T., Takaoka, C., Mukai, Y., Yamashita, A., Hattori, M., Ogasawara, N., Fukusaki, E.and Kobayashi, A. (2008) Cloningand characterization of mevalonate pathway genes in a natural rubber producing plant, Hevea brasiliensis.Biosci.Biotechnol.Biochem.72,2049-206).In the rubber tree of normally adopting glue, the latex dust in the stem portion bark is a storehouse of enlivening that utilizes sucrose, and sucrose provides energy and carbon skeleton (Silpi for latex regeneration, U., Lacointe, A., Kasempsap, P., Thanysawanyangkura, S., Chantuma, P., Gohet, E., Musigamart, N., Cl é ment, A., Am é glio, T.and Thaler, P. (2007) Carbohydrate reserves as acompeting sink:evidence from tapping rubber trees.Tree Physiol.27,881-889).Understand the transhipment of sucrose and distribute mechanism to help to improve rubber tree production efficiency, yet research in the past mainly concentrates on Physiology and biochemistry field (Tupy, J. (1989) Sucrose supply and utilization for latex production.In:Physiologyof Rubber Tree Latex, J.d ' Auzac, J.L.Jacob and H.Chrestin (eds), C.R.C.Press, BocaRaton, FL, pp.179-199).Clone and evaluation participate in the key gene that the sucrose latex dust is supplied with, and are not only the foundation stone of illustrating sucrose absorption and molecular regulation mechanism, also will be that the carbohydrate distribution based theoretical between glue tissue and other carbon storehouse (as nourishing and growing) is produced in artificial reasonable regulation and control.

Sucrose transporter (SUT) belongs to main easily diffusion superfamily, and 12 typical membrane spaning domains are arranged, and is divided into two portions (Barker, L. by a big kytoplasm ring in middle part, K ü hn, C., Weise, A., Schulz, A., Gebhardt, C., Hirner, B., Hellmann, H., Schulze, W., Ward, J.M.and Frommer, W.B. (2000) SUT2, aputative sucrose sensor in sieve elements.Plant Cell, 12,1153-1164).So far, mostly the plant SUT that is identified is to be positioned at need power consumption on the plasma membrane and carries out the proton/sucrose of the sucrose transhipment body that cotransports, and their mediation sucrose enters cell through striding film.These albumen are in time exported from synthesising part (chloroplast(id) of source leaf) at sucrose, long-distance transportation, and in the distribution of numerous storehouses tissue, bring into play central role (Sauer, N. (2007) Molecular physiologyof higher plant sucrose transporters.FEBS Lett.581,2309-2317; Braun, D.M.and Slewinski, T.L. (2009) Genetic control of carbon partitioning in grasses:roles of sucrose transportersand tie-dyed loci in phloem loading.Plant Physiol.149,71-81).In rubber tree, lack synplasm contact H é bant between latex dust and its peripheral cell, C. (1981) Ontog é nie des laticiferes du systemeprimaire d ' Hevea brasiliensis:une é tude ultrastructurale et cytochimique.Can.J.Bot.59,974-985), therefore SUT played an important role in ought to absorbing at the sucrose of latex dust cell, these more existing Physiology and biochemistry experimental evidences: it is not by freely spreading that sucrose enters latex dust, but rely on a kind of system of power consumption, the activity of this system is regulated and control by some inhibitor simultaneously, shown carrier mediated characteristic (Tupy, J. (1985) Some aspects ofsucrose transport and utilization in latex producing bark of Hevea brasiliensis (M ü ll.Arg.) .Biol.Plant.27,51-64).The most direct evidence is from the electric Physiological Experiment (Bouteau that utilizes sugared mark to carry out, F., Dellis, O., Bousquet, U.and Rona, J.P. (1999) Evidence of multiple sugar uptake across theplasma membrane of laticifer protoplasts from Hevea.Bioelectrochem.Bioenerg.48,135-139), this experiment shows and has a plurality of proton/sugar body that cotransports on latex dust plasma membrane that they are to the avidity of sucrose higher (1mM).In early-stage Study, we utilize degeneracy RT-PCR and RACE technology to clone six total length SUT cDNA (HbSUT1,2A from rubber tree, 2B, 3,4 and 5) (Yang, J.H., Huang, D.B., Liu, S.J.and Tang CR. (2007) Molecular cloning and sequence analysis of six sucrose transportergenes from Hevea brasiliensis (para rubber tree) .Chinese J.Trop.Crops, 28,32-38).Recently, the Frenchman has also separated seven SUT cDNA, and thinks that wherein two are new cDNA clone (HbSUT1B and 2C).Simultaneously, they have studied these SUTcDNA at low but ethrel (the 2-chloroethyl phosphoric acid of metabolic activity, a kind of ethene releasing agent) expression in the rubber tree strain (PB217) that effect of stimulation is good, main research is cut ethrel in the tree and is stimulated influence (Dusotoit-Coucaud to these genetic expressions not opening, A., Brunel, N., Kongsawadworakul, P., Viboonjun, U., Lacointe, A., Julien, J.L., Chrestin, H.and Sakr, S. (2009) Sucrose importation into laticifers of Hevea brasiliensis, in relation to ethylenestimulation of latex production.Ann Bot (Lond). (Epub ahead of print)).

Summary of the invention

The purpose of this invention is to provide the application of a kind of sucrose transporter and encoding gene thereof.

Sucrose transporter provided by the present invention derives from the Para rubber tree (Heveabrasiliensis) of Euphorbiaceae genus hevea, and name is called HbSUT3, is following (a) or protein (b):

(a) by SEQ ID № in the sequence table: the protein that the aminoacid sequence shown in 1 is formed.

(b) with SEQ ID №: 1 aminoacid sequence is through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with transhipment of plant sucrose or transportation by sequence 1 deutero-protein.

SEQ ID № in the sequence table: 1 is made up of 535 amino-acid residues.

The replacement of described one or number several amino acid residues and/or disappearance and/or interpolation are meant replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.

In order to make the HbSUT3 in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1 label

Label	Residue	Sequence
			??Poly-Arg	5-6 (being generally 5)	??RRRRR
??Poly-His	2-10 (being generally 6)	??HHHHHH
			??FLAG	??8	??DYKDDDDK
??Strep-tag?II	??8	??WSHPQFEK
			??c-myc	??10	??EQKLISEEDL

Above-mentioned (b) but in the HbSUT3 synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of HbSUT3 in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The encoding gene of described sucrose transporter (HbSUT3) also belongs to protection scope of the present invention.

The encoding gene of described sucrose transporter (HbSUT3) is following 1) or 2) or 3) dna molecular:

1) SEQ ID № in the sequence table: the dna molecular shown in 2;

2) under stringent condition with 1) the dna sequence dna hybridization that limits and the dna molecular of the described sucrose transporter of encoding;

3) with sequence table in SEQ ID №: 2 nucleotide sequence has a homology more than 80% and proteins encoded has the nucleotide sequence with transhipment of plant sucrose or the relevant correlation function of transportation.

SEQ ID № in the sequence table: 2 are made up of 1819 Nucleotide.

The genomic gene of described sucrose transporter (HbSUT3) is following 1) or 2) or 3) dna molecular:

1) SEQ ID № in the sequence table: the dna molecular shown in 3;

2) under stringent condition with 1) the dna sequence dna hybridization that limits and the dna molecular of the described sucrose transporter of encoding;

3) with sequence table in SEQ ID №: 3 nucleotide sequence has a homology 80% or more and proteins encoded has with the transhipment of plant sucrose or transports the nucleotide sequence of correlation function.

SEQ ID № in the sequence table: 3 are made up of 3689 Nucleotide, from SEQ ID №: 5 ' end 97-1410 position Nucleotide of 3 is first exon, from SEQ ID №: 5 ' end 2333-2396 position Nucleotide of 3 is second exon, from SEQ ID №: 5 ' end 2490-2533 position Nucleotide of 3 is the 3rd exon, and from SEQ ID №: 5 ' end 3389-3574 position Nucleotide of 3 is the 3rd exon.From SEQ ID №: 5 ' end 1-96 position Nucleotide of 3 is 5 ' end non-translational region of gene; From SEQ ID №: 5 ' end 3575-3689 position Nucleotide of 3 is 3 ' end non-translational region of gene.

Described stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.

The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain described sucrose transporter encoding gene all belong to protection scope of the present invention.

Sucrose transporter of the present invention, yeast expression system confirm its sucrose avidity height, have the cotransport characteristic feature of body of sucrose/proton, are that decision latex dust sucrose is supplied with and a key protein of rubber output.Sucrose transporter gene of the present invention can change in microorganism or the plant, improves microorganism and the plant utilising efficiency to sucrose, and improves the output of its microorganism or plant.This sucrose transporter gene changes in the yeast saccharomyces cerevisiae, can be by improving its assimilated efficiency to sucrose, the efficient thereby raising is made wine.

Description of drawings

Fig. 1 is the genome structure and the copy number analysis of HbSUT3 gene; (A) be genome structure, with the genome structure of HbSUT3 gene same seven Arabidopis thaliana SUT genes (AtSUC1, At1g71880; AtSUC2, At1g22710; AtSUC3, At2g02860; AtSUC4, At1g09960; AtSUC5, At1g71890; AtSUC8, At2g14670; AtSUC9 At5g06170) compares.Square frame is represented exon, and solid line is represented intron.(B) be the Southern hybridization analysis of the HbSUT3 gene under the high rigorous condition.

Fig. 2 is the time curve that recombination yeast SEY6210-pDR196-HbSUT3 sucrose absorbs.

The influence that Fig. 3 absorbs recombination yeast SEY6210-pDR196-HbSUT3 sucrose for the pH value.

Fig. 4 is the Michaelis-Menten curve of recombination yeast SEY6210-pDR196-HbSUT3 sucrose concentration and transport speed when pH4.0 and pH5.5.

The Lineweaver-Burk curve of sucrose concentration and transport speed when Fig. 5 is pH4.0 and pH5.5.Michaelis-Menton constant=1.30mM during pH 4.0, maximum reaction velocity=1.1nmol Suc min ^-1(10 ⁸Cells) ^-1Michaelis-Menton constant=5.19mM during pH 5.5, maximum reaction velocity=2.01nmol Suc min ^-1(10 ⁸Cells) ^-1

Fig. 6 is the sxemiquantitative RT-PCR expression analysis result of HbSUT3 gene in the different tissues.

Fig. 7 is the influence of different concns ethrel to HbSUT3 genetic expression.(A) in rubber tree, using different concns (0,0.1%, 0.5%, 1.0%, 2.0%, 4.0%and 6.0%) ethrel, analyze to reach before ethrel is handled with sxemiquantitative RT-PCR then and handle back 24h and 96h HbSUT3 expression of gene, do interior mark with 18S-rRNA.(B) handle the output of latex afterwards for the different concns ethrel.

Fig. 8 is a HbSUT3 expression of gene behind 1.5% (w/w) ethrel (Ethrel) the processing different time.(A) analyze ethrel for real-time quantitative RT-PCR and handle back HbSUT3 expression of gene, 18S rRNA does internal standard gene. (B) handle the variation of back different time latex sucrose concentration for ethrel.

Fig. 9 is the influence of rubber tapping to HbSUT3 genetic expression.(A) do not open cut gummy milk production with variation (B) the HbSUT3 gene expression dose that cuts time with the variation of cutting time.

Figure 10 is the influence of injury to HbSUT3 genetic expression; 18S rRNA is as interior mark.

Figure 11 is HbSUT3 genetic expression and rubber relation of yield; (A) the latex output of three groups of different PR107 rubber trees. (B) the HbSUT3 expression of gene compares in the flat PR107 rubber tree of different product glue, and 18S rRNA does interior mark

Figure 12 be sxemiquantitative RT-PCR analyze (A) ethrel, (B) rubber tapping and (C) injury handle, and (D) HbSUT3 expression of gene in the horizontal PR107 rubber tree of different output, 18S rRNA does confidential reference items.

Figure 13 handles the rubber tree picture for injury.

Figure 14 for jasmonic handle normally open cut the tree different time (hour) back HbSUT3 expression of gene.

Embodiment

Method described in the following embodiment if no special instructions, is ordinary method.

The acquisition of embodiment 1, sucrose transporter and encoding gene thereof

One, the acquisition of sucrose transporter and encoding gene thereof

By right, identified the peptide section (GWALQLS and FDTDWM) of two high conservatives of plant sucrose transporter to a plurality of multiple ratios of having delivered plant sucrose transporter sequence.Design degenerated primers 5 '-GGNTGGGCNYTNCARCTNTC-3 ' and 5 '-CCCATCCARTCNGTRTCRAA-3 ' (R=A/G wherein on this basis, Y=C/T, N=A/G/C/T), the latex cDNA that grinds 7-33-97 with the rubber tree kind heat of Rubber Institute, Chinese Academy of Agricultural Science is a template, utilize degeneracy RT-PCR to obtain the conservative section of hevea brasiliensis transporter gene, and then utilize the RACE technology to obtain the full-length cDNA of hevea brasiliensis transporter gene.

When carrying out 5 ' RACE, it is synthetic to be with Oligo (dT) that primer carries out cDNA first chain, and reaction product utilizes terminal enzyme (DNA) TdT catalysis to add the polyA tail after ethanol sedimentation is removed unnecessary dNTP and primer.First round amplification is a template with the cDNA of tailing, (sequence is referring to Dieffenbacher CW to use primer QT and Q0, the moral Vicks VapoRub is reined in GS.PCR technology experiment guide. and Huang Peitang translates. Beijing: Science Press, 2000.p268-277) and first round special primer (5 '-GCAGCAT-AACCGATAAGG-3 '), in 25 μ l reaction systems, carry out pcr amplification.Amplification program is: at first, 94 ℃ of sex change 5min, 50 ℃ of annealing 2min, 72 ℃ are extended 40min; Then, 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 30 circulations; At last, 72 ℃ are extended 10min.

After 100 times of above-mentioned acquisition PCR product dilutions, get 1 μ l cut back as template, (sequence is referring to Dieffenbacher CW to use primer Q1, the moral Vicks VapoRub is reined in G S.PCR technology experiment guide. and Huang Peitang translates. Beijing: Science Press, 2000.p268-277) take turns special primer (5 '-ACATAGCCAGATGAAAGCAG-3 ') and carry out second and take turns nested amplification with second, the pcr amplification program is: behind 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 30 circulations; Last 72 ℃ are extended 10min.1.0% agarose gel electrophoresis separates pcr amplification product, reclaims amplified fragments, and is cloned on the pMD18-T carrier, send company's order-checking.

When carrying out 3 ' RACE, it is synthetic to be with the QT of 50ng that primer carries out cDNA first chain.With the cDNA product is template, (sequence is referring to Dieffenbacher CW to utilize Q0, the moral Vicks VapoRub is reined in G S.PCR technology experiment guide. and Huang Peitang translates. Beijing: Science Press, 2000.p268-277) and first round primer (5 '-CCAGAGCGAGGAAACACC-3 ') carry out first round pcr amplification, amplification program is: 94 ℃ of sex change 5min, 50 ℃ of annealing 2min, 72 ℃ were extended 40 minutes; Then, 94 ℃ of sex change 1min, 56 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 30 circulations; At last, 72 ℃ are extended 10min.After 100 times of the first round PCR product dilutions, get 1 μ l cut back as template, (sequence is referring to Dieffenbacher CW to use primer Q1, the moral Vicks VapoRub is reined in G S.PCR technology experiment guide. and Huang Peitang translates. Beijing: Science Press, 2000.p268-277) take turns primer (5 '-CGATGCCGTTCTTCAGTG-3 ') and carry out second and take turns nested amplification with second, it is identical that amplification program and second of 5 ' RACE take turns pcr amplification.According to resulting rubber tree HbSUT3 gene conservative district, 5 ' end and 3 ' terminal sequence, utilize DNAman software to carry out sequence assembly, finally obtain the splicing sequence of HbSUT3 full length gene cDNA.

According to the sequences Design primer that splicing obtains, use primer 5 '-GA CCCGGGCGACCCAACTATGTACAAAAC-3 ' and 5 '-CA CTCGAGAT-GCCCTATTAGAACACCAAC-3 ' (the underscore sequence is respectively the restriction enzyme site Sma I and the Xho I of introducing).The latex cDNA that grinds 7-33-97 with the rubber tree sale kind heat of Rubber Institute, Chinese Academy of Agricultural Science is that template is carried out pcr amplification.

Amplification program is: 95 ℃ of sex change 3min; 95 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 2min, totally 30 circulations; At last, 72 ℃ are extended 10min.。

Amplification system is: primer 0.2 μ mol/L, dNTP 100 μ mol/L, the reverse transcription product of the total RNA of 2.5 μ g latex of 1% volume, Taq plus archaeal dna polymerase 2U, 2.5 μ L 10X PCR buffer, total reaction system 25 μ L.

Amplification is obtained the fragment of 1827bp, show through order-checking, the nucleotide sequence that the sucrose transporter gene that obtains has sequence 2 in the sequence table is HbSUT3 with this unnamed gene, and above-mentioned amplification has added at the primer two ends and makes things convenient for carrier construction to carry out the enzyme recognition site Sma I and the Xho I of functional verification.From 5 of sequence 2 ' end 97-1704 position Nucleotide is encoding sequence, the sucrose transporter (HbSUT3) shown in the sequence 1 in the code sequence tabulation.Sucrose transporter HbSUT3 is made up of 535 amino acid, and molecular weight is 56.88kDa, and iso-electric point (pI) is 8.82.The mass spectrometry analysis shows that HbSUT3 comprises 12 proteic membrane spaning domains of typical plant SUT.

5 ' and 3 ' non-translational region design primer according to the HbSUT3cDNA sequence, primer sequence is 5 '-CGACCC-AAC TATGTACAAAAC-3 ' and 5 '-ATGCCCTATTAGAACACCAAC-3 ', the leaf DNA of grinding 7-33-97 with rubber tree kind heat is a template, obtain the HbSUT3 genomic gene by pcr amplification, wherein amplification program and amplification system are with the amplification of HbSUT3cDNA sequence.

The fragment that amplification obtains shows that through order-checking the HbSUT3 genomic gene has the nucleotide sequence of sequence 3 in the sequence table, is the sucrose transporter genomic dna.From 5 of sequence 3 ' end 97-1410 position Nucleotide is first exon, from 5 of sequence 3 ' end 2333-2396 position Nucleotide is second exon, from 5 of sequence 3 ' end 2490-2533 position Nucleotide is the 3rd exon, is the 3rd exon from 5 of sequence 3 ' end 3389-3574 position Nucleotide.From 5 of sequence 3 ' end 1-96 position Nucleotide is 5 ' end non-translational region of gene; From 5 of sequence 3 ' end 3575-3689 position Nucleotide is 3 ' end non-translational region of gene.

The genome of HbSUT3 gene by having determined the exon of its gene with the comparison of its cDNA sequence, and is compared with seven SUT genes of Arabidopis thaliana.(A) shows among Fig. 1, and the gene structure of HbSUT3 is similar to AtSUC2, and they all comprise 4 exons that size is close, and just intron varies in size.

Utilize high preciseness dna molecule hybridize to analyze the copy number of HbSUT3 gene, Fig. 1-(B) show that this gene is a single copy gene, with LeSUT2 (Barker, L., K ü hn, C., Weise, A., Schulz, A., Gebhardt, C., Hirner, B., Hellmann, H., Schulze, W., Ward, J.M.and Frommer, W.B. (2000) SUT2, aputative sucrose sensor in sieve elements.Plant Cell, 12,1153-1164), TaSUT1 (Aoki, N., Whitfeld, P., Hoeren, F., Scofield, G., Newell, K., Patrick, J., Offler, C., Clarke, B., Rahman, S.and Furbank, R.T. (2002) Three sucrose transporter genes are expressed in the developinggrain of hexaploid wheat.Plant Mol.Biol.50,453-462) and nine SUT genes (Arabidopis thaliana information database, the http://www.arabidopsis.org/index.jsp of Arabidopis thaliana; Sauer et al., 2004)) unanimity.

The functional verification of embodiment 2, sucrose transporter of the present invention and encoding gene thereof

One, sucrose transporter improves the functional verification of microorganism to the utilising efficiency of sucrose

Utilize yeast SEY6210 expression system to analyze the rubber tree HbSUT3 gene sucrose transporter of function of whether encoding, and understand the dynamics of its transhipment sucrose.The SEY6210 yeast strain is carried a sucrose inversion enzyme mutant gene, is fit to carry out ¹⁴The absorption measurement of C-sucrose.

The latex cDNA that grinds 7-33-97 with the rubber tree kind heat of Rubber Institute, Chinese Academy of Agricultural Science according to the method for embodiment 1 is a template, with primer 5 '-GA CCCGGGCGACCCAACTATGTACAAAAC-3 ' and 5 '-CA CTCGAGAT-GCCCTATTAGAACACCAAC-3 ' (the underscore sequence is respectively the restriction enzyme site Sma I and the Xho I of introducing) amplification obtains the full-length cDNA of HbSUT3 gene.

The full-length cDNA of HbSUT3 gene is cut rear clone to Yeast expression carrier pDR196 (Rentsch D. with Sma I and Xho I enzyme, Laloi M., Rouhara I., Schmelzer E., Delrot S.and Frommer WB.1995.NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis.FEBS Lett.370:264-268, Rubber Institute, Chinese Academy of Agricultural Science's preservation) between the Sma I and Xho I restriction enzyme site, obtain recombinant vectors, recombinant vectors is carried out sequence verification, the recombinant vectors called after pDR196-HbSUT3 that checking is correct.

PDR196-HbSUT3 is imported Wine brewing yeast strain SEY6210 (Reinders A., Ward J.M.2001.Funtional characterization of the alpha-glucoside transporter Sutlp fromSchizosaccharomyces pome, the first fungal homologue of plant sucrose transporters.Molecular Microbiology 39:445-454) obtains the reorganization bacterium in, will identify the correct reorganization bacterium called after SEY6210-pDR196-HbSUT3 that has changed pDR196-HbSUT3 over to.

Utilize SEY6210-pDR196-HbSUT3 to carry out proteic sucrose transport function of HbSUT3 and dynamics mensuration, concrete grammar is as follows:

1, the time curve of recombination yeast SEY6210-pDR196-HbSUT3 sucrose absorption

Recombination yeast SEY6210-pDR196-HbSUT3 is detected it to the sucrose receptivity according to following method:

1) recombination yeast SEY6210-pDR196-HbSUT3 being grown to OD600 in SD/-Ura liquid nutrient medium (Clontech company) is 1.0～1.2,4 ℃, centrifugal 5 minutes collecting cell precipitations of 2000g;

2) the ice-cold distilled water wash of cell precipitation that step 1) is obtained is 1 time, ice-cold then 25mMNaH ₂PO ₄Damping fluid (pH4.0) washing 2 times is resuspended in same NaH at last ₂PO ₄In the damping fluid, to OD600 be 10, obtain the resuspended liquid of cell;

3) get 5 1.5mL centrifuge tubes, respectively with 975 μ L steps 2) the resuspended liquid of cell that obtains is added to the 1.5mL centrifuge tube, and 30 ℃ of water-baths were shaken incubation 2 minutes; The glucose solution that adds the 1M of 10 μ L then respectively, to the glucose final concentration be 10mM; Adding 15.0 μ L sucrose solutions (wherein 100mM 12C sucrose 10 μ L, radioactive intensity is the 14C sucrose 5.0 μ L of 1.0 μ Ci) to sucrose final concentration after 1 minute is 1mM, and 30 ℃ of water-baths cultivate 2,4,6,8,10 minutes respectively.

4) cell culture fluid that step 4) is obtained is transferred to two-layer mixed nitrate cellulose filter membrane (diameter 25mm is housed, aperture 0.8 μ m) in the suction filtration device, the suction filtration device includes the ice precooling 10mM sucrose of 5mL, piping and druming mixing stopped reaction, rapid suction filtration is used the ice precooling 10mM sucrose rinsing 2 times of 5mL again.

5) filter membrane is rolled with the ophthalmology tweezers put into the 2.0mL centrifuge tube, after treating the filter membrane drying, add 1.5mL scintillation solution (the OptiPhase Supermix of U.S. Perkin Elmer company scintillation solution), with liquid scintillation instrument (U.S. Perkin Elmer company model: MicroBeta) counting.According to the scintillometer numerical value of 1 μ Ci 14C-sucrose solution, sucrose absorbed dose (nmol sucrose/10 of conversion yeast cell ⁸Cell).

Aforesaid method is contrast with the Wine brewing yeast strain SEY6210 that changes the pDR196 empty carrier.

The result can make yeast cell absorb the ability of sucrose apparently higher than the cell that changes the pDR196 empty carrier as shown in Figure 2 after HbSUT3 expresses in yeast strain.

2, the pH value is to the influence of recombination yeast SEY6210-pDR196-HbSUT3 sucrose absorption

Sucrose absorption experiment under the different pH condition:, wherein use the 25mM NaH of specific pH value (3.0,4.0,5.0,6.0,7.0,8.0) according to the method for step 1 ₂PO ₄Damping fluid, rinsing and resuspended yeast cell; Reaction times is 10 minutes.Other reaction conditionss are with step 1.

The result as shown in Figure 3, SUT protein similar with most plants, the transport activity of HbSUT 3 also is subjected to the regulation and control of pH value, optimum pH is about 5.0 (Fig. 3), this characteristic shows that these HbSUT 3 proteic sucrose transhipments may be driven by proton, after this this supposition has obtained checking, and promptly their transport activity is to uncoupler sensitivity (table 3).

3, the Michaelis-Menten curve and the Lineweaver-Burk double reciprocal curve of recombination yeast SEY6210-pDR196-HbSUT3 sucrose concentration and transport speed when pH4.0 and pH5.5

Km experiment: the 25mM NaH of pH 4.0 or pH5.5 ₂PO ₄The resuspended yeast cell of damping fluid.The glucose that adds the 1M of 10 μ L, to the glucose final concentration be 10mM.The 12C sucrose (25,50,100 that adds the different concns of 5 μ L 14C sucrose (radioactive intensity is 1 μ Ci) or 10 μ L 14C sucrose (radioactive intensity is 2 μ Ci) and 10 μ L after one minute, 200,400,800,1200,1600,2000mM), be respectively 0.25,0.5,1.0 to the sucrose final concentration, 2.0,4.0,8.0,12.0,16.0,20.0mM.Reaction times is 10 minutes.Other reaction conditionss are with step 1.

The above-mentioned result who measures the affinity of 3 pairs of sucrose of HbSUT in the scope of sucrose concentration 0.5～8mM is used to make up the Michaelis curve, and the result as shown in Figure 4.The Lineweaver-Burk double reciprocal curve shows (Fig. 5), and HbSUT3 has similar sucrose avidity (Km=1.59mM) when pH4.0, but the maximum rate of transhipment sucrose be 1.10 nmole sucrose/minute/10 ⁸Cell.When the pH value of measuring system brought up to 5.5, HbSUT3 albumen descended to some extent to the avidity of sucrose, the transport velocity of HbSUT3 then bring up to 2.01 nmole sucrose/minute/10 ⁸Cell.

4, competitive sugar experiment:

The 25mM NaH of pH4.0 ₂PO ₄The resuspended yeast cell of damping fluid.The glucose that adds the 1M of 10 μ L, to the glucose final concentration be 10mM.The competitive sugar of different sorts that adds 10 μ L 1M after one minute, to final concentration be 10mM.After 1 minute, add the sucrose solution (wherein 100mM 12C sucrose 10 μ L, radioactive intensity is 1.01 μ Ci 14C sucrose, 5.0 μ L) of 15.0 μ L, the sucrose final concentration is 1mM, and the reaction times is 10 minutes.When carrying out the sucrose absorption measurement, add unmarked different types of sugar (as shown in table 2) of ten times of concentration, determine the specificity of HbSUT3 transhipment substrate with this.Other reaction conditionss are with step 1.

The result shows in the multiple sugar of being measured, to have only sucrose and maltose obviously to compete ¹⁴The absorption (table 2) of C-sucrose shows that HbSUT 3 transhipment substrates have stronger specificity.Lactose could stimulate the sucrose of HbSUT3 to absorb.

The influence that the different competitive sugar of table 2. absorb the yeast sucrose of expressing the HbSUT3 gene

Mark ^ACarry out analysis of variance (ANOVA) with SAS 6.12 softwares, different treatment is all made comparisons with the contrast that does not add competitive sugar, and the upper right corner has the processing of A to have significant difference (P＜0.01) with contrast.

5, inhibitor experiment

The 25mM NaH of pH4.0 ₂PO ₄The resuspended yeast cell of damping fluid.The glucose that adds the 1M of 10 μ L, to the glucose final concentration be 10mM.The certain density inhibitor (as shown in table 3) that adds 10 μ L after one minute.After 1 minute, add the sucrose solution (wherein 100mM 12C sucrose 10 μ L, radioactive intensity is 1 μ Ci14C sucrose, 5.0 μ L) of 15.0 μ L, the sucrose final concentration is 1mM, and the reaction times is 10 minutes.Other reaction conditionss are with step 1.

The result is as shown in table 3, and the result shows that when existing, the sucrose transport activity of HbSUT 3 significantly descends by (table 3) at uncoupler dinitrophenol (DNP), confirms that further it is sucrose/proton body that cotransports.In addition, HbSUT 3 proteic transport activities also are subjected to the inhibition of 1mM N-ethyl Malaysia alkene imines (NEM), but they are all insensitive to sulfhydryl reagent PCMBS, this with in the past the report be not inconsistent.In the former studies, PCMBS can effectively suppress the transport activity (Riesmeier of all other plant sucrose transporters, J.W., Willmitzer, L.and Frommer, W.B. (1992) Isolation and characterization of a sucrose carrier cDNA from spinach by functionalexpression in yeast.EMBO J.11,4705-4713; Noiraud, N., Delrot, S.and Lemoine, R. (2000) The sucrose transporter of celery.Identification and expression during salt stress.PlantPhysiol.122,1447-1455; Barth, I., Meyer, S.and Sauer, N. (2003) PmSUC3:characterization of a SUT2/SUC3-type sucrose transporter from Plantago major.Plant Cell, 15,1375-1385).

The influence that the different inhibitor of table 3. absorb the saccharomycetic sucrose of expressing the HbSUT3 gene

Inhibitor	The % activity
			??HbSUT3
Unconstrained dose	??100
		50μM?2，4-DNP	??70±4.2 A
50μM?PCMBS	??100±2.5

50μM?NEM	??63±3.2 A
		1mM?NEM	??18±1.1 A

Carry out analysis of variance (ANOVA) with SAS 6.12 softwares, different treatment is all made comparisons with unconstrained dose contrast, and the angle has the processing of A to have significant difference (P＜0.01) with contrast on the stone.

Above-mentioned experiment shows that HbSUT3 can improve the utilize ability of microorganism to sucrose, improves the culture efficiency of microorganism.Above-mentioned experiment has proved that also HbSUT3 can improve the utilising efficiency of yeast saccharomyces cerevisiae to sugar, fully improves the fermentation efficiency of yeast saccharomyces cerevisiae.

Two, the expression of sucrose transporter gene in different tissues

Select the age of tree for use 10 years, cut the rubber tree kind heat in 2 years ages and grind 7-33-97, get latex respectively, the trunk bark, the green branches skin, bud, bronze phase blade, variable color phase blade, stationary phase blade, female flower, male flower, mature seed, rootlet (diameter 0.5-1.0cm), feeding root utilizes sxemiquantitative RT-PCR to analyze the tissue specific expression characteristics of HbSUT 3 genes.The primer is 5 '-CACCCGCTTCAAGCAAGCTTAG-3 ' (forward) and 5 '-GCAGCATAACCGATAAGG-3 ' (oppositely), 55 ℃ of annealing temperatures, amplification cycles several 28, with 18S-rRNA is interior mark (primer sequence is 5 '-AAGCAAGCCTACGCTCTGG-3 ' and 5 '-GCTCCACCAACTAAGAACGG-3 '), 58 ℃ of annealing temperatures, amplification cycles several 18.

Detected result as shown in Figure 6, the result shows that HbSUT 3 genes all have expression (Fig. 6) in various degree in above-mentioned 12 kinds of tissues of being tested.The expression level of HbSUT3 in latex and female flower is the highest, and expression level is lower in stem bark and mature seed, and the expression level in feeding root is minimum, almost detect less than.

Three, sucrose transporter improves the functional verification of rubber output

1, ethrel stimulates the influence to HbSUT3 genetic expression

Use ethrel (2-chloroethyl phosphoric acid, the ethene releasing agent) can increase latex output, stimulate the partly cause of volume increase and the relevant (Coupe of enhancing of latex dust sugar supply capacity, M.and Chrestin, H. (1989) Physico-chemicaland biochemical mechanisms of the hormonal (ethylene) stimulation.In:Physiology ofRubber Tree Latex, J.d ' Auzac, J.L.Jacob and H.Chrestin (eds), C.R.C.Press, Boca Raton, FL, pp.295-319).Whether by the ethrel processing these HbSUT genes are studied in the influence of genetic expression stimulates the effect of volume increase relevant with ethrel.

At first, measure the influence of the ethrel processing of different concns (mass percentage concentration is 0.1～6.0%, and concrete concentration of treatment as shown in Figure 7) to HbSUT3 genetic expression.7-33-97 is ground in 10 years, the rubber tree kind heat of cutting the normal rubber tapping in 2 years ages to select the age of tree for use, amount by every tree 1.5g spreads upon on the face of 1cm on secant and the secant then, handle back 24h and twice rubber tapping of 96h sampling, extract latex RNA, and untreated tree is the contrast sampling, utilize sxemiquantitative RT-PCR to analyze the HbSUT3 expression of gene, with 18S-rRNA is interior mark, and method is as described in the step 2.Measure the different concns ethrel simultaneously and handle the latex output of back gum.

The result shows that the HbSUT3 gene is subjected to ethephon-induction, the 0.5%th, and the minimum concentration of ethephon-induction HbSUT3 genetic expression (among Fig. 7 (A)) research shows that simultaneously 0.5% is to stimulate the gelatigenous minimal effective concentration of latex (among Fig. 7 (B)) simultaneously.

Then, we utilize real-time quantitative RT-PCR to measure the speed that HbSUT3 gene response ethrel stimulates.Utilize real-time quantitative RT-PCR to measure the influence of different concns ethrel processing to HbSUT3 genetic expression, analyzing used HbSUT3 primer is 5 '-CACCACAACCACCATCAC-3 ' (forward) and 5 '-GTGGAAGAGGTTCAGAAGAG-3 ' (oppositely), and 18S-rRNA (interior mark) primer is 5 '-GTGGAAGAGGTTCAGAAGAG-3 ' (forward) and 5 '-TTCAGCCTTGCGACCATAC-3 ' (oppositely).The total RNA that handles with 2 microgram DNase I makees synthetic cDNA first chain of template, and concrete operations are with reference to the test kit explanation (RevertAid of company ^TMFirst Stand cDNA Synthesis Kit, Fermentas).Real-time quantitative RT-PCR utilizes the SYBR-Green fluorescence dye of TaKaRa company and the instrument of Roche Holding Ag, and (LightCycler 2.0system, Roche Diagnostics Germany) carries out.PCR reaction conditions: 95 ℃ of sex change of elder generation 30 seconds, carried out amplification cycles then (94 ℃ 5 seconds, 60 ℃ of 20 seconds and 72 ℃ 20 seconds), utilize the Ct value of instrument detecting gene amplification, the relative expression who utilizes instrument software kit (LightCycler Relative Quantification Software 4.05) to carry out gene analyzes: expression level=E ^{Ct (18S-rRNA)-Ct (HbSUT3)}The right amplification efficiency of HbSUT3 and 18S-rRNA primer is respectively 2.010 and 1.951, sequence verification amplified band verity.

The result shows, ethrel handles that the HbSUT3 expression of gene significantly raises after 12 hours, and it is expressed with the increase of the time of processing and further improves (among Fig. 8 (A)).Ethrel handles that this expression of gene level improves 7 times after 24 hours.

Latex sucrose concentration (measuring method reference: Ashwell is analysis of sugars.Methods in Enzymology 3:73-105. G.1957.Colorimetric) is handled the back at ethrel and is continued to descend, and handles 1/7th (among the Fig. 8 (B)) that were reduced in 24 hours before handling.This result shows that latex sucrose may produce a kind of reverse feedback regulation to HbSUT3 genetic expression.Studies show that more than HbSUT3 is the gene that decision latex dust sucrose is supplied with.

2, jasmonic (Jasmonic acid) handle difference normally open cut the tree different time (hour) back HbSUT3 expression of gene

Grind 7-33-97 with the different HORMONE TREATMENT age of trees 10 years, the rubber tree kind heat of cutting the normal rubber tapping in 2 years ages, amount by every tree 1.5g spreads upon on the face of 1cm on secant and the secant then, handle back 2h and 12h rubber tapping sampling, extract latex RNA, and untreated tree is the contrast sampling, utilize sxemiquantitative RT-PCR to analyze the HbSUT3 expression of gene, with 18S-rRNA is interior mark, and method is as described in the step 2.The expression (Figure 14) that the result has only 2% methyl jasmonic can significantly induce HbSUT3 hints that this gene participates in the demand of in latex dust differentiation sucrose being supplied with.

3, the influence to HbSUT3 genetic expression is handled in rubber tapping and injury

Research is adopted the heat reach the rubber tapping standard to grind 7-33-97 and is not opened and cut tree and be material, enclosing the system of cutting by three days one cuttves, 1/2nd trees taps rubber, abandon 20 post-samplings after cutting, carry out the analysis of HbSUT3 expression of gene with the real-time quantitative RT-PCR method, the real-time quantitative RT-PCR method is as described in the step 1.

The result shows, do not open when cutting rubber tree and tapping rubber for the first time and only produces a small amount of latex usually, and along with the increase of regularity rubber tapping number of times, latex output improves constantly, and generally will reach a metastable level (among Fig. 9 (A)) after rubber tapping 7～10 times.The latex regeneration associated genes transcribe and/or translation skill on should be subjected to rubber tapping regulation and control.

HbSUT3 obviously is subjected to rubber tapping regulation and control (among Fig. 9 (B)).When cutting first cutter, the expression level of HbSUT3 is very low, expresses constantly with the increase of rubber tapping number of times to strengthen, and reaches the highlyest when the 3rd cutter, though expression level descends to some extent and fluctuation is arranged in rubber tapping subsequently, expresses the level that all is higher than first cutter.

Rubber tapping produces two kinds of effects: storehouse effect and hinder effect.The former is produced by binder removal, and the latex of discharge needs replenished before the rubber tapping next time, and the latter may produce injury ethene (d ' Auzac, J., Jacob, J.L., J.C., Cl é ment, A., Gallois, R., Crestin, H., Lacote, R., Pujade-Renaud, V.and Gohet, E.. (1997) The regulation of cis-polyisoprene production (natural rubber) from Hevea brasiliensis.In:Pandalai S.G. (ed.) .Recent research developments in plant physiology.Vol.1.Trivandrum:Research Singpost, pp.273-332).

Be the better influence of understanding rubber tapping to HbSUT3 genetic expression, further do not done injury processing (not binder removal) (Figure 13) opening the bark that cuts tree.Method according to step 1 is carried out the real-time quantitative PCR analysis, and Figure 10 shows that the expression of only slightly raising HbSUT3 is handled in injury.Above result shows that the injury effect is little to the influence that HbSUT3 expresses in rubber tapping, illustrates that the expression of HbSUT3 and storehouse effect are closely related.

4, the expression of HbSUT3 in the different output rubber tree

For further studying the function of this gene, we have compared different output PR107 strain rubber tree and (have been cultivated by Indonesia, introducing the 1950's, now is that one of glue district main breed is planted by China, sells all nursery stocks of Chinese Academy of Tropical Agricultural Sciences's rubber) this expression of gene in the latex.Although these rubber trees are bud grafting trees of same strain, but the output difference is very big, the mean yield of first group of tree (107A) is for whenever cutting time 0.32 kilogram of latex, the output of second group of number (107B) is 0.83 kilogram, and the output of a strain super high-yielding rubber tree (SY107) is up to 4.1 kilograms (among Figure 11 (A)).

Utilize the relatively more different flat elastomer gum of the glue Ruzhong HbSUT3 expression of gene levels of producing of real-time quantitative RT-PCR, the real-time quantitative RT-PCR method is with step 1.Shown in (B) among figure Figure 11, the expression level of HbSUT3 is very high with the dependency of rubber output, and expression level is the highest in SY107, take second place among the 107B, and minimum among the 107A.

Above-mentioned experiment shows, confirms that further HbSUT3 is the key gene of sucrose supply of decision latex dust and rubber output.

5, rubber tree HbSUT3 expression of gene under the sxemiquantitative RT-PCR independent analysis different situations

With the real-time quantitative RT-PCR in the sxemiquantitative RT-PCR alternative steps 1-step 4, analyzed rubber tree HbSUT3 expression of gene under the different situations, the result as shown in figure 12, the result shows the real-time quantitative RT-PCR analytical results unanimity (Figure 12) of sxemiquantitative RT-PCR with us.

Discuss:

In the rubber tree of normal rubber tapping, HbSUT3 all has expression in the rubber tree tissue of being studied, there is notable difference again in expression abundance in different tissues, expresses abundance the highest (Fig. 6) in latex and female flower, shows that this gene works in latex regeneration and flower development.Use ethrel at stem portion bark and can strengthen the latex metabolic capacity, improve rubber production power, become the indispensable integral part of modern rubber production technology.Above-mentioned experiment shows that ethrel significantly raises the expression (Fig. 8 (A)) of HbSUT3.Above-mentioned experiment discovery 0.5% (mass percentage concentration) ethrel is not only and is stimulated the effective concentration of rubber production (Fig. 9 (B)), also is the minimal effective concentration (Fig. 9 (A)) of inducing HbSUT3 genetic expression simultaneously.The effect that these results increase production the stimulation of ethrel clearly connects with the influence to HbSUT3 genetic expression.

New carbon storehouse (latex dust) of the final formation of latex dust differentiation is to keep the limiting factor that rubber tree continues productivity.In the various plants hormone and growth regulator except that ethene that we measured, have only jasmonic can significantly induce the expression (Figure 14) of HbSUT3, show that the HbSUT3 gene participates in the demand of in the latex dust differentiation sucrose being supplied with.

Rubber tapping can stimulate latex production, cuts this effect more obvious (Fig. 9 (A)) in the rubber tree not opening.It is low not open the latex dust metabolic activity that cuts in the tree stem portion bark, and along with the increase of rubber tapping number of times, latex dust sucrose absorbs and quickens, and metabolic activity strengthens, and final latex output has also increased.Not opening this specific character of cutting tree makes it become the ideal material identifying and study the latex regeneration associated genes.Above-mentioned experiment shows, do not open cut HbSUT3 in the tree expression to the rubber tapping strong reaction, but (Fig. 9 (B)) induced in the expression of HbSUT3.Rubber tapping produces two effects to gelatigenous stem portion bark, i.e. storehouse effect and hinder effect, but the influence little (Figure 10) that HbSUT3 is expressed is handled in single injury (not binder removal), show rubber tapping to the influence of its expression mainly by the storehouse effect, promptly the latex regenerated needs.

The vital role of HbSUT3 gene in latex regeneration obtains the support of another experimental evidence, i.e. the output positive correlation (Figure 11) of the expression level of HbSUT3 and the different individual plants of same rubber tree strain (PR107).

Rubber tree SUT expression of gene under the different situations that we have also utilized sxemiquantitative RT-PCR independent analysis, experimental result is analyzed consistent (Figure 12) with our real-time quantitative RT-PCR.According to above research, we can confirm that HbSUT3 is the key gene of sucrose supply of decision latex dust and rubber tree production efficiency.

Sequence table

<160>3

 

<210>1

<211>535

<212>PRT

＜213〉Para rubber tree of Euphorbiaceae genus hevea (Hevea brasiliensis)

<400>1

 

Met?Glu?Thr?Asp?Pro?Thr?Lys?Glu?Ser?Lys?Leu?Pro?Ser?Ser?Glu?Pro

1???????????????5???????????????????10??????????????????15

Leu?Pro?Pro?Pro?Pro?Pro?Leu?Val?Pro?Pro?Pro?Pro?Ala?Ser?Ser?Lys

20??????????????????25??????????????????30

Leu?Arg?Lys?Ile?Val?Met?Val?Ala?Ser?Ile?Ala?Ala?Gly?Ile?Gln?Phe

35??????????????????40??????????????????45

Gly?Trp?Ala?Leu?Gln?Leu?Ser?Leu?Leu?Thr?Pro?Tyr?Val?Gln?Leu?Leu

50??????????????????55??????????????????60

Gly?Ile?Pro?His?Thr?Trp?Ala?Ala?Phe?Ile?Trp?Leu?Cys?Gly?Pro?Ile

65??????????????????70??????????????????75??????????????????80

Ser?Gly?Met?Leu?Val?Gln?Pro?Ile?Val?Gly?Tyr?His?Ser?Asp?Arg?Cys

85??????????????????90??????????????????95

Thr?Ser?Arg?Phe?Gly?Arg?Arg?Arg?Pro?Phe?Ile?Ala?Gly?Gly?Ala?Leu

100?????????????????105?????????????????110

Ala?Val?Ala?Val?Ala?Val?Phe?Leu?Ile?Gly?Tyr?Ala?Ala?Asp?Leu?Gly

115?????????????????120?????????????????125

Gln?Leu?Ser?Gly?Asp?Pro?Val?Ala?Lys?Ser?Pro?Lys?Pro?Arg?Ala?Ile

130?????????????????135?????????????????140

Ala?Val?Phe?Val?Val?Gly?Phe?Trp?Ile?Leu?Asp?Val?Ala?Asn?Asn?Met

145?????????????????150?????????????????155?????????????????160

Leu?Gln?Gly?Pro?Cys?Arg?Ala?Leu?Leu?Ala?Asp?Leu?Ser?Gly?Ala?Asn

165?????????????????170?????????????????175

Gln?Lys?Lys?Thr?Arg?Ile?Ala?Asn?Ala?Leu?Tyr?Ser?Phe?Phe?Met?Ala

180?????????????????185?????????????????190

Val?Gly?Asn?Val?Leu?Gly?Phe?Ala?Ala?Gly?Ser?Tyr?Thr?His?Leu?Tyr

195?????????????????200?????????????????205

Lys?Ile?Phe?Pro?Phe?Thr?Lys?Thr?Lys?Ala?Cys?Asp?Val?Tyr?Cys?Ala

210?????????????????215?????????????????220

Asn?Leu?Lys?Ser?Cys?Phe?Phe?Ile?Ser?Ile?Val?Leu?Leu?Leu?Thr?Leu

225?????????????????230?????????????????235?????????????????240

Thr?Val?Leu?Ala?Leu?Ile?Tyr?Val?Arg?Glu?Lys?Pro?Trp?Ser?Pro?Glu

245?????????????????250?????????????????255

Arg?Gly?Asn?Thr?Ala?Ala?Gly?Asp?Glu?Glu?Glu?Glu?Asp?Glu?Gly?Ala

260?????????????????265?????????????????270

Ser?Glu?Ser?Ser?Pro?Met?Pro?Phe?Phe?Ser?Glu?Ile?Phe?Ala?Ser?Leu

275?????????????????280?????????????????285

Lys?Asn?Leu?Gln?Lys?Pro?Met?Trp?Ile?Leu?Leu?Leu?Val?Thr?Cys?Leu

290?????????????????295?????????????????300

Asn?Trp?Val?Ala?Trp?Phe?Pro?Phe?Leu?Leu?Phe?Asp?Thr?Asp?Trp?Met

305?????????????????310?????????????????315?????????????????320

Gly?Arg?Glu?Val?Tyr?Gly?Gly?Asp?Ser?Asn?Gly?Asn?Pro?Val?Gln?Val

325?????????????????330?????????????????335

Arg?Leu?Tyr?Asp?Arg?Gly?Val?Arg?Ala?Gly?Ala?Leu?Gly?Leu?Met?Leu

340?????????????????345?????????????????350

Asn?Ser?Val?Val?Leu?Gly?Phe?Thr?Ser?Leu?Gly?Val?Glu?Val?Leu?Ala

355?????????????????360?????????????????365

Arg?Ala?Val?Gly?Gly?Val?Lys?Arg?Leu?Trp?Gly?Ile?Val?Asn?Phe?Ile

370?????????????????375?????????????????380

Leu?Ser?Phe?Cys?Leu?Phe?Met?Thr?Ile?Leu?Ile?Thr?Lys?Met?Ala?Glu

385?????????????????390?????????????????395?????????????????400

Ser?His?Arg?Arg?Phe?Ala?Thr?Val?Gly?Gly?Gly?Ala?Thr?Val?Pro?Leu

405?????????????????410?????????????????415

Pro?Pro?Pro?Gly?Asp?Val?Lys?Ala?Gly?Ala?Leu?Ala?Leu?Phe?Ala?Val

420?????????????????425?????????????????430

Met?Gly?Val?Pro?Gln?Ala?Ile?Thr?Tyr?Ser?Ile?Pro?Phe?Ala?Leu?Ala

435?????????????????440?????????????????445

Ser?Ile?Phe?Cys?Asn?Thr?Ala?Gly?Ala?Gly?Gln?Gly?Leu?Ser?Leu?Gly

450?????????????????455?????????????????460

Val?Leu?Asn?Leu?Ser?Ile?Val?Ile?Pro?Gln?Met?Val?Val?Ser?Val?Val

465?????????????????470?????????????????475?????????????????480

Ser?Gly?Pro?Trp?Asp?Ala?Leu?Phe?Gly?Gly?Gly?Asn?Leu?Pro?Ala?Phe

485?????????????????490?????????????????495

Val?Val?Gly?Gly?Val?Ala?Ala?Ala?Ala?Ser?Gly?Ile?Phe?Ala?Phe?Thr

500?????????????????505?????????????????510

Leu?Leu?Pro?Ser?Pro?Gln?Pro?Asp?Ala?Pro?Ser?Ala?Lys?Thr?Ser?Arg

515?????????????????520?????????????????525

Ala?Val?Thr?Ala?Ala?Phe?His

530?????????????????535

 

<210>2

<211>1819

<212>DNA

＜213〉Para rubber tree of Euphorbiaceae genus hevea (Hevea brasiliensis)

<400>2

 

caaatccgac?ccaactatgt?acaaaaccac?ctctctactt?ctaccaccac?cacaaccacc????60

atcaccgccg?ccacactctt?ctcaccacca?actcccatgg?agactgatcc?tacgaaagaa????120

tccaaactcc?cctcttctga?acctcttcca?cctcctccgc?ccctagttcc?tccaccaccc????180

gcttcaagca?agcttagaaa?gattgtaatg?gtggcctcca?tagctgctgg?tatccagttt????240

ggctgggctt?tgcagctctc?tcttttgaca?ccttacgttc?agcttcttgg?tatacctcac????300

acatgggctg?ctttcatctg?gctatgtgga?cccatttcag?gaatgcttgt?ccagccaatt????360

gtcggctatc?atagtgatcg?ttgcacctcc?cgttttggcc?gtcgccgccc?ttttattgca????420

ggcggtgctt?tggccgttgc?tgtagctgtt?ttccttatcg?gttatgctgc?tgaccttggt????480

caactctctg?gtgaccctgt?tgccaagagc?cccaaaccac?gagctatagc?cgtgtttgtc????540

gttgggttct?ggattcttga?cgtcgctaat?aacatgctcc?agggtccttg?ccgtgctctc????600

ctcgccgatc?tttctggtgc?caatcagaag?aagacgcgta?tagctaatgc?actgtactcc????660

ttcttcatgg?ccgtcgggaa?cgttcttggc?tttgccgctg?gatcctacac?tcatttatat????720

aaaatatttc?cattcacaaa?gacgaaagct?tgtgacgttt?actgtgcaaa?tctgaaatct????780

tgtttcttta?tctccattgt?cttgctttta?actctcactg?tattagctct?catttacgtt????840

cgtgaaaagc?cttggtcccc?agagcgagga?aacaccgctg?ccggtgacga?ggaggaggag????900

gacgaggggg?cgtcggagtc?ttctccgatg?ccgttcttca?gtgagatatt?tgcatctttg????960

aagaatctgc?aaaaacccat?gtggatcctt?cttctggtga?cgtgcctgaa?ctgggttgca????1020

tggtttccat?tcttgttgtt?cgatactgat?tggatgggta?gagaggtata?cggcggagat????1080

tcaaacggaa?atcccgttca?agtgaggttg?tatgatcgtg?gggtgcgcgc?tggtgcactg????1140

ggtctgatgc?ttaactcggt?ggttttgggg?ttcacctccc?ttggtgtgga?ggtgctggcg????1200

cgtgctgtcg?gaggagtgaa?aagattgtgg?ggtatagtga?atttcattct?ttcattttgt????1260

ttgttcatga?cgatcttgat?caccaaaatg?gctgaatccc?acagaaggtt?cgccaccgtg????1320

ggaggcggcg?ccacggttcc?cttgccacca?cctggagacg?tcaaggctgg?tgcattggcc????1380

ctttttgcgg?tgatgggtgt?acctcaagct?ataacttaca?gtattccttt?tgctttggca????1440

tcaatatttt?gtaacactgc?tggtgctggt?caaggacttt?ctttgggagt?tctgaatctt????1500

tcaattgtta?taccacagat?ggtggtgtca?gtagtaagcg?gaccatggga?tgcattattt????1560

ggagggggta?atctaccagc?ttttgtggtg?ggaggggttg?ctgccgcggc?cagtggaata????1620

tttgcattca?ccttactccc?atctccgcag?cctgatgccc?cctccgccaa?gacttcaagg????1680

gctgtcacag?ctgccttcca?ttgatcttca?tcacctccat?gtgtatgcgg?aaatgtgatc????1740

atgtagatgt?gccttttgtt?ttcgtgtgga?aaggccagaa?tggccatgtc?ttgtgttagt????1800

tggtgttcta?atagggcat?????????????????????????????????????????????????1819

 

<210>3

<211>3689

<212>DNA

＜213〉Para rubber tree of Euphorbiaceae genus hevea (Hevea brasiliensis)

<400>3

 

caaatccgac?ccaactatgt?acaaaaccac?ctctctactt?ctaccaccac?cacaaccacc????60

atcaccgccg?ccacactctt?ctcaccacca?actcccatgg?agactgatcc?tacgaaagaa????120

tccaaactcc?cctcttctga?acctcttcca?actcctccgc?ccctagctcc?tccaccaccc????180

gcttcaagca?agcttagaaa?gattgtaatg?gtggcctcca?tagctgctgg?tatccagttt????240

ggctgggctt?tgcagctctc?tcttttgaca?ccttacgttc?agcttcttgg?tatacctcac????300

acatgggctg?ctttcatctg?gctatgtgga?cccatttcag?gaatgcttgt?ccagccaatt????360

gtcggctatc?atagtgatcg?ttgcacctcc?cgttttggcc?gtcgccgccc?ttttattgca????420

ggcggtgctt?tggccgttgc?tgtagctgtt?ttccttatcg?gttatgctgc?tgaccttggt????480

caactctctg?gtgaccctgt?tgccaagagc?cccaaaccac?gagctatagc?cgtgtttgtc????540

gttgggttct?ggattcttga?cgtcgctaat?aacatgctcc?agggtccttg?ccgtgctctc????600

ctcgccgatc?tttctggtgc?caatcagaag?aagacgcgta?tagctaatgc?actgtactcc????660

ttcttcatgg?ccgtcgggaa?cgttcttggc?tttgccgctg?gatcctacac?tcatttatat????720

aaaatatttc?cattcacaaa?gacgaaagct?tgcgacgttt?actgtgcaaa?tctgaaatct????780

tgtttcttta?tctccattgt?cttgctttta?actctcactg?tattagctct?catttacgtt????840

cgtgaaaagc?cttggtcccc?agagcgagga?aacaccgctg?ccggtgacga?ggaggaggag????900

gacgaggggg?cgtcggagtc?ttctccgatg?ccgttcttca?gtgagatatt?tgcatctttg????960

aagaatctgc?aaaaacccat?gtggatcctt?cttctggtga?cgtgcctgaa?ctgggttgca????1020

tggtttccat?tcttgttgtt?cgatactgat?tggatgggta?gagaggtata?cggcggagat????1080

tcaaacggaa?atcccgatca?agtgaggttg?tatgatcgtg?gggtgcgcgc?tggtgcactg????1140

ggtctgatgc?ttaactcggt?ggttttgggg?ttcacctccc?ttggtgtgga?ggtgctggcg????1200

cgtgctgtcg?gaggagtgaa?aagattgtgg?ggtatagtga?atttcattct?ttcattttgt????1260

ttgttcatga?cgatcttgat?caccaaaatg?gctgaatccc?acagaaggtt?cgccaccgtg????1320

ggaggcggcg?ccacggttcc?cttgccacca?cctggagacg?tcaaggctgg?tgcattggcc????1380

ctttttgcgg?tgatgggtgt?acctcaagct?gtaagttctt?ctggcctacc?ttccctgaat????1440

ctctttcttc?tttctctatt?tttctctttt?gtcctaattt?gtaggttaac?agtcaacaat????1500

atttcctgct?gtgattatgc?aatacctacc?tttctcttta?ctttagaaaa?aaaaaaatta????1560

cgtgtgattt?ctgtctttta?tattttaatt?attcataatt?attaattatt?taaaagggta????1620

aatttaatgt?gggcccttgt?gcatttatta?gaatttttat?tattgtatta?tatccaatca????1680

cttttaattt?tttatattta?agttttatat?gataattaaa?aaataattta?ataattttat????1740

tttcataaaa?atacaataat?taattaaatt?aatatttaat?agaaataaaa?aagtaaaatt????1800

aaaaaaatta?atatattttt?attttttatt?aatttttaaa?atgataaatg?attttgaata????1860

aaataaaaat?tttaatatga?tagataaatt?agacgaatat?agataaattt?tttttgtcta????1920

aatttaattt?atcactgatt?taagattcaa?gatatatgat?gagacttacc?tattaaatat????1980

ataaatacta?cgtattgaga?tattaaatct?aaaaaacaat?ttctcaatcc?aaataatatt????2040

ttttaattga?gattgaagaa?atttcaattt?tatctaaaaa?tgaagattgt?tctgacttct????2100

aatgtcttat?ttgctttaag?aaacagactt?actatttgtt?attatatatt?atatataggt????2160

atactatata?ttaggttgct?taattaggaa?tatcaaagaa?acattttacc?ttattttttt????2220

ttccttaatt?aactttaatt?ttgtatgcag?gtttttcttt?ttttcttttt?ggtccctgat????2280

attaattcta?attaattagt?ggcacatttc?tttgtttatt?taacttgtgc?agataactta????2340

cagtattccc?tttgctttgg?catcaatatt?ttgtaacact?gctggtgctg?gtcaaggtaa????2400

ttaattaata?tattttttaa?attataaaat?aaatcctctt?aaaatattaa?gttttattct????2460

tctgacccat?ttcaatgttt?caatgttagg?actttctttg?ggagttctga?atctttcaat????2520

tgttatacca?caggtattat?tattattatt?atacccattt?gtgtttccct?aagaagattt????2580

ggcaacttat?taaaaatagg?attaaaaaaa?aaaaactata?tcatagctag?ctctaagacc????2640

tatttggcat?tgaattatta?aaattgcttt?tttttttaaa?tattgttaaa?aaataatttt????2700

aaaaaaataa?ttttaatttt?ttatgattaa?aagttattaa?atttaatttt?taattatttt????2760

tttaattaat?taatatattt?aaaaaatatt?ttttgtggtg?gtactagcag?gctggtggtg????2820

cgttagaaac?aagataggta?gttgaaaaag?aataataatt?gatctgcaaa?atttgaccca????2880

tgtgaggaat?attttcttgg?ggaagaacat?atgtaaggac?aacttagttg?agtatggcca????2940

acaatacctt?gcttttgaaa?ttattgaata?agtcaaaaat?aaactctttt?ctctttacta????3000

ccattgcatc?ccatacagtg?atggctttgg?aaattattat?ttttttagta?caattgtatc????3060

ttattggtat?tcaaacttga?tatatcacag?ctaccattct?aatattactg?acttaaaact????3120

tatcaataac?tttggaaatt?actaagaaaa?ataaaggttc?tatccacttt?ctcaatctgt????3180

tttgttcttt?ctttctatta?ttctctctgg?tgtatcatat?aatgaaattc?tttgatttgt????3240

agaaaaagaa?aaaagaaaaa?gagaaaggaa?aaggaaaagg?aaaagggttt?gttgcagctt????3300

tgattataag?aatattgtta?gttaagttga?ttggcattga?tttgaaacat?tttcatgtgt????3360

tcatgctttt?ggaaaaacat?tgttgcagat?ggtggtgtca?gtagcaagcg?gaccatggga????3420

tgcattattt?ggagggggta?atctaccagc?ttttgtggtg?ggaggggttg?ctgccgcggc????3480

cagtggaata?tttgcattca?ccttactccc?atccccgcag?cctgatgccc?cctccgccaa????3540

gacttcaagg?gctgtcactg?ctgccttcca?ttgatcttca?tcacctccat?gtgtatgcgg????3600

aaatgtaatc?atgtagatgt?gccttttgtt?ttcatatgga?aaggccagaa?tggccatgtc????3660

ttgtgttagt?tggtgttcta?atagggcat??????????????????????????????????????3689

Claims (10)

1. sucrose transporter is the protein with one of following amino acid residue sequences:

1) amino acid residue sequence of the SEQ ID № .1 in the sequence table;

2) with the SEQ ID № .1 amino acid residue sequence in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the sucrose transport function by SEQ ID №: 1 deutero-protein.

2. the encoding gene of the described sucrose transporter of claim 1.

3. encoding gene according to claim 2 is characterized in that: the encoding gene of described sucrose transporter has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 nucleotide sequence;

2) can be with 1 under stringent condition) nucleotide sequence of described dna sequence dna hybridization.

3) with sequence table in SEQ ID №: 2 nucleotide sequence has a homology more than 80% and proteins encoded has the nucleotide sequence of sucrose transport function.

4. encoding gene according to claim 2 is characterized in that: the genomic gene of described sucrose transporter has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 3 nucleotide sequence;

2) can be with 1 under stringent condition) nucleotide sequence of described dna sequence dna hybridization.

3) with sequence table in SEQ ID №: 3 nucleotide sequence has a homology more than 80% and proteins encoded has the nucleotide sequence of sucrose transport function.

5. the recombinant expression vector that contains claim 2 or 3 or 4 described sucrose transporter encoding genes.

6. the transgenic cell line that contains claim 2 or 3 or 4 described sucrose transporter encoding genes.

7. the transgenosis reorganization bacterium that contains claim 2 or 3 or 4 described sucrose transporter encoding genes.

8. claim 2 or the 3 or 4 described sucrose transporter encoding genes application in the transgenosis rubber tree of cultivating the output raising.

9. claim 2 or 3 or 4 described sucrose transporter encoding genes are improving microorganism to the application in the sucrose utilising efficiency.

10. application according to claim 9 is characterized in that: described microorganism is a yeast saccharomyces cerevisiae.

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